Clinical features of culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia OBJECTIVE: This retrospective chart review describes the epidemiology and clinical features of 40 patients with culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. METHODS: Patients with positive M. pneumoniae cultures from respiratory specimens from January 1997 through December 1998 were identified through the Microbiology records. Charts of patients were reviewed. RESULTS: 40 patients were identified, 33 (82.5%) of whom required admission. Most infections (92.5%) were community-acquired. The infection affected all age groups but was most common in infants (32.5%) and pre-school children (22.5%). It occurred year-round but was most common in the fall (35%) and spring (30%). More than three-quarters of patients (77.5%) had comorbidities. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common symptoms, and crepitations (60%), and wheezes (40%) were the most common signs. Most patients with pneumonia had crepitations (79.2%) but only 25% had bronchial breathing. Immunocompromised patients were more likely than non-immunocompromised patients to present with pneumonia (8/9 versus 16/31, P = 0.05). Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications, 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities. CONCLUSION: our results were similar to published data except for the finding that infections were more common in infants and preschool children and that the mortality rate of pneumonia in patients with comorbidities was high. Mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections. It remains one of the most frequent causes of atypical pneumonia particu-larly among young adults. [1, 2, 3, 4, 5] Although it is highly transmissible, most infections caused by this organism are relatively minor and include pharyngitis, tracheobronchitis, bronchiolitis, and croup with one fifth of in-fections being asymptomatic. [6, 7] Only 3 -10% of infected subjects develop symptoms consistent with bronchopneumonia and mortality from infection is rare. [6, 7] The organism is fastidious and difficult to grow on cultures. Therefore, diagnosis of infections caused by this organism is usually confirmed with serological tests or polymerase chain reaction-gene amplification techniques. At King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, the facility to perform Mycoplasma culture has been available since January 1997. As published information concerning M. pneumoniae infections in Saudi Arabia is scarce, [8, 9, 10] we wished to study the epidemiology and clinical features of cultureproven infections caused by this organism at this hospital.
KAUH is a tertiary care teaching hospital with a bed capacity of 265 beds and annual admissions of 18000 to 19000 patients. Patients with M. pneumoniae positive cultures from respiratory specimens were identified over a 24-months" period from January, 1997 through December, 1998 for this review.
During the study period, respiratory specimens (sputum, nasopharyngeal aspiration, endotracheal secretion, and bronchoalveolar lavage) for M. pneumoniae culture were obtained from patients with upper or lower respiratory tract infections seen as inpatients or in the outpatient or emergency departments. Respiratory specimens were aslo Gram-stained and cultured for bacteria and viruses. M. pneumoniae serological tests for IgG or IgM were not available at KAUH during the study period. All positive culture results were obtained from the Microbiology laboratory records. Charts of patients were reviewed with standardized data collection. Information collected included patients' demographics, comorbidities, clinical manifestations, complications, and outcome.
M. pneumoniae was cultured using the classic M. pneumoniae agar medium (M.P. agar) and the Pneumofast tray (Pneumofast ® , International Microbio, Signes, France). Specimens were processed according to the instructions of the manufacturer. The M.P. agars and Pneumofast trays were incubated anaerobically at 37°C and inspected daily for 4 weeks. The organism was identified based on typical colonial morphology (granular colonies, rarely fried-egg-like, 10-150 ∝ in diameter) on the M.P. agar medium and the change in the Pneumofast broth color from red to orange then to yellow (glucose fermentation) in the absence of turbidity of the broth. Antibiotic sensitivity profile on the Pneumofast tray was also used for identification according to the instructions of the manufacturer. Bacterial and viral cultures were performed using standard methods.
M. pneumoniae isolates were considered community-acquired if they were recovered from unhospitalized patients or within 72 hours of admission to the hospital, and nosocomial if they were recovered beyond that period.
Pneumonia was diagnosed based on clinical symptoms and signs, along with radiographic evidence of pneumonia when possible. Severe pneumonia was defined as pneumonia associated with tachycardia (>140 /minute), tachypnoea (>30/minute), hypotension (Systolic blood pressure <90 mmHg), hypoxemia (arterial oxygen partial pressure <8 kPa or oxygen saturation <90%), and/or more than 2 areas of consolidation.
Outcome of patients with M. pneumoniae infection was classified into 4 categories; uneventful recovery, recovery following complications, death due to M. pneumoniae infection, or death unrelated to M. pneumoniae infection.
The Statistical Package for Social Sciences (SPSS) program was used for data analysis. Comparison of categorical data was by Chi-square statistic or Fisher's exact test for small expected values.
A total of 40 respiratory specimens from 40 patients were positive for M. pneumoniae over the 24-months study period. The demographic and epidemiological characteristics of the patients are summarized in Table 1 . Of all isolates, 37 (92.5%) were community-acquired and 3 (7.5%) were nosocomial. Thirty-three (82.5%) patients required admission to the hospital and the remaining 7 (17.5%) were treated as outpatients. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Of the 24 cases of pneumonia, 21 were confirmed radiologically and the remaining 3 were diagnosed clinically. The two cases of bronchiolitis occurred in 2 children, one and three years old. Thirty-one patients (77.5%) had comorbidities. Eleven patients (27.5%) had cardiopulmonary comorbidities (asthma, 8, lung fibrosis, 1, congestive heart failure, 1, congenial heart disease, 1), 9 patients (22.5%) were immunocompromised (malignancy, 7, steroid therapy, 3, Human immunodeficiency virus infection, 1), and 11 patients (27.5%) had other comorbidities (premature newborns, 2, and one each of myelodysplastic syndrome, myelopro-liferative disorder, sickle cell anemia, Evan's syndrome, Down syndrome, sarcoidosis, demyelinating disease, cerebral palsy, and spinal muscle atrophy). Organisms concomitantly isolated with M. pneumoniae from the respiratory tract included herpes simplex virus type 1 (2 occasions), adenovirus (2 occasions), cytomegalo virus (1 occasion), respiratory syncytial virus (1 occasion), and bacterial isolates (2 occasions: Acinetobacter species, 1, and Enter obacter cloacae, 1).
Clinical manifestations associated with M. pneumoniae infections are summarized in Table 2 . Pneumonia was more common than upper respiratory tract infections (57.5 % versus 27.5%, respectively). Immunocompromised patients were more likely to present with pneumonia as opposed to upper respiratory tract infection or bronchiolitis than non-immunocompromised patients (8/9 versus 16/31, P = 0.05). Similarly, there was a tendency for patients 60 years of age or older to present with pneumonia more frequently than those below 60 (4/4 versus 20/36, P = 0.1). Of the 24 patients with clinically or radiologically confirmed pneumonia, 19 (79.2%) had crepitations and only 6 (25%) had bronchial breath sounds on physical examination. Of the 16 patients in whom wheezes were detected, 9 (56.3%) were not known to have asthma or other obstructive airway disease. Table 3 . Of the 24 patients with pneumonia, 21 (87.5%) were admitted to the hospital, and 20 (83.3%) had comorbidities. All patients with upper respiratory tract infections (11 patients) or bronchiolitis (2 patients) had uneventful recovery. Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications (acute respiratory distress syndrome, 2, respiratory failure, 1, septic shock, 1), 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities; one had congestive heart failure, the second had congenital heart disease, and the third was a 3months old infant born prematurely at 32 weeks of gestation who previously had 3 episodes of pneumonia due to other pathogens.
Mycoplasma pneumoniae is one of the most common causes of atypical pneumonia accounting for 5-23% of community-acquired pneumonia, [1, 2, 3, 4, 5] In a study of 511 children with acute respiratory tract infection in Riyadh, Saudi Arabia, Mycoplasma pneumoniae was found to be the second most common causative agent after Respiratory syncytial virus (RSV) accounting for 9% of all cases, [8] In a study of 112 adult patients with community acquired pneumonia admitted to a military hospital in Riyadh, Saudi Arabia, this organism accounted for 6% of all cases, [9] In another retrospective study of 567 pneumonic episodes in adult patients from Al-Qassim area, the organism accounted for 23% of all episodes, [10] The organism also causes other relatively minor infections such as pharyngitis, tracheobronchitis, bronchiolitis, and croup. It is transmitted from person-to-person by infected respiratory droplets during close contact. It is most common in school-aged children, military recruits, and college students. [11] Most cases occur singly or as family outbreaks. Larger outbreaks can also occur in closed populations such as military recruit camps or boarding schools, [12] Infection occurs most frequently during the fall and winter in temperate climates but may develop year-round, [13] The average incubation period is 3 weeks following exposure, [6] Although rare, complications are protean and may involve virtually any organ system such as the respiratory system (e.g.: pleurisy, pneumothorax, acute respiratory distress syndrome, lung abscess), the hematologic system (e.g.: hemolytic anemia, intravascular coagulation, thrombocytopenia), the dermatologic system (e.g.: maculopapular or urticarial rashes, erythema multiforme, erythema nodosum), the musculoskeletal system (e.g.: myalgias, arthralgias, arthritis), the cardiovascular system (e.g.: pericarditis, myocarditis), the nervous system (e.g.: meningoencephalitis, Guillain-Barre syndrome, neuropathies, acute psychosis), or the eye (optic disc edema, optic nerve atrophy, retinal exudation and hemorrhages). [6, 7, 14, 15, 16, 17, 18] Immunity following infection is not long lasting. [11] In our study, the infection affected all age groups but was most common in infants (32.5%) and preschool children (22.5%), and least common in adults aged 15 to 30 years (2.5%) and elderly above 70 years of age (5%). This contrasts with data from temperate countries where the infection is most common in school-aged children, and young adults. [11] One possible explanation for this difference is that infants and preschool children perhaps had more severe infections than did school-aged children, and young adults which prompted presentation of the former group to the hospital. The infection occurred year-round but was most common in the fall (35%), and spring (30%), and least common in the summer (10%). Most infections were community-acquired (92.5%).
More than one half of patients (57.5%) presented with pneumonia, and about a third (27.5%) presented with upper respiratory tract infection, Immunocompromised patients and patients 60 years of age or older were more likely to present with pneumonia as opposed to upper respiratory tract infection than non-immunocompromised patients or those below 60 years of age. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common presenting symptoms. Cough was usually dry or slightly productive of white sputum and mild to moderate in severity. Most febrile patients had mild to mod- erate fever of 39°C or less; high-grade fever of more than 39°C was rare. Crepitations (60%), and wheezes (40%) were the most common signs. Wheezes were as common in patients with no history of obstructive airway disease (9 patients) as it was in those with such a history (7 patients). Bronchial breathing as a sign of consolidation was detected in only one fourth of patients with pneumonia, which is consistent with the known disparity between clinical and radiological signs of M pneumoniae pneumonia. Crepitations, however, were detected in the majority (79.2%) of patients. Pleuritic chest pain and pleural effusion were rare.
More than half (56.5%) of the patients with pneumonia had uneventful recovery. Mortality from M. pneumoniae pneumonia was high (12.5%) and occurred only in patients with underlying comorbidities. None of the 9 patients with no underlying comorbidities died of M pneumoniae pneumonia. The relatively high complications rate (16.7%) and mortality (12.5%) related to M. pneumoniae pneumonia are likely due to selection bias as most patients with pneumonia were sick enough to require admission to the hospital (21/24 or 87.5%) and most of them had comorbidities (20/24 or 83.3%).
In conclusion, our data shed some light on the epidemiology and clinical features of M pneumoniae infections in one of the Saudi tertiary care centers. The data are comparable to those of other countries except for the finding that infections were more common in infants and preschool children than in school children and young adults. Additionally, mortality attributable to M. pneumoniae pneumonia was relatively high in patients with comorbidities. It is hoped this information will assist clinicians in their approach and management of respiratory tract infections. Nitric oxide: a pro-inflammatory mediator in lung disease? Inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO•) and increased indices of NO• -dependent oxidative stress. Although NO• is known to have anti-microbial, anti-inflammatory and anti-oxidant properties, various lines of evidence support the contribution of NO• to lung injury in several disease models. On the basis of biochemical evidence, it is often presumed that such NO• -dependent oxidations are due to the formation of the oxidant peroxynitrite, although alternative mechanisms involving the phagocyte-derived heme proteins myeloperoxidase and eosinophil peroxidase might be operative during conditions of inflammation. Because of the overwhelming literature on NO• generation and activities in the respiratory tract, it would be beyond the scope of this commentary to review this area comprehensively. Instead, it focuses on recent evidence and concepts of the presumed contribution of NO• to inflammatory diseases of the lung. Since its discovery as a biological messenger molecule more than 10 years ago, the gaseous molecule nitric oxide (NO • ) is now well recognized for its involvement in diverse biological processes, including vasodilation, bronchodilation, neurotransmission, tumor surveillance, antimicrobial defense and regulation of inflammatory-immune processes [1] [2] [3] . In the respiratory tract, NO • is generated enzymically by three distinct isoforms of NO • synthase (NOS-1, NOS-2 and NOS-3) that are present to different extents in numerous cell types, including airway and alveolar epithelial cells, neuronal cells, macrophages, neutrophils, mast cells, and endothelial and smoothmuscle cells. In contrast with the other two NOS isoforms (NOS-1 and NOS-3), which are expressed constitutively and activated by mediator-induced or stress-induced cell activation, NOS-2 activity is primarily regulated transcriptionally and is commonly induced by bacterial products and pro-inflammatory cytokines. As such, inflammatory diseases of the respiratory tract, such as asthma, acute respiratory distress syndrome (ARDS) and bronchiectasis, are commonly characterized by an increased expression of NOS-2 within respiratory epithelial and inflammatory-immune cells, and a markedly elevated local production of NO • , presumably as an additional host defense mechanism against bacterial or viral infections. The drawback of such excessive NO • production is its accelerated metabolism to a family of potentially harmful reactive nitrogen species (RNS), including peroxynitrite (ONOO -) and nitrogen dioxide (NO 2 • ), especially in the presence of phagocyte-generated oxidants. The formation of such RNS is thought to be the prime reason why NO • can in many cases contribute to the etiology of inflammatory lung disease [4] [5] [6] . Despite extensive research into both pro-inflammatory and anti-inflammatory actions of NO • , the overall contribution of NO • to inflammatory conditions of the lung is not easily predicted and seems to depend on many factors, such as the site, time and degree of NO • production in relation to the local redox status, and the acute or chronic nature of the immune response. In addition, our current understanding of the pro-inflammatory or pro-injurious mechanisms of NO • or related RNS is incomplete; this commentary will focus primarily on these latter aspects.
To explore a role for NO • (or NOS) in infectious or inflammatory diseases, two general research approaches have been taken: the use of pharmacological inhibitors of NOS isoenzymes, and the targeted deletion of individual NOS enzymes in mice. Both approaches suffer from the shortcoming that animal models of respiratory tract diseases generally do not faithfully reflect human disease. The use of NOS inhibitors to determine the contribution of individual NOS isoenzymes is also hindered by problems related to specificity and pharmacokinetic concerns. However, the unconditional gene disruption of one or more NOS isoforms, leading to lifelong deficiency, can have a markedly different outcome from pharmacological inhibition at a certain stage of disease, as the involvement of individual NOS isoenzymes can be different depending on disease stage and severity. Despite these inherent limitations, studies with the targeted deletion of NOS isoforms have led to some insights, indicating a role for NO • and NOS-2 in the etiology of some inflammatory lung diseases. For instance, mice deficient in NOS-2 are less susceptible to lethality after intranasal inoculation with influenza A virus, suffer less lung injury after administration of endotoxin, and display reduced allergic eosinophilia in airways and lung injury in a model of asthma, than their wild-type counterparts [7] [8] [9] . However, although the contribution of NOS-2 is expected in inflammatory conditions, recent studies have determined that NOS-1, rather than NOS-2, seems to be primarily involved in the development of airway hyper-reactivity in a similar asthma model [10] . The linkage of NOS-1 to the etiology of asthma was more recently supported in asthmatic humans by an association of a NOS-1 gene polymorphism with this disease, although the physiological basis for this association remains unclear [11] .
Despite the potential contribution of NOS-2-derived NO • to lung injury after endotoxemia, the sequestration of neutrophils in the lung and their adhesion to postcapillary and postsinusoidal venules after administration of endotoxin were found to be markedly increased in NOS-2-deficient mice, and NOS-2 deficiency did not alleviate endotoxininduced mortality. It therefore seems that the 'harmful' and 'protective' effects of NOS-2 might contend with each other within the same model, which makes the assessment of the potential role of NOS in human disease even more difficult. In this context, it is interesting to note that humans or animals with cystic fibrosis have subnormal levels of NOS-2 in their respiratory epithelium, related to a gene mutation in the cystic fibrosis transmembrane conductance regulator [12] . This relative absence of epithelial NOS-2 might be one of the contributing factors behind the excessively exuberant respiratory tract inflammatory response in patients with cystic fibrosis, even in the absence of detectable respiratory infections. Overall, the apparently contrasting findings associated with NOS deficiency, together with concerns about animal disease models used, make interpretations and conclusions with regard to human lung disease all the more difficult.
Pharmacological inhibitors of NOS have also been found to reduce oxidative injury in several animal models of lung injury, such as ischemia/reperfusion, radiation, paraquat toxicity, and endotoxemia (see, for example, [13] [14] [15] ). However, results are again not always consistent, and in some cases NOS inhibition has been found to worsen lung injury, indicating anti-inflammatory or protective roles for NO • . All in all, despite these inconsistencies, there is ample evidence from such studies to suggest a contributing role of NO • in various respiratory disease conditions, which continues to stimulate research into mechanistic aspects underlying such pro-inflammatory roles and modulation of NO • generation as a potential therapeutic target.
Although the pro-inflammatory and injurious effects of NO • might be mediated by a number of diverse mechanisms, it is commonly assumed that such actions are largely due to the generation of reactive by-products generated during the oxidative metabolism of NO • ; these are collectively termed RNS. One of the prime suspects commonly implicated in the adverse or injurious properties of NO • is ONOO -, a potent oxidative species formed by its almost diffusion-limited reaction with superoxide (O 2
•-), which is a product of activated phagocytes and of endothelial or epithelial cells [4, 5, 13] . The formation of ONOOseems highly feasible under conditions of elevated production of both NO • and O 2
•in vivo, and its oxidative and cytotoxic potential is well documented [5, 6] . However, because the direct detection of ONOOunder inflammatory conditions is virtually impossible because of its instability and high reactivity, the formation of ONOOin vivo can be demonstrated only by indirect methods. Thus, many investigators have relied on the analysis of characteristic oxidation products in biological molecules, such as proteins and DNA, most notably free or protein-associated 3-nitrotyrosine, a product of tyrosine oxidation that can be formed by ONOO -(and several other RNS) but not by NO • itself (see, for commentary review reports primary research http://respiratory-research.com/content/1/2/067 example, [5] ). Indeed, elevated levels of 3-nitrotyrosine have been observed in many different inflammatory conditions of the respiratory tract [16] , which illustrates the endogenous formation of ONOOor related RNS in these cases. However, without known evidence for functional consequences of (protein) tyrosine nitration, the detection of 3-nitrotyrosine should not be regarded as direct proof of a pro-inflammatory role of NO • . Moreover, although the detection of 3-nitrotyrosine has in most cases been interpreted as conclusive evidence for the formation of ONOOin vivo (see, for example, [17] ), it should be realized that other RNS formed by alternative mechanisms might also contribute to endogenous tyrosine nitration. Indeed, it has recently become clear that the presence of inflammatory-immune cells, and specifically their heme peroxidases myeloperoxidase (MPO) and eosinophil peroxidase (EPO), can catalyze the oxidization of NO • and/or its metabolite NO 2 to more reactive RNS and thereby contribute to protein nitration [16, 18, 19] . This notion is further supported by the fact that 3-nitrotyrosine is commonly detected in tissues affected by active inflammation, mostly in and around these phagocytic cells and macrophages, which can also contain active peroxidases originating from apoptotic neutrophils or eosinophils. Hence, the detection of 3-nitrotyrosine in vivo cannot be used as direct proof of the formation of ONOO -, but merely indicates the formation of RNS by multiple oxidative pathways, possibly including ONOObut more probably involving the activity of phagocyte peroxidases [16, 20] . In this regard, a preliminary study with EPO-deficient mice has recently demonstrated the critical importance of EPO in the formation of 3-nitrotyrosine in a mouse model of asthma [21] . Future studies with animals deficient in MPO and/or EPO will undoubtedly help to clarify this issue.
Given the considerable interest in 3-nitrotyrosine as a collective marker of the endogenous formation of NO •derived RNS, the crucial question remains of whether the detection of 3-nitrotyrosine adequately reflects the toxic or injurious properties of NO • . The formation of ONOO -(or of other RNS that can induce tyrosine nitration) might in fact represent a mechanism of decreasing excessive levels of NO • that might exert pro-inflammatory actions by other mechanisms. For instance, NO • can promote the expression of pro-inflammatory cytokines or cyclo-oxygenase (responsible for the formation of inflammatory prostanoids) by mechanisms independent of ONOO - [22, 23] , and the removal of NO • would minimize these responses. Furthermore, although ONOOor related NO •derived oxidants can be cytotoxic or induce apoptosis, these effects might not necessarily relate to their ability to cause protein nitration (see, for example, [16]). For instance, the bactericidal and cytotoxic properties of ONOOare minimized by the presence of CO 2 , even though aromatic nitration and other radical-induced modifications are enhanced [5] . Similarly, the presence of NO 2 in the incubation medium decreases the cytotoxicity of MPO-derived hypochlorous acid (HOCl) toward epithelial cells or bacteria, despite increased tyrosine nitration of cellular proteins (A van der Vliet and M Syvanen, unpublished data). Thus, it would seem that the cytotoxic properties of NO • and/or its metabolites might instead be mediated through preferred reactions with other biological targets, and these might not necessarily be correlated with the degree of tyrosine nitration. The extent of nitrotyrosine immunoreactivity in bronchial biopsies of asthmatic patients was correlated directly with measured levels of exhaled NO • and inversely with the provocation concentration for methacholine (PC 20 ) and forced expiratory volume in 1 s [24] . However, an immunohistochemical analysis of nitrotyrosine and apoptosis in pulmonary tissue samples from lung transplant recipients did not identify patients with an imminent risk of developing obliterative bronchiolitis [25] . It is therefore still unclear to what degree tyrosine nitration relates to disease progression.
Several studies with purified enzymes have suggested that nitration of critical tyrosine residues adversely affects enzyme activity, but there is as yet no conclusive evidence in vivo for biological or cellular changes as a direct result of tyrosine nitration [16, 20] . For instance, tyrosine nitration was suggested to have an effect on cellular pathways by affecting cytoskeletal proteins or tyrosine phosphorylation, thereby affecting processes involved in, for example, cell proliferation or differentiation [16, 26] . Recent studies have provided support for selective tyrosine nitration within certain proteins [27, 28] and of selective cellular targets for nitration by RNS (see, for example, [29, 30] ), and such specificity might indicate a potential physiological role for this protein modification. However, in none of these cases could tyrosine nitration be linked directly to changes in enzyme function. Chemical studies have indicated that tyrosine nitration by RNS accounts for only a minor fraction of oxidant involved, and reactions with other biological targets (thiols, selenoproteins, or transition metal ions) are much more prominent [5, 6] . Indeed, the extent of tyrosine nitration in vivo is very low (1-1000 per 10 6 tyrosine residues according to best estimates [16]), although different analytical methods used to detect 3-nitrotyrosine in biological systems have often given inconsistent results. It is important to note that recent rigorous studies have unveiled substantial sources of artifact during sample preparation, which might frequently have led to an overestimation of tyrosine nitration in vivo in previous studies [31] .
On the basis of current knowledge, the formation of 3-nitrotyrosine seems to be merely a marker of NO •derived oxidants, with as yet questionable pathophysiological significance. In view of the low efficiency of tyrosine nitration by biological RNS, and the endogenous presence of variable factors that influence protein nitration (antioxidants or other RNS scavengers), it seems unlikely that tyrosine nitration is a reliable mechanism of, for example, enzyme regulation. Nevertheless, the recent discovery of enzymic 'denitration' mechanisms that can reverse tyrosine nitration [32] merits further investigation of the possibility that tyrosine nitration might reflect a signaling pathway, for example analogous to tyrosine phosphorylation or sulfation.
The biological effects of NO • are mediated by various actions, either by NO • itself or by secondary RNS, and the overall biochemistry of NO • is deceptively complex. Moreover, the metabolism and chemistry of NO • depend importantly on local concentrations and pH; the recently described acidification of the airway surface in asthmatics [33] might significantly affect NO • metabolism in these patients. It is well known that interactions with the ion centers of iron or other transition metals are responsible for many of the signaling properties of NO • ; the activation of the heme enzyme guanylyl cyclase and the consequent formation of cGMP is involved not only in smooth-muscle relaxation but also in the activation of certain transcription factors, the expression of several pro-inflammatory and anti-inflammatory genes (including cytokines and cyclo-oxygenase), and the production of respiratory mucus [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . In addition to such direct signaling properties, many actions of NO • might be due largely to secondary RNS that can react with multiple additional targets, in some cases forming nitroso or nitro adducts as potentially unique NO • -mediated signaling mechanisms. As discussed, the formation of protein nitrotyrosine has been postulated as a potential RNSspecific signaling pathway. Even more interest has been given to the reversible S-nitros(yl)ation of protein cysteine residues, which has been proposed to affect a number of redox-sensitive signaling pathways, for example by the activation of p21 ras or the inhibition of protein tyrosine phosphatases [35, 36] . Similar modifications of reactive cysteine residues in transcription factors such as nuclear factor-κB or of caspases contribute to the regulation of gene expression and apoptosis [37] [38] [39] . The precise mechanisms leading to protein S-nitrosylation in vivo are still not clarified, but might involve dinitrogen trioxide (formed during the autoxidation of NO • ), iron-nitrosyl complexes, and perhaps ONOO - [16] ; changes in NO • metabolism during inflammatory lung diseases undoubtedly affect such NO • -dependent signaling pathways. In addition, S-nitrosylation can be reversed by either enzymic (thioredoxin or glutaredoxin) or chemical (metals or oxidants) mechanisms, and evidence is increasing that this reversible modification is complementary to more widely accepted oxidant-dependent redox signaling pathways [40] . The reported alterations in S-nitrosothiol levels in tracheal secretions of patients with asthma or cystic fibrosis further point to altered NO • metabolism in these cases, and might provide new clues to the role of S-nitrosylation in controlling such disease processes [41, 42] . Unfortunately, technical limitations to detect S-nitrosylation in specific protein targets in vivo have limited a full understanding of this potential signaling pathway; further research in these areas can be expected to establish more clearly its significance in the pathophysiological properties of NO • .
Despite the by now overwhelming evidence for the increased formation of NO • and NO • -derived oxidants in many different lung diseases, the exact contribution of NO • or its metabolites to inflammatory lung disease is still unclear. Indeed, NO • might have distinctly different roles in different stages of respiratory tract inflammatory diseases, being pro-inflammatory or pro-injurious in acute and severe stages but perhaps being protective and antiinflammatory in more stable conditions; it is uncertain whether NOS is a suitable therapeutic target in the management of inflammatory lung disease. Caution is clearly needed when interpreting observations of tyrosine nitration in animal models of disease or in human tissues, which does not automatically implicate ONOO -(as often thought), but rather indicates the formation of RNS by various mechanisms. Furthermore, animal models of chronic lung disease that usually reflect short-term or acute inflammation might not always be applicable to chronic airway diseases in humans. For instance, phagocyte degranulation, a common feature observed in association with human airway inflammatory diseases such as asthma, does not seem to occur in mouse models of asthma [43] . Therefore the importance of granule proteins, such as heme peroxidases, in the pathology of human airway diseases might not be adequately reflected in such animal models. More work with animal models more characteristic of human diseases or with biopsy materials from human subjects will be required to unravel the precise role of NO • in inflammatory lung disease, and might establish more clearly whether the pharmacological inhibition of NOS isoenzymes can be beneficial. This brings up the interesting paradox that, despite presumed adverse roles of NO • in such inflammatory lung diseases as septic shock and ARDS, NO • inhalation has been suggested as a potential therapeutic strategy to improve overall gas exchange [44] . Intriguingly, in a rat model of endotoxemia, inhalation of NO • was found to reduce neutrophilic inflammation and protein nitration [45] , again supporting the crucial involvement of inflammatory-immune cells in this protein modification.
For a better assessment of the role of NO • in respiratory tract diseases in humans, the production of RNS and/or characteristic markers would need to be more carefully monitored during various disease stages. Care should be given to analytical techniques, their quantitative capacity and the possibility of artifacts. The monitoring of exhaled NO • , although convenient and non-invasive, does not reflect the actual production or fate of NO • in the respiratory tract and is not well correlated with NOS activity in the lung [46] . We therefore need to continue research into the local biochemistry of NO • in the lung, taking into account the presence of secreted or phagocyte peroxidases and possible changes in local pH, as in asthmatic airways [33] , that might modulate NO • activity and metabolism. This might result in a better understanding of relationships between the various metabolic endproducts of NO • (NO 2 -, NO 3 -, or nitroso and nitro adducts) and its pro-inflammatory or injurious properties. Surfactant protein-D and pulmonary host defense Surfactant protein-D (SP-D) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. SP-D is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. SP-D, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. SP-D also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. In addition, SP-D binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. Consistent with these diverse in vitro activities is the observation that SP-D-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. The phenotype of macrophages isolated from the lungs of SP-D-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. The expression of SP-D is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized SP-D might contribute to the pathogenesis of a variety of human lung diseases. Surfactant protein-D (SP-D) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein A (SP-A) and the serum mannose-binding lectin [1] [2] [3] . Collectins inter-act with a wide variety of microorganisms, lipids, and organic particulate antigens, and can modulate the function of immune effector cells and their responses to these ligands. This article reviews what is currently known about the sites of production, structure, function, and regulated expression of SP-D. Emphasis will be placed on functional attributes, known ligand interactions, and structure-function relationships believed to be important for host defense. For additional information on SP-A and other members of the collectin family, the reader is referred to other recent reviews [4] [5] [6] .
SP-D is synthesized and secreted into the airspaces of the lung by the respiratory epithelium [1] . At the alveolar level, SP-D is constitutively synthesized and secreted by alveolar type II cells. More proximally in the lung, SP-D is secreted by a subset of bronchiolar epithelial cells, the non-ciliated Clara cells. Because SP-D is stored within the secretory granules of Clara cells [7, 8] , it seems likely that SP-D is subject to regulated secretion via granule exocy-tosis at this level of the respiratory tract. In some species, SP-D is also synthesized by epithelial cells and/or submucosal glands associated with the bronchi and trachea [9] . Although many alveolar macrophages show strong cytoplasmic and/or membrane labeling with antibody against SP-D, they do not contain detectable SP-D message.
The lung seems to be the major site of SP-D production. However, there is increasing evidence for extrapulmonary sites of expression as assessed with monoclonal or affinity-purified antibodies, reverse-transcriptase-mediated PCR (RT-PCR), and/or hybridization assays of tissues from humans and other large mammals [10 • ,11-14] (summarized in Table 1 ). It is difficult to entirely exclude crossreactions or amplification of related sequences; however, localization to many of these sites in human tissues was confirmed by using monoclonal antibodies in combination with RT-PCR with sequencing of the amplified products [10 • ]. Non-pulmonary expression seems to be largely restricted to cells lining epithelial surfaces or ducts and certain glandular epithelial cells that are in direct or indirect continuity with the environment. Notable exceptions to this generalization might include heart, brain, pancreatic islets, and testicular Leydig cells. SP-D has also been identified in amnionic epithelial cells by immunohistochemistry [15] ; however, it is unclear whether this is synthesized locally or derived from the lung by way of the amniotic fluid. Interestingly, in many of these sites SP-D microscopically co-localizes with gp-340, an SP-D binding protein and putative SP-D receptor [10 • ].
Sites of extrapulmonary expression have also been described in small mammals. In the rat, SP-D message was identified in RNA extracted from skin and blood vessel [16] , and both protein and message were identified in gastric mucosa [17] and mesentery [13] . Using RT-PCR, SP-D message has also been identified in mouse stomach, heart, and kidney [14] .
SP-D (43 kDa, reduced) consists of at least four discrete structural domains: a short, N-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide or coiled-coil neck domain, and a C-terminal, Ctype lectin carbohydrate recognition domain (CRD). Each molecule consists of trimeric subunits (3 × 43 kDa), which associate at their N-termini (Fig. 1) . Although most preparations of SP-D contain a predominance of dodecamers (that is, four trimeric subunits), the proportions of various oligomers vary between species. For example, rat lavage and recombinant rat SP-D are almost exclusively assembled as dodecamers (four trimers), whereas recombinant human SP-D is secreted as trimers, dodecamers and higher-order multimers [18] . SP-D isolated from the lavage of some patients with alveolar proteinosis consists predominantly of higher-order multimers, which can contain up to 32 (or more) trimeric subunits (Fig. 1 ).
Recent crystallographic and mutagenesis studies suggest that the structural determinants of saccharide binding are similar to those originally described for mannose-binding lectin [19,20,21 • ,22 • ]. At least two bound calcium ions and two intrachain disulfide crosslinks stabilize the required tertiary structure, and Glu321 and Asn323 within the CRD participate in glucose/mannose type recognition. Interactions with at least one glycolipid ligand, phosphatidylinositol (PI), require the participation of the C-terminal end of the protein [23, 24] .
A trimeric cluster of CRDs is necessary for high-affinity binding to carbohydrate ligands [21 • ,25]. The crystal structure of human SP-D suggests that the spatial distribution of CRDs within a trimeric subunit permits simultaneous and cooperative interactions with two or three glycoconjugates displayed on the surface of a particulate ligand [21 • ]. Furthermore, solid-phase binding studies have shown that monomeric CRDs have an approximately 10-fold lower binding affinity for multivalent ligands than trimeric CRDs.
Crystallographic studies of human SP-D further suggest that the spatial organization of CRDs within a trimer is stabilized by interactions of the C-terminal sequence with the trimeric neck domain [21 • ,26]. Interestingly, the three CRDs show a deviation from threefold asymmetry, suggesting some flexibility of the CRDs in relation to the neck. Thus, the dependence of the binding of PI on the C-terminal sequence could reflect conformational effects, rather than the direct participation of this sequence in ligand interactions.
The collagen domain length of SP-D is highly conserved and lacks interruptions in the repeating Gly-X-Y sequence (in which X and Y are different amino acids). As for other collagenous proteins, this domain is enriched in imino acids and contains hydroxyproline. Unlike SP-A, SP-D also contains hydroxylysine. Although the collagen domain of rat, human, bovine, and mouse SP-D lacks cysteine residues, cDNA sequencing has identified a codon for cysteine within the collagen domain of pig SP-D [27 • ]; this suggests the possibility of alternative patterns of chain association and oligomeric assembly for pig SP-D.
The first translated exon of SP-D contains a highly conserved and unusually hydrophilic Gly-X-Y sequence that shows little homology with the remainder of the collagen sequence. The functional significance of this region is unknown. However, it has been suggested that this region contributes to oligomer assembly or mediates interactions with cellular receptors.
The collagen domain determines the maximal spatial separation of trimeric, C-terminal lectin domains within SP-D molecules, but might also contribute to normal oligomeric assembly and secretion. For example, deletion of the entire collagen domain of rat SP-D results in the secretion of trimers rather than dodecamers [28] . In addition, 2,2-dipyridyl, an inhibitor of prolyl hydroxylation that interferes with the formation of a stable collagen helix, causes the intracellular accumulation of 43 kDa monomers and dimers [29] . In any case, the complete conservation of the number of Gly-X-Y triplets suggests that the spatial separation of trimeric CRDs is critical for normal SP-D function.
The N-terminal peptide of the mature protein contains two conserved cysteine residues at positions 15 and 20. These residues participate in interchain disulfide crosslinks that stabilize the trimer, as well as the N-terminal association of four or more trimeric subunits. Stable oligomerization of trimeric subunits permits cooperative or bridging interactions between spatially separated binding sites on the same surface or on different particles.
The process of forming interchain disulfide bonds is complex, and appropriate crosslinking of the N-terminal domains might be rate limiting for secretion [30] . Subcellular fractionation studies suggest that interchain bonds form initially between the three chains of a trimeric subunit. Subsequent rearrangements within the rough endoplasmic reticulum might allow the covalent crosslinking of a single chain from one subunit and two crosslinked chains of another, with the associated elimination of free thiol groups. Mutant proteins that contain unpaired N-terminal cysteine residues are not secreted. However, it is unclear whether this results from abnormalities in disulfide bonding itself, or the failure to stabilize the required N-terminal conformation.
The collagen domain contains hydroxylysyl-derived glycosides and a single N-linked oligosaccharide. In most species (human, rat, mouse, and cow) the site of N-linked glycosylation is located near the N-terminal end of the collagenous domain. Recently, it was shown that pig SP-D has an additional potential site of N-linked glycosylation within the CRD [27 • ]. Although rat and human lung lavage SP-D seem to be sialylated, as suggested by charge heterogeneity and cleavage with highly purified neuraminidase, preparations of human amniotic fluid and bovine lavage SP-D recovered from amniotic fluid showed predominantly complex type biantennary structures and no sialic acid [31] .
A variant form of SP-D (50 kDa) has been identified in lavage from a subset of human lavage samples; this protein shows O-linked glycosylation of threonyl residues within the N-terminal peptide domain [32 • ]. At present, the functional significance of these sugars is not known. The presence of O-linked glycosylation within the N-terminal domain might be predicted to interfere with normal dodecamer assembly. In this regard, the O-glycosylated 50 kDa form of human SP-D is recovered as trimeric subunits or smaller species.
As for many glycoproteins, the functional role of the attached carbohydrate is unknown. Mutational analysis has shown that the N-linked sugar on rat SP-D is not required for secretion, for dodecamer formation, or for interactions with a variety of microorganisms [29,33].
Consistent with its designation as a 'mannose-type' C-type lectin, SP-D preferentially binds to simple and complex saccharides containing mannose, glucose, or inositol [34, 35] . SP-D also interacts with specific constituents of pulmonary surfactant including PI [36-38] and glucosylceramide [39] . Binding to glucosylceramide involves interactions of the carbohydrate-binding sequences of the CRD with the glucosyl moiety. However, the interaction of SP-D with PI involves interactions with the lipid, as well as CRD-dependent interactions with the inositol moiety [24, 40] .
Microorganisms are surfaced with a diverse and complex array of polysaccharides and glycoconjugates, and most classes of microorganism contain one or more sugars recognized by SP-D. However, the outcome of this interaction depends on the specific organism and can be modified by the conditions of microbial growth. The potential consequences of this interaction include the following: varying degrees of lectin-dependent aggregation (namely, microbial agglutination), enhanced binding of microorganisms or microbial aggregates to their 'receptors' on host cells, phagocyte activation, and opsonic enhancement of phagocytosis and killing, potentially involving one or more cellular receptors for SP-D. Binding to organisms in suspension is often -but not always -accompanied by some degree of aggregation.
SP-D binds to purified lipopolysaccharide (LPS) isolated from a variety of Gram-negative organisms [35, 41] . In addition, LPS is the major cell wall component that is labeled on lectin blotting of outer membranes isolated from Escherichia coli [41] . The latter interactions involve the recognition of the core oligosaccharide domain, which contains glucose and heptose [41] . SP-D interacts preferentially with purified LPS molecules characterized by short or absent O-antigens and preferentially agglutinates bacterial strains expressing a predominance of rough (O-antigen-deficient) LPS [41, 44] .
Although the core oligosaccharide domain of LPS constitutes the major ligand for SP-D on at least some Gram-negative bacteria, the mechanism of interaction with this group of microorganisms is probably heterogeneous. SP-D binds to some smooth, unencapsulated strains of Gram-negative bacteria by immunofluorescence. The mechanism is uncertain; the quantity or quality of binding differs from that observed for rough strains and does not necessarily result in agglutination. LPS molecules on the surfaces of bacteria show heterogeneity in the extent of maturation, so it is possible that this interaction is mediated by a subpopulation of LPS with deficient O-antigens and that the density of binding sites is too low for high-affinity binding.
The recognition of the surface glycoconjugates on Gramnegative bacteria by SP-D depends not only on the expression of lectin-specific residues by a given strain or species, but also on the accessibility of these residues [1, 45] . For example, SP-D binds inefficiently to the core region of LPS of encapsulated Klebsiella, but efficiently agglutinates the corresponding unencapsulated phase variants. Interactions of SP-D with the core oligosaccharides of Gram-negative organisms are also influenced by the number of repeating saccharide units associated with the terminal O-antigen of the LPS [41,44].
Other potential ligands include the O-antigen domain of LPS, certain capsular polysaccharides, and membraneassociated glycoproteins. In this regard, SP-D can bind to di-mannose containing O-antigens expressed by a subset of Klebsiella serotypes (I Ofek, H Sahly and EC Crouch, unpublished data). Although other C-type lectins, specifically SP-A and the mannose receptor, can interact with specific capsular polysaccharides [46], a specific interaction of SP-D with capsular glycoconjugates or exopolysaccharides has not been described.
The mechanism of interaction with Gram-positive organisms has not been elucidated. Lipoteichoic acids, which are the major glycolipids associated with the Gram-positive cell wall, do not detectably compete with LPS for binding to SP-D (I Ofek, A Mesika, M Kalina, Y Keisari, D Chang, D McGregor and EC Crouch, manuscript submitted). In preliminary studies we observed that binding was competed only partly with maltose and/or EDTA, raising the possibility that binding might be more complex than for some Gram-negative organisms. . However, similar effects were observed when the neutrophils were preincubated with SP-D, and there was only a slight enhancement of uptake when bacteria were incubated with human SP-D and washed before their addition to neutrophils. Notably, the extent of binding and internalization was dependent on the extent of multimerization, with human SP-D multimers demonstrating the highest potency. Differences in cell type, the extent of SP-D multimerization, or differences in size or organization of bacterial aggregates could account for some of the apparent inconsistencies.
Although LPS mediates the binding of SP-D to at least some Gram-negative bacteria, SP-D can also bind to spe- In the latter study the authors suggested that fungal aggregation inhibits phagocytosis. Interestingly, SP-D binding directly inhibited fungal growth and decreased the outgrowth of pseudohyphae, the invasive form of the fungus, in the absence of phagocytic cells [57] . It is possible that these effects are also secondary to agglutination, possibly as result of nutrient deprivation.
Purified rat and human SP-D inhibit the infectivity and hemagglutination activity of influenza 
SP-D can interact with host cells, both directly and indirectly. As indicated above, SP-D can enhance the phagocytosis and killing of certain microorganisms and enhance the oxidant response to microbial binding. However, at present there is only one study that suggests that the enhancement of phagocytosis by SP-D might involve the participation of an opsonic receptor. Furthermore, the enhanced uptake of IAV seems to be mediated by viral aggregation, with enhanced interactions of the virus with its natural receptors on the host cell.
In any case, SP-D can interact directly with host cells, and in some cases can influence their behavior. SP-D is chemotactic and haptotactic for neutrophils and certain mononuclear phagocytes [59 • ,67-69] and can elicit directional actin polymerization in alveolar macrophages [69] . In this regard, SP-D is considerably more potent than SP-A. Early studies with natural proteins isolated from silicotic animals reported directed effects on the oxidant metabolism of isolated alveolar macrophages [70] . However, such effects can probably be attributed to endotoxin contamination and/or aggregation. Purified dodecamers do not significantly increase the production of nitric oxide [71] or of proinflammatory cytokines such as tumor necrosis factor-α (Y Kesari, H Wang, A Mesika, E Crouch and I Ofek, unpublished data). Interestingly, purified SP-D has been reported to increase the production of several metalloproteinases in the absence of a significant effect on proinflammatory cytokine production [72] .
Despite the ability of SP-D to modulate a variety of cellular functions, little is currently known about potential cellular receptors for this protein. compartments [73] , but it is unclear whether the uptake is receptor dependent and whether SP-D is being internalized in association with specific ligands.
There are at least two classes of binding to host cells: CRD-dependent and CRD-independent. Some studies have demonstrated CRD-dependent binding to phagocytes that can be inhibited with EDTA or competing saccharides, both in vitro and in vivo.
As indicated above, the ability of SP-D to elicit the chemotaxis of neutrophilic and monocytic cells depends on the lectin activity of SP-D [68] . In addition, Kuan and coworkers reported that extracting formaldehyde-fixed alveolar macrophages with detergents largely eliminates the binding of purified SP-D, suggesting a membrane-associated ligand or glycolipid receptor [73] . Dong and Wright have extended these findings and suggest that PI can contribute to SP-D binding by alveolar macrophages [74] .
It is of interest that SP-D can bind to recombinant sCD14 through interactions with N-linked oligosaccharides [51 • ].
Given that the membrane-associated form of CD14 is widely expressed on host cells, it is possible that CD14 can serve as a binding site on macrophages and other cell types.
The phagocytic uptake of certain bacteria by neutrophils is also inhibited by calcium chelation or competing sugars [42]; however, this could result from the inhibition of microbial agglutination rather than lectin-dependent interactions with the phagocyte. Wang et al suggested that SP-D can bind to lymphocytic cells by a lectin-dependent mechanism [75 •• ] . In this regard, it is interesting to note that glucosylceramide, a ligand for SP-D in vitro, is one of the most abundant neutral glycolipids expressed by lymphoid cells.
Reid and co-workers were the first to present evidence for lectin-independent binding [76] . These and other studies suggested that binding does not involve known C1q or collectin receptors. The only putative receptor protein, gp-340, is a widely expressed member of the scavenger receptor superfamily [77,78 • ]. It binds to the CRD of SP-D in a calcium-dependent manner that does not require the lectin activity of SP-D. Although the protein has been immunolocalized to alveolar macrophage membranes and distributes together with SP-D in many different human tissues [10 • ,77], it has not yet been shown to mediate the binding of SP-D to these cells or to participate in signal transduction events. The cDNAs isolated from lung have not shown a membrane-spanning region [77] , and the protein is abundant as a soluble component in BAL. Given that gp-340 is a highly multimerized protein that contains numerous potential ligand binding domains (Fig. 1b) , it is possible that the protein cooperates with SP-D in the neu-tralization or clearance of certain ligands rather than specifically mediating the interactions of SP-D with host cells.
Wright and co-workers have demonstrated the binding of SP-D to isolated type II pneumocytes. The mechanism seems distinct from the binding to macrophages [79 • ]. The binding was dependent on concentration, time, and temperature and required calcium; it was not sensitive to protease treatment or to PI-phospholipase C. Although the internalized SP-D was degraded or recycled to lamellar bodies, SP-D binding did not alter the uptake of surfactant lipids.
SP-D has demonstrated comparatively few direct effects on the metabolism of host cells, at least in situations where self-aggregation and endotoxin contamination have been excluded. One possible explanation is that modulation of cellular function requires the prior interaction of SP-D with a ligand. This would have numerous potential physiological advantages, because the presence of 'active' protein might be restricted to sites of microbial or antigenic deposition. The binding of complex, multivalent, particulate antigens to two or more CRDs could markedly alter the conformation of SP-D molecules, with respect to the spatial orientation of the arms in relation to the N-terminal crosslinking domain and/or with respect to the spatial orientation of the CRDs within a given trimeric subunit. Thus, the 'charging' of SP-D with a particulate ligand could lead to local or distant conformational changes that expose 'cryptic' binding sites for cellular receptors.
There is some preliminary evidence consistent with the notion that the interaction of SP-D with a ligand alters its capacity to activate host cells. Table 3 and discussed below.
SP-D can be isolated in different multimeric forms from proteinosis lavage [32 • ] and are produced by Chinese hamster ovary K1 cells transfected with human SP-D cDNA [18] . As described previously, the effects of SP-D on the neutrophil response to influenza virus are highly dependent on the ability of SP-D to agglutinate the viral particles, and the agglutination activity is directly correlated with the extent of multimerization. Trimers can bind to the virus but have little capacity to modulate neutrophil interactions. By contrast, highly multimerized proteins show greater activity than dodecamers [81] .
Given these observations, factors that favor enhanced oligomerization or lead to the accumulation of trimeric subunits promote might influence SP-D function. For example, the liberation of active trimers by a hypothetical microbial protease could lead to the accumulation of molecules that might inhibit the aggregation-dependent activities of SP-D.
In contrast, recombinant trimeric CRDs can stimulate chemotaxis [67] and decrease viral infectivity [65 • ]. Although higher-order oligomers of SP-D can self-aggregate and precipitate in the presence of calcium in vitro, the functional consequences are not known.
The lectin activity of SP-A is decreased after the nitric oxide-dependent nitration of tyrosine residues [82] , and nitration decreases the ability of SP-A to enhance the adherence of Pneumocystis to alveolar macrophages [83] . However, similar findings have not yet been reported for SP-D. Conditions of mildly acidic pH, as might be found in endocytic compartments, are predicted to disrupt the lectin-dependent activities of SP-D [34]. Proteolytic degradation remains an important possibility. However, SP-D is highly resistant to degradation by a wide variety of neutral proteases in vitro, and degradation products have not yet been shown to accumulate under pathological conditions in vivo.
Glucose concentrations at levels encountered in diabetes can interfere with SP-D's ability to interact with specific strains of IAV or other microorganisms in vitro [84 • ]. Many microorganisms release cell wall polysaccharides or glycoconjugates, which might interfere with the binding of collectins to the same or other organisms. In this regard, SP-D recovered from rats after the instillation of LPS into the airway shows decreased lectin activity, which is attributed to occupancy of the CRD with LPS [49 • ]. It seems reasonable to speculate that some organisms might compete with other organisms for binding to SP-D. Such a situation could conceivably predispose to secondary infections. Lastly, the potential inhibitory effects of competing saccharide ligands presents important methodological considerations for experiments using carbohydrate-containing cell culture medium or buffers. Non C-type lectins (such as ficolins)
It is difficult to predict the functions of SP-D within the airspace. Other lectins with overlapping specificity are also present. Although the levels of mannose-binding lectin are probably low in the absence of increased vascular permeability, SP-A and the macrophage mannose receptor could conceivably interact with the same ligands in the distal airways and alveoli. Such interactions could lead to antagonistic or cooperative effects. Furthermore, we have little knowledge regarding the microanatomic distribution of these molecules in specific circumstances in vivo.
Although most SP-A is probably associated with the insoluble phase of the alveolar lining material, and the macrophage mannose receptor is membrane-associated, the distribution might be altered in the setting of lung injury.
Models of SP-D deficiency show no detectable anatomical or physiological abnormalities at birth. However, the animals gradually develop a patchy, subpleural alveolar lipidosis with associated type II cell hypertrophy, the accumulation of enlarged and foamy macrophages, and an apparent expansion of peribronchial lymphoid tissue [85 • ,86 • ]. Interestingly, the mice eventually develop distal-acinar emphysema and areas of subpleural fibrosis, which could reflect a continuing inflammatory reaction associated with abnormal oxidant metabolism and metalloproteinase activity [87 • ]. By contrast, SP-A-deficient mice (-/-) show essentially normal respiratory function and surfactant lipid metabolism [88, 89] but numerous apparent host defense abnormalities [90] .
The capacity of SP-D to bind to specific strains of influenza A in vitro is highly correlated with the capacity of the virus to proliferate in mice in vivo [62] . Specifically, strains with more oligosaccharide attachments on the HA are preferentially neutralized by SP-D in vitro and show decreased proliferation in mice. Because the administration of mannan together with the virus increased the replication of IAV in the lung, the involvement of a mannose-type, C-type lectin was implicated.
SP-D-sensitive IAV strains also replicate to higher titers in the lungs of diabetic mice than in nondiabetic controls [84 • ]. Replication of the virus is positively correlated with blood glucose level, and decreases in response to insulin treatment. Significantly, blood glucose levels comparable to those measured in the diabetic mice were sufficient to inhibit the interaction of SP-D with these viral strains in vitro. PR-8, a strain that does not interact with SP-D but does interact with SP-A, replicated to the same extent in diabetic and control mice.
SP-D levels increase in association with certain infections. For example, SP-D levels, but not the levels of serum mannose-binding lectin, increase markedly after IAV infection [62] . Impressive increases in SP-D have also been observed in murine models of Pneumocystis carinii [91] and P. aeruginosa infection [92] .
SP-D-deficient mice have not yet been extensively characterized with respect to host defense function. However, they show decreased viral clearance and enhanced inflammation after challenge with respiratory syncytial virus [93] and IAV (AM Levine, personal communication). In addition, they show increased inflammation, increased oxidant production, and decreased macrophage phagocytosis in response to intratracheally instilled group B streptococcus and Haemophilus influenzae (AM Levine, personal communication). Although the overexpression of wild-type SP-D in type II pneumocytes with the SP-D-deficient mice can prevent the lipidosis and inflammatory changes [94] , the ability of overexpressed wild-type SP-D or exogenous SP-D to ameliorate these abnormalities has not yet been described. The coexisting pulmonary abnormalities also complicate the interpretation of challenge models. For example, macrophage activation might enhance killing and offset any decrease that results more directly from SP-D deficiency. SP-D deficiency modifies the host response to instilled LPS with decreased lung injury and inflammatory cell recruitment [50].
Molecules that can bind to potential antigens and deliver them to macrophages and other antigen-presenting cells might contribute to the development of acquired immunity. In this regard, a few published observations suggest possible roles in the development of humoral and/or cellular immunity in response to microorganisms or complex organic antigens. For example, SP-D can decrease interleukin-2dependent T-lymphocyte proliferation [95 • ]. Interestingly, single-arm mutants were at least as potent as intact dodecamers in mediating this effect. SP-D also binds to oligosaccharides associated with dust mite allergen [96 • ], and can inhibit the binding of specific IgE to these allergens, possibly through direct, CRD-dependent binding to lymphocytes [96 • ]. Thus, alterations in the level of SP-D (or the state of oligomerization) might influence the development of immunological responses and contribute to the pathogenesis of asthma and other hypersensitivity disorders.
There are other potential interplays between humoral immunity and collectins with regard to antimicrobial host defense. For example, increased glycosylation of IAV coat proteins, an adaptation that is believed to help the virus to evade antibody-mediated neutralization, is associated with increased reactivity with SP-D and other collectins [62]. Thus, the relative potential importance of antibody and collectin-mediated host defenses might be influenced by subtle variations in the structure of the microbial surface.
There is little recent information on the developmental regulation of SP-D expression. In general, SP-D increases rapidly late in gestation [97] [98] [99] [100] . The production of SP-D increases during the culture of fetal lung explants, and expression can be increased with glucocorticoids [98, 100, 101] . The exposure of fetal rats to glucocorticoids in vivo leads to precocious expression with increased numbers of SP-D-expressing cells and increased cellular levels of SP-D message [98, 101, 102] .
Although SP-D is produced constitutively within the lung, protein accumulation and gene expression are inducible and increases in SP-D expression have been observed in a number of disease states or models (Tables 4 and 5 ). In general, the synthesis and secretion of SP-D increase in association with lung injury and activation of the respiratory epithelium [1] . For example, levels of SP-D mRNA and SP-D accumulation are increased within 24-72 h after intratracheal instillation of LPS [103 • ], and SP-D expression by alveolar and bronchiolar epithelial cells increases after exposure of rats to 95% O 2 for 12 h [104] . Keratinocyte growth factor (KGF) increases SP-D expression and protein production in association with pneumocyte hyperplasia and after injury caused by bleomycin [105] . In addition, the levels of SP-D can increase markedly in response to the overexpression of certain cytokines, such as interleukin-4, or in response to microbial challenge [91, 92] .
Studies of the upstream regulatory region of the SP-D gene have demonstrated increased promoter activity in the presence of glucocorticoids, which is consistent with the findings in vivo and in lung organ culture [106] . However, no functional glucocorticoid response elements have been identified, and the effects of dexamethasone seem to be secondary and involve the effects of other transregulatory molecules.
The activity of the human SP-D promoter is dependent on a conserved activator protein-1 (AP-1) element (-109) that binds to members of the fos and jun families of transcriptional factors [107] . In addition, the promoter contains multiple functional binding sites for CCAAT-enhancer-binding protein (C/EBP) transcription factors. Mutagenesis experiments suggest that these are required for basal and stimulated promoter activity, and promoter activity is markedly increased in H441 cells after co-transfection with C/EBPβ cDNA (YC He and E crouch, unpublished data). The importance of the conserved AP-1 element and the presence of multiple binding sites for C/EBP transcription factors is consistent with the observed modulation of SP-D expression in the setting of tissue injury.
SP-D promoter activity is not dependent on the binding of thyroid transcription factor 1 (TTF-1) [107] . However, promoter activity is dependent on two interacting forkhead binding sites, upstream and downstream of the AP-1 element; these sites bind to hepatic nuclear factor-3α and apparently other forkhead box proteins in H441 lung adenocarcinoma nuclear extracts [107] .
Initial comparison of genomic and cDNA sequence suggested the existence of genetic polymorphisms in the SP-D coding sequence, including one in the N-terminal propeptide domain (Thr11 compared with Met11 in the mature protein) and three additional differences within the collagen domain at positions 102, 160, and 186 [108] . The latter substitutions are conservative to the extent that they are not expected to disrupt the collagen helix. Floros Table 5 Increased SP-D accumulation or expression in animal models
Silicosis Rat [118] Hyperoxia Rat [104] Endotoxin (LPS) Rat [103] Challenge with P. aeruginosa Mouse [92] Challenge with IAV Mouse
[62]
Challenge with Pneumocystis carinii SCID mouse [91] Rat [119] Overexpression of interleukin-4 Mouse [120] SCID, severe combined immunodeficiency. and co-workers have recently confirmed the existence of polymorphisms at positions 11 and 160 of the mature protein [109] . The potential biological significance, if any, is not known. Interestingly, the 50 kDa variant of SP-D showed O-linked glycosylation of Thr11 [32 • ], suggesting that this polymorphism might be associated with altered glycosylation. Interestingly, the 50 kDa variant was recovered as trimeric subunits, raising the possibility that differences in the glycosylation of residue 11, which is immediately N-terminal to Cys15, could influence multimerization and the capacity of SP-D to participate in bridging interactions.
There is increasing evidence that SP-D interacts specifically with a wide variety of respiratory pathogens, modulates the leukocyte response to these organisms, and participates in aspects of pulmonary immune and inflammatory regulation (Table 6) . SP-D can influence the activity of phagocytes through CRD-dependent and CRD-independent interactions. At least some of the effects of SP-D result from aggregation with enhanced binding of the agglutinated ligand to their natural 'receptors'. Although the lung is the major site of SP-D expression, it is likely that the protein has more generalized roles in host defense and the acute response to infection and tissue injury. 16  Role of endothelin-1 in lung disease Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease. from Xenopus laevis [16] . ETA receptors in normal lung are found in greatest abundance on vascular and airway smooth muscle, whereas ETB receptors are most often found on the endothelium. Clearance of ET-1 from the circulation is mediated by the ETB receptor primarily in the lung, but also in the kidney and liver [17] .
Activation of both ETA and ETB receptors on smooth muscle cells leads to vasoconstriction whereas ETB receptor activation leads to bronchoconstriction. Activation of ETB receptors located on endothelial cells leads to vasodilation by increasing nitric oxide (NO) production. The mitogenic and inflammatory modulator functions of ET-1 are primarily mediated by ETA receptor activity. Binding of the ligand to its receptor results in coupling of cell-specific G proteins that activate or inhibit adenylate cyclase, stimulate phosphatidyl-inositol-specific phosholipase, open voltage gated calcium and potassium channels, and so on. The varied effects of ET-1 receptor activation thus depend on the G protein and signal transduction pathways active in the cell of interest [18] . A growing number of receptor antagonists exist with variable selectivity for one or both receptor subtypes.
Regulation of ET-1 is at the level of transcription, with stimuli including shear stress, hypoxia, cytokines (IL-2, IL-1β, tumor necrosis factor α, IFN-β, etc), lipopolysaccharides, and many growth factors (transforming growth factor-β, platelet-derived growth factor, epidermal growth factor, etc) inducing transcription of ET-1 mRNA and secretion of protein [18] . ET-1 acting in an autocrine fashion may also increase ET-1 expression [19] . ET-1 expression is decreased by NO [20] . Some stimuli may additionally enhance preproET-1 mRNA stability, leading to increased and sustained ET-1 expression. The number of ETA and ETB receptors is also cell specific and regulated by a variety of growth factors [18] . Because ET-1 and receptor expression is influenced by many diverse physical and biochemical mechanisms, the role of ET-1 in pathologic states has been difficult to define, and these are addressed in subsequent parts of this article.
In the airway, ET-1 is localized primarily to the bronchial smooth muscle with low expression in the epithelium. Cellular subsets of the epithelium that secrete ET-1 include mucous cells, serous cells, and Clara cells [21] . ET binding sites are found on bronchial smooth muscle, alveolar septae, endothelial cells, and parasympathetic ganglia [22, 23] . ET-1 expression in the airways, as previously noted, is regulated by inflammatory mediators. Eosinophilic airway inflammation, as may be seen in severe asthma, is associated with increased ET-1 levels in the lung [24] . ET-1 secretion may also act in an autocrine or paracrine fashion, via the ETA receptor, leading to increased transepithelial potential difference and ciliary beat frequency, and to exerting mitogenic effects on airway epithelium and smooth muscle cells [25] [26] [27] [28] .
All three endothelins cause bronchoconstriction in intact airways, with ET-1 being the most potent. Denuded bronchi constrict equally to all three endothelins, suggesting considerable modulation of ET-1 effects by the epithelium [29] . The vast majority of ET-1 binding sites on bronchial smooth muscle are ETB receptors, and bronchoconstriction in human bronchi is not inhibited by ETA antagonists but augmented by ETB receptor agonists [30] [31] [32] . Since cultured airway epithelium secretes equal amounts of ET-1 and ET-3, which have equivalent affinity for the ETB receptor, bronchoconstriction could be mediated by both endothelins [33] .
While ET-1 stimulates release of multiple cytokines important in airway inflammation, it does not enhance secretion of histamine or leukotrienes. ET-1 does increase prostaglandin release [32] . Inhibition of cyclo-oxygenase, however, has no effect on bronchoconstriction suggesting that, despite the release of multiple mediators, ET-1 mediated bronchoconstriction is a direct effect of activation of the ETB receptor [32] . ETA mediated bronchoconstriction may also be important following ETB receptor desensitization or denudation of the airway epithelium, as may occur during airway inflammation and during the late, sustained airway response to inhaled antigens [31, 34, 35] . Interestingly, heterozygous ET-1 knockout mice, with a 50% reduction in ET-1 peptide, have airway hyperresponsiveness but not remodeling, suggesting the decrease in ET-1 modulates bronchoconstriction activity by a functional mechanism, possibly by decreasing basal NO production [36, 37] .
Asthma is also an inflammatory airway disease characterized by bronchoconstriction and hyperreactivity with influx of inflammatory cells, mucus production, edema, and airway thickening. ET-1 may have important roles in each of these processes. While ET-1 causes immediate bronchoconstriction [38] , it also increases bronchial reactivity to inhaled antigens [35] as well as influx of inflammatory cells [39, 40] , increased cytokine production [40] , airway edema [41] , and airway remodeling [28, 42, 43] . Airway inflammation also leads to increased ET-1 synthesis, possibly perpetuating the inflammation and bronchoconstriction [44] . ET-1 release from cultured peripheral mononuclear and bronchial epithelial cells from asthmatics is also increased [45, 46] . Inhibition of ETA or combined ETA and ETB receptors additionally leads to decreased airway inflammation in antigen-challenged animals, suggesting that the proinflammatory effects of ET-1 in the airway are mediated by ETA receptors [39, 47] .
Children with asthma have increased circulating levels of ET-1 [48] . Adult asthmatics have normal levels between attacks but, during acute attacks, have elevated serum ET-1 levels that correlate inversely with airflow measurements and decrease with treatment [49] . Bronchoalveolar lavage (BAL) ET-1 in asthmatics is similarly increased to concentrations that cause bronchoconstriction and inversely correlates with forced expiratory volume in 1 s (FEV 1 ) [29, 50, 51] . As in cultured epithelial cells, ET-1 and ET-3 are found in equal amounts in BAL fluid from asthmatics [33, 52] . There is also a relative increase in ETB versus ETA receptor expression in asthmatic patients, which may contribute to increased bronchoconstriction [53] . Not all asthmatics, however, have increased ET-1 as patients with nocturnal asthma have decreased BAL ET-1 levels [54] . Treatment of acute asthma exacerbations with steroids, beta-adrenergic agonists or phosphodiesterase inhibitors resulted in decreased BAL ET-1 [52, 55] . Immunostaining and in situ hybridization for ET-1 in biopsy specimens from asthmatics have shown an increase in ET-1 in the bronchial epithelium that correlates with asthma symptoms [46, 56] .
Cigarette smoking leads to increased circulating ET-1 [57] but patients with chronic obstructive pulmonary disease, in the absence of pulmonary hypertension and hypoxemia, do not have increased plasma ET-1 [58] [59] [60] . Increases in urinary ET-1 instead correlate with decreases in oxygenation, possibly through hypoxic release of ET-1 from the kidney [61, 62] . Smokers also have impaired ET-1 mediated vasodilation that correlates with bronchial hyperresponsiveness and may contribute to pulmonary hypertension [63, 64] .
ET-1 has been implicated in the pathogenesis of bronchiectasis by its ability to promote neutrophil chemotaxis, adherence, and activation [65] [66] [67] [68] [69] . Sputum ET-1 levels are increased in patients with cystic fibrosis [59] , and sputum ET-1 correlated with Pseduomonas infection in noncystic fibrosis related bronchiectasis [70] .
ET-1 has also been implicated in the pathogenesis of bronchiolitis obliterans (BO), which is characterized by injury to small conducting airways resulting in formation of proliferative, collagen rich tissue obliterating airway architecture. BO is the leading cause of late mortality from lung transplantation, and ET-1 is increased in lung allografts [71] . The pro-inflammatory and mitogenic properties of ET-1 in the airways has led to speculation that ET-1 may be involved in formation of the lesion [28] . This is further supported by the increase in BAL ET-1 in lung allografts [72, 73] . The in vivo gene transfer of ET-1 to the airway epithelium using the hemagglutinating virus of Japan in rats recently resulted in pathologic changes in the distal airways identical to those seen in human BO specimens [74] . These changes were not due to nonspecific effects of the hemagglutinating virus of Japan itself, but could be attributed to the presence of the ET-1 gene, which was localized to the airway epithelium, hyperplastic lesions, and alveolar cells.
Pulmonary hypertension is a rare and progressive disease characterized by increases in normally low pulmonary vascular tone, pulmonary vascular remodeling, and progressive right heart failure. ET-1 has been implicated as a mediator in the changes seen in pulmonary hypertension. In the pulmonary vasculature, ET-1 is found primarily in endothelial cells and to a lesser extent in the vascular smooth muscle cells. The endothelium secretes ET-1 primarily to the basolateral surface of the cell. ET-1 secretion may be increased by a variety of stimuli including cytokines, catecholamines, and physical forces such as shear stress, and decreased by NO, prostaglandins, and oxidant stress [20, [75] [76] [77] [78] . Hypoxia has been reported to increase, have no effect, or decrease ET-1 release from endothelial cells [79] [80] [81] [82] [83] .
Activation of the receptors for ET-1 in the pulmonary vasculature leads to both vasodilation and vasoconstriction, and depends on both cell type and receptor. In the whole lung, ETA receptors are the most abundant and are localized to the medial layer of the arteries, decreasing in intensity in the peripheral circulation [84, 85] . ETB receptors are also found in the media of the pulmonary vessels, increasing in intensity in the distal circulation, while intimal ETB receptors are localized in the larger elastic arteries [85] . This distribution of receptors has important implications in understanding ET-1 regulation of vascular tone. Vascular ET-1 receptors may be increased by several factors including angiotensin and hypoxia [80, [85] [86] [87] .
ET-1 can act as both a vasodilator and vasoconstrictor in the pulmonary circulation. Generation of NO or opening of ATP-sensitive potassium channels leading to hyperpolarization results in vasodilation mediated by ETB receptors on pulmonary endothelium [88, 89] . In hypertensive, chronically hypoxic lungs with increased ETB receptor expression, augmented vasodilation is due to increased ETB mediated NO release that is inhibited by hypoxic ventilation, while inhibition of NO synthesis leads to increased ET-1 mediated vasoconstriction [85, [90] [91] [92] . Both ETA and ETB receptors, conversely, acting on vascular smooth muscle, mediate ET-1 induced vasoconstriction. In the normal lung, ET-1 causes vasoconstriction primarily by activation of the ETA receptors in the large, conducting vessels of the lung [93, 94] . In the smaller, resistance vessels of the lung, ETB receptors in the media predominate and are responsible for the ET-1 induced vasoconstriction [93] . Interestingly, preconstriction of the pulmonary circulation resulted in a shift from primarily ETA mediated to ETB mediated vasoconstriction [94] .
The overall effect of ET-1 on vascular tone depends on both the dose and on the pre-existing tone in the lung. ET-1 administration during acute hypoxic vasoconstriction will result in transient pulmonary vasodilation [89] . This effect is dose dependent, with lower doses leading to vasodilation while higher or repetitive doses cause vasoconstriction following an initial, brief vasodilation [89] . The role of ET-1 in the acute hypoxic vasoconstriction in the lung is not certain. ETA receptor antagonism attenuates hypoxic pulmonary vasoconstriction in several species [95] , and ET-1 may be implicated in the mechanism of acute hypoxic response by inhibition of K-ATP channels [96] .
Several lines of evidence have suggested the importance of ET-1 in chronic hypoxic pulmonary hypertension. ET-1 is increased in plasma and lungs of rats following exposure to hypoxia [80, 97] . Treatment with either ETA or combined ETA and ETB receptor antagonists additionally attenuates the development of hypoxic pulmonary hypertension [98, 99] . ET-1 has also been implicated in the vascular remodeling associated with chronic hypoxia through its mitogenic effects on vascular smooth muscle cells [98, 100] .
ET-1 has also been implicated in other animal models of pulmonary hypertension. ET-1 is increased in fawn hooded rats that develop severe pulmonary hypertension when raised under conditions of mild hypoxia and in monocrotaline treated rats [101, 102] . The increase in ET-1 in both of these forms of pulmonary hypertension may be contributing to increases in vascular tone as well as in vascular remodeling [103] [104] [105] [106] 114] . Interestingly, transgenic mice overexpressing the human preproET-1 gene, with modestly increased lung ET-1 levels (35-50%), do not develop pulmonary hypertension under normoxic conditions or an exaggerated response to chronic hypoxia [107] .
Human pulmonary hypertension is classified as primary, or unexplained, or secondary to other cardiopulmonary diseases or connective tissue diseases (ie scleroderma). Hallmarks of the disease include progressive increases in pulmonary vascular resistance and pulmonary vascular remodeling, with thickening of the medial layer small pulmonary arterioles and formation of the complex plexiform lesion [108] . Circulating ET-1 is increased in humans with pulmonary hypertension, either primary or due to other cardiopulmonary disease [109] . Levels are highest in patients with primary pulmonary hypertension. Since the lung is the major source for clearance of ET-1 from the circulation, increased arterio-venous ratios as seen in primary pulmonary hypertension suggest either decreased clearance or increased production in the lung [17, 109] . ET-1 is also increased in lungs of patients with pulmonary hypertension, with the greatest increase seen in the small resistance arteries and the plexiform lesions [110] , and may correlate with pulmonary vascular resistance [111] . Interestingly, treatment with continuous infusion of prostacyclin resulted in clinical improvement and a decrease in the arterio-venous ratio of ET-1 [112] , possibly by decreasing ET-1 synthesis from endothelial cells [76] . Studies using ET-1 receptor antagonists in the treatment of primary pulmonary hypertension are underway and may offer hope to patients with this disease by inhibiting this pluripotent peptide's effects on vascular tone and remodeling.
Several lines of evidence suggest the importance of ET-1 in lung allograft survival and rejection. The peptide has been implicated as an important factor in ischemia-reperfusion injury at the time of transplant as well as in acute and chronic rejection of the allograft.
Circulating ET-1 is increased in humans undergoing lung transplant immediately following perfusion of the allograft. Plasma ET-1 increased threefold within minutes, remained high for 12 hours following transplantation, and declined to near normal levels within 24 hours [113] . This increase in ET-1 correlated with the increase in pulmonary vascular resistance occurring about 6 hours post-transplantation, suggesting that the release of ET-1 in the circulation may have mediated this event. ET-1 in BAL fluid from recipients of lung allografts is similarly increased several fold and remains elevated up to 2 years post-transplant [72, 73] . In recipients of single lung transplants, ET-1 was increased 10-fold in BAL fluid from the transplanted lung compared with the native lung, suggesting that the increase in ET-1 was due to the graft and not the underlying disease requiring transplant [72] . ET-1 in BAL fluid did not, however, correlate with episodes of infection or rejection.
The cellular source of ET-1 in lung allografts is unknown. The expression of ET-1 in nontransplanted human lungs is low and found primarily in the vascular endothelium [114] . Transbronchial biopsy specimens obtained either for surveillance or for clinical suspicion of infection or rejection following transplantation revealed the presence of ET-1 in the airway epithelium and in alveolar macrophages [115] . ET-1 was occasionally seen in lymphocytes but not in the endothelium or pneumocytes. ET-1 localization was no different in surveillance specimens compared with infected or rejecting lungs, or changed over time from transplantation. This study suggests that the source of the increased BAL ET-1 in transplanted lungs is due to the increased number of alveolar inflammatory cells and de novo expression in the airway epithelium. The biologic importance of the ET-1 from inflammatory cells is supported by the observation that peripheral mononuclear cells from dogs with mild to moderate lung allograft rejection cause vasoconstriction in pulmonary arterial rings, which is attenuated by the ETA blocker BQ123 [116] .
Analysis of ET-1 binding activity in failed transplanted human lungs suggested that ET-1 binding activity was not different compared with normal lung in the lung parenchyma, bronchial smooth muscle, or perivascular infiltrates. ET-1 binding was, however, decreased in small muscular arteries (pulmonary arteries and bronchial arteries) in the failed transplants, suggesting a role for ET-1 in impaired vasoregulation of transplanted lungs [117] .
Ischemia-reperfusion injury is the leading cause of early post-operative graft failure and death. In its severest manifestation, increased pulmonary vascular resistance, hypoxia, and pulmonary edema lead to cor pulmonale and death [118] . ET-1 has been implicated as a mediator of these events. The increase in pulmonary vascular resistance observed in human recipients of lung allografts follows an increase in circulating ET-1 and falls with decreases in circulating ET-1 [113] . A similar pattern is seen in dogs subjected to allotransplantation [119] . Conscious dogs with left pulmonary allografts demonstrate an increase in both resting pulmonary perfusion pressure and acute pulmonary vasoconstrictor response to hypoxia [120] . Administration of ETA selective or combined ETA and ETB receptor blockers did not change the resting tone. ETB receptor mediated hypoxic pulmonary vasoconstriction appeared, however, to be increased in allograft recipients. In another study, administration of a mixed ETA and ETB receptor antagonist (SB209670) to dogs before reperfusion of the allograft resulted in a marked increase in oxygenation, decreases in pulmonary arterial pressures and improved survival compared with control animals [121] . In a model of ischemia reperfusion, inhibitors of ECE additionally attenuated the increase in circulating ET-1 and the severity of lung injury [122] . ET-1 receptor antagonists did not, however, completely eliminate the ischemia-reperfusion injury, suggesting that changes in other vasoactive mediators, such as an increase in thromboxane, a decrease in prostaglandins, or a decrease in NO, may also contribute to the increased pulmonary vascular resistance. Administration of NO donor (FK409) to both donor and recipient dogs before lung transplantation reduced pulmonary arterial pressure, lung edema, and inflammation, and improved survival. This suggests that reductions in NO following transplantation may be partly responsible for early graft failure [123] . Treatment with NO donor was also associated with a decrease in plasma ET-1 levels.
Acute rejection is manifested by diffuse infiltrates, hypoxia, and airflow limitation, and may lead to respiratory insufficiency and death. BAL ET-1 was increased in dogs during episodes of acute rejection that decreased with immunosuppressive treatment [124] . Acute episodes of rejection in humans, however, are not associated with further increases in BAL ET-1 [72] . Chronic rejection of allografts, manifested as BO, is the major cause of morbidity and mortality in long-term lung transplant survivors [71] . The etiology of BO following transplant is unclear but may be related to repeated episodes of acute rejection, chronic low-grade rejection, or organizing pneumonia [125] . As discussed earlier, a chronic increase in ET-1, as seen in lung allografts, may contribute to bronchospasm and proliferative bronchiolitis obliterans due to the bronchoconstrictor and smooth muscle mitogenic effects of ET-1 [28, 126] . This is further supported by the increase of BAL ET-1 in the transplanted lung, which is susceptible to BO, but not the native lung in recipients of single lung transplants [72] .
The mitogenic effects of ET-1 may play a role in the development of pulmonary malignancy as well as metastasis to the lung. Many human tumor cell lines, including prostate, breast, gastric, ovary, colon, etc, produce ET-1. The importance of the ET-1 may lie in its mitogenic effects on tumor growth and survival. This has been suggested by blockade of ETA receptors resulting in a decrease in mitogenic effects of ET-1 in a prostate cancer and colorectal cell lines [127, 128] . ET-1 receptors in tumor cells may also be altered with increases in the ETA receptor and downregulation of ETB receptors [129] . Other tumors may have an increase in ETB receptors, however, and blockade of ETB results in a decrease in tumor growth [130, 131] . Tumor cells may, as a result of this altered balance, lose the ability to respond to regulatory signals from their environment. ET-1 may additionally protect against Fas-ligand mediated apoptosis [132] .
ET-1 has been detected using immunohistochemistry and in situ hybridization in pulmonary adenocarcinomas and squamous cell tumors and, to a lesser extent, small cell and carcinoid tumors [133] . In situ hybridization also demonstrated a similar pattern of ET-1 mRNA expression in non-neuroendocrine tumors. ET-1 receptors have also been found in a variety of pulmonary tumor cell lines. ETA receptors were found in small cell tumors, adenocarcinomas and large cell tumors, while ETB receptors were expressed primarily in adenocarcinomas and small cell tumors [134] . ECE, which converts big ET-1 to ET-1, the committed step in ET-1 biosynthesis, was also found in human lung tumors but not in adjacent normal lung [135] . These findings, combined with the presence of ET-1 in lung tumors, suggest a possible autocrine loop that sustains and supports the growth of lung tumors. A recent study, however, suggested that, while ETA and ECE-1 were detectable in lung tumors, these genes were downregulated compared with normal bronchial epithelial cell lines [136] . It was proposed that the role of ET-1 in lung tumors is not that of an autocrine factor, but that of a paracrine growth factor to the stroma and vasculature surrounding the tumor allowing angiogenesis.
Tumor angiogenesis is necessary for continued growth of the tumor beyond the limits of oxygen diffusion. The growth of vessels into the tumor is also important to metastatic potential of the tumor. ET-1 may play an important role in angiogenesis and tumor growth and survival Available online http://respiratory-research.com/content/2/2/090 commentary review reports primary research through induction of vascular endothelial growth factor expression and sprouting of new vessels into the tumor and surrounding tissue [137, 138] . ET-1 binding activity was found in blood vessels and vascular stroma surrounding lung tumors at the time of resection, most markedly surrounding squamous cell tumors [139] . ET-1 production may be further augmented by the hypoxic environment found within large solid tumors [140] . Since metastasis is dependent on neo-vascularization, ET-1 may also be an important mediator of this phenomenon. ET-1 receptor antagonists may have a useful role in the treatment of neoplastic disease by inhibiting growth as well as metastatic potential of human tumors.
Experimental lung injury of many different types results in increased circulating ET-1, BAL ET-1, and lung tissue ET-1 [18] . ET-1 levels in humans are also increased in sepsis, burns, disseminated intravascular coagulation, acute lung injury, and acute respiratory distress syndrome (ARDS) [141] [142] [143] [144] [145] [146] [147] . ET-1 increases also correlate with a poorer outcome with multiple organ failure, increased pulmonary arterial pressure, increased airway pressure and decreased PiO 2 /FiO 2 , while clinical improvement correlates with decreased ET-1 levels [144, 145, 147] . The arterio-venous ratio for ET-1 is increased in patients with ARDS but it is not clear whether this is due to increased secretion of ET-1 in the lungs or decreased clearance [142, 144] . In patients who succumbed to ARDS, there was also a marked increase in tissue ET-1 immunostaining in vascular endothelium, alveolar macrophages, smooth muscle, and airway epithelium compared with lungs of patients who died without ARDS. Interestingly, these same patients also had a decrease in immunostaining for both endothelial nitric oxide synthase and inducible nitric oxide synthase in the lung [148] . ARDS is also characterized by the presence of inflammatory cells in the lung. Since ET-1 may act as an immune modulator, an increase in ET-1 may contribute to lung injury by inducing expression of cytokines including tumor necrosis factor and IL-6 and IL-8 [149] . These cytokines may in turn stimulate the production of many inflammatory mediators, leading to lung injury. ET-1 additionally activated neutrophils, and increased neutrophil migration and trapping in the lung [65] [66] [67] [68] [69] .
Another hallmark of ARDS is disruption and dysfunction of the pulmonary vascular endothelium leading to accumulation of lung water. The role of endothelin in formation of pulmonary edema is uncertain. Infusion of ET-1 raises pulmonary vascular pressure, but it is uncertain whether ET-1 by itself increased pulmonary protein or fluid transport in the lung [150] [151] [152] . ET-1 may rather be acting synergistically with other mediators to lead to pulmonary edema [153, 154] .
Pulmonary fibrosis is the final outcome for a variety of injurious processes involving the lung parenchyma. The final common pathway in response to injury to the alveolar wall involves recruitment of inflammatory cells, release of inflammatory mediators, and resolution. The reparative phase occasionally becomes disordered, resulting in progressive fibrosis.
ET-1 in the lung may be important in the initial events in lung injury by activating neutrophils to aggregate and release elastase and oxygen radicals, increasing neutrophil adherence, activating mast cells, and inducing cytokine production from monocytes [65] [66] [67] [68] [69] 149, 155] . Among the many cytokines induced by ET-1 that are important in mediating pulmonary fibrosis are transforming growth factor-β and tumor necrosis factor α [156, 157] . ET-1 is also profibrotic by stimulating fibroblast replication, migration, contraction, and collagen synthesis and secretion while decreasing collagen degradation [158] [159] [160] [161] [162] . ET-1 additionally enhances the conversion of fibroblasts into contractile myelofibroblasts [43, 163] . ET-1 also increases fibronectin production by bronchial epithelial cells [164] . Finally, ET-1 has mitogenic effects on vascular and airway smooth muscle [126, 28] . ET-1 may thus play an important role in the initial injury and eventual fibrotic reparative process of many inflammatory events in the lung.
Several lines of evidence regarding the importance of ET-1 in pulmonary fibrosis are available. Plasma and BAL ET-1 levels are increased in idiopathic pulmonary fibrosis [50, 165] . Lung biopsies from patients with idiopathic pulmonary fibrosis have additionally increased ET-1 immunostaining in airway epithelial cells and type II pneumocytes, which correlates with disease activity [166] . Scleroderma is commonly associated with pulmonary hypertension and pulmonary fibrosis. Plasma and BAL ET-1 is increased in these patients [160, 167, 168] , but it is unclear whether the presence of either pulmonary hypertension or pulmonary fibrosis increases these levels further [167] . BAL fluid from patients with scleroderma increased proliferation of cultured lung fibroblasts, which was inhibited by ETA receptor antagonist. This suggests that the ET-1 in the airspace may be contributing significantly to the fibrotic response [160] . An increase in ET-1 binding has also been reported in lung tissue from patients with scleroderma associated pulmonary fibrosis [169] . Pulmonary inflammatory cells also appear to be primed for ET-1 production because cultured alveolar macrophages from patients with scleroderma and lung involvement secrete increased amounts of ET-1 in response to stimulation with lipopolysaccharide [170] . These observations collectively suggest that augmented ET-1 release may contribute to and perpetuate the inflammatory process. Bleomycin-induced pulmonary fibrosis in animals is associated with increased ET-1 expression in alveolar macrophages and epithelium [171] . The increase in ET-1 proceeds the development of pulmonary fibrosis. The use of ET-1 receptor antagonists has produced mixed results in limiting the development of bleomycin-induced fibrosis. A decrease in fibroblast replication and secretion of extracellular matrix proteins in vitro but not a decrease in lung collagen content in vivo has been shown using ETA or combined ETA and ETB receptor antagonists after bleomycin [172] . Another group did, however, observe a decrease in fibrotic area in lungs of rats following bleomycin that were treated with a mixed ETA and ETB receptor antagonist [173] .
While ET-1 seems to correlate with pulmonary fibrosis, it remains uncertain whether the increase in ET-1 is a cause or consequence of the lung disease. Pulmonary fibrosis was recently reported in mice that constitutively overexpress human ET-1 [107] . These mice were known to develop progressive nephrosclerosis in the absence of systemic hypertension [174] . The transgene was localized throughout the lung, with the strongest expression in the bronchial wall. In the lung, the mice developed age-dependent accumulation of collagen and accumulation of CD4+ lymphocytes in the perivascular space. This observation suggests that an increase in lung ET-1 alone may play a causative role in the development of pulmonary fibrosis [107, 175] .
Since its discovery 12 years ago, much evidence has accumulated regarding the biologic activity and potential role of ET-1 in a variety of diseases of the respiratory track. As compelling as much of this evidence is, the causal relationship between ET-1 activity and disease is not complete. The increasing use of ECE and endothelin receptor antagonists in experimental and human respiratory disorders will help to clarify the role of this pluripotent peptide in health and disease. Gene expression in epithelial cells in response to pneumovirus infection Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner. RSV and PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus; they are enveloped, singlestranded, nonsegmented RNA viruses that can cause intense viral bronchiolitis in humans and mice, respectively. In its most severe form, the lower respiratory tract infection caused by pneumoviruses is associated with the development of peribronchiolar infiltrates that are accompanied by submucosal edema and bronchorrhea, and ultimately leads to bronchiolar obstruction and compromised oxygen transfer. As the infection is confined to the respiratory epithelium, the responses of these cells are clearly of primary importance in determining the nature and extent of the resulting inflammatory process.
Most of our understanding of responses to pneumovirus infection has emerged from studies of RSV infection of human epithelial target cells in vitro; a list of genes and/or gene products produced by epithelial cells in response to RSV infection in vitro is provided in Table 1 . At the cellular level, epithelial cells initially respond to RSV infection by reducing their ciliary beat frequency. Production and release of chemoattractant cytokines (chemokines) can be observed as early as 12 h after infection, leading to the recruitment of specific leukocyte subsets to the lung tissue. RSV-infected epithelial cells become resistant to tumor necrosis factor (TNF)-α-induced apoptosis, but later fuse to form giant-cell syncytia and die by cellular necrosis. We review the molecular bases (to the extent that they
are understood) of these specific responses, and discuss several novel strategies that may permit us to study the responses to RSV and PVM infection in a more coherent and systematic manner.
Tristram et al [1] observed that explanted respiratory epithelial cells slow their ciliary beat frequency almost immediately after exposure to RSV, with complete ciliostasis seen as early as 6 h after the initial infection. The molecular basis of ciliostasis remains completely unknown.
The chemokines and cytokines with production and release that has been associated with RSV infection of human epithelial cells are listed in Table 1 . Much of this work was also recently reviewed elsewhere [2, 3] . We focus here on the three chemokines whose molecular mechanisms and physiologic implications are best understood.
The earliest reports on this subject described production of the neutrophil chemoattractant IL-8 from tissue culture supernatants from RSV-infected cells [4] [5] [6] and in nasal secretions from patients with viral rhinitis [7] . IL-8 has since been detected in lower airway secretions from patients with severe RSV bronchiolitis [8] , and the neutrophil influx observed in response to this infection is probably due, at least in part, to the activity of this chemokine.
At the cellular level IL-8 production can be observed in response to inactivated RSV virions, whereas IL-8 production in response to active infection was inhibited by ribavarin, amiloride, and antioxidants [9, 10] . Several groups have demonstrated activation of the transcription factor nuclear factor-κB (NF-κB) in response to RSV infection, and NF-κB is recognized for its central role in eliciting the production of IL-8 [9, 11, 12] . The transcription factor NF-IL-6 is also produced in response to RSV infection [13] , and participates in a co-operative manner with NF-κB in the regulation of IL-8 gene expression [11] , although later studies suggest that activator protein-1 may function preferentially in this role [14] . Interestingly, the NF-κB regulator IκBα, which functions by inhibiting NF-κB activation in response to TNF-α, was produced with different kinetics and does not promote a reversal of NF-κB activation in response to RSV infection as it does in response to TNF-α [15] . Most recently, Casola et al [16] demonstrated that the IL-8 promoter contains independent response elements, with nucleotides -162 to -132 representing a unique RSV response element that is distinct from elements necessary for IL-8 production in response to TNF-α. This concept of a stimulus-specific response will probably make an important contribution toward our understanding of how pneumoviruses promote transcription of unique and specific sets of independent gene products.
The pleiotropic chemokine regulated upon activation, normal T-cell expressed and secreted (RANTES) has also been detected in supernatants from RSV-infected epithelial cells in culture [17, 18] , as well as in upper and lower airway secretions from patients infected with this virus [7, 8] . RANTES acts as a chemoattractant for eosinophils and monocytes in vitro, although its role in vivo is somewhat less clear. Similar to IL-8, RANTES can be produced in vitro in response to inactivated virions [8] , and involves NF-κB activation, binding, and nuclear translocation [19] . However, Koga et al [20] demonstrated that stabilization of RANTES mRNA, a response to RSV infection mediated in part by nucleotides 11-389 of the RANTES gene, is probably the primary mechanism underlying increased production and secretion of RANTES protein. Further studies will determine whether a similar mechanism is also in place for IL-8 and other RSV-mediated responses. 
Several groups have recently shown that macrophage inflammatory protein (MIP)-1α is released from RSVinfected cells in culture [7, 21] ; MIP-1α was also detected in upper and lower airway secretions from RSV-infected patients [7, 8] . Interestingly, of the three aforementioned chemokines, MIP-1α is the one that is most closely correlated with the presence of eosinophil degranulation products; this, together with data from our PVM model of pneumovirus infection [22] , has suggested to us that MIP-1α plays a pivotal role in eosinophil recruitment in response to primary pneumovirus infection. Interestingly, production of MIP-1α in cell culture requires active viral replication [8] , which suggests that this response may proceed by a mechanism that is completely distinct from that which elicits production of RANTES and IL-8. However, no reports to date have addressed the molecular mechanism that underlies the RSV-mediated MIP-1α response.
A list of cell-surface molecules that have been reported as expressed in response to RSV infection is shown in Table 1 . We focus here on the expression of intercellular adhesion molecule (ICAM)-1 (CD54) and the leukocyte integrin CD18.
Increased expression of this cell-surface adhesion protein was observed in both respiratory epithelial cell lines [23, 24] and in human nasal epithelial cells [25] in response to infection with RSV in vitro. Chini et al [26] demonstrated that the expression of ICAM-1 mRNA, similar to IL-8 and RANTES, was dependent on an intact NF-κB site in the gene promoter, and demonstrated a role for the consensus binding site for the factor CCAAT/ enhancer-binding protein. Stark et al [27] demonstrated that ICAM-1 and CD18 expressed in response to RSV serve to enhance neutrophil and eosinophil binding to epithelial cells.
CD18 is a polypeptide of the integrin family that functions in mediating cell-cell interactions. Several groups have observed expression of CD18 on epithelial cells in response to RSV infection [27, 28] , with CD18 shown to enhance the degranulation of eosinophils in this specific setting [28] . Of particular interest are the recent findings relating expression of CD18 (along with CD14) to earlier literature on bacterial superinfections in the setting of viral infections. Earlier studies [29, 30] reported enhanced binding of bacteria to respiratory epithelial cells that were infected with RSV, findings that had clinical implications relating to acute bacterial otitis media in infants.
Two more recent studies addressed the question of binding sites. Saadi et al [31] determined that two strains of the pathogen Bordetella pertussis bound more efficiently to RSV-infected cells, and that the binding was reduced upon pretreatment of the cells with anti-CD14 or anti-CD18 antibodies. Similarly, Raza et al [32] reported that both CD14 and CD18 on RSV-infected epithelial cells contributed to the binding of nonpilate Neisseria meningitidis. In vivo testing is required before the clinical significance of these intriguing findings can be appreciated.
RSV-infected epithelial cells in culture do not show features that are suggestive of apoptosis (ie no evidence of membrane blebbing, fragmentation of chromosomal DNA, or characteristic changes in nuclear morphology). Takeuchi et al [33] showed that, although RSV-infected epithelial cells express a number of apoptosis-associated genes, including interferon regulatory factor-1, IL-1β-converting enzyme and caspase 3, they do not undergo formal apoptosis.
As part of our attempts to understand mucosal responses in a more systematic manner (see below), we discovered that RSV-infected epithelial cells express the recently described antiapoptosis gene IEX-1L [34] . In our studies, we found that expression of IEX-1L is a response to active virus; no gene expression was observed in response to irradiated, replication-incompetent virus. Moreover, expression of IEX-1L is not observed in response to adenoviral infection, suggesting that expression of this gene is not a universal response to cellular perturbation, or indeed to all viral infections. Functionally, we also demonstrated that RSV infection protects epithelial cells from TNF-α-induced apoptosis, an effect that is temporally associated with the expression of IEX-1L.
Apoptosis is generally considered to be a highly efficient self-defense mechanism employed by host target cells, because it permits the infected host to dispose of viral proteins and nucleic acids on a single-cell basis without inducing an inflammatory response. It is thus not surprising that many viruses have evolved strategies to circumvent this response. Of interest, Krilov et al [35] demonstrated that monocytes and cord blood mononuclear cells are similarly protected from apoptosis when infected with RSV.
Although virus-induced protection from apoptosis appears advantageous to the virus alone, another interpretation may be considered. Because respiratory epithelial cells are now recognized as a major source of leukocyte chemoattractants, and because leukocyte recruitment to the lung has been associated with enhanced viral clearance and prolonged survival in pneumovirus infection [22] , the ability to maintain chemoattractant production from viable cells may ultimately benefit the host organism as well.
Available online http://respiratory-research.com/content/2/4/225
In tissue culture, RSV-infection is characterized by the formation of giant-cell syncytia. The mechanisms for the formation of these fused masses of cells depend in part on the expression of the RSV-specific fusion (F) protein on the surface of infected host cells, and in part on virusmediated changes in cytoskeletal architecture. It is important to note that RSV-induced changes in cytoskeletal architecture are not restricted to cell lines grown in vitro, as giant-cell syncytia have also been found in pathologic lung specimens obtained from both humans and animals that were infected with RSV.
Again, as part of our systematic study of gene expression in response to pneumovirus infection, we found that human respiratory epithelial cells respond to RSV infection with increased expression of the cytoskeletal protein cytokeratin-17 [36] . Cytokeratin-17 is a 46-kDa cytoskeletal protein that belongs to the class I acidic cytokeratin family. In the lung, expression of cytokeratin-17 is normally restricted to basal epithelial cells of the larynx, trachea, and bronchi. In RSV-infected cells, we found expression of Ck-17 predominantly at sites of syncytia formation, and thus provided the first description of a unique component of these pathognomonic structures at the molecular level. Similar to what has been reported for the production of IL-8, expression of Ck-17 is dependent on the activity of the transcription factor NF-κB, and future studies will determine the role of the NF-κB consensus site (-200 to -208 of the cytokeratin-17 promoter) in mediating this response.
To date, efforts to study pneumovirus-induced alterations in gene expression have relied heavily on in vitro models of virus-infected cells and cell lines. The intrinsic value of characterizing the genes identified in this artificial system is by definition limited, and the clinical and physiologic sig-nificance of any findings must ultimately be tested in vivo.
To some extent, the study of gene products in clinical specimens is possible, but this approach is limited, cumbersome, and dictated by sample availability. It is clear that an appropriate animal model of inflammatory bronchiolitis is required to characterize the alterations in gene expression discovered using the available in vitro models. Although RSV has been used for the study of specific allergic reactions to viral antigens, it is not a natural pathogen of mice, and intranasal inoculation of virus at high titer results in, at best, a minimal primary infection with a correspondingly minimal inflammatory response. In order to study gene expression in response to primary pneumovirus infection in vivo, we developed a novel mouse model of inflammatory bronchiolitis using the natural rodent pneumovirus pathogen and closest phylogenetic relative of RSV [37] -PVM. We presented our initial findings on PVM infection in mice in three recent publications [22, 38, 39] . A summary of these findings is presented in Table 2 and Fig. 1 .
To begin, we described the cellular and biochemical pathology observed in response to PVM infection in mice [38] . We found that infection could be established with as few as 30 plaque-forming units (pfu) of PVM in the inoculum, with infection resulting in significant morbidity and mortality, and viral recoveries in the order of 10 8 pfu/g lung tissue. We also noted inflammatory bronchiolitis as among the immediate responses to this infection, with bronchoalveolar lavage fluid containing virtually 100% neutrophils and eosinophils obtained as early as 3 days after inoculation. Furthermore, we found that infection was accompanied by the production of the proinflammatory chemokine MIP-1α, which was previously shown by Cook et al [40] to be an important component of the inflammatory response to the orthomyxovirus influenza virus.
We also described the role played by MIP-1α in the pathogenesis of PVM-induced bronchiolitis [22] . Specifi- cally, we explored the responses of gene-deleted mice to infection with PVM, and found no inflammatory response in mice deficient in MIP-1α expression (MIP-1α -/-) and only minimal virus-induced inflammation in mice that lacked the major MIP-1α receptor on granulocytes chemokine receptor (CCR)1 (CCR1 -/-). Although the inflammatory response is often considered to be unnecessary and indeed detrimental, we demonstrated that the absence of granulocytic inflammation was associated with enhanced recovery of infectious virions, as well as with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of viral infection.
Our most recent report on this subject [39] presents a direct comparison between the responses of mice to challenge with PVM and RSV. Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of human infection because a limited, or 'semipermissive' infection can be established via intranasal inocula-tion of virus at very high titers. In this regard, we found (as have others) that RSV infection did not result in any measurable degree of morbidity, and that viral recovery was significantly lower than that found in the inoculum; these results suggested that there was no significant viral replication in mouse lung tissue. We further demonstrated that the inflammatory response to RSV challenge was minimal, as few leukocytes were recruited to the lungs (Fig. 1) .
Taken together, our results suggest that infection of mice with PVM provides a superior model for the study of acute inflammatory bronchiolitis in response to pneumovirus infection in vivo. The advantages of this model include the following: clinical parameters -morbidity and mortalitythat can be measured clearly and specifically; clear evidence of viral replication in lung tissue, with incremental recoveries that, at peak, are in excess of 10 8 pfu/g in response to as few as 30 pfu in the inoculum; and a dramatic granulocytic response that is modulated at least in part by the proinflammatory chemokine MIP-1α and its receptor CCR1. 
Traditionally, analysis of gene expression through measurement of steady-state levels of individual mRNAs could be conducted only one gene at a time using northern blotting, dot blots, or quantitative reverse transcription-PCR. Differential display, serial analysis of gene expression, and total gene analysis offer great promise, because they are multiplex technologies that provide simultaneous analysis of multiple mRNAs isolated under conditions of interest via PCR amplification techniques. DNA hybridization arrays are theoretically the most efficient of the gene expression analysis techniques. Although many skeptics have described these genome-based approaches as expensive, nonhypothesis-driven 'fishing expeditions', we view them as broad-based screening techniques that will enable us to identify patterns of gene expression that can then be subjected to careful characterization and analysis.
Differential display is a semiquantitative, reverse transcription-PCR-based technique that is used to compare mRNAs from two or more conditions of interest. Both increased and decreased expression of specific amplicons will be evident -an obvious advantage to this approach. Total RNA can be isolated from virus infected versus uninfected cells or mouse lungs both before and during infection, and differential display is performed using degenerate T11(XY) anchoring primers and random upstream oligomers, as described elsewhere [34, 36] . The resulting PCR products are separated by electrophoresis, and the gel is dried and exposed to film. An example of our results comparing cDNA amplicons from RNA extracted from RSV-infected epithelial cells daily for 4 days is shown in Fig. 2 . Differentially expressed bands are cut from the gel, eluted and reamplified using the same primers that generated the original signal, and northern blots generated from RNA extracted from pneumovirus-infected cells or tissue over time and probed with the differentially expressed amplicons serve to confirm differential expression of the identified sequence.
The DNA sequences of the newly identified differentially expressed amplicons are compared with sequences present in the GenBank database. Viral sequences are expected to be upregulated over time and can be identified immediately, because the entire genomes of both PVM and RSV are present in GenBank. In cases in which the amplicon represents a newly discovered gene, potential openreading frames are compared with sequences that are present in the Swiss protein database; motifs that share homologies with known proteins represent important clues to the identity of the differentially expressed gene.
With the help of differential display, we have identified and characterized several genes that are upregulated in RSV-infected respiratory epithelial cells. Two specific examples of genes that were found to be induced during RSV infection, and later characterized as playing independent roles in the pathophysiology of RSV infection, are the antiapoptosis gene IEX-1L [34] and the gene that encodes the cytoskeletal protein cytokeratin-17 [36] .
Unlike DNA viruses, which are known to encode virus-specific antiapoptosis genes, RSV -an RNA virus with a small (approximately 15.2 kb) viral genome -was shown to alter host cell expression of the apoptosis inhibitor IEX-1L. After demonstrating that IEX-1L mRNA was present at sevenfold higher concentrations in RSV-infected respiratory epithelial cells when compared with uninfected cells, we concluded that this cellular response protected against TNF-α-induced programmed cell death during viral infection. Further efforts to determine which of the 11 RSV proteins participate in the trans-activation of the IEX-1L gene (either directly or indirectly) are ongoing.
A second example of a gene that is specifically upregulated in RSV-infected respiratory epithelium, as identified by differential display, is that which encodes cytokeratin-17 [36] . Upon characterizing the molecular events that are important for cytokeratin-17 induction, we demonstrated a link to an NF-κB signaling pathway. Above, we discussed the importance of this transcription factor in the regulation of proinflammatory cytokine gene expression, and because of this involvement we were not surprised to discover its role in virus-induced cytokeratin-17 gene regulation. Perhaps the most interesting observation made during these experiments was the in situ localization of cytokeratin-17 protein to areas of cytopathic syncytia formation, suggesting a role for this cytoskeletal protein in their formation. Of note, we observed a dramatic decrease in RSV replication and in syncytia formation when we blocked cytokeratin-17 expression, suggesting that blocking syncytia formation, at least in part, impairs the direct cell-cell spread and productive replication of virus.
Although there are several companies that market these systems and components, the cytokine gene macroarray systems recently developed by R&D Systems (Sigma Genosys ® ; Minneapolis, MN, USA) and Clontech (Atlas ® ; Palo Alto, CA, USA) represent some of the newer opportunities available that have a focus on gene products that are known to be involved in inflammation. These arrays consist of different cDNAs printed as PCR products onto charged nylon membranes. An example of our experience with the Sigma Genosys array is shown in Fig. 3 . For this example, total RNA was extracted from RSV-infected HEp-2 cells and uninfected controls at day 3 after infection. Three micrograms of total RNA was used in a cDNA synthesis reaction, using a proprietary mixture of 378 primer pairs and trace amounts of 32 P-radiolabeled dCTP.
The resulting radiolabeled products were hybridized to the macroarrays overnight at 65°C, and then washed and exposed to film. The arrow highlights one of the most obviously upregulated sequences from this experiment, which was identified as the gene encoding human MIP-1α. The physiologic importance of MIP-1α upregulation during human RSV infection and during rodent PVM bronchiolitis has already been described.
Microarrays can be differentiated from macroarrays in several ways. Among these differences, the microarray matrix is a glass or plastic slide, probes are labeled with fluorescent dye rather than via radioisotopes, and, most significantly, microarrays generally include a larger number and a higher density of imbedded sequences than do macroarrays. Although this may seem to be highly appealing at first, the massive amounts of data generated by microarray technology poses new challenges with respect to data normalization, management, and development of mathematical models to assist in data interpretation.
The pneumoviruses RSV and PVM enter respiratory epithelial cells via a receptor-mediated event. During hostcell attachment and internalization, the target cell begins to alter its gene expression, which, among other events, involves the transcriptional upregulation of cytokine and chemokine genes. As RSV replication progresses, additional changes in cellular gene expression can be observed, including induction of the potent antiapoptosis gene IEX-1L and increased expression of the otherwise quiescent gene that encodes cytokeratin-17. What we know regarding the physiologic importance of these genes and their gene products has been described, but there is more to be learned. As the available technologies evolve, we can continue to capitalize on the use of Display of amplicons generated from RNA extracted from RSV-infected cells at daily intervals following infection (days 0-4) using a single anchoring primer, T11GC (downstream primer 8) and (A-H) eight random 10mers. Two differentially expressed sequences are highlighted by arrows (the black arrow shows an upregulated amplicon, and the white arrow highlights a downregulated amplicon). Several other differentially expressed signals are also seen.
genomic approaches as large-scale screening tools to identify genes that play important roles in the pathophysiology of pneumovirus infection. These elegant and simple tools will provide us with the means for thorough and systematic exploration of gene expression, both in the estab- Cytokine macroarray probed with radiolabelled cDNA generated from total RNA extracted from epithelial cells 48 h after RSV infection (upper panel) or 48 h after exposure to conditioned medium (lower panel). Signal intensity of each gene under each condition is compared. The arrow highlights the signal for human MIP-1α present at 12-fold higher concentration in infected epithelial cells compared with the uninfected controls. Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis. The genetic information of RNA viruses is organized very ef®ciently. Practically every nucleotide of their genome is utilized, either as protein-coding sequence or as cis-acting signals for translation, RNA synthesis or RNA encapsidation. As part of their genome expression strategy, several groups of positive-strand RNA (+RNA) viruses produce subgenomic (sg) mRNAs (reviewed by Miller and Koev, 2000) . The replication of their genomic RNA, which is also the mRNA for the viral replicase, is supplemented with the generation of sg transcripts to express structural and auxiliary proteins, which are encoded downstream of the replicase gene in the genome. Sg mRNAs of +RNA viruses are always 3¢-co-terminal with the genomic RNA, but different mechanisms are used for their synthesis.
Some viruses, such as brome mosaic virus, initiate sg mRNA synthesis internally on the full-length minus strand RNA template (Miller et al., 1985) . Others, exempli®ed by red clover necrotic mosaic virus (RCNMV), may rely on premature termination of minus strand synthesis from the genomic RNA template, followed by the synthesis of sg plus strands from the truncated minus strand template (Sit et al., 1998) . Members of the order Nidovirales, which includes coronaviruses and arteriviruses, have evolved a third and unique mechanism, which employs discontinuous RNA synthesis for the generation of an extensive set of sg RNAs (reviewed by Brian and Spaan, 1997; Lai and Cavanagh, 1997; Snijder and Meulenberg, 1998) . Nidovirus sg mRNAs differ fundamentally from other viral sg RNAs in that they are not only 3¢-coterminal, but also 5¢-co-terminal with the genome ( Figure 1A) . A 5¢ common leader sequence of 65±221 nucleotides, derived from the 5¢ end of the genomic RNA, is attached to the 3¢ part of each sg RNA (thè mRNA body').
Various models have been put forward to explain the cotranscriptional fusion of non-contiguous parts of the nidovirus genome during sg RNA synthesis ( Figure 1B and C). Central to each of these models are short transcription-regulating sequences (TRSs), which are present both at the 3¢ end of the leader and at the 5¢ end of the sg RNA body regions in the genomic RNA. The TRS is copied into the mRNA and connects its leader and body part (Spaan et al., 1983; Lai et al., 1984) . Synthesis of sg mRNAs initially was proposed to be primed by free leader transcripts, which would base-pair to the complementary TRS regions in the full-length minus strand, and would be extended subsequently to make sg plus strands ( Figure 1B ; Baric et al., 1983 Baric et al., , 1985 . This model, however, was based on the report that sg minus strands were not present in coronavirus-infected cells (Lai et al., 1982) . The subsequent discovery of such molecules (Sethna et al., 1989) resulted in reconsideration of the initial`leader-primed transcription' model. Sawicki and Sawicki (1995) have proposed an alternative model ( Figure 1C ), in which the discontinuous step occurs during minus instead of plus strand RNA synthesis. In this model, minus strand synthesis would be attenuated after copying a body TRS from the plus strand template. Next, the nascent minus strand, with the TRS complement at its 3¢ end, would be transferred to the leader TRS and attach by means of TRS±TRS base pairing. RNA synthesis would be reinitiated to complete the sg minus strand by adding the complement of the genomic leader sequence. Subsequently, the sg minus strand would be used as template for sg mRNA synthesis, and the presence of the leader complement at its 3¢ end might allow the use of the same RNA signals that direct genome synthesis from the fulllength minus strand.
Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis The EMBO Journal Vol. 20 No. 24 pp. 7220±7228, 2001 Using site-directed mutagenesis of TRSs of the arterivirus equine arteritis virus (EAV), we have shown previously that base pairing between the sense leader TRS and antisense body TRSs is crucial for sg mRNA synthesis (van Marle et al., 1999a) . However, base pairing is only one step of the nascent strand transfer process and is essential in both models outlined in Figure 1 . The EAV genomic RNA contains several sequences that match the leader TRS precisely, but nevertheless are not used for sg RNA synthesis (den Boon et al., 1996; Pasternak et al., 2000) . This suggests that leader±body TRS similarity alone is, though necessary, not suf®cient for the strand transfer to occur.
To gain further insight into the cis-acting signals regulating sg RNA synthesis, we performed a comprehensive site-directed mutagenesis study of the EAV leader and body TRSs. Every nucleotide of the TRS (5¢-UCAACU-3¢) was substituted with each of the three alternative nucleotides. Our analysis revealed a number of striking similarities with the process of copy-choice RNA recombination, as it occurs in RNA viruses. Whereas the leader TRS plays only a targeting role in translocation of the nascent strand, body TRS nucleotides appear to ful®l diverse position-speci®c and base-speci®c functions. In addition, the sequence of the leader±body junctions of the sg mRNAs produced by these mutants provided strong evidence for the discontinuous minus strand extension model.
EAV genome replication is not signi®cantly affected by leader TRS and body TRS mutations To dissect EAV RNA synthesis, we routinely use a fulllength cDNA clone (van Dinten et al., 1997) , from which infectious EAV RNA is in vitro transcribed. Following transfection of the RNA into baby hamster kidney (BHK-21) cells, intracellular RNA is isolated and analysed by northern blot hybridization and RT±PCR (van Marle et al., 1999a) . Due to differences in transfection ef®ciency, the total amount of virus-speci®c RNA (genomic RNA and sg mRNA) isolated from transfected cell cultures is somewhat variable. Thus, the accurate quantitation of sg mRNA synthesis by TRS mutants requires an internal standard for transfection ef®ciency. The amount of viral genomic RNA can be this standard, but only if its ampli®cation is not dramatically affected by the TRS mutations. To prove that this is the case, we used the previously described mutants L4, B4 and LB4 (van Marle et al., 1999a) , in which ®ve nucleotides of the TRS (5¢-UCAAC-3¢) were replaced by the sequence 5¢-AGUUG-3¢, either in the leader TRS (L4), RNA7 body TRS (B4) or both TRSs (LB4).
The three mutants were tested in three independent experiments. Intracellular RNA was isolated at 14 h posttransfection, early enough to prevent spread of the wildtype control virus to non-transfected cells (®rst cycle analysis). Transfection ef®ciencies were determined by immuno¯uorescence assays (see Materials and methods) and varied between 10 and 23% (data not shown). Prior to RNA analysis, the amount of isolated intracellular RNA was corrected for the transfection ef®ciency of the sample, so that each lane in Figure 2 represents EAV-speci®c RNA from an approximately equal number of EAV-positive cells. Phosphoimager quantitation revealed that genomic RNA replication of mutants L4, B4 and LB4 varied by not more than 30% (Table I) . These differences could re¯ect, for example, a slight in¯uence of RNA secondary structure changes in the TRS regions on genomic RNA synthesis. Remarkably, however, the genomic RNA level of the leader±body TRS double mutant LB4 was not affected by more than 10%. In view of the results obtained with these pentanucleotide TRS mutants, we assumed that the amount of genomic RNA could indeed be used as an internal standard during the analysis of mutants containing only single nucleotide replacements in leader TRS and/or RNA7 body TRS. The regions of the genome specifying the leader (L) sequence, the replicase gene (ORFs 1a and 1b) and the structural genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2±7) is depicted below. The black boxes in the genomic RNA indicate the position of leader and major body TRSs. (B and C) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus strand (B) or minus strand (C) RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus strand template. For details see text. Northern analysis of EAV-speci®c RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from TRS pentanucleotide mutants (UCAAC to AGUUG). The results of two independent experiments are shown.
The RNA±RNA interaction between the leader and body TRSs is not the only factor that regulates EAV sg RNA synthesis There are numerous examples of regulatory RNA±RNA interactions in both eukaryotic and prokaryotic cells, as well as in RNA viruses. Essential processes such as translation, replication and encapsidation of RNA virus genomes frequently depend on RNA±RNA interactions and higher order RNA structures. Regulation of sg RNA synthesis of +RNA viruses by RNA±RNA interactions is also not without precedent. In tomato bushy stunt virus, an RNA element located 1000 nucleotide upstream of the sg RNA2 promoter base-pairs with the promoter and is necessary for sg RNA production (Zhang et al., 1999) . Similarly, base pairing interactions between complementary sequences in the 5¢ end of the potato virus X genomic RNA and sequences upstream of two major sg RNA promoters are required for ef®cient sg RNA synthesis (Kim and Hemenway, 1999) . In RCNMV, an intermolecular RNA±RNA interaction is required for sg RNA synthesis (Sit et al., 1998) .
Recently, we have established the pivotal role of an interaction between sense and antisense RNA sequences in the life cycle of EAV (van Marle et al., 1999a) . In that study, the role of TRS nucleotides C 2 and C 5 was tested by substituting them with G. It was concluded that base pairing between the sense leader TRS and the antisense body TRS plays a crucial role in nidovirus sg RNA synthesis. We now took a more systematic approach and performed an extensive site-directed co-variation mutagenesis study of the entire leader TRS and RNA7 body TRS, which directs the synthesis of the most abundant EAV sg RNA. Every nucleotide of the TRS (5¢-UCA-ACU-3¢) was replaced with each of the other possible nucleotides. As in the study of van Marle et al. (1999a) , every mutation was introduced into leader TRS, RNA7 body TRS and both TRSs, resulting in 54 mutant constructs. Each mutant was given a unique name: e.g. BU 1 A refers to a mutant in which a U has been changed to A at position 1 of the body TRS; LU 1 A refers to the same substitution in the leader TRS; and DU 1 A means that these two substitutions were combined in one double mutant construct. The amount of sg RNA7 was quantitated by phosphoimager scanning of hybridized gels and was corrected for the amount of genomic RNA in the same lane (as outlined above). Figure 3 shows the relative sg RNA7 level of the 54 mutants, compared with the RNA7 level of the wild-type control. For a selection of 11 interesting mutants (see below), the analysis was repeated three times (Figure 4 ), without observing signi®cant variations in sg RNA synthesis.
The comprehensive analysis of the effects of TRS mutations considerably expanded our understanding of van Dinten et al., 1997) was taken along as a positive control. For every mutant, the level of sg RNA7 synthesis was calculated as [(sg/g)/(sg/g) wt ] 3 100%: it was corrected for the level of genomic RNA (used as an internal standard; see text) and subsequently was related to the level of sg RNA7 produced by the wild-type control in the same experiment, which was also corrected for the corresponding genomic RNA level. The relative sg RNA7 level of the wild-type control was set at 100%.
A.O. Pasternak et al. discontinuous sg RNA synthesis. Remarkably, the effects of single (leader or body) TRS mutations were mostly base speci®c, i.e. different nucleotide substitutions at the same position affected sg RNA7 synthesis to different extents. For example, at position 1, the BU 1 A mutant retained 44% of the wild-type RNA7 synthesis level, whereas both the BU 1 C and BU 1 G mutants lost RNA7 synthesis almost completely. Conversely, when U 1 of the leader TRS was changed to A or G, RNA7 synthesis was completely abolished, whereas 13% of the wild-type level was still maintained by LU 1 C. For position 2, only the BC 2 U mutant retained 30% of the wild-type RNA7 synthesis level, while all the other position 2 single mutants have lost 90% or more of wild-type RNA7 synthesis. Another example is position 6: BU 6 C left only 5% of wild-type RNA7 synthesis, whereas BU 6 A produced much higher RNA7 levels. This implied that for some positions (1, 2 and 6), certain mismatches in the duplex between plus leader TRS and minus body TRS, such as U±U (BU 1 A and BU 6 A) or C±A (LU 1 C and BC 2 U), are allowed to a limited extent. In contrast, no mismatches were allowed for position 5, where all single nucleotide substitutions abolished RNA7 synthesis almost completely. Surprisingly, both body TRS U to C substitutions at positions 1 and 6 (BU 1 C and BU 6 C) resulted in low levels of RNA7, despite the fact that these mutations allow the formation of a G±U base pair between the plus leader TRS, providing the U nucleotide, and the minus body TRS, providing the G. On the other hand, for positions 3 and 4, G±U base pairing was shown to be functional, because mutants LA 3 G and LA 4 G, which can form G±U base pairs between the G in the plus leader TRS and U in the minus body TRS, were the only position 3 and 4 single mutants that produced reasonable levels of RNA7. Taken together, these ®ndings suggest that other factors, besides leader± body base pairing, also play a role in sg RNA synthesis and that the primary sequence (or secondary structure) of TRSs may dictate strong base preferences at certain positions. Our analysis of the degree of complementation by the double mutants provided strong support for this assumption.
Differentiating between effects at the level of primary TRS sequence and the level of leader±body duplex formation For some TRS nucleotides (2, 5 and 6, except in the case of DU 6 C), the RNA7 level of double mutants was clearly higher than that of the corresponding single mutants. This means that base pairing between these leader and body TRS nucleotides is involved in sg RNA synthesis. However, none of these double mutants reached the wild-type sg RNA7 level. In the other double mutants (all position 1, 3 and 4 mutants, and DU 6 C), in clear contradiction to the predictions of the`base pairing model', RNA7 synthesis was not signi®cantly restored. Moreover, a comparison of the values for the B and D mutants in Figure 3 showed that, for almost all of these mutants (e.g. the position 1 mutants), the amount of sg RNA7 produced by the double mutant appeared to be limited by the level allowed by the body TRS mutation. Sometimes the RNA7 level of the double mutant was even less than that of the leader mutant (DU 1 C, DA 3 G, DA 4 G or DU 6 C). Clearly, for these substitutions, restoration of the possibilities for leader±body duplex formation did not restore sg RNA synthesis. Apparently this is because the effect of body TRS mutations at the level of primary sequence or secondary structure can be`dominant' over the duplex-restoring effects of the double mutations.
Body TRS mutants thus fell into two distinct types, determined by the position and chemistry of the substitution. In mutants of the ®rst type, sg RNA synthesis was impaired mainly because of the disruption of the leader± body TRS duplex. This effect could be compensated for by introduction of the corresponding mutation in the leader TRS and, in the double mutant, sg RNA synthesis was restored compared with the corresponding single mutants. In mutants of the second type, sg RNA synthesis was down-regulated as a consequence of both TRS duplex disruption and disruption of the primary sequence (or secondary structure) of the body TRS. Obviously, the latter effect could not be compensated for by mutating the leader TRS, and the corresponding double mutants did not show restoration of sg RNA synthesis.
In contrast to our ®ndings with the body TRS mutants, we did not obtain leader TRS mutations that appeared to determine the level of sg RNA7 synthesis of the corresponding double mutant (Figure 3) . Thus, effects of mutations in the leader TRS were not`dominant' over the duplex-restoring effects of the double mutations, suggesting that they only affected duplex formation. This indicated that the leader TRS probably does not have an additional, sequence-speci®c function in sg RNA synthesis in addition to its participation in TRS±TRS base pairing. The fact that single leader TRS mutations at all six Nidovirus discontinuous subgenomic RNA synthesis positions severely repressed RNA7 synthesis indicated that base pairing of every TRS nucleotide contributes to sg RNA production. In this respect, it was signi®cant that the two leader TRS mutants with the highest RNA7 levels, LA 3 G and LA 4 G, can form G±U base pairs to maintain the duplex.
The observation that leader TRS mutations could bè rescued' by introducing complementary mutations in the body TRS, but that many body TRS mutations could not bè rescued' by corresponding changes in the leader TRS, is clearly illustrated by the U 1 A mutants. Due to the restoration of TRS base pairing possibilities, the RNA7 synthesis of double mutant DU 1 A was signi®cantly increased compared with that of LU 1 A, but not above the level of BU 1 A. Thus, restoration of the leader±body duplex in DU 1 A exerted a clear effect on sg RNA7 production compared with LU 1 A, but had no effect on sg RNA synthesis compared with BU 1 A. This exempli®ed the dominant nature of a mutation in the primary sequence of a body TRS. In contrast, for instance, the BC 2 U mutation probably affected duplex formation only, because RNA7 synthesis was restored almost to wildtype levels in the DC 2 U double mutant.
These results indicate that there are strong base preference constraints for some body TRS positions. To interpret these base preferences accurately, it is necessary to limit the analysis to the double mutants only, because in these mutants the down-regulation of sg RNA synthesis was only due to the sequence changes in the body TRS, and not to the disruption of the leader±body TRS duplex. There were strict preferences for positions 1, 3 and 4 of the body TRS: at position 1, only the U to A substitution allowed for a signi®cant RNA7 level (~40% of wild-type); and at positions 3 and 4, only the A to U mutants retained 15±20% of the wild-type level. For positions 2 and 5, the sequence constraints were less stringent (all substitutions allowed for >20% of wild-type level), but still only DC 2 A and DC 2 U reached >50%. At position 6 of the body TRS, only U to C was not allowed, whereas the other two double mutants still produced 50% or more of RNA7. In other words, the functional EAV RNA7 body TRS (based on the analysis of our single nucleotide substitutions) can be described as U 1 (C/u/a) 2 A 3 A 4 C 5 (U/a/g) 6 , with wild-type nucleotides shown in upper case and nucleotides that allowed for at least 50% of the wild-type RNA7 level shown in lower case. Remarkably, TRS nucleotides A 3 , A 4 and C 5 are conserved in the TRSs of all other arteriviruses (Snijder and Meulenberg, 1998) . Also the fact that DC 2 U retained 80% of RNA7 synthesis corresponded nicely to the presence of a U at this position in other arteriviruses.
Until recently (Almazan et al., 2000; Thiel et al., 2001) , infectious cDNA clones were lacking for coronaviruses. Consequently, most studies on coronavirus sg RNA synthesis were carried out using defective interfering (DI) RNAs. These replicons carried body TRSs from which moderate levels of sg mRNAs could be produced in the presence of helper virus. Using this system, Joo and Makino (1992) and van der Most et al. (1994) performed body TRS mutagenesis studies for the murine coronavirus (MHV). Joo and Makino systematically mutagenized the core of the MHV body TRS. In contrast to our results, they found that in only two of 21 body TRS mutants was sg RNA synthesis from the DI RNA genome abolished, whereas all others supported normal levels of sg RNA production. Thus, it is possible that the MHV TRS which was used in that study is more tolerant to single-nucleotide mismatches than the EAV sg RNA7 TRS.
In a similar study, van der Most et al. (1994) observed that U to C substitutions at positions 1 and 3 of the MHV body TRS, which maintained the duplex by changing a U±A base pair into a U±G base pair, reduced sg RNA levels more strongly than substitutions that disrupted the duplex (van der Most et al., 1994) . This implies that, as in the case of EAV, leader±body TRS duplex formation is not the only factor that determines coronavirus sg RNA synthesis. However, because of the limitations of the DI RNA system, the leader TRS could not be mutagenized in these studies, and body TRS-speci®c effects could not be distinguished from effects at the level of leader±body duplex formation.
The discontinuous step in nidovirus sg RNA synthesis occurs during minus strand RNA synthesis Due to recent studies of arterivirus and coronavirus sg RNA synthesis (van Marle et al., 1999a; Baric and Yount, 2000; Sawicki et al., 2001) , the discontinuous minus strand extension model ( Figure 1C ) has been gaining more and more ground. This model predicts that the TRSderived sequence that forms the leader±body junction in the sg mRNA is a copy of the body TRS, and not of the leader TRS. The leader-primed transcription model predicts the opposite ( Figure 1B) . Therefore, determining the origin of the leader±body junction of sg mRNAs would help to distinguish between the two models. However, in the wild-type situation, EAV leader and body TRSs are identical and consequently one cannot determine the origin of the sg mRNA leader±body junction. This problem could be overcome by tracing the mutations introduced in leader or RNA7 body TRS mutants, most of which retained part of their ability to produce mRNA7. In a previous study (van Marle et al., 1999a) , we found that nucleotides 2 and 5 of the mRNA7 leader±body junction sequence were derived exclusively from the body TRS, and not from the leader TRS. This was shown by direct sequencing of RT±PCR products obtained from the residual mRNA7 produced by mutants BC 2 G, LC 2 G, BC 5 G and LC 5 G ( van Marle et al., 1999a) .
Using the same approach, we analysed mRNA7 from mutants BC 2 A and BC 2 U, and these transcripts also contained the mutated nucleotide derived from the body TRS (data not shown). Assuming that only one crossover event occurs during leader±body joining, we could thus map this crossover between positions ±1 and +2 of the sg RNA junction sequence. This left the intriguing question of whether the crossover site could be mapped even more precisely. In other words, was nucleotide +1 of the junction sequence derived from the body TRS or the leader TRS?
Using the position 1 mutants described above, we could answer this question ( Figure 5) . The most striking result was that mRNA7 of mutants BU 1 A, BU 1 G and LU 1 C contained exclusively the body TRS-derived nucleotide at position +1. Thus, for these mutants, the crossover site could be mapped precisely between TRS nucleotide positions ±1 and +1, meaning that the complete leader± body junction sequence in an EAV sg mRNA can be body TRS derived. On the other hand, sg RNAs from mutants LU 1 A, BU 1 C and LU 1 G contained mixed populations of leader TRS-and body TRS-derived nucleotides at position +1 ( Figure 5 ): A and U for LU 1 A, C and U for BU 1 C, and G and U for LU 1 G. Remarkably, this pattern correlated with the relative amounts of sg mRNA7 produced by these mutants (Figure 3 ). Mutants that produced populations of sg RNAs that were mixed with respect to the origin of the nucleotide at position +1 of the leader±body junction had lost RNA7 synthesis almost completely. On the other hand, mutants that contained exclusively the body nucleotide at position +1 retained higher levels of RNA7 synthesis. This observation may be explained as follows: in the wild-type situation, the large majority of the crossovers probably occur between positions ±1 and +1, leading to a body TRS-derived nucleotide at position +1 in the sg RNA; however, a low number of crossovers take place between nucleotides +1 and +2, resulting in a leader TRS-derived nucleotide at position +1. Mutants in which almost all sg RNA synthesis is blocked by a substitution at position +1 may somehow be de®cient in the crossover between ±1 and +1, but may have retained the ability for crossovers between +1 and +2, which were detected by sequence analysis. Conversely, in position +1 mutants that retain reasonable sg RNA synthesis, most crossovers occur between positions ±1 and +1, and they obscure the minority of crossovers between +1 and +2 in the sequencing electropherogram. Alternatively, position +1 TRS mutations that strongly interfere with sg RNA synthesis may force a shift of the crossover site in the remaining molecules.
We believe that our present ®ndings strongly support the discontinuous minus strand extension model. Indeed, the fact that a complete body TRS can be copied into the sg RNA is very dif®cult to reconcile with the alternative model, in which sg RNA synthesis from the genomic minus strand template is primed by free plus strand leader transcripts that contain the leader TRS at their 3¢ end ( Figure 1B) . To explain the presence of a complete copy of the body TRS in the sg mRNA in this model, one would have to assume that a 3¢±5¢ exonuclease activity trims back the free leader transcript prior to its extension into an sg mRNA (Baker and Lai, 1990) . Note that there would not be a single base pair left to hold these`trimmed' leader molecules on the template. Such an enzymatic activity, which is unprecedented in +RNA viruses, exists in yeast retrotransposon Ty5 (Ke et al., 1999) , in which reverse transcription is primed by an internal region in a tRNA. However, in this system, it is not a part of the duplex that is removed, but the single-stranded 3¢ tail of the tRNA, which cannot base-pair with the Ty5 RNA.
Removal of the TRS at the 3¢ end of the nidovirus leader, which has already base paired with the template, would be very energetically unfavourable for the RdRp. Instead of starting elongation using the intact and properly positioned leader as a primer, it would have to disrupt the newly formed duplex, degrade part of the leader RNA and then reinitiate polymerization, without any base pairing between primer and template. It has been shown that in¯uenza virus transcription does not require a sequence match between the (cellular) RNA primer and the (viral) template (Plotch et al., 1981) . However, if in the nidovirus system the`trimmed' leader RNA could also be ®xed on the template solely by RNA±protein interactions, the targeting of the nascent strand by TRS base pairing would be extremely puzzling.
Sequence data of sg RNA leader±body junctions from other arteriviruses are also dif®cult to reconcile with the leader-primed transcription model. For the porcine and simian arteriviruses (Meulenberg et al., 1993; Godeny et al., 1998) , the leader±body junctions of some sg RNAs mapped two nucleotides upstream of the body TRS, which again would not leave a single nucleotide to hold the putative free leader on the template after the hypothetical back trimming'. On the other hand, these ®ndings and our data can be explained readily by the discontinuous minus strand extension model ( Figure 1C ). The six-nucleotide Fig. 5 . Sequence analysis of mRNA7 leader±body junctions from position 1 TRS mutants. Sequences were determined directly from sg mRNA7-speci®c RT±PCR products. For the U 1 A and U 1 C mutants, the sequence shown corresponds to the plus strand of sg RNA7. For sequencing-related technical reasons, the minus strand sequence was determined for the U 1 G mutants; a mirror image of the electropherogram is shown with the corresponding plus strand sequence listed at the top of the panel. For every mutant, a sequence alignment of the leader (red) and body (blue) TRSs and surrounding sequences is shown (TRSs are boxed). The mRNA7 leader±body junctions detected by our sequence analysis are shown in yellow. duplex formed between the body TRS complement at the 3¢ end of the leaderless sg minus strand and the leader TRS in the genomic RNA template should suf®ce to position the nascent minus strand properly for subsequent elongation to add the complement of the leader sequence. In most cases, the nascent minus strand contains the entire body TRS complement at its 3¢ end at the moment of strand transfer, leading to a body TRS-derived leader±body junction sequence in the sg mRNA molecule. In a small number of transcripts, however, minus strand synthesis appears to be interrupted before nucleotide +1 of the body TRS is copied and, after strand transfer, resumes by incorporating the complement of the +1 nucleotide of the leader TRS. As stated above, we postulate that the detection of this phenomenon is determined by the level of crossovers between the ±1 and +1 position that is allowed by the mutations introduced at the +1 position of body TRS or leader TRS. We cannot, however, formally exclude that a`back trimming' activity degrades the 3¢-terminal nucleotide of the minus strand before or after strand transfer. However, note that in the discontinuous minus strand extension model ( Figure 1C ), such an activity would not disturb the proper positioning of the nascent minus strand on the leader template, because the TRS± TRS duplex would be shortened by one nucleotide only.
Nidovirus discontinuous minus strand extension resembles similarity-assisted, copy-choice RNA recombination Due to their discontinuous sg RNA synthesis, nidoviruses occupy a special`niche' in the +RNA virus world. Their mode of sg RNA production is clearly different from that of other +RNA viruses and resembles another welldocumented +RNA virus feature: RNA recombination (for recent reviews see Nagy and Simon, 1997; Aaziz and Tepfer, 1999; Worobey and Holmes, 1999) . Most of the experimental evidence supports an RdRp template switch (Kirkegaard and Baltimore, 1986) as the main mechanism of RNA recombination. Mechanistically, such a template switch involves the transfer of a nascent strand from one RNA template (donor) to the other (acceptor). Also, nidovirus discontinuous sg RNA synthesis involves transfer of a nascent RNA strand, the sg RNA, but now from one site to another in the same template.
Based on the data currently available, we refer to the discontinuous minus strand extension model as our working model for nidovirus sg RNA synthesis. If one applies the`recombination terms' to this model (Chang et al., 1996; Brian and Spaan, 1997; van Marle et al., 1999a) , the donor strand would be the body part of the genomic RNA template, the acceptor strand would be the leader part of the genomic RNA template and the nascent strand would be the discontinuously synthesized minus strand. Nagy and Simon (1997) have de®ned three main classes of RNA recombination: similarity-essential, similarity-non-essential and similarity-assisted recombination. The latter is de®ned as a mechanism in which strand transfer is determined by both sequence similarity between the parental RNAs and additional RNA determinants, present in only one of the parental RNAs.
The results of our present study strongly suggest that nidovirus discontinuous sg RNA synthesis can be considered a special case of high-frequency similarity-assisted RNA recombination. While the only obvious function of the leader TRS is to ensure the ®delity of the strand transfer by base pairing with the 3¢ end of the nascent strand, the body TRS in the donor template indeed has additional, sequence-speci®c functions. One of these functions apparently is to pause (or terminate) nascent strand synthesis and thereby provide the opportunity for strand transfer. In addition, body TRS-derived nucleotides may play a role in the reinitiation of nascent strand synthesis on the acceptor template. Given the compact nature of the EAV TRS, it is quite possible that some nucleotides ful®l multiple tasks.
RNA secondary structure of the body TRS may regulate sg RNA synthesis The sequence-speci®c function of the body TRS, revealed in this study, may be exerted at the level of either primary sequence or secondary structure. For a number of +RNA viruses, RNA secondary structure motifs located in the (proximity of) sg RNA promoters are vital for sg RNA synthesis. In alfalfa mosaic virus (Haasnoot et al., 2000) , turnip crinkle virus (TCV) (Wang et al., 1999) and barley yellow dwarf virus (Koev et al., 1999) , stem±loop structures in sg RNA promoter regions of the template strand are required for sg RNA synthesis. The sg RNA1 promoter of the latter virus is especially interesting, since it contains two stem±loop domains. For one of them, secondary structure, but not the primary sequence, is important for sg RNA synthesis, whereas the other domain acts through primary sequence, and not secondary structure (Koev et al., 1999) . Similarly, RNA secondary structure may play only a minor role in the sequence-speci®c recognition of the BMV sg RNA promoter by the RdRp Siegel et al., 1997) .
We have suggested previously that RNA secondary structure of body TRS regions contributes to their attenuating potential and thereby determines the relative portion of the nascent minus strands that is transferred to the leader TRS in the template (Pasternak et al., 2000) . At present, it is unknown whether EAV body TRSs are part of an RNA structural motif that is essential for body TRS function, or whether they are recognized by a protein factor in a sequence-speci®c manner. However, the latter seems less likely than the former, since even LB4 (Figure 2 ), in which ®ve TRS nucleotides were substituted, still produced some sg RNA7, although~30-fold less than the wild-type control. The fact that some sequences in the EAV genome match the leader TRS perfectly, but are not used for sg mRNA synthesis, also argues against the recognition of a speci®c sequence (Pasternak et al., 2000) . More probably, mutagenesis of the RNA7 body TRS disturbed an RNA structure that is necessary for its function. This could, for example, explain the fact that the BU 6 C substitution reduced the amount of RNA7 by 20-fold (and could not be rescued by the same mutation in the leader TRS), whereas the wild-type RNA6 body TRS contains a C at the same position. If a protein factor were involved in sequence-speci®c TRS recognition, then one would expect it to recognize all TRSs similarly. If RNA structure is important for recognition by such a protein, then the BU 6 C substitution probably disturbs a structural motif of the RNA7 TRS, which is not present in the RNA6 TRS. On the other hand, conservation of part of the TRS in other arteriviruses suggests a sequence-speci®c recognition. Further studies are required to distinguish between these possibilities.
In the TCV satellite RNA recombination system, the hairpin structure in the acceptor strand, as well as the donor±acceptor homology region, are necessary for the template switch . The hairpin has been postulated to bind the RdRp, whereas the homology region targets the nascent strand to the crossover site. The TCV RdRp probably recognizes the secondary and/or tertiary structure of the hairpin, while individual nucleotides play a less important role . In EAV, the leader TRS in the acceptor template is predicted to reside in the loop of an extensive hairpin, and its base pairing interaction with the body TRS complement at the 3¢ end of the nascent minus strand would resemble certain antisense RNA-regulated control mechanisms that are based on interactions between single-stranded tails and hairpin loops (van Marle et al., 1999a, and references therein) . It is possible that the EAV RdRp, or its accessory proteins, also binds to the stem of the long hairpin that presents the leader TRS. In any case, the leader TRS itself does not seem to be recognized by a protein in a sequence-speci®c manner.
The body TRS is a better candidate to serve as a protein recognition site. This protein would then mediate the pausing of the nascent strand synthesis and/or nascent strand transfer. This would resemble the DNA-dependent RNA polymerase I termination system, in which speci®c DNA-binding terminator proteins bind to termination sequences (Reeder and Lang, 1997) , or a function of the HIV nucleocapsid protein, which promotes the minus strand strong-stop DNA transfer (Guo et al., 1997) . The EAV replicase component nsp1, which recently was shown to possess an sg RNA synthesis-speci®c activity (Tijms et al., 2001) , may be a good candidate for such a regulatory role. Residues predicted to form a zinc ®nger structure in nsp1 were shown to be necessary for sg RNA synthesis. Interestingly, zinc ®nger structures in the HIV nucleocapsid protein facilitate strand transfer (Guo et al., 2000) . Finally, it should be noted that the RNA structure of the nascent strand may also in¯uence pausing, strand transfer or reinitiation, as illustrated by the fact that stable hairpin structures in the nascent strand promote termination of transcription by Escherichia coli RNA polymerase (Wilson and von Hippel, 1995) .
Site-directed mutagenesis, RNA transfections and immuno¯uorescence analysis Site-directed mutagenesis of EAV leader and body TRSs was carried out as described by van Marle et al. (1999a) , and all mutant constructs were sequenced. Following in vitro transcription from infectious cDNA clones, full-length EAV RNA was introduced into BHK-21 cells by electroporation, as described by van Dinten et al. (1997) . Immuno¯uorescence assays with EAV-speci®c antisera were performed at 14 h posttransfection as described by van der Meer et al. (1998) . To visualize the nuclei for cell counting, nuclear DNA was stained with 5 mg/ml Hoechst B2883 (Sigma). Cells were counted using the Scion Image software (Scion Corporation) and the percentage of transfected cells was calculated on the basis of the number of cells positive for the EAV replicase component nsp3 (Pedersen et al., 1999) .
For RNA analyses, cells were lysed at 14 h post-transfection. Intracellular RNA isolation was performed using the acidic phenol method as described by Pasternak et al. (2000) . Total intracellular RNA was resolved in denaturing agarose±formaldehyde gels. Hybridization of dried gels with the radioactively labelled oligonucleotide probe E154, which is complementary to the 3¢ end of the EAV genome and recognizes all viral mRNA molecules (genomic and subgenomic), and phosphoimager quantitation of individual bands were performed as described by Pasternak et al. (2000) . To determine the leader±body junction sequence of sg mRNA7, mRNA7-speci®c RT±PCRs were carried out as described by van Marle et al. (1999b) using an antisense (RT and PCR) primer from the RNA7 body region and a sense PCR primer matching a part of the leader sequence. RT±PCR products were sequenced directly as described by Pasternak et al. (2000) using the leader-derived primer, an ABI PRISMÔ sequencing kit (Perkin Elmer) and an ABI PRISMÔ 310 Genetic Analyser (Perkin Elmer). Debate: Transfusing to normal haemoglobin levels will not improve outcome Recent evidence suggests that critically ill patients are able to tolerate lower levels of haemoglobin than was previously believed. It is our goal to show that transfusing to a level of 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. Although many questions remain, many laboratory and clinical studies, including a recent randomized controlled trial (RCT), have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. In addition, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of red blood cells (RBCs), save blood overall, and decrease health care costs. Anaemia is a common condition in critically ill patients, and RBC transfusions are often used in the treatment and management of this patient population. In fact, one study [1] reported that 25% of all critically ill patients received RBC transfusions. Many laboratory studies [2] [3] [4] [5] [6] [7] [8] have examined the physiological responses (ie compensatory mechanisms) of the body to anaemia, which include the following [9] : increased cardiac output, decreased blood viscosity, capillary changes, increased oxygen extraction, and other tissue adaptations to meet oxygen requirements. Although critically ill patients are affected by a number of factors that predispose them to the adverse consequences of anaemia, persistence of this condition is of particular concern because it may cause the compensatory mechanisms in these patients to become impaired, risking oxygen deprivation in vital organs [9] . However, critically ill patients may also be at increased risk from the adverse effects of RBC transfusions, such as pulmonary oedema from volume overload, immune suppression resulting in increased risk of infection, and microcirculatory injury from poorly deformable RBCs.
It is the aim of the present commentary to justify the statement 'Transfusing to normal haemoglobin concentration will not improve outcome.' If we define normal haemoglobin as being greater than 115 g/l for women and greater than 125 g/l for men, then there is no evidence in the literature to justify maintaining such high concentrations by the use of RBC transfusions in any anaemic patient. There may, however, be some debate about adopting a transfusion threshold of 100 g/l, which is well below 'normal'. transfusion threshold would, obviously, reduce the number of allogeneic RBCs transfused. It is our goal to impress upon the reader that transfusing to a level equal to or greater than 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. We first explore the reasons why a reduction in the total number of allogeneic blood transfusions would be beneficial. Second, we examine the current evidence for using a lower transfusion strategy, specifically that employed in the Transfusion Requirements In Critical Care (TRICC) trial.
RBC transfusions have inherent risks that may be categorized as follows [11] [12] [13] [14] [15] : transfusion-transmitted infections; immune-related reactions (acute or delayed haemolytic reactions, febrile, allergic, anaphylactic reactions and graft-versus-host disease); and nonimmunerelated reactions (fluid overload, hypothermia, electrolyte toxicity and iron overload).
Major improvements in donor screening procedures and laboratory testing have dramatically improved the safety of the blood supply [16] . Currently, the risk of transmitting an infectious agent through blood transfusion ranges from 1:100,000 for hepatitis B virus to 1:1,000,000 for HIV (Canadian Blood Services, personal communication, 2000). The most important threats to the blood supply remain new and unknown pathogens. More recently, concern has focused on the potential transmission of prions through RBCs. Also, infectious agents with long latency periods pose particular risks to young individuals who require RBCs, such as multiple trauma victims. The risk : benefit ratio for a 24-year-old trauma victim with a 50-year life expectancy differs markedly from that for a person aged 80 years who is undergoing coronary artery bypass surgery. In summary, because there is a risk of transmitting diseases through the blood supply, we should always strive to use RBCs according to the best available evidence in order to ensure that we do more good than harm to our patients.
It is a long-standing observation [17] [18] [19] [20] [21] that blood transfusions are associated with immune suppression. This clinical phenomenon was first observed in renal transplant patients who had received blood transfusions while on dialysis before the transplant [22] , and has been observed repeatedly in transplant centres around the world [23, 24] . Recently, Opelz et al [25] reported a multicentre clinical trial in which all renal allograft recipients received modern immunosuppressive regimens. Those patients who were allocated to receive three allogeneic RBC units before renal transplant had a 1-year graft survival rate of 90%, as compared with 82% for patients who were not transfused (P = 0.02). These data suggest that there are long-term immunosuppressive effects following transfusion of nonleukocyte-reduced allogeneic RBCs.
A large number of studies [26] [27] [28] [29] [30] [31] [32] [33] [34] have also suggested that allogeneic transfusions accelerate cancer growth, perhaps due to altered immune surveillance. These altered immune responses after allogeneic RBC transfusions may also predispose critically ill transfusion recipients to nosocomial infections [35] [36] [37] [38] [39] [40] and increased rates of multiplesystem organ failure [41] , which may ultimately result in higher mortality rates. However, most studies that examined the association between cancer recurrence and postoperative infection after transfusion [42, 43] only provided weak causal inferences because of poor study design and the lack of independence between allogeneic RBC transfusions and the potential complication.
A recent meta-analysis [44] combined the results from seven RCTs, and was unable to detect clinically important decreases in mortality and postoperative infections. We added the results of a new RCT by van de Watering et al [45] to the above meta-analysis. The relative risk for allcause mortality was 1.05 (95% confidence interval 0.88-1.25), and was 1.10 (95% confidence interval 0.85-1.43) for postoperative infections. However, this meta-analysis excluded two positive RCTs [40, 46] because of the significant statistical heterogeneity introduced by these two studies. If all available RCTs are combined, ignoring heterogeneity, then the relative risk difference for postoperative infections across all studies is 1.60 (95% confidence interval 1.00-2.56; P = 0.05). Thus, the available evidence suggests that universal prestorage leukoreduction could have clinical effects that range from none to decreasing rates of infection by as much as 50% in high-risk patients. In summary, despite convincing laboratory evidence and some supportive clinical studies, the clinical significance of the immunosuppressive effects of allogeneic RBC transfusions have not been clearly established [47] . More importantly, the impact of a leukoreduction programme has not been studied in a large population of patients who are expected to have significant exposure to allogeneic RBCs.
The majority of complications from allogeneic RBC transfusion, however, are no more frequent in the intensive care setting than in other patient populations, with the possible exception of pulmonary oedema, hypothermia and electrolyte disturbance. Hypothermia and electrolyte disturbances occur most frequently with massive transfusions. In critically ill patients, the optimal effective circulatory volume may be difficult to determine, and as a consequence pulmonary oedema may be a much more frequent complication of RBC transfusion than in other patient populations. This may explain the significantly higher rate of pulmonary oedema in patients transfused using a threshold of 100 g/l (5.3% in the restrictive transfusion group versus 10.7% in the liberal transfusion group; P < 0.01), as reported in the TRICC trial [10] . As an alternative explanation, the more frequent use of RBCs might have resulted in more frequent episodes of transfusion-related acute lung injury in the liberal strategy group (7.7% in the restrictive strategy versus 11.4% in the liberal strategy; P = 0.06), as reported in the TRICC trial.
Clinical evidence is also insufficient to definitively establish a correlation between the age of RBCs being transfused and patient mortality; however, laboratory evidence has shown many storage-related changes that may result in impairment of blood flow and oxygen delivery at the microcirculatory level. Marik et al [48] demonstrated an association between a fall in gastric intramucosal pH and transfusion of RBCs stored for longer than 15 days. In addition, there is ample laboratory evidence that prolonged RBC storage adversely affects RBCs, potentially results in the generation of cytokines, and alters host immune function. In another study, Fitzgerald et al [49] , using an animal model of transfusion, consistently observed a lack of efficacy of transfused, stored rat blood to improve tissue oxygen consumption as compared with fresh cells or other blood substitutes.
Three retrospective clinical studies tested the association between the age of transfused blood and duration of stay in the intensive care unit (ICU) [50] and mortality [51, 52] . Martin et al [50] observed a statistically significant association between the transfusion of aged blood (>14 days old) and increased duration of ICU stay (P = 0.003) in 698 critically ill patients. In patients who received a transfusion, aged RBCs was the only predictor of duration of stay (P < 0.0001). In survivors, only median age of blood was predictive of duration of stay (P < 0.0001). Purdy et al [51] demonstrated a negative correlation (r = -0.73) between the proportion of RBC units of a given age transfused to survivors and increasing age of RBCs in patients admitted to the ICU with a diagnosis of severe sepsis (n = 31). Those investigators also noted that these latter units were more likely to be older. A recently reported study by Vamvakas and Carven [52] evaluated the effect of duration of RBC storage on postoperative pneumonia in 416 consecutive patients undergoing coronary artery bypass grafting. Those investigators noted an adjusted increase of 1% in the risk of postoperative pneumonia per day of average increase in the duration of RBC storage (P < 0.005) in transfused patients. Each of these three studies also noted that patients who received a large number of RBC units had a higher mortality. Although these risks are relatively small when viewed collectively, they become significant when one considers that 25% of all critically ill patients in Canada are transfused during their ICU stay [1] .
Until recently, physicians have depended on clinical judgement when deciding at what haemoglobin level to transfuse a critically ill patient. As a result, significant variation has been shown to exist in transfusion practice among Canadian critical care physicians [53] , which is due largely to a lack of published data on the subject. An arbitrary haemoglobin level of 100 g/l has historically been used as a threshold to transfuse critically ill patients.
Six observational studies investigated the importance of anaemia on transfusion practices in various settings. Of these, three large cohort studies, which were performed in different patient populations (intensive care [1] , coronary artery bypass surgery [54] and hip fractures [55]), reached different conclusions. RBC transfusions in particular improved outcome in critically ill patients with cardiovascular disease, but increased the risk of myocardial infarction in coronary artery bypass surgery patients. Transfusion had no impact on short-term or long-term mortality in hip-fracture patients. Three smaller studies [56] [57] [58] evaluated the relationship between anaemia and adverse outcomes in vascular disease patients. Although increased numbers of ischaemic events were observed in anaemic patients, the validity of these studies is uncertain, given that the decision to transfuse a patient was often correlated with illness burden of the patient. It is also possible that comorbidity was not adequately adjusted for in those studies.
Transfusion thresholds were compared in 10 randomized clinical trials [10, [59] [60] [61] [62] [63] [64] [65] [66] [67] . Although the clinical settings varied, each trial randomized patients to be transfused on the basis of a 'conservative' or a 'liberal' strategy. The definitions of conservative and liberal strategies varied, and actually overlapped between studies. Of these 10 trials, only three included more than 100 patients and only one trial evaluated the impact of transfusion on symptoms. In the first trial of patients undergoing elective coronary artery bypass surgery [65] , the differences between perioperative haemoglobin levels were small, event rates were very low, and there were no differences in any outcome. In the second trial [67] , patients undergoing knee arthroplasty were randomly assigned to receive autologous blood transfusion immediately after surgery or to receive autologous blood if haemoglobin level fell below 9 g/dl [67] . Again, no differences in outcome were observed. The third trial of hip fracture patients undergoing surgical repair [64] found no differences in outcomes; however, five deaths were recorded at 60 days after surgery in the symptomatic group, and two deaths occurred in the 10 g/dl group. The numbers of patients in these trials were too small to evaluate the effect of lower transfusion triggers on clinically important outcomes such as mortality, morbidity and functional status.
In 1999, Hebert et al [10] reported the results of the TRICC trial. Patients (n = 838) were randomized either to a restrictive strategy (haemoglobin concentration maintained between 70 and 90 g/l, with a trigger set at 70 g/l) or to a liberal strategy (haemoglobin concentration maintained between 100 and 120 g/l, with a trigger at 100 g/l). To date, the TRICC trial is the only large study that has investigated these parameters. The groups were comparable at baseline. The average daily haemoglobin concentration ranged from 85 ± 7.2 g/l in the restrictive group to 107 ± 7.3 g/l in the liberal group (P < 0.01). The average number of transfusions was reduced by 52% in the restrictive group (2.6 ± 4.1 versus 5.6 ± 5.3 RBCs/patient; P < 0.01). Cardiac events, primarily pulmonary oedema and myocardial infarction, were more frequent in the liberal strategy (P < 0.01; Table 1 ). On examination of composite outcomes, the number of patients with multiorgan failure was found to be substantially increased in both groups, with the results being marginally better in the restrictive strategy group (20.6% versus 26.0%; P = 0.07; Table 2 ). Overall, the restrictive transfusion group showed a lower 30-day mortality (18.7% versus 23.3%; P = 0.11; Fig. 1 ). Kaplan-Meier survival curves, however, were significantly different in the subgroup of patients with an Acute Physiology and Chronic Health Evaluation II score of 20 or less (P = 0.02; Fig. 2 ). In addition, although 60-day mortality (22.8% versus 26.5%; P = 0.23) and ICU mortality (13.9% versus 16.2%; P = 0.29) were not statistically significant, they did show a consistent trend in terms of absolute values that favoured the restrictive strategy. The key observation from the TRICC trial is not that the restrictive strategy is better, but rather that it is at worst equivalent to the liberal strategy and at best superior to the liberal strategy.
At this juncture, preclinical and clinical evidence support the adoption of a more restrictive transfusion strategy in most critically ill patients. However, there remain divergent views regarding the risks and benefits of treating anaemia in patients with cardiovascular disease. Laboratory-based studies [68, 69] suggest that patients with cardiovascular disease may require higher haemoglobin concentrations to maintain oxygen delivery in partially occluded or diseased coronary arteries. Studies to demonstrate how anaemia affects contractile function of the left ventricle have rarely shown important effects above haemoglobin concentrations of 70 g/l. Indeed, it is more important to address the underlying pathophysiological causes of the acute coronary syndrome with proven therapy such as aspirin and β-blockers, rather than treating mild-to-moderate anaemia as an initial step. If the effects of RBC transfusion were either limited or increased then there would be no debate; however, the use of allogeneic RBCs has been shown to be associated with immunomodulation [12, 47] and/or alteration in the delivery of oxygen in the microcirculation [70, 71] , resulting in increased rates of infections and organ failure.
Few clinical studies have attempted to elucidate the risk : benefit ratio of anaemia and transfusion in cardiac patients. Two small RCTs [62, 72] examined transfusion practice in patients undergoing coronary artery bypass grafting, and concluded that a conservative approach to the administration of RBCs may be safe. However, two recent cohort studies suggested that anaemia may increase the risk of mortality in critical illness [73] and following surgery in patients with cardiovascular disease [74] . There were 418 and 420 patients in the restrictive and liberal transfusion groups, respectively. *Difference calculated by subtracting mean values of restrictive group from those of liberal group. † Three patients were lost to 60-day mortality rate; therefore n = 835. ‡ Nonsurvivors are considered to have all organs failing on date of death. § Changes in MOD score from baseline, while also incorporating adjustment for death. Data from Hébert et al [10] .
In a study of Jehovah's Witnesses (a group that refuses RBC transfusion on religious grounds) undergoing surgical procedures [74] , it was noted that mortality was significantly increased in patients with cardiac disease after a decrease in haemoglobin levels from 100-110 g/l to 60-69 g/l. In that study, patients with no cardiac disease and similar changes in haemoglobin levels showed no increase in mortality, which is in accordance with the results of the TRICC trial [10] . In the study by Hébert et al. [73] of 4470 critically ill patients, a correlation between Critical Care Vol 5 No 2 Alvarez et al [10] .
Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (all patients). Data from Hébert et al [10] .
Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (patients with APACHE II score ≤20). Data from Hébert et al [10] .
anaemia and mortality rates was observed. Those investigators also found that the risk of anaemia appeared to decrease with RBC transfusion in patients with cardiac disease. In patients with cardiac disease, mortality increased when haemoglobin concentrations were below 95 g/l, as compared with anaemic patients with other diagnoses (55% versus 42%; P = 0.09). In the subgroup of patients with cardiac disease, increasing haemoglobin values in anaemic patients was associated with improved survival (odds ratio 0.80 for each 10 g/l increase; P = 0.012). One possible explanation for the discrepancy between the TRICC trial and this observational study may be that the attending physicians who recruited patients into the study did not enter those patients who were considered to have severe cardiac disease.
Hébert et al. [73] sought to examine further whether a restrictive transfusion strategy was at least as effective as a liberal strategy in critically ill patients with cardiac disease. In the subgroup of patients with cardiovascular disease from the TRICC trial, those investigators suggested that most haemodynamically stable critically ill patients with cardiovascular disease may be transfused when haemoglobin concentrations fall below 70 g/l, and that the hemoglobin concentration should be maintained between 70 and 90 g/l. Average daily haemoglobin concentrations were 85 ± 6.2 g/l in the restrictive transfusion group and 103 ± 6.7 g/l in the liberal transfusion group (P < 0.01). In the 357 patients with cardiovascular disease, the 30-day mortality rate was 23% in the restrictive transfusion group versus 23% in the liberal group (95% confidence interval of the difference -8.4% to 9.1%; P = 1.00). Other mortality rates, including 60-day (26% versus 27%; P = 0.90), ICU (19% versus 16%; P = 0.49) and hospital mortality (27% versus 28%; P = 0.81), were not significantly different between groups. Kaplan-Meier survival curves comparing time to death demonstrated similar trends in the two groups ( Fig. 3 ; P = 0.98). The multiple organ dysfunction (MOD) scores, during the entire study period, were also not significantly different between groups (8.6 ± 4.9 versus 9.0 ± 4.4; P = 0.40), but the change in MOD score from baseline values was significantly lower in the restrictive group than in the liberal group (0.2 ± 4.2 versus 1.3 ± 4.4; P = 0.02).
Combined measures of morbidity and mortality, or composite outcomes, were also examined. When all patients who died were given a score of 24, the total MOD score between groups was not different (P = 0.39), or were the changes in MOD scores significantly different from baseline (2.7 ± 6.9 versus 4.0 ± 7.3; P = 0.08). Among the specific subset of cardiac patients with ischaemic heart disease (n = 257), there were no discernible differences in 30-day and 60-day as well as ICU mortality rates. However, a nonsignificant (P = 0.3) decrease in overall survival rate in the restrictive group was noted in those patients with confirmed ischaemic heart disease, severe peripheral vascular disease or severe comorbid cardiac disease (Fig. 4) .
In conclusion, a restrictive RBC transfusion strategy generally appears to be safe in most critically ill patients with cardiovascular disease, with the possible exception of patients experiencing acute myocardial infarction or unstable angina. 
Survival over 30 days in patients with ischemic heart disease in the restrictive and liberal allogeneic RBC transfusion strategy groups. This graph illustrates Kaplan-Meier survival curves for all patients with ischemic heart disease in both study groups. There is no difference in mortality in patients in the restrictive group (dashed line) as compared to the liberal group (solid line) (P = 0.30).
The need to reduce the amount of allogeneic blood transfusions in order to reduce the associated risks has been firmly established. RBCs are associated with clinically important immune suppression, and stored RBCs have been shown to cause adverse microcirculatory effects that result in increased organ failure.
The question for some time has been whether critically ill patients are able to tolerate lower levels of haemoglobin without deleterious effects, thus reducing the amount of exposure to allogeneic transfusions. In the only large RCT, Hébert et al [10] established that there was no difference in mortality rates between restrictive and liberal transfusion strategies in noncardiac, critically ill patients. Although those investigators were able to show convincing trends that the liberal strategy may in fact be deleterious in terms of absolute values, statistical significance was not achieved. However, the fact that no difference between the two strategies was achieved is of great importance, because this means that the total number of transfusions can be reduced by approximately half without any impact on mortality. In addition, these findings are easily put into clinical practice. Although many questions remain, the TRICC trial and many laboratory and clinical studies have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. As such, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of RBCs, save blood overall, and decrease health care costs. The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 The 21st International Symposium on Intensive Care and Emergency Medicine was dominated by the results of recent clinical trials in sepsis and acute respiratory distress syndrome (ARDS). The promise of extracorporeal liver replacement therapy and noninvasive ventilation were other areas of interest. Ethical issues also received attention. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of a high standard. The meeting was marked by a sense of renewed enthusiasm that positive progress is occurring in intensive care medicine. This year's symposium was dominated by the results of recent clinical trials. After 10 years of 'magic bullet' trials in sepsis, a number of successful therapeutic options are now emerging. In addition, recent advances in our understanding of the soup of mediators observed in sepsis offer yet more tantalizing targets for new therapies.
In contrast, the eagerly awaited results from Italy of the prone positioning trial in ARDS were disheartening. The epidemiology of both sepsis and ARDS, and their impact on clinical studies and the future provision of critical care were also hot topics. The era of extracorporeal liver replacement therapy is upon us, with considerable early promise and the probability of wide availability. Finally, as always, ethics remained an area of interest.
This report summarizes and discusses the presentations on the above topics.
Angus (Pittsburgh, PA, USA) presented his group's work on the epidemiology of sepsis in the USA (accepted for publication in Critical Care Medicine). They developed a method for identifying hospitalized patients with sepsis based on ICD9 criteria, the most widely recorded coding system used in US hospitals. Prospective testing of the method found it to be both sensitive and reliable. They then applied it to a representative selection of US hospitals. Their results indicated that about 50% of intensive care unit (ICU) patients have systemic inflammatory response syndrome, and that approximately 20% of these progress to severe sepsis. Mortality for severe sepsis was greater than 30%. Demographically, those at the extremes of age represent the most at-risk groups, in whom the mortality is also the highest. These data provides yet another reminder that the increasing demands on health care resources caused by the ageing population is predicted to exceed intensive care provision within the next The 21st International Symposium on Intensive Care and  Emergency Medicine, Brussels, Belgium, 20-23 March 2001 10-20 years. Finally, those investigators found a striking demographic peak in patients aged 20-30 years, which they attributed largely to human immunodeficiency virus.
The long-standing debate between the two schools of sepsis theory -microcirculatory dys-autoregulation versus cellular dysfunction -shows signs of resolution. New techniques for studying tissue oxygen tension, presented by Ince (Amsterdam, The Netherlands), provide more evidence that microcirculatory dys-autoregulation results in significant shunting. This occurs predominantly in the submucosal and serosal portions of organs, and is an early event. These studies show that the macroscopic restoration of global oxygen delivery fails to improve oxygen consumption as the mucosa becomes hyperoxic, whereas the submucosa and serosa remain hypoxic. Somewhat counterintuitively, this can be reversed in the face of resistant hypotension with vasodilators, at least in animal models.
The cellular dysfunction camp, although still somewhat doubtful as to the importance of these microcirculatory findings, have now clearly established that their championed mechanism of mitochondrial failure is a late but crucial event in the evolution of sepsis. Fink (Pittsburgh, PA, USA) presented evidence that mitochondrial failure in septic cells is triggered by the activation of the enzyme poly-adenosine diphosphate ribose polymerase [1] . This enzyme represents a significant target for novel therapies, which are apparently already in development. The debate regarding the toxicity of oxygen and the formation of free radicals continues despite the absence of demonstrated effectiveness of scavenging therapies, and is a testament to the incomplete understanding of this area.
The round-table conference preceding this year's symposium concentrated on distilling current knowledge on the microscopic events in critically ill patients into an explanation of the macroscopic multiorgan failure that is so commonly encountered. The conclusions of the conference appeared to relate mostly to future directions for research, in particular the study of organ-organ interactions. Marshall (Toronto, Canada) proposed the development of an alternative to the much-maligned physiological scoring systems, based on the staging systems widely used in the field of oncology. He proposed that mediator levels, in addition to physiological variables, will soon be used usefully to characterize septic patients. He also suggested that, in the light of the recent successful mediator trials in sepsis, future therapies will be directed in a manner analogous to the control of glucose in diabetic patients.
The natural anticoagulants antithrombin III (AT III), tissue factor pathway inhibitor (TFPI) and activated protein C (APC), and the cytokine tumour necrosis factor (TNF)-α are the latest inflammatory mediators to be targeted in large multicentre clinical trials in an attempt to improve the current dismal outcome for patients with severe sepsis.
The KyberSept AT III study recruited over 2300 patients from 200 centres, with high Simplified Acute Physiology Scale scores (median 50), and a mortality of nearly 40% [2] . Unfortunately, no overall benefit was shown between AT III and placebo, although results were more encouraging in an analysis of the subgroup of patients who received AT III but no heparin, which is known to inhibit AT III. Interestingly, improvements in quality of life scores were seen in survivors who received AT III in comparison to those who received placebo, suggesting that morbidity may be reduced, although again this was an analysis of a subgroup. Patients in the AT III group who received concomitant heparin had a significantly higher incidence of bleeding events, and outcome worsened as the dose of heparin increased. Explanations for the failure of this study included the inhibitory effects of heparin and the failure to achieve AT III activity levels of greater that 200% from baseline in the treatment population, a level established as required for therapeutic benefit in phase II trials.
Phase II clinical trial results using TFPI (TFPI n = 141, placebo n = 69; unpublished data) show a mortality benefit in the sicker sepsis patients who already have coagulation problems. Results of the phase III multicentre study are expected to be presented at the 22nd International Symposium on Intensive Care and Emergency Medicine, in Brussels in 2002.
Human trials of various anti-TNF-α formulations have been variable to date, and include North American sepsis trial (NORASEPT) I [3] , International sepsis trial (INTERSEPT) [4] and NORASEPT II [5] . Possible reasons have included a lack of biological activity of the anti-TNF-α formulation studied, inappropriate timing of therapy, redundancy of proinflammatory mediators and hetereogeneity of patient populations. The Monoclonal Anti-TNF, A Randomized controlled Sepsis Trial (MONARCS) study used a different anti-TNF-α formulation (F[ab′]2 fragment of a murine monoclonal antibody to human TNF-α), and stratified patients based on demonstrable abnormalities in immunological pathways (highly elevated interleukin-6 levels -a circulating cytokine that is induced by TNF-α). Unpublished results revealed 28-day mortality rates of 44 and 48% in the anti-TNF-α and placebo groups, respectively, in those patients who had high interleukin-6 levels on recruitment to the study (n = 488 anti-TNF-α, n = 510 placebo). This represented a relative mortality reduction of 14%. Relative mortality reduction in all patients (n = 1305 anti-TNF-α, n = 1329 placebo), independent of baseline interleukin-6 levels, was only 10%.
The Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study is hot off the press [6] , and presentation of the results at the congress allowed those of us who still carry the unopened New England Journal of Medicine issue in our briefcases to catch up! A total of 164 sites from 11 countries recruited 1690 patients with severe sepsis, before the trial was prematurely stopped following the second safety analysis. Twenty-eight-day all-cause mortality rates for placebo and APC were 31 and 25% respectively, with a relative risk reduction of 19%. Resolution of cardiovascular and respiratory function was more rapid in survivors who received APC, although ICU and hospital stay did not differ. There was a trend towards an increase in serious bleeding events in the APC group (3% APC versus 2% placebo), but these events were primarily due to trauma or instrumentation. Although this is an exciting breakthrough, we all recognize that when APC reaches the market place it will seriously stretch ICU finances, especially because there appear to be other mediators on the horizon that we will be encouraged to use, in combination, to fight the inflammatory 'soup'.
Two opposing epidemiological views of ARDS were presented by Lemaire (Créteil, France) and Evans (London, UK). Broad agreement does seem to exist as to the incidence of this condition, which is in the order of 10/100,000, although there is significant variation between countries. It was argued that this variation results from the availability of ventilated beds, with higher incidences apparent in countries with greater provision, emphasizing that this condition can be considered the result of intensive care intervention or, as one speaker put it, 'physician-induced lung injury'.
Early results from the Acute Lung Injury Verification of European Epidemiology (ALIVE) study (unpublished data), sponsored by the European Society of Intensive Care Medicine, are at odds with recent trial findings. The ALIVE study, which included over 6000 patients surveyed in 1998, found a 50-60% 28-day mortality, which compares to only 20-30% in the control groups of recent trials. Pneumonia was the commonest cause, responsible for 50% of cases, with sepsis identified as the cause in a further 20-30%. Astonishingly, this study found the ratio of arterial oxygen tension to fractional inspired oxygen at ICU admission was highly predictive of mortality, despite continuing controversy regarding this measurement.
A diverse range of views were presented from the Third International Consensus Conference on ARDS (unpublished data), held in Barcelona late last year. The decision as to how to change the defining criteria for this condition remains unresolved. The debates surrounding chest X-ray criteria, the use of the ratio of arterial oxygen tension to fractional inspired oxygen, and the level/utility of pulmonary artery wedge pressure measurements continue.
In addition, a debate has arisen as to whether ARDS can be a unilateral process, and whether it can coexist with cardiac failure. There appears to be increasing recognition that ARDS represents only a small subset of patients with acute lung failure (approximately 30%). Surprisingly little is known about the remainder of this larger group. In contrast to the ALIVE study, several centres have reported their 28-day mortality at 40%, which represents an improvement from the 60% of 10 years ago. However, it was argued that a 28-day follow-up period is too short for clinical trials, as the long-term quality of life for patients with ARDS is poor compared with that of critically ill patients without this condition. Results suggest that the recovery of lung function is good overall, but is dependent on severity. Treatment recommendations include the universal adoption of the US National Institutes of Health protective lung ventilation strategy [7] . There was general agreement that recruitment manoeuvres are beneficial, but how and when to employ them remains controversial.
Rouby (Paris, France) put forward a new classification for ARDS based on computed tomography findings. He observed that patients can be split into three groups, depending on the appearance of the upper lobes. In group 1 the upper lobes are normal, and positive end-expiratory pressure (PEEP) is of little benefit and results in significant over-distension. Survival in this group is approximately 60%. In group 2 the upper lobes are abnormal, PEEP is of dramatic benefit, but survival is only approximately 25%. In group 3 there are mixed/patchy abnormalities, the effects of PEEP are less predictable, but, as in group 1, survival is approximately 60%.
Gattinoni (Milan, Italy) presented the results of the longawaited Italian multicentre randomized controlled trial of prone positioning in ARDS (unpublished data). This trial was terminated after 3 years despite having only recruited 304 patients; enrollment of 750 patients was originally planned, in order to achieve sufficient statistical power. At trial outset, recruitment was encumbered by the lack of familiarity with and scepticism regarding this procedure in many of the centres. However, by the end of the study many participants were unwilling to enter patients into the trial, because they felt it unethical to deny them this intervention. The trial protocol resulted in patients in the treatment group being prone for an average of only 7 out of 24 h for a 10-day period. Overall there was no difference in mortality between the control and treatment groups at day 10, time of ICU discharge, or at 6 months. Interestingly, analysis of subgroups revealed a significant difference in the outcome at 10 days for patients with the most severe disease, although this disappeared by ICU discharge. In retrospect, the design of this ambitious trial was flawed by its failure to establish the optimal utilization of this manoeuvre. 
The opening session reported that we are making progress in supporting the failing liver (Wendon, London, UK). Current optimism should probably be limited to extracorporeal methods, because the molecular adsorbent recirculating system (essentially extracorporeal albumin dialysis) has been shown to have beneficial clinical effects as well as improved survival in two small randomized controlled trials [8, 9] . The equipment is familiar to all those who use dialytic therapies, and we will undoubtedly hear more about this system in the next few years.
The slide of a patient reading the newspaper through a transparent helmet, while receiving noninvasive ventilation (NIV) resembled pictures of a NASA astronaut! However, it was reported to be well tolerated for prolonged periods, and significantly reduces the complications associated with NIV (pressure areas, tolerance of mask). The recent Consensus Conference [10] examined weaning aspects of NIV and emphasized the reduced weaning time and avoidance of reintubation, but called for more randomized controlled trials. Finally, although continuous positive airway pressure has been shown to be beneficial in pulmonary oedema, caution is still advised with the use of bilevel positive airway pressure because of the reporting of myocardial infarction in several studies. However, the groups studied were unmatched and starting points were different, so conclusions should not be drawn until randomized controlled trial results are available in this area.
This was a well-attended session, which, according to Levy (Providence, RI, USA), was in complete contrast to the interest shown in the USA for the subject. Although there were few new data in the session, the emphasis on a strategy for lawsuits was welcome. Suggestions included statements from scientific societies at a national and international level, open reporting in medical files of decisions to withdraw or withhold treatment, and family involvement in decision making that will ultimately involve better media education.
The last day of this year's symposium was sadly abandoned by many due to the Belgian rail strike. Despite this, the usual convivial atmosphere, both in and around the congress, was as abundant as ever. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of the usual high standard, although, yet again, access to many of the symposium's venues was limited by the lack of capacity of the secondary rooms. The 21st International Symposium was marked by a sense of renewed enthusiasm that real positive progress is occurring at the coal face of intensive care. Heme oxygenase-1 and carbon monoxide in pulmonary medicine Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states. The heme oxygenase-1/carbon monoxide (HO-1/CO) system has recently seen an explosion of research interest due to its newly discovered physiological effects. This metabolic pathway, first characterized by Tenhunen et al. [1, 2] , has only recently revealed its surprising cytoprotective properties [3, 4] . Research in HO-1/CO now embraces the entire field of medicine where reactive oxygen/nitrogen species, inflammation, growth control, and apoptosis represent important pathophysiological mechanisms [3] [4] [5] [6] . Indeed, the number of publications in recent years concerning HO-1 has increased exponentially, while the list of diseases and physiological responses associated with changes in HO-1 continues to expand [5] .
Until now, relatively few studies have addressed the role of HO-1/CO in pulmonary medicine. Several investigators have focused on the diagnostic application of the HO-1/CO system, by measuring exhaled CO (E-CO) in various pathological pulmonary conditions, such as asthma or chronic obstructive pulmonary disease (COPD) [7] . In another experimental approach, investigators have examined the expression of HO-1 in lung tissue from healthy or diseased subjects [8, 9] . This review will highlight the actions of HO-1/CO in the context of heme degradation have antioxidant properties [18, 19] . The liberated heme iron undergoes detoxification either by extracellular efflux or by sequestration into ferritin, an intracellular iron-storage molecule with potential cytoprotective function [20] [21] [22] [23] . Of the three known isoforms of HO (HO-1, HO-2, and HO-3), only HO-1 responds to xenobiotic induction [24] [25] [26] [27] . Constitutively expressed in many tissues, HO-2 occurs at high levels in nervous and vascular tissues, and may respond to regulation by glucocorticoids [25, 28, 29] . HO-1 and HO-2 differ in genetic origin, in primary structure, in molecular weight, and in their substrate and kinetic parameters [25, 26] . HO-3 displays a high sequence homology with HO-2 but has little enzymatic activity [27] . This review will focus on the inducible, HO-1, form.
In addition to the physiological substrate heme, HO-1 responds to induction by a wide variety of stimuli associated with oxidative stress. Such inducing agents include hypoxia, hyperoxia, cytokines, nitric oxide (NO), heavy metals, ultraviolet-A (320-380 nm) radiation, heat shock, shear stress, hydrogen peroxide, and thiol (-SH)-reactive substances [3] . The multiplicity of toxic inducers suggest that HO-1 may function as a critical cytoprotective molecule [3, 4] . Many studies have suggested that HO-1 acts as an inducible defense against oxidative stress, in models of inflammation, ischemia-reperfusion, hypoxia, and hyperoxia-mediated injury (reviewed in [3] ). The mechanisms by which HO-1 can mediate cytoprotection are still poorly understood. All three products of the HO reaction potentially participate in cellular defense, of which the gaseous molecule CO has recently received the most attention [30, 31] . The administration of CO at low concentrations can compensate for the protective effects of HO-1 in the presence of competitive inhibitors of HO-1 activity [32] [33] [34] . While HO-1 gene transfer confers protection against oxidative stress in a number of systems, clearly not all studies support a beneficial role for HO-1 expression. Cell-culture studies have suggested that the protective effects of HO-1 overexpression fall within a critical range, such that the excess production of HO-1 or HO-2 may be counterprotective due to a transient excess of reactive iron generated during active heme metabolism [35, 36] . Thus, an important caveat of comparative studies on the therapeutic effects of CO administration versus HO-1 gene delivery arises from the fact that the latter approach, in addition to producing CO, may have profound effects on intracellular iron metabolism.
HO-1 expression is primarily regulated at the transcriptional level. Genetic analyses have revealed two enhancer sequences (E1, E2) in the murine HO-1 gene located at -4 kb (E1) and -10 kbp (E2) of the transcriptional start site [37, 38] . These enhancers mediate the induction of HO-1 by many agents, including heavy metals, phorbol esters, endotoxin, oxidants, and heme. E1 and E2 contain repeated stress-responsive elements, which consist of overlapping binding sites for transcription factors including activator protein-1 (AP-1), v-Maf oncoprotein, and the cap'n'collar/basic-leucine zipper family of proteins (CNC-bZIP), of which Nrf2 (NF-E2-related factor) may play a critical role in HO-1 transcription [39] . The promoter region of HO-1 also contains potential binding sites for nuclear factor κB (NF-κB), though the functional significance of these are not clear [40] . Both NF-κB and AP-1 have been identified as regulatory elements responsive to oxidative cellular stress [40, 41] . In response to hyperoxic stress, AP-1 factors mediated the induction of HO-1 in cooperation with signal-transducer and activator of transcription (STAT) proteins [41] . Furthermore, a distinct hypoxia-response element (HRE), which mediates the HO-1 response to hypoxia, represents a binding site for the hypoxia-inducible factor-1 (HIF-1) [42] .
The toxic properties of CO are well known in the field of pulmonary medicine. This invisible, odorless gas still claims many victims each year by accidental exposure. CO evolves from the combustion of organic materials and is present in smoke and automobile exhaust. The toxic actions of CO relate to its high affinity for hemoglobin (240-fold greater than that of O 2 ). CO replaces O 2 rapidly from hemoglobin, causing tissue hypoxia [43] [44] [45] . At high concentrations, other mechanisms of CO-induced toxicity may include apoptosis, lipid peroxidation, and inhibition of drug metabolism and respiratory enzyme functions [44] .
Only recently has it become known that, at very low concentrations, CO participates in many physiological reactions. Where a CO exposure of 10,000 parts per million (ppm) (1% by volume CO in air) is toxic, 100-250 ppm (one hundredth to one fortieth as much) will stimulate the physiological effects without apparent toxicity [4] . The majority of endogenous CO production originates from active heme metabolism (>86%), though a portion may be produced in lipid peroxidation and drug metabolism reactions [46] . Cigarette smoking, still practiced by many lung patients, represents a major source of chronic lowlevel exposure to CO. Inhaled CO initially targets alveolar macrophages and respiratory epithelial cells.
The exact mechanisms by which CO acts at the molecular level remain incompletely understood. CO potentially exerts its physiological effects by influencing at least three known pathways (Fig. 2 ). By complexation with the heme moiety of the enzyme, CO activates soluble guanylate cyclase (sGC), stimulating the production of cyclic 3':5'guanosine monophosphate (cGMP) [47] . The sGC/cGMP pathway mediates the effects of CO on vascular relaxation, smooth muscle cell relaxation, bronchodilation, neurotransmission, and the inhibition of platelet aggregation, coagulation, and smooth muscle proliferation [48] [49] [50] [51] . Furthermore, CO may cause vascular relaxation by directly activating calcium-dependent potassium channels [52] [53] [54] . CO potentially influences other intracellular signal transduction pathways. The mitogen-activated protein kinase (MAPK) pathways, which transduce oxidative stress and inflammatory signaling (i.e. response to lipopolysaccharide), may represent an important target Possible mechanism(s) of carbon monoxide action Figure 2 Possible mechanism(s) of carbon monoxide action. Endogenous carbon monoxide (CO) arises principally as a product of heme metabolism, from the action of heme oxygenase enzymes, although a portion may arise from environmental sources such as pharmacological administration or accidental exposure, or other endogenous processes such as drug and lipid metabolism. The vasoregulatory properties of CO, including its effects on cellular proliferation, platelet aggregation, and vasodilation, have been largely ascribed to the stimulation of guanylate cyclase by direct heme binding, leading to the generation of cyclic GMP. The anti-inflammatory properties of CO are associated with the downregulation of proinflammatory cytokine production, dependent on the selective modulation of mitogen-activated protein kinase (MAPK), such as the 38 kilodalton protein (p38MAPK). In addition to these two mechanisms, CO may potentially interact with any hemoprotein target, though the functional consequences of these interactions with respect to cellular signaling remain poorly understood.
Anti-Platelet Aggregation Anti-Proliferation 
?
Inhibition of pro-inflammatory cytokine production
Modulation of hemoprotein function of CO action [32, 34, 55, 56 ]. An anti-apoptotic effect of CO and its relation to MAPK has recently been described. The overexpression of HO-1 or the exogenous administration of CO prevented tumor necrosis factor α (TNF-α)induced apoptosis in murine fibroblasts [57] . In endothelial cells, the anti-apoptotic effect of CO depended on the modulation of the p38 (38 kilodalton protein) MAPK pathway [34] . The role of the remaining heme metabolites, (i.e. Fe and biliverdin IXα) in the modulation of apoptosis is currently being investigated and is beyond the scope of this review. Recent studies have reported a potent anti-inflammatory effect of CO, involving the inhibition of proinflammatory cytokine production after endotoxin stimulation, dependent on the modulation of p38 MAPK [32] . The clinical relevance of p38 MAPK lies in the possibility of modulating this pathway in various clinical conditions to downregulate the inflammatory response [58] .
Oxidative stress arising from an imbalance between oxidants and antioxidants plays a central role in the pathogenesis of airway disease [59] . In lung tissue, HO-1 expression may occur in respiratory epithelial cells, fibroblasts, endothelial cells, and to a large extent in alveolar macrophages [41, 60, 61] . HO-1 induction in these tissues, in vitro and in vivo, responds to common causes of oxidative stress to the airways, including hyperoxia, hypoxia, endotoxemia, heavy metal exposure, bleomycin, diesel exhaust particles, and allergen exposure [4, 41, 61] . Induction of HO-1 or administration of CO can protect cells from these stressful stimuli [10, 41] . In one of the experiments that best illustrate the protective role of CO in vivo, rats were exposed to hyperoxia (>98% O 2 ) in the absence or presence of CO at low concentration (250 ppm). The CO-treated rats showed increased survival and a diminished inflammatory response to the hyperoxia [11] . As demonstrated in a model of endotoxin-induced inflammation, the protection afforded by CO most likely resulted from the downregulated synthesis of proinflammatory cytokines (i.e. TNF-α, IL-1β) and the upregulation of the anti-inflammatory cytokine interleukin-10 (IL-10) [32] . Furthermore, increases in exhaled CO (E-CO) have been reported in a number of pathological pulmonary conditions, such as unstable asthma, COPD, and infectious lung disease; these increases may reflect increased endogenous HO-1 activity [7] . Elevated carboxyhemoglobin (Hb-CO) levels have also been reported in these same diseases in nonsmoking subjects, where both the E-CO and Hb-CO levels decrease to normal levels in response to therapy [62] .
E-CO in humans originates primarily from both systemic heme metabolism, which produces CO in various tissues, and localized (lung) heme metabolism, as a result of the combined action of inducible HO-1 and constitutive HO-2 enzymatic activity. Endogenously produced or inspired CO is eliminated exclusively by respiration [63] . Elevation of E-CO may also reflect an increase in exogenous sources such as smoking or air pollution. In addition to changes in environmental factors, elevations of E-CO in lung diseases may reflect an increase in blood Hb-CO levels in response to systemic inflammation, as well as an increase in pulmonary HO-1 expression in response to local inflammation [9, 62, 64] .
The diagnostic value of measuring E-CO remains controversial due to many conflicting reports (i.e. some reports indicate differences in E-CO measurements between disease activity and controls, and some reports do not). The possible explanations for these discrepancies include large differences in patient populations and in the methods used for measuring E-CO, and undefined corrections for background levels of CO. Furthermore, remarkable differences arise between studies in the magnitude of the E-CO levels in the control groups as well as in treated or untreated asthma patients. When active or passive smoking occurs, or in the presence of high background levels of CO, the measurement of E-CO is not particularly useful for monitoring airway inflammation. In patients who smoke, E-CO can be used only to confirm the smoking habit [65, 66] . Comparable to the beginning era of measurements of exhaled NO, a standardization in techniques and agreement on background correction should be reached for E-CO measurements, to allow proper conclusions to be drawn in this area of investigation.
Asthma, a form of allergic lung disease, features an accumulation of inflammatory cells and mucus in the airways, associated with bronchoconstriction and a generalized airflow limitation. Inflammation, a key component of asthma, involves multiple cells and mediators where an imbalance in oxidants/antioxidants contributes to cell damage. Several pathways associated with oxidative stress may participate in asthma. For example, the redox-sensitive transcription factors NF-κB and AP-1 control the expression of proinflammatory mediators [59, [67] [68] [69] .
In light of the potential protective effects of HO-1/CO on inflammatory processes, the study of HO-1 in asthma has gained popularity. In a mouse model of asthma, HO-1 expression increased in lung tissue in response to ovalbumin aerosol challenge, indicating a role for HO-1 in asthma [70] . In a similar model of aeroallergen-induced asthma in ovalbumin-sensitized mice, exposure to a CO atmosphere resulted in a marked attenuation of eosinophil content in bronchoalveolar lavage fluid (BALF) and downregulation of the proinflammatory cytokine IL-5 [10] . This experiment showed that exogenous CO can inhibit asthmatic responses to allergens in mice.
Recent human studies have revealed higher HO-1 expression in the alveolar macrophages and higher E-CO in untreated asthmatic patients than in healthy nonsmoking controls [71, 72] . Patients with exacerbations of asthma and patients who were withdrawn from inhaled steroids showed higher E-CO levels than steroid-treated asthmatics or healthy controls [73] . Higher levels of E-CO may also occur in children with persistent asthma than in healthy controls [74] . E-CO levels may correlate with functional parameters such as peak expiratory flow rate. A low rate in asthma exacerbations correlated with high E-CO, whereas normalization of the rate with oral glucocorticoid treatment resulted in a reduction of E-CO [75] . Furthermore, increased E-CO was associated with greater expression of HO-1 in airway alveolar macrophages obtained by induced sputum in untreated asthmatic patients than in controls. These asthma patients also showed higher bilirubin levels in the induced sputum, indicating higher HO activity [71] . Furthermore, patients with asthma show an increased Hb-CO level at the time of exacerbation, with values decreasing to control levels after oral glucocorticoid treatment [62] . In human asthmatics, E-CO and airway eosinophil counts decreased in response to a one-month treatment with inhaled corticosteroids [73] . In direct contrast to such studies promoting E-CO as a useful noninvasive tool for monitoring airway inflammation, other studies reported no difference in E-CO levels of asthma patients versus healthy controls, or between patients with stable and unstable asthma. In one such report, no further change in E-CO occurred in asthma patients after a one-month treatment of inhaled corticosteroids, despite observed decreases in airway eosinophil content and bronchial responsiveness to metacholine [76] . A recent study accentuates this finding in asthma excerbations, where no decrease in E-CO of children with asthma could be detected after oral prednisolone treatment [77] . In human allergic responses, results on elevation of E-CO are also inconclusive. A clear elevation of E-CO after allergen exposure occurred in patients with asthma during the late response, and during the early response immediately after the inhalation [78] . However, another report showed that no elevation of E-CO occurred in allergen-induced asthma within 48 hours after allergen challenge [79] . Finally, increases in E-CO were measured in allergic rhinitis, correlating with seasonal changes in exposure to allergen (pollen) [80] .
Airway inflammation plays an important role in the development of COPD, characterized by the presence of macrophages, neutrophils, and inflammatory mediators such as proteinases, oxidants, and cytokines. Further-more, the inflammatory consequences of chronic microbiological infections may contribute to the progression of the disease. The current paradigm for the pathogenesis of COPD involves imbalances in protease/antiprotease activities and antioxidant/pro-oxidant status. Proteases with tissue-degrading capacity, (i.e. elastases and matrix metalloproteinases), when insufficiently inhibited by antiproteases, can induce tissue damage leading to emphysema. Oxidants that supersede cellular antioxidant defenses can furthermore inactivate antiproteases, cause direct injury to lung tissue, and interfere with the repair of the extracellular matrix. Smoking plays an important role in both hypotheses. Cigarette smoke will act primarily on alveolar macrophages and epithelial cells, which react to this oxidative stress by producing proinflammatory cytokines and chemokines and releasing growth factors. Nevertheless, smoking cannot be the only factor in the development of COPD, since only 15-20% of smokers develop the disease [81, 82] .
Exposure to reactive oxygen species (from cigarette smoke or chronic infections) and an imbalance in oxidant/antioxidant status are the main risk factors for the development of COPD. To defend against oxidative stress, cells and tissues contain endogenous antioxidant defense systems, which include millimolar concentrations of the tripeptide glutathione (GSH). A close relation exists between GSH concentration and HO-1, whereby depletion of GSH augments the transcriptional regulation of HO-1 by oxidants, suggesting that the HO-1/CO system acts as a secondary defense against oxidative stress [83] [84] [85] [86] . Accumulating clinical evidence suggests that HO-1/ CO may also play an important part in COPD. Alveolar macrophages, which produce a strong HO-1 response to stimuli, may represent the main source of CO production in the airways [60, 64] . Patients with COPD have displayed higher E-CO than healthy nonsmoking controls [87] . Furthermore, much higher levels of HO-1 have been observed in the airways of smokers than in nonsmokers [64] . Among subjects who formerly smoked, patients with COPD have lower HO-1 expression in alveolar macrophages than healthy subjects [88] . A microsatellite polymorphism that is linked with the development of COPD may occur in the promoter region of HO-1, resulting in a lower production of HO-1 in people who have the polymorphism. Thus, a genetically dependent downregulation of HO-1 expression may arise in subpopulations, possibly linked to increased susceptibility to oxidative stress [89] [90] [91] . Future studies on both genetic predisposition and possible therapeutic modalities will reveal the involvement of the HO-1/CO system in COPD.
Cystic fibrosis (CF) involves a deposition of hyperviscous mucus in the airways associated with pulmonary dysfunc-tion and pancreatic insufficiency, which may be accompanied by chronic microbiological infections. E-CO readings were higher in untreated versus oral-steroidtreated CF patients [92] . Furthermore, E-CO increased in patients during exacerbations of CF, correlating to deterioration of the forced expiratory volume in one second (FEV 1 ), with normalization of the E-CO levels after treatment [93] . E-CO levels may correlate with exhaled ethane, a product of lipid peroxidation that serves as an indirect marker of oxidative stress. Both E-CO and exhaled ethane were higher in steroid-treated and untreated CF patients than in healthy controls [94] . E-CO was higher in children with CF than in control patients. In addition to the inflammatory and oxidative stress responses to continuous infectious pressure in these patients, E-CO may possibly respond to hypoxia. E-CO increased further in CF children following an exercise test, and correlated with the degree of oxyhemoglobin desaturation, a finding suggestive of an increased HO-1 expression in CF patients during hypoxic states induced by exercise [95] .
In patients with pneumonia, higher Hb-CO levels can be measured at the onset of illness, with values decreasing to control levels after antibiotic treatment [62] . E-CO levels were reported to be higher in lower-respiratory-tract infections and bronchiectasis, with normalization after antibiotic treatment [96, 97] . Furthermore, E-CO levels in upper-respiratory-tract infections were higher than in healthy controls [74, 80] . The relationship between higher measured E-CO in these infectious states and higher Hb-CO levels cannot be concluded from these studies.
The role of HO-1 in the development of interstitial lung disease remains undetermined. Comparative immunohistochemical analysis has revealed that lung tissue of control subjects, patients with sarcoidosis, usual interstitial pneumonia, and desquamative interstitial pneumonia, all showed a high expression of HO-1 in the alveolar macrophages but a weak expression in the fibrotic areas [98] . The antiproliferative properties of HO-1 suggest a possible beneficial role in limiting fibrosis; however, this hypothesis is complicated by a newly discovered relation between IL-10 and HO-1. IL-10 produced by bronchial epithelial cells promotes the growth and proliferation of lung fibroblasts [99] . HO-1 expression and CO treatment have been shown to increase the production of IL-10 in macrophages following proinflammatory stimuli [32] . Conversely, IL-10 induces HO-1 production, which is apparently required for the anti-inflammatory action of IL-10 [100] .
A recent report clearly shows the suppression of bleomycin-induced pulmonary fibrosis by adenovirus-mediated HO-1 gene transfer and overexpression in C57BL/6 mice, involving the inhibition of apoptotic cell death [101] .
Overall, more research is needed to elucidate the mechanisms of HO-1 in interstitial lung disease and its possible therapeutic implications.
HO-1 action may be of great importance in solid tumors, an environment that fosters hypoxia, oxidative stress, and neovascularization. HO-1 may have both pro-and antagonistic effects on tumor growth and survival. HO-1 and CO cause growth arrest in cell-culture systems and thus may represent a potential therapeutic modality in modulating tumor growth [16] . The overexpression of HO-1 or administration of CO in mesothelioma and adenocarcinoma mouse models resulted in improved survival (>90%) as well as reduction in tumor size (>50%) [17] . Furthermore, HO-1 expression in oral squamous cell carcinomas can be useful in identifying patients at low risk of lymph node metastasis. High expression of HO-1 was detected in groups without lymph node metastasis in this report [102] . In contrast to growth arrest, HO-1 may protect solid tumors from oxidative stress and hypoxia, possibly by promoting neovascularization. In one study, zinc protoporphyrin, a competitive inhibitor of HO-1 enzyme activity, suppressed tumor growth [103] .
CO may represent a critical mediator of the body's adaptive response to hypoxia, a common feature in pulmonary vascular disease [104] . Since CO can modulate vascular tone by inducing cGMP and large, calcium-dependent potassium channels, HO-1 and CO probably play important roles in pulmonary vascular diseases [54] . A NOmediated HO-1 induction occurred in the hepatopulmonary syndrome during cirrhosis, associated with enhancement of vascular relaxation [105] . In portopulmonary hypertension, elevated levels of cGMP and inducible nitric oxide synthase (iNOS) expression in the vascular endothelium, and HO-1 expression in macrophages and bronchial epithelium have been described [106] . In transgenic mice models, ho-1 -/and ho-1 +/+ mice did not differ in their development of pulmonary hypertension following chronic hypoxia treatment, despite the development of right ventricular dilation and right myocardial infarction in ho-1 -/mice [107] . The preinduction of HO-1 protein with chemical inducers, however, prevented the development of pulmonary hypertension in the rat lung as a consequence of chronic hypoxia treatment [108] . Transgenic mice overexpressing HO-1 in the lung were resistant to hypoxia-induced inflammation and hypertension [109] . Further research is needed to elucidate the potential role of HO-1 and CO in primary human lung vascular diseases such as primary pulmonary hypertension.
Supplemental oxygen therapy is often used clinically in the treatment of respiratory failure. Exposure to high oxygen tension (hyperoxia) may cause acute and chronic lung injury, by inducing an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema [110, 111] . Hyperoxia-induced lung injury causes symptoms in rodents that resemble human acute respiratory distress syndrome [112] .
Hyperoxia induced HO-1 expression in adult rats but apparently not in neonatal rats, in which the expression and activities of HO-1 and HO-2 are developmentally upregulated during the prenatal and early postnatal period [113] .
Both HO-1 and HO-2 potentially influence pulmonary adaptation to high O 2 levels. In one example, the adenoviral-mediated gene transfer of HO-1 into rat lungs protected against the development of lung apoptosis and inflammation during hyperoxia [114] . In vitro studies showed that the overexpression of HO-1 in lung epithelial cells or rat fetal lung cells caused growth arrest and conferred resistance against hyperoxia-induced cell death [15, 16] . An oxygen-tolerant variant of hamster fibroblasts that moderately overexpressed HO-1 in comparison with the parent line resisted oxygen toxicity in vitro. The treatment of this oxygen-tolerant strain with HO-1 antisense oligonucleotides reduced the resistance to hyperoxia. In contrast, additional, vector-mediated, HO-1 expression did not further increase oxygen tolerance in this model [115] .
In vivo studies with gene-deleted mouse strains have provided much information on the roles of HO-1 and HO-2 in oxygen tolerance. Dennery et al. demonstrated that heme oxygenase-2 knockout mice (ho-2 -/-) were more sensitive to the lethal effects of hyperoxia than wild-type mice [116] . In addition to the absence of HO-2 expression, however, the mice displayed a compensatory increase in HO-1 protein expression, and higher total lung HO activity. Thus, in this model, the combination of HO-2 deletion and HO-1 overexpression resulted in a hyperoxiasensitive phenotype. Recent studies of Dennery et al. have shown that HO-1-deleted (ho-1 -/-) mice were more resistant to the lethal effects of hyperoxia than the corresponding wild type [117] . The hyperoxia resistance observed in the ho-1 -/strain could be reversed by the reintroduction of HO-1 by adenoviral-mediated gene transfer [117] . In contrast, mouse embryo fibroblasts derived from ho-1 -/mice showed increased sensitivity to the toxic effects of hemin and H 2 O 2 and generated more intracellular reactive oxygen species in response to these agents [118] . Both ho-1 -/-and ho-2 -/strains were anemic, yet displayed abnormal accumulations of tissue iron. Specifically, ho-1 -/accumulated nonheme iron in the kidney and liver and had decreased total iron content in the lung, while ho-2 -/mice accumulated total lung iron in the absence of a compensatory increase in ferritin levels [116, 119] . The mechanism(s) by which HO-1 or HO-2 deletions result in accumulation of tissue iron remain unclear. These studies, taken together, have indicated that animals deficient in either HO-1 and HO-2 display altered sensitivity to oxidative stress conditions. Aberrations in the distribution of intra-and extra-cellular iron, may underlie in part, the differential sensitivity observed [116, 117] .
Otterbein et al. have shown that exogenous CO, through anti-inflammatory action, may protect the lung in a rat model of hyperoxia-induced lung injury. The presence of CO (250 ppm) prolonged the survival of rats in a hyperoxic (>95% O 2 ) environment, and inhibited the appearance of markers of hyperoxia-induced lung injury (i.e. hemorrhage, fibrin deposition, edema, airway protein accumulation, and BALF neutrophil influx) [11] . Furthermore, in a mouse model, CO inhibited the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice induced by the hyperoxia treatment. Using genedeleted mice, Otterbein and colleagues also observed that the protection afforded by CO in this model, similar to a lipopolysaccharide-induced model of lung injury, depended on the p38 MAPK pathway (Otterbein et al., unpublished observation, as reviewed in [3] ).
In direct contrast to these studies, the group of Piantadosi and colleagues reported no significant difference in the hyperoxia tolerance of rats at CO doses between 50 and 500 ppm [120] . In their model, CO did not alter the accumulation of fluid in the airway. Furthermore, CO, when applied in combination with hyperoxia, increased the activity of myeloperoxidase, a marker of airway neutrophil influx. This study also suggested that inhalation of CO (50-500 ppm) did not alter the expression of HO-1 or other antioxidant enzymes such as Manganese superoxide dismutase (MnSOD) in vivo [120] . Furthermore, Piantadosi and colleagues were able to induce oxygen tolerance in rats and HO-1 expression with hemoglobin treatment, but this tolerance also occurred in the presence of HO inhibitors, thereby not supporting a role for HO activity in oxygen tolerance [121] . Although no consensus has been reached as to the protective role of CO inhalation and/or HO-1 induction in hyperoxic lung injury, human studies will be required to show if CO will supersede NO in providing a significant therapeutic benefit in the context of severe lung diseases [122] . While antioxidant therapies have been examined, until now no human studies exist on the role of HO-1 and CO in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia [123] .
Lung transplantation is the ultimate and often last therapeutic option for several end-stage lung diseases. After lung transplantation, there remains an ongoing hazardous situation in which both acute and chronic graft failure, as well as complications of the toxic immunosuppressive regimen used (i.e. severe bacterial, fungal, and viral infections; renal failure; and Epstein-Barr-virus-related lymphomas), determine the outcome [124] . The development of chronic graft failure, obliterative bronchiolitis (OB), determines the overall outcome after lung transplantation. OB, which may develop during the first months after transplantation, is the main cause of morbidity and death following the first half-year after transplantation, despite therapeutic intervention. Once OB has developed, retransplantation remains the only therapeutic option available [124, 125] . Little is known about the pathophysiological background of OB. The possible determinants of developing OB include ongoing immunological allograft response, HLADR mismatch, cytomegalovirus infection, acute rejection episodes, organ-ischemia time, and recipient age [125] . OB patients displayed elevated neutrophil counts in the BALF, and evidence of increased oxidant activity, such as increased methionine oxidation in BALF protein and decreases in the ratio of GSH to oxidized glutathione (GSSG) in epithelial lining fluid. [126, 127] .
So far, only very limited research data are available on the possible role for HO-1 in allograft rejection after lung transplantation. Higher HO-1 expression has been detected in alveolar macrophages from lung tissue in lung transplant recipients with either acute or chronic graft failure than in stable recipients [128] . The protective role of HO-1 against allograft rejection has been shown in other transplantation models, in which solid organ transplantation typically benefits from HO-1 modulation. A higher expression of protective genes such as HO-1 has been observed in episodes of acute renal allograft rejection [129] . Furthermore, the induction of HO-1 alleviates graft-versus-host disease [130] . Adenoviral-HO-1 gene therapy resulted in remarkable protection against rejection in rat liver transplants [131] . The upregulation of HO-1 protected pancreatic islet cells from Fas-mediated apoptosis in a dose-dependent fashion, supporting an anti-apoptotic function of HO-1 [132, 133] . HO-1 may confer protection in the early phase after transplantation by inducing Th2-dependent cytokines such as IL-4 and IL-10, while suppressing interferon-γ and IL-2 production, as demonstrated in a rat liver allograft model [134] .
Beneficial effects of HO-1 modulation have also been described in xenotransplantation models, in which HO-1 gene expression appears functionally associated with xenograft survival [135] . In a mouse-to-rat heart trans-plant model, the effects of HO-1 upregulation could be mimicked by CO administration, suggesting that HOderived CO suppressed the graft rejection [136] . The authors proposed that CO suppressed graft rejection by inhibition of platelet aggregation, a process that facilitates vascular thrombosis and myocardial infarction.
HO-1 may also contribute to ischemic preconditioning, a process of acquired cellular protection against ischemia/ reperfusion injury, as observed in guinea pig transplanted lungs [137] . HO-1 overexpression provided potent protection against cold ischemia/reperfusion injury in a rat model through an anti-apoptotic pathway [138, 139] . The induction of HO-1 in rats undergoing liver transplantation with cobalt-protoporphyrin or adenoviral-HO-1 gene therapy resulted in protection against ischemia/ reperfusion injury and improved survival after transplantation, possibly by suppression of Th1-cytokine production and decreased apoptosis after reperfusion [140, 141] . Until now, no reports have addressed E-CO measurements in lung transplantation, where it is possible that differences in E-CO will be found in patients with acute and chronic allograft rejection.
The evolution of CO in exhaled breath may serve as a general marker and diagnostic indicator of inflammatory disease states of the lung, though more research will be required to verify its reliability. Increases in exhaled CO presumably reflect changes in systemic and airway heme metabolic activity from the action of HO enzymes. Evidence from numerous in vitro and animal studies indicates that HO-1 provides a protective function in many, if not all, diseases that involve inflammation and oxidative stress. Thus, the exploitation of HO-1 for therapeutic gain could be achieved through the modulation of HO-1 enzyme activity or its up-and downstream regulatory factors, either by gene transfer, pharmacological inducers, or direct application of CO by gas administration or chemical delivery [142] [143] [144] [145] . The CO-releasing molecules (transition metal carbonyls) developed by Motterlini et al. [144] show promise in the pharmacological delivery of CO for therapeutic applications in vascular and immune regulation. The CO-releasing molecules have been shown to limit hypertension in vivo and promote vasorelaxation in isolated heart and aortic rings [144] .
Ultimately, the challenge remains in applying the therapeutic potentials of HO-1 to the treatment of human diseases. In vivo models of transplantation have shown that HO-1 gene therapy protects against allograft rejection [129, 134] . Given the toxic therapy that every transplant patient receives, especially after lung transplantation, the field of transplantation medicine may bring the first frontier for human applications of HO-1 gene therapy or exogenous CO administration. The potential use of inhalation CO as a clinical therapeutic in inflammatory lung diseases has also appeared on the horizon. In one promising study, an inhalation dose of 1500 ppm CO at the rate of 20 times per day for a week produced no cardiovascular side effects [146] . Cigarette smoking and CO inhalation at identical intervals produced comparable Hb-CO levels of approximately 5%. The question of whether or not CO can be used as an inhalation therapy will soon be replaced by questions of "how much, how long, and how often?" The fear of administering CO must be weighed against the severe toxicity of the immunosuppressive agents in current use, and the often negative outcome of solid organ transplantation.  Technical Description of RODS: A Real-time Public Health Surveillance System This report describes the design and implementation of the Real-time Outbreak and Disease Surveillance (RODS) system, a computer-based public health surveillance system for early detection of disease outbreaks. Hospitals send RODS data from clinical encounters over virtual private networks and leased lines using the Health Level 7 (HL7) message protocol. The data are sent in real time. RODS automatically classifies the registration chief complaint from the visit into one of seven syndrome categories using Bayesian classifiers. It stores the data in a relational database, aggregates the data for analysis using data warehousing techniques, applies univariate and multivariate statistical detection algorithms to the data, and alerts users of when the algorithms identify anomalous patterns in the syndrome counts. RODS also has a Web-based user interface that supports temporal and spatial analyses. RODS processes sales of over-the-counter health care products in a similar manner but receives such data in batch mode on a daily basis. RODS was used during the 2002 Winter Olympics and currently operates in two states—Pennsylvania and Utah. It has been and continues to be a resource for implementing, evaluating, and applying new methods of public health surveillance. Unfortunately, conventional public health disease surveillance-which relies on physician and laboratory reporting and manual analysis of surveillance data-is ill equipped for timely detection of such threats. 3 The reportable disease system relies on health care professionals to recognize, diagnose, and report cases and suspected outbreaks to public health officials 4, 5 ; however, it is unlikely that without an event or alert to raise his or her index of suspicion, a physician will attribute the early symptoms and signs of disease in a bioattack victim appropriately and report the case. 6 A key limitation of the current system is that the lone physician is blind to the cases his or her colleagues in a nearby hospital are seeing-knowledge that might lead the physician to consider uncommon diseases more strongly in his or her diagnostic reasoning. Mandatory laboratory reporting 4 is also illequipped for early detection, because it takes time before tests are ordered and specimens are obtained, transported, processed, and resulted.
Sufficiently early detection of a biological attack may be accomplished through surveillance schemes that can detect infected individuals earlier in the disease process. For completeness, we note that biosensors are being developed (and deployed) that detect organisms in the air and that this type of detection, if feasible, occurs fundamentally much earlier, because the delay introduced by the incubation period of the disease is eliminated from the surveillance system. 7 However, such approaches face unsolved technical problems in the analysis of contaminated specimens (the norm in air sampling). Biosensors also need to be in the right place-on every person's lapel or every street corner and hallway-to provide complete surveillance coverage.
Surveillance methods that can detect disease at an earlier stage are an important research direction for public health surveillance. These methods are generally referred to as syndromic surveillance because they have the goal of recognition of outbreaks based on the symptoms and signs of infection and even its effects on human behavior prior to first contact with the health care system. 8 Because the data used by syndromic surveillance systems cannot be used to establish a specific diagnosis in any particular individual, syndromic surveillance systems must be designed to detect signature patterns of disease in a population to achieve sufficient specificity. For example, it would be absurd to use only the symptom of fever to attempt to establish a working diagnosis of inhalational anthrax in an individual, but it would be very reasonable to establish a working diagnosis of anthrax release in a community if we were to observe a pattern of 1,000 individuals with fever distributed in a linear streak across an urban region consistent with the prevailing wind direction two days earlier. It would be beyond reasonable and, in fact, imperative to establish a working diagnosis of public health emergency if presented with such information.
One recent example of a form of syndromic surveillance is drop-in surveillance-the stationing of public health workers in emergency departments (EDs) and special clinics during high-profile events such as the Super Bowl to capture data on patients presenting with symptoms potentially indicative of bioterrorism. The major disadvantage of this approach is the cost of round-the-clock staffing for manual data collection.
A less expensive approach-and the one taken in the Realtime Outbreak and Disease Surveillance (RODS) system-is detection based on data collected routinely for other purposes. Examples of such data include absenteeism data, sales of over-the-counter (OTC) health care products, and chief complaints from EDs. 9 The expenses of manual data collection are avoided; however, the data obtained typically are noisy approximations of what could be obtained by direct interviewing of the patient (in the case of individual level data). Both approaches may play complementary roles with current methods of public health surveillance 10-12 by assisting the physician and public health official with a continuously updated picture of the ''health status'' of a population. 13, 14 A focus of our research has been syndromic surveillance from free-text chief complaints routinely collected by triage nurses in EDs and acute care clinics during patient registration. We have deployed this type of surveillance at the 2002 Winter Olympics and in the States of Pennsylvania and Utah. We described a previous version of the RODS system, 12 but the system has undergone considerable subsequent development both architecturally and functionally. This report provides a detailed description of the current version of RODS, an example of a computer-based public health surveillance system that adheres to the National Electronic Disease Surveillance System (NEDSS) specifications of the Centers for Disease Control and Prevention (CDC). 15, 16 Background
The role of public health surveillance is to collect, analyze, and interpret data about biological agents, diseases, risk factors, and other health events and to provide timely dissemination of collected information to decision makers. 17 Conventionally, public health surveillance relies on manual operations and off-line analysis.
Existing syndromic surveillance systems include the CDC's drop-in surveillance systems, 8 Early Notification of Community-based Epidemics (ESSENCE), 10,18 the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS), 19 the Rapid Syndrome Validation Project (RSVP), 20 and the eight systems discussed by Lober et al. 11 Lober et al. summarized desirable characteristics of syndromic surveillance systems and analyzed the extent to which systems that were in existence in 2001 had those characteristics. 11 A limitation of most systems (e.g., ESSENCE, 10 Children's Hospital in Boston, 11 University of Washington 11 ) was batch transfer of data, which may delay detection by as long as the time interval (periodicity) between batch transfers. For example, a surveillance system with daily batch transfer may delay by one day the detection of an outbreak.
Some systems required manual data input (e.g., CDC's dropin surveillance systems, RSVP, 20 and LEADERS 19 ), which is labor-intensive and, in the worst case, requires round-theclock staffing. Manual data input is not a feasible mid-or long-term solution even if the approach is to add items to existing encounter forms (where the items still may be ignored by busy clinicians).
A third limitation for existing surveillance systems is that the systems may not exploit existing standards or communication protocols like Heath Level 7 (HL7) even when they are available.
The data type most commonly used among surveillance systems is symptoms or diagnoses of patients from ED and/or physician office visits. Other types of data identified in that study include emergency call center and nurse advice lines. Other types of data being used include sales of over-thecounter health care products, prescriptions, telephone call volumes to health care providers and drug stores, and absenteeism. We have conducted studies demonstrating that the free-text chief complaint data that we use correlate with outbreaks. 21, 22 Design Objectives
The overall design objective for RODS is similar to that of an early warning system for missile defense; namely, to collect whatever data are required to achieve early detection from as wide an area as necessary and to analyze the data in a way that they can be used effectively by decision makers. It is required that this analysis be done in close to real time. This design objective is complex and difficult to operationalize because of the large number of organisms and the even larger number of possible routes of dissemination all requiring potentially different types of data for their detection, different algorithms, and different time urgencies. For this reason, our focus since beginning the project in 1999 has been on the specific problem of detecting a large-scale outbreak due to an outdoor (outside buildings) aerosol release of anthrax. Additional design objectives were adherence to NEDSS standards to ensure future interoperability with other types of public health surveillance systems, scalability, and that the system could not rely on manual data entry, except when it was done in a focused way in response to the system's own analysis of passively collected data.
This report describes RODS 1.5, which was completely rewritten as a Java 2 Enterprise Edition (J2EE) application since the previous publication describing it. RODS 1.5 is multidata type enabled, which means that any time series data can be incorporated into the databases and user interfaces. The deployed RODS system currently displays and analyzes health care delivery site registrations and separately monitors sales of OTC health care products.
Overview RODS uses clinical data that are already being collected by health care providers and systems during the registration process. When a patient arrives at an ED (or an InstaCare in Utah), the registration clerk or triage nurse elicits the patient's reason for visit (i.e., the chief complaint), age, gender, home zip code, and other data and enter the data in a registration computer. The registration computer then generates an HL7 ADT (admission, discharge, and transfer) message and transmits it to the health system's HL7 message router (also called an integration engine). There usually is only one message router per health system even if there are many hospitals and facilities. These processes are all routine existing business activities and do not need to be created de novo for public health surveillance. Figure 1 shows the flow of clinical data to and within RODS. The hospital's HL7 message router, upon receipt of an HL7 message from a registration computer, deletes identifiable information from the message and then transmits it to RODS over a secure virtual private network (VPN), or a leased line, or both (during the 2002 Winter Olympics we utilized both types of connections to each facility for fault tolerance). The RODS HL7 listener maintains the connection with the health system's message router and parses the HL7 message as described in more detail below. It then passes the chief complaint portion of the message to a Bayesian text classifier that assigns each free-text chief complaint to one of seven syndromic categories (or to an eighth category, other). The database stores the category data, which then are used by applications such as detection algorithms and user interfaces.
Data about sales of OTC health care products are processed separately by the National Retail Data Monitor, which is discussed in detail in another article in this issue of JAMIA. 23 The processing was kept separate intentionally because, in the future, the servers for the National Retail Data Monitor may operate in different physical locations than RODS. The RODS user interfaces can and do display sales of OTC health care products as will be discussed, but other user interfaces can be connected to the National Retail Data Monitor as well.
Prior to September 2001, RODS received data only from hospitals associated with the UPMC Health System, and efforts to recruit other hospitals met with resistance. After the terrorist attacks (including anthrax) in the Fall of 2001, other hospitals agreed to participate. Although data in this project are de-identified, certain information such as the number of ED visits by zip code were considered proprietary information by some health systems. Health Insurance Portability and Accountability Act (HIPAA) concerns also were very prominent in the discussions. Data-sharing agreements were executed with every participating health system that addressed these concerns. As an additional precaution, all RODS project members meet annually with University of Pittsburgh council to review obligations and are required to sign an agreement every year stating that they understand the terms of the data-sharing agreements and agree to abide by the terms. RODS began as a research project at the University of Pittsburgh in 1999 and has functioned with IRB approvals since that time.
Health care facilities send admission, discharge, and transfer (ADT) HL7 messages to RODS for patient visits in EDs and walk-in clinics. A minimal data set is sent, as shown in Figure  2 , which qualifies as a HIPAA Limited Data Set. 24 Currently the data elements are age (without date of birth), gender, home zip code, and free-text chief complaint.
The HL7 listener receives HL7 messages from the message routers located in each health system. The HL7 listener then passes the received HL7 message to the HL7 parser bean, an Enterprise JavaBean (EJB) in the RODS business logic tier. The HL7 parser bean uses regular expressions to parse the fields in an HL7 message. The HL7 parser bean then stores the parsed elements into a database through a managed database connection pool.
Although nearly all health systems utilize the HL7 messaging standard, the location of individual data elements in an HL7 message may differ from health system to health system. For example, some care providers' systems record free-text chief complaint in the DG1 segment instead of the PV2 segment of an HL7 message. To resolve this mapping problem, a configuration file written in eXtensible Markup Language (XML), a standard protocol often used to define hierarchical data elements, defines where each of the data elements can be found in the HL7 message. When an HL7 listener starts up, it reads the hospital-dependent configuration file and passes the configuration information to the parser bean.
We also use this configuration file to define the database table and field in which the HL7 parser bean should store each data element. This approach is useful because it allows the HL7 data to be stored to an external database. We anticipate that health departments with existing NEDSS or other public health surveillance databases may wish to use just this component of RODS for real-time collection of clinical data.
For hospitals that do not have HL7 message routers (two of approximately 60 in our experience to date), RODS accepts ED registration data files through either a secure Web-based data upload interface or a secure file transfer protocol. In general, these types of data transfers are technically trivial and for that reason are used by many groups but do not have the reliability of a HL7 connection (and have very undesirable time latencies).
RODS checks the integrity of the data in the HL7 messages that it receives. This processing is necessary because hospital data flows may have undesirable characteristics such as duplicates. RODS identifies and deletes duplicates by using a database trigger that creates a composite primary key before inserting the data. RODS also filters out scheduling messages, which are identified by the fact that they have future admitted date and time.
RODS monitors all data feeds to ensure continuous connections with health systems. If RODS does not receive data for six hours, it sends an alert to the RODS administrator and the sending health system's administrator. Because the commercial message routers that hospitals use queue up HL7 messages when encountering networking or system problems, data integrity is preserved.
RODS uses an Oracle8i database to store ED registration data.
(Oracle, Redwood Shores, CA). To ensure fast response for an online query (e.g., the daily counts of respiratory syndrome in a county for the past six months), we developed a cache 
For connectivity with the HL7 message routers, we utilize hardware-based routers. The VPN router is a Cisco PIX 501 and the leased-line routers are a pair of Cisco 2600s (Cisco Systems, Inc., San Jose, CA).
All of the RODS processes can be run on a single computer, but in our current implementation-serving Pennsylvania F i g u r e 2. Sample HL7 admission, discharge, and transfer (ADT) message from an emergency department. The circled fields are age, gender, home zip code, admitted date and time, and free-text chief complaint, respectively.
and Utah as an application service provider-we use five dedicated servers: firewall, database, Web server, a geographic information system (GIS) server, and computation. The processes are written in Java code and can run on most platforms, but here we describe the specific platforms we use to indicate approximate sizing and processing requirements. 
We developed RODS applications using the Java 2 Enterprise Edition Software Toolkit (J2EE SDK) from Sun Microsystems for cross-platform Java application development and deployment. 26 We followed contemporary application programming practices-a multitiered application consisting of a client tier (custom applications such as HL7 listeners and detection algorithms), business logic tier, database tier, and Web tier.
Business logic such as the HL7 parser bean was implemented as Enterprise JavaBeans (EJBs). NEDSS specifies EJB as the standard for application logic. RODS uses Jboss, an opensource J2EE application server, to run all EJBs. 10 The Web tier comprises the graphical user-interface to RODS and uses Java Server Pages (JSP), Java Servlets, and ArcIMS. The database tier was implemented in Oracle 8i.
RODS uses a naive Bayesian classifier called Complaint Coder (CoCo) to classify free-text chief complaints into one of the following syndromic categories: constitutional, respiratory, gastrointestinal, neurological, botulinic, rash, hemorrhagic, and other. CoCo computes the probability of each category, conditioned on each word in a free-text chief complaint and assigns a patient to the category with the highest probability. 27 The probability distributions used by CoCo are learned from a manually created training set. CoCo can be retrained with local data, and it can be trained to detect a different set of syndromes than we currently use. CoCo runs as a local process on the RODS database server. CoCo was developed at the University of Pittsburgh and is available for free download at <http://health.pitt.edu/rods/sw>.
Over the course of the project, RODS has used two detection algorithms. These algorithms have not been formally field tested because the emphasis of the project to date has been on developing the data collection infrastructure more than field testing of algorithms.
The Recursive-Least-Square (RLS) adaptive filter 28 currently runs every four hours, and alerts are sent to public health officials in Utah and Pennsylvania. RLS, a dynamic autoregressive linear model, computes an expected count for each syndrome category for seven counties in Utah and 16 counties in Pennsylvania as well as for the combined counts for each state. We use RLS because it has a minimal reliance on historical data for setting model parameters and a high sensitivity to rapid increases in a time series e.g., a sudden increase in daily counts. RLS triggers an alert when the current actual count exceeds the 95% confidence interval for the predicted count.
During the 2002 Olympics we also used the What's Strange About Recent Events (WSARE 1.0) algorithm. 29 WSARE performs a heuristic search over combinations of temporal and spatial features to detect anomalous densities of cases in space and time. Such features include all aspects of recent patient records, including syndromal categories, age, gender, and geographical information about patients. The criteria used in the past for sending a WSARE 1.0 alert was that there has been an increase in the number of patients with specific characteristics relative to the counts on the same day of the week during recent weeks and the p-value after careful adjustment for multiple testing for the increase was #0.05. Version 3.0 of WSARE, which will incorporate a Bayesian model for computing expected counts rather than using unadjusted historical counts currently, is under development.
When an algorithm triggers an alert based on the above criteria, RODS sends e-mail and/or page alerts to its users. RODS uses an XML-based configuration file to define users' e-mail and pager addresses. The e-mail version of the alert includes a URL link to a graph of the time series that triggered the alarm with two comparison time series: total visits for the same time period and normalized counts.
RODS has a password-protected, encrypted Web site at which users can review health care registration and sales of OTC health care products on epidemic plots and maps. When a user logs in, RODS will check the user's profile and will display data only for his or her health department's jurisdiction. The interface comprises three screens-Main, Epiplot, and Mapplot.
The main screen alternates views automatically among each of the available data sources (currently health care registrations and OTC products in Pennsylvania and Utah and OTC sales only for other states). The view alternates every two minutes as shown in Figure 3 . The clinic visits view shows daily total visits and seven daily syndromes for the past week. The OTC data view shows daily sales for five product categories and the total, also for the past week. Users also can set the view to a specific county in a state. If the normalize control box is checked, the counts in the time series being displayed will be divided by (normalized by) the total daily sales of OTC health care products or ED visits for the region.
The Epiplot screen provides a general epidemic plotting capability. The user can simultaneously view a mixture of different syndromes and OTC product categories for any geographic region (state, county, or zip code), and for any time interval. The user also can retrieve case details as shown in Figure 4 . The Get Cases button queries the database for the admission date, age, zip code, and chief complaint (verbatim, not classified into syndrome category) of all patients in the time interval and typically is used to examine an anomalous density (spike) of cases. The Download Data button will download data as a compressed comma separated file for further analyses.
The Mapplot screen is an interface to ArcIMS, an Internetenabled GIS product developed by Environmental Systems Research Institute, Inc. Mapplot colors zip code regions to indicate the proportion of patients presenting with a particular syndrome. The GIS server also can overlay state boundaries, county boundaries, water bodies, hospital locations, landmarks, streets, and highways on the public health data as shown in Figure 5 . Similar to Epiplot, Mapplot also can display case details for a user-selected zip code.
RODS has been in operation for four years and, like most production systems, has acquired many fault-tolerant features. For example, at the software level, HL7 listeners continue to receive messages and temporarily store the messages when the database is off-line. A data manager program runs every ten minutes and, on finding such a cache, it loads the unstored messages to the database when the database is back on-line. In addition, the data manager program monitors and restarts HL7 listeners as necessary.
The database uses ''archive log'' mode to log every transaction to ensure that the database can recover from a system failure.
The hardware architecture also is fault tolerant. All servers have dual power supplies and dual network cards. All hard drives use Redundant Arrays of Inexpensive Disk configurations. In addition to dual power supplies, all machines are connected to an uninterrupted power supply that is capable of sending an e-mail alert to the RODS administrator when the main power is down.
An important component of RODS that currently is used only at the UPMC Health System in Pittsburgh is the Health System Resident Component (HSRC). The HSRC is located within the firewall of a health system and connects directly to the HL7 message router. The HSRC currently receives a diverse set of clinical data from the HL7 message router including culture results, radiology reports, and dictated F i g u r e 3. Health care registrations view in the Main screen of RODS. The Main screen alternates views every 2 minutes among data types available in the public health jurisdiction. The figure shows eight plots of health care registration data-total visits, botulinic, constitutional, gastrointestinal (GI), hemorrhagic, neurological, rash, and respiratory. After 2 minutes, over-the-counter data will be displayed. The Main screen can be used as a ''situation room'' display. emergency room notes. Its purpose is to provide additional public health surveillance functions that would not be possible if it were located outside of the firewall due to restrictions on the release of identifiable clinical data. The HSRC uses patient identifiers to link laboratory and radiology information to perform case detection. In the past, we have used HSRC to monitor for patients with both a gram-positive rod in a preliminary microbiology culture report and ''mediastinal widening'' in a radiology report. The HSRC is a case detector in a distributed outbreak detection system that is capable of achieving much higher specificity of patient diagnostic categorization through access to more information.
HSRC also removes identifiable information before transmitting data to the RODS system, a function provided by the health system's message router in other hospitals that connect to RODS.
The HSRC at UPMC Health System functions as an electronic laboratory reporting system, although the state and local health departments are not yet ready to receive real-time messaging from the system. Currently, it sends email alerts to the director of the laboratory and hospital infection control group about positive cultures for organisms that are required to be reported to public health in the state of Pennsylvania. 30 It also sends messages to hospital infection control when it detects organisms that cause nosocomial infections. These organisms include Clostridium difficile, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus.
We have been able in HSRC to prototype one additional feature, which is a ''look-back'' function that facilitates very rapid outbreak investigations by providing access to electronic medical records to public health investigators as shown in Figure 6 . This feature requires a token that can be passed to a hospital information system that can uniquely identify a patient, and the reason we have prototyped this feature in the HSRC and not in RODS is simply that HSRC runs within the firewall so an unencrypted token can be used. The lookback is accomplished as follows: when a public health user identifies an anonymous patient record of interest (e.g., one of 20 patients with diarrhea today from one zip code), HSRC calls the UPMC Health System Web-based electronic medical record system and passes it the patient identifier. UPMC Health System then requests the user to log in using the UPMC-issued password before providing access to the record directly from its own secure Web site. This approach is not intended to be implemented in HSRC, but rather in the RODS system outside of the firewall of a health system. It is intended to use encrypted identifiers that the health system would decrypt to retrieve the correct record. The HSRC could provide the encryption-decryption service or it could be provided by another data system in the hospital. We estimate that the prevalence of health systems that have Web-based results review in the United States is 30% to 50% and growing so that this approach could very quickly improve the efficiency of outbreak investigations. For these reasons, we have moved to an application service provider model for dissemination in which we encourage state and local health departments to form coalitions to support shared services. We also have been fortunate to have sufficient grant funding from the Commonwealth of Pennsylvania to be able to support these services on an interim basis while sustainable funding models evolve.
Our original design objectives for RODS were real-time collection of data with sufficient geographic coverage and sampling density to provide early syndromic warning of a large-scale aerosol release of anthrax. Although we have not achieved all of our initial design objectives, progress has been substantial. The research identified two types of data-freetext chief complaints and sales of OTC health care prod- ucts-that can be obtained in real time or near real time at sampling levels of 70% or higher for most of the United States. These results were obtained through large-scale deployments of RODS in Pennsylvania and Utah and through building the National Retail Data Monitor described in the accompanying article in this issue of JAMIA. The deployments also provided insights about organizational and technical success factors that would inform an effort to scale the project nationally.
The project established the importance of HL7 message routers (also known as integration engines) for public health surveillance. HL7 message routers are a mature, highly prevalent technology in health care. We demonstrated that free-text triage chief complaints can be obtained in real time from most U.S. hospitals through message routers and that these data represent early syndromal information about disease. Many other clinical data of value to public health are transmitted using the HL7 standard (e.g., orders for diagnostic tests, especially microbiology tests, reports of chest radiographs, medications, and test results) and can be integrated into RODS or other surveillance systems capable of receiving HL7 messages.
As a result of our efforts to disseminate this technology by giving it away, we have learned that most health departments do not have the technical resources to build and maintain real-time electronic disease surveillance systems. Our application service provider model has been much more success-ful, and we now recommend that states form coalitions to share the costs of such services.
The project very early identified the need for a computing component to reside within the firewall of a health system, connected to the hospital's HL7 message router. This component would function as a case detector in a distributed public health surveillance scheme linking laboratory and radiology data to increase the specificity of case detection. It has proven very difficult to disseminate this technology, perhaps due to the complexity of the idea. Nevertheless, the threat of bioterrorism has created a need for such technology, and this approach, or something with equivalent function, must be deployed.
Adherence to NEDSS architectural standards was an early design objective that we have met. RODS 1.5 closely follows NEDSS architectural, software, messaging, and data specifications. Our success is a strong validation of those standards. We will gain further understanding of the standards as we attempt to use RODS components including HL7 listeners, natural language parsers, message parsers, databases, user interfaces, notification subsystems, and detection algorithms with other NEDSS compliant systems. An ongoing project will use RODS to collect chief complaints and integrate them into the Utah Department of Health's planned NEDSS system.
We have demonstrated the ability to rapidly deploy RODS in a special event with the added advantage that the system F i g u r e 6. Look-back function of RODS. The user has selected one patient to investigate using the screen that is in the background and partly hidden by overlap. RODS has logged the user into the results-review function of an electronic medical record and requested that patient's chart, which is shown on the screen in the foreground.
persisted after the event. This experience suggests strongly that RODS or similar systems be considered an alternative to drop-in surveillance.
Our future plans are to meet our initial design objective to develop early-warning capability for a large, outdoor release of anthrax, especially ensuring that the data and analysis produced by RODS are reviewed by public health. This goal will require improvements in the interfaces and the detection algorithms to reduce false alarms and to vastly improve the efficiency with which anomalies are evaluated by use of multiple types of data, better interfaces, and implementation of the look-back function. We would like to enlarge as quickly as possible the application service provider to include more states and more types of clinical data so that states will be in a position to prospectively evaluate the detection performance from different types of data on naturally occurring outbreaks.
Our long-term goals are to add additional disease scenarios to the design objectives such as detection of in-building anthrax release, vector-borne disease, food-borne disease, and a communicable disease such as severe acute respiratory syndrome (SARS).
RODS is a NEDSS-compliant public health surveillance system that focuses on real-time collection and analysis of data routinely collected for other purposes. RODS is deployed in two states and was installed quickly in seven weeks for the 2002 Olympics. Our experience demonstrates the feasibility of such a surveillance system and the challenges involved.
Outbreaks, emerging infections, and bioterrorism have become serious threats. It is our hope that the front-line of public health workers, astute citizens, and health care workers will detect outbreaks early enough so that systems such as RODS are not needed. However, timely outbreak detection is too important to be left to human detection alone. The notion that public health can operate optimally without timely electronic information is as unwise as having commercial airline pilots taking off without weather forecasts and radar. Conservation of polyamine regulation by translational frameshifting from yeast to mammals Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals. The ef®ciency of +1 ribosomal frameshifting at a speci®c codon is used as a sensor to regulate polyamine levels in mammalian cells. The frameshifting occurs in decoding the gene antizyme 1, which has two partially overlapping open reading frames (ORFs). Protein sequencing showed that the reading-frame shift occurs at the last codon of ORF1, causing a proportion of ribosomes to enter ORF2 to synthesize a transframe protein (Matsufuji et al., 1995) . ORF2 encodes the main functional domains (Matsufuji et al., 1990; Miyazaki et al., 1992) of antizyme but has no ribosome initiation site of its own. The antizyme 1 protein binds to ornithine decarboxylase (ODC) (Murakami et al., 1992a; Cof®no, 1993, 1994) , inhibits it (Heller et al., 1976) and targets it for degradation by the 26S proteosome without ubiquitylation (Murakami et al., 1992b (Murakami et al., , 1999 . ODC catalyzes the ®rst and usually ratelimiting step in the synthesis of polyamines, conversion of ornithine to putrescine. Putrescine is a substrate for the synthesis of spermidine and spermine. Because of its inhibition of ODC, antizyme 1 is a negative regulator of the synthesis of polyamines. In addition, antizyme 1 is a negative regulator of the polyamine transporter (Mitchell et al., 1994; Suzuki et al., 1994; Sakata et al., 1997) . As discovered by Matsufuji and colleagues (Gesteland et al., 1992) and Rom and Kahana (1994) , increasing polyamine levels elevate frameshifting in decoding antizyme 1 mRNA and so increase the level of antizyme 1. Since antizyme 1 negatively regulates the synthesis and uptake of polyamines, the frameshifting is the sensor for an autoregulatory circuit. A second mammalian paralog of antizyme, antizyme 2, has very similar properties to antizyme 1, including the regulatory frameshifting, but does not stimulate degradation of ODC under certain conditions where antizyme 1 is active (Ivanov et al., 1998a; Zhu et al., 1999; Y.Murakami, S.Matsufuji, I.P.Ivanov, R.F.Gesteland and J.F.Atkins, in preparation) . Just like antizyme 1, antizyme 2 mRNA is ubiquitously expressed in the body but is 16 times less abundant than mRNA of antizyme 1 (Ivanov et al., 1998a) . In addition to antizyme 1 and 2, mammals have a third paralog of the gene, antizyme 3 (also encoded by two ORFs), which is expressed only during spermatogenesis (Ivanov et al., 2000) . Zebra®sh also have multiple antizyme genes, which differ in their expression patterns and activities (Saito et al., 2000) .
Numerous studies have addressed the regulation of fungal ODC in response to exogenously added polyamines. In the cases examined, Physarum polycephalum (Mitchell and Wilson, 1983) , Saccharomyces cerevisiae (Fonzi, 1989; Toth and Cof®no, 1999) and Neurospora crassa (Barnett et al., 1988; Williams et al., 1992) , added polyamines, especially spermidine, result in signi®cant repression of ODC activity. The mechanisms of repression seem to vary from fungus to fungus and are apparently different from the mechanism of polyamine-dependent regulation of ODC in higher eukaryotes. In some cases, the existence of an antizyme-like protein has been suggested but has either been disproved, as in the case of N.crassa (Barnett et al., 1988) , or has never been substantiated, as is the case with S.cerevisiae.
As expected from their small cationic nature and ability to neutralize negative charges locally, polyamines play key roles in processes ranging from the functioning of certain ion channels (Williams, 1997) , nucleic acid packaging, DNA replication, apoptosis, transcription and translation. The role of polyamines can be complex as illustrated by the transfer of the butylamine moiety of spermidine to a lysine residue to form hypusine in mammalian translation initiation factor eIF-5A, the only known substrate for this reaction (Tome et al., 1997; Lee et al., 1999) . Spermine negatively regulates the growth of prostatic carcinoma cells at their primary site (Smith et al., 1995) , but at later stages of tumor progression it fails to induce antizyme, which correlates with cells becoming refractory to spermine (Koike et al., 1999) . Lack of antizyme function is also important in the early deregulation of cellular proliferation in oral tumors (Tsuji Conservation of polyamine regulation by translational frameshifting from yeast to mammals The EMBO Journal Vol. 19 No. 8 pp. 1907±1917, 2000 ã European Molecular Biology Organization et al., 1998) and probably others. The levels of polyamines are altered in many tumors, and inhibitors of polyamine synthesis are being tested for antiproliferative and cell death effects. The synthesis of ODC varies during the cell cycle in normal cells (Linden et al., 1985; Fredlund et al., 1995) . It is induced by many growth stimuli and is constitutively elevated in transformed cells (Pegg, 1988; Auvinen et al., 1992) with some phosphorylated ODC being translocated to the surface membrane where it is important for mitotic cytoskeleton rearrangement events (Heiskala et al., 1999) .
Antizyme is one example of certain mRNA-contained signals that can elevate speci®c frameshifting >1000-fold above the background level of normal translational errors. In addition to antizyme, frameshifting is also involved in the decoding of some bacterial and yeast genes and especially in many mammalian Retroviruses and Coronaviruses, plant viruses and bacterial insertion sequences (Atkins et al., 1999) . The site of frameshifting in both mammalian antizyme 1 and 2 mRNAs is UCC UGA, where quadruplet translocation occurs at UCCU (underlined) to shift reading to the +1 frame, immediately before the UGA stop codon of the initiating frame (Matsufuji et al., 1995; Ivanov et al., 1998a) . For the frameshifting to occur with an ef®ciency of 20% or more, it is important that the 3¢ base of the quadruplet is the ®rst base of a stop codon. Other important features are a pseudoknot just 3¢ of the shift site and a speci®c sequence 5¢ of the shift site (Matsufuji et al., 1995; Ivanov et al., 1998a) . A pseudoknot 3¢ of the shift site is a common stimulator for eukaryotic ±1 frameshifting, but the synthesis of antizyme is the only known case utilizing +1 frameshifting.
Comparative analysis of RNA sequences from different organisms is informative about important features and the different options selected by evolution. Since most of the known examples of programmed frameshifting are in viruses or chromosomal mobile elements, the opportunity for comparison of frameshift cassettes in divergent organisms where the time of divergence can be approximated is limited. A start has been made with the frameshifting required for bacterial release factor 2 expression (Persson and Atkins, 1998) , but antizyme provides the ®rst opportunity for such a comparison in eukaryotes. Antizyme genes in genetically tractable lower eukaryotes would be helpful for understanding the functionally important interactions responsible for autoregulatory programmed frameshifting.
Identi®cation of an antizyme gene in Schizosaccharomyces pombe A search for DNA sequences encoding protein sequences homologous to Drosophila melanogaster antizyme (Ivanov et al., 1998b) and Homo sapiens antizyme 1 identi®ed the same S.pombe anonymous cDNA clone (DDBJ/EMBL/GenBank accession No. D89228). The similarity is limited (~10% identity, 24% similarity to both human antizyme 1 and D.melanogaster antizyme); however, it is highest in regions that are most highly conserved among the previously identi®ed antizymes ( Figure 1A ). Closer examination of the cDNA nucleotide sequence provided further evidence that it encodes an S.pombe homolog of antizyme. The initiating AUG codon for the ORF that is similar to higher eukaryotic antizymes (ORF2 of those genes) is not the 5¢-most AUG in this cDNA. In fact, there are eight AUGs closer to the 5¢ end. The ®rst or the second AUGs would initiate translation of an ORF (ORF1) that overlaps the longer downstream ORF (ORF2) such that a +1 translational frameshifting event in the overlap would generate a protein product analogous to the products of antizyme genes from higher eukaryotes. Furthermore, the last 12 nucleotides of ORF1 (UGG-UGC-UCC-UGA) are identical to the last 12 nucleotides of mammalian antizyme 1 ORF1s, including the frameshift site. Eleven of these 12 nucleotides are identical to the corresponding regions of all previously identi®ed antizyme genes ( Figure 1B ). Previous experiments with the mammalian frameshift sequence tested in S.pombe have shown that this short 12 nucleotide sequence, by itself, is suf®cient to stimulate measurable levels (up to 0.5%) of +1 frameshifting (Ivanov et al., 1998c) . To con®rm the ORF con®guration of the putative S.pombe antizyme gene, a region corresponding to the two overlapping ORFs plus~80 nucleotides of the 5¢ UTR and 370 nucleotides of the 3¢ UTR, was ampli®ed from both S.pombe genomic DNA and a cDNA library. The sequence of the ampli®ed DNA con®rmed that there are indeed two overlapping ORFs with the deduced con®guration. This sequence (DDBJ/EMBL/GenBank accession No. AF217277) differs from the previously sequenced cDNA clone by three nucleotides (two in the coding region and one in the 3¢ UTR); one changes an alanine codon to proline, another is a silent mutation within a proline codon. Since the sequences from the cDNA library and genomic DNA are identical, we conclude that the differences with clone No. D89228 are most likely due to strain variation. This gene contains no introns within the ampli®ed region.
The S.pombe protein was tested for antizyme activity using a gene fusion with glutathione S-transferase (GST). In this construct, ORF1 and ORF2 of antizyme are fused in-frame by deleting the T nucleotide that encodes U of the stop codon of ORF1. This GST±antizyme fusion gene was expressed in Escherichia coli and the protein was puri®ed by af®nity chromatography. ODC inhibitory activity was tested by incubating the recombinant antizyme protein with an S.pombe crude extract and then assaying the mixture for ODC activity. The results ( Figure 2) show that the recombinant protein can inhibit S.pombe ODC. GST alone (1 mg) does not inhibit S.pombe ODC (data not shown). In light of these results, the S.pombe gene will be called S.pombe ODC antizyme (SPA). Interestingly, the S.pombe ODC was also inhibited by mouse antizyme 1 and antizyme 2 (both expressed as GST fusions); however, the yeast fusion protein did not inhibit mouse ODC (data not shown).
Deletion and overexpression of SPA Although the effects of overexpression of antizyme on cellular physiology have been tested previously in mammalian cells, the physiological changes associated with complete absence of antizyme activity have not yet been investigated because of the complication of multiple antizymes. The single S.pombe antizyme provides the chance to explore a knockout. SPA deletion strains were I.P. Ivanov et al. generated by replacing the two ORFs of the gene with the ORFs of either URA4 or LEU2 (see Materials and methods). Complete deletion of SPA (both ORFs) did not affect the viability of S.pombe cells in rich (YE) or minimal (MM) media. Temperature had no differential effect on mutant and wild-type cell growth. Similarly, the growth rates, mating ef®ciencies and overall morphology of the knockout strains are apparently indistinguishable from those of wild-type cells (results not shown).
In wild-type S.pombe cells the most abundant polyamine is spermidine followed by putrescine ( Figure 3 ). Spermine and cadaverine are found in much smaller amounts. This distribution of polyamine content is very similar to that in other fungi for which polyamine concentrations have been measured (for references, see review by Tabor and Tabor, 1985) . The effect of SPA deletion on cellular polyamine contents was examined in both exponentially growing and stationary phase cells ( Figure 3 ). The cellular concentrations of putrescine, spermidine and cadaverine (but not spermine) were higher in the knockout strains than in wild-type cells. The greatest effect was seen on putrescine and cadaverine content, with smaller effects on spermidine, presumably because eukaryotic ODC activity directly catalyzes decarboxylation of both ornithine and lysine to produce putrescine and cadaverine, respectively (Pegg and McGill, 1979) , but subsequent regulatory events affect homeostasis of spermidine and spermine. The effect of inactivating antizyme on the polyamine contents in exponentially growing cells is modest (<2-fold in all cases). The effect becomes very pronounced in cells in stationary phase with up to 40-and 10-fold increases of putrescine and cadaverine contents, respectively, in the knockout strains.
To test overexpression of SPA, two versions of the gene were cloned into pREP3 expression vector behind a strong, thiamine-repressible promoter (nmt1). One had the wild- type SPA sequence while in the second, ORF1 and ORF2 are fused in-frame. SPA wild type and an SPA deletion strain were transformed with each of the overexpression constructs. Derepression of the nmt1 promoter is a gradual process since it requires dilution of the intracellular pool of thiamine (the repressor) through cell division. After 2.5 days of exponential growth under derepressed conditions, yeast strains transformed with either SPA overexpression construct show signi®cant increases in doubling time ( Figure 4A ). The growth inhibition is greater with the construct expressing the in-frame version of SPA and after prolonged incubation (5±7 days); these cells cease growth and accumulate in G 1 as determined bȳ ow cytometry (data not shown). The fact that the inframe overexpression construct, which differs by a single nucleotide from the wild-type construct, confers a more severe phenotype is consistent with the hypothesis that translational frameshifting is required for expression of SPA. The growth phenotype associated with SPA overexpression is only partially relieved by adding 100 mM putrescine to the media (1 mM had no further effect) (data not shown). To see whether the slower growth is correlated with aberrant polyamine levels the polyamine contents of the deletion strain carrying in-frame SPA overexpression vector were measured under derepressed and repressed conditions, in both cases after 2 days of exponential growth ( Figure 4B ). As expected, overexpression of SPA results in signi®cant reduction in the intracellular levels of all four polyamines. After longer (4±5 days) incubation under derepressed conditions, no putrescine and cadaverine can be detected (data not shown).
Translational frameshifting during expression of SPA Previously, we developed an assay for measuring antizyme translational frameshifting in both S.cerevisiae (Matsufuji et al., 1996) and S.pombe (Ivanov et al., 1998c) . Brie¯y, the nucleotide sequence to be assayed is inserted between GST and lacZ, such that ORF1 of the assayed sequence is fused in-frame to GST, while ORF2 is fused in-frame to lacZ. b-galactosidase activity provides a measure of frameshifting ef®ciency. To determine whether translational frameshifting occurs in the overlap of ORF1 and ORF2 of SPA, a region of SPA including all but the ®rst codon of ORF1 plus 180 nucleotides downstream of the ORF1 stop codon was tested. +1 frameshifting occurred at 2.2% compared with a construct in which ORF1 and ORF2 are fused in-frame. This result is consistent with +1 frameshifting being crucial for expression of SPA.
Previous experiments have shown that the frameshift cassette of mammalian antizyme 1 can direct ef®cient +1 frameshifting when tested in S.pombe. The reverse experiment was conducted here. The SPA gene was translated in vitro in rabbit reticulocyte lysate and its resulting frameshift ef®ciency measured. With no addition of polyamines, frameshifting ef®ciency is~1.5%. Addition of spermidine to the translation mixture to a ®nal concentration of 1 mM results in a 3.7-fold increase in frameshifting to~5.5%, a level even higher than that observed in the endogenous system in vivo (autoradiogram not shown). The observed ef®ciency of frameshifting with the SPA frameshifting cassette in vivo in S.pombe is signi®cantly more than that expected from its limited nucleotide similarity to the antizyme frameshift sites of higher eukaryotes. This prompted a search for additional stimulatory elements within the SPA frameshift cassette.
The following experiments were done in a strain carrying deletion of SPA (high polyamines) because it gives higher frameshifting and higher b-galactosidase activity in general; however, we obtained similar ratios for mutant to wild-type frameshifting ef®ciency in a strain with the intact SPA gene. Deleting 5¢ sequences up to the third to last sense codon of ORF1 has little or no effect on frameshifting ef®ciency. Deleting all but the last sense codon (UCC) of ORF1 leads to a 4-to 5-fold reduction in frameshifting ef®ciency ( Figure 5A ). This implies that the conservation of the six nucleotides 5¢ of the UCC-UGA frameshift site is due to their importance for stimulating +1 frameshifting. It also suggests that no additional ORF1 sequences of SPA stimulate the +1 recoding event. The 180 nucleotide 3¢ region was searched for possible structure by computer RNA folding algorithms plus visual inspection. The algorithms predicted several minimal structures in that region. 3¢ deletion constructs (constructs del.3,3¢±81,3¢) tested the importance of any putative structure on the frameshifting ef®ciency. The results ( Figure 5B and C) show that all of these deletions lead to a signi®cant (~10-fold) reduction in +1 frameshifting, indicating the presence of a major 3¢ stimulatory element in the 180 nucleotide region immediately following the frameshift site of SPA. However, the results indicate that none of the putative RNA structures in this region are suf®cient for the activity of this element. Several additional 3¢ deletions delineated the boundaries of this stimulatory element from the frameshift site to 150 and 180 nucleotides downstream (since construct del.150,3¢ stimulates 5.5-fold more +1 frameshifting than del.129,3¢, 150 nucleotides downstream probably contain most of the 3¢ stimulator).
In the experiments described above, two of the characteristics of the autoregulatory circuit of mammalian antizyme 1 were con®rmed: SPA inhibition of ODC and the +1 translational frameshifting. The key question left is whether the recoding event is responsive to polyamine levels in cells. As shown above, overexpression of SPA leads to signi®cant reduction of polyamine levels in S.pombe. An SPA + strain was co-transformed with an SPA wild-type overexpressing plasmid (cells overexpressing wild-type SPA grow slowly but continuously) and a construct that monitors the +1 frameshifting from an SPA frameshift sequence. The +1 frameshifting was compared with that in SPA non-overexpressing cells (in both cases frameshifting was measured relative to in-frame control). The results ( Figure 6 ) show a signi®cant reduction (6.5-fold) in frameshifting ef®ciency in SPA-overproducing cells that correlates with a decrease of polyamine content (4.5-fold for putrescine and 3.9-fold for spermidine). This indicates that polyamines modulate the frameshifting ef®ciency of SPA. An alternative but less likely possibility is that SPA overexpression reduces frameshifting because high levels of SPA transcript titrate some factor limiting for frameshifting.
The SPA frameshift signals direct 2-fold more frameshifting in Dspa::LEU2 cells (4.4%) than in SPA + cells (in both cases the measurement is done during stationary phase); however, the relatively high standard deviations for both measurements make it dif®cult to draw ®rm conclusions from this particular result.
A search of Caenorhabditis elegans expressed sequence tag (EST) sequences with mammalian antizyme 1 sequence identi®ed 20 clones. These sequences could be deconvoluted into a contiguous cDNA sequence. Primers designed on the basis of this sequence were used to PCR amplify and subclone this cDNA from a C.elegans cDNA library. The sequence of the subcloned cDNA was con®rmed (DDBJ/EMBL/GenBank accession No. AF217278); the subsequently released genomic sequence of this C.elegans gene (DDBJ/EMBL/GenBank accession No. AF040659) con®rms our cDNA data. The amino acid sequence deduced from the cDNA sequence revealed that the longer ORF has similarity to previously reported antizyme sequences (overall 27% identity, 39% similarity to human antizyme 1; 19% identity, 34% similarity to Drosophila antizyme). These similarities are higher than that of SPA to these two antizyme genes and again are concentrated in the regions most highly conserved among previously identi®ed antizymes ( Figure 1A ). Just like mammalian antizymes, the longer ORF (ORF2) lacks an appropriate in-frame initiation codon, and expression could be provided by initiation in a short upstream overlapping ORF (ORF1) leading to +1 ribosomal frameshifting in the overlap. The putative C.elegans antizyme frameshift site (the nucleotides proximal to the end of ORF1) has 18 of 26 nucleotides identical to the consensus sequence for antizyme frameshift sites ( Figure 1B) .
Frameshifting for expression of C.elegans antizyme was investigated in heterologous systems. Two constructs containing the entire antizyme cDNA, one with the wildtype sequence and one with a single nucleotide deletion that fuses ORF1 to ORF2 in-frame (in-frame control), were transcribed in vitro and the RNA was translated in rabbit reticulocyte lysate. The products were examined by SDS±PAGE (Figure 7) . The main product from both constructs has an apparent M r of 21 kDa, slightly greater than the predicted M r of 17.7 kDa [aberrant, slower than expected, mobility is observed with antizyme proteins from other species (Ivanov et al., 1998a) ]. From the ratio of wild-type to in-frame product, we estimate that the ef®ciency of frameshifting of C.elegans antizyme in reticulocyte lysate is~0.8%, which is somewhat lower than SPA frameshifting in the same system. Addition of spermidine to the translation reactions almost doubles the ef®ciency of frameshifting to~1.5% (the exact numbers are not easy to determine because of dif®culty in de®ning background values). The frameshifting properties of C.elegans antizyme mRNA were also tested in vivo in S.pombe cells. A sequence including all but the ®rst codon of ORF1 plus 180 nucleotides downstream was inserted between GST and lacZ of the PIU-LAC plasmid. Comparison of the b-galactosidase activity of cells (Dspa::LEU2 strain) transformed with the wild-type construct and the in-frame control constructs indicated 3.5% +1 frameshifting. From the frameshifting observed in the heterologous systems, as well as the sequence considerations discussed above, we conclude that expression of this C.elegans gene requires ribosomal frameshifting.
Searching the EST database with the newly discovered C.elegans antizyme identi®ed antizyme orthologs in four other nematode species. In two cases (Necator americanus and Haemonchus contortus), the cDNA sequences in the database were suf®cient to make contigs of the complete coding regions. In the other two cases [Onchocerca volvulus (DDBJ/EMBL/GenBank accession No. AF217279) and Pristioncus paci®cus (DDBJ/EMBL/ GenBank accession No. AF217280)] the complete cDNA sequences were obtained by PCR amplifying and sequencing the full genes from cDNA libraries. As with the previously identi®ed eukaryotic antizyme genes, the ORF con®guration of the newly found nematode orthologs implies the necessity for +1 frameshifting for synthesis of full-length protein.
The C.elegans antizyme mRNA frameshift site UUU-UGA is unique, differing from the UCC-UGA of previously known antizyme mRNAs. The C.elegans antizyme gene shares this feature with N.americanus and H.contortus but not with P.paci®cus and O.volvulus antizymes. The phylogenetic tree of nematode antizyme protein sequences matches exactly the phylogenetic relationship (Blaxter, 1998) of the nematodes expressing them, indicating that these gene sequences are the result of divergent evolution within the nematode lineage (data not shown). These results also show that the UUU-UGA frameshift site evolved after the last common ancestor of P.paci®cus and C.elegans but before the divergence of C.elegans, N.americanus and H.contortus (probably 450± 500 million years ago).
The ability of UUU-UGA sequence to direct +1 frameshifting was further tested in a mammalian system in the context of the mammalian antizyme mRNA (i.e. in the presence of the 3¢ RNA pseudoknot and 5¢ stimulator). A BMV-coat-protein±antizyme 1 gene fusion construct, which has a TCC-TGA to TTT-TGA substitution, was transcribed and then translated in a rabbit reticulocyte lysate. Eleven percent frameshift ef®ciency was seen in the absence of exogenously added polyamines, 2.2 times the ef®ciency seen with the UCC-UGA transcript. The frameshift ef®ciency becomes 18% when 0.6 mM spermidine is added, which is 1.3 times that with the wild type (Matsufuji et al., 1995) . Similar results were obtained in cultured mammalian (Cos7) cells transfected with TTT-TGA mutant construct, the frameshift being higher than that of wild-type construct in both high-and lowpolyamine conditions (our unpublished results). These results demonstrate that the putative C.elegans frameshift site (UUU-UGA) is, if anything, shiftier than UCC-UGA in the antizyme 1 context and is subject to polyamine stimulation.
The results presented show that the yeast S.pombe has a homolog of mammalian antizyme. This is the ®rst documented example of antizyme-type regulation of ODC in a lower eukaryote.
Deleting SPA from the yeast genome has no detectable effect on viability or any other overt phenotypic effect but, as expected, it results in altered accumulation of polyamines in the cell. Interestingly, the effect is most pronounced in cells in stationary phase, where the knockout cells accumulate up to 40 times more putrescine than wild-type counterparts. This compares with a <2-fold increase of putrescine in exponentially growing cells. A likely explanation for this observation is that the usual rate of ornithine decarboxylation in exponentially growing cells is close to capacity given`normal' concentrations of substrate, enzyme and product. At the same time, all newly synthesized polyamines are continuously diluted through Fig. 6 . Effect of polyamine depletion on SPA +1 frameshifting. Polyamine depletion is achieved by overexpression of the wild-type version of SPA. The same cultures were assayed both for frameshifting and polyamine content. Numbers above columns indicate fold reduction of frameshifting and polyamine content compared with cells that do not overexpress SPA. Antizyme genes in S.pombe and C.elegans cell growth and division at a rate that is almost identical to the rate of maximum capacity synthesis. Cells in stationary phase can no longer dilute newly synthesized polyamines, and more importantly lack an effective antizymeindependent mechanism of shutting off ODC. This suggests that SPA is the primary regulator of ODC activity in S.pombe, not only during cell growth (short term regulation) but also in non-dividing cells (longer term regulation).
Overexpression of SPA (5±7 days derepression) leads to complete depletion of intracellular putrescine. This result implies that in S.pombe ornithine decarboxylation is the only source of putrescine synthesis (the pathway from arginine via agmatine is not utilized). The complete depletion of cadaverine in SPA overexpressing cells suggests that ODC is the only enzyme in S.pombe that can decarboxylate lysine, which is also the case in rat tissues (Pegg and McGill, 1979) .
It is somewhat perplexing that addition of putrescine to the media leads to only partial relief of the growth phenotype associated with SPA overexpression. There are two likely explanations. (i) Perhaps S.pombe imports putrescine poorly. (ii) Alternatively, like the mammalian system, maybe SPA inhibits not only ODC but also the polyamine transporter. Further experiments will help to distinguish between these two models.
It is unclear how widespread the antizyme gene is within the fungal kingdom. We have identi®ed and cloned antizyme homologs from two other ®ssion yeasts (Schizosaccharomyces octosporus and Schizosaccharomyces japonicus) and from two distantly related fungi (Botryotinia fuckeliana and Emericella nidulans) (our unpublished results). The antizyme frameshift site of the latter two fungi has evolved in a unique way different from all other known antizymes, but nevertheless even these two distantly related fungi have conserved the autoregulatory +1 frameshifting. The fact that the yeast S.pombe has an antizyme gene suggests the possibility that the higher eukaryotic metazoans may all have an antizyme gene.
The only previously reported antizyme activity in unicellular organisms is from E.coli, but recent analyses suggest that E.coli does not have a true antizyme (Ivanov et al., 1998d) . This makes SPA the ®rst bona ®de antizyme in a unicellular organism.
The remarkable similarity of the core sequence important for antizyme frameshifting from S.pombe to humans could be due to convergent or divergent evolution. The near identity of this sequence in worms, Drosophila, Xenopus, zebra®sh and humans argues against convergent evolution, as if antizyme frameshifting arose in a common ancestor perhaps more than one billion years ago.
Three cis-acting RNA elements are known to stimulate mammalian antizyme 1 frameshifting. One is a 50 nucleotide sequence immediately 5¢ of the shift site (Matsufuji et al., 1995; our unpublished results) . A second stimulator is the UGA stop codon of ORF1 and the third is an RNA pseudoknot starting 3 nucleotides 3¢ of the UGA stop codon. Among frameshift sites of the previously identi®ed antizymes from mammals all the way to Drosophila, there is substantial similarity in the sequences immediately 5¢ of the shift site. Sixteen of the last 18 nucleotides of ORF1 are completely conserved in these genes. Schizosaccharomyces pombe and C.elegans antizymes have 9 of 9 and 6 of 9 (14 out of 19 in O.volvulus) nucleotides identical to the consensus, respectively. For the 5¢ sequences, generally, the more distantly related two antizymes are, the more the similarity is con®ned to the 3¢ end of that region. Our SPA ORF1 deletion data show that mutation of nucleotides that are part of the 5¢ consensus sequence leads to reduced frameshifting ef®ciency. This is another indication that conservation of nucleotide sequence in this region is because of its importance for stimulating ef®cient +1 frameshifting. It is quite striking that in all antizyme gene sequences identi®ed so far, including a number of unpublished ones, ORF1 ends with a UGA stop codon. This is particularly surprising since any of the other two stop codons can substitute for UGA to stimulate antizyme 1 frameshifting, although slightly less ef®ciently, in vitro (Matsufuji et al., 1995) and in vivo (our unpublished results).
The 3¢ pseudoknot that stimulates frameshifting in antizyme 1 is highly conserved in all known vertebrate antizymes, including mammalian antizyme 2 ( Figure 1B) . None of the invertebrate antizyme mRNAs identi®ed so far, including those presented here, has a sequence in the equivalent region that can be simply folded to a comparable RNA structure. However, sequences immediately 3¢ of the frameshift site are conserved between invertebrates and vertebrates. The conservation of this region between Drosophila and the vertebrate counterparts has already been noted (Ivanov et al., 1998b) . The C.elegans antizyme gene contains the sequence YGYCCCYCA (Y = pyrimidine) in this region, which is identical to the consensus. The antizyme genes from the other four nematodes also have a similar sequence ( Figure 1B) . The signi®cance of this similarity is not clear [in fact, sequences in this region appear to play no role in antizyme 1 in vitro frameshifting outside of the RNA pseudoknot context (Matsufuji et al., 1995) ].
Only two examples are known where RNA elements 3¢ of the frameshift site stimulate +1 frameshifting. One is the RNA pseudoknot of mammalian antizyme 1 and the second is a short RNA sequence immediately following the frameshift site of Ty3 (Farabaugh et al., 1993) . Additional examples would be very helpful in deciphering the role such elements play in the mechanism of +1 frameshifting. It is currently not known how many and which of the invertebrate antizyme genes contain 3¢ frameshift stimulators. The results presented here show that an S.pombe 3¢ stimulator enhances frameshifting 10-fold. This stimulator appears completely different from the 3¢ RNA pseudoknot in vertebrates. Our deletion experiments indicate that none of the predicted RNA structures contained within the minimally required 3¢ region [up to 150±180 nucleotides downstream of the frameshift site ( Figure 5C )] are suf®cient to confer the stimulatory effect. The SPA 3¢ stimulator may act directly through sequence or may have an unusual RNA structure involving non-Watson±Crick base pairing. More detailed mutagenesis combined with phylogenetic analysis would be required to discern the nature of the 3¢ stimulator of SPA.
The nematode antizymes were analyzed for the presence of possible 5¢ or 3¢ stimulators¯anking the core frameshift site. Computer RNA folding programs did not identify any potentially interesting structure. More importantly, phylogenetic analysis with the ®ve identi®ed nematode antizymes failed to identify any conservation of primary RNA sequence (or for that matter potential secondary structure) outside of the core region that is shared between two or more members. This could indicate that no such extra cis-acting stimulators exist in nematode antizymes or that they are located in a very different place within the mRNA, for example the 3¢ untranslated region (the latter suggestion is not supported by our sequence analysis).
A common mechanism for frameshifting is re-pairing of the peptidyl tRNA in the new reading frame. However, an alternative mechanism whereby the peptidyl tRNA merely occludes the ®rst base of the next codon, has been documented for yeast Ty3 frameshifting (Farabaugh et al., 1993) . Results of experiments with some mutants of the mammalian antizyme 1 shift site pointed to an occlusion mechanism (Matsufuji et al., 1995) . However, the mechanism with the wild-type, UCC-UGA, shift site is not clear. For C.elegans antizyme the UUU-UGA sequence would be an obvious candidate for a re-pairing since Phe-tRNA could pair perfectly with UUU in both frames. But with UCC-UGA the Ser-tRNA ®rst reading UCC could at best pair two out of three with CCU. This important problem warrants further investigation.
The frameshift ef®ciency of SPA frameshift site is lower than that observed with mammalian antizyme 1 even when both are tested in the same organism (S.pombe) [for the frameshift ef®ciency of antizyme 1 cassette in S.pombe, see Ivanov et al. (1998c) ]. It is possible that the observed ef®ciencies for S.pombe antizyme are arti®cially low because the constructs do not include all the cis-acting stimulatory elements. On the other hand there is no reason why a lower level of frameshifting does not correctly re¯ect the evolved balance with the other characteristics of the complex system such as relative protein stabilities.
Like other core cellular processes, the antizyme polyamine regulatory scheme is conserved from yeast S.pombe to human. It is not obvious why this very special mechanism is so exquisitely preserved over vast evolutionary time. Perhaps there is another whole aspect to the system that our experiments do not yet detect. From this viewpoint it would seem very important to exploit the genetics systems of S.pombe and C.elegans to understand more thoroughly the physiological effects of perturbing the antizyme system.
The SPA gene was ampli®ed using the following primers: 5¢-CAAAACAAGTTTTCATTATTGGTTTTTTTTAAATCAATCCCC (sense) and 5¢-CGTAAATCCAATCTAAATTTAATCTTCAACTAA-ATCATGAAAAGCCTC (antisense). The S.pombe cDNA library used as a template in the ampli®cation was kindly provided by R.Rowley (University of Utah). The C.elegans antizyme gene was ampli®ed using the following primers: 5¢-CCCAGGAATTCCTCGAGTATTTTGA-GTATAATTTTAC (sense) and 5¢-CGGCCGCTCGAGTTAGACCTT-GTAGCTCATGATG (antisense). This same ampli®ed DNA was used to make the constructs for in vitro transcription and translation of C.elegans antizyme by cloning it into pTZ18U plasmid using the SacI and HindIII sites incorporated in the two primers. The in-frame construct was made using a two-step PCR. The cDNA sequences of O.volvulus and P.paci®cus antizyme genes were obtained by performing 5¢ and 3¢ RACE PCR with cDNA libraries, which were kindly provided by Ralf Sommer (P.paci®cus) and Susan Haynes (O.volvulus). The SPA overexpression constructs were made by amplifying the gene with the primers 5¢-GCATCCGAATTCCCAAATCCAAGCATCATACGCC (sense) and 5¢-GCATCCGGATCCGCCAGTGTTCTTACTTTGAGA-TGC (antisense), and then inserting BamHI-digested product between the MscI and BamHI sites of pREP3 plasmid. The in-frame construct was made by two-step PCR and subsequently all in-frame SPA constructs described below were made by one-step PCR using this plasmid's DNA as a template. To make the constructs for frameshift assays in S.pombe, DNA fragments with a given nucleotide length (as described in the main text), were ampli®ed from both the SPA and C.elegans antizyme constructs described above. These fragments were then cloned between the KpnI and BstEII sites of PIU-LAC plasmid (Ivanov et al., 1998c) . The PCR primers included an`AC' spacer between the 5¢ cloning site (BstEII) and the antizyme sequences in order to correct the reading frame. The in vivo frameshifting assays in S.pombe (strains ura4-D18 leu1-32 ade6-M216 h ± and Dspa::LEU2 ura4-D18 leu1-32 ade6-M216 h ± ) were done as described (Ivanov et al., 1998c) . The plasmid for GST±SPA expression was made by PCR amplifying SPA (all but the ®rst codon of ORF1 through the downstream ORF2) from an in-frame template and cloning the product into the EcoRI and XhoI restriction sites of pGEX-5X-3 plasmid. The antizyme frameshift site in the BMV-coatantizyme fusion construct (C3NE) (Matsufuji et al., 1995) was mutated with a two-step PCR. To generate the two knockout strains, Dspa::URA4 and Dspa::LEU2, both ORFs of SPA were replaced exactly with the ORF of either URA4 or LEU2. To accomplish this, two pairs of primers ampli®ed URA4 and LEU2 such that 50±60 nucleotides, which normallȳ ank the two ORFs of SPA,¯ank the ORFs of the two genes. The ampli®ed DNA products were gel puri®ed and 2 mg of each were used to electroporate into ura4-D18 leu1-32 ade6-M216 h ± cells. URA + and LEU + transformants were selected by growth on URA ± and LEU ± media, respectively. PCR screen and partial sequencing, with primers¯anking the regions used for the homologous recombination, con®rmed the SPA disruptions. All DNA clones were sequenced with automated sequencing machines (ABI 100).
Schizosaccharomyces pombe ODC active crude extracts were prepared as follows: S.pombe (strain 1519, leu1-32, h ± ) provided by R.Rowley was grown to OD 600 0.7 in 50 ml of minimal media + LEU. Ten milligrams of lysing enzymes (Sigma) were added, followed by continued incubation for 30 min at 30°C. Cells were harvested and washed once with cold homogenization buffer [25 mM Tris±HCl pH 7, 0.25 M sucrose, 1 mM dithiothreitol (DTT), 20 mM pyridoxal-5-phosphate, 2 mM EDTA] then resuspended in 0.75 ml of homogenization buffer. Cells were broken open and the lysate was clari®ed by centrifugation at 10 000 r.p.m. for 15 min at 4°C. Extracts were dialyzed overnight in dialysis buffer (25 mM Tris± HCl pH 7.4, 1 mM DTT, 20 mM pyridoxal-5-phosphate, 0.1 mM EDTA). A volume of 25 ml of extract was used for each ODC assay. ODC activity was assayed by measuring the release of 14 CO 2 from L-[1-14 C]ornithine (Amersham) as described (Nishiyama et al., 1988) . Each reaction took 1 h. Pre-incubation of S.pombe extract with 0.1 mM di¯uoromethyl ornithine (DFMO) for 15 min led to >99% inhibition of 14 CO 2 release.
The cells were collected by centrifugation, washed twice with 1 ml of phosphate-buffered saline (PBS) and then the pellet was frozen at ±80°C until use. The pellet was resuspended in 0.1 ml of PBS. An aliquot of the suspension was mixed with an equal volume of 8% perchloric acid, vortexed for 1 min, kept on ice for 5 min and centrifuged at 15 000 r.p.m., 4°C for 5 min. Ten microliters of the supernatant were subjected to polyamine analysis using¯uorometry on high-performance liquid chromatography as described previously (Murakami et al., 1989) . Protein concentrations were determined with the BCA protein assay kit (Pierce).
The experiments with the BMV-coat-antizyme fusion constructs were performed as described previously (Matsufuji et al., 1995) . All other plasmid DNA templates were prepared using QIAGEN Miniprep Kit and then digested with HindIII. Transcripts for SPA in vitro translation were made from PCR templates that had a T7 promoter incorporated into the PCR primers. Linearized DNA (1 mg) was used as a template for in vitro transcription with Ambion MEGAshortscript TM T7 Kit. The DNasetreated RNAs were recovered and resuspended in 40 ml of RNase-free water. One microliter of each speci®ed transcript suspension was used in each in vitro translation reaction [0.5 ml of 1 mM amino acid mix ±Met, 7 ml of reticulocyte lysate (Promega), 0.5 ml of [ 35 S]Met (Amersham)] to a total volume of 10 ml. The reactions were stopped by adding 1 ml of RNase (10 mg/ml). The frameshift ef®ciencies were quanti®ed as described (Ivanov et al., 1998a) . Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis. Introduction hnRNP A1 is an RNA-binding protein that contains two RNA-binding domains (RBDs) and a glycine-rich domain responsible for protein±protein interaction. It is involved in pre-mRNA splicing and transport of cellular RNAs (reviewed by Dreyfuss et al., 1993) . It is predominantly located in the nucleus, but also shuttles between the nucleus and the cytoplasm (Pin Äol-Roma and Dreyfuss, 1992) . The signal that mediates shuttling has been identi®ed as a 38 amino acid sequence, termed M9, located near the C-terminus of hnRNP A1 between amino acids 268 and 305 (Michael et al., 1995; Siomi and Dreyfuss, 1995; Weighardt et al., 1995) . Yeast two-hybrid screening with M9 as bait resulted in the discovery of a novel transportin-mediated pathway for nuclear import of hnRNP A1 (Pollard et al., 1996; Fridell et al., 1997; Siomi et al., 1997) . The function of the cytoplasmic hnRNP A1 has not been well de®ned. Studies have shown that cytoplasmic and nuclear hnRNP A1 exhibit different RNA-binding pro®les. Cytoplasmic hnRNP A1 is capable of high-af®nity binding to AU-rich elements that modulate mRNA turnover and translation (Hamilton et al., 1993 (Hamilton et al., , 1997 Henics et al., 1994) . It has also been shown to promote ribosome binding to mRNAs by a cap-mediated mechanism, and prevent spurious initiation at aberrant translation start sites (Svitkin et al., 1996) .
MHV belongs to the Coronaviridae family of positivesense, single-stranded RNA viruses. MHV replication and transcription occur exclusively in the cytoplasm of infected cells via the viral RNA-dependent RNA polymerase (RdRp) (reviewed by Lai and Cavanagh, 1997) . Initially, the 5¢-most gene 1 of the viral genome is translated into the viral RdRp, which then replicates the viral genomic RNAs into negative-strand RNAs. Subsequently, the negative-strand RNAs are used as templates to transcribe mRNAs, which include a genomic-sized RNA and a nested set of subgenomic mRNA transcripts, all with an identical 5¢ non-translated leader sequence of 72±77 nucleotides and 3¢ co-terminal polyadenylated ends. The subgenomic mRNA transcription of MHV utilizes a unique discontinuous mechanism in which the leader sequence, often derived from a different molecule, is fused to RNAs at the intergenic (IG) sites (i.e. transcription initiation site) to generate subgenomic mRNAs (Jeong and Makino, 1994; Liao and Lai, 1994; Zhang et al., 1994) . The exact mechanism of how these mRNAs are made is still controversial. However, it has been shown that the process of discontinuous RNA transcription is regulated by several viral RNA elements, including the cis-and trans-acting leader RNA Zhang et al., 1994) , IG sequence (Makino et al., 1991) and 3¢-end untranslated sequence (Lin et al., 1996) . There is considerable biochemical evidence suggesting possible direct or indirect interactions between the various RNA regulatory elements. hnRNP A1 binds MHV negative (±)-strand leader and IG sequences (Furuya and Lai, 1993; Li et al., 1997) . Site-directed mutagenesis of the IG sequences demonstrated that the extent of binding of hnRNP A1 to the IG sequences correlated with the ef®ciency of transcription from the IG site (Zhang and Lai, 1995; Li et al., 1997) . Immunostaining of hnRNP A1 showed that hnRNP A1 relocated to the cytoplasm of MHV-infected cells, where viral RNA synthesis occurs (Li et al., 1997) . hnRNP A1 also mediates the formation of a ribonucleoprotein complex containing the MHV (±)-strand leader and IG sequences . These results suggest that hnRNP A1 may serve as a protein mediator for distant RNA regions to interact with each other. Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus The EMBO Journal Vol. 19 No. 17 pp. 4701±4711, 2000 ã European Molecular Biology Organization Many cellular proteins, including calreticulin (Singh et al., 1994) , polypyrimidine tract-binding protein (PTB) (Hellen et al., 1994; Wu-Baer et al., 1996) , La protein (Pardigon and Strauss, 1996), Sam68 (McBride et al., 1996) , poly(rC)-binding protein (Parsley et al., 1997) and nucleolin (Waggoner and Sarnow, 1998) , have been implicated to be involved in viral RNA transcription or replication. In addition to MHV, hnRNP A1 has also been reported to interact with human cytomegalovirus immediate-early gene 2 protein, which plays an important role in the regulation of virus replication (Wang et al., 1997) . Furthermore, a yeast protein related to human core RNA splicing factors, Lsm1p, has been shown to be required for the ef®cient replication of brome mosaic virus RNA (Diez et al., 2000) . Recently, Reddy and colleagues demonstrated an inhibition of HIV replication by dominant-negative mutants of Sam68 (Reddy et al., 1999) . However, none of these cellular proteins has been shown experimentally to participate directly in RNA-dependent RNA synthesis.
In order to demonstrate the involvement of hnRNP A1 in MHV RNA replication and transcription, we established several DBT cell lines stably expressing either the wildtype (wt) hnRNP A1 or a C-terminus-truncated mutant lacking the M9 sequence and part of the glycine-rich domain. We showed that the mutant hnRNP A1, which was localized predominantly in the cytoplasm, exhibited dominant-negative effects on viral genomic RNA replication and subgenomic mRNA transcription. In contrast, overexpression of the wt hnRNP A1 accelerated the synthesis of all viral RNAs. Our results provide strong evidence that hnRNP A1 is directly or indirectly involved in MHV RNA synthesis in the cytoplasm and that the C-terminal part of the protein is important for its function. This ®nding thus reveals a novel function for hnRNP A1 in the cytoplasm.
Characterization of stable cell lines expressing the wt and a C-terminus-truncated hnRNP A1 To explore a potential role for hnRNP A1 in MHV RNA synthesis, we established murine DBT cell lines stably expressing the Flag-tagged wt hnRNP A1 (DBT-A1) or a mutant hnRNP A1, which has a 75 amino acid deletion from the C-terminus (DBT-A1DC) ( Figure 1A ). This mutant lacks part of the glycine-rich domain and the M9 sequence responsible for shuttling hnRNP A1 between the nucleus and the cytoplasm. Immunoblot of the whole-cell lysates with an anti-Flag antibody detected a 34 kDa protein in DBT-A1 cells and a 27 kDa protein in three independent clones of DBT-A1DC cells ( Figure 1B ), whereas no protein was cross-reactive to the anti-Flag antibody in the control cell line stably transfected with the pcDNA3.1 vector (DBT-VEC). The amounts of the Flagtagged wt and truncated hnRNP A1 were comparable in these cell lines. A chicken polyclonal antibody against hnRNP A1 detected two endogenous hnRNP A1 isoforms or hnRNP A1-related proteins in the whole-cell lysates of all of the cell lines. The bottom band (34 kDa) overlaps the Flag-tagged wt hnRNP A1 in DBT-A1 cells. There was only a slight increase in the overall amount of hnRNP A1 in DBT-A1 cells as compared with DBT-VEC cells, indicating that the exogenous hnRNP A1 constituted a small fraction of the total hnRNP A1 in the cells. In DBT-A1DC cells, an additional band of smaller size (27 kDa) corresponding to the mutant hnRNP A1 was detected. The overall expression levels of the exogenous hnRNP A1 and hnRNP A1DC were~3-fold lower than that of the endogenous hnRNP A1 in whole-cell lysates ( Figure 1B ). Similar to the endogenous hnRNP A1 protein (Pin Äol-Roma and Dreyfuss, 1992) , the Flag-tagged wt hnRNP A1 was localized almost exclusively in the nucleus ( Figure 1C ). The mutant hnRNP A1, however, was localized predominantly in the cytoplasm ( Figure 1C) , consistent with the previous ®nding that the M9 nuclear localization signal is necessary to localize hnRNP A1 to the nucleus Weighardt et al., 1995) . Thus, hnRNP A1DC was much more abundant than the endogenous hnRNP A1 in the cytoplasm. The expression levels of the wt or mutant hnRNP A1 varied among individual cells based on immuno¯uorescent staining ( Figure 1C ). The growth rate ( Figure 1D ) and cell morphology (data not shown) were similar among the different cell lines.
The effects of overexpression of the wt and mutant hnRNP A1 on syncytium formation and virus production We ®rst assessed the effects of hnRNP A1 overexpression on the morphological changes induced by MHV-A59 infection using several different clones of DBT cell lines. Virus infection was performed at a multiplicity of infection (m.o.i.) of 0.5 to detect the subtle morphological differences among the different cell lines. Syncytia appeared at~7 h post-infection (p.i.) in DBT-VEC cells and~1 h earlier in DBT-A1 cells. At both 8 and 14 h p.i., syncytia were signi®cantly larger and more spread out in DBT-A1 cells than those in DBT-VEC cells ( Figure 2A ). Similar differences were observed with two additional clones of DBT-A1 cells (data not shown). In contrast, no syncytium was observed in three different clones of DBT-A1DC cells, even at 14 h p.i. At 24 h p.i., almost all DBT-A1 cells detached from the plate, but~10±20% of DBT-VEC cells still remained on the plate (data not shown). Remarkably, there was no sign of syncytium formation in DBT-A1DC cells until 24 h after virus infection, when the overall morphology of the cells was similar to that of DBT-VEC cells at 7 h p.i. (data not shown). All of the DBT-A1DC cells were eventually killed at~48 h p.i., suggesting that the inhibition of viral replication was not a result of the disruption of the MHV receptor. Correspondingly, virus production from these cell lines was signi®cantly different. Between 6 and 14 h p.i., virus production from DBT-A1DC cells was 100-to 1000-fold less than that from DBT-VEC and DBT-A1 cells ( Figure 2B ). DBT-A1 cells produced twice as many viruses as those from DBT-VEC cells during that time period.
Relocalization of hnRNP A1 during MHV infection MHV RNA synthesis occurs exclusively in the cytoplasm of infected cells. In order for hnRNP A1 to participate directly in viral transcription, it has to be recruited to the site of RNA synthesis. Although hnRNP A1 shuttles between the nucleus and the cytoplasm in normal cells (Pin Äol-Roma and Dreyfuss, 1992) , the level of cytoplasmic hnRNP A1 is very low. We have demonstrated previously that hnRNP A1 relocates from the nucleus to the cytoplasm of MHV-infected cells (Li et al., 1997) . To determine whether the overexpressed hnRNP A1 may participate in MHV RNA synthesis, we performed immunostaining experiments using an anti-Flag antibody to localize Flag-tagged hnRNP A1. In DBT-A1 cells, a signi®cant increase in the cytoplasmic level of hnRNP A1 and a corresponding decrease of nuclear hnRNP A1 were observed in virus-infected cell syncytia at 7 h p.i.
( Figure 3B ); these cells express the MHV nucleocapsid (N) protein in the cytoplasm ( Figure 3A ). By comparison, in the uninfected cells, which did not have N protein staining, hnRNP A1 was predominantly localized to the nucleus (arrow in Figure 3B ). In DBT-A1DC cells, very few cells were stained positive for the MHV N protein at 7 h p.i. ( Figure 3C ). Signi®cantly, the viral N protein was detected only in the cells that were stained weakly or not at all for Flag-hnRNP A1 ( Figure 3D ), suggesting that the expression of a high level of hnRNP A1DC interfered with viral replication. The effects of wt and mutant hnRNP A1 on MHV protein production We further investigated the effects of the wt and mutant hnRNP A1 on the production of MHV structural and nonstructural proteins. Cytoplasmic protein was extracted from infected cell lines at different time points after infection for immunoblot analysis to detect an open reading frame (ORF) 1a product, p22 (Lu et al., 1998) and the N protein. p22 expression in DBT-VEC cells was clearly detected at 6 h p.i. and peaked at~16 h p.i. ( Figure 4A ). In DBT-A1 cells, p22 appeared at 5 h p.i. and peaked at~8 h p.i. In DBT-A1DC cells, no p22 protein was detected until 16 h p.i. Similar patterns of differences were observed for the N protein in these three cell lines. Actin levels in different cell lines remained relatively constant throughout the infection, except that, in DBT-A1 cells, actin was not detected at 16 and 24 h p.i. due to the loss of the dead cells ( Figure 4A ). These results clearly demonstrated that overexpression of the wt hnRNP A1 accelerated viral protein production, whereas expression of the mutant hnRNP A1 delayed it. We also performed immuno¯uorescent staining of the N protein at 7 h p.i. to further con®rm the western blot results. As represented by images shown in Figure 4B , there were more DBT-A1 cells stained positive for the N protein than DBT-VEC cells. Very few cells were found to express the N protein in DBT-A1DC cells. The p22 and N proteins appeared as doublets in some of the lanes of Figure 4A , but the results varied from experiment to experiment. The N protein is known to be phosphorylated (Stohlman and Lai, 1979) . Whether p22 is post-translationally modi®ed is not known. Figure 5A ). DBT-A1 cells showed a signi®cantly higher level of [ 3 H]uridine incorporation, which peaked at~8 h p.i. DBT-A1DC cells did not show any detectable level of incorporation of the radioactivity. These results suggest that hnRNP A1 regulates MHV RNA synthesis. We further assessed the production of genomic and subgenomic MHV RNAs in these cell lines by northern blot analysis. The genomic and the six subgenomic RNA species were detected at 8 h p.i. in both DBT-VEC and DBT-A1 cells; there were signi®cantly higher steady-state levels of all of the RNA species in DBT-A1 cells ( Figure 5B ). In contrast, no viral RNA was detected in DBT-A1DC cells at that time point. At 16 h p.i., MHV RNA levels in DBT-VEC and DBT-A1 cells decreased generally because of the loss of the dead cells, while the smaller subgenomic RNAs became detectable in DBT-A1DC cells. By 24 h p.i., most viral RNA species became detectable in DBT-A1DC cells ( Figure 5B , lane 10), while most of the DBT-A1 cells were dead (lane 9). These results con®rmed that the synthesis of all of the viral RNA species is accelerated by overexpression of the wt hnRNP A1 and delayed by a dominant-negative mutant of hnRNP A1.
In this analysis, we also detected an additional RNA species (arrow in Figure 5B ), which was determined to be a defective-interfering (DI) RNA by northern blot analysis using a probe representing the 5¢-untranslated region (without the leader), which is present only in genomic and DI RNAs (data not shown). Interestingly, this DI RNA was inhibited to a greater extent than other RNA species in DBT-A1DC cells. This result suggests that the replication of DI RNAs is more sensitive to the dominant-negative inhibition by cytoplasmic hnRNP A1.
To demonstrate further that MHV RNA transcription machinery is defective in cells expressing the mutant hnRNP A1, we studied transcription of an MHV DI RNA, 25CAT, which contains a transcription promoter (derived from the IG sequence for mRNA 7, IG7) and a chloramphenicol acetyltransferase (CAT) reporter gene . CAT activity can be expressed from At 1 h p.i., serum-free medium was replaced by virus growth medium containing 1% NCS and 5 mg/ml actinomycin D. [ 3 H]uridine (100 mCi/ml) was added to the infected cells at 2, 3, 4, 5, 6, 7, 8, 9, 16 and 24 h p.i. After 1 h labeling, cytoplasmic extracts were prepared and precipitated with 5% TCA. The TCA-precipitable counts were measured in a scintillation counter. (B) Northern blot analysis of MHV genomic and subgenomic RNA synthesis in DBT cells. Cytoplasmic RNA was extracted from MHV-A59-infected cells at 8, 16 and 24 h p.i. for northern blot analysis. The naturally occurring DI RNA of MHV-A59 is indicated by an arrow. this DI RNA only if a subgenomic mRNA containing CAT sequences is produced . The 25CAT RNA was transfected into MHV-A59-infected cells 1 h after infection. At 8 h p.i., CAT activity in DBT-A1 cells was signi®cantly higher than that in DBT-VEC cells ( Figure 6A ). On the other hand, CAT activity was very low in DBT-A1DC cells. At 24 h p.i., CAT activity in DBT-A1 cells became slightly lower than that in DBT-VEC cells because of the loss of the dead DBT-A1 cells. The CAT activity in DBT-A1DC was still signi®cantly lower than that in DBT-VEC or DBT-A1 cells. These results established that mRNA transcription from the DI RNA was also inhibited by hnRNP A1DC.
The results shown above ( Figure 5B ) also suggest that DI RNA replication is more sensitive to the inhibitory effects of the hnRNP A1 mutant. To con®rm this result, we further studied replication of another DI RNA during serial virus passages. DBT cells were infected with MHV-A59 and transfected with DIssE RNA derived from JHM virus (Makino and Lai, 1989) ; the virus released (P0) was passaged twice in DBT cells to generate P1 and P2 viruses. DBT cells were infected with these viruses, and cytoplasmic RNA was extracted for northern blot analysis using glyoxalated RNA for a better resolution of smaller RNAs. For DBT-A1DC cells, RNA was extracted at 36 h p.i. since viral RNA synthesis was delayed in this cell line. Cells infected with P0 viruses did not yield detectable amounts of DIssE, but contained the naturally occurring A59 DI RNA, whose replication was inhibited more strongly than the synthesis of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figures 5B, lanes 8±10 and 6B, lanes 1± 3). However, this A59 DI RNA was not detectable in cells infected with P1 and P2 viruses ( Figure 6B , lanes 4±9). In contrast, DIssE appeared in cells infected with P1 viruses and further increased in cells infected with P2 viruses, indicating that the replication of the smaller DIssE may have an inhibitory effect on the replication of the larger A59 DI RNA (Jeong and Makino, 1992) . Similar to the A59 DI RNA, the replication of DIssE RNA was much more strongly inhibited than that of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figure 6B , lanes 6 and 9). Our results thus suggest that MHV DI RNA replication is more dependent on the function of cytoplasmic hnRNP A1.
The mechanism of dominant-negative inhibition by the C-terminal deletion mutant of hnRNP A1 To understand the underlying mechanism of the inhibition of MHV RNA transcription by the C-terminal-deletion mutant of hnRNP A1, we ®rst examined the RNA-and protein-binding properties of this mutant protein.
Electrophoretic mobility shift assay demonstrated that hnRNP A1DC retained the ability to bind the MHV (±)strand leader RNA and to form multimers with itself, similar to the wt hnRNP A1 (data not shown); this is consistent with the fact that both of its RBDs are intact ( Figure 1A) . Furthermore, UV-crosslinking experiments showed that increasing amounts of puri®ed glutathione S-transferase (GST)±hnRNP A1DC ef®ciently competed with the endogenous hnRNP A1 for the binding of the MHV (±)-strand leader RNA ( Figure 7A ), indicating that the binding of hnRNP A1DC to RNA was not affected. These results suggest that the RNA-binding properties of hnRNP A1DC were intact.
We next examined the protein-binding properties of hnRNP A1DC. Since hnRNP A1 has been shown to interact with the N protein, which also participates in MHV RNA synthesis (Compton et al., 1987; Wang and Zhang, 1999) , we ®rst determined whether the dominantnegative mutant of hnRNP A1 retained the ability to interact with the N protein in vitro. GST pull-down assay using various truncation mutants of hnRNP A1 showed that the N protein bound the N-terminal domain (aa 1±163) of hnRNP A1 ( Figure 7B) ; thus, the binding of hnRNP A1DC [equivalent to hnRNP A1(1±245)] to the N protein was not affected. We next examined the in vivo interaction of the wt and mutant hnRNP A1 with an MHV ORF 1a product, p22, which has been shown to co-localize with the de novo synthesized viral RNA (S.T.Shi and The viruses were passaged twice in wt DBT cells to obtain P1 and P2 viruses. Cytoplasmic RNA was extracted from the DBT cells infected with P0, P1 and P2 viruses and treated with glyoxal before electrophoresis and northern blot analysis using a 32 P-labeled (±)-strand mRNA 7 as a probe. The A59 DI RNA and DIssE RNA are indicated by arrows.
M.M.C.Lai, unpublished results) and associate with the viral replicase complex (Gibson Bost et al., 2000) . Cytoplasmic extracts from MHV-A59-infected cells were immunoprecipitated with anti-Flag antibody-conjugated beads, followed by western blotting with a rabbit polyclonal antibody against p22. At 8 h p.i., p22 was co-precipitated with the Flag-tagged hnRNP A1 from DBT-A1 cells, whereas no precipitation of p22 was observed in DBT-VEC cells ( Figure 7C ). For DBT-A1DC cells, co-immunoprecipitation was performed at 24 h p.i., when abundant MHV proteins were synthesized. p22 was shown to co-precipitate with hnRNP A1DC, indicating that hnRNP A1DC still formed a complex with the viral polymerase gene product. These results suggest that the ability of hnRNP A1DC to interact with the N and polymerase proteins was not altered.
We next investigated whether the mutant hnRNP A1 is de®cient in the interaction with any other cellular proteins in this RNA±protein complex. We labeled proteins in MHV-infected cells or mock-infected cells at different time points after infection and immunoprecipitated with the anti-Flag antibody. Signi®cantly, a cellular protein of 250 kDa was shown to be associated only with the wt hnRNP A1, but not the mutant hnRNP A1 ( Figure 7D ), suggesting that hnRNP A1 binds to this protein through its C-terminal domain. We propose that this cellular protein is another important component of the MHV RNA transcription/replication complex.
There is an accumulating body of evidence signifying the importance of cellular factors in RNA synthesis of RNA viruses (reviewed by Lai, 1998) . Previous studies have shown that hnRNP A1 binds to the cis-acting sequences of MHV template RNA and that this interaction correlates with the transcription ef®ciency of viral RNA in vivo (Zhang and Lai, 1995; Li et al., 1997) . In addition, hnRNP A1 is also implicated in viral RNA replication by the recent ®nding that hnRNP A1 interacts with the 3¢-ends of both positive-and negative-strand MHV RNA (P.Huang and M.M.C.Lai, unpublished results). However, hnRNP A1 modulates cytoplasmic viral RNA synthesis the functional importance of hnRNP A1 in viral RNA synthesis has so far not been directly demonstrated. In the present study, we established that MHV RNA transcription and replication were enhanced by overexpression of the wt hnRNP A1 protein, but inhibited by expression of a dominant-negative hnRNP A1 mutant in DBT cell lines.
Our results suggest that hnRNP A1 is a host protein involved in the formation of a cytoplasmic transcription/ replication complex for viral RNA synthesis. This represents a novel function for hnRNP A1 in the cytoplasm.
Our results indicate that the inhibitory effects on MHV replication exhibited by the dominant-negative mutant of hnRNP A1 were relatively more prominent than the enhancement effects by overexpression of the wt hnRNP A1. This is consistent with the subcellular localization patterns of the wt and mutant hnRNP A1 proteins. The overexpressed exogenous wt hnRNP A1 in DBT-A1 cells was predominantly localized in the nucleus, similar to the endogenous hnRNP A1 ( Figure 1C ). The C-terminal-deletion mutant, however, was localized mainly in the cytoplasm. Thus, the level of hnRNP A1DC was much higher than the endogenous wt hnRNP A1 in the cytoplasm of DBT-A1DC cells, where MHV replication occurs. This result explains why hnRNP A1DC could have a strong dominant-negative inhibitory effect, despite the fact that it was expressed at a lower level than the endogenous hnRNP A1 ( Figure 1B) .
The effects of the expression of the wt and mutant hnRNP A1 on virus production ( Figure 2B ), viral protein synthesis ( Figure 4A ) and viral RNA synthesis ( Figure 5A ) correlated with each other. Furthermore, hnRNP A1DC caused not only a global inhibition of genomic RNA replication and subgenomic mRNA transcription, but also a preferential inhibition of at least two DI RNA species. These results suggest that the inhibition of MHV replication by the hnRNP A1 mutant was most likely a direct effect on viral RNA synthesis rather than an indirect effect on other aspects of cellular or viral functions. Since hnRNP A1 binds directly to the cis-acting MHV RNA sequences critical for MHV RNA transcription (Li et al., 1997) and replication (P. Huang and M.M.C.Lai, unpublished results) , it is most likely that hnRNP A1 may participate in the formation of the transcription/replication complex. Indeed, our data show that hnRNP A1 interacts directly or indirectly with the N protein and a gene 1 product, p22, both of which are probably associated with the viral transcription/replication complex (Compton et al., 1987; Wang and Zhang, 1999; Gibson Bost et al., 2000) . hnRNP A1 may participate directly in viral RNA synthesis in a similar role to that of transcription factors in DNAdependent RNA synthesis, e.g. by maintaining favorable RNA conformation for RNA synthesis. Alternatively, hnRNP A1 may modulate MHV RNA transcription or replication by participating in the processing, transport and controlling the stability of viral RNAs. It has been reported that RNA processing of retroviruses, human T-cell leukemia virus type 2 (Black et al., 1995) and HIV-1 (Black et al., 1996) , is altered by the binding of hnRNP A1 to the viral RNA regulatory elements. It is also possible that hnRNP A1 may participate in MHV RNA synthesis indirectly by affecting the production of other host cell proteins, which may, in turn, regulate MHV RNA synthesis. Since hnRNP A1 is a dose-dependent altern-ative splicing factor (Caceres et al., 1994) , even small changes in the intracellular level of hnRNP A1 can alter the splicing of other cellular proteins. Regardless of the mechanism, our study established the importance of cellular factors in viral RNA-dependent RNA synthesis.
The transcription from 25CAT RNA was strongly inhibited by the dominant-negative mutant of hnRNP A1, as shown by CAT assays ( Figure 6A ). In addition, the replication of the naturally occurring A59 DI RNA and the arti®cial DIssE RNA was completely abolished ( Figure 6B) . Surprisingly, the replication of MHV DI RNAs suffered a stronger inhibition by the dominantnegative mutant of hnRNP A1 than the synthesis of MHV genomic and subgenomic RNAs, suggesting that DI RNA replication may be more dependent on hnRNP A1. Although DI RNAs contain all of the cis-acting replication signals that are essential for their replication in normal cells (Kim and Makino, 1995) , the small size of DI RNA may cause it to require more hnRNP A1 to maintain a critical RNA structure. It has been shown that different DI RNAs require different cis-acting signals for RNA replication (Kim and Makino, 1995) .
Our results demonstrate that the C-terminal domain of hnRNP A1, including the M9 sequence and the glycinerich region, is important for MHV RNA transcription and replication, but the mechanism of the dominant-negative effects of hnRNP A1DC is still not clear. hnRNP A1DC retains the RNA-binding and self-association ability and is capable of binding the viral proteins N and p22, which are associated with the transcription/replication complex. It is possible that hnRNP A1DC is not productive due to its inability to interact with other viral or cellular proteins that are involved in MHV RNA synthesis. We have found a protein of~250 kDa that binds only the wt, but not the mutant hnRNP A1 ( Figure 7D ). It remains to be shown whether this cellular protein is involved in MHV RNA synthesis.
In our preliminary study, we found that MHV could replicate in an erythroleukemia cell line, CB3, which was reported to lack detectable hnRNP A1 expression as a result of a retrovirus integration in one allele and loss of the other allele (Ben-David et al., 1992) . Since hnRNP A1 protein is involved in a variety of important cellular functions, including RNA splicing, transport, turnover and translation, it is conceivable that other redundant gene products may substitute for the function of hnRNP A1 in CB3 cells. Indeed, UV-crosslinking assays using CB3 cell extracts detected two proteins comparable to hnRNP A1 in size that could interact with the MHV negative-strand leader RNA (data not shown). These proteins may represent hnRNP A1-related proteins, since many of such hnRNPs exist in the cells (Buvoli et al., 1988; Burd et al., 1989) . Therefore, multiple cellular proteins may have the capacity to be involved in MHV RNA synthesis.
Based on previous ®ndings (Kim and Makino, 1995; Zhang and Lai, 1995; Li et al., 1997) and the results from this study, we propose a model for the regulation of transcription/replication of MHV RNA by hnRNP A1. We hypothesize that hnRNP A1 is one of the components of the MHV RNA transcription or replication complex, and the crosstalk between hnRNP A1 and another viral or cellular RNA-binding protein (designated X in Figure 8 ) is essential for MHV replication and transcription. The X protein binds to the C-terminus of hnRNP A1 and cooperates with hnRNP A1 to recruit more proteins to form the transcription or replication complex. The C-terminaldeletion mutant of hnRNP A1 loses the ability to interact with the X protein and to bring it into the initiation complex, resulting in an inhibition of MHV RNA transcription and replication. The residual replication and transcription activities of MHV RNA in the absence of functional hnRNP A1 may be due to a limited af®nity of the X protein to a cis-acting signal that is only present in MHV genomic RNA (site B). On the other hand, DI RNAs may lack this cis-acting signal. When the crosstalk between the X protein and hnRNP A1 is abolished by the dominant-negative mutant of hnRNP A1, the X protein can no longer participate in the formation of the initiation complex, resulting in a complete loss of DI RNA replication.
In summary, our data provide direct experimental evidence that hnRNP A1 is involved directly or indirectly in MHV RNA synthesis, probably by participating in the formation of an RNA transcription/replication complex. This ®nding reveals a novel cytoplasmic function for hnRNP A1.
Cells and viruses DBT cells, a mouse astrocytoma cell line (Hirano et al., 1974) , were cultured in Eagle's minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS) and 10% tryptone phosphate broth. MHV strain A59 (Robb and Bond, 1979) was propagated in DBT cells and maintained in virus growth medium containing 1% NCS.
Plasmid construction and establishment of DBT stable cell lines The cDNA of the murine hnRNP A1 gene was ampli®ed by RT±PCR using RNA extracted from DBT cells and a set of primers representing the 5¢-and 3¢-ends of hnRNP A1-coding region, and cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). The 8 amino acid Flag tag was attached to the N-terminus of hnRNP A1 by including the Flag tag in the forward PCR primer. The truncated hnRNP A1DC was similarly constructed using a PCR-ampli®ed fragment that represents hnRNP A1 (aa 1±245).
For the establishment of permanent DBT cell lines, pcDNA3.1 alone or the plasmid containing the Flag-tagged hnRNP A1 or hnRNP A1DC was transfected into 60% con¯uent DBT cells using DOTAP according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN) . After 4 h, the transfected cells were selected in DBT cell medium containing 0.5 mg/ml Geneticin (G418) (Omega Scienti®c, Tarzana, CA) for 10 days. Single colonies were then collected and cultured individually for 10 additional days before screening for the expression of Flag-tagged proteins.
The polyclonal rabbit antibody against p22 was a gift from Dr Susan C.Baker at Loyola University, IL. The chicken polyclonal antibody against hnRNP A1 was produced by Aves Labs, Inc. (Tigard, OR) by immunizing chickens with the puri®ed mouse hnRNP A1 protein expressed in bacteria. The polyclonal anti-Flag antibody was purchased from Af®nity Bioreagents (Golden, CO). The goat polyclonal antibody against actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse monoclonal antibody against the N protein has been described previously (Fleming et al., 1983) .
Examination of growth rate of permanent DBT cells Equal numbers (1 3 10 5 ) of DBT-VEC, DBT-A1 and DBT-A1DC cells were plated in 10-cm culture plates and maintained in culture medium for 4 days. Cells were trypsinized, stained with Trypan Blue (Gibco-BRL, Grand Island, NY) and counted at 24-h intervals with a hemacytometer (Hausser Scienti®c, Horsham, PA).
Plaque assay DBT cells in 10-cm plates were infected with MHV-A59 at an m.o.i. of 2. After 1 h for virus adsorption, the cells were washed three times with serum-free MEM, which was then replaced with virus growth medium containing 1% serum. At 1, 6, 8, 10, 14 and 24 h p.i., 1 ml of medium was taken from each plate for plaque assay.
[ 3 H]uridine labeling of MHV RNA Cells plated in 6-well plates were infected with MHV-A59 at an m.o.i. of 2. At 1 h p.i., 5 mg/ml actinomycin D was added to the virus growth medium to inhibit cellular RNA synthesis. To label newly synthesized MHV RNA, 100 mCi/ml of [ 3 H]uridine (NEN, Boston, MA) were added to the medium at hourly intervals. After 1 h of labeling, the cells were washed twice in ice-cold PBS and scraped off the plates in 1 ml of PBS. The cells were then collected by centrifugation and incubated in 200 ml of NTE buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA) containing 0.5% NP-40, 0.5 mM dithiothreitol (DTT) and 400 U/ml of RNasin on ice for 15 min. After centrifugation, 5 ml of the cytoplasmic extract were spotted on a piece of 3 mm paper and incubated with 5% trichloroacetic acid (TCA). The radioactivity remaining on the 3 mm paper was measured in a scintillation counter.
Northern blot analysis DBT cells were infected with MHV-A59 at an m.o.i. of 2. At 8, 16 and 24 h p.i., cytoplasmic extract was prepared as described above and subjected to phenol/chloroform extraction and ethanol precipitation to purify cytoplasmic RNA. Approximately 10 mg of RNA were separated by electrophoresis on a 1.2% formaldehyde-containing agarose gel and transferred to a nitrocellulose membrane. For a better resolution of the DIssE RNA ( Figure 6B ), RNA was glyoxalated before being electrophoresed on a 1% agarose gel. An in vitro transcribed, 32 P-labeled negative-strand mRNA 7 of MHV-JHM was used as a probe to detect MHV genomic and subgenomic RNAs. For detecting DI RNA species, RNA blots were probed with an RNA representing a sequence complementary to the sequence of the 5¢-untranslated region of MHV-JHM RNA, but excluding the leader sequence.
Western blot analysis DBT cells in 6-well plates were infected with MHV-A59 and cytoplasmic extracts were prepared as described previously (Li et al., 1997) at various hnRNP A1 modulates cytoplasmic viral RNA synthesis time points p.i. The extracts were electrophoresed on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane for western blotting.
Immuno¯uorescence staining Cells were washed in phosphate-buffered saline (PBS) and ®xed in 4% formaldehyde for 20 min at room temperature, followed by 5 min in ±20°C acetone. Primary antibodies were diluted in 5% bovine serum albumin and incubated with cells for 1 h at room temperature. After three washes in PBS,¯uorescein-conjugated secondary antibodies were added to cells at 1:200 dilution for 1 h at room temperature. FITC-or TRITCconjugated secondary antibodies were used to generate green or red uorescence. Cells were then washed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA).
UV-crosslinking assay UV-crosslinking assay was performed as described previously (Huang and Lai, 1999) . In brief, DBT cell extracts (30 mg protein), 200 mg/ml tRNA and 10 4 c.p.m. of an in vitro transcribed, 32 P-labeled negativestrand MHV 5¢-end RNA (182 bp) were incubated for 10 min at 30°C. Increasing amounts of puri®ed GST (0, 0.5, 1.5 and 5 ng) or recombinant GST±hnRNP A1 fusion protein (0, 1, 3 and 10 ng) were included in the reaction to compete with the endogenous hnRNP A1 for binding. The reaction mixture was placed on ice and UV-irradiated in a UV Stratalinker 2400 (Stratagene) for 10 min, followed by digestion with 400 mg/ml RNase A for 15 min at 37°C. The protein±RNA complexes were then separated on a 10% SDS±polyacrylamide gel and visualized by autoradiography.
GST pull-down assay GST pull-down was performed as described previously (Tu et al., 1999) . In brief, GST±hnRNP A1 fusion proteins on glutathione beads (Pierce, Rockford, IL) were incubated with the in vitro translated, 35 S-labeled N protein in 0.3 ml of GST-binding buffer containing 0.1% NP-40 for 2 h at 4°C. The beads were washed ®ve times with the GST-binding buffer containing 0.3% NP-40. Proteins bound to beads were eluted by boiling in Laemmli buffer for 5 min and separated on a 10% polyacrylamide gel.
[ 35 S]methionine labeling and immunoprecipitation DBT cells were infected with MHV-A59 at an m.o.i. of 2. The cells were incubated with methionine-free medium for 30 min before labeling and were labeled in 100 mCi/ml [ 35 S]methionine starting at 1.5, 7 or 24 h p.i. After labeling for 2 h at each time point, the cells were harvested for protein extraction as described previously (Li et al., 1997) . The protein extracts were immunoprecipitated with anti-Flag antibody-conjugated beads (Sigma, St Louis, MO) in Tm 10 buffer (50 mM Tris±HCl pH 7.9, 0.1 M KCl, 12.5 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% NP-40, 1 mM phenylmethylsulfonyl¯uoride) at 4°C for 2 h. The immunoprecipitates were washed and separated on a 4±15% gradient SDS±polyacrylamide gel and visualized by autoradiography.
Plasmid 25CAT was linearized by XbaI and in vitro transcribed with T7 RNA polymerase to produce the DI RNA . The DI RNA was transfected into MHV-A59-infected DBT cells using DOTAP as described previously (Huang and Lai, 1999) . In brief,~80% con¯uent DBT cells were infected by MHV-A59 at an m.o.i. of 10. At 1 h p.i., the cells were transfected with 5 mg of in vitro transcribed DI RNA and incubated at 37°C for the desired lengths of time. To amplify the DI RNA, viruses (P0) were passaged twice in wt DBT cells to generate P1 and P2 viruses.
Cells were harvested at 8 or 24 h p.i. and lysed by freezing and thawing for three times. After centrifugation at 12 000 r.p.m. for 10 min, the supernatant was used in a CAT assay as described previously (Lin et al., 1996) . A Method to Identify p62's UBA Domain Interacting Proteins The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays. p62 is a novel cellular protein which was initially identified in humans as a phosphotyrosine independent ligand of the src homology 2 (SH2) domain of p56 lck (1, 2) . p56 lck is a member of the c-src family of cytoplasmic tyrosine kinases that is found predominantly in cells of lymphoid origin (3, 4) . In addition to the interaction with p56 lck , p62 also associates with the Ser/Thr kinase (1, 2) , atypical protein kinase C (5, 6) , and ubiquitin (7) . In addition to the SH2 domain, p62 possesses several structural motifs, including a ubiquitin associated (UBA) domain that is capable of binding ubiquitin nonconvalently (8, 9) . Ubiquitin (Ub) is a small polypeptide of 76 amino acids that can be convalently attached to other proteins at specific lysine residues through chains composed of one (mono) or several ubiquitin moieties (poly). In addition to its classical role in protein degradation, ubiquitin is emerging as a signal for protein transport and processing (10) (11) (12) . Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin activating enzyme E1, a ubiquitin-conjugating enzyme E2, and a ubiquitin ligase E3. Initially, E1 activates ubiquitin by forming a high energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2, then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and ε-amino group of a lysine residue of the substrate. Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin are attached to lysine side chains of the previously conjugated moiety to form branched polyubiquitin chains. The fate of ubiquitinated substrates depends on the number of ubiquitin moieties conjugated, as well as, the lysine linkage of Ub-Ub conjugation. The conjugation of ubiquitin to eukaryotic intracellular proteins is one way in which those proteins are targeted to the proteasome for subsequent rapid degradation. This mechanism is particularly important for short-lived regulatory proteins such as cyclins, cyclin-dependent protein kinase-inhibitors, p53, the nuclear factor kappa B precursor, and IκB (13) . The ubiquitinproteasome system consists of two steps: 1) the target protein is conjugated with polyubiquitin molecules, which mark the substrate for degradation; 2) the target protein is transferred to the 26S proteasome, unfolded and degraded.
The UBA domain is a conserved sequence motif among proteins that can bind polyubiquitin. It is comprised of ~45 amino acids (13) . The amino acids 386-434 of p62, which bind polyubiquitin, has been shown to possess homology to other recently described UBA domains (9) . Interestingly, proteins with UBA domains are more likely to bind polyubiquitin chains over monoubiquitin, such as the yeast UBA protein Rad23, a highly conserved protein involved in nucleotide excision repair (13) . Recently, it has been shown that yeast cells lacking two UBA proteins (Dsk2 and Rad23) are deficient in protein degradation and that the UBA motif is essential for their function in proteolysis (14) .
In addition to the important role in recycling of amino acids from damaged or misfolded proteins, ubiquitin-protein conjugation also has functions unrelated to proteasomal targeting. For example, polyubiquitination is required for the internalization of several yeast and mammalian cell surface proteins into the endocytic pathway (15, 16) . Interestingly, p62 appears to sequester ubiquitinated substrates into a cytoplasmic structure referred to as a sequestosome, into which excess ubiquitinated proteins are segregated (17) . In addition, p62 is an immediate early response gene product for a variety of signals (18) . Thus, p62 appears to play a novel regulatory role for polyubiquitinated proteins and may have an essential function in cell proliferation and differentiation. We have developed a method that will enable identification of protein(s) that interact with p62's UBA domain.
Human adult brain library 10×96 well plates with 100 cDNAs per well and Gold TNT SP6 
To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system. The human adult brain library 96 well plates with 100 cDNAs per well was transcribed and translated employing the Gold TNT SP6 Express 96 plate and [ 35 S] methionine. The TNT Quick-coupled transcription-translation system contained a rabbit reticulocyte lysate pre-mixed with most of the reaction components necessary to carry out transcription/translation in the lysate, including all of the amino acids except methionine. [ 35 S] Methionine was used to label newly synthesized proteins. The reactions were set up according to the manufacturer's instructions. Rabbit reticulocyte lysate has been shown to be capable of carrying out ubiquitination of proteins that were translated in such an in vitro translation system (19, 20) . The reactions mixtures also contained ubiquitin so that the newly synthesized proteins could be ubiquitinated. The reactions were incubated at 30°C for 2 hours. The resulting proteins were assayed to determine their binding ability with p62's UBA domain. Potential positive "hits" were further subdivided and reassayed to link individual clones to the protein of interest (Fig. 1 ). 
Each translated pool was resuspended in binding buffer (25 mM Tris pH 7.5, 125 mM NaCl, 0.1% NP-40) and used as a source of protein in p62 UBA pull down assays. Proteins that specifically interact with the UBA domain of p62 were isolated by interaction with agarose-immobilised p62-UBA peptide (amino acid 387-436 of p62) (5 µg) for 2 hours at 4ºC, then washed three times in washing buffer (25 mM Tris pH 7.6, 100 mM NaCl, 1% NP-40). Bound proteins were released by addition of SDS-sample buffer and separated by SDS-PAGE. The SDS-PAGE gels were fixed in 50% methanol, 10% acetic acid for 30 min, stained in 0.2% Commassie Brilliant Blue R-250, 45% methanol, 10% acetic acid for 15 min, destained in 10% acetic acid, 50% methanol overnight, and enhanced in autoradiography enhancer En 3 HANCE for 1 hr and exposed to X-Ray film.
By combining 4 pools as one mixed pool, 96 protein pools were divided into 24 mixed protein pools for use in p62 UBA pull down assays. Positive mixed protein pools were selected and individual pools were retested for its ability to bind p62's UBA domain. The individual cDNA pool from which the positive protein pool was generated was transformed into JM109 competent cells and plated on LB ampicillin plate. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNA was purified from the cell culture and used for TNT Quick coupled in vitro transcription/ translation. The individual protein synthesized from each plasmid DNA chosen was screened for its ability to bind p62's UBA domain.
To confirm the interaction with p62's UBA domain, the final resulting individual proteins were used in the coupled TNT/p62 UBA pull down assays. The cDNA inserts were sequenced in the Genomics Core Facility at Auburn University and the sequences were compared with known sequences in NCBI database by BLAST analysis.
Human embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and transfected with myc-tagged HSP70 plasmid using the Mammalian Cell Transfection Kit. Cells were harvested and lysed in 1 ml of SDS lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% TX-100, 1 mM Na 3 VO 4 , 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 1% SDS) for 30 min on ice, followed by centrifugation at 14000 rpm for 15 min at 4°C to remove the insoluble fraction. The protein concentration of the supernatant was determined using the Bio-Rad DC protein assay reagent with bovine serum albumin (BSA) as standard. Equal amount of protein (750 µg) was immunoprecipitated with anti-myc and collected with agarose-coupled secondary antibody. To the agarose beads containing the immunoprecipitated HSP70, 50 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP) was added containing 100 ng E1, 200 ng E2 (UbcH7), and 100 µg of E3 (Flag-tagged TRAF6) along with 5 µg GST-WT-Ub, GST-K29R Ub, GST-K48R Ub, GST-K63R Ub, or K63 Ub. Control samples without HSP70, E1, E2, E3, or GST-WT-Ub were also included. Reactions were carried out by continuous shaking at 37°C for 2 hours and then washed three times with reaction buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and Western blotted for anti-ubiquitin.
To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system from Promega (Cat. No. L6500). The human adult brain library 96 well plates with 100 cDNAs per well were transcribed and translated employing the Gold TNT SP6 Express 96 plate in the presence of [ 35 S] methionine and ubiquitin (25 µg/µl, Sigma). By combining 4 protein pools as one mixed pool, 96 protein pools were divided into 24 mixed pools ( Fig. 2A,  2B ). Each lane contained more than 100 proteins (theoretically 400) with different molecular weight. Therefore, each lane appeared as a smear, indicating that the in vitro transcription/translation system from Promega worked successfully. In order to examine whether proteins synthesized in the IVEC system are also ubiquitinated, Western blot analysis was performed by blotting the newly synthesized proteins (in the presence of cold methionine instead of 35 S methionine) with ubiquitin monoclonal antibody. In the mixed protein pools, each of the 24 lanes appeared as a smear, indicating that proteins synthesized by the IVEC system are also ubiquitinated (Fig. 3A) . Furthermore, the rabbit reticulocyte lysate in the IVEC system can utilize different lysine linkages of ubiquitin (i.e., Ub K29, Ub K48, and Ub K63) for ubiquitination (Fig. 3B) . In order to investigate whether the agarose-immobilised p62 UBA peptide has binding specificity, a mixed protein pool synthesized by IVEC system was tested in a pull down assay in the presence of agarose beads alone or in the presence of p62 UBA agarose beads (Fig. 3C ). Our results revealed that proteins that bound to p62's UBA domain could not be pulled down by agarose beads alone, indicating that the agarose-immobilised p62 UBA peptide had binding specificity. 
In order to identify proteins that bind to p62's UBA domain, p62 UBA pull down assays were performed. Out of the 24 mixed protein pools, several pools contained [ 35 S] methionine-labeled bands in the primary p62 UBA pull down assays (Fig. 4) . We chose 6 pools (pool # 2, 4, 8, 14, 20, 21) because of their stronger signal to specifically identify which individual protein pool in the mixed pools has the ability to bind to p62's UBA domain. Therefore, a secondary screen was conducted on the 6 positive individual mixed pools (representing 24 individual protein pools) which bound with p62's UBA domain (Fig. 5) . Mixed protein pool # 2 generated a positive protein with molecular weight of 51 KDa (Fig. 4) , and only individual protein pool "c" out of the four protein pools (a, b, c, d) that comprised protein pool #2 had a protein with the same molecular weight (Fig. 5) . Depending on the size of the protein pulled down in the secondary screen compared to the primary screen (Fig. 4) , individual protein pools "c", "h", "i", "o", "t", and "v" were identified (Fig. 5) . To specifically identify which protein in the individual protein pool has the ability to bind p62's UBA domain, the cDNAs from the positive individual protein pools were then transformed into JM109 competent cells and plated out on LB ampicillin plates. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNAs were purified from the cell culture and used for TNT Quick coupled in vitro transcription/translation. The individual protein synthesized from each plasmid DNA was retested for its ability to bind p62's UBA domain. By synthesizing individual protein from individual plasmid using the Gold TNT Quick coupled in vitro transcription/translation system and subjecting them to p62 UBA pull-down assays, 11 positive clones were isolated from the 6 positive individual pools. It is not surprising that 5 more clones showed binding ability with p62's UBA domain since there are 100 cDNAs in each positive individual pool and some of them could have lower binding ability and therefore showed weak signal in the mixed protein pool. It is also possible that they are not as efficiently synthesized in the mixed TNT reaction as in the individual TNT reaction in which only one cDNA was used as template. The 11 positive plasmids were sequenced and compared with known cDNA sequences in NCBI database using BLAST analysis with results shown in Table 1 .
Interestingly, the proteins identified in the screen fall into three distinct categories. One set are proteins that are associated with Alzheimer's disease, including myelin basic protein, 14-3-3 protein, syntaxin binding protein munc18, transketolase, heat shock protein HSP70, reelin, and calcium/calmodulin kinase II (Table 1 ). Significant decrease in the amount of myelin basic protein has been reported in the white matter of Alzheimer's disease patients, accompanied by increased quantities of βamyloid peptides (21) . The presence of β-amyloid peptides containing senile plaques and neurofibrillary tangles are the two major pathological features in the brain of patients with Alzheimer's disease (22) . Interestingly, 14-3-3 proteins have also been demonstrated to be components of neurofibrillary tangles of Alzheimer's disease brains (23) . Syntaxin binding protein munc18 can powerfully regulate amyloid precursor protein metabolism and β-amyloid secretion through direct and indirect interactions with X11 proteins (24) . The activity of transketolase has been reported to be reduced in dementia of Alzheimer's type brain (25) . Heat shock protein HSP70 expression is significantly increased in the temporal cortex of patients with Alzheimer's disease (26) . Besides HSP70, other heat shock proteins are also linked with Alzheimer's disease. For example, increased synthesis of HSP27 has been suggested to play a role in preventing neuronal injury in AD (27) , and alpha-crystallin heat shock protein has a close relationship with neurofibrillary tangles of AD brains (28) . Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation (29) . Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease (30) . Enhanced activity of calcium/calmodulin kinase II has been suggested to contribute to phosphorylation of tau protein and lead to neurofibrillary tangle deposition and neuronal death in Alzheimer's disease (31) . Although the relationship between p62 and neurofibrillary tangles or neuritic plaques is unclear, both neurofibrillary tangles and dystrophic neuritis of neuritic plaques are associated with ubiquitin (32) , suggesting that dysfunction in ubiquitin-mediated proteolysis and the resulting accumulation of ubiquitinconjugated proteins may contribute to the origination of dystrophic neuritis and neurofibrillary tangles. Furthermore, p62 has been recently reported to accumulate early in neurofibrillary tangles in Alzheimer's disease (33) , suggesting that p62 may play an important role in Alzheimer's disease by interacting with those proteins through its UBA domain.
A second set of proteins identified in the screen that bind to p62's UBA domain are associated with brain development, including homeobox protein Meis2 and unc51 like kinase II (Table 1) . Although Meis proteins are not extensively studied in humans, these proteins have been shown to be required for hindbrain development in the zebrafish (34) . Unc51 like kinase II has been demonstrated to play a role in axonal elongation (35, 36) , which is needed for the formation of complicated neuronal networks. The third set of proteins that exhibit ability to bind p62's UBA domain are proteins that are linked with other neurodegenerative diseases, including FK506 binding proteins and nuclear receptor corepressor I (Table 1) . FK506 (tacrolimus) is a potent immunosuppressive drug used in the treatment of patients after organ transplantation and in selected autoimmune disorders (37) . FK506 is activated upon binding to members of the immunophilin family of proteins, which were designated as FK506 binding proteins (38) . Immunophilins are chaperone proteins and FK506 binding proteins have been suggested as therapeutics for neurological disorders (39, 40) . Nuclear receptor corepressor I has been suggested to play a role in Huntington's disease because it is able to interact with huntingtin (41) . The proteins identified here suggest that p62's UBA domain has the ability to interact with multiple proteins that play important roles in neurodegenerative diseases. Further screening from the whole genome-wide perspective will be necessary to define the important role that p62's UBA domain plays. 
It has been reported that polyubiquitin chains assembled through lysine 48 of ubiquitin act as a signal for substrate proteolysis by the 26S proteasome (42) (43) (44) . In order to understand whether the proteins identified in our screen bind to the p62's UBA domain through lysine 48 (K48), polyubiquitin K48 chains were added to the p62 UBA pull down assay (Fig. 6) . Inclusion of polyubiquitin K48 chains in the assay should compete for the binding of substrate to the p62's UBA domain and reduce the interaction of those proteins with the p62's UBA domain if those proteins are assembled through K48 chains. An alternative interpretation for polyubiquitin K48 chain competition is that the ubiquitin chains are competing for the same binding site as the binding partners which are either ubiquitinated or non-ubiquitinated. We randomly chose five proteins out of the 11 binding partners for the competition pull down (Fig. 6) . Out of the five proteins, four proteins (# 2, 3, 4, and 5) showed reduced binding ability with p62's UBA domain when polyubiquitin K48 chains were included (Fig. 6A, 6B ). However, K48 chains failed to compete with HSP70, suggesting that p62's UBA domain binds to HSP70 through a ubiquitin lysine linkage other than K48. Interestingly, it has been reported that heat shock protein 70 cognate (HSP70) is ubiquitinated by CHIP (carboxyl terminus of Hsc70-interacting protein) via ubiquitin chain synthesis that uses either K29 or K63 (45) . In order to examine which lysine linkage utilized by HSP70 binds to p62's UBA domain, in vitro ubiquitination assay was performed by incubating lysates from HEK cells expressing HSP70 with E1, E2, and E3 in reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP). As control, the ubiquitination of HSP70 utilizing the rabbit reticulocyte lysate was also investigated by Western blot analysis. Our results revealed that HSP70 was ubiquitinated in the IVEC system (Fig.  7A, 7B) , and the rabbit reticulocyte lysate contained enzymes such as TRAF6 (E3) and UbcH7 (E2) for in vitro ubiquitination (Fig. 7C ). TRAF6 was chosen as an E3 in this in vitro ubiquitination assay due to its RING domain, a common feature of E3 ligases, and the observation that p62 is a scaffold for TRAF6 interaction (46) . Therefore, in vitro ubiquitination assays using the E1-E2-E3 system were performed in the presence of either ubiquitin wild type or ubiquitin mutants (K29R, K48R, and K63R). If one lysine mutant blocks the ubiquitination of HSP70, it would suggest that the ubiquitination of HSP70 utilizes that specific lysine linkage.
Our results revealed that HSP70 utilizes K63 linkage to assemble polyubiquitin chains to bind to p62's UBA domain since only the K63R ubiquitin mutant blocked the ubiquitination of HSP70 (Fig. 8A) . A similar result was also observed when reactions were conducted with wild type ubiquitin or mutant ubiquitin with all lysines mutated to arginines except K63 and the ubiquitination of HSP70 occurred only in the reaction that has either intact K63 ubiquitin or wild type ubiquitin (Fig. 8B ). This finding is consistent with previous reports (45) , demonstrating that HSP70 is K63-polyubiquitinated. Furthermore, the in vivo interaction of HSP70 and p62 was confirmed by transfecting myc-tagged HSP70 into HEK 293 cells in the presence of the proteasome inhibitor MG132 and subjecting cell lysates to p62 immunoprecipitation and Western blot with anti-myc antibody (Fig. 8C) . The interaction between HSP70 and p62 in vivo took place only when MG132 was included, suggesting that the interaction in vivo is dependent upon the ubiquitination of HSP70. The specific type of polyubiquitin chain recognized by p62's UBA domain is not yet known and studies are underway lab to determine p62's interaction with specific polyubiquitin chains, however, our preliminary studies suggest that p62's UBA domain may recognize K63 linked polyubiquitin chains. Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin, resolved on 10% SDS-PAGE gels, transferred to nitrocellulose membrane and western blotted with ubiquitin monoclonal antibody. B: HSP70 Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin and 35 S-methionine, resolved on 10% SDS-PAGE and exposed to X-ray film. C: Western blot of rabbit reticulocyte lysate with TRAF6 (E3) and UbcH7 (E2). In summary, for the first time, we demonstrate a systematic approach to identify UBA domain binding proteins from a proteome wide perspective. This approach could be readily adapted to high throughput screening. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins in the human adult brain that interact with the UBA domain of p62, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer's disease. This is a very interesting finding since 9600 cDNAs have been screened and only 11 of them showed binding specificity with p62's UBA domain. Studies are underway to unfold the functional roles of p62 in the ubiquitin system. Our approach provides an easy route to the characterization of UBA domain binding proteins at the level of the whole proteome, its application will unfold the important roles that p62's UBA domain plays. This method could be easily adapted to identify proteins that interact with other UBA domains as well. Vaccinia virus infection disrupts microtubule organization and centrosome function We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function. Intracellular bacterial and viral pathogens have evolved numerous mechanisms to appropriate and exploit different systems of the host during their life cycles in order to facilitate their spread during entry and exit from the host (Cudmore et al., 1997; Finlay and Cossart, 1997; Dramsi and Cossart, 1998) . In the case of viruses, perhaps the best studied example is the exploitation of the actin cytoskeleton by vaccinia virus during its exit from infected cells (Cudmore et al., 1997) . Vaccinia virus is a large DNA virus with a genome of~191 kb encoding 260 open reading frames (ORFs) that is a close relative of variola virus, the causative agent of smallpox (Johnson et al., 1993; Massung et al., 1993) . Vaccinia virus morphogenesis is a complex process which occurs in the cytoplasm of infected cells and results in the formation of the intracellular mature virus (IMV) and the intracellular enveloped virus (IEV). IMV consist of a viral core of DNA and protein enveloped in a membrane cisterna derived from the intermediate compartment (Sodeik et al., 1993) . The IMV core contains ®ve major proteins, A3L, A4L, A10L, F17R and L4R (Vanslyke and Hruby, 1994; Jensen et al., 1996a) , while 12 proteins, A12L, A13L, A14L, A14.5L, A17L, A27L, D8L, G4L, G7L, H3L, I5L and L1R, are associated with the membranes around the virus particle (Jensen et al., 1996a; Betakova et al., 2000) . Depending on the virus strain and cell type, a proportion of IMV can become enwrapped by a membrane cisterna derived from the trans-Golgi apparatus to give rise to IEV particles (Schmelz et al., 1994) . To date, six IEV-speci®c proteins, A33R (Roper et al., 1996) , A34R (Duncan and Smith, 1992) , A36R (Parkinson and Smith, 1994) , A56R (Payne and Norrby, 1976; Shida, 1986) , B5R (Engelstad et al., 1992; Isaacs et al., 1992) and F13L (Hirt et al., 1986) , have been identi®ed. Studies using recombinant viruses have shown that A33R, A34R, B5R and F13L play an important role in IEV assembly (Blasco and Moss, 1991; Engelstad and Smith, 1993; Wolffe et al., 1993 Wolffe et al., , 1997 Roper et al., 1998; Sanderson et al., 1998a; Ro Èttger et al., 1999) . Vaccinia virus is thought to leave the cell by fusion of the outer IEV membrane with the plasma membrane, to give rise to the extracellular enveloped virus (EEV) (Morgan, 1976; Payne, 1980; Blasco and Moss, 1991) or the cell-associated enveloped viruses (CEV) which remain associated with the outer surface of the plasma membrane (Blasco and Moss, 1992) .
During the complex vaccinia infection process, the actin cytoskeleton is dramatically reorganized and numerous actin comet-like tails are induced by IEV particles (Cudmore et al., 1995; Ro Èttger et al., 1999) . Using actin polymerization as the driving force, IEV particles are propelled on actin tails until they contact the plasma membrane and extend outwards, thereby facilitating infection of neighbouring cells (Cudmore et al., 1995) . In addition, vaccinia infection results in stimulation of cell motility, loss of contact inhibition and changes in cell adhesion (Sanderson and Smith, 1998; Sanderson et al., 1998b) . Vaccinia virus-induced cell motility can be subdivided further into cell migration and extension of neurite-like projections, the latter of which is dependent on microtubules (Sanderson et al., 1998b) . The dependence of neurite-like projection formation on microtubules suggests that the microtubule cytoskeleton may also play a role during the life cycle of vaccinia virus. Indeed, recently, the vaccinia A27L protein and microtubules have been shown to be required for ef®cient IMV dispersion (Sanderson et al., 2000) . Furthermore, in the absence of vaccinia actin-based motility, cell to cell spread still occurs although it is less ef®cient (Wolffe et al., 1997 Sanderson et al., 1998a) , suggesting that additional transport mechanisms must exist.
Given these observations, we wondered whether the microtubule cytoskeleton has a function during the life Vaccinia virus infection disrupts microtubule organization and centrosome function The EMBO Journal Vol. 19 No. 15 pp. 3932±3944, 2000 cycle of vaccinia virus. We now report that the microtubule cytoskeleton and the dynein±dynactin complex play an important role during the early stages of vaccinia infection. However, later during the infection cycle, loss of centrosome function and accumulation of viral-encoded microtubule-associated proteins (MAPs) result in a dramatic rearrangement of the microtubule cytoskeleton.
Vaccinia localization in the vicinity of the MTOC depends on microtubules and the dynein±dynactin complex Indirect immuno¯uorescence labelling shows that by 6 h post-infection the majority of vaccinia virus particles are concentrated in the area coinciding with the centre of the microtubule aster ( Figure 1A and C). To examine whether this localization is indeed microtubule dependent, we infected cells pre-treated with nocodazole to depolymerize microtubules. In the absence of microtubules, virus particles were distributed throughout the cytoplasm ( Figure 1B and D) . The accumulation of virus particles in the area around the centre of the microtubule aster suggested that a microtubule minus end-directed motor may be involved in establishing the position of the virus in this location. To examine this possibility, we infected cells overexpressing p50/dynamitin which acts as a dominantnegative for dynein±dynactin function (Echeverri et al., 1996) . We found in cells overexpressing p50/dynamitin that virus particles did not accumulate at the centre of the microtubule aster but rather throughout the cytoplasm, as occurs in the absence of microtubules (compare Figure 2B with Figure 1D ).
As vaccinia morphogenesis involves wrapping by host membranes, it was possible that the effects of nocodazole and p50/dynamitin on virus localization were in fact due to disruption of the intermediate compartment and Golgi apparatus by these reagents (Burkhardt et al., 1997) . However, two independent experiments showed that this is not the case. First, in cells infected in the absence of microtubules, the Golgi apparatus as well as vaccinia virus particles are dispersed throughout the cytoplasm but do not co-localize ( Figure 3F and O). Secondly, vaccinia particles remain in the vicinity of the microtubule-organizing centre (MTOC) when the Golgi but not the microtubules was disrupted by treatment with brefeldin A ( Figure 3G and P). Similar results were obtained using other markers: A17L for vaccinia, galactosyltransferase for the Golgi or ERGIC53 for the intermediate compartment (data not shown). Taken together, our data indicate that the microtubule cytoskeleton is required for the localization of newly assembled virus particles in the vicinity of the MTOC during vaccinia infection.
Formation of functional IEV, but not IMV, is microtubule dependent Given the requirement for microtubules in vaccinia localization, we subsequently examined whether this localization has a role in morphogenesis of the two different intracellular forms of vaccinia virus, IMV and IEV. From electron microscopic examination of cells infected in the presence of nocodazole, it became clear that IMV particles which are morphologically indistinguishable from controls are formed ( Figure 4 ). Although IMV particles are assembled in the absence of microtubules, we wondered whether their number is reduced and whether those that are formed are infectious, since the integrity of the intermediate compartment depends on microtubules (Burkhardt et al., 1997) . To address this question, three independent virus stocks were prepared in the presence or absence of nocodazole. To simplify the interpretation of the data, we used the recombinant vaccinia virus mutant DF13L, which is unable to form IEV (Blasco and Moss, 1991) . The ®nal concentration of virus particles produced, Vaccinia uses and abuses the microtubule cytoskeleton as determined by the method of Joklik (1962) , was 30.2 6 5.2 3 10 10 particles/ml in the presence of microtubules and 9.0 6 6.7 3 10 10 particles/ml in the absence of microtubules. Although there is a 3-fold decrease in the number of virus particles formed in the absence of microtubules, the particles that are formed are infectious (data not shown).
While infectious IMV are formed in the absence of microtubules, we found no evidence for IEV formation, based on electron microscope examination of cells infected in the presence of nocodazole ( Figure 4 ). We did, however, observe IMV particles partially wrapped in trans-Golgi membranes most probably in the process of abortive IEV formation ( Figure 4D ). Given these data, we examined by indirect immuno¯uorescence whether low amounts of IEV particles are formed in the absence of microtubules. However, we could ®nd no evidence for colocalization of the IEV protein markers A36R, A34R or A33R with vaccinia particles formed in the presence of nocodazole ( Figure 5F ). We also found no evidence for IEV formation, based on their ability to nucleate actin tails ( Figure 5O ). As IEV particle assembly involves wrapping by the Golgi apparatus (Schmelz et al., 1994) , we examined the effects of only disrupting this membrane compartment using brefeldin A. We could ®nd no evidence for IEV formation, based on co-localization of IEV protein markers with virus particles and actin tails in cells infected in the presence of brefeldin A ( Figure 5G±I and P±R). Indeed, in brefeldin A-treated cells, the IEV membrane proteins required for assembly were observed in the endoplasmic reticulum and not the trans-Golgi ( Figure 5H ). In summary, our data indicate that the microtubule cytoskeleton is required for ef®cient IMV assembly and is essential for IEV formation.
In the course of our experiments, it became obvious that the Golgi apparatus becomes progressively dispersed during infection co-concominantly with disruption of the microtubule network ( Figure 6 ). Further analysis showed that during infection the normal morphology of the microtubule cytoskeleton is replaced by morphologically aberrant microtubule forms, which vary among each other but have in common the absence of a discrete MTOC ( Figure 7 ). These aberrant forms can be broadly classi®ed into three types: (i) cells with a disorganized microtubule network where microtubules seem randomly oriented ( Figure 7E ); (ii) cells in which microtubules form rings around the nucleus and throughout the cytoplasm ( Figure 7H ); or (iii) cells with long projections consisting of microtubule bundles ( Figure 7K ). We quanti®ed the appearance of the different morphological forms in ®ve independent infection experiments, in which 200 cells were counted for each time point for each experiment ( Figure 7C , F, I and L). Small compact cells, representing 20.7 6 2.6, 21.8 6 12.4 and 29.7 6 15.2% for 5, 8 and 24 h post-infection, respectively, in which the microtubule cytoskeleton morphology was not evident were not included in the analysis. Already by 5 h post-infection, when virus particle assembly has occurred, the normal aster microtubule con®guration has been disrupted and replaced in the majority of cells by microtubules without obvious organization from the MTOC ( Figure 7F ). Furthermore,~10% of cells have microtubule rings and 5% of cells have long projections by this time point ( Figure 7I and L). As the infection proceeds, microtubules become progressively more disrupted and bundled ( Figure 7I and L).
From our observations, there seems to be no obvious connection between the disruption and changes in the actin and the microtubule cytoskeletons ( Figure 7) . Moreover, the same reorganization of the microtubule network occurs in cells infected with the vaccinia deletion mutants DF13L and DA36R which do not make actin tails (data not shown). The effects of vaccinia virus infection on the reorganization of the microtubule cytoskeleton were also observed in all cell lines we examined (BHK-21, C 2 C 12 , PtK2, RK 13 and Swiss 3T3) to varying degrees (data not shown). Our data show that vaccinia infection results in severe disruption of the normal morphology of the microtubule cytoskeleton.
The formation of microtubule bundles and the loss of organization from the MTOC in vaccinia-infected cells is strongly reminiscent of the phenotype observed in cells overexpressing a MAP (Weisshaar et al., 1992; Togel et al., 1998) . As overexpression of MAPs stabilizes microtubules, we examined whether the microtubule cytoskeleton in infected cells was more resistant to depolymerization by nocodazole or cold treatment ( Figure 8 ). This was indeed the case, suggesting that the virus genome may encode viral proteins with MAP-like properties. To identify viral proteins which exhibit microtubule-binding properties, we performed microtubule co-sedimentation assays using extracts prepared from uninfected and vaccinia-infected cells (Figure 9 ). Initial experiments, however, revealed that intact virus particles in the extracts were prone to pellet even in the absence of microtubules, making identi®cation of viral MAPs impossible. To avoid this problem, we prepared extracts from cells infected in the presence of rifampicin, a drug that inhibits vaccinia virus particle assembly but does not affect viral protein expression (Moss et al., 1969; Tan and McAuslan, 1970) . The morphological effects of vaccinia infection on the microtubule cytoskeleton were the same in the presence or absence of rifampicin (data not shown). Comparison of the proteins present in pellets from microtubule co-sedimentation assays reveals that a number of additional prominent and minor bands are present in extracts prepared from vaccinia-infected but not from uninfected cells (Figure 9 ). Co-sedimentation assays Vaccinia uses and abuses the microtubule cytoskeleton performed in the presence of nocodazole or with coldtreated extracts reveal that the majority of these additional bands disappear in the absence of microtubules. To identify the viral proteins co-sedimenting with microtubules, we performed in-gel protease digestion followed by analysis of the resulting peptides by MALDI mass spectrometry. Using this approach, we identi®ed a number of potential vaccinia-encoded MAPs: A10L (a structural protein), I1L and L4R (which are DNA-binding proteins), all of which are associated with viral cores (Vanslyke and Hruby, 1994; Jensen et al., 1996a; Klemperer et al., 1997) , and A6L which is conserved in all poxvirus genomes but is of unknown function (Figure 9 ).
A10L and L4R associate with microtubules in vivo and mediate binding of viral cores to microtubules in vitro Using available antibodies, we examined the localization of A10L, L4R and I1L in infected cells to see whether they associate with microtubules in vivo, in addition to their essential role in the virus core (Vanslyke and Hruby, 1994; Jensen et al., 1996a) . As a negative control, we also examined the localization of the A3L core protein which was identi®ed as the prominent 70 kDa protein pelleting in the absence of microtubules (Figure 9 ). Indirect immunouorescence analysis showed that A10L and L4R are associated with microtubules, in both the presence and absence of rifampicin ( Figure 10 ). As expected, A10L and L4R were also associated with viral particles (data not shown). In contrast, I1L and A3L were never observed in association with microtubules, regardless of the ®xation conditions, but were localized to viral factories and viral particles, respectively (data not shown). Interestingly, A10L and L4R were not associated with all microtubules but were co-localized with a subset of acetylated microtubules ( Figure 10) .
The association of A10L and L4R with virus particles and microtubules raises the question of whether there is a role for this microtubule-binding activity during infection. We wondered whether these two proteins mediate the interaction of incoming viral cores with microtubules at the beginning of infection, as cores and not virus particles are released in the cytoplasm at the start of the infection cycle (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) . To examine this possibility, we investigated whether puri®ed viral cores would bind microtubules in vitro. We found that viral cores were able to bind microtubules, while protease-treated cores showed no association ( Figure 11A and B) . Pre-incubation of puri®ed viral cores with antibodies against A10L and L4R speci®cally inhibited the interaction of viral cores with microtubules ( Figure 11C and D); in contrast, IgG or antibodies against A3L had no inhibitory effect ( Figure 11E and F). Taken together, our data suggest that A10L and L4R have MAP-like properties and may play a role in mediating interactions of incoming viral cores with microtubules.
The dramatic rearrangement of the microtubule cytoskeleton which occurs during vaccinia infection is unlikely to be attributed exclusively to the action of A10L and L4R since they only associate with a subset of microtubules ( Figure 10) . Furthermore, the loss of microtubule organization precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. We therefore wondered whether vaccinia infection disrupts centrosome function, given the loss of microtubule aster con®guration during infection (Figure 7 ). Since microtubules are nucleated by the centrosome in animal cells, we examined whether vaccinia infection affects g-tubulin, which is critically required for this process (Stearns and Kirschner, 1994) . We observed that g-tubulin labelling of the centrosome is greatly reduced from as early as 2 h post-infection ( Figure 12 ). The same result was obtained when we infected PtK1 cells stably expressing green¯uorescent protein (GFP)-labelled g-tubulin (Khodjakov and Rieder, 1999) . In addition, the centrosomal and centriolar components pericentrin, C-Nap 1, Nek 2 and centrin are reduced by immuno¯uorescence in the centrosomes/centrioles of vaccinia-infected cells ( Figure 12) . Furthermore, the reduction of centrosomal markers requires viral protein synthesis as their levels are not affected when cells are infected in the presence of cycloheximide (data not shown).
The dramatic reduction of g-tubulin from the centrosome implies that vaccinia infection perturbs centrosome Vaccinia uses and abuses the microtubule cytoskeleton function. To test this hypothesis, we examined whether the centrosome in vaccinia-infected cells could re-nucleate microtubules, following their depolymerization by nocodazole. We found that by 2 h post-infection, when we already see a reduction in g-tubulin, microtubule nucleation from the centrosome was very inef®cient, as compared with uninfected controls, indicating that vaccinia has disrupted`normal' centrosome function (Figure 13 ). At later times post-infection, microtubule re-nucleation ef®ciency from the centrosome was even lower (data not shown). However, following nocodazole washout, microtubules eventually are repolymerized throughout the cytoplasm of infected cells but do not display any organization from the MTOC, as do controls (compare Figure 13I and K).
The size of virus particles is such that they are unlikely to move within and between cells by diffusion alone, suggesting that their movements will require interactions with the host cytoskeleton. Previous data have shown that vaccinia virus both disrupts and hijacks the actin cytoskeleton to facilitate movement of the intracellular enveloped form of vaccinia virus (Cudmore et al., 1995; Ro Èttger et al., 1999) and of the infected cell itself (Sanderson and Smith, 1998; Sanderson et al., 1998b) . The data described here now show that vaccinia also uses and subsequently disrupts the microtubule cytoskeleton during its infection cycle. It is clear from our experiments and the previous observations of Ulaeto et al. (1995) that microtubules are required to maintain the integrity of the Golgi apparatus which is in turn required for IMV wrapping to form IEV (Schmelz et al., 1994) . In contrast, IMV are assembled in the absence of microtubules, albeit at reduced levels. While microtubules are not required for IMV assembly, they are required together with the dynein±dynactin complex for virion accumulation in the vicinity of the microtubule aster. One can envisage that minus enddirected microtubule-dependent movements of IMV particles from their site of assembly in the viral factory towards the MTOC, by the dynein±dynactin complex, would enhance the possibility of wrapping with the Golgi apparatus and subsequent IEV formation. Recently it has been shown that the IMV protein A27L and microtubules are required for ef®cient IMV dispersion from the viral factories (Sanderson et al., 2000) . In the absence of A27L, mature IMV particles accumulate at the periphery of the virus factory but do not subsequently wrap to form IEV, presumably because they are unable to move on microtubules (Sanderson et al., 2000) .
The microtubule-and dynein±dynactin-dependent accumulation of vaccinia in the vicinity of the MTOC is analogous to the microtubule-dependent movements required for herpes simplex virus 1 (HSV-1) and adenovirus to reach their site of replication in the nucleus (Sodeik et al., 1997; Suomalainen et al., 1999; Leopold et al., 2000) . In the case of HSV-1, the UL34 protein, 9 . Vaccinia encodes proteins that co-sediment with microtubules. Analysis of pellets from in vitro microtubule co-sedimentation assays performed with protein extracts from vaccinia-infected (inf.) and uninfected (uninf.) cells. Twice the amount of pellet has been loaded in control assays performed in the absence of microtubules (nocodazole or 4°C). Proteins co-sedimenting with microtubules that were only present in extracts from infected cells are indicated by an asterisk. The identity of proteins determined by in-gel proteolysis MALDI mass spectrometry is indicated (arrowheads).
which is associated with the incoming nucleocapsids, interacts with the intermediate chain of cytoplasmic dynein (IC-1a) (Ye et al., 2000) . It has also been reported that incoming nucleocapsids of pseudorabies virus, an alphaherpes virus closely related to HSV-1, are associated with and dependent on microtubules for their movement to the nucleus (Kaelin et al., 2000) . This interaction may be mediated by the UL25 protein, a minor but essential component of the capsid, which co-localizes with microtubules and accumulates at the MTOC (Kaelin et al., 2000) . The accumulation of UL25 at the MTOC is consistent with a possible interaction with the dynein± dynactin motor complex which is known to be localized at the MTOC (Echeverri et al., 1996) . It would not be surprising, based on observations with HSV-1 and pseudorabies, if microtubules and dynein±dynactin were also involved in establishing the infection cycle of cytomegalovirus (CMV), Epstein±Barr virus and varicella-zoster virus, all of which are herpes viruses. The other clear example of microtubule-dependent virus movements during the establishment of infection is that of incoming human foamy virus (HFV) which is dependent on microtubules and presumably a minus end-directed microtubule motor to get to its nuclear replication site (Saib et al., 1997) . In the absence of protein expression, HFV Gag proteins, which are associated with the viral genome, accumulate at the centrosome in a microtubuledependent fashion prior to nuclear import (Saib et al., 1997) . The centrosomal accumulation of Gag proteins of HFV, however, appears to be unique for this class of retroviruses as no similar localization has been reported for human immunode®ciency virus (HIV) or other retroviruses. On the other hand, the Gag protein of murine leukaemia virus and HIV has been shown to interact with KIF4, a microtubule plus end-directed kinesin motor, both in vitro and in vivo (Kim et al., 1998; Tang et al., 1999) , suggesting that additional roles may exist for microtubules and motors during the outward movement of virus particles. Indeed, vaccinia virus particles are able to reach the cell periphery in the absence of actin-based motility (see images in Wolffe et al., 1997; Sanderson et al., 1998a Sanderson et al., , 2000 Ro Èttger et al., 1999) , suggesting that viral particles can also move out on microtubules (Sanderson et al., 2000) . Microtubule-dependent motordriven movements of virus particles represent an ef®cient mechanism to achieve a peri-nuclear localization, required to facilitate entry into the nucleus during establishment of infection. They also provide an excellent way for newly assembled virus particles to reach the cell periphery, facilitating the continued spread of infection.
Our data show that although vaccinia virus uses the microtubule cytoskeleton to achieve a peri-nuclear localization, microtubule and Golgi organization becomes disrupted later during the infection process. Interestingly, HSV-1 and CMV have also been reported to disrupt the microtubule cytoskeleton and Golgi organization in their infection cycles (Avitabile et al., 1995; Fish et al., 1996) . While disruption of the microtubule network might at ®rst sight not appear to be bene®cial to the virus, it may not actually hinder viral spread but could enhance it. First, extensive virus assembly and spread to the cell periphery have already occurred by the time the microtubule cytoskeleton and Golgi organization are disrupted. Secondly, disruption of microtubule organization may overcome potential microtubule motor anchoring effects at the MTOC, thus allowing viral spread to the periphery to occur more easily. Lastly, the formation of long projections of up to 200 mm supported by extensive microtubule bundles provides a means to achieve long range spread of virus particles (Sanderson et al., 1998b) .
It is clear that disruption and reorganization of the microtubule cytoskeleton by vaccinia virus is mediated by the combined effects of viral proteins with MAP-like properties and loss of microtubule-organizing function from the MTOC. The same may also be true for HSV-1, although disruption of centrosome function remains to be established, as late in infection microtubules are organized in bundles around the nucleus and do not show MTOCorchestrated organization (Avitabile et al., 1995) . The identi®cation of viral proteins with MAP-like properties is not unique to vaccinia virus. The VP22 tegument protein from HSV-1 co-localizes with microtubules in infected cells and induces microtubule bundles when expressed in uninfected cells (Elliott and O'Hare, 1998) . Other examples of viral MAPs based on their in vivo localization Fig. 11 . Vaccinia cores bind directly to microtubules in vitro. Puri®ed viral cores labelled by DAPI (green) bind to rhodamine-labelled microtubules (red) in the absence of ®xation (A). Binding to microtubules is not observed if cores are pre-treated with protease (B) or pre-incubated with antibodies against the A10L (C) or L4R (D) proteins. In contrast, pre-incubation of puri®ed viral cores with control IgG (E) or antibody against the A3L protein (F) does not inhibit their interaction with microtubules. Scale bar = 5 mm.
Vaccinia uses and abuses the microtubule cytoskeleton or in vitro association with microtubules are the N protein from murine coronavirus (Kalicharran and Dales, 1996) , the movement protein from tobamovirus (Heinlein et al., 1995) , the aphid transmission factor from cauli¯ower mosaic virus (Blanc et al., 1996) , the UL25 protein from pseudorabies virus (Kaelin et al., 2000) , the VP4 spike protein from rotavirus (Nejmeddine et al., 2000) and the M protein of vesicular stomatitis virus (VSV) (Melki et al., 1994) . The identi®cation of A10L and L4R, two viral core proteins, as MAP-like proteins was, however, unexpected given their previously characterized role in viral morphogenesis (Vanslyke and Hruby, 1994) . The interaction of A10L and L4R with microtubules in vivo, together with the in vitro microtubule-binding data, suggest a potential mechanism for the association of viral cores with microtubules. One could envisage that viral cores which are released into the cytoplasm at the beginning of infection (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) bind directly to microtubules in a manner analogous to adenovirus or HSV-1 nucleocapsids. Further work is required to determine whether incoming cores do in fact move towards the MTOC by the dynein± dynactin complex and/or use the complex for anchoring on microtubules.
The loss of centrosome function must enhance disruption of the microtubule cytoskeleton during infection. Indeed, the loss of microtubule organization from the MTOC precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. Vaccinia-induced loss of centrosomal proteins is inhibited by cycloheximide, indicating that viral protein expression is required for disruption of the centrosome microtubule nucleation activity. To our knowledge, vaccinia virus infection represents the ®rst example of virus-induced disruption of centrosome function, although we would predict that HSV-1 may have a similar effect. The mechanism by which vaccinia virus disrupts the centrosome requires further study; nevertheless, it is clear that understanding the molecular basis of this disruption will provide important insights into the regulation and stability of centrosome function which currently is the subject of intense research (Ohta et al., 1993; Lane and Nigg, 1997; Karsenti, 1999) .
HeLa cells (ATCC CCL2) were infected with the wild-type vaccinia virus strain Western Reserve (WR) or with the vaccinia deletion mutants DF13L (vRB12) (Blasco and Moss, 1991) or DA36R (Parkinson and Smith, 1994) at a multiplicity of infection of 1 p.f.u. (plaque-forming unit) per cell, as described previously (Ro Èttger et al., 1999) . Nocodazole dissolved in dimethyl sulfoxide (DMSO) and brefeldin A dissolved in ethanol were added to the culture medium to ®nal concentration of 10 mM and 5 mg/ml, respectively unless otherwise stated. In non-treated controls, an equal volume of DMSO or ethanol was added. Cells transfected with a myc-tagged p50/dynamitin expression construct (Echeverri et al., 1996) were infected 24 h later with WR and subsequently ®xed 6 h postinfection. All experiments described have been repeated 3±10 times.
The following antibodies were kindly provided: anti-a-tubulin by Dr E.Karsenti, anti-centrin (20H5) by Professor J.L.Salisbury (Sanders and Salisbury, 1994; Paoletti et al., 1996) , anti-Nek2 and anti-C-Nap1 by Professor E.Nigg (Fry et al., 1998a,b) , anti-myc and anti-gp27 by Dr T.Nilsson (Fu Èllekrug et al., 1999) and antibodies against the corresponding vaccinia proteins: A3L, A10L and L4R by Professor D.Hruby (Vanslyke and Hruby, 1994) , I1L by Professor P.Traktman (Klemperer et al., 1997) , A27L (C3) by Dr M.Esteban (Rodriguez et al., 1985) , A33R, A34R and A36R (Ro Èttger et al., 1999) . In addition, the following antibodies were obtained from commercial sources: anti-a-tubulin (N356) (Amersham International, UK), anti-acetylated a-tubulin (6-11B-1) (Sigma, USA), anti-g-tubulin (GTU-88; Sigma), anti-pericentrin and anti-TGN46 (BAbCO, USA), and rabbit IgG (Sigma). Actin was visualized with¯uorescently labelled phalloidin derivatives (Molecular Probes, USA).
Cells were ®xed in ±20°C methanol or in 5% paraformaldehyde in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) followed by 0.1% Triton X-100 permeabilization. Fixed cells were processed for immuno¯uorescence, viewed and images recorded as described previously (Ro Èttger et al., 1999) .
HeLa cells were pre-incubated with 25 mM nocodazole in the medium for 1 h to depolymerize microtubules, prior to infection with vaccinia DF13L at 1 p.f.u./cell. Nocodazole was kept in the medium throughout the infection, while an equal volume of DMSO was added to the controls. At 24 h post-infection, the cells were scraped from the¯asks into the medium and sedimented by centrifugation (300 g, 7 min, 4°C). The cell membranes were disrupted and the nuclei were removed by centrifugation. The resulting post-nuclear supernatant was centrifuged through a 36% sucrose cushion (76 000 g, 30 min, 4°C). The virus pellet was resuspended in 10 mM Tris pH 9; the virus was collected by centrifugation (76 000 g, 30 min, 4°C), resuspended in 10 mM Tris pH 9 and stored at ±80°C. The concentration of the virus (elemental bodies) was determined by OD 260 measurement (Joklik, 1962) . Fig. 13 . Vaccinia infection reduces centrosome microtubule nucleation ef®ciency. In uninfected cells, microtubules (A, E and I) nucleate from centrosomes (B, F and J) after nocodazole washout for the times indicated. In contrast, 2 h after infection with vaccinia, microtubules (C, G and K) are nucleated inef®ciently from centrosomes (D, H and L). All images were collected with identical camera settings, to allow comparison of uorescence intensity between centrosomes. Inserts (B, D, F, H, J and L) are adjusted as in Figure 12 to facilitate visualization of the weak g-tubulin centrosomal labelling. Arrowheads indicate the position of the centrosome. Scale bar = 10 mm.
In vitro microtubule binding assays Puri®ed EEV particles were prepared as described previously (Ro Èttger et al., 1999) and subsequently were used to prepare virus cores following the method of Cudmore et al. (1996) . Rhodamine-labelled microtubules were prepared according to Hyman et al. (1991) . Vaccinia virus cores were incubated with rhodamine-labelled microtubules in BRB80 buffer containing 10 mM taxol for 5 min at room temperature. 4¢,6-diamidino-2phenylindole (DAPI) was added subsequently to a ®nal concentration of 0.1 mg/ml to label the virus cores. Finally, the mixture was diluted 1:1± 1:10 with antifade solution (0.1 mg/ml catalase, 0.1 mg/ml glucose oxidase, 10 mM glucose) and viewed without ®xation. Proteinase K or trypsin treatment of core particles prior to incubation with microtubules was performed as described previously (Roos et al., 1996) . Anti-A3L, A10L, L4R or control IgG antibodies were incubated with puri®ed cores for 1 h at room temperature prior to incubation with microtubules.
Cell extracts and microtubule co-sedimentation assay Extracts from HeLa cells infected for 24 h or uninfected controls maintained in the presence of 0.1 mg/ml rifampicin were prepared as described previously (Ro Èttger et al., 1999) . The extract was clari®ed by centrifugation at 150 000 g for 20 min at 4°C and cytochalasin D added to a ®nal concentration of 1 mg/ml to depolymerize actin ®laments. Endogenous tubulin in the extract was polymerized in a two-step procedure. First, the extract supernatant was supplemented with protease inhibitors, 2 mM MgGTP and 5 mM taxol and incubated for 5 min at room temperature; subsequently, an additional 15 mM taxol was added to the mix and the reaction incubated at 33°C for 30 min. For controls, no taxol was added at any stage and microtubule polymerization was inhibited either by the addition of nocodazole to a ®nal concentration of 40 mM or by maintaining the extract at 4°C throughout the experiment. Following microtubule assembly, each 400 mg extract reaction was diluted 5-fold in BRB80 buffer (containing protease inhibitors and 20 mM taxol) and centrifuged through a 10% sucrose cushion containing protease inhibitors and 20 mM taxol at 165 000 g for 20 min at 25°C. The microtubule pellet was solubilized in SDS±PAGE sample buffer and analysed by SDS± PAGE.
In-gel proteolytic cleavage was performed automatically in the`Progest' as described (Houthaeve et al., 1997) [Genomic Solutions Cambridge (http://www.genomicsolutions.com)] and the peptides obtained were analysed on a Bruker REFLEX MALDI mass spectrometer (Bruker Analytik, Germany) (Jensen et al., 1996b) . Proteins were identi®ed by peptide mass ®ngerprinting (Jensen et al., 1997) using the program PeptideSearch (http://www.narrador.embl-heidelberg.de/Services/ PeptideSearch/PeptideSearchIntro.html.
At 1 h post-infection, nocodazole was added to the culture medium to a ®nal concentration of 25 mM to depolymerize microtubules. At 2 h postinfection, the cells were washed 3±4 times in warm medium to remove nocodazole. Washed cells were incubated in medium without nocodazole for the indicated time at 37°C to re-initiate microtubule polymerization; they were then washed brie¯y in warm phosphate-buffered saline (PBS) and immediately ®xed. In parallel, samples were also removed at the same time point, brie¯y rinsed in ice-cold PBS, ®xed and processed for immuno¯uorescence to con®rm complete microtubule depolymerization before initiation of microtubule assembly. Uninfected control HeLa cells were treated and processed in an identical fashion. The same numbers of images were integrated using identical camera settings to allow direct comparison between infected and uninfected samples from the same experiment. The site of origin of the 1918 influenza pandemic and its public health implications  The 1918-1919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.
It has never been clear, however, where this pandemic began. Since influenza is an endemic disease, not simply an epidemic one, it is impossible to answer this question with absolute certainty. Nonetheless, in seven years of work on a history of the pandemic, this author conducted an extensive survey of contemporary medical and lay literature searching for epidemiological evidence -the only evidence available. That review suggests that the most likely site of origin was Haskell County, Kansas, an isolated and sparsely populated county in the southwest corner of the state, in January 1918 [1] . If this hypothesis is correct, it has public policy implications.
But before presenting the evidence for Haskell County it is useful to review other hypotheses of the site of origin. Some medical historians and epidemiologists have theorized that the 1918 pandemic began in Asia, citing a lethal outbreak of pulmonary disease in China as the forerunner of the pandemic. Others have speculated the virus was spread by Chinese or Vietnamese laborers either crossing the United States or working in France.
More recently, British scientist J.S. Oxford has hypothesized that the 1918 pandemic originated in a British Army post in France, where a disease British physicians called "purulent bronchitis" erupted in 1916. Autopsy reports of soldiers killed by this outbreak -today we would classify the cause of death as ARDS -bear a striking resemblance to those killed by influenza in 1918 [2] .
But these alternative hypotheses have problems. After the 1918-1919 pandemic, many investigators searched for the source of the disease. The American Medical Association sponsored what is generally considered the best of several comprehensive international studies of the pandemic conducted by Dr. Edwin Jordan, editor of The Journal of Infectious Disease. He spent years reviewing evidence from all over the world; the AMA published his work in 1927.
Since several influenza pandemics in preceding centuries were already well-known and had come from the orient, Jordan first considered Asia as the source. But he found no evidence. Influenza did surface in early 1918 in China, but the outbreaks were minor, did not spread, and contemporary Chinese scientists, trained by Rockefeller Institute for Medical Research (now Rockefeller University) investigators, stated they believed these outbreaks were endemic disease unrelated to the pandemic [3] . Jordan also looked at the lethal pulmonary disease cited by some historians as influenza, but this was diagnosed by contemporary scientists as pneumonic plague. By 1918 the plague bacillus could be easily and conclusively identified in the laboratory [3] . So after tracing all known outbreaks of respiratory disease in China, Jordan concluded that none of them "could be reasonably regarded as the true forerunner" of the pandemic [3] .
Jordan also considered Oxford's theory that the "purulent bronchitis" in British Army camps in 1916 and 1917 was the source. He rejected it for several reasons. The disease had flared up, true, but had not spread rapidly or widely outside the affected bases; instead, it seemed to disappear [3] . As we now know a mutation in an existing influenza virus can account for a virulent flare-up. In the summer of 2002, for example, an influenza epidemic erupted in parts of Madagascar with an extremely high mortality and morbidity; in some towns it sickened an outright majority -in one instance sixty-seven percent -of the population. But the virus causing this epidemic was an H3N2 virus that normally caused mild disease. In fact, the epidemic affected only thirteen of 111 health districts in Madagascar before fading away [4]. Something similar may have happened in the British base.
Jordan considered other possible origins of the pandemic in early 1918 in France and India. He concluded that it was highly unlikely that the pandemic began in any of them [3] .
That left the United States. Jordan looked at a series of spring outbreaks there. The evidence seemed far stronger. One could see influenza jumping from Army camp to camp, then into cities, and traveling with troops to Europe. His conclusion: the United States was the site of origin.
A later equally comprehensive, multi-volume British study of the pandemic agreed with Jordan. It too found no evidence for the influenza's origin in the Orient, it too rejected the 1916 outbreak among British troops, and it too concluded, "The disease was probably carried from the United States to Europe [5] ." Australian Nobel laureate MacFarlane Burnet spent most of his scientific career working on influenza and studied the pandemic closely. He too concluded that the evidence was "strongly suggestive" that the disease started in the United States and spread with "the arrival of American troops in France [6] ."
Before dismissing the conclusions of these contemporary investigators who lived through and studied the pandemic, one must remember how good many of them were. They were very good indeed.
The Rockefeller Institute, whose investigators were intimately involved in the problem, alone included extraordinary people. By 1912 its head Simon Flexner -his brother wrote the "Flexner report" that revolutionized American medical education -used immune serum to bring the mortality rate for meningococcal meningitis down from over 80% to 18%; by contrast, in the 1990s at Massachusetts General Hospital a study found a 25% mortality rate for bacterial meningitis. Peyton Rous won the Nobel Prize in 1966 for work he did at the institute in 1911; he was that far ahead of the scientific consensus. By 1918 Oswald Avery and others at Rockefeller Institute had already produced both an effective curative serum and a vaccine for the most common pneumococcal pneumonias. At least partly because of the pandemic, Avery would spend the rest of his career studying pneumonia. That work led directly to his discovery of the "transforming principle"his discovery that DNA carries the genetic code.
The observations of investigators of this quality cannot be dismissed lightly. Jordan was of this quality.
More evidence against Oxford's hypothesis comes from Dr. Jeffrey Taubenberger, well-known for his work extracting samples of the 1918 virus from preserved tissue and sequencing its genome. He initially believed, based on statistical analysis of the rate of mutation of the virus that it existed for two or three years prior to the pandemic. But further work convinced him that the virus emerged only a few months prior to the pandemic (personal communication with the author from J Taubenberger, June 5 th 2003).
So if the contemporary observers were correct, if American troops carried the virus to Europe, where in the United States did it begin?
Both contemporary epidemiological studies and lay histories of the pandemic have identified the first known outbreak of epidemic influenza as occurring at Camp Funston, now Ft. Riley, in Kansas. But there was one place where a previously unknown -and remarkable -epidemic of influenza occurred.
Haskell County, Kansas, lay three hundred miles to the west of Funston. There the smell of manure meant civilization. People raised grains, poultry, cattle, and hogs. Sod-houses were so common that even one of the county's few post offices was located in a dug-out sod home. In 1918 the population was just 1,720, spread over 578 square miles. But primitive and raw as life could be there, science had penetrated the county in the form of Dr. Loring Miner. Enamored of ancient Greece -he periodically reread the classics in Greek -he epitomized William Welch's comment that "the results [of medical education] were better than the system." His son was also a doctor, trained in fully scientific ways, serving in the Navy in Boston.
In late January and early February 1918 Miner was suddenly faced with an epidemic of influenza, but an influenza unlike any he had ever seen before. Soon dozens of his patients -the strongest, the healthiest, the most robust people in the county -were being struck down as suddenly as if they had been shot. Then one patient pro-gressed to pneumonia. Then another. And they began to die. The epidemic got worse. Then, as abruptly as it came, it disappeared. Men and women returned to work. Children returned to school. And the war regained its hold on people's thoughts.
The disease did not, however, slip from Miner's thoughts. Influenza was neither a reportable disease, nor a disease that any state or federal public health agency tracked. Yet Miner considered this incarnation of the disease so dangerous that he warned national public health officials about it. Public Health Reports (now Morbidity and Mortality Weekly Report), a weekly journal produced by the U.S. Public Health Service to alert health officials to outbreaks of communicable diseases throughout the world, published his warning. In the first six months of 1918, this would be the only reference in that journal to influenza anywhere in the world.
Historians and epidemiologists have previously ignored Haskell most likely because his report was not published until April and it referred to deaths on March 30, after influenza outbreaks elsewhere. In actuality, by then the county was free of influenza. Haskell County, Kansas, is the first recorded instance anywhere in the world of an outbreak of influenza so unusual that a physician warned public health officials. It remains the first recorded instance suggesting that a new virus was adapting, violently, to man.
If the virus did not originate in Haskell, there is no good explanation for how it arrived there. There were no other known outbreaks anywhere in the United States from which someone could have carried the disease to Haskell, and no suggestions of influenza outbreaks in either newspapers or reflected in vital statistics anywhere else in the region. And unlike the 1916 outbreak in France, one can trace with perfect definiteness the route of the virus from Haskell to the outside world.
All Army personnel from the county reported to Funston for training. Friends and family visited them at Funston. Soldiers came home on leave, then returned to Funston.
The Monitor reported in late February, "Most everybody over the country is having lagrippe or pneumonia (Santa Fe Monitor, February 21 st 1918)." It also noted, "Dean Nilson surprised his friends by arriving at home from Camp Funston on a five days furlough. Dean looks like soldier life agrees with him." He soon returned to the camp. Ernest Elliot left to visit his brother at Funston as his child fell ill. On February 28, John Bottom left for Funston. "We predict John will make an ideal soldier," said the paper (Santa Fe Monitor February 28 th , 1918).
These men, and probably others unnamed by the paper, were exposed to influenza and would have arrived in Funston between February 26 and March 2. On March 4 the first soldier at the camp reported ill with influenza at sick call. The camp held an average of 56,222 troops. Within three weeks more than eleven hundred others were sick enough to require hospitalization, and thousands morethe precise number was not recorded -needed treatment at infirmaries scattered around the base.
Whether or not the Haskell virus did spread across the world, the timing of the Funston explosion strongly suggests that the influenza outbreak there did come from Haskell. Meanwhile Funston fed a constant stream of men to other American locations and to Europe, men whose business was killing. They would be more proficient at it than they knew.
Soldiers moved uninterrupted between Funston and the outside world, especially to other Army bases and France. On March 18, Camps Forrest and Greenleaf in Georgia saw their first cases of influenza and by the end of April twenty-four of the thirty-six main Army camps suffered an influenza epidemic [3] . Thirty of the fifty largest cities in the country also had an April spike in excess mortality from influenza and pneumonia [7] . Although this spring wave was generally mild -the killing second wave struck in the fall -there were still some disturbing findings. A subsequent Army study said, "At this time the fulminating pneumonia, with wet hemorrhagic lungs, fatal in from 24 to 48 hours, was first observed [8] ." (Pathology reports suggest what we now call ARDS.) The first recorded autopsy in Chicago of an influenza victim was conducted in early April. The pathologist noted, "The lungs were full of hemorrhages." He found this unusual enough to ask the then-editor of The Journal of Infectious Diseases "to look over it as a new disease" [3] .
By then, influenza was erupting in France, first at Brest, the single largest port of disembarkation for American troops. By then, as MacFarlane Burnet later said, "It is convenient to follow the story of influenza at this period mainly in regard to the army experiences in America and Europe [6] ."
The fact that the 1918 pandemic likely began in the United States matters because it tells investigators where to look for a new virus. They must look everywhere.
In recent years the World Health Organization and local public health authorities have intervened several times when new influenza viruses have infected man. These interventions have prevented the viruses from adapting to man and igniting a new pandemic. But only 83 countries in the world -less than half -participate in WHO's surveillance system (WHO's flunet website http:// rhone.b3e.jussieu.fr/flunet/www/docs.html). While some monitoring occurs even in those countries not formally affiliated with WHO's surveillance system, it is hardly adequate. If the virus did cross into man in a sparsely populated region of Kansas, and not in a densely populated region of Asia, then such an animal-to-man cross-over can happen anywhere. And unless WHO gets more resources and political leaders move aggressively on the diplomatic front, then a new pandemic really is all too inevitable. Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients. There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients.
Microarray technology has been proven to be a powerful tool with great potential for biological and medical uses. In this technique, recombinant DNA fragments or synthesized oligonucleotides affixed on the surface of glass slides or nylon membranes are used for detecting complementary nucleic acid sequences (frequently representing a few hundred to >10,000 genes/expressed sequence tags) as well as for genotyping microorganisms and for profiling the gene-expression patterns in cells from higher organisms [1].
A new report by Ghedin, et al. [2] in Retrovirology describes the successful use of a multi-virus array (termed multivi-rus-chip) to detect multiple viral co-infections in cultured cells as well as to study viral gene expression and promoter activities (Figure 1 ). Ghedin's multivirus-chip contains genes from eight human viruses including human immunodeficiency virus type 1 (HIV-1). Conceptually, this chip can be used to detect viral co-infections in AIDS patients who are frequently rendered susceptible to additional opportunistic infections. In developing their multivirus-chip, Ghedin, et al. tested more than 250 ORFs from HIV-1, human T cell leukemia virus types 1 (HTLV-1) and 2 (HTLV-2), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human herpesvirus 6A (HHV6A) and 6B (HHV6B), and Kaposi's sarcoma-associated herpesvirus (KSHV) which were PCR-amplified and spotted on glass slides. They then hybridized their slides with Cy3-or Cy5labeled genomic DNA or cDNAs derived from various virus-infected cells. Their multivirus-chip was found to be highly specific and sensitive for detecting different viral genomic sequences in cell lines. Moreover, the chip could also detect the effect of various drugs on viral gene expression. In such instance, cell lines latently infected with HIV-1 and KSHV were used to generate profiles of viral gene expression in the presence of cyclin-dependent Schematic drawing of the multivirus-chip that possesses multiple functions kinase inhibitor (CKI), Roscovitine, which was applied to cells to suppress the reactivation of latently infected viruses.
Ghedin, et al. [2] also studied the role of cellular chromatin structure on viral gene expression using their multivirus-chip.
They employed the chromatin immunoprecipitation technique (ChIP) [3] to isolate cellular DNA fragments that were bound to phosphorylated histone H3 (P-H3). These DNA fragments were hybridized to the viral ORFs contained on the multivirus-chip to investigate the role of phospho-H3 on viral gene expression. They showed that whether transcriptionally active or silent the chromatin state played a role in regulating the expression of KSHV genes under the different cellular context. Current routine clinical diagnostics employ PCR, Southern blotting, Northern blotting, DNA sequencing and microarray hybridization to detect and characterize genes of interest in biomedicine. PCR is generally regarded as the most sensitive diagnostic method. However, Iyer, et al. [4] have shown that the sensitivity of cDNA-chip hybridization is comparable to that of TaqMan-driven quantitative PCR assay, and that the microarray hybridization technique is less likely to be complicated by high false positive rates due to carry-over contaminations. Furthermore, using microarrays, the viral gene transcripts in infected cells can be easily detected by hybridization without any prior amplification steps, and the microarray technique requires much less experimental material when compared to Southern or Northern blotting and can provide high sensitivity in the setting of large throughput.
In view of the above, the multivirus-chip described in Retrovirology [2] holds several advantages over other more commonly used techniques (e.g. PCR, DNA sequencing) for the diagnosis of viral infections. First, this chip provides a more accurate diagnosis of viral infection by simultaneously evaluating the transcription of all viral genes, and can use such cumulative data to correlate infection with clinical disease manifestations. Second, the high throughput and flexible synthesis nature of DNA microarray construction can allow scientists to tailor-make and rapidly alter arrays to match evolving emergence of new pathogens. The SARS genome chip made by the US NIAID, NIH is a good example [5] of how diagnostic arrays can be developed quickly and be used in a timely manner.
Finally, the most novel application described by Ghedin, et al. is their use of microarrays to correlate the cellular "histone code" [6] with the promoter activity of KSHV. Usually the transcription of a gene located on chromosomal DNA is influenced not only by the cis-acting ele-ments (or DNA-binding motifs), but also by the structure of chromatin. The latter can be vary depending on the post-translational modifications of histone proteins. Methylation, acetylation, and/or phosphorylation of certain amino acid residues at the amino terminal "tails" of histone H3 and/or H4 can indeed influence chromatin structure. Thus accumulating evidence has shown that chromatin-associated proteins and their modifications play vital roles in many physiological processes such as growth, differentiation, and development in mammals, plants and fungi [6, 7] . Many studies have used DNA array technology to investigate viral gene expression or to genotype viral isolates; however, none has used this technique to study the influence of cellular chromatin structure on viral gene expression [1]. Ghedin, et al. [2] demonstrated that only DNA fragments derived from ChIP of latent BCBL-1 cell genomic DNA captured using phospho-H3 antibody bound specifically to the KSHV ORF on the multivirus-chip. This result suggests that latent KSHV genome in BCBL-1 cells is packed into a nucleosomal structure and that histone H3 proteins near the viral promoter can be phosphorylated at serines to make the DNA at the promoter region less tightly packed with histones and more easily accessible to transcription factors.
In conclusion, the multivirus-chip improvements developed by Ghedin, et al. [2] provide versatile clinical and basic uses. In the near future, such chips are likely to be used to detect viral co-infections in many different clinical settings.  Herpes simplex virus type 1 and normal protein permeability in the lungs of critically ill patients: a case for low pathogenicity? INTRODUCTION: The pathogenicity of late respiratory infections with herpes simplex virus type 1 (HSV-1) in the critically ill is unclear. METHODS: In four critically ill patients with persistent pulmonary infiltrates of unknown origin and isolation of HSV-1 from tracheal aspirate or bronchoalveolar lavage fluid, at 7 (1–11) days after start of mechanical ventilatory support, a pulmonary leak index (PLI) for (67)Gallium ((67)Ga)-transferrin (upper limit of normal 14.1 × 10(-3)/min) was measured. RESULTS: The PLI ranged between 7.5 and 14.0 × 10(-3)/min in the study patients. Two patients received a course of acyclovir and all survived. CONCLUSIONS: The normal capillary permeability observed in the lungs argues against pathogenicity of HSV-1 in the critically ill, and favors that isolation of the virus reflects reactivation in the course of serious illness and immunodepresssion, rather than primary or superimposed infection in the lungs. In some critically ill patients herpes simplex virus (HSV)-1 is isolated from the upper or lower respiratory tract [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Immunodepressed patients may be susceptible to transmission and acquisition of viral diseases; alternatively, viral reactivation may occur and may contribute relatively little to morbidity and mortality. Indeed, reactivation of human herpesvirus-6 is common in critically ill patients and does not worsen outcome [16, 17] . In immunocompetent patients, however, isolation of HSV-1 may be associated with viral pneumonia, even if reactivation rather than primary infection is responsible [6, 8, 18] . HSV-1 has been associated with acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia in the critically ill [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] , as either a primary or a superimposed infection. However, there are few reports of the virus eliciting an infectious host response, as demonstrated by a rise in serum antibodies, by bronchoscopic airway disease, by 'typical' findings on computed tomography of the lungs, or by the presence of giant cells or nuclear inclusion bodies on cytology or biopsy of the lower respiratory tract [3, 5, 9, 10, 18] . Indeed, Tuxen and coworkers [4] observed that prophylactic antiviral therapy in ARDS prevented respiratory HSV-1 emergence but it had no impact on duration of mechanical ventilation or on patient outcome. The pathogenicity of the virus therefore remains unknown, and the rare association in the critically ill of HSV-1 isolation with mortality may represent reactivation of the virus in immunodepressed patients with multiple organ failure and poor outcome [1, 2, 11, 14, 15] , rather than a symptomatic primary infection or superinfection contributing to death.
Assessing pulmonary capillary protein permeability noninvasively at the bedside to yield the pulmonary leak index (PLI) could help in determining the extent of tissue injury, as was previously described [18] [19] [20] . This radionuclide technique involves gallium-67-labelled transferrin ( 67 Gatransferrin) and technetium-99m-labelled red blood cells ( 99m Tc-RBCs). In bacterial pneumonia, for instance, the PLI is elevated and the increase above normal directly relates to the severity of pneumonia, expressed as the lung injury score (LIS) [19] . In patients with acute lung injury (ALI) or ARDS during the course of bacterial pneumonia, the PLI is uniformly and greatly elevated above normal (up to 14.1 × 10 -3 /min) when LIS is greater than 2.5; the PLI is also elevated in 80% of patients with mild injury and a LIS between 1.5 and 2.5 [19] . Hence, the technique is a direct measure of permeability and an indirect measure of capillary injury in the lungs. The PLI is also elevated in interstitial lung disease [21] .
In order to help differentiate between symptomatic and asymptomatic viral shedding and spread, which could inform the decision regarding whether to institute antiviral therapy and help in determining the pathogenicity of the virus, we measured the PLI in four consecutive critically ill patients with persistent pulmonary infiltrates of unknown origin on ventilatory support, in whom a HSV-1 had been isolated.
We studied a small series of consecutive patients in whom respiratory secretions, sent for viral culture because of persistent pulmonary infiltrates of unknown origin, were found to be positive for HSV-1 (Table 1) . Tracheal aspirates or bronchoalveolar lavage fluid were transported directly to the microbiology laboratory or placed in viral transport medium (Copan Diagnostics Inc., Corona, CA, USA). For isolation of HSV-1, specimens were inoculated using standard procedures in triplicate flat bottom tubes on human embryonal lung fibroblasts and incubated at 37°C. Cultures were studied three times weekly for 10 days to identify the presence of a cytopathic effect. If a cytopathic effect, indicating the presence of HSV-1, was apparent or otherwise at days 2 and 7, the cells were fixed in methanol:acetone (1:1) and typed by immunofluorescence with labelled specific HSV-1 and HSV-2 antibodies (Syva Mikrotac HSV-1/HSV-2 typing kit, Palo Alto, CA, USA). In the four patients studied, the results were available within 3 days after samples had been inoculated in culture medium.
On the day of specimen collection for viral culture, demographic, chest radiographic and respiratory data were recorded, as were clinical features. In three out of four patients on mechanical ventilation after intubation, the total respiratory compliance was calculated from ventilator settings as follows (ml/cmH 2 O): tidal volume/(plateau -end-expiratory pressure). From the radiographic score (ranging from 0 to 4 depending on the number of quadrants with radiographic opacities), the ratio of arterial oxygen tension to fractional inspired oxygen, the level of positive end-expiratory pressure and the compliance, the LIS was calculated [22] . (LIS ranges between 0 and 4, with values up to 2.5 denoting ALI and those above 2.5 ARDS.) None of the patients had visible oropharyngeal vesicles.
To characterize further the persistent pulmonary infiltrates, the PLI was measured using a modification to a method described previously [19, 20] . Because this is a routine procedure, informed consent was waived. Autologous RBCs were labeled with 99m Tc (11 MBq, physical half-life 6 hours; Mallincrodt Diagnostica, Petten, The Netherlands), using a modified in vitro method. Ten minutes after injection of the labelled RBCs, transferrin was labelled in vivo, following intravenous injection of 67 Ga-citrate (6 MBq, physical half-life 78 hours; Mallincrodt Diagnostica). Patients were in the supine position, and two scintillation detection probes were positioned over the right and left lung apices. The probe system (manufactured by Eurorad C.T.T., Strasbourg, France) consists of two small cesium iodide scintillators (15 × 15 × 15 mm 3 ), each in a 2-mm tungsten and 1-mm aluminium housing cover (35 mm in diameter and 40 mm in height). The front end of each probe has an aluminium flange attached (3 mm in thickness and 70 mm in diameter) to facilitate easy fixation to the patient's chest with tape. Each probe weighs approximately 255 g. The probe signals are led into a dual amplifier, from which the output is fed into a multichannel analyzer system connected to a personal computer. Because the probes have separate channels, there is no electronic crossover.
Starting at the time of the intravenous injection of 67 Ga, radioactivity was measured each minute for 1 hour. For each measurement interval, the entire spectrum of photon energies was stored on disk. During processing, the 99m Tc and 67 Ga -and plotted against time. The PLI was calculated, using linear regression analysis, from the slope of increase of the radioactivity ratio divided by the intercept, in order to correct for physical factors in radioactivity detection.
By taking pulmonary blood volume and thus presumably surface area into account, the radioactivity ratio represents the ratio of extravascular to intravascular 67 Ga radioactivity. The PLI represents the transport rate of 67 Ga-transferrin from the intravascular to the extravascular spaces in the lungs, and it is therefore a measure of pulmonary capillary permeability to transferrin [19, 20] . The mean PLI from the two lungs was taken. The upper limit of normal PLI is 14.1 × 10 -3 /min. Where appropriate, numbers are summarized as median (range).
Patient data are presented in Table 1 . The patients had stayed for some time in the hospital or intensive care unit before HSV-1 was isolated, and they had been admitted primarily because of respiratory insufficiency during the course of pneumonia. Patient 4 was admitted into the coronary care unit a few days before intensive care unit admission for cardiogenic pulmonary oedema. All patients had been dependent on mechanical ventilatory support for some time before sampling. They had received adequate antibiotic therapy for pneumonia and had ALI at the time of sampling, which was of otherwise unknown origin. Table 1 shows that patients had radiographic abnormalities but without an increased PLI. Central venous pressure was not elevated, which suggests that the persistent pulmonary infiltrates were not caused by overhydration. In patients 1 and 3 a high-resolution computed tomography scan of the lungs with contrast was obtained; the findings were nonspecific, however, with alveolar consolidations and pleural fluid, even in the presence of interstitial abnormalities with a ground glass appearance in patient 3. In patient 1 a bronchoscopy was performed and there were no mucosal lesions. There was a normal distribution of lymphocyte subtypes in the lavage fluid. A transbronchial biopsy revealed interstitial inflammation with many macrophage deposits, and immunohistochemical staining for HSV-1 was negative. No multinucleated cells or cell inclusions were observed, either in bronchoalveolar lavage fluid from patient 1 or in tracheal aspirates from the other patients. In patients 1-3 concomitant isolation of bacteria by culture was regarded as bacterial colonization. Antibody testing was not done in patients 2-4 but was found to be positive for anti-HSV-1 IgG in patient 1, which is indicative of prior HSV-1 infection.
The antiviral agent aciclovir (10 mg/kg three times daily) was started when cultures became positive in two patients, at the discretion of the treating physician. Aciclovir was withheld in the other two patients because it was presumed that the pulmonary infiltrates were not caused by HSV-1, on the basis of a normal PLI among other findings. In patient 1, who had a normal PLI, a course of steroids was initiated on the day after the PLI was measured, and was continued despite positivity for HSV-1, reported 5 days later. All patients survived until discharge from the intensive care unit.
The 67 Ga-transferrin PLI is a sensitive and specific measure of pulmonary capillary permeability, which is utilized for noninvasive assessment of severity of a broad range of pulmonary conditions [19] [20] [21] . The PLI roughly parallels clinical severity (i.e. the LIS) [19, 20] . Although it involves the use of relatively routine equipment, the diagnostic method has not gained broad application, partly because of its laborious nature [20] . It has the advantage that bedside measurements are possible in mechanically ventilated critically ill patients, who cannot easily be transported. Pulmonary inflammation, of whatever cause, increases the PLI up to four times normal values in the most severe forms of lung injury, including ARDS. In less severe injury, such as impending ARDS and interstitial lung disease, the PLI is also elevated, albeit to a lesser extent, as reported by us and other groups [20, 21] .
The patients had in common a prior infectious episode, followed by a relatively prolonged period of respiratory insufficiency. They had persistent and nonspecific pulmonary infiltrates of unknown origin, after treatment of their primary disease, which prompted viral culture. The normal PLI observed suggests the involvement of a relatively harmless reactivation of HSV-1, rather than the presence of a primary and damaging infection. Indeed, critically ill patients with sepsis may have late immunodepression, with lymphocytic apoptosis, lymphocytopenia and T-cell anergy, promoting viral reactivation [23, 24] . Apparently, the virus must have been latent in the nerve endings of the mucous membranes of the upper respiratory tract in these patients [2, 15] . Herpesviruses (HSV-1) have frequently been isolated in vivo from respiratory secretions of patients with ARDS [3, 4] and detected in surveillance cultures from the respiratory tract of patients following burns, trauma, transplantation, major surgery and others. However, these viruses are detected in only 3% of lung biopsies from patients with prolonged and unresolving ARDS [3, 7, [9] [10] [11] [12] [13] 15] . The literature is thus widely divergent on the precise role of the virus in pulmonary disease in the critically ill and its contribution to patient morbidity and mortality [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .
We believe that the tracheal aspirates were representative of lower respiratory tract secretions, in the absence of herpes orolabialis and oral epithelial cells in smears for Gram stain of the secretions. Concurrent colonization with other pathogens has previously been described [5, 13] . Because there was no overlap in the duration of stay of the patients, transmission of the virus from one patient to another can be excluded. This further suggests that respiratory HSV-1 infections in the critically ill may result from relatively harmless endogenous reactivation. Although the normal PLI argues against pulmonary parenchymal pathogenicity, tracheobronchitis caused by the virus [18, 25] cannot be ruled out, even in the absence of orolabial lesions, because bronchoscopy was not performed in three of the four patients, even though it was unremarkable in patient 1. The persistent pulmonary infiltrates in our patients may thus relate to slow radiographic resolution of prior bacterial or aspiration pneumonia, rather then superimposed infection. Moreover, computed tomographic images of the lung may be largely nonspecific [26] , and so the precise diagnostic criteria for HSV-1 pneumonia remain unclear. When properly standardized, for instance with respect to cell numbers in bronchoalveolar fluid or tracheal aspirates, quantitative cultures, viral RNA and DNA by polymerase chain reaction, could be helpful together with the PLI in further studies to quantitate viral load and the ratio of replication to shedding, and therefore the pathogenicity of the virus in the lower respiratory tract.
In conclusion, the anecdotal data presented here suggest that isolation of HSV-1 from respiratory secretions in the critically ill patient with a persistent pulmonary infiltrate may warrant evaluation of tissue injury potentially caused by the virus to judge its pathogenicity. This could be done using a radionuclide PLI measurement, and would help to inform decisions regarding antiviral therapy, which may have adverse effects. In some patients a normal PLI may argue against viral pathogenicity, and withholding of aciclovir in such patients may be safe. Logistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model BACKGROUND: Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. METHODS: We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. RESULTS: We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. CONCLUSIONS: Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1, 3, 4, 2] . Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine.
The Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5] ), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6] ). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderatesized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination.
Mathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7] [8] [9] [10] [11] [12] [13] . The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14] ). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vac-cinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11] , a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9] ; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts.
Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9] , we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: exposed individuals and vaccinate them in time, resulting in a "race to trace" [9] .
Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize.
Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11, 23, 24] , although it may also result in more mild cases which may be harder to detect.
Public awareness may lead to more rapid detection of cases.
We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9] .
Natural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17, [25] [26] [27] 8, 28] . Following infection by the variola virus, individuals exhibited an incubation period of approximately 7-19 days with 10-14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2-3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22-27 of illness [26] .
The course of smallpox varied widely between individuals, and several different clinical classifications were developed [29] [30] [31] 17, 26] . Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32, 31, 26] ; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre-or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1].
Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17] . However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22, 28] . In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17] , and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26] , except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox.
Smallpox was largely a disease of close contacts [17, 26, 33] , spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26] . However, patients were believed to be most infectious during the first week of the rash [26] ; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26, 33] . The more severe the case, the more infectious they appeared to be [34] ; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34] . Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35, 36] . Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29] , but often approaching 100% [37] . Drier conditions were often believed to favor transmission [17, 27] , so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38] . The number of secondary cases resulting from a given importation into Europe varied widely [39] , with most importations yielding few cases, but with the occasional large outbreak being seen.
Mathematically, we represent the course of smallpox according to Figure 1 . We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible.
We simulate the transmission of smallpox on a "smallworlds" network (highly clustered, but with short characteristic path lengths) [40] . Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts).
In general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41, 42] (for a simple example, see [43] ), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined "containment" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure   Smallpox stages used in the simulation model Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and "mild" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table  6 . 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and nonrenewing population, epidemic extinction always occurs in finite time.
We assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable.
High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%).
In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9] ; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of K r per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts.
The contact structure of the network is illustrated in Figure  2 . Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p. 
We analyzed the model in three ways. First, we selected a Latin Hypercube sample [44] [45] [46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2] , and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through "sickbed" routes which would not occur in a modern setting [47] , so that in this sense our model errs considerably on the side of caution and pessimism.
To determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2 . Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2 )). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well.
To explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances.
We used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A . In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Table 1 .
Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A , we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure  3A assuming no diagnosis during the prodromal period.
In Figure 3B , we illustrate control of an epidemic for which all parameters are identical to Figure 3A , except that the workplace/social group size is 12 (instead of 8, as in Figure 3A ), and the probability of finding workplace/ social contacts is 60% (instead of 80%, as in Figure 3A ). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur.
Finally, in Figure 3C , we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure  3A . Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing.
In Figure 3D , we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin Figure 5A , 5B φ
Prob. of finding household contact 0.95 Delay, tracing household contacts 1-5 days Expanding severe smallpox epidemic Figure 3 3A -Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1 ; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2] . Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B -An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A , except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8).
3C -A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A , except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D -Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A ; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial.
Finally, in Figure 4 , we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process.
Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20) , and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), Table 3 : Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A . As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table 3 , support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing.
Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days.
(Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window).
One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the "calibrated" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ 2 ), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (K r ). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ 2 and φ, and for each value of K r we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions).
In Figure 5A , we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day -1 and 8 day -1 ). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50-60 per day (5-6 per index case per day), the average containment fraction Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A , except that contacts of contacts are not traced in these replications. 
The mean containment probability Figure 5 5A -The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 "calibrated" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by "high contact isolation"), and moderate levels (corresponding to a one day delay, and indicated by "less contact isolation"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 "calibrated" parameter sets). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1 , Table 8 ). 5B -The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A . Whereas in Figure 5A , we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A : a. high workplace/ social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, c.
d.
became close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B , we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices.
Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure  3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/ social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%.
While Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 "calibrated" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6 ) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6 ). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6 ). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occa-Faster contact tracing Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A ); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/ social contacts. This figure shows the average of one hundred realizations starting with ten index cases. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7 . We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance.
In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A ). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate.
We have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A , we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/ social contacts -even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown).
Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) ( Table 4 ). In these scenarios, we assumed that all traceable contacts were followed up very More rapid diagnosis Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A , and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced).
For the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A ), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic.
Transmission prior to the rash makes epidemic control more difficult. In Figure 8 , we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure   3A ) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control.
Finally, in Figure 9 , we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A , assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure) . Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), Table 4 : Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, "Contacts"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, "Contacts of Contacts"). All other parameters are the same as in Figure 3A . that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below.
Scenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown).
Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior Transmission prior to the rash Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A ). Additional scenarios, assuming 40 ring vaccinations or con-tact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A) ; for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A ); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure  3A , we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9 , we assumed this fraction was zero.
Scenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A ) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment).
Finally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone -because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection.
Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10 , we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5 , and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination cam- Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 "calibrated" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. paigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown).
We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release.
We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen.
We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox.
One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17, 22] ), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48] ). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12, 49] , and is beyond the scope of our model.
While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22] . Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control.
More serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic.
Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis).
Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination.
Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox -leading to prompt identification of cases -as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15] . However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash.
Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11] , despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for highrisk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic.
While the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant prep-arations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible. Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase BACKGROUND: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH. METHODS: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H(2)O(2). Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. RESULTS: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O(2 )toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. CONCLUSION: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O(2 )and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O(2 )and its metabolites. Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, druginduced lung toxicity, cancer and aging [1] [2] [3] [4] . Although the use of oxygen may be clinically indicated in hypoxemic situations, one must consider the potential longterm toxic side effects. For example, we know that oxygen creates cellular damage by a variety of mechanisms. Normal cellular metabolism of oxygen involves the transfer of electrons from NADH to O 2 molecules to form water (H 2 O). At normal partial pressure, 95% of oxygen molecules (O 2 ) are reduced to H 2 O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [5] . These metabolites include the superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals ( • OH) all of which make up what are known as Reactive Oxygen Species (ROS) [6] . Exposure to conditions of hyperoxia as well as ionizing radiation (IR) leads to increased amounts of these ROS and their damaging effects.
ROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues [5, 7] . Lipids, including pulmonary surfactant, react with ROS to produce lipid peroxides, which cause increased membrane permeability, inactivation of surfactant, and inhibition of normal cellular enzyme processes. Proteins reacting with ROS result in decreased protein synthesis due to inhibition of ribosomal translation or destruction of formed proteins. This ultimately leads to inactivation of intracellular enzymes and transport proteins resulting in impaired cellular metabolism and accumulation of cellular waste products. Lastly, ROS cause damage to nucleic acids by leading to modified purine and pyrimidine bases, apurinic (AP) / apyrimidinic sites, and DNA protein cross-links which can lead to single strand breaks [8] .
Several defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of H 2 O 2 directly to H 2 O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H 2 O 2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B 2 ), vitamin A, and ceruloplasmin [1, 2, 9] . These systems usually provide enough protection against oxygen metabolism under normal conditions, but may become depleted under conditions of increased oxidative stress [7, 10] .
The defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes Base excision repair pathways for Oxidative DNA damage oxidative DNA damage [11] . Damaged bases are excised and replaced in a multi-step process. Lesion-specific DNA glycosylase repair genes initiate this process. After removal of the damaged base, the resulting AP site is cleaved by APendonuclease generating a 3'OH and 5'deoxyribose phosphate (dRP). β-polymerase, which possesses dRPase activity, cleaves the dRP residue generating a nucleotide gap and then fills in this single nucleotide gap. The final nick is sealed by DNA ligase [12] [13] [14] ( Figure 1A ).
The oxidative repair genes that we have analyzed in this study include 8-oxoguanine DNA glycosylase (hOgg1), human Mut Y homologue (hMYH), human Mut T homologue (hMTH), and endonuclease III (hNTH) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [15] . If repair of C/GO does not occur, then G:C to T:A transversions may result [5, [15] [16] [17] . The repair enzyme hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [5, [18] [19] [20] . The repair enzyme hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA ( Figure 1B ) [21] . Lastly, the repair gene endonuclease III (hNTH) is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [22] . hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [23, 24] . Subsequent steps following hNTH are identical to those following hOgg1 ( Figure 1A ).
A previous study has shown that over-expression of the DNA repair gene hOgg1 leads to reduced hyperoxiainduced DNA damage in human alveolar epithelial cells [25] . The primary goal of our present study was to compare the protective effects of the four main lesion-specific DNA glycosylase repair genes by individually overexpressing each in lung cells and determining which of these provides the greatest degree of protection under conditions of increased oxidative stress.
The human alveolar epithelial cell line A549 (58 year old Caucasian male), was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/ streptomycin (100 µg/ml) (Gibco, Grand Island, NY).
Passaging of cells was performed every 3-4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator.
The retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described [26] .
Four DNA repair genes were individually ligated into the retroviral vector pSF91.1.
hOgg1-6pcDNA3.1 was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end [27] . Digestion of this product with EcoRI and SalI was performed and then hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene.
hMYH/PGEX4T-1 and hMTH/PGEX4T-1 hMYH was a gift from Dr. A. McCullough (University of Texas Medical School, Galveston, TX) and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes.
PGEX-6PI-hNTH1-wild type this gene was a gift from Dr. S. Mitra (University of Texas Medical School, Galveston, TX). Digestion with BamHI and SalI was performed and the hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene. 2.5 × 10 5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression.
Cell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to hOgg1 (Novus Biologicals, Littleton, CO), hMTH (Novus Biologicals, Littleton, CO), hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected.
Sorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% O 2 and 5% CO 2 . Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO 2 incubator. O 2 concentrations were monitored with a MAXO 2 analyzer (Maxtec, Salt Lake City, UT). Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay).
The SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells [28] . Cold 10% TCA was used to fix the cells to the plate. After incubation for 1 hour at 4°C, the individual wells were rinsed with water. After air-drying, SRB solution was added to each well and cells were allowed to stain for 20-30 minutes. 1% acetic acid wash was used to rinse off unincorporated dye. Incorporated dye was then solubilized in 100 µl per well of 10 mM Tris. Absorbance was measured by a tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 565 nm. Background absorbance measured at 690 nm was subtracted from the measurements at 565 nm.
Sorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0. well. All the plates were placed into a 37°C humidified-5% CO 2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells.
All drug exposure experiments were performed at least three times and individual drug doses included 6-8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's t test with significance being accepted for p < 0.05.
The DNA repair genes hOgg1, hMYH, hMTH, and hNTH were ligated into the retroviral vector pSF91.1 ( figure 2 ). This vector, derived from a murine stem cell virus backbone, along with each individual repair gene, was used for transfection of phoenix amphotropic cells. Viral supernatant was then collected and used to stably infect A549
Western analysis of A549 cells over-expressing individual repair genes and effect on endogenous glycosylase level contained the genes of interest integrated into their DNA (data not shown).
Western blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. hOgg1, hMYH, hMTH, and hNTH were all detected at their correct position on western blots (data not shown).
Western analysis was also utilized to assess whether overexpression of each individual repair gene resulted in altered endogenous expression of the other repair genes under both non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1, hMYH, hMTH, and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells ( Figure 3A ,3B,3C and 3D). hOgg1's endogenous expression was below the level of detection. The pattern of endogenous expression of hNTH was consistent for each condition when comparing cells over-expressing hOgg1, hMYH, hMTH, and pSF91.1. Reduced expression of hNTH after exposure to 95% O 2 was noted.
Lastly, we assessed endogenous expression of each individual repair gene in cells infected with pSF91.1 following non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad) at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding ( Figure 4A and 4B). Two or more replicates were performed for each western analysis to determine consistency of the results.
A549 cells expressing hMYH demonstrated increased survival after exposure to conditions with elevated levels of oxygen compared to cells expressing only the pSF91.1 vector ( Figure 5A ). Results were highly significant at all time points except after 12 hours O 2 where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12% after 12 hours O 2 exposure to 7% after 72 hours O 2 exposure. A549 cells expressing hMYH also demonstrated increased survival after exposure to all doses of radiation in comparison to pSF91.1 ( Figure 5B ). These results were also highly significant at all doses of radiation except at 250 Rads where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12%-14% for all doses of radiation. Also noted in these experiments was that vector control cells demonstrated no Experiments looking at the effects of H 2 O 2 on cells expressing the repair genes did not demonstrate increased survival for any of these repair genes when compared to vector control cells ( Figure 5C ). This data demonstrates that over-expression of hMYH has the ability to improve cellular survival under conditions of hyperoxia and radiation but may not be able to overcome the toxicity of H 2 O 2 .
Cell growth under normal conditions was ascertained to determine if over-expression of any of the repair genes caused an alteration in the growth of cells in the absence of oxidative stress. Wild type A549 cells and cells expressing pSF91.1, hNTH, hOgg1, and hMTH appeared to grow at similar rates with doubling times within the same range. A549 cells expressing hMYH did show a slower growth rate that resulted in significant differences in cell number by day 3. The calculated doubling time for the cells over expressing hMYH is > 3 hrs longer than the cells with the other repair genes and vector alone ( Figure 6 ). This slowing of growth may allow for more time to repair Cell survival analysis following O 2 , IR, and H 2 O 2 treatments 
Oxidative stress to the lung leads to cellular DNA damage as evidenced by the release of specific gene products known to regulate DNA base excision repair pathways such as p53 and p21 [29] [30] [31] . Alterations in pro-inflammatory mediators, transcription factors, and other related gene products are also observed [32] . This injury has been shown to be associated with features of both cellular necrosis and apoptosis [33] [34] [35] . The resultant cellular inflammation and death from oxidative stress has a dramatic impact on the outcome of patients in the clinical setting [7, 36] .
Most of our current clinical therapy towards oxidative stress in the lung involves both supportive measures and prevention. Research dealing with oxidative lung injury has focused mainly on enhancing antioxidant enzymatic processes and free radical scavengers [37] [38] [39] [40] . The ability to alter cellular survival by increasing specific DNA repair mechanisms may add another approach to the treatment of oxidant-mediated lung injury.
Many investigators have used hydrogen peroxide as a substitute for hyperoxia since it is known to be one of the metabolites produced by the metabolism of oxygen. ROS such as H 2 O 2 and those produced by hyperoxia clearly lead to DNA damage but questions exist as to whether H 2 O 2 leads to the same deleterious effects upon DNA as hyperoxia. Analysis of our growth curves after exposure to H 2 O 2 in comparison to hyperoxia and IR clearly indicates that cellular protection by oxidative DNA repair genes is specific to the agent used. Because no protection was observed with over-expression of any of the repair genes following exposure to H 2 O 2 , we speculate that the damage it causes is dissimilar. It may be that its damage not only involves oxidized bases, but may also include other forms of DNA, lipid, and protein damage that are not corrected by oxidative DNA repair genes. Alternatively, the amount and type of damage evoked by H 2 O 2 could be beyond that which can be corrected by over-expressing these repair genes.
Another form of stress known to induce damage through the formation of ROS is IR. Radiation induced free radical damage to DNA has substantial overlap with that of oxidative damage [41] [42] [43] . The protection provided by specific oxidative DNA repair genes under conditions of IR, was notable throughout our experiments only with the repair enzyme hMYH.
The primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and H 2 O 2 by mimicking the clinical situation where constant exposure to hyperoxia leads to cumulative cellular damage which further compromises repair. We determined that survival of A549 cells was also enhanced to a small degree with increased expression of the repair enzyme hMYH. This was an unexpected finding as we anticipated the repair gene hOgg1 would demonstrate the greatest protection in response to oxidative stress based on previous studies, however these experiments utilized the colony forming assay (CFA) to detect improvements in survival [25] . Additionally, the CFA may provide different results compared to the SRB assay, which allows for growth analysis over a shorter window of time. Furthermore, their study did not look at the repair enzyme hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [44] .
An interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of Cell growth curve and associated doubling times (DT) Figure 6 Cell growth curve and associated doubling times (DT hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1. Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [45] . We also have demonstrated endogenous activity of hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress.
As noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under nontoxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant.
Recent studies have discovered hereditary variations of the glycosylase hMYH that may predispose to familial colorectal cancer [46, 47] . Others have looked for hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an overrepresentation of hMYH polymorphisms [47] . The A549 cell line has not demonstrated somatic mutations in hMYH, but a single nucleotide polymorphism (SNPs) has been noted [45] . The impact on function by this SNP is unknown. It would appear that the function of hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments.
We additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of hOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [48] . It has also previously been reported that treatment of A549 cells with sodium dichromate, a prooxidant, leads to a reduction of hOgg1 protein expression that was not observed with H 2 O 2 [49] . One additional study demonstrated a dose dependent down regulation of hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [50] . In our experiments we were able to demonstrate that both hyperoxia and IR do not appear to impact the endogenous expression of hOgg1, hMYH, and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O 2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O 2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments.
Furthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the overexpression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions. Some limitations may exist in using a lung carcinoma cellline, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/ death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this.
It is difficult to know how this will translate to pulmonary epithelial cells in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O 2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells.
In summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites. Bioinformatic mapping of AlkB homology domains in viruses BACKGROUND: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes. RESULTS: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system. CONCLUSIONS: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment. The purpose of this study has been to identify domains with homology to AlkB in viral genomes, in order to get a better understanding of distribution and possible function of such domains. The AlkB protein of E. coli, and probably most of its homologues, is involved in repair of alkylation damage in DNA and RNA. It repairs 1-methyl-adenine and 3-methylcytosine by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Recently the protein was identified as a member of the 2-oxoglutarate (2OG)-and Fe(II)dependent oxygenase superfamily [1] [2] [3] . The catalytic reaction requires molecular oxygen, Fe 2+ and 2-oxoglutar-ate, which is subsequently converted into succinate, CO 2 and formaldehyde [4] .
The 2OG-FeII oxygenase superfamily is widespread in Eukaryotes and bacteria [1] , and is currently the largest known family of oxidising enzymes without a heme group [5] . The 3D structure of several of these oxygenases is known, and they share a common fold with a structurally conserved jelly roll β-sheet core with flanking α-helices. Very few residues are totally conserved across these structures, basically just the residues involved in coordination of the Fe(II) ion and the 2-oxoglutarate.
AlkB-like genes are widespread in most types of organisms except Archaea. However, whereas bacteria normally have just one or at most two AlkB homologues [6] , multicellular Eukaryotes tend to have several homologues. In the human genome at least 8 different AlkB homologues (ABHs) have been identified [7] . These homologues seem to have slightly different properties with respect to substrate preference and subcellular localisation, and this may be a reason for the proliferation of ABHs e.g. in humans. However, a detailed functional mapping of all ABHs has not yet been carried out.
A sequence alignment of known ABHs identifies very few residues as totally conserved, basically just a HxD motif, a H and a RxxxxxR motif. These residues are also conserved in the more general 2OG-FeII oxygenase superfamily as described above, except for the final R. The first three residues (HxD and H) are involved in Fe(II)-coordination, whereas the first R is involved in 2OG-coordination. The final R is most likely involved in AlkB-specific substrate binding.
In addition to DNA repair, it has been shown that E. coli AlkB and the human AlkB homologue hABH3 may be involved in RNA repair. When expressed in E. coli both AlkB and hABH3 reactivate methylated RNA bacteriophage MS2 in vivo. This illustrates that direct repair may be an important mechanism for maintenance of RNA in living cells [4] . RNA repair proceeds by the same mechanism as DNA repair. Repair of damaged RNA was previously considered very unlikely, due to the natural redundancy of RNAs in a cell [8] . However, RNA is essential for cell function: unrepaired RNA can lead to miscoded or truncated proteins, and alkylated RNA could signal cell cycle checkpointing or apoptosis [9] . Consequently the occurrence of RNA repair does not come as a great surprise. The mechanism of direct reversal of methylation as used by AlkB homologues is particularly important for RNA repair, as it means that single-stranded regions may be repaired without introducing strand breaks. Repair of alkylation damage in DNA and RNA has recently been reviewed [10] .
AlkB homologues have also been found in plant viruses. It has been suggested that methylation may be used in host-mediated inactivation of viral RNAs, and that AlkB homologues in some plant viruses may be used to counteract such defence mechanisms [1] . However, no detailed study of this has been published.
The research project reported here has focused on a better understanding of the distribution and potential function of putative AlkB homology domains by using in silico mapping of viruses in which such domains have been found, as well as related viruses.
The general mapping strategy of the project was to identify viral genomes with AlkB homology domains, identify common features of these genomes, and subsequently find additional genomes with similar features, but without AlkB homology domains. This data set could then be used to analyse the properties and distribution of AlkBlike domains in viruses, as a basis for generating hypotheses about the evolution and function of these domains.
The PSI-Blast search for viruses in the NCBI nr protein sequence database was initiated with ALKB_ECOLI (NCBI gi113638), restricted to residues 110 to 210 and using the default inclusion threshold of 0.005 on E-values. The [11] .
In all of these viruses the AlkB domain is a part of the replicase polyprotein, which normally consists of a viral Other Pfam domains -Peptidase_C21, C23, C33, C34, C35 and C41, A1pp and OTU -were also identified in subsets of sequences. A1pp is a member of the Appr-1-p processing enzyme family, and the domain is found in a number of otherwise unrelated proteins, including non-structural proteins of several types of ssRNA viruses. OTU is a mem-ber of a family of cysteine proteases that are homologous to the ovarian tumour (otu) gene in Drosophila. Members of this family are found in Eukaryotes, viruses and pathogenic bacteria.
The MT, HEL and RdRp domains identified by Pfam as described above were extracted from the library sequences, aligned by ClustalX, and combined into a new alignment consisting of only these domain regions. This turned out to be necessary in order to get robust alignments. The intervening regions between the conserved domains are extremely variable in these sequences, and this tended to confuse alignment programs in the sense that conserved regions were not correctly aligned. The combined sequence alignment of domains from Closteroviridae, Flexiviridae and Tymoviridae was then used as input for building a phylogenetic tree with MEGA2. The final tree is shown in Figure 2 , with polyproteins containing AlkB-like domains indicated.
A second alignment was generated from all sequences with AlkB-like domains, using only the regions corresponding to MT, AlkB, HEL and RdRp Pfam domains. The domains were aligned individually, and the combined alignment was used as input for MEGA2. However, this data set did not give a reliable phylogeny (data not shown), and the separate domains of this alignment were therefore analysed individually and compared. This analysis is summarised in Tymoviridae measures (including SJA) for comparison of random trees [12] . The SJA values shown in Table 2 for comparisons between MT, HEL and RdRp NJ trees were 14.2 -17.1 standard deviations from the expectation value of 0.665 for a tree with 22 nodes, whereas the corresponding values for the AlkB NJ tree were 4.4 -5.4 standard deviations from the expectation value. Similar ranges were observed for the ML trees as well as for alternative distance measures, e.g. the Symmetric Difference (SD) measure (data not shown). Although this means that the SJA value for comparing AlkB trees to MT, HEL and RdRp trees were significantly better than for random trees, it also shows that the MT, HEL and RdRp trees were clearly more similar to each other than to the AlkB tree.
The alignment of the AlkB domain seemed to be of comparable quality to the other alignments. In fact the AlkB domain had the highest average pairwise sequence identity, as seen in Table 2 (see Figure 3 for the actual alignment). In other words, these AlkB domains were as similar to each other as the other three domains with respect to sequence identity, but they did not represent a consistent evolutionary history when compared to the other domains of this polyprotein. This may indicate that the AlkB domains have evolved separately from the other domains, and possibly as several independent instances.
The degree of co-evolution was analysed by computing pairwise distances between sequence regions in the alignment of MT, AlkB, HEL and RdRp domains described above. In Figure 4 selected results are shown as scatter plots, where the Blosum 50 score value between e.g. the MT domains in a pair of sequences is plotted against the score value for AlkB domains in the same pair of sequences. Plots for the MT, HEL and RdRp domains show that they are strongly correlated for MT vs. RdRp (r 2 = 0.95), MT vs. HEL (r 2 = 0.87) and HEL vs. RdRp (r 2 = 0.81). The plot of the AlkB domain vs. these three domains for the same set of sequences shows a very low degree of correlation for AlkB vs. RdRp (r 2 = 0.10), AlkB vs. MT (r 2 = 0.12) and AlkB vs. HEL (r 2 = 0.16).
As mentioned above the genome organisation of these replicase polyprotein sequences seems to be very flexible. In order to analyse domain organisation the location of identified Pfam domains were plotted for a number of sequences, as shown in Figure 5 .
The results described above may indicate that the AlkB domains have been integrated into the replicase polyprotein relatively recently (see Discussion). In order to test for potential sources selected AlkB domains were compared to non-viral sequences. PSI-Blast was used to search the NCBI nr database, removing all viral hits in the final search report. Most of the remaining top-scoring hits were from bacteria. This included two different strains of Xanthomonas, X. axonopodis pv citri and X. campestris pv campestris. Xanthomonas attacks plants such as citrus, beans, grapevine, rice and cotton [13] . The search also returned high-scoring hits from another plant pathogen, Xylella fastidiosa. This bacterium infects a great variety of plants, including grapevine, citrus, periwinkle, almond, oleander and coffee [14] .
Pfam searches obviously will only identify known domain types in protein sequences. In order to identify potential similarities in regions that were not recognised by Pfam, systematic PSI-Blast searches were performed, using the polyprotein regions between the MT and HEL domains and searching against the NCBI database of reference sequences [15] , excluding all viral entries. A maximum of 5 PSI-Blast iterations were allowed, with an inclusion threshold of 0.005. The expected homologues of the AlkBdomain were found with high confidence, as most of the E-values were < 1 × 10 -50 . Homologues of typical viral domains like the viral peptidases were obviously not found, as all viral database entries were excluded. Very few Multiple alignment of sequence regions corresponding to the AlkB domains Figure 3 Multiple alignment of sequence regions corresponding to the AlkB domains. The alignment was generated with ClustalX. The residues involved in coordination of the essential Fe 2+ ion are completely conserved, except in one of the Vitivirus sequences. These residues are the HxD motif, a single H, and the first R in the RxxxxxR motif. The function of the remaining conserved residues is unclear, but at least some of them may be involved in coordination of the substrate [10] . Pairwise distances between sequence regions corresponding to methyltransferase (MT), RdRp and AlkB domains. Each data point corresponds to e.g. RP-RP and MT-MT distances for the same pair of sequences, and sequences showing similar evolutionary distance in these two regions will fall on the diagonal. The pairwise distances were estimated from multiple alignments using the Blosum50 score matrix [47] . Trend lines were estimated with Excel. The trend line for AlkB vs. RdRp is heavily influenced by the point at (675, 670). It represents two Foveavirus sequences (NCBI gi3702789 and gi9630738), they are 98% identical over the full polyprotein sequence. Alignment score (AlkB) Alignment score (RdRp) r 2 = 0.10 new similarities were found by these searches. Pepper ringspot virus (Tobravirus, NCBI gi20178599) showed significant similarity to site-specific DNA-methyltransferase from Nostoc sp (E = 1 × 10 -74 ), as well as other cytosine 5Cspecific DNA methylases. Bamboo mosaic virus (Potexvirus, NCBI gi9627984) showed similarity to aggregation substance Asa1 from Enterococcus faecalis (E = 6 × 10 -34 ). A small number of additional similarities seemed to be caused by biased sequence properties (e.g. proline-rich regions), and were probably not significant. This included matches against mucin and cadherin-like proteins from Homo sapiens and multidomain presynaptic cytomatrix protein (piccolo) from Rattus norvegicus. In general the variable regions seemed to be truly variable, with very little similarity to other proteins, except for the Pfam domains already identified.
As seen in Figures 2 and 5 , some closely related sequences are lacking specific domains in the sense that HMMER does not find a significant similarity to the Pfam entries for these domains. In order to understand the degree of sequence variation associated with this domain loss, as well as the general sequence variation in conserved vs. non-conserved regions of typical polyproteins, several dot plots were generated. The dot plot for two Carlavirus sequences, Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191), is shown in Figure 6 . The dot plot confirms that these two sequences are closely related in the MT, HEL and RdRp domains. However, there are significant differences in the region between MT and HEL. Potato virus M is lacking the AlkB domain whereas Aconitum latent virus is lacking the OTU domain. As seen from the dot plot, short regions of similarity close to the diagonal shows that both domains may have been present in an ancestral sequence. However, this region shows a high degree of sequence variation, and as indicated by the dot plot they are almost exclusively mutations. Non-essential or non-functional domains are probably rapidly lost. In this particular case, none of the typical AlkB motifs seem to be conserved in Potato virus M, indicating that this indeed is a non-functional AlkB domain.
The N-terminal domains of Flexiviridae and Tymoviridae are methyltransferases As described above the Pfam methyltransferase motif (Vmethyltransf) did not match any of the putative methyltransferase domains of Flexiviridae and Tymoviridae, despite the fact that they had been identified via PSI-Blast searches starting with known methyltransferases. Therefore an additional Pfam-type profile was generated. It is obviously a possibility that these domains in Flexiviridae and Tymoviridae are not methyltransferases, and that they are false positives from PSI-Blast. However, the essential residues of a typical viral methyltransferase motif are conserved in the alignment of these domains (data not shown) [16] . In Bamboo mosaic virus, which belongs to Flexiviridae, the residues H68, D122, R125 and Y213 have been identified as putative active site residues with similarity to the Sindbis virus-like methyltransferase [17] , and it has been demonstrated that this region of the Bamboo mosaic virus has methyltransferase activity, as it catalyses the transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP or guanylylimidodiphosphate (GIDP). The corresponding sequence positions are almost completely conserved in the alignment of Flexiviridae and Tymoviridae N-terminal domains. This is most likely significant, as only 7 positions in total are completely conserved in this alignment, which means that the majority of the conserved positions are known to be essential for methyltransferase activity. Work e.g. by Hataya et al. seems to support the assumption that this sequence region is a methyltransferase domain [18] . It therefore seems likely that all the sequences with AlkB domains also contain functional MT, HEL and RdRp domains. The MT Location of Pfam domains in the variable region of Flexiviridae 2 sequences Figure 5 Location of Pfam domains in the variable region of Flexiviridae 2 sequences. The regions have been extracted directly from Pfam output, and sequences and regions are drawn to scale. The black bar at each end of a motif indicates that a fulllength motif has been found, for partial motifs the bar at the truncated end would be missing. domains are probably involved in capping of genomic and subgenomic RNA [19] .
Based on the bioinformatic evidence generated here, it seems reasonable to assume that the viral AlkB domains identified by Pfam are functional. All the essential residues found in 2-oxoglutarate-and Fe(II)-dependent oxygenases are conserved, in particular the putative Fe 2+ coordinating H, D and H residues at alignment positions 19, 21 and 91 of Figure 3 , and the 2-oxoglutarate coordinating R at position 100. The conserved R at position 106 is also very characteristic of AlkB homologues [10] . The fact that all AlkB-like domains identified in these viral genomes are full-length, compared to the Pfam profile, also seems to support the hypothesis that these domains are functional.
The Pfam searches show that AlkB domains are found only in a subset of the viral genomes. This subset is phylogenetically consistent (see Figure 2 ), as it is mainly restricted to the Flexiviridae, and in particular to a subset of the Flexiviridae consisting of Viti, Capillo, Tricho, Fovea and Carlavirus. This subset is well separated from the remaining Flexiviridae in the phylogenetic analysis. The split seems to be robust from bootstrap analysis, therefore this family will be discussed here as two subfamilies, Flexiviridae 1 and 2. The same split was observed by Adams et al. in their recent analysis of the Flexiviridae family [20] . Most of the AlkB domains (15) are found in Flexiviridae 2. The remaining AlkB domains are found in Flexiviridae 1 (5) and Closteroviridae (2) . In general, all the Flexiviridae 2 sequences have at least one extra domain in addition to MT, HEL and RdRp: either AlkB, OTU-like cysteine protease or a peptidase. Most other plant viruses that are included in this survey do not have additional domains, except for Tymoviridae where a peptidase domain seems to be common. For the remaining plant virus families included here (excluding Tymoviridae and Flexiviridae 2), only 14% seem to have additional domains.
The observed distribution of AlkB domains could most easily be explained by assuming that an ancestral AlkB domain was integrated into the genome of the last common ancestor of the Flexiviridae 2 subfamily. Subsequent Figure 6 Dot plots for Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191). To the left the full sequences are shown, using the program default for similarity threshold, and to the right the region with AlkB, OTU and peptidase integration, using a slightly lower (more sensitive) threshold for sequence similarity. The Pfam regions corresponding to MT (magenta), AlkB (red), OTU (green), peptidase (blue), HEL (yellow) and RdRp (cyan) domains are indicated. virus generations derived from this common ancestor would then also contain an AlkB domain, except in those cases where the domain was lost again. This scenario could also include subsequent transfer to a small number of other virus families e.g. by recombination.
If this scenario was correct, then one would expect the different domains of the polyprotein to have a similar evolutionary history. From the phylogenetic analysis (Table 2) this seems to be confirmed for the MT, HEL and RdRp domains, but not for the AlkB domain. This indicates that the AlkB domain may not have co-evolved with the other domains, at least until relatively recently. This seems to be confirmed by looking at the degree of co-evolution, which was analysed by computing pairwise distances between alignment regions representing the relevant domains ( Figure 4 ). In the case of perfect co-evolution all points should fall on a diagonal. This seems to be the case for the MT, HEL and RdRp domains. However, the plot of the AlkB domain vs. these three domains for the same set of sequences does not show a similar correlation. Only some of the closely related sequence pairs in the upper right quadrant of the plot in Figure 4 show some degree of correlation for AlkB vs. RdRp. The most likely explanation seems to be that most of the AlkB domains have not coevolved with the other domains for any significant period of time. This seems to rule out the possibility of ancient integration of the AlkB domain, except if we assume that an ancient viral AlkB domain has frequently recombined with other AlkB domains. However, it is difficult to distinguish a scenario with frequent recombination of AlkB domains from de novo integration, and the net effect on the properties observed here would be the same.
As seen in Figure 4 , the range of score values is generally smaller for the AlkB domains than e.g. the RdRp domains, particularly if we exclude a couple of very high-scoring cases (see figure caption) . On the other hand, the degree of sequence variation within the collection of AlkB domains is significant, average sequence identity for pairwise alignments is 38%, and only 10% of the positions are totally conserved. This can be consistent with a recent integration if we assume that several different AlkB-type vectors have been used for integration (see below for details). An increased mutation rate after integration could also have contributed to sequence diversity in this region. Moving the AlkB domain into a novel structural and functional context would have removed many of the original evolutionarily constraints, as well as introduced some new ones. This could have created a "punctuated equilibrium" type of situation, potentially leading to a very rapid evolution that could have introduced significant differences between the AlkB domains, independent of the evolution in the other domains. A high mutation rate seems to be the case for this region in general, as indi-cated in Figure 6 . Although the MT, HEL and RdRp domains seem to be well conserved from the dot plot, there are very large sequence variations in the intervening region. One sequence in Figure 6 has a well conserved AlkB domain, the other an OTU domain. The fact that there are very weak sequence similarities in these two domains in the dot plot indicates that both sequences originally had both domains. However, the fact that this similarity now is very weak and without any of the typical AlkB active site motifs also indicates a high mutation rate where non-essential domains are rapidly lost. Therefore the conservation of AlkB domains is a strong indication that they are functional, as already mentioned.
If we assume that AlkB domains have been integrated relatively recently, then either de novo integration or recombination (horizontal gene transfer) may have been the main driving force for spreading the AlkB domain to new genomes. In the first case a large number of individual integrations could have lead to the present situation. If horizontal gene transfer was the main driving force, the initial number of integrations might have been quite small. It is not easy to differentiate between these two situations.
The map of Pfam motifs in the variable region between the MT and HEL domains in Flexiviridae 2 polyproteins ( Figure 5) shows that they have a very similar domain organisation, basically an AlkB domain followed by an OTU domain and a peptidase domain, located towards the C-terminal part of the sub-sequence. The relatively constant domain organisation seems to be consistent with a small number of initial integrations that were subsequently diffused to related genomes e.g. by homologous recombination. However, this is not fully consistent with the fact that the viruses with AlkB domains have been collected from hosts at very different locations, e.g. Canada, USA, Russia, Italy, Germany, France, India, Taiwan, China and Japan. Although import of virus-infected species or transmission by insects may transport viruses over significant distances, it is not obvious that this is enough to explain the observed distribution of AlkB-like domains. Therefore several independent integrations, mainly from closely related hosts, have to be considered as an alternative explanation. This explanation seems to be supported by the apparent lack of any consistent evolutionary relationships between the various AlkB domains, as seen in Table 2 . It is not easy to see how this model can be consistent with the observed similarities in domain organisation in Flexiviridae. Assuming that this region has a high degree of variability, one would expect the variability to affect localisation of integrated domains as well. However, it is possible that conserved regions e.g. in the polyprotein play a significant role in integration of novel domains. It may be relevant in this context that preliminary simulations indicate that e.g. the AlkB domains tend to form independent folding domains in the folded RNA structure of the polyprotein RNA (F. Drabløs, unpublished data). This property may possibly facilitate the insertion of such domains into the viral genome.
There are many groups of organisms that can act as vectors and spread viruses, including bacteria, fungi, nematodes, arthropods and arachnids. The plant viruses may have acquired the AlkB domain either from the vector or from the host itself. As already mentioned, searching with viral AlkB domains in protein sequence databases resulted mainly in bacterial sequences, including the plant pathogens X. fastidiosa and campestris. It is therefore a reasonable possibility that AlkB domains in plant viruses have originated from bacterial mRNA. It is also possible that the mRNA originated from other vectors or from the host itself, but at the present time this is not easily verified or disproved because of the limited number of insect and plant genomes that have been sequenced.
It has previously been suggested that the viral AlkB domain may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system of the host [1] . PTGS is known as one of a plant's intrinsic defence mechanisms against viruses [21] . Gene silencing can occur either through repression of transcription (transcriptional gene silencing -TGS) or through mRNA degradation, PTGS. The PTGS-mechanism in plants shows similarities to RNA interference (RNAi) in animals [22] . This mechanism results in the specific degradation of RNA. Degradation can be activated by introduction of transgenes, RNA viruses or DNA sequences homologous to expressed genes [23] . Many viruses have developed mechanisms to counteract PTGS in order to successfully infect plants [24] . Two of these suppressors of PTGS have been identified as Hc-Protease and the 2b protein of Cucumber mosaic virus [25] . Although both proteins suppress PTGS, it is likely that they do so via different mechanisms. Could the AlkB-like domain found in some of the plant viruses also be a suppressor of PTGS? Previously reported research indicates that methylation of transcribed sequences is somehow connected with PTGS, and the methylation can be mediated by a direct RNA-DNA interaction [26] . This RNA-directed DNA methylation has been described in plants, and leads to de novo methylation of nearly all cytosine residues within the region of sequence identity between RNA and DNA [27] . Both RNA methylation and methylation of host proteins that are essential for viral replication would be detrimental to the virus. It has already been mentioned that AlkB repairs 1methyladenine and 3-methylcytosine by oxidative demethylation. It is therefore possible that AlkB demethylates the nucleotides methylated by the PTGS mechanism, helping the virus to overcome one of the major defence mechanisms of the plant.
As shown here, only a subset of plant viruses have the AlkB domain. However, other viruses may be utilising naturally occurring AlkB proteins in the host. Viruses have to rely on a number of host proteins in order to replicate [28] . In some cases it is probably beneficial for the virus to integrate such genes into their own genome in order to ensure that they are accessible, although there will be a trade off between this advantage and the increased cost of maintaining a larger genome [29] .
However, there is an alternative hypothesis with respect to the AlkB integration that also has to be considered. As discussed above, the AlkB domain seems to have been integrated relatively recently in viruses found at very different geographical locations, and the only obvious connection seems to be that most viruses belong to a subset of the Flexiviridae. However, the source of these viruses points at another common feature. As seen from the table given in Additional file 1, AlkB domains are often found in viruses associated with grapevine, apple, cherry, citrus and blueberry -crops where the usage of pesticides is common. It is known that several common pesticides (e.g. methyl bromide and some organophosphorus compounds) may cause methylation of DNA and RNA [30] [31] [32] [33] . An integrated repair domain for methylation damage as part of the viral replication complex would therefore give the virus a competitive advantage in a highly methylating environment. The application of such pesticides would probably also stimulate AlkB production e.g. in co-infecting bacteria, giving these viruses easy access to AlkB mRNA for integration into their RNA genome.
It could be argued that a more active PTGS system in these plants would give a similar effect. However, in that case we would expect to see more ancient integrations of AlkB domains. It could also be argued that the presence of AlkB domains may be an artefact caused by promiscuous viral domains picking up available mRNA sequences during cultivation of viruses in the laboratory. However, given the large number of different laboratories involved, and the number of different hosts used (data not shown), this seems to be a very unlikely explanation.
The hypothesis that environmental compounds, in particular pesticides, may have provoked the integration of AlkB domains into the viral genomes depends upon a high mutation rate and frequent integrations of non-viral domains. The integrations have to be recent, not only in relative terms, compared to other domains in the same genome, but also in absolute terms, compared to the progress of modern agriculture. The integrations also have to be frequent, in the sense that it is likely that integration could have happened several times, in different biotopes.
It is difficult to estimate mutation rates in RNA viruses. They evolve very rapidly, and it is often difficult to assign reliable phylogenies. However, recent studies indicate that most ssRNA viruses have a mutation rate close to 10 -3 substitutions per site per year [34] , e.g. the SARS virus has 1.16-3.30 × 10 -3 non-synonymous substitutions per site per year, which is considered to be a "moderate" ssRNA mutation rate [34] . If we assume that most ssRNA viruses have effective mutation rates within the same order of magnitude, a realistic mutation rate for the viruses included here might be something like 2.0 × 10 -3 . In that case, the MT, HEL and RdRp trees shown in Additional file 2 represent approximately between 325 and 750 years of evolution. In general the NJ trees estimate a slightly shorter evolutionary history (between 325 and 450 years) compared to the ML trees (between 550 and 750 years). In this estimate the Ampelovirus sequences have not been included, as they seem to have diverged from the remaining AlkB-containing viruses at a much earlier stage. If we believe that the AlkB integrations happened after the divergence of most sequence included here, as indicated by the lack of co-evolution in Figure 4 , it does not seem unrealistic to assume that most of these integrations happened within the last 50 -100 years or so. This estimate is of course very approximate, in particular since we do not know the true mutation rate of these viruses. However, it shows that a likely time span for AlkB integration is compatible with the evolution of modern agriculture. Unfortunately, because of the lack of any robust phylogeny for the viral AlkB sequences it does not make sense to do a similar estimate for that domain.
Although it is generally accepted that viruses frequently use recombination to acquire functionality [35] , it is less well known how often this includes nonviral sequences. However, there are some well-documented examples, and in particular the properties of the ssRNA positive-strand Pestivirus may be relevant in this context. There are two biotopes of the pestiviruses, cytopathogenic (cp) and noncytopatogenic (noncp). The host is infected by the noncp form which is converted into the cp form by integration of a fragment of a cellular gene into the viral genome [36] . This introduces a protease cleavage site in the polyprotein. However, the important point here is that this happens as part of the normal infection process. It has been suggested that the integration is facilitated by the viral polymerase undergoing two subsequent template switches during minus-strand synthesis [37] , although nonreplicative RNA recombination also may be a possibility [38] . Inte-gration of cellular sequences have also been observed in other viruses, e.g. in influenza virus [39] . This shows that at least some viruses do have efficient mechanisms for recruitment of host genes into the viral genome. Therefore a recent and rapid integration of AlkB domains into selected plant virus genomes does not seem to be an unlikely scenario.
This study has focused on the AlkB domain, mainly as an attempt to get a better understanding of potential functions associated with this domain. However, it is likely that additional information about integration patterns and the relative importance of de novo integration vs. recombination can be achieved by a closer investigation of the other variable domains, e.g. by looking at how they correlate with the evolution of the AlkB domains.
We believe that the viral AlkB-like domains are conventional repair domains targeted towards the viral RNA. The integration of AlkB domains into viral genomes may have been provoked by environmental methylating agents, e.g. the introduction of DNA/RNA-methylating pesticides in farming. The hypothesis [1] that the domain interferes with the PTGS system of plants can not be excluded, but seems to be less consistent with observed features of the AlkB integration.
and Tymoviridae was generated from a ClustalX alignment, using hmmbuild and hmmcalibrate from the HMMER package. Visualisation of motif positions in viral sequences was generated directly from the HMMER output files using a local tool as an interface to the GNU [50] groff software. Systematic large scale searches with polyprotein subsequences were done locally with PSI-Blast and the NCBI reference sequence database [15] . Dot plots for comparison of viral protein sequences were generated with Dotter version 3.0 [51] .  Managing emerging infectious diseases: Is a federal system an impediment to effective laws? In the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries. The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge.
In the 1980's and 1990's HIV/AIDS was the latest "emerging infectious disease". Considerable thought was put into the legislative response by a number of Australian jurisdictions. Particular attention had to be given to the unique features of the disease such as the method of transmission, the kinds of people who were at risk, and the protections needed by the community and the infected population to best manage the care of those infected and to minimize new infections. Health workers and researchers began to find that "the most effective strategies that we have so far found to help promote reduction of the spread of HIV involve the adoption of laws and policies which protect the rights of people most at risk of infection" [1] . A good example of a legislative response which adopts this approach is found in section 119 and 120 of the Victorian Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach [2] .
In 2003 the community has been faced with the emergence of two new infectious diseases, SARS and Anthrax. Whilst there were no cases of either disease in Australia, the threat of a possible outbreak had to be acknowledged and a response planned. Anthrax is not a new infectious disease. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals. However, its manufacture and use as a weapon for bioterrorism forces us to rethink its management in a new context. These two infectious diseases have very different features from HIV which spreads only via transmission of infected bodily fluids such as blood or semen. SARS, by contrast is transmitted via droplets from infected cases which, as a result of coughing, carry the virus to close contacts [3] Thus, the infection profile of SARS requires planning for the possible overrun of Intensive Care Units and the likely infection of a number of ICU staff affecting both morale and capacity to cope. Anthrax raised different problems. These include the possible investigation of terrorist suspects alongside investigation of the outbreak of the infectious disease. Difficulties are also raised by likelihood of public panic, and the flooding of public health officials with reports of suspicious white powder.
In early 2004 the media reported the spread of avian influenza across South East Asia. This disease has different features from HIV/AIDS and SARS and an approach to an Australian outbreak would also be different. The main difference is in the source of transmission of the virus, that is, from infected birds to humans. There is very little difference [from ordinary influenza] in the symptoms (though these may vary in severity) or treatment of the virus [4] It is too early to predict whether this may be the next "emerging infectious disease", but its current spread has given rise to concern about such a possibility [5] Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult.
Without a specific head of power, the Commonwealth has limited ability to legislate with respect to health. "That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health" [6] For this reason, there are no Commonwealth emergency health powers except quarantine powers. Quarantine powers are currently restricted to isolation at the border of the country of people, plants, and animals to prevent the spread of disease. There is a real possibility that quarantine laws could have a broader scope. It depends on how widely the High Court would interpret section 51(x) of the Commonwealth Constitution. A quarantine law could override state laws as long as it remained a law "with respect to quarantine". However, "the power is potentially a colossus so far as the expansion of legislative authority in the fields of public health is concerned". [6] The quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty.
States and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers.
It is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism.
Currently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident.
It is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response.
It is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory?
It is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks.
Despite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis.
A review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers?
The States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions.
There has been work done internationally in this area. A model State Emergency Health Powers Act has been developed in the US in 2001 [7] In the preamble to this Act a rationale for its development is set out: "In the wake of the tragic events of September 11, 2001, our nation realizes that the Government's foremost responsibility is to protect the health, safety and wellbeing of its citizens. New and emerging dangers including emergent and resurgent infectious diseases and incidents of civilian mass casualties -pose serious and immediate threats to the population. A renewed focus on the prevention, detection, management and containment of public health emergencies is thus called for." The US, like Australia, is a Federal system. The model was intended to be taken up by those US states which wished to do so. To date, it has been passed in over half the US states. This bill would be an excellent starting point for development of an Australian model. There are a number of legislative mechanisms which could be used to support a nationally uniform approach to health emergency powers legislation in Australia.
The development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model [8] If a decision is made in favor of amendment, States and Territories will use their best endeavors to submit to their respective Parliaments, legislation which gives effect to the amendment.
The law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza. Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS (Generally Regarded As Safe) status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively.
In the last two decades, genetic tools for the model LAB, Lactococcus lactis, were developed: transformation protocols, cloning-or screening-vectors [1, 2] , and mutagenesis systems [3] are now available. Moreover L. lactis genome is entirely sequenced [4] . Many protein expression-and targeting-systems have also been designed for L. lactis [5] [6] [7] . These systems have been used to engineer L. lactis for the intra-or extra-cellular production of numerous proteins of viral, bacterial or eukaryotic origins (Table 1) . To produce a protein of interest in fermentors, secretion is generally preferred to cytoplasmic production because it allows continuous culture and simplifies purification. To use L. lactis as a protein delivery vehicle in the digestive tract of humans or animals, secretion is also preferable because it facilitates interaction between the protein (e.g. enzyme or antigen) and its target (substrate or immune system).
In LAB, like in other Gram positive bacteria, secreted proteins are synthesized as a precursor containing an N-terminal extension called the signal peptide (SP) and the mature moiety of the protein. Precursors are recognized by the host secretion machinery and translocated across the cytoplasmic membrane (early steps). The SP is then cleaved and degraded, and the mature protein is released in the culture supernatant (late steps). Sometimes, secreted proteins require subsequent folding and maturation steps to acquire their active conformation [8] .
In most of the works describing heterologous protein production by recombinant lactococci, only one cellularlocation (i.e. cytoplasm, external media or surface anchored) is described. Only a few works report the production of a given protein in different locations using the same backbone vector, the same induction level and or promoter strength, allowing thus a rigorous comparison of the production yields of cytoplasmic and secreted forms.
Here, six examples of different heterologous proteins produced in L. lactis in both secreted and cytoplasmic forms are reviewed and discussed. Our major conclusion is that the best production yields are observed in most of these cases with secretion (up to five-fold higher than with cytoplasmic production). Moreover, engineering the expres-sion cassette to enhance the secretion efficiency (SE, proportion of the total protein detected as mature form in the supernatant) resulted in increased overall amounts of the protein. L. lactis is able to secrete proteins ranging from low-(< 10 kDa) to high-(> 160 kDa) molecular mass through a Sec-dependant pathway. Altogether, these observations suggest that i) heterologous proteins produced in L. lactis are prone to intracellular degradation whereas secretion allows the precursor to escape proteolysis, and ii) conformation rather than protein size is the predominant feature that can impair SE. New perspectives are now opened in the studies of heterologous protein production in L. lactis. Indeed, there is a need for food grade systems and for a better understanding of the host factors influencing heterologous protein secretion in L. lactis . For example, HtrA-mediated proteolysis (HtrA is the unique housekeeping protease at the cell surface) is now well-characterized in L. lactis [9] and can be overcome by use of a htrA L. lactis strain designed for stable heterologous protein secretion [10] . However, intracellular proteolysis (involving Clp complex -the major cytoplasmic housekeeping protease-, and probably other cellular components) remains poorly understood and is also discussed here.
Genetic tools to target a given protein in different cellular compartments were developed using several reporter proteins [6, [11] [12] [13] (Table 1 ). The staphylococcal nuclease (Nuc) is a well-characterized secreted protein whose activity is readily detectable by petri plate assay and it has been used as a reporter protein for secretion studies in several Gram positive hosts [14] [15] [16] . In L. lactis, Nuc was used to develop protein targeting- [6] and SP screening-systems [1, 2] . Nuc was chosen to develop the pCYT and pSEC vectors for controlled production in L. lactis of cytoplasmic or secreted forms of a protein of interest, respectively ( Fig. 1 ) [5] . The pCYT and pSEC plasmids, where expression is controlled by a nisin inducible promoter, should be used in L. lactis NZ9000 (hereafter referred to as NZ) strain bearing a nisR,K chromosomal cassette, required for the nisin signal transduction [17] . In each case described below, protein sample concentration was adjusted to the cell density of the producing culture (for details see [18] ). At similar induction levels in lactococcal strains containing pCYT:Nuc and pSEC:Nuc vectors, the highest production yields were observed with the secreted Nuc form ( Table 2) . Similar results were obtained with constitutive nuc expression cassettes for cytoplasmic and secreted forms. Nuc was the first heterologous protein where highest protein yields were obtained with the secreted form.
Similar results were obtained for the production of a Brucella abortus ribosomal protein. B. abortus is a facultative intracellular Gram negative bacterial pathogen that infects Unpublished results Bacteriocins human and animals by entry through the digestive tract. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate for the development of oral live vaccines against brucellosis using L. lactis as a delivery vector. L7/L12 was produced in L. lactis using pCYT and pSEC vectors [19] . Similarly to Nuc production, the production yield of secreted L7/L12 was reproducibly and significantly higher than that of the cytoplasmic form (Table 2) .
Another example of higher protein yields in secreted vs cytoplasmic form is the production the human papillomavirus type 16 (HPV-16) E7 antigen, a good candidate for the development of therapeutic vaccines against HPV-16 induced cervical cancer. The E7 protein is constitutively produced in cervical carcinomas and interacts with several cell compounds. E7 was produced in a cytoplasmic and a secreted form in L. lactis [20] . Using similar induction level in exponential phase cultures, E7 production 1: protein samples were adjusted to the cell density and protein quantification was performed as described in the references either by western blot or by ELISA. *: E7 was not quantified but ratio was calculated by scanning the western blot signals and comparing their intensity as described in the corresponding reference. nd: not determined was higher for the secreted form than for the cytoplasmic form (Table 2) . This difference was even higher when induction occurred in late-exponential phase, where intracellular E7 was detected at only trace amount whereas secreted E7 was accumulated in NZ(pSEC:E7) culture supernatant (see below). Thus, production of E7 clearly illustrates the fact that secretion results in higher yields in L. lactis.
Production of ovine interferon omega (IFN-ω) further illustrates this observation. In the case of poorly immunogenic antigens, co-delivery of an immuno-stimulator protein can enhance the immune response of the host. In order to optimize the use of lactococci as live vaccines, the production of cytokines was investigated in L. lactis [5, 21, 22] . IFN-ω is a cytokine able to confer resistance to enteric viruses in the digestive tract by reduction of viral penetration and by inhibition of intracellular multiplication of the viruses. Delivery of ovine IFN-ω in the digestive tract by recombinant L. lactis strains could therefore induce anti-viral resistance and could protect the enterocytes. Ovine IFN-ω cDNA was cloned into pCYT and pSEC plasmids for intracellular (pCYT:IFN) and secreted (pSEC:IFN) production respectively [5] . Induction of recombinant NZ(pCYT:IFN) and NZ(pSEC:IFN) strains were performed at equal level and IFN-ω production was measured. The levels of IFN-ω activity showed that i) an active form of IFN-ω was produced in both strains, and ii) the activity of IFN-ω found in the supernatant and cell fractions of NZ(pSEC:IFN) strain was about two-fold higher than that observed for the cytoplasmic form (Table 2) . Similarly to what was observed for Nuc and E7, secretion leads to higher heterologous protein yields.
L. lactis has been engineered to secrete of a wide variety of heterologous proteins from bacterial, viral or eukaryotic origins (Table 1) . There are reports about secretion bottlenecks and biotechnological tools for heterologous secretion in model bacteria such as Escherichia coli and Bacillus subtilis [23, 24] , but only few data are available concerning this aspect in L. lactis. Protein size, nature of the SP and presence of a propeptide are parameters that may interfere with protein secretion. Some data available about these features are compiled here.
To optimize secretion and thus production yields, the nature of the SP was the first parameter to modify on heterologous precursor as previously shown using Nuc as a reporter protein. The replacement of the native staphylococcal SP Nuc by the homologous lactococcal SP Usp45 to direct the secretion of Nuc in L. lactis led to an increased SE [25] (Table 3) . On the other hand, the replacement of SP Nuc by SP Usp45 did not enhance the SE of NucT (a truncated mature moiety of Nuc devoid of N-terminal propeptide) suggesting the importance of the propeptide in the SE for Nuc [25] (Table 3) . However, in several cases, the use of a homologous SP (and especially SP Usp45 ) allows a better SE compared to a heterologous one. Screening vectors were thus developed to search for new homologous secretion signals in L. lactis [1, 2] . These screening works offer now a panel of SPs that are suitable for heterologous secretion. However, when compared to SP Usp45, the newly described SPs were less efficient to direct secretion of Nuc [1] . Even after a direct mutagenesis on SP310, one of these new SPs identified using a screening strategy [1] , the enhanced SE was still lower than the one measured with SP Usp45 [26] . However, a recent study by Lindholm et al. showed that a Lactobacillus brevis SP (originated from a Slayer protein) drove the secretion of the E. coli FedF Schematic representation of Nuc cassettes for controlled and targeted production in L. lactis adhesin more efficiently than SP Usp45 [27] . High SE might thus result, at least in part, from good adequacy between the mature protein and the SP used to direct secretion.
The fusion of a short synthetic propeptide between the SP and the mature moiety is another innovative biotechnological tool to enhance protein secretion. One such propeptide (composed of nine amino acid residues, LEISSTCDA) was developed and was shown to enhance the SE of several heterologous proteins in L. lactis: NucB, NucT, (Table 3 ) [18] , the B. abortus L7/L12 antigen (Table  3 ) [19] , and the α-amylase of Geobacillus stearothermophilus (Table 3 ) [18] . Directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety [25] . Furthermore, the enhancement effect does not depend on the nature of the SP, since the secretion of NucB fused to either SP Nuc or SP Usp45 was enhanced by LEISSTCDA insertion [25] . Strikingly, the enhancement of SE was reproducibly accompanied by an overall increase of protein yields as determined in Western blot experiments. This observation suggests that heterologous precursors are degraded by intracellular proteases when they are not efficiently secreted and that a higher secretion could be a way to escape proteolysis.
Proteins with molecular mass ranging from 165 kDa (size of DsrD, the Leuconostoc mesenteroides dextransucrase, [28] ) to 9.8 kDa (size of Afp1, a Streptomyces tendae antifungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L.
lactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [29] . Derivatives of pCYT and pSEC plasmids were constructed to target NSP4 into cytoplasmic or extracellular location. The highest level of production was obtained with the secreted form. However, no secreted NSP4 was detected in the supernatant and both SP Usp45 -NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SP Usp45 protected NSP4 protein against intracellular proteolysis.
Similar results were obtained when pCYT and pSEC vectors were used to produce the B. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SP Usp45 :GroEL was detected in Western blot experiments (V. Azevedo, unpublished data). In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SP Usp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production.
Another example is the production of the bovine β-lactoglobulin (BLG) in L. lactis [30, 31] . BLG, a 162 amino acid residues globular protein, is the dominant allergen in cow's milk and was produced in L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [30] . Western blot and ELISA showed that BLG production was significantly higher when BLG was fused to SP Usp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [30] . Further studies revealed that a fusion between the LEISS propeptide and BLG could not enhance the SE of BLG above ~5%, as determined by ELISA [31] .
For rotavirus NSP4, B. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 (a structural protein), and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [32, 33] . Altogether, these results suggest that protein conformation rather than protein size is a major problem for heterologous protein secretion in L. lactis as well.
It was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc [20, 34] . Nuc is reportedly a stable protein and its use, as a fusion partner, does not affect its enzymatic activity. The production of the resulting chimerical protein is thus easy to follow. The cytoplasmic form of E7 was stabilized by the fusion to Nuc even when the production was induced in stationary phase ( Fig. 2A) , whereas cytoplasmic E7 alone was degraded (see below; Fig. 3 ). Thus, fusion to the stable Nuc could rescue E7 production in L. lactis and allowed higher protein yields compared to E7 alone [20] . Stabilization by fusion to Nuc was observed for several secreted proteins as well. First, a Nuc-E7 fusion on a pSEC backbone resulted in higher production yield although the SE was altered (Fig. 2B) . Fusion to the synthetic propeptide LEISSTCDA in a pSEC:LEISS:Nuc:E7 construction restored an efficient secretion yield [34] . Second, in an attempt to increase the protein yield of the secreted L7/L12, a fusion to Nuc (pSEC:Nuc:L7/L12) resulted in a 2.5-fold increase in production yield (Fig.  2B ) [19] . Recent results concerning the production of BLG provide a third example of yield enhancement by fusion to Nuc. A pSEC:Nuc:BLG construction allowed a 2-fold increase in BLG yields compared to pSEC:BLG [31] . These results show that Nuc is a stable carrier protein and has a protective effect on labile heterologous chimerical proteins by reducing its sensitivity to intracellular proteolysis. To our knowledge, Nuc is the fusion partner most commonly tested so far for stabilization in L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [13] . It is thus difficult to postulate any rule concerning the stabilization effect. Different results (i.e. no stabilization) could perhaps be observed with a different partner and thus could help to determine the mechanism of the stabilization effect. In biotechnological use of recombinant L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [22] or with heterodimeric enzymes in E. coli [35] . In both cases, the resulting fusion had the expected properties. In other cases however, such fusions might dramatically interfere with the conformation of one or both of the proteins, which might be deleterious for the expected activity. Nevertheless, when L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [10, 20, 34] and are immunogenic [36] .
Several of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production [20] . E7 was indeed degraded when intracellular production was induced in late exponential or early stationary growth phase (Fig. 3) . E7 production was then tested in a clpP deficient strain (ClpP is reportedly the major house keeping protease in L. lactis; [37] ) and in a dnaK deficient strain (DnaK is an intracellular chaperone that may promote proteolysis by maintaining the protein in an unfolded state; [38] ). In exponential or stationary phase cultures, no significant difference in E7 patterns was observed between wild type and clpP - (Fig. 3 ) or dnaK -(not shown) strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP-and DnaK-independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [39] , have been identified in L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [40] . E7 may thus be a useful screening target to identify a putative L. lactis protease that, as suggested by our data, is activated in stationary phase.
Besides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE (Fig. 4) . Research efforts are now focusing on the analysis of host factors involved in protein production and secretion by either directed or random mutagenesis in L. lactis [41] .
Although L. lactis possesses a wide range of enzymes (peptidases, housekeeping proteases) dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [9] and its major extracel-lular scavenger protease, PrtP, is plasmid encoded [42] . Thus, a plasmidless strain does not present any protease activity in the medium. Better production yields could then be expected when secretion is used versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed Fusion to Nuc rescue E7 in intracellular production and increase protein yields for the secreted forms of E7 and L7/L12 
Native E7 production in wt L. lactis depends on growth phase Figure 3 Native E7 production in wt L. lactis depends on growth phase. E7 production and secretion were analyzed by Western blot from cultures induced at different times so that, 1 hour after nisin induction, the samples are harvested at exponential (OD 600 = 0.5-0.6, upper panels) or stationary phase (OD 600 = 1.5, lower panels). wt/pCYT-E7, NZ(pCYT-E7) strain (encoding native E7, cytoplasmic form). wt/pSEC-E7 NZ(pSEC-E7) strain (encoding the precursor preE7). Positions of E7 mature and precursor forms are given by arrows. C, cell lysates; S, supernatant fraction. ClpP is not involved in the intracellular degradation of E7 in L. lactis. Analysis by western blot shows that a strain of L. lactis deficient in the intracellular protease ClpP cannot rescue cytoplasmic E7 production. Induced cultures samples of wt L. lactis or L. lactis clpP mutant strain containing pCYT-E7 (clpP/pCYT-E7) or pSEC-E7 (clpP/pSEC-E7) taken at exponential-(upper panel) or stationary-(lower panel) phase.
Stationary-phase 
higher protein stability at the cell surface for several heterologous proteins [10] .
Current research works are now focusing on other host factors that affect protein production and secretion in L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein (Nouaille et al., submitted) . Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [41] . Similar approaches allowed the identification and characterization of genes of unknown functions specifically involved in production yields of the secreted proteins in L. lactis (Nouaille et al., in preparation) .
Many molecular tools are now available to direct heterologous protein secretion in L. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous pro-tein in L. lactis Figure 4 Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous protein in L. lactis. Thicknesses of the arrows are proportional to the final production yields. All the host factors involved in the cellular events are not identified and or characterized yet. SP, signal peptide (encoded in pSEC constructions), +Nuc, fusion between the protein of interest and the stable Nuc protein. production; and allow a better understanding of the protein production and secretion process in L. lactis.
Future works should investigate the L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [5, 22, 27] . However, no equivalent of E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements (i.e. PPIases, so-called maturases...) are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated.
Altogether, these works will contribute to the development and the improvement of new food-grade systems for L. lactis [43] and should lead, in a near future, to the construction of lactococcal strains dedicated to high-level production of proteins of interest. The GRAS status of L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins. Detection and characterization of horizontal transfers in prokaryotes using genomic signature Horizontal DNA transfer is an important factor of evolution and participates in biological diversity. Unfortunately, the location and length of horizontal transfers (HTs) are known for very few species. The usage of short oligonucleotides in a sequence (the so-called genomic signature) has been shown to be species-specific even in DNA fragments as short as 1 kb. The genomic signature is therefore proposed as a tool to detect HTs. Since DNA transfers originate from species with a signature different from those of the recipient species, the analysis of local variations of signature along recipient genome may allow for detecting exogenous DNA. The strategy consists in (i) scanning the genome with a sliding window, and calculating the corresponding local signature (ii) evaluating its deviation from the signature of the whole genome and (iii) looking for similar signatures in a database of genomic signatures. A total of 22 prokaryote genomes are analyzed in this way. It has been observed that atypical regions make up ∼6% of each genome on the average. Most of the claimed HTs as well as new ones are detected. The origin of putative DNA transfers is looked for among ∼12 000 species. Donor species are proposed and sometimes strongly suggested, considering similarity of signatures. Among the species studied, Bacillus subtilis, Haemophilus Influenzae and Escherichia coli are investigated by many authors and give the opportunity to perform a thorough comparison of most of the bioinformatics methods used to detect HTs. It is now widely admitted that actual genomes have a common ancestor (LUCA, Last Universal Common Ancestor). Their current diversity results from events that have modified genomes during evolution. While some of these events happen at the nucleotide level (point mutation, indel of few nucleotides), others [strand inversion, duplications, repetitions, transpositions and horizontal transfers (HTs)] may concern significant parts of the genome. It has been postulated that HTs (exchange of genetic material between two different species) were very frequent during the first stages of evolution and are essentially subsisting nowadays in prokaryotes (1) (2) (3) (4) . As a consequence, the detection of HTs appears crucial to the understanding of the evolutionary processes and to the qualitative and quantitative evaluation of exchange rate between species (5) (6) (7) (8) (9) .
The recent complete sequencing of several genomes allows to systematically search for the presence of DNA transfers in species, especially in prokaryotes where the probability of occurrence is higher (10) (11) (12) (13) (14) . It has been reported in particular that (i) HTs in bacteria account for up to 25% of the genome (8, (14) (15) (16) ; (ii) archaebacteria and non-pathogenic bacteria are more prone to transfers than pathogenic bacteria (15, 16) ; and (iii) operational genes are more likely transferred than genes dealing with information management (15) (16) (17) .
The HT concept has been originally coined to explain the dramatic homologies between genes of unrelated species (18, 19) . An 'unusual' match is subsequently the criteria for the detection of HTs (20, 21) . While this approach allows detection of gene transfers with only a partial knowledge of genomes, it requires the sequencing of homologous genes in a number of species and consequently cannot be used for HT screening.
Genes from a given species are very similar to one another with respect to base composition, codon biases and short oligonucleotide composition (15, 16, (22) (23) (24) . As a general rule, usage of oligonucleotides varies less along genomes than among genomes (24) (25) (26) (27) . In addition, it has been observed that transferred DNA retains (at least for some time) characteristics from its species of origin (8, 14) . These particularities are used alone or in conjunction to detect DNA transfers between species (8, 12, 13) . Transferred DNA is consequently detected on the basis of some of its singularities with respect to the sequence characteristics of the recipient species. However, these techniques suffer several drawbacks and weaknesses (28) (29) (30) that led us to consider generalizing the above approach for the screening of atypical regions in sequences. In fact, the genomic signature that accounts for all possible biases in DNA sequences has been shown to be speciesspecific (26, 27, 31, 32) . The signature is approximately invariant along the genome in such a way that the species of origin of DNA segments as small as 1 kb could be identified with a surprisingly high efficiency by means of their signatures (25, 27) . As a consequence, the sequence signature may be most often (at least in bacteria) considered a valuable estimation of the genomic signature. Assuming that (i) transferred DNA fragments exhibit signature of the species they come from and (ii) recipient and donor signatures are different, the screening of local variations of signature along genomes is expected to reveal regions of interest where HTs might be located. In addition, the status of HT is strongly suggested if the signatures of these regions of interest are found close to the signature of other species.
The sequence signature is defined as the frequencies of the whole set of short oligonucleotides observed in a sequence (26, 31) . It can be easily obtained thanks to a very fast algorithm derived from the Chaos Game Representation (CGR) (33) , which allows coping with a 1 Mb sequence in a few seconds on a laptop computer. Signatures may be visualized as square images where the color (or gray level) of each pixel represents the frequency of a given oligonucleotide (called word thereafter) (31) (for examples of signatures, see Supplementary Materials 2, 4 and 6).
DNA sequences are gathered from GenBank. The genomes of 22 prokaryotes are scanned for HTs, B.subtilis, E.coli and H.influenzae genomes being given a special attention to illustrate our approach. In particular, B.subtilis and E.coli provide valuable benchmark thanks to the set of previous works addressing that very issue (12, 14, 16, (34) (35) (36) (37) . Signatures of about 12 000 species are obtained from genomic sequences longer than 1.5 kb. Sequences derived from the same species are concatenated for accuracy purposes. Species from the three domains of life, archaea ($260 species), bacteria ($3950 species) and eukarya ($6750 species) as well as viruses ($1300 species), are represented for a total amount of 1.0 Gb.
The detection of atypical regions is based on the observation of deviation of local signatures (i.e. signature of small fragments of DNA) from the genomic signature of the recipient species. Genomes are consequently sampled by means of a sliding window with an appropriate size. In fact, it would be interesting to have windows the smallest as possible for highest sampling accuracy. However, intra-genomic variability of signature increases for small windows. In addition, variability depends on species and word length. Base composition (1-letter word), 2-and 3-letter words are poorly speciesspecific: they do not allow a good discrimination between species (25, 27) . As a general rule, the longer the words (up to 9-letter long), the higher the specificity of the signature (25, 27, 31) . However, counts of long words in small windows are too low to allow a reliable estimation of the parameters. In our hands, the analysis of 4-letter words in a sliding window of 5 kb (with a 0.5 kb step) offers a good trade-off between reliability of count, file size and computational charge, whatever the species. In addition, a double-strand signature (called local signature thereafter) is computed for each window to get rid of variations induced by strand asymmetry (38) (39) (40) (41) (42) .
For illustration purposes, local signatures are developed as vertical vectors and stacked together in genome order to give an overall picture of word usage variations along each genome. In such plots, horizontal lines show the variation in frequency of words along the genome, whereas local changes in word usage appear as vertical breaks ( Figure 1 ). Figure 1 . Signatures (4-letter words and 5 kb windows) along genome for Clostridium acetobutylicum, Deinococcus radiodurans and Mycobacterium tuberculosis. In this kind of displays, lines represent the frequency of words along genome, columns represent signature of windows.
Considering that the greatest part of the genome is speciestypical, the signature of the recipient species might have been estimated from the analysis of the whole sequence. Although the vast majority of local signatures look mostly the same (believed to be instances of the recipient species signature), some of them may greatly differ. In order to avoid potential biases linked to these outliers, it has been subsequently decided to select typical local signatures on the basis of their similarities, observed after clustering. The underlying idea is that typical local signatures aggregate in few large groups, whereas outliers are found in small complementary groups at a great distance from the recipient genome signature. Groups were consequently determined with the K-means clustering tool, using every scheme of clusters between 3 and 8 for each species. Finally, the best scheme of clusters was obtained by a decision tree-based partition [CART algorithm (43) ]. The purpose of the CART algorithm is to predict values of a categorical dependent variable (clusters of local signatures in this work, each signature being characterized by its distance to the estimated genomic signature) from one or more continuous and/or categorical predictor variables [the different clustering schemes (3-8 clusters) in this work]. The CART algorithm thus provides an optimal split between groups collecting signatures close to the estimated recipient genome signature and the others groups. For each species, a clustering scheme is selected (e.g. the 5-group clustering) and a partition offered (continued example: group 2 and 3 on one side; 1, 4 and 5 on the other). The recipient species signature is subsequently calculated as the mean of the signatures of the groups belonging to the partition with the smallest distance to the estimated genomic signature.
Comparison of signatures is made possible, thanks to an Euclidian metric, accounting for differences in word usage. It must be pointed out that distances between signatures are calculated for high dimensional data (256 dimensions corresponding to the 256 different 4-letter words) and are consequently subjected to the so-called 'concentration of measure phenomenon' (44) . All distances in a high dimension space seem to be comparable since they increase with the square root of the dimension of the space, whereas the variance of their distribution remains unchanged. In fact, the radius of the hyper sphere holding 99% of the signatures of our database is only seven times the nearest neighbor distance (smallest distance between two species). Small differences in distance may consequently be considered highly significant.
For each species, a set of recipient-specific distances is obtained, every local signature belonging to the large clusters being given a distance to the host signature. In order to select outlying signatures, a cut-off distance is chosen on the basis of the distribution of distances observed for each species. It appears that the 99% percentile offered a good trade-off between sensibility and specificity for outlier detection (for impact of the threshold on detection of atypical regions, see Results). Most signatures from minority clusters are detected in this way. Isolated signatures are detected as well, while very few signatures from the recipient species clusters are selected (1%). Outliers together with the flanking regions on the genome are later on reanalyzed with smaller window and step (1/10 th of the original size typically) in order to more accurately determine their limits, when signal-to-noise ratio allows it.
Finally, the gene content of all detected regions is analyzed with the help of species dedicated databases [Genome Information Broker, http://gib.genes.nig.ac.jp/]. A BlastN search (GenBank, default settings) is carried out for each atypical region in order to identify the origin of potential HTs if homology is high enough.
Search for the origin of atypical regions About 12 000 species (including chromosomal, plasmidic, mitochondrial and chloroplastic DNA) from GenBank are found eligible for a genomic signature. Given the signature of an atypical DNA fragment, species with a close signature might be considered as potential donors. Such a screening is performed for every atypical region of the 22 species under consideration. The first five nearby species are retained when their distance to the outlier was donor-compatible.
A total of 22 genomes are screened for atypical regions (Table 1 and Supplementary Material 1). On the average, the 6-cluster scheme offers the best partition. However, in a single case (Aeropyrum pernix), nine clusters are required. In general, a single cluster is devoted to rRNA. The mean distance of windows to host varies over species from 121 to 145 (mean = 132, coefficient of variation = 3%). It is tightly correlated (P-value for the Pearson correlation coefficient <10 À4 ) with the cut-off distance that varies from 178 to 289 (mean = 234, coefficient of variation = 14%). Such large variations can hardly be explained on the mere basis of statistical fluctuations. As already observed (31, 45, 46) , variation of oligonucleotides usage along genome depends on species and can consequently be considered as a species property.
Segmentation quality of atypical regions can be tested using rRNA genes. About 94% of rRNA is detected as atypical ( Table 1) . Borders of rRNA genes are accurate to within 130 nt (0.5 kb window and 50 bp step, threshold 99%). Meanwhile, adjacent tRNAs are identified as well. As a general rule, it can be concluded that rRNA has a specific signature that is consistently at variance with the host signature. In this context, it is worth noticing that rRNA and the remaining outliers lie at comparable distances from the species they belong to, but they are clearly different from one another, rRNAs being consistently found in their own cluster.
The percentage of RNA-free outliers (at the nucleotide level) varies from 1.3 to 13% as a function of species (threshold 99%, Table 1 ). B.subtilis shows the highest percentage of atypical regions, whereas Pyrococcus abyssi has the lowest. Percentages among species are found correlated with the cut-off distance: the higher the cut-off distance, the lower the percentage of outliers (P = 0.007). In fact, a high cutoff distance takes place in species that display a high intragenomic variability, also expressed by a high mean distance to the host (Table 1) . Whether the actual percentage of atypical DNA is an intrinsic property of the species or a mere consequence of the resolution power of nucleotide biases-based methods remains consequently an open question. In addition, as already observed (13, 14) , the percentage of outliers is significantly higher for longer genomes (P = 0.004), whereas the cut-off distance is not related to the length of the genome (P = 0.69).
The mean cut-off distance for the 22 species is 234 (Table 1) . This value is chosen to select credible donors. About 50% of atypical regions are subsequently given credible donors (Supplementary Material 1). Each species has it own set of (Table 1) . Many plasmids and viruses are also found in agreement with the known molecular mechanisms of horizontal transfer (Table 1 and Supplementary Material 1).
A clustering with three classes allows assessing the signature of B.subtilis. The most populated class (collecting 84% of the segments) is chosen to represent B.subtilis. For this subpopulation, the mean distance (arbitrary unit) to the recipient (centroid of the class) and the cut-off distance are 126 and 204, respectively ( Table 1 ). Runs of contiguous outlying windows sharing the same cluster are considered as single transfer events. As a consequence, 58 regions (Figure 2a and Supplementary Material 2) fall beyond the cut-off distance and are thus potential candidates for hosting foreign DNA (for a segmentation of the B.Subtilis genome in terms of genes, see Supplementary Material 3). Figure 2b illustrates the accuracy of segmentation of an atypical region obtained by using a sliding window of 0.5 kb with a 50 bp step. rRNA genes make up $1.1% of B.subtilis genome ( Table 1) . All rRNA genes are found in the outlier population. In addition, all windows containing rRNA are assigned to a specific cluster. In fact, it is known that rRNA has its own signature, which is at variance from the host signature (12) . rRNA genes account for 7% of the outliers (tRNAs are not considered in this study, because their size is too small to generate a significant deviation from the host signature if they are isolated).
A total of 86% of the B.subtilis genome should be considered as B.subtilis typical (Table 1) . When looking for the origin of B.subtilis segments in the 12 000 signature database, B.subtilis appears in the 10 first potential donors for 84% of the whole set of 5 kb sequences that can be derived from its genome. This result confirms that segments having signatures belonging to the predominant clusters are good representatives of the recipient species signature.
The 49 rRNA-free atypical regions vary in size from 1.5 to 135 kb and make up 13% of the total genome (Table 1) . About 50% of atypical regions are less than (or around) 6 kb long. Distances of outlier from first potential donor often fall within the intra-genomic range ( However, in some instances, the outlier-to-donor distance is too great to consider the 'closest' species as potential donor. In contrast, unusual small values deserve a specific attention. In particular, the very small distance between bacteriophage SPBc2 and '2150751-2285750' atypical region (d = 2) allows to spot the part of B.subtilis genome where bacteriophage SPBc2 is incorporated (12, 47) . Other regions in the genome are also found similar (in terms of signature) to bacteriophage SPBc2. Most of them correspond to bacteriophages, imbedded in B.subtilis genome, whose free forms are not sequenced (12, 47) . Observed similarities with SPBC2 are, however, expected since signatures of phages usually share some characteristics with the species they infect (48) . The SPBc2 sequence is the only foreign sequence identified in B.subtilis, using homology as criterion (BlastN, with parameters set to default). In fact, Blast analysis of B.subtilis outliers leads to contrasted results. Besides SPBc2 and 7 out of 9 prophages imbedded in the genome, the only atypical regions identified are those containing the 30 rRNA genes coded in B.subtilis genome. The only few genes that are homologous to parts of atypical regions are found in species belonging to the Bacillus genus. It is interesting to note that no house-keeping genes (except rRNA) are detected in atypical regions. In fact, a great number of genes in atypical regions (except bacteriophage genes and rRNA) have no known function.
A clustering with five classes is required to determine the recipient species signature of H.influenzae. The three most populated classes (collecting 94% of the segments) are chosen to calculate the H.influenzae signature. Mean distance to host and cut-off distance is subsequently found equal to 130 and 239, respectively (Table 1) . Similarly to B.subtilis, one cluster (1.5% of H.influenzae genome) is devoted to the 18 rRNA gene copies (Table 1) . A total of 91% of rRNA is labeled atypical and account for 29% of the outliers.
Analysis of Table 1 shows that 95% of the H.influenzae genome should be considered as H.influenzae typical. In fact, H.influenzae is one of the 10 first potential donors for 92% of all 5 kb sequences that can be derived from its genome. As already observed for B.subtilis, the concordance of these two percentages corroborates the partition procedure used for the selection of typical/atypical fragments.
The 13 rRNA-free atypical regions vary in size from 1.5 to 19.5 kb and make up 3.3% of the genome (Table 1 , Annex 4 and Figure 3 , see Annex 5 for a segmentation of the H.influenzae genome in terms of genes). About 50% of atypical regions are less than (or around) 2.5 kb long. Numbers for H.influenzae are clearly at variance with those for B.subtilis: a smaller percentage of the genome qualifies as atypical and the average size of atypical regions is also smaller. This result is examined below in the context of intra-species signature variability (see Discussion).
A clustering with six classes is required to determine the recipient species signature of E.coli. The main features are summarized in Table 1 . The potential donors of the 84 RNAfree atypical regions are given in Annex 6 (for a segmentation of the E.coli genome in terms of genes, see Annex 7). It is worth noticing that 56% of E.coli potential donors belong to the Enterobacteriales family. Segmentation in terms of genes is displayed in Annex 7. The analysis of this genome is particularly useful for the comparison with literature (see below).
Numerous approaches for detecting horizontal gene transfers have been proposed in the last 2 decades. Phylogenetic trees of protein or DNA sequences, unusual distribution of genes, nucleotide composition (including codon biases) are some of the HT features that are considered within the framework of these models (16, 34) , Hidden Markov Models (HMMs) (12, 14, 35) and Factorial Correspondence Analysis (FCA) (37) are some criteria that are currently employed. Each of the resulting models has its own advantages and caveats (28) (29) (30) . As it has been recently pointed out by Ragan (49) and Lawrence and Ochman (50) , each approach deals with a particular subset of HTs, being for example more efficient for detecting recent transfers, or more effective for the detection of ancient HTs. Our approach, which is clearly based on oligonucleotide composition, assumes that different species have different signatures but does not rely on any other assumption. It is not surprising, therefore, that the genomic signature approach provides results (in terms of % of DNA transferred) in reasonable agreement with those proposed by Garcia-Vallve (16) and Nakamura et al. (14) for the 22 species that were analyzed in common. Correlations between percentages of HTs found by these three methods are highly significant Two species are extensively studied for HT content: B.subtilis (five methods including ours) and E.coli (six methods including ours). H.influenzae is also analyzed by Garcia-Vallve (16) and Nakamura (14) . Comparisons of methods are presented in Tables 2-4 and detailed in Supplementary Materials 3, 5 and 7. A voting procedure (majority rule) has been implemented to determine the status of genes with respect to atypicality. For that task, our initial analysis is converted in terms of genes (Supplementary Materials 3, 5 and 7). Degree of agreement between methods is subsequently observed using the statistical Kappa coefficient (51) . Kappa measures the degree of agreement on a scale from minus infinity to 1. A Kappa of one indicates full agreement, a Kappa of zero indicates that there is no more agreement than expected by chance and negative values are observed if agreement is weaker than expected by chance (a very rare situation). (14, 13, 11, 13 and 15%, respectively). The number of detected genes per method is close, ranging from 457 for Nakamura (14) to 599 for this work (median 537). Detailed votes are given in Table 2 . Among the 4100 genes of B.subtilis genome, 1011 genes are detected by at least one method (about 25% of B.subtilis genes). The number of 'single vote' genes ranges from 116 for Garcia-Vallve (16) to 47 for Nicolas (12) . A total of 470 genes make up the majority consensus set and we detected 453 of them, which is the best score of the five methods. The best agreement with the majority consensus (in terms of Kappas) is reached by Nicolas (12), followed by our method and Moszer (36) ( Table 2 ). Our method gets the best agreement with Nicolas (12) and the worst with the other HMM method used by Nakamura (14) (pairwise Kappa comparison, Table 2 and Supplementary Material 3). In fact, Nakamura approach is at variance with every other approach (14) . It gets the lowest Kappa with the Garcia-Vallve (16) Hayes (35) Lawrence (34) Nakamura (14) Medigue (49) This work majority consensus or with whatever other methods. From Table 2 , the probable number of HT genes in B.subtilis would range from 230 to 1011 with a 'reasonable' estimation around 470 corresponding to the majority consensus. It is to be noted that our method is unable to find two genes that are detected by every other methods (Supplementary Material 3) . These genes are 338 and 236 nt long, respectively, as compared with 2500 nt, the median size of atypical regions detected by our method (Table 1) . Clearly, our method is not appropriate for detecting short isolated atypical genes.
H.influenzae. Garcia-Vallve (16), Nakamura et al. (14) and we are the voters concerned with the analysis of the H.influenzae genome (Supplementary Material 5 and Table 3 , H.influenzae). The originality of results obtained by Nakamura (14) is the salient feature of this comparison. The number of detected HT genes is more than twice higher for Nakamura et al., whereas the part belonging to the majority consensus is the smallest ( Table 3) . Eleven genes are detected both by Garcia-Vallve and Nakamura (14, 16) but not by our method; however, the small number of voters precludes any specific comment in this respect. The probable number of HT genes in H.influenzae would range between 11 and 273, with a 'reasonable' estimation around 60 (majority consensus of 57) ( Table 4 ).
The results obtained by Hayes and Borodovsky (35) are clearly at variance with the others (Table 4 ). Although the proportion of claimed outliers is within the range of published numbers for E.coli (14, 16, 24, 34, 35, 37) , 37% of them are method-specific, and the agreement with other methods is weak (Table 4 ). Hayes and Borodovsky have obviously developed an approach based on HMM dealing with specific outliers. Lawrence and Ochman (34) also get a poor rating especially because they detect about twice as many genes as the other authors do (Table 4) .
It is worth noting that if the cut-off distance for our method is lowered, i.e. 95% instead of 99% for instance, some of the 'single vote' genes are dug out (for details about the impact of the cut-off distance, see Supplementary Material 7). Meanwhile, the percentage of outliers as reported by our approach rises to 20% and the percentage of 'single vote' genes reaches 24%. As expected, a high cut-off distance provides few single vote genes at the risk of missing some potentially transferred genes. Lowering the cut-off increases the proportion of single vote genes with the advantage of detecting most of the potential transfers (Supplementary Material 7) . There is obviously a continuous grading in gene 'atypicality'. It is suggested to first consider most 'consensual' genes as potential HTs and then apply amelioration models to explain the grading.
It is difficult to assess the relevancy of proposed donors, because genes detected as potential HT have generally undergone amelioration (8) . The comparison of recently diverged genomes (species or strains) provides the opportunity to find recent HTs, for which corresponding homologous genes in the donor species may be detected (52) . Such a study is performed for five E.coli strains (two K12 strains: E.coli MG1655, E.coli W3110, one uropathogenic strain: E.coli CFT073, two enterohaemorrhagic strains: E.coli O157-H7 RIMD 0509952, E.coli O157-H7_EDL933) and two Shigella flexneri strains (S.flexneri 2a 2457T, S.flexneri 2a 301). These seven strains/ species have recently diverged, genome sizes are different and the proportion of horizontally transferred genes varies from one strain/species to another (14, 52) . For instance, only $40% of the non-redundant set of proteins is common to E.coli strains CFT073, 0157-H7 EDL 9333 and MG1655 (53) . These strains/species can be clustered in four groups with respect to phylogeny (Table 5) .
Two criteria are used to searching for 'recent horizontally transferred genes': atypical regions (window size 1 kb, step 0.5 kb) (i) must have a signature that differs greatly from that of the host [distance to host must be at least >325, 2.5 times the E.coli intrinsic mean distance (Table 1) ] and (ii) must be present in a limited number of strains/species to ascertain their recentness. In fact, outliers meeting the first criterion generally aggregate into several heterogeneous clusters (K-means clustering) that usually include samples from each strain/species. In some instances, however, some strains/species were absent from the cluster. It was subsequently considered that the corresponding regions might have been recently acquired by the relevant strains/ species. Table 5 shows a selection of potential recently transferred genes. Each cluster of atypical regions contains genes present in a specific set of strains. Some atypical genes are strainspecific, some are only absent in the non-pathogenic K12 strains and intermediate situations are also encountered.
FASTA and Blast searches confirm that these genes are absent from some of the tested strains as already observed in the analysis complete genomes (53) (54) (55) . In a large number of cases, we are able to find a well-conserved homologous gene in another species (Table 5) . It is interesting to note that some of the suggested donors using our 12 000 signature database are in agreement with the species found by alignment methods. When no homologous gene is found, the proposed donors give credit to the known mechanisms of gene transfer (bacteriophages or plasmids) ( Table 5) .
It is worth noticing that most of the selected genes that are absent in K12 strains are involved in the pathogenicity of the other strains (52) . E.coli 0157-H7 is the strain exhibiting the greatest number of genes absent in K12 strains [about 1400 (54) ]. It has the greatest number of genes for which no homolog can be found (Table 5) . Moreover, we are unable to propose a donor for a great part of these genes (Table 5) . Many selected genes for E.coli 0157-H7 lie in the Ter region of the genome (between positions 2 000 000 and 2 500 000) in agreement with the published results (56). 
We have observed that most genomic regions are typical of the genome they belong to, using the signature as endpoint.
Considering that the genomic signature is species-specific, atypicality of a region in terms of oligonucleotide usage has been promoted as a criterion for the detection of HTs.
However, atypicality-based methods suffer several caveats that reduce their effectiveness in such a way that only a part of HTs can be detected. In fact, transfers between species with close signatures cannot be detected: significant differences between characteristics of transferred DNA and recipient species DNA are required. For similar reasons, HTs that were drastically ameliorated following their introduction cannot be detected either (8, 14) . The most stringent constraint, however, results from the size of the screening window. On the one hand, ideally, the best signal-to-noise ratio would be obtained when windows and HTs have a comparable size.
On the other hand, the window size must be large enough to provide significant word counts, a requirement that strengthens with the size of the words under consideration and the intrinsic variability of the genomic signature along the genome. All together, the trade-off that has been implemented in this paper allows detecting atypical regions as small as 1 kb. In fact, rRNA regions sharing this characteristic were consistently detected. It must be pointed out that smaller fragments can be eventually detected if their signatures are radically atypical. G+C% atypicality has often been considered as criterion for detecting HTs (8, 24) , but this approach suffered several drawbacks (28) (29) (30) . It is to be noted that our signature-based method detects regions for which the G+C% lies within one standard deviation from the mean G+C% of the species (for instance, regions 2675251-2676250 in B.subtilis or 534751-535250 in H.Influenzae, see also Supplementary Materials 2 and 4).
As already observed by Nicolas et al. (12) for B.subtilis, rRNA has definitely an atypical signature. It is systematically classified as outlier, whatever the species (Table 1) . Although transfer of rRNA from one species to another is unlikely (11, 57) , it cannot be firmly ruled out. However, it is clear that the atypical signature of rRNA does not imply that they are horizontally transferred.
The signature approach has an interesting property (that it shares with HMM) (7, 12, 28) : detection is not bound to any specific function in the genome. In contrast with most other methods, the signature approach not only detects genes, but whole transferred regions as well, in agreement with the described mechanisms of DNA exchange between species. It is to be noticed that the method allows detecting several atypical non-coding regions (Supplementary Materials 3, 5 and 7). One major difference between HMM and signature method lies beyond the time required for the learning process, in the few resources that HMM can mobilize to deal with a short 'one of its kind' HT. On the other hand, HTs shorter than 1 kb can hardly be detected by a signature-based approach. An innovative HT detector is likely to result from an adequate fusion of both methods.
Several factors contribute to the efficiency of the search for donors. Of course, distance between putative HT and donor signatures is essential. Accuracy of signatures, linked to the length of available sequences, density of signatures in the 'vicinity' of HT, amount of amelioration sustained by HT during its presence in the host are also of importance [P. Deschavanne, S. Lespinats and B. Fertil, unpublished results; (25, 27, 31) ]. Distance between the signature of a putative HT and the closest species varies to a large extent, but usually the shortest ones fall within the intra-genomic range ( Table 1 , Supplementary Materials 1, 2, 4 and 6) . In some cases, the distance between the closest donor signature and the atypical segment signature is so great that no potential donor can be proposed (Supplementary Materials 1, 2, 4 and 6) .
When strong similarities between a given DNA sequence and a foreign species are observed, the hypothesis for an underlying transfer is highly strengthen. However, the 'true' donor has to be previously sequenced and included in our bank of signatures to allow such a situation to occur. Moreover, we must take into account the intrinsic variability of short DNA segment signature (which is a function of their size, but also species-specific) when compared with the signature of a complete genome or any other large species sample (25, 27, 31) . In the present state, our signature database is in no way representative of the diversity and richness of life. However, it must be noticed that there is already an obvious structure (in terms of distances between signatures) expressing taxonomy relationships between species in our signature database (31, (58) (59) (60) (61) . Related species are often found close to one another. Clusters of potential donors may consequently provide pertinent information about the origin of HTs.
The diversity of signatures of putative HTs that can be observed for most of the species analyzed in this paper reveals the multiplicity of transfer events and donors (Supplementary Materials 2, 4 and 6). However, several outliers, not necessarily neighbors in the genome, are given the same set of potential donors (Table 1 , Supplementary Materials 1, 2, 4 and 6). In general, the potential donors belong to few sets of taxonomically close species (Table 1 ) and share the biotope of the host (Supplementary Materials 1, 2, 4 and 6). For instance, B.subtilis, H.Influenzae and E.coli live in distinct biotopes; their potential donors do so as well. It is particularly encouraging to find that most of the potential donors that our approach has pointed out have had the opportunity to exchange DNA material with the recipient species.
Numerous viruses and plasmids qualify as potential donors (Tables 1 and 5 , Supplementary Materials 1, 2, 4 and 6 ). It is not really surprising since they are known as HT vectors. They are often totally or partially inserted together with transferred genes in the host genome (14) .
Some atypical DNA segments are particularly peculiar. They are isolated, have a specific signature (distances from neighbors are great), so that they cannot be given a credible set of donors (Supplementary Materials 1, 2, 4 and 6) . Lack of data in the search domain, shift of signature features after a substantial amelioration process, structural constraints serving special functions or roles (14,62) (as it is for rRNA coding regions) are some of the tracks that remain to explore in these circumstances.
It would be interesting to localize the region the transfer may come from when the complete genome of the donor is available. However, homology (at the DNA level) is not a pertinent criterion for the comparison of sequences as soon as amelioration has taken place (8, 14) . In fact, homology is sometimes weak, e.g. between genes of Escherichia and Salmonella although these species have 'recently' diverged (34) . It is clear that a more powerful search for the origin of putative HTs would have to embody models of amelioration [such as the one designed by Lawrence and Ochman (8) ].
When searching for very recent horizontally transferred genes, in different strains of a species for instance, it was possible to find a great homology between detected genes and some genes from other species (Table 5 ). In numerous cases, the selection of donors is consistent with FASTA results ( Table 5 ). This confirms the pertinence, beyond the similarity of signature between putative HTs and donors, of the proposed method to retrieve the species of origin of a transferred region. It seems that the search for origin of HTs on the basis of genomic signature is a powerful approach to understand some of the mechanisms of evolution (13, 63) .
Oligonucleotide usage is known to be species-specific and to suffer only minor variations along the genome (25, 27) . Considered together, these properties allow searching for atypical local signatures that may point out DNA transfers. Results obtained with the 22 genomes analyzed in this paper are found in good agreement with literature (Tables 2-4 , Supplementary Materials 3, 5 and 7) (12, (14) (15) (16) 24, 34, 35) .
The species specificity of signature allows searching for donor species. Quite often, sets of donor species with common taxonomic features are obtained. With the help of environmental considerations, it is subsequently possible to identify (or collect clues about) potential donors. The search for donor makes use of non-homologous sequences. Partially sequenced species become consequently eligible, inasmuch 1.5 kb of the genome is available (25, 27) . Thanks to the exponentially growing rate of nucleotide databanks, the search for donor species by means of the sequence signature will turn more and more pertinent and fruitful in the future. In this context, it is worth noticing that computational power is clearly not an issue since the CGR algorithm described in this paper is fast and of 0 order (calculation time is proportional to the number of nucleotides).
Several methods are proposed to look for HTs. The signature method, based on different hypotheses, is complementary to those already described. It seems that each method detects preferentially certain types of HTs (49, 50) . In agreement with many authors (1, 16, 49, 50, 64) , it appears that the conjunction of several methods is required to obtain an overview of HT extent in a genome.
The signature method described in this paper generalized many approaches that ground the detection of outliers on the basis of the bias in oligonucleodides. The strong species specificity of the signature not only allows detecting various kinds of outliers but also provides clues about their possible origin. Obviously, the detection of HTs remains an open question; a consensus has still to emerge.
Additional materials and experimentation with the genomic signature are available from the GENSTYLE site (http:// genstyle.imed.jussieu.fr). Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis. Oligonucleotide microarrays are a powerful technological platform for large-scale screens of common genetic variation and disease-causing mutations (1) (2) (3) (4) (5) . In most published studies (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) , oligonucleotide microarrays are designed to screen specific sequence tracts, up to megabases in length (11, 15, 22, 23) , for all possible single nucleotide substitutions. With some exceptions (24) (25) (26) (27) (28) (29) (30) (31) , the same emphasis was not placed on identifying all possible small insertions and deletions in the heterozygous state. Nevertheless, it is crucial to detect such small insertions and deletions since they can play a major role in inactivating or altering gene function by disrupting functional elements (e.g. splice junctions, cis-acting elements and open reading frames) and also represent another class of common genetic variation.
Two fundamental approaches are commonly used to analyze data sets from oligonucleotide microarrays tailored to identify genetic variation in specific DNA segments purely by hybridization (1, (3) (4) (5) 9) . One approach involves identifying statistically significant gains of target hybridization signal to oligonucleotide probes complementary to specific sequence variants (9) . In theory, the gain of signal approach has the advantage of both detecting the presence of genetic variation and identifying the nature of the sequence change in the target. However, it is not feasible to screen for virtually all possible insertions and deletions due to the overwhelming The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org number of mutation-specific probes needed for this analysis. Furthermore, little effort has been made to systematically access the hybridization properties of probes complementary to these small insertions and deletions. The second approach involves identifying losses of hybridization signal to perfect match (PM) probes that are fully complementary to the DNA segment of interest (8, 25, 27, 30, 31) . In theory, the loss of signal approach allows one to screen for all possible sequence changes, including insertions and deletions, that cause a given target nucleic acid sequence to contain mismatches with specific PM probes. However, this necessitates the sequencing of specific DNA regions to identify the nature of the sequence changes (8, 25, 27, 30, 31) . Thus, a combination of the gain and loss of hybridization signal analysis could provide the most robust means of identifying and characterizing mutations using non-enzymatic oligonucleotide microarray assays.
Here, we analyze the specificity and reproducibility of nucleic acid hybridization to oligonucleotide microarrays used in the large-scale mutational analysis of the ATM (ataxia telangiectasia mutated) gene that is responsible for autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition and is also commonly mutated in certain lymphoid malignancies (32, 33) . These microarrays include 25mer oligonucleotide probes complementary to all possible single base substitutions and insertions as well as one and two base deletions on both strands of the ATM coding region. This provides the first comparative analysis of the hybridization properties of substitution, insertion and deletion probes in an oligonucleotide microarray-based mutational analysis of a large gene.
A series of 120 DNA samples derived from biopsies of lymphoma patients were previously screened for all possible ATM mutations using oligonucleotide microarrays (30) . Here, we have selected a total of 68 samples that showed robust amplification signals in all 62 coding exons for further analysis (30) . A total of 17 unique mutations, each in a one-to-one mixture with wild-type sequence, occurred once in these samples. The impact of any given mutation in a single sample is minimal given that 67 other samples with wild-type sequences in the region encompassing a given mutation are included in this analysis. Several single nucleotide polymorphisms (SNPs) were present multiple times: 735 C/T, 2572 T/C and 4258 C/T in two samples; 3161 C/G in four samples; and 5557 G/A in five samples. Likewise, these SNPs have a minimal effect on our global analyses given the large number of samples and bases interrogated in this study.
As previously described (30) , individual ATM coding exons were amplified from genomic DNA using primers containing T3 and T7 RNA polymerase tails, pooled, and then in vitro transcribed using T3 or T7 RNA polymerase to create biotinlabeled sense and antisense strand targets, respectively. Fluorescein-labeled reference target was made using genomic DNA from an unaffected individual. Reference and test sample targets were fragmented, diluted in hybridization buffer [3 M TMA-Cl (tetramethylammonium chloride), 1· TE, pH 7.4, 0.001% Triton X-100] and hybridized to the ATM microarrays as described previously (30) . Afterwards, the microarray was stained with a phycoerythrinstreptavidin conjugate and digitized hybridization images from both reference and test targets were acquired using the Gene Array Scanner (Hewlett Packard, Palo Alto, CA) equipped with the appropriate emission filters.
Custom software was used to quantify hybridization signals for each probe and subtract background hybridization signals. We exclusively focused on raw data from the biotin-labeled test targets since they provide approximately seven times the hybridization signal of the fluorescein-labeled wild-type reference target in this system (28) . This enhanced signal provides greater sensitivity toward detecting weak hybridization.
For each sample, for each base and for each potential type of mutation (i.e. substitution, one or two base deletion or one base insertion), the specificity was calculated as the ratio of the PM probe hybridization signal of the wild-type target to their cognate insertion, deletion or single base substitution probes on each strand. The logarithm of these ratios was plotted as a function of the position within the gene. To illustrate the special patterns and to smooth out random variation, running averages of data from 10 bases were used. To capture the variability, at each base, the sample-to-sample standard deviation was again calculated using data derived from a running average of 10 bases for each sample.
To estimate the mean hybridization specificity for each type of mutation, the geometric mean (i.e. the antilog of the average of the logged ratios) over all bases and over all specimens was calculated (Table 1) . To further examine the variability of the specificity ratios, the coefficient of variation (cv) was calculated in two ways. The cv is the ratio of the standard deviation divided by the mean; it is useful for understanding the amount of variability relative to the magnitude of the mean or typical value. For the intra-sample cv, the cv was calculated for each of the 68 samples (using the running average of 10 at each ATM base) and the average of the 68 coefficient of variations was taken. For the inter-sample cv, at each of the bases, the cv a Hybridization specificity ratio is defined as the ratio of PM probe hybridization signal to that of the brightest mismatch probe within a given category. The global average of all hybridization specificity ratios for each base in all samples for a given probe type is provided. b Determined for hybridization specificity ratios averaged across windows of 10 bases either within (intra) or across (inter) samples.
was calculated using the 68 samples, and the average of the coefficient of variations was taken. For both calculations, the moving average of 10 was used, instead of the original value, since the goal was to understand how the specificity varied over bases and across samples, rather than to estimate the experimental (or measurement) error.
In order to determine the relative specificity of the hybridization of complex nucleic acid targets to oligonucleotide probes complementary to single base substitutions, insertions and deletions, we analyzed data generated from oligonucleotide microarray-based mutational analysis of the 9168 bp ATM coding region (30) . These studies used a pair of oligonucleotide microarrays (Affymetrix, Santa Clara, CA) containing over 250 000 probes (25 nt in length) specifically designed to screen the sense and antisense strands of the ATM coding region for genetic variation (27, 30) . Collectively, the ATM sense and antisense microarrays contain 55 008 probes complementary to all possible single base substitutions, 73 344 probes complementary to all possible one base insertions, and 18 336 probes complementary to all possible one base deletions and 18 336 probes complementary to all possible two base deletions in the ATM coding sequence (Figures 1 and 2 ). These microarrays have been used to screen for sequence variation in the ATM gene in over 100 DNA samples (30) . SNPs and gene inactivating mutations were uncovered by screening for localized losses of hybridization signal to PM probes complementary to every 25 nt segment of the ATM coding region (8, 25, 27, 30) . However, hybridization data from deletion and insertion probes were not relied upon in this analysis. Therefore, this data set provides a unique opportunity to examine the relative hybridization specificity of nucleic acid targets to each of these classes of mismatch probes.
In order to gain a global overview of hybridization specificity, we determined the average ratio of PM probe hybridization signal of wild-type target (see Materials and Methods) to their cognate insertion, deletion and single base substitution probes on each strand (Table 1 ). In these calculations, we considered data for all 9168 interrogated bases in all 68 DNA samples (see Materials and Methods). For example, we report the ratio of the PM probe signal to the signal from its cognate 1 or 2 bp deletion probe. However, for single base substitutions, we report the ratio of the PM probe signal to that of the cognate substitution probe with the highest hybridization signal. This provides the most rigorous assessment of cross-hybridization to single base substitution probes. Likewise, for single base insertion probes, we report the ratio of the PM probe signal to that of the cognate insertion probe with the highest hybridization signal.
For both sense and antisense strands, we found that the two base deletion probes had the highest average PM to cognate MM hybridization specificity ratio (3.26-fold sense and (Table 1) .
To provide a finer-scale analysis of hybridization specificity, we determined the relative frequencies of hybridization specificity ratios in defined bins. There was a similar distribution of specificity ratios for single base substitution and two base deletion probes on both strands ( Figure 3 ). The overall lower hybridization specificities of single base deletion and insertion probes are reflected by the increased frequencies of probes within the lower specificity bins (i.e. <2-fold ratio) and decreased frequencies of probes within higher specificity bins (i.e. >3-fold ratio) on both strands.
Next, we sought to uncover underlying trends in the hybridization specificity of different classes of mismatch probes across the entire ATM coding region within a given sample (intra-sample variation). This provides insights into sequence context effects that may influence the hybridization specificity of each class of mismatch probe. To approach this problem, we plotted the average hybridization specificity ratios of substitution, deletion and insertion probes for all 1168 bases across the 68 samples (Figure 4 and Supplementary Figure 1) . We analyzed data determined over running averages of 10 bases in order to maximize our ability to detect trends and minimize the effect of randomly dispersed confounding factors (e.g. intra-or intermolecular secondary structure) that may skew data for any given base.
As expected from Table 1 and Figure 3 , the two base deletion probes consistently showed a higher average hybridization specificity ratio followed by single base substitution, single base deletion and single base insertion probes on both strands of exon 50 ( Figure 4) . Nevertheless, the hybridization specificity ratios for all classes of mismatch probes fluctuate across the exon 50 sequence (Figure 4 ). For example, two base deletion probes showed a peak value of 6.76 (unlogged) centered at base 7071 and a trough value of 1.90 (unlogged) centered at base 7002 on the sense strand. We also found similar fluctuations in specificity ratios for all mismatch probe types in the remaining 61 ATM coding exons (Supplementary Figure 1 ).
To assess intra-sample variability in hybridization specificity by a different means, we determined the average cv for substitution, deletion and insertion probes within a given experiment (Table 1) . Again, we analyzed data from running average of 10 bases in order to maximize our ability to detect trends and maintain consistency in our data analysis. Substitution probes had the lowest average intra-sample cv, 0.31 and 0.23 for sense and antisense strands, respectively. One base deletion, two base deletion and insertion probes showed comparable intra-sample coefficients of variation on the sense strand, 0.37, 0.39, and 0.38, respectively. However, insertion probes showed relatively higher variability than the deletion probes on the antisense strand. Coupled with plots shown in Supplementary Figure 1 , it is evident that of all the mismatch probe types, the hybridization specificities of base substitution probes were least affected by target sequence context. Intrigued by the above observations, we next searched for specific target sequence tracts that produced the lowest hybridization specificity among and between the different classes of mismatch probes. To approach this problem, we determined how many mismatch probes within running windows of 10 bases gave poor hybridization specificity, previously defined as a hybridization specificity ratio <1.2 (26). In Table 2 , we report nucleotide tracts where at least 8 probes within a given 10 base window showed poor hybridization specificity ratios. A comprehensive listing of probes with poor hybridization specificity is provided in Supplementary Table 1 .
Repetitive sequence tracts, including homopolymer, homopurine and homopyrimidine, are highly represented in Table 2 . Upon closer inspection, it became apparent why the cross-hybridization is strong for probes in homopolymeric regions. In these sequence contexts, substitution and deletion probes can form duplexes with wild-type target that are longer than 12 bp in length. For example, the probe designed to detect a single base deletion at position 633 is designed to form one 12 bp and one 13 bp duplex with wild-type target. However, this probe can form duplexes that range from 12 to 18 bp in length with wild-type sense strand target due to slippage ( Figure 5 ). This type of ambiguity leads to increased stability of these DNA-RNA heteroduplexes (34) .
In principle, the homopurine and homopyrimidine tracts uncovered have the capacity to form higher order structures, such as triple helices (35) . These tracts are known to alter the conformation and stabilities of RNA-DNA heteroduplexes (36, 37) , such as those formed between RNA targets and DNA probes in our system. Finally, we expect the ATM target to be especially rich in such sequence tracts given that both strands of the 3 0 -splice acceptor sequences, typically containing homopyrimidine tracts, for all 62 coding exons are included in the ATM target. This increases the likelihood that highly related sequence tracts in the ATM target can cross-hybridize to probes interrogating a particular homopurine or homopyrimidine sequence tract and reduce the overall hybridization specificity in this region.
Next, we screened for potential structures that can form in the PM probes listed in Table 2 or their targets that could explain their poor hybridization specificity. To do this, we used Mfold (38) to calculate Gibbs free energies for intramolecular structures that can form in these PM probes and targets. Based on these Gibbs free energy values, we classified the probes and targets as having strong (S) [DG < (À3 kcal/mmol)], medium (M) [(À1 kcal/mmol) > DG > (À3 kcal/mmol)] and weak (W) [G > (À1 kcal/mmol)] potential for secondary structure. We found that several target and probe sequences could form substantial secondary structures, as displayed in Figure 6 . This could artificially lower the affinity of target to PM probes and thus lower the hybridization specificity. It is more difficult to model intermolecular structure in the solution-phase complex target and in the solidphase oligonucleotide probes. However, it appears likely that such structures could also have a similar negative impact on hybridization specificity.
The relative variability in hybridization specificity ratios across samples (inter-sample variability) represents another important issue that should be considered in resequencing analysis (9) . To uncover general trends in inter-sample variability for each type of mismatch probe, we calculated an average cv for mismatch probe hybridization specificity ratios determined over running windows of 10 bases (Table 1) . Interestingly, on both strands, the single base substitution probes showed the lowest inter-sample cv. The one and two base deletion probes showed at least 2-fold higher coefficients of variation on both strands, relative to the substitution probes. Surprisingly, the one base insertion probes showed significantly higher coefficient of variations than any of the other classes of mismatch probes across samples. In fact, they are 3.5-fold higher than the corresponding substitution probes on each strand.
The relative levels of inter-sample variation for all mismatch probes across exon 50 are displayed graphically in Figure 4 . The error bars represent one standard deviation from the mean of the hybridization specificity ratio determined over a running window of 10 bases in each of the 68 samples. Note that the substitution probes show lower inter-sample variability than one base deletion, two base deletion and . Hybridization specificities of mismatch probes. A 10-base running window of the log 10 hybridization specificity ratios of substitution (red), one base deletion (green), two base deletion (blue) and one base insertion (black) was plotted for the sense (A) and antisense (B) strands of ATM exon 50. The light red, light green, light blue and gray shaded areas represent -1 SD of the log 10 hybridization specificity ratios for the substitution, one base deletion, two base deletion and one base insertion probes, respectively. one base insertion probes, in agreement with Table 1 . The variability in hybridization specificity measurements is consistent across all 62 ATM coding exons (Supplementary Figure 1) .
Overall, our analyses indicate that, on average, single base insertion probes show substantially lower reproducibility across experiments than base substitution, one base deletion and two base deletion probes. The increased inter-and intrasample variability in hybridization specificity of single base insertion and deletion probes relative to single base substitution and two base deletion probes should be considered when designing and interpreting microarray-based screens for genetic variation. For a given microarray design, substantially more control hybridization experiments may be needed to determine baseline fluctuations in the hybridization specificities of insertion and deletion probes relative to those of substitution probes.
In contrast to single nucleotide mismatches, detailed thermodynamic analyses of double helical nucleic acids with bulged nucleotides have only recently been conducted (34, (39) (40) (41) . In such cases, the bulged nucleotide is unpaired on only one of the nucleic acid strands. These studies are relevant to understanding the properties of the deletion and insertion probes since they can form duplexes containing bulges with target nucleic acid. For deletion probes, the bulged nucleotide is located on the target strand ( Figure 7) . Conversely, the insertion probes contain the bulged nucleotide in duplexes with wild-type target (Figure 7) .
Although subject to sequence context effects, duplexes containing a single base bulge are predicted to be more stable than those containing single nucleotide mismatches (34, (39) (40) (41) . This is reflected in the lower average hybridization specificity of single base deletion and insertion probes relative to that of substitution probes (Table 1 and Figure 4) . Conversely, duplexes containing two base bulges are predicted to be generally less stable than those containing a single base mismatch (40, 41) . In part, this is due to the assumption that helical stacking is interrupted by bulges of two or greater bases in length while it is preserved for one base bulges (40, 41) . The higher average hybridization specificity ratios of two base (38) was used to predict the intramolecular structures with the lowest Gibbs free energy (DG) for either the 25-30 base stretches that encompass each listed sequence tract in the target or for the PM probes complementary to each sequence tract. We use these DG values to predict the stability of these structures. DG > (À1 kcal/mmol) = weak (W); (À1 kcal/mmol) > DG > (À3 kcal/mmol) = medium (M); and DG < (À3 kcal/mmol) = strong (S). c Type of mismatch probe that provided poor hybridization specificity ratios. d Low hybridization specificity found on both sense and antisense strands. e Immediately following the 3 0 end of this segment is a (T) 5 sequence tract.
deletion probes relative to substitution probes are in agreement with the predicted properties of these probes ( Table 1) . The considerably lower average inter-sample variability of substitution probes relative to deletion and insertion probes was unexpected given that the same target was hybridized to all mismatch probes simultaneously in the same experiment. The sources of inter-sample variation include sample preparation, hybridization conditions and the microarrays themselves. It is reasonable to assume that the microarrays themselves are not the major source of variability since the combinatorial manufacturing processes should lead to roughly equivalent synthesis quality for all the arrayed probes (42, 43) . It seems more likely that the insertion and deletion probes are more sensitive to subtle changes in target preparation (e.g. amount of fragmentation and dye incorporation) and hybridization conditions (e.g. target concentration, temperature and wash conditions) than the substitution probes. However, a definitive explanation for our observations will require further investigations (44) (45) (46) (47) (48) (49) (50) (51) (52) .
In addition to their potential value, it is important to note some of the caveats when relying upon mismatch probes for mutation detection. For example, it is important to screen for all possible sequence changes, including multiple base insertions and deletions, in mutational analyses of disease-related loci, such as the ATM, BRCA1 and BRCA2 genes. Given that 4 N probes per base per strand are needed to screen for insertions of length N in a mixed sequence, it is unlikely that oligonucleotides complementary to insertions of two or more base pairs will be represented on microarrays screening large sequence tracts for mutations in the near future. Deletions represent a more tenable situation since only one probe per base per strand is needed to screen for a deletion of a given length in a mixed sequence. Nevertheless, there will still be limitations as to the number of deletion probes that can be realistically represented in a given microarray.
Finally, it is often critical to precisely determine the nature of a sequence change within a given sample in order to properly assess its functional significance. Thus, it is important to consider error rates when assigning the identity of a mutation based on mismatch probe data. When dealing with clinical samples, it will be especially important to confirm the identity  A Gene Encoding Sialic-Acid-Specific 9-O-Acetylesterase Found in Human Adult Testis Using differential display RT-PCR, we identified a gene of 2750 bp from human adult testis, named H-Lse, which encoded a putative protein of 523 amino acids and molecular weight of 58 kd with structural characteristics similar to that of mouse lysosome sialic-acid-specific 9-O-acetylesterase. Northern blot analysis showed a widespread distribution of H-Lse in various human tissues with high expression in the testis, prostate, and colon. In situ hybridization results showed that while H-Lse was not detected in embryonic testis, positive signals were found in spermatocytes but not spermatogonia in adult testis of human. The subcellular localization of H-Lse was visualized by green fluorescent protein (GFP) fused to the amino terminus of H-Lse, showing compartmentalization of H-Lse in large dense-core vesicles, presumably lysosomes, in the cytoplasm. The developmentally regulated and spermatogenic stage-specific expression of H-Lse suggests its possible involvement in the development of the testis and/or differentiation of germ cells. Sialic acids are a diverse family of acidic nine-carbon sugars that are frequently found as terminal units of oligosaccharide chains on different glycoconjugates in higher invertebrates and vertebrates [1, 2] . As a part of determinants in many glycoproteins [3, 4] , sialic acids play an important role in intercellular and/or intermolecular recognition [5] . The 9-O-acetylation and de-Oacetylation are the most common modifications of sialic acids found in mammalian cell surface sialoglycoconjugates, which can alter its size, hydrophobicity, net charge, and antigenicity [2, 6, 7] . These modifications can regulate a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation [8, 9] .
Enzymes specifically capable of removing O-acetyl esters from the 9-position of sialic acids are sialic-acidspecific 9-O-acetylesterase. The enzymes in mammals have two forms, one is cytosolic sialic-acid-specific 9-O-acetylesterase (Cse) in the cytosolic fraction and another is lysosome sialic-acid-specific 9-O-acetylesterase (Lse) in the lysosomal/endosomal compartment [10] . Lse is likely to participate in the terminal lysosomal degradation of 9-O-acetylated sialoglycoconjugates, while Cse is likely to salvage any 9-O-acetylated molecules that escape the initial action of the Lse enzyme. The process of de-O-acetylation of sialic acid, which is catalyzed by sialicacid-specific 9-O-acetylesterase, has been implicated in organogenesis and cellular differentiation [2, 5] .
Spermatogenesis is a complicated process of germ cell differentiation in adult testis, which is established during testicular development. There are five types of germ cells, each at a specific developmental stage, found in the seminiferous tubules: spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperms. They can be divided into three groups according to their DNA content: 4N DNA content cells (4C cells), 2N DNA content cells (2C cells), and 1N DNA content cells (1C cells). The separation of these cells enables researchers to investigate the molecular mechanisms underlying testicular development and/or spermatogenesis. In the present study, we separated the 2C and 4C cells of seminiferous tubules in human adult testis by flow cytometry, and identified human H-Lse by differential display RT-PCR. The expression pattern of H-Lse was found to be developmentally regulated and stage-specific, indicating its possible role in testicular development and/or germ cell differentiation.
Human testes were obtained from Donation Center of Nanjing Medical University with consent of relatives. The seminiferous tubules were collected in DMEM/F12, which contained collagenase, and washed to remove the Leydig cells as well as interstitial cells. Trypsin treatment and a brief treatment with DNase I were used to release the spermatogenic cells from seminiferous tubules. The suspension of cells was filtered with nylon mesh.
Disaggregated spermatogenic cells were suspended at 1 × 10 6 cells/mL in 0.5 M sodium citrate solution (PH 2.35) with fresh 0.1% DEPC overnight at room temperature and at 4 • C for two days; they were centrifuged and resuspended in 0.5 M sodium citrate solution (PH 4.5) with fresh 0.1% DEPC for at least 1 day. The day before use, the cells were centrifuged and resuspended in PBS with 10 mM HEPES (PH 7.0), 0.1% BSA, and fresh 0.1% DEPC. Then the cells were spun down and resuspended in PBS with 100 µg/mL PI (propidium iodide) and fresh 0.1% DEPC. The cells were stained overnight at 4 • C [11] .
The flow cytometry (FCM) used in this research was FACSVantage SE (Becton Dickinson, Calif) equipped with argon laser (power: 200 mW, wavelength: 488 nm); a 585 nm/42 nm filter set was used before the FL2 detector. Cellquest (Becton Dickinson) was used for sorting and the sorting mode was Normal-R. Drops per sort were 3 and drop delay was 13.6. The density of cells for sorting was about 1 × 10 6 cells/mL.
Isolation of total RNA from 2C cells and 4C cells was performed with Trizol Reagent (Gibco BRL, Ontario). One hundred nanograms of total RNA was used for differential display RT-PCR [12] . The first chain cDNA was synthesized by using T12G, T12C, and T12A oligo (dT) primers, and then was used as template in PCR. PCR was performed as follows: 94 • C, 1 minute; 37 • C, 1 minute; 72 • C, 2 minutes for 40 cycles. Ten microlitres of the PCR products from the two cells were run on a 1.5% agarose gel. The fragments highly or specifically displayed in 4C cells were excised and purified. This DNA was reamplified with the same combination of primers and then subcloned into Pinpoint Xa1-T vector (Promega, USA).
The colonies of full-length cDNA were screened by PCR. Human Testis Large-Insert cDNA Library (Clontech, Calif) was first converted into plasmid cDNA Library, and then an arrayed cDNA library in 96-well plates was made according to the method of Munroe [13, 14] . In this arrayed cDNA library 1.54 × 10 6 colonies were screened by PCR.
Multiple tissue northern (MTN) blots (Clontech) were hybridized with the 32 P-labeled probes. The probe corresponding to 1378-1634 bp of H-Lse was used for hybridization. After stringent wash, the blot was placed on the storage phosphor screen (Packard, USA) and exposed for 3 hours in the dark. The signal was detected at the Cyclone storage phosphor system (Packard).
The Stanford TNG Radiation Hybrid Panel (Research Genetics, Huntsville, Ala) was used to map the chromosomal localization of HSE with primers HSEmapF (5 -ATGAACACCGTCTCCACC-3 ) and HSEmapR (5 -AAATCTGAAGGACCCATC-3 ), according to the manufacturer's instructions. After 35 cycles of amplification, the reaction products were separated on a 1.5% agarose gel. The positive amplification was labeled as 1 and the negative one was labeled as 0. The results were analyzed through the Stanford genome center web server to determine the probable chromosomal location.
RNA DIG-labeled probes were made by in vitro transcription. T7 and SP6 promoter sequences were incorporated into the two sides of the templates (195-553 bp of H-Lse) by PCR, sense and antisense probes were made using DIG-RNA labeling mix (Roche, USA) according to the manufacturer's instructions. After fixation, paraffin embedding, mounting, and sectioning, sections of human embryonic and adult testes were prehybridized in hybridization buffer (DIG Easy Hyb, Roche, Germany) at 42 • C for 2 hours. Hybridization was carried in hybridization buffer containing appropriate probes at 65 • C for 16 hours in humidity chamber.
Subcellular localization of HSEI and HSEII was performed by the method of green fluorescent protein. pEGFP-C2-HSEI AND pEGFP-C2-HSEII were constructed using two sets of primers (HSEI: 5 -GGGGAATT CAATGATATGGTGCTGCAG-3 and 5 -GGGGTCGACAT TTAGCAACATTGCTCTG-3 ; HSEII: 5 -GGGGAATTCA TGGTCGCGCCGGGGCTTG-3 and 5 -GGGGTCGACA TTTAGCAACATTGCTCTG-3 ) and EcoRI/SalI restriction sites of pEGFP-C2. Recombinant vectors were transfected into BxPC-3 cells (BxPC-3 cell is a cell lineage of adenocarcinoma from pancreas) by Lipofectin reagent (Gibco BRL). Cells were imaged 40 hours after transfection on the fluorescence microscope.
After being stained with PI and measured by the FCM, three groups of cells in seminiferous tubules of human adult testis were detected (Figure 1 ), 2C and 4C cells were subsequently sorted. A clone was identified by differential display RT-PCR, which was highly expressed in the 4C cells ( Figure 2 ) and with high homology (86%) to a mouse lysosome sialic acid 9-O-acetylesterase. The clone was named H-Lse.
In the two rounds of screening in the arrayed cDNA library, the plasmid containing full-length H-Lse (GenBank accession number: AF303378) was found. H-Lse is 2750 bp in length, encoding a putative protein of 523 amino acids with a molecular weight of 58 kd. Its isoelectric point is 7.19. The N terminus (1-18 aa) of the protein is a region containing hydrophobic amino acid residues, which may be a signal peptide. By comparison of the protein sequences (Figure 3 ), we hypothesized that H-Lse is the human counterpart of mouse lysosome sialic acid 9-O-acetylesterase.
After PCR amplification, the results can be shown as a pattern (00000000100010100000011000001000000011 000001000001000001001000000010000000000100100100 0001). Retrieving results from the Stanford genome center web server shows that HSE is localized in the human 11q24 ( Figure 4) .
The distribution of H-Lse in various human tissues was analyzed by Northern blot ( Figure 5 ) and the results showed the presence of three distinct mRNA species at approximately 2.7 kb, 6.0 kb, and 7.5 kb. The expected transcript of H-Lse was approximately 2.7 kb and it was consistently expressed in all the tissues examined with high expression found in the testis, prostate, and colon. The transcript of approximately 7.5 kb was exclusively expressed in the colon. The transcript of approximately 6.0 kb was distributed in the testis, colon, small intestine, prostate, and thymus, with the highest level of expression found in the testis.
To examine a possible role of H-Lse in testicular development and/or spermatogenesis, in situ hybridization experiments were conducted to compare H-Lse expression in human embryonic and adult testes since spermatogenesis is not initiated in the embryo and there is no meiosis in embryonic seminiferous tubules. The results showed that no signal was detected in the embryonic testis, while positive signals were detected in spermatocytes but not spermatogonia in the seminiferous tubules of adult testis. Signals were associated with germ cells but not other somatic cells in the testis, that is, Sertoli and Leydig cells. Negative control of sense probes confirmed the specificity of the results ( Figure 6 ).
The subcellular localization of H-Lse fusion proteins was visualized by transiently transfecting H-Lse gene fused with GFP into BXPC-3 cells. As shown in Figure 7 , the control cells transfected with GFP protein alone exhibited fluorescence evenly distributed throughout the cytoplasm, while GFP-H-Lse fusion protein was compartmentalized in numerous large dense-core vesicles in the cytoplasm.
Spermatogenesis is a developmental program that occurs in mitotic, meiotic, and postmeiotic phases. In the mitotic phase, spermatogonia proliferate to expand the quantity of germ cells; in the meiotic phase, spermatocytes accomplish chromosomal synapsis and genetic recombination before two meiotic divisions; and in the postmeiotic phase, haploid spermatids are remodeled into spermatozoa by the processes of acrosome formation, nuclear condensation, flagellar development, and loss of the majority of cytoplasm. Under the control of intrinsic and extrinsic factors, spermatogenesis is characterized by the expression of a spectrum of genes that are celltype-specific or stage-specific. They are thought to play an essential role in spermatogenesis at particular stages. For example, MutS homologue 5 is required for chromosome pairing, CPEB and SCP3 are required for synaptonemal complex assembly and chromosome synapsis in primary spermatocytes [15, 16, 17] .
In the present study, we have identified a gene, H-Lse, from human adult testis with high homology to m-Cse 1 [19] . Similarly, it can inhibit binding of sialoadhesin, a macrophage-restricted and sialic-acid-dependent adhesion molecule [20] . On the other hand, 9-O-acetylation of sialic acids can form novel epitopes. Influenza virus C haemagglutinin specifically requires 9-O-acetylated sialic acids for binding to host cells [21] . Incubation of red blood cells with sialate 9-Oacetylesterase rendered the erythrocytes resistant against agglutination by influenza C virus [22] . O-acetylation of disialoganglioside GD3 by human melanoma cells has been reported to create a unique antigenic determinant [23] . Modifications of sialic acids may be an important mechanism underlying the interaction/cross-talk between different types of cells. The essential role of sialic acids modification in cellular communications may explain the presently observed wide distribution of H-Lse in all examined tissues. The present study suggests that the expression of H-Lse is developmentally regulated and spermatogenic stage-specific. The evidence for this includes: (1) lack of expression in embryonic testis; (2) association of high level of mRNA detected by DD-RT-PCR with the 4C but not 2C cells in adult testes; and (3) detection of in situ hybridization signal in spermatocytes but not spermatogonia or other somatic cells. In the absence of spermatogenesis, embryonic testis contains only two distinct cell types, spermatogonia and Sertoli cells, while the seminiferous epithelium of adult testis consists of germ cells at different stages of spermatogensis. The 4C cells found in adult testis include the primary spermatocytes and spermatogonia of G 2 /M stage, while 2C cells include spermatogonia of G0/G1 stage, secondary spermatocytes, and Sertoli cells. The absence of H-Lse mRNA in embryonic testis and the high level of its mRNA in the 4C cells of adult testis suggest that its expression is restricted to spermatocytes, particularly the primary spermatocytes. Together with the in situ hybridization results showing mRNA of H-Lse restricted to spermatocytes, but not spermatogonia, Sertoli cells or interstitial cells, these data suggest that H-Lse is likely to be involved in the process of spermatogenesis, although its role in testicular development cannot be entirely ruled out. Unfortunately, due to the deformation of the available human testes, we were not able to make further distinction between primary and secondary spermatocytes. What has been clearly shown by the present data is that H-Lse is only present at a stage beyond spermatogonia, suggesting its possible role in the differentiation of germ cells.
G N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 143 m-Lse 181 A G N L G H G N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 240 h-Lse 181 S E N L G H G Y F K Y M S A V C W L F G R H L Y D T L Q Y P I G L I A S S W G G T P I E A W S S G R S L K A C G V P K Q 240 m-Cse 144 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 203 m-Lse 241 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 300 h-Lse 241 G S _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I P Y D S V T G P S K H S V L W N A M I H P L O N M T L K G V V 274 m-Cse 204 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 263 m-Lse 301 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 360 h-Lse 275 W Y Q G E S N I N Y N T D
Interestingly, the processes of 9-O-acetylation and de-O-acetylation of sialic acid have been implicated in organogenesis and cellular differentiation, since alteration of these processes could lead to interruption of cellular development such as embryogenesis. Transgenic mice constitutively overexpressed the 9-O-acetyl-sialicacid-specific esterase of influenza C that has been found to arrest embryo development at the two-cells stage. It has also been reported that in vitro development of embryonic stem cells shows that the expression level of Lse is low at the initiation of the development, and followed by an increase at later stages [24] . In transgenic mice with selective expression of 9-O-acetyl-sialic-acid-specific esterase in retina and the adrenal gland, these organs showed various abnormalities in organization, while all other tissues appeared normal [25] . Lse has also been considered to play a key role in the differentiation of B lymphocyte [2] , since it is expressed in late but not early B lymphocyte. The presently observed developmentdependent pattern of H-Lse expression is consistent with that found in other cell types: absence or low expression at early stage of differentiation but high at later stages. Taken together, 9-O-acetyl esters in sialic acids appear to be important for development or cellular differentiation.
Spermatogenesis is a multiple-staged continuous progress of cellular differentiation. It has been reported that some cell surface glycoconjugates are modified during the early steps of spermatogenesis, and influence the differentiation of spermatogenic cells [26] . As ninecarbon sugars commonly found in many glycoproteins of spermatogenic cells, sialic acids represent a target for cell surface modification, that is, removal of 9-O-acetyl esters by enzymes such as Lse. Modification of sialic acids may result in alteration in cell-cell communication, that is, Sertoli cells and germ cells interaction, thereby influencing the differentiation of spermatogenic cells. Thus, future studies on the presently identified H-Lse may provide insight into molecular mechanisms underlying testicular development and/or germ cell differentiation during spermatogenesis in humans. The role of mast cells in the pathogenesis of pain in chronic pancreatitis BACKGROUND: The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis. We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM). METHODS: Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted. The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis. RESULTS: Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis. WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM. CONCLUSION: Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis. Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood [1] . In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain [1] . More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis [2] . Mast cells are also increased in both acute and chronic pancreatitis [3, 4] but their role in the generation of pain in pancreatitis has not been investigated.
We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings [5, 6] . In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively [5, 6] . Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder [7] [8] [9] . This has also been observed in the pancreas -the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls [3] . One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves [10] [11] [12] . Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation [10] . Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation.
Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit W /Kit W-v mice, a strain deficient in mast cells (MCDM).
Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixtysix patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26) . Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/ 26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch.
All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v (MCDM) and the respective littermate control mouse strain, Kit W-v -+/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25-30 gram.
Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health.
Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [13] ). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure 1 ). Mice were sacrificed 8 weeks after surgery.
VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain.
More recently, VFF testing has been used as a surrogate marker for visceral pain [14, 15] .
Mice were placed in a cage with a mesh floor and habituated to the environment for 30-60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery ( Figure 1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1-2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing.
Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2 at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [16] .
Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test.
For each behavioral experiment (see figure 1), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The "post-pre response frequency" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the postpre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the postpre response frequency was greater for TNBS than for saline infusion.
Fisher's least significant difference procedure was used for multiple comparisons of least squares means with Experimental design Figure 1 Experimental design All mice underwent pre and post surgical VFF testing. For the VFF testing, 4 measures were taken for each mouse. WT and MCDM were randomized to either saline or TNBS perfusion into the pancreatic duct. 
Patient demographics are summarized in Table 1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8),
(33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) ( Figure 2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space. Figure 3 shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) ( Figure 3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level ( Figure 3B ). There was no significant TNBS effect on VFF response in the left A g e 
Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis ( Figure 5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM ( Figure  5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline Histology (Giemsa) of mice with chronic pancreatitis (Figure 6 ). As to be expected, no mast cells were present in pancreas of MCDM.
Chronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [17] . The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis.
There are clearly limitations to a retrospective, autopsybased study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig-Divi et al. [13] . Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation.
Next we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stim-ulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity [14] as well as rat models of acute necrotizing pancreatitis [15] and chronic pancreatitis [18] . The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis [18] . These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes.
Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product [19] [20] [21] [22] . NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation [23] . We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain [24] [25] [26] . Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function [27] [28] [29] [30] [31] [32] . Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis [33, 34] . Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure 7 ).
However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it [35] . Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype.
Our data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory
Proposed involvement of mast cells in nociceptive signaling in pancreatitis Figure 7 Proposed involvement of mast cells in nociceptive signaling in pancreatitis In pancreatitis, mast cells may migrate to sites of inflammation, in response to release of mast cell chemoattractants. Mast cell degranulation products may modulate neurotransmission directly by activating proalgesic receptors and channels such as trka (NGF), TRPV1 (NGF) and PAR-2 (tryptase and trypsin). 
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-230X/5/8/prepub Recombination Every Day: Abundant Recombination in a Virus during a Single Multi-Cellular Host Infection Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10(−5) to 4 × 10(−5). This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus. As increasing numbers of full-length viral sequences become available, recombinant or mosaic viruses are being recognized more frequently [1, 2, 3] . Recombination events have been demonstrated to be associated with viruses expanding their host range [4, 5, 6, 7] or increasing their virulence [8, 9] , thus accompanying, or perhaps even being at the origin of, major changes during virus adaptation. It remains unclear, however, whether recombination events represent a highly frequent and significant phenomenon in the everyday life of these viruses.
Viruses can exchange genetic material when at least two different viral genomes co-infect the same host cell. Progeny can then become hybrid through different mechanisms, such as reassortment of segments when the parental genomes are fragmented [10] , intra-molecular recombination when polymerases switch templates (in RNA viruses) [11] , or homologous or non-homologous recombination (in both RNA and DNA viruses). Quantification of viral recombination in multicellular organisms has been attempted under two distinct experimental approaches: in vitro (in cell cultures) [12, 13, 14, 15] , and in vivo (in live hosts) [16, 17, 18] . The in vitro approach, which has so far been applied only to animal viruses, allows the establishment of the ''intrinsic'' recombination rate in experimentally co-infected cells in cell cultures [14, 15, 19] . However, it does not necessarily reflect the situation in entire, living hosts, where the frequency of coinfected cells is poorly known and depends on many factors such as the size of the pathogen population, the relative frequency and distribution of the different variants, and host defense mechanisms preventing secondary infection of cells. The in vivo experimental approach is closer to biological conditions and may thus be more informative of what actually happens in ''the real world.'' However, as discussed below, numerous experimental constraints have so far precluded an actual quantification of the baseline rate of recombination. First, many experimental designs have used extreme positive selection, where only recombinant genomes were viable (e.g., [13, 20, 21] ). Other studies did not use complementation techniques but detected recombinants by PCR within infected hosts or tissues [18, 22, 23, 24, 25] , which provides information on their presence but not on their frequency in the viral population. So far, no quantitative PCR or other quantitative method has been applied to evaluate the number of recombinants appearing in an experimentally infected live host. Finally, recent methods based on sequence analysis inferred the population recombination rate, rather than the individual recombination rate [1, 26, 27] . While results from these methods certainly take in vivo recombination into account, there are other caveats: isolates have often been collected in different hosts-sometimes in different geographical regions-and sometimes the selective neutrality of sequence variation on which these estimates are based is not clearly established. Estimates from such studies by essence address the estimation of the recombination rate at a different evolutionary scale.
Taken together, the currently available information indicates that no viral recombination rate has ever been estimated directly at time and space scales corresponding to a single multi-cellular host infection, although this level is most significant for the biology and evolution of viruses. This study intends to fill this gap by evaluating the recombination frequency of the cauliflower mosaic virus (CaMV) during a single passage in one of its host plants (the turnip Brassica rapa).
CaMV is a pararetrovirus, which is a major grouping containing hepadnaviruses (e.g., hepatitis B virus), badnaviruses (e.g., banana streak virus), and caulimoviruses (e.g., CaMV). Pararetroviruses are characterized by a non-segmented double-stranded DNA genome. After entering the host cell nucleus, the viral DNA accumulates as a minichromosome [28] whose transcription is ensured by the host RNA polymerase II [29] . The CaMV genome consists in approximately 8,000 bp and encodes six viral gene products that have been detected in planta ( Figure 1 ) [30] . Viral proteins P1 to P6 are expressed from two major transcripts, namely a 19S RNA, encoding P6, and a 35S RNA corresponding to the entire genome and serving as mRNA for proteins P1-P5 [31] . Using the pre-genomic 35S RNA as a matrix, the protein P5 (product of gene V) reverse-transcribes the genome into genomic DNA that is concomitantly encapsidated [30] .
The detection of CaMV recombinants in turnip hosts has been reported numerous times. Some studies have demonstrated the appearance of infectious recombinant viral genomes after inoculation (i) of a host plant with two infectious or non-infectious parental clones [21, 32, 33, 34, 35] or (ii) of a transgenic plant containing one CaMV transgene with a CaMV genome missing the corresponding genomic region [36] . While the former revealed inter-genomic viral recombination, the latter demonstrated that CaMV can also recombine with transgenes within the host's genome. Another study based on phylogenetic analyses of various CaMV strains has clearly suggested different origins for different genomic regions and, hence, multiple recombination events during the evolution of this virus [37] . Indirect experimental evidence has indicated that, in some cases, CaMV recombination could occur within the host nucleus, between different viral minichromosomes, presumably through the action of the DNA repair cellular machinery [21, 35] . Nevertheless, the mechanism of ''template switching'' during reverse transcription, predominant in all retroviruses, most certainly also applies to pararetroviruses. For this reason, and on the basis of numerous experimental data, CaMV is generally believed to recombine mostly in the cytoplasm of the host cell, by ''legal'' template switching between two pre-genomic RNA molecules [21, 35, 36, 38, 39] , or ''illegal'' template switching between the 19S and the 35S RNA [36, 40] . Under this hypothesis, recombination in CaMV could therefore be considered as operating on a linear template during reverse transcription, with the 59 and 39 extremities later ligated to circularize the genomic DNA (position 0 in Figure 1 ). The above cited studies clearly demonstrate that CaMV is able to recombine. However, since these studies are based on complementation techniques, non-quantitative detection, or phylogenetically based inferences of recombination, they do not inform us on whether recombination is an exceptional event or an ''everyday'' process shaping the genetic composition of CaMV populations.
In the present work, we aimed at answering this question. To this end, we have constructed a CaMV genome with four genetic markers, demonstrated to be neutral in competition experiments. By co-inoculating host plants with equal amounts of wild-type and marker-containing CaMV particles, we have generated mixed populations in which impressive proportions of recombinants-distributed in several different classes corresponding to exchange of different genomic regions-have been detected and quantified. Altogether, the recombinant genomes averaged over 50% of the population. Further analysis of these data, assuming a number of viral replications during the infection period ranging from five to 20, indicates that the per nucleotide per replication cycle [44] ) indicates the origin of replication via reverse transcription, which occurs in the direction indicated by the dotted outermost circle-like arrow. Reverse transcription is accomplished by the viral reverse transcriptase, using the 35S RNA as template [49] . DOI: 10.1371/journal.pbio.0030089.g001 recombination rate of CaMV is of the same order of magnitude, i.e., on the order of a few 10 À5 , across the entire genome. We thereby provide the first quantification, to our knowledge, of the recombination rate in a virus population during a single passage in a single host.
From Figure 1 , and supposing that all marker-containing genomic regions can recombine, we could predict the detection and quantification of seven classes of recombinant genotypes: þbcd/aþþþ, aþcd/þbþþ, abþd/þþcþ, abcþ/þþþd, þþcd/abþþ, aþþd/þbcþ, and aþcþ/þbþd. Indeed, all classes were detected, and their frequencies in the ten CaMV populations analyzed are summarized in Table 1 .
Altogether, the proportion of recombinant genomes found in the mixed viral populations was astonishingly high and very similar in the ten co-infected plants analyzed (Table 1 , last column), ranging between 44% (plant 5) to 60% (plants 7, 12, and 20), with a mean frequency (6 standard error) of 53.8% 6 2.0%. This result indicates that recombination events are very frequent during the invasion of the host plant by CaMV and represents, to our knowledge, the first direct quantification of viral recombination during the infection of a live multi-cellular host.
The inferred per generation recombination and interference rates, assuming that CaMV undergoes ten replication cycles during the 21 d between infection and sampling, are given for each of the ten plants in Table 2 . Recombination rates between adjacent markers are large, on the order of 0.05 to 0.1. Taking the distance in nucleotides between markers into account yields an average recombination rate per nucleotide and generation on the order of 4 3 10 À5 .
Interestingly, this recombination rate does not vary throughout the genome (Kruskal-Wallis test, p = 0.16).
To relax the assumption of the number of replications during the 21 d, we calculated the recombination parameters assuming five or 20 generations. The effect of the number of generations on the estimates is linear: doubling the number of generations results in a halving of the recombination rate (detailed results not shown). For example, the average recombination rates r 1 , r 2 , and r 3 assuming 20 generations were equal to 0.05, 0.04, and 0.025, respectively (compare with values in Table 2 ), yielding per nucleotide per generation recombination rates of 1.9 3 10 À5 , 2.2 3 10 À5 and 1.6 3 10 À5 .
Inspection of Table 2 also shows that first-order interference coefficients were in general negative, indicating that a crossing over in one genomic segment increases the probability that a crossing over will occur in another genomic segment, while the second-order coefficient parameter had an average value close to zero with a large variance. The mechanism leading to these results will be discussed in the following section.
One major breakthrough in the work presented here lies in the space and time scales at which the experiments were performed. Indeed, the processes occurring within the course of a single infection of one multi-cellular host are of obvious biological relevance for any disease. Previous studies on viral recombination suffered from major drawbacks in this respect, basing their conclusions on experiments relying on complementation among non-infectious viruses or between viruses with undetermined relative fitness, on phylogenetically based analyses, or on experiments in cell cultures. For reasons detailed in the Introduction, the first two methods either do not provide information on the frequency of recombination, but only its occurrence, or address the question at a different temporal, and often spatial, scale. Results from cell cultures, on the other hand, impose cell coinfection by different viral variants, potentially overestimat- ing the frequency of recombination events. Our study circumvents these limitations by analyzing viral genotypes sampled from infected plants after the course of a single infection, and therefore the invasion and co-infection of cells in various organs and tissues is very close to natural. More than half of the genomes (53.8% 6 2.0%; see Table 1 ) present in a CaMV population after a single passage in its host plant were identified as recombinants, and these data allowed us to infer a per nucleotide per generation recombination rate on the order of 2 3 10 À5 to 4 3 10 À5 . The time length of one generation, i.e., the time required for a given genome to go from one replication to the next, is totally unknown in plant viruses. The only experimental data available on CaMV are based on the kinetics of gene expression in infected protoplasts, where the capsid protein is produced between 48 and 72 h [40] . The reverse transcription and the encapsidation of genomic DNA being two coupled phenomena [30] , we judged it reasonable to assume a generation time of 2 d and, thus, an average of ten generations during our experiments. In case this estimate is mistaken, we have verified a linear relationship between r and the number of generations, thereby allowing an immediate adjustment of r if the CaMV generation time is more precisely established. At this point, we must consider that all cloned genomes may not have been through all the successive replication events potentially allowed by the timing of our experiments. It was previously shown that about 95% of CaMV mature virus particles accumulate in compact inclusion bodies [41] , where they may be sequestered for a long time, as such inclusions are very frequent in all infected cells, including those in leaves that have been invaded by the virus population for several weeks. The viral population may thus present an age structure that could bias the estimation of the recombination rate. In order to minimize this bias, the clones we analyzed were collected in one young newly formed leaf, where the chances of finding genomes from ''unsequestered lines'' were assumed to be higher. In any case, our data analysis is conservative, since this age structure can only lead to an underestimation of the recombination rate.
Our results show that interferences between pairs of loci are negative: a recombination event between two loci apparently increases the probability of recombination between another pair of loci. We believe that the most parsimonious explanation of these negative interferences is based on the way the infection builds up within plant hosts. Indeed, one can divide infected host cells into those infected by a single virus genotype and those infected by more than one viral genotype. In the former, analogous to clonal propagation, recombination is undetectable. In the latter, recombination is not only detectable but, as our results indicate, very frequent. Samples consisting of viruses resulting from a mixture of these two types of host cell infections will thus contain viruses with no recombination and viruses with several recombination events, thus yielding an impression of negative interference. These conceptual arguments are supported by mathematical models. It is indeed easy to show (detailed results not shown) that if a proportion F of the population reproduces clonally, analogous to single infections, while the remaining reproduces panmictically, negative interferences could be inferred even if they do not exist. For example, assuming a three-locus model with real recombination rates r 1 and r 2 and interference i 12 , the ''apparent'' recombination and interference parameters, would be r 1 = (1 À F)r 1 , r 2 = (1 À F)r 2 , and i 12 = À(F À i 12 )/(1 À F). Interestingly, this example also shows that our estimates of the recombination rate are conservative: that a fraction F of host cells are singly infected while others are multiply infected leads to an underestimation of the recombination rate. As judged by r 1 , r 2 , and r 3 , calculated between markers a-b, b-c, and c-d, respectively, we found evidence for recombination through the entire CaMV genome. The values for r 1, r 2 , and r 3 are remarkably similar, hence the recombination sites seem to be evenly distributed along the genome. We considered the template-switching model as the major way recombinants are created in CaMV. As already mentioned in the Introduction, hot spots of template switching have been predicted at the position of the 59 extremities of the 35S and 19S RNAs [21, 36, 42] . If other recombination mechanisms, such as that associated with second-strand DNA synthesis or with the host cell DNA repair machinery, act significantly, hot The various parameters are as follows: r1, recombination rate between markers a and b; r2, recombination rate between markers b and c; r3, recombination rate between markers c and d; i12, interference between crossovers in segments a-b and b-c; i23, interference between crossovers in segments b-c and c-d; i13, interference between crossovers in segments a-b and c-d; i123, second-order interference accounting for residual interference. The recombination rates are the maximum likelihood estimates (6 95% confidence intervals). The interference parameters were obtained numerically as explained in the Materials and Methods. DOI: 10.1371/journal.pbio.0030089.t002 spots would be expected at the positions of the sequence interruption D1, D2, and D3 [43] . Due to the design of our experiment and the position of the four markers, we have no information on putative hot spots at positions corresponding to the 59 end of the 35S RNA and to D1 (at nucleotide position 0). Nevertheless, the putative hot spots at the 59 end of the 19S RNA and at D2 and D3 (nucleotide positions 4,220 and 1,635, respectively) fall between marker pairs c-d, b-c, and a-b, respectively. Our results indicate that either these hot spots are quantitatively equivalent-though predicted by different recombination mechanisms-or, more likely, that they simply do not exist. Whatever the explanation, what we observe is that the CaMV can exchange any portion of its genome, and thus any gene thereof, with an astonishingly high frequency during the course of a single host infection. To our knowledge, the viral recombination rate has never previously been quantified experimentally for a plant virus [3] . In contrast, retroviruses and particularly HIV-1 have been extensively investigated in that sense. As we have already discussed for these latter cases, the quantification of the intrinsic recombination rate was carried out in artificially coinfected cell cultures. The estimated intrinsic per nucleotide per generation recombination rate in HIV-1 is on the order of 10 À4 [14, 15, 19] , less than one order of magnitude higher than our estimation for CaMV. Because for various reasons detailed above we probably underestimate the within-host CaMV recombination rate, we believe that the intrinsic recombination rate in CaMV is higher and perhaps on the order of that of HIV.
Other pararetroviruses such as plant badnaviruses or vertebrate hepadnaviruses have a similar cycle within their host cells, including steps of nuclear minichromosome, genomic size RNA synthesis, and reverse transcription and encapsidation. Nevertheless, vertebrate hepadnaviruses (e.g., hepatitis B virus) infect hosts that are very different from plants in their biology and physiology, and this could lead to a totally different frequency of cell co-infection during the development of the virus populations. Thus, even though our results can be informative for other pararetroviruses because of the viruses' shared biological characteristics, they should not be extrapolated to vertebrate pararetroviruses without caution.
Viral isolates. We used the plasmid pCa37, which is the complete genome of the CaMV isolate Cabb-S, cloned into the pBR322 plasmid at the unique SalI restriction site [44] . To analyze recombination in different regions of the genome, we introduced four genetic markers: a, b, c, and d, at the positions 881, 3,539, 5,365, and 6,943, respectively, thus approximately at four cardinal points of the CaMV circular double-stranded DNA of 8,024 bp ( Figure 1 ). All markers, each corresponding to a single nucleotide change, were introduced by PCR-directed mutagenesis in pCa37, and resulted in the duplication of previously unique restriction sites BsiWI, PstI, MluI, and SacI in a plasmid designated pMark-S. Because, in this study, we targeted the possible exchange of genes between viral genomes, all markers a, b, c, and d were introduced within coding regions corresponding to open reading frames I, IV, V, and VI, respectively. Another important concern was to quantify recombination in the absence of selection, i.e., to create neutral markers. Consequently all markers consist of synonymous mutations (see below).
Production of viral particles and co-inoculation. To generate the parental virus particles, plasmids pCa37 and pMark-S were mechanically inoculated into individual plants as previously described [33] . All plants were turnips (B. rapa cv, ''Just Right'') grown under glasshouse conditions at 23 8C with a 16/8 (light/dark) photoperiod.
Thirty days post-inoculation, all symptomatic leaves were harvested and viral particles were purified as described earlier [45] .
The resulting preparations of parental viruses, designated Cabb-S and Mark-S, were quantified by spectrometry using the formula described by Hull et al. [46] . We fixed the initial frequency of markers to a value of 0.5, and a solution containing 0.1 mg/ml of virus particles of both Cabb-S and Mark-S at a 1:1 ratio was prepared. Plantlets were co-infected by mechanical inoculation of two to three leaves with 20 ll of this virus solution, using abrasive Celite AFA (Fluka, Ronkonkoma, New York, United States). The mixed CaMV population was allowed to grow during 21 d of systemic infection.
Estimation of marker frequency within mixed virus populations. We designed an experimental protocol for quantifying marker frequency within a mixed Cabb-S/Mark-S virus population after a single passage in a host plant. Twenty-four individual plants, inoculated as above with equal amounts of Cabb-S and Mark-S, were harvested 21 d post-inoculation, when symptoms were fully developed. The viral DNA was purified from 200 mg of young newly formed infected leaves according to the protocol described previously [47] . After the precipitation step of this protocol, the viral DNA was resuspended and further purified with the Wizard DNA clean-up kit (Promega, Fitchburg, Wisconsin, United States) in TE 1X (100 mM Tris-HCl and 10 mM EDTA [pH 8]). Aliquots of viral DNA preparations were digested by restriction enzymes corresponding either to marker a, b, c, or d and submitted to a 1% agarose gel electrophoresis, colored by ethydium bromide and exposed to UV. Each individual restriction enzyme cut once in Cabb-S DNA and twice in Mark-S, thus generating DNA fragments of different sizes attributable to one or the other in the mixed population of CaMV genomes. After scanning the agarose gels, we estimated the relative frequency of the two genotypes in each viral DNA preparation and at each marker position, by densitometry using the NIH 1.62 Image program. The statistical analyses of the frequency of the four markers are described below.
Isolation of individual CaMV genomes and identification of recombinants. To identify and quantify the recombinants within the CaMV mixed populations, aliquots from ten of the 24 viral DNA preparations described above were digested by the restriction enzyme SalI, and directly cloned into pUC19 at the corresponding site. In each of the ten viral populations analyzed, 50 full-genome-length clones were digested separately by BsiWI, PstI, MluI, and SacI, to test for the presence of marker a, b, c, and d, respectively. In this experiment, with the marker representing an additional restriction site, we could easily distinguish between the Cabb-S and the Mark-S genotype at all four marker positions, upon agarose gel (1%) electrophoresis of the digested clones. Clones with none or all four markers were parental genotypes, whereas clones harboring 1, 2, or 3 markers were clearly recombinants. Due to the very high number of recombinants detected, markers eventually appearing or disappearing due to spontaneous mutations were neglected.
Statistical analysis. Here we present the different methods we used to quantify recombination in the CaMV genome. Because all these methods assume that the different markers are neutral, we first discuss assumption.
We used two datasets to test the neutrality of markers, both resulting from plants co-infected with a 1:1 ratio of Mark-S and Cabb-S. The first consisted of viral DNA densitometry data derived from 24 plants (described above), where for each plant we have an estimate of the frequency of each marker in the genome population. The second consisted of the restriction of 50 individual full-genome-length viral clones obtained from one co-infected plant (described above), yielding an estimate of the frequency of each marker, and this was repeated on ten different plants. The frequencies of the different markers were 0.508, 0.501, 0.516, and 0.507 for markers a, b, c, and d in the first dataset and 0.521, 0.518, 0.514, and 0.524 in the second dataset. We tested whether these frequencies were significantly different from the expected value under neutrality, 0.5, using either t-tests, for datasets where normality could not be rejected (seven out of eight cases), or Wilcoxon signed-rank non-parametric tests otherwise (marker c in the first dataset). In all cases p-values were larger than 0.05.
There are several cautionary remarks regarding these analyses. First, in all cases we found an excess of markers. Unfortunately, the two datasets cannot be regarded as independent because, even though the methods through which the frequency estimates were obtained were different, the plants used in the second dataset were a subset of the plants of the first. We thus have only four independent estimates in each case, and there is minimal power to detect significant deviations from neutrality with such a small sample size. It should be noted at this stage that deviations from the expected value could also be caused either by slight deviations from the 1:1 ratio in the infecting mixed solution, or by deviations from that ratio in the frequency of the viral particles that actually get into the plants. Second, because of the relatively small sample sizes and low statistical power, the tests presented above could have detected only large deviations.
The results clearly show, however, precisely that the markers do not have large effects, if any, and that therefore recombination estimates would be affected only very slightly by any hypothetical selective effects of the markers. Because of this, along with the fact that the introduced markers provoke silent substitutions in the CaMV genome, we assumed that markers were effectively neutral in the rest of the analysis.
The dataset used to estimate the recombination frequency consisted of the 500 full-genome-length viral clones (50 from each of ten co-infected plants) individually genotyped for each of the four markers. As discussed in detail in the Results, recombination was very frequent and concerned all four markers. Indeed, approximately half of the genotyped clones exhibited a recombinant genotype. It was therefore meaningful to try to obtain quantitative estimates of recombination from our data.
Our aim was to analyze viral recombination in a live host. Consequently, we had to deal with the fact that more than one viral replication cycle occurred during the 21 d that infection lasted in our experiment (we had to wait that long for the disease to develop and to be able to recover sufficient amounts of viral DNA from each infection). Based on the kinetics of gene expression [40] , we postulate that each replication cycle lasts between 2 and 3 d, and that therefore seven to ten cycles occurred between infection and the sampling time. In case this assumption is incorrect, we did calculations assuming five, seven, ten, or 20 replication cycles during these 21 d. As shown, the results were not affected qualitatively, and only slightly quantitatively. It is important to note that we assumed that recombination occurred through a template-switching mechanism, and that therefore, from a recombination point of view, the CaMV genome is linear. The reverse transcription starts and finishes at the position 0 in Figure 1 , which is the point of circularization of the DNA genome. This implies that changes between contiguous markers a-b, b-c, and c-d can be considered as true recombination whereas those between a and d cannot, as they may simply stem from circularization of DNA, during the synthesis of which the polymerase has switched template once anywhere between a-b, b-c, or c-d.
To estimate the recombination rate between markers, we wrote recurrence equations describing the change in frequency of each genotype over one generation, assuming random mating and no selection (i.e., the standard Wright-Fisher population genetics model). We then expressed the frequency of all possible genotypes n generations later as a function of their initial frequency and of the recombination parameters. Subsequently we calculated the maximum likelihood estimates of the recombination parameters and their asymptotic variances given initial frequencies (we assumed that the two ''parental'' genotypes, Mark-S and Cabb-S, had equal initial frequencies of 0.5 and that all other genotypes had initial frequencies of zero) and frequencies after n generations (the observed frequencies; as stated above we used different values of n). All algebraic and numerical calculations were carried out with the software Mathematica.
The recombination parameters are the recombination rates between two adjacent loci, e.g., r 1 for the recombination rate between markers a and b, and the interference coefficients, e.g., i 12 for interference between recombination events in the segments between markers a and b and b and c. To define these parameters we followed Christiansen [48] , and in particular the recombination distributions for two, three, and four loci (respectively, Tables 2.7, 2.8, and 2.9 of [48] ). It is important to realize that given the definitions of these parameters, the estimator of the recombination rate between two loci is not affected by the number of loci considered. In other words, we obtain the same estimation of the recombination rate between markers a and b whether we consider genotypic frequencies at just these two loci, or the frequencies at these two loci plus a third locus, or the complete information to which we have access, the fourmarker genotypes. Information on additional loci only affects the estimates of the interference coefficients.
It proved impossible to carry out the calculations for four loci algebraically. Instead, we used a computer program to calculate the expected genotypic frequencies at all four loci after n generations, given the above stated initial frequencies and specified recombination parameters. For each combination of recombination parameters we calculated a Euclidean distance between the vector of the expected genotypic frequencies and the observed genotypic frequencies, and considered that the estimated recombination parameters were those yielding the minimal Euclidean distance. In all cases, the estimated recombination rates between pairs of loci were equal to the second decimal to those estimated algebraically from data for three or two loci. Torsional restraint: a new twist on frameshifting pseudoknots mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot. The structure of an RNA molecule is widely recognized to play a role in many processes, including structurally organizing complex RNAs, the assembly of ribonucleoprotein complexes, and in translational recoding and regulation [reviewed in (1) ]. One common RNA folding motifs is a pseudoknot, the folding back of a single-stranded RNA onto itself to form two helical structures with single-stranded loops joining them (2) . Many such structures can be inferred from RNA sequences and frameshifting function has been demonstrated for some of these [reviewed in (3) (4) (5) ]. However, though much theoretical progress has been made in understanding how mRNA pseudoknots promote efficient À1 ribosomal frameshifting (6), a complete understanding of this mechanism remains untested.
Programmed À1 ribosomal frameshift signals are typically divided into three components. From 5 0 to 3 0 these are (i) a 'slippery site' in the form N NNW WWH, where N must be a stretch of any three identical nucleotides, where W is either three A or U residues, and H is A, C or U (spacing indicates the unshifted zero frame), (ii) a spacer region and (iii) an mRNA structural element, most often a pseudoknot. The general model posits that upon encountering the mRNA pseudoknot, an elongating ribosome is forced to pause such that the anticodons of its A-and P-site tRNAs are base-paired with the zero-frame codons of the slippery site. The nature of the tRNA-mRNA interactions is such that a relative slip of À1 nucleotide still allows base-pairing in the non-wobble positions. The slippage occurs during the ribosomal pause, and it has been shown that changes affecting ribosome pause times affect frameshift efficiencies [reviewed in (7) ]. An important observation is that even though mRNA pseudoknots and energetically equivalent stem-loop structures appear to promote ribosome pausing with equal effectiveness, mRNA pseudoknots are more efficient at promoting À1 PRF (8). Our '9 Å ' model (6) provided a refinement of the original 'simultaneous slippage' (9, 10) model of frameshifting by suggesting that rather than the entire ribosome having to slip one base in the 5 0 direction, slippage could be accomplished by moving the small section of mRNA in the downstream tunnel by one base in the 3 0 direction. We have proposed that this is accomplished by the bulky and difficult to unwind mRNA pseudoknot structures becoming wedged in the downstream entrance tunnel of the ribosome, preventing the downstream region of the mRNA from being pulled into the ribosome by the equivalent of one base during the accommodation step of elongation. This blockage would introduce tension into the spacer region, which could be resolved by unpairing the mRNA from the tRNAs, allowing the mRNA to slip 1 nt backwards, resulting in a net shift of reading frame by À1 base.
Though the 9 Å model provides a partial explanation for why mRNA pseudoknots promote programmed À1 ribosomal frameshifting (À1 PRF) more efficiently than simple *To whom correspondence should be addressed. Tel: +1 301 405 0981; Fax: +1 301 314 9489; Email: dinman@umd.edu ª The Author 2005. Published by Oxford University Press. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org stem-loop structures, it does not answer the question of how the mRNA pseudoknot directs the ribosome to pause at the correct position along the mRNA. A complementary 'torsional restraint' model addresses this issue (11) . When a stem-loop structure is unwound by an elongating ribosome, unwinding of the stem forces the loop to rotate. Since a simple stem-loop is not restrained, the loop can rotate freely and only the base pairs within the stem resist ribosomal movement, and thus the potential energy of unwinding should be distributed along the length of Stem 1 ( Figure 1A ). However, if the loop is anchored or restrained, as it is in a pseudoknot by Stem 2, since the intrinsic ribosomal helicase is processive (12) , Stem 1 cannot be fully unwound until Stem 2 is first denatured. Mechanically, as the ribosome begins to unwind the base of Stem 1, Stem 2 forces the supercoiling in the remainder of Stem 1, providing extra resistance to ribosome movement. At some specific point, the resistance to ribosome movement provided by the supercoiling counteracts the forward movement of the ribosome, increasing the likelihood that the ribosome will stop at a precise point along the mRNA. Energetically, since full unwinding of Stem 1 is dependent on complete denaturation of Stem 2, the potential energy of unwinding of the pseudoknot structure should similarly be directed toward one point. Viewed either mechanically or energetically, this point is where ribosomes will be directed to specifically pause on the mRNA. If it occurs with the tRNAs in ribosomal A-and P-sites positioned at the slippery site, then frameshifting is stimulated. This is summarized in Figure 1B . The efficiency of À1 PRF can thus be viewed as a function of (i) the fraction of ribosomes paused over the slippery site and (ii) the rate at which the structure can be denatured. There is increasing evidence from single molecule experiments that unfolding occurs in quick 'rips' at a particular force (13) , suggesting that in the case of unfolding pseudoknots, frameshifting efficiency is related to both the energy barriers to unfolding the pseudoknot structure and the resistance of the structure against the force of the ribosome. In the context of the torsional restraint model, this resistance is dependent on the ability of Stem 2 to remain intact while Stem 1 is being unwound.
There is experimental data that indirectly support this model: (i) disruption of the first 3 bp of Stem 1, which would displace the ribosome's pause site to a point 3 0 of the slippery site, has been shown to eliminate frameshifting (14) ; (ii) destabilizing Stem 2, which would allow it to be unwound more readily, has been shown to result in decreased frameshifting efficiency (15) (16) (17) ; (iii) replacing bulges in Stem 1 with base pairs would increase the energy required to unwind the first three bases, and a longer ribosomal pause over the slippery site would follow, yielding increased efficiencies in À1 frameshifting (15, 18) ; (iv) destabilizing the base of Stem 1 by replacing G:C base pairs with A:U pairs decreases À1 frameshifting efficiencies (19, 20) ; (v) the model eliminates the need for a 'pseudoknot recognizing factor', the evidence of which has not been forthcoming in either competition assays in in vitro translation systems (21) or by gel retardation assays (J. D. Dinman, unpublished data); and (vi) elimination of a potential torsion-restraining Stem 2, but not of a non-torsion-restraining Stem 2 in HIV-1, resulted in decreased À1 PRF efficiencies (22) .
Though all of the cited studies support the torsional restraint model, none has directly addressed it. In the experiments presented in this study, a series of 'pseudo-pseudoknot' containing reporter constructs were used to test the torsional restraint hypothesis. In vitro frameshifting assays show that frameshifting can be significantly stimulated by limiting the rotational freedom of the loop region of a stem-loop structure, and that the degree of rotational freedom of Stem 1 is important in determining the extent of À1 PRF. Furthermore, mRNA toeprint analyses reveal a pseudo-pseudoknot-specific strong stop 16 nt 3 0 of the slippery site, consistent with this structure being able to direct ribosomes to pause with their A-and P-sites positioned at the slippery site.
All synthetic DNA oligonucleotides were purchased by IDT (Coralville, IA). The modified L-A viral À1 PRF signal containing the G GGU UUA slippery site followed by a simple stem-loop was amplified from pJD18 (23) using the primers luc5 0 b (5 0 -CCCCAAGCTTATGACTTCTAGGCAGGGTTT-AGG-3 0 ) and luc3 0 b (5 0 -CCCCCCATGGGACGTTGTAAA-AACGACGGGATC-3 0 ). These were digested with HindIII and NcoI (restriction sites are underlined) and cloned into the firefly luciferase reporter plasmid pT7-LUC minus 3 0untranslated region-A50 (24) . In the resulting reporter construct (pJD214-18), expression of firefly luciferase requires a À1 frameshift, and the 5 0 sequence of the Stem 2 is not able to base pair with the 3 0 sequence, so that only a stem-loop rather than a pseudoknot is able to form. The same primers were used to amplify DNA from pJDRC (23) to make pJD214-Ry. In this construct, complementary mutations (5 0 -GCUGGC-3 0 to 5 0 -CGACCG-3 0 ) in the 3 0 acceptor sequence of the pseudoknot-forming region of Stem 2 allow the formation of an mRNA pseudoknot that has previously been shown to promote frameshifting at the same frequency as the wild type (23) . The primer luc5 0 CON (5 0 -CCCCAAGCTTATGACTTC-TAGGCAAGGGTTTAGG-3 0 ) contains an additional A nucleotide upstream of the slippery site and was used to make pJD214-0, the zero-frame control. To eliminate the possibility of internal initiation occurring at the luciferase initiation codon downstream of the frameshift signals, the AUG codon was changed to AUA. The Stratagene Quik-Change kit was used to mutate pJD214-18 and pJD214-Ry into pJD336-18 and pJD366-Ry, respectively, using the oligonucleotides 5 0 -GGCGTTCTTCTATGGGACGTTGTA-AAAACGGATC-3 0 and 5 0 -GATCCGTCGTTTTTACAACG-TCCCATAGAAGACGCC-3 0 (the mutated codon is underlined). pJD366-18 was further mutated to make a zero-frame control by placing an A upstream of the slippery site using the oligonucleotides 5 0 -TGACTTCTAGGCAAGGGTTTAGGAG-TG and 5 0 -CACTCCTAAACCCTTGCCTAGAAGTCA (the inserted base is underlined). A series of synthetic DNA oligonucleotides were designed to join the loop acceptor region of mRNA transcribed from pJD366-18 to the downstream region that forms the pseudoknot in the wild-type L-A À1 PRF signal. In the J-oligos, the 3 0 sequences base pair with the loop of the mRNA transcribed from pJD366-18, and the 5 0 regions of these oligos base pair with the downstream sequence. This orientation is reversed for the R-oligos. These general orientations are shown in Figure 3B . The naming of the oligonucleotide names refers to the number of additional residues placed between the regions of complementarity. The bases complementary to the pJD366-18 sequence are underlined.
Plasmid DNAs were prepared using the Qiagen mini-prep kits and were linearized with DraI in a total volume of 20 ml. Proteins were eliminated by the addition of 2 ml of 1 mg/ml proteinase K and SDS to a final concentration of 0.5% followed by digestion at 50 C for 30 min. Volumes were then increased to 100 ml, extracted twice with phenol/chloroform, and DNA was precipitated with 10 ml NH 4 Ac and 250 ml ethanol. The purified DNA was resuspended in DEPCtreated H 2 O. To prepare synthetic mRNAs, 2 ml of purified linear DNAs were used for in vitro transcription using the Ambion T7 mMachine mMessage kit. RNAs were precipitated using 30 ml DEPC H 2 O and 25 ml LiAc. The RNA was resuspended in 11 ml DEPC H 2 O (1 ml in 500 would give an OD 260 of 0.05-00.1; 1-2 mg/ml).
To anneal the oligonucleotides with the mRNA, J-oligos, R-oligos or the equivalent volumes of dilution buffer alone (20 mM Tris, pH 7.4, 2 mM MgCl 2 and 50 mM EDTA final concentration) were added to synthetic mRNA (0.5 mg), and the mixtures were first incubated in a 70 C heating block for 10 min; the block was then removed and allowed to cool to 37 C (30 min), after which they were briefly spun down and incubated on ice. In all experiments, the molar ratios of J-and R-oligos to synthetic mRNAs were 100:1. In experiments using the competing oligonucleotide (C-oligo), this was added to either 0.5:1 or 1:1 molar ratios with either J-or R-oligonucleotides. Reticulocyte lysates were thawed on ice, 15 ml of Àmet and 15 ml of Àleu master mixes plus 20 ml of H 2 O were added to 400 ml of lysate, and 19 ml of this was added to each annealed reaction to start the in vitro translation reactions. These were incubated at 30 C for 60 min (the reaction reached a plateau after 30-35 min where the greatest difference was seen between the zero-frame controls and the frameshifting plasmids) (data not shown), and the reactions were then placed on ice. An aliquot of 7.5 ml from each in vitro translation reaction was added to 50 ml of the prewarmed luciferase reagent, and luminescence readings were taken after a 3 s delay for 15 s in triplicate using a Turner 20/20 Luminometer.
Synthetic transcripts generated from DraI-digested pJD366-18 ($1.7 kb) were 5 0 end labeled using [g-32 P]CTP. These RNAs (4 ml) were incubated with 1 ml of annealing buffer and either 1 ml of H 2 O or 1 ml of an oligo (0.25 ng) at 70 C. The heating block was allowed to cool at room temperature for 40 min before 8 ml of RNaseH buffer was added (20 mM HEPES, 50 mM KCl, 10 mM MgCl 2 and 1 mM DTT). An aliquot of 1 ml of enzyme was added (mung bean nuclease, RNaseH or RNaseT1) and the reactions incubated at 37 C for 1 h. The reactions were stopped by adding 4 ml of stop solution, the products separated through a 6% polyacrylamide-urea denaturing gel and visualized by autoradiography.
mRNA toeprinting JD366-18 mRNA (1 mg in 8 ml) was annealed with 2 ml of 3 0 end-labeled toeprinting primer (5 0 -CGTACGTGATCTTCA-CC-3 0 , complementary to sequence 240 bp 3 0 of the slippery site) as described above. This was added to 15 ml of lysate (200 ml Ambion retic lysate, 7.5 ml of each master mix [Àleu and Àmet] and 70 ml of 250 mM KCl), except for 2 ml, which was added to 15 ml of RT buffer [50 mM Tris-HCl (25), 40 mM KCl, 6 mM MgCl 2 , 5 mM DTT and 575 mM dNTPs] to be used as a no-ribosome control. In vitro translation reactions were incubated at room temperature for 10 min, which was empirically determined to provide the optimum amount of time to allow ribosomes to initiate translation and pause at the frameshift signal. Subsequently, 15 ml of RT buffer containing RNasin inhibitor and cycloheximide (to a final concentration of 100 ng/ml) was added to stop translation. To this, 2 ml of Superscript II (Invitrogen) was added and the reaction incubated at room temperature for 10 min. Reactions were terminated by phenol:chloroform extraction and 15 ml of stop solution added. The toeprinting primer was also used in conjunction with pJD366-18 to produce sequencing ladders by standard dideoxynucleotide chain termination methods using Sequenase (USB). Products were separated though 6% polyacrylamide-urea denaturing gels and visualized using a Storm phosphorImager (Pharmacia).
Pseudo-pseudoknots stimulate frameshifting, and frameshifting efficiency changes with the degree of pseudo-pseudoknot rotational freedom
We previously showed in intact yeast cells that the pseudoknot containing mRNA produced from pJDRC was able to promote efficient À1 PRF, whereas one in which only a stem-loop can form, transcribed from pJD18, could not (23) . As a first step in this study, we tested the ability of synthetic mRNAs produced from pJD366-RC and from pJD366-18, two plasmids derived from these parental constructs, to promote À1 PRF. Total luciferase activities produced from these synthetic mRNAs were divided by the luciferase activity produced from the zero-frame control plasmid, pJD366-0, and multiplied by 100% to determine À1 PRF efficiencies. The results show that the trends observed in yeast were replicated in vitro, i.e. JD366-RC mRNA promoted $8% efficiency of À1 PRF as compared with $1.1% promoted by JD366-18 mRNA (Figure 2 ).
The 'torsional restraint' model predicts that conditions that would inhibit the rotational freedom of the loop region of the pJD366-18-derived mRNA should result in enhanced À1 PRF efficiency. The strategy used in this study was to anneal this mRNA with synthetic oligonucleotides complementary to both the loop region and to the sequence downstream that is normally involved in pseudoknot formation. These 'pseudopseudoknots' would be predicted to restore a pseudoknot-like structure to the mRNA. This is diagrammed in Figure 3A . Two different classes of oligonucleotides having different orientations relative to the mRNA were used to this end: 'joining' (J-) and 'reverse' (R-) oligos. The orientation of the J-oligos promotes the formation of a structure containing the equivalent of a Loop 2 region, while that of the R-oligos promotes a Loop 1 equivalent. The model also predicts that pseudo-pseudoknots having different degrees of rotational freedom should promote different frequencies of ribosome pausing over the slippery site, resulting in different efficiencies of À1 PRF. In order to control this parameter, increasing numbers of nucleotides were inserted between the mRNA hybridizing regions of the J-and R-oligos. The additional non-complementary bases in the J-oligos are 3 0 to the stem-loop residues involved in Stem 2, thus effectively increasing Loop 2. Similarly, the additional non-complementary bases in the R-oligos are 5 0 to the loop acceptor residues and correspond to an increased Loop 1. The structure of the stem-loop of pJD366-18 and its maximum base-paired interactions with representative J-and R-oligos are shown in Figure 3B . To demonstrate that an oligonucleotide-mRNA hybrid was capable of forming under the assay conditions, the J1-oligo was incubated with 5 0 [ 32 P]labeled JD366-18 mRNA and subjected to RNaseH digestion. Digestion of the RNA-DNA hybrid resulted in a labeled 110 nt fragment, demonstrating that the oligonucleotide bound to the mRNA at the position of the pseudoknot (Figure 4) .
Having demonstrated the utility of the in vitro frameshifting assay and that the J-and R-series of oligonucleotides were able to hybridize with synthetic mRNA produced from pJD366-18, the next step was to monitor frameshifting efficiencies promoted by these hybrid species. Significant increases in frameshifting were observed with the incubation of pJD366-18 mRNA with oligonucleotides J1 ($10%) and J2 ($35%), while only modest increases were seen with J3 and J4 ( Figure 5 ). These findings are consistent with the notion that changes in the degree of rotational freedom of the structure would affect the distribution of paused ribosomes in the vicinity of the slippery site. One potential complication with the J-oligos is the possibility that they could interact with the Loop 2-Stem 1 region. In the R-oligos, the additional bases are distal to any possible Loop 2-Stem 1 interactions and would be more analogous to increasing Loop 1. The R-oligos stimulated À1 PRF to an even higher extent than the J-oligos ( Figure 5 ). Importantly, increasing the length of the bridging regions in these oligonucleotides (R1 to R3), which is predicted to increase the rotational freedom of the stem-loop, resulted in decreased frameshifting activity as predicted by the torsional resistance model. However, addition of three residues between the two binding regions of the R-oligo (R4) resulted in an unexpected increase in frameshifting with a very large amount of variation.
In a series of control experiments, 8 nt oligos complementary to the 5 0 (Loop 1) and 3 0 Stem 2 forming regions of the pseudo-pseudoknot were hybridized to the SL mRNA and À1 PRF assays were performed. Neither of these were able to stimulate À1 PRF, even at concentrations in 100-fold molar excess to the mRNA template (data not shown). Though supportive of our central hypothesis, it is also possible that these results were due to the thermodynamic instability of the RNA:DNA duplexes through the course of the experimental protocol.
To determine whether the stimulation of frameshifting was specifically due to the bridging of the stem-loop with downstream sequence (the pseudo-pseudoknot), as opposed to the nonspecific presence of an RNA:DNA hybrid, the competing oligonucleotide (C-oligo) was designed to form a 15 bp duplex with JD366-18 mRNA, including the 3 0 Stem 2 forming region, which was expected to significantly out compete either the J-or R-oligos from binding to this site, thus disrupting formation of the pseudo-pseudoknot (see Figure 3A ). Additionally, in the presence of the C-oligo, the J-and R-oligos were still predicted to hybridize with the 5 0 Stem 2 forming region, enabling us to address the question of whether this interaction alone was able to stimulate frameshifting. The results demonstrate that the addition of the C-oligo severely inhibited the abilities of both the J-and R-oligos to promote efficient frameshifting ( Figure 6 ). These findings demonstrate that (i) frameshifting was specifically stimulated by bridging of the 5 0 and 3 0 Stem 2 forming regions by the J-and R-oligos, and (ii) that the presence of an RNA:DNA hybrid at the 5 0 Stem 2 forming region was not sufficient to stimulate frameshifting by itself.
The torsional restraint model predicts that pseudoknots should direct elongating ribosomes to pause at one specific location 1 2 3 4 5 6 7 8 . Efficient frameshifting is stimulated by pseudo-pseudoknots. In vitro translation assays were performed in retic lysates with mRNAs derived from pJD366-18 (SL) to which J-or R-oligos were annealed. Luciferase activities were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting. The averages of three independent experiments performed in triplicate are shown. Error bars denote standard deviation. on the mRNA, rather than being distributed along Stem 1. We used mRNA toeprint assays to test this hypothesis. In mRNA toeprint reactions, the movement of reverse transcriptase is blocked by paused ribosomes, resulting in a strong stop positioned $16-18 nt 3 0 of the P-site of eukaryotic ribosomes (25) . Synthetic JD366-18 mRNAs were annealed with the sequencing oligonucleotide and either J1, R1 or no second oligo, and these were then used for in vitro translation reactions. After a period of time (10 min were empirically determined to be optimal), elongation reactions were stopped by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation. The results are consistent with the model, showing that the J1-and R1-oligos specifically promoted one strong reverse transcriptase stop 16 nt 3 0 of the P-site of the slippery site only the in the in vitro translation reactions ( Figure 7 ). As further predicted by the model, a broad distribution of stops of equal intensities was observed in this region with JD366-18 mRNA alone (Figure 7, lane 1) . Importantly, the +16 stop was not observed when toeprint reactions were carried in the absence of ribosomes. Additional strong stops were also of interest. One corresponding to the 3 0 end of the base of Stem 1 was observed in all samples, consistent with the presence of this structure. Both J-and R-oligo-specific pauses were also observed. The reason for the strong pause in the J-oligo is unknown. The R-oligo-specific pause is perhaps more revealing. It occurs at the 3 0 end of the RNA:DNA hybrid formed by this oligo and the mRNA, a structure that should also promote pausing of reverse transcriptase.
The results presented in this study provide strong support for the torsional restraint model of programmed À1 frameshifting. Specifically, we demonstrated that RNA:DNA hybrids that mimic mRNA pseudoknots can significantly stimulate frameshifting. As predicted by the model, changing the rotational freedom of the structure by altering the lengths in the J1-and R1-oligos between the 5 0 and 3 0 mRNA hybridizing regions resulted in changes in their abilities to stimulate À1 frameshifting. The demonstration that these 'pseudopseudoknot' structures cause elongating ribosomes to specifically pause with their A-and P-sites positioned at the slippery site provides independent evidence in support of the model. In the case of the J-oligo series, frameshifting was best stimulated by J2, suggesting the structure created and the rotational freedom allowed by it was optimal for À1 PRF. The experimental design is such that we assume a similar rate of unfolding for each oligo as the predicted maximum base pairing is the same for them all. However, we do note that the type of nucleotides separating the two, separately paired regions of the oligos, and their presentation, may play a role in À1 PRF efficiency. The recent NMR structural solution of the SRV-1 pseudoknot revealed a highly structured Loop 2-Stem 1 interface including base triples involving an A residue at the 3 0 end of Loop 2 (26) . The additional base in the J2oligonucleotide is also an A. Mutagenesis experiments in this region by other groups showed, for example, that replacing the 3 0 base in Loop 2 of IBV with an A residue promoted a significant increase in frameshifting efficiency (27) , and mutation or removal of the A residue at the 3 0 base in Loop 2 of the BWYV pseudoknot reduced frameshifting levels (17) . This part of the pseudoknot has been proposed to be important in a frameshifting model where differential transition state energy barriers (due to small differences in local structure, stability or dynamics) are the primary determinant of frameshifting efficiency (3). Indeed, a Loop 2-Stem 1 triplex interaction seen in smaller frameshifting pseudoknots from luteoviruses has been shown to be critical for À1 PRF, and that similar pseudoknots lacking the triplex are less efficient at frameshifting [(28) and references therein]. This extra structural feature would limit the rate of unfolding and provide extra anchoring of Stem 2 as the ribosome attempts to unwind Stem 1, i.e. it too would help to provide additional torsional restraint. It is also possible that although the J3-and J4oligonucleotides also help to form a pseudo-pseudoknot, the additional bases may interfere with the stabilization of Stem 1.
With the R-oligos, a general correlation was observed between minimization of rotational freedom and frameshifting efficiency, though this was not the case of the R4-oligo. Since the stability of the pseudo-pseudoknot generated with R4 should be similar to that of the other oligonucleotides based on the base-pairing, this result suggests that there are additional considerations to be uncovered with regard to the Frameshifting (% stimulated by R1) Figure 6 . Competition for J-or R-oligo binding sites inhibits its ability to promote efficient frameshifting. mRNA transcribed from pJD366-18 (SL) was annealed with either J-or R-oligos alone, or in combination with different concentrations of competing (C-) oligos (in ratios of 2:1 or equimolar as indicated). Sample marked SL is mRNA alone. Luciferase activities generated from in vitro translation reactions in rabbit reticulocyte lysates were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting.
pseudoknot structure influencing frameshifting. Addition of residues in the R-oligos was analogous to lengthening Loop 1, which is typically short in À1 frameshifting pseudoknots. Limited and conflicting data are available on the importance of Loop 1 in À1 frameshifting pseudoknots. In one study, addition of three A bases to Loop 1 did not affect frameshifting efficiency (15) , while in another all the mutations made in this region were detrimental to frameshifting efficiency (17) . Given the complex interactions occurring between the helices and loops in this region, we cannot yet account for why the R4-oligo stimulated frameshifting so efficiently and with such variable results. Examination of the RNA toeprint data presented here reveals that both of the pseudo-pseudoknot structures formed by the J1-and R1-oligos promoted strong stops of the reverse transcriptase $16 nt 3 0 of the P-site codon of the slippery site, consistent with the hypothesis that the presence of Stem 2 forces ribosomes to pause with their A-and P-sites positioned over the slippery site. Previous studies mapping the lagging edge of paused ribosomes, i.e. mRNA heelprint studies, did not reveal any striking differences between the effects of pseudoknots versus stem-loops (8, 16) . Interestingly, using this method, the ribosomal pauses appeared distributed over a broader stretch of mRNA ($4 nt) than observed here. It is possible that some critical level of resolution is lost in the requirement for many additional manipulations of substrates using the mRNA heelprint as compared with the toeprint methods.
A remaining question centers on whether the role of the RNA pseudoknot in À1 PRF is passive or active. In the '9 Å solution' (6), the frameshift mechanism is activated by movement of the A-site codon-anticodon complex by 1 base in the 5 0 direction upon accommodation. As currently described, the mRNA pseudoknot merely passively blocks entry of the downstream message into the ribosome, resulting in stretching of the segment of mRNA located between the codon-anticodon complex and the pseudoknot. By this model, all of the energetic input for the frameshift is derived from hydrolysis of GTP by eEF1A. However, it is possible that the pseudoknot may also actively contribute to the frameshift mechanism. Specifically, pulling the downstream message into the ribosome at accommodation could result in unwinding of Stem 1 of the pseudoknot by one additional base pair. The energetic cost of so doing would be to introduce an equivalent amount of torsional resistance into Stem 2. If Stem 2 were to release this resistance by 'pulling back', the base pair in Stem 1 would be re-formed, which in turn would contribute to the energy required to dissociate the A-and P-site codonanticodon complexes from the zero-frame. This would be followed by slippage of the mRNA by 1 base in the 3 0 direction relative to the ribosome, followed by the formation of À1 frame codon-anticodon complexes. As such, the proposed active role for the mRNA pseudoknot would further reduce the energetic barrier to À1 PRF. In sum, we suggest that the 'torsional restraint model' can be combined with the '9 Å solution' to mechanistically explain the original 'simultaneous 
3' mRNA + Ribos. mRNA Figure 7 . Pseudo-pseudoknots direct ribosomes to pause over the slippery site. mRNAs generated from pJD366-18 (SL) were annealed with the sequencing oligonucleotide and either J1-, R1-or no oligo (lanes 1, 2 and 3, respectively), and these were then used for in vitro translation reactions. Reactions were stopped after 10 min by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation (lanes 4-6). The positions of the slippery site, loops and stems of the pseudo-pseudoknots are indicated next to a sequencing reaction. Arrowheads indicate positions of reverse transcriptase strong stops and these are mapped to a representation of the stem-loop structure of pJD366- 18. slippage' model of À1 PRF (9, 10) . In other words, the 9 Å solution + torsional restraint = simultaneous slippage.
Two recent publications have also shown that oligonucleotide:mRNA duplexes can stimulate efficient À1 ribosomal frameshifting (29, 30) . These studies differed from the present one in a number of ways, particularly insofar as they examined the effects duplex structures immediately 3 0 of the slippery site rather than addressing mRNA pseudoknot related questions. The findings support the notion that the specific location of ribosome pausing on the mRNA plays a critical role in determining frameshifting, though they do come with caveats, e.g. neither study directly mapped ribosomal pausing, and the use of different slippery sites and downstream contexts likely contributed to disparate findings for the optimal distances between the 3 0 ends of slippery sites and 5 0 ends of frameshift-stimulating oligonucleotides. Although potentially useful therapeutically there are no known natural examples of frameshifting stimulated in this manner, and thus these results do not affect the hypothesis presented here. However, these studies are important in that they raise the possibility for a new role for micro-RNAs in regulating gene expression, and for therapeutic approaches to correcting inborn errors of metabolism due to the presence of frameshift mutations. Correcting errors in synthetic DNA through consensus shuffling Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ∼60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ∼1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences. Methods for the automated chemical synthesis of oligonucleotides (1, 2) and their assembly into long double-stranded DNA (dsDNA) sequences by PCR (3, 4) and LCR (5) have enabled the chemical synthesis of genes and even entire viral genomes (6, 7) . These technological advances have helped spur the formation of the new field of synthetic biology (8) , which aims at defining the functional units of living organisms through the modular engineering of synthetic organisms. In addition, the demand for fully synthetic gene length DNA fragments of defined sequence has dramatically increased in recent years for use in applications such as codon optimization (9), construction of DNA vaccines (10) , de novo synthesis of novel biopolymers (11) , or simply to gain access to known DNA sequences when original templates are unavailable. The future demand for long synthetic DNA is likely to dramatically increase when it becomes cheaper/faster to synthesize a desired sequence than to obtain it by other means.
The assembly of DNA is currently limited by the presence of random sequence errors in synthetic oligonucleotides that arise from side reactions during synthesis (incomplete couplings, misincorporations, etc.) and resulting in 1-3 errors/kb (7, 12, 13) . The deleterious impact of these errors becomes more significant as the desired lengths of synthetic DNA increase. Indeed, in the remarkable assembly of the PhiX 174 bacteriophage genome (5386 bp) using gel-purified, synthetic oligonucleotides, the products contained an average of $2 lethal errors/kb resulting in 1 plaque-forming genomes per 20 000 clones (7) . A functional selection (plaque formation) was required in this study to identify a clone with the correct sequence. Thus, error reduction/correction is a requirement for the efficient production of long synthetic DNA of defined sequence. However, the process of sequencing multiple clones and manual correction of errors is both costly and time consuming.
Several methods have been reported for the removal of error-containing sequences in populations of DNA. These methods rely upon the selective destruction (14, 15) or physical separation (16, 17) of mismatch-containing heteroduplexes. Smith and Modrich (14) reported the selective destruction of error-containing sequences in PCR products by generating dsDNA breaks upon overdigestion with the Escherichia coli mismatch-specific endonuclease MutHLS (18) . Gel purification and cloning of the remaining full-length DNA resulted in an apparent 10-fold reduction in the error rate for PCR products. However, the existing approaches are not well suited for error removal in long synthetic DNA sequences where virtually all members in the population contain multiple errors. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org Error correction with MutS is outlined in Figure 1 . The population of DNA molecules containing random errors is first re-hybridized to expose synthesis errors as mismatches ( Figure 1A ). Duplexes containing mismatches can then be removed from the population by affinity capture with immobilized MutS ( Figure 1B) , a process we term coincidence filtering, since both strands of the duplex must match to pass this filtering step. For long synthetic DNA sequences or for sequences with high error rates, coincidence filtering is ineffective, since the likelihood of both strands being perfectly matched after re-hybridization is very low. To generalize MutS error filtering for application on synthetic DNA, the synthetic DNA is cleaved into small overlapping fragments before MutS filtering. Fragments containing mismatches are selectively removed through absorption to an immobilized maltose-binding protein (MBP)-Thermus aquaticus (Taq) MutS-His 6 fusion protein (MBP-MutS-H6) (18) (19) (20) . The remaining mixture of fragments (enriched with fragments of the correct sequence) serves as a template for assembly PCR to produce the full-length product ( Figure 1C ). This process can be iterated until the consensus sequence emerges as the dominant species in the population. This approach is equivalent to DNA shuffling (21) with additional mismatch exposure and removal steps.
In this report, we assemble GFPuv from synthetic oligonucleotides and apply both coincidence filtering and consensus shuffling protocols to reduce errors in the resultant DNA populations. The error rates are characterized by gene function (fluorescence) and by DNA sequencing. We also provide a mathematical model describing the error reduction protocols to aid predictions about parameters influencing their effectiveness.
Chemicals were from Sigma. Restriction enzymes were from Promega and New England Biolabs. KOD Hot Start DNA Polymerase was from Novagen. Amylose resin was from NEB (catalog no. E8021S). Ni-NTA resin was from Novagen (catalog no. 70666). Ultrafiltration device from Millipore (catalog no. UFC900524). Slide-A-Lyzer dialysis membrane was from Pierce (catalog no. 66415).
Full-length Taq MutS was amplified from template pETMutS (22) with primers 5 0 -AAA AAA CAT ATG GAA GGC ATG CTG AAG G-3 0 and 5 0 -AAA AAT AAG CTT CCC CTT CAT GGT ATC CAA GG-3 0 and cloned into the Nde1/HindIII sites of vector pIADL14 (23) to give plasmid pMBP-MutS-H6.
E.coli strain BL21(DE3) transformed with pMBP-MutS-H6 was grown to OD 600 $1.0 and induced using 1 mM isopropylb-D-thiogalactopyranoside for 4 h at 37 C. Cells from 4 l of culture were pelleted and resuspended in 60 ml of buffer A (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 mM phenylmethlysulfonyl fluoride). Cell suspension was sonicated on ice and insoluble material was removed by centrifugation at 50 000 g for 10 min at 4 C. Supernatant was applied to 5 ml amylose resin pre-equilibrated in buffer A. Bound MBP-MutS-H6 was washed three times using 20 ml buffer B (20 mM Tris-HCl, pH 7.4, 300 mM NaCl) and stored The re-hybridized gene synthesis products are fragmented, and error containing fragments are precipitated by MBP-MutS-H6 immobilized on amylose support. Error reduced fragments (orange, blue and red) are reassembled into the full-length gene followed by PCR amplification to generate error reduced products. Primers: black lines.
overnight at 4 C. MBP-MutS-H6 was eluted using 20 ml buffer B + 10 mM maltose. Eluate was applied to $4 ml of Ni-NTA resin pre-equilibrated in buffer B. Bound MBP-MutS-H6 was washed four times using 20 ml buffer B + 25 mM imidazole. Bound MBP-MutS-H6 was eluted using buffer B + 1 M imidazole. Eluate was concentrated via ultrafiltration using Amicon Ultra 5 kDa MWCO at 4 C. Concentrated sample was dialyzed extensively against 2· storage buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.2 mM EDTA and 0.2 mM DTT) using a Slide-A-Lyzer 10 kDa MWCO cassette at 4 C. Protein concentration was determined using A 280 and a calculated extinction coefficient of 119 070 M À1 cm À1 . Dialyzed sample was diluted using an equal volume of glycerol and stored at À20 
Oligonucleotides were purchased from Qiagen with 'salt-free' purification. Sequence 261-1020 of pGFPuv (GenBank accession no. U62636 with T357C, T811A and C812G base substitutions) was assembled using 40mer (37) and 20mer (2) oligonucleotides with 20 bp overlap (Supplementary Table 1 ). Assembly reactions contained the following components: 64 nM each oligonucleotide, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. Assembly was carried out using 25 cycles of 94 C for 30 s, 52 C for 30 s and 72 C for 2 min. PCR amplification of assembly products contained the following components: 10-fold dilution of assembly reaction, 25 mM of 20 bp outside primers, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. PCR was carried out using 35 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 1 min followed by a final extension at 72 C for 10 min. PCR products were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O followed by speed-vac concentration. Assuming an error rate of 1 · 10 À6 /bp/duplication for KOD DNA polymerase (24) , 35 cycles of PCR would be expected to introduce $0.053 mutations per assembled GFPuv molecule.
Assembled GFPuv was diluted to 250 ng/ml in 10 mM Tris-HCl, pH 7.8, 50 mM NaCl and heated to 95 C for 5 min followed by cooling 0.1 C/s to 25 C. Heteroduplex for consensus filtering was split into three pools and digested to completion with NlaIII (NEB), TaqI (NEB) or NcoI plus XhoI (Promega) for 2 h following the manufacturer's protocols. Digests were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O. Samples were pooled and the concentration was determined by measuring A 260 .
MBP-MutS-H6 binding reactions contained $11.5 ng/ml DNA and $950 nM MBP-MutS-H6 dimers in 1· binding buffer (20 mM Tris-HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl 2 , 1 mM DTT and 5% glycerol). Reactions were allowed to incubate at room temperature for 10 min before incubation for 30 min with an equal volume of amylose resin pre-equilibrated in 1· binding buffer. Protein-DNA complexes were removed by low-speed centrifugation and aliquots of supernatant were removed for subsequent processing.
Supernatant (50 ml) from consensus filtering experiments was desalted using Centri-Sep spin columns (Princeton Separations) and concentrated. Purified and concentrated DNA fragments were reassembled as above with aliquots removed at varying cycles. Aliquots of assembly reactions were resolved on 2% agarose gels to monitor the reassembly process. Aliquots showing predominantly reassembled fulllength GFPuv were PCR amplified as above. Aliquots of supernatant from coincidence filtering experiments were diluted 10-fold and PCR amplified as above. PCR products were digested with BamHI/EcoRI and ligated into the 2595 bp BamHI-EcoRI fragment of pGFPuv. Ligations were transformed into E.coli DH5 and fluorescent colonies were scored using a handheld 365 nm ultraviolet (UV) lamp.
Preparation of substrate for consensus shuffling from 10 non-fluorescent GFPuv clones Ten non-fluorescent GFPuv clones were pooled in equal amounts. The nature and location of the mutations in these clones is shown in Figure 4 . The GFP coding region was PCR amplified from the mixture and submitted to the consensus shuffling protocol with and without the application of the MBP-MutS-H6 error filter.
To create an error filter, we constructed a fusion protein between MBP (19) and the mismatch binding protein from T.aquaticus (22) with a C-terminal His 6 tag (MBP-MutS-H6). MBP-MutS-H6 was overexpressed and purified from E.coli to >95% purity (Supplementary Figure 1) . MBP-MutS-H6 immobilized on amylose resin was shown to selectively retain a 40mer heteroduplex containing a deletion mutation over wild-type homoduplex (Supplementary Figure 2) .
To demonstrate error correction, unpurified 40mer oligonucleotides were assembled by PCR (3) to produce a 760 bp gene encoding green fluorescent protein (25) (GFPuv). Two independent preparations of GFPuv containing typical gene synthesis errors (Figure 3 and Table 1 ) were re-hybridized and subjected to two iterations of coincidence filtering or consensus shuffling. For consensus shuffling, the GFPuv assembly product was split into three pools and digested into sets of overlapping fragments using distinct Type II restriction enzymes ( Figure 2 ). The digests were pooled and subjected to error filtering with or without added MBP-MutS-H6. The unbound fragments were reassembled into full-length products and PCR amplified. For coincidence filtering, unbound fulllength GFPuv was PCR amplified following treatment with the error filter. After cloning in E.coli, error rates were estimated by scoring colonies for fluorescence under a handheld UV lamp (Figure 3) . The actual error rates of the input and consensus shuffled populations were determined by sequencing plasmid DNA from randomly selected colonies (Figure 3) . The results show that two rounds of consensus shuffling increased the percentage of fluorescent colonies from $60 to >90% and Table 1 . Sequence errors in input and consensus shuffled DNA Table 1 .
Although DNA shuffling has traditionally been used to create diversity through the combinatorial shuffling of mutations in a population, DNA shuffling also creates a sub-population of sequences with a reduction in diversity, as correct fragments can recombine to produce error-free sequences. Indeed, with consensus shuffling, it is possible to start with a population of DNA molecules wherein every individual in the population contains errors and create a new population where the dominant sequence is error free. To demonstrate this, 10 nonfluorescent GFPuv clones, each containing a deletion mutation (Figure 4) , were pooled and subjected to either DNA shuffling alone or two iterations of consensus shuffling. Products were cloned in E.coli, and the percentage of fluorescent colonies was monitored as an indication of progress toward the consensus sequence. DNA shuffling alone (no MBP-MutS-H6) increased the percentage of fluorescent colonies to 30% (387 colonies total) similar to a previous report (26) . Two rounds of consensus shuffling gave a new population that was 82% fluorescent (551 colonies total), indicating that the dominant species was likely the consensus sequence of the input population.
Both consensus shuffling and coincidence filtering protocols were effective in reducing errors in synthetic GFPuv populations ( Figure 3 ). In both cases, two iterations of either consensus shuffling or coincidence filtering increased fluorescent colonies from average values of $60 to >90%. Sequencing data from two independent experiments showed a 4.3-and 3.5-fold reduction in the error rate for the consensus shuffled populations compared with the input populations giving final error rates of 0.3 and 0.28 errors/kb, respectively. These results demonstrate the usefulness of the MBP-MutS-H6 error filter in both consensus shuffling and coincidence filtering protocols. Taq MutS has previously been shown to bind to deletion mutations with high affinity (27) , a mutation common in synthetic DNA. However, it is important to note that Taq MutS has lower affinity for specific point mutations and binds weakly to homoduplex DNA (27) . These factors may limit the stepwise efficiency of the error filter. Moreover, specific point mutations may be refractory to removal even after multiple rounds of consensus shuffling. Two rounds of consensus shuffling using the MBP-MutS-H6 error filter proved most effective in reducing deletions and G/C to A/T transitions, consistent with previous reports for the selectivity of Taq MutS (27) . However, it must be emphasized that each synthetic oligonucleotide point mutation would generate two heteroduplex DNA molecules containing unique mismatches after PCR amplification and re-hybridization ( Figure 1A and Table 1 ). For example, a G to A transition mutation in a synthetic oligonucleotide would generate heteroduplexes with G-T or A-C mismatches after PCR amplification and re-hybridization. For consensus shuffling, either of these mismatch containing heteroduplexes could evade precipitation by the MBP-MutS-H6 error filter and participate in the reassembly of full-length GFPuv. Therefore, Table 1 lists the pair of mismatches that could give rise to the observed transition or transversion mutation. These results show that the MBP-MutS-H6 error filter was most effective at removing insertion/deletion loops and G-T/A-C mismatches from the population.
It should be possible to generalize the consensus shuffling protocol to a large number of synthetic DNA constructs. GFPuv was chosen as the synthetic construct in this study for its advantages as a fluorescent reporter gene. This allowed easy optimization of our protocol without the need to sequence thousands of base pairs of DNA. We expect the results reported here for consensus shuffling to readily translate to synthetic DNA constructs of varied sequence, greater overall length and/ or higher initial errors/kb. Synthetic DNA constructs of varied sequence can be digested into a defined set of fragments using Type II restriction enzymes or fragmented into any desired size range using controlled DNase I digestion (26) . Digestion and reassembly of a large number of different genes is expected to be as robust as the protocol of DNA shuffling (28) , which has been broadly applied to a variety of gene sequences. Synthetic DNA constructs larger than GFPuv are expected to be amenable to error correction by consensus shuffling, as the error filtering is conducted on gene fragments before reassembly of the full-length gene. Thus, the errors/kb data presented in this study are expected to translate to larger genes with similar initial errors/kb (excepting mutations introduced by PCR amplification following the final application of the error filter). Synthetic DNA constructs of higher initial errors/kb are expected to be amenable for error correction by consensus shuffling. However, these constructs will require digestion into smaller sized gene fragments that may affect the efficiency of error correction. In contrast to consensus shuffling, an increase in the size of the synthetic DNA product or an increase in errors/kb would preclude the use of the coincidence filtering protocol, as every molecule in the population would contain one or more errors. As proof of the utility of the consensus shuffling protocol, 10 non-fluorescent GFPuv clones containing one or more errors (Figure 4 ) were converted into a population where 82% of the clones were fluorescent. It is important to note that DNA shuffling alone shows an improvement in percent fluorescent colonies in this example (from 0 to 30%). For synthetic DNA populations, DNA shuffling alone shows no improvement in percent fluorescent colonies (see Figure 3 'no MutS' treatments). DNA shuffling alone improves the overall number of correct sequences only for small DNA populations with low error rates. For example, when shuffling 10 clones with a unique mutation in each clone, one would expect the fraction of correct products to be (9/10) 10 = 35% (26), very close to the value of 30% that we observed. A mathematical model describing the error rates for shuffling and error filtering of synthetic DNA populations is presented below.
To estimate some parameters of consensus shuffling and coincidence filtering, a simple mathematical model (Equations 1-6) was constructed. An input population of dsDNA molecules of length N, containing E errors/base is re-hybridized, fragmented into shorter dsDNA fragments of average length S, error filtered and reassembled. P(F) is the probability a fragment of length S will have a correct sequence. We determine the probability that re-hybridized duplexes will have zero (C), one (H ) or both (I ) strands with errors.
Equation 5 estimates the probability that a fragment will be correct after a cycle of MutS filtering, P(F 0 ), by applying a MutS selectivity factor (M ) to adjust the relative amounts of mismatch containing duplexes (I, H ) while accounting for the total fraction of correct strands in the re-hybridized duplexes. The probability of obtaining an error free assembly product, P(A), is then given by Equation 6 .
From our consensus shuffling error rate data (Figure 3 ), we estimate the MutS selectivity factor M to be $2.2. Figure 5 shows some predictions that emerge from this model assuming typical length (2 kb), fragment sizes (200 bp) and error rates (1.8 errors/kb). Consensus shuffling is predicted to be most effective with smaller fragment sizes ( Figure 5A ). As mentioned above, smaller fragment sizes could be obtained by controlled digestion with DNase I (21) . In addition, multiple iterations of MutS filtering can have dramatic results on populations with few correct sequences ( Figure 5B ), although the model does not account for the differing specificity of MutS toward the various types of mismatches. The model also predicts that even modest improvements in the MutS selectivity factor through optimization of the MutS-DNA binding conditions and/or the use of a combination of MutS homologs with varying mismatch specificity (29) could dramatically improve the consensus shuffling protocol ( Figure 5C ). Coincidence filtering (N = S) is predicted to be effective for populations with low error rates per clone ( Figure 5D ) but becomes ineffective when the majority of re-hybridized duplexes contain mismatches.
We have demonstrated consensus shuffling and coincidence filtering as experimental methods to significantly reduce errors in synthetic DNA. Consensus shuffling should be generally applicable for error correction on synthetic genes of typical lengths and error rates. Two iterations of consensus shuffling ($6 h/iteration) generated a population with $1 error/3500 bp. This reduction in error rate will allow the identification of a correct clone after sequencing DNA from a reduced number of colonies. Coincidence filtering is a simple and effective procedure to reduce errors in synthetic DNA populations with low error rates per clone. These methods should significantly increase the speed and decrease the cost of production of synthetic genes.
Note: While this manuscript was under review, Carr et al. (30) independently reported the application of Taq MutS in protocols for error reduction on synthetic DNA. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data. Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses, including gene expression profiling methods to assess the expression levels of thousands of genes in a single assay. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labor-intensive, time-consuming and highly expensive. However, in spite of the need for standardization of RNA sample quality control, presently there is no real consensus on the best classification criteria. Conventional methods are often not sensitive enough, not specific for single-stranded RNA, and susceptible to interferences from contaminants present in the sample. For instance, when using a spectrophotometer, a ratio of absorbances at 260 and 280 nm (A 260 :A 280 ) greater than 1.8 is usually considered an acceptable indicator of RNA purity (1, 2) . However, the A 260 measurement can be compromised by the presence of genomic DNA leading to over-estimation of the actual RNA concentration. On the other hand, the A 280 measurement will estimate the presence of protein but provide no hint on possible residual organic contaminants, considered at 230 nm (3) (4) (5) . Pure RNA will have A 260 :A 230 equal to A 260 :A 280 and >1.8 (1) . A second check involves electrophoresis analysis, routinely performed using agarose gel electrophoresis, with RNA either stained with ethidium bromide (EtBr) (6) (7) (8) (9) , or the more sensitive SYBR Green dye (10) . The proportion of the ribosomal bands (28S:18S) has conventionally been viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of 'high quality' intact RNA (1) . However, these methods are highly sample-consuming, using 0.5-2 mg total RNA and often not sensitive enough to detect slight RNA degradation. Today, microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer (Agilent Technologies, USA) has become widely used, particularly in the gene expression profiling platforms (11, 12) . It requires only a very small amount of RNA sample (as low as 200 pg), the use of a size standard during electrophoresis allows the estimation of sizes of RNA bands and the measurement appears relatively unaffected by contaminants. Integrity of *To whom correspondence should be addressed. Tel: The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org the RNA may be assessed by visualization of the 28S and 18S ribosomal RNA bands ( Figure 1A and B); an elevated threshold baseline and a decreased 28S:18S ratio, both are indicative of degradation. A broad band shows DNA contamination ( Figure 1C ). As it is apparent from a review of the literature, the standard of a 2.0 rRNA ratio is difficult to meet, especially for RNA derived from clinical samples, and it now appears that the relationship between the rRNA profile and mRNA integrity is somewhat unclear (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) . On the one hand, this may reflect unspecific damage to the RNA, including sample mishandling, postmortem degradation, massive apoptosis or necrosis, but it can reflect specific regulatory processes or external factors within the living cells. Altogether, it appears that total RNA with lower rRNA ratios is not necessarily of poor quality especially if no degradation products can be observed in the electrophoretic trace ( Figure 1D ).
For all these reasons, the development of a reliable, fully integrated and automated system appropriate for numeric evaluation of RNA integrity is highly desirable. Standardized RNA quality assessment would allow a more reliable comparison of experiments and facilitate exchange of biological information within the scientific community. With that prospect in mind, and with the aim of anticipating future standards by pre-normative research, we identified and tested two software packages recently developed to gauge the integrity of RNA samples with a user-independent strategy: one open source, the degradometer software for calculation of the degradation factor and 'true' 28S:18S ratio based on peak heights (24) and the freely available RIN algorithm of the Agilent 2100 expert software, based on computation of a 'RNA Integrity Number' (RIN) (25) . Both tools were developed separately to extract information about RNA integrity from microcapillary electrophoretic traces and produce a userindependent metrics. Using these tools, we assessed the purity and integrity of 414 RNA samples, derived from 14 different human adult tissues and cell lines, many of which representing tumors. Those results were compared with conventional RNA quality measurement approaches as well as with highly expert human interpretation. We evaluated the simplicity for users and examined the potential, accuracy and efficiency of each method to contribute to standardization of RNA integrity assessment upstream of biological assays. These procedures were further validated by real-time RT-PCR quantitation of the expression levels of three housekeeping genes, using the same RNA samples, at different levels of degradation.
Total RNA was prepared from human cell lines (especially from the ATCC bio-resource center, N = 50) and tissue samples (clinical samples, N = 285) from 13 different human adult tissue types, i.e. blood, brain, breast, colon, epithelium, kidney, lymphoma, lung, liver, muscle, prostate, rectum and thyroid. RNA purification was performed by cesium chloride ultracentrifugation according to Chomczynski and Sacchi (26) , by phenol-based extraction methods (TRIzol reagent, Invitrogen, USA), or silica gel-based purification methods (RNeasy Mini Kit, Qiagen, Germany; Strataprep kit, Stratagene, USA or SV RNA isolation kit, Promega, USA) according to the manufacturer's instructions with some modifications. Material was maintained at À80 C with minimal handling. RNA extraction was carried out in an RNase-free environment (see Supplementary Table 1 online) .
The commercially available RNA samples were the 'Universal Human Reference' (N = 75) distributed by Stratagene (USA), and human brain (N = 2) and muscle (N = 2) RNAs supplied by Clontech (USA).
Once extracted, RNA concentration and purity was first verified by UV measurement, using the Ultrospec3100 pro (Amersham Biosciences, USA) and 5 mm cuvettes. The absorbance (A) spectra were measured from 200 to 340 nm. A 230 , A 260 and A 280 were determined. A 260 :A 280 and A 260 :A 230 ratios were calculated. For microcapillary electrophoresis measurements, the Agilent 2100 bioanalyzer (Agilent Technologies, USA) was used in conjunction with the RNA 6000 Nano and the RNA 6000 Pico LabChip kits. In total, 39 assays were run in accordance with the manufacturer's instructions (see Supplementary Notes online). To evaluate the reliability of the classifier systems described in this study, replicate runs were done on a set of 56 RNA samples loaded on different chips, resulting in 2 (N = 41), 3 (N = 12), 7 (N = 2) and 50 (N = 1) data points per sample.
Human RNA integrity categorization RNA integrity checking was performed by expert operators who classified each total RNA sample within a predefined discrete category from 1 to 5, examining the integrity of the RNA from electropherograms (see Supplementary Table 2 online). A low number indicates high integrity. Reference criteria parameters include ribosomal peaks definition, baseline flatness, existence of additional or noise peaks between ribosomal peaks, low molecular weight species contamination and genomic DNA presence suspicion. A smearing of either 28S and 18S peaks, or a decrease in their intensity ratio indicate degradation of the RNA sample and results in the classification into the higher categories. To evaluate the robustness of this human interpretation, five highly experienced operators, trained in these cataloging steps, separately classified a subset of 33 samples from breast cancers. It included samples with varying levels of integrity: intact RNA (33%), low quality samples (20%) and a wide range of degradation (47%).
Bioanalyzer electrophoretic data were exported in the degradometer software folder (.cld format). For comparison of samples, the original data were re-scaled by the classifier system, first along the time-axis to compensate for differences in migration time, then along the fluorescence intensity-axis to compensate for variation in total RNA amount. As a result, fluorescence curves that have the same shape will have the same peak heights after re-scaling. Then, Degradation Factors (DegFact) and corrected 28S:18S ratios were calculated (see Supplementary Table 3 online) using the mathematical model developed by Auer et al. (24) , examining additional 'degradation peak signals' appearing in the lower molecular weight range and comparing them to ribosomal peak heights. Calculation of the DegFact is based on a numbering of continuous metrics, ranging from 1 to ¥; increasing DegFact values correspond to more degradation, and a new group of integrity is defined after 8 graduation steps. Once the classification of the RNA samples is completed, 4 groups of integrity are displayed, 3 showing an alert warning indicative of some measurable degradation (Yellow: 8-16, Orange: 16-24 and Red: >24), while all non-reliable data come together and form the fourth group (Black). We introduced a fifth class labeled White (<8), when no alert was produced by the software.
Software and manual are freely available at http://www. dnaarrays.org/downloads.php. Degradometer version 1.4.1 (released in May 2004) of the software was used.
Bioanalyzer electrophoretic sizing files (.cld format) collected with biosizing software version A.02.12.SI292 (released in March 2003) were imported in the Agilent 2100 expert software (RIN beta release). The RIN algorithm allows calculation of RNA integrity using a trained artificial neural network based on the determination of the most informative features that can be extracted from the electrophoretic traces out of 100 features identified through signal analysis. The selected features which collectively catch the most information about the integrity levels include the total RNA ratio (ratio of area of ribosomal bands to total area of the electropherogram), the height of the 18S peak, the fast area ratio (ratio of the area in the fast region to the total area of the electropherogram) and the height of the lower marker.
A total of 1300 electropherograms of RNA samples from various tissues of three mammalian species (human, mouse and rat), showing varying levels of degradation and an adaptive learning approach were used in order to assign a weight factor to the relevant features that describe the RNA integrity. A RIN number is computed for each RNA profile (see Supplementary Table 4 online) resulting in the classification of RNA samples in 10 numerically predefined categories of integrity. The output RIN is a decimal or integer number in the range of 1-10: a RIN of 1 is returned for a completely degraded RNA samples whereas a RIN of 10 is achieved for intact RNA sample.
In some cases, the measured electropherogram signals are of an unusual shape, showing for example peaks at unexpected migration times, spikes or abnormal fluctuation of the baseline. In such cases, a reliable RIN computation is not possible. Several separate neural networks were trained to recognize such anomalies and display a warning to the user or even suppress the display of a RIN number. Combining the results of the neural network for the RIN computation and the neural networks to detect anomalies, the RIN algorithm achieves a mean square error of 0.1 and a mean absolute error of 0.25 on an independent test set.
The beta release of the software and manual are freely available at http://www.agilent.com/chem/RIN. Agilent 2100 expert version B.01.03.SI144 (released in November 2003) of the software was used.
Expression levels of three housekeeping genes (HKG)-GAPD, GUSB and TFRC-were measured by quantitative PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Sixteen aliquots of a unique batch of RNA sample (Universal Human Reference RNA, Stratagene, USA) of various levels of integrity (cf. Table 1 ) were used to test the influence of RNA quality on the relative expression of those three genes. In parallel, a 5 0 to 3 0 comparison was done using two separate GUSB and TFRC TaqMan probes.
An homogeneous quantity (0.8-1 mg) of the RNA samples was subjected to a reverse transcription step using the highcapacity cDNA archive kit (Applied Biosystems, USA) as described by the manufacturer. Single-stranded cDNA products were then analyzed by real-time PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Single-stranded cDNA products were analyzed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems, USA). The efficiency and reproducibility of the reverse transcription were tested using 18S rRNA TaqMan probes. Five assays were used, GAPDH-5 0 (Hs99999905_m1), GUSB-5 0 (Hs00388632_gH), GUSB-3 0 (Hs99999908_m1), TFRC-5 0 (Hs00951086_m1) and TFRC-3 0 (Hs00951085_m1). In each case, duplicate threshold cycle (Ct) values were obtained and averaged; then expression levels were evaluated by a relative quantification method (27) .
The fold change in one tested HKG (target gene) was normalized to the 18S rRNA (reference gene) and compared to the highest quality sample (calibrator sample), using the following formula: Fold change = 2 ÀDDCt , where DDCt = (C t-target À C t-reference ) sample-n À (C t-target À C t-reference ) calibrator-sample . Sample-n corresponds to any sample for the target gene normalized to the reference gene and calibrator-sample represents the expression level (1·) of the target gene normalized to the reference gene considering the highest quality sample. Mean 2 ÀDDCt and SD were calculated, considering the samples either individually or grouped by quality metrics categories, based on RIN metrics or DegFact values, together with the lower and upper bound mean of 95% Intervals of Confidence (IC). Using this analysis, if the expression levels of the HKG are not affected by the RNA degradation, the values of the mean fold change at each condition should be very close to 1 (since 2 0 = 1) (27) .
Descriptive statistics were executed using the XLSTAT software, version 7.1 (Addinsoft, USA), P = 0.05. Mean, SD and coefficient of variation (variation or CV) between and within groups of samples were calculated, together with a measure of the dispersion (range), inter-quartile range (1st and 3rd quartiles, Q1-Q3) and evaluation of the lower and upper bound mean of 95% Interval of Confidence (IC). Comparative statistical analyses between groups were completed, P = 0.05, using non-parametric statistical tests: two-independent Mann-Whitney U-test and k-independent Kruskal-Wallis test.
We analyzed 414 total RNA sample profiles from various human tissues (69%) and cell lines (31%) of either tumoral (85%) or normal (15%) origin, with varying levels of RNA integrity. Supplementary Table 1 online for details). Significant differences in A 260 :A 280 ratios were observed between specific groups of samples (i.e. tumoral versus normal or tissues versus cell lines). For instance, RNA extracted from normal samples displayed an improved ratio of 1.97, with 97% falling within the desired range ( Figure 2A ). In contrast, the distribution of A 260 :A 280 ratios was not found to correlate with either purification methods or tissues of origin.
RNA integrity was further assessed by resolving the 28S and 18S ribosomal RNA bands using the Agilent 2100 bioanalyzer and the RNA 6000 protocol. The analysis was done on 399 RNA profiles; data from 15 samples was not obtained due to device problems during the runs. The system automatically provided 28S:18S ratios for 348 (87%) of the 399 profiles. Figure 2B shows the distribution of the 28S:18S computed values, with a median ratio around 1.7 and a variation of 54% from the mean (IC 1.9-2.1 and Q1-Q3 1.4-2.5). In addition, a significant degree of variability of the 28S:18S ratio (19-24%) was found for identical samples from replicate runs (2-50 times). Among those RNA samples, 28S:18S ratios of 2.0 or greater were rare, less than 44% of the values measured being within the theoretically desired range, except for the samples prepared from cultured cells ( Figure 2B ). The integration failed in the remaining 51 cases, displaying an atypical migration, with no clear 28S and 18S rRNA bands, and no 28S:18S ratio was computed (data not shown). 
Expert operators categorized the set of RNA samples by inspecting the electrophoretic traces of successful assays. Over the 399 RNA profiles checked, 379 (95%) were scored within predefined categories ( Figure 2C ), namely good [Human Categorization (HC)-level 1], regular (HC-level 2), moderate (HC-level 3), low (HC-level 4) and degraded (HC-level 5). The remaining 20 (5%) were flagged as displaying a temperature-sensitive profile: RNA samples initially found intact became highly degraded when heated, although no RNase contamination was observed (data not shown).
Estimation of the robustness of this cataloging was done through comparison of qualifying criteria using a set of 33 breast cancer samples (see Materials and Methods). Integrity of the samples was evaluated independently by five expert operators, and categorization was found highly reliable with a coefficient variation (CV) $16%. This is low considering that individual interpretation is involved, but can be explained by the fact that very experienced operators accomplished the scoring based on a clearly defined set of instructions, thus limiting frequently observed subjective visual interpretation and inconsistency of human categorization. Predictably, a 28S:18S ratio of 2.0 denoted high quality for a majority of RNA samples, 91% being classified in HC-levels 1 to 3. However, 83% of total RNAs with 28S:18S > 1.0 but a low baseline between the 18S and 5S rRNA or front marker were also classified in HC-levels 1-3 (see Figure 1D ) and could be considered suitable for most downstream applications.
RNA degradation was first assessed using the degradometer software (see Materials and Methods). Over the 399 RNA profiles checked, all were scored in one of the five predefined classes ( Figure 3A) . Altogether, 334 (84%) Degradation Factors (DegFact) values were computed, the remaining 65 RNA samples (16%) displaying profiles that could not be interpreted reliably; no DegFact values could be scored, and samples were flagged in the Black category ( Figure 3A ). Most of them (80%) correspond to samples previously classified by our operators as degraded (HC-level 5). The remaining cases had an average degradation factor of 7.5 (IC 6.7-8.3) with large variations over the entire set of samples (over 103% from the mean, range 1-52). A lower variability was persistently found when identical samples from replicate runs were considered, resulting in observed DegFact values with a 26-32% CV. In addition, statistically significant differences were found between DegFact values of samples sorted by types. The highest DegFact values were found characteristic of tissue samples, 41% of them displaying a DegFact > 8, as compared with 6% for the cell lines (data not shown).
Remarkably, we found a significant linear relationship between the DegFact values distribution and the explicit human categorization. Most HC classes corresponded to an unambiguous DegFact distribution ( Figure 3B ), while HClevels 2 and 3 form a single class: HC-level 1, mean DegFact of 3.3, SD of 2.8 (IC 2.8-3.7); HC-level 2 and 3, mean Deg-Fact of 8.8, SD of 6.8 (IC 7.5-10.2); HC-level 4, mean DegFact of 15.9, SD of 7.8 (IC 12.7-19.1); HC-level 5, mean DegFact of 26.0, SD of 7.5 (IC 21.9-30.1). It is worth mentioning that the normalized heights of 18S and 28S peaks, and the interval between them after rescaling gradually decrease and then reverse with increasing degradation ( Figure 3B ).
Integrity of RNA samples was measured in parallel based on the RNA Integrity Number metrics using an artificial neural network trained to distinguish between different RNA integrity levels by examining the shape of the microcapillary electrophoretic traces (see Materials and Methods). Over the 399 RNA profiles checked, 363 (91%) were scored successfully ( Figure 4A) , with an average RIN of 7.7 (IC 7.4-8.0). The remaining 36 (9%) samples were associated with various unexpected signals, disturbing computation of the RIN using default anomaly detection parameters. In each case, a flag alert was added corresponding to critical anomalies including unexpected data in sample type, (or) ribosomal ratio, (or) baseline and signal in the 5S region (data not shown).
RIN categorization was found regular, variability between replicate runs, compared to the other methods, being consistently very small (CV 8-12%). As expected, the highest RIN were characteristic of cell line samples, 72% of them displaying a RIN > 9, as compared with 47% for the tissue samples (data not shown).
A first group, corresponding to 295 (82%) of the 363 RNA profiles, was analyzed using the default settings of the RIN system, but with a lower threshold of RNA quantity loaded (20 ng) for reliable detection of anomalies than that recommended by the manufacturer (50 ng). A significant linear relationship was found between the RIN number and both the explicit human classification provided by our operators, Figure 3 . RNA degradation characterization. Integrity of 399 RNA sample profiles was scored using the degradometer software. (A) A total of 334 RNA profiles were successfully categorized into 5 predefined alert classes using a mathematical model that quantifies RNA degradation and computes a degradation factor (DegFact). Four classes (White, Yellow, Orange and Red) are associated with different levels of degradation. A fifth class, Black alert corresponds to samples that the system was not able to qualify with accuracy (n.d.). The distribution is represented by the number of records in each class. (B) Comparative analysis was done using human evaluation (x-axis) based on electrophoresis analysis as a reference for RNA integrity classification; observations of rRNA peak heights and DegFact values were taken at each of the 5 HC levels. Histograms refer to the mean 28S and 18S rRNA peak heights and 95% confidence intervals (fluorescence intensities; left scale). Mean DegFact values and 95% confidence intervals (arbitrary unit, right scale) are plotted with the means joined. and the DegFact values calculated by the degradometer software ( Figure 4B ). Each distinct HC class corresponds to an explicit RIN number, with HC-levels 2 and 3 forming once again a single class: HC-level 1, mean RIN of 9.6, SD of 0.7 (IC 9.5-9.7); HC-level 2 and 3, mean RIN of 8.6, SD of 0.9 (IC 8.4-8.9); HC-level 4, mean RIN of 6.1, SD of 1.5 (IC 5.2-7.1); HC-level 5, mean RIN of 3.7, SD of 2.0 (IC 2.9-4.5).
For the remaining 68 samples (assay done with <20 ng of RNA), two separate groups were considered: 41 samples with a computed RIN below 5.0, and 27 above 7.0. All samples in the first group were derived from RNA 6000 Nano assays, with mean RNA quantities loaded below 10 ng (Q1-Q3, 5-12 ng), i.e. below the lower limit of quantitation indicated by the manufacturer. All but 8 of these samples were estimated by our operators to be of poor quality (HC-level 4; N = 3) or degraded (HC-level 5; N = 30), and all but 4 were flagged Black by the degradometer software and no DegFact values were scored. These RNA profiles could not be interpreted reliably, possibly due to either the low RNA concentration or the unusual migration behavior and shifted baseline values of degraded samples. Thus, the two automated systems were in disagreement for these samples; while human interpretation was in most cases in agreement with the RIN system, with less than 20% of inconsistency. In the second group of 27 samples, 20 of the profiles were derived from RNA 6000 Pico assays with RNA quantities loaded being on average below 4 ng (Q1-Q3, 0.5-0.8 ng), which is within the manufacturer specifications. All but 3 of them were estimated by our operators to range from high (HC-level 1; N = 12) to correct (HC-level 2 and 3; N = 12) quality levels. In addition, all RNA profiles except 1 were scored by the degradometer software, most of them displaying an alert flag (N = 20); some slight degradation was detected, associated to a low mean DegFact value of 9.7 (IC 8.1-11.3; Q1-Q3, 6.2-12.6). Thus, both automated systems and human interpretations agreed in most of these cases, with <11% of inconsistency.
The influence of RNA quality categorization obtained with both user-independent classifiers on gene expression profiling was explored using real-time RT-PCR. The expression levels of three housekeeping genes (HKG)-GAPDH, GUSB and TFRC-were measured in 16 aliquots of a unique RNA displaying various integrity metrics ( Table 1 ). The mean correlation coefficient (r) between the threshold cycle (Ct) among the 16 samples and both quality metrics was found high: r = À0.87 considering the RIN metrics and r = 0.85 considering the DegFact values. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ). Considering that HKG expression was measured relative to the reference sample, an obvious decline of the relative expression levels was observed, up to 24, 70 and 82%, in samples categorized according to the RIN metrics ( Figure 5A) and DegFact values ( Figure 5B ). These results indicate that 2-to 7-fold differences may be expected in the relative expression levels of genes in samples that differ only by their quality (Table 2 ). These fold differences are much larger than those measured for RNA samples of comparable integrity, consistently lower than 1.6 (Table 2 and Figure 5 ). In addition, an unambiguous gap in the distribution may be defined ( Figure 5A and B) , distinguishing the RNA samples of the higher quality categories (RIN > 8 and DegFact values < 7) from those of the lower categories (RIN < 8 and DegFact values > 12).
It would be expected that measuring expression of an intact mRNA would yield approximately equal results regardless of the region being probed, and if mRNA fragmentation had occurred, then some sequences may be more abundant than others. We thus tested the effect of PCR probe location on the RNAs. The 5 0 and 3 0 GUSB probes, separated by 1209 nt, were associated with highly similar threshold cycle (Ct) measures (r = 0.98, b parameter = 0.88) ( Figure 5C ). Similar results were obtained for TFRC, with probes separated by 2066 nt (r = 0.84, b parameter = 0.92, data not shown). It seems therefore that the region being probed is not a source of variation in our results.
It is universally accepted that RNA purity and integrity are of foremost importance to ensure reliability and reproducibility of downstream applications. In the biomedical literature (PubMed, November 2004), from the 485 090 articles that relate to RNA, and the 287 515 or 40 395 including respectively the 'quality' or 'integrity' term, less than 100 were found to contain 'RNA quality' or 'RNA integrity' terms. Interestingly, half of them were published between 2001 and 2004; but none is proposing a standard operational procedure for RNA quality assessment to the scientific community. Except for two studies (24, 25) , those reports are based on 10 to 15 years old methods (1), indicating that they represent the established and currently mostly used methods. Our results strongly challenge the reliability and usefulness of those conventional methods, demonstrating their inconsistency to evaluate RNA quality.
First, the A 260 :A 280 and A 260 :A 230 ratios are reflecting RNA purity, but are not informative regarding the integrity of the RNA. Available RNA extraction and purification methods yield highly pure RNA with very little DNA or other contaminations, resulting most often in both ratios )1.8, although 18% of the samples were found degraded and 7% more of poor quality. The high A 260 :A 280 ratios are indicative of limited protein contaminations, whereas high A 260 :A 230 ratios are indicative of an absence of residual contamination by organic compounds such as phenol, sugar or alcohol, which could be highly detrimental to downstream applications. Nonetheless, samples displaying low A 260 :A 230 ratios ((1.8) did not exhibit any inhibition during downstream applications, such as cDNA synthesis and labeling or in vitro transcription (data not shown). Second, due to a lack of reliability, the 28S:18S rRNA ratios may not be used as a gold standard for assessing RNA integrity. When ribosomal ratios were calculated from identical samples but through independent runs, a large degree of variability (CV 19-24%) was observed. Moreover, using the biosizing software, we found 28S:18S rRNA ratios evaluation compromised by the fact that their calculation is based on area measurements and therefore heavily dependent on definition of start and end points of peaks. In 13% of the cases, the system was unable to localize the ribosomal peaks, and therefore no 28S:18S ratios were computed. For the remaining samples, no clear correlation between 28S:18S ratios and RNA integrity was found although RNAs with 28S:18S >2.0 were usually of high quality. Most of the RNAs we studied (83%), displaying a 28S:18S > 1.0, could be considered of good quality. Interestingly, Auer et al. (24) in a study on 19 tissues from seven organisms, reported that an objective measurement of the RNA integrity may possibly be done through comparison of re-scaled 28S and 18S peak heights, but not of the corresponding areas. Actually, we observed a linear relationship between RNA integrity and differences in normalized 28S and 18S peak heights. Increased degradation resulted in a significant decrease in the scaled corrected heights of the ribosomal peaks, with inversion of the ratio at the highly degraded stages (cf. Figure 3B ). In comparison to the area computation, 28S:18S rRNA re-scaled peak height measurement produced more consistent values, with a CV reduced to 12-14%, and displayed clear concentration-independent values (see Supplementary Tables 1 and 3 online) . Human evaluation of the integrity of RNA through visual inspection of the electrophoresis profiles provided very consistent data. Variability between classifications produced by five independent expert operators (CV 16%) was lower than with automated management of more conventional control 28S:18S area values (CV 19-24%). It is, however, very time-consuming and strongly dependent on individual competence. Even with highly trained specialists, 5% of the set of RNA samples could not be allocated to any of the five predefined categories; their corresponding profiles were considered by our experts as atypical, displaying a temperature-sensitive shape (data not shown).
These strategies appear unsuitable for standardization and quality control of RNA integrity assessment, which require simple but consistent expert-independent classification, facilitating information exchanges between laboratories. The N-value corresponds to the number of samples by category. The mean quality metrics, i.e. RIN and DegFact and the mean fold change (2 ÀDDCt ) relative to the reference sample are indicated, together with the 95% confidence intervals. Observed technical variation (IC-rep, P = 0.05) is also specified, considering duplicate (two per gene per target sample) and replicate (six per gene per calibrator sample) measures. The reference sample exhibits a RIN of 9, a DegFact value of 4.9 and by default mean fold change set to 1. The observed decrease in the expression (relative expression, %) relative to the reference sample is calculated. The fold differences refer to the fold-ratios that are expected in the expression levels for a gene, across categories (between categories), given that the samples only differ by their quality, and within each category (within categories), considering RNA of comparable integrity. The fold-ratios (technical variation) that may be expected by chance in the gene expression levels, P = 0.05, from some technical reasons, are also considered.
We therefore investigated the performance of two recently developed user-independent software algorithms (24, 25) . The degradometer software provided a reliable evaluation of RNA integrity based on the identification of additional 'degradation peak signals' and their integration in a mathematical calculation together with the ribosomal peak heights. It allowed characterization of the integrity of 84% of the samples tested, one-third with an alert flag, which was first found to be fairly informative, as it strongly reduces the complexity of the metrics by introducing three distinct classes labeled Yellow, Orange and Red, and can be used as a first straightforward simple filtering step. However, degradation factors (DegFact) metrics yield precise measures with less than 32% CV and are much more valuable than flag alerts for the purpose of standardization. The same is true for the RNA Integrity Number 'RIN' software which allowed the characterization of the integrity of 91% of the RNA samples tested, with a RIN value for 363 RNA sample profiles with less than 12% CV. In general, there was a good agreement between the human classification, the degradation factor and the RIN (see Figure 4B ). This provided a cross-validation of the user-independent qualification systems tested. Both resulted in the refinement of human interpretations, validating four statistically relevant classes of samples, namely good (HC-level 1), regular/ moderate (HC-level 2 and 3), poor (HC-level 4) and degraded (HC-level 5). Moreover, the 5% RNA samples previously flagged by the operators as displaying an atypical temperature-sensitive shape were unambiguously assigned to one or the other category of samples [RIN = 7.3 (IC 6.8-7.8); DegFact = 11.9 (IC 9.5-14.2); data not shown]. Altogether, we found the degradometer and RIN algorithms to be highly reliable user-independent methods for automated assessment of RNA degradation and integrity. The RIN system is a slightly more informative tool, able to compute assessment metrics for 91% of the RNA profiles, compared to 84% with the degradometer software; the remaining being flagged respectively as N/A or Black alert. For samples available below a low limit of 20 ng (N = 80) the RIN system provided Figure 6 . Workflow of operational procedure for RNA quality assessment. Integrity of the RNA, once extracted and purified from cell lines, clinical or biological tissues samples, is controlled from the widely used bioanalyzer electrophoretic traces. As standard part of the Agilent analysis software (25), a RIN metrics is first calculated, scoring each RNA sample into 10 numerically predefined categories of integrity (RIN, from 1 to 10; N is a threshold value). As an independent control, a degradation factor metrics (DegFact, from 1 to ¥; N 0 is a threshold value) may optionally be allocated to each RNA sample using the bioanalyzer-independent degradometer software (24) . In a standard operating procedure, RIN and/or DegFact metrics will first be used as a standard exchange language to document RNA integrity and degradation, second to classify the RNA in homogeneous groups, and finally to select samples of comparable RNA integrity to improve the scheme of meaningful downstream experiments. The standard operating procedure will benefit from feedback information that will help users to define threshold integrity metrics values based on the results of RNA-based analyses. metric values for 85% of them, compared to only 46% with the degradometer software. Similarly, the RIN system was able to provide metric values for 81% of poor quality samples (including low quality and degraded samples; N = 96), whereas the degradometer software could classify only 44% of them. Another advantage with the RIN classifier is that, if there are critical anomalies detected (including genomic DNA contamination, wavy baseline, etc.), threshold settings may be changed and a reliable RIN value computed. This was the case for 25 of the 363 RNA sample profiles successfully classified by the system.
While intact RNA obviously constitutes the best representation of the natural state of the transcriptome, there are situations in which gene expression analysis may be desirable even on partially degraded RNA. Some studies report collection of reasonable microarray data from RNA samples of impaired quality (28) , leading to meaningful results if used carefully. Moreover, Auer et al. (24) recently concluded that degradation does not preclude microarray analysis if comparison is done using samples of comparable RNA integrity. We confirmed the direct influence of the RNA quality on the distribution of gene expression levels, by detecting using Q-PCR a significant (up to 7-fold) difference in the relative expression of genes in samples of slightly decreased RNA integrity, which is much larger than the variation within comparable RNA quality categories (cf. Figure 5 and Table 2 ). This may correlate with ratio discrepancies in gene expression experiments, and therefore with false positive and false negative rates of differential gene expression when comparing two samples. Therefore, computing reliable metrics of RNA integrity, even if the RNA is found to be partially degraded, may be highly valuable. The straight and unambiguous relationships established between human interpretations and both RIN and DegFact distributions indicates that, using these metrics, it should be possible to distinguish specific samples that are too disparate to be included in comparative gene expression analyses without compromising the results. Although the information provided by these user-independent classifiers is not a guarantee for successful downstream experiments, it gives a more comprehensive picture of the samples and can be used as a safeguard against performing useless and costly experiments.
Thus, the RIN system may be used as simple metrics that can be easily integrated in any sample tracking information system for definition of standard operating procedures under quality assurance following a scheme such as the one described in Figure 6 . In this context, we suggest that the growing number of laboratories performing RNA Quality Control by microcapillary electrophoresis should be offered the option to report objective RNA quality metrics as part of the 'Minimum Information About a Microarray Experiment' MIAME standards (29) . Through registration of RNA profiles in a public electronic repository, such standardized information should enable and facilitate comparisons of RNA-based bioassays performed across laboratories with RNA samples of similar quality, in much the same way as sequencing traces are compared. Factors affecting translation at the programmed −1 ribosomal frameshifting site of Cocksfoot mottle virus RNA in vivo The ratio between proteins P27 and replicase of Cocksfoot mottle virus (CfMV) is regulated via a −1 programmed ribosomal frameshift (−1 PRF). A minimal frameshift signal with a slippery U UUA AAC heptamer and a downstream stem–loop structure was inserted into a dual reporter vector and directed −1 PRF with an efficiency of 14.4 ± 1.9% in yeast and 2.4 ± 0.7% in bacteria. P27-encoding CfMV sequence flanking the minimal frameshift signal caused ∼2-fold increase in the −1 PRF efficiencies both in yeast and in bacteria. In addition to the expected fusion proteins, termination products ending putatively at the frameshift site were found in yeast cells. We propose that the amount of premature translation termination from control mRNAs played a role in determining the calculated −1PRF efficiency. Co-expression of CfMV P27 with the dual reporter vector containing the minimal frameshift signal reduced the production of the downstream reporter, whereas replicase co-expression had no pronounced effect. This finding allows us to propose that CfMV protein P27 may influence translation at the frameshift site but the mechanism needs to be elucidated. The principal mechanism of translation is the accurate decoding of the triplet codon sequences in one reading frame of mRNA. Specific signals built into the mRNA sequences can cause deviations from this rule. Viruses exploit several translational 'recoding' mechanisms, including translational hopping, stop codon readthrough and programmed ribosomal frameshifting (PRF) [reviewed in (1, 2) ], for regulating the amount of proteins produced from their polyproteins. For positive-stranded RNA viruses, À1 PRF is the prevailing recoding mechanism and an essential determinant of the stoichiometry of synthesized viral proteins. Most viral À1 PRF signals are regulating the production of replication-associated proteins. Depending on the virus, the efficiency of À1 PRF can vary between 1 and 40% (3) , and changes in the efficiency can inhibit virus assembly and replication (4) (5) (6) . Therefore, À1 PRF can be regarded as a potential target for antiviral agents (4, 7) . However, the development of efficient antiviral drugs is still hindered, since little is known about the trans-acting factors and the biophysical parameters affecting the À1 PRF efficiencies. Database searches have identified putative frameshift signals from a substantial number of chromosomally encoded eukaryotic mRNAs (8) . Thus, À1 PRF may also have an impact on the complexity of the proteome of several eukaryotic organisms.
Two cis-acting signals, a slippery heptamer X XXY YYZ (the incoming reading frame indicated) and a downstream secondary structure, direct the slippage and are therefore essential for this event (9) . À1 PRF takes place after the accommodation step in the slippery sequence by simultaneous slippage of both tRNAs into the overlapping À1 frame XXX YYY (9, 10) . The sequence of the heptamer allows postslippage base-pairing between the non-wobble bases of the tRNAs and the new À1 frame codons of the mRNA. Downstream RNA secondary structures [reviewed in (11) ] force the ribosomes to pause, and place the ribosomal A-and P-sites correctly over the slippery sequence (12) . However, the pausing of the ribosomes is not sufficient for À1 PRF to occur (13) ; in fact, the duration of the halt does not necessarily correlate with the level of the À1 PRF observed (12) . Crystallographic, molecular, biochemical and genetic studies suggest that a pseudoknot restricts the movement of the mRNA during the tRNA accommodation step of elongation by filling the entrance of the ribosomal mRNA tunnel (14) . This restriction can be eased either by unwinding the pseudoknot, which allows the mRNA to move forward, or by a slippage of the mRNA one nucleotide backwards. Chemical agents such as *To whom correspondence should be addressed. Tel: +358 9 19158342 ; Fax: +358 9 19158633; Email: kristiina.makinen@helsinki.fi ª The Author 2005. Published by Oxford University Press. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org antibiotics, certain mutations in the translation apparatus, and in translation elongation factors that change the translation fidelity and kinetics, have been shown to influence À1 PRF efficiency [(10,15) ; reviewed in (16) ].
The parameters known to contribute to the efficiency of À1 PRF are the sequence of the slippery heptamer, the downstream secondary structure, and the length and sequence of the spacer between the two cis-acting signals. Up-and downstream sequences such as termination codons in the vicinity of the À1 PRF signals, or even several kilobases away from them, can affect the À1 PRF efficiencies (3, (17) (18) (19) (20) (21) (22) . A specific sequence in the Barley yellow dwarf virus (BYDV) 3 0 untranslated region (UTR), 4 kb downstream from the slippage site, is vital for À1 PRF (6, 19) . A stimulating effect is achieved through the formation of a tertiary structure, where complementary nucleotides from the 3 0 UTR base pair with a single-stranded bulge in the cis-acting stem-loop (6). Human immunodeficiency virus (HIV) was also shown to require a more complex secondary structure instead of a simple stemloop for optimal À1 PRF in vivo (21, 22) . These investigations suggest that À1 PRF studies carried out with minimal frameshift signals may lead to inaccurate estimates of the stoichiometry of synthesized viral protein products during infection.
Cocksfoot mottle virus (CfMV; genus Sobemovirus) infects a few monocotyledonous plant species such as barley, oats and wheat. It has a monopartite, single-stranded, 4082 nt long, positive-sense RNA genome (23, 24) . The polyprotein of CfMV is translated from two overlapping open reading frames (ORFs) 2A and 2B by a À1 PRF mechanism (25) . In this study, we wanted to determine the in vivo À1 PRF efficiency guided by the CfMV U UUA AAC heptamer and the stem-loop structure. In addition to the minimal signal (18) , we decided to test the effect of flanking CfMV sequences for their ability to contribute to À1 PRF. We found that the surrounding viral sequences promoted more efficient À1 PRF than the minimal signal sequence in vivo when measured with the dual reporter vector system developed by Stahl et al. (26) . Therefore, we carried out an expression pattern and deletion analysis to understand the molecular basis of the observed upregulation. In addition, we critically analysed the suitability of the implemented experimental system for this type of a recoding study. An interesting possibility is that the viral proteins produced via À1 PRF could regulate À1 PRF. This hypothesis was tested by co-expressing the CfMV proteins P27 and replicase together with the dual reporter vectors.
Three regions from the CfMV polyprotein ORFs ( Figure 1A) were cloned into the NheI and BclI sites between the lacZ and the luc ORFs in pAC74 (26) . This dual reporter vector was a generous gift from Dr J. Rousset of the Universite Paris-Sud, France. The inserted sequences 1602-1720 (A region), 1386-2137 (B region) and 1551-1900 (C region), were amplified by PCR using pAB-21 as a template (18) . Primers were used to introduce NheI and BglII sites to the flanking ends of the inserts. Since NheI digestion removed lacZ ORF, it was reintroduced into the plasmids as a final cloning step. The resulting plasmids were named pAC-A, pAC-B and pAC-C.
Corresponding inframe controls, where one nucleotide was added in front of the slippery heptamer, were generated by PCR-based mutagenesis (Exsite, Stratagene) and named as pAC-Am, pAC-Bm and pAC-Cm, respectively. Deletion plasmids pAC-AB/ABm (1602-2137), pAC-AC/ACm (1602-1900), pAC-BA/BAm (1386-1720) and pAC-CA/ CAm (1551-1720) were also generated. The target sequences are shown in Figure 1B . The base numbering refers to the CfMV genome as in (23) . Transcription was driven from SV40 promoter. Plasmids encoded leucine (LEU2) and b-lactamase (ampicillin resistance) as selective markers. Plasmids were transformed into Saccharomyces cerevisiae H23 [MATa hsp150::URA3 ura3-1 his3-11 15leu2-3 112trp1-1 ade2-1 can100]. Dual reporter plasmid pAC1789 and the inframe control pAC1790 containing a 53 bp sequence from the HIV-1 frameshift region (26) were used as a positive control for monitoring the À1 PRF efficiency.
To analyse the proteins produced during À1 PRF, lacZ-A/ Am/B/Bm/C/Cm-Fluc fragments were cloned inframe with the N-terminal 6xhistidine-tag in pYES2/NT KpnI and XhoI sites (Invitrogen). Reporter fusions were amplified by PCR using pAC-A/Am, pAC-B/Bm or pAC-C/Cm as templates. The resulting plasmids were named pYES2/NT-A/ Am, B/Bm and C/Cm. Protein expression was regulated from GAL1 promoter. Two CfMV encoded proteins, P27 (C-terminal end of ORF2A) and replicase (ORF2B), were cloned into pYES2 (Invitrogen). Translation initiation codons were introduced within the oligonucleotides during PCR. The resulting plasmids were named pYES-P27 and pYES-Rep. Control plasmids, which lacked the translation initiation codons were prepared by PCR-based mutagenesis (pYES-P27DAUG and pYES-RepDAUG) and the resulting plasmids were verified by sequencing. Plasmids encoded auxotrophic marker for uracil (URA3).
All cloning steps were performed using standard protocols. Plasmids were amplified either in Escherichia coli DH5a or JM110, and purified with Qiagen columns. Inserts were verified by sequencing. Yeast transformations were done using the LiAc method (27) , and transformants were selected on a synthetic minimal defined medium (SC) lacking the corresponding auxotrophic marker(s) encoded by the used plasmid(s). Bacteria (E.coli DH5a) were grown in LB-medium containing ampicillin, whereas yeast cells were grown either in YPD, or in an SC medium. Protein expression from GAL1 promoter was repressed during growth at SC medium containing 2% glucose. Expression was induced by replacing glucose with 2% galactose and 1% raffinose.
Reporter fusions were expressed in S.cerevisiae INVSc1 (his3D1/his3D1, leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52) (Invitrogen) overnight. Protein fusions were purified in denaturing conditions using Ni-NTA agarose (Qiagen), and analysed in 6% SDS-PAGE gels. Proteins were visualized either by Coomassie staining, or by using antisera raised against the CfMV polyprotein region 1386-1724 encoding CfMV VPg (28) . Protein antibody complexes were visualized with horseradish peroxidase-conjugated anti-rabbit antibodies (Sigma) and ECL chemiluminescent reagents (Amersham).
Plasmids pYES-P27, pYES-Rep, pYES-P27DAUG, pYES-RepDAUG, or empty pYES2 were co-expressed with pAC-A or with the corresponding pAC-Am inframe control in S.cerevisiae EGY48 strain (MATa, ura3, trp1, his3, 6lexAop-LEU2) (Invitrogen). Transformants were grown overnight in SC-Leu-Ura media in non-inducing conditions, and used to inoculate induction medium. Cells were harvested at late logarithmic phase. Expression of the CfMV proteins was confirmed by western blotting using polyclonal antisera against the CfMV ORF 2a and 2b proteins (28) . Determining the enzymatic activities as described below monitored the effect of CfMV P27 and replicase on À1 PRF.
For the in vitro analysis, the lacZ-gene of pAC-A/Am, -B/Bm and -C/Cm vectors was replaced with PCR-amplified Renilla luciferase (Rluc) gene from pRLnull vector (Promega). The resulting pACRF plasmids were used as templates for PCR in order to add T7 promoter upstream of the Rluc gene. These PCR products were used for RNA synthesis with RiboMax kit (Promega). Transcripts were treated with RQ1-DNase (Promega), purified with Qiagen RNeasy columns, and quantified spectrophotometrically. The integrity of the transcripts was checked in agarose gels. In vitro translations were carried CfMV À1 PRF test and control sequences were cloned between the b-galactosidase (LacZ) and firefly luciferase (Luc) genes into a dual reporter vector pAC74 (26) . Inframe control constructs had one extra nucleotide inserted in front of the slippery heptamer, which fused the reporters into the same reading frame. Thus, translation of the inframe control results in the production of a b-galactosidase-CfMV-firefly luciferase fusion. Translation of the test constructs in the incoming 0-frame yields a b-galactosidase-CfMV fusion, whereas À1 PRF produces a b-galactosidase-CfMV-firefly luciferase fusions identical to those produced from the inframe controls. À1 PRF efficiencies were calculated from the firefly luciferase activities after b-galactosidase normalization with the given formula. (B) CfMV polyprotein is encoded by two overlapping ORFs, 2A and 2B via À1 PRF. Sequence regions tested in the dual reporter vectors for their activity to promote À1 PRF are indicated. The numbering refers to the CfMV RNA sequence as published in (23) . out in wheat germ extract (WGE) according to the manufacturer's protocols (Promega). Reactions were incubated in room temperature for 60 min, and stopped on ice prior to enzymatic measurements.
Cell cultures were started from at least three independent clones and grown until the late exponential phase. Cells were collected by centrifugation, frozen in liquid nitrogen and stored at À70 C. Bacterial cells were lysed by sonication (3 · 15 s), and yeasts by vortexing with glass beads (0.5 vol) in +4 C for 30 min. Lysates were cleared by centrifugation, and enzymatic activities were determined immediately. Total protein concentrations were measured by using a Bradford protein assay reagent (Bio-Rad). b-Galactosidase (LacZ) and firefly or Renilla luciferase (LUC or RUC) activities were measured with commercial kits from Promega according to the manufacturer's instructions. LacZ activity was determined as the colour intensity at A414 nm. Luciferase activities were measured as relative light units (RLUs) with luminometer (Biohit or ThermoLabsystems). À1 PRF efficiencies were calculated from normalized firefly luciferase activities with the following formula: [(LUC activity from the test construct)/(LacZ or RUC activity from the test construct)]/[(LUC activity from the inframe control)/(LacZ or RUC activity from the inframe control)] · 100%.
In CfMV, the motif for À1 PRF is the slippery heptamer U UUA AAC and a stem-loop structure 7 nt downstream (25) . The efficiency of À1 PRF directed by CfMV cis-acting signals was assayed in vivo using a dual reporter vector system ( Figure 1A ). Since reporters are produced from one single mRNA, factors that affect the stability of the mRNA as well as the rate of translation initiation have a similar influence on the expression of both reporters, and these variations can be monitored as changes in the activity of the upstream reporter. We quantified À1 PRF by comparing the b-galactosidase normalized firefly luciferase activities derived from the test constructs via À1 PRF to those obtained from the inframe controls, in which identical b-galactosidase-CfMV-firefly luciferase fusions are produced without À1 PRF due to the added nucleotide in front of the slippery heptamer (see Figure 1A ). Similar vectors have been shown to detect even small changes in the recoding efficiencies resulting from alterations in the cis-or trans-acting factors (26, (29) (30) (31) (32) .
Three inserts of varied lengths from the CfMV polyproteinencoding region (ORF2A/2B) were introduced between the two reporters ( Figure 1B) . The A-region, which at 119 bp was the shortest, represented approximately the minimal frameshift signal proven to be functional in vitro (18) . The longest region was the B-insert. At 752 bp, it started from the 5 0 -terminus of the 12 kDa viral genome-linked protein (VPg) gene and continued to the end of ORF2A. This region encodes CfMV protein P27 with an unknown function (28) . Since the minimal requirements for the functional frameshift signal in vivo were not known, an intermediate 349 bp C-sequence was also selected for the analysis. A well-characterized 53 bp frameshift cassette derived from HIV-1 RNA was used as a positive control. Our results regarding the HIV À1 PRF efficiency, 0.7 -0.1% in bacteria, and 4.5 -1.1% in yeast (Figure 2) , are corroborated by those published earlier (26, 33, 34) indicating that our dual reporter system was fully functional.
b-Galactosidase has been shown to retain its specific activity well, irrespective of the C-terminal fusions (35) . This is important, since the first reporter serves to control the variations among the abundance and translation rates of the studied mRNAs (26, 30) . In addition to changes in specific activities, heterologous fusions can cause alterations in the solubility and conformation, which can expose cryptic protease target sites and reduce the stability of the proteins (36) . Therefore, for a reliable quantification of À1 PRF, it was important to test that equimolar amounts of fusions produced from the corresponding test and control constructs had similar enzymatic activities. Most inframe controls and the analogous test constructs had equal absolute b-galactosidase activities ( Table 1) . Comparable results were obtained, if activities were normalized with total protein concentration (data not shown). These results indicated that the length of the fusion as such did not affect the specific activities. The b-galactosidase activity from pAC-Am inframe control was also comparable to activity obtained from an empty pAC74, where this enzyme has no fusion (data not shown). This further supported the view that the few observed variations in the b-galactosidase activities more likely resulted from the changes in translatability or stability of the transcripts. In addition to pAC-Cm, two inframe controls pAC-Am and pAC-ACm showed $25% lower b-galactosidase activities when compared to the equivalent test constructs ( Table 1 ), indicating that the productivity from these constructs was reduced. Taken together, b-galactosidase seemed to fit well to be used as the first reporter and thus normalization factor in the in vivo experiments of this study.
CfMV frameshift signals generated significant À1 PRF in yeast. À1 PRF level measured from pAC-A was 3-fold higher than from HIV RNA (Figure 2A and B) . The extent of À1 PRF directed by the minimal region A in yeast, 14.4 -1.9%, was at the same level as that reported for the CfMV minimal frameshift signal in vitro (12.7%) (18) . In contrast to our earlier in vitro observations (18) , the longer CfMV sequences upregulated À1 PRF in vivo. In yeast, the level of upregulation was 2-fold for pAC-B, the À1 PRF frequency being 26.3%, and almost 5-fold for pAC-C resulting in efficiency close to 70% (Figure 2A) , which is an extremely high value, if compared to the other values published earlier (3). CfMV frameshift signals directed À1 PRF at a lower level in bacteria than in yeast ( Figure 2B ). The extent of À1 PRF directed by region A in bacteria was 2.4 -0.7%. As in yeast, the longest B region stimulated À1 PRF 2-fold in bacteria when compared with pAC-A. However, region C did not further improve À1 PRF, but programmed À1 PRF to similar levels as pAC-B, the percentages being 4.7 -1.6% for pAC-C and 5.5 -1.5% for pAC-B.
To identify the sequence(s) responsible for the enhancement of À1 PRF in vivo, a deletion analysis was carried out. The 5 0 -or the 3 0 -sequences flanking the A-region were deleted from pAC-B/Bm or pAC-C/Cm as indicated in Figure 1B , which generated vectors pAC-AB/ABm, pAC-BA/BAm, pAC-AC/ ACm and pAC-CA/CAm. À1 PRF frequencies were determined in yeast ( Figure 2C ). Increased À1 PRF was observed in all deletion constructs in comparison to the À1 PRF directed by the A region. The BA and AB regions promoted À1 PRF as efficiently as the B region, whereas regions CA and AC were better than region A, but not as good as region B. In other words, the presence of nucleotides 1386-1720, or downstream nucleotides 1602-2137, was sufficient to increase À1 PRF to the level directed by the region B. Thus, the deletion analysis did not identify single specific sequence region as being responsible for the increased À1 PRF frequencies.
The expression pattern of the test and control constructs was analysed to understand the basis for the observed upregulation in yeast. Cassettes containing the reporters and the studied intercistronic sequences were expressed and purified as N-terminal histidine fusions. This allowed us to capture all the N-terminally intact products. The affinity-purified proteins were separated in SDS-PAGE gels, and visualized either by Coomassie staining (data not shown), or by western blotting with the CfMV-specific anti-VPg antibodies. The expected b-galactosidase-CfMV fusions terminating at the end of the 0-frame in the test constructs were detected. Also, the longer transframe b-galactosidase-CfMV-firefly luciferase fusion proteins were present in both the test and the inframe constructs ( Figure 3) . Comparison of the Coomassie-stained gels with the western blots revealed that the antisera recognized the products terminating at the CfMV-encoding regions better than the transframe products. Furthermore, the small size of the CfMV-specific region in the pYES2/NT-Am decreased the binding of the antibodies to these inframe control fusions. Thus, this data were not suitable for quantitative analysis of À1 PRF. Interestingly, an additional protein, which reacted with CfMV-specific antisera, was co-purified from the cells expressing pYES2/NT-Bm and pYES2/NT-Cm inframe controls ( Figure 3 ). The size of these fusions suggested that translation had terminated approximately at the site for À1 PRF signals. If such putative termination products were also present in cells expressing the test constructs, the correctly terminated 0-frame products in the western blots masked these products.
A closer look at the absolute b-galactosidase and firefly luciferase activities revealed that firefly luciferase expression from pAC-Cm was clearly reduced (data not shown). In fact, expression from the inframe control was comparable to the corresponding pAC-C test construct. This was also obvious when the firefly luciferase activities were normalized with the total protein amount. After setting the activity from pAC-Am to a relative value of one, the corresponding values from pAC-Bm and pAC-Cm were 0.80 and 0.28. Although the b-galactosidase measurements (Table 1) suggested that the overall translatability of the pAC-Cm mRNA was also reduced to some extent, it explained the decrease in firefly luciferase expression only partially. In the light of these findings, the extremely high À1 PRF frequency estimate calculated for the C-region could be explained with more frequent translation termination at the frameshift signals of the pAC-Cm mRNA, which reduced firefly luciferase activity in relation to b-galactosidase.
À1 PRF was also assayed in vitro in WGE. Although LacZ-encoding gene is suitable for the in vivo studies, it is an unsuitable first reporter for the in vitro determination of À1 PRF efficiencies due to its big size (30) . In good agreement with this, we observed several unexpected products in the in vitro translations programmed with LacZ-CfMV-luc mRNAs (data not shown). Renilla luciferase has been shown to retain its specific activity irrespective of the C-terminal fusions (30) . Therefore, we decided to use Rluc-CfMV-luc transcripts to determine the À1 PRF efficiencies in the cellfree system. First, we verified the suitability of Renilla luciferase for the intended in vitro experiments as described in (30) . Transcripts encoding monocistronic Renilla luciferase and Renilla luciferase fused to firefly luciferase (Rluc-Am/ Cm-luc) were mixed in different ratios and used to program the in vitro translations. Increasing concentrations of transcripts encoding the Rluc-Am-luc fusion resulted in linearly growing firefly luciferase activities. At the same time Renilla luciferase activities remained constant, which showed that its enzymatic activity was not sensitive to the C-terminal fusions ( Figure 4A ). Similar results were obtained with Rluc-Cm-luc mRNA (data not shown). À1 PRF efficiencies were then determined with transcripts that contained CfMV regions A, B and C, and their corresponding inframe controls. In all cases, slightly higher À1 PRF frequencies were obtained than in vivo. In nice correlation with the in vivo results, enhanced À1 PRF was observed with the region B, although the effect was weaker than in vivo. In this context, region C did not differ from the minimal region A in its capacity to program À1 PRF ( Figure 4B) .
The ratio between the CfMV P27 and replicase is regulated by À1 PRF during CfMV infection (28) . We studied whether these proteins could regulate the À1 PRF process. P27, replicase, or an empty expression vector was co-expressed in yeast together with the dual reporter vectors containing the minimal À1 PRF test and inframe control regions as intergenic sequences (pAC-A and -Am). P27 and replicase expression was verified by a western blot analysis ( Figure 5) . A faint band having nearly the same mobility as the replicase was detected in cells grown under repressing conditions. However, due to the small size difference, this protein was not regarded as replicase.
Enzymatic activities were measured from yeast lysates prepared from induced cultures. Measurements showed comparable levels of b-galactosidase in all the samples, indicating that P27 or replicase expression did not affect the stability of the dual reporter mRNA or the translatability of the first reporter ( Table 2 ). The effect of P27 or replicase expression was monitored by comparing the reporter activity ratios to those measured from cells harbouring the empty expression plasmids (Table 2) . Co-expression of CfMV replicase did not affect the normalized firefly luciferase expression (LUC/LacZ) from the inframe control, whereas slightly increased luciferase expression from the test construct was observed. In contrast, P27 expression reduced firefly luciferase expression both from the test and the inframe constructs. The effect was stronger in the presence of inframe control as normalized firefly luciferase levels reached only 54% of expression measured from the empty vector control.
To verify that the observed differences in firefly luciferase production depended on the studied CfMV proteins, we co-expressed the dual reporter vectors with plasmids having the first translation initiation codons of P27 and replicase deleted (pYES-P27DAUG and pYES-RepDAUG). Western blot analysis with antisera against ORF2A or 2B did not detect any proteins produced from these vectors (data not shown). The obtained LUC/LacZ ratios were compared to those measured from cells expressing the CfMV proteins (pYES-P27 or pYES-Rep). LUC/LacZ ratios measured from cells expressing replicase were slightly lower than the ratios calculated from cells harbouring pYES-RepDAUG plasmids, being $90% when co-expressed with pAC-A and $84% when co-expressed with pAC-Am. In the presence of P27, LUC/ LacZ ratio of pAC-A reached $81% of expression measured from cells transformed with pYES-P27DAUG. Again the effect of P27 expression was more evident with pAC-Am inframe control as P27 expression reduced LUC/LacZ ratio to half ($48%) when compared to the corresponding value measured from the cells harbouring pYES-P27DAUG. This verified that CfMV P27 was able to reduce the downstream reporter expression from dual reporter mRNAs. Since CfMV P27 had a proportionally stronger effect to firefly luciferase production from the inframe control mRNAs in comparison to the test mRNAs (Table 2) , the calculated À1 PRF efficiency increased from 14.7 to 22.4%.
Since À1 PRF studies are affected by a huge number of different parameters, it is not an easy task to determine the real ratio between the proteins produced via this mechanism in vivo. However, in viral systems, the efficiency of À1 PRF is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus. For example, frameshifting in retroviruses determines the ratio of structural (Gag) to enzymatic (Gag-Pol) proteins, and plays a critical role in viral particle assembly (5) . In this study, the capacity of CfMV frameshift signals to direct efficient À1 PRF was analysed in vivo by using dual reporter vectors. The length of the CfMV sequence clearly affected the actual efficiency percent in vivo. The PRF efficiency was elevated when longer viral sequences were directing the À1 PRF, but the deletion analysis did not identify any specific region as being solely responsible for the enhancement. Up-and downstream sequences nearby or far away from the cis-acting signals have been reported to enhance À1 PRF in other viruses, such as HIV, human T-cell leukaemia virus and BYDV (6, 19, 20) . Also out-of-frame stop codons have been shown to influence À1 PRF frequency in vitro in retroviruses (17) and in CfMV (18) . A study on the spacer sequences located between the cis-acting signals showed that high slippage frequencies were obtained when the first three nucleotides were G/U, G/A and G/A, the first two being the most important (37) . In CfMV, the spacer starts with UAC, which partially explains the capacity of the CfMV sequence to promote high slippage levels. In this study, the observed enhancement of À1 PRF was, however, caused by sequences that were not in the immediate vicinity of the Figure 5 . Co-expression of CfMV P27 or replicase simultaneously with the minimal frameshift signal construct pAC-A or the corresponding inframe control pAC-Am in yeast. Yeast total protein samples were separated in 12% SDS-PAGE gels, transferred onto PVDF membranes, and immunocomplexes detected by ECL chemiluminescent system. CfMV P27 expression was verified by western blotting with antisera raised against ORF2A (A), and CfMV replicase expression was detected with antisera raised against ORF2B (B). Abbreviations: À, repressed; +, induced; C1, pMAL-VPg $53 kDa; and C2, baculovirus expressed CfMV replicase. slippery sequence thus indicating that CfMV sequences further away also have an influence on the level of frameshifting in vivo. We conclude that the most reliable estimates for À1 PRF and consequently for the amount of replicase versus the 0-frame translation product P27 can be obtained only by using the full-length viral sequences. In reality, such a study would however be hampered by the non-quantitative nature of the western blot analysis, the presence of different polyprotein processing intermediates, and the differences in the stabilities of the end products in the infected cells.
The overall competence of CfMV signals to direct À1 PRF was high, when compared to related plant viruses, such as Potato leaf roll virus and BYDV. À1 PRF values of $1% have been reported for these viruses when measured with reporter-based assays (6, 38) . We can hypothesize that one reason for the high efficiency is the slippery tRNA Asn encoding the AAC triplet of the CfMV heptamer. Equal U UUA AAC slippery heptamer has been measured to induce 20-40% of À1 PRF in a diversity of animal viruses [(39); reviewed in (3)]. The low fitness of CfMV À1 PRF signals in bacteria is in agreement with the poor functioning of the eukaryotic slippery heptamers of the order X XXA AAC in prokaryotes (40) (41) (42) . IBV RNA, having an identical shifty heptamer, has been shown to direct À1 PRF at similar 2-3% level in bacteria (41) . A recent study reported that XXXAAAC heptamers dictate À1 PRF to occur via the slippage of two adjacent tRNAs placed over the heptamer, irrespective of whether the host is an eukaryote or a prokaryote (42) . Therefore, the inability of prokaryotic translation systems to direct efficient À1 PRF from this heptamer is not an inherited property of prokaryotic tRNA Asn , but results from differences in the ribosomes (42) .
Paused ribosomes can pass the À1 PRF site by À1 frameshifting, resumption of 0-frame translation, or termination (43) . Transient polypeptide intermediates that result from the pausing of ribosomes in the slippery sequences have been observed during IBV and S.cerevisiae L-A virus polyprotein synthesis (12, 13, 43, 44) . A pseudoknot structure formed by IBV mRNA causes a translational pause at fixed position upstream the secondary structure regardless of whether the slippery heptamer is present or absent (12) . Based on the findings of this study, we propose that also here a certain percent of ribosomes stalled at the secondary structure of the frameshift site in our inframe control and test mRNAs in yeast, and this led to the prematurely terminated products observed with the inframe control constructs pAC-Bm and -Cm. Although not unambiguously proven by this study, high frequency of termination of translation especially at the frameshift site of the pAC-Cm mRNA would nicely explain the extremely high calculated À1 PRF efficiency.
Factors that change the translation fidelity and kinetics have been shown to influence À1 PRF efficiency [ (10, 15) ; reviewed in (16) ]. Autoregulation of +1 frameshifting by mammalian ornithine decarboxylase antizyme has been reported (45) . This mechanism allows modulation of frameshifting frequency according to the cellular concentration of polyamines. One could speculate that such a regulation mechanism could also be useful to adjust the amounts of the replicationassociated proteins to match the requirements of different phases in viral replication cycle. This hypothesis was studied by expressing CfMV proteins P27 and replicase together with pAC-A and pAC-Am in yeast cells. Since b-galactosidase production remained constant regardless of the presence or absence of CfMV proteins, they did not interfere with translation initiation from pAC-A/Am mRNAs per se. However, P27 expression caused a reduction in the firefly luciferase production especially from the inframe control, whereas replicase production only slightly increased the firefly luciferase production from pAC-A, but not from pAC-Am. Since replicase expression had only a faint effect on the normalized firefly luciferase production via À1 PRF, our conclusion is that CfMV replicase had no pronounced effect on translation at the frameshift site. Co-expression of the non-translatable form of P27 with the dual reporter vectors verified that P27 truly affected firefly luciferase expression on the protein level. Therefore, we propose that CfMV protein P27 may influence translation at the frameshift site. If CfMV P27 indeed interferes with viral protein synthesis during CfMV infection, the mechanism, its specificity and the possible biological role needs to be elucidated in the future. Australian public health policy in 2003 – 2004 In Australia, compared with other developed countries the many and varied programs which comprise public health have continued to be funded poorly and unsystematically, particularly given the amount of publicly voiced political support. In 2003, the major public health policy developments in communicable disease control were in the fields of SARS, and vaccine funding, whilst the TGA was focused on the Pan Pharmaceutical crisis. Programs directed to health maintenance and healthy ageing were approved. The tertiary education sector was involved in the development of programs for training the public health workforce and new professional qualifications and competencies. The Abelson Report received support from overseas experts, providing a potential platform for calls to improve national funding for future Australian preventive programs; however, inconsistencies continued across all jurisdictions in their approaches to tackling national health priorities. Despite 2004 being an election year, public health policy was not visible, with the bulk of the public health funding available in the 2004/05 federal budget allocated to managing such emerging risks as avian flu. We conclude by suggesting several implications for the future. Public health is a small component of the health system, both in terms of budgetary allocation at either state or national level and in terms of the number of practitioners. It incorporates a myriad of activities; legislation and regulation for health protection, preventive services directed at specific diseases and populations, and health promotion programs geared towards particular risk factors and vulnerable groups in the community. As such, it looks like a disparate collection of programs and investments.
In Australia, there is also confusion about the very terminology of 'public health'. Despite its extensive history and global understanding, in Australia the term is used variously; to refer to publicly funded health services, and interventions (regardless of the funding source) which are aimed at primary prevention and the promotion and protection of the public health ('rats and drains'). This has led to an increasing number of jurisdictions adopting the label 'population health'.
Renovation of the public health system has been on the international agenda for some years. In the US, the Institute of Medicine released reports during 2003 about the public health workforce required for 21 st century challenges [1], as well as re-visited and updated its landmark report, The Future of Public Health in the 21st Century [2] . In the UK, following the path-breaking review of the NHS by Derek Wanless [3] the Treasury commissioned him, in 2003, to undertake a review of whole-of-government effort in public health. Arising in part from the challenges that confronted Canada during the outbreak of sudden acute respiratory syndrome (SARS) in 2003, a new public health agency, at arms length from government, is being created.
Public health in Australia, meanwhile, remained fragmented -by programs, across jurisdictions (particularly the states and territories) -and without a systematic approach to funding, organisation, or conceptualisation. In 2003/04, the gap between rhetoric and funding continued to be noticeable, along with the tension between framing priorities for popular appeal versus the technical language of the evidence base.
This article will examine some of the indicative developments of public health in Australia in 2003/04. The key developments are identified, and a number of them are selected for in-depth analysis. In this article, we use the traditional meaning of the term 'public health' and focus on activities which are usually designed to promote and protect the health of the population. The drivers for these developments, their short term implications and some signposts for the future are suggested.
While early global anxiety over SARS occupied headlines between February and May, the more persistent popular headline in 2003 focused on obesity. Summits were held in NSW and Victoria, while the National Obesity Taskforce was convened under the auspice of the Australian Health Ministers Council (AHMC).
When Kay Patterson was the Federal Health Minister, she declared that prevention was the fourth pillar of Medicare and she wanted to be 'Minister for Prevention'. Indeed, the 2003/04 federal budget, although limited, contained a bundle of initiatives entitled "Prevention on the Health Agenda". In particular, a number of immunisation and health promotion programs were included.
Significant amongst the funding initiatives for public health announced in 2003/04 was government support for the meningococcal vaccine. Although this was the culmination of many months of careful planning, a perception existed that this only occurred after considerable public interest in and anxiety about deaths from outbreaks of this disease.
Further changes to the recommended schedule in 2003 were made by the Australian Technical Advisory Group on Immunisation (ATAGI), in particular the inclusion of pneumococcal and varicella vaccines; however, these did not result in similar prescribed vaccine programs or in similar funding. These three developments are reviewed in greater detail in the next section.
The National Public Health Partnership (NPHP) and the AHMC adopted the influenza pandemic plan in October 2003, and with the advent of the newly-identified disease SARS, as well as outbreaks of meningococcal disease, management and prevention of communicable diseases was prominent. Following on from the significant funding boost for bioterrorism preparedness in 2002/03, public health preparedness became a more generic theme.
The arrival of SARS occupied the national popular and political imagination as well as tested the infrastructure capacity of public health. Australia fared well during the outbreak. Apart from escaping with only six Australian cases, it provided an opportunity to establish a coordinated approach between the Commonwealth and the states/territories and also contributed to the global epidemiological investigation and prevention effort. SARS also prompted amendments to the Quarantine Act [4] .
While the recall following the Pan Pharmaceutical crisis put the Therapeutic Goods Administration (TGA) under the spotlight, it also managed to conclude negotiations that had been in train for several years on a Trans-Tasman regulatory regime and authority. Also on the regulatory front, the Australian New Zealand Food Regulation Ministerial Council endorsed a nutrition, health and related claims policy guidelines and established a review of genetically modified (GM) labeling of foods [5] . All these developments pointed to the global nature of public health, and the intersection between public health activities and the economy.
Policy development in public health has never been confined to a set of health programs, and in 2003/04, the lead was often taken from outside the health sector. Most significant was the adoption of the National Agenda for Early Childhood [6] , pushed by public health advocates for child health since the mid 1990s. The National Public Health Partnership responded by coordinating a scoping of child health strategies across Australia. Elsewhere in Government, "Promoting and Maintaining Good Health" was adopted as one of the National Research Priorities [7] . Healthy ageing also emerged as a policy theme in Ageing Research.
Public health workforce development was pursued outside the mainstream education and training arrangements for public health in universities. The Community Services and Health Training Board commissioned a consultative process to develop population health competencies for the Vocational Education and Training (VET) sector [8] . New population health qualifications and competencies were proposed for incorporation into the Health Training Package -including certificates in population health and in environmental health, and diplomas in population health and in indigenous environmental health.
The release in 2003 of the report "Returns on Investments in Public Health: an epidemiological and economic analysis" [9] (often referred to as the Abelson report), may have a significant impact in subsequent years. Commissioned several years earlier by the Population Health Division of the Department of Health and Ageing (DoHA), the report experienced a relatively low profile until Derek Wanless visited from the UK. Having chaired a review that contributed to a significant budgetary increase for the NHS, Wanless had been commissioned by the British Treasury to examine prevention across government. In September 2003, at a meeting in Canberra with senior officials across key agencies, Wanless marveled at the value of the Abelson report, described in more detail below.
Although 2004 was an election year, public health policy was neither visible during the campaign or in policy development more generally. The Federal Government's initiative to wind up the National Occupational Health and Safety Commission received little publicity and comment, even though it indicated the Commonwealth's increasing tendency to pursue its own pathway, separate from states and territories, and to bring the functions of statutory bodies into departments.
Jurisdictional and annual reports show that across the states and territories, there were multiple plans, draft guidelines, meetings, episodic training and programs across a broad range of areas. Some health issues are being taken up across jurisdictions -particularly tobacco control, sexually transmitted infections, Aboriginal health, and vaccination. Innovative activities were reported in some jurisdictions, such as a new Health Impact Assessment Branch and a new public health training program in Western Australia. There was, however, no apparent consistency in health priorities across the nation, and an apparent divergence in the interests of the states/territories and the federal government.
While the "prevention and management of overweight and obesity" agenda may have appeared to many observers as a new issue in 2003, its arrival was preceded by several years of intensive work. The NHMRC had released Acting on Australia's Weight: Strategic plan for the prevention of overweight and obesity in 1997 [10] , the same year the ABS published the findings from the 1995 National Nutrition Survey, revealing that 45% of men and 29% of women in Australia were overweight, with an additional 18% of men and women classified as obese [11] . Furthermore, overweight and obesity were more common in lower socio-economic groups, in rural populations, in some immigrant groups, and in Aboriginal and Torres Strait Islander (ATSI) peoples.
Despite longstanding national cooperation on nutrition (since the days of the National Better Health Program in the late 1980s), and even more recent national cooperation on physical activity, public and political imagination was not captured until the same issues were recast as 'obesity', with a focus in particular on childhood obesity. Following from the NSW Childhood Obesity Summit in late 2002, the Australian Health Ministers agreed that a national approach was required and established a National Obesity Taskforce [12] .
In 2003, NSW Health released it's response to the Summit recommendations and supported the vast majority of the 145 resolutions [13] . The Victorian Department of Human Services also held a summit [14] , while Healthy Weight 2008 -Australia's Future was released by the Commonwealth [15] . The NHMRC joined in with release in late 2003 of clinical practice guidelines for general practitioners and other health professionals [16] .
While the specifics vary, the major themes and strategies are captured in Healthy Weight 2008. These are summarised in the Table 1. The Commonwealth strategy is, however, relatively weak on intersectoral policy and regulatory measures. As an illustrative example of the contrast at the state level, implementation in NSW now ranges from school physical activity and nutrition survey, to a school canteen strategy, to negotiating with Commercial Television Australia about their code of practice on advertising in peak children's viewing hours. The Commonwealth apparently chose not to consider how it might exercise its relevant taxation or legislative powers, despite the history of health promotion pointing to the importance of public policy measures beyond the health system.
An examination of the manner in which the obesity issue was framed, and the details contained in the national strategy, raises a number of issues and questions:
-Why was framing the issues as 'obesity' more successful than the focus on 'nutrition' and 'physical activity'? Why did 'obesity' gain traction while the other terms did not?
-Why did the Commonwealth opt for the softer programmatic approach, rather than tackle obesity with stronger public policy measures (such as taxation and regulation), and demonstrate its national leadership capacity? -Was the absence of stronger public policy measures because 'obesity' is regarded as largely a health issue, rather than a whole-of-government issue? Or was the Government waiting to see if the US opposed the WHO Global Strategy on account of the strength of the industry lobby?
-After a number of years of public concern about eating disorders and whether they arise in part because of promotion of certain types of body image, was the 'obesity' label a backward step for mental health and a return to traditional images of beauty?
-Is there a risk that people, including children, who are labeled as 'overweight and obese' will be stigmatised? To what extent have the voices of affected communities been incorporated into the development of national strategies, if at all? -Given the correlation between obesity and socioeconomic disadvantage, how would the proposed strategy not exacerbate those inequalities? -Were children targeted because they are a "captive audience" and therefore easy targets or did the evidence suggest the best return on investment (in terms of health gain and managing demand on the health care system) would come from a focus on children?
-Was the move to appeal to a populist agenda, while simultaneously progressing the longer-term agenda of tackling health inequalities through multi-sectoral partnerships, a triumph for public health advocates?
These complex threads are interwoven. For the moment, the publicly enunciated agenda represents a confluence of a number of rationales.
During 2003-4 three new vaccines were added to the schedule of recommended vaccines for Australians (an additional change to the schedule, recommending that polio immunisation be changed from oral to injected (IPD) vaccine, will not be discussed here). These vaccines protect against serogroup C meningococcal disease, some strains of Pneumococcal disease, and chicken pox [17] . For the first time, not all of these recommended vaccines will be funded by Government.
Prior to the introduction of these vaccines, the quality of information about the epidemiology and burden of disease caused by these three infections was extremely variable. Meningococcal disease has been notifiable for many years, and in Australia almost all is caused by serogroups B and C. Whilst serogroup B predominantly occurs in young children, a new strain of serogroup C [18] was causing increasing anxiety amongst public health professionals, microbiologists, staff of accident and emergency departments, intensive care units and of course the public and media.
The cause of anxiety amongst health professionals was based on the fact that this new strain carried a high fatality rate with severe after-effects in a high proportion of survivors. The attack rate, although still small, was increasing exponentially each year and reaching an important trigger point, and the majority of cases were now healthy teenagers and young adults. Although an initial accelerated catch-up programme was introduced for teenagers (the major risk group), the new conjugated vaccine was also introduced to the childhood schedule at age one, as from that age, only one dose (at a cost of $30-$60) was considered necessary for full protection from serogroup C disease.
Pneumococcal disease became notifiable in 2001, however, with such a short surveillance history, not much is certain locally, epidemiologically speaking, about risk groups and effects (although there is no reason to suppose that it has a different epidemiological pattern from other developed countries). Pneumococcal disease is thought to occur at least four times as often as meningococcal disease, is known to carry major sequelae and has a high case fatality rate. For some time it has been known to be even more common amongst the indigenous Australian population with attack rates of up to 1 in 500 each year, knowledge which underpinned the 1999 decision to target Aboriginal people for free vaccination as soon as the new vaccines became available. Unfortunately at about $120 per dose, conjugate pneumococcal vaccine is very expensive and, for the protection of the very young children who bear the brunt of this disease, it is licensed only to be given as a three dose course, making provision of this vaccine to all Australian children prohibitively expensive.
Varicella, predominantly a childhood disease, is caused by a Herpes virus known as herpes virus 3 or varicella-zoster virus or VZV. It is not notifiable in Australia; therefore no epidemiological population data are available. A reliable varicella vaccine has been available since the mid 1990s in the USA and is part of American routine immunisation schedule. This vaccine became available in Australia in 2000, at a cost of about $75-$90 per dose, with two doses being required for full protection.
In 2003 the Commonwealth provided its periodic update on the Australian Standard Vaccination Schedule, the list of vaccines it provides as appropriate at no cost to all Australians [19] . For the first time it differed from the National Immunisation Program recommendations in that besides meningococcal serogroup C conjugate vaccines, pneumococcal vaccine, varicella vaccine and also inactivated polio (injected) vaccine were also recommended: however, funding was only secured for meningococcal conjugate vaccines, with a continuation of the provision of pneumococcal vaccines for indigenous children. As a result, although recommended, pneumococcal and varicella vaccines were not funded and parents would have to decide whether or not to pay for them.
These funding decisions had important implications. Vaccines protect most of their recipients from unpleasant and sometimes life-threatening disease. One view, subscribed to in the UK, is that ethically, children should not be denied access because of their parents' inability to pay. These vaccines have been the subject of several cost-benefit studies, with generally favourable to extremely favourable pro-vaccination results. Table 2 summarises the various models for framing policy.
The policy of funding meningococcal serogroup C vaccine was built on a sustained program of epidemiological evidence, ethical decision-making and public support (and was arguably honed by public pressure). Pneumococcal disease and varicella vaccination programs however, were neither supported by good local epidemiological evidence nor respectable levels of public awareness about these diseases. There had not been a similar program of sustained policy building to support or drive a decision to fund these vaccines. As a funding policy, this was noteworthy in that it marked a departure from previous policies where all recommended vaccines were fully funded by governments. National vaccination policy is designed to advise vaccination policy makers and practitioners of the most up-to-date thinking about optimal vaccination schedules for Australian children, and is not therefore proscriptive, unlike the United Kingdom (UK). Changing or adding vaccines to the recommended schedule is therefore an advisory matter, and the question of funding the vaccination program is decided separately.
Cost benefit studies indicate pneumococcal polysaccharide and conjugate vaccines can be cost-effective although vaccine costs clearly affect ratios of cost to benefit greatly [20, 21] . Varicella vaccine is more contentious, because this disease is more severe in older cases, and it is possible that one result of a vaccination program could be an increase in older cases (and therefore severe disease). Whilst the vaccine undoubtedly works, there is no consensus about precisely who should be vaccinated for maximum population health as well as cost benefit, and again potential financial savings are highly dependant upon vaccine costs [22, 23] .
The costs of preventive vaccine programs and curative medicine are funded from different sources. Vaccines are currently funded by the Commonwealth and subsidised through the states according to local vaccination policies, whilst the costs of curing cases of these diseases is broadly funded through the Medicare and private health insurance systems. Savings to Medicare and health insurance funds, as a result of successful vaccination programs, are not automatically transferred to the Commonwealth to fund the vaccine programs. Savings -or costs -in one area are of little interest or importance to other program areas.
In 2004 the Government revised this funding policy, providing funding for conjugate pneumococcal vaccines population immunisation program for all children under seven years of age (as well as specific people in other risk groups) to commence in January 2005. The Australian Technical Advisory Group on Immunisation (ATAGI) completed Ministerial reports on both varicella and polio (injected as well as oral) vaccination late in 2004, and it is possible that programs for these vaccines will also be funded in the future.
The 2002/2003 Federal Budget papers stated that "the Government is committed to making disease prevention and health promotion a fundamental pillar of the health system": however, this was not evident in the subsequent 2003/2004 budget. The Government's Focus on Prevention Package in 2002/03 aimed to incorporate disease prevention into the core business of the primary health care system and was reflective of how the public health agenda was evolving at the national level [24] . The package was comprised largely of a range of measures directed at specific diseases, plus a bundle of initiatives for general practitioners, also referred to as the "primary health care system".
Amongst health conditions affecting Australians, breast cancer received the most attention, with the National Breast Cancer Centre being funded to develop a partnership approach to the review and dissemination of new information, along with information, support and management initiatives for rural women diagnosed with breast cancer. Hepatitis C also received some attention, with funding for national education and prevention projects. Financial support was offered for the SARS efforts that had been undertaken by states and territories, in particular for providing medical personnel at international airports. A clear process for assessing priorities under the broad banded National Public Health Program was also flagged.
For purposes of the budget, primary health care was defined as general practitioners, and the measures funded included:
• "Lifestyle prescriptions" to help GPs "raise community awareness and understanding of benefits of preventive health";
• Collaborative approach to learning, training education and support systems;
• Coordinated care plans for people with chronic or terminal conditions; and
• Involvement in multidisciplinary case conferencing.
The budget did not adopt a comprehensive approach to the primary health care system, perhaps because many community health services, which represent the other important arm for delivery of public health services, are the responsibility of states. The timetable for renewing Public Health Outcome Funding Agreements (PHOFAs) between the Commonwealth and states and territories in 2004 raised in the minds of some stakeholders, the possibility that the Commonwealth might adopt a more comprehensive and strategic approach, linking public health and primary health care funding streams.
Judging by the actual quantum of funds made available in the 2003/2004 budget, it would seem that most elements from the package did not actually receive additional funding, as shown in Table 3 . Indeed, many of the GP initiatives, previously cast as improving primary health care, were subsequently packaged as 'prevention'.
The combination of these measures reflected a tight fiscal climate, with little growth in the overall health budget, as well as that of other portfolios. It was also a package that demonstrated relatively limited imagination, with support for established issues (such as breast cancer) and repackaging general practice measures that were already in train. With Medicare spending "uncapped" (and targeted public health programs "capped"), attaining more prevention dollars through the GP sector may appear to be one of the few ways to 'grow' dollars for prevention. Although this could be considered to be consistent with the Ottawa Charter of "reorienting health services", many GPs are not trained in a population-based approach to practice, and simply providing new for payments to all represents an undifferentiated, uncoordinated and untargeted approach to prevention. If there is limited support to GPs, and little monitoring, then these measures are unlikely to translate into improved health outcomes. Funding for the Tough on Drugs strategy was announced outside the Focus on Prevention package; perhaps due to Source: [28] the fact that the Tough on Drugs was the responsibility of the Parliamentary Secretary therefore requiring a separate communications strategy, or because the Prime Minister has a strong personal interest in the illicit drug strategy. The range of measures funded (which included introduction of retractable needle and syringe technology, addressing problems related to increased availability and use of psycho stimulants, establishing a research fund, supporting alcohol and drug workforce development needs, promoting access to drug treatment in rural areas, and tackling problems faced by drug users with concurrent mental health problems) certainly suggested more serious government interest and commitment to illicit drugs. 
During the course of the Howard Government, there has been a gradual process of re-casting the "landscape" of interest groups and policy constituencies. Strong support for breast cancer and zero-tolerance on illicit drugs contrasts sharply with the delays experienced in renewal of the National HIV/Hepatitis C Strategy. The new prominence given to meningococcal vaccine, child health and obesity creates space for other interest groups: even if the re-framing was shaped by nutrition and physical activity lobbies, other clinical interests have been brought into the picture. These developments illustrate how 'political' considerations are important in determining 'public health policy'.
It was interesting however, to observe the interest in prevention from outside the health portfolio, particularly from Treasury. This was motivated in part by the Intergenerational Report and concerns about both the sustainability of Medicare as well as the social and economic cost burden arising from an ageing society. This helped to ensure interest in the Abelson Report [9] .
Few countries have conducted research on return of investment from prevention efforts. Australia was praised by Derek Wanless at a high-level consultation for completing such an analysis, during his visit to Canberra while conducting a review for the UK Treasury, "Securing Good Health for the Whole Population" [26] . His final report pointed to Australia and Netherlands as two countries that were increasingly using economic evaluation in public health programs. It will be interesting to see if public health policy analysts and Treasury officials draw on this report in future years. In the future it will be interesting to see if the focus on high-visibility programs can demonstrate short-term economic returns.
Given 2004 was an election year, the "political economy" of prevention programs could arguably have become a focus of future public health policy, with the 2003/4 agenda providing the Government with the opportunity to gauge public reaction to this new positioning and design their election campaign appropriately. This was, however, not the case. The American emphasis on 'preparedness' appears not to resonate with the Australian public in the same way.
From the perspective of public health policy advocates, some lessons that can be drawn from 2003/04 are:
• Government's response to public health proposals are shaped by its understanding of the popular interest and desire to communicate directly with the general public;
• Longer term public health issues which have struggled to gain support can be progressed if they are cleverly shaped to fit the Government's "formula";
• Develop and nurture new advocates, particularly in seeking to engage with the broader health system; and
• Work with the media as partners rather than adversaries
These lessons need to be learned well and quickly, to assist with moving the forum for public health policy debate more into the public domain; beyond an essentially "in house" discourse between politicians, researchers and public health advocates. If a more engaged and informed community takes up a public health issue, government will be more likely to respond.  GIDEON: a comprehensive Web-based resource for geographic medicine GIDEON (Global Infectious Diseases and Epidemiology Network) is a web-based computer program designed for decision support and informatics in the field of Geographic Medicine. The first of four interactive modules generates a ranked differential diagnosis based on patient signs, symptoms, exposure history and country of disease acquisition. Additional options include syndromic disease surveillance capability and simulation of bioterrorism scenarios. The second module accesses detailed and current information regarding the status of 338 individual diseases in each of 220 countries. Over 50,000 disease images, maps and user-designed graphs may be downloaded for use in teaching and preparation of written materials. The third module is a comprehensive source on the use of 328 anti-infective drugs and vaccines, including a listing of over 9,500 international trade names. The fourth module can be used to characterize or identify any bacterium or yeast, based on laboratory phenotype. GIDEON is an up-to-date and comprehensive resource for Geographic Medicine. As of 2005, the world is confronted by 338 generic infectious diseases, scattered in a complex fashion across over 220 countries and regions. Each new day confronts health care workers with unexpected outbreaks, epidemics and heretofore unknown pathogens. Over 2,000 named bacteria, viruses, fungi and parasites are known to cause human disease; and are confronted by 328 anti-infective agents and vaccines. Experts working in Health Geographics share an obvious and immediate need for comprehensive and timely data on the status of infection around the globe. A recent outline of GIDEON addressed uses for the Infectious Diseases clinician [1] . This review will focus on the Global Health aspect of the program.
In 1990, we initiated a project to design computer systems to follow all diseases, outbreaks, pathogens and drugs. The initial DOS-based program was written in Paradox for floppy disks, later evolving through a compact disk-based program in Windows. A commercial web-based program was eventually released under the name, GIDEON (Global Infectious Diseases and Epidemiology ON-line, Gideon Informatics, Inc, Los Angeles, California) at http:/ /www.GideonOnline.com. The current version is available on CD (updated every three months) or web subscription (updated every week).
The program consists of four modules: Diagnosis, Epidemiology, Therapy and Microbiology. Program modules of peripheral interest in Health Geographics (Therapy and Microbiology) will be discussed only briefly.
The Diagnosis module is designed to generate a ranked differential diagnosis based on signs, symptoms, laboratory tests, incubation period, nature of exposure and country of disease origin. Figure 1 depicts the data entry screen for a patient suffering from fever and joint pain following a trip to Indonesia. The lower 'Personal notes' box is used to record additional case data, and can be written in the user's own language. The differential diagnosis list for this case (figure 2) indicates that this patient may be suffering from Chikungunya. The appearance of many diseases on the list indicates that the user failed to enter all positive, and negative findings. For example, the fact that cough was absent would have reduced the likelihood of the second disease listed (Mycoplasma infection) and increased the statistical probability of Chikungunya.
At this point, the user can generate a hard copy or e-mail report, access a table comparing the clinical features of the diseases listed, or examine the ranking or omission of specific diseases. If the user clicks on a specific disease name, clinical and epidemiological data on the disease in question are depicted (figure 3). The differential diagnosis list is generated by a Bayesian formula which compares the product of disease-incidence and symptom incidence, for all compatible infectious diseases. In the above example, a number of diseases known to occur in Indonesia were capable of producing fever, and joint pain. The statistical likelihood of Chikungunya in this case can be computed by a simple Bayesian formula, as follows: Two spreadsheets in the GIDEON database respectively follow the incidence of all symptoms for every disease, and the incidence of all diseases for every country. When a clinical case is "entered" into GIDEON, the program identifies all compatible diseases and ranks their relative likelihoods as determined by the above formula, ie: P-(C/ S) vs. P-(D2/S) vs. P-(D3/S) ... vs. P-(Dn/S).
A blinded study of 500 cases conducted by this author found that the correct diagnosis was listed in the differential list in 94.7% of cases, and was ranked first in 75% [2] . A second study of hospitalized patients in Boston found that the correct diagnosis was listed in only 69%, and was ranked first in 60% [3] . It is likely that inclusion in the differential diagnosis list may be more important than disease ranking in such systems [4] .
A "Bioterrorism" option generates the differential diagnosis for diseases associated with suspected bioterror scenarios. In Figure 4 , "<bioterrorism simulator>" has been substituted for Indonesia, given the above constellation of fever, joint pain, etc. The resulting differential diagnosis lists Ebola (42.9% probability), followed by Crimean-Data entry screen for a bioterrorism scenario Figure 4 Data entry screen for a bioterrorism scenario.
Congo hemorrhagic fever (12.6% probability). A similar "Worldwide" option can be used to explore all of the worlds diseases consistent with given clinical features, and access text on the global status for individual diseases.
In theory, data entry by users can be monitored at the server level for purposes of surveillance. For example, if one or more users in China were to enter cases of fatal pneumonia, a "red-flag" at any monitoring agency (i.e., the World Health Organization) could indicate the possible appearance of SARS -long before submission of specimens or reporting of the case to local authorities. Similarly, the appearance of multiple cases of "dysentery" by users in a given community could indicate a possible outbreak of shigellosis.
The Epidemiology module presents detailed country-specific information on the status of each disease, both globally and within each relevant country. The current version contains over two million words in 12,000 notes. All data are derived from Health Ministry publications, peer-review journals, standard textbooks, WHO and CDC websites and data presented at conferences. The user may also access over 30,000 graphs which follow disease incidence, rates and other numerical data. The main Epidemiology screen is shown in Figure 5 . Note that the user can append custom "personal notes" -in any national language or font-regarding the status of every disease in their own institution. Such notes would be accessible by all colleagues using GIDEON on the local network.
Maps which depict the global distribution of each disease can be accessed through the 'Distribution' tab ( Figure 6 ).
Epidemiology module, main screen Figure 5 Epidemiology module, main screen. The 'images' tab has been pressed, to access thumbnail images of Plague. These can be maximized and copied to PowerPoint, etc. Note addition of 'Personal notes' by the user, at lower right.
Text outlining country-specific data for the disease ( Figure  7) is available through either a list of countries displayed in this module, or by clicking the relevant 'red dot' on the map. These text boxes also include data sets which automatically generate incidence / rate graphs (Figure 8) , a chronological account of all disease outbreaks, and numbered reference links to relevant journal publications and reports of ongoing outbreaks from ProMed http:// www.promedmail.org. A separate 'Graphs' option allows the user to generate custom-made graphs comparing multiple disease rates, or rates in multiple countries. (Figure  9 ).
Additional tabs access the descriptive epidemiology and clinical background of each disease. Synonym tabs generate lists of alternative terms for diseases and countries in Spanish, German, Norwegian, etc. Historical data record the incidence of individual diseases and significant outbreaks spanning decades. An additional "Fingerprint" option generates a list of diseases compatible with any set of epidemiological parameters. For example, in Figure 10 we see that ten parasitic diseases are transmitted by fish in Japan.
The Therapy module follows the pharmacology and application of all drugs and vaccines used in Infectious Diseases. The current version contains 264 generic drugs and 64 vaccines. Various sub-modules present the mechanism of action; pharmacology, dosages, drug-drug interactions, contraindications, spectrum, and susceptibility testing standards. An international synonym lists contains over 9,500 trade names. As in other modules, users may add Epidemiology module Figure 6 Epidemiology module. Map depicting the global distribution of plague. Specific map areas can be expanded, and all elements can be copied for reproduction as necessary. Country-specific notes regarding plague appear when corresponding red dots are clicked.
custom notes in their own language for each drug or vaccine: prices, resistance patterns, local trade names, etc.
The Microbiology option is similar to the Diagnosis module. Users may enter any combination of phenotypic tests, and obtain a ranked probability list of compatible bacteria. The current version incorporates more than 1,300 taxa. The Microbiology module is also designed to list the phenotype, prior names, ecology and disease association for any organism, or compare the phenotypes of any combination of organisms selected by the user.
Since the graphic and mapping functions of GIDEON treat individual countries as whole units, data presentations lack a certain degree of "granularity." Thus, the dif-ferential diagnosis of fever in Venezuela will include malaria, even if the patient is living outside of the endemic, southern region. This problem is corrected to a large extent by text in the associated country-specific notes and the general knowledge base of the treating physician. In theory, the manufacturer could follow the incidence of each disease for every state, district, province and oblast; but variability would still exist according to occupation, rural vs. urban setting, season, etc.
An additional problem relates to the availability and quality of valid epidemiological data. Disease reporting varies widely from country to country. For example, AIDS reporting statistics from sub-Saharan Africa are generally inadequate. Where necessary, the spreadsheets used by GIDEON record published true estimates rather than questionable reports. In other instances, Health Ministry Figure 7 Plague in Tanzania. Clicking on relevant data sets will generate incidence and rates graphs. Note several numbered links to journal publications.
Plague -Worldwide incidence and rates per 100,000 Figure 8 Plague -Worldwide incidence and rates per 100,000.
data conflict with reports of the World Health Organisation, a fact which is recorded in relevant GIDEON country notes. Occasionally, major diseases are not reported at all. For example, several recent cases of cholera in Japan originated from Thailand; but Thailand has not officially reported a single case in many years. Where possible, the GIDEON data base relies on published best estimates, and at times 'educated guesses' when data are entirely lacking.
Thus, there are few published data for disease incidence in Togo, and the program is forced to rely on publications for neighboring Ghana.
The reader is referred to the GIDEON website http:// www.GideonOnline.com for an extensive listing of data sources, published reviews, technical background and pricing information.
Graph contrasting AIDS rates among user-selected countries Figure 9 Graph contrasting AIDS rates among user-selected countries.
Publish with Bio Med Central and every scientist can read your work free of charge  Globalization and Health This debut editorial of Globalization and Health introduces the journal, briefly delineating its goals and objectives and outlines its scope of subject matter. 'Open Access' publishing is expected to become an increasingly important format for peer reviewed academic journals and that Globalization and Health is 'Open Access' is appropriate. The rationale behind starting a journal dedicated to globalization and health is three fold: Firstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health – be they a SARS virus or a predilection for fatty foods – have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing. Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve. And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic. A balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first. Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve.
And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic.
A balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first.
I am pleased to introduce 'Globalization and Health', a peer reviewed, open access (free to the end user) journal. In this, the début editorial, I will briefly outline the purpose and scope of this journal highlighting our intention to publish a balanced mixture of opinion on the subject.
That the journal be 'Open Access' is entirely appropriate. Knowledge, at its best utility, is a 'public good' i.e. nonrival, non-excludable. While this journal will deal with the subject matter of creating 'global public goods for health', it will also by virtue of its very existence, contribute toward that process. Globalization and Health's 'Open Access' policy changes the way in which articles are pub-lished. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [1] . Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. Globalization and Health's articles are archived in PubMed Central [2], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [3] in Germany, at INIST [4] in France and in e-Depot [5], the National Library of the Netherlands' digital archive of all electronic publications. Importantly, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [6]. Added to this, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter [7] ).
The rationale behind starting a journal dedicated to globalization and health is three fold:
Firstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health -be they a SARS virus or a predilection for fatty foods -have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing.
Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve.
And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic.
A balanced view would propose that the effects of globalization on health (and health systems) are neither univer-sally good nor bad, but rather context specific. The extent to which individual states are able to engage the process of globalization on their own terms differs widely from one country to the next. Child mortality, for example, changes quickly in response to subtle changes in purchasing power in impoverished communities. In affluent communities however, a small change in income has little effect on utility in either direction. As we consider the effects of globalization on wellbeing it becomes apparent that we need to consider both the long term scenarios for populations as a whole, and the immediate effects for the more vulnerable within those populations who are dependent on fragile local economies.
If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first. The 'polysemous' codon--a codon with multiple amino acid assignment caused by dual specificity of tRNA identity. In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNA(Ser)CAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m1G37 is critical for leucylation activity. This finding was supported by the fact that the tRNA(Ser)CAGs possessing the leucylation activity always have m1G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNA(Ser)CAGs possessing m1G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNA(Ser)CAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a 'polysemous codon'. (termed tRNA Ser CAG), and revealed its decoding mechan-Bioscience and Biotechnology, Tokyo Institute of Technology, ism by means of an in vitro translational assay system Nagatsuta, Midori-ku, Yokohama 227, Japan (Yokogawa et al., 1992; Suzuki et al., 1994) . Furthermore, 2 Corresponding authors when we investigated the distribution of this non-universal genetic code in fungi, as well as C.cylindracea, eight other In some Candida species, the universal CUG leucine
Candida species-C.albicans, C.zeylanoides, C.lusitaniae, codon is translated as serine. However, in most cases, C.tropicalis, C.melbiosica, C.parapsilosis, C.guilliermonthe serine tRNAs responsible for this non-universal dii and C.rugosa-were found to utilize the codon CUG decoding (tRNA Ser CAG) accept in vitro not only serine, for serine instead of leucine, all having tRNA Ser CAG as but also, to some extent, leucine. Nucleotide replacethe mediator in the unusual decoding (Ohama et al., 1993 ; ment experiments indicated that m 1 G37 is critical for Ueda et al., 1994) . Several other investigators have also leucylation activity. This finding was supported by the shown that the codon CUG is actually translated as serine fact that the tRNA Ser CAGs possessing the leucylation in vivo in C.albicans and C.maltosa (Santos and Tuite, activity always have m 1 G37, whereas that of Candida 1995a; Sugiyama et al., 1995; Zimmer and Schunck, 1995) .
One of the most remarkable structural features observed A37. Quantification of defined aminoacetylated tRNAs in most of these tRNA Ser CAGs is that the nucleotide 5Јin cells demonstrated that 3% of the tRNA Ser CAGs adjacent to the anticodon (position 33) is occupied not by possessing m 1 G37 were, in fact, charged with leucine the conserved U residue (U33) but by a G residue (G33).
It has been speculated that U33 is necessary for forming of C.maltosa possessing this type of tRNA Ser CAG also the U-turn structure of the anticodon loop in all tRNAs suggested that the URA3 gene inactivated due to the reported so far (Quigley and Rich, 1976 ; Sprinzl et al., translation of CUG as serine was rescued by a slight 1996) . Moreover, the nucleotide at position 37, 3Ј-adjacent incorporation of leucine into the polypeptide, which to the anticodon CAG, is 1-methyl guanosine (m 1 G) in demonstrated that the tRNA charged with multiple almost all tRNA Ser CAGs except for that of C.cylindracea amino acids could participate in the translation. These (A37), while all the serine tRNAs in fungi corresponding findings provide the first evidence that two distinct Introduction Normanly and Abelson, 1989; Shimizu et al., 1992; McClain, 1993; Schimmel et al., 1993) . This line of study The universality of the genetic code was once considered began with the artificial conversion of leucine tRNA of to be one of the essential characteristics of life, which led Escherichia coli to serine tRNA by Abelson's group 10 to the conception of the 'frozen accident theory'. This years ago (Normanly et al., 1986) . Recently, tRNA identity theory proposes that all extant living organisms use the elements of Saccharomyces cerevisiae leucine tRNA were universal genetic code, which was born by accident and elucidated using unmodified variants synthesized by T7 'frozen', and that they originate from a single, closely RNA polymerase (Soma et al., 1996) , indicating that in interbreeding population (Crick, 1968) . However, in recent addition to the discriminator base, A73, the second letter years a number of non-universal genetic codes have been of the anticodon, A35, and the nucleotide 3Ј-adjacent to reported in various non-plant mitochondrial systems, as the anticodon, m 1 G37, are important for recognition by well as in several nuclear systems (reviewed in Osawa leucyl-tRNA synthetase (LeuRS). The majority of Candida et al., 1992; Osawa, 1995) , which contradict the frozen tRNA Ser CAGs have A35 and m 1 G37, while the discriminaccident theory.
Among these deviations from the universal codes, ator is occupied by a nucleoside other than adenosine (mostly G73). In this respect, tRNA Ser CAG seems to be a potentially chimeric tRNA molecule capable of being recognized not only by seryl-but also by leucyl-tRNA synthetases.
Previously, we showed that these tRNA Ser CAGs would have originated from the serine tRNA corresponding to codon UCG . This suggests an evolutionary pathway in which conversion from A to m 1 G would have taken place at position 37 just after the emergence of tRNA Ser CAG had brought about a change in the universal code. Since such a mutation at position 37 might potentially result in the leucylation of tRNA Ser CAG, we attempted to elucidate the charging properties of these tRNA Ser CAGs both in vitro and in vivo. Based on the results of in vitro aminoacylation reactions using tRNA variants constructed by the microsurgery method, the direct analysis of aminoacylated tRNAs in cells and a genetic approach, we demonstrate here that these serine tRNAs are actually leucylated both in vitro and in vivo. Furthermore, m 1 G at position 37 was found to be indispensable for the leucylation of tRNA Ser CAGs. In fact, the tRNA Ser CAG of C.cylindracea, which has A at position 37, exhibits no leucylation activity. C.cylindracea has a high GϩC content (63%) and utilizes CUG as a major serine codon. However, the other Candida species have no such high GϩC content and utilize the CUG as a minor serine codon (Kawaguchi et al., 1989; Lloyd and Sharp, 1992; our unpublished observation) . Considering the relationship between the usage of the codon CUG as serine and the leucylation properties of tRNA Ser CAG, it seems that only Candida species with a genome in which the incidence of the CUG serine codon is very low possess serine tRNA Ser CAG that can be leucylated. Furthermore, such tRNA Ser CAGs charged with heterogeneous amino acids should be utilized equally in the translation process. This is the first demonstration that a single tRNA species is assigned to two different amino acids in the cell. We propose designating this type of codon having multiple amino acid assignment as a 'polysemous codon'. The correlation between the dual-assignment state and the pathway of genetic code diversification is also discussed. 
and C.cylindracea (Yokogawa et al., 1992; Ohama et al., 1993 ). The numbering system and abbreviations for modified nucleotides conform Candida zeylanoides tRNA Ser CAG is leucylated to Sprinzl et al. (1996) and Crain and McCloskey (1996), respectively. in vitro (B) Time-dependent aminoacylation with SerRS or LeuRS from First the leucylation of tRNA Ser CAGs from C.zeylanoides C.zeylanoides cells. Aminoacylation reactions were carried out with and C.cylindracea was examined using LeuRS partially 0.7 µM tRNAs and with same amounts of enzyme activities calculated using cognate tRNAs. Serylation and leucylation are shown by dotted purified from C.zeylanoides, since it is known that leucine and solid lines, respectively. The right-hand frame shows the solid tRNAs of yeast have one of their identity determinants at curves from left-hand frame plotted with an enlarged ordinate. The position 37 (Soma et al., 1996) and tRNA Ser CAGs of aminoacylation of C.zeylanoides tRNA Ser CAG (s) and of C.zeylanoides and C.cylindracea have different nucleo-C.cylindracea tRNA Ser CAG (u) are compared; C.cylindracea tRNA Ser GCU (j), having no leucylation activity, is shown as a tides at this position (m 1 G and A, respectively) (Figure control. (C) TLC analysis of acetylleucyl-tRNA fragments derived 1A). Both tRNAs showed almost full serylation activity from leucylated tRNA Ser CAGs. After leucylation with [ 14 C]leucine, (~1200-1500 pmol/A 260 unit), as shown in Figure 1B . The leucyl-tRNAs were acetylated with acetic anhydride. Acetyl-tRNA Ser CAG of C.zeylanoides was evidently leucylated leucylated at all, as was the case when another species tRNA Ser CAG by gel-electrophoresis under acidic con- observed with LeuRSs from both C.cylindracea and S.cerevisiae (data not shown). of serine tRNA specific for codon AGY (Y: U or C) m 1 G37 is responsible for recognition by (tRNA Ser GCU) was employed as a control substrate leucyl-tRNA synthetase ( Figure 1B , right-hand graph). The K m value of C.zeylan-Among the tRNA Ser CAGs of several Candida species, oides LeuRS towards tRNA Ser CAG (5.0 µM) is only one that of C.cylindracea is unique because it alone possesses order of magnitude larger than that of the serylation of no leucylation capacity. A sequence comparison of these this tRNA (0.22 µM) as well as that of leucylation toward tRNAs ( Figure 1A ) prompts us to speculate that the the cognate leucine tRNAs of S.cerevisae (0.34 µM; Soma nucleotide at position 37 is strongly associated with et al., 1996) .
leucylation, because all tRNA Ser CAGs possessing leucyl-In order to verify that the leucylation activity observed ation activity have m 1 G in common, while only the for the tRNA Ser CAG of C.zeylanoides actually came from tRNA Ser CAG of C.cylindracea, which possesses no leucylthe tRNA Ser CAG itself, and not from a trace amount ation activity, has A at this position. of leucine tRNA contaminating the tRNA sample, the To examine the validity of this speculation, a series of leucylated 3Ј-terminal RNA fragment derived from leucyl-tRNA Ser CAG variants was constructed by the in vitro tRNA Ser CAG was analyzed in the following manner. 14 Ctranscription method using T7 RNA polymerase, as well leucylated tRNA Ser CAG from C.zeylanoides was first as by the microsurgery method, and the leucylation activity acetylated with acetic anhydride to prevent deacylation, of each variant was measured. When the tRNA Ser CAG of and then digested with RNase T1. The resulting 3Ј-C.zeylanoides synthesized by in vitro transcription was terminal fragment with 14 C-labeled acetylleucine was employed as a substrate, no leucylation activity was analyzed by cellulose TLC. The results are shown in detected, not even for the tRNA transcript having G37 Figure 1C . If leucylated tRNA Ser CAG were digested ( Figure 3A ). On the other hand, as shown in Figure 3A , with RNase T1, 14 C-labeled acetylleucyl-CCA should be serylation activity exceeded 1000 pmol/A 260 unit. These released as a labeled fragment ( Figure 1C , lane 3), because results strongly suggested that some nucleoside modifica-G is located at position 73 of the tRNA Ser CAG (Figure tion is necessary in tRNA Ser CAG for recognition by 1A, left-hand structure). Any contaminated leucine tRNAs, LeuRS. We thus attempted to replace the m 1 G37 of if they exist, will give some 14 C-labeled fragments larger C.zeylanoides tRNA Ser CAG with G (the variant is symbolthan the tetramer ( Figure 1C , lane 4), because all the ized as m 1 G37G) or A (m 1 G37A), by the microsurgery leucine tRNAs of yeasts so far analyzed (Sprinzl et al., method (Figure 2A and B; for details, see Materials 1996) including those of C.zeylanoides (T. Suzuki, unpuband methods) to examine the contribution of m 1 G37 to lished result) are known to have A73 at their 3Ј-ends, leucylation and the contribution of A37 of C.cylindracea which are resistant to RNase T1. The mobility of the tRNA Ser CAG to the prevention of leucylation. acetylleucyl-oligonucleotide derived from tRNA Ser CAG When aminoacylation of m 1 G37A and m 1 G37G was from C.zeylanoides ( Figure 1C , lane 1) was identical to examined ( Figure 3A ), the results indicated that both that of acelylleucyl-CCA prepared from the RNase U2 substitutions lead to complete loss of leucylation (Figure digests of leucyl-tRNA Leu s from C.zeylanoides (lane 3).
3A, right-hand graph), although no apparent influence was This observation clearly demonstrates that leucine is observed on serylation ( Figure 3A , left-hand graph). These definitely attached to the tRNA possessing G73; the tRNA findings strongly indicate that the methyl group of m 1 G37 therefore must be tRNA Ser CAG and not tRNA Leu . Thus, plays a crucial role in enhancing the leucylation activity it is concluded that the tRNA which incorporated leucine of tRNA Ser CAG. in vitro is in fact tRNA Ser CAG. This deduction is supported
The slight reduction in leucylation activity observed in by the results of an additional experiment: incorporation the control variant z-G33G ( Figure 2A ) compared with of [ 14 C]leucine into the tRNA Ser CAG sample with LeuRS native tRNA ( Figure 3A , right-hand graph) was found to was reduced by the addition of SerRS and non-labeled have resulted from the partial deacetylation of 4-acetyl serine to the reaction mixture (data not shown), which cytidine (ac 4 C) due to acid treatment of the 5Ј-half clearly indicates that the same tRNA molecule is comfragment of tRNA Ser CAG (see Materials and methods). petitively aminoacylated by these two enzymes. This is considered further in the Discussion. To conclude that tRNA Ser CAG is aminoacylated with leucine, we carried out a further experiment. The G33 acts as a modulator of leucylation tRNA Ser CAG was charged with serine and serylated In addition to m 1 G37, another unique feature of the serine tRNA Ser CAGs in these Candida species is the presence tRNA Ser CAG was separated from non-aminoacylated The effect of mutation at position 33 in these two each variant was confirmed to have been replaced as expected (shown tRNAs was found to be quite different. In the case of the by arrows).
C.cylindracea tRNA, none of the mutations at position 33 caused leucylation of the tRNA, as was observed with the native tRNA Ser CAG, and there was no reduction in of G at position 33, where a pyrimidine (mostly U) is completely conserved in usual tRNAs (Sprinzl et al., serylation activity ( Figure 3C ). In contrast, the replacement of G33 by pyrimidines in C.zeylanoides tRNA Ser CAG 1996). Since we considered it is possible that this notable feature may be in some way related to the unusual considerably enhanced the leucylation activity ( Figure 3B , right-hand graph), while no significant difference was aminoacylation characteristics described above and/or to the translation of non-universal genetic code, we examined observed in the serylation activity ( Figure 3B , left-hand graph). The kinetic parameters of leucylation for the the effect of residue 33 on the aminoacylation and transla- show the spots corresponding to acetylleucine and acetylserine as markers, respectively. (D) Analysis of acetylamino acids attached to tRNA fragments on a TLC plate. Lane 2 shows the spot corresponding to the acetylamino acids derived from the RNase T1 fragment of C.zeylanoides tRNA Ser CAG. Lanes 1 and 3 indicate the spots corresponding to acetylleucine and acetylserine, respectively. Ten micrograms of [ 14 C]acetylaminoacyl-tRNA Ser CAG from C.zeylanoides was digested with RNase T1 and developed on cellulose TLC plates under the same conditions as (C). CCA fragments with [ 14 C]acetylamino acids were scraped from the plate from which the fragments were eluted with H20 and desalted by Sep-pak C18 under the conditions described in the literature (Wang et al., 1990) .
[ 14 C]acetylamino acids discharged from the fragments were developed on TLC and visualized by an imaging analyzer (BAS-1000, Fuji Photo Systems). variants of C.zeylanoides tRNA are shown in Table I . It
Evidence for leucylation of C.zeylanoides tRNA Ser CAG in vivo is notable that the K m values of the two pyrimidine At this point, we had established that the tRNA Ser CAG mutants, z-G33U (1.4 µM) and z-G33C (1.3 µM), are of C.zeylanoides is actually able to accept leucine in vitro. clearly lower than those of the two purine mutants, z-G33A However, considering the facts that SerRS and LeuRS (6.7 µM) and z-G33G (5.6 µM). The V max value of z-G33U coexist in cells and, judging from their K m values, that (1.2 pmol/min) is 39% of that of z-G33C (3.1 pmol/min), the affinity of tRNA Ser CAG toward SerRS is one order of which could explain why z-G33U shows lower leucylation magnitude higher than that toward LeuRS, we needed to activity than z-G33C despite having nearly the same K m ascertain whether the tRNA Ser CAG of C.zeylanoides is in value ( Figure 3B , right-hand graph). Judging from the fact leucylated in vivo. For this purpose, we adopted a sequence analysis (data not shown), the slight reduction newly developed method for quantifying an individual in the leucylation of z-G33G (5.6 µM) compared with aminoacyl-tRNA in cells (Suzuki et al., 1996) . that of the native tRNA Ser CAG (5.0 µM) is probably due Aminoacyl-tRNAs separately prepared from cells of to the partial deacetylation of ac 4 C at position 12, as C.zeylanoides and C.cylindracea were immediately submentioned above. This was confirmed by the observation jected to acetylation using [1-14 C]acetic anhydride to label of a slight reduction in leucylation activity also in acidthe amino acids as well as to stabilize the aminoacylated treated native tRNA Ser CAG (data not shown). It is thus tRNAs. From each of the acetylated aminoacyl-tRNA concluded that replacement of a pyrimidine by a purine mixtures, tRNA Ser CAGs from C.zeylanoides and C.cylindat position 33 has a repressive effect on leucylation of the racea were fished out by a solid-phase-attached DNA tRNA Ser CAG of C.zeylanoides.
probe as described previously (Tsurui et al., 1994 ; Wakita The translation efficiencies of the variants with a muta et al., 1994) . A single band for each of the aminoacyltion at position 33 were also examined in a cell-free tRNAs was detected by staining ( Figure 4A ) with which translation system of C.cylindracea (Yokogawa et al., the radioactivity coincided in each case ( Figure 4B ). 1992; Suzuki et al., 1994) , to evaluate the effect of G33.
Acetylated amino acids attached to these tRNAs were A change from G to U at position 33 apparently enhanced deacylated by alkaline treatment and analyzed by TLC. the translation activity 2.5-fold, although their decoding As shown in Figure 4C , acetylserine was observed as a properties did not change at all (data not shown). We thus major amino acid derivative in both tRNA Ser CAGs, but consider that G33 serves as a modulator of leucylation of acetylleucine was detected only in the C.zeylanoides tRNA Ser CAG, despite a slight disadvantage in transla-tRNA Ser CAG; the acetylserine and acetylleucine spots were identified as described previously (Suzuki et al., tion activity. 1996) . The radioactivities remaining on the origins probably came from the direct acetylation of some nucleotides in the tRNAs, as discussed previously (Suzuki et al., 1996) . From comparison with the radioactivity of acetylserine, it was calculated that~3% of the tRNA Ser CAG was attached with acetylleucine. These results were reproducible.
Digestion of purified acetyl-aminoacyl tRNA Ser CAG with RNase T1 also gave only a 14 C-labeled CCA fragment, as shown in Figure 1C . When the acetylated amino acid released from the fragment purified from the corresponding spot on TLC was analyzed by TLC, the ratio of acetylleucine to acetylserine was also found to be 3% ( Figure  4D ), indicating that acetylleucine is covalently attached to the tRNA Ser CAG fragment with G73. It thus became clear that the tRNA Ser CAG of C.zeylanoides was in fact charged with leucine by 3% of the amount of serylation of the same tRNA Ser CAG in C.zeylanoides cells.
Aminoacylation has generally been considered to be the final stage determining translational accuracy (reviewed in Parker, 1989; Kurland, 1992; Farabaugh, 1993) . However, in the case of tRNA Gln charged with glutamate in the chloroplast, Glu-tRNA Gln is rejected by an elongation factor so that the chloroplast translation machinery does not employ the mischarged aminoacyl-tRNA (Stanzel et al., 1994) . It is likely that this is an exceptional case due to the lack of glutamyl-tRNA synthetase in the chloroplast.
In order to prove that leucylated tRNA Ser CAGs actually participate in the translation process in Candida cells without such a rejection mechanism, we utilized a URA3 gene expression system derived from S.cerevisiae in C.maltosa, which was developed by Sugiyama et al. (1995) . Candida maltosa utilizes the codon CUG as serine and possesses the relevant tRNA Ser CAG gene (Sugiyama et al., 1995; Zimmer and Schunck, 1995) . Since the et al., 1995) . In the present study, this URA3 gene, with the CTG codon replaced by various leucine or serine codons, was utilized as a marker gene ( Figure 5A ). First, ADE1/ura3::C-ADE1) (Ohkuma et al., 1993) , the growth of which was monitored on minimal medium SD plates a plasmid in which the S.cerevisiae URA3 gene was inserted downstream of a C.maltosa-specific promoter in the presence and absence of uracil. When uracil was supplied to the SD plate for the (C-p) was constructed and designated as pCSU-CTG (Sugiyama, 1995) . As controls, mutant plasmids of pCSU-positive control experiments, all the transformants grew normally ( Figure 5B , middle row). However, in the absence CTG, in which the codon CTG was replaced by either the serine codon TCT or the leucine codon CTC, were of uracil, cells harboring pCCU and pCSU-CTC showed normal growth, whereas no growth was observed in those constructed and named pCSU-TCT and pCSU-CTC, respectively. In addition, a plasmid (pCCU) consisting of harboring pCSU-TCT and pUTH18 that contained no URA3 gene insertion. Cells harboring pCSU-CTG showed the URA3 gene of C.maltosa having a CTT leucine codon at the corresponding site, combined with the C.maltosa-weak but significant growth ( Figure 5B, uppermost row) . These results demonstrate that if the codon at position 45 specific promoter, was also used as a positive control. These variant plasmids were introduced into a URA3-is translated as leucine, active ODCase will be produced and the cells will be able to grow, but translation of the defective C.maltosa strain CHU1 (his5, ade1, ura3::C-codon with serine will produce inactive ODCase and the We believe that tRNA Ser CAG is the only molecule responsible for the leucine insertion corresponding to cells will be unable to grow. The result with cells harboring pCSU-CTG clearly demonstrates that the URA3 mutation codon CUG in C.maltosa cells, based on the following obervations. We have purified and sequenced a number on the C.maltosa chromosome was in some way complemented by the introduced pCSU-CTG plasmid, suggesting of leucine and serine tRNAs from Candida species, in which codon CUG is translated as serine, and failed in that the CTG codon was read at least partially as leucine in C.maltosa cells possessing tRNA Ser CAG.
finding tRNA with the anticodon sequence potentially complementary to codon CUG other than tRNA Ser CAG In order to quantify the growth rate of the cells harboring pCSU-CTG, the viability of the cells was examined in (Yokogawa et al., 1992; Ohama et al., 1993; Suzuki et al., 1994; Ueda et al., 1994; our unpublished observation) . liquid medium without uracil. As shown in Figure 5C , whereas translation of the CTG codon as serine completely Futhermore, tRNA genes for serine and leucine from these Candida species were sequenced following the blocked cell growth in the case of pCSU-TCT, and full complementation was observed in the case of pCSU-CTC amplification by cloning and/or PCR methods, and we found that only tRNA Ser CAG is able to translate codon in which the CTC codon was read as leucine, intermediate cell growth was observed in the case of pCSU-CTG, CUG (Yokogawa et al., 1992; Ohama et al., 1993; Suzuki et al., 1994; Ueda et al., 1994 ; our unpublished observ-indicating that ODCase was expressed in an active form, albeit at a low level, when there was a slight incorporation ation). Thus, it could be concluded that only the tRNA Ser CAG species inserts leucine into polypeptide of leucine at the CTG codon. The slow growth of the cells harboring pCSU-CTG was not due to the spontaneous corresponding to codon CUG. reversion of the CTG codon to another leucine codon or due to any other mutation, because the cells harvested Discussion from the colony on the SD-plate show the same growth phenotype. These results are unlikely to reflect the different The observations presented here clearly demonstrate that, in certain living organisms, a single codon can be simul-expression levels of the URA3 gene variants because the URA3 mRNA level is not altered by mutations at position taneously assigned to two distinct amino acids. Most codons in the genetic code degenerate, but our findings 45 (Ohkuma, 1993) . Furthermore, the possibility that the URA3 gene with CTG at position 45 is translated more show that some amino acids are also able to degenerate with respect to a particular codon. Such codon ambiguity efficiently than the gene with TCT at the same site due to codon preference (Ikemura, 1982) is excluded by the is governed by a tRNA acceptable to two amino acids simultaneously, as described above. We propose to desig-fact that the TCT codon is the most preferred of all the serine codons, including the CUG codon, in C.maltosa nate a codon corresponding to multiple amino acids a 'polysemous codon'. (Sugiyama et al., 1995) .
ODCase activity resulting from the translation of the A high degree of accuracy in tRNA aminoacylation has been considered crucial for preserving fidelity in protein URA3 gene was examined in the presence of a pyrimidine analog, 5-fluoroorotic acid (5FOA), an inhibitor in synthesis. It has been established that aminoacyl-tRNA synthetase is able to discriminate precisely its cognate pyrimidine biosynthesis. Incorporation of 5FOA with ODCase results in the formation of 5-fluorouridylate, amino acid from other structurally related amino acids at the adenylation reaction step, and its cognate tRNAs from which is harmful to cell propagation (Boeke et al., 1984) . Thus, URA3-defective strains grow normally on a medium non-cognate ones (reviewed in Parker, 1989; Kurland, 1992) . The misacylation error in this process has been containing 5FOA, whereas cells possessing the active URA3 gene are unable to grow on this medium. Cells estimated to range between 10 -4 and 10 -5 (Lin et al., 1984; Okamoto et al., 1984) . Discrimination of cognate harboring the respective plasmids were cultivated in the presence of 5FOA in addition to uracil. tRNA from non-cognate tRNAs is mediated by positive and negative identity determinants localized on the tRNA As shown in the bottom row of Figure 5B , cells harboring pCSU-CTG exhibited similar growth on the molecule (Yarus, 1988; Normanly and Abelson, 1989) . The only exception reported so far is that tRNA Gln is agar plate to those with pCSU-TCT and pUTH18, although the transformants with pCSU-CTC and pCCU were unable aminoacylated with glutamate in Gram-positive bacteria and in some organelles (Lapointe et al., 1986 ; Schön to grow. These results indicate that the CTG codon at position 45 was mainly translated as serine in C. maltosa, et al., 1988) . However, this differs from misaminoacylation in that this process is indispensable to compensate for the so as to produce the inactive ODCase. However, when the liquid medium was supplied with 5FOA, a slight lack of glutamyl-tRNA synthetase in these organisms. In general, high fidelity in the aminoacylation process is reduction in the growth rate was observed in the case of pCSU-CTG, compared with pCSU-TCT ( Figure 5C ), considered to be indispensable for translating genes into functionally active proteins with a high degree of accuracy. while very slow growth was observed in the case of pCSU-CTC used as a control. In order to detect a low
The discovery of a polysemous codon in a Candida species contradicts the established notion of aminoacyl-level of ODCase activity arising from a slight incorporation of leucine at the CUG codon in the 45th position, we ation with high fidelity. We have shown that a single tRNA is acceptable to two different amino acids, and adjusted the ratio of 5FOA and uracil as shown in Materials and methods. This growth rate reduction clearly suggests that it can therefore transfer two different amino acids corresponding to a particular codon. The expression that the slow growth observed in the SD medium was due to low expression of active ODCase. Thus it is concluded experiment using the ODCase-encoding URA3 gene containing codon CUG at the site essential for its activity that the CUG codon is partially translated as leucine in C.maltosa cells.
(see also Sugiyama et al., 1995) suggested that leucine could be incorporated into the gene product corresponding on experiments using an artificial mutation, and it does not reflect experimental observation in an extant living to codon CUG in C.maltosa, as judged from the complementation tests with the URA3 mutation. Although the organism.
On the basis of peptide sequences, several research amount of leucine incorporated per CUG codon was not quantitatively determined, it is clear that the incorporation groups have reported that codon CUG corresponds only to serine in C.maltosa (Sugiyama et al., 1995) and was mediated by the leucyl-tRNA Ser CAG. We thus concluded that codon CUG was simultaneously assigned to C.albicans (Santos and Tuite, 1995a; White et al., 1995) . No leucine-inserted peptide was detected in these studies. serine and leucine in the normal translation process in C.maltosa. A quantitative analysis of the amino acids However, we consider that any peptide with a leucine which was inserted for the codon CUG might have been attached to the tRNA indicated that 3% of tRNA Ser CAG is leucylated in C.zeylanoides cells. Such a high level of missed during purification or was undetectable in the peptide sequencing, because the amount of leucine-inserted leucylation is far beyond conventional misacylation, whose rate is estimated to be less than 10 -4 . Unless a proofreading peptide (~3%) would have been too low to be positively identified in sequencing experiments. mechanism exists on the ribosome, incorporation of leucine at CUG codon sites may reflect the relative ratio of
We have shown that tRNA Ser CAG in Candida species is a chimera of tRNA Ser CAG and tRNALeuCAG in so far tRNA Ser CAG leucylation, which is two orders of magnitude higher than that of conventional mistranslation.
as it is the substrate for both SerRS and LeuRS. The K m value for LeuRS is 5.0 µM, which is only one order of To date, artificial manipulations of molecules participating in the translation process, such as the overproduction magnitude larger than that for SerRS (0.22 µM). In an in vitro aminoacylation experiment Ͼ30% of tRNA Ser CAG of aminoacyl-tRNA synthetase (Swanson et al., 1988) , mutations of tRNAs etc. and/or control of growth condi-subjected to the reaction could be converted to leucyl-tRNA Ser CAG using an increased amount of LeuRS and a tions, such as deprivation of amino acids in the medium (Edelmann and Gallant, 1977; O'Farrell, 1978; Parker and longer incubation time (data not shown). We observed that while the presence of SerRS and non-radioactive Precup, 1986), have been found to increase the error rate in translation (reviewed in Parker, 1989) . However, our serine reduced leucylation, complete loss of leucylation could not be achieved (data not shown), indicating that the observation is based on experiments using wild-type cells grown in a rich medium suitable for high viability. In affinity of LeuRS toward tRNA Ser CAG is relatively high. In proliferating cells of C.zeylanoides, the leucyl-these respects, the polysemous codon is a phenomenon completely different from these artificial translational tRNA Ser CAG in the cells was estimated to be 3% of the seryl-tRNA Ser CAG, which is much lower than that errors. It is known that many examples exist for alternative decoding of universal codons-initiation codons other obtained in the in vitro experiments. We consider that such a reduction in leucylation is due to the competition than AUG (Gold, 1988; Kozak, 1983) , leaky stop codons caused by nonsense suppresser or native tRNAs (Murgola, for the tRNA Ser CAG between SerRS and LeuRS in the cells. Despite this competition, the distinct detection of 1985), the UGA codon used for incorporation of selenocysteine (Leinfelder et al., 1988) and so on. However, leucylated tRNA Ser CAG in vivo supports the existence of an ambiguous aminoacylation reaction toward the single because of strong dependence on the context effects or possible secondary structures of mRNAs, these recoding tRNA Ser CAG species. The polysemous codon results from the coexistence of events are those which are programed in the mRNAs (Gesteland et al., 1992) . We have sequenced several genes tRNA identity determinants for serine and leucine in a single tRNA molecule. Construction of tRNA Ser CAG in Candida genomes, but we could not find any secondary structure around the codon CUG in these genes. Con-variants by the microsurgery method led to the finding that a single methyl moiety of m 1 G at position 37 sidering that the polysemous codon is mediated by a single tRNA, it is unlikely that a polysemous codon occurs under is involved in the leucylation process. In contrast, the tRNA Ser CAG of C.cylindracea, which has A at the same the influences of the neighboring regions in mRNAs. Alternative decoding of a polysemous codon CUG is position, is deprived of such leucine-accepting activity. Himeno and his co-workers noted that three nucleotides possible, assuming that LeuRS is overexpressed under a certain physiological condition. Depending on the of leucine tRNAs were strongly recognized by S.cerevisiae LeuRS using unmodified variants transcribed by T7 RNA increased amount of the LeuRS in cells, incorporation of leucine corresponding to codon CUG may occur fre-polymerase (Soma et al., 1996) . Although the discriminator base, A73, is the strongest recognition site among them, quently, which causes the production of polypeptides with new functions. This possibility should be examined in A35 and G37 in the anticodon loop also play roles as determinants in tRNA. They were able to compare the further experiments.
The idea of a polysemous codon also differs from the activities of variants mutated at position 37 with A or G using the variants with A at the discriminator position 'near-cognate' concept proposed by Schultz and Yarus (1994) . They claimed that ambiguous decoding may occur which effectively elevates leucylation activity. In our work, we utilized serine tRNA with a modified nucleoside as a consequence of an irregular codon-anticodon interaction induced by the 27-43 base pair at the anticodon and with G at the discriminator position as a substrate for LeuRS, because the T7 transcript of tRNA Ser CAG showed stem of the tRNA, resulting in a genetic code change transition state. The polysemous codon found in our study no activity for leucylation. Our experiments using microsurgery methods indicated that m 1 G is of great importance is caused by the tRNA aminoacylation process of tRNA with codon-anticodon interaction proceeding precisely in in leucylation, despite the fact that the presence of G at the discriminator position is unsuitable for the recognition the conventional manner . Furthermore, since the hypothesis of Schultz and Yarus is based of LeuRS. Some modified nucleotides in tRNA are known to be involved in recoginition of some synthetases (Muramatsu et al., 1988) . Pütz et al. (1994) showed that m 1 G at position 37 of yeast tRNA Asp is one of the negative determinants for arginyl-tRNA synthetase.
We have also demonstrated that the nucleotide at position 33, where only tRNA Ser CAG uniquely possesses G, modulates the leucine-accepting activity. G33 may prevent tRNA Ser CAG from excessive leucylation, which S.cerevisiae cells, but that the viability of the cells decreased substantially. This finding suggests the polysemous state may be tolerated only when the ambiguous recognizes its cognate leucine tRNA from the 3Ј-side of the anticodon loop, which is afforded by the uridine-turn translation is under a strict constraint. We consider that G33 functions as a negative modulator in the leucylation structure due to U33 ( Figure 6A ). The methyl moiety of m 1 G37 is directly recognized by LeuRS. In the case of tRNA Ser CAG, thereby controlling the relative seryl-to leucyl-tRNA Ser CAG ratio.
of C.zeylanoides, the anticodon loop distorted by G33 decreases the affinity toward LeuRS, judging from the Several lines of experiment have suggested that U33 is involved in the tRNA function on ribosomes, such as in observation that G33 increased the K m value for leucylation approximately 4-to 5-fold in comparison with that with rigid codon-anticodon interaction, proper GTP hydrolysis of the ternary complex and the efficient translation of prymidine bases at the position ( Figure 6B ). In C.cylindracea, m 1 G is replaced by A, which means that the tRNA termination codons (Bare et al., 1983; Dix et al., 1986) . Indeed, the replacement of G33 by U in C.cylindracea has lost the two major determinants for LeuRS, m 1 G and the discriminator base ( Figure 6C ). Consequently, LeuRS tRNA Ser CAG increased the efficiency of in vitro translation by 2-to 3-fold (data not shown). The negative effect of is unable to recognize tRNA Ser CAG at all, and G33 concomitantly loses its function as a modulator. LeuRS G33 on translation may indicate involvement in some mechanism for decoding the polysemous codon. This is, of course, unable to recognize other serine isoacceptor tRNAs corresponding to universal codons, because they possibility needs to be clarified by further study. Nevertheless, we have shown here that one of the roles of G33 is have modified A at position 37. How did this interaction between LeuRS and the suppression of leucylation, and we consider that the nucleotide at position 33 is not directly involved in tRNA Ser CAG evolve? Candida species utilizing CUG as serine can be classified into two distinct groups: group 1 recognition by LeuRS. On the basis of our observation that no leucylation was detectable in the C.cylindracea contains the species that have tRNA Ser CAG with leucylation activity, and includes C.zeylanoides, C.maltosa and tRNA Ser CAG variants in which G33 was replaced by a pyrimidine base (c-G33U and c-G33C), we speculate that others (see Figure 6B ); group 2, which is represented solely by C.cylindracea, contains species that have tRNA Ser CAG G33 influences the location and/or conformation of m 1 G37, accompanied by the alteration of the anticodon loop without leucylation activity ( Figure 6C ). A plausible evolutionary process is that group 1 would have arisen structure, decreasing the affinity of LeuRS toward tRNA Ser CAG.
prior to group 2 after the genetic code change, which is speculated on the basis of the following observations. It has been generally considered that reconstructed tRNA does not lose its activity during the several reaction First, the homology between tRNA Ser CAGs in group 1 and its isoacceptor tRNAs for codon UCG is higher than steps needed in the microsurgery method, such as cleavage of the tRNA strand and ligation of tRNA fragments that between the tRNA Ser CAG from C.cylindracea and its isoacceptor . Second, C.cylindracea (Ohyama et al., 1985) . However, a slight reduction of leucylation activity was observed in the control variant, (group 2) possesses high copy numbers of the tRNA Ser CAG genes (~20 copies) on the diploid genome (Suzuki z-G33G, compared with that of the native tRNA ( Figure  3A , right-hand graph), which turned out to result from et al., 1994) , while low copy numbers (two or four copies) are observed for group 1 tRNA Ser CAG genes (Santos the partial deacetylation of 4-acetyl cytidine (ac4C) due to acid treatment of the 5Ј-half fragment (see Materials et al., 1993; Sugiyama et al., 1995; T.Suzuki, personal observations) . Third, the codon CUG is utilized as a major and methods). Nevertheless, it is reasonable to deduce the effect of base replacement on the aminoacylation activity serine codon on several genes in C.cylindracea, such as lipase (Kawaguchi et al., 1989) and chitin synthase by comparing the activities of these reconstructed tRNAs, because the same 5Ј-half fragments were used for all the (unpublished results) , while CUG appears infrequently on the genomes of other species belonging to group 1 (Lloyd manipulated tRNA molecules of C.zeylanoides.
A plausible mechanism by which LeuRS could recog-and Sharp, 1992; Sugiyama et al., 1995; T.Suzuki, personal observations) . During the course of the change in the nize cognate leucine and serine tRNAs specific for codon CUG is illustrated in Figure 6 . LeuRS contacts and genetic code, the genome should pass through a state group 2 (Ohama et al., 1993) . Fourth, the phylogenetic dextrose) and minimal medium SD [0.67% yeast nitrogen base without tree of these species and relatives constructed by using amino acids (Difco) and 2% dextrose] supplied with 24 mg/ml uracil were used for the cultivation of yeast cells.
several genes also supports this evolutionary pathway SD-plates with or without uracil were prepared by adding agar at a (manuscript in preparation (Boeke et al., 1984) .
for codon UCG Pesole et al., 1995) . Thus, the nucleotide at position 37 seems likely to have
In order to introduce mutation at the 45th codon in the reading frame mutated in the direction modified A→m 1 G- Alternative splicing generates a multiple protein pUTH18 containing an autonomously replicating sequence of C.maltosa (Takagi et al., 1986) and C-HIS5 (Hikiji et al., 1989) were used as sequence from a single gene at the mRNA level. In when the codon appears infrequently, as observed in group was mutated from CTG to CTC, and pCCU (Sugiyama et al., 1995) synthesis caused by a polysemous codon. We speculate instruction manual. The electrified cells were spread on a SD-plate that such ambiguity could have given rise to proteins containing uracil and incubated at 30°C.
with multiple amino acid sequences in non-house-keeping genes, which may have conferred multifunctionality on
In vitro aminoacylation assay Seryl-or leucyl-tRNA synthetases were partially purified from C.zeylan-the proteins. Since the C.cylindracea strain was developed oides cells as described previously , both of the industrially for the production of lipase, such multifunc- Large-scale purification of tRNA Ser CAGs from C.zeylanoides and C.cylindracea mmol) and leucine (11.5 MBq/mmol) were from Amersham. 5-fluoroorotic acid monohydrate (5FOA) was from PCR inc. 3Ј-Biotinylated Candida cylindracea cells (3.1 kg) were treated with phenol, from which 150 000 A 260 units of unfractionated tRNA were extracted. Eighty DNA probes were synthesized by Sci. Media, Japan. Synthetic RNA oligomers and a chimeric oligonucleotide composed of DNA and 2Ј-O-thousand A 260 units of tRNA mixture were obtained by DEAE-cellulose chromatography with stepwise elution, which was then applied onto a methyl RNA were synthesized by Genset Co. Ltd. Most of the enzymes used for the microsurgery were from Takara Shuzo (Tokyo, Japan).
DEAE-Sephadex A-50 column (6ϫ100 cm). Elution was performed with a linear gradient of NaCl from 0.375 to 0.525 M in a buffer Other chemicals were obtained from Wako Chemical Industries.
consisting of 20 mM Tris-HCl (pH 7.5) and 8 mM MgCl 2 . The fraction as CZE-37 (5ЈGmCmCmCmAmAmUmGmGmAmAmdCdCdTdG-CmAmUmCmCmAmUm3Ј), possessing a cleavage site between posi-rich in tRNA Ser was applied onto a RPC-5 column (1ϫ80 cm) and eluted with a linear gradient of NaCl from 0.4 to 1 M NaCl in a buffer tions 37 and 38 of C.zeylanoides tRNA Ser CAG. Two hundred micrograms of purified tRNA Ser CAG from C.zeylanoides was incubated at 65°C for consisting of 10 mM Tris-HCl (pH 7.5) and 10 mM Mg(OAc) 2 . As a result of these chromatographies, 300 A 260 units of purified tRNA Ser CAG 10 min with 14.4 nmol CZE-37 in a buffer consisting of 40 mM Tris-HCl (pH 7.7), 0.5 mM NaCl, 0.1 mM DTT, 0.0003% BSA and 0.4% were finally obtained.
One hundred and fifty thousand A 260 units of tRNA from C.zeylanoides glycerol (500 µl), and then annealed at room temperature. Magnesium chloride was added to the mixture up to a final concentration of 4 mM cells (3.7 kg) were fractionated on DEAE-Sepharose fast-flow column (3.5ϫ130 cm) with a linear gradient of NaCl from 0.25 to 0.4 M in a and the reaction was carried out at 30°C for 2 h by the addition of 600 units of RNase H (Takara Shuzo). About 60 µg of the cleaved 3Ј-half buffer consisting of 20 mM Tris-HCl (pH 7.5) and 8 mM MgCl 2 . About 300 A 260 units of C.zeylanoides tRNA Ser CAG were finally obtained by fragment was obtained by purification using 10% PAGE containing 7 M urea. Either of two synthetic oligo-RNAs, pCAGAp or pCAGGp, was further column chromatography with Sepharose 4B in a reverse gradient of ammonium sulfate from 1.7 to 0 M with a buffer consisting of ligated with the same 5Ј-half fragment digested by RNase T1 as the variants mutated at position 33 under the conditions described above. 10 mM NaOAc (pH 4.5), 10 mM MgCl 2 , 6 mM β-mercaptoethanol and 1 mM EDTA.
The ligated and dephosphorylated 5Ј-half fragments were annealed and ligated with the 3Ј-half fragment digested by RNase H. About 50 µg of each of the two variants from C.zeylanoides mutated at position 37-Construction of tRNA variants with mutation at position 33 The microsurgery procedures were basically carried out according to the m 1 G37A and m 1 G37G-was obtained by the phosphorylation of the 5Јend and purification by 12% PAGE containing 7 M urea. literature (Ohyama et al., 1985 (Ohyama et al., , 1986 . Limited digestion of 4 mg purified tRNA Ser CAG from C.zeylanoides with RNase T1 was performed at 0°C for 30 min in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), Identification of amino acids attached to tRNA Ser CAGs in 100 mM MgCl 2 , 0.5 mg/ml of the tRNA and 25 000 units/ml RNase the cells T1 (Sigma). After phenol extraction, the resulting fragments were treated Identification of aminoacyl-tRNA Ser CAG from Candida cells was carried with 0.1 N HCl at 0°C for 12 h in order to cleave the 2Ј, 3Ј cyclic out by a new method developed recently by us (Suzuki et al., 1996) . phosphate of the 3Ј-end of the fragments formed in the limited digestion,
The experimental conditions were the same as those reported. To and then the 5Ј-and 3Ј-half fragments were separated by 10% PAGE fish out the aminoacyl-tRNAs, we designed two 3Ј-biotinylated DNA containing 7 M urea (10ϫ10 cm). Four hundred and thirty micrograms probes: 5ЈAGCAAGCTCAATGGATTCTGCGTCC3Ј for C.cylindracea of the 5Ј-half and 520 µg of the 3Ј-half fragments were recovered from tRNA Ser CAG and 5ЈGAAGCCCAATGGAACCTGCATCC3Ј for the gel. The purified 5Ј-half fragment was dephosphorylated with C.zeylanoides tRNA Ser CAG. These probes were immobilized with strepbacterial alkaline phosphatase (Takara Shuzo), and G33 at the 3Ј-end of tavidin agarose (Gibco BRL) as reported previously (Wakita et al., 1994) . the 5Ј-half fragment was removed by oxidation with sodium periodate as described in the literature (Keith and Gilham, 1974) . After dephos- A universal BMV-based RNA recombination system—how to search for general rules in RNA recombination At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication. RNA recombination is a very common phenomenon. It has been observed in all types of viruses using RNA as a carrier of genetic information: in positive-sense, single-stranded RNA viruses (1) (2) (3) (4) , in negative-sense, single-stranded RNA viruses (5, 6) , in double-stranded RNA viruses (7, 8) and in retroviruses (9) (10) (11) . Moreover, it has been shown that RNA recombination enables the exchange of genetic material not only between the same or similar viruses but also between distinctly different viruses (12) . Sometimes it also permits crossovers between viral and host RNA (13) (14) (15) (16) (17) . Taking into account the structure of viral genomic molecules and the location of crossover sites, three basic types of RNA recombination were distinguished: homologous, aberrant homologous and non-homologous (3, 4, 18) . The former two occur between two identical or similar RNAs (or between molecules displaying local homology), while the latter involves two different molecules. Most of the collected data suggest that RNA recombinants are formed according to a copy choice model (4, 18) . A viral replication complex starts nascent RNA strand synthesis on one template, called RNA donor and then switches to another template, called RNA acceptor. Accordingly, two main factors are thought to affect RNA recombination: the structure of recombining molecules and the ability of the viral replicase to switch templates.
To gain more knowledge of the mechanism of RNA recombination, several model experimental systems have been created. They provided us with some specific data describing homologous and/or non-homologous recombination in particular viruses, e.g. in poliovirus, (19) mouse hepatitis virus (20, 21) , brome mosaic virus (BMV) (4, 22) , turnip crinkle virus (23, 24) or tomato bushy stunt virus (25) . As a result, the involvement of viral replicase proteins in recombination has been demonstrated (26, 27) and a wide spectrum of RNA motifs supporting recombination have been identified (4, 23, (28) (29) (30) . In general, the collected data suggest that there exist two major types of RNA structural elements that induce recombination events: (i) universal ones mediating template switching by different viral replicases, e.g. regions *To whom correspondence should be addressed. Tel: +48 61 8528503; Fax: +48 61 8520532; Email: marekf@ibch.poznan.pl The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org of local homology (28, 31) or complementarity (32) (33) (34) (35) and (ii) virus-specific ones, e.g. promoter-like structures (36, 37) . Unfortunately, up till now there has been no in vivo recombination system that could be used to test the recombination activity of any given RNA sequence and consequently to verify the above hypothesis and find some general laws governing the studied process.
In our studies on genetic RNA recombination we have used the well-characterized in vivo system developed in BMV (30, 33) . BMV is a model (+)RNA virus of plants (38) . Its genome is composed of three segments called RNA1, RNA2 and RNA3. RNA1 and RNA2 encode BMV replicase proteins 1a and 2a, respectively. RNA3 encodes movement (3a) and coat proteins (CP) (38) . All three BMV RNAs possess an almost identical 3 0 -untranslated region (3 0 -UTR). The first BMV-based recombination system was created by Nagy and Bujarski (33) . They constructed a recombinationally active BMV mutant whose genome is composed of wtRNA1, wtRNA2 and modified RNA3 (PN0-RNA3 called the recombination vector, for details see Figure 1 ). Only 3 0 -UTR was modified in PN0-RNA3, while its 5 0 -UTR, intergenic and coding regions were unchanged. Despite the introduced changes, the recombination vector is stable and replicates when used together with wtRNA1 and wtRNA2 to infect plants. It starts to recombine if a recombinationally active sequence (RAS) is introduced just between the CP coding sequence and the modified 3 0 region (into the RAS-cloning site). Non-homologous recombination was observed when a 140-60 nt sequence complementary to RNA1 between positions 2856 and 2992 was inserted into PN0-RNA3 (a sequence from the 3 0 -portion of RNA1 was introduced in antisense orientation) (30, 33) . Interestingly, the same RNA1 fragment inserted in sense Figure 1 . The BMV-based recombination system. White, black and gray boxes represent coding, noncoding and recombinationally active sequences, respectively. The location of the primers (A and B) used for specific RT-PCR amplification of the 3 0 -portion of BMV RNA3 (parental or recombinant) is indicated by arrows. (A) BMV genome. The BMV genome consists of three RNA segments: RNA1, RNA2 and RNA3. All three BMV RNAs share an almost identical 3 0 -noncoding region with a tRNA-like structure at the very end. (B) Recombination vector. The PN0-RNA3 vector is a wtRNA3 derivative with a modified 3 0 -noncoding end [(for details see ref. (33) ]. The latter includes (i) the RAS cloning site, (ii) a 197 nt sequence derived from the 3 0 -noncoding region of cowpea chlorotic mottle virus RNA3 (marked as CCMV), (iii) the sequence of wtRNA3 between nt 7 and 200 (counting from the 3 0 end-marked as the region B) and (iv) the last 236 nt from the 3 0 end of BMV wtRNA1 (marked as A). (C) Non-homologous recombination. Non-homologous recombination was observed if an 140 nt sequence, called RAS1as (shown in A), complementary to wtRNA1 between positions 2856 and 2992 was inserted into the PN0-RNA3 vector. The presence of the RAS1as sequence in RNA3 derivative (called Mag1-RNA3) allows local RNA1-RNA3 hybridization that mediates frequent non-homologous crossovers. It is thought that polymerase starts nascent strand synthesis on the 3 0 end of RNA1 and then switches to RNA3 within a local double-stranded region. (D) Non-homologous recombinant. Non-homologous recombination repairs Mag1-RNA3 by replacing its modified 3 0 end with the 3 0 -noncoding fragment coming from RNA1. orientation did not support homologous crossovers (33) . Non-homologous recombination repaired the RNA3 vector by replacing its highly modified 3 0 end with 3 0 -UTR derived from RNA1. The resultant recombinants replicated and accumulated better than the parental RNA3 molecule, and so the latter was out competed from the infected cells. The above system is extremely efficient, since it employs selection pressure to support the accumulation of RNA3 recombinants. Because RAS is placed in two different segments of the BMV genome, we have proposed to name this system heteromolecular.
Unfortunately, Nagy and Bujarski's BMV-based recombination system has one serious limitation. It was designed in such a manner that viable RNA3 recombinants can easily form only if a sequence derived from the 3 0 -portion of RNA1 or RNA2 is used as a RAS. Consequently, the heteromolecular system could not be applied for testing the recombination capacity of various RNA motifs. Olsthoorn et al. (39) attempted to solve that problem by inserting examined sequences into the 3 0 -noncoding region of BMV RNA2 and RNA3. This system was not further developed, since any changes in RNA2, which encodes BMV polymerase, could strongly affect the studied process.
Here, we describe a new BMV-based recombination system. It has been constructed in such a way that both tested RASes are placed in the same segment of the BMV genome (in the modified RNA3 molecule); therefore, we have called this system homomolecular. To prove the usefulness of the homomolecular system, we have employed it to examine the recombination activity of sequences derived from the hepatitis C virus (HCV) genome. The examined sequences have been inserted into RNA3 as direct or inverted repeats. This demonstrated that the 101 nt hypervariable region of HCV efficiently supports both homologous and non-homologous crossovers, while the most conservative 98 nt portion of HCV's 3 0 -UTR induces only non-homologous recombination events. Moreover, a direct comparison of the hetero-and homomolecular systems revealed crucial differences between the mechanisms of homologous and non-homologous recombination. The former involves preferentially two different segments of the BMV genome and the latter occurs more easily between the same genomic RNAs.
Plasmids pB1TP3, pB2TP5 and pPN0-RNA3 containing fulllength cDNA of BMV RNA1, RNA2 and modified RNA3 (recombination vector), respectively, were the generous gift from J. J. Bujarski (Northern Illinois University, DeKalb, IL).
Restriction enzymes (EcoRI, SpeI and XbaI) T7 RNA polymerase, RNasine, RQ DNase RNase free, MMLV-reverse transcriptase, Taq polymerase and pUC19 cloning vector were from Promega.
The following primers were used for the construction of pMatNH-pMatH-, pMatNH-HVR-, pMatH-HVR-, pMatNH-X-, pMatH-X-RNA3: 
Plasmids pMag1-and pMagH-RNA3 contain full-length cDNA of the RNA3 vector carrying the recombinationally active sequence RAS1 inserted in antisense or sense orientation, respectively. Both plasmids were constructed in the same way: pPN0-RNA3 was linearized with SpeI endonuclease and ligated with SpeI cut RAS1 cDNA. Then plasmids carrying RAS1 in antisense (pMag1-RNA3) and sense (pMagH-RNA3) orientation were identified (30) .
To prepare pMatNH-RNA3 and pMatH-RNA3 plasmids (containing full-length cDNA of MatNH-RNA3 and MatH-RNA3), pMag1-RNA3 and pMagH-RNA3 were digested with KpnI and EcoRI endonucleases. Then, the deleted fragment was replaced with a KpnI-EcoRI cut 379 nt cDNA fragment corresponding to the BMV RNA1 3 0 end (containing the entire 3 0 -UTR and RAS1). The latter were obtained by PCR involving primers 1, 2 and pB1TP3 as a template.
To construct pMat0-RNA3, i.e. a plasmid containing cDNA of the universal recombination vector Mat0-RNA3, the following modifications were introduced into pMatNH-RNA3. First, it was digested with SpeI endonuclease and religated. This way 5 0 RAS1as was removed and the 5 0 RAS cloning site (including only one restriction site SpeI) was created. Next, the plasmid was cut with KpnI and EcoRI to remove RNA1 3 0 -UTR and 3 0 RAS1s. Instead, a 295 nt fragment of RNA1 3 0 end (between positions 2940 and 3234) followed by the 3 0 RAS cloning site (including KpnI, MluI, BamHI and EcoRV restriction sites) was ligated into pMatNH-RNA3. The inserted sequence was obtained by PCR using primers 2, 3 and pB1TP3 as a template and digested with KpnI and EcoRI, prior to ligation.
To test the recombination activity of HCV-derived sequences (hypervariable region 1, abbreviated HVR and sequence X, abbreviated X) cDNA of the corresponding fragments of the virus' genome was obtained by RT-PCR method (40, 41) and cloned into the pUC19 vector. Then, both tested sequences were amplified by PCR with primers introducing an SpeI restriction site. Primers 4, 5 and primers 6, 7 were used to obtain HVR and X cDNA, respectively. PCR products and pMat0-RNA3 were digested with SpeI and ligated. Then pMat0-RNA3 derivatives bearing HVR and X in sense and antisense orientation were identified. HVR and X were amplified again by PCR involving primers introducing MluI and EcoRV restriction sites (primers 8, 9 and 10, 11 to amplify HVR and X, respectively). PCR products were cut with MluI and EcoRV and ligated into the 5 0 RAS cloning site of previously identified pMat0-RNA3 derivatives (carrying HVR and X in sense and antisense orientation). As a result four plasmids were obtained: (i) pMatH-HVR-RNA3-containing cDNA of MatH-HVR-RNA3 in which two HVRs were inserted in sense orientation; (ii) pMatNH-HVR-RNA3-containing cDNA of MatNH-HVR-RNA3 possessing two HVRs, 5 0 HVR in antisense and 3 0 HVR in sense orientation; (iii) pMatH-X-RNA3containing cDNA of MatH-X-RNA3 in which two Xes are in sense orientation; (iv) pMatNH-X-RNA3-containing cDNA of MatNH-X-RNA3 carrying two Xes, 5 0 X in sense and 3 0 X in antisense orientation. Their structure was confirmed by sequencing.
To test the recombination activity of the BMV mutants, the previously described procedure was applied (30, 33) . Infectious BMV genomic RNAs were obtained by in vitro transcription for which EcoRI linearized plasmids pB1TP3, pB2TP5, pMag1-RNA3, pMagH-RNA3, pMatNH-RNA3, pMatH-RNA3, pMat0-RNA3, pMatH-HVR-RNA3, pMatH-HVR-RNA3, pMatHN-X-RNA3 and pMatH-X-RNA3 were used. Five-leaf C.quinoa plants (local lesion host for BMV) were mechanically inoculated with mixtures containing BMV RNA1, RNA2 and one of the RNA3 derivatives. Two weeks post-inoculation, the number of lesions developed on each inoculated leaf was counted to establish the infectivity of the tested BMV mutant. Then, individual local lesions were excised and total RNA was extracted separately from every lesion. The isolated RNA was subjected to RT-PCR involving primer A (the first strand primer) and primer B (the second strand primer) specific for RNA3 3 0 fragment amplification (the region where recombination crossovers occur). As a control identical reactions involving either parental RNA3 transcript (positive control) or water (negative control) were carried out. RT-PCR products were analyzed by electrophoresis in a 1.5% agarose gel. The formation of 800 nt or shorter 500 nt products indicated that parental or recombinant RNA3 accumulated in the analyzed lesion, respectively. Next, RT-PCR products were cloned into the pUC19 vector and sequenced to determine the location of recombinant junction sites. Finally, the presence of recombinants in the selected local lesions was additionally confirmed by northern blot analysis.
The main question that we had to answer during our studies was how to design a vector that could be used for examining the recombination activity of any RNA sequences in vivo. As a result, the idea arose to construct a BMV-based homomolecular recombination system. In such a system, both tested sequences are supposed to be present within the same segment of the BMV genome (either in RNA1, RNA2 or RNA3). Thus, a new vector should possess two separately located RAS cloning sites, be replicable and stable during infection. It has to be capable of generating viable recombinants, which have selective advantage over parental RNA molecules. Consequently, recombinants ought to be able to out compete the vector with inserted RASes. Assuming that RNA recombination occurs according to a copy choice mechanism, we decided that RNA3, being dispensable for BMV replication, is the best candidate for a new vector. Any changes in RNA1 and RNA2, which encode BMV replicase proteins, would strongly affect the studied process. The next important question was whether the location of RASes within the same (homomolecular system) or within two different segments of the BMV genome (heteromolecular system) influences the recombination activity of the examined RNA sequence.
To address both issues, we decided to construct a so-called mixed system, homo-and heteromolecular at the same time.
To this end two RNA3 molecules, prototypes of a new vector carrying two RASes, were prepared. To obtain them we used PN0-RNA3, described earlier, and a well-characterized recombinationally active sequence from BMV RNA1 (RAS1, see Figure 1 ). The 137 nt RAS1 corresponding to RNA1 between positions 2856 and 2992 was inserted into the PN0-RNA3 RAS cloning site, in antisense (RAS1as) and sense (RAS1s) orientations ( Figure 2 ). As a result, we obtained Mag1-and MagH-RNA3 derivatives (30) . Then the 356 nt portion of Mag1-and MagH-RNA3 3 0 end was replaced with a 379 nt sequence representing the wtRNA1 3 0 end (fragment encompassing the entire 3 0 -UTR and RAS1s sequence) ( Figure 2 ). In addition, a marker mutation (called DXho) was introduced within the RNA1-derived fragment to make it distinguishable from an analogous region present in wtRNA1. To this end, the XhoI restriction site (2988-2994) was disrupted by a 4 nt insertion (GATC) between C-2991 and G-2992. Resultant RNA3 derivatives, called MatNH-and MatH-RNA3, have unchanged 5 0 -UTR, intergenic and coding regions and a highly modified 3 0 -UTR. The latter includes 3 0 -UTR coming from wtRNA1 and two RAS1 sequences (3 0 RAS1 and 5 0 RAS1) separated by a 338 nt spacer (sequence CCMV and B1). In MatNH-RNA3, 3 0 RAS1 is located in sense and 5 0 RAS1 in antisense orientation, while in MatH-RNA3 both RAS1 sequences are in sense orientation ( Figure 2 ).
Having these two RNA3 derivatives, we were able to construct two variants of the mixed system: one for homologous (MatH-BMV mutant) and the other for non-homologous (MatNH-BMV mutant) recombination studies. The MatH-BMV genome is composed of wtRNA1, wtRNA2 and MatH-RNA3 and the MatNH-BMV genome of wtRNA1, wtRNA2 and MatNH-RNA3 (Table 1 ). In genomes of both BMV mutants three copies of RAS1 are present: two in the RNA3 derivative (RAS1s-RAS1s or RAS1as-RAS1s in MatH-and MatNH-RNA3, respectively) and one in wtRNA1 (RAS1s). Thus, in the mixed systems two identical RASes or RAS and its complementary counterpart were capable of supporting, respectively, homologous or non-homologous (heteroduplex-mediated) recombination between the same or between different BMV genomic RNAs. As a result, we could directly compare homo-and heteromolecular recombination systems in one in vivo experiment and examine whether our presumptions concerning the new recombination vector are correct.
Homologous recombination in the mixed homo-heteromolecular system Earlier, Nagy and Bujarski (33) demonstrated that the 66 nt portion of RAS1 did not support homologous recombination in heteromolecular system. We repeated this experiment using MH-BMV mutants. Recombinants also did not form although the entire RAS1 sequence was present in wtRNA1 and MagH-RNA3 molecules (Table 1 and Figure 3 ). To test RAS1 activity Figure 1 , white, black and gray boxes represent coding, noncoding and recombinationally active sequences, respectively, in sense (RAS1s) or antisense (RAS1as) orientation, dashed line squares encompass replaced parts of wtRNA1 and modified RNA3 molecules. Mag1-and MagH-RNA3 were created by inserting the RAS1 sequence from wtRNA1 into the RAS cloning site of PN0-RNA3 in antisense (Mag1-RNA3) or sense (MagH-RNA3) orientation. To construct MatNH-RNA3 and MatH-RNA3, the 356 nt very 3 0 end of Mag1-RNA3 or MagH-RNA3 (between KpnI and EcoRI sites) was replaced with a 379 nt portion of the wtRNA1 3 0 end (fragment containing the entire 3 0 -UTR and RAS1). Thus, both constructs contain two copies of RAS1 sequence-MatNH-RNA3 includes 5 0 RAS1as and 3 0 RAS1s, while MatH-RNA3 comprises 5 0 RAS1s and 3 0 RAS1s. Furthermore, a marker mutation DXho (marked as a white dot), removing the XhoI restriction site, was introduced into the 3 0 end of MatNH-and MatH-RNA3, to make it distinguishable from an analogous region present in wtRNA1. in the mixed homologous recombination system, a previously used, well-established procedure was applied (30, 33) . C.quinoa plants (local lesion host for BMV) were inoculated with a mixture containing in vitro transcribed wtRNA1, wtRNA2 and MatH-RNA3. After 2 weeks, when infection symptoms were well developed, the number of lesions formed on every inoculated leaf was counted to determine the infectivity of the MatH-BMV mutant. Individual local lesions were excised and total RNA was extracted separately from each of them. Then, the 3 0 -portion of RNA3 progeny accumulating in examined lesions was selectively amplified by RT-PCR involving RNA3 specific primers A and B (for their location see Figure 1 ). Reaction products were separated in a 1.5% agarose gel and their length was determined. The formation of an 800 or 400-500 nt DNA fragment indicated that the lesion contained parental or recombinant RNA3, respectively. In this way, we were able to determine the number of lesions in which a viable RNA3 recombinant was generated. The presence of recombinants in analyzed lesions was confirmed by standard northern blot hybridization. DNA fragments obtained during selective RT-PCR amplification of RNA3 were cloned and sequenced. Finally, the results obtained with our new mixed homologous recombination system were compared with analogous data previously got using the heteromolecular system (33) (see Table 1 and Figure 3 ). The data presented in Table 1 indicate that the exchange of MagH-RNA3 (carrying a single RAS1) into MatH-RNA3 (bearing two RAS1 sequences) did not affect the infectivity of the BMV mutants. The average numbers of lesions appearing on the leaves inoculated with MH-and MatH-BMV were similar: 18 and 17, respectively. Interestingly, although RAS1 did not support homologous crossovers in the heteromolecular system represented by MH-BMV, it was very active in the mixed system. About 85% of the local lesions developed during MatH-BMV infection accumulated the RNA3 recombinant instead of parental MatH-RNA3. In all of them, one RAS1 and a spacer were deleted. This indicates that crossovers occurred either within 3 0 end 5 0 RAS1 present in MatH-RNA3 (inter-or intramolecular crossovers) or within RAS1 and 5 0 RAS1 located in wtRNA1 and MatH-RNA3, respectively. Recombinant junction sites were placed in identical regions; therefore, their location could not be precisely established.
The data presented till now also could not answer which molecules, exclusively MatH-RNA3 or wtRNA1 and MatH-RNA3, participated in recombination. In the heteromolecular system, only RAS1-mediated crossovers between wtRNA1 and MagH-RNA3 were permitted. The situation seems to be more complicated in the mixed homologous system where three copies of RAS1 are present, all in sense orientation: two of them in MatH-RNA3 (3 0 RAS1 and 5 0 RAS1) and one in wtRNA1. Consequently, RAS1-mediated homologous recombination may happen according to four different scenarios ( Figure 3 ). It can engage MatH-RNA3 only and occur as intra-or intermolecular process or it can involve wtRNA1 and MatH-RNA3. In the latter case, recombination can be mediated by RAS1 present in wtRNA1 and either 5 0 -or 3 0 RAS1 located in MatH-RNA3.
To learn according to which scenario homologous recombination occurred, we checked whether mutation DXho introduced into MatH-RNA3 (just behind 3 0 RAS1) is still present in RNA3 recombinants. In this way, we were able to determine if their 3 0 -UTR was derived from MatH-RNA3 or wtRNA1 molecules. The undertaken analysis revealed that the mutation was present in 20% of recombinants. This result suggested that homologous crossovers preferentially occur between wtRNA1 and MatH-RNA3.
However, there are other explanations why DXho was absent in a large fraction of recombinants. It is possible that the mutation was removed either from MatH-RNA3, due to homologous recombination between its 3 0 RAS1 and wtRNA1, or from the RNA3 recombinant (carrying a single copy of RAS1) because it could also have recombined with wtRNA1. To examine the first possibility, progeny RNA3 extracted from the local lesions accumulating MatH-RNA3 (lesions in which recombinant was not generated) was analyzed. About 800 nt RT-PCR products obtained during selective amplification of RNA3's 3 0 -portion were cloned and sequenced. In all of 20 analyzed clones DXho was present. To test the second possibility, a full-length cDNA clone of RNA3 recombinant containing DXho (RNA3-DXhoR) was obtained. It was inserted into the pUC19 vector under the T7 polymerase promoter. The resultant plasmid named pRNA3-DXhoR was used after linearization to produce an infectious RNA3-DXhoR molecule by in vitro transcription. Then, RNA3-DXhoR was used together with wtRNA1 and wtRNA2 to inoculate C.quinoa plants. After 2 weeks, total RNA was extracted from individual lesions and a 3 0 -portion of the progeny RNA3 was amplified by RT-PCR. Obtained products were cloned and sequenced. As described previously, DXho was present in all of the analyzed 20 clones. These two experiments proved that homologous recombination between either 3 0 RAS1 of MatH-RNA3 or RAS1 present in RNA3 recombinant and wtRNA1 does not occur frequently enough to explain why most recombinants lack DXho. Altogether, these results supported our initial thesis that DXho was removed from 80% of homologous recombinants, since most of the crossovers occurred within 5 0 RAS1 from MatH-RNA3 and RAS1 from wtRNA1.
Earlier we showed that RAS1 can effectively support nonhomologous recombination if inserted into PN0-RNA3 in antisense orientation (30, 33) . The heteromolecular system used in our experiment was composed of wtRNA1, wtRNA2 and Mag1-RNA3 (M1-BMV mutant). Crossovers occurred within the local double-stranded region (local heteroduplex), which wtRNA1 and Mag1-RNA3 were capable of forming. In order to test RAS1 activity in the mixed non-homologous recombination system, C.quinoa plants were inoculated with the MatNH-BMV mutant (its genome is composed of wtRNA1, wtRNA2 and MatNH-RNA3). Two weeks later progeny RNA3 were analyzed as described above. The number of lesions developed on each leaf was counted and total RNA was extracted from individual local lesions. After RT-PCR amplification, the 3 0 -portion of BMV RNA3 accumulating in each lesion was analyzed in an agarose gel, cloned and sequenced. The presence of recombinants was confirmed by a standard Northern blot. The results obtained were compared with analogous data we had got using the heteromolecular system (30) (Table 1 and Figure 4) .
As described previously, we observed that the exchange of Mag1-RNA3 (with a single RAS1as sequence) for MatNH-RNA3 (with two sequences: RAS1as and RAS1s) did not influence the infectivity of the BMV mutants. The average numbers of lesions developed on each leaf during infection with M1-BMV and MatNH-BMV were 19 and 18, respectively. There was also no difference between the recombination activity of M1-BMV and MatNH-BMV. RAS-1 (in fact RAS1s and RAS1as) supported non-homologous recombination . Non-homologous recombination in the heteromolecular and mixed homo-heteromolecular systems. As in Figure 3 , light and thicker lines represent viral genomic RNAs and nascent recombinant RNA, respectively. WtRNA1 is red (the recombinationally active sequence RAS1s which it contains is additionally boxed), wtRNA2 is black and modified RNA3 is blue. When inserted into RNA3 the wtRNA1-derived RAS1s sequence is also shown as a red box, whereas the complementary RAS1as sequence is shown as a green box. The portion of the RNA3 recombinant synthesized on wtRNA1 is red, the portion synthesized on the RAS1as sequence is green and the fragment synthesized on RNA3 is blue. The black dot symbolizes the DXho mutation present in MatNH-RNA3. RF, recombination frequency. Dashed line squares encompass the region identical in both systems [the region where crossovers occur, shown in detail in (E)]. (A) Heteromolecular system (M1-BMV). A detailed description of the M1-BMV genome is presented in Figure 1 . All nascent RNA3 molecules accumulating in M1-BMV infected plants were recombinants (RF = 100%). (B) Mixed system (MatNH-BMV). Three copies of RAS1 are located in the MatNH-BMV genome, two in MatNH-RNA3 (3 0 RAS1s and 5 0 RAS1as) and one in wtRNA1 (RAS1s). The recombination frequency observed during infection with MatNH-BMV was 95%. Of the identified recombinants, 10% were without the DXho marker, and 90% with the DXho marker. (C) Putative scenario of RAS1s/RAS1as-mediated non-homologous recombination in the heteromolecular system. Owing to the presence of RAS1s and RAS1as sequences in wtRNA1 and Mag1-RNA3, respectively, they are capable of forming a local double-stranded structure supporting non-homologous crossovers (for details see Figure 1 ). (D) Putative scenarios of RAS1s/RAS1as-mediated non-homologous recombination in the mixed system. The presence of 3 0 RAS1s and 5 0 RAS1as sequences in MatH-RNA3 and RAS1s in wtRNA1 creates several opportunities of heteroduplex formation: between wtRNA1 RAS1s and MatNH-RNA3 5 0 RAS1as (intermolecular), between two pairs of RAS1s/RAS1as sequences of two different MatNH-RNA3 molecules (intermolecular) and between 5 0 RAS1as and 3 0 RAS1s of the same MatNH-RNA3 molecule (intramolecular). Recombinants are generated if BMV replicase initiates nascent strand synthesis at the 3 0 end of wtRNA1 or MatNH-RNA3 and then switches to MatNH-RNA3 within the local double-stranded region. (E) Recombinants identified during M1-and MatNH-BMV infection. Boxed fragments of recombining wtRNA1/Mag1-RNA3, wtRNA1/MatNH-RNA3 and MatNH-RNA3/MatNH-RNA3 molecules are practically identical in both systems (except for the DXho mutation present in MatNH-RNA3). The locations of the junction sites are marked with arrows and letters. The numbers indicate how many recombinants of the same type were isolated. Upper case letters refer to M1-BMV, lower case letters refer to MatNH-BMV. equally in both systems. Recombination events occurred with a similar frequency (100 and 95% for M1-and MatNH-BMV, respectively) and recombinant junction sites were located within the same region of the heteroduplexes, which recombining molecules were capable of forming.
In the heteromolecular system, only one type of heteroduplex supporting non-homologous crossovers could possibly form: between wtRNA1 and Mag1-RNA3. In the mixed system, recombining molecules were capable of forming three types of heteroduplexes: (i) intermolecular, between 5 0 RAS1as from MatNH-RNA3 and RAS1s from wtRNA1, (ii) intermolecular, between 5 0 RAS1as and 3 0 RAS1s located in two MatHN-RNA3 molecules and (iii) intramolecular, between 5 0 RAS1as and 3 0 RAS1s located in the same MatNH-RNA3 molecule (Figure 4) . To determine the molecules that participated in non-homologous recombination, the RT-PCR amplified 3 0 -portions of RNA3 were checked for DXho. It was present in 90% of recombinants. This result clearly showed that non-homologous crossovers almost always involve one (intramolecular recombination) or two (intermolecular recombination) MatNH-RNA3 molecules.
The results presented above indicated that the homomolecular system can provide new interesting data concerning the mechanism of RNA recombination, especially if it could be used for testing the recombination activity of RNA sequences derived from other RNA-based viruses. Consequently, we attempted to construct a universal BMV RNA3-based recombination vector called Mat0-RNA3 (for details see Materials and Methods and Figure 5A ). In Mat0-RNA3, as in the former PN0-RNA3 vector, only 3 0 -UTR was modified. It is composed of the 295 nt very 3 0 end of RNA1 followed by the 3 0 RAS cloning site, a 338 nt spacer and the 5 0 RAS cloning site.
To determine the infectivity and stability of the new vector, C.quinoa plants were inoculated with a mixture containing wtRNA1, wtRNA2 and Mat0-RNA3 (Mat0-BMV mutant). After 2 weeks, the number of lesions developed on inoculated leaves was counted and then standard analysis of progeny RNA was carried out. Twenty separate lesions were excised, the total RNA was isolated and used for the selective RT-PCR amplification of the 3 0 -portion of progeny RNA3. The length of RT-PCR products was established by electrophoresis in a 1.5% agarose gel. In addition, reaction products were cloned and sequenced. This demonstrated that the Mat0-BMV mutant is infectious (usually 20 lesions were developed on each leaf, see Table 2 ) and Mat0-RNA3 is stable during the whole period of infection and thus it can be used as a recombination vector.
In order to demonstrate that Mat0-RNA3 can be used as an effective tool in recombination studies, we applied it to examine the recombination activity of two specific sequences derived from the HCV genome. The first, 101 nt sequence is placed within the 5 0 -portion of the HCV genome (within the fragment encoding E2 protein) and is named HVR (40, 42) . The second, called the sequence X (X) constitutes a 98 nt 3 0 end of HCV genomic RNA. It has been shown that X represents the most conservative fragment of HCV genome (43) . Both sequences were obtained by a standard RT-PCR method involving viral RNA isolated from the blood of infected patients as a template (40, 41) . Amplified fragments were inserted into the 5 0 -cloning site of Mat0-RNA3 in two different orientations (sense and antisense), then only in sense orientation into the 3 0 -cloning site (for details see Materials and Methods). As a result, four different Mat0-RNA3 derivatives were generated: (i) MatH-HVR-RNA3, possessing two copies of HVR in sense orientation (3 0 and 5 0 HVRs); (ii) MatH-X-RNA3, with two copies of X in sense orientation (3 0 and 5 0 Xs); (iii) MatNH-HVR-RNA3, with two copies of HVR, the 3 0 -copy in sense and the 5 0 in antisense orientation (3 0 HVRs and 5 0 HVRas); (iv) MatNH-X-RNA3, with two copies of X located in different orientation (3 0 Xs and 5 0 Xas) ( Figure 5B ). The former two were applied to test HVR's and X's ability to support homologous crossovers while the latter two to examine Xs/Xas' and HVRs/HVRas' capacity to induce nonhomologous, heteroduplex-mediated recombination. Unlike previously tested mutants (MatNH-and MatH-BMV), in MatH-HVR-, MatNH-HVR-, MatH-X-and MatNH-X-BMV, the examined sequences were present only in the recombination vector. They were absent in the two other genomic RNAs, so that recombination crossovers could involve only RNA3 molecules.
To determine the recombination activity of HCV-derived sequences, C.quinoa plants were inoculated with four BMV mutants: MatH-HVR-BMV, MatNH-HVR-BMV, MatH-X-BMV and MatNH-X-BMV. Their genomes were composed of wtRNA1, wtRNA2 and one of the newly generated Mat0-RNA3 derivatives (either MatH-HVR-, MatNH-HVR-, MatH-X-or MatNH-X-RNA3) ( Table 2) . After 2 weeks, the standard procedure of BMV RNA3 progeny analysis was applied. The number of lesions developed during each infection was counted. The 3 0 -portion of progeny RNA3 was amplified by the RT-PCR method. The length of RT-PCR products was established (by electrophoresis in a 1.5% agarose gel), then they were cloned and sequenced.
We found that BMV mutants carrying HVRs/HVRs and HVRs/HVRas sequences are as infectious as Mat0-BMV; usually they developed 14-18 lesions on each inoculated leaf. The two others, MatH-X-and MatNH-X-BMV mutants, are visibly less infectious and developed 3-5 and 4-8 lesions/leaf, respectively. Despite differences in their infectivity, BMV mutants carrying 3 0 HVRs and 5 0 HVRas as well as 3 0 Xs and 5 0 Xas supported non-homologous, heteroduplex-mediated crossovers very efficiently. An RNA3 recombinant was generated in 100 and 90% of lesions developed during infection with MatNH-HVR-and MatNH-X-BMV, respectively. Recombinant junction sites were located within the left portion of the local double-stranded region that could potentially be formed either by HVRs and HVRas or by Xs and Xas. As a result, both sequences supporting non-homologous crossovers were almost entirely deleted, together with the whole spacer ( Figure 5D and E) .
Interestingly, homologous recombinants were generated only during infection involving MatH-HVR-BMV. Fiftyfive percent of analyzed lesions contained the RNA3 recombinant. In all sequenced recombinants, one HVR and the spacer were deleted ( Figure 5C ). Their 3 0 -UTR was composed of a 295 nt RNA1-derived sequence and HVRs followed by To test the recombination activity of HCV-derived sequences X and HVR the following Mat0-RNA3 derivatives were prepared: MatH-X-RNA3-with two copies of X in sense orientation (3 0 and 5 0 Xs), MatNH-X-RNA3-with two copies of X located in different orientation (3 0 Xs and 5 0 Xas), MatH-HVR-RNA3-containing two copies of HVR in sense orientation (3 0 and 5 0 HVRs) and MatNH-HVR-RNA3-with two copies of HVR, 3 0 -copy in sense and 5 0 in antisense orientation (3 0 HVRs and 5 0 HVRas). MatH-X-and MatH-HVR-RNA3 were applied to test X's and HVR's ability to support homologous crossovers while MatNH-X-and MatNH-HVR-RNA3 were applied to examine X's and HVR's competence to induce non-homologous, heteroduplex-mediated recombination. RF, recombination frequency observed during infection involving each RNA3 derivative. (C). Homologous recombinants generated during infection involving MatH-HVR-BMV. RNA3 recombinants were only formed during MatH-HVR-BMV infection. In all of them, one recombinationally active sequence HVRs and a spacer were deleted and their 3 0 -UTR was composed of RNA1 derived sequence and HVRs followed by the CP coding region. Because crossovers occurred within identical regions, the location of recombinant junction sites could not be precisely The presence of homologous and non-homologous recombinants in the examined lesions was always confirmed not only by RT-PCR but also by northern blot analysis ( Figure 6 ). This revealed the same tendency as that observed earlier using the heteromolecular system (30, 33) . BMV accumulated to a very low level in lesions containing parental RNA3 (original molecules with duplicated sequences- Figure 6A , lane 5 and Figure 6B , lane 2). However, this changed in lesions where a recombinant was generated ( Figure 6A , lanes 1-4 and Figure 6B, lanes 1 and 3) .
Earlier it was shown that the BMV-based heteromolecular system can be used as an effective tool for investigating the mechanism of homologous and non-homologous recombination, although it is only suitable for testing the recombination activity of the sequences derived from the 3 0 -portion of BMV RNA1 or RNA2 (30, 33) . To overcome this problem, we attempted to create a new universal recombination in vivo system. The collected data suggested that a BMV RNA3-based homomolecular system would best fulfill our expectations. To confirm the correctness of the above presumption, to determine the efficacy of the homomolecular system and to compare it with the heteromolecular one, two mixed homoheteromolecular systems were constructed-one to study homologous (MatH-BMV) and the other non-homologous (MatNH-BMV) recombination.
The mixed systems were prepared in such a way that two identical or two complementary sequences were capable of supporting homologous or non-homologous crossovers, respectively, either between molecules representing the same segment of the BMV genome (modified RNA3) or between molecules representing two different segments of the BMV genome (wtRNA1 and modified RNA3). Experiments involving MatH-and MatNH-BMV showed that recombination can occur both in homo-and heteromolecular systems and proved that the former should be at least as effective as the previously utilized heteromolecular one. Interestingly, the RAS1s sequence did not support homologous recombination during infection with MH-BMV (heteromolecular system) and it was Infectivity was defined as the average number of lesions per leaf. b Recombination frequency was defined as the ratio between the number of lesions that developed recombinants and the total number of analyzed lesions. quite active in the mixed system. This clearly demonstrates that not only primary and secondary structure but also the location of RAS within the viral genome affects its ability to mediate homologous crossovers. The undertaken experiments also revealed that homologous recombination occurs more often between two different RNAs (RNA1 and RNA3), while non-homologous recombination usually involves molecules representing the same segment of the BMV genome (RNA3). The obtained results constitute yet another piece of evidence that the mechanisms of homologous and nonhomologous recombination are different. The same conclusion was reached by us earlier while studying the influence of specific mutations in BMV-encoded protein 2a (26, 27) . We identified among other the mutation in the 2a protein, which inhibits non-homologous crossovers without affecting the frequency of homologous ones. At present, it is difficult to judge at which stage of the recombination process the observed differences occur. One can only suppose that the structural requirements of transfer of the replicase-nascent strand complex from the donor to the acceptor molecule must be different in homologous and nonhomologous recombination. In the case of the former, a basic factor facilitating this process is complementarity between the acceptor and the nascent strand. Consequently, there is no necessity for the replication complex to be stable during homologous crossovers (22) . Replicase can leave the donor template alone or together with a nascent strand. Then, the 3 0 end of the newly synthesized RNA molecule can function as a guide; it can find a complementary sequence in the acceptor RNA, hybridize and serve as a primer allowing viral replicase to reinitiate RNA synthesis. In non-homologous heteroduplexmediated recombination, a factor enhancing crossover seems to be the interaction between the donor and the acceptor (the formation of a local double-stranded region) (30, 33) . Considering that BMV genomic RNAs are copied within spherules (44) , intramolecular hybridization between RAS1s and RAS1as, located in MatNH-RNA3, is much more likely. Thus, the results presented here indicate that the way the virus replicates can strongly affect the recombination process. However, further detailed studies are necessary in order to explain this phenomenon.
Based on results obtained using the heteromolecular (30, 33) and mixed systems, we constructed a new homomolecular one. Its most crucial element is the Mat0-RNA3 vector, into which both tested sequences can be introduced. In order to show the usefulness of this system, we employed it to test the recombination activity of two distinctly different sequences deriving from the genome of an RNA virus not related to BMV. Our choice was the 98 nt sequence X and an HVR both from HCV genome. There are many reasons as to why the two sequences can be deemed drastically different. The most important of them are (i) sequence X is placed in a noncoding region, while HVR in a coding one, (ii) sequence X is the least variable and HVR the most variable fragment of the HCV genome (42, 43, 45) , (iii) unlike to HVR, sequence X possesses a very stable and well-defined secondary and tertiary structure (42, 45) .
We ascertained that the introduction of HVR into the Mat0-RNA3 vector (in sense/sense and antisense/sense orientation) does not influence BMV infectivity. The latter was, however, reduced if HVR was replaced with sequence X. We found that HVR supports homologous recombination and HVRs and its complementary counterpart HVRas mediate non-homologous crossovers. The frequency of homologous recombination amounted to 55% and of non-homologous to 100%. Sequence Xs did not support homologous crossovers but Xs and complementary sequence Xas were capable of inducing non-homologous ones (their frequency reaching 90%).
The obtained results testify that the local double-stranded structures induce non-homologous recombination crossovers very efficiently. This may reflect the capacity of RNA viruses to remove inverted repeats from their genomes. Viruses lacking such ability would be an easy target for double-stranded RNA-induced RNA silencing, which is known as the plant antiviral mechanism (46) . Moreover, the data presented suggest that sequence X, which adopts a very compact and stable structure (45), is not able to mediate homologous recombination. It occurs efficiently within AU-rich HVR sequences whose structure is more labile and dynamic (42) . Earlier research on homologous recombination in BMV led to similar conclusions. It was shown that homologous recombination occurs effectively in AU-rich regions (47) and is not observed within highly structured 3 0 -and 5 0 -UTR (48) . These observations concur with the proposed mechanism of homologous RNA recombination (22) . It assumes that AU-rich regions facilitate the detachment of the polymerase-nascent strand complex from donor RNA. On the other hand, it is thought that the stability of RNA structure makes the hybridization of the nascent strand and/or replicase to the acceptor difficult.
Currently, it is becoming increasingly clear that RNA recombination plays a very complex role in a virus' life cycle. Not only does it permit the exchange of genetic material between viruses (3, 4, 22) , frequent homologous crossovers between molecules representing the same segment of the virus genome also stabilize genetic information (48) . Moreover, here we showed that homologous and non-homologous recombination might control the organization of the virus genome by removing direct or inverted repeats, which affect the virus' ability to replicate or accumulate in the infected cells. Interestingly, we observed that complementary sequences are more effectively deleted than homologous ones. It seems that some of the latter can prevail in the viral genome probably due to their compact stable structure that prevents recombination events.
Altogether, the data presented here prove that the newly created BMV-based homomolecular recombination system can be used to examine in vivo recombination activity of various RNA sequences derived from the genomes of related or unrelated viruses. However, there are other factors which, in addition to RNA structure, can affect the course of the studied process. Specific properties of the viral replicase and the host proteins that are necessary for recombination events can be of equally great importance. Therefore, there is a need to create similar universal recombination systems in other viruses. We believe that these systems will be very helpful in finding some general rules in RNA recombination and will provide us with knowledge which is indispensable to understand how new RNA viruses or retroviruses are generated. Neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in U937 promonocyte cells BACKGROUND: Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein σ3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating. RESULTS: The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis. CONCLUSION: Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections. Mammalian reoviruses are the prototypic members of the Reoviridae family, which also includes the pathogenic rotaviruses, coltiviruses, seadornaviruses and orbiviruses. These viruses share elements of their replication cycle as well as structural features, including a non-enveloped multi-layered capsid that surrounds a segmented dsRNA genome. In humans, mammalian reoviruses are typically associated with mild and self-limiting enteric and respiratory infections. However, studies in neonatal mice reveal that reoviruses can spread to distant tissue sites in immunocompromised hosts (reviewed in [1] ). The factors that determine reovirus cellular host range are poorly understood. Because reovirus attaches to cells through interactions with broadly expressed receptors, one or more subsequent steps in the viral life cycle must help to regulate host range and pathogenesis. Our recent studies suggest that one such step is proteolysis of the capsid protein σ3 [2, 3] .
In cell culture, the first step in infection is attachment to cellular receptors through interactions with the viral protein σ1 [4, 5] . σ1 interacts with two known receptors: sialic acid and junctional adhesion molecule 1 [6] [7] [8] . Following binding, virions are internalized by receptor-mediated endocytosis [9] . Endocytosis is an essential step in the viral life cycle under standard infection conditions [10] . Within the endosomal and/or lysosomal compartment, proteases convert virions into particles that resemble in vitro-generated intermediate subvirion particles (ISVPs) [10] [11] [12] [13] [14] . These uncoating intermediates, typically prepared using chymotrypsin or trypsin, lack σ3 and have a cleaved form of µ1. Studies using ISVPs and ISVPs recoated with recombinant outer capsid proteins reveal that σ3 plays a key role in regulating reovirus cell entry by interacting with, protecting, and controlling the conformational status of the underlying penetration protein µ1 [15] [16] [17] [18] . In cells that cannot efficiently mediate σ3 degradation during uncoating, reovirus infection is slow or blocked; these cells can be productively infected by particles that lack σ3 [2] . In vitro, ISVP-like particles can be generated by a variety of proteases in addition to chymotrypsin and trypsin, including proteinase K, thermolysin, endoproteinase lys-C, Cat L, Cat B and Cat S [3, [19] [20] [21] .
Recent work has provided insight into the cellular determinants of reovirus uncoating. In murine fibroblasts, where reovirus entry has been best studied, the cysteine proteases Cat L, and to a lesser extent Cat B, are required for σ3 removal, whereas the aspartyl protease Cat D is not [14, [21] [22] [23] [24] [25] . Virion disassembly in murine fibroblasts also requires acidic pH [10, 26, 27] . Recently, we demonstrated that reovirus uncoating in the macrophage-like cell line P388D is mediated by the acid-independent lysosomal cysteine protease Cat S [3] . This finding revealed that in different cell types, distinct proteases can facilitate reovirus uncoating. Our results suggested a model in which infection in some cells is acid-dependent because the proteases that mediate σ3 removal in those cells require acidic pH for maximal activity. Thus, in fibroblasts or other cells in which the acid-dependent proteases Cat L and Cat B mediate σ3 removal, infection is acid-dependent [21, 23, 28] , whereas in Cat S-expressing cells it is not [3] , because Cat S maintains its activity at neutral pH [29] . Insight from the analysis of reovirus cell entry facilitated the recent discovery that activation of the Ebola virus glycoprotein also depends on the activity of the acid-dependent endosomal proteases Cat B and Cat L [30] .
The role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread, and disease in animals is largely unknown, except in the murine intestinal tract where pancreatic serine proteases have been shown to mediate σ3 removal [31, 32] . Reovirus also naturally infects hosts via the respiratory tract [33] [34] [35] . One protease with well-described effects in the respiratory tract is elastase 2 (GenBank NM_001972), an inflammatory serine protease of the chymotrypsin family, which is predominantly expressed by neutrophils [36] . NE plays a prominent role in wound repair [37] [38] [39] and in controlling microbial infections [38] [39] [40] . NE expression can also promote pathogenesis; it has been implicated in smokeinduced emphysema [41] , respiratory syncytial viral bronchiolitis [42] and in the respiratory syndrome ARDS [4] . The fact that reovirus replication in the rodent lung causes an influx of neutrophils [35, 43] and that reovirus infection can recapitulate ARDS [44] , led us to ask whether NE could mediate productive reovirus uncoating. We investigated reovirus infection in the monocyte-like cell line U937, because it is known to express NE [45] . Experiments described in this report demonstrate that reovirus infection in U937 cells does not require cysteine protease activity and is not blocked in the presence of agents that raise vesicular pH. Studies using protease inhibitors suggest that, in the absence of cysteine protease activity, NE is largely responsible for productive infection of U937 cells. NE can directly mediate σ3 removal from reovirus virions; the resultant particles are infectious and do not require additional intracellular proteolysis. Our data raise the possibility that NE is involved in reovirus replication in the respiratory tract.
Analysis of viral replication in L929 and U937 cells treated with E64 Figure 1 Analysis of viral replication in L929 and U937 cells treated with E64. A. 3 × 10 6 L929 and U937 cells were untreated (-; black) or treated (+; grey) with 300 µM E64 for 3 h or 3 d. Cysteine protease activity was assessed using the fluorogenic substrate Z-Phe-Arg-MCA (Sigma) and plotted in arbitrary units. Activity levels in treated cells were so low (in L939 cells, 254 units at 3 h and 231 units at 3 d; in U937 cells, 200 units at 3 h and 115 units at 3 days) that they cannot be visualized on this graph. B. L929 (L; black bars) and U937 (U; grey bars) cells were treated with 300 µM E64 for 3 h prior to infection. Cells were then infected with reovirus strain Lang virions or ISVPs at an MOI of 3. Infectious virus present at 3 d p.i. was determined by plaque assay on L929 cell monolayers. Each time point represents the mean (+/-SD) derived from three independent samples.
Virion ISVP we first established conditions under which lysosomal cysteine protease activity was inhibited. Cells were treated with 300 µM E64, a broad-spectrum cysteine protease inhibitor [46] , and protease activity was assessed using the Cat L and Cat B-specific fluorogenic substrate Z-Phe-Arg-MCA. We analyzed enzyme activity at two time points: first after 3 h of treatment, because we typically pre-treat cells with inhibitors for 3 h prior to infection, and second at 3 d, the time point at which viral yield would be quantified. As shown in Fig. 1A , treatment with 300 µM E64 completely abolished cysteine protease activity in U937 cells. Consistent with our previous findings [3] , E64 also completely blocked cysteine protease activity in L929 cells. Raw values are provided, to illustrate the relative difference in Cat L/B enzyme activity levels between U937 cells and L929 fibroblasts. In the absence of inhibitor, Cat L and B activity was significantly lower in U937 cells than in L929 cells. This may be a consequence of high expression in U937 cells of cystatin F, an intracellular cysteine protease inhibitor with specificity for Cat L and papain [47] .
Next, we compared reovirus replication in E64-treated U937 and L929 cells. Cells were pre-treated for 3 h and infected with Lang virions or ISVPs at a multiplicity of infection (MOI) of 3. The results of a representative experiment are shown in Fig. 1B . In the absence of E64, both L929 and U937 cells supported reovirus replication, consistent with the fact that these cells express Cat L. As expected, E64 blocked virion infection of L929 cells; however, viral yields in E64-treated U937 cells were only slightly reduced relative to untreated cells. ISVPs, which lack capsid protein σ3, replicated efficiently in treated cells, indicating that 300 µM E64 was not toxic to either cell type. These results demonstrate that productive infection of U937 cells by Lang virions does not require the activity of E64-sensitive, papain-like cysteine proteases.
Acidic pH is required for productive reovirus infection of murine L929 fibroblasts [10, 27] , in which the aciddependent proteases Cat L and Cat B mediate uncoating [21, 23] . Serine proteases, including NE, and metalloproteases function over a broader pH range. Therefore, to gain insight into the nature of the protease(s) that can promote reovirus uncoating in U937 cells, we investigated the requirements for acidic pH. L929 and U937 cells were left untreated or pre-treated with E64 in the presence or absence of bafilomycin A1 (Baf) or NH 4 Cl. These latter agents raise vesicular pH by blocking the vacuolar H + -ATPase pump or by acting as a weak base, respectively [48] [49] [50] . After pre-treatment, cells were infected with Lang virions at an MOI of 3 and viral yields were determined at 3 days post infection (d p.i.). A representative experiment is shown in Fig. 2 . Treatment with either Baf or NH 4 Cl did not inhibit viral replication in U937 cells; yields reached 2.9 and 2.7 logs, respectively. Furthermore, these agents had little effect on viral replication in U937 cells even when the cells were also treated with E64 to inhibit cysteine protease activity. In contrast, Baf or NH 4 Cl alone completely blocked reovirus replication in L929 cells, consistent with the requirement for Cat L/B-mediated σ3 removal in these cells. Given that reovirus uncoating is an essential step in the viral life cycle [10] , these findings revealed that a non-cysteine protease that functions at neutral pH can facilitate this step in U937 cells.
Treatment of the promonocytic U937 cells with phorbol ester derivatives results in their differentiation into macrophage-like cells [51, 52] . This differentiation is characterized by several major phenotypic changes, including increases in expression of urokinase plasminogen activator receptors, upregulation of collagenase activity and a significant decrease in the expression of NE and Cat G [51, 52] . We predicted, therefore, that PMA treatment might decrease the capacity of reovirus virions to replicate in U937 cells when cysteine proteases were inhibited. To confirm that there was a significant decrease in NE expression in U937 cells differentiated with PMA, U937 cells were treated with 150 nM PMA for 72 h and expression of NE was analyzed by immunoblotting. As shown in Fig.  3A , NE was expressed in untreated U937 cells, but its expression was dramatically reduced following PMAinduced differentiation.
To examine the effect of U937 cell differentiation on reovirus infection, PMA-treated and untreated U937 cells were left untreated or were treated with E64 for 3 h and infected with Lang virions or ISVPs at an MOI of 3. Yields were measured at 3 d p.i. and the results of a typical experiment are shown in Fig. 3B . In the absence of E64, PMAtreated U937 cells were permissive to infection by virions. PMA treatment only decreased yields by ~0.5 log relative to untreated cells. In contrast, when PMA-differentiated U937 cells were treated with E64 to inhibit cysteine protease activity, they no longer supported productive infection by Lang virions. Because these results could be explained if E64 was toxic to PMA-treated U937 cells, we examined the replication of ISVPs. In the presence of E64, ISVPs replicated to high yields in both undifferentiated and differentiated U937 cells. Since PMA-induced differentiation of U937 cells caused a substantial decrease in NE expression, these results are consistent with the hypothesis that NE or another similarly regulated neutral protease facilitates productive reovirus infection in promonocytic (pre-differentiated) U937 cells.
Effects of agents that raise vesicular pH on reovirus replication in U937 and L929 cells A.
Analysis of reovirus replication in U937 cells differentiated with PMA Figure 3 Analysis of reovirus replication in U937 cells differentiated with PMA. A. Lysates generated from 10 5 U937 cells that were untreated (-) or treated with 150 nM PMA for 72 h were resolved on SDS-12% polyacrylamide gels and electrophoretically transferred to a nitrocellulose filter. The filter was subsequently incubated with a polyclonal goat antibody against human NE (1:400) (Santa Cruz Biotechnology). The filter was washed and incubated with a secondary anti-goat antibody conjugated to horseradish peroxidase (1:5000) (Santa Cruz Biotechnology). Protein bands were detected using reagents that generate a chemiluminescent signal (Amersham). B. U937 cells that were undifferentiated (-; black bars) or differentiated (PMA; grey bars) with 150 nM of PMA for 72 h were left untreated (-) or were treated with 300 µM E64. Following pre-treatment with the protease inhibitor, cells were infected with Lang virions or ISVPs at an MOI of 3. Viral yield was quantified at 3 d p.i. as described in the legend to Fig. 1B. A.
B. 
We directly examined the capacity of NE to facilitate reovirus infection by using the irreversible elastase inhibitor, N-(methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone [53] . This inhibitor is highly specific for NE and does not inhibit the activity of the related serine protease, Cat G [53] . First, we established the efficacy and specificity of inhibitor treatment under our experimental conditions. U937 cells were treated with the NE inhibitor, E64, Baf or NH 4 Cl for either 3 h or 2 d and the activity of NE in cell lysates was examined using a colorimetric substrate. As shown in Table 1 , the NE inhibitor was active at both time points. In cells treated with the specific inhibitor, NE activity was less than 9% of that in untreated U937 cells.
In contrast, in U937 cells treated with E64, Baf or NH 4 Cl, NE activity was only modestly reduced, remaining above 80% even after 2 d. These results are consistent with the capacity of NE to function at neutral pH. To verify the specificity of the NE inhibitor, we also examined its effect on Cat L/B activity using the fluorogenic substrate Z-Phe-Arg-MCA. As expected, Cat L/B activity was completely inhibited by E64 but largely unaffected by the NE inhibitor.
To examine the effect of the NE inhibitor on reovirus replication in U937 cells, we pre-treated them for 3 h with E64 in the presence or absence of the NE inhibitor, infected them with Lang virions or ISVPs at an MOI of 3, and quantified viral yields at 2 d p.i. A representative experiment is shown in Fig. 4 . Consistent with the results shown in Fig 1, virion replication was not blocked in E64treated U937 cells. However, in the presence of both E64 and the NE inhibitor, yields were significantly reduced. ISVPs replicated to high yields in treated cells, indicating that the combination of inhibitors was not toxic to U937 cells. These results demonstrate that NE plays a critical role in reovirus infection of U937 cells when cysteine proteases are inhibited.
NE, like many cellular proteases, is expressed as a proenzyme that becomes activated only after its pro-region is removed [54] . We envisioned two models by which NE could facilitate reovirus infection of U937 cells. In the first, NE could directly mediate σ3 degradation, leading to the generation of an ISVP-like particle. In the second, NE could act indirectly by activating another protease. To try to distinguish between these models, we examined the capacity of purified NE to directly mediate σ3 removal from Lang virions in vitro. Purified Lang virions were treated with NE for 1 and 4 h and the treated virus particles were analyzed by SDS-PAGE. As shown in Fig. 5A , NE efficiently removed σ3 from Lang virions; after 1 h very little intact σ3 remained on viral particles. After 4 h of NE treatment, σ3 was completely removed and the underlying µ1C was cleaved to the δ and φ fragments (φ was not retained on the gel). When we assayed the infectivity of the resultant particles by plaque assay we found that NE treatment did not negatively affect the titer of Lang particles (data not shown).
To determine if NE-generated SVPs required further proteolytic processing of σ3, L929 cells were pre-treated with E64 to block cysteine protease activity and infected at an MOI of 3 with Lang virions, ISVPs or NE-generated subviral particles (NE-SVPs). Viral yields were determined at 1 d p.i. As expected, E64 blocked infection of L929 cells by virions. In contrast, both ISVPs and NE-SVPs replicated efficiently in the presence of the cysteine protease inhibitor (Fig. 5B) . Because virion disassembly in L929 cells requires acidic pH [10] , we also examined the capacity of NE-SVPs to infect L929 cells treated with Baf, NH 4 Cl or monensin, three agents that raise vesicular pH by distinct mechanisms. Cells were treated with these agents and then infected with virions, ISVPs or NE-SVPs at an MOI of a U937 cells were treated with the indicated inhibitors for 3 h or 2 d. b NE activity was assessed using the colorimetric substrate MeOSuc-Ala-Ala-Pro-Val-ρNA and percent activity relative to untreated cells was calculated. c Cathepsin L and B activity were assessed using the fluorogenic substrate Z-Phe-Arg-MCA and percent activity relative to untreated cells was calculated.
10. At 18 hours post infection (h p.i.), cell lysates were harvested and expression of the reovirus non-structural protein µNS was analyzed by immunoblotting (Fig. 5C ). As expected, when treated cells were infected with virions, viral protein expression was blocked. In contrast, µNS expression was evident even in the presence of agents that raise pH when infections were initiated with ISVPs or NE-SVPs (Fig. 5C ). Together, these results demonstrate that NE can directly mediate σ3 removal from virions to generate infectious particles that do not require further proteolytic processing by acid-dependent cysteine proteases in L929 cells.
Serine proteases are involved in reovirus infection in the mammalian intestinal tract [31] and in this report we pro-vide evidence that they can mediate uncoating and promote infection in U937 cells. This expands the range of proteases that promote reovirus infection in cell culture to include NE as well as the cysteine proteases Cat L, Cat B, and Cat S. Several lines of evidence now support the notion that protease expression is a cell-specific host factor that can impact reovirus infection. For example, some reovirus strains are inefficiently uncoated by Cat S and thus do not replicate to high yield in P388D macrophages [3] . In this report we demonstrate that PMA-induced differentiation influences the type of protease that mediates reovirus uncoating in U937 cells. In these cells, PMA treatment is reported to increase Cat L expression [55] and decrease expression of the serine proteases NE and Cat G [56, 57] . Accordingly, when we used PMA to induce U937 cell cultures to differentiate, reovirus infection became sensitive to the cysteine protease inhibitor E64. We suspect that Cat L is largely responsible for uncoating in these PMA-differentiated cells, but the acid-independent protease Cat S may also play a role. We are currently addressing this question by analyzing infection in PMAdifferentiated cells treated with either Baf or NH 4 Cl.
Our data do not completely resolve this question. Cat G is expressed by U937 cells and, like NE, it is down-regulated by PMA treatment. Furthermore, we found that in vitro treatment of reovirus virions with purified Cat G generates SVPs that behave like NE-SVPs in that they are infectious in the absence of further proteolytic processing (data not shown). Results of our experiment with the NE-specific inhibitor suggest that NE is largely responsible for the E64-resistant infection in U937 cells. While this inhibitor is reported not to inhibit Cat G [53] , we have not independently confirmed this. Another approach to assess the role of Cat G in reovirus infection of U937 cells would be to examine the effect of Cat G-specific inhibitors on infection. We tried one such inhibitor, Cathepsin G Inhibitor I (Calbiochem) [58] , but found that it was cytotoxic to U937 cell cultures. Given that both NE and Cat G can generate infectious reovirus SVPs, more work needs to be done in order to understand the role that these two proteases play in infection in these cells.
Previously, we reported that virion uncoating mediated by Cat S does not require acidic pH [3] . These results were consistent with the acid-independence of Cat S activity [37] . Together, the results in Fig. 2 and Fig. 4 reveal that, like Cat S, NE-mediates infection in an acid-independent manner. This finding thus provides further support for a model in which the requirement for acidic pH during reovirus infection of some cell types reflects the requirement for acid-dependent protease activity in those cells rather than some other requisite acid-dependent aspect of cell entry. The small effect of Baf and NH 4 Cl on E64-resistant reovirus growth (Fig. 2 ) may reflect the participation of one or more acid-dependent proteases (such as Cat D) in the activation of NE.
Elastase is stored in azurophilic granules that are the major source of acid-dependent hydrolases in neutrophils [59] . Although these granules do not contain LAMP-1 or LAMP-2 [60] they contain the lysosomal markers LAMP-3 [61] and CD68 [62] and are accessible to endocytosed fluid-phase markers under conditions of cellular stimulation [63] . NE can be released from neutrophils during degranulation [64] and its cell surface expression can be induced upon PMA treatment [65] . However, studies in U937 cells have shown that NE is predominantly retained intracellularly and that little if any activity is present in the extracellular medium [45] . Consistent with this, we have been unable to generate ISVP-like particles by treatment of virions with U937 culture supernatants (data not shown). This observation, together with our finding that PMA treatment decreases the capacity of E64-treated U937 cells to support reovirus infection, leads us to favor a model in which NE-mediated virion uncoating in U937 cell cultures occurs intracellularly.
In vivo, a number of viruses, including dengue and respiratory syncytial virus, induce the release of IL-8, a cytokine that serves as a chemoattractant for neutrophils and promotes their degranulation [66, 67] . Reovirus replication in the rat lung results in neutrophilic invasion [35, 43] and studies in cell culture indicate that reovirus infection can induce IL-8 expression [68] . Thus, the capacity of reovirus to induce IL-8 secretion in vivo might facilitate the release of neutrophilic lysosomal hydrolases, including NE, into the extracellular milieu. In this report, we have shown that mammalian reovirus can utilize this acid-independent serine protease for uncoating. Our data suggest that, in vivo, one consequence of reovirus-induced IL-8 expression would be the generation of infectious NE-SVPs. Like ISVPs, these particles would be predicted to have an expanded cellular host range because they can infect cells that restrict intracellular uncoating [2] . Thus, inflammation might be predicted to exacerbate reovirus infection by promoting viral spread. Future studies using mice with deletions in the NE gene will be required to elucidate the role this protease plays during reovirus infection in the respiratory tract and other tissues. Finally, given the recent finding that endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection [30] , our results raise the interesting possibility that NE or other neutrophil proteases may play a role in cell entry of other viruses. [Furlong, 1988 #81] . ISVPs were prepared by treating purified virions with chymotrypsin as described elsewhere [Nibert, 1992 #95] .
Cysteine protease activity was measured as described previously [23] Samples were frozen and thawed three times and titrated by plaque assay on L929 cells as described elsewhere [69] . Viral yields were calculated according to the following formula: log 10 (PFU/ml) t = x hrlog 10 (PFU/ml) t = 0 +/-standard deviation (SD). 
To analyze NE expression, cell lysates were generated from U937 cells, either treated or untreated for 48 h with 150 nM PMA as described for the analysis of viral protein expression. Lysate from the equivalent of 1 × 10 6 cells was run on SDS-12% polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked overnight in TBST containing 10% nonfat dry milk. NE expression was analyzed using a polyclonal antibody against NE (1:400 in TBST) (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated anti-goat IgG (1:5000 in TBST). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemiluminescence (ECL) detection reagents (Amersham) and exposing them to Full Speed Blue X-ray film (Henry Schein, Melville, NY).
Cells were plated at 10 6 /well in a 6-well plate 18-24 h prior to infection. Virus was allowed to adsorb to cells for 1.5 h at 4°C. At this temperature, virus binds to cells but is not internalized [70] . After adsorption, the cultures were incubated at 37°C in fresh medium. Prior to some infections, cells were pre-treated for 3 h with 300 µM E64, 100 nM Baf, 25 µM monensin (Sigma), or 20 mM NH 4 Cl.
In those instances inhibitors were also included in the post-adsorption culture medium. At the indicated times p.i., cells were collected by centrifugation at 179 × g, washed twice in chilled PBS and lysed in TLB. After centrifugation at 179 × g to remove cellular debris, samples were resuspended in sample buffer. Protein samples (representing 1 × 10 5 cells) were analyzed by electrophoresis on SDS-12% polyacrylamide gels and transferred to nitrocellulose membranes for 2 h at 100 V in 25 mM Tris-192 mM glycine-20% methanol. Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) were blocked overnight at 4°C in TBST (10 mM Tris [pH 8.0], 150 mM NaCl and 0.05% Tween) containing 5% nonfat dry milk, rinsed with TBST, and incubated with a rabbit anti-µNS polyclonal antiserum [71] (1:12500 in TBST) for 1 h. Membranes were subsequently washed with TBST and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit immunogloblin G (IgG) (1:7500 in TBST) (Amersham, Arlington Heights, Ill.). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemilumescence (ECL) detection reagents (Amersham) and exposing the filters to Full Speed Blue X-ray film (Eastman Kodak, Rochester, N.Y.).
Purified virions (1.4 × 10 11 ) were incubated with 25 µg/ ml of purified neutrophil elastase (Calbiochem) in 40 µL of VDB at 37°C for 3 h. Reactions were terminated by adding 1 mM PMSF and 200 µM NE inhibitor to the reaction mixture. 5.0 × 10 10 particles were run on SDS-12% polyacrylamide gels stained with Coomassie Brilliant Blue to confirm the removal of σ3. Viral infectivity was determined by plaque assay on L929 cell monlayers.
Purified Lang virions (1.4 × 10 11 ) were treated with 25 µg/ ml of NE in 40 µL of VDB at 37°C for the times indicated. Reactions were terminated as described above. To verify σ3 removal, the proteins from 5.0 × 10 10 particles were separated on SDS-12% polyacrylamide gels and visualized with Coomassie Brilliant Blue staining. Viral infectivity for each time point was determined by plaque assay on L929 cell monolayers. The influence of locked nucleic acid residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA. Understanding the thermodynamics of nucleic acid duplexes is important for many reasons. For example, such knowledge facilitates design of ribozymes (1), antisense and RNAi oligonucleotides (2) (3) (4) (5) (6) (7) (8) (9) , diagnostic probes including those employed on microarrays (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) and structures useful for nanotechnology (24) (25) (26) (27) . Many modified residues have been developed for such applications. Examples include propynylated bases (28) (29) (30) , peptide nucleic acids (5, (31) (32) (33) , N3 0 -P5 0 phosphoramidates (34-38) and 2 0 -O-alkyl RNA (39) (40) (41) (42) (43) . A modification that is particularly stabilizing in DNA and RNA duplexes (44) (45) (46) (47) (48) (49) (50) (51) is a methyl bridge between the 2 0 oxygen and 4 0 carbon of ribose to form a 'locked nucleic acid' or LNA as shown in Figure 1 . McTigue et al. (48) have shown that the enhanced stability due to a single LNA residue in a DNA duplex can be predicted from a nearestneighbor model.
Hybridization of oligonucleotides to RNA is important for applications, such as antisense therapeutics (4, 8, 21, 46, (52) (53) (54) , diagnostics (32, 33, 42, 55) , profiling gene expression with microarrays (18) (19) (20) 56) , identifying bands by Northern blots of gels (57, 58) and probing RNA structure (1, 3, 15, (59) (60) (61) . Oligonucleotides with 2 0 -O-alkyl modifications can be particularly useful for these applications because they are easily synthesized (39, 43) , chemically stable and bind relatively tightly to RNA (39) (40) (41) (42) . However, for many applications, it is desirable to modulate the binding affinity. For example, sequence independent duplex stabilities would benefit applications that involve multiplex detection, such as microarrays.
Here, we show that introduction of LNA into 2 0 -O-methyl RNA oligonucleotides can increase stabilities of 2 0 -O-methyl RNA/RNA hybrid duplexes and that the enhancements in stability can usually be predicted with a simple model.
High-performance liquid chromatography (HPLC) was performed on a Hewlett Packard series 1100 HPLC with a reverse-phase Supelco RP-18 column (4.6 · 250 mm). Mass spectra were obtained on an LC MS Hewlett Packard series 1100 MSD with API-ES detector or on an AMD 604/402. Thin-layer chromatography (TLC) was carried out on Merck 60 F 254 TLC plates with the mixture 1-propanol/ aqueous ammonia/water ¼ 55:35:10 (v/v/v).
Oligoribonucleotides were synthesized on an Applied Biosystems DNA/RNA synthesizer, using b-cyanoethyl phosphoramidite chemistry (62) . For synthesis of standard RNA oligonucleotides, the commercially available phosphoramidites with 2 0 -O-tertbutyldimethylsilyl groups were used (Glen Research). For synthesis of 2 0 -O-methyl RNA oligonucleotides, the 3 0 -O-phosphoramidites of 2 0 -Omethylnucleotides were used (Glen Research and Proligo). The 3 0 -O-phosphoramidites of LNA nucleotides were synthesized according to the published procedures with some minor modifications (44, 47, 63) . The details of deprotection and purification of oligoribonucleotides were described previously (64) .
Oligonucleotides were melted in buffer containing 100 mM NaCl, 20 mM sodium cacodylate, 0.5 mM Na 2 EDTA, pH 7.0. The relatively low NaCl concentration kept melting temperatures in the reasonable range even when there were multiple LNA substitutions. Oligonucleotide single-strand concentrations were calculated from absorbencies above 80 C and single-strand extinction coefficients were approximated by a nearest-neighbor model (65, 66) . It was assumed that 2 0 -Omethyl RNA and RNA strands with identical sequences have identical extinction coefficients. Absorbancy versus temperature melting curves were measured at 260 nm with a heating rate of 1 C/min from 0 to 90 C on a Beckman DU 640 spectrophotometer with a water cooled thermoprogrammer. Melting curves were analyzed and thermodynamic parameters were calculated from a two-state model with the program MeltWin 3.5 (67) . For almost all sequences, the DH derived from T m À1 versus ln (C T /4) plots is within 15% of that derived from averaging the fits to individual melting curves, as expected if the two-state model is reasonable.
Free energy parameters for predicting stabilities of 2 0 -O-methyl RNA/RNA and 2 0 -O-methyl RNA-LNA/RNA duplexes with the Individual Nearest-Neighbor Hydrogen Bonding (INN-HB) model (64) were obtained by multiple linear regression with the program Analyse-it v.1.71 (Analyse-It Software, Ltd, Leeds, England; www.analyse-it. com) which expands Microsoft Excel. Analyse-It was also used to obtain parameters for enhancement of stabilities of 2 0 -O-methyl RNA/RNA duplexes by substitution of LNA nucleotides internally and/or at the 3 0 end when the LNAs are separated by at least one 2 0 -O-methyl nucleotide. Results from T m À1 versus ln (C T /4) plots were used as the data for the calculations. 3 show typical data from optical melting curves, and Table 1 lists the thermodynamic parameters for the helix to coil transition with either no or one LNA nucleotide in the primarily 2 0 -O-methyl strand of a hybrid with a Watson-Crick complementary RNA strand.
Single LNA substitutions at the 5 0 end of heptamer duplexes have little effect on stability
The effects of single LNA substitutions at the 5 0 end of the 2 0 -O-methyl strand were studied in duplexes of the form,
where superscript M denotes a 2 0 -O-methyl sugar, N is A, C, G, or U with a 2 0 -O-methyl or LNA sugar, r denotes ribose sugars, and Q is the Watson-Crick complement to N. As summarized in Table 1 , 5 0 terminal LNA substitutions make duplex stability more favorable by 0.3-0.6 kcal/mol at 37 C with an average enhancement of 0.45 kcal/mol. Thus, 5 0 terminal LNA substitutions increase the binding constant for duplex formation by $2-fold at 37 C.
The effects of single LNA substitutions at the 3 0 ends of heptamer duplexes is idiosyncratic
The effects of single LNA substitutions at the 3 0 end of the 2 0 -O-methyl strand was studied in duplexes of the form, Table 1 ). If N is A, C or G, then LNA substitutions have similar effects. On average, an LNA substitution makes duplex stability more favorable by 1.2 kcal/mol at 37 C. In the two sequences with a 3 0 terminal LNA U on the 2 0 -O-methyl strand, duplex stability is, however, affected little, averaging a destabilization of 0.08 kcal/ mol at 37 C. In both cases, the terminal U is preceded by a GC pair, but both orientations of the GC pair give similar destabilization upon LNA substitution at the 3 0 terminal U.
Single LNA substitutions in the interior of A M C M U M A M C M C M A M enhance the stability of the duplex formed with its complementary RNA by $1.4 kcal/mol
The effect of interior position on the free energy increment for a single LNA substitution for a 2 0 -O-methyl RNA was studied for the duplex 5 0 A M C M U M A M C M C M A M /3 0 r(UGAUGGU). As summarized in Table 1 , a single interior LNA substitution makes duplex stability more favorable by 1.2-1.7 kcal/mol at 37 C, with an average of 1.4 kcal/mol. This corresponds to roughly a 10-fold increase in binding constant. Thus, interior and 3 0 terminal LNA substitutions usually improve binding more than 5 0 terminal LNA substitutions. Table 1 . For 13 of 16 sequences, the LNA substitution makes duplex stability more favorable by 1.0-1.5 kcal/mol at 37 C, with an average enhancement of 1.3 kcal/mol. The enhancement for the other three sequences averages 2.1 kcal/mol at 37 C.
The dependence on the 5 0 nearest-neighbor nucleotide of effects from substituting U L for U M was studied in duplexes of the form, neighbor that is preceded by A M and U M , respectively. In both cases, the LNA substitution enhances duplex stability by 1.14 kcal/mol at 37 C. Thus, for seven duplexes, the enhanced stability from an LNA substitution is relatively independent of the nearest-neighbor nucleotide 5 0 to the LNA. The one exception is for the nearest neighbor 5 0 G M U L /3 0 r(CA). Interestingly, this nearest-neighbor combination is also destabilized by LNA substitution at a 3 0 terminal U (Table 1) . Evidently, an LNA substitution in the middle of a 2 0 -O-methyl strand usually affects heteroduplex stability with an RNA strand by about the same amount as an LNA substitution at a 3 0 terminus.
The effects of LNA substitutions are approximately additive when LNA nucleotides are spaced by at least one 2 0 -O-methyl nucleotide Table 2 contains thermodynamic parameters measured for duplexes having more than one LNA substitution and Table 3 compares the stabilities at 37 C with those predicted from four simple models. The first model, labeled 'additivity', predicts the DG 37 for duplex formation in the 5 0 ACUACCA/ 3 0 UGAUGGU series by adding the free energy increments measured for single LNA substitutions in the same context to the DG 37 for duplex formation in the absence of LNA nucleotides. The second model predicts the DG 37 (kcal/mol) for duplex formation with the following equation as deduced from fitting the data in Tables 1 and 2 Here, DG 37 (2 0 -O-MeRNA/RNA) is the free energy change at 37 C for duplex formation in the absence of any LNA nucleotides, n 5 0 tL is the number of 5 0 terminal LNAs, n iAL/UL and n iGL/CL are the number of internal LNAs in AU and GC pairs, respectively, n 3 0 tU and n 3 0 tAL/CL/GL are the number of Here, T m À1 is the inverse melting temperature in kelvin, R is the gas constant, 1.987 cal K À1 mol À1 , C T is the total oligonucleotide strand concentration, and both strands have the same concentration. Table 1 . Thermodynamic parameters of duplex formation between RNA and 2 0 -O-methyl oligoribonucleotides with and without a single LNA substitution a Oligonucleotides RNA Average of curve fits 3 0 terminal LNAs that are U or not U, respectively. Both methods that use experimental data for DG 37 (2 0 -O-MeRNA/RNA) provide reasonable predictions that are within 1 kcal/mol of the measured value (Table 3) . Two other methods that use nearest-neighbor models to approximate DG 37 (2 0 -O-MeRNA/RNA) provide somewhat less accurate, but still reasonable predictions as described below. The duplex with the worst prediction, 5 0 G M U L U M C L G M G L /3 0 CAAGCC has a 5 0 G M U L /3 0 CA nearest neighbor, consistent with this motif being unusually unstable by $1.2 kcal/mol. Thus, it is likely that the DG 37 of Equation 1 should be made less favorable by 1.2 kcal/mol for every internal 5 0 G M U L /3 0 CA nearest neighbor in a duplex. Evidently, the effects of multiple LNA substitutions are approximately additive when the LNAs are spaced by at least 1 nt.
The data may also be fit to a nearest-neighbor model containing 30 of the LNA enhancement parameters associated with duplexes of RNA strands bound to 2 0 -O-methyl RNA/ LNA chimeras. These parameters are listed in Supplementary Material. The number of occurrences for each nearest neighbor is limited, however, so the values are only roughly determined.
Predictions for RNA/RNA duplexes at 1 M NaCl can be used to approximate stabilities of 2 0 -O-methyl RNA/RNA duplexes at 0.1 M NaCl
The stabilities of RNA/RNA duplexes at 37 C and 1 M NaCl are predicted well by an Independent Nearest-Neighbor Hydrogen Bonding (INN-HB) model (64) . In this model, the stability of an RNA/RNA duplex is approximated by:
Here, DG init is the free energy change for initiating a helix; each DG j NN ð Þ is the free energy increment of the jth type nearest neighbor (see Table 4 ) with n j occurrences in the sequence; m term-AU is the number of terminal AU pairs; DG termÀAU is the free energy increment per terminal AU pair; DG sym is 0.43 kcal/mol at 37 C for self-complementary duplexes and 0 for non-self-complementary duplexes. 
À0.73 ± 0.26 5 0 AU3 0 À1.10 ± 0.08 Similar sequence dependent parameters may also be applicable to 2 0 -O-methyl RNA/RNA heteroduplexes because they are expected to have A-form conformations similar to those of RNA/RNA homoduplexes (68) . This was tested by comparing the predicted stabilities of RNA/RNA duplexes in 1 M NaCl at 37 C with those measured for 2 0 -O-methyl RNA/RNA duplexes in 0.1 M NaCl at 37 C. The predicted thermodynamics are listed in parentheses in Tables 1 and 2 . On average at 37 C, the RNA/RNA duplexes in 1 M NaCl are 0.12 ± 0.01 kcal/mol of phosphate pairs more stable than the 2 0 -Omethyl RNA/RNA duplexes in 0.1 M NaCl. Presumably, much of this difference is due to a sequence independent effect of salt concentration, which would primarily affect the DS for duplex formation (22, 69) . Thus, a reasonable approximation for the first term on the right hand side of Equation 1 is:
Note that DG sym from the RNA/RNA calculation is subtracted because a 2 0 -O-methyl RNA/RNA duplex cannot be selfcomplementary because the backbones differ. For the duplexes studied here, the number of phosphate pairs is one less than the number of base pairs. The effects of LNA substitutions are likely not very dependent on salt concentration. Thus, it is probable that in 1 M NaCl or in the presence of Mg 2+ (70) that DG 37 (2 0 -O-MeRNA/ RNA) can be approximated by DG 37 (RNA/RNA, 1 M NaCl). Table 3 compares measured values for duplexes with more than one LNA to predictions from combining Equation 1-3. The measured DG 37 values average À10.5 kcal/mol and the root-mean-square difference between measured and predicted DG 37 values is 0.6 kcal/mol with the largest difference being 1.7 kcal/mol. Again, the sequence with the largest difference contains a 5 0 G M U L /3 0 CA nearest neighbor so the prediction would be improved if Equation 1 was corrected for the apparent instability of this motif.
The results for 2 0 -O-methyl RNA/RNA duplexes provide preliminary nearest-neighbor free energy increments for predicting stabilities of such duplexes
The comparison of predicted RNA/RNA stabilities with those measured for 2 0 -O-methyl RNA/RNA duplexes suggests that the INN-HB model will also be applicable to 2 0 -O-methyl RNA/RNA duplexes (71) . The results in Tables 1 and 2 Table 4 ). Three nearest neighbors are only represented once or twice in the database, and these parameters are in parentheses. The parameters for 2 0 -O-methyl RNA/RNA and RNA/RNA duplexes are similar, especially if the RNA/RNA Watson-Crick nearest-neighbor parameters are each made less favorable by 0.12 kcal/mol, which largely accounts for the difference in salt concentration as suggested above. Evidently, the first term on the right hand side of Equation 1 can also be approximated by: Table 3 compares predictions from combining Equations 1 and  4 with measured values for duplexes with more than one LNA. The root-mean-square difference between measured and predicted DG 37 values is 0.6 kcal/mol with the largest difference being the 1.7 kcal/mol associated with the duplex containing a 5 0 G M U L /3 0 CA nearest neighbor. Undoubtedly, this model can be expanded and refined by more measurements, but it appears sufficient to aid sequence design for many applications.
Complete LNA substitution is no more stabilizing than substitution at every other nucleotide starting at the second nucleotide from the 5 0 end
The effect of complete LNA substitution for a 2 0 -O-methyl RNA backbone was studied for the sequences 5 0 A L C L U L A L C L C L A L /3 0 r(UGAUGGU) and 5 0 G L C L U L A L C L U L G L / 3 0 r(CGAUGAC). As summarized in Table 2 , the stabilities of these duplexes at 37 C are within experimental error of those measured for 5 0 A M C L U M A L C M C L A M /3 0 r(UGAUGGU) and 5 0 G M C L U M A L C M U L G M /3 0 r(CGAUGAC), respectively. Evidently, the most effective use of LNA nucleotides is to space them every other nucleotide with the first LNA placed at the second nucleotide from the 5 0 end.
Internal mismatches make duplex formation less favorable Table 5 contains thermodynamic parameters measured for the formation of duplexes containing single mismatches and the difference in stabilities relative to completely Watson-Crick complementary duplexes (Tables 1 and 2 ). All internal mismatches make duplex formation less favorable by at least 2 kcal/mol at 37 C corresponding to at least a 25-fold less favorable equilibrium constant for duplex formation. In general, terminal mismatches destabilize much less than internal mismatches. In fact, when the 3 0 terminal U L of 5 0 A M C M U M A M C M C M U L makes a GU pair, the duplex is stabilized by 0.14 kcal/mol at 37 C relative to a terminal AU pair.
For four cases, the effect of a mismatch with an LNA nucleotide was compared with that for the equivalent 2 0 -O-methyl nucleotide. In each case, the mismatch penalty for the LNA was less than that for 2 0 -O-methyl RNA. However, for an A M -G mismatch flanked by LNAs in the context 5 0 A L C M U L A M C L C M A L /3 0 r(UGAGGGU), the LNAs enhanced the mismatch penalty by $1 kcal/mol relative to a completely 2 0 -O-methyl RNA strand. Thus, oligonucleotides containing LNA may discriminate best against mismatches flanked by LNAs.
Oligonucleotide hybridization to RNA has many applications, ranging from quantifying gene expression (18) (19) (20) 56) to designing therapeutics (4, 8, 21, 46, (52) (53) (54) . LNA nucleotides have characteristics useful for these purposes. For example, LNA usually stabilizes duplexes (4, 44, 48, 51) and is more resistant than RNA and DNA to nuclease digestion (4, 6, 51) . The results presented here provide insights that are useful for designing 2 0 -O-methyl RNA/LNA chimeric oligonucleotides for various purposes. Some trends may be general for RNA A-form helixes and thus may also be relevant to other chimeras with nucleotides that favor A-form conformations. The results suggest several principles for the design of 2 0 -O-methyl RNA/LNA chimeras for hybridization to RNA
The database in Tables 1 and 2 is too small to The magnitude and sequence dependence of the stabilization due to LNAs are surprising. Ribose and therefore probably 2 0 -O-methyl ribose sugars in single strands are typically found in roughly equal fractions in C2 0 -endo and C3 0 -endo conformations. If the methylene bridge of an LNA only locks the sugar into the C3 0 -endo conformation, then the expected stabilization due to preorganization would be: DDG ¼ ÀRT ln 2, which is À0.4 kcal/mol at 37 C (310.15 K). The stabilization observed for a 5 0 terminal LNA is roughly À0.4 kcal/ mol, but the average stabilizations for internal LNAs and 3 0 terminal A L , C L and G L are more favorable at À1.3 and À1.2 kcal/mol, respectively. Moreover, if stabilization was only due to preorganization of an LNA sugar, then the effect would not saturate when alternate sugars are LNA. Evidently, the LNA substitution also affects the 5 0 neighboring base pair in a way that enhances the stabilization beyond that expected from preorganization of a single sugar. Interestingly, NMR structures of DNA/LNA chimeras bound to RNA show that only the DNA sugar 3 0 of the LNA is driven to a C3 0endo conformation for the sequence d(5 0 CTGAT L ATGC)/ 3 0 GACUAUACG, but all non-terminal DNA sugars are C3 0 -endo when all three Ts are LNAs (76) . The free energy increments at 37 C for LNA substitutions ranged from +0.83 to À1.90 kcal/mol with an average of À0.55 kcal/mol. This compares with a range from +0.18 to À2.17 kcal/mol and an average of À1.32 kcal/mol for the single internal LNA substitutions in Table 1 . The comparision suggests that single LNA substitutions are on average more stabilizing to 2 0 -O-methyl RNA/RNA duplexes than to DNA/DNA duplexes. This may reflect the expectation that LNA substitutions do not have a large effect on the conformations of 2 0 -O-methyl RNA/RNA duplexes, but alter the conformations of DNA/DNA duplexes.
LNA substitutions should be useful for probing RNA with short 2 0 -O-methyl RNA oligonucleotides RNA structure can be probed with short oligonucleotides on microarrays (3) . To optimize such methods, it is necessary to have tight binding that is sequence independent and that discriminates against mismatches. It appears that LNA nucleotides can be used to achieve this. For example, free energy increments for 2 0 -O-methyl RNA/RNA nearest neighbors range from À0.7 to À3.5 kcal/mol, corresponding to 5 0 A M U M /3 0 UA and 5 0 G M C M /3 0 CG, respectively ( Table 4 ). The average increment of À1.3 kcal/mol of internal and 3 0 terminal LNA can help compensate for such less favorable stability of AU relative to GC pairs. The stability enhancement from LNA can also allow the use of shorter oligonucleotides.
The potential disadvantage to LNA substitutions in 2 0 -Omethyl RNA oligonucleotides is that discrimination against mismatches containing an LNA may be less than with a complete 2 0 -O-methyl RNA backbone. This was clearly true for three of the four cases where such direct comparisons were made. Nevertheless, internal mismatches with LNA nucleotides are considerably destabilizing, averaging a penalty of 4.1 kcal/mol at 37 C (Table 5) , which translates to almost a 1000-fold weaker binding due to a single mismatch. When LNAs flanked an A M -G mismatch, the mismatch penalty at 37 C was 4.4 kcal/mol compared with 3.3 kcal/mol in the absence of LNAs. Such an effect may reflect enhanced rigidity due to LNA, which thereby prevents a mismatch from adopting a favorable conformation. Thus, it may be advantageous to use LNAs to flank nucleotides likely to give small mismatch penalties. Draft versus finished sequence data for DNA and protein diagnostic signature development Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(−3)–10(−5) (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. Draft sequencing requires that the order of base pairs in cloned fragments of a genome be determined usually at least four times (4· depth of coverage) at each position for a minimum degree of draft accuracy. This information is assembled into contigs, or fragments of the genome that cannot be joined further due to lack of sequence information across gaps between the contigs. To generate high-quality draft, usually $8· coverage is optimal (1). Finished sequence, without gaps or ambiguous base calls, usually requires 8· to 10· coverage, along with additional analyses, often manual, to orient the contigs relative to one another and to close the gaps between them in a process called finishing. In fact, it has been stated that 'the defining distinction of draft sequencing is the avoidance of significant human intervention' (1) , although there are computational tools that may also be capable of automated finishing in some circumstances (2) .
While some tabulate the cost differential between highquality draft versus finished sequences to be 3-to 4-fold, and the speed differential to be >10-fold (1), others state that the cost differential is a more modest 1.3-to 1.5-fold (3) . In either case, draft sequencing is cheaper and faster. Experts have debated whether finished sequencing is always necessary, considering the higher costs (1, 3, 4) .
Thus, here we set out to determine whether draft sequence data are adequate for the computational prediction of DNA and protein diagnostic signatures. By a 'signature' we mean a short region of sequence that is sufficient to uniquely identify an organism down to the species level, without false negatives due to strain variation or false positives due to cross reaction with close phylogenetic relatives. In addition, for DNA signatures, we require that the signature be suitable for a TaqMan reaction (e.g. composed of two primers and a probe of the desired T m s). Limited funds and facilities in which to sequence biothreat pathogens mean that decision makers must choose wisely which and how many organisms to sequence. Money and time saved as a result of draft rather than finished sequencing enables more target organisms, more isolates of the target and more near neighbors (NNs) of the target to be sequenced. However, if draft data do not facilitate the generation of highquality signatures for detection, the tradeoff of quantity over quality will not be worth it.
We used the Sequencing Analysis Pipeline (SAP) (5, 6) to compare the value of finished sequence, real draft sequence and simulated draft sequence of different qualities for the computational prediction of DNA and protein signatures for pathogen detection/diagnostics. Marburg and variola viruses were used as model organisms for these analyses, due to the availability of multiple genomes for these organisms. We hope that variola may serve as a guide for making predictions about The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org bacteria, in which the genomes are substantially larger, and thus the cost of sequencing is much higher than for viruses. Variola was selected as the best available surrogate for bacteria at the time we began these analyses because:
i. it is double-stranded DNA; ii. it has a relatively low mutation rate, more like bacteria than like the RNA or shorter DNA viruses that have higher mutation rates and thus higher levels of variation; iii. it is very long for a virus, albeit shorter than a bacterial genome; iv. we have access to many genomes, which were sequenced by our collaborators at the US Centers for Disease Control and Prevention in Atlanta, GA; v. there are finished genomes available, so we can compare actual finished data with simulated draft data. Only recently have a fairly large number of Bacillus anthracis genomes become available to us. However, since only some of these are finished, currently we cannot compare finished with draft results for this bacterial genome.
The sequencing analysis pipeline uses the DNA and protein signature pipelines
The draft SAP simulations are nearly identical to those using finished genomes, described previously (5, 6) . The SAP ( Figure 1 ) performs stochastic (Monte Carlo) simulations and includes our DNA and Protein Signature Pipelines as components, which will be summarized briefly below. It is necessary to describe what the signature pipelines do before the SAP can be clearly described, so signature pipelines will be discussed first. As a step within the DNA and Protein Signature Pipelines, DNA sequence alignments of multiple draft genomes are required. For this we use the WGASA software, also summarized below. Once each of these components of the SAP has been presented, the SAP itself will be described.
The DNA Signature Prediction Pipeline, described in detail elsewhere (7) (8) (9) (10) , finds sequence regions that are conserved among target genomes by creating a consensus based on a multiple sequence alignment. WGASA is the software used in the analyses here to create an alignment and will be discussed below. Next, the DNA Signature Pipeline identifies regions that are unique in the target sequence consensus relative to all other non-target bacterial and viral sequences that we have in a >1 Gb database, which is frequently updated from the NCBI GenBank sequence database (11) and other sources (e.g. our collaborators at the CDC, USDA and other public sources, such as TIGR, Sanger Institute and the Joint Genome Institute). From the conserved, unique regions, signatures are selected based on the requirements of a particular technology, in this case, TaqMan PCR. These signature candidates may then proceed for further in silico screening (BLAST analyses to look for undesired inexact matches) before undergoing laboratory screening. For an SAP run, first a pool of target genome and a pool of NN genomes are collected. Then many random subsamples of target and NN genomes are selected from the pool, and each subsample is run through either the DNA signature pipeline or the protein signature pipeline, which identify regions conserved among target genomes and unique relative to non-target genomes, where unique regions are evaluated by comparing to a large sequence database of all currently available bacterial and viral complete genomes or the non-redundant protein database, excluding NNs from the NN pool that are not in that random subsample. Thus, each run of the SAP requires many runs of the DNA or protein signature pipelines with different random samples, generating a range of outcomes that are plotted on range plots.
Protein signature prediction and SAP methods have previously been described in detail (6) . The following briefly describes the procedure. First, target genomes are aligned using WGASA. A set of gene (start, end) pairs for both the plus and minus strands relative to the reference genome is required. This implies that coding frames for the translation of nucleic acid codons into amino acids for each protein of the target organism's genome have been correctly determined. From the aligned genomes, nucleotide codons are translated into amino acid sequence based on the gene locations, and conserved strings of six or more amino acids among all the target genomes are recorded. These conserved fragments are then compared with the NCBI GenBank non-redundant (nr) database of amino acid sequences, unveiling peptides that are unique to the target species. For our computations, we require that if a peptide signature is longer than six amino acids, then every sub-string of length six amino acids is also conserved and unique. There may be many conserved and unique peptide signatures on the same and on different proteins. The resulting conserved, unique peptides that are at least six amino acids long from open reading frames are considered to be protein signature candidates.
Signature peptides may be used as targets for antibody or ligand binding and may be developed for use in detection, therapeutics or vaccines (12, 13) . Since the signature regions are highly conserved within a species, it is likely that they are functionally important to the organism's survival or reproduction. Those signatures that land on or near protein active sites may be developed into therapeutics, since antibody or ligand binding may interfere with protein function. Signature regions may even be considered as vaccine targets, since these unique peptides may evoke a specific response in the host (14, 15) .
For draft genomes, WGASA, or Whole Genome Analysis through Scalable Algorithms, is used to align multiple sequences. This is the only available tool that enables multiple sequence alignment of draft genomes and that is capable of aligning large or many genomes. WGASA requires at least one finished reference genome and the others may be draft.
Only recently has it become possible to use the DNA Signature Pipeline to predict signature candidates for draft genomes. This capability is due to the invention of software for multiple sequence alignment of draft genomes with at least one completed full genome. WGASA was developed by David Hysom, Chuck Baldwin and Scott Kohn in the Computations directorate at Lawrence Livermore National Laboratory. They designed the software in close communication with members of our bioinformatics team, and it is tailored for our needs of generating diagnostic and forensic pathogen signatures.
WGASA can efficiently align large (e.g. bacterial) genomes. In addition, the developers have created a parallel version that runs in minutes, allowing the SAP simulations, involving thousands of calls to WGASA, to complete in a feasible time frame. In addition to the SAP analyses, this tool has enabled us to revisit signature predictions for several important organisms, such as the food-borne pathogen Listeria, that were previously problematic because some of the sequences were available only in draft format.
The tool requires that there be one or more complete, finished genome, and any number of draft sequences. It is based on suffix-tree algorithms (16) . It requires that anchors, identical sequence fragments of user-specified length, be found in each of the genomes to be aligned. Thus, there must be some level of sequence conservation among the genomes in order to discover anchors of sufficient length (e.g. 35-60 bp) that are present in all the genomes. Then the regions between the anchors are aligned using a tool, such as clustalw or HMMer. The algorithm functions most efficiently if anchors are frequent and dispersed across the genomes to provide even coverage. If substitutions, deletions, insertions or gaps in sequence information (e.g. between contigs) result in an anchor's absence in one or more of the genomes, then those regions must be aligned using clustalw, which is slower and more memory intensive for large amounts of sequence data. Similar to all anchor-based alignment algorithms, WGASA is dependent upon a high degree of co-linearity across all input genomes.
The SAP for DNA signature analyses operates as follows. First, all available complete genomes of target were gathered into a pool with the total genome count called T. A second pool was created of all available NN complete genomes, with the total count of sequences called N. Next, we selected 10 random samples of size t targets and n NNs, for all t ranging from 1 to T and all n ranging between 1 and minimum(10,N). We ran the DNA Signature Prediction Pipeline for each sample, with signature prediction based on conservation among the t target strains and uniqueness relative to a >1 Gb database minus those NNs in the NN pool that were not chosen in that sample. Thus, for each sample, signature candidates were predicted as though we had only t target and n NN sequences, as well as the rest of the less-closely related organisms in our database that are not considered NNs. In addition to the number of TaqMan signature candidates, the fraction of the genome that is conserved among the t target sequences was also calculated. Based on the combined results of the many signature pipeline runs using random target samples of size t and n, we assessed how much sequence data, that is, the values of t and n, was required to approximate the number of signature candidates c that were predicted when the full data set (all target and NN sequences, t ¼ T and n ¼ N) was analyzed with the signature pipeline. Using the full data set will yield the fewest signatures, because lack of conservation or uniqueness will winnow away all unsuitable candidates.
Thus, the SAP performs Monte Carlo sampling from the target and NN genomes, runs each sample through the signature pipeline and summarizes the results of the hundreds of signature pipeline runs in a single plot. On our 24 CPU Sun server, up to seven signature pipeline simulations may be run in parallel, each requiring $15-22 min for viral genomes. All of the SAP analyses of dozens of bacteria and viruses to date have used a total run time of 6.26 years (operating in parallel), with an average pipeline run time of 0.522 h, and a process time span of 2.32 years.
The span of predictions generated by different random samples of genomes is illustrated using range plots (Figures 2-8 ). Along the y-axis, whole numbers represent the number of target strains t and the incremental values between the integers represent the number n of NN genomes. Only Figures 3, 5 and 6 have the incremental n values, because for the other plots of target sequence conservation only, the number of NNs was not relevant, and for the protein analyses NN comparisons were not made (described below). Outcomes of the number of signature candidates or the fraction of the target genome that is conserved are plotted along the x-axis as horizontal lines spanning the range (of predicted numbers of signatures or fraction conserved) for the s random samples of size (t,n) with the median and quantiles of the range indicated by colored, short vertical lines. If a random sample of t target strains and n NN strains were sequenced, there would be a 90% chance that the number of signature candidates for that sample would be less than or equal to the 90% quantile mark. The expected outcome is a reduction in the number of signature candidates or the fraction of the genome that is conserved as the number of target and NN sequences used in the simulations increases, due to a reduction in conservation from additional targets and a reduction in uniqueness from additional NNs.
The SAP analyses for proteins proceed much like that for DNA signatures. Random samples of size t target sequences are generated, where t ranges from 1 to T, the total number of target sequences in the pool. Either finished data, actual draft data, or draft data simulated as described below are aligned using WGASA. The protein signature prediction pipeline is run on each random sample, and the range, median, 75th and 90th quantiles of the number of protein signature candidates for the samples of a given target size t is plotted in range plots as described above.
Our DNA SAP analyses examined the effects of both the number of target as well as the number of NN sequences, but To discriminate samples in which zero NNs were used, the range is drawn as a horizontal gray line, and when n > 0, the range is drawn as a black line. The best estimate of the true value is the quality measure determined using the entire target and NN pools, and is represented by a vertical black line. This best estimate plus a constant c ¼ 20 is at the location of the vertical dashed line and was selected to indicate a reasonable distance from the true answer. The 75% quantile for each range is shown with a black, vertical tick mark. our protein SAP analyses investigated the effects of only the number of target sequences. This is because composing the lists of NN proteins for random, temporary exclusion from the protein nr database (to estimate the value of that NN sequence data) would be difficult to automate for rapid, high-throughput computations. Thus, we compared the target proteins with all the proteins in nr, regardless of their phylogenetic relationship to the target. This was comparable with DNA SAP results using all available NN data.
We had sequence data for four strains of Marburg virus, both the actual draft and the finished versions of those same isolates, provided for these analyses by a colleague working at Lawrence Livermore National Laboratory. The draft sequence was of $3· to 6· coverage, which enabled us to compare SAP results using the same strains in finished form. The identities of these sequences are provided in the Appendix. For the draft Marburg analyses, we selected one finished strain, the reference strain from GenBank (gi|13489275|ref|NC_001608.2|
Marburg virus, complete genome), as the WGASA reference genome, and then used random sub-samples from the four draft genomes. Marburg was the only organism for which we could obtain a sufficient number of draft genomes for the SAP Monte Carlo simulations. A total of 814 simulations for DNA signatures, i.e. individual runs of the DNA signature pipeline, and 48 simulations for protein signatures were performed using Marburg finished and draft data, requiring an average of 15 min per simulation.
We used finished sequence data generously provided by collaborators at the US CDC for 28 variola major genomes and 22 NN genomes from the Orthopox family. The sequence identities are provided in the Appendix. Since we did not have real draft data available, we developed a program to simulate draft sequence from finished sequence, based on guidance from two colleagues who have been involved in sequencing efforts and the finishing process in the Biology and Biotechnology Research Program at Lawrence Livermore National Laboratory. In outline, the draft simulator program randomly cuts a genome into contigs of a size randomly selected from an exponential distribution. Stochastic simulation also determines whether there are gaps or overlaps between contigs, as well as the size of the gap or overlap. Sequencing errors are also simulated.
The following paragraphs describe the draft simulation process in greater detail. First, the 5 0 end of the sequence is simulated as missing or present according to a random (Bernoulli) trial based on the probability of there being a gap in the sequence data. If simulations randomly determine that the first part of the sequence is missing, then the size of the missing segment is selected randomly from a uniform distribution ranging from the minimum gap size to the maximum gap size. The length of the first contig is selected randomly from an exponential distribution with a non-zero minimum contig size and a maximum contig size that is a fraction of the mean genome length for the species. The mean of this exponential distribution is also specified as a fraction of the mean genome length.
Next, a random Bernoulli trial again determines whether there is a gap or overlap between the first and second contigs, and the size of the gap or overlap is chosen from the appropriate uniform distribution (range for gap size ¼ 1-2000 bases, range for overlap size ¼ 20-40 bases). The size of the contig is selected from the exponential distribution as described above. Additional contigs are simulated in a similar manner.
Within each contig, sequencing errors are simulated based on the size of the contig, and whether the base position is at an end (first or last 100 bases) or in the middle of the contig. For long, double-stranded DNA viruses (e.g. variola) and bacteria, the sequencing error rates are larger at the beginning and end of a contig than in the middle, and small contigs are more likely to contain sequencing errors than are large contigs. In contrast, due to differences in generating the products for Sanger sequencing that are employed for smaller RNA and DNA viruses, there are often more sequencing errors in the middle of contigs for such smaller viral draft genomes. Although we did not specifically simulate draft for RNA and short DNA viruses, our simulator should work with minor modification to a few parameters. Thus, there are four parameters that must be specified for simulating sequencing errors: (i) the size cutoff for small versus large contigs, (ii) the probability of errors in the middle portion of small versus large contigs, (iii) the length of the contig ends where sequencing is either less accurate (bacteria and long doublestranded DNA viruses) or more accurate (small viruses, RNA viruses) and (iv) the probability of sequencing errors at the contig ends. If there is a sequencing error at a particular base, we assumed that that base is randomly changed to one of the other three bases with equal probability. Although additional features could be added to the draft simulation tool, the stochastic features that we have incorporated capture the main features of draft sequence and produce data that are suitable for SAP analyses.
We performed six sets of analyses using simulated variola draft. Three sets of simulated variola draft runs of the SAP used the following parameters: probability of a gap between contigs ¼ 0.95; probability of overlap between contigs ¼ 0.05; minimum gap size if there is a gap (uniform distribution) ¼ 1 bp; maximum gap size ¼ 2000 bp; minimum overlap if there is overlap (uniform distribution) ¼ 20 bp; maximum overlap ¼ 40 bp; minimum contig size (exponential distribution) ¼ 2000 bp; maximum contig size ¼ 0.5 · (mean genome length) bp mean contig size ¼ 0.05 · (mean genome length) bp cutoff size for small versus large contigs ¼ 10 000 bp; probability of sequence errors inside large contigs ¼ 0.01; probability of sequence errors inside small contigs ¼ 0.05; We will refer to the above set of simulations as those with a high probability of sequencing errors, or low-quality draft. The other three simulated variola draft runs used all the same parameters as above, except that the sequencing error rates were dramatically lower, more in line with error rates of 10 À5 / base that the US Centers for Disease Control and Prevention (CDC) has indicated for their draft variola genomes: probability of sequence errors inside large contigs ¼ 10 À5 ; probability of sequence errors inside small contigs ¼ 10 À4 ; probability of sequence errors in the contig ends ¼ 10 À3 ; These runs were referred to as low error rate, or highquality draft. Finally, we performed SAP runs using high error rate (low quality) simulated draft of the NN sequences and intermediate quality simulated draft of target genomes, using the following probabilities of sequencing errors: probability of sequence errors inside large contigs ¼ 10 À3 ; probability of sequence errors inside small contigs ¼ 10 À3 ; probability of sequence errors in the contig ends ¼ 10 À3 ;
The intermediate quality simulated draft is consistent with error rates for draft sequencing cited in the literature (1,3) .
For the parameter values specified above, three SAP experiments were simulated. In the first, only the target sequences were simulated into draft, and the NN sequences remained as finished sequences. In the second, the NN sequences were converted to simulated draft and the target sequences remained as finished. In the third, both target and NN sequences were simulated into draft. In the second and third cases, all the NNs were run through the draft simulator each time they were chosen, so that the draft sequences (i.e. location and extent of gaps and sequence errors) differ for the same genome among samples. In the first and third cases, the target sequences must be aligned, and WGASA requires that one of the sequences be a finished genome for reference. Thus, for each random sample from the pool of target genomes, one genome was randomly selected to be the finished genome, and so was left as finished sequence, and the other genomes in the sample were replaced with simulated draft sequence (by running through the draft simulator) before alignment. As with NNs, target draft sequences differ for the same genome among samples due to the randomness of the draft simulation each time it is run. In addition, the target genome that is chosen to be the finished, reference genome differs between samples, and the other target genomes in the sample simulation are replaced with simulated draft versions of the actual finished sequences. Then these sequences were aligned using WGASA and the SAP process was run as described above. A total of 1101 stochastic simulations per 'experiment' were performed, requiring $18 min per simulation. Each simulation involved randomly selecting the subset of target and NN sequences to be included, simulating the draft data based on the finished genomes, aligning the target sample, and finally running the DNA Signature Pipeline. There were four combinations examined: (i) finished variola and finished NN, (ii) draft variola and finished NN, (iii) finished variola and draft NN and (iv) draft variola and draft NN, with each of the draft runs repeated for both low and high sequencing error rates. The combination (iv) was also run with intermediate quality simulated draft variola and low-quality simulated draft NNs. In total, there were eight computational experiments for the finished and simulated draft variola data.
We have used the following function to estimate viral sequencing costs, based on discussion with our laboratory colleagues involved in sequencing and finishing. This is merely a rough estimate, and the actual costs of sequencing any given organism may differ substantially from this rule-of-thumb calculation. In Equation 1, it is assumed that the cost of sequencing viruses does not decline for sequencing second and subsequent isolates. While this may be a false assumption in cases where isolates are similar to one another, in other cases where the new sequences are divergent, as isolates from different outbreaks or for viruses with rapid mutation rates, the cost is especially unlikely to decline. In addition, the $0.40/bp figure for draft of 6· to 8· coverage could range from $0.30 to $0.50/bp using shotgun sequencing, but may be as low as $0.10-$0.20/bp if primer walking works well (i.e. known primer sites are found in new isolates). Finishing could be 1-3 times more than the cost of draft, so we used a factor of 2 times more (draft $0.4/bp, finished $0.4 + 0.8/bp) in the equation above as a reasonable estimate. With rapidly evolving sequencing technologies and costs, these are only rough guides that may quickly become outdated.
It may be substantially less expensive, on the order of 3-fold, to generate draft compared with finished sequence data for an organism like Marburg virus, according to estimates using Equation 1. For example, for $45K, one could sequence either two finished genomes or one finished and three draft. However, draft sequencing of this low quality (3· to 6·) for Marburg causes a dramatic decline in the ability to computationally eliminate regions of poor conservation, and thus to exclude poor signature regions ( Figure 2 ). This occurs because gaps in the draft data of some of the sequences mask sequence variation among strains. Using the best available data, all six finished genomes, there is 75.2% sequence conservation. The deficiencies of draft data give a false impression that there is 92.6% sequence conservation (Table 1) . Each additional finished genome reduces the conserved fraction by $5%, compared with a reduction of only 2% per genome for the draft data.
The overestimation of conservation using draft Marburg data also results in overestimation of the number of signature candidates (Figure 3 ). Samples of four draft targets plus one finished reference yield 43 signature candidates. A smaller sample size of only two draft targets and one finished reference generate upwards of 80 candidates. These results differ from those using finished genomes, where the lack of sequence conservation is more evident and there are zero TaqMan signatures conserved among all strains. Most combinations of four finished genomes are sufficient to eliminate nonconserved signatures ( Figure 3A ). Although predictions that there are 0 signature candidates shared among all finished strains may seem to argue against TaqMan methods, in fact this information provides important guidance for the development of TaqMan signatures with degenerate bases or a set of signatures that will, in combination, pick up all sequenced strains. Other analyses indicate that there are TaqMan signatures conserved among five of the six strains, so that two signatures would form a minimal set that could detect both the one divergent and the other five strains.
Estimated sequencing costs of draft variola and draft NNs indicate that draft may require only one-quarter to one-half the costs of finished sequencing. Simulations of high-quality draft data indicated that it is as good as finished data for diagnostic signature prediction. The conservation range plots ( Figure 4A and B) are virtually identical for finished and high-quality draft, and indicate that $98% of the genome is conserved among sequenced isolates. For intermediate quality draft ( Figure 4C ) the conservation range plot is also similar to that for finished sequence, showing that $97% of the genome appears to be conserved. The range plots for the number of TaqMan signature candidates are very similar for finished sequence data, high or intermediate quality draft target, and high or low-quality draft NNs ( Figure 5A-D) .
In contrast to the results using high-quality simulated draft or actual Marburg draft, simulations of low-quality variola draft target illustrate that sequence conservation may be underestimated compared with results with finished sequence data, due to sequencing errors (Table 1 and Figure 4D ). With lowquality draft target, it appears that only 58% of the genome is conserved among isolates.
Low-quality (high error rate) draft NN data, however, yield results that are very similar to those with high-quality draft or finished NN data, as long as the target sequence information is of intermediate to high quality ( Figure 5C -D andFigure 6): At least four NN sequences are necessary to ensure that signature regions are unique, whether the NN data are low-or highquality draft or finished. That is, our simulations indicate that low-quality draft NN data are adequate for predicting DNA signatures, as long as there is good quality target sequence data. This results because errors in the NN sequences occur at random locations that differ in each NN sequence. As long as at least one of the NNs has enough correct sequence to eliminate each of the non-unique target regions, then the unique regions of the target can be determined.
The results illustrated in the figures are emphasized by the data in Table 1 . This table shows the fraction of the target genome that is conserved and conserved+unique, the number of conserved+unique regions that are at least 18 contiguous base pairs long and the number of base pairs in the largest of these regions, since these are the sections that are of sufficient length for one or possibly more primers to be located. The number of these regions is similar for finished data and for draft with a low error rate. Low-quality draft (with a high rate of sequencing errors) for the target data, however, gives the false impression that there are fewer and shorter regions that are conserved and suitable as signature regions than is actually the case.
There is an artifact in some of our results that is a consequence of the order in which we calculate conservation and then uniqueness, although this does not affect the signatures that are predicted. First, a conservation gestalt is generated from the sequence alignment, in which non-conserved bases are replaced by a dot ('.'). Then uniqueness is calculated based upon perfect matches of at least 18 bp long between the conservation gestalt and a large sequence database of non-target The percent of the target genome that is conserved varies slightly among the runs using finished target sequences because different genomes were randomly selected to be the reference strain in each multiple sequence alignment.
sequences. Non-conserved bases in the conservation gestalt may break up a region into conserved fragments of <18 bases long, and as a result these short fragments are not tested for uniqueness. Consequently, if there is a low level of conservation, then we may overestimate the fraction of the genome that is unique. For example, in Table 1 the conserved+unique fraction is 4% with finished variola target data, but is overestimated at 58% with low-quality draft. This artifact does not, however, affect TaqMan signature prediction, since the regions suitable for primers and probes must have at least 18 contiguous, conserved bases, and all of these are tested for uniqueness, i.e. there is no underestimation of uniqueness in conserved fragments that are at least 18 bp in length, and thus no underestimate of uniqueness in the predicted signatures.
We are working to eliminate this issue in future versions of the software.
Protein results show a large disparity between finished and draft data. There are 113 protein signature candidates for finished Marburg data compared with only two protein signature candidates for Marburg draft (Figure 7 ). For variola, using all available target data, 97, 14, 6 and 0 protein signatures are predicted using finished, low error, intermediate error and high error draft target data, respectively ( Figure 8 ). Thus, sequencing errors substantially reduce the detection of amino acid sequence conservation, even if sequencing errors occur at the low rate of 10 À4 -10 À5 across most of the genome. The pattern of how additional sequences reduce the number of protein signature candidates also differs for draft compared with finished sequence data. With finished data, there is a large range in the number of peptide signature candidates predicted with 17 or fewer variola genomes, and this range narrows around the lower bound with >17 genomes. With 16 genomes, the 75% quantile mark approaches the final predicted number of 97 signatures ( Figure 8A ). This pattern indicates that there is a set of 97 peptides that are highly conserved among all currently sequenced variolas, which are unlikely to be eroded even as more sequence data are obtained. In other words, additional sequence data are probably not needed at this time in order to computationally predict good peptide signature targets, and as few as 16 finished target sequences would most likely have been adequate to generate this same list of $100 peptide signatures.
Draft data, in contrast, whether they are of low or high quality, mask the above pattern ( Figure 8B and C): the range and 75th quantile of the number of peptide signatures gradually decline with each additional target sequence (rather than a sudden, sharp drop as is seen with the finished data), suggesting that additional target sequences would continue to erode the number of peptide signatures. This occurs since sequencing errors occur at random, in different locations in each of the draft target genomes, and obscure the truly conserved peptides. One might falsely infer from peptide SAP results based on the draft data that additional sequencing (beyond the 28 variola major genomes used here) would be useful in generating peptide signature candidates. In actuality, however, SAP analyses using the finished sequence data indicate that there are already ample sequence data for peptide signature prediction.
The failure of draft sequencing for Marburg at 3· to 6· coverage or of simulated variola draft with a high error rate to facilitate the prediction of detection signatures highlights a need for finished viral sequences, or at least for draft of high quality such as 8·. Otherwise, a large number of signature candidates either will fail in screening because they are incorrectly designated as conserved among strains (as observed with the Marburg results), or too few regions will be classified as conserved (as observed with variola), and thus not be considered for signatures.
The variola simulations with intermediate to high-quality draft (that is, a low error rate, approximating what one might observe with 8· coverage) target and/or NN genomes deliver virtually the same results as finished genomes. Considering that it costs approximately three times as much to generate finished sequence as it does draft, our analyses indicate that investing in more high-quality draft target genomes is better than investing in fewer finished genomes. For our analyses, only one target strain must be finished, and the remaining target sequences and all the NNs may be provided as draft.
Our results indicate that NN sequencing may be of low coverage, and thus of low quality, without serious detriment to signature prediction, as long as there are at least four NN draft genome sequences.
If high-quality draft sequence is used, and it appears that there is too little sequence conservation among target strains, one might relax specifications for 100% conservation among strains for diagnostic signature prediction. Calculations indicate that it is often possible to generate signatures if one allows a base to be considered 'conserved' if it is present in only a fraction of the genomes (e.g. 75%) rather than the standard requirement for 100% conservation when finished sequence data are used. We have used this 'ratio-to-win' option to generate signature candidates for some highly divergent RNA viruses (for which we have finished sequence), although usually our preference is to include degenerate bases, especially when there are only a few bases with heterogeneity among strains in a given signature candidate. Using a ratio-to-win approach may be particularly important for the generation of protein signature candidates, since draft target data severely compromises the ability to detect conserved strings of amino acids.
In summary, intermediate to high-quality draft sequencing of target genomes, combined with low-quality draft sequencing of close phylogenetic relatives, is sufficient for the prediction of DNA diagnostic signatures. Prediction of peptide/ protein signature candidates, in contrast, requires finished sequencing to avoid substantial underestimation of conserved peptide regions. An ontology for immune epitopes: application to the design of a broad scope database of immune reactivities BACKGROUND: Epitopes can be defined as the molecular structures bound by specific receptors, which are recognized during immune responses. The Immune Epitope Database and Analysis Resource (IEDB) project will catalog and organize information regarding antibody and T cell epitopes from infectious pathogens, experimental antigens and self-antigens, with a priority on NIAID Category A-C pathogens () and emerging/re-emerging infectious diseases. Both intrinsic structural and phylogenetic features, as well as information relating to the interactions of the epitopes with the host's immune system will be catalogued. DESCRIPTION: To effectively represent and communicate the information related to immune epitopes, a formal ontology was developed. The semantics of the epitope domain and related concepts were captured as a hierarchy of classes, which represent the general and specialized relationships between the various concepts. A complete listing of classes and their properties can be found at . CONCLUSION: The IEDB's ontology is the first ontology specifically designed to capture both intrinsic chemical and biochemical information relating to immune epitopes with information relating to the interaction of these structures with molecules derived from the host immune system. We anticipate that the development of this type of ontology and associated databases will facilitate rigorous description of data related to immune epitopes, and might ultimately lead to completely new methods for describing and modeling immune responses. An epitope can be defined as the molecular structure recognized by the products of immune responses. According to this definition, epitopes are the specific molecular entities engaged in binding to antibody molecules or specific T cell receptors. An extended definition also includes the specific molecules binding in the peptide binding sites of MHC receptors. We have previously described [1] the general design of the Immune Epitope Database and Analysis Resource (IEDB), a broad program recently initiated by National Institute of Allergy and Infectious Diseases (NIAID). The overall goal of the IEDB is to catalog and organize a large body of information regarding antibody and T cell epitopes from infectious pathogens and other sources [2] . Priority will be placed on NIAID Category A-C pathogens (http://www2.niaid.nih.gov/Biodefense/ bandc_priority.htm) and emerging/re-emerging infectious diseases. Epitopes of human and non-human primates, rodents, and other species for which detailed information is available will be included. It is envisioned that this new effort will catalyze the development of new methods to predict and model immune responses, will aid in the discovery and development of new vaccines and diagnostics, and will assist in basic immunological investigations.
The IEDB will catalog structural and phylogenetic information about epitopes, information about their capacity to bind to specific receptors (i.e. MHC, TCR, BCR, Antibodies), as well as the type of immune response observed following engagement of the receptors (RFP-NIH-NIAID-DAIT-03/31: http://www.niaid.nih.gov/contract/archive/ rfp0331.pdf).
In broad terms, the database will contain two general categories of data and information associated with immune epitopes -intrinsic and extrinsic (context-dependent data). Intrinsic features of an epitope are those characteristics that can be unequivocally defined and are specified within the epitope sequence/structure itself. Examples of intrinsic features are the epitope's sequence, structural features, and binding interactions with other immune system molecules. To describe an immune response associated with a specific epitope, context information also needs to be taken into account. Contextual information includes, for example, the species of the host, the route and dose of immunization, the health status and genetic makeup of the host, and the presence of adjuvants. In this respect, the IEDB project transcends the strict boundaries of database development and reaches into a systems biology application, attempting for the first time to integrate structural information about epitopes with comprehensive details describing their complex interaction with the immune system of the host, be it an infected organism or a vaccine recipient [1] [2] [3] .
For these reasons, it was apparent at the outset of the project that it was crucial to develop a rigorous conceptual framework to represent the knowledge related to the epitopes. Such a framework was key to sharing information and ideas among developers, scientists, and potential users, and to allowing the design of an effective logical structure of the database itself. Accordingly, we decided to develop a formal ontology. Over the years, the term "ontology" has been defined and utilized in many ways by the knowledge engineering community [4] . We will adopt the definition of "ontology" as "the explicit formal specifications of the terms in a domain and the relationships among them" [5] . According to Noy and McGuinness [6] , "ontology defines a common vocabulary for researchers who need to share information in a domain and helps separate domain knowledge from operational knowledge". Thus, availability of a formal ontology is relevant in designing a database, in cataloging the information, in communicating the database structure to researchers, developers and users, and in integrating multiple database schema designs and applications.
Several existing databases catalog epitope related data. We gratefully acknowledge that we have been able use these previous experiences in the design and implementation of the IEDB. MHCPEP [7] , SYFPEITHI [8] , FIMM [9] , HLA Ligand Database [10] , HIV Immunology Database [11] , JenPep [12] , AntiJen [13] , and MHCBN [14] are all publicly available epitope related databases. In general, these databases provide information relating to epitopes, but do not catalog in-depth information relating to their interactions with the host's immune system. It should also be noted that none of these databases has published a formal ontology, but all of them rely on informal or implicit ontologies. We have taken into account as much as possible these ontologies, inferring their structure by informal communications with database developers or perusal of the databases websites.
The ontology developed for IEDB and described herein complements two explicit ontologies that are presently available: the IMGT-Ontology and the Gene Ontology (GO). The IMGT-Ontology [15] was created for the international ImMunoGeneTics Database (IMGT), which is an integrated database specializing in antigen receptors (immunoglobulin and T Cell receptors) and MHC molecules of all vertebrate species. This is, to the best of our knowledge, the first ontology in the domain of immunogenetics and immunoinformatics. The GO project [16] provides structured, controlled vocabularies that cover several domains of molecular and cellular biology. GO provides an excellent framework for genes, gene products, and their sequences, but it does not address the specific epitope substructure of the gene products. The IMGT provides an excellent ontological framework for the immune receptors but lacks information relating to the epitopes themselves. Therefore it was necessary to expand the available ontologies and to create an ontology specifically designed to represent the information of immune receptor interaction with immune epitopes. Wherever possible, the IEDB ontology conforms to standard vocabularies for capturing values for certain fields. For capturing disease names, IEDB uses the International Classification for Diseases (ICD-10) [17]. The NCBI Taxonomy database nomenclature [18, 19] is used to capture species and strain names, and HLA Allele names are consistent with the HLA nomenclature reports [20] .
The IEDB is being developed as a web-accessible database using Oracle 10g and Enterprise Java (J2EE). Industry standard software design has been followed and it is expected that IEDB will be available for public users by the end of 2005.
Protégé http://protege.stanford.edu was used to design and document the IEDB ontology. Protégé is a free, open source ontology editor and knowledge-base framework, written in Java. It provides an environment for creating ontologies and the terms used in those ontologies. Protégé supports class, slot, and instance creation, allowing users to specify relationships between appropriate entities. Two features that IEDB ontology effort used extensively were Protégé's support for creating ontology terms and for viewing the term hierarchies and the definitions. The support for a central repository on ontologies, along with browsing support, is key in reviewing and reusing ontologies.
While there are several open source tools available [21] for developing ontologies, we selected Protégé because of its extensibility to a variety of plug-ins that are readily available for integration. It also has the ability to export to different formats including the Ontology Web Language (OWL) (http://www.w3.org/TR/owl-features/), which allows interoperability with other ontologies.
We have previously described some of the general concepts relating to the IEDB design [1, 2] . More information relating to various aspects of the project can be accessed at http://www.immuneepitope.org/. Herein, we report a detailed description of the novel aspects of the IEDB ontology. In designing our application architecture, we have followed the common system engineering practice of first determining the scope and nature of the data involved. A first essential step is to understand the semantics of the domain and to capture that knowledge in an agreed-upon format. Arranging the domain concepts in a taxonomy is one of the initial organizing steps in the ontology design process. The class hierarchy represents the generalization and specialization relationships between the various classes of objects in a domain [6] . Briefly, classes describe concepts in the domain. Subclasses represent concepts (classes) that are more specific than the superclass and these subclasses can have their own unique properties. Slots represent properties of the classes. For example, in Figure 1 , we see that there is a class named Reference and three more specific subclasses of Reference: Journal Article, Patent Application, and Direct Submission. Figure 1 also shows that the class Epitope has a number of properties (slots) associated with it such as "has Epitope Structure" and "has Epitope Source".
Our approach for creating the class hierarchy was a topdown development process where we defined each class in a domain and then identified its properties before building the hierarchy. The main classes identified for IEDB are Reference, Epitope Structure, Epitope Source, MHC Binding, Naturally Processed Ligand, T Cell Response, and B Cell Response (Figure 1 ). The Epitope class is the main class that encompasses all the individual concepts that were identified. The individual concepts are related to other classes. The primary relationships use the sub-class relationship or use a property (shown in the figures by the arcs labeled "has") that has a restriction on the type of the value that may fill that slot.
"Reference" is the class encompassing information related to the data source from which an epitope and its related information are extracted into the IEDB. We have identified three broad subclasses of References that describe where epitope information will be obtained. They are Journal Article, Patent Application, and Direct Submission. The complete listing of slots (fields) encompassed by the Journal Article, Patent Application, and Submission classes are provided in Figure 2 . The Journal Article class refers to manuscripts published in peer-reviewed journals. The Patent Application class captures all the reference fields for a patent application that contain epitope information. The Submission class captures information about sources that contribute data to the IEDB directly. Data deposited by the Large Scale Antibody and T Cell Epitope Discovery contracts [3] and those transferred from other websites fall into this class.
The Epitope Structure and Epitope Source classes capture intrinsic features of an epitope. The Epitope Structure class captures the physical and chemical features of an epitope. Virtually any molecular structure may provoke an immune response, such as proteins, carbohydrates, DNA, and lipids. In the Epitope Structure class, structural information relating to linear sequences and 2-D structures of the epitope, if available, are catalogued. The Epitope Source class captures the phylogenetic source of an epitope, including species of origin, gene name, protein name, and links to other databases for more detailed information about proteins and genes. Figures 3A and 3B show the listings of properties (slots/fields) encompassing the Epitope Structure and Epitope Source classes.
The experimental data and information about specific experiments and the methodology utilized are captured in the Assay Information class. The name of the assay used, the type of response measured in the assay, and the readout of the assay are examples of information captured in the Assay Information class. This important class is used as a superclass of several other classes (and thus its properties are inherited by those classes). A complete listing the properties (slots/fields) in the Assay Information class is shown in Figure 3C .
As with Assay Information, the classes Immunization, Antigen, and Antigen Presenting Cell are used in multiple other class descriptions. Features relating to the induction of the immune response are captured in the Immunization class ( Figure 4A ). It has relationships to other classes like Immunized Species, Immunogen, In vivo Immunization, and In vitro Immunization. Immunized Species contains information relating to the host that is being immunized. The Immunogen class describes the molecules that induce the immune response and an associated carrier molecule, if present. Features relating to how the immunogen was introduced to the immunized species are captured under the In vivo and In vitro Immunization classes.
Similarly, antigens are defined as the whole molecules that react with the products of an immune response (as opposed to the epitopes which are the specific structures, contained within the antigen that engages the immune receptor). Information relating to the antigen and any associated carrier molecule is captured in the Antigen class ( Figure 4B ). During immune responses, antigen-presenting cells process antigens and present peptide epitopes complexed with MHC molecules. This information is captured in the Antigen Presenting Cells class, which has a relationship to the MHC Molecules and the Source Species classes ( Figure 4C ). The Source Species class describes the species information from which the antigen presenting cells are derived.
The MHC Binding class captures the details relating to the interaction of the epitope with specific MHC molecules and information relating to the MHC molecule along with any available Epitope-MHC complex structure details. This class also has a slot that is restricted to be an instance of the Assay Information class ( Figure 5A ).
Overview of IEDB Class Hierarchy Figure 1 Overview of IEDB Class Hierarchy.
classes. Extrinsic features are context-dependent attributes, being dependent upon specific experimental conditions. The Naturally Processed Ligand class captures data related to epitopes that are naturally processed and presented on the cell surface. This class has properties that are instances of classes including Antigen Presenting Cell, Antigen, and Assay Information ( Figure 5B ).
The Naturally Processed Ligand class differs from the MHC Binding class in that information related to the antigen that was processed and the cell types in which the processing occurred is represented. MHC Binding class captures data relating to in vitro MHC binding assays, which assess the epitope's binding capacity to the MHC molecule. Hence the MHC Binding class does requires neither the Antigen class not the Antigen Presenting Cells class. In general, naturally processed ligands are assessed in the absence of a T cell response, for example, identified by direct elution from MHC molecules extracted from infected cells or antigen presenting cells. Thus, the Immunization class is not used as a value restriction by the Naturally Processed Ligand class.
The T Cell Response class captures all of the T cell mediated immunity-related information ( Figure 6A ). It has properties that are of type: Immunization, Effector Cells, Antigen Presenting Cell, Antigen, Assay Information, and Epitope-MHC-TCR Complex. The Effector Cell class describes the cells that are elicited upon immunization and that acquire measurable functions as a result. The B Cell Response class describes antibody responses that are related to the epitope ( Figure 6B ). This class has properties that are of type: Immunization, Antibody Molecule, Antigen, Assay Information, and Antigen-Antibody Complex. Because B cell responses do not require MHC binding and 
There are three classes that capture information about the 3D structure of complexes: Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex. The Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex classes are used as restrictions on properties of the MHC Binding, T Cell Response, and B Cell Response classes respectively ( Figures 5A, 6A, and 6B) . These Complex classes capture the Protein Data Bank (PDB) Identifier, which provides detailed information about 3D structures. The Protein Data Bank [22, 23] contains approximately 1600 3D structures that are of immunological interest. Other information that is not available in PDB, such as the atom pairs that are involved in the interactions between molecules, the specific residues, the contact area of the molecules, and allosteric effect, is also captured here.
Each class has numerous slots that capture detailed information associated with epitopes. As mentioned above, a complete list of all the classes, their properties, and relationship, can be found at http://www.immuneep itope.org/ontology/index.html. One of the files provided as supplementary material contains two examples of how two literature references [24, 25] containing epitope information are extracted into the IEDB ontology (additional file 1). Along with the class hierarchy, the IEDB's data dictionary (additional file 2) provides more detailed information about the fields that are defined for the IEDB. The data dictionary contains a textual overview description and a listing of fields that are required to be completed for IEDB entries. The data dictionary also allows database users to provide comments and suggestions to IEDB team to enhance the formal ontology.
The IEDB will be a comprehensive resource pertaining to epitopes of the immune system. By extensively curating both intrinsic and extrinsic features associated with epitopes, the IEDB is expected to provide substantially greater detail about specific epitopes than any other databases presently available. The IEDB will be populated with data derived from three main sources, namely the peer-reviewed literature, patent applications, and direct submission. Epitope data published in the literature and patent applications are curated manually by the IEDB's curation team. Data from already existing epitope databases, whose authors have agreed to share their data, will also be imported into the IEDB. Apart from these, a main data source will be the direct submission of data from the Large-scale Antibody and T Cell Epitope Discovery programs [3] that are funded by NIAID. Presently, fourteen contracts have been awarded under this program, and all of them will submit their data to the IEDB. Direct antibody and T cell epitope submissions will also be sought from the broader research community, with an emphasis on antibody epitopes to NIAID Category A-C pathogens. Because of the large scale of the IEDB project, a formal ontology is critical to ensure consistency in the representation of data.
Communication between database developers, researchers, analysis tool developers, and team members is crucial, and can be performed in harmony only when a common vocabulary is established. An ontology, which is an explicit formal specification of the terms in the domain and relationships among them, is an effective way to share the knowledge contained in that domain. Accordingly, since the IEDB's domain is epitope-related data, we have created an ontology that captures detailed conceptual structure related to these data.
The development of this ontology has relevance for the expansion and modification of the epitope knowledge base. Our ontology design defines individual concepts as separate classes and then defined relationships between these classes and other objects in the domain. These classes serve to restrict the values that will describe properties of objects in the database. For example, the species is a separate concept defined in its own class. Depending on the context, this can refer to an immunized species or the species from which antigen presenting cells are derived. Similarly MHC Molecules is defined as a separate class, and it is used as a value restriction by concepts like MHC Binding and Antigen Presenting Cells. Defining concepts as separate classes and using them to restrict the values of properties in other classes facilitates the expansion and modification of our ontology. Adding properties (slots) to concepts is a task easily accomplished when there are well-defined class descriptions that may serve as value restrictions on the properties, and providing that High-level classification of Immunization (A), Antigen (B), and Antigen Presenting Cells (C) class these class descriptions are general enough to apply in all instances. We have ensured in our design that each concept is atomic and that it can be re-used by various classes.
The development of a formal ontology is valuable to database users and in particular to scientists contributing data to, and downloading data from, the IEDB. We anticipate that the availability of a formal ontology will ensure that a common language and shared understanding of concepts will inspire this type of communication, thus ensuring maximum efficiency and accuracy. The formal ontology developed will most likely require refinement and fine tuning when users provide suggestions and new technologies for performing experiments are discovered. The IEDB website will provide mechanisms for the users to provide suggestions and participate in the enhance- ment of the ontology. The IEDB Data Dictionary has a separate column for the users to provide comments on specific data fields. The IEDB website will also host web forms that will guide users to conform to the ontology definitions when submitting data. Apart from the web forms, an XML schema definition (XSD) will be available on the website for users to inspect and use in their data submission. Users will also be able to download epitope records from the website.
In the process of developing new ontologies, it is good practice to leverage existing community standards. In our initial analysis, we confirmed that there were no explicit ontologies that efficiently captured epitope details as per the scope of the IEDB program. As mentioned above, we have utilized, as much as possible, inferred ontologies from existing epitope databases. Among the ontologies that we analyzed, IMGT-Ontology and Gene Ontology were the only two formal ontologies that were related to the epitope domain. The IMGT-Ontology was designed for the ImMunoGeneTics database. IMGT is an integrated database specializing in antigen receptors (immunoglobulins and T-cell receptors) and the major histocompatibility complex of all vertebrate species. The ontology developed for this database has specific immunological content, describing the classification and specification of terms needed for immunogenetics. The IEDB does conform to IMGT's standards about receptors and MHC molecule chains in the sense that all the chain names follow IMGT's controlled vocabulary.
GO provides structured controlled vocabularies for genes, gene products, and sequences annotated for many organisms. The IEDB complements GO in terms of epitopes of immunological interest since GO is incomplete in this area. Antigens, which are primary sources of epitopes, are annotated in GO. Thus, in essence, the IEDB could be utilized to provide an extension of GO for antigens that contain epitope-related information.
Perhaps the most important element in the development of the IEDB ontology is that, to the best of our knowledge, this represents the first immunological ontology specifically designed to capture both intrinsic biochemical and extrinsic context dependent information. In this respect, it is similar in spirit, but different in approach, from other knowledge resources relating to systems biology. We anticipate that the development of this type of ontology and associated databases might lead to completely new methods for describing and modeling immune responses. Accordingly, this new program might represent a novel tool to assist in the design, testing, and development of new ways to combat infectious diseases and other immune related pathologies such as cancer and autoimmune diseases.
A complete listing of IEDB's class hierarchy and its properties is available at http://www.immuneepitope.org/ ontology/index.html  Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon. In real-time RT-PCR, the expression levels of the target genes of interest are estimated on the basis of endogenous controls. Various housekeeping genes, ribosomal RNA (rRNA) and total RNA are most commonly used as references in gene expression analysis today. The purpose of these controls is to remove or reduce differences due to sampling, i.e. differences in RNA quantity and quality.
The ideal endogenous control should be expressed at a constant level among different tissues of an organism, at all stages of development and should be unaffected by the experimental treatment. It should also be expressed at roughly the same level as the RNA under study [1] . However, data normalization in real-time RT-PCR remains a real problem, especially for absolute quantification [1] . Numerous studies have revealed that no single universal gene has a constant expression level under all developmental or experimental situations. The best choice of reference gene to use as an endogenous control varies, depending on the tissues of interest in the experiment. A large number of genes have for this reason been selected for normalization of mRNA expression data [2, 3] . If the selected reference gene fluctuates randomly between samples, small differences in expression between the genes of interest will be missed. Gene expression coefficient of variation (CV) between different groups of individuals should ideally be as low as possible [4] . In general, the stability of several potential reference genes should be tested in every examined tissue or cell, and under different experimental design [5, 6] . An increasing number of papers are discussing the selection of reference genes in real-time RT-PCR analyses [3, 7] .
Two of the most commonly used reference genes are those encoding glyceraldehyde-3P-dehydrogenase (GAPDH) and β-actin. Recently, the use of these two genes as endogenous controls has been scrutinized, and several studies have documented that the GAPDH and β-actin genes should be used with caution as controls [2, 8, 9] . GAPDH in mammals is known to play a role in a broad range of cellular mechanisms (for review see Sirover [10] ), including being a key enzyme in glycolysis. Overall, GAPDH mRNA levels might be regulated under a large number of physiological states, and its use as a reference is inappropriate for most experimental conditions. Actin is a major component of the protein scaffold that supports the cell and determines its shape, and is the most abundant intracellular protein in eukaryotic cells. Even though commonly used as a reference, the application of the β-actin gene has recently been characterized as a historical carryover from northern blots and conventional RT-PCR (for a general discussion on the use of 'classic' reference genes like GAPDH and β-actin, see Huggett et al. [7] ). Eukaryotic elongation factor 1A (eEF1A, formerly elongation factor 1 alpha) plays an important role in translation by catalyzing GTP-dependent binding of aminoacyl-tRNA to the acceptor site of the ribosome. However, the protein is involved in a broad diversity of functions and constitutes 1-3% of the total cytoplasmic protein content of the cell. In human, cDNAs of two actively transcribed isoforms have been cloned (eEF1A-1 and eEF1A-2) (for review see Thornton et al. [11] ). Two paralog EF1A genes (A and B) have recently been applied as references in real-time qRT-PCR of Atlantic salmon [12] . It is plausible to assume that the presence of these highly similar genes is a result of a tetraploidization event that occurred in a salmonid ancestor in the comparatively recent past [13, 14] .
Previously, the 18S rRNA gene was considered to be an ideal internal control in qRT-PCR analysis (Ambion [15] ). Ribosomal RNA constitutes up to 80-90% of total cellular RNA, and several studies have shown that rRNA varies less under conditions that affect the expression of mRNAs (discussed in Bustin & Nolan [16] ). However, questions have been raised against the use of ribosomal RNA genes as references. Vandesompele et al. [5] have stressed the fact that there sometimes might be imbalances in rRNA and mRNA fractions between different samples, making genes encoding ribosomal RNAs unsuitable as references.
To meet these challenges of accurate interpretation of realtime qRT-PCR data, the authors suggested that an index of the most stable housekeeping genes should be used for normalization, and developed the geNorm VBA applet for Microsoft Excel in this regard [5] . A similar software tool, the BestKeeper, has been developed by Pfaffl et al. [6] . These tools can be used to find the most stable reference genes under different experimental conditions. We used the geNorm software which determines the individual stability of a gene within a pool of genes [5] . The stability is calculated according to the similarity of their expression profile by a pair-wise comparison, using their geometric mean as a normalizing factor. The gene with the highest M, i.e. the least stable gene, is then suggested excluded in a stepwise fashion until the most stable genes are determined, and an index suggested, based on the best genes. geNorm has been used to select the most stable reference genes in several recent studies (e.g. [4, 17, 18] ).
The aim of this work was to evaluate the usefulness of six potential reference genes in the Atlantic salmon. Salmonid fish are among the most widely studied model fish species in general, and extensive basic information on many different aspects of their biology has been collected [19] . Large-scale DNA-sequencing projects on salmon have been initiated in several laboratories http://www.sal mongenome.no/cgi-bin/sgp.cgi; http://web.uvic.ca/cbr/ grasp/; http://www.abdn.ac.uk/sfirc/salmon/; http:// www.bcgsc.ca/gc/salmon. In this work we selected the two 'classic' reference genes encoding GAPDH and β-actin, two genes encoding 18S rRNA and S20 ribosomal protein and two paralog genes encoding elongation factor 1A (EF1A A and EF1A B ). To evaluate their usefulness as reference genes, RNA from eight tissues of six adult salmon were subjected to real time PCR. The relative transcription levels of the genes were also estimated in four phases of young salmon going through smoltification, in order to check their stabilities under physiological stressful conditions.
The ranking of the six examined genes analyzed by geNorm is shown in Table 3 . In six tissues (muscle, liver, gills, head kidney, spleen and thymus), the EF1A B gene emerged as the most stable, whereas the EF1A A gene was ranked number one in brain and the β-actin gene was ranked number one in intestine. The 18S rRNA and S20 genes were ranked among the worse genes in all tissues. Not surprisingly, the GAPDH gene was ranked worse in five tissues (liver, head kidney, spleen, brain and thymus), confirming the general skepticism against the use of this gene as reference [7, 16, 20] . Combined, the total ranking reads EF1A B >EF1A A >β-actin>18S rRNA>S20>GAPDH. We did not analyze our data with the Bestkeeper software. Analyzing reference genes in virus infected cells, Radonic et al. [4] concluded that the Bestkeeper tool gave results that slightly deviated from, but nevertheless corresponded to, those obtained using geNorm.
To be able to evaluate gene stability under stressful conditions, mRNA expression of the selected genes was examined in gills of salmon going through smoltification. Prior to seawater entry, juvenile anadromous salmon undergo a parr-smolt transformation, characterized by behavioral, morphological and physiological changes, known to be challenging for the fish. Physiological alterations include increased seawater tolerance, olfactory sensitivity, metabolic rate, scope for growth and changed hemoglobin and visual pigments [21] . We selected to examine the gills during smoltification, because this tissue plays a major role in ionic and osmotic regulation during adaptation to hyperosmotic seawater. Figure 1 shows the raw Ct values of the studied genes in gills before, during and after smoltification (smoltified in seawater and desmoltified in freshwater). In Figure 2 the same data are presented, but now normalized against an index calculated by geNorm of the three most stable genes (β-actin, EF1A A and EF1A B ). Based on the M values, geNorm ranks the stability of the six genes from 24 fish going through smoltification in the following order: EF1A B >EF1A A >β-actin>S20>18S rRNA>GAPDH ( Figure 3 ). In Figure 1 it can be seen that the 18S gene had the lowest individual raw Ct variation. Most individual raw Ct variation of the studied genes is seen in the presmolt and smolt groups. A characteristic drop in expression can be seen for all genes in the smolt group, compared to the presmolt group. After transfer to seawater, the individual raw Ct variation decreased for all genes. Overall, the raw Ct data suggest that the physiological challenging smoltification process affected the expression of all six genes. When the same data were normalized against an index of the three most stable genes, β-actin, EF1A A and EF1A B , the relative expression levels were altered for all genes. Now the ribosomal 18S gene emerges as the second worse, whereas the two paralog EF1A genes became the most stable. This might have to do with the fact that geNorm will top-rank co-expressed genes [22] , a weakness that has to be considered when evaluating paralog genes likely to be co-regulated. Even though the eEF1A-2 gene has been identified as an important oncogene and has been shown to be differently expressed in human tissues [11] , Hamalainen et al. [23] found the eEF1A-1 gene to be a good reference gene in real-time RT-PCR examinations. A similar finding was reported by Frost and Nilsen [24] in salmon louse, where they showed that the eEF1A and S20 genes were valid candidate references, whereas the 18S rRNA and GAPDH genes were unsuitable. The current findings based on geNorm evaluation question the recommended application of ribosomal genes as references (as suggested for example by Ambion (see reference [15] ), and are in line with earlier warnings against the use of rRNA genes as references [5, 6] . To avoid the normalization of the genes for β-actin, EF1A A and EF1A B against an index partly based on their own expression, the S20 gene was included in the index instead, and the mean normalized expression for these three genes calculated with the new index. The patterns of expression, however, were approximately the same for the three genes as seen in Figure 2 , suggesting that the gene-stability measure M can be used to find the most appropriate reference genes.
We see a correlation between the A260/230 absorbance on the NanoDrop and the PCR efficiency (data not shown). We tend to get PCR efficiencies that are too high in some samples with low A260/230 ratios. When the samples are treated with DNase solution, the A260/230 ratio usually drops. After DNase treatment, the A260/280 ratio increased from 1.8 to 2.1 (n = 45 samples). At the Table 3 : Evaluation of the usefulness of six potential reference genes in eight tissues of Atlantic salmon ranked by the geNorm software. 1 = best, 6 = worst. Six individuals were analyzed for six genes in eight tissues. qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater) Figure 1 qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater). Numbers indicate raw Ct values.
qRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon Figure 2 qRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon. The same data as in Figure 1 , but now normalized against an index of the three best genes (β-actin, EF1A A and EF1A B ) calculated with the geNorm software. The four groups were analyzed with Kruskal-Wallis test, and if significant, the overall p-value is given in the graphs. For β-actin, there were significant differences between the presmolt and the smoltified group (p < 0.05), the presmolt and the desmolt groups (n<0.01) and between the smolt and desmolt groups (p < 0.05). For EF1A A , there was a significant difference between the presmolt and the desmolt groups (p < 0.01). For EF1A B , there was a significant difference between the smolt and the smoltified groups (p < 0.05). An asterisk denotes significant differences between the groups.
same time, the A260/230 ratio dropped from 2.4 to 2.1. The DNase treatment therefore adds substances to the RNA solution that increases the absorbance at 230 nm more than it decrease the 260 nm absorbance. The added substance (salt or some other component) may inhibit the RT reaction or the PCR reaction, sometimes called PCR poisoning. We have seen that the A260/230 ratios are quite low in samples that give inadequate PCR efficiency slopes, especially with RNA from head kidney, thymus and intestine tissues, in which the gradient of the standard curve is less than -3.3 ( Table 2 ). The reason one obtain better amplification rate efficiencies with the more diluted samples is because the inhibitor has been diluted below its effective level. The obvious way around this problem is to dilute the amount of cDNA put into the PCR reaction. Alternatively, cleanup columns can be used to purify and concentrate the RNA.
Transcription levels of the examined genes and the coefficient of variance (CV) in different tissues varied considerably. mRNA levels in tissues are regulated by numerous endogenous and exogenous stimuli [16] . Transcription rates in metabolic active tissues might be up-regulated compared to those of less active tissues, whereas inter-tissue variation in degradation rates of mRNAs, for example, might affect mRNA stability [25] . The results revealed that muscle had the lowest CVs of the studied genes, compared to higher CVs in more active tissues like thymus, head kidney and spleen. In thymus, intestine, head kidney, gills, brain, liver and spleen, the 18S and S20 genes had the lowest CVs, based on raw Ct values. In all tissues, except intestine, the GAPDH gene had the highest CV. Except for thymus, the two elongation factor genes had relatively similar expression in all eight examined tissues. Their expression are most likely co-expressed in the examined tissues, and therefore favored in geNorm calculations [22] . The results also demonstrated that assays optimized for one tissue of an organism do not necessarily work equally well in other tissues. Of the tissues studied in this work, intestine, head kidney and spleen were the most troublesome.
Our data, based on geNorm calculations, suggest that the Atlantic salmon EF1A genes that have been tested in the present study may be good candidate reference genes. The GAPDH gene seems unsuitable as a reference in quantitative real-time RT-PCR. With regard to the 18S rRNA gene, this must be applied with caution. Tools like the geNorm applet for Microsoft Excel can be useful to help select the most stable genes in various experiments.
Tissues from 15 individuals were collected (852 ± 702 g, ranging from 254 to 1898 g). These individuals were not separated based on sex, size or sampling time, but treated as one heterogeneous group to examine the width of mRNA expression of the studied genes in eight different organs. This group of fish was handled and fed according to normal aquacultural management, and none of these individuals were exposed to any particular treatment. To examine if physiological stress may alter the gene expression in the gills, a total of 24 individuals were collected before (termed presmolt, 18.3 ± 0.9 g), during (termed smolt, 28.7 ± 3.7 g) and after smoltification. After smoltification, one group was kept and desmoltified in freshwater (termed desmolt FW, 30.0 ± 3.8 g), while the other group was transferred to seawater (termed smoltified SW, 30.2 ± 4.3 g) (n = 6 in each group). The Atlantic salmon examined during smoltification were from the anadromous population "Vosso" of the river Vosso in Southwestern Norway (see Nilsen et al. [27] for details on how these fish were treated). All fish were treated and euthanized according to Norwegian national legislation for laboratory animals.
Samples from eight organs, i.e. gills, liver, brain, head kidney, spleen, thymus, white muscle and posterior intestine, were dissected out and immediately frozen in cryo tubes in liquid N and stored at -80°C before RNA extraction. The RNA extracted from three spleen and four head kidney tissue samples were of low quality, and we had to redo the sampling from four individuals, These tissue samples Stability of six genes in gills of Atlantic salmon during smoltifi-cation calculated with the geNorm software were stored on RNA later (Ambion) at -20°C before further processing.
RNA was isolated with phenol-chloroform extraction as described by Chomczynski and Sacchi [28] , and stored in 100 µl RNase-free MilliQ H 2 O. Total RNA was extracted using Trizol reagent (Invitrogen, Life Technologies), according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by DNase treatment according to the manufacturer's description (Ambion). The RNA was then stored at -80°C before further processing. The quality of the RNA was assessed with the NanoDrop ® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A 260/280 nm absorbance ratio of 1.8 -2.0 indicates a pure RNA sample. The RNA 6000 Nano LabChip ® kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the integrity of the RNA. We used the RNeasy MinElute Cleanup kit from Qiagen to purify our most troublesome samples. With this kit the A260/230 ratio increased on average by 5 % (n = 10).
The PCR primer and TaqMan MGB probe sequences used for quantification of the genes encoding 18S rRNA, S20 ribosomal protein, β-actin, GAPDH, EF1A A and EF1A B , are shown in Table 1 . Four of these genes, 18S, β-actin, EF1A A and EF1A B , have also been used as references in real-time RT-PCR analyses of Atlantic salmon in other recent studies [12, 28] . The primers amplify PCR products between 57-98 basepairs (bp) long, which is within the range of 50-150 bp as suggested by Applied Biosystems for their Taq-Man assays. qPCR assays were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) to select appropriate primer and probe sequences from known Atlantic salmon genes. The mRNA sequences encoding S20 ribosomal protein and GAPDH were obtained from GenBank accession numbers BG936672 and BU693999, respectively (exon-exon borders were not considered). The EF1A A assay was based on a cDNA clone that we reported to the GenBank previously (AF321836), whereas the EF1A B assay was based on the EST BG933853.
An alignment with zebrafish indicated the exon-exon borders [29] . The chosen primers were subsequently used to confirm that the salmon genes contained an intron between the same sites as deduced from the alignment with zebrafish. The PCR products containing the introns were cloned into TOPO vector (Invitrogen) and sequenced (sequences can be provided upon request). PCR primers for β-actin were based on Atlantic salmon BG933897 and designed to span exon-exon borders of this gene, as deduced from corresponding genes in human and zebrafish (NW633959). For 18S rRNA the PCR primers and probe were designed from the Atlantic salmon sequence AJ427629, and placed in a conserved region of the gene based on comparison with the human gene. RNA samples were subjected to DNase treatment to avoid genomic DNA contamination. Amplified PCR products of all actual cDNAs were sequenced to ensure that the correct mRNA sequences were quantified. The fragments were sequenced with BigDye version 3.1 fluorescent chemistry (Applied Biosystems) and run on an ABI PRISM ® 377 DNA apparatus at the University of Bergen Sequencing Facility.
A two-step real-time RT-PCR protocol was developed to measure the mRNA levels of the studied genes in eight tissues of Atlantic salmon. For evaluation of the potential reference genes, raw Ct values are presented. The geNorm VBA applet for Microsoft Excel was used to determine the most stable genes from the set of tested genes [5] . The Ct values were transformed to quantities using standard curves, according to the geNorm manual. The gene expression stability (M) was calculated with the geNorm applet, and the genes were ranked from best to worst, based on the M value.
The GraphPad Prism 4.0 software (GraphPad Software, Inc.) was used for the statistical analyses in this work. Linear regression was used to determine PCR efficiency based on dilution curves. Non-parametric Kruskal-Wallis test was used to compare differences among four groups of salmon going through smoltification.
PAO was responsible for the experiment, data analysis and drafted the manuscript. KKL conducted the real-time RT-PCR analysis, and contributed throughout the experimental process. AEOJ constructed the qPCR assays for two of the genes. TON provided the cDNA from the smoltification experiment. IH participated as a supervisor in the study design, analyses and writing.  Relevance of human metapneumovirus in exacerbations of COPD BACKGROUND AND METHODS: Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR. RESULTS: We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10(5 )viral copies/ml in nasal lavage and 1.88 × 10(5 )viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD. CONCLUSION: HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely. Respiratory viruses play an important role in exacerbations of COPD and this has been increasingly recognised since the application of molecular detection methods [1, 2] . The most prevalent viruses detected by polymerase chain reaction so far were respiratory syncytial virus (RSV), Influenza A, Rhinovirus and Parainfluenza 3. Human metapneumovirus (hMPV) is a recently discovered respiratory virus first isolated from a dutch child with lower respiratory tract infection (LRTI) [3] . World wide distribution is probable since it has been isolated in North HMPV has been recognized as a member of the Paramyxoviridae like RSV and it is not only associated with bronchiolitis in most cases, but also with pneumonia, croup and exacerbations of asthma [14, 15] , diseases which share some features with COPD. Up to date reports about hMPV in adults are scarce. In a general Canadian population 14.8% of patients of all age groups with acute respiratory tract infections were hMPV positive. Thirty-three percent of hMPV-infected patients were hospitalized and the hospitalization rates were significantly higher among patients below 5 years and those over 50 years of age [16] . In another prospective cohort of adults, hMPV was detected in 4.5% of all illnesses but also in 4.1% of asymptomatic subjects. HMPV was most prevalent in young adults with children and in frail elderly [17] . HMPV infection can be severe since the virus was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . The role of hMPV in acute exacerbations of COPD (AE-COPD) has been studied recently in outpatients and only low frequencies have been observed [17, 19] . Up to now the prevalence of hMPV in patients hospitalized with AE-COPD is unknown. Our aim was therefore to investigate the frequency of detection of hMPV in a prospective cohort of hospitalized patients with AE-COPD and to compare these results to patients with stable COPD and to smokers without COPD.
Three different groups were studied. The first group consisted of hospitalized patients with an acute exacerbation of COPD (AE-COPD), the second group were subjects with stable COPD and the third group was composed of smokers without COPD. The groups were defined as previously published [20] . Briefly AE-COPD patients suffered from COPD as defined by GOLD [21] . Acute exacerbation was characterized by worsening in dyspnea, cough, and expectoration. A routine posterior-anterior chest radiograph was evaluated on admission by expert radiologists to exclude other other reasons for increased symptoms as pneumonia, tuberculosis, pulmonary fibrosis, bronchiectasis, bronchial carcinoma or congestive heart failure.Stable COPD patients did not have an exacerbation within the last 30 days prior to hospital admission and had no changes in therapy within the last 14 days (including inhaled and oral medication) and had been admitted for other medical reasons into departments of internal medicine other than pulmonary care. COPD subjects were recruited in a 2:1 ratio each month in order to prevent seasonal selection bias. Smokers have been smoking more than 10 pack-years, could have chronic symptoms like cough and phlegm but did not report dyspnea and did not have bronchial obstruction (FEV 1 /FVC>70%, FEV 1 >80% predicted). None of the smokers had a history of COPD or asthma, nor was using systemic or topic pulmonary medication. The smokers were recruited either from our smoking cessation initiative or by newspaper advertisement.
The study was approved by the ethical committee of the Ruhr-University of Bochum, Germany. Written informed consent was obtained from all patients and control subjects before inclusion in the study.
Clinical evaluation, spirometric tests, nasal lavage, induced sputum, specimen processing and viral ribonucleic acid (RNA) extraction were carried out as described by Rohde et al [2] . Elution volume was 100 µl. cDNA was generated with random-hexamer primers as previously published[2].
A hMPV-specific real-time RT-PCR designed and evaluated by Maertzdorf et al was used [22] . Primers and probe are localized within the nucleoprotein gene (NL-N) and the presence of a degenerate base within the probe allows detection of all four genetic lineages of hMPV.
The assays were performed using the TaqMan ® PCR Core Kit. The final volume was 25 µl containing 500 nM of the forward primer (NL-N-forward (5'-CATATAAGCAT-GCTATATTAAAAGAGTCTC-3')), 250 nM of the reverse primer (NL-N-reverse (5'-CCTATTTCTGCAGCATATTTG-TAATCAG-3')) and 500 nM of the probe (NL-N-probe (5'-FAM-TGYAATGATGAGGGTGTCACTGCGGTTG-TAMRA-3', in which Y is either a C or a T residue). Nuclease-free water was used as negative control and a plasmid containing the N gene of hMPV (kindly provided by James Simon, VIRONOVATIVE, EUR Holding, Erasmus University Rotterdam) was used as a positive control in all PCR runs. Cycling parameters were as follows: 5 min at 95°C, 45 cycles of 30 s at 95°C and 1 min at 60°C. Amplification and detection of RNA from virus isolates or clinical specimens were performed using the GeneAmp ® 5700 Sequence Detection System (Applied Biosystems). The real-time PCR product was cloned with the QIAGEN ® PCR cloning kit (QIAGEN, Hilden, Germany) and this standard plasmid DNA was used for absolute quantification of hMPV viral load. Calculations were performed as previously described for absolute quantification of RSV viral load [23] .
The primary objective of this study was to compare the frequency of hMPV detection in respiratory specimens between COPD patients with or without an acute exacerbation and smokers without COPD.
Continuous data were checked for normal distribution using the Kolmogorov-Smirnov test. The data were of non-parametrical distribution and results were expressed as median and range. Differences between groups were assessed by Kruskal-Wallis test. To further analyse significant differences between two individual groups a pair wise comparison by two-sided Mann-Whitney U-test was performed. All significance levels were set to 5%. Data were analysed and processed using SPSS Version 12.0 on a Windows XP operating system.
A total of 229 subjects were investigated between October 1999 and June 2004: 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. The clinical characteristics and lung function measurements are summarized in table 1. FEV 1 , FEV 1 in % of predicted value and FEV 1 /FVC were normal in smokers, significantly decreased in stable COPD patients (all) and further significantly decreased in AE-COPD patients (all p < 0.05 compared to stable COPD and all p < 0.001 compared to smokers).
HMPV could be detected in three subjects. All these subjects were AE-COPD patients. The prevalence of hMPV in AE-COPD patients was 2.3%. The virus was simultaneously detected in nasal lavage and induced sputum in one patient only. The viral load was about 100 times higher in nasal lavage than in induced sputum in this patient. Overall the viral load in nasal lavage was about 3.5 times higher compared to induced sputum (for details see table 2). The hMPV positive patients did not differ significantly from other AE-COPD patients when clinical parameters and lung function were analysed. hMPV was detected in the winter season only.
The main finding of this controlled study investigating the incidence of hMPV in subjects with COPD and smokers without COPD is that this recently discovered respiratory virus was detectable only during exacerbation of COPD. The frequency of detection was very low but in positive cases the viral load was considerable. There was no detection in patients with stable COPD or smokers without COPD.
Recently Vicente et al [19] reported about the incidence of hMPV in 89 COPD patients. Five patients (5.5%) were hMPV positive. Two of these patients had to be transferred to hospital. Although this was not a controlled study and not all details of the study are available due to the fact that the data were published in form of a letter, these results support our findings. The incidence of hMPV in this and in our study is low compared to other respiratory viruses. In a similar previous study we found that Picornaviruses were detectable in 36% of AE-COPD patients, Influenza A in 25% and Respiratory syncytial virus in 22%[2]. There is another prospective cohort study of adults in which hMPV was detected in 4.5% of all illnesses. HMPV was most prevalent in young adults with children and in frail elderly from long term care facilities [17] . Unfortunately this report does not specify how many of the elderly patients suffered from COPD. In our asymptomatic smokers without COPD hMPV could not be detected. A recent study investigating nasal secretions from adults with and without respiratory illnesses found hMPV in 5 of 146 ill patient and in none of 158 control subjects, strongly supporting our data [24] . A further recent study found hMPV in two out of 111 adult patients (1.8%) who presented to the emergency department for AE-COPD during 2 winter/ spring seasons in Quebec, Canada, also in support of our findings [25] . In a US American study investigating clinical samples collected between 1991 and 1995, hMPV could not be detected at all in 196 patients indicating important geographical and seasonal differences in hMPV prevalence [26] . Taken together the results presented here are in keeping with other studies in adults and add important information on the prevalence of hMPV in hospitalized AE-COPD.
To our knowledge this is the first study analysing the viral load of hMPV in COPD patients. We found a mean of 6.5 × 10 5 viral copies/ml in nasal lavage and 1.88 × 10 5 viral copies/ml in induced sputum. These values indicate that hMPV may have been the infectious agent triggering exacerbation in these patients. Viral load cut-off values for infectivity in COPD exacerbations have not been studied in detail yet and need further investigation. However, viral loads between 1120 copies/ml in Cytomegalovirus infection in lung-transplant patients [27] and 5.8log 10 copies/ ml in SARS [28] have been considered to indicate infectious disease. Moreover hMPV was only found in acute exacerbation and not in stable disease or in smokers without COPD supporting a triggering role in AE-COPD.
HMPV infection can be severe since it was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . Our hMPV positive patients did not differ in their clinical characteristics or lung function from the other AE-COPD patients which does not indicate a more severe course of AE-COPD in these patients.
Taken together this is the first controlled study on the relevance of hMPV in hospitalized AE-COPD. HMPV was detected in a very low frequency but with noticeable viral load in AE-COPD patients. Given that asymptomatic carriage of hMPV is very unlikely it should be considered as another possible trigger of AE-COPD. Since every AE-COPD has considerable impact on the course of the disease and regional outbreaks of hMPV are possible it should be included into future diagnostic and therapeutic considerations. 
The author(s) declare that they have no competing interests. Bioethical Implications of Globalization: An International Consortium Project of the European Commission The BIG project looks at some of the ethical concerns surrounding globalization and health. T he term "globalization" was popularized by Marshall McLuhan in War and Peace in the Global Village . In the book, McLuhan described how the global media shaped current events surrounding the Vietnam War [1] and also predicted how modern information and communication technologies would accelerate world progress through trade and knowledge development. Globalization now refers to a broad range of issues regarding the movement of goods and services through trade liberalization, and the movement of people through migration.
Much has also been written on the global effects of environmental degradation, population growth, and economic disparities. In addition, the pace of scientifi c development has accelerated, with both negative and positive implications for global health. Concerns for national health transcend borders, with a need for shared human security and an enhanced role for international cooperation and development [2] . These issues have signifi cant bioethical implications, and thus a renewed academic focus on the ethical dimensions of public health is needed.
Future developments in science and health policy also require a fi rm grounding in bioethical principles. These core principles include benefi cence; nonmalefi cence (to do no harm); respect for persons and human dignity (autonomy); and attention to equity and social justice. According to the World Health Organization [3] , global ethical approaches should (1) monitor and update ethical norms for research, as necessary; (2) anticipate ethical implications of advances in science and technology for health; (3) apply internationally accepted codes of ethics; (4) ensure that agreed standards guide future work on the human genome; and (5) ensure that quality in health systems and services is assessed and promoted.
The Bioethical Implications of Globalization (BIG) Project is a 42-month dialogue funded by the European Commission that involves a series of expert panel discussions on four specifi c globalization and health subject areas: (1) mobility of people;
(2) technological globalization; (3) liberalization of trade; and (4) new global health threats (bioterrorism). In addition, BIG includes a multipleround Delphi Process (Box 1) to solicit input on these issues from a broad, interdisciplinary audience.
The project's purpose is both to raise short-term, practical considerations about globalization and health and
Delphi is a group communication technique designed to obtain opinions from a panel of selected experts on specifi c issues through the sending of questionnaires to be completed within a specifi ed time. The experts are contacted individually and they do not know other group participants and their opinions-the aim is to submit the group participants to the same conditions. Participants do not meet personally, thereby avoiding undue infl uence. The process foresees the following points: The process is repeated a number of times, until a convergence of all group members is obtained. The process ends with analysis of the answers and formulation of the fi nal report.
(Adapted from http://www.bigproject. org/dephi.htm) Thomas E. Novotny*, Emilio Mordini, Ruth Chadwick, J. Martin Pedersen, Fabrizio Fabbri, Reidar Lie, Natapong Thanachaiboot,
to provide a longer-term, strategic perspective on the four selected public health-related issues. The fi nal conclusions will be presented to a high-level meeting of European Union (EU) policy makers in 2006; these conclusions may then inform future research directions and stimulate additional critical thinking about globalization and its bioethical implications for health policy. This article presents preliminary results from the BIG Project.
Mobility results from the increasing ease of domestic and international travel as well as from instantaneous access to information through the Internet and other electronic resources. Mobility may involve the pursuit of a better quality of life, development of markets for traded goods and services, return of resources to home countries, and improvement of professional and business networks.
However, migration may also affect psychological and physical health as a result of confl ict, famine, poverty, and the insuffi cient cultural or economic integration of migrants within their new home society. It may contribute to the spread of infectious diseases across borders ( Figure 1 ). The recent epidemic of SARS was a classic example of an infectious disease propagated through the movement of people across borders; it required attention from the original site to control migration (quarantine) as well as vigilance by secondary sites to protect their populations ( Figure  2 ) [4] . For these and other reasons, the International Organization for Migration is increasingly concerned with migratory patterns and their health consequences in a globalizing world (for an illustration of the emerging confl ict of ideas, see http:⁄⁄www.iom. int and http:⁄⁄www.noborder.org/ iom/index.php).
Cross-border health commerce is related to mobility. In Europe, this commerce is likely to increase as the EU enlarges to include Eastern and Central European nations. Such commerce may include the movement of health providers from East to West as well as "medical tourism" in pursuit of less costly or more accessible high-quality health care. In addition, international trade in illegal health products and inconsistent regulatory and safety standards for exports may threaten public health, especially in unregulated pharmaceutical markets.
Ethical concerns may also result from the vast growth in international tourist travel. Such travel now accounts for a twelfth of world trade, supporting an economy the size of a middle-income country [5] . Tourism may provide substantial economic benefi ts to many developing countries, and it may improve cultural understanding among travelers. However, these benefi ts require an ethical concern for the environment and for persons employed in the tourist industry.
The rights of nations to protect against infectious disease and unsafe medical practices, as well as the rights of human beings displaced by war, traffi cking, and economic and cultural disruption, are critical concerns for health policy makers. Poverty and social disparities are key factors in the growth of global migration. Therefore, it is timely to consider whether mobility is a human right, and whether those who migrate have rights to health care in their new country. These questions should be considered by health policy makers within the ethical contexts of individual autonomy, social justice, nonmalefi cence, and benefi cence.
Technology drives globalization and in turn is driven by globalization. However, there is considerable ambiguity as to the value of technological globalization, especially for health in low-income countries. The "digital divide" may be important in improving health or income disparities as the electronic revolution provides scientists and health workers in both the developed and developing world with unprecedented access to information. Much could be done to reduce information inequities for the developing world through collective international action, but new global governance mechanisms may be needed to achieve this goal for information technology [6] .
Interestingly, the Internet is a structural necessity for fi nancial and corporate globalization, but the same technology is used by nongovernmental organizations, political groups, and cultural movements to support grassroots social justice and human rights campaigns against these globalizing corporations. Neither side in this struggle would advocate limitations to the expansion of Internet technology, but both sides need to consider the bioethical implications of increased information access.
On the other hand, the ethical issues surrounding genomics (with both environmental and human concerns) are quite ambiguous. While there may be signifi cant benefi ts to identifying genetically benefi cial products or genetic determinants of disease, there are also concerns about altering natural environments and about collecting routine genetic information from general populations [7] .
For example, some experts assert that genetically modifi ed (GM) crops will signifi cantly increase crop yields without requiring any additional farmland, thus preserving valuable rain forests and animal habitats. herbicides, and pesticides. Farmers are not allowed to trade or save GM seed from one harvest to the next, and "terminator technology" (producing grains that are genetically modifi ed so that they cannot be used to generate new crops) is under development. (See http:⁄⁄www.globalissues.org/ EnvIssues/GEFood/Terminator.asp for more information on this technology.) Thus, ethical considerations of distributive justice and benefi cence must be considered in the debate about the global applicability of GM crops.
For the pharmaceutical and health care industries, genetic testing could provide information about the shape of future markets and the possible tailoring of specifi c pharmacotherapy to genomic susceptibility. For governments, genetic testing may provide predictive information on a population basis that could aid future health care planning. Genetic information might also be similarly used by the insurance industry, but the identifi cation of genetically "high-risk" individuals would likely interfere with their autonomy, in that they may not be able to purchase health insurance. For example, the Apolipoprotein E test may indicate that an individual has two copies of one form (allele e4) of the gene that leads to Alzheimer disease [9] . Could this information be used by insurance companies or possible employers to deny insurability, despite no current adverse health effects?
In the post-genomic era there is potential to both reduce and increase health inequities, but much will depend on how ethical issues are addressed. If interventions to increase the life span for those with access to high quality health care must compete with expensive investments in genetic research on infectious diseases (which affect the poor most of all), health inequalities may be amplifi ed between those with access and those without access to health care. If research participants or patients in low-income countries have unequal access to information, they may not be properly informed about genetic testing and the counseling needed if adverse genetic information is found. Population-based genomic research may characterize groups of people in such a way that encourages discrimination. Such research may also lead to disputes about ownership of genetic resources from participant populations. Health professionals must have a solid grounding in bioethical issues as they make clinical decisions based on genetic information. However, health policy makers and global governance structures must also be accountable for the potential adverse consequences such decisions might engender.
One may ask: will genomic science really help developing nations? To what extent can benefi ts be shared? Will pharmaceutical and biotechnology companies invest in poor countries if they can make more money working on therapeutics for high-income countries? Thus, concern for the bioethical issues of social justice and benefi cence arises. Genomics has the potential to be a global public good, but there is considerable uncertainty as to its bioethical justifi cation in all cultures [10] .
In general, globalization helps liberalize trade through removal of import restrictions and tariffs, through removal of restrictions on trade in services, and through linkage of trade sanctions to the protection of intellectual property rights. All these activities may have an impact on population health.
Defenders of trade liberalization claim that this process is one of the most effective means of increasing a country's wealth and, by extension, population health. Even if this were always true, there may be specifi c policies that have particularly detrimental effects on health (such as opening markets to trade in manufactured tobacco products). Further, there may be an ethical argument based on social justice against some trade liberalization policies. If, for example, trade liberalization between rich and poor countries produces proportionally more wealth in rich countries compared with poor countries, this may suggest a socially unjust result of liberalization; poor countries' economies may not grow as fast as rich countries' economies in this situation.
The relationship between wealth and health is actually somewhat controversial: the so-called Preston curves demonstrate a dramatic relationship between health and economic prosperity up to about a Purchasing Power Parity of US$3,000 per capita per year [11] . However, there are cheap, cost-effective approaches to population health (such as vaccination, clean water, and sewage disposal) that may not be affected by the increase in Purchasing Power Parity. These approaches were relatively more important than economic development per se in early 20thcentury interventions in developed countries, and they are likely to be more important for infl uencing health among developing country populations today than simple economic growth. On the other hand, high-intensity technological improvement rather than economic growth may be more important to health in rich countries compared with developing countries.
The concern for intellectual property rights in trade has been an extraordinarily contentious issue in recent years. Newer drugs that are effective against diseases in resourcepoor but highly impacted countries, such as antiretroviral drugs against HIV, have been prohibitively expensive in these countries, in part because of patent protections. With the Trade-Related Intellectual Property agreement, patent protection became linked to trade policy; if countries in need of cheaper essential drugs did not conform to patent rules, trade retaliation from exporting countries might ensue. However, restrictions on poor countries' responses to legitimate public health emergencies may be unethical on the basis of distributive justice, nonmalefi cence, and benefi cence. Exceptions for public health emergencies (such as HIV/AIDS) under the Trade-Related Intellectual Property agreement include the right to compulsory licensing (local companies produce patented medicines in exchange for a royalty payment to the patent holder) or parallel importing (importing patented drugs sold more cheaply elsewhere) that will make essential medicines more available to highly impacted countries without fear of trade retaliation from the originating country [12] .
The General Agreement on Trade in Services is a relatively new treaty that covers trade in health services [13] . The agreement has been severely criticized by some, who claim that it increases privatization of health care services and undermines public health care systems. However, given its ambiguities, the actual impact of the agreement on the health sector will be largely determined by the way in which the agreement is further specifi ed in multinational commitments [14] . Social justice, equity, benefi cence, and nonmalefi cence will all come into play in the implementation of this treaty.
Concerns for security against biological weapons have recently arisen among both poor and wealthy nations. Some, however, question the enormous sums now being spent to address the perceived threats due to bioterrorism even without strong evidence for actual threats. Even without such evidence, global bioethical principles at least suggest the need for a framework for consideration of distributive justice in this arena.
For example, should a nation with a limited supply of a vaccine against weaponized smallpox offer its stockpiles to a neighboring country that is under direct attack? This case is complicated by the fact that the infection could spread to its own territory. In the case of widespread biological attacks, which global governing agency, country, or other entity would be responsible for global resource allocation? Clearly, risks from bioweapons are trans-border, but resources may be unevenly and inequitably distributed, requiring a bioethically based policy determination on a global basis [15] .
A further concern with respect to biomedical research is the issue of dual-use technology development for health benefi ts as well as for possible bioweapons. Governments must balance the secrecy necessary for security with the need for disclosure of information that is essential for research and development in health. It is very diffi cult to sequester new knowledge that might be applied to building biological weapons without simultaneously impeding research on defense against those bioweapons and on other benefi cial biomedical advances. Most BIG Project scientists agree that the benefi ts of releasing scientifi c information in general outweigh the risk of its misuse. However, the scientifi c community needs to consider whether new codes of conduct are necessary or whether existing governance is suffi cient to support a bioethical approach to research on possible dualuse technologies.
Global bioethical challenges require careful theoretical deliberation and practical considerations for international health policies [16] . The BIG Project seeks to guide these processes in four selected areas of interest to the EU, so that the project results may be helpful to policy makers at local, national, and international levels.
The BIG Project has found that bioethical principles are important in considerations of migration, trade, information technology, genomics, and bioweapons threats. Globalization in these arenas is neither a right nor a wrong process, but it demands careful consideration of bioethical principles including social justice, benefi cence, nonmalefi cence, and individual autonomy. These concerns may not be immediately obvious to health policy makers, and thus the BIG Project results may help clarify the larger goals and purposes of bioethically based health policy development within the EU and elsewhere. More information about the BIG Project can be found at http:⁄⁄www. bigproject.org/project.htm. Public awareness of risk factors for cancer among the Japanese general population: A population-based survey BACKGROUND: The present study aimed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. METHODS: A nationwide representative sample of 2,000 Japanese aged 20 or older was asked about their perception and level of concern about various environmental and genetic risk factors in relation to cancer prevention, as a part of an Omnibus Survey. Interviews were conducted with 1,355 subjects (609 men and 746 women). RESULTS: Among 12 risk factor candidates, the attributable fraction of cancer-causing viral and bacterial infection was considered highest (51%), followed by that of tobacco smoking (43%), stress (39%), and endocrine-disrupting chemicals (37%). On the other hand, the attributable fractions of cancer by charred fish and meat (21%) and alcohol drinking (22%) were considered low compared with other risk factor candidates. For most risk factors, attributable fraction responses were higher in women than in men. As a whole, the subjects tended to respond with higher values than those estimated by epidemiologic evidence in the West. The attributable fraction of cancer speculated to be genetically determined was 32%, while 36% of cancer was considered preventable by improving lifestyle. CONCLUSION: Our results suggest that awareness of the attributable fraction of cancer causes in the Japanese general population tends to be dominated by cancer-causing infection, occupational exposure, air pollution and food additives rather than major lifestyle factors such as diet. In Japan, cancer has been recognized as a major component of the overall pattern of disease for decades. Thus, the importance of cancer prevention by lifestyle modification should now be strongly acknowledged.
Internationally, several studies have estimated the proportion of total cancer deaths attributable to various risk factors based on epidemiologic evidence [1, 2] , and various international guidelines and recommendations derived from these have appeared [3] [4] [5] [6] . Not surprisingly, domestic guidelines and recommendations for cancer prevention in Japan such as the 'Twelve recommendations for cancer prevention [7]' and 'Healthy People Japan 21 [8] ' have been significantly influenced by these reports.
Public awareness of risk factors in relation to cancer prevention has been surveyed in only a few countries [9, 10] , and results have demonstrated poor awareness. Other studies focusing on specific cancers only have also appeared [11] [12] [13] [14] . However, none of these studies quantitatively evaluated public awareness of the attributable fraction of individual risk factors.
In Japan, it appears that most people are aware of the major risk factors of cancer. Although we are unaware of any published evidence, however, public knowledge and information on cancer prevention now seems influenced largely by the mass media and other sources, rather than by information provided directly by health professionals, resulting in a distorted picture of causation. Cancer control policy therefore urgently requires a clarification of the discrepancies which now exist between ideal levels of public concern about risk factors and the current reality, particularly public health policy makers in their formulation of cancer control measures. To address this need, the present study was designed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. Since we are interested in quantitatively estimating the awareness of preventability, we placed special emphasis on gauging awareness by attributable fraction of cancer.
The study was conducted as a part of an omnibus survey in December, 2003, by commission to a polling agency. The omnibus survey is a monthly multipurpose cross-sectional survey which includes public opinion research, social research, scientific research, market research, and others. Using a stratified two-stage sampling method, a total of 2,000 people aged 20 or older were randomly selected as study subjects, from 160 districts selected from area units representing 12 geographical blocks (Hokkaido, Tohoku, Kanto, Keihin, Koshinetu, Hokuriku, Tokai, Kinki, Hanshin, Chugoku, Shikoku, Kyushu) and 3 types of city scale (14 metropolises, other cities, towns and villages) in proportion to the population distribution as at March 2002. After an initial visit to obtain oral informed consent and schedule a visit for the interview, the survey was conducted by face-to-face interview using trained interviewers in each district. The omnibus survey does not collect any personally identifiable information such as name, date of birth or address details at interview. For the present report, we obtained the electronic data file for the relevant interview component, with no personal identifiers. Ethical approval was not applicable to the present study under the Japanese ethical guidelines for epidemiologic studies, which comply with the declaration of Helsinki.
Among the 2,000 people selected for survey (977 men, 1,023 women), interviews were successfully obtained The questionnaire of this survey comprised questions on the awareness of various environmental and genetic risk factors in relation to cancer prevention by enquiring about the attributable fraction of cancer. Fractions were: 1) 12 risk factor candidates, namely alcoholic beverages, unbalanced diet, use of food additives and pesticide chemicals, charred fish and meat, tobacco smoking, obesity, physical inactivity, endocrine-disrupting chemicals, air pollution such as diesel emissions, occupational exposure, cancer-causing viral and bacterial infection, and stress; 2) genetic factors in general; and 3) the preventable fraction of cancer occurrence by lifestyle modification [see Additional file 1].
The first question asked about the preventable fraction of cancer which would result in Japan if each factor were completely and totally eliminated, using the fine categories of <5%, 5 to <10%, 10 to <15%, 15 to <20%, 20 to <25%, 25 to <30%, 30 to <40%, 40 to <50%, 50 to <60%, 60 to <70%, 70 to <80%, 80 to <90%, and 90 to 100%. These categories were exhibited together on a pie chart.
These risk factor candidates were selected with reference to previous international and domestic recommendations and guidelines [1] [2] [3] [4] [5] [6] [7] [8] . The second question asked about the fraction of cancer genetically predetermined using the same categories as the first, while the third asked about the preventable fraction of cancer by modification of lifestyle using estimation of an actual percent value. In addition to these questions, subjects were also asked about their smoking and drinking practices, and occupational and educational status.
Mean values of the attributable fractions were calculated for each risk factor of cancer and compared by demographic and habitual smoking and drinking status. For analyses, the mid-values of each category were assigned for categorical variables. All analyses were performed using Stata statistical software, S/E Version 8 [15] .
A total of 1,355 (67.8%) subjects responded to the survey, with a higher response rate in women (72.9%) than in men (62.3%). Response rate was lower in the 20s age strata than in the other age groups, but no trend to an increase in response rate with increasing age was observed. Overall, no significant difference in area and age distribution was seen between the sampled population and survey respondents. Response rate tended to be lower among subjects who reside in the Kanto region and in cities other than the 14 metropolises than among other subjects ( Table 1) .
Characteristics of the 1,355 respondents (609 men, 746 women) are presented in Table 2 . The proportion of current smokers was 44% in men and 15% in women, and decreased with age in both genders. In female subjects aged in their 20s, 26% currently smoke and 49% drink alcohol beverages at least 4 times a week.
Awareness of the attributable fraction of cancer causes among the Japanese general population is presented in Table 3 . Among the 12 risk factor candidates, the attributable fraction was considered highest for cancer-causing viral and bacterial infection (51.3%), followed by tobacco smoking (43.0%), stress (39.0%), and endocrine-disrupting chemicals (37.1%). In contrast, the attributable fraction of charred fish and meat (21.4%) and alcohol drinking (21.7%) were considered low compared with other risk factor candidates. The attributable fraction of other risk factor candidates such as occupational exposure, air pollution, food additives and pesticides, unbalanced diet, obesity and physical activity ranked between the high and low fractions. The attributable fraction responses tended to be higher in women than in men, and were increased among inhabitants of larger cities and in homemakers and decreased in those engaged in agriculture, forestry and fisheries. In contrast, risk factor candidate rankings were similar by gender, age group, city scale, and educational and occupational status. In men, those who neither smoke nor drink tended to consider the preventive fraction of the risk factors higher than those who both smoke and drink, whereas in women, the former subjects considered the values lower than the latter.
The speculated fraction of cancer which is genetically determined was 31.5% as an average (Table 3 ). This fraction was higher in current heavy smokers and former drinkers, and lower in homemakers and students. On the other hand, an average 35.5% of cancer were considered preventable by lifestyle improvement, with this ratio being higher in homemakers, former smokers, and never and former drinkers.
The present survey, targeted at the Japanese general population, showed that the attributable fraction of cancer among Japanese tended to be higher for cancer-causing infection, occupational exposure, air pollution and food additives than major lifestyle factors such as dietary factors. In addition, the attributable fraction of cancer estimated by the Japanese general population was higher than that derived from epidemiologic evidence in the West, which is frequently quoted as 30% for tobacco smoking and 30% for food as a whole [1, 2] .
Some of the major cancers in Japan, including gastric and liver cancers, are known to be related to cancer-causing viral and bacterial infection, and a higher level of concern about such infection among Japanese than in Western populations would therefore be understandable [9] . Notwithstanding the validity of such concern, however, the high level of concern for infection, as well as for endocrine-disrupting chemicals, identified in the present survey was most likely due to the severe acute respiratory Likewise, a high level of concern for tobacco smoking was also observed, in spite of a relatively dull reduction in the rate of male current smokers in past decades compared with the U.S. This was probably due to recent enactment of the Health Promotion Law, which curbs passive smoking in public spaces.
Respondent estimates for attributable fractions were generally high. This may be in part due to anchoring and adjustment effects of the response categories used and the tendency of people to respond near the middle of the scale. Given that responses tended to be generally high, concern over the present results should probably be focused on rankings rather than absolute values per se. Although tobacco smoking ranked among the top factors, risk factor candidates whose actual contribution is considered to be low, such as endocrine-disrupting chemicals, occupational exposure, air pollution such as diesel emissions and the use of food additives and pesticide chemicals ranked higher than previous estimates of the attributable fraction of cancer causes [1, 2] . In contrast, this should be compared with the results for unbalanced diet, which ranked at only 8th among the 12 risk factor candidates despite an actual ranking which is estimated to be as high as that for tobacco smoking. Particularly in light of findings on long-term exposure to common lifestyle factors such as diet as a cause of cancer, these results suggest that public awareness of cancer prevention is still insufficient.
We are unaware of any previous studies aimed at determining public awareness of the attributable fraction of cancer as a whole or at gauging the level the awareness of cancer prevention by attributable fraction. Accordingly, to our knowledge, this is the first attempt to discover the level of awareness for each risk factor candidate, and the questionnaire used has hence not been fully validated. In addition, as indicated above, responses to this type of cross sectional survey are subject to social conditions such as information from the mass media and other sources on disease epidemics and other putative risk factors. Thus, the results might not necessarily reflect actual public awareness. However, the study subjects were recruited from among a nationally representative random sample, and the response rate was similar to that of recent omnibus surveys in other countries [16] [17] [18] [19] . Nevertheless, the exclusion of non-respondents may have distorted the results.
In conclusion, awareness of the attributable fraction of cancer causes among the Japanese general population tended to be dominated by infection, occupational exposure, air pollution and food additives rather than dietary factors. The results of the present survey provide valuable clues and perspectives toward the formulation of relevant cancer prevention strategies in Japan. Sequence specific visual detection of LAMP reactions by addition of cationic polymers BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device. Loop-mediated isothermal amplification (LAMP) is a unique gene amplification method in which DNA can be isothermally amplified using only one enzyme [1] [2] [3] .
Since the advent of the LAMP method, many researchers have been engaged in basic research from a variety of perspectives. As a result, it is currently being put to practical use in the reagents for detecting various pathogens such as SARS [4] and the West Nile virus [5] and reagents for identifying the sex of fertilized eggs in cow in vitro fertilization [6] . Furthermore, LAMP is a gene amplification method with a variety of characteristics and applications in a wide range of fields, including SNP typing [7] and quantification of template DNA [8] . In particular, LAMP is considered to be effective as a gene amplification method for use in gene point-of-care testing (g-POCT) devices, which are used for simple genetic testing whenever and wherever necessary. First, since LAMP can amplify genes isothermally, the amplification reaction can be carried out with a simple heater. There is no need for the special device used Pattern diagram of LAMP reaction and hybridization of fluorescently labeled oligo DNA probes Figure 1 Pattern diagram of LAMP reaction and hybridization of fluorescently labeled oligo DNA probes. The LAMP reaction takes place in three steps (starting material production step, cycling amplification step, and elongation and recycling step) by the primers depicted in the enclosure. In the starting material production step, the starting material (6) is generated by primers (forward inner primer (FIP) and backward inner primer (BIP)). A complementary strand (11) of the starting material (6) is synthesized from the starting material (6) by a reaction that uses itself as a template and by a reaction from an FIP annealed to the loop segment, thus making up the cycle amplification step. During this step, probes (probe F and probe B, respectively) designed for the region between the F1 and F2 region or the B1 and B2 region can hybridize to the loop segment. As the cycle reaction progresses, an elongation and recycling step takes place, during which elongated products (8, 13, etc.) with an inverted repeat structure are generated. Numbers 14 and 15, which have a cauliflower structure, are also generated. They have many loop structures to which probes can hybridize.
for polymerase chain reaction (PCR) to rapidly control the temperature [3] . Next, a large amount of DNA (10-30 µg/25 µl) can be synthesized in a short time (15-60 min) while maintaining high specificity. This characteristic greatly facilitates detection of the LAMP reaction [9] . Moreover, since the LAMP reaction progresses by generating a characteristic stem-loop structure, LAMP products have a single-stranded segment in the molecule (loop segment; see Figure 1 and reference No. 1). By using oligo DNA probes designed to recognize the sequence of the single-stranded segment, it is possible to carry out hybridization assay without performing heat denaturation after amplification. This means that all processes, from the amplification reaction to the detection reaction, can be carried out completely isothermally.
If these characteristics of the LAMP method are used effectively, we believe it will be possible to develop simple genetic testing devices that have not been realized yet despite a strong awareness of their necessity, in a wide range of fields, including infectious disease testing, food inspection, and environmental testing. The key to developing such simple devices will be figuring out how to simply and clearly present the final amplification results. The objective of this research is to establish new techniques for sequence-specific visual detection of amplification results by means of the LAMP method. To that end, we made use of a reaction that has been known for a long time, i.e., cationic polymers like polyamines form an insoluble complex with DNA [10] . It is well known that one of such the polyamine, polyethylenimine (PEI), strongly interacts with DNA. PEI is widely used as a nucleic acid precipitant for nucleic acid purification [11] and as an in vivo and in vitro non-viral vector [12, 13] . We discovered that an insoluble PEI-LAMP product complex was generated under certain optimized conditions when PEI was added to LAMP reaction solution. Using this precipitation titration, we investigated whether it was possible to perform bound/ free separation of fluorescently labeled probes. In this paper, we report the novel visual detection methods of the presence of sequences of HBV and HCV cloned to plasmid as a model experiment using this method and the results of an investigation of the basic reaction conditions required to achieve this.
The primers used for LAMP reaction is schematically depicted in enclosure of Figure 1 . Forward Inner Primer (FIP) consists of F2 and the complementary sequence of F1, and Backward Inner Primer (BIP) contains of B2 and the complementary sequence of B1 when each sequences (F1, F2, B1, and B2) are defined on the template sequence as shown in Figure 1 . In some references such as Notomi et al. [1] or Parida et al. [5] , a spacer of few thymidines was inserted between F1c or B1c and F2 or B2 in the inner primer (FIP or BIP) so that one and two thymidine spacers were inserted in FIP and BIP of HBV, respectively. However, the spacer was not used in this study because the LAMP reaction can progress with the use of inner primers without the spacer as shown by Hong et al. [4] , Hirayama et al. [6] , and Iwasaki et al. [7] . The LAMP reaction takes place isothermally in the three steps shown in Figure 1 , i.e., starting material production step, cycling amplification step, and elongation and recycling step, by using of polymerase with strand displacement activity. First, the starting structure (structure 6) is generated from the template nucleic acid in the starting material production step. Next, the starting structure becomes structure 7 with a stem-loop structure by self-primed DNA synthesis. When the forward inner primer is hybridized to the loop segment and strand displacement synthesis takes place, structure 11, which is a complementary strand of structure 6, is generated. This means that an auto cycle reaction was established between structure 6 and structure 11. In addition, products bound by an inverted repeat with two amplified regions, like structures 8 and 13, are generated in association with the auto cycle reaction (cycling amplification step). Then, with these structures as starting points, products elongated to a length of several kbp and products with complex structures with cauliflower-like structures (14, 15) are ultimately generated.
Since the LAMP products have a loop structure, oligo DNA probes (green arcs in the figure) in the reaction solution can sequentially hybridize to products as the LAMP reaction proceeds. The cauliflower structures, in particular, contain two or more loops to which the probes can hybridize. This characteristic of the LAMP reaction plays an important role in the detection method described here.
Plasmid DNA cloned with HBV or HCV sequence was added to a LAMP reaction solution containing both HBV primers and the probe (FITC-labeled) and HCV primers and the probe (ROX-labeled) and amplified, after which low molecular weight PEI (Mw = 600; 0.2 µmol as a monomer) was added. As shown in Figure 2A , precipitate emitting green fluorescence characteristic of FITC was obtained in LAMP reaction solution containing the HBV template, precipitate emitting red fluorescence characteristic of ROX was obtained in LAMP reaction solution containing the HCV template, and precipitate emitting a color (orange) that was a combination of FITC green and ROX red fluorescence was obtained in LAMP reaction solution containing both templates. They could be observed using an ordinary UV illuminator or UV-LED (light emitted diode). When LAMP products (lambda DNA) not related to HBV and HCV were present, precipitate with no fluores-Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI Figure 2 Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. (A) Results of sequence-specific visual detection after adding PEI to LAMP reaction solution. After LAMP reaction in the presence of both FITC-labeled HBV probes and ROX-labeled HCV probes followed by addition of the prescribed amount (0.2 µmol as monomer) of PEI (Mw = 600), it was centrifuged for several seconds using a desk-top, low-speed centrifuge. The tube was then visually observed as is on a UV illuminator (365 nm). It was possible to differentiate the LAMP reaction by visualizing the presence of precipitate fluorescence and the color of the fluorescence. 1, LAMP reaction negative. 2, When LAMP reaction with PSA amplification (unrelated LAMP reaction) occurred. 3, When it contained HBV template nucleic acid. 4, When it contained HCV template nucleic acid. 5, When it contained both HBV and HCV template nucleic acids. (B) Diagram of principle of sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. First, a LAMP reaction is carried out using a LAMP primer set for two types of template nucleic acid and fluorescently labeled probes, which can hybridize to loop segments of each LAMP products. When a LAMP reaction corresponding to a certain fluorescently labeled probe progresses, the probe will sequentially hybridize to the loop segment generated during the reaction. On the other hand, an unrelated probe remains free in the solution. When an optimized amount of PEI is added after reaction for a set length of time, the positive charge of PEI neutralizes the negative charge of the DNA to form an insoluble LAMP product-PEI complex. At this stage, fluorescently labeled probes hybridized to LAMP products are taken up by the LAMP product-PEI complex together with the LAMP products. Since most of the PEI added is used for formation of the LAMP product-PEI complex, free oligo DNA probes cannot form a complex with PEI. When the generated insoluble complex is pelletized by centrifugation and the pellet is irradiated with excitation light, the labeled fluorescent dye hybridized to LAMP products produces fluorescence. Figure 3 Precipitation titration of LAMP products by addition of PEI. (A) Effect of amount of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex (Mw of PEI is 600). When 0.2 µmol to 1.0 µmol of PEI was added as a monomer, almost 100% of labeled probes hybridized to the LAMP products for lambda DNA was taken up by the DNA-PEI complex. On the other hand, when 0.4 µmol to 0.8 µmol of PEI was added as a monomer to a reaction solution in which an amplification reaction did not take place, a small amount (<20%) of labeled probes precipitated. Precipitation of this nonspecific probe did not take place when unrelated LAMP products (PSA) were present. (B) Effect of the Mw of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex. When PEI with Mw 600 was used, the fluorescence intensity of supernatant decreased only when LAMP product for lambda DNA was present. As the Mw of the PEI used increased from 1,800 to 10,000, the fluorescence intensity of the supernatant decreased in the absence of a LAMP reaction since the formation of insoluble PEI-oligo DNA probe complex occurred. (C) Effect of amount of KCI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by LAMP product-PEI (Mw = 600) complex. Normal LAMP reaction solution (control) contains 10 mM of KCI. Since formation of the PEI-LAMP product complex was inhibited by an increase in the amount of KCI added, the fluorescence intensity of supernatant increased regardless of the sequence of the LAMP product. (D) Effect of amount of LAMP product for lambda DNA on sequence-specific incorporation of ROX-labeled lambda DNA recognition probe by LAMP product-PEI complex (Mw of PEI = 600). Even when the amount of LAMP product was 1 µg per 25 µL, almost 100% of labeled probes (1 pmol) was taken up by PEI-DNA complex.
cence was obtained, and visible precipitate was not generated in a sample not containing a template (LAMP reaction negative). This means that it was possible to assess whether the HBV template nucleic acid was in the reaction solution, HCV template nucleic acid was in the reaction solution, or both were in the reaction solution by visualizing the fluorescent color of the precipitate. The LAMP method is a nucleic acid amplification method that is so sensitive that it is possible to create an amplification reaction from only six copies of template nucleic acid [1]. Therefore, a combination of LAMP amplification and this detection method makes possible sequence-specific visual presentation of the presence of trace amounts of nucleic acid, i.e., only several copies, found in a sample.
A model that represents the principle of this method is shown in Figure 2B . Oligo DNA probes labeled with fluorescent dye are hybridized to a specific LAMP product. When an optimized amount of low-molecular-weight PEI (Mw = 600) is then added, the positive charge of PEI neutralizes the negative charge of the DNA, which results in formation of an insoluble DNA-PEI complex. When this solution is left to stand for a few minutes or it is centrifuged with a small, desk-top centrifuge for a few seconds, the complex is deposited at the bottom of the tube. When the precipitate is observed on an illuminator (365 nm), the fluorescence of the dye from the probe taken up by the precipitate is visualized. On the other hand, probes unrelated to the sequence of the amplified LAMP product are not taken up by the LAMP-PEI complex because they are not hybridized to the LAMP product. When the molecular weight of the PEI used is small, oligo DNA probes and PEI cannot interact sufficiently to form an insoluble complex. Therefore, unrelated probes remain in the supernatant. Since the fluorescent probes in the supernatant are dispersed, the fluorescence cannot be visualized. Consequentially, bound/free separation of labeled oligo DNA is achieved as a result of insolubilization by PEI of LAMP products.
The following experiments were conducted to confirm this principle. First, we investigated the effect of the added amount of PEI on this precipitation titration ( Figure 3A ). Oligo DNA probes for lambda DNA were captured in the precipitate when 0.2 to 1 µmol of PEI was added to 25 µL of LAMP reaction solution for lambda DNA. If the amount of PEI added is too high or too low relative to the optimal range, the PEI-DNA complex precipitate is not formed, resulting in oligo DNA probes remaining in the solution. This phenomenon is characteristic in ionic interaction between cationic polymers and anionic polymers [14] . Namely, when the amount of the cationic polymer PEI is too low, the PEI-DNA complex becomes anionic. In contrast, when the amount of PEI is too high, the PEI-DNA complex becomes cationic. In both cases, the PEI-DNA complex is solubilized as a result. This characteristic, which is shown in Figure 3A , indicates that this precipitation titration is based on neutralization of the negative electric charge of the DNA by the cationic polymer PEI. In the case of LAMP negative, a small amount of free probe was deposited under certain conditions when 0.4 µmol to 0.8 µmol of PEI was added as a monomer (< 20%). However, the amount of precipitate in this case was so small that it was impossible to confirm it visually. On the other hand, when LAMP products (PSA) unrelated to the probe sequence were present, no free probe at all was deposited. This is because almost all of the PEI molecules added were consumed in the precipitation of unrelated LAMP products, an excess of which was present relative to the amount of probe. In other words, more reliable detection is achieved by first confirming whether the LAMP reaction has occurred by checking whether white DNA-PEI complex precipitate is generated as a result of addition of PEI and then determining for which nucleic acid template the LAMP reaction occurred based on the fluorescent color of the precipitate. Because of the above results, the amount of PEI added was fixed at 0.2 µmol for the following experiments in order to avoid generation of free probe precipitate as much as possible.
We investigated the effect of the Mw of PEI on this detection system ( Figure 3B ). When PEI with different Mw was added to the LAMP solution so that the amount per monomer of each was the same, we found that sufficient BF separation occurred if LAMP amplicons were present, even if the Mw of PEI increased up to 10,000. That is, the fluorescence intensity of the supernatant of LAMP solution with specific amplification (lambda DNA) is lower than that with unrelated amplification (PSA). As the Mw of PEI increased, however, almost all probes in LAMP reaction negative solution formed an insoluble PEI-oligo DNA complex. This result means that the LAMP reaction negative solution cannot be distinguished from the LAMP reaction positive solution, which successfully amplifies the targeting sequence if PEI with a high molecular weight is used. As was also observed in Figure 3A , when LAMP amplicons are present, almost all of the added PEI reacts with LAMP amplicons, so there is little opportunity for interaction with free oligo DNA probes, but when LAMP amplicons are not present, all of the added PEI interacts with oligo DNA probes. Under conditions where a large amount of PEI with a high molecular weight can strongly interact with oligo DNA, an undesirable insoluble PEIoligo DNA probe complex forms because the molecular weight of PEI is high. Therefore, the average molecular weight of PEI in this detection system should be about 600. We conducted a similar experiment using spermine, which is a polyamine with a lower molecular weight. In that experiment, an adequate amount of insoluble complex was not generated compared with PEI of an average molecular weight of 600 under conditions used for the present research (data not shown). It is well known that spermine can also make DNA insoluble, as indicated in many other reports [10] . Therefore, optimal conditions in the case of spermine as a precipitant might exist, but since further investigation would have gone beyond the scope of this paper, no further investigation was carried out.
Insolubilization of LAMP products by PEI was inhibited by addition of an excessive amount of KCl to the LAMP reaction solution after amplification ( Figure 3C ). This was because excessive amounts of potassium ions and chloride ions inhibited the electrostatic interaction between DNA and PEI. This finding is further indication that this detection method is based on neutralization of the negative electric charge of DNA by the positive electric charge of PEI. The effect of ionic strength on the LAMP reaction has been investigated and found that the presence of 200 mM or more of KCl markedly delayed the LAMP reaction (data not shown). Therefore, it can be said that this detection method will function without trouble if the solution used has a composition that is optimized for the LAMP reaction.
We investigated the sensitivity of this detection method ( Figure 3D ). Almost all probes were taken up by the precipitate in the case of up to 1 µg of LAMP product. Moreover, we were able to visualize the fluorescence in the precipitate even if the specific LAMP product was 0.2 µg. We found that this detection system was sensitive enough as a detection system for visual assessment to be used in simple g-POCT devices. We can see from the results shown in Figure 3C that all of the 1 pmol of probe added hybridized to 1 µg (= 3 nmol nucleotide) of LAMP product. In other words, one molecule of probe bound to every 1,500 base pairs of LAMP products. The LAMP product is a mixture of products of several different sizes, with an average molecular size of 2 kbp [1]. This finding that one molecule of probe binds to every 1,500 base pairs reflects well the fundamental characteristic of this LAMP reaction.
The new detection method described above utilizes the unique nature of low-molecular-weight PEI, i.e., it cannot form an insoluble complex with a single-stranded anionic polymer with a low molecular weight such as an oligo DNA probe, but it can form an insoluble complex with DNA with a high molecular weight such as LAMP product. Until now, not much attention appears to have been paid to the complex consisting of oligo DNA and PEI with a molecular weight of 1,000 or lower, which was used in this study. This is probably because the interaction between low-molecular-weight DNA and oligo DNA is very weak and because its practical utility as a vector or nucleic acid precipitant is low. However, Kunath et al. reported that the complex formed by PEI (5 kDa) and plasmid DNA was more unstable than that formed by PEI (25 kDa) [15] . Osland and Kleppe reported that spemidine, which has a structure similar to that of PEI, formed an aggregate with double-stranded oligo DNA, but it did not form an aggregate with single-stranded DNA [10] . Drawing upon these results, the low-molecular-weight PEI used in this study is thought to interact with DNA with high selectivity to differences in DNA concentration and structure (number of strands and molecular weight). The fact that we took advantage of this nature of lowmolecular-weight PEI as a nucleic acid precipitant for detection led to the establishment of this detection method.
This detection method effectively utilizes the characteristics of the LAMP method, i.e., a large amount of amplification product can be synthesized in a short time while maintaining high specificity. Since a large amount of amplification product is created by the LAMP reaction, precipitate of a size that can be easily confirmed with the eyes is generated when PEI is added to the LAMP reaction solution. Moreover, the fact that the amplification is highly efficient means that the amount of labeled probe for detection that can be added is large. As a result of these characteristics, the LAMP reaction followed by addition of PEI yields precipitate with a clear color and in a size that can be identified visually, as shown in the photograph in Figure 2 . Furthermore, the fact that the amount of the amplification product is large means that the range of the optimum amount of PEI necessary to generate the insoluble complex is wide. Thus, a precise system for adding the PEI solution is not necessary. This will contribute to simplification of g-POCT devices that use this detection method. In the case of PCR, the most widely used gene amplification method, the amount of amplification product is usually 1/20 or less that of LAMP [9] . Consequently, in order to apply this method to PCR, a device for rapidly cycling the temperature to perform PCR, a high-luminance fluorogenic reagent, a system for washing unreacted probes, and a system for accurately dispensing PEI would be necessary. This would likely be an obstacle to putting to practical use a simple, inexpensive g-POCT device for genetic testing.
There are several ways to make LAMP products insoluble besides addition of a cationic polymer like PEI. We tried carrying out the BF separation using chilled ethanol. Since the addition of chilled ethanol lowered the temperature of the LAMP solution, there was a strong tendency for nonspecific probes to weakly hybridize to LAMP products and precipitate (data not shown). Moreover, isopropanol and PEG are believed to be inferior to a PEI solution because it is necessary to add several times the amount of LAMP reaction solution and they are not as easy to handle. Therefore, we believe that low-molecular-weight PEI, which is efficiently insolubilizes amplicons with only a small amount, is the best option for high BF separation, as was shown in this study.
If the 5' end of the inner primer is fluorescently labelled, the LAMP product should be visible as in the current study. However, visualization using inner primers fluorescently labelled at the 5' end is not preferred, because the possibility of false positives from self-extension of the labeled primer cannot be excluded. There is no risk of false positives with the oligo DNA probes fluorescently labeled at the 3' end as in this study, so that highly accurate genetic testing can be established.
It is necessary to add PEI to the LAMP reaction solution after the LAMP reaction takes place since PEI strongly inhibits the LAMP reaction. However, opening the reaction tube after amplification is generally avoided to prevent carry-over contamination. Therefore, development of a technique for adding PEI in a closed system is needed to put this method to practical use. Some possible solutions could be to apply PEI to the lid of the reaction tube beforehand and turn the reaction tube upside down after LAMP amplification or use wax that responds to heat. Furthermore, we should be able to use technology like micro-Total Analysis system or Lab-on-a-Chip, for which much research is being conducted in recent years, to solve this problem.
We established an extremely simple method for visually detecting LAMP products in a sequence-specific manner by simply adding a small amount of low-molecularweight PEI to the LAMP reaction solution. The biggest feature of this technique is the ability to visually present sequence information of amplicons without using an expensive source of light or a detector. In contrast, conventional genetic tests require expensive reagents, complex and skillful manipulation, and large devices equipped with an expensive optical system. These are the main reasons why genetic testing is kept within the walls of specific institutions or university laboratories with special equipment and trained engineers. The combination of the LAMP method and the new detection method described here can overcome several factors that have been preventing true practical application of super-simple g-POCT devices for genetic testing. If a simple, inexpensive g-POCT device that is small and light enough to be held in one hand and whose main components are disposable can be developed, the day when parents will be able to genetically test for a pathogen while sitting next to the bed of their child who has a fever will not be far off [16] .
Viral DNA of the hepatitis B virus (GenBank accession number: V00867) digested with BamHI was cloned to pBR322. Reverse transcriptase PCR was conducted for HCV viral RNA (GenBank accession number: AB031663) from patient serum purified using a QIAmp viral RNA Mini kit (Qiagen K.K.) with the PCR primers designed to the sequence of 5' non-coding region and core. The PCR product was cloned to pBR322 according to the established method. Concentrations of the respective plasmids obtained were determined by 260-nm spectrophotometer (Ultrospec 20000, Pharmacia Biotech). The template DNA solutions for LAMP reactions were prepared by serial dilution of the plasmid solutions and purchased lambda DNA solution (Takara Bio, Inc.) with Tris-HCl buffer (10 mM, pH8.0) until it contained 1,000 copies/5 µl.
The LAMP reaction was carried out on a scale of 25 µl based on reference 1. Briefly, a forward inner primer (FIP) and a backward inner primer (BIP) at a final concentration of 1. The LAMP primers (Sigma Genosis Japan K.K., HPLC purification grade) were as follows. The hyphens were added between F1c or B1c and F2 or B2. The LAMP reaction for Lambda DNA was used as a LAMP reaction for a basic investigation to clarify the fundamental characteristics of the reaction that occurs between the LAMP product and PEI. A well-established LAMP product [1] for mRNA of prostate-specific antigen (PSA) was used as a LAMP reaction product unrelated to the three LAMP reaction products above. A solution was prepared by serially diluting a LAMP reaction solution with a known concentration (28 µg DNA/25 µl) using LAMP reaction buffer in order to systematically investigate the reaction between the LAMP product and PEI. The concentration of DNA synthesized by the LAMP reaction was determined according to the attached protocol using a PicoGreen dsDNA Quantitation Kit (Molecular Probes, Inc.). The spectrofluorophotometer used was the RF-5000 spectrofluorophotometer (Shimadzu Scientific Instruments, Inc.).
The loop segments within LAMP products, i.e., sequences complementary to sequences between either F1 and F2 or B1 and B2, were used as probes for detection, as shown in Fig. 1 . The probes were designed such that the sequences between F1c or B1c and F2c or B2c had a melting temperature (Tm) of 1 to 5°C lower than LAMP reaction temperature (62°C). All fluorescently labeled oligo DNA probes, which were HPLC purification grade, were purchased from Sigma Genosis Japan KK. The sequences of the probe used were as follows. The numbers in parentheses indicate the length and melting temperature (Tm) from supplier's information. 
Commercially available PEI (Wako Pure Chemical Industries, Ltd.) was used without further purification. The average molecular weight of PEI used was 600, 1,800, and 10,000. In this study, the concentration of the PEI aqueous solution is expressed as the concentration of the monomer unit (-C 2 H 5 N-, 46 g/monomer). The PEI stock solution (2.0 mol/l) was prepared by dissolving 4.6 g of PEI in 50 mL of deionized distilled water in a graduated cylinder. At the time of use, the stock solution was diluted with water to the desired concentration.
White precipitate of magnesium pyrophosphate forms in the LAMP reaction solution as a by-product of the amplification reaction [5] . Therefore, after amplification, we first precipitated the magnesium pyrophosphate to the bottom of the reaction tube by centrifugating the LAMP reaction solution for 10 seconds using a small desk-top centrifuge (6,000 rpm). Then, 4 µl of PEI solution adjusted to the desired concentration was added to the supernatant to form an insoluble DNA-cationic polymeric polymer complex at room temperature. A pellet was formed by immediately centrifuging it for 10 seconds using the same centrifuge. A fluorescent image of the pellet was photographed with a fluorescence microscope (VB-G05, Keyence Corporation) equipped with a Handy UV Lamp (wavelength: 365 nm; Vilber Lourmat) and a UV blocking filter (NEO Dynamic L-400, MARUMI Optical Co., Ltd.). Since the magnesium pyrophosphate precipitate was nonfluorescent, it had no effect at all on visualization of pellet fluorescence. To investigate the effect of ionic strength on precipitation titration, aqueous KCl was added to the reaction solution after the LAMP reaction so that it was the prescribed concentration.
After centrifugation, 20 µl of supernatant of the PEI-DNA complex solution was aliquoted to a 384-well assay plate (Corning, Inc.) for fluorometry using a fluorescence plate leader (Polarion; Tecan Japan Co., Ltd.). The percentage of probes captured in the precipitate of the PEI-LAMP product complex was calculated according to the following formula based on the obtained results.  Injection drug use and HIV/AIDS in China: Review of current situation, prevention and policy implications Illicit drug abuse and HIV/AIDS have increased rapidly in the past 10 to 20 years in China. This paper reviews drug abuse in China, the HIV/AIDS epidemic and its association with injection drug use (IDU), and Chinese policies on illicit drug abuse and prevention of HIV/AIDS based on published literature and unpublished official data. As a major drug trans-shipment country with source drugs from the "Golden Triangle" and "Gold Crescent" areas in Asia, China has also become an increasingly important drug consuming market. About half of China's 1.14 million documented drug users inject, and many share needles. IDU has contributed to 42% of cumulatively reported HIV/AIDS cases thus far. Drug trafficking is illegal in China and can lead to the death penalty. The public security departments adopt "zero tolerance" approach to drug use, which conflict with harm reduction policies of the public health departments. Past experience in China suggests that cracking down on drug smuggling and prohibiting drug use alone can not prevent or solve all illicit drug related problems in the era of globalization. In recent years, the central government has outlined a series of pragmatic policies to encourage harm reduction programs; meanwhile, some local governments have not fully mobilized to deal with drug abuse and HIV/AIDS problems seriously. Strengthening government leadership at both central and local levels; scaling up methadone substitution and needle exchange programs; making HIV voluntary counseling and testing available and affordable to both urban and rural drug users; and increasing utilization of outreach and nongovernmental organizations are offered as additional strategies to help cope with China's HIV and drug abuse problem. Illicit drug abuse has become an increasing public health and social concern in the past decades worldwide. Drug abuse causes many problems both to individuals and to societies, including loss of productivity, transmission of infectious diseases, crime, family and social disorder, and excessive health care expenditures [1] . Human immuno-deficiency virus (HIV)/AIDS, associated with injection drug use (IDU) and needle sharing to a large extent, has become one of most stunning tragedies in human history. It has caused more than 20 million deaths, and about 40 million people are living with HIV worldwide thus far, with Africa as the most afflicted continent [2] . As the most populous country in the world, China has also observed rapidly increasing drug abuse and HIV/AIDS occurrence in the past 10 to 20 years. China can still shape the course of its epidemic, but it needs to move swiftly and with great resolve [2] . This paper reviews global illicit drug trafficking, drug abuse and its association with HIV/AIDS epidemic in China, and Chinese policies on illicit drug abuse and prevention of HIV/AIDS, and offers additional strategies to governmental crack down on drug smuggling and drug use prohibition to help cope with China's HIV and drug abuse problem. We searched English and Chinese language literature via Medline and the China National Knowledge Infrastructure and reviewed unpublished official data, including national reports on illicit drug control and HIV/AIDS sentinel surveillance data. More than 100 papers and reports were reviewed. Key databases included: 
Drug abuse in China can be traced to the late Qing Dynasty (1644-1911 A. D.), when British colonists forcefully brought Indian opium into China for exchange of silk, tea, and cash. Opium was then locally planned. By the founding of new China in 1949, more than 20 million Chinese people were opium addicts, representing 5% of the total population [3] . After a short nationwide antidrug campaign, drug abuse was reported to be eliminated from the mainland in the early 1950s, and for the next three decades China was believed to be a drug-free nation [3] .
Illicit drugs reemerged in China in the 1980s as China adopted an open-door policy, and the reemergence was mainly connected with global drug trafficking activities. In Asia, there are two major opiate-producing regions: the "Golden Triangle," comprising three Southeast Asian countries of Myanmar (Burma), Laos and Thailand, and the "Golden Crescent" that includes the three Southwest Asian nations of Afghanistan, Iran and Pakistan [4, 5] . The majority of heroin and opium in the current Chinese market is brought from Myanmar into Yunnan Province or from Viet Nam into Guangxi Province, and then it is transshipped along inland trafficking routes to Sichuan, Guizhou, Gansu and Xinjiang or to Guangdong, Shanghai and Beijing [6] [7] [8] . A small potion of heroin/opium is trafficked into Xinjiang from the "Golden Crescent" [8] . These drugs further penetrate into other provinces. Since the late 1990s, increasing amounts of amphetamine-typestimulants (ATS) and other chemically related synthetic drugs including amphetamine, methamphetamine and ecstasy have been locally manufactured and consumed in China [4] .
The number of drug users documented officially by Chinese public security departments increased from 70,000 in 1990 to 1.14 million by 2004 [9] while the estimated number is 3.5 million [1] . The lifetime prevalence rates of illicit drug use among residents age 15 years or older in high-prevalence Chinese cities increased from 1.1% in 1993 to 1.6% in 1996, and the 1-year prevalence rate increased from 0.9% to 1.2% during this period [10, 11] .
The main drug of choice in China is heroin. According to a report of the National Narcotic Control Commission (NNCC), 87.6% of drug users abused heroin in 2002 [12] . The abuse of ATS and MDMA (methylenedioxymethamphetamine or ecstasy) has become popular in city night clubs in recent years [1] . In the 1980s, farmers living in rural bordering areas in Yunnan and Guangxi provinces constituted a large fraction of drug users. Since the early 1990s, more and more urban residents use illicit drugs. The majority of drug abusers are young people with a low education level and limited job skills, although a small proportion of urban users regard using drugs as an indicator of "high social class." NNCC data showed that 74% were aged 17-35 years in 2002 [12] . Experimentation (90%), peer pressure (44%), and relaxation (42%) are commonly cited reasons for beginning to use drugs. Initially, drugs were taken primarily through sniffing/snorting (55%) or smoking cigarettes mixed with drugs (38%) [13] . Injection of drugs became increasingly common among drug users, probably as a result of increasing prices of illicit drugs and greater cost effectiveness of injecting to achieve the desired effect. National behavioral surveillance data showed that the median prevalence of IDU among drug users increased from 35% in April 1995 to 49% in April 2004, and median prevalence of needle sharing among IDUs also increased from 26% to 43% during this period [14, 15] . "Buying from drug or grocery stores" and "buying from hospitals" were the two most common routes for obtaining injection needles and syringes [16] . Reasons for sharing a needle included: "do not care about anything else when hooked," "thought it had been cleaned up," "difficult to buy or obtain," "only sharing with selected persons," "no money to buy," and "following other drug users" [16] . Besides IDU and needle sharing, other risk behaviors including unprotected commercial sex put drug users and ultimately non drug users at high risk of HIV [17, 18] . It is believed that many female drug users sell sex for drugs, and limited published research also provides supporting evidence [18] [19] [20] . However, the impact of the interaction between IDU and commercial sex on HIV risk is not clear. Considering the dramatic increase in the commercial sex industry in China, and the potential bridging role of those with dual risk behaviors in transmitting HIV from high risk groups to the general population, research on the interaction of IDU and commercial sex should be given high priority.
Since the first AIDS case was detected in 1985 [21] , China has cumulatively reported about 100,000 HIV infections by 2004 [22] . Thus far, the HIV/AIDS epidemic is mainly concentrated in specific geographical areas and sub-group populations [23] . IDU has been the largest contributor to the reported epidemic since 1989, when the first HIV outbreak among IDUs was observed in Ruili City of Yunnan Province bordering with Myanmar [24] . All 31 provinces, autonomous regions and municipalities on mainland China reported HIV infections among IDUs by 2002, and 42% of infections in China were estimated to be due to IDU by 2004 [22] .
In 1995, only one out of 8 national sentinel sites for IDU detected HIV infections. The detected prevalence was 0.02% [14] . By 1997, 3 out of 22 sites detected positives, and the average prevalence increased to 6.6%. After that, national average HIV prevalence among IDUs varied between 5.4% and 8.2% [15, 22] . However, dramatic geographic differences in HIV prevalence have been observed. Yunnan and Xinjiang provinces have the most severe HIV infection rates among IDUs. Other provinces along or close to drug trafficking roads, such as Guangxi, Sichuan, Guizhou, Hunan, and Jiangxi, have moderate epidemics [25] . HIV began to spread rapidly in Yunnan province in the late 1980s and early 1990s [26] . High prevalence among IDUs was first detected in 1998 in Urumqi City (28.8%) and in Yining City (82.2%) of Xinjiang Province and later on in selected sentinel sites for IDU in other provinces [27] . These include Guangxi (>10%, 1998 sentinel data), Jiangxi (14.5%, 2000), Sichuan (16-20%, 2002) , Guizhou (17-19%, 2002) , and Hunan (15-20%, 2003) [23] . In 2004, 75% of HIV national sentinel sites for IDU detected HIV infection. More than 20% of HIV prevalence was reported in selected sites in Xinjiang, Sichuan, Guizhou, Guangxi and Hunan [22] . Official HIV prevalence rates in other geographic areas remain <10%; these reports may reflect reality or may be due to failure in detection.
At least 7 HIV-1 subtypes (A, B/B', C, D, E, F, and G), and 3 major circulating recombinant forms (CRF01_AE, CRF07_BC, and CRF08_BC) have spread in China [28] [29] [30] [31] . However, the most prevalent HIV-1 strains are subtype B' (44%), C (29%, usually CRF08_BC) and CRF01_AE (13%) [30] . Subtype C and B'/B are the main subtypes among IDUs [30, 32] . Co-circulating of multiple subtypes of HIV-1 in China implies the possibility of interclade recombination. Characterization of genetic variability of HIV-1 may help track the epidemic, generate subtype-specific immunological reagents, develop vaccines, and choose antiretroviral therapy regimens.
Drug trafficking and abuse are illegal in China. Offenders may be sentenced to prison if smuggling 10 grams or more of heroin and could receive the death penalty for smuggling more than 50 gram of heroin. In 1990, the "Regulations on Prohibition Against Narcotics" were enacted with three levels of penalty to be applied to drug users. First-time offenders may be fined and/or allowed to go to voluntary detoxification centers, where they receive 10-day methadone treatment managed by the Ministry of Public Health. The cost of treatment is about 2,000 to 5,000 Chinese yuan -a cost considered expensive for many drug users and their families. If drug users who have gone through a voluntary detoxification program are caught again using drugs, they are sent to compulsory rehabilitation centers (CRC) administered by the Ministry of Public Security for 6 to 12 months. Drug users who relapse users after going through CRC are sent to reeducation-through-labor-centers (RELC) administered by the Justice Department for two to three years [33].
In Chinese history, drug abuse and prostitution have been considered "social evils." The Chinese government typically takes "crackdown" measures and tries to eradicate these phenomena. China did achieve a success story in the 1950s. Illicit drug abuse and prostitution were eradicated through national anti-drug and anti-prostitution campaigns [3, 34 ]. However, this success has not been repeated in the past two decades. One possible explanation is that China has expanded its market economy and opened further to the outside world. Under this situation, it seems impossible to completely stem manufacture of highly profitable illicit drugs and import of drugs along China's long porous land borders with drug-producing countries.
Currently, the Chinese government adopts more pragmatic policies and takes measures targeting both the root and surface of the drug abuse problem. The measures targeting the root include continuously cracking down on drug smuggling activities and discouraging new users through anti-drug education campaigns. The Chinese government actively seeks to collaborate with neighboring countries to prevent drug smuggling across borders and to help Myanmar, for example, to reduce opium poppy cultivation by replacing with crop plantation. Chinese mass media have increased anti-drug education to the general population. Anti-drug education has been included in the curricula for primary and secondary school students [9] . The measures targeting the surface include providing drug detoxification and harm reduction services to drug users. China has about 300 voluntary detoxification clinics, 700 CRC, and 200 RELC. Each can accommodate 100 to 3,000 patients [9] . Both western medications and traditional Chinese medicines are used for detoxification, and community therapy has been provided in some heavily affected areas since the late 1990s [9] . The National Working Group for Community-based Methadone Maintenance Therapy was established collectively by the Ministry of Health, the Ministry of Public Security, and the State 
Whether or not China can shun a generalized epidemic of HIV/AIDS may be largely dependent on how China deals with IDU risk factors and breaks the bridge between IDU and heterosexual transmission. Past experience in China suggests that solely cracking down on drug smuggling and prohibiting drug use can not prevent or solve all illicit drug related problems in the era of globalization. Governmental support, harm reduction programs, voluntary counseling and testing, and utilization of non-governmental organizations are recommended.
China has a strong central government. Without government support, it would be not imaginable to achieve success in the campaigns against drug use and the spread of HIV/AIDS. Since the late 1990s, the Chinese central government has stepped up HIV/AIDS control efforts, including setting out national policy framework for responding to HIV/AIDS, increasing funding inputs, expanding collaborations with international organizations. The Chinese National Medium-and-Long-Term Strategic Plan for AIDS Prevention and Control (1998-2010) was formulated in 1998 and set one goal that "by 2002, health education on preventing HIV/AIDS and STDs should be carried out at all detoxification centers and re-education centers as well as in 80% of jails..." [37] . The Action Plan (2001-2005) calls for creating "drug-free communities" through drug prohibition education and drug detoxification activities, together with active promotion of healthy life styles and behaviors and harm reduction for drug users.
On the other hand, there is substantial autonomy at provincial level in some areas. Responses to drug use and HIV/AIDS epidemic vary significantly at provincial and lower administrative levels. For example, Yunnan and Guangxi provinces have done far more than other provinces in supporting, implementing, and advocating for harm reduction interventions for IDUs. Some local governments are not fully motivated to confront drug abuse and HIV/AIDS problems. Some government leaders still ignore or even cover up these problems. They are far more interested in economic growth than HIV/AIDS control and wish for their administrative areas to become "economy provinces" or "economy cities" rather than "AIDS provinces" or "AIDS cities," which is believed to be helpful for their career promotion. Advocacy to and support from government leaders at all administrative levels for harm reduction and community-based prevention are needed.
Harm reduction includes many strategies, such as methadone maintenance, needle exchange, dispensing other drugs, and outreach services [38] . Harm reduction has been a controversial issue as compared to abstinencebased philosophies [39, 40] . Harm reduction seems to encourage tolerance of social phenomena that are undesirable and hazardous and that may result in social turpitude. Instead, abstinence is considered to be the proper way to address drug problems [39] . In the United States, law and policy restrict the use of federal funds in supporting needle and injection equipment distribution projects. However, many studies refute the concern that access to sterile syringes is an endorsement of IDU and is likely to result in increases in injection and initiation of injection [41, 42] . Harm reduction projects in China are in the pilot phases. Some scholars and health officials still have similar concerns as in some Western countries. They believe that needle exchange services may send a wrong signal of encouraging drug abuse to drug users and the public, and they consider methadone substitution unethical because it uses one drug to replace another drug [43] . As drug use and HIV/AIDS spread rapidly across the country, it is urgent to find supporting domestic evidence that harm reduction will reduce HIV transmission by evaluating harm reduction projects and ultimately scaling up harm reduction efforts.
It is easy to access sterile needles and syringes in urban areas of China because they are legally sold and available at pharmacies and medical clinics [16, 44] . However, many drug users live in rural areas; in addition, drug users may share needles and syringes because they can not buy them during night time or do not have money to buy them [16] . Methadone is orally administered, and metha-done substitution can reduce injection and needle sharing of opiate drug addicts. But methadone maintenance therapy is costly and requires drug users to attend clinics on a regular basis. Therefore, methadone substitution and needle exchange services should be made available and affordable at convenient times and in both urban and rural settings, especially in the communities with heavy drug use.
In addition to cost and availability, other factors might also affect acceptability of methadone maintenance therapy, such as concerns about the safety and efficacy of the therapy [45] . Greater retention in treatment has been found to result in greater decreases in drug use, criminal activity, and unemployment [46] . The length of drug treatment has a positive association with better post-treatment outcome [46] . However, limited experience with methadone maintenance therapy (MMT) in China shows a high rate of dropouts. International studies have shown that motivational enhancement therapy or motivational interviewing enhances treatment initiation, retention and outcomes in MMT program [47, 48] , and adding behavioral intervention components into MMT programs increases abstinence and reduces HIV risk behaviors [49] . Policy-oriented operational research is needed in China to better understand how to increase the effectiveness of MMT and other harm reduction interventions in the Chinese context.
There are still persistent conflicts in the policy and legal landscape. The central government has given explicit support to harm reduction, for example, as stated in the Medium-and Long-Term Strategic Plan and the Action Plan. Some programs have been implemented successfully [36,50,51]. However, in China, as in many other countries, public health and public security authorities frequently approach drug abuse from different perspectives, leading to conflicting approaches at local levels. The crackdown philosophy and detention of drug users in China reflect inconsistent interpretations of "harm reduction" and present a challenge to public health officials in implementing methadone substitution and needleexchange programs [52] . Drug users may be reluctant to participate in these programs due to fear of being caught by police officers [50] . It might be impossible to completely solve the dilemma in the near future, but this conflict is expected to gradually reduce for the following reasons. First, Chinese national policies for HIV prevention and control have become much more pragmatic in the past years. MMT and needle exchange programs were almost unimaginable several years ago, but now they are ready to be expanded across the country. We expect that the open policy trend will continue as the Chinese economy is increasingly merged with international markets, and this trend will favor harm reduction programs. Fur-thermore, China's centralized government may achieve an advantage in promoting public health policies if these policies are believed to be correct. Second, inter-agency coordination on public health crisis has been enhanced at both central and local governmental levels since SARS outbreak in 2003, which reduces potential conflict of public health policies. Public health workers should provide policy advocacy to public security authorities and help them change their traditional norms about illicit drug control and obtain their supports for harm reduction. Third, operational research is needed to provide evidence on the benefits of harm reduction programs and convince policy enforcers and lead to revision of unfavorable policy components.
HIV voluntary counseling and testing (VCT) is often considered the first step for initiating prevention and/or therapy. One of the strategies for addressing the AIDS epidemic is to give people an opportunity to know their HIV status so that they can take precautions to avoid further spread and receive early therapy if they are infected [53, 54] . However, even in developed countries, many atrisk people do not take VCT. A national British survey in 2000 showed that only one-third of IDUs had VCT in the past 5 years [55] . About one-fourth of the 0.8 to 0.9 million infected people in the United States remain unaware that they are HIV positive [54] . In China, there is a large discrepancy between reported (about 100,000) and estimated (about 1 million) cumulative HIV/AIDS cases thus far. Many at-risk individuals do not seek out standard HIV counseling and testing services. The stigma associated with drug use and HIV/AIDS and fear of arrest or knowing a positive result can be major barriers to access to VCT. A survey among 840 pregnant women and 780 health professionals in Yunnan Province -an epicenter of the HIV/ AIDS epidemic in China -found prevalent negative attitudes toward HIV/AIDS. Twenty-three percent of health professionals and 45% of pregnant women thought HIV was a disease of "low class and illegal" people; 48% of health professionals and 59% of pregnant women thought that HIV positive individuals should not be allowed to get married; and 30% of the health professionals were not willing to treat an HIV-positive individual [56] . Cost of traditional VCT, low awareness of risk factors for HIV infection, distance, and inconvenience in time also may prevent access to VCT. Possible solutions include development of outreach programs to offer anonymous testing and counseling to those at heightened risk of HIV infection and adoption of new technologies such as rapid saliva testing and counseling strategies to improve the outreach and efficacy of programs.
Non-governmental organizations (NGO) can play a critical role in the delivery of HIV prevention services and other assistance to persons living with AIDS. The flexibility of NGOs enables them to respond quickly to fill in gaps in health care and social services. NGOs can do what government agencies cannot do or are not willing to dofor example, reaching out without perceived threat to IDUs and other marginalized sub-groups whose behaviors are often stigmatized and also put them at higher risk of HIV/AIDS. A recent survey of 29 NGOs in Central and Eastern Europe showed that most NGOs targeted injection drug users; provided needle exchange and HIV prevention peer education; and delivered AIDS presentations and distributed educational materials [57] . In Africa, where the main transmission occurs via heterosexual activity, NGOs are most likely to direct their attention to the general public and to youth; they provide peer-education or community outreach [58] . In both Thailand and Brazil, where success has been observed in controlling the HIV/AIDS epidemic or reducing AIDS mortality, NGO are believed to play a key role, but their programs lack rigorous and systemic evaluation [59, 60] . NGOs often face several difficulties: lack of financial resources [57, 58] ; lack of communications with governmental organizations [61] ; governmental indifference or opposition; and AIDSrelated stigma [57] .
China has large number of government organized NGOs (GONGOs), including Family Planning Associations, Women's Federation, Red Cross, Youth League, trade unions, and diverse academic associations. The members in these GONGOs have formal positions in governmental organizations while they volunteer at GONGOs. The "true" NGO that has no relationship to the government is just emerging in China. More and more of existing Chinese GONGOs are getting involved in sexually transmitted disease/AIDS prevention. Since the 1990s, the Chinese government has encouraged them to participate in HIV/ AIDS control. These government-sponsored NGOs support HIV/AIDS education and academic publications and participate in AIDS research and education with foreign governmental and non-governmental partners, and they can serve as a powerful aid to the Chinese government to achieve the goal of stopping further spread of drug use and HIV/AIDS epidemic.
In spite of its potential for greatly contributing HIV prevention in China, there is little literature in this area. An exception is a recent study (Chen and Liao, 2005 ) of a Women Federation's HIV prevention program in south China. The study showed that the Women Federation was able to deliver a culturally oriented, multi-level intervention program targeted at female drug users. The data also indicated that the program was successfully in increasing knowledge about HIV/AIDS, increasing condom use, and decreasing needle and syringe sharing among the female drug users in the project. Studies which systematically evaluate the implementation and effectiveness of NGO based intervention programs are greatly needed in the future. 
The author(s) declare that they have no competing interests.
HZQ and JES conceived of the study and wrote the first draft of the manuscript, HZQ, HTC and YHR collected the data, and all authors participated in the data interpretation and manuscript revisions. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nm1240) contains supplementary material, which is available to authorized users. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus E protein by nickel-affinity chromatography (data not shown). After immunization and screening 2,000 hybridomas, we isolated 46 new monoclonal antibodies that recognized WNV E protein (Supplementary Table 1 online).
We evaluated the antibodies for their ability to block WNV infection in BHK21 cells using a standard plaque-reduction assay 23 . Twelve had strong neutralizing activity that greatly exceeded the potency of immune human γ-globulin, with 50% plaque reduction neutralization titers (PRNT 50 ) below 2 µg, whereas immune human γ-globulin had a PRNT 50 value of 500 µg 5 . The inhibitory activity of two neutralizing antibodies, E16 and E24, was reproduced in J774.2 mouse macrophages and SW13 human adrenal carcinoma cells ( Supplementary Fig. 1 online) and thus was not specific to fibroblasts.
One of the potent neutralizing monoclonal antibodies, E16, inhibited infection of genetically diverse WNV lineage I strains that were isolated from mosquitoes, birds and horses in New York. E16 neutralized all WNV strains with PRNT 50 values of 4-18 ng and PRNT 90 values of 53-297 ng (Supplementary Table 2 online) . Notably, Fab fragments of E16 inhibited WNV (PRNT 50 , 23 ng), suggesting that neutralization does not require bivalent E protein binding. E16 potently blocked infection with strain 956, the original lineage II strain isolated in 1937 (ref. 24 ), yet was virus specific, as it neither recognized nor neutralized other flaviviruses including distantly related dengue and yellow fever viruses (data not shown) and closely related Japanese and St. Louis encephalitis viruses (Supplementary Table 2 Figure 1 Mapping of monoclonal antibodies to DIII with yeast. (a) Surface display of WNV E protein on yeast. A fusion protein is composed of the ectodomain or DIII of WNV E protein and the yeast Aga2 protein, which becomes attached to the Aga1 protein on the yeast cell wall. Yeast were transformed with the vector alone (pYD1; top row), the entire WNV E ectodomain (amino acids 1-415; middle row), or DIII alone (amino acids 296-415; bottom row). 24 h after induction, yeast were stained with the indicated monoclonal antibodies (negative control, anti-SARS ORF7a) and processed by flow cytometry. Data for a representative neutralizing (E16) and non-neutralizing (E18) antibody are shown. (b) Flow cytometric enrichment for DIII-expressing yeast variants that lose binding of E24. After each round, an increased percentage of DIII expressing yeast are recognized by the polyclonal WNV E-specific antibody but not by E24. After the final round, DIII-expressing variants (boxed region) were harvested. a b Figure 2 Fine epitope mapping of DIII neutralizing and non-neutralizing monoclonal antibodies. (a) Flow cytometry profiles for immunoreactivity by nonneutralizing (E2), weakly neutralizing (E1) and strongly neutralizing (E34) monoclonal antibodies with yeast expressing wild-type and variant DIII. The red, yellow and green arrows point to mutations that abolish yeast surface binding of individual monoclonal antibodies and correspond to distinct regions of DIII (see b). (b) Mapping of neutralizing and non-neutralizing monoclonal antibodies on the surface of WNV DIII. A molecular surface representation is depicted based on the nuclear magnetic resonance structure of WNV DIII 16 . The indicated amino acid residues associated with binding of neutralizing, weakly neutralizing and non-neutralizing monoclonal antibodies are shown in red, yellow and green, respectively.
To map our strongly neutralizing monoclonal antibodies, we developed a strategy using yeast surface display 25 . The ectodomain (amino acids 1-415) or DIII (amino acids 296-415) of WNV E protein were expressed as fusion proteins on the yeast cell surface (Fig. 1a) .
Monoclonal antibodies that recognize DIII alone are considered DIII specific. Monoclonal antibodies that recognized the E ectodomain but not DIII alone may contact residues that map to domain I or II or both, although cooperative contacts with DIII cannot be ruled out. Most of our 46 monoclonal antibodies recognized yeast that displayed the entire ectodomain of E ( Fig. 1a and Supplementary Table 1 online) . Sixteen antibodies recognized yeast that displayed DIII alone, and 10 of 12 strongly neutralizing antibodies localized to DIII. Only two neutralizing antibodies (E53 and E60) recognized the E ectodomain but not DIII alone.
We used error-prone PCR mutagenesis of DIII of WNV E protein and yeast surface expression to map antibody contact residues in a highthroughput manner. We performed individual screens to identify DIII mutants that lost binding selectively to strongly neutralizing (E16, E24 and E34), weakly neutralizing (E1), and non-neutralizing (E2 and E22) monoclonal antibodies. To eliminate mutants that abolished surface expression of DIII, yeast were stained sequentially with an Alexa Fluor 647-conjugated individual monoclonal antibodies and an Alexa Fluor 488-conjugated oligoclonal antibody derived from a pool of individual monoclonal antibodies. After cell sorting, we identified yeast that selectively lost expression of an individual monoclonal antibody epitope but retained surface expression of DIII (Fig. 1b) . Multiple independent yeast that lost binding of individual monoclonal antibodies were subjected to plasmid recovery and sequencing. Monoclonal antibodies that localized to DIII and strongly neutralized WNV had reduced binding when residues S306, K307, T330 or T332 were altered ( Fig. 2a and Table 1 ). These are located on adjacent loops and form a contiguous patch 16 on the solvent-exposed surface at the lateral tip of the DIII (Fig. 2b) . Only two other mutations caused considerable loss of binding of any of the ten neutralizing monoclonal antibodies tested: K310E or P315R reduced binding only of E49. No two neutralizing monoclonal antibodies had identical loss-of-binding patterns. For example, S306L reduced binding of E16, E27, E40, E43 and E49 but not E24, E33, E34, E47 and E58. K307R abolished binding of E34, E40, E43, E47, E49 and E58 but affected E16, E24, E27 and E33 less strongly. In contrast, K307E decreased binding of all neutralizing monoclonal antibodies yet did not affect non-neutralizing or poorly neutralizing monoclonal antibodies. Changes in residues T330 and T332 also abolished binding of neutralizing but not non-neutralizing monoclonal antibodies. T330I or T332M strongly reduced binding of all neutralizing monoclonal antibodies with the exception of E27, and T332V or T332A weakened binding of only E24, E27, E33 and E58.
Six monoclonal antibodies that recognized DIII were either poorly neutralizing or non-neutralizing, and none engaged the dominant neutralizing epitope defined by S306, K307, T330 or T332. E2 and E9 were abolished or reduced by mutation of D381 and H396, and binding of E22 was weakened by a change in P315. D381 and H396 are proximal to one another but physically distinct from the four residues that affect binding of neutralizing monoclonal antibodies (Fig. 2b) . None of the mutations identified by loss-of-binding sorts for E2 or E22 had any effect on two other non-neutralizing monoclonal Individual WNV-specific monoclonal antibodies (25 µg/ml) were mixed with yeast that displayed wild-type or mutant DIII on their surface. After washing, and incubation with an Alexa-647 goat-anti mouse IgG secondary antibody, yeast were analyzed for antibody binding by flow cytometry. The two values represent the percentage of yeast that were positive for DIII expression with a given monoclonal antibody, and in parentheses, the mean linear fluorescence intensity of the positive cells. Yeast were analyzed at 24 h after induction with galactose, which gives a baseline surface expression of DIII of 60-80% positive cells (Fig. 1a) . Bold values indicate an almost complete (>80%) loss of binding, whereas underlined values show a marked (50-79%) reduction in either the percentage or the mean fluorescence intensity of the positive cells. Results are representative of at least two independent experiments for each antibody and DIII variant.
antibodies, E21 and E23. E1, a monoclonal antibody with weak neutralizing activity, mapped to a site between the non-neutralizing and neutralizing monoclonal antibodies, as mutation of K310 and N394 strongly inhibited binding.
To determine whether human antibodies specific for WNV recognize the neutralizing epitope on DIII during infection, plasma was obtained from WNV-positive individuals. Samples from convalescent individuals were negative for WNV RNA but positive for neutralizing WNV-specific antibodies. The individuals reported mild systemic illness, though none progressed to severe disease. To determine whether these samples contained antibodies that localized to the neutralizing epitope on DIII, we tested whether E16 Fab or IgG could compete binding to recombinant, wildtype and mutant N394K and K307N forms of DIII ( Supplementary  Fig. 2 online); N394K retains wild-type binding to E16, whereas K307N has markedly reduced binding. E16 equivalently inhibited binding of patient WNV-specific antibodies to wild-type (Fab, 35% ± 8; IgG, 40% ± 12) or N394K DIII (Fab, 32% ± 9; IgG, 34% ± 11), whereas E53 IgG, a WNV-specific monoclonal antibody that recognizes an epitope outside DIII, did not compete binding to wild-type (1% ± 4) or N394K
(-2% ± 5) DIII. As expected, E16, which only weakly recognizes K307N, poorly competed (Fab 6% ± 3; IgG 9% ± 4) binding to K307N DIII. These data suggest that humans, who clear WNV infection, develop antibodies that recognize an epitope in close proximity to that defined by E16.
To evaluate the correlation between neutralization, epitope localization and in vivo protection, we assessed the therapeutic activity of different neutralizing monoclonal antibodies in an established mouse model 5 . Studies were performed with 5-week-old wild-type C57BL/6 mice, which have a ∼10% survival rate 5 . Mice were inoculated subcutaneously with 10 2 plaque-forming units (PFU) of WNV and administered a single dose of monoclonal antibody at day 2 after infection. Notably, 500 µg of the non-neutralizing monoclonal antibody E2 provided no protection (data not shown). In contrast, 100 µg of any of three different neutralizing monoclonal antibodies that map to K307 (E16, E24 or E34) protected greater than 90% of mice from lethal infection ( Fig. 3a-c) . Even a single 4 µg treatment of E16 or E34 on day 2 after infection prevented mortality. Given that humans can present with WNV infection of the CNS, we evaluated the therapeutic efficacy of monoclonal antibodies at later , or (c) E34 monoclonal antibodies. As controls, mice were independently administered saline (PBS) or a negative control monoclonal antibody (anti-SARS ORF7a, 500 µg). The survival curves were constructed using data from two independent experiments. The number of animals for each antibody dose ranged from 20 to 30. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown (P < 0.0001). (d) WNV burden in the brain of 5-week-old wild-type mice. At days 4, 5 and 6 after WNV infection, brains were harvested and viral burdens were determined by plaque assay. The following percentage of mice had viral burdens below detection (<20 PFU/g): day 4, 33%; day 5, 22%; day 6, 17%. (e,f) Efficacy of WNV-specific monoclonal antibody therapy at days 4 (e) and 5 (f) after infection. A single dose (0.5 mg at day 4 or 2 mg at day 5) of monoclonal antibody (E16, E24, E34 or anti-SARS ORF7a) was administered either 4 or 5 d after WNV infection. Data reflect approximately 20 mice per condition. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown at day 4 (P < 0.0001) and day 5 (E16, P = 0.0009; E24, P = 0.027). (g) Effect of E16 therapy on viral burden. Mice were treated with 2 mg of E16 or PBS on day 5 after WNV infection. On day 9, brains were recovered, homogenized and subjected to plaque assay. For a subset (6) that received PBS treatment, brains were harvested at days 7 and 8 from moribund mice. The data is expressed as PFU/g. Of 16 mice treated with E16 68% (11) had undetectable viral loads in the brain at day 9 whereas all mice (14 of 14) treated with PBS had detectable virus at the time of harvest. The dotted line represents the limit of sensitivity of the assay and the dark bars represent the mean of the log values. The differences were statistically different (P < 0.0001).
time points. At days 4, 5 and 6 after WNV infection, we detected virus in the brains of 67%, 78% and 83% of mice, respectively (Fig. 3d) . A single 500 µg dose of E16 or E34 at day 4 resulted in an 80-90% survival rate (Fig. 3e) . A single 2 mg dose of E16 at day 5 resulted in 90% survival (Fig. 3f) and complete clearance of WNV from the brain in 68% of mice by day 9 (Fig. 3g) . Thus, administration of neutralizing monoclonal antibodies to mice with active CNS infection improved survival and induced a virologic cure. As expected, lengthening the interval before treatment was associated with decreased benefit. Administration of E16 at day 6 did not enhance survival (data not shown), although average survival time was increased (9.5 d ± 0.4 to 11.5 d ± 0.4; P = 0.003).
We considered humanizing E16 or E34 as a possible therapeutic measure. Humanized monoclonal antibodies have substantially longer half-lives in humans than their mouse counterparts 26, 27 . Sequencing studies indicated that E16 had greater homology to human framework regions, making it simpler to construct a humanized version of E16. We amplified the cDNA encoding the heavy (VH) and light (VL) variable domains from the hybridoma cellular RNA by a 5′ rapid amplification of cDNA ends (RACE) procedure. The VH belongs to mouse heavy chain subgroup II (J558 family) and the VL belongs to mouse κ chain subgroup V. The complementarity-determining regions of E16 were grafted onto the human VH1-18 backbone (Fig. 4a) and human Vκ-B3 backbone (Fig. 4b) to create Hm-E16.1. One (VL-Y49S) and two framework back-mutations (VH-T71A and VL-Y49S) were introduced to create two variants, Hm-E16.1 and Hm-E16.3, respectively. The resulting humanized VH and VL were combined with human γ1 and κ constant regions, fused to an IgG signal sequence and inserted into expression plasmids. To construct the chimerized antibodies, the mouse VH and VL sequences were combined with the human γ1 and κ constant regions.
We expressed humanized (Hm-E16) and chimerized (Ch-E16) E16 in HEK-293 cells, and purified them from supernatants by affinity and size-exclusion chromatography (data not shown). The affinity was analyzed by surface plasmon resonance using purified antibody in the solid phase. Mouse E16 binds DIII with an affinity of 3.4 nM and a half-life of 3.9 min. The affinity of the Ch-E16 and Hm-E16 was similar with K D ranging from 7.1 to 21 nM (Fig. 4c) . Hm-E16, Ch-E16, and the parent E16 all had similar PRNT 50 values (Fig. 4d) .
We hypothesized that E16 could also control infection in mice through effector functions including antibody-dependent complement fixation and cytotoxicity. To test this, we generated a Ch-E16 N297Q aglycosyl variant that neutralizes WNV (Fig. 4d) but does not efficiently fix complement or bind Fc γ receptors 28 . Mice were administered Ch-E16 or Ch-E16 N297Q at day 2 after WNV infection. Although high doses of Ch-E16 and Ch-E16 N297Q provided virtually complete protection, lower doses of the aglycosyl variant afforded less protection (Fig. 4e) . Administration of 4 µg of Ch-E16 resulted in 84% survival, whereas 4 µg of Ch-E16 N297Q provided only 31% protection.
To test which effector function enhanced the activity of E16, we performed studies with 8-week-old C1qa, C4, or Fcgr1 and Fcgr3-deficient C57BL/6 mice (Fig. 5) . These mice all show increased susceptibility to lethal WNV infection compared to wild-type controls. In C1qa or C4-deficient mice, which cannot activate complement by the antibody-dependent classical pathway, E16 had a similar potency compared to wild-type mice. In contrast, in Fcgr1-and Fcgr3-deficient mice, although high doses afforded complete protection, lower doses resulted in higher mortality rates. A dose of 20 µg at day 2, which strongly protected wild-type, C1qa or C4-deficient mice, did not improve the survival rate of Fcgr1-and Fcgr3-deficient mice (P = 0.4). Thus, the Fc region enhances the potency of E16 in mice, by virtue of its ability to bind to Fc γ receptors.
To confirm the efficacy of humanized E16, wild-type mice were administered three different versions of purified Hm-E16 at day 2 after infection. Although Hm-E16 variants protected mice against lethal infection (Fig. 4f) , at a dose of 4 µg, the variant (16.3) that showed the highest affinity for DIII (Fig. 4c) was more protective (67% versus 46% survival; mean survival time of 14 ± 1 d versus 11 ± 2 d, P = 0.04).
We generated a panel of 46 monoclonal antibodies against WNV E protein, and applied a new high-throughput epitope-mapping strategy to identify a dominant epitope that was recognized by the majority of neutralizing monoclonal antibodies in DIII. This epitope was also detected by convalescent antibodies from individuals who had recovered from WNV infection without clinical consequence. Three neutralizing monoclonal antibodies protected against WNV mortality in a postexposure therapy model. One of these, E16, was humanized and confirmed as therapeutically effective in mice.
Previous studies have mapped amino acid contact residues of neutralizing monoclonal antibodies by sequencing in vitro neutralization escape variants, through site-specific substitution of specific charged or polar residues, and by performing binding assays with overlapping peptide libraries. We used error-prone PCR mutagenesis and yeast surface expression to identify contact residues in a high-throughput manner. Although this technique has been used previously 29 , this is the first highthroughput epitope-mapping application. By having a large panel of DIII monoclonal antibodies and selecting only variants that abolished or markedly reduced binding of a few monoclonal antibodies, we minimized the possibility that mutations altered protein folding. We have recently confirmed the recognition sites on DIII for E16 and the validity of the yeast display strategy by solving the crystal structure of the E16 Fab-DIII complex (G.N., T.O., M.D, & D.F., unpublished data).
Of 12 neutralizing monoclonal antibodies, 10 localized to the distal lateral surface of DIII, results that are consistent with prior studies that mapped three neutralizing monoclonal antibodies against WNV using in vitro escape variants 15, 30 . Our weakly neutralizing and non-neutralizing monoclonal antibodies did not recognize this epitope but localized to distinct regions. E16 recognized the dominant epitope and neutralized all strains that were tested. Sequence analysis of 124 WNV strains in public databases showed almost complete (98.4-100%) conservation of the contact residues S306, K307, T330 and T332. Only two clinically attenuated lineage II isolates had mutations at these residues. Because of the structural homology among flaviviruses 7, 8, 16 , the analogous amino acids that map to the distal lateral surface of DIII can be readily identified. Although this region is highly variable among flaviviruses, most mutations that abolish binding of virus-specific neutralizing antibodies map here 15, 18, 30, 31 , suggesting the existence of an analogous dominant neutralizing epitope for other flaviviruses. We speculate that successful vaccines against WNV or other flaviviruses should induce potent humoral responses against this neutralizing epitope.
Although passive administration of immune human γ-globulin after WNV infection improved survival in mice 4,5 , it may be limited by its low-titer neutralizing activity, variability and risk of transmission of infectious agents. Only two prior studies have shown postexposure therapy of neutralizing monoclonal antibodies with flaviviruses: 6B5A-2 reduced mortality 3-4 d after infection with St. Louis encephalitis virus 6 ; and 503 reduced mortality resulting from infection with Japanese encephalitis virus 5 d 32 . Here, we show that three different neutralizing monoclonal antibodies improved survival even when administered 4 and 5 d after WNV infection. Moreover, therapy with E16 at day 5 completely cleared WNV from the brain at day 9 in 68% of mice. Thus, inhibitory WNV monoclonal antibodies improve clinical and virologic outcome even after viral spread through the CNS, results that agree with studies showing that antibody can mediate viral clearance from infected neurons 33, 34 .
Our experiments are consistent with a model in which the therapeutic efficacy of monoclonal antibodies is determined by properties in addition to neutralization: (i) the monoclonal antibody (E24) with the strongest neutralizing activity in vitro did not have the greatest efficacy in vivo; (ii) an aglycosyl version of E16 that lacked the ability to fix complement or bind to Fc γ receptors had equivalent neutralizing but reduced therapeutic activity; (iii) E16 was less potent in mice that lacked Fc γ receptors. E16 was humanized as a possible therapeutic measure for humans. Hm-E16 bound DIII with similar affinity and showed efficacy as postexposure therapy. Moreover, it may be possible to improve Hm-E16 by a b c introducing mutations that enhance affinity, creating forms of E16 that more readily cross the blood-brain barrier, and combining monoclonal antibodies that neutralize WNV infection through independent mechanisms. Our results are the first successful demonstration of a humanized monoclonal antibody as postexposure therapy against a viral disease, and suggest that antibody-based therapeutics may have more broad utility than previously appreciated, especially in the treatment of CNS infections in which an effective antibody response is important for limiting virus dissemination and injury to neurons.
We cultured BHK-21, Vero and C6/36 Aedes albopictus cells as previously described 35 24, 36) . We also performed neutralization experiments with prototype strains of St. Louis (59268 (Parton)) and Japanese encephalitis (Nakayama) viruses 37 . For in vivo experiments, viruses were diluted and injected into mice as described 23 .
Purified WNV E protein expression. WNV E protein ectodomain was generated using a baculovirus expression system according to previously described methods for related flaviviruses 38 . The last 45 nucleotides of prM (endogenous signal sequence) and the first 1,290 nucleotides of WNV E protein from the New York 1999 strain 39 were fused downstream of the polyhedrin promoter and upstream of a histidine repeat in a baculovirus shuttle vector (pFastBac, Invitrogen) by PCR using a high-fidelity Taq polymerase (Platinum Taq, Invitrogen). Three days after baculovirus infection of Hi-5 insect cells at a multiplicity of infection (MOI) of 1, supernatants were harvested, filtered, buffer-exchanged and purified by nickelaffinity chromatography according to the manufacturer's instructions. The purified WNV E ectodomain lacks the C-terminal 71 amino acids that are associated with the membrane proximal, transmembrane and cytoplasmic domains.
Purified WNV DIII. The construction, expression, purification and refolding of DIII of WNV E protein is described in greater detail elsewhere (G.N., T.O., M.D. & D.F., unpublished data). Briefly, wild-type, N394K and K307N DIII were generated from an infectious cDNA clone of the New York 1999 strain of WNV (gift of R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO) using PCR and Quik-change mutagenesis (Stratagene). After cloning into a PET21 vector (Novagen) and sequence confirmation of the mutations, we transformed plasmids into BL21 Codon Plus E. coli cells (Stratagene). Bacteria were grown in Luria broth, induced with 0.5 mM isopropyl thiogalactoside (IPTG) and pelleted. Subsequently, we lysed bacteria after the addition of lysozyme, sonicated them and recovered DIII as insoluble aggregate from the inclusion bodies. DIII was denatured in the presence of guanidine hydrochloride and β-mercaptoethanol and refolded by slowly diluting out the denaturing reagents in the presence of L-arginine, EDTA, PMSF, reduced glutathione and oxidized glutathione. We separated refolded DIII from aggregates on a Superdex 75 16/60 size-exclusion column (Amersham Bioscience) and concentrated it using a centricon-10 spin column into 20 mM Hepes pH 7.4, 150 mM NaCl and 0.01% NaN 3 . After refolding, wild-type DIII reacted with all domain III-specific monoclonal antibodies including those that recognized conformationally sensitive epitopes.
Generation and purification of monoclonal antibodies. BALB/c mice were primed and boosted at 3-week intervals with insect cell-generated, purified, recombinant WNV E protein (25 µg) that was complexed with adjuvant (RIBI Immunochemical). Approximately 1 month after the last boost, we harvested serum and tested it for immunoreactivity against solid-phase purified E. Mice with high titers (>1/10,000) were boosted intravenously with purified E protein (5 µg) in PBS. We harvested splenocytes 3 d later and fused them to P3X63Ag8.653 myeloma cells to generate hybridomas according to published procedures 40 . We purified monoclonal antibodies against WNV or other control antigens by stan-dard protein A or protein G chromatography according to the manufacturer's instructions (Pharmacia).
For WNV infection experiments, all wild-type C57BL/6J mice were purchased from a commercial source (Jackson Laboratories). We obtained the congenic C1qa-deficient and C4-deficient mice from G. Stahl (Beth Israel Deaconess Medical Center, Boston, MA) and M. Carroll (The CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA), respectively. We obtained the congenic Fcgr1-and Fcgr3-deficient mice commercially (Taconic). We used mice between 5 and 8 weeks of age depending on the particular experiment and inoculated them subcutaneously with WNV by footpad injection after anesthetization with xylazine and ketamine. Mouse experiments were approved and performed according to the guidelines of the Washington University School of Medicine Animal Safety Committee. For passive-transfer experiments, we administered to mice a single dose of purified monoclonal antibody by intraperitoneal injection at a given time point (day 2, 4 or 5) after infection. To analyze virus production in the brain, infected mice were killed on a given day after inoculation. After cardiac perfusion with PBS, we removed the brains, weighed and homogenized them, and performed plaque assays as previously described 23 .
Expression of WNV E protein on yeast. The ectodomain or DIII of WNV E protein was expressed on the surface of yeast using a modification of a previously described protocol for surface expression of T cell receptors 29 . Amino acid residues 1-415 (ectodomain) or 296-415 (DIII) of WNV E protein were amplified with BamHI and XhoI sites at their 5´ and 3´ ends, respectively, by PCR from the New York 1999 infectious cDNA clone (R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO). The resulting products were digested with BamHI and XhoI, and cloned as downstream fusions to the yeast Aga2 and Xpress epitope tag genes in the yeast surface display vector pYD1 (Invitrogen). An upstream GAL1 promoter controls fusion protein expression. These constructs were transfected into the S. cerevesiae yeast strain EBY100 (refs. 25,41) resulting in yeast that expressed the WNV E ectodomain or DIII. Yeast that only expressed the Xpress epitope tag linked to Aga2 were prepared in parallel by transfecting EBY100 cells with the parent vector pYD1. Individual yeast colonies were grown to log phase overnight in tryptophan-free media containing 2% glucose at 30 °C and harvested in log phase. Fusion protein expression was induced on the yeast surface by growing yeast for an additional 24 h in tryptophan-free media containing 2% galactose at 25 °C. We harvested yeast, washed them with PBS supplemented with 1 mg/ml BSA and immunostained them with 50 µl of monoclonal antibody (25 µg/ml) against the Xpress tag or WNV E protein. After 30 min, we washed yeast three times and stained them with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (Molecular Probes). Subsequently, the yeast cells were analyzed on a Becton Dickinson FACSCaliber flow cytometer.
Library construction and screening. We mutated DIII of the WNV E protein using an error-prone PCR protocol 25 that included Mn 2+ and Mg 2+ at concentrations of 0.3 and 2.0 mM respectively. Subsequently, the cDNA library was ligated into pYD1 and transformed into XL2-blue ultracompetent cells (Strategene). The colonies were pooled and the plasmid DNA was recovered using the Qiagen HiSpeed Maxi kit.
For each individual antibody, we screened the yeast library of DIII mutants according to the following protocol. To identify yeast that selectively lost binding to a given monoclonal antibody epitope, the library was initially stained with an Alexa Fluor 647-conjugated WNV-specific monoclonal antibody for 30 min at 4 °C. To control for the surface expression of DIII, after washing, yeast were subsequently stained for 30 min at 4 °C with an Alexa Fluor 488-conjugated oligoclonal antibody that was derived from a pool of individual monoclonal antibodies (E1, E2, E9, E16, E24 and E34). After immunostaining, we subjected yeast to flow cytometry and identified the population that was single monoclonal antibody negative but pooled oligoclonal antibody positive. The yeast cells were sorted at an event rate of ∼4,000 cells/s and this population (monoclonal antibody-negative and oligoclonal antibody-positive) was enriched after three rounds of sorting. After the final enrichment sort, we plated yeast and selected individual colonies and tested them for binding to individual monoclonal antibodies. For individual clones that had lost only the desired monoclonal antibody epitope, the DIII-pYD1 plasmid was recovered using the Zymoprep Yeast Miniprep kit (Zymo Research). The plasmid was then transformed into DH5α cells, purified using the Qiaprep Spin Miniprep kit (Qiagen) and sequenced.
In some cases, DIII variants with two independent mutations were isolated. To determine which mutation conferred the loss-of-binding phenotype, single independent mutations were engineered by site-directed mutagenesis of DIII-pYD1 using mutant oligonucleotides and the Quik Change II mutagenesis kit (Strategene). All mutations were confirmed by sequencing.
We determined the titer of neutralizing antibodies by a standard plaque reduction neutralization titer (PRNT) assay using either BHK21 or SW13 cells 23 . Results were plotted and the titers for 50% (PRNT 50 ) and 90% inhibition (PRNT 90 ) were calculated. The inhibition assay with J774.2 mouse macrophages was performed as follows: we mixed medium and E16 or E24 (2.5 µg of monoclonal antibody) with 5 × 10 2 PFU of WNV, incubated the mixture for 1 h at 4 °C, and then added to 5 × 10 4 J774.2 mouse macrophages in individual wells of a 24-well plate. After 1 h, cells were washed four times with PBS to remove free virus and monoclonal antibody, DMEM with 10% FBS was added, and the cells were incubated for an additional 24 h. We subsequently harvested supernatants for a viral plaque assay on Vero cells.
Competition ELISA with human anti-WNV antibodies. After purification and refolding, wild-type, K307N and N394K DIII were diluted (5 µg/ml) in 0.1 M Na carbonate buffer (pH 9.3) and adsorbed to 96-well plates overnight at 4 °C. After blocking with PBS, 2% BSA and 0.05% Tween 20 (PBS-BT), wells were preincubated for 1 h at 23 °C with PBS-BT containing no antibody, E16 IgG (50 µg/ml), E16 Fab (50 µg/ml) or E53 IgG (50 µg/ml). E53 serves as a negative control as it recognizes an epitope in domain I and II of WNV E protein. Subsequently, human plasma (1/40 dilution in PBS-BT, heat-inactivated) was directly added for an additional 1 h at 23 °C. We obtained the human samples with informed consent from seven different WNV-infected patients (gift of M. Busch and L. Tobler, Blood Systems Research Institute, San Francisco, CA). Because the samples were sequentially numbered and not linkable back to the original subjects, they satisfied the criteria for exemption from approval from the Human Studies Committee at Washington University. After six washes with PBS-BT, plates were serially incubated with biotin-conjugated goat anti-human IgG (1 µg/ml), streptavidin-horseradish peroxidase (2 µg/ml) and tetramethylbenzidine developing substrate (DAKO). We determined optical densities at 450 nm with an automatic ELISA plate reader (Tecan) and adjusted them after subtraction of the value obtained from nonimmune human plasma.
Surface plasmon resonance. Antibody affinity analysis for DIII of WNV was performed by surface plasmon resonance (BIAcore 3000, Biacore, Inc). Binding curves and kinetic parameters were obtained as follows: we captured E16 antibodies by flowing (300 nM, rate of 5 µl/min for 2 min) them over immobilized F(ab)'2 fragment specific for goat anti-human or mouse IgG with Fc region specificity. Subsequently, DIII of the New York 1999 strain of WNV E protein (amino acids 296-415), which was generated in E. coli, was injected (6.25-200 nM, flow rate 70 µl/min for 1.5 min and then allowed to dissociate over 5 min). The F(ab)'2 surface was regenerated by pulse injection of 10 mM glycine (pH 1.5) and 100 mM NaOH before each E16 injection. We analyzed curves with a global fit 1:1 binding algorithm with drifting baseline.
Cloning and humanization of E16. E16 heavy-and light-chain RNA was isolated from hybridoma cells after guanidinium thiocyanate and phenol-chloroform extraction, and converted to cDNA by reverse transcription. The VH and VL segments were amplified by PCR using the 5′ rapid amplification of cDNA ends (RACE) system (Invitrogen). Gene-specific primers (GSP) for VH and VL were as follows: VH-GSP1: 5´-GGTCACTGTCACTGGCTCAGGG-3´; VH-GSP2: 5´-AGGCGGATCCAGGGGCCAGTGGATAGAC-3´; VL-GSP1: 5´-GCACACG ACTGAGGCACCTCCAGATG-3´; and VL-GSP2: 5´ CGGATCCGATGGATAC AGTTGGTGCAGCATC-3´. The PCR products were inserted into the plasmid pCR2.1-TOPO using the TopoTA kit (Invitrogen). We then subjected the resulting plasmids to DNA sequencing to determine the VH and VL sequences for E16. The cDNA sequences were translated and the predicted amino acid sequence determined. From these sequences the framework and CDR regions were identified as previously defined 42 . We joined the mouse VH to a human C-γ1 constant region and an Ig leader sequence, and inserted it into pCI-neo for mammalian expression. We joined the mouse VL to a human Cκ segment and an Ig leader sequence and also cloned it into pCI-neo for mammalian expression of chimeric E16 (Ch-E16). For Ch-E16, site-directed mutagenesis was also performed to change residue 297 from asparagine to glutamine of the heavy chain to eliminate the single glycosylation site on the γ1 Fc.
Humanized E16 VH consists of the framework segments from the human germline VH1-18 VH segment and JH6 segment 43, 44 , and the CDR regions of the E16 VH, respectively. The humanized E16 VL consists of the framework segments of the human germline VK-B3 VL segment and JK2, (refs. 45-47) segment and the CDR regions of E16 VL. The humanized VH segments were assembled de novo from oligonucleotides and amplified by PCR. The humanized VL segments were assembled by PCR and overlapping PCR. The resulting VH and VL segments were subsequently combined by overlapping PCR with a leader sequence and the appropriate constant region segment and cloned into the expression vector pCI-neo as NheI-EcoRI fragments. We confirmed the DNA sequence of the resulting plasmids by sequence analysis. Site-directed mutagenesis was then performed to substitute mouse for human residues at key framework positions VH-71 (T71A) and VL-49 (Y49S). The resulting plasmids were cotransfected into human 293 cells using lipofectamine-2000 and humanized antibody was recovered from the resulting conditioned medium and purified by protein A and size-exclusion chromatography.
Statistical analysis. All data were analyzed with Prism software (GraphPad Software). For survival analysis, Kaplan-Meier survival curves were analyzed by the log-rank and Mantel-Haenszel test. For viral burden experiments, we determined statistical significance using the Mann-Whitney test.
Note: Supplementary information is available on the Nature Medicine website. Local public health workers' perceptions toward responding to an influenza pandemic BACKGROUND: Current national preparedness plans require local health departments to play an integral role in responding to an influenza pandemic, a major public health threat that the World Health Organization has described as "inevitable and possibly imminent". To understand local public health workers' perceptions toward pandemic influenza response, we surveyed 308 employees at three health departments in Maryland from March – July 2005, on factors that may influence their ability and willingness to report to duty in such an event. RESULTS: The data suggest that nearly half of the local health department workers are likely not to report to duty during a pandemic. The stated likelihood of reporting to duty was significantly greater for clinical (Multivariate OR: 2.5; CI 1.3–4.7) than technical and support staff, and perception of the importance of one's role in the agency's overall response was the single most influential factor associated with willingness to report (Multivariate OR: 9.5; CI 4.6–19.9). CONCLUSION: The perceived risk among public health workers was shown to be associated with several factors peripheral to the actual hazard of this event. These risk perception modifiers and the knowledge gaps identified serve as barriers to pandemic influenza response and must be specifically addressed to enable effective local public health response to this significant threat. Local health departments are considered the backbone of public health response plans for any and all infectious disease outbreaks. An influenza pandemic is considered increasingly likely, and is now considered one of the most significant and urgent threats to the nation's public health preparedness infrastructure. It has been argued that of the 12 disaster scenarios recently assessed by the U.S. Department of Homeland Security, pandemic influenza is the most likely and perhaps the most deadly [1] . The United States pandemic influenza plan released in November 2005, lays out a critical role for local and state public health agencies during a pandemic, including: providing regular situational updates for providers; providing guidance on infection control measures for healthcare and non-healthcare settings; conducting or facilitating testing and investigation of pandemic influenza cases; and investigating and reporting special pandemic situations [2] . These specified activities would require an extensive prompt response by local health departments. Current contingency plans account for possible personnel shortages due to influenza morbidity, but previous studies have shown that during extreme scenarios, a varying proportion of healthcare workers may be unable or unwilling to report to duty [3] [4] [5] . This may be even truer for health departments, where unlike more "traditional" first responder agencies (such as law enforcement, fire services, and emergency medical services), the capacity and willingness to respond 24/7 to crises is not historically ingrained in the workforces' professional cultures and training. Even in the post-9/11 environment, recent data indicate inconsistent and sometimes slow after-hours response by health departments to urgent events involving communicable disease [6] .
Risk perception theory provides a revealing framework for better understanding response limitations and needs of the public health workforce. The perceived risk, according to this theory, is a multifactorial phenomenon, involving the summation of actual risk and other peripheral influences independent of the actual risk, such as perceived authority, trust, and situational control; these peripheral influences have been termed "outrage" [7] or "dread." [8] .
Based on these models, it was previously suggested that contributing factors peripheral to the actual risk will have a considerable practical impact on how public health employees would respond in a crisis [9] . Aside from physical and circumstantial barriers such as availability of transportation or dependency of family members, we have identified specific risk perception issues whose impact may be markedly high and of unique importance for the public health workforce's response to a crisis. These factors, or modifiers, stem from a number of features previously suggested to have been associated with elevated risk perception, including manageability of the threat; risk to future generations; direct personal impact; and sense of control over events.
Based on these modifiers, several major barriers to effective public health workforce emergency response were suggested; these include uncertainty regarding working environment safety, unclear expectations of role-specific emergency response requirements, safety and well being of family members, inadequate emphasis on the critical value of each employee to the agency response efforts, and insufficient emphasis on stress management techniquesall of which may heighten employees' sense of dread due to a lack of personal control [9] .
In light of the projected impact of an influenza pandemic, health departments must optimize the response rate of their employees in this crisis scenario. Based on the emer-gency response principle that all disasters are "local" [10] , we have set out to assess local public health employees' risk perception and likelihood of reporting to duty during a local outbreak of pandemic influenza, and to uncover the variables that affect these outcomes, thus providing a needed evidence base for health departments' planning and training efforts.
We conducted the study in Carroll, Dorchester, and Harford county health departments between March 2005 and July 2005. All three health departments are located in Maryland, and range in size from 132 employees to 225 employees. We selected these health departments because of their location in communities ranging from 30,000 on Maryland's Eastern Shore (Dorchester County) to 235,000 in the greater Baltimore/Towson metropolitan area (Harford County) [11] , thus reflecting the 96% of the nation's local public health agencies serving communities with populations of 500,000 or fewer [12] . Self-administered anonymous surveys were sent to all health department personnel by their respective health departments. Completed surveys were directly mailed to investigators at the Johns Hopkins Center for Public Health Preparedness.
The survey included questions on personal characteristics such as job classification, gender, and age. The respondents used a 5-point Likert scale for questions pertaining to a possible flu pandemic: probability of them reporting to work ("very likely" to "not at all likely"); possibility of being asked by their health department to respond to an emergency ("very likely" to "not at all likely"); how knowledgeable they thought they were about the potential public health impact of pandemic influenza ("very knowledgeable" to "not at all knowledgeable"); how confident they were about being safe in their work roles ("very confident" to "not at all confident"); how likely was their family prepared to function in their absence ("very likely" to "not at all likely"); how likely they felt their health department would provide them with timely updates ("very likely" to "not at all likely"); how familiar they were with their role specific response requirements ("very familiar" to "not at all familiar"); how well they thought they could address the questions of a concerned member of the public ("very well" to "not at all"); how significant a role they thought they would play in the agency's overall response ("very significant" to "not at all significant"); how important would be pre-event preparation and training ("very important" to "not at all important"); how important it was for them to have psychological support available during the event ("very important" to "not at all important"); and how important it was for them to have psychological support available after the event ("very important" to "not at all important").
The job classification variable was collapsed into technical/support staff (such as computer entry staff, clerical staff (e.g. receptionists), computer specialists, health information systems data analysts etc.), and professional staff. The latter included public health officials, clinical staff (e.g., nurse, dentist, physician), public health communicable disease staff, environmental health staff, public information staff, and other public health professional staff (e.g., health educator, legal professional, financial officer, other).
We dichotomized the responses to the job classification question into professional and technical/support categories. Questions about likelihood of reporting to work and pandemic influenza-related attitudes and beliefs were dichotomized into responses with a score two or less, and all other responses. We used logistic regression to compute Odds Ratios to evaluate the association of demographic variables and attitudes and beliefs with selfdescribed likelihood of reporting to work. We used multivariate logistic regression to explore associations between attitudes and beliefs related to pandemic influenza preparedness and self-described likelihood of reporting to work. The model included adjustment for age, gender, and job classification. Similarly, we used bivariate and multivariate (adjusted for age, gender, and job classification) logistic regression models to evaluate the association between the various attitudes and beliefs. In order to assess non-response bias, we compared age, gender, and job classification distributions for the respondents and for all health department personnel.
We used TeleForm Version 8 (Cardiff, Vista, CA) and Stata Version 9 (Stata Corporation, College Station, TX) for data capturing and analysis respectively.
We received 118 out of 205 (57.6%), 74 out of 128 (57.8%), and 116 out of 198 (58.6%) surveys fromCarroll, Dorchester, and Harford county health departments respectively, resulting in an overall response rate of 58.0% (n = 308). We did not find a statistically significant difference in age and gender distribution between the respondents and all health department personnel. A small yet statistically significant difference in the proportion of technical/support staff (vs. professional staff) was detected (22.4% vs. 32% in the study group and all personnel respectively, p = 0.003), yet no significant difference in the proportions of professional staff subgroups was detected.
Of the 303 who responded to the question about their likelihood of reporting during a pandemic influenza related emergency, 163 (53.8%) indicated they would likely report to work during such an emergency. Age and gender did not have an association with likelihood of reporting. Clinical staff indicated a higher likelihood of reporting (Multivariate OR: 2.5; CI 1.3-4.7) than technical/support staff (Table 1) .
Only 40% of all respondents-45.1% professional staff and 26.1% technical/support staff -felt it was likely they would be asked by their health department to respond to a pandemic influenza related emergency. Perception of Table 2) . Perception of one's existing knowledge about pandemic influenza, and perception of having an important role in the agency's overall response were significantly higher among professional staff compared to technical/support staff (Figure 1 ), In multivariate analysis, increased self-described likelihood of reporting to work during an influenza pandemic emergency was significantly associated with agreement with several constructs, most notably perception of the capacity to communicate risk effectively, perception of the importance of one's role in the agency's overall response, and familiarity with one's role-specific response requirements in a pandemic influenza related emergency. ( Table  2 ).
The vast majority (83%) of the respondents felt they would benefit from additional training activities. A lower perceived level of familiarity with one's role was not significantly associated with a higher perceived need for additional training ( (Figure 2 ).
The World Health Organization has urged all countries to prepare for the next influenza pandemic, which it termed . The federally adopted U.S. model of all-hazards emergency readiness has presented local health departments with new organizational challenges and learning curves. The all-hazards approach entails an ability and willingness to respond to a broad spectrum of disasters, ranging from the intentional (e.g., chemical, biological, or radiological terror) to the naturally occurring (e.g., weather-related crises or non-bioterrorism related infectious disease) [14] .
Current national contingency plans account for possible personnel shortages within the healthcare and public health settings, mainly due to the expected influenza morbidity among workers. Yet our data suggest that regardless of the expected morbidity among personnel during an influenza pandemic, nearly half of the local health department workers are likely not to report to duty during such an extreme public health crisis. In fact, most of the workers (and nearly three out of four technical/support workers) do not believe they will even be asked to report to work.
We have found that the willingness to report to duty during a pandemic varies considerably according to the individual's job classification. Clinical staff state they are significantly more likely to report to duty, compared with all other workers. This difference correlates well with the single most influential construct associated with willingness to report to duty -the perception of the importance of one's role in the agency's overall response. Less than a third of the respondents believed they will have an important role in the agency's response to local outbreaks of pandemic influenza, but within this subgroup, willingness to report to duty was as high as 86.8%. Belief in the importance of one's role was lowest among technical/support staff, environmental health staff, and other non-clinical professional staff (15.1%, 18.4% and 18.8% respectively), groups in which willingness to report was shown to be lowest. We therefore believe further efforts must be directed at ensuring that all local public health workers, but most notably non-clinical professional staff, understand in advance the importance of their role during an influenza pandemic -otherwise they will fail to show up when they are most needed.
Our findings fit well in the theoretical framework emphasizing risk communication needs of public health workers, who themselves serve as risk communicators [9] . Several factors, previously suggested to be risk perception "modifiers" [9] of substantial impact on public health
Proportion of individuals who agreed with each of the attitude and belief constructs by staff type Figure 1 Proportion of individuals who agreed with each of the attitude and belief constructs by staff type. 
Technical/Support Staff Professional Staff * * workforce's response to a crisis, indeed proved to be important in this context. Lack of knowledge, ambiguity regarding one's exact tasks, and questionable ability in performing one's role as risk communicator were all significantly associated with a higher perceived personal risk and a two-to ten-fold decrease in willingness to report to duty; these factors proved to be more influential even than the perceived level of family preparedness to function in one's absence. It is therefore important to recognize that public health employees, who are intended to serve as purveyors of risk communication for their communities, themselves represent a community with specific perceptions that must be addressed in the context of emergency readiness training.
The threat of an impending influenza pandemic is not a new one -pandemics have been taking place once every several decades for over 300 years. Yet it was only in the last couple of years, as highly pathogenic H5N1 strain became increasingly endemic in southeast Asia and as lethal infections with the virus occurred in an alarmingly increasing rate among humans, that the urgency of the situation was openly declared by national and international health authorities. The rapidity of this evolving situation may serve to explain why only one third of the respondents felt they were adequately knowledgeable on pan-demic influenza, and why only one in five respondents felt capable in effectively communicating pandemic risks. This finding is especially noteworthy, in that members of the public health support staff may become frontline telephone risk communicators in a crisis, serving as the first points of interface for concerned callers contacting a health department. Only one of the 35 technical/support staff workers who felt incapable of effective risk communication was willing to report to duty, even though most of them believed the health department will have the ability to provide timely information.
The study has some relevant limitations that must be factored into the overall analysis. First, the sample was limited to three non-randomly selected health departments, none of which serves a community larger than 250,000 residents, and all of which have staff sizes under 250. The sample size of 308 survey employees limited this study's power. As the study includes Maryland health departments only, it does not account for potential jurisdictional or regional variations nationwide in response capacity or risk perceptions toward pandemic influenza response. Furthermore, the job classifications -based on those used to develop the CDC-adopted emergency preparedness competencies [15] -do not necessarily map neatly onto functional responsibilities in disaster Odds Ratios of reporting to work in case of a pandemic-influenza-related emergency by staff and attitude or belief construct Figure 2 Odds Ratios of reporting to work in case of a pandemic-influenza-related emergency by staff and attitude or belief construct. 
Technical/Support Staff Professional Staff response. For example, health educators may play as frontline a role as clinical staff, in terms of their degree of interface with the public in a disaster. Our job categories therefore do not necessarily reflect the relative impacts of job-specific cohorts on disaster response in the event that they do not report to work.
We assessed the presence and the direction of nonresponse bias by comparing the distribution of personal characteristics for the respondents and for all health department personnel. The lack of significant difference in age and gender distribution, as well as the lack of significant difference in job classification other than technical/ support staff indicates that the extent of such a bias in the study is probably limited. The small yet statistically significant over-representation of technical/support staff in our study group may potentially have caused a slight underestimation of overall willingness to report. However, as the internal associations between the various variables were also studied separately for the technical/support staff and professional staff (Figure 2) , this over-representation should not impact the general conclusions presented above.
Having accounted for these limitations, it is important to note that the findings were internally consistent among the three surveyed health departments. Although none of the health departments served large metropolitan areas and all had fewer than 250 employees, it must also be recognized that only 4% of the nation's local health departments serve populations of 500,000 or more, and that local public health agencies tend to have small staff sizes (with a median of 13 full time employees) [12] .
Interestingly, our findings show similar patterns to data on the willingness of urban healthcare workers from nonpublic health settings to respond to emergencies: a survey of 6248 employees from 47 healthcare facilities in the New York City area revealed that these workers were least willing (48%) to report to duty during an untreatable naturally-occurring infectious disease outbreak affecting their facility (SARS), compared to other disaster scenarios [5] . In our study we have detected similar rates of likelihood to report to duty, although lower rates could have been expected in our study population since the New York City survey focused on healthcare workers whose organizational cultures are historically much more accustomed than that of local public health workers to emergency response, in a city with a heightened awareness of disaster preparedness in the wake of the World Trade Center attacks and subsequent anthrax attacks [5] .
In the face of a pandemic influenza threat, local health department employees' unwillingness to report to duty may pose a threat to the nation's emergency response infrastructure. Addressing the specific factors associated with this unwillingness is necessary to help ensure that existing local health department preparedness competencies [15] will translate into the scope of response described in the nation's pandemic influenza plans [2] . Interventions suggested to enhance the willingness of healthcare workers in non-public health department settings to report to duty in disasters include workforce preparedness education [5] , provision of appropriate personal protective equipment, [4, 14] crisis counseling, family preparedness and social support [5, 16] .
These recommendations fit well within the framework of our findings, and we further recommend that such education programs include specialized training emphasizing the specific nature of, and guidelines for, one's role in response to pandemic influenza; the relevance of each worker's role in the effectiveness of an overall public health response; and the workers' ability to provide effective risk communication. Additional research must further focus on best practice models for addressing the above described gaps in local public health response to this urgent public health threat.
These data offer a current, evidence-based window into the needs of public health workers who would serve as a backbone of locally-driven emergency response in an influenza pandemic setting. We found that most of these workers feel they will work under significant personal risk, in a scenario they are not adequately knowledgeable about, performing a role they are not sufficiently trained for, and believing this role does not have a significant impact on the agency's overall response. These specific perceptions and needs must be attended, and specific intervention programs must be initiated. In order to reduce the perceived risk associated with the worker's role in an influenza pandemic, each worker must have better understanding of the scenario and importance of his or her personal role within these settings, confidence that the agency will provide adequate protective equipment for its employees, psychological support and timely information, and a belief of being well-trained to cope with emergency responsibilities including the ability to communicate risk to others. In view of what is currently considered to be an impending influenza pandemic, a wide gap between these desired targets and current status exists, that may lead to significant hindrance in the ability of local health departments to function adequately. On pandemics and the duty to care: whose duty? who cares? BACKGROUND: As a number of commentators have noted, SARS exposed the vulnerabilities of our health care systems and governance structures. Health care professionals (HCPs) and hospital systems that bore the brunt of the SARS outbreak continue to struggle with the aftermath of the crisis. Indeed, HCPs – both in clinical care and in public health – were severely tested by SARS. Unprecedented demands were placed on their skills and expertise, and their personal commitment to their profession was severely tried. Many were exposed to serious risk of morbidity and mortality, as evidenced by the World Health Organization figures showing that approximately 30% of reported cases were among HCPs, some of whom died from the infection. Despite this challenge, professional codes of ethics are silent on the issue of duty to care during communicable disease outbreaks, thus providing no guidance on what is expected of HCPs or how they ought to approach their duty to care in the face of risk. DISCUSSION: In the aftermath of SARS and with the spectre of a pandemic avian influenza, it is imperative that we (re)consider the obligations of HCPs for patients with severe infectious diseases, particularly diseases that pose risks to those providing care. It is of pressing importance that organizations representing HCPs give clear indication of what standard of care is expected of their members in the event of a pandemic. In this paper, we address the issue of special obligations of HCPs during an infectious disease outbreak. We argue that there is a pressing need to clarify the rights and responsibilities of HCPs in the current context of pandemic flu preparedness, and that these rights and responsibilities ought to be codified in professional codes of ethics. Finally, we present a brief historical accounting of the treatment of the duty to care in professional health care codes of ethics. SUMMARY: An honest and critical examination of the role of HCPs during communicable disease outbreaks is needed in order to provide guidelines regarding professional rights and responsibilities, as well as ethical duties and obligations. With this paper, we hope to open the social dialogue and advance the public debate on this increasingly urgent issue. In 2003, the world witnessed the spread of a novel and deadly virus, namely SARS CoV. The health care workers (HCWs) and hospital systems that bore the brunt of the SARS outbreak continue to struggle with the aftermath of the crisis. Indeed, HCWs -both in clinical care and in public health -were severely tested by SARS. Unprecedented demands were placed on their skills and expertise, and their personal commitment to their profession was severely tried. Many were exposed to serious risk of morbidity and mortality; indeed, approximately 30% of reported cases were among HCWs, some of whom died from the infection [1] .
As a number of commentators have noted, SARS exposed the vulnerabilities of our current health care systems and governance structures [2] [3] [4] . The aftermath of SARS and the spectre of pandemic avian influenza make imperative the need to consider the obligations of HCWs for patients with severe infectious diseases, particularly diseases that pose risks to those providing care. It is of pressing importance that organizations representing HCWs -professionals and non-professionals alike -give clear indication of what standard of care is expected of their members in the event of a pandemic.
Many experts believe that the SARS outbreak was merely a preview of the next flu pandemic that is soon to arrive, possibly from an avian influenza virus [5] . Quite clearly, avian flu threatens to be more widespread than SARS, with the potential to become a truly global pandemic. An honest and critical examination of the role of HCWs during such a crisis is needed in order to provide guidelines regarding professional rights and responsibilities, as well as ethical duties and obligations [6] .
In this paper, we address the issue of special obligations of health care professionals (HCPs) during an infectious disease outbreak. We contend that there is a pressing need to clarify the rights and responsibilities of HCPs, especially in the current context of pandemic flu preparedness. Moreover, we argue that these rights and responsibilities would best be codified in professional codes of ethics. Finally, we present a brief historical account of the treatment of the duty to care in professional health care codes of ethics with the intention of opening the social dialogue and advancing the public debate on this increasingly relevant issue.
Given that the response by HCPs to the SARS crisis was generally regarded as exemplary, one might ask whether an ethical problem truly exists. There is little doubt that the vast majority of HCPs performed their jobs admirably under considerable stress and significant personal risk. Many HCPs provided exemplary care, and still others behaved in truly heroic fashion. So why, then, formally problematize something that is not a problem?
As noted, many HCPs acted in a supererogatory manner during the SARS outbreak [7], none more so than Dr. Carlo Urbani of the World Health Organization, who himself died of SARS after being exposed to the yet unknown virus in the course of carrying out his professional duties. Likewise, scores of nurses, doctors, respiratory technicians, and other professional and nonprofessional health workers laboured extremely long hours at personal risk. This demonstration of going above and beyond the call of duty, which proved necessary to control the disease, was highly morally commendable.
At the same time, however, serious concerns did surface during SARS about the extent to which HCPs would tolerate risks of infection [8, 9] . Some baulked at providing care to those infected with the unknown virus. In some circumstances, staffing became an issue in SARS wards and assessment centres; indeed, failure to report for duty during the outbreak resulted in the permanent dismissal of some hospital staff. As a consequence, the risk that was faced during SARS was not distributed equitably, and those HCPs who volunteered to provide care faced the greatest exposure.
Following the outbreak, many of those who treated SARS patients raised concerns about the protections that were provided to safeguard their own health and that of their family members. Conflicting obligations were another significant concern. HCPs are bound by an ethic of care, therefore, obligations to the patient's well-being should be primary. At the same time, however, HCPs have competing obligations to their families and friends, whom they feared infecting, in addition to obligations to themselves and to their own health (particularly those with special vulnerabilities, such as a co-morbid condition). During SARS, some HCPs questioned their choice of career; indeed, some decided to leave their profession and pursue new ventures, indicating an unwillingness or inability to care for patients in the face of risk. Recent survey data from the U.S. indicate that there exists mixed views on the duty to care for patients during infectious disease outbreaks [10] .
What is clear is this: the issue of duty to care has emerged as a matter of paramount concern among health care professionals, hospital administrators, public policy makers, and bioethicists [11] [12] [13] [14] .
The ethical foundations of the duty to provide care are grounded in several longstanding ethical principles. Foremost among these is the principle of beneficience, which recognizes and defines the special moral obligation on the part of HCPs to further the welfare of patients and to advance patients' well-being. In modern health care, it is commonly understood and generally accepted that the principle of beneficence constitutes a foundational principle of the patient-provider relationship [15] .
For the HCP in general, and for the physician in particular, there are a number of compelling reasons to provide care in the context of an infectious disease outbreak. Clark [12] has recently outlined three such reasons:
1. The ability of physicians and health care professionals to provide care is greater than that of the public, thus increasing the obligation to provide care Although self-care and self-protection, as well as the care and protection of friends and family members, are acknowledged in pandemic plans, it is evident that the expertise of HCPs is an integral and principal component of the response to a pandemic. There is no other sector of society that can be legitimately expected to fulfil this role and to assume this level of risk.
Arguably, HCPs have consented to greater than average risk by their very choice of profession. While it may be granted that the risk of contracting an infectious disease was likely not a concern for a generation of prospective health care workers, any informed reading of the medical literature in the last 20 years has shown that infectious diseases remain ubiquitous and problematic -notwithstanding overly-optimistic statements regarding the future threat of infectious diseases. It is therefore not unreasonable to argue that HCPs were aware of the greater than average risks posed by their choice of profession.
In publicly-funded health care systems, such as those found in many Western societies, there is a strong claim for a social contract between the HCP and society. It is a reasonable and legitimate expectation by the public that HCPs will respond in an infectious disease emergency. Society has granted and permits professions to be self-regulating on the understanding that such a response would occur.
One of the characteristics of a self-regulating profession is the development of standards of practice, sometimes referred to as best practice guidelines. These standards are articulated in professional codes of ethics, which are developed on the basis of the fundamental principles and values of the particular profession, as is the case, for instance, with respect to the codes of ethics that were developed long ago in medicine and nursing. Indeed, the code of ethics has a long and respected tradition in the health professions and today most, if not all, the various health and social care professions have codes of ethics in place to provide guidance to their members.
The code of ethics is sometimes referred to as an instrument of "soft law," owing to its non-legislative nature [16] . As such, in the health care professions, codes of ethics should be interpreted as guides for ethical reasoning and frameworks for the treatment of individual patients, rather than as substitutes for such reasoning or as an absolute mandate [17] . At the same time, a code that is too vague can render it ineffectual and irrelevant. In an era in which health care and technology are evolving at a rapid pace, efforts are necessary to ensure that codes of ethics remain current, practical, and concordant with public expectations.
An informative and comprehensible code of ethics has numerous tangible benefits. Perhaps the greatest benefit would be to dispel confusion and uncertainty for HCPs concerning their professional rights and responsibilities as regards the duty to care. Of course, a detailed treatment of the issue in professional codes of ethics would also serve to increase awareness and comfort levels, perhaps resulting in increased willingness to provide care in uncertain and risky conditions [18] . Additionally, codes guiding professional conduct may effectively serve as norms of standards recognizable and enforceable by law, acting as the foundation of legal obligations and decisions [16] .
Finally, codes of ethics also serve as potent forms of symbolic communication to the public that is served by the professions. By making explicit the values that health care professions represent, professional codes of ethics can reassure the public that the trust invested in the professions is justified and legitimate, as is properly noted in the following excerpt from the College of Nurses of Ontario Practice Standard on Ethics:
"Nurses have a commitment to the nursing profession. Being a member of the profession brings with it the respect and trust of the public. To continue to deserve this respect, nurses have a duty to uphold the standards of the profession.... As members of a self-regulating profession, nurses also have a commitment to help regulate nursing to protect the public's right to quality nursing services. It is in the public's interest that the profession continue to regulate itself by developing and changing the methods of self-regulation to meet the changes in health care and society. Nurses have an obligation to participate in the effective evolution of self-regulation. Self-regulation is a privilege and each nurse is accountable for the responsibilities that accompany this privilege." [19] What do current codes of ethics say regarding duty to care during epidemics? It is of no small concern that many current professional codes of ethics fail to provide explicit guidance sufficient to set policy or assure the public in the event of an infectious disease outbreak. The Canadian Medical Association, for instance, released a revised Code of Ethics in 2004, one year after the SARS pandemic in which Canada was particularly affected [20] . Despite the seemingly fortuitous timing of the publication, the revised Code is, quite astoundingly, altogether silent on physicians' duty to care, which might be described as the first among equals of the myriad ethical dilemmas that emerged during the global outbreak.
The key revision in the 2004 edition of the CMA Code was the addition of the following item to the 'Fundamental Responsibilities' section: "Consider the well-being of society in matters affecting health" [20] . This addition, however, does little to address, in any substantively meaningful way, the duty to care obligations of HCPs in the context of an infectious disease outbreak. Does the addition of this responsibility obligate physicians to provide treatment even when doing so would put their own health in peril? The wording is too vague to be of any significant guidance in clinical practice.
In contrast, the American Medical Association (AMA) appears to have recognized the present need to address the issue of duty to care. In the wake of the 9/11 terrorist attack, the AMA has adopted several new ethics policies that focus specifically on the medical profession's obligations and responsibilities in the context of a public health emergency. The following passage is from the AMA policy document "Physician Obligation in Disaster Preparedness and Response" that was adopted in June 2004: "National, regional, and local responses to epidemics, terrorist attacks, and other disasters require extensive involvement of physicians. Because of their commitment to care for the sick and injured, individual physicians have an obligation to provide urgent medical care during disasters. This ethical obligation holds even in the face of greater than usual risks to their own safety, health or life. The physician workforce, however, is not an unlimited resource; therefore, when participating in disaster responses, physicians should balance immediate benefits to individual patients with ability to care for patients in the future." [21] While the AMA has taken a step in the right direction by stating the obligations of its members, and it is to its great credit for initiating this process, it remains to be seen whether other national medical associations and other health care professions will follow suit and redress the silence of codes of ethics on the duty to provide care.
To some extent, codes of ethics can be seen as reflections of enduring professional values. At the same time, they are also clearly influenced by and are the product of historical circumstances. The CMA, for instance, previously included in its Code of Ethics a strongly worded statement explicitly addressing the obligations of physicians in infectious disease outbreaks. The 1922 version reads as follows: "When pestilence prevails, it is their [physicians'] duty to face the danger, and to continue their labours for the alleviation of suffering, even at the jeopardy of their own lives" [22] . This is the only appearance in the CMA Code of this type of strong categorical language regarding the professional duty to care. Interestingly, the specific text cited above appears for the first time in the revision following the 1919 influenza pandemic and then, conspicuously, disappears from the next revision released in 1926.
The AMA included the very same provision in its Code of Ethics from 1846 through until the 1970s when it was likewise excised. This marked professional retrenchment from a strong obligation to provide care -as reflected in current codes of ethics -is attracting increased interest of late. A number of explanations have been proposed by academic commentators. For instance, the retrenchment has been linked to the rise of government and corporate intrusions into medical practice [23] . Others, including Clark [12] , have pointed to an increasing general belief originating circa 1950 that infectious diseases had been vanquished. It is most likely the case that both these factors played a significant role in the observed retrenchment over time.
Irrespective of the reasons underlying the current silence, there can be little doubt that infectious diseases are an increasing clinical reality in the developed world, and have long been a tremendous challenge in the developing world. For this reason alone, the continuing silence of codes of ethics is greatly problematic, both clinically and normatively.
There is no current consensus as to how explicitly and stringently the requirements for the duty to care should be stated [14] . In a 2003 survey of 1000 American physicians, respondents reported decidely mixed views on whether they would continue to care for patients in the event of an outbreak. Given that only a narrow majority of the surveyed physicians reported believing in a professional duty to treat patients in epidemics, the authors of the study concluded that there should be a reinforcement of "the [medical] profession's ethical duty to treat" in the event of a public health crisis [10] .
This call for the reinforcement of the duty to care echoes the 1922 CMA Code of Ethics that clearly stipulates that physicians have a duty to provide care, even at the jeopardy of their own lives. This statement may indeed be considered too strong and too categorical by many today. To require the provision of care even when doing so entails significant risk to the provider would appear to be demanding that all HCPs behave like "supreme Samaritans" [12] . Is this reasonable? Is this ethical? These remain open questions, but it is doubtful that all HCPs would adhere to such stringent obligations when faced with a SARS-like crisis. As Emmanuel [24] has instructively noted, the historical record of physicians is decidedly mixed in this regard; indeed, it was in response to the vocal and mounting opposition to treating seropositive patients at the height of the HIV/AIDS epidemic in the 1980s that the medical profession reconsidered the ongoing retrenchment of the duty to care. With the threat of a new epidemic, another round of full and open discourse is required as to whether the acceptable standard of professional engagement should occur at the level of "supreme", "good", or "merely decent" Samaritan [12] .
There is presently a need to address the professional duties of HCPs to their patients with the risks to the well-being of society, which may include family, friends, co-workers, and other patients, in addition to the population at large. This is a matter of balance. The content of current professional codes of ethics offers little guidance or reflection of consensus in the health care community. In the wake of SARS and with the current threat of avian influenza, it is clearly time for medicine, nursing, and other self-regulating health care professions to address the issue head on.
A number of options are open to the professions. One option, which we have already argued is unacceptable, would be to remain silent. On the other extreme, codes of ethics could be revised with a strong emphasis on the professional obligation and duty to provide care during infectious disease emergencies; that is, assume a position leaning towards supererogation, or performing acts that are 'above the call' of duty [7] . Several other options exist between these two extremes. For instance, the codes could reflect a strong but limited duty to care, with the limits clearly specified. Alternatively, there could be a weak emphasis on duty to care -an option more sympathetic to the self-regarding concerns of HCPs [8] -although this may run the risk of dissolution of the generally high regard for the health care professions that exists in society today.
We maintain that, with respect to the duty to care, it is not acceptable for codes of ethics to be vague, ambiguous, or otherwise avoid explicit statements of position. This is particularly true in light of calls for additional protections for HCPs during infectious disease outbreaks, including a position statement to that effect issued by the Ontario Medical Association [25] . Such calls for danger pay and/or enhanced disability insurance could be justified if the professions expressed a strong commitment to the ethical duty to provide care during public health emergencies. In the absence of such commitment, however, any additional measures to protect and safeguard the well-being of HCPs would appear self-serving and misplaced.
In the current context of pandemic influenza planning, as with other public health emergencies, there is an acute need for strategies to encourage greater discussion and dialogue among all interested parties and stakeholders [26] . A first step would be for the professional colleges to create a forum to engage their memberships and encourage the exchange of views on the issue. Such an exchange could then inform the development of formal position statements on the duty to care during communicable disease outbreaks, as well as the development of clear and unambiguous guidelines regarding the professional rights and responsibilities and the ethical duties and obligations of HCPs during such outbreaks. Such statements ought to be made publicly available (e.g., prominently posted on the websites of professional health care colleges and associations) in order to encourage sustained dialogue on the issues raised by professional colleges. A next step would be to foster public debate and dialogue on the positions taken by the various health care professions. To promote this public debate, a working group at the University of Toronto Joint Centre for Bioethics has produced an ethical framework to guide preparedness planning for pandemic influenza, based in part on experiences and study of the SARS crisis [27] . The framework presents a 15-point, value-based ethical guide for pandemic planning, including the value of duty to care. This report has been made publicly available via the internet and the use of webcasting and electronic town hall meetings are being planned to facilitate an open exchange. It is of utmost importance to promote a public discourse on these issues and, most importantly, to give a voice to all those who would be directly affected by a communicable disease outbreak. Together, health care professionals and the general public should participate in discussions to determine whether and when it is legitimate for HCPs to eschew the duty to care in the face of personal risk.
In light of the recent experience of Canadian physicians, nurses, and other HCWs on the frontlines of the SARS outbreak, we submit that the Canadian health care community should lead the charge to address issues of duty to care and ethical obligations in times of public health emergencies. In place of open and honest discussion, we currently have vagueness and ambiguity. In our view, health care codes of ethics should speak specifically to this issue in order to guide professional behaviour during infectious disease outbreaks. Indeed, the time to address the ethical duty to provide care is at hand -before the arrival of the next public health emergency. Epicatechins Purified from Green Tea (Camellia sinensis) Differentially Suppress Growth of Gender-Dependent Human Cancer Cell Lines The anticancer potential of catechins derived from green tea is not well understood, in part because catechin-related growth suppression and/or apoptosis appears to vary with the type and stage of malignancy as well as with the type of catechin. This in vitro study examined the biological effects of epicatechin (EC), epigallocatechin (EGC), EC 3-gallate (ECG) and EGC 3-gallate (EGCG) in cell lines from human gender-specific cancers. Cell lines developed from organ-confined (HH870) and metastatic (DU145) prostate cancer, and from moderately (HH450) and poorly differentiated (HH639) epithelial ovarian cancer were grown with or without EC, EGC, ECG or EGCG. When untreated cells reached confluency, viability and doubling time were measured for treated and untreated cells. Whereas EC treatment reduced proliferation of HH639 cells by 50%, EGCG suppressed proliferation of all cell lines by 50%. ECG was even more potent: it inhibited DU145, HH870, HH450 and HH639 cells at concentrations of 24, 27, 29 and 30 µM, whereas EGCG inhibited DU145, HH870, HH450 and HH639 cells at concentrations 89, 45, 62 and 42 µM. When compared with EGCG, ECG more effectively suppresses the growth of prostate cancer and epithelial ovarian cancer cell lines derived from tumors of patients with different stages of disease. There is accruing evidence that green tea may have anticancer activity (1) , but the mechanisms for this action are poorly understood. Green tea is produced from the shrub Camellia sinensis (Fig. 1) ; leaves are dried but not fermented so that the green coloration attributed to polyphenols is retained. Commercially prepared green tea extracts contain $60% polyphenols (1) . These polyphenols are the source of bioflavonoids, which have strong antioxidant activity.
The major bioflavonoids in green tea are epicatechins. Like all bioflavonoids, the tea catechins have three hydrocarbon rings; hydroxyl molecules are found at the 3, 5, and 7 positions (Fig. 2) . The four major tea catechins are epicatechin (EC), EC 3-gallate (ECG), epigallocatechin (EGC) and EGC 3-gallate (EGCG). The relative proportions of EC, ECG, EGC and EGCG in non-decaffeinated green tea are 792 ± 3, 1702 ± 16, 1695 ± 1 and 8295 ± 92 mg 100 g À1 dry wt, respectively; corresponding proportions in non-decaffeinated black tea are 240 ± 1, 761 ± 4, 1116 ± 24 and 1199 ± 0.12 mg 100 g À1 dry wt (1) .
Epicatechins have apparent activity against human cancer: they reportedly may promote apoptosis (2) (3) (4) (5) (6) , arrest metastasis by inhibiting metalloproteinases (7, 8) , impair angiogenesis (9, 10) and reverse multidrug resistance (11, 12) . Although all epicatechins except EC can potentially suppress cell proliferation (13) (14) (15) (16) (17) (18) , EGCG appears the most promising and is therefore under clinical investigation in chemoprevention trials (19) . However, given the wide range in physiologic potency of the different catechins, an exclusive focus on EGCG is probably short-sighted. EGCG is reportedly more effective than EGC in decreasing the intestinal absorption of cholesterol (20) and it is the most potent catechin inhibitor of HIV-1 reverse transcriptase (21) , but ECG has the strongest collagenase inhibitory effect (22) and the highest antioxidant potential (23) . By contrast, only EGC is a potent mediator of oxidative modification and an inhibitor of xanthine oxidase during hepatic catabolism of purines (24) .
We hypothesized that the in vitro anticancer action of the various catechins varies with the type and stage of malignancy. We tested this hypothesis by examining proliferation of catechin-treated cell lines derived from organ-confined or metastatic prostate cancer (CaP) and from moderately or poorly differentiated epithelial ovarian cancer (EOC). The goal was to obtain data that would be useful for developing chemopreventive and therapeutic clinical trials in patients with gender-specific and non-specific solid tumors.
Four gender-specific human cancer cell lines were used. The HH870 androgen-receptor-negative CaP cell line was developed at Hoag Cancer Center, Newport Beach, CA, from an organ-confined primary tumor that had been resected from a 56-year-old, previously untreated Caucasian (25) . This tumor was Gleason Grade 3/4, with no evidence of vascular or perineural invasion or extracapsular extension (stage T2b). The DU145 metastatic CaP cell line (American Type Culture Collection line HTB-81) was derived from a brain lesion of 69-year-old male Caucasian. It is androgen insensitive and does not express prostate-specific antigen. Two EOC cell lines developed at Hoag Cancer Center were also used: HH639 was from a poorly differentiated clear cell, Grade 3 carcinoma in the omentum and left ovary of a 56year-old Caucasian female; HH450 was from moderately differentiated metastatic cells recovered from the abdominal fluid of a 52-year-old Asian female.
All four cell lines were cryopreserved in liquid nitrogen freezer at À70 o C. For recovery of cryopreserved cells, the vials were transferred to a 37 o C water bath for 15-30 s, further thawed at room temperature and then transferred to a 15 ml polypropylene tube with a Pasteur pipette. An aliquot of 9 ml of RPMI-9% fetal bovine serum (FBS) was added in drops. The cells were allowed to settle for 5 min and then centrifuged at 4 o C for 10 min at 300 g. Supernatant was removed, and cells were suspended in fresh RPMI, gently tapped and vortexed. Cell viability was monitored by 0.2% trypan blue dye exclusion, and cell count was determined using a hemocytometer. Cells recovered from cryovials were grown in RPMI-1640 with glutamine (Invitrogen, Carlsbad, CA) supplemented with 9% FBS, HEPES buffer, gentamycin (5 mg%) and fungizone (0.5 mg%), at 37 C in a humidified atmosphere of 5% CO 2 . Upon confluency, cells were detached with sterile EDTA-dextrose (137 mM sodium chloride, 5.4 mM potassium chloride, 5.6 mM dextrose, 0.54 mM ethylene diamine tetra acetate (EDTA), 7.1 mM sodium bicarbonate) at 37 C for 5-15 min (or $45 min for HH639), recovered with cold RPMI-1640-9% FBS and resuspended in the same medium. Use of trypsin was avoided for harvesting the cells. Cell viability and cell count were reassessed before cells were seeded in culture flasks.
All epicatechins used in this study (Fig. 2) with 50, 60 or 100 mM of each epicatechin or with no epicatechin (control) in RPMI-1640 with glutamine (Invitrogen), 9% FBS, 0.54% HEPES buffer, gentamycin (5 mg%) and fungizone (0.5 mg%).
All experiments used 25 ml sterile polystyrene tissue culture flasks with a vented blue plug seal cap (Beckton Dickinson, Franklin Lakes, NJ, Cat. No. 353107). Each flask contained stock solution with or without epicatechin in concentrations of 50 mM (five flasks for each epicatechin and five flasks for control) and 25, 75 and 100 mM (three flasks for each epicatechin and three flasks for control). Cells (0.25 · 10 6 ) suspended in 10 ml of the RPMI-1640-FBS solution described above were transferred to each flask and allowed to grow until control cells reached confluency. The cells were detached with sterile EDTA-dextrose at 37 C for 5 min, recovered with cold RPMI-1640-FBS medium and resuspended in the same medium.
Cells were counted using a hemocytometer; trypan blue dye exclusion was used to determine the number of viable versus dead cells. The interval between seeding and confluent growth of control cells was used to calculate the doubling time and the number of cell cycles. The 50% inhibitory concentration (IC50) of each catechin in each cell line was calculated using a software program (Microcal Origin Corp, OriginLab Corporation, Northampton, MA). The cells were photographed directly from the flask using light microscopy (Olympus IX-70, Japan).
Analyses of variance and Fisher's least significant difference (LSD) method were used for pairwise comparisons of values significant at the 0.05 level. 
Organ-confined prostate cancer cell line HH870 and primary and metastatic epithelial ovarian cancer cell lines (HH450 and HH639) seeded (2.5 · 10 5 cells) in flasks with or without various concentrations (25, 50, 75 or 100 mM) of ECG or EGCG were photographed under a light microscope after the untreated control cells reached confluency (Fig. 3 ). Both ECG and EGCG significantly affected the density of each cell line at or above 75 mM. The decrease in cell density at higher concentrations is much pronounced for ECG than for EGCG, a finding significant considering recommendations of clinical trials with EGCG (19) .
The mean density or viable cell number (in millions) (n ¼5 per treatment) of different cell lines was examined with or without catechins (50 mM) (Fig. 4) . The cell density was measured when growth of untreated cells reached confluency. Statistical analysis by ANOVA as well as by pairwise comparison showed that both ECG and EGCG significantly affected the cell density. ECG decreased the cell density of prostate cancer cells DU145, HH870 and ovarian cancer cell line HH639 more potently than EGCG. But EGCG inhibited the growth of ovarian cancer cell line HH450 better than ECG, suggesting the need to determine relative efficacy of ECG and EGCG in clinical trials for different cancers.
Tumor Cell Doubling Time: ECG versus EGCG Figure 5 shows the influence of the four epicatechins on cell doubling time. ECG and/or EGCG prolonged the doubling (14) EGC and ECG inhibited the growth of a human lung cancer cell line, PC-9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell-cycle regulation. EGCG (50 and 100 mM) increased the percentages of cells in the G 
The relative cytotoxicity (CTX) of ECG to carcinoma HSC-2 cells and normal HGF-2 fibroblasts cells from the human oral cavity, as compared with other polyphenols in tea, was evaluated. For the HSC-2 carcinoma cells, ECG, CG and EGCG grouped as highly toxic, EGC as moderately toxic, and C and EC as least toxic. For the HGF-2 fibroblasts, ECG and CG grouped as highly toxic, EGCG as moderately toxic, and EGC, C and EC as least toxic. The CTX effects of the polyphenols were more pronounced to the carcinoma, than to the normal, cells. Table 1 summarizes the effects of EC, ECG, EGC and EGCG on viability, doubling time and cycling of the four cell lines. Untreated cells from each line reached confluency in about 2.5 cell cycles. EC did not affect the proliferation of DU145, HH870 or HH450 cells but it reduced the proliferation of HH639 cells by half (P < 0.05) and prevented their confluent growth (Table 1) . EGC did not affect the proliferation of any cell line (Table 1) , whereas EGCG arrested proliferation of all four lines. ECG, followed by EGCG, was the most potent inhibitor of cell growth and cycling.
Proliferation of each cell line (n ¼ 3 per treatment) was monitored with or without ECG or EGCG at concentrations of 0, 25, 50, 75 and 100 mM. The dosimetric results plotted in Fig. 6 shows concentration-dependent suppression of cell growth by ECG and EGCG. The suppressive effect on cell density was striking at higher concentrations of ECG and EGCG. ECG was a more potent inhibitor of cell growth than EGCG. At 25 mM of EGCG, cell numbers for HH870 and DU145 were significantly higher than control values. Based on the results plotted in Fig. 6 , IC50 values were calculated. The IC50 values are 24-30 mM for ECG, versus 42-89 mM for EGCG (Table 2 ). ECG suppressed growth at all higher concentrations tested (Fig. 6) , whereas EGCG significantly (P < 0.05) enhanced proliferation of CaP cells at 25 mM, a finding relevant to chemoprevention trials with EGCG only.
Green tea is widely consumed in Japan and China and its polyphenolic components have a chemopreventive effect against cancer in vitro and in vivo (39) . A cup of green tea contains 100-150 mg catechins, of which 8% are EC, 15% are EGC, 15% are ECG and 50% are EGCG (40) . Although numerous investigations have shown the role of EGCG in cancer chemoprevention, only a few studies have attempted to compare the relative antitumor efficacy of all four catechins (Table 3) . When we used a systematic approach to assess the effect of various catechins on cell lines derived from gender-based cancers, we found that each catechin's antitumor activity depended on the type of tumor. EGCG was not always the most potent chemopreventive agent.
Most of the earlier literature (Table 3) indicates that EGCG is the most potent growth inhibitor of cell lines from glioblastoma, melanoma and cancers of the breast, colon, lung, prostate (androgen-receptor-positive), pancreas, liver and mouth. EGCG prevents proliferation of DU145 cells by arresting the cell cycle at G 0 /G 1 -phase (19) . Gupta and others (26) have documented that G 0 /G 1 -phase arrest is independent of p53 mutation, and EGCG treatment of DU145 induces the cyclin kinase inhibitor WAF1/p21. These observations suggest that EGCG imposes a cell-cycle checkpoint (19) . However, our results showed that ECG may be more potent than EGCG for inhibition of primary and metastatic CaP and EOC cells (Fig. 4, Tables 1 and 2) . ECG significantly reduced cell proliferation (Table 1, Figs 2 and 3) and increased mean doubling time (Table 1, Fig. 4) .
The in vitro effect of chemopreventive agents can be studied when tumor cells are in a matrix (1, 4, 27) or in a suspension (28, 29) . We used the suspension method because it exposes the entire cell surface to the chemotherapeutic agent. Our findings confirm an earlier report that used the matrix method to show that ECG is more potent than EGCG in suppressing the proliferation of DU145 CaP cells (4) . Thus reported differences in the relative efficacy of different catechins may not be due to differences in methodology.
Not all tumor cells are killed by catechins. In our study, ECG (50 mM) induced death of most but not all HH639 cells. Doubling ECG's IC50 concentration might increase the tumor kill rate if ECG does not epimerize to CG. Our in vitro dose of 100 mM is equivalent to 29 mg (EC/EGC) to 45 mg (EGCG/ ECG), far less than the 100-150 mg (50% of which is EGCG) in one cup of green tea. However, Lee et al. (41) reported that plasma levels of EGCG and EGC in healthy volunteers increased to 78 and 223 ng ml À1 , respectively, 20 min after drinking brewed green tea (1.2 g of tea solids in 200 ml hot water). This suggests that drinking more than 10 cups of green tea may be necessary to maintain a plasma concentration of EGCG equivalent to that used in vitro by a dose of 50 mM or 22.5 mg. Kaegi (42) suggested a daily intake of 13 cups of green tea as a chemopreventive measure. Because this level of tea consumption is impractically high, chemoprevention of cancer with catechins may require administration of the appropriate catechin in a purified form.
In conclusion it may be stated that both green and black tea polyphenols are important components of antitumor aspect of complementary and alternative medicine (CAM), which play a significant role in the American health care system and in patients who suffer from chronic problems (43) . While green tea catechin gallates such as EGCG and ECG possess potent antitumor activities, their epimers, commonly found in black tea, act as potent inhibitor of proteases involved in replication of viruses, including coronoviruses (44) . There is a need to understand preventive and therapeutic potential of catechin gallates from both green and black teas. We are currently designing a phase I chemopreventive study to examine the effects of purified EGCG and ECG in patients who have been chosen observational management of organ-confined prostate cancer. Markers of exacerbation severity in chronic obstructive pulmonary disease BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) can experience 'exacerbations' of their conditions. An exacerbation is an event defined in terms of subjective descriptors or symptoms, namely dyspnoea, cough and sputum that worsen sufficiently to warrant a change in medical management. There is a need for reliable markers that reflect the pathological mechanisms that underlie exacerbation severity and that can be used as a surrogate to assess treatment effects in clinical studies. Little is known as to how existing study variables and suggested markers change in both the stable and exacerbation phases of COPD. In an attempt to find the best surrogates for exacerbations, we have reviewed the literature to identify which of these markers change in a consistent manner with the severity of the exacerbation event. METHODS: We have searched standard databases between 1966 to July 2004 using major keywords and terms. Studies that provided demographics, spirometry, potential markers, and clear eligibility criteria were included in this study. Central tendencies and dispersions for all the variables and markers reported and collected by us were first tabulated according to sample size and ATS/ERS 2004 Exacerbation Severity Levels I to III criteria. Due to the possible similarity of patients in Levels II and III, the data was also redefined into categories of exacerbations, namely out-patient (Level I) and in-patient (Levels II & III combined). For both approaches, we performed a fixed effect meta-analysis on each of the reported variables. RESULTS: We included a total of 268 studies reported between 1979 to July 2004. These studies investigated 142,407 patients with COPD. Arterial carbon dioxide tension and breathing rate were statistically different between all levels of exacerbation severity and between in out- and in-patient settings. Most other measures showed weak relationships with either level or setting, or they had insufficient data to permit meta-analysis. CONCLUSION: Arterial carbon dioxide and breathing rate varied in a consistent manner with exacerbation severity and patient setting. Many other measures showed weak correlations that should be further explored in future longitudinal studies or assessed using suggested mathematical modelling techniques. Chronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by an airflow limitation and inflammation of the lower airways [1] . As the disease worsens, some patients experience 'exacerbations' of their principal symptoms of dyspnoea, cough and sputum. These exacerbations frequently result in a visit to a general practitioner's office or to a local hospital for treatment. Exacerbations occur in COPD patients at a median of three times a year with half of them being unreported [2] [3] [4] . The heterogeneity of COPD exacerbations make them difficult to define, classify and manage due to their range of symptoms, varied treatment requirements, seasonal occurrence, and ambiguous aetiology [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] .
To address this problem, attempts have been made to develop a consensus definition for COPD exacerbations [15] . Recently, the American Thoracic Society (ATS) and European Respiratory Society (ERS) adopted the following definition: 'an event in the natural course of the disease that is characterised by a change in the patient's baseline dyspnea, cough and sputum beyond day-to-day variability sufficient to warrant a change in management' [1] .
The severity of an exacerbation has been also difficult to classify despite the various schemes that have been proposed to deal with this issue [4, [15] [16] [17] . The ATS and ERS have also jointly suggested a classification based upon severity and the type of medical management used, i.e., Exacerbation Level I is home treatment, Level II is hospitalization, and Level III is specialised care [1] . The aim of this scheme is to improve the existing management of exacerbations and to serve as an aid in the assessment of treatment efficacy. Different operational definitions for COPD exacerbations have been proposed in the past and these have helped determine their relative importance, in particular their relationship to COPD progression [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . However, these definitions have relied primarily on symptoms, and this along with the absence of a standard classification for the degree of symptom severity, has delayed the development of new therapies for this condition. The current therapies for exacerbations have been evaluated based on their ability to reduce symptoms, and to improve a patient's forced expiratory volume in one second (FEV 1 ) since the latter is strongly correlated with COPD mortality. However, FEV 1 does not discriminate well between the stable and exacerbative states of COPD, particularly during the later stages of this disease. Hence, the development of biological markers, or biomarkers that are more sensitive and specific to the severity of COPD exacerbations would provide investigators with new insights and directions for further research.
At this time, only a few clinical variables or inflammatory mediators have been shown to be associated with COPD exacerbations and their related morbidity and mortality. Some of those include: age [18] [19] [20] ; FEV 1 , forced vital capacity and peak expired flow [19, 21, 22] ; body mass index [20] ; albumin [20, 22, 23] ; sodium [23] ; pH [24, 25] ; eosinophils [26] [27] [28] [29] ; interleukins 6 and 8 [29] [30] [31] [32] ; fibrinogen [31] ; and C-reactive protein [33] . Significant clinical events such as the number of exacerbations per year, the number of hospital admissions per year, time to relapses, and days in hospital have been regarded as useful measures in clinical studies designed to assess drug efficacy and cost-effectiveness as well as to standardize existing hospital support programs for COPD [34] [35] [36] [37] [38] [39] . However, it is not known how these measures change with increasing severity of COPD exacerbations. Therefore, we have surveyed the medical literature to identify which of the commonly accepted variables and suggested markers for COPD exacerbations change according to the ATS/ERS' levels of exacerbation severity. The long-term aim of our work is to assess the sensitivity and specificity of potential markers for use in future COPD studies as well as to determine how such markers can be further studied and fully integrated into the development of new drugs for COPD.
We searched standard databases since 1966 using medical search headings and related terms as obtained from major consensus documents related to COPD exacerbations. The major keywords were 'exacerbation', 'unstable', 'acute', 'bronchitis', and variants of the term 'COPD'. This phase of our search retrieved a total of 843 citations. For these citations, we read the title and abstract of each citation so as to exclude citations that concerned exacerbations of coronary artery disease, myocardial infarction, cystic fibrosis, asthma, pulmonary emboli, and community pneumonia. Citations for case studies, letters, reviews, meta-analyses, and animal studies were also excluded.
After this initial screening, we identified 387 citations to papers that were of possible interest. We retrieved the original articles in electronic and hard copy forms, and then critically read each article. As a result of this step, we arrived at a total of 268 studies in our final review and analyses. We selected these studies based on the availability of demographics, spirometry, clear study eligibility criteria, and the potential markers being used to assess exacerbations.
The objectives of this literature review and data analyses were to determine which of the baseline measures com-monly used in COPD exacerbation studies change with the extent of the exacerbation and disease severity, and to determine whether COPD exacerbations can be modelled as 'events' or 'time-to-event' in future investigations.
Initially, we considered various exacerbation definitions and classification schemes, in particular, those suggested by Rodriguez-Roisin [15] as well as those described by Pauwels and colleagues [17] . However, we determined that the ATS/ERS' operational classification of exacerbation severity [1] was the most sensible and feasible system for systematically assessing the patient baseline characteristics and biomarker information from the majority of published studies. We therefore used this classification scheme and the related clinical history, physical findings and diagnostic procedures for managing exacerbations to perform our data abstraction. From each study, we retrieved the reported demographics, spirometry, smoking status, clinical, cytological and biochemical variables as well as suggested markers of the severity of the exacerbation at baseline conditions, i.e., immediately prior to, or during the exacerbation event but before the time in which the intervention of interest was investigated (Table  1 ). Whenever such variables were measured in stable conditions, we also abstracted this information. For each study, we noted the type of definition used to define an exacerbation such as symptom-or event-based as well as the research question asked, the experimental design used, any sponsorship, and the presence or absence of data from individual study patients. Data was then further organized according to sample size and smoking status when available. Cytological and biochemical data were also classified according to their collection methods. These included sputum induction, bronchial biopsy, bronchoalveolar lavage (BAL), exhaled breath sampling, and blood sampling.
We were also aware of the possibility that for some study groups in severity Levels II and III (as per the ATS/ERS criteria) included in this review may have experienced a similar quality of care or medical management that was not reported adequately in the original publication. In attempt to correct for this problem, we combined the exacerbation data from Levels II and III into an 'in-patient' category and then compared it to Level I that we regarded as the 'out-patient' category.
We collected and calculated study means, medians, standard errors, standard deviations, 95% confidence interval, and inter-quartile ranges using the statistical algorithms in Microsoft Excel 2002. We then conducted fixed effect meta-analyses to obtain mean point estimates, 95% confidence intervals, and two standard deviations for each exacerbation level [40] . Exacerbation Severity Levels I and II, II and III, and I and III were each compared using a twotailed Z-test. The alpha level of p < 0.05 was adjusted for multiple testing according to the Bonferroni correction procedure [41] . In the event that a specific exacerbation severity level had a large number of studies in which only median data were available, the data were considered to be normally distributed and medians were treated as means. Since many studies did not publish data for individual patients, we were limited in addressing non-normality in the data by using a log 10 -transformation.
We again performed a fixed effect meta-analysis to obtain mean point estimates, 95% confidence intervals, and two standard deviations for in-patient and out-patient categories of each measure. We then compared each category using a two-tailed Z-test and a p-value of 0.05.
Our search strategy yielded 268 suitable studies that met our selection criteria. These studies were published between 1979 and July 2004 -Week 2. (The references for these studies can be found at the LACDR Division of Pharmacology website [42] ). The total number of study subjects included in this review was 142,407. Of this group, 18% fell in Exacerbation Severity Level I, 78% in Level II, and 4% in Level III. When we re-analysed the data according to out-or in-patient settings, 18% were out-patients and 82% in-patients.
Meta-analyses of typical study demographics showed that there was significant overlap in 95% confidence intervals and study data distributions for the three exacerbation severity levels except for age where study patients in Level II had a mean age of 64.2 years (95% confidence interval (CI): 62.9 to 65.5 years) compared to 68.0 years (95% CI: 65.9 to 70.1 years) for patients in Level III (p = 0.002) ( Table 2 ). When the demographics were re-analyzed according to patient settings, we determined that only body mass index was statistically different between the out-patient setting (mean point estimate: 26.2 kg/m 2 ; 95% CI: 23.8 to 28.7 kg/m 2 ) and the in-patient setting (mean point estimate: 23.4 kg/m 2 ; 95% CI: 22.5 to 24.3 kg/m 2 ) (p = 0.038) ( Table 3) .
The spirometry measures Forced Expired Volume in 1 Second (FEV 1 ) and Forced Vital Capacity (FVC), both in percent predicted, decreased from Exacerbation Levels I to II (p < 0.017) but remained unchanged from Levels II to III ( Figure 1A and 1C, respectively). However, when Levels II and III were combined to create an 'in-patient' category for each of these variables, there was a statistically signifi-Respiratory Research 2006, 7:74 http://respiratory-research.com/content/7/1/74 cant decrease for the in-patients versus the out-patients (p < 0.05) ( Figure 1B and 1D, respectively). We also observed the same trend for FEV 1 /FVC (Figure 2A and 2B ). For all other spirometry measures, there were too few studies available in Level III for meta-analysis.
We found for smoking that pack years increased with exacerbation severity, but only Levels I and II were statistically different (p = 0.015) ( Figure 2C ). When we compared pack years between patient settings, it was statistically higher for the in-patients than the out-patients (p = 0.010) ( Figure 2D ).
In terms of the hemodynamic measures, only heart rate showed a statistically significant difference being higher in Level II than Level I (p = 0.014) with no difference Many study variables were measured at or around the time of the exacerbation. If these variables were measured in the stable condition of these COPD patients, i.e., measurements were taken weeks or months prior to the exacerbation, then these were also obtained.
between Levels II and III ( Figure 3A ). Heart rates were also higher for in-patients than out-patients (p = 0.011) (Figure 3B ).
The clinical measures of dyspnoea, i.e., the breathing rate ( Figure 3C ) and Borg dyspnoea score, tended to increase from Levels I to II and then decrease from Levels II to III. However, only breathing rate demonstrated clear statistical differences between the three levels (p < 0.017). Only Levels II and III of the Borg Dyspnoea Score were statistically different (p < 0.001); a statistical comparison of these levels with Level I was not possible due to lack of data. When patient settings were compared, only breathing rate showed a clear statistical difference being statistically lower for in-patients than out-patients (p = 0.003) ( Figure 3D ).
Exacerbation Levels II and III were statistically different with respect to pH (p = 0.003) and bicarbonate (p = 0.002) in that pH decreased from Level II to III whereas bicarbonate increased. However, there was insufficient Level I data for each variable to allow for statistical comparisons with the other Levels. There was also insufficient data available to compare out-patients with in-patients.
In terms of blood gas measures studied, only arterial carbon dioxide tension (PaCO 2 ) showed a statistically significant increase with increasing exacerbation severity (p < 0.017) ( Figure 4A ) as well as out-versus in-patients (p < 0.05) ( Figure 4B ). In the case of oxygen saturation, it gradually decreased with increasing exacerbation severity with statistically significant differences between Levels I and II (p < 0.001) as well as Levels I and III (p = 0.011) ( Figure  4C ). It also decreased going from an out-patient to an inpatient setting (P < 0.001) ( Figure 4D ).
The six minute walking distance challenge test seemed to show a decreasing trend with increasing exacerbation severity but such changes did not reach statistical significance. This was also the case when the out-and in-patients were compared. Many other variables related to spirometry, respiratory status, exacerbation and hospital event categories also did not change significantly with exacerbation severity or out-and in-patients (See additional file 1). There was not enough data in the bacteriology and virology categories to permit any meta-analyses. Of the 268 studies sampled, only half contained data about the biochemical variables.
We conducted this review of the COPD exacerbation literature to determine which commonly-accepted baseline variables and suggested markers changed in a consistent manner with the severity of COPD exacerbations. As our index of COPD severity, we used the recently published ATS/ERS operational classification of exacerbation severity for medical management. This is because most of the published literature rarely provides sufficient details to characterise the severity of a patient's exacerbation. In addition, we also analyzed the same data according to out-and in-patient settings so as to account for possible overlaps in medical management between Levels II and III but were not reported in the original publication.
The long-term aim of our work is to improve the quality and applicability of exacerbation management through the identification of sensitive and specific markers that can be used for the assessment of treatment effects. This review identified a few potential markers of exacerbation severity.
When we assessed the spirometry measures FEV 1 and FVC in % predicted, as well as FEV 1 /FVC, we observed statistically significant differences with exacerbation severity, and between out-and in-patients ( Figures 1A-D and 2A -B). One draw-back was the paucity of such information in Level III studies. This confirms the clinical situation that as exacerbations worsen and more specialised care is required, spirometry measurements are less likely under baseline conditions or during an exacerbation [14] . Thus, such data is rare in many published studies.
The number of smoking-related pack years increased with exacerbation severity and showed a clear difference between out-and in-patient settings ( Figures 2C and 2D) , a finding that is consistent with the idea that the more a COPD patient smokes, and for longer, the higher the likelihood that COPD exacerbations will be more severe.
According to the mean point estimates obtained in this In-patient (N=81;n=5768) PaCO2 (mmHg) * B study, COPD patients with 40 to 60 pack-years of smoking will experience an increase in the severity of COPD exacerbations. However, our conclusion regarding this finding is limited by there being data from only two studies at Level III.
Although heart rate varied little between Exacerbation Levels II to III, it is important to note that it was substantially elevated in patients ( Figure 3A ) with the clearest difference being between in-and out-patients. This is possibly associated with the anxiety and dyspnea that experienced when an exacerbation occurs. The increase in heart rate of course increases the oxygen requirements of the heart. The increased heart rate may also be the result of underlying cardiovascular disease that is more prominent in severe COPD patients [43] .
The relationship of pH and bicarbonate to exacerbation severity are consistent with the signs of respiratory acidosis evident in COPD patients with exacerbations [1, 24, 25] . However, due to the shortage of data in Level I, proper statistical conclusions about each of these variables are difficult to make. In relation to this, breathing rate significantly increased from Levels I to II and then decreased from Levels II to III ( Figure 3C ). The first observation may reflect components of the exacerbation episode (i.e., anxiety and dyspnea) as well as the physiological need to breathe more to maintain adequate blood gas levels. The reduction at Level III possibly reflects the results of the specialized care where patients are given ventilatory support so as to return the breathing rate to normal. The Borg Dyspnea Score showed the same trend as breathing rate, although insufficient data in Level I did not allow for further comparisons. When out-and inpatient data were compared for each of these variables, only breathing rate demonstrated a clear statistical difference ( Figure 3D ). The Borg Dyspnoea Score on the other hand did not have enough studies in the out-patient category to perform any statistical test. Overall, the observed trends were consistent with the fact that management of dyspnoea is one of the main factors generating the high hospital costs associated with COPD exacerbations [44] . In keeping with the direct measures of dyspnoea, arterial carbon dioxide tension showed a clear relationship with exacerbation severity and patient management settings ( Figures 4A and 4B ) that is consistent with the conclusions reported in the medical literature [20, [45] [46] [47] . Arterial oxygen tension in contrast did not change with exacerbation severity or patient setting. Possibly this lack of correlation reflects the immediate administration of supplemental oxygen given to hypoxaemic patients in a hospital setting. There was however a decreasing trend in oxygen saturation with increasing exacerbation severity and clear differences between out-and in-patient settings ( Figures 4C and 4D ) that are consistent with the present thinking on blood gas changes.
Most of the other commonly accepted measures and suggested biomarkers poorly reflected exacerbation severity, or the fact that there was not sufficient data to undertake a meta-analysis (See additional file 1). This finding recalls a 2001 US Department of Health and Human Services report on exacerbation treatment outcomes from over 200 randomised controlled trials [14] . The aim of that study was to create new guidelines to improve the management of COPD exacerbations. That study also concluded that the current literature was limited in terms of the number of studies and the amount of detail available as well as the reliability and accuracy of the clinical assessments used to discriminate between COPD exacerbations and other causes of worsening respiratory status. Thus, our observations agree with previous observations regarding the assessment of the unstable COPD literature.
As previously discussed, most of the studies used for this review were predominately with hospitalized patients (Level II). However, most COPD occurs in an out-patient setting (Level I) [48] [49] [50] [51] [52] [53] . This has implications for our study since the latter population was poorly represented.
Our basic categorisation was according to the ATS/ERS' operational scheme for classifying the severity of COPD exacerbations as well as to out-and in-patient categories. To our knowledge, we are the first to undertake this type of literature review and thus we were faced with a lack of consistency in the definition of exacerbations as used in the various studies. We tried to overcome this difficulty by selecting and ranking clinical studies so as to improve the comparability of subjects between studies.
We were also aware that the clinical studies we analysed differed with respect to which comorbidities or identifiable causes for exacerbations were reported. Most patients were elderly and therefore were more likely to be suffering from one or more co-existing diseases such as asthma or cardiovascular disease. Such co-morbidity makes interpretation of our findings more difficult with respect to the true causes of exacerbations. If their aetiology could be determined, then susceptible patients such as those in Level I could be identified and new treatments developed to help prevent their onset and related hospital costs.
Finally, the compatibility between the studies of COPD exacerbation that we analysed may have been limited by substantial variations in the time and location of studies. Exacerbations are more likely in summer [5] but many studies failed to report the time of year or the time period for study implementation. Thus, seasonal effects, combined with the low incidence of exacerbations per patient, could represent an inherent bias. In addition, different institutions probably had different standards with respect to diagnosis and management of COPD exacerbations when these studies were performed. Such variations may also explain any observed inconsistencies in our findings. However, we attempted to overcome this possible bias in Exacerbation Levels II and III by the subsequent re-analysis of this data on the basis of out-patient and in-patient settings.
As observed in The additional online file, there was a scarcity of information particularly for biomarkers at different exacerbation levels. It is also unclear to us whether any of the variables that changed with exacerbation severity are causally-related. Hence, longitudinal studies and/or less restrictive eligibility criteria would be needed to address all these questions. One difficulty in tackling such problems is the enormous amount of time and expense involved in implementing such studies. In addition, the current methods for data analysis in clinical studies have limitations imposed by the assessment of the reduction in frequency or total suppression of exacerbation episodes (i.e. rare event or "non-event").
To overcome these drawbacks and obtain more accurate evaluation of treatment effect on COPD exacerbations, alternative analytical methods based, for example, on predictive mathematical models such as hidden Markov chains or Bayesian forecasting should be tried. Such models can characterise and predict rare events without undertaking a full-scale, long-term longitudinal study. This approach to predicting rare events has been used previously in studies of migraine, epilepsy and various cardiovascular diseases where the size of treatment effect is measured in terms of a reduction in the frequency of the repetition of an event within a given probability or within a given time period [54, 55] . One example of a mathematical model development includes the use of a Markov model to predict COPD exacerbation rates in a clinical trial of the inhaled anticholinergic bronchodilator tiotropium [56] . In this example, the model was developed on the basis of prior knowledge of the exacerbation rate as estimated from meta-analyses of randomised controlled trial data. This gave the probabilities for COPD exacerbations for different stages of COPD. In another study, a proportional hazards model was used to identify risk factors for COPD patients hospitalised due to an exacerbation [44] . The current ATS/ERS guidelines for exacerbations do not consider the implications of using probabilistic models as a means of assessing the severity of COPD exacerbations or the effect of treatment [1] . A modelling approach may offer new insights into which variables related to COPD exacerbations should be investigated.
From a research planning perspective, our study findings have generated some hypotheses and related considerations that could be evaluated in future clinical trials. One hypothesis is that the combination of variables that we observed to change in our study (i.e., FEV 1 , FVC, FEV 1 / FVC, arterial carbon dioxide, breathing rate, heart rate, pack years, and oxygen saturation) could represent a new definition for a 'severe' exacerbation event. Most definitions in the literature, including the recent ATS/ERS definition, do not indicate any assessment of (patho)physiological variables as signs of an exacerbation. They simply regard the exacerbation as a worsening of the normal day-to-day symptoms and/or an adjustment in medical management [17] . A definition that encompasses a clear set of objective measures would be useful to medical practitioners who predominantly rely on clinical judgement or past experiences for diagnosing an exacerbation and its severity as well as for assessing treatment effect.
Another important consideration for future clinical trials is the assessment of treatment effect based on predictions of exacerbation frequency and intensity. In other words, the collection of data such as the rate of onset and resolution of an exacerbation from longitudinal studies could be used to determine probabilities of second, third, fourth, etc., exacerbation events in individual patients [54] . The alteration of such probabilities with an experimental treatment could be a more sensitive and reliable approach for assessing treatment effect in clinical trials than recording daily changes in symptoms or medical management.
Lastly, our findings were obtained from COPD patients that had experienced at least one exacerbation during the study assessment period. In the same studies, there were also patients who did not experience an exacerbation. This indicates that a fraction of COPD patients may be regarded as being susceptible to an exacerbation whereas another fraction is 'exacerbation-free'. It would be interesting to determine how the variables we identified in our study change in the latter patient group according to FEV 1 . Some published studies have stratified COPD patients on the basis of exacerbation frequency; this is generally done by categorising patients as having either 'infrequent' or 'frequent' exacerbations if they had less than or greater than a mean of three exacerbations per year, respectively [57] . In our study, we were unable to make this distinction between COPD patients since many of the published studies did not provide individual patient data on exacerbation frequency. We are currently investigating a commercial database of clinical trials that will enable us to look at patients with 'infrequent' or 'frequent' exacerbations. The results of this work could help us better select patients as well as identify potential markers for future longitudinal studies.
The current management and treatment of COPD exacerbations is primarily dependent on the evaluation of the symptoms rather than the signs related to the exacerbation event. We found that arterial carbon dioxide tension and breathing rate consistently varied with the severity of COPD exacerbations and with in-versus out-patients. Other commonly-accepted measures and suggested biomarkers for exacerbations failed to show consistent trends or lacked sufficient data to permit any meta-analysis. We recommend the design of longitudinal studies looking at the frequency of exacerbations as well as the use of more advanced modelling techniques to improve the selection of potential markers for the categorization of the severity of COPD exacerbations and the assessment of treatment effect in future studies.
COPD -Chronic Obstructive Pulmonary Disease  Scientific Abstracts   Society of General Internal Medicine: 28th Annual Meeting New Orleans, LA May 11–14, 2005: Out of Chaos: The Critical Role of Generalists, ABSTRACTS OF SUBMISSIONS ACCEPTED FOR PRESENTATIOIN   CLINICAL VIGNETTES   CLINICAL VIGNETTES  LEARNING OBJECTIVES: Recognize that the presenting features of amyloidosis are protean and depend on the organ systems involved. Recognize that cardiac involvement is common in primary amyloidosis and that symptomatic heart involvement carries a poor prognosis. CASE INFORMATION: 66 year old man without PMH presented 8 months of progressive exertional dyspnea and fatigue. Evaluation revealed volume overload with anasarca and signs of biventricular heart failure. Laboratory studies revealed renal insufficiency with a creatinine of 1.8 and 2+ proteinuria. serum protein electrophoresis with immunofixation showed a monoclonal IgA kappa paraprotein; urine protein electrophoresis was unremarkable. Echocardiogram revealed an ejection fraction of 20%. Right ventricular function was moderately reduced and right and left ventricular hypertrophy was noted. ECG showed atrial fibrillation with a ventricular response of 105 BPM and low voltage. Pt. underwent right heart catheterization with endomyocardial biopsy. Tissue stained positive with crystal violet and Congo red with characteristic green birefringence under polarization microscopy. This result together with the paraprotein established the diagnosis of AL amyloidosis. IMPLICATIONS/DISCUSSION: Amyloidosis is a disease characterized by extracellular accumulation of insoluble protein fibrils in various organs and tissues. Classification of amyloidosis is based on the nature of the precursor proteins that form the fibril deposits. AL amyloidosis (primary) is a plasma cell dyscrasia in which clonal plasma cells in the bone marrow produce monoclonal immunoglobulin light chains. Clinical manifestations reflect the organ systems involved with kidney and heart involvement being most common. Clinically evident cardiac involvement occurs in up to 50% of patients with AL amyloid and often leads to significant cardiac dysfunction. Diastolic dysfunction is caused by diffuse infiltration of the left ventricular myocardium with resultant stiffening and impaired relaxation and usually precedes systolic dysfunction. Systolic dysfunction results from replacement of functional myocardial tissue with amyloid protein. The conduction system can be affected by amyloid infiltration as well, leading to conduction disturbances, arrhythmias or sudden death. ECG findings include low voltage, conduction abnormalities such as AV block, atrial fibrillation and pseudoinfarct patterns. This case demonstrates a classic presentation of AL amyloidosis with significant cardiac involvement. AL amyloidosis has a median survival of one to two years but patients with symptomatic heart involvement have a median survival of only six months. LEARNING OBJECTIVES: Recognize the importance of counseling patients who have received previous valve replacements that continued intravenous drug use may disqualify them from lifesaving interventions. CASE INFORMATION: A 44 year-old presented with progressive dyspnea, fevers and substernal tightness. He had a endocarditis requiring porcein valve replacement in 1993. He started using intravenous cocaine and heroin weekly one month prior. He had a fever of 40 C and a holosystolic murmur. Blood cultures grew steptococcus viridans. Transesophageal echocardiography showed aortic valve vegetations on all leaflets and a ring abscess. Toxicology screen was positive for cocaine. Ampicillin and gentamicin were initiated. After six weeks of therapy, the aortic vegetations and blood cultures persisted. Cardiothoracic surgery was consulted, but declined intervention, citing the likely recidivistic drug use and futility of care. IMPLICATIONS/DISCUSSION: Surgical intervention in active intravenous drugs users is an issue of ethical debate. Ninety percent of heroin users will reuse even after successful rehabilitation. Continued intravenous use represents a hundred-fold increased risk of recurrent endocarditis. The apparent futility of surgical intervention in this setting has prompted surgical services to adopt a``one strike and you are out policy.'' This is almost a certain death sentence for patients meeting surgical criteria. The ethics committee was involved but could not establish ethical grounds for challenging surgery's decision. The precedent of withholding organ transplantation from continuing drug users was applied. Physicians should counsel patients who have received previous valve replacements that continued intravenous drug use may disqualify them from lifesaving interventions. Added emphasis on rehabilitation following valve replacement may be the only sure method to avoid this dilemma. 
A 53 year-old woman presented with four months of weakness and a rash. The weakness was progressive, symmetric, and proximal. The muscles of both her upper and lower extremities were affected. The erythematous rash was located on the inner thighs and arms bilaterally. She could not raise her arms and had difficulty holding her head erect; her leg weakness prohibited standing independently. Sensation and reflexes were not affected. She had normal serum electrolytes and complete blood count. Her AST was 200 U/L, ALT 78 U/ L, ALKP 52 U/L, CK 7021U/L, and ESR 29 mm/hr. Her TSH, hepatitis panel, ANA, rheumatoid factor, anti-DS DNA, c-ANCA, and p-ANCA were normal. A muscle biopsy of left thigh showed vasculitis and perifascicular atrophy and necrosis. Methylprednisolone and methotrexate were initiated with resolution of her symptoms. An age-appropriate screen for cancer was negative. IMPLICATIONS/DISCUSSION: The diagnosis of dermatomyositis/polymyositis (DM/PM) requires a high degree of clinical suspicion, as it is established based on the combination of clinical, laboratory, and muscle biopsy findings. This patient demonstrated the proximal muscle weakness characteristic of DM/PM. Other causes of proximal muscle weakness, including electrolyte abnormalities, hypothyroidism, inclusion body myositis or drug induced (HMG CoA reductase inhibitors or steroids) were excluded by history and laboratory data. Perifasicular inflammation suggests dermatomyositis; perimysial inflammation is characteristic of drug-induced myositis. Diagnostic criteria for dermatomyositis include symmetric proximal muscle weakness, elevated serum muscle enzymes, perifasicular inflammation on muscle biopsy, and rash. The classic dermatologic findings of PM/DM include mechanic's hands (synthetase AB subset), heliotrope lids, Gottron's sign and shawl sign . Physicians should be aware that most cases of DM/PM present without the classic findings of DM/PM. Systemic corticosteroids are used for treatment. Immunosuppressive agents are used in cases refractory to corticosteroid treatment. Due to an association with underlying malignancy, an age and gender appropriate cancer screen should be performed in all patients diagnosed with dermatomyositis.
A CASE OF PREGNANCY, HYPOXIA, AND TOO MUCH PROTEIN. R.A. Andrews 1 , E.L. Salerno 2 ; 1 University of Connecticut, Farmington, CT; 2 University of Connecticut, Columbia, CT (Tracking ID #76994) LEARNING OBJECTIVES: 1) Discuss diagnosis, eitiology, and management of pulmonary alveolar proteinosis. CASE INFORMATION: A.G. is a 29 year old female 15 weeks into her fourth pregnancy who presents for diagnosis and treatment of severe hypoxia. Ten weeks prior to admission, she begins having progressive dyspnea on exertion, non-productive cough, weakness, fatigue, nausea, and diarrhea, but no fevers and occassional chills. Two days prior to admission she is referred to the emergency room for severe hypoxia. Patient denies problems with previous pregnancies and is HIV negative. Past medical history and social history are essentially unremarkable. At presentation, she is afebrile with a blood gas on room air that shows a PaO2 of 64. Cardiovascular exam is benign, but diffuse course rales are heard on lung exam. The patient has perioral and acral cyanosis and clubbing. Chest xray shows a diffuse patchy alveolar infiltrate. HCT is 49.3 with normal chemistries and negative D-dimer. Patient is placed on a non-rebreather mask at 100% and started on broad spectrum antibiotics. On day two, she is transferred to intensive care with a PaO2 of 54 on 100% oxygen. Video-assisted thorascopic biopsy is obtained providing the diagnosis of pulmonary alveolar proteinosis. Patient then undergoes whole-lung lavage with improvement in chest xray and PaO2 saturation. IMPLICATIONS/DISCUSSION: Pulmonary alveolar proteinosis (PAP) is a non-inflammatory disease of the lungs affecting men more than women (2:1) typically between 30 and 50 years of age. The primary defect is either a failure to degrade, or increased production, of surfactant causing an accumulation of amorphous proteinaceous material within the alveoli. The exact eitiology remains unclear. Patients generally present with dyspnea on exertion, 30% are asymptomatic. Common symptoms are dry cough, weight loss, malaise and fatigue. Rales are present in 50% of cases. Typical xray findings reveal perihilar alveolar infiltrates with typical``bat wing'' appearance. Diagnosis is made from BAL (bronchioalveolar lavage) or lung biopsy. Lavage produces a milky effluent composed of amorphous granular material that stains PAS (periodic acid-Schiff) positive. On biopsy, inflammatory cells are rare and there is no fibrosis. Alveoli are congested with proteinaceous material and dysfunctional macrophages. Standard of care is whole-lung lavage with 30±40% of patients resolving with only one lavage while the majority of patients require repeat lavage at 6±12 month intervals. Newer treatments include high doses of GM-CSF (granulocyte macrophage-colony stimulating factor) and possibly bone marrow transplant. Prognosis of PAP has been shown to be a 74% 5-year survival. LEARNING OBJECTIVES: 1. Recognize the long term complications after heart surgery for congenital defect in childhood. 2. Appreciate that patients living in precarious conditions need careful management that goes beyond an initial impression, although the risk of somatizing exists. CASE INFORMATION: A 25 year old male asylum-seeker, living in Switzerland for a month, with a history of surgical repair of a pentalogy of Fallot 20 years before with no subsequent complications, presented with breathing-related chest pain responsive to paracetamol, exertional dyspnoea and orthopnoea ± on the same day that five of his compatriots living in the same asylum-seeker centre located in a public bomb shelter with the same symptoms! He was in good general health, his blood pressure was 93/66 mmHg, and he had a regular heart rate of 100 beats/min and a respiratory rate of 25/min. His chest was slightly asymmetrical, with slight arching of the left hemithorax, the sternotomy scar appeared normal, the apical impulse was widened and pulsations were apparent across the whole sternal and left parasternal area (Harzer's sign). Auscultation revealed a holosystolic, non-radiating crescendo murmur of 3/6 in the left parasternal area. Diastole was free and peripheral arteries were normal. Laboratory tests were within normal limits, excluding acute conditions such as pulmonary embolism or cardiac ischaemia. The ECG revealed sinus tachycardia, a vertical frontal axis of the QRS complex and complete right bundle branch block. A transthoracic echocardiogram revealed a voluminous mass with an echo-dense border and an echo-poor centre adjacent to the free wall of the right ventricle, no vascularized (free of internal blood flow), exerting an obvious compression on the right ventricle, the intraventricular pressure being estimated at 80 mmHg based on the high-velocity severe tricuspid regurgitation. Magnetic resonance imaging and computed transverse tomography confirmed the presence of a hypodense mass, in close contact with the pericardium (Fig. 1 & 2) . The severe compression of the right ventricle prompted the surgical removal of this mass. The mass was adhering to the rib cage and the free wall of the right ventricle. Bacteriological investigation revealed the presence of an abscess with coagulase-negative staphylococci. Postoperative progress was uneventful, and the medium-term outcome favourable. No abnormal cavity was visualized during transthoracic echocardiography at the 3-month postoperative control. IMPLICATIONS/DISCUSSION: While infections are known to occur after sternotomy, usually involving coagulase-negative staphylococci, the formation of an abscess in the anterior mediastinum several years after the intervention appears to be exceptional. This diagnosis came to mind only after the more common postoperative complications, e.g. pseudoaneurysm or pericardial haematoma, and the wide range of possible diagnoses covering tumours, infections and malformations of the anterior mediastinum had been considered. Although the signs and symptoms were not very specific and potentially psychogenic in a patient living in precarious conditions, we were inclined to consider a wide range of differential diagnoses, given the patient's history.
care, her pulmonary status gradually returned to baseline and she was tapered off phenytoin without subsequent seizure activity. IMPLICATIONS/DISCUSSION: There are very few reports of a pulmonary embolus (PE) presenting as seizure. Given the acuity of onset of this patient's symptoms and the severity of her initial respiratory acidosis, as well as the lack of neurologic findings, we presume that her seizure represents the onset of her pulmonary embolus, though we can not definitively establish causal relationship. Clinical suspicion of PE may be compromised by the attribution of symptoms to the seizure, such as tachypnea, tachycardia, and hypoxia. The initial evaluation of seizure patients is primarily neurologic, delaying the diagnosis of a cardiopulmonary syndrome. In this particular case, the patient's medical history played a key role in the delayed diagnosis of PE, as her pulmonary symptomatology was attributed to asthma, despite the fact that she had been well controlled on minimal medication prior to admission, and that her clinical signs did not completely resolve with the treatment of her exacerbation. Furthermore, this young, active woman had low risk for thromboembolic disease, especially in the setting of a negative evaluation for hypercoaguability. She did use oral contraception, but this particular medication includes only 0.10mg levonorgestrel and 0.2mg ethinyl estradiol and the patient was a nonsmoker. Thromboembolism should therefore be considered in any patients with acute dyspnea, evidence of cerebral hypoperfusion, hypoxia, or unexplained seizure, even in the setting of pulmonary pathophysiology. year-old healthy female who presented to the emergency department after having her first seizure. She was found by her son in bed with jerking of her left upper extremity. When she regained consciousness she found that she bit her tongue and had urinary incontinence. She was well up until three days before when she developed a constant low-grade headache. Her last menstrual cycle was three months before presentation although she reports some light vaginal bleeding one week prior. Per the patient she had no sexual activity for six months. Physical exam on admission was significant for blood pressure of 145/82 and 1+ ankle edema. The neurological exam was completely normal. The patient did not know she was pregnant on admission but an initial beta-HCG was 12,700. Her urinalysis showed 3+ proteinuria. An ultrasound confirmed the presence of a 30 week gestation fetus. A head CT showed low attenuation lesions in both subcortical parietal lobes and in the left frontal lobe. The patient was transferred to Labor and Delivery with the presumptive diagnosis of eclampsia. She received intravenous magnesium sulfate and steroids to enhance fetal lung maturity. The next day after some fetal distress she delivered a viable male infant via cesarean section. Her vaginal bleeding one week prior to admission was due to placental abruption. IMPLICATIONS/DISCUSSION: Eclampsia is not commonly encountered in the practice of internal medicine but it should be considered in the evaluation of a new-onset seizure disorder in women of reproductive age. It has an incidence of approximately 5 cases per 100,000 live births. It is characterized by generalized seizures or coma in the presence of preeclampsia (hypertension, edema, proteinuria) . It is most common in nulliparous women in the teenage years and early twenties and in women over 35. More than 20% of cases occur before 30 weeks gestation, and 75% of cases occur at term or within 48 hours of delivery. It remains the second most common cause of maternal death in the United States. Treatment consists of oxygen, intravenous magnesium and antihypertensive medications. Anticonvulsant medications may be given for recurrent seizures. The fetus should be delivered promptly. Unfortunately this woman did not recognize that she was pregnant therefore she did not receive any prenatal care. This case underscores the importance of pregnancy testing in all women of reproductive age with a new-onset seizure disorder to expedite the diagnosis. Eclampsia should be part of the differential diagnosis in the evaluation of a new seizure disorder in all women of reproductive age unless she proves not to be pregnant. Early recognition and treatment of this disorder can prevent some potentially devastating consequences.
WHAT IS THE MEANING OF TRISMUS? H.A. Batal 1 , R.H. Harris 1 ; 1 Denver Health Medical Center, Denver, CO (Tracking ID #74878) LEARNING OBJECTIVES: 1) Identify the differential for trismus. 2) Recognize the acute presentation of tetanus. CASE INFORMATION: A 20 yo previously healthy Mexican native presented with 4 days of left cheek spasm associated with pain. He denied any trauma, abrasions or lacerations, thought that he had received his vaccines as a child, and denied any systemic symptoms such as fever or myalgias. He also denied sore throat or dental pain and did not have a personal or family history of neurologic problems, including tics. He had bitten his tongue a number of times due to his left cheek spasm and was finding it difficult to eat solid food, although his intake of liquids was adequate. On examination he had normal vital signs and did not appear to be in any distress. He spoke through slightly clenched lips, but was handling his secretions without difficulty. He had no apparent spasms or tics involving other muscle groups. His neurologic exam was normal, although he refused to protrude his tongue, as he was afraid that he would bite it. His exam showed poor dentition but no localizing dental infection and no evidence of pharyngeal abscess. Submandibular and parotid glands were non-tender. He was noted to have spasm of his left masseter muscle, and broke a tongue blade when an attempt was made to keep his mouth open for examination. He was only able to open his jaw approximately 2 cm. Laboratory examination showed a normal CBC, calcium, and electrolytes. A dental consultant was unable to find any underlying odontogenic etiology for the patient's trismus. Dental radiographs and head MRI were unrevealing. Because the diagnosis of acute tetanus was entertained, the patient was admitted for 48 hours of observation during which time his symptoms did not change. He was discharged with a diagnosis of trismus of unknown etiology and therapy with tegretol at 200 mg bid was initiated. IMPLICATIONS/DISCUSSION: Trimus is defined as spasm of the masticatory muscles, with difficulty in mouth opening, and is a characteristic early symptom of tetanus commonly referred to as lockjaw. The differential diagnosis of trismus is limited and besides acute tetanus includes odontogenic infections, peritonsilar abscess, strychnine poisoning, and drug-induced dystonic reaction. In addition, trismus can be confused with the jaw claudication seen in temporal arteritis as well as the pain of trigeminal neuralgia. Widsom tooth (3rd molar) infections are the most common odontogenic cause of trismus and notably can present without evidence facial swelling. Acute tetanus is important to recognize because it can progress to death as a result of respiratory failure, but the prognosis can be favorable if supportive treatment is initiated promptly. Predictors of inadequate immunity are older age, birth outside the US, low educational status, and poverty. LEARNING OBJECTIVES: To recognize the presentation and therapy of thyrotoxic periodic paralysis. CASE INFORMATION: A 26 yo Latino male was brought to the ER complaining of 12 hours of progressive upper and lower extremity weakness to the point that he could no longer get out of bed. The symptoms began with leg cramps shortly after a family picnic. He had no complaints of abnormal sensation, difficulty breathing or swallowing. He denied recent illness or vaccinations, or ingestion of canned food. He also denied past medical problems, medications, or illicit drug use. His mother and sister had hypothyroidism. The examination showed an alert, well developed male with a mildly enlarged thyroid, but no tremor or lid lag. Strength in the upper extremities was 3/5, and 2/5 in the lower extremities. Deep tendon reflexes were 1 plus bilaterally, and the cranial nerve exam was normal. Labs were remarkable for Na 142, K 1.8, Cl 111, CO2 20. A TSH was drawn. The EKG showed SR with flattened T waves and prominent U waves. After a total of 120 meq of combined IV and PO KCL, the serum K was 5.1 and the patient's symptoms and EKG findings had completely resolved. The following day, the TSH and FT4 returned at <0.03 and 2.48 respectively, consistent with hyperthyroidism. The patient was diagnosed with thyrotoxic periodic paralysis. Propranolol was initiated and a thyroid scan scheduled as an outpatient. Despite compliance with medications, the patient returned to the ER with a recurrence of symptoms after a large Christmas dinner. The iodine uptake scan showed a mildly enlarged gland with homogeneously increased uptake: 20 % (5±17%) at 4h, and 48 % (8±30%) at 24 hours, consistent with Graves disease. Therefore, the patient was treated with radioactive iodine ablation. Four months into followup, the patient was doing well, but had become hypothyroid. IMPLICATIONS/DISCUSSION: Thyrotoxic periodic paralysis is a well described disease in up to 2 percent of all Asian men afflicted with hyperthyroidism. However, it is being increasingly recognized in patients of other racial backgrounds including Native Americans, Latinos and rarely Caucasians or those of African descent. This disorder is caused by overactivity of the Na-K-ATPase channel in the presence of increased thyroid hormone, which shifts potassium intracellularly. The inital treatment involves modest potassium supplementation and non-selective beta blockers. The more common familial periodic paralysis is related to the Na-Ca channel, and presents in a similar fashion, frequently after a carbohydrate rich meal or exercise. It is important for clinicians to understand that thyrotoxicosis of any etiology is an underlying cause of hypokalemic periodic paralysis because treatment of the thyroid disorder will also prevent further paralytic episodes.
AN ATYPICAL CASE OF POLYARTERITIS NODOSA. B.N. Batson 1 , J.W. Blackston 1 , C. Subramony 1 ; 1 University of Mississippi Medical Center, Jackson, MS (Tracking ID #75156) LEARNING OBJECTIVES: Recognize the clinical and lab findings of polyarteritis nodosa. Perform an appropriate workup when polyarteritis nodosa is suspected. CASE INFORMATION: A 46 y/o BM was transferred complaining of abdominal pain for 4 days that had not improved with conservative treatment. Outside x-rays demonstrated dilated loops of bowel without obstruction. He denied fever, chills, nausea, and vomiting but reported fatigue, anorexia, and a 40-pound weight loss over the past 6 months. He also reported progressive weakness and numbness of both legs which limited ambulation. Physical was significant for BP of 178/114 and a diffusely tender abdomen without guarding, rebound or mass. He had faint bowel sounds and was heme positive on rectal. He had 2/5 strength in bilateral hip, knee, and ankle flexors and extensors. Sensation was diminished in both feet up to mid-shin. Reflexes and Babinski's were normal. CXR was negatvie except for blebs, but KUB showed multiple dilated loops of bowel with air-fluid levels. Lab included WBC 14.1, Hct 27, and plts 417. BUN was 23 and creatinine 1.2. Total protein was 6.7 with albumin 2.3. Hep BsAg was positive; p-ANCA was negative. On HD#2, CT abdomen showed a 9x5cm right lower quadrant mass, moderate ascites, and patchy kidney enhancement with wedged-shaped hypodensities. Renal ultrasound showed normal kidney size with inhomogeneous architecture. On HD#3, sural nerve biopsy was suspicious for mononeuritis multiplex. Later on HD#3, a mesenteric arteriogram was normal. By HD#4, the patient had decreased abdominal pain with stable hematocrit, but creatinine had risen to 2.7. On HD#5 colonoscopy was normal. He then had an MRI-guided biopsy of the RLQ mass which yielded only red blood cells. On HD#8 surgical exploration found bloody ascites and an 8x10cm mass of the ileocolonic junction requiring hemicolectomy. Ascitic cytology was negative for malignancy. Pathology of the mass showed extensive polyarteritis nodosa involving the small-and medium-sized submucosal and mesenteric arteries. IMPLICATIONS/DISCUSSION: While this patient exhibited many classic symptoms of polyarteritis nodosa, his workup of that suspected diagnosis was confounded by an abdominal mass and false-negative mesenteric arteriogram. One must remember that the diagnosis of PAN is best made with tissue biopsy of an involved organ system. While a positive p-ANCA is suggestive of PAN, and a mesenteric arteriogram showing pseudo-aneurysms is sufficient for diagnosis, both of these tests are positive in less than 50% of patients with known polyarteritis nodosa.
normalize. Magnetic resonance angiography of the abdomen revealed severe stenosis of the distal left renal artery consistent with FMD. The patient underwent contrast-enhanced renal artery angiography that confirmed the diagnosis. Balloon angioplasty of the stenotic left renal artery was performed. The day after angioplasty, the patient's plasma renin decreased to 50 microU/ml and BP to 130/70. IMPLICATIONS/DISCUSSION: Renovascular hypertension should be suspected in patients with age of onset before 20 or after 50 years, severe or refractory hypertension, acute rise in BP over previously controlled hypertension, rise in plasma creatinine concentration especially after starting an angiotensin-converting enzyme (ACE) inhibitor, abdominal bruit, absence of family history of hypertension, a unilateral small kidney, and recurrent acute pulmonary edema or unexplained heart failure. RAS may result from FMD or atherosclerotic disease. FMD most commonly affects women between the ages of 15 and 50 and accounts for less than 10% of cases of RAS. Atherosclerosis is responsible for the other 90% of cases. Gadolinium-enhanced magnetic resonance angiography is the best noninvasive imaging modality to diagnose RAS with 100% sensitivity and 95% specificity. Renal angiography is the gold standard to confirm the diagnosis. Percutaneous angioplasty is the treatment of choice for renovascular hypertension secondary to FMD. Patients with hypertension secondary to atherosclerotic RAS should be treated with aspirin, cholesterol-lowering agents, and smoking cessation. ACE inhibitors and diuretics can be used to control hypertension but should be avoided in the presence of bilateral stenosis. Balloon angioplasty is less successful in atherosclerotic RAS but placement of stents improves the success rates. Surgical revascularization should be considered in patients who fail angioplasty. LEARNING OBJECTIVES: 1. Identify a likely cause of recurrent painful blistering diseases in young adults 2. Identify the classical presentation, manifestations and prognosis of Epidermolysis Bullosa 3. Recognize the main management issues in caring for Epidermolysis Bullosa patients CASE INFORMATION: A 19 y/o latina woman presented for evaluation of painful blisters over her left hand and both feet after recent initiation of jogging. She reported a history of recurrent painful blisters affecting both her hands and feet since childhood. More severe blistering occured in areas where her skin was rubbed. Steroid creams provided no improvement. Lesions in the past healed over about a month, leaving hyper-pigmented scars. This current flare involved both her feet and left hand, but no other area. Systems review was negative. Other family members were not affected. She took Tylenol with hydrocodone for pain. On examination the patient had multiple blisters, 2±3 cm in diameter, mostly on the plantar surface of both feet. The soles of her feet were thickened and pigmented in circular patch-like forms. The blisters were exquisitely tender to touch. Initial differential diagnosis of this patient's condition included bullous pemphigoid, dermatitis herpetiformis, porphyria cutanea tarda and bullous SLE. A skin biopsy performed by a dermatologist demonstrated histologic separation of the basal layer. After immunohistochemistry, a diagnosis of epidermolysis bullosa, simplex form, was made. IMPLICATIONS/DISCUSSION: Epidermolysis Bullosa (EB) is the term given to three major groups of approximately 16 variants of rare dominant and recessive genetic diseases in which minor trauma causes non-inflammatory blistering (mechanobullous disease). There are as many as 150,000 Americans afflicted, mostly children, but variants present up to third decade of life. Those with medium to more severe kinds of EB can live into their thirties. Mortality is predominantly related to suprainfections. Management consists of all efforts to avoid trauma, particularly to the hands and feet via mittens and soft slippers. Adequate long-term pain control is a crucial aspect of taking care of EB patients, as is prevention and early treatment of supra-infections of the blistering lesions. Clasically all EB variants are resistant to all therapy, though literature reports exist of rare responders to treatment with Phenytoin, a known collagenase inhibitor, as well as IVIg. Genetic counseling is essential and fetal skin biopsy techniques have been developed for prenatal diagnosis. The diagnosis of glucagonoma was confirmed by pathologic examination. Upon removal of the tumor, her rash subsequently cleared without any complications. IMPLICATIONS/DISCUSSION: Glucagonoma syndrome is a rare syndrome with an estimated incidence of about 1 in 20 million people. It occurs with equal frequency in men and women and is usually detected between 40 to 60 years of age. The syndrome is caused by excessive production of glucagon by the alpha-secreting cells of the pancreas. Patients may have a characteristic rash, called``necrolytic migratory erythema'', that involves the perioral and perigenital regions. There may also be accompanying glossitis, angular chelitis, and blepharitis. The rash usually responds poorly to topical treatment. The only curative treatment for the syndrome is surgery. Glucagonoma syndrome should be considered in any patient with: 1) newly diagnosed diabetes and 2) a rash that does not resolve with proper topical treatment and is consistent with necrolytic migratory erythema. 10% of all strokes. There is a much broader differencial of disorders that cause infarction in younger people. Full evaluation is required to determine an underlying cause to treat patients accordingly and prevent recurrence. As opposed to stroke in the elderly which are often due to atherosclerosis and embolism, only 40% of strokes in the young are accounted for by these two etiologies. Other important causes include dissection, vasculitis, blood disorders, pregnancy, mitral valve prolapse, migraine, and patent foramen ovale. Given this patient had simulanteous anterior and posterior strokes, some of these etiologies are more likely, including: proximal emboli or a disseminated process such as vasculitis, blood disorder or drug use. This young person's stroke was a result of cocaine use. Proposed mechanisms by which cocaine causes stroke is that it induces vasoconstriction, vasospasm, hypertension and/or vasculitis. Case reports have documented stroke secondary to cocaine use in patients who had infarction in multiple distributions. Several of these cases were confirmed to have cocaine-induced vasculitis on autopsy despite having had negative MRA. LEARNING OBJECTIVES: To distinguish the toxicity of``crystal meth'' (methamphetamine) from other syndromes in a patient presenting with psychomotor agitation and tachycardia. CASE INFORMATION: A 22 year-old male with unremarkable PMH presented to the ED via ambulance with the acute onset of diaphoresis, tachycardia, and restlessness. He had been in police custody for approximately 48 hours, and charged with public intoxication. After his symptoms developed, he admitted to ingesting a``2 gram rock of crystal meth'' to avoid charges of illegal drug possession. Physical exam revealed an anxious but alert WM, BP 119/72 mm Hg, HR regular at 179 bpm, unlabored RR 16 per min., rectal temp T 99.3 degrees F, and O2 sat. 97% on RA. Auscultation of his chest was clear and his neurologic exam was nonfocal; but he was tremulous, pale, and his skin cold and clammy. He required substantial benzodiazepine doses to achieve sedation: diazepam 5mg IV en route to the ED, and a total of 8mg IV lorazepam and 14mg IV midazolam in the ED. The patient was electively intubated for``airway protection.'' His hospital course was uneventful; he was extubated within the first day of admission and transferred back to incarceration in 2 days. CPK levels were normal in the ED, but subsequently increased to ten times the ULN. A serum toxicology screen was positive for amphetamines and cannabinoids, and negative for salicylates, acetaminophen, methanol, ethanol, and ethylene glycol. Confirmatory testing revealed a serum methamphetamine level of 80,000 ng/dL. IMPLICATIONS/DISCUSSION: Delayed methamphetamine toxicity, defined as symptom onset 48 hours after ingestion, has not been previously reported in the medical literature. The crystalline form of methamphetamine, also known colloquially as``crank,'' is a stimulant drug of abuse that is usually nasally inhaled, snorted, or smoked. It is rapidly absorbed from any mucous membrane and produces almost immediate metabolic and psychotropic effects. This patient reported orally ingesting the drug in a plastic container, thus with gastrointestinal transit time and even partial digestion of the container, the drug may have been easily absorbed later in the distal GI tract. Alcohol withdrawal with incipient delirium tremens may also explain his presenting symptoms, but his markedly elevated CPK, a usual laboratory finding with acute methamphetamine overdose, and strongly positive serum methamphetamine level at the time of maximal symptomology, suggest methamphetamine toxicity as the etiology of his distress. This case stresses the need for medical personnel to obtain complete drug exposure histories from agitated patients, including explicit questioning with regard to drug ingestion stemming from attempts to conceal illegal activities.
CARDIAC TAMPONADE DUE TO SEVERE MYXEDEMA. J. Blonsky 1 , V. Shivaswamy 1 , K. Hoskison 1 , R. Sekhar 1 , D. Perry 1 ; 1 University of Oklahoma, Tulsa, OK (Tracking  ID #75122) LEARNING OBJECTIVES: Recognize hypothyroidism as cause for cardiac tamponade. Diagnosis of cardiac tamponade secondary to hypothyroidism. Treatment of cardiac tamponade caused by severe tamponade. CASE INFORMATION: We report a 53-year-old male with an enlarging right testicle, progressive dyspnea, severe dependent edema, and cold intolerance. Physical examination revealed faint heart sounds, ascites, ankle jerks with delayed relaxation, and an enlargement of the right scrotum consistent with a hydrocele, and a clinical diagnosis of cardiac effusion with tamponade was entertained. Further evaluations revealed low voltage complexes on the EKG, and marked cardiomegaly on a Chest Roentgenogram with bilateral pleural effusions. An echocardiogram showed a large pericardial effusion with signs of early tamponade, and a pericardial paracentesis drained 1400ml of exudate. Pericardiocentesis led to significant clinical improvement. To confirm a unifying diagnosis thyroid functions were diagnosed of profound hypothyroidism with a TSH of 52.5 iu/ml. The patient responded well to thyroid hormone replacement therapy, and demonstrated continued recovery at clinic follow-up visits. The scrotal swelling was confirmed as a hydrocele by ultrasound. Hypothyroid effusions involving the pleural, pericardial, and peritoneal cavities have been described, but uncommon clinically. Pericardial effusions tend to be large, and asymptomatic, as the slow temporal progression permits compensation due to pericardial stretching. The clinical presentation suggests chronic cardiac failure and constrictive pericarditis with pleural effusion, ascites, and anasarca. EKG findings include bradycardia and microvoltage complexes. Treatment with thyroid hormone replacement is nearly always followed by steady regression of the effusion. Pericardiocentesis is indicated for tamponade, after a confirmatory echocardiogram. This presentation illustrates the importance of detecting hypothyroidism as cause for cardiac tamponade. Prompt diagnosis is vital to prevent cardiogenic shock, and includes early suspicion on the basis of a detailed physical examination, and rapid diagnosis by thyroid functions and echocardiography. Evacuation of the pericardial fluid, and thyroid replacement therapy leads to complete regression and resolution of the condition. IMPLICATIONS/DISCUSSION: Hypothyroidism may lead to cardiac tamponade. Prompt diagnosis is vital to prevent cardiogenic shock. Complete resolution of pericardial fluid following evacuation and thyroid replacement therapy.
A HISPANIC MAN WITH LYMPHADENOPATHY AND EOSINOPHILIA. D. Bolger 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #76421)
LEARNING OBJECTIVES: 1. Recognize the clinical features of Kimura's disease. 2. Recognize that a constellation of nonspecific clinical signs can represent a specific entity when pursued. 3. Develop a differential diagnosis for lymphadenopathy and differentiate accordingly. CASE INFORMATION: A 63 year old man born in the Dominican Republic but residing in New York City for 30 years presented to a medical clinic with 2 years of right neck swelling. The gentleman initially noticed a small, painless bump in his neck. The bump enlarged to an eventual size of 8cm by 14cm. Similar bumps also arose in the right neck, left neck, and right axilla. Eventually the predominant lesion became painful and itchy. The patient felt healthy otherwise, denying fevers, fatigue, and weight loss. The patient had a history of hypertension, PPD positivity with negative CXRs, and remote microscopic hematuria. He was taking hydrochlorothiazide and prazosin for his hypertension. He was married with 2 children. A He traveled to the Dominican Republic every year for holiday. No other travel was noted. His physical exam was remarkable for a mobile nodular mass 8cm  14cm in the right anterior cervical neck. There were discrete subcutaneous nodules on both sides of the neck. There was lymphadenopathy in the left neck and right axilla. The nodules and lymph nodes were mobile and firm but without fluctuance. There was no hepatosplenomegaly or other lymphadenopathy present. Pertinent labs included a wbc count of 8100/mm3 with 38% eosinophils (absolute eos count 3078) IgE levels were high at >1800kIU/L. HIV test was negative. Excisional biopsy of lymph node revealed follicular hyperplasia with distinct lymphoid follicles with surrounding fibrosis, abundant eosinophils, post-capillary venules, no granulomas or necrosis, and preserved architecture. Cultures and smears were negative for AFB, fungi, bacteria, and Reed-Sternberg cells. Flow cytometry was negative for lymphoma. In addition, skin snips from the patient were negative for microfilariae. The patient was started on prednisone with rapid resolution of lymphadenopathy, however this recurred when prednisone was tapered. The patient was restarted on full dose prednisone and scheduled for curative radiotherapy. IMPLICATIONS/DISCUSSION: Kimura's disease is a rare disease usually seen in Asian men. They present with lymphadenopathy of the head and neck and eosilophilia. Pathology reveals preserved lymph node architecture with necrosis, predominant eosinophils, endothelial venules, and capsular fibrosis. Often lymphadenopathy is attributed to an infectious agent or malignancy. As this case illustrates, there are other causes to consider. A thorough evaluation and exclusion of important entities such as Hodgkin's disesase, extra-pulmonary TB, and Cat Scratch Disease is critical. Histological evaluation often provides a definitive diagnosis, but not always. The pathology along with the clinical signs (features) were required for the diagnosis in this case. osteomyelitis and an epidural phlegmon. Magnetic resonance imaging (MRI) showed cord compression. Needle biopsy of the abscess was performed, yielding prurulent material. In consultation with Infectious Disease and Pulmonary services, empiric treatment for TB was held as it could mask a pyogenic process. By the eleventh hospital day, needle biopsy failed to grow organisms. Polymerase chain reaction (PCR) results of the biopsy aspirate was positive for mycobacterium tuberculosis (M.Tb) and the patient began therapy with isoniazid, rifampin, pyrazinamide and ethambutol. He subsequently underwent surgery for spinal stabilization and was discharged on an 18 month course of antibiotics. IMPLICATIONS/DISCUSSION: Low back pain is the fifth leading reason for medical visits in the United States. A careful history identifies``danger'' signals of infection or malignancy, such as constitutional symptoms, immunosuppression, age and unremitting nocturnal pain unrelieved by recumbency. In Pott's disease, systemic symptoms are often absent, and patients do not undergo testing until the process is advanced. CXR is not useful in diagnosis since more than 50% of patients do not have lung disease. The primary focus starts in the anteroinferior aspect of the vertebral body of the thoraco-lumbar spine. Local destruction leads to collapse, disc herniation and cord compression. Likewise, a tuberculous abscess, seen in more than 50%, can impinge and compress the spinal cord. CT, myelography, and MRI are useful in diagnosis. While biopsy and culture are sensitive and specific, serology and PCR are useful for timely diagnostic confirmation. Literature reports culture sensitivity for mycobacterial detection is improved by culture media supplemented with oleic acid, albumin, dextrose, and catalase, however detection is not specific to M. Tb. PCR of fixed, paraffin-embedded tissue samples, was shown to have sensitivity and specificity of 94% and 83% respectively. In summary, TB remains a major public health problem. Before the disease can be treated it must be recognized. TB should be considered in cases of spinal osteomyelitis. Early institution of therapy reduces morbidity and mortality.
ASTHMA OR ARE WE MISSING SOMETHING. C. Borrego 1 , E. Flenaugh 1 , D. Norman 1 , J. Henao 1 ; 1 Morehouse School of Medicine, Buford, GA (Tracking ID #74416)
LEARNING OBJECTIVES: Diagnosis and Treatment of Pulmonary Blastoma. CASE INFORMATION: This patient is a twenty-seven year old black male who has a past medical history of asthma since eight years old. He is a non-smoker. He states that eight months ago he started to work at a police impound. Where he states he was exposed to a lot of dust. Six months later he started having shortness of breath, cough with whitish to yellowish sputum, and wheezing with associated left lateral chest pain. He then went to the hospital were he was treated for an asthma exacerbation with improvement and subsequently discharged. He then returned with chest pain, left sided rated 8/10 and admitted. On physical exam blood pressure was 90/43, pulse 97, respirations 20, and temperature 97.3 degrees Fahrenheit. Room air pulse oximetry was 97%. He has decreased breath sounds on the left hemithorax. Otherwise physical exam was normal. Chest x-ray showed left lung to be completely opacified by tumor mass or pleural effusion, with some cystic components. CT scan off chest showed a large soft tissue mass completely replacing the left lung of mixed density most likely representing a neoplastic process such as sarcoma, invading and obstructing the left main bronchus. The bronchoscopy showed the right lung to be patent and 3 cm below the carina going to the left main stem bronchus, the airway was completely occluded by a tumor mass in the lumen. Endo bronchial biopsy was performed at that time, which revealed pulmonary blastoma, monomorphic epithelial type (well differentiated fetal adenocarcinoma). IMPLICATIONS/DISCUSSION: Pulmonary blastoma is hard to be diagnosed before operation. The main symptoms can be cough, expectoration. This tumor is extremely rare and manifest's a mixture of epithelial and mesenchymal neoplastic cells. This is a low grade tumor that consists of immature embryonic epithelial elements forming tubules and glands and primitive lung structures, surrounded by a malignant-appearing mesenchymal stroma. Other tumors with better differentiated background mesenchymal elements or neuroendocrine cells in the stroma have also been reported and may represent part of this tumors spectrum. These tumors presents in children and adults and most commonly appear as parenchymal masses. Pulmonary blastoma is more common in women, and is usually diagnosed by surgical resection. This is usually curative, although if late relapses and metastasis occur they are usually not curable. For palliative treatment radiation therapy may be used, and a role for chemotherapy is not defined. LEARNING OBJECTIVES: Acute Myeloid Leukemia (AML) is an exceedingly rare diagnosis to find in association with Acute Intermittent Porphyria (AIP). Only one case report documents such an occurrence. CASE INFORMATION: We present the case of a 61-year-old man with a long-standing history of known Acute Intermittent Porphyria, (AIP) diagnosed in 1996, who was admitted to the internal medicine service for treatment of pharyngitis and severe dehydration. He had never undergone any chemotherapy and his AIP was fairly well controlled, with only four exacerbations noted including the sentinel episode. At presentation for his current admission the patient described fatigue and increasing retropharyngeal pain associated with deglutition, which had steadily progressed during the preceding three days. His CBC was significant for hemoglobin of 10.6 g/dl, WBC of 170.4/mm3, and a platelet count of 102,000/mm3. The differential was 24% segmented neutrophils, 0% bands, 3% lymphocytes, 57% monocytes, and 7% blasts. The patient had hepatosplenomegaly and a global petechial skin rash. Bone marrow studies were done to characterize the underlying etiology. Cellularity to fat ratio was 100:0 with normal bony spicules. Myelofibrosis was completely absent, and granulopoiesis was increased with an abnormal appearance. The blast estimate was greater than 20%. Over the subsequent week the patient experienced a rapid clinical decline with worsening coagulopathy. In accord with decisions made by the patient, his family, and consulting hematologists, chemotherapy was not instituted. AIP is the most common and most severe of the porphyrias.
IMPLICATIONS/DISCUSSION: Although there are case reports of AML occurring with other hepatic porphyrias, there is only one known prior case report of AIP and AML occurring in tandem. This represents the second known incidence of these two disorders occurring simultaneously.
SARCOIDOSIS PRESENTING AS CERVICAL CORD SYNDROME. D. Bradley 1 , E. Lower 1 , R. Baughman 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #73947) LEARNING OBJECTIVES: To recognize that sarcoidosis is a rare but treatable cause of spinal cord lesions that can mimic malignant tumors of the spine clinically and radiologically. CASE INFORMATION: A 53 year-old African American male presented with a six month history of worsening neck pain, progressive upper and lower extremity weakness, and difficulties with bowel and bladder function. He also noted severe fatigue and a 30 pound weight loss over the last eight months. Physical examination was remarkable for flaccid paralysis of his right upper extremity, 3/5 strength of his left upper extremity, and 3/5 strength of his lower extremities. He had decreased rectal tone. Significant laboratory findings included a normal CBC, chemistry panel, and liver function studies. Calcium was elevated at 11.5 mg/dL (nl 8.4±10.2), an ACE level was normal. Cerebral spinal fluid studies were unremarkable. MRI of the spine showed extensive abnormal signal involving the entire cervical cord extending through T3 with diffuse, patchy nodular enhancement and edema from C3±C7 suspicious for malignancy. CT of the chest revealed mediastinal adenopathy. Biopsy of subcarinal lymph nodes showed noncaseating granulomas consistent with sarcoidosis. The patient was treated with high dose corticosteroids and IV cyclophosphamide (off label use as a steroid sparing agent) with marked improvement. Repeat MRI six months later was normal. IMPLICATIONS/DISCUSSION: The spinal cord is involved in less than 1% of patients with sarcoidosis. It is rarely the first manifestation of disease. Sarcoidosis of the spinal cord can be can be clinically and radiologically indistinguishable from a malignant tumor of the spine. Definitive diagnosis requires pathologic examination of the spinal cord lesions or, as in our patient, other associated lesions such as hilar adenopathy. Prompt treatment with high dose corticosteroids is essential to a favorable outcome. Steroids may need to be continued indefinately. There has been some success using steroid-sparing agents such as cyclophosphamide to decrease the dose and duration of steroid therapy. Sarcoidosis is a treatable condition that should be included in the differential diagnosis of spinal cord lesions.
ZOSTER SINE HERPETE: A CASE OF MASKED DIAGNOSIS. J. Bratcher 1 , S. Patel 1 , K. Cynamon 1 , J. Andrieni 1 ; 1 Lenox Hill Hospital, New York, NY (Tracking ID #75529)
LEARNING OBJECTIVES: 1) Expand the differential for abdominal pain; 2) Diagnose an atypical presentation of herpes zoster in the absence of skin lesions; 3) Recognize that zoster sine herpete is a clinical diagnosis. CASE INFORMATION: A 58 year-old male with history of nephrolithiasis presented with abdominal pain, fever, and chills. Physical exam was significant for low-grade fever and left upper quadrant tenderness. Radiographic studies revealed a 5.3 Â 4.6 cm dissecting aortic aneurysm at the level of the diaphragm, and a separate dissection of the right common iliac artery. The patient was admitted with a diagnosis of an infected aortic aneurysm and evaluated by vascular surgery. The patient continued to complain of left upper quadrant pain, described in a definite dermatomal distribution along the left lower costal margin with radiation to the left flank. Despite absence of any physical evidence of rash, the description was suggestive of herpes zoster. Varicella titers were then ordered and positive IgM titers were revealed. Zoster sine herpete was diagnosed, and the pain spontaneously resolved without medical intervention. On follow-up, the patient remains asymptomatic, never having developed a rash, and repeat CT scans reveal stable aneurysms. IMPLICATIONS/DISCUSSION: Herpes Zoster (Shingles) is a reactivation of varicellazoster virus, which lies dormant in the dorsal or cranial nerve ganglia. Upon recurrence, patients typically have a prodrome of fever, along with pain or tingling in the affected dermatome (50% thoracic, 20% cervical, and 10% lumbosacral). In most cases, the virus creates vesicles along the dermatome after three days, which then crust and resolve within two to three weeks. A small percentage of patients, however, develop acute segmental neuralgia, with a concomitant rise in varicalla IgM antibodies in the absence of skin lesions. This is zoster sine herpete, and as our case illustrates, patients can be initially misdiagnosed with a host of other illnesses, including myocardial infarction, cholecystitis, appendicitis, or diverticulitis. Very little has been published concerning zoster sine herpete since many cases spontaneously resolve without ever having reached the diagnosis. In this particular case, a CT of the abdomen revealed a dissecting aortic aneurysm, which was unrelated to the patient's chief complaint. By relying more on physical exam, the patient's description of the pain, and prior radiographic studies, the diagnosis might have been made more efficiently. This case stresses the importance of history and physical exam in the clinical diagnosis of zoster sine herpete. Otherwise, the diagnosis can be masked by confounding information. ventricular response. CXR (photo to be included) disclosed severe tension pneumothorax with complete collapse of the left lung and slight mediastinal shift to right. Emergent placement of a chest tube resulted in re-expansion of the lung, and stabilization. The patient did well and was discharged from the hospital 2 days later. IMPLICATIONS/DISCUSSION: Use of complementary/alternative medicine, often in concert with``mainstream'' medical treatment, is common and widely practiced in the United States. Acupuncture is effective as sole or adjunctive therapy for management of many types of pain, and is generally regarded as safe. The current vignette describes a case of tension pneumothorax directly attributable to acupuncture treatment. Traumatic injury from acupuncture is rare, but review of the medical literature discloses numerous reports of adverse events resulting from acupuncture treatments. Pneumothorax is the most commonly reported traumatic injury, especially among patients predisposed by presence of COPD; other risks include cardiac tamponade, peripheral nerve injury, and injury to blood vessels including pseudoaneurysm formation. Non-traumatic adverse effects of acupuncture include infections (hepatitis and other blood-borne diseases, as well as reports of septicemia and endocarditis.) The overall safety profile of acupuncture is positive, but patients and physicians should be aware of potential risks of treatment. State laws vary widely regarding requirement for certification; referral to licensed or certified medical acupuncturists is recommended. year-old woman presented with one month of fevers, evanescent rash, myalgias and arthralgias, severe fatigue, and anorexia with 20 pound weight loss. Examination disclosed T 39.48C, tender posterior cervical lymphadenopathy, no obvious synovitis, and a salmon-colored macular exanthem on the lower extremities. CBC showed normocytic anemia (Hgb 9.8,) slight leukocytosis (WBC 11.1 with 82.3% granulocytes,) normal LFTs except for slight elevation of AST, normal BUN/Cr, UA, and CXR. Further tests revealed ESR 90, negative ANA and RhF, and ferritin >2000. Further diagnostic workup included negative PPD, negative blood cultures, normal echocardiogram, normal CT scan except borderline mediastinal lymphadenopathy and mild hepatosplenomegaly, and negative titers for HIV, EBV, CMV, and Lyme disease. Lymph node biopsy disclosed reactive node with no evidence of a clonal population of cells. The patient was diagnosed with AOSD and had minimal response to aspirin. She was treated with steroids and is presently minimally symptomatic on hydroxychloroquine and NSAID. IMPLICATIONS/DISCUSSION: AOSD is a rare disease (incidence 1.6 cases/million persons/yr) that highlights the importance of clinical history and physical examination, since there is no definitive test to make the diagnosis. Diagnostic criteria have been proposed (will be delineated.) ASOD usually follows one of three patterns: self-limited (monophasic) disease with complete resolution of symptoms within one year, intermittent (polyphasic) course with complete remission between flares, or persistent active disease, with chronic destructive arthritis. First line treatment is aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs;) patients with more severe disease may respond to steroids. There are no controlled trials of immune modulating drugs in ASOD. Reports exist of success using methotrexate to control systemic symptoms; other agents used (all off-label) include gold salts, azathioprine, cyclophosphamide, cyclosporine, sulfasalazine, and intravenous immune globulin. Recent reports suggest etanercept and infliximab may have benefit in patients not responding to other therapies. A 74 year old white female presented with a 3 day history of`d ifficulty getting out of a chair'', dyspnea on exertion and``tea colored'' urine. Her past medical history was significant for depression and hypercholesterolemia. Her past medications included simvastatin (40 mgs po qd) and nefazodone (150 mgs po bid). She had been started on the nefazodone 3 months ago for treatment of depression and shortly thereafter developed a fever, nausea/vomiting and muscle spasms. Physical examination revealed significant weakness of the proximal musculature: 3/5 strength in the shoulder abductors and 2/5 strength in the hip flexors. Her laboratory data revealed: CK 37,050, AST 1,744, ALT 1,029, BUN 17, Creatinine 0.8 , Urinalysis: SpGr 1.020, pH 6.0, protein 100, large blood, granular casts. The nefazodone and simvastatin were discontinued and a forced bicarbonate diuresis was initiated. Polymyositis was suspected and steroids were started empirically pending the outcome of testing. An EMG showed no evidence of an underlying myopathy). ANA and an anti-Jo-1 antibody tests were negative. A muscle biopsy showed age-appropriate atrophy but no necrosis of myocytes and no evidence of lymphocytic infiltrate, thus ruling out the diagnosis of polymyositis. Her renal function remained normal throughout the hospitalization. Her CPK was 1,776 eight days after admission and she gradually regained strength in her proximal muscle groups. She was thought to have developed rhabdomyalyis secondary to a drug interaction between simvastatin and nefazodone. IMPLICATIONS/DISCUSSION: To our knowledge, this is the second case report of an adverse event associated with the concomitant use of simvastatin and nefazodone. Simvastatin is metabolized via the CYP3A4 system and nefazodone is a direct inhibitor of CYP3A4 enzymes. While myositis and rhabdomyolysis are adverse events associated with simvastatin monotherapy, we speculate that the introduction of nefazodone into this patient's regimen sufficiently inhibited the CYP3A4 system to cause an increase in serum simvastatin concentration that was adequate to cause rhabdomyolysis. Therefore, we would recommend avoidance of CYP3A4 inhibitors such as nefazodone in those receiving simvastatin therapy.
COLON CANCER, WITH ANGIOGRAPHIC EVIDENCE OF VASCULITIS. I.D. Bucaloiu 1 , S. Dubagunta 1 , K.K. Pachipala 1 , N.R. Kamal 1 , F. Fata 1 ; 1 Geisinger Medical Center, Danville, PA (Tracking ID #76029) LEARNING OBJECTIVES: 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents in clinical oncology practice, alone or in different combinations. In stage III colorectal cancer, 5-FU based therapy has been shown to be associated with a survival benefit. We report a case of ischemic small bowel injury in a patient who received adjuvant chemotherapy with Oxaliplatin, 5-FU and LV and was found to have angiographic evidence of vasculitis in the superior mesenteric artery (SMA) territory. CASE INFORMATION: A 73 year-old male underwent a left hemicolectomy for Stage III colon cancer. He was enrolled in an adjuvant chemotherapy research protocol consisting of 5-FU 500-mg/m2 intravenous (IV) weekly boluses each over 1 hour, LV 500-mg/m2 IV weekly infusions each over 2 hours and Oxaliplatin (OP) 85 mg/m2 IV infusions every 2 weeks for 6 weeks followed by two weeks of rest. After the third weekly dose of cycle one of 5 FU/LV he presented with weakness, diarrhea, crampy abdominal pain and poor appetite. His abdomen was distended and he had heme positive stools. Stool cultures and clostridium difficile toxin antigen were negative. He was not neutropenic. Colonoscopy revealed dusky edematous changes in the ascending colon and focal ulceration with patchy submucosal hemorrhages in the distal ileum. Biopsies of the colon mucosa showed densely neutrophilic infiltrates of the lining epithelium and associated mild focal cryptitis. A SMA angiogram was performed and it revealed beading in ileal branches, consistent with the ischemic territory. Vasculitis serologies were negative. The patient was treated conservatively with hydration and IV antibiotics and later he was rechallenged with a 20% reduction of 5-FU dose with no recurrence of his symptoms in 12-month follow-up. IMPLICATIONS/DISCUSSION: Our patient had superficial erosions and ulcerations in the ileum as well as an ischemic appearance of the colon. The characteristic findings of``beading'' on the angiogram suggest an underlying ischemic mechanism related to 5 FU based therapy. This case will add to the series of patients reported with 5-FU induced vascular injury to the small bowel.
THROMBOTIC THROMBOCYTOPENIC PURPURA AND EXTENSIVE BONE MARROW NECROSIS SECONDARY TO METASTATIC SIGNET RING CELL ADENOCARCINOMA. I.D. Bucaloiu 1 , J. Brady 1 , S. Walker 1 , K.K. Pachipala 1 ; 1 Geisinger Medical Center, Danville, PA (Tracking ID #76352) LEARNING OBJECTIVES: Although rare, the association between Thrombotic Thrombcytopenic Purpura and extensive Bone Marrow Necrosis can be the presenting picture of metastatic adenocarcinoma. CASE INFORMATION: A previously healthy 49 years old male was transferred from another hospital with a two-week history of severe, intractable back pain, short-term memory loss fatigue and shortness of breath. On exam he was afebrile, icteric and had multiple ecchymoses on the trunk. Complete blood count revealed severe anemia and trombocytopenia, with serum hemoglobin of 4.6 g/dl, a platelet count of 16 Â 109/L and white blood bell count of 3.22 x 109/L. There was evidence of severe hemolysis with serum lactate dehydrogenase of 2659 U/L and Serum Haptoglobin of 7 mg/dL. Direct Coombs test was negative. The absolute reticulocyte count was 42.6 K/uL. The peripheral smear revealed schystocytes and thrombocytopenia. A CT scan of chest, abdomen and pelvis showed splenomegaly, and was otherwise unremarkable. The initial bone marrow aspirate and biopsy revealed extensive necrosis of bone marrow with marked reduction of normal hematopoietic elements. Flow cytometry was negative for a clonal lymphoid proliferation. The repeat contralateral iliac crest biopsy revealed among the extensive necrosis, small aggregates of highly atypical cells with pleomorphic hyperchromatic nuclei. Some cells had signet ring morphology and they stained positive with mucicarmin and cytokeratin stains, which indicate a carcinomatous origin. The patient received multiple blood transfusions, high dose steroids and plasmapheresis, but none of these interventions had a significant impact on the clinical course. His delirium worsened, developed respiratory failure and expired on the 7th day of hospitalization. IMPLICATIONS/DISCUSSION: Massive bone marrow necrosis is known to occur in the context of malignancy, usually hematopoietic, lymphomas, leukemias, but also solid tumors. At the same time TTP has been reported to occur in the setting of neoplasms, especially adenocarcinomas, usually gastric, pancreatic and breast. The association of TTP and massive bone marrow necrosis, although rarely reported in the literature may represent as illustrated in our case report the heralding picture of metastatic cancer. It is important for physicians to be aware of this association as it forces one to search for an unapparent neoplasm. and pelvis with subsequent abdominal MRI confirmed cavitary lesions in the left upper lobe and revealed a 12 Â 10 Â 7.5 cm adrenal mass. A serum ACTH was low at 3 pg/mL (normal 9± 52 pg/mL) and a serum free morning cortisol of greater than 50 mcg/dL (normal 6±20 pg/mL) confiming a diagnosis of Cushing syndrome. Urine catacholamines were normal. The adrenal mass was later surgically removed with pathology confirming a diagnosis of adrenocortical carcinoma. Sputum cultures obtained by bronchoscopy grew significant colonies of Nocardia Farcinica and Nocardia Nova. The patient was started on intravenous trimethoprimsulfamethoxazole for six weeks followed by oral therapy. IMPLICATIONS/DISCUSSION: Nocardiosis is a rare finding in immunocompromised patients with an estimate of 500±1000 new cases per year. Most cases involve the lung with inhalation as the mode of entry. Although an immunocompromised state predisposes patients to Nocardia infection, steroid use is considered to be an independent risk factor. Our patient, while not taking exogenous steroids, did have very high levels of endogenous cortisol. Definitive diagnosis is typically made by routine culture of an isolated specimen. The most effective treatment for Nocardiosis remains speculative and is based mostly on small retrospective studies and case reports. Trimethoprim-sulfamethoxazole demonstrates synergy against Nocardia and general consensus supports high doses of this drug initially followed by long-term treatment with lower doses for up to one year due to the high degree of recurrence of disease. As demonstrated in this case, one should consider Cushing syndrome as a risk factor for the development of Nocardia infections. year-old man presented with one day of abdominal and back pain. He was a daily heroin user and his last injection was two hours prior to presentation. During his evaluation, he became uncooperative and combative, subsequently leaving against medical advice. Two hours later he was found disoriented on the street. His temperature was 39 C; he had nuchal rigidity and tenderness to palpation over the lumbar spine. There were no murmurs, purpura, or petechiae. He rapidly developed respiratory failure and was intubated. Blood, urine, and CSF cultures grew methicillin-resistant Staphylococcus aureus. He received seven days of vancomycin, and rifampin, After stopping a seven day course of antibiotics, his fever returned. A repeat lumbar puncture revealed persistently positive MRSA. Vancomycin and rifampin were restarted , and his fever resolved. An MRI of the lumbar spine demonstrated pyomyosits of the paraspinal muscles. He received 42 days of vancomycin and rifampin with clinical improvement. IMPLICATIONS/DISCUSSION: Back pain in the intravenous drug user, especially when associated with fever, must prompt high suspicion for meningitis, discitis, oteomyelitis of pyomyositis. Although meningitis from Staphyloccocus aureus is usually secondary to neurosurgical procedures, intravenous drug users are susceptible to staph CNS infections due to their frequent innoculation and impaired immunity. With the increased availability of antibiotics on the street, methicillin-resistant Staphyloccocus aureus is increasingly becoming a community acquired pathogen. As this case illustrates, serious CNS infections in an intravenous drug user may warrant empiric vancomycin until definitive cultures return. Staphyloccocus aureus bacteremia requires at least two weeks of intravenous antibiotics; longer courses are required for deep-seated infections. A secondary focus should always be sought, as Staph is a rare primary infection. This case illustrates the importance of maintaining a high degree of suspicion for Methicillin-resistant Staphyloccocus aureus in intravenous drug users presenting with back pain and fever.
MITOCHONDRIAL TOXICITY AND HIV THERAPY. S. Burridge 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #75159) LEARNING OBJECTIVES: Recognize the clinical presentation of mitochondrial toxicity secondary to nucleoside reverse transcriptase inhibitors. CASE INFORMATION: A 43-year-old white male with AIDS presented with altered mental status, nausea, vomiting, right upper quadrant pain and myalgias. Medications included didanosine, efavirenz, lopinavir/ritonavir and stavudine. His temperature was 101.28F, HR 120, BP 135/85 and RR of 26. Physical examination was significant for bitemporal wasting, oral thrush, dry oral mucosa, ketotic breath, pain to palpation in the right upper quadrant and a liver span of 20cm. There were no identified sources of infection. Laboratory examination was significant for sodium 126mmol/L, potassium 5.1mmol/L, chloride 88mmol/L, and bicarbonate 13mmol/L. Lactate was markedly elevated at 9.9mmol/L. Transaminases were 111 IU/L and 128 IU/L, alkaline phosphatase was 209 IU/L and total bilirubin was 4.10 mg/ dL. Triglycerides were greater than 1,000 mg/dL. Abdominal CT revealed hepatomegaly with fatty infiltrate. Medication toxicity was suspected and HAART therapy was discontinued. Riboflavin supplementation was initiated. IMPLICATIONS/DISCUSSION: Mitochondrial toxicity is a rare syndrome that has been reported in patients treated with nucleoside reverse transcriptase inhibitors, particularly zalcitabine, didanosine, and stavudine. The diagnosis is clinical. Patients present with nausea/ vomiting, abdominal pain, weight loss, malaise, fatigue, dyspnea and sometimes fever. Laboratory evaluation reveals lactic acidosis, hypertriglyceridemia and elevated liver enzymes. Imaging studies demonstrate hepatomegaly with fatty infiltration. The mechanism of mitochondrial injury is postulated to be inhibition of the mitochondrial gamma polymerase, the enzyme that directs the replication of mitochondrial DNA. The mitochondria are unable to replicate, the citric acid cycle is blocked and lactate production increases. Fatty acid oxidation is inhibited leading to accumulation of lipid droplets. Treatment involves the administration of mitochondrial supportive therapy such as riboflavin, thiamine, ubiquinone and acetyl-carnitine in addition to the cessation of antiretroviral therapy. Unrecognized, this syndrome carries a 60% mortality attributed to worsening acidosis with resulting cardiovascular collapse or progressive hepatic failure. (Tracking ID #76589) LEARNING OBJECTIVES: To recognize the dilemma posed by an inconclusive MRI in cases of intracerebral mucormycosis and to emphasize the need for brain biopsy. CASE INFORMATION: A 42 year-old male with HIV presented with a five-day history of headache, fever, left sided weakness and multiple falls. Physical exam showed a left sided, central, facial nerve palsy, weakness and left sided hyperreflexia. The patient's CD4 count was 56. Cryptococcus, toxopolasmosis, tuberculous meningitis, atypical infectious agents and lymphoma were considered in the working differential diagnosis. CT scanning and CSF analysis were negative. MRI revealed a mass in the right basal ganglia with associated edema and a midline shift. An MRI guided brain biopsy showed mucormycosis. In spite of amphotericin B therapy he expired ten days later. IMPLICATIONS/DISCUSSION: Mucormycosis in an immunocompromised host with HIV has rarely been described in the literature. Notably, in such cases neither CT nor MRI scanning is sufficient to make the diagnosis. In these instances, surgical exploration is mandatory and can provide crucial diagnostic information.
DOWN BUT NOT OUT. S. Agresta 1 , J. Canterbury 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77016) LEARNING OBJECTIVES: 1. Recognize an under-diagnosed cause of rhabdomyolysis. CASE INFORMATION: A 55 year-old obese man presented with severe shortness of breath after being found at his nursing home. His morbid obesity (550 pounds) confined him to a wheelchair. On the evening prior to admission, he fell from his wheelchair without a loss of consciousness or traumatic injury. A nursing aid found him twelve hours later. At presentation, he complained of diffuse pain; he noted associated shortness of breath but no chest pain or palpitations. His heart and breath sounds were normal; pitting edema extended to the knees. His vital signs were normal. His urine was scant (less than 10 cc/hr) and dark brown in color. His CPK was 35,000 U/L, BUN 75 mg/dL and creatinine 2.1 mg/dL. IMPLICATIONS/DISCUSSION: Although rhabdomyolysis is usually seen with crush injuries or toxic myopathies, prolonged stasis and extended isometric contraction as occurs with seizures should also prompt immediate evaluation for the syndrome. A timely diagnosis is essential, as the degree of renal tubular necrosis is proportional to the time and duration of tubular exposure to the myoglobin pigment. Aggressive diuresis decreases this exposure time; this is the main focus of treatment. The color of the urine (brown as opposed to yellow or clear) predicts the pigment concentration in the tubules, and is a better measure of severity and treatment efficacy than the CPK level. Early diuresis, with or without alkalinization, should be maintained until the urine is consistently clear. Acute renal failure is often attributed to the primary disease (sepsis, MI) in patients found down. Rhabdomyolysis should be excluded as an independent cause of renal failure in all patients found down for an undetermined or prolonged period of time.
LISTEN TO ME, NOT MY THYROID. E. Chan 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77077) LEARNING OBJECTIVES: 1. Listen to the patient's previous history to establish appropriate management decisions. 2. Use the history of the patient's symptoms to distinguish Graves disease from propylthiouracil allergy. CASE INFORMATION: A 23 year-old woman presented with recurrent palpitations, muscle weakness and dyspnea. She had a past history of Graves disease but had refused propylthiouracil therapy. She had a heart rate of 130, and a temperature of 104 F. There was right eye exophthalmos, a diffusely enlarged thyroid. A venous hum was heard over the thyroid; she had proximal muscle weakness and hyperreflexia. The thyrotropin levelwas 0.06U/ ml and the FT4 was 17.01 g/dl. She was treated with with potassium iodide, propylthiouracil and propranolol with resoluton of her symptoms. Three days later, she developed severe generalized arthralgias and myalgias with progressively deteriorating muscle strength and swelling of her fingers. Her anti-nuclear antibody, anti-histone antibody, and acetylcholine receptor antibody were negative. The anti-striated muscle antibody, rheumatoid factor and p-ANCA were also negative. The propylthiouracil was discontinued based upon the hypothesis that the new symptoms were due to an allergic response, Her symptoms resolved the following day. Upon further questioning, she noted a similar reaction to a previous treatement with propythiouracil as the cause of her non-compliance with this therap. A subtotal thyroidectomy, resulted in complete resolution of her symptoms. IMPLICATIONS/DISCUSSION: The appropriate management decision is often obtained by listening to the patient. In the thrill of treating Graves's disease, the reason for the patient's previous non-compliance with propylthiouracil was ignored. Although an allergic reaction to propylthiouracil is rare, it is characterized by arthralgias, myalgias, anxiety and tachycardia when it occurs. Distinguishing this reaction from continued symptoms of Graves's disease requires a meticulous history. Thyroidectomy is the treatment of choice in patients who are unable to tolerate propylthiouracil therapy. Carefully listening to why patients have declined previous therapy is important in preventing recurrence of previous adverse effects.
included rolling and lifting weights on an exercise ball. After returning home he became nauseated, diaphoretic, weak, and experienced excruciating left flank pain followed by an episode of watery diarrhea. His symptoms worsened and he presented to the emergency room the following morning. Physical examination at that time revealed a well-nourished man in marked distress secondary to intractable pain. There was significant tenderness to minimal palpation and percussion in his lower left quadrant and over his left costovertebral angle region. An initial computerized tomography (CT) of the abdomen showed an insignificant left renal cyst. Laboratory evaluation was significant for leukocytosis with a left shift, mild normocytic anemia, slightly elevated creatinine and CPK, and urine which showed pyuria but was sterile. Overnight he experienced fever and worsening pain with apparent peritoneal signs. The next morning a chest x-ray was done which showed no evidence of pneumonia. CT of the abdomen was repeated. In view of the patient's declining condition, this subsequent CT was read as a possible abscess. Aspiration of the renal abscess grew Streptococcus pneumoniae, as did all blood cultures. Predisposing factors for pneumococcal bacteremia were excluded. He recovered fully after a two-week course of ceftriaxone and four weeks of levofloxacin. IMPLICATIONS/DISCUSSION: Streptococcus pneumoniae is a very uncommon pathogen in renal abscesses. Extrapulmonary pneumococcal infection is most often metastatic from the lung. No respiratory disease was noted in our patient. Only two published cases of Streptococcus pneumoniae renal abscesses have been reported. These will be discussed, one in full detail. The etiology of our patient's Streptococcus pneumoniae continues to be a mystery. Two hypotheses will be presented. LEARNING OBJECTIVES: Often uncommon diseases present initially in the primary care setting, and appropriate referral and diagnosis are essential. The following case illustrates an unusual skin condition seen in a primary care setting in order to expand the generalist physician's differential diagnosis of dermatologic conditions associated with renal failure. CASE INFORMATION: The patient is a 36-year-old woman with a history of type I diabetes complicated by renal failure for which she underwent a kidney-pancreas transplant ten years ago. Her transplanted kidney remained functional until three months prior to presentation, at which time hemodialysis (HD) was initiated. Within one month of starting HD, she developed thickening and hardening of the skin on her left hand, which rapidly expanded to involve the entire left arm, and the right hand and arm. Within two months, the feet, lower legs, buttocks, and areas of the back had areas of skin hardening. The face, abdomen, and chest were spared. These areas of skin hardening were associated with minimal overlying skin changes, but she developed burning paresthesias during this time for which she was treated with oxcarbazepine. A skin biopsy confirmed the diagnosis of nephrogenic sclerosing dermopathy (NSD), with the findings of increased numbers of dermal spindle cells and thick collagen bundles in the reticular dermis. IMPLICATIONS/DISCUSSION: NSD was first described in September 2000 in a case series of 14 patients with a fibrotic skin condition resembling scleromyxedema. Each patient had a history of renal failure and 12 of the patients were receiving HD at the time of diagnosis. Patients displayed skin thickening and hardening with overlying brawny skin discoloration, which was localized to the torso and limbs with sparing of the face. Several patients complained of painful paresthesias beginning with the onset of skin hardening. In each case, the skin condition has been progressive with no known effective therapy. The etiology of NSD is unclear, but infectious agents or toxic agents (toxins in the dialysate, endogenous toxins not cleared by dialysis) have been proposed. Resolution of the skin lesions occurred in one case when HD was stopped following resolution of acute tubular necrosis. Through accurate referral and diagnosis of patients, more can be learned about the etiology, prevention, and treatment of NSD. Early recognition of NSD may allow early referral for transplantation to prevent progression of disease on HD, although there is not definitive evidence to date that NSD regresses following transplantation. A 58 year old Mexican man presented with progressive shortness of breath and a dry cough for over 1 month. Review of systems was positive for dyspnea on exertion, three pillow orthopnea, and an eight pound weight loss. His past history is noncontributory, and he uses no medication, tobacco or alcohol. On physical exam breath sounds were decreased with bilateral rales 1/2 way up. He was tachycardic with a loud P2, a jugular venous pressure of 10cm and 2+ edema to the shins. Chest x-ray showed bilateral pleural effusions; right greater than left. Echocardiogram revealed an ejection fraction of 70% with pulmonary hypertension. Laboratories were significant for a normocytic anemia with low iron, TIBC and reticulocyte count; ESR 116; urinalysis with trace protein and 20 red cells; and negative serologies for hepatitis B/C, HIV, and coccidiodes. CT chest was negative for pulmonary embolism and infiltrate. Diagnostic thoracentesis removed 200cc of hazy fluid with pH 7.48, 1250 white cells (81% lymphocytes) and cultures negative for bacteria, fungi and acid fast bacilli (AFB). Cytology was negative for malignant cells. Rapid reaccumulation later prompted removal of another 1L of fluid. A tuberculin skin test was 8 mm but sputum AFBs were negative. Further work-up revealed a positive ANA (1:2560 titer) and strongly positive anti-Smith antibody with negative anti-dsDNA and ANCA. He was diagnosed with SLE and started on prednisone 40 mg daily with resolution of the effusion and improvement in pulmonary hypertension. IMPLICATIONS/DISCUSSION: Pleural effusion is the initial manifestation in 1%, and pleurisy in 3%, of SLE patients, more often in those with late-onset (after age 50). Lupus pleuritis usually presents as pleuritic chest pain with fever, cough and dyspnea but rarely with massive effusions. Pleural symptoms may precede other manifestations of SLE by months or years, particularly in men. To diagnose pleural effusions caused by lupus pleuritis, one must rule out other causes including pulmonary embolism, congestive heart failure, infections (i.e. parapneumonic or tuberculous), and malignancy. Effusion ANA titer detection is a very sensitive but nonspecific test while anti-DNA antibodies or LE cells in the pleural fluid are diagnostic but not very sensitive. Treatment includes NSAIDs for mild cases and systemic corticosteroids for more severe cases. Early diagnosis and prompt treatment can greatly decrease morbidity from this disease. year old man presents with complaints of progressive leg swelling, erythema, 30 lb weight loss, and severe bilateral hip pain over 3 months. He initially noted several fluid filled blisters and pedal edema that spontaneously resolved. Over the next several weeks he described progressive bilateral hip pain requiring the use of crutches for ambulation. The month prior to admission the patient was bed bound secondary to pain. He denied other constitutional symptoms. Of note, he had eliminated fruits and vegetables from his diet 3 years ago. The patient had emigrated from Ethiopia 35 years ago. Physical exam revealed poor dentition, a perifollicular petechial rash of his lower extremities, hyperpigmentation of his upper thighs and bilateral lower extremitity edema. MRI was significant for lower extremity soft tissue edema and osteoporotic femoral heads with abnormal marrow signal. Laboratory values were notable for a normocytic anemia, normal coagulation panel, and electrolyte panel and a Vitamin C level of <0.12 mg/dL. Punch biopsy of the petechial lesions demonstrated perifollicular hemorrhage and corkscrew hair follicles. Vitamin C supplemenation 1000mg per day was initiated with resolution of symptoms over several weeks. Repeat MRI of his LE noted resolution of soft tissue edema and improving osteoporosis. IMPLICATIONS/DISCUSSION: Symptoms of scurvy have been described as early as 1500 BC by the ancient Egyptians. In contemporary history, epidemics have been described in the Renaissance era, when sailors were frequently affected, and during the 19th century, by those who experienced the Great Potato Famine, American Civil War armies, and the California Gold Rush communities. A cofactor and strong reducing agent, vitamin C is crucial to numerous biochemical pathways including collagen synthesis, norepinephrine synthesis, prostaglandin synthesis, and antioxidant function. Collagen synthesis disruption in particular leads to the clinical syndrome of scurvy including ecchymoses, bleeding gums, petechiae, and arthralgias. Other signs and symptoms include muscle pain, microcytic anemia, decreased wound healing, mood disturbances, and anorexia. While scurvy occurs rarely in the US, typically it is seen in elderly or alcoholic patients and those with diets devoid of fruits and vegetables. Sudden cardiac death has been reported in these patients however, morbidity and mortality largely occurs as a result of hemorrhage which can occur in any organ. Skin biopsy findings of perifollicular hemorrhage and follicle corkscrewing are pathopneumonic for the disorder. Symptoms typically improve over days to weeks with the addition of Vitamin C to the diet. Prognosis is excellent with timely diagnosis and treatment.
NOT JUST ANOTHER DIARRHEA: A CASE OF SECONDARY AMYLOIDOSIS ASSOCIATED WITH LYMPHOMA. J. Chowdhury 1 , A. Gomez 1 ; 1 UCLA San Fernando Valley, Sylmar, CA (Tracking ID #76168) LEARNING OBJECTIVES: 1) Recognize lymphoma as a chronic inflammatory state that can be associated with secondary amyloidosis 2) Distinguish between primary and secondary amyloidosis. CASE INFORMATION: A 44 year old Hispanic male with non-Hodgkin's lymphoma, diagnosed and treated in Mexico with chemotherapy and radiation five years ago presents with five months of large volume, watery diarrhea. He had four to six episodes of blood-tinged diarrhea per day, with abdominal cramps, nausea and vomiting. The patient had anorexia with a twenty pound weight loss, orthopnea and bilateral lower extremity edema. His blood pressure was 60/palp, pulse 88, and he was afebrile. He had dullness to percussion and decreased breath sounds over the left lower lobe as well as 1+ pitting edema in BUE and 2+ pitting edema in the BLE. A CBC was normal, BUN 38, creatinine 1.3, PT 29.6, INR 2.47, PTT 47, albumin 1.4, AST 10, ALT 13, alkaline phosphatase 153, total bili <0.1, LDH 762. Urinalysis showed small blood, 3+ proteinuria, 2 rbc's and 3 wbc's. CXR showed a left-sided pleural effusion. The patient was admitted to the ICU and started on IV steroids and pressors. Thoracentesis revealed a transudate with many lymphocytes, but no malignant cells. CT scans revealed a mass in the left upper lobe of the lung and left hemithorax volume loss, colon dilation to 6 cm and small bowel dilation to 3 cm. Biopsy of the lung mass revealed Hodgkin's lymphoma. Bone marrow biopsy was positive for both crystal violet and Congo Red, suggesting amyloidosis. Rectal biopsy showed friable mucosa and revealed apple green birefringency when viewed with Congo Red stain, confirming the diagnosis of gastrointestinal amyloidosis. Unfortunately he succumbed to the disease a week later while on steroids and chemotherapy. IMPLICATIONS/DISCUSSION: Both primary and secondary amyloidosis present in a variety of ways, including heavy proteinuria, edema, hepatosplenomegaly, hematochezia, congestive heart failure, and carpal tunnel syndrome. Secondary, or AA, amyloidosis occurs in chronic inflammatory states, such as rheumatoid arthritis, inflammatory bowel disease, or osteomyelitis. However, reports of an association with lymphoma are rare. Obtaining a good past history for chronic inflammation is nevertheless important when diagnosing AA amyloidosis. The only disease linked to primary, or AL, amyloidosis is multiple myeloma. Once the diagnosis of amyloidosis is made from tissue biopsy, the distinction between primary and secondary amyloidosis is made based on the presence of a chronic inflammatory state in secondary amyloidosis or the presence of certain paraproteins, found in about 90% of primary amyloidosis. We diagnosed this patient as secondary amyloidosis based on his history of lymphoma.
``MY TOOTH IS KILLING ME!'': OVER THE COUNTER ANALGESIC RISKS AND HEPATITIS C. E. Coffey 1 , E. Schneider 1 ; 1 Hennepin County Medical Center, Minneapolis, MN (Tracking ID #76561) LEARNING OBJECTIVES: 1. Recognize increased risks of acetaminophen and nonsteroidal antiinflammatory drugs (NSAIDs) for patients with Hepatitis C. 2. Recognize importance of adequate medical and medication history, even for common complaints. CASE INFORMATION: 50 year old male presented to a walk-in clinic seeking medication for a two week old toothache. Nonprescription pills at home didn't help. He also reported 2 days of nausea, vomiting, loose bowel movements, diffuse myalgias, generalized weakness. He denied alcohol, infectious contacts. PMH included hepatitis C, alcohol and cocaine use. Physical exam remarkable only for an ill appearing male with an abscessed tooth and mild abdominal tenderness. Labs included K 3.8, BUN 32, creatinine 2.9, ALT 3460, AST 2575, bilirubin 5.1, alk p'tase 135, lipase 111, INR 1.4. Acetaminophen level 52. Salicylate level was not elevated. He later admitted to taking up to 20 tablets of naproxen and acetaminophen a day over the last few days. He was admitted to the medicine service, and improved with acetylcysteine and supportive care. He was counselled about acetaminophen use. A dentist extracted the abscessed tooth before discharge. IMPLICATIONS/DISCUSSION: Acetaminophen and NSAIDs are associated with increased transaminases in patients with hepatitis C. Renal failure has been associated with NSAIDs and with paracetamol poisoning.This case illustrates that the presenting problem isn't always the most serious. The non-pain symptoms, past history and specific information on his self medication were keys to diagnosis. Given potential toxicities of analgesics, patients with hepatitis C are well advised to seek advice for pain issues rather than self medicating.
DIAGNOSIS AND TREATMENT OF POST PRANDIAL NAUSEA, ALLERGY AND EOSINOPHILIA. D. Coleman 1 , J.S. Scolapio 1 , J.R. Cangemi 1 , J.C. Guarderas 1 ; 1 Mayo Clinic Jacksonville, Jacksonville, FL (Tracking ID #76262) LEARNING OBJECTIVES: Eosinophilic gastroenteritis, is a disorder characterized by infiltration of eosinophils into the mucosa of the gastrointestinal tract. As a clinical entity with recognizable symptoms it is a rare disorder. Here we discuss the extensive treatment course of a refractory case of eosinophilic gastroenteritis. CASE INFORMATION: A 16-year-old male patient who presented with post-prandial fullness and nausea. He had a history of seasonal allergies, asthma, and peripheral eosinophilia. Endoscopy of the stomach with mucosal biopsies revealed predominate eosinophils. A diagnosis of eosinophilic gastroenteritis was made. He was treated with a proton pump inhibitor (Prilosec) an oral cromolyn (for gastrointestinal symptoms), initially. After no response, he began on high dose prednisone with a gradual taper, with clinical response, to every other day prednisone indefinitely. Several years later, he again experienced symptoms of dysphagia, early satiety, nausea and occasional vomiting. He had been compliant with his medications. The oral steroids (20 mg every other day) had not been tapered because of recurrent symptoms upon dose reduction. As a result of his prolonged predisone use, bone mineral analysis was performed and showed signs of early osteoporosis. He was treated with Fosamax 5 mg a day, vitmain D 50,000 IU twice a week, and Calcium 1000 mg a day. A trial of an elemental nutrition formula was attempted in hopes of reducing his gastrointestinal eosinophilia. Although his peripheral eosinophil count significantly decreased on this elemental diet (Vivonex) he was unable to continue with it because of its poor taste. Given his refractory symptoms and continued risk of complications from chronic steroid use he was treated with Interferon Alpha, starting at a low dose and gradually increased to a dose of 1 million units per day. His peripheral blood eosinophils initially dropped from 5410 to 1020 but then slowly began to increase. In early 2002 his gastrointestinal symptoms recurred and his peripheral blood eosinophils began to rise. He had been compliant with medications. Because the majority of his symptoms were gastroesophageal, we elected to try oral flonase. Since this newest therapy was begun, he has experienced a marked drop in peripheral blood eosinophils (the lowest to date) and his gastrointestinal symptoms have markedly improved. IMPLICATIONS/DISCUSSION: This case is unique because it illustrates an unusual approach to a refractory case of eosinophilic gastroenteritis. Most known and reported therapies had failed in our patient with the exception of high dose steroids and, more recently, oral Flonase. Unfortunately his dependence upon corticosteroids resulted in the serious sequelae of osteoporosis. Although a systemic illness with marked peripheral eosinophils, the symptomatology of this patient was predominantly gastrointestinal. RAPIDLY PROGRESSIVE HEPATOSPLENOMEGALY. K. Cooke 1 , S. Rusch 1 ; 1 University of Illinois, Peoria, IL (Tracking ID #76040) LEARNING OBJECTIVES: Discuss the differential diagnosis of rapidly progressive hepatosplenomegaly while highlighting a typical presentation of an uncommon lymphoma. CASE INFORMATION: A previously healthy 32-year-old woman initially presented to her primary care physician with pleuritic left shoulder pain and a normal physical exam. Laboratory results then included platelets of 104,000/L; alkaline phosphatase of 125 U/L; alanine aminotransferase of 58 U/L; and aspartate aminotransferase of 65 U/L. Two weeks later, the patient returned with acute onset of fevers, fatigue and nausea. Physical examination was benign except for marked hepatosplenomegaly. The liver was palpable 11cm below the right costal margin and the spleen 5cm below the left costal margin. Laboratory results included platelets of 42,000/L; alkaline phosphatase of 1,045 U/L; alanine aminotransferase of 285 U/L; and aspartate aminotransferase of 610 U/L. Admission computed tomography showed diffuse homogenous enlargement of the liver, spleen and kidney with a few mildly enlarged retroperitoneal lymph nodes. Serologic evaluation was negative for hepatitis A, B and C viruses, cytomegalovirus, Epstein Barr virus, herpes simplex virus, toxoplasmosis and human immunodeficiency virus. Bone marrow biopsy on day two of admission was negative for malignancy, infection and amyloid. Screening for antinuclear antibody and extractable nuclear antigen antibodies was negative. The patient's history did not reveal risk factors for unusual infections or toxins. Noninvasive doppler ultrasound was not consistent with Budd Chiari. Blood cultures were negative and alpha-fetoprotein level was within normal limits. During the ensuing four days, the patient rapidly deteriorated with additional liver enlargement of 4cm, falling platelet count and metabolic acidosis. On day five of admission, a transjugular liver biopsy demonstrated alphabeta-hepatosplenic T-cell lymphoma. IMPLICATIONS/DISCUSSION: Rapidly progressive hepatosplenomegaly is not widely discussed in the literature. However, reviewing MEDLINE and EMBASE databases, one can conclude that it is most common in hematologic malignancies and infection. The above typical presentation of an uncommon peripheral T-cell lymphoma illustrates that lymphoma can present without lymphadenopathy or bone marrow involvement. Hepatosplenic T-cell lymphoma is an aggressive cancer with a poor prognosis. Despite aggressive treatment, our patient died on the 28th hospital day.
A PAIR OF PAINFUL HANDS. A. Cooper 1 ; 1 University of Pennsylvania, Philadelphia, PA (Tracking ID #73721) LEARNING OBJECTIVES: 1. To review the American College of Rheumatology diagnostic criteria for rheumatoid arthritis (RA). 2. To discuss initial treatment of newly diagnosed RA. CASE INFORMATION: A 39 y/o homeless woman with a past medical history significant for hypertension, diabetes, hepatitis C, and asthma on no medications was admitted to the inpatient psychiatric unit for depression with suicidal intent. The medicine service was consulted for evaluation of hand pain. The patient had been having 2 months of progressively worse pain in both her hands, as well as 1 month of swelling and morning stiffness lasting 90 minutes before improvement. The symptoms made it difficult for her to make a fist or hold her cigarette. Over the last 1 week, all of her symptoms markedly increased. Additionally, she recently noted a sensation of numbness and tingling in her first three fingertips of the right hand that wakes her from sleep. The patient denied any fevers, chills, night sweats or other joint involvement. She denied dry eyes or mouth, as well as urethritis/vaginitis. Review of systems in detail was otherwise negative. Social history was significant for a 20 pack year smoking history. Family history was noncontributory. Physical exam was significant for bilateral symmetric swelling, mild erythema and boggy tenderness of 8 proximal interphalangeal joints. There was no involvement of the distal interphalangeal joints or thumbs and minimal tenderness of the MCP joints. There was no wrist tenderness. Remainder of the joint exam was unremarkable. Remainder of the physical exam was significant for a blood pressure of 130/90, mild expiratory wheezes bilaterally at the lung bases, and a positive Phalen's sign. Pertinant negatives included a temperature of 97.9, moist mucous membranes, no cardiac murmurs, no rashes or nodules and a negative Tinel's sign. Laboratory results were significant for normal electrolytes, renal function, TSH, WBC and hemoglobin levels. The ESR was normal at 9. Anti-Scl 70, dsDNA, and complement levels were normal. The ANA was weakly positive at 1:160. The patient had a positive RF of 104 IU/mL (normal 0±29 IU/mL). Parvovirus IgM was negative, as were Hep B serologies and HIV. IMPLICATIONS/DISCUSSION: Discussion: This patient was diagnosed with early moderately severe RA. The American College of Rheumatology criteria for diagnosis of RA were initially developed to help classify patients for clinical studies. However, they have proved useful in diagnosis as well. The guidelines are as follows: A patient has RA if at least 4 of these criteria are met (the 1st 4 of which must have been present at least 6 weeks): 1) Morning stiffness, usually lasting at least 1 hour before maximal improvement. 2) Arthritis of at least 3 or more joint areas. 3) Arthritis of hand joints. 4) Symmetric arthritis. 5) Rheumatoid nodules. 6) Serum rheumatoid factor. 7) Radiographic changes±erosions, unequivocal bony decalcification adjacent to affected joints. Radiographic changes are present in 15% of patients in the 1st year of disease, in >90% of patients after the 1st 2 years. The initial diagnosis of RA may be difficult because early RA may satisfy few of the diagnostic criteria. Additionally, systemic lupus erythematosis, Sjogren's syndrome, and mixed connective tissue disease may also commonly present with a positive rheumatoid factor, and may be difficult to differentiate from RA early in the course of illness. Finally, seronegative RA, or RA presenting in a single large joint such as the knee, poses it's own obvious diagnostic dilemma. The diagnosis of RA is supported by findings of carpal tunnel syndrome, as seen in this patient, as well as weight loss, fever, anemia, serositis, pulmonary nodules and peripheral neuropathy. Therapy for RA is usually initiated with non-steroidal antiinflammatory drugs (NSAIDs). Prednisone is added for rapid improvement when severe joint symptoms are present, and is usually tapered after inflammation is controlled. Disease modifying anti-rheumatic agents (DMARDs) are added if symptoms are not controlled with NSAIDs, if symptoms are moderate or severe in nature, or if radiographic changes are present. DMARDS are medications that have been proven to reduce the rate of bony destruction in RA. The most common DMARD used for initial treatment is methotrexate, started at a dose of 7.5 mg/week. The maximum dose of methotrexate is 25 mg subcutaneously/week. It is important to monitor patients for methotrexate toxiticy including liver function tests (cirrhosis), pulmonary symptoms (interstitial lung disease), and bone marrow toxicity. LEARNING OBJECTIVES: 1) Apply epidemiologic factors to decisions of whether to screen for hepatocellular carcinoma (HCC) in chronically Hepatitis B-infected individuals. 2) Recognize the limitations of current screening modalities for HCC. CASE INFORMATION: An asymptomatic 23 year-old male student presents to clinic after being told by a former sexual partner that he had``given her an infection.'' He has no significant past medical or surgical history. His review of systems is negative. He was born in China and immigrated to the United States at two years of age. He uses a half-pack-per-day of tobacco, alcohol to intoxication 1±2 times per week, no recreational drug use. His physical examination is normal. His laboratory values are significant for being hepatitis B (HBV) surface antigen positive, surface antibody negative, core antibody positive, e antigen negative, and HBV viral load undetectable. Hepatitis C and Human Immunodeficiency Virus (HIV) are negative. His liver function tests are all within normal limits.
Observational studies show that among perinatally-infected individuals, incidence of HCC begins rising in young adulthood (20±30 years of age). Among those who are infected later in life the incidence of HCC begins rising at 40 to 50 years of age. Other major risk factors for HCC include presence of cirrhosis, e-antigenemia, exposure to other hepatotoxins including alcohol and tobacco, and co-infection with hepatitis C virus. HCC screening is usually done at 6±12 month intervals. Common modalities used include serum alpha fetal protein (AFP), ultrasound (US), and computed tomography (CT). The usefulness of AFP is limited by sensitivity (approximately 60%) and specificity (approximately 85%). Thus, US or CT must be used in conjunction with AFP. Ultrasound, the most widely-used modality, has a variable sensitivity determined by operator experience, reported to be 35% to 84%. CT, with less operator-dependent variability and sensitivity greater than 90%, is the most expensive of screening modalities. Given the limitations of existing screening methods and overall small survival-benefits of current widely-available treatments, the cost-effectiveness of HCC screening has been questioned. There are no randomized studies assessing cost-effectiveness of HCC screening. Non-randomized studies have shown conflicting results. Thus, although a recent survey of U.S. hepatologists showed that 84% of respondents screen for HCC in individuals with cirrhosis, no official guidelines have yet been provided by the National Cancer Institute (NCI).
ALONG CAME A SPIDER . . . . . . CAUSING UNUSUAL ULCERS. N. Correia 1 , R. Abou Jawde 1 , S. Baghdasarian 1 , S. Schmitt 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #76813) LEARNING OBJECTIVES: To emphasize the extensive differential diagnosis of lower extremity (LE) ulcers, the incidence of pyoderma gangrenosum (PG) following trauma and the importance of accurate diagnosis, we present a case in which lesions that appeared following a spider bite were intially treated as infection. CASE INFORMATION: A 59 year old man presented with non-healing painful left LE ulcers. He was bitten by a spider after which he noted a small red tender spot over his shin that progressed to an ulcer with purulent drainage. He was treated with oral and intravenous antibiotics, local wound care and surgical debridement without improvement. During treatment he developed a new ulcer on the same leg and an inflamed nodule over the contralateral malleolus. IMPLICATIONS/DISCUSSION: PG, a deforming ulcerative skin disease, usually presents with erythematous nodules or vesiculopustules on the legs. The lesions undergo a rapidly destructive necrotizing process to form large ulcers with surrounding erythema. Although frequently associated with inflammatory bowel disease or immunosuppression, in adults PG can occur spontaneously, following trauma (including spider bites), or in association systemic diseases. Biopsy confirmed the presence of PG in our patient and he improved dramatically on prednisone.
SARCOIDOSIS PRESENTING AS TESTICULAR MASS: CASE REPORT AND REVIEW OF THE LITERATURE. L. Corsino-Nunez 1 , M. Kam 1 , C. Kaye 1 ; 1 Wayne State University, Detroit, MI (Tracking ID #75657) LEARNING OBJECTIVES: To recognize sarcoid presenting as a testicular mass. CASE INFORMATION: Sarcoidosis a chronic multisystem disease of unknown etiology characterized by noncaseating granulomas. The most commonly involved organs are the lungs, lymph nodes, skin, uvea, liver and bone. Sarcoidosis of the male urogenital tract is uncommon. Physicians need to be aware of this occurrence especially in males with a known history of sarcoidosis. Case: A 31 y/o African American male presented to the Emergency Department complaining of dyspnea, fever and night sweats. Past medical history was significant for asthma. Admission physical exam was remarkable for bilateral, nontender, mobile, cervical lymph nodes and a right testicular mass. Chest-x-ray revealed mediastinal and bilateral hilar lymphadenopathy. Computarized Axial Tomography scan of the chest, abdomen, and pelvis was consistent for bilateral lymph nodes in the axillary and inguinal region. Ultrasonography of the right testicule was done and showed marked enlargement of the right epididymus and small hydrocele. Biopsy of one of the submandibular lymph node revealed non-caseating granulomas. A right orchiectomy was performed. The pathology report was consistent with non-caseating granuloma involving the epidiymis and focal testicular parenchyma. The spermatic cord was free of granulomatous inflammation. IMPLICATIONS/DISCUSSION: Discussion: Sarcoidosis with testicular involvement is rare. Only 0.2% of patients with sarcoidosis have urogenital involvement. Forty-three cases have been described in the English language medical literature. We want to enhance physicians' awareness of urogenital sarcoidosis and include in the differential diagnosis of a palpable testicular mass. The treatment for urogenital sarcoid is corticosteroids, identical to systemic sarcoid. Review of the literature shows that a great unnecessary percentage of patients with testicular sarcoid have undergone orchiectomy. Thus awareness of this condition may result in prevention of this radical approach in males who present with sarcoidosis and a testicular mass. LEARNING OBJECTIVES: To recognize Pott±Puffy Tumor as a complication of sinusitis. CASE INFORMATION: Pott±Puffy Tumor is defined as subperiostial abscess and osteomyelitis of the frontal bone seen as a complication of frontal sinusitis. With the advent of antibiotics, Pott±Puffy Tumor is a rarely recognized entity. A 31 y/o white female, previously healthy until one day prior to admission, presented with frontal headaches, dull in nature that progressively worsened, associated with remarkable forehead swelling, and dizziness. One week prior to admission she received a two-week course of cefuroxime for frontal sinusitis. Her past medical history was significant for fibrodysplasia of the ethmoidal sinus during childhood requiring five surgical interventions. Admission physical exam was remarkable for forehead swelling with tenderness over the frontal bone and absence of fever. Computerized axial tomography scan of the head revealed opacification of the frontal sinus. The frontal sinus was drained and a purulent discharge was obtained. Gram stain and culture showed no organisms or growth. Frontal headache and swelling improved after drainage. She was placed on ampicillin/sulbactam for a total of two months. She was scheduled for frontal sinus obliteration after completing the antibiotics. IMPLICATIONS/DISCUSSION: Pott±Puffy tumor represents a rare complication of frontal sinusitis. Only 21 cases have been reported after the antibiotic era, of wish 7 have presented in adults. The importance of making this diagnosis is to prevent expansion of the infection to the brain. We would like to highlight the importance of making an accurate diagnosis, especially in patients treated for frontal sinusitis. Five cases in which no organisms have been identified are reported in the literature. This entity can be successfully treated with antibiotics and drainage. LEARNING OBJECTIVES: Recognize the importance of secondary causes for hypertension. CASE INFORMATION: A 51 year old diabetic gentleman with obstructive sleep apnea treated with CPAP and a long-standing history of hypertension came to our clinic for a routine visit. Despite five anti-hypertensive medications, blood pressures remained near 180/100. This prompted a search for secondary hypertension. Studies were negative for renal vascular disease and pheochromocytoma as well as primary hyperaldosteronism. Despite preserved renal function, Nephrology consultation was obtained with the resulting suggestion that this may be a case of occult noncompliance. However, the patient regularly attended appointments and filled all of his antihypertensives in a timely manner. This convinced us to look further into additional possible rare causes for secondary hypertension. On one subsequent follow-up visit, the patient did complain of jaw changes and a thick tongue. He has coarse facial features, a large fleshy nose and large tongue along with multiple skin tags and large hands. The constellation of hypertension, sleep apnea, diabetes, and a documented history of carpal tunnel syndrome, along with his complaints and findings raised the possibility of acromegaly as a reason for refractory hypertension. For the initial workup, a screening insulinlike growth factor 1 level was then ordered, which returned at 2.5 times the normal range. A subsequent confirmatory glucose tolerance test with measurement of growth hormone levels then failed to show GH suppression with a glucose load, supporting the diagnosis of acromegaly. IMPLICATIONS/DISCUSSION: Acromegaly is a somatic growth and proportion disorder with elevated levels of GH and ILGF-1 found as hallmarks of this syndrome. It is most often caused by GH-secreting pituitary tumors. Some of its features include skeletal overgrowth, arthropathy, skin changes, cardiovascular disease and respiratory dysfunction. Hypertension is one important complication of acromegaly, with the prevalence of hypertension in acromegalic patients estimated to be about 35%, although there is no consensus in the medical literature regarding the exact mechanisms involved in its pathogenesis. While hypertension is a common condition treated by internists, fewer than 5% of cases are found to have secondary causes. This case highlights the need to consider rare causes of hypertension in patients resistant to treatment, and demonstrates why acromegaly should be considered in the differential.
THE TUMOR THAT MELTED AWAY. J. Cunningham 1 , M. Panda 1 ; 1 University of Tennessee±Chattanooga Unit, Chattanooga, TN (Tracking ID #76984) LEARNING OBJECTIVES: 1. To recognize the similarity in the radiographical and clinical presentation of pulmonary blastomycosis and neoplasms. 2. Stress the importance of a high index of suspicion to avoid misdiagnosis and unnecessary invasive diagnostic procedures. CASE INFORMATION: 57 year old white female with a 40 pack/year history of tobacco abuse presented with persistent cough, mucoid and occasionally bloody sputum for 2 months. She denied fever, chills, night sweats, dyspnea, or recent travel. Her vital signs and room air oxygenation were normal. Physical exam revealed diffusely diminished breath sounds with no accessory muscle use or clubbing. Labs were normal except for a normocytic anemia, and thrombocytosis. CXR showed a right upper lobe infiltrate. CT revealed a large right hilar structure surrounding the distal main stem bronchus with impingement, infiltrative changes surrounding the mass, and an enlarged precarinal lymph node. An endobronchial biopsy and aspiration of the mass revealed no neoplastic cells. Gram stain was negative, and cultures revealed no fungal growth after 28 days. Aspiration smears from the precarinal area were nondiagnostic, but smears from the postcarinal area revealed Blastomyces dermatitidis. She was treated with Itraconazole with complete resolution on repeat CT scan after 6 months. IMPLICATIONS/DISCUSSION: Blastomycosis is a pyogranulomatous infection that can involve many organ systems, but primarily affects the lungs. Infection results from inhalation of the Blastomyces conidia, which may be asymptomatic or present in ways indistinguishable from TB, bacterial pneumonia, ARDS, histoplasmosis, and most ominously bronchogenic carcinoma. Most cases occur in areas surrounding main waterways in North America. Demographics indicate no specific sex, age, race, occupation or seasonal predilection, but exposure to soil is a common factor linking infections. Blastomycosis radiographically mimics many lung diseases with carcinoma being the most common primary diagnosis. Definitive diagnosis is made by isolation in sputum smears or growth in sputum culture which may take up to 30 days. Bronchoscopy requires special considerations in order to obtain a higher yield of fungus. Treatment with lidocaine inhibits growth & limiting concentration to 1g/dL has been recommended. B. dermatitidis can be missed on H&E specimens, so silver or PAS stains should be used. If no diagnosis results invasive thoracoscopy or thorocotomy are often done hastily before the fungus has had time to grow in culture because of the common presentation of Blastomycosis and carcinoma. Blastomycosis infection has been known as``the great masquerader'', therefore, it is very important for physicians to be familiar with the epidemiology in order to develop an index of suspicion for the fungus in endemic areas. Thus, patients can be spared from the distress of undergoing unnecessary testing and invasive surgical procedures. year old Latino male presented to the emergency room with a five day history of severe sharp, pulsatile left sided neck pain, worsened with exertion and head movement with flexion and lateral rotation. This neck pain waxed and waned in intensity throughout the five days prior to admission. The patient reported weakness and numbness of the left arm, which he had used while driving to the emergency room. This weakness resolved with lowering of the left arm. He denied having associated headaches or visual disturbances. The patient was afebrile with a blood pressure of 127/76. Physical examination revealed marked tenderness upon palpation over the left carotid artery. Carotid artery bruits were not present on auscultation. No thyroid tenderness, enlargement, or nodularity was evident. Funduscopic examination, evaluation of cranial nerve function, and assessment of motor and sensory function in the upper and lower extremities were within normal limits. The patient was admitted for treatment of possible left carotid artery dissection and anticoagulation therapy with unfractionated heparin was initiated. An MRI/MRA of the neck was subsequently performed to evaluate for carotid artery dissection. No abnormal flow voids or evidence of dissection were noted. A sonogram of the neck failed to reveal evidence of dissection. ESR was 5, CBC was within normal limits. The diagnosis of carotidynia was made, and the patient's symptoms resolved with prednisone and NSAIDS. IMPLICATIONS/DISCUSSION: Carotidynia is an idiopathic neck pain syndrome arising from the cervical carotid artery resulting in unilateral neck pain that frequently radiates to the ipsilateral face and ear and sometimes to the head. Facial pain is a common primary symptom and carotid artery tenderness and overlying soft tissue swelling are the major physical findings. Two forms of this disorder have been described, an acute form that results in a monophasic illness usually lasting less than two weeks and tending not to recur, and a chronic recurring form which manifests as a vascular headache variant, responsive to antimigraine therapy. Both forms are responsive to the administration of corticosteroids. An evaluation for structural disease of the carotid arteries should be considered in patients with ipsilateral neck pain. MRI/ MRA of the neck has proven highly sensitive. Other imaging modalities to consider include ultrasound and angiography. The diagnosis of carotidynia should be considered only after careful evaluation for other etiologies of neck pain. Considerations for ipsilateral neck pain in the region of the carotid artery near its bifurcation include carotid dissection, migraine headache, thyroiditis, aneurysm of the carotid system, neck neoplasms, or rarely, temporal arteritis.
AN UNUSUAL CAUSE OF CHEST PAIN: SPONTANEOUS PNEUMOTHORAX. S. Daud 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73682) LEARNING OBJECTIVES: 1) Recognize spontaneous pneumothorax as an etiology of chest pain. 2) Describe the typical presentation of spontaneous pneumothorax 3) Recognize the treatment options and prognosis of spontaneous pneumothorax. CASE INFORMATION: A previously healthy seventeen year-old male was seen in the emergency department for complaints of chest pain. His pain was described as a sharp pain above his nipples, bilaterally, which initially had occurred at rest. The pain was neither exacerbated nor relieved by movement or deep inspiration. There were no associated palpitations nor diaphoresis or vomiting. He was taking no medications, but smoked marijuana weekly for the past few months. He occasionally smoked cigarettes, but never consumed alcoholic beverages. There was no family history of coronary artery disease, hypercoagulable states, or vasculitis. Examination revealed a 5 H 11 HH young man in mild distress. His weight was 60 kg, his respiratory rate was 16, and his heart rate was 84. He had no oral lesions and had normal dentition. The cardiac exam revealed normal heart sounds and no murmurs. The lungs were clear to auscultation and had a normal percussion note. The abdomen was scaphoid and non-tender. There was no peripheral edema. Electrocardiogram and pulse oximetry were normal. A chest roentgenogram revealed bilateral apical pneumothoraces. They were estimated to be 10% each. Bilateral apical drainage catheters were placed and the patient's symptoms resolved. The patient was admitted to the hospital for observation. IMPLICATIONS/DISCUSSION: Primary spontaneous pneumothorax is defined as a pneumothorax without a precipitating factor such as trauma, in a patient without clinically evident lung disease. It typically occurs in tall, thin males between 10 and 30 years of age. The use of smoking tobacco has been reported to increase the risk in men by as much as a factor of 20 in a dose dependent manner. Subpleural bullae are found in virtually all patients; their mechanism of formation is not completely understood, but is thought to be related to degradation of elastic fibers in the lung. This process is thought to be accelerated by smoking. The primary spontaneous pneumonthorax results in a decrease in vital capacity, and an increase in the alveolar-arterial oxygen gradient, leading to hypoxemia. Because underlying lung function is normal, hypercapnia is not usually seen. Most patients present with ipsilateral pleuritic chest pain occurring at rest. Tachycardia is frequently the only abnormal physical finding. The diagnosis is confirmed by the identification of a thin, visceral pleural line displaced from the chest wall on chest roentgenography. Treatment with observation, needle aspiration, or drainage through a chest tube all have been advocated. The average rate of recurrence is about 30%, with pulmonary fibrosis, asthenic habitus, and history of smoking all thought to be risk factors for recurrence. (Tracking ID #73698) LEARNING OBJECTIVES: 1) Recognize the common causes and workup of diffuse pulmonary infiltrates. 2) Recognize the usual presentation of desquamative interstitial pneumonitis. CASE INFORMATION: A 45 year-old male with a history of COPD and multiple pneumonias within the past year was admitted to the hospital for complaints of cough productive of yellow sputum, fevers, chills, and worsening shortness of breath. He was found to be febrile and hypoxemic, and have a leukocytosis with a predominance of neutrophils. A chest x-ray showed the presence of diffuse interstitial infiltrates. He was admitted to the hospital and treated for pneumonia with antibiotics and bronchodilators for 11 days without significant improvement. Sputum and blood cultures were negative for bacterial pathogens. Computed tomography (CT) of the thorax showed ground-glass opacities in all lung fields, worse on the right. Pulmonary function testing revealed mild restrictive disease with decreased lung volumes and decreased DLCO. He did not improve clinically and was scheduled for open lung biopsy. Pathology revealed the diagnosis of desquamative interstitial pneumonitis. He was started on Prednisone 60mg daily, had significant clinical improvement and was discharged from the hospital. CT of the thorax performed one month later showed near-complete resolution of the ground-glass infiltrates. IMPLICATIONS/DISCUSSION: The differential diagnosis of a patient with diffuse pulmonary infiltrates is broad. Acute diffuse lung diseases (less than 4±6 weeks in duration) include infection, pulmonary edema, pulmonary hemorrhage, hypersensitivity pneumonitis, and acute eosinophilic pneumonia. Chronic diffuse lung diseases include idiopathic pulmonary fibrosis, sarcoidosis, pneumoconioses, and interstitial lung disease associated with connective tissue diseases. Desquamative interstitial pneumonitis (DIP) is one of the distinct clinical and histopathological entities belonging to the family of idiopathic interstitial pneumonias. The average age of onset is about 45 years of age, and a male preponderance has been noted. Cigarette smoking has been linked consistently to this diagnosis. The typical presentation is an insidious onset of dyspnea and cough. Chest roentgeography often shows a diffuse pattern of reticulonodular opacities, with CT showing a ground-glass appearance. Pulmonary function testing shows a restrictive pattern. Diagnosis is best made with lung biopsy demonstrating the characteristic appearance of alveolar filling by mononuclear cells and thickening of the alveolar septa. Pateints are generally treated with corticosteroids until symptoms are improved at which point they are tapered slowly. Data are lacking regarding the optimal duration of treatment and prognosis of DIP. A 44 year old African American woman presented with the acute onset of shortness of breath and substernal chest pain with radiation of the pain to the neck and left arm. She was diagnosed with acute myocardial infarction with positive CK and isoennymes and troponin-T. Initial ECG did not indicate an acute injury pattern, however ST-T wave change in V5 and V6 suggested lateral ischemia. Platelet count on initial presentation was 36,000. Her initial Hgb was 9.2. No previous laboratory data were available to review. She was initially treated with aspirin, low molecular weight heparin, and GIIB/IIIA inhibitors. Subsequent to this her blood count was repeated and platelet count had dropped to 11,000. At this time her peripheral smear was reviewed and 20±25 schistocytes were identified per high power field. The case was discussed with the Hematology division and plasmapheresis was initiated within 12 hours. After two double volume exchange transfusions, her platelet count had rebounded to 50,000 and her LDH was falling as well. Her platelet count slowly continued to improve over the course of her hospitalization. At discharge on hospital day #8, her platelet count was 236,000. At no point did the patient have renal dysfunction, neurologic symptoms, or fever. IMPLICATIONS/DISCUSSION: TTP is characterized by arteriolar lesions in various organs that contain platelets and fibrin. This results in MHA, and thrombocytopenia. The tissue hypoxia that ensues is responsible for the symptoms. Myocardial vessel involvement may be a cause of sudden death in some patients. However the isolated involvement of myocardial vessels is rare, especially when there is no of involvement of renal and cerebral vessels. TTP is treated with plasmapheresis initially. The disease is now 90% survivable if therapy is initiated promptly. Before this therapy was available, TTP had a 90% mortality. Plasmapheresis is conducted until platelet count, LDH, and MHA resolves. At that time, pheresis is gradually weaned. Those patients who relapse can be treated with corticosteroids, cyclophosphamide, or vincristine. Splenectomy is also an option. LEARNING OBJECTIVES: 1. Diagnose B12 deficiency on clinical grounds 2. Recognize the relationship between H. pylori infection and B12 deficiency 3. Assess potential complications of B12 deficiency. CASE INFORMATION: A 65 year old male was admitted to our hospital with a two month history of progressive gait instability, weakness and bilateral lower extremity numbness. He was unable to walk without assistance, was totally dependant for activities of daily living, had recently become incontinent of urine and had developed severe constipation. He also complained of mild numbness, tingling and weakness in his upper limbs, and had difficulties with hand motor control. He denied fevers, myalgias, pain, involuntary limb movements, headaches, or trauma, however he did have a forty pound weight loss over the previous three months. He had no significant past medical history and was on no medications. Physical exam showed no cranial nerve defects, mild proximal weakness and proprioceptive loss, with severe, diminished lower extremity strength, proprioception, vibration and sensation. Lower extremity tendon reflexes were brisk with positive Babinskis. Bilateral crackles at mid lung fields were evident on chest exam. Laboratory values on admission were significant for hemoglobin of 10.8 g/dl, mean corpuscular volume of 106.8 fL, and B12 of 75 ng/L (190±900). Arterial blood gas on 2L of oxygen showed pH of 7.456, PCO2 of 40.4, and PO2 of 69.0. With the laboratory diagnosis of B12 deficiency, a homocysteine level was obtained and was significantly elevated at 188 mmol/L (0±13). Chest X-ray showed extensive bilateral reticular infiltrates and small bilateral effusions. Due to these findings, a computed tomography of the chest was done which revealed left apical bullae, extensive ground glass opacities, and multiple filling defects in the interlobar and branch arteries consistent with pulmonary emboli. Lower extremity Doppler ultrasound confirmed deep vein thrombosis (DVT). An esophagogastroduodenoscopy was ordered to help determine the cause of the B12 deficiency. This showed mild chronic gastritis and H. pylori organisms. Serum studies for intrinsic factor and parietal cell antibodies were negative. He was treated with intravenous B12, triple therapy for H. pylori, anti-coagulation and oxygen support, however his respiratory status declined over the next three to four weeks and he died approximately one month after admission. Autopsy confirmed sub acute combined degeneration in the spinal cord and organizing thromboemboli with diffuse alveolar damage of the lungs. IMPLICATIONS/DISCUSSION: This case demonstrates a cascade of events that most likely began with the relatively common finding of H. pylori gastritis. Our patient then developed B12 deficiency, which has a known association with this infection. This in turn contributed to elevated homocysteine by interfering with its conversion to methionine. This contributed to a hypercoagulable state and in combination with immobility from the B12 deficiency induced sub acute combined degeneration, led to the DVT and pulmonary emboli. This directly contributed to the patient's respiratory decline. This case demonstrates that when H. pylori gastritis is confirmed, the association between this infection, B12 deficiency and hyperhomocysteinemia should be considered, as this can have profound consequences. 3) Discriminate between opiate dependence and addiction. CASE INFORMATION: A 29-year-old male with a history of hemophilia and hepatitis C presented with recurrent episodes of crampy abdominal pain over the past seven months. He denied fevers, chills, nausea, vomiting. There was no change in bowel habits or alteration of the pain with food or defecation. He denied previous surgeries or intravenous drug use. Physical exam showed no fever, heart rate 106, dilated pupils, diaphoresis, and a soft abdomen with voluntary guarding. The patient had undergone an extensive prior workup for these symptoms including CT scanning, colonoscopy, and upper endoscopy-all of which were normal. On our further review we learned that the patient had been taking two narcotic medications prescribed from different doctors. He described occasionally taking these medications for pain control, but was unaware both were narcotics. His symptoms completely resolved after starting a low dose methadone taper, and the patient was grateful for ending a seven-month cycle of pain. IMPLICATIONS/DISCUSSION: The symptoms of opiate withdrawal can include sweating, headache, nausea/vomiting, abdominal cramping, diarrhea, dilated pupils, piloerection, insomnia, agitation, and anxiety. Opiate withdrawal can occur from chronic prescription narcotic use, but may go unrecognized by patients and physicians. In our patient with hemophilia, prescription narcotics rather than NSAIDS were given for minor pains. His abdominal pain along with dilated pupils and diaphoresis suggested withdrawal leading to a more extensive review of his medication use. Despite using his medications as prescribed, his chronic use led to physical dependence. Addiction is defined as the compulsive use of a substance despite negative consequences. Dependence, however, does not usually have the psychological associations that are seen in addiction, making it harder to recognize. Nonetheless, patients with physical dependence can experience severe distress, as did our patient. Opiate withdrawal causing abdominal pain remains a diagnosis of exclusion and other serious causes should always be ruled out. LEARNING OBJECTIVES: 1. Discuss approach to fever in travelers 2. Recognize Dengue fever presentation 3. Realize importance of controlling mosquito vectors. CASE INFORMATION: 23 year old male with no prior history presents with 3 days of fever, malaise, headache and body aches. He returned to the US 6 days prior after a month-long backpacking trip in Brazil. His fever has no pattern and is relieved with Ibuprofen. His headache is bitemporal with some associated photophobia and neck pain. His body aches are generalized but most severe in the lower back. He denied HIV risk factors and review of systems is negative. He did not receive any vaccines or prophylactic medications. His bp is normal, pulse 70, and temperature 38.6 degrees Centigrade with malaise and a maculopapular rash over the trunk. Otherwise exam was negative. At this point the differential included flu, malaria, typhoid fever, leptospirosis, acute HIV, and Dengue fever. Laboratory investigation revealed a mild leukopenia with normal platelet count. CSF, HIV p24 Ag, thick and thin smear, RPR were all negative. Dengue titer came back positive for serum ELISA IgM. He was treated symptomatically with resolution of symptoms within a week's time. IMPLICATIONS/DISCUSSION: Dengue fever is caused by a single stranded RNA virus in the family Flaviviridae (found throughout the tropics) and transmitted to humans by the Aedes aegyptia mosquito. With urbanization and subsequent inadequate waste and water management, Dengue incidence is increasing in South America and the Caribbean. There is a disease spectrum with Dengue ranging from a relatively non-specific illness, to Dengue fever (acute illness with leukopenia, headache, malaise, myalgias/arthralgias) to Dengue hemorrhagic fever (the above plus positive tourniquet sign, thrombocytopenia, and increase vascular permeability) to Dengue shock syndrome (the above plus hypotension and narrow pulse pressure). Diagnosis of Dengue is usually by isolation of the virus or serologic testing such as ELISA for IgM. Dengue fever is typically self-limited with symptoms lasting appoximately 7 days. Warning signs for Dengue shock syndrome include sustained abdominal pain, persistent vomiting, change of level of consciousness, and a sudden decrease in platelet count. Dengue has 4 serotypes and repeat infection with another serotype is more likely to casue severe disease. In addition to improved mosquito control, a vaccine is currently being developed.
BLOOD IS THICKER THAN WATER: THE MANAGEMENT OF HYPERVISCOSITY IN ADULTS WITH CYANOTIC HEART DISEASE. A. DeFilippis 1 , S. Aaberg 1 ; 1 Emory University, Decatur, GA (Tracking ID #76037) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of hyperviscosity syndrome in patient with chronic hypoxia. 2. To recognise the complications of phlebotomy in the treatment of erythrocytosis of cyanotic heart disease. 3. To recognise the appropriate use of hydroxyurea in the treatment of erythrocytosis of cyanotic heart disease. CASE INFORMATION: A 19-year-old male with uncorrected tetralogy of Fallot presented to the emergency room after a ground level fall after climbing two steps. The fall was preceded by two episodes of lightheadedness. He denied vertigo, bowel or bladder incontinence, chest pain, dyspnea, or palpitations. At presentation, he was afebrile with a pulse of 90 beats per minute, blood pressure of 117/63, 30 respirations per minute and an oxygen saturation of 79%. Physical examination was remarkable for a well developed, alert and oriented male in no distress, clubbing in all digits, normal S1 and S2 heart tones with a II/VI early systolic ejection murmur heard throughout the chest; a cardiac heave over the left side of the chest, no jugular venous distention, no peripheral edema or crackles. Chest radiograph revealed a large bootshaped heart and right-sided aortic arch. EKG was notable for a normal sinus rhythm with left axis deviation and right atrial enlargement. Remarkable laboratory values were hemoglobin of 24g/dL and a hematocrit of 76 %. The patient was phlebotomized 500ml of blood and discharged from the hospital after remaining in normal sinus rhythm and asymptomatic for 24 hours. IMPLICATIONS/DISCUSSION: Complications of chronic hypoxia including erythrocytosis, hyperviscosity, abnormalities of hemostasis, cerebral abscesses or stroke and endocarditis are among the most common consequences of cyanotic heart disease in adults. The compensatory erythrocytosis of cyanotic heart disease can become pathologic by causing an increase in blood viscosity, which decreases perfusion, resulting in a decrease in total oxygen delivery and an increased risk of veno-occlusive/hyperviscosity syndrome. Treatment of hyperviscosity secondary to erythrocytosis in cyanotic heart disease is controversial. Data suggest that phlebotomy has the potential to increase exercise capacity, reduce the symptoms of hyperviscosity syndrome and reduce the potential increased risk of vasoocclusive disease in selected patients with polycythemia secondary to cyanotic heart disease. Unfortunately, repeated phlebotomy can quickly lead to iron deficiency. Microcytic erythrocytes produce higher viscosity than normocytic erythrocytes at comparable hematocrits putting patients at high risk for vasoocclusive events. There is limited data on the use of hydroxyurea to suppress erythrocytosis in this patient population. Thus we concluded that until newer approaches to decreasing the hematocrit, without causing iron deficiency anemia, are shown to be safe and efficacious, phlebotomy should only be used for the acute resolution of hyperviscosity symptoms; and the use of hydroxyurea should be limited to recurrently symptomatic patients.
CARDIAC SARCOID AND RISK OF SUDDEN DEATH. A. DeFilippis 1 , A. Palacio 1 , D.W. Brady 1 ; 1 Emory University, Decatur, GA (Tracking ID #76794) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of cardiac sarcoid. 2. Treat cardiac sarcoid and its inherent risk for sudden cardiac death. CASE INFORMATION: A 58-year-old female with cutaneous sarcoid presents to the emergency room with a 3 month history of progressive dyspnea on exertion. She also reports a 2-week history of proximal nocturnal dyspnea, cough, 2-pillow orthopnea and lower extremity edema. She denies chest pain, fever, chills, or palpitations. Her past medical history includes hypertension, diabetes, hypercholesterolemia and obstructive sleep apnea. On presentation, she was afebrile, pulse 106, blood pressure 120/60, respiratory rate of 28 and oxygen saturation of 88% on room air. Physical exam was significant for S3 gallop, normal pulmonary exam, lower extremity edema and several silver dollar size hyperpigmented lesions on her legs. Routine laboratory testing was unremarkable, chest radiograph revealed mild cardiomegaly as compared to a normal radiography 1 month earlier. EKG revealed a new right conduction system block as compared to a normal EKG 2 years earlier. High resolution CT scan was negative for pulmonary embolus and deep venous thrombosis but revealed multiple enlarged mediastinal lymph nodes. Echocardiogram was notable for hypokinetic lateral and inferior wall motion, new from a normal dobutamine stress echocardiogram 2 years earlier. Cardiac catheterization revealed normal coronary angiography, an ejection fraction of 20% and regional wall motion abnormalities, which lead to a cardiac biopsy that revealed non-caseating granulomas consistent with active cardiac sarcoid. The patient improved with diuresis and systemic afterload reduction. She was started on a 6-month course of prednisone and equipped with an automated internal cardiac defibrillator with rescue pacing, prior to discharge from the hospital. IMPLICATIONS/DISCUSSION: Dyspnea and cough are amongst the most common clinical presentations of sarcoid but are attributed to pulmonary rather than cardiac pathology. Although less than 10% of sarcoid patients demonstrate symptoms secondary to cardiac sarcoid, cardiac involvement is found in up 76% of sarcoid patients at autopsy and accounts for 50% of the mortality in this patient population. The most common presentation of cardiac sarcoid, found in approximately 50% of patients, is arrhythmias or cardiac conduction abnormalities as a result of granuloma formation in the conduction system. Sarcoid granulomas in the ventricular myocardium may lead to ventricular arrhythmias in as many as 22% of patients with sarcoid. Granulomas in myocardium can result in diastolic or systolic dysfunction and has accounted for 25±75% of the deaths in some studies of cardiac sarcoid patients. Endomyocardial biopsy is highly specific but due to the non-homogeneous pattern of infiltration will only lead to a diagnosis of cardiac sarcoid in approximately 20% of cases. With arrhythmias or cardiac conduction abnormalities accounting for 30±65% of the deaths inpatients with cardiac sarcoid, automated internal cardiac defibrillators (AICD) with rescue pacing are standard of care in survivors of deadly arrhythmias. Although no trials have assessed the efficacy of AICDs in the absence of ventricular tachyarrhythmias, many advocate their use in all cases of known cardiac sarcoid; others reserve the use to patients with EKG abnormalities by standard of Holter monitor testing. Treatment of the underlying pathological process with immunosuppression is controversial and void of any large randomised trials to access efficacy. A small uncontrolled study demonstrated improvement and/or reversal of EKG abnormalities in greater than 50% of patients treated with corticosteroids. In one retrospective study of patients with cardiac sarcoid, 5-year survival was increased from 10% to 75% with the use of corticosteroids. Opponents to the use of corticosteroids raise concern that corticosteroids convert granulomas to scar tissue increasing the risk of aneurysms. Cardiac transplant has been successful with recurrent disease possible but rare.
A WOMAN FROM CAMEROON WITH FEVER, ABDOMINAL PAIN AND EOSINOPHILIA. K. Dendrinos 1 ; 1 Boston University, Boston, MA (Tracking ID #75960) LEARNING OBJECTIVES: Review the Differential Diagnosis of Eosinophilia, Evaluate Fever of Unknown Origin. CASE INFORMATION: A 70 year old woman, recently emigrated to the U.S., presents to this hospital complaining of fever and chills for the past 3 months. She also complained of right and left upper quadrant pain. These complaints originally began in her home country of Cameroon, she came to the U.S. one week prior to her initial admission. During her first admission, she was febrile daily to 101 degrees Fahrenheit. Her exam was notable for right upper quadrant abdominal tenderness. Her laboratory examinations were significant for a CBC with an eosinophilia of 43%, that would peak later that month at 60%. Suspicion was high for a parasitic infection. Serologies would later be positive for multiple parasites endemic to Cameroon. She was treated with a wide array of antibiotics, including anti-tuberculosis medications, anti-fungals, and anti-helminthics. Her clinical course deteriorated and she died in the intensive care unit. On autopsy, she was found to have widely disseminated lymphoma involving nearly every organ system. IMPLICATIONS/DISCUSSION: This case initially appeared to be a classic presentation of a parasitic infection: a patient with fever and eosinophilia from an endemic area with positive serologies. It presents a chance to review the differential diagnosis of eosinophilia, and to again rethink the work-up of a fever of unknown origin. This case again demonstrates that``common things occur commonly'' (even when the patient comes from an unfamiliar part of the world). It also demonstrates the importance of keeping the differential diagnosis open, especially when the therapy instituted according to the initial dianosis fails.`L OCKING'' IN THE CASE FOR PREVENTIVE IMMUNIZATIONS. J. Derby 1 , C. Manhart 1 , C. Murphy 1 ; 1 Creighton University, Omaha, NE (Tracking ID #74723) LEARNING OBJECTIVES: Recognize that there are many patients at risk who have not received a primary series in tetanus. Distinguish a differential diagnosis of tetany. Manage a tetanus infection. CASE INFORMATION: A 71 year old female with no significant past medicial history and on no medications presented to the ER complaining of waking up in the middle of the night and being unable to move for the last 24 hours due to muscle stiffness. On initial exam she had marked neck stiffness with muscle spasms triggered by light touch and deliberate speech. A CT scan was unremarkable. A trial of IV diphenhydramine was administered to rule out a dystonic reaction. Stychnine levels and tetanus antibodies were drawn ( both later reported as negative). Over the next few hours obvious trismus developed. Her muscle spasms spread inferiorly to involve her abdominal wall, and her upper and lower extremities demonstrated lead pipe rigidity. The patient was intubated prophylactically and IV diazepam was begun. The patient was given tetanus immunoglobulin as well as a Td vaccination (since the illness is not protective). Metronidazole was administered IV. IMPLICATIONS/DISCUSSION: The majority of reported cases of tetanus in the US are in patients over the age of 60 years old. Currently, nearly 50% of elderly patients in the United States have non-protective titers. This is attributed to waning immunity in patients who have not received booster shots. Additionally, many elderly never received the primary series as children (which was not available until the 1950's). This patient had received one booster without a primary series which did not protect her,but may have prevented her mortality. Many immigrants as well, may have never received the primary tetanus series. Physicians must be diligent in immunizing at-risk groups and ensure that all patients whos tetanus immunization status is unknown receive a primary series. LEARNING OBJECTIVES: To analyze an interesting presentation of rhinocerebral mucormycosis and review current diagnostic and treatment modalities. CASE INFORMATION: The patient is a 26 yo African American male with a past medical history significant for Type I Diabetes Mellitus who presented with a chief complaint of leftsided facial swelling for nine days. He stated that the onset of facial swelling occurred over few hours and that the progression was rapid. Pt reported subjective fevers and chills. He denied upper respiratory or sinus-related symptoms. He initially presented to an outside hospital after two days of facial swelling. There he underwent a sinus CT that revealed mucosal thickening involving the left ethmoid, maxillary and ethmoid sinuses. He was treated with a seven day course of Rocephin and Cleocin. The patient was in DKA at the time of presentation and was treated for two days per hospital DKA protocol. On the day of discharge from this outside hospital the patient presented to our institution reporting that the facial swelling had not improved with prior therapies. Additionally, he complained of bilateral blurry vision with increased sensitivity to light. On physical examination the patient was afebrile. HEENT examination was significant for left-sided facial swelling with infraorbital involvement, a greenish-yellow discharge from the left medial canthus and left nares, photophobia and a necrotic plaque of the hard palate. The patient was unable to medially deviate his left eye. Laboratory work revealed a normal white cell count and differential, and a normal chemistry panel with the exception of an elevated serum glucose of 256. The differential diagnosis at this point included among other conditions fungal sinusitis, bacterial sinusitis, lymphoma and Wegener's granulomatosis. MRI of brain revealed extensive paranasal sinus opacification with abnormal dural enhancement extending from the left sphenoid sinus to involve the left cavernous sinus, left middle cranial fossa and left fifth cranial nerve with no intracranial, parenchymal or orbital involvement. The patient underwent a nasal biopsy that revealed multiple hyphal elements consistent with Mucormycosis. The patient was started on Amphotericin B at 1.5/mg/kg/day and underwent extensive surgical debridement. Although no orbital involvement was confirmed on MRI, the patient's cranial nerve palsy and opthalmoplegia were understood to be a manifestation of rhino-orbital mucormycosis. The hospital course was complicated by acute renal failure, septic shock, diabetic ketoacidosis and cardiac arrest. While serial MRIs showed no regression of disease, the patient was medically stabilized, and showed clinical improvement over an eight week hospital course. However, the patient's vision loss and opthalmoplegia did not improve with treatment. IMPLICATIONS/DISCUSSION: Rhizopus oryzae accounts for 90% of rhinocerebral mucormycosis. Patients in DKA are highly susceptible because the acidic, glucose rich environment provides a perfect medium for growth. The clinical presentation includes classic sinusitis symptoms and signs along with nasal necrosis (38%) and palatal or gingival necrosis (14%) as in the case of our patient. The diagnosis of mucormycosis is histologic, and it is the only fungus that can be seen with H & E stain. The mainstays of treatment are surgical debridement and reversal of predisposing factors. While lipid containing formulations allow for higher doses to be given due to reduced toxicity there may be problems with delivering the complex to infarcted tissue and obtaining effective concentrations. Administration of intravenous Amphotericin B along with alternative routes such as aerosolized and CSF perfusion pathways is an aggressive treatment modality. Hyperbaric oxygen is an accepted, but not commonly utilized adjuvant to treatment. Even with early detection and treatment the overall mortality rate in patients with rhinocerebral mucormycois remains high. A 59 year old homosexual Caucasian male presented with complaints of b/l hearing loss, tinnitus and vertigo for several months prior to presentation. Patient had an audiogram performed which showed moderate to severe symetrical sensorineural hearing loss. Patient did not have a history of recent aminoglycoside use and was not a diabetic. He was worked up with thyroid stimulating hormone, sedimentation rate, coagulation tests, rheumatoid factor and anti-nuclear antibody titers which were all within normal limits. PPR was positive at 1:16 and MHA-TP was positive at 4+. He refused an HIV test. Patient subsequently received a lumbar puncture which was positive for VDRL . He was thus diagnosed with neurosyphilis and admitted to the inpatient medicine wards where he received a 14 day course of IV Penicillin. Hearing loss improved with treatment of the neurosyphilis. IMPLICATIONS/DISCUSSION: Neurological manifestations of syphilis can occur during any phase of this disease as the central nervous system is seeded during spirochetemia. Up to forty percent of patients infected with Treponema pallidum will have cns involvement. Although neurosyphilis can be aymptomatic, symptomatic neurosyphilis can present earlier as syphilitic meningitis or later in the infection, can have a meningovascular or parenchymatous pesentation. Cranial nerve involvement, especially of cranial nerves II±VIII, can be seen. Syphilitic otitis is a form of neurosyphilis that can present as deafness, vertigo and/or tinnitus and should considered in patients who present sensorineural hearing loss. If treated early, syphilitic otitis may be curable, but can lead to irreversible damage if untreated. CASE INFORMATION: 78 years old man with coronary artery disease, emphysema and alcoholism presents with short of breath and palpitations. He had clear chest film, but was found to be in rapid atrial fibrillation and controlled with diltiazem. On day 3 of hospital stay, his mental status deteriorated and rapidly went into delirium tremens. He subsequently had temperature spike to 103.0(F) with associated leukocytosis. His oxygen saturation became very unstable and eventually requiring mechanical ventilation. He repeat chest X-ray showed right lower lobe consolidation. His sputum gram stain showed 4+ small gram negative rods which was identified to be Pasteurella Multocida. He had been a long time avid deer hunter and has three dogs which he is in close contact with. He was treated with a third generation cephalosporin and his condition slowly improved. His subsequent CT of chest revealed left lung nodule. The biopsy of the nodule was consistent with squamous cell carcinoma of the lung. IMPLICATIONS/DISCUSSION: Pasteurella Multocida is common organism in many domestic and wild animals. Human Pasteurella is a major pathogen in wound infections due to animal bites. Greater than 70% of the reported cases of Pasteurella Multocida pneumonia occurred in patients with non-traumatic exposure to colonized animals.
Respiratory infection caused by Pasteurella Multocida can range from asymptomatic colonization to otitis media, sinusitis, epiglottitis, pneumonia, empyema and lung abscess. The degree of pulmonary infection depends on the immunity state of the host and can carry a very high mortality. Treatment of choice for Pasteurella Multocida infection is either penicillin, second or third generation cephalosporins, or doxycline. Prolonged antibiotic treatment is warranted in immnocompromised host. 2) To consider macrophagic myofasciitis in patients presenting with proximal muscle weakness and myalgias. CASE INFORMATION: Macrophagic myofasciitis is a syndrome of proximal muscle weakness, pain and fatigue. Clinical presentation is very similar to polymyositis, with proximal muscle pain, weakness, fatigue, fever and ternderness. Macrophagic myofasciitis is distinguished from polymyositis microscopically, with infiltration of the fascia with macrophages in MM. Cardiac involvement is found in over 70% of patients with polymyositis. We report a case of macrophagic myofasciitis presenting with proximal muscle weakness and ventricular tachycardia. A 49 year-old African American female with several months of progressive muscle weakness presents with palpitations and dizziness. Physical examination demonstrated significant proximal muscle weakness with no evidence of a skin rash. Laboratory data revealed a creatinine kinase of 1301 u/liter (normal <2oo), a CK-MB of 41.7 (normal 0± 60) and an aldolase of 40.2 (normal 1.5±8.1). An EKG showed a wide complex tachycardia with a rate of 220. A coronary angiogram was normal and electrophysiologic studies failed to induce ventricular tachycardia. Electromyopathy was consistent with a myopathy and her muscle biopsy revealed macrophagic infiltration of fascia, consistent with a diagnosis of macrophagic myofasciitis. Her ventricular tachycardia was postulated to have arisen from a myofasciitis. After initiation of high dose steroids, her muscle enzymes normalized and no further episodes of tachyarrhythmia occurred. IMPLICATIONS/DISCUSSION: MM is a relatively new muscle disease, with a first case report in 1993 in France. In several ways, it is similar to polymyositis, with bilateral muscle weakness and pain. Both are associated with an altered immune system. Cardiac arrhythmia in polymyositis is fairly common, occurring in over 70% of patients. These arrhythmias are due to an increase in spontaneous and disorganized myopathic contraction. There are no previous reports of cardiac disrhythmias in macrophagic myofasciitis. Most patients respond well to treatment with steroids and/or antibiotics in a few weeks. LEARNING OBJECTIVES: To recognize rare presentations of severe hypothyroidism. CASE INFORMATION: 18 year old African American female presented to the teen pregnancy center secondary to increased abdominal girth and amenorrhea. She also reported fatigue, hair loss, dyspnea on exertion, and sadness. Physical exam was significant for alopecia, a large goiter, markedly distended abdomen with fluid wave, tense edema of the hands, and delayed deep tendon reflexes. Labs were remarkable for a TSH of 240 with undetectable T4 and T3, marked microcytic anemia and an erythrocyte sedimentation rate of 140. CXR revealed a globular pericardial silhouette and ultrasound of the abdomen showed a large amount of ascites. Echocardiogram was unremarkable except for a moderate pericardial effusion. Pelvic ultrasound showed enlarged cystic ovaries, consistent with Van Wky Grumbach syndrome. Peritoneal fluid studies were consistent with myxedematous ascites. Upon further questioning, the patient stated that she had been treated for hypothyroidism as a young teen and had been lost to follow-up for two years. She also had a history of prior suicide attempts and depression. IMPLICATIONS/DISCUSSION: The manifestations of thyroid disorders are many, myxedematous ascites are a well-known but not often seen presentation. Serositis from hypothyroidism may be caused by several things: a capillary leak syndrome resulting from the dearth of thyroid hormones, an SIADH-like picture causing body cavity fluid accumulation, or high hyaluronate concentrations in the body fluid acting as an osmotic attractant. The anemia of hypothyroidism is thought to be caused by either direct bone marrow toxicity, lack of stimulation of erythropoiesis, decreased RBC survival time, and increased 2,3-DPG concentration. Van Wky Grumbach syndrome is a rare condition in which the TSH at high levels acts as an FSH analogue to cause ovarian hyperstimulation/cyst formation. This should be considered when a patient with no known thyroid disease presents with cystic ovaries. year old Hispanic male with no significant past medical history presents to our clinic with a one year history of intermittent bright red blood per rectum and two week history of mild dyspnea on exertion. He has previously been evaluated for the rectal bleed at various clinics and treated with topical hemorrhoidal ointments without relief. Patient denies chest pain, orthopnea, paroxysmal nocturnal dyspnea, abdominal pain, bowel changes, fever, chills, and weight loss. He is a sexually active homosexual. Family history is negative for colon cancer and polyps. Vital signs show temperature 100.3, BP 138/76, HR 75, RR 14. Physical exam is significant for mildly decreased breath sounds in left upper lobe and guaiac positive stool but no palpable rectal mass. Lab results show hemoglobin 10.9, MCV 78.0, and WBC 6.4. HIV test is negative. Colonoscopy with biopsy shows squamous cell cancer. Staging studies including chest x ray and a CT scan of the thorax display a 3.5 cm mass in the left upper lobe and CT pelvis confirms anorectal mass. Biopsy of the lung mass is consistent with a metastatic lesion. He is treated with chemotherapy and undergoes resection of the solitary lung mass. IMPLICATIONS/DISCUSSION: Anal canal carcinoma can be overlooked in young otherwise healthy patients as rectal bleeding is often attributed to hemorrhoids. Squamous cell cancer of the anal canal has a high association with human papilloma virus (HPV) particularly HPV-16. HPV DNA is isolated in 88% of anal cell cancers. Other risk factors include chronic immunosuppressive therapy, HIV, and smoking. Past treatment for anal canal carcinoma involved abdominoperineal resection with permanent colostomy. Cure rates with surgical resection ranged between 40±70%. However, in recent studies, chemotherapy with 5-FU and mitomycin combined with radiotherapy offer a 66±70% cure rate and lower rate of complications. Prognosis is dependent on the size of tumor, distant metastases, and nodal involvement. In conclusion, recurrent rectal bleeding in young patients with associated risk factors warrants a more thorough investigation. year old male presented with ten days of progressive difficulty walking, stating he had weakness in the lower extremities and in his right hand. He also reported slurred speech. Pt had no known medical problems and denied the use of medications, illicit drugs, tobacco or alcohol. Physical examination was significant for 3/5 right lower extremity strength with absent reflexes of the right leg. Sensory exam was grossly intact. The patient had poor performance of finger to nose testing and difficulty with rapid alternating movements. There was severe gait instability. Babinski's sign was bilaterally equivocal. CBC, chemistry panel, ESR, TSH, RPR, EKG were normal. After an unremarkable head CT, an MRI of the head showed a demyelinating pattern involving the middle cerebellar peduncles. The CSF protein, gram stain, culture, cell count, and glucose were within normal limits. On hospital day 4, HIV serology was positive and thus confirmed the clinical diagnosis of PML. CSF testing for JC virus later returned positive. IMPLICATIONS/DISCUSSION: PML is a demyelinating disease associated with infection of the JC virus. An AIDS defining condition, PML may present with rapid or progressive focal neurological deficits, including aphasia, ataxia, hemiparesis, hyporeflexia, and impaired cognition. In AIDS, PML implies a very poor prognosis with a survival expectancy of about six months. The diagnosis of PML is made through a clinical constellation which includes HIV positivity, characteristic MRI findings, and positivity for JC virus in the CSF. MRI findings of PML are similar to those seen in Acute Disseminated Encephalomyelitis (ADEM), a demyelinating process associated with infections or vaccinations in children, but they differ from those seen in Multiple Sclerosis (MS). The MRI with MS typically shows plaques in the periventricular region, corpus callosum, centrum semiovale, and less commonly in the deep white matter structures. There is no specific therapy for PML, and improvement of symptoms relies on increasing CD4 count via antiretroviral therapy. There has also been suggestion of improved clinical response with the addition of Cidofovir, a drug typically used to treat CMV retinitis.
ABDOMINAL PAIN: A CASE OF NARROWLY±FOCUSED DIFFERENTIAL. A.K. Duncan 1 , W.E. Wysokinski 1 , A.K. Ghosh 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #74267) LEARNING OBJECTIVES: 1) Recognize the need to expedite the work-up of poorly responding abdominal pain; 2) Recognize that vasculitis could present with diarrhea. CASE INFORMATION: A 72-year-old women was transferred to our hospital for management of her diarrhea and weight loss. Three months prior to admission, she had an episode of upper respiratory tract infection and was treated with a 10 days course of antibiotics. One month prior to entry, she developed profuse diarrhea and malaise. EGD reveled mild gastritis, small bowel follow through and colonoscopy was normal and she was empirically treated with metronidazole for pseudomembranous colitis. She complained of increasing abdominal pain and was found to have air on an upright abdominal film. Exploratory laparotomy revealed necrosis, inflammation and perforation in the small bowel and nodular lesions in the liver. She underwent extensive small bowel resection, cholecystectomy, and liver biopsy. Liver histology was reported to reveal stellate granulomata consistent with Yersinia. However, cultures and serology for Yersinia were negative. She was empirically treated with ciprofloxacin and transferred to our hospital. Examination revealed an elderly woman, with mild dyspnea, and atrial fibrillation with HR of 110/mt. She was mildly icteric, with skin breakdown in the sacral and inter-scapular region. Chest examination revealed bilateral basilar crackles. Abdomen was mildly tender and distended. Neurological examination revealed generalized but asymmetric neuropathy. Re-evaluation of the small bowel biopsy revealed transmural fibrinoid necrosis with granulomatous inflammation typical of polyarteritis nodosa (PAN). EMG was consistent with mononeuritis multiplex. ANCA was negative. Visceral angiogram revealed micro-aneurysms in the spleen, kidney, jejunal and ileal branches. She was initiated on prednisone and pulse cyclophosphamide therapy with rapid improvement in symptoms. On follow-up at 3 months she continues to gain weight and has no new symptoms. IMPLICATIONS/DISCUSSION: PAN affects the gastrointestinal tract in over 40% of cases. PAN cases presenting with acute abdomen has been associated with a high mortality (60%). GI bleeding, perforation, pancreatitis, renal insufficiency, proteinuria, cardiomyopathy and CNS vasculitis are poor prognostic factors. A high index of suspicion and re-examination of a broad differential diagnosis in cases of acute abdomen, could lead to earlier detection of PAN and successful therapy of this potentially fatal condition.
HYPOGLYCEMIA AND HYPONATREMIA: A HIGHER CAUSE. E.J. Dzielak 1 , N.C. Chiappetta 2 ; 1 Dunmore, PA; 2 Moses Taylor Hospital, Scranton, PA (Tracking ID #77122) LEARNING OBJECTIVES: DDX of hypoglycemia W/U of and DDX of primary, secondary and tertiary causes of adrenal insufficiency. CASE INFORMATION: A 74 year-old man with past medical history of hypothyroidism, prostate carcinoma, iron deficiency anemia, paroxysmal atrial fibrillation, and hyponatremia, presented nausea and vomitting. Physical exam was normal. Abnormal laboratory data included a sodium of 125, glucose of 40, and mild normocytic anemia. His hypoglycemia did not correct with exogenous glucose administration. An A.M. cortisol level returned as 1.3. ACTH level was then drawn and returned as 20 pg/ml. Further evaluation to distinguish between secondary and tertiary adrenal insufficiency was preformed with a corticotropin stimulation test. The results of this test confirmed the diagnosis of secondary adrenal insufficiency. IMPLICATIONS/DISCUSSION: Secondary adrenal insufficiency is a deficiency of ACTH from the anterior pituitary gland resulting in a decreased production of cortisol . In the adrenal gland, ACTH stimulates the zona fasiculata, while having minimal affect on the zona glomerulosa. Therefore, manifestations of aldosterone deficiency (hyperkalemia and hypotension) are not present, as this case demonstrated. To confirm adrenal insufficiency, an AM cortisol level of <1.3 is needed. If the cortisol level is greater than 1.3, the diagnosis of adrenal insufficiency could be confirmed by a cosyntropin stimulation test. To distinguish between primary and secondary adrenal insufficiency, an ACTH level should be drawn which should be >100 pg/ml in primary adrenal insufficiency. The Metyrapone and Insulin Induced Hypoglycemia tests confirm secondary adrenal insufficiency. To distinguish between tertiary and secondary adrenal insufficiency, the corticotropin stimulation test is indicated. An absent response from baseline with ACTH and cortisol confirms secondary adrenal insufficiency, while an prolonged and exaggerated ACTH repsonse is consistent with tertiary adrenal insufficiency.
A GASTROINTESTINAL LINK TO OSTEOPOROTIC FRACTURES. L. Eck 1 , C. Jachna 1 ; 1 Kansas University Medical Center, Kansas City, KS (Tracking ID #74458) LEARNING OBJECTIVES: To recognize that celiac disease is commonly associated with low peak bone mass and adult osteoporosis leading to disabling fractures. CASE INFORMATION: A 49 year old white male with a history of multiple atraumatic fractures including a hip fracture at age 22, wrist fracture at age 16, multiple rib fractures and a recent wrist fracture presented for evaluation of osteoporosis. Bone mineral density studies revealed a T-score of À3.1 at the spine, consistent with osteoporosis. On clinical history, the patient reported a long-standing history of approximately eight loose bowel movements per day associated with abdominal cramping. He also reported low body weight through young adulthood and lactose intolerance. Lab studies were notable for an elevated IgG antigliadin antibody of 132 Units, with a reference range of >30 Units being strongly postive for a diagnosis of celiac sprue. Follow up small bowel biopsy revealed villous atrophy consistent with the diagnosis of celiac sprue. IMPLICATIONS/DISCUSSION: Celiac disease is caused by a genetically based inability to digest gluten, a major protein commonly found in grains. Due to the availability of new highly sensitive and specific serologic diagnostic tests, it is increasingly recognized in the United States as a cause of malabsorption. Celiac disease is often asymptomatic but symptoms suggesting the diagnosis include bloating, flatulence, chronic diarrhea and lactose malabsorption. Other than gastrointestional symptoms, celiac disease also has a wide spectrum of extraintestinal manifestations including iron deficiency anemia, low bone mineral density, and dermatitis herpetiformis. In a study of North American adults with celiac disease, 70% of subjects had low bone mineral density, predisposing them to fractures. Other studies have suggested that in subjects with celiac disease, bone manifestations may be more severe in men than in women possibly due to estrogen's protective effects on the bone. Historically, initial screening for celiac disease involved detection of antigliadin antibody. However, more specific serologic tests, including antiendomysial and antitissue transglutaminase antibodies, are now available. Histologic identification of gluten sensitive enteropathy, with characteristic small bowel mucosal abnormalities including villous atrophy, crypt heperplasia, and an increased density of inflammatory cells, still remains diagnostic. The pathogenesis of low bone mass and osteoporosis in celiac disease is multifactorial. The reduction in surface area of the intestinal mucosa may contribute to calcium malabsorption and subsequent secondary hyperparathyroidism leading to increased bone resorption. Vitamin D deficiency is also believed to be common. Those who are undiagnosed during childhood and young adulthood may never achieve peak bone mass. In addition, some researchers suspect that systemic effects of inflammatory cytokines involved in the intestinal mucosa inflammation may also contribute to the development of bone disease. Not only should patients with celiac disease be screened for osteoporosis, but patients with low bone mineral density and no evident osteoporosis risk factors should be evaluated for celiac sprue.
A. El-Sharkawi 1 , S. Kripalani 1 ; 1 Emory University, Atlanta, GA (Tracking ID #75179) LEARNING OBJECTIVES: Hepatitis due to herpes simplex virus (HSV) is an uncommon, but potentially fatal cause of hepatitis that is often not considered in the differential diagnosis of acute hepatitis during pregnancy. Serological tests have limited value in establishing an early diagnosis and a high index of suspicion and early diagnostic tools, such as HSV DNA detection are essential. Early treatment with acyclovir decreases mortaility from herpes simplex hepatitis. CASE INFORMATION: A 19 y/o woman, G2 P1, with no significant PMHx, presented with premature labor at 32 weeks gestation. The initial symptoms were controlled, but over the next several days, the pt developed a cough, dyspnea, RUQ abd pain, and fever of 39C. Evaluation of blood, urine, and amniotic fluid cultures was unremarkable. CXR revealed bibasilar atelectasis versus infiltrate, and she received azithromycin and ceftriaxone for presumptive pneumonia. The pt remained febrile with increasing RUQ pain. Labs revealed declining Hb (7.1), WBC (5.6 with 56% bands), and Plt counts (62,000), with rising liver transaminases (AST 2490, ALT 796), INR 2.5, T. Bili 2.2. RUQ U/S showed hepatomegaly and fatty liver. Considering her worsening condition, obstetrics performed a C-section on hospital day 5.
Post-operatively, the pt was emergently intubated for acute respiratory failure. Physical exam revealed Temp 39.2, HR 146 (reg), BP 107/38, slight RUQ tenderness, liver span 13 cm, no fluid wave or shifting dullness, and 2 vesicular lesions on the Rt upper inner thigh. DDx at this point included HELLP syndrome, acute fatty liver of pregnancy, acute viral hepatitis, pyelophlebitis, portal vein thrombosis, and acute hepatic failure secondary to sepsis. Antimicrobials were changed to ceftazidime, vancomycin, and acyclovir. On hospital day 11, PCR for HSV type 1 and 2 returned positive. In addition, cultures of the vesicular lesions on the patient's right thigh grew herpes simplex. Further questioning of the patient's partner revealed a hx of genital herpes. With the initiation of acyclovir, the pt defervesced and her transaminases decreased. However, she suffered multiple ICU complications, as well as herpes encephalitis. The newborn infant was in the NICU with herpes meningitis. IMPLICATIONS/DISCUSSION: Hepatitis due to herpes simplex virus (HSV) is an uncommon, but potentially fatal cause of hepatitis that is often not considered in the DDx of acute hepatitis during pregnancy. HSV can affect the liver during primary or recurrent infection, although hepatitis during recurrent infection has not been documented in immunocompetent hosts. Review of the literature suggests that HSV hepatitis can present with wide clinical spectrum from mild symptoms to fulminant hepatic failure. However, the initial symptoms are often nonspecific. Therefore, a high index of suspicion and early diagnostic tools, such as HSV DNA detection are essential. Unfortunately, serological tests have limited value for establishing an early diagnosis, though they may be used retrospectively to support the diagnosis. Definitive diagnosis is made with a liver biopsy, but this is frequently not possible because of severe coagulopathy.`J UST A MED REFILL''. S. Elad 1 , G. Applebaum 1 ; 1 University of California, Los Angeles, Sylmar, CA (Tracking ID #74705) LEARNING OBJECTIVES: 1. To recognize the impact the closing of community clinics has had on uninsured and indigent patients. 2. To raise the possibility that short term economic gains resulting from the closure of primary care clinics may result in preventable hospitalizations. CASE INFORMATION:`Maria' is a 56 -year old woman with a past medical history of diabetes, hypertension and hyperlipidemia that came to the hospital urgent care for a`med refill'. She ran out medications 2 months ago after her community clinic was closed secondary to`budget cuts'. Today Maria reports she has noticed increased urination and thirst for the past two weeks and a frontal headache with blurry vision for the past three days. She denies chest pain or shortness of breath. She has not been febrile. Maria had been on two medications for her blood pressure and diabetes, and a pill for high cholesterol. Presently she is completely out of her medications. When questioned why she waited two months to get her medications refilled Maria stated the two hour bus ride and 6 hour wait in the emergency room required her to take time off work she could not afford. On exam, Maria's blood pressure was 190/108 with a pulse of 83. The rest of her cardiac, pulmondary and nuerologic exam was unremarkable except for bilateral decreased visual acuity. Her blood sugar was 275 with sugar and protien in her urine but no ketones. Her anion gap was normal as was her blood count. Maria was given a dose of lisinopril and her headache resolved once her blood pressure decreased to 160/90. She was restarted on her oral diabetes medication. Her medications were refilled and she was instructed to return to urgent care in one week to have her blood pressure and blood sugar rechecked. IMPLICATIONS/DISCUSSION: The national health care crisis has been the topic of many news programs and newspaper articles for the past year. According to the Kaiser Family Foundation approximately 19% of Californians are uninsured. In California, which faces a multi-billion dollar deficit, politicians have been discussing ways to trim costs which have included proposals to close hosptials and clincs. The recent closure of several primary care clinics in Los Angeles County has left many patients like Maria without access to health care. By the time Maria was able to visit the hospitals' urgent care clinic she had run out of medications and both her diabetes and hypertension were poorly controlled. Had Maria required hospitalization at the time of her visit the cost would have been much higher. Maria's primary care visit to the emergency room was entirely avoidable had her clinic not been closed. Much of the short term economic gains obtained by closing county subsidized clinics may later be lost by patients utlizing our emergency rooms for primary care visits. Even in the context of our current financial situation steps need to be taken to ensure that uninsured patients have reasnable access to primary care services outside the hospital setting. LEARNING OBJECTIVES: 1-To recognize fungal vertebral osteomyelitis as an etiology for worsening back pain in patients with history of multiple back surgeries. 2-To consider secondary bacterial infection in patients who fail to respond to appropriate antifugal therapy. CASE INFORMATION: A 48-year-old male presented in January 2001 with worsening back pain, bilateral leg weakness and urinary incontinence. Condition started in 1993 after back injury during demolition job that required multiple back surgeries. Examination was remarkable for bilateral leg weakness and local tenderness over lumbar spine. MRI examination revealed disc space infection at the L3-4 level with adjacent vertebral osteomyelitis. CT guided aspiration of L3-4 space was performed and cultures grew Candida albicans. Treatment was instituted with IV Amphotericin B via Mediport. Therapy was switched to oral fluconazole 400 mg daily. In March 2001, he developed Mediport related Staphylococcus aureus sepsis and was treated with cefazolin for two weeks. Patient was re-admitted in April with worsening back pain. MRI examination revealed worsening inflammatory changes within L3 and L4 vertebral bodies and small epidural abscess. CT guided aspiration was performed and cultures grew methicillin sensitive Staphylococcus aureus. We started treatment with Nafcillin 2 gm IV every 4 hours in addition to fluconazole. Surgical debridement followed by physical therapy and long term IV antibiotics controlled our patient's symptoms. IMPLICATIONS/DISCUSSION: Candidal vertebral osteomyelitis is a rare disease with 65 cases identified in literature. Risk factors have been associated with hyperalimentation, surgical procedures, abdominal surgeries, immunosuppressive therapy and IV drug abuse. The usual mode of spread is hematogenous during episodes of fungemia. The most frequent mode of presentation is severe back pain and diagnosis can reliably be made only when the organism is isolated from the lesion. Both medical and surgical treatment resulted in more favorable outcome for most patients. This case demonstrates the importance of recognizing fungal vertebral osteomyelitis as a cause of worsening back pain in patients with history of back trauma and multiple back surgeries even in the absence of risk factors.
A CASE OF STERNOCLAVICULAR JOINT SEPTIC ARTHRITIS. E.H. Elbadawy 1 , K. Gopal 1 ; 1 Fairview Hospital, Cleveland, OH (Tracking ID #76754) LEARNING OBJECTIVES: 1-To recognize the clinical picture of sternoclavicular septic arthritis. 2-To emphasize the importance of early diagnosis and treatment in preventing devastating complications. CASE INFORMATION: A 43-year old white male admitted with 2 days history of worsening dull aching pain, swelling and erythema at the right sternoclavicular joint (SCJ) area. Condition started 2 days after he sustained a fall as he was wrestling with his 8-year old son. Patient denied fever, chills or dyspnea. Past medical history was significant for hypothyroidism and depression. Patient denied IV drug abuse, alcohol intake or smoking. On examination he was febrile at 103 F with pulse of 125 and blood pressure of 146/81. He appeared to be in pain with significant soft tissue swelling, erythema and marked tenderness over the right SCJ. Range of motion of the right shoulder was severely limited due to pain. Initial laboratory evaluation revealed WBCs of 12.5 k/AL and ESR of 56. Therapy was initiated with imipenem 500 mg IV every 6 hours for possible SCJ septic arthritis because of history of penicillin allergy. SCJ aspiration was unsuccessful in obtaining any fluid. Computed tomography scan revealed soft tissue swelling around the right SCJ with no evidence of fluid collection. Three-phase bone scan showed increased soft tissue activity and delayed bony uptake involving the right SCJ area. Blood cultures drawn on admission grew methicillin-sensitive Staphylococcus aureus and therapy was switched to cefazolin 2 grams IV every 8 hours. Patient reported significant improvement is his symptoms after 3 days of intravenous antibiotics and was discharged home on cefazolin 2 gm IV for total of 4 weeks. Follow-up one week later revealed complete resolution of the soft tissue swelling, tenderness and erythema with painless full range of motion of his right shoulder. IMPLICATIONS/DISCUSSION: Sternoclavicular septic arthritis accounts for 1±9% of septic arthritis cases. Predisposing factors include IV drug abuse, DM, central venous catheters and trauma. Aspiration of the joint can be helpful in confirming the diagnosis and guiding treatment. Unfortunately, the failure rate with this method is high due to the technical difficulty in aspirating the small joint space, as in our patient. Clinical diagnosis requires a high index of suspicion in any patient presenting with swelling and pain around the SCJ. Spread of infection can lead to superior vena cava obstruction, mediastinitis and septic shock. Early diagnosis is critical in order to prevent life threatening complications.
M. Eskildsen 1 , K. Lasser 1 ; 1 Cambridge Hospital, Cambridge, MA (Tracking ID #74000) LEARNING OBJECTIVES: 1. To recognize the common side effects of atypical antipsychotics. 2. To learn about a previously unreported complication of these drugs. CASE INFORMATION: A 39-year-old man with a history of hepatitis C, schizophrenia, and past alcohol abuse was admitted with worsening abdominal pain and distension over one month, accompanied by vomiting and watery diarrhea. He stated that he had been sober for two years. His only medications were risperidone at 8 mg daily, and olanzapine at 10 mg daily. His psychiatrist had decreased the olanzapine dosage and attempted to discontinue his prescription one year prior to admission due to poor response, but the patient had refused to do so for unclear reasons. Physical exam revealed a temperature of 37.8C; and a distended, diffusely tender abdomen, without signs of ascites or peritoneal irritation. Laboratory evaluation was remarkable for triglycerides that were over 3800 mg/dl, our lab's upper limit of measurement, and a lipase of 250 U/l. Serum glucose was 553 mg/dl, with no prior readings in the diabetic range. A computed tomography of the abdomen indicated mild pancreatitis with no biliary obstruction. On admission, after discontinuing both medications, we gave him intravenous fluids and nothing by mouth, as well as patient-controlled analgesia with intravenous morphine. Upon discharge, one week later, he was pain-free, and his triglycerides and glucose decreased to 308 and 250 mg/dl, respectively. Of his antipsychotics, risperidone alone was resumed before discharge, up to a dose of 8 mg daily two months later. He has remained without abdominal pain, with serum triglycerides in the 300 range, and persistent diabetes mellitus treated with metformin. IMPLICATIONS/DISCUSSION: A MEDLINE search yielded two reports of pancreatitis associated with olanzapine, and two with risperidone. Hyperlipidemia, including elevated triglycerides, is a side effect of both drugs, although it is more commonly observed with olanzapine. However, this is the first described case of pancreatitis in relation to either of these two drugs that has been associated with massive hypertriglyceridemia, and we have reported it as an adverse event to the FDA. Because he has tolerated risperidone well after discharge, olanzapine seems more likely to have precipitated his pancreatitis, although a synergistic mechanism between the two drugs is another possibility. This case illustrates the need for close monitoring of lipids and glucose in patients taking atypical antipsychotics. 
A 28 year old white male with no significant past history was referred for preoperative clearance for left foot amputation. Patient had non-healing ulcers on his left heel and ankle for 1 month with no response to antibiotics. One year prior, he had a flulike illness, hematuria and painful left testicle which resolved spontaneously except for a persistent dry cough. CXR revealed a spot on the left lung. His TB skin test was negative. The patient denied hematospermia, weight loss, night sweats, trauma, neurologic or GI symptoms. On exam he was a healthy appearing male with previously unnoticed 2 crusted, raised lesions measuring 2.5 Â 2.5 cm on the left arm. These lesions had not been documented during his earlier visit notes though patient admitted to having these lesions for many months. He also had 3 left foot lesions, 1 on the heel and 2 draining lesions on the left malleolus. A plain film of left ankle revealed a lytic area in the distal tibial metaphysis. Bone scan was consistent with acute osteomyelitis. Biopsy of left bony lesion revealed chronic inflammation & giant cells but negative AFB stains and routine cultures. On H & E stain, a multinucleated, thick double contoured wall of yeast with single, broad-based buds was visualized consistent with BD. The patient had complete resolution of all skin lesions and CXR after 6 months of itraconazole treatment. IMPLICATIONS/DISCUSSION: BD is a fungus found in the soil and causes multisystemic pyogranulomatous disease. Blastomyces in endemic in areas bordering Mississippi and Ohio river basins and the Great Lakes. Initial infection is usually via the lungs, can be asymptomatic in 50% of patients or be mistaken for the flu or pneumonia. Pulmonary manifestations include involvement of upper lobes with cavitation, mass lesion with productive cough, hemoptysis, dyspnea, pleuritic chest pain, low grade fever, night sweats, and weight loss. Skin lesions are usually painless, nonpruritic, hyperkeratotic, ulcerative plaques on the face or extremities and may involve mucosa of nose, mouth and larynx. Other common sites of involvement include bone, genitourinary tract or CNS. If untreated, the clinical course can be months to years with remissions, exacerbations and progression in size. The rapid method of diagnosis is visualization of budding yeast in KOH mounts of pus from lesions. Definitive diagnosis is by growth of organism in culture. Treatment consists of 6 months of itraconazole in immunocompetent patients. Amphotericin B is used in patients who are pregnant, seriously ill & immunocompromised, with CNS infection or progressive disease on itraconazole. Our case stresses the importance of a thorough history and physical for appropriate management especially in a disease that is known as the great masquerader.
NECROTISING FASCIITIS. C. Eze 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77055) LEARNING OBJECTIVES: 1. Recognize the signs of deep fascial infections. 2. Use the physical examination to prompt timely surgical intervention for necrotizing fasciitis. CASE INFORMATION: A 21 year-old Hispanic-speaking man presented with four days of left leg pain, swelling and fever after a spider bite. He had a temperature of 103 F, a heart rate of 128, and a blood pressure of 128/70. His left lateral leg was warm, edematous and tender. There was a 1 cm ulcer without drainage or necrosis. The pulses, sensation and range of motion of were normal. A plain film of his leg showed no soft tissue air or bone involvement. His WBC count was 22,000; the remaining laboratory values were normal. Blood cultures were obtained and he was empirically started on gatifloxacin due to a penicillin allergy. After 48 hours, he showed no improvement. Bullae and ecchymosis were noted on the second day; he lost sensation of the overlying skin on the third day. His antibiotic coverage was broadened to include coverage for MRSA. An MRI revealed fluid collections between the muscle and fascia consistent with necrotizing fascitis. An emergent fasciotomy and posterior compartment release were peformed. Wound cultures grew group A Strep. and Strep. milleri. IMPLICATIONS/DISCUSSION: The timely diagnosis of necrotizing fasciitis is important to preventing tissue damage, systemic toxicity, limb loss or death. Cellulitis not responsive to antibiotics, or the appearance of ecchymosis, bullae or anesthesia should prompt consideration of this diagnosis. The physical examination reflects the deep fascial structured damaged by the advancing infection. Ecchymosis, bullae and anesthesia reflect the damaged vessels, skin planes and nerves. While MRI is useful, establishing a timely pre-test probability prompting surgical exploration is the gold standard for making the definitive diagnosis. The closed space of deep fascial infections renders them immune from systemic antibiotics; treatment requires immediate surgical release. Antibiotics only buy time for surgical intervention.
AUTONOMIC DYSFUNCTION AFTER HEAD TRAUMA. C. Eze 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77084) LEARNING OBJECTIVES: 1. Identify the subtle presentation of autonomic dysfunction in the elderly. 2. Recognize the association between autonomic dysfunction and spinal cord lesions. CASE INFORMATION: An 81-year-old man presented with acute confusion following six months of progressive lower extremity weakness. He had fallen two weeks prior resulting in a periorbital hematoma. A CT had been obtained on two prior ER visits to exclude a sub-dural hematoma, and was negative on both occasions. His initial blood pressure was 122/60 mmHg with a pulse of 84. Fifteen minutes later his blood pressure dropped to 64/42 with a pulse of 134. Intravenous fluids resulted in a blood pressure of 175/104 mmHg with fifteen minutes. Ten minutes later his blood pressure again declined to 55/39 mmHg He was placed on an epinephrine drip with an immediate and sustained response of his blood pressure to 169/101 mmHg. His examination was notable for flat neck veins, no S3, and normal lung sounds. His reflexes were absent in both lower extremities without a Babinski's response; his upper extremity reflexes were 1+. His laboratory values, including cortisol, were normal. The EKG revealed atrial fibrillation. There was no subdural hematoma on repeat head CT. Owing to the suspicion of autonomic insufficiency, cervical-spine X-ray was obtained, revealing compression fractures involving the C4 to C7 vertebrae. He was admitted to the ICU and neurosurgery was consulted. IMPLICATIONS/DISCUSSION: Wide hemodynamics variations should prompt consideration of autonomic dysfunction, that should in turn prompt suspicion for an occult spinal cord lesion. This is especially true in elderly patients who may not be able to verbalize cervical pain. Hypertention and bradycardia may result from spinal cord reflexes unopposed by central regulatory mechanisms. Support of vascular tone with vasopressors is critical to resuscitation. This case emphasizes the importance of a careful cervical spine evaluation in all falls involving the elderly. LEARNING OBJECTIVES: To recognize that high dose simvastatin monotherapy, although having an excellent safety profile, can cause significant rhabdomyolysis, acute renal failure and hepatitis in the right patient population. To recognize that the addition of a potent or weak cytochrome P450 inhibitor is not needed for the significant adverse effects of simvastatin to occur. CASE INFORMATION: A 62 y.o. woman developed increasing myalgias, malaise, shortness of breath and dyspnea on exertion over 1 week and left lower extremity swelling. Medications included simvastatin 80mg daily with normal baseline transaminase levels and diltiazem for the past year. CXR revealed mild bilateral pulmonary edema and an ultrasound of the left lower extremity revealed no deep vein thrombosis. Exam revealed in ill appearing female in moderate distress secondary to myalgias, mild rhonchi bilaterally, normal heart tones and bilateral lower extremity dependent edema (Lt>Rt). Laboratory studies: alkaline phosphatase 177, AST 1511, ALT 224, LDH 1396; creatinine 5.6 (baseline 1.4±1.7); BUN 92; potassium 5.6; phosphate 6.5; calcium 8.8; creatinine phosphokinase >20,000; troponin <0.05; CKMB 171; TSH wnl; uric acid 8.0. Urinalysis revealed a large amount of blood with only 4±10 red blood cells. Simvastatin was discontinued. Aggressive treatment with sodium bicarbonate, intravenous hydration and hemodialysis was begun. Over a sixty-plus day hospital course, the patient's renal failure resolved (baseline creatinine now 2.2±2.4) and transaminases normalized. The patient was eventually discharged to home to continue physical rehabilitation with no permanent requirement for hemodialysis. IMPLICATIONS/DISCUSSION: Simvastatin's long term safety profile is excellent. Simvastatin induced rhabdomyolysis is reported with low incidence (0.6% in one study involving 80mg of simvastatin) and it is seldom associated with acute renal failure or hepatitis. Myopathy and rhabdomyolysis increases with dose and with concomitant use or addition of potent cytochrome P450 3A4 (CYP450) inhibitors, i.e. fibrates, cyclosporine, azathiaprine. A baseline severe renal insufficiency (creatinine clearance by the Cockroft-Gault method, 17cc/ min) and a urinary tract infection treated with a fluoroquinolone 2 days prior, combined to increase the risk of this patient developing rhabdomyolysis. Published reports of simvastatin induced myopathy involve the addition of potent or weak CYP450 inhibitors. Our patient had been stable on high dose simvastatin and diltiazem (a weak CYP450 inhibitor) for over 1 year prior to presentation and did correlate with the addition of known CYP450 inhibitors. Additionally unique, this is the first reported case involving the combined toxicities of rhabdomyolysis, acute renal failure and hepatitis with high dose simvastatin monotherapy. 2) Recognize the dangers of rapid correction of hyponatremia. CASE INFORMATION: A 56 year-old man with a history of alcohol abuse was admitted to an outside hospital with altered mental status. On exam, the patient was disoriented, but neurological exam was otherwise nonfocal. Computerized tomography (CT) of the head was unremarkable. Laboratory results revealed hyponatremia with sodium of 104 mmol/L. The patient was given hypertonic saline, and the sodium was corrected to 123 within the first 24 hours. The patient was then transferred to our facility for continuation of his care. Physical examination on arrival revealed multiple focal neurological findings including bilateral eye deviation to the left, neck deviation to the left, absent gag reflex, and poor motor coordination with spastic movement of all extremities. The patient's speech was severely dysarthric and unintelligible. Magnetic resonance imaging (MRI) of the brain showed enhancement of the motor cortex of both cerebral hemispheres and enhancement of the frontal eye fields on the left with sparing of the adjacent sensory cortex. In consideration of the MRI findings, neurology consultation was sought. The patient's severe dysarthria, dysphagia, ataxia, and optic apraxia were attributed to extrapontine (as opposed to central pontine) myelinolysis from rapid correction of the patient's hyponatremia. IMPLICATIONS/DISCUSSION: Severe symptomatic hyponatremia should be treated with hypertonic saline, but with a correction in plasma sodium of not more than 1±2 mmol/liter/ hour and a maximum increase of 8 mmol/liter during the first 24 hours. Our patient's sodium was corrected from 104 to 123, with an increment of 19 within 24 hours. Rapid correction may result in osmotic demyelination or central pontine myelinolysis, which is characterized by flaccid paralysis, dysarthria, and dysphagia. Isolated extrapontine myelinolysis is extremely rare, with only two cases reported in the literature. The neurologic deficits depend on the anatomical location of osmotic demyelination. Treatment is supportive, and recovery is slow within weeks to months. Gradual correction of hyponatremia is important because aggressive correction can cause not only central pontine myelinolysis but also the unusual manifestation of extrapontine myelinolysis. LEARNING OBJECTIVES: 1) Construct a differential diagnosis of chronic ataxia. 2) Review the etiology, presentation, and prognosis of paraneoplastic cerebellar degeneration. CASE INFORMATION: A 55-year-old male ex-smoker with no significant past medical history presented to clinic with paresthesias of the right upper and lower extremities, followed by gradual onset of ataxia, diplopia, and dysarthria over a period of five months. The patient also reported weight loss and a chronic dry cough. The patient had no history of toxic exposures or family history of inherited ataxias. Physical exam revealed optical dysmetria, truncal ataxia, finger-to-nose dysmetria, and dysdiadochokinesis. The patient's speech was severely dysarthric, and sensation was decreased on the right upper and lower extremities. Magnetic resonance imaging (MRI) of the brain was normal. HIV and RPR serologies were negative, and TSH was within normal limits. Lumbar puncture revealed paraneoplastic antibodies, including anti-Hu, anti-P/Q type calcium channel, and anti-acetycholine receptor. Computerized tomography (CT) of the thorax demonstrated a 2 cm left hilar mass. Bronchoscopy and endobronchial biopsy were negative, but subsequent thoracotomy and biopsy revealed small cell lung carcinoma (SCLC). IMPLICATIONS/DISCUSSION: The differential diagnosis of chronic ataxia includes primary and metastatic neoplasms, paraneoplastic syndromes, progressive multifocal leukoencephalopathy, multiple sclerosis, hypothyroidism, tabes dorsalis, inherited ataxia syndromes, vitamin E deficiency, toxic exposures, and stable gliosis due to stroke or demyelination plaque. In approximately 80% of cases with paraneoplastic cerebellar degeneration, the underlying tumor is SCLC. Fifty-one percent of patients with cerebellar degeneration and SCLC have detectable anti-Hu antibodies. Other antibodies found in paraneoplastic cerebellar degeneration include anti-P/Q calcium channel, anti-Yo, anti-Ma1, anti-Tr, and anti-CV2, but their exact sensitivities and specificities are not yet known. Therapy is supportive, and symptoms usually do not improve with immunosuppression or treatment of the tumor. In conclusion, paraneoplastic cerebellar degeneration with paraneoplastic antibodies should be considered in all patients with chronic ataxia without other predisposing etiologies. LEARNING OBJECTIVES: 1. Discuss the presentation of primary bacterial peritonitis. 2. Recognize group A beta-hemolytic Streptococcus as a rare but serious cause of primary peritonitis. CASE INFORMATION: A 52 year old woman presented to the Emergency Room with a four day history of fevers, chills, and profuse watery diarrhea with associated crampy lower abdominal pain. She denied any nausea, vomiting, cough, dysuria, or headaches. She denied any recent travel or sick contacts, but noted that the symptoms began hours after having eaten at a Chinese restaurant. Her past medical history was remarkable for a prior appendectomy, she was on no medications, and her family history was significant only for colon cancer in her mother. Her exam revealed a temperature of 38.7 C, a blood pressure of 72/40, and a pulse of 112, with abdominal tenderness and guarding in both lower quadrants. Labs showed a white cell count of 5.7 (with a bandemia of 52), a potassium of 2.4, a bicarbonate of 15, a Blood Urea Nitrogen of 52, and a creatinine of 3.7. Liver function tests, amylase, lipase, and urinalysis were all normal. Fecal leukocytes were absent and her abdominal film showed only a few air-fluid levels. It was the impression of all clinicians involved that the patient's presentation was most consistent with acute infectious diarrhea and profound dehydration complicated by renal failure and electrolyte derangement. Following aggressive hydration with IV crystalloids, the patient's blood pressure improved to 120/80 and her heart rate slowed to 88. After blood, urine, and stool cultures were taken, she was started on IV ciprofloxacin for empiric treatment of her presumed acute infectious diarrhea. She continued to have diarrhea over her hospital course, but her abdominal pain and temperature curve lessened, and she appeared to be improving clinically. All cultures (including c. difficle and giardia screens) had returned negative. On hospital day four, her fever and abdominal pain worsened and her abdomen became distended, rigid, and equisitely tender. An abdominal CT showed diffuse thickening of her colon with ascites and dilated loops of small bowel. An emergent exploratory laparotomy was performed and several liters of cloudy yellow ascites were drained. A nectrotic mesenteric lymph node filled with pus was resected and her abdomen was irrigated with several liters of saline. Gram stains of both the pus from the lymph node and the ascitic fluid revealed gram positive cocci in chains, and she was promptly started on IV clindamycin. Cultures of the ascites and pus grew group A betahemolytic Streptococci, and she was continued on the clindamycin. She continued to improve over her hospitalization and made a full and healthy recovery. IMPLICATIONS/DISCUSSION: Primary bacterial peritonitis occurs as a result of bacterial infection of the peritoneal cavity and is distinguished from secondary peritonitis by the absence of any underlying cause or demonstrable abdominal source. The onset is often rapid and insidious, and is marked by the appearance of fever, nausea, abdominal pain, and diarrhea. The patient typically has abdominal rigidity and rebound with features of systemic inflammation (tachycardia, hypotension, tachypnea, etc.) which may progress to frank shock. Streptococcus pyogenes, or group A Streptococcus, is a very common human pathogen, with up to 15% of asymptomatic individuals carrying the bacterium (typically in the upper respiratory tract). However, primary bacterial peritonitis due to Streptococcus pyogenes is exceptionally uncommon with very few adult case reports documented in the literature. It may be slightly more common in women, with asymptomatic colonization of the genital tract acting as a portal of entry. The diagnosis is often made only at laparotomy and despite the profound morbiditiy associated with the disease, rapid initiation of the appropriate antibiotics usually results in a successful cure.
EUREKA! A TREATABLE CAUSE OF DIZZINESS. E.J. Favus 1 , R. Ambrosino 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73835) LEARNING OBJECTIVES: 1. To recognize and treat non-sustained ventricular tachycardia (NSVT). 2. To recognize the differential diagnosis of dizziness.
A 70 year-old male with a history of gout, hypertension, chronic renal insufficiency, and hypercholesterolemia presented to his primary care physician complaining of intermittent dizziness. He had approximately three episodes per week, each lasting ten minutes, and resolving spontaneously. He described feeling``lightheaded'' during each episode, and denied a positional component, vertigo, visual disturbances, tinnitus, history of seizures, aura, post-ictal state, loss of consciousness, palpitations, chest pain, and shortness of breath. An EKG shortly after the first episode showed no rhythm abnormalities, ischemia, or infarction. His chronic medications were simvastatin, allopurinol, and lisinopril. Physical examination revealed a blood pressure of 156/84mmHg, pulse 66, and respiratory rate 16. He did not have orthostatic hypotension. Cardiac exam revealed a non-displaced PMI, regular rate and rhythm, normal S1 and S2 with no murmurs, gallops, or rubs. Holter monitor testing revealed multiple episodes of multifocal ventricular ectopy including short runs of ventricular bigeminy, trigeminy, and quadrigeminy with a heart rate of 80 beats per minute. He was diagnosed with non-sustained ventricular tachycardia (NSVT). A review of the patient's log revealed that each tachyarrhythmic event correlated with an episode of dizziness. He underwent echocardiography, which showed a structurally normal heart and an ejection fraction of 55%. He was prescribed metoprolol 12.5mg po qam, and at a one-month follow-up visit reported improvement in his symptoms. IMPLICATIONS/DISCUSSION: Dizziness is a common complaint heard by primary care physicians, yet only a small percentage of patients receive a firm diagnosis and an effective treatment. In this patient, the discrete nature of these episodes hinted at the possibility of a cardiac arrhythmia. Clinical suspicion for a cardiac problem prompted an evaluation by Holter testing. The results showed a temporal relationship between NSVT and dizziness, and provided evidence for a firm diagnosis. Patients with NSVT and a structurally normal heart are not at risk for sudden death, and may be treated with daytime oral beta-blocker therapy to decrease symptoms. Conversely, large studies have shown that the combination of NSVT and a structurally abnormal heart is a risk factor for sudden death. Treatment of NSVT with a structurally abnormal heart requires the use of an Automatic Implantable Cardioverter-Defibrillator (AICD). Beyond cardiac arrhythmias, dizziness has many causes. A proper history of the dizzy patient should include questions about syncope, hyperventilation, anxiety, visual disturbances due to incorrect eyeglasses or diplopia, and concussion. Physical examination should evaluate orthostatic hypotension, hypertension, and peripheral neuropathy. Anemia and hypoxia should be evaluated by laboratory studies.
PUTTING IT ALL TOGETHER: A CASE OF PRIMARY HYPOGONADISM. E.J. Favus 1 , R.C. Brooks 2 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA; 2 Pittsburgh VA Healthcare System, Pittsburgh, PA (Tracking ID #73840) LEARNING OBJECTIVES: 1. To recognize the clinical presentation of male hypogonadism. 2. To recognize the differential diagnosis of acquired primary hypogonadism. CASE INFORMATION: A 66 year-old male presented with bilateral gynecomastia, osteoporosis, and erectile dysfunction. Three years ago he was seen by a urologist for erectile dysfunction and hesitency. He was diagnosed with a low testosterone state (laboratory values unavailable), and treated with testosterone. He had improvement of his erectile dysfunction but worsening of his hesitency and was switched to sildenafil. No cause was found for urinary hesitancy, but he was treated with tamsulosin without relief. Two years ago he fell and broke his left fourth metatarsal. Dual x-ray absorptiometry scan revealed a T-score of À2.7, and he was treated with alendronate. He denied a history of glucocorticoid use. One year ago he noticed increased breast tissue bilaterally and a mass in the right breast. A bilateral mammogram was negative and a breast biopsy was negative for malignant cells. His medical history is significant for tinea capitis, treated with 2% ketoconazole shampoo. His medications are sildenafil, alendronate, and ketoconazole shampoo. He has no history of alcohol, tobacco, or drug use. Physical examination reveals a normal thyroid, moderate bilateral gynecomastia without masses, no kyphosis, and normal size and shape of the penis, testicles, and prostate. Laboratory tests show total testosterone 239 ng/dL (241±827ng/dL), free testosterone 5.28 ng/dL (5±21ng/dL), FSH 32.1 mIU/mL (adult male normal range 1.55±9.70 mIU/mL), LH 14.7 mIU.mL (adult male normal range 0±9.0 mIU/mL), prolactin 13.0 ng/mL (adult male normal range 3.7± 17.9 ng/mL), TSH 4.13 uIU/mL (0.35±5.5 uIU/mL), PSA 1.68 ng/mL (0±4ng/mL). Given his low-normal testosterone and his clinical symptoms, he was re-started on testosterone therapy. IMPLICATIONS/DISCUSSION: The clinical presentation of osteoporosis, erectile dysfunction and gynecomastia should provoke a search for a unifying diagnosis of hypogonadism. The medical history of this patient is suggestive of a low testosterone state. His serum testosterone level is in the very low-normal range, and the FSH and LH levels needed to maintain this low-normal testosterone level are three times the upper limit of normal. High levels of FSH and LH exclude the diagnosis of secondary and tertiary hypogonadism. The low-normal level of testosterone has not been enough for this patient to avoid developing clinical disease. Male hypogonadism is defined as a failure of the testes to produce a sufficient amount of testosterone. Hypogonadism can be primary (testicular failure), secondary (pituitary failure), tertiary (hypothalamic failure), or combined primary and secondary. Primary hypogonadism can be either acquired or developmental. Our patient's laboratory values suggest primary hypogonadism. Since he had normal sexual development and fathered two children, he likely has acquired primary hypogonadism. Acquired primary hypogonadism may be the result of amyloidosis, autoimmune destruction, cryptorchidism, leprosy, leukemia, mumps, polyarteritis nodosa, radiation therapy, trauma, surgery (castration), or drugs such as cancer chemotherapy, ethanol, ketoconazole, marijuana, or spironolactone. In this patient, long term administration of ketoconazole shampoo for the treatment of tinea capitis may have resulted in acquired primary hypogonadism.
USING THE HEAD AND NECK EXAMINATION TO DIAGNOSE FOCAL NEUROLOGIC DEFICITS. D. Fotino 1 , N. Parekh 1 ; 1 Tulane University, New Orleans, LA (Tracking  ID #77089)   LEARNING OBJECTIVES: USE THE EXAM SKILLS TO DIAGNOSE FOCAL  NEUROLOGIC DEFICITS.  CASE INFORMATION: A 41 year-old diabetic man presented with a sudden onset of slurred speech and left-sided weakness. On examination, he had severe dental caries, slurred speech, and a left facial droop. His tongue deviated to left on protrusion, and he had left upper and lower extremity weakness. There was tenderness over the right mastoid process. His WBC was 27,000 with 14% bands. A CT showed subdural fluid collection in the right frontoparietal region. Vancomycin, ceftazidime and metronidzaol were empirically started for a presumptive diagnosis of subdural empyema. Shortly after admission, he deteriorated to persistent and refractory status epilepticus. The CSF protein was 149; WBC 630 (70% neutrophils); the gram stain was negative. A repeat CT showed an area of hypodensity and a midline shift, consistent with subdural empyema. He underwent drainage of the abscess; the cultures grew out pseudomonas aeruginosa. Ciprofloxacin was added to his regimen to double cover the pseudomonas. His seizure activity relented soon thereafter. IMPLICATIONS/DISCUSSION: Sinusitis is the leading cause of subdural empyema, and can present with focal neurological signs if intra-cerebral extension occurs. Diabetic patients are also at risk for malignant external otitis, and empiric coverage for pseudomonas is warranted at even the lowest level of suspicion. Although this patient's symptoms suggested a cerebrovascular accident, the finding of mastoid tenderness directed the diagnosis towards the infected mastoid sinus and the subdural abscess. In diabetic patients, empiric pseudomonas coverage should be initiated early; double coverage should be instituted when a pseudomonas infection is confirmed, as this reduces the mortality of deep tissue infections by 50% when compared to single coverage. A thorough examination of the oro and nasopharyngeal cavities can direct and expedite diagnostic testing and prevent grave sequelae of treatable diseases.
A ROCKY SITUATION. F. Francois 1 , J. Park 1 , C.T. Tenner 2 , S. Finkelstein 1 ; 1 New York University School of Medicine, Brooklyn, NY; 2 New York University School of Medicine, New York, NY (Tracking ID #74431) LEARNING OBJECTIVES: 1. Recognize the potential for bowel dysfunction in chronically ill patients 2. Consider the differential diagnosis of lower gastrointestinal bleeding in the elderly population 3. Review the management of stercoraceous masses. CASE INFORMATION: A 72-year-old HIV positive Hispanic male on highly active anti retroviral therapy (CD4 = 612 cells per cubic millimeter, viral load<50 copies per milliliter), was admitted for evaluation of hematochezia. The patient had a history of poorly controlled hypertension and had been hospitalized three weeks prior to this presentation with an acute change in mental status during which he was found to have a basal ganglia infarct. The family reported that the patient had been mainly bed bound since the stroke, and had intermittently complained of difficulty moving his bowels without any associated abdominal pain, nausea, or vomiting. On physical exam the patient was noted to have a right facial droop, right-sided hemiparesis, and normal active bowel sounds without abdominal distention, tenderness, or guarding. On rectal examination a freely mobile mass was palpated in the vault and bright red blood was seen on the glove. Laboratory evaluation was significant for a seven-point hematocrit drop from his baseline. On colonoscopy the patient was found to have 3 discontinuous areas of ulceration in the rectosigmoid region, with the largest measuring 6cm  2cm. The ulcers were associated with 3 large (4cm in diameter) hard fecal masses (fecalomas). The fecalomas were manually evacuated and the patient was started on a bowel regimen consisting of stool softeners and hydration. The patient's hematocrit remained stable with no further episodes of rectal bleeding and he was discharged to to the care of his family. IMPLICATIONS/DISCUSSION: Elderly patients may develop bowel dysfunction due to diabetes mellitus, hypothyroidism, renal failure, dementia, immobilization, cerebrovascular accidents, and medications. Once chronic constipation occurs the patients are at risk for fecal impaction and stercoral ulcers (a.k.a. Huntley syndrome). These ulcers result from pressure necrosis on the colonic mucosa by the rock-like fecal mass. Patients may present with rectal bleeding or colonic perforation with peritonitis. In the elderly population, the differential of hematochezia as a presenting sign in the setting of a change in bowel habits includes, malignancy, infectious colitis, and ischemic colitis. Initial management of a stercoral lesion involves manual evacuation of the stecoraceous mass followed by a bowel regimen with stool softeners along with adequate hydration. Patients with recurrent ulcer bleeding or perforation may require surgery as definitive therapy.
SEA-BLUE HISTIOCYTOSIS: A NEWLY REPORTED COMPLICATION OF PARENTERAL NUTRITION. S. Frost 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #73897) LEARNING OBJECTIVES: 1) Diagnose sea-blue histiocytosis syndrome as a complication of total parenteral nutrition (TPN). 2) Recognize that bone marrow sea-blue histiocytes are associated with many diseases in which the degradation of lipids and cellular lipid products is exaggerated. CASE INFORMATION: A 40 year-old man receiving chronic TPN with lipid emulsion after esophagogastrectomy following caustic ingestion was admitted to the hospital with fever for 5 days, jaundice, hepatitis, and pancytopenia. The patient did not appear ill. The physical exam was notable for a clean subclavian Hickman catheter site without erythema or tenderness, hepatomegaly, no heart murmur, and no peripheral stigmata of endocarditis. Laboratory revealed WBC = 3.8 K/uL, hemoglobin = 10 g/dL, platelet = 70 K/uL, AST = 223 U/L, ALT=160 U/L, alkaline phosphatase = 339 U/L, and bilirubin = 9.4mg/dL. Investigation for bacterial, fungal, mycobacterial, and viral infection was negative. Abdominal sonogram and CT scan revealed hepatomegaly without other abnormality. Echocardiogram revealed no vegetations. Upper extremity ultrasound was negative for catheter associated thrombosis. The Hickman catheter was removed and catheter culture specimens were sterile. Fever persisted despite therapy with piperacillin/tazobactam, vancomycin, fluconazole, and amphotericin B. Bone marrow biopsy revealed sea-blue histiocytes. TPN was held and improvement in liver function abnormalities and cell counts began on hospital day (HD) 6 and 7 respectively. Fever dissipated on HD 8, and antimicrobials were discontinued. TPN was restarted without lipid emulsion, and the patient has remained well with resolution of hepatitis and cytopenias. IMPLICATIONS/DISCUSSION: Sea-blue histiocytes (SBHs) are macrophages with lysosomal lipid accumulations that appear blue with Giesma staining. The sea blue histiocytosis syndrome is a typically benign idiopathic disorder characterized by hepatosplenomegaly, purpura, thrombocytopenia, and pulmonary infiltrates. SBHs have also been noted in association with hematologic disorders such as chronic myeloid leukemia, and metabolic illnesses such as Niemann Pick disease. SBHs in the bone marrow of patients with hepatosplenomegaly, jaundice, and pancytopenia receiving TPN containing lipid emulsions was first reported in 1996. Lowering the TPN lipid content has been shown to improve pancytopenia. Lipid induced hematopoetic cell membrane alterations triggering macrophage phagocytosis is one postulated mechanism accounting for TPN related sea-blue histiocytosis (TPN-SBH). This patient's illness is consistent with prior descriptions of TPN-SBH, except for fever, which has not been previously reported. However, the resolution of fever coincident with the improvement in pancytopenia and hepatitis after the discontinuation of parenteral lipids suggests an association with TPN-SBH.
IT'S BLACK, IT'S HAIRY . . . BUT IS IT A PROBLEM? C.C. Fu 1 , G. Applebaum 1 ; 1 University of California, Los Angeles, Los Angeles, CA (Tracking ID #74949) LEARNING OBJECTIVES: 1. Recognize the presentation of black hairy tongue. 2. Review possible predisposing conditions. 3. Emphasize benign nature of this phenomenon. CASE INFORMATION: A 53-year-old Mexican man with no significant medical history presented with one month of easy bruising and gum bleeding. The patient's only medicines were occasional aspirin for pain and bayberry extract. Lab results were significant for 2.2 WBCs, Hb 8.2, and 4 platelets. Bone marrow biopsy revealed a hypocellular and fatty marrow consistent with aplastic anemia. The patient's three-week hospital stay was complicated. He had extraction of an infected tooth and was started on a peroxide-based swish and spit regimen. Treatment for aplastic anemia was begun with anti-thymocyte globulin and cyclosporine. The patient experienced prolonged neutropenic fevers for which he was treated with ceftriaxone, tobramycin, metronidazole, and vancomycin. During this period, the patient's tongue was noted to develop a dark gray,``hairy'' appearance, the surface of which could actually be brushed with the end of a cotton swab. IMPLICATIONS/DISCUSSION: Black hairy tongue is a rare condition caused by the elongation of the filiform papillae associated with oral bacterial overgrowth. Black hairy tongue is idiopathic and has been associated with various predisposing factors including alcoholism, systemic illness, immunosuppression, cancer therapy, coffee, antibiotic use, and use of peroxide-based mouthwash. Our patient was a likely candidate to develop black hairy tongue as he had been given broad-spectrum antibiotics and used peroxide based mouthwash during immunosuppressive therapy. Treatments for black hairy tongue have included topical retinoin gel, podophyllum, and attempts at repeated tongue brushing. However, in consideration of our patient's underlying immunosuppression and profound thrombocytopenia, we chose not to treat the patient's tongue. It is important not to complicate the treatment of an ill patient with the unnecessary treatment of a benign, albeit strange, condition. A 45 year old Mexican female with a history of diabetes mellitus was brought to the emergency room by her family for increasing abdominal pain and a four day history of nausea, vomiting and diarrhea. There was no recent history of fevers, chills, or dysuria. Initial examination revealed an ill-appearing woman, moaning in response to painful stimuli, but not following commands. Although she was afebrile and normotensive, her resting heart rate of 119 beats per minute established systemic illness. Her other examination was remarkable for right lower quadrant tenderness in a soft abdomen, but no peritoneal signs. There was evidence of costovertebral angle tenderness bilaterally. Initial laboratory results were consistent with diabetic ketoacidsosis (DKA). Leukocytosis and pyuria led to the additional admitting diagnosis of pyelonephritis. Declining mentation led to rapid transfer to the intensive care unit on the day of admission. Computed tomography (CT) of the abdomen showed bilateral emphysematous pyelonephritis, emphysematous cystitis, bilateral perinephric abscesses, and air surrounding the inferior vena cava (IVC). Broad spectrum antibiotics were initiated. Emergent urology consultation recommended medical management due to high perioperative mortality risk. Bilateral percutaneous drains were placed under CT guidance. Nevertheless, the patient's condition rapidly deteriorated with development of thrombocytopenia and septic shock, requiring intubation and pressor support. She died the following hospital day. Blood, urine and perinephric fluid cultures grew Klebsiella pneumoniae. IMPLICATIONS/DISCUSSION: Emphysematous pyelonephritis (EPN) is a rare condition that causes an acute necrotizing parenchymal and perirenal infection due to gas-forming uropathogens. Bilateral disease is exceedingly rare. EPN occurs predominantly in diabetic patients and in patients with urinary tract obstruction. E.coli, Klebsiella and Proteus are the most frequently identified organisms. Computed tomography is used for diagnosis and evaluation of the extent of disease. Antibiotics and percutaneous drains can lead to successful outcomes in patients with limited disease. However, nephrectomy is often considered the treatment of choice, especially in patients with extensive disease or multiple poor prognostic factors. Bilateral disease makes the surgical decision even more difficult. Understanding the indications for surgical versus medical management may mean the difference between life and death in patients with EPN.
J.A. Garcia 1 ; 1 Wayne State University, Troy, MI (Tracking ID #74175) LEARNING OBJECTIVES: Physicians should be able to recognize early adverse events of commonly prescribed drugs. CASE INFORMATION: 56 y/o male, previously heatlhy presented to ER with fever, left calf pain and swelling which started after a brief hospitalization for a febrile illness. Lower extremity dupplex revealed thrombosis involving the Left popliteal and lesser saphenous veins. He was started on unfractionated heparin. Labs: WBC 10.2, Hemoglobin 11.7 and Platelets, 386k. Infectious work up was negative and he was eventually switched to coumadin and discharged. On the 4th day, he presented with fever and worsening left calf pain. A Lower extremity dupplex showed progression of the thrombus involving the posterior tibial vein. Heparin was restarted. WBC count 16.1, Hemoglobin 9.7 and platelets 240k . The fourth day of this admission Hb dropped to 6.5 with plts of 150k. On the 9th day, patient developed chest pain. EKG showed ST elevations and he had increased cardiac biomarkers compatible with acute lateral Myocardial Infarction. Cardiac catheterization revealed clean coronary arteries. On the 12th day, he complained of constipation, abdominal distension and pain. Abdominal Series showed ileus. Amylase 136, Lipase 73, ALT 21, AST 29, Alkaline Phosphatase 131, Tbili 0.9. WBC 27.2 Hemoglobin 11.7 and Platelets 72k. Because of the dropping platelets and the multiple thrombotic events, heparin was held, antibodies drawn, and liperudine was started. Heparin induced platelet aggregating antibodies were positive with ELISA. Abdominal CT showed focal areas of low attenuation with peripancreatic fat stranding compatible with necrotizing pancreatitis. IMPLICATIONS/DISCUSSION: Heparin is a widely used medication. Uncommon presentation of HITT should be early recognized to prevent unwarranted management. LEARNING OBJECTIVES: 1. Recognize the common toxicities of ecstasy 2. Diagnose water intoxication and its complications. CASE INFORMATION: KD is a 20-year old woman brought into the UCSF ED because she developed altered mental status after taking ecstasy. Seven hours prior to admission, she took 1.5 tablets of ecstasy, while her companion took 2 tablets of the same preparation. They remained at home and``drank lots of water.'' One hour prior to admission, KD complained of severe headache and multiple episodes of vomiting. She then``stopped making sense.'' In the ED, KD was alert and oriented to person and place but appeared agitated and confused. She then had a generalized tonic-clonic seizure, became apneic, and was intubated. A NCHCT showed bilateral uncal herniation with diffuse effacement of the sulci. Significant laboratory abnormalities included leukocytosis, serum sodium of 124, rhabdomyolysis, and evidence of SIADH. A urine toxicology screen was positive only for amphetamines. An ECG showed sinus tachycardia with diffuse ST elevations, and a chest radiograph showed bilateral patchy infiltrates consistent with pulmonary edema. Repeat physical exam three hours after admission documented the absence of brainstem reflexes. She expired 24 hours later, and her organs were harvested for transplantation. IMPLICATIONS/DISCUSSION: Ecstasy, or methylenedioxymethamphetamine (MDMA), is a recreational designer drug popular both for its pleasurable psychological effects and for its safe reputation. Acute adverse effects can range from nausea, headache, and myalgias to insomnia, paranoia, and depression. An ecstasy tablet often contains contaminants and other drugs. In addition, the dose of MDMA itself can range from 0 to 200 mg per tablet, and the toxic or even fatal dose range overlaps that of recreational use. The pattern of major physical toxicity is usually hepatic, cardiovascular, hyperpyrexic, or cerebral, although DIC, rhabdomyolysis, and acute renal failure also contribute to morbidity. Cerebral toxicity is manifested as seizures or cerebral edema, thought to result from hyponatremia. In most cases, the low sodium level is hemodilutional; users often sweat profusely and then drink large amounts of free water to rehydrate and to prevent overheating. Occasionally, SIADH contributes to hyponatremia in this setting. In addition to the physical toxicity, ecstasy can also lead to increased risk-taking behavior. Despite the widespread use of ecstasy and the range of potential toxicity, fatalities attributable to the drug are uncommon. KD's demise likely began with ingestion of MDMA, which led to water intoxication and SIADH. Hyponatremia followed, precipitating cerebral edema, increased intracranial pressure, and then uncal herniation. Respiratory arrest occurred with compression of the brain stem. The immediate cause of death was hypoxic encephalopathy. Several case reports suggest young women, such as KD, and children appear to be at the highest risk of poor outcomes associated with hyponatremia. This unfortunate case emphasizes the often overlooked and unpredictable morbidity and mortality associated with casual use of ecstasy.
GET YOUR COLONOSCOPY, GET YOUR CURE. D. Garrow 1 ; 1 New Hanover Regional Medical Center, Wilmington, NC (Tracking ID #74301) LEARNING OBJECTIVES: 1. Present a case of metastatic colon cancer. 2. Provide screening recommendations with colon cancer in a first-degree relative. 3. Discuss genetic factors implicated in colon cancer. CASE INFORMATION: The patient is a 57 year-old female who presented with dysequilbrium and hand tremor for one month. Head MRI revealed multiple intracranial lesions. ( fig. 1 ) Biopsy revealed poorly-differentiated adenocarcinoma. A colonoscopy was performed because of the above histology and a family history of colon cancer involving two brothers and a son. A near-obstructing mass was discovered in the right colon. Patient also had liver, spleen and multiple lymph node involvement via PET scan.
IMPLICATIONS/DISCUSSION: The majority of studies have shown a two-to four-fold increase in the incidence of colon cancer in patients with similarly afflicted first-degree relatives. Screening for colon cancer with a positive history in a first-degree relative is advised ten years prior to the index case. Hereditary nonpolyposis colon cancer (HNPCC) is a consideration in this patient. The genetic basis for HNPCC is heterogeneous involving chromosomes 2, 3, and 7. With the multiple genetic focus of HNPCC, genetic testing is seldom employed. The family is clinically positive for HNPCC as there are more than two cases of colon cancer spanning at least two generations. HNPCC is caused by defective DNA mismatch repair genes. Afflicted patients develop polyps at a rate similar to the normal population. However, because of genetic defects, precursor adenoma lesions undergo a more rapid progression to carcinomas than usual adenomas. HNPCC typically presents during the fourth and fifth decades of life. Affected patients are predisposed to a higher frequency of proximal colonic lesions with more advanced pathology, as seen in this case.
A. George 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking ID #76605) LEARNING OBJECTIVES: To recognize that shingles, although most commonly thought of as a sensory problem with a characteristic rash, can cause diaphragmatic paralysis. CASE INFORMATION: A 71 year-old white male with stable hypertension, CHF and COPD, presented to the emergency room with severe dyspnea. He had a 12-year history of chronic elevation of the left hemidiaphragm and had been well except for recent right-sided neck pain. There was no rash. He was intubated. The following day he developed a classic shingles rash involving the right side of the neck. An EMG revealed a right-sided C3-C4 motor neuropathy. Phrenic nerve function did not return. He died six weeks later when he extubated himself at a nursing facility. Autopsy revealed that the chronic elevation of the left hemidiaphragm was due to a benign angiolipoma and unrelated to the viral infection. IMPLICATIONS/DISCUSSION: Sir William Broadbent first described muscular paralysis in association with zoster in 1866. In 1949, Halpern and Covner published the first description of zoster induced diagphragmatic paralysis. Since then, anecdotal cases have been reported although complete historical data is frequently missing and it has been difficult to elucidate the natural history of the condition. Based upon cumulative data from 10 well-documented cases, several tentative conclusions can be made. The average patient is 69 years of age (range 54±79). The time from rash to paralysis can vary widely from almost immediately to 210 days. In 7/10 cases phrenic nerve function did not recover. In the remaining cases, recovery occurred between 30 and 540 days later. The right side was affected in 7/10 cases although this is not statistically significant. The ratio between male and female patients is 7/3 but not statistically significant in this series. Pathologically, zoster induces a myelitis that involves the dorsal root ganglia. Why certain individual develop this rare complication is unknown and will require many further observations. At present, though, the point is clear±herpes zoster can cause diaphragmatic paralysis in the elderly. In such cases the paralysis may develop as a late complication with a poor chance of recovery. CASE INFORMATION: A 63-year-old woman was referred to our center for evaluation of tinnitus in her right ear for 9 months. She described the sensation as,`like the sounds of a steam engine pumping in my ear,' and that it coincided with her heart beat. She noted that applying pressure with fingers over an area in the right retroauricular area would reduce this abnormal noise. Work-up at an outside hospital was negative including, carotid ultrasound, CT scan of mastoid and temporal bone, audiometry and MRI of the head and internal auditory canal. Examination was significant only for a loud continuos bruit over the retro-mastoid area. Complete blood counts, serum chemistry, and electrocardiogram was normal. Cerebral angiogram showed an extensive dural arteriovenous fistula (AVF) affecting the distal transverse and sigmoid sinuses with supply through both internal and external carotid arteries as well as right vertebral artery. There was no evidence of cortical venous thrombosis. Thrombophilia work-up was normal. She underwent a combined transarterial embolization and stereotactic radiosurgery using gamma knife. Tinnitus resolved completely after intervention. She remains asymptomatic at the 3-month follow-up visit. IMPLICATIONS/DISCUSSION: Pulsatile tinnitus resulting in audible retro-auricular bruit could result either from systemic diseases, causing alteration in hemodynamic, or due to local disorders within or close to the petrous bone. The most common causes of pulsatile tinnitus includes benign intracranial hypertension, atherosclerotic disease, and glomus tumor. Dural AVF is a rare cause of pulsatile tinnitus (2%).However, 90% of patients with dural AVF present with pulsatile tinnitus. Dural AVF has been reported following trauma, infection, pregnancy related thrombophlebitis, surgery on the sigmoid and ethmoid sinuses. Cerebral angiogram remains the diagnostic modality of choice. If untreated, a third of dural AVF's develop intracranial bleeding or infarction. Treatment of dural AVF involves transarterial particulate embolization and radiosurgery using a gamma knife .High index of suspicion and thorough physical examination remains crucial in the early recognition of dural AVF. 2) Recognize principles of management of diabetes mellitus during Ramadan. CASE INFORMATION: A 40-year old female immigrant from Somalia with the history of poorly controlled type II diabetes for the past seven years was enrolled in our continuity clinic. She had been on numerous oral hypoglycemic agents without adequate benefit. Her blood glucose ranged from 200±380 mg/dl and glycosylated hemoglobin was 10.7%. She was started on insulin 70/30, 100 units in the morning and 70 units in the evening with addition of oral thiaglitazone agent. During the month of Ramadan her insulin dose was decreased to 50 units in the morning and evening and she was advised to take her oral medication in the evening as most of her meals were spread over night till dawn. While accompanying her daughter for routine checkup she complained of feeling of generalized``shakiness'' which was new to her. We obtained a random blood glucose level, which revealed a blood glucose of 37mg/dl. She was treated with supplemental glucose with complete resolution of her symptoms. Her insulin regimen was subsequently adjusted further and dietary education and hypoglycemia education was given. Frequent telephone calls were made to adjust her insulin doses and monitor her blood sugars. IMPLICATIONS/DISCUSSION: Fasting in the month of Ramadan represents a recurring annual event in the life of a Muslim patient. Patients with brittle type I diabetes and poorly controlled type II diabetes could develop complications during month long fasting with change in dietary habits. Physicians should monitor their diabetic patients closely and adjustment in the insulin regimen should be made to prevent hypoglycemia. Long acting oral hypoglycemics and premixed insulin should be avoided. Dietary principles should be reinforced. Education regarding home monitoring of blood glucose, hypoglycemia episodes is recommended. After Ramadan, patient's therapeutic regimen will need to be changed back with repeat overall education. Obtaining detailed history could help in ascertaining additional cultural factors which could interfere with management of chronic illnesses.
ADULT ONSET RECALCITRANT PRURITIS Ð TIME FOR A SERIOUS LOOK. P. Chahal 1 , A.K. Ghosh 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #74142) LEARNING OBJECTIVES: 1) Recognize the differential diagnosis of worsening chronic pruritus.
2) Recognize that patients with adult-onset recalcitrant eczema may have underlying noncutaneous lymphoproliferative disorder. CASE INFORMATION: A 38-year old male presented with history of two and a half years of insidious onset of severe itching involving his feet and gradually spreading and becoming generalized in few months. Subsequently, he developed a rash, which consisted of hard bumps', intensely pruritic in nature. He was seen by several physicians and had multiple skin biopsies demonstrating folliculitis and wound cultures growing Staphylococcus species. Numerous treatment attempts were unsuccessful. Examination revealed hundreds of equally spaced excoriated crusted pink papules measuring up to 1 cm diameters. Rest of his systemic examination was normal. Investigations revealed a normal complete blood count, hepatitis profile, and complement levels. HIV and fungal serology and PPD test were negative. Skin biopsies showed chronic dermatitis and the direct immunofluorescence was negative. Chest radiograph revealed multiple lung masses and subsequent CT scan of chest revealed extensive anterior mediastinal, bilateral hilar, right paratracheal, and subcarinal adenopathy and multiple bilateral pulmonary nodules. CT abdomen was normal. He underwent mediastinoscopy and lymph node biopsy, which revealed the diagnosis of Hodgkin's Lymphoma, Nodular Sclerosing type. He was started on combination chemotherapy with ABVD. IMPLICATIONS/DISCUSSION: Chronic unexplained eczema and pruritus of adult onset might be associated with underlying lymphoproliferative malignancy. Although pruritus has been associated with Hodgkin's disease, the reports of prurigo nodularis' association with Hodgkin's lymphoma are rare. Prurigo nodularis is also associated with other malignancies like transitional cell bladder cancer, leukemia and HIV. When an obvious cause like drugs or atopy year old Asian male presented with complaint of inability to move his arms and legs. His symptoms began one day prior to admission after walking a significant amount while shopping at a local mall. After leaving the mall, he started to experience numbness and tingling of his arms, legs and abdomen. Later that day, he started to experience generalized weakness and went to sleep early due to fatigue. When he awoke the morning of admission, he could not walk or move his body. He had to be assisted out of bed and brought to the emergency room. He had no prior medical problems and was not taking any medications, drugs or supplements. Upon further questioning, he revealed that his mother has had problems with low potassium levels. His review of symptoms prior to this episode was completely negative. Physical exam was unremarkable except for neurological exam. He was alert and oriented. His cranial nerves and sensory exam were intact. Motor strength was 0/5 in upper extremities and 1/5 in lower extremities. The patient was areflexive. Labs: Na 145, K 2.0, Cl 106, CO2 16, creatinine .7, CPK 519, Ca 7.9, Phos 1.3, Mg 1.5, TSH 0.0, T4 total 13.8, T3 total 236.4. His urinary potassium excretion was normal. EKG: low voltage t waves and u waves. Patient was diagnosed with hypokalemic periodic paralysis and treated with oral and intravenous potassium chloride. Within a few hours, his potassium improved to 5.0 and his strength was 5/5. The next day, he was discharged and told to follow up with endocrinology for his hyperthyroidism IMPLICATIONS/DISCUSSION: The diagnosis of hypokalemic periodic paralysis is made most easily when hypokalemia is documented during an attack of weakness and other secondary causes are excluded. Attacks begin during the first three decades of life and occur during the rest period following exercise, stress or high carbohydrate meal. The condition can develop in patients with a familial predisposition (autosomal dominant inheritance) or in those with thyrotoxicosis. The latter form is more common in Asian males. Although, the mechanism is not entirely understood, it is thought to be related to sudden shift of potassium into cells. Patients have normal potassium values between episodes and may have decreased magnesium and phosphorus during episodes. Treatment includes oral potassium chloride and correction of thyrotoxicosis. Prevention may include beta blockers, oral supplements, potassium sparing drugs and low carbohydrate meals. Recognition is important because the condition can lead to respiratory muscle paralysis and subsequent death. LEARNING OBJECTIVES: 1. Emphasize the importance of draining an infected galactocele and describe a less-invasive modality to achieve this. 2. Highlight follow-up measures in women with a recurrent galactocele. CASE INFORMATION: A 37 year-old lady presented with a non-tender lump in her left breast 4 weeks after birth of her fourth child. She was lactating without difficulty and denied redness of the overlying skin or fever. Following clinical examination, she had an ultrasound of the breast revealing a 3.7cm cystic mass with internal echoes. Ultrasound-guided aspiration was performed and 30cc of milky fluid aspirated with complete resolution of the mass. However, the galactocele recurred within 24 hours but remained asymptomatic otherwise. Three weeks later, the patient presented with evidence of mastitis, was started on antibiotics, and the galactocele re-aspirated. With persistent signs of infection, galactocele-fluid cultures revealing Streptococcus viridans, and re-accumulation of the milk in the galactocele, incision and drainage was contemplated. This was subverted with a less invasive means by inserting an 8 french multiple side-hole pigtail catheter into the galactocele enabling complete drainage and healing of the underlying infection. The catheter was removed in 10 days and antibiotics given for a total of 2 weeks. To prevent development of a milk fistula through the drain site, fluid reaccumulation was monitored with ultrasound and re-aspiration performed at 4 and 16 days after drain removal. The patient recovered with complete healing of the catheter tract. The galactocele persisted even after cessation of breast-feeding. A mammogram was then performed which revealed no findings of underlying malignancy. IMPLICATIONS/DISCUSSION: A galactocele most commonly presents as a breast mass soon after the cessation of lactation, and an ultrasound evaluation is usually confirmatory. These masses usually disappear in few weeks to months. However, the persistence of a galactocele, particularly after repeated aspirations, necessitates further work-up to rule out ductal obstruction from an underlying malignant process. Our patient had another infrequent complication Ð an infected galactocele. The concern of incision and drainage of a galactocele was that of a high likelihood of milk fistula. On the other hand, if not drained, infection was likely to persist and progress to a breast abscess. An alternative was therefore considered of introducing a pigtail catheter into the galactocele to enable complete drainage while instituting appropriate antibiotic coverage. Catheter drainage of breast abscess has been described in the literature, but this is the first report, to our knowledge, of catheter drainage of an infected galactocele. A 78 year-old lady reported a``whitehead like'', non-irritating lesion on her right nipple. Watchful waiting was recommended and over the course of the next 3 weeks, this lesion progressed into a rash involving the nipple and areola with a painful central abrasion and crusting. With the use of antibiotic cream, the rash improved in 3±4 days but mild flaking of the skin persisted. Three months later, the patient presented to the Breast Clinic for evaluation of a mild, but persistent nipple rash. She denied breast lumps, pain or nipple discharge. Breast cancer risks included nulliparity, and use of hormone replacement therapy for nearly 40 years. On examination, there was mild scaling of the skin over the right nipple with no associated induration, erythema or excoriation. On palpation, no dominant mass, nipple discharge, axillary or supraclavicular lymphadenopathy was identified. Mammograms revealed increasing microcalcifications in the sub-areolar region of the right breast. Stereotactic biopsy of these calcifications was performed and demonstrated high-grade ductal carcinoma in situ (DCIS). Skin biopsy of the scaly region revealed Paget disease of the nipple characterized by tumor cells with large nucleoli distributed singly in the superficial epidermal layers with clusters in the basal portion of the epidermis. These tumor cells were immunoreactive to anti-cytokeratin 7 and carcinoembryonic antigen (CEA) antibodies confirming the diagnosis of Paget disease. In compliance with the patient's wishes, right mastectomy was performed and on pathologic evaluation, residual DCIS was noted around the biopsy site along with Paget disease of the nipple. IMPLICATIONS/DISCUSSION: Paget disease, with an incidence ranging from 0.5 to 2.6%, presents as a scaly persistent rash, and often mimics eczema, or contact dermatitis of the nippleareolar complex. It may or may not be associated with a breast mass. Diagnosis is established by a skin biopsy, revealing characteristic Paget cells. Delay in diagnosis may result when the rash is mild in intensity and if unassociated with a breast mass. Clinical management of a patient with a nipple, or areolar rash mandates continued follow-up. If the rash fails to resolve, a high index of suspicion for Paget disease of the nipple will enable appropriate work-up and optimal outcome.
BREAST NODULE AFTER PROPHYLACTIC MASTECTOMY. K. Ghosh 1 , K.R. Brandt 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #75726) LEARNING OBJECTIVES: 1. Build awareness among physicians that prophylactic mastectomy does not completely eradicate the possibility of breast cancer. 2. Emphasize that any breast lump after prophylactic mastectomy warrants complete work-up to rule out breast cancer. CASE INFORMATION: A 56 year-old lady with fibrocystic changes in the breast had undergone several benign right breast biopsies for mammographically detected abnormalities, and finally decided to have bilateral prophylactic mastectomy to prevent the anxiety associated with these biopsies. Following the mastectomies, she had bilateral pedicle transverse-rectusabdominis-myocutaneous (TRAM) flap reconstruction of the breasts with a smooth postoperative recovery. She was very satisfied with the outcome of the procedure. Four months after the surgery, the patient incidentally felt a lump in the left upper breast and when it persisted for about a month, the patient sought further evaluation. Examination revealed a firm, non-tender, fixed mass with ill-defined margins in the left upper breast. Ultrasound evaluation of the palpable mass revealed a heterogenic echotexture with no evidence of malignancy. Diagnostic left mammogram revealed no evidence of malignancy. Magnetic resonance imaging of the breast with dynamic gadolinium enhancement was then recommended and showed benign appearing localized collection of fat lobules and postoperative changes. The patient continues to do well on follow-up. IMPLICATIONS/DISCUSSION: The management of women at high-risk of breast cancer includes the options of surveillance, chemoprevention, prophylactic mastectomy and/or prophylactic oophorectomy. Prophylactic mastectomy is an option for risk reduction of breast cancer, and in women with a strong family history, has been associated with a 90% relative risk reduction. The procedure does not however, eradicate the risk of breast cancer as residual breast tissue may be left behind depending on the surgical technique. Therefore, any breast lump after PM must be evaluated to rule out breast cancer. In general, breast lumps after PM are often benign and related to fat necrosis as was the case in our patient, but the possibility of malignancy must be borne in mind when evaluating these patients.
A MAN NEWLY DIAGNOSED WITH HIV REFUSES TO INFORM HIS SPOUSE. C. Gibbs 1 , J. Tsui 1 ; 1 Emory University, Atlanta, GA (Tracking ID #74714) LEARNING OBJECTIVES: 1.) To appreciate a care giver's ethical and legal responsibilities when partner notification is refused by a newly diagnosed patient; 2.) To identify various partner notification strategies and weigh their advantages and disadvantages. CASE INFORMATION: A 36 year-old man presented to the emergency room complaining of fatigue, shortness of breath, severe dyspnea on exertion, and cough worsening over several weeks. The patient had no significant past medical history and denied tobacco, alcohol, or IV drug use. He denied any previous history of blood-product transfusions.He stated he had been in a monogamous marriage for the ten years and had had no other sexual partners. Further, the patient reported a negative HIV test three years ago. Chest X-ray revealed bilateral patchy opacifications without any focal consolidation. Laboratory results were significant for a WBC of 5.1, an LDH of 514, and an ABG of 7.46/39/55. The patient was admitted for communityacquired pneumonia and started on ceftriaxone and doxycycline; however, given his clinical picture, the patient's sputum was sent for silver stain. It returned positive for pneumocystis carinii. The patient's medications were changed to trimethoprim/sulfamethoxazole and prednisone, and he was counseled about the likelihood of immunocompromise. At this point, the patient agreed to HIV testing. He subsequently tested positive for HIV and had a CD4 count of 26. Patient education regarding HIV infection and AIDS was initiated. The patient acknowledged the possibility that his wife may have been infected and agreed that she should be notified. Initially, he wished to talk with her himself (self-referral). After a few days, it was discovered that the patient had not disclosed his HIV status to his wife. With much encouragement and further education, he agreed to a dual referral approach involving a healthcare provider being present when he notified his wife. However, when the appointed meeting day arrived, the patient once again refused. Patient education and support continued. As the patient's discharge approached, the medicine team feared that the patient's wife would never be informed of his seropositive status and her potential risk. These concerns were discussed with the patient and he was informed that if he did not notify his wife, the medicine team would act according to state and federal laws and notify her of her HIV exposure and need for testing. The patient informed his wife within 24 hours. IMPLICATIONS/DISCUSSION: This case demonstrates various approaches to partner notification and deals with the ethical and legal decisions physicians are forced to make when newly diagnosed HIV patients refuse to participate. Partner notification strategies can be classified into three basic categories: self-referral, dual referral, and provider referral. Each approach has its own advantages and disadvantages. Regardless of the strategy chosen, the patient's rights to autonomy, trust, confidentiality, and safety must be respected while balancing the physician's moral and legal responsibility to patients' partner(s), children, and community. Healthcare providers must recognize their responsibilities, know their federal and state laws, and act according to individual cases. A 50 year old African American man with no significant past medical history presented to the office with malaise, weakness, anorexia, watery diarrhea and mild headache of 2 days duration. He was treated conservatively for presumed viral gastroenteritis, until he presented to the emergency room 2 days later with increasingly severe frontal headache and fevers. Physical examination was remarkable for a temperature of 39.6 deg C and nuchal rigidity, but no focal neurological signs or impaired sensorium. Laboratory studies included a WBC count of 8800, and cerebrospinal fluid analysis which demonstrated a CSF WBC count of 150/mm3 with a differential of 11% neutrophils, 88% lymphocytes, 1% monocytes, and a CSF-protein level of 117 mg/dl. CSF gram stain was negative. The patient was admitted and placed on empiric ampicillin and cefrtriaxone. Blood cultures and CSF bacterial, fungal and protozoal studies remained negative. The patient slowly defervesced and his headache improved, and he was discharged to home. Notably, this case occurred at a time when public awareness of West Nile Virus infection in Allegheny County, Pennsylvania was just emerging; the virus had been isolated in several dead birds in the area, although there had been no reported cases of human infection. During the hospitalization, serum was therefore sent for West Nile Virus serologic testing. Following discharge, results of IgM and IgG antibody ELISA assays subsequently returned positive, and were later confirmed by plaque reduction neutralization test (PRNT). The patient gradually improved, although notably after developing a transient morbilliform rash on his chest, back and arms, observed during outpatient follow-up approximately one week after discharge. IMPLICATIONS/DISCUSSION: West Nile Virus infection is a mosquito-borne arboviral illness that is commonest in the United States in late summer and early fall. One of the early clues to an epidemic is the presence of dead birds in the surroundings. Infection is most often asymptomatic, and clinical illness is generally mild, presenting with constitutional symptoms much like those of other viral syndromes. Among patients with more serious CNS illness, isolated meningitis Ð such as occurred in our patient Ð is relatively less common than encephalitis or meningoencephalitis. Treatment is supportive, and advanced age is the biggest risk factor for severe neurologic disease. This case illustrates the need to consider West Nile Virus infection, however, in cases of aseptic meningitis occurring in endemic areas.Diagnosis is by serum or CSF IgM antibodies to West Nile Virus; first-line testing is by ELISA assay, and positive results are generally confirmed by the more specific PRNT.
AN UNEXPECTED CAUSE OF FEVER, HEADACHE AND ELEVATED ERYTHROCYTE SEDIMENTATION RATE. A.N. Githaiga 1 , E. Anish 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76143) LEARNING OBJECTIVES: 1) To recognize sarcoid as a cause of central nervous system dysfunction in a patient without other systemic evidence of sarcoidosis. 2) To appreciate the value of performing a brain biopsy to establish a diagnosis of neurosarcoidosis when CSF analysis and neuroimaging are nondiagnostic. CASE INFORMATION: A hypertensive 50-year-old African American woman presented to the hospital with a one-month history of retro-orbital headache, recurrent fevers, chills, sweats and weight loss. Temperature on admission was 38 deg and her neurologic exam was non-focal. Initial labs were significant for WBC-11,800,PLT-610, 000 and ESR-148. A brain CT scan showed old lacunar infarcts. Lumbar puncture showed protein of 111mg/dl, white cell count of 3 and 79% lymphocytes. Viral, bacterial and fungal cultures were negative. Autoimmune serological markers were negative. A bone marrow biopsy was negative for malignancy and granulomatous disease. Serum ACE level was normal ( 29 Units/L.). Chest CT scan was normal, and abdominal CT scan revealed numerous small mesenteric and retroperitoneal lymph nodes that were not accessible for biopsy. The patient was started on empiric prednisone therapy for temporal arteritis at 60 mg per day despite negative bilateral temporal artery biopsies. She noted rapid improvement of her headache and resolution of her fevers and was discharged home. Over the next 8 weeks her ESR dropped to 22 and she remained asymptomatic. However, as the steroid dose was being tapered, the patient began to experience recurrence of headache and fevers, as well as gait and cognitive dysfunction. An MRI was performed that revealed multiple subcortical areas with increased signal. Repeat CSF analysis showed a white cell count of 28 with 89% lymphocytes and increased protein (79 mg/dl). Brain biopsy performed revealed focal perivascular lymphocytic infiltrates with non-necrotizing granulomas. Special stains for AFB and fungus were negative. A diagnosis of neurosarcoidosis was made. Her symptoms steadily improved with high dose solumedrol and subsequent oral prednisone. IMPLICATIONS/DISCUSSION: This case illustrates the importance of considering a diagnosis of neurosarcoidosis as a cause of fevers and CNS dysfunction in an individual who does not have other overt manifestations of sarcoid. In 5% of cases, neurosarcoid is the initial presentation of the disease. CNS presentations typically include cranial nerve palsies, meningitis, optic nerve dysfunction and neuroendocrine disorders (e.g. diabetes insipidus and central hypothyroidism). CSF findings are nonspecific and include elevated protein and mononuclear pleocytosis. Contrast enhanced MRI may reveal meningeal/cortical masses or hydrocephalus. As demonstrated in the case presented, neural tissue biopsy may be useful, revealing perivascular non-caseating granulomas, which while not diagnostic, will be highly suggestive in the appropriate clinical context. FOOLED BY FAT/PERPLEXED BY PROPOFOL. E. Gjersvik Cichowski 1 , H. Sakowski 1 , H. Hashish 1 , R. Baltaro 1 ; 1 Creighton University, Omaha, NE (Tracking ID #74955) LEARNING OBJECTIVES: 1) Recognize laboratory error in the measurement of serum bicarbonate.
2) Utilize the Henderson-Hasselbach equation to indentify blood gas analysis errors.
3) Recognize interfering substances as potential causes of laboratory errors. CASE INFORMATION: A 72 year-old male was admitted for respiratory distress and confusion, and found to have a right upper lobe lung mass and hypercalcemia. He was intubated on the second hospital day due to worsening of his respiratory status. Propofol was initiated for sedation and methylprednisolone and levofloxacin were given for a presumed postobstructive pneumonia. His initial arterial blood gas after intubation showed a pH 7.38 pCO2 38 pO2 143 on an Fio2 of .60. His measured HCO3 was 26 meq/l. Over the next 4 days, his measured bicarbonate progressively dropped to 8 meq/l despite no change in his arterial blood gas (pH 7.38 pCO2 36 pO2 103 on an FiO2 of .45). His anion gap was calculated at 19. Serum lactate was normal, and serum ketones were absent. Consultation with the pathology department revealed the patient's serum to be grossly lipemic. A review of the chart revealed the patient did receive lipid infusions with TPN 36 and 18 hours prior to this discovery. A lipid panel was obtained and revealed marked hypertriglyceridemia at 4426 mg/dl. The lipid infusions were discontinued, and the propofol was weaned off. The bicarbonate level dropped to a low of 3 meq/l approximately 7 hours after the medication was discontinued. Four hours later, the bicarbonate had corrected to 21 meq/l. The serum, however, remained grossly lipemic. The patient's condition continued to decline with the development of septic shock, multi-organ failure and ventricular arrythmias. Results of a previous bronchoscopy demonstrated small cell carcinoma. The patient's family requested no further aggressive treatment and he expired later that day. IMPLICATIONS/DISCUSSION: This patient developed marked derangement in his measured bicarbonate levels that did not correspond to his arterial blood gas analysis. A laboratory error was hypothesized as the cause. Due to the finding of lipemic serum, the hypertriglyceridemia was initially suspected as the interfering substance. Upon discontinuing the propofol, the serum bicarbonate level normalized, the serum, however remained lipemic. In a review of the literature, neither propofol nor hypertriglyceridemia have been reported as potential causes of this lab error. Further testing is needed to determine the role of propofol as an interfering substance in bicarbonate laboratory analysis.
DYING TO BE THIN. R.E. Graham 1 ; 1 Lenox Hill Hospital, New York, NY (Tracking ID #75181) LEARNING OBJECTIVES: This case demonstrates the limitations of our current knowledge about herbal products, particularly in dose-related toxicities, and calls attention to the need for further studies. This case highlights the need for health care providers to be knowledgeable about the herbal products their patients may be using, and to be able to identify potential herbassociated adverse effects. I hope this case illustrates CAM's prevalence, benefits, and potential dangers but most importantly highlights the importance of obtaining a complete medical history; including asking our patients if they are using and/or incorporating alternative therapies into their general healthcare. CASE INFORMATION: 26 year old thin female presented to the ED at about 5:15 PM complaining of a frontal headache, nausea, and feeling weak after fainting. The patient stated to the triage nurse that she had eaten Mexican food at about 3:30 PM, went home and took``a fat burner pill'' at about 4PM. She has been on fat burner pills and herbal teas for about one year. Suddenly she began to feel nauseous, developed a worsening headache, then fainted which prompted her to seek medical attention. Initially, the patient was taken to the back of the ED. While awaiting physician examination, the patient syncopized and began to have a seizure. The patient was wheeled into trauma and found to be in Ventricular fibrillation with a potassium of 2.4. She was defibrillated and then became hypotensive and progressed into asystole, she was resuscitated and then intubated. She began posturing without corneal reflexes. After stabilization the patient was taken to CT; which revealed±Extensive subarachnoid hemorrhage with the presence of intraventricular hemorrhage. Saddly, over the next 48 hours she was placed on life support and an organ donation referral was made. Soon after she expired. IMPLICATIONS/DISCUSSION: Complementary and Alternative medicine (CAM) is one of the fastest growing sectors in health care today. In 1997 , Eisenberg & et al JAMA, 1998 :280:1569±1575, revealed that 42.1% of Americans (83million) used CAM over the past year. In the same article Eisenberg, showed that only 38% of patients disclose to their physicians their use of alternative therapies when not asked directly. As we continue to see an increase in the rate of obesity in the United States, our patients will incorporate both prescription and nonprescription products in hopes of obtaining their desired weight loss. This current vignette exemplifies the need for the health care provider to first, ask the unasked question, secondly, to have a basic understanding and knowledge regarding CAM, and finally to provide an important role in educating, advising and discussing the potential risks and benefits of CAM, and the need to closely monitor any use of them. CXR showed R mediastinal mass. CT showed diffuse LAN (mediastinal, peritoneal, retroperitoneal) and numerous liver metastases. HIV and hepatitis panel were negative. FNA of neck revealed atypical lymphoid cells. Excisional biopsy showed anaplastic carcinoma, no melanin but HMB-45 and melan A (melanocyte markers) were immunostain positive. IMPLICATIONS/DISCUSSION: Distinguishing between localized and generalized LAN helps guide the appropriate differential diagnosis. Diffuse LAN suggests HIV, mycobacterial infection, infectious mononucleosis, SLE, medications (i.e., phenytoin), metastatic carcinoma and lymphoma. The history and physical exam become crucial in the diagnostic work up of generalized LAN. The history should include signs and symptoms suggestive of infection or malignancy, exposures (cats±cat scratch disease; raw meat±toxoplasmosis; tick bite±lyme disease; unprotected sex or injection drug use-HIV), constitutional symptoms and medications. A complete physical exam should guide the appropriate diagnostic work-up. Patients with generalized LAN should have a CBC and CXR. If these are normal, consider PPD, HIV test, RPR, ANA, and heterophile test, although these tend to be low yield if not specifically indicated. An FNA for cytology is generally the first step due to ease and safety, especially for granulomatous versus malignancy and lymphoma versus carcinoma, yet an FNA fails to provide enough material to examine architectural detail or to perform immunostains. This is especially relevant with younger patients where hematologic malignancies or uncommon solid tumors are a consideration. Metastatic melanoma without a known skin primary accounts for around 3% of melanomas. Prior to melanocyte-specific immunostains, these cases were considered carcinoma of unkown primary. Cases of metastatic melanoma lacking primary skin lesion include primaries of mucosal surfaces (sinuses, oral mucosa, vulva, vagina and anorectum), occular and primary skin lesion which have regressed. Thus, the lack of skin lesion does not rule out metastatic melanoma.
No effective systemic therapy exists at this time for metastatic melanoma. Biotherapeutic approaches combining immunomodulators such as IL-2, INF, and vaccines with conventional cytotoxic chemotherapy are currenlty under active investigation.
A COURAGOUS CALL: SALINE FOR HEART FAILURE? M. Guidry 1 , J. Wiese 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77054) LEARNING OBJECTIVES: 1. Recognize the perils of over-diuresis in bi-ventricular heart failure. 2. Use disciplined methods of diagnosis to prevent errors in pattern recognition diagnosis. 3. Use the physical examination to predict cardiovascular function. CASE INFORMATION: An 80 year-old woman presented with two days of shortness of breath and orthopnea. Her blood pressure was 132/65 with a heart rate of 116. She had bibasilar crackles and 3+ pitting bipedal edema. There was an S3, S4, and an inferiorly displaced PMI. Her room-air ABG at presentation was pH: 7.44; pCO2:35; pO2:58. She was diuresed with a total of 320 mg of furosemide over one day. Her symptoms improved. On the third hospital day, she had a sudden worsening of her shortness of breath. Her neck veins were flat and there were no crackles. A repeat ABG was pH: 7.28; pC02: 57; p02: 81; HCO3:42 Her BUN tocreatinine ratio increased from 23/1.4 at presentation to 41/2.3 on the third hospital day. Diuresis was stopped, and hydration with normal saline was initiated. Her shortness of breath improved, with normalization of her acid base values. IMPLICATIONS/DISCUSSION: Dyspnea can result from hypoxia or hypercarbia. This patient initially presented with dyspnea due to congestive heart failure; she was appropriately managed with preload reduction via diuresis. Continuing diuresis was excessive however. In the setting of bi-ventricular heart failure, the over-diuresis resulted in an inadequate right ventricular preload, and a subsequent drop in her pulmonary artery pressure. With the alveolar pressure unchanged and the pulmonary artery pressure lowered, the percentage of dead space increased. This resulted in a ventilation to perfusion mismatch resulting in hypercarbia. The physical examination was the tell-tale sign, revealing that her volume status was low, instead of high. The ABG confirmed the diagnosis; the rehydration increased the pulmonary artery pressure, resolving the mismatch, and eliminating the dyspnea. In caring for patients with biventricular heart failure who are sensitive to preload reduction, it is important to remember that hypercarbia from dead space ventilation can result from aggressive lowering of the pulmonary artery pressure.
COLCHICINE±INDUCED MYOPATHY. K. Gupta 1 , T.A. Townley 1 ; 1 Creighton University, Omaha, NE (Tracking ID #76577) LEARNING OBJECTIVES: 1) Review the differential diagnosis of proximal muscle weakness in an elderly male 2) Review symptoms and signs of colchicine-myopathy 3) Review the risks factors for colchicine myopathy. CASE INFORMATION: INTRODUCTION: Colchicine has been used for 200 + years, but there are few reports of myopathy associated with its use. We present such a case. CASE: Patient is a 70-year-old male who was hospitalized for abdominal pain/diarrhea. He had fallen on his hip at home 1 week prior to his admission and became increasingly weak with considerable reductions in his ADL such as inability to get in and out of bed, stand from sitting and walk. His PMH includes COPD on home O2, CHF, HTN, DM, CRI (baseline Cr 2.2), obesity and gout. Medications include Oxycodone 5/325mg prn, metoprolol 50mg qd, colchicine 0.6mg qd, allopurinol 100mg qd, furosemide 80mg qd, glipizide 30mg qd, isosorbide dinitrate 30mg qd, fosinopril 40mg qd, ASA 325mg qd, KCl 20mEq qd, Insulin 70/30, MVI qd. His initial physical exam was normal except for reduced reflexes in upper limbs 2+/4 and lower limbs 1+/4 with no sensory deficits. Lab work revealed acute renal insufficiency on CRI (BUN 69/Cr 2.6), in addition LFT's were twice normal. CK was 1059 with MB fraction <5%. CXR and abdominal series were clear and EKG was unchanged. His abdominal pain resolved, and ARI improved with hydration and discontinuation of ACE-I. Initially his weakness was felt to be secondary to intercurrent illness superimposed on chronic illness. However weakness failed to improve and a repeat CK was 1825 with ESR of 36. His weakness localized to proximal muscles of upper and lower extremities, with strength 3/5. Distal muscle strength was 5/5. Colchicine was suspected as the cause of his myopathy and discontinued. He noticed a significant improvement in his proximal muscle of 4/5 on the following day. EMG showed myositis with predominantly proximal muscle irritability. Repeat labs 5 days after discontinuing colchicine, showed BUN 44/ Cr 1.4, and CK 958. There was a noticeable improvement in strength, with ability to stand from sitting, ambulate across room and get into and out of bed alone. IMPLICATIONS/DISCUSSION: DISCUSSION: Patients taking colchicine over an extended period of time have been reported to develop a subacute, often severe myopathy in conjunction with a relatively mild polyneuropathy. Chronic renal failure is a risk factor for this syndrome. Our patient had been on colchicine for more than a year. In this case an untreated myopathy may have resulted in nursing home placement versus a trial of steroids for polymyositis. In our patient, who had pre-existing renal insufficiency worsened by the dehydration, the clinical presentation and the improvement on discontinuation of colchicine strongly supports the diagnosis of Colchicine induced myopathy.
MANAGEMENT OF RAMSAY±HUNT SYNDROME. S. Gupta 1 , O. Melamed 2 ; 1 UCLA School of Medicine, Los Angeles, CA; 2 Olive View±UCLA Medical Center, Sylmar, CA (Tracking ID #75744) LEARNING OBJECTIVES: 1. Clinical presentation and pathophysiology of Ramsay±Hunt Syndrome 2. Lack of outcome benefit from head imaging, IV therapy, and surgery for RHS 3. Improved outcome with early treatment. CASE INFORMATION: A 30-year-old female presented to the urgent care clinic for evaluation of left ear pain, vertigo, left facial paralysis and small vesicles over the left pinna. The patient's past medical history was significant for chickenpox at the age of eight. On physical examination the patient had left ear swelling and erythema with multiple 0.5±1.0 mm vesicles around the pinna and marked erythema involving the external auditory canal. There was a positive Bell's phenomenon (the eyeball rolling up and outward on attempting to close the affected eye). A facial paralysis involved the left levator labii and left frontalis muscles. A left sensorineural deficit was observed with Weber test lateralizing to the right and a positive (normal) Rinne test bilaterally. The diagnosis of Ramsay±Hunt Syndrome was made. The patient was discharged from the clinic on a regimen of Acyclovir and Prednisone. She was instructed to avoid pregnancy, contact with pregnant women or neonates. IMPLICATIONS/DISCUSSION: Clinical presentation of unilateral facial paralysis and a vesicular rash on the ear (zoster oticus), often accompanied with hearing loss, tinnitus, nausea, vomiting, vertigo and nystagmus was first described by J Ramsay Hunt in 1907. Vestibulocochlear symptoms were explained by the anatomical proximity of the eighth nerve and the geniculate ganglion within the temporal bone. The pathophysiology of RHS involves the reactivation of varicella-zoster virus (VZV) within the geniculate ganglion and secondary edema in the facial nerve resulting in facial nerve palsy. VZV is considered a major etiologic agent of Bell's palsy. Use of contrast enhanced MRI in diagnosis of RHS does not appear to provide much information as enhancement of the geniculate ganglion, meatal fundus, and facial nerve is observed independent of etiology. Most patients with Bell's palsy tend to recover to complete or near normal function even without treatment. One prospective study found that 85% of patients showed signs of remission within the first 3 weeks, with 71% recovering complete function, 13% left with insignificant sequelae, and 16% left with permanently diminished function. Although most patients with RHS improve, many patients are left with functional and cosmetic deficits. Treatment of RHS with oral acyclovir and prednisone (AS therapy) may improve outcome. Two separate studies showed complete recovery in 75% of patients with RHS treated with AS. There has been no evidence of improvement in outcome with intravenous administration of either acyclovir or prednisone, though many patients are still admitted to the hospital for IV therapy. There is also insufficient evidence for efficacy of surgical facial nerve decompression. Evidence for improved outcomes with earlier administration of therapy have been observed. In one study 75% of patients beginning AS treatment within 3 days of onset of facial paralysis had complete recovery, compared with 30% of patients beginning therapy after 7 days. Earlier therapy decreased nerve degeneration and improved recovery of hearing.
SEIZURES ON POSTPARTUM DAY SEVEN. K. Gustafson 1 , R. Khurana 1 ; 1 University of Alberta, Edmonton, Alberta. (Tracking ID #76917) LEARNING OBJECTIVES: 1) To recognize that preeclampsia and eclampsia may occur in the postpartum period. 2) Diagnose eclampsia even when it presents without the typical signs of preeclampsia. We describe a case of postpartum eclampsia that was initially misdiagnosed. CASE INFORMATION: A 28 year old woman G2P2 was transferred to our tertiary case hospital on postpartum day 7 because of seizures. Her pregnancy had been uncomplicated and she had a vaginal delivery at term for a healthy 3515g female infant. Her past medical history was unremarkable and her blood pressure had remained normal throughout the pregnancy. She presented to a local hospital on postpartum day 7 complaining of severe headache, nausea and vomiting. Her blood pressure was 176/100. She was given analgesia, but then had two generalized seizures. She was given magnesium sulfate 5 g intravenously and transferred. When assessed, her blood pressure was 150/90. She had no focal neurologic findings, right upper quadrant tenderness, edema or hyperreflexia. Her liver enzymes and platelet count were normal. Urinalysis was negative for protein. Urate was slightly elevated. CT scan of the head was normal. EEG was normal. She was seen by the services of Neurology, Internal Medicine and the Intensive Care Unit. She was diagnosed with seizures of unknown origin and admitted for observation. The magnesium sulfate was discontinued. Her blood pressure varied over the next two days from 124/81 to 200/103. On postpartum day 10, she had another generalized seizure. MRI of her brain was consistent with hypertensive encephalopathy. She was given a diagnosis of postpartum eclampsia and treated with intravenous magnesium sulfate and antihypertensive therapy. She improved over the next few days and all therapy was discontinued. She remains well with no further seizure activity. IMPLICATIONS/DISCUSSION: Preeclampsia/eclampsia may worsen or even initially present in the postpartum period. Postpartum eclampsia represents 25% of all cases of eclampsia. The majority of these events occur in the first 24±48 hours postpartum, but have been reported to occur as late as 4 weeks postpartum. Eclampsia does not always occur in patients who have the classic triad of preeclampsia (hypertension, proteinuria and edema). About 20% of patients with eclampsia have no proteinuria. Computed tomography and EEG are often unremarkable, but should be performed to rule out other causes of seizure. When MRI abnormalities are present, hyperintense signals in cortical and subcortical areas on T2 weighted images may be seen. These are usually in the posterior parietal or occipital lobes. Management of eclampsia includes delivery of the fetus (if diagnosed antepartum), magnesium sulfate to prevent recurrent seizures and control of severe hypertension. A high index of suspicion is necessary to appropriately diagnose the woman with postpartum eclampsia and prevent further morbidity. LEARNING OBJECTIVES: To recognize focal muscle infarction as an unusual, and sometimes bilateral, cause of severe limb pain in patients with diabetes. CASE INFORMATION: A 55 year-old diabetic woman developed disabling pain in both upper legs, with thickening and tenderness of the soft tissues of the right lateral thigh. There was no evidence of infection. She had neuropathy but sensation in the thighs was normal. There was a history of disc disease but an MRI examination of the spine was unimpressive. She had arterial disease but ankle/brachial indices were only in the claudication range. There was a history of venous thrombosis but she was taking warfarin and duplex studies showed no clots. Plain films and a bone scan were negative. MRI examination showed diffuse edematous infiltration of the subcutaneous fat of both thighs, with increased signal in the right vastus lateralis muscle. These findings were consistent with infarction of the vastus lateralis, and conservative treatment was recommended. IMPLICATIONS/DISCUSSION: Diabetic muscle infarction causes pain, swelling, and tenderness, most commonly in the thigh, but sometimes in the calf. The opposite limb may be involved, either sequentially, or concurrently, as in this case. The MRI appearance is characteristic and allows the diagnosis to be made without the risks of biopsy. Treatment is supportive. This rare but important entity must be considered when diabetic patients suffer severe limb pain.
A CASE OF SPONTANEOUS BILATERAL CAROTID CAVERNOUS FISTULAS. S. Habib 1 , P.K. Han 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76354) LEARNING OBJECTIVES: 1) To recognize the clinical symptoms and signs of carotid cavernous fistula. 2) To describe an unusual presentation of carotid cavernous fistula. CASE INFORMATION: An 85-year-old woman presented with a 3±4 week history of progressive difficulty with eye opening, poor vision, and tinnitus. She denied headaches, ocular or orbital pain, diplopia, worsening of symptoms with activity, dizziness, focal weakness, or other recent illnesses. She had a history of recent falls, but no head trauma. Her past medical history was significant for hypertension, primary hyperparathyroidism, normocytic anemia, Paget's disease of bone, and a fifty pack-year smoking history. She was on ferrous sulfate but no other medications Physical examination revealed a blood pressure of 168/74 without orthostatic changes, mild conjunctival pallor, and bilateral complete ptosis. There was chemosis of the right eye and opthalmoplegia, with bilateral palsies of cranial nerves III, IV, and IV. Both pupils were mid-dilated and non-reactive to light. No other neurological deficits were noted. Diagnostic evaluation included a normal CT scan of the brain, while brain MRI demonstrated changes suggestive of bilateral carotid cavernous fistulas. Cerebral angiography was performed, which confirmed the presence of bilateral type A carotid cavernous fistulas. The patient then underwent trans-carotid endovascular embolization, which was unsuccessful. Unfortunately the patient developed right-sided hemiparesis a few days later, and subsequently declined further intervention. IMPLICATIONS/DISCUSSION: Carotid cavernous fistula is an unusual problem caused by an abnormal communication between the internal and/external carotid artery and the cavernous sinus. It is classically characterized by the triad of pulsating exophthalmos, ocular chemosis and an orbital bruit. Other features include orbital pain or tightness, pulsatile tinnitis, visual loss due to ischemia of optic nerve and retina, retinal vein congestion, and glaucoma. Ophthalmoplegia often results from pressure upon local cranial nerves; CN VI is affected most often, and CN III and IV are involved less frequently. Our patient did not present with the classic clinical triad, but with bilateral complete ophthalmoplegia and pulsatile tinnitis. Trauma is the most common cause of carotid cavernous fistula although 20 percent of cases are non-traumatic. Spontaneous bilateral carotid cavernous fistula, as observed in our patient, is a very rare entity. Risk factors for carotid cavernous fistulas include older women, mothers during childbirth, hypertension, atherosclerosis and collagen vascular diseases. A four-type classification of carotid cavernous fistulas based on the anatomy of the lesion has been proposed. Our patient had type A fistulas, presumably due to a rupture of an intra-cavernous carotid artery aneurysm. Early recognition and intervention is important to prevent permanent visual loss and ophthalmoplegia. Endovascular interventions (embolization and balloon occlusion) represent a successful treatment option although stroke is a well-recognized complication in 5±15 % of cases. LEARNING OBJECTIVES: 1, To recognize that autoimmune hepatitis is a rare complication following allogeneic stem cell transplantation. 2, To understand the pathophysiology of auto immune hepatitis folllowing stem cell transplantation. CASE INFORMATION: A 43 year-old white female developed Ph-chromosome positive chronic myeloid leukemia. She received cytoreductive therapy, followed by a 6/6 HLA matched allogeneic peripheral stem cell transplant (SCT). Tacrolimus and prednisone were continued for 18 months. One month after tacrolimus dose reduction, her serum AST and ALT levels were 723 and 965 respectively, but bilirubin and alkaline phosphatase were normal. An abdominal sonogram was normal. After the tacrolimus dose was increased to its baseline level, serum AST and ALT levels decreased to 45 and 60. Tacrolimus was then gradually reduced and eventually discontinued, which caused a rise in AST and ALT levels. She had no risk factors for viral hepatitis. Physical examination was normal except for Cushingoid appearance. CBC with differential were normal. Biochemical tests showed total bilirubin 0.7, AST 88, ALT 128, ALP 96, GGT 74, albumin 3.7, and total protein 9.8. Other biochemical profile including TSH, iron studies, ceruloplasmin, a 1 anti-trypsin and a feto protein were all within normal limits. ANA and anti thyroglobulin antibodies were positive whereas anti LKM, anti mitochondrial, anti smooth muscle antibodies were all negative. She had negative serology for EBV, CMV, HZV and hepatitis A, B, and C viruses. The histology was most consistent with autoimmune hepatitis. In situ hybridization study for Y chromosome established that immune reactive cells were exclusively of donor origin. There was no evidence of recurrent chronic myelogenous leukemia. Serum transaminase levels normalized with prednisone 20 mg a day but relapsed on dose reduction. Azathioprine was therefore added. Her transaminase levels have been normal for 28 months. IMPLICATIONS/DISCUSSION: This report documents a case of liver dysfunction, following SCT, which was indistinguishable from`definite' autoimmune hepatitis (baseline diagnostic score > 15) as defined by the modified criteria of International Autoimmune Hepatitis Group. To our knowledge, the present case represents the first report of this complication after allogeneic peripheral SCT. One intriguing feature of this case is that the target liver cells and immune reactive cells are of different origins. The donor origin of immune reactive cells was clearly established in our female patient by in-situ hybridization labeling of the Y chromosome. As the term`autoimmune hepatitis' does not accurately reflect the disease process, we propose the term`alloimmune hepatitis' for this syndrome. A 50 year old white male was admitted to our hospital in July with three days of fever, chills, headache, and neck stiffness. He worked as an air-conditioner repairman and spent a considerable amount of time shirtless, outdoors in East Texas. Physical examination was significant for fever of 102.4 degrees Fahrenheit, meningismus, and Kernig's sign. The patient was awake, alert, and oriented to person, place, and time. Initial CSF analysis revealed WBC 91 with 92% neutrophils and 8% monocytes; protein 81, glucose 68; gram stain revealed few WBCs, and was negative for bacteria; latex agglutination screen was negative for H. influenzae, S. pneumoniae, N. meningitis A, B, C and group B streptococcus. CBC revealed a WBC count of 10.6 with 70% neutrophils, 20% lymphocytes, and 10% monocytes. Within two days of admission, the patient became progressively more lethargic and delirious and was oriented only to person, month, and year. Repeat CSF analysis revealed WBC of 223 with 15% neutrophils, 59% lymphocytes, and 26% monocytes; glucose of 66 and protein of 81. CSF bacterial cultures remained negative. CSF studies for syphilis, tuberculosis, and herpes simplex virus were negative. CSF IgM was positive for West Nile virus. IMPLICATIONS/DISCUSSION: Until 1999, West Nile virus was found only in Africa, Asia, and the Middle East. Since that time, West Nile virus has infected over 3852 individuals in the U.S. in at least 39 states. West Nile virus is a single-stranded RNA Flavivirus virus that belongs to the Japanese encephalitis complex. Birds are the natural hosts of West Nile virus and mosquitoes, particularly of the culex species, act as vectors. Infection in humans typically occurs in late summer through early fall when mosquitoes are most predominant. Severe neurologic disease accompanying West Nile virus infection is rare. One out of every 150 infected patients develops meningitis or encephalitis. Age greater than 50, is the major risk factor for developing life-threatening neurologic disease. West Nile viral encephalitis is similar to other viral encephalitities and is characterized by fever, headache and an altered level of consciousness. CSF studies typically show a lymphocytic pleocytosis with elevated protein.
Early viral encephalitis, however, may demonstrate CSF with a predominantly neutrophilic CSF pleocytosis, as in our patient. Lymphopenia is a common feature of West Nile virus encephalitis and thus it may aid in distinguishing this disease from other forms of viral encephalitis. Definitive diagnosis is obtained through confirmation of blood or CSF IgM to West Nile virus. Treatment is supportive. The main intervention is prevention by promoting the use of mosquito repellant and protective clothing. year old white male presented to our hospital with complaints of fever, diarrhea, and fatigue. His admission was preceded by two days of watery, non-bloody stools, fever to 102, fatigue, malaise, and generalized weakness. The patient denied any sick contacts or travel, although he noted living in a mosquito-infested area of East Texas. On admission, the patient's temperature was 102.2, pulse 69, blood pressure 117/61 and respiratory rate 24. The patient appeared fatigued and exhibited a diffuse, erythematous rash and injected conjunctivae. The neurologic examination revealed normal tone and 5/5 strength throughout. Sensation was intact and reflexes were 2+ and symmetrical in all extremities. The rest of the physical examination was unremarkable. Within days, the patient developed severe generalized weakness with areflexia and ultimately, respiratory failure requiring mechanical ventilation. Analysis of CSF revealed a WBC count of 78 with 55% lymphocytes, 30% neutrophils, and 5% monocytes; glucose 154, and protein 116. Electrophysiologic studies were performed and were consistent with a diffuse axonal polyneuropathy. Serologic studies were positive for the West Nile virus. IMPLICATIONS/DISCUSSION: Before 1999, West Nile virus, a single-stranded RNA virus belonging to the Japanese encephalitis complex, was found only outside the western hemisphere. Since then, West Nile virus has infected over 3852 individuals in the U.S. in at least 39 states. Many clinicians are aware that West Nile virus is associated with encephalitis and meningitis, however, the association between West Nile virus infection and acute flaccid paralysis is less well known. CDC surveillance during the most recent outbreak of West Nile Virus in Louisiana revealed that almost 20% of patients described as having severe neurologic disease presented with acute flaccid paralysis. Early reports of patients with West Nile virus acute flaccid paralysis often confused the condition with Guillain-Barre syndrome. Clinicians should be aware that unlike most patients with Guillain-Barre syndrome, patients with West Nile associated acute flaccid paralysis typically have fever, CSF pleocytosis, and electrodiagnostic studies consistent with a predominantly axonal neuropathy. Most patients with Guillain-Barre syndrome are afebrile, have an elevated CSF protein, few if any WBCs in the CSF, and electrodiagnostic studies consistent with a demyelinating neuropathy. The presence of a maculopapular or morbilliform rash, such as our patient had, may also suggest the presence of West Nile virus infection. LEARNING OBJECTIVES: 1. Develop a differential diagnosis for thrombocytopenia in a well-appearing adult. 2. Learn to diagnose and treat ITP in adults using the best available evidence. CASE INFORMATION: A well-appearing 72 year-old Chinese-American man being treated with glipizide for type II diabetes presented to clinic with a petechial rash on his trunk, arms, and legs. A CBC revealed isolated thrombocytopenia (platelet count 23,000). Glipizide was discontinued but thrombocytopenia persisted. His platelet count normalized with a prednisone taper but in the ensuing months, despite splenectomy, he continued to require 5 mg of daily prednisone to maintain a normal platelet count. His diabetes became poorly controlled while on prednisone, and he had one episode of pneumonia requiring antibiotics. The decision was made to stop prednisone. After the initial petechiae he has had no further evidence of bleeding, but his platelet count remains between 10,000 and 40,000. IMPLICATIONS/DISCUSSION: Medications and ITP are common causes of isolated thrombocytopenia in well-appearing adults. Glipizide is among the many medications with published case reports of thrombocytopenia as a side effect. ITP is a clinical diagnosis made in the setting of isolated thrombocytopenia with a normal peripheral smear and no other identifiable cause of low platelets. Experts recommend a bone marrow biopsy in patients over age 60 to rule out myelodysplasia. Anti-platelet antibodies are not specific for ITP. Case series suggest that in adults spontaneous remission occurs less than 10% of the time. Splenectomy is curative in 50±65% of all cases, with better results in patients less than 40 years old. Lifethreatening bleeding rarely occurs with a platelet count of greater than 10,000. Prednisone is not curative and is most helpful as a supportive measure in patients with platelets under 10,000 or other high bleeding risks (ie: surgery). Asymptomatic patients with platelet counts above 10,000 and no other risks for bleeding can most likely be safely managed without steroids. Develop an approach for the evaluation of dyspepsia through review of the current evidence and recommendations 3. Review diagnostic testing and treatment for H. pylori. CASE INFORMATION: 46 y/o woman presented with epigastric discomfort and nausea 1±2 months after she ran out of her cimetidine. Symptoms were similar to ones she experienced 2 years prior when she may have had a``stomach bug'' for which she thought she was given antibiotics. The patient did endorse drinking 2 beers/day but denied dysphagia, anorexia, weight loss or melena. Physical exam was benign. Symptoms initially improved with reinstituting cimetidine and decreasing alcohol intake. Initial labs demonstrated a normal CBC, LFTs, amylase and lipase, but was impressive for an H. pylori titer 1:87 U/mL. Despite continued cimetidine, the patient's symptoms recurred within months and lansoprazole was initiated. An H. pylori breath test was not pursued due to the patient's reluctance to discontinue medications prior to the test. Subsequent endoscopy revealed 2 small, linear, cleanbased ulcers biopsy positive for H. pylori. Symptoms resolved after treatment with amoxicillin, clarithromycin and lansoprozole. IMPLICATIONS/DISCUSSION: Dyspepsia, or chronic and/or recurrent pain or discomfort of the upper abdomen, is a common complaint accounting for 5% of all PCP visits. H. pylori is implicated in several of the etiologies of dyspepsia, thus the management of dyspepsia must involve strategies to appropriately diagnose and treat H. pylori. Testing for H. pylori is recommended only if treatment is intended; however, guidelines regarding the indications for treatment of H. pylori put forth by the American College of Gastroenterology (1998) and the Maastricht 2±2000 (European) are conflicting. Both agree on the need to treat in cases of active ulcer disease or gastric/MALT lymphoma, but the Europeans are more aggressive in their recommendations advising treatment in entities such as non-ulcer dyspepsia or concurrent NSAID use. A recent meta-analysis (Laine, et. al. 2001) helps to clarify the issue of non-ulcer dyspepsia Ð which accounts for the majority of dyspepsia Ð suggesting that eradication of H. pylori does not improve symptoms in non-ulcer dyspepsia. Yet the uncertainty regarding the utility of eradication of H. pylori makes the appropriate work up of uninvestigated dyspepsia somewhat unclear. We will review several algorithms for the evaluation of dyspepsia and discuss the current data supporting the most often recommended``test and treat'' approach with regards to H. pylori and dyspepsia. In addition, we will discuss the range of diagnostic testing available for H. pylori and the appropriate use of these tests during the evaluation of dyspepsia. The topics of treatment and treatment failures will also be addressed. spells of altered mental status and syncope. The patient's behavior ranged from combative and agitated to lethargic. These variations in behavior usually occurred early in the morning, lasted one to three hours, and were associated with diaphoresis, marked confusion, and generalized weakness. The patient often had no recollection of these events and returned to baseline between episodes. This pattern increased in frequency and severity prior to admission. The spells were associated with blood sugar measurements in the low forties. The patient was hospitalized for further work up and underwent a monitored fast. Serial lab values were consistent with insulinoma. Imaging studies to locate a tumor included body CT, ultrasound, endoscopic ultrasound, and nuclear imaging, which were non-diagnostic. Initially the patient's glucose was maintained at normal levels with intravenous 10% dextrose and frequent snacking. Diazoxide therapy was initiated which allowed the intravenous dextrose to be discontinued; however, even with frequent snacks the patient still had occasional low sugars. With failure of medical management the patient underwent exploratory laparotomy with intraoperative ultrasound which did not detect a distinct mass. A distal pancreatectomy was performed, which demonstrated hyperplasia of islet cells. Post-operatively the patient was able to maintain normal blood sugars without diazoxide. IMPLICATIONS/DISCUSSION: Nesidioblastosis is a rare cause of hyperinsulinemic hypoglycemia that is differentiated by an insulinoma only by pathology. In this case, the patient's hypoglycemia was associated with syncope and altered mental status. LEARNING OBJECTIVES: 1) to review neutropenia as a presentation of SLE 2) to discuss strategies to avoid misdiagnoses and how to manage lupus neutropenia. CASE INFORMATION: A 53 y/o Egyptian woman presented with two weeks of fevers reaching 102±103 degrees F. She denied cough, headache, dysuria, diarrhea, abdominal pain, joint pain, oral lesions, rash, weight loss, sexual promiscuity, recent travel, or drug use. She last visited Egypt four years ago and worked as an apartment manager. She took occasional Tylenol, no other medication. On examination she had a temperature of 103 degrees F and appropriate tachycardia. No lymphadenopathy, breast masses, abdominal masses/pain, lung crackles, cardiac murmurs, or neurological findings were found. She had a WBC 1.7, 64% PMNs and mild anemia Hgb 11.1. LFT's, TSH, Fe/Ferritin, and chemistry normal. An extensive infectious workup was negative including blood and fungal cultures, AFB cultures, urinalysis, CXR, visceral leishmaniasis, malaria, brucellosis, EBV, CMV, HIV, RPR, PPD. CT of the chest, abdomen, pelvis, and head found only mild splenomegaly. Transesophogeal echo revealed no valvular vegetations. The patient continued with daily fevers to 102±103 degrees F, anemia worsened to a Hgb 8.4, WBC decreased over ten days to a nadir of 0.7 (ANC 60). GCSF was started for neutropenia and empiric Ceftazidime and Tobramycin were begun. Bone marrow biopsy showed normal cell elements and myeloid to erythroid ratio 1:1. Her mammogram was normal. At this point ANA returned +1:320 as well as Coombs positive. Antids DNA and Anti-Smith was sent. On second day of GCSF patient developed polyarthritis in hands, wrists, elbows, knees, ankles as well as faint malar rash. Follow up CXR revealed small left pleural effusion. GCSF was stopped and patient was started on Prednisone with subsequent resolution of fevers, rash, and arthralgias. Anti-ds DNA returned +1:80. IMPLICATIONS/DISCUSSION: Lupus can present in a variety of ways. In this case, the initial presentation was neutropenic fever. Only after receiving GCSF did the diagnosis become apparent as it precipitated a lupus flare. The diagnostic criteria for Lupus developed by the ACR are 96% sensitive and specific. Non-specific symptoms such as fever, fatigue, and weight loss are common presenting complaints and are often attributed to causes other than lupus leading to misdiagnoses. Leukopenia occurs in more than 50% of lupus patients with either granulocytopenia or lymphopenia. GCSF has been used in the treatment of lupus induced neutropenia resulting in a rise in neutrophils within 48 hours, however the elevation is transient with return to pretreatment levels within days after withdrawal of therapy. In addition treatment with GCSF has been associated with flares in lupus that resolve when the drug is stopped. A 63 year old male with hypertension, chronic renal insufficiency, and peripheral vascular disease presented to the Emergency Department with acute onset of bilateral lower extremity numbness and weakness. Six weeks prior, he had received an arteriovenous fistula in preparation for hemodialysis and was feeling well until the morning of admission, when, upon rising from bed, he fell to the ground because of numbness and weakness of his legs. Review of systems was positive for urinary incontinence,``sharp'' back pain, and shortness of breath which had developed at the same time as the paresthesia. Upon reviewing his medical records, it was discovered that his prostate specific antigen had been elevated at 24.9, but the patient had not followed up with the urologist for further evaluation. Physical exam showed a well-developed male, alert and oriented, in moderate respiratory distress. His respiratory rate was 24, blood pressure 142/90, heart rate 82, and temperature 36 C. Other significant physical findings included bilateral crackles, a pericardial friction rub, absent sensation to light touch at an L3 level, 2/5 bilateral lower extremity strength, absent ankle and patellar reflexes, absent pedal pulses (previously documented), diminished but equal femoral pulses, and an enlarged prostate. Labs showed BUN of 71mg/dl and creatinine of 9.3 mg/dl. Chest radiograph demonstrated mild pulmonary edema. Thorax and abdomen CT without contrast showed a normal aorta. A vascular catheter was placed, and emergent hemodialysis started. The neurology and neurosurgery services were consulted, and plans were made to get an emergent MRI of the lumbosacral spine to rule out cord compression after the patient was stabilized. An hour after dialysis had begun, the patient lost consciousness, and ACLS for pulseless electrical activity was initiated. After 25 minutes of unsuccessful resuscitation, the patient was declared deceased. An autopsy was performed which showed hemopericardium from type A aortic dissection which descended to the bifurcation of the aorta. IMPLICATIONS/DISCUSSION: In this case, aortic dissection caused peripheral arterial ischemia which presented as paresthesia and weakness. Stroke is the most common neurologic presentation of aortic dissection, although 10% of patients present with ischemic peripheral neuropathies. Although pain describing dissection has classically been taught to be``ripping or tearing'', most patients classify the pain as``sharp.'' Given the high mortality associated with aortic dissection, a high index of suspicion should be maintained for patients presenting with paresthesia and back pain. year-old white male with unremarkable medical history presented with acholic stools, dark urine, and constitutional symptoms. Physical examination revealed a diffuse erythematous, maculo-papular rash of trunk and legs, profound jaundice, liver span of 14cm with a firm edge, no splenomegally and no stigmata of chronic liver disease. His initial outpatient laboratory tests revealed: alkaline phosphatase 413 U/L, total bilirubin 3 mg/dl, SGOT 510 U/L, and SGPT 515 U/L. Viral hepatitis serologies, an abdominal ultrasound and CT, and an endoscopic retrograde cholangiopancreatography were unremarkable. Three weeks later, the patient underwent a percutaneous liver biopsy at which time his laboratory values were: WBC 1.9 Â 103/ 3 , Hgb 12.6 g/dl, Platelets 62,000, alkaline phosphatase 706 U/L, total bilirubin 26.9 U/L (direct 21.8 U/L), SGOT 190 U/L, and SGPT173 U/L. The liver biopsy showed a high-grade malignant leukemic infiltrate. Bone marrow biopsy confirmed the diagnosis of B cell/ Burkitt's type acute lymphoblastic leukemia with flow cytometry showing CD19+/CD10+/CD20-/CD34-/CD13+/HLA DR+ cells. IMPLICATIONS/DISCUSSION: Acute lymphoblastic leukemia is primarily a disease of children; adults only account for approximately 20% of cases and Burkitt's B cell ALL, in turn, makes up only 5% of the cases of ALL. Unfortunately, over 60% of adults with ALL are not cured of their disease. The clinical features of ALL are often nonspecific and primarily constitutional as well as manifestations of cytopenias secondary to the bone marrow infiltration. Also, ALL has a predilection for the CNS and symptoms of meningeal leukemia or hyperleukocytosis are not uncommon at presentation. Cholestatic jaundice, however, is a much less common presenting feature with a limited number of case reports in the literature. As demonstrated in this case, one should entertain the diagnosis of acute lymphoblastic leukemia in those presenting with acute cholestatic jaundice. He also had intermittent episodes of gum bleeding, anorexia and shortness of breath. His medications included Atenolol. On physical exam, his vital signs were stable and he was noted to have pale conjunctiva, minimal gum bleeding and a non-radiating systolic murmur. His white blood cell count was 1,600 with a normal differential. He had a hematocrit of 14.9 and a platelet count of 7,000, and lactate dehydrogenase of 794. His remaining lab findings were normal. His peripheral smear showed decrease red blood cells and platelets with normal morphology and no immature white blood cells. Two bone marrow biopsies were both dry taps and confirmed a diagnosis of Acute Myelogenous Leukemia (AML) of the Megakaryocyte subtype (FAB classification M7). His course was complicated early on by neutropenic fever and broadspectrum antibiotics were initiated. Chemotherapy was started after a 48-hour period feverfree period with Idarubicin for 3 days and Cytosine-arabinoside for seven days. He tolerated the treatment well, but remained pancytopenic. Two days after completing induction, he became febrile and complained of severe abdominal pain and diarrhea. Broad-spectrum antibiotics for both bacterial and fungal pathogens were started. Imaging of his abdomen showed bowel wall thickening consistent with neutropenic colitis (typhlitis). Treatment initially consisted of broad-spectrum antibiotic coverage and supportive care. His condition progressively worsened, and both he and family did not want any aggressive intervention. The patient passed away shortly thereafter. IMPLICATIONS/DISCUSSION: Acute leukemia accounts for about 10% of human cancers and is the leading cause of death in adults younger than 35 years of age. In our case, the patient had poor prognostic factors of age greater than 60 years old and poor functional status. The treatment of AML consists of supportive care and chemotherapy with induction therapy conducted with an anthracycline and Cytosine-arabinoside (except for subtype M3). Complications from leukemia include those related to immunosuppression and pancytopenia. Typhlitis represents inflammation and/or necrosis of the cecum, appendix, and/or ileum and is a frequent complication of the treatment of AML. Patients usually present with abdominal pain, diarrhea and fever. The etiology of typhlitis is unknown, but profound neutropenia and mucosal injury from cytotoxic drugs play important roles in the pathogenesis. The mortality rate from typhlitis in leukemic patients can be high and the treatment consists of broad antibiotic coverage, nasogastric suction and supportive care. Surgical intervention is indicated if there is bowel perforation or persistent bleeding. CASE INFORMATION: A 74-year-old female, who was previously independent and ambulating well, presented with a four day history of progressively worsening bilateral knee pain and swelling. Her past medical history is significant for degenerative joint disease with chronic pain in her knees. Her medications included Rofecoxib, Oxycontin, Oxycodone, and Calcium with vitamin D. The patient had received an injection of Hylan G-F 20 in both knees four days prior to presentation for chronic knee pain. A similar injection had been performed one year previously with relief of pain. The next day she developed fever, chills and the knees became warm, erythematous, and swollen, with worsening pain, 10/10 in severity. Upon presentation, the patient was afebrile and both knees were swollen and tender. The patient was unable to bear weight, range of motion of the knees was limited to about 30 degrees bilaterally, and there was tenderness in the lateral joint line bilaterally. Laboratory results showed a normal white blood cell count, ESR 70, and C Reactive Protein 20.6. Venous doppler ultrasound was negative for DVT. Knee x-rays revealed bilateral effusions and evidence of severe osteoarthritis. Bilateral knee arthrocentesis showed (left/right) white blood cell count, 11300/10500 with a normal differential, red blood cells 475/38000, and no crystals. Gram stain and cultures were negative. The patient was admitted to the hospital for pain management and transferred to sub-acute rehabilitation for 3 weeks where she responded well to corticosteroid injections and physical therapy. IMPLICATIONS/DISCUSSION: Visculosupplementation involves using intra-articular injections of high elastoviscous solutions of hyaluronan and hylans in an attempt to restore the natural mechanical properties of synovial fluid. Complications of intra-articular injections with visculosupplementation occur in 2±4% of injections. Local reactions include joint pain, effusion, and rarely, warmth. Acute pseudogout, granulomatous inflammation, and severe systemic reactions have also been reported. Rest, analgesics, corticosteroid injections, cold packs, and therapeutic arthrocentesis may help relieve symptoms. The presence of fever, debilitating pain, and joint effusions in our patient suggested both a systemic and local reaction to the supplementation. This impacted her functional status as she became bedridden and she required a prolonged rehabilitation to regain her independent ambulatory status. LEARNING OBJECTIVES: 1. Recognize myxedema coma as rare, but important diagnosis in differential for obtunded patient 2. Recognize emergent need for treatment of myxedema 3. Recognize mortality due to the condition and its therapy. CASE INFORMATION: 80 year old Caucasian female with history of congestive heart failure, chronic obstructive pulmonary disease, hypothyroidism was found by neighbors to be in obtunded state. In emergency room, her temperature was 95.9 F, blood pressure 108/56, pulse 50, respiratory rate 11, glasgow coma scale score 3. Her skin was cool to touch, with induration and 1+ non-pitting edema of the lower extremities. She had peri-orbital swelling. Labs: Na 142, K 4.3, Glc 142, creatinine 2.9, Hgb 9.3, CPK 286, CPK MB 5, troponin I .1, TSH 309, T4 total < 1, T4 free .1. ABG (after intubation, 100% FIO2) pH 7.26 pCO2 52 pO2 624. EKG:sinus bradycardia at 50 beats/minute, low voltage. CT head was normal. Blood cultures were negative. After labs were drawn, patient was started on nasogastric levothyroxine but continued to deteriorate. The next day, she was switched to intravenous therapy and started on steroids. Her mental status improved markedly and she was extubated. However, a few days later, she was found in asystole and expired. IMPLICATIONS/DISCUSSION: Myxedema coma is a rare, deadly condition. It should be suspected in patients who present with hypothermia and altered mental status. They may also have hypotension, hypoventilation, hyponatremia, hypoglycemia, and cardiovascular abnormalities. It may be caused by severe longstanding hypothyroidism or precipitated by infection, infarction, cold weather, narcotics/sedatives. Elderly females are most commonly affected. Therapy should be based on clinical suspicion and started right after drawing TSH, free T4 and cortisol levels. Glucocorticoid therapy prevents adrenal crisis in cases with associated adrenal insufficiency. Type of thyroid therapy is controversial. Some prefer T3 because of its rapid onset and greater biologic activity. Others prefer T4, which should be given intravenously to avoid malabsorption. Careful dosing based on age and weight may prevent cardiac complications of thyroid therapy. The mortality rate is 30±40% despite adequate therapy. Even though intravenous therapy was delayed in this patient, she showed significant improvement prior to her demise. Thus, her death may have been due to cardiac complications of therapy rather than the delay in intravenous therapy. However, definitive cause of death was not determined. Since therapy is controversial and associated with complications, an additional lesson is to focus on prevention via recognition and treatment of hypothyroidism. In this tragic case, the patient had been lost to follow up.
INTESTINAL T-CELL LYMPHOMA PRESENTING AS SMALL BOWEL PERFORATION. P. Hulick 1 , H. Tun 1 ; 1 Mayo Clinic, Jacksonville, FL (Tracking ID #74266) LEARNING OBJECTIVES: 1. Recognize the association of primary gastrointestinal T-cell lymphoma and celiac disease 2. Recognize the poor prognosis, especially with +CD56 marker 3. Treatment must include systemic chemotherapy. CASE INFORMATION: A 73-year-old white female presented to the emergency department following an acute exacerbation of abdominal pain that had progressed over the past two months. The pain was originally diffuse in nature but became sharp in character with the acute presentation. The patient denied radiation to the back while movement exacerbated the pain. The patient reported decreased appetite with a fifteen-pound weight loss over the preceding six months, nausea but no vomiting, and intermittent fevers to a maximum of 105F over the preceding two months. The patient denied other constitutional symptoms. On admission, the patient was afebrile with normal vital signs. Physical exam was notable for diffuse abdominal discomfort on palpation, decreased bowel sounds of normal pitch, and the absence of generalized lymphadenopathy Laboratory studies were Hgb 11.1g/dL, MCV 85 fL, and platelet count of 385,000. Electrolytes, creatinine and liver profile were unremarkable. LDH was 185 u/L. Computed tomography (CT) scan of the abdomen/pelvis revealed a pneumoperitoneum without identification of a specific site of perforation. Exploratory laparotomy revealed a perforated small bowel mass (7.0 Â 4.0 Â 1.0 cm) with infiltrating neoplastic lymphocytes positive for CD 3, 43, and 56 staining. Flow cytometry showed a T-cell phenotype with anomalous expression of CD 103 (T-cell mucosal homing antigen receptor). Staging evaluation did not reveal evidence of metastatic disease. The pathology findings in our case are consistent with enteropathy associated T-cell lymphoma. Our patient presented with the rarer T-cell CD56 positive variant which has a poorer overall prognosis and aggressive course. IMPLICATIONS/DISCUSSION: Primary gastrointestinal lymphomas account for 30±40% of extranodal lymphomas, but they are usually of B-cell lineage. Celiac disease, a malabsorption disorder related to gluten intolerance, is thought to be a risk factor for developing primary intestinal T-cell lymphoma. Intestinal T-cell lymphoma continues to have a poor prognosis. CD56 (neuronal cell-adhesion molecule) positive tumors have been associated with a more aggressive course. Additional poor predictors include nutritional status at presentation and presence of a bowel perforation. Survival data for cases presenting with bowel perforation has been estimated at 28% and 0% six months and one year post diagnosis. Treatment consists of surgical resection of the tumor and adjuvant chemotherapy which consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) which our patient received. Systemic chemotherapy is necessary, regardless of stage, because of the malignant clonal population of T-cells that exists in the normal gastrointestinal mucosa. Relapse rate is thus high, even in patients with initial response to chemotherapy. year-old woman with recurrent ovarian cancer was hospitalized and conservatively managed for a partial small bowel obstruction. After a second unsuccessful attempt to anti-coagulate her right su-bclavian central venous catheter, the patient developed acute left-sided weakness associated with slurred speech. She denied chest pain but stated she was increasing short of breath overnight. Vital signs included a heart rate of 117 beats per minute and a respiratory rate of 26 breaths per minute. Physical exam was remarkable only for a left facial droop, left arm and leg weakness, and dysarthria. An emergent CT scan revealed an acute, non-hemorrhagic right-sided cerebral infarct. ECG, duplex scan of the carotid arteries and chest radiograph were normal. Cardiac isoenzymes were negative for myocardial infarction. An arterial blood gas analysis revealed severe hypoxia and an alveolar-arterial oxygen gradient of 42. A ventilation-perfusion scan showed high probability for pulmonary embolism and a transesophageal echocardiogram revealed a patent foramen ovale. IMPLICATIONS/DISCUSSION: Indwelling catheters can serve as a nidus for thrombus formation. Emboli may produce pulmonary sequela with concomitant increased right sided circulatory pressures. This results in paradoxical emboli entering the arterial circulation though a right to left shunt, such as a patent foramen ovale, with resultant cerebral ischemia. Patients who demonstrate symptoms of both a cerebral infarct and a pulmonary embolism should be evaluated with a CT scan, carotid duplex, ventilation-perfusion scan and transesophageal echocardiogram to identify the source and location of the embolus. Treatment involves immediate evaluation for emergent anticoagulation if not contraindicated. Operative closure of a patent foramen is not indicated if the elevation of right sided pressure is related to an acute event. year old Asian female without significant medical history presented with ten days of rash, fever, joint pains and stiffness. Rash, which was erythematous and non-pruritic, began on her thighs and spread to her back and chest. Joint pains spread from her hands and wrists to her shoulders, ankles and knees. Her course of illness included high fevers and a sore throat. Physical exam revealed temperature of 103.9 F and erythematous, salmon colored maculo-papules on her trunk and proximal extremities. Palms and soles were spared. The rash was exacerbated by fever spikes, which usually occurred in the afternoon. Koebner's phenomenon occurred when rubbing caused the rash to appear on previously uninvolved skin. Metacarpophalangial joints, wrists and left shoulder were tender, but without erythema, warmth or effusions. There was no lymphadenopathy or splenomegaly. Labs: WBC 22,000 (85% neutrophils, 5% bands). Blood cultures and tests for HIV, enterovirus, cytomegalovirus, Ebstein-Barr virus, hepatitis, parvovirus were negative. Anti-streptolysin O titers were negative. LFT, ANA, RF, chest xray, CT chest/abdomen/pelvis, echocardiogram were normal. Skin biopsy showed perivascular infiltrate, no vascultis. Patient was started on nonsteroidal anti-inflammatory medications and gradually responded to therapy. IMPLICATIONS/DISCUSSION: Adult Still's disease, although uncommon, should be considered in adults with fever, joint pains and rash. The diagnosis requires atleast 5 criteria, including atleast 2 major ones. The major criteria are temperature of > 39 C for atleast 1 week, arthralgias for atleast 2 weeks, typical rash (salmon, maculopapular, evanescent, koebners phenomenon), WBC > 10,000 (> 80% neutrophils). Minor criteria are sore throat, lymphadenopathy and/or splenomegaly, elevated transaminases or LDH, negative ANA and RF. Diagnosis also requires exclusion of infection, malignancy and other rheumatic diseases. Treatment is aimed at joint symptoms because extent and chronicity of arthritis is best predictor of long term consequences. Nonsteroidals, corticosteroids and immunomodulating drugs are therapeutic options. Choice of therapy depends upon organ involvement and severity of disease. LEARNING OBJECTIVES: 1) To recognize intussusception as a cause of intestinal obstruction, 2) To recognize that the treatment of adult intussusception differs from pediatric cases, 3) To recognize that adult intussusception is often a presentation of underlying pathology. CASE INFORMATION: A 53 year old male with a past history of small cell lymphoma treated three years prior with CHOP chemotherapy and radiation therapy presented to the emergency department with several days of diffuse, unremitting periumbilical abdominal pain. He denied melena or hematochezia, and, until recent constipation, his bowel habits were unchanged. His intake of food was reduced due to bloating, but he denied nausea or vomiting. He did not experience fatigue, night sweats, fevers, or weight loss. On examination, his abdomen was soft and non-distended. He had normal bowel sounds. Despite diffuse nonspecific tenderness, he did not exhibit guarding or rebound. The spleen and liver were normal size, and no masses were palpated. A rectal exam was normal, and stool hemoccult was negative. Because of his medical history, a CT scan of the abdomen was performed which revealed ileocecal intussusception and resulting obstruction. A barium enema confirmed an intussusception but was unable to reduce it. He was taken to the operating room where the surgeons found intra-abdominal lymphadenopathy and ischemia of the proximal colon requiring hemicolectomy. Pathology of the resected tissue revealed follicular B-cell lymphoma within the terminal ileum and cecum. IMPLICATIONS/DISCUSSION: Intussusception is a process of gastrointestinal invagination in which a proximal segment telescopes into a distal portion. While common in children as the second leading cause for acute abdomen, adult intussusception occurs in only 5±10% of all cases. Presentation may be either acute or of a chronic indolent nature. It is rarely idiopathic in adults as it is in children, and underlying pathology is readily identifiable in up to 90% of all cases. Common causes include polyps, Meckel's diverticulum, endometriosis, and inflammatory bowel disease, but 54%±69% of identified pathology are malignancies including lymphomas. The process of intussusception causes gastrointestinal circulatory obstruction which can lead to ischemia and bowel infarct. Avoiding delay in surgical intervention preserves tissue. While barium enema is both diagnostic and therapeutic in children, it is less efficacious in adults, and its role in adult therapy is actually controversial. Because of the likelihood of underlying pathology, primary resection is often the treatment of choice. Whether or not to attempt reduction before resection is arguable; some authors have suggested that time spent on barium enemas compromises the integrity of bowel tissue. While uncommon, intussusception should be readily recognized when it occurs. Treatment of the disease requires timely surgical intervention to preserve bowel tissue. It is paramount to recognize that treatment in adults differs from pediatric cases where barium enemas are often successful. Discovery of adult intussusception must arouse clinical suspicion of underlying pathology which should be addressed. He was taken to the local emergency department, which was unable to run ethylene glycol level assays. At the outside hospital, he had an anion gap of 25, but an osmolar gap was not measured. He was placed on an ethanol drip and transferred to a tertiary care center. A stat ethylene glycol level returned elevated at 432 mg/dL, and the osmolar gap was 140 mOsm/kg. At this time, although stuporous, he was arousable and conversed appropriately. Unfortunately, urine was not sent for analysis, but serum creatinine was 1.2 mg/dL. Instead of initiating hemodialysis, the patient was placed on intravenous fluids with dextrose and saline and given fomepizole every 12 hours. Treatment continued for the next 60 hours until his ethylene glycol level fell within a non-toxic range. Clinically, his mental status returned to baseline, and his serum creatinine improved to 0.8 mg/dL. The patient underwent psychiatric evaluation and after medical clearance was transferred to an inpatient psychiatric facility for treatment of his depression. IMPLICATIONS/DISCUSSION: Ethylene glycol is a sweet liquid found in antifreeze. The taste has led to accidental intoxication in young children, but ingestion also occurs during suicide attempts. It is metabolized into oxalate, which is excreted by the kidneys, through several enzymatic steps beginning with alcohol dehydrogenase. Early toxicity is manifested by mental status changes ranging from stupor to coma. Renal damage from direct toxicity to the kidneys develops later although the rate of occurrence depends on the severity of intoxication. Presumptive diagnosis, based on a history of ingesting solutions likely to contain ethylene glycol, such as antifreeze, is the first critical step in diagnosis. While ethylene glycol can be measured directly, the laboratory equipment may not be available readily in all emergency departments. However, diagnosis can be confirmed indirectly with basic laboratory tests. Ethylene glycol itself causes an increased osmolar gap, and its metabolite glycolic acid creates the anion gap metabolic acidosis. The end product of metabolism, oxalate, can precipitate in urine, and urinalysis may reveal calcium oxalate crystals. Treatment must occur expediently to preserve renal and neurological function. This should involve adequate hydration and inhibition of alcohol dehydrogenase to prevent breakdown of ethylene glycol into its toxic metabolites. Ethylene glycol is dialyzable, and severe intoxication may require hemodialysis. However, in most cases, fomepizole, a more efficacious inhibitor of alcohol dehydrogenase than ethanol, can be used alone without need for dialysis. If both methods are used the dose of fomepizole needs to be increased since it is a dialyzable agent. Fomepizole can preserve renal function without invasive vascular access required for hemodialysis. Recovery without permanent sequelae is possible but requires timely recognition of ethylene glycol intoxication and familiarity with the treatment methods. A 24 year old man with severe hemophilia A and AIDS was admitted to the medical service with new neurological deficits. Eight weeks prior to admission he was started on highly active retroviral therapy (HAART). Six weeks prior to admission he developed left facial droop and left upper extremity weakness. The patient's family described behavioral changes including mood disturbances and aggressive behavior. On the day of admission the patient experienced two witnessed generalized seizures. On physical examination he appeared chronically ill. Neurological examination revealed a left cranial nerve VII upper motor neuron palsy and left upper extremity weakness. There was no papilledema. An MRI of the brain revealed multifocal hyperintense signals in the right frontal, left frontal, and right parietal areas predominately affecting the white matter without enhancement or mass effect. A subsequent spinal tap revealed a normal opening pressure with 1 white blood cell, 0 red blood cells, total protein of 74, and glucose of 54. Evaluation of the cerebrospinal fluid for tuberculosis, bacteria, fungus, toxoplasmosis, syphilis, herpes, CMV, and cryptococcus were negative. JC virus DNA was detected by PCR from the CSF. Based on the clinical presentation, radiographic findings and positive JC virus DNA in the CSF, the diagnosis of progressive multifocal leukoencepholapthy (PML) was made. IMPLICATIONS/DISCUSSION: PML is a disorder characterized by multiple rapidly progressive focal neurological deficits. Hemiparesis, visual field defects, cognitive impairment, and cranial nerve deficits are most common. Intracranial pressure usually remains normal. Ninety-five percent of all patients with PML have an underlying immunosuppressive disorder such as HIV. Conversely, 1±3% of AIDS patients will develop PML. CT or MRI of the brain reveals multiple bilateral, usually asymmetric areas of demyelination. The lesions are usually localized to the periventricular area and subcortical white matter. There is no contrast enhancement or mass effect. The diagnosis is made based on the clinical picture of multiple neurologic deficits, characteristic radiographic findings, and isolation of the JC virus from the CSF. Detection of JC virus from the CSF has a sensitivity of 74±95% and a specificity of 92±100%. There is currently no proven effective therapy for PML. HAART therapy has been shown to slow the progression of the disease. However, most patients deteriorate rapidly within 6 months. LEARNING OBJECTIVES: 1. Recognize the importance of opthomologic exam in the differential diagnosis of aseptic meningitis 2. Understand the role of steroid therapy in Vogt-Koyanagi-Harada Syndrome. CASE INFORMATION: A 24-year-old previously healthy man was admitted to the medical service with progressively worsening generalized headaches, decreased visual acuity, and hearing loss for two weeks. The patient reported malaise, but denied nausea, vomiting and fevers. He had never traveled outside the United States and did not use drugs or alcohol. The patient was afebrile. HEENT examination revealed bilateral papilledema and decreased visual acuity and hearing. Neck examination was remarkable for nuchal rigidity. The neurological exam was otherwise normal. CT scan of the head and orbits confirmed the papilledema but did not reveal any mass effect. A subsequent spinal tap revealed normal opening pressure, 727 white blood cells with 70% lymphocytes, a protein = 81, and glucose = 53. The patient was begun on broad-spectrum antibiotics and anti-tuberculous therapy without significant improvement. RPR, HIV, ESR, and cryptococcal antigen were negative. Ophthalmologic consultation was obtained. Slit lamp examination revealed cells in the anterior chamber consistent with uveitis, choroidal inflammation, and depigmentation. The patient was diagnosed with Vogt-Koyanagi-Harada (VKH) syndrome based on the ophthalmologic findings, aseptic meningitis and hearing loss. The patient was started on high dose steroids with relatively rapid resolution of the headaches and visual and hearing loss. IMPLICATIONS/DISCUSSION: VKH is a uveomeningoencephaltic syndrome and appears to be an autoimmune reaction to melanocytes thus making it more common in Asians, Middle-Easterners, and Hispanics than whites. Melanocytes are located in the skin, uvea, retinal choriod, membrane of the inner ear, and leptomeninges, accounting for the particular pattern of involvement seen in this syndrome. The ocular findings may include cataracts, glucoma, and globe atrophy. Meningeal involvement may lead to encephalopathy, seizures, myelopathy, or other focal signs. CSF findings include a lymphocytic pleocytosis, elevated total protein, and normal opening pressure. Other manifestations of VKH include dysacousia, alopecia, poliosis, and vitiligo. Although the optimal steroid dosage for treating VKH has not yet been defined it is generally accepted to begin therapy with high-dose systemic steroids followed by oral steroids for at least 6 months. Patients typically have a rapid decrease in symptoms. In about half of the patients, disease recurs within six months of discontinuation of steroids.
NONSTEROIDAL ANTI-INFLAMMATORY DRUG ASSOCIATE D LOWER GASTROINTESTINAL TOXICITY. J.T. Jacob 1 , A.K. Jaffer 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #74333) LEARNING OBJECTIVES: 1) Recognize the lower gastrointestinal (GI) adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs). 2) Examine the differential diagnosis of multiple gastrointestinal ulcers. 3) Implement appropriate testing and treatment of NSAIDinduced lower GI pathology. CASE INFORMATION: An 80-year-old female with coronary artery disease, hypertension, dyslipidemia and osteoarthritis was admitted to the hospital with chest pain and two episodes of syncope. Each episode of syncope was preceded by use of sublingual nitroglycerin. Review of systems was positive for chronic fatigue, but negative for weight loss, nausea, vomiting, abdominal pain or GI blood loss. Medications were metoprolol, omeprazole, hydrochlorothiazide, indomethacin, and gemfibrozil. Exam revealed a BP = 113/60 without orthostasis, a Pulse = 95 and 97% O2 sat. The exam of the abdomen was normal except for guaiac positive stool on rectal exam. A CBC showed a hemoglobin of 8.9 g/dl with a normal MCV and RDW. An iron panel, B12, folate, LDH, bilirubin, and haptoglobin were also normal. A dipyridamole thallium stress test was negative. The syncope was felt to be related to transient hypotension from nitroglycerin in the setting of anemia. She was therefore transfused blood and underwent upper and lower endoscopies. Multiple ulcers were noted in the stomach, terminal ileum, ileocecal valve, transverse colon and descending colon. Biopsies demonstrated acute nonspecific inflammation. Gastrin levels were normal. Indomethacin was discontinued, acetaminophen was initiated and omeprazole continued. She was discharged home in stable condition with a two-month follow up endoscopy. IMPLICATIONS/DISCUSSION: Adverse gastrointestinal (GI) effects of NSAIDs are not limited to the stomach and duodenum. Although these sites are most commonly affected, adverse effects related to the large bowel and the distal small intestine are increasingly being recognized in practice. NSAIDs can cause a variety of pathology in the lower GI tract including erosions, ulcers, diaphragms, strictures, small bowel enteropathy, diverticular bleeding and colitis resembling or exacerbating inflammatory bowel disease (IBD). The pathophysiology of these changes has not been elucidated. Other etiologies of multiple GI ulcers include: viral and bacterial infections, vasculitides, IBD and Zollinger-Ellison syndrome. Treatment consists of discontinuation and avoidance of all NSAIDs. Gastrin levels should be checked to rule out a gastrinoma. A repeat endoscopy in 6±8 weeks is needed to confirm healing otherwise other etiologies need to be considered. An increase in reporting of these adverse effects will lead to a better understanding of NSAID induced lower gastrointestinal toxicity amongst physicians. -old male with a history of DM1, inflammatory bowel disease (IBD) and PG was admitted for ileostomy closure. The patient discontinued his insulin pump the night prior to surgery. The preoperative glucose was 500 and the surgery was canceled. He was well until three weeks prior when his shin lesions started to get worse after minor trauma. Review of systems was otherwise negative. On exam, he appeared lethargic, T = 38.3 8C, Pulse = 145, and BP = 110/50 mmHg. The patient had four necrotic shin and ankle ulcers with exposed bone and tendon. Labs revealed a serum bicarbonate of 9 mg/dl, potassium of 4.6 mg/dl, glucose of 527 mg/dl, anion gap of 26 and elevated serum ketones. A blood gas revealed a pH of 7.24, pCO2 of 17 mmHg, pO2 of 123 mm Hg and HCO3 of 7 mg/dl. Culture of the ulcers grew gram positive and negative bacteria. The patient was treated with intravenous (IV) Insulin, fluids and antibiotics. After the DKA resolved, therapy for PG was begun using infliximab, topical cromolyn, clobetasol and whirlpool therapy. During infliximab infusion the patient developed transient hypotensive and shortness of breath. He was treated with diphenhydamine and hydrocortisone with resolution of his symptoms. He was switched to oral cyclosporine and discharged home. IMPLICATIONS/DISCUSSION: There are two accepted alternatives in the preoperative management of DM1. Patients can hold short-acting insulin on the morning of surgery and take one-half to two-thirds of their normal long-acting insulin dose or omit their morning insulin regimen entirely and be administered regular insulin on arrival to the hospital based on their glucose level. Patients with insulin pumps should continue their pumps at a basal rate until the morning of surgery. PG is an idiopathic dermatosis associated with epidermal and/or dermal inflammation. It is most commonly associated with systemic diseases such as IBD, rheumatoid arthritis and lymphoproliferative disorders. These lesions typically involve the lower extremities and are painful. Treatment is directed at the underlying systemic disease. Systemic steroids were avoided in this patient's case due to concerns for recurrent hyperglycemia. Alternatives include infliximab, dapsone, thalidomide, azathioprine, cyclophosphamide, 6-mercatopurine, tacrolimus and cyclosporine. Adjunctive therapies include antimicrobials, topical cromolyn, clobetasol and whirlpool therapy. female with Crohn's disease on long term TPN presented with shallow breathing, paresthesias involving the extremities and progressive motor weakness over a period of 2 days. Two weeks prior to presentation she was diagnosed with linerelated candidemia; the port was removed and she was treated with intravenous fluconazole. Exam revealed a cachectic female who was afebrile, BP = 110/80, HR = 90, RR=22, O2 sat = 97%. Neurological exam revealed a right facial droop, dysarthria, motor strength was 2/5 in the upper extremities (UE) and 0/5 in the lower extremities (LE) bilaterally. All reflexes were absent. Brain and spine MRI were unremarkable. The CSF cell count and glucose were normal while the protein was elevated. Blood, urine and CSF cultures were negative. The initial vital capacity (VC) was decreased to 0.89 L and the negative inspiratory force (NIF) was unobtainable. EMG revealed features of generalized sensorimotor polyneuropathy with demyelination and axonal loss. Recurrent asymptomatic episodes of non-sustained ventricular tachyarrhythmia were observed on telemetry but cardiac evaluation revealed no structural heart disease. The patient was diagnosed with GBS with cranial nerve involvement, a GBS variant called Miller-Fisher syndrome. Plasmapheresis was instituted and after a couple exchanges improvement was noted in her motor strength, VC and NIF. She was discharged after completing five session of plasmapheresis. 6-weeks post-discharge, her neurological examination was remarkable only for mild residual diplegia. IMPLICATIONS/DISCUSSION: GBS is an acute ascending inflammatory demyelinating polyradiculoneuropathy. The management of GBS requires early recognition because the respiratory muscles can be involved. Elective intubation should be considered when the forced VC falls below 15 mL/kg or when the patient is unable to generate more than 20 cm H2O of maximal NIF. Initial symptoms include parasthesias with rapid progressive muscle weakness and areflexia. The Miller-Fisher syndrome is a variant of GBS in which the cranial nerves are affected. Autonomic dysfunction with cardiac dysrythmias occurs in one half to two thirds of patients. The disease generally has a good prognosis with close cardiopulmonary monitoring. Plasmapheresis and IVIG have been shown to alter the natural history of GBS if treatment is instituted quickly. year old male presented for further workup after suffering a seizure episode on two separate occasions. 10 days prior to his presentation at the university hospital, the patient experienced a witnessed tonic-clonic event and was taken to a local hospital. The patient was admitted to that hospital and found to have a normal CT, MRI, and EEG. While preparing for discharge home, he had another tonic-clonic event which was witnessed by hospital personnel, and he was transferred to the university hospital for further evaluation. He complained of feelings of mild generalized weakness over the past few months as well as some tingling and numbness in his hands and feet. He denied any recent trauma or seizure history. He also denied any chills, fevers, nausea, vomiting, or weight loss. Past medical history was noncontributory. He denied the use of illicit or prescription drugs, tobacco, ethanol, or herbals. On examination, his vital signs were pulse 101, blood pressure 146/90, and temp 35.4C. Physical exam was unremarkable including normal neurological exam. Labs on admission were significant for K of 3.1 meq/L (nl 3.5±5.0), Cl of 96 meq/L (nl 100±110), CO2 33 meq/l (nl 20±32), Ca 9.0 mg/dl (nl 8.5±10.5), and Mg 1.0 meq/l (nl 1.5± 2.4). Records received from the outside hospital showed low Mg and K levels during the time of both wintessed seizures. Patient was repleted with IV Mg and K and placed on phenytoin. Repeat MRI and EEG were negative. The patient continued to have low serum magnesium levels in spite of aggressive IV and PO repletion. 24 hour urine studies revealed a significantly elevated urine magnesium excretion of 25 MEq/24hr (nl 6±10 MEq/24hr), as well as a decreased urine calcium excretion of 94 mg/24hr (nl100±300 mg/24hr). Urine diuretic screen was negative. Renin and aldosterone were both elevated at 15.8 ng/ml/hr (nl 0.2±1.6 ng/ml/hr) and 38.4 ng/dl (nl 1.6±16) respectively. The patient was diagnosed with Gitelman's Syndrome and discharged home on magnesium oxide, amiloride, aldactone and potassium supplements. He suffered no further episodes of seizure activity and his weakness improved. IMPLICATIONS/DISCUSSION: Seizures presenting with normal MRIs and EEGs can present a diagnostic dilemma for internists. This case emphasizes the importance of considering metabolic disorders as a cause of seizures especially in the setting of normal MRIs and EEGs. Gitelman's Syndrome, an autosomal recessive disorder which usually presents in early adulthood, is a rare variant of Bartter's syndrome resulting from defects in renal tubular function. Unlike Bartter's, Gitelman's patients present with hypomagnesemia and hypocalciuria, along with complaints of severe fatigue, cramps, muscle spasms, and polyuria. with progressive failure to thrive. Three exploratory laparotomies over that time for acute abdomen revealed no pathologic diagnosis. PMH includes asthma, G1P1. P.E. reveals a cachectic, female, 4ft. 11in., 36.1 kg. Evidence of hypotelorism, bilateral ptosis, decreased lateral gaze with nystagmus at end gaze. Abdominal exam reveals distended and tympanic epigastrum. Neurological exam reveals normal visual acuity, decreased C.N. 8 bilaterally, strength of proximal muscle groups 4/5 with atrophy, while distal muscle groups 5/5 bilaterally. DTR's brisk through out. Laboratory studies included CBC, basic metabolic panel, thyroid function tests, Vitamin B12, folate, and lactic acid which were normal except hemoglobin 9.9g/dl, low iron saturation 11%, ferritin 51. She underwent EGD which was normal including duodenal biopsies, aspirates and electronmicroscopy of duodenal biopsies. Her esophageal manometry revealed hypotensive LES, gastric emptying scan showed prolonged liquid gastric emptying with T 1/2 = 136 minutes (mean is 90 minutes). SBFT showed slow emptying of barium. In consultation with neurology, work up included urine porphyrin studies that were negative, while MRI of brain showed diffuse leukoencephalopathy. LP was recommended but nondiagnostic. Fatty acid levels were normal. Ophthalmology evaluation revealed retinal pigmented degeneration while audiometry revealed bilateral moderate sensorineural hearing loss. Muscle biopsy would be indicated if genetic testing were not available. Pt. had chromosomal karyotyping, as well as mitochondrial DNA and thymidine phosphorylase levels, which are absent in specific mitochondrial disorders. These confirmed the diagnosis of a mitochondrial disorder, subtype called mitochondrial neurogastrointestinal leukoencephalopathy(MNGIE). IMPLICATIONS/DISCUSSION: Mitochondrial disorders certainly accounts for <0.1% of causes of nausea and vomiting. On review of the patient's history and physical exam she also had salient findings of proximal muscle weakness, hearing and eye abnormalities.
Gastrointestinal involvement is an unusual presentation for mitochondrial disorders with only 21 reported cases. More typical findings of mitochondrial myopathies are progressive ptosis, ophthalmoplegia and proximal muscle weakness in young patients. All these findings can be found on an internists thorough physical exam, as this case illustrates.`I CAN'T TIE MY SHOES. '' ADENOCARCINOMA OF THE LUNG AND PARANEOPLASTIC ANTIBODY ENCEPHALOPATHY: FOLLOW UP 2 YEARS LATER. K.S. Jorn 1 ; 1 Mayo Community Internal Medicine, Jacksonville, FL (Tracking ID #74452) LEARNING OBJECTIVES: 1. To describe symptoms that may be seen in primary care that should prompt evaluation for paraneoplastic limbic encephalopathy 2. To describe the clinical course of and paraneoplastic antibody levels in this patient over time 3. To present the concept that paraneoplastic antibody levels may not be cost-effective for surveillance after cancer is treated, but additonal study is needed. CASE INFORMATION: A 47 yo nurse presented for primary care in May 2000 after recovering from a massive pulmonary embolus in February 2000. Previous medical history was significant only for tobacco abuse and history of depression and anxiety, stable with medical therapy. Evaluation for causes of the embolus revealed only a 1cm coin lesion in her right lung, stable on follow-up CT through June 2001. In Fall-Winter of 2000, the patient, co-workers, and her supervisors noted a decline in the patient's concentrating ability and documentation efficiency. An accusation of diversion of narcotics triggered severe depression and anxiety in December 2000. The patient was receiving medical and psychological therapy for depression and anxiety when apraxic symptoms such as inability to tie her shoes, loss of direction sense, fecal and urinary incontinence, and absence-seizure type episodes were noted in the spring of 2001. She was felt to have symptoms of limbic encephalopathy. A paraneoplastic antibody panel revealed an elevated P/Q calcium channel antibody. Thoracotomy with removal of the coin lesion July 2001 revealed it to be bronchoalveolar cell carcinoma. Patient's cognitive function has gradually improved to near-normal levels. Though the elevated P/Q antibody led to her cancer diagnosis, the antibody levels have not paralleled her clinical course. IMPLICATIONS/DISCUSSION: Neurologic paraneoplastic antibody syndromes are more typically associated with small cell lung carcinoma and often involve brain stem, cerebellar, or neuromuscular junction dysfunction. There is little literature to guide the follow-up use of paraneoplastic antibody testing after cancer has been treated. This unusual case is characterized by cognitive dysfunction in the presence of lung adenocarcinoma and suggests that follow-up paraneoplastic antibody testing does not add much to clinical assessment after treatment of cancer. Additional studies are needed to determine if there is prognostic information to be gained by surveillance with paraneoplastic antibody testing.
EVALUATION AND SIGNIFICANCE OF PERSISTENT ASYMPTOMATIC ELEVATED BONE ALKALINE PHOSPHATASE. K.S. Jorn 1 ; 1 Mayo Community Internal Medicine, Jacksonville, FL (Tracking ID #74454) LEARNING OBJECTIVES: 1. To review a series of cases in which asymptomatic elevated bone alkaline phosphatase was discovered, evaluated, and followed. 2. To propose an algorithm for evaluation of asymptomatic elevated bone alkaline phosphatase. 3. To suggest that additional study is needed to determine if this algorithm is cost-effective. CASE INFORMATION: Asymptomatic elevated liver enzymes are commonly encountered in primary care and are well documented in medical literature. Asymptomatic elevated alkaline phosphatase (bone subtype) also appears to be common in primary care but there is a lack of guidance in the literature for further evaluation of the abnormality once it is discovered. The cases presented provide examples of persistently elevated bone alkaline phosphatase and the outcome of the evaluation and followup over three to five years. Diagnoses revealed during evaluation of asymptomatic elevated bone alkaline phosphatase include metastatic lung cancer, male osteoporosis, and an apparently benign elevation of the enzyme. An algorithm for evaluation of an asymptomatic elevated bone alkaline phosphatase is proposed. IMPLICATIONS/DISCUSSION: The author hopes that this limited case series will help to focus additional attention on this relatively common clinical problem, ultimately providing more robust guidance to the clinician in its evaluation and management. -old female with a history of fibromyalgia and penicillin allergy presented several weeks after rectal abscess drainage with malaise, generalized myalgias and subjective low grade fever. On the first visit to her physician, the patient was sent home with the presumptive diagnosis of fibromyalgia exacerbation. Having continued to experience symptoms that she described as different from her usual``fibromyalgia pain'', she returned to the office to seek additional medical care. She was afebrile and her physical exam was notable for a diastolic murmur and generalized muscle tenderness. Pertinent labs included H/H of 11/ 39, WBC 14 and ESR 66. Blood cultures were drawn and returned 24±48 hours later, being positive for Enterococcus faecalis in 3 out of 3 bottles. TEE demonstrated an aortic valve vegetation with regurgitation. The diagnosis of IE was made and she was treated initially with vancomycin and gentamicin. She was successfully desensitized to penicillin and her treatment course with IV penicillin and gentamicin continued for 6 weeks. The patient had gradual resolution of her presenting symptoms during her treatment course. IMPLICATIONS/DISCUSSION: Patients with fibromyalgia or chronic pain syndrome can be challenging as they present frequently with multiple nonspecific complaints. It can be tempting to attribute all of their symptoms to these underlying disorders. However, physicians should objectively evaluate the patient and entertain other diagnoses when these patients present with new or worsening symptoms. In our case, this finally led to the diagnosis of IE.
The symptoms and signs of IE often are constitutional and, when localized, result from a complication of IE. Consequently, if physicians are to avoid overlooking the diagnosis of IE, a high index of suspicion must be maintained. This is especially true in cases of IE that have a subacute course. Subacute endocarditis is characterized by an insidious onset and by nonspecific signs and symptoms of chronic illness, such as low grade fever, fatigability, and malaise. Immunologic phenomena occur when the disease is protracted. Musculoskeletal symptoms, as was described in this patient, are common. Although fever is the most common sign in patients with IE, it is typically low grade. Heart murmurs are noted in 80 to 85 percent of patients with native value endocarditis (NVE) and are emblematic of the lesion predisposing to IE. Enterococci, which comprise normal gut flora, account for 5 to 15 percent of cases of NVE. There are no specific clinical manifestations nor is there a single lab test that establishes the diagnosis of IE unequivocally. A combination of major and minor clinical findings, outlined in the Duke criteria, makes the diagnosis highly probable. In the absence of surgical indication, optimal therapy for enterococcal endocarditis requires synergistic bactericidal interaction of antimicrobial agents targeted against the bacterial cell wall (penicillin, ampicillin, or vancomycin) and an aminoglycoside. Because of her overall clinical and laboratory presentation, the pituitary-adrenal axis was evaluated. The AM Cortisol was level 1.4 and ACTH was 5. Brain MRI and CT abdomen were unremarkable. The diagnosis of secondary adrenal insufficiency, potentially secondary to Megace 1 , was made. Patient was started on hydrocortisone 20 mg PO qAM, and 10 mg PO qPM. She had marked improvement in her symptoms. Megace 1 was discontinued and hydrocortisone was ultimately tapered. IMPLICATIONS/DISCUSSION: Findings in adrenal failure are nonspecific, and, without a high index of suspicion, the diagnosis of this potentially lethal but readily treatable disease is missed easily. Symptoms due to cortisol deficiency include anorexia, nausea, vomiting, weight loss, weakness, and fatigue. Although orthostatic hypotension is more marked in primary than secondary adrenal insufficiency because of aldosterone deficiency, it does occur in the latter. Hyperpigmentation (due to increase ACTH), hyperkalemia and volume depletion (due to aldosterone deficiency) occur only in primary adrenal failure. Hyponatremia is seen in both. In patients in whom adrenal insufficiency is merely to be ruled out, plasma cortisol is measured between 8 and 9 AM. Concentrations of <3 Ag/dl are indicative of adrenal insufficiency and obviate the need for other tests. Concentrations greater than 19 Ag/dl rule out the disorder. The short cosyntropin stimulation test is the most commonly used test for the diagnosis of primary adrenal insufficiency. Secondary adrenal insufficiency is characterized by low blood cortisol and ACTH levels, a low baseline rate of steroid excretion, and abnormal ACTH and metyrapone responses. Megace 1 is a progestational agent with activity in advanced breast and endometrial cancer and AIDS-related cachexia. Long-standing Megace 1 therapy can result in marked suppression of serum cortisol levels and decreased ACTH .The depressing effect of megestrol acetate on the pituitary-adrenal axis does not cause clinically evident problems in the overwhelming majority of cancer patients. However physicians should be aware of the possibility of adrenal insufficiency while taking or upon withdrawal of this drug. This is especially important in severely ill patients, in whom the subtle signs and symptoms of adrenal insufficiency can be attributed to their primary disease. LEARNING OBJECTIVES: 1) To recognize some causes of gait disorder in the elderly 2) To recognize the importance of a detailed history and 3) To recognize the role of genetic testing in appropriate elderly patients with gait disorders. CASE INFORMATION: 64-year-old female patient with past medical history of diabetes, mild osteoarthritis, and hypercholesterolemia developed gait problems over the last year and a half. She started losing balance around that time and her gait had become slow. She denied headaches, seizures, swallowing problems, memory disturbances or any sensory involvement. Her family history is significant for one sister and all male members in her father's family who developed gait disorders in their sixties. On examination, slurring of speech was present. Extraocular movements were intact. Strength in all extremities was normal and there was no pronator drift. Sensory examination was intact. Reflexes were symmetric and brisk. Gait was wide-based and she could not walk on her heels or toes. Her handwriting was small and she had difficulty with rapid alternating movements. An MRI showed mild cerebellar atrophy. Tests for Lyme disease, rheumatoid factor and anti-nuclear antibody were negative. Vitamin B12 level and sedimentation rate were normal. Genetic testing revealed that she possessed the CAG/ CAA repeat expansion mutation associated with spinocerebellar ataxia 17. Genetic counseling has been offered to her family members. IMPLICATIONS/DISCUSSION: There are many potential causes for gait disorders in the elderly. The most common are related to the presence of a sensory-motor polyneuropathy such as seen with diabetes, or strokes related to cerebrovascular disease. Also common are disorders secondary to compressive myelopathy. In this patient the presence of slurred speech made myelopathy and neuropathy unlikely and the absence of structural lesions on MRI suggested a progressive degenerative disorder. Spino-cerebellar ataxia(SCA) type 17 is a rare autosomal dominant neurodegenerative disease caused by an abnormal CAG expansion in the TATAbinding protein (TBP) gene. SCA 17 was identified in 4 Japanese pedigrees and first reported in 2001. Most individuals present between the ages of 20 and 30 with gait ataxia and dementia, progressing over several decades to include bradykinesia, dysmetria, dysdiadokokinesis, hyperreflexia, and paucity of movement. Neuroimaging shows cortical and cerebellar atrophy. Even the small number of SCA 17 patients identified so far suggests that there would be substantial phenotypic variability, probably secondary to incomplete penetrance. This case emphasizes the details of the differential diagnosis of gait disorders in the elderly and the importance of the family history even in the elderly patient. This has become especially important with the greater availability of genetic testing that will provide the basis for genetic counseling. 2) The importance of a complete history in pinpointing a rare diagnosis; CASE INFORMATION: A 37 yo white female with no significant PMH presented to the ER with a chief complaint of constant, progressive weakness over the past ten days. The patient initially noted swelling, paresthesias, and muscle tenderness in her legs, followed by progressive weakness from the ankles to the hips. She described difficulty in standing from a seated position, climbing stairs, and ambulating. She denied any alleviating or exacerbating factors or similar past symptoms. She had taken only acetaminophen as needed and had no known drug allergies. She denied any ingestions, exposures, or recent travel. On ROS, she denied fever, changes in appetite, weight, urine output or color, any GI upset, recent illness, or rashes. She was a smoker, but denied any alcohol or drug use. Exam revealed a thin, well-developed female laying in bed in no apparent distress. Her temperature was 98.28F, pulse was 100, BP was 139/ 82, RR was 20, weight was 115 lbs., and height was 5 H 7 HH . HEENT exam revealed poor dentition, otherwise normal. Cardiac, lung, neck, and abdominal exams were normal. Musculoskeletal exam showed strength to be 5/5 in the upper extremities, 3/5 in the proximal lower extremities, 4/5 in distal lower extremities. A positive Trousseau's sign (carpopedal spasm) was noted upon inflation of BP cuff. DTRs were slightly brisk in all 4 extremities but no spasticity was noted. Otherwise, neurologic exam was normal. Skin exam showed no rashes. Laboratory evaluation revealed significant hypokalemia of 1.7 mmol/L (normal 3.5±5.0), plasma chloride of 94, plasma CO2 of 31, serum calcium of 7.4, serum magnesium of 1.3, and serum phosphorus of 3.3. Other electrolytes, BUN/Cr, and glucose were normal. Serum CPK was significantly elevated at 5,098 U/L (normal 24±170). Urine sodium was 177 mmol/L, urine potassium was 8 mmol/L, and 24 hour urine potassium was 14 mmol/L. MCV was 104 fL, folate level 3.3 ng/mL, and albumin 3.7 g/dL. Toxicology and diuretic screens were negative. The profound hypokalemia, hypokaliuria, macrocytic anemia, and hypoalbuminemia were consistent with the suspicion of a 18 dietary disorder; i.e, low intake vs. eating disorder. The patient consistently denied an eating disorder or distorted body image. Upon obtaining further dietary history, the patient's daily consumption of 5±6 L of Pepsi 1 stood out as an apparent etiology. The patient stated that she had been drinking caffeinated colas heavily for several years. Upon cessation of cola consumption and replacement with a total of over 820 meq of KCL, the patient's weakness improved. IMPLICATIONS/DISCUSSION: Colas and other caffeinated beverages have been reported to be causes of significant hypokalemia, likely due to caffeine toxicity and/or phosphatepotassium binding. Although a rare diagnosis, this case of cola induced myopathy was made by using fairly simple, bedside physician-patient discussion.
PAINFUL RASH. K. Kamjoo 1 ; 1 University of California, Los Angeles, Sylmar, CA (Tracking ID #76685) LEARNING OBJECTIVES: 1-To recognize the clinical presentation of sarcoidosis and Lo È fgren's syndrome. 2-To understand that sarcoid is a systemic disease with multi-organ involvement and 3-To discuss treatment modalities for sarcoid and sarcoid arthritis. CASE INFORMATION: A 24 year old male without significant past medical history presented to Emergency Department (ED) with complaint of painful rash over both legs for 2±3 weeks. Patient is an Argentinean exchange student who traveled to the US approx. six months prior to this presentation. Patient was seen at an outside clinic for the same complaint and was treated with Keflex and Ibuprofen without improvement. Patient reported that few days after appearance of rash; both of his ankles became swollen and painful. Although patient reported that walking was very painful, he denied any weakness or limitation of movement. In addition to the rash, patient admitted that he has been having night sweats and subjective fevers for the past couple of weeks. Patient also reported a slight dry cough. Initial vitals in ED were: temp of 38.2, blood pressure of 132/70, pulse of 95, and respiratory rate of 16. On physical exam patient appeared alert but in mild distress due to leg pain. Heart exam was regular sinus without any murmurs and lungs were clear to auscultation bilaterally. Abdomen was benign. Examination of skin revealed large nodules that were raised, red and tender to touch. Nodules were warm to touch and distributed randomly all over both legs. Both ankles had non-pitting edema but they had full range of motion. Ankles were tender to touch and passive range of motion was painful. Initial lab results showed a serum calcium level of 9.4 and a sedimentation rate of 47. Chest x-ray examination revealed bilateral hilar lymphadenopathy and EKG showed a normal sinus rhythm without any conduction abnormalities. Patient was subsequently admitted to hospital and placed in respiratory isolation. Patient had negative coccidiomycosis titers and ruled out for pulmonary Tuberculosis. HIV test was also negative. Patient remained clinically stable but continued to have a low-grade fever. CT scan of chest showed bilateral hilar and sub-carinal lymphadenopathy as well as multiple 1 cm nodules within lung fields. Subsequently, patient had bronchoscopy with biopsy and pathology report confirmed presence of non-caseating granulomatous lesions. Patient was started on 40 mg of prednisone per day and made a remarkable recovery. IMPLICATIONS/DISCUSSION: Sarcoidosis is a multi-system disorder of unknown cause characterized by a non-caseating granulomatous reaction in affected organs. Lo È fgren's syndrome is a variant of sarcoidosis in which acute sarcoidosis often presents with constitutional symptoms, polyarthritis, and erythema nodosum. The diagnosis of sarcoidosis should be based on a tissue biopsy, but a patient with typical Lo È fgren's syndrome may not need biopsy proof. Non-steroidal anti-inflammatory agents usually effectively alleviate sarcoid arthritis and joint symptoms associated with erythema nodosum. In severe acute arthritis and in chronic arthritis, corticosteroids may be required to control the symptoms. with a productive cough of white sputum and intermittent fevers, chills and night sweats for 1 month. She also complained of dizziness, weakness, a decreased appetite and a 5 pound weight loss over 4 weeks. She had been treated with antibiotics for 14 days which provided no relief. She denied visual changes, head pain, neck stiffness, chest pain, shortness of breath, dyspnea on exertion, nausea, vomiting, dysphagia, abdominal pain, diarrhea, urgency, dysuria, arthralgias or myalgias. Her medications included hydrochlorothiazide (HCTZ) and lisinopril. On physical exam, she appeared cachectic with dry mucous membranes and decreased skin turgor. Temperature was 38.4. Lung exam was clear bilaterally. Cardiac exam revealed a II/VI systolic murmur. There was no lymphadenopathy or skin rash. Abdominal exam revealed no masses or tenderness. She was hydrated and her HCTZ was held. She had multiple episodes of fever of up to 39.1. At the end of 7 days, her diagnostic testing revealed the following: normal CBC with differential; normal LFT's; 6 blood and 2 urine cultures were negative; Legionella antigen was negative; non-reactive PPD and a TSH of 2.6. Chest x-ray revealed biapical pleural thickening. ESR was 85. ANA, Rheumatoid factor (RF) and Anti-ds DNA were negative. Transthoracic echocardiogram (TTE) was negative for vegetation. Chest and abdominal CT scans revealed no significant findings. Bone marrow biopsy, aspirate and culture were negative. Bilateral temporal artery biopsies revealed severe temporal arteritis (TA). She was discharged on prednisone and her symptoms resolved after 1 week of therapy. IMPLICATIONS/DISCUSSION: FUO is defined as a temperature greater than 38.3 on several occasions for more than 3 weeks or after 1 week of thorough diagnostic testing performed in the out-or inpatient setting. Earlier studies reported that infections represented 41% of all cases. Collagen vascular diseases represented 30% and tumors represented 13%. Miscellaneous causes represented 2% and in 13% of cases the etiology was not identified. More recent studies reveal that infections are less common than multisystem disease and this trend was even more pronounced in patients over 70 years of age. A cost-effect approach in the elderly consists of confirmation of fever and a thorough history and physical exam. Laboratory analysis should consist of a CBC with differential, ESR, TSH, chemistry and liver panel, urine analysis and multiple blood and urine cultures. HIV, EBV, CMV, ANA and RF should be considered in certain cases. Stop all nonessential drugs. Imagining should include a TTE, chest and abdominal CT scans. A temporal artery biopsy should be considered if the ESR is more than 40 mm/hr, even in the absence of symptoms. Gallium scan for hidden abscesses and liver and bone marrow biopsies should be considered only in certain cases. Explorative laparotomy is a final resort. LEARNING OBJECTIVES: Introduction: Typhoid fever is a common disease in the developing countries.
Step-wise rising fever, bacteremia, diarrhea associated with abdominal pain and hepatosplenomegaly are the usual manifestations. We report a case of typhoid fever with sore throat as an integral part of the clinical picture. CASE INFORMATION: A 21-year-old immigrant male from Ecuador presented with a seven-day history of sore throat associated with a foreign body sensation and dysphagia for solid foods. Three days prior to presentation, in addition to his sorethroat, the patient complained of vomiting, diarrhea, mild abdominal discomfort, myalgias, fatigue and subjective fevers. On examination, his temperature was 94.68F with a pulse rate of 66 beats/minute. The oropharynx was markedly erythematous. The monospot test and rapid streptococcal screen were non-reactive. A diagnosis of acute viral syndrome with pharyngitis was made and he was treated symptomatically with Ibuprofen. He returned to the emergency department two days later with worsening sore throat, fever, new onset of low back pain, and mild abdominal discomfort with nausea. Physical examination revealed a temperature of 103.28F, pulse rate of 60 beats/minute, mild peri-umbilical tenderness and splenomegaly. The patient was hospitalized and started on intravenous fluids and oral Ciprofloxacin 750 mg twice daily. His fever subsided and he showed significant symptomatic improvement on antibiotic therapy. On the fourth day of hospitalization, blood cultures yielded Salmonella Typhi. He was discharged home on day five with two weeks of ciprofloxacin. IMPLICATIONS/DISCUSSION: Conclusion: Once considered an integral part of the symptomatology of typhoid fever, sore throat is infrequently reported, perhaps due to differences in causative strains, less emphasis on documentation of all symptoms and early and effective treatment in the antibiotic era. Physicians should be aware that typhoid fever could present as pharyngitis and should be considered especially in immigrants, to aid in early diagnosis and treatment. 
A 27 year old African American female with a past history of the human papilloma virus, gonorrhea, and chlamydia presented with two weeks of diffuse joint pains. The pain started in her knees and progressed sequentially to include her elbows, shoulders, back, and hips. Her associated symptoms were nausea, fever, and a sallow complexion. She denied any history of intravenous drug use, but did endorse a history of multiple sexual partners in the past. Physical exam was remarkable for a temperature of 38.3 C, scleral icterus, and jaundiced skin; However, there was no cardiac murmur or hepatosplenomegaly. Musculoskeletal exam revealed no joint effusions, warmth, erythma, or tenderness. A pelvic exam was normal with negative gonorrhea and chlamydia testing. Labs demonstrated a normal CBC with differential, and an erythrocyte sedimentation rate was 11; An antinuclear antibody was less than 1:40; Rhuematoid factor, hetrophile antibody, cytomegalovirus (CMV) IgM, and rapid plasma reagin tests were all negative; AST and ALT were elevated at 605 and 1303, respectively; Bilirubin was 3.3, alkaline phosphatase 204, and albumin 3.3. At this point, work up was initiated for acute hepatitis. While hepatitis A and C serologies were negative, hepatitis B surface antigen and anti-hepatitis B core antigen were positive. HIV testing was negative. A diagnosis of acute hepatitis B was made. It was presumed that the aquisition of hepatitis B in this patient was from sexual contact. Over the next 10 days her AST and ALT peaked at 4500 and 5500 respectively, bilirubin at 10, albumin decreased to 2.0, and INR was 1.9. Treatment was supportive in nature including fluids and pain control. At time of discharge her AST and ALT were trending down toward normal levels and her symptoms were resolving. She was educated about transmission of hepatitis B with attention to safe sex practices. IMPLICATIONS/DISCUSSION: Migratory polyarthralgia has a challenging and broad differential diagnosis. Infectious causes are many and include gonorrhea, syphilis, CMV, Epstein-Barr virus, Lyme disease, and bacterial endocarditis. Viral hepatits is a possible but uncommon infectious cause. Inflammatory causes are a major category and include rhuematoid arthritis, systemic lupus erythematosis, scleroderma, and polymyositis. Postinfectious causes include rheumatic fever and Reiter's syndrome. Crystalline diseases namely gout and psuedogout can also cause polyarthralgias. Joint aspiration and analysis is the key to diagnosis, however our patient had no joint effusions. With the advent of vaccination, acute hepatitis B virus (HBV) infection is now much less common in the United States. However, it remains a major global problem, particularly in developing countries. Signs and symptoms include malaise, fatigue, myalgias, anorexia, nausea, vomiting, abdominal pain, fever, and jaundice. Migratory polyarthralgia, particularly with a benign joint examination, constitutes an atypical presentation of acute hepatitis B. The incubation period for HBV is 1 to 6 months. While 30% of patients who acquire HBV present with acute symptoms, the majority of patients remain asymptomatic with anicteric hepatitis. Ninety percent of cases of acute hepatitis B clear infection and resolve symptoms within 6 months. They remain asymptomatic carriers of HBV. This is demonstrated by disappearance of the heptitis B surface antigen and appearance of the hepatitis B surface antibody. Less than 1% of patients develop acute fulminant hepatitis. The potential outcome of this subgroup is poor and therefore close follow up of synthetic function is warranted during the initial period. Approximately 5 to 10% progress to eventually develop symptomatic chronic HBV infection. Sequelae can include cirrhosis, hepatic decompensation, and hepatocellular carcinoma. Diagnosis of acute HBV is confirmed with serology, and quantification can be done with the polymerase chain reaction. Treatment is mainly supportive in nature, and patients should be educated about disease transmission. Antiviral therapy is not currently recommended or approved for the treatment of acute HBV.
A. Kapoor 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76642) LEARNING OBJECTIVES: 1.) Recognize an unusual cause of persistent hypokalemia; 2.) Diagnose an atypical presentation of Cushing syndrome; 3.) Recognize the clinical presentation of an ectopic ACTH-producing tumor. CASE INFORMATION: A 77-year-old male with hypertension and Type II diabetes mellitus presented with a 2 month history of fatigue and weakness. He subsequently developed a gait dysfunction and had frequent falls. Outpatient potassium (K) was 1.9 and he was admitted for management of hypokalemia. Physical exam revealed a BP of 180/90, facial plethora and bilateral diffuse rales at the bases of the lungs. Proximal muscle strength was 4/5 in the upper and 3/5 in the lower extremities. Distal strength was 5/5. No moon facies, buffalo hump or abdominal striae were found. His K remained low despite aggressive replacement and secondary causes were investigated. Results of diagnostic testing included a urinary Na 17, K 52, pH 6 (4.8±8), serum Na 140, Cl 95, HCO3 40, BUN 28, Cr 1.4, renin 0.6 (<5 mcg/ml) and aldosterone 5.7 (2±9 mcg/ml). Urinary metanephrines were normal. Cortisol at 8 am was 100 (7±20 mcg/ml) and ACTH 380 (9±52 pg/ml). Post high-dose (8mg) dexamethasone suppression test (DST) cortisol was 118 and ACTH was 126 (2±8 pg/ml). FSH, LH, prolactin and IGF-1 were normal. Brain MRI revealed a normal sized pituitary. Chest CT showed pleural effusions, no masses or lymphadenopathy. Abdominal CT revealed a 3 cm renal mass and bilateral adrenal hypertrophy. Biopsy of the renal mass showed poorly differentiated small cell carcinoma (SCC). Treatment with ketoconazole failed to suppress his cortisol. The patient decompensated and expired. Autopsy revealed numerous nodules in the liver, the right renal mass with focal hemorrhage and no lung masses. Pathology revealed SCC of renal origin with metastasis to the liver. IMPLICATIONS/DISCUSSION: Cushing syndrome is an unusual cause of hypokalemia. Other causes include primary or secondary hyperaldosteronism, Type 2 renal tubular acidosis, Bartter syndrome, Liddle syndrome, diuretic use and VIPoma. Patients with ectopic Cushing secondary to carcinomas often lack the clinical manifestations associated with classic Cushing syndrome±the centripetal fat distribution, moon facies and striae. The metabolic abnormalities, however, are more common in ectopic ACTH syndrome namely hypokalemia, metabolic alkalosis, excessively high cortisol and elevated ACTH levels. Differentiation of ectopic from pituitary dependent Cushing is made by non-suppression of cortisol with the 8 mg DST. There have been reported cases of ectopic ACTH with lung SCC, bronchial carcinoid, medullary carcinoma of the thyroid, gastrointestinal and prostatic adenocarcinoma and pheochromocytoma. However, there are no reported cases of SCC of renal origin causing ectopic ACTH production as was present in our patient.
A CATASTROPHIC COMPLICATION OF GIANT CELL ARTERITIS. P. Kapoor 1 , S. Parikh 1 , P. Radhakrishnan 1 , M. Yungbluth 1 , D. John 1 ; 1 St. Francis Hospital, Evanston, IL (Tracking ID #75985) LEARNING OBJECTIVES: 1. Awareness of the association of aortic aneurysm (AA) with giant cell arteritis (GCA) will aid prompt recognition and therapy. 2. To highlight the significance of using Chest XRay (CXR) as a screening modality in patients of GCA for timely detection of thoracic AA.
A 75-year-old male presented with acute, severe right upper quadrant (RUQ) pain, radiating to the right shoulder and associated with nausea and vomiting. He denied any fever, chest pain, shortness of breath, change in bowel habits or similar episodes in the past. He had a history of coronary artery disease, well-controlled hypertension and biopsy-proven GCA, diagnosed 15 months ago. Physical examination: afebrile;pulses: 86/min, regular and symmetric; BP: 170/108 mm;tenderness in the RUQ with palpable gall bladder 4 cm below the right costal margin; an ejection systolic murmur in the aortic area. The rest of the physical examination was essentially unremarkable. Laboratory data: WBC-10000, Hb-13.8 and platelets-251,000. The CT scan of abdomen was suggestive of acute cholecystitis. A dissection of the superior mesenteric artery (SMA) was also noted. CXR showed aneurysm of ascending aorta. CT scan of thorax showed a Stanford Type A aortic dissection (AD) with aneursymal dilation of 7 cm. Communication of the true and false lumen suggested chronicity (see figure) . After stabilization of BP, he underwent an emergency laparoscopic cholecystectomy which led to a resolution of his abdominal pain. Subsequently, he was referred for an elective repair of his asymptomatic, chronic aortic dissection. IMPLICATIONS/DISCUSSION: Horton's syndrome or GCA is the commonest primary systemic vasculitis in the United States, associated with diverse manifestations and outcomes. The true incidence of aortic involvement is unknown, but is estimated to be 3±15 %. Patients with giant cell aortitis may be asymptomatic or may present with aortic arch syndrome, dilation of aorta, aortic aneurysm, AD or aortic incompetence. Although, in this patient AD may well be attributed to atherosclerosis,the history of GCA and segmental involvement of ascending aorta and SMA implicate GCA as a predisposing condition.A high incidence of thoracic aortic aneurysm has led Evans et al to recommend annual CXR, including lateral views in patients of GCA. Since the diagnosis of GCA is commonly made by the internists, awareness of it's possible complications helps us manage these patients better. A 37 year old Mandarin speaking woman with no known PMH presented with chest palpitations for 6 months. Her history was obtained in a crowded, noisy ER through a telephone translator, with her husband as a middle translator. She states having symptoms for over 6 months with acute decompensation over the last 2 months involving constant fast heart rate, swelling in her face, abdomen, and extremities for 2 months, dyspnea when walking 2±3 blocks, a 20 lb unintentional weight loss, poor concentration, and constipation. She went to a doctor 1 month ago and was told she has a thyroid problem, but has not been taking the prescribed medications. More than 10 years ago in China, she had problems with her thyroid and was given Western medications for 6 months with improvements. Otherwise, review of systems was unremarkable. On exam, her pulse was irregular, atrial fibrillation, at 125±145, BP 98/64. She was anicteric with swollen eyelids, no proptosis. She had an enlarged thyroid, JVD, a hyperdynamic precordium, no heart murmurs, a distended abdomen, and non-pitting edema in all extremities. Lab studies showed a mild transaminitis, negative hepatitis serologies, and positive thyroid stimulating antibodies. She was diagnosed with Graves disease. Echocardiography showed severe tricuspid regurgitation and right heart failure without evidence of left sided dysfunction, pulmonary artery hypertension or septal defects. Her atrial fibrillation was treated with beta blockers and anticoagulation, and she was scheduled for repeat echocardiogram in 2 months to evaluate for valve disease that may be contributing to her right sided failure. She would like more children, so she opted for medical therapy with propythiouracil to treat her hyperthyroidism and would consider radioactive iodide or surgical therapy after her thyroid disease is under control. IMPLICATIONS/DISCUSSION: 1. Obtaining a complete history in non-English speaking patients can be difficult. Despite seeing a private doctor, this patient came to the ER without her primary doctor's support, without her medications, and without the ability to communicate with hospital doctors. Studies have shown that intepreter services increase cost-effectiveness and quality of care as well as patient understanding and participation in healthcare, despite potential increases in the time and resources to provide such services. 2. Recurrent hyperthyroidism, most commonly due to Grave's disease, is treated similarly to the initial episode. Anti-thyroid medications are the drug of choice in pregnant women and are not known to effect reproductivity. Radioactive iodide is contraindicated in pregnancy, although has been shown to have no effect on reproductivity in the long term. Surgery is effective, although has no advantages and has been associated with increased fetal losses if done during pregnancies. A 74 year old male was brought by his friend to the emergency room with a four week history of gradually progressive shortness of breath that began 2±3 days after dual-chamber pacemaker placement for Mobitz II heart block and symptomatic bradycardia. The procedure was without any immediate complications. CXR post-precodure was negative for pneumothorax. He currently denied chest pain, cough, fevers, chills, sweats, lightheadedness, or dizziness. PMH was significant for, in addition to Mobitz II heart block, coronary artery disease, severe aortic stenosis and hypertension. Medications included felodipine 10 mg PO qd, simvastatin 10 mg PO qd, and lisinopril 20 mg PO qd. He had a remote history of tobacco use and was a former steel mill worker. On physical examination, VS: HR 105, RR 26, BP 150/90, pulse ox 92% (on room air). Pertinent findings included no JVD, a II/VI SEM at the right upper sternal border radiating to carotid arteries, laterally displaced PMI, decreased breath sounds on the left 2/3 up the lung fields, no organomegaly and 1+ pretibial edema. CXR showed a free flowing large left pleural effusion. The patient was admitted to the hospital and a thoracentesis was performed from which serosanguinous fluid was obtained. CT of the chest demonstrated a perforated right ventricle from the pacer wire. Surgical repair was successful but the patient had a very complicated postoperative course and expired. IMPLICATIONS/DISCUSSION: Pacemaker insertion is a relatively common and safe procedure and can be lifesaving. Ventricular rupture is a rare, albeit potentially serious, complication. The elderly, in whom most pacemakers are inserted, are at increased risk of complications. The Pacemaker Selection in the Elderly (PASE) study, a prospective multicenter trial involving 407 patients age 65 years and older, documented a 6.1% complication rate; 4.4% of patients required a repeat procedure. The most common complications were lead dislodgement (2.2%), pneumothorax (2.0%and observed only in those with subclavian venous access) and cardiac perforations (0.98%). Most perforations occur intraoperatively and rarely cause symptoms. However, as in our patient, serious complications, including death, have been reported postoperatively. Complications of pacemaker insertion may masquerade as other less serious entities with symptoms of dyspnea, chest pain, fever and weakness. It is paramount that internists be alert to the potential serious complications of cardiac rupture and pneumothorax in the patient presenting with these symptoms postprocedure. Symptomatic patients require surgical intervention. A 53 year-old Puerto Rican male presents with a chief complaint of two years of increasing dyspnea on exertion. Past medical history was notable for multiple episodes of pneumonia. Although the patient had been successfully treated as an outpatient during these episodes with oral antibiotics, his chronic baseline dyspnea and decreasing exercise tolerance continued to worsen. For one of these exacerbations, the patient was admitted to the hospital, where physical exam revealed nystagmus, poor visual acuity, oculocutaneous albinism, and a bleeding diathesis. Radiological studies revealed bilateral lower lobe infiltrates and diffuse pulmonary honeycombing fibrosis. The patient improved following a ten-day course of IV cefotaxime, gentamycin, and chest physical therapy. IMPLICATIONS/DISCUSSION: The combination of oculocutaneous albinism, a bleeding diathesis, and pulmonary fibrosis in this Puerto Rican patient raised the suspicion for Hermansky-Pudlak Syndrome. HPS is an inherited disease which results in a platelet storage pool defect and lysosomal accumulation of a substance called ceroid lipfuscin. HPS is an autosomal recessive syndrome found primarily in individuals from the northwestern quadrant of Puerto Rico. Patients have legal blindness, nystagmus, iris transillumination, foveal hypoplasia, decreased number of melanocytes, pulmonary fibrosis, renal disease and granulomatous enteropathic disease. Lab studies in patients with HPS may show an increased bleeding time with a therapeutic response to desmopressin, decreased bone density, and genetic testing showing a mutation on the HPS1 gene on chromosome 10q23. The most accurate diagnosis, however, is made by a pathognomic platelet morphology diagnosed only by electron microscopy. Management consists of recognizing the disease, having a lower threshold for admitting individuals for inpatient management, and administration of immunosuppressants and steroids. Current trials of a test drug called pirfenidone are being conducted and offer patients some hope for a treatment. Back to our patient: A careful history taking revealed that the patient presented above had all of the above manifestations, even decades prior to his official diagnosis being made. Management of this patient's disease ultimately consisted of various trials of steroids, interferon, and methotrexate. While the inpatient stay focused primarily on his pulmonary pathology, outpatient management consisted of patient education to minimize complications of the disease and carefully orchestrated care with numerous specialists. week history of progressive pain and swelling in his left index finger after an attempt to straighten an iron pipe with this left hand. Past medical history was significant for a similar episode involving his right index finger after trauma that resolved with symptomatic treatment. Patient had a 50pack year history of smoking. The rest of his review of systems was unremarkable. Physical examination was unremarkable except for a swollen and tender left index finger with a gangrenous tip. The left ulnar pulse was absent. His radial pulses were intact in both extremities. Laboratory data including ANA and sedimentation rate were within normal limits. An arteriogram of the left upper extremity revealed thrombosis of the distal ulnar artery and occlusion of digital arteries of the second digit that were thought to be secondary to embolic events from the ulnar artery thrombus. Duplex ultrasound also revealed a thrombosed pseudoaneurysm in the left ulnar artery at the hypothenar eminence. Diagnosis of Hypothenar Hammer Syndrome (HHS) was made. IMPLICATIONS/DISCUSSION: HHS is a rare clinical entity that results from repetitive palmar trauma leading to damage of the ulnar artery as it passes over the hammate bone. The syndrome usually manifests as unilateral digital ischemia caused by embolic digital artery occlusion from the thrombosed palmar ulnar artery. It is predominantly seen in males around the age of 40. In a small study of 21 patients, 12 out of 13 who had arteriography of both upper extremities had bilateral palmar ulnar disease (1). Diagnosis is made on clinical features and confirmed by angiography that shows a palmar ulnar artery abnormality and digital artery occlusion. Treatment may include surgery involving excision of abnormal palmar ulnar artery segment. LEARNING OBJECTIVES: To recognize and manage pustular psoriasis and to differentiate it from other causes of erythroderma. CASE INFORMATION: DP is a 58-year-old African American male who presented with an exfoliative erythroderma and pustules in the palmar-plantar areas that had coalesced to form`l akes of pus.'' The patient noted a similar but milder eruption 3 months earlier in which he was treated at another facility with oral prednisone with some improvement. He reported that the pustules had started on his hands, progressing rapidly over several days to affect the entire body. Erosions later appeared in the mouth. He complained of an intense pruritus and burning sensation along with a fetid odor. At the time of presentation he was noted to be febrile with a leukocytosis. A skin biopsy showed spongiform pustules in the upper epidermis with numerous PMNs present. Inpatient management included a combination of actretin, IV vancomycin, and topical mid-potency steroids, with rapid improvement in the patient's condition. IMPLICATIONS/DISCUSSION: A presentation of erythroderma should prompt the physician to consider pustular psoriasis, pemphigus foliaceus, pityriasis rubra pilaris, underlying leukemia/lymphoma, severe atopic dermatitis, ichthyoses, drug induced erythroderma, diffuse seborrheic dermatitis, scabies, and staphylococcal scalded skin. Pustular psoriasis can be differentiated by sudden onset of acral pustular lesions, fever, intense pruritis, and occasionally, hypocalcemia and mucous membrane lesions. Patients will often have a personal or family history of plaque psoriasis and/or psoriatic arthritis. Treatment of choice is acitretin (a second generation retinoid). Topical steroids are often helpful, but systemic steroids should be avoided.
WITH KLIPPEL-TRENAUNAY-WEBER SYNDROME. M. Kennedy 1 , C.V. Mueller 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #77195) LEARNING OBJECTIVES: 1) Learn about the vascular malformations seen in Klippel-Trenaunay-Weber Syndrome. CASE INFORMATION: A 25 yo Caucasian male originally presented to an outside hospital with bright red blood per rectum. He denied associated abdominal pain, nausea, vomiting, fever or prior history of rectal bleeding. Initial systolic blood pressures were in the 80-90's with a heart rate in the 130's. Work up at the outside hospital included flexible sigmoidoscopy that revealed nonbleeding internal hemorrhoids, a negative tagged RBC scan and an unremarkable CT of the abdomen. Upon transfer to our facility, he was hemodynamically stable, but found to have a hemoglobin of 7.5 with a baseline of 13.5. The morning after admission, the patient developed massive hematochezia associated with diaphoresis and tachycardia. He was urgently sent for tagged RBC scan that showed distal ileal bleeding. Subsequent mesenteric angiography did not identify any active bleeding or vascular malformation. The patient again decompensated and was taken for exploratory laparotomy that revealed bleeding ileal and colonic varices. He underwent ileocecectomy without complications and had no further episodes of bleeding. He required a total of 20 units of PRBC's during his hospitalization. IMPLICATIONS/DISCUSSION: KTWS is classically described as a venous monoangiodysplasia, but is actually associated with a variety of vascular malformations including: subcutaneous hemangiomas, cavernous hemangiomas, AV fistulas, deep vein malformations and/or aplasias, varices and a variety of lymphatic abnormalities. Vascular malformations may involve visceral organs such as the bowel, liver, kidneys, and lungs and can, uncommonly, cause complications in those organs. Treatment of KTWS is supportive and/or cosmetic unless orthopedic or bleeding complications arise, at which point surgery is generally indicated.
LEARNING OBJECTIVES: 1) Consider the potential for a fatal overdose of Metformin when treating diabetic patients who are at risk of commiting suicide. 2) Recognize the constellation of signs that are consistent with a Metformin overdose.
3) Recognize the need to quickly and aggressively treat a Metformin overdose and its medical consequences. CASE INFORMATION: A 42 year-old woman with a history of diabetes, hypertension and schizoaffective disorder was found obtunded in her apartment and admitted to the MICU. She had recently been hospitalized for suicidal ideation. Empty pill bottles of a benzodiazepene, an atypical antipsychotic, a SSRI and Metformin were found. Six hours after admission, the patient developed lactic acidosisand a severe sepsis-like syndrome consistent with Metformin overdose. She subsequently became critically-ill, and had a clinical course which included severe ARDS and anuric renal failure. Support was withdrawn at the request of her family following a prolonged course of aggressive treatment. IMPLICATIONS/DISCUSSION: Metformin is commonly used in primary care and its potential for lethal side effects is well known in the setting of medical illness. This case demonstrates how Metformin also has potential for great harm in the setting of an overdose. Physicians should be aware of the risks of prescribing Metformin to diabetic patients with depression or a history of impulsive behavior. A 49 year old female with recent diagnosis of pulmonary embolism due to oral contraceptive use presented with acute onset of left sided abdominal pain associated with nausea but no vomiting, diarrhea or constipation or changes in urinary habits. Patient's medications were warfarin, ibuprofen prn, home O2 and amitryptiline. Had 25 pack year smoking history. On examination, she seemed to be in moderate distress with severe tenderness on left side of midline from epigastrium to suprapubic region, worse with any movement associated with mild fever. Vital signs were stable otherwise. Stools hemoccult negative. Lab studies revealed a wbc count of 12,700, Hb of 10.7, noted to be reduced from previous admission. Creatinine 0.7 mg/dl, lipase 94 mg/dl, PT 23.1, INR 3.73 with normal liver function tests and normal UA. Contrast enhanced CT revealed acute hematoma of left rectus sheath, 20cm  6cm with active bleeding. Patient's Hb further dropped so eventually transfused, coumadin stopped and surgery consulted. They suggested conservative treatment with analgesia and rest and later on, IVC filter placement. Repeat CT Scan showed stable hematoma. Patient clinically better with stable Hb. So, again started on low dose coumadin and sent home with caution not to smoke and avoid trauma as on further questioning, eventually, patient provided history of fall. IMPLICATIONS/DISCUSSION: Left sided abdominal pain and fever can be mistaken for number of causes of acute diseases of abdomen. But, due to advent of CT, an unusual diagnosis is relatively easy to make. But, since rectus sheath hematoma is one of the reported complications of anticoagulation therapy, this diagnosis should always be kept in mind. Once diagnosis is established and if this hematoma is not causing severe symptoms, the condition can be managed nonoperatively with bed rest and analgesics. Ideally, anticoagulation should be discontinued. It is rarely necessary to reverse the coagulation defect if operation is not undertaken. As in our case, patient was clinically improved so it was decided to manage her conservatively and avoid surgery. She was restarted on anticoagulation because risk of pulmonary embolism is greater than hematoma. It was made sure to counsel her about any trauma as it increases risk of hematomas in future. Patient continued to do well on further follow ups.
M. Khalil 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73745) LEARNING OBJECTIVES: 1. To recognize ascites as a complication of eosinophilic gastroenteritis (EG) 2. To diagnose and treat EG. CASE INFORMATION: A 50 year-old female with no significant PMH presented with a 2 month history of epigastric pain that was associated with nausea and decreased appetite. She reported the pain to be constant and exacerbated with food, but mildly relieved with a bowel movement. She denied any vomiting, melena, hematochezia, fever, chills, recent travel, change in diet or alcohol abuse. Two weeks prior to admission, she developed non-bloody, watery diarrhea, increased abdominal girth, and 12 lb. weight gain, despite the poor appetite. Outpatient evaluation with abdominal ultrasound was unremarkable and an upper GI series revealed dysmotility of the lower esophagus with tertiary contractions. Treatment with rabeprazole provided minimal relief. Physical examination upon admission was remarkable for a mildly distended and diffusely tender abdomen with positive shifting dullness but no rebound, guarding or organomegaly. The remainder of physical exam was normal including heme negative stool. Lab work was significant for WBC of 14,000 with 30% eosinophils. Liver function tests were normal. Serum albumin was 2.4 and ascitic fluid analysis showed: pH 7.31, albumin 1.8, negative cytology, and WBC of 4700 with 96% eosinophils. Stool culture and sensitivity, ova and parasites were negative. Further workup with abdominal ultrasound with Doppler showed patent vessel and normal direction of flow. Upper endoscopy revealed erythema of the antrum. Colonoscopy was normal. CT scan of the abdomen and pelvis showed diffusè`L akes of pus'' small bowel thickening without fat stranding and moderate ascites. Trans-vaginal ultrasound was negative for ovarian mass. Bone marrow biopsy was normal. The patient refused surgical bowel biopsy and diagnosis of EG was established on the basis of exclusion. She was started on oral dose of prednisone for 6 weeks and discharged to home. Follow up visit and blood work showed 0% eosinophils, resolution of ascites, resolution ofabdominal pain, and good appetite. IMPLICATIONS/DISCUSSION: EG is an uncommon disorder characterized by gastrointestinal symptoms, blood eosinophilia, and eosinophilic infiltration of the gastrointestinal wall. The signs and symptoms of EG are related to the layer and extent of the bowel involved with eosinophilic infiltration. Patients with subserosal infiltration may develop eosinophilc ascites. The diagnosis of EG is confirmed by a full thickness biopsy of the bowel in the absence of intestinal parasitic infection. Treatment of EG is empiric because no prospective, randomized clinical trials are available to guide treatment. Prednisone (20 to 40 mg/day) is recommended for two weeks and should be tapered over the next two weeks. Improvement usually occurs within two weeks of therapy. Despite the rarity of EG, it should be suspected in a patient with any GI symptoms associated with peripheral eosinophilia because this treatable disease can disguise itself as the irritable bowel syndrome. LEARNING OBJECTIVES: 1) Recognize that pulmonary hypertension is not uncommon patients with SLE; 2) Recognize that primary pulmonary hypertension in SLE patients often presents earlier than pulmonary hypertension from a secondary cause. CASE INFORMATION: A 21-year-old woman was transferred to our hospital for management of her pulmonary hypertension (PHT) and systemic lupus erythematosus (SLE). She initially presented a year ago with progressive cough and pleuritic-type chest pain. 8 months prior to admission, she underwent an echocardiogram which revealed a pulmonary artery pressure (PAP) of 27 mm Hg. Four months later, she was admitted to a community hospital for congestive heart failure and was found to have positive anti-Smith, antiphospholipidand anti-nuclear antibodies, and was diagnosed with SLE. Her symptoms gradually progressed to dyspnea on exertion, paroxysmal nocturnal dyspnea and Raynaud's phenomenon. She was subsequently readmitted to that hospital 2 weeks prior to transfer to us for worsening of her symptoms. There, she had a generalized seizure and was initiated on plasmapheresis treatment. Examination revealed a thin, anxious lady in mild dyspnea and an obvious malar rash. Cardiac auscultation revealed a loud and palpable P2, and a II/VI systolic murmur at the tricuspid area. She had a non-tender hepatomegaly and mild bilateral pedal edema. Echocardiogram revealed a PAP of 76 mm Hg, with evidence of right heart enlargement and mild tricuspid regurgitation. A chest CT scan with angiography and a ventilation-perfusion scan showed no signs of a pulmonary embolism. A right heart catheterization was done as well showing moderate PHT and a moderate response to epoprostenol challenge. Workup for lupus cerebritis was negative. She was initiated on prednisone and Cytoxan for SLE. Plasmapheresis was discontinued. Diltiazem, Valsartan and Warfarin were initiated to control her pulmonary hypertension. She tolerated the medications well and was discharged with outpatient follow-up, with the consideration of using epoprostenol in the future should her condition worsen. IMPLICATIONS/DISCUSSION: Prevalence of PHT in SLE ranges from 5% to 14%. A third of SLE patients have positive antiphospholipid antibodies, although the significance of this in the development of PHT is unknown. In one study of SLE patients, 5.9% were found to have pulmonary hypertension, of which 48% of them had no identifiable cause, the rest found to have a secondary cause. For the subgroup with secondary hypertension, 50% of them were due to valvular abnormalities. The primary pulmonary hypertension tended to present far earlier after diagnosis of the SLE compared to secondary PHT, 8.8 versus 43.2 months. Raynaud's phenomenon in secondary hypertension is associated with a more severe pulmonary artery pressure. These patients seem to respond to epoprostenol therapy although there have been documented cases of fatal thrombocytopenia. year-old female with rheumatoid arthritis, hypertension, and seizure disorder presented with two days of hematuria. She denied fevers, dysuria, urinary frequency, headaches, visual changes, or cough. Her last seizure was two weeks prior to presentation. Medications included gabapentin, phenytoin, methotrexate, and amlodipine. There was no fever and her physical examination was otherwise negative for lymphadenopathy, hepatosplenomegaly or petechiae/purpura. Initial workup revealed a platelet count of 7,000, H/H of 7.3/24, WBC of 8.2, LDH of 900, and Cr of 3.5. Peripheral smear demonstrated schistocytosis. The patient was diagnosed with TTP-HUS and began daily plasmapheresis and prednisone, during which her platelet count improved to 130,000 and her LDH normalized. She was discharged to continue outpatient pheresis but was readmitted one week later with fever (1038 F) and a platelet count of 78,000. She was begun on plasmapheresis with cryoprecipitate-poor plasma, vincristine, and steroids. She responded well; her pheresis was decreased to every other day and prednisone was tapered. She was discharged with a platelet count of 398,000 to continue outpatient pheresis and vincristine, which were discontinued after one month. One month later, the patient had a platelet count of 70,000, LDH of 1,186, and schistocytes on peripheral smear. Plasmapheresis and prednisone were restarted, and a splenectomy was performed. Discharge platelet count was 199,000. Two weeks later, her platelet count was 57,000. Pheresis and vincristine were restarted with good response. IMPLICATIONS/DISCUSSION: TTP and HUS are diseases with abnormalities in multiple systems. Classically, TTP is described as having more severe neurologic disease, whereas HUS has more of a renal component. Clinically, however, patients are described as having TTP-HUS because most present with the same symptoms, have identical pathologic changes, and receive the same treatment. The classic pentad of thrombocytopenia, microangiopathic hemolytic anemia, neurologic abnormalities, renal abnormalities and fever is present in few patients. The diagnosis is made primarily by the presence of thrombocytopenia and microangiopathic hemolytic anemia. The etiology is a deficiency of von Willebrand factor (vWF) cleaving protease, which results in the accumulation of unusually large vWF multimers, causing platelet aggregation. Causes of TTP-HUS include E. coli 0157:H7 and medications such as ticlopidine, cyclosporine, and quinine. Most adult cases, however, are idiopathic. Treatment is plasmapheresis, the mechanism of which is not completely understood. 10 to 20% of patients have an incomplete response; steroids, vincristine or IVIG can be added in these cases, but are not alternatives to pheresis. Some studies show improvement with splenectomy, especially in conjunction with pheresis and steroids. 30 to 50% of patients have exacerbations of thrombocytopenia and hemolysis when pheresis is tapered. Relapse is defined as recurrence after 30 symptom-free days without treatment. Both are treated with reinstitution of daily pheresis. It is essential that physicians involved in the care of the patient with TTP-HUS recognize that many patients may have worsening disease with tapering of pheresis and to be aware of alternate life-saving treatments if this occurs. year-old female presented with 4 weeks of malaise, a dry cough, and recent high fevers. She also had night sweats for the 6 months since she started hormone replacement therapy. She had no pets and no recent travel history. Though exposed to tuberculosis as a child, she had never been tested or treated for it. She had begun minocycline just over a month earlier for rosacea and was on loratidine for seasonal allergies. She did not drink alcohol and quit smoking 5 years prior. Physical exam was notable for high fever, tachypnea, pulse oximetry of 96% on room air, inspiratory crackles at the right base and dullness at the left base. Labwork: WBC 15.2 with 84% neutrophils; blood eosinophilia increased over several days to 15%. ESR was 70. Chest radiograph showed small bilateral effusions, no cardiomegaly, and bilateral, peripheral, apical alveolar infiltrates. PPD was negative. Her fevers persisted and the left pleural effusion increased on intial therapy for community-acquired pneumonia. The effusion was transudative, with 1755 WBCs, 12% neutrophils and 10% eosinophils. A bronchoalveolar lavage (BAL) and transbronchial biopsies were performed; BAL fluid contained 63% eosinophils and biopsies revealed focal aggregates of eosinophils around the airways, consistent with eosinophilic pneumonia. Oral prednisone was started and both symptoms and eosinophilia resolved 48 hours after her first steroid dose. IMPLICATIONS/DISCUSSION: EP is characterized by eosinophilic pulmonary infiltrates. It presents as an acute febrile illness with dyspnea, night sweats, weight loss, dry cough, crackles or wheezing on lung exam, increasing blood eosinophilia and bilateral, peripheral patchy infiltrates on chest radiograph. The ESR is often elevated. Etiologies include common medications, including minocycline, parasitic infections, HIV, and fungal infections. A high index of suspicion, confirmed by BAL fluid eosinophilia and diffuse eosinophilic infiltration of the lung on biopsy, leads to the diagnosis. The response to steroids is dramatic, as was seen in our patient, with symptoms, eosinophilia, and lung infiltrates decreasing within 48 hours. Treatment is continued for 3 months to prevent relapses, which respond to re-intitiation of steroids.
A POTENTIAL BLOOD DONOR. J. Kimberly 1 , R. Watkins 2 ; 1 Wake Forest University Baptist Medical Center, Winston-Salem, NC; 2 Wake Forest University, Winston-Salem, NC (Tracking ID #76222) LEARNING OBJECTIVES: 1) Be able to counsel patients with evidence of infection with HTLV-I and 2) appreciate associated diseases. CASE INFORMATION: A 43 year old African American female with a history of hypertension, diabetes, and treated syphillis presents to clinic reporting that she fears she may have contracted HIV disease. She recently donated blood and subsequently received a letter stating she could no longer donate blood``because of an infection like AIDS.'' The patient presented the letter which notified her of the presence of HTLV-I/II antibodies with Western Blot confirming HTLV-I infection. ELISA for HIV 1 and 2 was performed and was negative. The patient was initially fearful and had many questions regarding HTLV-I infection: How did she get infected and was it sexually transmissible or contagious via other routes? Would she develop immunosuppression similar to that seen with HIV disease? Were there any other possible adverse effects on her health? IMPLICATIONS/DISCUSSION: HTLV-I is a retrovirus endemic in parts of Japan with other clusters of high prevalence including the southeastern US. The virus is transmitted by three known routes: 1) from mother to child, especially through breast feeding, 2) through sexual activity, and 3) through blood, either from contaminated needles or transfusion. In areas where HTLV-I is endemic, many inflammatory and autoimmune diseases have been attributed to the virus though causation has only been shown for two diseases. HTLV-I is clearly linked to adult T-cell leukemia/lymphoma (ATL), classically an aggressive, usually deadly, hematogenous malignancy characterized by rapid progression of skin lesions, pneumonitis, hypercalcemia, and lymphocytosis. The second disease with a clear association with HTLV-I infection is tropic spastic parapesis or HTLV-I-associated myelopathy (HAM) which, unlike ATL, is characterized by an insidious onset with symptoms often including stiffness or weakness in one or both legs, back pain, urinary incontinence, and mild sensory changes. Onethird of patients are bedridden by ten years. Both ATL and HAM occur in 2-5% of HTLVinfected patients. ATL is slightly more common in infected males and usually occurs after 20-30 years of infection whereas HAM is more prevalent among females and can occur just a few years after infection. More than 95% of patients with ATL and HAM have evidence of HTLV-I infection. The patient under discussion was educated about the nature of HTLV-1 and how it differed from HIV, and counselled to practice``safer sex'', avoid donating blood, and, should she become pregnant, avoid breast feeding. She was also informed of the risk, albeit a fairly low one, of ATL and HAM. 2) Identify the diagnostic criteria for eosinophilic gastroenteritis.
3) Understand the management and prognosis of eosinophilic gastroenteritis. CASE INFORMATION: A 38 year-old woman presented to the ER with vomiting and diarrhea for four weeks. She experienced 6-8 watery, non-bloody stools per day; vomiting after consuming any food or liquid; and a``pulling sensation'' across the lower abdomen. One week after the onset of symptoms the patient was admitted to the hospital, rehydrated, and discharged with a diagnosis of acute gastroenteritis. The symptoms worsened soon after discharge, and the patient was readmitted to the hospital. Evaluation included routine stool cultures and ova and parasites. The patient was treated empirically with levofloxacin and metronidazole and was discharged home. Stool cultures were later found to be negative. The patient's symptoms quickly worsened and she presented again to the ER. Past medical history was remarkable for asthma and lactose intolerance. On physical examination, BP = 115/62, P = 110, and T = 99.18F. Abdominal examination revealed a moderately distended abdomen with shifting dullness and mild tenderness to palpation in the upper abdomen. The remainder of the examination was unremarkable. The white blood cell count was 15,600 with 55.3% eosinophils; the hemoglobin was 8.9. CT of the chest, abdomen, and pelvis revealed a thickened terminal ileum and cecum, ascites, and bilateral small pleural effusions. Paracentesis was performed. The peritoneal fluid revealed 1,920 white blood cells with 3 neutrophils, 12 lymphocytes, and 85 eosinophils. Ascites fluid albumin was 3.3; the serum-ascites albumin gradient was 1.2. Upper and lower endoscopy revealed only melanosis coli. Biopsy of the duodenal mucosa revealed lymphocytic and eosinophilic infiltration. The patient was treated with prednisone. Her symptoms improved and she was discharged home. IMPLICATIONS/DISCUSSION: Eosinophilic Gastroenteritis (EG) is a rare disease whose etiology is unknown, though it may be a Type I hypersensitivity reaction. Patients with EG present a range of symptoms from nausea, vomiting, and diarrhea, to ascites and bowel obstruction. The symptom complex is determined by the degree of eosinophilic infiltration of the gastrointestinal tract. The diagnosis of eosinophilic gastroenteritis requires the presence of gastrointestinal symptoms, eosinophilic infiltration of one or more areas of the gastrointestinal tract on biopsy, absence of eosinophilic involvement of multiple organs outside the gastrointestinal tract, and absence of parasitic infection. Eosinophilic gastroenteritis without ascites is treated with cromolyn; the presence of ascites requires glucocorticoid treatment. More than 90% of patients respond quickly to treatment. The relapse rate is high with cessation of treatment (>33%). Those who relapse often require maintenance therapy with glucocorticoids.
A CURIOUS CASE OF CYANOSIS. C.T. Ko 1 , N. Spell 1 ; 1 Emory University, Atlanta, GA (Tracking ID #76425) LEARNING OBJECTIVES: 1. Recognize methemoglobinemia as a cause of cyanosis, even without G6PD deficiency. 2. Diagnose and treat methemoglobinemia by understanding its mechanism of action. 3. Recognize the drugs that can cause methemoglobinemia. CASE INFORMATION: A 26-year-old African-American female with HIV presented complaining of worsening shortness of breath, headaches, and generalized weakness. Her CD4 count was 11/mm3. She had been discharged from the hospital one week prior with treatment for presumptive Pneumocystis carinii pneumonia. Her discharge medications included clindamycin and primaquine. On physical exam, she was a thin, cachetic, pleasant female in mild respiratory distress. Vital signs included a temperature of 102.5F (39.2C), blood pressure 90/50 mm Hg, pulse 124 beats/minute, and respiratory rate 24 breaths/minute. Her pulse oximeter reading was 85% saturation on room air and 89% saturation with a 100% nonbreathing oxygen mask. She had conjunctival and mucosal pallor as well as cyanosis of her nail beds, diffuse shoddy lymphadenopathy, and tachycardia with no associated murmurs/rubs/ gallops. Lung fields were clear to auscultation with adequate airflow. Laboratory evaluation revealed white blood cells 3400/mm3 (47N, 46L, 6M), hemoglobin 7.0 g/dl, hematocrit 20.2%, platelets 130,000/mm3, and reticulocytes 1.4%. Her hematocrit one week prior was 35.5%. Chemistry profile showed total bilirubin 1.0 mg/dl, LDH 1374 U/L, and G6PD 10.6. All other chemistry values were within normal limits. Chest X-ray was unchanged from her previous films with minimal bilateral reticular prominence likely secondary to mild interstitial scarring. Arterial blood gas on room air showed pH 7.40, pCO2 28 mm Hg, pO2 73 mm Hg, oxyhemoglobin 73%, carboxyhemoglobin 0.2%, and methemoglobin 25%. Primaquine was discontinued, and methylene blue was administered. Methemoglobin level was 13.6% within one hour. One week later in outpatient follow-up, methemoglobin level was 2.6%. IMPLICATIONS/DISCUSSION: Methemoglobinemia causes cyanosis that is unresponsive to oxygen therapy. In addition, pulse oximeter readings are not accurate when there is methemoglobin in the blood. Etiologies of methemoglobinemia include medications, chemical agents, and hereditary causes. The goal of therapy in methemoglobinemia is to reduce heme iron in order to restore normal oxygen transport. year-old Asian-American woman presented with three weeks of progressive muscle weakness. She had difficulty combing her hair, lifting her arms or getting out of a chair. At presentation she could not walk. She had a dusky, almost dirty appearing rash on her chest and around her eyes. An erythematous rash was noted on the dorsal surface of the MCP and PIP joints of her hands. She could not lift either thigh off the bed. Laboratory values: CK 14,245, RF 273, ANA + >1:640 (speckled pattern), AST 605, ALT 207, ALK PHOS 95, LDH 150. Hepatitis B CAb +, Hep Sab +. Anti-RNP, Anti-Jo, Anti-SSA, Anti-SSB and anti-DS DNA were all negative. A presumptive diagnosis of dermatomyositis was made, a confirmatory muscle biopsy performed and prednisone begun. Malignancy workup was negative. She improved slightly and was discharged to a rehabilitation facility. IMPLICATIONS/DISCUSSION: Dermatomyositis (DM) is a rare idiopathic inflammatory myopathy that has a 2:1 female preponderance and presents most commonly in the fifth decade. The incidence of DM is 1:100,000. Pathogenetically, DM is associated with immune complex deposition in a perivascular and perifasicular distribution. The etiology is unknown although viruses have been implicated. The typical presentation of DM is proximal muscle weakness in association with classic dermatological findings. Gottren's papules, as seen in our patient, are the most commonly seen skin rash. The classic heliotrope rash is the most specific manifestation of DM but is only rarely seen. Other findings include acanthosis nigricans,`m echanics hands,'' diffuse flat erythema in a shawl like distribution, and periungual erythema. The elevated liver enzymes in this patient are problematic since DM alone can raise parenchymal liver enzymes. The hepatitis B profile is not suggestive of an acute infection and unlikely to be causing the elevated liver enzymes. Hepatitis B, however, has been implicated in the pathogenesis of some cases of DM and raises the question of a delayed slowly progressive humorally mediated immune reaction. The relationship between the two conditions is unknown although a few select reported cases suggest it may be more than coincidental. There was no history of smoking, travel, or prolonged immobilization. His ROS was otherwise negative. His medications were aspirin, metaprolol, lisinopril, and sulfasalazine. After initial treatment for pulmonary edema at an outside hospital, he was transferred to our institution. He was intubated due to worsening hypoxia. A bronchoscopy was performed: bronchoalveolar lavage (BAL) was bloody. VS: Temp -378C, BP -124/60 mmHg, HR -110/min, SaO2 -98% on assist control ventilation. Exam was notable for bilateral rales and absence of S3 or JVD. Initial labs revealed Na-127, creatinine -1.0, CK-MB -15, Troponin I -10.2. EKG was normal; CXR showed interstitial pulmonary edema. Echocardiogram showed EF of 35%. A highresolution chest CT showed ground glass appearance consistent with ARDS. He was treated with ceftriaxone and azithromycin. His hospital course was complicated by right middle and anterior cerebral artery ischemic stroke, ARF (BUN/Cr 118/3.3), anemia and ultimately shock. He expired on the ninth hospital day. Autopsy reports confirmed thrombotic microangiopathy consistent with HUS. BAL culture subsequently grew influenza A. IMPLICATIONS/DISCUSSION: Influenza, occurring as outbreaks in winter seasons, is usually self-limited; but causes increased morbidity and mortality in high-risk populations. Such high-risk group includes patients with underlying cardiac, pulmonary, and immunosuppressive diseases, nursing home residents and older than 65. Primary viral pneumonia is the most severe pulmonary complication that tends to occur in individuals with elevated left atrial filling pressures, and pulmonary diseases. Systemic complications include rhabdomyolysis, Reye syndrome, transverse myelitis, and myocarditis. Other rare complications, such as hemolytic uremic syndrome and toxic shock syndrome worsen the clinical outcome. Apart from the tissue culture, the virus can be detected in throat swabs and by serologic methods. This high-risk patient presented with acute severe respiratory failure from primary influenza A pneumonia complicated by HUS leading to renal failure and anemia. Increasing the pretest probability by clinical evaluation will be helpful in initiating treatment earlier in the course rather than be fooled by the flu. LEARNING OBJECTIVES: 1. To recognize the clinical and pathologic presentation of talcosis, also known as foreign body granulomatosis. To recognize the sequelae of talcosis, specifically panlobular emphysema, acute pulmonary hypertension, ARDS, and spontaneous pneumothorax. 2. To recognize signs and symptoms of factitious disorders (Munchausen's). 3. To discuss differences between factitious disorders, somatoform disorders and malingering. CASE INFORMATION: A 50 year-old Oregon man with an extensive past medical history including chronic pain syndrome involving his hip, knee, and groin, total hip replacement surgery with multiple revisions and post-surgical complications presented with a recent history of hypertensive episodes and syncope. He was referred to VMMC for evaluation of a possible pheochromocytoma. In addition to syncope, the history and physical revealed an antecubital abscess and fevers. The patient was admitted and an Infectious Disease consult ordered. He reported that In March, 2002, he developed increasing frequency of attacks characterized by chill, abrupt fever, hypertensive episodes, and syncope. In mid-March, he noted swelling, redness and warmth in his right antecubital fossa and continued to have fever every few days. The patient's hospital course was complicated by multiple episodes of fever >39 oC, witnessed and unwitnessed syncopal episodes, frequently infliltrated intravenous lines, polymicrobial blood cultures, increasing oxygen needs, infected PICC, and atrial fibrillation. His medical work-up was extensive. He ultimately underwent a chest CT which showed multiple 2-3mm, bilateral pulmonary nodules. The patient had thoracoscopic lung biopsy of several nodules. The pathology revealed near complete occlusion of small and medium vessels by extracellular refracticle material. The patient was confronted with these findings and confessed to having crushed and injected oxycontin on several hundred occasions.
Occurs when tablets are pulverized and injected. The most common fillers used in tablets are talc, cornstarch, cellulose. These fillers cause an initial arteritis which is associated with rapid influx of neutrophils around the intravascular foreign body. The influx of neutrophils is followed shortly thereafter by granuloma formation as talc particles migrate through the vessel wall to the surrounding perivascular and interstitial tissue. The most common radiographic finding is widespread, 2-3mm well defined nodules typically in the mid lung. The clinical presentation is typically non-specific and may mimic URI with: cough, dyspnea, increased sputum. Sequelae of talcosis includes: panlobular emphysema, acute pulmonary hypertension, ARDS, and spontaneous pneumothorax. Treatment options are limited but include: Dexamethasone and D-penicillamine. Steroids appear to have a limited (if any) role. Vasodilatation with hydralazine may help those with pulmonary hypertension. FACTITIOUS DISORDER-A form of feigned illness which differs from malingering (which has a external incentive-avoiding work, financial gain) in that it has no other incentive other than to be a patient and experience the sick role. Both factitious disorder and malingering differ from somatoform disorders in that they are voluntarily produced. Munchausen's Syndrome is the most extreme (and highly publicized) form of factitious disorder. Patients with factitious disorder often appear more comfortable than their condition would warrant. They may be pleasant, cooperative and receptive to ANY intervention-even if painful and risky. Nurses often note that they often have no visitors/phone calls suggesting anti-social or isolating behaviors. They may also give a medical history that is unbelievable or``too good to be true''. Be suspicious when pt's deny you the right to access old records or speak with previous providers. Yesterday she had been seen in the clinic and diagnosed with trichomonas vaginitis. The resident had dutifully told her that this was a sexually transmitted disease and that both she and her husband needed to be treated simultaneously. Unfortunately, because of her husband's health they had not had sex in over ten years. Now her husband was accusing her of infidelity, a fact she denied vehemently. The Case: The patient is a 59 year old white woman with a 1 week history of burning on urination and a mild vaginal discharge, described as thin and clear. She denied fever or flank pain. Her social history was significant for part-time work as a housekeeper which took her out of the house periodically. Her husband had been disabled for 10 years with a back injury and there had been no sexual activity since the injury. The pelvic examination was remarkable only for a thin watery discharge with a mild odor. Cervix and adnexa were benign. A wet prep revealed numerous trichomonads. IMPLICATIONS/DISCUSSION: Trichomonas vaginalis infection classically presents as a vaginitis/cervicitis with a thin, yellow, frothy, foul smelling discharge often times associated with dysuria. Nevertheless, up to 40% of infections are asymptomatic. The association with sexual activity is well documented. As an illustration of this, the incidence of infection in the general population has been reported as high as 5-10%, while in venereal disease clinics the incidence jumps to 25% and up to 70% among prostitutes. However, unlike other sexually transmitted diseases, the age distribution of of trichomonas infection is bimodal. For example, most women with gonorrhea are in their early 20's, with less than 2% over the age of 40. Trichomonas infections also peak during years of maximal sexual activity but there appears to be a second peak around the time of menopause. Indeed, >30% of patients are over the age of 40. It has been hypothesized that this represents women infected at an early age who harbored the trichomonas asymptomatically but present with symptoms at a time when the vaginal milieu changes (eg. menopause) allowing the trichomonas to propagate. There are several reported cases of newborns and apparent virgins with trichomonas. These likely represent either transmission from infected mothers at the time of birth or a nonsexual source. Studies from the British literature (Burgess,1963; Whittington 1957) establish that trichomonas can survive for up to 45 minutes on a toilet seat, although transmission by this route has never been proven. Has she been faithful? Clearly there is valid justification to believe that she is telling the truth. year-old man presented with a five weeks of bowel and bladder incontinence and parethesias of the lower extremities. He had a history of diabetes mellitus and HIV. Two weeks prior he had presented for evaluation of urinary hesitancy and paresthesias of the lower extremities. He was diagnosed with diabetic peripheral neuropathy and released. On this admission, his neurological examination revealed decreased sensation in the lower extremities, absent ankle reflexes, an ataxic gait, saddle anesthesia, and decreased anal sphincter tone. An MRI of the spine revealed an extradural mass extending from L4 to S4. Biopsy confirmed the neoplasm to be Burkitt's lymphoma. IMPLICATIONS/DISCUSSION: Although spinal cord compression is a medical emergency, access to an MRI may be limited. The physical examination is instrumental in establishing the diagnosis and expediting appropriate therapy. Although diabetes and HIV are common causes of peripheral neuropathy, they are not causes of the cauda equine syndrome (CES). A methodical physical examination detected saddle anesthesia and sphincter impairment, establishing the diagnosis of CES. Compression of the roots can be caused by a ruptured disk, infection, fracture, or tumor. HIV patients are at increased risk for tumors associated with co-sexually transmited viral infections such as HHV6 and HHV8. CES is a neurosurgical emergency, requiring immediately operative decompression of the spinal canal. A timely diagnosis of this syndrome is essential, as patients with paraneoplastic impingement may not regain function beyond the deficit at presentation.
CORONARY DISEASE. E.J. Lee 1 , K. Barnard 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #75596) LEARNING OBJECTIVES: Recognize gender differences in the presentation of myocardial ischemia and consider microvascular spasm as a cause for chest pain in women with angiographically normal coronary arteries. CASE INFORMATION: A 54 year-old Asian-American woman with history of chronic back pain presented with retrosternal chest pain. The pain was sharp, radiated to her left arm, awoke her from sleep, and lasted for one hour. She had had similar pain the previous day, lasting an hour before spontaneously resolving. Neither episode was associated with nausea, vomiting, diaphoresis, or exertion. Another similar episode occurred one year ago. Echocardiogram at that time showed mild hypertrophy and normal overall function, while coronary catheterization revealed normal coronary arteries and an ejection fraction of 65%. The patient had no history of diabetes, hypertension, hyperlipidemia, smoking or family history of coronary artery disease. On initial evaluation in the Emergency Department, the patient was without chest pain. Her electrocardiograph showed normal sinus rhythm with no ischemic changes. Chest radiograph was normal. Total CPK was 71 and troponin was less than 0.2. She was admitted to rule out myocardial infarction. Shortly after being admitted, the medicine house staff were called to evaluate the patient for``seizure-like activity.'' The patient was found in severe emotional distress with taut extension of her upper and lower extremities, and clawing of her hands bilaterally. She was alert, oriented, and able to follow commands throughout this episode which resolved within minutes. Electrolytes showed a calcium of 9.7, magnesium of 1.7, phosphate of 3.3, and potassium of 3.8. An electrocardiograph at this time showed ST depression in leads II, III, and AVF. Repeat electrocardiograph showed a similar pattern of ST depressions. The patient was transferred to the cardiac step-down unit with a presumptive diagnosis of unstable angina and started on anticoagulation therapy. Aspirin and beta-blockers were held due to reported allergies. Subsequent electrocardiographs showed normal ST segments in all leads. Second and third sets of cardiac enzymes were negative. Cardiac catheterization the following day revealed normal coronary arteries and an ejection fraction of 60%. The patient was discharged home the following day in good condition. IMPLICATIONS/DISCUSSION: There is abundant data to suggest there are gender differences in the risk factors, presentation, and management of cardiac disease. It has been shown that negative affect and emotional distress may increase risk of ischemic heart disease and interfere with effective cardiac rehabilitation. Mental stress has been known to cause ischemic changes on electrocardiograph and abnormalities in regional and global left ventricular function. These effects have been associated with microvascular spasm. A mental stress-induced ischemic episode may have occurred in this patient. A coronary catheterization that showed normal coronary flow would not be uncommon in such a case. It is important to realize that despite angiographically normal coronary arteries, this may be an atypical presentation of true underlying coronary disease.
THROMBOTIC THROMBOCYTOPENIC PURPURA-HEMOLYTIC UREMIC SYNDROME (TTP-HUS) AFTER A SINGLE DOSE OF QUININE. J. Lee 1 , M. Krasnoff 1 ; 1 Dartmouth Hitchcock Medical Center, Lebanon, NH (Tracking ID #74449) LEARNING OBJECTIVES: 1. Identify a potentially fatal complication of a commonly prescribed therapy for nocturnal leg cramps. 2. Recognize quinine as the most common cause of drug-associated TTP-HUS. CASE INFORMATION: The patient is a 66 year old previously healthy female who presented to the emergency department with the acute onset of nausea, vomiting, abdominal pain, diarrhea, and disorientation 2.5 hours after taking a single dose of 260 mg of quinine for nocturnal leg cramps. Over the next few hours, she developed severe hypotension, requiring pressors. Labs were notable for WBC 3.6 Plt 123,000 Bun 25 Cr 1.9 T bili 1.7 PTT 75 PT > 100. A CT scan of the abdomen and pelvis was unremarkable. The patient was admitted to the ICU with a presumed diagnosis of sepsis of unclear etiology; she was started empirically on IV ampicillin/sulbactam. Over the next few days, the patient developed microangiopathic hemolytic anemia, severe thrombocytopenia (Plt 12,000), and acute renal failure (Cr 8.4). Cultures of sputum, urine and blood showed no growth. Her clinical diagnosis was felt to be TTP-HUS; her antibiotics were stopped and she was begun on daily plasmapheresis. Dialysis was instituted. After two weeks of therapy, the patient's anemia, thrombocytopenia, renal failure, disorientation, and coagulopathy were all improving or resolved. An adverse event report was sent to FDA MedWatch. IMPLICATIONS/DISCUSSION: Nocturnal leg cramping is a common complaint encountered in the primary care setting. Despite quinine being frequently prescribed, its potential for severe adverse reactions is largely underrecognized. Quinine is the most common cause of drug-associated TTP-HUS. Common presenting symptoms include the acute onset of abdominal pain, nausea, vomiting, diarrhea, fevers and chills. The symptoms usually begin abruptly, several hours after the ingestion of quinine. A single tablet is sufficient to cause TTP-HUS. In addition to the expected findings of TTP-HUS, low-grade DIC and leukopenia can also be seen. The mechanism of quinine-associated TTP-HUS is presumed to be caused by drug-dependent antibodies. Treatment involves avoidance of quinine exposure, plasmapheresis and supportive therapy. While available by prescription, quinine is also found in beverages, such as quinine water, tonic water, and gin and tonic. In addition, there is an extensive list of herbal remedies containing quinine including cinchona tree, peruvian bark, and china bark. Physicians should carefully question any patient with TTP-HUS for quinine exposure, especially since patients may not report it among their medications. In addition, physicians need to counsel patients regarding future avoidance of all quinine-containing compounds, since repeat ingestion of quinine may cause relapse of TTP-HUS.
PATIENT. D. Lehman 1 , J. Mills 1 , C. Sorensen 1 , J. Whitesel 1 , R. Reves 1 ; 1 Denver Health and Hospital Authority, Denver, CO (Tracking ID #75102) LEARNING OBJECTIVES: To recognize that tuberculosis can present with unusual clinical manifestations, often sub-acute or chronic, especially in the immunocompromised context, such as HIV infection. CASE INFORMATION: 48 y/o male with heterosexually acquired HIV infection and a CD4 count of 306, presented with a 1-month history of penile and rectal pain and 1 week of no urethral voiding. He noted urine coming from the rectum with feces. Voiding cystourethrogram showed a prostatic-rectal fistula and a Foley catheter was placed. He was born in Mexico and had a negative PPD 10 years earlier. Four months earlier he had presented with dysuria, an enlarged prostate, pyuria and microscopic hematuria. CT scanning then showed a 2.7 by 3.0 cm hypodensity within an enlarged prostate that, in the absence of fever and toxicity, appeared clinically not consistent with a prostatic abscess. No urine culture was done, but he received 10 days of levofloxacin. Fistula evaluation by GI with colonoscopic biopsy showed chronic inflammation and necrosis. Transrectal ultrasound showed a large prostate with prostatic calculi with no cyst or abscess. Granulomas and acid-fast bacilli (AFB) were found in biopsies obtained during an unsuccessful surgical attempt to repair the fistula. Bilateral infiltrates were found on a post-operative chest X-ray. AFB were found in urine and sputum and cultures grew Mycobacterium tuberculosis (TB). Standard 4-drug therapy for TB was successful and the fistula closed without further surgery after 6 months of directly observed treatment and Foley drainage. IMPLICATIONS/DISCUSSION: Delayed and missed diagnoses of active pulmonary and extrapulmonary TB are common due to the relative rarity of the disease and a lack of familiarity with its protean manifestations. TB of the genitourinary tract accounts for about 1% of reported cases of TB in the US. Extrapulmonary forms (27% of US cases) are more common among children and patients with HIV-infection or other disorders of immunity. The keys to earlier recognition of active TB are awareness of: 1) the manifestations of the disease, 2) risk factors for previous exposure and latent TB infection, and 3) risk factors for progression from latent TB infection to active disease. In retrospect this patient had all three of these features. The clinical features of active TB, both pulmonary and extrapulmonary are variable, but chronic and sub-acute presentations are common. Signs of systemic toxicity are helpful if present but are commonly absent. TB of the urinary tract should be suspected in patients with unexplained``sterile'' pyuria and/or hematuria and in those with unexplained chronic inflammation and fibrosis. This patient had a common risk factor for infection, having been born in Mexico, and had HIV infection as a risk factor for progression to active TB. Although the GU tract is a relatively common extra-pulmonary site for TB, there is little mention in the literature of fistula as a complication of TB prostatitis. The most likely etiology of a prostaticrectal fistula is surgery, rarely by infection. Radiological studies revealed what appeared to be a solid posterior occipital mass consistent with a malignant lesion and surgical intervention was pursued. At craniotomy he was found to have an abscess; smears revealed yeast forms and cultures grew Cryptococcus neoformans. CSF results were WBC 255, protein 112, glucose 27 and cryptococcal antigen 1:64. Serum cryptococcal antigen was 1:128. He disclosed no risk factors for immunodeficiency which was confirmed by negative studies for HIV-1 and 2 antibody, HIV-1 and 2 PCR, HTLV-1 and 2 antibody and HTLV-1 and 2 PCR. Of note, his CD4 count was 79 and his total IgG, IgG1 subclass and IgG2 subclass levels were low. IgA and IgM levels were at the lower limits of the normal range. He received two weeks of amphotericin B and improved appropriately. Lumbar puncture at that time revealed: WBC 440, protein 111, glucose 30 and cryptococcal antigen 1:2. Repeat CD4 count was 191 and serum cryptococcal antigen was 1:64. His therapy was changed to fluconazole and he continued to improve. IMPLICATIONS/DISCUSSION: Although possible in immunocompetent hosts, cryptococcal meningoencephalitis presents most commonly in patients with HIV infection, drugassociated immunosuppression, leukemia and complement deficiency. This infection has rarely been reported in cases of hypogammaglobulinemia. Cryptococcus neoformans infects through aerosolization producing a primary respiratory infection that spreads hematogenously to distant sites. In hypogammaglobulinemia of the type shown in this patient, the proposed host defect stems from dysfunctional T-cell activity resulting in fundamental difficulties with complement fixation, opsonization and reduction of circulating antigens. We propose the possibility that the hypogammaglobulinemia facilitated the development of this patient's CNS disease. These immunosuppressed patients present with fever, headache and malaise similar to, but typically more intense and shorter in duration than infections in the immunocompetent host. Response to treatment with amphotericin B and fluconazole are similar to those in immunocompetent hosts.Close monitoring and medication adjustments per clinical response are essential. Thus, this case provided an unusual initial presentation of hypogammaglobulinemia as cryptococcal meningoencephalitis. LEARNING OBJECTIVES: To recognize filariasis in the differential diagnosis of nephrotic syndrome. CASE INFORMATION: A 46 year old man who had recently emigrated from West Africa presented with a three month history of lower extremity pitting edema. His physical examination was significant for anasarca. Laboratory data revealed a white blood cell count of 9900 with 58% eosinophils, an albumin level of 1.2 g/dl as well as a BUN and creatinine of 16 and 1.0 respectively. An initial urinalysis revealed protein level of greater than 300 mg/dl and microhematuria. A subsequent 24-hour urine collection revealed 7.6 grams of protein confirming a diagnosis of nephrotic syndrome. The differential diagnosis of nephrotic syndrome with eosinophilia includes lymphoma and various parasite infections. While parasitic serologies were pending, chest and abdominal CT scans were normal ruling out a diagnosis of lymphoma. A renal biopsy showed membranous glomerulonephritis with diffuse IgG deposits in the subepithelium. Subsequent peripheral blood smears showed obvious microfilariae of the Loa loa species, indicating active infection. Further laboratory studies revealed the absence of other conditions associated with membranous glomerulopathy. Therefore, it was concluded that the nephrotic syndrome and the membranous glomerulonephritis were most likely caused by the filarial infection. The patient was treated with metolazone and lisinopril to treat the anasarca/ nephrotic syndrome and given ivermectin to treat the filariasis. At follow-up two months later, the edema had resolved and his urinalysis had almost normalized. IMPLICATIONS/DISCUSSION: Loa loa is one of eight filarial species and occurs mainly in the rain forests of Africa. Adult worms develop and mate to form microfilariae, which appear in the blood. Whereas other filarial species cause the classic symptoms of lymphedema, the loaisis species is more commonly associated with pruritus and localizd areas of subcutaneous swelling. Various nephropathies, including nephrotic syndrome, are rare and likely result from immune complex deposition within the glomerulus. In tropical countries, infectious diseases are common causes of the nephrotic syndrome. As demonstrated in this case, one should consider the diagnosis of filariasis in patients that present with anasarca and/or nephrotic syndrome and who have a history of travel to endemic countries for filariasis. year-old white female with a history of von Willebrand disease presented with 3 weeks of severe fatigue. She denied history of trauma or abnormal bleeding although she reported easy skin bruising. On physical examination, her temperature was 37.4 degrees Centigrade and BP 102/72 mmHg. She appeared thin and pale. Neck was supple, without thyromegaly. Lungs were clear. Heart was regular, without murmurs or gallops. Abdomen was benign without masses, tenderness or hepatosplenomegaly. Extremities had no cyanosis, clubbing or edema. Neurological examination was non-focal. CBC revealed a hemoglobin of 10g/dl, platelet of 33,000/mm3, and WBC of 3500/mm3 with 13% blasts. Bone marrow biopsy found 40% blasts with positive CD33 and CD34. She was diagnosed with acute myeloid leukemia. Chemotherapy with cytarabine was started. Five hours later, she developed severe stabbing left upper quadrant abdominal pain radiating to the back and left shoulder, aggravated by body movement, only slightly relieved by intravenous hydromorphone. One hour later she became hypotensive with a BP of 90/50 mmHg. Hemoglobin dropped from 9.0 to 7.1g/dl in 4 hours. Electrocardiogram showed sinus tachycardia and no acute ischemic changes. Abdominal series was unremarkable. An abdominal CT scan was reported unremarkable initially, but further review demonstrated blood surrounding the spleen. Emergent laparotomy found acute rupture of a normal sized spleen (8.0 Â 5.5 Â 2.3 cm). Splenectomy was performed. She was transfused a total of 6 units of packed red blood cells. Hemoglobin remained stable after the surgery. She developed acute respiratory distress syndrome and died of respiratory failure and cardiac arrest within a week. IMPLICATIONS/DISCUSSION: The spleen is an immunologic organ commonly involved in hematologic diseases. Splenic rupture can be classified into 1) traumatic; 2) spontaneous; and 3) pathologic. Pathologic splenic rupture occurs in a spleen affected by a disease without previous trauma. The presentations of splenic rupture are the result of intra-abdominal hemorrhage, hypotension, tachycardia, peritoneal irritation signs, and fever. The abdominal pain could radiate to the left shoulder (Kehr's sign). Pathologic rupture of a spleen affected by a hematologic malignancy is a rare event and it is especially unusual for a normal sized spleen to rupture. The risk of splenic rupture increases with age and size of the spleen. The release of the enzymatic content of cells shortly after induction chemotherapy may lead to splenic damage, followed by rupture of the spleen. The present patient had received cytarabine just 5 hours before the abrupt onset of abdominal pain. Other risk factors of bleeding such as thrombocytopenia and von Willebrand disease might also have contributed to her catastrophic event. The diagnosis of pathologic splenic rupture begins with a high index of suspicion. A patient with risk factors presenting with left upper abdominal pain should be observed closely and investigated. Plain abdominal X-ray, ultrasound and CT scan are valuable tests. Aspiration of blood by abdominal puncture could confirm the diagnosis. For insidious rupture, liverspleen scintigraphy is of value. The only therapy is splenectomy. A 67-year-old male was well until 6 weeks prior to presentation, when he developed diffuse extremity deep aching pain and difficulty with walking, maintaining his balance and doing buttons. He denied diplopia, but had trouble looking to the left. He denied weakness, numbness, sensory loss, trouble with speech or articulation, or bowel or bladder dysfunction. On physical examination, he had normal vital signs. He was alert, cooperative, but somewhat slow. A 2 Â 2 cm non-tender supraclavicular lymph node was palpable. Heart, lungs and abdomen were unremarkable. Extremities had no cyanosis, clubbing or edema. Eyes could not move past the midline on leftward gaze. Motor strength was 5/5 in all muscle groups tested. Vibratory sense was diminished distally in the right lower extremity. A heel-to-shin testing was ataxic. He could not sit nor stand unaided. Romberg's sign was noninterpretable even with the eyes open. Plantar responses were flexor bilaterally. Cerebral spinal fluid reveaked glucose 93, lymphocyte 4, IgG 15.1, and protein 100. Anti-Hu antibody was positive. Brain MRI showed a small old lacunar infarct. Chest CT scan identified a 1 ± 2 cm right suprahilar pulmonary nodule, 3 right upper lobe nodular densities less than 1 cm each, and a prominent 3.5 cm nodule in the right pretracheal area. EMG and nerve conduction studies demonstrated a sensory motor polyneuropathy. Lymph node biopsy showed neoplastic cells staining with synaptophysin and chromogranin, supporting the diagnosis of large cell neuroendocrine carcinoma. The patient was treated with carboplatin and etoposide for three doses and IVIgG for five doses. The symptoms were stabilized. IMPLICATIONS/DISCUSSION: Paraneoplastic encephalomyelitis (PEM) is a frequent remote effect of cancer characterized by neuronal loss and inflammatory infiltrates in the nervous system. The onset is usually subacute and causes severe neurological dysfunction which antedates the diagnosis of cancer. The presenting neurological syndrome is usually sensory neuropathy, cerebellar dysfunction and cortical encephalitis. Diagnosis of the primary cancer remains challenging and requires a high index of suspicion. Usually, it is made by the demonstration of specific paraneoplastic antibodies. Anti-Hu antibody recognizes a family of RNA-binding proteins expressed in the nuclei of neurons and cancer cells. It has been used as a diagnostic marker, but no evidence suggests it causes neuronal damage. Effective treatment of the tumor with chemotherapy is an independent predictor for stabilization of PEM. Large cell neuroendocrine carcinoma is a highly aggressive tumor that usually occurs in smokers, and may be either central or peripheral. It has been suggested that surgically resectable tumors be excised and that advanced stage lesions be treated with combination chemotherapy. Immunotherapy has been proven effective in some patients in tumor regression and PEM stabilization or improvement. The prognosis is poor, with a median survival of 11 months. LEARNING OBJECTIVES: Recognize that the clinical manifestations and radiologic findings of peritoneal tuberculosis (TB) may mimic ovarian carcinoma. Recognize that an elevated CA-125 level may be more suggestive of tuberculous peritonitis than ovarian carcinoma in certain populations such as young women, HIV-infected women and women from areas endemic for tuberculosis. CASE INFORMATION: A 29 year-old Vietnamese woman with no significant past medical history presented to the emergency department with a 10 day history of fever, non-productive cough, abdominal pain, and increasing abdominal girth. A PPD placed as an outpatient was reactive to 16mm. She emigrated from Vietnam ten years prior, had no recent travel history, and had no recent sick or TB contacts. Family history was negative for cancer. On physical examination, the patient was febrile to 102.6F with a pulse of 111 BPM. Pulmonary exam was clear to auscultation. Abdominal exam was significant for mild distention, and tenderness in the epigastric and right upper quadrant regions without rebound or guarding. Gynecologic exam was normal. Chest x-ray was normal. A CT scan of the abdomen and pelvis showed diffuse intraabdominal ascites with omental caking, strongly suggestive of metastatic disease from ovarian carcinoma. Pelvic ultrasound was negative for adnexal masses. Ascitic fluid analysis showed a predominantly lymphocytic exudate. There were no malignant cells, acid-fast bacilli (AFB) or other organisms detected in the fluid. Fluid culture yielded no growth. Multiple sputum smears were negative for AFB. A CA-125 level was elevated to 546 U/ml. A CT guided biopsy of the omentum revealed granulomatous inflammation and well-defined granulomas with central necrosis ± histology consistent with tuberculous peritonitis. The patient was started on a regimen of isoniazid, rifampin, ethambutol and pyrazinamide. On follow-up two weeks after discharge, the patient reported feeling well with complete resolution of the fever and abdominal pain. IMPLICATIONS/DISCUSSION: Peritoneal tuberculosis is an uncommon entity. The common clinical presentation is ascites or an abdominal mass. The elevated CA-125 and radiologic appearance of the omentum, as in the case of our patient, can be highly suggestive of ovarian carcinoma. However, it is well established that many other conditions, including peritonitis can elevate the CA-125. When these features are present in a patient who is at high risk for developing tuberculosis and who is at low risk for developing ovarian cancer then the diagnosis of tuberculosis must be considered. Establishing the diagnosis of peritoneal tuberculosis can be difficult as ascitic fluid is rarely positive for AFB and even culture of biopsy specimens may be negative. Diagnosis is often made on the basis of suggestive histopathology and clinical features. Clinicians must maintain a high index of suspicion for tuberculous peritonitis in patients presenting with ascites and abdominal masses, especially in young women (in whom ovarian carcinoma is uncommon), HIV-infected patients and patients from areas endemic for tuberculosis. In these cases, empiric antituberculosis therapy can be considered while further workup is pending. CA-125, while nonspecific for peritoneal tuberculosis, can be useful in monitoring response to therapy and would be expected to return to normal levels.
CHRONIC SINUSITIS AND BEYOND. A. Loewen 1 ; 1 University of Calgary, Calgary, Alberta, Canada (Tracking ID #77075) LEARNING OBJECTIVES: To review an approach to the patient with chronic infection. To discuss the manifestations of Wegener's Granulomatosis. CASE INFORMATION: A 33 year old male with a 4 month history of chronic sinusitis which is refractory to antibiotics, and an episode of suppurative otitis media, presents with left sided Bell's palsy and multiple boils. There is microscopic hematuria with normal renal function. Drainage and biopsy of sinuses demonstrates necrotizing granulomatous inflammation. Anti PR3 level is 18.5 kEu/L. A diagnosis of Wegener's granulomatosis is made. The patient later develops hemoptysis while on therapy with prednisone and cyclophosphamide. IMPLICATIONS/DISCUSSION: An approach to the patient with apparently chronic infection must include infectious causes, underlying immunosuppressed states and noninfectious causes. Wegener's granulomatosis is a vasculitis of small arteries and veins and usually presents as a pulmonary-renal syndrome. In this case it presented with upper airway involvement, pyoderma gangrenosum and cranial nerve palsy, butpulmonary and renal symptoms were minimal. A broad differential diagnosis can lead to the correct diagnosis, avoiding unnecessary interventions, and hasten administration of appropriate therapy.
G. Loukatous 1 , J. Wiese 1 , J. Aliota 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77080) LEARNING OBJECTIVES: 1. Identification of the clinical signs and symptoms of Vibrio Vulnificus infection 2. Recognition that early and aggressive intervention is essential to optimal patient outcome CASE INFORMATION: A 42-year-old Vietnamese man presented with a two-day history of fever and a painful left leg. He denied trauma or stasis involving the leg. His pulse was 110; blood pressure 110/50; temperature 38.0 C. There was a 10 cm eccyhmotic lesion on his left lower leg. Despite therapy with cefazolin his condition deteriorated. Over 12 hours, the ecchymosis extended as the skin lesion rapidly developed multiple fluid-filled bullae. General surgery was consulted for surgical debridement. Upon further questioning, the patient noted a recent history of raw oyster ingestion. Wound and blood cultures returned positive for Vibrio vulnificus. He was started on tetracycline. Although he subsequently developed multi-organ failure, he recovered after 22 days of aggressive medical treatment and continued surgical debridement. IMPLICATIONS/DISCUSSION: Early diagnosis and treatment is the critical component of a successful outcome from a Vibrio vulnificus infection. The key to diagnosis is combining an astute history with an observant physical examination. In sea-side cities, the consumption of oysters or exposure to the water may be so common as to preclude the patient from thinking to offer this historical clue unless prompted. Recognizing the appearance of Vibrio, then, is the key to diagnosis. Erythematous and ecchymotic skin lesions that rapidly evolve to hemorrhagic vesicles and bullae should alert the physician to the possibility of a Vibrio infection. Like necrotizing faciitis, vibrio invades the deep fascia, destroying the facial structures as it rapidly extends. Ecchymosis, bullae and anesthesia reflect the loss of vessels, skin partitions and nerves. Early intervention with appropriate antibiotic therapy and aggressive wound debridement is essential in such cases. Even with early intervention, bacteremia from V. vulnificus carries a 50% mortality that increases to 90±100% in the presence of hypotension. A 34 year old non-smoking, HIV-negative Hispanic male, who received his last dose of chemotherapy for lymphoma 1 week prior, presented with 3 days of increasing cough, dyspnea on exertion, and fever. He denied any sick contacts, TB exposure, recent travel, or pet exposures. His vital signs were:T 99.6 F, BP 115/78, P 100, R 20, and Pulse Ox 96% on room air. Respiratory exam showed unlabored breathing and clear lungs. His studies showed: WBC 1.9 (N 51%, L 12%, M 35%), Hb 9, Hct 25.9, Plt 44, and LDH 1461. Chest xray showed bilateral hilar fullness not seen on prior films. Differential diagnosis on admission was pulmonary infection (community acquired, atypical, and fungal pneumonia; TB; PCP) versus recurrence of lymphoma. Hospital course:Ceftriaxone and azithromycin were empirically started on Day 1. By Day 2, the patient remained febrile and showed signs of decompensation with decreasing BP and increasing oxygen need to 4 liters. A room air ABG at that time showed: pH 7.49, pCO2 36, PO2 43, saturation 83%. Chest CT showed progression to diffuse bilateral alveolar infiltrates without adenopathy. Trimethoprim/sulfamethoxazole was added empirically for PCP. The patient was then transferred to the ICU for prophylactic intubation and diagnostic bronchoscopy showing positive Pneumocystis carinii direct fluorescent antibody on lavage. All other studies (biopsy, brush, and sputum/blood samples) were negative for bacterial, fungal, and malignant processes. Prednisone was subsequently added. The patient improved dramatically with extubation on Day 3 and discharge within the week. IMPLICATIONS/DISCUSSION: PCP is usually seen in AIDS patients with CD4 counts <200 without chemoprophylaxis. Unfortunately, it may not be readily considered in the differential in immunosuppressed, non-HIVpatients. The above vignette serves to remind us to not exclude PCP in the differential because a delay in evaluation could be fatal. Our patient fared well because we recognized his predispositions (hematologic malignancy and immunosuppression from chemotherapy) early in his hospital course which led to prompt treatment. The calculated absolute lymphocyte count was 228, making the CD4 count likely <200. LEARNING OBJECTIVES: Formulate a differential diagnosis of facial swelling using clinical and laboratory data. Initiate early and aggressive treatment based on the presumed diagnosis of facial swelling. Recognize that individuals with recurrent severe infection should be tested for immunodeficiencies. CASE INFORMATION: A 19 year old male was admitted in August, 2001 with face and lip swelling and pain. He has no significant past medical history or family history. He smokes cigarettes, drinks alcohol, and uses cocaine and marijuana. On physical exam he is afebrile, with a pulse of 132, respirations of 20 and a BP of 110/70. Oxygen saturation initially was 100% on room air. His left face, mouth, and neck are swollen and firm, his left eye was swollen shut and his tongue was swollen. He had no cervical adenopathy and clear lung fields. Initial impression was angioedema and treatment involved epinephrine, solumedrol, and with worsening respiratory status, intubation. Fever climbed to 103.3, treatment was started with ampicillinsulbactim and clindamycin. With positive blood cultures the diagnosis was now thought to be necrotizing fasciitis. Supportive care including TPN continued until he was extubated and discharged after 3 weeks. Patient never followed up and was readmitted in May, 2002 with a similar presentation and was found to have common variable immunodeficiency. Upon discharge, he still has not followed up and is not being treated. IMPLICATIONS/DISCUSSION: Necrotizing infections of the skin and fascia involve tissue destruction, systemic toxicity, and a high mortality rate. There is a tendency to underestimate the severity and extent of infection. We now see more angioedema and might miss this clinical presentation. Normal complement studies make hereditary angioedema unlikely and normal tryptase makes angioedema from other causes unlikely. Treatment of necrotizing fasciitis involves airway control, IV antibiotics, and early, aggressive surgical debridement. Individuals with common variable immunodeficiency frequently present with recurrent infections with encapsulated bacterial pathogens as in our patient. Those with recurrent infections should be checked for the disorder and treated with IV immunoglobulin. LEARNING OBJECTIVES: This case report illustrates a rare diagnosis and an often overlooked complication of a benign therapy. Prompt diagnosis is essential to preventing long term sequelae. CASE INFORMATION: A fifty-four-year-old man presented with nausea and vomiting for one month. He was having more than five non-bloody emeses per day, often within 10 minutes of eating. Emesis was not always associated with eating. He stated that he was unable to keep down any food. He denied fevers, chills, rigors, or night sweats. He described low-grade headaches with associated tinnitus, an 18-pound weight loss and generalized weakness with increased somnolence as well as new onset erectile dysfunction. He complained of intermittent epigastric abdominal pain, and had had a previous laparotomy for abdominal pain approximately five years ago. He is a lifetime non-smoker and denied taking any medications. Evaluation revealed total calcium of 16.6 with ionized calcium of 7.7 and a creatinine of 4.9. Unenhanced CT revealed a band of calcification in the midline of the anterior abdominal wall measuring approximately 6 cm in length and 1.4 cm in diameter. In addition, there were numerous small mesenteric and retroperitoneal lymph nodes along with a 1cm node in the suprapubic anterior abdominal tissue. He had a tiny stone in the left kidney and all the bones were noted to be hyperdense. His hemoglobin was 11.7 with a normal peripheral smear, erythrocyte sedimentation elevated at 38. B12/folate, LDH and T-and B cell surface markers were normal. PTH-RP was non-detectable and PTH was appropriately suppressed. He was treated with IV fluids, furosemide and pamidronate, with normalization of his calcium at time of dismissal. Upon further questioning, discovered that he was ingesting 6 to 7 grams of calcium per day for the last 20 years. IMPLICATIONS/DISCUSSION: These findings were consistent with milk-alkali syndrome. The recent emphasis on calcium therapy for prevention of osteoporosis and the availability of calcium carbonate has made milk-alkali syndrome the third leading cause of hypercalcemia.
This presentation is consistent with the subacute form of milk-alkali syndrome referred to as Cope's Syndrome. These patients are exposed intermittently for many years and present with symptoms of both acute and chronic hypercalcemia. This patient responded to therapy and his creatinine remained minimally elevated at 1.8 at a six-month follow-up visit.
A FIFTY-SEVEN YEAR OLD FEMALE PRESENTING WITH AGRAPHIA FROM CEREBRAL EMBOLISM OF UNUSUAL ETIOLOGY. E. Martorell 1 , P. Abraham 1 ; 1 Mayo Clinic, Jacksonville, FL (Tracking ID #75540)
LEARNING OBJECTIVES: 1) To describe an unique presentation of a cardiac myxoma.
2) To recognize cardiac myxomas in the differential diagnosis of acute embolic cerebrovascular accident in adult patients. 3) To assess, diagnose and manage cardiac myxomas early in order to prevent potential mortality and morbidity. CASE INFORMATION: A 57-year-old right-handed woman presented to the emergency department with a three-hour history of new onset writing difficulty without additional symptoms. On physical exam her vitals were normal except for an elevated blood pressure of 156/73. Her head, eye, mouth, neck, skin, cardiovascular and pulmonary examinations were unremarkable. Her neurological examination was completely unremarkable with the exception of a mild impairment of finger to thumb movements (apraxia) on the right hand. She was able to write her own thoughts, but had difficulty due to significant agraphia. MRI demonstrated multiple acute and sub-acute ischemic as well as hemorrhagic lesions without surrounding edema. Electrocardiogram and chest films were normal. Baseline CBC, coagulation studies, chemistry studies and rheumatologic serologies were within normal limits. Carotid duplex showed mild stenosis of the carotid arteries bilaterally. A tranesophageal echocardiogram revealed a large multilobulated, motile left atrial mass affixed via a stalk to the left interatrial septum consistent with atrial myxoma. The patient subsequently underwent urgent thoracotomy. A mucinous dark red polypoidgelatinous 5cm  2cm  1cm mass was resected from the left atrium. Biopsy revealed a benign cardiac atrial myxoma. The patient remained neurologically stable and was discharged home on day #10. IMPLICATIONS/DISCUSSION: Myxomas are twice as common in women as in men and most frequently develop between the third and sixth decades of life. About 1% of ischemic strokes in young people are caused by atrial myxomas. Although embolic complications from myxoma most often affect the brain in more than two third of cases, our patient is unique because of her mild, single neurological deficit and absence of the typical presenting signs and symptoms characteristic of this tumor such as obstructive cardiac signs (54±95%) and constitutional symptoms (90%). Systemic emboli occurs in 10±45% of myxoma patients. Cardiac myxoma should always be considered in the differential diagnosis of embolic stroke in adult patients because rapid diagnosis allows curative surgical resection and prevents further morbidity and mortality. heparin. An IVC filter was placed on day #2 because of large clot burden and concern for further pulmonary emboli. Given his right ventricular hypertrophy and likelihood of future pulmonary emboli, the patient was discharged on life-long therapy with coumadin. IMPLICATIONS/DISCUSSION: The incidence of pulmonary emboli in the US approximates 400,000 cases a year, with another 800,000 cases remaining undiagnosed. In one large series of patients with no pre-existing pulmonary disease, the incidence of dyspnea was 73%, pleuritic chest pain 66%, cough 37%, tachypnea 70%, tachycardia 30% and rales 51%. Laboratory, EKG and CXR findings are routinely nonspecific. The untreated mortality for PE is~30%, while the mortality in treated cases ranges from 2 to 8%. It is imperative that clinicians maintain a high index of suspicion for pulmonary emboli when evaluating the patient with dyspnea. Cerebral aneurysms are an extrarenal complication of APKD, with rupture causing 11% of deaths in theses patients. The prevalence of cerebral aneurysms increases with age, averaging around 10% in this population. Patients with a family history of subarachnoid hemorrhage or cerebral aneurysm are at greatest risk. In one study, asymptomatic cerebral aneurysms were found in 22% of patients with a family history, versus 5% of patients with no family history. Risk of rupture increases with size of the aneurysm and with location in the posterior circulation, with values ranging from 0.5 to 4% per year. As the size of the aneurysm is directly related to age, it is rare to detect an aneurysm in patients younger than age 30. Magnetic Resonance angiography is the recommended method of screening. APKD should be considered in patients with a strong family history of renal disease. Screening for cerebral aneurysms in patients with APKD is controversial. Many investigators recommend screening after the age of 30. Other authors recommend screening only for patients with a family history of cerebral aneurysms. Recommended screening intervals vary from every 3 to every 10 years. LEARNING OBJECTIVES: 1. Transient myocardial dysfunction has been described in many non-cardiac illnesses, including sepsis, subarachnoid hemmorhage (SAH), Acute Respiratory Distress Syndrome (ARDS), and and post operatively in non cardiac patients. 2. Mechanisms include oxygen supply and demand mismatch, circulating myocardial depressants, and calcium overload secondary to catecholamine surges. 3. Resolution is usually complete over ten to fourteen days, but elevated troponins may be a predictor of imcomplete recovery. CASE INFORMATION: The patient is a 20 year old previously healthy college student who presented to the emergency department with a three day history of increasing myalgias, sore throat, and diffuse erythematous rash with desquamation of her finger tips. She rapidly developed multi system organ failure, and required temporary mechanical ventilation, continuous veno-venous hemodialysis, and high doses of three pressors for blood pressure support. We had high clinical suspicion for toxic shock syndrome, which was later confirmed by positive TSST-1 antigen. On hospital day #3, she developed a wide complex tachycardia. Her ejection fraction was estimated at 15±20% by echocardiogram. Serial troponins peaked at 33.3. By the time of discharge, 2 1/2 weeks later, her ejection fraction had increased to 34±40%. Her ejection fraction was 30% at four weeks, and 30% at 4 months after initial presentation. She is maintained on a beta blocker, and an agiotensin converting enzyme inhibitor. IMPLICATIONS/DISCUSSION: Transient myocardial dysfunction has been described in a multitude of non-cardiac illnesses, including subarachnoid hemmorage (SAH), Acute Respiratory Distress Syndrome (ARDS), trauma, sepsis, and post operatively in non-cardiac surgical patients. It is a common complication of sepsis. A variety of non-coronary artery related mechanisms have been proposed, icluding oxygen supply and demand mismatch, circulation of various myocardial depressants such as cytokines, and high catecholamine surges leading to calcium overload. While transient myocardial dysfunction has been described in both ARDS and severe sepsis, there is only one other case report in the literature specifically related to toxic shock syndrome. The usual course of sepsis-related myocardial dysfunction is complete resolution over ten to fourteen days. The fact that this patient had an elevation in troponin, and that she still had a depressed ejection fraction four months after presentation, suggests that she may have experienced some degree of ischemic myocardial injury. The magnitude of the persistence of the myocardial insult attests to thd dangers of toxic shock syndrome, even in patients who recover from the acute sepsis and respiratory failure. A 52 year old homeless male presents with multiple complaints. He last saw a doctor ten years ago. His main complaints include right hand numbness and weakness, particularly while he's working. Additionally, he reports occasional dizziness and headaches. On further review of systems, he describes claudication symptoms, difficulty swallowing, especially solid foods, but denies weight loss. His past medical history includes diabetes and hypertension. However, he last received medications over ten years ago. He does drink alcohol daily and smokes cigarettes. On exam, his blood pressure reads 150/100 in the left arm and 130/70 in the right arm. His heart rate is 76, respiratory rate is 6, and he is afebrile. Significant findings on exam include right carotid, right subclavian, and right and left femoral bruits. He also has diminished pulses in the right radial and right brachial arteries, as well as decreased pulses in his feet. An EKG suggests enlarged left ventricle hypertrophy. He eventually received an angiogram, specifically to see if his subclavian bruits and right hand symptoms were significant. His right subclavian artery was aberrant and appeared to wrap behind his esophagus. He also had a severe stenosis of his right subclavian artery proximal to the right vertebral take-off. Furthermore, an exam of the lower extremities revealed severe stenosis in his right iliac artery that received angioplasty. Given the stenosis on angiogram and evidence of flow-reversal, it appeared that subclavian steal syndrome accounted for several of his symptoms, such as the dizziness, headaches, right hand claudication, and exam findings. Coincidentally, the aberrant subclavian seemed to explain his symptoms of dysphagia. Although the diagnosis of subclavian steal syndrome can be deduced by his history and exam, the aberrant subclavian was unexpected, but along with his stenosis, accounts for many of his symptoms. IMPLICATIONS/DISCUSSION: DISCUSSION: Subclavian stenosis can cause symptoms related to subclavian steal. The vertebral artery on the side of the stenosis creates retrograde blood flow from the brain stem by essentially stealing blood from the contralateral vertebral artery. The prevalence of an aberrant right subclavian has been reported as 0.5%-2.9%. The finding of dysphagia has also been reported. This is termed dysphagia lusoria, and was originally described in the 18th century. It translates as jest of nature. The treatment is surgical resection if it causes significant symptoms. He admitted to intravenous drug use, using his right thigh for access. His temperature was 101 F, pulse 110, blood pressure 90/60. His right thigh was erythmatous, edematous and tender, with 10  8 centimeter posterolateral area of fluctulance. The rest of the exam was unremarkable. Labs: WBC 50,000 (93% neutrophils), hemoglobin 7.1, CPK 104, creatinine 5.9. Blood and wound cultures were drawn. MRI showed necrotizing fascittis of posterior/lateral thigh with overlying cellulits, no osteomylelitis. Surgical exploration revealed necrotic skin and subcutaneous tissue extending down to muscle and fascial level, involving half the circumference of thigh. Wide excisional debridement of thigh was performed. Blood and wound cultures grew group A streptococcus sensitive to cefazolin. After antibiotic treatment and two subsequent debridements, the patient improved. Upon discharge, his WBC and creatinine were normal. IMPLICATIONS/DISCUSSION: There are two types of necrotizing fascittis. Type one, which is caused by mixed aerobic and anaerobic bacteria, is associated with diabetes mellitus, cervical necrotizing fasciitis, and fournier's gangrene of perianal area. Type two necrotizing fasciitis, due to group A streptococcus, is associated with history of blunt trauma, varicella, intravenous drug use, penetrating injury and possably with nonsteroidal antiinflammatory drugs. Necrotizing fasciitis should be suspected in patients with severe unexplained pain increasing over time, skin changes (erythema, red-purple discoloration, blisters, bullae), fever, systemic toxicity, myalgias, diarrhea and anorexia. Lab tests show leukocytosis with marked left shift and elevated CPK and creatinine. Imaging studies are most helpful in type one, where gas formation occurs. MRI may overestimate deep tissue involvement. Thus, the only way to make a definitive diagnosis is via surgical exploration. Surgery is indicated for severe pain, toxicity, fever and elevated CPK. Early surgery may improve the outcome, but mortality rates are high even in cases of optimal therapy. Repeat explorations and debridement should be performed until all necrotic tissue has been removed. Additional therapies include antibiotics and intravenous immune globulin.
NON-HODGKIN'S LYMPHOMA PRESENTING AS FLANK PAIN. K.Y. Miskel 1 ; 1 Hospital of the University of Pennsylvania, Philadelphia, PA (Tracking ID #73961)
LEARNING OBJECTIVES: 1) Recognize an atypical presentation of lymphoma 2) Recognize that systemic non-Hodgkin's lymphoma is a common HIV-related malignancy CASE INFORMATION: A 33 year old healthy male with no significant past medical history presented with three weeks of bilateral flank pain. His flank pain was intermittent but had progressively worsened, with radiation to his groin. The pain was associated with movement. He denied fevers/chills, nausea/vomiting, dysuria, or hematuria. Social history was negative for tobacco, alcohol, or illicit drugs. He was homosexual and involved in a long-term monogamous relationship. He had a family history of nephrolithiasis. On initial exam, he was afebrile with a benign abdominal exam, no costovertebral angle tenderness, a normal rectal exam, and guiac negative stool. A urinalysis was significant for +++blood and no leukocyte esterase or protein.
An abdominal xray showed a normal bowel gas pattern with no renal calculi. He was presumed to have radiolucent nephrolithiasis and was advised to follow-up closely. Five days later, he presented to the emergency room with unremitting flank pain, new suprapubic pain, dysuria and fevers to 103±104 F. His only medications were ibuprofen and percocet (prescribed recently for his flank pain). On exam, he appeared uncomfortable, had a T 99.2 F, oral thrush, and bilateral cervical/axillary lymphadenopathy. His abdomen was soft, mildly distended, and diffusely tender to palpation, particularly in the suprapubic area. He had scattered papules with central umbilication on his face, buttocks, and thighs. Laboratory studies were significant for a WBC 5.6, hemoglobin 11.9, creatinine 2.6, AST 124, ALT 158, alkaline phosphatase 627, and total bilirubin 1.5. Abdominal CT scan showed bulky retroperitoneal and pelvic lymphadenopathy, bilateral hydronephrosis, and a circumferentially thickened bladder wall. Bone marrow biopsy and bladder wall biopsy confirmed the diagnosis of stage IV immunoblastic non-Hodgkin's lymphoma involving the bone marrow, GI tract, liver, spleen, and bladder. HIV was positive with a CD4 count of 138 and a viral load of >500,000. IMPLICATIONS/DISCUSSION: Non-Hodgkin's lymphoma occurs in 10-30% of AIDS patients, the most common type being high-grade diffuse large B cell (immunoblastic variant) or Burkitt's-like. Patients usually present with stage IV disease and frequently have involvement of the bone marrow, CNS, liver, and gastrointestinal tract. Although HIV associated systemic lymphoma frequently presents with rapid nodal enlargement and B symptoms, it has no pathognomonic features. This previously healthy male had HIV risk factors as well as molluscum contagiosum and oral thrush that suggested the diagnosis of HIV infection. In addition to chemotherapy, immune reconsitution with highly active anti-retroviral would be necessary for effective treatment.
ALPHA-INTERFERON THERAPY AND DELIRIUM. D. Misra 1 , S. Young 1 , C. Schleupner 1 ; 1 New Hanover Regional Medical Center, Wilmington, NC (Tracking ID #75477) LEARNING OBJECTIVES: Recognize delirium as one of the neuro-psychiatric adverse effects of alpha interferon therapy. We intend to share our experience because it may pose as a diagnostic challenge for clinicians. CASE INFORMATION: We present the case of a 57 yr old caucasian male who had previously been diagnosed with malignant melanoma. He had undergone local excision and was found to have positive lymph nodes. He was started on adjuvant alpha interferon therapy, initially at a dose of 40 million units a day which he could not tolerate primarily because of constitutional symptoms. His symptoms included severe arthralgias, myalgias and headaches. He received this dosage for only 4 days, which was then discontinued and subsequently started back on a reduced dose of 20 million units a day after a drug free interval of one week. He tolerated the lower dose without any side effects. The following week, he was started back on 40 million units a day of interferon which he received for five days. Within a couple of days, pt was experiencing severe headaches along with myalgias and arthralgias. This became worse and patient presented with delirium, agitation and combativeness. He required intravenous sedation and eventually endotracheal intubation for airway protection. His workup including blood counts, basic metabolic panel; urinalysis, drug screen, CT scan of the head and CSF examination were all within normal limits. Serum transaminases were mildly elevated but this was unchanged since the beginning of interferon treatment. His stay in the intensive care unit lasted 24 hours. The following day, he was back to his baseline cognitive level and was discharged home. IMPLICATIONS/DISCUSSION: Alpha interferon is a glycoprotein produced by leukocytes which has broad spectrum antiviral, immunologic and antiproliferative properties. Therefore, it is commonly used in treatment of hairy cell leukemia, hepatitis, renal cell carcinoma and malignant melanoma. The spectrum of side effects is wide and depends on various factors including the dose of the drug, age of the patient and the type of interferon. Our literature search revealed very few case reports of interferon induced delirium. Neuro-psychiatric adverse effects occur with doses higher than 30 million units per day. The temporal relationship of the onset of delirium with high dose interferon treatment in our patient stands out prominently. We believe that this occurrence should be brought to the notice of physicians prescribing interferon so that patients presenting with neuro-psychiatric manifestations are diagnosed and managed appropriately. A 51 year-old man with an ischemic cardiomyopathy presented to the emergency department after experiencing multiple shocks from his automatic implantable cardiac defibrillator (AICD). In the ED, the patient developed ventricular tachycardia at a rate of 162 bpm with a BP of 132/102. His AICD began antitachycardia pacing and DC countershocks without success. The patient was loaded with amiodarone and placed on a 2mg/ min infusion of amiodarone. Sinus rhythm was restored. Lab values included normal liver function tests and a creatinine of 2.1. The patient stabilized. After 24 hours, the amiodarone infusion was reduced to 1 mg/min. Twelve hours later, the patient developed confusion and severe dyspnea. Lasix was given without improvement. The patient developed fever, hypotension, and became lethargic. The exam showed a quiet, tender abdomen. The HCO3 had fallen to 6 meq/L, and the creatinine had risen to 3.2 mg/dl. The ALT was 1947 U/L, the AST was 2629 U/L, the bilirubin was 4.2, and the PT was 16.9s (INR 2.8) . The patient was diagnosed with fulminant hepatic failure. Dopamine and a bicarbonate infusion were begun. Antibiotics were started. The patient was intubated and vitamin K was given. Amiodarone was considered to be a possible cause of the fulminant liver failure. Thus, 72 hours after admission, the amiodarone infusion was stopped despite persistent ventricular ectopy. The patient's status declined and he died on the fifth day from complications from a liver biopsy. IMPLICATIONS/DISCUSSION: Fulminant hepatic failure develops in less than 2 weeks after an insult to the liver. The diagnosis of liver failure should be entertained in the presence of encephalopathy associated with jaundice, coagulopathy, acidosis, fever and hypotension. In this patient, clinical suspicion was confirmed by the rapid change in the liver function tests. The viral hepatidities and drug/toxin exposure are the most common etiologies of fulminant liver failure. In this patient, the leading considerations were ischemic and drug/toxin exposure. A review of the literature revealed at least twenty fatalities from acute hepatic failure following the administration of high dose parenteral amiodarone over the last twenty years. The chronology of exposure and onset of hepatic failure were consistent with amiodarone induced hepatotoxicity in this patient. Regardless of the cause, the treatment of acute liver failure requires removal of the exposure and supportive care for the expected complications. Despite aggressive support, mortality remains at 60-90% without transplantation. LEARNING OBJECTIVES: To recognize that brown recluse spider bites may cause pyoderma gangrenosum and pathergy due to persistent neutrophil mediated injury. CASE INFORMATION: A 47 year-old woman presented with a six month history of recurrent, painful, nonpruritic ulcers on the dorsa of both wrists and the right calf. Past history revealed that six months prior, while cleaning debris from an old tavern, she was bitten on the hand by a spider. The spider was captured and identified as a brown recluse spider. Within hours she was ill with nausea, vomiting, malaise and fever. She improved with supportive treatment. The lesion cleared but weeks later, began to reoccur following minor trauma such as that caused by mosquito bites. On physical examination we discovered pyoderma gangrenosum like lesions that were confirmed by biopsy. Long-term therapy with dapsone was started. IMPLICATIONS/DISCUSSION: Pyoderma Gangrenosum was first described by Brunsting et al in 1930. The etiology is unknown in fifty percent of the cases but associated with Ulcerative Colitis and Behcet's Disease. Spider bites rarely lead to pyoderma gangrenosum and even more rarely lead to recurrent lesions and pathergy. Brown recluse venom has been purified and studied and is known to persist in some wounds for a relatively long time. The precise mechanism of the lesions is not known although neutrophil inhibitors such as nitrogen mustard and dapsone can mitigate or prevent the reaction. The development of new lesions at distant sites from the inciting lesion is unexplainable and suggests an immune mediated response and not a local reaction. The observed clinical response to dapsone suggests an ongoing neutrophil mediated injury. Currently dapsone is being carefully tapered. It is unknown if the lesions will persist and argue for a permanent ongoing cell mediated injury or cease and suggest a self limited condition as remaining venom is cleared.
PLASMAPHERESIS FOR THE TREATMENT OF HYPERTRIGLYCERIDEMIA. C. Carosella 1 , C. Karmen 1 , S. Warshafsky 1 , Y. Murray 1 ; 1 New York Medical College, Valhalla, NY (Tracking ID #76460) LEARNING OBJECTIVES: To recognize the value of plasmapheresis in the treatment of triglyceride-induced acute pancreatitis. CASE INFORMATION: A 50-year-old Hispanic woman with a history of insulin-dependent diabetes mellitus, coronary artery disease and hypertriglyceridemia presented to the emergency room with severe abdominal pain, nausea, vomiting and poor oral intake all gradually worsening over the prior two weeks. She had been treated with a combination of atorvastatin, gemfibrozol, and niacin with only marginal success in controlling her lipid levels. Physical exam revealed an ill-appearing woman complaining of abdominal pain. Bowel sounds were hypoactive, and the abdomen was diffusely tender. The serum was grossly lipemic. Laboratory studies revealed a glucose of 1321 mg/dl, amylase of 1008 mg/dl, lipase 2001 mg/dl, and triglycerides 9198 mg/dl. CT scan of the abdomen was consistent with acute pancreatitis. Her condition rapidly deteriorated with respiratory failure, requiring ventilator support, and renal failure, requiring dialysis. She was transferred to the University Hospital for plasmapheresis. One plasmpheresis treatment with continuous infusion of heparin lead to a substantial and sustained reduction of triglyceride level to less than 150 mg/dl. The patient steadily improved with decreased dependence on the ventilator, increased urine output, and decreased swelling of the pancreas. Unfortunately, the patient suffered a cardiac arrest on the sixteenth hospital day. IMPLICATIONS/DISCUSSION: Plasmapheresis has been used to treat hypertriglyceridemia when adequate diet and drug therapy fail. In this case plasmapheresis was highly effective in removingthetriglyceridesand mayhave ledto thepatient'ssteady,albeit temporary,improvement. (1) to recognize hyperammonemia due to acute valproic acid intoxication (2) to manage hyperammonemia due to acute valproic acid intoxication by understanding its mechanism.
CASE INFORMATION: The patient is a 42 year-old male who ingested 90 500 mg tablets of valproic acid following an argument with his wife. The exact time of ingestion was unclear, but was felt to be 6 to 8 hours prior to presentation. The patient's wife assumed that he was sleeping, but when found to be unresponsive again several hours later, he was brought to the emergency department. On physical examination, the patient was unresponsive, hypotensive, and without hyperreflexia or clonus on neurological examination. Laboratory values revealed normal liver function tests, hypocalcemia, valproate level of 1157 (ref 50±150 ug/ml), and an ammonia level of 270 (ref 9±33 umol/L). The patient's electrocardiogram was notable for QT prolongation. CT scan of the head and lumbar puncture were unremarkable. Intravenous fluids were administered and dopamine was initiated for blood pressure support. The patient's respiratory status declined, he was unable to protect his airway and therefore intubated. Carnitine was administered for the hyperammonemia on a daily basis. The valproic acid and ammonia levels declined over the next 48 hours and his mental status improved. He was extubated after approximately 72 hours. The patient did well and was transferred to psychiatry. IMPLICATIONS/DISCUSSION: Hyperammonemia results when valproic acid is metabolized to propionic acid, which inhibits carbamyl phosphate synthetase, the first enzyme involved in the urea cycle. Valproic acid also binds to carnitine, a molecule important in the metabolism of long-chain fatty acids and ketoacid analogues of various amino acids, thereby depleting serum concentrations of carnitine. Valproic acid also causes increased renal production of ammonia by reducing the synthesis of glutamine. Administration of exogenous carnitine is thought to decrease ammonia levels by binding to valproic acid, and also by relieving the inhibition of carbamyl phosphate synthetase and thus urea synthesis. Management of valproic acid intoxication is largely supportive. Patients who present early may benefit from gastric lavage and a single dose of activated charcoal; however, multiple-dose activated charcoal does not increase the elimination of valproic acid. Other interventions may involve blood pressure support with intravenous fluids and vasopressors, correction of electrolyte abnormalities, and correction of acid/base disorders (commonly an anion gap metabolic acidosis). Mechanical ventilation may be necessary in patients who require airway protection. In the majority of cases, this management strategy results in favorable outcomes. Hemodialysis and charcoal hemoperfusion are additional treatment options. Typically, extracorporeal removal is employed in patients with renal abnormalities, hypotension refractory to all supportive measures, severe metabolic abnormalities, active seizure, or those who are persistently comatose. However there are no controlled trials that demonstrate an improvement in outcome with these measures. year old female initially presented to her primary physician 5 years ago with a blood pressure of 159/98. Repeat readings were similar and she was started on hydralazine 25 mg. There was a modest effect from hydralazine, but control was never acheived. Four months later hydralazine was changed to losartan 50 mg. No significant lowering was obtained. Losartan was increased one month later to 100mg and two months later hydrochlorothiazide 25 mg was added. Her blood pressure stabalized to 140/90 for 6 months. Then a gradual increase in her blood pressure was noticed, so diltiazem was added. Her physical exam and lab values were unremarkable. Due to failure of multiple antihypertensive medications,a magnetic resonance arteriogram of the renal artery was conducted which revealed renal artery stenosis. An angiogram was conducted showing segments of stenosis and dilatation of the right renal artery, consistent with the diagnosis of fibromuscular dysplasia. Renal artery angioplasty was performed, and follow up blood pressure normalized on two medications. IMPLICATIONS/DISCUSSION: This is an elderly female with hypertension refractory to medications. A secondary cause was sought out with an imaging study, which showed a unilateral fibromuscular dysplasia of the right renal artery. It is unusual for a women in this age group to present with this type of secondary hypertension. Patients with fibromuscular dysplasia respond well to angioplasty. LEARNING OBJECTIVES: 1. The reader should be able to list three common causes for hypophosphatemia. 2. The reader should be able to explain why phosphate repletion is typically not be necessary for hypophosphatemia from respiratory alkalosis. CASE INFORMATION: Hyperventilation can precipitate life-threatening alkalosis and severe hypophosphatemia that does not require phosphate repletion. A 36 yo female who was status post cadaveric tracheal transplant and multiple stent placements presented with suspected stent re-stenosis, complaining of dyspnea, increased work of breathing, and inability to clear secretions. Her respiratory rate was 30±34 with normal oxygenation. Emergent bronchoscopy revealed only minimally occlusive concretions, however peri-procedure labs revealed an arterial blood pH of 7.71 and a phosphate of 0.6 mg/dL. The patient reported weakness and paresthesias in all four extremities plus arthralgias in her hands. The patient was started on a NaPO4 infusion @ 0.6 mmol/kg over 6 hrs. She was given supportive care and reassurance for her hyperventilation. After only three hours, the PO4 was 3.3 mg/dL and the infusion was stopped. Repeat ABG showed a pH of 7.49, with improvement in pCO2 from 14 to 27 mmHg. The patient's symptoms resolved. IMPLICATIONS/DISCUSSION: Severe hypophosphatemia, defined as a serum PO4 <1.5 mg/dL, has been reported in 0.22±2.15% of adult hospital admissions. It occurs more commonly in alcoholics and malnourished patients and has a variety of causes, including respiratory alkalosis. In respiratory alkalosis, carbon dioxide readily diffuses from the intracellular space resulting in higher intracellular pH. This activates glycolysis and the formation of phosphatecontaining compounds. Phosphate is used from the circulating inorganic phosphate pool, depleting extracellular stores. This process induces relative hypophosphatemia. This contrasts with metabolic alkalosis where excess extracellular bicarbonate is only poorly diffusible and does not significantly raise intracellular pH. Severe hypophosphatemia can be a lifethreatening disturbance but may not represent total body phosphate depletion or require supplementation. An understanding of the basic physiology guides therapy. 67 year old male initially seen in the emergency room five days before admission for right lower extremity redness, pain and swelling. He was given pain medication and sent home. He returned the following day and two days later with persistent symptoms and was given appropriate oral antibiotics for presumed cellulitis. An ultrasound showed no thrombosis. After his fourth ER visit he was admitted for intravenous antibiotics. Additionally, over the past three months he had noted bilateral lower extremity pain with walking over one block. Work up to date included normal resting and exercise ankle/brachial indices a CT scan of the lumbar spine, which showed disc bulges at L3-4 and L4-5 with mild spinal stenosis. When we met him, he complained of severe pain despite oral narcotics and also endorsed difficulty with his gait over the past week. On physical exam, temperature was 98.60 and the right lower extremity had diffuse anterior erythema without distinct borders. There were no breaks in the skin but onychomycosis was present on bilateral toes. There was 2+ edema on the right and none on the left. Dorsalis pedis pulse was noted only by doppler on the right, but was intact on the left. Neurologic exam revealed significant difficulty with dorsiflexion of the right foot which was presumed to be secondary to pain or possibly related to his known spinal stenosis. Labs revealed a normal white blood cell count, elevated C-reactive protein, small blood in the urine with 0±5 red cells and a creatinine slightly above his baseline. Plain radiographs were normal. Broad spectrum intravenous antibiotics, elevation and pain control were initiated at admission. After two days, the redness and swelling improved but a foot drop and pain persisted. Neurology felt his foot drop could be consistent with his known disc disease but also suggested surgical evaluation. Anterior compartment syndrome was subsequently diagnosed based on a direct compartment pressure reading of 45 mmHg. Because of the delay in diagnosis, the fact that pulses were present and that there was no tissue necrosis or abscess seen on subsequent scanning, the decision was made not to operate. The patient's pain and swelling gradually resolved over weeks but he was left with a permanent foot drop. IMPLICATIONS/DISCUSSION: Compartment syndrome is a surgical emergency that must be detected early to avoid potential serious sequelae. Causes are many and include closed trauma (most commonly), vascular injury and reperfusion, burns, prolonged surgery or traction, overexertion/exercise, and infection. It occurs most commonly in the arm or leg but can be seen in the foot, abdomen, retroperitoneum and mediastinum. Recognition depends on clinical suspicion and can be remembered as the five P's: pain (severe and out of proportion to injury); pulselessness, paralysis (excluding concomitant nerve injury); paresthesia and pallor. Laboratory may show myoglobin in the urine or elevated creatine kinase. Imaging studies may help rule out other causes but direct measurement of compartment pressures are needed for diagnosis. Also well described is the syndrome of chronic compartment syndrome, which is typically thought of as an overuse injury in runners but remains as an interesting possible explanation for this patient's claudication history. LEARNING OBJECTIVES: Early recognition and treatment of Alcohol Withdrawal Syndrome (AWS) may reduce perioperative morbidity and mortality. Intravenous Ethanol (IVE) is still used for the peri-operative management AWS. This case illustrates the importance of 1) identifying patients who require AWS treatment and using evidence-based treatments early to prevent AWS peri-operative complications, 2) exploring the use of alcohol to treat perioperative AWS, and 3) discussing optimum treatments of AWS with patients and family prior to surgery. CASE INFORMATION: T.O. is a 65 year-old man with history of hypertension, congestive heart failure and peripheral vascular disease with bilateral thigh claudication admitted for aortobifemoral bypass. Initial documentation of alcohol use consisted of an admission nursing note stating that the patient drank alcohol. After an uncomplicated surgery, he was successfully extubated and on post-operative day (POD) 2 was transferred to a general surgical bed. On POD 3, he became increasingly agitated and combative. AWS was suspected and Lorazepam 1±2 mg PRN was ordered. Over the next three days he did not improve and was returned to the SICU for re-intubation for airway protection and IV benzodiazapine treatment. After being informed of his clinical condition, the patient's son requested his father receive alcohol to prevent delirium tremens since his father had not consented to``detoxification''. A 10% ethanol drip was begun to 100 cc/h on POD 8 and on POD 9 he was successfully extubated. Although he became more alert and interactive, attempts to wean his IVE caused increased agitation. On POD 11 he was transferred to a general surgical bed, his IVE was discontinued, and he was ordered 12 ounces beer PRN. On POD 12, he continued to be confrontational and was discharged to home. IMPLICATIONS/DISCUSSION: AWS is a complex constellation of signs and symptoms related to autonomic hyperactivity in alcohol dependent patients whose alcohol intake is decreased or discontinued. Severe AWS includes life-threatening delirium tremens that may be exacerbated by the stress of surgery. Pre-or peri-operative detoxification from alcohol may also increase surgical morbidity. Symptom triggered, appropriate dosing of benzodiazapines are effective first line treatment for AWS. However, intravenous and oral alcohol use continues to be used in the perioperative period. Although multiple case reports exist detailing the use of IVE or oral alcohol to prevent withdrawal symptoms, no clinical trials exist to demonstrate its efficacy in management of peri-operative AWS. The use of IVE in the perioperative period is complicated by electrolyte disturbances, pancreatic complications, and difficulty in objectively measuring treatment efficacy. Use of oral alcohol may also reinforce unhealthy outpatient alcohol drinking behavior. Insufficient evidence-based studies exists to guide clinicians regarding the optimum management of peri-operative AWS and until such time, alcohol treatments will continue to be used.
Center, San Jose, CA (Tracking ID #75786) LEARNING OBJECTIVES: To diagnose nephrotic syndrome in a diabetic. To assess the clinical complications of nephrotic syndrome. To recognize the clinical challenges of the obese patient. CASE INFORMATION: A 52 year old morbidly obese male presented to his primary care physician with four days of facial and upper extremity edema. He had developed orthopnea, paroxysmal nocturnal dyspnea, and dyspnea with exertion. His past medical history included poorly controlled diabetes, chronic lower extremity edema, and untreated obstructive sleep apnea. An initial chest CT demonstrated pleural effusions and a soft tissue mediastinal mass thought to be compressing the superior vena cava. Mediastinoscopy of the mass demonstrated no tissue findings; a repeat chest CT, with dye injected in both arms simultaneously, revealed the mass was likely lipomatosis. Further workup of the pleural effusion included an unremarkable echocardiogram and an ultrasound-guided thoracentesis. The pleural effusion was transudative and negative for cytology. The patient's urine revealed 3+ proteinuria and oval fat bodies, and a spot urine protein/creatinine ratio demonstrated over 4 grams of protein.
These findings, along with hypoalbuminemia, were consistent with nephrotic syndrome. The patient's symptoms improved with furosemide diuresis. However, the patient was rehospitalized a week later, complaining of acute shortness of breath. He was already known to be hypoxic, probably from obstructive sleep apnea. A ventilation perfusion scan was performed, and demonstrated a new lingular mismatch of intermediate probability for a pulmonary embolism. The patient stayed in the hospital until five days of oral coumadin overlapped with a heparin drip, as his obesity prevented accurate dosing of low-molecular-weight heparin. IMPLICATIONS/DISCUSSION: Diabetes is a common cause of nephrotic syndrome. The diagnosis can be made by quantification of urine protein excretion; oval fat bodies suggest hyperlipidemia from a fall in oncotic pressure and decreased clearance of very low density lipoproteins. Clinical symptoms include peripheral edema, pleural effusions, and hypercoagulable conditions like pulmonary embolism. The pathophysiology of these clinical symptoms may be due to sodium retention from renal disease and hemostatic abnormalities such as urinary loss of antithrombin III. The diagnostic workup was made more difficult for this patient due to complicating conditions related to his morbid obesity, such as the lipomatosis and obstructive sleep apnea. As obesity increasingly becomes a public health epidemic, generalists need to recognize the extensive clinical challenges for the obese patient. LEARNING OBJECTIVES: 1) To discuss the differential diagnosis of hypoglycemia.
2) To recognize that accidental sulfonylurea ingestion is a cause of hypoglycemia in adult nondiabetics.
3) To recognize that polypharmacy issues are common and are not limited to a patient's own prescriptions. CASE INFORMATION: An 80 year old male on multiple medications presented with severe hypoglycemia. He had no prior history of diabetes and never used alcohol. During his initial hospitalization, the patient was found to be hypogonadal and diagnosed with cholangitis. Diagnosis was based on hypothermia, choledocholithiasis and ductal dilatation although blood cultures were negative and initially there was no leukocytosis. Cortisol levels were normal and no abdominal tumors were found by CT. Insulin levels and c-peptide levels were elevated. The patient's medications did not include any associated with hyperinsulinism. The patient's glucose normalized after two days of treatment with a glucose drip, antibiotics, and stone extraction with sphincterotomy. Upon discharge, the leading diagnosis was sepsis-induced hypoglycemia. The patient was rehospitalized five months later with another episode of hypoglycemia. A sulfonylurea level was sent and returned positive for glipizide. It was then discovered that the patient was erroneously taking his wife's glipizide. The patient has had no further episodes of hypoglycemia. IMPLICATIONS/DISCUSSION: The differential diagnosis of hypoglycemia includes hormone deficiencies, enzyme defects, acquired liver disease, malnutrition, sepsis, malignancy, medications, and hyperinsulinism. Hyperinsulinism can be caused by sepsis, insulinoma, autoantibodies and medications. These medications include exogenous insulin, sulfonylureas and other sulfa drugs, and also quinine and pentamidine. Hypoglycemia due to accidental oral sulfonylurea ingestion in non-diabetics is more common in the pediatric rather than the internal medicine literature. Drug-induced hypoglycemia should be considered whenever the patient has potential access to hypoglycemia-inducing agents. Polypharmacy is a wellrecognized cause of adverse drug events among the elderly. However, estimates of polypharmacy are limited to an individual's medications obtained by prescription or overthe-counter. It should be recognized that individuals with multiple medications may inadvertently consume medications other than their own.
A CASE OF ASCITES AND UNILATERAL LEG SWELLING. B. Taqui 1 , D. Oxman 1 , L. Kaplan 1 ; 1 Temple University, Philadelphia, PA (Tracking ID #76436)
LEARNING OBJECTIVES: 1. Recognize iliac vein compression (May-Thurner) syndrome as rare cause of left leg swelling in patient with negative venous dopplers 2. Recognize potential for endovascular damage and thrombosis resulting from condition 3. Recognize endovascular stenting as potential cure CASE INFORMATION: 38 year old Caucasian male with history of alcohol abuse presented with three days of abdominal pain and increasing abdominal girth. Serum lipase was markedly elevated and paracentesis showed ascitic fluid consistent with pancreatic ascites. He was started on total parenteral nutrition. He subsequently developed left leg and scrotal swelling. Lower extremity venous duplex imaging was negative for deep venous thrombosis, but contrast venography revealed compression of the iliac vein by the right iliac artery. The patient underwent endovascular stenting of the femoral vein with complete resolution of leg and scrotal swelling. His swelling has not recurred since then. His pancreatitis and ascites also gradually resolved. IMPLICATIONS/DISCUSSION: The potential for obstruction of the left common iliac vein by the overlying right common iliac artery against the pelvic brim was first noted by Virchow. Later, May and Thurner described the``iliac compression syndrome.'' In this patient, it was believed that the ascites coupled with intravascular volume burden of parenteral nutrition led to the patients symptoms. The syndrome, which can be acute or chronic, predisposes patients to thrombosis. Thus, contrast venography should be considered in patients with unilateral left lower extremity swelling and negative duplex imaging. Therapy with endovascular stenting of femoral vein offers patients the chance of cure. angiogram showed a beaded segmental narrowing involving the right internal carotid artery from the bifurcation to the base of the skull. Fibromuscular dysplasia was considered, but a repeat angiogram 3 days later showed that the vessels had returned to normal. The patient was discharged on a calcium channel blocker and coumadin. IMPLICATIONS/DISCUSSION: Stroke in young adults is usually due to cardiac embolism, dissection of arteries, vasculitis, and conditions associated with hypercoagulable states. Reversible cerebral segmental vasoconstriction' also known as Call-Fleming syndrome is a rare cause of stroke or TIA and is characterized angiographically by multiple areas of reversible segmental arterial narrowing. Symptom onset can be spontaneous, but has also been associated with pregnancy, migraine, and use of sympathomimetic drugs. Most patients present with headache. Symptoms can last from few minutes to 6 months. The mechanism of vascular narrowing is not understood. Repeat angiogram may be needed to demonstrate the reversible nature of the angiographic changes. LEARNING OBJECTIVES: 1. Consider scurvy in the differential diagnosis when patients appear to have vasculitis, especially in populations at high risk. 2. Appreciate that early diagnosis, facilitated by taking a good dietary history, is important, as scurvy is potentially fatal. CASE INFORMATION: A 54-year-old schizophrenic man presented to rheumatology clinic with a vasculitis-like skin rash, joint swelling and tenderness, edema of a lower extremity and poor dentition. He was a chronic smoker and had a history of alcoholism. He was not on any medication other than haloperidol on a prn basis. An extensive work up for vasculitis was negative. The patient was referred for biopsy of his skin lesions and scurvy was clinically suspected and confirmed with a low vitamin-C level. Biopsy did not show evidence of vasculitis. The patient's symptoms and signs resolved rapidly with oral vitamin-C. IMPLICATIONS/DISCUSSION: Scurvy is a disorder caused by vitamin C deficiency. Its protean manifestations include petechiae, ecchymoses, hyperkeratoses, arthralgias, malaise, fatigue, impaired wound healing, hair with corkscrew deformity and swollen, red purple gums. Our case is unusual because our patient presented predominantly with joint symptoms and a vasculitic-appearing rash. The diagnosis was missed by the rheumatologist, but was later made based on a dermatologist's suspicion. Scurvy is rare in industrialized nations but can occur in certain high-risk groups because of poor intake of vegetables and fruits or an increased need for vitamin C. High-risk groups include the elderly, pregnant and lactating women, infants, the urban poor, the malnourished, food faddists, alcoholics and those with cancer. Scurvy has also been reported in psychiatric patients with depression, schizophrenia and anorexia nervosa. Schizophrenic patients are predisposed to abnormal dietary patterns, food fads and self neglect which subject them to a risk of scurvy. Our patient was at risk because of underlying schizophrenia, a history of alcoholism, and malnutrition, which predisposed him to poor intake. His smoking history probably contributed to his deficiency by increasing vitamin C needs. LEARNING OBJECTIVES: Recognize that pulmonary edema is one of the many causes of localized upper lobe infiltrates, especially when patients at risk for heart failure have been in dependent positions for prolonged periods of time. CASE INFORMATION: A 52-year old man with type-2 diabetes and severe atherosclerotic cardiovascular disease, who had had a toe amputated for an infected ulcer two days before, was admitted with a 3-day history of orthopnea and dyspnea on exertion. He had been on prolonged bed rest to facilitate healing of his foot. On admission he was hypoxic, with coarse crackles at the apices of the lungs and decreased breath sounds at the lung bases. There was no elevation of JVP and the rest of the cardiovascular exam was unremarkable. CXR showed upper lobe alveolar shadows bilaterally with bilateral pleural effusions and Kerley B lines. Spiral CT of the chest done to rule out pulmonary embolism showed bilateral pleural effusions and bilateral nodular upper lobe infiltrates without evidence of embolism. Atypical pulmonary edema was suspected and the patient was propped up in bed, and given furosemide. His symptoms and CXR abnormalities resolved in 2 days. Natriuretic peptide was found to be markedly elevated at 857 pg/ml and an echocardiogram confirmed LV dysfunction with an EF of 20%. The atypical presentation of this patients pulmonary edema was explained by a gravitational effect on the pulmonary circulation, caused by his prolonged assumption of the Trendelenberg position as part of the management of his diabetic foot problems. IMPLICATIONS/DISCUSSION: Pulmonary edema presents classically with bilateral parahilar alveolar shadows producing a bat wing appearance on CXR. In situations characterized by gravitational effects or alterations in lung perfusion, as in COPD or pulmonary embolism, pulmonary edema may present in less typical ways. Acute mitral regurgitation has been reported as causing right upper lobe pulmonary edema and unilateral pulmonary edema has been attributed to the gravitational effects of posture. These atypical presentations can be confused with pneumonia and aspiration. To make the correct diagnosis in patients with atypical CXR's, clinicians must rely on other clinical and radiologic signs of heart failure and take a careful history. Workup revealed pulmonary infiltrates, anemia with left-shift, and a sputum KOH preparation demonstrating round, non-staining elements felt to be consistent with blastomycosis. After four days of intravenous amphotericin, the patient developed progressive pulmonary infiltrates requiring ventilatory support. Subsequent evaluation with broncho-alveolar lavage revealed alveolar hemorrhage and red cell ghosts within macrophages; these findings were consistent with a vasculitic rather than an infectious disease process. Eleven years later, the patient presented with hemoptysis, fatigue and arthralgias leading to initiation of immunosuppresants for presumed autoimmune disease. After discharge, however, sputum cultures grew Blastomyces dermatitidis, and followup chest CT revealed bibasilar consolidation with cavitary changes prompting a reduction in steroid dose and initiation of anti-fungal therapy. IMPLICATIONS/DISCUSSION: The literature contains multiple cases of blastomycosis mimicking a variety of illnesses, from neoplastic to autoimmune disease. Certain confounding factors can limit the differential diagnosis: (i) the indolent course of blastomycosis with nonspecific presenting symptoms may catch the unwary off-guard, (ii) blastomycosis may present in a manner similar to a variety of disease processes, (iii) and finally, blastomycosis may be superimposed upon another illness, clouding both diagnosis and management.
ACUTE TUBULOINTERSTITIAL NEPHRITIS ASSOCIATED WITH CLOPIDOGREL. N. Palanichamy 1 , C. Kumar 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking  ID #76611) LEARNING OBJECTIVES: To describe clopidogrel induced acute renal failure, a previously unreported drug reaction. CASE INFORMATION: An 89 year-old white male presented with hematuria and acute renal failure. His Medications were aspirin, plavix, metaprolol, prinivil, lasix and flomax. He was placed on clopidogrel two weeks prior. He stopped the drug the day before admission. His creatinine level was 3.9 mg/dl (baseline creatinine 1.3 mg/dl). Urinalysis showed proteinuria, leukocytes and erythrocytes. 24-hour protein excretion was 756 mg. A renal ultrasound showed no obstruction. Over the course of the next week his renal function deteriorated (creat 8.9 mg/ dl). Renal biopsy showed a widened, edematous interstitium with moderate lymphocytosis, plasma cells and regenerative tubules and was compatible with tubulointerstitial nephritis (AIN). Dialysis was started and the patient was placed on steroids. With treatment the creatinine decreased to 2.6 mg/dl, the dialysis stopped and the steroids tapered. Based on the history and presentation clopidogrel was thought to be the etiology of the nephritis in this case. IMPLICATIONS/DISCUSSION: To our knowledge, this is the first case of clopidogrel induced acute tubulointerstitial nephritis. The rapid deterioration in the renal function two weeks after starting clopidogrel and the absence of confounding factors suggests the diagnosis in the presence of the biopsy findings. Clopidogrel affects platelet aggregation by inhibiting the ADP receptor and the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex. The precise mechanism of the renal dysfunction is unknown but, like ticlopidine, a chemically similar drug with known adverse renal effects, may be caused by cell-mediated injury. Guerciolini et al. (1985) demonstrated a positive lymphocyte stimulation test in explaining agranulocytosis associated with ticlopidine. The other known mechanism of drug induced AIN, antibody mediated injury and circulating immune complexes are less likely causes insofar as the immunoflorescent studies in our case were nonspecific. In addition to the more commonly known side effects of clopidogrel physicians now need to realize that in rare instances it may cause renal dysfunction. LEARNING OBJECTIVES: Think about malignancy in the differential diagnosis of leukemoid reaction. CASE INFORMATION: A 68 year old Caucasian male with no significant past medical history presented with complaints of swelling of both legs, vague abdominal pain, watery diarrhea and increased urinary frequency of 6 weeks duration. He also reported a weight gain of 50 pounds over the past 6 months. On physical examination, he was afebrile, pulse was 81/min and blood pressure was 145/82 mm Hg. He had pallor and massive lower extremity edema extending upto the scrotum. There was no lymphadenopathy. Abdominal examination revealed a large hard mass extending anteriorly from the right lumbar region to the opposite flank and pelvis. Laboratory data-Hb 8.7gm/dl, Total WBC 74,800/cu mm, Differential±40% neutrophils, 56% bands and 4% monocytes, platelets 195 K/cu mm, LAP score 217. Urinalysis was normal. Alkaline phosphatase 144 IU/L, total serum protein 6.3 g/dl, C-reactive protein 13.3 mg/dl. Both blood and urine cultures were negative. Peripheral smear±leukocytosis, mature granulocytes and band forms, no blasts. CT scan of the abdomen showed a large, lobulated retroperitoneal mass extending to the posterior aspect of the right kidney, measuring 9Â8Â15 cm and another left sided pelvic mass, 14Â10Â17 cm, contiguous with the urinary bladder and pressing on the IVC. Multiple low-density lesions were also noted in the liver and spleen. Bone marrow biopsy revealed a myeloid predominant hypercellular marrow, no blast cells. A CT guided biopsy from the hypodense lesion in the liver revealed a metastatic, poorly differentiated malignant fibrous histiocytoma (MFH), pleomorphic type, positive for vimentin and negative for cytokeratin and alpha-fetoprotein on immunostaining. The IL-6 and IL-2 levels were normal. The patient was started on chemotherapy with cyclophosphamide, dexamethasone, adriamycin and dacarbazine. He developed disseminated intravascular coagulation on the 5th day of chemotherapy and expired. IMPLICATIONS/DISCUSSION: This case is unique because of the association of malignant fibrous histiocytoma with leukemoid reaction (LR). Several cases of soft tissue sarcomas with leukocytosis have been reported, but less than 15 cases of MFH with this phenomenon were found in the review of literature. The relationship between cytokine levels and LR has not been consistently demonstrated in the cases reported. Although, in our patient, serum cytokine levels were noninformative and histologically, the metastatic component of the tumor was not significantly different from the``non-inflammatory'' fibroxanthosarcomas, the absence of any other cause of leukocytosis highlights its association with MFH. The prognostic significance of leukocytosis with these tumors is yet to be defined, an added survival benefit of the inflammatory component is debatable. year-old male with a history of 30 pack-year tobacco use and positive tuberculin test, presented with nonproductive cough and night sweats for several months. Physical exam was normal. Serum immunofixation tests and acid fast stains of sputum were normal. Pulmonary function test (PFTs) showed moderate obstructive airway disease, and transthoracic echocardiogram was unremarkable. Chest x-ray (CXR) exam revealed a rounded opacity in the right lung apex. Computed tomography (CT) showed a 2.5 cm right apical mass and multiple small nodules seen in both lung fields. Transbronchial biopsy of the apical mass was consistent with pulmonary nodular amyloidosis. Follow-up CT imaging showed no significant change after one year. Case 2: A 73 year-old Caucasian male with a history of 40 pack-year tobacco use, had a CXR for preoperative evaluation for blepharoplasty. He denied cough, weight loss, and night sweats. Physical exam and laboratories were all within normal limits. PFTs were consistent with mild obstructive airway disease. CXR exam showed an incidental finding of a round density in the right middle lung field. CT revealed a 1.5 cm round opacity in the right middle lobe. Transbronchial lung biopsy of the mass was consistent with pulmonary nodular amyloidosis. Follow-up CT imaging showed no significant change after one year. IMPLICATIONS/DISCUSSION: Pulmonay amyloidosis may present in one of three ways:
(1) diffuse, interstitial process associated with primary, systemic amyloidosis and a median survival of 16 months after diagnosis; (2) Endobronchial amyloid causing obstruction that is usually amenable to treatment with laser therapy; and (3) isolated, pulmonary nodular amyloidosis that is not associated with systemic disease and follows a benign course. Our two cases are examples of pulmonary nodular amyloidosis. Pulmonary disease is uncovered as an incidental finding with one or more nodular opacities seen on chest imaging suggesting a malignant process. Transbronchial lung biopsy may establish the diagnosis with light microscopy showing amorphous, extracellular deposits that stain pink with hematoxylin and eosin. Under polarized light, amyloid produces apple-green birefringence. In our two cases of primary nodular amyloidosis, elevated monoclonal proteins were not found, consistent with the absence of systemic involvement in this presentation of disease. Further, follow-up chest imaging has revealed no change in the pulmonary nodular opacities, reflecting the benign course of this disease. year-old homeless male with a history of untreated Schizophrenia was brought in for evaluation of fatigue and altered mental status. On presentation, the patient was noted to be disheveled and confused with initial BP 128/61, P88, RR 17, and T99.9. Physical exam was remarkable for numerous nits on hair and clothing, and skin with innumerate number of excoriated, hemorrhagic papules. Rectal exam revealed no masses but guaiac negative brownish stool. Initial laboratories included: WBC 13.7, HGB 3.7, HCT 11.5, PLT 375, MCV 72, RDW 16, and 71% PMN. Gastric lavage was negative for occult blood. Computed tomography of head and a diagnostic lumbar puncture were normal. Serum anemia work-up was remarkable for low ferritin (11) and iron (76). Folate and vitamin B12 levels were normal. The remaining laboratories, imaging studies, and cultures were all within normal limits. He was treated for pediculosis with permethrin and received blood transfusion with an appropriate increase in blood counts. His mental status and leukocytosis significantly improved as pediculosis and anemia were treated. Blood counts has remained stable after treating pediculosis. IMPLICATIONS/DISCUSSION: The 3 most common arthropod exoparasites in humans are Pediculus capitis (head louse), Pediculus corporis (body louse), and Phthirus pubis (pubic louse). They belong to the order Anoplura, the sucking lice that feed on blood approximately 5 times per day for approximately 40 minutes each time. Hemorrhagic macules or papules develop at the sites of feeding lice, and vertical excoriations due to scratching and postinflammatory hyperpigmentation are common (aka, vagabond's disease). These lice can be transmitted directly from person to person. They are also a potential vector for transmission of diseases such as typhus, louse-borne relapsing fever, and endocarditis. Bacterial superinfection at the sites of infestation is also common. Zoonotic pediculosis (louse infestation) has been reported as a major cause of anemia in domestic animals. This possibility, however, has not been reported in humans. Our case presents a homeless male wtih severe iron-deficient anemia associated with chronic pediculosis. This case supports the hypothesis that louse infestation is an uncommon cause of anemia in humans. To our knowledge this is the first case report of its kind (Internet and Medline search from 1960 to present). year-old woman presented with chest pain and syncope after exertion. The chest tightness was associated with shortness of breath, nausea, and diaphoresis. She first experienced exertional angina at age 18 during military training. The episodes had increased in frequency since. Her vital signs were normal. The cardiac examination was normal with the exception of a a left-sided heave and a loud, persistently split S2 that widened with inspiration. An electrocardiogram demonstrated normal sinus rhythm, right axis deviation, and T-wave inversion in the inferior and anterior leads. Three troponin assays were negative. Echocardiography revealed right ventricular enlargement, dilated pulmonary arteries with pressures of 50 mm Hg, normal chamber sizes, normal valves, and no septal defects. A ventilation/perfusion scan, ANA and RF were normal. A right heart catheterization confirmed pulmonary artery hypertension with a systolic pressure of 70 mmHg and a normal wedge pressure. A bubble study looking for small septal defects was normal. Angiography confirmed the presence of dilated pulmonary arteries without evidence of thromboembolic disease. IMPLICATIONS/DISCUSSION: Primary pulmonary hypertension is a diagnosis of exclusion, with a prevalence of one to two people per million. Because of its low incidence and protean nature, the most cost effective strategy is to exclude common etiologies of pulmonary hypertension such as pulmonary embolism, valve disease, lupus, CREST and left heart failure. Right heart catheterization is the best diagnostic test. The median survival after diagnosis is 2.5 years. Intravenous epoprostenol is the drug of choice, but oral vasodilators are also used. Anticoagulation has been shown to prolong life; some patients may also respond to oral vasodilators. Anticoagulation improves mortality by decreasing thombosis and thromboembolism. This patient was started on a trial of oral bosentan and warfarin. Since primary pulmonary hypertension has no cure, an exhaustive search for secondary causes is warranted in any patient with this diagnosis. Other than these findings, her neurological examination was unremarkable. MRI of the head revealed multiple foci of hyperintensities in the white matter; bilateral enhancement of the tentorium and cranial nerves III, V, VII; and unremarkable orbits (Fig 1,2,3) . Cerebrospinal fluid (CSF) revealed a protein level of 600 mg/dl; WBC 210/uL (lymphocytes 95%) and glucose 91 mg/dl. Serum and CSF were positive for Lyme antibodies on ELISA. The result was confirmed by western blot assay on the CSF. The patient was started on intravenous ceftriaxone 2 gm every 24 hrs. On the 4th day of antibiotic therapy, she developed left sided LMN facial palsy. After 4 weeks, a repeat CSF study showed significant improvement: the protein level fell to 82 mg/dl and WBC count to 33/ul. Six weeks after ceftriaxone therapy, the diplopia, trigeminal neuralgia and facial palsy resolved completely. IMPLICATIONS/DISCUSSION: Though facial palsy is a relatively common presentation of Lyme disease, multiple cranial neuropathies may be the initial manifestation and must be borne in mind. The diagnosis can be easily missed in such cases without any antecedent history of tick bite or rash. A history of travel or residence in an endemic area is a useful clue. In addition, MRI may play an important role in the diagnosis. Our patient's MRI at admission revealed bilateral enhancement of meninges and cranial nerves III, V and VII indicating evidence of inflammation that preceded the full clinical expression. To our knowledge, multiple cranial neuropathies as the initial manifestation and evidence of gadolinium enhanced MRI lesions of the involved cranial nerves have been rarely reported. Recognizing these subtle imaging findings, combined with high clinical suspicion is the key to diagnosis and allows early treatment which can help prevent further complications.
WALDENSTROM'S MACROGLOBULINEMIA AND HEART FAILURE. R. Pearl 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77006)
LEARNING OBJECTIVES: 1. Use the physical examination to determine cost-effective use of echocardiography 2. Use an understanding of physiology to guide diagnostic procedures 3. Diagnose infiltrative cardiomyopathy. CASE INFORMATION: A 73 year-old man presented with right-sided chest pain and shortness of breath. The shortness of breath had worsened over two weeks, with new onset three-pillow orthopnea and lower extremity edema. His vital signs were normal. He had a soft S1, a normal S2, and an S4. Although he had bilateral crackles and an elevated JVP, there was no S3 or inferiorly displaced PMI. His edema was initially ascribed to hypo-albuminemia (Albumin = 2.) although his pit recovery time was greater than 90 seconds. Based upon a suspicion of a restrictive cardiomyopathy, an echocardiogram was obtained. This showed left ventricular hypertrophy and an E-to-A mitral-flow reversal consistent with diastolic dysfunction. His ejection fraction was 20% despite ventricular hypertrophy. A bone marrow biopsy was performed that confirmed the diagnosis of Waldenstrom's macroglobulinemia. IMPLICATIONS/DISCUSSION: The physical examination in concert with an understanding of the physiology underlying disease is the foundation for cost-effective identification of occult diagnoses. The silent S1 suggested a low ejection fraction, and while the S4 without an S3 or inferiorly displaced PMI suggested a non-compliant hypertrophic ventricle. Both of these conclusions were confirmed by echo. A low ejection fraction despite ventricular hypertrophy suggested an infiltration of the ventricular wall. LaPlace's law states that wall tension is a function of the pressure and radius of a chamber, divided by the thickness of the chamber's wall. Because wall tension is proportional to afterload (like a spring, the ventricle must overcome the weight that has stretched it) a thick ventricle wall should lower wall tension, thereby decreasing the ventricular afterload. The ejection fraction should increase proportional with the wall thickness. Failure to do so suggests that the thickness is due to infiltration with a non-contractile substance. The history of chronic renal insufficiency and anemia suggested the diagnosis of amyloidosis due to Waldenstrom's macroglobulinemia. This clue was instrumental in prompting the bone marrow biopsy that confirming the diagnosis. The finding of a speckled myocardium on echocardiogram is a classic, though late state finding of the disease. A 54-year-old male with a history of leukocytoclastic vasculitis was noted to have a diffuse erythematous rash while being admitted for an unrelated endoscopic procedure. The rash first developed two weeks prior in the patient's right groin and gradually spread to include both legs and his right arm. It had not responded to a course of levofloxacin or over the counter topical ointments. In the month prior to admission the patient had been tapered from 15 milligrams to 12.5 milligrams of prednisone. On admission the patient had warmth, erythema, and mild tenderness to the affected areas. Numerous 1 to 5 millimeter pustules also were present on the erythematous base. The patient was afebrile and had a mildly elevated white count of 11 thousand. Gram stain and culture of the pustules revealed no bacteria. Fungal elements were not seen. A punch biopsy was taken and returned consistent with pustular psoriasis. Acitretin therapy was initiated. IMPLICATIONS/DISCUSSION: Psoriasis is a relatively common dermatologic disorder that occurs in approximately 1% of the North American population. While generally benign, psoriasis can present in an atypical and potentially life threatening manner. Pustular psoriasis can occur in varying degrees of severity with distinct subtypes. These include the relatively limited form of palmoplantar pustulosis to the more severe generalized von Zumbush type. If untreated, disruption of the skin's barrier can lead to large amounts of volume loss and secondary bacterial infections. Systemic involvement including arthritis, heart, liver, and kidney failure has been associated with advanced cases. Triggering events are often unknown, but there has been an association with pregnancy, concurrent infections, and medications. Given the nonspecific somatic complaints and laboratory abnormalities, successful diagnosis requires recognition of the pustular areas on the erythematous base. Our patient had two associated medication risk factors of hydroxychloroquine therapy and corticosteroid withdrawal that likely acted as the inciting event. IMPLICATIONS/DISCUSSION: t is a challenge to manage pregnant patients who require anticoagulation for prosthetic valves. Coumadin is the drug of choice for anticoagulation, however because it freely crosses the placenta it is not considered safe to use in pregnant patients. The teratogenic effects of coumadin usually occur between the sixth and ninth weeks of gestation. The most common abnormalities occur in bone and cartilage development. In the second and third trimesters one can develop abnormalities of the fetal central nervous system, optic atrophy, microcephaly, mental retardation, spastisity and hypotonia. Unfractionated heparin does not cross the placenta because of its high molecular weight. Since it does not cross the transplacental barrier it lacks teratogenic effects and will not anticoagulate the fetus. Low molecular weight heparin is another anticoagulant that should be considered in these patients. However, its use in pregnancy is not well studied. The ideal treatment for a pregnant woman requiring anticoagulation would be to switch from coumadin to heparin prior to conception. The patient shold be hospitalized until she is therapeutic on heparin. The three approaches that are recommended include: 1. Dose adjusted unfractionated heparin throughout pregnancy. Heparin is given subcutaneously every 12 hours and then dose adjusted to maintain two times the normal PTT, or anti-Xa levels of 0.35 to 0.70 U/ml. 2. Dose adjusted low molecular weight heparin throughout the pregnancy so as to keep a four hour post injection anti-Xa level at 1.0 U/ml. 3. Unfractionated or low molecular weight heparin therapy until the thirteenth week, with a change to coumadin until the middle of the third trimester and then restarted on unfractionated or low molecular weight heparin until delivery. LEARNING OBJECTIVES: 1) Recognize the cutaneous manifestations of sarcoidosis 2) Review the differential diagnosis of a nodular cutaneous lesion in a immigrant from subsaharan africa 3)Review the pathophysiology of cutaneous sarcoidosis CASE INFORMATION: A healthy 21 year old South Sudanese woman, 4 weeks post-partum, who had spent 2 years in an Ethiopian refugee camp before immigrating to the U.S one year ago, presented with a rash on her left cheek of over 2 years duration. She had noticed the rash first back in Sudan and had persisted during her refugee camp stay. She reported that the rash had been non-pruritic, non-exudative, indolent and asymptomatic. She had no history of photosensitivity, exposure to chemicals or insect bite. A week's trial of anti-fungal topical application was unsuccessful. Her recent pregnancy was uneventful including negative HIV, Hep.BSAg and VDRL serology. Her PMH was negative for major medical illnesses and prior eruptive lesions. She did have a history of positive PPD 1 year ago with negative CXR following immigration to the U.S. She had not been treated with anti-tuberculous agents. Her ROS was otherwise unremarkable. Exam revealed a hyperpigmented nodular lesion approximately 1cm wide and 4 cms long in a semi-circular distribution over the left malar area. The rash was further noted to be non-tender, normothermic with mild induration. Physical exam revealed a healthy 21 year old woman with normal cardiac, pulmonary, gastrointestinal and neurological exam, with no evidence of lymphadenopathy and a similar rash elsewhere. Her CBC was within normal limits. Given the patient's ethnicity and travel history, leishmaniasis and cutaneous mycobacterial infections were included in the differential. Consideration was also given to Sarcoid and variant discoid lupus. (Syphilis was considered unlikely, given recent negative serology). A diagnostic excisional biopsy revealed sarcoid-like granulomas surrounded by mild chronic lymphocytic infiltration. Special stains (including AFB, giemsa, PAS, Fite, Giemza and warthin-starry) did not reveal pathogenic microorganisms. Patient was initiated on a local topical steroid application (clobetasol propionate) for two weeks with dramatic resolution of the rash. A diagnosis of Sarcoidosis was therefore made by exclusion and dramatic response to treatment. IMPLICATIONS/DISCUSSION: The protean manifestations of Sarcoidosis include a) Erythema nodosum (most common, although non-specific lesion). b)Lupus pernio (classically involving the rim of the nose and associated with infiltration of the nasal mucosa causing ulcerations or even fatal airway obstructions). c) Papules, nodules or plaques (as seen in the above patient). d) Psoriasis like rash. e) lesions in scars and tattoos. The learning objectives of this presentation are to review the cutaneous manifestations of Sarcoidosis, the differential diagnosis of nodular cutaneous lesions in an imigrant population and the pathophysiology of Cutaneous Sarcoidosis. Considering the growing immigrant population in the U.S, the differential of an indolent chronic rash should include parasitic infestations. However, in patients of African decent, the extremely variable masquerading rash of sarcoidosis should be considered as an important part of the differential. Hemochromatosis is the most common adult genetic disorder. Early diagnosis enables rapid treatment and an improved prognosis. The first clinical manifestation is represented by arthropathy in 45% of the cases. The articular features of are often nonspecific and associated with a delay in diagnosis that may compromise outcome. Treatment of the arthritis with analgesics is often disappointing, but identification of the underlying disease permits institution of life-saving phlebotomy therapy. The aim of this report is to highlight the fact that patients with premature osteoarthritis should alert the physician to screen for hemochromatosis in order to formulate the correct diagnosis before the development of severe internal organ involvement. MRI of the head was done to rule out pituitary masses and was normal. After extensive review it was found that she received an injection of Kenalog 40 mg to the right shoulder from another MD six weeks prior to presentation. Without any other exogenous sources of steroid, it was concluded the pt had experienced cushingoid symptoms from the Kenalog injection followed by secondary adrenocortical insufficiency at presentation. IMPLICATIONS/DISCUSSION: Therapy with pharmacological doses of glucocorticoids is the most frequent cause of secondary adrenocortical insufficiency. The usual presentation is similar to that of primary adrenocortical insufficiency, with two important exceptions. Since pituitary secretion of ACTH is deficient, the characteristic hyperpigmentation of Addisons disease is absent. The clinical features of mineralocorticoid deficiency are usually absent in the unstressed state, and therefore volume depletion, dehydration, and electrolyte abnormalities are usually absent. Hypotension is also less severe, except in acute presentations. Hyponatremia can be present, and is usually due to water retention and inability to excrete a water load rather than to sodium loss. The clinical features of ACTH and glucocorticoid deficiency are nonspecific and consist predominantly of weakness, lethargy, easy fatigue, anorexia, nausea, and occasionally, vomiting. Patients may also describe arthralgias, myalgias, and exacerbation of allergic responses. With acute decompensation, severe hypotension or shock may occur and be unresponsive to vasopressors unless glucocorticoids are administered. At that time he also described shortness of breath, insomnia, anorexia, and a sense of anxiety. The exam revealed an agitated but otherwise well-appearing man with tachycardia. Mr J had a positive urine cocaine screen. He was admitted and ruled-out for myocaridal infarction (MI) with serial troponins and electrocardiogram. Two months later, Mr J returned with similar complaints. Again his urine was positive for cocaine and his serum was negative for troponins. Over the next three months he returned to the ED four more times with a new resting tremor and a weight loss of over sixty pounds. Cocaine screens remained positive. The patient refused treatment for substance abuse and expressed dissatisfaction with the quality of care he received. Finally in September of 2002 a Thyroid work-up was performed, demonstrating a TSH <0.1 and a free thyroxine >6.5. A radioiodide uptake scan confirmed the diagnosis of Graves' disease. The patient had symptomatic relief with beta blockade and propylthiouracil. He is currently scheduled for thyroid ablation. IMPLICATIONS/DISCUSSION: An estimated 1.5 million Americans (0.7%) abuse cocaine chronically. In 1998, cocaine contributed to Emergency Department visits on 152,000 occasions (1). Cocaine abuse is a potentially life-long chronic disease. Acute cocaine intoxication may mimic other diseases, preventing or delaying their diagnosis. An easy explanation for Mr. J's tremor, weight loss and agitation were already available, thus a thyroid work-up was delayed. Cocaine abuse may draw the physician's attention to conditions caused by chronic abuse rather than unrelated medical problems. MI should be ruled out, but not without considering hyperthyroidism. In one study of 165 patients with cocaine intoxication and psychotic symptoms, only 18% were given the proper diagnosis of schizophrenia on the initial evaluation (2) . Finally, some physicians may perceive substance abuse as a primary and overriding health care problem. Mr. J did not wish to address his cocaine abuse and still rightly expected treatment of his other medical problems. In conclusion, many misperceptions may cloud the diagnostic process with patients who abuse cocaine: confusion of symptoms with drug effects, distraction from unrelated medical conditions and prioritizing abstinence above other medical care. LEARNING OBJECTIVES: Recognize the potential for endocarditis after an endometrial sampling procedure. CASE INFORMATION: Antibiotic prophylaxis is not recommended prior to endometrial sampling procedures or even dilation and curettage because of the very low risk of infectious complications. We report a case of acute bacterial endocarditis caused by Streptococcus agalactiae following endometrial sampling in a woman with no known valvular abnormalities. A 59-year-old woman with a history of abnormal uterine bleeding who had undergone office based endometrial biopsy about two weeks earlier was found unresponsive at home and was brought to the hospital. She was noted to have a temperature of 102.5 degrees Fahrenheit; she was awake, but did not speak and withdrew her left side more than her right side to painful stimuli. She also had petechiae in her conjunctiva, fingers and toes. Laboratory evaluation showed white blood cell count of 21,400 cells/mm3, normal electrolytes and glucose, and normal liver enzymes. CT scan of the head was normal at presentation. On hospital Day 3, blood cultures from admission grew beta-hemolytic Group B Streptococcus from all samples. Transthoracic and transesophageal echocardiograms demonstrated a 4Â3 cm lesion on the posterior leaflet of the mitral valve with moderate mitral regurgitation. Repeat CT of the head demonstrated a large left middle cerebral artery infarction, but no evidence of abscess. The patient responded to antibiotics, but died of complications before valve replacement surgery could be performed. IMPLICATIONS/DISCUSSION: Endometrial assessment by means of biopsy or sampling of endometrial cells is a minimally invasive alternative for dilation and curettage or hysteroscopy. Techniques using the Pipelle endometrial sampling device are the most popular due to high sensitivity and specificity compared to other office based procedures, and low rates of complications. In dilation and curettage, studies show transient bacteremia in 5% of patients and up to 0.34 in 100 patients have fever complicating their procedure. We hypothesize that our patient's endocarditis was caused by bacteremia resulting from her endometrial sampling, making this a very unusual complication of a common procedure. There is no data supporting the use of antibiotic prophylaxis for office based (i.e. Pipelle) or operating room (i.e. dilation and curettage) endometrial sampling. Unfortunately for our patient, mortality rates for Group B Streptococcus endocarditis approach 40%, similar to those associated with staphylococcal species.
WEIGHT fatigue, and night sweats. He denied using tobacco or intravenous drugs. On physical examination, he had orthostatic hypotension and a new 2/6 systolic ejection murmur. He did not demonstrate peripheral stigmata associated with endocarditis (Osler's nodes, Janeway lesions, or splinter hemorrhages). Pertinent labs for the hospitalization included a hemoglobin of 10.2 g/dL, hematocrit of 30.1%, and mean corpuscular volume of 81.9 fL (further labs confimed iron deficiency). An erythrocyte sedimentation rate (ESR) was 35; C-reactive protein was 12.5 and albumin was 2.0. WBC, BUN, creatinine, liver function tests, chest radiograph, and EKG were normal. PSA, TSH, and morning cortisol levels were normal. PPD and HIV screening were negative. Esophagogastroduodenoscopy and colonoscopy did not show any abnormal pathology. CT scans of the head, neck, chest, abdomen, and pelvis were unremarkable except for a small pericardial effusion and an old ischemic left cerebellar infarct. On day six of his hospital stay, the patient had a fever with a Tmax of 39.0 and a bandemia of 14% with a normal WBC count. Two sets of blood cultures were drawn and diphtheroids, identified as species other than Corynebacterium jeikeium, grew in all specimens. A transthoracic echocardiogram revealed an aortic valve vegetation, moderate aortic regurgitation, and an ejection fraction of 60 to 65%. A diagnosis of subacute bacterial endocarditis was made and the patient was treated with a four week course of intravenous ampicillin. Surveillance cultures drawn five days after starting antibiotics were negative and the patient remained afebrile for the rest of his hospital stay. IMPLICATIONS/DISCUSSION: Bacterial endocarditis is a common entity encountered in the hospital setting. The diagnosis may be missed as it can mimick a variety of conditions due its protean clinical manifestations and the diversity of pathogens associated with it. Recognition of these signs and symptoms can enable the clinician to make an early diagnosis and pave the way for intervention and further reduction in morbidity and mortality. These include constitutional symptoms such as fever, fatigue, and weight loss; new and/or changing murmurs; embolic phenomenon such as Janeway lesions, mycotic aneurysms, and peripheral emboli to multiple organs; and immune complex mediated phenomena such as glomerulonephritis, Osler's nodes, and arthritis. Elevated ESR and C-reactive protein, and a positive rheumatoid factor often accompany these presentations. Anemia is present in 70 to 90% of cases. Weight loss is found in up to 25% of cases and often leads to an exhaustive work up for malignancy, tuberculosis, and other chronic diseases. If there is a high index of suspicion, diagnosis can be made by positive serial blood cultures or the presence of valvular vegetations on echocardiogram. Streptococci, enterococci, and staphylococci are isolated in the vast majority of cases. Other pathogens are rather uncommon and include the HACEK group (Hemophilus, Actinobacilus, Cardiobacterium, Eikenella, and Kingella), Coxiella, Neisseria, Pseudomonas, Salmonella, Streptobacillus, Bacteroides, Corynebacterium and other anaerobic gram-negative bacilli. There are case reports of native and prosthetic valve endocarditis caused by diptheroids including C. jeikeium, C. urealyticum and C. striatum. Of the nondiphtheriae corynebacteria, C. jeikeium is the most worrisome as it is resistant to multiple antibiotics and warrants treatment with vancomycin. Diagnosis of the causative organism is imperative for initiation of therapy with appropriate antibiotics and possible surgical intervention. LEARNING OBJECTIVES: After listening to this vignette presentation the leaner will: 1. Recognize the importance of NSAID-induced adverse effects in the lower GI tract. 2. Add NSAIDs (including the selective COX-2 inhibitors) to the differential diagnosis of rectal bleeding and intestinal strictures. CASE INFORMATION: A 58 year old female with a history of GERD treated with a proton pump inhibitor, hypertension, hyperlipidemia, and hypothyroidism was prescribed celecoxib 200mg/day for mechanical low back pain. After approximately a month of therapy she presented with bloody diarrhea. She denied fever or chills. There was only minor abdominal cramping. The patient has a strong family history of colon cancer and had undergone a surveillance colonoscopy 2 years ago. This was normal except for a few small diverticula.
Abdominal examination was unremarkable. Rectal exam revealed a small external hemorrhoid and no masses, red blood was found on the exam glove. Stool culture, ova and parasites, and WBCs was negative. The patient discontinued the celecoxib and the bleeding diminished. A flexible sigmoidoscopy demonstrated a few 5 mm ulcers in the descending and transverse colon. Biopsies revealed nonspecific acute and chronic inflammation. No granulomas or crypt abscesses were seen. The bloody diarrhea completely resolved. The patient remains off of the celecoxib. Her back pain remains problematic. IMPLICATIONS/DISCUSSION: Upper GI tract ulceration with hemorrhage is a wellrecognized complication of NSAIDs. This serious side effect prompted the development of the selective COX-2 inhibitors which are touted as being safer in this regard. (However, this claim has recently been questioned.) Less known important GI adverse events attributed to NSAIDs include strictures and ulcerations of the small and large intestine. This case demonstrates symptomatic ulcerations of the colon attributed to celecoxib, a COX-2 specific NSAID. The patient's clinical presentation, and histology are consistent with that described with nonselective NSAIDS. Clinicians must consider NSAIDs in the differential diagnosis of lower GI tract bleeding, ulceration, or strictures. Studies are needed to determine if COX-2 specific inhibitors are safer than non-specific NSAIDs regarding the lower GI tract. This may become especially relevant in assessing the role of COX-2 inhibitors in the prevention of colon cancer. Although she had a family history of diabetes, a previous fasting glucose before beginning olanzapine was 85 mg/dL. Her exam was significant only for a non-obese, dehydrated appearing female who was orthostatic and tachycardic. Total glucose was 1122 mg/dL and measured serum osmolality was 320 mOsm/kg. A venous blood gas showed pH 7.38 and pCO2 41. Other labs included positive serum ACE test, trace urine ketones, and (in mEq/L) serum HCO3 23, sodium 122 and potassium 5.4 (anion gap of 19). IMPLICATIONS/DISCUSSION: As early as 1926, patients with schizophrenia have been known to have an increased risk of developing hyperglycemia, a process which was exacerbated with the introduction of phenothyiazines in 1956 and the atypical anti-psychotics in 1994. Increasingly, non-schizophrenic patients taking atypical anti-psychotics are demonstrating increased episodes of hyperglycemia and the complications thereof. One proposed mechanism for this hyperglycemia is through drug interference with cellular glucose transport proteins leading to increased insulin resistance and impaired glucose utilization in the periphery. This effect is not dose dependent and has its onset from 10 day to 18 months after the initiation of the drug. The literature is replete with examples of DKA in schizophrenic patients taking atypical anti-psychotics but does not specifically report the development of non-ketotic hyperosmolar syndrome. Additionally, patients taking atypical anti-psychotic medicines for diagnoses other than schizophrenia±including bipolar disorder and major depression with psychosis±risk developing hyperglycemia.
A CASE OF STAPHYLOCOCCAL COLITIS PRESENTING AS TOXIC SHOCK SYNDROME. T. Rutkoski 1 , C. Mueller 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #77132)
LEARNING OBJECTIVES: 1) Recognize the presentation of toxic shock syndrome.
2) Realize its occurrence in males and nonmenstrual females. CASE INFORMATION: MZ was an 18 y.o. caucasian female with a PMH of depression and PTSD who presented to the ER with complaints of fever, rash and abdominal pain. Outpatient medications included only quetiapine. The patient was currently living in a women's shelter b/c of reported sexual abuse and she denied tobacco, alcohol, and drugs. Initial PE was significant for fever, hypotension, conjunctival injection, rash, diffuse abdominal tenderness, and a negative pelvic exam. Labs revealed an elevated WBC count, a renal panel with elevated AG, mild LFT abnormalities, negative CSF tap, negative tox. screen, and a contaminated U/A with ketones. The patient was initially treated with ceftriaxone and aggressive fluid management. She did develop anemia and mild pulmonary edema. Her illness gradually improved. She was discharged home and follow up in the office 5 days later revealed desquamation of her hands. Her infectious w/u was negative except for a stool cx growing S. aureus. Toxic Shock Staphylococcal Toxin-1 was negative, but Staph aureus B toxin was positive. IMPLICATIONS/DISCUSSION: The initial cases of staphylococcal TSS were described in young menstruating females in the late 1970's. Much was learned about this disease in a short time. Soon an association was found between highly absorbent tampons and TSS. These tampons were removed from the market in the early 1980's. Since then the number of cases of TSS has declined greatly. Today, however the number of reported cases is relatively stable. These cases can be found in the menstruating and nonmenstruating female, the young and old, and the male and female patient. There are very few cases of Staph TSS secondary to colitis in the literature. Overall disease mortality has also improved throughout the years but remains as high as 15% in some studies (although more likely 2±5% by most accounts). It is therefore important to think of this disease in the differential diagnosis and treat it appropriately. The patient was started on a diltiazem drip for rate control and advanced to metoprolol for long term management. She was also started on aspirin for thrombus prophylaxis. No underlying etiology for her arrhythmia was identified. However, an extensive review of the patient's family history revealed that her father had been diagnosed with heart failure secondary to Chagas' disease back in El Salvador. A sample of the patient's blood was sent for serology for Chagas' disease and returned positive with a titer of 1:512. The patient was placed on nifurtimox. Although it was believed that she may have been in the subacute phase of her illness, treatment was initiated as a means of preventing advancement of her disease to chronic illness. IMPLICATIONS/DISCUSSION: Chagas' disease is a protozoan infection due to Trypanosoma cruzi and is primarily related to three problems: megaesophagus; megacolon; and cardiac disease. Chagas' myocarditis is the most common form of cardiomyopathy in Latin-American countries and therefore has become a considerable public health problem for many areas. It is estimated that over 750,000 years of productive life are lost annually because of premature deaths due to this disorder. The presentation of Chagas' disease consists of an acute and chronic phase, separated by an indeterminate phase. The latter describes a patient who has positive se-rology, but no symptoms, physical signs, or laboratory evidence of organ involvement. Biopsy was consistent with candida without evidence of aspergilus. IMPLICATIONS/DISCUSSION: Aspergillosis encompasses a variety of manifestations of infection. The major entities include invasive disease, acute bronchopulmonary aspergillosis (ABPA) and pulmonary aspergilloma. Disease due to aspergillus organisms is uncommon and usually occurs in the setting of immunosuppression for which neutropenia (ANC < 1500 TH/ MM3) and corticosteroids are the most common predisposing factors. The most common manifestation of invasive aspergillosis involve the lung and contiguous structures. The central nervous system and gastrointestinal system (especially esophagus and bowel) are the first and second most common target of dissemination respectively. Symptoms of aspergillus esophagitis are usually consistent with severe odynophagia similar to those of candidiasis. Isolated esophageal aspergillosis is very rare. There are two case reports published describing this unusual presentation, one involving a patient with acute leukemia and another occurred in a patient 25 days post bone marrow transplantation. This case is of interest because though our patient was steroid dependent she was not neutropenic prior to and at the time of presentation (ANC > 1500 TH/MM3). Further she was asymptomatic in regards to her fungal infection and did not have evidence of aspergillus involvement at other sites. In addition esophageal involvement usually occurs as a secondary complication of severe invasive lung disease. To our knowledge, this is the first case to describe isolated esophageal aspergillosis in a patient without the overt risk factors for invasive aspergillosis. Health is now moving forward in fostering high quality language programs that will help eliminate racial and ethnic disparities in healthcare and will promote access to optimal healthcare for all. Legislation has been introduced into the State legislature regarding cultural competency training and additional recommendations to the State are being drafted to expand this model program. Our hospital is now implementing an on-going training program for all interpreters and staff prior to enlisting their services, have hired bilingual greeters for the hospital, and a bilingual patient advocate. We have also initiated a cumulative and ongoing series of research projects in the field of cross-cultural -old Oriental man presented with complaints of cramping abdominal pain, tenesmus (immediately after eating) and constipation with pellet like stools of 3 weeks duration. His past history included Coronary Artery Disease and Radical Prostatectomy for Prostate Carcinoma 8 years ago with regular follow up. Physical examination was normal except for an irregular rectal mass partially occluding the lumen of the rectum. A preliminary clinical diagnosis of rectal carcinoma was made. Flexible sigmoidoscopy showed a cauliflower like ulcero-nodular mass occupying the lumen of the rectum. Biopsies of which were consistent with Adenocarcinoma. CT scan of the pelvis showed a mass adherent to the posterior wall of urinary bladder in the region of trigone, partially obstructing lumen of the rectum and also causing bilateral hydroureters and bilateral hydronephrosis. A subsequent serum PSA was 138 ng /ml, the previous value being 22 ng/ml. Based on this data a diagnosis of prostate carcinoma was made. Because of the extent of disease and underlying heart disease palliative measures were chosen for management including the stents in rectum and bladder to relieve obstruction. IMPLICATIONS/DISCUSSION: Finding a rectal mass on clinical examination is not uncommon. Our case is unusual in that the rectal mass was of prostatic origin, presenting 8 years after initial diagnosis. Though rectal involvement by prostatic carcinoma is present in 0.56% to 11.5% of cases on postmortem studies, the presence of Denonvillier's Fascia usually inhibits it. Commonest modes of involvement of rectum are a bulging prostate mass with intact rectal mucosa or an annular stricture, ulcerating masses being the rarest and accounting for less than 1% of these cases. Knowledge of this is important as it can avoid a misdiagnosis of primary rectal cancer, especially in patients who do not have a history of Prostate cancer or those with a history of prostate cancer who have received radiotherapy. LEARNING OBJECTIVES: 1. To recognise that superwarfarin causes prolonged coagulopathy needing high dose Vitamin K treatment for weeks to months 2. To suspect superwarfarin overdose as a cause of coagulopathy resistant to vitamin K and fresh frozen plasma treatment CASE INFORMATION: A 50 years old gentleman with a past medical history of depression and recurrent suicidal attempts presented to emergency room (ER) with history of hematuria, epistaxis and bruising of skin after suicidal attempt with anticoagulant ingestion. Physical examination revealed widespread ecchymosis of skin and heme positive stool but was otherwise unremarkable. Laboratory studies revealed hemoglobin (Hbg) 4.8 g/dl (13.7±16.5 g/dl), prothrombin time (PT) >50 sec (11.5±14.3 sec), INR too high to be reported, and activated partial thromboplastin time (APTT) >150 sec (21±37 sec). Patient was thought to have ingested warfarin, the most commonly available anticoagulant. His anemia was corrected with blood transfusion and coagulopathy treated by fresh frozen plasma (FFP) and Vitamin K. After 6 days of treatment his Hbg was12.2 g/dl, PT = 14.7sec, INR = 1.26, and APTT = 50 sec. Vitamin K was discontinued and he was discharged home after psychiatry consultation. His epistaxis and bruising of skin returned after 1 week resulting him to revisit ER. Patient denied any further intake of anticoagulant since discharge, however confirmed that the anticoagulant he had ingested 4 weeks ago with suicidal intent was infact superwarfarin (rodenticide) subsequently identified as brodifacoum. His blood studies revealed Hbg 7.9 g/dl, PT >200 sec, INR = 397.5 and APTT = 95.3 sec. He was again treated with FFP, blood transfusion and Vitamin K. This time he was discharged on long term high dose (100 mg/day) oral Vitamin K and out patient follow-up. IMPLICATIONS/DISCUSSION: Superwarfarins were developed in 1970's to overcome warfarin resistance in rats. They are long acting, fat-soluble anticoagulants, and 100 times more potent than warfarin. Their half-life varies from 16 to 69 days compared to 37 hours for warfarin. Brodifacoum is the most commonly used superwarfarin thus the commonest cause of superwarfarin poisoning. Its poisoning is mostly accidental however it can result from suicidal attempt, industrial exposure or deliberate self-poisoning with denial (Munchausen syndrome). Clinical features of brodifacoum poisoning are varied and include epistaxis, hematurea, gastrointestinal bleeding, ecchymosis, hemoptysis, spontaneous abortion and intracranial bleeding. PT, INR and PTT are usually severely prolonged but correct on mixing study. The diagnosis may be confirmed by detecting brodifacoum in blood however it is usually missed on the initial screening test for superwarfarin and may need a special assay. Physicians must have a high index of suspicion of superwarfarin exposure when patients have prolonged unexplained coagulopathy resistant to vitamin K replacement and FFP, along with undetectable warfarin levels and depletion of vitamin K dependent coagulation factors. After correction of initial life threatening coagulopathy by transfusion of blood products these patients should be placed on long-term high dose Vitamin K therapy and closely followed in outpatient clinic.
TRANSPLANT PATIENT. M. Schaeffer 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74228) LEARNING OBJECTIVES: 1) To understand that patients undergoing solid organ transplantation require immunosupressive medications and should undergo thorough evaluation for infectious diseases with consideration of subclinical parasitic infections. 2) To understand that patients taking glucocorticoids have compromised cell-mediated immunity and are susceptible to life-threatening infections by organisms that typically cause mild or no disease in healthy individuals. CASE INFORMATION: Solid organ transplant patients require immunosuppression and are susceptible to many life-threatening infections. We report a case of fatal fulminate infection with Strongyloides stercoralis 3 months following orthotopic heart transplantation. A 54-yearold male underwent orthotopic heart transplantation for ischemic cardiomyopathy. Over the next 6 weeks he required increasing doses of prednisone, tacrolimus and mycophenolate mofetil for graft rejection. 8 weeks after transplant he suffered a perforated colonic diverticulum, and underwent emergent surgery. On postoperative day 14 he developed hypotension, respiratory failure and decreased mental status. S. stercoralis was identified in stool and bronchoalveolar aspirate, and Gram negative bacteremia was found. LP revealed motile S. stercoralis larvae in CSF and Gram negative meningitis. Brain MRI revealed multiple areas of signal change consistent with extensive new infarcts. After 10 days of supportive care and antibiotics, treatment was withdrawn at the family's request and the patient died. IMPLICATIONS/DISCUSSION: Strongyloides Hyperinfection Syndrome (SHS) occurs when massive infestation by S. stercoralis occurs in the lungs, GI tract and rarely in the central nervous system (CNS). Strongyloides stercoralis is a helminthic parasite endemic to the southeastern region of the United States. S. stercoralis has a complex life cycle in the human involving the skin, circulatory system, lungs, and gastrointestinal tract. Compromised host defenses leads to susceptibility to SHS. Along with this massive invasion of S. stercoralis, secondary Gram-negative bacteremia and sepsis are common. CNS invasion is not common but has devastating consequences, as seen in our patient. Perhaps the most damaging aspect of CNS invasion by S. stercoralis occurs when bacteria cross the blood-brain barrier attached to the parasite or within the gut of the worm leading to secondary bacterial meningitis. Although pre-transplant patients infection screening occurs, thorough assessment for parasites is often lacking. In patients in endemic areas and with risk factors for subclinical parasitic infestation, consideration should be given to further evaluation prior to surgery and initiation of immunosupressive medications. Overall mortality of SHS is greater than 80%.
NEUROSYPHILIS PRESENTING AS FECAL INCONTINENCE. E. Schmidt 1 , B. Mathis 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74057) LEARNING OBJECTIVES: 1) Recognize various manifestations of tertiary syphilis. 2) Recognize treatment benefits of neurosyphilis. 3) Recognize that cases of tertiary syphilis may increase in prevalence in the clinic setting following the rise in cases of early syphilis seen in the past two decades. CASE INFORMATION: A 66 year-old African American male with history of treated syphilis at age 25 presented to an outpatient clinic following a hospital stay for recent subcortical stroke and non-Q wave myocardial infarction. He reported fecal and urinary incontinence and subjective memory deterioration for an undetermined amount of time. On exam he was found to have no anal sphincter tone and was unkempt. The patient was admitted to the hospital, and had a spine MRI that was negative for any lesions. He was found to have a SED rate of 117 mm/h, a positive RPR with a 1:1024 titer, and a positive serum FTA-Abs. Lumbar puncture revealed a pleocytosis with a predominance of lymphocytes, an elevated protein at 152 mg/dL, and a positive VDRL at 32 dilutions. The patient was treated for neurosyphilis with 14 days of intravenous penicillin G, 24 million units per day. He subsequently was found to be HIV positive. On clinic follow up, the patient had improved anal sphincter tone, improved physical appearance, and improved subjective cognition. His weight improved by 13 pounds and his RPR had decreased to a titer of 1:512. IMPLICATIONS/DISCUSSION: The patient described above had clear benefit of treatment with apparent reversal of some of the neurologic sequelae of his tertiary syphilis. There is lack of data in the literature supporting reversibility of the neurologic complications of this disease, especially the dementia, that is thought to be related to fibrosis, and therefore the thought of the RPR as a screening test for reversible causes of dementia has been questioned. It has been proposed that there may be an increase in the number of cases of tertiary syphilis in light of the increased number of primary syphilis cases seen in the past two decades, as well as an increase in the HIV population. There must be a heightened level of suspicion for this process in anyone with neurologic findings and history consistent with this diagnosis.
ENDOCARDITIS AS A RED-HERRING. N. Schmidt 1 , R. Witzig 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77065) LEARNING OBJECTIVES: 1. Develop a thorough diagnostic strategy for fever in patients who use intravenous drugs. 2. Diagnose splenic abcess. CASE INFORMATION: A 25 year-old woman presented with lethargy and fever. She reported a history of intravenous drug use and a recent forearm cellulitis. She had a temperature of 38.28C, a systolic murmur, diffuse abdominal pain, and a purulent pustule on the left leg. The WBC was 19,000. An Abdominal CT scan demonstrated several hypodense lesions in the spleen. Subsequent laparatomy confirmed splenic abscesses, and a splenectomy was performed. Blood and splenic tissue cultures grew methicillin-resistant Staphylococcus aureus. Transesophageal echocardiogram did not show evidence of endocarditis. The patient was diagnosed with impetigo and splenic abscesses with subsequent sepsis. IMPLICATIONS/DISCUSSION: Staphlococcal bacteremia is common in intravenous drug users due to unsterile injection techniques. Not all patients with bacteremia, however, will develop endocarditis. The evaluation of a fever in a patient who uses intravenous drugs should extend past the heart if the patient does not meet the Duke criteria for endocarditis. The local skin infection in this patient led to bacteremia and subsequent splenic abscess formation and sepsis. Spleenic abscesses with or without endocarditis are a frequent complication of patients who use intravenous drugs. Almost all patients have fever and abdominal discomfort, but signs and symptoms of a splenic abscess may be vague and overshadowed by underlying endocarditis. The careful attention to the historical complaint and a methodical and thorough physical examination led to the appropriate diagostic test. Treatment requires splenectomy and antibiotics directed against the involved pathogens. Patients with IVDU and fever require careful evaluation not only for endocarditis, but also for bacterial seeding of other organs. Abdominal signs should prompt the search for intraabdominal abscess formation. We present an unusual case of a patient with progressive and evolving postpartum neurologic changes thought secondary to a pregnancy related vasculitic process. A healthy 30 year-old female (G1 P0) underwent a Cesarean section after unsuccessful induction of labor. The pregnancy was uneventful without signs or symptoms of pre-eclampsia. Six hours postpartum she presented with right-sided hemiparesis and language difficulties. A head CT scan showed a hypodensity in the left caudate and anterior internal capsule. The patient was anticoagulated and evaluated for thromboembolic source. By hospital day 4, the patient's symptoms had nearly resolved. On day 5 she developed recurrence of symptoms and was found to have new areas of infarction including the left cerebellum and posterior frontal lobe. She was started on high dose steroids and cerebral angiogram was done which showed findings consistent with vasculitis. Subsequent imaging showed continuous evolving infarcts and intravenous cyclophosphamide was then added. A complete rheumatologic and hypercoagulable work up was performed and unremarkable except for an erythrocyte sedimentation rate (ESR) of 98 mm/hr on admission that decreased to 52 mm/hr prior to initiation of steroids. The patient was continued on steroids and cyclophosphamide, as repeat imaging 6 months postpartum showed new evolving infarcts. 9 months postpartum, no new areas of infarction were noted and repeat angiogram showed some resolution of changes. 18 months postpartum, the patient continues to improve and is slowly being tapered off of immunosuppressive therapy. The patient has never had any other manifestations of vasculitis other than the cerebral findings as above. IMPLICATIONS/DISCUSSION: The pathogenesis of postpartum cerebral vasculitis remains unclear. It is postulated that vasospasm of the vessels, as seen with migraine headaches, may be triggered by hormonal changes unique to the postpartum period possibly producing an autoimmune reaction resulting in a vasculitis. Contributing factors may also include altered hemodynamics, and the relative coagulopathy associated with pregnancy. Diagnosis of this rare but potentially neurologically devastating disease should be confirmed with angiogram as current therapy with high dose steroids and cyclophosphamide is not without risk. Recurrence of disease with subsequent births has been documented and women diagnosed with postpartum cerebral vasculitis should be counseled on potential risks with subsequent pregnancies. LEARNING OBJECTIVES: 1. Recognize the importance of eliciting a careful review of systems when evaluating patients with fever. 2. Consider prostatitis in the differential diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. CASE INFORMATION: A 53-year-old truck driver with diabetes presented with a three-day history of subjective fever and rigors. On review of systems the patient disclosed urinary urgency and dysuria. Physical examination was remarkable for a slightly enlarged, exquisitely tender prostate. Pertinent laboratory testing showed a mild microcytic anemia with a normal leukocyte count, glucose 275 mg/dl, hemoglobin A1C 11.7%, and a normal PSA. Urinalysis revealed no pyuria and urine cultures were negative. However, cultures of prostate secretions, and four blood cultures obtained before the initiation of antibiotics, were positive for MRSA. A transrectal ultrasound showed large, solid, nodules in the right and left prostate likely representing an infectious process. A biopsy of the prostate revealed severe acute and chronic prostatitis. Cultures of the prostate tissue grew MRSA. Notably, sensitivities from all MRSA cultures were identical. The patient was treated with intravenous vancomycin and his clinical status improved. IMPLICATIONS/DISCUSSION: Coagulase negative Staphylococcus is known to cause chronic prostatitis. Additionally, methicillin-sensitive Staphylococcus aures can cause chronic prostatitis, especially prostatic abscesses. However, we are unaware of prior cases of MRSA bacteremia resulting from MRSA prostatitis. The patient's prostate biopsy showing prostatitis, along with the identical antimicrobial sensitivities of MRSA from both the prostatic tissue and peripheral blood, implicate MRSA prostatitis with hematogenous spread. This is a unique case of MRSA bacteremia, which appears to have resulted from MRSA prostatitis. Furthermore, this case highlights the importance of eliciting a careful review of systems and performing a thorough physical examination when evaluating the source of a fever. LEARNING OBJECTIVES: To recognize Hypokalemic Thyrotoxic Periodic Paralysis and its precipitating factors, especially since it is a rare disorder that is treatable if diagnosed. We describe a case of HTPP likely precipitated by a test. CASE INFORMATION: The patient is a 59-year-old African-American female admitted for hypokalemia after a colonoscopy. The colonoscopy was for evaluation of her microcytic, hypochromic anemia and a recent history of a 30 lb. weight loss. The colonoscopy was negative, and a CT scan of the chest, abdomen, and pelvis was done for further evaluation of possible malignancy which was also negative. Review of systems was notable for bilateral lower extremity weakness in addition to the weight loss. Two months prior, she had a persantine thallium stress test for evaluation of chest pain which was positive and was followed by an angiogram which showed only moderate coronary artery disease requiring no intervention. Since the stress test, she had progressive lower extremity weakness associated with muscle cramps and had difficulty walking, standing at length, and had difficulty climbing stairs. Physical exam revealed tachycardia, thyromegaly, 3/5 motor strength of bilateral lower extremities, and areflexia. The remainder of the examination was unremarkable. Labs revealed a potassium level of 2.9 mg/dL, and oral and intravenous potassium replacement was given. ECG revealed a sinus tachycardia with left atrial enlargement. A thyroid workup revealed a TSH of <0.01 and a free T4 of 8.33. We diagnosed hypokalemic thyrotoxic periodic paralysis likely precipitated by iodine in the dye. The patient was started on propylthiouracil 150 mg three times daily and metoprolol. The patient was referred for radioactive thyroid scan and possible radioablation therapy. At discharge, the patient's hypokalemia and symptoms had resolved. IMPLICATIONS/DISCUSSION: HTPP is a rare, sporadic, acquired disorder usually seen in Asians. The incidence is <1% in Americans. The disease is associated with an underlying thyrotoxicosis (commonly Grave's disease or thyroiditis) and hypokalemia. The serum potassium level is low, but the total body potassium remains normal. Thyroid hormone mediates an increase in sodium-potassium ATPase activity and a transcellular potassium influx. This interferes with potassium's involvement in promoting muscle contraction Treatment of HTPP includes antithyroid drugs, beta-blockers, and cautious use of potassium. Definitive treatment involves radioablation therapy. The current vignette helps one to recognize the signs and symptoms of HTPP, and describes a case that was precipitated by dye used in an angiogram. LEARNING OBJECTIVES: 1) Review the differential diagnosis of an enlarged uterus in a non-pregnant woman.
2) Recognize intravenous leiomyomatosis as a rare cause of intracardiac tumors. CASE INFORMATION: A 56 year-old postmenopausal woman with a history of uterine fibroids presented with two days of exertional shortness of breath and constant retrosternal chest pressure, worse with exertion and lying flat. She denied orthopnea, paroxysmal nocturnal dyspnea or cough. Physical exam revealed normal vital signs, elevated JVD, clear lungs, regular rhythm with II/VI holosystolic murmur at the left lower sternal border, no extremity edema, and an enlarged uterus palpable 4 cm below the umbilicus. Basic labs, cardiac enzymes, and EKG were normal. Chest x-ray showed cardiomegaly and vascular prominence. An echocardiogram revealed normal left ventricular ejection fraction, dilated right atrium (RA) and right ventricle (RV), and tricuspid regurgitation. There were two linear mobile echodensities in the inferior vena cava (IVC) protruding into the right atrium and ventricle. Cardiac and abdominopelvic MRI showed multiple uterine leiomyomas, with a lobulated mass arising from the uterine fundus and extending up the right gonadal vein into the IVC, RA, and RV. The patient underwent total hysterectomy and total excision of the mass through the infrarenal IVC. Pathology of the mass showed intravenous leiomyomatosis. IMPLICATIONS/DISCUSSION: The differential diagnosis of an enlarged non-pregnant uterus includes endometrial and myometrial processes. Advanced endometrial carcinoma is the main endometrial cause. Myometrial causes are divided into benign and malignant. Benign conditions include leiomyomas (i.e. fibroids), adenomyosis, and stromal adenomyosis. Malignant causes include leiomyosarcoma and endometrial stromal sarcoma. Uterine conditions capable of extending into the IVC and heart are endometrial stromal sarcoma and intravenous leiomyomatosis. Intravenous leiomyomatosis is a rare benign condition in which nodules of benign smooth muscle tissue grow within the veins of the myometrium and may extend into the uterine and hypogastric veins. Rarely, these tumors extend up the IVC into the heart. Only 43 cases of intravenous leiomyomatosis with cardiac extension have been reported in the English literature. Symptoms of cardiac extension include venous obstruction, right-sided heart failure, and rarely, sudden death. Successful therapy entails total excision of the tumor, as residual tumor could result in recurrence. 3) Identify Salmonella as a rare infectious cause of spondylodiscitis. CASE INFORMATION: A 65-year-old woman presented with four days of low back pain and fever. The pain radiated down her left leg and was aggravated by movement. She denied trauma, weakness or numbness. She had a temperature of 104, blood pressure of 154/76, and heart rate of 115. Physical exam showed lumbar paraspinal spasm with no focal tenderness. Neurological exam was normal, including straight leg test. White cell count was 14000 with 90% neutrophils. ESR was 64. Blood and urine cultures were negative. Lumbar x-rays were normal. An indium scan done as part of the workup for fever and leucocytosis showed increased uptake at L4-L5. MRI of the spine revealed disc herniation at L4-L5, but no spondylodiscitis. Even though the source of fever was uncertain, the patient was treated empirically with vancomycin with resolution of fever. However, the pain persisted and she underwent discectomy. Pathological exam showed acute discitis, but cultures were not done. Despite the surgery, her back pain worsened, and repeat imaging showed destruction of L4 and L5 vertebral end plates, with prevertebral, epidural and paraspinal abscess. A CT-guided biopsy was done, and Salmonella javiana grew from the drained fluid. The patient had hemoglobin C trait but no immunocompromising conditions. She was treated with fluoroquinolones and slowly improved. IMPLICATIONS/DISCUSSION: Most cases of back pain are due to simple benign etiologies. However, certain red flags prompt the search for more serious causes. These include advanced age, malignancy, signs of infection, and progressive neurological symptoms. Spondylodiscitis is the simultaneous infection of the vertebral body and disc space. MRI is the most sensitive and specific imaging modality, with a sensitivity of 91% during the first two weeks of symptoms. Radionuclide scanning is done when MRI cannot be performed. It is very sensitive but not as specific. CT-guided biopsy should be pursued in cases of suspected spondylodiscitis, even with a normal MRI. Most cases of spondylodiscitis are due to Staphylococcus aureus. Other organisms include Streptococcus, E. coli and Salmonella, with the most frequent serotypes being Salmonella typhimurium and S. choleraesuis. Predisposing factors for salmonellosis include sickle cell disease, HIV, immunosuppressive therapies and previous trauma. Spondylodiscitis due to S. javiana has not been previously reported. Hemoglobin C disorders have not been associated with Salmonella osteomyelitis.
THE YOUNG WOMAN WITH NUMB FEET. J.J. Silverman 1 ; 1 University of Pennsylvania, Philadelphia, PA (Tracking ID #75939) LEARNING OBJECTIVES: 1) Recount the most common causes of a symmetric polyneuropathy; 2) Perform the initial evaluation of a polyneuropathy 3) Recognize an uncommon presentation of hypothyroidism. CASE INFORMATION: A 28 year-old female with no significant past medical history presents complaining of bilateral foot and hand numbness for the past six weeks. The sensation begins mid-foot, occurs intermittently throughout the day, and she specifically notes worsening symptoms walking on the treadmill at the gym. She reports similar but milder symptoms in her hands, which are not related to activity. Review of systems is otherwise notable for chronic constipation and longstanding irregular menses (since menarche at age 14). Family history is negative for polyneuropathies, cancer and thyroid disease. She smokes two packs of cigarettes per week, does not drink alcohol or use drugs, and denies toxic exposures. She has no history of blood transfusions, reports regular condom use with her two prior sexual partners, and is not presently sexually active. She takes fexofenadine as needed for seasonal allergies. Physical exam revealed a well-appearing young woman, who was afebrile, with a blood pressure of 130/80 and a heart rate of 74. Neurologic exam was notable for decreased sensation to light touch and pinprick in the plantar aspect of her right foot, as well as decreased vibratory sense in her right foot. The remainder of the physical exam was unremarkable. Labs were notable for a TSH of 109, FTI of 2.9, Thyroxine of 2.7 and T3U of 1.06. IMPLICATIONS/DISCUSSION: Polyneuropathy is not an uncommon problem in the primary care setting. The differential diagnosis is quite broad, but chronic systemic disorders, hereditary causes and inflammatory diseases account for the majority of cases. Chronic medical conditions commonly associated with polyneuropathies include diabetes mellitus, malignancy, collagen vascular disease, organ failure, B12 deficiency, alcoholism, AIDS, toxic exposures and plasma cell dyscrasias. Though the history can direct the initial evaluation, for most cases it will include a CBC, basic metabolic panel, liver panel, ANA, SED rate, B12, folate, lead level, RPR, HIV, hepatitis serologies and TSH. If this initial evaluation is inconclusive, an EMG and neurology consult are warranted. Finally, it is important to realize that hypothyroidism is protean in its manifestations, and can present without its most commonly recognized signs and symptoms.
SHORTNESS OF BREATH: A``NEGATIVE'' LOWER EXTREMITY DOPPLER STUDY. C.W. Simpkins 1 , C. Passaretti 1 , A. Zaas 1 , J. Cofrancesco 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #76910) LEARNING OBJECTIVES: 1. Assess subacute progressive dyspnea in a young woman. 2. Recognize the presentation of pulmonary sarcoidosis. 3. Recognize the pathology and pathophysiology of early pulmonary sarcoid. CASE INFORMATION: A 48 year old African American female with hypertension, gastroesophageal reflux disease, bipolar disorder, a remote history of lithium therapy and history of total abdominal hysterectomy and bilateral salpingoophorectomy on hormone replacement therapy (HRT), presents with slowly progressive dyspnea on exertion, orthopnea, and paroxysmal nocturnal dyspnea for one year. Lower extremity duplex ultrasounds were negative for blood clot but did show inguinal adenopathy. Review of systems included loud snoring, possible apneic episodes during sleep, nonrestful sleep, intermittent lower extremity swelling, nonproductive cough for 1 week, but no daytime somnolence, fevers, chills, or sweats. She had an 18 pound intentional weight loss over the past year. No history of alcohol, tobacco, or illicit drug use. Physical exam: temperature 100.5 degrees F, blood pressure 146/57 mmHg, pulse 67 bpm, respiratory rate 20, pulse oximetry 95% on 2 liters supplemental oxygen. Patient was a mildly obese woman in no acute distress, with no palpable adenopathy, slightly elevated jugular venous pulse, normal air movement except for rare bibasilar crackles, a prominent P2 heart sound, and no peripheral edema, cyanosis, or clubbing. Arterial blood gas on room air: pH 7.44/PaCO2 35/PaO2 55/bicarb 23. Ventilation/perfusion (V/Q) scan was low probability for pulmonary embolism. Pulmonary function tests showed moderate combined obstructive and restrictive physiology with severe gas transfer defect. Computed tomography (CT) of the chest showed enlarged right atrium, bulky mediastinal adenopathy, and diffuse patchy ground glass infiltrates without effusion or consolidation. Echocardiogram showed normal left ventricular (LV) size and function, moderate dilatation of the right ventricle (RV) and right atrium (RA) with flattened septum. A diagnostic procedure was performed. IMPLICATIONS/DISCUSSION: Initial differential diagnosis included pulmonary embolus (young woman on hormones), sleep apnea (given the pulmonary hypertension), and hypertensive cardiomyopathy with heart failure. However, the negative V/Q and CT, and the bulky lymphadenopathy and patchy ground-glass infiltrates were suspicious for pulmonary sarcoid. Bronchoscopy with lavage and biopsy confirmed this diagnosis with negative cultures, peri-mucosal granuloma and reactive histiocytes. The patient was diuresed, started on high dose steroids with slow taper, and is doing well. The diagnosis, pathophysiology and treatment of pulmonary sarcoid will be discussed. LEARNING OBJECTIVES: 1) Review the differential diagnosis of normocytic anemia. 2) Recognize the cause of normocytic anemia by mixed processes of microcytic and macrocytic anemia.
3) Appreciate the wide and unusual manifestations of Vitamin B12 deficiency. CASE INFORMATION: A 56 year-old black male with history of hypertension presented to the outpatient clinic with six month history of intermittent low grade fever. On review of systems, he denied cough, chest pain or shortness of breath, palpitations, gastrointestinal bleed, urinary or bowel symptoms, arthritis, weight loss, numbness or weakness. Vital signs revealed temp 100.5, pulse 116, blood pressure 158/96, respirations 18. Physical exam was significant for pale conjunctiva, guaiac negative stool, and slight decreased sensation to light touch, pinprick, and vibration in the lower extremities. Labs showed Hgb 6.7, MCV 87 with normal WBC and platelet with blood smear displaying nucleated rbcs, anisocytosis, poikilocytosis, and isolated macrocytes. A work up including chemistries, cultures of blood and urine, ANA, ESR, and TSH was unremarkable. A vitamin B12 level was 108 pg/mL (normal 180±914 pg/mL) and hemoglobin electrophoresis showed beta thallasemia. With Vitamin B12 supplementation, the patient's fever resolved. IMPLICATIONS/DISCUSSION: The differential diagnosis of normocytic anemia includes chronic diseases, inflammation, endocrinopathies and renal diseases. In some cases, the simultaneous processes of microcytic and macrocytic anemia should also be in the differential diagnosis of normocytic anemia. The abnormal blood smear with size variation of erythrocytes and neuropathy led to the work up and concomitant diagnosis of beta thallasemia and Vitamin B12 deficiency. The clinical manifestations of Vitamin B12 deficiency include fatigue, glossitis, vomiting, diarrhea, dementia, and neuropathies, but fever is not considered a typical feature. From the literature, fever was found in approximately 40% of patients with megaloblastic anemia caused by folate and Vitamin B12 deficiencies in two case studies. The mechanism of the fever is uncertain, but one proposed theory is the increased activity and ineffective erythropoiesis in the bone marrow. Fever resolves rapidly with treatment and failure to do so should prompt a search for other causes. In summary, normocytic anemia can be caused by dual processes of microcytic and macrocytic anemia and fever should be recognized as an atypical feature of Vitamin B12 deficiency.`D LEARNING OBJECTIVES: 1) Differentiate between gynecomastia and breast cancer 2) Recognize the causes and treatment of gynecomastia. CASE INFORMATION: The patient is a 67 year-old male with alcoholic cirrhosis who presented with a painful lump in his right breast. Medications included spironolactone. Social history was significant for one year of sobriety and current tri-weekly use of marijuana. On physical exam, a 3 by 3 centimeter subareolar, tender, mobile, rubbery right breast mass was palpated. A smaller, similiar mass was palpated on the left. There was no nipple discharge or retraction, overlying skin changes, or lymphadenopathy. His testosterone was normal. A diagnosis of bilateral gynecomastia was made, felt to be due to his cirrhosis, compounded by his use of spironolactone and marijuana. The patient stopped both drugs. Two months later, the patient had significant improvement in his pain and decrease in breast tissue. IMPLICATIONS/DISCUSSION: Gynecomastia is common in adult males. The highest prevalence is between 50 to 80 years, with an incidence of 24 to 65 percent. Patients present with a unilateral or bilateral breast mass that may be painful. The most important differentiation is between gynecomastia and breast carcinoma. Carcinoma is less common, usually unilateral, and eccentric rather than symmetric to the nipple/areola. It is firm, fixed and may be associated with skin dimpling, nipple retraction or discharge, and lymphadenopathy. The conditions associated with gynecomastia are felt to represent an imbalance between estrogens and androgens. The most common causes in the adult male are as follows: idiopathic, drugs, cirrhosis, malnutrition, primary hypogonadism, testicular tumors, secondary hypogonadism, hyperthyroidism, and chronic renal insufficiency. Causal drugs include antiandrogens, antibiotics, antiulcers, chemotherapeutics, cardiovascular drugs, and drugs of abuse. The history, physical, and certain diagnostic tests can identify the cause in the majority of patients. Patients with a pain, acute onset, or without an obvious etiology should be evaluated with serum hCG, luteinizing hormone, testosterone, and estradiol levels. An asymptomatic patient found to have gynecomastia incidentally, deemed to be idiopathic, does not require further tests and should be reevaluated in six months. Otherwise, treatment should be directed at removing the underlying cause. Active treatment with androgens, antiestrogens, and aromatase inhibitors is indicated in persistent gynecomastia associated with severe pain, tenderness, or embarrassment. Surgical therapy, including liposuction or excision, is considered in patients who do not respond to medical therapy.
CAN'T WE JUST LET HER LEAVE? T. Spector 1 , P.P. Balingit 1 , A.G. Gomez 2 ; 1 UCLA San Fernando Valley Program, Sylmar, CA; 2 University of California, Los Angeles, Sepulveda, CA (Tracking ID #76525) LEARNING OBJECTIVES: 1. To recognize and avoid counter-transference with difficult patients. 2. To maintain a professional attitude in difficult circumstances. 3. To discuss when a patient has capacity to refuse treatment. CASE INFORMATION: A 36 year old female with a history of polysubstance abuse presented with 2 weeks of fevers and painful forearm lesions after skin-popping heroin. The patient was an ill-appearing, disheveled woman. Temperature was 38.4C, BP 114/68, heart rate 112. Multiple abscesses were noted on her forearms. A II/VI holosystolic cardiac murmur over the tricuspid area, not heard on previous exams, was also present. EKG and CXR were normal. The patient was admitted with the diagnosis of probable infective endocarditis. Within minutes of arriving to the monitored unit, she demanded to be moved to a room with a TV and swore at the nurses when they refused to give meperidine for her pain. She also demanded that the intern be paged until methadone was ordered for her heroin withdrawal. The intern arrived to the unit after the patient had ripped off her monitor leads, pulled out her IV line and was dressing to leave the hospital. A panicked intern told the resident that the patient was leaving because she felt her needs were being ignored. A psychiatrist was urgently called to determine her capacity to refuse treatment, but she left before the psychiatrist arrived. The resident found the patient outside the hospital. The patient was allowed to vent her frustrations with much vehemence and profanity. Satisfied that her grievances were heard, she allowed the resident to fully explain the benefits of treatment and the risks of refusing care, including death. The patient was alert, oriented, and able to repeat the risks clearly prior to leaving. IMPLICATIONS/DISCUSSION: Most internists are enthusiastic about finding solutions to difficult medical problems. However, a patient's psychological or social problem may not be greeted with the same enthusiasm. It is not uncommon for a patient's personality trait or attitude to invoke anger, disdain or frustration in health workers. Caring for such patients with compassion becomes a challenge. It is important to understand one's own response to difficult patients in order to recognize and prevent counter-transference from interfering with sound and appropriate medical decisions. One must also recognize that the patient's decision to leave without treatment, although a poor one by medical standards, was certainly within her rights. It was important to appease the patient's anger to assure that she made an informed decision to leave against medical advice rather than out of rash frustration and anger. By clearly repeating the risks and consequences, she demonstrated legal capacity to leave and was empowered with the knowledge to return or seek care elsewhere. Definitive guidelines exist that dictate when a physician can treat a patient against his will.
AN UNCOMMON CAUSE OF HYPOGLYCEMIA. S. Agresta 1 , B. Springgate 1 ; 1 Tulane, New Orleans, LA (Tracking ID #75148) LEARNING OBJECTIVES: 1. Recognize ethanol ingestion as an important cause of sustained hypoglycemia. 2. Provide appropriate treatment for ethanol-induce hypoglycemia. CASE INFORMATION: A 37 year-old man presented after a syncopal episode. Seven months earlier he was diagnosed with alcoholic liver disease. On presentation, he was tremulous and diaphoretic. The remainder of the physical exam was normal. An initial blood glucose was 30 mg/dL. He received one ampule of intravenously D50. Twenty minutes later the symptoms returned. His glucose was 28. Despite additional intravenous dextrose, his glucose continued to drop to between 30 and 40 mg/dl. His AST was 175 U/L, ALT 73 U/L, Hgb A1C 4.6%, and C peptide 15.5 ng/mL (nl). Drug and ethanol screens were negative. After two days of continuous dextrose-containing intravenous fluids, his blood glucose level returned to normal. IMPLICATIONS/DISCUSSION: Alcohol-induced hypoglycemia is an important but infrequently diagnosed sequella of ethanol ingestion. Symptoms are precipitated by a period of fasting followed by ingestion of alcohol. Ethanol inhibits the normal hepatic gluconeogenesis response to starvation-induced hepatic glycogen depletion. As this inhibition may be gradual, hypoglycemic symptoms may develop slowly. Patients may not demonstrate acute alcohol toxicity at the time of presentation. 1 Continuous intravenous glucose is required to prevent recurrent hypoglycemia and to replete hepatic glycogen stores. Chronic alcoholics may also have inhibition of pituitary ACTH, creating a hypo-cortisolemic state, exacerbating the hypoglycemia. surpassed those of the squamous cell type. The general locations of adenocarcinoma metastasis are lymph nodes and liver, and occasionally lung and bone. We report a case of adenocarcinoma of the esophagus with leptomeningeal carcinomatosis. A 56 year old thin white male presented with dysphagia, and a 40 pound weight loss over six months. He also had nausea, vomiting, abdominal pain, rectal bleeding, and diplopia during this period. On exam, he was alert and oriented. He had sluggish reaction of his left pupil, diplopia on downward gaze, guaiac positive stool, and a microcytic anemia (MCV 61). Esophagogastroduodenoscopy showed a mass that extended from the 28th cm of the esophagus to the 37th cm including the GE junction. Whole body CT scan showed an isodense mass in the foramen of Monro, midesophageal thickening, multiple hepatic lesions, and celiac lymphadenopathy. Brain MRI showed leptomeningeal carcinomatosis surrounding the pons and multiple cranial nerves. The patient received urgent whole brain radiation. Upon completion of radiation an Ommaya reservoir was suggested for intrathecal methotrexate. However, his overall quality of life deteriorated rapidly and he chose to receive hospice care. IMPLICATIONS/DISCUSSION: Metastatic sites for esophageal cancer typically involve the liver and lymph nodes and rarely lung and bone. Direct CNS involvement is an extremely rare occurrence. On identification of the leptomeningeal carcinomatosis our patient was able to undergo whole brain radiation to prevent any immediate risk of herniation, but given the extent of his disease his prognosis was extremely poor. The reasons why this adenocarcinoma spread to involve the CNS are unclear, and further study needs to be undertaken. As esophageal adenocarcinoma becomes more common, this type of presentation may be seen more frequently, and clinicians should be aware of this. week. The test took place at 6 AM with an ambient temperature of 75F. At the end of the test the patient collapsed complaining of``complete muscle failure''. He was found to be hypotensive and tachycardic. The patient's history was significant for the use of an herbal supplement called Energel. He had been taking this consistently for the past month to try improve his performance time. Laboratory results initially revealed severe rhabdomyolysis with a creatinine kinase (ck) of 11,000. Acute renal failure secondary to pigment-induced ATN was demonstrated by a serum creatinine (cr) of 2.4 mg/dl and urine positivity for myoglobin. The patient's ck peaked at 240,000 during his hospital course. After 8 days his ck was 45,000 and his cr returned to normal. IMPLICATIONS/DISCUSSION: Energel is a herbal supplement that contains Ma Huang. Ma Huang is the Chinese version of Ephedra which grows in many parts of the world and is used predominantly as a stimulant. Ephedra alkaloids are structurally similar to ephedrine and pseudoephedrine. They stimulate both alpha and beta-adrenergic receptors as well as release norepinephrine from storage sites. Each tablet of Energel contains 330 mg of Ma Huang extract, which is 6% (19.8 mg) of Ephedra. The dose is 2 gelcaps a day. Our patient took this dose. The FDA has banned the sale of Ephedra in large amounts (over 24 mg a day and does not recommend taking this product in small amounts for more than 7 consecutive days. Our patient was taking 40 mg a day for 30 days. Ephedra has caused 17 deaths and hundreds of serious side effects including stroke, myocardial infarction and seizures in previously healthy young adults. To this date there have been no reported cases of rhabdomyolysis associated with Ma Huang. This is an extremely important health concern since Ephedra, in its many forms, is readily available in unscrutinized and easily obtainable health supplements.
IATROGENIC STROKE AND UNDIAGNOSED DELIRIUM: OPPORTUNITIES TO IMPROVE CARE FOR HOSPITALIZED ELDERS. R. Sudore 1 , C. Landefeld 1 ; 1 University of California, San Francisco, San Francisco, CA (Tracking ID #73989) LEARNING OBJECTIVES: To describe the risk of stroke after cardiac catheterization and to recognize iatrogenic impediments to stroke rehabilitation in elderly patients. CASE INFORMATION: An independent 73 yr-old man with a history of NIDDM and HTN was transferred for cardiac catheterization (cath) after a recent non-Q-wave MI. An uneventful cath revealed 5 vessel disease and an ejection fraction of 30%. 90 minutes post cath he developed right hemiparesis and receptive and expressive aphasia. Head CT was negative and TPA was administered within 2 hours of symptom onset, but without clinical improvement. Two weeks later, neurologic function had not improved; agitation developed and hand restraints and diazepam were started. On consultation, we confirmed the sequelae of stroke, identified ischemic lesions in three toes, and diagnosed delirium. Serum sodium was 128, but other lab studies were normal. After discontinuing diazepam and restricting free water, his agitation resolved, and his dysphagia and receptive aphasia improved. Physical therapy was started. Once his restraints were discontinued his strength improved, though other than feeding, he remained dependent in all activities of daily living. IMPLICATIONS/DISCUSSION: This case highlights the importance of iatrogenic stroke and of undiagnosed delirium. Stroke occurs unpredictably in 0.1% of persons undergoing cath. Stroke is most often due to emboli, as evidenced by this man's cerebral and toe ischemia. His risk factors for stroke from cath included advanced age, extent of coronary artery disease, and depressed ejection fraction. In-hospital delirium incidence on medical wards varies from 11± 33%; the diagnosis is often missed in elderly patients. Delirium increases the risk of death 8fold and the frequency of nursing home placement 5-fold. Diazepam likely led to this man's delirium and slowed his rehabilitation. Also, restraints have been shown to exacerbate delirium, adding to deconditioning and slow recovery. In addition, the incidence of delirium is decreased from 15% to 10% by a multicomponent inter-vention including avoidance of benzodiazepines and restraints, but this intervention is not implemented in most hospitals, including ours. As more elders undergo cath, iatrogenic stroke may occur more often. Simple clinical strategies are indicated to prevent delirium, which is often missed and may slow or prevent rehabilitation from stroke.
A. Sura 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73858) LEARNING OBJECTIVES: 1. To recognize the impact of severely elevated triglycerides 2. To recognize the common causes of acute pancreatitis 3. To evaluate and manage the patient presenting with acute pancreatitis CASE INFORMATION: KD is a 17 year old female with a history of hypertriglyceridemia who presented to the emergency department with severe abdominal pain, nausea, and vomiting. She also complained of dizziness and lightheadedness. She denied alcohol use, medication use, family history of pancreatitis, history of gallstones or trauma. Although her symptoms were similar in character to her previous episodes of pancreatitis, they were much more severe. At the time of presentation, she was febrile to 103 degrees Fahrenheit and orthostatic. Her examination revealed significant abdominal distention, absent bowel sounds, and exquisite diffuse tenderness to palpation. There was no rebound tenderness. Her lab work revealed a hemoglobin of 6.8 (normal for her was 15), WBC 15.8, albumin 2.3, calcium 6.5 and triglyceride level of 11,000. Lab work also showed Na 133, K 3.3, Cl 100, CO2 22, BUN <2, Cr 0.4, glucose 217, lipase 1221, amylase 117, triglycerides >11,000, total cholesterol 245, HDL 14, Ca 7.4, albumin 2.3, Tbili 1.6, AST 25, ALT 22, GGT 83 and alkaline phosphatase 96. CT scan of the abdomen revealed severe necrotizing pancreatitis with necrosis of the pancreatic duct, large amount of ascites, and a large retroperitoneal phlegmon. She was started on imipenem, given blood transfusions, and maintained NPO. She was admitted to the ICU for severe necrotizing pancreatitis. IMPLICATIONS/DISCUSSION: Acute pancreatitis is an acute inflammatory process of the pancreas. It is associated with severe acute upper abdominal pain and elevated serum levels of pancreatic enzymes. Most cases are associated with gallstones or alcohol, but the precise pathogenetic mechanisms are not understood completely. Other causes of acute pancreatitis include biliary sludge, post-procedure, hypertriglyceridemia, hypercalcemia, drugs, trauma, HIV, and hereditary causes (pancreas divisum or a genetic mutation). The diagnosis is made via clinical exam, laboratory findings, and, if needed, imaging studies. CT scan is the most helpful imaging test for the diagnosis and its intrabdominal complications. Serum triglyceride concentrations above 1000 mg/dl can precipitate attacks of acute pancreatitis and may account for 1.3±3.8% of cases. The clinical manifestations of hypertriglyceridemia associated pancreatitis are similar to those seen with other causes with the exception that, for poorly understood reasons, the serum amylase may not be elevated substantially. Clinical assessment of severe pancreatitis is as accurate as most scoring systems. Treatment of acute pancreatitis is aimed at correcting predisposing factors and at the pancreatic inflammation itself. General management consists of supportive care with intravenous hydration, pain management, antibiotics, parenteral or enteral (jejunal) feeding, and in severe cases, necrosectomy.
ACUTE-ONSET ADULT TRACHEOBRONCHOMALACIA. A.C. Sura 1 , M. Ghajarnia 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73859) LEARNING OBJECTIVES: 1) To recognize the clinical presentation and diagnostic workup of tracheobronchomalacia in adults. 2) To manage the patient with severe hypoxemia secondary to tracheobronchomalacia. CASE INFORMATION: A 74 year-old male with hypertension and degenerative joint disease presented for preoperative evaluation prior to hip replacement. He endorsed progressively worsening dyspnea with exertion in the past three weeks, orthopnea and lower extremity edema. His oxygen saturation was found to be 69% on room air. He denied fever, chills, chest pain, or chronic cough but endorsed occasional wheezing and cough productive of white sputum in the past two months. He had a distant history of 20 pack-years tobacco use as well as a 50 year history of work in a coal mine. On physical, blood pressure was 147/84, pulse of 69, and respiratory rate of 25; oxygen saturation improved to 91% on non-rebreather facemask. ABG revealed respiratory acidosis with metabolic compensation. The patient developed ventilatory failure with a drop in O2 saturation to 45% and PCO2 of 140. He was intubated, and chest CT revealed left lower lobe collapse. Bronchoscopy showed complete collapse of the left mainstem bronchus. The patient's subsequent hospital course consisted of intermittent respiratory failures requiring repeat intubations. Once extubated, he was maintained on supplemental oxygen and BiPAP. Repeat bronchoscopy found similar findings of flaccid airways. The patient was deemed ineligible for stent placement and high risk for distal collapse given extensive airway involvement. The patient was discharged on oxygen and BiPAP but subsequently weaned off of supplemental oxygen completely. IMPLICATIONS/DISCUSSION: Hypoxemia and hypercarbia refractory to standard treatments for obstructive pulmonary disease may result from airway collapse without any immediately identifiable causes. Well recognized in infants, tracheobronchomalacia (TBM) may also be present in 4.5±15% of all adult patients with respiratory complaints and can mimic chronic bronchitis. It is a disease of middle to late age, more common in males, and in those with a history of chronic respiratory irritation or inflammation but without a clear association to obstructive lung disease. Identified pathology has been limited to a decrease in the longitudinal elastic fibers of the trachea. Bronchoscopy under local anesthesia is the diagnostic gold standard, demonstrating airway narrowing greater than 50% during expiration. Also supportive are pulmonary function tests showing a low FEV1/EIV1 ratio and characteristic notching in the forced expiratory spirogram. Bronchodilators, antibiotics, and mucolytics may be beneficial in patients with concomitant obstructive airway disease or asthma; however, definitive treatment in TBM involves reversing the airway collapse. Avoidance of airway irritants and cough suppression are first line measures. Surgical stenting has been used with success in many cases, but in patients too frail to undergo such procedures or in those with extensive airway involvement, positive airway pressure appears to be the best means of medically managing patients with TBM. Moreover, airway inflammation may play a role in acute airway collapse, and the element of reversibility noted in our patient may point to a role for adjuvant anti-inflammatory medications.
YOUNG WOMAN. G. Szabo 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74064) LEARNING OBJECTIVES: 1) Recognize the potential for lindane toxicity in adults.
2) Diagnose the signs and symptoms of lindane toxicity in adults. CASE INFORMATION: The toxicity of lindane is well known for the pediatric population, but is generally under appreciated among adults. Inappropriate application of lindane by adults can lead to significant neurologic changes and even death. A 22-year-old thin female without significant medical history was brought to the emergency room by her mother after one day of agitation, visual and tactile hallucinations, paranoia and a five-day history of rash. The rash consisted of pruritic erythematous macules on her abdomen and body. The patient was seen at an outlying emergency room, diagnosed with scabies and given lindane cream. Her rash persisted and she went to another emergency room for evaluation; again she was diagnosed with the scabies and prescribed lindane. 2 days later she presented with the above symptoms. Her exam showed numerous crusted, mildly erythematous erosions and excoriations throughout her body. No vesicles or burrows were noted. She was agitated with paranoia and visual hallucinations. An extensive workup was essentially negative and included a normal lumbar puncture, negative blood cultures and herpes simplex PCR, an unremarkable head CT, no vasculitic change on skin biopsy, and negative serologic studies for RMSF, varicella zoster, syphilis, cryptococcus and HIV. After her sensorium cleared she acknowledged that she left the lindane cream on for 12 to 16 hours, applied it all over her body and did not wash her sheets. She was discharged in stable condition without any neurologic deficits. IMPLICATIONS/DISCUSSION: Although lindane deposits in all lipid tissues, it has a high propensity for the white matter of the brain. Generally, children are at higher risk for toxicity because of their smaller size and lipid deposits. However, thin adults like the one described above, are also susceptible. Lindane absorption is enhanced by its contact with irritated skin, another risk factor also present in our case. Lindane toxicity is a clinical diagnosis and as such must be kept in mind especially in patients presenting with a history of ascabies-like rash and mental status changes.
S. Tadic 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76487) LEARNING OBJECTIVES: 1.) Recognize an unusual presentation of a hypertensive emergency in a young person; 2.) Diagnose an atypical presentation of a pheochromocytoma; 3.) Evaluate and treat a catecholamine-producing tumor. CASE INFORMATION: A previously healthy 27-year-old Vietnamese male was brought to the emergency room with an acute onset of an expressive aphasia and left hemiparesis. He was unresponsive upon arrival and required intubation. A head CT revealed a large subcortical intracranial hemorrhage (ICH) involving the right basal ganglia. The patient underwent an emergent right craniotomy with evacuation of the intracerebral hemorrhage and placement of an extraventricular drain. Postoperatively, he was admitted to the Neuro Intensive Care Unit (NICU) for monitoring. Over the next 72 hours the patient was observed by nursing staff to have periods of intermittent hypertension, treated with prn IV medications and hypotension, treated with fluid boluses (SBP range 88±210). After 4 days in the NICU, the patient was observed to have facial and chest flushing. A pheochromocytoma was suspected. Abdominal MRI revealed bilateral large adrenal masses with increased T2-weighted signal. Results of a 24hour urine collection for metanephrines and catecholamines included: metanephrines 6,320 (26±230 mcg/24 hr); normetanephrines 24,760 (44±540 mcg/24 hr); VMA 16.5 (6 or less mg/ 24 hr); and total metanephrines 31,080 (90±690 mcg/24 hr). Urine dopamine 2,136 (80±440 mcg/24 hr); epinephrine 423 (0±25) and norepinephrine 3,071 (0±100). Calcitonin level was 5 (0-8) and genetic testing for von Hippel-Lindau (vHL) syndrome was negative. The patient developed a right femoral DVT and tested positive for the Lupus Anticoagulant. He underwent a bilateral adrenalectomy and was discharged to home 2 months later on Coumadin and Hydrocortisone. Pathology revealed malignant pheochromocytoma and a metastatic workup was negative. His post-surgical evaluation revealed normal levels of urine metanephrines and catecholamines. IMPLICATIONS/DISCUSSION: Although pheochromocytoma is a rare disease, this case represents the value of bedside evaluation by a physician. Paroxysmal symptoms (i.e. intermittent) can often be treated with prn interventions leading to misdiagnosis or delay in diagnosis. A rare presentation of pheochromocytoma is one of cyclic fluctuations of HTN and hypotension that occurs via an uncertain mechanism. While most pheochromocytomas are sporadic, bilateral tumors are often associated with 2 primary familial disorders: MEN-2 and VHL. Measurement of plasma or urine catecholamines and metabolites are diagnostic in 95% of patients with symptoms. MRI scans are sensitive (98±100%) but are only 70% specific. On T2-weighted images, however, pheochromocytomas appear hyperintense while other adrenal tumors appear isointense. While there is no universally accepted method of preparation for surgery, alpha-adrenergic blockade followed by beta-blockers with surgery 2 weeks later, is common practice. Recurrence rates are 14% at 10 years. LEARNING OBJECTIVES: This clinical vignette discusses the ethical implications of using medication and housing contracts in voluntary case management programs. The Medical High User Case Management Program provides intensive case management, assisting patients with medical adherence, entitlements and housing. When appropriate, case managers will draft housing and medication contracts for opiate pain medications Contracts are done in consultation with primary care providers and the program internist psychiatrist and nurse.
As part of these contracts, the program will assist with the refilling of opiates and provide temporary housing. Patients must agree to make scheduled medical and substance abuse appointments. CASE INFORMATION: The patient is a 47-year-old woman with a history of admissions for CHF secondary to cocaine dependence. Her care has been complicated by a history of borderline personality disorder, chronic back pain, a history of upper GI bleeds, major depression and homelessness. The patient's stated goals on enrollment include housing and sobriety. The patient signed a contract that included opiate pain medications, temporary housing, substance abuse treatment and money management. Over the course of the next few months the patient would repeatedly binge on cocaine after receiving her general assistance check at the beginning of each month. The case management program terminated the housing and medication contract and asked the patient to see her primary care physician for all opiate refills. While the patient continued to binge on cocaine, the case manager continued to assist the patient with non-opiate medication refills, appointments with her primary care physician and applications for both housing and a money management. The patient eventually enrolled herself in a residential drug treatment program. She was restarted on her pain medication contract after seeing her primary care provider and is awaiting housing. IMPLICATIONS/DISCUSSION: While we believe that contracts for housing and pain medication refills may be useful in providing external control for seriously medically ill patients, these contracts poses important ethical questions. These contracts may challenge the principle of patient autonomy in patients who retain the legal ability to make medical decisions. These contracts also use housing and pain medications as incentives, items that many feel should be viewed as rights. Therefore these contracts should never deny patients access to the usual standard of care. Instead they should be voluntary and provide additional services that are contingent on the patient's adherence to a care plan. It is also important that contracts be done as part of an ongoing patient provider relationship to insure patient safety. Finally these contracts have the potential to reduce morbidity and mortality, which remains an important ethical imperative. A 48 year old male presented with crushing chest pain of several hours duration and a worsening of his chronic headaches (which had started 8 months ago after a closed head injury). In the previous 30 hours, he had taken 14 tablets of clonidine to try to relieve the headache which he thought was due to his hypertension. On review of systems, he noted a 35 pound weight loss over the past 8 months and admitted to sexual activity with several prostitutes. The only other medication he takes is ibuprofen. His vitals revealed he was afebrile with a pulse of 42 and a blood pressure of 205/133. His exam showed some temporal wasting and grade III hypertensive retinopathy without papilledema. He had an S4 present, but the remainder of his initial cardiac, respiratory, abdominal, neurologic and musculoskeletal exam was unremarkable. Serial ECGs showed sinus bradycardia, left ventricular hypertrophy, and T-wave inversions in the lateral and inferior leads. Initial electrolytes, CBC and serial cardiac biomarkers were also unremarkable. A head CT without contrast showed grey-white matter changes consistent with hypertension. The patient was admitted to the cardiac care unit. A CT scan with IV contrast of the abdomen and thorax showed no evidence of an arotic dissection. His blood pressure was controlled with IV nitroglycerine initially, but then required fenoldopam and nicardipine drips, parenteral hydralazine, procardia, clonidine, and labetolol. He developed a third cranial nerve palsy on the right and his serum sodium dropped from 137 mmol/liter on admission to 121 mmol/liter three days later. CT scans of the head were repeated twice with and without contrast with no change from the initial CT scan. A lumbar puncture was performed and cryptococcal meningitis was diagnosed. A subsequent HIV test was positive. His CD4 count was 12. Amphotericin B was started. Elevated intracranial pressure was controlled by serial lumbar punctures. The patient's cranial nerve palsy showed minimal improvement, but his hypertension improved. IMPLICATIONS/DISCUSSION: The presence of intractable hypertension, bradycardia, and headache suggests increased intracranial pressure as a cause for his hypertension. It is a wellknown cause of hypertension but rarely considered. In this case, his bradycardia was initially thought to be secondary to clonidine. He also lacked any focal neurologic deficits or papilledema on initial presentation to suggest an intracranial pathology. A retrospective casecontrol study published in 1992 showed an increased incidence of diastolic hypertension in AIDS patients with cryptocccal meningitis.
SIADH. CAUSING OR CAUSED BY PSYCHOSIS? A. Tendler 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77076) LEARNING OBJECTIVES: 1. Recognize organic psychosis as a correctable cause of SIADH 2. Distinguish SIADH from from psychogenic polydypsia. 3. Know other causes of SIADH in the psychiatric patient. CASE INFORMATION: A 56 year-old woman with a history of schizophrenia was admitted to the inpatient psychiatry service with paranoid delusions of her brother trying to kill herself and all religious ministers. Her physical examination was normal, including normal neurologic and pulmonary examinations. Her serum sodium was 124 mmol/L and her measured osmolality was 261 mosm/kg. The urine osmolality was 522 mosm/kg. She was treated with risperidone and free water was restricted. She was allowed a liberal salt-containing diet. Her psychosis improved over the next week, with normalization of her serum sodium. IMPLICATIONS/DISCUSSION: Hyponatremia in the schizophrenic patient should not be reflexively attributed to psychogenic polydyspia. Because ADH is liberated from the hypothalamus through the posterior pituitary, anti-psychotic medications acting at surrounding structures may cause SIADH, as may primary psychosis. The urine osmolarity is the best test to distinguish SIADH from psychogenic water drinking, as the concentrating effects of ADH should increase the urine osmolarity. Hyponatremia may cause neurologic impairment, but this impairment is usually in the form of seizures as opposed to psychosis. Treatment of psychosis-induced SIADH is with neuroleptics designed to control the psychosis. Medical consultation for hyponatremia in the psychiatric ward is a common occurrence . Physicians should be aware of this underdiagnosed cause of hyponatremia, and of its successful resolution with anti-psychotic therapy. received no relief, however, and he re-presented with persistent back pain and fevers. Further questioning revealed he had a past history of mitral regurgitation and had had a tooth extraction six months prior. He had a 3/6 systolic murmur at the apex and point tenderness over the lumbar spine. His blood cultures grew Streptococcus viridans. An MRI of the lumbar spine showed edematous changes at T12-L1 with loss of disc space, consistent with discitis. An echocardiogram confirmed endocarditis. IMPLICATIONS/DISCUSSION: Although the diagnosis of urinary tract infection and lower back strain adequately explained his symptoms, each of the two diagnoses was insufficient in explaining all of the symptoms. William of Occam suggested that,``one should not increase, beyond what is necessary, the number of entities required to explain anything.'' Known as Occam's Razor, this principle guides physicians to search for one diagnosis to explain all symptoms, instead of invoking multiple diagnoses. The fever, bacteremia, discitis and immunemediated glomerulonephritis in a patient with valvular abnormality and poor dentition can all be attributed to infective endocarditis.
AN OIL PIPE CLEANER OUT OF BREATH. D. Tran 1 , J. Wallace 1 ; 1 UCLA±San Fernando Valley Program, Sylmar, CA (Tracking ID #75804) LEARNING OBJECTIVES: 1. Recognize silicosis as a cause of pulmonary fibrosis. 2. Discuss the differential diagnosis of silicosis CASE INFORMATION: 25 y/o Hispanic male presented to ER for increasing dyspnea on exertion. His symptoms began insidiously about a year prior to admission, and have gradually worsened to the point where he can walk less than 1 block. His clinical course, thus far, has been punctuated by periods of exacerbation for which he was treated with antibiotics and prednisone with moderate success. He denies fever, chills, weight loss. There is no history of wheezing, asthma, chest pain, orthopnea, PND, or edema. The patient emigrated from Mexico three years ago. He denies any current or past of tobacco, drug abuse, or alcohol. On further questioning, the patient states that he spent three years in Mexico working as an oil pipè`c leaner''±which involved sandblasting. Physical exam and labs are only significant for a respiratory rate of 32 and paO2 of 70 mmHg at rest. CXR showed diffused bilateral infiltrates. High Resolution CT showed extensive, bilateral, coalescing pulmonary nodules with an upper lobe predominance and non-calcified hilar adenopathy. Patient is PPD and HIV negative. ACE level was 73 (9±67). He underwent video assisted thoroscopic lung biopsy. The pathology showed non-casseating granulomas with bifringent spicules consistent with sicilosis. Fungal and AFB cultures were negative. IMPLICATIONS/DISCUSSION: Silicosis is a type of pneumoconiosis caused by the inhalation of silica dust leading to pulmonary fibrosis. Individuals who work in metal mining or cutting, polishing, or carving stone are at risk. Symptoms usually develop after 3 to 20 years, depending on exposure level. Silicosis can either be simple: asymptomatic and non-progressive, identified by small, round opacities on chest x-ray; or complicated: symptomatic with progression despite discontinuance of the insults. Differential diagnosis includes sarcoidosis, coal worker's pneumonconiosis, neoplasia, TB, and fungal pneumonia. The diagnosis of silicosis is based on careful occupational history, radiographical evidences and pathology. Radiographically, silicosis is characterized by small, round opacities with an upper lobe predominance that can form extensive coalescing masses in advanced cases. The presence of eggshell calcifications on hilar lymph nodes may help distinguish silicosis from other pneumoconiosis. ACE level is elevated in sarcoidosis but can also be elevated in miliary TB, berylliosis, asbestosis, and silicosis. When the diagnosis is in doubt, biopsy can help establish the diagnosis and exclude other causes. On pathology, hyalinized silicotic nodules can be seen. They are formed by macrophages engulfing the particles, releasing cytotoxic enzymes causing fibrosis. There is a high incidence of TB co-infection in individuals with silicosis. Co-infection can be confused with a progression of the silicosis and should be excluded first. There is no effective treatment aside from preventing further exposure.
A 92 YEAR-OLD WOMAN WHO TOOK A TURN FOR THE WORSE. V. Tsai 1 , R. Ziegelstein 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #75756) LEARNING OBJECTIVES: To call attention to the possibility that moving a patient soon after a cardiac procedure can result in malposition and malfunction of a permanent pacemaker. CASE INFORMATION: A 92-year-old woman was found unresponsive in her nursing home bed. She was in complete heart block with a ventricular rate in the 20s±30s. Soon after arrival to the hospital, she sustained an asystolic arrest and required emergency temporary pacemaker therapy. The patient did not regain consciousness after electrical pacing at 60 beats per minute. The patient's health care agent consented to the implantation of a permanent pacemaker. After the procedure, a chest radiograph showed an appropriately placed dual chamber permanent pacemaker. The patient did not regain consciousness nor spontaneous movement. Nursing staff turned the patient every two hours to avoid pressure sores. Several days later, a chest radiograph showed that the pacemaker generator was rotated and in the opposite configuration from that noted on the original film. There was no evidence of electrode dislodgment or pacemaker malfunction. IMPLICATIONS/DISCUSSION: Pacemaker-twiddler's syndrome is an unusual complication of permanent pacemaker implantation, first described by Bayliss, et al. in 1968 . It is usually characterized by partial or total loss of pacemaker function secondary to rotation of the pulse generator in its subcutaneous pocket and subsequent electrode dislodgment. The rotation is related to manipulation of the generator by the patient, either consciously or unconsciously. The present report differs from previous cases, since the pacemaker generator rotation could not have been induced by the patient, but was likely the result of routine turning of the patient by the nursing staff in an effort to prevent pressure ulcers. The possibility that turning immobile patients soon after device implantation will result in malposition and malfunction of pacemaker or cardioverter-defibrillator generators is important to recognize, since these devices are being used in an increasing number of elderly patients. Caution should be exercised while moving elderly bedbound patients in the first few days after their cardiac procedure, in order to prevent a potentially life-threatening condition.
THE USUAL SUSPECT IN AN UNUSUAL SPOT. J.T. Tseng 1 , P.P. Balingit 1 ; 1 UCLA San Fernando Valley Program, Sylmar, CA (Tracking ID #76488) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of liver abscess. 2. Consider mycobacterial and other atypical organisms as etiologies of liver abscess in the immunocompromised host. CASE INFORMATION: A 41 year old Mexican male with no significant past medical history presented to an urgent care clinic with 1 month of gradually worsening right upper quadrant abdominal pain associated with fevers and night sweats. He described the pain as a dull, constant discomfort without radiation, exacerbated with movement and without change with food intake. Several episodes of nausea and vomiting occurred in the last two days. Intermittent watery diarrhea was present over the past two weeks. He sought medical attention for his abdominal pain one week ago and was prescribed levofloxacin and metronidazole without relief of symptoms. The patient reported frequently visiting Mexico, his last trip being 2 months prior. He denied HIV risk factors including homosexual or promiscuous sexual behaviors, IV drug use, or transfusions. Exam was significant for temperature of 38.8 C and heart rate of 106. Tenderness was elicited upon palpation of the right upper abdominal quadrant. No hepatomegaly was apparent, and a Murphy's sign was not present. CBC was remarkable for lymphopenia. Liver transaminases were within normal limits. No organisms were identified from blood cultures, stool cultures, and studies for ova and parasites. HIV antibody test was positive. Abdominal CT scan revealed a 2 Â 2 cm low-density irregular lesion consistent an with abscess within the right hepatic lobe. Tissue obtained from percutaneous drainage of the liver lesion revealed necrosis and granulomatoid inflammation. AFB stain was positive, and TB was identified in culture. TB was also isolated from the patient's sputum, despite the absence of cough, dyspnea, or other pulmonary symptoms. Four-drug antitubercular therapy was initiated with resolution of the patient's presenting symptoms. IMPLICATIONS/DISCUSSION: Liver abscesses of varying etiologies tend to have similar clinical presentations. Patients typically present with fever, night sweats, and right upper quadrant abdominal pain. Identification of the causative agent becomes critical when patients fail to respond to empiric therapy against pyogenic or amebic abscesses. Serologies are often not helpful in identifying an organism as assays often lack sufficient sensitivity or specificity and blood cultures may not reveal the offending agent. While CT, MRI, or ultrasonography may help differentiate liver abscesses from neoplastic processes, they are unable to identify a specific organism. Drainage of the abscess remains the mainstay of diagnosis. For immunocompromised patients, laboratory analysis of drainage fluid should include evaluation for atypical organisms as well as Gram stain, culture, and ova and parasites. In these patients, mycobacterial and fungal organisms may form abscesses in addition to typical granulomatous lesions when they invade the liver. LEARNING OBJECTIVES: 1. Acromegaly can present in younger patients 2. The role of insulin-like growth factor 1 in diagnosis and therapy 3. The importance of early diagnosis CASE INFORMATION: 22 y/o latino male presented to our urgent care clinic with a history of headache for over 2 years. The headache was intermittent in nature, mostly frontal and without exacerbating, relieving factors or associated symptoms and with minimal relief from analgesics. On systems review he admitted to an increase in the size of his hands and feet. His family also reported a gradual change in his appearance. On examination, the patient was normotensive. He had prominent supraorbital ridges and a large lower jaw with poor occlusion of teeth and lower teeth overbite. His facial wrinkles were exaggerated and lips full. The nose tongue and ears were enlarged, the hands were large, doughy spade like and the skin over them thickened. Neurological exam showed grossly intact bitemporal visual fields. His appearance was consistant with the diagnosis of acromegaly. A work up consisting of growth hormone (GH), insulin-like growth factor 1 (IGF-1), TSH and random glucose was initiated with MRI imaging of the brain. The patient was also referred to ophthomology for visual field testing. IMPLICATIONS/DISCUSSION: Acromegaly is a disease caused by secretion of excessive amounts of growth hormone, almost always occurring as a result of a benign adenoma of the anterior pituitary gland. The disease occurs most frequently in middle age with an incidence of 3±4 per million. Somatotroph adenomas of the pituitary may be large enough to cause visual impairment or headaches as in our patient. Acromegaly leads to decreased life expectancy with a 2±3 fold increase in mortality from cardiovascular causes, cancer and all causes. The diagnosis is made clinically and confirmed by finding a high levels of GH and IGF-1. Growth hormone binds to receptors resulting in the stimulation of production of IGF-1 which mediates most of the actions of GH. Growth hormone release is pulsitile with concentrations varying throughout the day. Random GH is therefore not a reliable diagnostic marker and is only useful when correlated with the glucose tolerance test. IGF-I is present at more steady concentrations and is a reliable marker of disease and its control. Most patients are treated with transphenoidal surgery which usually results in clinical improvement with decreased GH and serum IGF-I concentrations. Other options include radiation therapy, medical therapy with dopamine agonists or somatostatin analogues. Pegvisomant is a newly developed GH receptor antagonist that leads to decreased IGF-I levels and clinical improvement. Reducing growth hormone levels improves symptoms and complications of the disease and increases life expectancy.
JUST ANOTHER CASE OF UNSTABLE ANGINA? G.J. Van Londen 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76750) LEARNING OBJECTIVES: 1.) Distinguish between Type A and B aortic dissections; 2.) Recognize an uncommon clinical presentation of an aortic dissection; 3.) Manage and treat a dissecting aorta. CASE INFORMATION: A 56-year-old male with a history of a 3-vessel coronary artery bypass graft in 1991, HTN and hyperlipidemia presented to the Emergency Room (ER) with bilateral jaw and retrosternal chest pain accompanied by an occipital headache and dyspnea on exertion. The chest pain was waxing and waning in intensity, non-radiating with a maximum pain score of 5/10. There was diaphoresis but no nausea or vomiting. The night prior, he noted some parathesias in his fingers and blurred vision that resolved within two hours. On exam, the patient's pulse was 110 with a BP of 130/80 on the left and 124/70 on the right. Room air pulse ox was 96%. No pulsus paradoxus, JVD or bruit was appreciated. Lung exam revealed decreased breath sounds in the left lower lobe. Cardiac exam was regular rhythm with a 2/6 diastolic murmur. Exam of the upper and lower extremities revealed equal and symmetric pulses. EKG showed sinus tachycardia with no ischemic changes. His first set of cardiac enzymes was negative. Portable chest x-ray revealed a left pleural effusion and a widening of the mediastinum with the patient rotated to the right. The patient was diagnosed in the ER with unstable angina and admitted. Based on the diastolic murmur and left pleural effusion on chest x-ray, an aortic dissection was suspected and a stat MRI was performed. This revealed a Type A dissection. Emergent surgery with replacement of the ascending aorta, valve and evacuation of a left hemothorax was performed. Cardiac arrests and a CVA complicated the perioperative course. The patient expired nine days after admission. IMPLICATIONS/DISCUSSION: A dissecting aorta must be recognized quickly as an acute Type A dissection (involves the ascending aorta) is a surgical emergency, whereas an uncomplicated Type B dissection (all others) can be treated with pharmacotherapy. Acute management consists of blood pressure control while confirming the diagnosis and type. Aortic dissection is relatively common with over 2000 cases reported each year. A recent study showed that 100% of dissections were diagnosed when all of the following clinical features were present: 1.) Acute onset of tearing or ripping-like chest or back pain; 2.) Mediastinal widening on chest x-ray and 3.) Variation in pulse and/or pressure in the upper extremities. A diastolic murmur, EKG without ischemic changes and a left-sided pleural effusion (hemothorax) are uncommon presentations of a dissection. Transesophageal echocardiogram (TEE) is recommended for unstable patients with acute chest pain. MRI is preferred in stable patients with chronic chest pain. CT scan or aortography should be reserved for situations in which both TEE and MRI are unavailable.
A. Venditto 1 , J. Wiese 1 , M. Landry 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77081) LEARNING OBJECTIVES: 1) Utilize effective diagnostic methods in cases of encephalitis. 2) Recognize the importance of early diagnosis of West Nile virus to control disease outbreaks. CASE INFORMATION: A 58-year-old man presented with two days of fever, headache, unsteady gait, and a macular rash. Although initially lucid, he deteriorated into a state of delerium within the first day of hospitalization. His vital signs were normal. He had meningismus with passive and active neck flexion; he had an ataxia gait and a macular rash over his extremities. There were 6 white blood cells /mm3 in the CSF and a normal protein and glucose. Owing to the degree of impairment, a serum immunofluorescent assay for flavivirus IgM was ordered. The titer was 1:32, increasing to 1:64 over the next five days. An assay for West Nile virus was sent to the CDC for public health monitoring. IMPLICATIONS/DISCUSSION: Most non-herpetic viral encephalitities are due to arboviruses that cycle with the lifecyle of the tick or mosquito that is the primary vector. While all are endemic to warm climates, viruses such as the Eastern Equine Virus, West Nile and St. Louis encephalitis can become epidemic when the mosquito vector greatly exceeds its primary host. For all three viruses, there is little more than supportive care to be offered. Contrary to most medical interventions, however, testing for a definitive diagnosis has merit in providing community outbreaks. The best test for distinguishing St. Louis from West Nile is an enzyme-linked immunosorbent assay (ELISA). A positive serum ELISA indicates probable infection with West Nile virus, whereas a positive cerebrospinal fluid ELISA confirms the diagnosis. Early identification of West Nile virus is important for mosquito population control and public education to prevent further disease spread. LEARNING OBJECTIVES: To Illustrate potential clinical presentations that must be considered in a patient with sarcoidosis CASE INFORMATION: History: 49 year old African American female with history of obesity, asthma and possible fibromyalgia presented with worsening fatigue, SOB, DOE over 2 months. She has been tobacco-free for the past 4 years and compliant with her asthma regimen. Over the ensuing 6 months she continued to have worsening symptoms which required episodic reinstitution of systemic steroids for presumptive asthma exacerbation. Further review revealed a past medical history of a 2 cm right upper lobe nodule with bilateral hilar adenopathy and interstitial lung disease 2 years ago. CT guided-biopsy of the nodule yielded non-caseating granuloma. Serum labs, such as CBC, Chem 10 (including calcium), TSH, ESR and ACE levels, were normal. LFTs were mildly elevated. Subsequent viral hepatitis serologies were negative. PFTS were consistent with combined obstructive and restrictive lung disease with mildly reduced DLco. The patient returned for an urgent care visit complaining of worsening DOE, bilateral lower extremity edema, polyuria, nocturia, blurry vision and severe persistent daily headache and stiff neck for the past 2 months. Prednisone 60 mg was started empirically for pulmonary sarcoidosis exacerbation and possible sarcoidosis involvement of the eyes, CNS, liver and heart. Repeat of the above serum labs were normal. Fasting glucose level was not diagnostic for diabetes mellitus or glucose intolerance. Urinalysis and urine C&S were normal. Urine osmolality was ordered but not performed. A water deprivation test was deferred until other test results were known. PT (INR) and albumin did not suggest cirrhosis. PFTs were unchanged from the prior study. PPD test was <5 cm. Gallium scan showed no uptake in the lung, salivary and lacrimal glands (e.g.``panda bear'' sign) or any other part of the body. ECG and echocardiography were also normal. MRI of CNS showed dural enhancement but no pituitary or other parenchymal lesions. CSF fluid analysis revealed only a lymphocytic pleocytosis and negative cytology for malignancy. Ophthalmology referral showed no uveitis, cataracts or retinal abnormalities. The patient's symptoms improved but she complained of increasing weight, generalized edema, low back pain and bilateral knee pain after 6 months of Prednisone therapy. The patient reported no further polyuria, headaches, stiff neck, and blurry vision but still had residual DOE and fatigue. Repeat MRI showed no dural enhancement. Prednisone was tapered off and the resultant decrease in weight and edema lead to resolution of her low back and knee pain. IMPLICATIONS/DISCUSSION: Discussion: Sarcoidosis can have numerous extrapulmonary involvement. CNS and cardiac involvement portend worse prognosis than hepatic, pulmonary or arthritic involvement. SOB and DOE is most likely due to pulmonary involvement (i.e., due to endstage parenchymal fibrosis with or without acute inflammation) but concomitant restrictive cardiomyopathy due to sarcoidosis may exist synchronously. Echocardiography is essential in ruling out this possibility. Hepatic sarcoidosis involvement can progress from mild elevations in LFTs to frank cirrhosis, which fortunately was not present in this patient. The LFTs elevation may, however, be simply due to fatty liver of obesity. Uveitis due to sarcoidosis must be a consideration in patients with vision complaints, but retinitis, episcleritis, glaucoma and premature cataracts, as well as the sicca syndrome due to involvement of the lacrimal and salivary glands, can occur. Symptomatic CNS sarcoidosis classically manifests as diabetes insipidus due to posterior pituitary infiltration. Other CNS syndromes include aseptic meningitis and diffuse white matter disease. Diabetes insipidus is a distinct possibility in this patient. Although comparison of urine and serum osmolality and water deprivation test were not done to confirm diabetes insipidus, the patient's polyuria readily responded to glucocorticoids therapy. The MRI and CSF findings clearly support an aseptic meningitis picture and her headache symptoms responded to glucocorticoids therapy. LEARNING OBJECTIVES: To recognize a hypersensitivity reaction to dialyzers in a patient on hemodialysis CASE INFORMATION: A 67 year old female with end stage renal disease secondary to chronic uncontrolled hypertension was admitted to the hospital with fevers and chills. On admission, the potential etiologies of her fever were line sepsis or lower extremity cellulitis. She was begun on appropriate antibiotic therapy. Within 5 minutes on her routine hemodialysis, she suddenly became bradycardic, hypotensive and unresponsive. On examination before dialysis, vitals were HR-80/min, BP-215/110 mm Hg, and RR-16/min. Lungs were clear to auscultation. Cardiovascular exam revealed a normal S1 and S2 and a 3/6 holosystolic murmur at the apex. After 5 minutes on dialysis, her vitals were HR-48/min, BP-64 mm Hg systolic; respirations became agonal, followed by apnea. The patient became obtunded and unresponsive, with twitching of her lips. She sustained cardio-respiratory arrest and required CPR with endotracheal intubation and mechanical ventilation and oxygenation. Dialysis was aborted during this episode. Her EKG revealed pulseless electrical activity followed by junctional rhythm. Once dialysis was stopped, it converted to normal sinus rhythm with normalization of hemodynamic parameters. Her WBC was 8000, H/H 8.2/ 25.1, electrolytes were normal, BUN 72 and Cr 14.6, BNP-1300, CPK 20, Troponin I-0.21. Chest X-ray showed mild congestion and her subclavian catheter tip was in right atrium. Echocardiogram revealed concentric left verntricular hypertrophy with normal LV function. 72 hours later, at her next routine hemodialysis, a similar episode occurred. Heparin was discontinued from her dialysis regimen due to possibility of heparin sensitivity as she was thrombocytopenic with presence of heparin associated antibodies. However, a third episode occurred on the next dialysis. At this time, the patient's dialyzer was changed from B3-1.3 to F-8. Thereafter, no further episodes occurred and she tolerated subsequent hemodialysis without complication. IMPLICATIONS/DISCUSSION: Dialyzer-induced acute hypersensitivity reaction is a rare entity (5/100,000 dialysis cases), but is a potentially life threatening condition. Presentation varies from mild urticarial reaction to cardiorespiratory failure. The onset of symptoms due to dialyzer-induced hypersensitivity reaction is usually 5±30 minutes after initiation of hemodialysis. Possible causes are bio-incompatible membrane (i.e. cellulose), ethylene oxide use for sterilization, use of ANP69 membrane in patients on ACE inhibitors, bacterial contamination of dialysate in high flux dialyzer, heparin allergy, acetate bath (acetate is a vasodilator and myocardial depressant), and inappropriate cytokine and complement activation. Differential includes severe hypotension, acute MI, arrhythmias, air or pulmonary embolism, or severe pulmonary edema. A high index of suspicion is necessary to recognize this entity. Treatment is standard care for any anaphylactic reaction and includes intravenous steroids, antihistamines and supportive care. Removal and avoidance of the culprit inciting agent is of paramount importance. A 67 year-old Filipino male presented to the ED with right facial erythema, edema, and subjective fever. The lesion progressed from a pimple 2 days prior. His past medical history was significant for Lepromatous Leprosy diagnosed in 1998. He had been treated with rifampin and ofloxacin since then. In the ED, he was treated with amoxicillinclavulanate for presumed cellulitis. Nevertheless, his facial lesion worsened, and he developed diffuse lymphadenopathy and a reddish rash on his upper extremities. Upon admission, the patient was afebrile with normal vital signs. His exam demonstrated left facial erythema, edema, and induration with central ulcertation extending from the maxillary area to the left ear and mandible and exhibited normal sensation. He had diffuse lymphadenopathy of the head and neck, and erythematous plaques on both upper extremities. His chronic findings of a left hand claw deformity and decreased sensation in the lower extremities were unchanged. Lab studies were notable for a WBC of 13 and blood and wound cultures were negative. Antibiotics were changed to vancomycin and imipenem without improvement. Subsequent skin biopsy revealed numerous acid-fast rods within macrophages and a dense neutrophilic infiltrate. His clinical presentation and biopsy findings were consistent with a Type II lepromatous reaction, or Erythema Nodosum Leprosum (ENL). He was treated with prednisone and thalidomide with ultimate resolution of his symptoms within days of treatment. IMPLICATIONS/DISCUSSION: ENL is characterized by fever with multiple erythematous tender nodules, sometimes accompanied by neuritis, edema, arthralgias, leukocytosis, iridocyclitis, pretibial periostitis, orchitis, and/or nephritis. It is associated with Borderline and Lepromatous forms of Leprosy and occurs in up to 25% of patients. ENL is most common during treatment, but can also occur before or after therapy. It is thought to be an immunecomplex disorder with tumor necrosis factor alpha playing a role in its pathogenesis. Acute treatment consists of steroids and/or thalidomide; the latter is highly effective, generally controlling the reaction within 48 hours. Clofazimine may be effective for chronic reactions. Prompt detection and treatment of ENL has decreased the number of patients with resultant chronic disabilities. Our patient had a clinical picture consistent with ENL which was initially difficult to distinguish from cellulitis. He responded to treatment with prednisone and thalidomide. As international travel and immigration continue to increase, it is important to recognize the reactions associated with Mycobacterium Leprae. year-old man was admitted for elective T12-L2 posterior spinal fusion. His past medical history included CAD, s/p CABG, and idiopathic scoliosis with prior spinal surgeries. Six hours post-operatively, the patient developed hypotension, fever and hy-poxemic respiratory failure. Intra-operatively, he received multiple blood products including 6 units of whole blood, 5 units of PRBC, 12 units of FFP, and 3 6-packs of platelets. On exam in the ICU post-operatively, he had a temperature of 398C and a BP of 70/ 40. He had a normal cardiac exam including a normal JVP, lungs had coarse breath sounds, and his extremities were warm without a rash. Chest x-ray revealed diffuse bilateral infiltrates and a chest CT was negative for PE. Echo showed no focal wall motion abnormalities. Labs were notable for a new low WBC count (0.4). Successive blood and sputum cultures were negative. The blood bank was notified to investigate a possible transfusion reaction. There was no evidence of hemolysis and a direct Coombs' test was negative. The patient was treated with vasopressors, including epinephrine, and broad-spectrum antibiotics. On post-operative day 7, the patient suffered a cardiac arrest and expired despite full resuscitative efforts. Post-mortem analysis revealed that 1 of the 32 total blood product donors was a multiparous female who donated a platelet pheresis. She carried an HLA-I antibody against one of the patient's neutrophil antigens (Bw4 public antigen). This confirmed the cause of death as transfusionrelated acute lung injury (TRALI). IMPLICATIONS/DISCUSSION: TRALI is a clinical syndrome characterized by fever, severe hypoxemia, non-cardiogenic pulmonary edema, and hypotension occurring one to six hours post transfusion. Case reports have identified transient leukopenia as another manifestation. 80% of patients recover within 48-96 hours with little permanent sequelae. Despite being the third most common cause of death from transfusions in the developed world (estimated incidence of 1/5000 transfusions, with a mortality rate of 5±14%), it is probably significantly under diagnosed. The majority of cases are associated with transfused complement-activating antibodies, either HLA (class I or II) or granulocyte specific. All blood components containing plasma have been associated with the injury. Risk factors include multiparous donors, underlying recipient conditions such as recent surgery, cytokine treatments, massive blood transfusions, active infection, and prolonged storage of transfused products. Current treatment is limited to respiratory and hemodynamic supportive measures. Diuretics and steroids have been used with variable efficacy and have not been tested in clinical trials. Our patient had an unusual course for TRALI in that his symptoms developed relatively late post-operatively and he did not recover despite maximal support. TRALI should be considered in all patients with hypoxemia following transfusions. Previously, the patient was playing golf 3 times a week without difficulty. His hypoxia began post surgery to relieve a small bowel obstruction. He was treated with antibiotics for pneumonia and ruled out for PE with 2 low probability V/Q scans. The patient was discharged home on oxygen, but his symptoms worsened. He had increasing malaise with shortness of breath upon minimal exertion, such as sitting up in bed. Eventually, he was bed bound. He denied cough, fever, wheezing, chest pain, or leg swelling. Upon admission the patient was afebrile, BP 130/70, RR 18 and supine O2 saturation of 93% on 10 liters of oxygen. Upon sitting, the patient was dyspneic with RR 40 and O2 saturation 70% on 10 liters of oxygen. His exam was normal including a normal cardiac exam including JVP, slightly decreased breath sounds at the right base, and his extremities were warm without edema. Laboratory exam revealed a normal EKG, CXR with unchanged right opacity, chest CT negative for PEor A-V fistula, electrolytes, CBC, and cardiac enzymes were also normal. PA02 on 4 liters of oxygen was 54 mm Hg and on 100% oxygen 84 mm Hg. Transthoracic echo showed normal left ventricular size and function with an inter-atrial septum hypermobility and a large patent foramen ovale with normal right sided pressures. Injection of agitated saline revealed a large amount of immediate right to left shunting. Transesophageal echo confirmed the PFO with right to left shunting. The patient underwent transcateter closure of his defect. One day post procedure, the patient was markedly improved with O2 saturation on room air of 95% supine and sitting. The patient continued to improve as an outpatient and was playing golf within 2 weeks after discharge. IMPLICATIONS/DISCUSSION: Our patient exhibited the platypnea-orthodeoxia syndrome. This is defined as dyspnea and arterial desaturation in the upright position and improved by recumbency. This syndrome occurs with an intracardiac or intrapulmonary shunt. Our patient had the most common etiology, a patent foramen ovale, with a right-to-left shunt. Shunting in the face of normal right sided pressures, as in this case, is unusual but described in the literature. Diagnosis is made by visualization of the PFO with shunting on transesophageal echocardiogram with agitated saline (``bubble study''). Percutaneous closure of the PFO is safe and effective. Cardiac shunts should be suspected in all patients with persistent hypoxia, especially in those who exhibit worsening symptoms and hypoxia in an upright position.
SMOKING CHINESE BROCCOLI? J.L. Yuh 1 , E. Yee 2 , A.G. Gomez 3 ; 1 Olive View±UCLA SFVP, Sylmar, CA; 2 VA Albuquerque±University of New Mexico, Albuquerque, NM; 3 University of California, Los Angeles, Sepulveda, CA (Tracking ID #75461) LEARNING OBJECTIVES: 1. Recognize acute nicotine in toxication 2. Discuss the management and treatment of acute nicotine intoxication CASE INFORMATION: A 56 year old Thai nun, non-smoker, with no known medical history was brought in for an acute onset of confusion, lethargy, and diffuse body weakness. Following breakfast, the patient was found to be minimally responsive next to a bowl of soup. By report, this soup contained many leaves of Chinese broccoli self-grown at the temple. In the ED, the patient's presenting GCS was 9, T 37.1F, pulse 49, BP 91/39, respirations 16, and room air O2 sat 94%. She was minimally responsive and diffusely flaccid on motor exam. IV fluids, NG charcoal lavage, and sorbitol were administered. CBC, CHEM 10, LFTs, and UA were all normal; serum/urine toxicology screen, acetaminophen and alcohol levels were negative. CXR was clear, and head CT did not show any shifts, masses, or bleeds. EKG showed only sinus bradycardia at 50 bpm. On repeat exam, pt was slightly more responsive since presentation, with pupils dilated and sluggish, but not following commands in English or Thai. She was admitted to the ICU for monitoring. Several leaves of the Chinese broccoli used in the soup were brought in later. Upon calling poison control and researching botanical atlases, it was determined to be a tobacco plant. Her serum nicotine level was low, while her serum cotinine level was high, indicating the metabolism of nicotine to cotinine, and confirming an ingestion of 15+ tobacco leaves. The patient regained her strength and alertness gradually and was discharged in normal health on day 3. The temple members subsequently removed all remaining`Chinese broccoli' plants! IMPLICATIONS/DISCUSSION: Nicotine overdose can result from overuse of cigarettes, nicotine gum or patches, and plant ingestion. It is seen most commonly in patients smoking while on a patch. Patients can exhibit weakness, convulsions, coma, respiratory distress/apnea, pupillary dilatation, abdominal cramps, vomiting, initial tachycardia/hypertension, followed by bradycardia/hypotension. Diagnosis is by careful history and physical exam as there are no set serum toxicity levels. The half-life of nicotine is 0.5±2 hours while that of cotinine (a better marker due to its slower clearance) is 12±30 hours. Management includes airway protection, charcoal lavage, sorbitol, hydration, and circulatory support. Symptoms can last several hours, and survival after 4 hours of intoxication usually prognosticates complete recovery. Lethargy may remain for several days. Caution needs to be emphasized for patients ingesting unknown plants and to remind patients of the dangers of using nicotine replacements while continuing to smoke.
PREECLAMPSIA AND PSEUDO POSTPARTUM CARDIOMYOPATHY. H. Zakariya 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking ID #76568) LEARNING OBJECTIVES: To recognize that volume overload in the preeclamptic patient can lead to pulmonary edema and the misdiagnosis of postpartum cardiomyopathy. CASE INFORMATION: A 29 year-old, G5P3AB2 woman presented with severe dyspnea, orthopnea and peripheral edema five days after delivering a baby. Her pregnancy had been complicated by preeclampsia but managed without difficulty. Vital signs were: BP 157/96, P 110, R 22, T afebrile. Oxygen saturation was 94% on 100% O2. Bilateral rales, jugular venous distention, an S3 and S4 with 3+ pitting edema and a mitral regurgitation murmur were noted on physical examination. Cardiac enzymes were normal. Hgb was 9.7 g/dl. An EKG showed sinus tachycardia. Spiral CT was negative for pulmonary embolus. She was admitted with a diagnosis of postpartum cardiomyopathy. An echocardiogram showed severe mitral regurgitation, trivial tricuspid regurgitation, a small pericardial effusion and an ejection fraction of 50-55%. She was treated with furosemide and rapidly improved. Repeat echocardiography, done two weeks later when she was asymptomatic and off medications, showed that the mitral regurgitation had cleared, there was no pericardial effusion and her ejection fraction was unchanged. Further history revealed that in the last months of pregnancy and immediately postpartum she had been advised to``drink a lot of fluids.'' IMPLICATIONS/DISCUSSION: In preeclampsia the expected fall in colloid oncotic pressure is greater than that seen in normal pregnancies. Cardiac output increases 40% during normal pregnancy and extravascular volume is expanded. Following delivery, significant amounts of extravascular fluid move into the intravascular space and are cleared. In our patient, increased fluid intake led to over expansion of the extravascular volume with pulmonary edema occurring as the fluid was mobilized. Notably, this is not a case of postpartum cardiomyopathy. Her ejection fraction was normal on both initial and repeat echocardiograms and does not show primary muscular dysfunction. The mitral regurgitation cleared with diuretics and is not suggestive of a structural lesion. Small pericardial effusions may occur as a normal variant during pregnancy. Instead, her condition is explained by extreme fluid shifts in a preeclamptic woman. Diagnostic confusion occurs if the physician interprets the findings in terms of the non-pregnant state. year old female with no PMH except appendectomy, admitted with a five days of abdominal pain. It began as``indigestion'' in the epigastrum for several days but progressed to a sharp, constant, right-sided abdominal pain. Associated symptoms included anorexia, three days of constipation, with no bowel movement but some flaitus. She had no dysuria, normal menses, and no bloody bowel movements. She had one episode of``sweats'' and chills two days prior. On exam, she was afebrile and in moderate distress. Her vital signs were normal and abomen was soft with hypoactive bowel sounds and voluntary guarding in the right mid abdomen. Rectal exam was guiac negative. The remainder of the exam was normal. UA, CBC, LFTS, amylase, and lipase were normal. CT of the abdomen revealed a 5.3 cm mass( with a small air pocket) adjacent to the hepatic flexure with mesenteric stranding and sigmoid diverticulosis. Bowel rest and ampicillin -sulbactam were implemented. By the third day, her exam markedly improved. She was discharged on amoxicillin-clavulanate with follow up colectomy planned. IMPLICATIONS/DISCUSSION: The frequency of diverticulosis in younger patients is thought to be increasing. Most people will be asymptomatic. Overall, 15% will be right sided in western countries. Symptoms include bloating and abnormal bowel habits. About 20% develop clinical diverticulitis and of those, 10 to 20% will require surgery. Complications of diverticula include simple inflammation, abscess, bleeding, and fistula formation. Abscess may cause abdominal pain for several days duration, nausea, constipation and fever. WBC can be normal in up to 45% of patients. Treatment of complicated diverticulitis often requires combined medical and surgical approach. Evidence suggests more virulent disease in young people. Often these patients are treated with early colectomy. They have a higher incidence of right sided disease that can be mistaken for appendicitis. Longer lifespans lead to increased potential for morbidity. With CT guided drainage, the two part surgeries (with colostomy and then take down) decreased dramatically. Contained perforation is managed with antibiotics followed by partial colectomy. Offering colectomy after the second attack of uncomplicated diverticulitis has recently been challenged. Conservative treatment after the second attack is now considered a reasonable option in older patients. However, for younger individuals, with no medical comorbidities, early surgery may be the preferred option. The patient is a 33 year old female, gravida two para one, at eight weeks gestation who presents to the office. Her first pregnancy was complicated by a deep venous thrombosis at fifteen weeks gestation. At that time hypercoaguability work up was performed due to a positive family history. The patient had elevated protein C resistance and she was placed on full dose heparin through the duration of her pregnancy. Her third trimester she developed hypertension. She delivered at thirty-five weeks and remained on anticoagulation six weeks following delivery. Factor V leiden mutation was confirmed by gene testing following delivery. She had a seizure disorder as a child and took dilantin and multivitamin prior to pregnancy. Her father had a pulmonary embolus and has antithrombin III deficiency. She is a pharmacist and does not smoke or drink alcohol. Comprehensive physical exam is normal. The patient was placed on continuous subcutaneous infusion of heparin because she declined lovenox therapy. IMPLICATIONS/DISCUSSION: Factor V Leiden is the leading cause of thomboembolism in pregnancy (44%). Pulmonary embolus contributes greatly to maternal mortality rates. Venous thromboembolism occurs in one in 1500 pregnancies. The most prothrombotic time is immediately post partum. In addition factor V Leiden has been associated with HELLP, intrauterine growth retardation, miscarriage, and pregnancy induced hypertension. The activated protein C resistance test may be falsely positive because of changes that take place in the coagulation system during pregnancy. Diagnosis must be confirmed with gene testing. If factor V positive, patients have a three to five times greater risk of stillbirth. Factor V Leiden is present in 20±40% of abruptions. There have been no randomized control trials to demonstrate that full dose anticoagulation will reduce the risk of any of these complications. Treatment is based on extrapolation of data from nonpregnant patients. There is controversy over treatment of pregnant patients with factor V and a prior DVT. At present, patients are offered prophylactic dose heparin or full dose heparin or lovenox based on individual risk stratification (i.e homozygous vs. heterozygous). For individuals with factor V but no prior event, a variety of options exist: prophylactic subcutaneous heparin throughout the pregnancy with full dose anticoagulation postpartum for six weeks versus postpartum treatment only versus clinical surveillance alone. Lovenox will likely replace heparin because of its favorable side effect profile. Risks include a 2% major bleeding risk, heparin induced thrombocytopenia, and osteoporosis. (2) To review the presentation, treatment, and prognosis of CMV colitis among immunocompetent adults. CASE INFORMATION: A 17-year-old female was admitted to the hospital for a complaint of bloody diarrhea. She had been in good health until 4 months earlier when she noted intermittent passage of bright red blood and reddish tissue with her stool. Ten days before admission, she developed cramping periumbilical pain, soon followed by fever and watery diarrhea with hematochezia. Pertinent findings on examination included a temperature of 100.4 F, slightly pale conjunctivae, a soft abdomen with normoactive bowel sounds and minimal tenderness periumbilically, and bright red blood on digital rectal exam. Her white cell count was 8,200/mm3 with 37% neutrophils and 55% lymphocytes. Atypical lymphocytes were present. Stool samples were negative for ova, parasites, C. difficile toxin, and other bacterial enteropathogens. An abdominal CT scan showed thickening of the wall of the entire large bowel. On colonoscopy, there were diffuse inflammation in the colon, mucosal nodularities, and diminished haustral markings. Colonic biopsies demonstrated active colitis with CMV infection, but no architectural distortion to suggest an underlying chronic inflammatory bowel disease (IBD). A CMV antigenemia test came back positive, and anti-CMV IgM and IgG titers were both elevated. HIV serology, however, was negative and CD4 count was 354. Markers for IBD, including p-ANCA and ASCA, were likewise negative. The patient was treated with valganciclovir for 21 days. ABSTRACTS   Clinical Vignettes  LEARNING OBJECTIVES: 1) Raise index of suspicion for severe metabolic derangement in elderly patients. 2) Recognize clinical signs of hypernatremia. 3) Recognize limitations of using concentrated supplements as meal replacement. CASE INFORMATION: A 79 year old woman with advanced Alzheimer's disease was brought to the E.D. due to a 4-week progressive decline in her overall level of function. She had been hospitalized 8 weeks prior for an aspiration pneumonia. Her daily fluid intake since that time consisted of six 8oz cans of a``balanced nutrition shake'', along with some soup. 4 days prior she was given antibiotics by her primary physician. She showed no improvement and developed diarrhea. Physical exam revealed an obtunded woman. Her pulse was 120bpm and rectal temperature was 38.9 Celsius. She was normotensive. She had dry oral mucosa and skin tenting. Her neurological exam was nonfocal. Lab studies were significant for sodium of 185mEq/L, chloride of 148mEq/L, BUN of 124mg/dL and a creatinine of 3.0mg/dL. Urine osmolality was 819mOsmol per kg with urine sodium of 22mMol/L. Her WBC was 13K. Labs from her prior admission revealed sodium of 143mEq/L; chloride of 106mEq/L; BUN and creatinine were 7mg/dL and .7mg/dL respectively. Her free water deficit was calculated at 9 liters, and she was placed on D5W1/2NS at 150cc/hr. Her tachycardia and fever resolved. She began keeping her eyes open and speaking some words. On the sixth hospital day serum sodium was 146 with normal BUN and creatinine. The patient was discharged on a diet of purees and thickened liquids. DISCUSSION: Patients with dementia are at a risk for hypernatremia due to their inability to verbalize a need for water or to obtain it for themselves. Hypernatremic dehydration can lead to mental status changes that are easily missed in a severely demented patient. While canned high calorie shakes are an attractive supplement for patients with poor oral intake, they are highly concentrated and contain little water. This renders them a poor substitute for total meal replacement.
A. Aggarwal 1 , J. Brainard 1 , D. Brotman 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1. Recognize that glucagonomas can present with spinal metastases. 2. Appreciate that the absence of cutaneous, endocrinologic and gastrointestinal manifestations does not rule out the presence of a glucagonoma. 3. Know that tissue staining for neuroendocrine peptides can confirm that a tumor is of pancreatic origin, even when there is no radiographically identifiable pancreatic mass. CASE INFORMATION: A 38 year-old African-American woman presented to the emergency department with low back pain of three months duration. The pain involved both flanks and radiated to the buttocks and posterior thighs bilaterally, as well as to the right groin and medial right thigh. By exam, she was thin and appeared uncomfortable. Shotty nodes were palpable in multiple regions, but none were fixed or enlarged. There was percussion tenderness of the lumbar spine, but motor function and reflexes were normal in the lower extremities. Serum chemistries were notable for an elevated alkaline phosphatase but otherwise normal liver enzymes. Serum calcium and glucose were normal. CT of the abdomen and pelvis showed peripancreatic lymphadenopathy and numerous hepatic lesions suggesting metastases. MRI of the spine demonstrated a destructive lesion in the second lumbar vertebra with soft-tissue extension compressing the spinal cord (figure). Whole body bone scan showed diffuse osseous metastases to the upper and lower extremities as well as the axial skeleton. Ultrasound-guided biopsy of the peri-pancreatic adenopathy revealed a low-grade neuroendocrine tumor that stained positive for glucagon, but stained negative for serotonin, pancreatic polypeptide and gastrin. The serum glucagon level was normal. Due to widespread metastases, the patient was deemed a poor surgical candidate, however she did undergo radiation therapy to the spine and to painful lesions in the right humerus and left femur. Subsequently she was treated with systemic chemotherapy consisting of adriamycin and streptozocin with no initial reduction in tumor mass. DISCUSSION: Glucagonomas have previously been reported to metastasize to bone, but to our knowledge there is only one other reported case of bony metastases as the initial manifestation of the tumor. This may be because most glucagonomas come to early clinical attention via the effects of high serum levels of glucagon. It has previously been reported that systemic and endocrine manifestations of glucagonomas are more common in advanced disease and are directly related to tumor size. This is clearly not the case in our patient. Notably absent were the typical skin rash (necrolytic migratory erythema), diarrhea, hyperglycemia, stomatitis and chelosis associated with the classic glucagonoma syndrome. Another important clinical feature in our patient was the absence of a radiographically identifiable pancreatic mass despite widespread metastases. While the absence of a clear primary source is not an uncommon situation in cancer patients, the identification of the primary tumor carries particular importance for neuroendocrine malignancies. Metastatic small-cell carcinoma of the lung has a more rapid progression than both carcinoid and islet cell tumors, and the treatment is different for these three types of malignancies. In our patient, tissue staining for neuroendocrine markers allowed a specific diagnosis to be made and appropriate treatment to be instituted. We conclude that the absence of clinical evidence of hormonal activity should not dissuade the physician from considering the possibility of a pancreatic islet cell primary when a patient presents with metastatic neuroendocrine malignancy, even when there is no radiographic evidence of pancreatic mass. Stains for neuroendocrine peptides are of crucial importance to making the diagnosis in such patients.
DISCUSSION: Lyme disease involves multisystems with dermatological, rheumatological, neurological and cardiac manifestations. It is caused by Borrelia burgdorferi and transmitted by Ixodes. Several neurological syndromes had been described including lymphatic meningitis, cranial neuropathy (commonly unilateral or bilateral Bell's palsy), painful radiculoneuritis, optic neuritis, mononeuritis multiplex, Guillain-Barre syndrome, encephalomyelitis, peripheral neuropathy and encephalopathy. Myelitis has recently been reported as an unusual complication of Lyme disease. Our case represents one of the few cases of this type. The unique feature in our case is that Lyme disease was first presented as myelitis.
CEREBRAL AUTOSOMAL DOMINANT ARTERIOPATHY WITH SUBCORTICAL INFARCT AND LEUCOENCEPHALOPATHY (CADASIL) SYNDROME. A. Alghadhan 1 , A. Hamad 2 ; 1 SUNY at Stony Brook, Port Jefferson, NY; 2 NUMC, Eastmeadow, NY LEARNING OBJECTIVES: -recognize cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy (CADASIL) in the differential diagnosis for normal pressure hydrocephalus. CASE INFORMATION: 53 years old white female presented with progressive cognitive dysfunction and dementia, gait disturbance, urinary incontinence over the last 3 years. She has long standing history of migraine. 3 years ago, she had a computed tomography (CT) of the brain that showed dilated ventricles and labeled as normal pressure hydrocephalus and the patient refused treatment. Family history was significant for early dementia and migraine in the mother. Her physical exam showed severe dementia, severe ataxia on standing or walking despite normal muscular strength. Sensory exam was normal. Laboratory tests were unremarkable. Head CT and MRI showed severe atrophy with severe dilated ventricles and severe periventricular white matter disease. Skin biopsy showed characteristics PAS positive granular material in the media and narrowing of the lumen of the vessels which is typically seen in cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy (CADASIL). The patient was placed in a nursing home after explaining the nature of the disease to the family. DISCUSSION: CADASIL can be considered as a new disease that had been described recently (1996) in few families. It predominantly affects the small vessels of the brain. The transmission is autosomal dominant linked to chromosome 19. The clinical features include migraine with or without aura (the gene for familial type of migraine is located on the same chromosome), strokes or stroke-like episodes, psychiatric symptoms and dementia. In our case the presentation was similar to the normal pressure hydrocephalus symptoms. We want physicians to be aware of this similarity as the management is completely different. year-old women presented to the hospital with nonhealing ulcers on her breast, thighs and toes. She developed systemic lupus at a young age that ultimately caused complete renal failure. She underwent renal transplant; however, she reinitiated hemodialysis after failure of the transplant. Her examination revealed necrotic ulcers in the left upper breast region. Radiologic evaluation of the breast, hands and chest revealed significant calcifications throughout the body. This pattern revealed systemic calciphylaxis. She underwent debridement and nerve block for pain control. She was dismissed; however, she eventually died three months after dismissal. DISCUSSION: Nonhealing ulcers are a common source of morbidity in hospitalized patients. Common etiologies include ischemia from vascular causes, chronic edema, and small vessel disease. Uncommon etiologies like calciphylaxis differ from traditional chronic ulcers. The skin appears violaceous with a plaque-like or a nodular appearance. The ulcers cause extreme pain independent of size or appearance. The pathogenesis of calciphylaxis remains unknown. Biochemical tests often reveal abnormal calcium or phosphorus levels. Treatment options often include subtotal parathyroidectomy in some patients. Normalization of calcium and phosphorus remains the primary focus of medical treatment. The chronic wounds are often aggressively de Âbrided and antibiotics are used in cases with infections. The use of immunosuppression for calciphylaxis has had mixed results. Despite aggressive wound care and calcium management, the prognosis of calciphylaxis remains poor at 60% morality. Further research needs to be performed on this uncommon yet potentially lethal illness.
A. Almahameed 1 , S. Baghdasarian 2 , S. Frost 2 ; 1 Cleveland Clinic Foundation, Shaker Heights, OH; 2 The Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1) Recognize the potential for severe complications in diabetic patients with urinary tract infection. 2) Diagnose renal emphysema. 3) Recognize that prompt medical and surgical treatment is essential to decrease the high rate of mortality associated with emphysematous pyelonephritis and pyelitis. CASE INFORMATION: A 48 yo black woman with multiple sclerosis and diabetes mellitus presented with abdominal pain, fever, and dysuria 3 days after starting ciprofloxacin for presumed urinary tract infection (UTI) . Physical exam demonstrated T=39C, HR=130, BP=150/80, and tenderness in the left lower quadrant without peritoneal signs. Laboratory investigation revealed WBC=9.91, Cr=1.2, and BUN=25. Urinalysis showed trace leukocyte esterase, WBC=6-10, 2+ blood, and RBC=0-3. Ampicillin and gentamicin were administered. CT scan of the abdomen was obtained demonstrating slight enlargement of the left kidney with stranding of the perinephric fat, and a dilated left renal pelvis and proximal ureter containing pockets of gas. Hydronephrosis, pyelonephritis, and emphysematous pyelitis were diagnosed and the patient promptly underwent intraoperative cystoscopy to evaluate for ureteral obstruction. Debris occluding the left ureter was visualized and a ureteral stent was deployed. Urine and blood cultures revealed Proteus Mirabilis and Aerococcus Urinae. The patient improved quickly and was discharged on the fourth day of hospitalization to complete antibiotic treatment with Augmentin. DISCUSSION: UTI is a significant problem in diabetic patients, and complications such as pyelonephritis, renal or perirenal abscess, focal or multifocal bacterial nephritis, xanthogranulomatous pyelonephritis, and renal papillary necrosis can occur. Emphysematous pyelonephritis (air in the renal parenchyma), and emphysematous pyelitis (air in the collecting system) are complications that occur more frequently in diabetic than in non-diabetic patients. Early diagnosis of emphysematous complications is crucial due to the high rate of mortality in patients whose diagnosis is delayed. Radiographic confirmation is essential (preferably by abdominal CT scan) as the history, physical exam and laboratory findings are rarely diagnostic. Emphysematous pyelonephritis responds poorly to antibiotics alone, with mortality rates as high as 80%. Surgical intervention is virtually mandatory, with nephrectomy lowering mortality to 20% or less. Emphysematous pyelitis is less critical, with an overall mortality of 20%. It is often associated with urinary tract obstruction, and responds well to intravenous antibiotics and obstruction relief.
A 55-YEAR-OLD PHYSICIAN WITH LYMPHOCYTOSIS, FEVER, ABDOMINAL PAIN, AND PROFOUND THROMBOCYTOPENIA. A. Almahameed 1 , A. Absi 2 , D. Farray-berges 2 , S. Rehm 3 ; 1 Cleveland Clinic Foundation, Shaker Heights, Ohio; 2 The Cleveland Clinic Foundation, shaker hts, Ohio; 3 The Cleveland Clinic Foundation, Cleveland, Ohio LEARNING OBJECTIVES: 1-Recognize infectious mononucleosis as a rare cause of severe thrombocytopenia. 2-Utilize EBV-specific antigens to diagnose infectious mononucleosis in monospot-negative patients. 3-Treat EBV-induced severe thrombocytopenia. CASE INFORMATION: A previously healthy 55-year-old male physician reported 2 weeks of fatigue, malaise, and mild abdominal discomfort. On initial evaluation, a right submandibular lymph node was moderately enlarged and the CBC was normal. Several days later, he became febrile (39 C). He had a normal WBC count with lymphocytosis (50%) and atypical lymphocytes (25%), as well as transaminitis (ALT 239 U/L and AST 198 U/L). Monospot tests, CMV titers, HIV antibodies, and blood and urine cultures were all negative. Self-prescribed oral therapy with empiric clarithromycin and ciprofloxacin produced little improvement. One week later, his platelet count was 90,000/mm 3 . He was admitted three days later with petechial rash involving the upper extremities and the upper part of his trunk, and a platelet count of 8,000/ mm 3 . He was afebrile, had diffuse non-palpable, non-blanching petechiae on his upper extremities, ecchymoses on his buccal mucosa, 3 mildly enlarged non-tender lymph nodes in the right submandibular area, and a palpable spleen tip. The WBC count was 10,000/mm 3 with 26% polymorphs and 63% lymphocytes, of which 20% were atypical. His hemoglobin was 13.4 g/dl, and his platelet count was less than 1,000/mm 3 . Blood chemistries and coagulation parameters were normal. Occasional atypical, large lymphocytes and rare platelets were evident on a peripheral blood smear. Bone marrow biopsy showed normocellular composition with severe thrombocytopenia and adequate numbers of megakaryocytes. Flow cytometry of the blood found no malignant cells. Serologies were negative for toxoplasmosis, hepatitis C, hepatitis B, herpes simplex, and herpes virus 6, but the patient had elevated levels of IgM and IgG antibodies to Epstein-Barr virus viral capsid antigen (VCA) (IgG VCA: 152 IU/ml, normal < 18, and IgM VCA 71 TV (normal < 18). The patient was treated with high-dose prednisone and IVIG 1 g/kg/day for 2 days, and his platelet count rose gradually (11,000/mm 3 on discharge and 180,000 mm 3 3 weeks later). DISCUSSION: Infectious mononucleosis (IM) should be included in the differential diagnosis of severe thrombocytopenia in adults even when the initial monospot test is negative. Lowgrade neutropenia and thrombocytopenia are common during the first month of illness. Heterophile antibodies are useful in diagnosing disease in many cases but are often not detectable in children under age 5, in the elderly, and in patients presenting with symptoms not typical of IM. An alternative is EBV-specific antibody testing. Titers of IgM and IgG antibodies to viral capsid antigen are elevated in the serum of more than 90% of patients at the onset of disease. IgM antibody to VCA is useful for the diagnosis of acute IM because levels are elevated only during the first 2 months of the disease. We can not entirely exclude the possibility that ciprofloxacin or clarithromycin therapy caused the thrombocytopenia. Given the clinical illness, the results of serologic testing, and the rarity of profound antibioticassociated thrombocytopenia, we believe that acute Epstein Barr virus infection is the more likely cause of the abnormalities. DISCUSSION: Vitamin B12 comes from the diet and is present in all food products of animal origin. Liver stores of B12 can last for several years after dietary ingestion ceases. Although dietary B12 deficiency is extremely rare, it can occur in vegans who abstain from all such foods for prolonged periods of time.
WHEN A PATIENT PRESENTS WITH WEAKNESS: IT MAYBE HELPFUL TO REVIEW YOUR PRESCRIPTION. A. Arshad 1 , R. Keating 1 ; 1 Fairview Hospital, Cleveland, OH LEARNING OBJECTIVES: 1) Diagnose drug-induced myositis from its presentation. 2) Recognize how adding multi drug antihyperlipidemic therapy increases the incidence of myositis. CASE INFORMATION: A 72 year old white female with past medical history of diabetes mellitus, hypertension and hyperlipidemia who was in relatively good health presents with progressive weakness for the last one week. A week ago she had a yeast infection and took two tablets of diflucan. Shortly after she started feeling weak and the weakness progressed gradually. She is now unable to stand or walk has trouble feeding due to weakness in her arms and also occasionally gets short of breath. She denies any headache, double vision, blurred vision, fevers, chills, pain in her muscles or joints or any recent falls. She is currently on cerivastatin, insulin, lansoprazole, gemfibrozil, atenolol, aspirin, nortriptyline, sertraline, and lisinopril. On physical examination she is afebrile with stable vital signs. The muscle strength was severely decreased in all extremities, upper greater than lower and especially in the shoulder and pelvic girdles. No skin rash or mouth ulcers were seen. Laboratory work shows a creatine kinase of 4132 U/L, myoglobin titer of 3634. Liver enzymes were mildly elevated. Patient was started on steroids and she adequately recovered. DISCUSSION: The onset of generalized progressive myopathy in a patient taking cholesterol lowering drugs should alert us to the diagnosis of drug induced myopathy. Several antihyperlipidemic drugs have caused myopathy including the statins, gemfibrozil, nicotinic acid and clofibrate. Lovastatin has been reported several times in literature to induce myositis. The concomitant use of gemfibrozil in patients taking statins increases the incidence of myositis to five percent. The creatine kinase and sedimentation rate is markedly elevated in these patients. Creatine kinase elevations are very specific and consist of only the MM fraction. Discontinuation of statin therapy in these patients is advisable because of the potential development of rhabdomyolysis and acute renal failure. Cautious use of statins is recommended in patients with prior history of liver disease, those with active liver disease and in heavy alcohol users. Hepatic and muscle enzyme levels should be tested periodically during statin treatment. Other medications known to increase the incidence of myopathy in patients taking statins are cyclosporine, nicotinic acid, prednisone and azathioprine. Treatment includes discontinuation of the medication and use of steroids. The use of diflucan was most likely incidental as there have not been cases of myopathy attributed to diflucan. 2) Recognize Propionibacterium acnes as a cause of intra-cranial abscess.
3) Manage Propionibacterium abscess with surgical drainage and antibiotic monotherapy. CASE INFORMATION: A fifty-two year old white male presented with a lump on the head for over a year. Magnetic resonance imaging (MRI) showed a mass lesion in the parietal lobe. He subsequently underwent craniotomy for an epidural neoplasm and left parietal reconstruction with duragen. He then received two doses of radiotherapy. About three months later he presents with right sided weakness, focal seizures in the right hand and a fight facial palsy. After recurrent seizures on anticonvulsant therapy a repeat computed tomography scan and MRI scan was done which showed an epidural lesion with mass effect. The patient was afebrile and had a normal peripheral blood count. The patient was taken to surgery and an epidural abscess was drained. The abscess aspirate on gram stain revealed pleomorphic gram positive bacteria. Aerobic cultures were negative and so were the anaerobic cultures initially but after several days of incubation they grew out a pure culture of a pleomorphic gram positive organism which was identified as Propionibacterium acnes. He was started on intravenous ceftriaxone for 6 weeks and he recovered fully after completion of therapy. DISCUSSION: This case illustrates a case of post-surgical epidural abscess caused by Propionibacterium acnes. Propionibacterium acne is a corynebacterium usually isolated from the human skin where it plays a role in the pathogenesis of acne vulgaris. There have been reports in literature of clinically significant infections related to CNS shunts, intracranial abscesses related to dental sepsis, sepsis related to indwelling vascular catheters, and also a few cases of intracranial abscesses unrelated to foreign bodies. The above case is an addition to these rare cases of intracranial abscesses caused by this unusual pathogen. They are slow growing organisms and as their identification presents problems, they have been frequently overlooked as contaminants. It has been suggested that grain positive rods on gram stain and negative aerobic cultures of suspected infected fluids without organisms on gram stain should be cultured anaerobically. Most infections reported in literature were treated with a combination of antibiotics, though there have been reports of successful treatment with penicillin and surgical drainage alone. The above case also shows how ceftriaxone monotherapy is an effective treatment for such infections along with surgical evacuation of the abscess. year-old male with HIV (CD4 of 343, undetectable viral load) was admitted to the hospital with shortness of breath. He was diagnosed with pneumocystis pneumonia vs cocci pneumonia and treated with Bactrim and Fluconazole. One week later he returned with continuing dyspnea. A VQ scan showed high probability for pulmonary embolism. He was treated with heparin then coumadin and discharged. Three days later he returned with new left lower extremity swelling and pain. He had been taking Coumadin with an INR of 5.1. He denied shortness of breath, chest pain, IV drug use, smoking, ETOH, or recent travel. His medications included Abacavir, Efavirenz, Nelfinavir, Fluconazole, and Bactrim. Physical examination revealed a tender, swollen left lower extremity with a positive Homan's sign. An ultrasound showed thrombosis in the left popliteal vein. The swelling and pain resolved with heparin and he was discharged on Lovenox. A hypercoagulable evaluation revealed normal levels of protein S, activated protein C resistance, anti-thrombin III, and homocysteine. Protein C was slightly elevated. Lupus anticoagulant and anti-cardiolipin antibody were negative. An abdominal CT revealed no mass lesions. DISCUSSION: Thrombosis is an uncommon complication of HIV infection that many primary care providers may not be aware of. Anecdotal evidence in the literature relates thrombosis and HIV, especially in patients with IV drug use, Kaposi's, cytomegalovirus (CMV) disease, megestrol use, protease inhibitor use (including nelfinavir), and hypercoagulable states such as anticardiolipin antibody and protein S deficiency. A recent epidemiologic study showed that HIV patients with thrombotic events had the following characteristics: age greater than 45 years, CMV disease, AIDS-defining opportunistic infection, hospitalization, megestrol use, and indinavir use. The only risk factors for thrombosis that this patient had were hospitalization and nelfinavir use. HIV itself may be a risk factor for thrombotic events and should be considered in cases where shortness of breath remains unresolved despite maximal medical therapy. The mechanism by which HIV causes thrombosis remains unknown but several theories have been proposed. This case illustrates that the link of HIV as a possible cause of thrombosis may have implications for prophylaxis in HIV patients. There are no consensus studies and prophylaxis remains at the discretion of the clinician.
LEARNING OBJECTIVES: 1. To illustrate clinical features of hypoglycemia from inadvertent administration of a sulfonylurea compared to an insulinoma. 2. To demonstrate the importance of carefully reviewing medications that patients with hypoglycemia are taking. CASE INFORMATION: Two patients were referred for recurrent episodes of hypoglycemia thought to be due an insulinoma. The clinical presentations were characterized by precipitous drops in blood glucose (BG) associated with marked adrenergic symptoms. The first patient was a 69 yo man with a coronary artery disease, heart failure, hypertension, chronic renal insufficiency, and peripheral vascular disease. He had recurrent acute hypoglycemic reactions (including 3 hospital admissions) accompanied by adrenergic symptoms. He also had a recent myocardial infarction. Medications included warfarin, metoprolol, nifedipine, isosorbide dinitrate (IsoD), furosemide, famotidine, lisinopril, potassium, pentoxyphylline, and colchicine. His chemistry panel was normal. Prior workup included normal cortrosyn stimulation test and abdominal CT scan, and negative serum sulfonylurea levels. A 36 hour fast ( the patient refused to go beyond 36 hours) showed a BG of 59 mg/dl, a corresponding cpeptide of 3.5 ng/ml (0.7±3.0) , and absent insulin antibody. Inspection of his medications revealed a bottle labeled for IsoD contained two types of light green pills: IsoD and glyburide 5 mg. The second patient was an 87 yo woman who was referred for surgical treatment for a presumed insulinoma. Over a 2 mo period she has had several episodes consisting of confusion, diaphoresis, and syncope with confirmed hypoglycemia(BG levels < 40mg/dl). Medications included cardizem, ranitidine, acetaminophen, and diazepam. Evaluation included a normal chemistry panels, somatomedin, and cortrosyn stimulation test. Serum insulin and c-peptide levels at the time of hypoglycemia (BG= 37 mg/dl) were 36.38 pmol/L and 1600 pmol/L respectively. Serum and urine SFU screens were negative. CT and octreotide scans of the abdomen showed no abnormalities. Identification of the patient's home medications was done. In a bottle labeled diazepam were light blue-green tablets identified as generic glyburide 5mg. To further confirm SFU induced hypoglycemia, a repeat 72 hour fasting was done. At a glucose level of 94mg/dl, the insulin and insulin antibody levels were 3.2uU/ml (4.0±24.0) and < 5 % respectively, and at a glucose level of 58mg/dl, the levels were < 2 uU/ml and < 5% respectively. An exploratory laparotomy was canceled. In each case, no further hypoglycemic episodes occured following discontinuation of the SFU. DISCUSSION: The biochemical features of hypoglycemia due to sulfonylureas and insulinomas are similar. Insulinomas are commonly associated neuroglycopenic symptoms that are insidious in onset, and recipitous hypoglycemia with adrenergic symptoms is uncommon. Presence of such symptoms mandates a search for other causes. SFU-induced hypoglycemia may mimic an insulinoma biochemically. Our two cases illustrate the importance of careful attention to history, and a thorough inspection of all medications in patients presenting with hypoglycemia before extensive, diagnostic tests, including invasive procedures or surgery are undertaken LEARNING OBJECTIVES: 1) Diagnose chronic lung disease in patients with sickle cell disease.
2) Recognize the risk of pulmonary hypertension during pregnancy. CASE INFORMATION: A 30-year-old G2P1 with a history of sickle cell disease presented to our institution at 26 weeks gestation with the complaint of preterm contractions. She was admitted and placed on magnesium as a tocolytic. She then began to complain of back and leg pain leading to a diagnosis of a sickle crisis. Her medical history was notable for 3 to 4 inpatient admissions for sickle crises a year. She had a port-a-cath placed 11 months prior to the admission. Her gall bladder was removed two years prior to admission. Her evaluation included room air PaO2 of 68 mmHg and pulse oximetry of 93%, and a chest xray revealed bilateral pulmonary infiltrates in an alveolar pattern. Given a normal urinalysis and urine culture, and no other identifiable trigger for the crisis or the preterm contractions, she was treated for pneumonia. Magnesium was discontinued. The improvement in her oxygenation seemed slower than expected prompting the high-risk team to order an echocardiogram. Her left ventricle and valves were normal, but the right-sided chambers were enlarged and her estimated pulmonary artery systolic pressure was 55mmHg. V/Q scan was normal. This is the first time she was diagnosed as having pulmonary hypertension. Review of prior chest x-rays from another institution revealed that the pulmonary infiltrates had been present for at least 18 months. DISCUSSION: A review of the literature revealed many series of sickle cell patients with chronic lung disease. These patients may be asymptomatic, but abnormalities on pulmonary function testing are an early and reliable marker of this complication. Patients with abnormal chest radiography, pulmonary function testing,x or hypoxia during stable periods have an average life expectancy of 2.6 years. They are at risk for sudden death from subendocardial infarction without epicardial arterial disease. We are surprised that more pregnant patients with long standing sickle cell disease do not have routine screening for lung disease by their physicians. This is particularly important during pregnancy because severe pulmonary hypertension (pressures > 80mmg) from any cause has a post-partum mortality rate as high as 50%. The precise risk with less severe elevations is not known. Given the prevalence of sickle cell disease in the general medical population we recommend that all patients with long standing sickle disease have pulmonary function testing and/or echocardiography to evaluate pulmonary arterial pressures. If chronic pulmonary disease is diagnosed in a woman considering pregnancy then consultation with experienced internists, pulmonary specialists, and perinatologists is warranted.`H complaints or symptoms suggestive of cardiopulmonary diseases. On a follow up visit he reported dyspnea on minimal exertion of two months duration. He also reported chest pressure, dizziness and nausea with the dyspnea. He told his doctor that he has dyspnea even after speaking few sentences. His exam revealed BP= 146/90 Pulse 80 regular. Heart auscultation revealed a normal S1, split S2, and I-II/VI systolic murmur. Lungs were clear to auscultation. Abdomen was soft, no hepatosplenomegaly. No pedal edema. EKG was done in the clinic showed left ventricular hypertrophy. Chest X-ray was normal. Stress echocardiogram revealed a small apical wall motion abnormality worsening with stress. A cardiac catheterization revealed normal coronaries, no wall motion abnormality and left ventricular end diastolic pressure of 35 mmHg. The aortic root was bicuspid with severe coarctation of the aorta at the isthmus. DISCUSSION: Coarctation of the aorta is an unusual cause of hypertension in young adults and is often unrecognized by general practitioners. Although the left ventricle is usually hypertrophied because of the hypertension, the heart is usually normal in size or only slightly enlarged until cardiac decompensation occurs late in the course of the disease. Our patient left ventricular end diastolic pressure was 35 mmHg, which might account to his dyspnea. In retrospect knowing the diagnosis of coarctation our patient has the body habitus of a chronic coarctation of the aorta with a large head and upper extremities and is otherwise short. He had diminished leg pulses. His blood pressure was comparable in upper extremities; however, there was a difference of 50 mmHg between upper and lower extremities. Coarctation of the aorta is a potentially lethal condition at all ages. Many patients first come to medical attention because of signs and symptoms related to hypertension. The diagnosis should be considered in patients with hypertension and diminished leg pulses, and we recommend the routine examination of leg pulses in young adults with hypertension.
HEMOLYSIS IN A RECENTLY HOSPITALIZED PATIENT. L. Chang 1 ; 1 UCLA/San Fernando Valley Program, Sepulveda, CA LEARNING OBJECTIVES: 1) To recognize drug-induced hemolysis as a common cause of hemolytic anemia 2) To recognize that 2nd and 3rd generation cephalosporins are the most common cause of drug-induced hemolytic anemia 3) To recognize that cephalosporin-induced hemolytic anemia is often serious, and can be fatal. CASE INFORMATION: A 62-year-old female discharged 7 days ago after being treated for Streptococcus pharyngitis presents to a follow-up clinic complaining of a 5-day history of nausea, nonbilious and nonbloody vomiting, and anorexia. She also complains of a generalized headache and subjective fevers. In her previous hospitalization, she presented to the emergency room complaining of fever, sore throat, and epigastric pain. At that time, she was found to have a WBC of 16 with 89%neutrophils, a Hct of 44, and a clean urinalysis. A CT abdomen was done revealing diverticulosis only. Her temperature was 38.4. She was given one dose of Cefotetan in the ER and admitted for further workup. On the medicine wards, further evaluation revealed an exudative tonsillitis with anterior cervical lymphadenopathy. She admitted to a history of recurrent pharyngitis. She was treated with penicillin VK, improved clinically, and sent home on penicillin. She had no known drug allergies. Physical examination: Temp 38.5, BP 141/70, pulse 97. She was moderately ill appearing. Eyes were icteric. Oropharynx revealed tonsillar swelling, but was significantly improved from her previous hospitalization exam. Neck was supple. Heart and lung exams were normal. Abdomen revealed mild epigastric tenderness without peritoneal signs. Neurological exam was normal. Laboratory studies: WBC 18.2, Hgb 11, Hct 30.6, Plt 300, MCV 89. Total bilirubin 13.2, direct bilirubin 0.5, AST 34, ALT 28, alkaline phosphatase 93. PTT 23, INR 1.04. LDH 1578, corrected reticulocyte count 1.8, haptoglobin < 50. Direct Coombs was markedly positive. DISCUSSION: The positive direct Coombs test was further investigated by the blood bank that, along with assistance from the Red Cross, determined that this patient had developed a Cefotetan-induced autoimmune hemolytic anemia (HA). It should be recognized that 1stgeneration cephalosporins rarely cause HA, but 2nd and 3rd generation cephalosporins are the most common cause of drug-induced HA. Most cases of cephalosporin-induced HA revealed moderate-to-severe hemolysis with fatalities reported. Most patients required treatment with blood transfusion and steroids. Because of the common use of 2nd and 3rd generation cephalosporins by physicians, and the severity of the hemolysis that can be induced by them, recognition of this entity is of significant importance to the practicing clinician.
A CASE OF HEMOPTYSIS FOLLOWING PERCUTANEOUS TRANSLUMINAL CORONARY ANGIOPLASTY. S. Chebrolu 1 , J. Addagatla 1 , F. Ali 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize the occurrence of bleeding complications including pulmonary alveolar hemorrhage following the use of Group IIb-IIIa receptor inhibitors in acute coronary syndromes. 2) Diagnose and manage pulmonary alveolar hemorrhage. CASE INFORMATION: A 68 year-old woman with a past medical history of diabetes mellitus and hypertension presented to the emergency room with chest pain. She was a non-smoker and had no known lung disease. Physical examination was normal. EKG showed an acute anteroseptal myocardial infarction. Emergent angiogram revealed a 100% proximal left anterior descending artery lesion. Percutaneous transluminal coronary angioplasty with placement of a multilink stent of the left anterior descending artery lesion was done. During the procedure, she was given 10,000U of heparin and 0.25 mg/kg of abciximab boluses followed by infusion of heparin at 700U/hr and abciximab at 0.125 mcg/kg/min after the procedure. An intra-aortic balloon pump was also placed. One hour later, she had hematemesis and hemoptysis and developed respiratory distress with hypoxia. Heparin and abciximab were discontinued and emergent intubation was done. A copious amount of blood was aspirated from the endotracheal tube. CXR revealed diffuse bilateral interstitial infiltrates. Hemoglobin dropped from 12.6g/dl to 10g/dl. A diagnosis of pulmonary alveolar hemorrhage was made. Bright red blood was suctioned from the endotracheal tube for a few hours. Hypoxemia improved rapidly with complete clearing of the CXR over the next two days. She was extubated on day three and was discharged home on day seven on aspirin, clopidogrel, metprolol and ramipril. She received two units of packed red blood corpuscles during her hospital course. DISCUSSION: Pulmonary alveolar hemorrhage is an infrequent and rarely reported hemorrhagic complication of the use of Group IIb-IIIa receptor inhibitors in acute coronary syndromes. It should be considered in the differential diagnosis of a patient who receives these agents and presents with respiratory distress, pulmonary infiltrates and a drop in hemoglobin, with or without hemoptysis. It is usually seen in older patients and in those with increased pulmonary capillary wedge pressure and evidence of pulmonary edema. Management is mainly supportive with discontinuation of all anticoagulants and with transfusions of packed red blood corpuscles and platelets as needed. An 82 year-old man was admitted with complaints of dysphagia and constipation for one week. It was associated with anorexia and significant weight loss over one month duration. He denied any history of abdominal pain, hematemesis or melena. Past medical history included COPD and coronary artery disease. Past surgical history included CABG, appendectomy and bilateral inguinal hernia repair. Abdominal examination was benign but he was found to be fecal occult blood positive. Laboratory work up revealed evidence of iron deficiency anemia and a gastrointestinal malignancy was suspected. He underwent gastroduodenoscopy that revealed hiatal hernia and a colonoscopy was planned. A few hours after the procedure, he complained of severe lower abdominal pain when he was noticed to have right lower quadrant tenderness and fullness but with no guarding or rigidity. CT scan of the abdomen was done which revealed a mass in the terminal ileum and a 5-cm mass in the left lobe of liver. It also revealed superior MVT with ischemic changes in the small bowel. He underwent emergent angiography that confirmed the presence of MVT and was given local t-PA for dissolution of the MVT. Following this, his abdominal pain worsened with development of peritoneal signs and he underwent exploratory laparotomy with the resection of terminal ileum, right hemicolectomy and biopsy of the liver mass. Histopathology of the mass and liver biopsy revealed malignant carcinoid tumor with mesenteric node and liver involvement. DISCUSSION: MVT is characterized by pain out of proportion to physical findings, nausea, vomiting, constipation and occasional bloody diarrhea. Previous abdominal surgery, hypercoagulable states, cirrhosis and gastrointestinal malignancies are the most common conditions associated with MVT. Abdominal distention, though nonspecific, is the most frequent sign. Leukocytosis may be found in half of the patients but laboratory tests are not helpful in the diagnosis. CT is the diagnostic test of choice and demonstrates thrombus in the superior mesenteric vein with evidence of bowel ischemia. Anticoagulation with heparin followed by warfarin in the absence of contraindications is the mainstay of medical management. All patients with localized or diffuse peritonitis should undergo immediate exploratory laparotomy with resection of nonviable bowel. MVT has not been reported previously as a presenting feature of malignant carcinoid tumor as seen in our patient here.
MYELOMA IN A HYPERCALCEMIC PATIENT. S. Chebrolu 1 , F. Zar 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize that Primary Hyperparathyroidism (PHP) and Multiple Myeloma (MM) are the most common causes of hypercalcemia in the elderly population and diagnose them. 2) Recognize that more than one independent cause of hypercalcemia is very rare and should be considered in a patient unresponsive to treatment. CASE INFORMATION: A 76 year-old male was admitted with weakness, lethargy and occasional confusion of three months duration. His past medical history included sick sinus syndrome and valve replacement for aortic incompetence. His primary doctor discontinued chlorthalidone four weeks prior to this admission after he was found to be hypercalcemic (13.9mg/dl). Examination was unremarkable. Initial studies revealed calcium 11.8mg/dl (normal 8.8±10.4mg/dl), ionized calcium 6.77 mg/dl (normal 4.6±5.3mg/dl), and phosphorus 2.3 mg/dl (normal 2.4±4.5mg/dl). CT scan of head was normal. Patient was hydrated with normal saline and later diuresed with furosemide. An intact parathyroid hormone level by immunochemiluminescent assay was 12.1 pmol/l. C-terminal peptide was 323 pmol/l. Serum osteocalcin was 21U/l. A CT scan of the neck showed a mass suggestive of a parathyroid adenoma, confirmed by Technetium-99m sestamibi scan showing a persistent crescentric area of increased activity in the left lower neck in both immediate and delayed films. In addition, urine protein electrophoresis showed monoclonal kappa light chains. Serum protein electrophoresis revealed a monoclonal spike and IgG kappa monoclonal protein (2770mg/dl). Beta-2 microglobulin was 6.4mg/dl. A skeletal survey revealed multiple lytic defects throughout the skeletal system. Bone marrow aspiration showed focal increase in plasma cells consistent with MM. The patient was given pamidronate and started on chemotherapy with melphalan and he responded well. DISCUSSION: Although a variety of conditions can give rise to hypercalcemia, nearly 90% of the cases can be attributed to either PHP or MM. PHP was diagnosed in our patient by finding elevated intact parathyroid hormone levels associated with elevated C-terminal peptide levels. Technetium-99m-sestamibi scan confirmed its presence. IgG kappa monoclonal paraprotein on serum and urinary protein electrophoresis associated with multiple lytic defects on the skeletal survey was highly suggestive of MM which was confirmed by bone marrow biopsy. However, more than one independent cause of hypercalcemia in a patient is rarely seen. Only eighteen cases of simultaneous occurrence of both PHP and MM were reported and our patient will be the nineteenth such case. Considering the rarity, it appears that this is a chance association. Persistent and unresponsive hypercalcemia may occur if only one of causes is recognized in such rare instances and in a MM patient, this may falsely lead to a higher tumor grading.
METASTASIS-INDUCED ACUTE PANCREATITIS. S. Chebrolu 1 , A. Naidu 1 , F. Zar 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize that metastatic tumors to pancreas may rarely induce acute pancreatitis referred to as Metastasis-Induced Acute Pancreatitis (MIAP). 2) Diagnose and manage MIAP. CASE INFORMATION: A 37-year-old woman presented with a one-day history of epigastric pain radiating to the back associated with nausea and decreased appetite. One and half years ago, she had a missed abortion followed by a Placental Site Trophoblastic Tumor (PSST) which was treated with total abdominal hysterectomy and chemotherapy with methotrexate. Her beta-HCG was less than 2 IU/l six months prior to this presentation. She had no history of alcoholism. On examination, she was found to have epigastric tenderness. Laboratory investigations revealed amylase 309 IU/l, lipase 154 IU/l, lactate dehydrogenase 544 IU/l, calcium 8.5mg/dl, glucose 221mg/dl and hemoglobin 13.6g/dl. She was diagnosed as acute pancreatitis and treated medically. Her pain persisted and she had CT scan of the abdomen that showed a swollen and enlarged pancreas with multiple rounded lesions of variable sizes. There was also a lobulated 3x4cm mass behind the sigmoid colon with a surgical clip adjacent to it associated with retroperitoneal and inguinal lymphadenopathy. Beta-HCG was found to be 2320.6 IU. ERCP was unremarkable. Exploratory laparotomy with bilateral salpingoopherectemy and tumor dissection was done showing histologic evidence of recurrent PSTT. A diagnosis of MIAP secondary to metastatic PSST was made and chemotherapy with etoposide, methotrexate and adriamycin was started with resolution of her symptoms. DISCUSSION: MIAP refers to non-pancreatic tumors metastasizing to the pancreas and resulting in acute pancreatitis. It is a very rare cause of acute pancreatitis and only 42 such cases have been reported in the literature. Most of them were associated with bronchogenic carcinoma and lymphoma. Mechanisms implicated in the causation of MIAP include mechanical ductal obstruction and rupture with direct parenchymal tumor invasion or ischemia secondary to vascular occlusion and encasement of pancreatic vessels. The clinical and laboratory presentation is similar to the other causes of pancreatitis and MIAP remains a diagnosis of exclusion. An accurate history, supportive clinical evidence and radiological findings of pancreatic and peri-pancreatic metastatic lesions in the presence of a known primary tumor aid in the diagnosis. MIAP is managed conservatively with nasogastric tube suction, intravenous fluids and analgesics and this approach is helpful in most cases. If it persists, systemic chemotherapy for the metastatic tumor or palliative abdominal radiation may help. Overall, MIAP portends a bad prognosis and many patients succumb to the metastatic tumor sooner or later. PSST, a rare form of Gestational Trophoblastic Disease, has never previously been reported as a cause of MIAP and this is the first such instance. year old female Stanford graduate student presented with three days of fever and headache with associated nausea, neck stiffness, and photophobia. Past medical history significant for migraine headache and menstrual irregularity which responded well to oral contraceptives. Current medication includes three days of leuprolide injections for commercial egg donation. On exam, patient was febrile to 39.6 degrees Celsius with otherwise normal and stable vital signs. Patient was slightly lethargic and had slight photophobia and mild posterior neck tenderness on palpation, but otherwise completely normal exam. Ophthalmologic and neurologic exams were completely normal. Labs were normal except for Sodium 126 and Potassium 3.3. Lumbar puncture revealed opening pressure of 13cm. CSF studies revealed Glucose 44, Protein 51, WBC 27, RBC 510, and negative microscopic evaluation. Bacterial, viral and fungal cultures were eventually all negative. Empiric antibiotics of ceftriaxone, vancomycin and acyclovir were started. Patient continued to have headache, nausea, and fever with no clinical improvement or worsening except for Sodium continue to drop to 115 over next three days. On hospital day three, patient developed seizure and head MRI revealed 2.3cm  1.8cm sellar mass with hemorrhage and compression on optic chiasm. Surgical decompression of mass was complicated by right ACA/MCA territory infarct secondary to vasospasm and great sodium fluctuation due to SIADH and adrenal insufficiency. Patient eventully was discharged on thyroxine, cortisone, and DDAVP. She has made incredible recovery and will be resuming her PhD studies. DISCUSSION: The risk of pituitary apoplexy with use of leuprolide has been described in the literature but not well screened. Detailed clinical history, exam, and laboratory investigation might identify patients at higher risk of pituitary apoplexy for selective imaging evaluation. In this patient, the history of menstral irregularity controlled by oral contraceptives, the hyponatremia and loss of body temperature control could have been the starting point of investigation for pituitary adenoma and apoplexy. With increasing number of patients receiving elective leuprolide for fertility purpose, the medical community should be aware of potential risk in order to assess and advise patients accordingly. CASE INFORMATION: A 63-year-old male with a history of diabetes mellitus, hypertension, heavy alcohol consumption, and a T6 vertebral fracture was admitted to hospital with a one year history of progressive lower extremity proximal muscle weakness. Concurrent with his worsening weakness, the patient noticed increasing central obesity and easy bruising. Physical examination revealed a classic cushingoid appearance. The neurological examination demonstrated symmetric polyneuropathy as well as lower extremity proximal muscle weakness and wasting. A T6 sensory level was present. The reflexes were 1+ and the plantar responses were flexor bilaterally. Laboratory investigations confirmed Cushing's syndrome and suggested a pituitary source of hypercortisolism. An MRI of the spine showed multiple vertebral fractures and epidural lipomatosis in the posterior epidural space, from T2 to T8, compressing the spinal cord maximally at T6. Given the fragility of this patient's spine and the ongoing investigations to determine the cause of hypercortisolism, the patient did not undergo emergent epidural fat debulking. A pituitary microadenoma was diagnosed and resected by a transphenoidal approach. Although repeat imaging was not performed to document regression of the spinal lipomatosis, there was clinical and laboratory evidence of resolution of the hypercortisolemia. The post-operative course was complicated by pulmonary emboli and recurrent sepsis; the patient suffered a fatal cardiac arrest 96 days after surgery. DISCUSSION: Epidural lipomatosis has been described in steroid use and obesity, and more rarely in association with endogenous hypercortisolism such as Cushing's disease. This case emphasizes the difficulty in suspecting epidural lipomatosis in a patient with Cushing's syndrome in the setting of other neurologic processes. The diagnosis of epidural lipomatosis needs to be systematically considered in any patient with Cushing's syndrome who presents with back pain, lower extremity weakness, radiculopathy, or subtle signs of myelopathy. MRI of the spine is the diagnostic test of choice. Treatment modality (surgical debulking of epidural fat versus correction of the hypercortisolic state or weight loss) should be individualized based on the severity of neurological signs, the potential reversibility of the causative factor, and the estimated operative morbidity.
S. Christopoulos 1 , A. Szilagyi 1 , S.R. Kahn 1 ; 1 SMBD Jewish General Hospital, McGill University, Montreal, Quebec LEARNING OBJECTIVES: 1) To recognize the importance of a careful medication history and a thorough review of side effects; 2) To recognize ergotism as a cause of mesenteric ischemia. CASE INFORMATION: Over a two-year period, a 43-year-old woman presented to the emergency department on five separate occasions with complaints of severe abdominal pain. Extensive investigations including gastroscopy, colonoscopy, two CT scans and ultrasounds of the abdomen, small bowel follow-through contrast study, enteroclysis, as well as a complete gynecological work-up were all negative. Over this period of time, the patient's treating psychiatrist diagnosed her as having Somatic Pain Disorder. The patient was admitted to the medical service of our institution with severe malnutrition. Her past medical history included a longstanding history of migraine headaches treated with caffeine/ergotamine tartrate enemas, weekly or more often, since the age of 18. The patient described her abdominal pain as sharp, located primarily in the epigastric region, and radiating to the back. The episodes occurred one hour postprandially, and were associated with nausea and vomiting. She reported a 60 lb weight loss over a one-year period. Physical examination revealed cachexia. Serial abdominal examinations were unremarkable. Stool occult blood testing was negative. Tube feeding was initiated. A CT angiogram was requested, which revealed significant narrowing of both the celiac and superior mesenteric arteries with the presence of collaterals. A diagnosis of mesenteric ischemia was established. An angiogram was requested prior to intestinal bypass surgery, however the patient developed worsening abdominal pain, significant leucocytosis, a lactate level three times normal, and evidence of peritoneal irritation. She underwent urgent laparotomy, which revealed extensive ischemia of the small bowel and diffuse arterial disease of the abdominal aorta, the iliac, superior mesenteric, and celiac arteries. An aorto-superior mesenteric bypass was performed. The patient underwent a second look laparotomy within 24 hours and 30 cm of dusky small bowel were resected. The post-operative course was uneventful. Following the discontinuation of ergotamine use, the patient no longer experienced abdominal pain and gained a significant amount of weight. DISCUSSION: Despite new modalities to treat migraine headaches, ergotamine is still used chronically by some migraine sufferers. The most recognized complication of ergotism is arterial vasospasm of the upper and lower extremities. More rarely, vasospasm is recognized in the carotid, retinal, renal, and mesenteric arteries. In our patient, failure to suspect this unusual cause of mesenteric ischemia led to delayed diagnosis, severe weight loss, and advanced intestinal infarction. The importance of a careful drug history cannot be overemphasized.
MRI demonstrating thoracic epidoral lipomatosis with cord compression.
LEARNING OBJECTIVES: 1. Diagnose polycystic ovary syndrome (PCOS). 2. Restore fertility in the setting of PCOS and subclinical hypothyroidism. 3. Recognize and manage the potential long-term sequelae of PCOS. CASE INFORMATION: A 28 year-old G0P0 Asian woman presented for a new patient appointment concerned about 7 months of amenorrhea. She recalled that her menstrual cycles since menarche at age 13 had often been irregular-varying in length from 28 to 45 days-but she had never gone more than two months without a period. Her last menstrual period was shortly before an extended and stressful trip back to her family in Taiwan. After several months without a period, a basic workup in Taiwan revealed a negative pregnancy test, elevated blood pressure, and a slightly increased TSH. She returned to the U.S. and had no subsequent followup. She made her appointment when she and her husband had become increasingly concerned about her fertility. Her past medical history was remarkable only for elevated triglycerides several years ago. She took no medications and denied any use of tobacco, alcohol, or drugs. Born in Taiwan, she emigrated to the U.S. as a child. She lived with her husband and worked as a psychiatric nurse. Her family history was unremarkable. Review of systems was notable for weight gain of about 15 pounds over the last year, intermittent fatigue and mood swings, frequent frontal headaches and lower abdominal cramping, bilateral hand numbness at night, and increased hair loss. She denied heat or cold intolerance, fevers, night sweats, nausea, vomiting, diarrhea, dysuria, vocal or visual changes. On exam, she was mildly obese and well-appearing. Compared with her driver's license photo from two years before, she had apparently gained some weight but showed no obvious changes in her facial structure. Her blood pressure was 110/70 and her heart rate was 80. Her BMI was 30. Her thyroid gland was slightly enlarged. There were terminal hairs present on her upper back but no other evidence of hirsutism. Her abdomen was slightly obese without striae. Bimanual exam revealed slight enlargement of her ovaries and associated mild tenderness. Initial laboratory data included normal CBC and renal panel, undetectable serum beta-HCG, TSH 5.02, free T4 14, FSH 6.4, LH 11.5, prolactin 9.3, fasting glucose 103, total cholesterol 167, triglycerides 314, LDL 66, and HDL 38. Pelvic ultrasound revealed bilateral polycystic ovaries. Additional blood tests were obtained. Daily administration of 10mg of medroxyprogesterone acetate yielded a normal menstrual period on day nine. She was also started on thyroxine and a program of weight reduction. DISCUSSION: PCOS is the most common endocrinopathy among women of reproductive age with an estimated prevalence of 4±7%. Though a clear consensus about diagnostic criteria for PCOS has not been achieved, the clinical hallmarks include evidence of hyperandrogenism, menstrual dysfunction, and polycystic ovaries. The diagnosis also requires the exclusion of other functional and neoplastic disorders with similar clinical features. Fertility may be restored in obese patients with PCOS through weight reduction alone. Metformin and clomiphene are other therapeutic options. In the unusual circumstance in which subclinical hypothyroidism may be contributing to menstrual disturbances, treatment with thyroxine has been demonstrated to restore normal function. Proper management of women with PCOS requires recognition of the long-term health risks associated with unopposed estrogen, insulin resistance, and lipid abnormalities including endometrial cancer, overt type 2 diabetes mellitus, and coronary artery disease.
EPHEDRINE NEPHROLITHIASIS ASSOCIATED WITH EPHEDRINE USE. B.A. Costello 1 ; 1 Mayo Clinic, Rochester, MN LEARNING OBJECTIVES: 1) Awareness of nephrolithiasis as a potential complication of ephedrine overuse. 2) Recognition of importance of asking patients about over-the-counter (OTC) supplements. CASE INFORMATION: A 34 yo female was referred to our instution following a diagnosis of nephrolithiasis. She reported 2±3 similar episodes/yr over the past 20 years. Prior to transfer, she was found to have a 5mm stone in her left ureter. Upon admission, analgesia and hydration were provided; urine was strained for debris. 24h urine for citrate, oxalate, calcium, uric acid, and cystine was normal. She passed a stone which was analyzed and found to be composed of ephedrine. Upon further questioning, she reported long-term use of ephedrine and pseudoephedrine for nasal congestion. In addition, she had recently been using herbal supplements for weight loss. Although she was unaware, we found an additional ephedrine stone on this patient in our referral lab files. We advised her to discontinue all these agents. She has not had further episodes after 6 months of follow-up. DISCUSSION: Ephedrine nephrolithiasis has previously been reported in patients consuming ephedrine or ephedrine-containing herbal preparations (1, 2) . Our case suggests that chronic use of ephedrine can lead to recurrent nephrolithiasis. Herbal supplements used for dietary purposes commonly contain ephedrine and we suspect our patient was likely receiving additional ephedrine through the supplement. In 1997, the FDA did call into question the safety of supplements containing ephedrine (3), however, they remain readily available and are sometimes 3D Surface Shaded display showing high-grade stenosis of the celiac axis and superior mesenteric artery origins.
with hoarseness, dyspnea and sore throat. Since the morning of admission, the patient had drooling as he was unable to swallow saliva. His symptoms were aggravated when supine. He denied fever or cough. His vital signs were normal. Temperature was 36.8 C. Remainder of the examination was remarkable only for an erythematous posterior pharynx and stridor on auscultation of the neck. Laboratory evaluation revealed a white cell count of 21,700 with 87% neutrophils and a normal hematocrit, platelets and normal routine serum chemistries. Lateral view of the neck showed an edematous, thumb shaped epiglottis. Blood cultures revealed no bacterial growth, but throat culture grew Haemophilus influenza type b. The patient received intravenous methylprednisone and cefuroxime. His symptoms improved and he was discharged on the fifth hospital day. DISCUSSION: Acute epiglottis has increasingly become an adult disease, probably as result of immunization of children against Haemophilus influenza type b. A high index of suspicion is required in any patient with sudden onset of " 4 Ds" dyspahgia, dysphonia, dyspnea and drooling. The frequency of positive blood culture is lower in adults than in children. A lateral cervical soft tissue x-ray is a useful test with sensitivity of 79±90% and specificity of 86±90%. Indirect laryngoscopy is the confirmatory test needed only if diagnosis remains in doubt after initial evaluation. Conservative medical management consisting of antibiotics and corticosteroids with vigilant monitoring are the mainstay of therapy in adults. Prophylactic intubation is not recommended. Prognosis of adults with acute epiglottitis is good with early recognition and prompt treatment.
TOO WARM FOR COMFORT±HEAT STROKE IN MINNESOTA. S. Dubey 1 , J.W. Leatherman 2 ; 1 Hennepin County Medical Center, Minneapolis, MN; 2 University of Minnesota LEARNING OBJECTIVES: 1) To recognize the morbidity and mortality from heat stroke 2) To emphasize that heat stroke is preventable and screening should be incorporated into seasonal health care maintenance. CASE INFORMATION: An 80 year old healthy male was found unresponsive by his wife in their sauna at home when he failed to come out in four hours time. Paramedics rushed him to the hospital where his core temperature was found to be 103 degrees F. He developed multiorgan damage including, respiratory failure requiring ventilatory support, myocardial injury, impaired hepatic and renal function, rhabdomyolysis and disseminated intravascular coagulation. With supportive treatment he made remarkable recovery and was discharged form the hospital on day six with a diagnosis of heat stroke. DISCUSSION: Heat stroke is a medical emergency manifesting with core temperature greater than 106 degrees F and multiorgan injury. Heat stroke can be fatal with almost 200 deaths occurring during an average summer in the United States alone. Risk factors include advanced age, lower socioeconomic status, chronic illness, dehydration, strenuous exercise and hot weather. Prevention is of utmost importance. Treatment consists of cooling, which must begin in the field, and supportive care. The mortality rate is as high as 10% in spite of aggressive management. Since it is easily preventable, screening and counseling need to be integrated into health care maintenance during high risk seasons to reduce the burden inflicted by this illness.
IT QUACKS LIKE A DUCK, BUT IT ISNT A DUCK. S. Dubey 1 , B. Marinelli 1 ; 1 Hennepin County Medical Center, Minneapolis, MN LEARNING OBJECTIVES: 1) To recognize precordial changes on EKG as a manifestation of massive pulmonary embolism 2) To emphasize the importance of screening for major alternative diagnoses in patients who present with apparent acute coronary syndromes CASE INFORMATION: A 44-year-old man, with history of coronary stent placement in the left anterior descending artery five months previously, presented with sudden chest pain and dyspnea. Relevant past medical history included a motor vehicle accident several months ago, which resulted in multiple lower extremity procedures and infections leading to reduced ambulation. EKG on admission was significant for precordial T wave inversion, and preliminary trans thoracic echocardiogram done in the emergency department showed normal left ventricular function. Considering stent closure, a coronary angiogram was done which showed patent coronaries. At that time, the formal reading from the already done echo was obtained and showed right ventricular enlargement and reduced right ventricular function. Immediate pulmonary angiogram was done and revealed extensive thrombus in the left pulmonary arterial system. Thrombolysis was initiated with t-PA. DISCUSSION: This is an example of acute pulmonary embolus very convincingly masquerading as coronary stent closure. An important clue, namely, reduced ambulation due to recent leg injuries, lay hidden in the past medical history. Precordial T wave inversions on EKG in the setting of pulmonary embolism, has been well described in medical literature. Various theories, including true coronary insufficiency and catecholamine mediated global T wave inversions have been proposed. The presence of such T wave inversions correlates with increased severity of pulmonary embolism, and resolution over a few days correlates with improved prognosis. Treatment includes anticoagulation with or without thrombolysis. Such EKG changes warrant at least a detailed history and physical exam focusing on deep vein thrombosis and pulmonary embolism risk factors.
EVALUATION AND DIAGNOSIS OF APPENDICITIS. S. Elad 1 ; 1 UCLA San Fernando Valley/ Olive View Medical Center, Los Angeles, CA LEARNING OBJECTIVES: 1. Recognize the clinical feautures of early appendicitis. 2. Identify atypical presentation of appendicitis. 3. Review common signs and symptoms of appendicitis. CASE INFORMATION: A 30 year old African American male with no significant past medical history presents with a one day history of right lower quadrant pain and constipation. Pt denied nausea, vomiting and anorexia. Pt was able to eat and tolerate both breakfast and lunch without incident. He denied fevers, chills. He was not taking any medications. Family history was noncontributory. He denied allergies and did not use tobacco, alcohol or drugs. On exam the patient was afebrile, pulse rate was 80 and blood pressure was 120/70. The cardiovascular exam was normal and his lungs were clear bilaterally. Abdominal exam revealed normoactive bowel sounds and mild right lower quadrant tenderness to palpation. He had no psoas and no murphys sign. No rebound, no guarding. His stool was heme negative. Neurological exam was within normal limits. Abdominal x-ray revealed right colon full of stool with no air fluid levels and no fecalith. Lab studies: NA 138 K 3.8 Cre 1.2 ALT 29 WBC 8 Hgb 12 Hct 39 Alk P 73 AST 36 TBili 0.5 Pt was sent home with diagnosis of constipation and given milk of magnesium and fleets enema. Pt returned to ER the following morning with complaints of worsening abdominal pain and anorexia. CT of abdomen revealed para appendiceal stranding. Pt was taken to the operating room for emergent appendectomy. Pt was found to have a gangrenous appendix with micro-perforations. Pt required a five day hospitalization and was discharged on oral antibiotics. DISCUSSION: The diagnosis of early appendicitis is often difficult to make. Classically appendicitis presents as a pentad of signs and symptoms consisting of RLQ pain, anorexia, vomitting, low grade fever and leukocytosis. Typically, there is a history of pain beginning in the peri-umbilical region that migrates to the RLQ. However, this presentation occurs in only 50% of patients. The location of the appendix may also alter the presentation of pain. A retrocecal appendix may cause flank pain whereas an appendix in the pelvis may elicit tenderness on rectal exam. Although the WBC is elevated in 80% of patients, patients with early appendicitis may have a normal WBC. The diagnosis of acute appendicitis remains a challenge to clinicians with up to a 20% missed diagnosis rate. Recently, studies have suggested that the introduction of focused appendiceal CT would reduce the cost of caring for patients with suspected appendicitis and aid in diagnosis. Prompt diagnosis of appendicitis can avert the development of complications such as perforation and subsequent septicemia. Physicians should rely on both their clinical judgement and the aid of radiological evaluation in the diagnosis of acute appendicitis. year-old male presented in clinic requesting genetic testing for Huntington's Disease (HD). Several of his family members had developed late-onset choreiform disorders. His nearest affected relative was an uncle who developed symptoms after age 60, progressively worsening until his death 5 years later. The patient's parents died of other causes and his siblings were well. The patient's physical exam was unremarkable. He demonstrated no involuntary movements; no abnormalities in sensation, motor strength, coordination, or gait; and no deficits in concentration, memory, or intellect. The patient was counseled as to the risks and benefits of testing. In particular, he was informed that no treatment exists to cure or forestall the course of the disease nor could the advent of symptoms be predicted. The patient continued to express a desire for testing, primarily to alleviate his anxiety with possibly harboring a genetic predisposition to HD. Laboratory testing revealed alleles with approximately 28 and 40 CAG repeats, consistent with high genetic burden for HD. 28 CAG repeats indicated a normal mutable HD allele which may expand upon transmission to offspring (27±35) while 40 CAG repeats was consistent with individuals who typically show features of HD ( > 39). The patient was informed of the results and their interpretation and referred to a neurologist for further discussion and counseling. DISCUSSION: Huntington's disease is a degenerative brain disorder with autosomal dominant inheritance manifested by chorea and dementia. Initial involuntary movements may not be noticed by the patient, but symptoms progress to irregular, sudden movements of the limbs and trunk, which can be disabling. Deficits in attention, memory, and judgment can be present as well as depression, social withdrawal, and disinhibition. Genetic testing for HD carries with it several important clinical and ethical implications. A positive test result implies an inherited mutation that will lead to the disaese at some unknown future date, with no available cure or even effective treatment. Protocols for pre-and post-test counseling have been proposed to address the potential psychological consequences, including anxiety, depression, family and marital stress, survivor's guilt, and even suicide. Anonymous testing has also been suggested to alleviate fears of discrimination in employment and insurance. year-old female presented with several days of increasing cough, wheezing, and dyspnea. She was in good health until 18 months prior when she first developed a non-productive cough. Six months later, she noted intermittent wheezing that increased in frequency and intensity. Recently she developed dyspnea at night when lying down. Inhaled steroids and bronchodilators gave little improvement. She denied fevers, chills, abdominal pain, and symptoms of acid reflux. Her past medical history was significant only for hypothyroidism treated with thyroxine. She had never smoked and had no pets. On examination, the patient was afebrile and not tachypneic. She was able to speak in complete sentences and coughed frequently. A loud, monophasic, inspiratory wheeze was audible without a stethoscope. Auscultation of the lungs revealed stridor. Prior pulmonary functions tests were reported as normal. Inspiratory and expiratory PA and lateral radiographs of the chest revealed a 2 cm diameter soft tissue mass in the tracheal air column. This mass moved from the carina at inspiration to the level of the clavicular heads at expiration. A subsequent CT scan of the thorax confirmed a distal tracheal mass. The patient was admitted for bronchoscopy to remove the tumor. Pathological examination found that the tumor did not fit with any known entity and was conjectured to be a hamartoma-like lesion of the seromucinous glandular tissue normally present in the distal trachea. DISCUSSION: Primary tracheal neoplasms are extremely rare, accounting for fewer than 0.1% of cancer deaths. They occur with equal frequency in men and women, most often between the ages of 30±50. The most common presenting symptoms relate to airway obstruction, notably cough, wheezing, dyspnea, and recurrent pneumonia. A "tracheal syndrome" of dyspnea, wheezing, stridor, hemoptysis, and voice change is present in up to 85 percent of patients with primary tracheal neoplasms. As this case illustrates, the diagnosis of primary tracheal neoplasms is typically marked by significant delay between the onset of symptoms and proper diagnosis. One study reported an average delay of eight months for malignant lesions and 25 months for benign tumors. Most patients are unsuccessfully treated for presumptive diagnoses of asthma or chronic bronchitis, with little improvement, before a diagnosis of a tracheal neoplasm is made. Other diagnoses, including tracheal neoplasms, should be considered when the usual treatment for asthma does not improve the symptoms.
DIFFICULT DYSURIA. S.B. Fazio 1 ; 1 Harvard University, Brookline, MA LEARNING OBJECTIVES: 1. Characterize the differential diagnosis of dysuria 2. Distinguish prostatitis from other causes of rectal pain 3. Recognize atypical presentations of appendicitis CASE INFORMATION: A 21 year old male presented to urgent care complaining of three weeks of dysuria and rectal pain. The pain was sharp and intermittent, brought on by urination or defecation, and radiated into the right inguinal area. He denied fever, nausea, vomiting, abdominal pain or tenesmus. Past medical history was unremarkable; he was on no LEARNING OBJECTIVES: (1) Recognize thyroid disease as an extrahepatic manifestation of chronic hepatitis C. (2) Manage thyroiditis associated with hepatitis C when the disease is either subclinical or mild. (3) Realize that thyroiditis can occur in patients with hepatitis C with or without the use of interferon therapy. CASE INFORMATION: A 29 year old woman with a 15 year history of hepatitis C (HCV) presented to general internal medicine clinic to establish primary care. She had undergone initial evaluation of her HCV disease 3 years earlier but had declined liver biopsy or interferon therapy at that time and discontinued followup. She had recently returned to hepatology and had proceeded with liver biopsy. On questioning and complete review of systems, the patient had no complaints. Notable findings on exam included a symmetric, smooth, nontender goiter, 6 cm with no palpable nodules. Exam of the liver was normal. Studies from 3 years and 6 months earlier were reviewed. At both time points, TSH was below normal, ALT was moderately elevated and HCV viral quantitative measurements were low. Genotype was 1A. Liver biopsy revealed minimal disease with Grade I lobular inflammation and Stage 0 fibrosis. Updated studies were ordered and revealed a normal TSH, mildly elevated total T3, normal free T4 and elevated anti-thyroid antibodies. No treatment was prescribed. At followup 1 month later, she remained asymptomatic and her goiter had markedly resolved to a minimally palpable and visible gland. Repeat laboratories revealed an isolated mildly elevated total T3. DISCUSSION: Chronic HCV infection has been associated with several extrahepatic conditions including thyroiditis. The mechanism may be related to HCV infection of mononuclear cells but the exact mechanism is still largely unknown. Recent case-control studies have found the prevalence of thyroid abnormalities and anti-thyroid antibodies significantly higher in patients than controls. Most commonly, there is no associated thyroid enlargement. Approximately 50% of those with anti-thyroid antibodies have subclinical thyroid disease and 50% have overt symptoms. Thyroid dysfunction has also been noted during interferon-alpha therapy in up to 12% of HCV patients but studies have found disease in both interferon-treated and untreated patients. Prospective studies show thyroid dysfunction may develop in patients on interferon therapy with or without antibodies. Those with antibodies do not show a significantly higher propensity to develop thyroid dysfunction during interferon therapy. In summary, as in the general population, management includes timely recognition of signs and symptoms of thyroid disease. In the HCV population, these symptoms can often be subtle or may be attributed to HCV itself. HCV patients with thyroiditis should be followed closely; however, they may not necessarily require intervention if thyroid disease is subclinical and the thyroiditis or goiter may remit spontaneously. Finally, pre-existing thyroiditis is not a contraindication to interferon therapy.
SPACE OCCUPYING LIVER LESIONS WITH FAMILIAR OCCUPANTS. F. Francois 1 , L. Yatskar 2 ; 1 New York University, Brooklyn, NY; 2 New York University, New York, NY LEARNING OBJECTIVES: 1. Develop a high index of suspicion for tuberculosis in refugees and immigrants 2. Recognize immunocompromised hosts as being at risk for extrapulmonary tuberculosis 3. Assess space occupying hepatic lesions in the setting of possible tuberculosis CASE INFORMATION: A 35 year old Tibetan male with the Acquired Immune Deficiency Syndrome (AIDS), CD4 = 32/mm 3 and viral load = 102,000 copies/ml, presented complaining of fever, chills, abdominal pain, diarrhea, and malaise of increasing severity over five days. Six years prior to this clinic visit the patient was treated with Isoniazid for 4 months after a positive purified protein derivative skin test. Two years prior to the visit he was diagnosed with AIDS and completed treatment for multiple episodes of infectious diarrhea. Two weeks prior to this current evaluation he began to experience abdominal discomfort associated with loose non bloody stools. A course of Metronidazole did not improve his symptoms. He had never been on antiretroviral therapy, and on examination was noted to be a thin febrile male in no acute distress with bitemporal wasting, oral thrush, nonicteric sclerae, moderate right upper quadrant tenderness to deep palpation, and hyperactive bowel sounds. The patient was admitted to the hospital for evaluation, at which time laboratory analysis was notable for a white blood count of 5.8, an hematocrit of 26.6, and an INR of 1.59. An abdominal CT revealed low attenuated lesions in the liver, the largest measuring 6cm in diameter, with a smaller lesion in the porta hepatis extending into the head of the pancreas. Percutaneous drainage of one of the lesions revealed acid fast organisms subsequently identified as Mycobacterium Tuberculosis (MTB) which was pan sensitive. A bronchoscopy did confirm concurrent pulmonary TB. There was radiographic evidence of resolution of the hepatic lesions over several months after initiating treatment with Isoniazid, Rifampin, Pyrazinamide, and Ethambutol. DISCUSSION: One in three individuals worldwide is infected with MTB. In the U.S., 40% of cases occur among foreign born persons. Tuberculosis most commonly presents as pulmonary or lymph node disease, but can also affect other organs. Immunosuppressive therapy, malignancy, and AIDS are known risk factors for disseminated complications with the organism. Extrapulmonary disease is seen in 60% of AIDS patients with lung disease, and of those patients, 7.5% have tuberculosis of the liver. Hepatic tubercular involvement is most commonly described as granulomatous, but can also be seen as discrete space occupying lesions such as tuberculomas, and abscesses. Patients typically present with fever, night sweats, abdominal discomfort, weight loss, and hepatomegaly. Diagnosis is made by aspirating a lesion, and treatment is based on drug sensitivities. her abdominal wall, breasts, thighs, and calves. The lesions were associated with central necrosis and peripheral mottling. She was alert, oriented, afebrile, and without leukocytosis. She had a calcium-phosphorus product of 75 and a parathyroid hormone level of 438. Despite aggressive wound care and antibiotics, her condition worsened precipitously over 72 hours. Her skin lesions became necrotic, ulcerative, and suppurative. She became febrile, hypotensive, and stuporous. Despite broadened antibiotics and aggressive fluid resuscitation, she became unresponsive, pulseless, apneic, and died. DISCUSSION: Calcific Uremic Arteriolopathy (CUA) is characterized by calcification of subcutaneous arterioles and infarction of skin and subcutaneous adipose tissue. It occurs in a minority of chronic renal failure patients. In CUA, calcification of the media of arteriolar vessels leads to regional ischemia and then infarction of skin and subcutaneous tissue, especially in areas of high adipose content. Skin necrosis and ulceration develop and patients usually die as a result of ischemic or infectious complications. While the true pathogenesis of CUA is unknown, several factors have been associated with a higher incidence. A high calcium-phosphorus product and secondary hyperparathyroidism are often associated with a increased incidence of CUA. Other features such as female gender, white race, obesity, recent significant weight loss, insulinrequiring diabetes mellitus, and use of calcium carbonate as a phosphorus binder have been reported as predisposing factors. It is likely that modification of these factors in our patient, such as management of high calcium-phosphorus product with non-calcium containing phosphorus binders, reduction of calcium content in dialysate fluid, and gradual weight loss may have prevented this deadly outcome.
DISCUSSION: HMG-CoA reductase inhibitors are popular lipid lowering agents. The most common adverse drug reaction is a transient chemical hepatitis of little clinical significance. Clinically significant rhabdomyolysis is extremely rare with an incidence of < 0.5%. HMG-CoA reductase inhibitors require metabolism by P-450 enzymes before they become active. Inhibition of the P-450 system leads to decreased metabolism of the statin drugs and higher plasma levels. The resultant increased delivery of the HMG-CoA reductase inhibitors to muscle cells decreases the production of cholesterol and cytochrome Q10 and makes muscles susceptible to oxidative damage and rhabdomyolysis. Cerivastatin is a third-generation HMG-CoA reductase inhibitor that is marketed as a``safer'' drug because it is metabolized by two different P-450 isoenzymes, CYP3A4 and 2C8. Cases of rhabdomyolysis in patients treated with cerivastatin and gemfibrozil have been reported; however, there have been no published reports of cerivastatin alone causing rhabdomyolysis. The etiology of rhabdomyolysis in this patient is somewhat puzzling. He was on no potent inhibitors of CYP3A4 nor did he have pre-existing hepatic or renal dysfunction. It may be that he had an intrinsic deficiency of CYP2C8. The treatment of HMG-CoA reductase inhibitor induced rhabdomyolysis involves withdrawal of the drug and supportive care. The myositis usually resolves completely although renal failure has been reported.
AN UNUSUAL CASE OF FLANK PAIN. F. Green 1 , R. Granieri 1 ; 1 University of Pittsburgh School of Medicine, Pittsburgh, PA LEARNING OBJECTIVE: Identify and treat acute renal infarction. CASE INFORMATION: J.C. is a 63 year old female with ischemic cardiomyopathy, mitral regurgitation, hypertension, and rheumatoid arthritis who developed spontaneous right flank pain that was constant, 8/10, sharp, and sufficient to wake her from sleep. The pain was associated with nausea, vomiting,and intermittent diaphoresis. There was no radiation to the groin, abdomen,or chest. There was no history of chest pain, palpitations, dysuria, abdominal trauma, vaginal discharge, or invasive vascular procedures. She has never had thromboembolic disease or atrial fibrillation. Medications included digoxin, losartan, prednisone, furosemide, hydroxychloroquine, aspirin, and methotrexate. Physical examination was notable only for compensated biventricular heart failure, mitral regurgitation, and right flank tenderness. Electrocardiogram revealed sinus tachycardia. Laboratory data was notable for BUN 35, Cr 1.2 WBC 14.1 (94% PMN), ALT 28, AST 37, GGT 180, Alk Phos 192, and LDH 386 . Urinalysis revealed 4 RBC, 0 WBC, and trace protein. CT scan of the abdomen and pelvis with contrast showed an acute infarct of the inferior pole of the right kidney. The patient was admitted and anticoagulated. Echocardiogram demonstrated an ejection fraction of 25% and severe mitral regurgitation with no evidence of mural thrombus. DISCUSSION: Renal infarction is an often overlooked clinical syndrome than can be mistaken for more common causes of flank pain. The etiology of most cases is embolic disease from underlying atrial fibrillation, valvular disease, or cardiomyopathy. No historical variable aside from risk factors for thromboembolic disease has been shown to be associated with renal infarction. Physical examination is non-specific although an incomplete infarction may cause a hyper-renin state with secondary hypertension. LDH is the best marker for renal infarction although initial measurements may be normal. Other lab abnormalities are inconsistently present. CT scans have replaced IVP and angiogram in making the diagnosis of renal infarct. While areas of infarction can be seen on non-contrasted CTs, contrast is necessary to increase sensitivity, better define the infarct, and distinguish acute from completed infarcts. Surgery, angioplasty, fibrinolysis, and conservative medical management are therapeutic options. Revascularization appears to be most successful in early infarction (first 3 hours). Conservative therapy with anticoagulation (and additional antihypertensive therapy if needed) is thought to have lower morbidity than invasive therapy in most cases. Although the mortality from the renal infarct itself is low, these patients have a dismal one year prognosis because of further embolic disease and underlying cardiac disease. year-old male roof repairman presented with shortness of breath for the past two weeks. He had no history of alcoholism, smoking or previous lung disease. He had been taking Azithromycin during the week prior to admission but had become worse. He had right-sided, pleuritic chest pain, fever, chills, yellow-greenish sputum, night sweats, and mild weight loss. Examination revealed moderate respiratory distress, a temperature of 101.1 8F and a pulse of 115. The mouth, teeth and oral pharynx were normal. The cardiac exam was normal except for tachycardia. Diminished breath sounds were noted on the right side of the chest. His WBC was 19.2 with 89% PMN's. A CXR and subsequent CT revealed a large loculated pleural effusion with right upper and middle lobe atelectasis. A sputum gram stain showed gram positive cocci in chains. A 2D echo showed a large peri-cardial effusion without evidence of tamponade. 1,200 cc of yellowish-greenish pus was drained following thoracentesis and placement of a chest tube. Cultures grew only Streptococcus constellatus which was sensitive to Clindamycin and Ampicillin. He recovered following these antibiotics with chest tube drainage. DISCUSSION: Streptococcus constellatus is a gram positive microaerophillic coccus that grows in chains or pairs and is a member of the Streptococcus intermedius group. It is usually present as a harmless commensal organism in the mouth and GI tract but like other members of the S. intermedius group has the propensity to form abscesses. Intrinsic virulence factors (hydrolytic enzymes, different adhesins on cell surfaces and a polysaccharide capsule) facilitate adherence, decrease phagocy-tosis and increase pathogenicity. S. constellatus is frequently associated with tho-racic infections and more prone to occur in males, alcoholics, people with previous pneumonia and in those at risk for aspiration. Mortality ranges between 15±30%. Although this patient had few risk factors for the development of this infection, the presence of a 3D Surface Shaded display showing high-grade stenosis of the celiac axis and superior dense empyema and pericardial effusion suggested S. costellatus or other organisms in the S. intermedius group as a possible cause.
A. Hamad 1 , R. Clark 1 , J. Singh 1 ; 1 Nassau University Medical Center, Eastmeadow, New York LEARNING OBJECTIVES: 1-deep venous thrombosis (DVT) can cause of fever of unknown origin (FUO). 2-the importance of computed tomography of the abdomen during work up for FUO.3-AIDS is associated with high incidence of thrombosis. CASE INFORMATION: A 45 years old Haitian female presented with generalized weakness, weight loss, and low-grade fever. She had a history of AIDS with wasting syndrome and cerebral toxoplasmosis, and recurrent deep venous thrombosis in the lower extremities. She had a temperature of 100 0F, with no swelling in the lower extremities. Plain radiograph of the chest and Urinalysis were normal. Blood, stool, and urine cultures were negative. Stool for Clostridium difficile, ova and parasite, Cryptosporidium, acid-fast bacilli and Isospora belli was negative. Sputum for acid-fast bacilli smear and culture was negative. Hepatitis profile was negative. Sputum for acid-fast bacilli and Pneumocystis carinii were negative. Few days later, she complained of severe perineal pain, rectal exam was very painful and frank blood was found on a gloved finger. Sigmoidoscopy was not done because of the pain. She was treated for an anorectal fissure and the pain improved after few days but she continued to have fever. Computed tomographic scan of the head with contrast and cerebrospinal fluid analysis were unremarkable. Computed tomography of the abdomen was done as work up for FUO. It showed massive thrombosis extending from the right renal vein to the inferior vena cava up to the liver. Hematologic work up was significant for decreased total protein S (28%) with normal protein C (64%) and antithrombin III (102%). Anticoagulation therapy was started and fever improved after more than a week. DISCUSSION: Our patient has massive thrombosis, which we think caused her prolonged fever, as an extensive work up ruled out other causes of fever. Total protein S is normal in AIDS patients with decrease in free protein S level. We believe that our patient has two separate disorders predisposing her to thrombosis (HIV infection and primary protein S deficiency). This belief depends on the fact that her total protein S was < 50% of the normal, consistent with the classic type of the hereditary protein S deficiency. Although acute thrombosis can decrease antithrombin III, it is less likely to cause a decrease in protein S or protein C. The decrease of protein S in our patient is not secondary to the thrombosis, as her antithrombin III and protein C levels were normal. We found only few reports of patients with HIV infection with protein S deficiency who presented with significant DVT. Weakness started the night before presentation and deteriorated rapidly in the next day. He reported having polydipsia and polyuria in the last few days. The patient was afebrile with blood pressure of 110/70 and heart rate of 90 bpm. Physical exam was significant for short stature (155cm) and moderate weakness of all extremities. Laboratory tests showed severe hypokalemia (1.3 meq/dl) and hypophosphatemia (1meq/dl), low bicarbonate (9 meq/dl) and normal anion gap. Arterial blood gas showed acidosis (pH = 7.12) and pCO 2 of 28 mmHg. After receiving 280 meq of potassium supplements over 16 hours his potassium level improved to 2.7 meq/dl and he regained his power completely. The patient received replacement of potassium, phosphorus, and bicarbonate for 4 days period till his numbers became normal and was discharged home on potassium citrate supplements only. Work up during hospitalization revealed urine pH repeatedly > 6.5, urine specific gravity of 1.010, urine anion gap of +40, urine pCO 2 of 50 mmHg after correction of the acidosis, daily requirement of 20 to 40 meq of bicarbonate only to maintain normal serum level, and fractional excretion of bicarbonate of 2.5% and phosphorus of 22%. Renal ultrasound revealed echogenic shadows distinct between the medulla and the cortex, consistent with calcification. DISCUSSION: Distal and proximal renal tubular acidosis are uncommon diseases. The proximal type characterized by reduction in reabsorpsion of bicarbonate while the distal type results from inability to secrete hydrogen ion. This patient represents a typical case of type I RTA (distal) with all the manifestations and complications. Short stature, severe hypokalemia with secondary paralysis and polyuria (loss of urine concentrating ability), renal calcifications, positive urine anion gap, inability to acidify the urine despite severe acidosis (oppose to proximal type), decreased urine pCO 2 (which is usually higher than the plasma because of secretion of H+ in the distal tubule that binds the bicarbonate and become CO 2 ), fractional excretion of bicarbonate of 2.5% (in proximal RTA this number is > 15%) and minimal amount of bicarbonate replacement was enough to keep normal blood level. We want physicians to be aware of this rare disease that is very easy to treat but if left untreated can have detrimental consequences.
QT PROLONGATION SECONDERY TO HYPOCALCEMIA AFTER CORRECTION FOR ELEVATED SERUM ALBUMIN. A. Hamad 1 , M. Zihlif 1 , M. Salameh 1 , A. Alghadban 2 , D. Feinfeld 1 ; 1 Nassau University Medical Center, Eastmeadow, New York; 2 SUNY at Stony Brook, Port Jefferson NY LEARNING OBJECTIVE: 1-recognize that correction of calcium level for albumin should be done also if albumin is high. CASE INFORMATION: 42 years old white male with no significant past medical history except alcoholism came to the emergency room with vomiting of small amount of blood associated with mild epigastric pain. He was not taking any medications at home. He was afebrile, blood pressure was 123/83, heart rate was 126 bpm. Physical exam was unremarkable except for mild dehydration and tachycardia with no added sounds or murmur. Laboratory tests were significant for normal calcium level (9 mg/dl), magnesium of 2 meq/dl, serum albumin of 5.3 mg/dl, potassium of 3.3 meq/dl. Arterial blood gas showed normal pH of 7.44. Electrocardiogram showed regular sinus rhythm with prolonged QT interval (QTc = 44 msec). Because of this prolongation, the patient was given calcium gluconate intravenously as corrected calcium with elevated albumin was low (7.9 mg/dl). Ionized calcium was low (1.04 meq/dl. Repeat EKG showed return of QT interval to normal limit. The patient was treated with vitamins and anxiolytics for his mild alcohol withdrawal and was discharged home after 2 days. DISCUSSION: QT prolongation can occur as a congenital or an acquired disorders. The acquired form is secondary to pharmacologic agents (mainly antiarrhythmatics, tricyclic antidepressants, antihistamines and erythromycine), and electrolytes disturbances (hypokalemia, hypomagnesemia and hypocalcemia). We are presenting this case to stress on fact of correcting calcium level to the serum albumin, this correction can be by adding 0.8 mg to each 1 mg of albumin below the normal level (4mg/dl) or by subtracting 0.8 mg to each 1 mg of albumin above the normal limit.
NEUROMYELITIS OPTICA. I.P. Haque 1 ; 1 University of Virginia at Roanoke-Salem, Roanoke, VA LEARNING OBJECTIVE: Diagnose the Different Variants of Multiple Sclerosis including Neuromylelitis OpticaTreatment of Neuromyelitis Optica CASE INFORMATION: Neuromyelitis optica or Devic's syndrome is an uncommon neurological illness characterized by optic neuropathy and myelopathy. This case presents a 39 year old African-American female with new onset of visual changes, periocular pain, headache, malaise, lower extremity weakness, and back pain. The patient's symptoms began one week prior to admission. She previously presented with lower extremity pain and weakness six months prior and was suspected clinically to have multiple sclerosis and had resolution of her symptoms with corticosteroids. She had no changes on magnetic resonance imaging of brain and cerebral spinal fluid findings were nonspecific at the time. Her symptoms of visual changes and loss with periocular pain were not present upon this prior admission. On physical exam, she had papillitis, severe visual field defect, profoundly decreased lower extremity weakness and sensation, and Lhermitte's sign. On laboratory exam, cerebral spinal fluid showed increased protein and she had an elevated ESR. Magnetic resonance imaging of spine showed diffuse cervical cord swelling and cervical signal changes. This patient presented with bilateral optic neuritis and myelitis, known as Devic's syndrome or Neuromyelitis optica. She was treated with intravenous methylprednisolone and had gradual improvement of her symptoms. DISCUSSION: Devic's syndrome generally has a rapid onset of optic neuritis and myelitis. Patients with acute or subacute Devic's syndrome often respond to corticosteroids as this patient did. In Devic's syndrome, acute spinal cord lesions can demonstrate diffuse swelling extending over several levels or the entire cord in either a continuous or patchy distribution. The optic nerve and chiasm can show either demyelinating lesions and/or necrotizing lesions. Approximately 35 percent of patients have a monophasic illness and 55 percent develop relapses, and rarely patients have a fulminantly progressive course without relapses or a course typical of multiple sclerosis. (2) Identify the indications for valve replacement in IE. CASE INFORMATION: A 41-year-old white male with past medical history of migraine headaches presented to the emergency department for a seizure occurring in a restaurant. While sitting, the patient suddenly developed stiffness and ridgidity in the right arm then the left arm. He then collapsed and had a generalized seizure. According to an eyewitness the patient lost consciousness for 3±4 minutes, after which he was confused but without incontinence or focal weakness. Prior to this event the patient experienced low-grade fever as high as 1018F and myalgias for one week but no other symptoms. Physical examination revealed a young ill looking male; temperature was 378C. He was tachycardic and the blood preasure was 164/78 mmHg. Significant findings included abrasions over the left temple, evidence of an old infarct in the left parietal region thought to be embolic. Echocardiogram showed a dilated left ventricle with borderline left ventricular hypertrophy and good systolic function, severe aortic regurgitation and suspicion of an aortic valve vegetation. A transesophageal echocardiogram showed a bicuspid aortic valve with prolapse of one of the leaflets, 3+ aoritic incompetence and evidence of a vegetation 4±5 mm in size on one of the leaflets of the aortic valve. Blood cultures initially reveled Gram + Cocci in clusters later identified as coagulase negative Staphylococcus. All the cultures drawn later were negative for any growth. Because of evidence of embolic disease on the CT scan of the brain and the recent generalized seizure (representing multiple embolic events), aortic valve replacement was indicated. The patient had a St. Jude HB mechanical valve replacement. He had an uneventful post-operative course. He was started post-operatively on heparin and then later on Coumadin. Blood cultures and cultures from the aortic and mitral valves were negative for any growth. The culture from the aortic vegetation was also negative. The patient was continued on cefrtiaxone for 4 weeks to treat likely atypical organisms. DISCUSSION: When considering the differential diagnosis of a new onset seizure, IE is a relative uncommon cause, but should be considered when fever and/or a heart murmur is present. When IE is associated with seizures, the seizures usually occur later in the course of the disease rather than as part of the initial presentation as in this case. The bicuspid aortic valve in our patient was a risk factor of IE but it is important to recognize that one third of patients with IE have no identifiable predisposing cardiac lesions. Negative cultures occur in 2±5% patients with IE. The indication for valve replacement in this case was evidence of recurrent embolic events. Mechanical valve was chosen because of the patient's young age.
DOCTOR, CAN YOU HELP ME DIE? J.M. Hauser 1 ; 1 University of Chicago, Chicago, IL LEARNING OBJECTIVES: 1. Explore reasons for a request for physician-assisted-suicide 2. Learn how to respond to a requests for physician-assisted-suicide CASE INFORMATION: Mr. Hayes [name changed] was a 57 yo man with advanced esophageal cancer who requested assistance in dying. He initially presented with dysphagia and underwent endoscopy which revealed esophageal cancer. After an abdominal CT scan showed multiple liver masses consistent with metastases, he was referred to hospice. His predominant symptoms were abdominal pain treated with morphine, anxiety treated with lorazepam and dysphagia which required he stop eating solid foods. Mr. Hayes initially resisted medications because of possible drowsiness. It soon became clear, however, that his refusal was because medication could not address his most significant suffering: as a relogious person, he told me, he was angry at God for not curing his illness and for letting him die without a wife. He did not know how to cope with this anger and did not think that either hospice or his own priest could help address it. At the end of our second visit, after discussing his symptoms and his continuing spiritual distress, I asked Mr. Hayes``Is there any other way I can help you?'' He answered:``You can get put a needle in my vein and help me end it.'' I asked what he meant. "You can help me die. It's in your oath to stop suffering." I explored with him how he was suffering, whether there were things he looked forward to in his future and whether there were ways we could help him short of ending his life. After discussions with many members of the hospice team, aggressive treatment of his symptoms, and help from two of his brothers helping care for him, his desire that we help end his life waned but never completely disappeared. DISCUSSION: Requests for physician assisted suicide (PAS) are troubling, partly because they highlight intense and unrelieved suffering. This case illustrates how such a request can be a window into inadequately addressed suffering, can reveal misunderstandings about what physicians are legally permitted to do; and how palliation of many symptoms may not be adequate for some patients' suffering. Rather than a debate about the ethics of PAS, this case discussion will consider the reasons behind such requests and our possible responses to them.`I CEBERG LETTUCE AND CARROTS'': A MEAL IN SEARCH OF A DIAGNOSIS. R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the varied presentations of Pica as well as its clinical significance. CASE INFORMATION: A 57 year-old woman presented with memory loss, a fear of becoming obese and a recent four-month history of cravings for``iceberg lettuce and carrots.'' She was eating two heads of lettuce and a pound of carrots daily. Her past history included hypothyroidism (1970) , treated with oral replacement; a hysterectomy (1970) ; severe osteoarthritis with chronic left hip pain; fibromyalgia treated with amitriptyline; a gastric exclusion procedure for weight loss (1994) ; and severe personal stress due to the recent suicide of her 27 year old son six months prior. Her weight loss procedure was a partial gastrectomy with creation on a six-ounce pouch and anastomosis of the distal duodenum to the terminal ileum. This resulted in a``four foot common channel.'' She lost 117 pounds without incident. Physical exam was unremarkable except for mild memory loss, obvious osteoarthritis with a left sided limp and well healed surgical scars. She weighed 170 pounds. DISCUSSION: Although there was obvious depression and a profound fear of weight gain, the cravings for``iceberg lettuce and carrots'' were of recent onset. Because of the history of the gastric bypass procedure and its expected result of intestinal mal-absorption, a Pica variant was suspected. Although Pica has been associated with such bizarre food cravings as laundry starch, clay, chalk, ice, and paint chips, it has been associated with normal foodstuffs such as apricots, almonds, chocolate, carrots, etc. Because of the association between Pica and iron deficiency, laboratory tests for anemia were ordered. The results were compatible with iron deficiency anemia. Additional studies revealed zinc deficiency, early vitamin B12 deficiency, and vitamin D deficiency with hypocalcemia and secondary hyperparathyroidsim. With these findings, the patient revealed that although she had taken nutritional supplements following her operation, she stopped them following the death of her son. With reinstitution of nutritional supplementation her behavior and laboratory studies have been improving. 1. Recognize (DI) diabetes insipidus clinically. 2. Confirm DI using standard diagnostic testing. 3 . Through thorough review of its possible causes, establish the etiologic lesion for DI in a patient. CASE INFORMATION: A 23 year old male came to the emergency room with a chief complaint of a nine day history of blood in his stool, lightheadedness, nausea, diarrhea, and profound weakness. He was admitted for dehydration, anemia, and bright red blood per rectum. On review of systems, it was noted that he had been drinking several gallons of water a day since 5 months prior to admission and had been urinating hourly. Diabetes insipidus (DI) was suspected when it was observed that he had a clear dilute urine despite his tachycardia and orthostatic hypotension. His urinalysis revealed a low specific gravity of 1.002 and no glucose; serum chemistries were normal. A water deprivation test was consistent with diabetes insipidus. Subsequent workup revealed panhypopituitarism and ultimately the causative lesion, a midline germinoma. The rectal bleeding, which might have lead us toward a different evaluation, was hemorrhoidal. The diarrhea was most likely secondary to his hypoadrenal state. DISCUSSION:
CERVICAL SPINE OSTEOMYELITIS CAUSED BY STREPTOCOCCUS MILLERI. M.R. Huber 1 , P.S. Mueller 1 ; 1 Mayo Clinic, Rochester, Minnesota LEARNING OBJECTIVES: #1 Recognize that poor dental hygiene can lead to serious complications, such as cervical spine osteomyelitis.
#2 Distinguish Streptococcus milleri (S. milleri) from other viridans streptococci by its tendency for suppurative infections. #3 Recognize S. milleri as a rare but known cause of vertebral osteomyelitis. CASE INFORMATION: A 61 year old male was found to have fractures of C5 and C6 with associated left sided radiculopathy after falling down stairs. The patient had no chronic medical conditions except for recurrent tonsillitis and tonsilloliths. The patient was admitted to an outside hospital and found to have a WBC of 13,300 Â 10 9 /L and a WESR of 67 mm/hr. Surgery was performed to stabilize the fracture and revealed cervical osteomyelitis with associated phlegmon and epidural abscess. The patient underwent debridement and internal fixation. Five of six cultures grew only pan-sensitive S. milleri. With ampicillin, the patient's WBC had normalized and WESR had improved. However, he experienced two internal fixation rod failures and developed a mediastinal abscess with associated cutaneous fistula. He was then transferred to our hospital. Tissue cultures from the neck and mediastinal abscess grew several organisms, and as such, antibiotic coverage had to be broadened. The patient required three additional spine stabilizing operations before a satisfactory result was achieved. He had a number of complications including drug induced neutropenia, thrombocytopenia, rash, and atrial fibrillation. At the time of this writing, the patient has made a nearly full recovery. He has a WBC of 4,300 Â 10 9 /L and WESR of 0 mm/hr. He has been maintained on gatifloxacin and minocycline while he has metal hardware in the previously infected region. DISCUSSION: Streptococcus milleri represents several biologically diverse, yet clinically homogenous, types of viridans streptococci. They are characterized by their tendency for suppurative infections. They can be found in abscesses of the mouth, brain, and liver as well as empyemas. Several cases of vertebral osteomyelitis have been previously reported. Of the two cases of cervical osteomyelitis, one involved an intradiscal steroid injection and the other had known severe periodontal disease. The patient in this case had known palatine tonsillar disease which may very well have led to his cervical spine osteomyelitis.
M.T. Hughes 1 ; 1 Johns Hopkins University, Baltimore, Maryland LEARNING OBJECTIVES: 1. Assess the role of weak paternalism for patients lacking decision-making capacity, when no readily identifiable surrogate decision-maker is available. 2. Weigh the obligations of beneficence, respect for autonomy, and an ethic of care. 3. Determine the goals of care for cognitively impaired patients with a life-limiting illness. CASE INFORMATION: 82 year old male with multiple medical problems including coronary artery disease, severe chronic obstructive pulmonary disease, and moderate, multi-factorial dementia presented with poor appetite after being lost to medical follow up for six months. Previous physicians had prescribed megestrol for anorexia. Left renal mass had been detected five years earlier and``watchful waiting'' approach had been arranged with urologist two years prior to presentation. Six months prior to presentation, CT scan demonstrated enlargement of mass and findings consistent with renal cell carcinoma. Patient had been referred back to urology but appointment did not occur. Over course of current evaluation, when asked whether to pursue repeat CT scan, patient stated,``It don't bother me, so don't bother with it.'' Patient lived with his 84 year old wife, who likewise had moderate dementia and had recently undergone surgical resection of Stage III gastric carcinoma. Patient had lost contact with distant cousins in another state and denied any other family members who could help with decision-making. His day consisted of "chewing the fat" with friends at the local market. He enjoyed his life and wanted to stay at home with his wife as long as he could. CT scan and urology consultation were not pursued. One year later, the patient suffered a major stroke. Home hospice was arranged, but his wife was unable to care for him at home. On presentation to the emergency room, she had no recollection of his stroke and no recognition of his dense hemiplegia. The patient was transferred to an inpatient hospice, where he died two weks later. His wife underwent psychiatric hospitalization for her dementia and was subsequently transferred to an assisted living facility. DISCUSSION: Informed consent rests upon the principle of respect for autonomy and thus requires voluntariness and competence. This patient had impaired judgment, poor recall, and an inability to comprehend complex information. Informed consent was not possible. His wife lacked decision-making capacity. Together, both could live independently in the community, compensating for each other's deficits. Neither could weigh the benefits and burdens of a major surgery. In acting out of beneficence, the physician in this case needed to determine whether definitive treatment for the renal mass (nephrectomy with significant perioperative risk) was a benefit or a harm. Goal setting and understanding the patient as person resulted in a decision that did not strictly adhere to an autonomy model. An ethic of care, focusing on maintenance of the relationship between husband and wife, was judged to be more determinative than a principle-based ethic. This case demonstrates that in certain well-defined situations, the physician may be justified in invoking weak paternalism.`P ANCREATITIS, HEPATITIS, AND DERMATITIS'' IN A 70 YEAR OLD FEMALE. L. Humphreys 1 , E. Yee 1 , A. Gomez 1 ; 1 UCLA San Fernando Valley Program, Sepulveda, Ca LEARNING OBJECTIVES: 1) Recognize the clinical presentation of dermatomyositis, 2) Discuss the work up and treatment of dermatomyositis, 3) Discuss screening for occult malignancies associated with dermatomyositis. CASE INFORMATION: A 70 yo Latina female with a history of cholecystectomy presented with 3 days of nausea, vomiting, and epigastric pain. She denied fever, chills, diarrhea and constipation. She took no medications and denied ETOH use. She was a thin female with T 36.1 C, BP 96/52, P 68, R 18. A violaceous rash was present on her forehead, eyelids, and nose. She had epigastric tenderness with guaiac negative stool. Extremities had subcutaneous calcifications with diffuse weakness proximally. Lab work was remarkable for AST 842, ALT 467, T bili 1.3, D bili 0.3, Alk Phos 352, lipase 9,759, and serial CK's of 5640, 4811, 3835. EKG was normal and troponin negative x 2. An U/S showed no biliary dilation, gallbladder, or stones, and a poorly visualized pancreas. She was hospitalized with the presumptive diagnosis of "pancreatitis, hepatitis, and dermatitis". Subsequent workup found normal triglyceride and calcium levels, negative viral hepatitis serologies, and an unremarkable abdominal CT scan. An elevated aldolase of 24.9, EMG with myopathic changes, and muscle biopsy demonstrating myositis confirmed the diagnosis of dermatomyositis. She improved clinically with steroids. Work up for occult malignancy including CXR, PAP smear, mammography, CA-125 level, endoscopy, and colonoscopy were all normal. An etiology for her pancreatitis was never found. DISCUSSION: Dermatomyositis (DM) is an idiopathic inflammatory myopathy that usually presents with proximal muscle weakness. Distinctive skin manifestations include a psoriatic rash over the knuckles (Gottron's papules), periorbital discoloration (heliotrope rash), and subcutaneous calcifications. DM occurs in approximately 5/1,000,000 people. Diagnosis is confirmed by elevated muscle enzymes, myopathic EMG findings, and muscle inflammation on biopsy. Presence of anti-Mi-2 antibody is specific for DM, but is found in only 25% of cases. First line therapy is systemic corticosteroids, with methotrexate, azathioprine, or cyclophosphamide reserved for refractory cases. Malignancy occurs between 7% and 34% in DM patients, with ovarian and colon cancers found most often. Cancer associated disease is more common in older patients and is associated with a worse prognosis. There may be a delay of up to five years before any associated malignancy develops. Although this patient had an extensive workup because of her symptoms, asymptomatic patients with DM should routinely undergo age specific screening tests for occult malignancies. LEARNING OBJECTIVE: To recognize the early signs of cerebral aneurysms and make an early diagnosis CASE INFORMATION: 59-year-old male with history of well controlled hypertension and COPD presented with a severe headache 10/10 in intensity, throbbing, constant and gradual in onset which began 11 days ago in the occipital area and then extended to the right temporal/ posterior orbital area. Two days prior to admission he started having diplopia, ataxia and nausea/vomiting. The patient denied fevers, chills, photophobia, phonophobia, neck stiffness, numbness, weakness, vertigo or seeing flashing lights. Past surgical history includes splenectomy after gun shot wound in 1965. Patient also with history of polysubstance abuse, last used 2 years ago. Family history is non-contributory. On initial examination blood pressure was 167/90, temperature 36.5, heart rate 72 and respiratory rate 18. The patient was alert and oriented and in no acute distress but did report a 10/10 headache. Eye examination was significant for a right pupil which was 5 mm and sluggish to react as opposed to a left pupil which was 2 mm and reactive. There was ptosis of the right eyelid, a deficit in medial and upward gaze on the right and a deficit in accommodation on the right side. There was no papilledema and pressures were 12 bilaterally. Visual acuity was normal and equal bilaterally. The neck was supple and there were no carotid bruits. Neurologically there were no other cranial nerve deficits and no other neurological deficits. CBC and chem.-7 were normal and patient had an ESR of 10. The patient had an MRA which confirmed the diagnosis of posterior communicating artery aneurysm compressing the third cranial nerve. The patient was then transferred to neurosurgery and underwent clipping of the aneurysm. DISCUSSION: 90%of all intracranial aneurysms are congenital. Common locations include: anterior communicating artery, bifurcation of the middle cerebral artery, posterior communicating artery, internal carotid artery bifurcation and the basilar circulation. Unruptured aneurysms are usually < 6±7 mm and 20% of patients present with oculomotor nerve palsy. These patients usually have aneurysms of the internal carotid artery at the posterior communicating artery. Rupture most commonly causes subarachnoid hemorrhage. Symptoms of rupture include sudden severe headache, altered mental status, meningeal irritation, vomiting, diaphoresis, fever, focal neurological deficits, seizures and visual changes (diplopia, homonymous hemianopsia, miosis or mydriasis). The incidence of sudden death is greatest in the first week. Potential complications include rebleeding, cerebral swelling, hydrocephalus, and infarction secondary to vasospasm. The diagnosis can be confirmed with lumbar puncture where the CSF will show red blood cells, low glucose, elevated protein and a pressure usually > 150. A CT is also useful in detecting aneurysms > 5 mm, subarachnoid blood, infarction and edema. MRA is more sensitive and detects 80% or aneurysms. The treatment is usually surgical but medical therapy can be used to delay surgery and is surgery is contraindicated. Medical therapy includes lowering of blood pressure, bed rest, steroids, anticonvulsants and antifibrinolytic agents. year-old male presented with a one week history of abdominal pain, nausea, and vomiting. The night prior to presentation, he was noted to be disoriented and lethargic. Several hours later, he was febrile and diaphoretic and was taken to the hospital. On physical exam, he was alert and responsive. His BP was 70/36, heart rate 132, respiratory rate 40, and temperature 35 degrees celsius. Significant findings also included edematous sclerae with petechiae, diffuse rales, a distended abdomen, and diffuse purpura over his extremities. ABG revealed a pH of 7.08, pCO 2 of 71, pO 2 of 77, and bicarbonate of 20. His WBC was 25,600 (28% bands), platelet count 18,000, BUN 36, Cr 4.2, and INR 3.2 . Chest xray showed bilateral alveolar edema. Despite receiving several liters of saline, he continued to be hypotensive and became more hypoxemic, requiring intubation. His cardiac rhythm deteriorated to pulseless electrical activity from which he was successfully resuscitated. The diagnoses of septic shock, acute renal failure, disseminated intravascular coagulation, and adult respiratory distress syndrome (ARDS) were made. He was started on Penicillin G for suspected meningococcemia, dopamine, norepinephrine, continuous veno-venous hemodiafiltration (CVVHD), fresh frozen plasma and antithrombin III. His blood cultures grew Neisseria meningitidis. After 2 weeks, his renal function and coagulopathy resolved. His left lower extremity became ischemic and nonviable and was amputated. One week later he was discharged to a rehabilitation facility. DISCUSSION: Infections with Neisseria meningitidis range from mild upper respiratory infections to fulminant meningococcemia in 10±20% of cases. Few other diseases present as catastrophically as fulminant meningococcemia and prompt recognition and early treatment are crucial. Symptoms include sudden onset of fevers, chills, nausea, vomiting, and myalgias, progressing to shock and multisystem organ failure within hours. Purpura fulminans is present in 75% of patients, characterized as maculopapular or petechial lesions which can become hemorrhagic. In addition to antibiotics and coagulation factors, patients may also require ventilatory support. In order to remove toxic substances and cytokines, CVVHD is beneficial. The mortality rate with fulminant meningococcemia is as high as 50%, but with prompt recognition and treatment, up to 80% of patients can have favorable outcomes.
HEPATIC ENCEPHALOPATHY MASKING A DEEPER ABNORMALITY. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1) Identify the clinical presentation of hemochromatosis (HC). 2) Recognize the diagnosis and management of this condition. CASE INFORMATION: A 53-year-old female with hypertension, diabetes, and cirrhosis presented with worsening lethargy and confusion. Social history was remarkable for alcohol abuse, which ended two years before presentation. On physical, she had scleral icterus, hepatosplenomegaly, generalized jaundice with palmar erythema and spider angiomatas. Neurologically, she was alert but intermittently confused. Asterixis was present. Laboratory studies were significant for elevated transaminases and ammonia levels. Subsequent lab work revealed a ferritin of 2000, Fe of 250, and transferrin saturation of 89%. A liver biopsy demonstrated increased iron deposition in the hepatocytes, confirming the diagnosis of HC. A consequent family discussion revealed that her younger brother (age 41) and nephew (age 36) both complained of arthralgias. Genetic testing confirmed HC in both of them. DISCUSSION: Hemochromatosis is the most common inherited disorder in the U.S. It is an autosomal recessive condition in which there is increased absorption of iron that causes damage to the liver, heart, pancreas, joints, and other organs. The mean age of presentation is in the fifth decade. The classic triad of cirrhosis, diabetes, and skin hyperpigmentation is not usually the presenting sign; rather it represents late-stage disease. Arthropathy, especially in non-weight bearing joints, can be a common presenting complaint, as it is prevalent in 60% of symptomatic patients. Other early signs and symptoms include erectile dysfunction, amenorrhea, depression, and fatigue. Diabetes and cardiomyopathy may also be presenting signs in younger individuals. The liver is the most affected organ with iron deposition leading to functional abnormalities, fibrosis, and eventually cirrhosis. Liver failure is the most common cause of death. A combination of increased transferrin saturation ( > 50%) and an elevated ferritin is about 95% accurate for the diagnosis. Confirmation is made with a liver biopsy. Genetic testing has also shown promise, especially to test the first-degree relatives of patients with HC. Treatment involves phlebotomy, which depletes iron stores. Given the high prevalence of the condition and effectiveness of early intervention, early recognition of the disease is critical for successful treatment and prevention of premature morbidity and mortality. Therefore, one should consider the diagnosis in those cases presenting with abnormal transaminases, idiopathic diabetes, or unusual presentations of arthritis.
AN UNWANTED FAMILY INHERITANCE: THE CASE OF THE RAPIDLY PROGRESSIVE DEMENTIA. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1) Recognize the clinical presentation of Creutzfeldt-Jakob disease (CJD). 2) Recognize the diagnosis of this condition. CASE INFORMATION: A 44-year-old white female with no significant past medical history was in her usual state of health until two months prior to presentation when she began to experience an unsteady gait and problems with memory. One month prior to presentation, she experienced a seizure and then, two weeks later, she developed a rapid decline in her function, starting with short-term memory loss and progressing to unresponsiveness. Family history was significant for a paternal aunt who, at the age of 49 years, had a similar decline in cognitive function over 14 months. Physical exam revealed an awake woman who was unresponsive to commands. Vital signs were within normal limits. Neurological exam revealed dystonic posturing of the head with spontaneous conjugate eye movements, myoclonic activity of the upper extremities, and increased motor tone bilaterally. DTR's were normal bilaterally in the upper and lower extremities, with downgoing plantar reflexes. Bilateral grasp and glabellar signs were elicited. ANA, ESR, RPR, and Lyme titers were negative. An LP and a MRI were non-revealing. An EEG revealed periodic sharp-wave complexes. CSF was positive for a mutation of the prion protein gene, confirming familial CJD. DISCUSSION: The prion diseases constitute a family of subacute, neurodegenerative diseases referred to as transmissible spongioform encephalopathies. Incubation periods last from months to years and symptoms gradually increase in severity and result in death over a period of months. CJD is the major transmissible spongioform encephalopathy in humans. While a sporadic form tends to occur after the sixth decade and has a rapidly progressive course of under a year, an inherited form usually manifests at a younger age and has a more protracted course. Cardinal clinical features include confusion and memory loss progressing to severe cortical dementia, ataxia, and myoclonus. The EEG typically demonstrates bilateral periodic discharges. Neuroimaging is helpful to exclude other etiologies, since there are usually no abnormalities. Elevated levels of the 14-3±3 protein in the CSF, which has a 96% sensitivity and specificity for CJD, are also helpful for the diagnosis. The defining pathology is the presence of "spongy" degeneration of cortical gray matter. At present, there is no effective treatment for CJD. The diagnosis of prion disease should be considered as part of the differential in cases presenting with dementia and an atypical movement disorder, especially if the rate of disease progression is rapid.
BEYOND THE OBVIOUS ABNORMAL LABORATORY VALUE. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, Pennsylvania LEARNING OBJECTIVES: 1) Recognize secondary causes of hypercholesterolemia. 2) Recognize the association between hypercholesterolemia and hypothyroidism. CASE INFORMATION: A 47-year-old male with hypertension and diabetes presented for a new visit. He endorsed occasional myalgias and constipation. He denied fatigue, weakness, cold intolerance, or dry skin. On physical, blood pressure was 150/86 and pulse was 74. The remaining exam was unremarkable, including the absence of tendinous xanthomas and delayed DTRs. Old records revealed a diagnosis of hypercholesterolemia (295 mg/dL) and hypertriglyceridemia (1200 mg/dL), with subsequent initiation of gemfibrozil. His lipid levels continued to be elevated 6 months after initiation of the medication. Subsequent testing revealed an elevated TSH and abnormal TFTs, consistent with a picture of hypothyroidism. Levothyroxine was started. Upon follow-up in 2 months, the TSH levels had decreased; cholesterol levels had also declined modestly. Further follow-up 4 months later demonstrated normal TSH levels and significantly decreased cholesterol (220 mg/dL) and triglyceride (480 mg/dL) levels. DISCUSSION: Significant hyperlipidemia may be present in the absence of clinical features; thus, secondary causes need to be considered before the initiation of lipid-lowering therapy. These include Type II diabetes, chronic renal insufficiency, nephrotic syndrome, cholestatic liver disease, obesity, smoking, alcohol, steroid, and oral contraceptive use. Hypothyroid patients also display a variety of patterns of lipid abnormalities, including elevated cholesterol, triglyceride, LDL levels. The main mechanism is a decrease in the number of LDL cell receptors, resulting in an increase in LDL levels. Other mechanisms include a diminished secretion of cholesterol into bile and reduced lipoprotein lipase activity, resulting in hypertriglyceridemia. The severity of the hypothyroidism may play a role in treatment, since many studies show a more significant improvement in the lipid levels in those with a more severe form of hypothyroidism. The evidence is less clear regarding the significance of the relationship between subclinical hypothyroidism and elevated cholesterol levels. In general, those with overt hypothyroidism seem to respond best to levothyroxine treatment and subsequently show a decline in their cholesterol levels, whereas those with subclinical hypothyroidism seem to need additional lipid-lowering agents. In conclusion, this case underscores the importance of identifying secondary causes of hypercholesterolemia before initiating treatment for an apparent lipid abnormality.
ITS NOT ALWAYS DKA. A. Tabas 1 , G. Tabas 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1.Recognize lactic acidosis as a complication of metformin use. 2.Differentiate between diabetic ketoacidosis and metformin induced lactic acidosis. CASE INFORMATION: A 22 year old black female was admitted with three days history of nausea, vomiting, headache and abdominal pain, preceded by two days of rhinorrhea, and diarrhea. She was diagnosed with insulin requiring diabetes three years ago. There was no history of pre-existing renal insufficiency, chronic pulmonary disease or heart disease. Her medication was changed from insulin to metformin 500 mg bid a week prior to hospitalization. Her physical exam revealed tachycardia, and tachypnea. Pulse oximetry revealed 100% saturation on room air. The rest of the exam was unremarkable. Laboratory data were significant for ABG on room air - . Sodium-139 meq/L, Potassium-4.3 meq/L, Chloride-106 meq/L, CO 2 -7 meq/L, BUN-5 mg/dl, Creatinine-0.8 mg/dl, Glucose-276mg/dl, serum acetone-positive. The anion gap was 26. CBC & differential, and chest x-ray were within normal limits. Lactate and metformin levels were not checked. The patient was treated with normal saline, insulin drip, sodium bicarbonate and metformin was held. The metabolic acidosis resolved and the patient was discharged on long acting insulin. DISCUSSION: We do not know whether our patient had atypical diabetic ketoacidosis or metformin induced lactic acidosis. The presenting serum glucose in patients with diabetic ketoacidosis is usually between 450 mg/dl and 750 mg/dl. This patient's initial serum glucose of 276 mg/dl is somewhat uncommon for diabetic ketoacidosis, and suggests that other causes of high anion gap acidosis must be considered. The incidence of lactic acidosis is about 9 per 100,000 person-years in patients taking metformin. It usually occurs in patients who have preexisting cardiac disease, congestive heart failure, chronic pulmonary disease with hypoxia, sepsis or old age. Metformin induced lactic acidosis in patients such as ours, with no pre-existing risk factors, is rare but has been reported in literature. The mechanism is unknown. In patients taking metformin, presenting with ketoacidosis, metformin induced lactic acidosis should be considered. Assessing risk factors, appropriate laboratory workup (arterial lactate level, metformin level), withdrawing metformin and supportive care are key components of management.
A. Jha 1 , K. Bernard 1 , D. Wilson 1 , R. Quinlin 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1) To recognize rhabdomyolysis due to morphine. 2) To consider rhabdomyolysis in the differential diagnosis of post-operative renal failure. CASE INFORMATION: A 33-yr. old female underwent a laparoscopic`Roux-en-Y' gastroplasty under general anesthesia for morbid obesity. The anesthesia was induced by Midazolam, Fentanyl, Rocuronium and Succinylcholine. There was no masseter rigidity, or hyperthermia noted. Maintenance of anesthesia was done by Nitrous oxide, Propofol and Fentanyl. The patient had an uneventful perioperative course. There was no hematoma, or muscle tenderness following surgery. Morphine PCA was started. She has a past medical history of sleep apnea, hypertension, tubal ligation and hysterectomy without any complications. Her medications include Sertraline, Hydrochlorothiazide, Enalapril, and Albuterol inhaler. Her family history was unremarkable. The next morning she was found unresponsive and hypoventilating. An ABG revealed acute respiratory acidosis. She received Naloxone and the ABG normalized. Lab data revealed BUN -4 mg/dl, creatinine-2.0mg/dl (baseline -0.8), calcium -6.7 mg/dl, phosphorus-5.7mg/dl, magnesium-1.6mg/dl, sodium-138meq/L, potassium-4.1 meq/L, chloride-103 meq/L, C02-21meq/L, CPK-658 IU/L, CPK-MB-5.8 IU/L, Urine myoglobin-374( < 27mcg/L). EKG showed sinus tachycardia and an echocardiogram was normal. EEG, thyroid function, and serum Cortisol were normal. CPK levels increased to around 9400 IU/L and CPK-MB increased to 26 IU/L within two days. A diagnosis of morphine-induced rhabdomyolysis was made and patient was treated with D5Wand NaHCO3. Her Creatinine normalized the following day, and CPK, CPK-MB became normal on the 7th day. DISCUSSION: Prolonged surgery, electrolyte disturbances, infection and drugs are common causes of rhabdomyolysis and myoglobin-induced renal failure in the post-operative period. Morphine infrequently causes rhabdomyolysis. Proposed mechanism for morphine-induced rhabdomyolysis is myohypoxic necrosis and myocompression due to sedation related immobility. Reversal of narcotic effect with naloxone, withdrawing morphine, and hydration are the key components of management.
PARKINSONS TREMOR MIMICKING MULTIPLE ARRYTHMIAS. M.F. Jhandir 1 , D. Brotman 1 ; 1 Cleveland Clinic Foundation, Clevland, OH LEARNING OBJECTIVES: 1. Rule out Parkinson's tremor first when pateints with Parkinson's disease present with symptoms or electrocardiogram changes suggestive of tachyarrythmia. 2. Appreciate that holding anti-Parkinon's medications can be a simple provocative manuever to determine whether the electrocardiogram abnormalitites are of noncardiac origin in such patients. CASE INFORMATION: A 75 year old nursing home resident with a history of coronary artery disease and Parkinson's disease presented to the emergency department for evaluation of pre-syncopal episodes and hypotension. He had a three day history of feeling lightheaded and unsteady while standing but had no true syncope. He denied chest pain, dyspnea and palpitations. There was no prior history of documented arrhythmia. His medications included aspirin, donepezil, levodopa/carbidopa, and lisinopril. On physical examination, he was not in acute distress. Heart rate was 68 with occasional premature beats. Blood pressure was 65/45 bilaterally with orthostatic changes. Cardiac exam was without murmurs or gallops. Neck veins were flat, lungs were clear and there was no peripheral edema. He had cogwheel rigidity but no tremor. The admission ECG showed sinus rhythm with occasional premature ventricular contractions. After intravenous crystalloids were given, the blood pressure normalized, and he was admitted to a monitored hospital bed for further observation. The following morning, the intern was called to the patient's bedside because of an apparent supraventricular tachycardia (SVT)(figure 2). Despite his abnormal ECG, the patient was asymptomatic, with a pulse of 64, and a blood pressure of 160/90. He had developed a pronounced bilateral upper extremity resting tremor at about 5 Hz, but otherwise his examination was unchanged. After the patient received his morning anti-Parkinson medications, the tremor resolved and his ECG returned to baseline. In order to prove that the tremor was responsible for the ECG changes, the next dose of levodopa/carbidopa was held; his tremor returned as did his pseudo-atrial flutter. DISCUSSION: Because the classic Parkinson's tremor has a frequency of 4±5 Hz (260±300 cycles/min), it is not surprising that its electrical potentials on ECG can mimic atrial flutter, as in our case. Furthermore, Parkinson's disease is commonly associated with orthostatic hypotension and syncope, which may in turn prompt cardiac evaluations. Although there are previously documented cases of Parkinson's tremor leading to ECG artifacts resembling wide complex tachyarrhythmias such as ventricular tachycardia, we know of no other reports of Parkinson's tremor mimicking atrial flutter or SVT. Careful review of our patient's rhythm strip reveals that there are buried QRS complexes within the pseudo-SVT complexes (figure 2). The importance of correlating the ECG with the clinical exam cannot be overemphasized in this case, as the heart rate during the pseudo-SVT was normal, and the ECG artifacts were present only when the patient was tremulous. Finally, our case illustrates that if a patient with Parkinson's disease has transient ECG abnormalities suggestive of tremor-induced artifact, holding medicines can be a simple provocative maneuver to determine whether the ECG abnormalities are of non-cardiac origin. We conclude that Parkinson's tremor should be thoroughly ruled out when a patient with Parkinson's disease presents with symptoms and ECG changes suggestive of a tachyarrhythmia.
STRONGYLOIDIASIS AND THE HYPERINFECTION SYNDROME. H. Jneid 1 , N. Correia 1 , F. Michota 1 ; 1 Cleveland Clinic Foundation, Cleveland, Ohio LEARNING OBJECTIVES: 1. List the differential diagnoses for immunocompromised patients with chronic obstructive pulmonary disease and progressive dyspnea. 2. Recognize the risk factors and clinical features of strongyloidiasis. 3. Recognize the hyperinfection syndrome of strongyloidiasis. CASE INFORMATION: A 65-year-old retired coal miner presented with a 2-month history of progressive shortness of breath and nonproductive cough. He had a history of emphysema and coal worker' pneumoconiosis and had been using an inhaler and oxygen therapy. He had grown up in a rural Appalachian area of Ohio and stopped using tobacco 20 years ago. He had been started on cyclophosphamide and high-dose prednisone therapy for rapidly progressive glomerulonephritis 3 months previously. He was morbidly obese, with T 37.48C, BP 134/65 mm Hg, Pulse 100, RR 22/min, and SpO2 91% on 4 L of oxygen. Lung auscultation showed decreased air entry and diffuse wheezing. Initial chest x-ray showed no parenchymal lung disease. Review of his kidney biopsy revealed chronic interstitial nephritis, cyclophosphamide was stopped, and prednisone was gradually tapered. His dyspnea and oxygen requirement increased despite aggressive therapy with bronchodilators. Venous duplex ultrasound of the upper and lower extremities showed no thrombosis. V-Q scan revealed no mismatches. Computed tomography of the chest subsequently showed diffuse alveolar ground-glass appearance. The patient was started on broad-spectrum antibiotics, including intravenous Bactrim and amphotericin. Bronchoscopy was done, and Strongyloides stercoralis was diagnosed by transbronchial biopsy. The patient was started on intravenous thiabendazole. Prednisone was tapered quickly and stopped. His respiratory status deteriorated, and the patient eventually died. Autopsy found disseminated infection, with panlobar pulmonary infestation and patchy small and large bowel strongyloidiasis. DISCUSSION: Our immunocompromised patient had worsened respiratory symptoms despite inhaler therapy, which prompted us to seek other diagnoses. Initial work-up was negative for congestive heart failure and thromboembolism. His previous diagnosis of simple coal worker' pneumoconiosis was not supported by initial radiologic findings and would not have explained his rapid clinical deterioration and radiologic evolution. In this setting, opportunistic infections should be strongly suspected. Strongyloides is an intestinal parasite that is common in tropical and subtropical areas. Infection rates are highest among residents of the southern states and Appalachia, and among immigrants from endemic areas. Transmission occurs when infective larvae come in contact with the skin. The parasite has an autoinfection cycle that is usually contained by the host immune system; therefore, larval forms may persist in the body up to 50 years. Clinical manifestations of strongyloidiasis include abdominal upsets, lung manifestations such as wheezing and cough, and a pathognomonic skin eruption called larva currens. The autoinfection cycle accelerates inversely with the strength of the immune system and may yield to the highly lethal hyperinfection syndrome in immunosuppressed patients, characterized by massive dissemination to multiple organs and systems, such as the central nervous system, heart, liver, and endocrine glands. People at risk for Strongyloides infection should be screened before being given immunosuppressive therapy. Recognize the predisposing conditions and the treatment of cerebral arterial air embolism. CASE INFORMATION: A 50-year-old white woman presented with two transient 15-minute episodes of left upper extremity parasthesia, slurred speech, and diplopia. She had had inflammatory breast cancer 3 years previously, and had undergone right modified radical mastectomy, chemotherapy, and radiotherapy. Liver metastasis had recently been discovered. Neurologic exam on presentation was nonfocal. Computed tomography (CT) of the brain and lumbar puncture fluid analysis were normal. Magnetic resonance imaging found no leptomeningeal enhancement or focal lesions. Duplex ultrasound of her extracranial arteries showed no significant atherosclerotic stenoses, but 15 minutes of continuous Doppler monitoring showed active embolization (>1000 emboli /minute) to the brain. Venous duplex ultrasound of her upper and lower extremities showed no thrombosis, and CT scan of her abdomen showed no pelvic or inferior caval thrombosis. Holter monitoring showed no cardiac arrhythmias. Transesophageal echocardiography showed no valvular vegetations, intracardiac thrombi, or patent foramen ovale, but revealed spontaneous air bubbles in the left atrium. The patient' neurological status progressively worsened until she became comatose, entered respiratory arrest, and died. No autopsy was performed.
DISCUSSION: Cerebral arterial air embolism has been described with cardiac surgery, mechanical ventilation, underwater diving and bronchoscopic laser operations. Arterial embolization results from direct passage of air into the arterial circulation or from paradoxical embolization. The effect of an air embolus depends on its size as well as its rate of entry into the circulation. Arterial embolization is dangerous in that it can cause ischemic damage to organs. Because absorption of arterial emboli is inversely related to the arterial pressure of nitrogen, treatment with hyperbaric oxygen has been effective. This treatment also helps reduce the air bubble size and increases the oxygen content of blood. In our patient, no bubbles were detected in the right heart chambers, which makes paradoxical embolization unlikely. A plausible biological mechanism is cerebral air embolism through bronchovenous fistula, with possible pathogenetic implication of either micrometastases to the lung or the prior thoracic radiation therapy, but this remains to be proven. Recognize the possibility of multiple opportunistic infections in immunocompromised patients. CASE INFORMATION: A 40-year-old man presented to a hospital with a 5-month history of progressive dyspnea on exertion, productive cough, and gradual weight loss. He had a 52 packyear history of smoking. He was treated with several courses of antimicrobials for bronchitis and was maintained on high-dose prednisone (60 mg daily) for several months. He presented to an outside hospital with respiratory distress, requiring 50% FiO2. Chest x-ray showed bilateral alveolar infiltrates, and on computed tomography (CT), the chest had a diffuse ground-glass appearance. Bronchoscopy was nondiagnostic, so he underwent an open lung biopsy, which showed pulmonary alveolar proteinosis. He was transferred to our tertiary care facility where he was given normal saline lavage (17 L per lung) under general anesthesia. His respiratory status improved and he was discharged home on 6 L/min of oxygen and tapering doses of prednisone. He returned few days later with worsening dyspnea, fever, and increased cough, now productive of copious brown rusty sputum. His vitals: T 101.08F, BP 95/56 mm Hg, Pulse 120, and RR 30/min. His lung auscultation revealed diffuse crackles. His PaO2 on 6 L of oxygen was 57 mm Hg. He was intubated and started on broad-spectrum antibiotics. Sputum staining showed branching gram-positive filaments with weak acid-fast stain, and intravenous Bactrim (trimethoprim and sulfamethoxazole) was added for treatment of Nocardia pneumonia. Ophthalmologic exam and CT of the abdomen and brain showed no evidence of nocardial dissemination. His sputum stain subsequently showed septate hyphae, which was targeted with amphotericin B. The fungus was identified as Trichosporon beigellii. The patient eventually died of septic shock. Autopsy showed panlobar bilateral necrotizing pneumonia and the presence of silver-positive organisms consistent with Nocardia, Trichosporon, and Aspergillus. DISCUSSION: Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease characterized by dense accumulation of PAS-positive (periodic acid-Sciff) phospholipid material in the alveoli, with preservation of the lung architecture and absence of inflammation. Patients usually present with a subacute prodrome of cough and progressively decreasing exertional tolerance. Tissue biopsy, by open lung or thoracoscopy, is the gold standard for diagnosis, though transbronchial biopsy and fluid obtained by bronchoalveolar lavage may also be used. Whole lung lavage is the treatment of choice, and recently interest has grown about the potential therapeutic role of GM-CSF (granulocyte/macrophage colony stimulating factor) because defective alveolar macrophages are involved in the pathophysiology. There is no role for immunosuppressants in treatment, and, in fact, some suspect that corticosteroids may increase mortality. The association of pulmonary nocardiosis with PAP has been described frequently. Nocardia asteroides tends to disseminate from a pulmonary or cutaneous focus to any organ, notably the central nervous system, retina, heart, kidneys, and joints. The new onset of fever and change in the quality of sputum in our immunocompromised patient prompted us to think of superimposed opportunistic infections. A thirty-three year old Caucasian man presented with history of recurrent spells. He described episodes of palpitations and hot flushes, followed by tunnel vision, pre-syncopal feeling, and loss of consciousness. He had spontaneous recoveries, with no incontinence or postictal state. He reported loose stools, intermittent urticarial rash, and occasional wheezings associated with these episodes. He had an AICD (automatic implantable cardioverter-defibrillator)placed after the second spell for complete heart block that degenerated into ventricular fibrillation. Subsequent interrogation of the AICD showed no evidence of dysrrhytmias despite recurrent spells. 24-hour EEG monitoring showed no epileptiform activity. He underwent a cardiac catherization, which showed normal coronary arteries. CT scans of the head and the abdomen were non-revealing. Pulmonary angiography showed no evidence of pulmonary embolism. On presentation, his physical exam, including neurological exam, was completely normal, except for urticarial rash on the dorsum of his hands. Telemetry monitoring didn't reveal any arrhythmia. A tilt table test showed asymptomatic postural tachycardia. Urine metanephrine and 5-HIAA levels were within normal. Echocardiography showed no valvular lesions. Glucose levels and CBC were normal. A MIBG scan was also negative. The patient was discharged home on a beta-blocker. His spells recurred. Further work up included a twenty-four hour urine histamine collection, which was normal. Punch biopsy of his urticarial skin lesions, revealed urticaria pigmentosa, with perivascular and interstitial mast cells infiltration of the superficial dermis. Colonoscopy showed mast cells infiltration of the lamina propria of the ileum and right colon. A bone marrow aspirate revealed 15% mast cell cellularity. The diagnosis was consistent with systemic mastocytosis. The patient was switched to a combination of H1 and H2 blockers, and later had a leukotriene receptor antagonist added to his regimen. He remained spell-free afterwards. DISCUSSION: One needs to exclude other causes of spells, like cardiovascular, neurologic, endocrine, pharmacologic, and psychologic etiologies. Systemic mastocytosis is a disease characterized by mast cell infiltration of multiple organs. It presents with episodic flushing, diarrhea, abdominal pain, bronchospasm, alcohol intolerance, and syncope. Triggers include emotional stress, iodinated contrast agents, aspirin, and exercise. Urticaria pigmentosa is a characteristic skin lesion. Therapeutic armamentorium includes H1 and H2 blockers, cromolyn sodium and prostaglandin synthesis inhibitors. Epinephrine is used in severe cases associated with anaphylaxis, while interferon therapy is reserved for proliferative disease. Our patients had a dramatic presentation and a dramatic response after addition of a leukotriene antagonist. -old white woman presented with a 4-week history of progressive shortness of breath and persistent dull right-sided chest pain. She had never been a smoker. On admission, she was afebrile, RR 14, and had oxygen saturation of 95% on room air. Lung auscultation in the right lower lung fields found decreased breath sounds and decreased tactile fremitus. Chest x-ray confirmed the presence of right pleural effusion. Thoracentesis showed a milky fluid consistent with chylothorax (pH = 7.26, RBC = 79, WBC = 4.4, LDH = 398 u/l, protein = 1.6 g/dl, glucose = 51 mg/dl, triglyceride = 715 mg/dl, cholesterol = 26 mg/dl, and positive chylomicron screen). CT of the chest and abdomen revealed a large pleural mass emanating from the posterior hemithorax, with extension below the right diaphragm. Thoracoscopic biopsy of the mass revealed malignant mesothelioma. The patient had no occupational exposure to asbestos, but her father (who had no lung disease) had worked in the steel industry. The patient was switched to a medium-chain fatty acid diet, and her pleural drainage became serous, although it persisted. Eventually, a Pleurex catheter was inserted for self-administered therapeutic drainage as needed, and the patient was discharged home with hospice care. DISCUSSION: Malignant mesothelioma is an insidious neoplasm that usually afflicts patients in their fifth to seventh decades of life. Patients usually present with dyspnea, nonpleuritic chest pain, and unilateral pleural effusion. Exposure to asbestos is elicited in only 70% to 90% of cases. Domestic exposure to asbestos through family members is well documented and could have been the cause in our patient. Multimodality therapeutic approaches (surgery with chemotherapy +/-radiotherapy) produce a slight improvement in quality of life and survival. Our patient' tumor extended across the diaphragm and was therefore nonresectable (stage IV). Chylothorax is usually due to either trauma such as surgery or nontraumatic causes such as malignancies, with lymphoma accounting for the majority of cases. Prolonged chyle loss can lead to nutritional depletion and immunologic deficiency. A triglyceride level above 110 mg/dL in a pleural effusion is very suggestive of chylothorax, and the presence of chylomicrons confirms the diagnosis. The presentation of a young patient with no asbestos exposure, with malignant mesothelioma and a chylothorax is extremely rare.
COMBINED ADRENAL CRISIS AND MYXEDEMA COMA DUE TO HYPOTHYROIDISM. K. Kamjoo 1 , N. Michail 2 ; 1 University of California, Los Angeles, Sherman Oaks, Ca; 2 UCLA LEARNING OBJECTIVES: 1-To recognize the clinical presentation of severe hypopituitarism. 2-To understand the physiology of pituitary hormones. 3-Patient compliance as a cause of serious illness. CASE INFORMATION: A 37 years old female with a history of pituitary adenoma status post trans-sphenoid resection in 1990, was brought to Emergency Department (ED). Pt was not responsive and paramedics' report indicated that patient had gotten worse over the past month with progressive lethargy, vomiting, and altered mental status. Patient had stopped her medications approximately one year prior to admission. In ED patient was comatose and her initial vitals were: Rectal temperature 33.3 C, pulse 64, respiration rate 14, blood pressure 80/ 44, and blood sugar of 26. On physical exam patient appeared pale and had marked facial puffiness. She had no heart murmur but was bradycardiac. Patient's neuro exam showed bilateral hyperreflexia with a definite delay in the relaxation phase of her deep tendon reflexes. Patient's skin was very dry and she had no axillary or pubic hair. CXR only showed mild cardiomegaly. EKG showed sinus bradycardia with diffuse low voltage and flat T-waves. Patient was given 2L of intravenous (IV) D5W and 100 mg of IV hydrocortisone. After administration of IV steroids, patient's core temperature increased to 37.2 C and her blood pressure was stable at 89/50. She had an O2 saturation of 100% on room air and became conscious and alert. Subsequently, she was admitted to step-down unit with a diagnosis of adrenal crisis due to untreated hypopituitarism. Patient was started on DDAVP, and hydrocortisone. Serum TSH and free T4 were pending at this time and patient was started on 0.1 mg of oral Synthroid. Patient was doing well after the first day of hospitalization. However, on the second day her mental status deteriorated again and became obtunded. She had increased puffiness in her face and marked edema of her lower and upper extremities. Patient's blood gas showed CO2 retention. Patient was diagnosed with myxedema coma and tranferred to medical ICU. Patient was intubated and a single 300 mcg dose of IV Levothyroxine was administered, followed by 100 mcg of IV Levothyroxine daily. At this time serum TSH and free T4 drawn on admission showed TSH = 0.1 and free T4 < 0.40. The patient subsequently improved markedly and was discharged from hospital after 7 days. DISCUSSION: Myxedema coma is a very rare disease occuring primarily in elderly patients with primary thyroid failure. This case illustrates the importance of early recognition that myxedema coma can occur in combination with adrenal crisis in patients with decompensated hypopituitarism. Non-compliance in this disease could lead to fatal consequences. A 56 year old male presented for routine primary care evaluation. His past medical history was significant only for mild COPD and tobacco use. He specifically denied any history of coronary artery disease,diabetes,hypertension or dyslipidemia. Review of systems identified long standing complaints of erectile dysfunction and fatigue. Specifically, the patient noted a frequent inability to reach orgasm and lower extremity fatigue when climbing stairs. Physical examination was notable for a blood pressure of 110/65,an arm span greater than height,intact lower extremity pulses,sparse male pattern hair distribution,gynecomastia,small penis size and small,firm testes. Pulse volume recordings of the lower extremities and electrocardiogram were unremarkable. Laboratory evaluation found LDL=68, fasting glucose=104,Hct=48,TSH=1.08,free testoster-one=2.6,LH=14,FSH=26 and prolactin=3.5. Subsequent chromosomal karyotyping indicated the presence of an 82% XXY pattern consistent with Klinefelter Syndrome. The patient was treated with intramuscular testosterone. On follow-up, this patient reported improved energy levels and sexual performance. Indeed, the patient presented with new complaints consistent with bilateral epicondylitis. Apparently, following diagnosis and therapy, this patient was now hard at work buffing combines! DISCUSSION: Klinefelter syndrome,a primary form of testicular failure, is a common disorder that affects 1 in 500 male patients. It results from nondisjunction during parental gametogenesis and yields an individual with an XXY karyotype. Individuals with Klinefelter syndrome do not progress through puberty and ultimately develop the clinical features of eunuchoidism. These features of androgen deficiency include:arm span greater than height,long legs,sparse male pattern hair distribution,gynecomastia,small testes and penis and infertility. Endocrine evaluation of patients with this syndrome indicates low free testosterone together with elevated gonadotropin levels (ie LH and FSH). Klinfelter syndrome is treated with testosterone supplementation. This produces positive changes in mood,behavior,muscle mass and bone density. Once testosterone supplementation is begun, patients must be routinely screened for erythrocytosis and lipid abnormalities. year-old man with history of recurrent spontaneous pneumothorax (he underwent pleurodesis 5 years ago), presented to our service complaining of 2 months of progressive abdominal swelling, leg edema, jaundice, and worsening shortness of breath. The patient is a chronic smoker who rarely consumes alcohol. On exam the patient was afebrile with a regular pulse of 110, blood pressure of 118/78, respiratory rate of 18/min, distended neck veins, S4 gallop, massive ascites and +3 leg edema. Other than high total/direct bilirubin 2.2/3.6, the labs were unremarkable. Echocardiogram revealed a markedly dilated right ventricle with moderate pulmonary hypertension, severe tricuspid regurgitation and left ventricle of normal size and function. Spiral chest CT revealed pulmonary embolism of both left lower and upper divisions of the pulmonary artery. Doppler ultrasound of both legs was negative. The patient was anticoagulated (heparin, then warfarin) and once stable discharged home. DISCUSSION: CTEPH results from single or recurrent pulmonary emboli arising from sites of venous thrombosis. Most emboli completely resolve spontaneously, or they have minimal residual effects. In CTEPH, for unclear reasons, the emboli do not resolve completely. Rather, the emboli undergo abnormal organization and recanalization, leaving endothelium-covered residua that obstruct or significantly narrow major branches of the pulmonary artery. This is very different from primary pulmonary hypertension, where obstructive lesions appear in the small distal pulmonary arteries. Most patients with CTEPH have history of venous thrombosis with embolism; however this history may not be elicited unless actively sought for. After the initial embolic event the patients' condition gradually improves and they usually resume their normal activities. This period of improvement is however a temporary``honeymoon period' frequently followed by more subclinical thromboembolic events. Recurrent pulmonary embolism destroys the pulmonary vascular bed and causes pulmonary hypertension. With time patients deteriorate and become dyspneic at lower levels of exertion. Clinically, they present with a triad of dyspnea, syncope and right heart failure. Initial treatment includes chronic anticoagulation; consider embolectomy for massive embolism. and thrombotic and vasculitis work up. Transesophageal echocardiography (TEE) with agitated saline demonstrated a PFO, with significant right to left shunt, with transcranial Doppler demonstrating contrast reaching cerebral circulation. The patient underwent surgical closure of PFO by direct suturing. The patient now presented with the fourth episode of transient right-sided numbness and paresthesia in the last three years. TEE with agitated saline showed no residual shunt. Patient was anticoagulated (heparin, then warfarin) and once stable sent home. DISCUSSION: Stroke causes more than 100,000 deaths in USA each year, and leaves many others with major disabilities. In 25 to 40% of strokes in young adults, an extensive evaluation fails to identify the cause; these are classified as crypotogenic strokes. Notably, patients with crypotogenic strokes have a higher incidence of PFO than patients with stroke of determined cause, even after correcting for presence of recognized stroke risk factors. Potential mechanism for PFO related ischemic event includes paradoxical embolism in which a thrombus dislodges from a venous thrombotic site, traverses the interatrial septum and reaches the brain. Other mechanisms may include a thrombus formation secondary to atrial arrhythmias or atrial septal aneurysm. The incidence of stroke will be higher as the size of a PFO increases and if it is associated with an atrial septal aneurysm. Treatment options for PFO may range from no therapy, to medical therapy, to surgical closure of PFO. Choosing the type of treatment would depend on the patient's age, surgical and anticoagulation risks. The average annual risk of recurrence of cardiovascular events is 3.8% for medical therapy (aspirin 250 ± 500 mg/d, warfarin with target INR 2 ± 3, or both together) and 2.5 % after percutaneus transcatheter closure of the PFO. Closure done through open heart surgery by direct suture or patch closure is the gold standard of therapy and offers permanent closure of the defect with minimal risk and avoidance of long term anticoagulation. Recurrence of cerebrovascular ischemic events is still possible after successful surgery. Having multiple neurological events before surgery is the only significant risk factor of recurrence after the procedure. One should try to identify causes other than paradoxical embolism in patients who experience recurrent cerebrovascular events after surgical closure of PFO.
A. Karcic 1 , J. Dell'orfano 1 ; 1 Departments of Internal Medicine and Cardiology, State University of New York at Stony Brook and Nassau University Medical Center, East Meadow, NY LEARNING OBJECTIVE: 1. To understand that ischemic myocardium has a decreased capability to conduct pacemaker beats. CASE INFORMATION: A 73 years-old hypertensive man, had one year ago a pacemaker inserted for symptomatic sinus bradycardia (fainting spells). Coronary angiography performed at that time showed only mild nonobstructive coronary disease, The pacemaker was a dual chamber device, with leads in the night atrium and night ventricle, and it was functioning appropriately until a week ago, when the patient noticed chest pressure while shoveling snow. At that time his pulse rate, on self-exam was 40 bpm, so the patient went to see his internist, knowing that``there is something wrong with the pacemaker, since it was programmed to keep the heart rate above 60 bpm.'' During hospitalization the patient had slightly positive cardiac enzymes. The ECG revealed sinus bradycardia and inferior wall ST depressions with regular vertical pacemaker spikes at 60 bpm, but failure of these paced stimuli to``capture'' the myocardium and lead to ventricular beats. Coronary angiography revealed a 100% obstructed night coronary artery (RCA) as the cause of ECG changes and ischemia. Angioplasty was performed on the RCA, and a stent inserted to keep the vessel open. Several hours after the procedure a repeat ECG revealed no ST depressions, with all the pacemaker spikes 'capturing' the myocardium, and pacing the heart at the set rate of 60 bpm. DISCUSSION: Our patient had a complete RCA obstruction, leading to ischemia of the inferior areas of the heart, as seen on the ECG. It also caused ischemia of the myocardium where the night ventricular pacemaker lead was inserted. Ischemic myocardium (or dead myocardium Ð as a scar from an old myocardial infarction) has a decreased ability to conduct electrical stimuli, as those received from a pacemaker. It effectively acts as an obstacle to the conduction of these pacemaker beats to the surrounding healthy nonischemic myocardium. For at least some paced beats to be conducted through the ischemic myocardium, impulses at much higher energy levels need to be delivered by the pacemaker. This not only leads to faster pacemaker battery depletion, but also usually does not guarantee that all beats delivered by the pacemaker will be conducted, which is needed. The solution to this problem is relieving the ischemia, if possible (like in our patient), or by changing the position of the night ventricular lead, if it was located in myocardial scar tissue. (8 years), risk factor HIV positive hemophiliac husband, with history of recurrent respiratory infections, was admitted for worsening dyspnea of several months duration, and two weeks of abdominal and leg swelling and oliguria. On exam she was tachypneic with a blood pressure 100/60 mm Hg and pulse rate of 100/ min. Lungs were clear to auscultation, while a right-sided gallop was audible. The jugular veins were distended, the liver painfully enlarged, and there was bipedal pitting edema. EKG revealed an incomplete RBBB. Chest radiograph showed mild cardiomegaly, and a prominent pulmonary artery, with clear lungs. Sonogram of the heart revealed large right-sided chambers, paradoxical septal motion and pulmonary hypertension. VQ scan was low probability for pulmonary embolism. Finally a right-sided cardiac catheterization excluded a left to right shunt and revealed severe pulmonary hypertension, with preserved left ventricular function.
DISCUSSION: PPH is a diagnosis of exclusion. In order for it to be diagnosed, no cause for the pulmonary hypertension should be found. Frequently, in HIV positive patients, pulmonary hypertension develops secondary to HIV related cardiomyopathy and left ventricular failure. Early reports of PPH in HIV involved hemophiliacs receiving large amounts of factor VIII concentrates, which over a period of time can cause pulmonary vascular insults leading to PPH. Only later came reports of non-hemophiliac HIV positive patients with PPH. Intravenous drug users can develop pulmonary hypertension as tale emboli cause pulmonary fibrosis and vascular disease. Progressive pulmonary thromboembolism has also been reported to cause pulmonary hypertension in HlV positive patients. While mild and moderate pulmonary hypertension can develop in many cardiac and pulmonary diseases, it is proposed that a genetic predisposition (rare familial PPH), in concord with pulmonary vascular insults (shear stress, chronic hypoxia and chronic inflammation), leads to severe and fatal PPH. Patients with AIDS have a multitude of pulmonary vascular insults (infections, emboli, hypoxia etc.), but only 0.5% of them develop severe PPH. Our patient for example had frequent bronchitis. It appears that PPH in patients with HIV is multifactorial in origin, rather than being merely a coincidental finding. As a conclusion, one could mention that pulmonary hypertension in HIV which is labeled as primary may in fact be secondary to a multitude of factors that may remain unrecognized, year old woman with a history of smoking and hypertension was admitted to the hospital with syncope. She was found to have episodes of significant sinus bradycardia for which she needed a pacemaker. A dual chamber pacemaker was inserted, with pacemaker leads placed in the right atrium and right ventricular apex. ECG, fluoroscopy and a chest radiograph confirmed the correct position of the leads, while postoperative pacemaker interrogation revealed that the right atrial and right ventricular pacing and sensing thresholds were within normal limits. Initially after the procedure the patient was feeling fine, but the same night she started complaining of persistent, rhythmic, frequent and annoying contractions on the right side of her upper abdomen, feeling somewhat like hiccups. There was nothing the patient could do in order to bring upon or abort these symptoms. After having interrogated the pacemaker, the cardiologist abandoned right atrial pacing and the symptoms resolved completely. DISCUSSION: This patient had right hemidiaphragmatic contractions that were induced by pacemaker beats. The diaphragm is a muscle and hence can be stimulated by electrical stimuli from the pacemaker. In order to understand how a pacemaker induces diaphragmatic contractions, one should recall that the right phrenic nerve traverses the right atrium (atrial electrode location), and the left phrenic nerve the right ventricular apex (ventricular electrode location). If the pacemaker stimuli are of adequately high energy, not only the surrounding myocardium wall be paced, but the paced beats will also be conducted through the overlying right or left phrenic nerve to cause contractions of the right or left hemidiaphragm respectively. In our patient the right hemidiaphragm was paced, telling us that the right phrenic nerve was paced via the right atrial electrode. Frequently there is a therapeutic window that enables us to pace the myocardium without pacing the overlying phrenic nerve. This is due to the fact that usually lower energy levels are needed to pace the myocardium. Gradual decrease of pacemaker energy output below that needed to pace the phrenic nerve, but still high enough to pace the myocardium is the perfect solution for this problem. If such a therapeutic window does not exist, the culprit electrode must be abandoned (atrial), or a new one can be placed in a different location (ventricular).
DELAYED DIAGNOSIS OF PERFORATED SIGMOID STERCORAL ULCER. M. Kasubhai 1 , M. Singh 1 , T. Saleem 1 , A. Singh 2 , V. Dimitrov 1 ; 1 Lincoln Medical and Mental Health Center., Bronx, NY; Cornell University LEARNING OBJECTIVES: 1) Recognize that stercoral ulcers result from pressure necrosis of the mucosa by the direct effect of a mass of retained feces. 2) Fecal Impaction is commonly diagnosed and rapid and gentle care can prevent this morbid complication CASE INFORMATION: 67 year old male presented with history of seizures, lower abdominal pain and constipation. Past history was relevant for seizure disorder, diabetes, stroke and bilateral above knee amputation. He was afebrile and hemodynamically stable. Left lower quadrant tenderness was noted. Rectal examination revealed fecal impaction and was manually disimpacted. The hemotocrit was 40 with leucocytosis. X-rays of chest and abdomen were normal. The patient subsequently developed fever and was treated for diverticulitis. Fever resolved with antibiotics. CT scan revealed thickening of the wall of the rectum and sigmoid, colonic dilatation and small bowel ileus. Colonoscopy performed two weeks later to evaluate the cause of constipation revealed an ulcer at the recto-sigmoid junction with biopsy revealing dysplastic changes. The other considerations of the ulcer being viral, bacterial, fungal or tuberculous were excluded by biopsy and culture. A stercoral ulcer was also considered and treated conservatively. After two weeks a repeat colonoscopy showed a persistent ulcer with acute and chronic inflammatory exudate. An exploratory laparotomy was performed because of persistent colonic dilatation and ileus. A sigmoid tear was noted over the ulcer, which was plugged by matted loops of small bowel. Sigmoid resection and colostomy was done. The patient recovered completely and was discharged home. DISCUSSION: Stercoral ulcers are usually found in the rectum and sigmoid. Their size is variable and margin usually irregular. When symptomatic, they usually reveal themselves by bleeding or perforation. Relieving the fecal impaction manually can result in perforation of the ulcer. Fever and leucocytosis, as in our patient, should suggest perforation. Our patient also had unexplained localised ileus and colonic dilatation, which should have led to an early exploratory laparotomy. Fecal impaction is commonly diagnosed and the rapid and gentle care can prevent its morbid complications HYPOKALEMIC PARALYSIS: PURSUING A DIAGNOSIS. J. Kent 1 , W.P. Moran 1 , L.B. Jones 1 , J. Stopyra 1 ; 1 Wake Forest University School of Medicine, Winston-Salem, NC LEARNING OBJECTIVES: 1) Recognize an uncommon presentation of SjÐgren's Syndrome. 2) Use a literature search as a diagnostic aid. 3) Demonstrate the importance of pursuing a diagnosis to the end. CASE INFORMATION: A 69 yo w female with past medical history of gastroesophageal reflux disease, hypertension, anxiety, arthritis and hypokalemic paralysis presented to the emergency department with a one-week history of progressive right-sided weakness. Her symptoms included difficulty using her right hand, impairment of gait, and a decrease in appetite. She had a history of right-sided paralysis due to hypokalemia nine months earlier requiring hospitalization, which had resolved with aggressive potassium replacement. She received neurologic and endocrine workups at that time without any etiology found, and she was discharged on oral potassium and spironolactone. With this second episode, her CT scan was negative for an acute CVA, and her potassium level was found to be 2.1. On exam, her oral mucous membranes were dry. Her lab values were suggestive of renal tubular acidosis. After doing a MEDLINE search with keywords including hypokalemia, paralysis, and acidosis, it was found that there have been over 20 case reports of patients with SjÐgren's syndrome presenting with a hypokalemic paresis or paralysis. Upon further questioning, the patient did note that her eyes and mouth had felt unusually dry for the past year. Diagnostic testing included a positive Schirmer's test and a minor salivary gland biopsy that was strongly suggestive of Sjo È gren's syndrome. The hemiparesis completely resolved with replacement of her potassium, and she was discharged on potassium supplementation with spironolactone, as well as artificial tears and lozenges for treatment of her Sjo È gren's symptoms. DISCUSSION: This patient's episode of hemiparesis/ paralysis due to hypokalemia was the second such episode within 9 months. During her first episode, it was also noted that she had symptomatic dryness of mouth and eyes and that her mucous membranes were dry. Sjo È gren's syndrome itself was not mentioned in a differential diagnosis. The main clinical problem appeared to be the hypokalemia and hemiparesis, which are not traditionally associated with Sjo È gren's. When the patient's hypokalemia and paralysis resolved, she was discharged with outpatient follow up. During this second episode, the patient's main symptoms were again relieved before a diagnostic etiology was found. However, the diligent search for a reason for her hypokalemia by the team resulted in the discovery of her underlying problem, Sjo È gren's Syndrome, which then allowed for more complete treatment. The use of a literature search and its finding of a series of case reports was a significant aid to the team in reaching the diagnosis. An 86 year old woman with no significant past medical history and taking no medications was admitted to the intensive care unit after a one day history of weakness, dizziness, nausea and vomiting. The patient was hypotensive with a blood pressure of 90/50 mmHg, without orthostatic changes. The pulse was 64; she was afebrile, and had dry oral mucosa. The remainder of the physical examination was normal. Laboratory workup was significant for pyuria and mild anemia (Hgb = 11.4 g/dL). Intravenous fluids were started and antibiotics were given for her urinary tract infection. During the hospital stay, the patient developed disorientation, hallucinations, and incoherent speech interpreted as delirium secondary to her infection and ICU psychosis. When her delirium did not resolve with hydration, antibiotics and transfer to a regular floor, a hepatic profile was ordered as part of the workup for metabolic causes for her delirium. This testing revealed an isolated elevated alkaline phosphatase (650 U/L). Abdominal ultrasound showed marked dilation of the common bile duct and subsequent abdominal CT scan showed no masses in the area of the gall bladder and liver. A 1.5 cm common bile duct stone was successfully extracted by endoscopic sphincterotomy and her mental status returned to baseline. DISCUSSION: The causes of delirium include medication side effects or withdrawal, dehydration, infections, hypoxia, hypoglycemia, and other metabolic abnormalities. Many systemic illnesses may produce delirium in elderly patients, especially in combination with new medications, fever, or sleep deprivation. In this patient, a comprehensive evaluation for metabolic causes of delirium (despite lack of localizing symptoms or signs on physical examination led to a diagnosis of unsuspected common bile duct stone and institution of appropriate treatment. Since delirium is associated with adverse outcomes, making the correct diagnosis is important. It is essential to do a complete workup for delirium in order to determine all treatable causes, particularly in hospitalized elderly patients.
ANTICONVULSANT HYPERSENSITIVITY SYNDROME: A CALL FOR INCREASING AWARENESS. N. Khan 1 , B. Cheong 1 , A. Jaffer 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1. Diagnose the distinct entity of hypersensitivity reaction to anticonvulsants. 2. Recognize the prevelance of cross-reactivity between different anticonvulsant medications. CASE INFORMATION: A 27 year-old lady with a history of multiple sclerosis was initiated on oral phenytoin for tonic spasms of extremities. Two weeks later she noted a mild maculopapular erythematous rash on her extremities. Concerns for drug induced rash and minimal response to treatment, led to a change in regimen from phenytoin to carbamazepine. One week later the patient was hospitalized with worsening pruritic maculopapular rash, more prominent on the trunk and extremities, and recurrent fevers. Except for a temperature of 101.5 Å F, her vital signs were within normal limits. Notable changes on physical exam included mild facial edema, with 1.5 to 2cm bilateral, tender, mobile jugulodigastric and submandibular lympadenopathy (LAD). Laboratory abnormalities on admission included peripheral eosinophilia and elevated hepatic enzymes: AST=271 (7 ± 40 U/L), ALT=324 (5 ± 45 U/L). Other hepatic enzymes were normal. A CBC with differential revealed WBC=15.63 (4 ± 11.0 K/uL) with neutrophils 59% (40 ± 70%), lymphocytes 27% (22 ± 44%), eosinophils 11% (0 ± 4%), monocytes 2% (0 ± 7%), and atypical lymphocytes 1%. Carbamezepine level= 9.0 (8 ± 12 ug/mL) Blood and urine cultures were normal. Titers for Epstein-Barr virus and cytomegalovirus were normal. Given the rash, pyrexia, eosinophilia, lymphadenopathy and recent treatment with anticonvulsants the patient was diagnosed with anticonvulsant hypersensitivity syndrome (AHS). Progressive resolution of the dermatological changes and hepatitis was noted with discontinuation of anticonvulsants and treatment with corticosteroids for 1week. DISCUSSION: AHS is an adverse drug reaction associated with medications such as phenytoin, carbamezepine and phenobarbital. Originally cited prevelance of 15% is increasing with broader spectrum of use of anticonvulsants, such as for treatment of neuropathic pain. This patient exhibited a typical presentation including hepatitis and eosinophilia. The pathogenesis of AHS involves inadequate function of epoxide hydroxylase enzyme, leading to decreased inactivation of toxic metabolites of anticonvulsant drugs. Current data indicates allergic cross-reactivity between these medications to be as high as 80%. Treatment involves discontinuing the medication and initiating steroid therapy. With a mortality rate suggested at 40% (mostly due to hepatic necrosis), it is important for general practitioners to recognize AHS; its presentation, cross-reactivity of medications, and treatment options. A high index of suspicion based on the patient's history and exam will enable an astute primary care provider to make the diagnosis early.
HYPERPARATHYROIDISM IN PREGNANCY. R. Khurana 1 ; 1 University of Alberta, Edmonton, AB LEARNING OBJECTIVES: 1) Recognize the changes in calcium metabolism that occur with pregnancy. 2) Manage hyperparathyroidism in pregnancy. CASE INFORMATION: A 38-year old pregnant woman, G4P1 at 21 weeks gestation was seen for assessment of hypertension. Her blood pressure had been normal at the first prenatal visit, but had started to rise and had been high for 4 weeks. She was asymptomatic. Past medical history was significant for two miscarriages and one full-term pregnancy, all in Nigeria. There was no prior history of hypertension. Her only medications were prenatal vitamins. Physical exam was normal other than a blood pressure of 168/94 and a grade II/VI ejection systolic flow murmur. Laboratory screening for preeclampsia was normal. Laboratory screening for secondary causes of hypertension revealed hypercalcemia at 2.79 mmol/L (11.16 mg/dl) with an albumin of 36 g/L (3.6 mg/dl). An elevated ionized calcium confirmed hypercalcemia. A 24 hour urine calcium was elevated at 9.0 mmol/24h (360 mg/24h). 25-OH Vitamin D levels were normal, but her Parathyroid hormone (PTH) level was elevated at 15.0 pmol/L (normal 1.1 ± 6.8 pmol/L). She was told to keep herself well hydrated with oral fluids and started on oral phosphate 500 mg bid. A 24 hour urine for metanephrines was ordered to rule out pheochromocytoma as part of a multiple endocrine neoplasia (MEN) syndrome in combination with the hyperparathyroidism. It was normal and she proceeded to exploratory surgery with removal of a right superior parathyroid adenoma. Post-operatively, her calcium levels normalized. DISCUSSION: Primary hyperparathyroidism is rare during pregnancy. It may be masked because total calcium levels decrease secondary to a decrease in albumin from the volume expansion that occurs with pregnancy. However, ionized calcium levels remain unchanged despite decreases in PTH levels. This is likely due to the effects of PTH related peptide (PTHrp) which is found to be increased during pregnancy. PTH-rp may also help regulate fetal calcium levels which are higher than maternal levels. Maternal hyperparathyroidism can cause significant morbidity for the fetus and neonate. There is an increased risk for stillbirth, intrauterine growth restriction, premature labor and neonatal hypocalcemic tetany. The diagnosis is often made on a retrospective basis postpartum after the development of neonatal tetany. Surgical management is usually preferred as it causes less morbidity than medical management. However, hyperparathyroidism can be associated with pheochromocytoma in Type 2 MEN and this should be ruled out prior to surgery.
A. Kim 1 , Y. Braver 1 , A. Jaffer 1 ; 1 The Clevelend Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1) Recognize the clinical presentation of diabetic neuropathic arthropathy. 2) Review the management of acute-onset Charcot arthropathy. CASE INFORMATION: A 51-year old African American female insulin-dependent diabetic of 12 years presented with a 2 month history of right lower extremity (RLE) swelling. This was associated with erythema, warmth, and minimal pain. She also reported a 3-day history of lowgrade fever and generalized weakness. She had previously been treated with oral antibiotics for presumed cellulitis with no improvement. Diagnostic studies at that time revealed a normal WBC of 10 K/cmm with an elevated sedimentation rate (WSR) of 31 mm/hr. X-ray of the right foot/ankle showed soft tissue swelling without evidence of bony destruction. Lower extremity duplex revealed no deep vein thrombosis. CT scan of the extremity revealed only subcutaneous edema. A subsequent right ankle joint aspiration was performed which was negative for crystals and infection. The physical exam on admission was notable for an afebrile, obese female with 2+ pitting edema extending from the dorsal aspect of the right foot to the knee associated with warmth, erythema, and tenderness. There were no areas of skin breakdown or ulcers. Decreased sensation to pain, temperature, and proprioception was noted. The patient was admitted for cellulitis and started on IV antibiotics. Laboratory studies showed WBC of 8 K/cmm with an elevated WSR of 64 mm/hr and a CRP of 3.8 mg/dl. Blood cultures were negative. The patient remained afebrile with mild improvement of her symptoms. A MRI of the extremity revealed changes in the midfoot and forefoot consistent with an acute neuropathic arthropathy. Orthopedic consultation fitted the patient with a total-contact cast, instructed her on no weight bearing in the RLE with follow-up in 4 weeks. DISCUSSION: Diabetes mellitus is now the most common cause of neuropathic (Charcot) arthropathy. The foot and ankle are the most commonly affected joints. Unilateral swelling, warmth, and erythema are typical clinical findings and it is usually painless. The pathogenesis of this disease is unclear but may be related to a combination of peripheral neuropathy and neurovascular insufficiency allowing repetitive microtrauma. This can result in chronic destruction of bones and joints ultimately resulting in deformities and disabilities. In early disease, it may be difficult to differentiate from cellulitis, osteomyelitis, septic arthritis, crystalline arthritis, and osteoarthritis. Therefore, having a high clinical suspicion and obtaining studies such as MRI or In-111 labeled leukocyte scan can help make a prompt and accurate diagnosis so that aggressive immobilization can be initiated.
NOT JUST A RASH. B. Kinder 1 , P. Basaviah 1 ; 1 University of California at San Francisco, San Francisco, CA LEARNING OBJECTIVES: 1) Recognize the clinical features of Anticonvulsant Hypersensitivity Syndrome (AHS). 2) Manage a potentially fatal adverse drug reaction. CASE INFORMATION: A 36 year old Caucasion female reported a four day history of a progressive maculopapular rash associated with a mouth ulcer. On day 2 of the rash, she developed mucosal sloughing and presented to an emergency room (ER). At that time she denied drug allergies or use of new detergents or skin products. Of note, three weeks prior to this presentation, the patient had been prescribed phenytoin by her psychiatrist for treatment of refractory bipolar affective disorder. In the ER she was treated with hydroxizine, prednisone, and analgesics and told to discontinue the phenytoin. Progression of her skin lesions to blisters over the next two days prompted a medical admission. Exam revealed a temperature of 38.2, pulse of 100, injected, desquamating conjunctivae, oral and vaginal erosions, a confluent maculopapular rash involving her face and trunk, and no hepatosplenomegaly or lymphadenopathy. Data were remarkable for a WBC of 2.4, AST of 70, and ALT of 81. Intravenous hydration, methylprednisolone, morphine, diphenhydramine, and local wound care were initiated. The rash progressed to bullae affecting her face and upper body,were limited to 10 ± 15 % of body surface area, and gradually improved over several days. Her elevated transaminases normalized within twenty-four hours of steroid initiation. Steroids were tapered. The patient was discharged with outpatient follow-up. DISCUSSION: AHS is a potentially fatal drug reaction induced by arene oxide-producing anticonvulsants, including phenytoin, carbamazepine, and phenobarbital. It occurs in 0.01%-0.1% of the population and is fatal in 5 ± 50% of cases if toxic epidermonecrolysis (TEN) or hepatitis develop. The mechanism apparently involves an inability to detoxify arene oxide metabolites which then bind to proteins and elicit an immune response. AHS may be genetically determined, conferring increased risk to siblings. The hallmark clinical features include fever, rash, and lymphadenopathy which can be associated with hepatitis, hematologic abnormalities, and interstitial nephritis. The skin eruption typically begins as a morbilliform rash involving the upper trunk, face and upper extremities. The cutaneous manifestations can be variable, including target lesions, bullae, and skin sloughing and mucosal involvement resembling Stevens-Johnson Syndrome and TEN. AHS usually occurs 2 ± 6 weeks after initial drug exposure, later than most other skin reactions. Steroid therapy is controversial and has not demonstrated benefit in randomized control trials. However, some experts recommend steroid use if there is evidence of hepatic or renal involvement and if skin desquamation is limited. Essentials of management include prompt discontinuation of the offending drug, supportive management, avoidance of cross-reactive drugs in the future, and counselling regarding potential genetic implications to family members. LEARNING OBJECTIVE: To recognize that in the face of renal dysfunction, azotemia may reflect non-renal causes. CASE INFORMATION: A 76 year-old white male presented with shortness of breath and hypoglycemia. He had vomited that morning and had nothing further to eat or drink. His history was significant for diabetes mellitus (27 years), COPD, CHF, and stable chronic renal insufficiency (BUN 54, creatinine 1.6). The patient' home medications included aspirin qd, acetominophen #3 prn pain, potassium 20 mg qd, glyburide 2.5 mg qd, furosemide 20 mg bid, prednisone 10 mg qd, and lisinopril/HCTZ 20/25 mg qd (started three weeks previously). Physical exam revealed bilateral wheezing. Lab studies showed BUN 93, creatinine 3.2, Hgb 9.4, U/A showed 2+ protein and no casts. On ultrasound, the kidneys were normal size with no hydronephrosis. The patient had good urine output. The patient was admitted with an exacerbation of COPD, hypoglycemia and renal failure. Hypoglycemia was considered to be secondary to inadequate clearance of glyburide and poor oral intake. Glyburide, furosemide, and lisinopril were held. The patient was treated with albuterol, ipratropium, fluticasone, salmeterol inhalers and IV solumedrol for the COPD exacerbation. Over several days the BUN increased to 115 while the creatinine declined to 1.9 . Because of persistent azotemia, other non-renal causes were considered. Colonoscopy revealed several large, hyperemic polyps which were removed. Eventually the BUN declined to 46 and the creatinine returned to baseline. DISCUSSION: This case illustrates the multifactorial nature of illnesses in the elderly and emphasizes the importance of considering alternate or confounding hypotheses when making a diagnosis. Although this patient had underlying renal failure, focusing on this alone would have missed such potentially life-threatening problems as dehydration, steroid-induced azotemia, and GI bleeding from colonic polyps, all of which could have contributed to this patient' condition.
A HIGH RISK EXPOSURE TO MENINGOCOCCAL MENINGITIS. L.M. Kosseim 1 , C.M. Stoltz 1 ; 1 University of Pennsylvania, Philadelphia, PA LEARNING OBJECTIVES: 1. Recognize patients at high risk for meningococcal meningitis 2. Understand options for prophylaxis for meningococcal meningitis 3. Identify candidates for the meningococcal vaccine CASE INFORMATION: A 20 year-old male college student made an urgent appointment at his student health service for evaluation of possible meningococcal meningitis. He had returned the day before from a 14 day trip to Israel with other college students. A student with whom he had spent time in Israel became ill prior to boarding the flight home. This contact student died from meningococcal meningitis within hours of landing in the United States. The patient was advised by the airline company to see his physician for evaluation. History was remarkable for sharing a drinking glass at a party with the young man who later died. A thorough physical exam was unremarkable. Because of his potentially high risk exposure to meningococcus, the patient was given rifampin 600 mg PO bid for two days and counselled on the symptoms of meningococcal meningitis. He was also advised to receive the meningococcal meningitis vaccine. As this patient had been on a school trip, the other students from his group were also counselled and given prophylaxis. Additionally, the student health service used this as an opportunity to educate the student body on the availability of the meningococcal vaccine and on recognizing the signs and symptoms of meningitis. DISCUSSION: Meningococcal meningitis is a rare but often deadly infection affecting approximately 2,800 Americans annually. Chemoprophylaxis with antibiotics is recommended for all close contacts of an index case of meningococcal meningitis. Close contacts include household members, daycare workers, and anyone directly exposed to the case patient's oral secretions. Internists should be comfortable prescribing chemoprophylaxis for meningococcal meningitis and also know which patients are advised to get the meningococcal vaccine. The Centers for Disease Control has recently changed its guidelines, and now advises that all college students be aware of the increased risk of meningococcal meningitis in this population and in the availability of the meningococcal vaccine. CASE INFORMATION: A 32-year-old female hotel banquet manager with no signficant past medical or surgical history presented with nausea, diffuse myalgias, clear rhinorrhea, and frontal headache. She took oral contraceptives and just finished her menses. The patient was afebrile and had frontal sinus tenderness. Her physician prescribed trimethoprimsulfamethoxazole for sinusitis. The patient stopped the sulfa after five days because of worsening nausea with diarrhea. She also developed arthralgias of the knees, ankles, and elbows. Two days later she returned to the doctor with a maculopapular rash over her anterior shins with a few urticarial lesions. The elbows appeared discolored. The knees were warm, tender, and swollen. The tissue around the ankles was tender, but they were not swollen or warm. A serum sickness like reaction secondary to sulfa was suspected. A CBC and LFTs were normal, and an antihistamine was prescribed. Over the next week her condition worsened with fever, more extensive arthralgias (now including the hips and wrists) and the development of tender nodules along the anterior tibias and elbows characteristic of erythema nodosum. A chest radiograph showed right hilar and mediastinal lymph nodes. Prednisone 20 mg. daily was begun with rapid improvement of the arthritis, erythema nodosum, fatigue, and fever. The prednisone was tapered over the next month. A CXR three months later showed resolution of the hilar and mediastinal adenopathy. Nine months later the patient remains well without recurrence. DISCUSSION: This case illustrates that some episodes of``serum sickness" are due to an underlying disease for which an antibiotic is used and then blamed. In retrospect, the symptoms of fatigue, myalgias, and URI that lead to the antibiotic prescription were actually the prodrome for acute erythema nodosum (EN). While half of cases of EN are idiopathic, it is important to rule out secondary causes. Infectious etiologies can include streptococcal pharyngitis, non-streptococcal URIs, TB, intestinal pathogens, and systemic fungi such as coccidiomycosis. Drugs such as sulfonamides and oral contraceptives can precipitate EN. One of the most common etiologies of secondary EN is Lo È fgren's syndrome, a triad of EN, bilateral hilar adenopathy, and arthritis. If EN is not clearly due to an infection, medication, pregnancy, or inflammatory bowel disease, a CXR and PPD should be performed. LÐfgrens is an acute, self-limited form of sarcoidosis, most common in young women. EN is a good prognostic sign, in which case an invasive diagnostic procedure for the hilar adenopathy is not necessary. NSAIDs are the treatment of choice; corticosteroids are indicated for severe, debilitating arthritis. A 51-year-old man was seen in the Emergency Department for one week of sore throat, fever, and hoarseness; he was diagnosed with a respiratory virus. Two days later, he was evaluated in a primary care clinic for persistent fever, fatigue, and a new, nonpruritic rash. He was ill-appearing and had a maculopapular rash over his upper back, chest, and thighs; anterior cervical lymphadenopathy was present. Laboratory data revealed WBC 4.2, and a negative monospot and throat culture. HIV testing was obtained two days later when risk factors were identified. Intial HIV antibody was negative, but his viral load was 300,000. His symptoms resolved after two weeks. One month later, his HIV antibody test became positive. CASE 2: A healthy, 35-year-old man was seen for urethral discharge. History revealed recent unprotected intercourse with a new partner. Urethral culture was negative but urine PCR was positive for Chlamydia trachomatis. He was treated with a one-time dose of azithromycin. Two days later he developed a rash over his face and trunk accompanied by chills, fatigue, and headache. He was afebrile with a diffuse maculopapular rash over his face, chest, and abdomen. Although a drug reaction was suspected, HIV serologies were sent. His HIV antibody was indeterminate but his viral load was over 500,000. DISCUSSION: Acute HIV infection is often underdiagnosed, but should be routinely considered in clinical care. More than 80% of people with acute infection seek medical care for their symptoms, yet the diagnosis is considered less then 25% of the time. Nonspecific symptoms such as fever, myalgias, rash, and lymphadenopathy are typical. Initial testing should include HIV antibody as well as HIV RNA viral load or HIV DNA. Early treatment decreases viral burden as well as improves, and perhaps restores, host immune responses. HIV is highly contagious during the acute infectious stage. Early diagnosis is critical in decreasing HIV transmission and has significant impact on public health, treatment, and possibly prognosis. Because the majority of patients present to primary care physicians, they have an important role in recognition of early infection. Further education regarding symptoms and serologic testing, as well as screening strategies, may be needed to increase awareness of acute infection in primary care settings.
AN UNUSUAL CASE OF HYPERTENSION. S. Kripalani 1 ; Emory University, Atlanta, GA LEARNING OBJECTIVES: 1) Explore causes of secondary hypertension in a young man. 2) Recognize the appearance and pathophysiology of Page kidney. CASE INFORMATION: A 27 year-old narcotics agent presented for evaluation of previously untreated high blood pressure. Manual readings over the past year ranged from 140 ± 151/96 ± 110. He complained of daily bifrontal headaches, as well as occasional palpitations and diaphoresis, but only when working in high-stress situations. Review of systems was otherwise negative, as were past medical history and medication use, including OTC products. He denied smoking and illicit drug use, but he used chewing tobacco and had 2 ± 3 alcoholic drinks per week. He used to play high school football and had been in a car accident years ago, with no apparent injury. His father received a heart transplant for idiopathic cardiomyopathy. Family history was otherwise negative. Blood pressure was 162/108 in the left arm, 152/104 in the right arm, and 154/106 in the right leg. Heart rate was 64, height 71 inches, and weight 196 pounds. He appeared comfortable and well-developed. Physical examination was unremarkable. EKG, chest x-ray, blood count, basic chemistry panel, TSH, urinalysis, and drug screen were normal. The patient was started on amlodipine, to avoid interfering with anticipated testing, and his blood pressure responded appropriately. Initial 24-hour urine catecholamines were elevated, prompting a CT scan to help localize a potential pheochromocytoma. The CT showed normal adrenals, but unexpectedly, the left kidney had a 5x5x11cm subcapsular fluid collection, known as Page kidney. An MRI/MRA confirmed the CT findings and demonstrated normal vasculature. Renin levels were slightly elevated Ð supine 1.7 ng/mL/hr (0.2 ± 1.6) and upright 4.9 ng/mL/hr (0.5 ± 3.3) . Cortisol, aldosterone, and upright aldosterone/renin ratio were within normal limits. Head CT and repeat urine studies were normal. Given the imaging results, the initial catecholamine elevation was deemed a false positive. DISCUSSION: Potential causes of secondary hypertension included renal parenchymal and renovascular diseases, coarctation of the aorta, mineralocorticoid excess (either through hyperaldosteronism or use of chewing tobacco or licorice), renin-producing tumors, pheochromocytoma, Cushing's syndrome, congenital adrenal hyperplasia, substance abuse, exogenous hormones or sympathomimetics, sleep apnea, hypo-and hyperthyroidism, hyperparathyroidism, acromegaly, carcinoid, and increased intracranial pressure. Page kidney is rare cause of hypertension resulting from compression of the renal parenchyma. High blood pressure is mediated by elevated renin levels, which are thought to be secondary to local ischemia or parenchymal scarring. In the model which he described in 1939, Page used cellophane wrapping to constrict the kidneys. However, most cases in clinical practice are due to a subcapsular hematoma resulting from blunt trauma, usually associated with contact sports or an accident. If blood pressure cannot be controlled medically, percutaneous drainage or nephrectomy can be curative.
NOT``JUST ANOTHER GI BLEED''! A. Krishnamurthy 1 , R. Ruffner 1 , D. Weber 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1) Consider small bowel tumors in the evaluation of GI bleeding with the upper endoscopy and colonoscopy are negative. 2) Small bowel series, enteroclysis and enteroscopy have a role in their diagnosis. CASE INFORMATION: 84-year-old Asian male presented with his second episode of melena. He also complained of``fullness of the abdomen after meals''. He had no nausea, vomiting, abdominal pain, constipation or diarrhea. The patient had been admitted two weeks before with melena and anemia requiring blood transfusions. He had both upper DI endoscopy (showed mild esophagitis) and colonoscopy (showed two polyps). PMH of hypertension. His medications included iron, famotidine and Metoprolol. He did not smoke or abuse alcohol. ROS: Negative. Examination: His temperature was 36.8, pulse 72 bpm, BP 120/60 and was not orthostatic. His conjuntiva was pale. Abdomen was soft with normal bowel sounds, no tenderness, no masses, liver/spleen not palpable. Rectal exam reveled heme positive stool. Examination of all other systems was normal. Labs: WBC 5.4, Hemoglobin 8.9, BUN 42 & Cretinine 1.2, Iron 28, TIBC 258 Ferritin 25. Small bowel entersocopy was normal upto the duodenum but the scope could not be advanced beyond the fourth part of duodenum because of narrowing. Small bowel follow through shoed an apple-core defect at the duodeno-jejunal junction. Surgical resection of lesion confirmed a 4-cm adenocarcinoma of jejunum without lympnode involvement. DISCUSSION: Small bowel neoplasms are relatively uncommon. Small bowel accounts for less than 3% of GI malignancies despite contributing to more than 90% of the surface area of GI tract. The possible reasons are:
1. in November 1999 complaining of weight loss, bulky stools, fatigue, abdominal distention, and anorexia. Review of those hospital records revealed an extensive evaluation that yielded a diagnosis of``protein losing enteropathy". She improved with total parenteral nutrition (TPN). Small bowel biopsy and serologies were not reported, nor was there mention of treatment with a gluten-free diet. In May 2000, the patient presented to our institution with similar complaints and worsening lower extremity edema and weight loss. Physical examination revealed an extremely cachectic woman with flat affect, hypotension, tachycardia, inspiratory crackles, hepatomegaly, and 2+ pitting lower extremity edema. Laboratory exam showed severe hypoalbuminemia, hyponatremia, and hypocalcemia, mildly elevated transaminases and alkaline phosphatase, and iron deficicency anemia. Antigliadin and antiendomysial antibodies were elevated. Histologic examination of biopsies obtained from the proximal small bowel was consistent with collagenous sprue. Due to her severe presentation, short-term corticosteroids and TPN were initiated. She improved and was discharged with instructions to continue a gluten-free diet. DISCUSSION: Adult patients with celiac sprue may present with a variety of gastrointestinal as well as extraintestinal symptoms, many of which were present in our patient. The diagnosis is usually made by histological evidence of villous atrophy in the small intestine along with symptomatic improvement on a gluten free diet. Serological tests (antiendomysial and antigliadin antibodies) further aid in diagnosis and screening of celiac sprue. Collagenous sprue was first described (in 1970) as continued malabsorption, failure of a gluten-free diet, and a subepithelial collagen layer on small bowel biopsies in a patient previously diagnosed with celiac sprue. Most published case reports of collagenous sprue have not detected these serological markers typical in celiac sprue. Recently, a relationship was postulated when serological markers were present in a patient with collagenous sprue. Our patient exhibited biopsy proven collagenous sprue, but also demonstrated positive serology for adult celiac sprue, supporting the notion that collagenous and adult sprue may be related. Whether this simply reflects an improvement of antibody assays remains to be established as more cases are evaluated. American female presented with abdominal pain, nausea and emesis for two days, and 10 lb. weight loss over a month. Physical examination revealed a cachectic female in moderate distress, hypotension, tachycardia, and diffuse abdominal pain with rebound tenderness. Laboratory exam showed an anion gap metabolic acidosis with a mild lactic acidosis, acute renal failure due to acute tubular necrosis, hyponatremia, leukocytosis with bandemia, and elevated Troponin associated with anteriorlateral ischemic EKG changes. Swan-Ganz readings indicated cardiogenic shock. Emergent cardiac catherization revealed diffuse``beading'' of small and medium coronary vessels consistent with vasculitis. Renal angiography revealed a similar picture. A primary vasculitis workup was negative. Hepatitis studies were consistent with acute HBV: + Hb SAg, À Hb SAb, + Hb Core IgM. Quantitative HBV-DNA was 6290 pg/ml. Hepatitis A, C, and D studies were negative. Initially, high-dose parenteral steroids were started for suspected PAN, but when the HBV serologies became known steroids were discontinued and Lamivudine initiated. Hemodialysis was required. Clinically, the patient improved and was discharged home. Three months later the patient' condition deteriorated acutely, and an acute cardiorespiratory arrest lead to her death. Histological examination revealed a multisystem, segmental, necrotizing vasculitis of medium and small-size arteries (most notably the coronary and renal vasculature) in various phases of progression consistent with classic PAN. DISCUSSION: HBV-related PAN is most commonly described with chronic HBV infections. In our case, laboratory evidence indicated acute HBV infection. Cardiopulmonary involvement of PAN is fairly common, but rarely associated with cardiogenic shock. Traditional treatment of PAN cannot be utilized when PAN is associated with an acute HBV infection. Good therapeutic outcomes have been reported with immunosuppressive agents along with plasma exchanges, or treatment with two weeks of high-dose steroids followed by immnuosuppresives and plasma exchanges. 
A 45 year old woman with a history of hypertension, s/p total hysterectomy was admitted for unstable angina. She had a positive stress test and underwent a left heart catheterization (LHC) during her hospitalization that was normal. Her catheterization site was sealed with a vascular sealing device and was without external hematoma. Her precatheterization Hgb was 13.3 mg/dL. One day post-LHC, she complained of dysuria. A urinalysis was normal and she was discharged for outpatient evaluation of her chest pain. The patient developed worsening dysuria, frequency, and urgency and was prescribed antibiotics over the telephone for the treatment of a presumed UTI. Her symptoms improved but did not resolve and she was seen in the office 2 weeks later now also complaining of new constipation. Examination revealed normal vitals, a shuffling gait and rebound tenderness on abdominal exam. Pelvic exam revealed a tender, non-pulsating mass in the right lower quadrant. The catheterization site was identified as above the inguinal ligament and did not show any external signs of hematoma. An ultrasound and CT of the pelvis and abdomen revealed a 12Â7Â8cm mass overlying the iliac artery consistent with a hematoma and blood tracking up the right paracolic gutter. The bladder and colon were compressed by the mass. Doppler studies did not reveal active arterial flow from the iliac arteries. Her hemoglobin was 9.6 mg/dL. The patient was admitted for supportive care and her symptoms improved. DISCUSSION: Cardiac catherization is a common procedure with serious potential complications including a 1% risk of vascular injury including intraperitoneal bleeding. Risk factors for complications include female gender, congestive heart failure, percutaneous transluminal coronary angioplasty, valvuloplasty and peripheral vascular disease. The recent increased use of vascular sealing devices may decrease the risk of hemorrhage from anterior arterial perforation, but will not provide hemostasis from a posterior wall puncture. Thoughtful surveillance for post-LHC complications is essential. Management of peritoneal hematomas should include serial hemoglobins, diagnostic imaging and surgical consultation when necessary. In this patient, her post-LHC bleeding was misdiagnosed as a urinary tract infection. The Internist must recognize that post-LHC complications may masquerade as more common pathology leading to delayed diagnosis and potentially poor outcomes. presented to an outpatient clinic seeking primary care. Her complaints were depression and high blood pressure. Her primary language was Korean, but she spoke moderately fluent English. On exam she was oriented to person and place, but was hard to interview as she shifted from topic to topic. Depressive symptoms were not present, nor were symptoms of psychosis. Over the course of a few months she came often but erratically to clinic with vague complaints. She often discontinued her medications without any clear reasons. The nurses noted that although they understood her English, they could never tell what she wanted. They wondered if she was schizophrenic. After struggling to understand her clearly a Korean interpreter was obtained. This interpreter said that in Korean she was difficult to understand as well. After psychiatric evaluation which confirmed lack of mental illness, neuropsychometric testing was done with an interpreter. This revealed mental retardation with developmental disability especially in areas of language. DISCUSSION: Mental retardation is often defined by a composite IQ of less than 70. Developmental disabilities are very prevelent and encompass overt mental retardation as well as less pervasive cognitive deficits. As in this case, these patients can have difficulty with reading and communicating clearly, which can be a barrier to effective healthcare. Most people in the US are screened for this in school or childhood clinic visits. Many adult immigrants likely were not screened. When this diagnosis is known accomodation can be made to improve compliance and effective communication. Also, a diagnosis of mental retardation may qualify a patient for government benefits and outpatient social services. This case highlights the need to diagnose developmental disabiltiy and differentiate it from mental illness especially in adult immigrants. To recognize the preliminary management for IPA.  CASE INFORMATION: A 28 year old female presented requesting prenatal vitamins and  offered no complaints. PMH was significant for exploratory laparoscopies for abdominal pain and endometriosis. The patient was in graduate school and had been married for 1.5 years. When asked``do you always feel safe at home?'', she was noted to hesitate, whereas on previous questions, she answered rapidly. This question was immediately followed by``does your husband ever hit you, yell at you, or make you feel afraid?'' She replied that while she knows her husband loves her, on occasions he does get``upset''. They have frequent arguments, during which he breaks furniture and hits her. She stated``he never hits me hard'', he``always apologizes immediately'', and his hitting has``never gotten out of hand''. She denied having a plan of action should that happen. There were no weapons in the house. Patient was immediately referred to a psychologist for counseling and who helped her to formulate a safety plan. DISCUSSION: Intimate partner abuse (IPA) is common, with an estimated prevalence of 5 ± 20% women per year. It is defined as a pattern of intentionally coercive and violent behavior toward an individual with whom there is or has been an intimate relationship. These behaviors can be used to establish control over an individual and include physical and sexual abuse, psychological abuse with verbal intimidation, progressive social isolation or deprivation, and economic control. IPV transcends social classes and so all women should be asked about safety in their current setting as part of routine primary care and when presenting for emergency care with traumatic injuries. A screening question can be``have you been hit, kicked, punched, or otherwise hurt by someone within the past year? If so by whom?'' The physician must then make an assessment of the patient's safety, ascertaining the presence of weapons in the house, violence that is increasing in severity or intensity, threats to kill the woman or her children, and the woman having told her partner she is``planning to leave.'' It is important to give the patient validation and support and help her to develop a safety plan. Physicians should be aware of resources within their institution, which include social workers or domestic violence advocates. If these are unavailable the physician can provide counseling and give referral to a local domestic violence hotline. A 32 year old woman presented with bright red blood per rectum associated with tenesmus and urgency. She then developed left lower quadrant pain. There was no fever, rash, eye or joint pain. There was no foreign travel, unusual food intake, or history of antibiotic, NSAID or oral contraceptive use. Her past medical history was unremarkable. She had no known drug allergies, used alcohol on occasion, and did not use illicit drugs. Examination revealed postural hypotension, scant bowel sounds, and tenderness in the left lower quadrant. Rectal exam showed gross blood. Investigations revealed hemoglobin 141, platelet count 270, and wbc of 14.7. Electrolytes, INR, PTT, liver profile, amylase, and beta-HCG was negative. Abdominal films were normal. Micrologic examination of the stool was negative. Colonoscopy showed erythematous, friable mucosa from 75 cm (mid descending colon) to 35 cm distal to the anus. Pathology revealed patchy disease with inflammatory exudates on the surface, consistent with ischemic colitis. No crypt abscesses or granulomas were identified. A vasculitic workup was performed, with negative ANA, pANCA, cANCA, and normal complement levels. Hepatitis B and C serology were negative. Antithrombin III, Protein C, Protein S, homocysteine, Factor V Leiden genotype, and anticardiolipin antibody were all normal. Her diarrhea improved within 3 days of admission with conservative treatment, and she was discharged on ASA. She has had no recurrence. DISCUSSION: Internists frequently consider ischemic colitis in the differential diagnosis of the elderly patient who presents with bloody diarrhea. However, ischemic colitis in young patients is more common than realized. Case series in younger patients have described an increased risk in women, and a lack of vascular risk factors, when compared to the typical older patient with ischemic colitis, who has multiple vascular risk factors. Etiologic agents that have been implicated include estrogen, collagen vascular disease, diabetes, hypercoagulable states, cocaine use and even long distance running. It is also common to find no identifiable etiologic cause. In an analysis of 68 consecutive cases diagnosed by early colonoscopy, 23 (34%) of the patients were younger than 50 and nineteen of those were women. Habu found that the associated factors for ischemic colitis were an increased incidence of chronic constipation and a prior history of abdominal surgery. Our case illustrates the importance of consideration of ischemic colitis in the differential diagnosis of bloody diarrhea in the younger patient.
BATS AND RABIES: IT DOESNT HAVE TO BE A BITE. D.S. Lindes 1 ; 1 University of California, San Francisco, San Francisco, CA LEARNING OBJECTIVES: 1) Recognize the risk of rabies from non-bite exposure to bats. 2) Provide post-exposure prophylaxis against rabies infection. CASE INFORMATION: A 40 year old woman living in the San Francisco Bay Area presented to her primary care physician with concern about the risk of rabies from contact with a bat. Three weeks prior to presentation, she had found a bat flying around in her living room. Her two cats had tried to catch it, without success. She used a window screen, held in front of herself and above her head, to gradually guide the bat towards an open window. The bat collided with this screen several times before it flew out the window, and the patient spent approximately 45 minutes in the room with the bat. She was not bitten, nor did she recognize any direct contact with the bat's saliva. At the time of presentation, she had no signs or symptoms suggestive of rabiesvirus infection. DISCUSSION: Rabies is a rare but nearly always fatal viral infection. Its clinical course is characterized by a nonspecific prodrome, with associated paresthesias or fasciculations at the inoculation site, followed by an encephalitis with agitation, confusion, hyperesthesia, autonomic dysregulation, paralysis, and progressive brainstem dysfunction. Death generally results from respiratory center depression. Wild animals, including racoons, skunks, and bats, are the principal vectors for human rabies. Since 1980, 21 (58%) of the 36 human rabies cases diagnosed in the United States have been associated with bats. Of these cases, there was definite history of a bite in only one or two. In another 10 ± 12, contact but no bite was reported; the other 7 ± 10 cases had no clear contact, but an undetected bat bite was the most plausible route of exposure. Rabid bats have been documented in all 49 Continental U.S. States. Current Centers for Disease Control and Prevention guidelines recommend consideration of post-exposure prophylaxis (PEP) against rabies whenever direct contact between human and bat has occurred, unless the exposed person can be certain that there was no bite, scratch, or mucous membrane exposure. Rabies PEP consists of the following measures: 1) thorough cleansing of all wounds with soap and water, as well as povidone-iodine irrigation if possible; 2) administration of rabies immune globulin, 20 IU/kg, with as much as possible infiltrated around the wound(s) and the remainder given intramuscularly at a site distant from vaccine administration (only needed for individuals not previously vaccinated); 3) rabies vaccine (human diploid cell vaccine [HDCV] , rabies vaccine adsorbed [RVA], or purified chick embryo cell vaccine [PCEC]), 1.0 ml administered intramuscularly on days 0, 3, 7, 14, and 28 . Because incubation periods of over one year have been reported, PEP is indicated regardless of the amount of time after exposure. If at all possible, the involved animal should be collected for rabiesvirus testing. In the above case, because of prolonged contact and possible mucous membrane exposure, the woman underwent PEP, as did her previously unvaccinated cats. Recognition of the significance of non-bite exposure to bats is important for primary care physicians because they are likely to encounter patients with such exposures, many of whom may be candidates for post-exposure prophylaxis.
PERIPARTUM CARDIOMYOPATHY: DIAGNOSIS AND IMPLICATIONS. C.L. Long 1 , R. Deversa 1 ; 1 University of Tennesee College of Medicine-Chattanooga unit, Chattanooga, TN LEARNING OBJECTIVES: 1-Identify theories regarding etiology of peripartum cardiomyopathy. 2-Recognize physical findings of peripartum cardiomyopathy. 3-Recognize the morbidity and mortality associated with peripartum cardiomyopathy. CASE INFORMATION: A 23 year old caucasian female, G 1 P1, who is status post caesarean section two weeks prior presented with increasing shortness of breath. She had elevated JVP, bibasilar crackles, and a S3 gallop. ECG showed no acute changes. CXR revealed cardiomegaly with pulmonary venous congestion. An echocardiogram revealed a dilated left ventricle and an ejection fraction of 15%. Despite appropriate treatment during admission, patient became hypotensive, and went into asystole. Aggressive resuscitation was unsuccessful. DISCUSSION: Peripartum cardiomyopathy is defined as the development of heart failure in the last month of pregnancy or within five months after delivery without any determinable cause and without previous heart disease before the last month of pregnancy. The etiology of peripartum cardiomyopathy is unknown. Some actually question whether this is a distinct disorder or another form of idiopathic cardiomyopathy. Several ideas have been proposed including an immunologic response to the fetus, increased hemodynamic load of pregnancy, nutrition, and hormone response. Several studies have actually shown that these patients have a high incidence of myocarditis, possibly implicating a viral trigger like enterovirus. Signs and symptoms are typical for congestive heart failure. However, these can sometimes be confused with normal pregnancy. Therefore, peripartum cardiomyopathy may not be diagnosed until postpartum. Evaluation would include ECG, chest xray, and an echocardiogram. Cardiac catheterization should be considered in those at risk for atherosclerosis. Endomyocardial biopsy remains controversial. Serum can be tested for bacterial and viral cultures and Coxsackie B virus titers. Therapy consists of digoxin, diuretics, sodium restriction, and afterload-reducing agents. Thromboembolic phenomena are common and anticoagulation should be strongly considered. Cardiac transplantation may be considered as well. The mortality rate ranges from 25 ± 50%. Nearly half of these deaths occur within the first 3 months postpartum. Death is often caused by chronic progressive congestive heart failure, an arrhythmia, or a thromboemolic event. If the patient's heart is to recover, it willusually do so within the first 6 months. If ventricular function does not improve, it is often recommended that the patient not become pregnant again due to the high risk of death.
WHEN ANTIBIOTICS DONT WORK, TIME TO THINK BEYOND BACTERIUM. S. Malhi1 1 , K. Gopal 1 ; 1 Fairview Hospital, Fairview Park, OH LEARNING OBJECTIVES: 1) Work up of non resolving pulmonary infiltrate. 2) Recognize the clinical manifestations of Disseminated Histoplasmosis. 3) Suspecting HIV disease in the presence of opportunistic infection CASE INFORMATION: A 54 yrs. old white male presented to the ER with complaint of fever, shaking chills, myalgias, fatigue, and episodes of profound sweating. He had been sick for the past 4 weeks. His primary care physician ordered a chest radiograph that revealed an infiltrate & he was treated with Levofloxacin for 10 days. Patient continued to do poorly with fevers in the range of 100 ± 104 F and was then treated with another 7 ± 10 days of antibiotics. He lost 20 lb. of weight and hence presented to the ER. His other medical problems included hypertension and remote history of alcohol abuse. He denied smoking, current alcohol or drug use. He was divorced, had 3 children and worked as a social worker. Physical exam was remarkable for a fever of 102 F and mild tachycardia. A CXR on admission showed worsened left lung infiltrate. Initial Lab studies revealed WBC 3.0, HGB 11.0, HCT 31.8, MCV 81, PLT 192 DISCUSSION: A lung biopsy may be the last recourse to obtain a diagnosis and ascertain the correct treatment for a non resolving pulmonary infiltrate. Disseminated Histoplasmosis occurs in elderly and about 5% of AIDS patients in endemic areas and is characterized by fever, malaise, pulmonary infiltrates, hepatosplenomegaly, lymphadenopathy and weight loss. Where as asymptomatic infection is most common; CXR of patients with acute infection show focal mid lung infiltrates (30%), hilar or mediastinal lymphadenopathy (30%) or both (30%).
A COUGH THAT LINGERED: DIAGNOSING LEGIONELLA PNEUMONIA. R. Mann 1 , D.W. Brady 1 ; 1 Emory University, Decatur, GA LEARNING OBJECTIVES: 1. Diagnose legionella pneumonia from its clinical manifestations. 2. Recognize the limited utility of the legionella urinary antigen in excluding the diagnosis of legionella in a patient with a pneumonia that has been refractory to treatment. CASE INFORMATION: A 40 year old black male presented complaining of a 3-week history of cough (productive of dark yellow sputum), fevers, chills, nightsweats, and malaise. He initially was treated at an out of town facility with a 5 day course of azithromycin, with some improvement in symptoms; however, within 2 ± 3 days after completing the medication, the symptoms returned. He presented to a second emergency room, where he received a chest xray and was discharged home with a prescription for guafenesin. One week later, he presented again. A second chest xray revealed interval worsening of a right middle and right upper lobe pneumonia with a patchy airspace opacity in the left upper lobe consistent with pneumonia. On exam his blood pressure was 156/75, pulse 119, respiratory rate 30, temperature 36.9C, with bilateral rales throughout. Pulse ox-70% on room air ( ABG on 100% FiO2-pH 7.44; pC02 30; pO2 100); WBC count 16.5 (77%N,6%Mono,16%L), ; HIV test was negative. He was admitted and treated with IV ceftriaxone and erythromycin for the first 72 hours with worsening hypoxemia. Erythromycin was replaced with IV Azithromycin for the next 48 hours with no improvement. Levofloxacin 750mg IV qd was added to replace azithromycin to cover Legionella. A Legionella urine antigen was negative. After 48 hours of high dose Levofloxacin, the patient showed little clinical improvement. Legionella, mycoplasma, and influenza serologies were sent, as preparations were being made for bronchoscopy. On the 3rd day of high dose Levofloxacin the patient began to require lower FiO2's and subsequently the Legionella Pneumophilia IgM serology was found to be positive (1:64) . Within the next week, the patient was discharged home to complete a 21 day course of high dose Levofloxacin for treatment of Legionella pneumonia. DISCUSSION: This patient's final diagnosis was made after a protracted outpatient and subsequent inpatient course of therapy. His pneumonia ultimately was found to have been caused by Legionella pnuemophila; however, the legionella urinary antigen was negative. Clinicians should be aware that the legionella urinary antigen only tests for serogroup I of L. pnuemophila, and a negative urinary antigen does not, in fact, rule out infection caused by L. pneumophila (the most common Legionella species causing community-acquired pneumonia in the U.S.). Often, serum antibodies are needed to confirm or exclude the diagnosis.
THROMBOTIC THROMBOCYTOPENIC PURPURA FOLLOWING THE FLU VACCINE. A. Markarian 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize thrombotic thrombocytopenic purpura (TTP) as a potential complication of the influenza vaccine. CASE INFORMATION: A 58 year-old man with COPD, diabetes mellitus, and hypertension presented with mental status changes and oliguria. He had felt fatigued for several days and had noted a rash on his shins. His chronic medications were Metformin, Glipizide, Nitrobid, Metoprolol, Omeprazole, and Quinine. He received the flu vaccination two weeks prior to admission. He was afebrile but tachycardic. Physical exam showed flaccid paralysis, diminished deep tendon reflexes and bilateral Babinski signs. The heart, lungs and abdomen were unremarkable. A petechial skin rash was noted on both shins. Laboratory studies revealed mild anemia (Hgb 11.7), schistocytes on the peripheral smear, LDH (1927) and a low haptoglobin. The platelet count was 13,000. PT, PTT, INR, fibrinogen and fibrin degradation products were normal. The BUN was 84 and the creatinine 3.4. ANA was positive with a titer of 1:160; anti ds DNA and Lupus anticoagulant were negative. Total compliment levels were normal. SSA/RO and SSB/LA were negative. Antiglo-merular basement membrane Ab, ANCA, ACLA-IgG and IgM, and C1 Esterase inhibitor were normal. HIV was non reactive. Cultures were negative for enteric pathogens. Von Willebrand' factor activity was normal. Quinine dependant antibodies against platelet glycoprotein were negative but non drug dependant IgM antibody was positive. The patient was treated with plasmapheresis and hemodialysis and recovered. DISCUSSION: The classic pentad of TTP includes microangiopathic hemolytic anemia, thrombocytopenia, neurologic symptoms, renal disease and fever. The etiology is poorly understood although underlying immune complex formation is frequently found. Plateletassociated antibodies and serum reactivity against endothelial cells are positive in some cases. Most cases are idiopathic. Associated conditions include infections (bacterial, viral, and fungal), autoimmune diseases, AIDS, cancer, vaccinations and drugs such as quinidine, quinine and ticlopidine. Cases of TTP following influenza vaccine have been reported (Brown et al, Br Med J 1973; 2(861):303; Symmers, Br Med J 1973; 2(866) :614). In the present case, it is likely that the influenza vaccine triggered the antibody formation and immune complex deposition and led to the development of TTP. CASE INFORMATION: A 55-year old male post myocardial infarction was transferred for cardiac catheterization from a small community hospital. His past medical history per family included closed head trauma as a child, with residual cognitive impairment, hypertension, gout, and some hearing loss over the past 2 months. He has worked as a janitor for a local school for the past 13 years. He was described by his family as``slow'' and had a 6th grade education. On hospital day 6, after stabilization, his disposition became a concern. That morning, he sat in bed watching cartoons, laughing, with the sound off. When examined, he became confused and cried. His answers to questions were intermittently unintelligible. He frequently answered questions not asked. An uncle confirmed that this was his baseline, and that he had exhibited odd behavior since a child; his family had attributed it to falling off a truck and hitting his head. His primary team asked psychiatry if evaluating the level of his cognitive functioning would help, and his supervisor at work was called. She said that he was an excellent and very dependable employee as long as he was shown exactly what to do. When asked if he would follow verbal orders, she again stated that he needed to be shown what to do. Suspecting severe hearing impairment, a stethoscope was placed in his ears. With the communicator shouting into the diaphragm, he could hear clearly and responded appropriately. Audiometry on day #10 revealed complete sensory hearing loss on the right, severe loss on the left, with 10% speech discrimination on the left, which increased to 20% when allowed to lip-read. Further questioning revealed that he remembered being able to hear to some degree as a very young child. He was not aware that his family did not know how profoundly deaf he was. DISCUSSION: Chartlore, the process by which diagnoses are assigned via undocumented notes in the chart, is usually a product of loosely applied terms that, once used, tend to``stick.'' This particular example of chartlore is interesting in that the original misdiagnosis/assumption appears to have been made, not by the medical staff, but by the patient's own family, and tragically for him, perpetuated over decades, preventing its correction. Physicians must recognize the often deceptive nature of chartlore and the need to confirm history gleaned from medical records and family members. This fact is especially true when the``facts'' obtained conflict. 1) Recognize that McArdle disease is the number one glycogen storage disease diagnosed in adults, 2) Recognize the typical history of a patient with McArdle disease, and 3) Increase awareness of the disease for possible earlier detection. CASE INFORMATION: CASE: CC is a 31-year-old male who presented with a one-day history of dark urine. CC had to swim strenuously when a barge he was on sank. He then developed diffuse body aches and dark urine. He had a long history of exercise intolerance, easy fatigability, and on several occasions had dark colored urine after exertion. CC's younger brother had a similar history but his symptoms were not as severe. Meds: none, otherwise negative PMH. Pertinents on PE were BP 131/78, P 101, RR 24, WNWD male in moderate distress from diffuse whole body pain. CV: RRR with mild tachycardia, abd was normal with no HSM. The patient had diffuse muscular tenderness over the thighs, arms, forearms, anterior chest, and back. Labs BUN of 14, Cr of 1.2, AST of 461, ALT of 138, ALK PHOS of 97, LDH of 7,090, and a CK of 87,000. UA showed specific gravity of 1.015, pH 5.0, 4+ prot, 3+ blood, + LE, + NIT, 0 ± 3 RBC's, and +3 myoglobin. The patient had a biceps muscle biopsy which showed the absence of myophosphorylase. DISCUSSION: McArdle Disease or muscle phosphorylase deficiency results in limited ATP production from glycogenolysis and results in glycogen accumulation. It is the most common glycogen storage disease in adults. Inheritance is autosomal recessive, and the abnormal allele is on chromosome 11. Patients usually are diagnosed in the second or third decade but most have had symptoms since childhood. Exercise intolerance with muscle cramps, myoglobinuria, and a second wind phenomenon are common. Most patients have baseline elevated CK's. Diagnosis can be made by after ischemic exercise testing by measuring lactate levels and ammonia. Definitive diagnosis can be made by muscle biopsy and demonstrating myophosphorylase deficiency. DNA based diagnosis and carrier detection are available. Prognosis is generally good since longevity is not affected. Patients should avoid strenuous exercise and know the symptoms for seeking medical care during an exacerbation. High protein diets and oral glucose or fructose may increase exercise endurance.
OH NO, NOT``NPO''. UNCOVERING OCCULT DIABETES INSIPIDUS. S. Mcelhattan 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1. To recognize that occult diabetes insipidus can be unmasked when a patient is no longer able to maintain adequate free water intake. 2. To recognize diagnosis and discuss pathophysiology of lithium-induced diabetes insipidus. CASE INFORMATION: An 81 year-old male was admitted with aspiration pneumonia and dehydration two weeks after head and neck surgery. Postoperatively, he was maintained on nasogastric tube feedings but otherwise kept NPO. His past medical history was notable for bipolar disorder requiring lithium for 20 years. On physical, he was lethargic and orthostatic with dry mucous membranes. His sodium was 157 and creatinine was 2.6. The free-water deficit was calculated, and appropriate IVF therapy was initiated. However, despite increasing IVF administration and free-water boluses, hypernatremia and polyuria persisted. Urine osmolality was 171 and serum osmolality was 324. A water restriction test was not performed as the clinical occurrence of nephrogenic diabetes insipidus was suspected. The patient was given larger and more frequent free-water boluses with improvement. DISCUSSION: Nephrogenic diabetes insipidus (DI) is resistance to endogenous ADH at the level of the kidney. Up to 20 percent of patients on chronic lithium therapy develop nephrogenic DI. Patients generally adjust their lifestyle and maintain normal fluid balance to keep up with the polyuria. Although the etiology is not totally understood, it is thought that lithium enters the renal collecting tubule cells via a sodium channel and inhibits adenylate cyclase. Lithium may also reduce the density of ADH receptors or downregulate the expression of aquaporin-2 molecules. The diagnosis is substantiated by a water restriction test. As water is restricted, urine and plasma osmolality are measured hourly. If a steady urine osmolality is measured for three successive hours or if the plasma osmolality reaches 300 and the urine osmolality remains below normal, exogenous ADH is given. If urine osmolality increases, central DI is diagnosed but if the urine osmolality does not change, a diagnosis of nephrogenic DI is made. In our patient, it may have remained clinically silent for many years if he had not been NPO. It is essential that all patients on lithium who are made NPO be monitored for the development of hypernatremia and fluid imbalance. Abdominal pain is one of the most common ambulatory patient complaints. Its etiologies can be numerous, leaving the meticulous physician to pursue leads by the history, physical examination and laboratory or imaging tests. We present an unusual cause of abdominal pain that presented in the outpatient setting. CASE: A previously healthy 58 yearold male presented to the office with a two-day history of``heartburn.'' His abdominal pain worsened post-prandially and he noted early satiety, but denied nausea, vomiting, melena, or hematochezia. Alka-Seltzer and aspirin provided no relief. He denied usage of tobacco or illicit drugs, and reported occasional alcohol use, though none over the preceding days. His exam was significant only for mid-epigastric tenderness, without rebound or guarding. His rectal exam revealed brown colored stool, negative for occult blood. Laboratory data was significant only for a mildly elevated ALT. He was treated empirically with cimetidine for possible dyspepsia, gastritis, or peptic ulcer disease. Two days later, the patient was emergently seen for worsening abdominal pain. His exam was unchanged and laboratory data continued to be unremarkable. An urgent abdominal ultrasound surprisingly revealed acute portal, superior mesenteric, and splenic vein thromboses and an enlarged prostate. He was admitted for treatment and diagnostic work-up, which failed to reveal an underlying etiology for the thromboses. DISCUSSION: Portal vein thrombosis (PVT) is a rare condition associated with inherited hypercoagulable states, underlying myeloproliferative disorders, neoplasms, infections, and other inflammatory processes. It remains idiopathic in 8% to 15% of cases. Abdominal pain is commonly reported when thrombosis also involves the superior mesenteric vessels and produces bowel ischemia, though not present in our patient. The treatment has traditionally focused on complications, most commonly acute gastrointestinal bleeding. The role of anticoagulation therapy remains controversial, but appears to be indicated in patients with acute thrombotic events without intestinal ischemia, as was the case with our patient. While rare, PVT remains an important cause of abdominal pain. A high index of clinical suspicion is necessary for successful diagnosis.
J. Messler 1 , V. Nunez 1 , T. Jacobson 1 ; 1 Emory University, Atlanta, GA LEARNING OBJECTIVES: 1. Learn the differential diagnosis of pulmonary cavities in HIV patients. 2. Recognize the importance of bronchoscopy for diagnosis. 3. Recognize the various pulmonary manifestations of cryptococcus. CASE INFORMATION: A 41yo homeless man presents to the walk-in clinic with a chief complaint of cough. He complains of blood-tinged sputum for two months, worsening over the past three weeks. He denies fever or chills but reports night sweats, dizziness, decreased appetite, and weight loss. Except for an episode of``bronchitis'' treated 2 months earlier, his past medical history is unremarkable. A former intravenous drug user, he has lived in and out of shelters for three years. A recent PPD was negative. He admits to multiple, unprotected bisexual contacts in the past year. He has never been tested for HIV. On admission, his temperature is 39.1C, pulse 98, respirations 16, and blood pressure 100/60, sitting and lying down. Significant findings on his exam include bitemporal wasting, pale conjunctiva and oral thrush. Chest x-ray on admission reveals a 3cm cavity in the right posterior upper lobe. His PPD is negative and sputum cultures for AFB are negative. He tests positive for HIV with a CD-4 count of 6mm3. Bronchoscopic washings grow budding yeast. Serum cryptococcal antigen returns positive at a titer of greater than 1:512. The opening pressure from the lumbar puncture is normal. Cerebrospinal fluid (CSF) analysis contains 4 WBC, glucose 40, and protein 50. CSF yields a positive india ink stain and CSF cryptococcal antigen titer is positive. Subsequently, he begins therapy for cryptococcal pneumonia and cryptococcal meningitis. DISCUSSION: In HIV patients, the differential diagnosis for pulmonary cavities is extensive. The most common etiology is tuberculosis. However, as the CD-4 count falls below 200mm3, tuberculous cavities decrease in prevalence. Various other etiologies include atypical pneumocystis presentation, toxoplasmosis, invasive fungal infections, bacterial infections, and neoplasms including Kaposi's sarcoma. Sputum samples are a start to diagnosis, but bronchoalveolar lavage or biopsy would likely be required to obtain a definitive diagnosis for a cavitary process. A cavitary lesion involving cryptococcosis is a rare occurrence. Usual radiographic presentations for pulmonary cryptococcosis include interstitial infiltrates or pleural effusions. Additionally, there is no typical clinical picture. Interestingly, although the majority of cryptococcal patients are initially diagnosed with disseminated CNS disease, the pulmonary complaints of cryptococcosis often precede the diagnosis of meningitis by months. The variety of clinical and radiographic presentation underscore the need for definitive diagnosis, especially in the patient with clinically silent cryptococcal meningitis. 
A 17 year old incarcerated male presented to our emergency department complaining of headache, nausea, vomiting, diarrhea, subjective fever and weight loss for one week. There were no sick contacts or recent travel. The patient had no prior medical history, and had tested negative for HIV six months before presentation. He was taking no medication and denied any family history. Sexual history revealed prior homosexual intercourse. On presentation, the patient's blood pressure was 99/44 mm/Hg with a temperature of 39.4 8C. Physical exam revealed seborrhea over the face and cervical, axillary and inguinal lymphadenopathy. Laboratory studies revealed a WBC count of 2,000, hematocrit of 48% and platelet count of 48,000. Urinalysis showed 2+ protein. Cerebrospinal fluid analysis, stool studies, and blood cultures were negative. Chest X-ray was normal. The patient underwent bone marrow aspiration, which revealed a hypoplastic marrow with decreased erythroid precursors, consistent with viral infiltration. The patient clinically improved and remained afebrile with resolution of all of his symptoms. His platelet count returned to normal. Parvovirus serologies were equivocal, and Epstein Barr serologies did not reveal acute infection. The patient underwent HIV testing which revealed a positive ELISA with an indeterminate Western blot. The CD4 count was 420. After consultation with the infectious disease team and the pathology department, we were permitted to perform viral load testing in the hopes of confirming the diagnosis of acute HIV infection. The viral load was greater than 750,000 copies per mL. DISCUSSION: Acute HIV infection presents with a variety of non-specific symptoms, including fever, lethargy, rash, myalgias and headache. Symptoms are present in 50 ± 90% of patients with acute infection, and the complex is known as acute antiretroviral syndrome. Because of the non-specific symptoms, acute HIV infection in adolescents often resembles, and may be mistaken for, more common viral illnesses such as infectious mononucleosis. The diagnosis should be suspected in any patient at risk for HIV infection who presents with this constellation of symptoms or fever of unknown cause. Early diagnosis of HIV, particularly during acute infection, is crucial so that antiretroviral therapy may be initiated without delay. The diagnosis is based on a HIV-1 RNA level of greater than 50,000 copies per mL or positive p24 antigen in the absence of a positive ELISA and confirmatory Western blot test for HIV. If a patient is HIV enzyme immunoassay-negative and HIV RNA-positive, follow-up antibody testing should be performed 2 ± 4 weeks after the resolution of symptoms to document seroconversion.
COMMON COUGH,UNCOMMON CAUSE. F. Millhouse 1 , L.J. Schultz 1 ; 1 Emory University, Atlanta, GA LEARNING OBJECTIVES: 1. Learn when to evaluate further young patients with``recurrent pneumonias.'' 2. Recognize the clinical presentation of pulmonary sequestration. 3. Learn the pathology and pathophysiology surrounding the formation of sequestration cysts. CASE INFORMATION: A 26-year-old male presented with the complaint of a 2 ± 3 week history of cough productive of approximately one cup of yellow green sputum per day. On the day prior to admission, he noted 1 ± 2 teaspoons of blood with each cough. Five years earlier with a similar presentation, he was noted to have a cavitary lesion on chest x-ray. He was treated with antibiotics for a presumed lung abscess and was asymptomatic until this admission. Vital signs on admission revealed a blood pressure of 137/76, heart rate of 137, respirations of 22, temperature of 38.4 degrees Celsius, and a pulse ox of 88%. On physical exam he was a young well-developed male in no apparent distress with dullness to percussion and tubular breath sounds at the right base. Chest x-ray showed an interval increase of the air-fluid level within a large right middle and lower lobe cystic lesion and a left mid lung infiltrate. The patient was admitted and started on antibiotics for pneumonia and presumed lung abscess. The patient was unable to tolerate a CT scan or bronchoscopy secondary to uncontrollable coughing and hemoptysis. With a high suspicion for pulmonary sequestration, an aorto-bronchial arteriogram was obtained that confirmed the diagnosis. It showed an aberrant vessel arising off the infradiaphragmatic aorta just above the level of the celiac artery supplying a right lower lobe sequestration. DISCUSSION: Pulmonary sequestration is defined as an area of nonfunctioning lung tissue that lacks normal communication with the tracheobronchial tree and derives its blood supply from systemic vessels. Pulmonary sequestration is divided into two types: intralobar and extralobar sequestration. The former usually presents in early adulthood. The clinical presentation may range from asymptomatic to chest pain, chronic cough or hemoptysis. Radiographic findings can include either consolidation with irregular margins, multicystic lesions, or cavitation. The diagnosis should be considered in young patients with recurrent or persistent pneumonias. The treatment is surgical removal of the sequestration via segmentectomy or lobectomy. SEVERE POSTURAL HYPOTENSION AS A DELAYED SEQUELA OF LIGHTNING INJURY. V. Mukerji1 1 , R. Nonneman 1 ; 1 Southern Illinois University, Springfield, Illinois LEARNING OBJECTIVES: 1) Review the acute and delayed injuries that may result from lightning strike. 2) Recognize postural hypotension as a possible delayed sequela of lightning injury. CASE INFORMATION: A 27-year-old woman was referred to us for increasing dizziness and palpitations. About 8 years ago while sitting indoors with her foot on the fireplace grill she was struck by lightning. She suffered a brief syncopal episode followed by severe lower extremity weakness and inability to walk. With physical therapy her weakness had almost cleared but she continued to experience increasing dizziness and palpitations. Her physical examination was unremarkable except for a resting heart rate of 110 beats per minute. The EKG and echocardiogram were normal. On tilt table testing the patient became dizzy and lightheaded as the blood pressure fell from 116 to 76/42 mm Hg and her heart rate rose from 94 to 150 beats per minute. Her symptoms cleared and the heart rate and blood pressure returned to normal as soon as the table was brought back to the horizontal position. The tilt table test was then repeated with both lower extremities wrapped with Ace bandage. This time there were only minor changes in her blood pressure and heart rate and the patient remained asymptomatic. Compression stockings were recommended for the patient with significant improvement in her symptoms.
DISCUSSION: Lightning is the second most common cause of environmental death in the United States. It is estimated that the number of human injuries may be as high as 5000 per year with 300 deaths. Permanent sequela may occur in 75% of survivors. The serious injuries from lightning strike are usually cardiovascular or neurologic. Most cases develop burns but they are rarely serious. Eye and ear injuries each occur in 50% of cases. Cardiovascular problems include asystole, dysrhythmias, cardiac contusion, pericardial effusion, EKG changes and cardiac enzyme elevations. Neurologic problems include syncope, confusion, paresthesias, and neuropsychiatric disorders. Respiratory center paralysis may occur. Less commonly seizures, cerebral edema, cranial nerve dysfunction and cerebellar ataxia have been reported. This patient developed autonomic dysfunction with severe disabling postural hypotension. The onset was gradual over several years after the actual lightning strike. A 61 year old man was admitted with atrial fibrillation and congestive heart failure. Pharmacologic treatment to limit the ventricular rate was complicated by episodes of bradycardia. Heart rates as low as 30 beats per minute in the presence of continued tachycardia necessitated the implantation of a pacemaker. A pacemaker was implanted, and anticoagulants were restarted twenty-four hours after the surgery. A hematoma was noted at the site. Warmth and erythema developed, yet the patient was afebrile. Empiric antibiotics were started and an aspirate culture was drawn. The culture grew Corynebacterium Urealyticum. The patient was treated with vancomycin, and the pacemaker was explanted. Cultures of the pacemaker also grew Corynebacterium Urealyticum. DISCUSSION: The incidence of pacemaker infections post-implantation ranges from one to six percent, with the majority of cases being caused by staphylococcus species. This case represents an unreported etiologic organism of pacemaker pocket infections. Corynebacterium Urealyticum is a slow growing, multi-drug resistant, gram positive aerobe that is often found in skin flora in hospitalized patients. 2. Generalized erythroderma is rare in Psoriasis and any such a change should prompt a biopsy and further evaluation. CASE INFORMATION: Peripheral T-cell lymphoma is classified among aggressive group of non-Hodgkin lymphomas by R.E.A.L. classification and accounts for < 15% of lymphomas in US. It presents as generalized disease with pruritis. Lymph nodes, skin, liver spleen and other viscera maybe involved. Presentation with generalized erythroderma is rare with peripheral Tcell lymphomas and is usually seen with primary cutaneous lymphomas. Case History: 78 year old Hispanic female with past medical history of hypertension, psoriasis and eczema for the last ten years presented to dermatology service with two month history of gradually worsening generalized erythematous macular rash with mild itching. Patient denied HIV infection or risk factors. Home medications included plendil, monopril, aspirin and lidex oint. There was no change in her medications recently. Physical exam. revealed generalized erythmatous non scaly macular rash with violacaeous discoloration at some areas and cervical lymphadno -pathy . CBC revealed leukocytosis with eosinophillia. LDH was 441. CXR was unremarkable. Skin biopsy was performed which revealed atypical lymphocytic infiltrate with abundant eosinophills. These cells were positive for CD3, CD4, CD5 (T cell markers) and negative for CD20 & CD30 (B cell markers). Lymph node and bone marrow biopsies were performed to further characterize the disease. Both specimens showed neoplastic infiltrates of lymphoid cells, which were positive for CD3, CD4, and CD5. Review at NIH confirmed the above findings and a diagnosis of peripheral T cell lynphoma-unspecified was made. Patient, who was visiting US from Santo Domingo, declined chemo-herapy and elected to return to her country. DISCUSSION: Peripheral T cell lymphomas-unspecified is a rare kind of lymphoma. Whereas erythro-derma is not an uncommon presentation of primary cutaneous T cell lymphomas especially Sezary syndrome, it is very rare in perpheral T cell lymphoma-unspecified. In a patient with psoriasis, such as ours, generalized erythroderma is rare and such an evolution of the disease should prompt skin biopsy and further workup. Treatment of peripheral T cell lymphomas-unspecified is not clear due to rarity of this disease. These lymphomas are usually very aggressive and have only 20% cure rate.
S. Narreddy 1 , M. Snyder 1 , P. Khan 1 , N. Lekas 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the dilemma posed by multiple enhancing ring lesions in the brain and emphasize the need for brain biopsy in equivocal cases. CASE INFORMATION: A 67 year-old Arabic male presented with a one-month history of progressive weakness and sensory changes of the left arm and leg. He had lived in the US for thirty years before returning to Yemen two years ago to work on a farm. On examination, he had right homonymous hemianopia, left hemipareisis and left hemianesthesia but nothing that would suggest either an infectious process or a malignancy. Basic work-up including HIV test was negative. An MRI revealed multiple enhancing ring lesions in the left and right occipital lobes, the right parietal lobe, and left cerebellar hemisphere. The findings were interpreted as metastatic lesions from an unknown primary. When no primary focus was found a brain biopsy was performed. Gross pus was aspirated. Histology was consistant with abscess. Smears revealed filamentous gram-positive organisms and cultures grew Nocardia asteroides. DISCUSSION: Multiple ring enhancing lesions in the brain typically represent either metastatic lesions (3 ± 15% of brain tumors; bronchogenic, breast, melanoma, renal, lymphoma are most common primaries) or abscesses. Differentiating between the two by clinical or radiographic means has been difficult, even with the development of CT and MRI scanning. Although advanced radiographic technologies (i.e., diffusion-weighted echo planar imaging, technetium-99m labeled leukocyte scintigraphy) accurately image brain appearance, to clarify treatment it is important to recognize that a brain biopsy is warranted when the diagnosis is in question and a primary focus cannot be identified. A 44 year old Hispanic male presented to clinic with multiple complaints including: red itchy eyes, productive cough with occasional blood, rhinorrhea, nasal congestion, myalgias, abdominal pain, and subjective fevers and chills. Except for a gastric ulcer diagnosed 1 month earlier, he was otherwise healthy. He was diagnosed with a viral syndrome, reassured, and given symptomatic treatment. The patient returned for a second visit 2 weeks later without improvement in his symptoms. A CXR, PPD, and HIV test were all negative. An ophthalmology exam confirmed the diagnosis of viral conjunctivitis. The patient, concerned that his symptoms were not improving, returned a week later, appearing very ill, and was admitted for further work up. On physical exam, notable findings included a T = 38.8 C, scleral injection, nasal and oropharyngeal erythema, decreased breath sounds at both lung bases, and mild abdominal tenderness in the right upper quadrant. Labs were significant for proteinuria, hematuria, and elevated liver function tests. To work up his abdominal symptoms, a CT of the abdomen was obtained which showed gastric thickening and a right lung nodule. Subsequent testing included an EGD showing diffuse erythematous mucosa; a repeat CXR and CT of the chest revealing multiple opacities in both lungs with hilar lymphadenopathy; bronchoscopy showing erythematous friable mucosa; and a repeat slit lamp examination revealing bilateral episcleritis. C-ANCA titers were 1:160. A nasal biopsy was performed showing granulomatous disease with giant cells and chronic vasculitis confirming the diagnosis of Wegeners Granulomatosis. The patient was treated with Cytoxan and Prednisone with resolution of symptoms. DISCUSSION: Wegeners Granulomatosis is a form of small vessel vasculitis with prevalence of 3:100,000. The mean age of onset is 40 years, with men affected slightly more than women. It classically involves upper and lower respiratory tracts and kidneys, but can involve virtually any organ system. This case illustrates how Wegeners can mimic colds and sinusitis, obscuring the diagnosis and delaying treatment. Early diagnosis is crucial because untreated Wegeners has a poor prognosis with median survival of 5 months and up to 90% mortality. Furthermore, renal failure can progress rapidly even in the absence of symptoms. Timely initiation of therapy with Cytoxan and Prednisone can be life saving and may markedly decrease morbidity and mortality. On the morning of admission, he was unable to arise from bed. The weakness involved both upper and lower extremities and was associated with mild abdominal pain. After 5 hours, he alerted a family member who assisted him to his feet. Upon standing the patient lost consciousness. He denied vomiting or recent illness but noted decreased food intake for the 2 days prior to admission. His only past medical history was a penetrating knife wound to the forehead 20 years prior to admission. He was taking no medications and did not use intravenous drugs. The blood pressure was 90/54 mmHg, the heart rate 110 beats/min and the temperature 36C. He had a normal thyroid exam and there were no carotid bruits. Examination of the heart, lung and abdomen were normal. Reflexes were decreased throughout; proximal and distal muscle strength was diminished. There were no focal deficits; sensation and cerebellar functions were normal. His sodium, potassium, calcium, magnesium, and creatinine were normal. The blood glucose was 54 mg/dL, and despite multiple ampules of dextrose he remained hypoglycemic. Persistent hypotension and refractory weakness prompted an evaluation of his adrenal function. The random cortisol was 1.6 mg/dl; the ACTH was low. DISCUSSION: The diagnosis of adrenal insufficiency should be considered when refractory hypoglycemia, orthostatic hypotension, and weakness simultaneously present. The causes of acute adrenal insufficiency include iatrogenic withdrawal of steroids, adrenal hemorrhage, tuberculosis and autoimmune disease. An important and under-recognized cause of adrenal insufficiency is inhibition of the hypothalamic-pituitary-adrenal axis by heavy alcohol ingestion. A repeat history revealed that the patient had a 6-day period of heavy alcohol and cocaine use. The normal electrolytes were clues that the adrenal insufficiency was not due to Addison's disease. The functional aldosterone axis and lack of hyperpigmentation implied a pituitary etiology which was confirmed by the low ACTH and robust rise in cortisol following cortrosyn stimulation. The weakness and hypoglycemia corrected with administration of hydrocortisone. Clinical reasoning identified the specific etiology of this patient's Addisonian syndrome and prevented unnecessary therapy.
PROGRESSIVE PNEUMONIA IN AN IMMUNOCOMPETENT HOST. L. Orlando 1 ; 1 Tulane Medical Center, New Orleans, LA LEARNING OBJECTIVES: Suspect fungii in antibiotic refractory pneumonias and treat the presence of hyphae as invasive aspergillosis in these patients. Recognize that chronic diseases can increase the risk for invasive aspergillosis.
A 71 year-old man with severe COPD (not on steroids) presented with one week of a non-productive cough, fever and dyspnea. His exam revealed crackles and egophony over the right upper lobe (RUL). The chest X-ray confirmed a community acquired pneumonia (CAP), and he was started on Azithromycin. His symptoms did not improve. A chest CT showed extension of the RUL consolidation, but no cavities or endobronchial lesions. Blood and sputum gram stain, AFB, KOH, and cultures were negative, except one sputum with branching hypae, which was dismissed as a contaminant. Fluconozole and Vancomycin were added, but his condition worsened. Acute renal failure occurred on day seven. Bronchoscopy was performed on day eight, and revealed black mucus and white nodules within the bronchioles. Lavage and biopsies were negative for organisms or cancer. The patient died from an acute lung hemorrhage on day ten. Autopsy revealed invasive Aspergillus Niger with an acute lung hemorrhage. DISCUSSION: A progressive CAP despite antibiotic therapy implies one of the following: the wrong antibiotic selection, a post-obstructive pneumonia (POP), a fungal pneumonia, immunosuppression, alveolar proteinosis or BOOP. In our patient the acute presentation, imaging studies without fleeting infiltrates, and bronchoscopy without obstruction were clues against BOOP, alveolar proteinosis, and POP. The patient's brisk immune response, severe COPD and negative bacterial cultures were important hints towards a fungal infection. Although Aspergillus pneumonia usually occurs in the immunocompromised, it can occur in immunocompetent patients so the presence of branching hyphae on cultures should not be disregarded as colonization or contamination when a clinical pneumonia is present, especially in those with chronic diseases. Empiric Amphotericin B and lung biopsy should be considered when clinical evidence suggests Aspergillus since cultures and even bronchoscopy are frequently negative. This patient had Aspergillus Niger which produces oxalic acid creating a pathognomonic black acidic sputum and an immune mediated glomerulonephritis. Sputum and BAL cultures are positive in less than 10% and dissemination occurs in 25% of patients.
A. Pai 1 , A.L. Riba 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the unique radiologic and therapeutic features associated with impending paradoxical emboli. CASE INFORMATION: A 61 year old female presented with an acute onset of intractable headaches, vertigo, dyspnea and melena. A CT scan confirmed an embolic CVA involving the right occipital lobe. The presence of an asymptomatic, swollen right leg, and an acute axis change on EKG lead to the suspicion of a DVT with PE. Large thrombi were seen in both pulmonary arteries on spiral CT. Additionally, a TEE demonstrated a large embolus straddling a patent foramen ovale. Therapy was problematic due to the patient's history of complications following a CABG in the past and active GI bleeding. A conservative treatment approach involved careful anticoagulation with heparin and Greenfield filter placement. The patient success-fully recovered without further embolic episodes or complications. DISCUSSION: Although 25% of patients at autopsy have a patent foramen ovale that can be detected by probing, the actual occurrence of paradoxical emboli (as with most pulmonary emboli) can safely be assumed to be under-diagnosed. Although newer technology (spiral CT, TEE) has increased our ability to diagnose such conditions, impending paradoxical embolus is still rarely reported in the literature. The optimal therapy is unknown although embolectomy with closure of the patent foramen ovale, thrombolysis and anticoagulation are the commonly available options. Treatment should be individualized depending on the patients age, existing co-morbidities and the availability of cardiothoracic surgical services.
POLYARTERITIS NODOSA PRESENTING AS AN ACUTE CEREBROVASCULAR ACCIDENT IN A YOUNG MALE. P.J. Bennett 1 , J.J. Yium 1 , M. Panda 1 ; 1 University of Tennesee -Chattanooga, Chattanooga, TN LEARNING OBJECTIVES: To understand the differential diagnosis of CVA in young patients. To recognize clinical features and proper diagnosis of polyarteritis nodosa (PAN) CASE INFORMATION: A 24-year-old black male with a questionable history of multiple sclerosis presented to the ER with acute onset of right-sided weakness, slurred speech and dysphagia. The patient denied any visual symptoms, headache, fevers or recent illnesses. On exam the patient was afebrile, pulse 60 beats per minute, and BP 180/128mm/Hg, neck was supple without bruits, and a 2/6 systolic murmur was noted at the apex. No skin lesions were noted. On neurologic exam the patient had dysarthria, right facial droop and right hemiplegia. Routine lab work was unremarkable with a normal CBC, metabolic panel and sedimentation rate. Urinalysis revealed 2+ proteinuria with benign sediment. LP revealed no oligocional bands. An MRI of the brain revealed a recent deep left-sided infarct involving the internal capsule as well as a small aneurysm of a temporal branch of the right middle cerebral artery. Hypercoaguable work up was negative, p-ANCA was negative but myeloperoxidase antibody was positive. Echocardiogram revealed mild left ventricular hypertrophy with normal LV function. Cerebral and renal angiography revealed innumerable aneurysms involving almost all branches of medium-sized arteries. A biopsy of the sural nerve with accompanying vasculature confirmed the diagnosis of PAN. The patient was treated with steroids, monthly pulse dose Cytoxan, and aggressive physical therapy. Nine months later he shows marked improvement and no further neurologic insults. DISCUSSION: PAN is a systemic vasculitis involving small and medium sized arteries and is more common in middle-aged men. Any organ can be affected but skin, peripheral nerves, joints, intestinal tract and kidneys are most commonly involved. The onset of disease can be abrupt or gradual and usually involves symptoms such as fever, malaise, palpable purpura, infarctive ulcers, joint pain and livedo reticularis, although multiple mononeuropathies are the most typical neurologic manifestation. The sedimentation rate is usually elevated but, as in our case, can be completely normal. Antibody to hepatitis B surface antigen can be positive. Tissue biopsy confirms the diagnosis. Untreated the 5 year mortality rate is greater than 85%. High dose steroid therapy has improved the five-year mortality to 30 ± 45% and the addition of immunosuppressive has reduced relapses. Plasmapheresis offers no additional advantages. Treatment of any associated viral illness (hepatitis, HIV) should be part of the initial therapy.
RAPIDLY PROGRESSIVE CARCINOID SYNDROME IN AN IMMUNOSUPPRESSED MALE. J.R. Pederson 1 , J.J. Yium 1 , M. Panda 1 ; 1 University of Tennessee±Chattanooga, Chattanooga, TN LEARNING OBJECTIVES: 1. Recognize the increased risk for malignancy in an immunosuppressed patient 2. Recognize the signs and symptoms of carcinoid syndrome. CASE INFORMATION: A 61 year old male, status post renal transplantation in 1995 for chronic renal insufficiency, was admitted in 9/1999 with complaints of weight loss, diarrhea, anorexia and early satiety. The patient noted a gradual onset of symptoms since 1996, with worsening over a six month period prior to presentation. He was on immunosuppressive therapy since 1995, Physical exam revealed a cachetic white male with redness and telangectasias of the face and neck. His physical exam was otherwise unremarkable. CBC, electrolytes and liver function tests were all within normal limits. Ultrasound of the abdomen, CT pelvis and colonoscopy in 1996 and panendoscopy in 1999 were normal Work up was negative for malabsorption, H. pylori, Giardia and C.difficile. He was readmitted a month later for right hip fracture. He still had diarrhea and weight loss but also noted prominent bilateral facial, neck, and arm flushing. Punch biopsy from the skin of the neck was essentially unremarkable. Post-op CT of the abdomen revealed innumerable hepatic lesions in all lobes of the liver. Ultrasound guided liver biopsy revealed metastatic carcinoid tumors. Subsequent 24 hour urine collection revealed elevated 5-HIAA levels. The patient was treated with sandostatin and antihistamines. He rapidly deteriorated and died within several months of diagnosis. DISCUSSION: Carcinoid accounts for 30 ± 40% of all small intestinal tumors. Over 95% of all GI carcinoids occur in one of 3 sites: the rectum, appendix or small intestine (usually the ileum). Tumors less than 2 cm are less likely to metastasize. These tumors secrete vasoactive materials responsible for the clinical manifestations of carcinoid syndrome which is seen only in patients with hepatic metastasis. With advanced disease, treatment is symptomatic and true cure is seldom achieved. Rapid progression of indolent carcinoid has been reported inpatients who are immunosuppressed. As our patients preliminary extensive GI work-up did not reveal an obvious primary, we postulate that our patient may have primary hepatic carcinoid which rapidly progressed due to his immunosuppressed state. There has been only one case reported in the literature of primary hepatic carcinoid in a renal transplant patient. With the world wide increase in the incidence of tumors among immunosuppressed patients, physicians should have a low threshold for early thorough workup of suspected tumors. A 34 year old black male presented with a 2 week history of low grade fever, night sweats, generalized fatigue, weakness and myalgias. He had a waxing and wanning rash on his face and arms. Past medical history was significant for hypertension since age 26 and a similar rash and fever about 12 years ago. On examination he was febrile (temperature of 103F) and tachycardic. Physical examination was unremarkable except for a 2Â2cm right submandibular lymph node, a few lymph nodes in the right posterior cervical triangle and a hyperpigmented maculopapular skin rash involving the upper extremities and the face. The laboratory data showed WBC: 2th/mm3, Hbg./Hct.À8.4gm/dl 25.8%. The blood cultures, urine cultures, chest x-ray, imaging studies of the head, neck, thorax, and abdomen were all negative. ESR: 98, CRP 8.4mg/dl. Rheumatologic workup, viral and rickettsial serology, RPR and HIV were negative. Bone marrow biopsy and culture was also negative. The right submandibular lymph node biopsy showed histiocytic necrotizing lymphadenitis. He was started on Vioxx for the fever and myalgia. The patient's fever subsided after a total of 5 weeks. He returned with vision problems and was found to have retinal hemorrhages bilaterally with normal disc margins. Steroids were initiated for ocular symptoms with some improvement. DISCUSSION: Kikuchi's disease was first described in Japan in 1972 by Kikuchi and Fujimoto. It is a self limiting disease involving different ages, races and geographic regions. The etiology of this disease is uncertain, although its association with certain viruses like CMV, EBV, HSV, Parvo B-19, HIV, Yersinia and Toxoplasmosis has been described. These viruses or other antigens can induce immunological abnormalities in a susceptible individual with progression to autoimmune diseases like SLE and Still's disease. The most common clinical features of Kikuchi's disease are fever, weakness, fatigue, myalgia, arthralgia and lymphadenopathy (especially cervical). Skin rash is non-characteristic particularly involving the upper regions of the body. Hepatosplenomegly and neurological manifestations like aseptic meningitis and ataxia have also been described. However this is the first case described with retinal changes. Kikuchi's syndrome needs to be differentiated from other diseases like malignant lymphoma, SLE, tuberculosis, sarcoidosis, and cat scratch disease. As the course of Kikuchi's disease is generally self limited, and histopathology is diagnostic it is important to include it in the differential diagnosis of FUO.
A CHALLENGING CASE OF DYSPNEA IN A PATIENT WITH BREAST CARCINOMA. P.J. Bennett 1 , M. Panda 1 ; 1 University of Tennesee, Chattanooga, TN LEARNING OBJECTIVES: Use of appropriate diagnostic testing to differentiate the pulmonary tumor emboli syndrome from a thromboembolic event. Understand the diagnosis and management of pulmonary tumor emboli CASE INFORMATION: A 75 year old white female with a past history of poorly differentiated ductal adenocarcinoma of the right breast diagnosed in 7/95 presented with progressive shortness of breath for 4 ± 5 days. On exam, she was afebrile, tachypneic and tachycardic. She had decreased breath sounds bilaterally with crackles posteriorly in the right lower hemithorax. ABG revealed: pH 7.46, PCO2 26mmHg, PaO2 43mmHg, HC03 19meq, 82% saturation on room air. Chest films and CT were unchanged from previous of 8/98. There was no evidence of lymphangitic carcinomatosis. Echocardiogram revealed an EF of 72%, evidence of new moderate pulmonary hypertension and new tricuspid regurgitation, without tamponade. VQ scan revealed small unmatched peripheral deficits in the left upper lobe consistent with intermediate probability of pulmonary embolus. Subsequent pulmonary angiogram revealed no evidence of pulmonary embolism. Due to worsening hypoxia, bronchoscopy with transbronchial biopsy was performed. Biopsy revealed metastatic poorly differentiated carcinoma with prominent intravascular growth consistent with a breast primary. DISCUSSION: Pulmonary tumor embolism is more often a postmortem diagnosis in patients with known solid tumors such as breast, liver, prostate, and kidney. Potential mechanisms of tumor emboli to the lung include direct extension into the IVC, extension via the thoracic duct to pulmonary lymphatics or via the SVC & right side of the heart to the pulmonary arterioles. The tumor cells occlude the smaller arterioles without parenchymal involvement. Resulting intimal proliferation leads to increased pulmonary pressures and right ventricular strain. Progressive dyspnea is the most common symptom. Chest X-ray is often unremarkable. VQ scans may be normal, indeterminate, or reveal focal defects. Pulmonary angiography may only reveal slow blood flow. Occasionally, CT/MRI scans may reveal a tumor mass occluding the pulmonary artery. Diagnosis is proven by tissue biopsy. Treatment of patients with pulmonary tumor emboli is difficult. Tumor embolectomy may be an option in large proximal emboli. Chemotherapy and radiotherapy usually produce a poor response. Together with fat and amniotic fluid embolism, tumor embolism cause hypoxia with a normal pulmonary angiogram. As in our patient unrelenting hypoxia and new development of hypertension may be important diagnostic clues.`I A 40 year old white female with no significant medical history presented with a 5 month history of a slowly expanding rash on her lower trunk and proximal extremities associated with generalized fatigue. The rash was painless, nonpruritic and not associated with arthralgias, fever or chills. The patient admitted to taking several herbal medications which she had recently stopped. Her physical exam was unremarkable except for a pink, blanchable, nontender, confluent, macular rash involving the lower abdomen and proximal portions of the arms and legs. There were no bullae or pustules. Laboratory evaluation revealed a negative ANA, rheumatoid factor, SS-A and SS-B antibodies. The patient had normal levels of aldolase, CPK and sedimentation rate. Her CBC was unremarkable except for the presence of 26% eosinophils. A deep fascial biopsy of one of the left thigh lesions revealed an inflammatory infiltrate of the fascia consistent with eosinophilic fasciitis. Upon review of the patient's herbal medication list and available literature, the probable causative agents were glucosamine sulfate and the fever few leaf. The patient was started on steroids with slow improvement. DISCUSSION: Eosinophilia-myalgia syndrome was first described in 1989 when it was observed in 3 group of patients who developed scleroderma-like skin changes, myalgias, eosinophilia, and histologic findings similar to eosinophilic fasciitis after ingestion of Ltryptophan manufactured by a single company. However, as this case illustrates, other agents, including herbal medications can precipitate a similar syndrome. Other clinical manifestations include low grade fever, fatigue, dyspnea, cough, arthralgias/arthritis, erythematous rashes and myalgias. Myocarditis and pulmonary hypertension can occur. Usually significant peripheral eosinophilia is noted and striking tissue eosinophilia may be noted as well. Diagnosis is dependent on histologic findings from a deep biopsy including fascia and muscle. Response to treatment is variable but patients may respond to steroids, plaquenil, methotrexate, Dpenicillamine and cimetidine. The course of the disease is poorly defined but many patients spontaneously regress or remain unchanged for years. Use of herbal medications is becoming very common as these products are widely advertised and readily available. However clinical trails and adequate information on efficacy and adverse reactions are limited. This case stresses the importance of incorporating questions on use of herbal medications routinely on medication history and the need to have a hightened awareness of their possible implication in disease. A 74 year old previously healthy male presents complaining of a 2 month history of progressively worsening scrotal swelling. The patient denied urinary hesitancy, frequency, hematuria, dysuria, or incontinence, but admits to mild decrease in urine output with occasional urgency. Past medical history was negative, and the patient denied using any medications including NSAIDS. Physical exam was significant for marked scrotal, suprapubic, penile shaft, and foreskin edema with normal testicular size. No masses or hernias were detected. Routine labwork showed BUN 67, Creatinine 9.6. When seen for the same complaint 5 weeks earlier, the patient had normal electrolytes and creatinine 1.2. Foley catheter was successfully placed with approximately 50cc of residual volume obtained. Renal ultrasound showed mild to moderate right hydronephrosis, mild prominence of the left collecting system without hydronephrosis. Percutaneous nephrostomy of the right kidney failed to improve renal function, and hemodialysis was initiated. Left renal biopsy revealed normal glomerular structure. CT scan of pelvis showed calcification of the prostate, with thickened mucosa of the sigmoid colon and rectum and bilateral hydroceles. Prostate biopsy was negative for malignancy. Rectal biopsy revealed poorly differentiated carcinoma of probable uroepithelial etiology. After 7 days, urine output began to improve, and dialysis was discontinued. Hepatitis serologies, urine and serum protein electrophoresis, ANCA, and anti-GBM antibodies were negative. Cystoscopy showed bladder thickening with locally invasive disease and bilateral ureteral obstruction. Results of bladder biopsy showed high grade papillary transitional cell carcinoma with vascular invasion and extension to the pelvic wall and rectum. DISCUSSION: Approximately 4% of cases of renal failure due to obstructive uropathy present without dilation of the urinary tract. Of these cases, 60% are due to intrapelvic malignancy (prostate, bladder, colorectal). Proposed mechanisms for lack of dilation include ureteral encasement by tumor, ureteral edema, reabsorption, and interrupted peristalsis. Although renal ultrasound is often used to rule out urinary tract obstruction, the absence of hydronephrosis does not exclude obstructive uropathy as the cause of acute renal failure. In the presence of high clinical suspicion, additional studies such as cystoscopy, intravenous pyelogram, or retrograde pyelography must be pursued to evaluate patency of the upper and lower urinary tracts.
ACQUIRED HEMOPHILIA IN SLE. J. Pedersen 1 , J. Paty 1 , M. Panda 1 ; 1 University of Tennessee College of Medicine -Chattanooga Unit, Chattanooga, TN LEARNING OBJECTIVES: 1. Recognize the entity of acquired hemophilia secondary to antifactor VIII antibodies and its association with autoimmune diseases and occult malignancy. 2. Understand the indications for treatment of acquired hemophilia. CASE INFORMATION: A 76 year old white female with a past history of SLE and polymyalgia rheumatica, in remission on daily prednisone and Plaquanil, presented to the office with a one week history of spontaneous, extensive bruising of the right hip and thigh after arising from a chair. She denied trauma, a personal or family history of easy bruising or prolonged bleeding. Physical examination was unremarkable except for an extensive area of ecehymosis along the right inner and outer thigh. MRI revealed iliospoas hemorrhage. Initial lab revealed a normochromic, normocytic anemia with a hemoglobin of 6 (one week prior to admission her hemoglobin was 10). She had a normal platelet count, prothrombin, thrombin and bleeding times, but an elevated partial thromboplastin time (57.1 seconds). The PTT partially corrected with the addition of normal plasma, so Factor VIII assay was done which revealed decreased activity at 8% with positive titers for factor VIII inhibitor (72 Bethesda, n1 0.1 ± 0.8). Lupus anticoagulant and antiphospholipid antibodies were negative. Subsequently the patient had a spontaneous right retroperitoneal and left upper extremity hemorrhage and required factor VIII transfusions, high dose corticosteroids and oral cytoxan. Six months later she remains symptom free on 5mg of prednisone and Plaquenil 400mg daily. Her hemoglobin is 11.8; PTT-28.6 and there is no detectable Factor VllI inhibitor. DISCUSSION: Hemophilia A, as a result of Factor VIII deficiency, can be inherited or acquired. Acquired inhibitors to Factor VIII are endogenous IgG/IgM immunoglobulins which interfere with normal blood coagulation. Acquired Factor VIII inhibitors can occur in patients with inherited hemophilia A, postpartum women, patients with collagen vascular diseases such as SLE, or idiopathicafly, especially in older patients. They are also associated with occult malignancy such as lymphoma and solid organ tumors. The syndrome usually presents with significant bleeding and soft tissue ecehymoses with minimal or no trauma. Treatment of active episodes of bleeding may involve administration of exogenous porcine or human Factor VIII or treatment with activated prothrombin complex concentrates as in our patient. Prophylactic treatment with steroids, with or without immunosuppressive agents, is indicated in patients with significant bleeding or with high titers of inhibitor. year-old African-American woman with no significant past medical history presented to clinic with complaints of bilateral lower extremity swelling, easy fatiguability and generalized weakness of 2 weeks duration. She also reported increased sensitivity to cold, dryness of skin and menorrhagia. On exam, she was moderately obese, did not appear acutely ill. Vitals signs were normal. Physical exam remarkable for mild conjunctival pallor, soft heart sounds, slow relaxation phase of deep tendon reflexes and bilateral lower extremity non pitting edema. EKG showed low voltage complexes in all leads. Echocardiogram demonstrated large pericardial effusion with no tamponade. Labs revealed normocytic, normochromic anemia. Thyroid profile with TSH 2.07 (0.4 ± 6) and free thyroxine 0.30 (0.6 ± 1.7) pointed towards the diagnosis of central hypothyroidism. Other hormonal assays were done which also revealed coexisting adrenal insufficiency. MRI of brain showed flattened pituitary along the floor of sella and majority of the sella was filled with CSF.These findings were consistent with empty sella . No focal lesion in the pituitary gland were identified. Pt was diagnosed to have hypopituitarism due to primary empty sella syndrome . She was treated with thyroxine and steroids to which she responded appropriately. The pericardial effusion was presumed to be secondary to the hypothyroidism and pericardiocentesis was not performed as she was hemodynamically stable. DISCUSSION: Primary empty sella syndrome has been classically described in obese hypertensive women. It is characterized by the presence of an arachnoid herniation filled with fluid that compresses the pituitary against the sellar wall.It is often asymptomatic but may be associated with endocrine disorders. Less than 30 percent of these patients presents with symptoms suggestive of hormonal deficiency. Recent literature reports only about 10 percent of these patients present with hypopituitarism as in our patient. The most common causes of central hypothyroidism are pituitary adenomas,pituitary apoplexy, infiltrative lesions. Pericardial effusion is one of the common cardiovascular manifestation in hypothyroid patients but most of these cases are described in patients with primary hypothyroidism. There are only few case reports describing pericardial effusion as presenting manifestation in patients with central hypothyroidism. These pericardial effisions are usually large but rarely progress to tamponade. They usually respond well to thyroxine supplementation. for unexplained anemia (Hb 6 g/dl). The patient had a history of chronic diarrhea with weight loss secondary to a diagnosis of CVID in 1994. Multiple colonoscopies revealed only nonspecific colitis. CT scan showed extensive mesenteric lymphadenopathy and thickening of the small bowel wall. He underwent EGD which showed healed duodenal erosions with copious bile in the esopaghus and stomach. A small bowel follow-through revealed proximal bowel dilatation and a partially obstructing process in the mid-jejunum (see Figure) . Exploratory laparotomy with partial small bowel resection revealed malignant lymphoma (large B-cell type). Bone marrow biopsy was normal. Tests for HIV antibody were repeatedly negative. The patient was discharged for outpatient oncological treatment. DISCUSSION: GI lymphomas have been reported rarely in patients with CVID, but are not usually considered among the initial differential diagnoses. We present this case to call attention to non-specific symptomatic presentation of lymphomas of the GI tract (Lai Ping So and Mayer, Semin Gastrointest Dis 1997; 8:22; Gottesman et al., Leuk Lymphoma 1999; 32: 589) . Figure  Legend . Barium failed to progress rapidly to the distal jejunum (left panel), but was present after 15 minutes (right panel), indicating a partially obstructing process with considerable proximal bowel dilatation. An 84 year old woman with end-stage renal failure, congestive heart failure, insulin-dependent diabetes, depression, and osteoarthritis begins to express a desire to discontinue hemodialysis after many years. She feels sad, exhausted, and a burden to her family. The patient has been widowed for five years. Her medications include Celexa and Vicodin. She has four attentive children who all live in the same city. She is cared for primarily by her youngest daughter, who lives with the patient, administers medications and transports the patient to dialysis. Intent on caring well for her mother, and worried that pain was interfering with the patient's appetite, the patient's daughter has been administering increasing doses of Vicodin. While the patient has made a number of comments about discontinuing dialysis to each of her children, none of the children have talked with each other, hoping to avoid such discussions, as well as differences of opinion between them. The patient's thoughts have not been assessed during the last year by either her nephrologist or primary care physician. An endof-life consultation team met with the patient and her daughter. Clarification of the appropriate use of pain medicine allowed the patient to control her pain with only occasional use of Vicodin, leading to increased energy, independence, and enthusiasm. Assessment of the patient's CESD depression score showed an increase from 14 (six months earlier) to 22. The patient's Celexa dose was adjusted. The patient's daughter was invited to attend support classes for family taking care of patients with end-stage illness and grew more comfortable talking about her fears and sense of responsibility. With renewed energy and improved mood, the patient withdrew her desire to discontinue dialysis for now. In a family meeting, all four children were able to recognize their own feelings and to better coordinate care. DISCUSSION: Even among closely-knit families and well-supported patients, miscommunication is possible, especially around the highly-charged issues of advance care planning for patients with end-stage illness. Assessing for medication over-and underprescribing, and ruling out depression as an etiology for withdrawal of support requests, are good first steps during discussions. Understanding the motivations and influences of relevant family members is necessary as well. The key to good advance care planning is to begin the discussions well in advance of the decisions as there is often much to learn and minor clinical interventions can have major effects on planning variables.
HYPERTROPHIC GASTROPATHY -ANEMIA. S. Ramalakshmi 1 , J. Lloyd 1 , R. Gregorio 1 , H. Dubner 1 ; 1 UPMC Shadyside Hospital, Pittsburgh, PA LEARNING OBJECTIVES: Hypertrophic gastropathy is a diverse disease characterized by giant mucosal hypertrophy of gastric rugae. The disease is usually confined to gastric body and fundus and can manifest with hypoalbuminemia. We present a case of an extensive hypertrophic gastropathy involving the antrum and body of the stomach associated with iron deficiency anemia. CASE INFORMATION: A 41year-old male patient during a routine physical, with symptoms of fatigue was diagnosed with iron deficiency anemia. Physical exam was unremarkable with guiac negative stools. Hemoglobin 6.0, Ferritin 1, Protein 4 and Albumin .9. Work-up of anemia included endoscopy, which showed diffuse nodularity of the body and a nodular mass in the antrum, which obscured pylorus and extended into second portion of the duodenum. Biopsies of stomach and duodenum were inconclusive. Upper GI series confirmed endoscopic findings. Due to the extensive involvement of the lesion as well as uncertainty of the diagnosis, gastrotomy was done. Large polypoid lesions were seen in the gastric antrum prolapsing into the duodenum and were excised. The mucosal surface of the duodenum was normal. Pathology was consistent with hypertrophic gastropathy. Stains and serum antibodies were negative for H. pylori. Patient was treated with acid suppressive therapy and iron supplementation. On follow-up patient was free of symptoms with Albumin 3.7 and Hemoglobin 12.2. DISCUSSION: This case is a relatively new variant of the already known hypertrophic gastropathy, in regards to its site and clinical presentation.
HIV RELATED HEMOLYTIC UREMIC SYNDROME. Physical exam in the ER showed a distressed lady with tachycardia and tachypnea. She had petechiae over the legs, 3+ bilateral pedal edema and a right pleural effusion. Labs revealed severe anemia (hematocrit 21); normal platelet count (227k/ml), increased BUN and creatinine (81/5.3); increased LDH (579) and near normal PT and PTT (14.8/28.6) . Peripheral smear showed schistocytes. Factor V and VIII were normal. Hospital stay was initially represented by persistent anemia, worsening thrombocytopenia (down to 29k/ml), bleeding from multiple sites, worsening renal and pulmonary function. Neurological exam remained normal. LDH remained high whereas PT and PTT remained mostly unchanged. CD4 count and viral load sent at this time came back as 89 (Â106/L) and 42 (k copies/ml) respectively. A diagnosis of HUS was made on clinical and laboratory parameters. No known risk factors were identified. Patient underwent plasmapheresis and hemodialysis. Her condition however continued to deteriorate and she died seven weeks post admission. Autopsy confirmed HUS and ARDS. DISCUSSION: HUS has been seen mostly in terminal AIDS patients and thrombotic microangiopathy in AIDS patients is increasingly being considered a consequence of direct pathogenic effect of the virus over glomerular capillaries and arterioles. We were unable to identify any other risk factor for HUS in our patient as well. Prior to this admission our patient did not have any AIDS defining illness. We concur with others that HUS may be considered as an AIDS defining illness. Plasmaphersis was not associated with survival in our patient. The role of this mode of therapy in HIV patients needs to be studied further. MRI showed mild periventricular white matter disease and audiometric testing showed severe bilateral vestibular defects. In late 1997 she presented with recurrent pulmonary infections and was diagnosed with common variable immunodeficiency (CVID). In October 1998 she began having 10 ± 15 minute episodes of ascending right or left hand tingling and weakness followed by slurred speech and a reported mild facial droop that resolved with verapamil in December. In April 2000 she presented with declining memory, concentration problems and diarrhea. Workup revealed secondary adrenal insufficiency, hyperprolactinemia and decreased insulin-like growth factor 1 (IGF-). Extensive testing over six years included three head MRIs, two lumbar punctures, audiology testing, psychosocial evaluation, bronchoscopy, muscle biopsy, abdominal CT and numerous blood tests. Positive studies included high CSF protein of 64, oligoclonal bands, cortisol base 3.2 "g/dL (6 ± 25) after stimulation 14.6 "g/dL (!18 at 60 minutes after injection) and ACTH 5, prolactin 109ng/mL (1 ± 24) , and IGF1 50 ng/mL (71 ± 240). The lack of linear plaques and atrophy of the corpus callosum on the MRI scans were inconsistent with MS and neurology and otolaryngology consultants concluded that her progression of symptoms, imaging studies, and lab testing were most consistent with a diagnosis of Susac Syndrome. DISCUSSION: There have been less than 50 reported cases of Susac syndrome in the literature. This is the first reported case of immune deficiency and endocrine abnormalities accompanying Susac's syndrome which is described as a clinical course of hearing loss, encephalopathy, and visual changes consistent with a microangiopathy of the cochlea, brain and retina. High suspicion and extensive testing is required to rule out other etiologies of this triad of symptoms.
A REFRACTORY NEUTROPENIC PNEUMONIA. S. Regenbogen 1 , A. Rosen 2 ; 1 University of California, San Francisco, CA LEARNING OBJECTIVE: 1) Diagnose and treat Bronchiolitis Obliterans Organizing Pneumonia (BOOP) in patients with hematologic malignancies who present with pneumonia. CASE INFORMATION: A 59-year-old man with cryptogenic pancytopenia was admitted with 3 weeks of cough, fatigue and fever. Bone marrow was markedly hypocellular, but nondiagnostic. He had no hematuria, rheumatic symptoms, or toxin or radiation exposure, except for 6 months on a Naval nuclear submarine. He attributed his condition to myelosuppression from chronic ibuprofen use. Admission vitals were temp 39C, BP 133/71, pulse 78, 02 sat 97% on room air. He had no bleeding or bruising, and no lymphadenopathy. Lung exam showed crackles and egophony at the right mid-chest. Spleen and liver were not palpable. CBC showed WBC 0.4, Hct 34, Plt 102, and ANC 0.2. CXR showed dense right-sided infiltrates. Blood and induced sputum cultures were repeatedly negative and broncho-alveolar lavage was nondiagnostic. Despite treatment with broad-spectrum antibiotics and antifungals, his lung exam and CXR continued to worsen. His neutropenia persisted despite 3 weeks of stem cell growth factors. On day 15, immunoperoxidase staining confirmed the diagnosis of hairy cell leukemia. He underwent open splenectomy, which increasd his WBC to 3.13, and ANC to 2.35. Open lung biopsy (OLB) on day 22 was consistent with BOOP. He was treated with high dose steroids and discharged on a prolonged prednisone taper. DISCUSSION: BOOP is an inflammatory response to lung injury, with edematous plugs of granulation tissue and interstitial fibrosis. It is associated with infection, autoimmunity, hematologic cancers, and other conditions. Patients present with a subacute, febrile illness with dry cough and dyspnea that is unresponsive to antibiotics. CXR shows patchy alveolar infiltrates with airspace consolidation, and PFTs reveal a mixed restrictive/obstructive pattern. Transbronchial biopsy is often nondiagnostic, therefore requiring OLB for definitive diagnosis. BOOP is usually responsive to steroids, but relapse is common. In one series (Am J Resp Crit Care Med 161:723, 2000), among patients with hematologic cancers undergoing OLB for an unknown pulmonary process, BOOP was the most common finding±20% of cases where a diagnosis was made. BOOP accounted for 30% of the diagnoses missed by bronchoscopy but found on OLB. Identification of a specific diagnosis on OLB significantly increased survival. In the setting of refractory pneumonia in a patient with a hematologic malignancy, the diagnosis of BOOP should be considered. OLB is advised if other procedures fail to identify a cause. LEARNING OBJECTIVES: 1. Participants will learn to recognize skeletal muscles as a rare metastatic site for the cancers. 2. Participants will learn diagnosis and treatment of esophageal cancer. 3. Participants will learn the incidence, pathophysiology, diagnostic strategy and treatment of skeletal muscle metastasis. CASE INFORMATION: Introduction: Although comprising 50% of body mass with a very rich blood supply, skeletal muscle is rarely the site of metastatic disease with only 242 cases having previously been reported (1) . Although primary cancers of the lung, colon, genitourinary tract and blood are most frequently involved (1, 35) , gastroesophageal adenocarcinoma has never been reported. We report a case of thigh muscle metastasis from primary adenocarcinoma of the gastroesophageal junction. CASE REPORT A 71-year-old African American male presented with severe right thigh pain, causing him to be unable to walk. His medical diagnoses included Stage IV Adenocarcinoma of the Gastroesophageal junction, hypertension, COPD (chronic obstructive pulmonary disease), and GERD (gastroesophageal reflux disease). He was an active smoker and Ex-alcoholic. Physical examination was remarkable only for his cachectic state and an extremely tender right upper thigh. Despite NSAID (non-steroidal anti-inflammatory drug) and full dose narcotic analgesia, the patient continued to complain of severe pain in his thigh. Thigh X-rays showed no lytic or blastic lesions. Bone scan did not show any metastasis. A MRI was done and showed a deep 2 x 4-cm soft tissue muscle mass in the right thigh without bony involvement, just anterior and medial to the femur. Diagnostic considerations included a soft tissue metastasis or a sarcoma. A CT guided needle biopsy of the right thigh mass was positive for metastatic adenocarcinoma consistent with the primary esophageal cancer. Radiation therapy was started to the patient's right thigh with a good response. At the time of discharge, the patient was walking without pain receiving narcotic analgesia. Upon returning to the clinic after 2 weeks for follow-up, he continued to walk without difficulty receiving outpatient radiation therapy. DISCUSSION: The incidence of adenocarcinoma of the esophagus and esophagogastric junction has been increasing over the past 25 years (2, 3, 4, 5) , with cancer of the esophagus (used to be predominantly squamous cell carcinoma) ranking among the ten most frequent cancers in the world. Although direct muscle invasion by carcinoma is well recognized, distant metastasis to skeletal muscle is uncommon (6). The present case is unique in that the localized thigh pain was produced by metastatic involvement to the thigh musculature without any osseous or perineural lymphatic extension. Even though autopsy series report a 0.8% to 20% (8,9,10,11,12) incidence of microscopic intramuscular metastasis, only a few cases of visible metastasis to the muscle have been described in the literature (7, 13, 14, 15, 16) . Accordingly, muscle metastasis often remains asymptomatic often undetected by both physical examination as well as diagnostic imaging procedures. The reported incidence, therefore, might be infrequent because of either a lack of recognition, underreporting, or infrequent autopsy evaluation for muscle metastasis. (7). Furthermore, it may be that only a fraction of patients with metastases to muscle survive long enough to allow clinical detection (17). Just why metastases to skeletal muscle is so rare is still unknown (17, 18) . Multiple factors, such as blood flow, intramuscular blood pressure, blood flow per unit of weight (millilitres/minute per gram); local changes in pH, as well as local temperature distribution may be involved. (6, 9, 11, 19, 20, 21, 22) . Organs with a high incidence of metastatic carcinoma such as lung, liver and bone have a constant blood flow. Although equally rich in its vasculature, it has been suggested that the blood flow in skeletal muscle is variable, is under the influence of Betaadrenergic receptor control, and is subject to varying tissue pressure that may affect tumor implantation (20,23). Although some have suggested that protease inhibitors in the muscle's extracellular matrix may resist tumor cell invasion (24), it may be the production of lactic acid and other metabolites by skeletal muscle that inhibits tumor cell growth. (20, 18) Whereas two thirds of all cancers metastatic to muscle are carcinomas, about one third are from leukemias and lymphomas with rare cases originating from melanoma. (25). Accordingly, although factors in the recipient site may be responsible for the relatively low rates of metastasis to muscle, properties of the primary tumor may also be involved. Furthermore, the differentiation between a primary soft tissue sarcoma and metastatic carcinoma to muscle is important as the treatment and prognosis is markedly different. Although the presence of a soft tissue mass caused by metastatic carcinoma may be misdiagnosed easily as a soft tissue sarcoma on physical examination and imaging studies (10), the current literature does not provide any clinical or radiographic characteristics that helps distinguish the two (17). Whereas 50% of carcinomas and sarcomas metastatic to muscle occur in the lower extremity, a greater percentage of upper extremity (26%) carcinomatous metastases occurs then has been reported for soft tissue sarcomas (10%). In addition, most of the cases reported are located in one muscle group or in one part of the body (26, 27, 28) . Although various imaging studies were used to identify metastases to muscle, none were specific for differentiating among carcinoma, sarcoma, or other muscle disorders. Plain radiographs, radionuclide scanning, and angiography have not been beneficial in differentiating carcinoma from sarcoma (17) although it has been thought that MRI imaging is superior to CT scanning (29, 30) . CT guided fine needle aspiration provide a rapid, minimally invasive means of diagnosis (17). Treatment options include radiation therapy, surgery or a combination of the two. Reports suggest surgical resection followed by adjuvant radiation or chemotherapy provides excellent palliative results (31,32,33). Although solitary metastases less then 4 cm in diameter can be treated by excisional biopsy (34), other reports suggest a less invasive approach may be better due to a low incidence of functional disability and because of the poor survival of these patients. Surgical resection is often reserved for those lesions that fail to be controlled locally with radiation or when the tumor growth results in neurologic deficit. SUMMARY: Although metastasis to skeletal muscle is extremely uncommon, physicians must remain aware of its occurrence, as its detection often requires specific evaluation. Differentiation between a primary soft tissue sarcoma and metastatic carcinoma to muscle is important, as their treatment and prognosis are so markedly different. The current literature does not provide any clinical or radiographic characteristic (plane x-ray or bone scans) that distinguishes metastatic carcinoma to muscle from soft tissue sarcoma (17). MRI, however, appears promising, and it should be considered earlier in the diagnostic work up. Treatment continues to remain palliative as metastatic carcinoma to muscle continues to remain a late event, with an overall poor prognosis.
SHOULD WE SCREEN FOR HEPATOCELLULAR CARCINOMA? G. Roehrig 1 ; 1 UCSF Primary Care Internal Medicine Program, San Francisco, CA LEARNING OBJECTIVES: 1)Recognize screening methods for Hepatocellular carcinoma (HCC) and the evidence for or against them. 2)Identify who should be screened for HCC. CASE INFORMATION: A 46 year old woman with a history of injection (IV) drug use and alcoholism presents for establishment of primary care, without specific medical complaints. Her last IV drug use was over 10 years ago, is recently HIV negative, and doesn't recall hepatitis testing. She reports 15 yrs of intermittent heavy drinking. Her past medical history is also significant for Diabetes mellitus Type 2 and obesity. Her medications include methadone and metformin. Reveiw of systems: negative history of jaundice, ascities, bruising, or gastroentestinal bleeding. Exam: Obese woman with normal vital signs and no scleral icterus. She had normal cardiac and pulmonary exams and her abdomen was obese few spider angiomata. Initial lab values: Hepatitis C antibody positive, B negative, Hct: 35.8, Platelets: 109, INR 1.1, Albumin 3.0, AST: 94 (Normal < 40), ALT: 87 (Normal < 40), Total Bili 1.1, Alk Phos 174 (Normal < 130). Given the lab results and spider angiomata, an abdominal ultrasound (U/S) was ordered which showed signs of cirrhosis as well as a 1.4 cm liver``hemangioma''. An alpha-fetoprotein (AFP) subsequently obtained was 42.0 (normal < 20). The patient was referred to Hepatology, but then lost to follow-up. Repeat AFP 6 monts later was 316. An abdominal CT was obtained to better characterize the lesion showed growth to 2.7cm. A biopsy of the lesion was not done due to its location but a routine liver biopsy confirmed cirrhosis. The patient underwent transarterial chemoembolization for presumed HCC, her AFP at this time was 551. DISCUSSION: The major risk factor for development of HCC is cirrhosis, at a rate of 1 ± 7% per year. The most common etiologies of cirrhosis are viral Hepatitis (B or C) and alcohol. Data for time to development of HCC range from 15 ± 25 years. Survival rates are 50% at 3 years for resectable lesions and 20% at one year for non-resectable HCC. Screening tools: AFP > 20 sensitivity of 39 ± 70%, U/S = 58 ± 85%, CT = 46 ± 84%, AFP and U/S combined = 79 ± 90%. Unofficial screening guidelines, followed by many surveyed by Chalasani et al, aren't necessarily supported through evidence-based medicine (EBM). The 6 month screening periods initially arose from data on tumor doubling time. Currently, most hepatologits do every 6 month AFP or U/S in patients with documented cirrhosis, with CT imaging alternatively being used. A recent cost-effective analysis by Sarasin et al concluded that screening offered minimal survivial benefits for the majority. The most significant benefits were found in well-compensated Child's A cirrhosis, especially with resectable tumors. There is a logical argument for screening liver transplant candidates. In summary, there is currently no EBM to support screening beyond the select group of patients mentioned above. There is a need for more research to better establish the mortality benefit and cost-effectiveness of HCC screening. A 52 year old man with a history of low back pain and alcoholism presented to his PMD with severe low back pain. He was given Vicodin, but returned to clinic 5 days later with worsening pain, constipation, difficulty walking, and a fever. Pt was sent to a local hospital where he was found to be confused and febrile with bilateral chest infiltrates; last drink had been 2 days prior. He was treated for pneumonia and presumed alcohol withdrawal. 4/4 blood cultures grew out S. aureus, but TTE showed no valvular vegetations. 4 days later, pt was transferred to our hospital where he was febrile to 103, agitated, disoriented, unable to follow commands, but spontaneously moving all extremities. To the extent testable, neurologic exam was nonfocal except for marked low back pain on neck flexion and possible nucchal rigidity. He had no spinal tenderness. He was pan-cultured and a head CT showed no acute event. MRI and TEE were planned for the following morning to rule out SEA and endocarditis, respectively. Lumbar puncture was initially deferred because of concerns for possible SEA and because pt was already on meningitis-dose Nafcillin. The following morning (5 hrs after admission) the patient no longer spontaneously moved his lower extremities and his toes were upgoing bilaterally. Emergent MRI showed a large T9 epidural abscess with posterior and anterior extension. Patient underwent emergent neurosurgical evacuation and had slow but progressive improvement in lower extremity function over the next 2 weeks. One week after transfer to rehabilitation, he expired in the middle of the night. Autopsy showed reaccumulation of the epidural abscess with infarction of nearly half of his spinal cord. IMPLICATIONS/DISCUSSION: Spinal epidural abscess is a rare but serious infection with potentially disasterous consequences, as highlighted in this case. Entertaining the diagnosis early is the key to prompt discovery and treatment. Severe back pain in a febrile patient, particularly with other risk factors for SEA (IVDU, past back surgeries, bacteremia), warrants rapid workup. Spinal MRI should be done as soon as the diagnosis is entertained, ideally before any neurologic deficits develop. In most cases, spinal cord damage occurs suddenly secondary to cord infarction from thrombosis or interruption of the local venous or arterial supply rather than via cord compression. Management requires neurosurgical evacuation and antibiotics (etiology is S. aureus in > 60% of cases). Early surgical intervention ( < 24hrs) is associated with markedly better prognosis.
WHEN THE DATA ARE NOT ENOUGH: SHARED MEDICAL DECISION MAKING. A. Rosen 1 ; 1 University of California, San Francisco, CA LEARNING OBJECTIVES: 1) Increase awareness of decision analysis as a tool for shared medical decision making (SMDM), 2) Recognize the importance of patient preferences in making medical decisions. CASE INFORMATION: A 77yo male with paroxysmal atrial fibrillation and recent diagnosis of colon cancer with hepatic metastases presented to my clinic. His quality of life (QOL) and functional status were excellent and he desired no adjuvant chemotherapy unless his QOL significantly worsened. He presented questioning his need for coumadin. We constructed à`b ack-of-the-envelope'' decision tree to address this question.
In conjunction with a review of the literature and discourse with the cardiology and oncology services, his risk of significant bleeding on coumadin with known liver metastases was thought equal to his risk of stroke off coumadin. In discussing his preferences for health states, his fear of mental incapacity and other potential sequelae of stroke far outweighed his disutility for a bleeding complication of the coumadin. These preferences were so strong that coumadin remained the preferred treatment modality even when aspirin was presented as an option. While the patient initially desired cessation of coumadin, this decision changed following the presentation of a simple decision tree and open discussion of his preferences. DISCUSSION: Decision analysis allows for the implicit decisions made everyday in clinical medicine to be made more explicit. The process of SMDM is often very illuminating, as it encourages better communication between patient and physician as they consider the consequences of a particular medical decision together. These consequences include the probabilities of health outcomes and the values, or utilities, that the patient places on these outcomes. Explicit knowledge of patient utilities may have a major impact on the medical decision particularly in a setting such as this one in which quality of life may be valued over life expectancy. SMDM is an excellent way to engage patients in the important dialogue needed to best inform medical decision making. Heparin infusion was discontinued and warfarin was continued. On the same day he had a burning pain over his right shoulder and forearm. Day 6, well demarcated edema and erythema of his right shoulder and forearm and later purpura and skin necrosis occurred. Warfarin was discontinued and he was offered lepirudin. Biopsy of the skin confirmed anticoagulant related skin necrosis. Day 11, platelet count increased to 150. Platelet factor 4 antibody was positive confirming heparin induced thrombocytopenia (HIT). The liver biopsy showed metastatic adenocarcinoma. DISCUSSION: Warfarin skin necrosis is a rare complication but could lead to extensive skin loss with its associated problems such as is seen in major burns. The patient usually complains of pain of the affected area and this should prompt the physician to consider it in any patient on anticoagulation who has unexplained cutaneous pain. It may be minimized by discontinuing the warfarin and substituting with heparin, heparinoids or lepirudin if anticoagulation is needed. If INR is high, reverse anticoagulation. Avoid initial``loading'' dose of warfarin because this will lead to rapid depletion of protein C leading to a relatively hypercoagulable state. This risk appears to be particularly high in inherited hypercoagulable states and in patients with HIT. Heparin skin necrosis may occur with all types of heparin and could be at or away from the site of injection. Antibodies to heparin-platelet-factor 4 is suggestive. Give warfarin or lepirudin if needed. It is not possible to tell if this patient had warfarin or heparin skin necrosis. A method of distinguishing these two conditions is needed as treatment is different. week history of increasing confusion and 3 day history of right-sided frontal headache. His medications included thyroxine, oxybutynin, and theophylline. One week following recent knee surgery, he stated that his energy level was decreased and he was having persistent memory difficulties. Physical examination was unremarkable except for delirium and a resting tremor. The patient was alert, but oriented only to self and location. He had difficulty cooperating with the examination and was easily distracted from tasks. He was admitted to the hospital, where outpatient medications were restarted. Chemistry panel, CBC, LFT's, thyroid function tests, blood cultures, and cardiac enzymes were all normal. Theophylline level was high normal (19.9 mg/L). An EKG was read as sinus arrhythmia. A head CT showed ventriculomegaly with no intracranial bleed or mass. Results of lumbar puncture, EEG, MRI of the brain, and overnight pulse oximetry were nondiagnostic. On day 4 of hospitalization, confusion persisted. The patient experienced a 9 beat run of ventricular tachycardia and a cardiology consult was obtained. Review of EKG's and a rhythm strip revealed multifocal atrial tachcardia. A theophylline level was obtained and reported at 39 mg/L. Theophylline was discontinued. Over the next 7 days, the patient's mental status gradually cleared and he was discharged. DISCUSSION: Theophylline has a narrow therapeutic index (10 to 20 mg/L). Hypotension, cardiac arrhythmias, and seizures have been reported in association with toxicity. Levels often do not correspond to symptoms in chronic toxicity. Treatment is mainly supportive, although dialysis is often used in management of severe toxicity. The case illustrates: 1) the broad differential diagnosis and resource intensive evaluation of delirium in the elderly and 2) the insidious nature of chronic poisoning from a prescribed medication. A bladder scan demonstrated a residual urine of greater than 800 cc; bladder catheterization produced one liter of clear, yellow urine. Urinalysis was within normal limits. A review of the patient's medication list revealed that he had most recently been prescribed phenylpropanolamine and guaifenesin and had also been taking oxybutinin for``incontinence''. DISCUSSION: Benign prostatic hypertrophy (BPH) is a common problem, ranging from 40% ± 50% in males 51-60 years old, to over 80% in males above 80 years of age. Males with BPH have symptoms suggestive of overflow incontinence. Pharmacologic therapy includes antiadrenergic agents to assist with detrusor contraction and relaxation of the urinary sphincter. In addition, current literature provides some evidence for 5-alpha reductase inhibitors (finasteride) to decrease testosterone-dependent prostate gland enlargement. Phenylephrine, a popular over the counter and prescription medication, is commonly used as decongestant. It has been associated with acute urinary retention. It would follow that phenylpropanolamine (PPA), also a sympathomimetic, has similar capabilities. In this scenario, the patient's BPH in combination with PPA and the anticholinergic effect of the oxybutinin precipitated acute bladder outlet obstruction and anuria. Although PPA has been taken off the market recently, other sympathomimetic drugs can be inferred as equally hazardous in these patients. One of the many ways to prevent adverse outcomes from polypharmacy is to take a medication history every time one sees a patient and to be coginzant of potential adverse medication interactions. By reviewing all current medications, we may reduce iatrogenesis. DVT but an MRI demonstrated extensive soft tissue bleeding. Anticoagulation was withheld and her coagulopathy resolved as the hyperthyroid state was treated. DISCUSSION: Atrial fibrillation (AF) is a common arrhythmia, found in 1% of patients over age of 60. It is the most common cardiac condition associated with cerebral embolism. Underlying hyperthyroidism occurs in up to 12% of patients with this arrhythmia and therefore, a TSH is recommended in patients presenting with new onset AF. Our patient had an embolic right middle cerebral artery stroke due to AF and her AF was undoubtedly due to unrecognized hyperthyroidism. In addition, an atypical manifestation of hyperthyroidism was present. As has been reported in the literature, the hypermetabolic state of hyperthyroidism, exacerbated by a recent iodine load from her CT scan, led to consumption of clotting factors and a clinically relevant coagulatopathy which resolved with treatment of the hyperthyroid state. Finally, in this patient, the extrinsic compression of the venous system by the bleed was initially incorrectly interpreted as a DVT. Only by careful physical exam and attention to pathophysiologic and laboratory details of the case was potentially harmful treatment with heparin avoided.
PHEOCHROMOCYTOMA RELATED CARDIOMYOPATHY. Papules that progress to hemorrhagic pustules develop on the trunk and extensor surfaces of the distal extremities. Asymmetric joint involvement of the knees, elbows, wrists, MCPs and ankles occurs. The cutaneous and articular findings are due to an immune reaction to circulating gonococci and to immune-complex deposition. Synovial fluid cultures are consistently negative and blood cultures are positive in less than 45% of patients. If cultures are negative, alleviation of symptoms in 12 to 24 hours after antibiotic therapy supports a clinical diagnosis. Initial treatment is IV ceftriaxone. A similar arthritis-dermatitis syndrome may occur with N. meningitides. If cultures are negative, this must be considered in the differential. However, gonococcemia and meningococcemia respond to similar antibiotic treatment.
CASE INFORMATION: C.D. is a 44-year-old female with Grave's disease treated with propylthiouracil for two years. She presented with a 2 week history of productive cough, fevers, pleuritic chest pain and fatigue. She was diagnosed with right lower lobe pneumonia and admitted for IV antibiotics. Despite appropriate therapy, the patient's condition deteriorated and she developed hemoptysis and progressive hypoxemia that required intubation. CXR showed bilateral alveolar infiltrates. Bronchoscopy was consistent with diffuse alveolar hemorrhage. Admission CBC revealed pancytopenia. Her PTU was discontined. Additional labs included serum perinuclear antineutrophil cytoplasmic antibody (p-ANCA) > 160, proteinase 3 antibody 10 ( > 2 positive), myeloperoxidase antibody 8 ( > 6 positive), decreased C3 and C4, microscopic hematuria and negative ANA, anti-Smith, anti-RNP and HIV. The patient was diagnosed with p-ANCA vasculitis secondary to PTU and was treated with pulse dose steroids with stabilization of alveolar hemorrhage. She was extubated several days later. DISCUSSION: PTU is the most common antithyroid drug prescribed for Grave's disease.
There have been 5 reported cases in the English literature of PTU-associated ANCA-positive syndrome with the development of diffuse alveolar hemorrhage. All patients had a preceding influenza-like prodrome, and all improved with discontinuation of PTU. Two were treated with corticosteroids and cyclophosphamide, and one with steroids alone. The mechanism of PTUinduced ANCA-positive vasculitis is unknown. It has been proposed that activated neutrophils in the presence of hydrogen peroxide release myeloperoxidase (MPO) which converts PTU into cytotoxic products. Alternatively, in the presence of MPO, PTU is converted to PTU-sulfonate which is immunogenic for T cells. CASE INFORMATION: CASE: A 23-year-old G8P1 female with diet controlled gestational diabetes presented at 33 weeks gestation with dyspnea and productive cough. She was febrile, tachypneic and tachycardic. Her chest film revealed bilateral perihilar infiltrates and left lower lobe airspace disease. She was empirically treated for a community-acquired pneumonia. Initial laboratory results showed a HCO3 of 9, an anion gap of 20, and a glucose of 94. Urinalysis showed greater than 80 ketones and no glucose. Arterial blood gas was 7.32/18/68. Subsequent investigation revealed a lactic acid level of 0.6 and large serum ketones. Shortly after admission, the patient exhibited worsening respiratory distress and polydipsia. Repeat labs showed a HCO3 of 5, an anion gap of 24, a glucose of 113, and an ABG of 7.16/17/68. The patient was started on D5 1/2 NS and a continuous insulin infusion. Continued therapy led to resolution of the anion gap acidosis and clearance of serum ketones. DISCUSSION: Pregnancy is a state of relative insulin resistance marked by enhanced lipolysis and ketogenesis. Under certain circumstances, this hormonal milieu may lead to DKA. DKA can present differently in the pregnant patient, making the diagnosis difficult. The mechanism responsible for insulin resistance in pregnancy involves human placental lactogen, progesterone, cortisol, and prolactin, all of which impair glucose uptake by insulin sensitive cells. The cause of DKA with euglycemia is not well understood, but may result from constant fetal removal of glucose, the expanded blood volume of pregnancy, or the rapid clearance of glucose due to increased glomerular filtration. As seen in our patient, DKA in pregnancy may not present classically. As such, high clinical suspicion of DKA is required in ill gestational diabetics to prevent adverse maternal and fetal outcomes. LEARNING OBJECTIVES: 1). Review the newly described association of osteoporosis and HIV disease 2). Identify the secondary causes of osteoporosis in AIDS patients. CASE INFORMATION: A 39 year old man with AIDS presented to the emergency department with the acute onset of back pain over his thoracic spine. He denied trauma or radicular symptoms. His past medical history included Kaposi's Sarcoma of the lower legs and esophagus, CMV esophagitis, and adrenal insufficiency. He had received multiple antiretroviral regimens over the past 10 years. His current combination therapy included abacavir, lamivudine, and nelfinavir. His exam revealed marked tenderness over the thoracic spine from T4 to T10. His WBC was 3.1, hematocrit was 31%, electrolytes, renal and liver function tests were normal. A recent CD4 count was 10 cells/ul with a viral load of 85,000 RNA copies/ml. Spine radiographs revealed multiple new vertebral fractures at T5, T6 and T9 confirmed by MRI. Bone biopsy at T6, however, revealed normal cellularity without infiltrate, granuloma or obvious infection. A subsequent lumbar DEXA bone mineral density demonstrated a T score of -2.9, consistent with severe osteoporosis. Laboratory evaluation of secondary causes of osteoporosis demonstrated hypogonadism with a free testosterone of 0.8pg/ml (NL 47-244) and a total testosterone of 7ng/dl (NL 400-1080). The patient was managed with opioid analgesics, calcitonin, and calcium with vitamin D supplimentation. DISCUSSION: Metabolic bone disease may represent an important complication of HIV antiretroviral therapy. A few published reports have noted a prevalence of osteopenia or osteoporosis as high as 50% in some groups of HIV patients. This case, however, illustrates the multifactorial nature of osteoporosis and the importance of identifying all of the potential secondary causes. Since AIDS patients have a higher rate of GI malabsorptive syndromes, poor PO intake, immobility, and hypogonadism they are already at a substantial risk for osteoporosis regardless of the potential risk from antiretroviral therapies. As AIDS patients continue to live longer, the cumulative consequences of these risks may lead to a substantial rise in complications from osteoporosis. It is important for clinicians caring for HIV patients to be cognizant of this risk. Recognize accessory spleen as a rare cause of a pancreatic mass 3. Identify strategies to differentiate accessory spleen from a pancreatic hypervascular tumor CASE INFORMATION: A 41 year old female with a past medical history of uric acid nephrolithiasis presented to the Emergency Department complaining of right flank pain and blood in her urine. Her only medication was allopurinol 300 mg daily. Physical exam revealed right flank tenderness to palpation. A spiral CT of the abdomen revealed two renal calculi in the right and one in the left kidney. In addition, a mass was seen in the tail of the pancreas. A CT scan of the abdomen with contrast confirmed a 4.1 Â 4.8 cm hyperenhancing, hypervascular, well defined mass within the tail of the pancreas consistent with an islet cell tumor. Upon further questioning, the patient denied symptoms of peptic ulcer disease (gastrinoma), hypoglycemia (insulinoma), diarrhea and renal failure (VIPoma), hyperglycemia (glucagonoma), or diabetes, steatorrhea and cholelithiasis (somatostatinoma). A presumptive diagnosis of a nonfunctioning islet cell tumor was considered and the patient was referred to a surgeon. A distal pancreatectomy was performed and pathology revealed an intrapancreatic accessory spleen. DISCUSSION: Accessory spleen has been recognized as a rare cause of an intrapancreatic mass. It is seldomly detected clinically due to its lack of symptoms. Due to the rise in abdominal imaging procedures performed, though, the detection of accessory splenic tissue in the pancreas can be expected to increase. Its importance lies in the fact that it can mimic a pancreatic hypervascular tumor. Strategies to differentiate an accessory spleen from a pancreatic hypervascular tumor include angiographic demonstration of splenic blood supply, similar enhancement of the accessory spleen and the spleen on CT or MRI, radionuclide testing or biopsy. In this case, the possibility of an accessory spleen should have been considered and potentially could have avoided unnecessary surgery. (1) to recognize that thyrotoxicosis can potentiate warfarin's anticoagulant effects; (2) to manage warfarin therapy in thyrotoxic patients. CASE INFORMATION: A 59 yo woman with hypertension noted new-onset palpitations and "shakiness" at a routine clinic visit. She had no prior history of cardiac or thyroid disease and denied dizziness, loss of consciousness, chest pain, use of new medications, dietary supplements, drugs or alcohol. Current medications included benazepril and aspirin. Physical examination revealed a thin, anxious woman with a HR of 128 and BP of 100/60. She had a prominent, smooth, non-tender thyroid gland. Cardiopulmonary exam was notable for an irregularly irregular tachycardia, a normal S1 and S2 without gallops, and a II/VI short, systolic ejection murmur at the apex. An ECG showed rapid atrial fibrillation, prompting transfer to the emergency department, where work-up revealed a normal PT/INR, creatinine, and hepatic function. After cardiopulmonary stabilization, she was discharged on metoprolol and warfarin 2.5 mg/d. Subsequent TSH < 0.03 mIU/L, free T4 > 71 pmol/L, total T3 of 667 ng/dL, elevated anti-TSH receptors, and abnormal thyroid uptake scan confirmed Graves' disease. The patient's palpitations resolved, but anti-coagulation proved challenging, with INR' s above 4 on average warfarin doses of < 1.0 mg/d (Table I) . She exhibited exquisite warfarin sensitivity until her free T4 levels normalized three weeks after starting methimazole. DISCUSSION: Warfarin exhibits many drug-drug interactions, including an increased effect with l-thyroxin administion. Warfarin potentiation by endogenous thyroid hormone is a less appreciated phenomenon documented in case reports dating to 1972. As this patient's initial INR illustrates, thyrotoxicosis alone does not prolong bleeding times. Rather, thyroxin enhances the metabolic clearance of vitamin K-dependent clotting factors II, VII, IX, and X, resulting in relative hypoprothrombinemia and warfarin potentiation. As in this case, potentiation closely parallels free T4 levels and can occur after one dose. Thus, in thyrotoxic patients, appropriate initial warfarin doses may range from 1.0 mg QOD and require upward titration as hyperthyroidism improves. Awareness of such patients' potential enhanced warfarin sensitivity and judicious anti-coagulation may minimize hospital stays and prevent bleeding complications. A 17-year-old male presented to the emergency room with a history of myalgias for two weeks. Two weeks prior to admission he underwent a dental extraction that required general anesthesia with succinylcholine. He developed symptoms of myalgias four hours after procedure that persisted until readmission. While in the emergency room, he described severe muscle pain in the upper and lower extremities. Enzyme analysis revealed a creatine kinase of 22, 000, and he was admitted for rhabdomyolysis. The patient was treated with IV fluids and rest. His urinalysis was negative for myoglobinuria after hydration. He subsequently underwent a muscle biopsy that revealed a generalized myopathy. After hydration, pain control and initial rest, he gradually improved over the ensuing weeks. DISCUSSION: Growth hormone excess in childhood results in an uncommon condition of giantism. Gigantism causes inappropriate growth of both bones and muscles that results in a large individual. With the continued muscle growth, gigantism can cause an underlying myopathy. Succinylcholine provides muscle relaxation during anesthesia; however, it can cause rhabdomyolysis. This normally occurs in patients with an underlying muscle disorder. Succinylcholine may damage muscle in patients with muscular dystrophy or other primary muscle disorders. This is the first case of succinylcholine-induced rhabdomyolysis in a patient with primary myopathy from gigantism. While unusual, rhabdomyolysis from succinylcholine must be considered in all patients with potential myopathy. Treatment involves IV fluids and supportive care. Sugar water test was negative. Occult mediastianl lymphadenopathy was not found with contrasted chest CT. Bone marrow biopsy showed normocellularity with blasts of less than 5%, myeloid with left shift and one lymphoid aggregate, with adequate numbers of megakaryocytes. The iron was absent. CD59 and CD55 cell markers were normal. Comprehensive studies for lymphoma/leukemia were normal. Autoimmune pancytopenia was confirmed. He was treated with platelet pheresis transfusion initially, which hemolyzed shortly. He responded to IVIG and solumedrol with normalized WBC and platelet count. He was discharged home on prednisone taper. One month follow up showed normal WBC and platelet, but with persistent iron deficient anemia. Pt remained asymptomatic. DISCUSSION: There are three general causes of pancytopenia: splenic sequestion, insufficient production, and excessive destruction. In our case, drug-induced autoimmune processes were suspected after thorough work-up. Treatment for autoimmune hemolytic processes includes steroid, IVIG. If not effective, splenectomy may be an option. Immunosuppressive drugs such as azothioprine and cyclophosphamide, have been used with satisfactory results. Recently, we have had two patients present with autoimmune hematologic disorders within one to two weeks after using Levofloxacin. This association has not been reported previously in the literature. Submission of these cases may assist primary care providers recognize this adverse effect of Levofloxacin.
PROLONGED MOOD DISORDER AFTER 3,4-METHYLENEDIOXYMETH-AMPHETAMINE (MDMA)USE: A CASE REPORT. S.D. Thakur 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Recognize the potential for prolonged neuropsychiatric illness after methylenedioxymeth-amphetamine use. CASE INFORMATION: A 23-year-old male with no prior psychiatric history except polysubstance abuse, presented with sudden onset of anhedonia, mutism, avolition, feelings of guilt and worthlessness, and hypersomnia after an MDMA binge. The patient had severe paranoid delusions about eating and ultimately quit eating altogether. Hospitalization and a feeding tube were required for his malnutrition. The patient was diagnosed with major depressive disorder with psychotic features, and was started on venlafaxine, olanzapine, haloperidol, and benztropine. His symptoms have continued to persist for several months. He was discharged once but decompensated and attempted suicide within the week. Upon rehospitalization lithium augmentation therapy was implemented. It is postulated that his prolonged psychiatric illness was precipitated by his MDMA use. MDMA ingestion has been linked to acute psychosis, mood disorder, memory and cognitive disorders. Recent reports have implicated its involvement in prolonged mental disturbances as well.
MDMA is thought to cause potent and possibly permanent neurotoxicity in the serotonin (5HT) pathways. These pathways are the same ones that underlie chronic neuropsychiatric illnesses such as major depressive disorder, psychosis, and panic disorder, supporting the view that MDMA can lead to prolonged neuropsychiatric problems. As in our patient, recovery from the neuropsychiatric complications of MDMA tends to be slow and tedious. Improvement may be explained by compensatory metabolic changes within remaining 5HT and non 5HT neurons, or regeneration of damaged neurons. These mechanisms serve to reverse the 5HT deficit and lead to clinical improvement. MDMA has gained popularity recently as a recreational drug in the high school and college populations. It is commonly referred to as``ecstasy'',``X'',``E'', or``Adam''. It has hallucinogenic and stimulant properties. Due to its potential for severe and chronic complications, it must be considered in the differential diagnosis of new onset psychiatric illness, especially in adolescents and young adults. DISCUSSION: He was discharged home after alcohol counseling. DISCUSSION: Hyponatremia is the most common electrolyte abnormality in hospitalized patients. Symptoms are related to CNS dysfunction and include headache, nausea, vomiting, anorexia, disorientation and depressed reflexes. Symptom severity is correlated with how rapidly the hyponatremia developed. Coma and seizures can occur with acute drops to less than 120 meq/L. The initial approach to diagnosis is measurement of serum and urine osmolality and urine sodium. The most common finding is hypotonic hyponatremia, which is further subdivided by volume status. Our patient was hypovolemic, and low urine sodium implied appropriate renal sodium retention. The diagnosis of beer potomania was also considered, although this is classically a euvolemic state with a low urine osmolality. Treatment should be initiated in all symptomatic patients and those with levels less than 120mEq/L. For hypovolemic hyponatremia, the treatment of choice is normal saline. Overly rapid correction is avoided to prevent central pontine myelinolysis. year-old male with a longstanding history of steroid-dependent asthma and recurrent sinusitis presented with bilateral upper extremity weakness, beginning during a steroid taper. Symptoms then progressed to generalized weakness, malaise, anorexia, and a peripheral eosinophilia. He was given stress dose steroids with prompt resolution of both symptoms and eosinophilia and kept on prednisone 10 mg a day. He then began to develop wasting of his forearms, difficulty in performing tasks using his hands, and difficulty arising from a chair. Medications included salmeterol, fluticasone 220 mcg MDI, and prednisone. On physical examination, he was cushingoid. Lungs were clear. Pertinent neurologic findings included moderate proxminal muscles weakness and motor weakness with atrophy in distribution of the left and right median and ulnar nerve, left radial nerve and bilateral peroneal nerves. Deep tendon reflexes were absent in the right biceps, brachioradialis and left achilles. White count was 22,000 with 30% eosinophils. ACTH stim test was consistent with adrenal insufficiency. Chest X-ray was clear. EMG was consistent with a mixed motor and sensory loss in the ulnar and median nerve distribution. Peroneal muscle biopsy showed eosinophilic infiltration of a blood vessel consistent with a vasculitis. DISCUSSION: Our patient presented with adult-onset asthma, peripheral eosinophilia, and mononeuropathy multiplex. These features are consistent with Churg-Strauss Syndrome (CSS). The muscle biopsy showing eosinophilic infiltration of a blood vessel ensured the diagnosis. The American College of Rheumatology has developed classification criteria by comparing 20 patients who had CSS to 787 control patients with other forms of vasculitis. The six criteria more typical of CSS include: asthma, eosinophilia of greater than 10% , mononeuropathy or polyneuropathy, nonfixed pulmonary infiltrates on CXR, paranasal sinus abnormalities, and biopsy containing a blood vessel with extravascular eosinophils. The presence of any four or more yielded a sensitivity of 85% and a specificity of 99.7%. The natural history of the syndrome follows three clinical phases: (1) prodromal allergic phase (2) allergic rhinitis, nasal polyposis, and asthma and (3) peripheral and tissue eosinophilia. The differential diagnosis includes Wegener's granulomatosis, hypereosinophilic syndromes, polyarteritis nodosa, and microscopic polyangiitis. LEARNING OBJECTIVES: 1) Recognize that an asymptomatic mass on an extremity can be a soft tissue sarcoma. 2) Consider an incisional biopsy in an extremity lesion, particularly if it is larger than 5 cm in diameter. CASE INFORMATION: A 32-year-old African American woman with no significant past medical history presented with a 4 week history of right thigh pain, swelling and hemoptysis. She noted that her right thigh had been swollen without pain for several weeks prior to her presentation. On examination she had a swollen, tender, firm right thigh with right inguinal adenopathy. She had no skin lesions, clear lungs, and a normal pelvic exam. A pap smear was normal. Laboratory data were notable for a hematocrit of 27.7% and a serum iron of 24 ug/dl. Doppler ultrasound studies of the legs were negative for deep venous thrombosis. Chest x-ray showed multiple pulmonary nodules of variable sizes in both lungs, confirmed by a CT scan of the chest which also showed no mediastinal or hilar lymphadenopathy. MRI studies revealed a 6.5Â4.5Â4.5 cm soft tissue mass in the right thigh with lymphadenopathy in the right inguinal, external iliac and common iliac chains, and retrocaval region at the level of the renal veins. A biopsy of the right thigh mass revealed sheets of large monomorphic epithelioid cells which were positive by immunohistochemical studies for cytokeratin and vimentin. These findings were consistent with a diagnosis of epithelioid sarcoma metastatic to the lungs. DISCUSSION: Soft tissue sarcomas account for fewer than one percent of all malignancies and often present as an asymptomatic mass on the extremities of young adults, most often in the third decade of life. Fewer than 20% present as metastatic disease. Because of their slow-growth (over months) and initial presentation, they may be mistaken for other entities (eg. lipomas). Compression of a nerve or blood vessel can cause symptoms. Sarcomas usually spread hematogenously to the lungs, although epithelioid sarcomas can also metastasize through the lymphatic system. Patients with soft tissue sarcomas < 5 cm in diameter have a metastasis-free 5year survival rate of 81%, regardless of tumor grade, depth, and location. In contrast, there is only a 20% 5-year survival rate for patients with lung metastasis. Diagnostic workup should include radiographs, MRI, referral for an incisional biopsy, and chest x-ray or chest CT to search for possible metastatic disease. Because sarcomas are rare and histological interpretation can be difficult, a primary care physician should refer patients to surgeons or centers experienced in the diagnosis of soft tissue sarcomas. The early consideration of this diagnosis can greatly improve patient outcome.
HYPOGLYCEMIA PRESENTING AS BIZARRE BEHAVIOR. On the day of admission, he was found in his home unresponsive. Emergency medical services were called, and they found his glucose to be 34. Glucose was administered en route to the hospital with an increase in his glucose to 154 and return of consciousness. He had no history of diabetes and denied use of exogenous insulin. Physical exam at the time of admission was normal. Routine labs were all normal except for a glucose of 52. The patient was admitted for a supervised 72-hour fast. Several hours into the fast, the patient's accucheck was < 50, and he again began to exhibit bizarre, child-like behavior. Stat chemistries, serum insulin and C-peptide level were drawn revealing a glucose of 36, insulin 97.1 mU/ml and C-peptide 7.6ng/ml, values consistent with excess endogenous insulin production. A sulfonylurea level was ordered, and a CT scan was scheduled. The CT revealed a 5Â2.5 cm mass in the body of the pancreas and numerous masses throughout the liver. DISCUSSION: This case illustrates the importance of recognizing the signs and symptoms of hypoglycemia in the context of a low glucose value. Low glucose without neuroglycopenia or sympathoadrenal activation may not require further evaluation. However, the presence of these symptoms in an otherwise healthy-appearing, non-diabetic patient with low glucose necessitates a work-up for a hypoglycemic disorder. Careful measurement of insulin, C-peptide, glucose, and sulfonylurea levels can quickly narrow the differential. Whipples Triad (symptoms of hypoglycemia, a glucose of < 50, and relief of symptoms with glucose) is present in the initial presentation of most hypoglycemic disorders.
CEREBRAL HERNIATION AND FATAL HYPERAMMONEMIA WITH NORMAL HEPATIC FUNCTION. R. Tripathi 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Determine differential diagnosis and treatment of severe hyperammonemia and microvesicular steatosis, 2) Describe the mechanism of action of a previously unreported enzyme deficiency (Hepatic Glutamine Synthetase Deficiency) that has fatal consequences, and 3) Discuss appropriate screening for this deficiency. CASE INFORMATION: A 60 year-old white male with a past medical history significant only for chronic obstructive lung disease was admitted to the hospital with shortness of breath and pneumonia. He was treated with piperacillin/tazobactam, steroids and inhalers. On day 2 of hospitalization, the patient developed mental status changes and had an ammonia level of 41. By day 3 he developed seizures, and on day 4 he was comatose with an ammonia level of 2808. At that point, family withdrew all support. Throughout his course, the patient had essentially normal liver enzymes. Autopsy showed pulmonary embolism with infarction, ischemic colitis, cerebellar tonsil herniation, and multifocal areas of microvesicular steatosis in less than 50% of liver. DISCUSSION: Microvesicular steatosis has a limited differential including Reye's syndrome and it is usually not associated with normal liver function enzymes and tremendous increases in ammonia. Similar histopathological and clinical findings as in our patient have been described in patients with heart-lung transplantation. A case series of these orthotopic lung transplant patients with fatal hyperammonemia have shown them to have a hepatic glutamine synthetase deficiency with normal urea cycle and liver function enzymes. The increased ammonia is shunted to the cerebrospinal fluid. Initial increase in ammonia is thought to be secondary to an increased protein load (i.e. gastrointestinal bleeds, TPN) and/or increased catabolic processes (i.e. major surgery). Patients with intact glutamine synthetase enzymes are able to handle this increased ammonia production and there is a pulmonary process in patients with the deficiency for clearing the excess ammonia. A combination of hepatic enzyme deficiency, increased protein load and acute pulmonary disease could be postulated to be the cause of rapid hyperammonemia, cerebral herniation and death in our patient. This is a recently reported entity that may have been underdiagnosed in the critical care setting in the past. In the future, screening for this disorder may be important before patients are candidates for heart-lung transplantation or other pulmonary surgery. It may be important currently to screen patients with mental status changes and no known liver disease with ammonia levels because the only proven treatment modality for severe hyperammonemia is emergent hemodialysis. LEARNING OBJECTIVES: Identify the causes of acute paralysis in the patient who uses drugs.Illustrate the importance of using the history and physical exam to quickly discern the correct diagnosis while definitive studies are pending CASE INFORMATION: A 20 year-old man presented with acute onset of bilateral lower extremity weakness and anesthesia shortly after arriving in New Orleans. He reported passing out for 12 hours after injecting heroin and cocaine. When he regained consciousness, he could not ambulate and had to pull himself to the telephone to call for help. On physical exam, he was afebrile with normal vital signs. He had no heart murmurs and there were no skin ulcerations. He had bilateral loss of sensation in the L4-S2 distribution. He was unable to dorsiflex, plantarflex, invert or evert his ankles. Patellar reflexes were normal; ankle reflexes were absent bilaterally. Flexion of the hips and knees, and sphincter tone were normal. The remaining neurologic exam was normal. The urinalysis showed large blood, but few red blood cells. His serum creatinine was 4.1 mg/dL and the creatinine kinase was 43,900 U/dL. All other laboratory values were normal. The patient was diagnosed with rhabdomyolysis and a compression neuropathy of the peroneal nerves. He received aggressive intravenous hydration; his creatinine decreased to 1.3mg/dL at discharge. He regained sensation and progressive motor function in his lower extremities. DISCUSSION: Acute paralysis is a medical emergency, which requires prompt diagnosis and treatment. The differential diagnosis in the intravenous drug user includes wound botulism, paradoxical embolism with stroke, HTLV-1 with spastic paraparesis, spinal cord compression from epidural abscess, nerve compression and rhabdomyolysis. The history and physical exam can quickly discern the correct diagnosis while definitive studies are pending. The abscence of skin wounds and cranial nerve deficits excluded botulism; the distribution of nerve deficits were inconsistent with a cerebral stroke and there was no murmur on cardiac exam. The time course for the paralysis was too rapid for HTLV-1 infection, and the normal sphincter tone and hip function argued against spinal cord compression. Deductive reasoning led to the diagnosis of nerve compression and rhabdomyolysis, which was confirmed by laboratory testing. Upon further questioning, the patient reported finding himself with his buttocks on the floor, his knees draped over the metal railing of the shower doors and his torso slumped over his lower extremities. This resulted in compression of the peroneal and tibial nerves proximal to the popliteal fossa with subsequent paralysis.
SHOULD WE SCREEN YOUNG HYPERTENSIVE PATIENTS WITH ACUTE ISCHEMIC STROKES FOR HYPERHOMOCYSTEINEMIA? L. Vaidyanathan 1 , K. Barnard 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: There is data suggesting that increased plasma homocysteine confers an independent risk for vascular disease (cardiac, cerebral and peripheral). It also (powerfully) increases the risk associated with smoking and hypertension. Therefore, consider obtaining homocysteine levels in young patients with strokes despite the presence of other risk factors. CASE INFORMATION: A 40 year old hypertensive African American male was admitted to the hospital with sudden onset right sided weakness and slurred speech of 8 hours duration. Past medical history includes hypertension (diagnosed at 24 years). Patient is a nonsmoker. Initial evaluation revealed a blood pressure of 200/110 mmHg, right upper and lower extremity weakness with an upgoing right plantar reflex and dysarthria. MRI of the brain showed small vessel ischemia in the lenticulostriate distribution in the posterior limb of the left internal capsule extending into the anterior aspect of the thalamus and corona radiata. EKG showed normal sinus rhythm and there were no arrhythmais on the monitor. Transthoracic echocardiogram only showed left ventricular ejection fraction of 50%. Carotid and transcranial Dopplers were normal. Fasting homocysteine level was increased at 14.4 Umoles/liter (reference < 9Umoles/liter). DISCUSSION: More data is required to determine if hypertensives and smokers under 60 years with acute ischemic stokes, should be screened for hyperhomocysteinemia and if treating these patients with folate and vitamin B6 will decrease their risk of a recurrent cerebrovascular event. We treated our patient with asprin, folate and Vitamin B6 for secondary stroke prevention. LEARNING OBJECTIVES: 1. Recognize the increasing prevalence of Tuberculosis (TB) in the U.S. due to the immigration of high-tech workers. 2. Highlight the diagnostic dilemma a physician can face when the traditional work-up for TB is negative. 3. Highlight the Adenosine Deaminase Test (ADA) as a quick and highly sensitive and specific test to aid in the diagnosis of TB. CASE INFORMATION: A 26-year-old high-tech worker from Madras, India presented with new, severe headache. He denied cough or constitutional symptoms. The patient had a history of``Intestinal TB'' two years prior, with 16 months of medical therapy, the details of which were not known at the time of admission. His physical exam was completely normal, his chest X-ray showed a diffuse miliary pattern, and a head CT showed multiple ring-enhancing lesions. An extensive work-up for TB was undertaken, all of which was negative. Additionally, tests for fungi, HIV, bacteria, viruses and malignancy were negative. The patient's headache continued to worsen, so anti-tubercular therapy was started along with prednisone to cover for possible neurosarcoidosis. The patient did quite well and was discharged home soon thereafter. One week later, the patient returned with altered mental status. A new CT scan showed massive hydrocephalus. A ventriculostomy was placed and the CSF was analyzed for TB; the AFB smears and PCR were once again negative. An Adenosine Deaminase Test of the CSF was positive. Treatment was initiated to cover for multi-drug resistent TB. An open lung biopsy was performed to harvest tissue for culture and a few AFB were identified by smear. The original sputum culture finally turned positive for TB eight weeks after the patient's initial presentation. The patient improved slowly over the next several weeks and was eventually discharged in stable but neurologically impaired condition. DISCUSSION: CNS TB is uncommon, but is in the differential diagnosis of ring-enhancing lesions. It occurs in only 10% of immunocompetent patients and is highly lethal with mortality exceeding 25%. Even if a patient has widespread TB, the diagnosis of TB can be very difficult and require an extensive, invasive investigation. The ADA test is highly sensitive (100%) and specific (99%) for TB when compared to culture-proven disease in the CNS and lungs. It is an excellent test to aid in the diagnosis of TB and should be considered in the routine work-up whenever CSF or pleural fluid are analyzed.
WHEN NEW ONSET POLYURIA AND POLYDIPSIA DOES NOT EQUAL DIABETES. E.W. Vogel 1 ; 1 MCP Hahnemann University, Philadelphia, PA LEARNING OBJECTIVES: 1. Recognize severe hypercalcemia as a cause of polyuria and polydipsia, and check for hypercalcemia in patients with polyuria when diabetes has been ruled out. 2. Recognize parathyroid carcinoma as a cause of severe hypercalcemia. CASE INFORMATION: A 37 year old African American female with no significant past medical history presented with a two to three week history of increasing thirst, leading to intake of approximately three liters of water per day, associated with urination every 30 minutes. She also noted some polyphagia, dizziness, and mild blurred vision. She had a strong family history of type 2 diabetes, with her mother, brother, and uncle all having diabetes. On a brief initial physical examination, no significant abnormalities were detected. Somewhat surprisingly, her office finger stick glucose measurement was only 106 mg/dl. A basic sevenitem chemistry panel, urinalysis, and hemoglobin A1C were sent to the lab from the office, and the patient was released to home. Although not specifically ordered initially, a calcium level included with the chemistry panel came back at 14.5 mg/dl; her glucose and hemoglobin A1C were normal. The patient was admitted to the hospital for treatment, and on reexamination was found to have a hard left thyroid mass. Her parathyroid hormone level was markedly elevated at 485 pg/ml, with normal thyroid function tests. After resolution of her hypercalcemia with intravenous normal saline and pamidronate, she underwent surgical neck exploration, and was found to have a tumor extending from her thyroid to local skeletal muscle and the esophagus. Pathological examination revealed parathyroid carcinoma. The patient later was found to also have metastatic disease in her lungs. Despite primary tumor resection, local neck irradiation, chemotherapy, and multiple outpatient doses of pamidronate, the patient has required several admissions for management of recurrent, severe hypercalcemia. DISCUSSION: Severe hypercalcemia can have a number of clinical manifestations, including polyuria and polydipsia. Interestingly, this patient manifested none of the more common findings of hypercalcemia at the time of presentation, such as bone pain, renal colic from nephrolithiasis, or gastrointestinal or neurological symptoms. Clinicians should therefore consider hypercalcemia in the differential diagnosis of polyuria, especially when diabetes has been ruled out. In this case, the serum calcium was not ordered initially, but luckily this laboratory routinely includes a calcium level with the chemistry seven panel, which established the diagnosis. Parathyroid carcinoma is a rare cause of hypercalcemia; an underlying parathyroid cancer causes only 1 to 2 percent of cases of primary hyperparathyroidism. This patient had two of the warning signs for parathyroid carcinoma as the cause for primary hyperparathyroidism: severe hypercalcemia at the time of presentation ( > 14 mg/dl), and a palpable neck mass. This patient also typifies the clinical course of parathyroid carcinoma that is not cured by primary surgery, in that she has developed recurrent episodes of severe hypercalcemia. This persistent hypercalcemia eventually results in renal and cardiac complications, accounting for most of the morbidity and mortality associated with this disease.
NOT ALL VOMITING IN PREGNANCY IS HYPEREMESIS. S. Vora 1 , R.O. Powrie 1 ; 1 Brown University School of Medicine, Providence, RI LEARNING OBJECTIVES: 1. Recognize the differential diagnosis of hyperemesis gravidarum and illustrate the role of a general internist in diagnosis and management of medical illness in pregnancy. 2. Demonstrate the clinical presentation of mesenteric thrombosis. 3. Emphasize the fact the pregnancy is a hypercoaguable state and briefly outline the management of thrombosis in pregnancy. CASE INFORMATION: A 37 year-old primigravid Nigerian woman presented in her 19th week of an IVF pregnancy to the emergency room with complaints of two days of fever and constipation and three weeks of malaise, anorexia, and vomiting. She had been treated in the ER on three prior occasions with hydration and antiemetics for hyperemesis gravidarum. On this last presentation, she produced her photograph from one month prior to demonstrate the extent to which her appearance had deteriorated. Her past medical history was significant for only childhood malaria and sickle cell trait. She had not suffered from protracted nausea and vomiting of pregnancy during the first trimester. She was taking only prochlorperazine suppositories at the time of presentation. She had no personal history of smoking, alcohol or recreational drug use. Her examination was significant for a temperature of 101.6 F, a heart rate of 114 per minute, respiratory rate of 12 per minute, and BP 103/60. She was mildly jaundiced with sunken orbits. Her cardiopulmonary examination was normal. Her abdomen was markedly tender in the right upper quadrant with voluntary guarding but no rebound. No fluid wave was present and her rectal examination was normal. Her uterus was gravid and nontender to palpation. Her fetal heart tracing was reassuring. Neurologic examination was unremarkable. Her laboratory examination was remarkable for a leukocytosis of 36.6 with a left shift. Her chemistries revealed a hypochloremic metabolic alkalosis with a serum bicarbonate of 34 mEq/ L. She had a mild elevation of her serum AST to 40 U/L. The remainder of the laboratory data was unrevealing. A malarial blood smear was negative. An abdominal ultrasound revealed absent portal venous flow, but was otherwise normal. A subsequent CT scan of the abdomen demonstrated thrombosis of the portal vein and superior and inferior mesenteric venous branches. The patient was admitted, treated with intravenous unfractionated heparin, gentamycin and piperacillin and achieved significant improvement in pain. She defervesced by the second hospital day. Her liver enzymes normalized. Her hypercoaguable work-up revealed only a relative protein S deficiency. Once therapeutic on intravenous heparin, she was changed to low molecular weight heparin, which she continued until 36 weeks gestation. At that time her therapy was changed to adjusted dose subcutaneous unfractionated heparin until her uncomplicated cesarean delivery of a healthy 2475 g girl. DISCUSSION: Nausea and vomiting occurs frequently during pregnancy, especially during the first 20 weeks, afflicting 70-90% of all pregnant women. Causes can be physiologic, due to progesterone effects on the lower esophageal and pyloric sphincters, or due to mechanical compression of the diaphragm on the enlarging uterus. However pregnant women are also subject to all other causes of nausea and vomiting, such as severe gastroenteritis, cholecystitis, pyelonephritis, hyperthyroidism, primary hyperparathyroidism, liver dysfunction, and rarely, small bowel obstruction, mesenteric thrombosis or mesenteric ischemia. Internists can be instrumental in assisting in these less common medical diagnoses during pregnancy. Portal and mesenteric venous thromboses are rare conditions, even in nonpregnant patients. Most patients present with at least two weeks of abdominal pain, anorexia, vomiting, and change in bowel habits. Many patients are also febrile. In 20% ndash;40% of cases of mesenteric thrombosis no underlying cause can be found. Other investigated cases of mesenteric thrombosis during pregnancy have revealed inherited thrombophilias such as activated protein C resistance, antithrombin III deficiency, and protein C and S deficiency. The latter is a difficult diagnosis in the face of both active thrombosis and in pregnancy, since protein S normally decreases in both of these settings. The treatment of acute thrombosis involves heparin (either low molecular weight or unfractionated), as warfarin is contraindicated in pregnancy. Special care is required in the management of anticoagulation in the peripartum period. Recognize the importance of ductography and fiberoptic ductoscopy, the later being a new technology for nipple discharge. CASE INFORMATION: A 45-year-old woman presented with a 1 day history of a spontaneous amber right nipple discharge. Breast examination revealed fibrocystic changes. A small amount of clear, non bloody fluid could be expressed from one of the right breast ducts. There was no palpable lymphadenopathy. A mammogram revealed fibroglandular densities. Ultrasound (US) of the right breast showed slightly dilated ducts. Ductography was performed as follows: by applying focal pressure a single draining orifice was identified, cannulated and 0.2 cc of Conray 60 were injected. Post-mammographic images showed the presence of a cystically dilated duct that contained a small 3 ± 4 mm intraluminal filling defect, which now could also be visualized by repeat US examination. Multiple core biopsies were obtained under US guidance using a vacuum assisted biopsy (bx) needle. A small clip was deployed in the bx cavity following the procedure. Histology revealed high grade ductal carcinoma in situ (DCIS). The patient was seen in surgical consultation and underwent an attempted wide local excision, which revealed extensive DCIS, 5 cm in greatest extent. There were multiple microscopic foci of invasive cancer. The surgical resection margins were positive for DCIS after multiple re-excisions. After thorough discussion the patient elected to undergo bilateral mastectomies. Two sentinel lymph nodes (blue dye and lymphoscintigram) contained micrometastatic adenocarcinoma. The patient is presently undergoing adjuvant chemotherapy. DISCUSSION: This case exemplifies the importance of an extensive evaluation of a pathologic nipple discharge. While a bilateral, inducible nipple discharge involving multiple ducts is a physiologic finding in many women, patients presenting with a spontaneous, unilateral discharge (bloody or watery) confined to one duct need careful evaluation for an underlying neoplasm (papilloma, carcinoma). A small malignancy, which in our patient had already involved axillary nodes, can be easily missed on physical examination and mammogram. In this setting an US examination in the "trigger zone" may identify an occult lesion. Cytologic evaluation of the discharge or ductal lavage fluid may be helpful. An important diagnostic tool is ductography, as performed in our patient. With the recent introduction of fiberoptic ductoscopy direct, realtime intraductal images, allowing detection of lesions as small as 0.2 mm can be obtained offering a safe alternative to ductography in guiding subsequent breast surgery in the treatment of nipple discharge. (Shen K-W. Breast Cancer Research and Treatment, Vol. 64, No 1, Nov. 2000, p. 30) ACUTE METHEMOGLOBINEMIA SECONDARY TO TOPICAL BENZOCAINE SPRAY. J. Walker 1 , H. Houston 1 , S. Miller 1 , G. Rouan 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Define the pathophysiology associated with methemoglobinemia, and 2) Recognize the clinical settings likely to be explained by methemoglobinemia. CASE INFORMATION: JT is an 82-year-old white female with a history of HTN, paroxysmal atrial tachycardia, and hypothyroidism who was admitted for elective laparoscopic left adrenalectomy, which proved to be a benign adenoma. Post-operative course was uncomplicated until POD #3 when the patient developed nausea and vomiting with a distended abdomen and decreased bowel sounds. A diagnosis of paralytic ileus was made and a nasogastric tube was placed, preceded by a 3 second dose of Hurricane spray (20% Benzocaine, approximately 600 ± 885 mg total dose) to the oropharynx. Within several minutes, the patient developed lethargy and confusion. The patient then became dyspneic, syncopal, and developed a cyanotic appearance. She did not respond to 100% O 2 by non-rebreather mask despite an arterial blood gas (ABG) with a PaO2of 310 and an O2saturation of 99.7%. A simultaneous pulse oximetry revealed an O2sat of 86%. The arterial blood was extremely dark in appearance. An ECG revealed supraventricular tachycardia with a rate of 170 ± 180 and ST depression, which converted after 6mg adenosine to sinus rhythm, with a rate of 120 and resolution of the ischemic changes. A diagnosis of methemoglobinemia was confirmed by co-oximetry, which revealed a methemoglobin level of 30.9% (normal = 0%). A 50 mg dose of methylene blue was administered, and over the course of 1 hour, the patient developed dramatic improvement in mental status and cyanosis. Methemoglobin levels decreased to 6.4%, 3.7% and 2.4% over the next 1, 3, and 6 hours, respectively. DISCUSSION: Methemoglobinemia is a condition in which iron in hemoglobin becomes oxidized (Fe 2+ to Fe 3+ ) at an overwhelming rate, resulting in a drastic decline in the oxygen carrying capacity of the RBC. Pharmacological agents, such as benzocaine, are capable of inducing methemoglobinemia by direct or indirect oxidation of the hemoglobin molecule. The ABG is not an accurate indicator of oxygenation status in a patient suffering from acute methemoglobinemia. The treatment of choice is methylene blue (1mg/kg, IVP). This case of benzocaine-induced methemoglobinemia is similar to others that have been reported in the past and indicates the need for a high index of suspicion for this syndrome and a need for close supervision of patients receiving benzocaine spray for clinical signs and symptoms of cyanosis. 1) Recognize the importance of the life narrative in the comprehensive understanding of the adolescent who presents with somatic distress and chronic fatigue. 2) Distinguish between diagnoses that denote a disease process (eg anemia) and those that are strongly influenced by cultural``memes'' (eg chronic fatigue syndrome). CASE INFORMATION: CASE 1: A 17 y/o white teenager developed increasing fatigue beginning at age 15. Other symptoms included daily fever spikes, visual changes, myalgias, and headaches. Her grades suffered greatly as a result of repeated absences from school, & she also had to quit her part-time job. At the age of 5, she witnessed her 3 y/o brother drown in their pool while her mother was in the house. Her parents subsequently divorced when she was 12, & she has a strained relationship with her alcoholic father. The patient received counseling after the drowning and at the time of the divorce. CASE 2: A 18 y/o high school senior was diagnosed with chronic fatigue syndrome (CFS) at the age of 13. She was unable to attend school fulltime because of sleep disturbance, diffuse pain, & disabling fatigue. Both her parents were alcoholic, and her dad was verbally & physically abusive. She served as a caretaker for her wheelchair-bound mother. At the age of 12 she was raped by her half-sister's 29 y/o boyfriend. At the age of 17 while being treated with an antidepressant for depression, her psychiatrist would not allow concurrent oral contraceptives & she became pregnant and had an abortion. DISCUSSION: Many hypotheses have been put forth to explain CFS, an illness in search of an etiology. Characterized by a variety of somatic symptoms, CFS often has an abrupt onset. We postulate that it may be a form of "post-traumatic stress disorder," particularly in young patients. Children with CFS have been reported to exhibit more psychological morbidity, such as anxiety and depression, than those with chronic disease, but these psychological categories are not necessarily helpful in patient management. We have found that the life narrative which will often include antecedent traumatic events serves to illuminate the unique personal issues of the adolescent with CFS. This approach can create a doctor-patient relationship that will engender trust and perhaps lead to better clinical outcomes. LEARNING OBJECTIVES: 1) Recognize Salmonella Hepatitis as a cause of acute hepatitis in a patient with typhoid fever. 2) Mortality rate is as high as 20% particularly with delayed treatment. The prognosis is good if treated early with specific antibiotic therapy. CASE INFORMATION: A 22 year old female ,immigrant from Bangladesh with no past medical history was admitted with complaints of fever, nausea ,vomiting and headache of one week duration. The patient had visited Bangladesh three weeks back. Prior to this presentation she had two visits to the ER in the same week and was diagnosed as viral syndrome. On admission now the patient was delirious and agitated .Her BP was 112/62; Pulse-70; Temperature-105 deg. Farenheit. Physical Examination -Right hypochondriac tenderness, no hepatosplenomegaly, no neck rigidity, no icterus, no skin rash. Lumbar puncture and CT Scan of head were normal. USG Abdomen was normal and HIDA Scan showed no uptake. There was a moderate elevation in the Transaminase level with AST/ALT of 294/205 respectively, LDH 579,Total bilirubin of 1.3 and direct-0.5.Albumin PT/APTT and Reticulocyte counts were normal. WBC count was 8300 with 10% band cells. Blood cultures later came positive for Salmonella Typhi sensitive to ceftriaxone. Hepatitis serology was negative for A,B and C.The patient was started on ceftriaxone .LFT's improved and patient was discharged home after 7 days in stable condition. DISCUSSION: Salmonella hepatitis is a rare condition and 20% of the patients with salmonella hepatitis may not be bacteremic. A diagnosis of salmonella hepatitis was made on the basis of 1)ALT/LDH < 4 (0.35 in this case).This happens to be the best discriminator between Salmonella hepatitis and viral hepatitis as is appreciated in this case.2) Positive blood culture for Salmonella.3) Relative bradycardia. 4) High-grade fever.5) Left shift of WBC and 6) Positive travel history to an endemic area. The prognosis is good if treated early with specific antibiotic therapy, however the clinical course can be severe with a mortality rate as high as 20% particularly with delayed treatment. the malar eminences, neck and trunk with bilateral axillary and submental lymphadenopathy. Neurological exam was normal and no joint deformity was noted. There was generalised abdominal tenderness with no guarding or organomegaly. Investigations revealed Hb-10.6 gm/dl and total WBC-3.4K/ul, BUN-23mg/dl, creatinine-0.9 mg/dl. Liver function tests showed albumin-2.3 g/dl, AST-54, ALT-26, GGT-53U/L, LDH-485 U/L. Serum amylase was 401 and serum lipase 358. Serum calcium was 8.9 with triglycerides of 187mg/dl. Urine analysis showed specific gravity of 1015 with 3+ proteinuria. CT scan of abdomen showed fullness of pancreatic head and ultrasound showed a normal sized CBD with no evidence of cholelithiasis. Biopsy of skin rash showed intravascular thrombi, vasculitis consistent with early lupus with atypical lymphocytic infiltrate. Diagnosis of SLE with acute pancreatitis was made on the the basis of above and ANA of 1:1280 with homogenous pattern and Anti ds DNA positivity. Patient was started on intravenous steroids. Patient's clinical status continued to worsen and required mechanical ventilation. Repeat CTscan confirmed pancreatitis with bilateral pleural effusions and no evidence of necrosis or abscess formation. She improved over the next few days and was successfully extubated. However, two weeks later she developed diffuse and severe abdominal pain accompanied with fever. Repeat CTscan now showed Grade E pancreatitis(multiple fluid collections +/À gas in or adjacent to pancreas)with necrosis. CT guided aspirations of necrotic material was negative for gram stain but culture grew pseudomonas, MRSA and candida. The patient had an exploratory laprotomy with necrotectomy. Despite aggressive antibiotic and antifungal treatment with multiple abdominal explorations the patient's condition deteriorated. She died of multiple organ failure. DISCUSSION: Acute pancreatitis is an uncommon manifestation of SLE. It is even rarer for such patients to present with pancreatitis as the sole major organ affected. The role of corticosteroids in such setting is controversial. Review of literature suggests that corticosteroids do not cause pancreatitis in patients with SLE and they should be used during episodes of pancreatitis if required. He also noted pruritic, non-tender skin lesions on his face and arms. He denied the use of any medications. Review of systems revealed a 10 lb. weight loss, blurry vision, nonproductive cough, hoarseness and mild dyspnea on exertion. He denied any HIV risk factors. Physical examination revealed widespread, small, hyperpigmented, nodular and papular lesions. A 3Â2 cm subcutaneous nodule was noted on the left side of the neck. Breath sounds were decreased bilaterally, with dullness to percussion on the left. The testicles were each twice normal size and tender with enlarged epididymi. Vital signs and the remaining exam were unremarkable. Scrotal ultrasound confirmed the physical exam and showed no discrete masses. Chest x-ray and CT revealed a moderate left pleural effusion with mediastinal and hilar adenopathy but no infiltrates. PFTs were consistent with a mild restrictive pattern. Serum ACE level was elevated at 87. Skin biopsies revealed non-caseating granulomas, negative for AFB or fungi. Follow-up scrotal ultrasound after corticosteroid therapy showed regression of testicular abnormalities. DISCUSSION: Sarcoidosis is a disease of unknown etiology, which can present with a wide variety of symptoms and involve virtually any organ system. In this unusual case, the patient presented for care due to testicular involvement but also had dermatologic and pulmonary manifestations, which he had ignored. While many cases of sarcoidosis spontaneously remit within 5 years and do not need treatment, therapy is indicated in patients such as this one, with symptomatic disease or systemic involvement. The goals of treatment include a reduction in symptoms and the avoidance of systemic complications. Despite attempts with steroid-sparing agents, the mainstay of treatment remains systemic corticosteroids. Monitoring with chest x-ray and PFT's is often warranted and patient education remains essential. SARCOIDOSIS AND STONES. K. White 1 , U. Mason 1 ; 1 Denver Health Medical Center, Denver, CO LEARNING OBJECTIVES: 1) Diagnose renal manifestations of sarcoidosis, and 2) Recognize the need for monitoring calcium levels and renal function in sarcoidosis. CASE INFORMATION: A 43-year-old man with a long history of cystic sarcoidosis presented with acute renal failure. He had chronic renal insufficiency with a baseline serum creatinine of 1.4 mg/dL. He had been treated with prednisone for several years and his dose had been tapered recently because his pulmonary status appeared stable. Review of systems ascertained a 10 ± 15 pound weight loss and decreased energy for 2-3 months. Physical examination was unrevealing. Laboratory data was significant for serum creatinine of 2.5 mg/ dL and calcium of 11.3 mg/dL. Renal ultrasound showed right hydronephrosis and bilateral nephrolithiasis. Computed tomography showed left staghorn calculus with caliectasis and right ureteropelvic junction stone with hydronephrosis, hydroureter and a more distal stone. A right percutaneous nephrostomy was placed and serum creatinine decreased to 1.8 mg/dL. Evaluation of the etiology of hypercalcemia included normal serum PTH, protein electrophoresis and PSA levels. Urinary calcium excretion was 252 mg/24 hours. The hypercalcemia was felt to be secondary to sarcoidosis and therefore the prednisone dose was increased. He underwent right nephrolithotomy, which was successful only for the proximal stone. Bilateral stone removal is planned for the future. Serum creatinine is back to baseline and serum calcium is normal. DISCUSSION: Hypercalcemia -an uncommon complication of sarcoidosis ± results from endogenous overproduction of 1,25 dihydroxyvitamin D. Nephrolithiasis is more common, occurring in about 10% of patients, and more frequently associated with hypercalciuria than with hypercalcemia. Nephrocalcinosis is much less common and is probably related to chronic hypercalcemia. This patient had both nephrocalcinosis and nephrolithiasis, likely due to both long-standing hypercalcemia and hypercalciuria leading to chronic and then acute renal failure. Long-term management of patients with sarcoidosis includes frequent monitoring of calcium levels and renal function.
A PRESENTATION OF SHEEHANS SYNDROME 22-YEARS LATER. W. Whitwam 1 , D. Stuart 1 ; 1 Hennepin County Medical Center, Minneapolis, MN LEARNING OBJECTIVES: 1) Recognize complications of analgesics associated with unrecognized hypothyroidism. 2) Understand the complications of Sheehan's syndrome and panhypopituitarism. CASE INFORMATION: A 62-year-old African-American female, with a past medical history notable only for a prior cesarean section, received an uneventful laparoscopic repair of a ventral hernia. Post-operatively, she received morphine analgesia by a patient controlled analgesia (PCA) pump. After developing an unanticipated stupor, her morphine was discontinued. She received several doses of naloxone without response. A computerized tomography (CT) of the head demonstrated no acute changes. With an arterial pCO2 value of 62 mm Hg, she was placed on biphasic positive airway pressure (BiPAP) ventilation. She remained somnolent. A formalized evaluation of her delirium included a thyroid function panel and random cortisol level, both studies demonstrated abnormally low values. A magnetic resonance image (MRI) of her brain revealed a partially empty sella turcica. Her normal luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were discordant with her post-menopausal status, and her prolactin level was below the normal reference range. After receiving thyroxine and hydrocortisone replacement therapy, her depressed consciousness resolved within two days. Upon a later interview, she revealed that 22-years prior she had a cesarean delivery of her seventh child, resulting in a large blood loss and subsequent hypotension. Unlike her six previous pregnancies, she failed lactation after delivery, and subsequently never resumed menstruation. DISCUSSION: Sheehan's syndrome is an obstetric complication that occurs with excessive blood loss and ischemic shock following a complicated delivery. This results in an infarction of the pituitary's anterior lobe. As a result of her hypopituitarism, this patient developed secondary thyroid and adrenal failure as well as gonadal insufficiency. With an unrecognized secondary hypothyroidism, the exaggerated depressant effects of her analgesics resulted in a postoperative stupor. Careful attention to hypothyroidism should be noted with any patient who develops an excessive central nervous system response to anesthetics or analgesics.
EPISODIC HYPERTENSION ASSOCIATED WITH SITTING ON HARD SURFACES. R.M. Witteles 1 ; 1 Stanford University, Stanford, CA LEARNING OBJECTIVES: 1. Distinguish between the potential causes of episodic hypertension. 2. Recognize the practical consequences of the complex relationship between pain and elevated blood pressure. CASE INFORMATION: A 76-year-old Japanese-American male was referred to the hypertension clinic for a long-standing history of baseline normotension interrupted by hypertensive episodes associated only with sitting on hard surfaces. Home blood pressure measurements (consistent with measurements in clinic) ranged between 110 ± 120/70 ± 80 mm Hg, but rose to 180 ± 190/100 ± 110 mm Hg immediately after the patient sat on a hard surface such as an ordinary chair; no change in blood pressure from normal values was observed when the patient sat on a soft surface or was in any other position. Measured blood pressure was the same in both arms, and returned to a normal range immediately after the patient stopped sitting on a hard surface. The patient experienced no symptoms when sitting on a hard surface except for bilateral blurry vision; this too promptly returned to normal after getting up from the hard surface. At various times in his life, the patient had taken reserpine, lisinopril, verapamil, enalapril, atenolol, hydrochlorothiazide, and clonidine to control his hypertensive episodes, always without efficacy; he is currently taking no anti-hypertensive medications. Of possible relevance, the patient had chronic neck pain which was exacerbated by sitting on hard surfaces; however, the change in blood pressure reliably and reproducibly occurred before the pain. The patient had normal urinary catecholamines, vanillylmandelic acid, and metanephrine values, normal thyroid studies, no evidence of panic disorder, and no evidence of end-organ damage. A trial of opioid analgesics had no effect on the patient's blood pressure. DISCUSSION: Episodic hypertension is a relatively uncommon clinical entity, most often associated with the "white-coat" phenomenon, pheochromocytoma, ingestion of sympathomimetic drugs, withdrawal from alcohol and other drugs, thyrotoxicosis, panic disorder, and intermittent pain. Prior studies have discovered a complex relationship between pain and blood pressure, with involvement at multiple areas of the central nervous system. This patient demonstrated an unusual temporal relationship between pain and blood pressure, with the rise in blood pressure occurring first; the hypothesis of a conditioned response should be considered.
BELLS PALSY AND HYPERTENSION SECONDARY TO DISCONTINUATION OF ANTIHYPERTENSIVE TREATMENT. S. Yakoob 1 , K. Barnard 1 , J. Haretos 1 ; 1 UPMC Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1. Recognition of an association between facial nerve paralysis and accelerated hypertension.2. High dose corticosteroid therapy often prescribed for facial nerve palsy has serious consequences in hypertensive patients.3.Facial palsy resulting from severe hypertension may be distinguished from Bell's palsy by lack of pain and preservation of taste. CASE INFORMATION: An 84-year-old male who presented with a two-day history of rightside facial drooping, difficulty in chewing and inability to close his right eye. His past medical history is significant for hypertension controlled on quinapril and bisoprolol/HCTZ. Two days prior to the onset of symptoms, the patient had discontinued his medications. Physical examination was remarkable for a blood pressure of 190/96 and signs of right lower motor neuron facial nerve palsy. Diagnostic evaluation included CT scan of the brain that was negative for hemorrhage and a maxillofacial CT that showed thickening of paranasal sinuses. An EMG revealed decreased conduction over the right facial nerve.The patient's blood pressure was controlled by oral metoprolol and quinapril. High dose corticosteriod therapy was started and subsequently discontinued when the patient's blood pressure increased to 205/ 105 mm Hg. This case is similar to two other case reports in the literature of abrupt withdrawal of antihypertensive treatment resulting in an acute increase in the blood pressure and subsequent facial paralysis. DISCUSSION: LEARNING OBJECTIVES: To recognize toxic methemoglobinemia as a rare but potentially lethal complication of local anesthetics so that prompt diagnosis and treatment can be initiated. CASE INFORMATION: A 37 year old white female patient with negative past medical history, presented to our clinic with a complaint of epigastric pain and was scheduled for endoscopy. Benzocaine spray was administered topically in the oropharynx during endoscopy and the procedure was uneventful, but approximately 10 minutes after completion, the patient oxygen (O2) saturation began to fall and she became cyanotic despite administration of 100% O2 via a non-rebreathing mask. Intravenous Narcan and Flumazenil were administered without improvement. The O2 saturation continued to fall with the lowest level obtained was 81%. Arterial blood gas analysis showed PaO2 of 367mmHg and PaCO2 of 23mmHg so methemoglobinemia was suspected. Methemoglobin level was sent and later showed a level of 37.6%. Methylene blue was given intravenously, the patient started to improve gradually and within 20 minutes O2 saturation rose to 96% and cyanosis was resolved. The patient was transferred to intensive care unit, where her O2 saturation rose to 99% and methemoglobin level dropped to 1% . The remainder of hospital stay was unremarkable. DISCUSSION: Methemoglobinemia is a rare complication of local anesthetics that has been reported with procedures including bronchoscopy, endoscopy, tracheal intubation and dental procedures. Methemoglobin is formed normally in the body because small amount of ferrous iron in hemoglobin is continuously oxidized to ferric iron which can't carry oxygen or carbon dioxide. Red cell defense against accumulated methemoglobin is methemoglobin NADH reductase which is responsible for 99% of in vivo reduction of methemoglobin to form normal hemoglobin. Methemoglobinemia has both acquired and congenital forms. Acquired methemoglobinemia can be associated with toxic shock or can result from ingestion or skin exposure to oxidizing agents which oxidizes hemoglobin to methemoglobin. Although cyanosis may appear with levels as low as 15%, acquired methemoglobinemia is rarely symptomatic when levels are below 20%. Lethargy, dizziness, lightheadedness and anxiety are present between levels of 30 ± 40%. Coma, seizures, arrhythmias and acidosis could be caused with levels of50 ± 70%. Levels more than 70% are fatal. We were first alerted to the presence of methemoglobinemia by decrease in O2 saturation inspite of normal O2 tension (PaO2) which is almost a universal finding. Methylene blue given intravenously 1 ± 2mg/Kg over 5 minutes acts a cofactor in the transfer of an electron from NADPH to ferric iron and methemoglobinemia should resolve within one hour, if not the dose may be repeated. LEARNING OBJECTIVES: 1) To consider dural venous sinus thrombosis in the differential diagnosis of puerperal psychoneurotic symptoms. 2) Initiating management as soon as the diagnosis is recognized can cause major decrease in morbidity and mortality. CASE INFORMATION: 21 year old female patient with no significant past medical history, had normal vaginal delivery 3 weeks prior to admission, presented with depressed mood, poor concentration and headache. Physical examination was negative. Brain computed tomography scan (CT) was done and found to be negative so the patient was diagnosed as a case of postpartum depression and was admitted to psychiatric floor. One day after admission patient became drowsy and her level of consciousness dropped. Head and neck CT scan with contrast showed thrombosis of the right jugular vein. Magnetic resonance image (MRI), magnetic resonance angiography (MRA) and magnetic resonance venography (MRV) were done and showed thrombosis of the superior sagittal sinus, right transverse and deep venous sinus. Patient developed respiratory distress and was intubated and was started immediately on Intravenous Heparin. After few days she was extubated and transferred to medical floor where she was switched to oral anticoagulants. Patient depressive symptoms improved and her mood returned back to normal. DISCUSSION: Dural venous sinus thrombosis is an unusual disorder most often attributed to hematological disorders, oral contraceptives, association with pregnancy and puerperium, Bechets disease, cardiac disease and post operative conditions. Patients usually present with nonspecific symptoms as headache, seizures, hemiplegia or other neurological symptoms. Brain swelling and bilateral involvement can produce lethargy or stupor early in the course. Diagnosis depend on recognition of impaired venous flow. On contrast CT scan a nonenhanced triangular area surrounded by contrast in the posterior sinus (the empty delta sign), should suggest the diagnosis. Cerebral angiography has been considered the gold standard for the diagnosis but MRI, MRA and MRV are becoming the diagnostic studies of choice.Management increasingly relies on the use of anticoagulation even in the presence of superimposed parenchymal hemorrhage. Venous occlusions are serios and often fatal but acute anticoagulation started as soon as the diagnosis recognized appears to lessen substantially the morbidity and mortality of the condition. Nonanticoagulated venous sinus thrombosis that is not complicated by infection carries a mortality rate of 25 to 40% . Uncontrolled series suggest that early heparin treatment can reduce mortality by more than a half. OBJECTIVES: Reflective practice may enable physicians to become more competent and successful practitioners. This technique, however, is usually developed at the faculty level. The objective of this workshop is to begin to develop this skill at an earlier level. Students may then begin to utilize this skill for success at the student/resident level as well as honing this skill for use when at the faculty level, for both clinical practice and teaching. METHODS: Students will be given a brief (approximately 1 1/2 hour)skill session in the use of reflective practice. Intervention outcome will be assessed using the following:Pre/post test for knowledge, skills and attitudes regarding reflective practice.Randomization of the same class of students (Class of 2005)À 1/2 to intervention, 1/2 no intervention. Each group will then be given a reflective practice questionnaire following an OSCE exercise, to assess utilization of the skill. OBJECTIVES: The UCSF Division of General Internal Medicine (DGIM) faculty development program trains general internists to be effective teachers in primary care settings that serve a diverse patient population. During the first year of our program, we recruited general internists from community based health centers and faculty of the DGIM at UCSF. We developed and taught a 9-month long curriculum with six core content areas: teaching skills, evidence based medicine, cultural competency/caring for vulnerable patients, psychosocial medicine, population medicine, and leadership. Sessions were 3.5 hours long, two afternoons per month. METHODS: Prior to implementing our program we conducted a baseline needs assessment among trainees. An external evaluator met with the trainees during and at the end of the year to elicit feedback and to assess if the program was meeting its' objectives and the trainee's learning goals. RESULTS: Twelve trainees, 5 from under-represented minority groups, participated in the program last year. The evaluation concluded that the most valued aspect of the program for the trainees was the opportunity to learn and practice new teaching skills. The trainees also expressed interest in developing a tangible product, such as a new curriculum, at the completion of the program. RESULTS: Primary care physicians in the SF Bay Area have adequate access to new medical knowledge, but have limited opportunities to learn and refine new teaching skills. Now in year 2, the program has been modified to put greater emphasis on learning and practicing teaching skills, and less time is devoted to teaching the trainees new medical knowledge. Trainees are now required to develop a project, with the guidance of a program mentor, that is relevant to their own teaching setting. The faculty development program has enabled community and academically based general internists to form a common bond around teaching. METHODS: Instructional methods supporting individualized learning plans were enhanced by yearlong, on-site, one-on-one educational support from faculty development specialists. Curriculum has evolved to address the changing medical education environment. The program was examined within grant objectives, historical records, and archival data obtained through written surveys and focus groups. RESULTS: Focus groups and survey results have tentatively identified that the one-on-one consultation enhanced the 59 participants' ability to provide curriculum instruction within SCS member hospitals. 88% of graduates continue to teach in SCS institutions, using skills obtained through the program. 33% of the participants have received teaching awards with two winning national poster awards. 95% of the participants have remained in primary care. Some graduates have moved to higher positions within medical education. CONCLUSION: Developing part-time, community-based faculty is a multi-faceted endeavor. Faculty are challenged between providing service versus training others. The focus of providing a yearlong stipend supported program with one-on-one consultation support by specialists has proven to be a successful model in attracting and developing part-time, community-based faculty. OBJECTIVES: Primary care physicians are challenged to be clinically productive and effective teachers. A critical review of the literature revealed limited evidence regarding effectiveness of four often cited teaching methods: 1-2 focal teaching points; priming; teaching in the patient's presence [TIPP] ; and feedback. To address this gap seven experienced clinician educator [CEs] faculty (4 general internists; 3 pediatricians), participated in a faculty development project to evaluate the effectiveness of the four selected clinical teaching methods. METHODS: During monthly faculty development sessions, CEs were trained on the teaching methods using multiple strategies including simulations and written exercises. Each CE recorded use of these methods over a 10-month period on a Palm Pilot(tm) using a specially designed form. Baseline (July 1997 ± June 1998) and study period clinical teaching evaluation ratings for CEs (treatment) and non-participating faculty in their specialties (control) were compared using ANOVA. RESULTS: CEs reported using all targeted teaching methods > 50% of time during recorded teaching sessions (N=1,176 sessions) with use of priming and feedback increasing over baseline while use of the other two methods remained constant. CEs reported that the teaching methods focused both the learner and the clinical teacher, making subsequent encounters more productive and efficient. CEs teaching evaluations were significantly higher (p < .001) on three items during the study period while control group's ratings showed no change (i.e., responded to student initiated learning issues; emphasized comprehension of concepts; instructor had sufficient data to assess learner performance). During the study period, two CEs were first time recipients of major department teaching awards with three CEs receiving highly competitive, college-wide teaching awards one-year post study period. CONCLUSION: Experienced CEs who participate in a faculty development program can improve their clinical teaching ratings using four literature-based clinical teaching methods. Recognition by learners and peers of the CEs educational excellence through awards was an unanticipated outcome. OBJECTIVES: The population of older persons is rapidly rising in developed countries like Japan. Oldest old patients aged 90-year-old and more had occasionally utilized acute care hospital emergency department in recent years. To our knowledge, there were no study evaluating clinical reasons and outcomes of these highly aged population. METHODS: To determine the short-term outcomes and predictors of outcome, we reviewed medical records of elderly patient aged over 90-year-old visiting emergency department in Okinawa Chubu Hospital, acute care community hospital in rural Okinawa, Japan, in 1999. RESULTS: Three hundred seventy one patients (95 men, 276 women: mean age 92.9 years old) were studied. Five common chief complaints were fever (65 cases),shortness of breath (46), consciousness disturbances (33), leg pain (29),and nausea or vomiting (21). Pulmonary diseases (94) were the commonest category of disorders, followed by orthopaedic diseases (43) and gastroenterologic diseases (42). Only 42(11% ) patients were dead during hospitalization following admission from emergency department except for 14cardiopulmonary arrests at arrival. In multiple logistic regression analysis, cognitive dysfunction was only significant predictor for short-term mortality. CONCLUSION: Majority of oldest old patients had survived to discharge following emergency department admission. Cognitive function was important in terms of survival prediction.
AN INTEGRATED PARTNERSHIP BETWEEN THE UNIVERSITY OF CINCINNATI AND COMMUNITY HEALTH CENTERS. T. Redington 1 ; 1 College of Medicine, Cincinnati, OH OBJECTIVES: 1. Identify the common interests of both academic health centers and community health centers that cause them to be natural partners. 2. Demonstrate the unprecented level of support that the University of Cincinnati has shown in preserving the capacity of community health centers. 3. Describe the team approach adopted by the providers of the Cincinnati Health Network, Cincinnati Health Department,University of Cincinnati Hospital clinics, and Southern Ohio Health Network, which included a common eligibility system and data sharing thoughout the delivery system. 4. Discuss this common approach to patient eligiblity and clinical care includes disease management protocols for asthma, diabetes, hypertension and depression. This common clinical approach is expected to improve if not eliminate health disparities, consistent with the Bureau of Primary Health Care's 100% access, 0% disparities goal. METHODS: The University of Cincinnati has budgeted 2 million dollars of inkind support for all the center sites of the Cincinnati Health Network. This support includes a pharmacy with pharmacist, radiology suite, midwives, and 12 full time providers, eleven are physicians. These physicians are extensively involved in medical student education at these center sites. Also the University led the successful effort to recieve a million dollar grant (from HRSA the Community Access Program) to improve the infrastructure around the indigent and uninsured in Southern Ohio. The major partners include the University, a hospital of the Health Alliance of Greater Cincinnati, a five hospital system in southerwestern Ohio, the Cincinnati Health Network, the Southern Ohio Health Network(both Federally Qualified Health Centers),and the Cincinnati Health Department. Together, these partners serve about 200,000 lives, 90% are at or below the minimum federal poverty level. RESULTS: The quality of care standards around depression, hypertension, asthma and depression have been installed. The information technology infrastructure and eligibility systems are being developed and linked between different computing platforms. The measurement of our quality of care will begin later this calendar year. Medical education in these clinic sites is extensive, about three quarters of the medical student class are being educated in these sites.
CONCLUSION: There is a major partnership underway in southern Ohio, linking the University of Cincinnati's Hospital and College of Medicine with four large, previously independent health care systems, that have traditionally provided for the same patients, the indigent and uninsured. By the adoption of common eligibility and disase management standards, access will be improved and disparities reduced.
A. Rubin 1 , T. Bertsch 1 ; 1 University of Vermont College of Medicine, Burlington, VT OBJECTIVES: To retain and recruit community-based preceptors in Vermont, we developed, distributed, and analyzed a survey to understand the needs of our community faculty and the barriers to precepting students in their offices. METHODS: We mailed questionnaires to all 570 primary care physicians in Vermont. We received replies from 251, of whom 160 were preceptors. We asked questions about their needs to become better teachers, the infrastructure needed to teach, rewards for teaching, and the desired format for faculty development. RESULTS: Areas of high importance for teaching include becoming more efficient, balancing student needs with patient time, increasing abilty to teach clinical skills, givng feedback to students, dealing with difficult students, and finding ways of orienting students to practice. In terms of infrastructure, preceptors want more time and space to teach. They want rewards in the form of CME or CME credit and in hearing feedback about their students. They are happy to have faculty development sessions once a year, either at a central site or near their practice site. CONCLUSION: As with our students, eliciting feedback from community based teachers can result in programmatic change. As a result of ths survey, we have developed a teaching skills exercise using standardized students and presented itin three sites. We are also changing the ways we award CME credits.
A TEACHING EXERCISE FOR COMMUNITY PRECEPTORS USING STANDARDIZED STUDENTS. A. Rubin 1 ; 1 University of Vermont College of Medicine, Burlington, VT OBJECTIVES: To assess the teaching skills needs of our community based teachers.To develop and present a workshop that would allow them to practice these skillls. METHODS: As part of our HRSA project we developed a needs asssessment for our community based teachers. Of the areas in which they wanted to enhance their teaching skills, we chose four to learn and practice: Teaching Efficiently, Framing a Clinical Question, Giving Feedback, and The Difficult Student. We developed scripts and videos for a four station teaching exercise using a rolling role play and standardized students. RESULTS: Preceptors rotated in groups of four through each half-hour station. In each, a brief didactic presentation was followed by a rolling role play with a standardized student, which gave each preceptor a chance to practice. A short debriefing followed. We have presented this workshop to 60 of our preceptors in three areas of the state. CONCLUSION: Preceptors rate the workshop format as effective and``couldn't be better.'' They find the topics appropriate and practical. They especially like the student participation and the ability to practice what they learn in small groups. We don't yet know whether preceptors are using the skills they have learned. This is an area for further inquiry. OBJECTIVES: Despite expansion of programs in geriatrics, it will not be feasible to train enough geriatricians to care for all elderly patients. Several organizations have suggested that improved training of primary care physicians in geriatrics is likely to have the greatest impact on improving medical care of geriatric patients. To address this need, we have enhanced several aspects of our curriculum in geriatrics to strengthen the clinical competencies of primary care residents. Our specific objectives are 1) to improve the didactic curriculum; 2) to increase the number and quality of ambulatory clinical experiences; and 3) to develop new methods for measuring the clinical competencies of residents in geriatrics. METHODS: Primary care residents spend six months both years on structured ambulatory rotations. Two half days per week are devoted to structured didactics and eight half days to ambulatory rotations. Portions of both of these experiences are now devoted to enhanced geriatric training. Didactic curriculum: We have developed twenty specific learning objectives in geriatrics. These are taught in ten seminars accompanied by a syllabus of recent and sentinel articles. Ambulatory experiences: Ambulatory geriatric training begins in the first year on a home care rotation directed by geriatrics faculty. Second and third year residents may also work on home care and/or in a variety of geriatric health centers. Selected residents spend one half day per week during each ambulatory block in geriatric health centers as a second continuity experience. We have also begun to increase geriatric teaching in the residents' own primary care continuity clinics by recruiting more geriatrics-trained faculty to serve as core resident preceptors. Enhanced evaluation: We have enhanced our efforts at evaluating resident experiences in geriatrics with the use of a web-based evaluation system. We will also evaluate resident attitudes toward the care of older patients and the extent to which learning objective have been met. Finally, we will develop new strategies in measuring resident competency in geriatrics with a geriatric clinical evaluation exercise (geri-CEX) and with structured medical record reviews. RESULTS: We hypothesize that these methods will increase the knowledge, attitude and skills of residents in geriatrics and will result in improved competency in geriatrics for primary care physicians trained in this manner. CONCLUSION: The curriculum of structured primary care residency programs can be modified to address specific deficiencies in health manpower. We have designed a strategy for specifically enhancing the competency of primary care physicians to care for the elderly. OBJECTIVES: 1. Promote community-oriented primary care. 2. Teach medical students and residents about COPC through service learning in diverse underserved communities. 3. Develop role models in public health practice. 4. Engage the academic health center in its community. METHODS: 1. We established an "Institute for Community Health" in Cambridge and Somerville, dedicated to research and education in health promotion and disease prevention. Three health care networks (Cambridge Health Alliance, CareGroup and Partners) found common cause in a scholarly collaboration to improve the health of these communities. 2. The Community-oriented Primary Care (COPC) curriculum was chosen as one of three priority initiatives. The curriculum contains: a) experiential (research) elements: community defined projects; teams of a preceptor, medical resident and one or several medical students; year-long longitudinal projects. b) Didactic elements include a seminar series taught by faculty drawn from collaborating institutions. 3. We expect to enroll the first students in September. RESULTS: 1. Trainees will be evaluated by faculty and their community partners -in accordance with defined competencies, the quality of their products (e.g., policies, guidelines, webpages, interventions, etc.) and the sustainability of their work. 2. The faculty and currriculum will be evaluated by the community on the basis of the contributions to the local community's health, spread to other communities (within the collaborators' catchment areas), and impact on students' careers; and by the trainees. CONCLUSION: This educational model has the potential to positively influence the health of underserved communities, the careers of health professionals, and the missions of health care institutions -in Cambridge and Somerville, and beyond.
RESIDENT PARTICIPATION AT NATIONAL SGIM MEETINGS. D.W. Brady 1 , L.J. Schultz 1 , W.T. Branch 2 ; 1 Emory University, Decatur, GA; 2 Emory Healthcare, Atlanta, GA OBJECTIVES: 1. Expose residents to general medicine career options by having them attend National SGIM meetings. 2. Encourage residents to participate in National SGIM meetings by submitting vignettes, abstracts, and workshops. METHODS: Our residency training grant affords us the opportunity to send all 10 of our second-year primary car residents to the national SGIM meeting each year. We encourage the residents to actively participate in the meeting by collaborating with faculty on workshops and submitting their own research abstracts or clinical vignettes. By exposing them to generalists from various institutions and supporting their own scholarly activity, we hope they will pursue generalist careers and remain active in SGIM. RESULTS: Beginning with the 1998 SGIM meeting in Chicago and including this year's meeting in San Diego, we have brought 40 second-year residents to national SGIM meetings. In 1998, all 10 residents collaborated on a workshop that won the David E. Rogers Award. In 1999, two of those residents returned as senior residents to present research posters. In 2000, three third-year residents presented research posters, and one second-year resident presented a clinical vignette poster. This year, one second-year resident is co-leading a workshop while the othe 9 second-year residents are all submitting clinical vignettes. So far, 90% of our graduates have remained in general internal medicine, with approximately 50% obtaining academicallyaffiliated positions. CONCLUSION: By exposing our residents to the myriad of people and opportunities afforded them at national SGIM meetings, we encourage them to remain excited about generalist careers and to pursue their own academic interests. We believe their attendance at the national SGIM meetings is an integral part of our internal medicine primary care residency program and our HRSA grant.
SELF-REFLECTION AS A TOOL TO ENCOURAGE CULTURAL COMPETENCY. D.W. Brady 1 , I. Genao 1 , J. Bussey-jones 1 ; 1 Emory University, Atlanta, GA OBJECTIVES: 1. To promote cultural competency through self-reflection. 2. To encourage residents to examine their own cultural biases and prejudices. 3. To provide a safe environment where residents can ask difficult questions about other cultures in a spirit of inquiry. METHODS: As part of our cross-cultural curriculum, we set aside one session to allow residents to self-reflect and ask questions they had about each other's cultures. The group consisted of 10 third-year primary care residents -six men and four women, including two African-Americans, two Asian-Americans, at least two evangelical Christians, one Jew, at least one atheist, and two homosexuals (these were some of the represented cultures known to the group). We asked them each to write down a question and place it in an envelope. They were asked to write questions that they had always wanted to ask a person of a different culture (race, ethnicity, religion, sexual orientation, etc.) but were either afraid to ask or thought was too stereotypical. The questions were not to be directed personally at any specific individual within the group. After collecting the questions, we passed the envelope back around the room and asked each person to take out one question and read it to the group. The intent was to create a safe environment where the residents could bring their "forbidden" questions into the open and to allow them to see the reaction of the group without being personally humiliated or attacked for asking their questions. Our hope was for each resident to become more aware of his or her own cultural competency and to learn to hear "the other" in a spirit of inquiry. RESULTS: The group rated this session higher than any other session in the curriculum. We originally intended for the group to just hear the questions and feel their impact. The group, however, at the beginning of the session decided by consensus to allow some discussion of each question by the group before proceeding. In evaluating the experience, the residents said that the discussion was integral to making the experience its most meaningful. They also said that late internship might be a good place to introduce this exercise as it would have helped them earlier in their training. They added, however, that introducing it too early, before the group has a chance to form, might have risked alienating members of the group, thus damaging their ability to develop cohesion. CONCLUSION: The depth of learning and the residents' evaluation of the exercise have encouraged us to retain it as a part of our multicultural curriculum. Whenever we as physicians can afford trainees the opportunity to be self-reflective, expecially in a cross-cultural context, we can enhance doctor-patient communication, learn more about ourselves and each other as human beings, and, ultimately, take better care of each individual patient and society as a whole. OBJECTIVES: To describe the efforts of one general internal medicine training program to promote the selection of generalist careers by residents. In the past couple of years, there has been a declining interest in generalist careers, yet primary care physicians play an important role in meeting the health care needs of the medically underserved. METHODS: Multiple strategies for promoting selection of primary care careers have been piloted in the Primary Care track of our internal medicine residency program: ÁMentorship: Each primary care resident is paired with a general internal medicine faculty member based on common career or personal interests and goals. Mentorship pairs meet at least twice a year to review clinical performance, research, educational plan, and career goals. ÁContinuity clinics: Primary care residents have two half days of clinic in settings which serve a large Medicaid and medically indigent population. ÁPrimary care rotations: Several primary care rotations including adolescent medicine, women's health, and outpatient HIV have been developed to improve the ability of residents to practice in a variety of outpatient settings. ÁPreceptorships: All primary care residents are required to do a preceptorship during the R2 year. Rotations in medically underserved and rural areas are strongly encouraged. ÁCareer development seminars: Topics such as Finding a Job, Evaluating Contracts, and interaction with a panel of internists who practice in various settings are incorporated into the three-year primary care curriculum. RESULTS: During HRSA funding (1997 ± 2000) , 23 of 25 graduates chose primary care careers; of the two choosing fellowships, one was in geriatrics. The career development sessions were consistently rated very positively; 4.85 to 5.00 on a 5-point scale. In focus groups, residents also reported high satisfaction with these sessions, primary care rotations, continuity clinics and the mentorship program. CONCLUSION: While we have successfully promoted primary care career choices, we have been less successful in placing graduates in medically underserved areas.In attempt to increase graduates choosing careers in medically underserved areas, we are focusing recruitment efforts on students with documented interest in care of the medically underserved and supporting this interest throughout residency training. Due to the national trend of declining interest in primary care careers, these efforts will be extended to include categorical residents and medical students.
A REQUIRED ROTATION IN HOMELESS MEDICINE -PROMOTING ALTRUISM TO RESIDENTS. D.R. Buchanan 1 , L. Rohr 1 ; 1 Cook County Hospital / Rush University, Chicago, IL OBJECTIVES: Although the American Association of Medical Colleges (AAMC) considers altruism to be one of the basic qualities that physicians should possess, residency programs rarely incorporate required rotations aimed at meeting this goal. Our objective was to design and implement a required rotation to promote altruism and careers with underserved populations through teaching residents about homelessness. METHODS: The course was designed to provide a balance between direct patient care and didactic sessions. Clinical experiences were supplemented by visits to providers of homeless services and by a lecture series. Residents worked in a variety of clinical settings, including performing medical outreach in locations around the city. The locations selected were meant to expose the residents to different facets of impoverished communities. In addition to the sites the residents visited as part of their clinical work, they also visited a number of medical and nonmedical sites related to homelessness. Specific sites included Chicago's only respite center for medically ill homeless people, Chicago's largest homeless shelter and its adjoining health center, and a smaller homeless shelter. A lecture series was included to help provide a context for understanding the medical and social issues unique to this population. The lectures focused on causes of poverty and homelessness as well as their effects on health and access to healthcare. The series was also used as a time for discussions, allowing the residents to reflect on their experiences and on assigned readings. RESULTS: The course was evaluated by pre and post course surveys. The surveys were designed to identify resident attitudes toward caring for homeless patients. We also used the surveys to identify barriers that residents feel toward pursuing careers in which they would care for underserved populations. Results of these surveys will be presented at the meeting. CONCLUSION: This required rotation,``Caring for Homeless Patients'', was implemented in the spring of 2001 for third year Primary Care Internal Medicine residents. Initial responses to the rotation have been positive. Future directions for this program include further assessment of whether the rotation is meeting its goal of promoting altruistic behavior among residents.
T. Cavalieri 1 , K. Welding 1 , J. Ciesielski 1 , J. Kaiser-smith 1 , H. Dombrowski 1 ; 1 University of Medicine and Dentistry of New Jersey, Stratford, NJ OBJECTIVES: To establish an inner-city community-based training site that will enhance primary care skills, build cultural competency, and improve internal medicine residents' skill in addressing the needs of an underserved population. METHODS: The University of Medicine and Dentistry of NJ-School of Osteopathic Medicine (UMDNJ-SOM) established an affiliation with an existing primary care practice founded by the Diocese of Camden and the Society of Jesus. This practice, St. Luke's Catholic Medical Services, serves the poor, uninsured and underinsured community of Camden, NJ. It has a fluent bilingual staff and attracts a predominately Latino population. The physician at St. Luke's was a primary care internist/Jesuit priest who was volunteer faculty in UMDNJ-SOM's Department of Medicine. Initially, a full-time faculty member from UMDNJ-SOM was assigned part-time to assist him in providing patient care and to establish a training site for internal medicine residents. The Department then recruited a full-time primary care internist with a special interest and prior experience in serving the underserved for this facility. As part of this collaboration between UMDNJ-SOM, the Diocese and Jesuit Order, the Order funded the physician's participation in a Spanish immersion program in Mexico to improve language skills and cultural awareness. The physician has since initiated care at St. Luke's and been named Medical Director. RESULTS: This new partnership with St. Luke's, attributable to the collaborative efforts of the University, Diocese and Jesuits, has led to the development of learning objectives for a primary care continuity experience for the residents in our HRSA funded primary care residency. The primary care residents have begun weekly half-day sessions at St. Luke's that sensitize them to the medical, cultural and psychosocial needs of this underserved community. As a result of this HRSA grant, the new affiliation with St. Luke's has created additional educational opportunities. It has provided a new training site for HRSA-funded geriatric fellows and UMDNJ-SOM's extern program as well as primary care residents. CONCLUSION: This collaboration has provided the University with a unique training site and rewarding learning experience. Internists intending to serve underserved populations must be able to appreciate cultural diversity both within and between cultures; understand the impact that psychosocial, educational, cultural and environmental issues may have on client utilization and compliance; and respect the distinct cultural needs of various client populations. Collaboration between UMDNJ, the community and church, through HRSA support, has created a primary care training site to enhance cultural competency.
L. Christophe 1 , P. Arnaud, P. Eliane 1 , A. Anne-franc Ëoise 1 ; 1 University Hospitals of Geneva. Dep Int Medicine, Geneva, Switzerland OBJECTIVES: The first years of professional activity are critical for doctors in training. However, little is known about the difficulties encountered by residents. METHODS: We put the following open question to 24 consecutive residents in a structured one year training in internal medecine: «Please, identify 2 to 3 major difficulties/concerns related to your practice of medicine within this hospital». Each resident gave confidential written answers. These were coded into 8 categories by content analysis by 3 researchers. RESULTS: Characteristics of the residents were: female: 37% , mean age: 28 2.2 years, mean duration of postgraduate training: 2.5 1.3 years. The total number of answers was 122, with an average of 5.1 1.3 per resident. 93% of the residents expressed difficulties in their relationships with their colleagues or felt not adequately supervised. 63% judged that they were not respected or recognized by senior staff. 63% admitted feelings of severe disarray and helplessness in dealing with patients. 58% complained about work overload. 50% felt burdened by the intensity of emotional investment and responsibility towards patients. Finally, 16% admitted fearing for their professional future and only 8% cited lack of theoretical knowledge about the diseases they were dealing with. Four answers did not fit into the 8 above categories. CONCLUSION: Most of the interviewed residents in training expressed major difficulties within their professional relationships. Feelings of inadequate practical and relational training by medical school, of lack of recognition by senior staff, and of work pressure and overload were common. These elements challenge the organisation/coaching in our institution and should lead us to improve our teaching. METHODS: Internal medicine residents are exposed to curriculum in cancer prognosis using several instructional strategies including didactic/lecture, small group case discussions, roleplaying and debriefing actual patient encounters. Additionally, some of the residents participate in an elective block rotation in end-of-life issues providing them with more intensive exposure by following patients in our hospice program. Along with relevant reading assignments for background, residents review sample case studies of terminally ill patients at latter stages of illness. After learning a system of estimating prognosis they discuss how this information may be used to design care plans so that the therapies that are most appropriate in light of known prognosis are chosen. Residents review basic principles of communicating with patients about prognosis and end-of-life issues that they then implement in role playing exercises. RESULTS: Formative and summative evaluation methods are utilized to assess progress toward the objectives. These include individual faculty preceptor assessments of residents caring for terminally ill patients, knowledge and attitude questionnaires regarding end-of-life issues, scenarios highlighting communication issues, and resident self-assessment. In addition, roleplays are rated on several criteria and feedback is given to residents in a post session briefing.
CONCLUSION: Although end of life issues are receiving increased attention and concern, it remains an area physicians tend to feel insecure in their skills and poorly prepared by their medical training. Developing curriculum to increase physician's confidence and comfort with these issues improves the quality of care to patients and family members in the dying process.
COMBINING OSCES WITH THE MINI-CEX FOR RESIDENT ASSESSMENT. R.S. Crausman 1 , J.P. Miskovsky 1 ; 1 Memorial Hospital of RI, Pawtucket, RI OBJECTIVES: To assure a high standard of clinical competence throughout the continuum of medical education, it is imperative that appropriate, task-relevant evaluative tools and methods be continuously developed and applied. In response to the perceived inadequacies of conventional evaluative measures, the use of test patients for structured, standardized evaluation (Objective Structured Clinical Examination, OSCE) has gained favor for the assessment of medical students and residents. However, the role of physician faculty observer, when present, in these exercises is unclear. Physician supervisors bring a clinical perspective to the encounter different from, and complimentary to that of the standardized patient. The challenge is to provide a structured evaluative framework to improve the quality and reproducibility of assessment. In 1995 the American Board of Internal Medicine developed and validated the Mini-CEX. These observed resident-physician patient encounters consist of a single faculty member observing a resident conduct a focused history and physical examination. Residents and evaluators report satisfaction with the format, and reliability of the evaluation is thought to be superior to traditional clinical evaluation exercises. Here we describe our early experience combining the Mini-CEX with outpatient OSCE's for enhanced resident assessment. METHODS: Second year medical residents (n = 10) in our Primary Care/General Internal Medicine Residency underwent a multi-station OSCE designed to assess their clinical skills. Each 20 minute encounter was observed by a faculty physician who structured observation and assessment using the Mini-CEX instrument which uses a two page form with a 9 point rating for assessment. After each encounter both standardized patient (five minutes) and faculty physician (five minutes) provided structured feedback to the resident trainee. A time keeper assured that encounters were kept to 20 minutes. RESULTS: A structured feedback session with all participants was conducted after the exercise and all reported high levels of satisfaction with the format. Standardized patients uniformly commented that the presence of a physician faculty evaluator represented an improvement and provided complimentary evaluative information. Faculty observers were pleased with the imposed structure that the Mini-CEX provided. Residents were satisfied that feedback was appropriate and valuable CONCLUSION: In response to the perceived inadequacies of conventional evaluative measures we have introduced a combination of outpatient OSCE's and the Mini-CEX towards developing a more flexible, reliable and task-relevant teaching and evaluation tool. Our early experience with this approach has been very positive. OBJECTIVES: One of the goals of our residency program is to improve residents' ability to care for the medically underserved. We expect that our graduates will not only provide excellent clinical care to individual patients, but will also integrate public health concerns into their medical care. In addition, we anticipate that our graduates will work with community residents, organizations and leaders to serve as advocates for the underserved. In order to expose our residents to this model of community-oriented primary care, we developed an eight-week course in community medicine. METHODS: The Community Medicine seminar series was held 1/2-day each week for eight weeks and was attended by first, second and third year residents. The curriculum included a community inventory exercise in which residents obtained information about the community in which they saw continuity patients, a community assessment in which residents toured through a neighborhood on foot and described the people, businesses, housing and transportation they encountered, a community treasure hunt in which residents shopped at local businesses and map exercises in which residents plotted the location of their continuity patients' homes. A key component of the curriculum was an intranet website designed specifically for the Community Medicine course. Residents used the website to participate in on-line discussions of reading material and to access public health information and resources. RESULTS: Residents were asked to complete an on-line assessment instrument at the conclusion of the course. 91% of the residents (10/11) found the seminar series to be very useful for their future practice of medicine. 91% (10/11) felt the course should be offered again in the future. 92% (11/12) felt they understood and could explain the concept of community-oriented primary care. 73% (8/11) of the residents felt the map exercises helped them understand where their patients lived and how they traveled to medical appointments. 100% (11/11) found the public health links on the intranet website useful for learning about COPC. 73% (8/11) residents found the community treasure hunt useful in terms of how to view a community from the standpoint of its people and institutions. CONCLUSION: This Community Medicine course increased residents' knowledge of the communities in which they and their patients live. Future directions for investigation include determining whether this course impacts residents' decisions to practice primary care in underserved areas and to implement community-oriented primary care concepts after graduation from the program. OBJECTIVES: Emergency contraception (EC) is an effective but underused method of preventing unwanted pregnancy. In a 1997 national study, only 25% of college-educated women and 6% of women with high-school degrees knew key facts about EC. Only 5% of women had heard about EC from their providers. While other studies have addressed barriers to EC use in the general population, few have focused on barriers in poor, urban populations. Three residents in a program designed to train physicians to care for the underserved looked at this issue in a community satellite clinic. METHODS: As part of a Quality Improvement/Research curriculum in a primary care residency program, we conducted a focus group with eight providers at an urban, publiclyfunded community health center in San Francisco. Provider beliefs/practices, perceived patient barriers, and possible interventions to increase emergency contraception use were discussed. The session transcript was then analyzed to identify a consensus surrounding specific barriers and interventions. RESULTS: Providers identified a significant problem with unintended pregnancy at their clinic, estimated at 85 ± 90% of all pregnancies. Despite this high rate, emergency contraception was prescribed only 2 ± 3 times a year at the clinic. Providers identified the following obstacles to emergency contraception use: lack of patient knowledge regarding the availability of EC; the prevalence of social and economic stressors that made contraception a low priority for patients; patients' fatalistic beliefs surrounding pregnancy; and operational obstacles to obtaining emergency contraception prescriptions. Identified interventions to increase use/access were: patient and community education about emergency contraception; arranging to have a prepackaged product on-site to give to patients; and routine counseling by providers at all reproductive health visits. CONCLUSION: In this urban, community-based primary care clinic, unintended pregnancy is common and emergency contraception rarely used. In the focus group session, providers clarified barriers and identified interventions that the clinic could employ to increase patient's access to and use of emergency contraception. Barriers to use by urban, underserved populations may need further study and targeted interventions before true access to emergency contraception becomes a reality for this vulnerable patient group. OBJECTIVES: Increase residents knowledge of common IM approaches (acupuncture, physical therapy/massage, mind-body medicine, herbal medicine, chiropractic) including what that approach entails, what conditions it is thought to be useful for treating, and any contraindications for its use; and ability to identify, access and critically evaluate evidencedbased research regarding treatment outcomes and efficacy of the particular approach. METHODS: Residents are assigned a faculty mentor who oversees and provides guidance in the month; helps to monitor and direct the residents' learning and to address their objectives. Residents spend several 1/2 days each week in the office of IM providers. Each provider prepares a reading list the resident is responsible for working through in that area. Residents see patients along with the provider and have opportunity to discuss the patients from that IM perspective. Residents also spend time with a general internist who is well versed in IM at our Center for Integrative Medicine. They learn how primary care physicians incorporate IM into their practice. Residents attend several one-on-one classes. For example, residents meet with the librarian to learn how to search the web to find IM information and research and are introduced to IM databases in the area (i.e., Natural Medicine Database). An oncologist talks with the residents about the use of alternative techniques cancer patients often use; how the oncologist approaches discussions about IM with patients and the contraindications that may exist for various IM approaches within oncology. The residents also go on several site visits (walk through a pharmacy with the pharmacist looking at OTC herbal remedies as well as visiting a wellness center providing patient education, support groups and counseling to cancer patients. RESULTS: Intensive interviews are conducted with each resident completing the rotation to evaluate quality of the experience and to elicit opinions for changes/additions to the experience. CONCLUSION: Setting up curriculum addressing nontraditional approaches in a western medical setting raises many issues regarding developing common standards for education, reimbursement for practitioner training time and credentialing, applying research standards to cross cultural scientific literature. The process is a challenging and yet a rewarding venture for both traditional and nontraditional medical learners and educators. OBJECTIVES: A one-month IM elective block rotation was developed for third year residents to more intensively, study IM approaches. It was hoped that this exposure would prepare residents to better integrate IM into their medical practice. Increase residents knowledge of common IM approaches (acupuncture, physical therapy/ massage, mind-body medicine, herbal medicine, chiropractic)including what that approach entails, what conditions it is thought to be useful for treating, and any contraindications for its use; and ability to identify, access and critically evaluate evidenced-based research regarding treatment outcomes and efficacy of the particular approach. METHODS: Residents are assigned a faculty mentor who oversees and provides guidance in the month; helps to monitor and direct the residents' learning and to address their objectives. Residents spend several 1/2 days each week in the office of IM providers. Each provider prepares a reading list the resident is responsible for working through in that area. Residents see patients along with the provider and have opportunity to discuss the patients from that IM perspective. Residents also spend time with a general internist who is well versed in IM at our Center for Integrative Medicine. They learn how primary care physicians incorporate IM into their practice. Residents attend several one-on-one classes. For example, residents meet with the librarian to learn how to search the web to find IM information and research and are introduced to IM databases in the area (i.e., Natural Medicine Database). An oncologist talks with the residents about the use of alternative techniques cancer patients often use; how the oncologist approaches discussions about IM with patients and the contraindications that may exist for various IM approaches within oncology. The residents also go on several site visits (walk through a pharmacy with the pharmacist looking at OCT herbal remedies as well as visiting a wellness center providing patient education, support groups and counseling to cancer patients. RESULTS: Intensive interviews are conducted with each resident completing the rotation to evaluate quality of the experience and to elicit opinions for changes/additions to the experience. CONCLUSION: Setting up curriculum addressing nontraditional approaches in a western medical setting raises many issues regarding developing common standards for education, reimbursement for practitioner training time and credentialing, applying research standards to cross cultural scientific literature. The process is a challenging and yet a rewarding venture for both traditional and nontraditional medical learners and educators. OBJECTIVES: In order to effectively care for patients and promote health, physicians must have a broader view of their role in society as``health promoters''. Knowledge of public health resources as well as the process of policy development is especially important when caring for vulnerable patients. Because of the crowded curriculum of a residency program, we have developed a public health curriculum that is integrated into the existing framework of the residency program. METHODS: A needs assessment was conducted via interviews with the Primary Care interns by an outside evaluator and a survey completed by Primary Care and traditional track house officers (80% response rate). Several areas within the existing curriculum were identified where public health concepts could be inserted Ð complementing, not replacing,``traditional medicine'' topics. All sessions are evaluated. Residents will complete yearly surveys and an exit interview to assess the impact of this new curriculum. RESULTS: The resident survey showed a significant level of interest in public health, greatest in the senior residents. Residents rated their own knowledge of many public health concepts as`a verage'' to``low'', and rated a broader knowledge of public health as``important'' to``very important'' to patient care. Faculty from the Boston University School of Public Health, Boston Public Health Commission and the Massachusetts Department of Public Health have agreed to participate, and run joint sessions with faculty from the Section of General Internal Medicine. Sessions include: 1) A monthly three-hour seminar during an ambulatory block, attended by all of the residents (both Primary Care and traditional track) that reviews the history of medicine and public health, highlighting areas of tension and opportunities for collaboration. 2) Ambulatory Morning Report: topics relevant to ambulatory medicine are presented by the Primary Care chief resident and internal medicine faculty, with monthly participation by public health faculty, adding the public health perspective with a 10 ± 15 minute discussion. 3 ) A threehour session in the Primary Care seminar, focused on the role of Departments of Public Health and an introduction to health policy and government. 4) An elective rotation at the Massachusetts Department of Public Health provides primary care residents with the opportunity to work directly with public health staff, participate in projects and learn how programs and policies are developed. CONCLUSION: Our survey found that the residents were interested in public health and felt that it was relevant to clinical care. Public health faculty were very willing to participate in resident education and collaborate with curriculum development. Important public health concepts can be incorporated into a Primary Care residency curriculum without displacing existing``traditional topics''. OBJECTIVES: Since Hepatitis B (HBV), Hepatitis C (HCV) and HIV share the same high-risk behaviors, coinfection is frequent in clinical practice. In the US, 30 ± 50% of HIV patients are coinfected with chronic HCV. Almost 95% of HIV patients have serological evidence of HBV exposure. In the era of highly active antiretroviral therapy, death from rapidly progressive viral hepatitis is increasingly common in HIV patients. Since early detection and treatment of HIV and viral hepatitis has been shown to improve the outcome of patients, reactive serologies to HBV and/or HCV should provoke discussion of HIV testing in a general medicine clinic to reveal coinfection. METHODS: Retrospective chart review of patients with serological exposure for viral hepatitis, identified or followed at a hospital based general medicine clinic in East Harlem, NY. A query was done on hepatitis serologies sent from 1/1/99 to 12/31/99 from the clinic to create 3 patient groups : 1) HBV exposure (Reactive HBc only) 2) HCV exposure (Reactive HCV only) 3) Both HBV and HCV exposure (Reactive HBc and HCV). Charts were reviewed to determine whether house staff physicians documented discussions of high risk HIV behavior after obtaining the hepatitis serologies. The number of HIV tests requested and results were noted. Patient return rate for HIV results was also noted. OBJECTIVES: Most guidelines for prevention and treatment of osteoporotic fractures are neither evidence-based nor well-suited to the needs of the diverse patient population found in urban public health settings. This Quality Improvement project aimed to create a clinical guideline adapted to San Francisco's Community Health Network and its diverse population with limited resources. The Guideline aims to be based on current evidence wherever possible, and is based in part on existing evidence-based guidelines such as the National Osteoporosis Foundation (NOF) Guideline. Implementation of the guideline will follow published effective approaches to changing practice patterns. METHODS: 1. Adaptation of currently existing evidence-based guidelines, such as the NOF guideline, and review of recent relevant literature to create a guideline appropriate for and tailored to San Francisco's Community Health Network (CHN.) 2. Distribution of these guidelines to CHN providers, ideally targeting those who see our at-risk patients, using a creative media approach with paper and electronic formats (including distribution of a file transferrable to handheld organizers such as the Palm OS) and additionally promoting the guideline in a didactic format. 3. Improving awareness of and adherence to these guidelines with such techniques as Clinical Alerts on the CHN computer record, and easy Web access on the CHN website. RESULTS: We designed a guideline that was tailored to meet the needs of the CHN patient population. This guideline was evidence-based. Distribution was achieved via CHN website, computer-based reminders and triggers where appropriate, handheld organizer files, and didactic sessions in local clinics and conferences. CONCLUSION: Few guidelines for prevention and treatment of osteoporotic fractures currently exist that are both evidence-based and tailored to meet the needs of an underserved urban public health community. Using good existing guidelines such as that published by the NOF, current literature can be adapted to design a guideline that is both based on good evidence and appropriate to the limited resources of an urban public health setting. Novel uses of computer-based and didactic tools can enhance distribution and awareness of clinical practice guidelines. OBJECTIVES: To develop and implement a curriculum in women's health for internal medicine residents in a community hospital based university program. METHODS: Participants are first year internal medicine residents in their ambulatory block month (N=16). The curriculum content was determined by a needs assessment conducted on previous and current residents. It consists of the following six units covered during three hourly didactic sessions: 1) Pretest and Introduction to Women's Health, 2) Selected Gynecological Conditions, 3) Obesity and Eating Disorders, 4) Menopause, Osteoporosis and Hormone Replacement, 5) Psychosocial Issues, 6) Resident Presentations and Posttest. Also included in the curriculum is a three-hour visit to the local domestic violence prevention center. All necessary reading materials are handed out during the first session after the pretest. The didactic sessions start with a brief case presentation by the participating resident(s); this is followed by an in-depth discussion of the topics of the day with expected resident participation. Each session except the introductory and resident presentation session ends with role-plays where the instructor plays the role of the patient and the resident the doctor. This enables the resident(s) to put into immediate practice what they have just learned. Clinical practice of women's health is expected to occur when residents see female patients in their primary care practices. To expand the scope of this curriculum beyond these limited topics both residents and faculty have been encouraged to incorporate into everyday teaching and learning how various conditions may differ or are unique in women. Participating residents are expected to make a formal thirty to forty-five minute presentation on a topic in women's health not covered in this curriculum. Learners are evaluated by the completion of a standard evaluation form used in the residency program. Evaluations are based on the residents' performance on the posttest, participation during the didactic sessions and their presentations. Learners evaluate the curriculum by completing a survey. RESULTS: The curriculum was well received by all residents. All residents surveyed either agreed or strongly agreed that they would now be able to evaluate, manage or appropriately refer female patients as a result of knowledge gained during the month. The mean posttest score was 81%, an improvement over the mean pretest score of 66%. Residents stated particularly that role-plays helped improve their understanding of the topics. A survey of these residents in their third year regarding their perceptions on the women's health education they received in this program and how well they think it has prepared them for their future practices year is planned. CONCLUSION: The curriculum improved the knowledge of residents in selected women's health topics. Using the same curricular model, topics can be adapted to meet the learning needs of residents in other residency programs.
IMPROVING THE HEALTH CARE RESPONSE TO DOMESTIC VIOLENCE: A SEMINAR SERIES FOR PRIMARY CARE RESIDENTS. K. Riordan 1 , S. Love 1 , C. Warshaw 1 , S. Glick 2 ; 1 Chicago Abused Women Coalition, Chicago, IL; 2 Cook County Hospital, Chicago, Illinois OBJECTIVES: Violence against women has been increasingly recognized as a major public health problem. Despite this, many physicians still find it difficult to ask patients about abuse. A major barrier that health care providers face in effectively identifying and treating victims of abuse is a lack of training on the prevalence and impact of domestic violence, ways to intervene appropriately and how to access available resources. We integrated an innovative 8-week seminar series on domestic violence into our primary care residency training. The goals of our curriculum were to enable residents to: 1) understand the prevalence and dynamics of domestic violence; 2) identify the impact of domestic violence on women's physical and psychological health; 3) develop culturally respectful intervention skills to identify and assist abused patients; and 4) identify resources available to victims of abuse.
METHODS: The seminar series was led by the Hospital Crisis Intervention Project (HCIP), a collaborative program of the Cook County Bureau of Health Services and the Chicago Abused Women Coalition (CAWC). The seminars were held 1/2-day each week for eight weeks. Topics included An Overview of Domestic Violence; Identifying, Assessing and Intervening with Domestic Violence Victims; Working with Diverse Populations; The Batterer; Intervening with Sexual Assault Patients; Community Violence; and Child Abuse, Elder Abuse and Legal Issues. A highlight of the course was a site visit to CAWC's domestic violence shelter where the primary care residents were able to speak candidly with survivors of abuse. Seminars were led by domestic violence advocates, physicians, social workers and attorneys. Instructional format included lectures, interactive discussions, videotape reviews, problem-based case discussions and a community site visit. RESULTS: Residents assessed each session and the course overall with written evaluations. 100% of the residents (9/9) found the course very effective at helping them learn more about the dynamics of domestic violence. 100% (12/12) found it very effective at helping them learn more about the impact of domestic violence. 100% (8/8) found it very effective at helping them work effectively with diverse populations. 92% (11/12) found the course very effective in helping them learn more about the resources available to victims of domestic violence. CONCLUSION: This 8-week course on domestic violence improved residents' understanding of the dynamics and impact of domestic violence, culturally appropriate interventions and available resources. Future directions for evaluation include the use of simulated patients to determine whether this course improves residents' ability to identify and care for victims of violence in the clinical setting. OBJECTIVES: We designed a 2 week rotation in addiction medicine to promote the role of the primary care physician in prevention, early identification, and treatment of substance abuse. The objectives are to: 1) support positive attitudes towards patients with addiction disorders, 2) define the responsibilities of the primary care physician for substance abuse intervention, 3) practice new skills and apply new knowledge of substance abuse in a general medical setting, 4) provide access to physician role models. METHODS: We adapted the structure and content of a successful 4 week elective rotation in addiction medicine into a 2 week core curriculum for all second year, primary care residents. Previous experience from the longer rotation informed us that residents unanimously chose as core learning objectives: screening and assessment, discussing substance abuse with a patient, and making a referral to community resources for substance abuse problems in general medical practice; and half also chose as a focus: developing a non-judgmental approach to addicted patients, providing preventive education, and management of withdrawal. We therefore made these the learning objectives of the brief course, adding: how to monitor and support patients in recovery. Modeled on the longer elective, the two weeks are structured around the residents' ambulatory primary care clinics, where they are responsible for practicing their new skills. They attend 4 half-day workshops, including case discussions of core topic areas and role play of office-based skills. Working with hospital-based substance abuse counselors, they perform substance abuse assessments and referrals for medicine and trauma patients; observe treatment groups of an out-patient treatment program for substance abusing persons with HIV; visit community-based residential treatment programs, a social-detox program, and a 12-step meeting. RESULTS: We used one experienced faculty-member to organize the curriculum and make community contacts for the 2 week rotation. Workshops are delivered by a group of faculty including internists, psychiatrists, and chemical dependency professionals. We found that community-based, substance abuse treatment programs were willing to sponsor visits by small numbers of residents, and several would permit residents to observe treatment groups. Treatment programs were generally willing to arrange for residents to interview individuals clients. CONCLUSION: A 2 week rotation in addiction medicine can be created that focuses on the role of the primary care physician in the prevention, early identification and treatment of substance abuse. Residency programs that do not have substance abuse services within their teaching hospitals or clinics can seek cooperation from community-based, substance abuse treatment programs to provide residents with clinical experiences. OBJECTIVES: Establish an ambulatory care morning report for residents, medical students, and other trainees in a multidisciplinary clinic, and encourage the integration of evidence based information into clinical practice. METHODS: McClennan Banks Adult Primary Care Center at the Medical University of South Carolina serves as an ambulatory teaching site for internal medicine residents, medical students, physician assistant students, and pharmacy graduate students. Clinic faculty have appointments in general internal medicine, pharmacy, the physician assistant program, and psychiatry. We organized a forty-five minute, twice-weekly morning report for all residents and students on their monthly ambulatory rotations. The report takes place prior to clinic hours and is attended by four to eight residents and students and two to three faculty or fellows. Each student or resident is assigned a date to give a presentation describing one of their patients from the monthly rotation. The presenter is encouraged to focus on a common primary care topic, and to search for available evidence (guidelines, systematic reviews, clinical trials) on topics such as screening, diagnosis, prognosis, or treatment of the condition being discussed. The report begins with a brief history and physical, followed by a discussion of the diagnostic evaluation and management alternatives, and concludes with an evaluation of the evidence presented. Informal discussions, observation and exit interviews at the conclusion of the ambulatory rotation have assessed educational value and trainee and faculty satisfaction.
RESULTS: The program has been functioning for one year, and is a valued educational experience according to feedback from the trainees. The students have had excellent attendance, while residents have needed greater encouragement to attend the sessions. Faculty participation has been excellent, as the sessions provide greater opportunity to teach than is possible during busy clinic hours. An additional benefit has been the increased interaction of learners from the variety of disciplines represented. The timing of the report has not interfered with clinic operations. CONCLUSION: Our ambulatory morning report enhances the quality of clinical education and is a valued addition to the ambulatory rotation. Plans are being discussed to include trainees from other clinical teaching sites. Future implementation should include prospective evaluation of impact on knowledge, attitudes, and the use of evidence in patient care. OBJECTIVES: As part of our HRSA-funded primary care grant, we increased the longitudinal ambulatory care requirement for our categorical internal medicine residents. With an increasing proportion of our residents having this ambulatory care experience in the community, we felt the need to determine whether the educational experience for these residents is comparable to that of residents in a more traditional hospital-based clinic setting. We developed a new evaluation tool to assess the experiences of residents in these two settings. METHODS: In developing our Evaluation of Continuity Preceptor and Site instrument, we sought to assess those aspects of the resident's experience most important for optimal ambulatory education in both community and hospital-based sites. The evaluation instrument is divided into a group of questions concerning the preceptor and another group of questions regarding the ambulatory site. The 15 preceptor items include questions about role modeling, supervision, teaching and an overall rating. The 18 site items include questions about office systems (patient flow, medical records, billing), patient characteristics, continuity of care and an overall rating. Items were rated on a 5-point Likert scale, assessing frequency of preceptor and site attributes, using terms varying from never/almost never to always/ almost always. RESULTS: The instrument was piloted and yielded good internal consistency (Cronbach's alpha .88 for preceptor items and .83 for site items). The survey was then distributed to all categorical residents semiannually for two years. The response rate was 82%, and 132 surveys were analyzed. When tested for differences using the Mann ± Whitney test, the group mean scores for site items were significantly higher for the community sites as compared to the hospital-based clinic site (4.08 vs 3.72; P=0.001). The group mean scores for the preceptor items were higher for the clinic-based faculty as compared with the community preceptors (4.58 vs 4.44), but the difference was not significant (p = 0.08). Site items rated higher by residents in the community included questions about exposure to a broad range of patients (p = 0.001) and to a full range of patient problems (p = 0.07). Residents in the hospital-based clinic felt they had a higher level of responsibility for their patients (p < 0.001) and they rated their preceptors slightly higher in providing explanations and asking questions to enhance learning (p = 0.13). CONCLUSION: Internal medicine residents are being increasingly assigned to community sites for their longitudinal ambulatory care experience. In our survey, we found that residents in the community rated site items higher and residents in the hospital-based clinic rated preceptor items slightly higher. Awareness of the differences between the two types ambulatory care experiences is important for planning future ambulatory educational activities. OBJECTIVES: A web-based modular curriculum for information literacy was developed to support evidence-based clinical practice and refine residents' skills for preparing professional conference presentation materials. This curriculum was designed to fill the gaps in informatics knowledge necessary to subsequent assignments supporting an Evidence Based Medicine (EBM) residency curriculum. METHODS: To assess readiness and assure curricular relevance, baseline data is gathered concerning essential EBM and information literacy skills. The initial one month curriculum consists of four two-hour class sessions supported by hands-on instruction and maintained on the open residency website. Content focuses on current and future informatics practice issues, orientation to significant information resources, emerging technology developments related to clinical practice, EBM searching strategies for the general Internet and quality medical sources, overview of quality patient education materials, government websites, and time saving shortcuts for common technology applications including browsers, word processing and presentation programs. The team of people who support and maintain this curriculum include a variety of Internal Medicine content experts, the medical librarian, a nursing informatics webmaster, and a research specialist. RESULTS: The initial series of courses is completed within the first three months of the first post-graduate year. Continued exposure to EBM skill usage and the website is required during all three years of the resident's outpatient clinic via topic sessions. Final assignments of the second and third residency years include a digital presentation of a clinical topic in an Internal Medicine research forum conference. CONCLUSION: Integration of information literacy skills into a residency curriculum must consider variations in learning curves and the need for changes in approaches to traditional problem solving techniques in clinical practice. Clinical faculty and residents need support and encouragement to adapt existing processes to include EBM approaches in daily practice. Traditional dogma states that a shift in institutional thinking may be needed to provide conveniently accessible Internet resources on the practice units and in the residency work areas. However, providing a readily accessible set of instructions and relevant EBM resources via a residency website removes barriers to implementation of evidenced based clinical practice for residents and attending physicians. The ACGME/ABMS vision is the integration of life-long learning for physicians with the maintenance of Board certification. Their collective goal is to ensure that physicians are better prepared to provide compassionate, appropriate, and effective patient care in the changing health care environment. DESCRIPTION OF PROGRAM/INTERVENTION: Purpose: Using this background as a framework, the ABIM Clinical Competence Program is exploring ways to foster self-reflection, self-assessment, and continuing professional development among internal medicine residents and subspecialty fellows throughout their training. Through a newly developed tool and template, the use of a portfolio designed and maintained by the physician, may facilitate achieving this goal and serve to chronicle medical professionalism. FINDINGS TO DATE: What Comprises A Portfolio?: Multidimensional, qualitative and quantitative, portfolios encompass a broad spectrum of activity from definitive learning goals, performance assessment and reviews, to personal narratives and community service. KEY LESSONS LEARNED: Conclusion: The professional portfolio is the tangible culmination of one's acquired and applied knowledge, learning experiences, and unique opportunities for self-assessment and self-reflection. The portfolio remains the practical and intellectual property of the creator. Through the creation of a portfolio, physicians are empowered to gain better insight into their own goals, personal and professional growth. and career development. We summarize our needs assessment of unique MD/MBA competencies and the integrated 5-year curriculum created in response to these needs. OBJECTIVES OF PROGRAM/INTERVENTION: Our goals were to 1) identify unique MD/MBA competencies through the input of private sector and academic physician leaders; and 2) develop new curricular opportunities to meet these needs. DESCRIPTION OF PROGRAM/INTERVENTION: Through a structured process, an advisory board comprised of 14 health care leaders identified six competency areas critical to MD/MBA graduates: clinician leadership skills in business environments, common business components of public and private health care delivery, a historical perspective on the US health care delivery infrastructure, the role of public policy in shaping health care delivery, an internal framework for ethical organizational decisionmaking and a population perspective in health care planning. These MD/MBA competencies represent additional skills required by graduates beyond the core competencies of each degree independently. During the first three years students complete all the standard MD curricular activities. MD students apply for the MD/ MBA Program during their third year. Once accepted, students spend the fourth year completing the entire core and some elective requirements in the Business school. The fifth year curriculum was developed as a summation year, integrating the two Schools' curricula while explicitly addressing the unique dual degree competencies. Fifth year curricular elements include a management component integrated into standard clinical electives, a broadened range of electives in other Schools within the University such as Public Health, an evening Physician Leadership Colloquia Series, and a clinically oriented field study/research experience. Students completed an evaluation that covered both School's core competencies as well as the unique dual degree competencies using a Likert scale. Information was also elicited about students' short and long term plans. FINDINGS TO DATE: All six members of the first cohort completed evaluations. Overall the curriculum addressed the majority of the unique MD/MBA competencies with a mean score of 3.0 or higher. Two competencies need further emphasis: developing an ethical framework for organizational decisionmaking (2.8) and an application of physician leadership skills in business environment (2.5 STATEMENT OF PROBLEM OR QUESTION: Statement of the Problem: Professional behavior is often not a structured topic in the standard medical school curriculum. Much of the socialization for medical professionals occurs in what we refer to as the hidden curriculum. Frequently medical students may not have the understanding of what is considered appropriate professional behavior, yet they are exposed to positive and negative role models from which they may choose to emulate. OBJECTIVES OF PROGRAM/INTERVENTION: Objectives: Our goal was to develop curriculum that begins to address some issues around the socialization that occurs for medical students during their clinical years of training. The purpose is to provide an informal, safe environment for students to discuss issues of professional behavior to which they are exposed during their third year of medical school. Information obtained from these gatherings is used to modify the experiences the students have during clinical training. In addition it serves the purpose of allowing students to share experiences with their colleagues. DESCRIPTION OF PROGRAM/INTERVENTION: Description: In a course entitled``The Hidden Curriculum'', junior medical students are divided into groups of 12 and have 6 scheduled meetings during the academic year with two facilitators (a faculty member and a senior medical student). For each session, the facilitators are provided with 2 to 3 questions concerning a variety of topics. Some of the topics include balance of professional and personal life, perceived medical student abuse and career goal exploration. A written summary report is generated from all of the data gathered from the small groups. The summary report is distributed to the clerkship directors, students and several of the Deans involved in medical student education. FINDINGS TO DATE: Findings to Date: Medical student experiences are generally positive but they are continually exposed to some negative role models. The results from the curriculum thus far have lead to creating a more formal evaluation of professional behavior. In some instances specific individuals have received feedback concerning egregious behavior. Students view this as a very positive experience, finally allowing them a format that facilitates critical feedback about their experiences. KEY LESSONS LEARNED: Key Lessons Learned: Medical students rarely receive formal training on what appropriate professional behavior entails. In many circumstances they do not feel comfortable providing honest, critical feedback to their superiors. Evaluation of professional behavior should be done in a more formal manner, addressing appropriate knowledge, skills and attitudes just as we do with clinical performance. OBJECTIVES OF PROGRAM/INTERVENTION: Conduct a workshop with residents using patients with life-threatening illness in order to: 1) educate residents about how to communicate bad news to patients and 2) change residents' attitudes about communicating bad news and maintaining hope for patients. DESCRIPTION OF PROGRAM/INTERVENTION: Four patients with cancer from the Wellness Community were recruited to participate in a 2 hour resident workshop on giving bad news to patients. The curriculum included patients using their own diagnoses and histories as the role play scenarios. Residents were instructed to give bad news as they usually do, then feedback from the patients, discussion about the role play and about the method as described by Buckman ensued. A second role play using the learned concepts was conducted and final debrief occurred. Prior to the workshop, residents completed an 11 item questionnaire about actions to be taken during the communication of bad news, with a similar post-test being used at the end of the workshop. FINDINGS TO DATE: Twenty-five residents participated, with 15 (60%) completing pre and post tests. Attitudes toward ensuring hope is conveyed to patients (p < 0.05), starting the discussion by ascertaining the patient's understanding of the condition (p < 0.01), and encouraging the patient to express his/her feelings (p < 0.01) all significantly improved with the use of the workshop. Most of the improvement occurred in residents with previous training. KEY LESSONS LEARNED: A role play workshop using untrained patients with cancer can significantly improve residents' attitudes about giving bad news. Volunteers are without cost to the institution, and have a positive influence in such workshops. Residents who are previously trained may hold counterproductive attitudes, and therefore all residents should participate in workshops using role play by actual patients about giving bad news. is predicated on the belief that humanism and professionalism come to students and others through understanding a number of core concepts and relationships complemented by self-reflection. The concepts, which have been described by ABIM's Project Professionalism, include ideas of integrity, respect and others; the relationships include those with patients and families and with other health care professionals. Talking Medicine offers a consistent (every other week for 10 weeks) opportunity to share experiences in small groups (6 ± 8), facilitated by two preceptors in a format driven by students' experiences. Although the focus is on students' experiences, readings are provided on basic topics and contexts in humanism and professionalism (e.g. end of life care, mistakes, spirituality in medicine, and boundaries between patients and doctors). FINDINGS TO DATE: Talking Medicine began in summer, 2000 and half of the third year class has taken it. We surveyed students (total 54, response rate 63%) and found 94% felt "very" or "somewhat" comfortable in the course. 73% of students reported that the course increased their "connectedness" to classmates and 61% favored it occuring during all rotations. 59% reported that their interest in caring for patients improved and 53% reported their interest in internal medicine as a field improved. Answers to open-ended questions highlighted the importance of Talking Medicine as a forum to connect with others Ð both students and faculty. KEY LESSONS LEARNED: These results suggest Talking Medicine may be most effective in helping classmates connect to and learn from each other, thereby setting a foundation for changes in how they interact with patients. STATEMENT OF PROBLEM OR QUESTION: End-of-life care is assuming increasing importance within medical education curricula. Traditional teaching methods may be inadequate to address the emotional, interpersonal, and spiritual aspects of palliative care. The performing arts offer an innovative approach to teach medical trainees in these essential but under-emphasized domains. OBJECTIVES OF PROGRAM/INTERVENTION: We instituted a program using the dramatic arts to foster empathy for the terminally ill person's experience of illness, and selfreflection with regard to personal practices in caring for dying persons. DESCRIPTION OF PROGRAM/INTERVENTION: The Wit Educational Initiative uses professional readings of the Pulitzer-Prize winning play``Wit'' at medical training sites throughout North America. "Wit" narrates the personal and medical care experience of a patient dying from metastatic ovarian cancer. Medical students, housestaff, and ancillary providers attend readings of the play followed by structured discussions of the play's themes. Program participants are asked to complete a survey evaluating the program's acceptability and relevance. FINDINGS TO DATE: To date, 9 of 14 program sites have returned surveys. An estimated 44% (614/1385) of program attendees completed the survey and 43% of respondents were medical students or residents. Eighty-four percent of trainees were emotionally moved``a great deal'' or``pretty much'' by the performance, (mean = 4.3; 1 =``not at all'', 5 =``a great deal'') and 64% reported that the play portrayed the emotions of dying patients in an``entirely real'' or`v ery real'' manner (mean = 4.3). Of trainees who provide direct patient care, 75% reported that the program was``extremely relevant'' or``very relevant'' to the care they provide (mean = 4.0). The program encouraged attendees to reflect on specific aspects of palliative care including helping patients live as long as possible (mean = 3.8, 1 =``not much at all'', 5 =``very much''), talking about prognosis (mean = 4.4), addressing physical pain (mean = 4.4), addressing emotional and spiritual suffering (mean = 4.4), and talking with patients about treatment wishes (mean = 4.4). The majority of trainees rated the program a more useful training tool than didactic lectures on palliative care (83%), journal article readings (85%), and bedside rounds (57%). KEY LESSONS LEARNED: The dramatic arts foster awareness of the patient experience of illness that is relevant to the care trainees provide terminally ill persons. Many trainees feel such experiences are more useful approaches to palliative care training than traditional educational methods. Medicine has partnered with a not-for-profit managed care organization, AvMed Health Plan, Inc. Students visit the administrative offices of AvMed where presentations cover general concepts of MC, physician profiling, practice guidelines, outcome management, and disease management programs. Students also``round'' in AvMed's four major departments; Pre-Authorization, Disease Management, Member Services, and Physician Services where they witness the daily operations of the health plan. A 14 item survey (5-point Likert scale) was completed by students at the beginning of the 2nd year, the beginning of the 3rd year and again immediately before (pre) and after (post) the day at the AvMed MCO. FINDINGS TO DATE: The program has been evaluated favorably by more than 200 thirdyear medical students. There were no significant differences between mean responses at the beginning of the 2nd year of school and the beginning of the 3rd year (t-test). Of the 14 items, 8 responses were neutral and 6 were negative towards MC. The responses to the pre-seminar survey were also not different from the responses at the beginning of the 2nd or 3rd years. However, post-seminar responses showed significant (p < .05) changes favorable towards MC for 7 items and > 20% of respondents abandoned their negative attitude towards MC for 5 survey items (e.g. MCO's have potential to improve quality, MCO's provide better care than the traditional system, I understand how MC functions). STATEMENT OF PROBLEM OR QUESTION: America's health care system is undergoing dramatic transformation in care management. Most medical schools, however, have not updated their curricula to prepare graduates for practice in this changing environment. OBJECTIVES OF PROGRAM/INTERVENTION: Strategies to enhance student acquisition of the knowledge, skills, values and attitudes needed to practice in intensely managed and integrated health care systems; fostering partnerships between academic centers and appropriate health care organizations; enhancing interdisciplinary primary care education in ambulatory/ community-based settings. DESCRIPTION OF PROGRAM/INTERVENTION: Project requirements include: leadership among departments/divisions of general medicine, general pediatrics and family medicine; cooperation of participating partner organizations; and establishment of learning objectives in nine content areas: health systems financing, economics, organization and delivery; evidence-based medicine; ethics; communication skills; leadership; quality measurement; systems-based care; medical informatics, and wellness and prevention. Instruction is required for all students in their clinical years, and must begin no later than the third year. FINDINGS TO DATE: The UME-21 projects within internal medicine include: Incorporating medical students in continuous quality improvement (CQI) measurements in community practices; students' use of palm pilots to monitor disease prevalence and adherence to practice guidelines in office settings; office-based instruction in physical diagnosis; web-based learning integrated into medical clerkships; utilization of simulated patients into medicine clerkships; interdisciplinary primary care clerkships; incorporation of managed care organization physicians and evidence-based medicine into medical clerkships; and epidemiology in community settings. KEY LESSONS LEARNED: With strong leadership from general internists and their colleagues, U.S. medical schools are responding creatively to the challenges of curricular innovations in this national demonstration project.
TEACHING RESIDENTS THE IMPORTANCE OF COMMUNITY TO HEALTH. E. Jacobs 1 , C. Kohrman 2 , D. Vickers 1 , M. Lemon 1 ; 1 Cook County Hospital, Chicago, IL; 2 Westside Health Authority, Chicago, IL STATEMENT OF PROBLEM OR QUESTION: Many residents care for patients from communities that are very different from their own, yet they rarely have any introduction to the lives, families, backgrounds, and cultural and social contexts of their patients. This is a crucial issue at Cook County Hospital (CCH) where many residents are international medical graduates who have had little exposure to the lives and socioeconomic struggles of our predominantly disadvantaged African American population. OBJECTIVES OF PROGRAM/INTERVENTION: All first-year internal medicine and pediatrics residents participated in the course with the goals of (1) increasing understanding of the US system of health care for disadvantaged groups; (2) improving communication with patients from diverse cultures; (3) increasing appreciation of the complexity of patients' daily lives and how it impacts their health and ability to access health care; (4) enhancing awareness of community and local services' impact on patients' well being. DESCRIPTION OF PROGRAM/INTERVENTION: The curriculum consists of 5 teaching modules taught by faculty from CCH and the Westside Health Authority (WHA), a community health advocacy organization, in 4 half-day sessions over a month-long ambulatory rotation. The first module is a case-based overview of the financing of care in the US with a focus on Medicaid, Medicare, and the impact of insurance status on health. The second module addresses problems language or culture can create in patient encounters. The third module uses a case study of opening an ambulatory clinic in a diverse neighborhood to teach about community based primary care. The fourth and fifth modules focus on the community and its citizens and are taught by citizen leaders and a sociologist. In the fourth session, the citizen leaders lead a discussion about their community and their experiences with the health care system. In the fifth session, residents have lunch with citizen leaders in the community and are given an introduction to the neighborhood and to the mission and activities of WHA. FINDINGS TO DATE: More than 50 internal residents participated in the curriculum. The evaluations of the program have been uniformly positive, with many residents indicating that it has changed the way they think about their patients and their health care. A majority has also asked that more teaching time be dedicated to these topics. An unexpected benefit has been the education and empowerment of the citizen leaders. They came to see physicians in a new light, as accessible human beings, and learned so much about health and health care that they became community health advocates. KEY LESSONS LEARNED: Residents are eager to learn about the lives, cultural and social contexts of their patients and value this kind of teaching. Communities and residency programs can create mutually beneficial relationships in the common pursuit of improved patient care and community health. national demonstration project Ð and the acquisition of a large regional primary care network (Clinical Care Associates) provided the opportunity to introduce students to the community practice environment. Our primary goal is to develop a cohesive academic primary care network for both clerkship and residency education. The specific objective of the present component of this broad initiative was to assess medical students' satisfaction with the community training environment by comparing their evaluations of newly-recruited community preceptors with those of more experienced hospital-based preceptors. DESCRIPTION OF PROGRAM/INTERVENTION: The core clinical clerkships in medicine (12 weeks) and pediatrics (6 weeks) are both equally divided between inpatient and outpatient experiences. Three weeks of the medicine clerkship are spent in a general internal medicine practice, three weeks in a family medicine practice, and six weeks in an inpatient setting. Pediatrics is comprised of three weeks in an outpatient setting and three weeks on an inpatient service. Outpatient preceptors work in 1:1 relationships with students. FINDINGS TO DATE: In the 1995 ± 96 academic year, primary care clerkship students spent a total 483 half-days in an outpatient environment, all as part of the internal medicine clerkship. Today, students spend 10,720 half-days annually in physicians' offices (internal medicine=3840, family practice=3840, and pediatrics=3040 half-days, respectively). Every student receives a minimum of 72 half-days of ambulatory (predominantly community-based) primary care training during their core clinical clerkship year. Using 9-point Likert scales in up to 15 different categories, students' ratings of newly recruited community internists and pediatricians compared very favorably with those of more experienced hospital-based preceptors. Community family physicians were more highly rated than a core group of residency-based family physicians. KEY LESSONS LEARNED: Despite the increasing demand for clinical productivity, community faculty accepted medical students with enthusiasm, and students were delighted with the opportunity to work closely with community primary care preceptors. The positive responses from students indicate that the integration of community practice experiences into the undergraduate curriculum will provide medical schools with the opportunity to expose students to diverse role models and a wide spectrum of patients in very favorable settings. The SP is a free health clinic that was started in 1996 by Tufts University medical students and volunteer physicians. The SP's mission is to improve the Chinatown community's access to health care by providing initial care and then facilitating referrals to ensure continuity of care. The clinic space is donated by a local church and laboratory services are donated by a local laboratory. A grant from the Massachusetts Medical Society, technical support from the New England Medical Center, and an annual, student-run auction, allows the SP to provide services free of charge. First and second year medical students handle the clinic's management and administration; they act as physician's assistants, case managers and medical interpreters. Students are supervised by volunteer physicians. Since 1996, the clinic has continued to expand and now offers the following: General medical care every Tuesday, 6 ± 10 PM, year round; Psychiatric and gynecological services; Onsite EKG machine and phlebotomy; Free medication samples; Referrals to primary health care providers, specialists, dentists, and optometrists; Adult and child immunizations; Anonymous HIV counseling and testing. FINDINGS TO DATE: Over the past 5 years, more than 400 medical students have volunteered at the SP. First-year medical students take over project management mid-year. The SP's success is a result of the dedication and innovation each class brings to the project. Medical students from Boston University and Harvard, as well as residents from the New England Medical Center have become involved and now contribute to the project. The following are demographics for the 1528 patient visits from 02/97 ± 12/00: Ethnicity(%) Chinese=81.7, White=9.8, Black=2.7, Hispanic=1.3; Age of Patient(%) 0 ± 19=4.7, 20 ± 39=29.2, 40 ± 59=32.2, 60+=33.9; Health Insurance(%) Yes=20, No=76. KEY LESSONS LEARNED: The SP provides an educational model for medical schools around the country. It is an example of how medical students can establish and manage a free health care clinic and actively participate in patient care during their pre-clinical years. We have learned that it is important to stress the mission of the SP to its patients. The SP is not a substitute for primary care delivered to Chinatown residents. The SP is a place where medical students can begin to take responsibility for patient care and patients can begin to access the American health care system. In an ideal teaching environment medical interns and residents are expected to assume total care of teaching patients. When the residents are bypassed in the decision making process, it is likely due to a communication problem which leads to attending physicians writing orders directly. Despite repeated efforts to dissuade attendings from writing orders at our community based program we decided to look at what other factors may contribute to this. OBJECTIVES OF PROGRAM/INTERVENTION: The goal was to improve communication between house staff and attendings and to ultimately decrease attending order writing on teaching patients. In addition we wanted to see an increase in satisfaction by the house staff, nursing staff, and attendings with the communication process. DESCRIPTION OF PROGRAM/INTERVENTION: A team consisting of house staff, nursing staff, and attendings met on a regular basis to identify key issues impeding good communication. Using an interrelationship diagraph the most important issue was identified as the nursing staff directly calling attendings on management issues, due to difficulty in identifying the house staff carrying their patient. The following interventions were developed to facilitate better identification and location of house staff. First, the intern's name and beeper number were entered on the nursing station board, as well as in the computer system which printed it daily on the nursing cardex. Second, the interns were issued personal non-cellular phones to provide direct communication with nurses and attendings. Third, the nursing station board was updated daily identifying the house officers on call. Fourth, the interns and residents were required to twice daily update their status on their beeper voice messages. Outcome measures used were orders written per patient by attendings as identified by a weekly log kept by the house staff. Second, there was a monthly survey of nursing staff, house staff, and attendings of their satisfaction with the communication process. FINDINGS TO DATE: The average number of orders written per chart by attendings was 1.7 and showed no substantial variation on a run chart since the beginning of the intervention. Poor return on the satisfaction surveys has limited any useful interpretation at this time although efforts are being made to increase their return. KEY LESSONS LEARNED: KEY LESSONS LEARNED: First, we learned that the major barrier to the communication process was the difficulty in identifying or locating the appropriate house staff. Second, we learned that the motivation for writing orders by attendings is multi factorial and often system related. We will continue to identify those other components that may contribute to order writing as we track the above outcomes. This program serves as a model that provides a proven approach to community-based learning for medical students which is translatable to traditional Western medical educational systems. DESCRIPTION OF PROGRAM/INTERVENTION: The COBES program implemented at MUFHS over the past ten years provides a valuable model for community-oriented education. The current program comprises approximately 27% of the curriculum for medical students at this nascent medical school in Eastern Africa. Under this program, students spend classroom time developing knowledge of research methodology, epidemiology and public health initiatives. During three years of the curriculum, they spend three to six weeks per year in rural communities learning community interaction skills, public health intervention, and health administration skills. In addition, they spend two years designing and implementing research projects within an urban community of their choice. FINDINGS TO DATE: Focus groups with students and faculty have demonstrated overwhelming support for the value of this community-based educational program in Kenya. Students have gained valuable training and experience within communities, experienced crosscultural interactions and have been able to conduct relevant descriptive and interventional research projects. COBES projects have won international awards for student research, but more importantly, the findings have been useful in creating public health initiatives for a variety of communities at a local level. KEY LESSONS LEARNED: The COBES program at Moi University provides an important model for community-based education that can also be applied to the Western system of medical education. The community interaction skills, public health knowledge and research techniques gained by the students will benefit them in any future endeavors, whether in a subspecialty, research or in community practice. While the immediate value of these types of programs is obvious, further observation and research will be needed to demonstrate the longlasting effects of this model of community-based education on the makeup and practice of medical practitioners in the future. Texas, provides educational services for clinicians. The medical education director of TOAETC served as consulting physician (CP) for this project. In July 2000, a 1800 number was established to accept requests for patient specific consultations from providers in Texas. The number was advertised by: 1. Mailing brochures to potential HIV medication prescribers (n=17,000); 2. Distribution of brochures at TOAETC sponsored lectures (n=421); 3. Two day clinical trainings in which the CP engaged in hands on patient care with community based HIV providers, and personally distributed the TOAETC brochure (n=24). Electronic consultations involved Email, faxes, websites, digital photography, as well as phone conversations. Examples will be displayed. All consultations were completed within 24 hours of the request. FINDINGS TO DATE: Forty-six patient specific consultations were performed between July 5, 2000 and January 5, 2001. The vast majority (87%) of requests for consultation came from community providers with whom the CP had engaged in a two day clinical training (n=40). Six requests came because of referrals from the CP's colleagues. No patient specific consultations stemmed from either the mass mailing or the direct appeals at TOAETC sponsored lectures. Total cost for the mass mailing was $36,545. Total cost for the clinical trainings was $6232. Interestingly, all requests came via the CP's private office number or his private email. The 1800 number was not utilized for its intended purpose of facilitating consultation. KEY LESSONS LEARNED: Community HIV providers are willing and able to link with providers at tertiary care centers via electronic consultation. However, our experience suggests that they are not likely to utilize electronic consultation methods without first establishing a professional relationship between consultant and consultee, as can be accomplished in a cost effective two day clinical practicum. STATEMENT OF PROBLEM OR QUESTION: Community-based educational programs pose challenges that include providing a high quality consistent curriculum across teaching sites and reducing student isolation. OBJECTIVES OF PROGRAM/INTERVENTION: To provide an interactive all day educational experience for students in our community-based medical school. Education Day is designed to: 1) Provide consistent high quality instruction for all students in key areas of the clerkship curriculum; 2) Allow students to learn from our best teachers; 3)Provide a recruiting opportunity for our community-based residency programs; and 4) Demonstrate the fun and intellectual challenge of internal medicine. DESCRIPTION OF PROGRAM/INTERVENTION: Students at our institution receive their clinical training in one of six community campuses located throughout the State. Students in the Medicine Clerkship travel to one campus for an all-day educational experience. The program includes three large group sessions and five small group workshops. Master teachers chosen from each community conduct the sessions. The three large-group sessions include: 1) Acid-Base Interpretation, an interactive lecture; 2)``Thieves Market'' where students try tò`s teal'' the diagnosis in a series of progressively disclosed cases; and 3)``Stump the Chump'' where the department chairman``thinks aloud'' working through a series of difficult clinical cases in a CPC-like format demonstrating the thought processes of an internist. Small groups of students rotate through each of five workshops, which are interspersed between the large group sessions. They include: 1) The Bedside Cardiac Exam using a Harvey (r) mannequin; 2) Starting IVs with Confidence using models and an interactive computer program; 3) Dynamic ECG Interpretation; 4) Chest X-Ray Interpretation; and 5) Utilizing the Peripheral Blood Smear. FINDINGS TO DATE: We have conducted two iterations of Education Day that included 70 students. Written and oral feedback has been overwhelmingly positive with some suggestions for minor changes. Students expressed specific appreciation for the quality of the program, a chance to see their colleagues at other campuses, and a break from the tedium of their clinical responsibilities. Several students requested expanding the program to two days. KEY LESSONS LEARNED: Education Day is one approach we have used to address the challenges of community-based education. Although it is not the entire answer, we are pleased with the initial results. While the feedback from students has been overwhelmingly positive, we look forward to assessing whether Education Day has achieved other desirable outcomes including improved shelf exam scores and an increased interest among our students in a career in internal medicine. STATEMENT OF PROBLEM OR QUESTION: Our Internal Medicine ambulatory rotation hosts a variety of learners with separate calendars and needs, and our many faculty members had their own sets of performance expectations. We had no consistent orientation process for these learners. OBJECTIVES OF PROGRAM/INTERVENTION: 1) to have an orientation program for our ambulatory site listing expectations for medical students, physician assistant students, Internal Medicine residents and Gynecology residents 2) to develop an orientation program using a minimum of faculty time and available to learners at any point during the rotation DESCRIPTION OF PROGRAM/INTERVENTION: Working with the Educational Technology Lab at MUSC, we developed an electronic, on-line orientation program for our Internal Medicine ambulatory block that can be navigated in a learner-specific fashion. This includes a virtual tour of the clinic, an overview of the patient population and expectations for medical students, physician assistant students, Internal medicine and Gynecology residents. We will present the orientation on computer notebook for perusal by attendees, and will discuss the development of the program. FINDINGS TO DATE: The orientation has been well received by faculty, staff and students. The amount of faculty time spent in orientation has been minimal. We are currently gathering formal feedback that will be available for the presentation. KEY LESSONS LEARNED: Learners must be given a set of expectations that should be communicated to them in a timely and consistent fashion. This on-line orientation is engaging, readily available and easy to update. STATEMENT OF PROBLEM OR QUESTION: With the increased demands on teaching faculty time, the need for standardized methods to assess the acquisition of auscultatory and observational skills of students is required. OBJECTIVES OF PROGRAM/INTERVENTION: This module was developed and administered to test clinical skills for medical students as part of their introduction to clinical medicine course, with focus on cardiac and chest auscultation as well as determination of radiological images findings. DESCRIPTION OF PROGRAM/INTERVENTION: A computer software (Question mark Perception) was used to present five clinical scenarios to the students. Each followed by five to six multiple choice, one-best answer questions. Radiological images (. jpg format) and sound files (. wav format) were used to present common cardiac and period in the fall of 2000. A total of nine computers were available in the Learning Resource Center of the library. Library staff members lung findings. The exam was administered to 143 second year medical students over a one-week proctored the exam. The exam was scored with the same software (Question Mark Perception) FINDINGS TO DATE: The average time the students used to complete the test was 47:35 minutes. Minimum time was 4:13 minutes and maximum time was 2 hours and 21 minutes. Of the total class, 84 % of the students took the exam in the last two days. There was virtually no waiting time, although one student where he had to wait for 5 minutes for a computer to be available. The maximum score obtained was 97%; minimum score was 58%. The software program reported the mean score, the standard deviation and degree of difficulty of each question. One question was eliminated from scoring because the sound quality was poor. KEY LESSONS LEARNED: Computer based testing of clinical skills needs further development, but is currently feasible. We plan in using the same software for an exam in spring 2001. This method of testing can also be expanded for use in clinical clerkships as well as assessing clinical skills of house staff. STATEMENT OF PROBLEM OR QUESTION: Clinical practice in community sites is an integral part of our primary care internal medicine training program. Despite this, many of our residents remained unfamiliar with the practice of community-oriented primary care (COPC). In order to teach them about and encourage them to practice COPC, we developed an eightweek course in community medicine. In conjunction with this course, we designed an intranet website to facilitate rapid learning of COPC. OBJECTIVES OF PROGRAM/INTERVENTION: The goals of our intranet site were to: 1) provide residents with readily accessible information about community medicine; 2) prompt residents to consider, discuss and learn about COPC on a near-daily basis; and 3) introduce distance learning concepts into the residency program. DESCRIPTION OF PROGRAM/INTERVENTION: We located an internet website (http:// intranets.com/) that supported the creation of free intranet sites. Once created, access to the intranet site was restricted to the primary care residents and selected faculty. The intranet site (http://pcimcommunitymed.intranets.com) featured discussion groups, documents and readings, links to important public health internet sites and a weekly course calendar.
Residents were oriented to the intranet site during the first session of the community medicine course. Each week, residents were asked to respond to an on-line discussion question. Homework exercises and additional reading assignments were also posted on-line. Residents were required to use several internet links to gather neighborhood public health data and to propose a COPC project. FINDINGS TO DATE: 73% of the residents who participated in the community medicine course (11/15) responded to an anonymous post-course evaluation posted on the intranet website. 73% of the respondents (8/11) found the intranet site enhanced their learning of community-oriented primary care. 9% (1/11) found it neither enhanced nor detracted from their learning experience and 18% (2/11) felt it detracted from their learning. 40% of the respondents (4/10) used the intranet site more than once per week; 50% (5/10) used it once per week; 10% (1/10) used the site less than once every two weeks. The median number of visits to the intranet site during the eight-week Community Medicine course was 11 per resident (range 3 to 60). Seven residents did not participate in the on-line discussion groups. Key reasons for lack of participation included the amount of work assigned for the course (33%), lack of internet access at home (27%) and difficulty navigating the intranet site (20%). KEY LESSONS LEARNED: Use of an intranet website shows promise as a tool to facilitate rapid learning of concepts in community-oriented primary care. STATEMENT OF PROBLEM OR QUESTION: A significant portion of graduate medical education is based on exposure to a variety of clinical experiences. For residents training in internal medicine the majority of these interactions occur in the inpatient setting. It is important not only to document the range of diagnoses that residents encounter, but also to track length of stay and readmission rates, two important markers of the quality of inpatient care. Quality markers may be linked to attendings supervising the residents and may indicate topics for faculty development. OBJECTIVES OF PROGRAM/INTERVENTION: We developed a computerized database to 1) track inpatient diagnoses that IM residents encounter on a general medical service; 2) track length of stay, readmission rates, and other performance quality measures for particular diagnoses; and 3) link resident practice measures with supervising attendings to identify areas for faculty development. DESCRIPTION OF PROGRAM/INTERVENTION: We created a computerized database in Microsoft Access. The database documents every patient admission to the internal medicine teaching service; the medical team (residents and attending) responsible for the patient's care; primary and secondary diagnoses listed for the patient (from the discharge summary); admission and discharge dates. The database user interface is organized with multiple data tables supporting``drop boxes'' to click on the appropriate physician names and diagnoses. An administrative assistant enters the data, and any diagnoses not listed in the data tables are referred to a physician for clarification. FINDINGS TO DATE: This computer-based information system allows tracking of all diagnoses seen by IM residents on a general medical service. The data can be used to compare each resident's exposure to specific diagnoses with his/ her performance on in-training exam sub-scale scores; educational interventions can be developed to enhance medical knowledge in those areas identified as having low exposure and low in-training score. The documentation of residents' experiences also fulfills the Residency Review Committee's requirement for documentation of IM residents' diagnostic experience. The database is used as part of hospital-wide disease-specific quality improvement initiatives to review length of stay and readmission rates on the teaching and non-teaching services, and to target physician education at all levels to improve patient outcomes. Future links between this database and pharmacy and laboratory utilization measures will permit even more detailed analyses of physician performance. KEY LESSONS LEARNED: An accurate computerized tracking system is a useful educational and quality improvement tool for residents and attendings in internal medicine residency programs. This database recognizes the value of integrating measures of quality improvement into residency training, and may be used by training programs in other disciplines to track clinical performance. STATEMENT OF PROBLEM OR QUESTION: When faced with teaching physical examination skills to a large class of medical students (as well as coordinate the teaching efforts of a diverse group of physician teachers), it is a challenge to ensure that students are exposed to a common set of physical exam findings and find techniques that optimize their opportunity to develop physical exam skills. OBJECTIVES OF PROGRAM/INTERVENTION: To expose students to certain common abnormal physical exam findings and enhance learning of physical examination skills through the integration of new technologies and traditional educational strategies. DESCRIPTION OF PROGRAM/INTERVENTION: Computer-based technology is becoming an integral part of curriculum innovation at Ohio State University. As a part of this pedagogical effort, an innovative, highly interactive website was developed to promote active learning at a level appropriate for undergraduate medical students and to be compatible with the current Physical Examination Course curriculum. This web-site makes use of Macromedia Flash, video streaming, and SMIL technologies to bring to life portions of the physical exam and to make available common abnormal findings often difficult to demonstrate to medical students at any specific time. It emphasizes learning through reading, hearing, seeing, and doing in a sequence designed to capture the learner's attention and to make information available through multiple sensory modalities. There are 6 core modules (HEENT, pulmonary, cardiac, abdominal, neurologic, musculoskeletal) under development. Each module includes the following: a Flash-based interactive component highlighting important anatomy and physiology; web text and video clips discussing and demonstrating essential physical examination skills; Flash-based interactive components where students hear findings or practice selected physical examination skills (e.g. taking a blood pressure and hearing Korotkoff sounds); a glossary of clinical terms; links to clinical pearls and additional explanations of techniques or abnormal findings; and clinical correlates where students practice integrating physical examination findings. This exhibit will showcase interactive portions of the modules via a CD-ROM presentation. FINDINGS TO DATE: Pilot data were collected and of the nine respondents, all reported that this site was an excellent learning tool, desired further development of similar technology, and would recommend this site to other students. Eight of the nine students reported being able to navigate the site and use the interactive Flash components without difficulty, and stated they would be more likely to use this site than the textbook to prepare for practice sessions with faculty tutors. Overall, students found it helpful to hear findings, liked the links built into the site, and enjoyed the clinical correlates component. The only weakness identified was the time to download the video clips if access was obtained from modems as opposed to a high-speed internet connection. KEY LESSONS LEARNED: Next Steps: Data are currently being collected with a larger sample concerning the use and effectiveness of this site to guide subsequent revision of current modules and to plan the design of future ones. Also being considered is the development of patient cases representing core disease presentations, as well as modules specifically designed for educators in this area. STATEMENT OF PROBLEM OR QUESTION: Advances in evidence-based research, together with unprecedented access to original sources of information, provide a unique challenge to medical decision-makers that may improve the quality of care provided to their patients Ð if they can find and correctly interpret this readily available information. OBJECTIVES OF PROGRAM/INTERVENTION: Our aim was to instruct clinicians accessing and interpreting online sources of evidence-based research using a cognitive theory that has been applied to clinical decision-making. DESCRIPTION OF PROGRAM/INTERVENTION: Decision-making tasks concerning chest pain evaluation in women were developed for medical students and internal medicine residents. The cognitive theory Ð fuzzy-trace theory Ð guided the selection of online sources (e.g. target articles) and decision-making tasks. FINDINGS TO DATE: Thiry-four participants (12 students and 22 internal medicine residents) attended didactic conferences emphasizing search, evaluation, and clinical application of evidence. A 17-item Likert scale questionnaire assessed participants' evaluation of the instruction. Ratings for each of the 17 items differed significantly from chance in favor of this alternative approach to instruction. KEY LESSONS LEARNED: Fuzzy-trace theory was found to be a useful guide for developing internal medicine learning exercises in medical informatics and decision making. However, studies with more learners and behavioral evidence of improved clinical decision-making skills and, ultimately, patient outcomes are needed. STATEMENT OF PROBLEM OR QUESTION: Scheduling conflicts prevent many residents, especially those on``non-ward'' rotations, from attending morning report. Even when able to attend, however, residents may miss salient learning points presented during those conferences. In order to circumvent these problems, we created eReports, electronic summaries of morning report distributed to all of the internal medicine residents by E-mail. OBJECTIVES OF PROGRAM/INTERVENTION: The goals of our intervention were: 1) to provide all of our residents with the learning opportunities of morning report even if unable to attend; 2) to model the practice of asking and answering patient-centered questions in an evidence-based way; and 3) to strengthen the educational impact of morning report by cogently synthesizing information presented, further emphasizing salient teaching points, and providing additional relevant information. DESCRIPTION OF PROGRAM/INTERVENTION: Each day, we developed a summary of the material presented in morning report and distributed it to all of the residents by Email. These summaries, which we called eReports, mirrored the structure of our morning reports. Each eReport began with follow-up information that included clinical follow-up to previously presented cases and literature-based answers to questions generated by those cases. We then summarized the two new cases discussed in morning report. At the end of each eReport, a task (e.g., view the peripheral smear) or a central question (e.g., how helpful would a positive result of your planned diagnostic test be?) was generated for each new case. FINDINGS TO DATE: Residents were asked to complete an anonymous survey prior to the dissemination of eReports. 81% of the residents responded. Of these, 71%``almost never'' or only``occasionally'' knew the content of morning report (cases or follow-up) when they were unable to attend. 88%``strongly agreed'' that eReports would be educationally helpful. Further investigation is underway to determine if eReports were, in fact, helpful. For example, did they successfully clarify teaching points, stimulate self-directed learning, or contribute to improved memory of cases and information presented in morning report throughout the rotations studied? KEY LESSONS LEARNED: eReports are a promising way to facilitate case-based learning among internal medicine residents. Not only do they ensure the dissemination of the material presented in morning report, they also afford a synthesis that strengthens the quality and content of that material. STATEMENT OF PROBLEM OR QUESTION: The ACGME has endorsed competencies in 6 core areas as a first step to emphasize educational outcome (rather than process) assessment for residency program accreditation. The guidelines require the residency programs to provide an appropriate educational experience in these areas, evaluate the residents' performance, provide feedback and achieve progressive improvements in residents' competence and performance. In addition the effectiveness of the educational program itself also needs to be evaluated. To achieve the ACGME goals, residency programs must develop a new educational model that will engage residents in a time efficient manner. In addition to their areas of clinical training, the new curriculum should cover areas such as doctor patient communication, medical ethics and professionalism. OBJECTIVES OF PROGRAM/INTERVENTION: We have designed and are now implementing a web-based interactive curriculum that would meet common and specific needs of various residency programs at our institution. The objectives of this web-based program are: 1. The curriculum is available at a convenient time and place convenient to the residents. 2. The residents' decide the order and pace of the curriculum. 3. The curriculum utilizes existing teaching materials as far as possible. 4. The new curriculum makes efficient utilization of educators' time and skills. 5. An efficient and effective evaluation and feedback system is an integral part of the new curriculum. DESCRIPTION OF PROGRAM/INTERVENTION: We designed the web-based curriculum to be based on interactive, question and answer scenarios. The key features of this program are: 1. The curriculum is on a password protected web site 2. The curriculum consists of brief interactive case scenarios that are grouped in the 6 core competency areas endorsed by the ACGME. 3. These case scenarios can be created by any educator by copying and pasting text from a text document into the text box of a separate development web site. Content is automatically converted into interactive HTML format and placed on the web site. Audio, video and graphic files can also be included. 4. Pretests, posttests, multiple-choice questions and free text entry areas can be included to capture the users responses, track performance and assess competency. In order to simulate a real-life scenario, the residents have an option to order consults and clinical tests to solve the cases. 5. The program provides extensive feedback including tracking amount of money spent while resolving a case scenario, amount of questions answered correctly at first attempt, number of attempts and the ability to compare themselves with other residents who have tried that particular scenario. 6. The program creates a customized recommended reading material based on the questions answered incorrectly. 7. If a user leaves a case scenario incomplete, the progress is saved and can be continued from that point when the user logs on the next time. 8. The program uses a database of house staff to create individualized reports for program directors on the participation and performance of their residents, and also is used to assign particular cases to a particular group of residents. FINDINGS TO DATE: We plan to formally implement the program beginning in the July 2001 academic year. Pilot testing has shown this system to be acceptable to the house staff, attractive to the program directors and effective and efficient method of teaching for the educators. KEY LESSONS LEARNED: As new requirements are placed on a medical education system strained for resources and funding, creative solutions are needed that will make efficient use of existing education material, to meet the needs of the students, educators and organizations using current technological advances in computers and the Internet. Using appropriate technology it is possible to create stimulating and educational material that also seamlessly combines tracking, feedback and evaluation and thus efficiently meet the ACGME goals.
WEB-BASED INTERACTIVE CURRICULUM FOR THE ACGME OUTCOMES PROJECT. N.B. Mehta 1 , A. Hull 1 , J.H. Isaacson 1 , A. Jain 1 , 1 Cleveland, Cleveland, OH STATEMENT OF PROBLEM OR QUESTION: The ACGME has endorsed competencies in 6 core areas as a first step to emphasize educational outcome (rather than process) assessment for residency program accreditation. The guidelines require the residency programs to provide an appropriate educational experience in these areas, evaluate the residents' performance, provide feedback and achieve progressive improvements in residents' competence and performance. In addition the effectiveness of the educational program itself also needs to be evaluated. To achieve the ACGME goals, residency programs must develop a new educational model that will engage residents in a time efficient manner. OBJECTIVES OF PROGRAM/INTERVENTION: 1. Implement a web-based interactive curriculum that would meet common and specific needs of residency programs 2. The curriculum be available at a time and place convenient to the residents. 3. The residents' decide the order and pace of the curriculum. 4. Utilize existing teaching materials as far as possible. 4. Make efficient utilization of educators' time and skills. 5. Incorporate an evaluation and feedback system seamlessly in the new curriculum. DESCRIPTION OF PROGRAM/INTERVENTION: 1. The curriculum is on a password protected web site and consists of brief interactive case scenarios (ICS) grouped in the 6 core competency areas. 3. The ICS can be created by an educator without programming knowledge by automatically converting text into interactive HTML format on the web site. Audio, video and graphic files can also be included. 4. Various question formats can be included to capture the users' responses and assess competency. The residents can order consults and clinical tests to solve the ICS. 5. Extensive feedback includes amount of money spent while resolving an ICS, number of attempts and questions answered correctly at first attempt and comparison with peers. 6. The program creates a customized recommended reading material based on performance. 7. If interrupted, the user's progress is saved for subsequent sessions. 8. The program creates individualized reports for program directors on the participation and performance of their residents. FINDINGS TO DATE: We plan to formally implement the program beginning in the July 2001. Pilot testing has shown this system to be acceptable to house staff, attractive to program directors and effective and efficient method of teaching for the educators.
KEY LESSONS LEARNED: As new requirements are placed on a medical education system strained for resources and funding, creative solutions are needed that will make efficient use of existing education material, to meet the needs of the students, educators and organizations. Using current technological advances in computers and the Internet, one can create stimulating and educational material that combines tracking, feedback and evaluation and thus efficiently meet the ACGME goals. STATEMENT OF PROBLEM OR QUESTION: There is interest in using the Internet to assist in teaching medical clerks ECG reading skills, but multiple technical barriers have impeded efforts thus far. These include inability to place high quality ECG images on the Internet and failure to utilize software that allows interactivity yet is supported by most web browsers. OBJECTIVES OF PROGRAM/INTERVENTION: To develop an internet-based, comprehensive ECG tutorial that is interactive and can be viewed by standard web browsers. DESCRIPTION OF PROGRAM/INTERVENTION: We developed a new method for digitizing paper ECG files. We then digitized our complete ECG library used for our four session, clinical clerkship ECG tutorial. These high quality images were placed in web pages using multimedia development software that can be viewed with most web browsers. FINDINGS TO DATE: An online ECG tutorial is now available to all internal medicine clerks. It covers 4 chapters of material and includes over 30 ECG cases. The interactivity allows users of various skill levels to choose the amount of assistance they receive in interpreting the cases. KEY LESSONS LEARNED: A large amount of effort was required to overcome technical barriers. Once these issues were overcome, further work was very efficient. The solutions to these technical issues can be easily shared. STATEMENT OF PROBLEM OR QUESTION: Evidence-based medicine and Internet resources have become increasingly important in the organization and philosophy underlying morning report. Electronic media is easily available to house officers in real time and this often supercedes the limited, outdated content in standard textbooks. Managing that data and integrating it into an educational culture is a new challenge for residents and faculty alike. OBJECTIVES OF PROGRAM/INTERVENTION: Prior to implementation of the new paradigm, the chief resident conducted morning report and the post-call senior resident presented the case. Faculty, other senior residents, interns, and medical students were present in the room. Qualitative surveys of residents at the time revealed concerns of knowledge stratification between interns and senior residents, need for more current handouts, and a desire for more involvement by the interns and medical students in discussion. In the new model, two chief residents conduct morning report and the two-day post call intern presents the case. No senior residents or faculty are present in the room. DESCRIPTION OF PROGRAM/INTERVENTION: We describe a novel morning report format that:
1. Separates intern and senior morning reports to alleviate pressures of knowledge stratification and to enhance the learning experience for each group. 2. 3. Facilitates learner interaction by optimizing learning climate and geography (e.g., location,
food, and open table settings). 4. 5. Implements two active facilitators to engage the group, maximize participation, and utilize complementary skill sets. 6. 7. Applies educational and computerized resources including electronic databases, networked computers, and data projectors to develop an efficient and educationally driven case selection process. 8. 9. Incorporates electronic tools (``E-tools'') to adapt quickly to learner goals and needs, based on time of year, learner experience and learner interest. 10. FINDINGS TO DATE: Anonymous intern evaluation data suggest that the new model has effectively addressed these issues. Based on current academic year data of almost 100 responses, all interns consistently felt either``challenged'' or``interested''. Almost 25% of interns in fact preferred a longer morning report. The use of electronic handouts has met with universal acceptance: 77% thought the handouts were``very good'' and 23% considered them``good''. Only one resident had a neutral opinion and there were no negative responses. Almost 96% of respondents agreed with exclusion of faculty and senior residents in morning report. Interns who were exposed to the morning report prototype (n=47) had a significant 9.7% increase in average ITE percentile scores (p = 0.015, unpaired t-test) compared with interns not exposed to the model (n=96). KEY LESSONS LEARNED: We conclude that our morning report model has increased the level of participation among interns, alleviated pressures of knowledge stratification between interns and residents, and successfully utilized electronic tools to enhance and adapt to a learnercentered environment. Areas for further study include successful adaptation of the model to the dynamic needs of report participants, correlation with the ITE, development of more specific outcome metrics, and impact on patient care.``I The third year of medical school can be particularly stressful for students. Some students are reluctant to access their school's mental health resources for fear of appearing``weak'' or unable to cope in a highly competitive environment. OBJECTIVES OF PROGRAM/INTERVENTION: To help students cope with the stresses of their clinical clerkships, the Program for the Humanities in Medicine at Washington University School of Medicine instituted the Clerkship Counseling Hotline. DESCRIPTION OF PROGRAM/INTERVENTION: The Clerkship Counseling Hotline was a confidential, anonymous service intended to provide third year students with a short-term mental health resource aimed at easing stress. The hotline was staffed by a masters level counselor around the clock. FINDINGS TO DATE: 17 calls were received by the Hotline from August 1999 through November 2000. Issues prompting calls, in order of frequency, included a) disillusionment with medical environment; b) anxiety over performance/evaluation; c) personal/relationship problems; d) requests for information/referral; and e) anxiety over decision to go into medicine. 15 of the 17 callers were female. A year-end survey assessing the Hotline was returned by 83% of the students. 75% of all students said that continuing the Hotline was``somewhat important'' or``very important,'' and 75% of students described the availability of the Hotline as`s omewhat reassuring'' or``very reassuring.'' 88% of women found the hotline reassuring, compared to 58% of men (P=.002). KEY LESSONS LEARNED: This Clerkship Counseling Hotline was regularly used by our 3rd year students, who strongly endorsed its continuation. Female students used the Hotline substantially more frequently than did male students. Such a Hotline may provide a service that is complementary to the traditional mental health resources offered by most medical schools. Comply with the ACGME-RRC scholarly activity requirement (SAR). 3. Evaluate the program. DESCRIPTION OF PROGRAM/INTERVENTION: Key components of our SAP include: precise delineation of requirements; dedicated time of a research director and coordinator; core curriculum with didactics (trial design, test interpretation, meta-analysis, bioethics, medical writing, informatics, computer graphics), journal club focused on trial design, optional clinical teaching seminars, and an elective 10-hour biomedical writing course; optional research modules; SAP manual; staff mentors and institutional support. The SARs include: participation in didactic program and journal club; completion of a pre-approved written project; completion of a (staff evaluated) 30 ± 60 minute oral presentation using computer-generated slides; and a 1:1 literature search session with a librarian. An annual research day includes: residents' poster presentations (in 2000: 33 posters) with a reception and a visiting professor lecture. Two selected research projects are presented by the residents at a medical grand rounds. Our 2001 budget of $26,340 includes: resident travel to present at meetings, printing of SAP manual, biomedical writing course, Research Day expenses, and general operating costs. The budget does not include salaries for Research Director (10%) or Coordinator (50%). FINDINGS TO DATE: Since inception of our SAP, resident publications and presentations have increased (in 2000: 10 published articles, 12 abstracts, 42 presentations at national or regional meetings). The SAP and the research mentor's department co-support residents' travel to present at meetings. The most common written project is an original research abstract; case reports and simple literature reviews are not acceptable. A survey of our graduating residents indicates acceptance of the SAP (20 of 29 PGY-3 residents felt the SAP to be``very valuable''). KEY LESSONS LEARNED: We believe the success/acceptance of our SAP are due to an established core curriculum, supportive personnel, dedicated funding (in particular for resident trips to meetings), availability of staff mentors, strong institutional resources, and meticulous tracking of residents' progress.~50% of our residents take 1 ± 2 research modules, which must be approved in advance. No relation exists between the type of project and whether residents took a research module. and an audience response system was also used to administer the questionnaire during a noon conference FINDINGS TO DATE: Thirty five HS participated in this survey; (48% GL-I, 24% GL-II, 14% GL-III, 7% GL-IV and 7% GL-V). Most of them thought the book was trustworthy (87%), accurate (86%), user friendly (57%), and overall useful for the management of patients (87%). Although most HS always carried the CCF book (73%), most also carried other commercially available antibiotics recommendations book(s) (68%). There was no consensus which of these books was more useful for them. The CCF Book sections on guidelines for specific diseases and dosing information were referred to most often (37% & 40%; respectively). Requests were made to add more microbiology information and other clinically important antimicrobial drug interactions. KEY LESSONS LEARNED: The CCF Guidelines for Antimicrobial Usage book is well respected among the HS. The book serves it's purpose well in helping HS with their daily care of hospitalized patients. This survey helped us in implementing some changes that we believe will improve these guidelines and consequently, improve the quality of care for our patients.
DIVING FOR PERLS: USING PORTFOLIOS FOR EVALUATION AND REFLECTION ON LEARNING. K. Edwards 1 , L.E. Pinsky 1 ; 1 University of Washington School of Medicine, Seattle, WA STATEMENT OF PROBLEM OR QUESTION: Lifelong learning is an important goal of medical training. However, objective tools for teaching skills of self-assessment and for giving feedback on professional development are lacking. Medical training is an experiential process and reflection is necessary to develop expertise and professionalism from the practical experiences. OBJECTIVES OF PROGRAM/INTERVENTION: Residents participating in the portfolio program will: 1) learn how to set achievable learning goals; 2) identify learning experiences that will facilitate achieving learning goals; 3) develop skills of self-assessment, identifying strengths and weaknesses 4) receive structured peer and supervisor feedback in a range of skill areas 5) develop important professional skills that support life-long learning, physician-patient communication, and excellence in patient care. DESCRIPTION OF PROGRAM/INTERVENTION: Portfolios constitute an integrated system of learner-directed evaluation. The primary outcome of the evaluation project is for each resident to create a portfolio that includes a comprehensive collection of different types of feedback and self-assessment. The components of the portfolio may vary, but are based on specific activities to assure broad coverage of skill areas. They include: worksheets on goal setting, tracking and self summary of learning, teaching self-evaluation, colleague feedback; critical incident type narratives; mini-CEX observations; CD-ROM compilation of the resident's videotaped encounters with patients, directed self-evaluation, a summary of resident/attending meeting, and overall department evaluation. There is a Working Portfolio for the resident's use and work-in-progress, and a Performance Portfolio for highlighting core competencies and strengths at the end of the year. FINDINGS TO DATE: After a pilot program with the internal medicine residents, a surprising number of the portfolio skills and objectives needed significant attention by the mentor faculty. Simple goal setting exercises presented challenges for some residents. This finding highlighted the need for the portfolio exercise within the residency program. KEY LESSONS LEARNED: We identify 5 elements critical to the success of a meaningful and effective portfolio intervention: 1) distinguishing the goals of the Working and Performance portfolios; 2) establishing a collegial educational climate; 3) teaching and refining selfassessment acumen; 4) promoting reflection on goals and progress over time; and 5) supporting structured autonomy via iterative discussions with mentors. Without these elements, trainees may view a portfolio simply as another tedious exercise and we risk trivializing an innovative tool. STATEMENT OF PROBLEM OR QUESTION: Community based ambulatory educational experiences have been increasingly emphasized in medical education due to the need to prepare medical students and residents to become more knowledgeable about primary care medicine in the era of managed care. At our institution, students start working in community based practices in the first year of medical school. Effectiveness of the students' educational experience in the community has frequently varied depending on the skills and interests in teaching of the physicians participating in the program. All physicians in our network are required to teach medical students at least 1/2 a day a week but little faculty development in teaching has been provided. How to effectively address the needs of these faculty is addressed in this innovative peer teaching program. OBJECTIVES OF PROGRAM/INTERVENTION: In order to improve the overall experience for both students and faculty, our institution has developed a``Teach the Teachers'' program. The primary objective of the program is having community based faculty associated with our institution's primary care network teach their peers skills in effective teaching and mentoring of medical students. It is also an objective to develop a core of community based faculty who will be able to provide leadership in the faculty development arena for our community based teachers of medical students. DESCRIPTION OF PROGRAM/INTERVENTION: Over the course of a year, the university staff collaborated with four affiliated community based primary care faculty to guide them in selecting the curriculum modules they felt would be most useful to office based teachers. The team also participated in the nationally organized General Internal Medicine Faculty Development Project (GIMGEL) where additional skills and training techniques were acquired. In order to develop a quality program, the community faculty selected four curriculum areas as well as a kick-off session for their program activities. They also worked with the Sr. Associate Dean for Medical education to make sure that the chairman of the Department of Medicine supported the program and would strongly encourage all of the university's primary care network of physicians (total of 91) to participate in at least 20 hours of faculty development over a two year time period. CME was also offered for all faculty development. The program selected includes the following modules:
1. The kick off program( one time session for all participants), Developing Teaching Skills for the Primary Care Physician A nationally known physician educator, Gary Ferenchick, MD, MS, was selected to present the basics of community based teaching to the entire group of primary care network physicians at one of their required dinner meetings. The speaker was chosen for his enthusiastic hands-on approach to office-based teaching. The speaker at the end of his talk then introduced the community based faculty development program and the four peer faculty. Each of the four peer faculty developed an interactive module to be presented to each of four groups of teaching physicians at dispersed sites around our large urban service area. They selected these topics based on a needs survey and their own personal experiences in teaching. These topics include the following:
2. Orienting and Organizing the Student Experience 3. The One Minute Preceptor-Teaching on the Run 4. Arrows in the Quiver: Using Multiple Strategies to Reach Your Student 5. Developing Effective Techniques for Student Evaluation and Feedback FINDINGS TO DATE: In an earlier program of training of community based faculty by our institution, training of community based faculty was very successful but the lack of departmental support for the training led to smaller numbers of faculty participating. This program reviewed the prior one and developed a different strategy. The four community based faculty have spent two hours each month for the past year in preparation for the training program in addition to attending the three day GIMGEL conference on faculty development. They have reviewed major articles and materials on curriculum development in order to select those they felt most appropriate based on the initial needs assessment of their peers. They have obtained the Chair of Medicine's support for the program as well as for the publication of the four curriculum articles. The program times are scheduled and the kickoff session is set. Success of the program will be reported on at the conference. KEY LESSONS LEARNED: Community based faculty need to be involved in leadership roles in faculty development for their peers. The topics may not be new to the field but having peers teaching peers is innovative and an effective strategy for encouraging the acceptance and adoption of new teaching skills. Materials Produced and Available A series of four curriculum articles written by the four community based faculty and workshop handouts will be available for those interested in implementing a similar type of peer training program. A video will also be available to demonstrate the interactive modules. For this reason medical schools are devoting time and resources to prepare faculty for providing students with effective feedback. In an effort to reach more faculty and allow members to learn at their own pace, we designed a web-based faculty development module to address the relevant skills. OBJECTIVES OF PROGRAM/INTERVENTION: -Improve the observation and feedback skills of faculty.
-Design an interactive web-based teaching module utilizing multimedia. DESCRIPTION OF PROGRAM/INTERVENTION: The web-based module is the first of a series of teaching modules planned to support the Macy Initiative in Health Communication at NYU (http://endeavor.med.nyu.edu/macy/nyumacy/). The Macy Initiative is a three school (New York University, University of Massachusetts and Case Western University Schools of Medicine) project funded by the Macy Foundation to improve the communication skills of physicians. Part of the faculty development efforts at NYU include plans for several on-line teaching modules. The observation/feedback module contains three sections. In the first section participants are asked to watch a 1st year student interview and then to complete a checklist. During the second section, the participant's checklist data is compared with that of several expert faculty. In the last section, participants are taken stepwise through the feedback process. They begin by orienting the student, then by asking for the student's own self-assessment, followed by giving feedback and finishing with the closing. At each step participants are asked to enter their own comments and then watch a video clip of the same 1st year student receiving feedback from an expert faculty member. Seminar leaders for the current Physician, Patient and Society Course as well as Primary Care Residents will be asked to take the module. Participants fill out a pre-post survey to assess changes in attitudes and confidence in giving feedback and to comment on the module. FINDINGS TO DATE: It is feasible to design an interactive web-based faculty development module. We are able to track which faculty have completed the module using a log-in page and retrieve faculty responses anonymously by having programmed the module to send them to a separate web-site without identifiers. Data on the changes in attitudes and confidence as well as feedback on the module will be available. KEY LESSONS LEARNED: -Helpful collaborations can take place among Clinical and Academic Computing Departments. -These collaborations often require learning each others`l anguages'' and involving project managers. -Interactive digital media is a very exciting, innovative way to teach the medical interview. DESCRIPTION OF PROGRAM/INTERVENTION: Our program was comprised of an interactive discussion entitled``What Every Clinician Needs to Know About Quality'' led by an international expert in QI, followed by a series of observed structured teaching experiences (OSTEs). These OSTEs were case-based teaching modules utilizing role-playing to address QI issues encountered in typical clinical cases. The program was evaluated through a formal discussion with participants at the conclusion of the seminar along with pre and post-program surveys. FINDINGS TO DATE: Twenty faculty from the Departments of Medicine and Family Medicine participated in the program. The faculty was surveyed pre and post-program on their knowledge, competency to lecture about, and competency to teach clinical applications of QI. A 10-point scale was utilized, with 0 indicating no knowledge or competency, 5 being neutral, and 10 indicating superior knowledge or competency. In addition, attitudes towards teaching about QI were surveyed pre and post-program, using a 5-point Likert scale. With an 85% response rate, perceived knowledge of QI increased from below neutral to above neutral in 29% of respondents. Competency to lecture on QI increased from below neutral to above neutral in 41% of respondents. Most importantly, competency to teach clinical applications of QI increased from below neutral to above neutral in 59% of respondents. The perceived value of teaching about QI also increased, (p = 0.04) and faculty uniformly commented on how this program reinforced the importance of (or value of) teaching this essential clinical competency. KEY LESSONS LEARNED: QI issues are seen in daily clinical practice and need to be addressed with medical students and residents. Faculty development through interactive role-playing and discussions can be influential in changing attitudes and self-perceptions of teaching abilities. Objectives of Part I were to enable faculty participants to understand the purpose of incorporating genetics into primary care teaching and practice, identify specific teaming needs for home faculty and available resources, develop a 6-month implementation plan and acquire teaching techniques to incorporate genetics into primary care teaching and practice. DESCRIPTION OF PROGRAM/INTERVENTION: Part I of the two-day GPC Training Program held in October 2000 included didactic and interactive plenary sessions, content-based breakout sessions, a poster session, and computer demonstrations. Twenty competitively selected faculty teams were paired to enhance interaction and to facilitate discussions with advisors (members of the GPC Advisory Committee or Genetics Education Consultant Committee) regarding specific implementation plans for faculty development. A draft curriculum manual, largely case-based in its organization with information concerning electronic and written resources, was prepared for use by participants and faculty in the course. FINDINGS TO DATE: Findings from the external evaluation of Part 1 of the GPC Training Program show that the greatest self-reported increase in knowledge and skills are in the areas of resources, development of a specific implementation plan, and in applying teaching techniques for addressing ethical, legal, and social implications of genetics in medicine. Participants say they need more help with genetics content and with around specific methodologies for delivery of genetics-based faculty development to primary care faculty. KEY LESSONS LEARNED: Meaningful incorporation of additional genetics content into Part 2 of the GPC Training Program is indicated. Additionally, more interaction time and opportunities for team members to observe and practice specific methods of delivering training may enhance participant teaming. students were randomly assigned to participate in a 2-week ambulatory block during their 2 month required IM clerkship; the remaining 6 weeks were spent on inpatient wards. The remaining 17 students completed 2 months of inpatient wards. The settings included: community private clinics, VA Clinics, and faculty practices. The 2-week period was comprised of clinic 5 half days per week, and 4 half days of small group interactive sessions covering general medicine topics. Students were required to present an interesting case they were exposed to during the ambulatory portion of the clerkship. FINDINGS TO DATE: Compared with inpatient students there was no difference on their performance. However, 60% were more likely to go into Internal Medicine, while only 33% stated it had no effect on their career choice. They enjoyed working in the outpatient setting, and saw on average 3 ± 4 patients per 1/2 day. Approximately 50% watched procedures, while 20% were able to participate in procedures such as pelvic exams, joint injections and mole removal. They saw a wide range of problems including chronic medical diseases, skin diseases, smoking related issues, as well as preventative medicine. Overall, the students enjoyed their outpatient experience and would have preferred more time in the clinic setting. KEY LESSONS LEARNED: Even though there was no statistically significant difference in how well the students performed on their examinations, they did view this two-week ambulatory block as a positive experience. In addition, we feel it is significant that 60% of the students reported they were more likely to go into Internal Medicine despite this very brief exposure to outpatient medicine. STATEMENT OF PROBLEM OR QUESTION: The most common deficiency among our graduating residents was a lack of preparation for dealing with the business side of medicine. What is the best way to introduce third party payment systems and the intricacies of running an office practice to residents? OBJECTIVES OF PROGRAM/INTERVENTION: We hope to create an ambulatory experience that would combine patient care with an educational focus on medical business. The residents will have an introduction to billing and coding. They will become familiar with the different payer sources and how they relate to patient care. The curriculum will allow residents to apply the basics of managed care (including formularies and referral) while taking care of patients. The residents should also develop a basic knowledge ethical conflicts that exists within insurance entities. DESCRIPTION OF PROGRAM/INTERVENTION: The educational curriculum consists of one month rotations in ambulatory clinics with significant managed care exposure. The clinic experience is supplemented with lectures on billing, coding, insurance basics, and ethics. Residents provide input on deficiencies in their knowledge and which topics should be expanded. Residents also receive hands on experience by observing ancillary office staff in their different duties (billers, referral staff, etc.) in order to help solidify their learning experience. FINDINGS TO DATE: Residents show improvement in their ability to bill and code appropriately. They also are satisfied with their learning experience and think it should be expanded. KEY LESSONS LEARNED: Knowledge of the business aspect of medicine has become an essential part of medical practice. Residency programs need to address this need in educating their residents. Residents are very receptive to lectures on medical business and see these topics to be an important supplement to medical education.
T. Houston 1 , R. Connors 1 , N. Cutler 1 , M. Nidiry 1 ; 1 Johns Hopkins University, Baltimore, Maryland STATEMENT OF PROBLEM OR QUESTION: Although a highly rated Rheumatology rotation exists, our residents rated their training in``primary care'' musculoskeletal medicine (knee pain, biceps tendonitis, epicondylitis, ankle sprain, injections, etc.) as less than optimal. OBJECTIVES OF PROGRAM/INTERVENTION: Success in``Primary Care'' Musculoskeletal Medicine training was limited by lacking primary care preceptors trained in musculoskeletal problems and by not having a concentrated collection of patients with musculoskeletal problems. Our OBJECTIVE was to establish an innovative primary care musculoskeletal medicine clinic precepted by general internists with additional training in joint issues. DESCRIPTION OF PROGRAM/INTERVENTION: In the planning year, faculty attended special musculoskeletal training sessions sponsored by SGIM and Rheumatology organizations, attended orthopedic clinics, and assembled a musculoskeletal patient population. Triage nurses were instructed to only schedule patients with acute and chronic joint problems to a special clinic session at a community-based practice site. One half-day per week, residents interviewed patients, discussed diagnosis and management with faculty, and then were supervised during procedures. The curriculum also included group discussions focusing on diagnosis and treatment (medications, physical therapy, etc) of musculoskeletal problems not covered in the Rheumatology rotation, skills practice with injection models, and a reference syllabus. FINDINGS TO DATE: Our evaluation of the musculoskeletal clinic within the first six months focused on the success in achieving a concentrated experience for the first six residents to participate in the rotation. Residents averaged seeing 4.3 patients with musculoskeletal complaints per half-day clinic. The most common musculoskeletal complaints were knee pain(25%), back pain(19%), shoulder pain(16%), and hip pain(9%). In treating these patients, residents performed a mean of 2.0 procedures per half-day clinic. The most common procedures included injections of the : knee, subacromial region, and trochanteric bursa. Residents have uniformly rated the usefulness of the clinic as very good/superb. An additional pre-post knowledge and skills assessment of residents will be completed at the end of the two-year curriculum. KEY LESSONS LEARNED: Our needs assessment indicated that residents wanted additional training in musculoskeletal medicine, but preferred to learn in a primary care setting. Despite the logistic challenges, our curriculum has demonstrated the feasibility of a concentrated primary care musculoskeletal medicine experience. STATEMENT OF PROBLEM OR QUESTION: Medical advice and nicotine replacement are effective smoking cessation interventions. However physicians have a limited impact as they miss many opportunities to help smokers quit and often lack the necessary counseling skills. As smoking cessation is a stepwise process, physicians should tailor counseling to each smoker's motivation to quit. OBJECTIVES OF PROGRAM/INTERVENTION: We designed a program based on active learning methods and the``stages of change model'' to train physicians in smoking cessation. At the end of training, physicians should be able to: (1) assess each smoker's motivation to quit; (2) advise all smokers with strategies matching their motivation to quit; (3) prescribe pharmacotherapy to smokers ready to stop. DESCRIPTION OF PROGRAM/INTERVENTION: In two 4-hours sessions, active educational methods enable participants to progressively learn these new skills. In the first session, learners use a checklist to observe 3 videotaped encounters with smokers and identify the 3 main stages of motivation to quit. An interactive workshop follows for presentation and discussion of basic concepts and strategies of smoking cessation in relation to videotaped cases. Then participants are involved alternatively as physician, patient and observer in role plays with smokers at various motivation stages. In the second session, learners practice counseling skills with trained standardized patients who portray smokers with different profiles and readiness to quit. Trainees also receive a reference document, pocket algorithms, a record sheet for smokers, 5 stage-matched brochures for patients and instructions to patients about use of nicotine replacement. FINDINGS TO DATE: We tested this training program in a randomized trial among residents in primary care clinics. Compared to the control group, trained residents provided smoking cessation interventions of higher quality (mean score: 4.0 vs. 2.7, p=0.01, range: 0 ± 14) and expressed higher confidence in their skills 3 months after training (mean score: 7.7 vs. 5.2, p=0.002, range: 0 ± 10). Moreover, 1-year smoking abstinence almost doubled among smokers visiting trained residents (13% vs. 7%, p=0.04). KEY LESSONS LEARNED: We could develop a physician training program in smoking cessation based on active and progressive learning of counseling skills. This program was effective to improve physicians' practices and smokers' cessation rate. If this training is implemented at a large scale, physicians could contribute more effectively to reduce smoking prevalence and its major health consequences. ; 2) To allow interested PGY-2 and -3 residents an opportunity to work closely with faculty in developing publicspeaking, advocacy, and teaching skills. DESCRIPTION OF PROGRAM/INTERVENTION: Two PGY-2 residents were identified who had an interest in DV. These residents helped to design a 3-hour curriculum which was implemented on a monthly basis for all interns during their Ambulatory Medicine rotation. The curriculum consisted of: 1) A portion of a video about DV survivors; 2) A lecture on the dynamics, screening, and management of DV; 3) A handout including articles on DV and an annotated bibliography; 4) A talk by a social worker on principles of counseling and local resources for survivors of DV; 5) A role play demonstrating screening strategies; 6) An interactive discussion of three cases of``positive``results of screening; 7) Small group sessions in which the interns were given case scenarios and asked to try the screening and management techniques they had learned. The lecture was given by the lead author, who is a faculty member. Elements 5 ± 7 of the curriculum involved the PGY-2 residents to an increasing degree over the course of the spring. FINDINGS TO DATE: The curriculum was well-received by the interns. Both the lecture and the portions led by the PGY-2's were consistently rated highly in feedback obtained at the conclusion of the sessions. The interns commented frequently on the benefits of the role playing. We were also able to identify several interns interested in DV who have been recruited to participate in the curriculum this spring. KEY LESSONS LEARNED: Residents respond well to learning skills of outpatient medicine from their peers, whom they may feel have a better sense of the limitations and realities of their own practice. Many housestaff have an interest in both the care of vulnerable patients and advocacy that is rarely put to use in the traditional IM residency. Curriculum design and implementation can be made easier and more appealing by involving residents with specific interests. Faculty can serve as mentors in order to facilitate resident academic growth and development. The educational intervention consisted of a 3-hour interactive seminar on DV, including discussion of video material, case discussion, an evidence-based literature review, and role-playing. The seminar was designed to heighten awareness of abuse as a problem in the primary care setting, and to teach the importance of screening and useful techniques for screening. After the seminar, residents were asked to screen all of their female patients for DV for the next 2 weeks, and return to discuss their findings in a follow up seminar. Six to 12 months after completing the intervention, all second and third year residents received a questionnaire containing eight modified essay questions concerning clinical scenarios. All PGY3 residents surveyed (intervention group) had received the educational intervention while none of the PGY2 residents (control group) had received it. The residents were unaware of the purpose of the questionnaire, and all responses were anonymous. Three of the eight cases described scenarios in primary care which were designed to stimulate a suspicion of DV, for which residents were asked to list up to five leading diagnoses. A response was counted as correct if it contained the words``violence'' or``abuse.'' Questionnaires with at least 2 correct responses (to the 3 suspicious questions) were graded as positive for demonstrating adequate suspicion of DV. FINDINGS TO DATE: The program was well-received, with an average rating of 4.6 on a 5point scale (n=33.) As of 12/29/00, response rates were 70% (26 of 37) in the intervention group and 51% (19 of 37) in the control group. The rates of "positive questionnaires" were 21% (4/19) for the control group and 58% (15/26) for the intervention group (p=.014) KEY LESSONS LEARNED: An educational intervention in domestic violence using video, case discussion, evidence-based review and role-playing was well-received, changed physician awareness of domestic violence in primary care, and had a lasting effect six to 12 months after the intervention. DESCRIPTION OF PROGRAM/INTERVENTION: We will demonstrate the session plans of two EBM courses. Course 1 consists of an introduction session in which staff present the fundamentals of asking clinical questions, searching for the best evidence, and critiquing articles. Afterwards, one resident prepares a patient case and presents it to the group. The resident then formulates a clinical question and searches for the best evidence to answer it. The resident must individually meet with a staff preceptor to review the answer. Each week a different resident presents a case, formulates the question, meets with a staff preceptor, and presents the findings to the group. EBM Course 2 consists of five didactic sessions. The first session is the same as for Course 1. The subsequent sessions include staff presenting methods on how to evaluate articles of diagnosis, therapy and prognosis. The students and staff then discuss cases, formulate clinical questions and go to the library together to search for answers. The group reconvenes to critique the search strategies. FINDINGS TO DATE: Residents (N=12) in both courses completed the same 32-item questionnaire on the first and last sessions. Residents were asked the amount of searching done, comfort in searching/critiquing findings, applying findings to patient care, and what data source they'd search first to answer a particular clinical question. Pre and post differences were tested with the Wilcoxon Signed Ranks test. Comments from Course 1 regarding the need for more time to learn how to search prompted formal didactic sessions for Course 2. Course 2 residents significantly increased their frequency of searching for answers to clinical questions/applying EBM(Z = 2.02, p = .028). They reported more confidence in their ability to conduct quality searches (Z = 2.21, p = .027) and to critique the articles(Z = 2.23, p = .026). Residents from both courses mentioned that they learned about many new sources of medical information on the Internet and library databases. KEY LESSONS LEARNED: These courses may be effective means to increase residents' confidence to conduct EBM searches and to evaluate results. These courses are important ways to recruit staff to actively further their own education and application of EBM. Subsequently, they spent an afternoon session in groups of four students, one faculty coach and four standardized patients (SP's). Each student interviewed a patient, received verbal and written feedback from his/her peers, from the coach, and from the SP. Then they would re-do parts of the interview based on suggestions. At the end of the year, students interviewed a different SP case for a final exam, and were rated by instructors, the SP and themselves. The videotapes of these evaluations are then reviewed one on one with a faculty member trained in this process. FINDINGS TO DATE: In contrast to group lectures and small group sessions, students were most highly engaged in the group practice sessions with the SP's. Many students used a high control interview, but with feedback, rapidly learned to use more patient-centered interview techniques. KEY LESSONS LEARNED: After trying several didactic methods (lecture, small group workshop of 10 ± 15 students, optional videotape review sessions), we found that students responded best to a carefully facilitated SP session within a very small group (4 students) with immediate feedback from student colleagues, coach and SP. STATEMENT OF PROBLEM OR QUESTION: The first 2 years of medical school are filled with a seemingly endless stream of new information. While the basic science classes are similar to prior learning experiences, clinical learning, which may be more satisfying (i.e.,``feeling like a doctor''), requires skills that are relatively new and different. History taking skills are often evaluated by Observed Structured Clinical Exams (OSCE) or by direct observation and feedback. The traditional method of understanding the medical history is often a static list of questions and categories that fail to direct the flow of the student-patient interaction. In addition, students often desire more feedback than is possible to provide in a busy office setting. OBJECTIVES OF PROGRAM/INTERVENTION: This project seeks to create and implement a system by which medical students can understand, self-evaluate, and improve their history taking skills. DESCRIPTION OF PROGRAM/INTERVENTION: The subjects were 8 first-year medical students at Dartmouth Medical School enrolled in the On Doctoring course ± a longitudinal clinical care experience for all students in years 1 and 2. The students alternate one half-day per week with a facilitator in a small group setting, then one half-day one-on-one with a preceptor in an ambulatory setting. In the small group, the facilitator (GSO) and the students together built a process flow diagram of the medical history placing a strong emphasis on the transitions of the process. This process diagram was then used as a template for students to evaluate and improve their history taking skills.``History scorecards'' were given to the students for use in their subsequent preceptor visits. At the preceptor's office, immediately after taking a history, the student rates several aspects of the interaction (e.g., introduction of self, agenda setting, use of silence) on a 10-point scale for each patient encounter. FINDINGS TO DATE: On the subsequent preceptor visits, 25% of students used the``history scorecards'' to evaluate their performance. Those who used the system found it easier to track their skills and to ask for directed feedback from their preceptor. Those who did not use the system either forgot to bring the scorecard, found it cumbersome, or did not realize that it was an``assignment.'' KEY LESSONS LEARNED: In addition to learning history taking skills, students design a process diagram, analyze the process to find aspects that can be changed/improved, and collect and analyze data so as to monitor improvement. This novel way of teaching and learning the medical history incorporates elements of evidence based decision making for directed feedback and quality improvement skills that are important for students to understand. Graduate Medical Education, and the results of needs assessments of internal medicine residents at our institution. Using Medline, an extensive literature search was performed on the following topics: osteoporosis, breast cancer, hormone replacement therapy, domestic violence, coronary artery disease in women, menopause, headaches, substance abuse in women, urinary incontinence, dementia, sexual dysfunction, and evidence-based medicine to create a bibliography of readings for residents who rotate through our women's health center. Peerreviewed journal articles were compiled. Priority was given to data published since 1990, and randomized, double-blinded, placebo controlled studies. Faculty and residents review and analyze two to four articles weekly on a given subject. Content experts provide context and clinical expertise to discussions. Clinical questions, such as``What is the risk of my patient developing breast cancer on hormone replacement therapy?''; and``Should I prescribe hormone replacement therapy to my post-menopausal patient to help prevent coronary artery disease?'' are addressed in each session. Evidence-based medicine core concepts are reviewed and applied, including the number needed to treat, absolute risk reduction, and relative risk. FINDINGS TO DATE: Previous work from a needs assessment of residents at our institution found a discrepancy in perceptions and actual knowledge in women's health. Ninety-one percent of the residents rated inadequacies in the women's health curriculum. Our evidencedbased curriculum serves to bridge this gap of knowledge. Residents participating in our curriculum have expressed increased knowledge in the subjects discussed in our weekly conferences. Also, residents have stated that much of what is taught in this curriculum has not been covered elsewhere in their residency curriculum. KEY LESSONS LEARNED: A gap exists to be filled between perceptions of curriculum adequacies in women's health and actual knowledge. Our curriculum serves as a forum for an update in women's health literature, an exchange of ideas for the improvement of women's health as it is taught in internal medicine, and for further elucidation of the evidence behind what we practice and teach. ; 2) individualized educational tutorials using the assessment videotapes and published guidelines; and 3) measurement and feedback of practice patterns relative to standards of care. The efficacy of this model was assessed through a randomized trial of educational interventions for two independent guidelines. After initial assessments with standardized patients, 28 internal medicine residents were randomly assigned to receive either the guideline intervention for elders at risk for depression or the guideline intervention for patients with diabetes. All residents in the study are reassessed through direct observation with standardized patients and through chart review of a panel of clinic patients, thus providing independent comparative measures of competence. The two randomized groups are being compared on a measure of the difference in performance with diabetic patients and elderly patients. This study design, using two interventions, has the potential to double the effect size producing a more powerful study than one with a single intervention for research involving small numbers of residents. FINDINGS TO DATE: To date, half of the residents in the intervention have been reassessed using standardized patients. These results show that residents with the diabetic intervention performed better with their diabetic patients than with their elder patients, and the reverse was found for the residents trained in elder care. Difference between the diabetes trained (n=7) and elder care trained (n=6) residents on their difference in performance on two diabetes cases versus two elder care cases was large. The effect size of the two interventions (combined) was .72 standard deviation units (+/-.27 standard errors) and significant (p = .02). These results were consistent for the component score differences for history taking, physical examination, and diagnosis, but inconsistent for management plan. KEY LESSONS LEARNED: The overall advantage of the intervention is an educational experience that involves faculty and residents in an interactive learning and evaluation setting. Added benefits are the development of new tools to measure quality and physician compliance with guidelines for patient care. Videotape review of standardized cases, tutorial outlines and chart review templates used in the study will be available at the exhibit. sheet``to model searching behavior that will assist the learner in executing effective searches for clinical decision-making activities for the case studies. To conclude the topic, the nursing informatics educator/webmaster identifies quality patient education resources for the specific outpatient topics. An online submission form sends case study answers via e-mail to the clinical faculty or chief resident for evaluation prior to the outpatient conference. The case study submission is also filed on the website server for future reference. FINDINGS TO DATE: This approach has a strengthened the preparatory behavior of Internal Medicine residents in relation to the outpatient curriculum. Year 1 of a 3-year follow-up prepost resident self-report suggests mosest changes in attitudes and EBM behaviors (p < .10). The lead resident submits information prior to the group presentation. The monitor receives the residents' answers via e-mail and addresses significant issues confidentially. KEY LESSONS LEARNED: The outpatient website has increased the use of EBM clinical decision-making tools in the clinical practice settings. It has provided opportunities for improving residents' information literacy skills and knowledge of quality patient education resources. Attending physicians and monitors are requesting additional education related to information literacy and evidence-based medicine. Clinical activities take place in 3 ambulatory settings: a primary care clinic serving mainly people of lower socio-economic status and migrants, a geriatric clinic delivering home care to fragile elderly patients, a unit caring for patients with alcohol or illicit drug abuse. Students attend 8 tutorials for clinical reasoning and problem solving regarding common ambulatory conditions. A workshop based on role plays sensitizes students to counseling for smoking cessation or alcohol use. All students must also present and comment a clinical case to their colleagues. Time is allocated for self-directed learning to prepare some activities. Students receive formative assessment based on their behavior in clinical activities and their ability to solve a new clinical problem. FINDINGS TO DATE: At the end of the clerkship, students evaluate its various aspects on a 5point scale. After the first year, 108 students expressed a high global satisfaction with the clerkship (mean score: 4.2). Mean scores were also high for key elements like achievement of learning objectives (4.3), organization (4.5) and quality of teachers (4.6). KEY LESSONS LEARNED: We successfully developed a structured clinical clerkship in community medicine enabling students to learn about ambulatory care and health of vulnerable populations. This program met students' expectations and was highly appreciated. Further research should assess the impact of this clerkship on clinical competences in ambulatory care and career choices at the end of undergraduate training. Medicine residents lack self-confidence and competence in end-of-life patient care, including breaking bad news, patient goal-setting, DNR discussions and death pronouncement. In 1999, we formed a working group to teach and evaluate these core communication skills in our Internal Medicine residency program.
Á Demonstrate how to give unwanted news Á Demonstrate how to conduct a discussion with thè`a ngry patient'' Á Demonstrate how to lead a discussion to establish goals/patient preferences, including DNR orders and changes in treatment from curative to palliative approach DESCRIPTION OF PROGRAM/INTERVENTION: All interns attend 2 required afternoon retreats (8 hours total contact time). The first retreat focuses on basic communication skills such as empathy and attentive listening and breaking bad news. The second retreat addresses goalsetting and DNR decisions with the terminally ill patient and death pronouncement. We used a variety of formats: large group role-plays and debriefing discussions, brief handouts, pre-tests and videotaped, standardized patient stations with immediate, individual feedback from faculty observers. FINDINGS TO DATE: 20 interns completed the first retreat training and 22 completed the second. Intern pre/post self-assessment shows significant improvement in degree of competency and knowledge. On a scale of 1 to 4 (4 being most competent/most knowledgeable), interns report greater knowledge in discussing goal setting (2.5 to 3.5; p < 0.05) and discussing DNR orders (2.4 to 3.5; p < 0.05) and self-perceived increased competence in giving bad news (3.1 to 3.7; p < 0.05 ) and death pronouncement procedures (2.9 to 3.7; p < 0.05). Interns praised the retreat for the open interaction with faculty experts, the opportunity to role play patient care situations and the exposure to topics not previously discussed in medical training. A few mentioned distaste for being videotaped. KEY LESSONS LEARNED: A retreat format, using large group role-plays and individual standardized patient encounters with feedback, is a useful method of teaching and evaluating important end-of-life communication skills. Interns who needed extra help in communication and interpersonal skills were identified and individually reviewed these skills with faculty mentors.
A RESIDENT-AS-TEACHER CURRICULUM DURING AMBULATORY BLOCK. T.L. Simon 1 ; 1 Mount Sinai School of Medicine, New York, NY STATEMENT OF PROBLEM OR QUESTION: Residents are expected to teach interns and medical students, yet receive no training to help improve their skills as teachers. An effective resident-as-teacher (RAT) program needs to utilize protected time and provide sufficient opportunity for skills practice, reflection, and reinforcement. OBJECTIVES OF PROGRAM/INTERVENTION: (1) To prepare residents for their role as teachers in the medical setting. (2) To enable residents to examine their own teaching skills and behaviors, and take active steps to improve their teaching effectiveness. (3) To improve clinical teaching in the Department of Medicine. DESCRIPTION OF PROGRAM/INTERVENTION: The RAT curriculum consisted of four consecutive 2.5 hour weekly workshops, repeated monthly, during the PGY2 ambulatory block rotation in the first half of the academic year. A group of 5-6 residents met with one faculty member for sessions utilizing interactive techniques such as facilitated discussion, videotape review, and role-play. The workshops covered the following content areas: (1) Introduction to Teaching Principles/Diagnosing the Learner, (2) Microskills of Clinical Teaching/Giving a Microlecture, (3) Resident as Team Leader, and (4) Evaluation and Feedback. Teaching homework assignments were completed between sessions and reviewed as part of the next workshop. Reinforcement materials were sent to the residents 1, 2, and 3 months after completion of the curriculum. FINDINGS TO DATE: The program was well-received, with a mean``overall rating'' score of 4.5 on a 5 point scale (n=33). A pre-test rating scale of teaching behaviors was also administered at the beginning of the curriculum, and a post-test will be distributed to each group four months after completion. Qualitative data was collected during workshop discussions, on topics such as`O vercoming Barriers to Teaching on the Wards'' and``Characteristics of the Ideal Resident''. In addition, residents completed self-assessment checklists of their own performance as clinic preceptors. Finally, there is a long term plan to analyze resident teaching evaluations from interns and medical students before and after the institution of this curriculum. KEY LESSONS LEARNED: RAT programs have traditionally been done in either a single 1 ± 2 day block, or``longitudinally'' with discrete sessions in each year of training. Utilizing the ambulatory rotation allows for protected time with small groups of residents in a more relaxed setting. Weekly workshops provide the opportunity for practice teaching exercises, review, and reflection. Residents are eager for this type of training, participate enthusiastically, and rate the program highly. Whether this will translate into measurable improvement in teaching months to years later will be assessed. Traditionally, faculty observe residents interviewing and examining patients and discuss further care with the resident. Time pressures have led to difficulty recruiting adequate numbers of faculty to participate in the traditional CEX at one institution. An alternative format became a necessity. OBJECTIVES OF PROGRAM/INTERVENTION: Our primary objective was to develop a more reliable and reproducible test using standardized patients that successfully assessed the same skill areas as the traditional CEX. We also wanted to obtain data on resident competency that would be better suited to use for program evaluation. DESCRIPTION OF PROGRAM/INTERVENTION: Four SP cases (chest pain/unstable angina, headache/tension, chest pain/pulmonary embolus and abdominal pain/dyspepsia) were developed. The residents were instructed to obtain a history of present illness, past medical and psychosocial history, perform a comprehensive physical examination (PE) and write an assessment and plan (AP). SPs completed the history (Hx) and PE checklists (yes/no, yes = done and done correctly, no = omitted or done incorrectly). The SP rated PE technique (6 questions) on a 5-point Likert scale e.g.``the resident minimized patient movements'', interpersonal skills (5 questions) on a 5-point Likert scale, e.g.``the resident demonstrates a professional demeanor'', and nonverbal communication skills (16 questions) e.g. "the resident's tone of voice was 1=unfriendly, cold, 7=friendly, warm". AP score was based on key developed by 5 faculty. FINDINGS TO DATE: 50/52 intern and senior residents participated. Mean (SD) percent sub-scale scores were Hx 58.4 (7.0), PE 72.2 (15.8), AP 42.7 (18.5), technique 94.0 (4.9), interpersonal skills 94.9 (5.1), and non-verbal communication skills 83.8 (16.7). The scores on the Hx, PE, and AP were lower than expected and ratings of technique, interpersonal and nonverbal communication skills were acceptable. KEY LESSONS LEARNED: The SP CEX is a feasible alternative to the traditional CEX. However, a considerable amount of time and effort is required to establish an examination using this format. A pre-existing SP program is a helpful to this process. The SP CEX enables residency programs to standardize the CEX and avoid variability from different raters (faculty). The SP CEX provides more detailed information on resident performance than the traditional format, which will be useful in providing more specific feedback to examinees and directing curricular development within a program. STATEMENT OF PROBLEM OR QUESTION: In the care of patients with chronic diseases, no doctor is an island. This is particularly true in group practices, clinics and health centers where a number of professionals and support staff interact with each patient. When a large resident component is added to the provider base, there is increased demand on all staff to understand the content and process of caring for individuals with common chronic diseases. Educating residents in the care of multisystem chronic conditions in outpatients is also vital to successful care, particularly with evolving changes in management.
Health Center (MSNHC) is a hospital operated health center with a staff of 9 attendings, 8 RN's, 3 NP's, 32 med/peds residents, and 8 MA's. It serves 600 diabetic patients and employs a "staged diabetes management" guideline for care. MSNHC is challenged with keeping staff knowledgeable in the many facets of diabetes in order to improve care and teamwork. It must do so without a devoted diabetes educator to run such an effort. DESCRIPTION OF PROGRAM/INTERVENTION: The authors developed a monthly diabetes lunch conference for all health center staff. Drawing on the resources of the hospital, each conference centered on one diabetes related specialty and featured a guest presenter. Topic areas have included vascular disease, pharmacology, renal, nutrition, etc. The format is casebased with 15 minutes left at the end for prepared comments in the presenter's field. Attendees are asked to complete a feedback form highlighting what they have learned in the session and listing questions for the next month's presenter. FINDINGS TO DATE: Over 1/2 years, the average attendance has been 17 (3 attendings, 2 NP's; 4 residents, 2 RN's, 2MA's, 1 student). The most often cited key elements of the programs have been: lively discussion, a practical handout, good visual aids, and lunch. Most attendees have been able to specify 1 ± 2 practice process changes they would engage in as a result of attending the conference. At one conference the group developed quality improvement goals for lipid management in diabetics. KEY LESSONS LEARNED: ISSUES AND LESSONS FOR CONSIDERATION: 1) We have brought specialized knowledge to an interdisciplinary health center staff with a minimum investment of any one person's time.
2) Time pressures make staff involvement a challenge to sustain.
3) Our next step is to measure the long term effects of this intervention. introductory workshop on the history and physical of the female patient using videotaped interviewing and surrogate patients, continuity clinics in gynecology and mental health, and clinical experience in metabolic bone clinic. A yearly community month at an interdisciplinary women's health care center and a breast health month with clinical exposure to oncology, pathology, radiology, and plastic surgery provide additional women's health training. Didactic components include a journal club on landmark women's health studies, women's health seminars and grand rounds, a primary care/women's health didactic month, and a CME women's health conference. Clinical research and community outreach projects are encouraged. A 36-month reading curriculum provides knowledge of core women's health topics. This consists of review articles from reputable journals, now accessible as a web-based curriculum. Clinical and didactic components are integrated into the Internal Medicine curriculum and all Women's Health residents are board-eligible in Internal Medicine at the completion of the program. FINDINGS TO DATE: In July 1997, the program was piloted. Subsequently, highly qualified applicants have filled all 4 annually allotted positions. The ratio of applicants to positions continues to increase with a current ratio of 15:1. Of the graduating residents, 3 are pursuing fellowships, 2 are joining community practices, 1 has become an academic faculty member, and 1 has become a chief resident. KEY LESSONS LEARNED: With both institutional support and interdepartmental collaboration, this program continues its success. It attracts highly motivated and qualified applicants, it is a vehicle of awareness for the medical community through the recognition of Women's Health residents, and it has begun to foster future leaders for the continued advancement of women's health research, writing, and education. This program is a model for integrated and comprehensive women's health training. national demonstration project ± provided the opportunity to introduce medical students to the foundations of population-based medicine upon entry to medical school. Our primary goal is to give students this fundamental skill set prior to their entry into the core clinical clerkships. In sequential fashion, students are introduced to: validity, uncertainty, resource allocation, financing and access, and managing care to enhance quality. DESCRIPTION (3) Managing Care. FINDINGS TO DATE: Students rate the curriculum in terms of content, presentation and teaching materials. Some courses are as highly rated as more traditional biomedical science courses. Unfavorable ratings correlate best with poor course organization. However, perceived``inappropriate'' course content also plays a role. Student assessment maps changes in knowledge, values and attitudes, and includes a variety of traditional and more innovative modalities. Overall, students perform as well in these courses as in biomedical science courses. Preliminary data suggests that students attitudes about managing care may also be changing. KEY LESSONS LEARNED: Interdisciplinary curriculum design, implementation and oversight ± and extensive and ongoing input from students ± have been critical elements in the success of this broad-based initiative. Additional interventions are planned during the clinical curriculum, where the principles introduced in the preclinical years will be reinforced and expanded. Future studies will assess whether this approach influences how students perform as clinical clerks and residents. -The evaluation team has completed the pre-and post-test of the control group and is collecting data on the intervention group. KEY LESSONS LEARNED: -It is possible to collaborate on a large curriculum development project across three medical schools. It is important to define which aspects of the project the schools will collaborate on and which will be done individually.
-The competency document has helped drive specific curriculum at each school and has emphasized the evidence behind our curriculum.
-The teaching of communication skills is best done if it is integrated into clinical content and it provides active``hands on'' experience.
-Rigorous, comprehensive evaluation is possible and integral to the success of the project.
TEACHING CROSS-CULTURAL COMMUNICATION. S. Mutha 1 , C. Allen 1 , M. Evaluations of the trainings have revealed high levels of agreement that the content was highly relevant to clinical practice and high satisfaction with the training sessions. A six-month followup evaluation is underway to assess the ways in which attendees have incorporated the contents of the training into their practices. KEY LESSONS LEARNED: The curriculum content and design allow successful interaction among multidisciplinary health professionals. Participant evaluations underscore that simulations and experiential exercises are an especially powerful way to increase sensitivity to and awareness of diversity in the clinical setting. The sensitive nature of issues surrounding diversity such as prejudice as well as discrepancies in power and authority emphasizes the need to develop a cadre of clinician/educators who are committed to providing this type of training in clinical settings. medical students between the M1 and M2 years, with each student linked directly to a senior administrator of a major academic medical center or private managed care organization. Each fellow is assigned a project, which is summarized into a presentation to all program faculty. Weekly journal club (focusing on health policy, administration, and financing) is also required. FINDINGS TO DATE: Evaluations by students of experience in the program have yielded uniformly positive results when asked to rate the quality of the program, as well as its importance to their careers as physicians. 6 of the 16 students participating in the program have applied for admission to UCLA's combined MD/MBA program upon completion of the fellowship. Students have gained increased understanding of the administrative aspects of health care organizations. KEY LESSONS LEARNED: 1) Dedicated and experienced mentors are critical. 2) Students must tailor work projects to normal operations of organization; 3) Feedback and discussion with students during program is a critical success factor; 4) Most medical students could benefit from some type of exposure to the administrative STATEMENT OF PROBLEM OR QUESTION: Current fiscal reality has resulted in increased demands for clinical/ economic productivity on the part of all physicians. This is perhaps most acutely felt in academic practice groups that devote part of their activity to nondirectly reimbursed teaching activity. With pressures to see more patients while teaching students and residents, time and excellence in teaching and research may suffer.
Education and Research Foundation (BMERF), at the strong urging of its membership, endeavored to develop a financial incentive program to reward and encourage excellence and productivity in research and teaching . As a multispecialty group, it was important to the group to treat all specialties fairly and equitably. DESCRIPTION OF PROGRAM/INTERVENTION: The chairs of each academic department (Emergency Medicine, Medicine, OB/Gyn, Pathology, Pediatrics, Psychiatry and Surgery) nominated one member from each department to a Committee on Academic Excellence. Committee members identified a set of achievement criteria and then weighted them to form a quantitative measurement scale.
In the first year, major weighted criteria included but were not limited to: article publication; editor or author of text or journal; receipt of large research grants; achievement awards from national organizations. Junior faculty received additional consideration for more modest grant receipt; advancement to associate professor; and chairmanship of major committees in medical societies or at the medical school. Criteria were further refined the second year based on BMERF member feedback. FINDINGS TO DATE: In the first year of 197 eligible members, 47 (24%) submitted material for consideration for compensation. 31 awards were given (66%), ranging upwards from $1,000, few going to junior faculty (defined by the committee as 7 or fewer years out of residency). The second year there were 77 submissions (39%) and 41 awards (53%), with a minimum award of $625. Three General Medicine Division members received awards, from a division membership of 24. KEY LESSONS LEARNED: LESSONS AND ISSUES FOR CONSIDERATION: 1) The concept and execution of the program were well received.
2) There will be pressure to allocate more money in future years.
3) We need to further refine criteria for junior faculty and generalists. 4) It can be difficult to identify and reward excellence in teaching. HCA serves 32,000 patients, 40% capitated, with 91,000 annual visits and is the primary ambulatory teaching site for 130 medical housestaff. We reward our clinicians with added compensation for high patient visit volume, panel growth, severity of illness, commitment to teaching (comanagement) and, most recently, panel management. DESCRIPTION OF PROGRAM/INTERVENTION: Funded annually by the hospital, the incentive pool derives from an estimated payout per faculty FTE for practice-wide projected increases in visit volume and panel size. The core clinical payout is based on a point system. Promoting both visit volume and panel growth, new patient encounters earn 3 points, while all others earn 1 point. Incentive is paid for all points above a threshold of 8 patients/session, averaged over the year. To promote teaching, we incentivize comanagement sessions more heavily than individual practice sessions. As a proxy for severity of illness, we pay a third component based on the inpatient activity of a faculty member's panel. We are now adopting a more diversified model, with 25 ± 35% of total incentive dollars linked to panel size, age and gender adjusted, to reflect the realities of clinical practice more accurately. We are also moving to include provider specific quality measures in the incentive system, and to extend incentive payments to non-physician and non-clinical staff. FINDINGS TO DATE: Faculty visit volume productivity increased 30% in 3 years and overall panel has grown at a rate of over 400 new patients/month. In academic year 99 ± 00, the faculty incentive paid a total of $310,000 for an average payout of $8,000 per faculty member, with a range of $0 ± $19,927, and we have been able to fill our comanagement teaching slots easily. System is a multi-site county health care system, with significant variability in clinical background and practice styles in primary care providers. System wide disease management initiatives and clinical practice guidelines have been created in an effort to standardize care but have been difficult to disseminate and implement. Traditional CME is individually chosen to accommodate the provider's preferences regardless of the system's needs. Furthermore, there is evidence that this approach rarely modifies physician behavior resulting in a delay of standard of care practices implementation. A program that combines disease management, continuing medical education, and peer review was designed to foster the creation, dissemination and evaluation of clinical practice guideline implementation for primary care issues, and improve system-wide provider communication.
Providers to exchange clinical interests and expertise.
To provide a forum to develop, implement and monitor quality improvement initiatives.
To develop a curriculum of clinical material relevant to primary care.
To promote collaboration among adult providers across the healthcare system.
To promote development of one standard of care throughout the system, that is both evidence based and cost effective. DESCRIPTION OF PROGRAM/INTERVENTION: Description: An initial curriculum was developed based upon a survey of provider's interests and system needs identified by the quality management department. Prior to meetings planning committee reviews or creates disease specific standard indicators for appropriate measurement of guideline implementation. Monthly meetings consist of a lecture given by a recognized expert and a discussion of the perceived barriers to achieving that care in our institution. The indicators are then presented followed by a review of charts of identified patients with the discussed medical illness. Results of previous peer reviews are presented with a discussion of what could be done to improve them. Individualized provider specific feedback is distributed confidentially. FINDINGS TO DATE: Results: Re-measurement of disease specific indicators have shown significant increase in provider compliance with established standards of care: a 24% increase in the use of ACE-Inhibitors in CHF, a 45% of increase in screening for microalbuminuria in diabetics. Yet, some of the newer disease management knowledge, such as use of beta-blocker in patients with CHF showed no significant change. KEY LESSONS LEARNED: Key Lessons: A program that combines disease management and a continuing medical education involving self-evaluation through peer-reviews more effective to disseminate and implement system based clinical practice guidelines, also improves providers communication and involvement in those initiatives. identified and a multi-disciplinary team consisting of a hospital pharmacist, dietician and discharge planner visit and educate the patient and family. Standardized education materials are provided. The GHC home-visiting nurse service, called Family Health Workers(FHW) also visits the CHF Project patient while in hospital. At discharge, the hospital pharmacy provides an updated medication list to the patient, the FHW, the patient's pharmacy and the GHC electronic medical record. The same FHW visits the patient within 48 hours of discharge, and arranges for further follow-up as needed. FINDINGS TO DATE: [1] Over the first six months, the CHF Project has been well received by all. A crucial and well-appreciated role of the FHW is to sort out discharge medications at the initial home visit. [2] There have been a total of 174 CHF patient admissions, of which 57 belong to the GHC and were exposed to the intervention. [3] There has been a 68%, [95% confidence interval (CI) 9% ± 96%] decrease in readmission rates compared to historical controls and a non-significant 57% [95%CI -5 to 89%] decrease compared to concurrent non-GHC controls. [4] There has also been a non-significant trend towards decreased mortality. KEY LESSONS LEARNED: A community-based collaborative to help CHF patients in the crucial discharge transition period can be effective in a non-tertiary care setting. Further efforts are needed to expand access to the program for all patients, to study and improve upon separate components of the program and to sustain the improvements already achieved. (range 23 ± 87); 69% of patients were African American; 60% were Female; 58% had less than a high school education; over 50% relied on Medicaid or pharmacy assistance for medications. Average duration of disease was 10.4 years (range 0 ± 42). Although 81% of patient had hypercholesterolemia and 88% had hypertension, only 30% were on a lipid lowering agent and only 60% were on or had ever taken an ACE-inhibitor. Baseline HgbA1c at enrollment was 10.8% and did not differ significantly from values obtained 6-12 months prior to enrollment (mean 10.2%). At three to four months follow-up, the mean reduction in HgbA1c was 1.7% points (p < .0001).Baseline diabetes knowledge score on our 11 question diabetes knowledge test (DKT) averaged 36%. Of 69 patients who have retaken the DKT, there was a 23% point improvement from baseline to follow-up (p < .0001). Blood pressure and total cholesterol did not change importantly from baseline to follow-up. Multiple regression analysis shows that higher educational status, when adjusted for baseline HgbA1c, is associated with a significant improvement in HgbA1c. Other socioeconomic factors, such as race and gender were not significant predictors. KEY LESSONS LEARNED: A pharmacist-assisted care program can improve patient's knowledge of diabetes and significantly reduce HgbA1c. We did not see improvement in blood pressure or lipid control -two areas that we did not specifically intervene upon. To further improve care and reduce the risk of macrovascular complications, we plan to test pharmacistassisted, algorithm-based, hypertension and cholesterol management in a randomized controlled trial. STATEMENT OF PROBLEM OR QUESTION: Nursing staff in VA primary care clinics are required to document numerous patient education and screening activities, and they are using progressively more advanced software in the electronic patient record to accomplish this documentation. However, time limitations make it difficult to accomplish all these educational and screening mandates while the patient is in clinic, and staff may miss documenting activities which were previous accomplished but not quickly found in the record. OBJECTIVES OF PROGRAM/INTERVENTION: We experimented with the process by which nurses documented that patients were taught the proper use of metered dose inhalers (MDIs). We chose to update our electronic documentation of this activity (1) at a time other than the patient visit and (2) by using a population based approach. DESCRIPTION OF PROGRAM/INTERVENTION: At our facility, two teams of similar provider and support staff composition provide primary care in non-teaching clinics. Of the 6129 patients in group practice A, 640 use MDIs, and 572 of 5596 group practice B patients use MDIs. Information about this population of MDIs users was compiled, and their names and telephone numbers were listed in the chronological order of their next appointment. Starting in May 2000, clerks began calling each patient as their next appointment approached, in order to remind patients to obtain MDI instruction when they attended clinic. In group practice B, nurses also used the list during lulls in routine clinic screening activities to contact patients, ask them about prior MDI education, and then update the electronic record accordingly. Five months later, electronic documentation of MDI patient education was extracted from the computer patient database. Documentation data was also obtained about one education/ screening activity not targeted in this study (depression screening) because this activity applies to all group practice patients and are electronically documented in the same manner as MDI teaching. FINDINGS TO DATE: In group practice B, MDI inhaler education and depression screening were documented on 84% and 60% of the 572 MDI patients. In group practice A, the same percentages were 52% and 65% of their 640 MDI patients. Chi square comparisons were made of patients receiving only MDI instruction or MDI teaching and depression screening. In each comparison, group practice B performance was significantly (p < .05) higher. Logistic regression also showed that group practice B was associated with a higher likelihood of MDI teaching documentation (odds ratio 3.54, p < .001). KEY LESSONS LEARNED: As the number of routine patient education guidelines expands, there is not sufficient time during each clinic visit to complete and document them all.
Documentation may be better accomplished when a population of patients is targeted for review at times when they are not actually in clinic. Americorps volunteers who serve as case managers, a part-time lay health worker, and four medical students who coordinate service-learning activities. We developed an Access database to monitor clinical services, pharmaceutical utilization, and case management with a focus on preventive care, referral data, Medicaid enrollment, and pharmaceutical costs and utilization. Students in pharmacy, medicine, and nursing conducted quality assurance projects, including a patient satisfaction survey and chart reviews to assess hypertension management and antibiotic usage. FINDINGS TO DATE: On-site dispensing was promoted over vouchers at a cost saving of $21.34 per prescription with a total saving of about $20,000 a year. Blood pressure control was achieved in 46% of hypertensive patients, but almost half failed to return for follow-up. Narrowspectrum antibiotics were selected as first-line therapy 66% of the time, but over-prescribing for URI was also noted. In a predominantly uninsured (73%) clinic population, 27% of all patient encounters resulted in referrals with 30% of all referrals for dental care, 17% to primary care providers and medical specialists, and only 1% to behavioral health. Sub-optimal documentation was noted in chart reviews. KEY LESSONS LEARNED: Based on our findings, we implemented an appointment reminder system, progress note templates, and a mechanism for tracking referrals. Providing routine feedback to volunteer providers on findings such as mental health referrals may promote more aggressive screening and counseling. In conclusion, quality assurance projects incorporated into volunteer clinic activities can identify unmet health needs and areas requiring improvement. Services Administration is the second and current stage of program development. Focus groups and advisory work groups with pharmacists, physicians and nurse practitioners, and counselors are being held to identify potential barriers to participation. Actuarial consultants are developing cost estimates and financial models for the proposed program. Project leaders work with state and federal regulatory and legislative officials to facilitate program planning and implementation. Phase III: Implementation of a pilot project is targeted for the summer of 2001. FINDINGS TO DATE: The phase I community planning created program framework with the San Francisco Department of Public Health as holder of a central Narcotics Treatment Program (NTP) licensure for the OBOAT Program, in which interested and eligible public and private-sector physicians, counselors, and pharmacists would be trained to offer methadone and other approved medicines to treat opiate addiction in the setting of their regular clinical practices. Pharmacists view the opportunity for professional development as an incentive for potential program participation. They view lack of lack of methadone education in pharmacy school and inexperience with methadone dispensing to addicts as barriers to potential program participation. The planning process has challenged physicians in the narcotic treatment arena and those in the primary care arena to learn about each other's`c ulture'' and communicate effectively to develop an innovative program that will serve patients in new way. KEY LESSONS LEARNED: The planning process has involved reframing opiate addiction treatment as a medical model, rather than a regulatory model. Close collaboration with existing methadone clinics has been critical in moving program planning forward. Close work with state legislature has resulted in recent passage of enabling legislation as well as allocation of additional resources. (CTI) is a computer-based application that automatically calls patients to remind them of their appointments. This program is interfaced with the scheduling system to automatically download patient and appointment information. It uses an interactive voice response technology to call patients 24 ± 72 hours before their appointment. If there is no answer, the patient will be called back up to 5 times. All patients with appointments scheduled at least 72 hours ahead of time at several Community Health Clinics over a period of 6 weeks were eligible for our study. 5,696 patients had appointments during this time and thus could have been called by the CTI system. During randomized alternating one week periods, patients at each of the participating clinics were assigned to receive automated appointment reminders. Patients not receiving calls were used as the control group. Kept and missed appointment rates were recorded for each of these groups. FINDINGS TO DATE: 5,696 patients (3, 038 in the appointment reminder group, 2,617 of whom consented to being called by the system; 2,658 in the control group) were prospectively followed over a 6 week period. The kept appointment rate in the appointment reminder group was 69.2%. The kept appointment rate in the control group was 64.1%. This result translated into an 8% increase in kept appointments when the automated system was used (p < 0.01). The increase occurred despite the fact that only 1,192 of 2,617 eligible patients were actually contacted. KEY LESSONS LEARNED: A computerized telephone reminder system increased the kept appointment rate in our Community Health Clinics. This increase may have a considerable economic impact. Assuming an increase in the kept appointment rate of 8% translates into 8% more patient visits, this could represent 9,000 more visits a year to our clinics. If the average charge for an outpatient visit is $177.35, 9,000 more visits represents $1.5 million in increased charges. Implementation of such a system could have a large positive economic and health impact in a public health system such as ours.
IMPROVED GLYCEMIC CONTROL IN DIABETIC PATIENTS UNDERGOING CORONARY ARTERY BYPASS SURGERY. N. Ashri 1 , R. Lippe 1 , N. Rao 1 ; 1 UPMC Shadyside, Pittsburgh, PA STATEMENT OF PROBLEM OR QUESTION: Management of blood sugar in the metabolically stressful postoperative period in a Diabetic patient undergoing major surgery such as coronary artery bypass (CABG)is often a challenging task. However, adequate glycemic control is necessary to prevent postoperative infections such as mediastinitis and delayed wound healing.Conventionally, the management involves insulin sliding scale and despite multiple calls to physicians and inconvenience to nursing staff, the glycemic control is not adequate. OBJECTIVES OF PROGRAM/INTERVENTION: 1) To develop a simple, effective insulin infusion protocol, which will maintain Diabetic CABG patients capillary blood sugars (CBS) below 200 mg/dl in the first 48 hours postoperatively. 2) To decrease the rate of mediastinitis in postoperative period. 
conducting end-of-life discussions is presented. The four steps are based on structured interviews at a major university hospital with five faculty clinicians experienced in the care of dying patients. Their experiences and actual words have been synthesized into this 4-step approach. The four steps are: 1) Initiating Discussions 2) Clarifying Prognosis 3) Identifying End-of-Life Goals 4) Developing a Treatment Plan FINDINGS TO DATE: Using the 4-step approach with your patients will result in improved communication. Good communication results in patient fears being allayed, pain and suffering minimized, and facilitates developing a treatment plan that is medically sound and concordant with the patient's wishes and values. KEY LESSONS LEARNED: Providing good end-of-life care requires both an understanding of how patients and famlieis experience the dying process, and a sensitive communication style. With these skills, physicians are able to conduct thoughtful discussions in which most decisions evolve comfortably and without controvery. Providing care for a dying patient is challenging, and when done well, is a meaningful and gratifying experience for the physician. To help someone die in comfort, in peace, and with dignity is to give one final gift of life. and an audience response system was also used to administer the questionnaire during a noon conference FINDINGS TO DATE: Thirty five HS participated in this survey; (48% GL-I, 24% GL-II, 14% GL-III, 7% GL-V). Most of them thought the book was trustworthy (87%), accurate (86%), user friendly (57%), and overall useful for the management of patients (87%). Although most HS always carried the CCF book (73%), most also carried other commercially available antibiotics recommendations book(s) (68%). There was no consensus which of these books was more useful for them. The CCF Book sections on guidelines for specific diseases and dosing information were referred to most often (37% & 40%; respectively). Requests were made to add more microbiology information and other clinically important antimicrobial drug interactions. KEY LESSONS LEARNED: The CCF Guidelines for Antimicrobial Usage book is well respected among the HS. The book serves it's purpose well in helping HS with their daily care of hospitalized patients. This survey helped us in implementing some changes that we believe will improve these guidelines and consequently, improve the quality of care for our patients. On an average there are close to 1,500 patients seen in this unit annually, with a wide variety of medical, surgical and rehabilitation problems requiring skills of a physician trained in the care of these patients. This presentation is based on our experience and observation. The Lecture format will be as follows: Introduction: Advances in technology and an increase in life expectancy for the general population have resulted in an increasing demand for medical services. In the early 1980s, for example, Medicare introduced the Diagnosis Related Group (DRG ) based system of payment for inpatient care. The DRG system created financial disincentives for the hospitals by imposing a fixed reimbursement rate for each condition unrelated to patient acuity or the actual costs of care incurred by the hospital. In response, many hospital systems created alternative discharge sites, thus shifting care to subacute units, nursing homes, and home-based care. Principles of subacute care The philosophy of Subacute or Transitional care units is the management of patients after acute exacerbation of an illness, providing a more healing environment and a change of focus away from high technologic interventions. While these patients do not need intense diagnostic work-up or invasive procedures, they still require frequent physician monitoring, nursing care, and rehabilitation. Patient Selection: The decision of appropriate referral to subacute unit is a key to both continuity of care and financial viability. Appropriate subacute care candidates must have a definitive goal and identified skilled needs. It is important to identify these patients as early as possible during their acute hospitalization in order to reduce the length of stay. Examples of patients appropriate for subacute units will be discussed in detail. Also difference between these patients and nursing home patients requiring custodial care will be discussed. Financial challenges: As the number and type of alternate care sites have increased, federal efforts at cost containment have shifted to limit financial reimbursement to post-acute care facilities as well. The Balance Budget Act of 1997 introduced the Prospective Payment System for these units, shifting the reimbursement from per-diem rate to a fixed rate which would include all the ancillary services received by a patient except physician visits. This fixed rate is calculated based on the Resource Utilization Group per Medicare guidelines. In many cases, the cost of providing care for medically complex patients far exceeds the reimbursement. Mounting economic pressure to contain treatment costs may threaten the financial health of many subacute units especially as hospitals seek to admit only the sickest of the sick and treat them in the shortest possible amount of time. This section will include discussion on the problems created by these changes in reimbursement and the possible solution to maintain quality of care. Educational opportunities: With the decrease in length of stay on the acute inpatient site and increasing medical complexity, subacute units continue to provide care for a variety of patients. Members involved in their care require both medical and surgical skills. Our subacute unit is a site for residency training program and continues to offer wide variety of experience ranging from routine postoperative care to caring for the elderly and medically sick patients. Residents also learn the importance of maintaining functional integrity in elderly patients, concepts of rehabilitation working in an interdisciplinary team model and dealing with end of life decisions. They also learn to provide care in face of financial cut-backs, without compromising on the quality of care. Outcomes research continues to remain an unexplored area in this arm of health care. The value of this cannot be underestimated, especially in view of financial cut-backs, with the goal of maintaining a high standard of care for an enlarging, vulnerable patient population in this new environment. FINDINGS TO DATE: KEY LESSONS LEARNED:
PHYSICIAN RECORD KEEPING WAS ALTERED BY FOCUSED PROFILING. R.E. White 1 , D. Gray 1 ; 1 Albuquerque VAMC, Albuquerque, NM STATEMENT OF PROBLEM OR QUESTION: Since 1998, attending physicians in our general medicine, continuity clinics have instructed resident practitioners to enter patient problem lists into the electronic medical record. After eighteen months, however, only 45% of patients had an electronic problem list (EPL). OBJECTIVES OF PROGRAM/INTERVENTION: We implemented a profiling system based exclusively on the electronic medical record database. It allowed one person to review 100% of patient and resident records and then provide peer compared feedback. DESCRIPTION OF PROGRAM/INTERVENTION: Data about resident entry of EPLs was extracted from the computerized, medical record database and recurrently fed back to them between February and June 2000. Profile reports were posted in clinic, and all practitioners could compare their patient panel sizes and EPL completion rates with their peers. Several times during the intervention, each resident also received a list of his or her assigned patients who needed EPL action. The profiling intervention was suspended July through September 2000. Since all practitioners in the teaching group practice were profiled, no control group existed. Therefore, the presence of an EPL for each patient and the entry dates of individual problems contained in those lists were analyzed and compared across three time periods: the seven months before profiling (July 1999 through January 2000), the five months during the profiling intervention (February through June 2000), and after profiling ceased (July through September 2000). FINDINGS TO DATE: During the five months of profiling, residents added Electronic Problem Lists to 1087 patients, raising the percentage of patients with EPLs from 45% in January to 88% in June. An unintended, side product of profiling was an 11% increase in the number of patients which residents identified as belonging to their panels, from a total of 2048 in January to 2272 in June. Besides starting more EPLs during profiling months, residents also entered significantly more individual problems into those lists during profiling. During the seven pre-profiling months, they entered an average of 321 individual patient problems per month, and during profiling they entered 757 per month (a 136% increase). Not only did individual residents increase their number of electronic problem entries, but also more residents entered problems. Prior to profiling, 18 of 71 residents did not enter a single problem over seven months, and only 30 residents each month entered problems. During profiling all 71 residents entered problems and 50 did so each month. Forty-two residents were present during all data collection months (July 1999 through September 2000). These 42 residents entered individual problems into EPLs at monthly rates of 220 before, 526 during and 285 after profiling.``Before'' and``after'' periods differed significantly from the profiling period (ANOVA p < .005) but not from each other. The number of residents adding problems each month averaged 20 before, 31 during and 26 after profiling (Chi-square p < .02 for the``before''/`d uring'' comparison). KEY LESSONS LEARNED: Profiling was associated with increased compliance with record keeping policy, and affected all residents. Repeated feedback to practitioners about their specific performance and with peer comparisons is more engaging and motivating than reminders by supervising attendings. Using a computer patient database to review 100% of all patient and physician documents offers an efficient means to enhance physician performance.
THE USE OF PALM HANDHELD COMPUTERS TO CARE FOR NURSING HOME PATIENTS. J.M. Previll 1 ; 1 East Carolina University, Greenville, NC STATEMENT OF PROBLEM OR QUESTION: Our medical school nursing home service has had difficulty monitoring the care of our nursing home patients because different attendings and residents were involved in the care of these patients. These difficulties included not having access to the patient's diagnoses, code status, current medications, pending labs, imaging, or procedures scheduled for the patient, or the subsequent results. There was also the question of when patients must be seen to comply with Medicare and institutional requirements. OBJECTIVES OF PROGRAM/INTERVENTION: The objective was to develop a system that would enable attendings and residents to track all the data on patients and provide better care with this information. DESCRIPTION OF PROGRAM/INTERVENTION: A Palm Pilot database called HandBase was used to develop a database that had information on the nursing home patients concerning their location, dates seen, diagnoses, code status, allergies, medications, reports on labs and x-rays, and CPTand ICD-9 codes for billing. HandJet, an interface program, linked the database in the Palm Pilot to a Microsoft Access database located in the Brody School of Medicine computer network. Patient data is protected by passwords in the Palm Pilot and the firewall in the Brody School of Medicine computer network. Changes to patient records can be made during time of visit or by telephone and disseminated to others by infrared beaming between Palm handhelds or hotsyncing to the computer network. FINDINGS TO DATE: This system allows database backup, printing of work lists, and patient data notebooks. It also enables all physicians and staff involved in patient care to have access to information needed in the ongoing care of the nursing patients. Having a current and complete profile on each patient has given the doctors the ability to respond and care for these patients in a more timely manner. Paperwork is simplified for admissions, discharges and scheduling. Documentation is immediate and more detailed, making billing more efficient and accurate, as it is now a part of the report generator. KEY LESSONS LEARNED: Maintaining a database that is mobile improves effectiveness and provides more timely patient care in the nursing care setting.
PATIENT PROFILE: A PRACTICE MANAGEMENT SYSTEM DEVELOPED USING AN OFF-THE-SHELF RELATIONAL DATABASE PROGRAM. T. Yackel 1 , B. Slater 2 ; 1 Oregon Health Sciences University, Portland, OR; 2 George Washington University Medical Center, Washington, DC STATEMENT OF PROBLEM OR QUESTION: The George Washington University Department of Health Care Sciences is part of a multi-specialty faculty practice with departmentally centralized paper medical records. An internal study revealed that patient chart availability at the time of care was as low as 66%. The use of electronic resources to obtain patient specific information was limited to laboratory results retrieval and patient scheduling. Laboratory results were available online only for tests drawn in the past 60 days. OBJECTIVES OF PROGRAM/INTERVENTION: 1) Improve availability of patient information at the time of care. 2) Implement an electronic disease management system for patients with HIV and diabetes mellitus. 3) Accomplish these objectives in a short period of time with little funding. DESCRIPTION OF PROGRAM/INTERVENTION: Using an off-the-shelf relational database product, Access 97 (Microsoft, Redmond, WA), we implemented an advanced clinical information system over a six-month period. The program was written during spare evenings and weekends by the authors, neither of whom have had formal training in database programming. The system includes sophisticated features such as a problem list linked to ICD-9 codes; a prescription manager that provides drug-drug interaction checking and patient-specific formulary information; templates for HIV and diabetes disease management; automatic importing of laboratory information with longitudinal views, including results from the past 3 years for HbA1C, PSA, cholesterol, HIV viral loads, and CD4 counts; a reminder system; the ability to import dictations; user-level security and encryption of data; and an easy-to-use interface. FINDINGS TO DATE: Patient Profile has been in continuous use since June 1998 by a group of 3-5 users and has required little maintenance. The availability of the system exceeds 99%. Due to the program's simplicity, low-cost, and popularity among care providers, it became a prototype for a practice-wide electronic medical record project. KEY LESSONS LEARNED: Using commercially available tools, clinicians with minimal experience in computer programming can develop and implement their own practice management systems for a fraction of the cost of commercial products. Because these programs are infinitively customizable, they may serve the specific needs of providers better than a system purchased from a vendor. All students were mailed a questionnaire requesting they rate the importance of various factors in choosing a residency program using a 5 point Likert scale (1 = not important, 3 = somewhat important, and 5 = very important). Underrepresented minority (URM) applicants were defined as African American, Latino, or Native American. RESULTS: Response rate was 36% (1005/2820), 55% were men, 30% were married and 61% self-rated in the top 25% of their class. The factors most important to all applicants were good housestaff morale (mean SD= 4.54 0.72), the academic reputation of the program (4.46 0.78), a positive interview experience (4.10 0.99), variety of clinical experiences by residents (4.05 0.72) and location near spouse/significant other (4.04 1.43). URM applicants (N=92 or 9%) were significantly more likely to identify ethnic diversity of the patients ( CONCLUSION: Applicants rate location and program-related factors as most important in influencing their decision to chose a particular Internal Medicine Residency Program. However, URM applicants also place significant importance on the ethnic diversity of patients, housestaff and faculty, and the support provided to ethnic minorities within the academic environment. Residencies must place an emphasis on improving these factors if they wish to recruit highly qualified minority applicants. Although there is some literature on violence in the medical workplace, little has been written about threats physicians experience on the job. Our study examines the epidemiology of threats to resident physicians in internal medicine by patients, their families, and non-physician staff. METHODS: We implemented a mass-administered survey to internal medicine residents in two large urban training programs. One program is in a public county hospital which serves predominately uninsured patients. The other is in a private, tertiary care, academic medical center whose patient base is predominately insured by Medicare or managed care entities. We defined a threat as an actual or implied action intended to harm or torment someone. The survey measures the frequency, type and setting of threats. It examines the support structures residents use to deal with threats. We also assess resident attitudes towards receiving threats, particularly as they relate to patient care. RESULTS: Our preliminary data analysis shows 24 of 69 (34.8%) residents at the public hospital and 15 of 40 (37.5%) of residents at the private hospital have experienced one or more threats (total number of threats 66, mean 1.7). The threats were most commonly from patients assigned to that resident (47%), 16% from patients they covered on call, 19% from family members of patients, and 18% from other non-physician staff. Eighty five percent of the threats were verbal in nature and 15% were physical. The location of the threat occurrence was usually on the wards (70%). The remainder occurred in the emergency department (24%), and 6% occurred in outpatient clinics. Residents reported that the threats made them feel angry, nervous, afraid, and helpless. Approximately three-quarters of residents (74%) sought some support for these episodes. This support came from a variety of sources including peers, family, attending physicians, and security. Of residents reporting a threat, one in five (21%) said the experience impacted on the care of the patient involved, while only 2 of 39 (5%) said the experience impacted on how they care for other patients. Eleven of 39 (28%) said that following the threat they are more likely to avoid certain situations at work to feel more safe. Residents strongly disagreed with the statement that being threatened by a patient or their family members is a part of their job. CONCLUSION: Residents commonly experience threats from patients, their family members and non-physician staff in the workplace. The prevalence of threats was similar at both a public and private hospital. The most common reactions residents reported to these experiences include negative emotions and an impact on their care of the involved patient. An in-house support system needs to be developed to prevent and handle threats residents experience in the workplace. Residency programs have used support groups and Balint groups to address some of the stresses faced by trainees. We developed a monthly, faculty facilitated, hourlong session for residents on a ward or coronary care unit rotation. The goal of this session was to promote an opportunity and a safe environment for residents to explore clinical issues that had an impact on the feelings, attitudes, and challenges of becoming a physician. In each session, ground rules for safety were reiterated, as was the voluntary nature of this session. The group was held during one half of a scheduled attending rounds session in the third week of the month. The teaching attending (different from the facilitator) was welcome to participate. METHODS: We developed a confidential and anonymous survey given to all medicine and medicine pediatrics residents at the end of the 1999-2000 academic year. The survey was designed to access resident attitudes towards participation, their acceptance of this discussion format and any potential behavior change as a result of participation in those groups. RESULTS: Overall response rate was 75% (65% medicine and 35% medicine pediatrics). 62% of respondents were under age 30; 27% were age 30-35. 62% of respondents were female. Residents had attended an average of 1.6 sessions on the wards, and 1.1 sessions in the CCU. 90% of respondents indicated the timing was appropriate. Residents rated their comfort in these discussions with fellow residents on a 1 (low comfort) to 5 (high comfort) scale, with the mean response of 3.83. Residents were asked the following: The issues discussed in the balance groups are ones you may have thought about or discussed in the past. As a result of the discussions this past year, please rate your current activity from 1 (less activity) to 3 (same activity) to 5 (more activity) in: self reflection (mean response = 4.41), discussions with peers (mean response = 4.33) and discussions with family and friends (mean response = 4.17). CONCLUSION: Balance groups in this Internal Medicine residency training program were well received regarding their timing, and the comfort felt among participants. Importantly, residents indicated that they continued to reflect privately and with peers, family, and friends. Timing and attendance are significant barriers to successful groups; we found that scheduling this during a regular educational session, reinforcing appropriate ground rules and the voluntary nature of this session were important to its success. We recognize that some groups are longitudinal and benefit from the increased trust gained by the group. We chose to focus on the small group of each ward or unit team, and planned this discussion later in the month when some group identity and cohesion was more likely to have taken place.
PREDICTORS OF EFFECTIVE PHYSICAL DIAGNOSIS TEACHING. K. Barnard 1 , D. Lescisin 1 , N. Armistead 1 , D. Elnicki 1 ; 1 University of Pittsburgh, Pittsburgh, PA PURPOSE: Interns play an important role in the teaching of medical students. We sought to identify demographic characteristics and teaching behaviors that predict effective teaching of physical diagnosis skills. METHODS: Self-administered surveys were completed by third year students at the end of their internal medicine clerkship at two medical schools from 7/00-11/00. Questions included demographic characteristics of interns and students, and occurrence of teaching behaviors, such as observation and feedback, reviewing cases, and establishing a comfortable learning climate. We also asked students to assess to what degree the intern contributed to their learning physical diagnosis skills (diagnosing heart murmurs, distinguishing pulmonary consolidation from pleural effusion, detecting hepatomegaly, examining the thyroid gland, detecting a joint effusion and lymphadenopathy). These items were selected from the CDIM-SGIM curriculum, and were combined to form a grouped physical diagnosis variable. A forward, step-wise linear regression model was built to determine which of the independent variables predicted effective physical diagnosis teaching. Adjusted p-values are shown here. RESULTS: A total of 70 students were surveyed (95% response rate). The teaching behavior,`m aking helpful suggestions to improve performance'', was most important in predicting effective teaching of physical diagnosis, as measured by the grouped variable, (p = 0.006). The addition of two other variables,``corrected mistakes without making you feel belittled'', and intern gender created a model with R2= 0.35. When the physical diagnosis variables were analyzed separately, the teaching behaviors,``observed you elicit physical examination findings'' and,``demonstrated a breadth of knowledge in internal medicine'' were important in predicting the ability to distinguish pleural effusions (p = 0.087 and 0.075 respectively). Observing students eliciting findings was important to examination of the thyroid gland (p = 0.07). The models for the individual physical examination skills explained 23%-59% of variances. CONCLUSION: Teaching behaviors involving giving feedback and female gender were most important to students learning physical diagnosis skills from interns. Other teaching behaviors were important for individual physical examination skills. The reasons for the gender differences remain unclear. PURPOSE: In order to prepare residents for primary care practice an understanding of what factors influence their satisfaction with the outpatient experience is necessary. However, few studies have addressed determinants of resident satisfaction in their continuity clinics. This study was conducted to determine these factors. METHODS: Over a 2-month period, resident satisfaction was assessed through a selfadministered questionnaire completed by each internal medicine resident in their outpatient continuity clinic. All 68 residents working in the clinic completed the questionnaire. The number of questionnaires completed by each resident ranged from 1 to 60 with a median of 23. The questionnaire was comprised of three 5-point Likert scale items that assessed resident satisfaction with each patient encounter. Resident satisfaction was defined as the mean of the 3 items. The resident satisfaction items had a Cronbach's alpha of 0.88. Other items assessed by the questionnaire included contextual aspects of the clinic, diagnoses of the patients, continuity, and demographic data. RESULTS: Overall, the majority of clinic visits were satisfying to residents. The mean satisfaction score of all resident clinic visits was 4.02 (5-point scale) with a standard deviation of 0.91. However, patient diagnosis had a significant impact on resident satisfaction. The mean resident satisfaction score for patients diagnosed with general medical problems was 4.30. The mean resident satisfaction score for patients diagnosed with pain complaints was 3.58 (p < 0.0001) and for psychiatric disorders it was 3.27 (p < 0.0001). Clinic visits with patients diagnosed with pain complaints or psychiatric disorders were more satisfying to residents if the patients were male rather than female (p < 0.05). 1st year residents were less satisfied with patients diagnosed with pain complaints or psychiatric disorders than 2nd and 3rd year residents (p < 0.05). On the other hand, 1st year residents found visits with patients diagnosed with general medical problems more satisfying than 2nd and 3rd year residents (p < 0.05). A number of factors were not associated with resident satisfaction including resident gender, rotation, and call status. CONCLUSION: Although resident satisfaction with patient encounters in the outpatient clinic is high, resident satisfaction is influenced by the patient diagnosis. Seeing patients with pain or psychiatric diagnoses is associated with decreased resident satisfaction in their continuity clinic. However, this appears to be influenced by the experience level of the resident. Future research can explore whether early resident training in the treatment of pain and psychiatric conditions may increase resident satisfaction with such patients. PURPOSE: Approximately 25% of Americans are functionally illiterate and low literacy has been associated with poorer physical health, psychological health, and higher health care cost. This study was conducted to see if residents could identify patients with potential literacy problems based solely on interactions with patients during a clinic visit. We hypothesized residents will overestimate patients' literacy abilities and not identify many patients at risk for potential literacy problems. METHODS: Residents completed a questionnaire measuring a number of constructs regarding continuity clinic in general, and their perception of their continuity patients' literacy. The residents were asked,``Do you feel this patient has a literacy problem?'' Patients in continuity clinic were given the Rapid Estimate of Adult Literacy in Medicine-Revised (REALM-R), a new instrument to quickly screen for potential literacy problems. The REALM-R has been previously correlated with the Wide Range Achievement Test-Revised (WRAT-R) and the Rapid Estimate of Adult Literacy in Medicine (REALM), two well-validated instruments but impractical for large scale screening in a busy clinic. The REALM-R asks patients to read a series of eight medical words and a correct response is given for each correct pronunciation. A score of six or less was used to identify patients with a potential literacy problem. Data were available from 182 patients who completed the REALM-R and whose residents completed the literacy question. RESULTS: Patients ranged in age from 17 to 93, and 85% were Caucasian. Scores on the REALM-R ranged from 0 to 8 with a mean of 6.8 and S.D. of 2.1. Twenty-three percent of patients read at the eighth grade level or less according to the WRAT-R. Despite these baseline characteristics, residents believed only 10% (18) of patients had a literacy problem based on their clinical interactions. Residents perceived 90% (164) of patients to have no literacy problem, but 36% (59) of these patients scored 6 or less on the REALM-R. Conversely, residents identified three patients as having a literacy problem whom scored higher than six on the REALM-R, while the other 15 all scored six or less. CONCLUSION: Residents perceived a significant number of patients to have no literacy problem, although these patients scored poorly on the REALM-R. Residents incorrectly identified only a few patients. Residents' overestimation of patients' literacy is concerning given that many patients will hide literacy problems from physicians and the poor health outcomes associated with poor literacy. A brief screening instrument like the REALM-R may help clinicians identify patients in whom literacy may complicate their health care. METHODS: This data is derived from the intake questionnaire for participants in the PSPA study. We obtained names and addresses from the AAMC roster for faculty that entered academic medicine as Assistant Professor June through December of 1995. In 2000, 183 faculty members from 35 different states volunteered to participate. Participants indicated they were clinician-educators (CE) or clinician-investigators (CI). If participants indicated``other'' or`c linician'', we assigned them a priori to one of the two former categories based upon their percent effort allotted to various work activities. RESULTS: Participants had a mean age of 40 years, and had been working at their current position for 4.7 years. 63% of the faculty were clinician-educators (CE) and 37% were clinician-investigators (CI). More CI's than CE's were minorities (30% vs. 14%, p < .05), but there was no significant difference in gender (35% female). Whereas 98% of CI's reported a publication expectation to be promoted, 25% of the CE's reported no such expectation (p < .001). Significantly more CI's had career mentors available than CE's (68% vs. 32%, p < .001), and 92% of CE's for whom they are not available indicated they would like one. 79% of CI's indicated >10% protected scholarly work time, compared to only 35% of CE's (p < .001). 53% of CI's vs 32% of CE's (p < .01) meet more often than yearly with their chief/ chair for performance review, and more CI's have seen written promotion guidelines (72% vs. 51%, p < .01). CE's and CI's assigned similar importance to clinical research, written scholarship, and reputation (top 3 out of 11 items in importance for both groups) as activities leading to their promotion. The top 3 important activities for CE's according to promotion committee chairs in our previous study (teaching skills, clinical skills, and mentoring), were ranked 4th, 10th, and 8th by CE's in this study. Both CE's and CI's agreed that CI's more likely get promoted (82% vs. 79%). CONCLUSION: CE's indicated fewer performance reviews with their chair/chief, fewer mentors, and less knowledge of written promotion guidelines than their CI colleagues. Although university promotion chairs have indicated that teaching and clinical skills are most important in the promotion of CE's, CE's believe research and written scholarship are most critical to their promotion. PURPOSE: The purpose of this study was to identify residents' perceptions of the socioculturally and linguistically based barriers they face in caring for a diverse patient population. We also attempted to determine whether, and in what ways, a required crosscultural curriculum helped them deal with these barriers. METHODS: We conducted structured interviews with the entire intern class of an urban academic internal medicine residency program (n=40). These were carried out with the stated goal of identifying general barriers to quality care for their patient population. Interviews were performed within six months of the completion of an eight-hour, case-based, cross-cultural curriculum. Residents were not aware of any connection between the research assistant who conducted the interview and the primary researchers who taught the curriculum. Probes for the interview focused on: general barriers to care, sociocultural and linguistic barriers to care, if and how they had been prepared to deal with these barriers and the type of training they had received, and whether the cross-cultural curriculum had helped them and affected their attitude towards these issues. Interviews were recorded, transcribed, and coded by two independent outside researchers experienced in qualitative analysis. RESULTS: Several barriers to delivering care were identified:
1. Language differences between patient and resident 2. Limited time to address important cross-cultural issues in medical encounter 3. Understanding socioculturally-based patient expectations and perceptions medical care (including mistrust) 4. Differences between patient beliefs and physician beliefs regarding disease and illness Effect of cross-cultural curriculum on attitudes and practice:
1. Greater acknowledgement of the role of sociocultural issues 2. More probing into patients' beliefs and practices 3. Overall very helpful in addressing barriers CONCLUSION: Residents cite several barriers to caring for their socioculturally and linguistically diverse patient populations. While they identified several benefits of a crosscultural curriculum, they also highlighted the need for effective interpreter services and increased time to care these patients.
D.E. Bonds 1 , R. Watkins 1 , J.C. Mychaleckyj 1 ; 1 Wake Forest University, Winston-Salem, NC PURPOSE: Previous studies have found that Internal Medicine faculty and residents rate their ability in ambulatory care procedures as poor. Our objective was to assess faculty and residents ability in those skills related to Women's Health, and whether that ability varies by gender or status (resident vs faculty). METHODS: A descriptive survey was administered to all Internal Medicine residents and teaching faculty at one academic medical center. Respondants were asked to rate their ability to perform 5 ambulatory skills (breast exam, pelvic exam, wet mount/KOH, pap smear, endometrial biopsy) and comfort in obtaining 4 areas of history/counseling (sexual activity history, domestic violence history, preconception counseling, contraceptive counseling). A 4point Likart scale was used. RESULTS: 88 Internists completed the survey: 91% of residents (72/79) and 80% of faculty (16/20). Women comprised 32% (32% residents, 31% faculty). Greater than 70% felt able to perform all physical exam and procedural skills except endometrial biopsy (0% able). Conversely, less than 40% rated their comfort level as high for domestic violence history, and areas of counseling. Women rated their ability higher than men, and faculty higher than residents. See table 1 for full results (* indicates p < =0.05). CONCLUSION: Faculty and residents rated their ability high for exam and procedural skills, but most felt uncomfortable in obtaining history or performing counseling. Women rated their skill level higher, as did faculty. Both faculty and residents need additional training in ambulatory skills related to women's health. To assess the relative degrees to which a variety of factors are perceived to contribute positively and negatively towards motivating internal medicine residents to improve their physical examination skills. METHODS: In May 2000, forty G1 internal medicine residents at the Mayo Clinic completed an objective, structured clinical examination (OSCE). Immediately following this exercise, they were anonymously surveyed regarding the degree to which certain factors tended to either increase or diminish their motivation for improving their skills in physical examination. Twelve potential motivators and thirteen potential barriers were ranked on a Likert scale from one to seven. RESULTS: Those factors ranked as the strongest motivators for improvement had to do with the role of the physical examination (PE) in patient care decisions (mean score = 5.9), the regret of missed findings in the past (5.9), and the perceived contribution of strong PE skills to one's overall excellence as a clinician (6.0) or teacher (5.3). The need to enhance one's performance on formal measures such as the OSCE (3.6) or to fulfill the expectations of faculty (4.6) or peers (4.0) on rounds were rated as weaker motivators. Perceived barriers to improvement included lack of time to study (5.2) or apply (5.1) the methods of PE and a lack of faculty emphasis on the importance of PE (4.8). Although the sense of exam findings being eclipsed by test results in making clinical decisions was rated as a moderately strong demotivator (5.1), other factors designed to look for overall``PE nihilism'' were ranked as only weak hindrances to improvement (2.7). Likewise, a sense of discouragement over one's past performance and future prospects for improvement seemed to be a minor barrier (3.4) , as did reliance on a perceived supervisorỳ`s afety net'' (2.0). CONCLUSION: Internal medicine residents in their first year of training seem to be most strongly motivated to improve their PE skills by factors which relate to their desire to improve the quality of patient care, to avoid regret, and to build personal confidence as an excellent clinician and teacher. The most significant demotivators are lack of time to study and apply the methods, and lack of faculty emphasis on the importance of these skills. The perception of test results eclipsing the contribution of the physical exam to clinical decisions also seems to demotivate at some level, and educational strategies which emphasize those situations wherein the exam makes an indispensable contribution to patient care may help to overcome this potential barrier. Few studies, however, explore fellows' training expectations or their satisfaction with the program they select. We sought to evaluate these factors and identify areas of training that lead to a productive and satisfying fellowship experience. METHODS: We developed a 43-item questionnaire that addressed fellows' initial training expectations, satisfaction with the quantity and perceived quality of training (teaching and research), availability of mentors, scholarly productivity (publications, grants) and anticipated career track. Question format included best answer and Likert scales. Directors of the 37 active fellowship programs listed in the 2000 SGIM Fellowship directory were contacted by e-mail and asked to forward our web-based survey to current fellows. To calculate an accurate response rate while maintaining respondent anonymity, each fellow used a program-specific code to initiate the survey. RESULTS: Forty-three percent (65/152) of fellows completed the survey, with at least 1 respondent in 65% (24/37) of the programs. Nearly three-quarters (74%) anticipated careers as clinician-investigators. Eighty-two percent had high or very high initial expectations for research training, 26% for teaching and 20% for learning leadership skills. The availability of mentors (95%), program flexibility (94%) and protected time for research (91%) were rated as the most important factors in fellowship training. Most fellows were satisfied or very satisfied with the actual availability of mentors (80%), program flexibility (85%) and protected research time (88%). Over 80% agreed that their expectations had been met for both the quantity and quality of research opportunities, while 63% reported this for the quantity and 40% for quality of teaching opportunities. The availability of mentors and the perceived quality of research opportunities were significantly correlated with fellowship scholarly productivity, 0.507 and 0.539 respectively (p < .001). CONCLUSION: This study suggests that the majority of GIM fellows enter training with the expectation for advanced research skills and are satisfied with most aspects of their experience. These data highlight the important correlation between mentoring, high quality research opportunities and scholarly productivity during fellowship. That relatively few fellows are satisfied with the quality of available teaching opportunities is concerning and deserves further exploration. METHODS: We designed a 10-page survey to determine the extent of faculty development (FD) activities that focused on``improving the teaching/educational skills'' of faculty. For those hospitals with ongoing activities we asked about the subjects covered, the methods of teaching, the formats offered, and the evaluation of the programs. In April 2000, we mailed the survey to Department of Medicine chairmen at the 390 teaching hospitals with internal medicine residency training programs in the U.S. We classified teaching hospitals with membership in the Association of Professors of Medicine as university hospitals (N=114), and those without membership as non-university. We performed descriptive analyses of hospitals with and without ongoing FD, and used chi2 to detect differences in categorical variables and t-tests for differences in continuous variables. RESULTS: Three hospitals responded that their training programs had closed. Of the remaining 387 hospitals, 279 responded for a response rate of 72%. Only 39% of teaching hospitals have ongoing FD, including 48% of university and 34% of non-university hospitals (p=.007). An additional 34% have occasional FD; 27% have no FD activities. At the hospitals with ongoing FD activities (N=109), an average of 10 content areas are covered, with university hospitals including more than non-university hospitals (11 vs. 9, p=.036). The most common content areas are general teaching principles (90%), giving feedback (88%), outpatient precepting (72%) and evaluation of learners (72%). FD is taught most commonly in small group sessions, with 65% of hospitals using them frequently/always, and in lectures, with 50% using them frequently/always. The most common format for FD is a 1/2 day workshop (78%), but 21% offer courses lasting more than a month. Compared to university hospitals, non-university hospitals are more likely to offer 1/2 day workshops only (p=.008), or courses < 7 days (p=.013), and less likely to offer longer courses (p=.006). Evaluation of FD programs is limited to forms filled out by participants at the end of the program. Most programs had trained: < 50% of hospital-based and < 25% of community-based faculty; < 50% of general internal medicine faculty and < 25% of subspecialist faculty. 2/3 of hospitals with FD, but only 37% of hospitals with no FD, arrange for faculty to go off-site for FD.
CONCLUSION: A minority of teaching hospitals have ongoing faculty development programs for improving their faculty's teaching skills and evaluations are primitive. University hospitals are more likely to have ongoing FD and to have programs that are more developed. Overall, only a limited percentage of faculty have been trained, especially community-based and subspecialist faculty. These gaps should be considered in future funding decisions to provide learning opportunities for all faculty. PURPOSE: This study was done to determine the relationship between students' ratings of faculty on a clinical teaching evaluation form and a medical lecture evaluation form. METHODS: Twenty-three faculty received ratings between July 1999 -December 2000 from medical students immediately after a didactic lecture on an 11-item lecture evaluation form that used a scale from 1 (not satisfied) to 7 (very satisfied) to assess lecture skills (Chronbach's alpha=.96) and at the end of a clinical rotation on a 15-item clinical teaching form that used a scale from 1 (never/poor) to 5 (always/superb) to assess attending/teaching behaviors in the patient care setting (Chronbach's alpha=.97). An average of 6 and 16 students per faculty completed the lecture and the clinical teaching forms respectively. Average students ratings on all items for each faculty member were calculated. Pearson correlation coefficients were computed between the average scores for each item on one form and the overall average scores on the other evaluation form. RESULTS: Overall there is a correlation of .69 (p < .01) between the average scores on the two forms. Only correlation coefficients above .65 (to explain >45% of the varaince) were considered meaningful. The following five items from the lecture form significantly correlated (p < .01) with the average clinical teaching scores: how well the course was organized (r=.69), how effectively time was used in class activities (r=.67), the clarity of the instructor's explanations (r=.70), the instructor's use of examples or illustrations to help clarify the material (r=.67), and having learned something which I consider valuable (r=.67). The following three items from the clinical teaching form significantly correlated (p < .01) with the average medical lecture scores: teaches diagnostic skills (r=.73), teaches effective patient/family communication skills (r=.72), and teaches principals of cost-appropriate care (r=.70). Interestingly, these three items were the only items that included the word``teaches'' suggesting direct didactic instruction as opposed to the teaching strategies suggested in the other items (i.e., adjusts teaching, coaches, asks). Items from the lecture form that did not correlate with the average clinical teaching scores (e.g., specifying objectives, summarizing material, encouraging class discussion) are often considered fundamental in classroom teaching and may not easily translate to clinical teaching. Yet, the larger number of related items from the lecture form suggests that lecture techniques do crossover to clinical teaching, especially with providing explanations and using examples. CONCLUSION: There appear to be some differences in the skills involved in clinical teaching and lecturing; only 47% of the variance is explained between them. Although requiring more work by students, using two forms to evaluate teaching may better enable instructors to identify specific strategies where they are strong or need improvement for the various teaching activities they perform.
AVAILABILITY OF SAMPLE MEDICATIONS AND RESIDENTS PRESCRIBING PATTERNS. J. Diaz 1 , M.J. Fagan 1 , A. Etienne 1 ; 1 Brown University, Providence, RI PURPOSE: The use of sample medications in clinical teaching settings is controversial. One concern is that the presence of sample medications may affect physician compliance with treatment guidelines. This project examined the availability of anti-hypertension sample medications and the selection of specific medications by internal medicine residents to treat hypertension. METHODS: From 12/4/00 to 12/20/00, charts of patients presenting to the residents' clinic at Rhode Island Hospital were reviewed if hypertension was listed as one of their diagnoses and the patients were receiving medications. All anti-hypertension medications prescribed, the date of visit and insurance status of each patient were recorded. During the same time period, inventory of the clinic's medication sample closet was taken for each clinic session and names of all antihypertension medications available were noted. RESULTS: During the 3 week period, 288 patient charts met criteria for review. 56% (161/ 288) of patients had no insurance coverage for medications. A total of 41 different blood pressure medications were documented as being prescribed and 18 (44%) of these were available as samples. The 5 most commonly prescribed medications were hydrochlorothiazide (86/288), Norvasc (amlodipine-73/288), Accupril (quinapril-66/288), Vasotec (enalapril-42/288) and Toprol XL (metoprolol-32/288). Three of these, Norvasc (amlodipine), Accupril (quinapril), and Toprol XL (metoprolol) were available in the sample closet during the study period and one, Vasotec (enalapril), had recently been available. Except for diuretics, for each class of antihypertension medication, the most commonly prescribed agent was also available as a sample. Of 146 notations of ACE-Is, 65 (44.5%) were Accupril (quinapril). Of 118 calcium-channel blockers (CCBs) prescribed, 70 (59%) were Norvasc (amlodipine). Of 65 notations of betablocker use, 31 (48%) were Toprol XL (metoprolol). No diuretics were available as samples, but the diuretic most commonly prescribed was a generic agent, hydrochlorothiazide (70%, 84/120). Stratifying by medication coverage, a sample closet CCB and a sample closet ACE-I continued to be the medications most commonly prescribed in their drug classes. Norvasc (amlodopine) accounted for 54% (38/70) of CCBs prescribed to patients without medication coverage and 67% (32/48) of patients with coverage. Accupril (quinapril) accounted for 42% (36/85) of ACE-I prescribed to patients without coverage and 47% (29/61) of patients with coverage. CONCLUSION: This pilot study suggests that residents selecting medications to treat hypertension may be influenced by the availability of sample medications. With the exception of hydrochlorothiazide, for each class of anti-hypertension medication, a drug available in the sample closet was the one most commonly prescribed. Although this reflects patterns at only one institution, residency programs should be aware of how sample medications may influence the prescribing patterns of physicians in training.
PURPOSE: Several studies over the past two decades have examined the issue of depression in house staff, but none have attempted to relate it to house staff performance. The ideal study to examine this question would be prospective and longitudinal, using validated psychiatric rating instruments and quality assurance data. Since IRB concerns have thus far precluded such a study, a pilot retrospective survey was developed to obtain preliminary data. METHODS: After IRB approval was obtained for the pilot, the survey instrument was distributed to interns, junior residents, and senior residents in the internal medicine residency program at our institution. Participants were asked whether they had worked with someone they thought was depressed, if they had worked with a colleague whose performance had been affected by depression, and if they had ever been depressed themselves during the residency. RESULTS: 45 members of the house staff (33%) replied to the survey. 34 respondents (76%) believed that they had worked with a depressed colleague during the residency, and 23 respondents (51%) believed that a coworker's performance had been affected by depression. Fifty-one percent of the respondents also reported having experienced depression themselves during their residency. CONCLUSION: Although the sample size is small and these data are only preliminary, depression appears to be prevalent in some internal medicine residency programs. Furthermore, house staff depression may affect patient care. The current pilot study indicates the need for further research in this area. format, the other 2/3rds was interactive. Each workshop included a case with a clinical question, the search strategy, and an article selected to answer the question. While in the small groups participants assessed the validity of the study, interpreted the results and applied the results of the study to the clinical question. We assessed the usefulness of the workshop, materials and effectiveness of the facilitators by asking participants to fill out an evaluation form using 5-point Likert scales to rate the individual items. The participants were asked to rate the workbook and its content, the facilitators, the format of the workshop, the didactic sessions, the interactive sessions, and if they would recommend the workshop. RESULTS: Sixty-one participants completed the evaluation sheets. The median scores for all items were 4 or 5 for all items in both years. The workshop``practice session'' was slightly higher rated in 2000, compared to 1999 (p = 0.047). There were no other significant differences between years. CONCLUSION: A 90 minute workshop can effectively teach the principles and methods for the development of a successful EBM-CDM journal club to faculty members. PURPOSE: Evidence based medicine (EBM) is part of the curriculum of most medical schools and residency training programs in the US. We hypothesized that the concepts and skills may not be integrated into clinical practice because there is little support outside the classroom setting. We surveyed academic internal medicine generalists and subspecialists to assess whether they have different attitudes about evidence based medicine and whether they rate the importance of specific EBM skills differently. METHODS: We surveyed a stratified random sample of academic internists and subspecialists from 11 medical centers in the Chicago area (n=330) in the Spring of 2000. Respondents completed a 39-item self-administered questionnaire, which was piloted and revised for internal consistency prior to use. The three survey questions required respondents to: 1) rate the importance of 9 core skills and 5 EBM skills on a 5-point Likert scale, 2) choose the five most important skills in residency training, and 3) indicate the level to which they agreed or disagreed with 15 attitudinal statements about EBM, also on a 5-point Likert scale. We also collected information on year of graduation from medical school, teaching responsibility, and exposure to a formal course on clinical epidemiology or evidence based medicine. RESULTS: The response rate was 63%. Core medicine skills (clinical reasoning skills, differential diagnosis skills, physical exam skills, interviewing skills, and doctor-patient interaction skills) were scored highest, with all EBM skills rating at least 1 point lower: average core skill score 4.4 vs. average EBM skill score 3.5 (p < 0.001). None of the EBM skills were listed in the five most important skills. EBM skills were rated higher among younger faculty (p = 0.01) and those who had previously taken an EBM course (p = 0.03), but generalists and subspecialists rated the importance of EBM skills similarly (p = 0.56). However, generalists had slightly more positive attitudes about EBM (p = 0.014). Neither year of graduation nor exposure to an EBM course predicted faculty attitudes toward EBM. CONCLUSION: Contrary to our initial hypothesis, generalists and subspecialists differ little in their attitudes about EBM and in their ratings of the importance of EBM skills in residency training. Although both groups rated EBM skills positively, the skills were prioritized much lower than all core skills. Perhaps EBM skills are not routinely reinforced outside formal teaching settings because they are not a high priority among academic faculty-generalists and subspecialists alike. PURPOSE: Few studies have examined the effectiveness of Internet-based medical educational programs for physicians. We wanted to determine whether a skin cancer triage intervention, developed and proved successful in a face-to-face, on-site application, could be effectively delivered over the Internet with the same positive results. METHODS: Physicians were randomly assigned to an Intervention or Control Group. Intervention Group physicians completed modules which included a Pretest, Pretest Scores with Individualized Feedback, Instruction (text and hyperlinks to digitized images of skin lesions), and two Posttests. Control Group physicians completed the Pretest and Posttest I. The program could be completed in 3-4 hours, at participants' convenience. There were 14 outcome measures including diagnosis and evaluation planning for malignant melanoma, basal cell carcinoma, and squamous cell carcinoma. RESULTS: 71 physicians completed the program through Posttest 1, and 46 (27 of 39 in the Intervention Group) completed the entire program. The Intervention Group showed significantly greater improvement than the Control Group in 9 of 14 outcome measures. By Posttest II, Intervention Group improvement was maintained in 5 of the 9 outcomes. CONCLUSION: Our brief, Internet-based, educational intervention with individualized feedback was successful in improving the skin cancer diagnosis and evaluation planning test performance of primary care physicians. The IOM's report on medical errors focused attention on the importance of measuring quality and outcomes. As part of the UME-21 project funded by HRSA, the UCSOM developed a Quality Improvement Curriculum. This abstract examines the impact of student CQI projects on the quality of care delivered at community practices. METHODS: Eighty second year students working in groups of 3-4, initiated a CQI study on Diabetes Mellitus at 23 community-based primary care practices collecting baseline data, implementing a results-specific intervention and re-measuring quality indicators 8 months later. Students attend community-based primary care continuity clinics one-half day/week, year I-III. Students were evaluated using Likert-scale rated attitudinal items as well as open ended questions. RESULTS: 512 charts were abstracted for the baseline sample with 383 charts abstracted postintervention. The number of documented foot and eye exams increased significantly from baseline to remeasurement (51% to 67.8%; p < .001 and 26.4% to 35.5%; p= 0.003, respectively). The mean value for glycohemoglobin dropped from 7.71% at baseline to 7.2% at remeasurment (p=.0001). Analysis of student attitudes revealed acknowledgement of the benefit of outcomes measurement in clinical practice despite frustration with the tedium of the chart abstraction process. Feedback was used to modify curriculum design for future classes. CONCLUSION: Medical student-driven CQI projects can have significant impact on improving the quality of care at the practices in which they participate while introducing them to the process of quality measurement and improvement. Formative input from students should be used to optimize CQI experiences. The use of medical students to lead CQI efforts in private practices may represent an underutilized resource in our efforts to improve the quality of care provided to the public. launched the first program to financially support community-based teaching by enhancing the capitation level of primary care preceptors. We studied the effects of these enhanced capitation payments on preceptors' attitudes towards student training and towards managed care's support of teaching medical students. METHODS: Participants were community-based preceptors who completed a questionnaire consisting of sections on attitudes towards teaching and attitudes towards managed care, given prior to the distribution of enhanced capitation payments (Time 1) and after receiving payments (Time 2). The instrument was given to three groups: AUSHC preceptors receiving enhanced capitation (86/93, 92.5% response rate) and control groups consisting of AUSHC preceptors who chose not to participate in the enhanced capitation program (57/66, 86.4% response rate) and non-AUSHC preceptors (62/68, 91.2% response rate). Responses were scored using a sixpoint Likert scale ranging from Strongly Disagree (1) to Strongly Agree (6). Statistical tests employed included chi-square for differences between the respondent groups, and principal factor analysis and t-tests for differences between Time 1 and Time 2. RESULTS: Factor analysis identified three underlying factors: 1.) Attitudes towards managed care support of physicians, which became less positive, decreased from 3.58 to 3.43 (p = 0.28); 2.) Attitudes regarding the impact of teaching on their practices, remained positive, although slightly decreasing from 4.70 to 4.57 (p=.043); 3.) Attitudes towards their roles as trainers, which was very positive, remained unchanged (5.06 to 5.06, p = .956). No significant difference was found in attitudes when comparing those who received enhanced capitation to the control groups. CONCLUSION: While the enhanced capitation program has provided community-based preceptors with additional financial support, it did not improve preceptors'attitudes concerning managed care's support of their teaching activities. We believe this program is an important first step but more is required. Just as attitudes towards managed care are the result of many factors, initiatives to change those attitudes should be multifactorial.
MODALITIES. E.H. Green 1 , R. Granieri 1 ; 1 University of Pittsburgh School of Medicine, Pittsburgh, PA PURPOSE: Internal medicine residency training involves exposure to different learning modalities including rounds, lectures, small group discussion, and independent study. Little is known about learners' views regarding different educational interventions. We administered a questionnaire to assess current medical residents' attitudes towards different learning modalities. METHODS: Second and third year medical residents from an academic residency program and closely affiliated community residency program were surveyed (n = 91). Respondents were asked to rate, using a Likert scale and list ranking, the contribution of various educational activities to their understanding of clinical medicine, preparation for standardized tests, and overall education. Subgroup analysis was done according to level of training, career goals (primary care or subspecialty medicine; academic or community), gender, college major, and additional education. RESULTS: A total of 50 residents (55%) responded.``Noon conference'' (core lecture series) and morning report were highly valued, with 82 and 84% of residents rating these endeavors as moderately or very important to their overall education. Attending rounds were less important, with 68% rating them as moderately or very important. Further, 44% of residents felt``too much'' time was devoted to attending rounds. Medical grand rounds had the least contribution to residency education with 33% rating it as moderately or very important. In contrast, 92% of residents rated independent learning with as moderately or very important. Independent study was the only educational activity that residents thought prepared them equally well for clinical medicine and standardized exams. Residents actively pursued independent study with a mean of 12.69 topics researched in the 3 months prior to the study. However, 52% of respondents felt they had``too little'' time for this endeavor. Independent study and morning report were independently identified as the two most important educational activities during residency. The only significant finding from subgroup analysis was that residents with advanced degrees valued independent study more than their colleagues (p = .038). CONCLUSION: In this study, internal medicine residents valued lectures and morning report over attending rounds or medical grand rounds. However, independent study is the most highly valued educational method, and house officers are very involved in this despite a perceived lack of time. Medical residents seem to be mastering the principles of adult, independent learning: future curricular reform should consider incorporating more time and opportunities for independent study. used; whether PDA's replaced medical texts; and subjective responses regarding the impact of PDA's on patient care and medical education. 114 of the 133 surveys were returned (86% response rate). RESULTS: 62% of medical residents owned PDA's (100%-based on the Palm operating system). The mean age of the cohort was 28 years, and 55% of PDA owners were male. Ownership was inversely correlated with both age and level of training. The most commonlyused medical applications were drug formulary references, medical formula calculators, medical note-taking, and reference texts. Interestingly, only 15% of PDA owners replaced their paperbased drug references with the PDA version. Relatively few residents used their PDA's for patient tracking, appointment scheduling, or to log procedures. The most-commonly used nonmedical applications included the built-in address book, memo pad, to-do list, calculator, local city guides, and games. 83% of owners felt that PDA's improve efficiency, and a majority felt that they were useful in medical education. 69% responded favorably when asked if PDA's improve patient care, but only 48% believed that PDA's decrease medical errors. 37% of nonowners were committed to purchasing a PDA within 12 months. CONCLUSION: The majority of internal medicine house officers at New York-Presbyterian Hospital already own PDA's. Most residents use drug references and medical calculators, perhaps because they provide point-of-care answers to direct questions. The majority of residents believe that PDA's are useful for improving efficiency, medical education, and patient care. However, more research is required to correlate these positive perceptions with objective improvements in clinical outcomes, as well as broadening the applicability of the results to other specialties. Furthermore, as PDA's and the availability of medical applications continue to proliferate, issues of medical privacy and quality-assurance of medical content will become increasingly important. PURPOSE: Despite calls to incorporate active learning strategies into medical education, many teachers believe that efficient delivery of content can only be achieved through didactic lectures. The goal of this study was to examine the effects of reducing didactic lecturing and adding small group work, a commonly employed active learning strategy, on learner participation, attitudes, and knowledge acquisition. METHODS: We recruited residents in internal medicine, pediatrics, and family medicine to attend a session on effective use of diagnostic tests, and assigned them to one of two groups. The control group received a standard 'slide and lecture' session where the lecturer spent a full hour delivering content. The intervention group received a session where residents were assigned to groups of 4-5 and completed a series of 3 small-group tasks with large-group lecturer-facilitated discussion between tasks; in this session, the lecturer delivered content only during large group discussions (approx. 30 minutes). Both sessions used the same lecturer and covered the same content. We measured resident demographics before and self-perception of active learning after both sessions. We measured attitudes toward the session content and knowledge before and after both sessions. We also employed trained observers, blinded to the study purpose, who used a standardized instrument to observe resident patterns of behavior during each session. RESULTS: 27 residents completed the control session and 36 completed the intervention session. The study groups did not differ significantly in demographic variables, baseline attitudes about, or baseline knowledge of the session content. As measured by the trained observers, during the intervention session more students were communicating with other students (p < .001) than during the control session. Students in the intervention group also selfrated their participation in the session higher (p=.001). While both groups improved in both knowledge and attitude scores from pre-to post-session (p < .001 for all comparisons), there were no significant differences in the amount of improvement between groups. CONCLUSION: In these sessions conducted in a controlled environment, we reduced the time spent 'lecturing' by the teacher by 50 percent and covered the same amount of content with no detrimental effects on knowledge acquisition or attitude enhancement. In addition, our incorporation of active learning strategies produced residents who both self-rated and were observed to be more actively engaged with each other and with the learning process. Our future work will measure the effects of this active engagement on long-term retention of content and attitudes. PURPOSE: Existing data suggests that physicians who demonstrate patient-centered behaviors enjoy high patient satisfaction. However, little data exists to link physician attitudes about medical care with patient-centered outcomes. In this study, we explored associations between student physicians' attitudes toward the physician-patient relationship and patients' perceptions of students' humanism during the medical interaction. METHODS: At the beginning of a primary care (PC) clerkship, we surveyed 293 third-year students using the Patient-Practitioner Orientation Scale (PPOS), a validated instrument that measures attitudes toward the physician-patient relationship. PPOS scores range from patientcentered (egalitarian, whole person-oriented) to physician-centered (paternalistic, less attuned to psychosocial issues). During the PC clerkship, all students performed a medical interview with a series of 5 standardized patients (SPs) who were trained to portray varying biopsychosocial issues. All SPs were unaware of the students' PPOS scores. After each interaction, each SP completed a 7-item validated instrument that measured their perceptions of the student physician's humanism. We calculated a total humanism score for each student and compared these to students' attitudes as measured by PPOS.
RESULTS: Mean humanism scores were lowest for students who scored in the most physiciancentered quartile (79.6 9.4) and highest for students who scored in the most patient-centered quartile (83.4 7.0) on PPOS. In linear regression analysis controlling for month of the thirdyear on the primary care clerkship, patient-centered PPOS scores (p=.01) and female gender (p=.05) were found to be associated with higher total humanism scores. CONCLUSION: Student physicians with more patient-centered attitudes demonstrated higher degrees of humanism as perceived by standardized patients. Patients' perceptions of humanism were also influenced by student gender. Further research is needed to explore the nature of associations between physicians' attitudes and patients' perceptions of care, as well as the impact of medical education on students' attitudes toward the physician-patient relationship. PURPOSE: Although training in the skills required to perform pelvic examinations is ubiquitous in medical schools, few residency programs assess or reinforce these skills, and only limited evidence justifies the substantial cost of post-graduate training. We report results of a controlled study of training for Internal Medicine interns utilizing professional instructors who also serve as models for the exam. METHODS: 48 interns from two university Internal Medicine residencies completed questionnaires about their experiences with pelvic examinations. 23 participated in a two-hour training session after which their skills were assessed. At follow-up four months later, the skills of 13 of the trained interns were compared with that of 24 interns who had not been trained. Assessments were based on the observation of verifiable behaviors. Chi-square tests were used to assess the statistical significance of comparisons. Inter-rater reliability was assessed with the kappa statistic. RESULTS: Mean age of the 37 interns in the controlled study was 28.4 (range 24-40), with 57% women and 89% graduates of U.S. medical schools. 70% of the interns described having received specific pelvic exam skills training in medical school, 78% felt comfortable performing the exam, and 91% felt they give patients clear instructions. Interns who participated in the current training displayed 10 of 12 key behaviors more frequently when compared with interns who had not been trained. Overall, trained interns demonstrated 87% of key behaviors, compared with 63% for untrained interns. Specific items for which the largest improvements occurred were in providing an outline of the exam (100% for trained interns vs. 38% for those not trained) and explicitly giving permission to ask questions (69% vs.17%; p < 0.01 for all three comparisons). Independent video and in-person observers agreed well with ratings of instructors (kappas > 0.7). CONCLUSION: Most interns described exposure to medical school pelvic examination training, but many overestimated their skills. A program employing professional trainers can reliably evaluate and improve key skills and improvements are demonstrable months after training. Further work is needed to demonstrate consistent benefits in larger groups of interns and residents, to ascertain whether training efficiency could be improved, and to measure the impact of training on subsequent encounters with actual patients. The mini-CEX can facilitate interactive feedback (FB) between faculty and residents to help residents correct deficiencies and reinforce good clinical skills. However, little is known about the nature of the FB that results after a mini-CEX is completed, and whether the FB is interactive. PURPOSE: To assess how frequently interactive FB is given after completion of a mini-CEX with PGY-1s in an outpatient setting. METHODS: Prospective cohort study of mini-CEXs performed with PGY-1s in the outpatient setting at three internal medicine residency programs: Yale University Primary Care, Washington Hospital Center, and the National Naval Medical Center. Feedback sessions were audiotaped after completion of the mini-CEX. All tapes were transcribed with all identifying information deleted. Transcripts were independently coded by two investigators (ESH, SH). Disagreements were resolved by consensus. Interactive FB was defined as: a) any recommendation(s) given to the PGY-1s; b) PGY-1s were asked to self-assess their mini-CEX performance (self-assessment); c) PGY-1s were given an opportunity to react to the FB (learner reaction); d) and faculty stated a specific plan to change PGY-1's future performance (action plan). RESULTS: For this preliminary analysis, 30 attendings provided FB sessions to 41 different PGY-1s at the 3 residency programs. Fifty sessions have been transcribed and analyzed. Forty-five (95%) FB sessions included at least one recommendation. The median number of recommendations per session was 2 (range 0-9). Recommendations were given most commonly for communication skills, notably patient interviewing (31 sessions, 62%) and counseling (21 sessions, 42%). Feedback on components of the physical exam was given in 13 sessions (26%). Attendings asked PGY-1s to self-assess performance in 19 sessions (38%) and to specifically respond to the FB (learner reaction) in 32 sessions (64%). Despite the high frequency of recommendations, only 3 sessions (6%) included an attending's discussion for an action plan to help the PGY-1 correct noted deficiencies. CONCLUSION: For this cohort, the mini-CEX effectively generated recommendations to improve clinical performance, especially in communication skills, and the majority of attendings asked PGY-1s to react to the FB. However, programs planning to use the mini-CEX should also consider strategies to encourage their faculty to provide more FB on other observed skills, promote more self-assessment, and establish definitive action plans to correct deficiencies.
A.L. Hull 1 , H. Copeland 1 , M.G. Hewson 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH PURPOSE: Clinicians' self-perceptions of teaching effectiveness will affect their interest and committment to improving teaching ability. This IRB-approved study investigates the relationships among self-perception and characteristics of clinical teachers including demographics, interest and satisfaction with teaching, and perceived institutional support for teaching. METHODS: Perceptions, attitudes, and characteristics of clinical teachers was obtained in a survey instrument sent to 164 Division of Medicine faculty who had taught and had teaching evaluation instrutments completed Internal Medicine residents in 1998 and 1999. 94 (57%) of the surveys were completed after 2 reminders. The responses were coded and matched to the respondents' average of a 15-item clinical teaching evaluation (which has been judged to be reliable and valid) completed by residents for any rotation during the 2-year period. The dependent measure was a 5-point Likert-scale survey item evaluating the self-perceived teaching effectiveness (TE). The independent measures included faculty characteristics (age, gender, years in practice), satisfaction with teaching (enjoy teaching, career satisfaction, perceived institutional support for teaching), percent time spent teaching, involvement in teaching improvement activities (workshops, reading), and average of self-ratings of the 15-item clinical teaching evaluation. SPSS/PC was used to compute descriptive statistics, correlations between the dependent and independent measures, and a step-wise regression. Self-ratings of the 15-item teaching evaluation were not included in the regression analysis in order to maximize the effects of the other independent measures. RESULTS: The average TE score was 3.85 (+À .63). Statistically significant positive correlations (p < .05) were found between TE and average resident evaluation score (r=.213), percent time teaching (r=.251), average self-rating of the 15-item teaching evaluation (r=.457), and satisfaction from teaching residents (r=.336). All other characteristics were not significantly correlated with TE.
Step-wise regression only resulted in reported enjoyment of teaching residents predicting TE (R=.414 [difference in N causes difference between r and R]). CONCLUSION: Internal Medicine faculty believe that they effectively teach residents, and self-perceptions of teaching effectiveness are correlated with actual resident evaluations of teaching (suggesting accuracy of the faculty member's perception), and time spent and enjoyment from teaching (faculty self-select activities that they enjoy and are good at). Faculty self-preceptions in general are not correlated with demographic characteristics, effort at improving educational competence, and perceived support for teaching, suggesting that faculty development efforts may need to be more individualized and focused on faculty who elect to participate rather than institution or department-wide programs.
RESIDENTS AS TEACHERS. R.W. Janicik 1 , M.D. Schwartz 1 , A. Kalet 1 , S. Zabar 1 ; 1 New York University School of Medicine, New York, New York PURPOSE: We sought to measure residents' attitudes, preparation, and confidence about teaching and how these relate to their plans to teach. METHODS: We conducted an anonymous, cross-sectional survey of Medicine and Psychiatry residents at NYU. The questionnaire assessed attitudes and preparedness to teach, selfperceived teaching knowledge and skills, and plans to teach. RESULTS: Sixty-one residents (42 Psychiatry, 19 Internal Medicine) completed the questionnaire (79% response). Factor analysis confirmed scales for measuring residents' attitudes (Teaching Enthusiasm, Important Role in Department), preparedness to teach (Teaching Exposure, Teaching Capability) and self-perceived teaching knowledge and skills (Clinical Reasoning, Patient Management, and Procedural Skills) . Scales were dichotomized to present the % of residents with high scores: Teaching Enthusiasm -67% agree; Important Role in Department -34% agree; Teaching Exposure -only 7% reported high amount; Teaching Capability -23% high confidence; Knowledge of Clinical Reasoning -30% confident; Patient Management -49% confident; and Procedural Skills -21% confident. When asked the frequency with which they plan to teach, 75% planned to directly observe learners, 71% planned to give feedback, and 74% planned to teach with the patient present, all at least once per week. Plans to teach was not associated with resident age, gender, year of training, or department. Nearly significant trends suggested that residents with high teaching enthusiasm were more likely to plan to observe their learners and those with high prior teaching exposure were more likely to plan to give feedback. Teaching capability and all 3 knowledge variables were correlated with plans to teach with the patient present (p < .05). CONCLUSION: Residents value and look forward to their teaching roles despite having little preparation for teaching, and only modest confidence in their ability and knowledge to teach. Resident attitudes and self-perceived capabilities influenced their plans to teach. Programs to prepare residents as teachers that address attitudes, skills, and knowledge in these areas may lead to more teaching by residents. were general internists, 24% family practitioners and 5%``other medical specialties.'' Forty percent of respondents received some form of spirituality training. Twenty-nine percent received this training while medical students compared to 18% as residents or as fellows and 23% as attending physicians. The training received in medical school was largely in the form of small group lecture series, but during residency and fellowship, the training consisted of informal clinic discussions. Attendings primarily received training through self-education (books and journals). Forty-two percent of respondents agreed that they would desire further spirituality training while 20% disagreed, and 38% were neutral. The physicians scoring highest (greater than 4 out of a possible 6 on the Spiritual Well-Being Scale) were more likely to desire further training. In addition, those physicians who attended worship service (48%) and those who agree to the statement``faith alone could cure disease'' (60%) were more likely to desire further training (all p-values < .05). Also family physicians had a greater tendency to desire further training compared to general internists. CONCLUSION: Formal spirituality training related to medical care is sparse in medical school and almost non-existent thereafter. Although physicians with stronger religious predilections desire formal training, nearly half of all primary care physicians who returned the survey felt that further training in spirituality and medicine was desirable. A curriculum concerning patient spirituality and medicine at the medical school and residency level appears to be warranted. PURPOSE: Chart audits are used to assess physician performance and quality of care. Typically, records are reviewed for practicing physicians. Only recently have resident report cards been described. The purpose of this study was to compare performances of interns, senior residents, and faculty on adherence to national guidelines regarding prevention and management of common outpatient medical conditions. METHODS: The study was conducted at the outpatient practices of a university based internal medicine training program. For interns (n = 50), rosters of all new general medicine patients with 2 or more visits between July 1999 and April 2000 were generated. For senior residents (n = 39) and faculty (n = 30), the time frame extended from July 1998 to April 2000. A random sample of 9 patients per physician was identified. All visits with the designated provider through June 2000 were abstracted by nurse abstractors using a 115 item form. 91% (n= 965) of targeted charts were abstracted: 366 for interns, 349 for senior residents and 246 for faculty. Performance scores (the percent of indicated actions taken) were determined for each chart in the areas of cancer screening, immunizations, counseling, management of diabetes (DM), hypertension (HTN), asthma, coronary disease (CAD), and depression. Differences in intern, resident and faculty performance were assessed with ANCOVA and correlations. RESULTS: The mean patient age was 52.9 (sd=17.6); 68% were women. Patients had a mean of 0.77 comorbidities and 4.1 visits with the provider. These factors were covariates in the ANCOVA due to group differences. Faculty performed better than interns and senior residents overall (p = .03) and in the areas of counseling (p = .0007), immunizations (p = .003) and HTN (p = .002). Unadjusted mean performance scores for interns, senior residents and faculty were as follows: overall (46%,49%,52%), counseling (26%,27%,37%), immunizations (36%,37%,43%), and HTN (44%,50%,57%). There were no differences between intern, resident and faculty performance in the other areas. Mean performances were: cancer screening (66%), management of DM (48%), asthma (56%), CAD (67%), and depression (41%). Higher performance scores were associated with more visits (r = .19, p=.0001), fewer comorbidities (r = À.08, p = .02) and younger age (r = À.12, p = .0002). CONCLUSION: On only a few domains did performance improve with experience. In those domains, there was steady improvement through training. Average performance rates fell short of national guidelines for all groups, though some actions were likely not documented. Future efforts should explore how to improve adherence in training programs, including among the teaching faculty.
IMPACT OF A NIGHT FLOAT SYSTEM ON HOUSESTAFF SATISFACTION. S. Kripalani 1 , M.V. Williams 1 ; 1 Emory University, Atlanta, GA PURPOSE: In an effort to reduce workload, a night float (NF) rotation was established in July 1999. Three of twelve ward teams were on call each day and stopped admitting patients at 2300. They remained overnight to complete work-ups and cover their own service. From 2300 until 0700, NF teams performed all medical admissions and covered the other 9 teams. Two or three NF teams (consisting of an intern and resident) worked each night. In the morning, they handed-off the new patients to ward teams and attended a teaching conference before going home. We conducted this study to evaluate the impact of the NF system on satisfaction and quality of care. METHODS: Ward residents (WR) and night float staff (NFS) completed a 2-page questionnaire at the end of each month from July 1999 to April 2000. Survey questions focused on the following domains: workload, educational activities, morale, and quality and continuity of care. RESULTS: The overall response rate was 81% (n=92 WR and 51 NFS). WR got an average of 4.5 hours of sleep on call, compared with 2.4 hours the previous year, p < .0001. Nearly all (89%) felt more rested post-call, and 71% thought they could take better care of patients during the day because of the NF system. According to 87% of WR, the workload was significantly better, and 63% had more time for educational activities. Approximately 90% agreed that NF had improved housestaff morale and the overall experience of the ward rotation. Regarding the NF admissions, most WR appeared satisfied with the quality of history and physicals (63%), choice of diagnostic testing (72%), and initial treatment (71%). However, many WR disagreed with the assessment and changed the treatment plan about 25% of the time. One-third expressed some concern that continuity may suffer when patients are handed-off in the morning, but only 2% felt that overall patient care suffered, and 94% were glad that the NF system was in place. Each NF team received a mean of 3.9 admissions per night (range 2-11) and a median of 4.8 crosscover calls, which required about 45 minutes to answer. Nearly 95% of NFS agreed that the overall workload and number of admissions were reasonable. However, only 66% were satisfied with the amount of learning/teaching on the NF rotation. Working nights for a month was difficult for 67% of NFS, and 80% felt it affected their social life. Daytime sleep was often unrestful, afternoon continuity clinics disrupted sleeping schedules, and NFS had limited time with friends and family. Nevertheless, 90% agreed that the month was a positive experience overall, and 96% were glad that the system was in place. CONCLUSION: Residents on the ward teams reported improved morale, increased sleep, more time to read and attend conferences, and better quality of care delivery. In spite of complaints about the nocturnal lifestyle and educational content of the rotation, housestaff on the NF teams favored the system as well. We implemented a clinic-based coaching model using rating forms and observed interviews. The purpose of this study is to describe the use and modification of the rating scales for coaching purposes, and to discuss the variations among specific communication skills by resident year and gender. METHODS: The Calgary-Cambridge Referenced Observation Guides were selected as a teaching tool because of their specificity and ease of use during observed interviews. To provide skill discrimination for each rating item, a 5 point Likert scale was created for each item ranging from poor to excellent performance. After obtaining patient consent, resident physicians were shadowed by the residency behavioral scientist (BRL) for one clinic afternoon or morning, and their skills were rated using the instrument. 25 resident physicians were observed for a total of 48 interviews. Likert scores were dichotomized into excellent (5) and less than excellent (1) (2) (3) (4) categories for each item; and mean scores generated for the following domains: Initiating the session (5 items); Exploration of problems (7 items); Understanding the patient's perspective (5 items); Structuring the consultation (4 items); Building the relationship (7 items); and Explaining and planning (5 items). General Linear Models procedures were used to compare mean differences in domain scores by gender and year of resident training (R1, R2, R3). RESULTS: No significant gender differences were found for any of the domains (p < .05).
Overall, the resident physicians demonstrated the least proficiency with the domaiǹ`U nderstanding the patient's perspective'' (p = 0.01, mean domain score (MDS) = 1.07). When scores were analyzed by resident year, significant maturation effects were observed for:`E xploration of problems'' (p = 0.01, MDS: R1 = 2.6, R2 = 4.2, R3 = 4.8);``Understanding the patient's perspective'' (p = 0.01, MDS: R1 = 0.28, R2 = 1.23, R3 = 1.94); and``Explaining and planning'' (p = 0.02), MDS: R1 = 1.5, R2 = 2.1, R3 = 3.4). Resident evaluations of the coaching were uniformly positive. CONCLUSION: The development of physician-patient communication skills is an essential part of resident education. The modified Calgary-Cambridge guides provide data that identifies areas of general strengths and weaknesses in communication skills. Using this instrument, we saw maturation effects in domains that require facilitation of emotional expression and shared decision-making. These results can be used to focus educational efforts, and may be useful for evaluating the impact of interventions to improve physician-patient communication. METHODS: Using a 58-item clinical teaching evaluation instrument previously employed to determine the construct validity of the framework for inpatient clinical teachers, a split sample of 4702 evaluations completed by 550 students rating 450 teachers was evaluated using factor analytic and standard item-reduction techniques. Students completed evaluations for any university-or community-based outpatient teacher with whom a minimum of three half-day teaching encounters had occurred during a one-month internal medicine ambulatory block rotation. Results were replicated using the second half of the data. Generalizability analyses were used to quantitatively determine the number of evaluations needed to obtain a stable estimate of a teacher's performance using the reduced instrument. RESULTS: As in the construct validity testing with inpatient clinical teachers, thè`k nowledge'' items statistically combined with``self-directed learning'' items and``learning climate'' items held up as a discrete construct; however, items from``control of session'',`c ommunication of goals'',``understanding and retention'',``evaluation'', and``feedback'' clustered into one remaining``global'' teaching construct. These three factors explained 67 % of the variance. Following item reduction, the three constructs were compressed to contain three items each. The items remaining in the``global'' construct were all``communication of goals'' items. Overall internal consistency of the reduced instrument was .90 with internal consistency of constructs ranging from .90-.97. The overall inter-item correlation of the reduced instrument was .55 for sample one and .54 for sample two (optimal inter-item correlations are approx =.6). A minimum of 5 evaluations are needed per teacher to obtain a generalizability coefficient > .90 using conservative estimates from both samples. CONCLUSION: For students having limited contact with outpatient teachers, setting the climate, communicating goals, and stimulating students' future learning emerge as focal areas. Further study is needed to see if this three-category framework persists as viewed by trainees with more extensive contact with outpatient preceptors. PURPOSE: Professional isolation, poor financial remuneration, and insufficient training to meet patient needs decreases career satisfaction, making rural and inner city areas less desirable for primary care practice. Changes to Internal Medicine (IM) and Family Practice (FP) residency curriculum have attempted to address health care disparities by better preparing physicians for generalist careers. The effect of these curricular changes on career satisfaction has not been examined. METHODS: We surveyed all IM (n=128) and FP (n=74) physicians 1 to 10 years after graduating from a large Mid-west community teaching hospital. The survey assessed physician and practice demographics (i.e., practice type and location, community size, case mix), career satisfaction, resource availability, and adequacy of curricular content for practice. RESULTS: Survey response was 84% (105 IM, 65 FP, p=ns). Respondents were primarily male (66%), Caucasian (88%), and in solo (13%), group (42%), or multi-specialty group practice (20%). Thirty-one percent practiced in a community of < 50,000 residents (9% < 10,000) and 28% noted some or all subspecialties were unavailable in their community. Most respondents (88.5%) were somewhat to highly satisfied with their present career, and 81% would choose medicine again as a career (78% IM, 85% FP, p=ns); those practicing in emergency room settings were least satisfied with their present career (p = 0.04). Greater career satisfaction was related to meeting expected financial remuneration (p = 0.046), satisfaction with training in palliative care (p < 0.01), faculty role modeling of compassion (p < 0.05), and support/assistance provided by residency faculty (p = 0.001). Career satisfaction was not related to community size (p = 0.6), subspecialty availability (p = 0.7), computer/internet searching skills (p = 0.11), or time spent in ambulatory training (p = 0.3). Training better focused to practice demands in differential diagnosis, neurology, orthopedics, and hematology were associated with greater career satisfaction (all p < 0.05). Using logistic regression, controlling for physician demographics, greater career satisfaction was associated with meeting financial remuneration expectations (OR=3.9, p=0.01), and better training in differential diagnosis (OR=5.8, p=0.03), and literature appraisal (OR=3.0, p=0.04). CONCLUSION: Results suggest that at this institution, nationally mandated curricular reform in several areas is associated with greater career satisfaction. Satisfaction was also related to residency faculty role modeling and support. Whether additional curricular reform further improves career satisfaction warrants investigation. The AAMC has mandated the inclusion of end-of-life (EOL) and palliative care (PC) in undergraduate medical curricula. A significant number of medical schools teach about death and dying, yet focus predominantly on ethical aspects rather than the development of clinical skills in EOL and PC. We report here on the design and implementation of an EOL curriculum directed towards specific communication skills. This curriculum was developed and initiated by students at Stanford Medical School. METHODS: A needs assessment of advanced clinical students demonstrated little formal training in EOL communication skills, while a literature review surveyed current EOL curricula on communication. These efforts were used to design a case-based curriculum that underwent faculty review. Next, faculty with national or local experience with EOL and palliative care training contributed to case development. Twelve of the faculty attended an orientation/training session on teaching the curriculum and then taught it in pairs to six groups of 10-14 medical students per group. RESULTS: The four-hour curriculum described here was taught to students at the start of their clinical training. Topics entailed breaking bad news and discussing treatment options in the setting of life-limiting illness. In two successive sections, students (1) discussed and critiqued a physician modeling these areas [Weissman DE], (2) role-played a prepared case in dyads, (3) evaluated successful elements of communication skills, and (4) assessed their performance in the role-playing dyads. CONCLUSION: Medical students require skill-based EOL and PC curricula that emphasize clinical skills specific to their level of training. This module taught early clinical students communication skills for the setting of PC and EOL care. Further interventions will be necessary to advance these students' communication skills as they progress in their clinical training. We propose next to evaluate the effectiveness of this curriculum by comparing students' EOL communication skills with standardized patients before and after this curriculum. The longitudinal clinic experience at Creighton University consists of participating in the same clinic one-half day every other week of their 2nd year of medical school in a primary care setting (general internal medicine, family practice, or pediatrics). The 4th year medical students were surveyed regarding their 2nd year longitudinal clinic experience and their 1st choice for their future career. Nonparametric correlations (phi and point-biserial coefficients) were used to assess the associations between the student's actual choice of specialty to their self-reported effect of the longitudinal clinic on their specialty choice and the specific specialty (internal medicine, family practice, or pediatrics) of the longitudinal clinic. Hometown and MCAT scores were used as control variables. RESULTS: Of the 106 students participating in the longitudinal clinic experience, 42% reported it had a positive effect on the career choice in primary care (e.g. internal medicine, family practice, or pediatrics), while 53% reported it had no effect, and the remaining 5% stated it had a negative effect. There was no association between the student's self-reported effect of the clinical experience on their actual choice of specialty (phi=.248, p=.05). There was also no association between a student's choice of specialty and whether they were involved in an internal medicine, family practice, or pediatric longitudinal clinic (p > .05 for all three comparisons). CONCLUSION: The clinic setting had no effect on the students' career choice. Other factors (e.g. market factors such as surplus/shortage of primary care physicians or clinical experiences during their 3rd year) may also be influential in such decisions. To determine if self study with a CD-ROM auscultation tool would increase internal medicine residents' knowledge and skills in cardiac auscultation. METHODS: The interactive computer-based auscultation program was developed by two of the authors. It includes 20 multi-media cases that center on common cardiovascular conditions, including valvular abnormalities, congenital heart disease, coronary artery disease and congestive heart failure. At 2 institutions, a total of 127 residents participating in their 1-month primary care rotation were enrolled in this fixed-group controlled trial from April 1999 through June 2000. Residents were assigned to the intervention or control arm based on their clinic site. Those allocated to the intervention group (n=56) were provided two hours per week during their month to use the program. Control residents (n=71) were exposed only to the content of their rotation.
Auscultation knowledge and skills were assessed by a computerized validated 40-item test administered before and after the rotation. Ten of these items tested the recognition and interpretation of heart sounds (skills component), and 30 tested facts about auscultation (knowledge component). Baseline and post-intervention scores were compared within groups using paired t-tests, and between groups with independent t-tests. When possible, residents who dropped out (n=10) were analyzed with their last outcome carried forward. RESULTS: Pretest and posttest scores are shown in Table 1 . At baseline, the two groups did not differ in demographic characteristics, pretest knowledge or total scores. The intervention arm had a lower mean pretest skills score than the control group. While both groups had improved scores after the rotation, residents in the intervention group had significantly higher improvements in skills, knowledge, and total scores than those not exposed to the CD-ROM based tool (* all p < 0.001). CONCLUSION: Protected time for internal medicine residents to pursue self-directed study with a multimedia auscultation tool during a primary care rotation enhanced both cardiac auscultation knowledge and skills. (1) self-reported use of information resources, (2) self-reported comfort with EBM skills, and (3) PURPOSE: Although thyroid examination is the least invasive and inexpensive method to detect thyroid pathology, most physicians lack the ability to detect thyroid abnormalities by examination. We developed a single and brief multimodality session to improve the thyroid examination skills of physicians-in-training and evaluated its efficacy in a controlled trial. METHODS: Thirty-nine first-year internal medicine residents were randomly allocated to participate in a multimodality learning session on thyroid physical examination or to receive no intervention (control group). The 50-minute learning session consisted of a slide presentation, a computer graphics animation, videotaped and hands-on demonstrations of the appropriate technique, participant examination of a patient with thyroid abnormalities and a normal volunteer under preceptor observation, and an evidence-based handout. All the participants were assessed in two stations of an objective structured clinical examination (OSCE) for appropriate thyroid examination technique (through a 2-way mirror) and identification of thyroid abnormalities (using a scored participant report of findings). The OSCE preceptors and results analysts were blinded to participant allocation. RESULTS: Of the 19 residents in the intervention group and 20 residents in the control group: 6 and 3 observed the neck for thyroid abnormalities (32% vs 15%; p = 0.3); 17 and 16 residents used proper hand position (90% vs 80%; p=0.7); 13 and 15 residents had the patient swallow while palpating the neck (68% vs 75%, p=0.7), respectively. There was a significant difference between the mean scores on thyroid physical findings between the intervention and control groups (100 vs 52.5, p=0.047) CONCLUSION: A single and brief multimodality learning session did not statistically improve the thyroid examination skills of first year Internal Medicine residents, but did positively affect their ability to detect abnormalities. Future research (with larger sample sizes) should test the effectiveness of iterative sessions or of increased emphasis on hands-on thyroid abnormality detection rather than appropriate technique. 
Questions ( Table I ). CONCLUSION: Conclusions : Clinical data and physical exams are essential for ID. However complementary tests are important to determine FD. Among the tests, it's important to highlight the importance of CT scan , although we must re-evaluate others like Chest X-ray and Electrocardiograms which were taken to all patients but they were of poor value in patients without specific symptoms. We need to rationalize others like biochemistry tests and consults to others specialist. The biopsies and therapy tests were very useful. These results reflect what has happened with Medicine in the last two decades, in which the technological progress allowed a better diagnosis, although it was initially established by physicians at the patient's bedside. (sixth) year medical students while they were rotating through general medical outpatient clinic for 2 weeks. Faculty internists evaluated student's clinical competence observing student's ability to assemble clinical information gathered through medical history taking and physical examination and to formulate working diagnosis. Students were rated as follows; A, able to integrate pertinent information for initial assessment, B, able to gather pertinent information, C, information gathering is passive and superficial, and D, detrimental to patient. A, B and C were further divided into three subgroups according to individual performances to develop a 10 point Competence Score. Patient satisfaction ratings for students were also obtained at the same time by using a modified Japanese version of Patient Satisfaction Questionnaire developed by the American Board of Internal Medicine (m-PSQ). Multiple regression analysis was used for statistical comparison. RESULTS: A total of 77 medical students were evaluated by 9 staff physicians and by 229 patients. The m-PSQ consisted of single factor with Cronbach's a coefficient of 0.90. Multiple regression test revealed that competence scores of students were strongly associated with the m-PSQ ratings (p < 0.001), but not with student's age and gender, Objective Structured Clinical Examination (OSCE) scores, patients' demographic characteristics such as age, sex, occupation status. CONCLUSION: Patient satisfaction ratings of medical students in outpatient clinics were strongly associated with student's ability to gather and integrate clinical information obtained through history taking and physical examination in a university hospital outpatient clinic. The use of Palm Pilots is rapidly expanding among residents and some training programs are even providing them for their housestaff. However, little is known about how residents view their utility. The purpose of this study is to determine 1) residents' perception of the utility of Palm, 2) potential obstacles toward their use, and 3) the influence of providing Palms to medical residents. METHODS: We conducted a survey of all medicine residents at two hospitals. One of the hospitals heavily subsidized and encouraged residents' purchase of Palms. The survey included questions regarding perceptions toward the benefits of using Palms to help them record patient data (record) and improve the quality of their signouts (signout). We also surveyed for potential obstacles to their use: cost, difficulty in learning how to use (learning), fragility and effort required to enter data (enter). A Likert scale (1=strongly disagree to 5=strongly agree) was used. The survey was piloted for reliability at a third hospital. RESULTS: 65 of 81 residents (80 %) in the hospital that did not subsidize the purchase of Palms (unsubsidized group) and 25 of 29 (86%) in the hospital that subsidized their purchase (subsidized group) returned the survey. 21 residents in the unsubsidized group (32%) and 24 residents in the subsidized group (96%) owned a Palm. The unsubsidized group rated questions on the utility to record patient data and signout higher than the subsidized group (table 1) . Regarding potential obstacles the unsubsidized group felt that cost was a greater obstacle while the subsidized group felt that the fragility of the device and the effort to enter data was a greater obstacle than the control group. Analysis by ownership of a Palm independent of the site also failed to reveal any significant results with exception to cost and learning (less of an obstacle to Palm users). CONCLUSION: Our study demonstrates that in a training program that subsidized the purchase of Palms, residents perceived them to be less useful and felt that the fragility of the device and the effort to enter data were greater obstacles to their use. These results imply that simply providing housestaff with Palms may not be beneficial. Studies are needed to see if providing medical software and additional training will increase their utility and decrease perceived obstacles. inquired about the most common uses in patient care as well as the use of any medical programs. Information on training level and whether they owned a Palm Pilot was also obtained. The survey was piloted for test ± retest reliability at a third hospital. Mann ± Whitney U tests were performed to compare groups and logistic regression was performed to adjust for resident genre, level of training, and program site. RESULTS: The survey was sent to 81 residents in the hospital that did not subsidize the purchase of Palm (unsubsidized group) and 29 residents in the hospital that subsidized their purchase (subsidized group). 65 residents (80 %) in the unsubsidized group and 25 (86%) in the subsidized group responded to the survey. 21 residents in the unsubsidized group (32%) and 24 residents in the subsidized group (96%) owned a Palm device. Palm owners in both groups responded that they used these devices to organize patient records (23%) and phone numbers used in patient care (82%), keep patient``to do'' lists (39%), schedule events and appointments (75%), and as an aid for clinical calculations (86%). The most frequently used programs were pharmacopoeias (93%), medical reference (44%), and clinical calculators (18%). Ownership of Palm and training sites were not independent predictors of using clinical prediction rules more frequently on logistic regression. CONCLUSION: Our study demonstrates that medical residents use Palms for a variety of purposes relating to patient care. However, owning Palm Pilots did not increase the likelihood of using clinical prediction rules and very few residents used them to organize patient records.
From an educational perspective, at this point the available evidence does not support providing Palms to medical residents. Further research is still needed to determine if the use of Palm Pilots will result in better patient outcomes or resident efficiency. , 4) Menopause, Osteoporosis and Hormone Replacement, 5) Psychosocial Issues, 6) Resident Presentations and Posttest. Also included in the curriculum is a three-hour visit to the local domestic violence prevention center. All necessary reading materials are handed out during the first session after the pretest. The didactic sessions start with a brief case presentation by the participating resident(s); this is followed by an in-depth discussion of the topics of the day with expected resident participation. Each session except the introductory and resident presentation session ends with role-plays where the instructor plays the role of the patient and the resident the doctor. This enables the resident(s) to put into immediate practice what they have just learned. Clinical practice of women's health is expected to occur when residents see female patients in their primary care practices. To expand the scope of this curriculum beyond these limited topics both residents and faculty have been encouraged to incorporate into everyday teaching and learning how various conditions may differ or are unique in women. Participating residents are expected to make a formal thirty to forty-five minute presentation on a topic in women's health not covered in this curriculum. Learners are evaluated by the completion of a standard evaluation form used in the residency program. Evaluations are based on the residents' performance on the posttest, participation during the didactic sessions and their presentations. Learners evaluate the curriculum by completing a survey. RESULTS: The curriculum was well received by all residents. All residents surveyed either agreed or strongly agreed that they would now be able to evaluate, manage or appropriately refer female patients as a result of knowledge gained during the month. The mean posttest score was 81%, an improvement over the mean pretest score of 66%. Residents stated particularly that role-plays helped improve their understanding of the topics. A survey of these residents in their third year regarding their perceptions on the women's health education they received in this program and how well they think it has prepared them for their future practices year is planned. CONCLUSION: The curriculum improved the knowledge of residents in selected women's health topics. Using the same curricular model, topics can be adapted to meet the learning needs of residents in other residency programs. (COPD)). Each physician subject saw a simple and a complex case for each disease. Fifteen physicians were randomly selected at each site, yielding a total of 120 visits. From these, we randomly selected 10 percent of these visits to audio record the SP physician interaction yielding 1 recording for each of the 12 SPs. To make the visits as realistic as possible, physicians consented not to be informed if a patient was an SP or if the visit was being recorded. SPs rated clinical care provided by physicians using closed-ended checklists that contained explicit quality criteria developed from national guidelines. Transcribed recordings were scored identically using the same checklists to determine if SP reports were valid. We calculated the percentage of criteria where SP checklists and recordings were in agreement and disagreement. McNemar's chi-square test for paired proportions was used to determine the statistical significance of differences between the two methods. RESULTS: The overall rate of agreement between SPs and recordings was 90.3 percent (p = 0.00). The rate of agreement ranged from 86.9 percent (p < 0.01) for the complex depression case to 95.3 percent (p = 0.375) for the simple COPD case. Eighty-five percent of the disagreements between SPs and recordings occurred when SPs recorded that physicians performed actions that were not verified by the recording. CONCLUSION: The high rate of agreement between SPs and recordings indicates that SPs are valid measures of the quality of outpatient care. The higher rate of agreement in the COPD cases as compared to depression may be related to two factors: 1) the larger number of items in the depression checklists, and 2) the subtlety of checklist items in the history section of these cases. The preponderance of overrecording vs. underrecording suggests that SPs may tend to give physicians the benefit of the doubt in the presence of ambiguity. Unannounced SPs are attractive quality measurement tools in the outpatient setting because they are inherently adjusted for case mix. These prospectively obtained results indicate that SP reports are valid measures of the quality of care, and may be useful for comparative evaluation of physician training across health care systems. Little is known about changes in end-of-life care attitudes among medical and pharmacy students following experiences working with patients at the end of life. METHODS: 8 medical or pre-medical students and 19 pharmacy students served as volunteer "patient advocates" (n = 27), providing social support in the form of weekly phone calls and monthly visits to patients with end-stage congestive heart failure, chronic obstructive pulmonary disease, and cancer. Students worked in pairs with an individual patient for a 4-month period. In addition, students participated in a 10-hour series of physician-led discussions about end-stage illness, the patient and family experiences of illness, and the emotional and spiritual dimensions of end-of-life care. Using a confidential, self-administered questionnaire with 10-point Likert responses (1 =very negative; 10 = very positive), student attitudes about the end of life were assessed before and following their work as advocates and participation in the class. The mean values among all respondents pre-and post-training were compared and analyzed using the two-tailed t-test. RESULTS: The response rate was 70% (n = 19). Students felt that the quality of their experiences with patients at the end of life was more positive after the course than before (mean = 7.0 post vs. 5.7 pre; p = 0.013). Students also felt more comfortable working with patients at the end of life (mean = 7.3 post vs. 6.4 pre; p = 0.045). Additionally, following their experiences, students estimated the average quality of life for patients at the end of life as higher (mean = 6.3 post vs. 4.7 pre; p = 0.028). Following their experiences, student belief in the possibility for growth at the end of life did not change significantly (mean = 8.7 post vs 8.9 pre; p = 0.48). Following their experience, students' rank of the professional importance of working with patients at the end-of-life remained high (mean = 9.0 post vs 9.6 pre; p = 0.077). CONCLUSION: Direct contact with patients at the end of life and formal physician-led discussions about end-of-life care led to improved quality in students' experiences with people at the end of life, as well as increased comfort working with patients at the end of life. While endof-life care experiences led students to estimate that patients' quality of life was higher than initially expected, following their work, students were no more likely to believe that growth at the end of life was possible. The influence of early end-of-life care experiences on preprofessional students is generally positive, but should be explored further as students receive increased training in end-of-life care. PURPOSE: Mentorship has been associated with greater career satisfaction among academic physicians, but has not been studied during residency training. While female physicians may benefit more from mentorship, little is known about mentorship among female residents. We compared the prevalence and characteristics of mentorship between male and female residents in medicine. METHODS: We developed a questionnaire to collect data on attitudes regarding mentorship, satisfaction with mentorship, and satisfaction with residency education. After pilot testing the questionnaire, we mailed it to residents in the five internal medicine residency programs affiliated with Harvard Medical School. We sent two additional mailings to nonrespondents. We compared responses between men and women, and then used logistic regression to identify factors significantly associated with having a mentor and being satisfied with mentorship or career guidance. RESULTS: Among 329 respondents (response rate 65%), 47% were women. Of the women, 57% were white, 29% Asian and 9% underrepresented minorities. Although 50% of both men and women had a current mentor, and both men and women were equally likely to report satisfaction with mentorship (46% vs 38%, p=0.16), women were more likely to report that having a mentor was important during residency training (96% vs 90%, p=0.04). While women were more likely than men to want a mentor of the same gender (35% vs 8%, p < 0.01) and were more likely to have a female mentor (52% vs 17%, p < 0.01), having a female mentor was not associated with satisfaction with mentorship. In addition, regardless of whether they had a preference for a female mentor, women were equally likely to have a mentor and to be satisfied with mentorship. In multivariable models adjusting for ethnicity, year of training, being assigned a mentor and history of mentorship, women and men were equally likely to have a mentor and to be satisfied with mentorship. However, after adjusting for ethnicity, year of training and having a mentor, women were more likely than men to report receiving inadequate guidance with career decisions [adjusted odds ratio 1.8 (95% confidence interval 1.1, 2.8)] and were less likely than men to think that their residency was doing the best possible job preparing them for a future career [0.6 (0.4, 0.9) ]. CONCLUSION: Although women in residency training developed mentoring relationships as often as men and were equally satisfied with mentorship, they were significantly more likely to judge guidance with career decisions as inadequate, and were less likely than men to believe that their residency was doing the best possible job preparing them for a future career. PURPOSE: Since mentorship has been associated with greater career satisfaction among academic physicians, some residency training programs assign mentors to all house officers. We compared characteristics of mentorship between residents who were assigned a mentor by their training program, and those who were not assigned a mentor. METHODS: We developed a questionnaire to collect data on attitudes regarding mentorship, satisfaction with mentorship, and satisfaction with residency education. After pilot testing the questionnaire, we mailed it to residents in the five internal medicine residency programs affiliated with Harvard Medical School. We sent two additional mailings to nonrespondents. Using logistic regression, we identified factors associated with three different outcomes: having a mentor, being satisfied with mentorship, and being satisfied with career preparation. RESULTS: Among the 329 respondents (response rate 65%), 34% were in residency programs that assigned mentors (assigned), and 66% were in programs that did not assign mentors (nonassigned). Most (87%) residents in programs that assigned mentors knew that they were assigned a mentor. Residents in assigned programs were significantly more likely to identify a mentor than those in nonassigned programs (82% vs 43%, p < 0.01). Residents with mentors in assigned programs were less likely than residents with mentors in nonassigned programs to report their mentor was helpful with professional development (64% vs 77%, p=0.05) or personal development (45% vs 69%, p < 0.01). However, residents with mentors in assigned programs were more likely to be satisfied with mentorship than residents with mentors in nonassigned programs (53% vs 36%, p < 0.01). In multivariable models adjusting for sex, ethnicity, year of training and history of mentorship, residents in assigned programs were more likely to identify a mentor [adjusted odds ratio 8.1 (95% confidence interval 4.3, 15.
3)] and to be satisfied with mentorship [2.2 (1.4, 3. 6)] than residents in nonassigned programs. Regardless of whether residents were in a program that assigned mentors, multivariable analyses showed that residents who had a mentor during residency were more likely to think that their residency was doing the best job preparing them for their future career [2.0 (1.2, 3. 2)] compared with residents who did not have a mentor. CONCLUSION: Internal medicine residents who were mentored were significantly more likely to report excellent preparation for future careers. Residents who were assigned a mentor were more likely to identify a mentor and to be satisfied with the mentorship they received. These data suggest that residency training programs should assign mentors to house officers. PURPOSE: Identifying potential mentors is an important part of residency training. Morning report (MR) provides exposure to a variety of attending physicians. We studied resident and program characteristics that were associated with the ability to identify a potential mentor at MR. METHODS: We performed a cross-sectional survey of 356 internal medicine residents from a convenience sample of thirteen residency programs. The instrument included questions about demographic characteristics, subspecialty fellowship plans and ability to identify a potential mentor in MR during the previous six months. RESULTS: The response rate was 63% (38% female). Women were significantly more likely to have plans to go into general internal medicine (GIM) (43% vs.58%, p < 0.001). Overall, 73% of residents were able to identify a potential mentor at morning report within the previous six months. Women were significantly less likely than men to be able to identify a mentor (63% vs. 79%, p < 0.001). Stratification by fellowship plans revealed that female residents not planning on subspecialty training were significantly less likely than male to be able to identify a potential mentor ( Figure) . Residents from programs with > 50% generalists at MR were less likely than those from programs with more specialists at MR to identify a potential mentor (57% vs 81%, p < 0.001).
CONCLUSION: Future generalists may face barriers to appropriate mentorship. Even though female residents were more likely to go into GIM, those who chose to do so were less likely to find a potential mentor at MR. Residents from programs with a majority of specialist attendings at MR were more likely to be able to identify a potential mentor, regardless of their career plans. completed a questionnaire at the end of each continuity clinic session which asked them to assess the number of patients they saw during that day's clinic using a 5 point Likert-type scale (1=too few, 3=just right, 5=too many). Chi-square analyses, Student's t-tests and linear regression modeling was used to evaluate the variables that contributed to optimal scheduling. RESULTS: Residents completed questionnaires for 73% of their clinic sessions (N=105). Overall, they saw an average of 4.5 (SD=1.6) patients per session. 75% of the time, patient volumes below 3 patients were considered too low and volumes above 6 patients too high. During sessions where they rated patient volume as "just right" interns saw an average of 3.7 (SD=1.2) patients, second residents saw 4.6 (SD=0.7) patients and third year residents saw 5.3(SD=1.2). Linear regression modeling adjusted for resident track, gender and study period, confirmed that second and third year residents saw more patients than interns, 0.7* and 1.5* patients, respectively; that contact with less than 1.2* patients per session below average constituted``too few'' patients; and that contact with more than 1.8* patients above average constituted``too many''. (*p-values < 0.0001) CONCLUSION: Residents at all levels of training were comfortable seeing the minimum volume of patients recommended by the Internal Medicine Residency Review Committee (1 new and 3 follow-up patients). Patient volumes below 3 were too low and volumes above 6 were too high. In between these apparent limits, ideal volume depended on year of training, increasing by about 1 patient per session per year. While information from the medical education literature suggests that some aspects of the clinic educational program may suffer when patient volumes exceed 4 per session, we found that residents at more advanced levels of training preferred patient volumes higher than this. While one explanation for this observation is the higher skill level of the third year residents, it may also be that more advanced trainees derive greater educational benefit from direct patient care than from supplemental educational activities such as reviewing patient care with attendings. Important next steps will be to improve our understanding of the relationships between volume, type and complexity of patient visits, and the acquisition of expertise in specified aspects of ambulatory care, as well as to further delineate the relative educational value of the different clinic activities residents engage in. year. The aims of our study are to evaluate whether this clinical experience: 1) serves as a bridge between the basic science years and the clinical years, maintaining clinical skills and confidence, and 2) improves students' knowledge of Ambulatory Medicine. METHODS: At the end of the clinic year, the students took a standardized knowledge test and completed a validated questionnaire assessing their skills. MD/Ph.D. students who chose not to attend clinic and MD non-Ph.D. students also took the test and answered the questionnaire, serving as reference groups. To compare the groups, ANOVA was performed, followed by Tukey testing.
RESULTS: The MD group had proportionally more women and started medical school 2 years later than either MD/Ph.D. group(p < .05). The MD/Ph.D. clinic students (n=20) were significantly more confident than the MD students (n=49) about their skills in every category of the questionnaire (history and general competencies, readiness to function as a doctor, physical exam, diagnosis and treatment) (p < . 05). They were more confident than the MD/Ph.D. nonclinic students (n=13) First-year students' perceptions of necessary professional skills focused upon behaviors relative to their current roles in their educational experience. The patient care related issues identified by students such as patient confidentiality and knowledge of limitations appeared to be appropriate for their level of training. Applying the ABIM format to the survey results shows that the majority of responses fall into the categories of excellence (dedication to learning), honor and integrity (honesty), and respect for others (respect). Presenting first-year students with a professionalism curriculum that is primarily focused upon patient-care related issues may not be as relevant as professionalism issues more closely related to their current level of training. Addressing issues such as dedication to learning, honesty, and respect may increase the relevance of professionalism to pre-clinical students. We are using this work to develop identify students' perceptions of stage-appropriate professional skills and behaviors to devise stage-appropriate educational interventions to teach professionalism. METHODS: Faculty members participated in a pre ± post study of a faculty development program consisting of three 90-minute interactive seminars teaching evaluation, feedback, and One-Minute Preceptor microskills. Audiotapes were collected of ambulatory teaching encounters with 3rd year medical students before and after the intervention. The audiotapes were transcribed and coded by individuals blind to the identity of the teachers and learners. Transcripts were coded using the Teacher Learner Interactive Assessment System, a qualitative tool designed to comprehensively code all utterances into mutually exclusive categories. Ten percent of audiotapes were double coded to assess inter-rater agreement. Written surveys after each encounter assessed learner, teacher, and patient satisfaction. Learner and teacher perceptions of the amount and quality of several aspects of the encounter, including feedback, were also evaluated. RESULTS: Nine teachers and 64 third year medical students participated, providing 45 encounters before and 48 encounters after the seminars. 17,859 utterances were coded. Coders achieved a high degree of agreement (Spearman's rho > 0.8). In the baseline encounters, 17% of teacher utterances were some form of feedback, predominantly (92%) minimal feedback statements such as "right" or "I agree". Only 8% of feedback utterances were specific and none were interactive. Most (91%) of the feedback was positive. After the faculty development workshops, the amount and quality of feedback increased; teachers were more likely to provide feedback (OR 1.21; 95% CI 1.07 ± 1.36) and that feedback was nearly twice as likely (OR 2.08; 95% CI 1.45 ± 2.99) to be specific. Subjectively, teachers reported a higher likelihood of allowing students to present their own plans (p = 0.004) and of providing specific feedback to students (p = 0.004) after the intervention. Students also reported greater ability to present their own plans (p = 0.02) and rated the encounters as more successful (p = 0.003 Results are presented as univariate descriptive statistics and using Pearsonchi ± square tests to assess the statistical significance of differences in proportions between groups on categorical variables. The level of significance for all tests was 0.05. All analyses were performed using SAS statistical software, version 6.11 (SAS Institute, Cary, NC, 1995) . RESULTS: A total of 55 of the 61 graduating third year residents were surveyed. Over their three years of training, residents reported caring for a median of two patients who died in outpatient primary care, and 10 patients who died during inpatient rotations. In addition, they reported caring for a median of only 3 patients who they felt might have had a life expectancy of 6 ± 12 months. They reported median``little/poor'' EOL teaching from faculty, and``little/ poor'' support from resident colleagues in their EOL clinical care. Only 6% rated oncology rotations as``relevant or helpful in learning about EOL care;'' only 10% rated intensive care rotations as helpful; and only 27% rated geriatrics rotations as educationally beneficial. CONCLUSION: These results suggest that residents are meaningfully and intensively involved in the care of remarkably few terminally ill patients, and when they are, these teaching opportunities are too often being squandered. They suggest that we must improve EOL education in acute inpatient settings, capitilizing on the opportunities to teach residents around the dying patients that they currently care for. They further suggest that we must increase residents' experience in EOL patient care in community settings, targeting primary care clinics, home care, nursing home and hospice services. PURPOSE: Although the ability to work with and interpret numbers is vital to patient care, little is known about the numeracy skills of health care professionals. To facilitate mastery of numeracy in medicine, we sought to (1) assess the numeracy skills of first-year medical students, and (2) determine how different risk presentations affect student ability to compare and calculate treatment benefits. METHODS: We surveyed 62 first-year medical students who were attending a seminar on risk communication. Students were asked to (1) answer three questions assessing their ability to handle numbers in a non-health setting (non-health numeracy; i.e. Imagine that we flip a fair coin 1000 times-what is your best guess about how many times the coin would come up heads?), (2) state which of two drug treatments for a hypothetical disease Y provided more benefit, and (3) calculate the effect of drug treatment for a patient with a given baseline risk of disease. Risk information was presented to each student in one of four randomly allocated risk formats-relative risk reduction (RRR), absolute risk reduction (ARR), number needed to treat (NNT), or a combination of all three of these risk presentation formats (COMBO). Students' abilities to compare and calculate drug treatment benefits were stratified according the number of correct answers they gave to the non-health numeracy questions: 0 or 1 correct=minimal numeracy, 2 correct=moderate numeracy, 3 correct=advanced numeracy. RESULTS: Fourteen (23%) students incorrectly answered one or more non-health numeracy questions. Lower non-health numeracy skills correlated with lower health numeracy skills. While more than 90% of students with advanced (n=48) or moderate (n=11) non-health numeracy skills correctly stated which of two treatments provided more benefit, only 33% of students with minimal (n=3) non-health numeracy skills correctly did so (p = 0.03). Similarly, 71% of students with advanced non-health numeracy skills, but only 36% with moderate and 0% with minimal non-health numeracy skills correctly calculated the effect of drug treatment on a given baseline risk of disease (p < 0.01). Regardless of the risk presentation format, students were able to correctly state which of two treatments provided more benefit (range 81 ± 93%). When asked to calculate the effect of drug treatment on a given baseline risk of disease, however, students had significantly more difficulty when risk was presented as NNT (25% answered correctly, compared to 75% for all other presentations; p=0.01). CONCLUSION: First-year medical students with lower non-health numeracy skills demonstrated less skill in estimating both relative and exact treatment benefits. For all students, information presented as NNT was interpreted less successfully than information presented in other risk formats. If these findings persist through training, educators may need to de-emphasize NNT relative to other formats that communicate the same information more successfully. Step 1 scores were available for 355 students (91%). Average total OSCE score was 59 6. Average Step 1 score was 234 17. Correlation coefficient for the total OSCE score with USMLE Step 1 score was 0.41 (p < 0.0001). In the linear regression model (R square = 0.52), 5 of the 16 station scores were significant (P < 0.05) predictors of USMLE Step 1 score, as were MCAT biological sciences and physical sciences sub-scores. The 5 predictive stations (alcoholism and abdominal exam, arthritis, calf pain, lung exam, and skin exam) were weighted toward identification of abnormalities (28% of all items on the 5 predictive stations, as compared with 17% of all 16 OSCE stations) and differential diagnosis (32% versus 19%). CONCLUSION: An OSCE in physical diagnosis taken by second-year medical students is correlated with scores on the USMLE Step 1 exam, with OSCE skills that foreshadow the clinical clerkships (identification of abnormality and development of differential diagnoses) most predictive of USMLE scores. This correlation suggests predictive validity of the OSCE and supports its use in identifying students who need remedial education. This OSCE provides an additional measure to predict students' success on subsequent national board exams. PURPOSE: Community-based experiences can help prepare medical students for addressing disparities in health care. Clerkship management and instruction can be difficult with multiple community-based teaching sites. To help address this challenge we have been developing Webbased components for our internal medicine clerkship. To date these components include forms evaluating the clerkship and individual preceptors, an interactive patient logbook with real-time faculty access to completed logs, clerkship calendar and, on a pilot basis, the ACP/ASIM clinical problem solving cases. This study addressed 1) if students were able to conveniently access these components from all of our six community-based campuses, each with multiple hospitals and clinics, 2) students' acceptance of these web-based components, 3) the efficiency gains of Webbased data collection and clerkship management. METHODS: We appended questions concerning the ease of accessing the Web-based components and students' preference for this format on the clerkship feedback form. Students completing the ACP/ASIM cases filled out a Web-based survey. Efficiency gains were based on comparison with the former paper-based evaluation system. RESULTS: To date we received feedback from 87 students who completed the third year basic and fourth year advanced internal medicine clerkship. 91% of the students indicated they had convenient access to the web-based components. 77% of the students preferred Web-based to paper evaluation forms while 60% preferred Web-based to paper logbooks. All eight students completing the ACP/ASIM cases found them useful or extremely useful and only one encountered any technical difficulty. CONCLUSION: Our students appeared to have little difficulty accessing the web-based components of the clerkship even though they were in a variety of community-based settings with no special provisions for Internet access. Students clearly preferred the Web-based to paper evaluation forms. While on the whole they preferred Web-based logbooks, they were less enthusiastic. The concerns they expressed to a large extent reflected design problems with the new system. Many of these have been resolved. The Web-based feedback forms and logbook dramatically streamlined the process of collating the data and producing feedback reports. These now take less than an hour to prepare and avoid the time consuming and error prone task of transcribing data from paper forms. Pilot results evaluating the use of the ACP/ASIM cases in the clerkship curriculum are encouraging and suggest these cases could provide a valuable adjunct learning experience. For the next academic year we plan to develop two Web-based instructional modules and a more comprehensive clerkship management system that provides additional real-time feedback to students and preceptors.
COMPARING CARDIAC AUSCULTATORY SKILLS USING REAL PATIENTS AND CD-ROM HEART SOUNDS. J. Solomonides 1 , D.S. Hatem 1 , M. Belanger 2 ; 1 University of Massachusetts Memorial Medical Center, Worcester, MA; 2 University of Massachusetts Medical School PURPOSE: Recognition of cardiac auscultatory events has been shown to be deficient. Whether the assessment method influences documented skills acquisition has not been determined. Our purpose was to compare second year medical students' abilities to recognize cardiac physical examination findings in patients with known murmurs with recognition of abnormal heart sounds produced by a CD-ROM. METHODS: Ninety-one second year medical students were evaluated on cardiac auscultation skills at the end of a Physical Diagnosis course. Students examined 1 Real Patient (RP) with a known murmur and listened to a CD-ROM (CD), to 2 murmurs and 2 extra sounds, then recorded their findings. Findings assessed included Heart Sounds (HS;5 items for RP and CD)and Murmur (M;5 items for RP, 4 items for CD) scores. RP murmurs included a systolic ejection murmur (SEM; n=44 students) and aortic stenosis with aortic insufficiency (AS/AI; n=47). Gold standard for the RPs was a recent exam by their cardiologist. The Criley CD-ROM was used to assess HS with abnormalities including S3 and S4 sounds (5 items; n=91), and murmurs with abnormalities including mitral regurgitation (4items; n=91) and aortic stenosis (4 items; n=91). Mean scores were compared using paired sample T-tests. RESULTS: Students' recognition of cardiac findings differed significantly with the two methods of evaluation. Overall mean scores for the CD-ROM cardiac findings (HS and M scores)were 77.5 while those for the RPs were 59.3 (p < .001). Mean HS scores were similar between groups (CD 81.7,RP 78.7; p=0.1) while M scores accounted for the differences in student performance for the two evaluation methods (CD 72.0, RP 37.7; p < .001). These mean score differences were similar when comparing single murmurs on CD-ROM to the RP with a single murmur (CD 68.5, RP 37.7; p < .001)and to the RP with two murmurs (CD 68.5, RP 38.1; p < .001). CONCLUSION: Medical students scored significantly higher when cardiac auscultation skills were assessed using a commercially produced CD-ROM compared to their skills in examining real patients. Differences may be due to examination technique, patient factors (body habitus), competing sounds (breath sounds), or patient complexity (2 murmurs v 1 murmur). The lack of difference in HS scores may be due to the small numbers of abnormal findings resulting in lack of discriminatory capacity as opposed to a true skills difference. The use of a CD-ROM alone to assess cardiac findings may overestimate student skills. This reinforces the need to focus our cardiac auscultatory skills teaching to enhance student examination skills acquisition. (3) increase the number of independent patient contacts by students in community teaching sites. Our current study looks at the impact of this mission based program. METHODS: We developed a survey evaluating six key components of an Active Learning Environment (ALE) (table 1) .Third year students rated their preceptors on each ALE component at the end of the 4-week internal medicine ambulatory clerkship using Likert-type scales to generate an ALE score (min.=6, max.=30). Faculty development programs were designed to improve preceptors' ALE teaching skills. Follow-up letters were sent to each participant reviewing program highlights and providing them with the ALE ratings they received from students for 6 months prior to their workshop. These were compared to preceptors' ratings received for 6 months after their faculty development participation. Student's t-test was used to compare differences in pre-and post-workshop mean survey results. RESULTS: To date 14 community preceptors have participated in three faculty development programs. Table 2 results show a trend towards higher post-workshop ALE scores. Their was a significant increase in the post-workshop independent patient contacts reported by students. CONCLUSION: A mission-based faculty development program can have a significant impact on the learning environment of community teaching sites in an ambulatory clerkship.
Components of an Active Learning Environment ( We hypothesized that residents could develop and implement a successful practice guideline as a way of gaining quality improvement experience. We studied their guideline development process and evaluated the effect of their guideline on patient care. METHODS: We asked a group of nine resident volunteers to develop a clinical guideline on a topic of their choice. The hospital funded the effort, extended after-hours availability to guideline-related diagnostic services and provided a nurse coordinator. We observed the process of guideline development, tracked guideline utilization, and measured physician satisfaction with it after six months of use. We also compared admission rate, length of stay and resource costs for a cohort of patients before and after guideline implementation. RESULTS: The residents selected acute chest pain evaluation and developed an evidence-based guideline. They focused on risk stratification and rapid diagnostic testing. They streamlined documentation by developing evaluation templates that substituted for written notes and orders. When applied to 319 patients 30 and older presenting with acute chest pain, 49% were classified as``possibly cardiac'' of which 41% were considered``high suspicion'' and 59%``low suspicion.'' An acute cardiac diagnosis was made in 12% overall (24% of possibly cardiac, 46% of high suspicion and 9% of low suspicion). This cohort of 319 patients was compared to 150 chest pain patients evaluated before the guideline was implemented. Median length of stay and cost of care both decreased by 34%. Admission rate was unchanged. Physician satisfaction with the guideline was uniformly positive six months after its implementation. Physicians rated 24-hour availability of a nurse ± coordinator and after-hours functional testing particularly highly. Guideline utilization remains high more than two years after its implementation. CONCLUSION: Resident physicians were the key participants in developing and implementing a successful clinical guideline that enjoys ongoing use. By experiencing the success of their own efforts, residents learned important lessons about quality improvement. Such experiences are an important addition to resident education and to the quality of care at academic health care centers. We asked students if they owned a computer with Web access, how many of their clinical sites had Web access, their level of comfort finding and using Web medical sources before and after the clerkship and the overall value of the site to their learning. A forward stepwise linear regression model was built to predict comfort and satisfaction in the use of the Web site as a learning resource. RESULTS: We surveyed 147 students (98% response rate) of whom 64% owned their own computer. On a scale of I to 5 (5= extremely valuable), 88% of students rated the value of the Web site > 3. While 76% were extremely or somewhat comfortable finding and using Web resources before the clerkship, 90% (p < .0001) reported the same at the end. The site was accessed on a weekly or greater basis by 58% of the students. Although 13% of the students experienced no clinical sites with Web access,61% had 1 or 2 sites with access and 26% had 3 or 4 sites with Web access. In a stepwise linear regression model, comfort in using Web medical resources at the end of the rotation was best explained by comfort in using the Web before the rotation, owning a computer and the number of clinical sites that had Web access (R2 =.48).
Overall satisfaction with the Web site was explained by the time of year the clerkship was taken, owning a computer and the number of sites with Web access (R2 = .067). CONCLUSION: Students found value in an ambulatory clerkship Web site as an information resource. It could be made more useful to them by assuring better access to a computer at their clinical sites and providing a personal computer. While comfort in using a Web site as a resource improved by the end of the clerkship, this can been enhanced by training prior to the beginning of the clerkship and again assuring better computer access. Given the relatively low frequency of use however, it cannot yet suffice as the sole curricular resource. The curriculum consisted of 10 one-hour case-based seminars, including two devoted to pain management. We reviewed consecutive billing and pharmacy records of patients of medical residents (n=733) and a comparison group of patients of neurology and rehabilitative medicine residents (n=273) who received an opioid during two 8-month periods before (1/1/ 97 ± 4/30/97) and after (1/1/99 ± 4/30/99) the implementation of the curriculum. The data abstraction protocol was validated by standard chart review of a random subsample of 50 charts (concordance = 89.9%). Three outcomes were measured:1) % opioid orders for meperidine in non-gastrointestinal patients (excluded because of a longstanding belief that meperidine should be used in biliary disease);2) % opioid orders accompanied by a bowel regimen (to prevent constipation); and 3) % opioid orders accompanied by adjuvant nonsteroidal anti-inflammatory drugs (to achieve the``additive effect''). RESULTS: We had a power of 80% to detect a 10% difference at a= .05, two-tailed. Percent change before/ after for each outcome for each group was assessed by t-testing. Logistic regression models were developed for each of the outcome measures in order to control for independent variables that could be confounders and to assess the magnitude of effects. The models used controlled for age and race. Significance is noted by * if p < .05. CONCLUSION: These data suggest that a palliative care curriculum can improve the opioid prescribing practices of medical residents, over and above a secular trend among other house officers. Larger samples and further research are needed to understand these findings and how palliative care education can improve patient care. Delayed and incorrect diagnosis can lead to needless suffering, be potentially life threatening, and also be costly to society as physicians spend significant health care resources to work-up presenting symptoms. Because of increased public awareness of DV, and reports of underdetection by practicing physicians, many have sought to improve DV education at the medical student and residency levels. Have these educational interventions improved residents'
abilities? In the current study, we examined the abilities of internal medicine residents to identify and care for a victim of domestic abuse. METHODS: Seventy-one internal medicine residents in four programs participated in a tenstation standardized patient-based Clinical Skills Assessment. One standardized patient (SP) portrayed a woman, complaining of headaches, who was a victim of domestic abuse. The SP assessed residents' elicitation of information and counseling interventions on a fifteen-item checklist. After the encounter, residents documented the patient's problems, etiologies, work-up and treatment recommendations. RESULTS: Forty (56%) residents correctly diagnosed domestic violence and discussed the diagnosis with the patient. Eighteen residents (25%) asked about immediate safety concerns. Twenty-three (32%) asked about concomitant child abuse. Forty-one (58%) did not refer the patient for DV counseling. Forty-eight (68%) made one or more incorrect recommendations. Some of these recommendations could be dangerous: recommending marital therapy (n=19, 27%), prescribing potentially addictive medicines (n=9, 12%), or suggesting that the patient bring her abusive partner in to the doctor (n=23, 32%). To work-up the patient's headache, 36 (52%) ordered unnecessary tests which would have cost $32,500. CONCLUSION: In our sample there were many deficiencies in the residents' abilities to diagnose and manage DV. The majority of residents provided inappropriate care that in some cases would be dangerous. In addition, the residents' ordering of unnecessary and costly tests is a drain to already-limited health care resources. There appears to be a need for more intensive training in DV at the undergraduate and residency levels. METHODS: Groups of 4 to 5 residents participate in 10 to 12 weekly 1-hour sessions during the rotation. After 2 didactic sessions to review basic biostatistics, clinical epidemiology, and other core EBM topics, the sessions follow a journal club format. One resident searches for, selects, and distributes an article relevant to the clinical question chosen for that session. They present a summary and initial evaluation of the article, which is followed by participatory discussion facilitated by the instructor. The Users' Guides to the Medical Literature series from JAMA is used to guide topic selection and as supplementary reading. Group discussions focus on the Users' Guides core concepts and evaluative criteria. The residents also do a more intensive final presentation in a similar format. Changes in EBM knowledge and skills were evaluated with a 38-item test given before and after the course, with blinding of participants and the instructor to the results. Participants also self-evaluated changes in their knowledge, skills, and practice on an anonymous evaluation form. . Pairwise comparisons revealed that the three categories within each teaching setting were all significantly different from one another (p < .04). Regression analysis revealed that time spent teaching in the IP setting was a significant negative predictor (p < .001) in the LC and CG ratings for IP teaching, while total years of teaching experience was a significant positive predictor (p < .001) in the SDL ratings for IP teaching. For the OP teaching ratings, time spent teaching in the OP setting was a significant negative predictor (p < .002) for CG and SDL, while LC ratings were unaffected by any of these variables. CONCLUSION: Our results suggest the need for focused faculty development programs to enhance CG skills-particularly for teachers who spend more clinical time in either the IP or OP setting, SDL skills for less experienced IP teachers and teachers who spend more clinical time in the OP settings, and LC skills for faculty with more inpatient clinical time. Internal Medicine residents at one urban, multi-center residency program were asked to complete a 71-item questionnaire. The survey was self-administered at conferences, on wards, and by e-mail. It was piloted on faculty, fellows, and chief residents for clarity of the questions and content validity. We solicited residents' observations of the impact of crosscultural issues on clinical care. Attitudes and self-perceived knowledge and skills were assessed with 6-point Likert scales. RESULTS: 71 of 133 residents (53%) responded. There were 35 PGY-1, 16 PGY-2, and 20 PGY-3. The mean age was 28 years, 52% were women, 60% white, 39% were fluent in a language other than English, and 49% had non-U.S. born parents. Residents reported experiencing a situation where a patient's care was negatively impacted due to sexual orientation (28%), gender (29%), ethnicity (38%), race (44%), socioeconomic status (56%), or a language barrier (90%). 76% of residents responded that cultural issues are``very''/``extremely'' important to patient care, but only 50% thought training in cross-cultural medicine could substantially improve health delivery. Residents viewed the following as major barriers to integrating cross-cultural issues in residency training: time constraints (60%), lack of experts (30%), lack of interest among learners (19%), lack of interest among faculty (16%), perception that these issues are already adequately addressed (12%), belief that these issues might offend some people (12%). Most residents rated themselves as being overall``somewhat'' tò`m oderately'' knowledgeable (69%) and skilled (81%) about cultural issues and medicine. A significant association was found between PGY level and perception of overall knowledge (p < 0.04%). Interestingly, 16 ± 18% of PGY-1 and 2 residents felt very knowledgeable about cultural issues and medicine, while no PGY-3 did. CONCLUSION: Successful development of curricula in cross-cultural medicine requires an understanding of trainees' attitudes and observations. Residents regard cultural issues as important to patient care and many report observing a direct impact. Despite diverse backgrounds, they do not consider themselves to be highly knowledgeable or skilled in this area. Based on these observations, a need for cross-cultural curricula exists. PURPOSE: Mentors are regarded as important for the academic success of junior faculty, but the availability and impact of mentors on the careers of junior faculty has not been well studied. The purpose of this study was to evaluate junior faculty perceptions of senior faculty mentors at one large academic medical center. METHODS: We surveyed all 154 physicians in the clinician ± educator (CE) and clinician ± scientist (CS) tracks from the Departments of Medicine, Surgery and Obstetrics/Gynecology who held the rank of Instructor or Assistant Professor at the University of Washington. We collected demographic information and asked respondents to describe their academic productivity and rate the adequacy of their mentors. RESULTS: Among 122 (79%) junior faculty who responded the mean age was 38.7 years (SD=4.3); 56 (46%) were women; 52 (43%) were in the CE track; and the mean years in rank was 2.5 (SD= 1.5). CEs reported authoring 2.6 (SD=3.7) book chapters and 4.5 (SD=8.1) original manuscripts whereas the means for CSs were 3.5 (SD=12.1) and 10.9 (SD=12.6), respectively. CEs and CSs had a mean of 1.1 (SD=1.8) and 3.1 (SD=2.6) funded grants, respectively. When asked if they had access to a senior faculty member in their department whom they trusted and who supported them in achieving their career goals, similar percentages of CEs and CSs answered affirmatively (75% and 74%, respectively). On the other hand, when asked if they had adequate mentoring, 36% of all respondents``strongly agreed'' or``agreed,'' (mentored faculty) versus 64% whom``neither agreed nor disagreed'',``disagreed'' or``strongly disagreed'' (nonmentored faculty). In bivariate analyses, mentored faculty were more likely to be men (44% vs. 27%, p=0.049), have completed fellowship training (41% vs. 21%, p=0.048) and to be CSs (51% vs. 15.4%, p < 0.0001). 93% of mentored respondents``agreed'' or``strongly agreed'' that they would achieve their short-term career goals vs. 65% of the non-mentored faculty (p = 0.0006). Gender (OR=3.6, p=0.02) and academic track (OR=9.9, p=0.0002) were independently associated with junior faculty perception of adequate mentoring in logistic regression adjusting for age, years at current rank, department affiliation and fellowship training. Mentored faculty reported more publications than non-mentored faculty. This difference did not reach statistical significance in linear regression (p = 0.1) adjusting for all independent variables entered in the logistic multivariate analysis. CONCLUSION: Over half of the junior faculty in this study did not feel that they were being adequately mentored. Women and CEs were more likely to perceive not being adequately mentored. Strategies to improve mentoring need to be developed and evaluated. Little is known about the quality of Pap smears obtained by internists. The presence of endocervical cells increases the quality of pap smears and has been previously evaluated in family physicians and ob-gyn. We examined the influence of gender, status (faculty vs. resident) and practice site (university vs. community) on rates of satisfactory smears as defined by the presence of endocervical cells. METHODS: Retrospective review of Pap smears evaluated by one cytology department over a 2 year period that were performed by internal medicine faculty and residents from 1 academic department at three clinic sites: two university-based clinics(one staffed by faculty and one staffed by residents) and one community-based clinic(staffed by both faculty and residents). Vaginal smears were excluded. RESULTS: 1086 smears were evaluated. 56% of smears were obtained at the faculty university clinic (FC), 18% at the resident university clinic (RC), 26% at the community clinic (CC). FC patients were older than RC or CC (mean age 56,40,42). 86 Internists were included: 65% males, 17% faculty (60% FC), 83% residents (52% CC). 75% of smears were satisfactory, 19% satisfactory but limited by no endocervical cells, 5.6% limited for other reasons, 0.18% unsatisfactory. Males and female internists had similar satisfactory rates (78% vs. 71%). Physicians at the university clinics had higher satisfactory rates (83%) compared to the community clinic (53%). University faculty had higher satisfactory rates than community faculty (83% vs. 43%). University residents had satisfactory rates similar to university faculty (83%) but higher rates than community residents (56%). Community residents had higher satisfactory rates than community faculty (56% vs.43%). Using the generalized estimating equations approach to control for volume of smears done by each physician we found that younger patient age (p = 0.02), faculty status (p = 0.03), and community clinic site (p = 0.0001) were all associated with lower satisfactory rates. The odds of a satisfactory reading were 1.13 for each additional decade of age, 2.04 for residents vs. faculty, and 6.22 for FC vs. CC, 3.49 for RC vs. CC, and 1.78 for FC vs. RC. CONCLUSION: Satisfactory rates among this sample of Internists were 11 ± 18% lower than published reports for family physicians and ob-gyn. Rates of satisfactory smears did not differ by gender. We did find higher satisfactory rates in older patients, residents and at university-based sites. These results suggest that academic institutions should place more emphasis on teaching Pap smear sampling technique to internists. METHODS: Mailed and web-based surveys were conducted of all 395 internal medicine residency programs regarding 45 procedural skills. We asked directors which methods of procedural skills training they used, which procedures residency graduates should master, and the amount of training needed to attain and maintain competency in each procedure. This paper reports analysis of responses of 269 (68%) program directors or their designees. RESULTS: As in the 1987 survey, the proportion of program directors who said all residents should master these procedures differed from the proportion who said that all residents do master them. For 21 of the 45 procedures in the survey, at least 30% more respondents said residents should master the procedure than said all their residents do master the procedure. This gap is larger in the new survey. For the 35 procedures that appeared in both surveys the new survey found 12% fewer reporting that all residents mastered those procedures. For example, although more than 90% of respondents in both surveys thought thoracentesis should be mastered by all residents, 79% in the 1987 survey vs.58% in the new survey thought it was mastered by all. In addition, estimates of the amount of training needed to master the procedures were lower in 31 of 35 procedures and were 30% lower overall. Some procedures were less strongly recommended, as might be expected from changes in practice or new regulations (e.g. bone marrow aspiration, gram stain). For 4 of 6 procedures required by both the ABIM and RRC, fewer than 60% of directors said that all of their graduates master them. CONCLUSION: Compared with 1987, similar numbers of IM program directors consider procedural skills' training necessary, but fewer think that their residents are mastering the skills. Program directors' estimates of the amount of training needed are 30% lower. Although residents appear to be getting less training in procedural skills, these skills remain important for internists to learn. Given the implications for patient care, this apparent mismatch between expectations and actual practice requires careful review and intervention by medical educators and policy makers alike. PURPOSE: Managed care curricula are being developed and implemented in graduate health professions education programs throughout the country. The purpose of our study was to measure the attitudes of resident physicians in three departments at a large academic medical center during 1999 and 2000. Specifically, we wished to compare the attitudes of incoming interns and graduating seniors, since differences between these groups could indicate effects of graduate medical education on physicians' attitudes towards managed care. METHODS: A 35-item questionnaire was administered to all first-and third-year residents in Family Medicine, Internal Medicine, and Pediatrics at a large midwest academic medical center through two cross-sectional surveys in 1999 and 2000 (n=320). Topics included the effects of managed care on physician autonomy, costs, and quality of care (7 items); practice guidelines (3 items); capitation (3 items); Medicaid managed care (4 items); factors necessary for success under managed care (7 items); and expected practice payor mix (4 items), geographic setting (6 items), and practice type (1 item). All items were 4-or 5-point Likerttype scales. RESULTS: Over 60% of residents agreed that managed care has restrictive effects on physician autonomy, compensation, referrals, and test ordering. Residents were relatively neutral regarding managed care's effects on quality and costs of care. Residents recognized ( > 60% of residents rated as important) several``non-traditional'' areas as important for success under managed care, including patient outcomes, assessing the needs of populations, and team building. First-and third-year residents' attitudes towards managed care were largely similar, though first-year residents were more positive about managed care's effectiveness in controlling costs (64% vs. 46% agreed, p < .04), and more likely to expect to care for underserved (79% vs. 66%, p < .005) and Medicaid patients (98% vs. 88%, p < .04). CONCLUSION: Resident physicians are concerned about managed care's threat to professional autonomy, are relatively neutral regarding other aspects of managed care such as quality of care or cost control. Differences between first-and third-year residents were consistent with greater idealism among interns than seniors. Residency curricula should directly address issues of autonomy under managed care and provide accurate information on the effects of managed care on quality and costs. Recognition of the importance of several``new'' physician competencies suggests a positive effect of recent changes in medical school and residency curricula. Future research should be directed towards whether differences in idealism between interns and seniors are due to the effects of training, or secular trends in attitudes towards managed care and practice intentions, or both. This questionnaire was sent to all US medical schools in May and the results tabulated at the national CDIM office. Questions asked 1) whether formalized courses on student professionalism existed at the school, 2) whether white coat ceremonies were held, 3) what percentage of third year students had been counseled by the CD concerning professionalism issues, 4) what percentage of students received unsatisfactory clerkship grades because of these deficiencies in professionalism and 5) whether separate forms or mechanisms existed to document and deal specifically with issues of misconduct. RESULTS: 74% (92 of 124) of US medical schools completed the survey. 25% of respondents stated that a formalized course for professionalism existed at their institution. 84% of schools hold a white coat ceremony or similar event. During the past year CDs counseled almost 4% (range 0 ± 15%) of third year students and 1% of all students received an unsatisfactory clerkship grade because of these deficiencies. Only 32% of medical schools reported a separate form or mechanism to document breeches of professionalism. CONCLUSION: The results of this national survey of Internal Medicine CDs confirm the increasing interest in professionalism in medical education. The vast majority of institutions have established white coat ceremonies to foster the values of the profession. CDs are counseling students for unprofessional behavior and for a small minority this has significantly impacted the grade they received. However, formalized courses and effective policies to evaluate and document student misconduct still need broader implementation. This study sought to examine and understand how highly respected physician role models think about role modeling. METHODS: In-depth semi-structured 30-minute interviews were conducted by the primary investigator in the offices of 29 of the 30 most highly regarded role models, as judged by the house officers, at two large teaching hospitals in Baltimore. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major categories of themes, which were examined and conceptually organized. RESULTS: The analyses revealed that role models have identifiable characteristics. Subcategories under the domain of personal qualities include a commitment to excellence and seeking continual improvement, integrity, a positive outlook, leadership, and being interpersonally skilled. Under the domain of teaching, the subcategories were establishing rapport with learners, being committed to their growth, and developing specific teaching philosophies and approaches. Subjects thought there was some overlap between teaching and role modeling, but role modeling was felt to be more implicit, more intimate, and more encompassing. In the clinical domain, a recurrent theme was that being a strong clinician was necessary but not sufficient for being considered a role model for medical learners. The informants identified barriers to effective role modeling and they included being over-extended, being passive, and being impatient, inflexible and overly opinionated. Although any given role model might embody a range of talents, subjects believed it was valuable for each medical learner to have multiple role models. CONCLUSION: Highly regarded role models shared their opinions about the critical components of role modeling in medicine and the barriers that can make it difficult. The identification of personal qualities and features of teaching and clinical work associated with effective role modeling by physicians advances our understanding in this area. PURPOSE: Due to the dwindling numbers of generalists available to teach interviewing and physical exam skills to small groups of medical students in the first and second year, all clinical departments at the University of Wisconsin Medical School agreed to supply faculty for small group teaching in the Patient, Doctor, and Society Course (PDS.) Much concern remained, however, as to whether specialist and generalists could teach pre-clinical students with equal effectiveness. METHODS: Specialists and generalists were randomly assigned to the third semester of PDS to teach groups of four students. All leaders were provided with the same verbal and written orientation materials. The specialists from nine clinical specialties were assigned to lead 18 groups of students and the generalists from general internal medicine and family medicine were assigned to lead 16 groups. To determine from the students' point of view, whether specialists and generalists teach with equal effectiveness, students were asked to evaluate their small group leaders on a seven point Likert scale on nine items that included: enthusiasm for teaching; fosters discussion; prepared/ knowledgeable; availability; constructive feedback; timely return of work; good role model; treats students with respect; and an overall rating of the group leader. Scores for leaders were compared using a chi square test. In addition, a questionnaire was distributed to the leaders to determine whether specialists and generalists were equally confident in teaching basic history and examination skills. The questionnaire asked leaders to evaluate their confidence in their ability to teach the abdomen, neurology, cardiovascular, pulmonary exams, and in providing feedback to students in the complete history and physical exam skills. Confidence ratings for leaders were compared using a chi square test.
To determine if there might be differences in specialist and generalists' teaching effectiveness, student scores on an objective, structured, clinical examination (OSCE) were compared. Finally, we compared small group grades assigned by specialist and generalist leaders to their students. RESULTS: Responses from 77% of the students indicated that specialists and generalists teach basic examination skills with equal effectiveness. Responses from 71% of the leaders indicated that they are equally confident in teaching basic examination skills. There were no differences in the generalists and specialists assignment of grades to their students. Most importantly, there were no differences in the scores on the OSCE between students taught by generalists and specialists. CONCLUSION: Medical schools can expand their pool of physicians teaching basic skills by utilizing specialists without decreasing teaching effectiveness. With changes in the organization, financing and delivery of health care, this strategy could help schools preserve their academic mission in the face of mounting pressure to increase clinical revenue. Novel point-of-care echo machines are becoming available and may provide a rapid and cost-effective method for accurately assessing LV function. It is unknown, however, whether physicians without prior echo experience can be trained to use echo to assess LV systolic function. METHODS: Physicians, without prior echo experience, completed a 3-hour training course including didactic and hands-on training in echocardiography. Patients scheduled for standard transthoracic echocardiography as part of their clinical care were eligible for enrollment. Enrolled patients had a point-of-care echocardiogram performed and interpreted for LV ejection fraction (EF) by a trained physician within 24 hours of their standard echo. LV EF was classified as 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%. Point-of-care echos were performed using the new Agilent``OptiGo'' cardiac ultrasound device. Each standard echo was read twice. All echocardiographic interpretations were performed blinded to one another. Agreement was defined as a difference in LV EF of 15%. Agreement rates between point-of-care echo and standard echo were determined and compared to standard echo interobserver agreement rates. RESULTS: Twenty-five physicians enrolled 510 patients. The mean age of the patients was 59.4 years and 52.8% were male. The indications for echocardiographic assessment was evaluate ejection fraction in 70.4% and evaluate murmur in 11.1%. A total of 57.9% of patients had an LV EF of > 55%, 14.2% had an LV EF of 45 ± 50%, 9.2% had an LV EF of 35 ± 40%, 7.2% had an LV EF of 25 ± 30%, and 11.5% had an LV EF of < 15 ± 20%. Rates of agreement for evaluation of LV function are shown in the table. CONCLUSION: Physicians without prior echo experience can be trained in 3-hours to assess LV function with a novel point-of-care echo device. Agreement between point-of-care echo and standard echo is similar to standard echo interobserver agreement. Wider availability of pointof-care echocardiography could improve medical care. CONCLUSION: Although only a minority of low income women report low levels of trust in their personal health care provider, lack of trust is strongly associated with lack of adherence to recommended cancer screening. Mistrust of health care providers in general and beliefs about unethical medical research practices are more common, but have less effect on use of preventive health services. Increasing trust of primary care physicians among low income populations may offer a novel approach to addressing socioeconomic health disparities.
universally useful markers of medical underservedness. The purpose of this study was to determine how well race/ethnicity and income identify women who do not receive screening mammography in the City of Philadelphia. METHODS: Telephone survey of adult residents of the City of Philadelphia. Households were sampled using random digit dialing stratified by service area. Interviews were conducted in English or Spanish between June and September 1998. Women ! 50 yo were asked``About how long has it been since you last had a mammogram?'' and women who had not had a mammogram in past year were asked the most important reason they had not had a mammogram. RESULTS: Of 6,329 eligible households, 4,114 individuals (65%) agreed to participate. 828 respondents were women between 50 and 75 years of age. Mean age was 61.9 yrs; 46% were Caucasian, 48% African-American and 4% Hispanic; 26% had not completed high school. Mean annual household income was $19,000 (range < $8,000 to > $100,000). 75% had undergone screening mammography in the prior year, 9% one to two years previously, 11% more than two years ago and 5% had never had a mammogram. In the overall sample, mammography in the prior year was associated with African-American ethnicity (RR 1.19, 95% 1.1 ± 1.3), but not with age, income or education. However, there were significant interactions between ethnicity and income and ethnicity and age. Income was associated with mammography among Caucasian women only (RR 1.28, 95% CI 1.0 ± 1.7 for above vs. below federal poverty level) and age was associated with mammography among African-American women only (RR 1.34, 95% CI 1.0 ± 1.8 for over 60 vs. 60 and under). Among women who had not had a mammogram in the prior year, African-American women were less likely to cite lack of motivation as their reason for not undergoing screening (RR 0.70, 95% CI 0.48 ± 0.99) and more likely to cite system barriers (e.g. insurance, transportation) than Caucasian women. CONCLUSION: In the city of Philadelphia, low income, minority women no longer represent the underserved for screening mammography. Furthermore, the characteristics of women underusing screening mammography differ between African-American and Caucasian women. These data suggest that the sociodemographic characteristics of the medically underserved may depend upon type of medical care, geographic location and even ethnic group. Generalist efforts to reduce medical underservedness should be based on accurate assessments of the communities they serve. Our purpose was to describe the relationship between both selfreported (SR) and electronically monitored (MEMS) adherence (ADH) and HIV viral load (VL), and to characterize drug resistance patterns in subjects who failed to sustain virologic suppression on potent HAART regimens. METHODS: Subjects on HAART were recruited from a longitudinal study of HIV-infected drug users. During a median of 166 days, VL and ADH with all antiretrovirals were assessed by SR and MEMS at each of 6 monthly visits. ADH (% doses taken/doses prescribed) was assessed for both 1 day and 1 week preceding each visit, and mean ADH was calculated for the entire study period. We also studied drug resistance in 11 patients with viral rebound (VL > 1000 copies/ml for > 2 consecutive mos. after previously maintaining VL < 400 copies/ml for > 2 consecutive mos.). RESULTS: 501 SR estimates and 417 MEMS estimates were obtained from 85 subjects. Mean 1 week SR ADH correlated with mean 1 day SR ADH (76% v. 77%; r=0.84, p=0.0001), as did mean 1 week and 1 day MEMS ADH (55% v. 57%; r=0.86, p=0.0001). Though SR ADH was greater than MEMS ADH, both were correlated with VL (r=0.6 for 1 week MEMS, r=0.5 for 1 day MEMS, r=0.5 for 1 week SR, r=0.4 for 1 day SR). The following factors were associated with poor ADH: active heroin use (64% for active v. 80% for former use, p=.02), active cocaine use (38% for active v. 60% for former use, p=.009), active alcohol use (60% for subjects who drank several days/week v. 80% for subjects who drank less, p=.002), side effects (73% for subjects with > 2 side effects v. 83% for subjects with < 2, p=.08), and female gender (44% for women v. 60% for men, p=.04). Among the 11 patients in whom we analyzed ADH, resistance and rebound, we found that median ADH during viral suppression was 54% (range 0 ± 83%), and median ADH after rebound was 8% (range 0 ± 36%). Only 2/11 developed high-level phenotypic resistance, and 4/11 developed significant mutations in either RT or protease; M184 in 3/11 on 3TC, K103N in 2/2 on NNRTIs, and various IDV mutations in 1/11 on PIs. In 2/4 patients, M184 mutations were present without corresponding phenotypic resistance. CONCLUSION: High correlation between 1 day and 1 week estimates suggest that ADH does not change in the day before a visit. While SR overestimates true ADH, repeated quantitative SR estimates correlate with VL. Our data further show that SR ADH > 90% is necessary to maintain virologic suppression, and that active drug/alcohol use and medication side effects lower adherence. Our analysis of drug resistance suggests that significant RT or protease mutations may be present as early indicators of phenotypic resistance. However, due to lack of sufficient selective pressure, high level phenotypic resistance is uncommon after viral rebound when ADH is poor. PURPOSE: Pulmonary infections, including bacterial pneumonia, cause significant morbidity and mortality for patients infected with HIV. The purpose of this project was to determine the predictors of inpatient mortality among HIV patients with community-acquired pneumonia admitted in the early highly active antiretroviral therapy era, between 1995 ± 1997. METHODS: Trained abstractors performed retrospective chart reviews of hospitalized HIV patients diagnosed or suspected of having community-acquired pneumonia, who were admitted between 1995 ± 1997 at 86 hospitals in 7 geographic areas. We used hierarchically optimal classification tree analysis to develop a pre-admission severity of illness staging system for predicting inpatient mortality. RESULTS: Among HIV patients with confirmed or suspected community-acquired pneumonia (n=1112), the inpatient mortality rate was 9.5%. Multivariate modeling indicated a 5-category staging based on four predictors (Table 1) . Bootstrap validity analysis (50% resample) supported the stability of this model. CONCLUSION: Our staging system identifies HIV patients with low, moderate, and high risk of inpatient mortality from community-acquired pneumonia and may be useful for case-mix adjustment while exploring variations in community-acquired pneumonia mortality rates among hospitals and across cities caring for HIV patients. The potential risks of herbal supplement use is of growing concern. Reports of serious side effects and drug interactions have prompted many physicians to incorporate questions of supplement use into their medical history. Despite these efforts, clinicians are often faced with inadequate information due to the recent boom of combination herbal products known only by their trade names. Supplements marketed to consumers as weight loss remedies are one such group of products that frequently contain a multitude of``natural'' ingredients. This study was designed to characterize the ingredients found in a number of popular weight loss products in order to assist physicians in preventing potential adverse effects. METHODS: A systematic search of the World Wide Web was conducted to identify twenty herbal weight loss products for sale on the internet. Three search engines were used (HotBot, Google and Excite) and the search term``herbal weight loss.'' A site abstraction form was developed to collect data on the ingredients contained within each product. Descriptive statistics were used to characterize the most common ingredients found among the twenty combination products. RESULTS: Nine ingredients were identified in six or more of the products. These included: MaHuang, Ginger, Guarana, Siberian Ginseng, White Willow Bark, Chromium, Garcinia Cambogia, Gotu Kola, and Zinc. The number and frequency of each of these ingredients among the herbal weight loss products characterized are included in the table below. CONCLUSION: Despite little evidence of efficacy and the potential for significant toxicity, ingredients such as Mahuang, Guarana, and Chromium are found in a majority of weight loss products. Physicians need to take this into consideration when faced with a patient who discloses the use of an unfamiliar over-the-counter weight loss supplement. HIV prevention education is a part of substance abuse treatment at many correctional facilities, but the effectiveness of these programs has not been evaluated. The goals of our study were to evaluate who receives HIV prevention services in correctional substance abuse treatment programs, and to determine the impact of these services on short term risk behaviors. METHODS: This study was a secondary analysis of data from the National Treatment Improvement Evaluation Study (NTIES), conducted during 1993 ± 1995. A total of 1,223 HIVfree adult inmates, enrolled in nine correctional substance abuse treatment programs were evaluated. A composite index modeled after the validated Risk Assessment Battery (RAB) measured HIV risk behavior at treatment entry and at twelve-month follow-up. We employed multivariable analysis of covariance to assess the influence of receiving HIV prevention services, controlling for potential confounders, on HIV risk behavior at twelve-month follow-up. Because of assumed differences between those who were in and those who were out of custody during the follow-up period, all analyses were stratified by this variable. RESULTS: Overall, 77.1% of the sample received HIV prevention services while in treatment.
Among the out of custody group, Hispanics, persons of younger age and those in outpatient substance abuse treatment programs were significantly less likely to received HIV prevention services. Among the continuously incarcerated group, women, persons of younger age and those in outpatient substance abuse treatment programs were least likely to receive these services. Both the in custody and out of custody groups exhibited HIV risk behavior reductions after twelve months of follow-up. However, controlling for baseline HIV risk behavior and other possible confounders, receiving HIV prevention services was associated with less risk behavior only among the out of custody group (=À.14, P=.04). No association was observed among the group that was continuously incarcerated throughout the follow-up period. CONCLUSION: This analysis suggests that HIV prevention services are beneficial in reducing risk behavior among inmates whose discharge is expected in the near future. The benefit of administering these services in individuals with longer sentences earlier in the incarceration period is unclear. Lifestyle changes, such as smoking cessation, can play a pivotal role in outcomes following the diagnosis and treatment of coronary heart disease (CHD). However, many patients with CHD are not motivated to make lifestyle changes even after undergoing coronary revascularization. Prior studies have shown that time orientation is a major motivational determinate of an individual' s behavior and more specifically as it relates to healthy lifestyle choices. Therefore, we sought to examine the association of future orientation with readiness to make healthy lifestyle changes in a diverse group of patients at high risk of CHD. METHODS: Forty patients scheduled for elective coronary angiography were identified in the fall of 2000. High risk patients were those with one or more of the following: diabetes, hypertension, or hyperlipidemia. Following informed consent, a baseline 20-minute structured bedside interview was conducted, which collected information on the following: demographics (e.g. age, race, and sex), high-risk behaviors (e.g. smoking) using the interview format of the Computerized Lifestyle Assessment (CLA), and future orientation using Carstensen and Lang' s Future Orientation Scale. This latter scale measures the extent that one perceives there is sufficient time to achieve his/her goals. A follow-up interview was conducted 3 months from baseline to assess readiness to change as measured by the CLA. RESULTS: Follow-up data were completed on 33 subjects (83%). Their mean age was 58 years (S.D.=10.4), 64% were women, 42% were Hispanic, 27% were White, 21% were Black, and 10% were Others. Most patients (79%) did not exhibit any movement in readiness to change their lifestyle or habits as measured by the CLA subscale. Overall, 15% of patients had movement toward adopting a healthier lifestyle following angiography, such as improving their eating habits and exercising; 6% regressed. Patients who were future oriented (n= 16) had the most progress in movement toward making healthy changes in exercise (25%), diet (10%), and smoking (10%), compared to 17%, 23%, and 6% respectively of patients who were present oriented. However, 13% of future oriented subjects had an overall decrease in their readiness to change score following angiography, compared to none (0%) of the 17 patients who were present oriented. CONCLUSION: These data suggest that patients at high-risk of CHD, as well as those who are future oriented, may be slow to make lifestyle changes following angiography. These findings also indicate that there is a need for more research to ascertain patients' perception of their personal coronary risks. Primary care and other health providers can use such novel information to implement individually relevant educational programs to address motivational factors related to health behavior. RESULTS: There were 1006 patients surveyed with a refusal rate of 20.3%. The study patients did not differ from the total population who accessed care during this time period in regards to age, insurance status, ethnicity, or gender. After controlling for several factors, including age, gender, and the presence of chronic medical conditions, Hispanic patients were more likely than white patients to report having a regular source of health care (p = 0.008) or a regular physician (p = 0.018). However, Spanish speaking patients were less likely to note a regular physician (p < 0.0001) than English speaking patients. Black patients were more likely than white patients to note a regular source of care (p = 0.04) but not a regular physician (p = 0.8108). Hispanic patients were 1.7 times more likely to have visited the ED or UCC in the past year than our white patients (p = 0.006). However, Spanish speaking patients had less use of these services (p = 0.01) than English speaking patients. CONCLUSION: Sociocultural issues such as race and English fluency likely contribute to utilization of resources such as EDs and UCCs. For example, in our population minorities tended to utilize EDs and UCCs more often than white patients, but were also more likely to report that they had a regular source of care. Given the racial disparities in health outcomes there is an imperative to address this problem with additional studies. Simply attempting to redirect patients who inappropriately utilize EDs and UCCs to their regular source of care is unlikely to succeed unless language and cultural issues are addressed. where an aggressive approach to care following acute myocardial infarction (AMI) is more commonly adopted in comparison to a conservative approach, the aggressive approach may be associated with small incremental (marginal) mortality benefits. We conducted this study to evaluate the effectiveness of aggressive care following AMI in Canada. We hypothesized that the marginal benefits should be larger in Canada, as the country is operating on a lower margin because the approach to care is more conservative overall. METHODS: We conducted a retrospective cohort study using administrative data of acute care hospital admissions and in-and out-patient services for all patients who sustained a first AMI in Quebec in 1988 (n=8674). We used differential distances to hospitals offering aggressive care as instrumental variables to control for unobserved case-mix variation when measuring mortality up to 4 years after first AMI. RESULTS: Of the 4422 subjects who were > 64 years old, 11% received cardiac catheterization within 90 days after admission. In a previous study that applied similar methodology to the 1987 United States (US) Medicare population of first AMI patients, 30% of subjects received catheterization within 90 days. As in the US study, we found that subjects living relatively close to hospitals offering aggressive care were more likely to receive aggressive care (26% of``close'' versus 19% of``far'' subjects received cardiac catheterization within 90 days; 95% CI: 5% to 9%). Unlike the US study, we found no differences in mortality across the``close'' versus``far'' differential distance groups (unadjusted differences at 1 year: 1%; 95% CI: À1% to 3%). This absence of association was found in elderly ( > 64 years) and younger age groups. Adjusted results also showed no differences between subjects receiving aggressive versus conservative care (at 1, 2 and 4 years: 4%, 2%, À4%; 95% CI: À11% to 20%, À15% to 18%, À26% to 8%, respectively). CONCLUSION: Contrary to our hypothesis but consistent with results from numerous randomized trials and observational studies, the aggressive approach to post-AMI care does not appear to be associated with marginal mortality benefits even in Canada, where the approach to post-AMI care is conservative overall. Although clinicians may prefer results that are reported as``normal'' or``abnormal,'' this usually requires the specification of an artificial diagnostic criterion. Diagnostic information is typically lost in this simplification process. One application of the mutual information function is the calculation of the amount of information lost as test results are dichotomized. Consider, for example, a population of individuals in which the probability of disease is 0.5. Assume that the``healthy'' and``diseased'' individuals in this population are normally and identically distributed with respect to some diagnostic marker, except that the means of the two populations are separated by one standard deviation. In this case, I(Dx;T) = 0.16 bits. If, however, the results are dichotomized using the optimal cutoff (the midpoint between the means of the two populations) then I(Dx;T) is reduced to 0.11 bits. CONCLUSION: Information theory provides a framework for understanding diagnostic testing. This perspective suggests that mutual information, rather than the AUC, provides the appropriate summary index of diagnostic test performance. PURPOSE: Racial disparities in procedure use for acute myocardial infarction (AMI) have been well documented in selected populations in the 1980s and early 90' s, but little is known about recent trends in disparities in the general population. METHODS: We conducted a series of cross-sectional analyses of data from the Nationwide Inpatient Sample (which includes race and demographic, diagnostic and procedure data on all discharges from over 900 representative hospitals in 18 states) for 1995 through 1997 to compare rates of catheterization performed prior to discharge for acute myocardial infarction (ICD9 code 410) in whites vs. blacks. RESULTS: We identified 298,496 discharges among whites and 23,496 discharges among blacks with AMI as the first listed diagnosis during 1995 ± 97. Age-adjusted catheterization rates by race are presented in the figure. From 1995 to 1997, age-adjusted peri-AMI catheterization rates increased more sharply in blacks (39% to46%) than in whites (48% to 52%; p < 0.01). After adjustment for age, gender, comorbidity, region, and rural locale using Poisson regression, the incidence rate ratio (IRR) for peri-AMI catheterization in blacks compared to whites in 1995 was 0.84 (95% confidence interval (CI) 0.78 ± 0.90). During the next two years, the gap progressively narrowed, such that in 1997 the adjusted IRR was 0.90 (95% CI 0.83 ± 0.96). Similar results were obtained when we restricted the analysis to discharges from hospitals that performed catheterizations. CONCLUSION: These recent nationwide data suggest that, while blacks remain less likely to undergo catheterization during a hospitalization for AMI than whites, this racial gap appears to be narrowing.
PURPOSE: Women veterans, like their male counterparts, are at increased risk for heart disease because they are more likely to be obese and smoke cigarettes than the general population. In order to develop clinical interventions that will improve risk behaviors among women veterans, we assessed their awareness of heart disease. The purpose of this study was to determine whether women veterans are worried about heart disease and to specify whether women with risk factors for coronary artery disease (CAD) are more likely to worry about heart disease compared to women without risk factors. METHODS: Cross-sectional survey of women veterans receiving primary care at the Durham VAMC. We measured demographics, CAD risk factors, other medical conditions, and worry about heart disease. Statistical analysis was performed using the chi-square test and logistic regression analysis using worry about heart disease as the outcome variable. RESULTS: Overall, 409 women veterans completed the questionnaire (response rate 71%). We excluded women with heart disease (n=64) and incomplete data (n=17 PURPOSE: Blacks are less willing to donate cadaveric organs, the most common type of transplantation, but whether the same race differences exist for other donations is unknown. We compared patterns of race differences in willingness to donate cadaveric organs with those of becoming a living kidney donor and donating blood. METHODS: We conducted a cross-sectional study in Maryland via standardized telephone questionnaire using random digit dialing to identify households. Persons age 18 ± 75 years were randomly selected within households. Subjects were considered willing to become cadaveric donors if they had designated themselves as an organ donor on their driver's license, livingrelated donors if they were willing to donate a kidney to a sibling, and blood donors if they had previously donated blood. Using logistic regression we analyzed the independent effects of race on willingness to donate adjusting for demographic, clinical and attitudinal characteristics.
RESULTS: Of 385 respondents (84% of randomized households), 19% were White men (WM), 30% White women (WW), 12% Black men (BM), and 31% Black women (BW). BM and BW were more likely than WM and WW to have < high school education (p < 0.01), to have annual income < $40,000 (p < 0.01), to have dependents (p < 0.01), to be unemployed (p = 0.01), to consider spirituality important (p < 0.01), to believe hospitals``want to know more about personal affairs or business than they really need to know'' (p < 0.01), and more likely to believè`h ospitals have done harmful experiments on patients without their knowledge'' (p < 0.01). BM were least willing to become cadaveric donors (17%) compared to BW (34%), WW (59%), and WM (65%) (p < 0.01). However, WM were least willing to become living related donors (60%) compared to BW(61%), BM(72%), and WW(75%) (p = 0.05). BW were least willing to donate blood (40%), compared to WW (58%), BM (65%), and WM (86%)(p < 0.01). Despite adjustment for differences in respondent characteristics, BM were still much less likely to become cadaveric organ donors [odds ratio (95% CI)]=[0.2 (0.1 ± 0.7)], equally as likely to donate blood [0.8(0.3 ± 2. 5)], and almost 3 times more likely to become living-related donors [2.8 (0.9 ± 8.7)] than WM. It is unclear whether BW were less likely to be cadaveric organ donors [0.6 (0.2 ± 1.5)], but they were much less likely to donate blood [0.1 (0.04 ± 0.3) ], and equally as likely to be willing to be a living related donor [1.3 (0. PURPOSE: Ethnic minorities with coronary artery disease have worse outcomes compared to Caucasians. Depression is known to be a major risk factor for poor outcomes among patients with coronary artery disease. However, among ethnic minorities with coronary artery disease, little is known about the prevalence of depression. The purpose of this study was to determine whether ethnic variations in pre-operative depression exist between Caucasians and ethnic minorities undergoing angioplasty. METHODS: Subjects in the study were patients who had recently undergone coronary angioplasty and were eligible for enrollment in a randomized trial to reduce cardiac risk factors. Baseline evaluation included an assessment of demographic characteristics, cardiac history, and severity of illness. Depression was assessed using the Center for Epidemiologic Studies Depression Scale(CES-D). A score of 16 or greater indicated the presence of depression. RESULTS: Of 542 patients enrolled, 33% had evidence of pre-operative depression. Their mean age was 61(+12), 34% were female, 14 % were African-American and 16% were Latino-American. Patients who were depressed were more likely to be younger in age (P=03). A greater proportion of women were depressed in comparison to men (P=.003). Mean CES-D scores were significantly higher among ethnic minorities compared to Caucasians( P=.002). The highest CES-D scores were observed among Latino-Americans. In general, African-Americans and Latino-Americans were 2 times more likely than Caucasians to have evidence of depression ( 95 % CI 1.3 ± 3.2). CONCLUSION: Among patients undergoing coronary angioplasty, minorities were 2 times more likely to have evidence of depression at baseline. This finding is clinically important since baseline depression among patients with coronary artery disease is a poor prognostic indicator. Future studies are needed to determine whether baseline depression among ethnic minority patients with coronary artery disease is associated with poor outcomes. PURPOSE: Patients often receive medical information from the media and internet, but little is known about how information sources affect risk perceptions. Our objective was to investigate whether the source of medical information affected perceived risks for common diseases. METHODS: Women treated for breast cancer at an urban hospital were recruited through flyers and an oncology social worker. Participants completed a health survey about their sources of health information (doctor, television (TV), or internet) and their perceived lifetime risk of heart disease, hip fracture, and recurrent breast cancer. Risk estimates among patients receiving vs not receiving information from each source were compared using Wilcoxon Rank tests. RESULTS: The mean age of the 51 participants was 52 years, 16% were non-Caucasian, and the median educational level was 2 years of college. Patients receiving medical information from either TV or the internet had higher perceived risks for both heart disease and hip fracture than those not receiving information from those sources. Patients receiving information from TV perceived breast cancer recurrence as being more likely even though fewer among them had late stage or node-positive disease. Patients who received information from their doctors did not have consistently higher or lower perceived risk estimations compared to patients not receiving information from their doctors. CONCLUSION: Patients' perception of risk may be affected by the source of health information, with those who rely on the TV or internet having higher risk perceptions than those who rely on other sources. PURPOSE: Alcohol abuse is a common but frequently unrecognized problem in surgical patients, with important implications for Medical consultants. Studies have shown that 10 ± 50 % of hospitalized patients on surgical services have alcohol problems, depending on the subspecialty service and the detection methods used. We screened a large cohort of VA surgical patients to determine the prevalence of problem drinking in a combined inpatient and outpatient population as the initial step in a study of alcohol consumption and surgical outcomes. METHODS: All patients at the VA Pittsburgh Healthcare System admitted to surgical services or scheduled for elective surgery requiring at least an overnight hospital stay were considered candidates for screening. Patients were asked to complete a confidential screen for alcohol problems using the Alcohol Use Disorders Identification Test (AUDIT) and quantity-frequency measures. Non-cirrhotic patients who met criteria for alcohol problems (AUDIT score 8 or weekly consumption > 15 drinks in men or > 11 drinks in women) and control patients who did not meet these criteria were invited to participate in a descriptive cohort study. Controls were matched by surgical service and admission status (inpatient versus outpatient at screening, planned overnight versus longer admissions for elective procedures) in a 2:1 control:study ratio. Participants received a baseline preoperative assessment including demographic information and the Alcohol Timeline Follow-Back to measure alcohol consumption. Descriptive statistics were reported as mean standard deviation. Current drinking and drinking over the last 6 months were analyzed using a non-parametric test for trend. RESULTS: Among 1624 patients presenting for surgical admission or preoperative assessment over one year, 1067 (65.7%) were screened for alcohol problems, of which 99 (9.3%) screened positive. The mean AUDIT score for screen-positive patients was 12.0 | 9.6 and the mean number of drinks per week was 33.2 | 37.2, compared with 1.1 | 2.1 and 2.0 | 8.5, respectively, for screen-negative patients. Forty-seven (47.5%) screen-positive patients agreed to further assessment and follow-up, as did 93 matched controls. Mean AUDIT and alcohol consumption scores were not statistically different between enrolled and non-enrolled study patients (p = 0.3). Among enrolled problem drinkers, there was a trend toward less alcohol consumption in a typical drinking day as surgery approached, from an average of 9.2 | 12.5 drinks/day over 6 months prior to their assessment to 5.4 | 4.4 drinks/day over the previous 1 month (p = 0.13 for trend). Binge drinking episodes ( > 6 drinks in one day) showed a similar reduction, with the average problem drinker reporting 5.6 | 8.7 episodes/month at 3 ± 6 months prior to assessment but only 3.4 | 6.7 episodes/month over the previous 1 month (p = 0.18 for trend). The problem drinkers were most commonly found on the Orthopedic (29.8%)and General surgery (25.5%) services. CONCLUSION: The prevalence of problem drinking in our large cohort of veterans undergoing surgery was lower than that found in prior studies of surgical inpatients but similar to that seen in published studies of medical outpatients. The inclusion of outpatients and patients undergoing elective procedures may shift the drinking profile of surgical patients towards that of the general population. Intensive screening of surgical patients for problem drinking by Medical consultants may be most effective on selected subspeciality services. Alcohol consumption levels among problem drinkers tended to decrease as the date of surgery approached, suggesting that the perioperative period may provide a good opportunity for brief interventions.
ALPHA BLOCKERS AND HYPERTENSION Ð HOW MANY ARE AT RISK FOR SUBOPTIMAL THERAPY? C.L. Bryson 1 ; 1 Puget Sound VA, Seattle, Washington PURPOSE: This study defines the number of people at risk for suboptimal therapy with hypertension who are only on alpha blockers. Results of randomized controlled trials have provided substantial evidence that antihypertensives differ in their ability to mitigate outcomes of CHF, CVA, MI and renal failure in the hypertensive population despite nearly equivalent blood pressure control. This is of particular interest because of the early termination of one arm of the ALLHAT study in which the alpha-1 blocker doxazosin was associated with a doubling of the rate of incident heart failure. Doxazosin is less effective for preventing the outcome of CHF than chlorthalidone in hypertensive patients. This result was unexpected, since doxazosin has favorable effects on other surrogate markers including serum lipids. Alpha-1 blockers are an effective treatment for prostatism, and fifty percent of men have histologic evidence of BPH by 60 years of age. It is difficult to quantitate the exposure of alpha-1 blockers for hypertension in elderly men on pharmacy data alone because information about the patient' s comorbid illnesses is necessary to interpret the indication for the agent. This is true not only for alpha-1 blockers but also for other antihypertensives. METHODS: The National Ambulatory Medical Care Survey (NAMCS) is a national survey of physician visits that records not only physician diagnoses but also patient therapy. It was a sampling study that was carefully performed and allows extrapolation to the national visits during the study times. The NAMCS data was analyzed with STATA. The provided weights were utilized to expand the results to projected national averages. Summary measures were computed for variables of interest while maintaining acceptable relative standard errors. RESULTS: This presentation or poster will present the results of the analysis of the 1998 NAMCS for antihypertensive use, with an accent on alpha blocker usage as a single drug. Approximately 3.5 to 4% of the hypertensive population appears to be treated with only an alpha blocker, which is between 840,000 to 1 million patients. Interestingly, about half of these are women. Only 15% of hypertensives were on diuretics. Time trends and usage by indication may also be presented from prior NAMCS data. A correlation with VA data will be available as well by the time of presentation as a result of an ongoing project. CONCLUSION: An estimation of the use of different antihypertensive agents correlated with indication is important, particularly in light of recent clinical trials. An estimate of the use of alpha-1 blockers as single agents prior to changes in recommendations will help enumerate the size of the population at risk for suboptimal pharmacotherapy with regard to cardiovascular endpoints. It will also provide context for the importance of findings from clinical trials of antihypertensive agents. (3) clinical cues found empirically to be used by clinicians to differentiate sinusitis from viral URIs. Agreement between diagnostic approaches was determined using the Kappa statistic. RESULTS: Of 91 eligible patients, 61 (67%) agreed to participate in the study. Mean age was 42 years (range 12 ± 78), 43 (70%) were women, and mean duration of symptoms was 4.7 days (range 1 ± 23). Forty-four patients (72%), 6 patients (10%), and 11 patients (18%) were diagnosed with sinusitis by each diagnostic approach respectively. Agreement between diagnostic approaches ranged from kappa=0.08 (approach 1 compared to approach 2) to kappa=0.39 (approach 2 compared to approach 3). CONCLUSION: Three published approaches to diagnosing sinusitis modified for telephone use exhibited poor to marginal agreement. More research is needed to determine a valid tool for telephone diagnosis of sinusitis before the diagnosis and treatment of acute sinusitis solely through telephone contact can be recommended. Increasingly, hospices are developing affiliations with academic medical centers. However, little is known about hospice patients referred from academic medical centers, and how their needs may differ from those of the general population of hospice patients. Therefore, the purpose of this study was to identify differences between patients referred to an urban, non-profit hospice from academic vs. non-academic medical centers. METHODS: In this retrospective cohort study, 1,691 patients admitted to an inpatient and outpatient hospice program were identified between January of 1997 and January of 1999, and data were gathered until discharge or death. RESULTS: Patients referred from academic medical centers were younger, had higher incomes, and were less likely to have Medicare or Medicaid. Patients referred from academic medical centers were less likely to have a Do Not Resuscitate order or a living will, and had more medical and nursing needs. Survival analysis revealed no difference in length of stay between patients referred from academic and non-academic medical centers. CONCLUSION: Patients referred to hospice from academic medical centers have greater needs for nursing and medical care than do patients referred from non-academic medical centers. These results have important implications for hospice planning and policy. If they are replicated in other hospice settings, they suggest that hospices that establish ties with academic medical centers may be caring for patients with more symptoms, and more needs for care. Such differences should be considered in drawing up plans for affiliations. They might also be considered on a more global scale in assessing reimbursements to hospices by Medicare and other payers. were younger than 65 years, and 8 had concomitant valvular disease leaving 145 patients for analysis. Data collection was complete. The mean age was 77 years, and women comprised 55% of the cohort. According to the AFI criteria, 38% of the population had an annual stroke risk of < 4.3% while 50% of the population had an annual stroke risk < 3.6% by SPAF (p < .05). There was agreement between the AFI and SPAF in estimating the patients with the highest stroke risk of 8% annually (43% and 50%, respectively). The OBRI estimated 91% of all patients to have a bleeding risk of 12% over two years while 9% of the cohort had the highest estimated bleeding risk of 53% over 48 months. In all but the highest stroke risk groups, the OBRI-estimated bleeding risk exceeded the expected benefits of anticoagulation. CONCLUSION: In this population of elderly patients with atrial fibrillation, the use of published risk stratification indices has limited clinical utility, mainly because of the difficulty in balancing the relative risks in the group with low and intemediate stroke risk. In particular, the validated OBRI does not seem applicable to the stable outpatient elderly population, and its use might actually deter the appropriate anticoagulation of patients. PURPOSE: Cold angina is explained with increased coronary``vascular resistance''. The temperature of the lower extremities in cold weather and the temperature of blood during hibernation is approximately 228C. It is suggested that when blood temperature decreases from 36.68C to 228C, blood viscosity increases 26.13%. The aim of this study is determination of the effect of the increased blood viscosity on circulatory system. METHODS: Changes in circulatory parameters due to increased viscosity can be calculated with Poiseuille' s hydrodynamics equation. In the equation, blood flow rate in m3/sec is Q=%a4/ 8 L(F1-F2) and peripheric resistance of a vessel is R=(F1-F2)/Q, where a is the radius, L is the length of vessel, is blood viscosity, F1 is the initial and F2 is the final pressure at the two ends of a vessel. RESULTS: According to these equations, flow rate decreases 20.72 % and peripheral resistance increases 20.72 % when viscosity increases 20.72 % due to cold environment. This state can be compensated by increased cardiac work as 20.72 % increase in blood pressure or 5.9 % increase in the vessel radius. CONCLUSION: The term``peripheral resistance'' is more correct for the explanation of cold angina than vascular resistance, because peripheral resistance can cover blood viscosity (resistance against blood flow) and vascular resistance (change in diameter, length and shape of vessel). These calculations show that decreased temperature and increased viscosity of the blood in the skin and lower extremities in cold weather results in increased peripheral resistance and circulatory work. Due to sclerotic vessel has no vasodilatation capacity, Y Â schemia risk is high for the coronary atherosclerotic patient. Unfortunately, similar circulatory changes caused by the cooling of the pulmonary blood in cold weather are unknown. The information above shows that the mechanism of cold angina gains an additional explanation with increased blood viscosity and peripheral resistance due to decreased blood temperature, for the first time. This method is important for further evaluation of the dynamics of the circulatory system. RESULTS: CC occurred in 13 patients (14.4%) including 8 patients who experienced 11 major complications. There was a trend toward more CC with age > 60, anesthesia duration > 3 hours, creatinine > 2.4, and vascular and emergency surgery. The ASA scale and revised cardiac index(Lee) performed better than the original Goldman and Detsky indices. The ACC and ACP algorithms were best at identifying low risk patients, but some of this effect was due to the results of non-invasive tests. (Table 1 ) Exercise capacity, Eagle criteria, and Larsen's index were not predictive of CC. CONCLUSION: Although our group of patients had relatively few major complications, we were able to validate the predictive ability of various indices to stratify perioperative risk; however, statistical significance of the differences could not be achieved due to the small numbers. The goals of risk indices are to identify low risk patients requiring no further workup and to select other patients potentially benefiting from additional testing or interventions. Other management strategies, including perioperative beta-blockers, will be needed to reduce complications in the intermediate and high risk groups, and in emergency surgery patients. The ACC and ACP each published guidelines for perioperative cardiovascular evaluation for noncardiac surgery. Both were evidence-based, but the ACC also used expert consensus opinion in the absence of strong evidence. Because of this difference, the guidelines may present conflicting opinions. The purpose of this study was to compare their recommendations with respect to non-invasive testing (NIT) and evaluate risk stratified clinical outcomes. METHODS: We retrospectively reviewed records of 90 patients (pts) undergoing 104 major intra-abdominal or vascular procedures in a large, urban teaching hospital. Patients were classified into low, intermediate, or high (including emergency) risk groups based on the algorithms. We then compared the ACC and ACP recommendations for NIT and subsequent clinical outcomes for each group. Major complications were defined as cardiac death, MI, ischemia, CHF, and VT. RESULTS: Eleven major complications occurred in 8 pts, 3 of whom had MI' s (1 cardiac death). Of the 104 procedures, 30 were emergent (1 MI). Additionally, 9 and 5 pts respectively were classified in a high risk group by ACC and ACP criteria. The remaining pts were divided into test or no test subgroups ( Table 1 ). The ACC recommended NIT for 11 of these lowintermediate risk pts, 2 of whom had postop MI' s, and no test for the other 54 pts, none of whom had major complications. The ACP recommended NIT for 6 of 14 intermediate risk pts, 1 of whom had an MI, and no test for the 55 low risk pts, 1 of whom had an MI. ACP guidelines recommended no test in 7 of the 11 pts (1 had an MI) in whom the ACC suggested testing . The guidelines agreed otherwise. CONCLUSION: Both ACC and ACP algorithms separated low risk patients from intermediate-high risk ones. Because of differing concepts regarding exercise capacity and NIT in non-vascular surgery, the ACP classified more patients as low risk and recommended fewer NIT' s; however, the clinical outcomes appeared comparable (despite the small numbers). Physician judgement and personal preference for the guidelines will determine the approach regarding NIT for an individual patient undergoing elective surgery. Future studies will need to clarify the best approach for high risk/emergency patients. were independent risk factors for lower extremity amputation in patients with PAD. Although the burden of certain atherosclerotic risk factors is higher in minority patients, the impact of this burden does not account for the increased risk of the outcome of lower extremity amputation in these two populations. As we do not have information on the severity of disease, race/ethnicity may be a marker for the severity of PAD. Further research is needed to better understand the reason (s) why race/ethnicity is independently associated with poor outcomes in PAD. PURPOSE: Whether patients and their providers make comparable assessments of the severity of pain, the likelihood of improvement and optimal treatment strategies when patients present with an acute episode of low back pain is not known. We sought to evaluate the level of concordance on these dimensions between patients and providers at the time that individuals with acute low back pain (ALBP) enrolled in a randomized controlled trial (RCT) of usual care versus choice of complementary and alternative medical (CAM) therapy. METHODS: Baseline data obtained by face to face interview at RCT enrollment. Eligible subjects were adult members of a multi-specialty medical practice who reported experiencing uncomplicated ALBP for less than 3 weeks with no co-morbid explanation for symptoms. Enrollees were randomized to either usual care or to a choice of chiropractic, massage, acupuncture or usual care. Enrollees' medical providers completed a written survey at the time they referred a patient to the study. RESULTS: Of the 80 enrolled in the study to date, the mean age was 42, 51% were women, and 58% were white. Sixty-seven of the enrollees' providers (84%) responded to a questionnaire about the enrollee's ALBP. The mean patient-reported severity score for back pain on a scale of 0 ± 10, with 10 representing the worst pain ever, was 8.0 (s.d. 1.6, range 4 ± 10); providers reported a mean score of 6.3 (s.d. 2.1, range 1 ± 9). Providers substantially underestimated patients' pain (t=5.48, p < .001), with 24% of the enrollees describing their pain as 10/10 and none of the providers rating pain higher than 9/10. Providers and patients had similar expectations for recovery. On a 0 ± 10 scale, with 10 representing complete improvement in 6 weeks and 0 representing no improvement, patients reported a mean score of 8.4 (s.d. 1.8, range 4 ± 10) and providers a score of 8.1 (s.d. 2.5, range 1 ± 10), t=.84, p=.40. The majority of patients (55%) expressed a preference for massage, 31% for chiropractic, 12% for acupuncture, and 1% for usual care. Thirty-two percent of the providers accurately anticipated the type of therapy the patient said s/he would choose, 18% thought that the modality the patient preferred was the best choice for that patient. Only 7% believed the therapy preferred by the patient was the best treatment for ALBP in general. Patients presenting to a university-based clinic were screened for MD using the PRIME-MD. Eligible patients also had a 17-item Hamilton Rating Scale for Depression (HRS-D) !12. Patient satisfaction with their PCP was assessed at baseline using a 5-point Likert scale and later dichotomized to``very satisfied'' or``not very satisfied'' for our analysis. The HRS-D was repeated at 6 months. Recovery was defined as a HRS-D 7. Information about depression treatment, such as pharmacotherapy, number of visits, and referral to a mental health specialist was abstracted from the EMR. RESULTS: Between 4/96 and 12/98, 211 depressed patients met all protocol eligibility criteria and completed a baseline interview. Of the 193(91%)patients who completed 6 month interviews, their baseline mean HRS-D was 20, 71% were women, 24% were African American and 119 (62%) reported being``very satisfied'' with their PCP. No differences in HRS-D or satisfaction were noted by gender, race or medical comorbidity. Although``very satisfied'' patients had significantly lower baseline HRS-D, the difference of only one point was not considered clinically significant. At 6 months 43(22%)of the patients recovered.``Very satisfied'' patients were more likely to recover at 6 months than those who were``not very satisfied'' (31% vs 8%, OR 5.1, 95%CI=2.0 ± 12.8). This association persisted after controlling for baseline HRS-D scores, medical co-morbidity and process measures for depression care (adjusted OR 5.3, 95%CI=2.0 ± 13.8). A baseline report of``very satisfied'' was not significantly associated with subsequent number of PCP visits, medications prescribed, or mental health referrals. CONCLUSION: Patients who are``very satisfied'' with their PCP are more likely to recover from a MD episode. Patient recovery has been found to be related to physicians engendering confidence in their patient's recovery. It is tempting to speculate that PCPs who stimulate greater optimism in their patients, improve satisfaction and subsequent recovery from depression. Assessment of patient satisfaction early in the therapeutic relationship may identify patients at risk for poor recovery from MD. Smoking is a significant issue among the homeless, but little is known about their willingness to quit or their interest in assistance with quitting. We therefore sought to determine what proportion of homeless persons smoked, whether they were ready to stop smoking, and whether they had preferences for smoking cessation treatments. METHODS: We anonymously surveyed homeless adults at nine sites in Pittsburgh, including emergency shelters, drop-in centers, transitional housing, a community health center, and residential substance abuse treatment programs. All persons at each site were approached during 2-hour blocks; 97% of those present completed the survey. Measures included demographic characteristics, living situation, smoking behavior, readiness to change smoking behavior (based on a stage-of-change model), self-efficacy to stop smoking (rated from 1 to 10), nicotine dependence (Fagerstrom scale), and preferred method of smoking cessation. RESULTS: 273 persons completed the survey, of whom 87% met criteria for homelessness. The sample was middle aged (mean age 42 years), male (81%), and minority (63%). Of the 68% who were current smokers, 87% had interest in quitting, and 39% indicated readiness to quit smoking within the next six months. Compared to those not ready to quit, persons were significantly (p < 0.05) more likely to be ready to quit if they had high self-efficacy (47% vs. 21%), knew persons who would support their smoking cessation efforts (47% vs. 13%), or smoked within 30 minutes of awakening (40% vs. 31%). Readiness to quit was not associated with gender, ethnicity, substance abuse, nicotine dependence, or type of shelter. Participants self-efficacy to quit smoking was significantly higher with assistance, compared to their selfefficacy without assistance (6.3 vs. 4.8, p = 0.000). Treatments preferred by persons ready to quit included nicotine replacement (33%), counseling (15%), bupropion (Zyban) (7.5%), and nicotine in combination with other treatments (12%); one-third (33%) indicated``nothing'' or`q uitting cold-turkey'' would help. CONCLUSION: The majority of homeless persons smoke, many are ready to stop smoking, and most believe they will be more successful with treatments such as nicotine replacement. There is an urgent need to develop and implement smoking cessation programs for the homeless. METHODS: We used data from a national telephone survey that examined attitudes and beliefs of Americans toward participation in clinical research. A distrust index, scores ranging from 0most trusting to 7-most distrusting, was created using the sum of the responses to a seven-item measure of trust. The dependent variable in these analyses, high levels of distrust, was categorized as 5 on the distrust index. Of 1000 survey respondents, there were 527 Black and 382 White respondents eligible for this analysis (n = 909). RESULTS: Black respondents were more likely than White respondents not to trust that``their doctor would fully explain research participation'' (41.7% vs. 23.4%, p < 0.01) and less likely to believe that they could ask their doctor questions (15.2 vs. 7.6%, p < 0.01). Black respondents were also more likely to disagree that``their doctor would not ask them to participate in research if the doctor felt there was harm" (37.2% vs. 19.7%, p < 0.01) and more likely to state that they felt their doctors sometimes exposed them to unnecessary risks (45.5% vs. 34.8%, p < 0.01). Blacks were more likely to believe that``someone like them'' would be used as a guinea pig without their consent (79.2 vs. 51.9, p < 0.01) and that their physician had given them treatment as part of an experiment without their permission (24.5% vs. 8.3%, p < .01). Black respondents has a significantly higher mean index score (3.1) than Whites (1.8) (p < 0.01). Nearly 30% of blacks compared to 9% of whites had scores 5 (p < 0.01) In unadjusted analyses, Blacks had more than 4 times the odds of whites of having a distrust index score 5. After controlling for other sociodemographic variables in the logistic regression model, race remained strongly associated with a higher distrust score (OR=3.6; 95% CI=2. 4,5.4) . CONCLUSION: We found important differences by race in distrust. Even after controlling for markers of social class, African Americans were less trusting. Investigators trying to engage African Americans in research must focus on developing interpersonal trust with community members by actively engaging the members in all aspects of research design development and dissemination of findings. We included clinical trials conducted in patients in the following areas: cardiovascular disease, diabetes mellitus, HIV/AIDS, and cancer. For each RCT we collected data on the number and percent of white and minorities (i.e., African Americans, Hispanic, Asian/Pacific Islander, and Native Americans) reported in the study samples. In addition to simple descriptive statistics, we used ANOVA to assess reporting of race before during and after 1993 ± 1995. RESULTS: A total of 253 RCTs met our inclusion criteria (29 diabetes, 84 cardiovascular and 50 cancer and 90 HIV/AIDS). Forty percent of RCTs did not report the race/ethnicity of the study sample. Of the RCTs that reported race (n=150), 29% of the study samples were minorities. There was no statistical difference in the percent of minorities in RCTs published before 1993 RCTs published before , between 1993 RCTs published before and 1995 RCTs published before , and after 1995 . In addition there was no difference in the percent minorities when stratified by disease. CONCLUSION: In diseases where there are significant disparities by race in health outcomes, a large number of RCTs did not report the race/ethnicity of the study sample. In addition, there has been no increase in the proportion of minorities in study samples before, during or after enactment of federal initiatives to increase the proportion of minorities in RCTs. For clinicians to have the highest level of evidence to effectively care for minority patients and reduce disparities in health outcomes, RCTs must consistently report race/ethnicity and the proportion of minorities included in study samples needs to increase. METHODS: 309 psychiatric outpatients underwent a personal interview assessing medical conditions and health status with the Medical Outcomes Study Short Form-36 (SF-36) survey. We assessed psychiatric severity with the Brief Psychiatric Rating Scale (BPRS) which rates psychiatric severity from 0 (none) to 6 (extremely severe) in 18 symptom constructs. Podiatric health was assessed using 9 items from the National Health Interview Survey (NHIS) and an additional item addressing foot pain. RESULTS: 57% of our sample were diagnosed with schizophrenia and the mean BPRS score was 22 reflecting moderate severity of psychiatric illness at the time of the interview. 70% of participants reported at least one of 8 medical problems. 82% of patients surveyed reported at least one podiatric problem and 40% reported at least 3 problems. The most common problems were foot pain (48%), nail disorders (35%), and corns/calluses (28%). The prevalence rates of podiatric disorders in our cohort were 4 ± 10 times higher than those reported by the general population in the 1990 NHIS. For example, 5.2% of the general population reported nail disorders, 5.0% reported foot infections, and 5.7% reported corns/calluses. In bivariate analysis, the total number of podiatric problems reported by the participants was inversely related to their scores in all eight domains and both summary scores of the SF-36 (all p < 0.001). After controlling for sociodemographic factors, psychiatric illness, and medical conditions, the total number of podiatric limitations remained significantly associated with lower patients ratings in 4 of the 8 SF-36 domains: bodily pain (p < 0.001), role limits-emotional (p = 0.002 ), social functioning (p = 0.03), and general health (p = 0.003) and both summary domains, physical component (p = 0.04) and mental component (p = 0.03). CONCLUSION: Persons with severe and persistent mental illness have markedly elevated rates of podiatric problems when compared to the general population. These problems are associated with worsened self-perceived health status. Addressing podiatric health may be a successful way to improve the overall health of this population. PURPOSE: HIV continues to be a destructive problem in the urban poor. The SRO hotel population, a unique subset of the urban poor, is inadequately described in the literature. Our purpose is to describe the level of comprehensive health care, perceptions of health care, and utilization of health care services in the SRO hotel population served by both the Department of Acquired Immunodeficiency Syndrome (AIDS) Services Income Support (DASIS), a division of New York City's Department of Welfare, and CitiWide Harm Reduction, a community-based organization in the South Bronx. METHODS: Following a pilot survey, we conducted an extensive community-based survey of SRO hotel residents in the South Bronx from 8/99 to 4/00. We went door-to-door in all 10 DASIS-and CitiWide-served SRO hotels administering anonymous surveys to residents. The 34-item questionnaire included demographic information, health care utilization patterns, status of HIV disease and treatment, perceptions of quality of and access to health care, and drug use. RESULTS: Of the 190 hotel residents approached, 97% completed the survey. Of these residents, 91% reported being HIV-positive, 63% male, 59% Black, 34% Latino, and 62% active drug users. The average age is 41 years (range 20 ± 74 years), and 56% are high school graduates. The median length of stay in a single hotel is 4 months (range 1 day ± 32 years), and 96% have Medicaid. Of the 167 HIV-positive residents, 67% report having primary care providers. Of these individuals, 71% report receiving care in an infectious disease (ID) or HIVspecialty clinic. However, only 22% of 18 eligible residents report taking Mycobacterium avium complex (MAC) prophylaxis, 65% of 57 eligible residents report taking Pneumocystis carinii pneumonia (PCP) prophylaxis, and 44% of 88 eligible residents report taking antiretroviral medications. In the previous 6 months HIV-positive residents report a mean of 1.5 Emergency Department visits, and in the previous year they report a mean of 1.7 hospitalizations. Quality of care and access to care are perceived to be less than``good'' by 44% and 36% of HIV-positive residents, respectively. CONCLUSION: Among South Bronx HIV-positive SRO hotel residents, a largely substanceusing population with Medicaid, two thirds report having primary care providers. Yet a significant proportion of these individuals are not receiving medically recommended HIV therapy. Approximately half are dissatisfied with their quality of care, and one third with their access to care. In light of these findings, steps are currently being undertaken to address this deficiency in health care among HIV-positive individuals living in SRO hotels. PURPOSE: Although use of electronic medical record (EMR) systems is increasing, the effects on actual communication between patients and physicians have not been studied extensively. We sought to assess physician-patient communication patterns associated with use of an EMR system in an outpatient setting and provide an empirical foundation for larger studies. METHODS: We conducted a cross-sectional, case-control study involving analysis of videotaped physician-patient encounters, surveys of physicians and patients, and medical record reviews. The setting was an academic general internal medicine practice. We studied three physicians who used an EMR system (EMR physicians) and three who used solely a paper record (control physicians). A total of 204 patient visits were included in the analysis (mean = 34 for each physician). Makoul's SEGUE instrument was utilized for content analysis of whether physicians accomplished communication tasks during encounters. A separate, qualitative analysis by the authors sought evidence of general patterns of EMR and paper chart use. RESULTS: Compared to the control group, EMR physicians adopted a more active role in clarifying information and providing patient education. Overall, there were no statistically significant differences between the physician groups in mean duration of encounters, number of treatment options offered, or number of lab tests ordered. The qualitative analysis showed that physicians in both groups tended to direct their attention to the patient record during the initial portion of the encounter. The physical orientation of the computer keyboard/monitor and physicians' facility in typing were important determinants of attentiveness to the patient throughout the EMR encounters. CONCLUSION: EMR physicians' communication behaviors may reflect styles established before they began using an EMR system. Coupling education on patient-centered communication when physicians are trained on the use of EMR systems could enhance the effectiveness of providers using this tool. that might deter women from recommended screening. Another possible explanation for avoidance of BCS might be that some women are not sufficiently concerned about the consequences of metastatic breast cancer to submit to screening. We, therefore, measured womens' perceptions of metastatic breast cancer and compared them to self-reported compliance with BCS. METHODS: A trained research assistant interviewed 106 women aged 50 to 75 years using a structured questionnaire that assessed patients' attitudes toward mammography and their compliance with BCS. Interviews were conducted in North Carolina and Florida. Patients preferences for the health state``life with metastatic breast cancer'' were measured using a standard gamble approach. Standard gamble results were converted to health utility scores(HUS) for which 1.0 = normal health and 0 = death. Descriptive statistics were compiled. Bivariate analysis was performed to identify possible associations with recent noncompliance with BCS. Demographic data, attitudes toward screening, and HUS were used as independent variables in this analysis. Due to the exploratory nature of this study and small sample size, a p-value of < 0.1 was used as the cutoff for statistical significance. RESULTS: Of the 106 women interviewed, 83% were high school graduates or above, 35% were African-American, and 89% had some form of health insurance. The mean age was 60 yrs. Two thirds of participants reported themselves to be at least in good health. The mean HUS for life with metastatic breast cancer was 0.39 with a median score of 0.25. Thirty-two of the one hundred-six women interviewed had not received BCS during the 2 years preceding this survey. Over 90% agreed that mammograms could save their life and that the actual x-ray would not lead to breast cancer or needless breast surgery. Bivariate associations with BCS non-compliance included worse self-reported health, education less that high school, African-American race, and high HUS (0.7 or above) for life with metastatic breast cancer. CONCLUSION: Poverty, African-American race, and lack of education have long been sited as sentinel characteristics for lower BCS rates. The above data suggest that optimistic perceptions of metastatic cancer might also contribute to these lower rates. If indeed, higher individual HUS, can be confirmed as an independent predictor of BCS non-compliance, then a standard gamble approach could be used to identify a group of patients that could benefit from a targeted educational intervention that aggressively portrays consequences of metastatic breast disease. PURPOSE: A recent analysis of secondary data revealed marked differences in lung surgery rates between African-Americans and whites who suffered from early stage, non-small cell, lung cancer. The disparity in treatment according to race led to an 8% reduction in 5 year survival for African-American patients. The authors were unable to sort out whether patient or physician factors explained the difference in surgical decision making. Because of this uncertainty, we decided to measure patient perceptions of progressive lung cancer and evaluate whether more optimistic perceptions of this disease could reasonably explain differences in surgical management. METHODS: We recruited a stratified, random sample of 160 individuals from church groups, health fairs, and medical clinics in urban areas of North Carolina and Florida. A trained research assistant administered an oral questionnaire to obtain demographic information and assess patient preferences using a standard gamble approach. Standard gamble results were converted to health utility scores (HUS) for which 1.0 = normal health and 0 = death. HUS for progressive lung cancer were analyzed according to race. We then constructed a decision model that evaluated lung cancer surgery vs. no surgery for stages I and II non-small cell cancer. The model accounted for racial differences in HUS for progressive disease. The model was intentionally biased against surgery by overestimating surgical risk and survival with progressive lung cancer. Sensitivity analysis was performed. RESULTS: The average age of the 160 patients interviewed was 59 yrs. Sixty-three percent were women, 42% were educated beyond highschool, 41% had incomes < $20,000, 15% had no health insurance, and 41% were African-American. The mean HUS for progressive lung cancer among African-Americans surveyed was 0.32 compared to 0.21 among whites. The decision model when applied to a 60 yr.old African-American male demonstrated a quality adjusted survival of 3.38 life years (QALY's) for the surgical therapy group compared to 0.48 QALY's for the``no surgery'' group. In the sensitivity analysis, if patient age was adjusted to 80 yrs., surgery still maintained a large advantage (1.58 QALY's for surgical treatment vs. 0.48 QALY's for``no surgery''). Even if surgical cure rate was reduced to 10% in the model, the advantage in QALY's remained for surgical patients(1.2 surgery vs. 0.48``no surgery''). CONCLUSION: Although African-Americans hold a much more optimistic view of progressive lung cancer than whites, the difference does not explain discrepancies in lung surgery decisions even when decision models are significantly biased against surgical intervention. Other issues, such as physicians' misinterpretation of patients' surgical risk and patients' mistrust or misunderstanding of physicians, need to be assessed in order to resolve discrepancies in lung cancer treatment and survival.
INDIVIDUALIZED TREATMENT FOR ALCOHOL WITHDRAWAL. J. Daeppen 1 , P. Gache 1 , U. Landry 1 , E. Sekera 1 , V. Schweizer 1 , S. Gloor 1 , B. Yersin 1 ; 1 Alcohol Treatment Center, Lausanne PURPOSE: To assess the effect of an individualized treatment regimen on the quantity and duration of benzodiazepine prescribed for alcohol withdrawal. METHODS: A randomized double-blind controlled trial including 117 consecutive patients admitted in an alcohol treatment program was conducted. Patients were randomized into two groups: i) 56 patients were treated with oxazepam only in response to the development of signs of alcohol withdrawal (symptom-triggered), and ii) 61 were treated with oxazepam every 6 hours with additional doses as needed (fixed-schedule). The administration of additional oxazepam was determined using the CIWA-Ar, a validated measure of the severity of alcohol withdrawal. RESULTS: 40% of the patients in the symptom-triggered group were treated with oxazepam, compared to 100% in the fixed-schedule group (p < .001). Mean oxazepam administered in the symptom-triggered group was 40 mg compared to 230 mg in the fixed-schedule group (p < .001). Mean duration of oxazepam treatment was 20 hours in the symptom-triggered group compared to 63 hours in the fixed-schedule group (p < .001). There were no differences in the incidence of complications and in the measures of comfort between the two groups. CONCLUSION: A symptom-triggered pharmacological treatment decreases the quantity and duration of benzodiazepine prescribed in alcohol withdrawal, with similar safety and comfort. Since both HIV/AIDS and its treatments commonly cause adverse somatic effects, symptom assessment is necessary for delivering and evaluating care for people with HIV. This study compared three methods to assess patient reported symptoms (presence, frequency, and bother) and to validate report of individual symptoms using longer patient-reported scales and clinical parameters in people with HIV disease. METHODS: Prospective cohort study. A questionnaire completed by 160 individuals on two occasions, four months apart. The questionnaire included 39 HIV-related symptoms, the Medical Outcomes Study-HIV Health Survey (MOS-HIV), and Health Transition items. Correlation coefficients were calculated to assess relationships between summary scores for the three symptom methods with health-related quality of life domains, CD4 counts, and HIV disease stage. Analyses were stratified by HIV disease stage to determine if correlations differed with respect to clinical stage. RESULTS: Sixty-seven percent of subjects were male, 63.5% were African-American, and 26% had less than a high school education. The percentage of participants with AIDS, symptomatic HIV, and asymptomatic HIV were 46.2%, 28.5%, and 25.3%, respectively with mean CD4 counts of 194, 386, and 400, respectively. The average number of symptoms reported was 15.2 (SD=8.4). There were statistically significant correlations between CD4 count and symptom frequency, symptom bother, and symptom presence. As hypothesized, correlations with the MOS-HIV Quality of Life domain were moderate and statistically significant whereas there were no significant correlations with the Quality of Life Transition score. Examination of individual symptoms revealed that fatigue and decreased memory were significantly correlated with both the Health Transition and MOS-HIV scores. Presence, frequency, and bother scores for trouble falling asleep were correlated with Sleep scale scores. Presence and frequency of fever correlated significantly with CD4 count. Bodily pain had statistically significant correlations with the Pain domain of the MOS-HIV. Hot/cold spells were not significantly correlated with CD4 count. CONCLUSION: The three symptom methods and their scores were strongly intercorrelated. Participants with more symptoms reported greater frequency and bother from symptoms.
Several individual symptoms correlated strongly and statistically significantly with longer measures of the same concept, like the MOS-HIV Health Survey. Overall, no one method is clearly superior; different individual symptoms were better measured using different methods. Studies are needed to develop a measure of symptoms commonly experienced by HIV-infected people. Medical College of Wisconsin, Milwaukee, WI PURPOSE: Women have been found to overestimate breast cancer risk. Perceptions are more accurate in higher educated and more numerate women. The objectives of this study are to evaluate the association between African-American (AA) race and breast cancer risk perceptions. METHODS: We assessed 5-year and Lifetime Risk Perceptions of AA and Caucasian women recruited from a primary care clinic. Actual risk was calculated using the Gail Model. Error in estimation (EE) was defined as perceived risk minus calculated risk. The following categories were defined. Under-estimators had an error of < À10%, Accurate Estimators had an error of > À10% but < 10%, Moderate Over-estimators had an error of > 10% and < 30%, High Overestimators had an error of > 30% and < 50%, Extreme Over-estimators had an error of > 50%. Nonparametric statistics were used to compare EE between groups. RESULTS: There were 254 subjects. Sixty-eight percent (68%) were Caucasian, 30% AA, and 2% other. AA were younger (mean of 55 yrs vs. 58 years, p = 0.03), of lower income (84% vs. 35% of Caucasians < $20,000/year, p < 0.0001), had lower Rapid Estimate of Adult Literacy in Medicine (REALM) scores (mean 53 vs. 65, p < 0.0001), and less likely to answer a set of three numeracy questions correctly (11% vs. 55%, p < 0.0001), compared to Caucasians. The calculated mean lifetime and five-year risk of AA women was lower than Caucasian women (lifetime risk: 5.5% vs. 9.7%, p < 0.0001, five-year risk: 0.89% vs. 1.8%, p < 0.0001). In univariate analyses, race, age, and reading level were not associated with accurate risk perception. A trend of increased accuracy of lifetime and five-year risk with higher numeracy was found (p = 0.08). The table shows error in estimation stratified by race (*p-values > 0.05 using a chi-square test). CONCLUSION: In a primary care population, AA women had similar levels of accurate breast cancer risk estimation as Caucasian women, despite lower education and numeracy levels. Both populations could benefit from education to improve the accuracy of risk estimation. PURPOSE: Deciding on anticoagulating the oldest-old (OO)with atrial fibrillation (AF)with warfarin is an increasingly common problem for general internists. Age is an important risk factor not only for AF, but also for stroke in patients with AF. In addition, other risk factors for stroke in AF, such as hypertension (HBP)and diabetes (DM), also increase with age. Though anticoagulation(AC)has been shown to be effective in preventing strokes in some patients with AF, these studies cover only short periods and include few OO. Also, contraindications and side effects of AC are greater in the OO, the fastest growing segment of the population. METHODS: A cost-effectiveness analysis was developed using 3 Markov states: 1ife without stroke, life with stroke residuals and death. Quality-adjusted life expectancy (QALE) was calculated for each state. Baseline probabilities and rates were obtained from systematic reviews and age-adjusted. Based on self-report, women were categorized into three subgroups Ð current, past, and never drinkers. A drinker was defined as someone who drank moderately ( < 2 drinks/day). Depression was evaluated by using the Geriatric Depression Scale (low score indicating less depression) and Social Networks were defined by using the Lubben Social Network Scale (higher score indicating worse social network). RESULTS: When compared to past drinkers, current drinkers are less likely to carry a previous diagnosis of depression, score low on the depression scale, and take antidepressants. Current users are more similar to never users for these measures. CONCLUSION: Women who currently drink a moderate amount of alcohol are less likely to be depressed and more likely to have a stronger social network than women who are past or never users. Questions regarding possible confounders and why past users quit using alcohol may be answered after analyzing a recent questionnaire describing this cohort's pattern of alcohol use. PURPOSE: Public health departments have traditionally provided episodic safety net services to the uninsured. Increasingly, these providers have taken up the role of providing primary care clinical preventive services as well, particularly to racial/ethnic minority populations. To assess the association of race/ethnicity and other patient and system characteristics with receipt of preventive health care services by low-income women, we studied patients cared for through the Los Angeles-County-Department of Health Services(LAC-DHS) Primary Care Network. METHODS: We sampled patients receiving primary care services at 50 facilities to represent all patients in care throughout the LAC-DHS Primary Care Network. 1,288adult females were interviewed for a response rate of 80%. The sample was racially and ethnically diverse-63% Latina/Hispanic, 22% white, 13% African American,1% Asian/Pacific Islander and 1% Mixed race/Other. We measured receipt of the following tests and services: Pap smear within 3 years, mammogram within 2 years among women age 50 years and older, flu shot among women at risk, and cardiovascular disease testing and counseling among women at risk. We used bivariate and multivariate logistic regression analyses to assess the association of patient characteristics (age, race/ethnicity, education, income, immigration status, health insurance status, health status) and system characteristics (type of facility-comprehensive health center, personal health center, hospital outpatient clinic and public/private partnership clinic) with receipt of preventive health care services by low-income women within a publicly funded health care system. RESULTS: Eighty-eight percent of women had undergone cervical cancer screening within 3 years and 82% of women age 50 years and older had undergone breast cancer screening within 2 years, with the highest rates of screening among Latina patients. Among women at increased risk for morbidity and mortality associated with influenza 41 % had received a flu shot within one year. For women at increased risk for cardiovascular disease 91 % had undergone cholesterol screening within 3 years, 82% had been counseled about the importance of exercise, 86% about nutrition and healthy eating and 46% about tobacco use. In multivariate regression analyses Latinas remained the most likely to have undergone cervical and breast cancer screening and among women at risk for influenza African Americans were the most likely to have received a flu shot. CONCLUSION: For low-income women who are able to gain access to a large urban publicly funded health care system for their primary care needs their level of receipt of preventive health care is comparable or better than that of women in other healthcare settings, although shortcomings remain. PURPOSE: Studies have demonstrated that people seek emergency department services for a variety of reasons, including not being able to otherwise access needed health care. To assess the use of emergency department services(emergent and non-emergent) by low-income patients with a regular source of care we interviewed a sample of patients who received their medical care through a large public health system. METHODS: We performed a stratified cross sectional study utilizing probability sampling techniques and survey methods. We sampled patients receiving primary care services through the Los Angeles County Department of Health Services Primary Care Network. 1819 adults were successfully interviewed for a response rate of 80%. The sample was racially, and ethnically diverse. We used bivariate and multivariate logistic regression analyses to assess the association of patient characteristics including insurance status and use of primary care services with visits ( 1 or more) to the emergency department within the preceding 12 months. RESULTS: Twenty-eight percent of the adults in this sample had made one or more visits to an Emergency Department for medical treatment within the preceding 12 months. Almost half of those who had used emergency department services reported that they felt their medical problem was an emergency (49%),but a substantial number of patients sought emergency care for financial reasons(10%) and ease of accessibility to needed medical care (12%). Multivariate analyses revealed that patient characteristics associated with a greater likelihood of having used emergency department services included being white, poor health status, having Medicaid rather than being uninsured, and having delayed seeking needed medical care. CONCLUSION: Patients within a publicly funded medical system have high rates of emergency department use. Type of health insurance and whether people had delayed receiving needed medical care during the preceding year were associated with use of emergency department services. Thus, access and barriers to needed medical care are related to use of emergency department services for emergent and non-emergent medical care. PURPOSE: Conflicting study results regarding the role of intranasal steroids in patients with rhinosinusitis create a dilemma for clinicians. Using a double-blind, randomized, placebocontrolled methodology, we examined whether the addition of an intranasal steroid to conventional antibiotic therapy speeds the recovery of patients with recurrent acute rhinosinusitis. METHODS: Ninety-five (95) patients, ages 18 or older, presenting with acute rhinosinusitis and a previous history of recurrent sinusitis or chronic rhinitis were enrolled from 22 primary care and otolaryngology practices nationwide. Either plain film sinus radiograph or nasal endoscopy supported the diagnosis of sinusitis for all patients. Patients with a history of chronic bacterial sinusitis, previous sinus surgery, and recent intranasal steroid or antibiotic use were excluded. Subjects were randomized to receive either 2 puffs of fluticasone proprionate (n = 47) or saline nasal spray (n = 48) in each nostril once daily for 21 days. All patients received 2 puffs of oxymetazoline hydrochloride in each nostril twice daily for 3 days initially and cefuroxime axetil 250 mg twice daily for 10 days. Patients recorded their daily symptom status in a diary during the 3-week treatment phase. Telephone follow-up was conducted at 10, 21, and 56 days after enrollment; 88 patients (92.6%) completed all telephone follow-up. The primary endpoint was time to clinical success over the 8-week follow-up period (defined a priori as cured or much improved). RESULTS: Patients using the regimen which included fluticasone propionate achieved higher rates of clinical success, 93% versus 74% in the placebo group (p = 0.009 by Chi-square test). The relative benefit increase was 26% with the inclusion of fluticasone to the regimen and the absolute benefit increase was 19%; the number needed to treat to achieve one additional treatment success was 6. Fluticasone-treated patients improved significantly more rapidly (median days to clinical success was 9.5 and 6.0 days, respectively; p = 0.012 by log rank test). Other covariates, including specialty vs. primary care study site, age, number of comorbidities, gender, race, recent upper respiratory infection, or history of allergy, were tested using a Cox proportional hazards model, but only the inclusion of fluticasone to antibiotic and decongestant therapy significantly influenced the clinical outcomes. CONCLUSION: The addition of a 3-week course of fluticasone propionate to oxymetazoline (3 days) and antimicrobial therapy with cefuroxime (10 days) improves clinical success rates and accelerates recovery of patients with a history of either chronic rhinitis or recurrent sinusitis who present for treatment of acute rhinosinusitis. PURPOSE: Disadvantaged populations are often the last to benefit from innovation, including quality improvement (QI) programs for depression. Public sector clinics may not have the resources to implement and maintain QI programs and QI programs designed for private patients may not address needs, treatment preferences, and resources available to depressed, indigent patients. Careful needs assessment can help formulate objectives of QI for such populations, but assessment may be limited by the fact that such populations have little prior exposure to appropriate treatments. We present the development of a unique approach that demonstrates respect for indigent patients and their providers by employing market research strategies to rigorously assess their preferences for and barriers to depression care, as the first step to developing a tailored QI program. METHODS: We conducted literature reviews of existing primary care depression interventions and reviewed methods for understanding patient treatment preferences, including the use of market research methods for assessing consumer preferences. Key informant interviews were conducted with 4 principal investigators of depression treatment interventions. RESULTS: The reviews indicated that few existing QI studies for depression explicitly incorporate assessment of and respect for patient treatment preferences in intervention design. Further, valid methods for assessing patient and provider preferences for and barriers to depression care have not been systematically applied to treatment interventions for the poor. Market research techniques including conjoint analysis surveys have been validated and used to design appealing and acceptable consumer products and services. Because market research methods, such as conjoint analysis, have not been commonly used in developing health services interventions, especially in populations with little treatment experience, we have developed an iterative process for assessing and validating patient and provider preferences and barriers to care. The method includes 4 phases: 1.) patient and provider focus groups, 2.) a market research survey (using conjoint analysis) 3.) a pilot depression intervention in which patient preferences, barriers to care, and opportunities for treatment are identified and results from step 2 are validated, and 4.) indepth qualitative interviews with patients and providers. For all phases of the design depressed patients from the general internal medicine clinic are identified using waiting room screening. Focus groups were held with depressed patients to inform the design of the conjoint analysis survey. Of 86 patients in the general internal medicine clinic screened for focus groups, 26 (36%) had current depressive disorders, 23 patients were eligible to participate in focus groups, and 15 (65% of those eligible) did so. The mean age of focus group participants was 52 (SD = 8.9); 14 of 15 were female; 73% had less than 6 years of education; and all were Spanish speaking. Only one participant reported prior treatment for depression. Patients preferred counseling as the first line treatment for depression, but would consider antidepressants if recommended by a caregiver. A majority preferred group over individual counseling. Patients preferred to receive treatment at the primary care clinic, but from a mental health specialist rather than a primary care provider. Patients reported that their lack of knowledge regarding depression and its treatment and their fear of treatment costs were the main reasons that they had not sought care. In the second phase, 175 depressed patients are recruited to complete a conjoint analysis survey. In this technique, key attributes of a proposed product and possible variations (called``levels'') of the attributes are defined. Hypothetical products with variations of the defined attributes are rated or ranked by patients according to their preferences. Conjoint analysis then determines the relative importance consumers attach to each of the attributes and the utilities they attach to each variation or level of the attributes. We assess the validity of conjoint analysis for determining preferences for and barriers to depression care by comparing survey results with actual treatment choices and barriers to care following a limited depression intervention. Finally, indepth qualitative interviews developed with a medical anthropologist are used to complement quantitative data gathered during the pilot intervention. CONCLUSION: Previous QI programs for depression in primary care have generally not included systematic assessment of patient and provider preferences for and barriers to care in their design. Rather than making assumptions about the needs of providers and consumers in disadvantaged settings, researchers should rigorously assess the preferences of those most affected by a planned QI intervention. A rational and respectful approach for assessing patient and provider preferences using market research methods is proposed for developing and adapting health care QI interventions for disadvantaged populations. PURPOSE: To use anti-Xa activity to monitor enoxaparin dosing in the following patient types: greater than 65 years old, with a creatnine clearance less than 30ml/min and in morbid obesity. To develop a weight adjusted dosing method for patients over 65 years old and for patients with a creatnine clearance less than 30mls/min. METHODS: All patients were dosed by their physician using 1mg/kg sub-cutaneous every 12 hours. Peak anti-Xa activity was obtained 3 ± 5 hours after the third dose, trough activity was obtained just prior to the dose. Anti-Xa activity was measured using Dioagnostica Strago instrumentation. aPTT was obtained simultaneously. RESULTS: Thirty-eight patients were involved in the evaluation. Thirty-six peak levels and 17 trough levels were obtained. Thirteen patients greater than 65 years old were able to be evaluated. All values are mean: age 77, Scr 1.29mg/dl, CrCl 42ml/min, peak anti-Xa 1.3IU/ml. aPTT was prolonged in the vast majority of patients who had anti-Xa activity > 1.0IU/ml. One patient experienced a major hemorrhage. CONCLUSION: Doses of 1mg/kg of enoxaparin in elderly patients, especially females, seems to produce higher than expected peak anti-Xa activity. This can result from a decrease in renal function and an altered volume of distribution. As a follow-up to this evaluation we developed a weight-adjusted dosing method for enoxaparin in our elderly patients. We realize anti-Xa acitvity is only one of several pharmacologic properties of enoxaprin. PURPOSE: Self-reports of chronic diseases and general measures of health status predict use of health services but it is not established how well they predict mortality or hospitalization. We developed the Seattle Index of Co-morbidity (SIC) using self-reported chronic illnesses and tested its ability to predict mortality and hospitalizations among general outpatients. METHODS: Using data from the Ambulatory Care Quality Improvement Project (ACQUIP), we conducted a prospective cohort study of patients from GIM clinics at 7 VA medical centers.
Our primary outcomes were all-cause mortality and first hospitalization. Of the 34,103 subjects who were eligible for ACQUIP, 12,388 subjects returned both a health inventory and the SF-36 at entry to the study and were eligible for this analysis. The health inventory asked whether a provider had told patients if they had any of 25 common chronic illnesses. We used Coxmodeling to estimate the hazard ratio for mortality and for first hospitalization. Because patients below 50 years of age violated the proportional hazards assumptions, the analysis was restricted to the 10,947 patients over 50 years of age. These patients were followed for a mean of 722.5 (plus or minus 84.3) days. RESULTS: Using a derivation set of 5,469 patients, the SIC was constructed using age, smoking status and 7 of 25 self-reported medical conditions that were univariately associated with increased mortality. The SIC was predictive of both mortality and hospitalizations when tested in a validation set of 5,478 patients. Multiple imputation methods were compared with a strategy of enhancing the clinical database by merging it with diagnoses derived from administrative ICD-9 codes in hospital discharge data and day procedure data (Norris et al., 2000) . Logistic regression models predicting death at one year were based on the different missing data strategies applied to the 1995 data and were evaluated using measures of discrimination and goodness of fit. The strategies were further evaluated by examining how well the logistic regression models predicted outcomes in data collected from patients in 1996.
The different methods produced similar logistic regression coefficients. The Cstatistics for the logistic regressions were 0.825, 0.819, 0.815 and 0.802 for the MICE, transcan, norm and data enhancement methods respectively. Decreases in deviance from the null model were 469.4 (MICE), 432.0 (transcan), 412.3 (enhanced data) and 412.2 (norm). When the logistic regression models were applied to 1996 data, the C-statistics were 0.806, 0.803, 0.803, and 0.800 for the MICE, transcan, enhanced data, and norm models respectively. Decreases in deviance from the null model were 236. 0, 229.5, 226.5, and 225 .5 for the MICE, transcan, norm, and enhanced data models. CONCLUSION: The performance of the data enhancement and multiple imputation strategies, although not identical, was generally similar. Multiple imputation methods require considerable statistical expertise, while data enhancement requires availability of data resources.
Researchers should therefore base their choice of methods on their team's expertise and data resources, rather than on performance considerations alone. Univariate and multiple-linear regression analysis showed that the level of fatigue after one month were significantly improved for the group who received iron in comparison with the placebo group (p = 0.0049). This improvement was independent of the depression or anxiety scores. Interestingly, among the women in the iron group, the best predictor of response (in a regression analysis) was the amount of iron consumed and not the serum ferritin at baseline (p= 0.007). After the first month , the patients who received iron because of a low level of ferritin were greatly improved at 3 months in comparison with no treatment at all (p = 0.0005). CONCLUSION: Iron supplementation even in the absence of anemia may be beneficial to women complaining of unexplained fatigue. PURPOSE: Some previous studies have found that depressive symptoms predict coronary artery disease. This association has been less strong and not as well studied in women, especially older women. Our aim was to analyze prospective data from a cohort of older women to determine if depressive symptoms predict myocardial ischemia or infarction. METHODS: The Fracture Intervention Trial (FIT) was a multicenter randomized control trial of alendronate vs. placebo among 6459 women with low bone mass 55-80 years of age. At baseline, each participant was examined and completed an extensive questionnaire that included the 16 item Geriatric Depression Scale (GDS). The 140 women with a previous myocardial infarction (MI) were excluded from this analysis. Over a mean follow-up of 4.25 years, 77 women had fatal or non-fatal MIs, and an additional 102 had ischemic events/unstable angina. Medical records, discharge summaries, and death certificates were collected from each event, and were blindly adjudicated by 3 cardiologists. Relative hazard models, adjusted for potential confounders, were used to model the association between GDS scores and documented cardiovascular events. Results are reported as relative risk and 95% confidence intervals. RESULTS: Mean GDS (SD) was 1.52.0, and 168 (5%) reported 5 or more depressive symptoms. In age-adjusted analyses, a GDS score of 5 or more was associated with a trend towards increased MI and acute myocardial ischemia. After adjustment for age, self-reported health, BMI, smoking, alcohol consumption, exercise, hypertension, stroke, diabetes, and use of estrogen, however, there was no longer an association between depressive symptoms and subsequent ischemia or MI (Table 1) . CONCLUSION: In this cohort of older women, there was no significant association between depressive symptoms and subsequent myocardial ischemia and/or infarction when the data was adjusted for potential confounders.
Relative Risk (95% CI) of Cardiac Events in Women with GDS score of 5 or greater. Johns Hopkins University, Baltimore, MD PURPOSE: While poverty and minority status have been associated with poor health and health care outcomes, research suggests that social networks may improve health status. However, the relationship between the degree of social integration into a community and heath care seeking behaviors is unclear. The objective of the study was to evaluate the relationship between social integration and positive health care seeking behaviors in a low-income African-American community.
METHODS: This secondary cross-sectional analysis used data from a door-to-door survey of 2,196 residents in a low-income, urban, and African-American community. The measures of social integration included 1) frequency of church attendance and 2) duration of residence in that community. Health care seeking measures were 1) Pap smear, 2) mammogram and, 3) dental visit all within 2 years, and 4) blood pressure measurement within 1 year, 5) having a regular source of care, and 6) not seeking care when needed. In multivariate analyses, we controlled for socioeconomic factors (age, gender, education, employment, marital status, and health insurance) and number of chronic diseases. Interactions were examined between measures of social integration and other covariates. RESULTS: The study population had a mean age of 44 years, 37% male, 63% completed high school, 21% married, 74% had health insurance, and 25% had > =2 chronic diseases. Eight hundred and eighteen (37%) went to church at least once a month and 1116 (51%) resided in that community for 10 or more years. In multivariate analyses, regular church attendance increased dental visits (OR heath care seeking behaviors is unclear. The objective of the study was to evaluate the relationship between social integration and positive health care seeking behaviors in a lowincome African-American community. METHODS: This secondary cross-sectional analysis used data from a door-to-door survey of 2,196 residents in a low-income, urban, and African-American community. The measures of social integration included 1) frequency of church attendance and 2) duration of residence in that community. Health care seeking measures were 1) Pap smear, 2) mammogram and, 3) dental visit all within 2 years, and 4) blood pressure measurement within 1 year, 5) having a regular source of care, and 6) not seeking care when needed. In multivariate analyses, we controlled for socioeconomic factors (age, gender, education, employment, marital status, and health insurance) and number of chronic diseases. Interactions were examined between measures of social integration and other covariates. RESULTS: The study population had a mean age of 44 years, 37% male, 63% completed high school, 21% married, 74% had health insurance, and 25% had > = 2 chronic diseases. Eight hundred and eighteen (37%) went to church at least once a month and 1116 (51%) resided in that community for 10 or more years. In multivariate analyses, regular church attendance increased dental visits (OR PURPOSE: Airline passengers are particularly vulnerable to the effects of cardiac arrest due to a lack of access to emergency medical services. To offset this relative isolation, airlines are installing automated external defibrillators (AEDs) on commercial aircraft. Our objective was to measure the cost-effectiveness of airline AED programs and estimate their value to the flying public. METHODS: A decision analytic model was constructed to estimate the clinical and economic effects of airline AEDs. Inputs were obtained from published data and the FAA. Utility estimates were derived from cardiac arrest survivors. The medical event rate was .0004 per flight in the base case; 15% of medical events were cardiac arrest. Of these cardiac arrests, 48% were ventricular fibrillation or tachycardia. Cardiac arrest survival with an AED on-board was 40%, compared to 8% without AED access. The AED cost (including training) was $2.51 per flight. Sensitivity analyses evaluated changes in AED cost, probability of cardiac arrest (and arrest survival), and the likelihood of airplane diversion. Since AEDs may provide utility gains through`p eace of mind'' for passengers not experiencing a medical event, the impact of this added passenger confidence was also evaluated. RESULTS: AEDs on commercial aircraft cost an incremental $5.16 per flight (approximately $.05 per passenger). AED deployment resulted in an estimated additional $162,000 per QALY gained (16 quality-adjusted minutes per flight). Sensitivity analysis of event probabilities and cost inputs did not substantially change the results. However, the cost effectiveness of AEDs was significantly enhanced by the inclusion of utility gain experienced by passengers from increased peace of mind. While the magnitude of this benefit is unknown, an incremental increase of .003 in utility over the flight duration would reduce the incremental cost-effectiveness of AEDs to less than $50,000 per QALY gained. CONCLUSION: Our model estimated that when the benefits of on-board AEDs are limited to patients experiencing a medical event, the incremental cost-effectiveness is inferior to most recommended medical interventions. However, if passengers gain utility from knowing an AED is on the aircraft, then these incremental expenditures may be justified. Utility gains from``peace of mind'' may have significant implications in determining the value of health care interventions. Thus, further research should be conducted into this potentially important area.
A CHF OUTPATIENT POPULATION. B. Fenster 1 ; 1 Stanford University, Stanford, CA PURPOSE: Angiotensin Coverting Enzyme (ACE) inhibitors have been well established as first-line therapy in a wide spectrum of congestive heart failure (CHF) patients (1, 2) . ACE inhibition has been proven to ameliorate symptoms, decrease hospitalizations, and decrease mortality. In patients in whom ACE inhibitors cannot be used due to contraindications, intolerances, or allergies, angiotensin receptor blockers (ARB's) or isosorbide dinitrate and hydralazine, particularly in combination, have been shown to provide similar therapeutic benefits (3, 4) . Although the use of ACE inhibitors in treatment of CHF has become standard of care, previous studies have demonstrated wide variations in utilization patterns. Furthermore, few studies have directly assessed ACE inhibitor utilization among CHF outpatients (5,6). Finally, little is known about the usage of ACE inhibitor-alternatives in the setting of ACE inhibitor intolerance. The purpose of this study is to determine the utilization patterns of ACE inhibitors relative to established ACE inhibitor-alternatives in an outpatient CHF population. METHODS: The Palo Alto Medical Foundation (PAMF) is a large multi-specialty clinic located in Palo Alto, California. The PAMF CHF Registry consists of a database listing demographic and clinical data for 229 PAMF outpatients who currently carry the diagnosis of CHF. A retrospective electronic medical record audit of the PAMF CHF Registry population was performed to assess current utilization and dose administration of ACE inhibitors. Patients not receiving ACE inhibitors were assessed for ACE inhibitor-alternative usage, specifically ARB's, hydralazine, or isosorbide dinitrate. Reasons for ACE intolerance, contraindications, or allergies were also recorded. RESULTS: Review of all 229 patients in the PAMF CHF registry revealed that 87% of patients were receiving with either ACE inhibitors or established ACE inhibitor-alternative therapy. Sixty seven percent of all patients were receiving ACE inhibitor therapy, and 68% of these patients were treated with doses considered to be within the target range. An additional 20% of the population was being treated with either ARB's or hydralazine and/or isosorbide dinitrate. Many patients (13%) who were treated with ACE inhibitors experienced allergies or intolerances. CONCLUSION: The overwhelming majority of PAMF CHF outpatients are receiving either ACE inhibitor or ACE inhibitor-alternative therapy. In addition, the majority of patients receiving ACE inhibitors are receiving doses within target range. Although current practice patterns are better than predicted, a small but significant population exists that may benefit from ACE inhibitor initiation and/or more aggressive titration of existing ACE inhibitors. However, such efforts may be limited by a significant number of ACE inhibitor intolerances, allergies, and contraindications. Patients with alcohol problems are common in primary care and there may be advantages to treating these patients in this setting. However, there are few systematic reviews that focus on the efficacy of treatment for alcohol problems in primary care. Our goal was to evaluate the literature on treatment of alcohol problems in primary care according to alcohol consumption outcomes and methodological standards. METHODS: We searched the published literature for years 1966 ± 1999 to identify studies on treatment of alcohol problems in primary care. We included studies from English language peer reviewed journals that were (1) performed in a primary care setting; (2) had as the main focus the treatment of alcohol problems; (3) had two or more treatment arms; and (4) reported alcohol consumption outcomes. Two reviewers appraised all articles for methodological content and results according to pre-specified coding criteria. RESULTS: We identified eleven studies on treatment of alcohol problems in primary care. Seven described treatments for at risk drinking, while four described treatment for alcohol abuse and dependence. The studies were performed in a variety of primary care settings and all reported on the efficacy of brief interventions (BI). There was marked variability in these brief interventions with 4 (36%) using a single session, 6 (55%) using a drinking diary and 5 (45%) discussing a treatment plan. BI were effective in reducing alcohol consumption compared to control treatments in only three of the eleven studies (27%). This finding was only demonstrated in studies of at risk drinking. The studies inconsistently adhered to methodological standards with only 3 (27%) using a single step recruitment procedure and reporting the participation rate, none (0%) providing a full description of patient demographics and medical/psychiatric comorbidity, 1 (9%) assessing prognostic susceptibility, 10 (91%) performing randomization, 1 (9%) assessing ancillary treatments, 0 (0%) assessing discrimination between the treatments, and 5 (45%) providing follow-up on all enrolled subjects. CONCLUSION: This systematic review, restricted to studies performed in primary care found that, in contrast to expert recommendations, there is contradictory evidence on the efficacy of brief interventions for at risk drinkers in primary care. Based upon evidence from the medical literature, patients in primary care with alcohol abuse or dependence are unlikely to decrease their alcohol consumption as a result of brief interventions alone. Finally, studies of brief interventions in primary care inconsistently adhere to methodological standards.
REPLACEMENT AMONG AFRICAN-AMERICANS WITH OSTEOARTHRITIS? M.K. Figaro 1 , P. Williams-Russo 1 ; 1 Cornell University, New York, NY PURPOSE: Total Knee Replacement (TKR) improves mobility, decreases pain, and improves quality of life for patients with severe knee osteoarthritis (OA). Despite the higher prevalence and higher reported disability of knee OA among Blacks, whites are over 1.5 times more likely to have TKR than Blacks. This study examined beliefs of Harlem Blacks with OA regarding TKR, including its efficacy and safety. METHODS: Community-dwelling older adults were recruited from churches, senior centers or other social networks in Harlem. In-person interviews elicited familiarity with others in the community who had TKR, perceived knowledge of TKR, referral to specialists, concerns and beliefs regarding surgery. We also assessed demographic data and severity of OA using the Western Ontario and McMaster Osteoarthritis Index (WOMAC). RESULTS: Women made up 84% of the sample of 65 patients. The mean age was 73, with a mean duration of symptoms of 9 years; 61% had at least a high school education and 45% had an income < $10,000 in 1999; 99% of subjects had Medicare insurance. Subjects had a mean WOMAC score of 56 +18, indicating high levels of OA disability; 45% had been referred to specialist. Table 1 summarizes major factors and concerns.
CONCLUSION: The most important of subjects' concern were regarding post-surgical pain, low expectations of the eventual efficacy of surgery, and concern over the cost of TKR. In addition, despite the high WOMAC disability score of the group, less than 50% of patients were referred to a specialist. Efforts to address the current disparity in use of TKR should attend to these potentially modifiable factors. We conducted a randomized effectiveness trial in the General Internal Medicine clinics at 7 Veteran Affairs facilities that had discrete firms. All patients who were assigned a primary provider and who had an appointment in the prior year were eligible. Randomization, intervention and analysis were by firm. Respondents to a baseline health inventory were regularly mailed the SF-36 and, as relevant, validated questionnaires about 6 chronic conditions: coronary disease, COPD, depression, diabetes, alcohol use and hypertension. From the SF-36, we computed physical and mental component scales (PCS and MCS). We also sent surveys about satisfaction with humanistic and organizational aspects of care and retrieved clinical data from computerized records. The information collected was presented to primary care providers of eligible patients at every visit for 2 years in graphical format (e.g., blood pressure, HbA1C, SF-36 scale scores and anginal frequency) plus interpretations (e.g.,``Patient unable to climb stairs due to shortness of breath'') and``tips'' derived from national guidelines. Clinicians also received summary reports comparing their patient panels with local and national norms and highlighting patients with potentially serious problems. ). There were also no significant differences in satisfaction in the humanistic or organizational domains between intervention and control firms. Similar results were found using several analytic strategies including restriction to patients who completed all forms. CONCLUSION: Routine collection and feedback of measures of general and conditionspecific health and satisfaction did not improve outcomes. It is likely that such data must be linked to specific management suggestions if they are to influence patients' outcomes. in several studies appears to be an independent risk factor for developing incident coronary artery disease (CAD). The biological mechanism explaining the increased risk is not certain. We wanted to determine if C-reactive protein (CRP), a marker of increased risk for CAD, is associated with major depression. METHODS: Our analysis is based on data from the third National Health and Nutrition Examination survey conducted between 1988-1994. The Diagnostic Interview Survey (DIS) was administered to 8451 individuals between 17 ± 39 years of age to assess current and past episodes of major depression. The analysis is based on 7091 individuals (3849 females and 3242 males) with complete data on CRP and other important covariates. Analysis was stratified by gender because of the influence of estrogens on CRP levels. Logistic models were used with detectable CRP as the dependent variable. Unadjusted and adjusted estimates for current (last year) and past major depression compared to never lifetime depression were calculated. Estimates are adjusted for sampling design and nonresponse. RESULTS: Men with current major depression (OR=3.29 95% CI 1.56, 6.92) and past major depression (OR=2.05 95% CI 0.84, 5.04) had elevated levels of detectable CRP after adjustment for age, African-American race, BMI, total cholesterol, log triglycerides, diabetes, systolic blood pressure, tobacco smoking (self-reported current or past or cotinine level above 10 ppm), and alcohol intake compared to never depression. A similar trend was found for single episode of depression (OR=1.13 95% CI 0.26, 4.90) and multiple episodes of depression (OR=3.87 95% 1.67, 8.96) compared to never depression after adjustment. There was a significant linear trend between number of lifetime depressive symptoms and elevated CRP (p < 0.001). The relationship between depression and elevated CRP was present for smokers, nonsmokers, and those with low or high total cholesterol. For women, there were no associations between any of the measures of depression and elevated CRP. Adjusting for current use of estrogens or ever use of oral contraceptive agents and excluding those who were pregnant did not substantially change the results. CONCLUSION: In young men, current major depression is associated with elevated CRP levels that appear to return toward normal when the depression remits. CRP may be one mechanism by which depression leads to increased coronary heart disease in men. The same associations were not present in women but we may not have been able to adequately account for their hormonal status. This cross-sectional data needs to be confirmed with prospective studies. PURPOSE: Understanding why people drink alcohol is important for the health and safety of individuals and the public. The aim of this study was to examine from a cognitive point of view the hypothesized link between drinking and stress. METHODS: 25 scenarios were constructed by combining two items, either two life-change events or a social situation and an emotional state. In the initial three experiments, 159 male and 43 female alcoholics and 157 male and 93 female non-alcoholics in France judged the degree to which these scenarios were stressful and subsequently the degree to which they stimulated an urge to drink. In the final experiment, 126 of the male alcoholics were studied at the beginning and end of an in-patient alcohol rehabilitation program. RESULTS: The alcoholics and non-alcoholics, regardless of gender, assigned similar stress values to the scenarios and used the same cognitive rules for combining the stress associated with two items (disjunctive rules for two life-change events and additive ones for a personal emotion combined with a social situation). They differed, however, in how they judged the urge to drink. The non-alcoholics reported little stimulus to drink from any combination of items, whereas the alcoholics not only perceived the individual items as stimulating an urge to drink, but also used the same cognitive rule in judging the combined urge to drink of two items as they used in judging the combined stress. After completing rehabilitation, the alcoholics judged the combinations of life-change events as stimulating less stress and less urge to drink; nevertheless, they continued to use a disjunctive combination rule. CONCLUSION: Stress and drinking are linked at a fundamental cognitive level among alcoholics, though not among non-alcoholics. Alcoholics should be helped to recognize this link, to reduce their feelings of stress, and to respond to stress in ways other than drinking. documented. Yet, widely used systems of monitoring quality such as the NCQA's HEDIS measures do not require plans to report results separately by patient sociodemographic group. This AHRQ sponsored project is investigating the need for and feasibility of plans reporting selected HEDIS measures stratified by key social groupings. Our initial analysis examines plans' performance on advising smokers to quit, stratified by social group. METHODS: We obtained HEDIS 2000 data for 3,663 commercially-insured and 2,824 Medicaid-insured members enrolled in 7 UnitedHealthcare plans located in 3 regions. As specified by the NCQA, the Consumer Assessment of Health Plans Survey (CAHPS 2.0) was used to collect information on whether the enrollee was a current or recent smoker (denominator) and advised to quit smoking (numerator). We obtained information on patients' sociodemographic characteristics, visits, and health from the survey. Response rates were 55% and 38% for commercial and Medicaid patients, respectively. We analyzed rates of advising smokers to quit separately for commercially insured and Medicaid patients as is customary with HEDIS measures. For comparisons between patients in different sociodemographic groups, we pooled data from the different plans. We used Chi-Square tests to compare the proportion of smokers advised to quit by gender (female vs. male), race/ethnicity [nonHispanic White vs. (nonWhites and Hispanics)], and education (high school or less vs. some college or more). Pending multivariate analyses will assess independent effects of these factors adjusting for age, number of visits, and self-reported health. RESULTS: Of the respondents, 693 commercially insured and 973 Medicaid patients were smokers. The average rate of advising smokers to quit was the same for commercial (66%) and Medicaid (66%) patients. Among commercially insured patients, men were less likely to be advised to quit than women (61% vs. 70%, P=.01) and a lower proportion of less educated patients were advised to quit than among more educated patients (70% vs. 64%, P=.10), however, the latter difference was not significant. In contrast, among Medicaid patients, nonHispanic Whites were more likely than other patients to be advised to quit (69% vs. 61%, P=.01). The proportion of commercially insured patients advised to quit was also higher for nonHispanic Whites than for others (67% vs. 60%), but the difference was not significant (P=.32 The study population consisted of 1394 diabetics with a mean age of 69.2 (+/À 7.2 SD), 98.4% male, and 1.6% female. The old-old diabetic population (n=360) comprised 13% of the Louisville VAMC patient population 75 years old and older in 1997 (equivalent to the national rate of diabetes in persons aged 75 and older (13.2%)). The mean cholesterol was 200 mg/dL (+/À 42.8 SD) and the mean LDL was 120 mg/dL (+/À34.3 SD). The oldest-old were least likely to have their cholesterol checked (p= < 0.01). They were also least likely to be placed on cholesterol-lowering medications when their LDL Cholesterol was greater than or equal to 160 mg/dL. However, in 1998 and 1999, the oldest-old were more likely to be treated with medication than the middle old. CONCLUSION: The oldest old are less likely to have their cholesterol checked, but once it is checked, they are more likely than the middle old to be treated. Since diabetes and cardiovascular disease are common, cardiovascular disease is more common in the elderly, and morbidity and mortality from cardiovascular disease is great, physicians need to attempt to reduce morbidity and mortality by adhering to the practice guidelines for cholesterol management Ð even in the oldest diabetics. Practice patterns demonstrated in this study suggest increasing use of cholesterol-reducing medications in older adults. . As indicated by a c-statistic significantly greater than 0.5, the two existing classification schemes predicted stroke better than chance: 0.78 for the scheme developed by the Atrial Fibrillation Investigators (AFI) and 0.82 for the Stroke Prevention in Atrial Fibrillation (SPAF) III scheme. However, the new CHADS2 index performed better with a c-statistic of 0.87. The stroke rate per 100 patient-years without antithrombotic therapy increased by a factor of 1.5 (95% CI, 1.3 ± 1.7) for each 1-point rise in the CHADS2 score (Table) . CONCLUSION: The two existing classification schemes and especially a new stroke-risk index, CHADS2, can quantify the risk of stroke for patients who have AF. Use of the CHADS2 index should aid in the selection of antithrombotic therapy for patients who have non-rheumatic AF. PURPOSE: Medication errors (MEs) and adverse drug events (ADEs) are common in the hospital setting. However, relatively little is known about the frequency of MEs and ADEs in outpatients. Therefore, we prospectively examined the frequency, type, severity, and preventability of MEs and ADEs in the ambulatory setting. METHODS: We studied 2 academic hospital practices and 2 community based clinics in the Boston area, and collected copies of prescriptions written by 24 primary care providers from Sept 1999 to March 2000 (6 weeks per clinic). Prescription copies were reviewed by a pharmacist to screen for MEs and potential ADEs. In addition, patients who received prescriptions were telephoned 2 weeks after their visit to ask about problems with their medications, with a response rate of 59%. Patient-reported problems were presented to 2 MD reviewers, who classified them as ADEs or not, and determined severity and preventability. Events were considered preventable if they persisted for an unnecessarily prolonged period (even if they could not have been completely prevented). RESULTS: Of 1173 prescriptions screened during this time period, 202 (17%) were rule violations (orders that violate strict standards but are generally understood and generate no additional work), 44 (4%) were MEs, 59 (5%) were potential ADEs. The most frequent rule violations were missing route, and the most frequent MEs were errors in route or dose. The most frequent potential ADEs were errors in frequency and dose. Of 661 patients surveyed, 178 (27%) reported a total of 206 ADEs. Of these, 29 (14%) were serious, 74 (36%) were preventable, and 13 (6%) were both. The most frequent types of ADEs and preventable ADEs were CNS-related (35%, 36%), gastrointestinal (21%, 9%), and cardiovascular (16%, 18%). The most frequent medications involved in preventable ADEs were antidepressants (22%), ACE-inhibitors (14%), beta-blockers (10%), and calcium-channel blockers (10%). Of preventable ADEs (n = 74), physicians were primarily responsible in 63% and patients in 33%. The main types of physician error were failure to act on results of monitoring or tests (50%) and inappropriate drug choice (24%). The main type of patient error was failure to inform their physician of problems (100% Unfortunately, educational brochures are often generalized, and do not address the different needs and barriers that impair the self-management required for care of this chronic disease. Few materials are available that cater to individuals of different ethnic backgrounds, especially those with low literacy or education backgrounds. This study evaluated a new multimedia software application for diabetic populations with these needs in mind. The target population of subjects included diabetic patients from an urban, minority community not accustomed to computer usage. METHODS: 22 diabetic African-American patients were consecutively interviewed and digitally videotaped in an urban general medicine clinic. These individuals provided testimonials regarding the barriers, fears, benefits, and myths surrounding the ophthalmologic eye examination including pupil dilation. Specific issues, such as faith, lack of insurance, and misconceptions about eye exams were addressed. The video clips were edited and integrated into a multimedia application that included simple activities to engage the user over 15 ± 20 minutes. The application was available near the waiting room of a clinic for patients to view when waiting for an appointment, or afterwards before leaving. The program uses a touch screen, so previous technological experience was not required. A separate sample of 26 patients viewed the multimedia program, and completed a brief questionnaire. RESULTS: 11 male and 15 female diabetic patients completed the multimedia program and survey, with 17 (65%) African-American, and 18 (69%) having elementary or high school education. 12 of 26 (46%) have never used a computer before, and 18 (69%) described the computer as``easy'' or``very easy'' to use. Preferences for medium of learning about diabetes were greatest for computers (11), followed by doctors and nurses (8), television (5), and brochures and articles (4) . CONCLUSION: A multimedia patient education program that addressed barriers related to literacy and culture was found to be acceptable by a convenience sample. Additional telephone surveys will provide data regarding knowledge and attitudes toward obtaining annual eye examinations, as well as actual compliance with visits. Given the growing interest in on-line consumer health information, the availability of similar applications for underserved populations may help to bridge the``digital divide'' (or, the gap between different communities in terms of access to technology). PURPOSE: While gender selection of physicians has been studied extensively, mostly by survey, selection of physicians by race has rarely been addressed. We used an experimental design and an actor-portrayed video doctor to investigate patients' preferences for physician gender and race. METHODS: Participants were asked which one of six video doctors (one male and female African American, Latino, and Caucasian) they would pick to be their physician after viewing each deliver a 45-second health prevention message. The videotapes were professionally produced and great care was taken to keep all variables constant, including the age, attractiveness, and clothing of the video doctors; the video setting; and the delivery of the prevention message (script, non-verbal behaviors, facial expressions, and interpersonal style Ð e.g., warm, empathic, collaborative). Each group of 20 ± 22 participants viewed one of 18 configurations of the presentation order of the six video doctors to control for any order effect. RESULTS: 400 participants were recruited in a shopping mall in the San Francisco Bay Area. The sample was diverse in gender (39% male, 61% female); race (29% Caucasian, 8% African American, 26% Latino, 30% Asian, and 7% Other); and age (18 years ± 87 years). When asked who they would choose to be their physician, both male and female participants were more likely to choose a female (82%) as their physician rather than a male (18%). Among female participants, 88% chose a physician of their own gender, while only 29% of males chose a male physician. When asked who they would choose to be their physician, 23% of participants chose an African-American physician, 32% chose a Latino physician, and 44% chose a Caucasian physician. Fifty-two percent of African-American participants chose an African-American physician; 44% of Latino participants chose a Latino physician; and 56% of Caucasian participants chose a Caucasian physician. Among Asian participants, 20% chose an African-American physician, 37% a Latino physician, and 43% a Caucasian physician. CONCLUSION: The vast majority of participants in our study chose a female video doctor as their physician, regardless of their own gender. Although approximately half of African-American, Latino, and Caucasian participants chose their own race, the other half were willing to choose a video doctor of a different race. Our data suggests that when selecting a physician, patients may strongly associate women, regardless of a standardized patient-centered delivery, with those qualities they want in their physician. Future studies may want to use video doctor technology to further investigate patients' preferences in selecting their physician, in particular the effect of physician race on patient choice. PURPOSE: Adolescent obesity continues as public health epidemic. Data from the National Health and Nutrition Examination Surveys (NHANES I, II, III) reveal that the percentage of youth above the 85th percentile for body mass index (BMI) increased to approximately 22% by the late 1980's. Further, since the beginning of these surveys, the prevalence of adolescent obesity has increased by 39%, with Hispanic, Native American, and African American children more affected than other populations. A 1997 survey conducted by the Allentown (PA) Health Bureau revealed a 31% obesity rate among adolescent students in the Allentown School District (ASD), where 57% of students are non-white and 62% are eligible for free or reduced lunches. Both decreased physical activity and poor eating behaviors contribute to obesity. General internists have a responsibility but limited opportunities to understand and influence teen nutrition, especially in minority populations. METHODS: To better understand students' attitudes toward nutrition and physical activity, as well as barriers to improving their choices, we conducted focus groups among representative students to determine their food preferences, and dietary and activity patterns. We developed a 57-question survey that we utilized in six focus group sessions with high school students in an alternative program within the ASD. Sessions were audiotaped, and responses to questions categorized into four areas: Food choices, Physical Activity, Self-Esteem/Identity, and Knowledge. RESULTS: In general, students expressed a willingness to eat healthier foods, if provided a variety including fresh fruits and vegetables, as well as culturally acceptable products. Adolescents desired daily gym class with a variety of activities, although admitted that much of their free time was spent engaged in sedentary activities. Groups also expressed indifference, although many had basic knowledge of the consequences of obesity.
CONCLUSION: Providing greater choices and opportunities for food and physical activity can promote healthier behaviors among adolescents, impacting upon the prevalence of obesity in this population. In particular, minority students may have different needs than others. General internists can have a large impact on adolescent obesity and the subsequent development of related diseases by working together with community leaders and other health care professionals in advocating policies to provide adolescents with these opportunities. PURPOSE: While some studies have suggested that there may be sex differences in access to cardiac procedures, others have found no evidence of`gender bias' in cardiac care. Possible explanations for these inconsistent results include the focus, in some studies, on nonrepresentative patient populations, and the occasional use of data sources that lack clinical detail. We used a clinically-detailed cardiac database to study crude and adjusted rates of cardiac revascularization for males and females in a population-based cohort of patients undergoing cardiac catheterization. METHODS: The Alberta Provincial Project for Outcome Assessment in Coronary Heart Disease (APPROACH) is an inception cohort of all patients undergoing cardiac catheterization in the province of Alberta, Canada. We studied 21,816 patients undergoing catheterization in calendar years 1995 through 1998, and determined rates of percutaneous coronary intervention (PCI) and/or coronary artery bypass grafting (CABG) for males and females in the year following cardiac catheterization. Cox proportional hazards models were then used in a two-step analysis to determine adjusted relative risks for revascularization and adjusted procedure rates. An initial`partial adjustment'; controlled for variables such as age and comorbidities that are routinely available in most databases, including administrative databases. A subsequent`full adjustment' additionally controlled for extent of coronary artery disease and ejection fraction, variables that are only available in detailed clinical databases. RESULTS: For the endpoint of any revascularization procedure (PCI or CABG), the unadjusted relative risk for revascularization for females relative to males was 0.67 ( 1, 1995, and December 31, 1998 . Cases were linked via postal codes to Canadian census data to determine population rates of cardiac catheterization by census-defined geographic regions. Individuals were flagged as being of aboriginal ethnicity when they resided in regions where greater than 95% of 1996 census respondents indicated that they were of aboriginal ethnicity. Such regions generally represent native reservations. Using the Alberta-wide population as a reference, we employed indirect standardization to adjust rates of cardiac catheterization to sequentially control for 1) age and sex, 2) income quintile, and 3) urban vs. rural residence. We also evaluated survival and the occurrence of revascularization procedures after catheterization (i.e., coronary artery bypass grafting [CABG] The prevalence of type II diabetes is much higher among Mexican Americans than among non-Hispanic Whites. Although much of this difference is thought to be genetic, environmental factors such as diet and exercise may also play a role. One way to try to separate genetic from environmental factors is to use acculturation data in immigrant populations. Prior studies on the effects of acculturation on the prevalence of diabetes in Mexican Americans have reported conflicting results. A study of Mexican Americans in San Antonio, Texas showed a decrease in diabetes rate with increasing acculturation. This decrease was hypothesized to be secondary to improved diet and increased levels of exercise in the more acculturated group. The Hispanic Health and Nutrition Examination Survey (HHANES) studied Mexican Americans in the Southwestern United States and found no effect of acculturation on rate of diabetes in Mexican Americans. METHODS: All patients seen in the adult clinic of a large community health center in Denver, Colorado over a fifteen month period were included in our analysis. For each patient seen, diabetes was considered present if the ICD9 code for diabetes was billed as the primary diagnosis on at least one visit during the 15 months. Level of acculturation was assessed by using language preference. Hispanic patients who spoke primarily Spanish were considered less acculturated than those who spoke English. Demographic and language preference data were obtained from registration records. RESULTS: Approximately 5,300 patients were included in our analysis. Of these, 1,500 patients were non-Hispanic, 2,600 were English-speaking Hispanics, and 1,200 were Hispanic who spoke primarily Spanish. By our current diagnostic criteria, overall diabetes prevalence was 14%. Hispanics had a higher prevalence of diabetes than non-Hispanics at all age groups and this difference was more pronounced with increasing age. In the 55 ± 64 yo age group, 31% of Hispanics vs. 20% of non-Hispanics had diabetes. There was no significant difference in diabetes prevalence between primarily Spanish-speaking and English-speaking Hispanics. CONCLUSION: We found no significant effect of acculturation on diabetes prevalence in this clinic population of Mexican Americans. Our findings also support previous reports of increased diabetes prevalence among Mexican Americans when compared to non-Hispanics, and a magnification of this effect in the higher age groups.
T. Gill 1 , C. Williams 1 ; 1 Yale University School of Medicine, New Haven, CT PURPOSE: When determining the incidence of disability, long assessment intervals maybe problematic because they do not account for the possibility of recovery not for losses to followup due to deaths. In this study, we tested two related hypotheses: (1) longitudinal studies with long assessment intervals underestimate the incidence of disability; and (2) these underestimates increase as the length of the assessment interval increases.
Project, an ongoing longitudinal study of nondisabled, community-living persons, aged 70 years or older. After a comprehensive, home-based assessment, participants were categorized into three groups according to their risk for disability (low, intermediate, and high) and were subsequently followed for up to two years with monthly telephone interviews (98% completion rate) to ascertain the presence of disability in four key activities of daily living (ADLs).We compared the rate of ADL disability obtained from a single follow-up assessment with that obtained from monthly assessments for intervals of 6, 12, 18, and 24 months. RESULTS: For any ADL and for each of the four individual ADLs (walking, bathing, dressing, and transferring), the rates of disability at 12 months were at least twice as high for the monthly assessments as for the single follow-up assessment. For any ADL disability, for example, the corresponding rates were 0.22 and 0.10. Although the overall rates were lower, the results for persistent disability, defined as a new disability that was present for at least two consecutive months, were similar. As the length of the assessment interval increased, the``true'' rate of disability, as determined by the monthly assessments, was increasingly underestimated by a single follow-up assessment. For example, the rates of any ADL disability for the monthly and single follow-up assessments were 0.28 and 0.09 at 18 months and 0.37 and 0.11 at 24 months, respectively. While these underestimates of disability were due almost exclusively to high recovery rates for low-risk participants, they were due increasingly to losses to follow-up from deaths for high-risk participants. CONCLUSION: The incidence of disability is substantially underestimated by longitudinal studies with long assessment intervals. These underestimates could lead policy-makers to inaccurately estimate the health care needs of community-living older persons. While adolescents in general face many serious health risks, these youth face worse and more chronic health problems, psychiatric and behavioral problems, alcohol and substance abuse, sexually transmitted diseases (STD) and teen pregnancies. School-based clinics have been reported to improve health among adolescents through increased access. We hypothesized that increased access to medical care within the shelter environment would improve health outcomes in this under served population. METHODS: Homeless youth were surveyed in 1992 and 1998 at the only Denver-area shelter dedicated to youth under 21. The first survey, in 1992, occurred when medical services were provided in the shelter 8 hours per week. At the time of the second survey in 1998, medical services had increased to 36 hours per week. The surveys were of structured interview form based on the Adolescent Health Survey instrument. Outcomes assessed included access to health care, health satisfaction, STD's and pregnancies. RESULTS: The two study samples differed somewhat with regard to demographics. In 1998, there were more males (62%) than in 1992 (55%). There were also substantially more African-Americans (16% vs. 7%). The proportion of African Americans among shelter users was twice the proportion of African Americans in the community.`Good' or`Excellent'; self-reported health increased from 65% to74% between 1992 and 1998. Endorsement of frequent difficulty accessing care decreased from 3 1% to 14% between 1992 and 1998. Agreement with the statement,``the last time I needed healthcare I took care of it myself'' decreased from 24% in 1992 to 8% in 1998. The number of self-reported STD's decreased from 26% in 1992 to 17% in 1998, compared with the 2.4% reported in Colorado high school students in 1996. This decrease was seen at the same time a Centers for Disease Control-sponsored STD screening project was working to reach homeless youth, and detection rates may actually have increased. The number of teen pregnancies decreased from 14% in 1992 to 6% in 1998. CONCLUSION: Among homeless youth, increased access to medical care through increased availability of services in the shelter environment was associated with improvements in perceived health, access to health care, and reductions in some common health problems. Continued expansion of services within the shelter could have a profound impact on problems such as mental health and substance abuse.
PURPOSE: Concussion has been associated with disturbance of physical well-being, thought process, and emotions, that may last 6 months or longer after the time of injury. Homeless adolescents are at higher risk for brain injury than adolescents from safer environments due to increased behaviors leading to injury. Impulsivity and affective liability are part of the DSM-IV definition of Postconcussional Disorder, These attributes have also been noted to be more common in homeless youth. We hypothesized an association between previous head injury and impulsivity and behavioral dyscontrol, as possible symptoms of postconcussional disorder. METHODS: Homeless adolescents accessing services at a shelter were surveyed about health histories, including the statement``Have you ever been hit on the head so hard you lost consciousness''. In the same survey a psychologic symptom battery, the SCL-90, was administered, The incidence of head injury was then compared with psychiatric symptom indices in the highest quartile. An odds ratio for the severity of psychologic symptoms in those with history of concussion was then calculated. RESULTS: Of the 97 youth interviewed, 38 endorsed a history of grade III concussion, generating a prevalence of brain injury which is 40% higher than that reported in one study of high school football players. Odds ratios for psychiatric symptom score in the 75h percentile or greater among those with history of grade III concussion were as follows:
Odds Ration (95% Ci) Hostility 3.5(1.5 ± 8.4) Phobia 21(0.8 ± 5.2) Impulsivity 2.0(0.6 ± 7.2) Anxiety 1.5(0.6 ± 5.4) Obsessive/Compulsive 1.5(0.6 ± 3.7) Depression 1.3(0.5 ± 3.4)
CONCLUSION: The symptoms of hostility, impulsivity, anxiety, and depression are included in the DSM-IV definition of Postconcussional Disorder. Impulsivity, anxiety, and depression were modestly, but not significantly, increased in those with history of grade III concussion. A significant association between hostility and concussion was found. As with other studies of brain injury in adolescents, the direction of the relationship between hostility and brain injury is unclear. However, the effect brain injury may have on the skills necessary to exit street life may be serious, and should be further investigated.
PURPOSE: Echinacea is one of the most popular dietary supplements with over $10 million in annual sales in the U.S. However, since dietary supplements are not regulated by the FDA in the same manner as other over-the-counter products, there is concern as to whether contents are consistent with the labeling. Three species of Echinacea are sold commercially, augustifolia (EA), pallida (EPA), and purpurea(EPU). Each species has a different biochemical footprint, analogous to different contents. Clinical trials have reported decreases in cold symptoms using EPU, decreases in cold symptoms and duration using EPA, and no effect on cold symptoms using EA. It follows that labeling of species content on Echinacea supplements is important consumer information. We hypothesized that species content in Echinacea preparations may not be consistent with labeling. METHODS: Ten single-herb liquid-only preparations of Echinacea from 5 Denver area stores were purchased and sent to a blinded independent laboratory. The preparations were analyzed by thin layer chromatography (TLC) for fingerprints unique to each species of Echinacea. Of the 10 samples sent, 3 were not analyzed due to difficulties with glycerol in the preparation. RESULTS: The following RESULTS: Of the 182 persons interviewed, 120 (66%) eventually enrolled into the program. Enrollees in the work component were middle aged (mean age = 39), minority race (92%), and male sex (64%) and abusing primarily alcohol (31%), crack/cocaine (21%), heroin (5%) alone or poly-substance abuse (44%). They lived in emergency shelters (23%), doubled-up (49%), and bridge-housing (25%) arrangements. Self identified co-morbid medical and psychiatric illness were common and a third were on at least one prescribed medication. Fifty-seven (48% of enrolled) completed the work component of the program. Of these, 95% were subsequently employed at completion of the work component. During the work component, clients were abstinent (40%), improved their housing status (47%), attended medical appointments (87%), reported career skill improvement (69%), and arrest free (84%). Six months after the completion of the program, over 33% continued to be employed full-time, 39% had further improved their housing, and 36% remained abstinent of substance use. A vast majority reported having medical (90%) and psychiatric (93%) service needs met. CONCLUSION: A work stabilization program, supported by a collaborative network of community and academic partners, decreased substance use, criminal behavior, and health care utilization for a drug and alcohol abusing homeless population. Community and academic partnerships can be a powerful impetus to promote health and social improvement for this distressed population. The majority of depressed patients initially present in primary care with physical symptoms rather than psychological complaints. We examined the outcome of physical symptoms with antidepressant treatment of depression during a 9-month period. METHODS: This clinical trial enrolled 573 depressed patients cared for by 40 physicians in two primary care research networks. Patients were randomized to receive one of three selective serotonin reuptake inhibitor (SSRI) antidepressants: paroxetine, fluoxetine, or sertraline. Data was collected from participants via computer-administered telephone interviews both at baseline and after 1, 3, 6, and 9 months of treatment. This data included validated measures for depression, anxiety, and 14 common physical symptoms (SCL-20 and PRIME-MD), healthrelated quality of life (SF-36), work functioning (Work Limitation Questionnaire), and Medical Outcomes Study measures for social functioning, positive well-being, sleep, concentration, and sexual functioning. The prevalence of each physical symptom was assessed at baseline and all follow-up intervals and symptom severity graded from 0 (none)to 2 (bothered a lot). Stepwise linear regression models were used to determine the independent effects of physical symptoms and depression on HRQoL and other domains at baseline and over 9 months of treatment. All models were adjusted for demographics and anxiety scores. RESULTS: Most of the individual symptoms had a baseline prevalence in depressed patients of 50% or greater. Moreover, the prevalence of symptoms that were graded as``bothered a lot'' exceeded 10% for 11 of the 14 symptoms. Physical symptom prevalence and severity dropped substantially during the first 4 weeks of antidepressant therapy with minimal improvement thereafter. While also showing the greatest improvement during the first month, depression and all other outcomes continued to improve over the 9-month trial. Physical symptoms had the strongest association with physical functioning (11% ± 20% of the variance), pain (23%), and overall health perceptions (19%), whereas depression had the greatest association with mental (32% ± 49%), social (11% ± 36%), and work functioning (9% ± 37%). CONCLUSION: Many physical symptoms are present in at least half of depressed patients presenting in primary care. The predominant benefits of antidepressant therapy occur during the first 4 weeks of treatment for physical symptoms, while depression and other HRQoL domains continue to improve for some months thereafter. Physical symptoms and depression have diffential effects on HRQoL domains. METHODS: We surveyed a stratified random probability sample representative of managed care members aged 45 and older in 2 health plan locations (n=2168). Respondents reporting doctor-diagnosed arthritis or chronic joint symptoms (pain, stiffness or swelling of a joint within the past year that is present on at least half the days of a typical month) were considered to have arthritis. Weighted descriptive analyses were conducted to estimate the age-and gender-specific arthritis prevalence, and to describe the impact of arthritis (i.e., health status, activity limitations). We also describe treatments respondents reported using for arthritis and the type of physician who provided the arthritis diagnoses. RESULTS: The prevalence of arthritis ranged from 32.0% for the 45 ± 54 age group to 36.8% for the !75 age group. Women were more likely to report arthritis than men (38.3% vs. 29.9%). Compared to those without arthritis, respondents with arthritis were more than twice as likely to report fair or poor health (24.9% vs. 11.1%). Of those with arthritis, 39.9% indicated activity limitations related to their joint symptoms. Respondents reported using prescription medicines (43.9%), over-the-counter pain medications (57.2%), complementary therapies (i.e., herbs, glucosamine) (32.8%), physical or occupational therapy (20.2%), and exercise (i.e., walking, swimming) (70.5%) to treat their arthritis symptoms. Just over half of those reporting physician-diagnosed arthritis received their diagnosis from a primary care physician. CONCLUSION: One-third of these managed care members have self-reported arthritis and 40% report arthritis-related activity limitations. The prevalence and impact of arthritis on health-related quality of life suggest an opportunity to explore interventions, including education on self-help activities, that may complement care provided by clinicians. None have explored these issues in a multi-culturally diverse population of young women. The purpose of this exploratory study was to assess knowledge, beliefs and behaviors about osteoporosis prevention in this target population. METHODS: We performed a cross-sectional study of multi-culturally diverse college women attending classes over a two week period in 2000. In this convenient sample (N=50), we collected data using a survey instrument of 20 questions designed to measure knowledge, beliefs and behaviors about osteoporosis prevention. Incorporating the selfreported data, we characterized the respondents by demographic information, medical conditions and family history; calculated Body Mass Index, general consumption of calcium, their level of alcohol and nicotine use and degree of physical activity; and tabulated à`B elief-about-Disease'' Score to capture their perception of osteoporosis in relation to other medical conditions. RESULTS: Nearly a third of the respondents were non-White and 14% were of Asian origin. Respondents were able to identify the risk factors of low calcium intake(96%), lack of exercise(86%), and menstrual irregularities(68%). However, other risk factors such as White race(20%), Asian race(21%), and excessive exercise (42%)were less likely to be identified. Ten percent had a medical condition putting them at risk. There was no relationship between correctly identifying lack of exercise and low calcium intake as risk factors and getting adequate osteoprotective exercise ( > 360 minutes/month)(p = 0.53) and dietary calcium( > 1200mg/daily) (p = 0.85). Osteoporosis (3.31) (on Likert scale from 1 ± 5, 1 indicates not at all; 5, extremely)was believed to be significantly less serious than heart disease(3.60) * , breast cancer(3.66) * , AIDS(3.79) * , and Alzheimer's disease(3.54) * ( * =p < 0.05). There were no associations between ethnicity and respondents' responses, knowledge and beliefs. CONCLUSIONS: Many college women, regardless of race, lacked knowledge about the relationship between osteoporosis and low calcium intake, use of tobacco, alcohol, physical inactivity, and excessive exercise. Increasing levels of osteoporosis knowledge was not associated with young women's beliefs and behavior.Our data suggest a need to educate young women. Comprehensive osteoporosis educational programs during college should be evaluated as an intervention to improve osteoporosis prevention. Recently published studies suggest that automated external defibrillators (AEDs) on commercial aircraft may save the lives of passengers who have out-of-hospital cardiac arrest (OHCA). However, AED equipment and training costs are high, while cardiac arrests onboard aircraft remain rare. We conducted a cost-effectiveness analysis to explore the economic and health impact of AED deployment in the U.S. passenger air fleet. METHODS: We developed a decision analytic model using a societal perspective for costs and benefits. Estimates of the incidence of OHCA onboard aircraft, effectiveness of AEDs in resuscitation, subsequent hospital survival rate, and annual mortality after surviving OHCA were derived from the medical literature. Equipment, maintenance, and flight attendant training costs were obtained from AED manufacturers, the Federal Aviation Administration, and the Air Transportation Association. Hospitalization and downstream medical costs were abstracted from published cost analyses. Published estimates of healthrelated utility after cardiac arrest were used to express effectiveness in terms of qualityadjusted life years (QALYs) gained. We compared four strategies: 1) AEDs on all aircraft with basic life support (BLS) training for flight attendants; 2) AEDs on wide-body aircraft only; 3) AEDs on no aircraft±but attendants trained in BLS; and 4) no aircraft AEDs and no BLS training. RESULTS: Placing AEDs on all passenger aircraft with concurrent BLS training would cost $49,800 per QALY gained compared to no AEDs and no BLS training. BLS training without AEDs was dominated by a combination of strategies #1 and #4. Deploying AEDs exclusively on wide-body aircraft would be cost-effective only if total training costs could be limited to less than 49% of the cost of training all attendants. Sensitivity analyses indicated that the ability of AEDs to improve survival was the most important influence on cost-effectiveness. AEDs must demonstrate an incremental survival rate of at least 14% to have a cost-effectiveness ratio less than $50,000/QALY. A Monte Carlo analysis, which varied all assumptions simultaneously, indicated that the cost/QALY value for strategy #1 would be less than $50,000 at a probability of 34%, while the probability of the cost-effectiveness being greater than $100,000/QALY was only 0.1%. CONCLUSION: The use of AEDs on passenger aircraft is similar in cost-effectiveness to many widely accepted medical interventions and health policy regulations. Sensitivity analysis suggests this result is robust, even when underlying assumptions are varied widely. This study implies that requiring the placement of AEDs on U.S. commercial aircraft would be a cost-effective use of resources. PURPOSE: Pressures to shorten length of stay increase the chance that patients (Pts) may be discharged`sicker and quicker.' Pts hospitalized with hip fracture are very frail and maybe at particularly high risk. We sought to measure prevalence of active clinical issues (ACIs)on discharge (DC) in Pts hospitalized with hip fracture and their associated clinical and functional outcomes. METHODS: Information on vital signs, eating status, mentation, mobility, incontinence, and wound status, and other active problems on DC, and in the 24 and 48 hours prior to DC was collected on 559 Pts hospitalized with hip fracture as part of a 4 hospital prospective cohort study. Deaths, readmission, and functional mobility within 60 days of DC were ascertained by telephone interviews and querying the NY State hospitalization database. Mobility was measured with the validated Functional Independence Measure-Locomotion scale (FIM). Logistic and linear regression assessed associations between the number of ACIs on DC and post-DC outcomes. ACIs on DC were defined as any of the following in the 24 hrs prior to DC (Temp > 100, 100 > HR > 60, SBP < 90, DBP < 60, RR > 24, O2 sat < 90%, altered mentation, inability to eat, incontinence, and not mobilized beyond a chair (all different from baseline), and other active medical problems [dyspnea, chest pain, arrhythmias, CHF, or wound infection]). We used a validated risk adjustment model to control for covariates known to influence clinical and functional hip fracture outcomes. RESULTS: Pts mean age was 81 yrs, 82% were female, 22% had dementia, and 12% came from a nursing home. The mean length of stay was 8.56.2 days. Overall, 74% of Pts had!1 ACI on DC (29% had 1, 28% had 2, and 17% had!3 ACIs), most commonly not mobilized beyond a chair (37%), new urinary incontinence (34%), Temp > 100 (11%), abnormal mentation (5%), DBP < 60 (5%), and new bowel incontinence (3%). Within 60 days of DC, 3.8% of Pts died, 18.8% were readmitted, and 20.2% died or were readmitted (major events). The mean FIM locomotion score at Day 60 was 6.1 4.0 (scale from 2 ± 14; higher is better). The greater the number of ACIs on discharge, the higher the risk of post-DC death, readmission, and major events and worse functional mobility [FIM score] (p < .001 for all). The odds of major events increased 40% for every additional ACI on DC (OR=1.4; 95% CI 1.1 ± 1.8). The number of ACIs on DC remained a significant predictor of all post-DC outcomes even after controlling for age, sex, comorbidities, initial APACHE score, and pre-fracture nursing home residence, dementia, home health assistance, and FIM score. Similar results were obtained for outcomes at 30 and 90 days as well as for analyses that considered only the last set of vital signs along with the other ACIs or ACIs in the 48 hours prior to DC. CONCLUSION: The greater the number of ACIs on DC, the worse the post-DC clinical and functional outcomes even after controlling for other important prognostic factors. Clinicians should factor this information into deciding appropriateness for DC and the type and intensity of post-DC care and medical observation. PURPOSE: Elevated HDL is considered a negative risk factor for ischemic heart disease and HDL level > 75mg/dl has been found to be associated with the``longevity syndrome''. We wanted to study people with extreme HDL elevation as no studies have been done to evaluate them before. METHODS: We identified all persons presented to our hospital with HDL level > 95mg/dl in the last 2 years. All subjects were contacted by phone. RESULTS: 102 subjects were contacted (83 females and 19 males). The majority were African Americans (52 subjects) (13 males and 39 females). The mean age was 59.3 + 1.5 years. The mean cholesterol was 234 + 5 mg/dl with a mean HDL of 108 + 2 mg/dl, triglyceride of 85 + 4 mg/dl, and LDL of 112 + 5 mg/dl. The total cholesterol to HDL ratio was 2.4 + 0.2. Mean Body Mass Index (BMI) was 25.8 + 0.6 kg/m2. (table) Only 15% were younger than 50 years old. We found an unusually high prevalence of hypertension in our subjects (53%) especially in the African American population (71%). Seven patients have coronary artery disease (CAD) but all had other cardiac risk factors. CONCLUSION: Our study showed a predominance of African Americans and postmenopausal females. This population had normal TG, LDL, and BMI, and a high prevalence of hypertension and smoking. Half exercised or reported following a special diet. Although elevated HDL is associated with longevity syndrome, it may not be protective against CAD in people with multiple other risk factors. were advised not to have or were not offered AVR by a cardiologist or cardiothoracic surgeon, and 2 declined further evaluation for AVR. For the remaining patients a decision was made to proceed with AVR or no decision had been documented by 60 days. At the time of the chart review (2 ± 4 years after echocardiogram), 37 patients (39%) had AVR and 13 (14%) had palliative aortic valvuloplasty. Compared with patients who did not have AVR, patients who had AVR were younger (78 vs 84, p < .0001), more likely to be male (46% vs 26%, p=.05), had fewer comorbid illnesses (mean number comorbidities 1.1 vs 1.7, p=.04), and were less likely to need assistance with ADLs (mean number of ADL dependencies 1.1 vs 1.7, p=.04). In a multivariable logistic regression model including age, sex, number of comorbid illnesses, and ADL dependency, these differences persisted, but only the effect of age was statistically significant at p < 0.05. Of the 33 patients who had AVR at one of the study hospitals, 19 also had coronary artery bypass surgery, 2 also had mitral valve repair or replacement, 2 died postoperatively, and 1 suffered a stroke. CONCLUSION: In this cohort of elderly patients with severe, symptomatic AS, the majority did not have AVR surgery. Patients who were younger and who had better baseline health status were more likely to have AVR. Few patients who were selected for AVR died postoperatively or suffered a major surgical complication. PURPOSE: African American women experience higher breast cancer mortality than Caucasian women despite increasing rates of screening mammography. One postulated factor in these worse outcomes is a lower rate of follow-up for diagnostic testing once a breast abnormality is detected. We examined factors associated with inadequate follow-up in a referral group of predominantly low-income minority women. METHODS: The study population consisted of women referred to a Breast Center at an urban medical center from January to June 2000. Demographic information was collected via medical charts, registration files completed at the time of appointment scheduling, and consultation/referral forms. Adequate follow-up was defined as patient arrival to the scheduled Breast Center appointment and inadequate follow-up was defined as failure to arrive on the designated appointment day. We analyzed factors associated with inadequate follow-up such as patient age, race, median household income by zip code, referral source, and insurance type. Maintenance dose was defined as being greater than one month out from initiation of warfarin therapy with at least two consecutive measurements one week apart within the target range. Potential predictor variables included: age, sex, weight, race, indication for anticoagulation, individual medications and total number of medications. A combined approach using recursive partitioning and multivariate logistic regression was used to develop and evaluate potential prediction rules. Two different rules were tested: one looking at low maintenance requirements (less than or equal to 3 mg per day), the other looking at high maintenance requirements (greater than or equal to 7 mg per day). RESULTS: 131 patients meeting entry criteria were identified with an average age of 68 years (range 27 to 93 years), 52% male sex, 70% Caucasian race, 75% anticoagulated for either atrial fibrillation (38%) or deep venous thrombosis/pulmonary embolus (37%), average weight of 184 pounds (range 100 to 350 pounds) and average number of chronic medications (in addition to warfarin) of 6 (range 0 ± 29). The simultaneous presence of 3 of the following variables predicted low maintenance requirements with 80% specificity (95% CI 73 ± 87%), 67% sensitivity (95% CI 59 ± 75%) and a positive predicted value of 92% (95% CI 87 ± 97%): age greater than 50, weight less than 200 pounds and being on 5 or more chronic medications. Although there were factors (age less than 50 years, and weight greater than 200 pounds) that on univariate analysis were associated with high maintenance requirements, a statistically meaningful rule could not be developed. CONCLUSION: A prediction rule that identifies those with the lowest warfarin maintenance requirements has been developed which could potentially aid in determining how to initiate warfarin therapy. This rule needs to be validated and refined using different patient populations. PURPOSE: Our objectives were three-fold: To conduct a pilot study of risk factors and screening behaviors relevant to breast and cervical cancer in rural lesbian women, to assess women's willingness to participate in a study of these factors, and to assess the feasibility of contacting women through regional lesbian organizations. METHODS: Using a brief self-administered questionnaire, we obtained information concerning major risk factors and screening behaviors relevant to breast and cervical cancer. The questionnaire, targeted to rural lesbian who were at least 40 years of age, was distributed at lesbian community events in rural New Hampshire and Vermont between June and August 1997. A separate survey, assessing willingness to participate in a future study, was distributed (between October and December 1998) by regional lesbian organizations to their membership. RESULTS: The first questionnaire was completed by 105 women. Of these, 82% reported at least one physician visit during the previous year. With regard to breast cancer risk factors and screening, 66% were nulliparous, 66% had at least one clinical breast exam during the previous year, 43% had had a mammogram, and 40% conducted breast self-exams. With regard to cervical cancer risk factors and screening, 80% had a history of heterosexual activity, and 62% had undergone a Pap smear during the previous year. Over the past 3 years, 21% had an abnormal pap smear. The later survey assessing willingness to participate in breast and cervical cancer research was mailed to 250 women; 47 surveys were undeliverable due to unknown address. Of the remaining 203 (62%), 108 women (53%) expressed willingness to participate. CONCLUSION: This pilot study suggests that breast cancer risk may be higher among lesbian women than non-lesbian women, and that lesbian women are at risk of cervical cancer despite of their current sexual practices. Our preliminary data also suggest that rural lesbian women are medically underserved with regard to breast and cervical cancer screening. The mailed survey also supports the feasibility of recruiting large numbers of rural lesbian women for health research.
PREVALENCE AND TREATMENT OF MENOPAUSAL SYMPTOMS AMONG BREAST CANCER SURVIVORS. P.F. Harris 1 , P.L. Remington 1 , A. Trentham-Dietz 1 , C. Allen 1 , P.A. Newcomb 1 ; 1 University of Wisconsin-Madison, Madison, WI BACKGROUND: Women diagnosed with breast cancer often experience early menopause secondary to treatment effects, yet physicians may be reluctant to prescribe hormone replacement therapy (HRT) in order to reduce their risk of cancer recurrence. The objective then is to assess the burden of menopausal symptoms, HRT use, and alternative treatments in recent breast cancer survivors compared to age-matched controls. METHODS: This is a population-based case-control study using breast cancer survivors and age-matched controls. Wisconsin women 18 ± 74 years old with a new diagnosis of breast cancer 8 ± 11 months prior to interview (n=110) and control subjects randomly selected from population lists (n=73). A standardized telephone questionnaire was administered to elicit information on menopausal symptoms, estrogen and alternative (prescription medications, vitamins, herbal preparations, soy products, acupuncture, chiropractic) therapies used to alleviate symptoms.
Multivariate logistic regression was used to obtain odds ratios between cases and controls. Main outcomes were symptoms of menopause, use of estrogen, and use of alternative therapies. RESULTS: Breast cancer survivors were 5.4 (95% CI 2.9 ± 10.4) times more likely to experience symptoms, 25 (95% CI 8.3 ± 100) times less likely to use estrogen, and 7.4 (95% CI 2.5 ± 21.9) times more likely to use alternatives than controls. Soy, vitamin E, and herbal remedies were the most common alternative therapies reported by participants; use was greater in cases compared to controls. Among cases, tamoxifen users (n=62) reported a higher prevalence of symptoms and a higher prevalence of alternative treatments. Most soy users reported increasing soy products specifically to reduce the chances of a diagnosis of recurrent breast cancer. CONCLUSION: This is the first population-based survey of menopausal symptoms and treatments that compares breast cancer cases with disease-free controls. Prevalence of menopausal symptoms and use of alternative menopausal therapies were higher in breast cancer survivors than in controls; the rate of estrogen use was much lower. The increased use of soy products in this population has not been previously documented. (2) Test the effect of a personalized letter directed to women who have not had a recent mammogram Ð reticent beneficiaries. (3) Assess the cost-effectiveness of the intervention. METHODS: HCFA data for Medicare beneficiaries were used to identify women living in Michigan continuously from 1993 ± 98, age !65 in 1993, with no obvious comorbidity affecting screening, and no mammogram for !5 years (1993 ± 97). A randomized design included paired intervention and control women matched on residential zipcode and race. The study sample of 2,458 women had 1,229 pairs of either African American (AA) or non-AA women and either urban or rural women. The intervention used principles from the Health Belief Model in a personally addressed letter from the Medical Director of Michigan Medicare noting the individual's lack of use of the mammography screening benefit, with additional breast cancer related information enclosed. Letters were sent in Nov. 1997 with Medicare mammography claims followed through 1998. RESULTS: All women were age !70, with a mean of 79 in both control and intervention groups. Overall, 5.2% of controls and 8.1% of the intervention group subsequently had mammograms, + 2.9%, OR 1.6 (p < .005). The findings were similar by urban/rural and AA/ non-AA subgroups. Rates and the effect were higher among the women age 70 ± 79: 6.5% of control and 10.6% of intervention, + 4.1%, OR 1.6 (p < .02). Projecting to the Medicare population in Michigan, a total cost of $117,000 to $278,000 for a state wide intervention would produce 3,700 to 5,000 mammograms at $32 to $54 per additional mammogram in this reticent group. CONCLUSION: This approach for measurement and intervention is feasible, effective, and can be directly implemented in other states and nationally. Targeting a reticent group of older women, this intervention demonstrated a significant improvement in subsequent mammography. This targeted approach is likely to be more cost effective than``blanket'' community-based approaches with significantly higher overall cost but the same target group of reticent older women. Future research can address variations to increase the communication's effectiveness (e.g., multiple communications). The approach to measurement and intervention can be tried for other preventive services in the Medicare or other defined populations. However, few data are available about how often patients do not know or misunderstand the reasons for their medications. We studied a group of general medicine outpatients to assess patient understanding of their medications. METHODS: Patients seen in four Boston general medicine practices from Sept 1999 to March 2000 (6 weeks per clinic) and who received at least one prescription from a study physician (6 MDs per clinic) were eligible for the study. Approximately two weeks after the index visit, patients were telephoned (response rate 59%) and asked to retrieve all of their pill bottles, read the name of each medication, and state their reason for taking it. Answers were coded``know'' or`d on't know,'' and the reasons were recorded verbatim. The accuracy of``know'' responses was coded by a physician-reviewer on a 5-point scale.
RESULTS: Of 598 patients, 58 (9.7%) did not know or gave definitely inaccurate indications for at least one medication. The drugs most often associated with inaccurate indications were blood pressure and lipid-lowering agents. An example of an inaccurate response was`a torvastatin is for my blood pressure.'' Education and younger age were directly related to knowledge (see Table) . There was a trend toward association of non-white race and male gender with poorer medication knowledge, but they were not statistically significant. Patients on more than one medication were far less likely to know the indication of all medications (p < .001). CONCLUSION: In this relatively well-educated population, knowledge about drug indications was high. However, older and less educated patients, as well as patients on multiple medications, were much less likely to know why they were taking all of their medications. Such patients may benefit from intensive education programs to enhance understanding of their medications, which could improve adherence. PURPOSE: Adherence to evidence-based practice guidelines is frequently suboptimal. We wished to determine whether an opinion leader quality improvement project would increase adherence to the Unstable Angina (UA) AHCPR guidelines compared to a traditional HCFA quality improvement model. METHODS: We designed a three-armed randomized controlled trial of 22 acute care hospitals in one state. The intervention arms were: 1) hospital-specific data-feedback of performance on quality indicators combined with a physician opinion leader-driven improvement intervention (OL); 2) hospital-specific data-driven feedback with traditional HCFA quality improvement model efforts (HCFA); and 3) no intervention (NI). We convened a national panel of experts to translate selected elements from the AHCPR UA guidelines into quality measures. We selected the following indicators: 1) ASA within 24 hours of admission, 2) ASA at discharge, 3) heparin during hospitalization, 4) beta-blockers at discharge, and 5) EKG within 20 minutes of presentation. For chart abstraction, we identified potential cases of UA using a stratified random sampling scheme based on ICD-9 codes from Medicare claims data. A computerized algorithm was developed to confirm UA cases from the abstracted data. Centrally trained abstractors reviewed the complete medical records. We used GLM to adjust for patients-nested within hospitals. RESULTS: Charts were abstracted for 2,516 patients; their average age was 72 11 years, of whom 45% were male and 85% were white. Average baseline performance for the indicators was 71.6 (ASA in 24hrs), 69. PURPOSE: Men with clinically localized prostate cancer have several treatment options including conservative management (C), radiation therapy (RT), or radical prostatectomy (RP). We used data from the population-based Prostate Cancer Outcomes Study (PCOS) to evaluate patient satisfaction with these treatments. METHODS: The PCOS evaluated 3,830 incident cases of prostate cancer reported in 6 regional Surveillance, Epidemiology, and End Results (SEER) programs between October 1, 1994 and October 31, 1995 . The current analysis was restricted to the 2,387 subjects with clinically localized prostate cancer who completed a 24-month post-diagnosis follow-up questionnaire. Weighted multivariate logistic regression analysis was used to determine factors associated with treatment satisfaction. Independent variables included demographics, socioeconomic status, comorbidities, tumor characteristics, additional cancer treatments, perception of being free of cancer, scales measuring self-reported bowel, urinary, and sexual function, and perception of problems with these three functions. RESULTS: Treatment selections were 431 C, 583 RT, and 1,373 RP. The cohort was 74% non-Hispanic white, 13% African American, and 13% Hispanic; 41% were 65 years and younger. Perception of being free of cancer was reported by 17% (C), 87% (RT), and 94% (RP), even though 16% (RT) and 14% (RP) required additional androgen deprivation therapy. Moderate/big sexual problems were reported by 33% (C), 44% (RT), 53% (RP); moderate/big urinary problems were reported by 4% (C), 3% (RT), 11% (RP); and moderate/big bowel problems were reported by 6% (C), 7% (RT), 3% (RP). Men undergoing radiation therapy were more likely to be delighted/pleased with their treatment choice (70%) than those undergoing either conservative management (55%) or surgery (59%), P < 0.0001. The following factors were independently associated with treatment satisfaction for conservative management: receiving androgen deprivation therapy (OR = 2.0; 95% CI 1. CONCLUSION: The majority of men with localized prostate cancer were very satisfied with their treatment decision. Sexual problems were common with all treatments but significantly decreased satisfaction for only RT and RP. RP most frequently led to urinary problems and this significantly decreased satisfaction. Androgen deprivation therapy increased satisfaction for C but decreased satisfaction for RT. For all treatments, the perception of being free from cancer was highly associated with satisfaction.
PACIFIC ISLANDER CANCER CONTROL NETWORK. This study aims to assess frequency, content and impact of weight and diet counseling provided by primary care physicians to such patients. METHODS: We conducted an observational study on clinical prevention in 2 university-based primary care clinics staffed by 35 residents. We included 893 consecutive patients of whom 396 (44%) were overweight or obese (body mass index > 25 kg/m2). We interviewed patients after the index visit to assess counseling performed by residents during the three last visits. At 1 year, we mailed a questionnaire to assess patients' self-reported behavior change and weight loss (60% responders). We compared outcomes according to the quality of physician counseling (4 ± 6 vs. 1 ± 3 strategies used). RESULTS: Physicians infrequently advised overweight and obese patients about weight and diet: informing of health risks (40%); recommending to lose weight (40%); assessing motivation to lose weight (34%). They rarely used strategies facilitating behavior change: planning specific dietary changes (24%), setting a target weight (14%) and written material (14% 5) ). We calculated descriptive statistics using the Chi-square test and used generalized linear models to determine the independent effect of adverse computing habits on UEMD symptoms and functional impairment. RESULTS: Our sample included 46% women and 38% racial/ethnic minorities. 29% reported never having UE pain related to computer use, 30% had experienced pain in the past, 36% had pain in the past and current pain (in preceding 2 weeks), and 5% only had current pain. We defined``intensive computer users'' as students who typically computed for > 4h/day or spent !4h computing without getting out of the chair at least once a month. 66% were intensive computer users. Women were more intensive users (p = 0.05), reported more functional impairment due to UEMDs (p = 0.013), and had more upper arm (p = 0.07), shoulder (p = 0.001), and neck pain (p < 0.0001) than men. Worse mental health scores were associated with increased reported UE pain (p = 0.01) and functional impairment (p < 0.0001). Controlling for sex, mental health, and overall health, intensive computer use independently predicted higher UEMD symptom (Type III SS p=0.03) and pain severity scores (p = 0.01). CONCLUSION: Most college students in this sample engage in intensive computer use and have UE pain with computing. We found a significant independent association between intensive use and UEMD symptomatology. Our results suggest that programs to foster healthy computing skills may prevent long-term disability from UEMDs as students enter the work force. with attitudes (r = À0.15, P = 0.000 and r = À.0.12, P = 0.004, respectively) at the bivariate level. When included in the final model, the relationship between ethnicity and attitudes toward joint replacement was no longer significant; adjusted OR 0.66 (95% CI 0.38 ± 1.14).
Responses on familiarity with JR did not mediate the relationship between ethnicity and attitudes toward JR. CONCLUSION: Differences in attitudes toward JR between AA and white patients is mediated by post-surgical concerns about pain and disability. If confirmed, our findings suggest an opportunity for generalists to address patient-level factors that contribute to racial disparity in the utilization of joint replacement. PURPOSE: Although racial and gender differences in mortality have been reported for systolic heart failure, little is known regarding diastolic heart failure prognosis; even though diastolic heart failure is the most common type of heart failure in the US. METHODS: Our sample consisted of 1058 patients 65 years of age or older who were admitted to 30 hospitals in Northeastern Ohio with a principal diagnosis of heart failure and had documented diastolic dysfunction by Echo. Diastolic heart failure in this cohort was defined as`h aving a principal diagnosis of heart failure and a LVEF of Ê 50% by Echo.'' Mortality data for all patients were obtained from Ohio MEDPRO files for Medicare beneficiaries. Logistic regression was used to compare 18-month mortality by ethnicity and by gender, adjusting for age, gender and comorbidities. RESULTS: Of the 1058 patients with documented diastolic heart failure (13% AA and 87% white), AAs and whites were comparable with respect to history of angina, stroke, being on dialysis, alcohol use, and the proportion of males. AAs were more likely to have hypertension (50% vs 36%; P = 0.001), diabetes (46% vs 29%; P = 0.000), history of tobacco use (27% vs 18%; P = 0.011), and high serum creatinine (1.99 2 vs 1.50 1; P = 0.003); they were also younger (76 7 vs 79 8; P = 0.000). Whites were more likely to have a history of ischemic heart disease (48% vs 32%; P = 0.000), metastatic cancer (3% vs 0%; P = 0.034), DNR status on record (14% vs 7%; P = 0.013), and atrial fibrillation (24% vs 14%; P = 0.002). The AA to white adjusted OR for 18-month mortality was 1.03 (0.66 ± 1.59). For men vs women (30% vs 70%), the above-mentioned comorbidities were comparable, except women were more likely to have DNR status (16% vs 7.3%; P = 0.000) and to be older (79.5 8 vs 77 7; P = 0.000). Males were more likely to have a history of tobacco use (30% vs 14%; P = 0.000), alcohol use (36% vs 15%; P = 0.000), and higher serum creatinine level (1.7 1.2 vs 1.4 1.1; P = 0.001). Men to women adjusted OR for 18-month mortality 1.06 (0.76 ± 1.46). CONCLUSION: In this cohort of elderly patients admitted with diastolic heart failure, there were no ethnic or gender differences in 18-month mortality.
ALTERNATIVE MEDICINE USE AMONG MEDICAL OUTPATIENTS. M. Herman 1 , L. Inouye 1 ; 1 Naval Medical Center Portsmouth, Portsmouth, VA PURPOSE: The use of complementary and/or alternative medicine (CAM) is increasing in popularity among patients in the United States. Hundreds of herbal products and homeopathic remedies are readily available to the consumer, but most of these have not been proven safe or effective. Potential side effects and interactions with traditional medications are often unknown. Studies suggest that between 30% and 50% of the adult population in industrialized nations use some form of CAM to prevent or treat a variety of health-related problems. The purpose of our study was to evaluate CAM use by Internal Medicine. The analysis would assess the proportion of patients in the clinic who are using alternative medicines, provide a description of the most common alternative medicines being used, and evaluate characteristics associated with alternative medicine use. METHODS: A convenience survey was completed by patients of the Internal Medicine clinic at Naval Medical Center, Portsmouth (NMCP) between 6DEC99 and 7JAN00. Information was gathered on the patients gender, age, race, medical problems and medications, educational background, military status, herbal and non-herbal usage to include confidence of efficacy and safety, and the source of information for herbal and non-herbal products. Chi-squared analysis was used to compare CAM use differences in gender and educational groups. RESULTS: There were 212 surveys completed. Of those responding, 15% were active duty or a dependent of an active duty member, and 60% were female. The mean age of respondents was 53 years. Complementary therapies had been used in 60% of the patients. The most common herbal products were garlic, ginseng, gingko biloba, and St. Johns wort. The most common non-herbal products were vitamins, chromium, glucosamine, and chondroitin sulfate. Sixty-five percent of females used CAM compared with 53% males, although this difference was not statistically significant (p = 0.125). The average age of CAM users was 53 years. Thirty percent of CAM users had a college degree, 15% were active duty, 35% were retired, and 50% were dependent. Sixty percent of CAM users had been using an alternative medicine for at least one year. Use of herbal CAM was associated with a higher educational background, although the difference was not statistically significant (p = 0.132). However, vitamin use was significantly correlated with a higher educational background. In those subjects with less education than a college degree, only 25% used vitamins. Among college graduates 54% used vitamins (p < 0.0001). Seventy-one percent of respondents felt that complementary therapy was safe and only 17% were very confident of its efficacy. CONCLUSION: CAM use among Internal Medicine patients at NMCP is higher than reported use in studies of civilian populations, which may reflect a difference in patient profile. Of interest is that a significant proportion of people using alternative medicines were not confident of its safety and only a few were very confident of its efficacy. This information may correlate with compliance issues pertaining to physician prescribed medications. While the social profile may reflect those most likely to use CAM, our data demonstrates widespread use of alternative medicine. This information has important implications for health care providers, who must consider safety, efficacy and possible interactions with standard therapies. Further analysis will include the results from over 400 respondents. METHODS: In 1995, 500 primary care patients presenting to a walk-in clinic with physical complaints were screened for mental disorders with the PRIME-MD. Five years later, patient surveys assessed symptom outcome, mental disorders (PRIME-MD), functional status (MOS SF-6) and whether they had been diagnosed or treated for a mental disorder. Vital status was assessed with the Social Security Index. RESULTS: Forty patients (8%) died. Among the remainder, 65% completed surveys, with follow up losses equal between patients with and without a mental disorders. The prevalence of mental disorders declined from 30% at baseline to 12%, though patients with a disorder at baseline were more likely to still have a disorder at 5 years (RR: 3.5, 95% CI: 2.0 ± 6.0). Only 32% of patients with a mental disorder at enrollment were diagnosed during the 5 years of follow up. The rate of diagnosis varied by disorder: 52% of patients with major depression, all patients with panic disorder and 66% of patients with generalized anxiety at baseline were diagnosed. For subthreshold disorders, diagnoses rates were lower: 32% for depression and 5% for anxiety. Patients with unrecognized depression (RR: 5.0, 95% CI: 1.2 ± 21.1) or anxiety (RR: 4.8, 95% CI: 1.1 ± 23.5) were more likely to still have their disorder 5 years later, compared to patients whose disorders were recognized. While most patients with minor depression or subthreshold anxiety (73 ± 83%) had their disorder at only one time point and no patient with subthreshold anxiety progressed to generalized anxiety or to panic disorder, 18% of those with minor depression progressed to major depression by 5 years. Patients whose mental disorder went undiagnosed over 5 years were more likely to have worsening of their initial symptom (RR: 3.6, 95%CI: 1.6 ± 8.4), greater stress (RR: 2.3, 95% CI: 1.9 ± 2.8), greater worry that their symptom represented a serious illness (RR: 2.9, 95% CI: 1.7 ± 4.9), a greater number of``other'' bothersome physical symptoms (p < 0.0001), and worse functional status (p < 0.0001). Outcomes among patients whose mental disorders were diagnosed during the intervening 5 years, were the same as among those without mental disorders. CONCLUSION: Patients with unrecognized major mental disorders were more likely to still be struggling with their disorder 5 years later, had less resolution of their presenting physical symptom and experienced worse health-related outcomes. METHODS: In 1995, 500 adults presenting with a physical complaint completed surveys that assessed symptom characteristics, functional status (MOS SF6) and mental disorders (PRIME-MD). Follow up surveys assessed symptom outcome, functional status and satisfaction at 2 weeks, 3 months and 5 years. RESULTS: Patients averaged 43 years in age (18 ± 92), were 52% female, 45% African American and 49% white. Patients presented with a myriad of complaints that we collapsed to 13 categories. Musculoskeletal symptoms were the most common category, present in a third and half had some sort pain complaint. Twenty ± one percent had experienced their symptom less than 3 days, 40% less than a week, 68% less than a month; 46% had seen a previous doctor for the same problem. Most patients experienced improvement, 70% at 2 weeks, 79% at 3 months and 75% at 5 years. There was no relationship between type of symptom and likelihood of improvement. However, there was good correlation between improvement in symptoms between each of the three time points. (Spearman's rho:0.40 ± 0.43). Among patients whose symptom had improved by 3months, most (83%) were still improved at 5 years. Conversely among those reporting no improvement, 52% were still not improved at 5 years. There was no relationship between likelihood of symptom improvement and age, sex, or baseline functional status. Patients without symptom improvement had worse functional status (p < 0.001), more illness worry (p < 0.001), a greater number of``other'' bothersome symptoms (p = 0.003) and were more likely to have an active mental disorder at 5 years (RR: 1.3, 95% CI:1.02 ± 1.7). Independent predictors of improvement included improvement at 3 months, symptom less than 3 days in duration at presentation and no patient worry that the illness could be potentially serious at presentation. CONCLUSION: Patients with symptoms present for more than 3 days at presentation,who are worried their symptom may be serious or who have not improved by 3 months are likely to still have problems with their symptom at 5 years. The 1994 AHRQ Low Back Pain Clinical Guidelines recommended treatment with tylenol in patients less than 55 years of age with no historical or physical examination findings suggestive of more serious causes. Nonsteroidal use was discouraged. Muscle relaxants, narcotics and routine radiographs were deemed inappropriate. Our purpose was to assess the impact of these guidelines on clinical practice. METHODS: Patients greater than 17 but less than 55 years in age, being seen in primary care settings (FP, IM, GP) with low back pain, without an inflammatory or secondary diagnosis to explain their back pain were abstracted from the National Ambulatory Medical Care Survey. Data from 1991 ± 1993 (prior to release of the 1994 AHRQ), were compared to data from 1995 ± 1997. RESULTS: A total of 11,854,653 encounters met criteria and were included in this analysis. The mean age of patients was 37 years, 57% were male and 34% of the visits were injury related. Prior to the guidelines, 0.33% of patients with back pain were treated with acetaminophen, 39% with a nonsteroidal, 27% with muscle relaxants and 4% with narcotics. Twenty-seven percent were referred for physiotherapy and 16% had an Xray associated with the visit. After the guideline release in 1994, acetaminophen use increased to 3%, muscle relaxant use declined to 17% and referrals to physiotherapy declined to 22%. Nonsteroidal (40%), muscle relaxants (20%), narcotic use (6%) and the rate that Xray's were obtained (19%) were unchanged. CONCLUSION: The AHRQ guidelines increased acetaminophen use, but only to 3%. Physicians were less likely to prescribe a muscle relaxant and were less likely to refer for physical therapy. There was no impact of the guidelines on clinician use of nonsteroidals, narcotics or radiographic evaluation. PURPOSE: Clinicians are often reluctant to recommend alcohol consumption among those with a history of stroke. We therefore sought to examine the relationship between alcohol intake and the risk of total and cardiovascular mortality in men with a previous history of stroke. METHODS: The study population consisted of the 104,353 physicians who comprised the enrollment cohort of the Physicians' Health Study. We focused our analyses on the 1,320 men who indicated a history of stroke at baseline and provided self-reported data on alcohol consumption. Cox proportional hazards was used to model the association between alcohol and mortality after adjustment for major lifestyle and clinical risk factors. Alcohol consumption was classified into four categories, with those reporting rarely/never used as the referent group. RESULTS: During a mean follow-up of 4.6 years, 369 men died, of whom 267 died from cardiovascular disease. Multivariate relative risks of total mortality were decreased in men with a history of stroke who consumed < 1drink per week, 1 ± 6 drinks per week, and at least 1 drink per day, at 0.88(0.60,1.28), 0.64(0.48,0.85) and 0.71(0.54,0.94) respectively (p, linear trend 0.028). Multivariate relative risks of cardiovascular mortality in men with a history of stroke were also decreased in men who consumed < 1 drink per week, 1 ± 6 drinks per week, and at least 1 drink per day, at 0.89(0.58,1.36), 0.56(0.40,0.79), and 0.64(0.46,0.88) respectively (p, linear trend 0.008). Adjustment for lifestyle and other clinical risk factors did not significantly change the risk estimates for total or cardiovascular mortality as compared with age-adjusted models. Because only fifty-five men died as a result of stroke, we were underpowered to examine this association, but the risk estimates were nonsignificantly lower among men consuming at least 1 drink per week.
CONCLUSION: These data suggest that alcohol may reduce the risks of total and cardiovascular mortality in men with a history of stroke. More data are needed to confirm or refute these results. PURPOSE: Historically, it has been suggested that the symptoms associated with benign prostatic hyperplasia can be aggravated by infrequent sex. We evaluated this by assessing the cross-sectional association between frequency of ejaculation and lower urinary tract symptom severity and general self-perceived health among participants in the Olmsted County Study of Urinary Symptoms and Health Status among Men. METHODS: In 1989 and 1990, 2,115 Caucasian men between the ages of 40 and 79 years were recruited from a random sample of Olmsted County residents (55% response rate). These men completed a self-administered questionnaire that assessed lower urinary tract symptom severity with questions similar to those in the American Urological Association Symptom Index (AUASI) and completed a similar questionnaire biennially thereafter through 1998. In 1994, a question was added to assess the frequency of ejaculation during the previous month. This was answered by 81 percent of participants in that round. RESULTS: Overall, men who reported ejaculating at least once a week were less likely to have moderate-severe (AUASI > 7) symptoms than men reporting no ejaculations (Odds ratio=0.62, 95% Confidence Interval=0.51, 0.75). There was a similar association with health-related quality of life (Poor-good vs. very good-excellent, OR=0.45, 95% CI=0.37, 0.60). The association with symptom severity did not exist within age decade, however. The odds ratios for ejaculating at least once a week were 0.93, 0.79, 1.21 and 1.12 for men in their forties, fifties, sixties and seventies, respectively. Thus, after adjusting for age, the odds ratio was 0.95 (0.75, 1.21). The odds ratio for health-related quality of life, however, remained virtually unchanged (OR=0.47, 95% CI=0.37, 0.60). CONCLUSION: While there has been speculation about the adverse effects of abstinence on prostate health, these cross-sectional data suggest that frequency of ejaculation has no effect on lower urinary tract symptoms; the apparent protective association appears to be an artifact caused by the confounding effects of age. This is not the case, however, for the cross-sectional association with quality of life. PURPOSE: Re-hospitalization after in-patient treatment for community-acquired pneumonia (CAP) is common. The aims of this study were to examine reasons and identify predictors of rehospitalization (rehosp). METHODS: This analysis was performed as part of the randomized, controlled trial,`D issemination of Guidelines for Pneumonia Length of Stay,'' implemented in 7 hospitals in Pennsylvania from February 1998 to March 1999. The trial assessed the effect of a guideline intervention to reduce the length of stay (LOS) for patients hospitalized with CAP. Inclusion criteria included a clinical diagnosis of pneumonia and a chest radiograph with a new pulmonary infiltrate. Severity of illness was quantified using the Pneumonia Severity Index (PSI), a predictor of 30-day mortality in patients with CAP. For this study, 2 physicians independently reviewed the records of patients re-hospitalized within 30 days and afterwards reached a consensus on reasons for rehosp. Demographic, clinical, laboratory, and radiological data were used to identify reasons for rehosp. Categorical and continuous variables were assessed using the Chi-square test and the Student's t-test, respectively. Any variable significant at the p < 0.05 level was entered into a multiple logistic regression model. RESULTS: Of the 608 patients (283 in the intervention arm and 325 in the control arm), 69 patients (11%) were re-hospitalized within 30 days. There was no significant difference in readmission rates between the 2 groups. The mean time to rehosp was 8.49 (+/À 0.96) days. The reasons for rehosp were CAP-related (n=14), combination of CAP and comorbid condition (n=3), or comorbid condition alone (n=52). The kappa statistic, assessing inter-rater reliability, was 0.784. The major comorbid conditions requiring rehosp were cardiovascular (n=19), neurologic (n=6), pulmonary (n=6), gastrointestinal (n=5), genitourinary (n=5), and others (n=11). Significant predictors of rehosp included age (p < 0.001), unemployment status (p = 0.04), congestive heart failure (p < 0.001), coronary heart disease (CAD) (p < 0.001), ventricular arrhythmias (p = 0.014), chronic obstructive pulmonary disease (COPD) (p < 0.001), chronic oxygen treatment (p = 0.001), and PSI (p = 0.05). Significant predictors in the multiple logistic regression model included CAD (odds ratio 3.2 with a 95% confidence interval of 1.8 ± 5.5), COPD (2.5, 1.5 ± 4.2), and unemployment status (2.4, 1.1 ± 5.3). CONCLUSION: This study describes the incidence and reasons for re-hospitalization in patients with CAP and identifies subsets of patients that could be targeted for interventions to decrease re-admissions after CAP. The value of hospice lies in its ability to provide continuous quality care until the time of death. However, some patients are discharged from hospice, usually because they no longer meet hospice criteria or choose more aggressive care. We sought to determine differences between patients who are discharged vs. those who die in the hospice setting. METHODS: We identified 48 patients who were discharged from a local hospice organization between the years 1996 and 2000. We then selected 47 control cases, matched on primary diagnosis and month of admission, who died while in the same hospice organization. Data were collected on these patients through restrospective chart review. RESULTS: Of the 95 patients, 66% were female, 87% were Caucasian, 37% were married and 74% had Medicare as the primary payor source. The mean age of admission was 70 years, mean length of stay 50 days, and mean Karnofsky status on admission 30%. The most common primary diagnosis was cancer (38%), followed by neurologic disease (14%) and dementia (14%). At the time of admission, 43% received care at home, 40% in assisted living or a nursing home (AL/NH), and 17% in the hospice facility. At the time of discharge/death, 33% were at home, 41% in AL/NH, and 26% in the hospice facility. The reason for discharge was stabilization of condition in 54%, revocation of hospice benefit in order to pursure more aggressive treatment in 13%, and transfer to different hospice in 11%. Discharge disposition was to a different hospice in 11%, to AL/NH in 48%, and to home in 23%. The discharged patients vs. deaths did not differ in age, sex, race, payor source, site of hospice care, admission weight, level of cognition, severity of pain, level of mobility at admission, number of ADL's performed at admission, or Karnofsky status at admission. Having a primary caregiver was also not significant. Compared to patients who died, those discharged had a longer length of stay (mean 62 and 38 days, p=0.05), were less likely to be married (25% and 50%), and less likely to be bedbound at discharge (23% and 76%, p = 0.000). CONCLUSION: Patients discharged alive from hospice, compared to case controls matched on diagnosis and date of admission who died in hospice, had similar demographics and level of functioning at the time of admission. The presence of a primary caregiver did not alter outcome and being married was associated with death. We speculate that hospice provides additional social contact or improved medical care that prolongs the life of some patients. PURPOSE: Recognizing patients' expectations is considered as an important objective for primary care physicians. However, few studies have investigated the influence on such matter of race and/or ethnicity, specially among immigrants. The aim of the study was to investigate whether patients' expectations among immigrants and natives are different and if physicians can perceive them. METHODS: The design is a two month pre-consultation patient survey and post-consultation physician survey (matched pairs) in an academic primary care center. Subjects were natives (Swiss French-speaking people) and immigrants (ex-Yougoslav, European, French-speaking African and other) consulting without appointment. The main outcome measures were patients' expectations (14-item questionnaire: receiving a prescription, a sickness certification, reassurance, seeing a specialist, etc...), patients' perception of their health status, physicians' perception of their patients' expectations (14-item questionnaire) and agreement between patient and physician. Questionnaires were available in 3 languages (French, Serbo-Croat and Albanian). RESULTS: 343 patient and 333 physician questionnaires were analyzed. Patients were separated into 5 groups: Swiss (n=167), European (n=50), Ex-Yugoslav (n=37), African (n=62) and other (n=27). Immigrants (n=176) do not consult more quickly than native patients and initiate a treatment at home as often (n=ns). However, more immigrants perceive their health as bad (22.7% vs 5.4% p < 0.01) and ask their doctor for reassurance (84.3% vs 62.7% p < 0.01). More Ex-Yugoslav patients expect to be referred to specialists (60% vs 30% p < 0.01) and more African patients wish to receive medication than Swiss patients (83% vs 59% p < 0.01). These results are confirmed by a stepwise logistic regression including independent variables such as age, sex, civil status and education. Finally, we found a poor agreement between patients' expectations and their perception by physicians, regardless of patients' origin (k < 0.36) CONCLUSION: Our study suggests that patients' expectations may differ according to their origin and that physicians certainly have a poor perception of their patients' expectations in an outpatient emergency setting, regardless of patients' origin. However, further investigation is needed to analyze whether the physicians' lack of knowledge of their patients' expectations influences the latter's satisfaction and the quality of medical care. shown that vaginal administration of hormones decreases UTI risk in post-menopausal women with recurrent UTIs, but evidence supporting a similar effect of oral hormones is mixed. We assessed the effects of oral hormone treatment in a randomized trial and evaluated potential risk factors for UTI frequency among post-menopausal women. METHODS: 2,763 postmenopausal women were enrolled in the Heart and Estrogen/progestin Replacement Study (HERS), a randomized, double-blinded, controlled trial to evaluate hormone treatment for the prevention of coronary heart disease events in women with established coronary heart disease. Participants were randomly assigned to daily 0.625 mg of conjugated estrogens plus 2.5 mg of medroxyprogesterone acetate or placebo and followed for a mean of 4.1 years. We analyzed results for 1,318 women randomized to hormone therapy and 1,336 women randomized to placebo who had complete data. Participant demographic information, reproductive history, sexual activity, vaginal symptoms, urinary incontinence symptoms, overall health status and chronic medical illnesses including diabetes were assessed at baseline and each annual visit. Number of UTIs diagnosed by a physician in the previous year was assessed by self-report at each annual visit. A proportional-odds model for repeated ordinal measures was used to test the treatment effect at all annual visits together, and risk factors for UTI were assessed using generalized estimating equation logistic models. RESULTS: UTI frequency tended to be a little higher in the group randomized to hormone treatment, although the difference was not statistically significant [Odds Ratio (OR) 1.2; 95% confidence interval 1.0 ± 1.4]. Among the 116 women who reported multiple UTIs in the previous year at baseline, there was no difference in the odds of UTI for the oral hormone vs. placebo group [OR 1.1 (0.7 ± 1.9)]. The average incidence of UTI at each annual visit was 12.3%. Among women with at least one UTI during a reporting period, 32.2% had two or more UTIs in the previous year. In multivariate analysis, risk factors significantly associated with UTI included diabetes requiring medications [OR 1.6 (1.3 ± 2.0)], urinary urge incontinence symptoms [OR 1.5 (1.3 ± 1.7)], frequent vaginal itching or dryness [OR 1.4 (1.1 ± 1.7)], history of child-birth [OR 1.4 (1.0 ± 1.9)], and self-reported poor health [OR 1.4 (1.2 ± 1.6)]. UTI in the previous year was strongly associated with incident UTI [single UTI: OR 6.9 (5.8 ± 8.1); multiple UTI: OR 18.2 (14.0 ± 23.5)]. After controlling for diabetes status, fasting blood glucose level was not associated with risk of UTI. Sexual intercourse was not a risk factor for UTI. CONCLUSION: Oral hormone therapy did not reduce the frequency of urinary tract infections. Potentially modifiable risk factors to decrease risk of urinary tract infection in postmenopausal women are different from those for younger women, and include diabetes, vaginal symptoms, and urge incontinence. PURPOSE: Thyroid dysfunction increases with age, is more common among women, and among whites is associated with lipid changes. There is limited information on racial differences in thyroid dysfunction and the association with cholesterol abnormalities. METHODS: Health ABC is a cohort of well-functioning whites and blacks, ages 70 ± 79, recruited for a population-based study in 1997. We analyzed thyrotropin (TSH) and serum cholesterol test results for 2,742 participants performed at the second annual visit. Free thyroxine (FT4) levels were performed on those with abnormal TSH results. Medical history and physical measurements were gathered at baseline and the second annual visit. The relationship between TSH and total cholesterol was analyzed with multivariable linear regression adjusting for multiple covariates. RESULTS: 267 (9.7%) participants were taking thyroid hormone medication and 223 (83.5%) of these men and women were biochemically euthyroid. Among those not taking thyroid hormone, 2,364 (95.5%) participants were euthyroid. Subclinical hypothyroidism was the most prevalent disorder among all race/gender groups; white women had a significantly higher prevalence of subclinical hypothyroidism than black women (4.5% vs. 1.6%, p = 0.004). Mean cholesterols were highest for those who were biochemically hypothyroid and lowest among those who were hyperthyroid. After adjustment for age, gender, race, body-mass index, smoking, alcohol use, diabetes, hormone-replacement therapy and thyroid hormone use, a TSH > 7.0 ulU/ml was associated with a 18 mg/dl increase in cholesterol and a TSH < 0.1 ulU/ml, with a 21 mg/dl decrease and did not differ between black and white participants. CONCLUSION: Healthy community-dwelling elderly blacks have a lower prevalence of thyroid dysfunction compared to whites. The association between increased TSH and increased cholesterol is similar in blacks and whites. PURPOSE: Atrial Fibrillation (AF) is the most common sustained arrhythmia, and is the cause of significant morbidity. Risk factors which increase the incidence of AF are: increasing age, ischemic heart disease, valvular heart disease, cardiomyopathy, congestive heart failure and chronic lung disease. AF may also occur in a normal heart. Ibutilide, a class III antiarrhythmic agent, has emerged as an effective intravenous agent for treatment of AF to normal sinus rhythm (NSR). However it is associated with a 4% chance of ventricular arrhythmias. In addition, Ibutilide remains a very expensive medication often requiring a second dose. Oral Propafenone, a class IC agent, has been shown to be an effective treatment for recent-onset AF in patients without structural heart disease. It is an inexpensive medication usually administered as an oral medication in the dose range of 150 to 300 mg. Recently it was shown that a single dose of 600 mg of Propafenone, was successful compared to control, in converting recent-onset AF to NSR. Although the effectiveness of both oral Propafenone and Ibutilide, individually, in converting recent-onset AF to NSR has been well established, the comparative studies of these agents are lacking. The aim of this study is to evaluate in a randomized fashion the efficacy of Intravenous Ibutilide versus a single 600 mg dose of oral Propafenone in treatment of recent-onset AF. METHODS: All eligible patients with recent-onset AF/atrial Flutter (AFL) were identified and placed in the telemetry unit. Recent-onset AF was defined as arrhythmia lasting less than two weeks duration. The onset of AF was established based on symptoms such as palpitation, dizziness, chest pain or electrocardiogram (ECG) finding. If timing of onset of AF/AFL was unclear or greater than 48 hours, before attempting cardioversion, patients underwent a transesophageal echocardiogram (TEE). All patients were started on heparin and their prothrombin time (PTT) was maintained at 1.5 ± 2.0 times control. If patients had a rapid ventricular rate of greater than 100 bpm, AV nodal blocking agents were used to control the ventricular response. Exclusion criteria included: age less than 18 year old; hemodynamically unstable patient; previous myocardial infarction; known coronary artery disease; unstable angina; NYHA functional class greater than 1 heart failure; ventricular rate less than 60 bpm; hypokalemia (less than 3.5 mEq/L); hyperkalemia (greater than 5.5 mEq/L); untreated thyroid disease; recent GI bleeding; history of adverse drug reaction; contraindication to heparin; second or third degree atrioventricular block; bifascicular block; Complete bundle branch block; sick sinus syndrome; other antiarrhythmic therapy used within 8 hours prior to the enrollment in this study. Patients were monitored, TSH was checked and all electrolytes including potassium and magnesium were supplemented prior to drug administration. After it was determined that AF/AFL has been stable with a documented duration of greater or equal to one hour, eligible patients were randomly assigned to receive a single 600 mg dose of oral Propafenone or an intravenous infusion of 1 mg of Ibutilide over 10 minutes. A second infusion of Ibutilide was given 10 minutes after completion of the first infusion if conversion did not occur. Serial ECG's were performed following drug administration. A 12-lead ECG was performed immediately upon cardioversion to NSR. Cardioversion was identified as a stable sinus rhythm lasting for at least one hour. In case of persistent AF/AFL, patients underwent electrical cardioversion after the 24-hour observation period. RESULTS: We identified a total of 100 patients with recent-onset AF/AFL. Of the 100 patients 37 were eligible for enrollment in this study. 19 patients (51%) converted to NSR spontaneously. 18 patients were randomized to either receive Propafenone(8) or Ibutilide(10). 6 of 8 patients converted to NSR following Propafenone(75% conversion rate) with a mean time to conversion of 4.5 hours. 7 of 10 patients converted to NSR following Ibutilide (70% conversion rate). No significant difference in conversion rate between drug arms. All patients except for one required two doses of Ibutilide for conversion. Mean time for conversion to NSR with Ibutilide was 2 hours. No significant ventricular tachyarrhythmias were noted. CONCLUSION: Propafenone is a safe, inexpensive and well-tolerated agent for the acute conversion of recent-onset AF/AFL to NSR, with the same efficacy as Ibutilide. In select patients(e.g. patients with no structural heart disease) Propafenone may be safe to use as an outpatient basis.
email to communicate with their doctor. These patients were more likely than those unwilling to use email to be younger (mean age 47.1 vs 55.4), have higher incomes ( > $75,000, 32.2% vs 17.0%) and to report better health (self assessed 10pt scale, 7.2 vs 6.5, All p < .01). Patients and physicians reported a number of barriers to use of email: 46.2% of patients who were email users`w ould rather speak to a real person'' and 31.5% thought email``would take too long'' but only 11.1% were worried about privacy at work or home. 47.4% of physicians were afraid they would be overwhelmed with patient emails, 30.2% did not feel they had enough time to check patient emails, and 44.8% felt patient email would add rather than substitute for other tasks. More physicians than patients thought that email could improve the patient-physician relationship (67.0% vs 54.3% of patient email users, p < .05) but physicians were more concerned than patients over security issues (43.2% vs 33.4% of patient email users, p < .05). CONCLUSION: Patients and their physicians are increasingly connected to email but few patients are connecting to their doctors. Our results suggest that patients perceive different barriers to use of email than their physicians. Patients may be more concerned about whether email will substitute for phone calls or visits versus concerns about privacy. Physicians appear to be optimistic about the role of email in improving the patient-physician relationship, but have concerns about privacy and workload. Research is needed to address these concerns about the efficiency, effectiveness and appropriate use of electronic patient-physician communication.
RESPOND TO STROKE SYMPTOMS. G. Kefalas 1 , S. Hazelett 1 , K. Hua 2 , K.R. Allen 1 , G.C. Wickstrom 3 ; 1 Summa Health System, Akron, OHIO; 2 Northeastern Ohio Universities College of Medicine, Rootstown, OHIO; 3 Summa Health System, Akron, OH PURPOSE: Lack of knowledge of stroke signs and symptoms is prevalent, even among stroke survivors. This lack of knowledge can translate into a delay in seeking treatment at the onset of stroke symptoms. Such delays limit the effectiveness of current treatments for stroke. It is assumed that the education stroke survivors receive on an acute stroke unit is sufficient to ensure that they will recognize and properly respond to stroke symptoms after discharge. This assumption has not been tested. METHODS: This study examined stroke survivors' knowledge of stroke symptoms and their anticipated response to these symptoms at the time of discharge from an acute stroke unit and after either a 3 month post-discharge comprehensive team management intervention or usual post-discharge care. This was a secondary analysis of data from a randomized controlled pilot study measuring the overall impact of the post-discharge intervention and included 96 patients discharged from an acute stroke unit. The investigator-generated``stroke knowledge test'' assessed 1) how patients would respond to specific stroke symptoms (i.e., call 911, call their Dr, call a friend, take care of themselves), 2) whether they could correctly label varied symptoms as stroke-related, and 3) if they would call 911 if they knew they were having a stroke. RESULTS: Overall, the percentage of correct responses declined over three months in the control group but increased in the intervention group. At three months 91% of intervention patients and 85% of controls recognized numbness in an extremity as a stroke symptom, and, of these, significantly more intervention patients (p = 0.02) reportedly would have called 911 if they experienced such numbness. Likewise, 100% of intervention and 97% of control patients recognized sudden speech difficulties as a stroke symptom at three months, but significantly more intervention patients would have called 911 if they experienced these speech difficulties (p = 0.002). Lastly, 94% of intervention patients and 88% of controls recognized sudden onset of dizziness as a stroke symptom at three months, but significantly more intervention patients (p = 0.005) would have called 911 if they experienced such dizziness. When asked at three months what they would do first if they knew they were having a stroke, 86% of intervention patients and 74% of controls said they would call 911. CONCLUSION: This study showed that stroke education incorporated in a comprehensive post-discharge intervention improves post-discharge knowledge of stroke symptoms compared to education on the stroke unit alone, and, in as much as self-reported anticipated behavior correlates with actual performance, results in more patients calling 911 at the onset of stroke symptoms. These results suggest that in-hospital education alone may not be as effective as that combined with education delivered in an outpatient setting. They also suggest that the emphasis of educational efforts should be on the appropriate response to stroke symptoms, not just on stroke symptom recognition. PURPOSE: In the PURSUIT trial, eptifibitide was found to reduce the risk of acute myocardial infarction (AMI) and mortality (composite) from 15.7% to 14.2% (relative risk reduction [RRR] = 9.6 percent) in patients presenting with non-ST elevation acute cardiac ischemia (ACI). A published cost-effectiveness analysis, based only on the more favorable United States (US) results of the trial, found that the cost-effectiveness ratio for eptifibitide for this indication was $16,491 per year of life saved. We sought to estimate the cost-effectiveness of eptifibitide based on the results of the entire trial and the expected risk of AMI/death in a community based-sample of patients. METHODS: Using logistic regression equations to predict patient-specific risks of AMI and death and assuming a constant RRR of 9.6 percent for AMI and death, we estimated the costeffectiveness of routine use of eptifibitide across a population of 2,780 consecutive patients with non-ST elevation ACI who were admitted to hospital, but were not treated with eptifibitide. RESULTS: Predictions were obtained on 2,766 (99.5 %) of patients who met inclusion criteria. Of these patients, 2.0 percent died at 30 days and 12.2 percent had the composite outcome of AMI or death. Given these overall risks and a constant RRR, the average cost per year of life saved in our sample was estimated to be $41,832 per year of life saved. Moreover, adjusting each patient's risk of AMI and death using individual patient characteristics, only 6 percent of patients were at sufficiently high-risk to warrant therapy under a threshold of $20,000 per year of life saved. More than half of admitted patients were at such low risk for a bad outcome, even without therapy, that the marginal cost-effectiveness in these patients was greater than $50,000 per year of life saved. According to our analysis, one in twenty admitted patients were at insufficient risk to warrant therapy even under a threshold of $100,000. CONCLUSION: Given a constant RRR, use of eptifibitide is economically attractive in highrisk patients, but less attractive in low risk patients. Strategies to risk stratify patients to optimize clinical and economic outcomes should be considered. PURPOSE: It has been demonstrated that primary angioplasty (PTCA) in high-PTCA-volume hospitals is more effective in reducing mortality in reperfusion-eligible patients with acute myocardial infarction (AMI) than thrombolytic therapy. However, the benefits of PTCA disappear completely at low-volume hospitals. These findings support the strategy of bypassing low-volume community hospitals to deliver reperfusion-eligible patients with AMI to highvolume cardiac centers. However, bypassing community hospitals, or transferring patients from their emergency departments, causes delays that may nullify the expected benefits of PTCA. Moreover, immediate thrombolytic therapy leads to excellent outcomes in most patients. Thus, it is not clear which patients might benefit from PTCA, in the face of additional transportrelated delay. METHODS: We used the results of ten published trials and meta-regression techniques to assess the relationship between the treatment benefit of PTCA (in terms of the reduction in mortality rate in a trial) and procedure-related time delay (i.e. the difference between the median``door-toneedle'' time with thrombolytic therapy and the median``door-to-balloon'' time with PTCA in a trial). The magnitude and statistical significance of the relationship was estimated using weighted least squared regression, weighting each trial by the inverse of the variance of its log odds ratio. We also assessed the relationship between treatment benefit and mortality risk (using the mortality rate in the control group treated with thrombolytic therapy as a measure of baseline risk). RESULTS: A statistically significant trend demonstrates that the treatment effect of PTCA decreases across trials as procedure-related delay increases (p = 0.014). The regression line crosses the x-axis at 48 minutes, suggesting that, where the median procedure-related delay is greater than about three quarters of an hour, PTCA may be no more effective than thrombolytic therapy and may be harmful, even at the high volume centers represented in these trials. Additionally, all trials in which the baseline mortality risk of the patients was moderate to high (reflected by a mortality rate in the thrombolytic treatment arm of greater than 5 percent), showed treatment benefit, while four out of five trials with low mortality risks demonstrated no benefit for PTCA, and potential harm. CONCLUSION: Our meta-regression suggests that high-risk patient populations get substantially greater benefit from PTCA than low risk patients with AMI. It also suggests that the benefits of PTCA, on average, are nullified where the procedure-related delay is about 45 minutes or more. How this``time interval to mortality equivalence'' varies with a patient's mortality risk, and with the time elapsed from symptom onset, requires modeling on individual patient data. PURPOSE: Studies suggest that health status influences patient satisfaction, but little work has examined the influence of general physical functioning versus disease severity health status measures on satisfaction. Using data from a survey of diabetic patients, our objectives were to examine 1) the association between health status and patient satisfaction using two different health status measures and 2) whether the associations differed with different dimensions of satisfaction. METHODS: We surveyed 2000 patients receiving diabetes care across four Veterans Integrated Service Networks in fiscal years 1998 and 1999. Diabetes severity was measured using diabetes-related components of the Total Illness Burden Index (TIBI), a measure of diabetes complications and co-morbidities. Physical health status was measured by the Physical Function Index (PFI10) from the Short Form 36. Using satisfaction with overall quality as the dependent variable, we constructed two separate multiple linear regression models that examined the association between physical health status (PFI10 or TIBI) and overall satisfaction, controlling for gender, race, income, education, age, number of primary care visits, and presence of mental health diagnosis. We re-ran these models using a scale of satisfaction with patient-provider communication as the dependent variable. RESULTS: 70% of eligible patients responded to the survey (n=1314). These patients reported low levels of physical function (PFI10 mean(SD) = 46.5(30.2)), and high levels of diabetes severity (TIBI mean(SD) = 40.5(18.8)). Overall, these patients were moderately satisfied, with 65% percent reporting excellent or very good quality of care. Lower levels of physical functioning were associated with less satisfaction (p < .01), but generic physical health status explained less than 1% of the variation in satisfaction (model r-squared =.03). In contrast, diabetes-related severity and co-morbidities were more strongly associated with lower levels of satisfaction (p < .001), with the TIBI explaining 4% of satisfaction variation (model r-squared =.05). Results were similar when we examined satisfaction with patient-provider communication. CONCLUSION: In this diabetes population, diabetes-related disease severity and comorbidities explained a greater portion of the variance in satisfaction than did a measure of physical functioning. Adjusting only for general physical function in studies comparing satisfaction among individual providers or organizations may not be sufficient if patients of these providers have similar physical functioning but differing degrees of disease severity. PURPOSE: Although attention to substance abuse is considered important in the care of the homeless, published data do not indicate whether homelessness carries a worse prognosis following detoxification. We studied the association between housing status and substance abuse relapse following detoxification (detox). METHODS: Subjects underwent inpatient detox for alcohol, cocaine or heroin from 6/97 to 3/ 99. Baseline interviews assessed substance use, homelessness during the 6 months prior to detox, and demographic, health and psychosocial variables. Six months later, subjects reported recurrent substance use (relapse) and post-detox treatment history. Survival analysis (proportional hazards regression) was used to assess the association between homelessness, other clinically relevant predictors, and tune to relapse follow4ng detox. We specified a single interaction term, homelessness with Inpatient Sobriety Stabilization Program (ISSP) exposure. ISSP's are voluntary, short-term transitional sobriety-oriented facilities, often called``holding programs.'' RESULTS: Of 254 subjects (54% of 470) available at 6 months, 72% reported relapse, occurring a median of 31 days after detox. Forty-nine percent of subjects had reported 1 homeless night before detox. Follow-up rates did not significantly differ between homeless and nonhomeless subjects. There was no difference in time to relapse for subjects stratified by housing status or by varying durations of homelessness. Controlling for demographic and psychosocial variables, subjects with full-time employment were at significantly lower risk of relapse ISSP-(Hazard Ratio=0.71, 95% CI .50 ± .98). There was no main effect for homelessness (p=.19) but there was a significant interaction between homelessness and ISSP exposure (p=.04). The Table shows the proportion relapsed at 6 months, broken down by housing status and ISSP exposure, and helps to demonstrate the interaction. Homeless persons who used ISSP's had the lowest relapse risk. In Navajo women, a population at high-risk for type 2 diabetes mellitus, depot medroxyprogesterone (DMPA) is a common contraceptive due to its ease of administration. However, recent evidence suggests that DMPA contraception may lead to weight gain and independently decrease insulin sensitivity. The objective of this study was to determine if DMPA was associated with development of diabetes in Navajo women. METHODS: We conducted a clinic-based case-control study. Eligible subjects were Navajo women aged 18 ± 50 years who had seen a healthcare provider at a Navajo Area Indian Health Service clinic at least once in 1998. Diabetic cases (n = 284) and non-diabetic controls (n = 570) were matched by age. Medical records were reviewed to determine contraception use before the diagnosis date of diabetes in each case and its matched controls. RESULTS: DMPA users were more likely to develop diabetes than patients who had used combination estrogen-progesterone oral contraception only (OR 3.8, 95% CI [1.8 ± 7.9]). The greater odds persisted after adjustment for body mass index (BMI) (OR 3.6, 95% CI [1.6 ± 7.9]). When DMPA use of 3 months vs. 4 ± 11 months vs. 12 or more months was compared, risk of diabetes was associated with longer use(p = 0.02). Longer DMPA use was also associated with greater BMI; each additional month of use was associated with a 0.12 kg/m2 greater BMI (95% CI 0.001 ± 0.24). CONCLUSION: DMPA contraception was associated with greater risk of diabetes compared to combination oral contraception, and risk was greater with longer use and persisted after adjustment for body mass index. Additional research is needed to confirm these results and elucidate the mechanism, but DMPA may have this previously unrecognized side effect that could influence choice of contraceptive method, especially in women at high risk for diabetes mellitus. PURPOSE: Diabetes mellitus affects an increasingly younger population. Although over 10 million women in the United States use oral contraceptives (OCs), the association between diabetes and current OC use in young adult women is unclear. We studied the associations between 1)current OC use and 2)glucose levels, insulin levels, and diabetes in young adult women. METHODS: Female participants (n=2,787) in the Coronary Artery Risk Development in Young Adults study (CARDIA), a multi-center, prospective observational study of African-Americans and whites, were aged 18 ± 30 years at enrollment. OC use, fasting glucose, fasting insulin levels, and diabetes diagnosis were assessed at study examination years 0, 7, and 10. Current OC use was defined as OC use at each time of examination; non-use (the reference group) was defined as combined past OC use and never use at each examination. Using generalized estimating equations, we analyzed the cross-sectional associations between 1)current OC use and 2)fasting glucose, fasting insulin, and presence of diabetes.
RESULTS: Current OC users differed from non-users on several factors. Current users were younger, had a lower mean body mass index (BMI), lower smoking prevalence, lower parity, and higher mean total cholesterol at all exams. By year 7, current users were more highly educated and by year 10 current users were more likely to be white and were more physically active than non-users. In unadjusted analyses, current use was associated with lower fasting glucose levels [À4.9 mg/dl, 95% CI (À5.7, À4. PURPOSE: Advanced lung cancer is a disease with little hope of long term survival. The two treatment options for patients are chemotherapy or supportive care. Several factors may influence the decision to undergo treatment. One poorly understood factor is the influence of a patient's faith on how they make medical decisions. We compared the effect of faith on treatment decisions among doctors, patients (PTs) and their caregivers (CGs). METHODS: 100 PTs with newly diagnosed advanced (stage IIIB) or metastatic (stage IV) lung cancer, their CGs, and 257 medical oncologists were interviewed separately using a standardized questionnaire. Participants were asked to rank, from 1 to 7, the factors that influenced their treatment decisions: (1)cancer doctor's recommendation, (2)faith in God, (3)ability of treatment to cure disease, (4)side effects, (5)family doctor's recommendation, (6)spouse's recommendation, (7)children's recommendation. Rankings were compared between the 3 groups of participants using restricted simultaneous ordered logistic regression models. Comparisons between those PTs who ranked faith as a high priority (1 or 2), and those PTs who ranked faith as a low priority (3 ± 7), were made using t-tests and chi-square tests. RESULTS: All 3 groups ranked the oncologist's recommendation as the most important factor. However, PTs and their CGs ranked faith in God second, while physicians placed it last (p < 0.001). The ability of the treatment to cure disease and side effects were important to all groups. PTs and CGs ranked their family doctor, spouse or child's recommendation as less important. Physicians were generally in agreement with PTs and CGs with the exception of physicians feeling the spouse's input was more important than did the PTs or CGs. PTs who placed a high priority on faith in God (n=53) were more likely to be African American (40% vs. 19%; p < 0.05). Irrespective of race, PTs who placed a high priority on faith in God were less likely to have graduated high school than those who ranked faith lower (47% vs. 72%; p < 0.001). There was a trend for PTs who ranked faith high to leave all decisions regarding treamtent to their oncologist. CONCLUSION: PTs and CGs are in complete agreement on the factors that are important in deciding on treatment options for advanced lung cancer but differ substantially from doctors. All 3 agree that the single most important factor is the oncologists's recommendation. However, faith in God is an extremely important factor for PTs and CGs but not physicians. PTs who ranked faith high are disproportionately African American and less likely to have graduated high school. Medical decision making incorporates tangible probabilities and utilities, (i.e. morbidity, mortality, side effects and costs of differing treatment options), into decision models that aid in a patient's treatment choice. This study suggests the previous model of decision making may be oversimplified. To our knowledge this is the first study to demonstrate that, for some, faith is an extremely important factor in medical decision making, more so than even the efficacy of treatment. If faith plays an important role in how some PTs decide treatment, and physicians are unaware of or do not account for it, the decision making process may be unsatisfactory to all involved. Future studies should clarify how faith impacts on individual decisions regarding treatment. Alcohol consumption decreased in all treatment arms. The 77 subjects (36%) who sustained a 30% decrease in drinks/month from baseline through 12 months had significant improvement in the SF-36 Mental Component Summary score (P = 0.037) and Physical Component Summary score (P = 0.058) and had fewer alcohol-related consequences (P < 0.001) when compared to those who did not sustain a decrease. In addition, the 34 subjects who sustained a decrease to non-hazardous levels (defined as < 16 drinks/week for men and < 12 drinks/ week for women) through 12 months had significant improvement in the SF-36 Physical Component Summary score (P = 0.002) and had fewer alcohol-related consequences (P = 0.047) when compared to those who continued a hazardous level of use. CONCLUSION: Outpatient problem drinkers who sustain a reduction in alcohol consumption have improved health-related quality of life and fewer alcohol-related adverse consequences. Benefits are seen across a wide spectrum of alcohol problems. These findings provide additional motivation for primary care physicians to identify patients with alcohol problems and to initiate intervention. Our overall response rate was 34.6%. We found that over half of the residents who responded had ever fallen asleep while driving, and 21.9% of respondents were involved in an automobile accident during residency. Over half of the respondents had received a moving violation, most often for driving fast and driving to or from clinic or the office. There was no correlation between frequency of call and hours of sleep to incidence of motor vehicle accidents and receiving a citation; however, there was a higher incidence of falling asleep while driving in patients who were on call every third night and slept fewer than four hours a night (p = 0.001 for both). If they did have an accident, it was most likely on a post-call day returning from the hospital. 89.9% of the residents got four or fewer hours of sleep on a typical on-call night. CONCLUSION: Residents-in-training are at high risk for motor vehicle accidents and falling asleep while driving when they are post-call and have less sleep. Mechanisms for reducing resident fatigue post-call such as a night-float system may reduce these incidences. . Descriptive statistics, t-tests, and ANOVA (SPSS version 10) were used to describe the population and determine associations between the variables of interest. RESULTS: 66/82 (80%) individuals from 14 PoPCRN sites consented to and were able to participate. 56% were female, 89% were white, 39% were cared for at home, and 53% had a cancer diagnosis. The median age was 76 years, median Karnofsky score 50%, and median time between hospice admit and interview was 49 days. MQOL responses (range 0 ± 10; 0=bad, 10=good) indicate that these patients had few physical symptoms, but the ones they had were problematic (single troublesome symptom score=4.4). The MQOL subscale scores indicate that these patients were less troubled by psychosocial and existential issues than by physical symptoms: Total MQOL score=7.1, Physical well-being score=6.0, Physical symptom overall score=5.9, Psychological symptom score=7.1, Existential well-being score=7.4, Support score=8.6. There were no significant associations between age, marital status, gender or length of hospice care and any of the QOL scores. A Karnofsky score < 50% was associated with worse Existential well-being (6.9 vs. 7.9, p=0.017) while a cancer diagnosis was associated with greater Existential well-being (7.8 vs. 6.9, p=0.047). Being cared for at home and a cancer diagnosis were both associated with a greater sense of Support ( From pharmacy data, we categorized the type of prescribed ART for > 50% of treatment time in each year into 4 intensity levels: none; 1 ART; 2+ ART but not highly active ART (HAART); or HAART including a protease inhibitor or non-nucleoside reductase inhibitor. Changes in ART from the base year to the next were classified as: suboptimal (ie, continuing or increasing to 1 ART or changing from a more intense to a less intense regimen); acceptable (ie, continuing or increasing to 2+ non-HAART drugs); or optimal (ie, continuing or increasing to HAART). Indicators were also created for visits (N=0, 1 ± 3, 4+) to a provider with a NYS contract to offer HIV-focused care in exchange for higher Medicaid payment. Each patient's outpatient care pattern in the base year was categorized as: regular medical care alone ( > 35% of visits to one provider); regular substance abuse treatment alone (6+ months with one provider); both; or neither. We estimated ordinal logistic regression models of ART pattern corrected for clustering of observations. RESULTS: Temporal changes in ART were: suboptimal (44%), acceptable (20%), and optimal (36%). Adjusted odds ratios (AOR) of greater intensity of ART were higher for persons with PURPOSE: Patients with limited English proficiency who are seen by language discordant providers may communicate through a variety of interpretation methods. We studied the effect of translation method on satisfaction among Spanish-speaking patients at a hospital-based walkin clinic serving primarily indigent patients. METHODS: All Spanish-speaking patients and a random selection of 10% of English-speaking patients presenting to the walk-in clinic at Denver Health Medical Center between July and October 2000 were approached for study entry. Participants completed a self-administered post-visit questionnaire assessing demographics, general health, and satisfaction with clinic visit.`L anguage concordant'' patients were defined as Spanish-speaking patients seen by Spanishspeaking providers and English-speaking patients. These patients were compared with`l anguage discordant'' patients who used AT&T interpreters, family, or other interpreters for translation. Patients were considered``satisfied'' if they rated their satisfaction as very good or excellent. Evaluation of satisfaction was by multiple logistic regression, controlling for age, sex, race, language spoken, education, and health. RESULTS: Of the 536 study participants, 67% were Spanish-speaking. Among Spanishspeaking patients, 42% were seen by language concordant providers. For patients who did not see language concordant providers, translation was through ATT telephone interpreters (19%), family members (23%), and other interpreters (16%). Language concordant and language discordant participants did not differ in age, sex, race, insurance status, or education level. Language concordant patients were less likely to be Hispanic (59% vs 95%, p=0.001) or describe their health as poor or fair (29% vs 51%, p=0.001). Language concordant patients and patients using ATT telephone interpreters reported identical overall visit satisfaction (77%), while patients using family or other interpreters were significantly less satisfied (54% and 49%, p < 0.01 and p=0.007, respectively). Compared to language concordant patients, patients who had family members translate were less satisfied with their provider in regards to listening to health concerns (62% vs 85%, p=0.003) and discussing sensitive issues (60% vs 76%, p=0.02). Patients who used other interpreters were less satisfied with provider skills (60% vs 83%, p=0.02), manners (71% vs 88%, p=0.02), listening (54% vs 85%, p=0.002), explanations (57% vs 84%), p=0.02), answers (57% vs 84%, p=0.05), and support (63% vs 84%, p=0.02). CONCLUSION: Patients utilizing less formal translation methods, such as family members and ad hoc interpreters, are less satisfied with care than those seeing language concordant providers or using AT&T telephone interpretation. METHODS: Women ages 35 ± 50 without a history of breast cancer were recruited from the waiting room of a university internal medicine practice. Following a baseline questionnaire about breast cancer risk factors, and benefits/harms of mammography, 179 women were randomized to one of three videos. For women ages 40 ± 49 who undergo 10 years of mammography, the videos described the number of lives extended(1/1000), the number of false positives expected(300/1000), and the number of women expected to worry after a false positive mammogram screening(100/1000). Information was numerically equivalent in each video but presented in a positive frame(1 woman would have her life extended) a negative frame(999 women would not have their lives extended) or a balanced frame(positive and negative information given). Participants completed a follow-up questionnaire immediately after viewing the video. RESULTS: Participants were predominately insured(80%), white(60%) and high school graduates(63%). At baseline, 3% endorsed the correct response(1 per 1000 women screened for 10 years would live longer because they had mammograms) and 76% thought 100 or 500 women would live longer. After the video, 53% endorsed the correct response with no differences by video (p=.21). For the number of false positives expected, 15% gave the correct response at baseline, but at followup the correct responses were given by more women in the negative(65%) and balanced(65%) frames than in the positive frame(40% P < .01). The results were parallel for the number expected to worry after a false positive (62%,56%, and 32% for negative, balanced and positive frames respectively). A majority of respondents in all three groups thought the benefits of mammography were more important than the harms before(75 ± 80%) and after seeing the the video(77 ± 81%). CONCLUSION: Women's baseline knowledge of the benefits of mammography was overly optimistic. All three videos improved women's knowledge of the benefits/harms of mammography. Despite this improvement in knowledge and exposure to varying frames of information, women's perceptions of the benefits/harms did not change. To determine if``numeracy '' affects women's ability to learn information about the potential benefits and harms of mammography. METHODS: In the waiting room of a university internal medicine practice we recruited 179 women ages 35 ± 50 without a history of breast cancer and randomized them to one of three videos. For women ages 40 ± 49 who undergo 10 years of mammography, the videos described the number of lives extended(1/1000), the number of false positives expected(300/ 1000), and the number of women expected to worry after a false positive mammogram screening(100/1000). Participants completed a questionnaire immediately after viewing the video. Information presented in each video was numerically equivalent, but to obtain the correct answers, subtraction was required for at least one question in each video. Numerate women were defined as being able to correctly note that 40% was equivalent to a 4 out of 10 chance. RESULTS: Participants were predominately insured(80%), white(60%), high school graduates(63%), and over half were numerate(65%).The table shows the percentage of correct responses for numerate and non-numerate women when the answers were given in the video compared to when subtraction was required to get the correct answers.
CONCLUSION: Most numerate women were able to learn numeric information from the video but had difficulty manipulating this information if they were not given the correct response in the video. Non-numerate women were unable to learn numeric information even when given the correct response. Although women in their forties are supposed to be informed of the benefits/harms of mammography to make decisions about screening, many women may have difficulty understanding this information if it is relayed numerically. The sample consisted of a primarily well educated group of 30 ± 50 year old (75%) professional men (37%) and women (63%). Almost all of the sample had been born in China (93%), claimed Mandarin Chinese as their native language (88%), and had resided in the United States an average of 8.9 (SD=7) years. Although most of the respondents had health insurance (93%) and more than half (69%) had a primary care provider, only 37% stated that they would have a routine check-up with this provider if they``felt fine.'' However 54% of the females in our sample reported having annual pap screening and breast exam by their physicians. Over half of the sample (63%) reported problems with understanding medical terms. Less educated respondents were more likely to be hesitant to see the doctor due to language difficulties (p = 0.001) and more likely to use western medicine for a cold (p = 0.023). Respondents with lower incomes were also more likely to be hesitant to see the doctor due to language difficulties (p = 0.036) but respondents with higher incomes were more more likely to have a primary care physician (p < 0.001). CONCLUSION: Although our sample was well educated and had adequate access to health care services, the inability to understand medical terminology may be a prime contributor to the under-utilization of health care services. A more diverse and larger sample is needed to further assess the utilization patterns from all socioeconomic levels. In addition, health literacy levels of Chinese Americans need to be assessed in order to provide culturally competent health care to this population. PURPOSE: Group A -hemolytic streptococci (GABHS) are cultured in 10 ± 25% of adults with sore throat. GABHS is the main cause of sore throat requiring antibiotic therapy. The Infectious Disease Society of America recommends either penicillin or erythromycin as first-line agents to prevent complications and reduce symptoms. Our goals were: 1) define the rate and type of antibiotics given to patients with sore throat by community primary care physicians over a ten-year period, and 2) determine predictors of antibiotic prescription and use of nonrecommended antibiotics for sore throat. METHODS: The National Ambulatory Medical Care Survey (NAMCS) collects data from office-based physician practices in the United States including patient demographics, patients' reasons for visit, physician diagnoses, and medications prescribed. Using NAMCS from 1989 to 1998, we analyzed 2,141 adult primary care visits with a chief complaint of sore throat. We estimated national antibiotic prescription rates over time and determined both the most frequent diagnoses made and the most frequent antibiotics prescribed. We evaluated significant predictors of antibiotic prescription and non-recommended treatment using multivariate logistic regression. RESULTS: An estimated 6.8 million adult patients each year made acute visits for sore throat to office-based, primary care physicians between 1989 and 1998. The most common diagnoses were acute pharyngitis (42%), acute upper respiratory tract infection (21%), acute tonsillitis (8%), and streptococcal infection (6%). Antibiotics were prescribed to 73% (95% CI 70 ± 76%) of patients. The most common antibiotics prescribed were aminopenicillins (23% of visits), cephalosporins (14%), penicillin (14%), and erythromycin (10%). Over ten-years the proportion of patients receiving any antibiotic decreased while the proportion of patients receiving non-recommended treatment increased ( 1989 ± 1992 1993 ± 1995 1996 ± 1998 NHANES included 157 women with a personal history of breast cancer. Eleven of these survivors (7.0%) were currently using HRT. CONCLUSION: A surprisingly large fraction of breast cancer survivors use HRT. The concordance between two estimates obtained using very different methods argues against random or data error. Other possible explanations include: 1) the patient opts to accept the putative risks of recurrence in an informed manner, 2) the prescriber does not know that HRT is contraindicated in breast cancer, 3) the prescriber rejects the theory of hormone induced recurrence, 4) the prescriber does not know the patient is a breast cancer survivor, or 5) the patient self-prescribes. We believe that HRT use in breast cancer survivors is a sentinel event that may represent medical error. In stage 1, multi-pattern regressions were performed to impute PCS and MCS using SF-12 items only (simple model), and then using SF-12 items plus patients demographics and comorbidities (enhanced model) in an attempt to reduce bias from the simple model. Distribution and variation of the imputed SF-12 values were evaluated. Based on confidence bands of the imputed SF-12 values, a cut point of number of missing SF-12 items was selected for using the simple or enhanced model to impute SF-12 PCS and MCS. In stage 2, patients with no missing SF-12 items who also had demographic and comorbidity data were randomly classified into 11 subgroups. Permutations of missing patterns (missing 1 to 11 out of the total 12 items) on SF-12 items were generated to simulate missingness (missing 1 to 11 SF-12 items) for these patients. The imputed PCS and MCS were compared to the observed values for these patients separately by the identified cut point. The analysis was performed separately for chronic and non-chronic subgroups. RESULTS: Results: In stage 1, the standard errors of means of imputed summary scores increased almost monotonically by the number of missing SF-12 items, ranging from 0.11 to 4.55 for PCS and 0.09 to 2.18 for MCS in the non-chronic subgroup, and from 0.28 to 5.84 for PCS and 0.24 to 5.62 for MCS in the chronic subgroup. A significantly consistent increase of the width of confidence bands (about 50% in standard error) occurred between 5 and 6 items missing. Of the 13,438 patients, 10,766 (8949 non-chronic and 1817 chronic) patients had non-missing data for all SF-12 items as well as demographic and comorbidities. Productmoment correlations between the imputed and the observed scores were large, ranging from 0.73 to 0.97. The correlations from imputation with the enhanced model were consistently higher than the correlations from the simple model. The increments of correlations by the enhanced model were statistically significant for patients with more than 6 missing SF-12 items (7%, P < 0.05). CONCLUSION: Conclusion: For patients with partially missing SF-12 items, direct imputation of summary SF-12 physical and mental scores is efficient. Adding patients' demographics and comorbidity information to the imputation model for PCS and MCS can improve the imputed value and reduce bias. The enhancement of adding patients' demographics and comorbidities to the imputation models for PCS and MCS is necessary and important for patients with 6 or more SF-12 items missing. PURPOSE: Most clinicians rely on the history to make an accurate diagnosis and prescribe appropriate treatment. However, as increasing administrative and financial pressures shorten the outpatient clinical visit, the opportunity to take a careful history is becoming more abbreviated. This study prospectively evaluated whether accurate history taking, under precisely controlled conditions, actually leads to better outpatient clinical practice. METHODS: We prospectively collected data in the primary care general internal medicine clinics at two VA-university teaching hospitals. Greater than 95% of residents and attendings consented to participate Ð and 20 were randomly selected as subjects. Eight unannounced standardized patients (SPs) were introduced into the clinic of each selected provider presenting four common conditions: COPD, Low Back Pain, diabetes mellitus and coronary artery disease (i.e., 2 SPs per condition). Thus, every physician saw an identical set of cases. All visits were new patient appointments. Visits were scored by the SP using a closed-ended checklist of quality criteria; other criteria were assessed by abstracting the medical record. Scoring was based on explicit criteria derived from national guidelines and expert panels. Separate scores were generated for History Taking, the Physical Examination, Testing (laboratory and imaging studies), and Diagnosis plus Treatment. Scores were expressed as a percentage of criteria that were correct. Multiple linear regression models were used to predict if better history taking led to more accurate diagnosis and treatment plans, more correct testing, and appropriate physical examinations. The models controlled for case, site, and level of training. RESULTS: Physicians who scored higher on history taking (that is, took more accurate histories) made more accurate diagnoses and more appropriate treatment decisions (p = 0.006). By contrast, more accurate physical examinations and diagnostic test ordering did not predict better diagnosis and treatment (p > 0.05). The quality of diagnosis and treatment varied between the two sites (p < 0.001) but not by level of training or case. Taking an accurate history did predict the appropriateness of the physical examination (p = 0.004), but it did not predict testing (p > 0.05). CONCLUSION: Under controlled conditions, where case mix was specified by experimental design, our results show that accurate history taking predicted when residents and attendings were more likely to make the correct diagnosis and prescribe the best treatment. While accurate history taking also guided the physical examination, the examination itself did not lead to subsequently higher process scores. The site effects were also strong. These findings demonstrate that successful completion of the history effectively predicts of diagnostic and treatment accuracy. Emphasis on development of history taking skills in residency programs, and adequate visits time allocations in practice settings, should contribute to higher quality clinical care. were able to recall their PCP's name. When asked,``When you are sick and need care right away, how quickly do they see you?'' 70% answered the same day or the next day. Regarding ED usage, 49% of patients called triage before coming to the ED; those with CMA insurance were less likely to call (42% vs 75%, p =. 04). Of those that called, 96% reported they were told to go to the ED (``callers''). ED nurses triaged 59% of``callers'' and 44% of non``callers'' to Level 1 or 2 (out of 4; 1 is high). Eighty per cent of respondents rated``How well doctor listens to you,''`D octors respect for you,'' and``Doctors ability to figure out what is the matter'' as excellent or very good. There were no significant differences in ratings between PCPs and ED physicians.
CONCLUSION: Our small pilot survey indicates that patient knowledge of, and access to, acute primary care was very good. However a large number of patients with non-urgent conditions still didn't call before presenting to the ED. Almost all that did call were sent to the ED, even though 41% were then judged to be of lower sickness acuity by the ED triage nurses. These results suggest that our CMA and other patients need more education and/or incentives to access primary care alternatives to ED use. Our results also suggest that our triage nurses could benefit with further training on appropriate ED referral. We hypothesized that physically active PAD persons with no exertional leg symptoms would have better leg functioning than other leg pain groups. METHODS: Study participants were 454 men and women with PAD identified from three medical centers in Chicago. Participants were categorized into one of six mutually exclusive leg symptom groups: Group 1:No exertional leg pain/active (walked > 6 blocks in the last week) (13%); Group 2: No exertional leg pain/inactive ( < = 6 blocks walked last week) (6%); Group 3: Atypical exertional leg pain and walk through leg pain (8%); Group 4: Intermittent claudication (37%); Group 5: Atypical exertional leg pain/stop walking with leg pain (17%); Group 6: Leg pain on exertion and rest (19%). Functional measures included six-minute walk, four meter walking velocity, time for five chair rises, and standing balance. All statistical analyses were adjusted for multiple comparisons using Bonferroni method. RESULTS: Group 1 and Group 3 had significantly less severe PAD, as measured by the ankle brachial index, than the other groups. Compared to all other groups, Group 6 had the highest prevalence of lower extremity arthritis, disc disease, diabetes, and depression. In general, functional performance was poorest in Group 6 and best in Groups 1 and 3. Compared to Group 1, Group 6 had slower walking velocity (0.75 m/sec vs. 0.89 m/sec, p < 0.05), achieved shorter distance in the six-minute walk (972 feet vs. 1201 feet, p < 0.05), had slower time for rising from a seated position five times (14.2 sec vs. 11.3 sec, p < 0.05), and was less able to hold the tandem stand position for ten seconds (32% vs. 51%, p < 0.05). Compared to Group 1, Group 4 was more likely to stop during the six-minute walk (38% vs. 16%, p < 0.05) and walked fewer blocks in the last week (35.7 vs. 53.0, p < 0.05). Group 2 had slower walking velocity than Group 1 (1.02 vs. 1.22 meters/sec p < 0.01). CONCLUSION: Comorbid disease may contribute to the nature of leg symptoms reported by PAD patients. Clinicians can use the leg symptom categories defined above to gauge the degree of functional impairment associated with PAD. PURPOSE: Understanding the natural history of weight gain on a population basis may be a critical step towards developing effective clinical or public health interventions. While the marked rise in overweight and obesity prevalence in this country has been well documented by the serial cross sectional data of the US National Health and Nutrition Examination Survey (NHANES), longitudinal description of the trend is lacking. This study assesses the development of body mass of a cohort of young US adults and identifies predictors including age, gender and ethnic group that may have implications for targeted intervention. METHODS: The National Longitudinal Survey of Youth (NLSY79) is a nationally representative sample of over 12,000 persons aged 14 ± 22 years at baseline, with oversampling of minority ethnic groups. Body mass index (BMI = kg/m2) was calculated from self-reported height and weight data at 12 sample points over 17 years.`Overweight' and obese' were defined as BMI > 25 and > 30, respectively. Mean BMI was calculated by age group, gender and ethnicity. In this study, unadjusted data are used to describe trends in BMI, and to calculate incidence of overweight and obesity using survival analysis. RESULTS: BMI trends were strongly related to age, race and gender. Women's BMI increased steadily with age, with average BMI reaching``overweight'' status by age 26 in Black women, age 29 in Hispanic women and age 35 in all others. The average BMI of Black women was in the obese range before age 40. Ethnic differences in weight prevalence were evident by age 17. Survival analysis quantified the incidence rate of obesity as 2.5 times faster for Black women as for Non-Hispanic/Non-Black women (CI 2.2 ± 2.8) and 2.1 times faster for Hispanic women versus those who were Non-Hispanic/Non-Black (CI 1.8 ± 2.4). Male average BMI showed less dramatic age trends and a different ethnicity pattern: Hispanic men consistently had the highest mean BMI values. Hispanic men developed obesity 2.0 times faster (CI 1.7 ± 2.3) and Black men developed obesity 1.8 times faster than Non-Black/Non-Hispanic men (CI 1.6 ± 2.0). CONCLUSION: This large longitudinal survey shows marked differences in the rate of weight accumulation for different ethnic groups, evident at a very young age. This pattern persisted through young adulthood with excess weight, on average, increasing gradually with age. To alter the trajectory of obesity in this country, interventions should target young adults, and focus on those of minority ethnic groups. have been shown to be as effective as antidepressant medications for treating patients with major depression. The success of these methods may be related in part to the use of specific counseling techniques. These analyses assess the extent to which primary care clinicians and mental health specialists use CBT techniques to treat primary care patients with major depression. METHODS: Cross-sectional descriptive evaluation of counseling received by 567 patients meeting criteria for 1-year major depressive disorder based on a structured diagnostic interview. The 567 enrolled participants are from a consecutive sample of patients attending one of nine participating primary care practices in California (3 based in a single academically-affiliated VA medical center and 6 in independent medical centers from 1 region of Kaiser Permanente). Participants completed an interviewer-administered survey of health perceptions and utilization patterns. Patients reported the frequency of visits to medical and mental health providers and the extent to which their providers used four different CBT techniques. These included helping the patient reduce negative thinking, encouraging enjoyable activities, assisting with problemsolving, and helping the patient feel better about their life as it is. RESULTS: Patients reported an average of 3.2 visits (median=1.0, range 0 ± 144, 61.3% with no visits) to medical providers in which they discussed their personal or emotional problems and 4.5 (median=0, range 0 ± 96, 60.7% with no visits) visits to mental health specialists. Of those patients with at least one visit to a medical provider during the past six months, between 42.1% and 57.6% per therapeutic technique reported that their medical providers had used it. The range was 78.9% to 84.9% per therapeutic technique for patients visiting mental health specialists. The most prevalent therapeutic technique was encouragement of more enjoyable activities. 59.3% and 85.5% of patients reported receiving counseling that included at least two of the four therapeutic techniques from medical and mental health providers, respectively. CONCLUSION: Although the mental health specialists used therapeutic counseling techniques more often than did the primary care clinicians in these practices, primary care clinicians used them more often than we expected given the competing demands of generalist practice. This optimistic result suggests that further evaluation of the efficacy and effectiveness of primary care clinician therapeutic counseling in improving outcomes is warranted. Little is known about the predictors of alternative medicine use in the veteran population. This study investigates whether veterans' sociodemographic characteristics, selfreported health status, health-related beliefs, and satisfaction with their VA health care providers, are related to their use of alternative medicine. METHODS: Participants included patients attending 7 VA general internal medicine clinics participating in the VA Ambulatory Care Quality Improvement Project (ACQUIP), a multicenter randomized controlled trial to test the effectiveness of an information feedback system on improving health status and satisfaction outcomes. This analysis is based on a random subsample of 1034 patients who also received the ACQUIP Health Beliefs Scale. This scale assessed patients' attitudes towards conventional medicine and adherence to health-conscious lifestyles. Patients' satisfaction with their provider was measured with the 12-item humanistic component of the Seattle Outpatient Satisfaction Questionnaire. Self-reported health status was measured with the mental, physical, and pain subscales of the SF-36. Additional data included patients' age, gender, ethnicity, level of education, and income. Alternative medicine use was determined with a questionnaire assessing the use of the following: biofeedback/relaxation, chiropractic, acupuncture, massage, herbs, homeopathy, naturopathy, spiritual healing, folk remedies, and other. Multiple logistic regression techniques were used to evaluate the association between alternative medicine use within the past year (yes or no), sociodemographic characteristics, health beliefs, and patient satisfaction. RESULTS: 495 patients returned the Health Beliefs Scale (48% response) and were included in this analysis. 124 patients (25%) reported the use of at least one alternative medicine therapy in the past year, with the most common modalities including herbs (13.5%), chiropractic (7%), and massage (6%). In multivariate analyses adjusted for sociodemographic characteristics and selfreported health status, the following were associated with alternative medicine use: more education (OR 1.5; 95% CI 1.3 ± 1.9); better physical health (OR 1.044; 95% CI 1.009 ± 1.081); and more pain (OR 1.022; 95% CI 1.005 ± 1.039). After adjustment for sociodemographic factors and subjective health status, alternative medicine use was associated with the following health-related attitudes and behaviors: believing more in the value of non-medical treatments (OR 1.4; 95% CI 1.1 ± 1.9); adhering to health-conscious lifestyles (OR 1.7; 95% CI 1.3 ± 2.2); and actively seeking health information from non-medical sources (OR 1.6; 95% CI 1.3 ± 2.1). In adjusted analyses, there were no significant associations between alternative medicine use and attitudes towards conventional medicine or patient satisfaction. CONCLUSION: A quarter of VA general internal medicine patients in this study reported the use of alternative medicine in the past year. Patients who used alternative medicine had more education, and reported better physical health but more pain. Alternative medicine users also believed more in the value of non-medical treatments, reported adherence to health-conscious lifestyles, and reported obtaining more health information from non-medical sources. Similar to previous studies, negative attitudes towards conventional medicine and lower levels of patients' satisfaction with their VA providers did not appear to influence use of alternative medical modalities. Procedures Ð Using established criteria, potentially inappropriate medications were identified in the hospital formulary and assessed by the central pharmacy database for frequency of use. The hospital admissions database was used to identify the total number of elders hospitalized in the previous year. The two databases were merged, sorting by patient age, non-intensive care unit location, and our defined inappropriate medications. A medical error rate was calculated by dividing the total of inappropriate medications dispensed by 100 patient-days. Patient days were calculated by length-of-stay data also obtained from the hospital admissions database. The study was repeated in the subsequent 6-months to assess for secular trends and the stability of the error rate. RESULTS: A total of 11,797 doses of inappropriate medications were dispensed to 15,011 patients aged 70 and over during a 12-month period. The most common of these prescribed medications were meperidine (3,652 doses), amitriptyline (1,736 doses) , and diazepam (1,644 doses) . The medical error rate was 0.12 per day, or 12 errors per 100 patient days. Findings for the subsequent 6 months were similar. CONCLUSION: The results of our study are troubling though not surprising. Academic teaching hospitals often require all patient orders to be written by resident physicians, with variable levels of senior supervision. Educational interventions targeted at internal medicine residents have the potential to reduce the medical error rate in elderly hospitalized patients. References of key articles were also reviewed for related studies. Studies were included if they enrolled postmenopausal women and reported deep venous thrombosis (DVT), pulmonary embolism (PE), or VTE as either part of a predetermined outcome or as a reportable adverse event related to HRT. Another Medline search from January 1991 to June 2000 identified studies addressing SERMs and VTE risk. Ten HRT studies (two randomized controlled trials, seven case-control studies, and one cohort study) and five SERM randomized controlled trials met inclusion criteria. Two investigators abstracted data on participants, intervention, VTE rates, and confounders. We used the Bayesian data analysis framework for the meta-analysis of HRT studies. The SERMs were not included in the meta-analysis. RESULTS: HRT was associated with an increased risk of VTE in current users (RR=2.34; 95% Cl, 1.83 ± 2.94). Five studies showed a significant increase in VTE with HRT. The highest VTE risk was reported with the first year of URT use (OR =2.9 to 6.7). Two of the SERM randomized controlled trials reported an increased risk of VTE (RR= 1.91 to 3. 1). CONCLUSION: Postmenopausal HRT is associated with an increased risk of VTE. SERMs may be similarly associated with VTE risk. These findings are pertinent to clinicians and patients considering HRT.
BREAST CANCER SCREENING IN AMERICAN SAMOAN WOMEN. S. Mishra 1 , F. Hubbell 1 , P. Luce 2 ; 1 University of California, Irvine, CA; 2 National Office of Samoan Affairs, Carson, CA PURPOSE: Little is known about breast cancer screening practices or predictors of age-specific screening for Samoan women. We determine population-based estimates of utilization of clinical breast exam (CBE) and mammography by Samoan women and examine predictors of recent age-specific utilization of screening exams. METHODS: Through systematic, random sampling procedures, we identified and interviewed 720 adult (!30 years) Samoan women residing in American Samoa, Hawaii, and Los Angeles. Multivariate logistic regressions were performed to determine independent predictors for recent age-specific screening RESULTS: Only 55.6% of women (!30 years) had ever had a CBE and 32.9% of women (!40 years) had ever had a mammogram. Furthermore, only 24.4% and 22.4% of Samoan women (!40 years) residing in Hawaii and Los Angeles, respectively, had an age-specific mammogram within the prior year. Independent predictors of age-specific CBE screening included age, education, health insurance, ambulatory visit, and being a resident of Hawaii or Los Angeles; and those for mammography included ambulatory visit and awareness of screening guidelines. CONCLUSION: Population-based estimates of age-specific breast cancer screening among Samoan women are lower than nation objectives and those reported for other minorities. Targeted efforts that address doctor patient communication on preventative behavior, improved access to health care services (especially in American Samoa), and focused educational awareness programs are needed to improve the dismal screening rates observed in this indigenous population. PURPOSE: Controversy exists on whether postmenopausal hormone replacement therapy (HRT) can lessen or avert cognitive impairment that may lead to dementia. The purpose of this study is to examine the association of HRT use and impaired cognition. METHODS: Postmenopausal women (n=1403) participating in the population-based Beaver Dam Eye Study and the Epidemiology of Hearing Loss Study in Beaver Dam, Wisconsin were included in these analyses. Data were from the 10-year follow-up for the eye study and the concurrent 5-year follow-up for the hearing study (1998 ± 2000) . HRT use was assessed by questionnaire and confirmed by participants' presentation of prescribed medications at the eye examination. Cognitive impairment is defined as less than 24 points, or 80%, on the minimental status exam, or doctor-diagnosed Alzheimer's disease. Odds ratios were obtained from logistic regression models. RESULTS: The average age of the women was 69 years (range 53 to 97). Twenty-five percent of participants were current HRT users. The average age of current users was 65 years; the average age of non-users was 72 years. More current users completed high school than nonusers, 91% and 78%, respectively. In preliminary analyses, the overall prevalence of cognitive impairment was 6%. HRT users were less likely to be cognitively impaired than non-users (5.7% vs 0.5%, p = 0.0003). After adjusting for age, education, and depression (as measured by Short Form-36 mental health subscale), the odds of being cognitively impaired were not statistically significantly different for HRT users versus non-users (OR = 0.40; 95% CI = 0.14 ± 1.15, p = 0.09). CONCLUSION: These preliminary analyses suggest that postmenopausal HRT use may be associated with a slighter lower odds of having impaired cognition. However, this association may be marking selection bias, as the relationship is no longer statistically significant after adjusting for the confounding effects of age, education, and depression. Longitudinal studies, adequately controlling for these confounders, are needed to determine the effect of HRT use on cognition. PURPOSE: Assessing patient satisfaction with care received is important to measure as it is a primary goal of providers and clinic practices. Studies done, to date, have been limited in that most measure patient satisfaction with experiences related to medical student education and have been retrospective in design. We designed a prospective study to find the differences in patient satisfaction with outpatient care provided by continuity clinic resident physicians compared to satisfaction with care provided directly by faculty physicians in the same practice. METHODS: During one week in June 2000, 1107 adult and pediatric clinic patients completed a patient satisfaction questionnaire following their visit, during which the patient was either seen by the faculty member or by the resident physician year 1 through 4. The data was collected from 5 primary care clinics; pediatrics, internal medicine and a combined medicine/ pediatric clinic. Four clinics were in community practices and one clinic was an academic hospital based practice. The questionnaire comprised of 26 questions addressing service characteristics of the office staff, provider and facility. Ten questions assessed satisfaction of care provided by the doctor. Differences in results for faculty vs. resident patients were analyzed for the entire set of collected responses, as well as for each clinic. RESULTS: 1772 total patients were seen during the study week. Sixty-three percent of the questionnaires were completed (1107); 824 from faculty patients and 283 from visits with the resident. 42% of the faculty patients and 24% of the resident patients saw the doctor they wanted to see on the day of the visit. 67% of the faculty patients and 60% of the residents patients``strongly agreed'' that the resident answered questions in a way the patient could understand. 52% of faculty patients and 43% of the resident patients indicated that the time spent with the physician was``excellent''. 62% of the faculty patients rated thoroughness of the exam as excellent versus 50% of the resident visits.47% of the faculty visits and 36% of the resident visits rated the care received overall as``excellent''. All differences reached statistical significance with p < 0.05. A similar distribution of small differences, all indicating better satisfaction with care by faculty occurred in all 5 participating practices. CONCLUSION: Patients were more satisfied with care received by faculty as compared with residents. Difficulty for the residents patients to schedule an appointment with their physician of choice is an important barrier to improving patient satisfaction. Overall access to and use of HIV care is lower for Hispanics than for whites, but little is known about how these vary within the diverse Hispanic population in the US. We examined access to, use of, and unmet need for care among HIV+ Hispanics according to level of acculturation, language in which subjects chose to complete surveys, citizenship status, gender, education and insurance status among the 415 Hispanic patients (15% of all subjects) in the HIV Costs and Services Utilization Study (HCSUS). METHODS: HCSUS is a nationally representative study of HIV-infected persons in care. Weights were used to adjust for sampling, multiplicity, and non-response. Standard errors were corrected for the multistage sampling design. Use of care was measured in four dimensions: ambulatory care, emergency care, hospital care and mental health services. Overall access was measured with a six-item scale (alpha = 0.72) asking about urgent care, emergency care, hospital care, clinic location, costliness of services, and access to specialists. Unmet need for care was measured by five items asking about foregone medical care due to: inability to get time off work, being too sick to seek care, lack of transportation, needing to care for someone else, or insufficient money to pay for care. All results reported are significant at a p < 0.05 level. RESULTS: In separate multivariate analyses controlling for CD4 count, Hispanics who were US citizens reported greater use of emergency care (Odds Ratio [OR]=1.92), hospital care (OR=2.59) and mental health services (OR=1.92) than non-citizens. Hispanics who were more acculturated (OR=1.76), who completed an English-language survey (OR=2.43), who were US citizens (OR=1.85), who were more educated (OR=1.93) and who were insured (OR=3.54) reported greater access to care. Women reported less use of ambulatory care (OR=0.66) and mental health services (OR=0.63) and worse overall access (OR=0.63) than men. Women were more likely to report unmet need for medical care (OR=2.17) than men (due to any reason of the five reasons asked about). CONCLUSION: Significant variation exists among HIV+ Hispanics in access to and use of care by language preference, acculturation, citizenship status and gender. Interventions to improve access for Hispanics should prioritize those Hispanics in greatest need. University of Pittsburgh, Pittsburgh, PA PURPOSE: Community-acquired pneumonia (CAP) is the leading infectious disease cause of death in the United States. Although risk factors for short-term mortality are widely studied, there is very little data on the risk factors for long-term mortality after surviving CAP. The aim of this study is to examine the predictors of long-term mortality after initial CAP. METHODS: This analysis was a follow-up of the 4 United States sites of the Pneumonia Patient Outcomes Research Team (PORT) prospective, multicenter cohort study. Baseline demographic and clinical data, including comorbid illnesses, laboratory and radiology results, were obtained for all patients. Long-term mortality was determined using the National Death Index. Patients were enrolled between October 1991 and March 1994, and mortality information was obtained up to December 1998. Charlson's comorbidity scale was used to assign a comorbidity score for preexisting comorbid conditions and age. All patients who died within 90-days of enrollment were excluded from the analysis in order to study the long-term mortality of those surviving after the acute illness. Statistical modeling included the use of Kaplan ± Meier survival plots and the log rank test for categorical variables, and univariate Cox proportional hazard modeling for continuous variables. Any variable that was statistically significant (p 0.05) was then entered into a step-wise forward multivariate Cox proportional hazard model. RESULTS: Of the 1555 subjects enrolled at the 4 U.S. centers, 1419 subjects survived past 90days after enrollment. Of those patients 472 (33.3%) died by December 1998. During the first year after enrollment 29.2% of patients died, in the second year 19.1%, third year 16.1%, fourth year 10.0% and !fifth year 25.4%. Significant baseline predictors of long-term mortality were being DNR (hazard ratio 2.1 with 95% confidence interval of 1.5 ± 2.9), poor nutritional status (1.9, 1.3 ± 2.8), pleural effusion (1.4, 1.1 ± 1.8), steroid use (1.6, 1.3 ± 2.1), nursing home residence (10.1, 3.2 ± 32.1), Charlson's score of 1 or 2 (2.5, 1.6 ± 4.0), Charlson's score 3 or 4 (5.8, 3.9 ± 8.9), and a Charlson's score !5 (14.6, 10 ± 21.5). Significant protective factors against mortality included !college education (0.6, 0.5 ± 0.8), female gender (0.7, 0.6 ± 0.8), and the symptom of fever (0.7, 0.6 ± 0.9). CONCLUSION: This study demonstrates that there is a significant long-term mortality burden after CAP, and that age, education, gender, comorbidity, nutritional status and nursing home status are important predictors of long-term mortality after CAP. PURPOSE: Although studies of tea consumption and mortality among healthy adults report conflicting results, the effect of tea consumption on patients with acute myocardial infarction is unknown. METHODS: In a prospective cohort study, we followed 1935 patients (601 women and 1334 men) hospitalized with a confirmed acute myocardial infarction between 1989 and 1994 at 45 community hospitals and tertiary care centers in the United States, as part of the Determinants of Myocardial Infarction Onset Study. During hospitalization, participants reported their usual intake of caffeinated tea during the year prior to infarction to trained interviewers. We excluded 35 patients with missing information on tea intake. We analyzed survival through 1995 using Cox proportional hazards regression, controlling for age, sex, body-mass index, previous myocardial infarction, diabetes, hypertension, education, income, current and former smoking, alcohol use, regular exertion, acute infarct complications, previous medication use, and receipt of thrombolytic therapy. RESULTS: Of the 1900 eligible patients, 1019 consumed no tea, 615 consumed less than 2 cups daily (moderate use), and 266 consumed 2 or more cups daily (heavy use). Heavy tea drinkers were older, less obese, and more likely to be female than non-drinkers, but their sociodemographic and clinical characteristics were otherwise similar. During a median followup of 3.8 years, a total of 313 patients (16%) died. Compared to non-drinkers, total mortality was lower among moderate tea drinkers (hazard ratio 0.78; 95% confidence interval (CI), 0.60 ± 1.01) and heavy tea drinkers (hazard ratio 0.65; 95% CI, 0.44 ± 0.97; p for trend = 0.01). When modeled continuously, the hazard ratio for each additional daily cup of tea was 0.89 (95% CI, 0.78 ± 1.01). The effect of tea was similar in men and women, in younger and older patients, and for total and cardiovascular mortality. Controlling for coffee intake did not change the results. CONCLUSION: Self-reported tea consumption during the year prior to acute myocardial infarction is associated with improved survival following infarction. PURPOSE: With recent changes in the financing of health care, home care patients may be discharged from home care prematurely, placing them at risk for adverse outcomes. Little is known about what happens to patients after home care discharge. We examined the frequency of adverse outcomes within 3 months of home care discharge, and identified factors associated with their occurrence. METHODS: Patients admitted to a single home care agency between April and September 2000 were invited to participate in the study. We collected data by chart review and telephone interview. We followed patients for 3 months after home care discharge and collected information on 5 adverse outcomes: death, readmission to home care, nursing home placement, emergency room use, and hospitalization. As potential correlates of adverse outcomes, we analyzed patient demographics, reason for home care, dependency in activities of daily living (ADLs) and instrumental activities of daily living (IADLs), cognitive function, depression, number of medications, caregiver support, and living alone. We developed a logistic regression model for adverse outcomes within 3 months of home care discharge. RESULTS: Of 217 eligible patients, 180 (83%) agreed to participate in interviews. To date, 155 have been discharged from home care (85% remained home, 13% were hospitalized, and 2% were transferred to hospice). We obtained 3-month outcomes on 108 (84%) of the 129 patients who remained at home after home care discharge. Of these patients, 60% were female, 78% were white, 74% were !65 years of age (mean age 74 years), and 72% had Medicare or Medicare managed care as their primary insurance. The median home care length of stay was 23 days. At the time of home care discharge, 57% had ! 1 ADL dependency; 95% had ! 1 IADL dependency; 37% lived alone; and 16% had no caregiver. The most common diagnoses were congestive heart failure (25%), chronic pulmonary disease (28%), diabetes (28%) and cancer (30%). Within 3 months of home care discharge, 34 patients (32%) had one or more adverse outcomes: 5% died, 4% were readmitted to home care, 26% had an emergency room visit, and 15% were hospitalized. Patients with ! 1 ADL dependency were more likely to have an adverse outcome compared to those with no ADL dependency (42% vs. 17%, p = 0.007). In a multivariable model including patient age and sex, factors associated with adverse outcomes within 3 months of home care discharge were ! 1 ADL dependency (Adjusted Odds Ratio 3.9 [95% CI: 1.3, 11.5]) and a history of cancer (4.7 [1.7, 12.9] ). CONCLUSION: Home care patients are at substantial risk for adverse outcomes within 3 months of home care discharge. Those with functional dependence or cancer are at highest risk. Further research is necessary to determine whether additional home care services would prevent adverse outcomes in these high risk patients. To determine if Enhanced QTc Dispersion occurs in ischemia during pharmacologic stress testing. This may serve as a non-invasive method to detect ischemia in patients suspected of having CAD. BACKGROUND: QTdispersion (Qtd), defined as the interlead variability in QT intervals on a 12 lead surface electrocardiogram (EKG) reflects regional inhomogeneity of ventricular repolarisation. Increased QTd has been reported in patients with ischemic heart disease (IHD) during exercise and after myocardial infarction (MI). Pharmacological coronary vasodilatation is known to alter regional myocardial blood flow. However, the effect of redistribution of coronary blood flow during pharmacological coronary vasodilatation on myocardial function is unclear. We hypothesized that increased disparity of myocardial blood flow can lead to nonhomogenous regional ventricular repolarisation. METHODS: We examined the effect of pharmacologic coronary vasodilatation on QTd in 81 subjects who received dypiridamole during myocardial stress imaging study (imaging). Patients with history of MI in the past 2 weeks, electrolyte imbalance, cardiac arrhythmia, long QT syndrome, those on pro-arrhythmia agents were excluded. Routine EKGs were performed at rest (0 Mts) and peak stress ( 7 ± 9 Mts after dipyridamole infusion). All EKGs were interfaced to a personal computer equipped with a digitizing tablet consisting of a magnifier used to enhance the accuracy of measurements. QT intervals were measured in all 12 leads and corrected for heart rate (QTc) using Bazett's formula. Intra & interpersonal variations were minimized by repeated blinded measurements. Patients were divided into three groups based on clinical probability and the results of the myocardial perfusion scan. 25 subjects with high pre-test probability for IHD and high risk reversible defects on imaging (reversible group) were compared with 33 other subjects with low clinical probability and normal imaging study (``normal'' group). 12 patients with low risk fixed defects on imaging and variable probability (``fixed'' group) were also studied. Mean Qtcd values at rest and during pharmacologic stress testing were compared in each group. Statistical analysis was done using t test. Results are expressed in mean+ SD.
Imaging results ( * n) * QTcd at rest Examination Survey, 1988 ± 1994 , a stratified, multistage sample of the US civilian non-institutionalized population. The study involved detailed home interviews, a careful physical exam, laboratory and other measurements. The study sample comprised 5,063 subjects with HTN. Ethnicity indicators were non-Hispanic Whites (NHW), non-Hispanic Blacks (NHB) and Mexican ± Americans (MA). Socioeconomic indicators were years of education and the poverty ± income ratio (PIR). The outcome measures were treatment (self-report), adherence (self-report) and control of HTN (SBP < 140 and DBP < 90). Treatment measures were medication prescriptions, counseling to restrict salt or sodium, Counseling to lose weight, or use of other methods like exercise, restrict alcohol intake, and to change diet. Adherence measures were self-report of taking medications, report of salt or sodium restriction, attempts to lose weight, and report of other approaches like exercise, alcohol restriction, and to change diet. BP control was measured by taking the mean of 6 BP measurements taken on 2 different days. Multivariate regression analyses, while accounting for the complex sampling design by using the appropriate sampling weights, adjusted for age, sex, access to health care, obesity, and urbanization. Significant results (p < .05) are reported as means or as odds ratios (OR) with the referent category being NHW, PIR less than or equal to 1 or < 12 years education. RESULTS: There were no racial or socioeconomic differences in likelihood of prescription of medicines or weight reduction counseling. NHB were more likely to be counseled for salt restriction (OR 1.4) . Subjects with > 12 years education were more likely (OR 1.5) to be counseled to exercise, restrict alcohol intake, and to change diet. In subjects who were counseled, NHB (OR 1.6) and subjects with > 12 years education (OR 1.6) were more likely to report salt restriction. NHB were also more likely to attempt to lose weight (OR American adults over age 25 who completed the household questionnaire and laboratory exam in NHANES III. All respondents were asked:``Have you ever had your cholesterol checked?'' If respondents answered yes, they were asked:``Have you ever been told by a doctor that your cholesterol level was high?'' and``Because of your high cholesterol, have you ever been told by a doctor to take prescribed medication?'' If a prescription medication was taken, the interviewer asked to see the medication container. Bivariate and multivariate analyses were used to examine the impact of race and ethnicity on cholesterol screening and on taking a cholesterol lowering medication. Unweighted counts and weighted percentages and odds ratios are presented. Odds ratios are adjusted to account for age, gender, income and, education, insurance status, comorbid illness and regular source of health care. RESULTS: African Americans and Mexican Americans were significantly less likely than whites to report ever having their blood cholesterol checked (Table 1) . Of those who were told to take medication, African Americans and Mexican Americans were significantly less likely to be taking a cholesterol-lowering agent ( Table 1 ). The mean total serum cholesterol (from the laboratory exam) of those who reported being told their cholesterol was high was 234 mg/dL as compared to a mean of 198 mg/dL of those who were not told their cholesterol was high (p < .001).
CONCLUSION: African Americans and Mexican Americans are less likely to report serum cholesterol screening than whites. Even when identified as requiring medication, African Americans and Mexican Americans were less likely than whites to be on cholesterol lowering agents. These disparities in primary prevention are likely to lead to an increase in the risk of coronary artery disease in these populations. To describe the outcome of the Institutional Review Board (IRB) process from a multi-site QI evaluation and to examine participation rates as a function of recruitment and informed consent procedures. METHODS: We analyzed participation rates from a patient survey and medical record abstraction from 1504 individuals with either congestive heart failure or diabetes treated at 13 clinical sites in the United States that had implemented an evidence based QI intervention. We used bivariate analyses to assess the effect of recruitment procedures and informed consent requirements on participation rates. RESULTS: Recruitment procedures mandating patient permission to be contacted about the study varied by clinical site (Table 1 ). Among the sites that required advanced permission, only 58% (584/1012) of potentially eligible participants granted permission to be contacted about the study. Participation rates varied significantly by the type of recruitment and informed consent procedures required ( Table 2) . CONCLUSION: We found substantial variation in provisions for subject recruitment and informed consent. These differences significantly impacted participation rates, and possibly the generalizability of this multi-site QI evaluation study. To describe the demographic characteristics of participants who completed a mailed survey as compared to those initial non-responders who completed the same survey over the telephone. METHODS: To better understand barriers to care for abnormal cervical cytology, we performed a cohort study of all women with an abnormal pap smear who received care at Kaiser Permanente Los Angeles Medical Center between July 1998 and October 1999. We administered a mailed survey with telephone follow-up for non-responders between April and August 2000. Potential participants were mailed an introductory letter and survey in both English and Spanish with a self-addressed stamped return envelope. If no response was obtained within three weeks, a second letter and survey were mailed. If no response was received within three weeks, a trained bi-lingual interviewer called potential participants and offered to complete the survey over the telephone. The survey asked questions regarding patient satisfaction, health beliefs, cancer knowledge, and socio-demographics characteristics. We used bivariate and multivariate analyses to describe the population characteristics of respondents who replied by mail as compared to those who completed the survey by telephone. RESULTS: Of the 1049 potentially eligible participants, 733 women completed the survey for an overall response rate of 70%. Thirty one percent (n = 226) of the surveys were received after the first mailing, 25% (n = 185) were returned after the second mailing, and 44% (n = 322) were completed over the telephone. Sixty seven percent of those who completed the survey in Spanish were obtained by telephone, compared to only 37% of the surveys completed in English. (p < .001) In multivariate analysis, Latinas who completed the survey in Spanish and African American women were significantly more likely to complete the telephone survey than their white counterparts. Telephone respondents were younger, had lower household incomes, and were significantly less satisfied with their health care than those who responded to the mailed survey. Women who completed the telephone survey had more misconceptions about cancer and were more likely to report no knowledge of having an abnormal pap smear. Harvard University, Boston, MA PURPOSE: Pharyngitis is the second most common presenting complaint in the primary care office. Since rheumatic fever has become uncommon in the United States while rapid streptococcal antigen test technology has improved and concerns over drug side effects have grown, we examined cost-effective diagnosis and treatment of patients with suspected GABHS pharyngitis. METHODS: We constructed a decision analysis model from a societal perspective to examine the short-term cost-effectiveness of five strategies for the management of adult patients with pharyngitis: 1) Observation without testing or treatment, 2) Empiric treatment with penicillin, 3) Throat culture using a two-plate selective culture technique 4) Optical immunoassay (OIA) followed by culture to confirm negative OIA tests, or 5) OIA alone. We obtained data on test characteristics and event probabilities from published studies. We estimated utilities from a published patient survey which used a time-tradeoff technique to measure perceptions of outcomes (mild penicillin reaction, severe penicillin rxn, acute rheumatic fever) expressed in pharyngitis day equivalents. Based on published studies in which patients assigned a utility of 0.95 to other common symptoms such as diarrhea and dyspepsia, we estimated phayngitis to be associated with a utility of 0.95. We estimated costs from our hospital's financial systems, prior publications, and Medicare reimbursement rates. We converted all costs to year 2000 dollars. In sensitivity analyses, we varied values for probabilities and costs across ranges that included all published values or across ranges of 50 ± 200% of our baseline estimates. RESULTS: At a baseline prevalence of GABHS pharyngitis of 9.7%, a culture alone strategy was both most effective and least expensive, resulting in an average of .2618 quality-adjusted life days (QALDs) lost and an average cost of $6.19 per patient. For our base-case analysis, average costs in dollars, effectiveness in lost QALDs, and incremental cost-effectiveness were as below (see table) . Our results were sensitive to the prevalence of GABHS, so that OIA followed by culture was most effective at a prevalence greater than 17%. Observation was least expensive at a prevalence less than 3% and empiric treatment least expensive at a prevalence greater than 75%. As the probability of allergic reactions was varied throughout a possible range from 0 to 5%, OIA with culture, culture, and observation were all optimal strategies. Our results did not change with variations in costs of diagnosis or treatment. CONCLUSION: We found that observation, culture, and two rapid antigen test strategies for diagnostic testing and treatment of suspected GABHS pharyngitis in adults were all very similar in terms of both effectiveness and costs, although culture was least expensive and most effective at our institution's GABHS prevalence of 9.7%. We did not find empiric treatment to be most effective or least expensive at any prevalence of GABHS seen in adult populations. METHODS: A population-based telephone survey of 1205 women from a combination of random-digit-dial and targeted listed household samples from lower income census tracts of Washington, D.C. was conducted from January ± March, 2000. Valid and reliable measures of specific aspects of primary care and of the physician ± patient relationship were used. RESULTS: The survey response rate was 85%. Socioeconomic characteristics of the respondents reflected success in reaching the population sought. Four attributes of primary care (continuity, organizational accessibility, comprehensiveness of services, and coordination of specialty care) were important positive correlates of all aspects of the patient ± physician relationship (trust, compassion and comprehension), regardless of insurance, demographics, socioeconomic or health status. For example, women with the highest level of comprehensiveness of services at their usual source of care, were 11 times as likely to trust their physician (p < .01) and 6 times as likely to find their physicians compassionate and communicative (p < .01), compared to those with the lowest level of comprehensiveness. Higher organizational accessibility was strongly associated with greater trust, compassion and communication aspects of the physician ± patient relationship. (OR 3.2, OR 7.4 and OR 6.9 respectively, p < .01 for each). CONCLUSION: Women in ambulatory care systems that most strongly exhibit the features of primary care report stronger physician ± patient relationships than women whose usual sources of ambulatory care lack those primary care features. Ambulatory care systems which are organized to be more accessible to consumers, which permit patients to see the same clinician for their visits, which provide more comprehensive services, and which allow clinicians to coordinate specialty services are more likely to foster strong relationships between their patients and physicians, and to have patients who are more satisfied with those relationships. We excluded studies based on previously published decision analyses. The articles were reviewed by 2 investigators who were blinded to author and journal. We extracted information necessary to determine if the search strategies and databases searched were reported, if reference lists were reviewed for additional studies, if a systematic effort was made to find relevant unpublished studies, and if an a priori selection criteria or any validity grading system was used. RESULTS: We identified 257 articles through our Medline search and 36 met our eligibility criteria. The search strategy, including databases searched, was reported in 9 (25%) of the articles. Reviewing the reference list for additional studies was reported by 5 (14%) of the articles. Though no article reported a systematic effort to locate unpublished studies, 3 articles did report using data from unpublished sources. Two studies (6%) reported using a selection criteria and one study (3%) a grading system for the quality of the evidence. CONCLUSION: Our study shows that only 25% of CDAs published in major medical journals reported their process for selecting and evaluating the data they used. This is very concerning in view of the importance these items play when readers must appraise the validity of a CDA.
Although it is possible that most studies were very rigorous in their selection of data, the lack of a clear report in the methods section makes it difficult for readers to identify stronger CDAs from ones that are less evidence-based. Our limitations include the fact that some of these data elements can only be obtained from databases like SEER, but all of these studies included probabilities that were obtained from the medical literature. We believe that the addition of evidence selection and appraisal in CDAs can strengthen these studies and give readers more confidence to evaluate their results. SD 3.8) ; the median number of months with the same counselor was 12. We found a high level of interest in quitting: 81% expressed the desire to quit, and 71% had plans to quit within the next six months. Sixty-nine percent agreed at least somewhat that counseling may aid people with nicotine addiction, and 73% that smoking cessation should be discussed in counseling sessions. However, 56% reported that their counselors had never asked about smoking. Over two-thirds reported that counselors never or rarely 1) discussed the adverse health effects of nicotine (68%), 2) advised them to quit (68%), nor 3) discussed smoking cessation options (72% We collected data on risk factors for coronary artery disease and type of surgery to be performed during the preoperative evaluation. All patients had CBC, routine chemistry, coagulation studies, electrocardiogram, and chest x-ray according to the protocol followed in the ambulatory surgery unit. A chart review conducted at least a month after the surgery was done to assess the incidence of postoperative cardiovascular complications. RESULTS: Average age of patients studied was 57, with a range of 28 ± 86 years. There were 108 men and 173 women. 30% had an abnormal electrocardiogram, 61% had hypertension, 30% had diabetes, 37% patients had at least one other risk factor for coronary disease, and 53% had two or more cardiac risk factors for surgery. The type of surgery included a variety of ambulatory procedure. The most common were eye 21%, breast 15%,cholecystectomies/ laparatomies 10%, hernias 7%, and ENT 5%. Overall outcome There were no patients in this cohort group who had postoperative cardiac complications. CONCLUSION: The incidence of cardiac complications after low-risk ambulatory procedures is low, even in the patients with risk factors. Additional testing is very unlikely to add to the ability to predict risk in this group of patients. À.13 ** À.21 ** À.08 * À.18 ** À.20 ** +.26 ** Fat Intake À.07 À.08 * À.06 À.08 * À.10 ** +.10 * Fr/Veg Intake À.14 * À.13 ** À.11 ** À.16 ** À.12 * +.18 ** Manage Stress À.33 ** À.31 ** À.15 ** À.32 ** À.25 ** +.38 ** Tob(Smokers only) À.23 À.11 À.12 À.03 À.03 +.12 Phys Activity À.19 * À.22 ** À.13 ** À.22 ** À.20 ** +.28 ** * P < .05; ** P < .001 PURPOSE: Diabetes mellitus is associated with abnormal autonomic function. Impaired glucose tolerance and diabetes mellitus are related to increased mortality. Abnormal heart rate recovery (aHRR), a measure of autonomic dysfunction, is also known to be associated with increased mortality. Whether impaired fasting glucose (IFG) is associated with aHRR has not been characterized. METHODS: 5190 healthy adults without medically treated diabetes (mean age 45, 39 % women) enrolled in the Lipid Research Clinics Prevalence study underwent exercise testing. Low physical fitness was defined according to the lowest quartile of peak METs. HRR was defined as the change from peak heart rate to that measured after 2 minutes of recovery, an abnormal value was < 42 BPM. RESULTS: 504 subjects (10%) had IFG and 131 (3%) were diabetics. An aHRR was found in 1699 (33%) adults; 1196 (23%) were unfit. Increasing levels of fasting plasma glucose (FPG) were strongly associated with aHRR (see figure) , even at glucose levels < 110 mg/dl. The association between increasing FPG and poor physical fitness was weaker. FPG remained an independent predictor of aHRR after adjustment for standard risk factors and resting heart rate (RHR) (adjusted OR for increase of glucose of 500/glucose by 1, 1.17, 95% CI 1.05 ± 1.30, chisquare = 9, P = 0.003). There was a strong interaction between FPG and RHR for prediction of aHRR. The association between FPG and aHRR was very strong with RHR 80 BPM (P for interaction = 0.0008); no association was present with RHR < 80 BPM. CONCLUSION: 1. FPG is strongly and independently associated with aHRR, even at nondiabetic levels. 2. The association between FPG and aHRR is substantially affected by resting heart rate. Internists. When practicing physicians are asked about the areas of medical practice where they feel the least prepared by their formal medical education and the most uncomfortable in clinical practice, they frequently cite scenerios around controlled drug prescribing. These include acute / chronic / and malignant pain management, management of anxiety vs. depression, management of insomnia, identification and management of addictive dissorders, and opioid and benzodiazepine pharmacology. Pilot data exist to suggest that the prescribing of controlled drugs in primary care clinic settings bear little if any resemblence to current practice recommendations. As part of a controlled clinical trial of alcohol screening and interventions in a primary care population, we performed a chart review in the Resident and Attending Primary Care Medical Clinic of a large urban teaching hospital. In an effort to assess controlled drug prescribing practices, part of the chart review included assessing whether there was addiction screening information in the patient record when controlled drugs were prescribed. METHODS: A total of eight hundred charts were randomly chosen from the three Internal Medicine Clinic resident firms as well as from the Attending clinic. Charts were categorized based upon 1) whether controlled drugs were prescribed at any time in the 12 months prior to the audit, 2) the type of controlled drug prescribied, 3) wheter there was any evidence of an alcohol or drug abuse histroy documented in the chart, and 4) the result of that history if documented. The charts were abstracted by trained research assistants using a data template and supervised by the Project Director and the Project Administrator. RESULTS: A total of 768 charts were completely reviewed, of which 135 indicated some prescribing of controlled drugs (17.6%). The percentage of patients receiving controlled drug prescriptions was not different based upon the gender of the patient (18%of men and 17%of women), and the type of controlled drugs showed no gender differences. 45%of women and 35%men had no evidence of a substance use history having been taken in their charts, and 3%of women and 13%of men had charts that documented hazardous or harmfull use of alcohol or drugs. Of the 70 male pateints prescribed controlled drugs, they represented 17.6%of those wit hno substance use assessment, 18.8%of those with a low risk substance use assesment, and 17%of those with active substance use problems documented in their charts. For the 65 women patients prescribed controlled drugs, they represented 18.5%of those with no substance use assessment, 14.7%of those assesed as low risk, and 33.3%of those wit hchart documentation of active addictive problems. There were no differences between the controlled drug prescribing patterns of attendings versus residents. CONCLUSION: The rate of prescribing controlled drugs and the appropriateness of prescribing decisions is becomming more and more closely monitored, bot hby regulatory agencies as well as advocay groups. Little is known about the controlled drug prescribing practices of practicing physicians, but they self-report it as an area of great un-ease. Although guidelines are not well established in this area of practice, there is general agreement that ambulatory controlled drug prescribing is relatively contraindicated in the face of active or past substance abuse on the part of the patient. This pilot data contains concerning information to suggest that a lack of any documentation of substance use histroy is common place when prescribing controlled drugs in primary care. Worse, the precentage of patients receiving controlled drug prescriptions who have a documented active substance abuse problem, is as high as those who receive a prescrition without any history taken, and atlease as high as those who both have an apropriate history AND whose hostory indicates that they are a low risk population.
Ominously, attending prescribing practice seems identical to house staff practice patterns. PURPOSE: Infliximab is an antibody to tumor necrosis factor-alpha. This drug has in several studies been shown to be effective in treating patients with inflammatory and fistulous Crohn's disease (CD) refractory to conventional therapy. However, treatment with infliximab is expensive and has associated toxicities. In addition, the mean duration of response to infliximab is approximately 12 weeks which necessitates re-dosing at regular intervals. Therefore identifying predictors of rate of response to infliximab could be of great benefit in selection of patients for this treatment. Furthermore, identifying factors that are associated with a prolonged duration of response could allow for modification of these factors to achieve a longer duration of response in treated patients. The purpose of this study was to identify factors predictive of rate and duration of response to infliximab in patients with CD. METHODS: 100 patients with refractory CD (59 with inflammatory CD and 41 with fistulous CD) and at least 3 month of follow-up following infliximab infusion were evaluated (mean follow up = 9 months; range = 3 ± 17 months). Clinical-response, duration of response, smoking history, gender, race, duration of disease, concurrent immunosuppressive or steroid use, age at diagnosis and age at infusion were analyzed. RESULTS: Rate of response: 67%of patients responded to infliximab. Rates of response were significantly higher in patients with fistulous disease (80%) compared to those with inflammatory disease (58%); p < 0.02. 77%of non-smokers responded compared to 49%of smokers (p < 0.004). The favorable response in non-smokers applied mostly to patients with inflammatory disease (p < 0.001). Concurrent use of immunosuppressive medication was also associated with higher rates of response in inflammatory disease(p < 0.007). Duration of response: There was a statistically significant longer duration of response among non-smokers compared to smokers both for inflammatory and fistulous disease. Among patients with inflammatory Crohn's disease, 87%on concurrent immunosuppressive medication had a duration of response longer than 2 months compared to only 45%not on any immunosuppressants (p < 0.04 The social norm for a larger body image may support the propensity for overweight and obesity, which has a prevalence of 66%, in black women. The goal of this study was to determine a psychometrically stable and socially acceptable Figure Rating Scale (FRS) for assessing body image in black women. METHODS: The study sample (n = 50)was selected sequentially from black women (mean age 52.3+/À 10 years) who were being screened for a larger randomized trial of nutrition and exercise education in urban black churches (Project Joy). Three standard published FRS were compared with a new culturally specific scale, the Reese FRS, developed from digitized photographs of black women and modified according to recommendations from focus groups. All four FRS consisted of nine ordered body images increasing in size from very thin to obese. Distributions of respondent' s selections on all scales were examined relative to anthropometric measures, including body mass index, BMI(weight in kg/m2) and categories of obesity and overweight (National Expert Panel on Overweight and Obesity Guidelines). Cultural identity, based on questions adopted from an African American Acculturation scale, and its relationship to FRS preference was also assessed. RESULTS: Body weight distribution for US black women over 40 years matched that found in NHANES III. All four FRS performed similarly and correlated significantly with BMI, r=À 0.70 to À 0.75, p < 0.0001. The percentage of obese women who identified with one of the three largest images in the FRS was 22%on three scales and 38%on one; this is markedly below 56%of women who were obese based on the national guidelines. Overall, 44%of the women preferred the new Reese FRS; among those women with cultural identity scores in the upper quintile, 72%preferred the new FRS. CONCLUSION: Overall, for all FRS, there is considerable overlap among images selected for all weight categories. There is a strong preference for the culturally specific Reese scale, especially among women with strong cultural identity scores. This new FRS appears to be more socially acceptable. The failure of a large percentage of obese black women to identify themselves as obese needs further investigation. PURPOSE: Evidence supports the effectiveness of collaborative care for depression in primary care, incorporating clinician education, proactive care management, and collaboration with mental health specialists. Little is known, however, about the influence of proactive care management on processes and outcomes of care for depression. The purpose of these analyses is to assess adherence to specific aspects of the nurse care management protocol, and evaluate the effects of adherence on quality of care and intermediate outcomes.
METHODS: We used a randomized encouragement design to study the effects of assisting 6 managed care organizations to implement collaborative care for depression in 30 experimental and 16 control practices. We identified a total of 920 depressed patients and referred them to their clinical practice site' s collaborative care program. Access to the program was through a nurse care manager. Care managers recorded all patient contacts on pre-structured forms. Patients completed self-administered surveys at baseline and every six months. Our evaluation uses bi-variable and multi-variable regression analyses. RESULTS: Overall, 73%of patients completed initial assessment visits. There was considerable variation among sites in adherence to the care management protocols, e.g., assessment of patient treatment preferences at the initial visit ranged from 18%to 100%at different sites (73%of patients assessed overall).
In bivariate regression analyses, greater performance of care management processes was strongly associated with higher quality of depression care, including the degree to which patients received and adhered to appropriate anti-depressant and psychotherapy regimens. Performance also related to improved levels of patient knowledge, probable depression, and active coping. When nursing assessment, proactive follow-up, and other self-management support processes were included together in regression analyses, the number of follow-up contacts had the greatest effect on patient outcomes (p < 0.001). CONCLUSION: Practices assisted in implementing collaborative care models for depression achieved varying levels of adherence to the care management design. Of all the processes studied, the amount of proactive follow-up had the strongest effect on care and outcomes.
PRIMARY CARE PHYSICIAN DEPARTURE: EFFECTS ON HEALTH CARE QUALITY. A.G. Pereira 1 , S.D. Pearson 1 ; 1 Harvard Medical School/Harvard Pilgrim Health Care, Boston, MA PURPOSE: Discontinuity of care is an important health policy issue, but few data exist to assess its effects on health care quality or outcomes. This study evaluated measures of health care quality in patients whose primary care providers (PCPs) left their medical practice. METHODS: This study was performed in a large, multi-site multispecialty group practice associated with a single insurer. We used a controlled pre-post design to compare measures of health care quality received by patients whose PCPs left the practice (LEAVEmds) with the measures of health care quality in patients whose PCPs did not leave the practice (STAYmds). For all patients, we compared rates of preventive care, and in patients with hypertension, we compared blood pressure control. The practice had a standardized process for reassigning patients of departing PCPs. Patients were eligible for analysis if their insurance and care within the practice was uninterrupted over a four-year study period. LEAVEmds were matched to eligible STAYmds by age, sex and practice site. RESULTS: During the study period, nine PCPs, caring for 3,931 patients, voluntarily left the group practice. These LEAVEmds were matched to 16 STAYmds, caring for 8,009 patients. Mean age of LEAVEmd patients and STAYmd patients was the same: 46 (13); 76%of the LEAVEmd patients were women, as compared to 80%of the STAYmd patients (p < 0.01). During the two baseline years prior to LEAVEmd departures, there were no differences in quality of care measures between the two groups of patients. In the two years following PCP departure there was no difference in rates of Pap smears among women aged 25 ± 65 (88%vs. 88%, p = 0.85), or in mammography in women 50 ± 65 (90%vs. 93%, p = 0.09). Rates of fecal occult blood testing for colon cancer in all patients aged 50 ± 65 were lower among LEAVEmd patients (51%vs. 56%, p = 0.02). There was no difference in the proportion of patients with hypertension who experienced a rise of !10%in mean systolic and/or diastolic blood pressure (6.0%vs. 7.0%, p = 0.53). CONCLUSION: Overall, PCP departure from this group practice was not associated with substantial decreases in rates of several important preventive screening measures for their patients who continued to receive care within the practice. Among patients with hypertension, we found no difference in rates of blood pressure control. Further research is needed to address two questions: first, whether these findings are consistent with the experiences of patients in a variety of practice settings, and second, whether PCP discontinuity may adversely affect subgroups of patients with other chronic conditions. PURPOSE: Helical computed tomography (CT) is commonly used to diagnose pulmonary embolism (PE), although its operating characteristics have been insufficiently evaluated. Therefore, we aimed to assess the performance of helical CT. METHODS: Two-hundred and ninety-nine consecutive patients admitted to the emergency ward for clinically suspected PE, with a D-dimer level above 500 "g/L (ELISA assay) were included. The diagnosis of PE was established by a validated algorithm including clinical assessment, lower limb compression ultrasonography, lung scan and pulmonary angiography. The CT scans were read by radiologists blind to all clinical data 3 months after image acquisition. RESULTS: Pulmonary embolism was present in 118 patients (39 %) in the study population. In 12 patients (4%), the CT scan was inconclusive, of whom two had a PE. Among patients with a conclusive CT, sensitivity of CT was 70%(95%CI 62 to 78), specificity was 91%(95%CI 86 to 95), positive predictive value was 84%(95%CI 76 to 91) and negative predictive value was 82%(95%CI 76 to 87). Sensitivity of a strategy adding lower limb ultrasound to helical CT increased to 79%(95%CI 71 to 86), and associating a lung scan in patients with normal ultrasound, helical CT being performed in patients with a nondiagnostic lung scan, would have a sensitivity of 95%(95%CI 89 to 98) and a specificity of 93%(95%CI 88 to 96). Interobserver agreement was high (kappa coefficients 0.82 to 0.90). CONCLUSION: Helical CT should not be used as a single test in suspected PE, but it could replace angiography in combined strategies including ultrasound and lung scan. PURPOSE: Helical computed tomography (CT) is increasingly used in the diagnosis of suspected pulmonary embolism, alone or in combination with other diagnostic tests, such as lung scan, lower limb ultrasound (US) or plasma D-dimer measurement (DD). Therefore, we assessed the cost-effectiveness of including helical CT in the diagnosis of suspected pulmonary embolism. METHODS: We performed a formal cost-effectiveness analysis by a decision model. Probabilities for test characteristics and clinical outcomes were obtained from the literature. Cost estimates were derived from our hospital's database. We compared 1) CT as a single test, and 2) inclusion of CT in a sequential strategy resting on clinical assessment, DD, US, lung scan and pulmonary angiography, either as a substitute for angiography or for lung scan. These strategies were compared with a reference strategy in which all patients with a nondiagnostic lung scan underwent an angiogram. Outcome measures were costs per patient, 3-month quality-adjusted survival, and incremental costs per quality-adjusted life years (QALYs) gained. RESULTS: Helical CT as a single test is associated with a 1%higher mortality than the reference strategy, and higher costs ($3,439 per QALY compared to $3,202 per QALY for the reference strategy). Replacing angiography by CT in the sequential strategy, was the most costeffective strategy ($2,447 per QALY gained). Replacing lung scan by CT was also cost-effective ($2,700 per QALY gained) provided an angiogram was performed in patients with a high clinical probability and a negative CT. Omitting DD measurement from the strategies increased costs but did not change effectiveness. The results were stable over the entire range of values tested in sensitivity analysis. CONCLUSION: Helical CT as a single test in suspected pulmonary embolism is not costeffective. However, CT may be cost-effective when included in combined diagnostic strategies.
TRUST OF PHYSICIANS AND SATISFACTION AMONG MINORITY PATIENTS WITH ISCHEMIC HEART DISEASE. L.A. Petersen 1 , T.C. Collins 2 , N.R. Kressin 3 ; 1 VAMC, Houston, Texas; 2 Baylor College of Medicine; 3 Boston University, Bedford, Massachusetts PURPOSE: Studies show that African ± Americans (AAs) are less satisfied with their health care. Since AAs have historically experienced bias within the health care system, and recent work suggests bias on the part of physicians toward minority patients, the goal of this study was to assess racial differences in patients' trust and satisfaction with their physicians regarding decision-making for coronary disease treatment. METHODS: We prospectively enrolled 431 males with nuclear imaging studies graded as positive for cardiac ischemia (71.6%self-reported white, 28.4%self-reported AA) at 5 VA hospitals (Houston, Atlanta, St. Louis, Pittsburgh, Durham). We collected survey data using the published Trust in Physician scale, Seattle Angina Questionnaire, and new items developed from focus groups. RESULTS: There was no difference in mean age of the groups. AAs were less likely than whites to be``completely satisfied'' that everything possible is being done for their cardiac symptoms (44.4%vs 51.4%, respectively; P = 0.05). There was a trend toward more AAs reporting dissatisfaction with explanations provided by the physician (14.8%vs. 8.0%; P = 0.12). Though all patients had positive imaging studies, equal percentages (33.4%vs 33.8%; P = 0.98) thought they``did not have heart disease''. There were no racial differences in the percentage reporting that their heart condition was``not at all serious'' (3.2%vs 3.1%; P = 0.97). There were no racial differences in the percentage of patients who preferred to leave all treatment decisions to their doctors, who agreed with statements that doctors knew the patient' s medical and personal situations, or who reported that doctors were respectful or were concerned about them. Despite racial differences in overall satisfaction with medical care, there were no differences in the percentage who agreed that they``trust the doctor' s judgments about medical care'' (85.3%vs 88.7%; P = 0.17) or who stated that the doctor was``well qualified to manage medical problems'' (83.0%vs 87.5%; P = 0.79). CONCLUSION: Though we found racial differences in satisfaction with care and with physician ± patient communication, there were no racial differences in patients' trust of their physicians. Further work should assess whether there are racial differences in the construct of trust not captured with current measurement scales as well as develop strategies to improve physician ± patient communication and satisfaction with care.
PURPOSE: H 376/95 is a novel, oral direct thrombin inhibitor that shows predictable pharmacokinetics and has not shown clinically significant food or drug interactions. Anticoagulants such as warfarin are used to lower the risk of stroke in patients with nonvalvular atrial fibrillation (NVAF). In the SPORTIF II study, the tolerability and safety of three doses of H 376/95 were compared with warfarin in NVAF patients. METHODS: This was a randomized, parallel-group study of NVAF patients who had at least one additional risk factor for stroke. The primary outcome was the number of adverse events, e.g., bleeding and thromboembolic events. The duration of treatment was 12 weeks, during which three groups received H 376/95 (n = 187) 20, 40 or 60 mg bid, given double blind. In a fourth group, warfarin (n = 67) was managed and monitored according to normal routines, aiming for an INR of 2.0 ± 3.0. RESULTS: A total of 257 patients were randomized to treatment and 254 patients received study drug. The median age was 70 years (range: 39 ± 95). In addition to NVAF, all patients had one additional risk factor for stroke, 75%had two additional risk factors, and 42%had three or more additional risk factors. The number of minor bleeds was comparable and low in all four groups. There was only one major bleed (vaginal) observed in a patient receiving warfarin and none in the H 376/95 groups. Out of 67 patients in the warfarin group, two patients had transient ischaemic attacks. Of the 187 patients in the H 376/95 groups, one patient had a transient ischaemic attack and one patient had an ischaemic stroke; both patients were in the H 376/95 60-mg bid group. CONCLUSION: Fixed doses of H 376/95 up to 60 mg bid were well tolerated, without the need for dosage adjustment or coagulation monitoring, during a 3-month treatment period in NVAF patients with medium-to-high risk for stroke and systemic embolism.
in mortality is a discrepancy in the rate of follow-up of abnormal Pap smears among African ± American women. The objective of this study is to determine the demographic factors associated with a delay to follow-up of abnormal pap smears. METHODS: Eligible subjects were a sample of women aged 18 years and older with an abnormal cervical cytology report between February 1999 ± April 2000 screened at an academic medical center or one of three neighborhood health centers. We obtained information on subjects from a registration database and a pathology database. We excluded subjects who did not have a race category specified (17%). We defined adequate follow-up conservatively, as cytology or pathology within 4 months for dysplasia (low or high grade squamous intraepithelial lesion or carcinoma in-situ) and within 7 months for atypia (atypical squamous or glandular cells of unknown significance). We analyzed differences in follow-up rates by race, age, insurance status, source of care (academic medical center or neighborhood health center), and type of pap abnormality (dysplasia or atypia). RESULTS: Of the 345 subjects, 57%were African American, 16%were Caucasian, and 26%were from other racial/ethnic groups. 49%of the subjects were under the age of 30. 62%were screened at the medical center and 38%at the neighborhood health centers. Only 18%of the subjects had private insurance. Overall, only 53%of the subjects had follow-up for an abnormal pap smear either by additional cytology or pathology within 4 months for dysplasia (50%) or 7 months for atypia (56%). 55%of African ± American women, 55%of Caucasian women, and 48%of women from other racial/ethnic groups received follow-up (not significant). The follow-up rate of 45%for subjects less than 30 was significantly lower than that of subjects aged 30 or older (61%, p-value 0.003). No differences in follow-up rates were seen according to the subject's race, insurance status, source of care, or type of pap abnormality. CONCLUSION: In a low income, predominantly minority population, the rate of follow-up for abnormal pap smears was low, only 53%. This may explain the higher mortality rate seen in African ± American women. Younger women were less likely to receive adequate follow-up. Race, source of referral, insurance status, and type of pap abnormality were not associated with follow-up rates in this cohort. Patients were also asked to express their preferences regarding physician involvement in their religious/spiritual life. Descriptive statistics were tabulated. Associations between patient characterisitics and patients' preferences for specific physician spiritual behaviors were assessed using bivariate and multivariate techniques. RESULTS: Two hundred-ninety nine patients were surveyed. Patients' ages ranged from 19 to 86 years. Fifty six percent were male, 46%were African American, 51%white, and 63%belonged to Protestant denominations. Fifty three percent had annual incomes of $20,000 or less. Fifty nine percent of African American patients (AA) felt it was important that their physician have strong spiritual beliefs compared with 40%of whites (W)(p < .001); 45%of AA vs 32%of W would want a different physician if their doctor did not believe in God or a higher power (p < .001). Forty five percent of AA felt their health would improve if their physician prayed for them vs 32%of W (p = .03). Fifteen percent of AA were willing to give up time spent on medical problems to discuss spiritual issues with their physicians while only 4%of W agreed with this viewpoint (p = .001). In bivariate analysis, significant associations with patient desires for more physician religious involvement in medical encounters (p < .05) were AA race,older age, education less than college, and higher score on the SWB scale. In logistic regression, only AA race remained significantly associated with willingness to give up time spent on medical care in exchange for spiritual discussion (OR 3.8, 95%CI 1.3 ± 11) . AA race (OR 1.9, 95%CI 1.1 ± 3.4) and higher SWB score (OR 1.8, 95%CI 1.3 ± 2.4) were associated with patients' desire for physicians to have strong religious beliefs. Patients who would change physicians if theirs did not believe in God/higher power were more likely to be AA (OR 2.1, 95%CI 1.1 ± 3.9), have higher SWB score (OR 1.5, 95%CI 1.0 ± 2.1) and education less than college (OR 1.9, 95%CI 1.1 ± 3.4). AA race (OR 2.0, 95%CI 1.1 ± 3.5) and being married (OR 2.1, 95%CI 1.2 ± 3.7) were associated with the belief that physician prayer would improve patients' health. Age, sex and physical function had no significant associations. CONCLUSION: Physician religious beliefs and behaviors are more important to AA patients than to white patients. Physicians should be aware that religious discussions might be important for effective communication with AA patients in making medical decisions. PURPOSE: It has previously been reported that many patients would like their physicians to engage in prayer with them as part of their medical care. We studied the relationship of care settings to this preference and physicians' willingness to respond to patients' religious/spiritual needs.
METHODS: We surveyed patients and physicians at 7 medical centers in 4 states (NC,GA,FL,VT). For the patient questionnaire, a trained research assistant administered a 112-item survey that included questions pertaining to demographics, health status, functional status (SF-36), and spirituality assessment (SWB scale). Patients were also asked to express their preferences regarding physician involvement in their religious/spiritual life. The physician questionnaire was administered to residents and primary care practitioners through the mail. Descriptive statistics were tabulated. RESULTS: Two hundred ninety-nine patients and 444 physicians were surveyed. Patients' ages ranged from 19 to 86 years. Fifty six percent were male, 46%were African ± American, 51%white, and 63%belonged to Protestant denominations. Fifty three percent had annual incomes of $20,000 or less. Of the physicians, 45%were practicing physicians and 55%were residents/fellows. Seventy three percent were internists and 21%family practitioners. Forty five percent of physicians were of Protestant denominations. Twenty percent of patients wanted their their physicians to pray with them during a routine office visit, but only 5%of physicians wanted to pray with their patients under the same circumstances. However, 55%of physicians said they would pray with their patients during a routine office visit if the patients requested. If patients were hospitalized and near death, 52%wanted their physicians to pray with them compared to 26%of physicians who felt this appropriate. This number increases to 77%of physicians if a patient requests such prayer behavior. CONCLUSION: Patients' preferences for prayer in medical settings increases in settings associated with more severe illness. Physicians' willingness to engage in prayer with patients rises in a parallel manner but does not match the level of patients' desires unless prayer by a patient is requested. These data suggest that patients should communicate their feelings about prayer to their doctor if they truly desire their physician' s participation. PURPOSE: Alcohol is known to affect HIV risk behaviors and adherence to antiretroviral medications, but its prevalence among HIV-infected persons and effective screening approaches are less well described. Our objective was to determine the prevalence of a history of alcohol problems in patients entering primary care for HIV infection and to assess the positive predictive value of the CAGE questionnaire for alcohol abuse or dependence. METHODS: Between 7/97 and 10/00, HIV-infected patients presenting to a multidisciplinary clinic for evaluation and linkage to primary care were assessed for alcohol problems using the standard threshold of 2 or more positive responses to the CAGE questionnaire. The predictive value of the CAGE was evaluated by administering the CIDI-SAM, an interview for DSM-IV lifetime diagnoses of alcohol abuse and dependence, to a sample of those with a positive CAGE screening test. RESULTS: Among the 715 patients who spoke English or Spanish, mean age was 38.6 years; 70%were male; 50%were Black, 25%Latino, 23%White, 2%other. Primary HIV risk factors were injection drug use 47%, heterosexual 37%, and men who have sex with men 16%. Most (673/715, 94%) were evaluated for alcohol problems: 41%(279/673) had a positive CAGE screening test (2+); 7%(47/673) had 0 or 1+ CAGE scores, but were deemed to have had an alcohol problem by physician clinical judgment. Of the 326 patients with reports of alcohol problems, 116 underwent a diagnostic interview: 80%(93/116) met DSM-IV criteria for diagnosis of lifetime alcohol dependence, 14%(16/116) for lifetime alcohol abuse, and 6%(7/ 116) did not meet criteria for either diagnosis. In the group of patients with 2 or more positive responses to the CAGE questionnaire (n = 102), 95%(97/102) met DSM-IV criteria for alcohol dependence or abuse. In the group not identified by the CAGE but by clinical judgment (n=14), 86%(12/14) met the same criteria. CONCLUSION: Nearly half of patients presenting for HIV care in an urban clinic had a history of past or present alcohol problems, and the positive predictive value of the CAGE questionnaire was 95%for lifetime diagnoses of alcohol abuse or dependence. Given the prevalence of alcohol problems in this population, the potential impact of such problems on treatment, and the high predictive value of this simple screening tool, alcohol screening should be routinely implemented for all patients initiating HIV medical care. PURPOSE: Previous research suggests that diabetic patients with low literacy have lower disease-specific knowledge. We sought to examine the prevalence of low literacy among high-risk diabetic patients and determine if literacy level affects the change in knowledge or average hemoglobin AIC (HBAIC) after a comprehensive intervention to improve diabetes care. METHODS: Using a well-validated literacy instrument, the Rapid Estimate of Adult Literacy in Medicine (REALM), we screened 91 diabetic patients who were referred for enrollment in a comprehensive, pharmacist-based diabetes care program in a university internal medicine clinic. The intervention involved direct verbal teaching, simple diagrams, and phone-based follow-up to help patients better manage their blood sugar levels. We obtained diabetes knowledge scores and HBAIC values at entry and after 3 ± 4 month follow-up. We then analyzed the effect of literacy level on change in knowledge and HBAIC from baseline to follow-up using nonparametric Wilcoxon rank sum tests. RESULTS: Mean age of subjects was 58 (range 27 ± 87); 63%were female, 65%were African ± American, and 38%completed high school. Average duration of disease was 9years (range 0 ± 35). Low literacy, defined as a reading level below 9th grade (REALM < 61) was present in 78%of patients (32%grade 3 or below, 22% grade 4 ± 6, 24%grad 7 or 8). The effect of literacy on the response to the intervention is shown in the Table. CONCLUSION: Low literacy is extremely common among high-risk adult patients with diabetes. Our intervention improved knowledge and outcomes for patients with high and low literacy, but the effect was greater for patients with higher literacy. To prevent disparities in health outcomes, specific interventions to assist low literacy patients should be developed. We prospectively evaluated patients with any viral symptoms (temp > 1008F, headache, fatigue, sore throat, myalgias, night sweats, rash, oral ulcers, or diarrhea) and any risk for HIV infection (sex or drug use) in the prior two months. Primary infection was defined as ELISA negative and RNA positive; chronic infection was defined as ELISA/Western Blot positive and RNA positive. RESULTS: 1010 patients who presented to the UCC with viral symptoms were screened. Of 413 interested and eligible patients, 375 (91%) enrolled and had antibody and RNA testing. HIV infection was diagnosed in 9 (2.4%, 95%CI: 1.1%, 4.5%). Primary infection was diagnosed in 4 (1.1%, 95%CI: 0.3%, 2.7%) and chronic infection in 5 (1.3%, 95%CI: 0.4%, 3.0%). Fever was associated with a higher prevalence of primary HIV infection (7.0%, OR = 24.8, p < 0.0001). Symptoms associated with a higher prevalence of chronic HIV were oral ulcers (5.7%, OR = 6.8, p = 0.018) and diarrhea (4.2%, OR = 12.3, p = 0.0047). The four patients with chronic HIV and available CD4 counts all had very advanced disease (CD4 < 50/uL). CONCLUSION: Undiagnosed primary and advanced chronic HIV infection are prevalent in patients presenting with viral illness to an urban urgent care clinic. Testing for both should be recommended for these patients. PURPOSE: Coronary artery calcium (CAC), a marker of coronary artery disease (CAD), may be detected and quantified using electron beam computed tomography (EBCT). Proposals have been made to use the CAC score to guide cholesterol-lowering therapy, but the costs and benefits of this strategy have not been quantified. We performed a cost-effectiveness analysis to determine whether measurement of CAC with EBCT is a cost-effective method of targeting patients for cholesterol-lowering drug therapy. METHODS: A Markov model was constructed to estimate the incremental cost-effectiveness, over 10 years, of five different strategies:``Treat none, ''``Treat all, ''or test with EBCT and treat if the CAC score is greater than 0 (``Test and treat>0"), 100 (``Test and treat>100"), or 400 (``Test and treat>400"). Overall rates of CAD events were based on Framingham risk equations. Distributions of CAC scores given risk factors were calculated from the literature. Summary relative risks for CAD events in each of four CAC score strata (0, 1 ± 100, 101 ± 400, > 400) were calculated with a random-effects model using data obtained from meta-analysis of articles identified through a systematic search of the literature. Therapy with HMG-CoA Reductase Inhibitors (STATINS) was assumed to reduce CAD event rate by 31%. Cost data were derived from literature review. Two base-case analyses were performed. Case 1 was a 50 year old man with low density lipoprotein cholesterol (LDL) of 175, high-density lipoprotein cholesterol level (HDL) of 45, and no other CAD risk factors. In Case 1, STATINS would not be recommended according to the National Cholesterol Education Program II (NCEP II) guidelines. Case 2 was the same 50 year old man with high cholesterol, but also Stage I hypertension, for whom STATINS would be recommended by NCEP II. RESULTS: Meta-analysis: Four studies met inclusion criteria. Relative risks (with 95%confidence intervals) for CAD events were 1.0 (reference), 3.9 (1.7 ± 8.8), 7.9 (2.5 ± 24.4), and 9.9 (2.8 ± 34.6), given CAC scores of 0, 1 ± 100, 101 ± 400, or > 400, respectively. Cost-effectiveness analysis: In each case, the``Test and treat ''strategies were more costeffective than the``Treat all ''strategy. This result held true while the cost of EBCT was less than $2400 (base case $400/scan), and the cost of STATINS was more than $150/year (base case $849.84/year). It was also insensitive to reasonable variation of other parameters, including the cost of CAD events, overall risk of events, relative risk given CAC score stratum, relative risk reduction with STATINS, and distribution of CAC scores. The``Test and treat > 400'' strategy failed by extended dominance in both cases. In Case 1, the incremental cost-effectiveness ratios (C/E' s) of all``Test and treat ''strategies, though lower than``Treat all,'' were greater than 300,000 $/life-year compared with``Treat none'' (the NCEP II recommendation). Given a reasonable C/E threshold of 50,000 $/life-year, this result was also insensitive to variation of parameters. In Case 2, the C/E of``Treat all'' (the NCEP II recommendation), in comparison to the``Test and treat > 0'' strategy, was 1,600,000 $/life-year. The per-year cost of STATINS would have to be less than $160/year, or the cost of EBCT greater than $2400, in order to make the NCEP II standard (``Treat all'') cost-effective or dominant in comparison to the optimal`T est and treat'' strategy. Note that the NCEP II standard (``Treat all'') in comparison with`T reat none'' also did not meet reasonable C/E standards, consistent with a recent costeffectiveness analysis of STATINS for primary prevention of CAD (Ann Intern Med 2000;132:769 ± 779). CONCLUSION: Screening of patients with EBCT who would not otherwise qualify for cholesterol-lowering therapy, based on NCEP II guidelines, does not meet reasonable costeffectiveness guidelines. On the other hand, using EBCT to identify patients at very low risk for CAD events among patients who would otherwise be treated with STATINS may be a costeffective strategy in selected patient populations. Research on the utilization of screening mammography for women has consistently shown that women are not taking advantage of recommended screening services. Most of the research on breast cancer screening has utilized retrospective crossectional survey designs, convenience samples and has not controlled for important confounding variables. These studies have limited themselves to describing characteristics of patients who do not get screened or listing patient-reported reasons for underutilization. Studies have implicated cost, insurance status and socioeconomic status as strong predictors of who will follow through with screening. METHODS: Randomized, unblinded, controlled interventional design. Consecutive clinic attendees who were > 40 years of age and eligible for a screening mammogram were enrolled in the study (N = 334). Patients were randomized within resident patient panels to be offered free screening mammograms or to be offered mammograms without mention of cost. Residents used a standardized script to offer all elegible patients a mammogram. Those who agreed were given appointments prior to leaving the clinic. The hypothesis was that those patients who were assigned to receive a free mammogram would be more likely to follow through with their mammogram appointment. RESULTS: Baseline characteristics between the control and intervention group were similar. Overall compliance rates were similar for both groups. Fifty four percent of the control group and 63%of the intervention group completed mammograms. This difference was not significant (chi square = 2.88; p = .237). Black women were 23% less likely than their non-black counterpoints to complete the mammogram (p = .069). There was no association between completion of mammogram and age, education, family history of breast cancer or type of insurance. CONCLUSION: Offering free screening mammograms and eliminating many of the known barriers to screening was not sufficient in this population to encourage compliance with screening mammography. PURPOSE: Low health literacy, a hidden obstacle to adequate health care, has significant impact on health services utilization. We set out to determine the association between health literacy of medical inpatients and subsequent emergency room visits, and hospital readmissions. METHODS: The study is a prospective cohort study with 3 months of follow-up, and took place in the medical service of an urban, teaching, hospital in Providence, Rhode Island. Patients admitted to the medical service at Rhode Island Hospital were eligible to participate in the study. A total of 293 hospitalized patients were approached over a seven week period for participation in the study, of which 161 (55%) completed the instrument. Patients who agreed to participate completed the short version of the Test of Functional Health Literacy in Adults (sTOFHLA), which was administered by a bilingual research assistant. RESULTS: Of the participants, 54%were female, and the mean age was 59 years old. 126 (78%) of the participants scored in the adequate health literacy range, which was defined as a score of 23 ± 36 (maximum score = 36). 35 (22%) patients scoring in the marginal health literacy range (17 ± 22), and those scoring in the inadequate range (0 ± 16) were combined into one group as low health literacy. Three months after discharge from the hospital, the hospital computer records were reviewed for the main outcomes-number of emergency room visits and hospital readmissions. There were 22 emergency room visits in the 126 patients with adequate literacy and 15 emergency room visits in the 35 patients with low health literacy (mean number of emergency room visits 0.17 (95%CI 0.082 ± 0.267) vs. 0.43 (95%CI 0.092 ± 0.765), p = 0.04). Patients with low health literacy had 2.5 times more visits to the emergency room than patients with adequate literacy. There were 52 hospital readmissions in the 126 patients with adequate literacy and 25 readmissions in the 35 patients with low health literacy (mean number of readmissions 0.413 (95%CI 0.237 ± 0.589) vs. 0.714 (95%CI 0.346 ± 1.083), p = 0.12). CONCLUSION: In our study of a medical inpatient population, patients with low health literacy had an increased number of emergency room visits within three months of discharge when compared to patients with adequate health literacy. Future studies should be focused on interventions aimed at patients with low health literacy to improve health services utilization. Higher * 10.5% 8.4% À2.1% (À3.5%, À0.7%) À1.1% (À2.5%, +0.4% Lower ** 9.6% 8.5% À1.1% (À1.6%, À0.5%) * Higher literacy is !9th level (n=19); ** Lower literacy is < 9th grade (n=72) ACCESS TO CARE ISSUES IN A PUBLIC HOSPITAL URGENT CARE CLINIC. S. Prock 1 , H. Batal 1 , S. Majeres 1 , L. Lasater 1 , R. Lundgren 2 , J. Adams 1 , P. Mehler 1 ; 1 Denver Health Medical Center, Denver, CO; 2 Colorado Prevention Center, Denver, CO PURPOSE: To describe access to care issues in a population of patients seeking care at a public hospital urgent care clinic. METHODS: 20%of patients presenting to the Denver Health Medical Center urgent care clinic (UCC) between June 15 and August 11, 2000 were randomly approached to participate in a pre-visit interview, offered in both English and Spanish. Patients were asked standardized questions regarding physical and mental health, their reasons for accessing care in the UCC, barriers to health care, history of prior Emergency Department (ED) and UCC usage, reasons for delaying health care, chronic medical conditions, and prior receipt of preventive health services. Study patients' charts and admission records were abstracted for the number of prior visits and documentation of receipt of preventive health services at our institution, ethnicity, age, gender, and insurance status. Data were analyzed using multiple logistic regression. RESULTS: There were 1006 patients surveyed with a refusal rate of 20.3%. The study patients did not differ from the total population who accessed care during this time period in regards to age, insurance status, ethnicity, or gender. A regular source of care, other than an ED or UCC, was identified by only 37.1%of patients. Those patients with insurance were 1.7 times more likely to note a regular source of health care (p = 0.0046) than those without insurance or those on the state indigent care discount program. Hispanic (p = 0.0075) and black (p = 0.04) patients were more likely than white patients to note a regular source of health care. Older patients (p < 0.0001)and female patients (p < 0.0001) were also more likely to report a regular source of health care. Patients accessing care in the UCC were likely to have delayed accessing care, 71%had been sick for more than two days, with 49%noting that their current medical problem had been present for a week or longer. Those patients without insurance or on the state indigent care program were more likely to report a delay in seeking care (p = 0.0279). Among those who delayed care for more than two days, 26.4%reported that a lack of insurance contributed to their delay. CONCLUSION: Patients presenting to a public hospital urgent care clinc often delay care and often do not have a regular source of health care. Having a regular source of care was associated with age, gender, ethnicity, and insurance status. Patients without insurance were more likely to report delaying health care.
H. Quan 1 , J.E. Arboleda-florez 2 , G.H. Fick 1 , H.L. Stuart 2 , E.J. Love 1 ; 1 University of Calgary, Calgary, Alberta; 2 Queen's University, Kingston, Ontario PURPOSE: Only a few small studies have explored the association between various physical illnesses and suicide in the elderly and they have produced inconsistent results. Thus, we undertook this larger study to more definitively assess the association between elderly suicide and physical illness. METHODS: This case-control study included all suicides (920 cases) and motor vehicle accident deaths (1,050 controls) for 1984 ± 95 in the Province of Alberta, Canada among Alberta residents aged 55 years and older. We reviewed Medical Examiner records and extracted sociodemographic information. Then, deterministic linkage was used to link subjects to the Alberta provincial health care registry to determine personal identifiers. Those identified in the registry (1766, 90%) were linked with hospital discharge and physician claims data. We extracted coded diagnoses for each subject in the two years prior to the date of suicide or accident. The accuracy of the administrative data diagnoses was assessed through patient chart review in a sub-set of the study population, revealing kappa values ranging from 0.70 for prostatic disorder to 1.0 for cancer. RESULTS: Compared to the motor vehicle accident victims, the elderly who committed suicide were more likely to be men (78.6% vs. 62.2%), aged 55 ± 64 years old (50.6% vs. 37.5%), unmarried (46.4% vs. 41.0%), white (96.4% vs. 91.7%), residents of high median income areas (43.7% vs. 26.9%) or urban areas (58.4% vs. 40.5%), and to have a history of depression (37.0% vs. 6.2%) or other psychiatric illnesses (57.1% vs. 26.0%). The two groups were similar in the proportion of unemployed (80.9% vs. 82.8% currently). When controlling for sociodemographic characteristics and psychiatric disorders, the elderly who committed suicide were more likely to have had cancer (odds ratio [OR]: 1.73, 95% confidence interval [CI]: 1.16 ± 2.58) and prostatic disorders excluding prostate cancer (OR: 1.70, CI: 1.16 ± 2.49) than were motor vehicle accident victims. Among those who were married, chronic pulmonary disease was more frequent among the elderly who committed suicide than among motor vehicle accident victims (OR: 1.86, CI: 1.22 ± 2.83). Ischemic heart disease, cerebrovascular disease, peptic ulcer disease, and diabetes mellitus were not independently associated with elderly suicide. CONCLUSION: Cancer, chronic pulmonary disease, and prostatic disorders appear to be associated with suicide among the elderly. Physicians and other clinicians should consider assessment and monitoring of patients' suicidal tendencies when such conditions are present. PURPOSE: Hypothyroidism (HYPO) is associated with increased atherosclerosis in autopsy studies. Higher plasma homocysteine (Hcy) levels, even within the normal range, are an independent risk factor for cardiovascular disease. A few studies have shown that patients with HYPO have increased Hcy levels. Some have suggested that this may be due to the slightly increased creatinine (Cr) level in HYPO but other factors affecting Hcy status were not examined. Therefore, we assessed Hcy, vitamin and Cr levels together in patients with HYPO. Because little comparative information exists for patients with hyperthyroidism (HYPER) and none for patients with subclinical hypothyroidism (SC-HYPO), these conditions were also studied. METHODS: Hcy, thyroid stimulating hormone (TSH), free thyroxine (FT4), vitamin B12 (B12), folate and Cr levels were measured in 11 (2 men, 9 women), 10 (2 men, 8 women), and 19 (5 men, 14 women) patients with HYPO, SC-HYPO and HYPER respectively. RESULTS: Mean (SD) Hcy level was higher in HYPO than in HYPER (9.63.0 vs 8.42.7 "mol/l) but not significantly so (p = .26) and only 1 patient had an abnormal Hcy (normal in women = 4.4 ± 12.1 "mol/l). Hcy in SC-HYPO (10.32.2 "mol/l) was also higher than in HYPER (p = .055). Plasma Hcy did not correlate with either TSH or FT4 levels. Although Cr levels were higher in HYPO than in the other groups (p = .0003), the Cr level did not correlate with Hcy. On the other hand, folate levels were significantly lower in HYPO than in HYPER (9.26.0 vs 17.611.2 "g/l, p = .013); folate levels were also lower in SC-HYPO (8.9 3.2 "g/l; p = .005). The folate levels correlated inversely with Hcy levels in the entire population (p = .001) and within the HYPO group (p = .06). B12 levels showed no significant patterns in thyroid disease. CONCLUSION: Our data show that Hcy levels are higher in HYPO than in HYPER but not significantly so. While Cr levels were significantly higher in HYPO than in HYPER, our data suggest that Cr levels are not the major determinant of Hcy changes in thyroid disease. Instead, folate status appears to play an important role in thyroid disease that was not appreciated because vitamin and renal status had not been examined together until now. Finally, our data raise the possibility that early changes in Hcy levels similar to those in HYPO may appear in SC-HYPO. If confirmed by our therapeutic trials, these data may provide an additional reason for intervention in SC-HYPO to mitigate atherosclerotic risk. PURPOSE: Female patients are less likely to undergo cardiac procedures after a myocardial infarction (MI) than male patients. We examined the use of cardiac catheterization and coronary revascularization post-MI to determine whether sex disparities in procedure use were associated with physician sex or were more pronounced when a patient and physician were of different sexes. METHODS: We evaluated data from the Cooperative Cardiovascular Project, a sample of Medicare beneficiaries hospitalized for MI in 1994 and 1995. Patients age 65 years and older with a confirmed MI who presented directly to the hospital with no prior history of revascularization (n = 111,319) were linked with physician data provided by the American Medical Association and evaluated for the use of cardiac catheterization (CATH) and coronary revascularization (REVASC, by PTCA or CABG) within 60 days of admission for MI. Separate multivariable logistic regression analyses were employed to ascertain the influence of patient sex and physician sex for CATH and REVASC use adjusting for patient sociodemographic characteristics, illness severity, physician factors (specialty, age, race, practice type), and hospital characteristics. A patient sex/physician sex interaction term was incorporated in multivariable analysis in order to test whether differences in procedure use were greater when a patient and physician were of different sexes. RESULTS: Female patients were less likely to undergo CATH (39.4% vs. 52.1%, p = 0.001) and REVASC (25.8% vs. 36.7%, p = 0.001) than male patients, while patients treated by male physicians were more likely to undergo CATH (45.9% vs. 39.5%, p = 0.001) and REVASC (31.4% vs. 25.8%, p=0.001) than patients treated by female physicians. Female patients remained less likely to undergo CATH (odds ratio [OR]: 0.82, 95% confidence interval [CI] 0.80,0.85) and REVASC (OR 0.80, 95% CI 0.77,0.82) in multivariable analysis, regardless of the treating physician' s sex. In contrast, patients treated by male physicians were more likely to undergo CATH (OR 1.15, 95% CI 1.07,1.23) and REVASC (OR 1.14, 95% CI 1.06,1.22) than those treated by female physicians, regardless of patient sex, in adjusted analysis. Differences in CATH (p = 0.66) and REVASC (p = 0.32) use were not greater when a patient and physician were of different sexes. CONCLUSION: Though male patients and patients treated by male physicians were more likely to undergo cardiac catheterization and coronary revascularization post-MI, differences in procedure use were not greater when a patient and physician were of different sexes, suggesting that factors other than sex bias account for sex differences in cardiac procedure use. PURPOSE: Patient satisfaction instruments are frequently used to measure the quality of care provided by physicians and practices. Because these instruments are used for diverse purposes, however, they often become long and difficult for patients to complete, especially those from vulnerable populations. We attempted to reduce the 63-item Veterans Administration (VA) Ambulatory Customer Care Satisfaction Survey into a concise instrument focused on patients' concerns. METHODS: Patients were recruited sequentially at two VA Medical Center clinics and two medical clinics at a nearby University. Patients were first asked an open-ended question,``What is important to you regarding your outpatient clinic visits?'' They were instructed to provide comments based only on their ambulatory care, not inpatient or specialty care. Comments with similar themes were grouped into categories. For the second part of the task, they were shown a list of 15 randomly chosen items from the VA instrument and asked to rate each on a 5-point scale from``not at all important'' to``extremely important.'' A range of content and complexity was represented within each set of 15 items. RESULTS: A total of 496 patients participated. Most were middle-aged (mean age = 50.5, sd = 17.8), male (67.1%), and African ± American (59.9%); 38.7%had a high school education or less. A total of 879 comments were provided about their outpatient clinic visits. The majority of patients gave at least one (80.6%) or two comments (53.8%). The five topics volunteered most frequently were the wait time for appointments (31%), doctor ± patient interactions (14%), getting good service and care (11%), overall speediness (11%), and friendliness/courtesy by all staff (10%). However, when patients rated a list of existing items, the average rating for every item was above 4.0 (i.e., between``very important'' and``extremely important'').
CONCLUSION: Reducing the number of items in patient satisfaction questionnaires is complex. Patients are willing to specify what is important to them in their ambulatory care, however, most patients volunteer only one or two issues, and these issues vary across patients. When shown a partial list of items from an existing instrument, ceiling effects limit the patients' ability to reveal the relative value of potentially competing items. The disparity in results with the two methods suggests researchers need to be aware of how existing instruments were generated, think about the ability and willingness of patients to discriminate among multiple items with similar themes, and find ways to capture what is important to patients without overburdening them.
COST-EFFECTIVENESS OF CANCER SCREENING IN THE ELDERLY. J.S. Rich 1 , J.D. Birkmeyer 1 ; VA Medical Center, White River Junction, VT PURPOSE: Although the benefits of continuing to screen for cancer in the elderly are unknown, the elderly are screened. We developed a Markov model to estimate the costeffectiveness of continuing to screen for cancer beyond 70 years of age. METHODS: For each of three cancers (breast, cervical and colon), we estimated the marginal cost-effectiveness of continuing to screen average risk elderly men and women who had previously undergone regular screening. All-cause and cancer-specific mortality data were obtained from the National Center for Health Statistics and the Surveillance Epidemiology and End Results survey. Assuming that the benefits of screening seen in younger patients extend to the elderly, we used a reduction in cancer-specific mortality of 27% for biennial mammography and 33% for annual fecal occult blood testing (FOBT) to model screening benefits. We assumed a 70% reduction in cervical cancer mortality to model benefits with triennial screening Pap smears. Three costs were included: cost of the screening test, cost to evaluate an abnormal, and the cost of cancer care. We assumed that there were no harms with screening. RESULTS: The table below shows the incremental gains in life expectancy and costeffectiveness of continuing to screen the elderly for cancer. The values for the 70 ± 79 year old interval are compared to stopping screening at age 69; the values for the 80 years and over interval are compared to stopping screening at age 79.
CONCLUSION: Under assumptions that are very favorable to screening, continuing to screen for cancer beyond 80 years of age does not appear to be cost-effective. Although screening for breast and colon cancer in 70 ± 79 year olds may be cost-effective, the gains in life expectancy are small and may be outweighed by potential harms.
AT HOME WITH HEART FAILURE & TRADE: EFFECT OF STANDARDIZED TELEPHONIC CASE MANAGEMENT FOR HEART FAILURE. B. Riegel 1 , B. Carlson 1 , Z. Kopp 2 , B. Lepetri 2 , A. Unger 3 ; 1 Sharp HealthCare, San Diego, CA; 2 Pfizer, Inc., New York, NY; 3 Science Applications International Corporation, Reston, VA PURPOSE: Case management that promotes heart failure (HF) self-care is thought to decrease the need for hospitalization but few randomized, controlled clinical trials have tested the approach. Since much of the effectiveness of case management depends on the unique abilities of the provider, decision-support software from Pfizer, Inc.,``At Home with Heart Failure TM '', was used to standardize care. METHODS: A prospective randomized controlled clinical trial was conducted to evaluate the effectiveness of software-supported telephonic case management in decreasing acute care resource use. 281 physicians from two hospitals in So. California were matched on specialty and practice size and randomized to intervention or usual care. Patients who were cognitively intact and spoke English or Spanish were identified during a HF hospital admission; 358 patients of the randomized physicians were included. Mean age was 72 years, 51% female, 56% unmarried, 72% class III or IV. The intervention group (n = 130) was telephoned within 5 days after hospital discharge and thereafter at a frequency guided by the software based on patient symptoms, knowledge, and needs. On average, patients received 17 calls (median = 14) from the case manager over the 6-month intervention period. Printed educational materials were mailed. Physicians were notified of patient progress in writing and telephoned as needed. Care for patients in the usual care group (n = 228) was not standardized. T-tests were used to test the hypothesis of equal mean acute care resource use in the two groups. RESULTS: Resource use was consistently lower in the intervention group: 6-month all-cause hospitalization rates were 27% lower (.62 vs. .86, p = 0.03), HF hospitalization rates were 48% lower (.22 vs. .41, p = 0.005), HF hospital days were 46%lower (1.1 vs. 2.1, p = 0.04). Multiple readmissions were 43% lower (.13 vs .23, p = .025). CONCLUSION: Standardized case management using telephonic decision-support software provided in the early months after a HF admission can augment care and significantly reduce acute care resource use. PURPOSE: In 1999, the Institute of Medicine reported that medical errors kill between 44,000 and 98,000 patients each year. There is little information on physician and public attitudes regarding disclosure of medical errors. METHODS: One thousand Colorado physicians were randomly selected to receive a mail survey describing three medical error scenarios (one minor, one moderate and one serious). The same three scenarios were presented to five hundred Colorado residents via telephone survey. For each scenario, the physicians and the public were asked if the error should be disclosed to the patient. The moderate error scenario was slightly modified from one previously tested and reported by M. Hingorani et al (BMJ 1999; 318:640 ± 1) . RESULTS: Response rates were 57%for physicians and 82%for the public. The public was less likely to desire disclosure of all three errors compared to physicians (p < 0.01) especially for the moderate error (p < 0.001) and the serious error(p < 0.045). Retired physicians (vs. active physicians, p < 0.05) and the over 65 public (vs. public under 65, p < 0.004) would not disclose the error for at least one of the scenarios. Primary care physicians (Internal Medicine, Family Medicine, Pediatrics, and General Practice) were more likely to disclose all three errors (p < 0.04) than non-primary care physicians. Physician response was not influenced by gender, years in practice, practice location or previous malpractice litigation. Desire for disclosure of the moderate error was roughly comparable for Colorado residents (88%) and United Kingdom respondents (92%). Gender, race, education, and income did not influence public response. CONCLUSION: Overall, 87% of Colorado physicians would disclose all medical errors to patients. The Colorado public was less inclined to disclose errors than physicians. Retired physicians and senior citizens were less likely to disclose one or more errors. Primary care physicians relative to all other physicians were most likely to disclose all three errors.
OBESITY AND ACTIVITY SELF-PERCEPTIONS AND BARRIERS AMONG OLDER, LOW-INCOME HIGH FUNCTIONING WOMEN. C.S. Ritchie 1 , B.A. Stetson 1 , K. Adams 1 , E. Rucker 1 ; 1 University of Louisville, Louisville, KY PURPOSE: Obesity is increasingly recognized as a highly prevalent condition among older women and is associated with multiple comorbidities. Physical activity is an important component of weight reduction and attempts to improve function. We sought to identify perceptions regarding physical activity among older, low-income high functioning women. METHODS: We performed prompted survey completion with a group of 38 older, low-income women who volunteered as senior companions and caretakers for homebound older adults. BMI classifications are described using WHO criteria. RESULTS: Mean age: 73.81 (range 63 ± 93). Seventy percent of subjects were African American; 30% Caucasian. Nearly 65 % were overweight or obese (2.9% underweight; 32.4% normal weight; 8.8% overweight; 41.2 %obese; 14.7% morbidly obese). Of the non-obese women, 85.7% were satisfied with their activity level; compared with only 26.3% of obese women (p < .01). Despite their mobility and involvement in volunteer activities, participants cited numerous barriers to health-promoting physical activity. In contrast to common assumptions regarding social and environmental obstacles to exercise, such as family caregiving responsibilities and weather; this sample indicated that their primary barriers were cost, experiencing pain, illness or injury, safety concerns and self-consciousness. Obese women were more likely to report barriers associated with illness and injury (p < .05) and self-consciousness (p < .01). Obese women also indicated a greater dislike of solitary,``lifestyle'' exercise (p < .05). Despite a high prevalence of obesity in this group, only half of the participants reported being encouraged by their physicians to engage in physical activity. CONCLUSION: Physical limitations and obesity are significant problems even among this relatively high functioning group of older low-income women. Perceived barriers including underlying illness and self-consciousness need to be addressed when considering interventions.
A CONTROLLED TRIAL OF COLLABORATIVE MEDICATION EDUCATION AND PHARMACEUTICAL CARE. M.S. Roberts 1 , K. Cholka 1 , J. Chang 1 , C. Amy 1 , W.N. Kapoor 1 ; 1 University of Pittsburgh, Pittsburgh, PA PURPOSE: To assess whether a multidisciplinary intervention designed to improve patient medication education and evaluate specific medication regimens can improve patient knowledge concerning medication use, increase compliance with medication use post-discharge, and decrease medication errors. METHODS: A multidisciplinary intervention was designed that included 1) a folder with medication information sheets placed at the bedside, 2) nurse-directed teaching about each medication with each administration throughout the hospital stay, 3) automatically triggered specialized teaching plans for anticoagulation, diabetes, and inhaled mediations, 4) protocoltriggered review of medications by a pharmacist to decrease administration complexity and identify potential medication-related problems (interactions, dosing problems, ADRs). The intervention was studied in 4 nursing units in a tertiary teaching hospital utilizing a non- repeated pre ± post/intervention-control design. The intervention was applied to all patients admitted to the intervention unit, and was not instituted on the control unit. Random samples of patients from both the control and intervention units before and after the implementation of the intervention were selected for participation in the evaluation component of the project. Evaluation consisted of baseline data collection and a structured survey that occurred 14 days post-discharge to assess knowledge and compliance. Multivariate logistic regression was used to test for significance. RESULTS: 2397 patients were admitted during the study period (1264 on the control unit, 1133 on the intervention). 1246 randomly selected patients (602 control, 644 intervention) were screened for participation in the evaluation component of whom 611would be responsible for their own medications post-discharge and were eligible. 302 patients agreed to participate and 239 (155 control, 124 intervention) completed the 14-day follow-up. Improvement in postdischarge assessment of knowledge regarding specific medications was significantly higher in the intervention group (7.4% for control vs 28.0% intervention, p = 0.0129), as was patient satisfaction with medication education. Protocol-initiated pharmacy consults were completed on 43 patients, of which 18 resulted in improvements in therapy. CONCLUSION: This pilot trial demonstrates that a multi-disciplinary intervention composed of nurse-directed education given at the time of medication administration linked with protocol-based consults to pharmacists and other health care providers can significantly increase patient knowledge regarding medication and satisfaction with medication education. Larger sample sizes are required to assess impact on medication error reduction, readmission and health status. ., yet little information exists on physician or public opinion regarding conclusions from that report. METHODS: We randomly surveyed 1000 Colorado physicians using a mail questionnaire and asked them to rate their agreement with several statements from the TOM report. A telephone survey of 500 Colorado residents asked respondents to rate their agreement with the same statements. Chi-square tests were used to compare proportions of physicians and the public who agreed with the statements and to assess for demographic differences by response. RESULTS: Response rates were 57% for physicians and 82% for the public. The following table shows percentages of physicians and the public who agreed with each statement:
Physicians 65 and older were more likely to feel that quality of health care is asignificant problem (p < 0.01), but there were no differences in physician responsesby gender, specialty, practice type, or by whether they had ever had a malpracticesuit filed against them. Of the public respondents, females, those over 65, and thosewith lower incomes were more likely to believe quality of health care is a problem(p < 0.01). Public responses did not differ by race or level of education. CONCLUSION: There is a marked disparity in public vs. physicians' perceptions of the quality and safety of health care in the U.S., as well as in the need for a national agenda directed towards reporting and reduction of medical errors. Physicians should recognize that a majority of the public believes that the quality of health care is a significant problem and that mandatory reporting of medical errors is widely supported. PURPOSE: Studies have found that African ± Americans (AA) tend to receive poorer quality care and experience poorer clinical outcomes than Caucasians (C) across a variety of medical conditions. However, little is known about racial differences in care and recovery from major depression (MD) in primary care settings. METHODS: We examined data collected as part of a clinical trial testing the effectiveness of disseminating a depression treatment guideline via an electronic medical record system (EMR). Patients presenting for routine care by a board-certified primary care physician (PCP) at a university-based clinic were screened for MD using the PRIME-MD and their PCPs were subsequently informed of this finding. Study patients had a Hamilton Rating Scale for Depression (HRS-D) score !12 and were not in treatment with a mental health specialist (MHS). The HRS-D was repeated at 6 months to ascertain recovery (HRS-D 7) . Data about depression treatment such as number of visits and discussions of depression, counseling, pharmacotherapy, and referral to a MHS, was abstracted from the EMR. Non AA/C patients were excluded from our analyses (3%) given the focus of this report on comparing differences between AAs and Cs. RESULTS: Between 4/96 and 12/98, 204 depressed patients (25% AA) met all protocol eligibility criteria and completed a baseline interview. They were cared for by 15 PCPs (100% C; 53% male; median age 39 years). At 6 months, 187 (92%) had follow-up information available including 47 (24%) AAs and 140 (73%) Cs. AA and C patients were similar at baseline on level of depressive severity (mean: HRS-D AA = 21 vs. C = 20), age (mean: 44 vs. 43), gender (female: 64% vs. 73%), employment (fulltime: 45% vs. 58%), medical comorbidity (2+ conditions: 57% vs. 59%), and history of depression (43% vs. 49%). AAs had a mean of 3.1 contacts with their PCP including 1.4 contacts discussing depression while Cs had 2.9 and 1.3 contacts, respectively over a 6-month period following study entry. PCPs documented counseling their AA patients for MD for 15%, recommending pharmacotherapy for 57%, and recommending a MHS referral for 28%. These rates were comparable for C patients (15%, 54%, and 35%, respectively). Overall, a similar proportion of AA and C patients recovered from their MD episode at 6-months (21% vs. 24%). CONCLUSION: Although inadequate caregiving and poor treatment outcomes are commonly found among patients with MD who are cared for by PCPs, we were unable to identify any disparities in care or recovery rates for MD among the AA and C patients enrolled in our clinical trial. Future studies should confirm our results and examine methods for improving the delivery of guideline-based treatments for patients of all races experiencing MD. PURPOSE: Hepatitis C viral infection represents a substantial clinical and public health burden. In New York City (NYC) with its diverse ethnic population and large proportion of immigrants, characterizing the subgroup-specific burden of HCV is important to target HCV diagnosis, treatment and prevention efforts. Our objective was to describe the clinical and epidemiologic features of HCV infection in NYC, and to assess the sub-population specific burden of HCV mortality. METHODS: We used data collected by the New York City Department of Health (NYCDOH) Vital Statistics office to identify all persons who died with a diagnosis of HCV coded as a primary or contributing cause of death between 1992 and 1997. Demographic data collected by this office was analyzed. To obtain information regarding cause of death, clinical characteristics, and risk factors for HCV infection, we performed medical record reviews on the subset who died during 1996 ± 1997. RESULTS: Between1992 and 1997, 1002 persons met the case definition for HCVassociated death. Eight hundred and ninety were identified as NYC residents. Median age at death was 55 (3 ± 97); 56% were male; 39% white non-Hispanic, 32% Hispanic, 22% black non-Hispanic, 4% Asian-Pacific Islander, 3% other and unknown. Forty eight percent were foreign born. Three hundred and nine (81%) of the 1996 ± 1997 medical records requested were received. Of these, 26 could not be reviewed because of insufficient information. Of the 283 medical records reviewed 156 (55%) had a risk factor for HCV infection reported. Of these, injection drug use accounted for 73%and blood product transfusion accounted for 26%. We calculated a median years of potential life lost (YPLL) of 12 for the entire study population. During 1997 the proportion of deaths attributable to HCV in comparison to other causes of death was 0.9% among Hispanics, 0.5% among Asian among black and 0.2% among white. CONCLUSION: Although HCV causes death among all segments of the NYC population, it has disproportionately burdened Hispanics and young adults. While additional research on incidence, prevalence, and distribution of risk factors is necessary, these data suggest that primary and secondary prevention efforts against HCV infection be directed at the most vulnerable populations. Figure 1 shows proportions of physicians'``optimal choice'' according to different specialties. Family medicine favored flex-sig + FOBT strategy, gastroenterologists-colonoscopy and internists any of these two. Only about 13% of all physician favoured FOBT strategy. Most physicians indicated patient preference (78%), clinical status (48%) and cost to the patient (41%) as determinants of using strategy other than their preferred. Factors that would, in the opinion of physicians, keep patients from undergoing colonoscopy were: apprehension of pain (77%) and embarrassment (39%) as well as patient financial cost (55%). We analyzed data from a national random-digit-dial telephone survey conducted in 1994. We measured respondents' satisfaction with their regular physicians, health care services, health insurance, and life in general, using 4-point Likert scales (4 indicating highest rating). We hypothesized that if low patient satisfaction ratings among Asians were attributable to response tendencies, we should observe a similar pattern of responses among Asians across domains related and unrelated to health care. We also asked if respondents had ever changed physicians due to dissatisfaction. RESULTS: Respondents included 627 Asian, 1005 black, 1000 Hispanic, and 1114 white adults. Asian respondents were mainly Chinese (33%), Vietnamese (32%), and Korean (32%). Asians reported lower satisfaction with their physicians and health care services than other ethnic groups, even after adjusting for demographic and health-related variables. Asians also reported lower satisfaction with their health insurance and with life in general (Table; p < .001 for all comparisons of Asians vs. non-Asians). Asians' responses to all satisfaction measures were similar and were more normally distributed than non-Asians' responses, which were more skewed toward the highest ratings. Asians were less likely than non-Asians to report having changed physicians due to dissatisfaction (24% vs. 31%, p = .001). CONCLUSION: Asians recorded lower satisfaction ratings not only for physicians and health care services but also for life in general and, despite lower reported satisfaction, were less likely than non-Asians to have changed physicians due to dissatisfaction. These findings suggest that lower satisfaction ratings among Asians are due to different response tendencies rather than worse experiences or higher expectations of health care. Further research is needed to explain this phenomenon and assess its potential impact on performance measurement for physicians and health care organizations providing care for large numbers of Asian Americans. PURPOSE: Vascular catheter-related blood stream infections are costly and associated with substantial morbidity and mortality. Central venous catheters coated with antibacterial agents have been shown to be superior to non-coated catheters. Trial results suggest, however, that central venous catheters impregnated with minocycline/rifampin, although more expensive, are clinically superior to catheters impregnated with chlorhexidine/silver sulfadiazine. It remains unclear if minocycline/rifampin catheters are cost-effective for all high-risk patients or only those requiring longer-term catheterization. METHODS: We developed a series of decision analytic models using patient-level clinical trial data to determine if minocycline/rifampin catheters are cost-effective for patients requiring various durations of catheterization. We calculated incremental cost-effectiveness ratios for patients catheterized for durations ranging from 1 to 25 days. We simulated the use of 10,000 catheters and calculated the proportion of times that the catheters were cost-effective or costsaving at each duration of catheterization. The perspective was that of the healthcare payer; the time horizon was the period of hospitalization. RESULTS: The data were too sparse to estimate cost-effectiveness for patients catheterized less than 8 days. The probability that minocycline/rifampin catheters were cost-effective or costsaving compared to chlorhexidine/silver sulfadiazine catheters in patients expected to be catheterized at least 8 days was 91%. The probability minocycline/rifampin catheters were costeffective or cost saving in patients expected to be catheterized 13 days or longer was over 95%. These findings remained relatively consistent during one-way and multi-way sensitivity analyses. CONCLUSION: The probability that minocycline/rifampin catheters are cost-effective or cost-saving increases relative to the expected duration of use. There is insufficient evidence to suggest that minocycline/rifampin catheters are clinically or economically preferable to chlorhexidine/silver sulfadiazine catheters in patients catheterized less than 8 days. However, minocycline/rifampin catheters are likely to be cost-effective for patients expected to be catheterized for periods longer than 1 week, and are clinically and economically preferable for patients expected to be catheterized for 2 or more weeks. year, with an associated mortality of 10 to 20%per episode. Though many recent studies of nosocomial bacteremia have been reported, there are limited data focusing on communityacquired bacteremia. Given the morbidity, mortality, and economic consequences of community-acquired bacteremia, we decided to: (1) describe the epidemiology and microbiology of community-acquired bacteremia; (2) determine the crude mortality associated with such infections; and (3) identify independent predictors of mortality. METHODS: This prospective study was conducted at the Seattle Division of the Veterans Affairs Puget Sound Healthcare System from January 1, 1994 to December 31, 1997. All patients with clinically significant community-acquired bacteremia or fungemia were evaluated. Data were collected on demographics, co-morbid conditions, clinical parameters, microorganisms, source of infection, and patient outcome. RESULTS: During the study period, 387 bacteremic episodes occurred in 334 patients. Staphylococcus aureus (18%), Escherichia coli (15%), and coagulase-negative staphylococci (12%) were the most commonly isolated organisms. The most frequent sources were the urinary tract, intravascular catheters, and pneumonia. Overall, almost one-third of bacteremia cases were directly related to indwelling catheters: either intravascular (20%) or urinary (10%). Approximately 14% of patients died. Patient characteristics independently associated with increased mortality included shock (OR 3.7, p = 0.02), renal failure (OR 4.0, p = 0.003), and a`D o Not Attempt Resuscitation ''order (OR 21.7, p < 0.001). The risk of death was also higher in those whose source was pneumonia (OR 6.3, p = 0.03) or an intra-abdominal site (OR 10.7, p = 0.02), or if multiple sources were identified (OR 13.4, p = 0.003). The presence of fever (temperature > 38.08C) was associated with a decreased risk of death (OR 0.4; p = 0.005). CONCLUSION: Vascular and urinary catheters were implicated in a substantial proportion of infections, emphasizing the need for appropriate and judicious use of such devices. We will likely observe an increase in the incidence of such device-related infections as healthcare expands into the outpatient setting. Fortunately, many of these infections are potentially avoidable via established infection control practices used to prevent hospital-acquired infections. Thus, strategies that have been useful in preventing nosocomial device-related bacteremia could prove successful if adapted into the outpatient setting. If this is borne out, community-acquired bacteremia may increasingly become viewed as a preventable disease. PURPOSE: Despite known interactions, the occurrence of inappropriate medication and alcohol use has not been well described. We sought to assess 1) the prevalence of potential alcohol and medication interactions in hazardous and harmful drinkers in a primary care setting, 2) the association between this medication use and alcohol consumption, and 3) whether users of medications known to interact with alcohol are counseled by physicians regarding drinking. METHODS: We interviewed current hazardous drinkers (at least one drink in the past month, and at least one positive response to the CAGE alcoholism screening questionnaire or > 14 drinks per week/ > 4 drinks per occasion for men, 7 and 3 respectively for women) just prior to a visit with a primary care physician to determine the self-reported current (30 day) prevalence of use of medications that can interact with alcohol. Alcohol consumption was assessed by trained interviewers using a validated calendar method. Immediately after the physician visit, patients reported whether there had been a discussion about alcohol with the physician during the visit that day. RESULTS: The 312 subjects were 63% male, mean age was 44, and 63%had a high school education. Subjects saw one of 41 physicians. Most (78%) reported current use of medication that can interact with alcohol: 59% used non-steroidal anti-inflammatory drugs, 44%acetaminophen, 16%antihistamines, 10% each narcotics, antidepressants, and medication for sleep, 6% anxiolytics, and 3% blood thinners (i.e. warfarin). Users of these medications drank more drinks per drinking day (median 4.0 vs. 3.5, mean 5.8 vs 4.6, p = 0.04) and binge drank more often (median 2 vs. 1 binges in the past month, mean 6 vs. 5, p = 0.03) than nonusers. There was no discussion about alcohol use with the physician for 37%of users and 40% of non-users of medications that can interact with alcohol (p = 0.68). CONCLUSION: Most hazardous and harmful drinkers in a primary care setting were using prescription and nonprescription medications that can interact with alcohol. Users of these medications drank more than non-users. Many had no discussion about alcohol with their 3.20 +/À .68 3.20 +/À .66 3.11 +/À .77 3.27 +/À .69
Non-Asians (mean +/À SD)
3.47 +/À .67 3.37 +/À .79 3.29 +/À .82 3.40 +/À .74 physicians. While the extent and impact of alcohol-medication interactions require further study, these results suggest that potentially dangerous alcohol and medication interactions are common and unrecognized by primary care physicians. (1966 ± 2000) , PsycLit, Cinhal, Embase, Aidsline, Healthstar, Cancerlit, the Cochrane library (clinical trials registry and the Database of Systematic Reviews), Micromedex and FEDRIP as well as references of reviewed articles. Inclusion criteria included English-language, randomized, placebo-controlled trials of antidepressant medication among adults with back pain. Data were abstracted independently by two reviewers. Two continuous outcomes, back pain severity and ability to perform activities of daily living, were measured. Study quality was assessed using the methods of Jadad and data were synthesized using a random effects model. RESULTS: Ten trials were included, the majority of which studied tricyclic antidepressants (8/ 10 studies) in the setting of chronic back pain (7/10 studies). Patients treated with antidepressants were not more likely to improve than those treated with placebo either in pain severity (Standardized Mean Difference: (SMD) 0.14, 95%CI: À 0.27,0.55) or in activities of daily living (SMD: 0.77, 95% CI: À 0.07, 1.61). Patients receiving antidepressants experienced greater side effects (24% vs. 16%, p = 0.03) than those receiving placebo.
CONCLUSION: Antidepressants are no more effective than placebo in the management of back pain. Further randomized controlled trials of antidepressants are needed before they can be routinely prescribed for treatment of back pain. PURPOSE: Older adults vary greatly in their expectations regarding aging, with some expecting to maintain high function, and others expecting to experience functional decline. Whether having low expectations regarding aging causes older adults to seek less health care for modifiable age-associated conditions is unknown. We set out to determine whether older adults with low expectations regarding aging are less likely to think that they should seek health care for age-associated conditions associated with functional decline. METHODS: We surveyed by mail a random sample of 588 English-speaking patients aged 65 and older cared for by 20 UCLA network physicians. We measured expectations regarding aging using a recently developed and validated 38-item Expectations Regarding Aging (ERA) Survey, which includes 10 domains of expectations such as physical health, cognitive function, and mental health. Additionally, to identify beliefs regarding care-seeking, we asked participants if they felt it was``very",``somewhat", or``not at all ''important to seek health care for 13 ageassociated conditions such as falling, urinary incontinence and pain. Participants also reported on their chronic medical conditions, ability to perform activities of daily living, level of depressive symptoms, religiosity, and generic physical and mental health status. We then constructed a multiple linear regression model to assess the independent relationship between ERA Survey scores and beliefs regarding care-seeking, adjusting for potential confounders. RESULTS: Surveys were returned by 72% (n = 429) of those surveyed; 54% were women; the mean age was 76 years; 80% were Caucasian, 9%Latino, 6%African American; 59%reported > 2 medical conditions, and 21%reported disability. After controlling for all covariates including age, comorbidity, activities of daily living, religiosity and generic physical and mental health status, having lower expectations regarding aging was associated significantly with lower beliefs regarding care-seeking for age-associated conditions (p = .01). CONCLUSION: In this community-based sample of older adults, having lower expectations regarding aging was independently associated with placing less importance on seeking health care for age-associated, potentially modifiable conditions. These results suggest that older adults with low expectations regarding aging may not receive health care for modifiable conditions, and thus may experience preventable functional decline. METHODS: For 1984 indigent patients, adherence was modeled using multivariable linear regression with an alpha of 0.01 for statistical significance. DA was defined as either mean DA for all drugs taken by each patient or minimum DA based on the lowest adherence drug using prescription refill data from a closed pharmacy system. Additional data were drawn from a clinical data repository. RESULTS: Based on mean DA, 31%of patients took < 80% of prescribed doses. Based on minimum DA, 52% of patients took < 80% of prescribed doses for at least one drug. Increasing age, ethnicity (white), and greater medication supply per prescription were independently associated with higher mean and minimum DA. Number of drugs taken had a positive mean but negative minimum DA association. Gender, number of primary care visits, dosage schedule, and copayment were not independently associated with DA. The model explained only 6% of the variance in mean DA. CONCLUSION: In an indigent population with HBP, DM or HC, DA was associated with ethnicity, age, and quantity of drug supplied. However these factors explained only a small amount of the substantial DA variability. To target DA efforts, better predictors must be developed to identify patients most in need of adherence intervention. Prescription refill claims data could serve this purpose. Cook County Hospital, Chicago, IL PURPOSE: Because of its high prevalence and cost, preventing emergency departments (ED) visits and hospitalizations for congestive heart failure (CHF) has become the focus of extensive`d isease management ''activities. As part of a national chronic disease improvement collaborative, we sought to better understand and improve the care for patients with CHF. Because virtually all admissions in our public hospital system are the result of an emergency department visit, and our finding that approximately one in three ED visits of patients in our CHF cohort resulted in a hospital admission (unpublished data), we sought to better characterize the frequency and patterns of ED visits in this patient population. METHODS: We assembled two cohorts of patients with the diagnosis of congestive heart failure ± -1) an outpatient cohort attending one of four outpatient sessions at an public hospital general medical clinic over a three month period (7/99 ± 10/99) who had CHF listed on their PURPOSE: Low levels of functional health literacy (FHL) are common among public hospital patients with chronic conditions. Identifying patients with low FHL may improve care at the risk of disclosing a stigmatizing issue. We evaluated the acceptability and utility of FHL screening among type 2 diabetes patients (DM2). METHODS: This study was a randomized controlled trial of FHL screening. We measured FHL for all patients with the short Test of Functional Health Literacy in Adults. We enrolled 155 patients who had low FHL (scores < 23), DM2, spoke English or Spanish, and were cared for by one of 61 primary care physicians (PCPs) at a public hospital. PCPs in the intervention group were alerted prior to the visit when the patient had low FHL ; control PCPs were not. We surveyed patients regarding the acceptability of FHL screening. We surveyed PCPs regarding their satisfaction with the visit, communication techniques, and self-rated effectiveness. We also asked intervention PCPs to report their prior estimates of the patient' s FHL and the extent to which knowledge of the patient' s FHL would impact future diabetes care. Post-visit survey data were available for both patients (141) and physicians (152). Post-visit differences between intervention and control patients and physicians were examined, controlling for the clustering of patients by physicians. RESULTS: Nearly all patients (95%) felt that FHL screening was useful; of these, 99%felt it was important to share this information with their PCP. After adjusting for clustering, PCPs who received FHL feedback felt less trained and confident in caring for their patient than control physicians(p=.02), were more likely to involve patient' s family or friend in decisionmaking (p=.03), or use pictures and diagrams to promote understanding (p=.08). However, intervention and control PCPs had similar self-rated effectiveness (p = 0.66). Intervention PCPs accurately estimated the FHL of patients with inadequate FHL only 30%of the time. Intervention PCPs believed that FHL screening was clinically useful (63%of visits), would result in improved medication adherence (63%), and would improve future DM2 care (58%). CONCLUSION: Patients with DM2 and low FHL found FHL screening acceptable. PCPs, despite working in a setting with high prevalence of FHL problems, frequently overestimated patients' FHL. FHL feedback was associated with greater feelings of PCP inadequacy and use of recommended communication strategies, but no differences in physician self-rated effectiveness. Most PCPs believed FHL screening was clinically useful. Further research should explore the impact of FHL screening combined with provider communication training on patient-centered measures and clinical outcomes. RESULTS: Only 2.2%of the sample reported eating a vegetarian diet was 43%(116) of selfreported vegetarians reported eating some meat (beef, pork, poultry or fish) during the 24-hour food recall surveys. Overall, reports of vegetarian diet were more likely in women (67.8%of vegetarians v. 49.0%of controls; p < .001) and``other ''races (12.6%of vegetarians vs. 7.7%of controls; p < .05). Medical history was strongly associated with reporting a vegetarian diet. Among those who were vegetarian, 22.3%, 16.3%and 6.7%reported a history of high cholesterol, heart disease and stroke compared to 16.6%, 9.2%and 1.7%of controls, respectively (all comparisons significant at p < .01). Self-report of other risk behaviors was also significantly associated with reporting a vegetarian diet; 16.7%and 59.5%of vegetarians reported current smoking and current vigorous exercise at least two times per week compared to 23.2%and 50.9%of controls, respectively. No differences were seen between vegetarian and non-vegetarian subjects with respect to age, body mass index, household income, education level, region of country and self-reported health status. CONCLUSION: In conclusion, self-report of a vegetarian diet is uncommon among U.S. adults and is strongly associated with gender, chronic medical conditions, and the self-report of other health-promoting behaviors.
Hospital and Brown University, Providence, RI PURPOSE: We analyzed a representative sample of 19,058 visits from the 1998 National Ambulatory Medical Care Survey (NAMCS) to examine the frequency that physicians reported counseling patients about their diet. METHODS: Patients with diabetes, coronary heart disease (CHD), hyperlipidemia, obesity, and hypertension were identified using a combination of visit codes, diagnoses and current medications from forms completed by physicians after patient encounters. Independent determinants of diet counseling were evaluated using multiple logistic regression. RESULTS: Overall, diet counseling was performed during 12.3% of all visits and was more common among patients who were middle-aged (45 ± 64 years) (13.4% vs. 11.8% for other age groups; p < .05) nonwhite (14.2% vs. 12.3%; p < .01), presenting for routine chronic care or a routine physical vs. acute care (14.7% and 18.5%, respectively: vs. 9.7%; p < .01), for patients with diabetes (33.8%), obesity (55.8%), hyperlipidemia (36.2%), and hypertension (36.%) (p < .01 for all comparisons), if the physician was a primary care provider (22.0%), cardiologist (27.0%) or ostetrician/gynecologist (16.0%) vs. other providers (5.8%, p < .001), and if the patient was also counseled about physical activity (67.5% vs. 11.5%; p < .001). These effects persisted after multiple logistic regression analyses controlling for potential confounders. Gender, coronary heart disease history, region of the country and insurance type were not associated with diet counseling. CONCLUSION: In conclusion, health care providers primarily use diet counseling as a treatment for diet-related illnesses, rather than as primary prevention, where it was noted that fewer than 1 in 5 patients were counseled about their diet during routine physicals. . This testing is typically with a sensitive activated partial thromboplastin time (aPTT) and dilute Russel viper venom time (dRVVT), followed by mixing studies and confirmatory tests. With decision analysis, we investigated the optimal testing strategy for detecting LA in three clinical settings. METHODS: A decision-tree was constructed with 12 strategies, using a combination of aPTT and dRVVT with confirmatory tests, tissue thromboplastin time (TTI), platelet neutralizing procedures, and mixing studies. Probabilities of adverse events, utilities of these events, and costs were obtained from a literature review. Sensitivity and specificity of each strategy was calculated by testing 90 healthy people and 77 patients, with true positives defined as per the algorithm recommended by the International Society on Thrombosis and Haemostasis. RESULTS: For healthy people with a prolonged aPTT, the optimal strategy is not to test for LA and assume that no patient has LA. For patients with systemic lupus erythematosus (SLE), it is cost saving to test with TTI alone if the cost of the test is less than $13 (base case -$9), or if the prevalence of LA in the population is > 33% (base case-35%). For patients with past thrombosis or fetal loss, it is cost saving to avoid testing and assume no patient has LA, or to use TTI alone if the expected prevalence of LA is > 33% (base case-9%). CONCLUSION: It is optimal in healthy people and in those with a low likelihood of LA to avoid testing and to assume that these patients do not have LA. However, for patients with SLE, or for patients with past thromboses and a high likelihood of LA, TTI is a cost saving test. These results suggest that the current strategy for detecting LA needs to be modified.
PURPOSE: symptoms of hyperthyroidism and hypothyroidism include weight changes, fatigue which can be confused with symptoms of cancer.very few data exit about the prevalence of thyroid disease in cancer patients. This was a prospective study testing thyroid functions in patients with various diagnosis of cancer in order to assess the prevalence of thyroid disease in those patients. METHODS: cross sectional study was conducted at the Comprahensive cancer center of Saint Vincents Hospital in NYC. Patients were recruited to have thyroid function done at the same time other blood work was done. Later data was collected from the patients chart regarding their type of cancer, the stage of their disease, and type of therapy they were receiving(chemotherapy, radiotherapy, hormonal therapy, etc)Thyroid function were performed by the hospital laboratory using standard kits.TSH normal range 0.49 ± 4.67Free T4 normal range 0.17 ± 1.85 RESULTS: Total of 158 cases analyzed to date.44 cases of breast cancer,25 cases of gastrointestinal cancer, 7 cases of lung cancer, 60 cases of hematological malignancies, and 22 cases of other types of cancer (ovarian, prostate, ks, etc).The mean age of patient was 57 y/ o(range 28 ± 86)There were 85 females and 73 males in the study.17 patients had abnormally low TSH suggesting subclinical hyperthyroidism and 1 patient had high T4 suggesting Thyrotoxicosis. 6 patients had elevated TSH and 3 had low T4 suggesting hypothyroidism. In total 26 cases (16%) of abnormal thyroid function in the population studied. CONCLUSION: The prevalence of thyroid disease in cancer patients is significant, and since the symptoms mimic those of cancer, both internists and oncologists caring for the patients should be aware of that. METHODS: We surveyed 2,500 adult patients seen in the ambulatory clinics of a university hospital. We asked patients about their desire for information regarding the side effects of prescription drugs. We asked 190 physicians practicing in the same clinics about their perceptions of patients' expectations for this information. We also collected information on age, gender and level of education for patients and age, gender and specialty for physicians. We found significant differences in responses of patients and physicians. RESULTS: Please see the Researchers have suggested that patients may understand treatment benefits better when they are presented as numbers needed to treat (NNT) rather than as absolute or relative risk reductions. We sought to determine if NNT helps patients interpret treatment benefits better than absolute risk reduction (ARR), relative risk reduction (RRR) or a combination of all three of these risk reduction presentations (COMBO). METHODS: We surveyed 357 men and women, ages 50 to 80, who presented for care at a university internal medicine clinic. After answering three questions assessing their ability to handle numbers (numeracy skill), subjects were asked to (1) state which of two drug treatments for a hypothetical disease Y provided more benefit, and (2) calculate the effect of drug treatment on a patient with a given baseline risk of disease. Risk information was presented to each subject in one of four randomly allocated risk formats±NNT (100 versus 250 people just like you would need to be treated for 5 years for a benefit against disease Y to be seen in one of you), ARR (treatment reduces the chance that you will develop disease Y by 10 versus 4 out of 1000 over the next 5 years), RRR (treatment reduces the chance that you will develop disease Y by 25% versus 10% over the next 5 years), or COMBO. RESULTS: Subject ability to interpret treatment benefits varied significantly with the risk presentation format they received. Patients correctly interpreted the treatment benefit most often when it was presented in the RRR format: when asked to state which of two treatments provided more benefit, subjects who received treatment benefit information in the RRR format responded correctly 60% of the time, whereas subjects receiving the COMBO, ARR, and NNT presentation formats responded correctly only 43%, 42%, and 30% of the time, respectively (p < 0.01). Most patients were unable to calculate the exact effect of drug treatment on a patient with a given baseline risk of disease, although subjects receiving the RRR and ARR formats responded correctly slightly more often (21% and 17%, respectively, compared to 7% for COMBO and 6% for NNT, p < 0.01). Higher numeracy skills were associated with increased ability to interpret treatment benefits: 88% of subjects who answered all three numeracy questions correctly stated which of two treatments provided more benefit versus only 63% of subjects who answered two questions correctly and 35% of subjects who answered one or no questions correctly (p < 0.01). This association persisted when subjects were asked to calculate the effect of drug treatment on a patient with a given baseline risk of disease. CONCLUSION: Patients are best able to interpret the benefits of treatment when they are presented in a RRR format. NNT is often misinterpreted by patients and should not be used alone to communicate risk to patients. PURPOSE: Depression is very common, particularly in primary care. While depression in otherwise healthy young patients is rather straightforward, it is less clear how to manage depressed patients with multiple medical problems. We assessed the level of physical and mental health impairment in primary care patients who had recently had depressive symptoms. METHODS: We identified two samples of veterans from the Primary Care Clinic at the VA Sepulveda Ambulatory Care Center who had depressive symptoms during a 7-month period: 290 patients whose primary care encounter form had a depression-related diagnosis (PC group) and 130 patients seen in primary care by Consultation L group). These patients all were sent a letter from their attending physician, inviting them to participate in a study of assisted referral back to primary care for patients who were still depressed. 19 PC group patients (7%) and 23 C&L group patients (18%) declined to participate and over half never responded (65% PC, 52% C&L). Respondents (82 PC, 39 C&L) were contacted 6 months after their initial diagnosis of depressive symptoms. The interviewer-administered initial survey included the Veterans Short Form-12 (SF-12V) and three questions on where they received health care. Respondents not currently seeing a mental health specialist were asked to complete a more detailed survey, which included measures of depression, anxiety, alcohol use and sociodemographics. RESULTS: Completed interviews were available for 86 patients (53 PC, 33 C&L). 14 patients who agreed to participate could not be reached and 20 interviews were still pending. The average Mental Component Score for respondents was 36.4. While 40% had no mental health impairment, 17% had impaired mental health (MCS 31 ± 40) and 43% had severely impaired mental health (MCS 30 or less). The average Physical Component Score for respondents was 37.3. The breakdown was similar to that for mental health: 38% had no physical health impairment, 24% had impaired physical health (PCS 31 ± 40), and 37% had severely impaired physical health (PCS 30 or less). 20% of respondents were both physically and mentally impaired and another 14% were severely impaired physically and mentally. Of those with current mental health impairment, the prevalence of physical health impairment was similar between the group seeing psychiatry and those not currently seeing psychiatry. CONCLUSION: Six months after having depressive symptoms identified, most veterans still have profoundly impaired mental health. Of note, the degree of physical health impairment is equally profound. This suggests that for many depressed patients in primary care, treatment models need to account for severe disability both physically and mentally. We assessed whether or not this same relationship holds among veterans, who themselves tend to be sicker and have more health care utilization than the general public. METHODS: As part of the baseline patient survey for a multi-site trial of evidence-based quality improvement for smoking cessation, we interviewed veterans at 18 sites in the Southwestern U.S. The survey, conducted by computer-assisted telephone interview among patients with at least 3 primary care visits in the prior 18 months, covered smoking history, health habits, health status and demographics. Questions were adapted from previously validated sources, including the California Tobacco Survey, Medical Outcomes Study, CES-D (depression), AUDIT (alcohol abuse), and others. Outpatient utilization data for FY 1999 were extracted from the VA Outpatient File at Austin and grouped by type of visit. Inpatient utilization data were obtained from the Patient Treatment File at Austin. Comparisons were done using chi-squared tests and ANOVA. For this preliminary analysis, we also analyzed the utilization data splitting subjects into those under 65 (n=3745) and those 65 or older (n = 3961). RESULTS: Of the 7,706 subjects interviewed, current smokers were more likely than former smokers or those who never smoked to be younger, less active, and divorced (all p < 0.001). Current smokers were more likely than the other two groups to have severe mental health impairment (21% vs. 12% vs. 14%, p < 0.001) and severe physical health impairment (55% vs. 48% vs. 46%, p=0.026). Current smokers were less likely than former smokers or those who never smoked to report being in excellent or very good health (16% vs. 22% vs. 26%, p < 0.001). Among current smokers, approximately half reported they never drink alcohol, but 18% met AUDIT criteria for alcohol abuse. Forty-three percent of current smokers met CES-D criteria for depression at the time of the interview. Current smokers, former smokers, and those who never smoked did not differ in the total number of outpatient visits or for emergency room or general medicine visits. Current smokers did average more mental health and social work visits and fewer medical subspecialty visits than former smokers or those who never smoked (p < 0.001). The rate and length of acute care hospitalizations did not differ among the three groups, but smokers had more nursing home admissions. When we analyzed the data separately for subjects over 65 and under 65, the utilization differences tended to occur predominantly in the half of the sample under age 65. CONCLUSION: While smokers had higher outpatient mental health use, they did not have higher utilization for most other categories. They tended to have worse functional and health status than former smokers and those who never smoked.
BENEFICIARIES WITH CANCER. L.R. Shugarman 1 , C. Bird 1 , J. Lynn 1 ; 1 RAND, Santa Monica, CA PURPOSE: Medical care at the end of life is quite costly; over one quarter of Medicare payments support care of beneficiaries who will die within a year. The aggregate costs and utilization patterns of men and women facing death may not be the same due to differences in their age, preferences, and cultural stereotypes. We analyzed Medicare services utilization and payments for men and women dying of cancer to determine whether differences occur within 5year age strata. We selected cancer because of its clear diagnosis, generally accepted standards of care and limited treatment options for which we do not expect gender differences. METHODS: We analyzed 1993 through 1998 Medicare claims and eligibility data for an overall 0.1% random sample (a 2% sample of an initial 5% sample) of beneficiaries who: were age 65 and over with a diagnosis of cancer but without a diagnosis of end stage renal disease, died in the target time interval and had one year of continuous data before death. Beneficiaries were identified as having a cancer diagnosis if cancer accounted for the plurality of physician spending in the year of death. Age strata were based on the beneficiary's age at death. RESULTS: Across all age strata, women were more likely to use skilled nursing facility care than men (35% vs. 27%, p < .10) and hospice care (42% vs. 37%, p < .10). With the exception of 80 ± 84 year-olds, Medicare total reimbursements for hospitalizations were higher for women than for men ($18,531 vs. $17,166, p < .05, overall) . The beneficiary's responsibility for Medicare costs (e.g., deductibles and co-payments) was higher for men aged 65 ± 69 ($5,188 vs. $4,863, p < .10) and for women in the 70 ± 74 age stratum ($5,163 vs. $4,403, p < .05) and in the 75 ± 79 age stratum ($4,501 vs. $4,298, p < .10). CONCLUSION: Observed gender differences in aggregate spending and utilization are attenuated by age stratification. The persisting differences in use of skilled nursing facilities and hospice and higher hospital costs represent both differences in care and coverage under Medicare. These findings require more study, such as analyses stratified by other primary diseases, type of cancer, and type of service. Our data support the contention that social setting, cultural setting, and patient preferences may combine to create modest but persistent differences in treatment patterns by gender. PURPOSE: Although cervical cancer deaths account for fewer than one percent of cancer deaths among women aged 65 and older, most elderly women report continuing to undergo periodic Pap smear screening. We sought to describe the burden of downstream testing following Pap smears in elderly women who undergo screening. METHODS: Using three years of Medicare Part B 5% Files (1995 ± 1997), we identified women over age 65 who underwent Pap smear screening during a 16-month period in 1996 and 1997. In order to exclude women undergoing follow-up Pap smears or surveillance smears (women with a previous procedure or diagnosis suggestive of cervical dysplasia or malignancy), we required a one-year observation period without such testing prior to the screening Pap smear for each woman. We measured downstream events including subsequent Pap smears, colposcopies, and other diagnostic and therapeutic surgical procedures (e.g. conization) during the 8 months following the screening Pap smear. RESULTS: In 1996, almost two and a half million female Medicare beneficiaries over age 65 underwent Pap smear screening. For every 100,000 women screened, 3020 had at least one additional Pap smear, 669 underwent colposcopy, and 183 had other surgical procedures during the eight-month period following the screening Pap smear. Rates of downstream interventions were similar for women aged 66 ± 75 and 76 ± 85, and declined only modestly in women age 86 and older. Overall, for every 100,000 women screened, 3497 experienced downstream interventions within 8 months of the initial Pap smear. For comparison, according to population-based surveillance data (SEER,1996) , 16 women per 100,000 aged 65 and older are diagnosed with cervical cancer each year, while nine are expected to die from the disease. CONCLUSION: Relative to the small number of expected cases of cervical cancer, women aged 65 and older continue to be screened in large numbers and undergo substantial follow-up testing in response to abnormal Pap smear results. Whether this testing reduces the subsequent risk of death from cervical cancer is unknown. There are no data, however, regarding the frequency at which women actually undergo screening. We sought to describe the frequency of cervical cancer screening in the United States. METHODS: We used the 1998 Behavioral Risk Factor Surveillance System of the Centers for Disease Control and Prevention (CDC), a cross-sectional population-based telephone survey conducted annually on a random sample of civilian non-institutionalized adults. We focused on female respondents aged 18 and older (n=86,715). For women who reported ever having had a Pap smear, we estimated screening intervals based on the timing of a woman's most recent Pap smear. We assumed that each woman either was not being regularly screening, or was being screened at one of three discrete screening intervals (every year, every 2 years, or every 3 years). RESULTS: The vast majority (93%) of American women report having had at least one Pap smear in their lifetime. Among women who have not had a hysterectomy and who have been screened at least once, 90% report having had a Pap smear within the past three years. Based on the reported time since the last Pap smear, we estimate that 59% of women undergo Pap smear screening annually, 18% have a two-year screening interval, 13% are screened every three years, and 10% are not being screened regularly. Even the very elderly report regular screening Ð an estimated 34% percent of women aged 75 ± 84 and 18% of women aged 85 and older undergo annual Pap smear screening. Extrapolating these data to the country as a whole, more than 45 million American women undergo Pap smear screening annually. CONCLUSION: The majority of American women are being screened for cervical cancer too frequently. Lengthening the screening interval would not only reduce the number of pelvic exams, but would also reduce follow-up testing for abnormal smears and the volume of specimens that cytotechnologists are required to read. We performed a study to determine the utility of routine screening of transaminase and CPK values in patients taking statin medications. METHODS: We performed a retrospective on-line medical record review of the primary care practice at Beth Israel Deaconess Medical Center. A computerized search identified all patients at our institution with a statin on their medication list, as well as their ALT, AST and CPK values for 1998. Patients were separated into four categories based on which laboratory data had been measured during the year: CPK only, transaminase only, both CPK and transaminase or neither. From each of these four categories, 45 patient records were randomly selected for review to verify that they were followed at this primary care practice and were on a statin. The random samples were used to identify the rate of physician monitoring of transaminase and CPK values within this practice. We reviewed the on-line medical records of all patients within this practice on statins who had significantly abnormal test results (defined in prior trials of statin therapy as greater than 3x the upper limit of normal for transaminases and greater than 10x the upper limit of normal for CPK) to determine the relationship to statin therapy and outcome. RESULTS: 4556 patients at our institution had a statin on their medication list. Of the 180 random charts reviewed, 26% (corresponding to 1189 of the total cohort) were receiving care from this primary care practice and were taking a statin. Among the patients whose charts were reviewed, 53% were female with a mean age of 61 years. These data also demonstrated that physicians within our practice monitored serum transaminase values (defined as measurement of either AST or ALT during 1998, or within six months of starting a statin) in 84% of patients; physicians monitored CPK values in 54% of patients. 29% of the patients within this practice were on a dose greater than the recommended starting dosage. Of all the patients in this practice who had been monitored, 12 (1.2%) had a significant elevation of AST or ALT and 6 (0.9%) had a significant elevation of CPK. However, after a detailed chart review, none of these abnormalities were attributable to statins. CONCLUSION: In this study of statin use in a primary care practice, no cases of significantly abnormal transaminase or CPK values attributable to statins were discovered through routine monitoring. This questions the necessity of routine measurement of transaminase and CPK in all patients taking statin medications. and had no risk of zoster during that time. Thereafter, vaccinated patients had half the usual zoster and postherpetic neuralgia (PHN) incidence rates (or post-boost effect=50%). Agespecific zoster incidence was that of Rochester, Minn. Half the zoster patients received antiviral therapy; 20 ± 40% of all zoster patients developed PHN, depending on their age. Multiple sensitivity analyses were performed. RESULTS: In the baseline analysis, vaccination gained 0.0004 QALY compared to no vaccination at a cost of $51.13 or $143,000/QALY gained. Vaccination is least expensive at age 66, costing $129,000/QALY gained. In 60-year-olds, costs/QALY gained is greater than $50,000 with wide variation of: immunologic boosting duration, post-boost effect, PHN probability or duration, herpes zoster duration or utility, or antiviral therapy or hospitalization parameters. Vaccinating 60-year-olds costs $103,000 or $61,500 per QALY when lifetime zoster risk (baseline 11.0%) increased to 1 in 7 (14.3%) or 1 in 5 (20%) respectively. If lifetime zoster risk is 20%, costs/QALY gained are less than $50,000 if post-boost effect is < 38%, median PHN duration is > 114 days (baseline 90), average PHN utility is < 0.76 (where 0=death and 1=perfect health), or vaccination cost < $54.21 (baseline $61.96, including $5 administration cost). CONCLUSION: Varicella vaccination of 60-year-olds is expensive compared to many accepted medical interventions in a model biased toward its use. Results are most sensitive to the lifetime risk of zoster, which varies significantly in different populations. 1994) is a valid and reliable assessment tool designed to enable rapid determination of current or past psychiatric disorders in primary care patients. The current analyses were conducted to assess whether or not smokers with current psychiatric diagnoses, as determined with the PRIME-MD, are at higher risk for early relapse to smoking after quitting. METHODS: The PRIME-MD was administered as part of the study screening procedures in an NIH-funded smoking cessation clinical trial that enrolled 677 smokers. Brief individual smoking cessation counseling was provided prior to quitting and on the quit day; nicotine patches were dispensed on the quit day. The outcome measure in the current analyses was biochemically-verified smoking status at one week post-cessation. PRIME-MD diagnoses included major depressive disorder (MDD), panic disorder (PD), generalized anxiety disorder (GAD), and probable alcohol abuse/dependence. RESULTS: A total of 182 smokers (27%) were diagnosed with at least one of the four disorders and 30 smokers had two or more diagnoses. 63% of the participants without any current diagnoses were abstinent at one week compared to 20% of participants with MDD (n=133), 5% with GAD (n=20), and 0% with PD (n=3). No statistically significant increase in risk of relapse was observed for 62 participants with alcohol abuse/dependence (50% abstinence rate). Only 7% of participants with two or more disorders were abstinent at one week post-quit. CONCLUSION: Smokers with current mood and anxiety disorders appear to be at significant risk for early relapse to smoking. The PRIME-MD can provide rapid assessment of these psychiatric diagnoses that are commonly seen in the primary care setting. Identification of smokers with significant depression and anxiety can aid in the development of smoking cessation treatment plans (e.g., including an anti-depressant such as bupropion SR for depressed patients) and can focus attention on the importance of aggressive relapse prevention in these individuals. Russian language interpreters who administered the survey to adults with a valid phone number, using a list provided by the resettlement agency. We report comparisons with the 1999 Behavioral Risk Factor and Surveillance System (BRFSS) data (www.cdc.gov). RESULTS: Of 426 available adults, completed interviews were obtained in 56% (n=241), with mean age 54.2 yrs (18 ± 94), 54% women, and subjects had resided in the county a mean of 7.5 yrs (1 ± 29) . 91% had some type of health care coverage 91% (n=220), comparable to reports for BRFSS. Self-rated health was rated poor/fair by 60% (n=241), compared with responses on the 1999 BRFSS of 13.1% National and 21.6% Kentucky. Preventive measures shown in Table [ To elucidate the impact of the individual and combined components of a physician's preventive``medical message'' on people's intention to adopt more healthy behaviors. The specific risk used in this study was that of developing manifestations of coronary artery disease. METHODS: 86 young people (ages 20 ± 30) and 64 older people (ages 60 ± 80) in France indicated their degree of intention to adopt a healthy behavior in response to a series of 64 hypothetical messages from their physician about their risk of developing a cardiac event. The messages were all possible combinations of 4 components: the type of cardiac event (angina or a heart attack), the probability of developing it (5, 10, 15, or 20%), the time frame (within 5, 10, 15, or 20 years), and the degree of the patient's control over developing it (``given your family history, it is probable that you could not do much to reduce this risk'' or``the reduction of this risk depends entirely on your behavior''). They were asked about adopting 1 of 3 behaviors: à`m edical treatment,'' a strict dietary regimen, or regular exercise. The results were examined graphically and by ANOVA. RESULTS: All components had a significant effect on the intention to change behavior, but these effects varied according to age. The young people were influenced most by the degree of controllability, the probability, and the nature of the cardiac event; the older people by the time horizon. Even without a promise of controllability, participants were inclined to adopt protective behavior. An increase in probability had less added effect as the probability got larger; nonetheless, the level of intent to change behavior associated with a large probability over a long time (e.g., 20% over 20 years) was higher than that associated with a smaller probability over a short time frame (e.g., 5% over 5 years). The type of protective action had no effect on intention. CONCLUSION: The preventive medical message should include the type of event, the probability of developing it, the time frame, and the degree to which the patient can prevent it. The physician should stress the time frame in talking with older people and the other factors with young people. TREATMENT, 1987 TO 1998 . R.S. Stafford 1 , E. Macdonald 1 , S. Finkelstein 2 ; 1 Massachusetts General Hospital, Boston, MA; 2 Massachusetts Institute of Technology, Cambridge, MA PURPOSE: Physicians' approach to the treatment of depression has changed drastically in the last 15 years with the availability of selective serotonin reuptake inhibitors (SSRIs) in 1988. We sought to closely examine trends in antidepressant use, as well as assess broader changes in treatment patterns. METHODS: Using data available from the National Disease and Therapeutic Index (NDTI), a physician survey conducted by IMS Health, we examined trends in antidepressant prescribing from 1987 ± 98. NDTI provides nationally representative diagnostic and prescribing information on patients treated by U.S. office-based physicians. We selected visits by patients reported to have depression. Annual sample sizes varied from 3,901 visits in 1987 to 6,639 in 1998. We examined annual prescribing data to measure: 1) the frequency of visits where depression was noted and 2) the likelihood of specific medications being reported as treatment. RESULTS: The NDTI data show a dramatic shift in the treatment of depression between 1987 and 1998. In 1987, tricyclic antidepressants (TCAs) were the predominant drug class prescribed to patients with depression (47% of patients). Among individual antidepressants, the most common were amitriptyline (14%), trazadone (12%), doxepin (8%), and desipramine (6%). In 1989, a year after its introduction, fluoxetine was prescribed to 21% of patients with depression. Despite the introduction and growing use of other SSRIs in the 1990s, fluoxetine has maintained a constant share of patients at around 25%. The introduction of sertraline in 1992, paroxetine in 1993 and more recent SSRIs led aggregate SSRI use to grow to 38% in 1992 38% in , 56% in 1994 38% in , 60% in 1996 38% in and 75% in 1998 38% in . In 1998 , sertraline (21%), paroxetine (17%) and bupropion (6%) were the leading antidepressants. TCAs accounted for only 8% of depression patients in 1998. The prescribing of non-TCA, non-SSRI antidepressants decreased from 13% (chiefly trazadone) in 1987 to 10% (chiefly buproprion) in 1998. The use of benzodiazepines in depression declined from 11% of patients in 1987 to 3% in 1998. The rate of patients with reported depression not receiving an antidepressant decreased from 30% in 1987 to 9% in 1998. The estimated national number of physician visits by patients with depression increased from 14.4 million visits in 1987 to 22.5 million in 1998. CONCLUSION: The increasing therapeutic dominance of SSRIs has been pivotal to changes in depression treatment. Perhaps because of their effectiveness and side-effect profile, increasing SSRI use also may have contributed to the declining use of benzodiazepines, increased aggregate antidepressant treatment rates, and the increasing frequency of physician visits where depression was reported.
NATIONAL TRENDS IN RECOMMENDED CARDIAC MEDICATIONS. R. Stafford 1 , C. Chaisson 1 ; 1 Massachusetts General Hospital, Boston, MA PURPOSE: Previous studies suggest that recommended cardiac medications are underutilized. We evaluated recent national patterns of medication use in the ambulatory care setting for warfarin use in atrial fibrillation (AF), beta blocker (BB) and aspirin (ASA) use in coronary artery disease (CAD), and ACE inhibitor use (ACEI, including angiotensin receptor blockers) in congestive heart failure (CHF). METHODS: We used the 1989 ± 1998 National Ambulatory Medical Care Surveys to identify nationally representative samples consisting of patients with AF, CAD and CHF who lacked specific contraindications to therapy. For AF, 1,370 office visits were identified; for CAD, 8,773 visits (for BB) and 8,808 visits (for ASA); and for CHF, 3,310 visits. We examined time trends in the proportion of visits reporting the selected medications, weighted to reflect national patterns. Logistic regression was used to evaluate the independent predictors of cardiac medication use in 1995 ± 1998, including patient and physician characteristics. RESULTS: In patients with AF, warfarin use increased steadily from 13% of visits in 1989 to 38% in 1994 to 50% in 1998 (see Table) . In CAD patients, BB use increased from 16% in 1989 to 19% in 1994 to 33% in 1998 . ASA use in CAD was 11% in 1989 increasing to 22% in 1992 where it plateaued, reaching 25% in 1998. In CHF patients, ACEI use increased slowly from 22% in 1989 to 28% in 1994 to 32% in 1998. Patterns of medication use for 1995 ± 98 varied by patient and physician characteristics. Women were less likely to be taking warfarin for AF (OR=0.35 P=0.06) or ASA for CAD (OR=0.79 P=0.04). Nonwhite CAD patients were less likely to take BB (OR=0.68 P=0.03) or ASA (OR=0.69 P=0.05) than were whites. The oldest and youngest patients tended to be less likely to receive medications than those aged 60 ± 69; this was most dramatic for BB in CAD in those 80+ (OR=0.57 P < 0.001). Patients with CAD visiting cardiologists were more likely to receive recommended drugs. CONCLUSION: Recommended cardiac medication use increased from 1989 to 1998, but some increases have not continued into the late 1990's and have not been uniform across subpopulations. Further adoption of these effective therapies could result in additional benefit for patients with cardiac conditions. identified by ICD-9 codes. Our outcome measure was the pattern of medication use for these conditions. In comparing indemnity and managed care, we used logistic regression to adjust for case-mix: patient age, gender, number of physician visits, Diagnostic Care Group (DxCG) risk score, and a series of DxCG co-morbidity indicators. RESULTS: With few exceptions, the use of chronic disease medications was more likely among managed care patients. For patients with DM, managed care plans had greater use of sulfonylureas than indemnity (42% vs. 37%), metformin (26% vs. 17%) and troglitizone (9% vs. 6%). In multivariate analysis to adjust for case-mix, these differences remained significant (p < 0.001). Insulin use was no different in managed care and indemnity plans. For CHF patients, managed care patients had greater use of loop diuretics (44% vs. 39%), ACE inhibitors/ARBs (50% vs. 39%), and beta-blockers (25% vs. 16%); differences that were significant in multivariate analysis (p < 0.001, except loop diuretics [p=0.003]). No differences in digoxin use were observed. For asthma patients, managed care had greater use of inhaled corticosteroids (33% vs. 28%), systemic corticosteroids (18% vs. 15%), short-acting betaagonists (41% vs. 31%), long-acting beta agonists (10% vs. 8%), and leukotriene modifiers (5.3% vs. 3.9%); differences that were significant in multivariate analysis (p < 0.001, except for long-acting beta agonists [p=0.03] and leukotriene modifiers [p=0.04]). No differences were found for cromolyn or methylxanthine use. CONCLUSION: Chronic disease patients in managed care plans are more likely to receive a broad range of medications. Three of 4 exceptions to this pattern are older medications whose use has been partly supplanted by newer therapies. The general pattern of greater medication use in managed care could reflect patient selection, reduced out-of-pocket cost barriers, or a strategy to avoid other, more costly services. Managed care plans do not appear to withhold expensive, newer medications. The emphasis on screening and minimally invasive breast techniques has led to an increase in detection of both benign and malignant breast disease. Optimal management for benign breast disease remains uncertain, and population-based studies of surgical intervetions are lacking. This study uses population-based administrative data to examine use of excisional breast procedures across 5 states and 7 years, comparing benign to malignant breast diagnoses. METHODS: Data are from the Healthcare Cost and Utilization Project, a federal-stateindustry partership in administrative data. Ambulatory surgery(AS) and inpatient(IP) data are used from CO, CT, MD, NJ and NY for 1990 ± 1996. All women with a lumpectomy(ICD-9 85.21) or subtotal mastectomy(85.22, 85.23) were extracted. Lumpectomy(LUMP) and subtotal mastectomy(STMAS) were further classified as either benign disease or cancer using diagnoses codes. Overall IP and AS age-adjusted rates per 100,000 women for each state, year and procedure were calculated using direct standardization. RESULTS: The rates of breast conserving surgery(LUMP+STMAS)for breast cancer increased across all five states, from between 33 ± 71/100,000 women in 1990 to between 70 ± 140/100,000 women in 1996. The rate of LUMP is 3 to 4 fold higher for benign disease as compared to that for cancer in every state. For instance, in 1996, the rate of LUMP in NY for cancer was 91/ 100,000 women, while for benign disease, 271/100,000. However, in contrast to cancer, the rate of LUMP for benign disease is relatively stable across all of the states and years. The overall rate of STMAS is one-tenth that of LUMP in all the states. Similar to LUMP, the rate of STMAS for benign disease was stable across time. However, there was a great deal of variation among the states in use of STMAS for benign breast disease. The rate varied from a negligible rate in CO, to as high as 50/100,000 women in CT in 1996. In addition, for CT and NJ there were equal rates of STMAS for benign and cancer diagnoses(50 and 27/100,000 respectively), while the STMAS rate for cancer was higher in the other 3 states. CONCLUSION: This is the first population-based study comparing interventions for benign breast disease to cancer. This study demonstrates unexpectedly high rates of surgical interventions (LUMP and STMAS) for benign breast disease. These rates may be explained by women and physician fear of missed cancer diagnoses, lack of best practices, upcoding of procedures, or AS data not capturing cancer at discharge for short stays. However, the latter would indicate a lack of two-stage decision-making process for women. PURPOSE: Anecdotal reports have highlighted the stories of patients who skip doses or otherwise avoid using their medications because they cannot afford the expense. However, little is known about which elderly patients without prescription coverage are at highest risk of not taking their medications because of cost, and how prescription coverage modifies this risk. METHODS: We performed a cross-sectional study of 4896 subjects in the 1995 ± 1996 wave of the Survey of Asset and Health Dynamics Among the Oldest Old (AHEAD), a population-based survey of Americans age 70 years and older. Subjects were asked the extent of their prescription coverage, and whether they had taken less medicine than prescribed for them because of cost over the prior 2 years. We used bivariate and multivariate analyses to identify risk factors for restricting one's use of medications because of cost in subjects who lacked prescription coverage. Among subjects with these risk factors, we then examined the effect of prescription coverage on rates of medication restriction. RESULTS: Of 4896 seniors who regularly used prescription medications, 39% had no prescription coverage, 44% had partial coverage, and 17% had full coverage. Not taking medications because of cost was reported by 8%, 3%, and 2% of patients with no, partial, and full prescription coverage, respectively (p < .01 for trend). Among subjects with no prescription coverage, the strongest independent predictors of foregoing medications because of cost were ethnicity (prevalence of restriction 21% for minority subjects, 6% for white subjects, P < .01), income (16% for annual income < $10,000, 8% for $10,000 to $19,999, 4% for !$20,000, P < .01), and out-of-pocket prescription drug costs (13% for monthly cost > $100, 7% for $20 to $100, 3% for < $20, P < .01). Prescription coverage markedly reduced the rate of medication restriction in these high-risk groups. For example, rates of medication restriction in minority and low-income subjects were 21% and 16% among those with no coverage, 8% and 8% among those with partial coverage, and 4% and 2% among those with full coverage, respectively (p < .001). Almost half (43%) of minority patients with low income, high drug costs, and no prescription coverage reported restricting their use of medications, compared with 12% of patients with these three risk factors and partial prescription coverage. CONCLUSION: Restricting one's use of medications because of cost is common in vulnerable groups of seniors who lack prescription coverage. Among these high-risk groups, prescription coverage markedly reduces the rate of medication restriction. PURPOSE: Hospital contact isolation policies are designed to prevent the nosocomial transmission of infectious diseases, but may inadvertently promote the neglect of isolated patients. We tested whether the frequency and quality of recorded vital signs differed between isolated and non-isolated inpatients. METHODS: We identified consecutive adults admitted to a large Canadian teaching hospital between January 1, 1999 and January 1, 2000 who were placed in contact isolation during their hospital stay (n=81). Controls were selected by identifying the two patients who occupied each isolated patient's hospital bed immediately before and after their admission (n=162). Vital signs recorded in the medical records were compared for the two groups using t tests. Adjustments for age, gender, Charlson Comorbidity Score, living status (nursing home vs. home) and admitting service (medicine vs. surgery) were done using propensity score matching. RESULTS: We found no difference in the frequency of daily vital signs ordered for isolated and non-isolated patients (2.8 vitals/day vs. 3.2 vitals/day, p > 0.20). However, isolated patients had a significantly higher percentage of their vital signs incompletely recorded (missing at least one of heart rate, blood pressure, respiratory rate or temperature) as compared to non-isolated patients (17.3% vs. 12.7%, p=0.03). Both groups had surprisingly high percentages of their respiratory rates recorded as twenty (53.2% vs. 50.9%, p > 0.20). CONCLUSION: Patients placed in contact isolation appear to have their vital signs ordered as frequently though not recorded as completely as non-isolated patients. Differences in the recording of vital signs may reflect underlying differences in the quality of care received by isolated patients and warrant further investigation. OBJECTIVE: Several studies have previously reported that patients want their physicians to address spiritual issues as part of their health care. It has also been reported that patients feel their individual spiritual faith can help them recover from illness. Surveys of physicians have shown that they often do not routinely address these issues with patients. We sought to examine patient and physician perceptions regarding inclusion of religiosity and spirituality in the medical encounter. METHODS: We surveyed primary care physicians and patients in primary care clinics at 7 sites in 4 states (NC, FL, GA, VT). For the patient survey, a trained research assistant verbally administered a 112 item survey that included questions pertaining to demographics, health status, utilization of health care, physical function (SF-36), and a religious/spiritual assessment (Spiritual Well Being, or SWB) scale. Physician surveys were mailed to primary care faculty, residents, and recent graduates at the same 7 sites. Bivariate analyses were done to examine associations between patient/physician characteristics and questions regarding the impact of prayer on health outcomes. RESULTS: 299 patients and 444 physicians were surveyed. Patients' ages ranged from 19 ± 86 years. Fifty-six percent were male, 46% were African-American, 51% Caucasian, and 63% were Protestant. Fifty-three percent of the patients surveyed had annual incomes of $20,000 or less. Of the physicians, 45% were in practice, and 55% were residents/fellows. Forty-five percent were Protestant. Fifty-three percent work in an academic setting. Of the patients, 30% felt that praying with their doctor would improve their own health. Thirty-nine percent of patients vs. 19% of physicians agreed that their doctor praying for them would improve their health. In addition, 42% felt that praying for their doctor would improve their own health, while 32% of physicians felt that patient prayer for them would help them provide better health care. Nearly half of physicians felt that patients who prayed for themselves or had strong religious/spiritual beliefs would have improved health outcomes. Fifty-three percent of patients as compared to 19% of physicians agreed that faith alone can cure disease. Bivariate analyses in the patient sample showed that the perception that prayer improves health outcomes is associated with higher attendance of organized worship services, hospitalization within the past year, higher score on the SWB scale, and lower total household income. In the physician surveys, bivariate analyses associated the equivalent perceptions with attendance of organized worship services and higher SWB score. CONCLUSION: A significant percentage of patients perceive that prayer (whether it is done by their doctor, with their doctor, or for their doctor) is linked to improvements in health. Although there are a significant number of physicians who perceive that prayer is linked to improved health outcomes, there is still a large disparity in the prevalence of physicians compared to patients who share this perception. These data suggest that a more reliable quantification of the benefit of spiritual behavior in medical encounters might be needed to bridge this gap. The prevalence of soft tissue infections among injection drug users (IDUs) is estimated between 11% ± 32% with 40% of those presenting for care requiring hospitalization. Risk factors for hospitalization from these infections are not known. This study sought to identify factors associated with increased health care utilization. METHODS: This was a cross-sectional study of all IDUs seeking initial care for soft tissue infections at an urban, public hospital emergency department (ED) from November, 1999 through April, 2000. Demographics, infection prodrome, and clinical measures were extracted from medical records. Systemic infection was defined as temperature !388C or WBC !15,000 per milliliter, prolonged hospitalization as !2 days, and delay in seeking care as !5 days of symptoms before the ED visit. RESULTS: Two hundred forty-two patients sought initial care for IDU-related soft tissue infections. Most patients were male (64%), Caucasian (69.4%), unemployed (78%), and uninsured (52%). Soft tissue infections included abscesses (74.4%), cellulitides (31%), and infected ulcers (5%) and were mainly located on the lower arm (49.6%), deltoid (14.1%), leg (22.7%), and buttock (19.8%). Ninety-seven (40.1%) patients delayed seeking care. Systemic signs of infection were identified in 118 (48.8%) patients. Of the 97 (40.1%) patients hospitalized, 70 (72.2%) stayed 2 or more days. Ethnicity, gender, age, employment status, and homelessness were not associated with having systemic infection, with hospitalization or with prolonged hospitalization. Compared to those with other types of infection, patients with abscesses were much less likely (OR 0.13, 95% CI 0.02 ± 0.73) to be hospitalized while those with cellulitides were 3.2 times more likely (95% CI 1.47 ± 6.79) to be hospitalized and 3.8 times more likely (95% CI 1.70 ± 8.57) to be hospitalized !2 days. Among patients who delayed seeking care, those with systemic signs of infection were 5.36 times (95% CI 1.67 ± 17.19) more likely to have a prolonged hospitalization compared to those without systemic signs of infection. Prolonged hospitalization was not associated with systemic infection among those who did not delay seeking care. CONCLUSION: Among IDUs seeking ED care for soft tissue infections, systemic signs of infection after delay in seeking care, and cellulitides, were each associated with increased use of health services. Gender, ethnicity, employment status, insurance status, and homelessness did not alter the risk of hospitalization, of prolonged hospitalization, or of having systemic signs of infection. PURPOSE: Stroke is the third leading cause of death and a major cause of disability among postmenopausal women in developed countries. Although postmenopausal hormone replacement therapy (HRT) is one of the most widely prescribed drugs, it is associated with increased rates of thromboembolic events and therefore may be important etiologically in stroke. We conducted a systematic evidence review and meta-analysis for the third US Preventive Services Task Force (USPSTF) to investigate the relationship between HRT and stroke. METHODS: We searched the MEDLINE database from 1992 to 2000 for all published English language studies reporting the association between HRT and stroke. In addition, reference lists of key articles were reviewed for related studies, including those predating the search. Thirty-three observational studies met inclusion criteria and were reviewed; however, only those studies considered good or fair quality based on criteria developed by the USPSTF were included in the detailed review and meta-analyses. We identified no randomized controlled trials. We used the Bayesian data analysis framework for the meta-analysis. RESULTS: The pooled relative risk of stroke mortality in women who had ever used HRT was 0.83 (95% Cl 0.64 ± 1.05). Stroke incidence was significantly increased among ever users, with a pooled relative risk of 1. 15 (1.03 ± 1.29). On subanalyses, the risk of thromboembolic stroke was significantly elevated among women who had ever used HRT (RR 1.30 [1.10 ± 1.58]); however, not subarachnoid hemorrhage (RR 0.93 [0.69 ± 1.25]) or hemorrhagic stroke (RR 0.71 [0.25 ± 1.29] ). CONCLUSION: Studies evaluating the association between URT and stroke mortality suggest no effect. Our meta-analysis suggests an increase risk of the incidence of total stroke, largely due to thromboembolic stroke, among women with exposure to HRT. These results are consistent with preliminary findings from the Women's Health Initiative. The results are limited by the observational nature of the data and randomized controlled trials will be the most valid way of clarifying the association between stroke and HRT. METHODS: A community-based, in-person survey of Chinese women was conducted in Seattle, Washington during 1999. The total estimated response rate was 64% and the cooperation rate was 72%. Four hundred and thirty-two women in the 20 ± 79 age-group were included in this analysis. The main outcome measures were a history of at least one previous Pap smear and Pap testing within the last two years. RESULTS: Nineteen percent of the respondents had never received cervical cancer screening and 36% percent had not been screened in the previous two years. Eight characteristics were independently associated with a history of at least one Pap smear: being married, thinking Pap testing is necessary for sexually inactive women, having concerns about embarrassment or cancer being discovered, having received a physician or family recommendation, having obtained family planning services in North America, and having a female provider. The following characteristics were independently associated with recent screening: thinking Pap testing is necessary for sexually inactive women, having concern about embarrassment, having received a physician recommendation, having obtained obstetric services in North America, and having a female provider. CONCLUSION: Pap testing levels among the study respondents were well below the National Cancer Institute's Year 2000 goals. The findings suggest that cancer control interventions for ethnic Chinese women are more likely to be effective if they address multiple barriers and facilitators. Results also indicate that efforts to increase Pap testing rates among Chinese should target the health care providers who serve Asian American communities. We have randomly assigned 252 women with at least 3 months amenorrhea who experience > = 35 hot flashes per week to 3 months of daily Promensil(r) (80 mg total isoflavones), Rimostil(r) (50 mg total isoflavones), or an identical placebo. At randomization and close-out visits, participants health related quality of life is evaluated using the Medical Outcomes Study 36 Item Short Form (SF-36). Non-parametric statistical tests were used as most of the SF-36 scale scores were not normally distributed. RESULTS: Participants' mean (SD) age is 52.3 (3.1) years and they average 3.3 (4.5) years since menopause. Ten percent of the participants are African American and 84% are Caucasian. Forty-two percent had completed college and all but 2 participants had completed high school. At baseline, women in the study scored higher than age and gender equivalent normative data on the Physical Component Summary (PCS mean 51.2, median 52.9) and the Mental Component Summary (MCS mean 51.9, median 54.8). Among the 8 domains of the SF-36, the participants scored lower than population norms on only the bodily pain scale. The weekly hot flash count decreased 36% for the 232 women who have completed the 12 week protocol. The PCS was unchanged over 12 weeks, while the MCS increased 6%. Change in hot flash count was not correlated with change in any of the eight SF-36 domains or with changes in the summary components. CONCLUSION: Since the blind has not been broken, it is not possible to definitively assess the effects of these dietary supplements on health related quality of life. The lack of association between the change in hot flash count and the SF-36 scales suggests that the SF-36 may not be a sensitive measure of menopausal quality of life. PURPOSE: Chronic heart failure (CBF) is costly, morbid, and common. CHF care is often suboptimal because appropriate drug regimens are complex and should be modified according to patients' symptoms. Standard measures of CHF symptoms and health status would be a valuable tool in providing CHF care. METHODS: To assess a measure of CHF symptoms and compare it with CHF-specific and generic health status and satisfaction with care, we studied 497 veterans(80% of those eligible) who had an outpatient diagnosis of CHF, objective left ventricular systolic dysfunction, and being actively treated for CHF by their primary care physicians (PCPs). We administered the Kansas City Cardiomyopathy Questionnaire (KCCQ) (to assess CHF-specific symptoms) and the SF-36 [to obtain generic summary measures: Physical Composite Score (PCS) and Mental Composite Score (MCS)]. Symptoms were used to assign New York Heart Association (NYHA5 functional class. We also assessed patients' satisfaction with their PCP and the most recent prior outpatient PCP visit using locally validated measures; higher scores denote better status or greater satisfaction. RESULTS: Patients' mean age was 69 years; 98% were men and 86% Caucasian. Patients' had moderate to severe limitations (means: NYHA=2.6, PCS=34, MCS=54). As shown in the table, KCCQ symptoms and NYHA class were highly correlated with CHF-specific physical limitations, functional status, and PCS (p < .0001 for each but less so with MCS (p < .005). KCCQ physical limitations and functional status scores and the PCS were highly correlated, but less with the MCS (p < .005 for each). There was no correlation between satisfaction with the PCP and any symptom or health status measure. Satisfaction with the most recent primary care visit was correlated modestly with PCS and MCS (p < .0001 for each) but less strongly with symptoms (p < .05). CONCLUSION: Measures of CHF-specific and generic health status are highly correlated with each other and with severity of CHF symptoms, but they tap different aspects of patient status. But there was little impact on satisfaction with their PCPs or primary care among these veterans with CHF. These health status measures can provide important data to assess and improve the care of patients with CHF. To determine the level of interest in taking tamoxifen for the primary prevention of breast cancer among women over age 50. METHODS: We conducted a cross-sectional, one-time structured interview of women over 50 years old in outpatient geriatrics and women's health clinics at an inner-city academic medical center. Data on age, race, socioeconomic factors, comorbidities using the Charlston comorbidity index, and objective/subjective risk of breast cancer and interest in taking a breast cancer chemopreventive agent were obtained. RESULTS: From the forty-eight (48) participants enrolled in this pilot study, 31 (65%) were black, 17 (35%) white, Hispanic or other, 35 (73%) were > 65 years old. A substantial proportion, 37.5%, of older women were interested in taking preventive medication for breast cancer. Interest in taking a cancer preventive agent appeared to be higher among white women (59% vs 39%), women who have had a breast biopsy (71% vs 35%), and those who believe their risk of cancer warrants taking an agent (93% vs 24%). Of women receiving Medicare, those with additional drug coverage were more willing to take a preventive agent than those without drug coverage (61% vs 38%). Interestingly, those women who paid the highest percentage of their monthly income for medications were also the most interested in taking a breast cancer preventive agent (87.5% vs 36%). No difference was observed by educational level, income, family history, number of comorbidities, objective breast cancer risk, or personal acquaintance with a person with breast cancer. CONCLUSION: A substantial number of women in our pilot study were interested in taking a chemopreventive agent for the primary prevention of breast cancer. Interest appeared to be associated with race, subjective cancer risk, breast biopsy history, and prescription drug coverage. Given our limited numbers, further study is warranted to test the robustness of these associations. To determine the level, pattern, and factors associated with preventive medication use among women over age 50. METHODS: We conducted a cross-sectional, one-time structured interview of women over 50 years old in outpatient geriatrics and women's health clinics at an inner-city academic medical center. Data on preventive medication use, age, race, socioeconomic factors, insurance status, and comorbidities using the Charlston comorbidity index were obtained. RESULTS: From the forty-eight (48) participants enrolled in this pilot study, 31 (65%) were black, 17 (35%) white, Hispanic or other, 35 (73%) were > 65 years old. All (100%) women in this study took at least one preventive agent every day (defined as a multivitamin, calcium, hormone replacement therapy, aspirin, cholesterol lowering drug or antihypertensive (to prevent myocardial infarction or stroke)). Over 55% took at least two preventive medications a day. The frequency of medications used were: antihypertensive (69%), aspirin (50%), multivitamin (50%), calcium (35%), cholesterol lowering agent (31%), and hormone replacement therapy (15%). Women with Medicare alone versus those with Medicare plus additional drug coverage were less likely to take: 1) a cholesterol lowering agent (23% vs 46%); 2) an aspirin (38% vs 61%); and 3) an antihypertensive (69% vs 84%). Lower income women were less likely to take calcium (25% vs 53%) or a multivitamin (43% vs 65%). Older women were less likely to take a cholesterol lowering agent than younger women (0% vs. 38%), even though the same older women were much more likely to be taking an antihypertensive agent (90% vs 62%). Women cited that physician recommendation was the most important factor in deciding to take a preventive medication. Conversely, women cited that cost and insurance coverage were the least important factors. CONCLUSION: There is a high level of interest in taking preventive medications among older women. However, low-cost, proven therapies such as aspirin and calcium are underutilized. Ability to pay appears to drive decisions about medication use despite the fact that women cited cost/insurance coverage as the least important decision-making factor. Women whose Medicare lacks supplemental drug coverage were less likely to take both expensive drugs (cholesterol lowering agents) and inexpensive drugs (aspirin). Lower income women were less likely to take over the counter preparations such as calcium and multivitamins. In addition, the pattern of use of some medications appeared to be inconsistent; the very elderly women in our study, at risk for coronary artery disease as evidenced by their high rate of antihypertensive use, were not taking cholesterol lowering agents. Additional study is needed to test the robustness of our findings and to help understand how women make decisions about their medication use so that inequalities due to age or ability to pay may be addressed. We performed independent duplicate review of each study for both inclusion and data extraction. Global improvement was abstracted as a dichotomous variable. Effect on pain, fatigue and sleep were abstracted as continuous variables at 4 time points (2, 4, 8 and 12 weeks) . Analysis was done using a random effects model. Quality was assessed using the methods of Jadad. RESULTS: Six randomized, placebo-controlled trials were identified of which all were appropriate for some data extraction. Overall the quality of the studies was fair (mean score: 4.7), range 0 ± 8. The odds ratio for improvement with therapy was 3.7 (95% CI: 2.2 ± 6.3). The pooled risk difference for these studies was 0.28 (95% CI: 0.16 ± 0.40), which calculates to 3.6 (95% CI 2.5 to 6.25) individuals needing treatment for one patient to experience symptom improvement. Patients experienced improvement in pain at 2 weeks (Standardized Mean Difference: 0.44, 95% CI: 0.18 ± 0.88) but none at 4, 8 or 12 weeks. There was no significant effect on patient fatigue at any time point. Patients experienced a statistically significant improvement in sleep at all time points with SMD's ranging from 0.35 ± 0.49. Eighty-five percent of patients given cyclobenzaprine reported at least one side effect.
CONCLUSION: While patients were nearly 4 times as likely to improve if given cyclobenzaprine, the effect was modest, with no effect on fatigue, and improvement in pain only at two weeks. Patients treated with cyclobenzaprine did report modest improvement in sleep at all time points as well as more side effects.
PREGNANCY AND FAMILY PLANNING SERVICES AMONG INCARCERATED WOMEN. I. Tong 1 , J.G. Clarke 2 ; 1 Brown University, Department of General Internal Medicine, Providence, RI; 2 Brown University, Providence, RI PURPOSE: Among incarcerated women, pregnancies are often unplanned and high-risk due to high rates of psychiatric disease and illicit drug use. Obtaining birth control can be costly and challenging for this underserved population given its lack of medical insurance and poor access to health care. Offering women an intramuscular injection of progesterone or a three-month supply of oral contraceptive pills prior to their release from prison could prevent or delay potentially high-risk and unwanted pregnancies. In addition to pregnancy prevention, birth control provision would also introduce control and stability into the lives of this disenfranchised population. The aims of this study are to obtain pilot data to determine 1) the number of pregnancies among incarcerated women in Rhode Island and 2) the number of pregnancies occurring within 12 weeks of a prior incarceration. METHODS: Charts of all patients with positive (+) serum beta-human chorionic gonadotropin (b-HCG) values from January 1998 to January 2000 were reviewed. Women who were postpartum or who had a recent termination of pregnancy were excluded from the study. Chi-square and t-tests were used where applicable. RESULTS: A total of 129 women and 140 pregnancies were included in the study. Of the 140 pregnancies, seventy-seven (55%) pregnancies occurred in women with prior incarcerations. Of these 77 pregnancies, 38 (27%) occurred within 12 weeks of a prior release. Women who conceived within 12 weeks of their prior release had a higher number of prior incarcerations (5.5 vs. 3.7, p = 0.0043) and a longer mean length of incarceration during their pregnancy (61.4 days vs. 26.7 days, p = 0.0016). Women who were incarcerated for greater than 30 days during their pregnancy had a higher number of incarcerations (5.3 vs. 3.7, p = 0.0043) and a longer stay during the prior incarceration (58.9 days vs. 21.4 days, p = 0.0082). CONCLUSION: Twenty-seven percent of conceptions among incarcerated women occurred within 3 months of a prior release, suggesting that initiating birth control prior to release from prison could prevent a significant number of pregnancies. Women with potentially preventable pregnancies had multiple prior incarcerations and longer lengths of stay, thus providing multiple opportunities for medical intervention and counseling. Family planning services may also provide cost savings as women with potentially preventable pregnancies have longer lengths of stay while pregnant, thus requiring more financial support for prenatal care. The establishment of a family planning program for incarcerated women would not only provide an important service to a medically underserved population, but it may also be a cost-effective measure for the Department of Corrections. Smoking is strongly related to most health outcomes. However the contribution of smoking behavior to mortality benefits in this group has not been investigated. PURPOSE: In a recent national study, women reported poorer adherence to ART than men but socioeconomic differences may explain this finding. We used a pharmacy-based measure to assess adherence in HIV-infected drug users enrolled in New York State (NYS) Medicaid, a population where men and women have similar socioeconomic backgrounds. METHODS: We analyzed longitudinal claims files for 9,557 non-pregnant HIV+ drug users enrolled in Medicaid > 10 months in each year of 1997 ± 98. In persons prescribed ARVs for > 6 months and on 2+ ARVs for > 2 months in each year, adherence was defined as > 90% days covered by 2+ ARVs from the start of combination therapy through the last prescribed ARV(s) in each year, with inpatient days judged adherent. Demographics and clinical conditions (i.e., HIV, substance abuse, and general medical) and health care factors were identified from eligibility/claims files in 1997. Outpatient care in 1997 was categorized as: regular medical care ( > 35% of visits to one provider); regular substance abuse treatment (6+ months with one provider); both; or neither. We created separate indicators for any care in 1997 from: a provider offering HIV-focused care under NYS Medicaid contract; an infectious diseases (ID) specialist; and a psychiatrist. Two logistic regression models were estimated for adherence to 2+ ARVs in 1997 and, to reduce possible confounding due to concurrent assessment of medical care patterns, adherence to 2+ ARVs in 1998. RESULTS: Of 9,557 persons on Medicaid in 1997 ± 8, 2+ ARVs were prescribed for 4,299 in 1997 4,299 in and 4,589 in 1998 4,299 in . In 1997 .4% of 1,564 women and 37.6% of 2,735 men were > 90% adherent while, in 1998, 34.6% of 1,730 women and 40.3% of 2,186 men were adherent. In both models, the adjusted odds of adherence were lower for women than men (adjusted odds ratio (AOR) 0.67 [CI 0.58, 0.77] Uncertainty about the diagnosis and fear of an episode taking place in a crowded or dangerous situation can influence social functioning. Few and only small studies on quality of life of patients with loss of consciousness have been performed. METHODS: As part of the Dutch Fainting Assessment Trial (FAST) we asked consecutive patients presenting to all departments of the AMC with loss of consciousness to participate in this study. All patients were asked to fill in the SF-36, a generic quality of life questionnaire, and the Syncope Dysfunction Scale (SDS), a previously validated disease-specific questionnaire which assesses specific areas of impairment due to syncope and fear or worry about syncopal spells. RESULTS: Of the first 144 patients 125 (87%) were included in analysis of quality of life. Disease specific impairment was moderate, with acknowledged impairment in 32% of listed activities (e.g. driving, physical activities and ability to work). Fear and worry due to syncope was also moderately high at 40 (SD 29) (possible range 0 ± 100, 0 = no fear or worry, 100 = terrified/ all I do is worry). CONCLUSION: Quality of life in patients with episodes of syncope is seriously affected. Knowledge of problems and fears which these patients encounter are vital knowledge for physicians that can improve patient-doctor understanding and guide (practical) medical advice to this group of patients. PURPOSE: African-Americans and Latinos are more likely to have diabetes and experience worse outcomes from diabetes than Whites. We compared rates of outpatient office visits, emergency ward (EW) visits, and hospitalizations for persons with diabetes by race and ethnicity. We studied Medicaid recipients in New Jersey, thus controlling roughly for income and financial access to care. METHODS: From the 1994 New Jersey State Medicaid Research Files, we identified persons with diabetes by ICD-9-CM diagnosis code or by prescriptions for sulfonylureas or insulin. The final database excluded patients who were: < 18 or > 64 years old; enrolled < 3 months on Medicaid; in Medicaid managed care or Medicare; or pregnant. We compared Black, Hispanic, and White patients, eliminating persons with other or unknown race/ ethnicity. We identified visits using CPT evaluation and management procedure codes for outpatient office visits and for EW services, verifying each by place of service documentation. Unique hospitalizations were identified based on date of admission. We used a Poisson regression multivariate model to examine the effect of race on service use, adjusting for age, sex, reason for Medicaid eligibility, selected co-morbidities, and duration of enrollment. RESULTS: Of 11,841 patients, 44% were Black, 15% were Hispanic, and 41% were White; 73% were women; mean age was 47years; and 66% had claims for sulfonylureas or insulin. Of Blacks 51% had no outpatient visits, compared to 47% of Whites and 42% of Hispanics; 35% of Blacks had at least one hospitalization, as did 31% of Whites and 19% of Hispanics. For outpatient visits, compared to Whites, Blacks had an adjusted relative risk (aRR) of 0.84 (95%CI, 0.79 ± 0.89), and Hispanics had an aRR of 1.00 (0.92 ± 1.08). For EW visits, compared to Whites, Blacks had an aRR of 0.95 (CI, 0.89 ± 1.02), and Hispanics had an aRR of 0.68 (CI, 0.61 ± .077). For hospital admissions, compared to Whites, Blacks had an aRR of 1.21 (CI, 1.14 ± 1.28), and Hispanics had an aRR of 0.91 (CI, 0.82 ± 1.01). Additionally aRR's for all three services differed significantly between Blacks and Hispanics. CONCLUSION: Among persons with diabetes, Blacks had significantly lower rates of outpatient visits and higher hospitalization rates than Whites despite similar incomes and identical insurance benefits. Hispanics had similar outpatient visits and admissions rates, but significantly fewer EW visits than Whites. These results suggest that factors other than income and insurance, such as availability of primary care services and physician-patient factors, contribute to differences in utilization by race and ethnicity. 18) . We defined South Asian as patients who state on registration forms that they speak Bengali, Gujarati, Hindi, Punjabi, Nepalese or Urdu in the home. We defined diabetic as any patient with a billing diagnosis of diabetes in two or more visits over the last two years. Europid controls are those who meet the same criteria for a diabetes diagnosis and state that they speak English in the home. We controlled for socioeconomic class by including only patients who use CHA Free Care or a Medicaid variant as their primary insurance. We compared mean HbA1c, mean LDL cholesterol, and prevalence of microalbuminuria between the two populations. We will continue the analysis with the comparison of BP and ophthalmologic changes, and will report statistical tests of significance for all variables. RESULTS: Sixty-eight of 937 (7.26%) South Asian patients were diabetic, compared with 1,698 of 49,206 (3.45%) Europid patients (RR: 2.10). Mean Hb A1c (8.71% vs. 8.47 %), mean LDL-C (124.17 mg/dl vs. 116.44 mg/dl) and prevalence of microalbuminuria (21% vs. 13%) were all higher in the South Asian patients than in the Europid patients CONCLUSION: The South Asian population is at higher risk of type 2 diabetes mellitus and its complications, and may exhibit poorer metabolic control as compared to the Europid population. Our findings warrant increased research into this population to determine the factors that influence observed differences, and to elaborate culturally appropriate interventions for this high-risk population.
Results SF-36 (scores 0 ± 100; 100 = perfect health) PURPOSE: The complications of type 2 diabetes result in substantial morbidity and mortality. Racial disparities in clinical outcomes of diabetes care are well documented; however, racial variations in disability rates due to diabetes have not been studied. We sought to evaluate the impact of higher complication rates in minorities on health status and rates of disability. METHODS: We used data from the 1992 wave of the Health and Retirement Study (HRS), a national household sample of people age 50 or older in the United States, to provide estimates of prevalence of disability in patients with type 2 diabetes. Logistic regression models were constructed to evaluate predictors of disability. Self-reported disability was the dependent variable. Independent variables included race and other sociodemographic characteristics and the presence or absence of common diabetes complications including stroke, heart failure, coronary artery disease, visual loss, and renal failure. Costs of disability were calculated by applying the median reported income in the HRS to the lost work time associated with disability. Results were extrapolated to the US population using survey weights. RESULTS: In bivariate analyses, we found that African-Americans with diabetes were substantially more likely to be disabled than non-Hispanic whites with diabetes (unadjusted OR = 2.38; 95% CI 1.79, 3.16), as were Hispanic-Americans (OR = 1.54, 95% CI 1.05, 2.26). However, after adjustment for age, education, and gender, only African-Americans were at higher risk of disability (adjusted OR = 2.09; 95% CI 1.78, 2.45). These effects persisted when adjusting for the microvascular and macrovascular complications of diabetes (OR 2.74, 95% CI 1.89, 4.00). There were no significant interaction effects between race and other demographic or clinical variables. In the US population, the total lost income since the onset of disability for African-Americans with type 2 diabetes is $26 billion, which represents an incremental lost income of $12 billion compared to non-Hispanic whites with diabetes. CONCLUSION: African-Americans with diabetes have substantially elevated risks of disability and loss of income compared to non-Hispanic whites. However, the reasons for this disability remain unclear, and do not appear to be solely related to the increased risk of microvascular and cardiovascular complications. More research is needed to better define the causes and effects of increased rates of disability in minorities with type 2 diabetes. PURPOSE: The complications of type 2 diabetes, such as cardiovascular disease, stroke, visual loss, and renal failure, result in high rates of morbidity and mortality. Disability from these complications is substantial and leads to dramatic losses in income and productivity. We sought to quantify the rates, causes, and costs of disability in patients with type 2 diabetes in the United States, which have not been well delineated. METHODS: We used data from the 1992 wave of the Health and Retirement Study (HRS), a national household sample of people 50 and older in the United States, to estimate risks of disability in patients with type 2 diabetes. Self-reported disability was the primary outcome variable; duration of disability was a secondary outcome. Predictors of disability in the population of patients with type 2 diabetes (n=1330) were evaluated using logistic regression models. Independent variables included sociodemographic characteristics, health status, and presence or absence of common diabetes complications including stroke, heart failure, coronary artery disease, visual loss, and renal failure. Costs of disability were calculated by applying the median reported income in the HRS to the lost work time associated with disability; these were estimated for the entire US population using survey weights. RESULTS: 23.1% of patients with diabetes were disabled, compared to 7.7% of subjects without diabetes (unadjusted OR = 3.62; 95% CI 3.14 ± 4.18). Although more patients with diabetes were disabled, they were not disabled for longer than patients without diabetes (2550 days versus 2847 days; p=.1663). In multivariate analyses, the strongest predictors of disability in patients with diabetes were the presence of poor visual acuity (OR = 3.48; 95% CI 1.23, 9.81), congestive heart failure (OR = 3.47; 95% CI 1.69 ± 7.14), coronary artery disease (OR = 2.41; 95% CI 1.26,4.64), or renal failure (OR = 2.41, 95% CI 1.25, 4.62); stroke was not a significant independent predictor of disability. After adjustment for these outcomes, diabetes was still an independent predictor of disability (adjusted OR=2.29; 95% CI 2.61, 3.55). Based upon a median reported income of $26,000 in this sample, the lost income in the current United States cohort of patients with type 2 diabetes is approximately $14 billion annually; the total incremental lost income attributable to diabetes is $9.3 billion annually. To date, the total lost income over the lifetime of the current US diabetes population is nearly $100 billion. CONCLUSION: Type 2 Diabetes is associated with a substantially increased risk of disability, and diabetes-related disability leads to staggering losses in income and productivity. Some, but not all of this increased risk is explained by their rates of microvascular and cardiovascular diabetes complications. Both health care and societal costs should be considered when setting health care policy. 4) and a survey that asked about socio-demographic and clinical characteristics and health status. Hearing was tested with a hand-held audioscope during the study's physical examination. Hearing impairment was defined as better ear threshold at 40dB at the 1kHz or 2kHz frequencies. Logistic regression models were constructed to evaluate the influence of hearing impairment on depressive symptoms. All models were adjusted for patient socio-demographic and clinical characteristics, health status, and system of care. RESULTS: There were 484 study participants (response rate 68%). Mean age was 75+6 years; 47% were women; 52% were white, 20% Hispanic, 18% African-American, 6% Asian/Pacific Islander, and 5% Other/Multiracial; 48% had annual income under $20,000 per year; 9% received Medicaid; 24% had not graduated from high school; and 24% were enrolled in FFS. Depressive symptoms were reported by 25%. Hearing aids were worn by 9%, 17% had hearing loss at 1 kHz, and 23% demonstrated hearing loss at 2 kHz. In the multivariate analysis, hearing impairment was significantly associated with depression (OR, 1.8; P=0.05). Although there was an association between depression and hearing impairment at a frequency of 2kHz, it was not statistically significant (OR=0.67; p=.09). Other characteristics that were significantly associated with depression were higher comorbidity score (OR=1.2; p=.005); worse physical well-being (OR=1.04; p=.009); low income (OR=1.8; p=.02); and male gender (OR=1.6; p=.05). CONCLUSION: Hearing impairment is significantly associated with symptoms of depression among older persons with diabetes, a group that is already at high risk for sensory impairment due to visual loss and diabetic neuropathy, and points to a potentially correctable cause of depression. BACKGROUND: Although traditional teaching has insisted the sensitivity and specificity of a diagnostic test should not change as the pre-test probability of disease changes, several authors have suggested this may not be true in practice as patients are referred from a setting of undifferentiated problems with lower pre-test probabilities in primary care to tentative diagnoses with higher pre-test probabilities in secondary and tertiary care. The purpose of this paper is to demonstrate how and understand why sensitivity and specificity change as a patient population is divided by the physician's decision whether to refer. METHODS: Using MEDLINE, a collection of published studies with various inclusion criteria was assembled{216}. Studies were included when a) the study provided enough information about the studied population to determine its position along the referral algorithm b) the article provided history or physical examination component data that could be combined with data from other articles, and c) the article provided enough information to construct 2 Â 2 tables to calculate sensitivity and specificity for those components of the clinical examination. The studies were divided into two groups, depending on whether the patient population was studied before or after referral for surgery. Group A consists of those studies evaluating all patients in whom appendicitis was considered as a diagnosis, regardless of whether they were referred for surgery. Group B consists of those studies evaluating all patients who had been referred to the operating suite for suspected appendicitis. RESULTS: In the metanalysis of the two-by-two table data, there were several trends observed as the population studied changed from group A to group B. Sensitivity tended to rise and specificity tended to fall across most of the exam components reported. These changes were found to be statistically significant for the specificity of migration and rebound pain. When all components of the examination were combined, the fall in specificity was statistically significant for the aggregate data. CONCLUSION: This study provides the first evidence that sensitivity and specificity change in populations of patients evaluated at different positions along the referral spectrum. These changes in the operating characteristics of the finding on clinical examination are important to recognize. These changes have implications for teaching the clinical examination as well as interpreting their presence or absence in varying patient populations. For example, a finding that is felt to be non-specific in tertiary care settings may be quite specific in primary care settings. To determine the rate of hip fracture and the risk factors associated with hip fractures in disabled elderly persons who choose to live in the community rather than a nursing home. METHODS: We assessed potential risk factors in 5,187 persons who enrolled 1/90 ± 12/97 in 12 nationwide sites of the Program of All-Inclusive Care for the Elderly (PACE), which provides comprehensive care to nursing-home-eligible men and women living in the community (mean age = 79, 71% women, 49% white, 47% with dementia). Functional status on enrollment was assessed by each site's nursing staff and included degree of dependence in walking and 5 Activities of Daily Living (ADL): bathing, dressing, toileting, transferring and eating. Cognitive status was assessed using a 10-item mental status questionnaire (SPMSQ). Demographics and comorbidities were also recorded on enrollment. The main outcome measure was new hip fracture that resulted in hospital admission. Average follow-up was 2 years. RESULTS: A total of 238 hip fractures (4.6%) occurred during follow-up. The rate of hip fracture was 2.3% per person-year. Four independent predictors of hip fracture were identified using Cox proportional hazard analysis: age !75 years (adjusted hazard ratio (HR) = 2.0, 95% CI 1.4 ± 2.8); white ethnicity (HR= 2.1, 95% CI 1.6 ± 2.8); ability to transfer independently from bed to chair (HR= 3.0, 95% CI 1.2 ± 7.2); and SPMSQ errors !5 (HR= 1.6, 95% CI 1.3 ± 2.1). Several risk factors, including gender and history of stroke, were not independently associated with hip fracture after adjustment for the above risk factors. PURPOSE: Missed clinic visits interfere with good medical care and have adverse effects on clinic efficiency. Explanations for patient non-compliance in keeping appointments generally emphasize practical physical issues such as finance, transportation and socioeconomic ones. We evaluated other factors, as well, in a community hospital's medical clinic in order to define ways to improve compliance with appointments. METHODS: A 10-item questionnaire covering demographic, medical conditions, socioeconomic and support conditions, missed visits, and reasons for missed visits, were filled out by 24 clinic visitors. All charts were separately analyzed for kept and missed appointments in the previous year. 72 patients were compliant ( < 20% appointments missed) and 36 were noncompliant ( > 30% appointment missed; range=30.7 ± 85.7% median=42.3%). RESULTS: Contrary to expectations non-compliant and compliant patients did not differ by medical insurance, availability of social support, transportation, education, age, or ethnicity. The sole significant difference in medical conditions was the presence of depression. The odds ration for depression was 3.18 (95% confidence interval(1.7 ± 8.7); p=.016) times greater among noncompliers, 39% of whom admitted to depression, than compliers. The non-compliers were aware of their noncompliance. The most common reason cited for missed appointments were forgetting (n=13), inadequate money for transportation (n=12), personal problems (n=9), and too long a wait (n=9). However, these did not differ significantly from the reasons cited by the others. CONCLUSION: The most striking, and we believe important, finding in our survey was the significant association of depression with noncompliance far outstripped all other characteristics, including financial and social ones. This observation suggests a very important problem, as well as a potential solution to the problem of noncompliance. We conclude that it is essential to address the possibility of depression actively in such patients as both a therapeutic goal in itself, and a potential means to improve compliance with medical care. PURPOSE: Although intentional weight(wt) loss is recommended to many patients, whether it improves survival, particularly among men, is unclear. We examined the association between intentional wt loss and mortality in a national sample of men. METHODS: We used data from the 1989 National Health Interview Survey where 20,131 adults (91% response rate) were asked about sociodemographic and basic health information including self-reported health and smoking status, health care utilization, height, current and maximum(max) wt, and whether they were trying to lose wt. Date of death was obtained by linking to the National Death Index through 12/31/95. We determined wt loss by subtracting wt in 1989 from max wt. We classified wt loss as intentional if respondents reported that they were trying to lose wt in 1989; otherwise, it was considered of unclear intention. We used Cox models to examine if intentional wt loss was associated with time to death. We limited our analyses to men only to avoid misclassification and confounding due to pregnancy. We weighted all percentages to reflect population estimates and adjusted standard errors to account for the complex sampling. RESULTS: Among 8395 eligible men, 604 had died by the end of follow-up. Among men who lost wtintentionally,thosewholost5 ± 10lbshadthelowestunadjustedmortality(2.3%)whencompared to those who lost up to 5 lbs (5.2%), p=.004. After adjusting for age, max. BMI, race, education, smoking, health status, number of doctor visits, hospitalized days, and days in bed, and intent to lose wt, intentional wt loss was associated with improved survival among men who lost 5 ± 10 lbs (see Table) . The benefits of weight loss were most consistent among men with BMIs of 25 ± 29.9 kg/m 2 . CONCLUSIONS: Modest intentional weight loss appears to be associated with mortality benefit among men. The benefit of greater weight loss is less clear.
ACUTE PANCREATITIS. Z. Weikang 1 ; 1 Union Hospital, Wuhan, Hubei PURPOSE: Glycoprotein ulinastatin is a newly discovered proteinase inhibitor extracted from human urine that can restrict the activity of pancreatin. This study was designed to evaluate its clinical effect on patients suffering from acute pancreatitis.
METHODS: 257 patients were divided into two groups. The patients in the experimental group (132 patients) received ulinastatin (300,000U/day for two weeks). The patients in the control group (125 patients) were treated with routine protocol (anisodamine 20mg/d). Both groups received similar supportive treatments. Observations of clinical symptoms and signs were made on daily basis and measurements of serum amylase, hepatic/renal function, and TNF-I Ã A Â were performed at the 3-day interval for three weeks. RESULTS: The results showed that the clinical symptoms and signs such as fever, abdominal distention and pain, belch, and constipation of the experimental improved faster than those of the control group. Analyses of serum amylase, glutamic pyruvic transaminase (GPT), creatinine, and TNF-I Ã showed that all these indexes were significantly lower in the treatment group than those of the control group (p < 0.01) at the 6th, 9th and 12th day, even though no such difference was observed at the 3rd day. CONCLUSION: Acute pancreatitis patients treated with ulinastatin recovered significantly faster than those patients treated by routing protocol. Thus, ulinastatin is an effective medicine for treating patients with acute pancreatitis. PURPOSE: Pharmacists may improve patient outcomes by engaging in pharmaceutical care (PC) activities (e.g., monitoring symptoms, counseling about medications, helping resolve drugrelated problems). We conducted a randomized trial in 36 community drug stores to evaluate the effectiveness of PC for patients with asthma or chronic obstructive pulmonary disease (COPD). METHODS: 36 drug stores were divided into 12 clusters of 3 stores matched on key criteria. Within each cluster, stores were randomized to PC or to 1 of 2 control groups.
Pharmacists in the PC group were provided with: recent patient-specific data [e.g., peak expiratory flow rates (PEFR), acute exacerbations resulting in hospitalization or emergency department visit] displayed on a computer in their stores; materials to facilitate implementation of PC; and training on the PC program. Customers of these stores were eligible if they: filled a prescription for a breathing medication during the past 3 months; reported having asthma or COPD; were 18 years or older; filled !70% of their prescriptions in a single study store; were able to be interviewed; and resided in the community. Primary outcomes (PEFR, health-related quality of life, acute exacerbations) were assessed at 6 and 12 months. We present 6-month interim data; differences at the p < 0.05 level are considered significant. RESULTS: Asthma subjects (N=660) had a mean age of 45 years, 80% were Caucasian, and 82% female. COPD subjects (N=453) had a mean age of 62 years, 86% were Caucasian, and 66% female. At the 6-month interim analysis, the PC group did not have significantly better health-related quality of life or PEFR than either control group. The proportion of patients in the PC group with an acute exacerbation (11.4%) was not significantly different from either control group (9.4%, 9.1%). Neither was their a difference in months until the first acute exacerbation between the PC group (2.91.8) and either control group (3.11.6 and 2.31.7). CONCLUSION: Our interim (6-month) analyses found that an intensive PC program in community drug stores had no significant effect on our primary outcomes. We are currently examining long-term (12-month) outcomes, secondary outcomes (e.g., patient satisfaction), and dose-response effects. Recent studies report inconsistent findings on the changes in the incidence of hospitalizations for ischemic heart disease. These reports have relied primarily on hospital discharge data. Preliminary data suggest that a significant percentage of patients suffering acute myocardial infarction in rural communities are transferred to urban centers for care. Patients transferred to a second hospital may be counted twice for one episode of ischemic heart disease. The purpose of this study is to describe the impact of double counting and transfer bias on the estimation of incidence rates and outcomes of ischemic heart disease, specifically acute myocardial infarction, in the U.S. METHODS: Methods: Analysis of state hospital discharge data from Kansas, Colorado, Nebraska, Arizona, New Jersey, Michigan, Pennsylvania, and Illinois for the years 1995 ± 1997 for patients reported to have suffered ischemic heart disease (ICD9 codes 410 ± 414, 786.5). We developed a matching algorithm for hospital discharges to determine patients counted twice for one episode of ischemic heart disease. We validate the matching algorithm with a sensitivity analysis and chart audit. RESULTS: We found that double count rates range from 10 ± 15% for all states and have increased over the past 3 years. Moderate sized rural counties had the highest estimated double count rates at 15 ± 20% with a few counties having estimated double count rates a high as 35 ± 50%. Older patients and females were less likely to be double counted (p < .05). CONCLUSION: Conclusions: Double counting patients has resulted in a significant overestimation in the incidence rate for hospitalization for acute myocardial infarction. Correcting for this double counting reveals a significantly lower incidence rate and a higher inhospital mortality rate for acute myocardial infarction. Transferred patients differ significantly from non-transferred patients, introducing significant bias into myocardial infarction outcome studies. Double counting and transfer bias should be considered when conducting and interpreting research on ischemic heart disease, particularly in rural regions. Jan 1, 1991 and June 30, 1996 for either PE or DVT were analyzed. For each record, the first hospitalization with a principal diagnosis of DVT (N= 51,233) or PE (N = 20,017) was considered the index hospitalization. Demographic and clinical characteristics (ICD-9 codes) were compared, as were the 6-month incidences of re-hospitalization for recurrent PE or DVT, bleeding and death. Surgery defined as operation within 61 days. RESULTS:
All differences p < 0.001 except: bleed Ð 6 month follow-up p= 0.05; death Ð 6 months p=0.52, NS. In multivariate logistic modeling, odds ratios (ORs) for recurrent VTE in 6 months: All differences significant with p < 0.001. Each race compared to others combined.
All differences p < 0.001 except: bleed Ð 6 month follow-up p= 0.05; death Ð 6 months p=0.52, NS. In multivariate logistic modeling, odds ratios (ORs) for recurrent VTE in 6 months: CONCLUSION: Patients with PE (versus DVT) were less likely to be Latino, more likely to be African-American, and more likely to have cardiopulmonary disease or recent surgery. Recurrent VTE manifested as PE was strongly associated with an initial diagnosis of PE, and conversely, recurrent DVT was strongly associated with an initial diagnosis of DVT. Coupled with recent data indicating that the prevalence of factor V-Leiden among patients with PE is significantly lower compared to patients with DVT, our findings suggest that there are undefined genetic or acquired factors that effect the phenotypic expression of VTE. complications after specific kinds of surgeries has been intensely studied, few studies have compared the 3-month incidence of VTE after different surgeries. METHODS: We analyzed the California linked discharge records of 2,159,747 patients hospitalized between Jan 1, 1991 and Oct 1, 1996 who underwent one of 75 selected surgical procedures on the first or second hospital day, and determined the 3 month incidence of rehospitalization for VTE. Data were stratified by age ( > 65 and < 65) and malignancy (present within 6 months or absent). A multivariate model was developed to compare the 3 month incidence of VTE using inguinal hernia repair (ICD-9-CM = 53.0) as the referent surgery, adjusting for age, sex, presence of malignancy, Charlson Index > 1, race (Asian vs. non-Asian), and prior VTE ( < 6 mo.). RESULTS: The table below shows the VTE incidence in a analysis restricted to age > 65 yrs, no cancer, > 10,000 procedures. In a logistic model that included all surgeries, parameters significantly associated with VTE included age > 65 (OR = 1.5, CI 1.4 ± 1.6), malignancy (OR = 2. (83%) returned questionnaires. Mean patient age was 42 years, 17% were female, 71% were white, and 66% were gay. In bivariate tests, general communication (r = 0.12, p = 0.003), ADH dialogue (r = 0.19, p < 0.001), overall satisfaction (r = 0.10, p = 0.01), willingness to recommend (r = 0.12, p = 0.006) and MD trust (r = 0.10, p = 0.01) were significantly associated with ADH. MCS (r= 0.226, p < 0.001), PCS (r = 0.160, p < 0.001), and age (r= 0.16, p = 0.0003) were significantly associated with ADH. Blacks reported worse ADH than whites (p < 0.05). In multivariable models, after controlling for MCS, age, and race, ADH dialogue was the only variable significantly associated with ADH (R2 = 0.12, p = 0.001). CONCLUSION: Only ADH dialogue was independently associated with adherence. Optimizing patients' ADH probably requires detailed and specific information gathering and problem solving directed at specific dosing times and/or specific medications. These data do not appear to be an automatic biproduct of good general MD-PT communication. The Surgeon General's Report on Oral Health highlights the inadequate oral health care provided to the vulnerable population of homebound older adults. Cost of dental care and transportation challenges serve as major barriers to dental care in this population. We sought to examine the impact of removing both cost and transportation barriers on dental care utilization in urban homebound older adults. METHODS: 225 older adults participating in a home and community based waiver program and/or a home-delivered meals program were invited to receive free nutritional and oral health assessments. Individuals with both poor oral health and nutritional needs were offered free dental care including free transportation. A dental hygienist and nurse coordinator made appointments on behalf of patients, provided appointment reminders and arranged transportation. RESULTS: Mean age of participants was 81.6. 81% were female, 53% were African American. Of 159 subjects who completed both nutritional and oral health assessments, one-third were eligible for free dental intervention and transportation. Of those offered free dental care, 80% agreed to receive care. Of those who agreed to receive care, 14% did not keep any dental appointments, 19% kept some of their dental appointments but did not complete their dental intervention, 67% fully completed their dental interventions. The only significant barrier to completing the recommended dental intervention was depression score (p=.03). Even when functional status and Mini Mental State Examination scores were adjusted in the model, depression remained an independent predictor for not completing the recommended dental care visits. CONCLUSION: When cost and transportation difficulties are removed as barriers to dental care, it is possible to complete needed dental interventions in a significant number homebound adults. Public policy changes would be needed to provide an infrastructure to facilitate this process. Depression remains an independent risk factor for incomplete interventions. PURPOSE: To avoid erroneous results in the estimation of the sensitivity and specificity of diagnostic tests, it has been recommended that a broad spectrum of disease be represented in the study population when evaluating the efficacy of a test. However, the actual effect of severity of disease on these indexes has not been evaluated on a clinically relevant population. The objective of this study was to assess the effect of the severity of disease on the diagnostic performance of a positive PPD and upper lobe disease on chest radiograph (defined as infiltrates and/or consolidation above the third rib posteriorly) for the diagnosis of pulmonary tuberculosis (TB). METHODS: Seventy five patients with positive sputum culture for TB and 75 subjects who were considered at risk of TB but whose sputum tested negative for TB were identified from the hospital's TB registry. Patients were divided into two groups, severe disease and non-severe disease according to the presence or absence of specific criteria that are known to be associated with worse clinical outcomes. Patients with TB were classified as having severe disease if the sputum smear tested positive for acid-fast bacilli. Patients without TB (most of which had pneumonia) were classified as having severe disease if they were older than 65 years of age, had a temperature greater than 408C, or had pleural effusion on chest radiograph. The sensitivity and specificity of a positive PPD and upper lobe disease were determined independently among the group of patients with severe and non-severe disease. RESULTS: The sensitivity of upper lobe disease for the diagnosis of TB was 41.6% and 21.6% among those with severe and non-severe disease respectively (P = 0.03). The specificity of this radiologic finding increased from 77.2% among patients with severe disease to 94.7% among those with non-severe disease (P = 0.05). Conversely, the sensitivity of PPD was lower among those with severe disease (22.2%) compared to those with nonsevere disease (42.0%, P = 0.05). The specificity of PPD was not statistically significantly different among this two groups (100.0% vs 89.0%, severe vs non severe disease respectively; P = 0.10). CONCLUSION: The spectrum of disease significantly modified the sensitivity and specificity of a positive PPD and upper lobe disease on chest radiograph for the diagnosis of TB. Interestingly, the effect of the severity of disease was not in the same direction in the two tests evaluated. This potential source of error in measuring test performance should be considered when translating the results of research studies to the clinical practice. Variables (and beta weights) negatively (p05) associated: age in decades (À0.9), being female (À2), being employed, retired, homemaker or student (À0.89) compared to being unemployed, having friends who provide encouragement when faced with a difficult situation (À.76), and frequency of physical problems interfering with social activities (À.26). Family CAGE, race, marital status, education level, having a close friend, losing a friend or relative, frequency of contact with those closest to you, self-report number of visits to a doctor in the past year were not significantly associated with AUDIT score. CONCLUSION: The associations presented do not necessarily suggest causality. As expected, being male, younger, having friends who are problem drinkers, and higher CAGE scores had the highest association with AUDIT scores. Counter to our a priori predictions, frequency of participating in spiritual activities and having a friend willing to help were associated with higher AUDIT scores. Interestingly, frequency of emotional problems is positively associated with AUDIT scores while frequency of physical problems is inversely associated. Also unexpected was the lack of association between Family CAGE and AUDIT score. Further investigation may help explain these unexpected findings. Patients were asked about their preference for first seeing their PCP or a specialist for 3 clinical scenarios and the health problem for which they sought medical care on the day on which they were surveyed. To identify the independent predictors of preferring to see a specialist, we combined patients' responses to the 3 scenarios and health problem and performed a multiple logistic regression, adjusted for clustering at the patient level using the Huber-White method. We also performed analyses separately for each of the 3 scenarios and the presenting health problem. RESULTS: The proportion of patients who preferred to see a specialist first for the 3 scenarios was 13% for chest pain, 15% for knee pain, 11% for a rash, and was 8% for the presenting health problem. In the multivariable analysis, blacks were less likely than whites to prefer a specialist (OR=0.45, p < 0.0001). Compared to those with fee-for-service insurance, those with VA coverage and Medicaid insurance were less likely to prefer a specialist (OR=0.55, p=0.04 and OR=0.42, p=0.01, respectively). Those who preferred a specialist had less trust in their PCP (OR=1.6, p < 0.0001), had less confidence in their PCP's ability to diagnose and treat their problem (OR=2.6, p < 0.0001), were more certain about their diagnosis (OR = 1.4, p < 0.0001), and perceived the health problem as being more serious (OR = 1.2, p = 0.02). Non-significant predictors included age, gender, employment, education, income, managed care insurance, health status and several attitudes including perceived susceptibility to illness, entitlement and assertiveness. These results were similar across the 3 scenarios and the presenting health problem. CONCLUSION: Controlling for other sociodemographic characteristics, health beliefs and attitudes, blacks were much less likely to prefer care from a specialist. Though blacks have generally had worse access to care, lower quality of medical care, and worse health outcomes, their satisfaction with medical care has not consistently been lower than that of whites. Our results support the hypothesis that differences in expectations for care might explain this incongruity and raise the question about whether differences in expectations might contribute to disparities in health care quality and outcomes. PURPOSE: Most providers say that they withhold protease inhibitors (PIs) from HIV-infected patients whom they believe will be non-adherent. We hypothesized that this prescribing attitude might influence patients' access to PIs and sought to determine whether this prescribing attitude explained why African Americans tend to receive PIs later than whites. METHODS: In the HIV Cost and Services Utilization Study (HCSUS), we prospectively studied a nationally representative sample of HIV-infected patients (n=2864) and their providers in the U.S. We examined data for those with completed provider and patient surveys (398 providers taking care of 1892 patients (66% of original patient cohort)). We asked providers whether they prescribe PIs to patients whom they believe will be non-adherent. Patients were followed over a 2-year period and were asked at baseline and both follow-up surveys whether and when they began using PIs. Using parametric survival models, we determined the impact of providers' PI prescribing attitude on time to first PI use and on racial differences in time to first PI use. RESULTS: Contrary to our hypothesis, having a provider who considers adherence before prescribing PIs (More Restrictive) predicted earlier PI use, adjusting for provider characteristics only (e.g. HIV knowledge and type of specialty) (p = 0.01). However, providers' PI prescribing attitude was not associated with time to PI use after additionally adjusting for patient CD4 count, symptoms, age, gender, HIV risk factor, education, income, insurance and perceived access to care. After adjusting for patient and provider characteristics, the median time to first PI use was 74.2 days later for African Americans than for whites (p = 0.001). This racial difference in time to PI use was similar for an individual with a More or Less Restrictive provider (adjusted racial difference in time to PI use was 74.7 and 73.3 days respectively, p > 0.20). CONCLUSION: Providers' PI prescribing attitude was not associated with patients' access to PIs. African Americans received PIs later than whites, but providers' PI prescribing attitude did not explain this racial disparity. Future studies will need to examine whether other patient or provider characteristics better explain why African Americans receive PIs later than whites. PURPOSE: Though blacks have greater mortality rates than whites, it is not known which diseases contribute most to this disparity. We examined mortality rates as well as years of potential life lost (YPLL), which is a more sensitive measure of loss of life due to premature death. METHODS: To examine nationally representative samples of adults in the U.S., we used data from the 1986 , 1988 and 1990 for participants greater than age 25 with corresponding National Death Index data on each cohort through 1995. YPLL was calculated as the difference between the age at death and the maximum years that a person could have lived, which we set at age 85. Mortality rates and YPLL due to all and specific causes of death were calculated for blacks and whites and standardized to the age, sex, and educational distribution of the total U.S. population. We then calculated the percent contribution of racial differences in YPLL from specific causes of death to racial differences in YPLL from all-cause mortality. RESULTS: Adjusted for age and sex only, YPLL due to all-cause mortality was 0.62 years/ person greater for blacks than for whites, averaging across the 3 NHIS cohorts. However, this disparity disappeared after adjusting for education in addition to age and sex (black-white difference was À0.08 years/person). Age and sex-adjusted YPLL was greater among blacks compared to whites for all of the specific causes of death that we examined and for all 3 NHIS cohorts. The contribution of specific causes of death to the racial disparity in YPLL was 25.6% for infections, 14.0% for cancer, 6.1% for diabetes, 4.6% for stroke, 4.0% for ischemic heart disease, 3.3% for renal disease, 3.1% for lung disease, and 4.0% for accidents, homicides, and suicides combined. An analysis of mortality rates revealed similar results, with the exception that cancer contributed most to the racial disparities in mortality (22.6%) followed by infectious diseases (14.6%). For deaths due to infections, the black-white mortality rate difference was more pronounced in those younger than 60 years old compared to those 60 years or older (6.0 vs. 0.6 deaths/10,000 person-years, respectively). In contrast, the racial disparity for deaths due to cancer was much greater in those age60 years than in those age < 60 years (black-white mortality difference=20.2 vs. 3.5 deaths/10,000 person-years, respectively). CONCLUSION: YPLL was greater among blacks than whites, though differences in socioeconomic status (using education as a proxy) appear to account for this disparity. Mortality rates and YPLL yielded slightly different results because YPLL accounts for the age at death and racial disparities in death due to infections were more pronounced at younger ages as compared to deaths from cancer. Future efforts to reduce the racial disparity in mortality may have the greatest impact if focused on infectious diseases and cancer, which we found contributed the most to the difference in YPLL and mortality rates between blacks and whites. OBJECTIVES: Multiple studies indicate that patient compliance with cardiovascular medications is poor. One possible explanation is that patients perceive medication use as difficult. The goal of this study was to assess characteristics of patients who found medication use difficult, including the physician-patient relationship, health beliefs, and socioeconomic factors. METHODS: We prospectively surveyed 273 White and African American patients who underwent nuclear imaging studies graded as positive for cardiac ischemia at 5 VA hospitals (Houston, Atlanta, St. Louis, Pittsburgh, and Durham). Of these, 33 (12.1%) patients rated medication use as difficult. Difficulty was assessed by asking patients``How bothersome is it for you to take your pills for chest pain, chest tightness, or angina as prescribed?'' RESULTS: Patients who found medication use difficult were less likely than those who did not find it difficult to agree that they were satisfied with their current treatment (59.4% vs. 77.0% respectively, P = 0.03) and that their doctors used words they could understand (68.8% vs. 88.3%, P=0.003). Among patients who found medication use difficult, there were also trends towards having less trust in their doctor's judgement about their medical care (78.8% vs. 87.5%, P = 0.17) and feeling that they were less able to get to know their doctor (59.4% vs. 70.6%, P = 0.20). There were no differences between patients who found medication use difficult and those who did not in the perception that their doctors were well qualified to manage their medical problems ( (SD 10.4) ]. In addition, 35/50 faculty and residents that provide DM care in the clinic completed physician surveys. Variables were coded as pt, provider or system barriers if 30% or > of respondents endorsed an item as a problem some or all of the time. Provider and system barriers were also identified from chart review when 30% or > of data was missing. RESULTS: Thirty-five (35) variables and 63 barriers to DM care were identified. Pt barriers (25.3%)included adherence with medication, glucose monitoring, diet and exercise. Noncompliance with appointments was affected by transportation, childcare, and work-related issues. Costs of care, pt literacy, lack of family support and other pt priorities were also cited as pt barriers. Provider barriers (45.2%)included knowledge of/agreement with ADA guidelines, reimbursement issues, prescribing uncertainty, and provider perceptions of pts with DM. System barriers (33.3%) included lack of insurance, DM care costs, reimbursement problems, lack of DM educators/nutritionists, poor record-keeping, difficulty obtaining lab data and provider communication issues. Chart review documented: HBA1c -67%, UA -52%, urine microalbumin -20%, total cholesteral -23.5%, LDL -11.8%, foot exam -25%,funduscopic exam -19%, influenza and pneumonia vaccine -18% and 7%, respectively. CONCLUSION: We found that provider and system barriers outweighed patient barriers to DM care in this high-risk population. In addition to pt empowerment, DM improvement programs must address provider and system obstacles that impede optimal DM management. Further clarification of specific barriers and their impact on diabetes care is needed, particulary in AA, underinsured and/or high-risk populations. Adult patients who had been cared for by the same physicians for more than 3 months and who had at least 4 visits during this period, were selected for this study. The patient trust of physician ratings were obtained by self-administered questionnaires, the Trust in Physician Scale ( modified by the Stanford Trust Study Group). In addition to patient trust, we obtained a General Trust Scale (Yamagishi, Japan), patients' demographic data, the lengths of the patientphysician relationships, the reasons for the patients' choice of physicians. 275 patients answered the questionnaire to rate 11 different physicians during their visits. Multiple regression analysis was used for this study. RESULTS: The Trust in Physician scale (5 Likert scales: 1 = totally disagree to 5 = totally agree) were transformed to a 0 to 100 scale, and it showed high internal consistency (Cronbach's alpha = .82) for Japanese patients. Mean scores of male and female were 86.3 (SD: 10.3) and 85.9 (SD: 10.5) respectively. The Trust in Physician scores were strongly associated with the General Trust scores (p = .017), the reasons for the patients' choices (p = .037) and the satisfaction with care (p < .001) after adjusted by patient characteristics. However,the Trust in Physician scores were not statistically associated with gender, age, education levels and the lengths of the relationships. CONCLUSION: The patient trust, a measure of the health care seeking behaviors, was associated with the societal trust in general in our study. In addition, the patient trust was a useful concept to assess the relationship between patient and physician, especially the satisfaction with care. Normal range (18.5 ± 24.9 kg/m 2 ) was the referent category in the analyses. RESULTS: Participants had a mean age of 54.6 years (SD=5.6); 69% were white, 29% were black and 94% were male. The distribution of BMI was: < 18.5 kg/m 2 (n=16, 1.3%), 18.5 ± 24.9 kg/m 2 (n = 254, 20%), 25 ± 29.9 kg/m 2 (n = 531, 42%), 30 ± 34.9 kg/m 2 (n = 308, 25%), 35 ± 39.9 kg/m 2 (n = 112, 8.9%) and > = 40 kg/m 2 (n = 32, 2.6%). Mean SF-36 subscale scores for the sample were lower than U.S. norms by a mean of À18.7 points (range: À32.9 on Role-Physical to À3.5 on Mental Health). After adjusting for covariates, individuals with BMI > = 40 kg/m 2 had significantly lower scores compared with normal weight individuals on the Role-Physical ( = À18.8, p=0.008), Vitality ( = À12.5, p=0.001) and Social Functioning ( = À9.3, p=0.017) subscales ( represents the deviation in mean score from the referent BMI group (3.9 vs. 2.8) than their male counterparts. Of the 652 calls during the study, female patients called more frequently than male patients (p < .001), and female patients generated more total calls than male patients (p < .001). Also, female physicians had significantly more calls and callers than male physicians did (p < .001). Physician age, specialty, and years in practice did not affect calling rate. CONCLUSION: Compared to male physicians, female physicians experience more demand for on-call service. In addition to slower advancement, factors intrinsic to their practice may contribute to higher reported job stress among female physicians. Physician gender may be an important factor when considering severity adjustments to determine reimbursement schedules.`W E'RE JINXED!'' -ARE RESIDENT'S FEARS FOUNDED? A.C. Ahn 1 , B. Nallamothu 2 , S.K. Saint 2 ; 1 Massachusetts General Hospital, Jamaica Plain, MA; 2 University of Michigan, Ann Arbor, MI PURPOSE: Many house officers believe that there is bad kharma bestowed on them when supportive remarks are expressed during call days. Remarks such as``Hope you have a great call!'' or``Hope you have no more admissions!'' are interpreted as ill-fortuned greetings that may do more harm than good. The purpose of this study is to determine whether this fear of being``jinxed'' is based on objective findings or mere psychological superstition. METHODS: We performed a randomized controlled trial using senior medical residents at 2 university-affiliated hospitals. On the morning of a call day, each resident was randomized by selecting either a Jinx (``You will have a great call day!'') or No Jinx message (i.e. blank form) from a collection of unlabeled envelopes. Primary outcomes measured included the number of total admissions and hours of sleep for each resident, and the subjective level of difficulty of the resident's call day (on a scale from 1 to 5 with 1 being easiest and 5 the most difficult). Some residents were randomized more than once during a call month. Student t-tests assessed for differences in outcomes between the Jinx and No Jinx groups, with robust variance estimates used to adjust for the effects of clustering at the resident level. RESULTS: Thirty-three Jinxes and 36 No Jinxes were performed on 30 residents. No statistically significant differences were seen between the 2 groups in important baseline characteristics such as a priori level of superstition or admitting service (i.e., general medical service, cardiology service). Relative to those in the No Jinx group, residents who were jinxed had significantly fewer admissions (5.1 versus 6.2;P=0.021), more hours of sleep (3.8 versus 2.9; P=0.034), and a lower subjective level of difficulty (2.9 versus 2.5;P=0.040) during their call day. CONCLUSION: There is no evidence that offering encouraging remarks to residents on a call day``jinxes'' them. Therefore, such remarks may be given safely to residents without fear of causing more admissions or less sleep, or worsening a call day's level of difficulty.
GAMING THE SYSTEM -PUBLIC PREFERENCES THAT DOCTORS PLAY? G. Alexander 1 , R. Werner 1 , A. Fagerlin 2 , P. Ubel 2 ; 1 University of Pennsylvania, Philadelphia, PA; 2 Ann Arbor Veterans Affairs Medical Center, Ann Arbor, MI PURPOSE: Efforts to contain rising costs have lead third party payers to restrict access to some healthcare services. Previous research suggests that the``hassle'' of the appeals process influences physicians' sanctioning of deception to obtain medically necessary services. However, little is known about whether the public supports such deception, and if so, whether this support is sensitive to the burden of the appeals process. METHODS: A clinical vignette was administered to 700 jurors and to a randomly selected national sample of 1617 physicians. Vignettes differed by condition severity (severe angina vs. moderate low-back pain), likelihood of successful appeal (95% vs. 50%), and length of appeals process (5-10 minutes vs. 1 hour). Participants were asked whether in response to a restriction on medically necessary healthcare the doctor in the vignette should (1) appeal the restriction, (2) accept the restriction, or (3) misrepresent the facts in order to obtain the desired service. RESULTS: Overall, the public was more likely than physicians to sanction deception (26% vs. 11% respectively, p < .001), and less likely to support accepting (4% vs. 12%, p < .001) or appealing (70% vs. 77%, p < .001) the restriction. Sanctioning of deception was greater among the public than physicians regardless of the vignette used. The public's support of misrepresenting (vs. appealing) was greater with a lower likelihood of successful appeal (Odds ratio [OR] 1.67, 95% confidence intervals [CI] 1.18, 2.37) but was not significantly related to the length of appeals process (OR 1.10, 95% CI .78, 1.55). Among physicians, the likelihood of sanctioning deception was significantly greater with both a lower likelihood of successful appeal and a longer length of the appeals process. CONCLUSION: Sanctioning of physician deception is even greater among the general public than among physicians, and unlike physicians, the public's support of deception is not sensitive to the length of the appeals process. Patients' preferences regarding deception of insurance companies may further pressure physicians who struggle to balance the tension between patient advocacy and honesty. There was no difference in the two groups in the percentage of patients discharged to Skilled Nursing facilities or to home with home health. However, for the patients needing SNF placement there was increased LOS for Friday admissions (mean LOS: Fri 4.7, Mon 3.7; p = 0.03). Similar trend was present for patients needing HH but did not reach statistical significance (mean LOS: Fri 4.4, Mon 4.0, p = 0.20) . There was no difference in mortality (% Expired: Fri 1.22%, Mon 1.88%; p = 0.45). Analysis of the yearly trend revealed decreasing cost and LOS through the four years, but the Mon-Fri difference was maintained. CONCLUSION: Patients admitted on Fridays have higher costs and LOS, despite similar case mix. They have more labs and diagnostic radiology testing done. They are admitted under`O bservation'' status less frequently and spend more time in observation. They wait longer for placement. Despite recent successes in decreasing cost and LOS, weekends continue to play a significant role in the cost of healthcare. Further reduction in cost will require attention to the factors we have identified including reduction in diagnostic testing and more efficient discharge planning over the weekend. this study was to determine the correlation between risk-adjusted hospital rates and rankings for postoperative mortality, postoperative pneumonia (POP), and respiratory failure (RF). METHODS: Cases were selected from those who underwent major noncardiac surgery at 44 VA hospitals participating in Phase I and II of the National VA Surgical Quality Improvement Program. Postoperative mortality was defined as death within 30 days postoperatively. POP was defined as a positive sputum culture with antibiotic treatment or an infiltrate on chest x-ray diagnosed as pneumonia or pneumonitis following surgery. RF was defined as mechanical ventilation greater than 48 hours postoperatively and/or reintubation and mechanical ventilation subsequent to postoperative extubation. Separate logistic regression models predicting mortality, POP, and RF were developed using Phase I cases (10/91 ± 12/93) and validated using Phase II cases (1/94 ± 8/95 PURPOSE: Due to a number of factors, many clinicians have experienced a decline in their income over the past few years. To compensate for this decline, some physicians have chosen to supplement their income through means not associated with their primary practice of medicine (e.g. drug trials, speaking engagements, malpractice case review, product sales). This is the first study to qualify and quantify the extent of participation in such supplemental income-generating activities (SIGAs). METHODS: We conducted a cross-sectional mailed survey of 1000 Maryland members of the American College of Physicians to determine the frequency of participation in various SIGAs. Members were sampled sequentially with a random start. The survey consisted of 24 items designed to collect information on demographics, practice patterns, income variables, and participation in nine different SIGAs. We used Chi-square analysis to determine whether relationships existed between personal and professional characteristics and involvement in SIGAs. RESULTS: After the first two mailings, our response rate was 40%. Sixty-three percent of those surveyed reported engaging in at least one SIGA. The three most common activities noted by these physicians are shown in the table below. Statistical analysis suggested a significant association between participation in SIGAs and male gender, married status, age between 41 and 50, having dependents, dissatisfaction with income, urban or suburban practice setting, and medical subspecialization. CONCLUSION: Physician participation in income-generating activities outside of their primary medical practices is widespread, perhaps reflecting an increase in financial pressures caused by decreasing reimbursements and managed care limitations. The impact of time diverted from the primary practice of medicine on continuity of care, as well as the ethical issues surrounding the enrollment of patients in clinical trials, the sale of medical and/or non-medical products from the office, and physician relationships with the pharmaceutical industry needs to be further examined. PURPOSE: Transthoracic echocardiograms (TTE) are frequently ordered by generalists to assess a heart murmur or screen for valvular heart disease. Little is known about interobserver agreement (IOA) in diagnosing aortic insufficiency (AI)or stenosis (AS)by TTE. The limited studies done have suffered from one or more of the following: 1) verification bias, 2) small sample size, 3) limited generalizability as observers were given a specific interpretation protocol and 4) possible Hawthorne effect. Despite these features fostering IOA, some studies have reported significant lack of IOA, particularly for AI. Our submission avoids the above deficiencies and appears to be the largest analysis of IOA in the diagnosis of AI and AS by TTE. METHODS: 143 patients undergoing TTE for any indication were prospectively enrolled in a blinded study assessing components of the physical exam for diagnosing AI. The TTE, incorporating color and continuous flow Doppler analysis, were performed by an experienced technician and officially read by one of three staff cardiologists. These studies were also interpreted by a blinded study cardiologist (SRB) for the presence of AI or AS. All observers were experienced in TTE interpretation and none (including SRB) were aware of each other's findings or the planned IOA analysis. No interpretation protocol was specified. SRB's findings were compared to the official readings. AI/AS was graded 0 (absent) to 3 (severe respectively. Data were not linked by respondent. The single item burnout measure used in this survey has previously been validated in this and other settings. RESULTS: Self-reported burnout increased in prevalence from 19% of the 439 respondents in 1991 to 41% of the 482 respondents in 1999 (p = .001). Between 1991 and 1999, the number of female and part time physicians increased as did the average age and organizational tenure. Burnout was higher in physicians in the middle range of tenure (6 ± 15 years) and in 1999 among physicians working part-time but was not associated with physician specialty, age, or gender. Burnout was associated with workload factors such as stress from long hours, inadequate time for patient visits, and the perception that the visit rate was too high to do a good job (p=.001).
Negative attitudes regarding relationships with colleagues, dissatisfaction or stressful relations with patients, and negative attitudes about organizational control of practice were also associated with burnout (p = .001). From 1991 to 1999, the number of physicians reporting great stress from long hours increased from 15 to 41%, physicians reporting great stress from inadequate time for patient visits increased from 37 to 48%, and the number of physicians reporting that the visit rate was too high to do a good job increased from 31 to 54% (p = .001 for all). The quality of professional relations was perceived to be worsening by 20% of physicians in 1991 but by 40% in 1999 (p = .001). Although satisfaction with patient relations and continuity of care remained stable, physicians experienced higher levels of stress from demanding patients in 1999 than in 1991 (p = .004). Measures of perceived organizational control of practice were unchanged. CONCLUSION: Key correlates of the increased burnout rates noted in 1999 appear to be stress from workload factors, stress from demanding patients, and perceptions of declining quality of professional relations. Further research needs to elucidate organizational and structural changes that can prevent burnout by modifying perceptions of physician workload and improving relationships with colleagues and patients. RESULTS: Most clinic encounters were satisfying to physicians and patients. The mean physician satisfaction score was 4.01, SD .92 (5-point scale) and the mean patient satisfaction score was 9.46, SD 1.13 (10-point scale). Continuity of care was associated with patient satisfaction. Patient satisfaction was 9.65 if they had seen the physician previously; whereas, it was 9.24 if they had not seen the physician previously (t = 3.61, p = .0004). However, physician satisfaction was not influenced by previously seeing the patient. Physician satisfaction for an encounter was greatest if the patient was the first of the clinic day compared to all others (4.15 vs. 3.96, t = 3.05, p = .002) . Similarly, patient satisfaction was greatest if they were the first patient of the clinic day (9.69 vs. 9.39, t = 3.13, p = .001). Physician satisfaction with the immediately preceding patient encounter also appears to influence satisfaction within a clinic visit. Physician satisfaction with a clinic visit is lower if the physician was dissatisfied with the patient from the immediately preceding clinic visit (p = .001). However, physician satisfaction with the preceding clinic visit does not influence patient satisfaction. CONCLUSION: The majority of clinic visits are satisfying to physicians and patients. However, clinic continuity, the order in which patients are seen and even the previous patient can influence satisfaction within a clinic visit. These insights can guide the development of educational strategies to improve the patient-physician interaction, which in turn should enhance the satisfaction of physicians and patients with clinical encounters. RESULTS: Overall unadjusted in-hospital mortality was 12.2%, and was similar during July ± September and October ± June in major (14.2% vs. 14.5%; p = .37) and minor (11.5% vs. 11.5%; p = .97) teaching hospitals, but differed in non-teaching hospitals (10.3% vs. 11.3%; p < .001).
Adjusting for severity of illness using logistic regression, the odds of death was similar (p > .1) in July ± September admissions, relative to October ± June admissions, in major (OR 0.96; 95% CI, 0.89 ± 1.03), minor (OR 1.06; 95% CI, 0.96 ± 1.19), and non-(OR 0.95; 95% CI, 0.88 ± 1.01) teaching hospitals. Results were similar when admissions in July, August, and September were examined separately, as shown in the table below (data reflect OR (95% CI)).
Likewise, the relative LOS of ICU admissions in July ± September was nearly identical to admissions in October ± June in major teaching hospitals (1.00; 95% CI, 0.98 ± 1.02). This was also true in minor teaching and non-teaching hospitals. CONCLUSION: We found no evidence to support the existence of a July phenomenon in ICU patients. Patients admitted to teaching hospital ICUs during July, August, and September, have similar adjusted mortality and LOS. These findings suggest that residency programs may compensate for the inexperience of new housestaff during the first 3 months of the academic year.
OUTCOMES? RESULTS OF A REGIONAL ANALYSIS. W.A. Barry 1 , G.E. Rosenthal 1 ; 1 University of Iowa Hospitals and Clinics and Iowa City VAMC, Iowa City, IA PURPOSE: Discontinuity of care in teaching hospitals may occur when teams rotate on and off service. The purpose of this study was to determine relationships between the timing of admission (during the month) and in-hospital mortality and length of stay (LOS) in patients admitted to intensive care units (ICUs). METHODS: We conducted a retrospective cohort study of 156,136 consecutive admissions (mean age 63.0 years, 51.7% male) to 38 ICUs in 28 hospitals in a large mid-western metropolitan area during 1991 ± 1997. Hospitals included 5 major (n = 48,853), 6 minor (n = 29,995), and 17 non-teaching (n = 77,288) facilities. Data were collected from patients' medical records. Admission severity of illness was measured using the APACHE III methodology. Multivariable analyses compared mortality for admissions on Days 1 ± 5, 6 ± 10, 11 ± 15, 16 ± 20, 21 ± 25 , and 26 ± 31 of each month, adjusting for severity of illness and month and weekday of admission. RESULTS: In-hospital mortality was higher in patients admitted on Days 1 ± 5, 6 ± 10, and 26 ± 31, compared to admissions on Days 11 ± 25 in major teaching hospitals (14.7%, 14.7%, 14.5%, and 10.9%, respectively; p < .01). Adjusting for severity of illness and other factors using logistic regression, the odds of death was higher in patients admitted to major teaching hospitals ICUs on Days 1 ± 10 or 26 ± 31, relative to patients admitted on Days 11 ± 25 (OR 1.11; 95% CI, 1.04 ± 1.18; p = .01). Adjusted odds of death were not higher during these periods in minor teaching (OR 1.03; 95% CI, 0.94 ± 1.13; p = .59) or non-teaching (OR 0.95; 95% CI, 0.90 ± 1.00; p = .07) hospitals. Results were similar when admissions on Days 1 ± 5, 6 ± 10, and 26 ± 31 were examined separately, as shown in the table below (data reflect OR (95% CI)).
In linear regression analyses, LOS was nearly identical in major, minor, and non-teaching hospitals (relative LOS 1.01, 1.00, and 1.00 respectively). CONCLUSION: Patients admitted to ICUs in major teaching hospitals early or late in a month have a modestly higher risk of death, even after adjusting for admission severity of illness. A similar pattern was not observed in minor teaching and non-teaching hospitals. These findings may reflect the discontinuity of care that typically occurs when housestaff and attending teams rotate on and off service. PURPOSE: To explore the extent to which physicians agree that their main responsibility is to individual patients rather than to society, to understand which physician and practice characteristics may influence this attitude, and to examine how this attitude affects physicians' career satisfaction. METHODS: We surveyed 500 primary care providers from 80 outpatient clinics in 11 managed care organizations (MCOs) and received 414 completed surveys (response rate 83%). We used t-tests, chi-squared tests and logistic regression to compare the socio-demographic and practice characteristics of physicians who strongly agreed versus all other responses with the statement``The practitioners main responsibility is to each individual patient rather than to society'' (the individual-patient ethic). We then examined the relationship between the individual-patient ethic and career satisfaction using chi-squared tests and logistic regression with career satisfaction as the dependent variable. RESULTS: 28% of physicians strongly agreed, 42% somewhat agreed, 13% were neutral and 17% somewhat or strongly disagreed that their main responsibility was to each individual patient rather than society. Physicians with the individual-patient ethic were older (43% physicians > 50 years vs 21% physicians < 35 years, p = .009), spent more hours/week in direct patient care (36 vs 32 hours, p = .048), and tended to practice in network rather than staff model MCOs (33% vs 24%, p = .077). There was no difference in gender, race, specialty, or geographic region between physicians with or without the individual-patient ethic. In multivariate analyses, physician age remained the only significant predictor of the individualpatient ethic after controlling for all other physician and practice characteristics. When examining the relationship of the individual-patient ethic to career satisfaction, physicians with the individual-patient ethic were more satisfied with their ability to provide good quality care (38% vs 19% very satisfied, p = .000) and more satisfied with their ability to serve enrollee needs (28% vs 12% very satisfied, p = .000) but were not significantly more satisfied with their overall careers (20% vs 14%, ns). After controlling for physician age, gender, specialty, number hours/ week in direct care, type of MCO and geographic region, physicians with the individual-patient ethic were more likely to be very satisfied with their ability to provide good quality patient care (AOR 3.16, 95% CI 1.88 ± 7.75), to serve enrollee needs (AOR 3.81, 95% CI 1.87 ± 7.75) and more likely to be very satisfied with their overall career (AOR 2.13, 95% CI 1.04 ± 4.39). CONCLUSION: Physicians who strongly agree that their main responsibility is to each individual patient rather than society are older and more satisfied with their careers. The extent to which this attitude impacts patient care is unknown. PURPOSE: Fragmentation of health services may hamper women's ability to obtain comprehensive, coordinated healthcare. The Veterans Administration (VA) has encouraged its medical facilities to create specialized women's health clinics (WHC) to meet these needs. This study compares patient perceptions of the quality of primary care delivery in WHC versus traditional VA general medicine or primary care clinics (PCC). METHODS: We mailed an anonymous survey to women veterans from the 10 VA facilities in the Mid-Atlantic region. For the 8 facilities with a WHC code, we randomly sampled 170 veterans who used the WHC, and 80 who used a PCC from March 1, 1999 , to March 1, 2000 At the other 2 sites, we selected 250 women who visited a PCC. The initial mailing was followed at one week intervals by a reminder postcard then a repeat survey. Patient perceptions of primary care were measured using the original 19-item Components of Primary Care Index (CPCI) by Flocke (1997 . Additionally, other provider characteristics significantly influenced patient ratings. The presence of a regular VA provider was associated with higher ratings on all four subscales (OR 2.53, 1.53, 2.44, and 3.26 respectively, p < 0.001). Among the women who had a regular provider, those who used a female practitioner also rated their care significantly higher on all subscales (OR 1.67, 1.80, 2.16, and 1.83, p < 0.001) . CONCLUSION: Female veterans using specialized women's clinics perceive a higher quality of primary care delivery even after controlling for demographics, health status, and provider characteristics. Further details about model of care, type of provider, and health outcomes for women veterans need to be examined. Since WHC appear to differ from PCC on some factors, one may infer that selection bias accounts for the differences in patient ratings between clinics. Further analyses will account for selection bias through Heckman models or other methods. PURPOSE: Waiting lists for coronary revascularization are frequently managed without any explicit queuing criteria. Patients may thus not receive priority based on the severity of their clinical symptoms. This is of particular concern in Europe. We therefore convened a European expert panel to develop criteria for maximum acceptable waiting times for coronary revascularization. We assessed the applicability of this criteria and compared Dutch patients' waiting times with the panel's recommendations. METHODS: A panel of 13 cardiothoracic surgeons and cardiologists from Spain, the Netherlands, Sweden, Switzerland and the United Kingdom was convened to assess the appropriateness of, and priority for, a set of hypothetical scenarios for coronary revascularization. They rated the appropriateness of these scenarios using a modified delphi process and then assigned a maximum waiting time, on a scale of 7 time frames, for 200 indications that were not judged inappropriate. We then measured the waiting time for coronary revascularization (i.e. the number of days between when a recommendation was made that a patient should undergo revascularization and the time the procedure was performed) for 1713 chronic stable angina patients who were treated at one of 10 hospitals in the Netherlands. We also collected data on how the patient's clinical data was presented at the meeting where the recommendation was made for revascularization: (1) direct presentations occurred when the referring cardiologist attended the meeting; and (2) indirect presentations occurred when the patient's clinical data was provided by telephone, letter or facsimile. We assessed the proportion of patients who underwent revascularization within the maximum recommended time. RESULTS: There was significant variation in the maximum recommended waiting time among the panelists (mean maximum recommended waiting time 96 days; standard deviation 85 days). Angioplasty patients waited fewer days than bypass patients (48 vs. 83 days, p < 0.001). Thirtyseven percent of patients waited longer than the maximum recommended waiting time. The majority of patients with excess waiting times were referred for bypass surgery rather than angioplasty (e.g., 83% vs. 17%, respectively). Patients whose cases were discussed during a direct presentation waited fewer days than those patients whose cases were presented indirectly (61 vs. 74 days). CONCLUSION: One-third of patients referred for coronary revascularization waited for periods longer than those recommended by a multinational panel. Two contributing factors were the type of revascularization procedure the patient was referred for and how the patient's case was presented, factors not considered by the panel as they felt a patient's waiting time should be determined by clinical symptoms. was convened to assess the appropriateness of, and priority for, a set of hypothetical scenarios for coronary revascularization. They rated the appropriateness of these scenarios using a modified delphi process and then assigned a maximum waiting time, on a scale of 7 time frames, for all non-inappropriate indications. They also assessed the impact of social factors on a subset of 45 scenarios in which the patient was considered to have mild-moderate angina, moderate left ventricular function, and had a low-moderate operative risk. The scenarios varied in the patients ability to work, live independently or care for dependents which was categorized as being: (1) not threatened, but more difficult; (2) threatened, but not immediately; and (3) immediately threatened. The time frame was converted to a linear scale and multiple regression was performed on the 540 individual panelist's ratings to assess the impact of each factor on waiting time. RESULTS: Twelve of the 13 panelists responded. Large shifts in (p < 0.001) occurred in ratings of waiting time, with the order of priority being those whose ability to work, live independently or care for dependents was immediately threatened first, those whose activities were threatened but not immediately second, and those whose activities were not threatened last. The overall mean shift in maximum recommended waiting time due to a threat to ability to work, live independently or care for dependents (37 days longer wait for those who were not threatened vs. those immediately threatened) was less than the mean shift due to extent of coronary disease (e.g., 93 days longer wait for those with 1 or 2 vessel nonproximal left anterior descending artery disease compared to those with left main disease) or stress test results (62 days longer for those with a negative compared to very positive stess test). CONCLUSION: Cardiovascular specialists may place considerable weight on the threat of cardiovascular disease on a patient's ability to perform their usual social activities. However, the impact of this factor varies according to clinical presentation. The impact of these factors should be assessed in actual practice. PURPOSE: Teaching hospitals, due to their size, expertise, and location may find themselves in a position to treat alcohol and drug related diagnoses more often than their community counterparts. Previous studies have shown the existence, in some cases, of a volume-to-outcome relationship. Thus, though past research has shown that overall teaching hospitals may be less efficient, they may experience efficiencies in uniquely qualified or niche areas. This study examines the relationship of hospital resource use and the treatment of alcohol and drug diagnoses. Specifically the study tests the hypothesis that teaching hospitals will have greater efficiency than other community hospitals in the treatment of alcohol or drug related diagnoses. This study also examines the hypothesis that hospitals with more experience in treating alcohol and drug related diagnoses would experience greater efficiencies than hospitals with less experience. METHODS: This is a retrospective cross-sectional study that examines the association of hospital resource use and the treatment of alcohol and drug related diagnoses. The population for this study comes from the 1996 Healthcare Costs and Utilization Project's National Inpatient Sample. The final sample consisted of patients with the appropriate alcohol and drug related diagnosis (n = 9,528 patients). Data from the American Hospital Association, the Area Resource File, InterStudy, and the Health Care Financing Administration supplemented the patient level data from NIS. Teaching hospitals were identified using the Council of Teaching Hospital designation. Hospital experience was measured by the number alcohol and drug related admissions relative to the hospital's total admissions. Hospital resource use was measured by taking both the log of length of stay and total hospital charges. Least square regression was used to analyze hospital resource use while controlling for hospital, patient, and market characteristics. RESULTS: The average age of the sample was 39 with 68% of the patients being male and 66% of them white and 23% black. Twenty percent of the admissions were to teaching hospitals. Teaching hospitals had significantly lower charges (p = 0.029) than non-teaching hospitals and shorter lengths of stay (p = 0.042) than their community hospital counterparts. When examining the hospitals relative experience, hospitals with greater experience had significantly lower total charges (p < 0.001); however, contrary to our hypothesis they had significantly longer lengths of stay (p < 0.001). CONCLUSION: Though previous studies have shown teaching hospitals, as compared to non-teaching hospitals, are less efficient overall, the results of this study show that they are able to experience certain efficiencies in niche areas. Specifically, teaching hospitals are more efficient at treating alcohol and drug related diagnoses. As teaching hospitals continually find themselves competing for managed care contracts, finding and documenting niche areas in which they excel becomes increasingly important. PURPOSE: Routine screening for domestic violence (DV) is increasingly recommended in outpatient healthcare settings. However, little is known about the screening process. The purpose of this study was to document the screening process for women counseled for DV in a primary care setting. METHODS: We identified pregnant women who were counseled for DV between 8/98 ± 6/99 at a university obstetrics clinic. The DV protocol at this clinic, similar to national guidelines, recommends that women be asked about DV at every visit. Identified women are offered counseling using a specific form (usually performed by a social worker). Data were collected from the DV counseling form and from medical records for the previous 6 months. We assessed how the women were screened, who had counseled the women, and what types of abuse led to the counseling. We compared demographic and health characteristics of counseled women to a control group of pregnant women, matched on race, age and type of visit, who were not counseled for abuse. RESULTS: Of 63 pregnant women counseled for abuse, 53 had complete medical records available for review. Fourteen (26%) of the women were identified as victims at their first clinic visit. Thirty-three (62%) of women denied abuse in at least one previous screening attempt. Eighteen (67%) of the 27 women who were asked about abuse by their physician on the same day that they received counseling denied abuse to their physician. Of the 48 women (91%) referred by an MD/RN to social work for counseling, only 25 (52%) were referred for DV. Twenty-three (48%) were referred for non-DV reasons with the social worker subsequently identifying the abuse. Women referred for DV were more likely to be seen within a month of referral than women referred for other reasons (72% vs. 39%, p=0.01). The majority of abuse reported was physical (96%) and recent (83% with event within past month); 37% had been experienced abuse for more than 2 years. In the 40 cases we were able to match to controls, women counseled for abuse were more likely to be depressed (51% vs. 29%, p < 0.05), to be currently using drugs (20% vs. 6%), p < 0. Bivariate analysis revealed that physicians with a higher score on the SWB scale and more frequent church attendance were more likely to hold beliefs that health outcomes are improved with patient and physician prayer and that faith alone can cure disease. There were no associations between physician sex, age, race, or spirituality training and these attitudes. CONCLUSION: A large number of physicians believe religious and spiritual behaviors by patients will improve health outcomes. A smaller but significant number believe that physician prayer will improve patient outcomes. These findings suggest that there is a need for more rigorous examination of the connection between health outcomes and spirituality as many physicians seem willing to incorporate aspects of spiritual behavior into their practices. 1995) . Little is known about the impetus to develop a separate WHC, especially within the VA, where issues of access to care, privacy, and quality of care for women veterans have often been of concern. We examined organizational, provider, and patient-level determinants of developing a separate WHC for delivery of gender-specific and primary care services. METHODS: We used data from the 1999 VHA Primary Care Practices Survey, a national survey of senior VA primary care leaders at all VA health care facilities with > 4,000 unique patients and > 20,000 outpatient visits during fiscal year 1998 (response rate 93%, n = 219). Analysis of facility characteristics associated with a self-reported WHC for delivering primary care was performed using bivariate analysis and independent predictors were determined using logistic regression. RESULTS: Nationally, 62% of facilities reported having a separate WHC to provide primary care for women. WHC's tended to reside in urban locations (p < 0.05) and in more complex facilities (p < 0.05). Facilities with WHC's had more female patients (p < 0.001) with more visits (p < 0.001), but comparable visits-per-patient (7.1 vs. 7.4). Facilities with team-based primary care that integrated providers from multiple disciplines were more likely to have developed a WHC (p < 0.05). WHC's were more likely where primary care leadership was distinct from subspecialty care (p < 0.001) and a separate budget existed for primary care (p < 0.05). In addition, WHC's were more common in academic facilities (69% vs. 56%, p = 0.09) with higher numbers of internal medicine houseofficers (43.5 vs. 26.9, p < .05) and longer outpatient block rotations (p < .05). Having separate primary care leadership, and higher women veteran caseload remained significant predictors of WHC development after adjusting for other variables. CONCLUSION: Separate WHC's were present in larger, complex, more intensively academic medical centers with more established and authoritatively independent primary care programs. As a cross-sectional study, it is unclear whether WHC development occurred as a result of or resulted in a higher female patient volume. The implications of separate service delivery for women with respect to clinical quality, and patient satisfaction are still unclear and somewhat controversial; assessing WHC performance is a crucial next step. 1.14 ± 1.25), and for all encounters combined (RR 1.07; 1.04 ± 1.09). As compared to their use of Outpatient services, PDs were especially more likely than non-PDs to use high cost Emergency Department and Inpatient services. CONCLUSION: Using CAGE results and objective encounter data from continuity patients in a large group practice, problem drinking was common and associated with higher overall utilization as well as use of higher cost sites for specific alcohol-related conditions. These findings support initiatives to screen and intervene for problem drinking in primary care practices. Respondents were asked to rate how important it is for asymptomatic men to know each of the 17 facts about PSA screening using a 5-point Likert scale ranging from``not at all important'' to``extremely important.'' Because internists and family physicians had similar responses, they were combined as non-urologists and compared to urologists. We used logistic regression to compare differences between urologists and non-urologists and adjusted for the effects of age and gender. RESULTS: Urologists and non-urologists differed in rating how important it is for men to know 9 of the 17 facts. Urologists considered it extremely or very important for men to know that it is unclear whether regular PSA screening will reduce prostate cancer mortality, that done together the PSA and digital rectal exam (DRE) can screen for prostate cancer, and that PSA and DRE are most appropriate for men whose life expectancy is at least 10 years. Non-urologists considered it extremely or very important for men to know that PSA screening is controversial, that there are risks and benefits to PSA screening, that prostate cancer may grow slowly without symptoms, that it is unclear whether treatment of early, localized prostate cancer is helpful, that there are side effects to treatment, and that a man over age 70 is less likely to die from prostate cancer even though he has a higher risk of having it. RESULTS: Faculty and resident patients were similar with respect to sex, race, weight, years of diagnosis of diabetes mellitus and type of diabetes therapy. However, resident patients were slightly older than faculty patients were (mean age 63 vs. 57, p = 0.008). Residents and faculty were no different in frequency of adherence to the ADA Clinical Practice Guidelines for visits per year, HbgA1C testing, lipid profile testing, and foot exam. Faculty were significantly more adherent to urine evaluation (44% vs. 26%, p = 0.04) and referral for dilated eye exam (54% vs. 39%, p = 0.02). There were no differences between residents and faculty patients with respect to mean HbgA1C levels, mean control of blood pressure, use of ACE inhibitors and use of lipid lowering therapy. Resident patients were more likely than faculty patients to have LDL levels controlled to less than 100 (42% vs. 25%, p = 0.03). CONCLUSION: Residents and faculty provide primary care for similar groups of diabetic patients at this academic health center. Adherence to ADA Clinical Practice Recommendations was similar between faculty and residents for many, but not all areas. Outcomes such as HbgA1C levels and blood pressure control were similar for both groups, though more resident patients had better LDL cholesterol control. Though resident continuity practices are faculty supervised and often in the same setting as faculty practices it cannot be assumed that quality outcomes are the same for patients of these two groups. Interventions to improve rates of adherence to specific recommendations should be geared towards both faculty and residents at this academic health center. Younger generation for females and males were significantly less likely to receive primary care compared to older generation males and females (females p < .001 and males p < .006). Male relatives are significantly less likely to access primary care compared to females, 57% vs. 70% (p < .015) and are less likely to access the Charity system compared to females 20% vs. 34% (p < .014). Male relatives are more likely to obtain the majority of their health care in the ER and Walk-in Clinic compared with female relatives 20% vs. 8% (p < .001). There was no difference between work status, age distribution, and health care coverage between male and female relatives. CONCLUSION: The barriers to health care access in both males and females were Full/Part time-work status, younger generation, and lack of health care coverage. Our patients' male relatives are less likely to have a regular source of primary care compared to female relatives. Offering evening and weekend hours in our indigent continuity clinics may possibly reduce this barrier. Further studies are needed to better assess this inequality of primary care access between males and females. PURPOSE: Decreasing waiting times to next available appointment involves evaluation of the physian appropriateness and effectiveness of practice in the clinic system. Some studies have shown that patients receive``better'' care from a sub-specialist than those followed by a generalist. The appropriateness and variability of this care, however, has not been well scrutinized. The purpose of this study, was to identify if patients enrolled in the cardiology clinic belong there based on a set of criteria agreed between internal medicine and cardiology. Also, to assess the variation among cardiology fellow's discharge rates and revisit interval assignment. METHODS: Data was extracted from 194 cardiology clinic notes between 1/14/00 ± 2/27/00. Providers were cardiology fellows practicing at an urban VA. The main outcomes of interest were appropriate discharge from the cardiology clinic (per criteria established by the authors) and assigned revisit interval. Predictors of interest included provider data and patient level information such as a severity of illness score and ejection fraction. RESULTS: Overall, discharge rate was 24/194 (12.4%) . Of the 161 remaining in the clinic, 74 (46%) could have been discharged to their primary provider by the agreed criteria. In logistic regression, disease severity was correlated with discharge from clinic (r =0 .69). The more stable patients were more likely to be discharged from clinic. Overall, average clinic revisit interval was 3.8 months. There was a large amount of variability among providers. Individual provider average follow up time ranged from 2.2 ± 7.7 months. Individual discharge rates ranged from 0 ± 26%. Individual clinic appropriateness ranged from 0 ± 90%. There was no correlation of revisit intervals with severity of illness score (r = .021, p = 0.794) or with ejection fraction (r = .013, p = 0.91). At best, patient severity of disease accounted for only 4% of the variance. Individual providers saw patients back at a consistent length of time regardless of the needs of the individual patients for instance, provider #10 saw every patient back in 6 months and provider #31 saw every patient back in 3 months.
CONCLUSION: There needs to be established criteria which patients must meet to remain in the clinic that will to be utilized by all providers in the clinic to reduce variability in care. There needs to be improved consistency and decreased variability in return to clinic times to improve access in the cardiology clinic setting. Return visit time should be based on severity and stablility of patient's illness and not on individual provider preference. Centers for Disease Control and Prevention, Atlanta, GA PURPOSE: Influenza vaccine is the primary method for preventing influenza. Delay in influenza vaccine distribution was expected for the 2000 ± 01 influenza season. We conducted a cross-sectional study to characterize practice adjustments and expectations of physicians regarding influenza vaccine delay. METHODS: We sent a two-page mail survey to a national random sample of 1606 internists (IM) and family physicians (FP). Participants answered questions regarding their usual influenza vaccine administration period, their practices' capacity for identifying high-risk patients, and modifications in their clinical practice for the 2000 ± 01 influenza season. The first mailing was conducted in September, 2000; non-respondents received another mailing in October, with a $2 incentive. Tests of significance were computed using likelihood ratio chisquare. RESULTS: Response rate was 60% after two mailings and did not differ significantly by respondent specialty. Of 952 respondents currently practicing, 756 (79%) typically administered influenza vaccine; family physicians were more likely than internists to administer influenza vaccine (82% vs. 76%; p < 0.05). Among physicians administering vaccine, 356 (47%) selfidentified as IM and the remainder as FP. At the time of survey completion, 13% of respondents had received their full order of vaccine; another 23% reported receipt of a partial order. Whereas 37% of physicians who had already received their full order had modified their practices this influenza season, 70% of physicians who had received either partial or no shipment reported practice changes (p < 0.001). The most commonly reported practice modification was to target high-risk patients for receipt of available doses. However, only one-quarter of physicians (28% IM vs. 24% FP; NS) had experience with mail or telephone reminder systems to contact high-risk patients. Of concern, 43% of providers were hesitant about administering vaccine after the onset of influenza season in their practice area. CONCLUSION: Facing delays in influenza vaccine distribution, many physicians reported an intention to vaccinate high-risk patients first. However, few physicians had experience using reminder systems, and nearly half of physicians were hesitant about administering vaccine after influenza season has begun. These findings raise concerns about the capacity for physicians to meet domestic vaccination goals during this influenza season and in the future, and the possibility of preventable morbidity and mortality attributable to influenza as a result. PURPOSE: Payers and institutions use chart abstraction to measure physician performance, despite its underestimation of the quality of care due to recording bias. We wondered if the medical record might also overestimate the quality of care through false, and potentially unethical, documentation by providers. To determine this, we compare the quality of care as documented in the medical record with the reports of actor patients. METHODS: Twenty physicians in the primary care clinics of two Veterans Affairs Medical Centers were randomly selected among consenting residents and faculty (97% agreed to participate). Data were collected from standardized (actor) patients, who served as the gold standard and presented undetected as patients to physician subjects, and from the medical record generated from these visits. Quality criteria were developed from national guidelines and a modified Delphi technique for four common medical conditions. These were then recorded by a standardized patient or abstracted from the medical record. Physician subjects completed 160 evaluations of standardized patients (8 cases  20 physicians). We determined the proportion of criteria reported in the medical record but not by the standardized patient (false positives). We also determined the distribution of false positives according to domain (history, physical exam, diagnosis, treatment), study site, physician subjects, and actor patients. False positive rates at the two study sites were compared by t-test.
RESULTS: Compared to the gold standard of standardized patients, false positives were identified in the medical record for 6.4% of measured items overall. False positives were higher for physical examination (13.5%) and diagnosis (14.6%) than for history (3.8%) and treatment (3.4%) . The difference in false positive rates between site 1 (7.1%) and site 2 (5.7%) was not statistically different (p = 0.34). The proportion of false positives for individual physician subjects ranged from 2.2% to 13.0% and for actor patients from 1.4% to 11.6%. CONCLUSION: These results suggest that chart abstraction may overestimate the quality of care due to false positives. The clustering of false positives in the domains of physical examination and diagnosis suggests that these are not incidental occurrences or underreporting by actor patients. Though false positives in the physical examination could result from careless documentation by physician subjects, they may indicate intentional misrepresentation of the process of care, perhaps to up-code a visit or save time by adding an exam element not performed. Such fabrication would violate ethical standards essential to the integrity of clinical practice, potentially putting patients at risk by including misinformation in the medical record. By contrast, documentation of diagnosis in the medical record but not by the actor patient would represent an important lapse in communication of essential information. Improved evaluation methods are needed to detect such irregularities as well as guidelines to determine appropriate actions when potentially unethical conduct is identified in studies of quality. University of Washington, Seattle, WA PURPOSE: For many physicians, the environment in which they trained differs from the current practice environment of managed care, declining revenues, patients on the Internet, and insurance paperwork. Studies of physician career satisfaction have suggested that unmet expectations may play a role in declining satisfaction, but this area remains largely unexplored. We designed a survey to examine career expectations and satisfaction, as well as evaluating trends in satisfaction over time. METHODS: We mailed surveys to half the ACP-ASIM membership in Oregon (n=550) in Fall 2000. Survey categories were developed from previous literature on career satisfaction and physician self-care. We asked respondents to rate 16 features as``not important, important, or very important'' to their current view of their career. The features included: having an adequate income, being valued by others, intellectual challenge, control of professional life, helping others, collegial relationships, being part of a team, doctor-patient relationships, working on the cutting edge of science, providing quality health care, managing own business, and others. We then asked whether expectations about these features were``not met, matched, or exceeded'' in their career currently. RESULTS: We had a 75% response rate (n = 412). We removed surveys returned from retired physicians and residents, analyzing 345 (67% male, 33% female). The highest number of physicians ranked``providing a high standard of care'' as a``very important'' feature of their career (81%), with 93% stating that this expectation was either matched or exceeded in their current work.``Physician-patient relationships'' and``helping others'' were also ranked as very important (68% and 63% respectively) and were matched or exceeded (84% and 94% respectively). 98% of respondents reported``autonomy/control over professional life'' as an important or very important to their career, yet a high proportion of unmet expectations appeared in the following areas:``having control over my professional life'' (52% unmet), and`h aving autonomy in current position'' (38% unmet). There was also high rates of unmet expectations in``making as much money as anticipated'' (42% unmet) and``work is fun'' (33% unmet). 71% report being``somewhat-very satisfied'' with their current practice situation, while 83% selected these satisfaction levels for their situations 10 years ago. 61% would go to medical school again. CONCLUSION: Overall career satisfaction appears to be declining with high unmet expectations regarding professional autonomy and control while, in this sample, internists reported that expectations regarding quality of care were generally met. Improved understanding of expectations and practice changes should help educators and professional groups in their efforts to educate and promote professional development. Part B in 1998 and 1999 . We utilized Medicare claims to measure quality of care based on whether or not beneficiaries had received an influenza immunization in 1999, a pneumococcal immunization between 1991 and 1999, whether females had a mammogram in 1998 or 1999, and whether persons with diabetes had an eye examination or a lipid profile in 1998 or 1999 or a hemoglobin A1C in 1999. We linked patients to primary care providers based on office visit claims and merged providers into practices based on common provider numbers. We restricted our analyses to 141 practices with at least 50 patients in the denominator for each quality indicator. We identified benchmarks as the 90th percentile in performance on each indicator and compared benchmark performance with median performance and performance at the 10th percentile. RESULTS: We observed large discrepancies in performance among practices on all quality indicators (see table) . Performance on certain quality measures was highly correlated (i.e. practices performing well on one measure were more likely to perform well on other measures). CONCLUSION: There is substantial variation among physician's practices in their performance on commonly utilized quality markers. Further efforts are needed to describe the characteristics of practices that are performing at the highest levels. PURPOSE: Previous attempts to improve the triage of patients with suspected acute cardiac ischemia in the Emergency Department (ED) have been disappointing. We developed and prospectively evaluated a decision aid based on a validated clinical prediction rule to determine if physicians' triage behavior would change and whether any changes would be beneficial. METHODS: We constructed a decision aid from Goldman and colleagues' published clinical prediction rule, which predicted major cardiac complications (N Engl J Med 1996) . After achieving over 80% compliance with use of the decision aid in our ED, we prospectively collected data on 1011 consecutive patients admitted from the ED with suspected cardiac ischemia during late 1999. Clinical data collected in the ED allowed us to adjust for possible differences in case mix when comparing decisions and outcomes before and after the intervention. We compared triage decisions (CCU vs. inpatient telemetry vs. observation unit or ward) and their safety and efficiency with similar patient cohorts studied prospectively in our institution during 1997 (n = 207) and 1998 ± 99 (n = 1033). Triage safety was defined as the proportion of all patients who had major cardiac complications within 3 days who were admitted to either the CCU or inpatient telemetry unit. Triage efficiency was defined as the proportion of all patients who did not have major cardiac complications who were not admitted to either the CCU or the telemetry unit. RESULTS: Among the 1011 patients, 1008 were eligible, with clinical follow-up on over 98%. Major complications occurred within 3 days for 3.5% (35/1008). A greater proportion of patients were admitted to an observation unit in 1999 compared to 1997: 35% vs. 21%; P < 0.001. In 1999, a smaller proportion of admissions to the inpatient telemetry unit were very low risk patients compared to 1998 ± 99: 41% vs. 52%; P < 0.001. Triage safety was slightly higher in 1999 compared to 1997: 94% (33/35) vs. 89% (8/9); P = 0.3. Triage efficiency was much greater in 1999: 36% vs. 21%; difference: 15 percentage points (95% CI: 8% to 21%); P < 0.001. CONCLUSION: A new decision aid changed physicians' triage decisions and improved triage efficiency without compromising triage safety. We therefore surveyed internists about how they inform patients about bad news. METHODS: A survey instrument asked how frequently physicians perform different activities in giving bad news to patients, based on a four point Likert scale. Categories included previously recommended activities involving emotional support of the patient (11 items) and those involving the proper setting (9 items). The average amount of time spent with patients while giving bad news was assessed. The impact of demographic variables on the number of emotional support items, setting and time items, and number of minutes spent informing patients were analyzed via ANOVA. All significant variables for each category were entered into a multiple linear regression model. RESULTS: Of the 961 surveys which were received by subjects, 461 (48%) were completed and returned. A majority of physicians (52 ± 93%) always or frequently performed 10 of the 11 recommended emotional support items and 6 (60 ± 92%) of the 9 proper setting items while giving bad news to patients. The average time spent in giving bad news was 27 minutes. Although training in giving bad news had a significant impact on the emotional support items provided to patients (p < 0.05), only 25% of respondents had any previous training in this area. A number of demographic factors were also associated with an increased number of emotional support items provided to patients, including being single (p < 0.05), being female (p < 0.005), and having personally had a life-threatening illness ( p < 0.05). CONCLUSION: By their own report, internists generally inform patients of bad news in an appropriate fashion. Some deficiencies do exist, but training in giving bad news can improve this important type of communication. Educational opportunities about how to give bad news should be provided to practicing physicians; and curricula designed for medical students and residents.
CARDS. P.D. Faris 1 , W.A. Ghali 1 , R.F. Brant 1 ; University of Calgary, Calgary, Alberta PURPOSE: In health outcome report cards comparing providers for binary outcomes such as mortality, a commonly used method of profiling providers is to use risk factor data from patients treated by the providers to construct logistic regression models. Such models are then used to obtain the expected number (E) of outcomes for each provider and the ratio of observed (O) to expected outcomes (O/E ratio) is used as a risk-adjusted measure of provider performance. To account for chance variation and to determine``outlier status'', confidence intervals (CI's) derived from standard deviations are placed around the O/E ratios. Here we compare two methods for calculating standard devations (SD's) and CI's. The typically used method is compared with a more complex method based on the propagation of errors (PE). METHODS: Typically, when calculating the variances and SD's of O/E ratios, only O is treated as a random variable, and variability in E is ignored. A variance estimate treating both O and E as random variables was derived using the propagation of errors (PE) method. The resulting PE-SD estimates were compared with typical SD estimates using a data set (N = 50,357) previously employed to profile CABG providers in Canada. O/E ratios were used to profile three levels of hospital patient volume (low, medium, high), 8 provinces, and 23 hospitals. For each O/E ratio, the ratio of the typical SD to the PE-SD was used to evaluate the relative sizes of the SD estimates. In addition, computer simulations based on a hypothetical data set with three providers were used to evaluate the SD estimates. Measures of performance included empirical coverage and bias. RESULTS: For the CABG data profiles, the ratios of the typical SD's to the PE-SD's were 1.04, 1.65, and 1.23 for the low, medium and high volume hospitals, respectively. For 7 of 8 provinces, the ratios of the SD's were only marginally larger than 1. However, for the province treating the largest proportion of patients (52%) the ratio was 1.40. For the 23 hospitals, the largest SD ratio was 1.09. The computer simulations confirmed that the typical SD's were less accurate than the PE-SD estimates, and the coverage probabilities indicated that confidence intervals based on the typical SD's are too wide. CONCLUSION: The typically used SD estimates and CI's were always larger than the PE-SD estimates that accounted for variability in E. The bias in variance and SD was greatest when one or more providers treated a large proportion of the patients. When only a few providers are being compared, or when some providers treat a large proportion of the patients, the use of typical SD's may lead to incorrect conclusions regarding the outlier status of providers. PURPOSE: Language concordance between physicians and patients is associated with greater patient satisfaction. However, it is not known what level of physician Spanish language competence is important to Spanish speaking patients. We used four measures to test the association between physician Spanish ability and the probability of these physicians having Spanish speaking patients in their practice. METHODS: Primary care physicians for Spanish and English speaking diabetics from eleven public health clinics completed a questionnaire containing four questions about Spanish language competence: 1) self-rated fluency 2) use of interpreters 3) confidence in conducting an H&P 4) confidence in discussing complex topics. We used chi-squared to test the association between measures of physicians' Spanish competence and the proportion of Spanish speaking patients in their practice. RESULTS: 105 MDs caring for 365 Spanish and 1023 English speaking diabetics participated. 17 (16%) MDs rated their Spanish fluency as excellent and they cared for 144 (40%) of monolingual Spanish speaking patients. Conversely 27 (26%) of MDs rated their fluency as poor or none and they cared for 26 (7%) of Spanish speaking patients. MDs whose self-rated Spanish was excellent, good, or fair were much more likely to treat Spanish speaking patients than those whose Spanish was self-rated as poor or none (OR 3.5, CI 2.3 ± 5.3) . Similarly, MDs who never, rarely, or occasionally use an interpreter were more likely to treat Spanish speaking patients than those who usually, or always use an interpreter (OR 3.8, CI 2.6 ± 5.4) . The two additional questions assessing clinican confidence in conducting a Spanish patient history or carrying out a complex discussion were less predicitive of treating Spanish speaking patients. CONCLUSION: While factors other than patient preference may affect the distribution of patients, our study demonstrates that a brief physician questionnaire can discriminate degrees of language competence that appear meaningful to patients in selecting their physician. University of California, San Francisco, CA PURPOSE: Prior research has shown that old age and chronic conditions such as congestive heart failure are associated with multiple admissions of patients to the medical wards of community hospitals. Risk factors associated with multiple admissions to public hospital wards are unknown, though poor access to primary care is known to be associated with preventable hospitalizations. We studied the demographic, medical, and utilization patterns of high utilizing patients of an urban public hospital to determine risk factors for multiple admissions. METHODS: Using an administrative data set, we obtained data on patient demographics, inand out-patient utilization, and in-patient and out-patient billing diagnosis on all patients discharged from a public hospital medicine/cardiology service between 7/97 ± 6/98. Patients with 3 or more discharges in a one-year interval were defined as high utilizers (HU). We used chi square to test differences between high utilizers and non-high utilizers (NHU) in utilization of emergency room and primary care services in the year following the index discharge, in diagnosis and in demographic characteristics. RESULTS: 3562 patients had 5573 discharges in the study period. 465 patients (13%) were HU who accounted for 35% of discharges, and 36% of hospital bed-days. Compared to NHU, HU were slightly more likely to be male (71% vs. 66% p = 0.02) and more likely to be African American (46% vs.30% p = 0.001) They were no different in age. HU had more visits to primary care clinics (mean 5.72 vs.2.5 p = 0.01), to the emergency department (mean 4.38 vs.2.5 p = 0.01) and to any network clinical site (median 24 visits vs. 5 visits p = 0.001). HU had more congestive heart failure (21% vs.12% p = 0.01), AIDS (30% vs.14 p = 0.01) and COPD (21% vs. 13% p = 0.02). HU were much more likely to abuse alcohol (47% vs.25% p = 0.01) or drugs (58% vs 30% p = 0.01) or either alcohol or drugs (71% vs. 42% p = 0.01). CONCLUSION: High users of urban medical wards in a clinical system with good access to primary care have high rates of use of primary care services. Our data suggests that interventions to reduce multiple hospitalizations in urban public hospitals need to integrate substance abuse services to traditional disease management programs. Indicator  Benchmark  50th Percentile  10th Percentile  Influenza  64  50  29  Pneumococcal  55  43  30  Mammograms  80  68  51  Eye examinations  88  81  69  HgbA1c  96  88  61  Lipid profile  80  57 PURPOSE: Heart Failure (HF) is a leading cause of hospitalization and re-admission in most hospital systems. Multidisciplinary``Discharge Transition'' programs aimed specifically at the education and close follow-up of HF inpatients have been evaluated in a number of relatively small randomized controlled trials (RCTs). This is the first meta-analysis to evaluate the effectiveness of peri-discharge, multidisciplinary HF patient management programs. METHODS: Electronic database searches were conducted on MEDLINE, HealthSTAR and EMBASE. Reference lists of identified articles and experts' opinions were also reviewed. All potentially relevant articles were obtained. Study selection criteria were: (1) RCTs of adult inpatients hospitalized for HF enrolled at the peri-discharge transition period; (2) HF-specific patient education intervention coupled with a post-discharge follow up assesment; (3) Primary outcome of unplanned all-cause readmission secondary outcomes of mortality, compliance and quality of life. Study inclusion and quality assessment using a modified Jadad Scale were independently assessed by all four authors. Agreement was rated by a weighted Kappa and final decision agreed upon by consensus. RESULTS: A total of 529 citation titles were identified: 199 from MEDLINE; 148 from HealthSTAR; 162 from EMBASE and 20 from personal files, reference lists, or communication with experts. Of these, 94 were deemed potentially eligible for pre-test selection. Two research trials in progress were identified and interim data obtained from the principal investigators. The four reviewers selected 10 papers and two studies``in progress'' for inclusion in the overview. The Kappa agreement statistic for multiple reviewers was 0.73 (SE 0.09). One study was subsequently excluded because it represented an earlier publication of the same trial, and another study was excluded because of substantial heterogeneity in methods, patient population and results. PURPOSE: Depression typically presents in a medical rather than mental health setting. VA and non-VA settings may differ in physician characteristics, training opportunities, and organizational factors. We sought to assess VA and non-VA medical physicians' recognition and treatment of depression. METHODS: We selected a random sample of VA and non-VA primary care physicians and medical specialists practicing in the Northeastern United States. Physicians viewed a 5 minute, professionally produced video of a patient with somatic symptoms, who described 5 symptoms meeting clinical criteria for major depression. After viewing the video, physicians answered interviewer-administered questions about differential diagnosis and management recommendations. Because of the experimental design, every physician reviewed identical clinical data, permitting direct comparisons of physicians' diagnostic reasoning and management approaches. Physicians also viewed two``control'' scenarios (a patient with chest pain and a patient with polymyalgia rheumatica), further masking the key study purpose.
RESULTS: 81 VA and 129 non-VA physicians participated. Regarding diagnosis, 85% of VA versus 95% of non-VA physicians listed depression as a possible diagnosis (p = .02), and 47% versus 62% assigned depression a probability of greater than 50% (p = .04). Regarding management recommendations, 15% of VA physicians versus 2% of non-VA physicians said they would recommend a mental health referral (p = .001), and 9% versus 13% would recommend an antidepressant (p = NS). 35% of VA physicians versus 53% of non-VA physicians would see the patient portrayed in the videotape for a return visit within 2 weeks (p = .01). CONCLUSION: Non-VA physicians were more likely to recognize depression, although many VA physicians also considered it. Neither group followed Agency for Healthcare Research and Quality guidelines for follow-up and for initiation of pharmaco-or psychotherapy, although non-VA physicians were more likely to see the patient back within 2 weeks and VA physicians were more likely to refer to mental health. Differences in management by VA versus non-VA physicians may point to systems issues underlying non-adherence to depression management guidelines.
DO HOSPITALISTS FOLLOW EFFICIENCY GUIDELINES BETTER THAN NON-HOSPITALISTS? S. Freer 1 , J. Cotter 1 , C. Pugh 1 , W. Smith 1 ; 1 Virginia Commonwealth University, Richmond, VA PURPOSE: The hospitalist model of inpatient care on general medicine wards offers the potential for a more efficient use of hospital resources, but limited formal evaluation has been conducted. The ability to effectively move patients out of the hospital on day of discharge is an important indicator of the improved use of hospital resources. This is the first study we know of to examine if hospitalists, when compared to non-hospitalists, could more efficiently discharge patients. Physicians have control over the initiation but not the completion of this process. METHODS: We studied discharge orders written by 11am and completion of discharge by noon as a measure for evaluating efficiency. These efficiency guidelines were established by the institution. We evaluated compliance with these guidelines through a retrospective analysis of clinical and utilization data on 3,040 patients admitted to general medicine wards of the Medical College of Virginia Hospital at Virginia Commonwealth University. We analyzed the number and percentage of discharge orders written before 11am and the number of patients discharged by noon by hospitalist and non-hospitalist physicians using chi-square to compare proportions. RESULTS: In FYOO the percentage of discharge orders written before 11am for the hospitalist teams (44.3%) was greater than that for the non-hospitalist teams (28.2%; p < 0.0 1). Very low percentages of patients were discharged by noon from either general medicine service. CONCLUSION: Hospitalist physicians performed significantly better than did non-hospitalist physicians on entry of discharge orders by 11am, a process measure of efficiency over which they have control. There was no difference between the groups on the completion of discharge, a process measure of efficiency over which physicians have less control. METHODS: Pharmacy records for all outpatient diabetes prescriptions during 1997 were obtained at four VA hospitals. Primary care providers (PCP) were defined as writing > 50% of prescriptions, and coded as staff attendings (AT), medicine house officers (HO), nurse practitioners (NP), endocrinologists (EN), or no PCP. Patients were grouped based on whether they received oral agents only, insulin only or both. ANOVA was performed to evaluate medication costs, glucose monitoring supply costs, total costs and glycemic control by site, provider type and treatment classification, controlling for demographics. Supply costs were calculated only across patients receiving supplies; but all patients were included in total monthly costs. Chart reviews were done on a subset from 2 sites to evaluate for differences in DM severity and comorbid conditions. RESULTS: 4544 patients were identified with complete information for cost analyses ( METHODS: In December 1998 we randomized 4 of 8 outpatient health centers that participated in a local HMO to have access to an ACS. The multi-disciplinary, telephone-based ACS evaluated the appropriateness of warfarin therapy, dosed the warfarin therapy, educated the patients, and monitored their international normalized ratios (INRs). At the control sites, physicians and their staff performed these tasks. We assessed the rate of hospitalization for a hemorrhagic or thrombotic adverse event by analyzing hospital claims submitted to the HMO. Using two-sided tests of the Poisson approximation, we compared the relative rates of adverse events between control and ACS sites using an intent-to-treat approach. RESULTS: Of the approximately 1100 patients taking warfarin at the 8 centers, approximately half (N = 503) were eligible and enrolled in the ACS. The remaining patients had their warfarin monitored and dosed by their physician. Using hospital claims data through July 30, 2000, the relative risk for an adverse event was 0.71 among patients who had access to the ACS, corresponding to a significant (p < 0.05) relative risk reduction of 29% compared with patients whose warfarin was managed by their physician. CONCLUSION: The telephone-based ACS was associated with a significantly decreased rate of hospitalization for warfarin-related adverse events. This finding suggests that multidisciplinary, disease-state management programs that empower nurses and pharmacists can improve medical care. RESULTS: Baseline patient satisfaction with their doctor was quite high. The 1,270 patients interviewed gave their physician a median rating of 9 on a scale of 0 (worst doctor possible) to 10 (best doctor possible). 47% of patients rated their doctor as a``10.'' Physicians also scored highly using a validated 10-item scale to designed assess communication skills and humanistic qualities; the median score was 46 of a possible 50 points, with 35% of patients rating their doctor as`e xcellent'' on all 10 items. Patients' ratings of physicians' communication skills were a powerful independent predictor of overall patient satisfaction (p < .001). However, analysis of variance showed that attending the workshop had no significant effect on patients' rating of their doctor's communication and humanistic skills (p = 0.32) or patients' overall rating of their doctor (p = 0.33). Physicians with baseline satisfaction ratings in the lowest quartile were no more likely to show improvement in their ratings than were physicians with higher baseline satisfaction ratings. PURPOSE: In the inpatient setting, physician order entry has been shown to significantly reduce serious medication errors (MEs). However little is known about the impact of computerized prescribing systems in the ambulatory setting on MEs and adverse drug events (ADEs). We compared the frequency of these events in outpatient clinics using handwritten versus computerized prescribing. METHODS: We prospectively studied 2 sites with handwritten prescribing and 2 with basic electronic prescribing in the Boston area. The computerized sites had printed prescriptions and required fields, but no defaults and optional or non-existent checks for allergies and drug interactions. We collected copies of prescriptions written by 24 primary care providers from Sept 1999 to March 2000 (6 weeks per clinic). Prescription copies were reviewed by a pharmacist to screen for MEs and potential ADEs. In addition, patients who received prescriptions were telephoned 2 weeks after their visit to ask about problems with their medications (response rate 59%), which were then classified as ADEs or not by 2 MD reviewers. RESULTS: Of 1173 prescriptions screened during this time period, 202 (17%) were rule violations (orders that violate strict standards but are generally understood and generate no additional work), 44 (4%) were MEs, and 59 (5%) were potential ADEs. Sites with computerized prescribing were significantly less likely to have rule violations (p < .02) and ME's (p < .01), but potential ADE rates were not significantly different. Of 661 patients surveyed, 178 (27%) reported a total of 206 ADEs, of which 74 (36%) were preventable. There was no significant difference between computerized and non-computerized sites in ADE rates and preventable ADE rates (37%, 35%). Of preventable ADEs (n = 74), 45 (62%) occurred in the ordering stage, and 26 (36%) in the patient administering stage. The main types of physician error were failure to act on results of monitoring or tests (50%) and inappropriate drug choice (24%). Improved computer ordering checks would have prevented only 20% of physician errors. CONCLUSION: Errors in the drug process were common in the outpatient setting. However, while basic computerized prescribing systems were associated with lower rates of rule violations and MEs, serious error rates (potential and preventable ADEs) were similar. Monitoring for ADEs was unexpectedly important. Prescribing systems with advanced decision-support, including monitoring and communication features as well as allergy/interaction checking, may be required to substantially reduce the frequency of serious errors. PURPOSE: Despite mandates for physicians to screen for and intervene with domestic violence as they should do for HIV/STD risk, alcohol abuse, and smoking, studies show that physicians face many barriers to doing so. We wanted to know how asking and intervening with domestic violence compares to asking and intervening with patients in these other three health-risk areas. METHODS: In November 2000, we mailed a questionnaire on physicians' screening and intervention behaviors for domestic violence, HIV/STD risk, alcohol abuse, and tobacco use to a national random sample of 1200 physicians (internal medicine and family practice). Our response rate to date, prior to our final mailing, is 49%. Chi-square tests with 3 degrees of freedom (p) and Bonferroni-Holm adjusted pairwise comparisons among topics (p * ) were used to test results. RESULTS: Sixty-six percent of our physician sample reported that intervening when domestic violence is identified is an essential part of their role as a physician. In contrast, 79% reported that intervening with patients identified for HIV/STD risk is an essential part of their role; 82% with alcohol abuse; and 85% with tobacco use (Chi-square=13.8, p=.003; p * < .032 for domestic violence vs. tobacco and vs. alcohol). Regarding screening, only 10% of physicians reported that they always ask new patients about domestic violence, compared to 22% for HIV/STD risk, 72% for alcohol use, and 84% for tobacco use (Chi-square=188, p < .001; p * < .035 for all 6 pairwise comparisons). When asked about their knowledge of and confidence in screening in these areas, only 19% strongly agreed that they knew how to assess patients for the risk of domestic violence, whereas 47%, 59%, and 70% strongly agreed that they knew how to assess patients for HIV/STD risk, alcohol abuse, and tobacco use respectively (Chi-square=68.3, p < .001; p * < .001 for all 3 pairwise comparisons with domestic violence and HIV/STD vs. tobacco). When asked if they would rather refer domestic violence victims to outside resources than provide counseling themselves, 79% agreed or strongly agreed that they would. This compares with 42%, 51%, and 23% who agreed or strongly agreed that they would rather refer patients identified with HIV/STD, alcohol, and tobacco risks respectively (Chi-square = 74.7, p < .001; p * < .006 for all pairwise comparisons except HIV/STD vs. alcohol). CONCLUSION: These data suggest that, despite physicians' view that screening for domestic violence is part of their role, physicians are not prepared or able to overcome barriers to ask their patients about abuse. It is even more difficult for physicians to screen their patients for the health risk of domestic violence than to screen for the other health risks, including the risk for HIV/ STD, which is also considered a sensitive and stigmatized topic. One solution may be simplifying the physicians' role and augmenting patient care with other experts and assessment tools.
THE COMPUTERIZED PATIENT RECORD SYSTEM: A TRADE-OFF BETWEEN PHYSICIAN TIME AND PATIENT BENEFIT. P.A. Glassman 1 , P. Belperio 2 , B. Simon 2 , K. Lim 1 , J. Sayers 2 ; 1 VA Greater Los Angeles, Los Angeles, CA; 2 VA Greater Los Angeles, LA, CA PURPOSE: Improving patient safety is a critical concern. As part of a larger evaluation study designed to reduce adverse drug events, we conducted a baseline survey on clinicians' attitudes towards and knowledge of the Computerized Patient Record System (CPRS) and associated drug interaction alerts.
We surveyed 319 clinicians at 12 sites within an integrated VA Healthcare System located in Southern California. Clinicians included attending-level physicians, nurse practitioners and physician assistants. The questionnaire, developed and tested locally, assessed extent of and comfort with CPRS. Questions assessed how CPRS in general, and clinician order entry and drug interaction alerts in particular, have affected clinician efficiency and patient care. Additionally, we assessed general knowledge about 21 common drug-drug and drug-disease interactions. This abstract reports the results of the first 63 (20%) respondents in this abstract. The survey period is expected to end in January 2001, with an anticipated response rate of 65% to 70%. RESULTS: In this preliminary analysis respondents were 71% male, 87% full-time employees and 72% Internists. Participants averaged 48 years of age, practiced 2.5 days per week in outpatient clinics and wrote 53 prescriptions per week. A majority of clinicians (range: 70% to 86%) preferred using CPRS to conventional written methods for entering patient notes, requesting consults, ordering radiological procedures, ordering laboratory tests, and prescribing medications. Overall, clinicians reported that CPRS improved the safety and quality of patient care but reduced clinician efficiency. For example, 81% of clinicians felt that drug interaction alerts increased the potential for prescribing safely but 70% perceived that clinician order entry increased the time required to write prescriptions. Also, while 71% felt that clinician order entry reduced errors in ordering laboratory tests, 68% reported that doing so required extra time. Clinicians recognized a median of 53% (range: 17% to 89%) of 21 common drug interactions. Eighty five percent (85%) of clinicians reported that they would have felt more confident about their answers had they had drug alerts to identify these interactions. In practice, however, respondents reported that they would be more likely to change a patient's medication based on personal interaction with a pharmacist (58%) rather than depend on a CPRS drug interaction alert (4%). Thirty-eight percent were equally likely to change medications based on an alert from either source. Clinicians reported the greatest barriers to effective use of drug alerts included non-relevant alerts (72%), system slowdowns and shutdowns (65%), and lack of time to review alerts (57%). CONCLUSION: Early analysis of an on-going survey suggests that clinicians perceive that CPRS improves the quality and safety of patient care but decreases their efficiency. Clinicians favor the CPRS drug alert system as a means to improve their recognition of drug interactions, an issue that needs to be further addressed based on our data, but note several implementation problems that impede effective utility. PURPOSE: Clinical practice guidelines (CPGs) are being implemented in many large health care systems; yet, little is known about clinician reaction. We evaluated clinician response to a hypertension (HTN) guideline implementation at a large, geographically-diverse VA medical center. METHODS: In the context of a facility-wide implementation of 10 CPGs, the HTN guideline was implemented with 36 attending physicians and nurse practitioners in a randomized trial comparing a general intervention (educational components and a list of the patient's antihypertensive drugs delivered to clinicians at each primary care clinic visit) with an individualized intervention (the general intervention plus patient-specific recommendations about drug therapy delivered to clinicians during primary care clinic visits). For the 4500 hypertensive patients of these study clinicians meeting study criteria, the individualized intervention improved guideline concordance of drug therapy (Med Dec Mkg 2000; 20:488) . At the end of the study period, two physicians, previously unaffiliated with the study, conducted structured interviews with 32 (89%) study clinicians. RESULTS: 31 (97%) clinicians were aware of CPG implementation at the facility and 28 (88%) were specifically aware of the HTN CPG. 29 (91%) and 28 (88%) clinicians, respectively, reported that they use CPGs and that CPGs help with patient care. 16 of 18 (89%) clinicians in the individualized group agreed with the recommendations, but only 3 (17%) reported that seeing the recommendations affected their management of patients. Barriers to following the recommendations included patient reluctance to change medication; concern about need for additional clinic visits and laboratory monitoring of recommended medications; previously demonstrated patient intolerance of the guideline drugs. Time constraints were mentioned frequently. CONCLUSION: Clinicians had a high level of awareness of the CPG implementation and a generally positive attitude toward CPGs. In the context of the randomized trial evidence of the impact of the guideline implementation, the small percent of clinicians who reported that the recommendations affected their management of patients suggests that clinicians may not be aware of the impact of guideline implementation. PURPOSE: Patient adherence to antiretroviral therapy (ART) is critical to effective treatment of HIV and may depend partly on health professionals' practices. However, little is known about providers' knowledge, practices, and barriers in promoting ART adherence. We sought to assess adherence counseling practices among the physicians (MD), pharmacists (PH), and case managers (CM) caring for HIV+ patients in NC and to identify barriers they faced in facilitating adherence. METHODS: In February 2000, using NC AIDS Drug Assistance Program records and commercial prescription records, we identified and surveyed, by mail, the 1,301 MDs, PHs, and CMs caring for HIV+ patients in NC. After 4 mailings, 440 (77%) of the PHs, 94 (85%) of the CMs, and 380 (63%) of the MDs responded. Among the responding MDs, only the 190 reporting having prescribed a protease inhibitor in the last year were included in the analytic sample. RESULTS: 31% of the MDs were general internists, 24% family practitioners, and 22% infectious disease specialists; half cared for < 10 HIV+ patients. 66% of PHs worked in chain pharmacies, 28% in independents and only 2% in hospitals; half cared for < 4 HIV+ patients. On average, CMs cared for 34 HIV+ patients. Regarding the average number of minutes counseling a patient on a new 3-drug ART regimen, PHs spent 7, MDs 13 and CMs 21. MDs seeing a higher volume of HIV+ patients spent no more time counseling but were more likely to: advise patients of side effects (SE), tailor the regimen, explain intake requirements, and provide a pill box (p < .01 for each). Over 90% of MDs explained dosing, asked for patients' questions and discussed drug resistance most or all of the time; < 60% discussed SE management, handling missed doses, ways to remember doses, or planning dose times. Over 70% of PHs explained dosing instructions and asked for questions but < 50% discussed SE management, handling missed doses, drug interactions or storage requirements. The most common CM adherence counseling behaviors were: praising adherent clients (84%), discussing nonadherence repercussions (74%), asking about clients' treatment concerns (74%) side effects (65%), and if medications were taken on time (71%). The vast majority of MDs, PHs, and CMs reported strong interest in doing adherence counseling and had positive attitudes toward ART, ART counseling, and HIV+ patients. The primary barriers reported were lack of space, time, and reimbursement for counseling. MDs viewed CMs' doing adherence counseling as acceptable but were dissatisfied with HIV CM availability. Both MDs and CMs indicated a high interest in improving collaborations with each other in the care of HIV+ patients. CONCLUSION: Innovative practice arrangements between all three provider groups may be needed and feasible to facilitate adherence counseling and address the multifaceted problem of antiretroviral adherence. Practice guidelines for sore throat, nasal congestion and cough illness developed by the Colorado Clinical Guidelines Collaborative were mailed to all primary care physicians belonging to the Colorado Medical Society in November 1999.`P rofiled physicians'' (having at least 10 office visit claims for adults with acute bronchitis in 1998) (n = 352) also received antibiotic prescribing profiles based on aggregated office visit and pharmacy claims from 7 HMOs participating in the Colorado Medical Society Joint Data Project; and``non-profiled'' physicians (n = 514) received summary data based on the entire cohort of patient visits. The office visit was the primary unit of analysis. Multivariate mixed effects models included patient age, physician specialty, HMO and time segments (baseline period = 1/98 ± 10/99; study period = 11/99 ± 2/00) as fixed effects, and unique physicians as random effects. Units of time were measured in months. RESULTS: There were 12,993 and 2,899 adult office visits for acute bronchitis in the baseline and study periods, respectively. The majority of patients were 18 ± 44 years old and treated by family physicians. Prescription rates for family physicians were about 5% greater than for internists (p = 0.006), and were positively associated with increased patient age. Adjusted monthly antibiotic prescription rates for acute bronchitis were stable during the baseline period for both physician groups (profiled: 65% to 63%, p = 0.10; non-profiled: 64% to 63%, p = 0.78), and declined significantly during the study period among profiled physicians (63% to 54%; p = 0.0005), but not among non-profiled physicians. The rate of decline in antibiotic prescription rates during the study period did not differ by patient age (p = 0.09) or physician specialty (p = 0.87). CONCLUSION: This study demonstrates that antibiotic treatment of adults diagnosed with acute bronchitis by physicians in private practice can be reduced using a combination of educational physician profiling and practice guideline dissemination. , 1994 ± 1998 . R. Gonzales 1 , J. Maselli 1 ; 1 University of Colorado Health Sciences Center, Denver, CO PURPOSE: Rising rates of S. pneumoniae resistant to penicillin and other antibiotics have been publicized in the US since 1994, and have led to numerous calls to limit excess antibiotic use in ambulatory practice. This study evaluates whether changes in antibiotic treatment of acute respiratory infections (ARIs) between 1994 and 1998 have occurred, and whether these changes are associated with specific patient and physician characteristics. METHODS: The National Ambulatory Medical Care Survey data files for years 1994 ± 98 were obtained from the National Center for Health Statistics web site. Office visits with a principal diagnosis of otitis media, sinusitis, pharyngitis, bronchitis and upper respiratory tract infections (URIs)/common cold were identified, and further limited to visits to a general or family practitioner (GFP), internist (IM) or pediatrician (PED). Antimicrobial treatment was assigned to the visit only if it was entered as the principal medication entry (out of 6 possible). Multivariate logistic regression analysis was used to evaluate independent associations between antibiotic treatment for ARIs and time (year); patient age, race and diagnosis; and physician specialty and practice location. To determine if a change in antibiotic prescription rates during this period were associated with one of the other variables, interaction terms were utilized. RESULTS: Overall, annual antibiotic prescription rates for ARIs declined between 1994 and 1998 (65%, 64%, 60%, 62% and 55%, respectively; p = 0.0001). This equates to 7 million fewer antibiotic prescriptions for ARIs dispensed by primary care physicians in 1998 compared to 1995 (the peak year). Independent of specific diagnosis and time, antibiotic treatment was less likely for patients age > 45 years (vs. children age < 5 years) (OR = 0.62, 95% CI = 0.51 ± 0.76), and of non-white race (OR = 0.75, 95% CI 0.65 ± 0.88). Because of a significant interaction between time and physician specialty, we stratified further analyses by specialty. Between 1994 and 1998, time was associated with a decrease in adjusted annual antibiotic prescription rates among PED and IM, but not among GFP (relative rate change = À8.9%; p = 0.0001; À9.3%; p = 0.043; and À4.5%; p = 0.17, respectively). Antibiotic prescription rates for specific diagnoses declined variably across specialties: sinusitis: no declines for any specialty; otitis media: PED; pharyngitis: PED; bronchitis: GFP; URIs/colds: IM decline > PED > GFP. CONCLUSION: Primary care physicians are heeding the call to limit antibiotic prescribing in ambulatory practice, although there remains much room for improvement. Changes in prescribing need to be correlated with trends in antibiotic-resistance rates. PURPOSE: Antibiotic treatment of the common cold and upper respiratory tract infections (URIs) by US primary care physicians declined between 1994 and 1998. This study evaluates the amount of public and professional media exposure provided to the topics of antibiotic-resistance and overuse of antibiotics, and the correlation between media exposure and changes in antibiotic treament of colds and URIs during this period. METHODS: Treatment related to primary care physician (general and family practitioner, internist or pediatrician) office visits with a principal diagnosis of the common cold or URI were analyzed using the National Ambulatory Medical Care Survey (years 1994 ± 98) . Key word searches relating to excess antibiotic use and antibiotic resistance were performed on all databases, and final tallies confirmed by manual review. Lexis-Nexis was used to identify US news stories from large-circulation newspapers corresponding to the northeast (NY Times), midwest (Chicago Sun-Times), south (Atlanta Journal-Constitution) and west (LA Times), and one national newspaper (USA Today). The Vanderbilt Television News Archive was used to identify evening news stories on ABC, CBS, NBC, and CNN. Articles from medical journals represented in the Abridged Index Medicus were identified using MEDLINE.``Sentinel articles'' were defined as receiving evening network news coverage by at least 1 network within 3 days of publication. Segmented time series analysis (logistic regression) was used to compare the change in 60-day (the smallest time unit providing adequate sample size) antibiotic prescription rates between time periods corresponding to increases in media exposure. RESULTS: From 1994 ± 98, there were 207 newspaper stories, 31 major network evening news stories, and 65 journal articles identified. The number of newspaper stories reported in 1994 ± 1996 was stable, but more than doubled in 1997, and remained high in 1998. Evening news stories doubled in 1996 and 1997 compared to the previous years, but reverted to baseline levels in 1998. In contrast, journal articles have increased linearly between 1994 and 1998. There were 6 sentinal publications during this period: June 94, January 95, August 95, January 96, September 97 and March 98. Inspection of 60-day antibiotic prescription rates for colds and URIs reveals only 2 major trends: 1) relatively stable rates between 1994 and 1996 (p = 0.73); and 2) declining prescription rates from 1996 through 1998 (parameter estimate = (À)0.036; p = 0.06). CONCLUSION: The change in antibiotic prescribing for colds and URIs that occurred in 1996 appears to be more strongly correlated with an increase in public media exposure than with medical journal publications. However, one can not rule out an impact of other forms of physician education (eg. local or national conferences) on prescribing behavior. PURPOSE: Home glucose monitoring is a common practice in diabetes (DM) treatment, but adds significantly to costs of care. Several studies have questioned the benefit of glucose monitoring in patients with type II DM. We examined the impact of glucose test strips on glycemic control in outpatient DM patients at four VA facilities. METHODS: All patients receiving outpatient DM medications during 1997 were evaluated at four VA hospitals in Pennsylvania. Administrative data identified demographic variables (age, sex, race, marital status), glycosylated hemoglobin (HBA1C), all medications and DM supplies, and type of clinician (resident, nurse practitioner, staff internist, endocrinologist, and other). We examined differences among patients who recieved glucose test strips (strip+) and those that did not (strip-). Simple descriptive statistics was performed comparing strip+ and strip-patients, and analysis of variance was done, including main effects (medication type [oral agents, insulin, and both oral agents and insulin], site of care, and physician type), interaction terms, and demographic variables. Chart review of a subset of patients examined Charlson comorbidity scores, and presence of diabetes complications (neuropathy, retinopathy, and nephropathy). RESULTS: There were 2912 strip+ patients, and 2221 strip-patients. Strips added an average of $22.56 each month to cost of diabetes care. Average HBA1C for strip+ patients was 7.92; for strip-patients, 7.34. There were significant differences for use of strips in patients for site (ranging from 26% to 73% strip+), race (favoring white race), and marital status (favoring married). Differences in HBA1C persisted after adjusting for type of medication, site, and demographic covariates. There was no difference in Charlson comorbidity scores between groups in the substudy, but there were more diabetic complications in the strip+ group (15% more had at least one diabetic complication). CONCLUSION: Glucose control was poorer in patients who received test strips. Differences persisted after controlling for important confounders. Thus, our study does not suggest benefit from home glucose monoitoring in terms of improved glycemic control, although presence of more DM complications in the strip+ group suggests that strips may be used more often in difficult to control patients. Because home glucose monitoring is costly, further study should evaluate the cost-effectiveness and clinical benefit of its use. used to exclude subjects who are likely to be non-adherent with therapy or to experience side effects. While this approach may increase the chances of finding a treatment effect by eliminating non-compliers and subjects likely to experience adverse events, it diminishes the generalizability of the results of the trial, as patients are less likely to be as adherent and more likely to experience side effects. This is especially so if measures such as the``Number Needed to Treat'' (NNT) are used, as the``true'' NNT is likely to be higher than that obtained in the trial if an appreciable number of patients are excluded during the run-in period. As run-in periods would seem to increase the chance of finding a positive result, we hypothesized that there might be an association between the use of run-in periods in the design of randomized trials and the presence of commercial sponsorship, particularly by the pharmaceutical industry. METHODS: We searched all randomized trials published in The New England Journal of Medicine, The Journal of the American Medical Association, The Lancet, and The BMJ, from January 1, 1998 through December 31, 2000 . We included trials that had at least one arm consisting of either PO, dietary, intranasal, or subcutaneous therapy administered by the study subjects, of at least 4 weeks duration. We excluded studies of treatments not administered by subjects (e.g., intravenous or intramuscular treatments) and studies of treatment of acute events (e.g., myocardial infarction or COPD exacerbations), in which setting a run-in period would not be feasible. However, studies of treatments following acute events that did not require acute treatment were included. Trials of treatments during pregnancy were also excluded. For the purpose of our study, we defined a run-in period as follows: Any period preceding randomization during which time subjects took active treatment, placebo, or a dietary intervention to which they might subsequently be randomized. Pre-randomization periods used to assess subjects' symptoms were not considered run-in periods, even if the authors of the studies designated them as such. This is because these screening or``baseline'' periods serve a very different function from the above defined run-in periods. Drug``wash-out'' periods were also not considered run-in periods in our analysis, for similar reasons. Sponsorship was determined based on stated funding source at the conclusion of the article. We considered a study to be industry sponsored if at least one of the funding sources was from a commercial entity (e.g., a pharmaceutical company). We did not consider the provision of medication or placebo to constitute commercial support. All studies were evaluated separately by two reviewers, and determination of the presence or absence, as well as type of run-in period was made while blinded to the source of funding. Likewise, ascertainment of funding source was made blinded to presence or absence of a run in period. Any disagreement regarding presence or type of run-in period was resolved by consensus among the three authors, while blinded to the funding source. RESULTS: There were 214 eligible trials. Of these, 138, or 65%, were industry sponsored. Runin periods as defined above were found in 39 of the trials. Twenty-eight of these were placebo runin, 3 were placebo and diet, 2 were treatment, 2 were diet alone, 2 were treatment and placebo, and in 2 reports the type of run-in was not stated. In only 12, or 31%, of the 39 trials was the number of subjects excluded because of non-compliance or adverse events included in the result. Of the 39 trials with run-in periods, 36, or 92%, were commercially funded. The odds that a trial with a run-in period was commercially funded was 12:1, compared to 1.4:1 for trials without run-in periods (OR = 8.6). CONCLUSION: Run-in periods in clinical trials appear to serve a commercial, rather than a scientific, purpose. As their presence limits the generalizability of the results of these trials, we question their use in the design of randomized clinical trials done for scientific purposes. . Despite demonstrated efficacy of medical therapy in clinical trials, effectiveness of CVD risk reduction in actual practice may be sub-optimal. Our goals were to: 1) determine patterns of hyperglycemia (HG), hypertension (HTN) and hyperlipidemia (HL) management among patients with DM2, and 2) assess whether effective HG management was associated with effective HTN and HL management. METHODS: 601 confirmed DM2 patients attending our outpatient clinics between 3/96 ± 8/ 97 were randomly selected; chart abstraction data were linked to laboratory testing results. We defined 3 components of effective CVD risk reduction: 1) risk marker testing for HbA1c, systolic blood pressure (SBP), and LDL cholesterol, 2) initiation of medical therapy if above goal, and 3) use of high dose therapy if above goal. To assess the relationship between HG, HTN, and HL management, we compared risk marker levels, testing frequency, and medication dose intensity, using linear and logistic regression. RESULTS: Cohort characteristics included: mean age 64.7, 58.4% male, median DM2 duration 6 years, with 73% HTN and 50% HL prevalence. Sub-optimal management effectiveness was greatest for HL compared to HG or HTN. Fewer patients were tested for LDL than HbA1c (Fisher's exact test, p < 0.0001), and among tested patients, significantly fewer were at LDL goal than HbA1c goal (p = 0.045) [Table] . Levels of HbA1c, SBP, and LDL were not significantly correlated after adjusting for BMI. Achieving goal HbA1c did not predict meeting either SBP or LDL goals. There were no significant correlations between HG medication dose intensity (oral HG agent or insulin U/Kg dose) and either HMG CoA reductase (statin) dose or number of HTN medications. To explore factors associated with physicians' decisions to use warfarin in patients with non-valvular atrial fibrillation (NVAF). METHODS: We mailed a self-administered questionnaire to a random sample of general internists, selected from the AMA Masterfile. The instrument included questions on physicians' demographic characteristics, prior experiences and beliefs about the natural history and treatment outcomes for patients with NVAF. Case scenarios described patients with varying degrees of risk of thromboembolism or hemorrhage. Respondents who were in the upper quartile of warfarin use, as defined by the proportion of cases in which they recommended warfarin, were``high-users''. Results were summarized as proportions; chi-square was used for comparisons with a significance level of 0.05. RESULTS: Surveys were returned by 120 of 427 eligible respondents. The mean age was 46 years (79% male; 38% academic). The median perceived risk for TIA and ischemic stroke (CVA) in a NVAF patient who was 76 year old (otherwise healthy, not receiving therapy) were 10% (Interquartile Range -(IQR): 5 ± 20%) and 7.5% (IQR: 5 ± 16%) per year, respectively. For NVAF patients on warfarin, the median risk estimates for TIA and ischemic stroke were 2% for TIA (IQR: 1 ± 5%) and 2% for CVA (IQR: 1 ± 5%); the median annual risk estimate of intracranial hemorrhage (ICH) for these patients was 3% (IQR: 1 ± 5%). Respondents indicated they would recommend warfarin for a median of 9 of the 14 cases (IQR: 7 ± 11 cases). Physician age, gender, perceived risk of ischemic stroke and estimated stroke risk reduction attributable to warfarin were not associated with warfarin use. However, respondents whose estimated risk of warfarin-associated ICH was in the highest quartile were significantly less likely to be high-users than those who provided lower estimates (6.9% vs. 30.8%; p = 0.01). Use of warfarin was also inversely related to the belief that an ICH was a``worse outcome'' than an ischemic strok for a NVAF patient or a``more regrettable'' outcome for their physician. CONCLUSION: There is substantial variability in beliefs about the natural history of NVAF and the risks and benefits of warfarin. Warfarin use was impacted more by estimated risk of intracranial hemorrhage and by the perceived severity and``regrettability'' of this complication than by the potential benefits. However, little is known about the recruitment and evaluation of potential subjects in published trials. We therefore performed a review of published randomized controlled trials (RCT's) to assess the rigor with which enrollment experience was reported, and to analyze the available data. METHODS: We selected all RCT's that were published in the Annals of Internal Medicine, The New England Journal of Medicine, Lancet, or JAMA between April 1, 1999 and March 31, 2000 . From each article, we abstracted the number of patients that were screened by the investigators to determine if they were eligible, the number who were eligible, and the number who were enrolled. Results were summarized as proportions; chi-square tests were used to compare groups with a significance level of 0.05. RESULTS: A total of 172 RCT's were reviewed; the median number of participants was 260 (range 18 to 54,654). Of these, 90 (52%) papers included an estimate of the number of patients that were screened by the investigators for eligibility. The number of patients who were eligible for participation was reported in 74 (43%) studies. Multicenter trials were significantly less likely to report the number of eligible patients (33.9%) than single center trials (58.7%; p = 0.002). Only 54 (31.4%) papers included information about both the number of patients screened and the number eligible. In these studies, the median proportion of screened patients who were eligible for participation was 58.8% (range 1.4% ± 100%); and the median proportion of eligible patients who enrolled was 91.8% (range 35.1 ± 100%). CONCLUSION: Many RCT's published in major medical journals provided incomplete information about their patient recruitment experience. In the papers that did include this information, there was marked variability in the proportion of patients who were eligible and the proportion that enrolled. Clinician-scientists should be encouraged to collect and report this information in order to allow readers to consider the external validity of their work. we considered the RR's to be discrepant when they were not both less than or greater than 1.0.
To provide some perspective on the disease burden, we also calculated the ratio of DSM to ACM in the control groups for each cancer type. RESULTS: The RR's were discrepant in 5 of 14 trials. See table for details of RR and disease burden for each screening test. In addition, in one of the trials in which the RR's were both less than 1.0 (Edinburgh mammography trial), the observed difference in ACM was far greater than could be expected on the basis of decreased breast cancer mortality (5 times larger than the observed difference in DSM). CONCLUSION: ACM should be considered in randomized trials of cancer screening because it helps to identify threats to validity (such as the randomization flaws in the Edinburgh trial) and because it provides needed perspective on the magnitude of the benefit of screening. RR's based on ACM and DSM are often discrepant, raising questions about the validity of DSM and the potential for unrecognized harm from screening. For each study, we calculated the absolute difference in mortality rate between the highest and lowest volume strata reported and the number needed to treat (NNT) to prevent one death attributable to low volume. Two investigators independently abstracted each article using a standard form. RESULTS: Of 257 studies reviewed, 128 met inclusion criteria covering 27 procedures and clinical conditions. The methodological rigor of most studies was modest. Few studies used clinical data for risk-adjustment or examined hospital and physician volume effects simultaneously. Overall, 70% of all hospital volume and 74% of physician volume studies reported a significant association between higher volume and better outcomes. The strongest associations were for surgery on pancreatic cancer, esophageal cancer, abdominal aneurysm, and pediatric cardiac problems, and treatment of AIDS (median, 8 to 14 excess deaths per 100 cases attributed to low volume; NNT, 7 to 12). Although statistically significant, the magnitude of the volume-outcome relationships for CABG, PTCA, MI, carotid endarterectomy, other cancer surgery, and orthopedic procedures was much smaller (median, 0.2 to 3 deaths per 100; NNT, 35 to 500). CONCLUSION: High volume is associated with better outcomes across a wide range of procedures and conditions, but the magnitude of the association varies greatly. The clinical and policy significance of this finding is complicated by many methodological shortcomings. Differences in processes of care between high and low volume providers may explain much of the relationship between volume and outcome. (10):769) met all important quality criteria. In that study, 90% of total costs were attributable to drug costs alone, and the cost-effectiveness ratios were > $50,000/QALY for all 240 sub-groups analyzed. The statin dosage range used in that study is achieved at our institution by using half tablets of currently available preparations, and the actual VA costs for statins were 14% of those drug costs assumed in this study. When the VA costs were applied to this analysis, the cost-effectivneness of using statins for primary prevention was < $50,000/QALY for the majority of patient subgroups. CONCLUSION: In at least one large health care system, statins are available at acquisition costs considerably lower than assumed in published CEAs. These acquisition costs permit statin use for primary prevention of CAD to have an acceptable cost-effectiveness ratio. If these drug prices were widely available to consumers, this important category of medication would have great potential for improving the nation's health at reasonable cost. Mechanisms for creating a uniform pricing structure for these widely used medications should be explored. . Qualitative studies suggest that DCs give patients more information and more control over their treatment. Among primary care patients, information and control generally lead to greater satisfaction. We estimate the effects of provider type, explanation of treatment, and self-care advice on satisfaction in a randomized trial of DC vs. MD treatment for low back pain. METHODS: 681 adult low back pain patients were randomly assigned to treatment by either a DC or a MD and were surveyed at baseline, 2, and 4 weeks; follow-up was 99.7%. We used ordinary least squares linear regression to estimate the effects of provider type, explanation, selfcare advice, and other factors on satisfaction with provider. Explanation of treatment was reported as``yes'' or``no.'' Self-care advice was the number of items of advice (0 ± 10) the patient reported receiving. Satisfaction was measured on a 10-item scale (5 = low to 50 = high). RESULTS: The mean satisfaction score was 36.1 for patients in the DC group and 30.6 in the MD group; the crude difference was 5.5 (95% confidence interval [CI] = 4.5, 6.5). Adjustment for sociodemographic and baseline illness characteristics alone did not substantially change these estimates. The mean number of items of self-card advice was 2.3 in the DC group and 1.6 in the MD group (p < 0.001). Sixty-one percent of patients in the DC group vs. 16% in the MD group received an explanation of treatment (p < 0.001). In a regression model that included provider type, explanation, self-care advice, improvement in disability, prior experience with and confidence in treatment, baseline sociodemographic and illness characteristics, and interaction terms, the DC-MD difference in satisfaction was 4. PURPOSE: Although patient demand is high, physicians have not widely adopted use of e-mail with patients. Our purpose was to explore the experiences of physicians frequently using e-mail with their patients, as a window to the future. METHODS: To identify the rare``vanguard'' physicians who frequently use e-mail with their patients, we recruited 11,859 physicians using Physicians' Online, an Internet professional information portal, to participate in a survey. To identify frequent users of e-mail, we asked``In a typical day, please estimate the average number of e-mails you and your immediate staff receive from patients who are currently in your care.'' Additional questions assessed common clinical topics in e-mail, physician's adherence to published guidelines from the AMA and American Medical Informatics Association, and physician satisfaction, measured by``Would you recommend to a colleague use of e-mail with patients.'' RESULTS: Among the 1,325 physicians responding (including individuals from all 50 states), we identified 204 frequent users who received one or more e-mails from patients daily. The mean age was 49 years, 82% were male, and 35% were primary care providers. Participants were from a variety of specialties including adult medical subspecialties(9%), pediatrics(11%), surgery(11%), and psychiatry(7%). Common clinical topics, received frequently or sometimes by over 40% of physicians, include: non-urgent new symptoms, questions about lab results, and questions about information on the Internet. Although infrequent, 7% of our sample did report frequently or sometimes receiving e-mails about urgent issues such as chest pain. The most common practice adherent with published guidelines was including the e-mail in the medical record (38%), but they rarely incorporated other guideline issues such as educating patients about appropriate topics (10%). Despite daily use of e-mail with patients, 25% of the 204 were not satisfied. Physicians who were not satisfied more frequently (80%) reported``patient request'' as the most important reason for using e-mail, compared with those who were satisfied (39%). (p < 0.01). Compared with dissatisfied physicians, physicians who were satisfied with e-mailing patients more frequently noted a decrease in their amount of telephone medicine (29% versus 2%; p < 0.001), and were more likely to use the emails to educate their patients (25% versus 14% p < 0.01). CONCLUSION: Experiences of these``vanguard'' physicians were mixed. Although patient demand is high, physician satisfaction of e-mail with patients may also be important to sustained use of e-mail with patients. Our data suggest that increased dissemination/adherence of published guidelines and increased perception of the efficiency of e-mail communication may improve acceptance. PURPOSE: Continuity of care is a highly desirable characteristic that may be threatened by recent changes in the health care system. We sought to assess patients' perceptions of continuity of care and the relationship between continuity and satisfaction in a managed care organization. METHODS: Within a randomized controlled trial of a patient-provider matching intervention in a group model health maintenance organization (HMO), we evaluated whether subjects reported usually seeing their own primary care provider (PCP), and the association of this perception with satisfaction and trust. Between October 1999 and September 2000, 2501 (64%) subjects completed a mailed, self-administered questionnaire, one year after receiving a new PCP. RESULTS: Among patients who had seen their PCP at least once during the study period, the majority (76%) reported usually seeing their own PCP during their visits. These subjects tended to be older (58 v. 52 yo, p < 0.0001), white (75% v. 69%, p < 0.02), and had more visits during the past year to their PCP (3.6 v. 3.2 visits, p < 0.04) than subjects who reported usually seeing another provider, i.e. lacking continuity of care. Moreover, compared to subjects without continuity, subjects perceiving that they had continuity reported greater satisfaction with their PCP and the health system. For example, subjects reporting that they usually saw their PCP had significantly higher levels of satisfaction with their PCP (72% v. 38% reporting excellent or very good overall satisfaction, p < 0.0001). These subjects were more likely to recommend their PCP to others (82% v. 52%, p < 0.0001), reported greater trust in their PCP (p < 0.0001), and in the health system (p < 0.0001). In addition, these subjects also were less likely to perceive that the PCP created barriers to access, such as with specialist referrals (p < 0.0001). CONCLUSION: In short, the majority of subjects in this study perceived that they usually saw their own PCP during visits. Furthermore, the perception of continuity of care with one's PCP was associated strongly to satisfaction and trust in the PCP, and also in the health system. Further research is needed to assess the direction of this association, as well as to identify potential areas for organizational improvements. Despite a growing focus on this problem many health care organizations provide inadequate interpreter services. A principal reason is the concern that uncertain benefits do not justify the costs of adequate services. The objective of this study was to assess the impact of an interpreter service program on the utilization and cost of health care services at a staff model HMO. METHODS: We conducted a 2-year cohort study of continuously enrolled adult members of a staff model HMO where new comprehensive interpreter services for Spanish and Portuguesespeaking ambulatory patients were implemented in year 2 of the study. Two groups were studied: an interpreter service group (ISG, n = 380) consisting of members who used the new interpreter services and a comparison group (CG, n = 4119) consisting of a 10% random sample of all other members who received ambulatory care in year 2. We abstracted demographic information and utilization of primary health care services (preventive services and outpatient services) and hospital-based services (ED visits and hospitalizations) from the HMO's administrative database. We calculated the unit cost per interpretation based on the cost of the services (salaries, fringe benefits, supervision, and overhead) and reported volume of use of services. We calculated induced costs of interpreter services by multiplying the change in utilization for each health care service by its 1997 fee-for-service reimbursement rate from the Massachusetts' Division of Medical Assistance. RESULTS: Utilization of primary health care increased in both groups after implementation of interpreter services. The changes (yr2-yr1) in utilization of preventive services (p < 0.05), utilization of office visits (p < 0.01), prescriptions filled (p < 0.01) and prescriptions written (p < 0.01) were significantly greater in the ISG compared to the CG. Utilization of hospitalbased services remained the same for both groups, except for a reduction in ED use by the ISG. The change in rate of ED use (yr2-yr1) was not significant when compared to the CG. However, relationships between patient satisfaction and other performance measures are poorly studied. The goal of this study was to examine the relationship between patient satisfaction and severity-adjusted mortality rates. METHODS: The current study used data from a regional initiative to measure performance in 31 hospitals in Northeast Ohio during 1991 ± 1995. Satisfaction was assessed using the Patient Judgement System, a 41 item survey mailed to patients after discharge. Responses were obtained from 40,344 medical patients (response rate 48%). We utilized 5 scales (physician care, nursing care, coordination of care, information provided, and discharge instructions) and 1 single item assessment (overall quality). For each hospital, mean scores were determined, adjusting for self-reported health status, age, gender, education and other demographic factors. Severity-adjusted mortality was determined for patients with 6 medical diagnoses. For each hospital, observed mortality was compared to predicted mortality derived from validated disease-specific multivariable models that were based on data abstracted from medical records. Comparisons of observed and predicted mortality rates were summarized using the z-statistic. RESULTS: Mean satisfaction scores for the 5 scales and 1 single-item indicator ranged from 68.0 (information provided) to 72.6 (physician care). Z-statistics for mortality rates ranged from -7.6 to 4.6. In analyses including data for all 5 years (1991 ± 95), patient satisfaction scores, at a hospital level, were inversely correlated with mortality for each of the 6 measures of satisfaction. from each group who met these criteria: > half-time practice for the last 2 years, > 40% minority patients, and providing primary care of HTN. The 2-hour sessions with 2 ± 4 observers were audiotaped. A facilitator elicited comments and discussion on JNC6 recommendations, perceived barriers, efficacy of traditional guideline interventions, and recommended solutions for future HTN care. One investigator analyzed tapes for emerging themes and concepts. The resulting summaries were distributed to the research team to discuss alternative interpretations. All focus group observers did a final reading of the summary to confirm the validity of the themes and conclusions. RESULTS: Similar themes emerged in the two groups. Physicians felt the JNC6 recommendations were realistic but that physician behavior was not the problem with implementation. Physicians outlined the barriers to optimal control of HTN: poor office systems for tracking care, inability to address lifestyle modification in office visits, impracticality of self-monitoring, the disincentive of co-pays, lack of generic samples for cost-effective prescribing. There was no interest in traditional physician directed interventions such as seminars, academic detailing, incentives for chart review to change therapy, pocket or wall versions of the guidelines, or practice profiling. Physicians suggested making monitoring more practical, providing generic samples, and utilizing group appointments, pharmacies, the internet, and ethnically sophisticated mass-media campaigns for patient education. CONCLUSION: Physicians agreed with the goals and recommendations of the JNC6 HTN guidelines but felt that existing efforts to improve guideline compliance by changing their behavior are bothersome and ineffective. They suggest intervening with the patient to address barriers outside the control of their practices. Future research should examine patients' perceptions of the acceptability and efficacy of these interventions. METHODS: This retrospective cohort study evaluated consecutive ICU admissions to a VA hospital (n = 1,142) and 27 private sector hospitals (n = 51,249) serving the same health care market in 1994 ± 95. Mortality and ICU LOS were adjusted for severity of illness using APACHE III, a validated method that considers age, comorbidity, ICU admission diagnosis, admission source, and abnormalities in 17 physiologic variables during the first 24 hours of ICU admission. We used two multivariable statistical methods to estimate the risk of death in VA patients, relative to private sector patients. 1) Logistic regression provided the odds of inhospital death in VA patients. 2) Cox proportional hazards regression was used to account for potential differences in LOS and in the timing of death by censoring patients at the dime of discharge. RESULTS: Unadjusted in-hospital mortality was higher in VA patients (14.5% vs. 12.0%; p = .01), as was hospital (28.3 vs. 11.3 days; p < .001) and ICU (4.3 vs. 3 .9 days, p < .001) LOS. Using logistic regression to adjust for admission severity of illness, the odds of death was similar in VA patients, relative to private sector patients (OR 1.16, 95% C.I. 0.93 ± 1.44; p = .18). However, a higher proportion of VA deaths occurred after 21 hospital days (33% vs. 13%; p < .001). Using proportional hazards regression, the risk of death was actually lower in VA patients (hazard ratio, 0.70, 95% CI, 0.59 ± 0.82; p < .001). This result was consistent in stratified analyses of medical (hazard ratio, 0.80, p = .02) and surgical (hazard ratio, 0.46, p = < .001) patients. After adjusting for severity using linear regression, differences in ICU LOS were no longer significant (p = .19). CONCLUSION: Severity-adjusted mortality in ICU patients was lower in a VA hospital than in private sector hospitals in the same health care market, based on proportional hazards regression. This finding differed from logistic regression analysis, in which mortality was similar. This difference suggests that comparisons of hospital mortality between systems with different hospital utilization patterns may be biased if LOS is not considered. Moreover, if generalizable to other markets, our findings suggest that ICU outcomes are at least similar in VA hospitals compared to private sector hospitals. BACKGROUND: The evidence to support colorectal cancer (CRC) screening has become definitive though it is well known that most adults do not receive the screening that is recommended. METHODS: We merged patient self-report data from 1998 for respondents > =50 years (n=20,199) with surveys of medical directors of 53 medical organizations [29 medical groups (MG) and 24 Independent Practice Associations (IPAs)] from 3 west coast states to evaluate rates of CRC screening and patient and organizational predictors of those rates. We used logistic regression adjusted for intra-organization clustering of patients within medical organizations to estimate CRC screening rates as a function of patient demographic and comorbid characteristics, receipt of care within a MG or IPA, receipt of care within an organization that uses their corporate structure to implement guidelines, tracking, or feedback of CRC screening results, and the unique identity of each of the medical organizations. RESULTS: Overall, screening rates were modest [45% for sigmoidoscopy or colonoscopy during the last 5 years (SIG), 43% for fecal occult blood tests by the patient at home (FOBT), 64% for either SIG or FOBT, and 24% for both]. Screening rates varied by demographic characteristics. After adjustment for comorbidity, use of any CRC screen was significantly lower for females (OR 0.69 *** ), Asians (OR 0.73 *** ), Hispanics (OR 0.86 * ), and those from other nonwhite race (OR 0.76 * ); screening rates were higher for African Americans (OR 1.3 ** ). Respondents receiving care in IPAs reported less screening than those in MGs (OR 0.83 * ) after adjustment for patient characteristics as well as use of guidelines, tracking and feedback pertinent to CRC screening, and clustering of patients within organizations. In addition to the amount of variation in CRC screening explained by patient characteristics alone, receipt of care in a MG or IPA and corporate policy for use of guidelines, tracking or feedback for CRC screening explained 8% of the unique variation in CRC screening rates, while the unique identity of the 53 medical organizations explained 51% of the unique variation. CONCLUSION: Although there is no clinical reason demographic characteristics should influence rates of CRC screening, they do. In addition to demographics, the corporate structure of the medical organization is associated with rates of patients receipt of CRC screening such that screening occurs more frequently within MGs than IPAs. Even after adjustment for patient characteristics, the use of guidelines, feedback, and tracking of guideline performance, and whether the patient received care in a MG or IPA, most of the variation in screening is explained by the unique identity of their medical organization. PURPOSE: State medical associations may choose to participate in public debates surrounding physician-assisted suicide (PAS). However, physicians lack consensus on the ethics of PAS and its legalization. We examined physician attitudes about PAS and the prospects for consensus regarding its legalization. METHODS: Anonymous questionnaires were mailed to all (1456) members of the Connecticut Chapter, ACP-ASIM. The survey instrument measured attitudes toward PAS; consensus on health policy issues generally; participation in public discussions about PAS; prospects for consensus on its legalization; demographic data including age, gender, experience caring for terminally ill patients, religious affiliation, and religious service attendance. The proportion of foreign-born residents in the United States is at its highest point in over 100 years. Provision of interpreter services for non-English speaking patients is now a federal requirement and general internists will need to use interpreters to care for these patients. We surveyed clinicians in three academic generalist practices to describe how interpreters are being used. METHODS: We surveyed all of the primary care internists and nurse practitioners (n=194) in three academic outpatient settings in San Francisco regarding their most recent patient encounter which involved an interpreter. Questionnaires were self-administered and took about 10 minutes to complete. RESULTS: 158 questionnaires were completed (81% response rate). Among the respondents, 53% were women, 64% White, 66% residents, 78% spoke and understood a language other than English, and 60% had some prior training regarding working with interpreters. Reflection of the most recent patient encounter requiring an interpreter showed that 67% used professional interpreters, 49% felt that not enough time was allotted for the appointment, 90% felt satisfied that they had accomplished their own goals during the encounter, and 83% felt satisfied that the patient had accomplished his/her goals. Most respondents (78%) were very satisfied or satisfied with the medical care they provided, 85% felt satisfied with their ability to diagnose a disease and treat a disease, but only 45% were satisfied with their ability to empower the patient with knowledge about their disease, treatment or medication. Even though 71% felt they were able to make a personal connection with their patient, only 33% felt they had learned about another culture as a result of the encounter. Clinicians had trouble eliciting exact symptoms (70%), explaining treatments (57%), and eliciting treatment preferences (51%). Almost half (45%) felt that too much time was spent on translation. A majority of clinicians perceived that lack of knowledge of the patient's model of disease and lack of knowledge of the patient's culture hindered their ability to provide quality medical care. However, only 18% felt they were unable to establish trust or rapport. CONCLUSION: Although most clinicians feel good about their encounters with patients in which they utilize an interpreter, many of them have received no training regarding that use, and their ability to elicit information, convey information, understand the patient in his/her cultural context, and empower the patient with regard to his/her own healthcare appears to be compromised. We conducted a controlled trial of a guideline-derived intervention, which included 3 components: 1) training of clinic nurses and medical assistants to use a modified vital signs stamp in identifying and counseling smokers at each visit, 2) feedback to clinic staff on performance of AHRQ guideline-recommended activities, and 3) proactive telephone counseling by a smoking cessation counselor (2 sessions) and free nicotine replacement therapy (NRT) for all pts who indicated willingness to make a quit attempt within 30 d of their clinic visit. After a period of baseline data collection (8/99 ± 1/00), the intervention was implemented at 1 pilot family practice (FP) clinic (2/00 ± 3/00); patterns of usual care were observed concurrently at 5 control FP clinics. We obtained exit interviews of consecutive adult smokers who presented for routine, non-emergency care. Abstinence (no cigarettes over prior 7 d) and quit attempts were determined by telephone interview during 6 mo follow-up. Odd ratios (OR) were computed for each outcome. RESULTS: There were no significant differences in demographic or smoking variables between baseline and intervention pts, 87 and 85% of whom completed 6 month follow-up. Concordance with guideline recommendations at the clinic visit and quit status are shown below ( * p < 0.05). For comparison, the 6 mo quit rate for pts at control clinics during baseline and intervention periods was 12 and 10%, resp. 37% of intervention group pts received telephone counseling plus NRT. CONCLUSION: Effective reduction of tobacco use requires redesigning health care systems to increase the identification of smokers and the delivery of smoking cessation advice in a timeefficient manner. The 9% absolute difference in 6 mo quit rates associated with this nursebased, primary care intervention is comparable to that observed in clinical trials of NRT. Cleveland between 1991 and 1997. Patient level data were obtained through the Cleveland Health Quality Choice (CHQC) program; mortality was obtained by linking CHQC data with MEDPAR files. Pneumonia was identified from secondary ICD-9-CM codes. Cox regression analysis was used to estimate the effect of pneumonia on mortality up to 30 days from admission after adjusting for severity of illness using a model of 30-day predicted mortality from admission data (c-statistic = 0.78). Patients dying or having a``Do Not Resuscitate'' order within 3 days of hospitalization were removed a priori from the regression analysis to focus on those for whom avoiding pneumonia was most pertinent. RESULTS: Pneumonia occurred in 6.9% (n = 985) of all patients hospitalized for acute stroke. There was a significantly higher incidence in patients: with hemorrhagic stroke (11.5% vs 6.3%,p < .001), men (9.0% vs 5.4%, p < 0.001), from nursing homes (11.4 vs 6.4%,p = 0.001), aged > 74 (7.2% vs 6.3%, p = 0.032), and with greater severity of illness on admission (predicted 30-day mortality 24.8% vs 13.2%, p < 0.001). Average length of stay for patients with pneumonia was 16.2 0.44 days compared to 7.5 0.05 days in patients without pneumonia (p < 0.001); fewer patients with pneumonia were discharged to home (12.0% vs 41.4%, p = 0.001). Crude 30-day mortality rates were 37.4% for patients with pneumonia and 13.0% for those without (p < 0.001). There was a steady increase in the adjusted hazard ratio for death among stroke patients with pneumonia throughout the 30-day period; adjusting for admission severity, the hazard ratio for death within 30 days was 4.31 (95% C.I. 3.55 ± 5.23). CONCLUSION: In this largest community-wide study of stroke complications, pneumonia conferred significantly higher severity-adjusted hazard for mortality within 30days of admission and was associated with dramatically longer lengths of hospital stay and reduced chances of discharge to home. Measures to reduce the incidence of pneumonia following stroke are warranted to reduce morbidity, mortality, and cost. , and personal experience with BRCA. The dependent variable, a sum score of attitudes towards BRCAS, was calculated such that a higher score indicated a more favorable attitude towards screening. Univariate and multivariate analyses with backwards ± stepwise regression assessed the relationship between the dependent and independent variables. RESULTS: The response rate for the survey was 82% (N = 140 responses); 120 physicians provided complete data for analysis. Of these physicians, 62% were male, 60% were under 30 years old, 12% worked in a private office, 42% worked in a health center, 46% worked in an outpatient setting, 38% had some personal experience with BRCA, and 68% had some professional experience with BRCA. In both univariate and (multivariate) models professional experience p = 0.006 (0.03) was associated with a more favorable attitude concerning BRCAS, but age p = 0.10 (0.54), gender p = 0.54 (0.59), practice type p = 0.23 (0.62), and personal experience 0.54 (0.37) were not. CONCLUSION: Prior professional experience of the physicians, but not demographic or personal variables, was significantly related to a favorable attitude towards BRCAS. Our data differs from previous studies, which found that BRCAS may be influenced by physician characteristics like age, gender, practice type, and personal experience. Increasing awareness through the education of physicians has been a focus of many efforts to increase the incidence of screening for BRCA. If professional experience is associated with a favorable attitude towards BRCAS, then medical trainees should be exposed to environments where BRCAS is a high priority in an effort to increase BRCAS in women. (4.4 ± 29.4, 95%CI) . Most commonly cited missing elements that were needed included current notes types that were not online (33%), notes predating the system (20%), EKGs (16%), and vital signs (12%). On the same Likert scale, 75% agreed that they could give up the traditional paper chart in favor of the mobile computer. CONCLUSION: Overall, internal medicine residents responded positively to the implementation of the mobile CPRS system. Based on surveys and computerized order logs, all residents voluntarily used the devices to enter nearly half of orders over available desktop computers. Reported difficulties with the technologies did not appear to significantly affect the mobile computer users ordering pattern, suggesting that the technical problems were limited and the technology was robust. Finally, feedback from this study was sufficiently positive to justify implementation to all internal medicine teams. After one year, we assessed their impact on patient length of stay as well as resident education and service from the viewpoint of both medical residents and community physicians. METHODS: Two clinician educators were hired to provide inpatient attending medical care and resident education, including 1:1 intern mentoring. Length of stay data was calculated for Medicaid and self-pay patients based on DRG groups where > 50% of patients were admitted to the clinician educator service and was compared to data on the same patient mix from the preceding year. Categorical medicine residents (N=36) were given an anonymous survey assessing the clinician educators' impact on resident education and service, including resource utilization and resident roles. Community internists (N=150 on staff, 100 who actively use hospital) also received an anonymous survey assessing their perceptions of hospitalist medicine and the clinician educators' impact on housestaff behavior. RESULTS: Length of stay: LOS was reduced from 5.3 to 4.4 days comparing the last 6 months of the clinician educators' first year with the last six months of the previous year. Reductions in LOS > 1 day were found with GI, pulmonary, infectious disease, endocrine and neurology admissions and 1/3 of a day for cardiology admissions. Resident survey: Return rate was > 90% with all residents noting improvement in the quality of attending rounds, bedside teaching and the overall inpatient experience. Increased use of evidence based medicine resources was noted by 90%. Residents' roles as teachers and team leaders were increased or unchanged for over two-thirds and none felt they had been reduced to unacceptable levels. A potential influence on these behaviors is the availability of pharmaceutical samples. A prospective blinded study was designed to evaluate this influence in an academic clinic. METHODS: The antibiotics in the drug closet of our teaching clinic were manipulated over 20 consecutive weeks. The following changes were made at 4 week intervals: 1) removal of all antibiotics, 2) removal of macrolides, 3) removal of quinolones, 4) removal of cephalosporins, 5) removal of amoxicillin/clavulanate and nitrofurantoin. Upon completion of the study, a chart review was conducted to identify acute infectious illnesses for which an antibiotic was likely to be prescribed. Datapoints collected include: clinical diagnosis, antibiotic prescribed, postgraduate year (if a resident), date of service, and patient's gender and age. Fishers Exact 2-sided tests were performed to determine whether the presence of antibiotics in the closet influenced their frequency of use. RESULTS: 465 encounters fulfilled our inclusion criteria. 77% of these encounters led to an antibiotic prescription. Of these, 73% had an antibiotic prescribed that could be found in the drug closet. Defining the relative risk as the risk of being prescribed a specific antibiotic if it was available in the closet versus if it were not, the relative risk ratios (RR) with their associated 95% confidence intervals (CI) for each class of antibiotics are listed in the following table. CONCLUSION: The prescribing practices of clinicians in our academic clinic do not appear to be directly influenced by the availability of antibiotic samples in a drug closet. Although a direct relationship between the drug closet and the prescribing practices of clinicians was not observed, it is notable that 73% of all prescriptions were for antibiotics that could be found in the drug closet. This still suggests that pharmaceutical marketing exerts an influence on clinician antibiotic prescribing practices. is important that primary care physicians be aware of the safety of drugs in pregnancy given to nonpregnant women, as well as the effect of common medical problems in pregnancy. For the same reasons, it is also important that counseling and prevention of risk factors known to affect pregnancy outcome occur prior to conception. The purpose of this study was to assess the knowledge and practice of preconception care by primary care attending physicians. METHODS: All primary care attending physicians in general internal medicine and family practice at our institution were given a written survey regarding their knowledge and practice of preconception care. Knowledge questions assessed were 1) safety of medication use in pregnancy 2) risk associated with diabetes in pregnancy, and 3) the rate of unplanned pregnancies and estimated gestational age at presentation to a pregnancy provider. Practice questions included counseling of the nonpregnant patient regarding 1) the hazards of smoking, alcohol and illicit drug use 2) folic acid supplementation, 3) rubella screening, and 4) the effect of chronic medical disorders were pregnancy to occur. Surveys were mailed. Responses were kept anonymous and a number was used to identify participants. RESULTS: Of 25 surveys mailed, 21 were returned. Analysis was done of the returned surveys. 80% of responding attending primary care physicians were aware that 50% of women present to their pregnancy provider after organogenesis and that similar rates of pregnancies are unplanned. 45% counseled women with substance abuse of potential effects on pregnancy, 40% recommended folic acid use, and 10% screened for rubella. 55% counseled women with chronic disease of deleterious effects on pregnancy. Safety of medications in pregnancy were correctly identified by 35% for pain relievers, 45% for antibiotics, 25% for asthma medications, and 10% for antihypertensives. Only 45% of providers were aware of the risk associated with diabetes in the first trimester. No statistical difference using Chi Square analysis was found when the results were computed by the sex of the provider or by primary care specialty, possibly due to the small sample size. CONCLUSION: Despite the knowledge that women often do not plan pregnancy and do not present to a pregnancy provider early enough to prevent the toxic effect of medications, chronic medical disorders, or personal behaviors, the majority of primary care providers were not knowledgeable regarding preconception care and did not incorporate it into the continuing care of women of childbearing age. PURPOSE: The quality of care provided to patients hospitalized for heart failure has been shown to vary by physician, hospital and region. Hospitalists are physicians whose primary professional focus is the general medical care of hospitalized patients. While hospitalists appear to reduce costs and length of stay, their impact on quality of care is less certain. We sought to compare the quality of care provided to patients with heart failure by hospitalists and nonhospitalist physicians. METHODS: We conducted a retrospective medical record review at a 550 bed communitybased teaching hospital in Massachusetts that is home to 3 hospitalist groups as well as numerous small group practices that provide care for their own hospitalized patients. We identified all patients cared for by hospitalists and by non-hospitalist general internists who were discharged with an ICD-9-CM principal diagnosis of heart failure between 4/1/99 and 3/30/00. We evaluated quality of care by measuring adherence to a set of process measures derived from the JCAHO's``Core Measures Set'' and HCFA's Health Care Quality Improvement Initiative. PURPOSE: There has been an increasing amount of research in the area of religion and spirituality and its effect on patient care and health outcomes, but how this should be incorporated into physicians' practice has been an area of debate. We evaluated the extent to which general internal medicine residents and faculty acknowledge a personal belief in spirituality and religion; and to what degree internists feel it is appropriate to incorporate religion and spirituality into health care. METHODS: A twelve-item survey of the general internal medicine residents and faculty at Wake Forest University. RESULTS: 87/112 physicians responded (response rate 77% of residents and 82% of faculty), and an impressive majority (90%) of respondents professed a belief in God or other higher power. 82% considered themselves to be religious or spiritual. An overwhelming 92% felt that patients' beliefs can influence their health. 71% agreed that it would be helpful to inquire about patients' religious/spiritual concerns relating to their health. However, less than half (47%) of respondents felt there is a role for direct physician involvement in meeting patients' religious or spiritual needs, and even less (43%) had actually been directly involved in patients' spiritual care. CONCLUSION: A vast majority of respondents profess their own personal religious and spiritual beliefs, and feel that religion and spirituality can have a positive effect on health outcomes. Yet only a minority feels that doctors should actively pursue any action in this area, and even fewer have actually done this. This is a striking disconcordance between physician attitude and action. Given the growing amount of evidence that suggests religion and spiritual factors can affect health outcomes, efforts should be made to determine what barriers lie between these attitudes and actions, and improve physician education on how to approach these important patient-centered issues. PURPOSE: HMOs' financial incentives to restrict medical care have generated public concern about the quality of care they provide. Public disclosure of quality of care data might improve public accountability, enhance informed consumer decision making and promote quality
improvement. Yet, public disclosure of this data is voluntary. We sought to determine the association between HMO quality of care and willingness to publicly disclose quality of care scores. METHODS: We analyzed data from the National Committee for Quality Assurance's annual Quality Compass databases, including the Health Plan Employer Data and Information Set (HEDIS), a standardized set of quality of care measures (e.g. % of women > 50 who received biannual mammography). We determined how frequently HMOs that publicly disclosed HEDIS scores in 1997 or 1998 withdrew from public disclosure the subsequent year. We also assessed whether withdrawal from public disclosure was associated with being in the lowest (compared with the highest) tertile of plans ranked by HEDIS scores for 9 clinical quality measures, after adjustment for the method of data collection (administrative vs. medical record review). RESULTS: Of the 329 HMOs which publicly disclosed HEDIS scores in 1997, 161 (48%) withdrew from public disclosure in 1998. Of the 292 HMOs which publicly disclosed in 1998 (including newly disclosing plans), 67 (23%) withdrew from public disclosure in 1999. Quality varied greatly between plans with absolute differences in HEDIS scores between lowest and highest tertile plans ranging from 14.6 to 42.3 percentage points. When HEDIS measures were analyzed individually, HMOs ranked in the lowest (compared with the highest) tertile in 1997 were significantly more likely to withdraw from public disclosure in 1998 for 7 of the 9 measures (RR = 1.6 ± 2.7, p < 0.05 for all). In 1999, HMOs ranking in the lowest tertile in 1998 for 8 of the 9 measures were significantly more likely to withdraw from public disclosure (RR = 1.9 ± 7.0, p < 0.05 for all). When examined by average rank across all 9 measures, plans in the lowest tertile were significantly more likely to withdraw from public disclosure in 1998 ( Reprints and monographs were excluded. Clinical information was reviewed by two general internists and compared to information in Goodman s 9th edition textbook of pharmacology, in the pharmacology section of the UpToDate version 8.2, or in both. References cited were reviewed for correct citation and accessibility in any of the four major medical libraries in Buenos Aires. RESULTS: Of the 64 pieces of printed promotional materials collected, 30 were randomly selected and evaluated. In 21 (70%) of the thirty promotional printed materials evaluated the therapeutic effect promoted in the advertisement appeared in the references used. Only 18 (60%) had statements supported by cited references. From a total of 131 references cited in printed promotional materials, 60 (46%) were incorrectly listed according to the International Committee of Medical Journal Editors. These references were also inaccessible. Of the 71 references correctly cited, 49 (69%) were not available in any of the four major medical libraries in Buenos Aires and 8 (11%) were available in only two of the libraries. Twenty-two references were read, and in only 12 (54%) of these, did the objective of the research study concur with the statement of the printed promotional material. Adverse drug reactions, warnings about drug interactions and drug contraindications were absent from all printed promotional materials. CONCLUSION: The printed promotional materials distributed by pharmaceutical sales representatives in Buenos Aires are biased and provide misinformation more often than not. We recommend that practicing physicians should routinely disregard printed promotional materials as a source of clinical information. PURPOSE: Hospitalist physicians who specialize in inpatient care are rapidly increasing in number but there is limited evidence from randomized trials concerning their effects on resource utilization and outcomes or how they achieve their effects. This project aims to determine the effect of hospitalists on resource use and outcomes on a general medicine service in an academic medical center and the mechanism for their effects. METHODS: A longitudinal trial from July 1997 ± June 1999 with all patients admitted every fourth day assigned to teams led by hospitalist physicians (HPs) who care for inpatients 6 months per year versus teams led by non-hospitalist physicians (NHPs) who care for inpatients 1 or 2 months per year. Resource utilization was measured by length of stay and costs. Patient outcomes were measured by 30-, 60-, and 365-day mortality rates, readmission rates, reported physical function, and patient satisfaction. RESULTS: Of 6511 admissions to the general medicine service, 24.8% were to HPs and 75.2% to NHPs. Patients cared for by HPs and NHPs did not differ in age, race, gender, diagnosis mix, Charlson index, or payer mix. Average length of stay for the general medicine service was 4.7 days and average cost was $8517. In multiple regression analysis controlling for diagnosis with DRG weight and for comorbidity with Charlson index, HPs did not have different length of stay or costs than NHPs in year 1, but HPs had 0.5 day lower length of stay (p < 0.01) and $740 lower costs (p < 0.01) in year 2. There were no differences in mortality in year 1, but in year 2 HPs had lower mortality at 30 days (4.2% vs. 6.0% for NHP, p < 0.04), and 60 days (8.8% vs. 6.8%, p < 0.07). There were no statistically significant differences between HPs and NHPs in 365-day mortality rate, readmission rate, physical function, or overall patient satisfaction, but the trends favored HPs in all these measures. In analyses controlling for diagnosis-specific fixed effects, month of admission, total volume of patients seen by the physician to date, and total volume of patients with the same diagnosis seen by the physician to date, the effect of hospitalists on length of stay and costs, but not mortality, was explained by diagnosis-specific clinician volume to date. (1) preference rankings of reasons for selecting drugs in the treatment of patients with CAP, (2) knowledge and attitude questions regarding antimicrobial drug prescribing, and (3) measurement of thresholds for switching from hypothetical older drugs with increasing resistance to newer drugs with much less current resistance. Physicians were sampled from the AMA Masterfile and surveyed by mail with two follow-up mailings. Responses were calculated for the overall sample as well as stratified by clinical specialty. RESULTS: 833 out of 1582 physicians (53%) completed the survey. Response rates did not differ across specialty, gender, or US region. Overall, physicians ranked efficacy, severity of illness, and prior experience as the top reasons for selecting antimicrobial drugs, and this ranking was consistent across specialty. Concern over contributing to rising antimicrobial resistance was ranked low by both ID specialists and generalists. Although 88% agreed that`a ntibiotic resistance is a major health problem'' and 89% agreed that``over-prescribing antibiotics is a major cause of antibiotic resistance,'' only 54% agreed that``before prescribing an antibiotic, I weigh the potential benefit to the patient against the potential harm to society.'' In a hypothetical scenario for the treatment of an outpatient with CAP due to S. pneumoniae, 55% of generalists and 67% of ID specialists were unwilling to accept a 20% or greater level of resistance with an older drug before switching to a newer drug without resistance (p=.01). Physicians reported a higher threshold for switching to a newer antibiotic if they agreed that before prescribing an antibiotic, they weigh the potential benefits to patients against the potential harms to society. CONCLUSION: Many physicians report that societal issues created by antimicrobial drug resistance have at most a small impact on their prescribing decisions. These attitudes were generally consistent across different response tasks and between generalists and ID specialists. Since efforts to reduce antibiotic resistance focus on increasing provider knowledge about the societal risks of these drugs, these results suggest that such efforts alone will have limited success. PURPOSE: Primary care physicians have been the focus of efforts to improve the quality of preventive health care such as smoking cessation counseling. Many primary care practices have not implemented systematic protocols to identify patients who smoke or to encourage clinicians to provide smoking cessation counseling because such interventions are thought to be too time and effort intensive. With collaboration from Blue Cross Blue Shield of Massachusetts, we designed a project to assess the relative effectiveness, as compared with no intervention (usual care), of two brief primary care interventions on patient smoking cessation rates. We hypothesized that improved documentation would increase smoking cessation among patients. METHODS: We performed the study at the adult primary care practice of a large urban tertiary care center. We chose interventions that could be used routinely in a busy primary care practice, and which differed in the level of patient and physician involvement. Each intervention was implemented on a geographically separate``team'' consisting of 5 ± 6 attending physicians, 2 nurse practitioners, and 3 ± 5 residents. Three teams served as controls (usual care). Thè`m inimal'' intervention consisted of a smoking status``vital-sign'' stamp, which identified smokers and documented patient smoking status. The``enhanced'' intervention consisted of a simple, one page, 5-question form which identified smokers, assessed level of cessation readiness and motivation, and provided smoking cessation counseling prompts for clinicians. Outcomes were collected 8 ± 10 months after implementation of the interventions. Patients who had identified themselves as current smokers at the beginning of the study period and who had seen a clinician at least once during the study period were contacted and their smoking status assessed by self-report. Two individuals unaware of study design or purpose performed structured chart reviews to assess clinician documentation of patient smoking status and cessation counseling. RESULTS: Four hundred seven charts were reviewed for documentation assessment.
Smoking status was documented at 84% of the visits on the minimal intervention team, 91% on the enhanced intervention team, and 49% on the control teams (p-value < 0.0005). Cessation counseling was documented at 36% of the visits on the minimal intervention team, 47% on the enhanced intervention team and 30% on the control teams (p-value = 0.015). The improvement in smoking status documentation was mainly noted 1) for routine and non-cardiac/pulmonary urgent care visits compared with new patient or cardiac/pulmonary urgent care visits, and 2) among nurse practitioners and a patient's primary care physician compared with residents or urgent care physicians. The trends were similar for cessation counseling documentation. Two hundred forty-five patients completed the smoking status assessment at the end of the study period: 58% were female, the mean age was 46 years, and 40% were evenly divided between the minimal and enhanced intervention teams. There were no significant differences between groups in mean length of follow-up, or median number of visits or cigarettes smoked. A total of 33 (13.5%) patients had stopped smoking by the end of the follow-up period: 4% in the minimal intervention team, 30% in the enhanced intervention team, and 11% in the control teams (p-value < 0.0005). The proportion of patients reporting increased awareness of the health risks associated with smoking or improved motivation to stop smoking was higher among the intervention (56 and 67%) than control teams (40 and 59%). CONCLUSION: An enhanced intervention that uses a short questionnaire to identify patients who smoke and assess their level of cessation readiness may significantly improve smoking cessation rates compared with minimal or no intervention. Both enhanced and minimal interventions can improve physician documentation of patient smoking status and cessation counseling. The rate of new applications to medical school in the United States has been declining over the past three years, possibly because of less interest in medicine as a career among young persons. It has been suggested, however, that even established physicians are leaving medicine for a host of reasons (e.g., financial issues, time factors). Our study attempts to qualify and describe this issue in relation to financial pressures of physicians. METHODS: We conducted a state-wide, randomized survey of 1000 Maryland chapter members of the American College of Physicians. Surveys were mailed, with a follow-up reminder letter and survey sent at one month to non-responders. The 24-item survey collected data on physician demographics, satisfaction with income, activities to generate income outside of medical practice, and plans to remain in medicine in 5 years. RESULTS: After initial mailings, the response rate was 40%. Mean age was 47.7 years, and 76.2% were male. Excluding the 11.9% of physicians who planned to retire, only 193 (62.1%) intended to remain in their current practice or position in 5 years. 11.6% felt they would still be in medicine, but in a different practice or setting, and another 6.5% expected to be in medical industry or management. A surprising 16.4% planned to be in a profession outside of medicine or were unsure about remaining in the medical field. In univariate analysis, factors associated with a desire to change positions were female gender, unmarried status, no dependents, generalist (vs. specialist), decrease in primary medical income over 5 years, and low level of satisfaction with primary medical income. If starting over, 25% of all responders would not have chosen medicine again as a career. CONCLUSION: In our survey of Maryland physicians, over 35% planned to be in a different practice or position, many not related to medicine. This documents further the growing dissatisfaction with medicine among physicians, much of which is related to financial pressures. The impact of these findings on continuity and quality of patient care deserves further study. The effects of maternal smoking on infant mortality and morbidity are well recognized. Brief physician advice has been shown to increase quit rates among pregnant smokers and to improve pregnancy outcomes. Frequent prenatal visits offer repeated opportunities for smoking cessation counseling. We evaluated how frequently physicians identify the smoking status of pregnant patients and how frequently physicians counsel pregnant smokers. METHODS: The National Ambulatory Medical Care Survey is an ongoing national survey of U.S. office based physicians. During the years 1991 through 1996, physicians answered questions both about patient smoking status and whether or not tobacco counseling was performed. We analyzed data from these six years to determine the frequency with which physicians identified the smoking status of pregnant patients and the frequency with which physicians counseled pregnant smokers. We compared smoking status identification and counseling rates for different specialties. RESULTS: 9807 physicians recorded data on 5622 visits by pregnant women. The Figure  presents is not yet widely used. A wide variety of physician order entry systems currently exists making it important to determine which systems physicians are likely to accept. While studies have evaluated physician satisfaction with institution-specific order entry systems, few published studies have assessed commercial products, even though nationally these products will represent the majority of systems that will be implemented. METHODS: We distributed the Questionnaire for User-Interface Satisfaction (QUIS) to Internal Medicine physician housestaff who use both a commercially available order entry system and the Veterans Affairs Computerized Patient Record System (CPRS). The QUIS is a validated questionnaire that has been used to measure user satisfaction with the humancomputer interface with electronic medical records. It is a twenty-seven-item survey and questions are scored on a 0 (lowest) to 9 (highest) scale. RESULTS: 90 and 84 surveys were returned giving a response rate of 63% and 64% for the commercially available product and the CPRS surveys respectively. Overall, residents were dissatisfied with the commercial system giving it an overall mean score of 3.67 (95% confidence intervals 3.37,3.97). In contrast, the Veterans Affairs CPRS had a mean score of 7.21 (95% confidence interval 7.00, 7.43) indicating the residents were more satisfied with the CPRS (pvalue < 0.0001). Overall satisfaction was strongly correlated (& = 0.71) in both surveys with the ability to perform tasks in a``straight-forward'' manner. Overall satisfaction was also correlated with terminology consistency (&=0.60, &=0.71) and screen sequence (&=0.61, &=0.67) for the commercial product and the CPRS respectively. CONCLUSION: Satisfaction with the user interface for the two order entry systems differed dramatically between the two systems, suggesting that all physician order entry systems are not equally accepted. Satisfaction was highly correlated with the ability to perform given tasks in a direct and consistent manner. These data support prior studies that have found correlations between user satisfaction and efficiency with order entry systems. If maximal benefit is to be gained from the implementation of such systems further studies are needed to investigate what specific order entry features physicians find most useful and least disruptive to daily workflow. PURPOSE: Nearly half of all California physicians still practice in solo or self-employed group practices. The recent and well-publicized demise of independent practice associations (IPA) and practice management organizations in the state has underscored the tenuous financial status of these practices. As part of a larger study about the state of physician practice in California, we interviewed community-based physicians and asked them to describe various aspects of their practice lives. We describe here the variety of approaches that physicians are taking to enhance or maintain the viability of their practices. METHODS: We interviewed fifteen physicians throughout California who spend > 50% time providing care in office-based practices. Participants were identified using the snowball sampling method. Each individual was interviewed by telephone by a study physician. The average age of participants was 41 years (31 ± 55), 43% were specialists, 36% were female and 30% practiced in community health center or public clinics. Only 13% expressed any dissatisfaction with their practices. Transcribed interviews were thematically analyzed using ATLAS 4.1 software.4.1 software. RESULTS: Physicians in solo or small group practices were more apt to discuss financial issues related to their practices than those affiliated with larger groups or with health maintenance organizations. Specific methods for improving the financial viability of their practices included improving``customer service'' for patients and referring physicians, limiting contracts with low paying insurers and or patients when feasible, lengthening work hours, shortening duration of office visits, offering supplemental patient services for out-of-pocket pay, streamlining office processes and staffing, optimizing billing and using physician extenders. A few physicians described colleagues whose response was to leave practice for administrative positions or early retirement. CONCLUSION: Physicians are experiencing financial pressures that in California have been amplified by the financial insolvency of IPAs and other practice organizations. The predominant response to improve the financial viability of their practices has been more efficient and effective utilization of existing resources (including personnel). Less common approaches include providing supplemental services that patients are willing to pay for out-ofpocket and leaving clinical practice. These experiences, to some degree, mimic those experienced by other professions such as dentistry. The frequency with which the approaches are used and their implications for access, patient outcomes and physician workforce requires further study. Virginia has the greatest number of free clinics of any state. We evaluated the type and dollar value of medical services provided in 1999. METHODS: Survey completed by the directors of all 32 clinics registered in 1999 with the Virginia Association of Free Clinics. Dollar value of services provided was estimated to be thè`u sual and customary'' charges for the region of each clinic; prescription value was estimated to be $45, the average retail price of a prescription for Virginia in 1999. Operating expenses were expenditures for clinic administration, fund raising, and direct services, excluding capital expenses.
RESULTS: See table. CONCLUSION: Free clinics in Virginia effectively leveraged $6.8 million operating expenses into $24.5 million worth of medical services. The average expense per visit, including prescriptions and health care provider services, compares favorably to private practice office benchmarks. Although many patients were served, they represented only the tip of the iceberg in Virginia in 1999, comprising only 4% of the approximately one million uninsured. While free clinics provide an outlet for patients who might not receive care otherwise, physicians and policy makers must continue to work for a more comprehensive solution.
M. Nadkarni 1 , D. Hoffman 1 ; 3 University of Virginia, Charlottesville, VA PURPOSE: To ascertain how many patients in a medically underserved population receiving continuity care at a University Clinic actually qualify for additional health and social assistance benefit programs but are not yet enrolled. METHODS: A random sample of 200 medically indigent patients at University Medical Associates, a combined resident-faculty practice with 9000 active patients which provides care to a primarily underserved population in central Virginia, was selected for analysis of healthcare benefits they were eligible for but were not currently receiving. Many patients without health insurance are actually eligible for assistance programs and potentially could receive some form of healthcare insurance. A significant number of patients in underserved populations who are already covered by Medicare may still qualify for additional benefits but have not accessed them. Aggressive attempts to overcome barriers for enrollment in important assistance programs could substantially increase the welfare of many patients in indigent or uninsured populations. Interventions to increase enrollment of eligible patients and their families should be undertaken on national, state, and county level. Additionally, individual clinics and practices can help increase enrollment with relatively simple procedural measures.
SURGEON VOLUME OF EARLY STAGE BREAST CANCER CASES. A. Nattinger 1 , R. Hoffmann 1 , R. Sparapani 1 ; 1 Medical College of Wisconsin, Milwaukee, WI PURPOSE: The percentage of U.S. women with early stage breast cancer who receive initial treatment in accordance with national guidelines has been declining. We are interested in whether physician volume of cases is related to processes or outcomes of care, and therefore determined for a population-based cohort the distribution of cases cared for by each surgeon operating on one or more cohort patients. METHODS: We used data from a population-based SEER-Medicare linked data base to study women aged 65 or older, who were diagnosed with a stage I or II breast cancer in 1993 ± 95, and underwent either breast-conserving surgery (BCS) or mastectomy. We determined the operating surgeons from the Medicare part B claims, and eliminated surgeons with an office zip code outside the SEER states. RESULTS: Over the three study years, 15,574 women were operated on by 3,265 different physicians. The median number of cases performed per physician per year was 1.0 with 1st/3rd quartiles of 0.0/4.0. Over three years, 35.3% of physician-years were zero-case years. We We developed a conceptual model for assessing the validity of existing guidelines using literature searches and expert guidance. Two physicians with expertise in literature retrieval and evaluation conducted limited literature searches for new evidence that could affect the validity of the guideline statements. We also searched for practice guidelines that had been published after the release of the AHCPR guidelines. Guideline statements were sent to original guideline panel members and other content experts, who were asked to evaluate the current validity of each statement and include evidence to support their assessment. The evidence collected was then used to determine the validity of the guidelines, which were classified as``still valid'' or``needs updating.'' Finally, we conducted a``survival analysis'' to estimate the rate at which guidelines become outdated using a Kaplan ± Meier survival curve and the Weibull parametric model. RESULTS: We reviewed 17 guidelines and surveyed 170 experts, 121 (71%) of whom returned their surveys. Our literature searches identified 6,994 titles, which were all reviewed.
We reviewed the abstracts of 610 of these articles and then narrowed our selection down to 173 full-text articles. We also reviewed 159 guidelines. There were 13 (76%) guidelines in need of an update, 3 (18%) guidelines that were still valid, and 1 (6%) guideline for which we could not make a determination due to insufficient information. The guidelines had an estimated``shelf-life'' of 3 years (90% of the guidelines were still valid) to 5.9 years (upper bounds of the 95% CI). CONCLUSION: Our study provides the first empirical evidence of the rate at which guidelines go out-of-date. More than 75% of the AHCPR guidelines are in need of updating. We recommend re-evaluation at a minimum every three years with modifications in this timeline based on whether advances in the field are rapidly or slowly evolving. Future guidelines could facilitate the updating process by including an estimate of the type of evidence necessary to prompt revisions. PURPOSE: Critics state that some of the cost reductions demonstrated by hospitalist services may be explained by``cost shifting'' to the outpatient setting. Timely and appropriate patient follow up is also of concern. The purpose of our study was 2 fold: 1)determine if a significant portion of the cost savings realized by our hospitalist service could be attributed to``cost shifting'' and 2)determine if follow up issues were addressed as an outpatient. METHODS: During the 1998 ± 99 academic year, WVU implemented a hospitalist service (H) which was compared with the two other medicine services, one staffed by General Internists (GIM) and the other by subspecialists (SS). Inpatient cost (COST) and length of stay (LOS) were the main outcomes. 956 charts (321 for H, 257 for GIM, 378 for SS) were retrospectively analyzed in a blinded fashion to determine the following for each group: 1) percentage of patients discharged with workups needing to be performed (% W/TJ) 2) percentage of patients with tests or procedures scheduled as outpatient (% TEST) 3) average costs of test/procedures specified at discharge (F/U COST)(elective surgeries excluded). Of patients who had follow up within our system, 25 charts in each group were reviewed to determine follow up rates (% F/U). RESULTS: See table. Among the 3 groups, there were no significant differences in the percentages of patients discharged with test procedures or workups pending (p > 0.6).Patients who had longer LOS or who were followed in our system tended to have more outpatient test and workups scheduled (p < 0.0 1). In terms of cost specified at discharge, there were no significant differences (p = 0.09). There were no significant differences in percentage of issues followed up, however the numbers were small (p = 0.2). CONCLUSION:``Cost shifting'' does not appear to explain the cost benefits realized within our hospitalist system. Most of the issues specified at discharge tend to be followed up in the outpatient setting. While alerts about life-threatening drug-drug interactions are integrated into many medical information systems, few data are available regarding whether these warnings affect prescribing behavior or prevent patient injury. METHODS: We examined all consecutive cases of alerts warning of life-threatening drug-drug interactions during a six month period at a 700-bed academic tertiary care hospital. The alerts examined were the 7 with the most severe consequences from a database of known drug-drug interactions. Data were automatically collected each time a physician entered an order that triggered an interaction alert. The hospital information system logged the drug pair involved in the interaction, whether the alert was overridden, and any subsequent orders to discontinue one of the drugs in the pair. Adverse events attributable to the drug interaction were ascertained by chart review. RESULTS: Over 6 months, there were 168 life-threatening drug-drug interaction alerts (see table) . After 65(43%) of the alerts, physicians immediately discontinued one of the medications. For the 85(57%) alerts where both medications were given despite the alert, there were 2 adverse drug events, both involving the drug cisapride, and neither resulted in subsequent disability. CONCLUSION: Physicians overrode alerts about life-threatening drug-drug interactions more than half the time. When they did so, few adverse drug events occurred. These data suggest that the severity level of some drug-drug interactions may be set too high. For the small number of drug pairs that should never be given jointly, stronger safety measures should be put in place. people. Some plans claim that providing higher quality care raises costs and lowers profits, and that plans withdraw because they cannot sustain high quality care under current payment levels.
We performed a study to assess whether higher quality performance by Medicare managed care plans was associated with withdrawal. METHODS: Taking each county where a contract was active as a unit of analysis, this was a cohort study of Medicare managed care plans active in 2310 contract-county combinations in 1997 and followed for three years. We measured quality using fourteen indicators from the Health Employer Data and Information Set which we collapsed into four summary measures. We used a separate Cox proportional hazards regression for each indicator and each summary measure to assess the association of high quality care with time-to-withdrawal from Medicare while adjusting for market, organizational, environmental, and demographic factors. RESULTS: Of 2310 managed care contract-county combinations, 877 (38%) withdrew. The withdrawal rate among plans with high scores on a summary clinical quality measure was onefifth that for low scorers (4.2% v. 20.5%). For the summary ambulatory care access measure, the corresponding ratio was two-fifths (12.8% v. 32.0%). Lower payment rates were associated with higher withdrawal risk, contrary to plan claims, with higher clinical and ambulatory care access quality performance. In separate multivariable analyses, both high summary clinical performance (HR 0.18, 95%CI 0.08 ± 0.42), and high summary ambulatory care access performance (HR 0.53, 95%CI 0.27 ± 1.07) were independently associated with lower withdrawal risk. There was no independent association between either physician qualification or primary care provider stability measures and withdrawal. CONCLUSION: Managed care plans that continue to provide care to Medicare beneficiaries have higher average performance on clinical and ambulatory care access measures than plans that withdrew. System (BI-RADS) category 4 assessments (suspicious for malignancy) or category 5 assessments (highly suggestive of malignancy) were considered positive and in need of biopsy. These mammograms were matched to a subsequent biopsy obtained by fine needle aspirate, core biopsy, or surgery. We calculated the proportion of patients who had biopsies performed within 30 days, had biopsies within 1 to 12 months, and not biopsied within 12 months after the positive mammogram. RESULTS: 5,713 mammograms met eligibility criteria. 57.8% of mammograms were followed by biopsy within 1 month, 18.5% within 1 month to 12 months, and 23.8% did not have a biopsy within one year. There was a significant variation (p < .001) in time to biopsy by facility with a range of 33.5% to 77.5% of patients biopsied within one month and 9.3% to 60.0 % not having had a biopsy within one year. CONCLUSION: Though a majority of women received an expedient biopsy after a positive mammogram, the large proportion of women having a delayed biopsy or apparent lack of biopsy is of concern.
DELAY TO BIOPSY AFTER A POSITIVE MAMMOGRAM. R.G. Pinckney 1 , B.M. Geller 2 , M. Coahran 2 , P. Vacek 2 , B. Littenberg 1 ; 1 University of Vermont, Burlington, VT PURPOSE: Delay from detection to diagnosis has been associated with more advanced stage at diagnosis and decreased survival in women with breast cancer. We sought to determine the delay to biopsy after mammographic detection of suspicious lesions at a statewide level. METHODS: The Vermont Mammography Registry (VMR) collects information on all mammograms and breast pathology obtained in Vermont. Mammograms performed on Vermont women between 1/1/1994 and 8/31/1999 with Breast Imaging Reporting and Data System (BI-RADS) category 4 assessments (suspicious for malignancy) or category 5 assessments (highly suggestive of malignancy) were considered positive and in need of biopsy. These mammograms were matched to a subsequent biopsy obtained by fine needle aspirate, core biopsy, or surgery. We calculated the proportion of patients who had biopsies performed within 30 days, had biopsies within 1 to 12 months, and not biopsied within 12 months after the positive mammogram. RESULTS: 5,713 mammograms met eligibility criteria. 57.8% of mammograms were followed by biopsy within 1 month, 18.5% within 1 month to 12 months, and 23.8% did not have a biopsy within one year. There was a significant variation (p < .001) in time to biopsy by facility with a range of 33.5% to 77.5% of patients biopsied within one month and 9.3% to 60.0% not having had a biopsy within one year CONCLUSION: Though a majority of women received an expedient biopsy after a positive mammogram, the large proportion of women having a delayed biopsy or apparent lack of biopsy is of concern. The short-term follow-up of marginally abnormal mammograms presents a challenge to both physicians and patients. A variety of systems and individual factors may impact on the adherence to mammographers' recommendations. We sought to assess the extent of this problem and to examine the factors associated with the provision of appropriate follow-up care. METHODS: Among a sample of 579 women with abnormal screening mammograms or breast complaints, we analyzed the clinical data of 126 women who had marginally abnormal mammograms for which the mammographers recommended 6-month repeat follow-up mammograms. All women were contacted for detailed telephone surveys at the time of their index mammogram, and 7 to 8 months later. The medical records of these women were reviewed to determine the breast care they received before and during the follow-up period. Women who received follow-up mammograms as recommended, saw breast specialists, or underwent breast biopsies within the 7 ± 8 month follow-up period were considered to have received appropriate follow-up care. We then studied the differences between women who did and did not receive appropriate follow-up care April 1, 1996 and March 31, 1997 . Each discharge record contains up to 16 diagnoses, 16 corresponding diagnosis type indicators (value of`2' = complication), and up to 10 procedures. A clinically trained chart reviewer examined the corresponding medical charts for evidence of diagnoses and complications. A complication was defined as a new diagnosis arising after the start of hospitalization. We defined 12 common conditions using the ICD-9-CM coding system: stroke, cystitis/pyelonephritis, atrial fibrillation, respiratory failure, post-hemorrhage anemia, pneumonia, bowel obstruction, congestive heart failure, acute renal failure, myocardial infarction, atelectasis, and pleural effusion. We then selected records with presence of a condition in both administrative data and chart review data. Finally, we determined the extent to which the diagnosis type indicator in administrative data agreed with the chart reviewer's assessment (criterion standard) of whether a diagnosis was a complication or not.
RESULTS: Observed agreement for flagging complications using diagnosis type indicators was generally high between the data sources (agreement ranging from 47 to 97%, and agreement > 80% for 8 of the 12 conditions studied). However, kappa varied greatly across conditions studied (range: -0.06 ± 0.87) and was low at < 0.20 for 6 conditions. Specificity was generally high (range: 68 ± 100%) and was > 90% for 8 conditions, suggesting that pre-existing conditions were usually appropriately coded as such in the administrative data. In contrast, sensitivity was lower (range: 0 ± 83%), with sensitivity < 50% for 7 conditions, indicating a tendency for complications to often be miscoded as baseline comorbidities. CONCLUSION: The validity of diagnosis type indicators in Canadian administrative discharge data appears to be sub-optimal for some types of complications. This is likely to be of greatest concern in studies that rely on diagnosis type indicators to define complications as outcomes. PURPOSE: Recent data suggest that there are substantial variations in the treatment of acute myocardial infarction (AMI) based on age, race, gender and socioeconomic status. Specifically, the use of proven pharmacologic interventions and lifestyle modification counseling have been shown to vary by patient characteristics. We evaluated the use of post-AMI therapies and counseling in patients with AMI treated in an academic referral center with special attention to those receiving medical management alone for AMI. METHODS: We reviewed the records of a convenience sample of 384 patients with AMI from 1997 ± 2000. Our outcomes of interest included use of post-AMI therapies such as aspirin (ASA), beta-blockers (BB), angiotensin converting enzyme inhibitors (ACEI) and other anti-coagulants (AC). We collected variables including use of primary angioplasty (PA), defined as a cardiac procedure within twenty-four hours of admission, demographics, comorbidities, do-notresuscitate (DNR) status, physician characteristics, and severity of illness using the ATLAS system. Univariate analyses were performed on the variables of interest, and subsequent logistic regression modeling incorporated all variables significant on univariate analyses. RESULTS: Unadjusted analyses of pharmacologic therapies revealed that severely ill patients were less likely to receive any post-AMI therapies (all p < .05). Older patients were significantly less likely to receive both ASA and BB post-AMI (p < .05), but were not less likely to receive ACEI or AC. Race and gender were not associated with differences in any medication use. Conversely, patients who had undergone PA were significantly more likely to receive all medications (p < .05). Unadjusted analyses of tobacco and cholesterol counseling, defined as any chart reference to tobacco use in current smokers and defined as any chart reference to serum cholesterol in all AMI patients, was significantly more likely to occur if one underwent PA, and significantly less likely to occur as severity of illness increased (p < .05). After adjustment for potential confounders, patients who underwent PA were significantly more likely to receive proven post-AMI therapies and counseling (p < .01), and this increased likelihood persisted across increasing levels of severity of illness. CONCLUSION: Our results indicate significant differences in the use of post-AMI pharmacologic therapies and counseling correlated with the use of primary angioplasty for AMI, despite evidence of proven benefit in AMI in patients undergoing medical management alone. These differences are not explained by severity of illness and suggest that standard therapies may be withheld from those who may benefit most. in the diagnosis and treatment of depression, PCC success in this area is problematic, and direct patient access to mental health care has been proposed as a solution. The purpose of these analyses is to learn how often PCCs in two large healthcare organizations that allow direct patient access to mental health services discuss depression diagnosis or treatment with depressed outpatients, and the relationship between PCC discussion and treatment rates. METHODS: We screened 19,793 consecutive patients attending one of nine primary care practices in one of two staff model managed care organizations in California: three practices based in a single academically-affiliated VA medical center and six in separate Kaiser Permanente (KP) medical centers. Of the 597 who met full criteria for current major depression by diagnostic interview and met other study inclusions criteria, 567 completed a 40-minute telephone-administered survey assessing their mental health care over the preceding six months.
Measures include PCC discussion of depression (the PCC asked about depression or gave the patient a treatment for depression or emotional problems at the last visit); recent depression treatment (the patient took an antidepressant or had at least 4 mental health specialty visits in the past 6 months); and high or low depression symptoms by CESD score. Significance is by Chi-Square. RESULTS: Of 567 patients with major depression, 414 (73%) had been recognized as depressed (64% HMO, 76% VA) as indicated either by PCC discussion of depression (333 or 59%) or by recent depression treatment in the absence of PCC discussion (81 or 14%). A total of 290 of the 567 (51%) reported recent depression treatment; treatment rates were significantly higher among the 333 patients who had discussed depression with their PCC than among the 233 who had not (63% vs 35%, p < .001). Excluding patients reporting recent treatment, PCCs who discussed depression were more likely to assess suicidality, mania, and alcoholism. Among the 567 patients, those whose PCC discussed depression were more likely to be treated with an appropriate antidepressant (35% vs. 18%, p < .001), and to visit mental health (51% vs 33%, p < .001). Results comparing treatment for patients with PCC discussion and those without remained similar and significant when analyzed separately for patients with high or low CESD scores. CONCLUSION: In two organizations with direct primary care patient access to mental health specialty, discussion with PCCs remains an important determinant of depression treatment quality. These findings support the importance of PCC recognition of depression, and the need for better integration of mental and physical health care. There are limited data about minority physicians' satisfaction with their professional lives. Previous studies suggest that among academic physicians, minorities may be less satisfied than whites. In this study, we describe, by race and ethnicity, satisfaction and job stress among a national sample of physicians. METHODS: We analyzed data from 2,326 respondents to a career satisfaction survey of physicians drawn from the AMA Physician Masterfile. Scales (from 1 ± 5) measuring overall job satisfaction, career satisfaction, and work-related stress were constructed from Likert-response items and tested using factor and reliability analyses. We examined the association between physician ethnicity and each of these scales. We also examined how the proportion of patients from ethnic backgrounds other than the physician's own affected satisfaction and job stress. RESULTS: Respondents included 56 black, 136 Hispanic, 409 Asian, and 1,651 white physicians. Black and Hispanic physicians, as compared with others, cared for higher proportions of minority, non-English speaking and uninsured patients. Scores for job and career satisfaction and stress by ethnicity are displayed in the table. Higher scores indicate higher satisfaction or stress, and p-values refer to comparisons of the highest and lowest scores. After adjusting for physician age, gender, practice setting, specialty, and patient panel characteristics (proportion of patients who were female, elderly, non-English speaking, medically complex, psychosocially complex, or substance-abusing), Hispanic physicians had higher job and career satisfaction than all other groups. The proportion of physicians' patients from ethnic groups other than their own was associated with lower job satisfaction and higher stress (p < .001). In adjusted analyses, these findings persisted but were most consistent among white and black physicians. CONCLUSION: Hispanic physicians were more satisfied with their jobs and careers than physicians from other ethnic groups. For white and black physicians, caring for ethnically dissimilar patients was associated with lower job satisfaction and higher stress. Future study should investigate how physician cultural factors and cross-cultural relationships with patients affect physician worklife and professional satisfaction. Family physicians were more likely (OR 1.254, 95% CI 1.253 ± 1.255) to see patients for acute problems unrelated to chronic medical conditions. Family physicians performed more pelvic exams (OR 1.447, 95% CI 1.446 ± 1.448) and Papanicolaou smears (OR 1.391, 95% CI 1.390 ± 1.392), and more frequently provided counseling on smoking cessation, mental health, injury prevention, family planning and sexually transmitted diseases (p < 0.001 for all). Family physicians were more likely to prescribe medication at patient visits (OR 1.229, 95% CI 1.228 ± 1.230) than internists, but internists more frequently ordered advanced radiologic tests such as CT, MRI, and ultrasound (p < 0.001 for all) even after adjustment for age, gender, and number of ICD-9 diagnoses. CONCLUSION: Significant differences exist between ambulatory practice patters of primary care physicians independent of patient age and gender.
LAB-PHARMACY LINKS: LEGAL LABELING LANGUAGE OVERLOAD. G. Schiff 1 , G. Shah 1 , H. Lubin 1 , D. Klass 1 , D. Bates 2 ; 1 Cook County Hospital, Chicago, IL; 2 Brigham and Women's Hospital, Boston, MA PURPOSE: Lab and pharmacy are intimately related. However their departments, functions and information systems are often disconnected. The extent of these clinical interrelationships, and expectations upon prescribing physicians to remember critical information (warnings, toxicity, monitoring) is vast and taxes the memory of busy clinicians. A number of drugs have recently been withdrawn because of operational failure in meeting such expectations (eg troglitazone-hepatic toxicity monitoring). To better understand the extent of such information, and because the Physicians Desk Reference (PDR) is the most widely used drug information source and represents legally-mandated prescribing labeling for drugs marketed in the U.S, we analyzed the content of tab-pharmacy interactions and warnings for frequently prescribed and recently approved drugs. METHODS: We developed a taxonomy of critical lab-pharmacy interaction categories, and reviewed all solid oral medications for the most frequently prescribed drugs (N = 40), plus new molecular entities (NME's) marketed in 1998 ± 99(N = 37). We conservatively counted multiple warnings as a single test whenever possible (eg thrombocytopenia, anemia, leukopenia = CBC). RESULTS:
CONCLUSION: Officially labeled lab-pharmacy interactions/warnings are frequent±totaling 249 (6.2/drug for commonly prescribed drugs, and 238 (6.4/drug) for NME's)±a number that exceeds the unaided prescribers' abilities to remember and monitor. Automated screening and reminders will need to be programmed into routine prescribing processes to prevent errors related to such interactions. A noteworthy discrepancy exists between number of toxicities and recommended monitoring, and relatively few drugs (especially NME's) appear to have lab interference evaluated, suggesting a need for additional research to ensure safe prescribing and error-free lab test interpretation. To assess racial disparities in influenza vaccination among Medicare beneficiaries and evaluate whether or not the magnitude of racial disparities is smaller for those enrolled in managed care than among those with fee-for-service insurance. METHODS: We analyzed 13,674 responses to the 1996 Medicare Current Beneficiary Survey of African American and white Medicare beneficiaries with managed care and fee-for-service insurance. We compared sociodemographic characteristics and health attitudes among the groups, the proportion receiving influenza vaccination, and differences in racial disparities in influenza vaccination between those with managed care and fee-for-service insurance. Using a propensity model based on sociodemographics, comorbid illnesses, attitudes toward health care, and their interactions with race, we adjusted for differences in the two insurance groups and compared the adjusted racial disparities. RESULTS: Eight percent were African American. Eleven percent were enrolled in managed care. On average, African Americans and fee-for-service enrollees were older, had worse health status, had a higher prevalence of diabetes, and were more likely to avoid doctor visits. Overall, 65.8% of Medicare beneficiaries received influenza vaccination. Whites were much more likely than African Americans to receive influenza vaccination (67.7% vs. 45.1%, absolute disparity = +21.6%; 95% C.I. 18.2% to 25.0%). Managed care enrollees were more likely to receive influenza vaccination than those with fee-for-service insurance (71.2% vs. 65.4%, difference = +5.8%; 95% C.I. 3.3% to 8.3%); however racial disparities in influenza vaccination were nearly identical among beneficiaries both with managed care and with fee-for-service insurance. These disparities were not significantly different after adjustment for propensity to enroll in managed care. CONCLUSION: While managed care is associated with higher rates of influenza vaccination, we found no evidence that managed care reduces racial disparities in influenza vaccination. Our results suggest that health plans are not yet adequately addressing racial disparities in the use of preventive services.
TEACHING HOSPITAL. J.L. Schnipper 1 , R.S. Stafford 1 ; 1 Massachusetts General Hospital, Boston, MA PURPOSE: To determine the extent to which outpatients with coronary artery disease (CAD) receive adequate secondary prevention measures, and to identify the factors that predict variations in the provision of these services. METHODS: Cross-sectional study of 3920 patients with coronary disease (ICD-9 codes 410.0 ± 414.99) who were seen in one of ten outpatient practices affiliated with Massachusetts General Hospital (MGH) during fiscal year 1997. Demographic and laboratory information were collected on all patients, and a medical record review was performed on a subset of patients to confirm CAD and to determine blood pressures, medical conditions, and medications during fiscal years 1997 and 1998 (N = 302). RESULTS: Our study showed evidence of variable short-comings among different CAD prevention strategies. 17% of patients never received a low density lipoprotein (LDL) cholesterol test during the study period, and only 19% of patients had an LDL level 100 mg/ dL. Among those with an LDL ! 130 mg/dL, 41% received no lipid-lowering medication and only 5% were on a standard maximum dose of an HMG CoA reductase inhibitor (statin). The average of the last two blood pressure readings was less than 130/85 mm Hg in 42% of patients, and 53% of patients met JNC VI guidelines for blood pressure control. Among hypertensive patients, 94% were on some type of anti-hypertensive medication. 75% of all patients were prescribed a beta-blocker during the time period, and 86% were prescribed aspirin or another antithrombotic medication.
In multivariable models adjusted for several patient and physician characteristics, having an LDL < 130 mg/dL was less likely for patients in the oldest age quartile (odds ratio (OR) 0.59, 95% confidence interval 0.49 ± 0.72), female patients (OR 0.60 [0.51 ± 0.69]), those without insurance coverage of medications (OR 0.54 [0.37 ± 0.79]), and those whose type of insurance indicated low socioeconomic status (OR 0.85 [0.70 ± 1.01]). Older patients and women were less likely to have a blood pressure < 140/90 mm Hg. Younger age was of borderline significance in predicting beta-blocker use. CONCLUSION: At outpatient practices affiliated with a major academic medical center, there is still much room for improvement in the secondary prevention of CAD. Deficiencies were greatest in the area of lipid management. Lipid management also was the most susceptible to practice variation on the basis of socioeconomic status. Older patients and women also were less likely to receive adequate secondary prevention. Interventions such as physician education, computerized reminder systems, and population management may be effective in correcting some of these shortcomings.
FOR-PROFIT HOSPITALS AND DRG``UPCODING''. E. Silverman 1 , J. Skinner 2 ;
1 Dartmouth Hitchcock Medical Center, Lebanon, NH; 2 Dartmouth College, Hanover, NH PURPOSE: One large for-profit hospital chain has admitted to inflating the severity of discharge diagnoses (``upcoding'') for economic gain. It is unknown how widespread this practice is, or whether it is endemic to for-profit hospitals. We examined the association between for-profit hospital ownership and upcoding for Medicare discharges for pneumonia, the most widely recognized condition under which upcoding seems to occur. METHODS: To analyze time trends in coding patterns, we used a 20 percent national sample of Medicare inpatient claims for 1989 through 1997 with the hospital as the unit of analysis. Hospital ownership was defined as for-profit, not-for-profit, or government based on the American Hospital Association's Annual Survey of Hospitals for each year. The relevant diagnosis related groups (DRGs) were for simple pneumonia with and without complications (DRGs 89 and 90), and for complex pneumonia with and without complications (DRGs 79 and 80). The dollar reimbursement for complex pneumonia is approximately 50 percent higher than for simple pneumonia. For each year and ownership group we calculated an upcoding ratio defined as the percentage of disharges coded for complex pneumonia out of the total discharges for simple and complex pneumonia combined. RESULTS: Crude upcoding ratios for pneumonia were highest among for-profit hospitals for each year between 1989 and 1997. In addition, between 1989 and 1996, the upcoding ratio increased from 0.20 to 0.30 in not-for-profit hospitals, 0.21 to 0.30 in government hospitals, and from 0.28 to 0.51 in for-profit hospitals. Between 1996 and 1997 the ratio fell slightly for each group, which corresponds to a period of great publicity and legal charges of fraud. Average thirty-day mortality rates for the DRGs under consideration were similar across ownership categories, and in fact declined over the study period, suggesting that differences in the percentage of pneumonia discharges coded as complex was not related to case-mix. CONCLUSION: For-profit hospital ownership was associated with higher upcoding ratios among Medicare discharges for pneumonia than in not-for-profit hospitals. PURPOSE: Physicians' attitudes are important because of the possible impact they may have on the structure and quality of patient care [1] . In 1982 Mathews et al [2] , surveyed San Diego County Medical Society's physicians about their attitudes toward homosexuality. They found significant differences in prevalence of homophobic attitudes by gender, year of medical school graduation, specialty and practice setting [2] . Our objective was to assess physicians' attitudes toward homosexuality and toward persons with HIV infection and to compare the current prevalence of homophobia to that measured seventeen years previously. METHODS: An anonymous, self-administered, 17-item survey was mailed to all 4,385 members of the SDCMS and 1,271 UCSD physicians. The survey included 8 likert-scaled items measuring general attitudes toward homosexuality (Cronbach's alpha = 0.88). Additional items assessed attitudes toward entry to medical school and consultative referral patterns, conditional on sexual orientation and HIV status of hypothetical referents. PURPOSE: Somatization is a common costly problem with great morbidity, but there has been no effective screening method to identify these patients and target them for treatment. We tested an hypothesis that we could identify high utilizing somatizing patients from a management information system (MIS) by total number of visits and what we termed`s omatization potential,'' the percentage of visits for which ICD-9 primary diagnosis codes represented disorders in the musculoskeletal, nervous, or gastrointestinal systems or ill-defined complaints. METHODS: We identified high utilizing patients from the MIS of a large staff model HMO as those having 6 or more visits during the year studied (65th percentile). A physician rater then reviewed the medical records of these patients and identified somatizing and nonsomatizing patients. In two-thirds of the population (the derivation set), we used logistic regression to refine our hypothesis and identify predictors of somatization available from the MIS: demographic data, all medical encounters, and primary diagnoses made by usual care physicians [ICD-9 codes]. We then tested our prediction model in the remaining one-third of the population (the validation set) to validate its utility. RESULTS: The derivation set contained the following significant correlates of somatization: gender, total number of visits, and percent of visits with somatization potential. The c-statistic was .90. In the validation set, the explanatory power was less with a c-statistic of .78. A predicted probability of .04 identified almost all somatizers, while a predicted probability of .40 identified about half of all somatizers but produced few false positives. CONCLUSION: We have developed and validated a prediction model from the MIS that helps to distinguish chronic somatizing patients from other high utilizing patients. Our method requires corroboration but carries the promise of providing health plan directors with an inexpensive, simple approach for identifying the common somatizing patient and, in turn, targeting them for treatment. The screener does not require clinicians' time. PURPOSE: High utilizing patients with medically unexplained symptoms often are believed to be somatizing patients, defined here as no organic disease explanation for symptoms of at least 6 months duration. High utilizing patients with unexplained symptoms of shorter duration have not been studied and there is no effective treatment for this group, which we call``minor acute illness.'' METHODS: Our aim was to determine how many high utilizing patients with medically unexplained symptoms met our chart-rating criteria for somatization and or minor acute illness and what the stability of these diagnoses was over time. We performed chart review at baseline; one and two years later, we re-rated the charts of patients initially rated as having somatization as well as a 15% sample of those with minor acute illness. Subjects were a random sample of high utilizing staff model HMO patients (6 or more visits/year) from 21 ± 55 years old, identified from the management information system (MIS). Chart review designations as organic disease, somatization, or minor acute illness were the measurements made. RESULTS: Among 883 high utilizing patients, 35% had organic diseases, 14% had somatization, and 51% had minor acute illness as their primary problems. No patients with initial minor acute diagnoses were reclassified as somatization one and two years later and all but 2 patients had minor acute illness in one or both follow-up years. Among somatizers, 54% were re-classified as minor acute illness the following year and, of these, 50% remained as minor acute illness in the second follow-up year. 70% of initial somatizers and 45% of initial minor acute patients continued to be high utilizers in one or both follow-up years. Persistent high utilization in both follow-up years occurred in 17% of minor acute patients and33% of somatizing patients. CONCLUSION: Minor acute illness appears more common among high utilizing patients than somatization and organic diseases combined. It has not previously been studied. Diagnoses of somatization were unstable over two years follow-up while minor acute diagnoses were stable, supporting the latter as a valid entity. Utilization did not remain high in either group. We recommend further study of minor acute illness to determine its relationship to somatization, to determine if it is a valid entity, and to better understand the broad spectrum of patients with medically unexplained symptoms.
AVOIDANCE OF REGRET AS A MOTIVE FOR ORDERING PROSTATIC SPECIFIC ANTIGEN TESTS. P.C. Sorum 1 , E. Mullet 2 , G. Chasseigne 3 , S. Bonnin-scaon 2 , J. Shim 4 , J. Cogneau 5 ; 1 Albany Medical Center, Schenectady, NY; 2 Ecole Pratique des Hautes Etudes; 3 Universite  Franc Ëois-Rabelais, Tours; 4 University at Albany, Albany, NY; 5 MG France, St Avertin PURPOSE: When making management decisions, physicians try to avoid the regret that would be provoked by unwanted consequences of their actions or inactions. We explored the strength and determinants of such anticipated regret in a study of decisions to order prostatic specific antigen (PSA) tests for hypothetical patients. METHODS: 32 U.S. and 33 French primary care physicians looked at 32 patient scenarios and estimated the probability each patient had prostate cancer and the likelihood the participant would order a PSA. They were then given 12 scenarios and were told to suppose they had performed routine exams on these patients, had not ordered PSAs, and now several years later found advanced prostate cancer in these patients. They were asked to indicate how much regret they would feel that they had not ordered PSAs. The 12 hypothetical patients differed according to age (55, 65, or 75) , the request to be screened for prostate cancer (no or yes), and the shape of the prostate on the earlier rectal exam (no irregularity or some irregularity). The assessments of regret were analyzed by ANOVA (with significance set at p < .02). For each group of physicians, a correlation was calculated between the mean rating for regret and the mean likelihood of ordering a PSA. RESULTS: The regret scores were correlated with the likelihood of ordering PSAs for both the French (r = .64, p = < .005) and the Americans (r = .42, p < .02). The level of regret was higher when the patient had requested a PSA, when the shape had some irregularity, and when the patient was younger. When the patient had requested a PSA, the shape of the prostate had less effect than when the patient had not requested it. The U.S. physicians indicated a higher level of regret than did the French. A patient request had a greater impact on U.S. than on French physicians. Increasing patient age reduced regret more among the French than among the Americans. CONCLUSION: Anticipated regret over a failure to have diagnosed an aggressive prostate cancer is correlated with a policy of ordering of PSAs. The extent and determinants of this regret appear to be culturally sensitive. PURPOSE: Several studies have suggested that a central medical organizational structure can influence processes and outcomes, for example with audit and feedback, economic incentives, and implementation of guidelines. One form of organizational structure is clinical structure, the application of organizational policies which, in aggregate, allow medical organizations to improve the efficiency of care and outcomes delivered to cohorts of patients. We used literature review supplemented by clinical and administrative input to design a model of clinical structure which was tested in a 45 minute telephone survey by physician interviewers of medical directors of 56 medical organizations. METHODS: Responses were obtained from the directors of 53 medical organizations (response rate 95%) providing data from 29 medical groups (MGs) and 24 independent practice associations (IPAs) from 3 west coast states. The survey queried directors about whether their organization had in place corporate-wide policies to standardize clinical operations across the offices and providers that define their corporate entity. The survey asked about each of 30 dimensions of clinical corporate structure distributed across 183 individual survey items We considered an organization to have corporate policy (CP) if the respondent indicated CP for at least one item associated with a dimension of clinical structure. RESULTS: There was substantial variation across organizations in the extent to which the directors reported corporate policies. On average, organizations had CP for 49% of the 183 items across 30 dimensions of corporate structure. The clinical corporate policies most frequently cited by respondents pertained to the use of clinical guidelines (94%); specifications regarding the exact content of the ambulatory medical record (92%), and whether or not prospective review was required prior to specialty consultation (91%). Organizations infrequently cited having clinical corporate policies for assigning new patients to providers according to clinical need (19%), for office staff counseling patients following MD visits regarding next steps (21%), and use of multidisciplinary team meetings (25%). Of the 30 dimensions of clinical structure studied, CP was more prevalent in MGs than IPAs (p < .05) for 17 (57%) dimensions. For example, 66% of MGs and 33% of IPAs reported CP regarding written educational materials being available to patients (p < .05). CONCLUSION: While some medical organizations have developed sophisticated systems to organize the delivery of clinical care to cohorts of patients using corporate administrative policies, most have not. For most dimensions for which the literature suggests care could be improved with organizational support, most organizations, especially IPAs, do not report corporate policies in place.
not accept a diagnosis of CHF based upon ejection fraction alone. Thus, it is important for both clinical and research purposes to estimate the agreement between quantitative measurements and the cardiologists' qualitative interpretation reported on echo METHODS: All patients at a university-affiliated VAMC who had an echo between 12/96 and 12/99 were identified by electronic medical records (N = 5,505). One record per patient was identified and for each echo, we abstracted both quantitative (fractional shortening and fractional area change) and qualitative (cardiologists' interpretation) data. We excluded reports with missing quantitative (N = 912) or qualitative (N = 85) data. Abnormal numeric data was defined as fractional shortening less than 0. 1 8 or fractional area change less than 0.35. Abnormal textual report was defined as left ventricular systolic dysfunction or generalized wall motion abnormality. We then calculated agreement from a 2Â2 table. RESULTS: Of 4,508 patients with echos in our study, 1237 had an abnormal echo by quantitative and/or qualitative data (Table) . Of these 1237 patients, an abnormality was identified by both strategies in only 548 (44%) of patients. Discrepancies rates between qualitative and quantitative reports were similar. The cardiologists' interpretation of the echo was normal when the numeric data were abnormal 41% (386/937) and the cardiologist's interpretation was abnormal with normal numeric data 35% (303/851).
CONCLUSION: There is significant discrepancy between qualitative and quantitative strategies to diagnose CHF, which may explain non-compliance with AHRQ recommendations. Physicians who use either standard alone, risk undertreating or overtreating patients which may lead to morbidity. We conclude that CHF guidelines must develop a clinically acceptable way of identifying patients with CHF in order to insure that appropriate care is delivered and that patient outcomes are maximized. PURPOSE: Studies examining some of the clinical and non-clinical determinants of outcomes of acute myocardial in the VA suggest that age; race, co-morbid illnesses. and availability of cardiac surgical services all play a role. However, few data are available on the regional variation in health care utilization and outcomes during and after acute myocardial infarction. The purpose of our study was to examine regional variation in utilization and outcomes during the acute and chronic care of patients with acute myocardial infarction (AMI). Examining these differences will lead to a better understanding of potentially modifiable variables and processes of care that influence patient outcomes METHODS: Using national VA databases, we identified all veterans who were hospitalized for AMI at any VA Medical center between 10/90 to 10/97. Demographic, inpatient, outpatient, mortality and readmission data were extracted from the VA's administrative databases for the four regions: Northeast, South, Midwest and West. Multivariable Cox proportional hazards models, controlled for comorbidity, were used to assess the effect of independent variables on time to death and readmission. RESULTS: We identified 67,889 patients with AMI who were 98% male at mean age of65 years. There were no significant regional differences in patients' demographic data. Patients in the Northeast had a greater prevalence of comorbid conditions and longer lengths of stay during the index AMI hospitalization (14 days in the NE Vs 10 days in the W). There was no variation in the rates of cardiac procedures (PTCA and/or CABG) among the regions during the index admission. Region of the country was an independent predictor of mortality. Subsequent adjusted all-cause mortality after discharge from the index hospitalization was lower in the Northeast (OR = .910 CI .86 ± .95) and the West (OR = 0.871 CI .87 ± . 95). Patients in the Northeast and West had greater cardiology or primary care follow up at 60 days and 1 year post discharge than patients in the South. Outpatient follow up appeared to account for a substantial amount of the regional variation in all-cause mortality.
CONCLUSION: There was substantial geographic variation in clinical care and outcomes among veterans with AMI. Outpatient follow-up was highly variable and associated with mortality. Further studies are needed to determine the most effective strategies for improving outcomes after AMI. METHODS: We recently described a new, valid, and reliable instrument called QUEST, which has 4 scales measuring patients' perceptions of quality and their satisfaction with care by physicians (MDs) and nurses (RNs). We administered QUEST and other standardized instruments to 84 medical inpatients (45 terminally ill and 39 comparison) at 2 hospitals. We used correlations, t-tests, and ANOVA to test associations between sociodemographic/clinical factors and QUEST scores. RESULTS: Mean patient age was 60, APACHE-III physiology score 16.6; 71% were white, 60% men, 54% Catholic, 28% Medicaid/uninsured, 38% had cancer or HIV, 41% moderate/ severe pain, 19% depressed, and 58% private (PVT). Age, APACHE, race, gender, religion, insurance, and diagnosis were unassociated with quality or satisfaction. Quality and satisfaction scores for MDs were lower among``service'' (SERV) than PVT patients and lower among terminal than non-terminal patients. Quality and satisfaction scores for RNs were lower among SERV patients and depressed patients. P-values for ANOVA models for QUEST were: MD Quality = .01, MD Satisfaction (adjusted for pain) = .007, RN Quality = .16, RN Satisfaction = .002. CONCLUSION: Terminally ill SERV patients rated MD quality and satisfaction lower than PVT patients and lower than SERV patients not terminally ill. RN satisfaction scores were high only among PVT patients who were not depressed. Understanding these apparent differences in end-of life care will require further investigation. PURPOSE: At the end of life, when cure is no longer possible, perhaps the most important medical intervention that can be offered is time. However, almost nothing is known about the time health professionals spend with dying inpatients. METHODS: We asked day-shift nurses to use a diary to record how much time they spent in various activities for 47 medical inpatients at 2 teaching hospitals (25 terminally ill patients with Do Not Resucitate (DNR) orders and 22 comparison patients). To minimize Hawthorne effects, nurses were told to record multiple categories of activities and were only informed that this was a``time-motion study for seriously ill patients.'' This method has been validated against video-recordings of actual time spent in the room (r = .70, p < .0001). Non-English speaking patients, those whose clinical condition prohibited interview, and those with a reduced set minimental status (MMS) score indicating dementia were excluded. Charts were reviewed and patients were interviewed using a battery of standardized instruments. Because nursing time was not normally distributed, we used log-transformed time as our dependent variable. We then used t-tests, correlations, and ANOVA to test associations between sociodemographic/clinical data and bedside nursing time. RESULTS: The mean patient age was 59, and the mean APACHE-III physiology score 16.3; 64% were white, 19% African-American, 11% Latino, and 6% Asian; 60% were men, 39% Protestant, 41% had cancer or HIV, and 23% were depressed. The mean amount of time nurses spent with patients per day shift was 27 minutes. In bivariate analyes, age (p = .23), APACHE-III physiology score (p = .78), gender (p = .81), religion (p = .27), diagnosis (p = .99), and DNR status (p = .26) were unassociated with nursing bedside time. Nurses spent more time at the bedsides of patients with lower reduced-set MMS scores (indicating worse mental function (r = .26, p = .08)), more time with white patients compared with non-white minorities (31 minutes vs. 22 minutes, p = .08), and more time with depressed compared with non-depressed patients (43 minutes vs. 23 minutes, p = .02). In an ANOVA model, lower reduced-set MMS scores (p = .02), white race (p = .02), depression (p = .15), and a race-depression interaction term (.045) were associated with more nursing bedside time. CONCLUSION: In this population, nurses spent more time with patients who had worse mental function (even after excluding those who were frankly demented), and more time with white patients who were depressed compared with both non-depressed white patients and minority patients regardless of depression score. DNR orders were not associated with less nursing contact. While some of these results appear to suggest unexpected differences in the time nurses give to patients, firm conclusions will require further study. Ryukyu University Hospital, Nakagami, Okinawa PURPOSE: General internal medicine (GIM) as an academic discipline was recently introduced to medical schools in Japan. Residency programs are still quite young and fully trained primary care physicians are in shortage. In May 1999, the Ryukyu University Hospital started a general medicine clinic to provide better care for its patients. However, since there were no staff trained in GIM, specialists from the Department of Medicine and Department of Psychiatry were assigned to the clinic. We conducted two surveys to assess the attitudes toward and perceptions of GIM practice among specialists at different academic disciplines in the hospital. METHODS: First a survey was carried out using a questionnaire that was distributed to all physicians who work at the Ryukyu University Hospital. We also conducted a 2nd survey which focused on the experience at the GIM clinic for specialty physicians who had ever practiced at the clinic. RESULTS: Overall response rates were 66.3% (n = 236) and 75% (n = 48), respectively. The clinic performance over the first year was evaluated and rated from À3 (extremely poor) to +3 (excellent). Respondents whose specialty was not in internal medicine perceived the GIM clinic more positively (average +1.0) compared to internists with and without GIM clinic experience(average À1.9 and À1.6, respectively), which reached statistical significance (p < 0.001). Among residents, fellows and faculties, there was no statistical difference regarding attitudes toward the competence of primary care physicians, encouragement and positive regard for GIM. Eighty percent of internists who had practiced at the GIM clinic agreed that their clinical competencies as a specialist were not sufficient to be a primary care physician. 92% of them disagreed with the hospital's continued operation of the GIM clinic under current administration, however, if GIM trained physicians were stationed at the clinic, the same number of internists agreed that the clinic would play an important role at their hospital. Psychiatrists were more willing to cooperate with GIM clinic compared to other specialties. CONCLUSION: Historically, the culture of academic health centers had not been hospitable toward primary care. However, our survey demonstrated that the chilly climate has been changing. Only very few physicians perceive primary care tasks as not requiring a high levels of expertise. In many schools, increased attention to education for primary care practice is evident in Japan. A more generalist workforce with competence and expertise in primary care is essential. PURPOSE: Previous work has demonstrated that African American elders experience higher functional and cognitive disability than White elders. The Program of All-Inclusive Care for the Elderly (PACE) provides comprehensive community-based long-term care services for nursing home eligible elders who remain in the community. Once enrolled patients have equal access to all needed medical and long-term care services at no additional cost. We examined the impact of enrollment in the PACE system on mortality differences between African American and White patients. METHODS: 2002 White patients and 859 African American patients enrolled at 12 nationwide PACE sites from 1990 ± 1996. All patients were followed from enrollment through death, or 12/31/97. On enrollment, measures of demographic characteristics, functional status, and comorbid medical diagnoses were obtained by social workers, nurses, and physicians. We used the method of Kaplan-Meier to describe mortality rates in African American and White patients. We used the log-rank test and Cox-proportional hazards models for bivariate and multivariate comparisons of mortality rates for African American patients vs. White patients. RESULTS: On enrollment into PACE, African American patients were younger than White patients (77.4 v 79.6, P < .01). However, African American patients had worse functional status (mean score on 10 point ADL scale, 6.5 v 7.2, p < .01) and were more likely to be diagnosed with dementia (56% v. 45% p < .01). 74% of both African American and White patients were women. A survival advantage for African Americans emerged after 9 months of enrollment in PACE.
Rates of mortality at 1, 2, and 3 years after enrollment were 13%, 24%, and 33% for African American patients and 16%, 29%, 39% for White patients (HR for African American patients = 0.76; 95% CI 0.66 ± 0.88). After adjustment for PACE site, age, gender, education, caregiver support, ADL function, presence of dementia and other comorbid conditions, African American patients continued to have a lower rate of mortality than White patients (HR = 0.79; 95% CI 0.66 ± 0.95). CONCLUSION: In a system providing equal access to comprehensive medical and long-term care services to frail elders, African American patients have substantially lower rates of mortality than White patients after adjustment for age, comorbidity, and ADL function on enrollment.
POTENTIALLY AVOIDABLE HOSPITALIZATION DAYS: A PROSPECTIVE ANALYSIS. A. Torn 1 , A. Pichon-Riviere 2 , C. Romero 1 ; 1 Sanatorio de la Trinidad, Capital Federal, Buenos Aires; 2 Programa de Efectividad Clinica -Argentina, Capital Federal, Buenos Aires PURPOSE: Analyze the distribution and causes of the potentially avoidable additional hospitalization days (PAAD) in an institution that has a hospitalist program. METHODS: From 06/99 to 01/00 the surgical and medical admissions of adult patients to a private hospital in Buenos Aires, Argentina were followed prospectively to detect PAAD. The additional days were defined using the Milliman and Robertson's Optimal Recovery Guidelines. The case management nurse assigned a cause from a set of 16 possibilities to each additional day and categorized the admissions in 5 severity levels. RESULTS: There were 1812 medical and surgical admissions of adult patients; 626 (34.55%) were medical and 1186 (65.45%) surgical from which 27.4% were emergency and 72.6% programmed procedures. The mean age was 56.3 years with a 25% of the patients older than 74 years; 58.5% were women. The average length of stay (ALOS) was 4.1 days with a median of 2.1; 5.8 for the medical admissions and 3.2 for the surgical ones. The difference between the programmed and emergency operations was significant: 2.6 vs. 4.9. There was also a significant increase of the ALOS with categories of age and severity. The inpatient mortality was 3.5%, 93.4% were discharged and 3% were transferred to a rehabilitation center or to home care. A total of 723 PAAD, representing a 9.64% of the hospitalization days, were generated by 600 admissions. The physicians were responsible of 668 PAAD (92.40%). Inappropriate admission (potentially ambulatory procedures), was the cause of 392 (54.22%) PAAD, 181 (46.17%) were due to gynecological procedures, 73 (18.62%) to general surgical and 62 (15.82%) to traumatological ones. Prolongation of hospital stay was the cause of 331 (45.78%) PAAD divided in 55 days (16.62%) attributed to the health care system and 276 (83.38%) to the physicians. Of the latter 106 (38.40%) were responsibility of traumatology and 82 (29.71%) of gynecology. A logistic regression model adjusted for severity, age, sex, type of admission and specialty was analyzed. The following specialties were positive predictors of PAAD compared to general surgery: gynecology (OR 7.84), traumatology (OR 2.75) and neurosurgery (OR 4.58) . Emergency surgery (OR 0.61) was a negative predictor of PAAD and age (OR 1.01) was a positive one. CONCLUSION: Even though this study was conducted in an institution where a hospitalist program is in place, there are still a significant number of potentially avoidable additional days. They are concentrated in programmed surgical procedures mainly gynecological and orthopedic, and in the lower levels of severity. PURPOSE: Primary care physicians (PCPs) are often required to act as gatekeepers and or patient advocates for mental health services in an extremely complex administrative environment that includes managed care organizations with and without mental health carve-outs. This analysis addresses two aspects of this environment: (1) number of managed care contracts per practice (2) percentage of patients for whom the physician serves as managed care gatekeeper. METHODS: Data from the 1996 ± 97 Physician Community Tracking Study performed by the Center for Studying Health System Change were analyzed. This cross-sectional survey consisted of telephone interviews of 12,385 physicians (5,583 primary care physicians caring for adults) and had a response rate of 65 percent. Access to high quality mental health services was defined as PCPs indicating that they always or almost always could obtain high quality mental health services for their patients. RESULTS: Perceived availability of high quality services was much lower for outpatient mental health than for outpatient medical specialists (30% vs. 81 %, p-value < 0.001). Perceived access to high quality outpatient mental health services declined with increasing numbers of managed care contracts: zero (44%, Odd Ratio 2.3, CI 1.8, 3.1), one (37%, OR=1.8), two to ten (27%, OR=1.1), eleven to twenty-five (29%, OR=1.2) and more than twenty five (25%, OR=1.0). These findings persisted after adjustment for patient demographics (Medicare percentage, Medicaid percentage), physician demographics (age, gender, foreign vs. US medical graduate and overall career satisfaction) and practice characteristics (type of practice and concern about financial penalties for practice decisions) using multiple logistic regression analysis. An adjusted OR of 1.9 (p-value of < 0.001) was observed for zero contracts compared to twenty-five or more. A U-shaped distribution in perceived access was observed for the percent of patients in the practice for whom physician served as gatekeeper: three percent or fewer patients in gatekeeper status (42%, OR 2.6), more than ninety percent of patients (43%, OR 2.6) and fiftytwo to sixty percent of patients were in gate-keeping status (22%, OR 1.0). These findings persisted after adjustment using the same model described above (p-value < 0.001). The same patterns were observed for inpatient psychiatric services. The same relationships between access to high quality services and number of managed care contracts and gatekeeper percentage were not observed for medical services. CONCLUSION: The mental health referral process may be uniquely vulnerable to the administrative burdens imposed by the current managed care environment. Paradoxically, while large numbers of managed care plans with a variety of options are thought to increase choice, they may actually decrease access to high quality mental health services. PURPOSE: For many chronic diseases, effective drug therapy is now available. However, patients often fail to adhere to prescribed regimens with suboptimal clinical results. We surveyed resident and faculty physicians (MD) of a university-based internal medicine clinic serving an indigent population to determine MD attitudes, beliefs and behaviors with respect to patient drug adherence (DA). METHODS: One hundred two MDs completed a 14 question survey asking them to definè good' adherence, estimate the proportion of their patients who achieve 'good' patient DA, and to respond to a series of statements regarding DA. Two mailings produced an 86% response rate. At least 20 responses were obtained from each of PGY1,2,3 and attending MD levels. RESULTS: Seventy nine percent of MDs defined`good' adherence' as patients taking at least 80% of prescribed doses. However, MDs estimated that only 66+/À15% (mean/SD) of their patients acheived`good' adherence. In the prior 6 months, 50% of MDs discussed DA with at least 80% of patients, and 91% of MDs reported discussing DA with at least 50% of patients. Despite these discussions, only 32% of MDs felt that at least half of their patients had a good understanding of the importance of drug therapy to avert adverse outcomes. Almost all (97%) MDs felt that DA assessment and intervention was an important component of the office visit and 100% felt that prescription refill feedback would help them target patients for adherence intervention. There was no association between year of training and DA responses. CONCLUSION: In this indigent care setting, MDs indicated that they frequently discussed DA with patients but felt that patients lacked a good understanding of the role of their medication and that many were not adherent to their prescribed regimens. All MDs felt that specific DA feedback based on prescription refill data would assist them to identify and manage non-adherent patients. Since automated pharmacy claims systems cover the majority of the US population, such feedback is feasible and worthy of future study in both indigent and more affluent populations.
PURPOSE: Despite mounting evidence that beta-adrenergic antagonists (beta-blockers) are beneficial immediately following acute myocardial infarction (AMI), a median of only 64% of Medicare beneficiaries nationwide receive beta-blocker within 24 hours of AMI. Measuring use of beta-blockers in AMI usually requires expensive chart reviews. Using a comprehensive electronic medical records system instead of chart review, we sought to measure use of betablockers in AMI as well as differences in charges between those who received beta-blocker and those who did not. METHODS: We examined data from all 15,610 Medicare-covered hospitalizations during 1995 ± 99 for 7,251 patients in an urban academic medical center, evaluating for International Classification of Diseases codes for AMI. Restricting study to patients 65 years of age or older, we collated and merged into this all available prescription drug data pertaining to these patients, separating inpatient from outpatient drugs based on date. We then calculated rates of betablocker use in AMI and differences in estimates of total hospital charges and length of stay depending on whether beta-blocker were prescribed. RESULTS: Of 9,191 hospitalizations among 4,490 older adults, 385 (4%) contained a diagnostic code for AMI. Beta-blockers were prescribed in 252 (65%) of the encounters, and 85% of prescriptions occurred on the date of admission or one day later. Overall, beta-blocker prescriptions occurred on one of these days in 55% of hospitalizations for AMI. Metoprolol was the beta-blocker prescribed most often (77% of beta-blocker use in AMI). Of those receiving any beta-blocker, 3% had a diagnostic code for obstructive lung disease, compared to 5% among those not receiving beta-blockers (p = 0.37). Those receiving beta-blockers had mean length of stay 1.5 days longer (p < 0.05) but mean charges not significantly different from those not receiving beta-blockers. CONCLUSION: A comprehensive, electronic medical records system can be used instead of resource-intensive chart review to measure use of beta-blockers after AMI. Uncertainty about the precise relationship between the two methods should prompt, as a next investigative step, a more direct comparison of chart review to the use of electronic records to measure indicators of quality of healthcare. shown that these agents are underutilized in practice. Contributing to this finding is the perception that other agents may work better or have fewer side effects. Under the assumption that a medication class switch represents a physician's belief that the medication was of little benefit, or was not well-tolerated by the patient, this study compares the degree to which each of the major classes of antihypertensive medications is discontinued in favor of another class of antihypertensive. METHODS: We examined pharmacy data from a population of Medicare patients enrolled in a managed care organization (MCO) between December 1996 and October 2000. An overall interval of antihypertensive medication usage was determined for each patient. The subset of this time interval in which the patient received a beta blocker (BB), calcium channel blocker (CCB), ACE inhibitor (ACEI), or diuretic was determined. A therapy class switch was defined as prescribing a new class of antihypertensive medication more than one month after the end of the original antihypertensive class interval ended. In this manner, the addition of a second antihypertensive would not be considered a class switch, unless the first medication was stopped permanently, and the second medicine continued for more than a month. RESULTS: Of the 9798 patients who filled at least 3 antihypertensive prescriptions over at least 90 days, 4260 received a BB, 4225 received a CCB, 4533 received an ACEI and 3480 received a diuretic (sum exceeds total since patients could have been on more than one class). Kaplan Meyer survival analysis indicates that after 1000 days, a higher percentage of patients continue to take BB (68.5%) than ACEI (63.8%), CCB (61.8%) or diuretics (53.4%). The length of time until 25% of patients had switched their medication class was longest for the group taking BB (760 days), then ACEI (589 days), CCB (568 days) and diuretics (387 days). This trend favoring BB is also observed when limiting the analysis to classes of antihypertensives that were initiated in the observation period. CONCLUSION: The results of the study suggest that, contrary to common perceptions, among a Medicare population, beta blockers are not more commonly discontinued than the more modern, and expensive antihypertensive agents. While the clinical factors that influence a physician's decision to start a particular medication class are not known, this potential bias is mitigated by the common tenets of good clinical practice. These tenets dictate that when choosing to prescribe any class of drug, the physician believes it would be safe, effective and well tolerated. The study results suggest that, regardless of the initial indications for choosing a particular therapeutic class of antihypertensive, or the reason for eventually changing the class (non-compliance, patient complaints, ineffectiveness, expense, etc.), BB seem less likely than ACEI and CCBs to be switched, while diuretics are more likely than the other classes to be discontinued. PURPOSE: Attention to issues of autonomy, surrogates and treatment preferences are important for vulnerable elders, who are at risk of decisional incapacity and death. We investigated these elements of end-of-life care among a cohort of vulnerable elders. METHODS: The Assessing Care of Vulnerable Elders project developed and tested quality indicators (QIs) for community-based personsÊ65 years at increased risk of death or functional decline. Based on a systematic literature review, an expert panel rated 14 QIs related to documentation about surrogates and preferences, and translation of preferences into care as valid process measures for end-of-life care. We measured these QIs by reviewing 13 months of inpatient and outpatient charts of patients in 2 managed health plans. RESULTS: Of 3206 seniors randomly selected from the plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. Of 400 (84%) who consented to study participation and had abstractable medical records, 376 were eligible for at least one QI. Fourteen elders (4%) had any documentation (formal or informal) about surrogate decision makers in their outpatient charts. Charts for 103 hospitalizations yielded 8 advance directives, 23 noted a surrogate or an attempt to find one, and 72 (70%) contained no documentation. When hospitalized with altered mental status, 5 of 20 elders had any documentation about a surrogate within the first 3 hospital days and 1 of 6 elders admitted to an ICU had any documentation about preferences within 3 days. Seventy percent of orders to limit care in the hospital considered patient participation in decision-making and 2 of 2 ventilated patients had documentation about preferences. CONCLUSION: Despite much recent attention to end-of-life care, surrogates and preferences are not elicited prospectively from vulnerable elders. Improvement in the quality of end-of-life care is needed, particularly for persons at risk of life-threatening events. PURPOSE: Measurement of quality of care (QOC) for mainstream medical conditions has received much attention, but no method exists to measure QOC for geriatric conditions. We developed a system to comprehensively measure QOC provided to vulnerable elders and compared QOC for geriatric and medical conditions. METHODS: Based on structured literature reviews, content experts developed 239 quality indicators (QIs) for the process of medical care provided to``vulnerable elders'', communitybased persons > 64 years old at increased risk of death or functional decline. These QIs assessed care for 22 conditions. We applied these QIs to a community sample of vulnerable elders by abstracting their outpatient and inpatient charts, comparing QOC provided for medical conditions (depression, diabetes, heart failure, hypertension, coronary disease, osteoarthritis, osteoporosis, pneumonia and stroke) to geriatric conditions (delirium, dementia, falls, geriatric assessment, incontinence, malnutrition and pressure ulcers) RESULTS: Of 3206 seniors randomly selected from two managed senior health plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. Of these, 400 (84%) consented to participate and had abstractable charts. For medical conditions, 52% of 1897 QIs were satisfied (e.g. diabetes 58%, heart failure 72%). This was significantly better (p < 0.01) than the 37% of 2036 QIs satisfied for geriatric conditions. Compliance rates for geriatric conditions included falls 26%, dementia 35% and incontinence 29%. CONCLUSION: QOC for geriatric conditions falls far below that of QOC for medical conditions. Quality assessment and assurance systems should incorporate QIs measuring geriatric syndrome care. Care for geriatric conditions ± common among vulnerable elders and associated with functional decline and mortality ± must be improved. PURPOSE: Continuity and coordination (CC) of care are core elements of general internal medicine. This is particularly important for``vulnerable elders'' who may be at high risk of adverse events due to poor CC. Yet there is no method of measuring CC in medical care. We developed and tested a system to measure CC of care among a cohort of vulnerable elders. METHODS: The Assessing Care of Vulnerable Elders project developed and tested quality indicators (QIs) for community-based persons > 64 years at increased risk of death or functional decline. Based on a systematic literature review, an expert panel rated 13 QIs as valid process measures of care CC. Six of these could be measured by abstracting information from medical records. QIs evaluated medication, test and information continuity, within and between providers, across the inpatient and outpatient settings.
RESULTS: Of 3206 seniors randomly selected from two managed health plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. For the 400 (84%) patients who consented to study participation and had abstractable medical records, 67% of 324 CC QIs were satisfied. Of 185 seniors started on new medications during the study, 67% had follow-up of this medication at the next physician visit. Of 72 patients who were hospitalized, 89% received postdischarge follow-up within 6 weeks; 6 of 11 had follow-up of a medication started in the hospital and 10 of 14 had follow-up of a test pending at hospital discharge, but only 41% had a copy of the discharge summary in the outpatient chart. CONCLUSION: Vulnerable elders at two managed care plans received a high level of CC of care. Measuring CC care identifies aspects that require intervention. In an effort to control health care costs, many third party payers limited physicians' abilities to order expensive tests for their patients, requiring the use of an appeals process to obtain restricted medical services. Studies suggest that, at times, physicians are willing to misrepresent clinical information to third party payers in order to obtain these services for their patients. Little is known about whether characteristics of the appeals process influence physicians' willingness to sanction deception. METHODS: We surveyed a random sample of 1,617 physicians in the U.S., and presented each with a hypothetical scenario in which an insurance company refused to pay for a medically important service. We varied the``hassle'' of the appeals process by varying the time required to make an appeal and the likelihood of having a successful appeal. In addition, we varied the severity of the patient's health condition from moderate low back pain to severe angina.
Physicians were asked if the patient's physician should accept the insurance company's restriction, appeal the restriction or misrepresent the facts to the insurance company in order to obtain coverage for the patient. RESULTS: Overall, most physicians responded that they would appeal (77%) rather than accept (12%) or misrepresent (11%) a restriction on medically necessary care. Physicians were more likely to misrepresent if the appeals process was longer (OR=1.68, CI 1.09 ± 2.59), less likely to succeed (OR=1.57, CI 1.02 ± 2.42), or involved a more severe condition (OR=2.19, CI 1.39 ± 3.45). Among physicians asked about severe angina, as the appeals process became more cumbersome, they were more likely to choose to misrepresent the facts to the insurer rather than appeal. When asked about a patient with moderate low back pain, physicians' decisions about how to respond to an insurance company restriction were not significantly affected by the hassle associated with the appeals process. CONCLUSION: Physicians' willingness to sanction deception of insurance companies varies according to length of the appeals process, the likelihood of a successful appeal, and condition severity. When the stakes are high, and the hassle of appeals is great, physicians become increasingly willing to sanction deception. Changes in the difficulty of appeals processes may ease the tension physicians' face between patient advocacy and honesty. PURPOSE: To assess general internists' (GIM) satisfaction and recruitment of medical students and compare these to family physicians (FP). METHODS: 5704 physicians were surveyed (adjusted response rate 52%) from a national random stratified sample-19% GIM (n=450, 108 women, 75 were``young'' (age < 45); 341 men, 109 were young) and 22% FP (n=502). Mean global job and specialty satisfaction as well as likelihood of recommending their specialty to medical students were compared by specialty. All were assessed on a 5 point Likert scale (3 = neither agree nor disagree). For general internists, further analyses were done by age and gender and logistic regression was used to model predictors of medical student recruitment. RESULTS: GIM had significantly lower job and specialty satisfaction vs FP (job: 3.52 vs 3.77; specialty: 3.18 vs 3.69, both p < .001). Global satisfaction did not differ by gender alone for GIM. However, younger women and older men had higher job satisfaction then their same gender counterparts (women 3.75 vs 3.28, p=.043, men 3.63 vs 3.28, p=.015) with no difference in specialty satisfaction. GIM was less likely than FP to recruit medical students to their specialty (3.26 vs 3.85, p < .001). Women GIM recruited with more enthusiasm than men (3.56 vs 3.16, p=.022) with younger women the most enthusiastic (young 3.67 vs old 3.35, p=.038). Global specialty satisfaction and female gender were positively associated with medical student recruitment by internists. CONCLUSION: General internists have lower job and specialty satisfaction than family physicians. This dissatisfaction leads to less recruitment of medical students into internal medicine. Increasing numbers of young women in GIM may change this trend but recruitment needs to be seen as an important outcome of physician satisfaction to keep the specialty viable. (27); plan cost rating, 64 (29); service restrictions 71 (24). TC rates were: HAART, 68%; PPD,14%; and flu shots, 32%. In multivariable models there were no significant differences between MC and FFS coverage for any of 7 IPC, 4 HPQ, or 4 TC quality indicators. Models controlling for race, education, sexual preference, and duration of physician ± patient relationship, which varied by plan type, were no different. Tests for an interaction between physician and FFS/MC differences were non-significant. CONCLUSION: For patients with HIV in Boston, managed care coverage was not associated with lower quality care. These experienced HIV providers do not appear to practice differently depending on insurance type. Our findings suggest that managed care coverage can be as effective as FFS coverage even for patients with a complex, expensive, chronic illness. We hypothesized that physicians would frequently field questions about CAM from their patients, but, due to a lack of education about these treatments, would feel uncomfortable discussing them. We therefore surveyed a metropolitan area group of physicians to ascertain their patterns of communication with patients regarding CAM and the factors that influence them to discuss CAM with their patients or refer them for CAM treatments. METHODS: Seven hundred fifty one randomly selected physicians in all specialties in the Denver, Colorado metropolitan area were asked about their exposure to, knowledge of, and belief in 16 CAM modalities and patterns of physician Ð patient communication regarding CAM. Analyses were conducted using the SAS system (version 6, fourth edition, Cary, NC, 1989) and included the two-tailed t-test, Pearson correlation coefficients, linear regression, and multivariate analysis. RESULTS: Three hundred and two of 705 deliverable surveys (43%) were returned. Responders averaged 44 years old (+/À 12 years), 63% were male, and 48% were selfdesignated primary care physicians. Seventy-six percent of physicians reported having at least one patient using a CAM therapy, 59% had received queries from patients about specific CAM treatments, 48% had recommended a specific CAM therapy to at least one patient, and 24% had used a CAM modality for themselves. While patients frequently sought out information about CAM modalities from their physicians, only 31% of physicians routinely ask their patients about their use of CAM modalities, and 17% never ask. Physicians most frequently recommended massage, relaxation, acupuncture, and biofeedback to patients, while the modalities physicians used most frequently for themselves were massage, relaxation, herbs, and yoga. Physician recommendation of CAM use to patients was most strongly associated with physician self-use (odds ratio 6.98,p=0.0001). Only 45% of physicians felt at least somewhat comfortable discussing the risks and benefits of CAM with their patients; the overwhelming majority (84%) felt they needed to learn more about CAM modalities to adequately care for patients. CONCLUSION: Education about CAM modalities is a significant unmet need among physicians residing in the Denver metropolitan area, and education may help alleviate the discomfort physicians have when fielding questions about CAM from patients. Physicians who already use CAM treatments for themselves are much more likely to recommend CAM for their patients than those physicians who don't personally use CAM. (1346) responded. The questionnaire included demographic items, the SF-12 to assess physical and psychological function and their satisfaction with, need for and importance of both their VA and non-VA sources of care. Non-VA care was identified using the HCFA enrollment database. RESULTS: Among the entire population, 303 (17%) belonged to a Medicare HMO for the entire 24 months, and 130 (7%) belonged to an HMO for part of the period. Questionnaire responders did not differ from non-responders by marital status, gender, service connected status or HMO enrollment. Among the HMO enrollees, 66% reported having a VA primary care provider and 46% reported receiving all their medical care from the VA. However, 72% reported having a non-VA primary care provider. When comparing VA users who belonged to a Medicare HMO to those who did not enroll in an HMO, the HMO members reported significantly higher levels of educational attainment and financial resources. The psychological and physical function scores did not differ amongst the various Medicare enrolled veterans. CONCLUSION: Users of VA medical care frequently belong to Medicare HMOs, and nearly half of these HMO enrollees report receiving most of their medical care from the VA. VA users who belong to HMOs tend to be better educated and more financially secure. VA care appears to provide a substantial financial benefit to Medicare HMO plans. Little is known about the extent to which characteristics of health care organizations foster or hinder such performance. We evaluated variations in tobacco counseling rates among VA medical centers (VAMC's) nationwide and assessed the medical center and primary care program attributes related to high and low performance. METHODS: We assessed VAMC organizational characteristics (e.g., region, urban location, primary care features) using a previously validated mailout survey fielded to VA primary care directors (100% response). We matched these survey-based measures to two centrally available databases: (1) organizational culture scores derived from the first wave of the National VA Quality Improvement Survey (G. Young, PI) administered to over 100 employees at each VAMC, and (2) tobacco counseling rates for each VAMC from externally-performed chart reviews of randomly sampled users with 1+ primary care and 1+ subspecialty visits (88% match). We used bivariate analyses to examine the relationships between discrete organizational features and counseling rates and multiple linear regression using a forward stepwise algorithm to assess the independent predictors of counseling rates. RESULTS: Counseling rates varied by region (p < .01), with lowest rates in the East (0.73) and highest in the Western states (0.83). More complex VAMC's had lower tobacco counseling rates (p < .0001), consistent with lower rates among urban (p < .0001), academic (p < .01) VA's, with more internal medicine house officers (p < .01) and larger patient caseloads (p < .001). VA's with higher self-reported levels of primary care implementation, higher proportions of veteran users receiving all or most of their care in primary care, and where primary care physicians had responsibility for their patients as inpatients had higher tobacco counseling rates (all p < .05).
Higher rates also occurred in VA's with higher staff ratings of leadership quality (p < .001) and marginally higher group (p=.054) and risk-taking cultures (p=.055), while those VA's with higher staff orientation to quality improvement (p < .05) and more primary care-based process action teams (p < .05) had higher rates. CONCLUSION: Achieving more guideline-adherent smoking cessation practices in large, academic medical centers may pose significant challenges for primary care managers, but appears more likely among medical centers with more highly organized primary care practice. Improving counseling rates requires systems approaches to address both structural and provider-related constraints. Our objective was to describe duration of visit to chiropractors (CP) and acupuncturists (AC), the factors associated with these visit times, and to compare visit duration among physicians (MD), CP, and AC. METHODS: A random sample of licensed CP (N=130) from Arizona and Massachusetts, and licensed AC (N=133) from Washington and Massachusetts, was surveyed on consecutive office visits in 1998 ± 99 regarding visit time, patient demographics, reason for visit, acuity of problem, payment source, diagnostic and therapeutic techniques performed. (N=2550 CP encounters, mean age 44.9 years, 57% female; N=2561 AC encounters, mean age 46.2 years, 68% female).
We used linear regression to analyze factors associated with visit duration and to calculate the minutes attributed to each factor. Models were adjusted for specific therapeutic and diagnostic techniques performed at the visit. MD visit duration was obtained from published 1995 ± 96 National Ambulatory Medical Care Survey results. RESULTS: Mean visit duration for CP was 21.5 minutes (SE=0.8) and for AC 56.6 minutes (SE=0.7), compared to 18.9 minutes (SE=0.5) for a visit to an internist (p=.006 for CP vs. MD, p < .001 for AC vs. MD). AC spent more time with patients at self-pay visits, new patient visits, and visits for flares of chronic problems, adding 4.7, 4.0, and 2.4 minutes respectively. AC visits for anxiety/depression were 2.6 minutes longer, with a mean duration of 50.2 minutes, compared to 32.6 minutes for MD visits for the same problem (p < .001). For CP visits, significantly more time was spent with new patients, when there was communication with other providers, and with patients receiving concurrent care from an MD, adding 6.7, 3.2 and 2.3 minutes to each visit, respectively. CP visits for wellness and chronic problems were shorter than acute visits, by 3.2 and 4.3 minutes, respectively. Patient age, sex, and reason for visit (i.e. back or neck symptom) did not affect CP visit duration. Visits in Arizona were, on average, 3.0 minutes longer than those in Massachusetts. CONCLUSION: Acupuncturists spend significantly more time with patients on an average visit compared to physicians. Chiropractors report spending more time than physicians, although this difference is small and may be due to differences in how the data were collected. CP visit duration suggests regional variation. Payment source, communication with other providers, psychosocial reason for visit, and acuity of visit are important correlates of CP or AC visit duration. These trends towards``vertical integration'' (VI) have been studied primarily in terms of their financial causes and impact. Effect on clinical care is unknown. This study examines whether hospital VI impacts continuity and timing of clinical process of care for acute stroke patients. METHODS: A survey was fielded in mid-2000 of the primary stroke providers at all acute care hospitals with acute stroke discharges in California in 1998. Survey findings were linked to California state hospital facility reports and individual patient discharge records for 1998. Multiple regression models were used to examine the association of highest level of integration (internal rehab unit (IR), formal control of external unit (FER), informal agreement of external unit (IER), and no agreement (NoR)) with service availability, reported interventions, and patient outcome, controlling for other hospital features and patient demographics. RESULTS: Surveys were completed for 256/374 (68%) of hospitals that admitted stroke patients in 1998 and that were still open in 2000. Non-respondent hospitals were smaller, rural, or independent. Among respondent hospitals, level of integration was 20% IR, 7% FER, 15% IER, and 58% NoR. Greater integration was significantly associated with stated greater use of acute care guidelines, hours of rehab care during acute hospitalization, continuity of providers between acute and post-acute care, management by specialists, and use of common medical information systems. When linked to patient records, level of integration did not predict initiation of rehab during hospitalization or in-hospital mortality, but was associated with increased likelihood of discharge to post-acute rehab.
CONCLUSION: VI appears to have clinical advantages of greater underlying consistency and continuity of care, but does not appear associated with early (acute) rehab intervention. Shortterm mortality does not appear to be associated with VI, but other outcome measures such as functional recovery may be more sensitive and appropriate. As trends towards vertical integration continue, a better understanding of VI's clinical impact is imperative. PURPOSE: One barrier to screening and disclosure of Intimate Partner Violence (IPV) is the patient's lack of trust in their health care provider. The objective of this study was to identify characteristics that facilitate trust in the patient-provider relationship among survivors of IPV. METHODS: Qualitative data were collected using semi-structured, open-ended interviews with a sample of 28 female survivors of IPV. Participants were recruited from community-based IPV organizations in the Boston area. A one hour face-to-face interview explored the woman's beliefs and attitudes about trust in her interactions with health care providers. Interviews were audiotaped and transcribed. Using Grounded Theory Methods, the transcripts were analyzed for significant themes, from which a conceptualization of trust was developed. RESULTS: Among the participants, ages ranged from 18 to 56, 36% were Black, 32% Hispanic and 18% white. All suffered physical violence, but only 60% ever sought medical care as a result. We identified five dominant concepts that described dimensions of provider behavior on which trust was based: 1) OPEN COMMUNICATION Ð listening to patients and keeping them well informed of their medical condition; 2) PROFESSIONAL COMPETENCY Ð appearing knowledgeable, performing thorough medical evaluations and being familiar with the medical and social histories; 3) PERSONAL COMMITMENT Ð being consistently accessible, both physically and emotionally (``always been there for me''), respecting confidentiality, spending ample time with participants and demonstrating personal concern outside the traditional provider role; 4) EMOTIONAL SAFETY Ð sharing personal information and establishing a climate of safety for the participants through nonjudgmental and compassionate gestures (``he had tears in his eyes'') or comforting nonverbal mannerisms; 5) EMPOWERMENT Ð promoting self-efficacy and shared decision-making. Gender and ethnic characteristics of the providers had variable effects on trust. CONCLUSION: These survivors of IPV reported several aspects of provider behavior that facilitate trust in their clinical relationship, including open and explicit communication, professional competence, personal commitment, establishing a safe emotional environment and patient empowerment. Strengthening these provider behaviors may increase trust with patients and thus improve screening and disclosure of IPV. PURPOSE:``Turfing'' is the transfer or triage of a patient to a physician for reasons the receiving physician deems inappropriate.``Turfing'' has been identified as a troubling source of professional and ethical conflict for housestaff and medical students. Prior work has reported that``turfed'' patients have a worse hospital experience than appropriately transferred patients. We wondered whether attendings' perceptions of``turfing'' supported patients' perspectives. METHODS: One trained research assistant conducted in-depth, semi-structured, open-ended individual interviews with a convenience sample of 16 attending physicians from internal medicine, emergency medicine and general surgery. The interviews were meant to elicit attendings' views on the quality of``turfed'' patients' care as well as other dimensions of this triage phenomenon. Audiotapes of interviews were subjected to content and thematic analysis. Results presented here include only attendings' comments about the quality of``turfed'' patients' care. RESULTS: All attendings interviewed in emergency medicine (n=4) and general surgery (n = 4) felt that``turfed'' patients received inferior care. Of the 8 internal medicine attendings interviewed, 5 felt``turfed'' patients received inferior care, 1 was equivocal, and 2 did not comment on this specific issue. Subjects considered quality of care to include length of stay, interpersonal interactions between patients and housestaff, and delays in management plans and bed assignments. Subjects stated:``There is always hostility toward the patient from the housestaff in these situations If you go watch [the housestaff] and see how they interact with the patient, they are abrupt with them, dismissive toward them.''``Patients are put in harm's way.''` [ Housestaff] probably don't take as inquisitive an approach [to history-taking] which is not in the patient's best interest.''``We avoid people who have been in the ED for too long [because no one will accept them on their service].'' Three themes emerged as causes for this inferior care: lack of``ownership'' of``turfed'' patients by physicians, the transient nature of physician relationships with``turfed'' patients, and feelings that``turfs'' are unwanted. CONCLUSION: Attendings' interviews substantiate that the care of``turfed'' patients suffers. Interviewees recommended remedies for these troubling findings, such as stronger moral leadership from department chairs and improved role modeling by clinical faculty. The purpose of this pilot study was to determine whether there are differences in the way Caucasians and African Americans would present facts about prostate cancer screening in a culturally appropriate brochure to men considering screening. Because screening with prostate specific antigen (PSA) is controversial, several professional organizations recommend informing men about the risks and benefits of screening. Compared to Caucasians, African Americans have a higher risk for prostate cancer and are less knowledgeable about screening. In a previous study, experts in prostate cancer and Caucasian and African American couples with screened and unscreened men identified 17 key facts about PSA they believe men ought to know. METHODS: We convened a focus group of 5 African American couples and a focus group of 5 Caucasian couples who each met twice. At the first meeting couples viewed a videotape about prostate cancer and screening and were asked to discuss how to present the 17 key facts in a culturally-sensitive way in terms of content and format. Mock ups of brochures were developed for discussion at the second meeting. We analyzed transcripts of the focus group meetings using content analysis to identify differences between African American and Caucasian groups in how to present this information. RESULTS: We found differences in content emphasis and in graphic design choices between the brochures that African Americans and Caucasians would design for members of their racial/ethnic group. Because of the perceived discomfort and embarrassment associated with the digital rectal exam (DRE), African American men felt strongly that it was important to emphasize the advantages and disadvantages of screening with the DRE, as well as the PSA. Caucasian men did not discuss the DRE at all. African Americans believed it was important to emphasize epidemiologic data specific to African American men, such as their higher risk of prostate cancer. They also preferred images and symbols rooted in African American culture (e.g. Kente cloth). CONCLUSION: African Americans and Caucasians differed in the way they chose to present facts about prostate cancer screening to members of their racial/ethnic group. Cultural differences in format and content need to be considered when designing educational materials such as brochures.
RACIAL PROFILING, CLINICAL EPIDEMIOLOGY, AND CULTURAL COMPETENCE: WHEN IS RISK STRATIFICATION BY RACE OR ETHNICITY JUSTIFIED? M.H. Chin 1 , C.A. Humikowski 1 ; 1 University of Chicago, Chicago, IL PURPOSE: Population-based, probabilistic approaches to medicine have become more prominent with the rise of the evidence-based medicine movement. Simultaneously, diverse patient populations have spurred development of curricula in cultural competence. Building upon lessons from the racial profiling debate in the legal and political realms, we aimed to develop a conceptual framework for exploring whether race and ethnicity should be used as clinical tools or if dangers of stereotyping are too great. METHODS: We reviewed key literature pertinent to racial profiling, race and medicine, and curricula in cultural competence. We explored issues of race and medicine through a regular seminar series with medical housestaff, and extensive feedback from colleagues. We developed a conceptual framework that balances clinical epidemiological principles with lessons learned from social and political racial profiling. RESULTS: We propose that viewing the patient within a wider population-based cultural setting can help guide the initial clinical approach, but individualized care is mandatory. The use of race and ethnicity as clinical tools is on a slippery slope Ð they may be useful or dangerous. Biological examples are rare in which race or ethnicity are relevant (e.g. Ð Tay ± Sachs, sickle cell). However, ethnicity may have a statistical association with a variety of factors. The potential benefit of using ethnicity as a tool is to aid an individual patient by making medical care better tailored to him or her. The potential detriments are reinforcement and perpetuation of partial myths, encouragement of undesirable behavior by creating negative expectations, and avoidance of addressing the underlying individual factors. Racial profiling has taught us that the costs of using ethnicity as a proxy for behavior are too high. But, ethnicity may appropriately be used as an initial proxy for history, language, culture, and health beliefs, as long as individualization of care is rapid. The predominant approach to cultural competence in medical education today teaches a consideration of individual patients as opposed to a rigid checklist of ethnic traits. For example, providers are advised to inquire about the meaning of illness to the patients, and the social context such as family, literacy, and finances. However, given that physicians have limited time and energy, context including race and ethnicity can be helpful in guiding initial inquiry and raising awareness that an issue (e.g. Ð strong distrust of the health care system) may be important.
CONCLUSION: In the specific situations outlined, race or ethnicity may be useful clinical tools as long as individualization of care is quick. However, vigilance is required as medicine has not been immune to overt or subconscious racism. Case examples are used to define our conceptual framework in more detail.
MOOD DISORDERS. W. Resnick 1 , T. Houston 2 , K. Swartz 2 , L.A. Cooper 2 ; DRADA, Baltimore, MD; 1 Johns Hopkins University, Baltimore, MD PURPOSE: To describe how mutual help support groups are used by individuals affected by mood disorders. METHODS: Six focus groups, including 39 participants (19 women, 20 men), were conducted. Participants were recruited through Depression and Related Affective Disorders Association (DRADA) support groups. Discussions addressed what if any influence support groups have had on their lives; and the relationship of the groups to medical treatment and/or therapy. Discussions were audiotaped, transcribed verbatim, and reviewed independently by two investigators to group distinct comments into categories with specific themes. Differences were resolved after a joint review of themes. Comments within categories were then checked for relevance and consistency by two second reviewers Ð a general internist, and a psychiatrist. Adjustments to categories were made until consensus was reached. RESULTS: Comments fell into 10 categories: 1) Patient Activation/Empowerment in their treatment, 2) Using support groups to complement treatment, 3) Obtaining education and information, 4) Learning to interact positively with family and co-workers, 5) Socializing with support group members, 6) Sharing Ð to understand each other, 7) Helping others, 8) Increasing self-esteem, 9) Emotional Support, and 10) General comments. All participants were positive about the influence of support groups in their lives. A sense of belonging, talking about problems, and feeling understood by others, were seen as a source of comfort and well-being. Participants felt their family and work relationships, and communication skills were improved by participation in support groups. They also reported an increase in self-esteem and participation in medical decision-making. All agreed that exchange of information about their illness and exchange of ideas with other people about coping, contributed to their health. Emotional support from peers, destigmatization of their illness, and a sense of hope were all benefits from the groups. Most individuals did not use the group as replacement for treatment. CONCLUSION: Health professionals should consider recommending mutual-help support groups as an adjunct to treatment. The combination of medical treatment and peer support may enhance patient participation in care, treatment adherence and social skills, and provide patients with positive coping strategies. Baltimore Mental Health System, Baltimore, MD PURPOSE: Studies document underutilization of outpatient specialty mental health services by African Americans (AAs). However, AAs with depression are just as likely as whites to receive care in primary care. Despite their use of primary care, AAs are less likely than whites to be recognized as depressed, offered pharmacotherapy, and to initiate or complete guidelineconcordant depression care. AAs express preferences for counseling and negative attitudes toward antidepressant medication, the most common form of depression treatment used by primary care physicians. Few depression education programs and materials target cultural beliefs and values of AAs. The purpose of this study was to determine the acceptability and usefulness of an educational videotape for AAs with depression. METHODS: After showing the videotape, we held four focus group discussions in two community settings and at an historically black university. Subjects included twenty-four AAs, aged 18 ± 76 years, who screened positive for depression. Thirty-eight percent reported a history of previous treatment. Focus group discussion questions addressed the usefulness of the videotape for helping viewers understand depression and its treatment, the most and least effective parts of the videotape, and the cultural appropriateness of the information presented. Participants took pre-and post-tests on attitudes about depression. Discussions were audiotaped, transcribed, and reviewed independently by two investigators to identify and group comments into specific themes. Two other investigators reviewed the themes and comments for consistency. Changes were incorporated to achieve consensus. RESULTS: In addition to comments specifically related to positive and negative aspects of the videotape, the following themes were identified: concerns about antidepressant medication; identification with people in the videotape; racial and cultural issues; stigma and stereotypes; spirituality; and validation and support from the focus group. The videotape was well received and rated by most viewers as effective in improving knowledge and alleviating concerns about depression and its treatment. After watching the videotape, attitudes improved in several areas: depression as a medical illness, effectiveness of treatment, negative perceptions of antidepressant medication and reliance upon spirituality alone to heal depression. CONCLUSION: This culturally tailored videotape about depression is deemed acceptable and effective to AAs with depression participating in focus groups. It also improves knowledge and attitudes about depression. Incorporating culturally-tailored educational materials and messages into community and primary care depression programs may reduce barriers to appropriate depression care for AAs.
THE SIGNIFICANCE OF LONG-TERM DOCTOR-PATIENT RELATIONSHIPS AMONG OLDER PATIENTS. C. Crenner 1 , K.A. Greiner 2 ; 1 University of Kansas Medical Center, Kansas City, KS; 2 Kansas University Medical Center, Kansas City, KS PURPOSE: Many older Americans have a personal stake in a relationship to a single physician that extends back decades. Changes in our healthcare system and in chronic care facilities may increasingly disrupt these connections. People who have preserved such long-term relationships have much to tell us about the significance of these relationships and their relevance to care. METHODS: We invited participation at four sites: a rural nursing home; an ambulatory clinic in an urban medical center; a suburban, assisted-living facility; and a private clinic in a lowincome, urban neighborhood. We enrolled subjects who could give an account of past medical care and who had kept the same primary physician for seven years or longer. We sampled to increase variability. We recorded and transcribed 17 semi-structured interviews and conducted a focus group with seven participants including 2 subjects not interviewed individually. We elicited open-ended discussion of each subject's relationship to a long-term doctor, asking about the features that interfered with or helped to sustain the relationship, and the significance that the relationship held. Immersion in the transcribed material and simultaneous reading in social history and anthropology produced a theoretical model based on the distinction between giftexchange qualities and commodity-exchange qualities in these relationships. RESULTS: Subjects were 6 men and 13 women, with a mean age of 75 years, and a mean duration of medical relationship of 19 years. Eighteen subjects were white and one was African American. Subjects attributed much of the durability of these relationships to the positive personal and professional characteristics of their doctors. There was variability in characteristics described as important. Few informants recalled their own active role in selecting their current physicians, and some had been in practices that were``handed-down'' from doctor to doctor. Distance and transportation were very important considerations for continuing care. Subjects were generally able to describe a rational process for selecting their next physician. CONCLUSION: Our theoretical model suggested that gift-exchange qualities in these relationships implied mutual obligations between recipients and donors that extended beyond the simple transaction of services. In contrast, commodity-exchange relationships existed solely for the exchange of specific services. Services in gift-exchange relationships had a value partly determined by the value attached to the relationship, while in commodity-exchange the value was independent. Our subjects favored characterizations of their long-term relationships as forms of gift-exchange, but they demonstrated an ability to shift to a commodity-exchange model. Statements consistent with a commodity-exchange model appeared especially in references to relationships of shorter duration, or to the conclusion of relationships to long-term providers. The day-to-day practice of medicine entails many ethical quandaries and yet little is known about practicing physicians' encounters with ethical dilemmas or how they resolve them. METHODS: A randomized, national telephone survey of internists including generalists was conducted to determine the ethical dilemmas encountered, the strategies used to address them, and the use and value of ethics consultation. Coding schemes were used to categorize verbatim descriptions of ethical dilemmas. RESULTS: Responding generalists (N=95) reflected the demographic characteristics of U.S. general internists (20% female, 23% non-white, 33% foreign born). One quarter of the ethical dilemmas encountered by general internists were related to justice issues concerning uninsured patients, practice in managed care, or allocation of limited resources. During the previous 2 years respondents had requested an average of 2 consultations. Physicians were most hesitant to request consultations because they are time-consuming. While many of the ethical dilemmas encountered were related to justice issues, no justice-related dilemmas were referred for ethics consultation. Physicians handled justice related dilemmas by strategies that included explicitly discussing limits with patients, acquiescing to limits, overstepping limits, searching for alternative sources, or providing free care. Physicians were often dissatisfied with resolution of justice questions and believed institutional or policy changes were required to achieve better resolution. CONCLUSION: General internists encounter a wide array of ethical dilemmas and address the vast majority of them on their own. Medical ethicists need to be aware that justice related dilemmas are often unsatisfactorily resolved and should explore ways to help in addressing them. Recent data suggest that this funding is becoming more concentrated at the medical schools most active in research, which tend to be in urban centers. I hypothesized that over the past 20 years, NIH research funding has increased in the states and regions with the highest per-capita NIH funding and highest population density, relative to the other states and regions. If present, the hypothesized shift in research funding may hinder efforts to reduce regional disparities in medical care. METHODS: Data from the NIH and US census bureau were used to calculate the per-capita NIH funding received by each state during 1980 ± 2000. The per-capita NIH funding amount for each state was obtained by dividing the annual funding amount by the population that year. To enable comparisons between years, per-capita funding for each state was standardized by dividing by the mean for all states in the same year. Regional disparities in per-capita NIH funding were evaluated in 2 ways: standardized funding amounts and their changes over 20 years were calculated for each formal US census region (Northeast, Midwest, South, and West), and standardized values and 20-year changes for each state were plotted on US maps. To determine whether per-capita funding shifted from rural to populous states over the past 20 years, the temporal trends in correlation between per-capita funding and population density were examined. RESULTS: Year 2000 per-capita NIH funding ranged from $3 in Idaho, to $247 in Massachusetts. Over the past 20 years, 7 of the 10 states with the highest per-capita NIH funding in 1980 had relative decreases, and 7 of the 10 states with the lowest per-capita NIH funding had relative increases in per-capita NIH funding. During this time, the Northeastern region of the US received the highest relative per-capita funding (1.6 times the national average), but the proportion (34%) of total research dollars did not significantly change during that time. Analysis according to formal US census region, and by US map inspection, suggested that per-capita funding was relatively stable for large regions, but considerable shifting of percapita funding occurred within states of every region. States with relatively higher population density tended to receive higher funding per capita (r=0.59, p < 0.01), but this relationship did not substantially change during the past 20 years, suggesting that per-capita NIH funding is not moving toward populous states at the expense of more rural states. CONCLUSION: During the past 20 years, disparities in relative per-capita NIH funding have shifted substantially among states, but changed little among major regions of the US. There is no evidence to suggest that NIH funding became more concentrated in the states with the highest per-capita funding or population density during that time. PURPOSE: Repeatedly, race has been shown to be a powerful predictor of health and access to care, but the reasons for racial disparities in health often remain obscure and speculative. Recognizing many concerns about vague and essentialist notions of race in epidemiological and clinical research, the Institute of Medicine, the American Academy of Pediatrics and the National Cancer Institute have recently called for more cautious use of the race variable. The goal of this paper is to provide a much-needed synthesis of key critiques of race, some of which go beyond the concerns expressed by the aforementioned medical bodies. Armed with more complete knowledge about race, investigators should be better able to evaluate, plan, and conduct studies on health differences among human populations. METHODS: In an iterative fashion, and on the basis of clarity and logic, important critiques of race were identified through online and bibliographic searches and reviews of biomedical and social science publications and through discussions with colleagues. Subsequently, recently published studies in highly-regarded medical journals were selected to illustrate major themes. RESULTS: Delineating the causes of health disparities is often limited by misunderstandings of race and inadequate attention to its fundamental confounders, including: BIOLOGY (e.g. race is a social construct, not a biological one); SOCIAL CLASS (e.g. emphasizing biology diverts attention from considerations of SES, and SES is frequently gauged using an inadequate number of measures); CULTURE (e.g. biomedical studies of patients' culturally-based preferences and practices may rely on notions of culture that are overly deterministic, ethnocentric, and unduly constrained by the use of questionnaire data); ETHNICITY (e.g. self-reported ethnicity has become a common substitute for race, but although it offers some advantages, researchers should bear the ultimate responsibility for specifying membership in groups, ethnic or otherwise); RACISM (e.g. the most disturbing explanation for health disparities, racism is also one of the most difficult to study because it is politically charged and open to competing interpretations). CONCLUSION: A great deal of important work has been accomplished in terms of documenting the variety and widespread nature of health disparities, generally, and racial disparities in health, more specifically. The next step is extremely challenging: we must discover more precise information regarding causes and mechanisms. This can be accomplished by remaining aware of the shortcomings of overarching categories of race/ethnicity, focusing on more narrowly defined subgroups, and giving more explicit attention to the confounders of race.
TRENDS OF ORGANOPHOSPHATE POISONING IN URBAN ZIMBABWE. X. Dong 1 , M.A. Simon 2 ; 1 Yale University, New Haven, Connecticut; 2 Yale Medical Center, New Haven, CT PURPOSE: There have been 3 million reported pesticide poisonings and 200,000 deaths worldwide. In developing countries, poisonings are mostly associated with prescription medications, agrochemicals and household chemicals which have posed major global heath problems. Organophosphates are the most commonly formulated, packaged and used pesticides in Zimbabwe. However, there are no studies elucidating the current trends of organophosphate poisoning. The objectives of this study were to examine the mortality, trends and causes of organophosphate poisoning in an urban hospital in Zimbabwe. METHODS: We conducted a cross sectional descriptive study to examine the occurrence and trends of admissions for organophosphate poisoning in Parirenyatwa, one of the two major urban hospitals in Harare, Zimbabwe. We examined a total 185,828 patients' records and 599 cases on organophosphate poisoning from January 1995 through November 2000. Trends in admissions of the organophosphate poisoning were recorded. Other variables such as sex, age, season and geographic area were examined. Total mortality rates were calculated. The intent of the poisoning was also compared. RESULTS: These data reveal a steady increase in organophosphate poisoning during these years with the exception of 1999. The numbers of admissions for organophosphate poisoning have increased by more than 320% compared to six years ago. There is no difference in the male and female ratio for admission(48% vs. 52%). The majority of the cases admitted were below age thirty-one (82%)and were from urban settings (86%). Suicide is the predominant reason for poisoning (74%). There was a marked peak in the number of cases of children under age eleven (62%) that were due to accidental organophosphate poisoning. Mortality of organophosphate poisoning is 8.3% over the last six years. CONCLUSION: These results reveal startling realities. Organophosphate poisoning has been escalating throughout the world, especially in developing countries, such as Africa, South America and Asia, where agriculture demands large supplies of pesticides. Our data, although representing a very small microcosm of the extent of poisoning and in particular organophosphate poisoning, are consistent with multiple prior studies. Organophosphate poisoning is increasing uncontrollably and is being used at high rates as a suicidal agent in the young urban population, and contribute to the accidental ingestion in the pediatric population. Further research should concentrate on behaviors and situations that lead to poisoning. PURPOSE: Patients with medically unexplained symptoms (MUS) present a major problem for healthcare, yet they have not been adequately described. Physician perspective is especially important in providing the context for these patients' health perceptions, behaviors and outcomes. METHODS: A focus group of 6 internal medicine resident physicians was conducted to provide preliminary data for subsequent focus groups of primary care physicians of a panel of managed care patients with MUS. The discussion was tape-recorded, transcribed and analyzed via content analysis. RESULTS: Participants defined high utilization in terms of time and resource utilization and high physician emotional investment and not number of visits. Most participants described a typical high utilizing patient as one with multiple visits for uncontrolled organic disease or chronic, MUS. These patients were considered extremely difficult, demanding and unpleasant with a sense of entitlement. They believed that patients with somatization were often condescending, unappreciative and litigious. Interaction with these patients left the participants feeling frustrated, helpless, inadequate and powerless. Other emotions expressed include anger, guilt and apprehension about the possibility of missing an organic disease. Participants reported positive reactions to high utilizing patients (defined as more than 8 visits/year) with transient, minor acute illness. These patients were described as nice people with simple, straightforward problems, which got better regardless of what they did. Although they believed that the real motivation behind their visit was something other than their physical complaints, (such as stress, or the need for support and validation), they neither saw these patients' visits as problematic nor perceived a real need to address their possible underlying problems. Some participants considered these patients a major source of practice income and not high consumers of resources. Caring for high utilizing patients with organic disease was most gratifying. Most participants would rather not see patients with somatization; and the possibility of having to care for these patients as primary care physicians is a major factor in choosing to pursue subspecialization. CONCLUSION: This group of internal medicine residents did not consider high utilizing patient with MUS to be problematic if they had multiple transient minor acute illnesses. Conversely they found chronic somatization patients to be extremely problematic and would rather not care for them. Caring for patients with documented organic disease was gratifying regardless of utilization.
Center, Denver, CO PURPOSE: Among patients with hypoxemic chronic obstructive pulmonary disease (COPD), supplemental oxygen provides significant physical and cognitive benefits. Despite these benefits to therapy, no trial has demonstrated an improvement in subjective quality of life and patient adherence to therapy is low. The purpose of this study was to describe the experience of using supplemental oxygen therapy to identify barriers to its use and aid in improving adherence to therapy and quality of life among oxygen users. METHODS: 27 participants with hypoxemic COPD underwent semi-structured interviews to explore their experiences with supplemental oxygen therapy. Interviews were taped, transcribed, and analyzed for thematic content in a manner informed by grounded theory. RESULTS: The most commonly described barrier to oxygen use among participants was a fear of dependency. Two metaphors were used to describe this fear: crutches and addiction. As described by participants, a crutch was an external device or a behavior used to compensate for some weakness. Achievements and activities accomplished while using a crutch were considered tainted and illegitimate. Subjects feared that using a crutch made them more dependent on the crutch and less able to function without it. They feared that accepting dependency on a crutch was accepting a lesser state of being and a sign of moral weakness. Respondents described addiction as dependency on some substance used to illegitimately induce a sense of well-being or to reduce physical or psychic discomfort. Oxygen was likened to alcohol, psychotropic prescription medications, and illicit drugs. Participants equated using oxygen to relieve breathlessness with using alcohol or other drugs to alleviate psychic or physical discomfort. They feared that their need for oxygen would increase if they allowed themselves to become dependent on it and they considered using a substance to relieve their symptoms a sign of moral weakness. CONCLUSION: Fear of dependency is a significant barrier to oxygen use among patients who will benefit from supplemental oxygen therapy. In many patients, improving adherence to oxygen therapy may require exploring the metaphors of crutches and addiction with patients to help them see the flaws in these analogies. PURPOSE: The current procedures for procuring tissues and organs for donation have resulted in both organ and tissue shortages and general dissatisfaction with the current system. A new system for allowing individuals to delineate the treatment of their organs and tissues after death could improve donation and assure patient autonomy. METHODS: We reviewed and analyzed current research and opinions on upholding the wishes of patients regarding the treatment of the body after death. This included a review of literature regarding advance directives, organ and tissue donation, and burial preferences. RESULTS: It has been shown that for psychological health, individuals need to believe that they can decide what will happen to their bodies after their deaths. Many proposed systems for the procurement of organs and tissues for donation, including both presumed consent and the requirement of familial consent, directly or indirectly deprive the individual of control over the fate of his or her body after death. In addition, the fear caused by misconceptions regarding organ donation leads otherwise willing people to refuse to donate their own or their relatives' organs. CONCLUSION: In order to improve the current system of organ and tissue procurement, we propose the concept of the``Dying Will,'' a legal document in which an individual may delineate his or her wishes regarding organ and tissue donation, funeral procedures, and the like. In order to alleviate the fears of the individual, physicians could be responsible for helping their patients to write their dying wills. This might increase the number of individuals willing to donate their organs or tissues. In addition, with such a system, the permission of family members would be unnecessary for organ donation. This could further increase the number of available donors and prevent family members from overriding the wishes of the deceased. To expedite the transferal of dying will information to medical personnel, computerized databases such as the one recently initiated by Medic Alert could be used. PURPOSE: Since the beginning of the practice of medicine, family members have played a role in the treatment of a patient. Although this role has traditionally been limited to providing emotional support, loved ones can also become directly involved in doctor-patient communication or in the medical treatment of a patient. But there may be detriment to patient care due to the involvement of friends or family. METHODS: We reviewed and analyzed both literary and scholarly opinion on the role of the family in the practice of medicine. RESULTS: Family members have been described as both helping and hurting the quality of care received by patients. They can provide health care in the home, especially for chronically or terminally ill patients, often facing emotionally or technically trying situations. When a patient is mentally or emotionally compromised, a loved one can speak on the patient's behalf. But this kind of involvement can easily be detrimental to the patient if a friend or relative tries to control the patient or makes decisions based on his or her own values rather than on those of the patient. CONCLUSION: Friends and relatives can help a patient receive the best care possible; but when these individuals have selfish motivations or feel the need to control their loved one, they can do more harm than good. Physicians should take steps to encourage positive family participation while preventing this involvement from interfering in the patient's care. The physician must be able to tell whether a friend or relative is truly helping the patient or merely acting in his or her own interests. In addition, efforts should be made to contact supportive friends or family members for patients. When no loved one is available, a nurse, social worker, or appointed guardian could play the part of a third party in the doctor ± patient relationship. As indicated by the literature, the doctor ± patient relationship can greatly benefit from the involvement of a patient advocate. University of Michigan, Ann Arbor, MI PURPOSE: Physicians may occasionally encounter an ethical dilemma about whether to protect the rights of an individual patient, versus acting in the interests of society. When physicians act on the behest of society, such as in cases of impaired airline pilots, it is usually on the basis of legal imperatives or clearly stated normative guidelines. However, there may be cases where no such guidelines exist, or those in which physicians act to help society despite professional values to the contrary. We examine these situations. METHODS: We have conducted several studies in which physicians were asked about hypothetical dilemmas involving the potential for harm to the society, but in which there are no clear guidelines; or in which guidelines state that physicians should not act in behest of the society due to harm to the individual. These cases involve the report of a past crime by the patient, the issue of patient-initiated health insurance fraud, and the issue of physician involvement in lethal injection for capital punishment. We also conducted a literature review on this subject. RESULTS: A number of physicians in each of these studies would be willing to act on behalf of society and against the individual patient despite no clear normative values or those actually contrary to the physicians' decisions. When analyzed for the factors being most likely to influence the respondents, duty to society and the values of the overall population were most associated with the decision to act on behalf of society rather than in the interest of the individual patient. CONCLUSION: In some settings, a number of physicians are willing to act on behalf of society rather than on behalf of the patient. In such circumstances, physicians rely on their personal and societal values, rather on those promulgated within the medical profession. Some of these decisions may actually be harmful to the patient, and therefore contrary to ethical guidelines established by the medical profession. The impact of societal values on physician decision making needs further exploration.
PERKS AND PITFALLS: THE EXPERIENCE OF PHYSICIAN ± PATIENTS WITH CANCER. E.K. Fromme 1 , R.S. Hebert 1 , J.A. Carrese 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: Physicians have been described as the`worst' patients. Despite this, several prominent medical journals routinely feature writings by physician patients about their illnesses. This qualitative study examined the experience of physician patients treated for cancer. METHODS: Physicians who had a previous diagnosis of cancer other than non-melanoma skin cancer were recruited by their oncologists or radiation oncologists. 24 physician subjects representing diverse spectra of race, age, specialty, career stage and practice setting were identified. Various disease stages and tumor types were represented, from incidentally identified carcinoma to imminently terminal metastatic carcinoma. Subjects participated in in-depth semistructured interviews lasting an hour and a half. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major categories of themes. RESULTS: 5 themes representing experiences specific to physician patients are presented. (1) Trusting others to provide care presented a particular challenge to our physician subjects who were well aware of the potential for error and were used to being in control in the medical environment. Physician subjects ranged from total trust in their providers and a desire to`just be a patient' to getting care only in institutions where they had access to their own lab results and computerized records. (2) Physicians have the ability to doctor themselves, and our physician subjects varied in their willingness to do so. In one instance, a physician found himself ordering his CT scans, referring himself to specialists and administering his own chemotherapy. Others, unable to wait for their physicians, broke their own bad news by checking lab results. (3) Physicians with a serious illness are faced with a choice about disclosing their illness to colleagues. Our physician subjects who chose to do so elicited reactions ranging from strong support to total avoidance. (4) Similarly, physicians are faced with a choice about disclosing their illness to patients. Our physician subjects who chose to tell their patients reported that their patients often responded with interest and support, and that they used self-disclosure to facilitate some aspect of the medical encounter. (5) Our physician subjects perceived clear advantages as patients such as easier access to their records, better understanding of their conditions, and particularly prompt and supportive follow up by their care providers. CONCLUSION: Our physician subjects identified advantages and challenges in their experiences as patients with cancer. In some cases, their illnesses affected the way that they interacted with colleagues and patients, whereas their physician status affected the way that they interacted with their physicians and the health care system. The insights of these physician patients may be helpful to physicians who are seriously ill and to those who are currently healthy.
THE INNER CURRICULUM: LEARNING AND CHANGE BY PHYSICIAN ± PATIENTS WITH CANCER. E.K. Fromme 1 , J.A. Carrese 1 , R.S. Hebert 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: A common notion, exemplified in the 1991 motion picture The Doctor, is that physicians who experience serious illness themselves can learn to be more caring and therefore better physicians. This qualitative study examined what physicians with cancer learned from their illnesses and whether they felt the way they practiced medicine changed as a result. METHODS: Physicians who had a previous diagnosis of cancer other than non-melanoma skin cancer were recruited by their oncologists or radiation oncologists. 24 physician subjects representing diverse spectra of race, age, specialty, career stage and practice setting were identified. Various disease stages and tumor types were represented, from incidentally identified carcinoma to imminently terminal metastatic carcinoma. Subjects participated in in-depth semistructured interviews lasting an hour and a half. The interview questions were based on a literature review of writings by physicians with cancer and other illnesses in journal citations identified in MEDLINE. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major learning themes. Relationships between themes were examined and organized conceptually. RESULTS: Physicians' learning led to perceived changes in 4 main areas: (1) Understanding what patients are going through. Physicians better understood the patient's perspective, for example the anxiety created by waiting for test results or delayed surgery. (2) In some cases, this understanding was accompanied by changes in empathy that manifested as a felt connection to patients or a greater sensitivity to patients' emotions. (3) Changes in communication such as repeating important information, being careful not to minimize illness, and striving to deliver bad news skillfully. (4) Changes in values and priorities such as setting limits with employers and difficult patients, appreciating family, and acknowledging the possibility of death. CONCLUSION: Experience with a serious illness is a powerful source of learning for many physicians. In some cases, their experiences caused changes in their attitudes, their communication, and other aspects of their medical practice. Educators interested in teaching about patient centered care might utilize the experience and insights of physician patients. PURPOSE: There is a well-documented interest in genetic susceptibility testing among firstdegree relatives of women with breast cancer. Interest in, and uptake of, testing has been shown to remain high despite education regarding risks and limitations of testing suggesting unknown factors influencing the decision to test. The purpose of this study was to explore factors influencing interest in genetic testing for breast cancer. METHODS: A qualitative study design exploring interest in genetic testing was chosen to allow us to establish the appropriate parameters for future intervention research. We conducted 4 focus groups with 10 ± 12 participants each. Participants were recruited from the general population. Women included in the study were 18 ± 74 years old and had at least one firstdegree relative with breast cancer by self-report. Focus groups were videotaped and transcribed. Content analysis was performed including independent extraction of themes by 2 authors and confirmation of themes by a third. A computerized software program was used to systematize the process. RESULTS: Two critical domains influencing interest in testing were identified: 1) Selfperceived risk, and 2) Perception of testing as loss or gain of control. Accurate self-perceived risk required an understanding of critical genetic concepts such as sporadic vs. hereditary cancer. Women who categorized themselves as low risk for hereditary cancer had less interest in testing while those who categorized themselves as moderate to high risk were more interested in testing. Women who were interested in testing viewed it as gaining control,``knowing what you're facing,'' and``taking measures to prevent it.'' Women who were not interested in testing were fearful of losing control over their lives, of genetic determinism. Women were able to understand the probabilistic nature of genetic concepts discussed. Paradoxically, understanding that genetic testing would provide probabilities rather than``black and white answers'' made testing more acceptable to some women by allowing them to maintain``a percentage of control'' over their lives. Willingness-to-pay for testing was explored as a proxy for interest in testing. The cost of testing was prohibitive for all but those women who considered themselves at high risk. If cost were not an issue many women would choose to get the test. Concern for genetic discrimination was raised. CONCLUSION: Interest in genetic susceptibility testing for breast cancer is influenced by selfperceived risk and knowledge and beliefs about genetic testing. Comprehension of critical genetic concepts is necessary to accurately assess risk and understand the risks and limitations of testing. Cost and concern for genetic discrimination will affect uptake of testing.
TRUST AS A FRAMEWORK FOR ETHICAL ANALYSIS. S.D. Goold 1 ; 1 University of Michigan, Ann Arbor, Michigan PURPOSE: Examine trust as a sociological and philosophical construct. Argue that trust can serve as a valid and useful conceptual framework to examine ethical issues in healthcare. METHODS: It is widely acknowledged that trust is a vital component of and basis for relationships between clinicians and patients and that these interpersonal trust relationships have moral content. Trust is especially important in health care, and is present in relationships with organizations (e.g., hospitals, insurers) as well as with clinicians. In this paper I will describe the nature and elements of trust-based relationships, both between individuals (i.e., interpersonal trust) and between individuals and organizations (i.e., institutional trust), and the relationship between interpersonal and institutional trust. I will argue that trust is a valid foundation for healthcare ethics because healthcare is relational and serves vulnerable parties. I will discuss conditions and actions that may increase or decrease actual trust. I will then compare and contrast these with actions and conditions Ð both for individuals and organizations Ð that may influence trustworthiness. Texas A&M University, College Station, TX PURPOSE: Anthropologists have proposed the concept of explanatory models of illness to explain discrepancies between patients' and physicians' views of the illness experience. Because large socioeconomic differences often exist between VA physicians and patients, we performed this qualitative study to explore variations and common tendencies among veterans' explanatory models of illness and the impact that these models have on veterans' perceptions of health and healthcare. METHODS: We developed an interview guide to elicit patients' perspectives about the cause of, meaning of, severity of, amount of control over, and perceived treatment efficacy for their illness. Using this guide, we conducted 10 in-depth, semi-structured interviews with patients at the Houston VAMC one hour prior to their regularly scheduled primary care visits. We audiotaped and transcribed the interviews. We analyzed these transcripts through 3 iterations of individual reading and team discussion to identify common themes that occurred in our 5 areas of inquiry. RESULTS: All participants identified themselves as Caucasian, with ages ranging from 52 to 81. Most participants were seeking care for at least one chronic illness. While some veterans' responses to our specific questions could be considered to fit a biomedical paradigm (e.g. Interviewer:``What do you think caused your diabetes?'', Patient:``My pancreas quit working.''), we discovered that broader, non-biomedical stories of experience, or metanarratives, existed across our categories of questions. These meta-narratives described themes pertaining to patient fatalism and self-efficacy and preferences for physician roles of acknowledgement, action, and communication during the healing process. In cases where veterans perceived their physicians to not attend to these meta-narratives, veterans were dissatisfied and perceived a poorer quality of life. CONCLUSION: While veterans' answers to specific questions about the experience of illness may follow a biomedical paradigm, broader, non-biomedical narratives emerge when these answers are viewed in aggregate. Physician understanding of these narratives during the medical interview is important for enhancing the patient's ability to cope with chronic illness. PURPOSE: Repeated calls have been made for educators to foster students' personal awareness of attitudes and perspectives that influence their professional practices. As part of an exercise intended to foster such personal awareness, all students at Baylor College of Medicine (BCM) complete several standardized instruments (SI) that measure their views toward the physician ± patient relationship, psychosocial care, and stress from uncertainty. Students complete these instruments in their first and third years and, after an explanation of the constructs that are measured by the instruments, receive feedback consisting of their own scores, the average scores of their classes, and published normative data from physicians in various specialties of medicine. We conducted this qualitative study to explore the meanings that students attribute to this feedback. METHODS: We conducted focus groups with students in each of the classes of 2000, -01, -02, and -03 at BCM (one group for each class). We developed guiding questions that explored the effects of the SI scores on students' perceptions of self-knowledge, abilities, and attitudes (a conceptual framework for personal awareness training proposed by Novack, et al). Focus groups were co-moderated by a sociologist and a physician. All focus groups were audiotaped and transcribed. We developed categories for transcript coding by noting the patterned occurrence of specific behaviors, attitudes, and values in the transcripts. RESULTS: We conducted 4 focus groups with 22 students; focus groups lasted an average of 90 minutes. Students from different classes framed the information provided by SI scores differently. Preclinical students expressed a lack of experience with patient care, and therefore completed the questionnaires relative to their best guess of the answers of an 'ideal' physician. This led to anxiety when these 'ideal' physician scores differed unexpectedly from the normative scores of the class or of practicing physicians. Clinical students completed the SIs relative to their own patient-care experiences and devalued scores that differed from norms that they saw as different from their own emerging professional identity. CONCLUSION: Using a 'trigger' such as SI scores may be a useful means to stimulate student self-reflection. Such feedback, though, should be framed in the context of student experience. Preclinical students struggle with the meaning of feedback given their perceptions of how an 'ideal' physician thinks and acts. Clinical students possess an emerging professional identity and may devalue feedback that is inconsistent with this identity. In order to foster professional attitudes and behaviors using feedback, educators should take into account differences in preclinical and clinical students' perceptions of the meaning and value of feedback.
OLDER HEMODIALYSIS PATIENTS PERSPECTIVES ON QUALITY OF LIFE. S.E. Hardy 1 , S.T. Crowley 1 , T.R. Fried 1 ; 1 Yale University, New Haven, CT PURPOSE: As the dialysis population has aged, quality of life (QOL) has become an increasingly important outcome for both clinicians and researchers in end-stage renal disease. Unfortunately, most QOL scales, whether generic or renal-specific, have been developed with limited input from patients, particularly older patients. The aim of this study was to identify those factors most important to older hemodialysis patients in determining their QOL. METHODS: Semi-structured interviews lasting 20 to 60 minutes were conducted with 27 hemodialysis patients, aged 65 years or older, who had been on dialysis for at least 90 days.
The interviews used open-ended questions, such as``How has kidney disease requiring dialysis affected your quality of life?'' Two coders independently reviewed the interview transcripts to identify themes, resolving disagreements by consensus. Themes were compared within and across subjects to identify and organize important concepts. Subjects were enrolled until the point of thematic saturation, when further interviews revealed no new themes. RESULTS: The 27 subjects interviewed had a mean(SD) age of 747 years and had been on hemodialysis for an average of 3.53.1 years. They identified five domains important to QOL: symptoms, burdens of treatment, benefits of treatment, information needs, and relationships with providers. The most common symptoms reported were fatigue, balance problems, weakness, cramps, itching, and nausea. Burdens of treatment included time spent at dialysis, difficulty traveling, problems with vascular access, and giving things up. Benefits of treatment included relief of symptoms, social interaction, and enhanced access to health care providers. Subjects noted the importance of adequate information about how to care for themselves and what symptoms or problems to expect, and of competent and caring providers who treat them with respect and acknowledge their individuality. Symptoms and burdens of treatment affected QOL negatively and benefits of treatment affected QOL positively, while the remaining domains could have either positive or negative effects. The magnitude of these effects was influenced by non-medical factors, such as social support, family relationships, formal instrumental support, finances, and spirituality. Subjects universally noted the necessity of dialysis for sustaining life, and preferred even a low QOL on dialysis to death. CONCLUSION: When asked about their perspectives, older hemodialysis patients, while acknowledging the necessity of dialysis, identified a variety of domains important to their QOL as dialysis patients. Several of these domains, particularly information needs and the benefits of treatment, are not found in existing generic or renal-specific QOL scales. In developing such scales, it is important to elicit the factors most important to patients and ensure that these are included.
TAKING CARE OF OUR OWN: WHEN A RESIDENT BECOMES A PATIENT. R. Harrison 1 , S. Desai 1 , D. Webster 1 , A.J. Hunter 1 , J.L. Bowen 1 ; 1 Oregon Health Sciences University, Portland, OR PURPOSE: To date there are no studies discussing the experiences of caring for residents who are hospitalized at their own training institution. While providing care for a hospitalized resident at our institution a great deal of turmoil developed among the housestaff and faculty over the issue of house officers providing care for their peers. Using the critical incident technique we assessed the experiences of the resident patient and the house staff taking care of their peer in the hospital. METHODS: In order to better understand the origins and nature of these conflicts focus groups were performed that included the resident patient and the house staff who provided the care. We convened 3 focus groups with the house staff and a separate session with the resident patient. All five residents who cared for the hospitalized resident were invited to participate in this study on a voluntary basis. Consent was obtained before audio recording each confidential session. Each session facilitated by the investigators consisted of one to three resident physician volunteers who were asked to describe feelings, both positive and negative surrounding care of their peer. The audiotapes were transcribed, independently analyzed and coded by each investigator. The coded transcripts were then compared and discussed generating a coding scheme. Disagreements were discussed and resolved by consensus. OHSU IRB approved this study. RESULTS: One hundred percent of eligible participants completed the study. Caring for a colleague had an overwhelmingly negative impact on the residents routine medical decision making, work structure, and team dynamics. Barriers to routine care of the hospitalized resident included intense emotional experiences, ambiguity in the care structure, and concern for confidentiality and privacy. This resulted in paralysis in care decisions leading to reactionary care or even care avoidance. Lack of communication amongst team members, faculty and patient was a commonly sited experience. The housestaff patient perspective was equally negative. Problems with communication and care delivery were emphasized particularly surrounding the issues of objectivity and confidentiality. Themes of vulnerability, frustration and powerlessness dominated the narratives of the resident patient. CONCLUSION: The critical incident technique offers a unique perspective into the experiences of residents taking care of peers in the hospital. Many of the themes were identical to both the residents and resident patient including issues of confidentiality, communication and intense emotional conflict. While many residents choose medical care at their own training institutions, this study demonstrates the potential difficulties with this approach and suggests the importance of determining a policy of care for the hospitalized resident.
WOULD YOU BE SURPRISED IF THIS PATIENT DIED: EXPLORING THE TRANSITION BETWEEN AGGRESSIVE AND PALLIATIVE CARE. D.C. Johnson 1 , J.S. Kutner 1 , J.A. Armstrong 1 ; 1 University of Colorado Health Sciences Center, Denver, CO PURPOSE: Multiple studies have shown that physicians inadequately address the suffering of severely ill patients at the end of life. This deficiency, in part, arises from the difficulty in recognizing the dying patient and an overly restricted focus on aggressive care. The purpose of this study was to examine the thought processes of resident physicians taking care of critically ill patients, with a goal to better understand how physicians transition between aggressive and palliative care, and to determine if the explicit consideration of the possibility of death improves attention to the suffering of patients and their families. METHODS: Internal medicine resident physicians (n=8) caring for severely ill patients were sequentially interviewed over a four week period during Medical Intensive Care Unit Ethics and Discharge Planning rounds. The residents were asked a series of questions based on their response to the core question``Would you be surprised if this patient died?'' The interviews were taped, transcribed and qualitatively analyzed for the purpose of identifying common patterns in the decision-making process of physicians when managing severely ill patients. RESULTS: When asked the core question, there were an equal number of cases in which residents responded that they would (n=4) versus would not (n=4) be surprised if their patient died. Reasons for being surprised that a patient would die included the presence of a reversible disease, the rapid onset of an acute illness, situations where a patient was``doing better'' and prior survival under similar circumstances. Among residents who indicated that they would not be surprised, most (n=3) reported that their management would not change knowing that their patient might die. Cited changes, when present, included clarifying goals, improving communication with families, spending more time with patients and ordering fewer labs. Stated or implied barriers to changes in management despite knowing that a patient might die included a lack of time and experience,``not knowing'' a patient, an improvement in medical condition (``doing better''), a lack of clear goals (``doing everything''), the presence of uncertainty and an exclusive focus on technical intervention. CONCLUSION: Asking the question``Would you be surprised if this patient died?'' encourages proactive reflection and dialogue about the potential for death in severely ill patients. An active acknowledgment of the possibility of death may lead to changes in management that would potentially diminish the suffering of patients and their families. To effectively impart such changes, it may be necessary to develop and implement techniques to overcome identified barriers. PURPOSE: A physician's effectiveness depends on the application of good communication, cognitive, and technical skills, guided by maturity, wisdom, compassion, and integrity. Development of the latter attributes requires growth in the awareness and management of one's feelings, attitudes, beliefs, and life experiences. Yet there is a dearth of empirical research related to physicians' personal growth. We used qualitative research methods to analyze stories of personal growth obtained from a selected group of medical faculty, in order to increase understanding of personal growth in this group and to develop a conceptual framework that might be useful to medical educators and researchers. METHODS: Questionnaire survey of facilitators, facilitators-in-training, and members of a personal growth interest group of the American Academy on Physician and Patient, who were chosen because of their interest, knowledge, and experience in the topic area. Respondents were asked to submit one or more stories of growth in their own self-awareness, and how that growth had affected them professionally and personally. Themes were identified in the submitted stories by 5 members of the research team and sorted into categories by 3 members. Slight further revisions were made following discussion by the entire research team and application of the proposed categories to a subset of stories. The findings were verified by two independent reviewers, who reviewed all of the stories. RESULTS: 32 of 64 subjects returned questionnaires containing 42 stories. There were no significant differences between respondents and non-respondents in age (mean 45.3 vs. 44.6 years), gender ( 75% vs. 69% male), or specialty (69% vs. 72% internists, 28% vs. 19% behavioral scientists / psychiatrists). Usually personal growth stories began with a powerful experience and/or helping relationship, proceeded to introspection, and ended in a personal growth outcome. Personal growth outcomes included: changes in values, goals, or direction; healthier behaviors; improved connectedness with others; improved sense of self; and increased productivity, energy, or creativity. CONCLUSION: Powerful experiences, helping relationships, and introspection are processes that preceded personal growth in a subset of medical faculty. The findings, if confirmed in other populations of physicians, may have implications for medical education and practice. The ABIM expects all physicians board certified in internal medicine to posses humanistic qualities of integrity, respect and compassion. To date, no studies have been done comparing humanistic qualities among physicians at different levels of training. We compared humanistic quality scores of fourth year medical students (SI), internal medicine residents (PGYI and II), and attending physicians on a general medicine ward of a teaching hospital. METHODS: A validated seventeen-item nursing survey to assess humanistic qualities among physicians was distributed to randomly selected nurses working day shifts on the medicine wards. The survey measured physician relationships with other medical staff, and the physician relationship with the patient and family. Each item was scored on a 5 point Likert scale. Composite scores for physician to staff relationships (PSR) and physician to patient/family relationships (PFR), as well as an overall evaluation score (OE), were compared across levels of physician training. Student's t-test was done to determine statistical significance across training levels. RESULTS: 33 nurses completed a total of 295 questionnaires. Surveys were performed for each of the 7 sub-interns, 8 PGYI, 4 PGYII, and 11 attending physicians rotating on the medical wards during a month of the ward service. SI's had higher PSR scores (PSR = 24.2) than PGYI (PSR = 21.3, p value = 0.013), PGYII (PSR = 21.9, p = 0.042) , and attending physicians (PSR = 23, p < 0.01). SI's also had higher PFR scores (PFR = 33) compared to PGYI (PFR = 29.4, p = 0.025), PGYII (PFR = 28.3, p < 0.001) , and attendings (PFR = 31.7, p < 0.01). Finally, SI's had higher OE scores (OE = 4.4) when compared to PGYI (OE = 3.8, p < 0.01), PGYII (OE = 3.8, p < 0.01), and attendings (OE = 4.2, p < 0.001). No statistically significant differences were found comparing PGY I, PGY II, and attending levels among each other. CONCLUSION: Sub-interns appear to have better perceived qualities of humanism compared to resident and attending physicians. Because resident and attending physicians play an important role in medical education, efforts should be made to better understand the mechanisms of these differences, and to improve the humanistic qualities of both resident and attending physicians. PURPOSE: Academic Medical Center (AMC) leaders must respond to numerous internal and external challenges. Self reports from leaders have documented these challenges; however, little is known of the daily workings of AMC leaders and the tactics used to run an AMC in the 21st century. METHODS: Ethnographic methods were used to explore the challenges faced by leaders in the top 3 levels of one institution's organizational chart. The study was conducted at a public Midwestern school, with 1600 faculty and $165 million in NIH funding. The data are: 1)10 hrs. of interviews with leaders and 2)field notes and minutes from over 300 hrs. of participant observations in meetings. Field notes captured process (e.g. deference, control, tolerance etc.) and content (e.g. parking, safety, and budget concerns etc.). The team then identified themes by iterative review of field notes, with validity assessed by comparison with interview responses and subsequent member checking. This report focuses on identified content themes. RESULTS: Six broad content domains are recurrent: 1)faculty morale, 2)business issues e.g. intellectual property rights and establishing frameworks for joint ventures between AMCs, business and/or government. 3)developing affiliations with others to remain competitive 4)creating plans of action to improve diversity 5)creating tools and metrics to demonstratè`a cademic productivity'' & assure society that graduates are competent. 6)maneuvering AMCs to adjust and survive a unpredictable environment. CONCLUSION: AMC's are currently facing instability. This study of leaders day to day activities reveals challenges in 3 broad areas: A)human resources, B)institutional fiduciary roles and C)vision setting. Human resources include faculty, student and staff morale, issues of workforce diversity and assessment of``academic productivity''. The institution has a fiduciary responsibility in ensuring financial stability, education and assuring graduate competency. Finally vision setting includes the ability to adjust and survive in an unpredictable environment. Improved understanding of leadership challenges will allow better preparation of new generations of leaders, and will better prepare AMCs for future challenges. Talking Medicine) developed for third year medical students to teach humanism and professionalism, we asked students to define these concepts and use these definitions to spark small group discussion. METHODS: At the beginning of each internal medicine clerkship we asked students to define humanism and professionalism anonymously on sheets of paper to be handed to the preceptors. We conducted a content analysis of 3 small groups' definitions (n=14). RESULTS: In Project Professionalism, the ABIM defines professionalism as: Altruism, Accountability, Excellence, Honor / Integrity, and Respect. The Students, however, saw a broader definition of professionalism. Themes that they identified included: 1) being a physician, student, teacher, philosopher and healer all at once, 2) doing no harm, 3) understanding that``respect for others'' must include tolerance of differences; as well as a focus on collegiality among practitioners at various levels of the medical hierarchy 4) having appropriate speech, dress and emotions'-which should often be held in``check'', 5) honoring the system, but not blindly and 6) stating when a good job has been performed. CONCLUSION: Third year medical students tend to agree broadly with the ABIM components of Professionalism. However, they focus more on tolerance of difference (e.g. race, socioeconomic status, and varying health beliefs), and the importance of collegiality and collaboration in the new environment of patient care. Their vantage point early in training allows them to look critically at the profession which they are joining and view its shortcomings and strengths. Future work will focus on how these definitions change as students progress through 3rd and 4th year and into residency.
MINORITIES AND EVIDENCE BASED MEDICINE, DO RANDOMIZED CONTROLLED TRIALS PROVIDE THE ANSWER? S. Malhi 1 , S.M. Sandhu 1 , H.S. Gurm 2 ; 1 Fairview Hospital, Fairview Park, OH; 2 Cleveland Clinic Foundation, Cleveland, OH PURPOSE: Race has a significant impact on prevalence, presentation and outcome of disease. While the randomized controlled trials (RCTs) have become the gold standard for evidence based medicine, there is a paucity of data on representation of minorities in RCTs. The purpose of this study was to review minority enrollment in RCTs published in a major medical journal. METHODS: We conducted a systematic search of all articles published in the original articles section of the New England Journal of Medicine from 1993 ± 2000. All randomized control trials done in North America were included for abstraction. We excluded all studies limited to certain ethnic groups, subset analysis of other trials and meta-analyses. Trials were characterized by primary speciality and source of funding. Data were analyzed using Students t test and ANOVA. RESULTS: 1799 original articles were published in the period between 1993 ± 2000 including 535 Randomized controlled trials. Of the 302 RCTs done in North America, only 106 specified the percentage of minorities enrolled. A total of 180,364 patients were enrolled in these 106 trials of which 37,653 were minorities (20.8%) . The maximum number of trials (80) were in cardiovascular medicine. The minority enrollment was specified in only 24, (30%) and in these the minorities constituted only 14% of the enrollees. (4766/34,132) . In contrast to cardiovascular medicine, 15 out of 22 trials (68%) in pregnancy related disorders and 12 out of 27 RCTs (44%) related to pediatric population specified minority enrollment. Similarly, the trials in these groups enrolled a larger percentage of minorities: 40.7% (13928/34220) in pregnancy related trials and 51.3% (3253/6343) in the pediatric trials. After trials in pregnant women and children are excluded only 6% (20,472/293,297) of enrolled subjects were minorities. CONCLUSION: Although minorities now make up almost one third of the American population, they are significantly underrepresented in randomized controlled trials. Most studies fail to specify the ethnic or racial mix of the population and thereby significantly limit the generalizability of the results. At a time when all research is held up to the gold standard of the RCTs, the RCTs fail to provide data on a significant segment of the population. Our data underscores the importance of an ongoing effort to improve minority enrollment and ethnic specific analysis in clinical trials. PURPOSE: Among the rehabilitation options for persons with substance abuse disorders are the prevalent and increasingly publicized faith-based programs. We sought to describe key structural aspects of substance abuse recovery programs within one major national Christian organization. METHODS: A convenience sample of 7 administrators, clinical directors, and counselors from 4 New England-based residential recovery programs was identified. Each subject was interviewed for 60 minutes using a semi-structured protocol. Using qualitative methods, key words and phrases were derived from all instances where aspects of the program structure were mentioned. The key words were placed into groups that categorized the major structural elements of the program RESULTS: The programs in this study provide long-term free services for men and women of all faiths. All programs are self-supporting and receive no governmental funding. Religious (or spiritual) components mentioned were Bible studies, worship and devotional services, vespers and prayer meetings, spiritual growth classes, retreats, and Alcoholics Anonymous or Narcotics Anonymous meetings. Individual and group therapy sessions also included spiritual references and themes. Secular components were medical and dental services, mental health services, Graduate Equivalency Diploma classes and job skills training, work therapy, sponsorship, access to physical fitness facilities, recreational activities, and aftercare program planning. CONCLUSION: Activities relating to religious study, worship, and spiritual growth are critical components of the faith-based recovery programs studied. However, the religious aspects of these programs appear to complement rather than replace the elements seen in secular programs. Further analyses of how their philosophy of faith is translated into practice are needed. PURPOSE: Tamoxifen was FDA approved for breast cancer risk reduction in 1998 for women who have a 5 year risk > 1.7% (average risk of a 60 year old white woman). Healthy women deciding whether to take it for prevention must weigh its potential benefits and adverse effects. Purpose: To develop an understanding of how ethnically diverse risk-eligible women weigh risks and benefits regarding tamoxifen prophylaxis. METHODS: We undertook a qualitative intervention study using a focus group-format to provide general information about the risks and benefits of tamoxifen for breast cancer risk reduction and to assess the concerns of ethnically diverse (Caucasian, African-American, and Latina) women about the use of tamoxifen prophylaxis. Group facilitators were matched to the ethnicity of participants. Focus group discussions involved: (1) exploration of knowledge of the risks and benefits of tamoxifen as a preventive therapy; (2) a standardized educational intervention about tamoxifen's risks and benefits based on findings from the Breast Cancer Prevention Trial; and 3) discussion about participant decision-making and tamoxifen's overall value in prevention. All participants completed a brief questionnaire to assess the impact of the intervention on their understanding of tamoxifen as preventive therapy. Focus groups were audiotaped and later transcribed for analysis. A multi-disciplinary team of investigators identified prominent themes related to health beliefs that emerged from iterative review of focus group transcripts. RESULTS: 27women (age range 61 ± 78) participated in the focus groups. Most reported that they felt more informed after the group. In general, fear of breast cancer was not prominent, and participants were not inclined to take tamoxifen as preventive therapy. Decisions about tamoxifen were based on participants' assessment of the balance of competing risks and competing benefits. Specifically, participants expressed limited willingness to take medication with serious side effects for breast cancer risk reduction, uncertainty about its interaction with other medications, and concern about the need to discontinue hormone replacement therapy. Caucasian and Latina groups expressed concerns about reliability of scientific studies. African ± American women described faith as important to prevention. CONCLUSION: Women were wary of taking a drug for a disease they might not develop. Women felt they had options other than Tamoxifen to reduce risk of breast cancer, including early detection, diet, faith, and alternative medicine. Information about tamoxifen decreased interest in use for breast cancer prevention. PURPOSE: Although shared decision-making has been advocated for screening mammography for women under age 50, little is known about how women decide whether to get screened, what are their preferred sources of information and what are their preferences for involvement in screening decisions. This study explored these issues in patient decision-making. METHODS: We conducted in-depth, semi-structured telephone interviews with 16 randomly selected English-speaking women ages 38-45 who were enrolled at a large New England HMO, and had no prior history of breast cancer. Interviews were transcribed and coded into major themes using QSR NUD*IST. RESULTS: Thirteen participants were aged 41 ± 45 and 3 were aged 38 ± 40. Nine were white and seven were African ± American. The majority (10/13) of women over age 40 had prior mammograms and all intended to have a screening mammogram before age 50. Factors in women's decisions to undergo screening included knowledge of the``recommendation to begin screening at age 40'' obtained either from their physicians or the media and``having a friend or acquaintance with breast cancer.'' Most women stated that screening has a number of benefits, including``early detection,''``peace of mind'' and``it's better knowing.'' Possible risks of mammography, although not considered important, included radiation, pain, and``not being able to catch everything.'' None of the women who heard of or experienced false positive mammograms (8/16) were or would be deterred from future screenings. While all women stated that the physician should be the primary source of information about screening, 11/16 had no discussions with their own physicians regarding the mammography procedure, its risks and/or benefits. Many (12/16) learned such information from the media. Preferences for involvement in decision making varied; 4/16 preferred to leave screening decisions mostly to the physician, 9/16 preferred to make them mostly themselves, and the rest were uncertain. We found no major thematic differences among the white and the African ± American women. CONCLUSION: Our results suggest that in this setting the decisions of women under 50 to undergo screening mammography are based on their own perceptions of risks/benefits or on the recommendation of their physicians. However, in these recommendations, discussion of specific information relating to screening rarely occurs, and shared decision-making is limited. These findings can guide future research and lead to the development of policy that facilitates patient ± physician communication in this area.
K.E. Olive 1 , J.K. Neumann 2 ; 1 East Tennessee State University, Johnson City, TN; 2 James H. Quillen VA Medical Center, Mountain Home, TN PURPOSE: A previous study indicated that the absolute vs relative ethical construct is a factor influencing physician decisions. The purpose of this study was to confirm, in a different physician population, the previous results Ð that physicians who believe in ethical values which do not change (absolute values) will respond differently to ethically sensitive clinical scenarios than those who affirm changing ethical values (relative values). METHODS: A national sample of family physicians and psychiatrists received a mailed survey. The survey included a four-item scale to determine relative vs absolute values, demographic and psychological measures, and three clinical scenarios involving contraception, physician-assisted suicide, and abortion. The primary analysis compared approval of the clinical scenarios for physicians with absolute values to those with relative values. Approval ratings were determined using a seven point Likert-type scale (1=do not approve, 7=do approve). Data were analyzed using analysis of variance. RESULTS: 317 of 1000 physicians surveyed (32%) responded. Of those responding, 99 met criteria for absolute values and 59 for relative values. Those endorsing absolute values were significantly less approving of all three scenarios than those endorsing relative values: Contraception (5.5 vs 6.4, F=10.17, p=.0017), Physician Assisted Suicide ( 2.1 vs 4.1, F=36.22, p < .00001), Abortion (3.0 vs 5.0, F=28.45, p < .00001). CONCLUSION: Physicians with absolute ethical values are less approving of contraception for single women, physician-assisted suicide, and abortion than those endorsing relative ethical values. The absolute vs relative values ethical construct may be useful in examining physician attitudes which impact health care delivery. These survey results replicate a previous study indicating the absolute vs ethical construct is a factor influencing physician decisions. This construct is directly relevant to clinical care and physicians need to be aware of their own biases in discussions with patients, families, and other health care providers.
PURPOSE: As of December 2000, 31 states passed mental health parity statutes, laws requiring a health plan, insurer, or employer to provide mental health benefits``equal'' to physical health benefits. With parity, lawmakers are for the first time legislating their own state definitions of mental illness rather than leaving the definition up to insurance plans or covering all disorders in the American Psychiatric Association's Diagnostic and Statistical Manual (DSM-IV) as legislation has traditionally done. My research examines definitions of``mental illness'' used in state parity laws and implications of these definitions including what populations are getting increased access to care. METHODS: I reviewed the statutory language in 31 state parity bills and conducted a series of interviews with stakeholders participating in the national and state mental health parity process. Interviewees included mental health advocacy groups, provider organizations, legislative bill sponsors, health plans, and employer groups. Interview data was supplemented with extensive literature review. RESULTS: The definition of mental illness varies significantly across states, but falls into one of three categories:``broad-based mental illness,''``serious mental illness,'' and``biologically-based mental illness.'' Broad-based mental illness includes all disorders listed in the DSM-IV and/or World Health Organization's ICD-9.``Serious mental illness'' and``biologically-based mental illness'' include from three to eleven specific DSM-IV mental disorders. Several factors influence a state's definition of mental illness including the cost of parity, the leadership role taken by state advocacy group(s), the strength of insurance interests, the reliance on antidiscrimination arguments, and the political feasibility of parity in the state. CONCLUSION: With parity, state lawmakers are not relying on clinically accepted definitions or previous federal and state definitions of mental illness. It appears that political and economic factors influence the statutory definition, rather than needs-based strategies or clinical judgement. Insurers argue that legislating parity for all mental disorders results in tremendous healthcare costs and necessitates the development of definitions that target only the most seriously mentally ill. However, using a``pick and choose'' approach to the DSM-IV as most states have done has little (if any) clinical basis, and can potentially limit access to care. Increased reliance on clinician judgement and further studies measuring the access implications of current parity laws are needed to identify those who would most benefit from parity. PURPOSE: Ethnic culture ± the basic beliefs an ethnic group uses to interpret experience ± affects patients' views on dying. But views within an ethnic group are not all alike. The genders within an ethnic group may share some views but differ on others and, thus, be distinct subcultures for advance care planning purposes. METHODS: We explored this possibility by asking Mexican Americans (14 men, 12 women), Euroamericans (7 men, 11 women), and African Americans (7 men, 7 women) their views on advance directives (ADs). All subjects were inpatients aged 50 to 79. RESULTS: While believing ADs improve the chances a patient's wishes would be followed, the genders within all three ethnic groups differed over the power and trustworthiness of the health care system, treatment wishes and the willingness to express them, and beliefs about ADs. Among Mexican Americans most men but few women believed the system controls treatment (79% versus 33%), believed patients should be allowed to die when treatment is futile (57% versus 42%), wanted no life support (71% versus 42%), had not told anyone their wishes (64% versus 42%), and believed ADs are needed for when a patient is brain dead or cannot function (57% versus 33% for both themes). But few men and most women trusted the system to honor written ADs and believed ADs help the staff know a patient's wishes (43% versus 67% for both themes). Among Euroamericans most men but few women distrusted the system (57% versus 18%) and disliked ADs (57% versus 36%). But few men and most women expressed wishes not to suffer during terminal illness (0% versus 55%), believed patients should be allowed to die when treatment is futile (0% versus 55%), and believed the system honors written ADs (43% versus 73%). Among African Americans most men but few women distrusted the system (57% versus 29%) and wanted to wait until very sick to express their wishes (57% versus 43%). And few men but most women said that the patient deserves a say in treatment (43% versus 57%), wanted no life support (29% versus 57%), and believed ADs help staff know the patient's wishes (0% versus 57%) and can prevent life support (14% versus 57%). CONCLUSION: While sharing some views, men and women of each ethnic group differed on others. Unlike men, women tended to trust the system and to believe ADs will help realize their wishes. Providers should consider gender-specific values within an ethnic culture when doing advance care planning with patients. PURPOSE: Ethnic culture ± the beliefs an ethnic group uses to interpret experience ± affects patients' views on dying. But views within an ethnic culture are not all alike. Age groups within an ethnic culture may share some views but differ on others and, thus, be distinct subcultures for advance care planning purposes. METHODS: To explore this possibility we interviewed Mexican Americans, Euroamericans, and African Americans. Younger subjects (ages 50 ± 64) numbered 15, 11, and 10 respectively; older subjects (ages 65 ± 79) numbered 11, 7, and 4. RESULTS: While all ethnic groups liked some aspects of advance directives (ADs), age groups within each ethnic group differed over trust in the health care system, treatment wishes, and beliefs about ADs. More younger than older Mexican Americans wanted to be allowed to die when treatment is futile (60% versus 36%), had not told their wishes to anyone (60% versus 45%), believed ADs are needed for when a patient cannot voice decisions (60% versus 27%), and trusted the system to honor written ADs (60% versus 45%). Fewer younger than older Mexican Americans expressed wishes unrelated to life support (for example, where or when they wished to die) (33% versus 64%). More younger than older Euroamericans described unacceptable outcomes (55% versus 43%) and believed that ADs help staff know a patient's wishes (91% versus 29%) and are needed for when a patient is brain dead (64% versus 29%). Fewer younger than older Euroamericans disliked some aspects of ADs (36% versus 57%), had not told anyone their wishes (18% versus 71%), and doubted ADs change treatment (27% versus 57%). More younger than older African Americans believed the system controls treatment (80% versus 25%), distrusted the system (50% versus 25%), said the patient deserves a say in treatment (60% versus 25%), wanted no life support (50% versus 25%), doubted ADs change treatment (50% versus 25%), and said ADs just mean the patient is dying (70% versus 0%). Fewer younger than older African Americans trusted the system to serve patients well (30% versus 50%), had not told their wishes to their physicians (10% versus 50%) but planned to tell someone before becoming too sick (0% versus 75%). CONCLUSION: While sharing some views, intraethnic age groups differed in their trust of the health system, their willingness to express treatment wishes, and their confidence in ADs. Providers must consider age-specific beliefs within an ethnic culture when conducting advance care planning with patients.
MAY IT PLEASE THE COURTS: THE LEGAL CAMPAIGN AGAINST HOSPITAL DISCRIMINATION. P. Reynolds 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: Hospital discrimination was widespread throughout the United States and in many jurisdictions legally sanctioned as late as the mid-1960s. Discrimination most commonly was experienced as the denial of staff privileges to minority physicians and dentists, refusal to admit minority applicants to nursing and residency training positions, and lack of medical and surgical services to minority patients in many urban private and public institutions. A national campaign to eliminate discrimination in hospital clinical services and education programs involved the collaboration among minority medical and activist organizations, and a direct attack against the hospital policies and practices through litigation culminating in two landmark decisions which both emerged from the 4th Circuit Court of the United States. METHODS: Research was conducted in national archives, in manuscript collections of minority and non-minority physicians, in legal documents with review of relevant court cases, and in primary and secondary law and medical journals. RESULTS: The first hospital discrimination case brought before the courts was filed in North Carolina in 1956 by three physicians, Dr. Hubert Eaton, Dr. Daniel Roane, and Dr. Samuel Gray, and their lawyer, Conrad Pearson. While the courts initially denied the plaintiffs the relief they sought, the NAACP lawyers used information emerging from Eaton vs James Walker Memorial Hospital when deciding the most essential elements in constructing a``test case''. Their attack centered on the federal government's use of public funds to construct``Separate but Equal'' hospitals throughout the south. That same year, the City of Chicago passed the Harvey ± Campbell Ordinance to prevent racial discrimination in Chicago hospitals. In 1962 the second hospital discrimination case in North Carolina was filed by George Simkins and the lawyers for the NAACP against the Moses H. Cone Memorial Hospital and Wesley Long Hospital. This case would determine ultimately the legal foundation of``Separate but Equal'' in hospital policies and practices. Two years later African ± American physicians in Chicago sued 56 hospitals throughout the city for refusal to adhere to the Harvey ± Campbell Ordinance charging they had been denied staff appointments to hospitals in the area because of their race. By the time the Medicare Hospital Certification Program was implemented in the summer of 1966, over 35 suits had been filed by physicians and citizens with the assistance of NAACP lawyers against hospitals and nursing homes located in communities throughout the south. The Medicare hospital racial integration guidelines themselves would be tested in the courts through Cypress versus Newport News Hospital Association. CONCLUSION: The legal foundation for``Separate but Equal'' in hospital policies and practices was eroded through a series of court battles developed and argued by NAACP lawyers culminating in Simkins versus Moses H. Cone Memorial Hospital (1963) and Cypress versus Newport News Hospital Association (1967). The first of these two cases challenged the federal government's use of public funds to expand and maintain segregated hospital care. The second case reaffirmed the federal government's use of public funds to force hospitals to open up patient admission policies, education programs and hospital staff privileges to all citizens and physicians regardless of race, gender, age or nationality. Successful pursuit of a legal strategy against racist hospital policies and practices was a critical cornerstone in a national campaign to eliminate discrimination in health care delivery.`I THINK MY HEART GOT A LITTLE STONE IN IT NOW: CONSEQUENCES OF VIOLENT VICTIMIZATION AMONG YOUNG AFRICAN AMERICAN MEN. J. Rich 1 , C. Grey 1 ; 1 Boston University School of Medicine, Boston, MA PURPOSE: Violence remains a leading cause of death and disability for young African American men. Recurrent violent injury is also well documented in the medical literature. The purpose of this study is to understand how the experience of violent injury changes the lives of young black men and predisposes them to recurrent victimization. METHODS: Participants in the study were 34 African American men, between the ages of 18 and 30, who were admitted to an urban medical center for violent injury. In ± depth, semistructured interviews were conducted within 2 weeks of the initial injury and, in a subset of participants, a follow-up interview was conducted 2 months later. Analysis was performed using grounded theory methods and narrative analysis to identify prominent themes common across interviews. RESULTS: The narratives of young black men who are victims of violence highlight the physical pain and suffering that they experience. More prominent, however, are accounts of negative interactions with the police, both prior to the injury and in the immediate aftermath, who treat them as perpetrators regardless of the circumstances of their injuries. Also prominent are vivid descriptions of disabling symptoms of traumatic stress, and profound feelings of fear and vulnerability. These extreme feelings of vulnerability, coupled with mistrust of the police and other community institutions, lead these young men to develop their own strategies to remain safe and to deal with their distress. Some of these strategies either hinder their ability to heal from their emotional trauma (such as self-treating with drugs and/or alcohol) or put them at risk for recurrent injury (such as acquiring a weapon). Few young men expressed knowledge of resources to address their psychoemotional issues. CONCLUSION: The physical and emotional consequences of violent injury, coupled with a social environment that is hostile and unsafe, places young black male victims of violence at risk for recurrent injury. Efforts to reduce recurrent violent injury will be more effective if they address both the emotional consequences of injury and the often oppressive social environments in which these young men live.
UNDERSTANDING TEAM-BASED QUALITY IMPROVEMENT FOR DEPRESSION IN PRIMARY CARE. L.V. Rubenstein 1 , L.E. Parker 2 , L.S. Meredith 2 , N.P. Gordon 3 ; 1 VA Greater Los Angeles, RAND and UCLA, Sepulveda, CA; 2 RAND, Santa Monica, CA; 3 Kaiser Permanente Division of Research, Oakland, CA PURPOSE: Team-based quality improvement (QI) methods are an attractive option for designing and implementing improvements in depression care in primary care (PC) practices. QI methods experts disagree, however, about whether teams should be centralized (the CT approach), or local (the LT approach). In CTs a few key experts, with input from local clinicians based in the target practice sites, are responsible for developing the QI intervention design. In LTs the local clinicians develop the QI design through on-site interdisciplinary team meetings. The qualitative analyses presented here compare the success of the two approaches and evaluate the characteristics of teams and organizational environments that affect success. METHODS: We partnered with two non-profit staff model managed care organizations ± VA Greater Los Angeles and Kaiser Permanente of Northern California ± to initiate and evaluate five QI teams tasked with improving care for depression. Teams were structured as either CTs or LTs. The three LTs involved three PC practices and the two CTs involved three PC practices. Trained observers transcribed process notes during all team meetings, and carried out structured interviews with team and clinical practice leaders and members at one and two years. Three social scientists with qualitative expertise independently reviewed all study data and rated teams. Six national depression experts independently rated team QI designs (plans). Analysis of team success used qualitative predictor-outcome matrices. RESULTS: The two CTs ranked higher for depression program planning than two of the three LTs, but one LT received equivalently high ratings. Across all teams, team leadership and primary care and mental health top management support for depression QI had the most effect on plan quality and implementation. CTs, however, had better success than LTs in less supportive environments. For example, PC top management for one of the two PC practices linked to one CT was relatively unsupportive, as was mental health top management for the practice linked to the other CT; the two CTs did better than the two LTs that were based in similarly low support organizational environments. The one LT with favorable team leadership and organizational environment did as well as the best performing CT. CONCLUSION: CT and LT approaches have equivalent success when team leadership and organizational support are strong, while CTs have better success than LTs in less supportive environments. Our results suggest that depression QI approaches that depend on local interdisciplinary team design and implementation should be reserved for favorable organizational environments. Less favorable environments are likely to be better served by a more centralized, expert directed QI approach. PURPOSE: Patients fail to recall and comprehend as many as half of the critical pieces of information conveyed during a clinical encounter. To maximize patients' recall, understanding, and ultimately their treatment adherence, it is recommended that clinicians limit the number of new concepts they present and always elicit patients' comprehension of these points. This educational technique may be particularly important for patients with low functional health literacy (FHL) and chronic conditions such as type 2 diabetes (DM2), since these patients grapple with complex clinical management regimens and often have difficulties processing oral communication. In this study, we measured the extent to which primary care physicians (PCPs) assess these patients' recall and comprehension of their advice. METHODS: The study was conducted in primary care centers at a public hospital. Outpatient encounters between 30 English-speaking, DM2 patients with low FHL (score < 23 on shortform Test of Functional Health Literacy) and 22 PCPs were audiotaped. 2 trained coders measured the duration of each encounter, and identified the following communication events: (1) new concepts raised by physicians (such as an explanation of how blood pressure affects the kidneys, or a change in medication regimen); and (2) whether each new concept was followed up by the physician with an attempt to ensure that patients comprehended the key points, e.g. by a request for patient restatements, or eliciting patient perceptions. The percentage of new concepts for which follow-up assessments occurred was then calculated. RESULTS: The mean number of new concepts conveyed by PCPs was 2.0 (range 0 ± 5). The mean number of follow-up assessments was 0.27 (range 0 ± 2) or 13%; in over half of these assessments the patient did not recall or comprehend the new concept, allowing the PCP to tailor subsequent information. Visits including one or more follow-up assessments did not differ in duration when compared to visits that lacked such follow-up (18 vs. 21 min, p=0.2). CONCLUSION: The vast majority of PCPs caring for DM2 patients with low FHL do not assess patient recall or comprehension of new concepts conveyed during the clinical encounter. Overlooking this critical step in the communication process reflects a missed opportunity to enhance patient care. Future studies should evaluate whether more consistent application of this communication technique improves patient recall, adherence, and health outcomes. PURPOSE: Breakdown can be described as a disruption in smooth unreflective practice that forces an individual to adopt a more explicit, deliberate and abstract perspective. This study examined a series of breakdowns in an ambulatory medical clinic. It examined the response of residents to these breakdowns and its effect on patient care and learning. METHODS: Trained observers collected ethnographic data from waiting areas, workstations and exam rooms. Real-time field notes from sixty-eight separate observations (total 130 hours) were immediately transcribed into the data set, 2919 paragraphs of text. Two analysts coded text units at the paragraph level into recurrent themes using template analysis. Breakdown emerged as the major theme, and was fine coded at the phrase level to create a taxonomy. Using this taxonomy, 156 vignettes were selected to represent the full spectrum of breakdown. Recursive analysis of the vignettes was used to create a structural model of breakdown and learning. Triangulation and member checking were used to validate the model. RESULTS: Breakdown occurred in 1255 (43%) of the text units, and was clearly important for learning. Breakdowns occurred both from individual actions and from clinic structure and process. Preliminary results indicate that the computer-based patient record is an important source of breakdown; it interferes with communication and structures the interview, more so for residents than for faculty. The structural model of breakdown has three levels: psychomotor, conceptual and cultural. Breakdown begins at the psychomotor level of automatic behaviors (e.g., withdrawal in response to patient anger). If the breakdown persists, it moves to the conceptual level, where models are shared to determine correct action. At conceptual level, breakdowns often involve heuristic biases (e.g., favor curable diagnoses over chronic ones). Unresolved breakdowns become obtrusive and progress to the cultural level, where the entire process of norms, concepts, actions and consequences are reflected upon. At the cultural level, breakdowns reflect failure to engage with cultural norms. CONCLUSION: Our observations reinforce previous research that indicates a need to provide time and training for communication, cognitive and reflective skills. In addition, new heuristic biases are described, and problems arising from physician interface with computer-based patient records may have implications for clinic structure, process and training needs. Finally, the data suggest a need to align norms, expectations and resources. PURPOSE: Surrogates are often entrusted to make decisions regarding cardiopulmonary resuscitation (CPR) for patients who lack decision making capacity. Not infrequently, surrogates refuse to authorize Do-Not-Resuscitate (DNR) orders, even when clinicians deem CPR futile in a strict biomedical sense. Few states besides NY have legislation to address these cases. In NY, patients or surrogates must generally sign complex DNR consent forms. However, by statute, NY physicians also have the authority to write DNR orders over the objections of surrogates. Before writing such an order, a physician must conduct a``dispute mediation'' meeting with the patient's surrogates. The purpose of this paper is to provide a description and moral analysis of a series of such cases. METHODS: We present a case series of 6 dispute mediation meetings in which surrogates refused to sign for DNR orders when clinicians thought CPR was futile, convened and moderated by the ethics consult service of a single academic medical center. We use casuistic moral analysis to compare what occurred in these cases with the paradigmatic practice of requiring surrogates to provide substituted judgments and to give formal consent for DNR orders. RESULTS: The average patient age in our series was 60. Two of these patients were from nonwhite minority groups, 1 from a recent white immigrant group, 2 had malignancies, 2 had HIV, and 2 had cardiovascular disease. In all 6 cases, surrogates expressed their refusals to sign DNR orders in similar terms. Some voiced religious concerns; others``could not give up hope.'' In 5/6 cases the surrogates refused to sign the DNR order form even after the dispute mediation meeting. Nonetheless, DNR orders were written in all 6 cases with the implicit assent of the surrogates even in the absence of formal consent. After being told that DNR orders would be written even over their objections, surrogates' comments included:``Well, you gotta do what you gotta do.''``I no make decision. Doctor decision.''``I know he is dying. Just don't make me sign. I feel like I'm signing a death warrant.'' A psychiatrist involved in 3 of these cases noted denial, anxiety, pathological ambivalence, and guilt in the surrogates. No attempts were made by surrogates to transfer the patients to other physicians or facilities. All of the patients expired within one week. To date, no lawsuits have occurred. CONCLUSION: These cases suggest that what appear to be``futility'' disputes may in fact represent the complex emotional states that surrogates experience when they believe that they are responsible for making these decisions. All of the surrogates seemed relieved when they were told that a DNR order would be written regardless of whether they had chosen to`sign' the formal document. These cases raise questions for policy and clinical ethics about the extent to which asking surrogates to consent to DNR when CPR is futile imposes unnecessary burdens and no benefits for either patients or their surrogates. PURPOSE: A variety of factors influence clinical ethical decision-making by physicians. The process by which physicians weigh these many factors against one another in order to make a treatment decision is poorly understood and documented. This study examines the influential factors involved in clinical ethical decision-making by using Q Sort by-person factor analysis. METHODS: A convenience sample of internal medicine attending physicians and housestaff (n = 35) at a university affiliated medical center were presented with a series of four hypothetical case vignettes and asked to select a specific treatment action for each case. Case vignettes involved clinical decision-making near the end-of-life. Based on the selected treatment action, participants arranged twenty-five self-referenced opinion items into a unique quasi-normal factor array according to a continuum of preferences from most to least salient. Q sort factor analysis was performed by means of standard factor extraction whereby individuals with correlated Q sorts are clustered into homogenous attitude groups. A second order factor analysis was used to cluster factors to reveal their underlying commonalities. Subjective interpretation of the salient factors in relation to the specific clinical case scenario was performed in order to explain the different ethical perspectives utilized in clinical ethical decision-making. RESULTS: Data analysis revealed four non-correlated salient factors which guided ethical decision making in the four hypothetical cases. The primary salient factors were defined by the perspectives of: 1) beneficence, 2) patient guided decision-making, 3) patient and family focused decision-making, and 4) legally guided decision-making. Normalized factor scores for statements included in each viewpoint had a Z score > 1.00. Non-influential statements noted across all four cases were the economic impact on the physician, expediency in resolving the situation, and the expense of medical treatment (Z < À1.00). CONCLUSION: Q sort factor analysis is a useful tool with which to study clinical ethical decision-making because it provides an analytical method for discerning salient factors selfidentified by physicians in making ethically-challenging treatment decisions. The four factors identified are grounded in current bioethical values. METHODS: A convenience sample of internal medicine attending physicians and housestaff (n = 35) at a university affiliated medical center were presented a series of four hypothetical case vignettes involving clinical decision making near the end of life, and were asked to choose between aggressive, conservative, and consultative courses of action. An aggressive course of action was one in which medical and/or surgical treatment was pursued in spite of poor prognostic outcome. A conservative course of action was a treatment decision that withdrew medical therapy and instituted palliative treatment. A consultative course of action was defined by a request for information and guidance from an ethics committee or family members. RESULTS: Twenty percent (7/35) of respondents demonstrated a consistent pattern of clinical course of action by choosing either an aggressive, conservative, or consultation course of action three or more times. 8.6% (3/35) of respondents chose an aggressive course of action three or more times, whereas 2.9% (1/35) of respondents chose a conservative course of action three or more times. Three respondents (8.6%) chose a consultative course of action three times or more. A majority (80%, 28/35) of respondents did not demonstrate an identifiable pattern of clinical ethical decision-making by choosing an aggressive, conservative, or consultative course of action for more than two out of the four cases presented. CONCLUSION: When confronted with an ethically challenging clinical case, most physicians adapted their responses to the particulars of the case presented by demonstrating variability in their treatment approaches. Only a minority of physicians demonstrated a consistent pattern of behavior by selecting either an aggressive, conservative, or consultative treatment option. Physician interpretation of the specific ethical situation of each case guides their clinical ethical decision-making.`T HEY TREATED ME LIKE A LEPER'' Ð STIGMATIZATION AND THE EMOTIONAL BURDEN OF HEPATITIS. C S. Zickmund 1 ; University of Iowa, Iowa City, IA PURPOSE: Hepatitis C is a chronic progressive disease that is typically acquired through contaminated blood products or needle sharing. While the widely known association between this illness and intravenous drug use may lead to stigmatization, no study has explored the consequences of such stereotyping. The goal of this investigation was to identify the prevalence and impact of stigmatization on patients with chronic hepatitic C virus (HCV) infection. METHODS: Patients with HCV infection attending the University of Iowa Health Care liver clinic were randomly chosen to participate. All patients completed a semi-structured interview and the Sickness Impact Profile (SIP). Two blinded coders analyzed the interviews, identifying the frequency of stigmatization, characteristics of relationships with friends, family and colleagues in the work environment, anxiety, depression, and helplessness. RESULTS: A total of 193 patients (44.80.7 years; 33% women) with HCV infection were enrolled. The analysis of qualitative data demonstrated an excellent intercoder reliability (kappa=0.86). Eighty-five patients (44%) experienced stigmatization which they attributed to the disease. Gender, education, professional status and mode of HCV infection did not influence the likelihood of stigmatization. Negative stereotyping was associated with a higher score of the psychological SIP subscore (11.11.5 vs. 15.61.9; p =0.05). The responses demonstrated that stigmatization affected all facets of social interactions from family life to the work environment, causing a significant emotional burden for the patients. Stereotyping was associated with increased expressions of depression and anxiety (depression: 58% vs. 37%; p < 0.01; anxiety: 72% vs. 47%; p < 0.01), problems with family (38% vs. 15%; p < 0.01), friends (35% vs. 9%; p < 0.01) or coworkers (34% vs. 7%, p < 0.001). These experiences led to a sense of loss of control (38% vs. 19%; p < 0.01) and problems coping with the disease (38% vs. 19%; p < 0.01). CONCLUSION: Stigmatization is a very common experience of patients with HCV infection and erodes important support structures. The resulting distance from family, friends and colleagues increases feelings of anxiety and depression, common emotional disturbances in this group of patients. These data support the need for broad-based educational efforts to reach patients as well as their relatives and friends. Lowering the emotional impact of negative stereotyping may enhance both the quality of life of patients as well as their compliance with the medical therapy and required life style changes. Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach BACKGROUND: The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. RESULTS: We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis. CONCLUSION: The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies. Microarray is a powerful technique for simultaneously determining the expression of thousands of genes [1] [2] [3] . Such studies can quickly yield a genome-wide description of mRNA expression, called transcriptomes, in a given cell or tissue at a given physiologic or pathologic condition [4] . Even though, one of the main challenges in such genome-wide gene-expression profiling is the difficult inspection of genes with rare transcripts.
On the other hand, PCR-based suppressive subtractive hybridization (SSH) techniques are highly sensitive for identifying differences in gene expression [5] [6] [7] [8] [9] . However, the potentials of SSH in assaying dynamic changes of gene expression in minute levels have never been addressed before. In addition, SSH techniques are also restricted in terms of limited specificity and difficulties in identifying enriched genes. Combining the SSH technique with highthroughput screening of the harvested clones through the use of cDNA microarrays could greatly reduce the tedious work for northern blot analysis, as well as the likelihood of false-positive clones enriched via SSH [10] . Such combined approaches by printing the clones obtained from the SSH amplicones on chips have been successfully used for profiling the differentiation of gene expression [10] [11] [12] [13] . Nevertheless, in such approaches, the genes of the subtracted clones remain to be sequenced for identification, and a large portion of redundancy in the enriched amplicons must also be identified. Moreover, since the targets used to hybridize the amplicon clones printed on chips were the un-enriched cDNA pools rather than the subtracted, enriched clones, such approaches would not increase the sensitivity for detection of the low-expression genes.
Herein, we report our modifications using the subtracted amplicons as the targets to hybridize the pre-prepared microarray chips for SSH/microarray analysis. Since all of the probes on the microarray chips have been well characterized, the hundreds and thousands of genes in the subtracted amplicons can be determined by a single hybridization. Moreover, the relative expression status between the compared tissues for each gene can be augmented and easily determined by combining the use of the targets prepared both from forward and reverse subtractions of SSH. We named this modified approach "SSH/microarray" and used human hepatocellular carcinoma as a model to demonstrate the feasibility of this approach.
The SSH/cDNA microarray versus regular cDNA microarray approaches To conduct the comparative transcriptomic studies on human hepatoma particularly for the genes at low-expres-sion levels, we set out an approach as shown in Figure 1 . We used the PCR-based suppressive subtractive hybridization to enrich the differentially expressed cDNA clones between human hepatoma and non-hepatoma liver tissues. The SSH was conducted both forwards using RNA prepared from hepatoma and non-hepatoma liver tissues as tester and driver, respectively, to yield the hepatoma (T-N) subtracted amplicons, and reversely using the mRNA prepared from non-hepatoma and hepatoma liver tissues as tester and driver respectively to yield the non-hepatoma (N-T) subtracted amplicons as well. Instead of the previously reported combination with cDNA microarray approaches, in which clones in the subtracted cDNA libraries were cloned and then printed on chips, we directly labeled the subtracted T-N and N-T amplicons and then used as targets to hybridize the pre-made microarray chips printed with the known 14,811 cDNA clones. We named this modified approach as "SSH/microarray". The same tissue RNA pairs and cDNA microarray chips were also used for the regular cDNA microarray assays for comparison. Since human hepatoma generally occurs in patients of chronic hepatitis with/without cirrhosis, to identify the genes implicated in the common biological processes leading to hepatocellular carcinogenesis, we selected three hepatoma patients with different underlying liver disease. The first was with chronic hepatitis C and cirrhosis, the second had chronic hepatitis B and cirrhosis, and the third had chronic hepatitis B but no clinical or histological evidence of cirrhosis. Figure 2 demonstrates the representative results obtained from the corresponding regular cDNA microarray and the SSH/microarray assays. Of note, in the SSH/microarray assays the genes in the subtracted T-N and N-T amplicons were readily identified without tremendous sequencing works, which were required for the original SSH and microarray combination assays. In addition, many of the clones, including those of the house-keeping genes, such as β-actin and GAPDH, with unremarkable difference in hybridization intensities by the regular cDNA microarray assays were efficiently excluded from the subtracted amplicons ( Figure 2 , indicated by white circles). This indicated the efficiency of subtraction hybridization. Most importantly, many of the clones with hybridization intensity below the evaluation threshold by the regular cDNA microarray approaches (hybridization intensities lower than or close to the noise) became detectable by the SSH/ microarray method ( Figure 2 , indicated with red circles). These observations suggested a more sensitive detection of the low abundance transcripts using the SSH/microarray assays. Figure 1 Experimental flowchart. Messenger RNA was prepared from hepatoma (T) and the corresponding non-hepatoma liver tissues (N), and then subjected to 1) PCR-based suppressive subtractive hybridization (SSH) followed by using the resulted subtracted cDNA libraries as targets for cDNA microarray analysis (the SSH/microarray), and 2) conventional cDNA microarray analysis. SSH was performed in both the forward (T as tester) and reverse (N as tester) direction to enrich up-regulated (T-N amplicon) as well as down-regulated transcriptomes (N-T amplicon) in human hepatoma, respectively. The two subtracted amplicons were labeled with fluorescent cy-dyes as targets for microarray analysis, in which dye-swapping approaches were used. The results thus obtained were then compared to those obtained from the conventional cDNA microarray assays. The differentially expressed genes were categorized into three groups: I) only detected by conventional cDNA microarray approaches, II) identified by both conventional cDNA microarray and SSH/microarray approaches, and III) only obtained from SSH/microarray assays. The results were further confirmed by qRT-PCR in 6 genes randomly selected from group III differentially expressed genes. 
Labeled and used to hybridize chips
To further address whether the SSH/microarray approach was able to assay the expression of those genes with rare transcripts, we selectively inspected those genes with low expression. Of the total of 14,811 clones on the chips, 9376 clones that were filtered out for further analyses in the regular cDNA microarray studies due to the low hybridization intensity (intensities <500 unit after the noise subtracted) ( Figure 3A ) were selected for examination of their hybridization intensity in the SSH/microarray assays ( Figure 3B ). Of these, 5015 clones were enriched via the SSH/microarray assays and could be analyzed for differential expression (intensities >500 unit after the noise intensity subtracted) ( Figure 3B &3C ). That is, the SSH/microarray approach allowed about 100% more of the genes, most of which were low abundant, for the subsequent analyses.
Since SSH specifically amplified the difference of gene expression, the SSH/microarray approach should also be able to identify more differentially expressed genes between hepatoma and non-hepatoma liver tissues, which were originally undetectable by the regular cDNA microarray methods. As shown in Fig 3C, of the 5015 lowexpression genes identified only by the SSH/microarray assays, we identified additional 512 and 1923 genes with at least a 2-fold decrease and increase in hepatoma tissues, respectively.
We further examined the sensitivity for detection of differential expression by both approaches. We compared the ratio of Cy5/Cy3 intensity of each clone obtained from the regular cDNA microarray assays to that obtained from the SSH/microarray assays. As shown in Figure 4 , a total of 3028 genes with a ratio lower than 2 folds (-1 < log 2 < 1) using the cDNA microarray assays had a ratio greater than 2 folds (log 2 > 1 or < -1) using the SSH/microarray assays The representative results of the regular cDNA microarray vs Figure 2 The representative results of the regular cDNA microarray vs. SSH/microarray assays. RNA were prepared from the hepatoma and para-hepatoma liver tissues of a patient with HCC. Left panels are the representative results obtained via the regular cDNA microarray assays using the Cy 5 and Cy3 labeled aRNA generated from hepatoma and non-hepatoma liver tissues, respectively. Right panels are the representative results obtained via the SSH/microarray assays using Cy3 and Cy5 to label the forward and reverse subtracted amplicons, respectively. Lower panels are the close views of the upper panels. White circles indicate the clones, which had equal Cy3 and Cy5 hybridization intensities in the conventional cDNA microarray assays but been subtracted away from the subtracted amplicons. Red circles mark the clones, which had low Cy5 and Cy3 hybridization intensities in the conventional cDNA microarray but presented with differential expression in the subtracted amplicons.
SSH/microarray ( Figure 4 , 1091 and 1937 genes indicated by the red box, respectively). That is, using the modified SSH/microarray approaches resulted in much more differentially expressed genes, particularly for those with rare transcripts that using the regular cDNA microarray approaches.
A total of 69 and 701 genes differentially expressed in human hepatoma were finally identified (based on the data derived from the three tissue pairs in duplicate with log 2 > 1 or < -1, and P < 0.01) by the regular cDNA microarray assays and by the SSH/microarray assays, respec-tively ( Figure 5 & see additional file 1). That is, the SSH/ microarray assays detected 10-fold more differentially expressed genes, particularly for those low-expression genes, than did the conventional cDNA microarray. To confirm the differential expression of genes with low expression identified by the SSH/microarray assays (including 446 and 255 up-and down-regulated genes, respectively), we quantified the relative expression level of nine randomly selected genes, whose differential expression was detected by the SSH/microarray assays, in 18 pairs of HCC and the matched non-HCC liver tissues. We found the results were consistent with those obtained from SSH/microarray assays ( Figure 6 ).
Detection of differential low-expression transcriptomes by the SSH/microarray Figure 3 Detection of differential low-expression transcriptomes by the SSH/microarray. A) Of the 14811 distinct genes on the chips, 9376 genes had hybridization intensities lower than 500 units (the defined low intensity threshold for exclusion from further microarray analysis) in a representative set of results of the conventional cDNA microarray assay. B)These low-expression genes were then subjected to re-calibration for their Cy3/Cy5 intensities in the SSH/microarray assay and 5015 genes became detectable in SSH assays (blue spots). C) The distribution of these 5015 low-expression genes identified only in the SSH/microarray assays, including 512 and 1923 genes down-and up-regulated in hepatoma tissues, respectively. The data presented in this figure were based on the results derived from patient 1, a case of chronic hepatitis C with cirrhosis and hepatoma. 
We compared the HCC up-and down-regulated transcriptomes identified by the SSH/microarray assays in accordance with their potential molecular functions, implicated biological processes and sub-cellular localization. As presented in Table 1 
Microarray techniques are limited in the detection of genes with low expression [14] , while subtractive hybridization methods are restricted by their specificity and the tremendous work needed for validating the results, as well as for sequencing to identify genes. There have been many reports using microarray techniques for rapid and high throughput validation of the subtraction specificity of SSH by spotting the enriched clones on the chips or membranes [10] [11] [12] [13] [15] [16] [17] [18] [19] [20] [21] [22] [23] . However, such approaches would not only require tremendously sequencing works to identify genes including a great number of redundant clones in the subtracted amplicons, but also lose the sensitivity of SSH techniques to detect the low abundance transcripts, since the sensitivity of microarray analysis is determined by the targets used to hybridize chips but not by the probes printed on chips.
Herein we report our modified approaches. Instead of using the subtracted clones as probes spotted on chips, we directly labeled the enriched amplicons and used them as targets to hybridize the pre-made microarray chips for microarray analysis (named as "SSH/microarray" in this report). This approach allowed us not only to readily identify genes without tremendous sequencing works in the subtracted amplicons regardless of many redundant clones, but also to easily evaluate the subtraction efficiency and specificity. Moreover, the SSH/microarray approach made it possible to conduct a transcriptomewide identification of differentially expressed genes particularly for those with low expression. It has been reported that the absolute expression level is not a crucial determinant for identifying genes, while the relative difference in expression levels does impact on whether or not a gene is recovered by subtractive hybridization [5, 24] . In this report using the SSH/microarray approach, we identified about ten times more of the differentially expressed genes, particularly for those with low expression, in human hepatoma. Our findings successfully demonstrated the transcriptome-wide assays of differentially expressed genes at low abundance. This simple but very powerful approach would greatly facilitate future researches on functional genomics [15, 19, 25] .
One of the main concerns about SSH techniques is their specificity. However, as combined with microarray tech- Figure 4 Comparison of the relatively expression by regular cDNA microarray vs. the SSH/microarray. The distribution of the ratios of Cy5/Cy3 intensities obtained by the conventional cDNA microarray assays vs. the SSH/microarray assays is shown. Only the clones with significant Cy3 and Cy5 intensities in both assays were included in the analysis. The clones inside the red-boxes were those with their log 2 ratios < 1 and > -1 via the conventional cDNA microarray assays, while their log 2 ratios > 1 or < -1 by the SSH/microarray assays. The data presented were based on those derived from patient 1, a case of chronic hepatitis C with cirrhosis and hepatoma.
Log 2 ( Cy5/Cy3) of SSH Log 2 ( Cy5/Cy3) of cDNA niques, the subtraction efficiency can be readily evaluated. As shown in Figure 2 , the subtraction efficiency was determined by the rare presence of clones with equal hybridization intensities between the forward and reverse amplicons, and by the consistent exclusion of the housekeeping genes in the subtracted amplicons. The specificity was further confirmed by quantification of the differential gene expression between hepatoma and para-hepatoma The differentially expressed genes Figure 5 The differentially expressed genes. The genes differentially expression in human hepatoma were identified by either only the conventional cDNA microarray assays (26 genes) or SSH/microarray assays (658 genes), or both (43 genes). The results were derived from three pairs of hepatoma liver tissues with at least two-fold difference in gene expression and P < 0.01. APEG1, TTN, HTN1, MEST, EVL3,  VLDLR, MYOG, TNNI2, HOXC11, IGF2, AHSG, TTID,  BIRC1, PAFAH1B1, SYK, LMNA, TNFRSF11B, FALZ,  BMP1, ETS2, NRD1, CXCL1, JAG1, SEMA4G, HEY1,  ZNF22, CRMP1, TEAD4, IGFBP1, IGFBP3, IGFBP7,  PTGS1 , NEUGRIN, CUGBP1) related to the regulation of cell differentiation and embryonic development, and 59 genes (ASGR2, CAP2, RGS5, ITGB4BP, NCK1, EVL3,  PLCG2, EPHA7, IGF2, IFNGR2, AHSG, CAP1, PTPRF,  CXCL2, GNAI1, TGFBR1, NCSTN, DOK1, FRBB3, SYK,  MC1R, CD79B, CSNKIE, CD69, AVPR1A, GNB2L1,  ACVR1, BIRC2, FLT4, CXCL1, FAG1, PNOC, SLC9A3R1,  LANCL1, GNB3, ADORA2B, FGR, NCK1, RAB11A, PLCG2,  LOC91614, PRKAR1A, FLJ22595, ARF3, TYK2,  MAP3K7IP1, MC1R, TNFSF10, ARF4, VAV2, AVPR1A,  GNB2L1, MAPK10, STMN1, SNX17, ADORA2B, ECT2,  RGS5, IGFBP3 ) associated with signal transduction in response to extra-cellular proliferation and growth stimuli were found. Our findings that differentially expressed genes were related to multi-biological processes suggest the complexity of the molecular mechanisms for hepatocellular carcinogenesis.
In this study, we modified the previously method of the combination of the suppressive subtractive hybridization and microarray techniques to differentially profile the low-expression transcriptomes of human hepatocellular carcinoma by directly labeling both of the reciprocal subtracted amplicons as target for cDNA microarray assays. Compared to SSH or previous SSH in conjunction with microarray approaches, this modified approach provided us with three additional advantages: 1) easy inspection of the subtraction efficiency, 2) avoidance of tremendous sequencing work for gene identification, 3) high sensitiv-ity for identifying the low-expression, differentially expressed genes. This approach allowed for the detection of about ten times more of the differentially expressed genes than did the regular cDNA microarray approach in human hepatoma, particularly for those with low expression. Using this approach, we identified many genes potentially implicated in human hepatocarcinogenesis, which were not identified before. For its high efficiency and high sensitivity, this SSH/microarray approach is powerful for the rapid differentially profiling the lowexpression transcriptomes, and most adequate for applying to functional genomic studies.
For SSH and cDNA microarray analysis, hepatoma and the corresponding non-cancerous liver tissues were obtained from 3 patients who had liver surgery at the Chang Gung Memorial Hospital. For reverse-transcription real-time PCR, hepatoma and the matched non-hepatoma liver tissues were obtained from additional 18 patients of hepatoma. Diagnoses of HCC and non-hepatoma liver tissues were based on histo-pathologic findings. The Internal Review Board for Medical Ethics of Chang Gung Memorial Hospital approved the specimen collection procedures and informed consent was obtained from each subject or subject's family.
Total RNA from hepatoma and the para-hepatoma liver tissues and the poly(A) RNA was prepared as described before [26, 27] . SSH was performed with the Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) as described by the manufacturer but with the following modifications. Starting material consisted of 2 μg hepatoma mRNA as tester and 2 μg nonhepatoma liver tissue mRNA as driver, and vice versa. Primary and secondary PCR conditions were altered to increase specificity of amplification according to either plan A or B. Both A and B reduced the extension time and the number of cycles of the primary PCR to 2 min and 26 cycles, respectively. The primary PCR products were diluted 1/50 prior to use in the secondary PCR. All other aspects of plan A were as per the instructions of the manufacturer. Plan B diverged from plan A in two ways. First, the initial cycle of primary PCR was performed using annealing and extension times that had been reduced to 15 s and 1.5 min, respectively. Second, for subsequent cycles, the denaturing time was increased to 10 s while the annealing and extension times were reduced to 15 s and 1.5 min, respectively.
In this study, we used the GMRCL Human 15 K set, Version 2 chips as previously described [28] , which contained 14,811 sequence-verified, human cDNA clones mapped to 12,530 distinct genes. All of the samples for the regular cDNA microarray or SSH/microarray assays were performed with the dye-swapping microarray design for minimizing labeling bias and statistical variances of data. For the regular cDNA microarray experiment, we used 2 μg of the total RNA for labeling and hybridization using a 3DNA Array 350RP Detection kit (Genisphere, PA, USA).
For the SSH experiment, 1 μl subtractive PCR products were labeled with Cy3 and Cy 5-dCTP (NEN, Boston, MA) using random primers. Unincorporated fluorescent nucle-otides were removed using a Qiaquick PCR purification kit (Qiagen). The fluorescent-labeled DNAs were mixed with 30 μg of human cot-1 DNA (Invitrogen) and 100 μg yeast tRNA, precipitated and then resuspended in 30 μl of Microarray Hybridization Buffer Version 2 (Amersham Pharmacia). The hybridization solution was heated to 80°C for 10 min to denature the DNA and was then incubated for 30 min at 37°C, allowing cot-1DNA and yeast tRNA to block the repetitive sequences in genome probes. The probes were hybridized to a human cDNA microarray (GMRCL Human 15 K). We scanned the slides with a confocal scanner ChipReader (Virtek, Canada) and acquired
Quantification of relatively gene expression in human hepatoma Figure 6 Quantification of relatively gene expression in human hepatoma. A total of nine differentially expressed genes including six (GBA, PTPRF, GNL3, ERBB3, OGDHL, FMO3 belonging to Class III) identified only by the SSH/microarray assays and three (C9, MT1F, MT1X belonging to class II) identified by both of the SSH/microarray and regular cDNA microarray assays were assayed for their relative expression between HCC and the corresponding non-HCC liver tissues of 18 patients of hepatoma using real-time semi-quantitative RT-PCR. The results are presented with the log 2 values of the gene expression ratio's between HCC vs. the matched non-HCC liver tissues. Left panel is the relatively expression level of these genes initially determined by the SSH/microarray and regular cDNA microarray assays, respectively. Of note, the difference of the expression for the six genes belonging to Class III was originally only identified by the SSH/microarray assays, but not by the regular cDNA microarray assays. the spot and background intensities with the GenePix Pro 4.1 software (Axon Instruments, Inc., CA, USA). The within-slide normalization was done using programs written with MATLAB 6.5 software (The MathWorks, Inc., MA, USA).
To validate the results obtained from the SSH/microarray assays, we randomly selected six differentially expressed genes identified only by the SSH/microarray assays and three differentially expressed genes identified by both the regular cDNA microarray and SSH/microarray assays for the comparison of gene expression between hepatoma and the corresponding non-hepatoma liver tissues in eighteen patients of HCC using reverse transcription realtime PCR (qRT-PCR). Total RNA was extracted from tissues with Trizol reagents and reverse transcribed using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA). qRT-PCR was conducted using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Pre-designed Assays on Demand TaqMan probes and primer pairs for these 9 genes were obtained from Applied Biosystems Incoporated (ABI) (Foster City, CA). For each gene, two to four sets of Taq-Man probes and primers were tested. The probes contained a 6-carboxyfluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end. These were designed to hybridize across exon junctions. As a result, no fluorescent signal was generated by these assays when genomic DNA was used as a substrate, which confirmed that the assays measured only mRNA. Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate, and 18S ribosomal RNAs were used as internal controls.
All data (derived from three pairs of HCC and non-HCC liver tissues in a dye-swapping approach) were filtered so that 446 up-regulated and 255 down-regulated genes in HCC with at least two-fold difference and p value less than 0.01 were included in the further studies. After removal of the un-annotated genes, a total of 360 and 202 HCC up-and down-regulated genes were subjected to the subsequent gene ontology analyses. The two lists of the differentially expressed genes were analyzed using the online software FatiGo for comparative gene ontology categories including molecular function, biological process and cellular component [29] . They were also imported into the on-line software KEGG2 for pathway mapping [30, 31] . The statistical significance was defined as P < 0.01 between the HCC up-and down-regulated gene groups using Fisher's exact test. From Functional Genomics to Functional Immunomics: New Challenges, Old Problems, Big Rewards The development of DNA microarray technology a decade ago led to the establishment of functional genomics as one of the most active and successful scientific disciplines today. With the ongoing development of immunomic microarray technology—a spatially addressable, large-scale technology for measurement of specific immunological response—the new challenge of functional immunomics is emerging, which bears similarities to but is also significantly different from functional genomics. Immunonic data has been successfully used to identify biological markers involved in autoimmune diseases, allergies, viral infections such as human immunodeficiency virus (HIV), influenza, diabetes, and responses to cancer vaccines. This review intends to provide a coherent vision of this nascent scientific field, and speculate on future research directions. We discuss at some length issues such as epitope prediction, immunomic microarray technology and its applications, and computation and statistical challenges related to functional immunomics. Based on the recent discovery of regulation mechanisms in T cell responses, we envision the use of immunomic microarrays as a tool for advances in systems biology of cellular immune responses, by means of immunomic regulatory network models. During the past decade, the highly successful field of functional genomics experienced huge growth as a result of the development of DNA microarray technology [1] [2] [3] [4] , which made it possible for the first time to measure the RNA expression of thousands of genes in parallel, in a single assay. Immune responses are complex phenomena that supervene on genomics, that is, immune responses ultimately depend on the expression of genes inside a variety of cells, but explaining the function of the immune system only in terms of gene expression in those cells would constitute a reductionist approach. While studying the immune system in terms of genomics is an important goal [5, 6] , the function of the immune system, from antigen processing to epitopespecific immune responses, may be better understood through an integrated approach that takes into account properties of the immune system as a whole.
We quote from [7] , ''The immunome is the detailed map of immune reactions of a given host interacting with a foreign antigen, and immunomics is the study of immunomes.'' Whereas functional genomics strives to identify the role of genes in cellular processes via the paradigm of hybridization of mRNA to complementary DNA, functional immunomics aims to identify the roles of chemical/biological targets involved in immunological processes via the paradigm of specific cellular and humoral immune responses elicited by antigens presented to the immune system [8] [9] [10] [11] . This is an effort that promises great rewards, both in terms of our basic understanding of the immune system and in terms of disease diagnosis/prognosis [12] and the design of vaccines [13] [14] [15] to combat a variety of human infirmities ranging from pathogenic infections to allergies and cancer.
Enabling technologies. Functional genomics was made possible by the significant advances that had previously been made in sequential genomics, including not only the massive efforts required to identify genome-wide DNA sequences [16] , but also the computational methods used to parse and align those sequences [17] . Sequential genomic data are deposited in large public-access databanks such as GenBank [18] , and researchers or companies who make DNA microarrays use the sequences in these databases as probes. In a similar fashion, the field of functional immunomics has now come of age as a result of advances in sequential immunomics, which consists of methods to catalogue the chemical/biological targets capable of eliciting an immune response, also known as epitopes. Computational and statistical methods are now available for automated large-scale epitope prediction (please see the next subsection), in addition to classical immunoassays such as ELISPOT [19] and tetramer staining by flow cytometry [20] that together enable high throughput identification of epitopes. Recently, a coordinated effort has been initiated by the National Institute of Allergy and Infectious Disease at the US National Institutes of Health, under the auspices of the Large-Scale Antibody and T Cell Epitope Discovery Program (in which the authors of this paper participate), to create an integrated immunome database and resources such as a toolbox of epitope prediction methods. This initiative is designed to identify immune epitopes from selected infectious agents; the information will be made freely available to scientists worldwide through the Immune Epitope Database and Analysis Resource (IEDB) [7, 21, 22] (http://www. immuneepitope.org). The sequencing information produced by this and other epitope mapping efforts being carried out will be essential for the construction of immunomic microarrays (discussed in detail below), leading to an experimental paradigm similar to that employed in functional genomics.
Computational epitope prediction methods. The immune system recognizes antigen via binding of antibody (humoral response) or T cell receptors (cellular response) to self or foreign proteins. B cell epitopes correspond in general to the 3-D features on the surface of antigen where recognition by the immune system occurs; a continuous or linear epitope is a sequential fragment from the protein sequence, while a discontinuous or conformational epitope is composed of several fragments scattered along the protein sequence and brought together in spatial proximity when the protein is folded [23] . Humoral response is targeted mainly at conformational epitopes, which may represent up to 90% of the total B cell responses. This makes prediction of B cell epitopes a hard problem [24] , even more so because B cell responses are virtually only restricted by immunoglobulin access to the epitope, B cell receptor activation, and self versus no-self discrimination rules of the immune system. Ideally B cell prediction systems would use 3-D surface models of the protein antigens and measure surface energy interactions of variable regions of the immunoglobulins that correlate with B cell activation. However, so far B cell prediction systems make estimations of the probability of a primary peptide sequence being present at the surface of a protein based on hydrophilicity and secondary structures [25] . Cellular responses, on the other hand, are restricted through the binding of T cell receptors to short linear peptides, which are bound by a specific groove in two main classes of major histocompatibility complex (MHC) molecules, and presented on the surface of cells to the T cell receptors of CD4 þ and CD8 þ cells [26] . Binding affinity between the peptide and the MHC molecule is therefore a necessary requirement for effective cellular immune response. A complicating factor is the highly polymorphic nature of the MHC molecule, which displays large variability in human populations. Using experimental affinity data deposited in public databases as training data, researchers have developed statistical methods to predict the MHC affinity of a given unknown peptide [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Typically, such computational epitope prediction methods scan the full length of pathogen or self-immunogenic protein sequences by taking consecutive overlapping peptides. In addition, such methods can predict ''promiscuous'' epitopes, that is, the ones that bind to a large class of different MHC alleles, know as supertypes [43] . Computational MHC-binding prediction methods have become essential for the systematic search for epitopes, in situations where techniques such as ELISPOT and flow cytometry are effectively impractical due to the large number of peptides to be assayed.
Early MHC-binding studies identified characteristic amphipathic chemical patterns on the binding peptides [44] , and enhanced versions of these systems continue in use in association with other methods [41, 44, 45] . Today, the most basic MHC-binding prediction methods are based on the identification of specific amino acids commonly found at particular positions, called binding motifs, within peptides that bind to a specific MHC molecule. However, all the amino acids of a peptide bound to an MHC groove (normally 8-10 amino acids for MHC I and 8-14 amino acids for MHC II) can potentially play a positive or negative role in binding, and more complex methods assign positive or negative values for each amino acid at each position of a peptide and combine these values to define scores that predict binding; these ''quantitative-matrix'' approaches have been very successful. One of the limitations of the ''quantitative-matrix'' approach is that it does not take into consideration the influences of interactions between amino acids at different peptide positions of the epitope. The value of these interactions are difficult to measure and have been explored only in a limited fashion by combining pair-wise interactions between two peptide positions [46] . The combination of independent binding calculated by quantitative matrices with coefficients derived from the pair-wise interaction provided better predictions. Moreover, quantitative-matrix scores have been generated for several HLA alleles, and studies using HLA sequence homology have allowed the development of virtual quantitative matrices to be applicable to many more HLA alleles [28, 39] . General methods for MHC-binding prediction systems, such as artificial neural networks, and statistical models such as Hidden Markov, can incorporate nonlinear complex interactions between the MHC molecule and the peptide epitope, and can evolve as more data is included in the training set. These general methods have shown to be potentially superior to the previous ones [27, 37] . The greatest challenge, however, of the general methods is that, to be reliable, they require a larger amount of peptide-binding data. Strategies using query-by-committee approaches to compare predictions from different training sets have been used to identify the most informative peptide-binding data to be determined in the biochemical binding assays in order to more efficiently build a representative training dataset [40] . Recently a collection of more than 48,000 peptide binding affinities of class I molecules were made public [47] . The development of prediction models will be greatly accelerated by this community resource benchmark. We remark that epitope recognition by the immune system involves more than a receptor-ligand problem between the MHC molecule and a peptide, as other biological processes are involved in preparation of the peptide for loading into the MHC molecule. Epitope prediction systems continue to evolve, and many steps in antigen processing and presentation required for development of cognate immune responses are being modeled and combined into rational prediction systems. The greatest current challenge is the development of models incorporating the rules of B cell and T cell receptor engagement and their possible outcomes.
The growth ''boom'' of immunomics. While the growth boom in genomics took place in the 1990s and this field has now begun to enter a mature stage of development, a similar growth boom in immunomics is likely to take place over the remaining years of the current decade. We recently searched the PubMed database (with Sente 2.3) using the following query: ''immunomics OR immunomic OR immunome OR (antigen AND microarray AND functional) OR (epitope AND microarray).'' After removing nine irrelevant articles from the output list and adding five articles for the new journal Immunome Research, we obtained a list of 71 articles covering the years from 1999 to the present (please see Figure 1 ). It is clear that interest in this field has accelerated, supporting the expectation of a continuing boom in growth. It is expected that the number of publications will increase at an exponential pace as immunomic microarrays became commercially available for research use. As immunomic array technology evolves, we expect that immunomic arrays with a small number of features will eventually be designed for specific clinical diagnostic purposes and used regularly in medical practice. However, these clinical applications might still be in the distant future.
The basic functioning principle behind all microarray technology is the binding, and subsequent measurement, of target biological specimens of interest to complementary probes arrayed in a spatially addressable fashion. Typically, a planar surface, such as a glass slide, is used to support an array of spots containing the probes. As a consequence of using spatially addressable probes, a large number of different targets can be measured in a single experiment. For example, in the case of DNA microarray technology, which provides the basic enabling technology for functional genomics, the targets are fluorescent mRNA molecules (indicators of genomic expression) that are hybridized to gene-specific DNA probes immobilized on a planar surface. In a similar fashion, the enabling technology for functional immunomics is the immunomic microarray. The basic technologies for immunomic microarrays that we consider in detail in this paper are antibody, peptide, and peptide-MHC microarrays (see Table 1 for a summary of these technologies). Other functional immunomic approaches include dissociable antibody microarrays [48] , cell microarrays [49, 50] , serum microarrays [51] , peptide libraries [52, 53] , and serological analysis of cDNA expression libraries (SEREX) [54] [55] [56] [57] [58] . There are significant technological challenges inherent in fabricating immunomic microarrays, including the identification of a workable surface coating for the glass, appropriate probe concentration and target incubation times, and suitable spot size and interdistance [59] [60] [61] [62] [63] [64] .
Antibody microarrays consist of antibody probes and antigen targets; thus, they can be used to measure concentrations of antigens for which the antibody probes are specific [65, 66] . As such, antibody microarrays are quite useful in proteomic applications, such as in the proteomic profiling of cancer antigens [67] [68] [69] . Antibody microarrays have also been proposed for post-translational functional genomics [70] . The rationale for directly measuring protein concentration, rather than using a traditional DNA microarray format, is the existence of evidence of poor correlation between concentrations of mRNA and its corresponding protein, which reflects post-translational modification of the protein [71] . Given the possibility of measuring antigens or proteins associated with ''foreign agents,'' antibody microarrays can be employed in functional immunomics applications [72, 73] (The application in [73] actually used cells, which display the target protein markers on their surfaces.) As a general rule, however, using antibody microarrays in data-driven functional immunomic applications may be problematic. One of the main reasons is that this approach requires the production of specific antibody sets for use in defining each of the antigen targets, and the development of large numbers of interrogative features (antibody sets) is a tremendous challenge, since humoral responses are much broader than MHC-restricted T cell responses, are highly conformationally dependent, and can be developed against a great variety of chemical/biological elements present in biological fluids, including small molecules. Peptide microarrays use the opposite technical approach; that is, they use antigen peptides as fixed probes and serum antibodies as targets [74] . This format is promising for functional immunomic applications. Published studies using peptide microarrays include applications to autoimmune disease [10, [75] [76] [77] [78] [79] , allergy [80] [81] [82] [83] [84] [85] , B cell epitope mapping [13, [86] [87] [88] , vaccine studies [89, 90] , detection assays [91, 92] , serum diagnostics [12] , characterization of weak protein interactions [93] , and analysis of antibody specificity [94] . Peptide microarrays essentially correspond to highthroughput parallelized ELISA assays [12, [95] [96] [97] [98] and thus can reveal the repertoire status of antigen-specific B cell antibody responses. However, B cell responses are highly dependent on CD4 þ T cell immune responses, and thus peptide microarrays should ideally be used in parallel with extensive analysis of T cell responses (e.g., by using peptide-MHC microarrays; see below). One of the pioneer studies that best depict the usefulness of peptide immunomic technology was performed using an array of 87 protein antigens to search for specific antibody reactivity patterns in the serum of 20 normal health volunteers; these were compared to the patterns of 20 type-1 diabetes mellitus patients, and simple classifiers were designed to discriminate between healthy and diabetic patients, with an overall sensitivity of 95% and specificity of 90% [78] . In a subsequent study, the 87-feature array was able to identify prognostic signatures that could predict the susceptibility of healthy animals to develop diabetes [10] . Another important report describes the use of a panel of 225 selected peptides of several protein antigens known to be recognized by autoimmune disease patients [79] . The autoimmune peptide array was used to study the profile of the autoantibody reactivity pattern of rheumatoid arthritis (RA) patients. The RA study used serum from 18 RA patients, 38 healthy controls, and 58 recently diagnosed RA patients, and found early clinical prognostic markers able to predict which patients are more likely to develop severe RA, and also markers to identify the group of patients with the milder form of the disease [77] . Moreover, an array developed from a panel of 213 peptides derived from allergenic peanut proteins established that patients responding to a greater diversity of peptide peanut epitopes had the worst allergic reactions [80] . The importance of the breadth of antibody response against the simian-human immunodeficiency virus (SHIV), a experimental nonhuman primate model for human immunodeficiency virus (HIV), was demonstrated through studies performed with an array of 430 peptides derived from simian immunodeficiency virus (SIV) and HIV amino acid sequences [90] . This study indicated that the reduction of the repertoire of the antibody response was associated with development of acquired immunodeficiency syndrome (AIDS). These examples underscore the great potential of peptide microarrays to identify several valuable clinical markers.
The most recent technology to be proposed is the peptide-MHC microarray or artificial antigen-presenting chip [9, 11, 99] ; in this case, recombinant peptide-MHC complexes and costimulatory molecules are immobilized on a surface, and populations of T cells are incubated with the microarrays, whose spots effectively act as artificial antigen-presenting cells [100] containing a defined MHC-restricted peptide. Different methods have been proposed for detecting T cells expressing receptors with affinity for specific peptide-MHC complexes on the microarray; these can include simple inspection of T cell clusters bound to a spot [9] or identification of activated cells secreting specific cytokines with cytokine-specific capture antibodies [11, 99] . Peptide-MHC microarrays correspond to high-throughput parallelized ELISPOT assays [19] , particularly when low enough densities of cells are used, in which case a direct counting of activated cells is possible [11] . Quantitation can involve cell counts alone, detected cytokine intensities alone, or a combination of both, as in [99] , which used a cell-count score adjusted by an intensity score. The benefit of using peptide-MHC microarrays is that it can map MHC-restricted T cell epitopes, which are involved in several helper and regulatory functions of the immune system, and can be used in conjunction with peptide-based B cell epitope microarrays to study the adaptive system as whole. Figure 2 illustrates the functioning of peptide-MHC microarrays. In Figure 2a , a peptide-MHC microarray is depicted, with an inset showing the probe molecules that are deposited on a microarray spot. Figure 2b depicts T cells that bind to, and are activated by, specific peptide-MHC complexes, with the help of co-stimulatory antibodies; these T cells secrete cytokines that are captured by specific detection antibodies. Finally, as depicted in Figure 2c , the T cells and excess cytokine are washed away, and the bound cytokine is revealed by fluorescent antibody (other methods can be employed to reveal the cytokine [11] ). Therefore, a peptide-MHC spot is designed taking into account two elements: a peptide-MHC complex, and the detection antibody specific to the particular cytokine one wants to measure. A third element can be the kind of T cell population (e.g., T helper or CTL) that is used as a target (i.e., incubated with the microarray). The choice among these three different elements will lead to a vast number of immunomic responses that can be measured.
An exciting feature that distinguishes immunomic from DNA microarray data is the possibility of measuring two or more signals simultaneously, determined by a single feature, the epitope. In the case of DNA microarrays, one response value is obtained for each gene per sample, namely the concentration of mRNA produced by the gene (note that twodye experiments use mRNA from two different samples). In the case of peptide-MHC chips, a single epitope can generate different response values corresponding to different cytokines or different target T cell populations, or even different antibody isotypes, in the case of peptide microarrays. In other words, in the case of genomic microarrays, one parameter is measured, namely the level of transcription of each individual gene, whereas in the case of immunomic microarrays, it is possible to measure several parameters regarding immune responses against a single epitope. For instance, a single B cell epitope can be recognized by different isotypes of immunoglobulins, such as IgE or IgG1. Therefore, in this case it is not only the intensity of the antibody response that can be measured, but also the quality of the antibody response. This aspect can be very relevant since a high IgE titer in relation to IgG1 may be associated with allergy, whereas the opposite, a high IgG1 titer in relation to IgE to the same epitope, is not. This situation is even more significant in the case of the peptide-MHC array, where the same peptide-MHC epitope can induce several different cytokine responses. These ''multicolor'' peptide-MHC microarrays have a counterpart in the multicolor ELISPOT assays currently in use [101] . It is known that the combined effect of multiple cytokines is essential to the control of immune responses; this is described by the suggestive term ''cytokine chord'' in [102] . Thus, given a family of epitopes, one may want to simultaneously measure both inflammatory (effector) and anti-inflammatory (regulatory) T cell responses, which are known to be associated with the concentrations of IFN-c and IL-10, respectively [103] . In this case one would have more than one spot on the microarray containing the same epitope (peptide-MHC complex) but use distinct cytokine antibodies for detection (see Figure 3 ). The result of this analysis is not a real-valued profile, as is obtained from functional genomics microarrays, but rather a vector-valued profile. Such profiles are sometimes called ''multispectral'' profiles (see the section on data analysis below). We will, however, adopt the term ''multicolor'' when referring to immunomic data, due to the fact that this term is already used in the similar setting of ELISPOT assays.
The technological challenges mentioned previously in connection with antibody and peptide microarrays are much more complex in the case of peptide-MHC microarrays, which in fact involve elements of the two previous technologies, namely presentation of peptide and antibody detection of secreted cytokine. The technology of peptide-MHC microarrays, though still in its infancy, is viewed as a simple and economical method for screening the T cell repertoire of a host [99] , and thus holds great potential. The first clinical research application of peptide-MHC microarray technology was the study of the correlates of protection regarding the effects of an experimental therapeutic cancer vaccine [104] . Ten patients with melanoma were immunized with a peptide vaccine, and their immune responses were examined with a peptide-MHC microarray, which contained seven types of peptide-MHC epitopes and probed for 26 secreted factors. This peptide-MHC array was shown to have the sensitivity to detect one peptide-MHC specific T cell in 10,000, and 10 6 CD8 þ cells were incubated with the array (so, in theory, this peptide-MHC array could detect up to 100 distinct reactive features that have reached minimum frequency of 1:10,000). Analysis of the peptide-MHC microarray response patterns demonstrated that patients who presented both IFN-c and TNF-a secretory responses against a specific epitope remained free of melanoma.
The complexity of the statistical analysis with regard to immunomic microarray data is on a whole different level than that of genomic microarray data. The total number of genes in humans is estimated to be ;30,000; in comparison, the total number of different T cell receptors in humans, generated by somatic recombination, which recognize peptides within the context of a major histocompatibility molecule, is estimated to be on the order of 10 7 to 10 15 , and the number of B cell clonotypes, generated by somatic recombination of V(D)J genes, is estimated to be on the order of 10 12 . DNA is based on a four-letter ''alphabet,'' consisting of the four nucleotide bases A,G,C,T, whereas peptide epitopes are based on a 20-letter alphabet, consisting of the amino acids known to be involved in life processes. Clearly, the combinatorial complexity in the case of functional immunomics is several orders of magnitude higher than that of functional genomics. In addition, for functional genomics the number of interrogative features that need to be built on microarrays is on the order of 10 4 to 10 5 . In functional immunomics, the total number of interrogative features included in microarray analysis can be much larger and may be estimated as follows, in the case of peptide-MHC microarrays: analysis of MHC peptide-binding motifs [26] suggests that a core of nine amino acids within a peptide is sufficient for characterization of a T cell epitope. The total number of interrogative features would thus correspond to the number of possible nine-letter words based on a 20-letter alphabet, which is 20 9 ' 10 11 ; fortunately, only ,1% of these are able to bind to MHC molecules, which makes the number of interrogative features more manageable. In addition, B cells and T cells go through a process of clonal selection, where leukocytes that either do not react or react too strongly are eliminated, and dangerous clones that react with self antigens are deleted or anergized as part of the process of immune tolerance. The number of features is still very large, however, and methods to further reduce this number are essential; such methods include the prediction methods and immunomic databases mentioned previously in connection with epitope mapping, as well as the selection of peptides from specific genomes of pathogens, allergens, and self-antigens involved in human infirmities, such as tumor antigens, diabetes, and autoimmune diseases.
One additional procedure that we and other groups have used after screening the genomes of pathogens for putative binding peptides is to compare those candidate epitopes with known host protein sequences, and in some cases we have found peptides that are identical to host peptides. This is very important because several critical diseases are caused by pathogen molecular mimicry, that is, some diseases, such as diabetes, dengue hemorrhagic fever, and Guillian Barret syndrome, are hypothesized to be the result of infections that induce self-reactive pathogenic immune responses.
It is important to state that the goal of the immunomic array is not to test all possible naïve T cell or B cell clones that can be generated by the somatic, VJ, or V(D)J recombinations against all possible combination of peptides-there simply would not be enough patient blood to do that, even if it were considered to be a relevant pursuit. The goal of the immunomic array is to identify primed cells that have reached a reasonable level of precursor frequency and are thus expected to have biological relevance. If the frequency of a circulating T cell in the peripheral blood is less than 1:100,000 we can expect that the biological relevance of it is small in contrast to a T cell that has a frequency of 1:1,000. In addition, a T cell has a binding affinity for the peptide-MHC that the T cell is specific for, and it should not bind to the ones with which it has no affinity. The current reported limit of detection of the peptide-MHC immunomic array is 1:10,000 cells when using 10 6 CD8 þ cells. So, in theory, a peptide-MHC array incubated with 10 6 purified CD8 þ cells (;10 ml blood) could detect up to 100 distinct reactive features that have reached minimum frequency of 1:10,000. However, if we expect to be able to detect rare clones, with very low frequencies, more cells would be needed. This requirement may be overcome with larger amounts of blood, and it is not unreasonable to collect 100 ml blood, or by adding a T cell expansion step to grow the cell population ex vivo before it is incubated with the array. This technique of T cell clonal amplification is commonly used to detect rare populations of cells by flow cytometry or by ELISPOT and may as well be applied in immunomic studies.
Normally, 250,000 human PBMCs are used in an ELISPOT assay with one peptide, and the limit of detection is estimated to be 4-fold to 5-fold more sensitive than flow cytometry. However, ELISPOT is quite distinct experimentally from immunomic microarrays. In ELISPOT, T cells and APCs are present in the same mixture, and for a T cell to be activated, it has to be in close contact with its APC. In the peptide-MHC array the spot surface is equivalent to a very large defined APC, completely loaded with one specific peptide epitope, for which a reactive T cell has a binding affinity, so that it adheres to its specific peptide-MHC spot and not to the other spots. In [104] , the authors used 10 6 cells and compared the limit of detection of the array with flow cytometry. It turned out that both had similar limits of sensitivity, approximately 1:10,000 cells or 0.01%. It is possible that in the future the peptide-MHC spot surface can be improved and the sensitivity of the peptide-MHC array may become even greater than the current ELISPOT assay.
Another great challenge is the polymorphism of HLA genes, in particular HLA class II, and the several combinations of different alpha and beta chains. Some approaches may be useful to limit the number of features, such as the selection of specific alleles most frequently found in a population to be used in broad screening arrays. A second such approach could be the use of supertype prototype HLA molecules compatible with a set of several HLA alleles. A third approach would be through customization of the arrays, by having many different arrays of single alleles and combining them according to the HLA types of the individuals being tested.
An issue that sets immunomic microarray data apart is the availability of vector-valued response profiles (in the case of peptide-MHC microarrays). The statistical challenge here is reminiscent of the data analysis problem in the engineering field of remote sensing [105] , where different materials have characteristic vector responses, called spectral signatures. In the case of immunomic data, the analogous notion to the spectral signature is the cytokine profile associated with a given epitope and T cell population; see Figure 4 for an illustration. One simple technique to address the data analysis problem for multicolor immunomic data is to combine the responses into one long feature vector, by juxtaposing the individual cytokine response profiles for each epitope, with the caveat that there may be systematic correlation among the features in the resulting feature vector.
The large number of features that can be measured simultaneously with microarray technology also presents a challenge. On one hand, it is likely that a large number of irrelevant features will be present; on the other hand, the scientist would like to work with a small number of strong, relevant features that can be used for diagnostic/prognostic panels, or as the basis for further biochemical validation studies of the mechanisms involved. This problem of feature selection also arises due to a fundamental limitation in statistics, sometimes called the ''curse of dimensionality'', according to which the existence of a large number of features necessitates an even larger (exponentially larger) number of samples to achieve consistent and accurate results. As the number of patients in microarray-based studies is severely restricted by factors such as the cost of the technology and difficulties in patient enrollment, it is almost always the case that only a small number of samples are available. Thus, only a small number of features at a time can be considered. The recommended approach to feature selection is to consider combinations of m features at a time, do classifier design based on the feature set under consideration, and use an estimate of its probability of error as the performance score. Application of this type of analysis to immunomic microarray data would allow the identification of sets of epitope-specific immune responses, associated for example with distinct disease states. However, feature selection presents an explosive combinatorial problem. For example, in an exhaustive selection of sets of three features among 1,000 initial features, the total number of feature sets to be assessed is equal to 166,167,000. If an initial set of 10,000 features is used instead, the number of feature sets of size three to be searched is larger than 10 11 . The complexity of feature selection is especially crucial in functional immunomics applications, since in this case the number of initial features to be considered is huge. The use of high-performance computing architectures, such as large computer clusters, is almost mandatory.
Given that a set of features has been selected, one should be able to design a classifier that takes as input microarray data for an unknown sample and generates as output a predicted class label (e.g., clinical outcome, kind of infection, or other conditions). Figure 5 illustrates this approach in a hypothetical functional immunomics application. In this case, there are two class labels, corresponding to control and protected patients, in a situation in which protection is achieved by immunization with an attenuated-virus vaccine for a given infectious disease. The objective is to identify the epitopes that show a discriminatory response between the two groups and are therefore prime targets for rational epitope-based vaccine design. A set of two features, corresponding to epitopes X and Y, have been identified via feature selection among the thousands of microarray probes. For instance, we can imagine a situation in which the protected individuals presented a higher TNF-a response to epitope X, as well as a higher IFN-c response to epitope Y. Based on the response values observed for each patient (note that a patient corresponds to a point in the plane), a linear classifier is designed. This classifier corresponds simply to two decision regions separated by a line. If a future unknown patient has response values that fall in the upper decision region, he/she is likely to be a protected patient, provided that the classifier has a small probability of error. In this case, the responses to epitopes X and Y (in terms of the TNF-a and IFN-c cytokines) characterize immunological memory induced by the attenuated-virus vaccine: large response values to both epitopes X and Y indicate protected patients (note that the response of neither epitope X nor epitope Y by itself is a good discriminator in this example, indicating the need to consider the multivariate, combined effect of both responses). Note, in Figure 5 , that the apparent error rate (i.e., the number of misclassified sample divided by the total number of samples) is 2 4 20 ¼ 10%; the actual probability of classification error on future data typically exceeds the apparent error rate [106] .
Systems biology makes use of mathematical modeling in order to provide a theoretical core for biology, analogous to the way that mathematical theories provided that core for physics in the 20th century. The success of engineering and computational methodology in the physical realm is due to the predictive capability of mathematical modeling. We quote from [107] : ''Predictive mathematical models are necessary to move biology in the direction of a predictive science. They are also necessary to the application of engineering methods to translate biological knowledge into therapies with a mathematical and computational basis.'' The large complexity of biological systems, in comparison with most physical systems, makes even more urgent the application of mathematical and computational modeling techniques to them. Dynamical systems provide ''the natural language needed to describe the 'integrated behavior' of systems coordinating the actions of many elements'' [108] , and are also capable of displaying emerging self-order from massively disorganized complexity, which is believed to be a fundamental feature of life. In what follows, we will describe the notion of immunomic regulatory networks, a dynamical system model for immune regulation.
An important recent development in immunology has been the discovery of regulatory T cells [103, [109] [110] [111] . These T cells suppress immune responses, helping to stem runaway inflammatory processes and to avoid autoimmune disease. It is thought that this beneficial suppression activity can turn deleterious when it is taken advantage of by pathogens, leading to chronic and abnormal infectious processes. It has been observed that some regulatory T cells are not antigenspecific; these are called natural regulatory T cells [103] . In addition, there exist regulatory T cells, both CD4 þ and CD8 þ , that are antigen-specific and thus epitope-driven [103] . Given the suppressive action of epitope-driven regulatory T cells in conjunction with the promoting activity of epitope-driven helper T cells, it follows that the immunological response to a given epitope may be suppressed or promoted by the immunological response to other epitopes. Thus the notion of regulatory networks arises as a fundamental concept in understanding the functioning of the immune system. In fact, most human diseases are the result of an unbalance in immune system homeostasis.
In functional genomics, DNA microarray data is used to infer genomic regulatory networks [112] . For biological and efficiency reasons, gene expression is often quantized to two levels: on and off [113] . The multivariate methods of classification and feature selection discussed in the previous section have proved to be essential in the inference of such Boolean (binary) regulatory networks. By the same token, immunomic microarray data can be used to infer immunomic regulatory networks. As is true for gene expression, one may quantize each epitope response measured with an immunomic microarray at one of two levels-on (immunogenic/responder) and off (nonimmunogenic/ nonresponder)-leading to the inference of Boolean immunomic regulatory networks.
In the general case, each node of an immunomic regulatory network represents a combination of the epitope, the cytokine response measured, and the T cell population used as the target; in most cases, each node is in a one-to-one relationship with a single physical spot on an immunomic microarray experiment with a given T cell population. The edges between nodes represent putative regulatory relationships between the cells that respond to the respective epitopes. Figure 6 depicts a simple example with quantized Boolean responses, where peptide-MHC microarrays are used in conjunction with three kinds of T cell targets: CD4 þ helper T cells, CD4 þ regulatory T cells, and CD8 þ cytotoxic T cells. Epitope A is specific to the CD4 þ helper T cells that promote the response to epitope C, which is specific to the CD8 þ effector T cells that produce the actual protective mechanism. In addition, there is an epitope B that activates the CD4 þ regulatory T cells that suppress the effector response to epitope C, thereby producing an antiinflammatory response. In practice, such a model would be derived from microarray data by automatic epitope (feature) selection and determination of predictive relationships (classifier design). In this example, the effector response is activated only in the presence of both help from epitope A and an absence of regulatory response to epitope B. The suppressing response to epitope B is itself promoted by the presence of a response to epitope C, providing a negative feedback mechanism. These relationships can be represented by a wiring diagram and transition rules, depicted in Figure 6a .
Since the responses have been quantized to two values, on and off, and there are three epitopes, the total number of possible states of the system is 2 3 ¼ 8. Figure 6b depicts the state transition table obtained from the transition rules in Figure 6a . Using this table, one can determine the attractors of the system, which are the states or sets of states in which the system stays in the long run in the absence of external disruptions. For each attractor, there is associated a basin of attraction, containing attractors and transient states, which are the sets of states that lead to the attractor [108] . In the context of biological systems, attractors are a mathematical model for homeostasis. In our minimalist example, we see that there are two different behaviors, corresponding to the two distinct basins of attraction in Figure 6c ; the respective attractors are indicated by dashed rectangles. As can be seen, which of the two behaviors the system is in depends only upon the state of the response to epitope A. If it is off (there is no help), then the system may pass through some irrelevant transient states but it will always tend toward the resting single-state 000 attractor and thus an absence of activity; see the diagram on the left in Figure 6c . If the response to epitope A is on (there is help), then the activity of the system corresponds to that of a cyclic attractor, with the effector DOI: 10.1371/journal.pcbi.0020081.g006 Figure 6 . Example of a Simple Immunomic Network, Consisting of Three Epitopes Epitope A is a promoter (A is specific to CD4 þ helper T cells), epitope B is a suppressor (B is specific to CD4 þ regulatory T cells), while epitope C produces the effector response (C is specific to CD8 þ cytotoxic T cells), while also promoting the suppressing response of epitope B (negative feedback). response being turned on and off cyclically; see the diagram on the right in Figure 6c . This situation corresponds to modulation of the effector response and regulation of the inflammatory response by means of a negative feedback mechanism. Note that when there ceases to be a response to epitope A, the system jumps to the other basin of attraction, and tends to the resting 000 state. The immune response to epitope A in this example determines the behavior of the system, and thus it functions as the master of the overall immunological response, with the individual immune responses to epitopes B and C being slave to it. The concept of master-slave regulatory units is quite important for the understanding of complex regulatory systems and has in fact been considered as a mechanism for genomic regulation (Michael Bittner, unpublished data).
Inference of immunomic regulatory networks from immunomic microarray data constitutes, after proper validation, computational knowledge discovery. There are subtle epistemological issues involved in using data-driven, computer-based methodology to obtain scientific knowledge. As Karl Popper explained in his classic book The Logic of Scientific Discovery [114] , every scientific theory consists of an initial irrational act of creativity (induction) followed by rigorous logical consequences (deduction) and testing of the initial hypothesis. Where is the initial act of creativity, in other words the scientific hypothesis, in computational knowledge discovery? Computers are clearly not capable of irrational creativity. Can this be considered science? We maintain that the answer is yes [115] . In fact, the initial irrational act of creativity is there; it is involved in all the steps of experiment design, selection of patients/samples, and choice of statistical methodology. Once these are settled, the actual data analysis is purely logic deduction via the machinery of mathematical operations, as prescribed by Popper. In this deductive stage the computer plays a critical role, as it facilitates the application of very complex computational methods. Therefore, scientist (and statistician) bias here is in fact unavoidable, as it is in all scientific disciplines. In particular, the term data-driven is really a misnomer in describing computational knowledge discovery.
Functional immunomics promises great rewards, both in terms of our basic understanding of the immune system and in disease diagnosis/prognosis and rational epitope-driven vaccine design. Research into the basic biology and statistical methods associated with functional immunomic experiments will lead to the advancement of medical science and public health. Functional immunomics is however still at an early stage of development. In this review we have attempted to provide a coherent vision of this nascent field, and have speculated on future research directions for this technology.
In our discussion we have often compared and contrasted immunomics with genomics. Immunomics supervenes on genomics, in the epistemological sense, since immunology ultimately depends on the functioning of genes inside cells, but immunomics has its own independent character and properties. In the same manner that each cell has its own pattern of gene expression that defines its unique cellular properties, each reaction of the cognate immune system to an antigen has its own pattern of epitope-specific responses that define its final outcome.
There exists a large collection of mathematical models to describe the immune system [116] . Here, we have proposed Boolean immunomic regulatory networks as a new mathematical model for immune system regulation. This is a dynamical system model, with parameters that can be estimated from immunomic microarray data. A somewhat similar concept was suggested in [102, 117] , where a network model for cytokine action was proposed, albeit without explicit reference to large-scale immunomic technology or regulatory T cell response. In addition, immune system regulatory networks have been previously discussed in the context of genomics [118] . The immunomic regulatory network model may be useful for computational knowledge discovery and simulation of regulatory mechanisms of the immune system in health and disease, which may lead to advances in practical applications (e.g., vaccine design) as well as in the basic scientific understanding of the immune system. " Transmission patterns of smallpox: systematic review of natural outbreaks in Europe and North America since World War II BACKGROUND: Because smallpox (variola major) may be used as a biological weapon, we reviewed outbreaks in post-World War II Europe and North America in order to understand smallpox transmission patterns. METHODS: A systematic review was used to identify papers from the National Library of Medicine, Embase, Biosis, Cochrane Library, Defense Technical Information Center, WorldCat, and reference lists of included publications. Two authors reviewed selected papers for smallpox outbreaks. RESULTS: 51 relevant outbreaks were identified from 1,389 publications. The median for the effective first generation reproduction rate (initial R) was 2 (range 0–38). The majority outbreaks were small (less than 5 cases) and contained within one generation. Outbreaks with few hospitalized patients had low initial R values (median of 1) and were prolonged if not initially recognized (median of 3 generations); outbreaks with mostly hospitalized patients had higher initial R values (median 12) and were shorter (median of 3 generations). Index cases with an atypical presentation of smallpox were less likely to have been diagnosed with smallpox; outbreaks in which the index case was not correctly diagnosed were larger (median of 27.5 cases) and longer (median of 3 generations) compared to outbreaks in which the index case was correctly diagnosed (median of 3 cases and 1 generation). CONCLUSION: Patterns of spread during Smallpox outbreaks varied with circumstances, but early detection and implementation of control measures is a most important influence on the magnitude of outbreaks. The majority of outbreaks studied in Europe and North America were controlled within a few generations if detected early. The anthrax attacks that followed the events of September 11th, 2001 focused attention on the threat of terrorism with biological agents including variola major, the causative agent of smallpox [1] . There is concern that an attack with smallpox could result in many deaths because of the susceptibility of the U.S. population [2] [3] [4] [5] [6] [7] [8] . A number of issues, such as the amount of virus released and the number of people exposed, need to be considered in order to estimate the impact of a bioterrorist attack with smallpox. However, one key factor in determining the size of an outbreak resulting from such an attack is the transmissibility of smallpox, which can be difficult to estimate. One measure of transmissibility is the effective reproductive rate (R), which is equal to the expected number of infections produced by an infectious host in a real world setting [5, [9] [10] [11] .
R can be determined in a straightforward manner for individual epidemics and is related to the circumstances, such as population vaccination status, under which the individual epidemics occur. When considering intentional attacks, the greatest interest lies in initial spread, much of which often occurs before an outbreak is recognized. For this reason, we focused on the transmission from index cases to the first generation of secondary cases, which we termed the initial R.
The data for this study was obtained from a systematic literature review for reports of smallpox outbreaks. We limited our review to reports of smallpox outbreaks occurring in Europe and North America after 1945 because these societies were much more similar to the modern United States in terms of their demographic, social, and physical structure, than were the pre-World War II western and the developing societies from which most smallpox outbreak reports have emanated. Moreover, population immunity was also relatively more similar to a contemporary US population than might be expected a priori. While early post-war European and North American populations were generally vaccinated in childhood, re-vaccination rates were generally low and few adults had more than remote vaccination [12, 13] . Immunity naturally acquired through infection was also similarly low in both populations because outbreaks were rare. Although terrorist use of smallpox in the 21 st century may have substantially different epidemiological characteristics (see discussion below), the outbreaks analyzed in this paper are the closest actual population experience that may help partially guide modern smallpox control efforts.
The following databases were searched for literature on smallpox through 2001: National Library of Medicine 1800 to 2001 (keywords: smallpox or variola), Embase 1974 to 2001, Biosis 1969-2001 (keywords: smallpox or variola virus and human and not in-vitro), Cochrane Controlled Clinical Trials Library (keywords: smallpox or variola) and Defense Technical Information Center 1947 to 2000 (keywords: smallpox). The search was later updated by a PubMed search from 2001 to 2004 (keywords: smallpox or variola). Duplicate titles were removed. Secondary searches included more specific searches for outbreaks in specific countries and sites, and a review of reference lists from publications identified in other searches (Figure 1 ). There was no language restriction. Two authors (SB and VB) independently scanned titles for relevance, and publications chosen by either reviewer were reviewed further. One of the authors (VB) scanned the selected articles to identify post-1945 smallpox outbreaks from Europe and North America. Two abstractors (RB or research associate and VB) independently extracted data from the identified outbreaks. Discrepancies between reviewers were resolved by consensus. Non-English publications were abstracted by one author (RB) or by a research assistant under an author's (VB) direction.
To be included in the analysis, the following had to be available: the date of occurrence, and the number of index cases, total cases, cases in each generation, and generations. The following outbreak details were also extracted if available: the number of cases acquired within a hospital, a household, or at a distance (cases not acquired by household or hospital contact); the number of missed cases (cases not diagnosed as smallpox at the time of illness) and deaths; the generation at which the outbreak was identified, and the clinical presentation of the index case. Index cases were classified as having had typical smallpox, atypical smallpox (a milder illness as seen in people with prior smallpox vaccination and/or a rash not characteristic of smallpox), or hemorrhagic smallpox (a usually fatal illness characterized by internal hemorrhage and/or petechiae) [14] . The following data regarding the efficacy of control measures were also extracted if available: the use of ring vaccination (the vaccination of people who were in close contact with a patient with smallpox), case isolation (the separation of suspected cases of smallpox from the general public), quarantine (the restriction of movement in or out of a region that has been exposed to smallpox), mass vaccination (the vaccination of an entire region), and/or prophylactic vaccinia immune globulin administration [15] [16] [17] .
The number of outbreaks over the period studied was small (total of 45 outbreaks) and the circumstances varied widely. Therefore, a descriptive and qualitative analysis was emphasized. For this analysis, the initial R was meas-ured by dividing the total number of first generation cases by the number of index (zero th generation) cases. In addition, several outbreaks had sufficient details to be classified by setting. Because hospitals were often the centre of outbreaks, setting was defined according to the proportion of secondary cases (non-index cases) acquired within a hospital [12, 13, [18] [19] [20] . After examination of the distribution of case locations across outbreaks, hospital outbreaks were defined those in which 70% or more of secondary cases were hospital acquired, mixed outbreaks as those in which 30% to 70% of secondary cases were hospital-acquired, and community outbreaks as those in which 30% or less of secondary cases were hospitalacquired.
Based on the results of the primary searches, 5,976 titles were scanned; 1,389 of these were selected. Despite initial de-duplication, 10 of these were duplicates (Figure 1 ). Secondary searches identified 14 additional publications. Of the resulting 1,393 publications, 1219 (87%) were obtained; 92 journal citations (7% of the selected publica-tions) and 41 government reports (3% of the selected publications) were unobtainable.
Forty-nine post-1945 European and North American outbreaks were identified. Thirty-six of these were included in a single World Health Organization (WHO) publication [21] and prior work by T.M. Mack [12] . Several outbreaks were documented in multiple publications (Tables 1 and  2 ). Four outbreaks were excluded. The source of the first outbreak could not be confirmed as the outbreak may have resulted from the importation of smallpox on a rug (Union of Soviet Socialist Republics or U.S.S.R, 1959). The second and third outbreaks occurred under unique circumstances; one occurred aboard a ship (Poland, 1962) and the other may have been the result of testing aerosolized smallpox (U.S.S.R, 1972) [21] [22] [23] . A fourth outbreak was excluded because the number of cases in each generation was not documented; therefore, the initial reproductive rate (R) could not be derived from the data (United States or U.S., 1946) [24] . The remaining 45 original outbreaks occurred over a thirty-year period in different countries and under different circumstances. The majority of reported outbreaks occurred in three coun- (Tables 1 and 2 ). Most resulted from involved index cases imported from endemic countries, but two outbreaks were the result of laboratory exposure [25, 26] .
Although published or listed as single outbreaks, four outbreaks included multiple epidemiologically distinct components, such as occurred when a case travelled to a different region resulting in a secondary outbreak in the new area (see reference column and footnotes for Table  2 ). The components of these four outbreaks were treated as 10 separate outbreaks so that an effective total of 51 outbreaks were analyzed [21, [27] [28] [29] . For all 51 outbreaks, available information included the total number of generations and cases, the number of cases in each generation, the duration of the outbreak, and the number of deaths (Tables 1 and 2 ). Thirty of the outbreaks could also be categorized by setting and had detailed information on the generation that the outbreak was identified, control measures, clinical presentation of the index case, and the number of missed cases ( Table 2) .
The median initial reproduction rate (R) across all 51 outbreaks was 2 with a range of 0 to 38 (Table 3, Figure 2a ). About half had an initial R of 1 or less, and over two-thirds had an initial R of 3 or less. The median duration, as measured by number of generations, was 1 with a range of 0 to 9 (Table 3, Figure 2b ). About a third did not extend beyond the index generation, and nearly three quarters lasted for 3 or fewer generations. The median outbreak size, as measured by the total number of cases, was 4 with a range of 1 to 134 cases (Table 3, Figure 2c ). About half involved 3 or fewer cases, and two-thirds involved 15 or fewer cases. The median number of deaths was 1 with a range of 0 to 26 (Table 3, Figure 2d ). About two-fifths involved no deaths, and three quarters involved 3 or fewer deaths.
Outbreaks with greater initial R-values tended to be larger and longer. For example, in outbreaks with an initial R of 5 or less versus more than 5, median values for the total number of cases and generations were 2 versus 23 and 1 versus 2.5 respectively.
There was no temporal trend in the amount of detail reported, but the thirty outbreaks reported in detail typically had a larger R (median 2 vs.0), lasted more generations (median 2 vs. 0), and had more total cases (median 13 vs. 1) and deaths (median 2 vs. 0) compared to lessdetailed outbreaks (Table 3 , Figures 2a-d) . In the detailed outbreaks, most of the index cases had atypical (mild) or hemorrhagic disease. Because these clinical presentations of smallpox are less common, many of these index cases were missed or not diagnosed with smallpox at the time of illness. The majority of index cases were infected 
The median initial R across the 30 detailed outbreaks was 2 with a range of 0 to 38 (Table 3 , Figure 2a ). About a third had an initial R of 1 or less, and about two thirds had an initial R of 3 or less. Outbreaks that remained in the community had a lower initial R (median 1) than those that were hospital based from the first generation (median 12) ( Table 3 , Figure 3a ). Mixed outbreaks, which generally started in the community and later spread through hospital contacts, had an intermediate initial R (median 2) ( Table 3 , Figure 3a ). The initial R was smaller when the index case(s) had a typical versus atypical or hemorrhagic presentation (medians 1 vs. 4 or 5, respectively), and when the index case was identified as smallpox versus unrecognized (median 1 vs. 5) ( Table 3 , Figures 3b and  3c) . The effect was most pronounced in hospital outbreaks -median initial R's for identified versus unidentified index cases in hospital outbreaks were 3 versus 15, but the same values for community and mixed outbreaks were 1 versus 4, and 2 versus 2, respectively.
The median duration for 30 outbreaks was 2 generations with a range is 0 to 9 generations (Table 3, Figure 2b ). About a third did not extend beyond the first generation, Table 2 ) analyzed by components:
(1) Outbreak from Marseille, France (1952) broken into two components: Entries 3 and 15 [27] .
(2)Outbreak from Cardiff, U.K.(1963)broken into three components: Entries7, 24 and 26 [21, 28] .
(3) Outbreak from Poland (1963) broken into three components: Entries 8, 17 and 27 [41] .
(4)Outbreak from Kosovo, Yugoslavia (1972) broken into two components: Entries 10 and 19 [21] . Footnotes for Table 2 : a Nature of the index cases was not clear from the text (limited information) [27] . b Total number of cases listed as 74, consistent with text [38] and second reference [27] . c Total number of cases listed as 5 [28, 34] ; 6 is listed in World Health Organization Table 23 .4 [21] . d Proportion of hospital acquired cases listed as 0.44 because three nurses infected as a result of household exposure and were setting excluded as hospital contacts [21, 36] . e Total number of cases listed as 1 [37] ; 2 is listed in World Health Organization Table 23 .4 [21] . and another third lasted for only through the second generation. Outbreaks that remained in the community lasted a median of 1 generation, while those that were hospital-based lasted a median of 2 generations and those that were mixed lasted a median of 3 generations (Table  3) . Outbreaks tended to be shorter when the index case was typical compared to atypical or hemorrhagic (median number of generations 1 vs. 3 vs. 2, respectively) and when the index case was identified (median number of generations 1 vs. 3) ( Table 3 ). The duration of hospital outbreaks was not affected by the identification of the index cases, but the duration of mixed and community outbreaks lasted up to 6 and 9 generations, respectively, when index cases were unidentified.
The median outbreak size, as measured by the total number of cases, was 13 with a range of 1 to 134 cases (Table 3, Figure 2c ). There were approximately equal proportions of small (total number of cases less than 5) and large (total over 20) outbreaks ( Table 2 ). Outbreaks that remained in the community involved a median of 2 cases, while those that were hospital based involved a median of 20 cases and those that were mixed had a median of 22 cases (Table 3) . Outbreaks tended to be smaller when the index case was typical versus atypical or hemorrhagic (median number of cases 2 vs. 27 vs. 16.5) and when the index case was identified (median number of cases 3 vs. 27.5) ( Table 3 ). The outbreak size was similarly small across all 3 settings when the index case was identified and similarly large across the settings when it was not.
The median number of deaths was 2 with a range of 0 to 26 (Table 3 , Figure 2d ). About one-fifth involved no deaths, and four fifths involved 4 or fewer deaths (Table  2) . Community, hospital based, and mixed outbreaks had medians of 1, 4, and 3 deaths, respectively (Table 3) ; however, case fatality rates were 0.24, 0.20 and 0.17. The number of deaths was smaller if the index case was identified (median equals 1 vs. 6, Table 3 ). The case fatality rate was highest if the index case presented with hemorrhagic-type smallpox.
There was a strong association between typical presentation and early identification of index cases: index cases were identified as smallpox in 9 of 9 outbreaks when the presentation was typical, 3 of 13 when it was atypical, and 2 of 6 when it was hemorrhagic. This relationship aids in the understanding of outbreak severity. For example, 93% of outbreaks had greater than 10 cases when the index case was not identified. Moreover, in all of these larger outbreaks, the index cases were atypical or hemorrhagic. When the index case was identified, most outbreaks had less than 10 cases regardless of whether the index case was typical, atypical, or hemorrhagic.
Descriptions of smallpox transmission are important to biosecurity planning, especially since some planning efforts have posited outbreaks resulting in numbers of cases -hundreds, thousands, or more -that are very large relative to the clinical experience shortly before eradication of the natural disease [32] . In a systematic review designed to provide an understanding of smallpox transmission patterns, we identified and analyzed what were effectively 51 smallpox outbreaks from post-1945 Europe and North America. In these outbreaks, we found a median of 4 total cases and 1 death.
The characteristics of the outbreaks varied greatly: one outbreak with an initial R of 11 involved 134 people and caused 26 deaths over only 3 generations of spread; another outbreak with initial R of only 2 involved 28 cases but lasted 9 generations. However, most outbreaks were small and contained within a few generations: 31 percent involved only one case, 41 percent caused no deaths, and 31 percent had an initial R of 0 (i.e., no first generation cases).
Examination of outbreaks within categories of setting, identification of the index case, and clinical characteristics provided additional insights. By reviewing original publications, we were able to categorize and analyze many outbreaks by setting. For example, were able to review the original publications for eight outbreaks documented by the WHO [25, 26, 28, [33] [34] [35] [36] [37] as well as, identify thirteen additional outbreaks [20, 27, [29] [30] [31] [38] [39] [40] [41] . Several of these outbreaks also had epidemiologically distinct components, resulting in 51 effective outbreaks for analysis.
Many (six out of eleven) of the hospital outbreaks were only found in the above-mentioned additional outbreaks [20, 27, 29, 30, 38, 39] . Hospital outbreaks had higher initial reproductive rates than either community or mixed outbreaks, and had more cases and deaths than did community types. Moreover, the interaction of identification and setting in the studied outbreaks reveals a pattern similar to that seen in frequently unrecognized infectious diseases, such as tuberculosis and, more recently, SARS [42] . When index cases were identified, control is established early and both the median initial R and the number of generations were similarly low in all 3 settings. When the index cases were unidentified, a large increase in the median initial R was seen only in the hospital setting and a large increase in the number of generations was seen only in community and mixed settings. This is consistent with the observation that, within the close quarters of an institution, the transmission of smallpox is relatively effective before identification and containment measures are quite effective after identification. This impression is reinforced by examination of the two shipboard out-breaks that were excluded from the analysis. In these outbreaks, the initial R's were 6 and 25, respectively, but recognition was followed by control within two generations [22] .
In community outbreaks, transmission is intrinsically less effective. The consequence of missed initial identification in the community was not an elevated initial R, but an increase in the length of the outbreaks. The longer outbreaks may be due to a decrease in the effectiveness of containment measures after initial spread in the community, possibly because of the greater difficulty of tracking and managing cases in that setting. Nonetheless, all of the 30 outbreaks reported in detail were brought under control, generally by case isolation and ring vaccination.
Other measures were rarely used (mass vaccination was used in the New York City, 1947 outbreak and mass vaccination and quarantine were used in the Kosovo, 1972 outbreak [21, 30] ) and there is limited information on the efficacy of these additional measures. It should also be noted that newer treatment modalities, such as Cidofovir, have been shown to be protective in animal studies against vaccinia and other orthopox viruses (monkeypox and cowpox) after exposure [43, 44] .
Outbreaks beginning with an atypical or hemorrhagic versus a typical presentation of the index case had more cases, a higher initial transmission rate, and were somewhat longer. This is related to the ease of initial diagnosis: only non-typical cases were mis-identified, and outbreaks in which the index case was mis-identified were generally larger, deadlier, longer, and had a median initial R of 5 compared to 1 if the case was identified. We speculate that the large difference in initial R is because, when the index case is identified, this parameter is effectively the initial reproductive rate with early institution of control measures. However, it is clear that the types of outbreaks seen in developed countries in the second half of the 20th century were well and quickly controlled.
Despite searching several databases, using broad searches, scanning nearly 6,000 titles, and reviewing papers in several languages, our requirement that included epidemics involve post-World War II western populations limited us to identifying only 45 separate outbreaks, some of which were divided for analysis. The majority of the outbreaks were from only three countries, suggesting that outbreaks from certain countries were more likely to have been documented. The 30 outbreaks reported in detail were larger than the 21 that were briefly reported; it is probable that smaller outbreaks were not routinely published in detail because outbreaks were still relatively common and smallpox was still endemic in many parts of the world. This apparent publication bias, however, suggests that there may have been many unpublished small outbreaks and that this analysis may overestimate the transmission potential of smallpox because it is based only on published reports.
The recent literature contains several estimates for the reproductive rate of smallpox. It is difficult to directly compare our findings and estimates for initial R, the initial effective reproduction rate for smallpox, to these other studies. Two of the studies do not use contemporary western outbreaks. One estimates the basic reproductive rate, (R 0, a theoretical parameter defined as the expected number of new infected hosts that an infectious host will produce in a large randomly mixed population of susceptible individuals [9] ), rather than effective reproduction rate. Specifically, Gani and Leach used epidemic modelling of data from 19th Century European smallpox outbreaks to estimate a R 0 of 3.5 to 6.0 [19] . Eichner and Dietz obtained an R of 6.9 from data on a 1967 outbreak in Nigeria [5] . Meltzer et al examined post-1961 outbreaks from countries around the world in estimating an R of 3 [8] .
Our estimate of the overall initial R is quite comparable to Meltzer et al., but use of any single point estimate for R 0 or initial R in policy analysis ignores a main lesson of this review. This work clearly shows that the pattern, speed, and duration of an outbreak's spread vary widely according to the specific circumstances surrounding an outbreak. Different smallpox release scenarios should be expected to yield different results in part because the parameters and modified natural history of the outbreak itself will vary with the scenarios. For example, our previous work modelled the outcome of terrorist attacks under different scenarios [4] . These scenarios included community based outbreaks resulting from infected cases riding a busy transit system and the intentional release of aerosolized smallpox in airport terminals, and a mixed outbreak resulting from the release of aerosolized smallpox in a large office building. Although adjustments had to be made for a poorly vaccinated population and healthcare workforce, the understanding of smallpox transmission patterns in hospital, mixed and community settings, provided important insights into how smallpox may spread under different attack scenarios.
In a contemporary US population, one might expect transmission from introduced smallpox to be higher than we found to be typical. Population density is higher; mobility is greater, and, though more similar that one might think, immunity in the contemporary U.S. population is likely somewhat lower than in the populations studied. The healthcare workforce is also poorly vaccinated and inexperienced with smallpox. This will decrease the human resources available for care and containment, and increase the likelihood that initial cases will be missed. Counterbalancing this is the fact that few index cases will be vaccinated recently so the likelihood of more easily recognized typical cases is much higher. As the outbreak scenarios of greatest concern involve intentional release, outbreaks could be larger and more obvious than those reviewed, or could involve strains selected or engineered to be more virulent or contagious.
Finally, the outbreak from Kosovo deserves notice because this region is not as developed or as wealthy as most of the other countries studied. This outbreak was the largest identified for this review (N = 176 for both combined hospital and mixed components). Moreover, the hospital and mixed component of this outbreak had the second highest (R = 38) and highest (R = 11) initial reproductive rate, and the hospital component lasted 9 generations (median number of generations for a hospital outbreak = 2). This suggests that a smallpox outbreak in a less developed country with limited resources for healthcare, disease surveillance, and case isolation could be potentially more devastating than a bioterrorist attack in a Western/industrialized country [44] .
It is clear that most post-World War II outbreaks in Western countries were small and had low transmission rates. This was almost uniformly the case when the index case presented with typical smallpox and was recognized early. However, exceptions were common and heterogeneous, particularly when (usually atypical or hemorrhagic) index cases were not initially recognized. Initially unrecognized community outbreaks lasted several generations despite low initial R values. This suggests that control measures were less effective in this setting, perhaps because of the difficulty tracing and vaccinating all cases and contacts. In contrast, unrecognized hospital outbreaks were controlled quickly despite high initial R values, suggesting that control measures worked very well in a closed, contained setting. Early detection, particularly of patients who do not present with typical smallpox, coupled with early implementation of control measures decreases both the duration and size of outbreaks in all settings.
Publish with Bio Med Central and every scientist can read your work free of charge  Reliability of case definitions for public health surveillance assessed by Round-Robin test methodology BACKGROUND: Case definitions have been recognized to be important elements of public health surveillance systems. They are to assure comparability and consistency of surveillance data and have crucial impact on the sensitivity and the positive predictive value of a surveillance system. The reliability of case definitions has rarely been investigated systematically. METHODS: We conducted a Round-Robin test by asking all 425 local health departments (LHD) and the 16 state health departments (SHD) in Germany to classify a selection of 68 case examples using case definitions. By multivariate analysis we investigated factors linked to classification agreement with a gold standard, which was defined by an expert panel. RESULTS: A total of 7870 classifications were done by 396 LHD (93%) and all SHD. Reporting sensitivity was 90.0%, positive predictive value 76.6%. Polio case examples had the lowest reporting precision, salmonellosis case examples the highest (OR = 0.008; CI: 0.005–0.013). Case definitions with a check-list format of clinical criteria resulted in higher reporting precision than case definitions with a narrative description (OR = 3.08; CI: 2.47–3.83). Reporting precision was higher among SHD compared to LHD (OR = 1.52; CI: 1.14–2.02). CONCLUSION: Our findings led to a systematic revision of the German case definitions and build the basis for general recommendations for the creation of case definitions. These include, among others, that testable yes/no criteria in a check-list format is likely to improve reliability, and that software used for data transmission should be designed in strict accordance with the case definitions. The findings of this study are largely applicable to case definitions in many other countries or international networks as they share the same structural and editorial characteristics of the case definitions evaluated in this study before their revision. between countries. One of the first case definitions used for national disease reporting was the case definition for AIDS published by the Centers for Disease Control and Prevention (CDC) in 1982 [2] . In 1985 Sacks published a survey among all 50 US states, Puerto Rico, and Washington, DC, that revealed important variations in the case definitions between the different states, and concluded the necessity to unify case definitions if surveillance data between states are to be compared [3] . In 1990 the CDC in collaboration with the Council of State and Territorial Epidemiologists published an edition of case definitions for public health surveillance [4, 5] .
Since then case definitions have become an important tool of other national surveillance systems and international surveillance networks. Koo and colleagues have analyzed surveillance data for Cholera in Latin America and have described the importance of uniform case definitions to make data comparable between countries [6] . In 2003 the European Union (EU) case definitions for the European networks have reached obligatory status for the member states reporting to the EU [7] . During the SARS epidemic the case definition had a major impact on whether and how countries were considered affected or not, resulting in severe political and economic consequences for a number of countries [8] .
Coggon and colleagues have demonstrated the difficulties of determining optimal case definitions if a satisfactory diagnostic gold standard is lacking [9] . In sharp contrast to the importance of case definitions hardly any research has been published on the performance of surveillance case definitions. Studies are rare on how local health departments and other health professionals are able to understand case definitions and to what extent case definitions are unambiguous enough to really assure reliability. To our knowledge, the only publication investigating this issue was focused on case definitions for nosocomial infections: Gastmeier and colleagues had investigated how uniform the case definitions of the nosocomial infections surveillance system in Germany had been applied by different investigators using a set of 60 case studies [10] . Due to the general importance of case definition for public health surveillance and the current need for harmonization in international surveillance systems we conducted a systematic evaluation of the national case definitions with the objective to identify general as well as specific criteria and recommendations for improvement of case definitions.
Germany is a federal republic with 16 states subdivided into 440 counties. As in many countries the local (county) health departments (total number: 425) are the primary recipients of infectious disease notifications made by physicians and laboratories. Local health departments verify the incoming notifications and assess the need for public health action. Local health departments use one of five software products on the market to classify the case reports according to the national edition of case definitions and to report these cases electronically to the state health department. From there the report is being forwarded to the Robert Koch Institute (RKI), the federal institution in charge of national infectious disease surveillance in Germany [11] .
The edition of national case definitions for all notifiable infectious diseases was introduced in Germany in 2001, following the implementation of a new law to control infectious diseases (Infektionsschutzgesetz, IfSG) [12] [13] [14] . The IfSG determines the set of diseases and pathogens to be notified by physicians and laboratories throughout the Federal Republic of Germany. The five eastern states, which formerly belonged to the Democratic Republic of Germany (East Germany) and the State of Berlin have enacted complementary rules that make certain diseases additionally notifiable within the state jurisdiction, that are not notifiable in all of Germany.
The case definitions were developed by the RKI, using the delphi method including the expertise of state epidemiologists, national reference laboratories and medical and scientific associations for the specific diseases. The case definitions for infectious conditions under public health surveillance published by the CDC were also taken into account [5, 15] . After having published the IfSG case definitions in the fall of 2000 to be implemented with the beginning of 2001 the RKI also published additional case definitions in January 2002 for some of the diseases exclusively notifiable in the eastern states jurisdictions [11, 16] . From June 2002 to September 2003 we had conducted a systematic evaluation of the case definitions with the purpose to revise them by the end of 2003.
The German case definitions are divided into three types of evidence: Clinical picture, laboratory detection, and epidemiological confirmation. The types of evidence are specifically defined for each disease (see table 1 ). Based on whether or not requirements for these three types of evidence are fulfilled a case is classified into five categories. In the revised 2004 edition of case definitions these categories are named: A) clinically diagnosed illness (neither epidemiologically nor laboratory-confirmed), B) clinically and epidemiologically confirmed illness (not laboratory-confirmed), C) clinically and laboratory-confirmed illness, D) laboratory-detected infection not fulfilling clinical criteria, E) laboratory-detected infection with unknown clinical picture. (In the 2001 edition of case definitions these five categories were named slightly differently) For most notifiable diseases only categories B, C, D and E are reportable from the local health department to the next level, requiring at least laboratory detection of the pathogen or epidemiological confirmation. For some exceptions (e.g. tuberculosis, polio, measles, Creutzfeldt-Jakob disease), cases are also reported from the local health department to the next level if category A -the clinical picture alone -is fulfilled.
In June 2002 we conducted a Round-Robin test in analogy to the established quality control procedure of laboratories and other testing units [17] . Round-Robin tests are mainly used in proficiency tests in order to determine laboratory performance by means of comparing tests on identical items by two or more laboratories in accordance with predetermined conditions [18] .
We asked each local and state health department to classify a selection of 68 written case examples on the basis of the case definitions that were implemented in 2001 (2002 respectively for disease only notifiable in East German States). While proficiency tests generally intent to assess the ability of laboratories in finding identical results, we applied this method to assess to which extend the case definitions were unambiguous enough to assure identical classification by the health departments.
We applied four different outcome variables in our analysis: 1) Disease identification: A disease was defined as being correctly identified if the participant of the Round-Robin test was able to identify the correct disease of the case example.
2) Case categorization: A case example was considered correctly categorized if the participant classified the case example with the correct disease and the correct case definition category as defined in the gold standard.
3) Reporting: The decision on reportability was considered correct if a case that should have been reported to the Clinical picture: Clinical picture compatible with salmonellosis, characterized by diarrhea, abdominal pain, malaise, vomiting, fever. Salmonella can also cause infections outside the intestinal tract (for example: arthritis, endocarditis, pyelonephritis, septicaemia). Laboratory diagnosis: Isolation (culture) of pathogen from stool or other clinical material (e.g. blood, urine). The identification of serogroup has to be attempted.
Clinical picture • Clinical picture of acute salmonellosis, defined as at least one of the following four symptoms: diarrhea* • cramp-like abdominal pain • vomiting • fever* additional information: Samonella can also cause generalized (septicemia) and localized infections outside the intestinal tract (for example: arthritis, endocarditis, pyelonephritis). These should in case of an acute infection also be reported. The reactive arthritis, which may also be caused by Salmonella infection, is not to be reported. Laboratory diagnosed Positive finding using the following method: • Direct detection of pathogen: isolation of pathogen (culture) Additional information: Results of identified serogroup and lysotype should also be reported. Epidemiological confirmation Epidemiological confirmation, defined as at least one of the following three constellations while taking into account the incubation period (about 6 to 72 hours):
• Epidemiological link to another laboratory-diagnosed human infection through ❍ Person-to-person transmission OR ❍ Same source of exposure (e.g. animal contact*, food*) • Consumption of food (including drinking water), for which Salmonella spp. was laboratory-detected in non-consumed food.
• Contact to animal (e.g. poultry) with a laboratory-detected infection, or contact to its secretions or consumption of its products (e.g. eggs). * terms marked with an asterix are defined in more detail in a glossary of the case definitions next level would have been forwarded according to the case definition category, given that the correct disease was identified. Inversely decision on reporting was also seen to be correct if a case that should not have been reported to the next level was in fact classified in a way that the case would have been held back. However, cases forwarded with wrong disease identification (see above) were a priori considered incorrect. Thus reporting was based on the question whether the case needed to be forwarded to the state level or not, which is a direct result of the disease identification and the case definition category. Sensitivity of reporting was defined as the number of cases that would have been correctly forwarded divided by the number of cases that should have been forwarded according to the gold standard. The positive predictive value of reporting was defined as the number of cases that should have been forwarded among those that would have been forwarded. Precision of reporting is defined as the number of cases that would have been either correctly forwarded to the state level, or would have been correctly held back at the local health department level, divided by the total number of case examples. Unless stated otherwise, reporting precision was the outcome parameter used in the following analysis.
To specifically assess the effect of different styles in formulating case definitions, a fourth outcome variable was used. The clinical classification was considered correct if the part regarding the clinical picture was classified according to the gold standard, regardless whether other parts of the case definition were correctly classified or not. This analysis was done to compare case definitions with narrative description of the clinical pic-ture (as in all former IfSG case definitions) to case definitions with a more explicit check-list format of clinical criteria, that was implemented for diseases additionally notifiable in specific states and for the new IfSG case definitions.
The case examples consisted in facsimile excerpts of one or more of the following sources: laboratory report form, physician form, and protocol of the patient interview [see additional file 1]. The case examples were created based on real cases that have come to the attention of the RKI in the quality control process and in the information service hotline that the RKI is offering to the health departments.
The case examples were pre-tested among epidemiologists within the RKI and among epidemiologists and public health nurses in the state and local health departments. 
After the data of the respondents had been analyzed, the classification originally intended while creating the case definition, was challenged with the results of the respondents. Three epidemiologists then reassessed each individual case example and re-examined whether the classification originally intended was still justified. Based on this process the gold standard was defined for each case example.
We compared the responses to the established gold standard and stratified by the following variables: health department being in an East German versus a West German state, disease of the case example, whether or not physicians participate in routine quality control of case reports (versus this being done exclusively by public health nurses), institutional level (local health depart-ment, state health department, RKI), acceptance and style of case definitions (check-list vs. text) and software used at local health department. Because of the selection and distribution of case examples described above, we conducted the individual analyses for each group. After univariate analysis we conducted a multivariate analysis using SPSS 13.0 for Windows (Version 13.0.1).
The distribution of the classifications was compared to the gold standard, in order to identify common discrepancies. Based on these discrepancies we identified which part of the case definition was affected and identified specific aspects of the case definitions that had repeatedly been interpreted differently by the participants, indicating failure of the case definition to be unambiguous and reliable. These aspects were then summarized in order to deduct commonalities which could then lead to specific recommendations on how to improve this particular case definition and also on how to improve formulation of case definitions in general.
In May 2002 -simultaneously with the Round-Robin test -we conducted a written survey addressed to all 425 local health departments in Germany. Among various questions on the structure and equipment of the local health departments, and their experiences with the new IfSG, we also asked about the profession of the person who had actually filled the questionnaire and about his or her attitudes and experiences towards the case definitions. 
The multivariate analysis was limited to data from the local health departments and without additional case examples for the East German states (n = 5995). Only statistically significant associations are mentioned in the following.
The disease of the case examples was for all groups significantly associated with reporting (p < 0.001 in group 1, 2 and 4, p = 0.022 in group 3).
Local health departments using the RKI-software showed a higher chance to identify the disease (disease identification) of the case example according to the gold standard compared to health departments using any of the commercially available software programs (group 2: OR = 
The administrative level at which the respondents worked, was significantly associated with the outcome reporting.
For the analysis we used all cases of set A and set E (without the additionally diseases for the East German States, n = 2213). Adjusted for the diseases the chance of correct reporting to the next level was 1.5 times higher in cases done by state level staff compared to those done by local health department staff (OR = 1.52, CI: 1.14 -2.02).
The following observations have been made in the qualitative analysis of the responses:
• The concept of epidemiological confirmation was not well understood. For example travel in endemic countries was equivocally seen as an epidemiological confirmation (e.g. haemorrhagic fever and travel to Egypt). Re-evaluation of the case definitions showed that in fact there was only a vague definition of the epidemiological confirmation.
• Participants appeared to have difficulties in deciding whether all clinical signs and symptoms mentioned in the case definition had to be existent in a case, or whether they were only listed as descriptive examples.
• Case examples of diarrheal disease without any evidence of a specific pathogen, were frequently classified as salmonellosis.
• Laboratory findings with only one elevated antibody value in serum were repeatedly classified as laboratory detection although the case definition required a rise in antibody level.
• In some case definitions detection of the pathogen is only accepted if the detection was done in specific materials (normally sterile material such as blood for detecting N. meningitidis). This limitation was frequently neglected.
• Some of the information in the case definition intended to serve as additional background information was mistakenly used as selection criteria (e.g. statement that clinician described rash as "very typical" for measles, but fever was missing).
When asked about the availability of the case definitions, 395 (99%) of 398 local health departments responded that the case definition were accessible at the work place.
The case definitions were seen as useful by 377 (95%) of 397 health departments who answered this question and not useful by 20 (5%). The clarity of the individual sections of the case definitions was rated differently: The section on the clinical picture of the case definitions was seen as unambiguous in all case definitions by 72 respondents (18%), in the majority of case definitions by 305 (76%), in the minority by 20 (5%), and in none of the case definitions by one (0.3%) of the respondents (n = 398 respondents). The section on the laboratory confirmation of the case definitions was seen as unambiguous in all case definitions by 137 respondents (34%), in the majority of case definitions by 248 (62%), and in the minority by 11 (3%) (n = 396).
Three-hundred and three (87%) of 347 health departments stated that case classifications were done exclusively or primarily by public health nurses. With respect to the case examples presented to the participants, 220 (55%) of 396 respondents (from the local health departments) stated that the case examples were realistic.
The results of our evaluation have shown that although case definitions may appear to be clearly defined, they may be interpreted quite differently by their users, which may result in severe misclassifications and reduced sensitivity and positive predictive value. This study is believed to be the first to systematically assess these effects Also the complexity of the case definition itself is likely to affect reporting precision. Unfortunately much of the complexity of the case definition is a result of methodological limitations of available laboratory tests and cannot be influenced. The case definition system with its three different types of evidence leading to five different categories may appear very complex and less intuitive that the classical categories of "suspect", "probable" and "confirmed". The detailed differentiation of the German case definitions however enables us to apply computer algorithms in order to translate these to the EU case definitions and thus make the data compatible to the standards of various European surveillance networks and to WHO reports.
Reassessment of the gold standard after receipt of the responses resulted in modifications of 5 of the 68 case examples. This procedure took place in an initial review process of gold standard before the actual analysis was done. We believe it was legitimate and necessary in order to correct for biases caused by unforeseen ambiguity of the case examples.
The software used at the local health department was significantly associated with the quality of the data in only some subgroups and outcomes. Apparently the software is not a very strong determinant in the given study design, although our experience in implementing the electronic surveillance system in Germany showed that commercially available software products often do not fully implement the standards published by the RKI for data transmission software or they do so with a delay of several years [21] .
The other interesting finding is that the administrative level of the participants was significantly associated with the outcome: Participants from state health departments had a significantly higher rate of agreement with the reporting gold standard than the participants from local health departments. This might be explained by the fact that staff at the state level is generally higher trained in epidemiology and infectious diseases than local health department staff and they are routinely involved in quality control of incoming case reports and also training and supervision of local health departments' staff. 
All the observed quantitative effects and their propagated explanations merge into the one main conclusion: Case definitions must be very carefully formulated in order to assure their unambiguous interpretation by local health department personnel. The detailed evaluation of our study has resulted in a substantially revised edition of the German case definitions [23,24]:
• We rephrased the case definitions in a check-list format indicating clearly how many of the symptoms and signs had to be fulfilled in which combination.
• Some diseases previously jointly described in one case definition were defined separately (Dengue was separated from other haemorrhagic fever; hemolytic uraemic syn-drome was created new, separately from EHEC and Shigella.)
• We rephrased the definitions in a way that for serologic confirmation the necessity for two samples is clearly apparent at the beginning of the phrase.
• The material in which the pathogen has to be detected is now highlighted and is only listed if it is relevant for the case definition.
• A glossary now defines the expressions that are being used repeatedly in the case definitions
• The case definitions are now limited to criteria relevant for the decision process. All additional explanatory information is clearly indicated as such in a separate section of the case definition • The evidence type "epidemiological confirmation" was completely redesigned and replaces the previously used term "epidemiological link". The accepted types of epidemiological links are now specified individually for each case definition.
One practical implication, that is supported by this analysis is, that software used at the local health department must be designed with strict accordance to the case definitions using identical terminology and structuring which would have been more easily archived if all local health departments had been equipped with one identical software system developed within or under supervision of one institution. Possibly other countries in the process of developing or implementing new electronic surveillance systems might want to learn form this experience [21,25].
The case example book, which resulted from this study, constitutes a detailed feed back for the participants of the study and is now being used as training material for public health nurses.
We have demonstrated that rigorous reduction of case definitions to testable yes/no-criteria in a check-list format is likely to improve their reliability. Reducing the differential diagnostic complexity of a disease to a limited number of yes/no-criteria, is a major challenge, but it also carries the benefit of facilitating computerized testing algorithms for quality control and for case classifications.
As the reliability of epidemiologic surveillance largely depends on the reliability of its case definitions, it is essential to create and revise case definitions based on systematic evaluations [9] . Most of the basic principles for the revision of the German case definition edition deducted from this analysis may also be applicable for case definitions in other countries (such as the United States, Ireland, Sweden, Mexico) or international networks (EU, WHO) as they share the same structural and editorial characteristics that we identified to be problematic in the first edition of the German case definitions [4, 7, 8, 26, 27] . We therefore believe that our findings are highly relevant for many national and international surveillance systems. Ventilator associated pneumonia and infection control Ventilator associated pneumonia (VAP) is the leading cause of morbidity and mortality in intensive care units. The incidence of VAP varies from 7% to 70% in different studies and the mortality rates are 20–75% according to the study population. Aspiration of colonized pathogenic microorganisms on the oropharynx and gastrointestinal tract is the main route for the development of VAP. On the other hand, the major risk factor for VAP is intubation and the duration of mechanical ventilation. Diagnosis remains difficult, and studies showed the importance of early initiation of appropriate antibiotic for prognosis. VAP causes extra length of stay in hospital and intensive care units and increases hospital cost. Consequently, infection control policies are more rational and will save money. Nosocomial pneumonia (NP) is defined as parenchymal lung infection, occurring after the first 48 hours of hospital admission [1] . It accounts for 13-18% of all hospitalacquired infections, but leading cause of death from nosocomial infections [2] . It is a major threat to patients admitted intensive care units (ICU) and receiving mechanical ventilation (MV). In the recent studies, it was shown that ventilator associated pneumonia (VAP) was the most common infectious complication among patients admitted ICU [3, 4] . It results in high mortality and morbidity, prolonged lengths of hospitalisation, and also increased cost of hospitalisation. The mortality rates for VAP range from 20% to 75% according to the study population [5] [6] [7] [8] [9] [10] [11] [12] [13] . The average excess cost of pneumonia was estimated to be U.S.$1255 per patient in 1982 (14) , U.S.$2863 per patient in 1985 [15] , and in a recent study in 1999, it was >U.S.$40 000 per patient [16] .
Despite the clinical experience and major advances in diagnostic techniques and management, VAP remains a significant problem for intensivists. In this review, epidemiology, diagnosis and, mainly, infection control of VAP were discussed.
In different studies, the incidence of VAP was reported different, depending on the definition, the type of hospital or ICU, the population studied, and the type of rate calculated and varies from 7% to 70% [17] [18] [19] [20] [21] [22] [23] . In a large database, 1-day point prevalence study, conducted in 1417 European ICUs, pneumonia accounted for 47% of nosocomial infections [4] . In the National Nosocomial Infections Surveillance System (NNIS), NP accounted for 31% of all nosocomial infections in ICU [24] and in another NNIS data in medical ICUs, it was accounted for 27% [25] . The recent studies reported the device-associated incidence rate 13.2-51 per 1000 ventilator days [20, 23, 26, 27] . Generally, the rates of VAP in surgical ICU were higher than in medical ICUs, depending on the differences in the patient population, surgical disorders, the proportion of patients that needed MV and the duration of ventilation. Kollef et al. [28] reported incidences of NP of 21.6% in patients admitted to a cardiothoracic ICU, 14% in other surgical ICU, and 9.3% in a medical ICU.
NP may occur by four routes; haematogenous spread from a distant focus of infection, contiguous spread, inhalation of infectious aerosols and aspiration. Aspiration of the pathogenic gram-positive and gram-negative bacteria, colonized on the oropharynx and gastrointestinal tract, is the main route. The role of other routes is very rare [1] .
Once microorganisms reach the distal lung, they multiply and cause invasive disease. The host defence, including filtration and humidification of air in the upper airways, epiglottic and cough reflexes, ciliary transport by respiratory epithelium, phagocytes and opsonins in distal lung, and systemic cell mediated and humoral immunity, prevent bacterial invasion [29] . In ICU, the host defence of patients are usually altered because of their underlying diseases, and devices that are used. They can not cough efficiently due to sedation or underlying disease. And also, when they are intubated, the endotracheal tube holds the vocal cords open and facilitates aspiration.
The most important risk factor for NP is tracheal intubation; associated with a 3 to 21 fold risk [30] [31] [32] [33] . It increases the risk by; 1) causing sinusitis and trauma to nasopharynx (nasotracheal tube), 2) impairing swallowing of secretions, 3) acting as a reservoir for bacterial proliferation, 4) increasing bacterial adherence and colonization of airways, 5) requiring the presence of a foreign body that traumatizes the oropharyngeal epithelium, 6) causing ischemia secondary to cuff pressure, 7) impairing ciliary clearance and cough, 8) causing leakage of secretions around the cuff, and 9) requiring suctioning to remove secretions [34] . Microorganisms can adhere to the surface of the endotracheal tube and some species exude an exopolysaccharide that acts as a slime-like adhesive. That microbial biofilm on the tube surface provides a reservoir of microorganisms, and they are greatly resistant to the action of antimicrobials and host defence [35] . Also, the patient requiring MV exposes to other devices, such as nebulizers, humidifiers, which can be the source of microorganisms.
The duration of MV increases the risk of infection. Cook et al. [22] reported a cumulative increased risk of VAP with time, with 3% per day in the first week of MV, 2% per day in the second week, and 1% per day in the third week. In other studies, similarly, it was shown that the risk of pneumonia increased by the duration of MV and the highest risk was during the first 8-10 days [36] [37] [38] . The need for reintubation, urgent intubation and documented massive aspiration are also associated with high incidence of VAP [1, 21, 29, 39] .
The effect of prior antibiotic therapy is still controversial. Sirvent et al. [40] reported that a short course of cephalosporin prophylaxis was associated with a lower rate of VAP in patients with structural coma. Also other investigators showed that antibiotics administered during the first days, reduced the risk of early-onset ventilator associated pneumonia [22, 37] . However, prior antibiotic exposure predisposes patients to subsequent colonization and infection with resistant pathogens [28, 41] .
Nasogastric tubes impair the function of the gastroesophageal sphincter and increase the risk of maxillary sinusitis, oropharyngeal colonization and reflux, all of which may lead to migration of bacteria and pneumonia [29, 39] . Enteral nutrition given by nasogastric tube is also associated with increased risk of VAP. Moreover, it may predispose to VAP by elevating gastric pH, leading to gastric colonization and increasing the risk of reflux and aspiration by causing gastric distension [22, 42, 43] . Also patient transportation was found risk factor for VAP by facilitating the aspiration of contaminated secretions from the upper airway and the ventilator circuits in the supine position [44] .
The other independent risk factors for VAP are shown in Table 1 [6, 7, 11, 22, 28, 33, 34, 45] . Identifying these risk factors will guide to prevention measures of VAP.
The causative organisms vary according to the patients' demographics in the ICU, methods of diagnosis, the durations of hospital and ICU stays, and the antibiotic policy. Gram negative bacteria are the most common pathogens cause VAP in several studies [7, 17, 44] . In NNIS data, although the most frequent reported isolate was Staphylococcus aureus (17%), 59% of all reported isolates were gram-negative. The most common gram-negative species were Pseudomonas aeruginosa (15.6%), Enterobacter species (10.9%), and Klebsiella pneumoniae (7.0%) (24) . In recent years, gram-positive bacteria have become more common in ICU and in the EPIC study, S. aureus accounted for 31% of the 836 cases with identified microorganisms (46) . NNIS data from medical ICU, also, reported high percentage (20%) of S. aureus [25] . Polymicrobial infection rate is usually high in VAP [12, 17, 47, 48] .
The duration of MV and the prior exposure to antimicrobials significantly influence the distribution patterns of etiologic agents. In early onset VAP (<5 days), methicillinsensitive S. aureus, Streptococcus pneumoniae, and Haemophilus influenzae are the most common pathogens, whereas methicillin-resistant S. aureus (MRSA), P. aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia are more frequent in late onset VAP (≥ 5 days) [49, 50] . Also, MRSA, P. aeruginosa, A. baumannii and the other multi-resistant gram negative pathogens are the most common pathogens in the patients expose to prior antibiotic.
The other special factors that predispose patients to infection with specific microorganisms are summarized in Table 2 [29, [51] [52] [53] [54] [55] [56] . Determining risk factors for microorganisms will help to select appropriate antimicrobial treatment, that improves the outcome.
The diagnosis of pneumonia in mechanically ventilated patients is difficult, and still there is no "gold-standard" diagnostic method. It is usually based on the combination of clinical, radiological, and microbiological criteria defined by Centers for Disease and Control (CDC) ( Table  3) . But these criteria have low sensitivity and specificity. The systemic signs (fever, leukocytosis, etc.) of infection can be seen by any condition in ICU (pulmonary edema, pulmonary infarction, after surgery, trauma, devascularized tissue, open wounds, etc.). Investigators reported that the clinical diagnosis of VAP is associated 30-35% falsenegative and 20-25% false-positive results [57] [58] [59] . And also, ICU patients do not always have systemic signs of infection due to their underlying disease (chronic renal failure, immunosuppresion, etc.). Radiological infiltration has limited value, mimicking by cardiogenic pulmonary edema, noncardiogenic pulmonary edema, adult respiratory distress syndrome (ARDS), atelectasis, pulmonary contusion, which are not uncommon in ICU [29] . In an autopsy-proven VAP study, Wunderink et al. [1] reported that no radiographic sign had a diagnostic accuracy greater than 68%. The presence of air bronchograms was the only sign that correlated well with pneumonia, correctly predicting 64% of pneumonias in the entire group. The upper respiratory tract of patients is colonized with potential pulmonary pathogens a few hours after intubation [48, 61] . Consequently, isolation of pathogens from tracheal secretions do not always indicate pulmonary infection. But a positive Gram's stain may guide the initial antibiotic therapy. However prior antibiotic and corticosteroid therapy can reduce the sensitivity of this technique [62, 63] .
Pugin et al. [64] proposed to combine the seven variables (temperature, leukocytes, tracheal aspirate volume and purulence of tracheal secretions, chest X-ray, oxygenation-PaO 2 /FiO 2 -and semiquantitative culture of tracheal aspirate) for the diagnosis of VAP, defined as clinical pulmonary infection score (CPIS) ( Table 4 ). The score varied from 0 to 12 points and was reported that a CPIS of more than six was associated with a sensitivity of 93% and a specificity of 100% for the diagnosis of pneumonia. In a post mortem study, Papazian and colleagues [65] reported a high diagnostic accuracy of CPIS at a threshold of 6 (72% sensitivity and 85% specificity). However, the original scoring system has some limitations; that it requires 24-48 hours for the results of tracheal aspirate cultures, and also identifying pulmonary infiltrates progression depends on intensivist experience. Singh et al. [66] used a modified CPIS (calculated at baseline from the first five clinical variables, and CPIS at 72 hours was based on all variables of the score) that antibiotics were stopped in patients with a persistent low score (<6) after 3 days of therapy, avoiding unnecessary use of antibiotics, and all patients who discontinued the therapy improved. In a recent study, Fartoukh et al. [67] reported that the modified CPIS does not perform better when the clinical suspicion of pneumonia is high, so they proposed incorporating the results of specimens gram stain (by adding two more points when gram stains were positive) to modified CPIS to increase the sensitivity of the score and the physicians' diagnostic accuracy.
Qualitative cultures of tracheal aspirate (TA) is not a specific diagnostic method because of the lower respiratory tract colonization and a high percentage of false-positive results [48, 68] . However, investigators reported that quantitative cultures of TA have equal diagnostic accuracy to the other invasive techniques [69] [70] [71] [72] [73] . In a recent study, quantitative cultures of TA were compared with plugged telescoping catheter (PTC). The specificity of TA was similar to PTC when a cut-off point of 10 6 cfu/mL or higher was used, although the sensitivity of TA at ≥ 10 6 cfu/mL was lower than PTC. But when a cut-off point of 10 5 cfu/ mL was used, the sensitivity of TA was not statistically different from that of PTC [74] . Although, quantitative cultures of TA is non-invasive, inexpensive and a simple method, it has some risks, that if the cut-off value ≥ 10 6 cfu/mL is used, sensitivity will be low and some patients with VAP may not be identified or when the cut-off value ≥ 10 5 cfu/mL is used, unnecessary antibiotic treatment will be given because of low specificity [75] .
In the recent years, many investigators favour invasive techniques for diagnosis of pneumonia (protected-speci-men brush -PSB-or bronchoalveolar lavage-BAL-) that may have more diagnostic accuracy [76] [77] [78] [79] [80] . In PSB, 0.001 mL of secretions are collected and the presence of >10 3 cfu/mL bacteria has 80-90% sensitivity and 95% specificity for the diagnosis of VAP. In BAL, larger proportion of lung can be sampled and the diagnostic threshold is >10 4 cfu/mL. The sensitivity and specificity of BAL are 86-100% and 95-100%, respectively [76, [81] [82] [83] . Heyland and colleagues [84] proposed that PSB or BAL may increase physician confidence in the diagnosis and management of VAP and allows for greater ability to limit or discontinue antibiotic therapy. Also in this study, patients who underwent bronchoscopy with PSB and BAL had a lower mortality rate compared with patients who did not undergo bronchoscopy. However, a recent meta-analysis concluded that regular use of bronchoscopy for the diagnosis of VAP does not alter mortality, because it does not directly affect the initial antibiotic prescription [85] . The disadvantages of these invasive techniques are [71, 86, 87] ; a) prior antibiotic use may decrease the sensitivity and accuracy of these methods. However, in a recent study, Souweine et al. [88] reported that if a current antibiotic treatment prescribed for a prior infectious disease other than VAP, the diagnostic accuracy of protected specimen brush or bronchoalveolar lavage is not changed, b) these techniques are based on quantitative culture and results of these cultures require 24-48 hours, and, therefore miss early cases, and also give no information about appropriate initial antibiotic therapy, c) these invasive tests may worsen the patient's status (cardiac arrhythmias, hypoxemia, bleeding, etc.), d) increase the costs of caring, e) it has not been proven that the use of these invasive techniques lead to a decrease in patients' mortality.
The spread of microorganism to blood or pleural space is <10%, so blood and pleural effusion cultures have low sensitivity and specificity. Luna and colleagues [89] demonstrated that the positive predictive value of blood cultures to detect the etiologic microorganism was 73% and the sensitivity of blood cultures was only 26%. They con- Three or more of the following criteria:
Rectal temperature >38°C or <35.5°C Blood leucocytosis (>10.10 3 /mm 3 ) and/or left shift or blood leukopenia (<3.10 3 /mm 3 ) More than ten leukocytes in Gram stain of tracheal aspirate (in high power field) Positive culture from endotracheal aspirate AND New, persistent, or progressive radiographical infiltrate cluded that blood cultures in patients with VAP are useful if there is suspicion of another probable infectious condition, but the isolation of a microorganism in the blood does not confirm that microorganism as the pathogen causing VAP. Therefore, two sets of blood samples for culture and tapping pleural effusions >10 mm should be performed in patients suspected VAP [50] .
In conclusion, microbiological testing should be always performed to decide the appropriate initial empirical antibiotic therapy. Clinicians can choose optimal diagnostic test for specific patients in their clinical setting.
Because of longer duration of mechanical ventilation, longer stay in the ICU, increased use of antibiotics, higher costs for healthcare, and most importantly, increased mortality, the prevention of VAP is the major priority. But, despite the advances in the pathogenesis of VAP, intensivists still struggle with the prevention strategy.
Basic hygiene principles of infection control (hand washing/disinfection just before and after each patient contact, the use of glove and sterile equipment) remain important for the prevention of VAP. Healthcare workers (HCW) can spread microorganisms from patient to patient by their hands easily. Although HCWs realize the importance of handwashing/disinfection, their compliance is still low (25-40%) [90] [91] [92] . Especially their compliance rate is lowest in activities that carried higher risk for transmission and in ICU. High workload decreases their compliance [93] . Wrist watches, bangles, and other jewellery act as reservoirs for organisms, and inhibit effective hand cleaning [94] . Therefore, staff have to take off wrist watch and jewellery to achieve effective hand cleaning. They have to use gowns and gloves when appropriate and must change and wash/disinfect their hands between patients [95] . Bedside hand antiseptics (alcohol-based handrub solution), easier access to sinks and availability of washing equipment, decrease in workload, communication and education tools (posters) and feedback improve compliance and decrease the cross-transmission of nosocomial infection [90, 96] .
The internal machinery of mechanical ventilators is not an important risk factor for VAP. Therefore, using a filter between the inspiratory phase circuit and the patient is not necessary. Furthermore, the importance of filters on the expiratory limb of the mechanical-ventilator circuit in preventing cross comtamination has not known and needs further investigation [34] .
The devices used on the respiratory tract come into contact with mucous membranes, therefore cleaning and highlevel disinfection (at 75°C for 30 minutes) of reusable equipments are required [97] . Resuscitation bags, spirometers, and oxygen analyzers must be cleaned and disinfected between patients to avoid cross-transmission [95] .
In several studies, routine changing of the ventilator circuits is not recommended [26, [98] [99] [100] [101] [102] [103] [104] . Replacement is required when there is gross soilage and mechanical malfunction [105] . The condensate fluid in the ventilator circuit, which contains high concentration of pathogenic bacteria and a risk factor for VAP, has to be removed regularly [106] . And accidental drainage of condensate into the patient's airway and contamination of caregivers during ventilator disconnection or during disposal of condensate should be avoided. In-line devices with one-way valves, put in place into disposable circuits and emptied regularly, are recommended for collecting condensate [75] . Humidification of the inspired air is an important care in the ventilator management. Humidification may be achieved by active humidifiers (bubble-through or wick) or passive humidifiers (hygroscopic condenser-artificial nose-or heat-moisture exchanger-HME-). In humidification, formation of condensate in tubing and colonization of this condensate with microorganisms is an important risk factor for VAP. HME recycle heat and moisture that reduces the condensate formation and also bacterial colonization in the circuit. Moreover they have bacterial filtration characteristics [34, 98] . They do not need to be changed daily and can be used for at least 48 hours, sometimes for up to 1 week [107, 108] . Also with other advantages (reduced nurses workload, reduced financial cost, and better safety), HMEs are favourable devices in many ICUs. Indeed several investigators [109] [110] [111] [112] reported lower rates of VAP in HME groups than conventional heated-water humidification systems, the effect of HME on the prevention of VAP is still controversial and a recent study showed no significant difference in VAP rates [113] . Furthermore, HMEs increase dead space and resistance to breathing, cause airway occlusion, and more tenacious secretions [34, 98, 104, 111] . Additional studies are needed to identify the benefits of HMEs on infection control of VAP.
Nebulizers are used for medication or humidification of air, and inserted into the inspiratory phase of the mechanical ventilator circuit. They can be contaminated by condensate in the tube or by using contaminated solutions, and inoculate infectious aerosol particles directly into the lung parenchyma and can cause outbreaks in ICUs. Recommendations for the infection control of nebulizers are; a) filling immediately before use, b) using sterile water and drugs, c) never refilling the liquid to be nebulised, d) cleaning and disinfecting the receptacle daily, d) using sterile water for rinsing and allowing to dry, e) using patient-specific large-volume nebulizers, f) using patient specific mask, mouthpiece, connecting pieces and medicine cups [34, 114] .
Suctioning the secretions in the trachea is another approach to VAP prevention. Two types of tracheal suction catheters are used on ventilated patients; the open, single-use catheters and the closed, multiple-use catheters.
In single-use system, HCWs have to use sterile solutions during rinsing these catheters and have to care aseptic technique when suctioning endotracheal secretions. In closed suctioning systems, secretions can be suctioned without removal of mechanical ventilation support. This may cause less hypoxia, hypotension and arrhythmias, and also less environmental contamination [115, 116] . However similar VAP rates with closed and open system were suggested in the earlier trials [115, 117] , Combes and colleagues [116] reported a 3.5 times greater risk of VAP in open suctioning system than closed suctioning system in a recent study. Indeed, closed suction catheter is an extension of the ventilator circuit, daily change of this catheter is not necessary for infection control, and in one study no significant difference in VAP rate was reported when daily changes were compared with no routine changes, that may decrease the costs [118] . The use of closed suction system is recommended as part of a VAP prevention program [104] .
The relationship between the use of invasive devices and nosocomial pneumonia directed the investigators to use noninvasive ventilation to reduce the VAP rates. In several studies, lower risk of VAP, with less antibiotic use, with a shorter length of ICU stay, and with lower mortality were reported in the use of non-invasive ventilation [119] [120] [121] [122] [123] . Therefore, care can be taken to use non-invasive mechanical ventilation more often, and to reduce the frequency of tracheal intubation.
Endotracheal tube alters host defences, impairs mechanical clearance from the respiratory tract, causes local trauma and inflammation, and allows pooling of secretions around the cuff. The pressure of the endotracheal tube-cuff should be sufficient to prevent the leakage of colonized subglottic secretions into the lower airway [37] . Also, continuous or intermittent suctioning of oropharyngeal and upper respiratory tract secretions above endotracheal cuffs can prevent aspiration. Endotracheal tubes with a separate dorsal lumen above the cuff, designed to suction the subglottic secretions continuously, were found able to decrease the rates of early-onset VAP [124, 125] . However, in another randomised trial, no benefit with continous subglottic suction on the overall VAP frequency was found [126] . It can reduce but not eliminate the volume of fluid aspirated into the lungs. The lack of effect on prevention of late-onset pneumonia and the high cost of these tubes restrict the usage of them.
Microbial biofilm on the endotracheal tube surface is a reservoir for the pathogens and prevent the microorganisms from the action of antibiotics [35] . Adair and colleagues [127] proposed that high concentrations of antibiotic on the endotracheal luminal surface, achieved either by nebuliser or endotracheal surface modification, would be expected to prevent biofilm formation on the endotracheal tube and may have a role in reducing the incidence of VAP, also minimising patient exposure to systemic antibiotics.
Nasotracheal intubation increases the risk of nosocomial sinusitis, that may predispose VAP by the aspiration of infected secretions from nasal sinuses [128, 129] . There- fore, endotracheal intubation should be preferred to decrease the risk of VAP.
As nasotracheal intubation, nasogastric tube can cause orophanryngeal colonization and nosocomial sinusitis. By impairing the function of the upper oesophagus sphincter, it may facilitate the reflux of bacteria from the gut. As a result, it increases the risk of VAP [130, 131] . In a randomized study, gastroesophageal reflux and microaspiration of gastric contents to the lower airways were not influenced by the size of the nasogastric tube. Because of their potential complications (tracheal malposition by coiling and clogging) and high cost, the small-bore nasogastric tubes are not routinely recommended for the prevention of VAP [132] .
Poor nutritional state and hypoalbuminemia contribute the development of VAP. For this reason, early initiation of enteral nutrition may have preventive effect in mechanically ventilated patients. Moreover, it helps to maintain the gastrointestinal epithelium and reduces the need for stress-bleeding prophylaxis. However, because of using nasogastric tubes and alkalinization of the stomach contents by these feeds; gastric colonization, gastrooesophageal reflux, aspiration and pneumonia might be promoted [29] . In a recent study, postpyloric enteral access placement improved tube-feeding tolerance and reduced the rates of VAP [133] . Heyland et al. [134] used acidified feeds in critically ill patients and demonstrated a dramatic reduction in bacterial growth from aspirates of stomach contents and a lower rate of gram-negative bacterial growth in tracheal secretions in patients receiving acidified feeds, but not a significant reduction in nosocomial pneumonia. They cannot be used in patients with active gastro-intestinal bleeding, acidemia, or renal failure. Furthermore, it was a small size study and further research on the effect of prevention is needed, before it is used in practice.
In the recent years, selective decontamination of the digestive tract (SDD) is one of the most extensively studied prevention strategy in VAP. In SDD, topical non-absorbed antimicrobials (usually combining polymyxin, aminoglycoside and amphotericin B) are used to prevent gastrointestinal colonisation by pathogenic microorganisms. It selectively eradicates the potential pathogenic microorganisms (gram-negative aerobic intestinal bacteria, S. aureus and fungi) and does not affect anaerobic flora, as elimination of anaerobic flora leads to increased colonization with gram-negative aerobic flora. Although some investigators used only topical antibiotics applied to the oropharynx and through a nasogastric tube, many of them added systemic therapy with a broad spectrum (e.g. cefotaxime) during the first few days, to prevent early infections with S. pneumoniae, H. influenzae and S. aureus [29] . In a recent meta-analysis that searched 33 randomized controlled trials published from 1984 to 1996, significant reductions in the incidence of respiratory tract infections (65%) and in total mortality (20%) were determined [135] . Also in this meta-analysis and in the other recent prospective, randomized studies, it was mentioned that using only topical antibiotics reduced respiratory infections, but did not influence the survival [135] [136] [137] .
The threat of SDD is to lead the selection and overgrowth of antibiotic resistant microorganisms. In a recent study from the Netherlands, where the incidence of MRSA and vancomycin resistant enterococcus (VRE) are very low, a reduction in the frequency of colonization with resistant gram-negative bacteria and no effect on the acquisition of MRSA were reported (138) . But the studies from the ICUs where MRSA was endemic, increased incidence of MRSA was reported (139, 140) . Therefore, in ICUs with high incidence of multi-resistant microorganisms, SDD cannot be used. On the other hand, in trauma and surgical patients, SDD seems more effective than in medical patients, may be due to less colonisation [141] . In conclusion the routine use of SDD in ICUs is not recommended, it should be decided according to the patient population studied and the characteristic of ICU.
In addition, the colonization of the oral cavity with pathogens is important risk factor for the develoment of VAP, it is unclear if oral care with chlorhexidine reduces VAP. Also the concern over a chlorhexidine-related increase in colonization of gram negative bacteria should be considered [142] .
Semirecumbent position (45°) prevents aspiration and the passage of bacteria into the airways, and should be preferred in ICU patients, if there is no contraindication [143] .
"Kinetic Beds" or Continous Lateral Rotational Therapy (CLRT) turn continuously and slowly and change the patient's position. Investigators believe that it helps the drainage of pulmonary secretions. However, these beds are so expensive and their effectiveness are not demonstrated. So, the routine use of these beds is not recommended [97] . Also, chest physiotherapy, to improve the clearance of secretions, for the prevention of VAP is not recommended because of its lack of proven benefits and the associated risks (e.g., arterial oxygen desaturation) [105, 144] .
Stress ulcer prophylaxis is proposed to be a risk factor due to the alkalinization of gastric content. The effect of stress ulcer prophylaxis with H 2 -antagonists or antacids on VAP is still controversial. In some studies [145, 146] , the use of sucralfate was associated with a decreased incidence of VAP, however the other reports did not support this [147, 148] . Also, H 2 -antagonists are more efficient for anti-ulcer prophylaxis than the sucralfate [148] . Therefore, the choice of agent for prophylaxis should be done according to the patient and cost-effectiveness.
To reduce the aspiration of oropharyngeal contents, over use of sedatives should be avoided. Kress et al. [149] reported that for reducing over use of sedatives, daily interruption of sedative-drug infusions until the patients were awake decreased the duration of mechanical ventilation and the length of stay in the ICU. Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years BACKGROUND: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region. METHODS: We established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs. RESULTS: During this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information. CONCLUSION: Knowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease. Hand, foot and mouth disease (HFMD) is a common acute viral illness that primarily affects infants and young children, and often occurs in clusters or outbreaks. It is characterized by rapid onset of fever and sore throat, accompanied by vesicles and ulcers on the gums, tongue, buccal mucosa and palate. Punctate and usually transient skin lesions appear on the palms, soles and occasionally on the buttocks, knees or other areas. While the fever and rash may subside rapidly, the mouth lesions may last more than a week, and virus may continue to be shed for several weeks [1] . In temperate countries HFMD occurs during the summer but in the tropics HFMD can occur at any time during the year.
The major causative agents of HFMD are coxsackievirus A16 (CVA16), human enterovirus 71 (HEV71) and coxsackievirus A10 (CVA10) of the genus Enterovirus in the family Picornaviridae [2] . Other enteroviruses isolated from HFMD cases are the other species A human enteroviruses such as coxsackievirus A (CVA) 4, CVA5, CVA6 and CVA7, and coxsackievirus B (CVB) 1, CVB2, CVB3 and CVB5 [2] [3] [4] . Unlike other aetiological agents of HFMD that normally cause mild disease, HEV71 infection has been reported to cause neurological disease manifesting as aseptic meningitis, encephalitis or poliomyelitis-like acute flaccid paralysis [5] . First isolated from a child suffering from encephalitis in California in 1969, HEV71 was further isolated from 23 cases with severe neurological disease in California during the next three years [3] . Historically, HEV71-associated outbreaks have been reported in Australia in 1972 [6] , Japan in 1973 and 1978 [7] , Bulgaria in 1975 [8] and Hungary in 1978 [9] .
In the past decade, countries in the Asia-Pacific region have experienced an increased occurrence of HEV71-associated HFMD outbreaks [10] . HEV71 outbreaks have been reported in Sarawak in 1997, Taiwan in 1998, Perth in 1999, then in Singapore, Korea, Malaysia and Taiwan in 2000 [11] [12] [13] [14] [15] [16] [17] .
In an outbreak of HEV71 in 1997 in Sarawak, a state of Malaysia on the island of Borneo, a cluster of unusual paediatric deaths due to encephalitis and cardiac failure was observed [12, 13] . This raised a lot of fear and anxiety and because of the heightened concern about HEV71 in Sarawak, we implemented a sentinel surveillance programme for HFMD beginning in March 1998. This programme was set up as part of the operational functions of the Sarawak Health Department and was approved by the Director of Health. The principles of the Helsinki Declaration were followed throughout the surveillance operation.
Our aims were to investigate the epidemiology of this common childhood disease in Sarawak, and to determine if there were any differences in the patterns of transmission of HEV71, CVA16 and other aetiological agents of HFMD. It was also the aim of this programme to provide data of practical value for doctors and public health personnel with a view to efficient and effective virological surveillance of HEV71, in particular, to provide an early warning system for HEV71 outbreaks. This paper describes the preliminary observations from our surveillance programme from March 1998 through June 2005.
In early 1998 after discussion with a number of community paediatricians, our team set up a protocol for a sentinel surveillance programme for HFMD. The doctors who had consented to actively participate were provided with a standard reporting and specimen collection form, sterile swabs and virus transport medium, a telephone number for obtaining assistance for transport of specimens to the laboratory and a facsimile number to report cases to the Health Department. All sentinel clinic doctors obtained parental consent before swabs were taken. Sentinel clinic doctors were provided with feedback on viruses isolated from their patients and contact was maintained through both outbreak and inter-outbreak periods to assure doctors that the surveillance programme was active and ongoing. Data obtained in this manner were expected to provide accurate information about disease trends and molecular epidemiology of the relevant viruses.
Three specialist paediatric clinics located in the towns of Kuching and Sibu in the state of Sarawak actively participated in this study from March 1998. In 2000 we included a fourth specialist clinic in Sibu. Two government polyclinics in Kuching and Sibu also participated as sentinel clinics. All children presenting to the sentinel clinics with a history of oral or other skin lesions typical of HFMD were enrolled into the surveillance study, and throat and rectal swabs were obtained from each child enrolled in the first 18 months. Where possible, swabs were also obtained from mouth ulcers, vesicles and other skin lesions. After a preliminary analysis of data from the first 18 months, the protocol was modified to require only throat swabs from sentinel clinics. Rectal swabs were optional and doctors were requested to provide vesicle swabs whenever possible. Specimens were to be transported on ice to the laboratory in 2 ml of viral transport medium (VTM) where they were vortexed, freeze-thawed and aliquoted.
Since the primary objective of programme was a sentinel surveillance system for HEV71 HFMD, we inoculated specimens into human rhabdomyosarcoma (RD) cells susceptible to both CVA16 and HEV71. It was not a particular objective of this exercise to identify the minor causative agents known to be associated with HFMD and hence we did not include multiple cell lines as part of our virus isolation protocol. RD cell cultures normally showed the characteristic enterovirus CPE in 2 to 10 days and were harvested after the monolayer showed extensive CPE. A blind passage was done with all cultures showing no CPE after 10 to 14 days.
RNA was extracted from all culture harvests using Tri Reagent LS (Molecular Research Centre, Cincinnati, OH, USA) according to the manufacturer's instructions. The dry RNA pellet was dissolved in 20 μl of sterile ultra high quality RNase-free water and stored at -80°C until use.
The presence of enterovirus RNA in culture fluids was determined by a previously described pan-EV RT-PCR method [18] with some modifications. The duration of all the steps in the PCR was reduced to one minute and the final extension was reduced to 5 minutes.
From 1998 through 2002, specimens positive using the pan-EV primers were tested for the presence of HEV71 genome by RT-PCR using the primers 159S and 162A, which anneal to the VP1 gene of HEV71. Dr. Mark Pallansch (Centers for Disease Control, Atlanta) generously made the primer sequences available to us prior to publication [19] . All PCR products were sequenced to confirm the identification. In 2002, we changed our protocol for identification of HEV71 due to problems of misidentification of local strains of CVA16 as HEV71 using the primer set 159S/162A [20] . Currently, HEV71 specific primers designed in-house are used for specific identification of HEV71 [20] . All primers used are listed in Table 1 .
Sequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Kit version 3.0 or 3.1 (Applied Biosystems, Foster City, CA, USA).
Molecular serotyping of non-HEV71 enteroviruses isolated was carried out using the methods and sequences published by Oberste and colleagues [21] and Chu, Ishiko and colleagues [22, 23] . Prior to 2002, serotyping of selected non-HEV71 enteroviruses was performed exclusively according to Oberste's method. When Ishiko's method [23] was published in 2002, we made a comparison of the methods by serotyping new isolates using both methods. We determined that for human species A enteroviruses circulating in our region, Ishiko's primers gave identical serotype identification to that obtained using Oberste's method [21] . Since there was no discrepancy between the methods, we modified Ishiko's primers to convert the method from a semi-nested to a non-nested method for ease of use. Our modifications were verified and described by Cardosa and colleagues [24] . We then retrospectively retested all non-HEV71 enteroviruses isolated prior to 2002 using the modified method.
A subset of isolates identified by sequencing of VP4 were subjected to confirmation by using VP1 specific primers and DNA sequencing of the PCR products, as described previously [24] .
DNA sequences of VP1 and VP4 gene products generated by RT-PCR from isolates were used in this analysis essentially as previously described [24] . The software package ClustalX [25] was used for alignment and to generate a bootstrapped phylogenetic tree using the neighbour joining method according to Saitou and Nei [26] .Primers and DNA sequences TTCCAATACCACCCCTTGGATGA Antisense (1195-1173) HEV71 VP4 gene. 159 [19] ACYATGAAAYTGTGCAAGG Sense (2385-2403) HEV71 VP1 gene. 162 [19] CCRGTAGGKGTRCACGCRAC Antisense (2869-2850) HEV71 VP1 gene. 161 [19] CTGGGACATAGAYATAACWGG Sense (2766-2785) HEV71 VP1 gene. NP1A [19] GCICCICAYTGITGICCRAA Antisense (3355-3336) HEV71 VP1 gene. MAS01S [20] ATAATAGCAYTRGCGGCAGCCCA Sense (2355-2377) partial VP1 gene. MAS02A [20] AGAGGGAGRTCTATCTCYCC Antisense (2731-2712) partial VP1 gene. MD91 [18] CCTCCGGCCCCTGAATGCGGCTAAT Sense (450-474) partial 5UTR. MD90 [18] ATTGTCACCATAAGCAGCCA Antisense (603-584) partial 5UTR.
*Position relative to the genome of HEV71 strain 7423-MS-87 (GenBank accession number U22522)
All primers used in the methods described above are listed in Table 1 . All new DNA sequences used in the phylogenetic analysis but hitherto unpublished have been deposited in GenBank and have the accession numbers AY794032, AY794033, AY794035 to AY794042. All other sequences used are from previous publications by our own as well as other groups [17, 20, [22] [23] [24] 27] . Detailed protocols, sample collection methods and other practical information have been placed in the public domain through our APNET (The Asia-Pacific Enterovirus Surveillance Network) website [28] .
Statistical analysis was performed using the software package JMP Statistics version 5.01(SAS Institute Inc., USA) and Prism 4 for Macintosh (Graphpad Software, Inc., USA).
The first provisional protocol we provided to the sentinel clinics for the collection of specimens required both throat and rectal swabs and vesicle or ulcer swabs where possible. Results from virus isolation studies of specimens obtained from both sentinel clinics as well as hospitals during this period were used to review the protocol that was originally implemented. A total of 579 specimens from 263 children with a clinical diagnosis of HFMD were received during the 18-month period from March 1998 through August 1999. The age of the children ranged from 6 months to 13 years, with 153 (58.2%) males and 110 (41.8%) females.
All specimens received were subjected to virus isolation. Fifty specimens from 44 children, of a total of 579 (8.6%) specimens, were too heavily contaminated with bacteria. Twenty four of the contaminated specimens were rectal swabs, 19 were throat swabs and 7 were from various skin lesions. All remaining uncontaminated cell culture harvests were tested for enteroviruses by using the pan-EV set of primers and 235 of the 529 (44.4%) specimens tested yielded an enterovirus, but only 15 of the 235 (6.4%) enteroviruses were HEV71. These specimens were from 259 children and an enterovirus was isolated from 153 (59.1%) children. Only 6 (3.9%) of the children had HEV71.
From this early set of specimens, we were able to isolate an enterovirus from 44% of the throat swabs, 40% of the rectal swabs, 44% of the mouth ulcers and 66% of the vesicle swabs. Clearly vesicle swabs are very useful specimens, but only 18% of the children had had vesicle swabs taken because not all children presented with skin lesions filled with abundant fluid. Since throat swabs provided a reasonably high yield of enterovirus isolates, we made the decision in 2000, to require throat swabs as the primary specimen from the sentinel clinics, with vesicle swabs where possible, while rectal swabs were not required. This served to reduce the laboratory workload during an outbreak.
Our laboratory received 4290 specimens from 2950 children from March 1998 through June 2005, with a male to female ratio of 1.35:1. The histogram in the top panel of Figure 1 shows the distribution of HFMD cases seen in our sentinel clinics during this period. There have clearly been two large outbreaks of HFMD in 2000 and 2003 (bottom panel of Figure 1 ), with some sporadic activity between these peaks. The dominant enterovirus serotype isolated during both the outbreaks was HEV71 as shown in the middle panel of Figure 1 . CVA16 was always isolated during HEV71 outbreaks as well but was also isolated in interoutbreak periods. Other species A human enteroviruses such as CVA2, CVA4, CVA5, CVA10 and CVA12 were also isolated in inter-outbreak periods.
Phylogenetic analysis of the HEV71 strains isolated in Sarawak from 1998 to 2005 show that both genogroup B and genogroup C strains circulated in Sarawak during this period (see Figures 2 and 3) . We have used both VP4 and VP1 genes in the phylogenetic analysis in order to be certain that there is no major discrepancy in genotyping associated with using VP4 and VP1 gene regions. Furthermore, we wish to provide both options to other groups who may wish to compare their data with ours since it is known that many groups may still use VP4 sequencing as a first step in molecular identification of human enteroviruses. Although both genogroup B and genogroup C HEV71 strains co-circulated in Sarawak, the predominant genogroup in both the HEV71 outbreaks of 2000 and 2003 was genogroup B. Besides co-circulating with genogroup B strains during outbreaks, genogroup C viruses also appeared sporadically between outbreaks along with other species A human enteroviruses. We never isolated a genogroup B HEV71 in non-outbreak periods. The distribution of genogroup B and genogroup C HEV71 strains during the surveillance period is shown in the bottom panel of Figure 1 .
The phylogenetic trees in Figures 2 and 3 
The HFMD epidemiological curves for the outbreak years 2000 and 2003 were plotted according to epidemiological week ( Figure 4 ) and show clearly that the first HFMD cases began to be seen early in the year. By week 7 a clear rise in the number of cases was seen. This early rise in cases differs from the summer outbreaks seen in temperate countries, and we suggest that the HEV71 outbreaks in Sarawak preceed the summer outbreaks of countries in the northern hemisphere in each year. In 2000 the HFMD outbreak stretched to the end of the year, peaking between Phylogenetic tree generated from the VP1 gene, showing relationships between HEV71 isolated in different years Figure 1 ). In 2003, the number of HEV71 cases declined sharply by the end of April (by week 18) and were no longer detected by the end of June, coinciding with the last HFMD cases seen that year. Interestingly this outbreak coincided with the SARS outbreak in the region and the public health measures put into place during this time evidently served to control the transmission of enteroviruses as well.
A detailed analysis was done on data collected from two sentinel clinics, coded S1 and S2, which had sent samples to our laboratory consistently and reliably throughout the seven-year study period. A total of 2570 specimens were collected from 1894 cases during the 7 years. Of the 1894 cases, specimens from 1804 (95%) were subjected to virus isolation. A total of 2272 specimens were subjected to virus isolation, thus ensuring that the majority of specimens from the majority of cases were tested (88.4% of specimens from 95% of cases).
An analysis of the proportion of the different types of specimens and the virus yield obtained is shown in Table  2 . More than 2000 specimens from outbreak and nonoutbreak periods were tested from 1998 to 2005. Enteroviruses were grown from 21.6% of those tested. Throat swabs comprised 72.3% of the total number of specimens tested and 25.4% of these yielded enteroviruses. Detailed information about the enterovirus serotypes isolated during this surveillance programme is also provided [see additional file 2] . Although on the whole, the virus isolation success rate was much lower than anticipated from the results for the first 18 months, it remained the case that throat swabs were more useful than the rectal swabs which yielded non-polio enteroviruses in only 5.8% of the samples tested. It should be noted however, that the first 18 months of the study coincided with an inter HEV71 epidemic period, with mostly CVA16 and non-HEV71 species A human enteroviruses causing HFMD. We have compared the virus isolation yields during HEV71 outbreak (2000 and 2003) years with the yields during an HFMD outbreak caused by non-HEV71 enteroviruses (2002) and we found that we successfully isolated virus from 40% of specimens collected in 2002 but only 20% of viruses during the HEV71 outbreak years, suggesting that HEV71 is more difficult to isolate than CVA16 and other species A enteroviruses. The virus isolation rate in the first 18 months (44%) is therefore comparable to that obtained later, when HEV71 was not circulating.
There were 491 children from whom a non-polio enterovirus was isolated. Of these, 8 were excluded from the analysis because of missing information on their age at presentation. The children ranged in age from 18 days to 155 months, with a mean of 32.2 months and a median of 27.5 months. There were 3 dominant serotypes of enteroviruses isolated from these 483 children and we asked the question if different serotypes of enteroviruses caused infection in children of different ages. Table 3 shows the mean ages of the children who had CVA16, HEV71 and CVA10 infection. Comparison of means for each pair using an unpaired t test at an alpha of 0.05, showed that there was no significant difference in the mean ages of the children in the different groups (CVA10 versus CVA16: P = 0.0872; CVA10 versus HEV71: P = 0.1800; CVA16 versus HEV71: P = 0.6992).
Following the 1997 outbreak of EV71 associated HFMD in Sarawak, Malaysia, the Health Department installed a sentinel surveillance programme with the expectation that we would be able to study epidemiological trends and begin to predict when to expect outbreaks with sufficient accuracy in order to implement public health interventions to reduce the burden of the disease. Although the surveillance programme is still ongoing in Sarawak, we have sought to glean some preliminary information from the data generated over the first 7 years of the programme. two HEV71 outbreaks. A recent report on a similar surveillance programme in Yamagata Prefecture in Japan (1998) (1999) (2000) (2001) (2002) (2003) suggests that in Yamagata there is frequent importation of HEV71 from surrounding countries seeding the clusters of cases seen annually in this community [10] . The HEV71 strains in this study were isolated from small clusters of cases that tended to be seen in the summer months while in our situation we observed outbreaks of HEV71 every 3 years with cases being seen much earlier in the year, well before the northern summer. In 2003 both Sarawak and Yamagata experienced a large outbreak and in both situations, a genogroup shift from C to B was noted.
It is interesting that in Sarawak, of the genogroup C viruses, only genogroup C1 strains have been observed, while genogroup B viruses appear to be changing from outbreak to outbreak, suggesting that it is likely that genogroup B viruses are evolving within Borneo and that the outbreaks we have experienced are being seeded from within rather than from imported viruses. Since the outbreaks in Sarawak typically begin early in the year, it is also possible that genogroup B strains generated in Sarawak may seed HEV71 outbreaks in the region, which typically occur later than the Sarawak outbreaks. This temporal sequence of regional outbreaks is also true of those occurring in Singapore and in Peninsula Malaysia.
The data we have obtained through 7 years of our sentinel surveillance programme for HFMD in Sarawak have provided useful clues to understanding the epidemiology of HEV71 in the state. It is clear that the appearance of HEV71 associated HFMD in sentinel clinics signals the start of an outbreak, but the rise in the number of cases is so rapid that this approach is not a suitable early warning system. In 2003 there were only 5 weeks between the time the first HEV71 cases were seen and the peak of the outbreak. Clearly this could be explained by rapid and effective response by the public health teams, but we have no way to know.
Alternatively, the 3-year cycle of HEV71 outbreaks we have observed could, if verified in the coming years, provide public health officials with the relevant information to plan and to implement their intervention programmes to reduce the disease burden in the years when an HEV71 outbreak is expected. Although this is not expected to prevent the outbreaks entirely, effective public health measures put into place early enough can limit the spread, reduce mortality and reduce the burden on the community and the health system.
It is important to note that epidemiological curves showing HFMD alone, without distinguishing the infecting agent for each case, can stretch broadly over many months, with non-HEV71 enteroviruses continuing to be isolated after cessation of HEV71 activity. This was especially evident in 2000, when HEV71 associated fatal cases were reported in neighbouring Singapore in September and October 2000 [29] , and the media attention surrounding these events generated a high index of suspicion in Sarawak as well. No HEV71 was isolated in Sarawak after August that year, but numerous CVA16 continued to be isolated until the end of 2000. Thus even though sociological factors affect the shape of the HFMD epidemiological curves in Sarawak, epidemiological curves specifically showing genogroup B strains of HEV71 were consistently sharp and well defined in 2000 and 2003. The mean and median age of children with HFMD was 36 months and 30 months respectively, but the mean ages did not differ between the groups infected with the different serotypes. It is thus intriguing that HEV71 has caused much larger and sharper outbreaks than either CVA16 or CVA10. This suggests that HEV71 has the capacity to spread rapidly through the susceptible population and then become quiescent in the community. In the third year after any HEV71 outbreak, the whole cohort of children under 3 years of age has not been exposed to HEV71 and all of these children are then susceptible, providing the conditions for another sweeping transmission of HEV71 through the community. The annual birth cohort in Sarawak is 48 to 49 thousand and thus in 3 years there are up to 150,000 susceptible children in the state.
According to the trends we have reported, we expect that the next outbreak of HEV71 in Sarawak will be in 2006. At the time of writing we have already begun to pick up HEV71 cases in our sentinel programme and from past experience, an outbreak in Sarawak is often followed by outbreaks in other countries in the region. We have therefore decided to put our data into the public domain in order that other public health practitioners in the Asia Pacific region may benefit from this experience and prepare for a spread of HEV71 in the region once again in the months to come.
The main conclusions arising out of this preliminary report are described below:
a. HEV71 outbreaks have occurred every 3 years in Sarawak starting in 1997. All the 3 outbreaks (1997, 2000 and 2003) have been caused by genogroup B viruses and furthermore, each of the 3 outbreaks has been associated with genogroup B viruses that are genetically distinct from each other.
b. HEV71 of subgenogroup C1 has been isolated throughout the 7 years of the surveillance programme and are closely related to each other and to genogroup C1 viruses isolated elsewhere. Sarawak has so far not experienced large HFMD outbreaks caused by HEV71 of genogroup C1. Indeed HEV71 of subgenogroup C1 behave much like other species A enteroviruses, occurring sporadically throughout the surveillance period.
c. In Sarawak, occurrence of HEV71 genogroup B infections is tightly clustered, with cases rising and falling very rapidly. 26th International Symposium on Intensive Care and Emergency Medicine, 21–24 March 2006, Brussels, Belgium   Model-Based Design of Growth-Attenuated Viruses Live-virus vaccines activate both humoral and cell-mediated immunity, require only a single boosting, and generally provide longer immune protection than killed or subunit vaccines. However, growth of live-virus vaccines must be attenuated to minimize their potential pathogenic effects, and mechanisms of attenuation by conventional serial-transfer viral adaptation are not well-understood. New methods of attenuation based on rational engineering of viral genomes may offer a potentially greater control if one can link defined genetic modifications to changes in virus growth. To begin to establish such links between genotype and growth phenotype, we developed a computer model for the intracellular growth of vesicular stomatitis virus (VSV), a well-studied, nonsegmented, negative-stranded RNA virus. Our model incorporated established regulatory mechanisms of VSV while integrating key wild-type infection steps: hijacking of host resources, transcription, translation, and replication, followed by assembly and release of progeny VSV particles. Generalization of the wild-type model to allow for genome rearrangements matched the experimentally observed attenuation ranking for recombinant VSV strains that altered the genome position of their nucleocapsid gene. Finally, our simulations captured previously reported experimental results showing how altering the positions of other VSV genes has the potential to attenuate the VSV growth while overexpressing the immunogenic VSV surface glycoprotein. Such models will facilitate the engineering of new live-virus vaccines by linking genomic manipulations to controlled changes in virus gene-expression and growth. Infections caused by viruses persistently threaten human health. For example, 40 million, 350 million, and 170 million people in the world are carrying human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV), respectively [1] [2] [3] . Annually 5% to 15% of the global population is infected with influenza, resulting in 250,000 to 500,000 deaths [4] . Protection against viral infections may be provided by inoculations with live-virus, killed-virus, or subunit vaccines. Live-virus vaccines offer advantages because they activate both humoral and cellmediated immunity, require only a single boosting, and generally provide longer immune protection than other forms of vaccines. However, they must be adequately attenuated in their growth to minimize the possibility of vaccine-induced pathogenic effects while retaining their immunogenicity. Attenuation of live viruses has traditionally been achieved by serially passaging viruses in tissue or cell culture and adapting them to grow well on new cell types or at elevated or reduced temperatures [5] , a process that tends to reduce their replicative ability and virulence in humans or animals [6] . Such attenuation has historically been a highly empirical process, where its mechanisms are often neither known nor elucidated.
During the last decade the emergence of reverse genetics techniques has created unprecedented opportunities to better control viral attenuation [7] [8] [9] . Reverse genetics enables the production of RNA viruses from cloned cDNA, so specific mutations can be relatively easily introduced into viruses. The challenge to engineering viruses for attenuation then shifts from creating the variants to predicting how specific genetic changes define or correlate with measurable effects on virus growth. Such a challenge can be addressed through the development of quantitative and mechanistic models that map genome-level changes to the dynamics of virus growth under different environmental conditions. Models of intracellular virus growth aim to predict how rapidly a virus-infected cell will produce virus progeny. Inputs to such models include rates of constituent processes such as entry of the virus into the cell, transcription of viral mRNAs, translation of viral proteins, replication of viral genomes, assembly of intermediates, and finally, production and release of viral progeny. Decades of detailed biochemical, biophysical, and genetic studies have, for diverse viruses, contributed toward a level of mechanistic understanding of viral functions and interactions. Various intracellular models of virus growth have been developed for phage Qb [10] , phage T7 [11, 12] , HIV-1 [13] , and influenza A virus [14] .
How can a detailed model for the intracellular growth of a virus be used to explore the behavior of mutant viruses that encode alternative designs? As a starting point, one can create alternative designs that reorder or rearrange the wildtype genes or regulatory elements. Such genomic changes can alter the timing and level of expression of different viral genes, and thereby impact growth because the production of viral progeny depends on the dynamic expression of viral genes. Preliminary models of such alternative genome designs can use the ''language'' of the wild-type virus. They retain the parameters that characterize wild-type molecular interactions, wild-type average rates of viral polymerase elongation, and wild-type composition of progeny viruses, but they apply them in a manner that reflects the reordering of wild-type components in the engineered genome. For example, the timing of expression for the genes of phage T7 during infection maps closely to their sequential order on the T7 genome [15, 16] . By relocating an essential early gene, encoding the T7 RNA polymerase to downstream positions, one delays initiation of transcription by the highly efficient T7 RNA polymerase and thereby attenuates phage growth [17] . Preliminary models for the growth of phage carrying the altered genomes, based on wild-type parameters, capture the overall observed trends in attenuation. Here we expand this approach to a mammalian virus of biomedical and agricultural relevance: vesicular stomatitis virus (VSV).
VSV is a prototype negative-sense single-stranded RNA (Mononegavirales, (-)ssRNA) virus and a member of the family Rhabdoviridae [18, 19] , which includes rabies virus. Each VSV particle has a single copy of an 11-kb genome carrying five genes that encode nucleocapsid (N), phospho (P), matrix (M), envelope (G), and polymerase (L) proteins. VSV is economically important because it can cause symptoms like those of foot-and-mouth disease in livestock [19] . It offers several advantages as a vaccine vector including low seropositivity in humans, a capacity to accommodate foreign genes up to 40% of its own genome size, and an established reverse genetics [18, 20] . Recombinant forms of VSV carrying foreign virus genes that encode immunogenic proteins have been proposed as potential vaccines against HIV, influenza, and respiratory syncytial virus [21] [22] [23] [24] [25] . Less pathogenic but more immunogenic VSV-based vaccines against infection by VSV or other viruses are being sought.
Here we develop an in silico model of a VSV infection cycle, incorporating known regulatory interactions and mechanisms and relevant quantitative data from the literature of the past 40 years. These interactions and the corresponding equation formulations are described in detail in the model development section of Materials and Methods. Using the model, we first quantitatively analyze how the intracellular growth of wild-type VSV directs host biosynthetic resources toward VSV gene expression, synthesis of progeny genomes, and pathway switching from the synthesis of VSV intermediates to the production of VSV progeny. We then reveal that the model captures experimental results showing progressive attenuation of virus growth associated with moving the N gene downstream from its wild-type position. Finally, we use the model to predict how altering the positions of other VSV genes and promoters may attenuate the growth of VSV while increasing its potential capacity to activate an adaptive immune response.
Using our model with the established parameter set (Tables  1 and 2) , we first analyzed quantitatively and systematically how the intracellular growth of VSV is regulated. The improved understanding of the virus infection by this model-based analysis may guide us to identify the key regulatory components to manipulate for developing virus mutants as possible vaccine or vector candidates.
Attenuation mechanism leads to unequally distributed synthesis of viral mRNAs and proteins. The partial transcription termination mechanism (or attenuation) is common in (-)ssRNA viruses. This mechanism is important to satisfy the different needs of each viral protein during its infection cycle. Five attenuation factors for each intergenic region of the VSV genome (Table 1 and Equation 8 ) were obtained from the literature [18, 19, 26, 27] and incorporated into our model.
Owing to the step-wise release of polymerases from each gene junction, our simulations estimated the gradual decrease of VSV mRNA synthesis in the order of N . P . M . G . L ( Figure 1A ). Compared with the most abundant N mRNA, L mRNA is very scanty in infected baby hamster kidney (BHK) cells (40 ; 140-fold less). The relative production level of each protein matched the relative availability of the corresponding mRNAs ( Figure 1B ) even though different proteins degrade at different rates ( Table 1 ). Because of the different level of incorporation of each protein into a single virion particle, as defined by the protein stoichiometry (Table 2 , [28, 29] ), the relative levels of free viral proteins in the cytoplasm develop differently from their mRNA levels ( Figure 1C ). P protein is most abundant owing to its low content in the virion, and L and N proteins are least abundant. The persistently low level of N protein is related to its immediate complexation with nascent genomes and antigenomes to make nucleocapsid particles during the replication step. Owing to the cyclic switching between transcription and replication by the encapsidation process, the N protein level is predicted to oscillate as shown in Figure 1C .
The engineering of viral genomes provides ways not only to explore viral regulatory mechanisms at a genomic level, but also to produce recombinant viruses that may serve as vaccines, gene delivery vectors, and oncolytic (tumor-killing) agents. However, the complexity of interactions among viral and cellular components involved in the life cycle of a virus can make it challenging to anticipate how altering viral components will influence the overall behavior of the virus. Lim, Lang, Lam, and Yin have developed a computer model that begins to mechanistically account for key virus-cell interactions in its predictions of viral intracellular development. Lim et al.'s model was able to capture experimentally observed effects of gene rearrangements on the levels and timing of viral protein expression and virus progeny production, aspects that are important for the design of live-virus vaccines. Refinement and extension of their approach to current and other virus systems has the potential to advance the application of viruses as therapeutic agents.
After 9 h post-infection, our simulation predicts a significant decrease of free M proteins. This arises from the dominance of the virion assembly process, which depletes M proteins, compared with transcription and replication. In infected murine delayed brain tumor (DBT) cells, similar distributions of viral mRNAs and proteins were obtained (unpublished data).
Higher demands for genomes are satisfied by the stronger promoter of the anti-genome template relative to that of the genome template. Anti-genome templates are only utilized to amplify genomes, while genome templates are used to amplify both anti-genomes and mRNAs, and they are also incorporated into virion progeny particles. The higher demands for genome by these multiple tasks are satisfied by the stronger promoter of the anti-genome template compared with that of the genome template [30] [31] [32] . More polymerases bind to the stronger promoter of the anti-genome, ultimately enhancing the production of genomes over anti-genomes ( Figure 2 ). In our model the parameter S prom measures the strength of the anti-genomic promoter relative to that of the genomic promoter ( Table 1 ). The production ratio of genomes to anti-genomes was estimated to be dynamically changed [33] , varying from one to 30 (wild-type VSV case (S prom ¼ 5.4), Figure S1 ). Such oscillatory changes in the production ratio shown in Figure S1 follow from the on-off use of the genomes for transcription or replication. They also arise owing to the staggered shifting of dominant templates between genomes and anti-genomes during replication. A large value of S prom favors use of anti-genome templates to replicate genomes. However, as genome templates accumulate in large excess relative to anti-genome templates, they successfully compete for replication resources. Synthesis of anti-genomes then dominates until they accumulate and serve again as the dominant templates.
The virion production rate in BHK cells is at maximum 5-10 h post-infection. In infected DBT cells, similar simulation results were obtained except that the synthesis of genomesized viral RNAs continued for longer time (active until 15 h post-infection, Figure 2 ).
Optimal utilization of genomic nucleocapsids. Genomic nucleocapsids can either be used as templates for RNA synthesis or they may be incorporated into progeny virions. Their fate depends on levels of polymerase and M protein, which respectively favor RNA synthesis or virion production pathways, as well as on the extent to which association of the nucleocapsid with M protein will dominate over association with polymerase, described with the parameter S cond in our model (Table 1) . Because both RNA synthesis and virion production are essential processes of the infection, extreme values of S cond that favor one process over the other will be [49] S pol ¼ 170 nt Strength of anti-genomic promoter relative to that of genomic promoter a S prom ¼ 5.43 Rate constant ratio of the associations of M protein and polymerase with the genomic nucleocapsid a S cond ¼ 6.2 3 10 À5
Fraction of M protein bound to the plasma membrane [51] cond M ¼ 0.1 Attenuation factors [18, 19, 26, 27] / N / / P / / M / / G / / L ¼ 0.0 / 0.25 / 0.25 / 0.25 / 0.95 Degradation rate constants N protein [58] k dp,N ¼ 3.5 3 10 À5 sec À1 P protein [59] k dp,P ¼ 1.4 3 10 À6 sec À1 M protein [58] k dp,M ¼ 1.5 3 10 À4 sec À1 G protein b k dp,G ¼ 5.7 3 10 À5 sec À1 L protein [59] (if concentration P protein ! 10 3 concentration L protein ) k dp,L ¼ 1.2 3 10 À5 sec À1 (if concentration P protein , 10 3 concentration L protein ) k dp,L ¼ 4.3 3 10 À5 sec À1 mRNA [60] k d,mRNA ¼ 1.9 3 10 À4 s À1 nucleocapsid c k d,nc ¼ 1.9 3 10 À5 s À1 Host-dependent parameters Number of ribosomes in host cell [61] nrib ¼ 5 3 10 6 Elongation rate of ribosome [62] k e,rib ¼ 6 aa/s Spacing between neighboring ribosomes a (BHK/DBT) Srib ¼ 238. 5 Owing to their increased stability by encapsidation, nucleocapsids are less degradable than naked mRNAs. It was assumed that the nucleocapsids of VSV are degraded 10-fold slower than its mRNAs, and our simulation results were insensitive to this parameter. DOI: 10.1371/journal.pcbi/0020116.t001 detrimental for growth. For excessively large S cond , newly synthesized genomic nucleocapsids would tend to be prematurely incorporated into virion particles before they could serve as templates for transcription and replication. On the other hand, for extremely small S cond , genomic nucleocapsids would be utilized primarily to produce viral RNA without being packaged into viral progeny. Hence, an intermediate parameter value is expected to be optimal for viral growth. We estimated a possible range for the wild-type value of S cond by fitting our simulation results to previous experimental observations by others (2.5 3 10 À5 to 1.0 3 10 À4 , Figure S4 ). Our simulations further indicate that this range of S cond is near-optimal and optimal for VSV growth on BHK and DBT cells, respectively (unpublished data). Diversion and inhibition of host translation machinery create a time window of opportunity for translation of viral proteins. During the infection cycle, virus actively and passively competes with the host for limited translation resources by inhibiting host transcription and by amplifying viral mRNAs, respectively. Viral leader-mRNA and M protein play key roles in this inhibition [19, 28, [34] [35] [36] . As viral components accumulate in the cytoplasm from the initiation of infection, an ever-increasing fraction of host ribosomes are available for viral mRNAs ( Figure 3A , the fraction of ribosomes associated with viral mRNAs is defined by 1-rib_host in Equation 26 ). However, the inhibition of host macromolecular synthesis causes a failure to supply accessory factors needed for initiation and elongation steps of translation, resulting in a reduction in the fraction of active ribosomes over time ( Figure 3A , as described by f dec in Equation 25 ). These two mechanisms create a time window when active ribosomes are maximally available for viral translation in infected cells ( Figure 3A , refer to the term (1-rib_host)*f dec in Equation 27 ). The abundance of viral mRNAs and the limitation imposed by ribosomal spacing determine the fraction of the active ribosomes involved in translating viral mRNAs (occupied ribosomes in Figure 3B , refer to Equations 11 and 27) . In our model, if the occupied active ribosomes are less than the available ones (in this case the number of free active ribosomes . 0), viral translation is fully supported without any limitation of host machinery. In the early infection stages up to 7 h and 13 h post-infection for BHK and DBT cells, respectively, the host machinery is in excess ( Figure 3B ). However, at later times viral translation becomes limited by the host resources (in this case the number of free active ribosomes ¼ 0). This limitation may cause a transition from replication-dominant to assembly-dominant infection stages because the replication requires the continuous protein synthesis. As shown in Figure 3B , a small fraction of ribosomes as active forms (less than 5% out of 5 3 10 6 ribosomes, Table 1 ) are utilized for viral translation. Experiments and simulations of VSV gene-order mutants. For vaccine applications, one seeks to minimize viral pathogenicity and maximize its immunogenicity. Based on observed correlations between in vitro and in vivo results, we assume here that the pathogenicity and the immunogenicity of a virus are directly linked to the levels of progeny production [20, 37, 38] and G protein expression in infected cells [39, 40] , respectively. In the previous section we have showed that various VSV regulatory mechanisms are involved in maintaining balances, during infection, among viral synthesis processes, which indirectly indicates the importance of such balances for viral growth. Perturbations of such balances by genetic or genomic manipulations could provide ways to obtain viral phenotypes favorable to vaccine applications. We first test the predictive ability of our model by comparing simulated protein expression and growth of several gene-rearranged VSV strains with experimental results. Later we employ the model to predict how various genomic manipulations could attenuate virus growth and increase G protein expression.
Protein expression rates of gene-rearranged viruses. The stepwise decline in the transcription of genes more distant from the 39-end region promoter highlights how gene order affects gene expression in VSV. Advances in reverse genetics have made it possible to create gene-rearranged virus strains where the transcriptional attenuation mechanism then creates altered levels of gene expression [7, 9, 18, 20] . In one study [18] the three internal genes, P, M, and G, were permuted, and the resulting six possible VSV strains were characterized. Relative rates of viral protein expression in BHK cells were experimentally measured based on their incorporation of [ 35 S]-labeled methionine for a one-hour window at 4 h post-infection [18] . We extended our model to simulate this experiment for mutants representing each geneorder permutation and compared the model prediction with the published results [18] , as shown in Figure 4 . All rates are expressed relative to the synthesis rate of N protein, whose corresponding gene was in position 1, closest to the 39 end of the genome in all strain cases. Expression of gene L, in position 5, was minimal in both simulations and experiments, and the expression of all other genes was above 40%, a feature of the experimental data that the simulation also captured. Most of the points fall close to the parity line, indicating agreement between the simulation and experiment. Noteworthy are two subsets of points. First, the four circled points are exceptions to the general rule that gene order determines the level of gene expression. These were genes in the second position of the genomes that were expressed essentially at the same rate as gene N, in the first position [18] . This result highlights that the expression rate of protein is affected not only by its gene order, or corresponding rate of mRNA production, but also by its length and degradation rate. For a fixed average rate of translational elongation, longer gene products will tend to be produced more slowly. Further, the net rate of protein production will reflect the rates of both protein synthesis and protein degradation. The model accounts for these contributions, and for the circled genes such accounting appears to capture unexpected high translation levels of genes in the second position, which were measured by Ball et al. [18] . The second sets of points, shown in two boxes, indicate mismatches ( Figure 4 ) that, in the most challenging scenario, could reflect unknown strain-specific mechanisms that are not present in our general gene-permutation model. However, one should also note that the experiment is based on labeling and quantifying proteins about 4 h post-infection. This relatively early time point allows one to minimize potentially confounding influences of virion particle assembly and production on cytoplasmic levels of viral proteins, but it also represents a point before the majority of viral proteins have been made ( Figure 1B) .
Growth of gene-rearranged viruses. We also employed our model to predict the growth of VSV strains having the N gene first position on the genome grows best, followed by N2, N3, and N4. This result is consistent with the previously suggested hypothesis that relocation of the N gene to 39-distal positions on the genome would be an efficient way to attenuate VSV for vaccine use [20] . The reduction in growth that follows from moving the N gene likely reflects, at least in part, an imbalance between replication and transcription. Insufficient production of N protein would reduce the extent of encapsidation of nascent anti-genome and thereby allow transcription to dominate over genome replication [41] . While the simulation matches the growth ranking, it did not quantitatively match the experimental data. The predicted variation in virion production (N1 ! N4: 4.7-fold decrease) is smaller than the experimentally observed variation (N1 ! N4: 16.7-fold decrease). A potential source of this quantitative difference was our neglect of mass action effects of N proteins on the encapsidation process in our model; encapsidation was simulated as an instantaneous process when free N proteins were available. As shown above, our model could capture the major effects of gene rearrangement on viral growth and protein expression.
Effects of relative promoter strength on viral growth. The genome and anti-genome of VSV are synthesized in unequal amounts, determined by the differing strengths of their promoters [19, 31, 33, 39] . The stronger promoter of the antigenome allows viral polymerases to produce more genomes to meet their demands as components of virion particles and as templates for transcription and replication. To explore how VSV growth is influenced by differences in the relative strength of the genomic and anti-genomic promoters, we predicted the yield of virus on BHK and DBT cells over a broad range of S prom , as shown in Figure 6A . Small S prom virus cannot grow well because most polymerases would be associated with genomes and tend to synthesize primarily anti-genomes. For example, for S prom equal to 0.1, infected BHK and DBT cells make 5-fold and 26-fold fewer progeny than wild-type VSV infected cells, respectively. However, large S prom virus also cannot grow well because most polymerases would preferentially bind to newly synthesized anti-genomes, producing few of the anti-genomes that are needed as templates to amplify genomes. Our simulations predicted that values of 30 and 50 for S prom would be optimal for VSV growth in BHK and DBT cells, respectively ( Figure 6A ). The estimated wild-type value of S prom of 5.4 gives VSV yields higher than 80% of their maximum yields for both cell types ( Figure 6A) .
We speculate that a rational way to attenuate the pathogenicity of the live wild-type virus would be to swap its two promoters, giving an S prom of (5.4) À1 . For this promoter swap we predict virion production would be decreased by 3.3fold and 14.5-fold for infected BHK and DBT cells, respectively, relative to wild-type. However, the extent of growth attenuation by the promoter swapping can be higher than the model prediction because the swapping may also perturb viral transcription and virion assembly and budding processes modulated by the signals encoded in the 39and 59 termini of the genome [42, 43] that are not yet sufficiently defined to be included in the simulation.
Rational vaccine attenuation by double genomic manipulations. Several variant VSV strains, including N1 through N4, have been made by Ball and Wertz [18, 20] . With an aim to generate a potentially broader diversity of growth phenotypes, we created and tested in silico VSV mutants by combining N gene relocations with a range of S prom . This is a computationally simple task, but experimentally nontrivial. For example, the VSV N4 with S prom ¼ 0.1 produced a simulated 38-fold fewer virus progeny than wild-type in BHK Figure 6B ). It is interesting to note that this 38-fold degree of growth attenuation is greater than the product of the constituent attenuations (4.7 3 5.4 ¼ 25) that one calculates by assuming that the effects were uncoupled. Such nonmultiplicative effects of double genomic manipulations on growth would be challenging to predict in the absence of a quantitative model.
Modulation of VSV immunogenicity by gene shuffling. To elicit a systematic immune response, live viral vaccines must present or display neutralizing epitopes, typically through the expression of viral surface proteins. Higher levels of antigen expression have been found to correlate with more rapid and potent induction of anti-viral antibodies [39, 40] . As Flanagan et al. suggested, the gene encoding G may be moved to other positions in the VSV genome to modulate the expression of the VSV surface glycoprotein G [39] . We generated in silico five gene-shuffled VSV strains, having gene orders for the three internal genes, MPG, MGP, PGM, GMP, and GPM, and simulated levels of G protein in BHK cells infected with those strains. Our simulation results were consistent with the idea that the location of G gene affects the production of G protein. The GPM strain gave the highest concentration of G protein in the cytoplasm (almost 2-fold higher than that of wild-type, Figure 7 ). However, in many cases effects of such gene rearrangements can be difficult to anticipate because of the complexity of the involved interactions among viral components. For example, the PGM strain showed only a level of G protein expression similar to those of PMG (wt) and MPG strains even though it has the G gene at an earlier position than the other two strains (Figure 7) .
For vaccine use we might aim to maximize the immunogenicity of VSV or a VSV-based vector through the expression increase of VSV G gene or inserted foreign gene while minimizing their potential pathogenicity by growth attenuation. Given such design goals, specifically for a VSV vaccine, we might prefer strain GPM, which showed the highest expression of G protein (Figure 7 ) and the lowest production of virions [18] . Toward such favorable features, Flanagan et al. previously constructed three VSV strains having the following gene orders: 39-G-N-P-M-L-59 (G1N2), 39P-M-G-N-L-59 (G3N4), and 39-G-P-M-N-L-59 (G1N4) [39] . These genome constructions were based on their intuitive idea that translocations of G gene and N gene to earlier and later positions, respectively, compared with wild-type, could not only increase the expression of G protein, but also attenuate virus growth [39] . This idea was supported by their experimental results [39] .
Seeking a more detailed correlation between locations of the two genes and the viral phenotypes relevant to vaccine application, we simulated in silico the growth of all mutants that retain the gene order P -M -L of the wild-type, but allow G and N to move, criteria that define 20 possible geneorder permutations. The viral growth and the level of G protein in infected BHK cells mainly depend on the locations of N gene and G gene, respectively ( Figure 8A and 8B) , which is consistent with the experimental results of Flanagan et al. [39] . Further, if gene G is fixed, then moving gene N closer to the 39 promoter is predicted to increase protein G expression ( Figure 8B ). Consistent with this prediction, Flanagan et al. also observed a higher G protein expression for the G1N2 strain than for the G1N4 strain [39] . Enhanced replicative ability of VSV strain by locating its N gene at an earlier position in its genome can contribute to increasing the level of G protein. If either gene N or gene G is located at the fifth position, then both levels of virus growth and G protein expression are very low (Figure 8) , because with such genome organizations the stoichiometric amounts of N and G proteins required for replication and assembly (Table 2 , [28, 29] ) cannot be reached. Our simulations with the BHK cell parameters (Table 1 ) overall captured the experimentally established relative growth of the VSV strains in BHK-21 cells, but the growth of the G3N4 strain was significantly overestimated compared with the experimental results [39] ( Table 3 ). The relevant mechanism for such a large discrepancy between the simulation and the experimental results remains to be elucidated.
The changes of protein expression levels by gene shuffling can be a rational means to modify the viral features for vaccine use. Robust synthesis of antigen by a highly attenuated strain appears to be an effective vaccine strategy as Flanagan et al. previously suggested. In addition to controlled attenuation of virus growth, a potent vaccine should ideally elicit a strong humoral or cell-mediated immune response.
In the era of highly advanced genetic technologies, we have witnessed a turning point for the development of live viral vaccines. Conventional empirical vaccine development processes are now being replaced by more rational reversegenetics-based ones. With this trend, much attention will be focused on mechanism-based design of less pathogenic and more immunogenic virus stains. Mathematical models for intracellular virus growth can support this design process by providing a tool to systematically analyze the viral infection regulatory network, identify critical regulatory mechanisms or components for redirecting viral phenotypes, and reverse engineer desirable phenotypes. One-step infection of cell monolayers. Cells were harvested, resuspended in growth medium, and plated into six-well plates at a concentration of 5 3 10 5 cells per 2 ml per well. Plated cells were returned to the incubator and allowed to grow overnight. The next day, two representative cell monolayers were harvested and counted to give an approximate number of cells per well. Each monolayer was then incubated with 200 ll of virus inoculum (MOI 3) for 1 h to allow virus adsorption. The plates were rocked gently every 20 min to evenly distribute virions on the monolayers during the adsorption step. After the adsorption period, the monolayers were rinsed twice with 1 ml of HBSS and then placed under 2 ml of infection medium for incubation. Medium samples of 200 ll including virion particles were taken from each well at 2, 3, 4, 6, 8, 10, and 20 h postinoculation. Samples were kept frozen at À90 8C until their analysis by the plaque assay.
Plaque assay. BHK cells were plated into six-well plates and cultured to 90% confluence. Culture medium was removed from each well and replaced with 200 ll of serially diluted viral samples. The inoculated monolayers were returned to the incubator for 1 h to allow virus adsorption. The plates were rocked gently every 20 min. At the end of the adsorption period, the inoculum was removed from each monolayer sample and then replaced with 2 ml of agar overlay. The agar overlay consisted of 0.6% weight/volume (w/v) agar (Agar Nobel, Difco, BD Diagnostic Systems, http://www.bd.com). 5-Bromo-29-deoxyuridine (B5002, Sigma) was added, at 100 lg/ml, to the agar overlay of N3-and N4-infected samples to enhance plaque formation. Following agar addition, the plates were allowed to cool at room temperature for 30 min, returned to the incubator and incubated for 24 h, and then each sample was fixed with 2 ml of fixative for 3 h at room temperature. The fixative consisted of 4% (w/v) paraformaldehyde (VWR) and 5% (w/v) sucrose (Sigma) in 10 mM phosphate buffered saline (PBS, Sigma) of pH 7.4. The agar overlay was then removed, and each sample was rinsed twice with 2 ml of PBS. Gentian violet diluted in methanol (0.01% (w/v), Sigma) was used, at 1 ml each, to stain the samples. Model development. Using algebraic and differential forms of equations, our mathematical model aims to account for established molecular processing steps in the development of VSV. Most model parameters were extracted from the literature. However, five parameters were obtained by fitting our simulation results to experimental data that were from the literature and our own experiments. Key model parameters are given in Table 1 , and detailed descriptions of the model and parameter estimation process are provided below, and in Protocol S1 and Figures S2 and S3 , respectively.
Virus binding and penetration. As shown in Figure 9A , VSV initiates an infection by binding to a receptor such as phosphatidyl serine, a lipid component in the plasma membrane [19, 44] . After the binding step, the VSV particle is endocytosed via a clathrin-coated pit, and then penetrates intracellular vesicles such as endosomes by membrane fusion [19, [45] [46] [47] . The penetration leads to the release of the encapsidated negative-sense viral genome and virus proteins into the cytoplasm of the host cell. By assuming the binding step is ratedetermining [48] , we lump these early steps from the binding to the penetration into a first-order expression:
where V b and V ex are the concentrations of bound and extracellular virus particles, respectively, t is time, and k b is the apparent rate constant for virus binding. After binding, we assume the bound virus is immediately endocytosed and fused, and its genome and protein components are instantaneously released into the cytoplasm at the expense of the fused virus particle. The protein stoichiometry of a single VSV particle and the lengths of each viral gene and protein are summarized in Table 2 , [28, 29] . Population distribution of polymerases and nucleocapsids. Following the release of the encapsidated genome and proteins into the cytoplasm, VSV transcription is initiated. The viral transcription was assumed to be independent of host-cell functions such as replication [33] . Instead, the viral complex of L and P proteins, with a stoichiometry of 1-3.6, was taken to function as polymerase in transcription and replication [49] . In the absence of P protein, L protein cannot bind to the genome or anti-genome [50] . After binding to the 39 promoter regions of the genomic and anti-genomic templates, the viral polymerase starts to synthesize its own RNA transcription and replication products by elongating along the templates. During transcription a fraction of elongating polymerases terminate transcription by dissociating from the templates as they encounter regulatory signals at intergenic regions [19, 26] . In addition to the regulated polymerase dissociation, time-dependent concentration changes of the polymerases and the viral templates in the cytoplasm influence the distribution of polymerases on the viral templates during transcription and replication. Hence, the distribution of polymerases continuously varies over the viral templates, ultimately determining the relative synthesis levels of mRNAs and genome-size RNAs.
We simulate the transcription and replication processes by considering the spatial-temporal distribution of template-associated polymerases. We first partition the viral genome and anti-genome templates into 40 segments, excluding their 39 and 59 end regions, which are the leader (Le) and trailer regions (Tr) for the genome, and the complementary trailer (Trc) and complementary leader regions (Lec) for the anti-genome, respectively ( Figure 9B ). For the genome template that is used for transcription as well as for replication, we specially grouped the segments into five genes ( Figure 9B ). We chose 40 as a minimum number for total segments which allows each gene to be split into a specific integer number of segments, proportional to the length of the gene. By considering the mechanisms for the interactions between polymerase and the intergenic regulatory sequences of the templates, as described below in the Transcription section, we simulated the polymerase flux into each segment over the time elapsed from the initiation of transcription on each template. Then we correlated the level of polymerase occupying each geneencoding section of the template with the synthesis rate of each corresponding viral mRNA. In a similar way, the distribution of polymerases on the replication templates was correlated with the synthesis rate of viral genome-sized RNA. Such explicit treatment of polymerase spatial distributions on the viral genome and antigenome templates was central to modeling the growth of wild-type and gene-rearranged virus strains. This treatment systematically accounts for polymerization-associated time delays and the polymerase fluxes into each template segment.
Before estimating the polymerase flux, we need to figure out how the polymerase complex and M protein compete with each other for binding to the genomic nucleocapsids as well as how the polymerases bound to nucleocapsids are subsequently distributed to one of three possible tasks: transcription, replication of genome, or replication of anti-genome. In our model we assume that the genomic templates (negative-sense nucleocapsids) whose promoters (leader regions) are free of polymerases are available for association with free polymerase or M protein. We further assume that the associations of the free genomic templates by M proteins or polymerases take place instantaneously:
where (-)nc, (-)nc M,new , and (-)nc pol,new are the concentrations of total genomic nucleocapsids and subsets of genomic nucleocapsids whose promoters are newly occupied by M protein and polymerase, respectively. S pol is the spacing between neighboring polymerases on the genomic or anti-genomic template, pol l is the concentration of polymerases bound to the promoter region (Le) of the genomic template, and l l is the length of the promoter region. Specifically, the second term in the left-hand side of the equation denotes the concentration of the genomic templates whose promoters are currently occupied by polymerases. In our model the concentration of the genomic nucleocapsids whose promoters are bound to polymerases and the concentration of the polymerases bound to the promoters of the genomic nucleocapsids are interchangeable with each other by the factors (l l /S pol ) and (S pol /l l ), respectively. The binding of M protein or polymerase initiates reactions leading to virion assembly or RNA synthesis, respectively. Because the initiation of RNA synthesis by the polymerase requires a finite time, a space between adjacent polymerases on the template (S pol ) would be maintained during infection, assuming a fixed elongation rate. With these considerations, one may expect that at any time the concentration of nucleocapsids available for the new binding of the free proteins is inversely proportional to the concentration of polymerases currently bound to the leader region of the genomic nucleocapsids (pol l ) and the polymerase spacing (S pol ) as shown in the second term of Equation 2.
The ratio of (-)nc M,new to (-)nc pol,new is determined by the ratio of the association rates of M protein and polymerase with the genomic nucleocapsid, which is further a function of the rate constants and relative amounts of the corresponding free components in the cytosol:
where r asso,M and r asso,pol are the rates of the associations of M protein and polymerase with the genomic nucleocapsid, respectively, and k M and k pol are the rate constants for each association reaction, respectively. S cond denotes the ratio of the two rate constants (¼k M / k pol ). Unlike L protein, 10% of synthesized M proteins are associated with the plasma membrane [51] . In Equation 3, cond M is the fraction of M proteins associated with the plasma membrane, trans is the fraction of L proteins satisfying the polymerase stoichiometry with P protein, pol total is the total concentration of polymerases associated at the time with nucleocapsids, and M and L are the total concentrations of M and L proteins not assembled into viral progeny. If the concentration of P protein (P) is larger than 3.6-fold concentration of L protein, then trans is equal to 1. Otherwise, trans is equal to P/(3.6L). In our model, M and L proteins compete for free genomic nucleocapsids, and the condensed nucleocapsids, owing to their association with M proteins, cannot be utilized for transcription or replication [19] . From Equations 2 and 3, the newly occupied nucleocapsids by polymerases ((-)nc pol,new ) can be calculated:
In the same way, given S pol , the concentration of positive-sense antigenomic nucleocapsids available for binding to polymerases would be ((þ)nc -pol trc (S pol /l trc )), where (þ)nc is the total concentration of antigenomic nucleocapsids, pol trc is the concentration of the polymerases bound to the promoter region (Trc) of the anti-genomes, and l trc is the length of the promoter region. Because the anti-genome has a stronger promoter than the genome [19, 31] , which is quantified by S prom in our model, S prom -fold, more polymerases bind to the promoter of the anti-genome than to that of the genome. Under the limitation of free polymerase complex, the concentration of the polymerases newly binding to the promoters of the genomes or the anti-genomes (pol term new ) could be described as follows: 
where pol trc new is the concentration of the polymerases newly binding to the complementary trailer region (promoter) of the anti-genome. The polymerases newly binding or already bound to the promoters of the genomes and anti-genomes start viral RNA synthesis as transcription or replication process.
Transcription. The viral polymerase on the leader region of the genome starts either transcription or replication. If there are sufficient N proteins, transcription is inhibited by the encapsidation of nascent positive-sense RNAs by N proteins; then replication dominates transcription [41, 52, 53] . In contrast, if there are insufficient free N proteins, then transcription dominates replication. In the model we correlate the extent of transcription dominance with the availability of N proteins by introducing a factor, encap. This factor is defined as the ratio of the number of free N proteins to the number required to encapsidate all available nascent genome-sized viral RNAs. Only nocap (¼ 1 À encap) of the polymerases bound to the genomic promoters can start the transcription: dpol N;1 dt ¼ k e;pol ðð1 À / N Þ nocap pol l l l À n sec;N l mRNA;N pol N;1 Þ ð 7Þ
where pol N,1 is the concentration of the polymerases located at the first segment of the N gene ( Figure 9C) , k e,pol is the elongation rate of polymerase, / N is the attenuation factor for N gene, n sec,N is the total number of the segments of N gene, and l mRNA,N is the length of N mRNA (Table 2 , [28, 29] ). The genome segments are continuously charged with incoming polymerases and discharged with outgoing polymerases with a rate of k e,pol (Equations 7-9). If the polymerase input to the leader region of the genome is decreased owing to a lack of free polymerases, then the polymerase concentrations downstream of the leader region will be subsequently reduced ( Figure 9C ). There are conserved intergenic sequences involved in letting a fraction of viral polymerases release from the genome template at intergenic sections during transcription, which is so-called partial transcription termination or attenuation ( Figure 9C ) [18, 19] . Because the transcription is initiated from the 39 end promoter, the attenuation mechanism causes genes more proximal to the 39 end to be more highly expressed, which ultimately leads to an unequal concentration distribution of viral mRNAs. The extents of partial transcription termination are quantified by the attenuation factors, / i , in our model ( Figure 9C ). These are 0/0.25/0.25/0.25/0.05 for leader-N/N-P/ P-M/M-G/G-L intergenic regions, respectively [18, 19, 26, 27] . / i fraction of polymerases are released at intergenic region i. With Equations 7-9, we simulate the polymerase flux into each gene segment, which is proportional to the elongation rate of polymerase, but inversely proportional to the extent of attenuation: dpol i;j dt ¼ k e;pol ðð1 À / i Þ n sec;iÀ1 l mRNA;iÀ1 pol iÀ1;nsec;iÀ1 À n sec;i l mRNA;i pol i; j Þ j ¼ 1; i ¼ P; M; G; L ð8Þ dpol i;j dt ¼ k e;pol n sec;i l mRNA;i ðpol i; jÀ1 À pol i; j Þ j 6 ¼ 1;
where pol i,j is the concentration of the polymerases located at the jth segment of gene i, and iÀ1 indicates the prior gene of gene i. The amount of newly synthesized mRNAs for each gene is determined by the concentration of polymerases occupying each gene section on the genome template and the decay rates of the mRNAs:
where mRNA i is concentration of mRNAs for gene i, k d,mRNA is the decay rate constant of mRNA that is the same for all five viral mRNAs [54] , and pol t,i is the total concentration of the polymerases occupying on the ith gene. Our formulation for transcription assumes that the synthesis of viral mRNAs is rate-controlled by the transcription initiation as well as the elongation of polymerase. Transcription initiation rate is parameterized by the spacing between neighboring polymerases in our model. At a given polymerase elongation rate, the larger polymerase spacing indicates the lower rate of transcription initiation. Transcription initiation modulates the input of polymerases to the leader region of the genome.
Translation. We consider that both translation initiation and polypeptide chain elongation contribute to the rate of viral protein synthesis. The translation initiation rate is parameterized by the ribosomal spacing. In our model we first calculated the number of ribosomes involved in viral translation by considering the maximum concentration of the ribosomes bound to viral mRNAs at a fixed ribosomal spacing:
where rib and n rib,avail are the concentrations of the ribosomes actually involved in viral translation and the ribosomes available for viral translation, respectively, and S rib is the spacing between neighboring ribosomes.
The ribosomes involved in viral translation (rib) are allocated to the five types of viral mRNAs according to their length and abundance, assuming that each viral mRNA has the same efficiency of translation initiation [55] :
where rib i is the concentration of the ribosomes assigned to mRNA i. The synthesis rate of each viral protein depends on the elongation rate of the ribosome, linear density of ribosomes on its corresponding mRNA, and its first-order decay rate: dp i dt ¼ k e;rib l p;i rib i À k dp;i p i i ¼ P; M; G; L ð13Þ
where p i is the concentration of protein i, k e,rib is the elongation rate of ribosome, l p,i is the length of protein i, and k dp,i is the decay rate constant of protein i. We also accounted for the consumption of free N proteins during the encapsidation of genome-length nascent RNAs and assumed that the degradation of nucleocapsids yielded intact N proteins: dp i dt ¼ k e;rib l p;i rib i À k dp;i p i À n i ðencap Á k e;pol pol tr l tr þ pol lec l lec
where n N is the stoichiometry of N protein in a single nucleocapsid or virion progeny (Table 2 , [28, 29] ), pol tr and pol lec are the concentrations of the polymerases located on the trailer and complementary leader regions of the genomes and the anti-genomes, respectively, l lec (¼ l l ) is the length of the complementary leader region, and k d,nc is the decay rate constant of nucleocapsid. As progeny virions are assembled, the concentration of each protein is reduced by the amount corresponding to its stoichiometry in a single virion particle.
Replication. We assumed that N protein regulates the switch of the role of polymerase between transcription and replication by encapsidating the newly synthesized RNAs [41, 52] . The polymerase that starts the replication at the leader region of the genome requires further supply of N proteins to skip the attenuation signals at each gene junction and thereby to complete each round of replication. Depending on the availability of N proteins, nocap(¼1-encap) fraction of polymerases terminate the replication at each gene junction in our model: dpol r;n;N;1 dt ¼ k e;pol ðencap pol l l l À n sec;N l mRNA;N pol r;n;N;1 Þ ð 15Þ
where pol r,n,N,1 is the concentration of the replicating polymerases on the first segment of the N gene section in the negative-sense genomic nucleocapsid. dpol r;n;i;1 dt ¼ k e;pol ðencap n sec;iÀ1 l mRNA;iÀ1 pol r;n;iÀ1;nsec;iÀ1 À n sec;i l mRNA;i pol r;n;i;1 Þ i ¼ P; M; G; L ð16Þ
where pol r,n,i,1 and pol r,n,i-1,nsec,i-1 are the concentrations of the replicating polymerases on the first segment of gene i, and on the last segment of gene iÀ1, respectively. The level of polymerases that scan through the whole genome (pol tr ) determines the amount of newly synthesized anti-genomic nucleocapsids, (þ)nc: dpol tr dt ¼ k e;pol n sec;i l mRNA;i pol r;n;i; j À pol tr l tr i ¼ L; j ¼ n sec;L ð17Þ dðþÞnc dt ¼ k e;pol encap pol tr l tr À k d;nc ðþÞnc ð18Þ
where l tr (¼ l trc ) is the length of the trailer region of the genome. We also considered the first-order kinetics for the decay of anti-genomic nucleocapsid.
The synthesis and decay of genomic nucleocapsids are described in the same way as for those of the anti-genomic nucleocapsids except that the polymerases on the anti-genomic templates are not released at intergenic regions: dpol r;p;j dt ¼ k e;pol ðencap pol trc l trc À n sec l À l trc À l lec pol r;p; j Þ j ¼ 1 ð19Þ
where pol r,p,j is the concentration of the replicating polymerases on the jth segment of the positive-sense anti-genomic nucleocapsids, l is the total length of the genome, and n sec is the total number of segments of the genome ( Figure 9B ). dpol r;p; j dt ¼ k e;pol n sec l À l trc À l lec ðpol r;p; j À 1 À pol r;p; j Þ j ¼ 2; . . . n sec ð20Þ dpol lec dt ¼ k e;pol n sec l À l trc À l lec pol r;p;nsec À pol lec l lec ð21Þ dðÀÞnc dt ¼ k e;pole ncap Á pol lec l lec À k d;nc ðÀÞnc ð22Þ
In our model, non-encapsidated nascent genome and anti-genome fragments are released from polymerases and immediately degraded.
As polymerases leave the promoter regions by moving toward the downstream sequences, the concentration of polymerases on the promoters will decrease. The dynamic changes of the polymerase concentrations on the promoters of the genomic and the antigenomic templates are finally described, respectively: pol l;nþ1 ¼ pol l;n þ pol new l;n À pol lÀleave;n ð23Þ
where pol l-leave is the concentration of the polymerases leaving the genomic promoters, O ,n and O ,nþ1 are the concentrations of a component (O) at time n and time nþ1 (in our numerical integration, time nþ1 À time n ¼ Dt), respectively. pol trc;nþ1 ¼ pol trc;n þ pol new trc;n À pol trcÀleave;n ð24Þ
where pol trc-leave is the concentration of the polymerases leaving the anti-genomic promoters. Assembly and budding. We assume that the condensation of negative-sense nucleocapsid by M protein initiates the virion assembly and the condensed nucleocapsids are not degraded in the same manner as virion progeny. Whenever the requirement for the stoichiometric amounts of proteins is satisfied, progeny virions are instantaneously assembled and released to the extracellular space. The time required for the condensation of the negative-sense nucleocapsid, the assembly, and the budding of progeny virion was assumed to be negligible relative to the preceding steps.
Host cell. In our model, the host cell provides unlimited building blocks such as nucleoside triphosphates and amino acids for the growth of virus. However, as viral components accumulate during the course of infection, some key host components for translation such as initiation and elongation factors may be depleted [28, 63, 56] . Two main viral products, leader-mRNA and M protein, contribute to the deficiency by inhibiting the synthesis of host macromolecules at the transcription level [19, 28, 35, 36] . Because leader-mRNA starts to accumulate soon after the initiation of infection, and a small amount of the component is enough to trigger the inhibition [28, 35] , the pool of host factors is continuously reduced from the onset of infection. We quantify this reduction with a single decay rate constant specific to the type of host cell:
where f dec is the level of host translation factors at time t, relative to that of the initial state of the cell before infection (at t ¼ 0), and k d,host is the decay rate constant. The inhibition by the leader mRNA causes a first-order decay of the host factors, resulting in a shortage of the ribosomes equipped with the accessory factors for viral translation in the late infection stage in our model. Unlike viral transcription and replication, viral translation is directly affected by the decay of host factors since it depends entirely on host machinery. In the early infection, host mRNAs outnumber viral mRNAs and thereby successfully compete for the host translation machinery. However, the newly synthesized M proteins inhibit the host transcription initiation and the export of host mRNAs from the nucleus to the cytoplasm [57] , thereby causing a gradual shift in translation from host mRNAs to viral mRNAs. For our model we assumed that the potency of the inhibition by the M protein was independent of the type of cell and its differentiation state [36] , and we developed an empirical formula using available experimental data from the literature [36] to account for the competition between host and viral mRNAs for ribosomes. Lyles at al. cotransfected the host cells with VSV M mRNA and chloramphenicol acetyl transferase (CAT) plasmid DNA, and then they quantified the expression of CAT based on its activity, as a function of the expression of VSV M protein [36] . In their experiment the gene expression of CAT was more reduced at higher M protein expression levels. We assume that the decrease of the expression of CAT (or its activity decrease) is proportional to the decrease of the occupancy of host mRNAs by the translation machinery. Using their experimental data, the occupancy of host mRNAs by the translation machinery is correlated with the number of newly synthesized M proteins in the cytoplasm:
where rib_host is the fraction of the translation machinery associated with host mRNAs, and M cell is the total number of newly synthesized M proteins per cell. Considering the decay of host factors and the competition between host and viral mRNAs, we could derive a formula to quantify the number of the fully functional ribosomes that are available for the viral protein synthesis over time post-infection (nrib avail ):
where nrib denotes the total concentration of ribosomes whether or not they incorporate all the required accessory factors for their translation function. Although the ribosomes distribute into membrane-bound and cytoplasmic forms, each class supporting the syntheses of the viral G protein and the other four viral proteins (N, P, M, and L), respectively, we treated the ribosomes in our model as one population.
Initial condition for simulation. The initial condition for our simulation is set by a fixed number of infectious extracellular virus particles per cell (V ex (0)). At time zero (t ¼ 0), the number of bound virus particles and the level of all viral components within cells are zero. In our model, binding of extracellular virus particles to cells reduces their level (Equation 1), and an encapsidated genome and stoichiometric amounts of viral proteins (Table 2 , [28, 29] ) are then immediately released from each bound virus particle to the cytoplasm. Specifically, we assume that all N proteins from a bound virus particle are released as a form of encapsidated genome complex. Downstream processes, beginning with transcription, are then initiated. In our simulation, viral infection starts with rib_host ¼ 0.99 (with rib_host ¼ 0.9, ; 0.9995 simulations showed the same results). Other key model parameters for simulation are summarized in Table 1 . In addition, a nomenclature list is shown in Table 4 . Figure S1 . Ratio of Genome to Anti-Genome During Simulated VSV Infection Depends on Relative Promoter Strengths The relative strength of the anti-genomic promoter relative to the genomic promoter is given by S prom . PI stands for time post-infection. Figure S4 . Productivity Ranking of Six Gene-Shuffled Viruses Provides Parameter Constraint For each value of S cond and its corresponding host parameter values (k d,host and S rib ), the virus productivity of each gene-shuffled virus in BHK cells was determined and normalized by the highest productivity. Simulated rankings of six strains matched experimentally observed rankings [9] over a narrow range of S cond , indicated by the bar. Initial number of infectious extracellular virus particles per cell was three. Found at DOI: 10.1371/journal.pcbi/0020116.sg004 (872 KB TIF).
Protocol S1. Parameter Estimation Delivery Systems for the Direct Application of siRNAs to Induce RNA Interference (RNAi) In Vivo RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described. Altered expression levels of certain genes play a pivotal role in several pathological conditions. For example, in many cancers the upregulation of certain growth factors or growth factor receptors, or the deregulation of intracellular signal transduction pathways, represents key elements in the process of malignant transformation and progression of normal cells towards tumor cells leading to uncontrolled proliferation and decreased apoptosis. Since these processes may result in the direct, autocrine stimulation of the tumor cell itself as well as the paracrine stimulation of other cells, including the stimulation of tumor-angiogenesis, many novel therapeutic strategies focus on the reversal of this effect, that is, the inhibition of these proteins or the downregulation of their expression. Likewise, several other diseases have been firmly linked to the (over-)expression of endogenous wildtype or mutated genes. Taken together, in addition to strategies based on the inhibition of target proteins, for example, by low molecular weight inhibitors or inhibitory antibodies, this opens an avenue to gene-targeting approaches aiming at decreased expression of the respective gene.
The first method to be introduced for the specific inhibition of gene expression was the use of antisense oligonucleotides in the late 1970s [1, 2] . Upon their introduction into a cell, antisense ODNs are able to hybridize to their target RNA leading to the degradation of the RNA-DNA hybrid double strands through RNAase H, to the inhibition of the translation of the target mRNA due to a steric or conformational obstacle for protein translation and/or to the inhibition of correct splicing. In the early 1980s, the discovery of ribozymes, that is, catalytically active RNAs which are able to sequence-specifically cleave a target mRNA, further expanded gene-targeting strategies [3] [4] [5] . Subsequently, both methods were extensively studied and further developed with regard to the optimization of targeting efficacies and antisense-ODN/ribozyme delivery strategies in vitro and in vivo.
Most recently, another naturally occurring biological strategy for gene silencing has been discovered and termed RNA interference (RNAi). Since RNAi represents a particularly powerful method for specific gene silencing and is able to provide the relatively easy ablation of the expression of any given target gene, it is now commonly used as a tool in biological and biomedical research. This includes the RNAimediated targeting in vitro and in vivo for functional studies of various genes whose expression is known to be upregulated as well as the development of novel therapeutic approaches based on gene targeting. double-stranded RNA molecules as described first in C elegans by Fire et al [6] who then introduced the name RNA interference. These findings also explained earlier observations in petunias which turned white rather than purple upon the introduction of the "purple gene" in form of dsRNA [7] , and on gene silencing by antisense oligonucleotides as well as by sense oligonucleotides in C elegans [8] . Subsequent studies demonstrated that RNAi, while described under different names (posttranscriptional gene silencing (PTGS), cosuppression, quelling), is present in most eukaryotic organisms with the response to dsRNA, however, being more complicated in higher organisms.
RNAi relies on a multistep intracellular pathway which can be roughly divided into two phases, that is, the initiation phase and the effector phase. In the initiation phase, double-stranded RNA molecules from endogenous or exogenous origin present in the cell are processed through the cleavage activity of a ribonuclease III-type protein [9] [10] [11] [12] into short 21-23 nucleotide fragments termed siRNAs. These effector siRNAs, which contain a symmetric 2 nt overhang at the 3 -end as well as a 5 -phosphate and a 3hydroxy group, are then in the effector phase incorporated into a nuclease-containing multiprotein complex called RISC (RNA-induced silencing complex) [13] . Several structural and biochemical studies have shed light on the processing of double-stranded RNA and the formation of the RISC complex (see, eg, [14] for a recent review). Through unwinding of the siRNA duplex by an RNA helicase activity [15] , this complex becomes activated with the single-stranded siRNA guiding the RISC complex to its complementary target RNA. Upon the binding of the siRNA through hybridization to its target mRNA, the RISC complex catalyses the endonucleolytical cleavage of the mRNA strand within the target site, which, due to the generation of unprotected RNA ends, results in the rapid degradation of the mRNA molecule. With the RISC complex being recovered for further binding and cleavage cycles, the whole process translates into a net reduction of the specific mRNA levels and hence into the decreased expression of the corresponding gene. For an overview of the RNAi pathway, see Figure 1 .
While from this mechanism it becomes obvious that siRNA molecules complementary to the target mRNA and thus being able to serve as a guide sequence for the RISC complex play a pivotal role in this process, they need not be derived from long double-stranded precursor molecules. Rather, omitting the initiation phase, they can be delivered directly into the target cell ( Figure 1 , upper right arrow).
Several studies have led to the development of guidelines for the generation of siRNAs which are optimal in terms of efficacy and specificity [12, 16] . This includes the initial definition of the preferable length (19-25 bp) combined with a low G/C content in the range between 36% and 52% and the requirement of symmetric 2 nt overhangs at the 3 -end [16] [17] [18] . Later studies on synthetic siRNA molecules, however, revealed an up to 100-fold higher targeting efficacy in the case of even longer duplexes (25-30 nucleotides) which act as a substrate for Dicer and which therefore allow the direct incorporation of the newly produced siRNAs into the RISC complex [19] . As to be expected, intramolecular foldback structures which can result from internal repeats or palindrome sequences decrease the numbers of functional siRNA molecules with silencing capability [20] . Additional silencing-enhancing criteria include an A in position 3 and a G at position 13 of the sense strand, the absence of a C or G at position 19 and, most importantly, a U in position 10 of the sense strand. Since nucleotides 10-11 represent the site of the RISC-mediated cleavage of the target mRNA, this indicates that RISC is comparable to most other endonucleases in preferentially cleaving 3 of U rather than any other nucleotide [20, 21] . Furthermore, it was shown more generally that the thermodynamic flexibility of the positions 15-19 of the sense strand correlates with the silencing efficacy and that the presence of at least one A/U base pair in this region improves siRNA-mediated silencing efficacy due to a decreased internal stability of its 3 -end [20] .
Still, different siRNA sequences may display differing efficacies, which suggest additional still unknown criteria for optimal siRNA selection and emphasize the influence of target mRNA accessibility. In fact, several studies also correlate the siRNA efficacy with the mRNA secondary structure [18, [22] [23] [24] [25] [26] [27] .
In conclusion, apart from the selection criteria defined above, the individual screening of different siRNAs for highly efficient and specific duplexes, or the pooling of multiple siRNAs, is the most effective approach to increase siRNAmediated targeting efficacy.
For the design of effective siRNAs, several algorithms on publicly accessible web sites are available (see [28] for review). To reduce the risk of nonspecific ("off-target") effects of the siRNAs, a homology search of the targeting sequence against a gene database is necessary and already incorporated in some of these web sites. Nevertheless, it has also been shown that siRNAs may cross-react with targets of limited sequence similarity when regions of partial sequence identity between the target mRNA and the siRNA exist. In fact, in some cases regions comprising of only 11-15 contiguous nucleotides of sequence identity were sufficient to induce gene silencing [29] . The prediction of these off-target activities is difficult so far.
An additional mechanism that may lead to nonspecific effects in vivo relies on the interferon system [30] [31] [32] [33] which is induced when double-stranded RNA molecules enter a cell activating a multi-component signalling complex. This effect is particularly true for long dsRNA molecules and essentially prevents them from being used as inducers of RNA interference in mammalian systems. The development of synthetic siRNAs [10, 12, 33, 34] largely circumvents this problem since they seem to be too small. However, some synthetic siR-NAs do induce components of the interferon system which seems to be dependent on their sequence [31, 32, 35] as well as, in the case of in vitro transcribed siRNAs, on the 5 initiating triphosphate [36] . Thus, strategies to avoid as far as possible the unwanted interferon response upon application of siRNAs in vivo will include a design of siRNAs without known interferon-stimulating sequences, the use of the lowest possible siRNA dose to still achieve the desired effect and optimized siRNA delivery methods.
Based on the known mechanisms of antisense technology, ribozyme-targeting or RNAi, small oligonucleotides or plasmid-based expression vectors can be used to specifically downregulate the expression of a given gene of interest or of pathological relevance in vitro. In principle, this also applies to the in vivo situation leading to novel, potentially relevant therapeutic approaches.
For the delivery of therapeutic nucleic acids, viral vectors have been used which have the advantage of high transfection efficacy due to the inherent ability of viruses to transport genetic material into cells. On the other hand, however, viral systems show a limited loading capacity regarding that the genetic material are rather difficult to produce in a larger scale and, most importantly, pose severe safety risks due to their oncogenic potential and their inflammatory and immunogenic effects which prevent them from repeated administration [37] [38] [39] [40] .
In the light of these problems, concerns, and limitations, nonviral systems have emerged as a promising alternative for gene delivery. Main requirements are the protection of their nucleic acid "load" as well as their efficient uptake into the target cells with subsequent release of the DNA or RNA molecules and, if necessary, their transfer into the nucleus. Several strategies can be distinguished, mainly lipofection and polyfection relying on cationic lipids or polymers, respectively (see, eg, [41] [42] [43] ).
The efficient protection against enzymatic or nonenzymatic degradation is particularly important for RNA molecules including siRNAs. In fact, while the therapeutic potential of siRNAs for the treatment of various diseases is in principle very promising, limitations of transfer vectors may turn out to be rate-limiting in the development of RNAi-based therapeutic strategies. One approach to solve this problem is the use of DNA expression plasmids which encode palindromic hairpin loops with the desired sequence. Upon transcription and folding of the RNA, the doublestranded short hairpin RNAs (shRNAs) are recognized by Dicer and cleaved into the desired siRNAs. Additionally, an in vitro method has been described recently which is based on the expression of shRNAs in E coli and their delivery via bacterial invasion [44] . While all these different DNAbased systems offer the advantage of siRNA expression with a longer duration and a probably higher level of gene silencing, they still rely on (viral or nonviral) delivery of DNA molecules and again raise safety issues in vivo. Hence, the direct delivery of siRNAs molecules, derived from in vitro transcription or chemically synthesized, offers advantages over DNA-based strategies and may be preferable for in vivo therapeutic use.
In the last years, a large body of studies has been published which describe different strategies for the systemic or local application of siRNAs in vivo. Tables 1-3 give an overview. The probably largest number of papers focuses the use of unmodified siRNAs (Table 1 ) whose administration is often performed IV by hydrodynamic transfection (high pressure tail vein injection). While this method is widely used and in some cases led to efficient target gene inhibition in the liver and, to a lesser extent, in lung, spleen, pancreas, and kidney, it may suffer from certain technical and practical limitations at least in a therapeutical setting since it relies on the rapid IV injection of a comparably large volume (>= 1 ml/mouse/injection, in theory equivalent to a ∼ 3 l IV bolus injection in man). Alternative strategies for the application of naked siRNAs include various delivery routes which, however, often provide an only local administration or rely on an administration at least close to the target tissue or target organ, thus restricting the number of target organs which may not be relevant for certain diseases. It should also be noted that several studies described here and below use rather large amounts of siRNAs and that upon intravenous injection of siRNAs the liver is the primary site of siRNA uptake. As an alternative approach for the application of siR-NAs in vivo, their delivery by liposomes/cationic lipids has been described. For liposome-based siRNA formulations, a wide variety of modes of application allowing local or systemic delivery has been used (Table 2) . Finally, several other strategies for local or systemic siRNA administration have been explored, including chemical modifications of siRNA molecules, electropulsation, polyamine, or other basic complexes, atelocollagen, virosomes, and certain protein preparations (Table 3 ).
An alternative approach relies on the complexation of unmodified siRNA molecules with a cationic polymer, polyethylenimine (PEI).
Polyethylenimines (PEIs) are synthetic polymers available in branched or linear forms (Figure 2 , upper panels) and in a broad range of molecular weights from < 1000 Da to > 1000 kd. Commercial PEI preparations, although labelled with a defined molecular weight, consist of PEI molecules with a broad molecular weight distribution [45] [46] [47] . PEIs possess a high cationic charge density due to a protonable amino group in every third position [48, 49] . Since no quarternary amino groups are present, the cationic charges are generated by protonation of the amino groups and hence are dependent on the pH in the environment (eg, 20% at pH 7.4, see [50] for review). Due to its ability to condense and compact the DNA into complexes, which form small colloidal particles allowing efficient cellular uptake through endocytosis, PEI has been introduced as a potent DNA transfection reagent in a variety of cell lines and in animals for DNA delivery (for review, see [51, 52] and references therein). In fact, in several studies PEI has been shown to be able to deliver large DNA molecules such as 2.3 Mb yeast artificial chromosomes (YACs) [53] as well as plasmids or small oligonucleotides [48, [54] [55] [56] into mammalian cells in vitro and in vivo. The N/P ratio, which indicates the ratio of the nitrogen atoms of PEI to DNA phosphates in the complex and thus describes the amount of PEI used for complex formation independent of its molecular weight, influences the efficiency of DNA delivery. A positive net charge of the complexes, resulting from high N/P ratios, inhibits due to electrostatic repulsion their aggregation and improves their solubility in aqueous solutions as well as their interaction with the negatively charged extracellular matrix components and thus their cellular uptake [57] . Additionally, the strong buffer capacity, described by the "proton sponge hypothesis" which postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H + buffering polyamines due to enhanced endosomal Cl − accumulation and osmotic swelling/lysis [48] , seems to be responsible for the fact that PEI-based delivery does not require endosome disruptive agents for lysosomal escape. This tight condensation of the DNA molecules as well as the buffering capacity of PEI in certain cellular compartments like endosomes and lysosomes also protects DNA from degradation [48, 49, 58, 59] . PEIs have been successfully used for nonviral gene delivery in vitro and in vivo. While initial publications showed increased transfection efficacies when using high molecular weight PEIs [45] , more recent studies demonstrated the advantages of certain low molecular weight PEIs [47, 60, 61] . The higher transfection efficacy of low molecular weight PEIs may be due to a more efficient uptake of the resulting PEI/DNA complexes, a better intracellular release of the DNA and/or lower in vitro cytotoxicity as compared to high molecular weight PEI [60] [61] [62] [63] . In fact, a decrease in the molecular weight of the PEI leads to an increase in complex size which may be favourable at least for in vitro use [64, 65] . On the other hand, other PEIs with very low molecular weight (< 2 kd) display little or no transfection efficacy even at very high N/P ratios which may be attributed to the fact that a decrease in the molecular weight of PEI has been shown to translate into an increasingly lower ability to form small complexes [63] . Therefore, low molecular weight PEIs require higher N/P ratios for optimal transfection efficacies as compared to higher molecular weight PEIs since higher N/P ratios lead to an increase in compaction with reduced complex sizes and a reduced tendency of the complexes to aggregate due to hydrophobic interactions [61, 63, 64] . Nevertheless, while several parameters have been extensively studied, some precise determinants for transfection efficacy remain to be elucidated (see [50, 66] for review). Also, the mechanism of the cytotoxic effects of PEI complexes is only poorly understood. It may rely on the formation of large aggregates in the range of up to 2 μm which, when formed on the cell surface, impairs membrane functions finally leading to cell necrosis [60] . Clearly, there is a trend towards low molecular weight PEIs as rather nontoxic delivery reagents in vitro and in vivo, which combine high biocompatibility and reduced side-effects thus also allowing to employ larger PEI/DNA complex amounts without significant cytotoxicity. More recently, the use of polyethylenimines has been extended towards the complexation and delivery of RNA molecules, especially small RNA molecules like 37 nt all-RNA ribozymes [67] [68] [69] and siRNAs [70] (Figure 2 ). While chemically unmodified RNA molecules are very instable and prone to rapid degradation, the PEI complexation has been shown to lead to an almost complete protection against enzymatic or nonenzymatic degradation. In fact, PEI-complexed siR-NAs, which are [ 32 P]-labeled for better detection, remain intact in vitro for several hours even in the presence of RNase A or fetal calf serum at 37 • C, while non-complexed siRNAs are rapidly degraded (Figure 3(a) ). This indicates that siRNA molecules are efficiently condensed and thus fully covered and protected by PEI. Indeed, the analysis of PEI/siRNA complexes by atomic force microscopy showed the absence of free siRNAs or siRNA molecule ends and thus confirms these findings regarding an efficient complexation (Grzelinski et al, submitted). However, while the complex stability seems to be sufficient for siRNA protection with all PEIs tested (Werth et al, in press; Aigner et al, unpublished data), several of these complexes do not show any targeting efficacy at all. In fact, only when using certain polyethylenimines, PEI/siRNA complexes are efficiently delivered into target cells in vitro, where siRNAs are released and display bioactivity (Figures 1 and 2) . In general and as seen before for PEI/DNA complexes (see above), the transfection efficacy is dependent on the PEI used, also indicating that the siRNA targeting efficiency mainly depends on the endocytotic uptake of the complex and/or its intracellular decomposition rather than on the in vitro complex stability. Good results were obtained with commercially available JetPEI [70] while the in vivo JetPEI from the same supplier showed only poor siRNA delivery efficacies [71] . Likewise, a novel low molecular weight PEI based on the fractionation of a commercially available polyethylenimine demonstrates high siRNA protection and delivery efficacies in vitro (Werth et al, in press). Under certain conditions, the PEI/RNA (siRNA or ribozyme) complexes retain their physical stability and biological activity also after lyophilization ( [72] and Werth et al, in press). Although the PEI transfection is only transient, data from our lab show that PEI/siRNA effects are stable for at least 7 days (Urban-Klein and Aigner, unpublished results). Finally, another study has explored the use of siRNA nanoplexes comprising of PEI that is PEGylated with an RGD peptide ligand attached at the distal end of the PEI. Again, siRNA nanoplexes protect siRNAs against serum degradation and show in vitro activity [73] .
The ultimate goal is the application of siRNAs in vivo which has been explored in some studies in different mouse models. Ge et al showed that PEI-complexed siRNAs targeting conserved regions of influenza virus genes are able to prevent and treat influenza virus infection in mice. Upon IV injection, PEI promoted the delivery of siRNAs into the lungs where, either given before or after virus infection, siRNA reduced influenza virus production in the lungs [74] .
Most biological effects of the systemic application of PEIcomplexed siRNAs, however, have been determined in different mouse tumor models and by targeting different proteins which have been shown previously to be tumor-relevant. This includes the epidermal growth factor receptor HER-2 (c-erbB-2/neu), the growth factor pleiotrophin (PTN), and vascular endothelial growth factor (VEGF) and its receptor (VEGF R2), and the fibroblast growth factor-binding protein FGF-BP.
The in vivo administration of PEI complexed, but not of naked siRNAs, through IP or subcutaneous injection resulted in the detection of intact siRNAs even hours after injection (Figure 3(b) ). Radiolabeled siRNA molecules were found in several organs including subcutaneous tumors, muscle liver, kidney and, to a smaller extent, lung and brain. It is important to note that the siRNAs were actually internalized by the tissues as indicated by the fact that blood was negative for siRNAs ( Figure 3(b) ).
Overexpression of the HER-2 receptor has been observed in a wide variety of human cancers and cancer cell lines. Since HER-2 displays strong cell growth-stimulating and antiapoptotic effects especially through heterodimer formation with other members of the EGFR family, its overexpression has been established as a negative prognostic factor and linked to a more aggressive malignant behaviour of tumors (eg, [75] ). Consequently, HER-2 qualifies as an attractive target molecule for antitumoral treatment strategies including anti-HER-2 antibodies, low molecular weight inhibitors, or HER-2-specific gene-targeting approaches. In fact, the relevance of HER-2 (over-)expression in tumor growth has been established in several in vitro HER-2 targeting studies including the use of ribozymes [76, 78, 79] or siRNAs [80, 81] . proposed mechanism of PEI-mediated siRNA transfer. Due to electrostatic interactions, PEI is able to complex negatively charged siRNAs leading to a compaction and the formation of small colloidal particles which are endocytosed. The "proton sponge effect" exhibited by PEI complexes leads to osmotic swelling and ultimately to the disruption of the endosomes. siRNAs are protected from degradation due to their tight condensation in the complex and the buffering capacity of PEI. Upon their release from the PEI-based complex, intact siRNAs are incorporated into the RISC complex and induce RNAi (see Figure 1 ).
It was demonstrated that HER-2 reduction in vitro leads, among others, to the inhibition of cell proliferation and increased apoptosis.
The systemic treatment of athymic nude mice bearing subcutaneous SKOV-3 ovarian carcinoma tumor xenografts through IP injection of PEI-complexed HER-2-specific siRNAs led to marked antitumoral effects as seen by a significant reduction tumor growth (Figure 4 ) [70] . PEIcomplexed nonspecific siRNAs or HER-2-specific, naked siR-NAs had no effects. This was paralleled by the detection of intact HER-2-specific siRNAs in the tumors of the specific treatment group already 30 min after administration and for at least 4 h, and by the downregulation of HER-2 on mRNA and protein levels [70] .
Another receptor, VEGF R2, was targeted in a study employing self-assembling nanoparticles based on siRNAs complexed PEI which is PEGylated with an RGD peptide ligand attached at the distal end of PEG. While the PEGylation allows steric stabilization and reduces nonspecific interactions of the complexes, the RGD motif provided tumor selectivity due to their ability to target integrins expressed on activated endothelial cells in the tumor vasculature. Upon IV administration into mice bearing subcutaneous N2A neuroblastoma tumor xenografts, a selective tumor uptake and a VEGF R2 downregulation were observed, resulting in decreased tumor growth and tumor angiogenesis [73] .
The receptor ligand, VEGF, is a mitogenic and angiogenic growth factor stimulating tumor growth and angiogenesis in several tumors including prostate carcinoma. Thus, it may represent attractive target molecule for RNAi-based genetargeting strategies also bearing in mind the double antitumoral effect due to reduction of tumor cell proliferation as well as tumor angiogenesis. The subcutaneous or intraperitoneal injection of VEGF-specific siRNAs complexed with a novel PEI obtained through fractionation of a commercially available PEI (Werth et al, in press) resulted in the reduction of tumor growth due to decreased VEGF expression levels (Höbel and Aigner, unpublished results). The same was true for PEI/siRNA-mediated targeting of FGF-BP (Dai and Aigner, unpublished results), which has been established Figure 3 : Protection and in vivo delivery of siRNAs upon PEI complexation. In [70] (a) in vitro protection of siRNAs against nucleolytic degradation. [ 32 P] end-labeled siRNAs, complexed (upper panel) or not complexed (lower panel) with PEI, were subjected to treatment with 1 % fetal calf serum at 37 • C. At the time points indicated, the samples were analysed by agarose gel electrophoresis, blotting, and autoradiography. The bands represent full-length siRNA molecules indicating that PEI complexation leads to the efficient protection of siRNAs while noncomplexed siRNAs are rapidly degraded. (b,c) In vivo delivery of intact siRNAs upon PEI complexation. [ 32 P]-labeled siRNAs, complexed (+) or not complexed (−) with PEI, were injected IP into mice bearing subcutaneous SKOV-3 ovarian carcinoma cell tumor xenografts, and after 30 min (b) or 4 h (b) total RNA from various organ and tissue homogenates was prepared and subjected to agarose gel electrophoresis prior to blotting and autoradiography. The bands represent intact [ 32 P]-labeled siRNA molecules which for several hours are mainly found in tumor and muscle as well as in liver and, time-dependently, in kidney. Only little siRNA amounts are detected in the lung and traces in the brain.
previously as "rate-limiting" for tumor growth and angiogenesis in several tumors ( [82, 83] , see [84] for review).
Finally, PEI/siRNA-mediated targeting of pleiotrophin (PTN) exerted strong antitumoral effects. PTN is a secreted growth factor which shows mitogenic, chemotactic, angiogenic and transforming activity [85] [86] [87] [88] [89] [90] [91] [92] [93] and which is markedly upregulated in several human tumors including cancer of the breast, testis, prostate, pancreas, and lung as well as in melanomas, meningiomas, neuroblastomas, and glioblastomas. The in vivo treatment of nude mice through systemic subcutaneous or IP application of PEI-complexed PTN siRNAs led to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerted antitumoral effects. This establishes, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM, and further extends the modes of delivery of PEI/siRNA complexes intrathecal strategies as employed in the therapy of glioblastomas with antisense oligonucleotides.
Only a few years after their discovery, siRNAs are catching up with ribozymes and antisense oligonucleotides as efficient tools for gene targeting in vitro and, more recently, also in vivo. This includes the exploration of their potential as therapeutics which will lead to the development of siRNA-based therapeutic strategies. Their ultimate success, however, will Figure 4 : Systemic treatment of mice with PEI-complexed HER-2-specific siRNAs leads to reduced growth of subcutaneous SKOV-3 tumor xenografts due to decreased HER-2 expression. In [70] athymic nude mice bearing subcutaneous tumor xenografts were injected IP with 0.6 nmoles HER-2-specific naked (open circles) or PEI-complexed (closed circles) siRNAs 2-3 times per week and tumor sizes were evaluated daily from the product of the perpendicular diameters of the tumors. Mean +/-standard error of the mean (SEM) is depicted and Student's unpaired t test was used for comparisons between data sets ( * * P < .03, * * * P < .01). Differences in tumor growth reach significance at day 5 indicating the antitumoral effects of the PEI-complexed HER-2-specific siRNAs. strongly depend on the development of powerful and feasible siRNA delivery strategies which need to address several issues including the stability/stabilization of siRNA molecules while preserving their efficacy and maintaining their genesilencing activity, an efficient delivery into the target organ(s) as well as a sufficiently long siRNA half life in the organism and particularly in the target organ. Thus, siRNA delivery strategies must provide siRNA protection and transfection efficacy, the absence of toxic and nonspecific effects, they must be efficacious also when using small amounts of siRNAs and must be applicable in various treatment regimens and in various diseases even when this requires to overcome biological barriers after their administration to reach their target tissue or target organ. The research done on DNA-based gene delivery, ribozyme-targeting, and antisense technology will facilitate this process since it already provides a basis of established technologies. This is also true for the complexation of siRNAs with polyethylenimine, which may represent a promising avenue for siRNA applications in vivo. This may eventually lead to novel therapeutic strategies. 
The work of A. Aigner is supported by the Deutsche Forschungsgemeinschaft (AI 24/5-1) and by the Deutsche Krebshilfe. The author would like to apologize to the authors whose primary works have not been cited due to length considerations. Electrochemical Molecular Analysis Without Nucleic Acid Amplification Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electro-chemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.  Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays BACKGROUND: The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. METHODS: Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. RESULTS: From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. CONCLUSION: We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. In fish, as in other lower vertebrates, the post-vitellogenic period is very important for the completion of the oogenetic process. During this step, the follicle-enclosed postvitellogenic oocyte undergoes several key events such as the final acquisition of the ability to resume meiosis in response to the maturation-inducing steroid (MIS), the resumption of the meiotic process and, finally, its release from the surrounding follicular layers. In addition, the whole follicle (oocyte and surrounding follicular cells) undergoes a progressive differentiation ultimately leading to the release of a metaphase 2 oocyte. The key hormonal and molecular events involved in the control of meiosis resumption have been thoroughly studied and many studies have been dedicated to the action of gonadotropins, the regulation of steroidogenenic events and the action of the MIS (see [1] [2] [3] [4] [5] [6] for review). However, the associated follicular or extra-follicular events involved in concomitant processes such as oocyte-follicular cells cross talk and ovulationmechanisms have received far less attention. Nevertheless, several researchgroups have studied the periovulatory ovarian physiology using classical biochemical or histological tools and, later, molecular approaches. Thus, several studies have dealt with ovarian proteases in their participation in the ovulatory process [7] [8] [9] . Differential display PCR and suppressive subtractive hybridization (SSH) approaches have also been developed in order to identify new differentially regulated genes in the fish periovulatory ovary [10] [11] [12] [13] . In addition, numerous candidate gene studies have also been performed in the fish periovulatory ovary. Apart from genes related to hormonal controls, these studies were mostly dedicated to some specific gene families such as TGF beta family [14, 15] or connexins [16, 17] . Finally, fewer studies have simultaneously analyzed the expression profiles of several genes belonging to different families [18, 19] . However, in contrast to other biological processes, such as immune response [20] , the post-vitellogenic period has never benefited from genome-wide transcriptomic studies that could provide a global view of the molecular events occurring in the post-vitellogenic ovary undergoing oocyte maturation. In this context, the present study aimed at performing a transcriptomic analysis of the postvitellogenic rainbow (Oncorhynchus mykiss) trout ovary. In order to do so, 9152-gene rainbow trout cDNA microarrays were hybridized using RNA samples originating from rainbow trout ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. A statistical analysis was performed in order to identify all the genes exhibiting a differential expression over this period. In addition, a supervised clustering analysis was performed using only the differentially expressed genes in order to identify groups (or clusters) of genes exhibiting similar expression profiles. Furthermore, as a first step in a long-term transcriptomic analysis of the rainbow trout post-vitellogenic ovary, we deliberately chose to focus on 3 gene clusters exhibiting the most remarkable expression patterns. Finally, specific genes were selected in each cluster based on the novelty of their putative identity and/or function. For each gene, a real-time PCR analysis of their ovarian expression profiles was performed using additional ovarian RNA samples.
Investigations were conducted according to the guiding principles for the use and care of laboratory animals and in compliance with French and European regulations on animal welfare. Two year old female rainbow trout (Oncorhynchus mykiss) were obtained during their first reproductive season from our experimental fish farm (Sizun, France) and held under natural photoperiod in a re-circulated water system in INRA experimental facilities (Rennes, France). The water temperature was kept constant at 12°C. Ovaries were sampled from individual females during late vitellogenesis (N = 6), post-vitellogenesis (N = 6) and during oocyte maturation (N = 6). Oocyte developmental stage was assessed under binocular microscope according to previously described criteria [21, 22] . Late vitellogenic samples were collected at the end of the vitellogenic process, approximately 3-4 weeks before expected ovulation. At this stage, germinal vesicle is not visible and no polarized cytoplasm area can be observed. Post-vitellogenic samples were collected 2-3 weeks later but before any noticeable morphological changes in yolk structure due to the process of meiosis resumption. At this stage, oocytes can display a subperipheral or peripheral germinal vesicle. When germinal vesicle is not visible, a dark mass of polarized cytoplasm can be observed. Oocyte maturation samples were collected after meiosis resumption. Those samples were thus collected after yolk clarification and around the time of germinal vesicle breakdown (GVBD). For tissue collection, trout were deeply anesthetized in 2-phenoxyethanol, killed by a blow on the head and bled by gill arch section. Ovaries were then dissected out of the body cavity under sterile conditions. Ovarian aliquots were frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Low oligonucleotide signals (lower than three times the background level) were excluded from the analysis. After this filtering step, signal processing was performed using the vector oligonucleotide data to correct each spot signal by the actual amount of DNA present in each spot. After correction, signal was normalized by dividing each gene expression value by the median value of the array.
A statistical analysis was performed in order to identify differentially expressed genes between late vitellogenic, post-vitellogenic and maturing groups using SAM software [28] . Three 2-by-2 statistical analyses were performed in order to compare each group with the two other ones. In addition, a comparison was performed between samples taken prior to meiosis resumption (from late and post-vitellogenic females, N = 7) and during oocyte maturation (N = 6). For each comparison, the lowest false discovery rate (FDR) was used to identify differentially abundant genes. All genes identified in at least one of the above comparisons were kept for clustering analysis in order to characterize the expression profiles of statistically relevant genes. For supervised clustering analysis [29] , data was log transformed, median-centered and an average linkage clustering was performed using CLUSTER software [29] . Clusters were visualized using TREEVIEW software [29] .
Rainbow trout sequences originating from INRA Agenae [24] and USDA [30] EST sequencing programs were used to generate publicly available contigs [31] . The 8th version (Om.8, released January 2006) was used for BlastX [32] comparison against the Swiss-Prot database (January 2006) [33] . The score of each alignment was retrieved after performing a BlastX comparison. In addition, for each EST spotted onto the membrane, the accession number of the corresponding rainbow trout cluster (Unigene Trout, January 2006), if any, was retrieved from the UniGene database [34] .
Real-time PCR was performed using all RNA (N = 18) samples including those used for microarray analysis. Several over and under expressed clones belonging to three selected remarkable clusters, were selected according to their putative identity and/or function for analysis. Reverse transcription and real time PCR were performed as previously described [19] . Briefly, 3 μg of total RNA were reverse transcribed using 200 units of Moloney murine Leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) and 0.5 μg random hexamers (Promega) per μg of total RNA according to manufacturer's instruction. RNA and dNTPs were denatured for 6 min at 70°C, then chilled on ice for 5 min before the reverse transcription master mix was added. Reverse transcription was performed at 37°C for 1 hour and 15 min followed by a 15 min incubation step at 70°C. Control reactions were run without MMLV reverse transcriptase and used as negative controls in the real-time PCR study. Real-time PCR experiments were conducted using an I-Cycler IQ (Biorad, Hercules, CA). Reverse transcription products were diluted to 1/25, and 5 μl were used for each real-time PCR reaction. Triplicates were run for each RT product. Real-time PCR was performed using a real-time PCR kit provided with a SYBR Green fluorophore (Euro-gentec, Belgium) according to the manufacturer's instructions and using 600 nM of each primer. After a 2 min incubation step at 50°C and a 10 min incubation step at 95°C, the amplification was performed using the following cycle: 95°C, 20 sec; 60°C, 1 min, 40 times. For all primer pairs, the relative abundance of target cDNA within sample set was calculated from a serially diluted ovarian cDNA pool using the I-Cycler IQ software. This dilution curve was used to ensure that PCR efficiency was within an 80-100% range and that amplification was linear within sample set. After amplification, a fusion curve was obtained using the following protocol: 10 sec holding followed by a 0.5°C increase, repeated 80 times and starting at 55°C. The level of 18S RNA in each sample was measured and used for target genes abundance normalization within sample set. In addition to the genes identified from the transcriptomic analysis, a widely used standard gene, elongation factor 1 alpha (ef1α), was monitored using the same sample set to validate the normalization procedure. GenBank accession number and primer sequences are shown in table 1. Statistical analyses were performed using Statistica 7.0 software (Statsoft, Tulsa, OK). Differences between ovarian developments stages were analyzed using non parametric U tests.
After signal processing, 8263 clones out of 9152 were kept for further analysis. From the statistical analysis, 310 clones were found to exhibit a differential abundance between at least 2 of the studied ovarian stages (late vitellogenesis, post-vitellogenesis and oocyte maturation). For all SAM analyses performed, the false discovery rate (FDR) was always lower than 0.7%. Among the 310 identified clones, 90 were up-regulated during oocyte maturation while 220 exhibited an opposite pattern. A clustering analysis was performed using only expression data of the 310 identified clones in order to characterize the expression profiles of those genes. The clustering analysis clearly separated the over from the under expressed genes ( Figure  1 ). The number of each clone in the clustering analysis ( Figure 1 ) was kept in subsequent tables 1, 2, 3, 4 and in the text. Within down-regulated genes, a cluster of 32 genes (cluster 1, Figure 1 ) was characterized by high expression levels during late vitellogenesis, low levels during oocyte maturation and intermediate or variable levels during post-vitellogenesis ( Figure 1 ). Within up-regulated clones, a cluster of 44 genes (cluster 2, Figure 1 ) was characterized by a strong over expression at the time of meiosis resumption while a cluster of 14 genes (cluster 3, Figure 1 ) exhibited a very low expression during late and post-vitellogenesis and an up-regulation before meiosis resumption ( Figure 1 ). 
The rainbow trout (Oncorhynchus mykiss) genome has not been sequenced and the number of characterized rainbow trout proteins and mRNAs is limited. The identity of studied transcripts was therefore based on the most significant hit obtained after performing a BlastX search against the SwissProt database. For the clones belonging to cluster 1-3, the results of this blast search is presented in tables 2, 3, 4. For each clone, the identity of the best hit in SwissProt and the score value of the BlastX comparison are given. However, this similarity search was performed using all EST sequences available in public databases and not using fully characterized cDNAs displaying the full coding sequence of the transcript. For some of the clones spotted on the trout array, the corresponding mRNA was previously characterized and made available in public databases. The identity of those clones is therefore unambiguous. In contrast, for some other clones, the best hit in SwissProt only gives significant, but incomplete, information. This is especially true for protein family members for which only a phylogenetic analysis will allow a more relevant identification of the gene. However, the name of the best hit was used in the text for clarity reasons.
This large cluster of 32 clones (# 189-220) was characterized by a clear under expression at the time of oocyte maturation. Among those 32 clones, 29 belonged to a UniGene cluster and 30 had a significant hit in Swiss-Prot ( (Table 3 ). In addition, 39 clones exhibited a significant hit in SwissProt while 5 clones had no significant sequence similarities with known genes (Table 3) . Within this cluster, several genes exhibited inflammation or ovulation-related functions. Thus some of the clones exhibited sequence similarities with human chemokine cxcl14 (clone # 250), clawed frog adam22 (clone # 258) and coagulation factor V (cf5) (clone # 235). In addition, one clone (# 245) exhibited strong sequence similarity with human angiotensin-converting enzyme 2 precursor (ace2). Two clones (# 238 and 239) exhibited strong sequence similarity with salmon (Oncorhynchus keta) vasotocin-neurophysin (avt) and isotocin-neurophysin respectively. Finally, cluster 3 also contained clones exhibiting sequence similarity with, human Forkhead box protein O3A and human pendrin, also know as solute carrier family 26 member 4 (slc26) (clone # 236). Within cluster 2, cxcl14, adam22, slc26, avt, ace2 and cf5 genes were kept for real-time PCR analysis.
This small cluster of 14 clones (# 296-309) was characterized by an over expression occurring earlier than for the genes belonging to cluster 3. Among those 14 clones, 12 belonged to a UniGene cluster and 11 had a significant hit in SwissProt (Table 4 ). Two clones (# 305 and 306) were most similar to rat and human aquaporin 4 (aqp4) respectively. These 2 clones belonged to the same UniGene cluster (Omy.23866). In addition, one clone (# 296) was most similar to mouse serine protease 23 (sp23). Within cluster 3, aqp4 and sp23 genes were kept for real-time PCR analysis. 
For all the genes selected for the real-time PCR analysis, a similar up or down regulation was observed between microarray and real-time PCR experiments.
We observed a dramatic under expression of aromatase (cyp19a1, clones # 196 and 198) in the ovary during the preovulatory period ( Figure 2 ). The mRNA abundance of cyp19a gene during oocyte maturation was more than 200 times lower than during late vitellogenesis. In addition, successive decreases of cyp19a gene expression levels were observed during post-vitellogenesis and during oocyte maturation ( Figure 2 ). The mRNA abundance of vitamin K-dependent protein S precursor gene (clones # 199 and 200) was lower during oocyte maturation than during late or post-vitellogenesis. In contrast, no significant differences were observed between late and post-vitellogenesis ( Figure 2) . A similar expression profile was observed for Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) gene ( Figure 2 ).
We observed a strong over expression of aquaporin 4 (aqp4) gene during post-vitellogenesis and at the time of oocyte maturation (Figure 3) . The mRNA abundance of aqp4 gene exhibited a 6-fold increase during post-vitellogenesis and a further 12-fold increase during oocyte maturation. In addition, the mRNA abundance of pendrin (slc26) gene exhibited a 1500-fold increase during oocyte maturation while no significant differences were observed between late and post-vitellogenesis. Similarly, vasotocin (avt) mRNA abundance exhibited a 500-fold increase at the time of oocyte maturation ( Figure 3 ). Angiotensinconverting enzyme 2 (ace2) gene expression levels exhibited a 215-fold increase between late vitellogenesis and oocyte maturation (Figure 3) . A similar profile was observed for the chemokine cxcl14 gene. The mRNA abundance of this gene exhibited a 35-fold increase between late vitellogenesis and oocyte maturation ( Figure  3 ). The mRNA abundance of coagulation factor V (cf5) gene exhibited a 177-fold increase between late or postvitellogenesis and oocyte maturation while adam22 mRNA abundance exhibited a 6-fold increase between late or post-vitellogenesis and oocyte maturation ( Figure  3) . Finally, the mRNA abundance serine protease 23 (sp23) gene monitored during oocyte maturation was higher than in the late vitellogenic ovary. However, this difference was not significantly different (p = 0.078).
The mRNA abundance of elongation factor 1 alpha (ef1α), a translation regulatory protein commonly used as a stable reference, did not exhibit any significant difference over the preovulatory period ( Figure 3 ).
The hybridization of radiolabeled cDNAs with cDNAs deposited on nylon membranes has been used for several decades. However, the use of nylon cDNA microarrays is not very common in comparison to glass slide microarray technology. Nevertheless, this technology has successfully been used for several years [27, 35] . In the present study we used similar cDNA manufacturing and hybridization protocols. While most of the 9152 clones used to generate the microarray putatively correspond to distinct genes, a small proportion of genes are represented by 2 distinct clones (e.g clones belonging to the same UniGene cluster). In our data, it is noteworthy that those clones are usually found in the same gene clusters (e.g clones #196 and 198, #199 and 200, #305 and 306). Since the position of clones in the clustering analysis is based on the correlation between their profiles, this indicates that they display very similar expression profiles. In addition, for all genes selected for real-time PCR analysis, the over or under expression observed was always consistent with microarray data. Furthermore, the expression of ef1α, a widely used reference gene, was stable over the preovulatory period. Together, these observations suggest that our overall microarray analysis is extremely robust and reliable.
In the present study, we identified 310 genes exhibiting a differential expression during the preovulatory period. Among them, 220 were down-regulated during oocyte maturation while 90 exhibited an opposite pattern. However, because we decided, as a first step, to focus our anal-ysis on the genes exhibiting the most differential regulation in the periovulatory period, we only present the identity of the 90 genes belonging to 3 specific clusters exhibiting the most remarkable patterns. Among those 90 transcripts we have chosen to discuss the most informative or novel genes based on their identities and/or putative involvement in the rainbow trout preovulatory ovarian functions.
Among the 32 clones belonging to cluster 1, two clones correspond to rainbow trout ovarian aromatase (cyp19a1). The real-time PCR study confirmed that cyp19a1 was dramatically under expressed during the preovulatory period. This observation is in total agreement with existing data on aromatase expression during this period [19, 36] . In addition, a clone putatively encoding for a NADPH-cytochrome P450 reductase (EC 1.6.2.4) was also located in cluster 1. The aromatase enzyme complex is formed from 2 principal protein components. CYP19a1 contains the catalytic domain that binds C19 steroid substrates in the proximity of the heme prosthetic group critical in the activation of molecular oxygen and subsequent substrate hydroxylation. The other essential component is the redox partner flavoprotein, NADPH cytochrome P450 reductase. Interestingly, present data show that both transcripts exhibited an under expression during the rainbow trout preovulatory period, although it should be confirmed that the identified clone is coding for the oxydoreductase protein involved in the aromatase complex.
Other cytochrome P450 genes Two other cytochrome P450 genes, exhibiting similar expression profiles were found in the same cluster. One clone (# 194) was most similar to rat cytochrome P450 2J3 while the other one (# 202) putatively corresponded to rainbow trout cytochrome P450 1A3 (cyp1a3). Cytochrome P450 1A proteins are ubiquitous proteins that have been associated with the detoxification of several organic compounds such as PCB (polychlorinated biphenyl), PAH (polyaromatic hydrocarbons), and dioxin [37] . In fish, these compounds are able to induce cyp1a gene expression in a variety of tissues. In the rainbow trout immature ovary, a constitutive expression of CYP1A protein was previously reported [38] . Together, previous and present observations suggest that a CYP1A-related detoxification activity in the rainbow trout ovary. From the under expression of cyp1a3 gene observed in the ovary immediately prior to ovulation we could speculate that a decrease of the detoxification activity of the ovary is required before the beginning of the ovulation process. In addition, it was previously shown in rat C6 glioma cells that epoxygenases could inhibit prostaglandin E2 production [39] . Interestingly, C6 cells express epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which convert arachidonic acid to epoxyeicosatrienoic acids; those epoxyeicosatrienoic acid being able to inhibit the activity of cyclooxygenase [39] . The role of prostaglandins in the ovulatory process has been thoroughly studied (see [40] for review). Thus, in rainbow trout, prostaglandin F2α was able to induce in vitro ovulation [21, 41] . Therefore, the observed down-regulation of cyp1a1 and cyp2j3 genes in the ovary prior to ovulation is therefore totally consistent with available data on the participation of prostaglandins in the ovulatory process.
In the present transcriptomic analysis, two aquaporin 4 (aqp4) clones were found in cluster 3. Real-time PCR data confirmed that rainbow trout aqp4 gene exhibited a strong over expression in the preovulatory ovary. In mammals, AQP4 is also known as mercurial insensitive water channel (MIWC). It was previously shown that water permeability was strongly increased in African clawed frog oocytes expressing MIWC [42] . In marine fish, a strong oocyte hydration occurs during oocyte maturation [43, 44] . In addition, it was recently shown that this oocyte hydration involves an aquaporin1-like protein in seabream [45] . In freshwater species, data on oocyte hydration is more controversial. However, a limited but
Ovarian expression profiles of aromatase (cyp19a1), vitamin K dependent protein S (proteinS) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) genes during rainbow trout late oogenesis (mean ± SEM) Figure 2 Ovarian expression profiles of aromatase (cyp19a1), vitamin K dependent protein S (proteinS) and cytidine monophosphate-Nacetylneuraminic acid hydroxylase (cmah) genes during rainbow trout late oogenesis (mean ± SEM). Ovaries were sampled from separate females during late vitellogenesis (LV, N = 6), post-vitellogenesis (PV, N = 6) and oocyte maturation (MAT, N = 6). The mRNA abundance of each gene was determined by real-time PCR and normalized to the abundance of 18S. Abundance was arbitrarily set to 1 for LV stage and data are expressed as a percentage of the transcript abundance at this stage. Bars sharing the same letter(s) are not significantly different (p < 0.05).
Relative mRNA abundance Ovarian expression profiles of angiotensin-converting enzyme 2 (ace2), coagulation factor V (cf5), CXC chemokine L14 (cxcl14), aquaporin 4 (aqp4), pendrin (slc26), vasotocin (avt), serine protease 23 (sp23), ADAM22 (adam22), and elongation fac-tor 1 alpha (ef1α) genes during rainbow trout late oogenesis (mean ± SEM) Figure 3 Ovarian expression profiles of angiotensin-converting enzyme 2 (ace2), coagulation factor V (cf5), CXC chemokine L14 (cxcl14), aquaporin 4 (aqp4), pendrin (slc26), vasotocin (avt), serine protease 23 (sp23), ADAM22 (adam22), and elongation factor 1 alpha (ef1α) genes during rainbow trout late oogenesis (mean ± SEM). Ovaries were sampled from separate females during late vitellogenesis (LV, N = 6), post-vitellogenesis (PV, N = 6) and oocyte maturation (MAT, N = 6). The mRNA abundance of each gene was determined by real-time PCR and normalized to the abundance of 18S. Abundance was arbitrarily set to 1 for LV stage and data are expressed as a percentage of the transcript abundance at this stage. Bars sharing the same letter(s) are not significantly different (p < 0.05). significant hydration was also observed in several freshwater species including rainbow trout [46] . Our data suggest that, similarly to marine species, the oocyte hydration occurring during oocyte maturation could also be aquaporin-mediated in freshwater species such as rainbow trout. In addition to aqp4 gene, we also observed a dramatic over expression of slc26 gene at the time of meiosis resumption. Solute carrier family 26 member 4 (slc26) is also known as sodium-independent chloride/ iodide transporters or pendrin. The over expression of slc26 gene at the time of oocyte maturation is dramatic, as demonstrated by real-time PCR. Together, the strong upregulation of aqp4 and slc26 genes at the time of meiosis resumption stresses the importance of water and ion transports in the rainbow trout preovulatory ovarian functions. In marine species, the major oocyte hydration occurring before ovulation is probably important for adjusting egg buoyancy. In contrast, in freshwater species laying demersal eggs such as rainbow trout, it has been hypothesized that the limited (25%) oocyte hydration occuring before ovulation could be necessary for the completion of the ovulation process [46] . Thus, the increase of oocyte volume could facilitate the rupture of the follicular walls and subsequently, the release of the oocyte from its follicular layers.
The neurophysial hormones arginine vasotocin (AVT) and isotocin (IT) are the fish counterparts of argininevasopressin and oxytocin respectively. Vasotocin precursor and isotocin precursor cDNAs were previously cloned in several fish species including chum salmon [47, 48] . In fish, AVT is involved in several physiological processes including water conservation and excretion of electrolytes [49] . However, existing data in fish correspond to the local effect, in various tissues, of circulating AVT [49] . Surprisingly, we observed that AVT precursor (avt) mRNA is expressed in the rainbow trout preovulatory ovary. To the best of our knowledge, there is no evidence of non-neural expression of avt mRNA in fish. In addition, it is noteworthy that we also observed a similar over expression of isotocin mRNA precursor in the ovary at the time of oocyte maturation. Further investigations are needed to elucidate the role of AVT and IT in the trout preovulatory ovarian functions.
Ovulation is a complex process resulting in the release of the oocyte from surrounding follicular layers. Since the early eighties, the similarities between ovulatory and inflammatory processes have been thoroughly discussed [50] [51] [52] and it is now well accepted that mammalian ovulation is an inflammatory-like reaction. In fish, despite numerous studies on the hormonal control of spawning, the ovulatory process has been far less documented.
In mammals, ovulation is accompanied by broad-spectrum proteolysis and the implication of several classes of proteases is well documented (see [53] for review). In salmonid fish, several proteases have been identified in the periovulatory ovary [54] . In mammals, there is evidence that mature ovarian follicles contain proteolytic enzymes, including serine proteases. Indeed, serine proteases have been implicated in both ovulatory and inflammatory reactions (see [50] for review). In the present study, serine protease 23 (sp23) gene appears progressively up-regulated during the preovulatory period. To our knowledge, sp23 gene expression was never reported in the periovulatory ovary of any vertebrate species. However, we could speculate that this protease participates in the rainbow trout ovulatory process. Interestingly, our data showed that adam22 metalloprotease-disintegrin gene was sharply up-regulated at the time of oocyte maturation. The metalloprotease-disintegrin protein family (also known as ADAMs: A Disintegrin And Metalloproteinases) is thought to function in cell-cell interactions and in the proteolysis of luminal or extracellular protein domains. In mammals, several ADAMs family members are involved in the ovulatory process. In brook trout (Salvelinus fontinalis), metalloprotease activity increases in the ovary prior to ovulation [8, 9] . Together, these observations also suggest that adam22 also participates in the rainbow trout ovulatory process.
Mammalian CXC chemokines, named after a conserved pattern of conserved cysteine residues, have been initially identified as potent mediators of neutrophil chemotaxis [55, 56] and are also involved in chemotaxis of monocytes and lymphocytes. They have also been implicated in angiogenesis and, later, in a large variety of functions [57, 58] . In mammals, 16 CXC have been described. In Fish, however, several CXC have been identified but only CXCL12 and CXCL14 exhibit unambiguous orthologues [59] . In the present study, we showed that cxcl14 gene expression strongly increases during the preovulatory period. In catfish, RT-PCR data showed that cxcl14 gene was expressed in a wide variety of tissues, including the ovary [60] . In carp, quantitative PCR data showed that cxcl14 was predominantly expressed in the brain [61] . Despite its good conservation throughout vertebrate evolution [59] , the number of studies addressing the in vivo role(s) of CXCL14 is limited. As a consequence, a lot of information is still unavailable in fish. In a murine model used to study Crohn's disease, cxcl14 expression is induced during inflammation [62] . Together, these observations suggest that cxcl14 gene expression induction contributes to the inflammatory-like events occurring in the rainbow trout at the time of ovulation. To date the participation of this gene in preovulatory ovarian functions was unsuspected.
In mammals, coagulation factor V participates in the coagulation process. In zebrafish, a coagulation factor V (cf5) cDNA was previously characterized [63] . According to these authors, several lines of evidences including biochemical and phylogenetic analyses suggest that the modern coagulation pathways found in mammals could also be functional in fish. Furthermore, it was previously shown that cultured rabbit macrophages were able to generate factor V procoagulant activity [64] . In the present study, we observed a dramatic increase of cf5 gene expression in the ovary during oocyte maturation. However, no significant difference was observed between late and postvitellogenesis. From these observations we could speculate that, immediately prior to ovulation, the trout ovary secretes coagulation factors in order to prevent bleeding from ruptured ovarian follicles at the time of ovulation. Interestingly, the transcriptomic analysis showed that a transcript exhibiting sequence similarity with clotting factor C (Clone # 251, Table 3 ) was also over expressed immediately prior to ovulation.
Angiotensin-converting enzyme (ACE) cleaves Angiotensin I (Ang I) to form Angiotensin II (Ang II). Angiotensin-converting enzyme 2 (ACE2) is a recently described ACE homolog [65] . Both ACE and ACE2 are zinc-dependent peptidases of the M2-metalloprotease family. Within the renin-angiotensin system (RAS), ACE2 competes with ACE because it is capable of hydrolyzing Ang I into the nonapeptide Ang(1-9) [65] . In humans, ace2 gene expression was predominantly detected by Northern blot analysis in kidney, heart and testis [65, 66] . In addition, a moderate expression was also observed in several other tissues including the ovary [66] . Using semiquantitative RT PCR, a wide distribution was observed in rat tissues [67] . In mammals, previous observations suggested that the renin-angiotensin system was functional in the ovary. In cattle, a greater expression of Ang II was observed in large follicles. In addition, several lines of evidence supported the idea of Ang II in blocking the inhibitory effect of theca cells on meiosis resumption of bovine oocytes [68] . In brook trout (Salvelinus fontinalis) salmon Ang I and human Ang II were both able to increase the level of in vitro spontaneous ovulation [69] . In the present transcriptomic study, we observed a dramatic increase of ace2 gene expression during the preovulatory period. This observation was confirmed by real-time PCR data. Together, these observations suggest that the dramatic upregulation of ace2 gene immediately prior to ovulation is important for the ovulatory process. In mammals, little is know about the role of ACE2 in the ovary. However, it is known in mammals that ACE2 can function as an Ang II degrading enzyme, forming the vasodilatator peptide Ang(1-7) [70, 71] . Interestingly, a local vasodilatation is a key characteristics of the inflammatory response that is also observed during the mammalian ovulatory process (see [50] for review). Therefore, it can be hypothesized that the observed increase of ace2 gene expression in the trout preovulatory participates in the vascular dynamics changes that are putatively occurring during the ovulatory process.
Genes involved in the synthesis of egg components Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) is the key enzyme for the synthesis of N-glycolylneuraminic acid. In salmonid eggs, cortical alveoli contain polysialoglycoproteins (PSGP). In rainbow trout, it was previously shown that those PSGP contain N-glycolylneuraminic acid residues [72] . In the present study we observed a significant decrease of cmah gene expression at the time of oocyte maturation. While the presence of cmah gene expression in the ovary is totally consistent with the presence of N-glycolylneuraminic acid in rainbow trout cortical alveoli content, it seems however difficult to speculate on the dynamics of PSGP accumulation in the oocytes.
Our observations further confirmed that a progressive shut down of estrogen synthesis genes expression occurs in the ovary prior to meiosis resumption. In addition to already well studied genes such as aromatase, the present work shows that other genes exhibit a similar down-regulation, thus suggesting their participation in the preovulatory decrease of circulating estrogen levels.
In addition, we observed a strong up-regulation of ion/ water transport genes in the preovulatory ovary. The identity of those genes is consistent with the recent identification of aquaporin mediated mechanisms in the fish oocyte hydration process and further supports the recent description of a limited but significant oocyte hydration occurring in the rainbow trout preovulatory ovary.
Finally, in addition to oocyte hydration-related genes, we also observed a strong over expression of several genes such proinflammatory factors, coagulation/clotting factors, vasodilatation factors and proteases in the ovary immediately prior to ovulation. Together, these observations suggest that, similarly to the theory developed in mammals, fish ovulation could also be compared ton an inflammatory-like reaction. In addition, the identification of those genes will allow specific studies leading to a better understanding of the ovulatory process in fish.
In the future, a global analysis of differentially regulated genes, based on their ontologies, is needed to satisfyingly describe preovulatory ovarian mechanisms. In addition, a cellular localization of gene expression will contribute in the understanding of their respective roles in the preovulaory ovarian physiology. Nevertheless, the present study clearly demonstrates that distinct (i.e. steroidogenic, proteolytic, proinflammatory) but concomitant events occur in the preovulatory ovary. Together, all those events concur to achieve the same goal which is the release, at the time of ovulation, of a fully competent oocyte, ready to be fertilized. An object simulation model for modeling hypothetical disease epidemics – EpiFlex BACKGROUND: EpiFlex is a flexible, easy to use computer model for a single computer, intended to be operated by one user who need not be an expert. Its purpose is to study in-silico the epidemic behavior of a wide variety of diseases, both known and theoretical, by simulating their spread at the level of individuals contracting and infecting others. To understand the system fully, this paper must be read together in conjunction with study of the software and its results. EpiFlex is evaluated using results from modeling influenza A epidemics and comparing them with a variety of field data sources and other types of modeling. EpiFlex is an object-oriented Monte Carlo system, allocating entities to correspond to individuals, disease vectors, diseases, and the locations that hosts may inhabit. EpiFlex defines eight different contact types available for a disease. Contacts occur inside locations within the model. Populations are composed of demographic groups, each of which has a cycle of movement between locations. Within locations, superspreading is defined by skewing of contact distributions. RESULTS: EpiFlex indicates three phenomena of interest for public health: (1) R(0 )is variable, and the smaller the population, the larger the infected fraction within that population will be; (2) significant compression/synchronization between cities by a factor of roughly 2 occurs between the early incubation phase of a multi-city epidemic and the major manifestation phase; (3) if better true morbidity data were available, more asymptomatic hosts would be seen to spread disease than we currently believe is the case for influenza. These results suggest that field research to study such phenomena, while expensive, should be worthwhile. CONCLUSION: Since EpiFlex shows all stages of disease progression, detailed insight into the progress of epidemics is possible. EpiFlex shows the characteristic multimodality and apparently random variation characteristic of real world data, but does so as an emergent property of a carefully constructed model of disease dynamics and is not simply a stochastic system. EpiFlex can provide a better understanding of infectious diseases and strategies for response. The most commonly used measure in public health, R 0 , is estimated from historical data and derived from SIS/SIR type models (and descendents) for forward projection [14, 15] R 0 is the basic reproductive ratio for how many individuals each infected person is going to infect [16] R 0 is often used on its own in public health as an indicator of epidemic probability; if R 0 < 1 then an epidemic is not generally considered possible, for R 0 > 1, the larger the value, the more likely an epidemic is to occur. R 0 is a composite value describing the behavior of an infectious agent. Hence, R 0 can be decomposed classically, for example, as: p d c, where p is probability of infection occurring for a contact, d is duration of infectiousness, and c is number of contacts [17] .
However, R 0 in the classical decomposition above, while it is one of the best tools we have, does not account for age segregation of response, existing immunity in population, network topology of infectious contacts and other factors.
These observations were significant in the motivation for developing EpiFlex.
The EpiFlex model was designed to create a system that could incorporate as much realism as possible in an epidemic model so as to enable emerging disease events to be simulated. There are limitations, described below in a separate section, but the model is quite effective as it stands. In most cases, the limitations of EpiFlex are shared by other modeling systems.
There are a variety of methods used for mathematical modeling of diseases. The most common of these are the SIR (susceptible, infected, recovered) of Kermack and McKendrick [15] , SIS (susceptible, infected, susceptible), SEIR (susceptible, exposed, infected, recovered), and SIRP (susceptible, infected, recovered, partially immune) as developed by Hyman et al. [18] and further developed by Hyman and LaForce [19] . The SIRP model was used as the starting point for development of the object model of Epi-Flex. In SIRP, the SIR model is extended to include partial immunity (denoted by P) and the progressive decline of partial immunity to allow influenza to be modeled more accurately. (See Appendix.)
There is a need for experimentation in more realistic discrete modeling, since the lattice type of discrete modeling is understood to skew in favor of propagation, as discussed by Rhodes and Anderson [20] and Haraguchi and Sasaki [21] . Others such as Eames and Keeling [22] and Edmunds et al. [12] have explored the use of networks to model interactions between infectable entities, and Ferguson et al. [23] and others have called for more balance in realism for epidemiology models. Since EpiFlex was completed, Lloyd-Smith et al. [17] have shown the importance of superspreading in disease transmission for the SARS epidemic. EpiFlex is designed to take these issues into account.
There are known weaknesses in SIS-descended models, some of which are discussed by Hyman and LaForce [14] . They suggested that a model dealing with demographics and their subgroups would be useful and described a start Theoretical Biology and Medical Modelling 2006, 3:32 http://www.tbiomed.com/content/3/1/32 toward conceiving such a model, creating a matrix of SIRP flows for each demographic group within a "city" and modeling contacts between these groups. Thus, the possibility of building an entirely discrete model using the object-oriented approach, essentially setting the granularity of the Hyman-LaForce concept at the level of the individual, together with Monte Carlo method, was attractive. The object method of design seemed to be a good fit, since object-oriented programming was invented for discrete simulations [24] . An Object-Oriented (OO) design defines as its primitive elements "black box" subunits that have defined ways of interacting with each other [25] .
The OO language concept originally was conceived for the Simula languages [24] for the purpose of verifiable simulation. Enforcement of explicit connections between objects is fundamental to OO design, whereas procedural languages such as FORTRAN and COBOL do not because data areas can be freely accessed by the whole program. OO languages wrap data in methods for accessing the data. If each "black box" (i.e. object) has a set of specified behaviors, without the possibility of invisible, unnoticed interactions between them, then the simulation can potentially be validated by logical proof in addition to testing. (It would take an entire course to introduce OO languages and concepts, and there is not space to do so here. Interested readers are suggested to start with an implementation of Smalltalk. There are excellent free versions downloadable. Smalltalk also has an enthusiastic and quite friendly user community. See: http:// www.smalltalk.org/main/.)
The design of EpiFlex is described more completely in the appendix. Design proceeded by establishing the definition of a disease organism as the cornerstone, then defining practical structures and objects for simulating the movement of a disease through populations. The disease object was assigned a set of definitions drawn from literature that would allow a wide spectrum of disease-producing organisms to be specified. The aim was to minimize the number of configuration parameters that require understanding of mathematical models.
The hosts that are infected became the second primary object. A host lives and works in some area, where hosts are members of some demographic group, which together determine what of n types of contacts they might have to spread an infectious disease. The hosts move about the area in which they live between locations at which they interact. In EpiFlex, an area contains some configured number of locations, and locations are containers for temporary groups of hosts. Since people travel between metro areas, the model supports linkages between areas to move people randomly drawn from a configurable set of demographic groups.
The remainder of this section presents the disease model adopted, an overview of each component, an overview of program flow, and a description of the core methods. This is followed by discussion of results from the EpiFlex software system.
This model has up to four stages during the infection cycle: the Incubation, Prodromal, Manifestation, and Chronic stages; to this is added a fatality phase. I have named this 'extended-SIRP'. Fig. 1 shows a diagram of this model.
The model of Fig. 1 allows us to track the different phases of the disease process separately, and to define variable infectiousness, symptoms, fatality, recovery and transition to chronic disease at each stage as appropriate. This allows us to model the progress of a disease in an individual more realistically. For diseases that have no identifiable occurrence of a particular stage, this stage can be set to length zero to bypass it entirely.
The 8 contact types designed into EpiFlex are drawn from literature in an attempt to model spread of infection more accurately. These contact types are: blood contact by needle stick, blood to mucosal contact, sexual intercourse, skin contact, close airborne, casual airborne, surface to hand to mucosa, and food contact. The probability of infection for a contact type is input by the user as estimated from literature or based on hypothetical organism characteristics.
Durations of disease stages are chosen uniformly at random from a user-specified interval [R low , R high ]. Random numbers, denoted by ξ, on [0, 1] are used to seed the determination of the infected disease stage periods (denoted I Incubation , I Prodromal , I Manifestation , I Chronic ). R low and R high are taken from medical literature and describe a range of days for each stage of an illness. These calculations are simply: (ξ × (R high -R low )) + R low = D, where D is days for a particular stage. (This may be extended in the future to include ability to define a graph to determine the flatness of distribution and the normative peak. This will make a significant difference in modeling of diseases such as rabies, which can, under unusual circumstances, have very long incubations.)
One of the following three equations describing immunity decay is chosen; L is the current level of partial immunity, P is the level of partial immunity specified as existing Random values on [0, 1] are then used to decide whether an infection occurs during the partial immunity phase P shown in the chart above. This decision uses the output of the immunity level algorithm, L, which is a number on [0, 1], as is the random value ξ:
EpiFlex uses a dynamic network to model the interactions between hosts at a particular location based on the skew provided and the demographic segments movement cycles. The networks of contacts generated in this version
Extended-SIRP disease model of Epiflex Figure 1 Extended-SIRP disease model of Epiflex. S: susceptible I: Infected R: recovered P: partially immune F: fatality Extended SIRP breaks the infected stage I into 4: I Incubation , I Prodroma l, I Manifestation , I Chronic , and adds a fatality terminating stage.
of EpiFlex are not made visible externally; they can only be observed in their effects. (See: Limitations of EpiFlex modeling.) Their algorithms were carefully designed and tested at small scales, observing each element.
A location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. A location can have a certain number of cells, which are used to specify N identically behaving locations concurrently. This acts as a location repetition count within an area when the location is defined. The user sets an average number of hosts inhabiting each cell, and a maximum. There is also a cell exchange fraction specifiable to model hosts moving from cell to cell. The algorithm for allocating hosts in cells is semi-random. It randomly puts hosts into cells in the location. If a cell hits the average, then it does another random draw of a cell. If all locations are at maximum, then it overloads cells.
Interactions are within the cell. So a host must be exchanged to another cell in order to be infective. See the appendix for 'Location component', and also with an open model look at how hospitals were defined. Households are modeled at this time using a cell configuration.
EpiFlex is implemented with a Monte Carlo algorithm such that each host in a location is assigned a certain number of interactions according to the Cauchy distribution parameter setting for that location. This distribution describes a curve with the y axis specifying the fraction of the maximum interactions for the location and x axis specifying the fractional ordinal within the list of hosts in the location. The distribution can be made nearly flat, or severely skewed with only a few actors providing nearly all contacts, as desired by the user of EpiFlex. Note that the structure of the network formed also depends on what locations are defined, what demographic groups are defined for the population, and how demographic groups are moved between locations. Each location has a maximum number of interactions specified per person, which is used as the base input. Initially, a Gaussian equation was used, but it was discarded in favor of a Cauchy function since this better fits the needs of the skew function and computes faster. The algorithm iterates for each infectious host, and selects other hosts to expose to the infected party in the location, by a Monte Carlo function. This results in a dynamically allocated network of interactions within each location.
The exposure cycle also makes use of Monte Carlo inputs. Each location has a list of contact types that can take place at a particular location, and a maximum frequency of interactions. This interaction frequency determines how many times contacts that can spread a disease will be made, and the contact specification defines the fractional efficacy of infection by any specific route. Modeling the effect of different types of contacts has been discussed in the literature, e.g. Song et al. [26] . EpiFlex attempts to make a more generalized version.
For each host infection source, target hosts are drawn at random from the location queue. A contact connection is established with the target as long as the contact allocation of that target has not been used up already. Contact connections made to each target are kept track of within the location to prevent over-allocation of contacts to any target. Thus, for each randomly established connection, a value is set on both ends for the maximum number of connections that can be supported. Once the maximum for either end of the link is reached, the algorithm will search for a different connection.
The location algorithm is described below in more detail. The user specifies the maximum number of connections for a location; the σ output from a Cauchy distribution function determines how many connections an individual will have. This allows variations in the degree of skewness for superspreading in a population to be modeled, which has been shown to be of critical importance by Lloyd-Smith et al. [17] .
If p = position in queue, q = number of hosts in queue for location: X = p/q, where X denotes the proportional fraction of queue for position.
If K is a constant chosen for the location to express skew distribution, the Cauchy distribution function is:
If κ is the number of contacts for a particular host and κ max is maximum number of contacts for any given host in the location:
When hosts move from one location to another within the model, they tend to maintain a rough order of ordinal position. Consequently, when there is a high σ for a location, the high connection host in one location tends to be a high connection host in another. This reflects real-world situations, (though not perfectly) and corresponds better than persistently maintaining high connection individuals from location to location, since host behavior changes from place to place.
The Cauchy distribution function is fairly fast in execution. The function can be used to approximate the often radical variations seen in epidemiology studies; as an extreme example, one active super-spreader individual might infect large numbers, when one or even zero is typical [17] . This type of scale-free network interaction has been explored by Chowell and Chavez [27] . The Cauchy function allows networks to be generated dynamically within each type of location in a very flexible manner, such as corresponding to super-spreader dynamics [17] . In addition to the specification of skew within a location, the network of contacts is also defined by (a) what locations are present and (b) the movement cycles defined for each demographic group within the model.
Processing time increases with population. This slowing is an expected characteristic of an object modeling system and is the price paid for the discrete detail of the EpiFlex model. The primary source of this increase in processing time is the sum of series of possible infectious events that are modeled for each iteration. It therefore scales as a series sum not as a log, based on the contagiousness of the disease and the number of potential hosts in a location with an infected host. This is minimized by only processing infectious host contacts. The increase stems from the characteristics of networks in which each node has n connections to other nodes. When iteration is done for a location containing infectable hosts, it is the number of infected hosts that creates an element of the series. The infected hosts are put into a list, and each one interacts randomly with other hosts (including other infected ones) in the location. Thus, considered as a network with m nodes, each of the m nodes is a host. A temporary connection to another host is made to n other nodes where n <m, and n<k. The value of k is determined by a randomized input that returns the number of contacts of this infected host in this location. Consequently, the series consists of all the temporary connections made for contact modeling for each cycle.
In the interest of completeness, the limitations of the Epi-Flex model are described here. The plan is to address these elements for implementation in future versions.
Only one infectious disease can be occurring at a time. Thus, competitive inhibition [28] and synergistic effects will not be seen. Hosts do not reproduce Hosts do not reproduce within a model. Removal and addition rates are defined for the population as a whole, and the basis is US Census data. To meet the specifications for removal and addition within the model, hosts are removed from randomly chosen locations, and similarly added to randomly chosen locations. Demographic group is also randomly assigned. For long-term modeling, and modeling of alternative short-lived hosts, a reproduction cycle is desirable. However, EpiFlex is a practical way of modeling periods of a few years.
There is no explicit definition of an age distribution for the host population, which can be quite significant [29] . To a degree, age is taken into account through the demographic segmentation of populations. A demographic can be defined with a fraction or multiple of baseline susceptibility. However, hosts do not age, nor do they move from one demographic to another as they age.
No provision is made to define a previous exposure profile for hosts [30, 31] . In real populations previous exposures can have significant effects on the spread of a disease and dramatic effects on mortality where infection does occur [32] . Proper implementation of previous exposure profiles is intertwined with age definition.
There is no implementation of mutation rate for diseases. Mutation rates vary considerably by type, particularly for viruses [33] . Decay of immunity is modeled, and immunity decay can act as a fair surrogate for antigenic change.
Pass-through events must be defined as part of surface contacts For efficiency, EpiFlex eliminates pass-through infection events from being modeled: for example, an infected A shakes the hand of a non-infected B, who then shakes the hand of another non-infected C, but B washes hands and does not become infected while C does. Therefore, the model definition must account for this through "Surface to hand to mucosa" contacts, where a person can also be a surface.
The model does not at this time record the contact network that is dynamically created except in the log file at this time. Those that are logged are only potential infectious contacts. To get at that data requires looking at the log file and writing an extract program. Making the network visible is an item for the future.
Currently, EpiFlex has no way of accounting for seasonal damping. Similarity of results is due to the settings of the rate at which immunity declines. Addition of a seasonal damping function would be expected to cause EpiFlex results to synchronize with a yearly cycle. Seasonal damping would result in loss of interesting epidemic behavior with an overriding function that would virtually be guaranteed to drown out other behaviors.
Public health response to epidemics is not optimally modeled A public health response definition component is present in EpiFlex. Testing of this component, and more thorough review of literature, indicate that the method used is not optimal. Current public health responses are centered on contact tracing, ring vaccination and quarantine [34] , with mass vaccination as a backup when it is available. Closures of schools, daycare and travel restrictions are also used. These methods are not modeled in EpiFlex' response component. Their importance has recently been underscored by Lloyd-Smith et al. [17] . As a consequence, results from the current system that defines across-theboard cuts in the probability of infection should be considered in this light. It is not clear whether any other object system can model all current techniques properly.
Diseases have ranges of times for each stage that can be drawn from literature. Probability of a specific disease stage time period for an infected host being chosen within the range is equal. This is reasonably adequate for most diseases where times are measured in a few days, however, some, such as rabies have a quite unequal distribution, and their very long tail makes a difference in modeling.
The discussion is presented in three parts: (1) a brief set of examples of native EpiFlex displays to develop a better feel for the system; (2) comparisons of EpiFlex results with real world data; (3) a set of examples of observations made using EpiFlex. The purpose of these examples is to serve as a guide to others who may want to experiment and analyze results.
Different views of the epidemic data for simulated influenza in two different populations are shown in Figure 2 . Figures 2a and 2b show graphs of the second and third epidemics in the population. These graphs show the kinds of commonly-seen deviations from a smooth curve that occur in real world data [35] . In the EpiFlex model, this is attributed to less synchronization of immunity combined with the formation of small world networks among demographic groups as they move from location to location. Figures 2c and 2d are alternate views of a simple influenza epidemic occurring within a naïve population (Figure 3 ).
Comparison of EpiFlex with WHO/NREVSS surveillance Comparing EpiFlex with surveillance data, we see that WHO/NREVSS surveillance data [36] have a qualitatively similar graph form to EpiFlex for influenza, as shown in Figures 4 and 5. The width of the primary curves per season for EpiFlex is 3.5 to 4.5 months while that of the NREVSS data is approximately 5 to 7 months, which can be explained by the NREVSS data being collected nationally from surveillance centers, whereas the EpiFlex data shown are for a single area. EpiFlex runs executed with multiple cities connected by transport, such as the 3.5 million population 35 city model, have a combined graph for all cities showing self-similarity to the graphs for individual areas, becoming wider, matching the NREVSS data graph formation.
The NREVSS data consist of diagnostics of samples sent in by physicians. Comparisons of absolute numbers in terms of quantity are therefore not applicable. A percentage of population comparison is done below.
In Table 1 , EpiFlex indicates that roughly 48% of the population has been infected before herd immunity stops the epidemic, though this depends on population size. Total morbidity is obtainable from EpiFlex by adding maximum immune level to deaths, although deaths contribute such a small amount to influenza morbidity that for practical purposes the immune level is used as a proxy for morbidity. Moreover, true morbidity itself is relatively prone to inaccuracy, whereas better measures of immune fractions for influenza are available. The California state average for 2000-2003 is 25.4% infected in a range from 12.7 to 44.6 depending on county [37] . Thus, EpiFlex is above the high end of the state of California estimated morbidity range.
Dowdle [32] gives serological influenza data categorized by age. For influenza A/Swine/15/30 H1, seroprevalence ranges from roughly 25% to over 95%. For influenza A/ Hong Kong/68 H3, the range is from 5% to 99%. EpiFlex figures fall within this latter pair of ranges, and EpiFlex immune fraction is more properly comparable. 
What is most notable in Figure 7 is the relationship between the rough sine wave form of the classically derived SIRP [19] once the initial startup period is over for influenza, a repeating wave develops that is similar in overall shape and variability to real world data such as those for Milwaukee, at a roughly similar scale. These two graphs refer to populations that differ in size by about 1 order of magnitude (i.e. Milwaukee is 9 times the size of the model run shown). We can also see a similar number of peaks. Owing to the need to compare these two graphs natively, these two figures are not optimum. However, they show essential features.
Total morbidity rate linked to population size The smaller a population over the range 1,000 to ~3.5 million, the higher the total morbidity rate, given identical organisms ( Figure 6 ). It is intuitively expected that population size will affect morbidity since, for any given network of contacts connecting individuals in populations, the chance of the epidemic spreading during the window prior to the development of immunity in parts of the population increases as the population size decreases. This This is the simulation that was imported for comparison Figure 3 This is the simulation that was imported for comparison. Vertical scale 1000 per demarcation. Horizontal scale one year per demarcation. Upper white line is total population with standard removal rate. effect is most striking when very small populations in the order of 1,000 are examined. Literature data regarding this in real world populations are sparse. However, there are indications from historical accounts of small populations in the new world that a link between population size and morbidity is observable in real world populations [38] [39] [40] [41] [42] [43] . The most recent such account is from Heyerdahl in the Pacific in the mid 20 th century [44] .
In the graphs of Figure 6 , the immune fraction at completion is used as a proxy for total morbidity on a log scale of population. The longer an epidemic takes to progress within an enclosed population, the greater the number of potentially infectious contacts that hit a dead end because the host is already immune. Since very small populations will mostly function within the window when there is no host immunity, the infection will spread to a larger fraction. This effect has public health implications because, clearly, the structure of the network is highly significant in determining the likelihood that an infected host will contact naïve hosts. Essentially what this EpiFlex result indi-cates is that during the period prior to the development of an immune subpopulation, a disease has a functionally higher R 0 . (i.e. R 0 is variable through the course of an epidemic.)
In the Figure 6 graphs, EpiFlex is also suggesting that there are more asymptomatic infected spreaders of influenza in our populations than surveillance data estimate. This is also suggested by the discussion above regarding comparison with seroprevalence.
A minor experimental result is that for a repeating illness such as influenza, when a continuously active initiating disease vector tries to infect 3 people per day, it will develop higher peaks after the initial event than a vector that tries to infect 30 people a day (where both are randomly distributed through the population.) This makes intuitive sense, because there is lower probability that a subpopulation of susceptible hosts will become large when there are more attempts to infect them. Similarly, in a system of cities interconnected by transport linkages, later peaks tend to be smaller and more variable than earlier ones. This is due to two things. First, a degree of lowgrade infection linking back through the system provides a higher total level of infection events in the whole system than the formally defined initiating disease vector. Second, as time passes, the mix of immune versus susceptible becomes unsynchronized for the population as a whole, since hosts that escaped infection during one epidemic may be infected during the next, and some whose immunity has declined may also become infected. Thus, it is expected that we would see the development of a complex non-repeating waveform with some similarity. This type of waveform is what EpiFlex shows with longer simulations in large populations, as illustrated in Figure 8 .
A variety of results is obtained when one or just a few index cases are provided to seed a single city's susceptible population when those index cases are not repeated. This is expected, owing to random interactions that break the chain of contacts in some percentage of cases. This effect would be expected to increase with a higher skew on super-spreaders. The significance of this for modeled epidemics, particularly in the light of recent work [17] , is that in some cases (the proportion would be expected to vary in rough accordance with R 0 ) the infection dies out owing to random chance. Thus, a Monte-Carlo model such as EpiFlex, when used in multiple trials, clearly reveals the potential range of variation in epidemics given apparently identical conditions.
EpiFlex shows wave propagation of epidemics through its transport network that are similar to real world epidemic studies such as those of Viboud et al. [45] . Viboud et al. state a mean duration of 5.2 weeks to spread across the United States, with a range from 2.7 to 8.4 weeks. The Epi-Flex results shown using a simplified city configuration of 35 major airline hub cities, with a total 3.5 million population among all 35 cities, shows a propagation wave of 1.8 weeks. While this is shorter than real world data, several factors account for the difference. First, in the current EpiFlex "vanilla" configuration, the transport network is flat in terms of the numbers of persons moved from city to city. Second, each city contains the same population of only 100,000 due to practical limitations. For the propagation histogram of Figure 9a , 1000 manifesting cases or more was used as the data point. Please note, however, this flatness of transport and population is purely a matter of the configuration of the specific model used. The Epi-Flex system allows separate specification of all parameters for each city, and any kind of transport level between any two cities that is desired. Figure 9a shows a histogram of cities in which 1,000 manifesting cases are first occurring. Figure 9b shows a histogram of cities in which the first occurrences of at least 10 incubating cases of influenza appear. Note that in Figure   Upper graph shows graphed points for population versus total morbidity as estimated from immunity Figure 6 Upper graph shows graphed points for population versus total morbidity as estimated from immunity. Lower two graphs show sample graphs for 3500 (lower left) and 35000 (lower right). The green line in the lower two graphs shows immunity. One vertical demarcation on the x axis is one month in the lower two graphs. 
EpiFlex is useful for doing in-silico experiments with epidemic behavior and easy to configure. It can run on an ordinary computer without special configuration. Data can be imported from it into other tools and worked with there. It is effective for showing factors that are invisible or difficult to access under normal conditions, such as visibility of incubation and prodromal stages, true morbidity and estimates of immunity. EpiFlex has capabilities that were not discussed owing to time and space constraints. This system is effective in duplicating the kind of multimodality and apparent stochastic variation [13] that are seen in real populations. EpiFlex results can provoke thought about the nature of epidemics and infectious disease spread in interesting ways by providing an experimental test environment that is not as abstracted from its subject as most mathematical models are.
Items in bold below are entities that are objects in the Epi-Flex system. They are the "nouns", each of which became a class when the program was written in C++. Items in italics are actions, the "verbs" that represent interactions between objects.
Hosts go through the infectious stages, are members of Demographic groups and move between locations
Locations contain hosts (For example a hospital, home or workplace.)
Areas contain locations (A city, typically) and contain special locations that can move hosts to locations in other areas.
Disease Vectors introduce disease into host populations at some location in some area, in either a limited way or on a regular cycle.
Epidemic responses modify the spread of an infection. They represent actions taken to respond to the epidemic that will damp the spread. 
SIRP is a simple flow chart, which is then elaborated into a multi-city system. Three graphics are reproduced in Figure 10 concluding with output of the model, because the SIRP paper is in a specialty publication and the work was important to the conception of EpiFlex.
As seen in the "SIRP diagram" of Figure 10 , a host begins as susceptible (S) in the upper left. They can become infected (I), and then they either recover (R) or are removed (removal not shown). After recovery (R), they have an immunity level that starts at or near complete as in SIR. This immunity declines, in the case of influenza, over a period of roughly 2 years, with a steep drop-off beginning after the first year, and hence is termed partial immunity(P). (It is not actually the case that the immune system's response to influenza antigens degrades so rapidly; rather, the virus mutates rapidly, and host immunity declines. The net result is similar.) This decline in immunity models the real world antigenic mutation of influ-enza viruses. In a more perfect model, the influenza virus would change and multiple viruses would be modeled. At some point the immunity declines so that it is effectively zero in the model (which corresponds to large antigenic change) and the person is returned to the fully susceptible(S) population. A person can become reinfected (diagonal arrow), though their probability of reinfection is lower, during this period of declining immunity.
In the "Multi-city SIRP" flows of Figure 10 center, m [x,y] denote movement between the cities in a four city example. Moving further to the right of Figure 10 and looking at the "Actual versus model graph", one can see how a sinusoid curve, with some general correlation with period, results from the conventional model.
There are four files used or generated by EpiFlex. All three generated files have the name of the configuration from A 5.5 year, 9 city simulation linked by transport corridors. 100,000 people per city Comparison of distribution of days when 1,000 manifesting cases or more first appear in a city (upper graph -9a) versus distri-bution of days when 10 or more incubating cases first appear in a city (lower graph -9b) Figure 9 Comparison of distribution of days when 1,000 manifesting cases or more first appear in a city (upper graph -9a) versus distribution of days when 10 or more incubating cases first appear in a city (lower graph -9b). 
Model files end in .EPDM. These files are an XML format. This is the file edited by Epiflex using the various panels available from the menu. An EPDM file can also be edited directly in WordPad or a good XML editor by more sophisticated users. Reading through them should be selfexplanatory to most users who familiarize themselves with the software.
Run record files end in .RPX. These files are in text format, comma, and semicolon delimited. This format is intended to be as easy to import as possible. RPX files can be imported into Excel, read by SAS, SPSS, etcetera. At the top of this file is a descriptor of the fields. The most common problem is to import using spaces as a delimiter and have spaces in area names define new columns.
Log files end in .LOG. Everything that happens in a model is logged. These files can get fairly large. Potentially, they could be parsed by a secondary piece of software to match them up with RPX files. However, that is an exercise for the user at this time. There is a viewer for log files under the View menu.
Snapshot files end in .SNAP. Whenever a run completes, a SNAP file is written that is read only. This file is a duplicate of the model that was used to run -as that model existed in memory at completion of the run. This is done as a "lab notebook" aid because it was recognized early on that remembering exactly what was contained in a model was well nigh impossible and a lot of work to document.
Note that if your model does not complete its run, the RPX file will still be there, but the SNAP file will not be. In that case it is up to the experimenter to make a record of the state of the model.
The SNAP file can be copied outside of Epiflex, manually changed from read-only and the extension renamed to .EPDM. The resulting model can then be used and run just like any other.
Software engineering details such as internal class definitions and structure of components will not be presented since the UI does a better job of educating, and is much more compact.
Disease component Looking at screen shots of the current UI in Figure 11 , one can see what the functional parametric elements of a disease specification are. This particular example is selected for influenza. The parameters set reproduce the known characteristics of the disease.
The disease stages of Figure 11 correspond to the model diagram shown in Figure 1 . One can see that this definition is superior in several ways to what is available with SIRP modeling as shown in the previous section. (See Figure 10 ). In addition to multiple stages, one can define the level of infectiousness for each type of contact that an infected host might have. On the lower right, the partial immunity stage is specified. Here, one is able to define a wide variety of immune responses. Asymptotic decline seems to fit available data and theory on immune system (from left) SIRP Diagram, Multi-city SIRP, Actual versus model Figure 10 (from left) SIRP Diagram, Multi-city SIRP, Actual versus model.
function the best for many diseases. Use of the slider allows one to "eyeball" the asymptotic curve to approximate what seems reasonable. In this case, immunity is set to begin dropping after approximately half the 730 day period has passed. It makes sense to do this because data in this area are fairly sparse, and one may want to perform multiple runs with slightly different immune drop characteristics. Keep in mind that the width of the graph is for the number of days entered on this panel.
A host is implicitly one of the number in the population of an area. In the EpiFlex system, there is one object defining each disease. A host infected by a disease receives a pointer to this archetypal disease object, and stores their disease stages internally as the disease progresses. Most of the functionality of hosts consists of an implementation of disease stage transition. Figure 12 is a screen shot of the UI for a set of demographic definitions selected from US Census data for the nation as a whole. In the resulting model, one can over-ride the default fractions later when using them in populations for specific areas. No fractions are represented that could not be derived from recent census data.
Each demographic group can be set up to specify variations in overall susceptibility to disease, or modify it by specific type of contact if that is desired. In the example, no fractional modifiers have been created for this demographic group. There is no necessity to stick to the demographic groups defined here. One can add new ones as desired.
A location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. A location can have a certain number of cells, which are used to specify N identically behaving locations concurrently. This acts as a location repetition count within an area when the location is defined.
A technical point with homes as cells is that the model cannot yet be set to create a number of homes based on population size with a distribution of household sizes. Figure 11 Disease definition panel.
Consequently, one must, at present, define the number of home cells appropriate to hold the population for each area. If a distribution function were available to specify household sizes and households were definable by population size, it would then be practical to provide canned profiles of household sizes based on US census data.
The types of contacts that are available among hosts in the location are defined, together with the average number of such contacts per host during one cycle of the model; see Figure 13 . To the right of the contact definitions is a skew function that enables one to specify the degree to which contacts vary. Within any host subpopulation, there are high contact individuals and lower contact individuals. The example shown sets the difference rather high to describe the way that service workers interact more strongly than customers. When running the simulation, during determination of which hosts become infected in this cycle, the position of the host in the queue is used together with the contact frequency to decide how many contacts an individual will get in a particular cycle. Think of this adjustable skew graph as the histogram of contact counts.
For each demographic group, a cycle of movement between locations in an area is defined as illustrated in Figure 14 . In the model used as an example, days are divided into three cycles. So a sequence of 21 movements defines a 7 day week. Once a cycle completes, it restarts at the top. This is how hosts in the model are moved from one location to the next.
An area is made up of a list of locations that it contains, as shown in Figure 15 . An area specifies a particular population level, and the population fractions for each demographic group making up the area are allocated at runtime. Demographic population, removals and additions can be overridden at the area level.
An area also has linkages to other areas, through special locations that are eligible as links. In the example of Fig- Group demographic population definition panel Figure 12 Group demographic population definition panel.
ure 16, the Anchorage airport is going to send 2% of its population to Chicago, Illinois, each cycle. In each cycle, 70% of the population of the Anchorage airport location is going to be shipped off somewhere.
A 'Vector' is defined in EpiFlex as n infection events to attempt at a specific location. A vector can repeat each cycle, or have a limited input. A vector can represent an arthropod or snail, or it can represent people arriving on aircraft from Hong Kong. The vector shown in Figure 17 is a startup vector for influenza used for these modeling runs. This initiating disease vector will operate for the first 3 cycles, then stop, attempting to produce 9 cases at a location in Chicago. An initiation disease vector can have delay parameters, run continuously, or run once. It can force the infection of a specific number of people or infect them at a particular rate.
When a disease occurs, the medical system will respond to it in some way. Some illnesses, such as influenza A, will result in very little intervention. Others, such as SARS or smallpox, would result in a great deal of intervention very rapidly. Consequently, a reasonable modeling system will allow response to an epidemic occurrence to be modeled.
In the example shown in Figure 18 , we see the response specification for an emergent illness. A response has an alert trigger, which in this case is the appearance of fatalities. This results in a period of heightened awareness of the disease and its symptoms, which is set in this example to 100 days. The probability of noticing the triggering event is set, to unity in this example, which may be overly optimistic. This specification says that after three fatalities, an alert would be triggered. Since this alert trigger is for fatalities, not symptoms or instruments, the detection fraction for each disease stage is not applicable.
Once the alert has been triggered, the detection method for the illness switches primarily to symptoms. We can then specify what fraction of each occurrence will be diagnosed and at what stage. If we detect an event, then we can mitigate it. In this implementation, one has to specify the degree of infectiousness remaining after intervention is in process. Some methods, for some diseases, can stop disease spread completely. This is not true for all diseases, so it must be specifiable. Figure 13 Location definition panel.
Note that this component is the one with which the author is least comfortable. None of the examples for this paper have used responses as a consequence. The primary reason is that the way response is modeled is over-simplified and does not conform well to the response that happens in the real world; see: Limitations of EpiFlex modeling in the introduction.
EpiFlex is now composed of 83 major classes, of which 45 are core internal model functionality, the rest being infrastructure and UI. A functional walkthrough of how the system operates is presented below.
A model starts running by verifying the model data. It looks for any references to things that were deleted and definitions that are impossible. It writes an error log file with this information that can be audited after the run. If errors are found, you will be informed of their severity and given the option to cancel. Many of these errors are non-fatal, and may be modified with a warning, but they may change the results of a run.
The EpiFlex system iterates through all areas in a model and allocates hosts, putting them in their initial locations, per the movement definitions for the demographic group.
Each group steps through its location movement list to determine whether the area to which it is attached has the locations the group needs. If it does not, EpiFlex will increment the pointer when it comes to this location, but it will leave the hosts in their previous real location until a good one is found. This was done for ease of practical use, allowing the same demographic to be used when a location may not exist in an area.
Step 1 -disease stage pass. For each infected host, it will update the stage of the illness for that host. This is also where the system checks to see if disease response conditions have been met yet.
Step 2 -vector pass. EpiFlex will iterate through each vector and apply the disease to the locations in each area in the vector according to the rules defined.
Step 3 -infection pass. Iterate through each location in each area, and figure out the contacts between infected and uninfected hosts. For each contact between an Group movement cycle definition panel Figure 14 Group movement cycle definition panel.
infected host and a host not infected with the disease, a probabilistic determination will decide whether or not this illness is communicated.
Step 4 -iterate areas looking for locations that have group draw factors. Randomly pull hosts from the groups specified, and move them to the group draw location. This feature is used to simulate various things; generally an airport will have this type of specification.
Step 5 -iterate all areas looking at area links. Area links push population from one location to another area's location as specified.
Step 6 -Areas will be iterated, and each location in each area will be iterated using the random exchange factor to move hosts assigned to cells of a location between cells in that location.
Step 7 -Normal addition and removal of population groups is applied to model. This allows the user to model normal birth and death rate plus immigration and emigration across the outer boundary of the model.
Step 8 -iterate all areas, and iterate through each location in each area. For each location, it will iterate each host attached to the location. Each host has a location pointer for its group that indicates where it is in its movement cycle. It increments the pointer, and if the location name is different from the one it is in, it will find a location of that name in the area to put itself into. If the location has N cells, it will put itself into the cells in a semi-randomized fashion according to the parameters defined. Note that currently the hosts do not remember which cell they are put into from one iteration to another. So they will not return to the same cell next time they come back around in their movement cycle. This is a limitation at present, which would require greatly increased space allocations to change.
Step 9 -An audit is done to verify that all hosts occur only once in a location.
Step 10 -Areas are iterated, locations are iterated, to total results. Results are then either written to the log file and/ or displayed on the main window in a graph form depending on how parameters were specified. Figure 15 Area definition panel. Area linkages panel Figure 16 Area linkages panel.
Initiating disease vector definition panel Figure 17 Initiating disease vector definition panel. Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet BACKGROUND: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. METHODS: MultiCode(®) PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in ~3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. RESULTS: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. CONCLUSIONS: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting. Few multiplexed technologies with sufficient specificity to identify small changes within the human genome are available for clinical use. Line probe or linear array technology uses nitrocellulose paper strips as the support matrix (1 ) . A benefit of the line probe is that expensive instrumentation is not required. A technology termed "oligonucleotide ligation assay" uses target-specific ligation to generate mutationspecific products that are analyzed by gel electrophoresis (2 ) . Other genetic analysis systems are coupled to microparticle flow cytometry (3) (4) (5) (6) (7) (8) . Limitations of these methods include complicated procedures that require extensive training (washings, centrifugations, and transfers), long completion times, expensive instrumentation, and technician-dependent result analysis. In addition, these technologies are not easily amenable to automation. Many hospitals and clinics currently send molecular diagnostics tests to large reference laboratories. This is in part attributable to the limitations described. Because there are benefits in having local laboratories perform diagnostic tests (9 -12 ) , there appears to be a need for molecular testing methods that circumvent the limitations posed by current testing methods.
We have constructed a three-step platform technology called MultiCode ® PLx that eliminates the most technically challenging steps involved in multiplexed genetic analysis and compared it with the line probe assay in a clinical setting. MultiCode PLx uses one additional nucleobase pair constructed from the complementary nucleobases 2Ј-deoxy-isoguanosine (diG) 5 and 5-Me-iso-cytosine (iC). These nucleobases specifically recognize each other based on a different pattern of hydrogen bondings. We chose this pair because no other such pair is available commercially and their chemistries have been well explored. For example, iG and iC have been successfully used for both molecular recognition (13) (14) (15) and for site-specific incorporation (16 -18 ) . In addition, because iG:iC recognition is "orthogonal" to the naturally occurring nucleobase pairs, a strand of DNA that contains several iC/iG components can be constructed so that it will not hybridize to natural DNA. In complex reactions in which high concentrations of natural DNAs of known or unknown sequence exist, this orthogonal attribute allows specific molecular recognition to take place without interference. These additional nucleobases are used in each step of the PLx process: PCR, extension labeling, and liquid decoding. PCR primers are first designed to be target-specific and contain single iCs. The amplicons act as labeling templates for the target-specific extension (TSE) step. During that step, labels attached to diG triphosphate (diGTP) are incorporated site specifically into coded target-specific extenders (Fig. 1 ). The tags are short sequences (typically 10 nucleotides in length) assembled by use of a mixture of natural and nonnatural bases. The tags are designed to hybridize only to their perfect complements encoded on color-addressed microspheres. In the final step, decoding of the extension reactions is accomplished at room temperature by capture of the coded extenders on the addressed microspheres and reading of the reporter signal on a Luminex 100 instrument. All steps are carried out in the same reaction vessel without transfers or washings.
To demonstrate the approach, we developed a screening test for carriers of cystic fibrosis (CF) that tests for all 25 mutations and the reflex targets in the widely used panel (19 ) and also tests for 394delTT and 3199del6. 394delTT is the second most prevalent mutation in populations with Nordic heritage and may be important in Wisconsin, where testing was performed. 3199del6 was added because several lines of evidence indicate that 3199del6 is a disease-causing mutation and may replace I148T (20 ) .
The Wisconsin State Laboratory of Hygiene, in conjunction with the University of Wisconsin, was chosen as the major testing site because of its long history of screening newborns for CF (21 ) and study of screening methods (22) (23) (24) (25) . In March 2002, DNA testing of the top 4% of the daily immunoreactive trypsinogen results expanded to include all 25 mutations recommended by the American College of Medical Genetics, the American College of Obstetricians and Gynecologists, and the NIH.
In this report, we first present MultiCode PLx testing data on a variety of genomic DNAs with various genotypes prepared from several sample types for the presence or absence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Finally, data are presented on a prospective study comparing testing results obtained for freshly prepared blood spots by the PLx assay and the commercially available CF Gold Linear Array TM system (Roche Molecular). ) . DNA was extracted from whole blood and dried blood spots on Guthrie cards. The dried blood spots were stored at room temperature for 2-3 days after receipt until routine testing was completed. For long-term storage, dried blood spots were stored at 4°C. DNA extraction from Guthrie cards was performed with the Generation Capture Card Kit (Gentra Systems) according to the manufacturer's guidelines for DNA purification and elution. DNA was extracted from whole blood by use of the Puregene DNA Isolation Kit (Gentra Systems) or the Whatman FTA system (Whatman Inc., Clifton, NJ). All genomic DNAs from these preparations were diluted 1:10 in water, and 1L was added to each PCR reaction (1-50 ng of total DNA). As a cross-reference and in the prospective study, genotypes were also determined by CF Gold linear array or DNA sequencing if discrepancies were detected.
PCR and TSE oligonucleotides were manufactured inhouse with a 48-column DNA Synthesizer (Northwest Engineering; now the 3900 from Applied Biosystems Inc.), using standard ␤-cyanoethyl phosphoramidite chemistry (for sequence information, see Tables 1, 2, and 3 in the Data Supplement accompanying the online version of this article at http://www.clinchem.org/content/vol50/ issue11/). Synthesis of sequences that contain iC and iG can also be purchased from IDT, Eurogentec, or EraGen. A demonstration assay encompassing this specific system can be purchased from EraGen Biosciences. Amino-labeled EraCode oligonucleotides were manufactured with an ABI 394 DNA Synthesizer (Applied Biosystems) using 3Ј-PT-Amino-Modifier C6 CPG (Glen Research). Target-specific extender primers were manufactured with the spacer phosphoramidite C3 (Glen Research) placed between the EraCode complement and the target-specific region. The isoG and isoC phosphora-
, respectively] were coupled and deprotected under the conditions used for the natural base phosphoramidites. Postsynthesis work-up consisted of ammonium hydroxide deprotection followed by ethanol precipitation (PCR and TSE oligonucleotides) or 2-propanol precipitation (EraCode oligonucleotides).
EraCode bead coupling For each conjugation reaction, a mixture (400 L) containing ϳ5 ϫ 10 6 carboxylated microspheres (Luminex Corporation) was centrifuged at 8000g for 1 min. The supernatant was removed, and the beads were resuspended in 50 L of 0.1 mol/L MES, pH 4.5. After resuspension, 3.3 L of 300 mol/L 5Ј-amine-modified EraCode DNA oligonucleotide and 5.0 L of freshly prepared 10 g/L EDC (Pierce Chemical) were added. The reaction was mixed thoroughly and incubated at room temperature in the dark for 30 min. A second 5.0 L of 10 g/L EDC was added, and the mixture was incubated for 30 min. Era-Code microspheres were washed by the addition of 1.0 mL of 10 mmol/L Tris (pH 8.0) containing 0.2 mL/L Tween 20 and pelleted by centrifugation at 8000g for 1 min; the supernatant was then removed. EraCode microspheres were washed a second time with 1 g/L sodium dodecyl sulfate in 10 mmol/L Tris (pH 8.0) and resuspended in 100 L of bead buffer (10 mmol/L MOPS, pH 7.5; 1 mmol/L EDTA, 0.5 g/L sodium dodecyl sulfate, 200 mmol/L sodium chloride, and 0.1 g/L sonicated herring sperm DNA). EraCoded microspheres were combined and diluted in bead buffer to generate a 50-code mixture at a concentration of 800 microspheres/L for each class of microsphere.
Capture at room temperature of a tag to its specific complementary EraCode was tested by use of a model extension system where one target with a 5Ј-iC was constructed and 50 tags were interchanged onto a targetspecific extender sequence. EraCodes were constructed by use of 9 -10 bases comprising 2-3 iCs. In an effort to minimize interference from naturally occurring sequences, EraCodes were designed to have no more than four naturally occurring bases in a row. In an effort to minimize code-to-code cross-hybridization, no two codes were allowed to contain the same series of more than four nucleobases in a row.
Fifty EraCodes were validated by use of 50 extenders, all containing the identical 3Ј target-specific sequence. The extenders were individually labeled using the target, standard concentrations of all four naturally occurring deoxynucleotide triphosphates, TiTaq, and diGTP-biotin at 1.5 mol/L. Each EraCode sequence was separately coupled to a specific color-coded fluorescent microsphere type (Luminex Corp). Each microsphere type contains a unique color or signature that can be determined by the Luminex 100 . After coupling, each EraCode bead conjugate was pooled to create an equalized bead master mixture (bead array: 50 different EraCode-labeled beads). The bead array was distributed into 50 separate reaction vessels. To each of the vessels, a specific extension reaction from above was added at room temperature along with streptavidin-phycoerythrin and was read on the Luminex 100 .
In a 10-L extension reaction, an excess of a template oligonucleotide (5Ј-iC-CGTATGGGCTCACCTCGCTGTG-3Ј) at 150 nmol/L was combined with a control extension sequence at 100 nmol/L in EraGen MC TSE solution 35 (prod. no. PN1236; EraGen Biosciences, Inc.) with 100 mol/L each deoxynucleotide triphosphate, 1.5 mol/L diGTP-biotin, and 1ϫ Titanium Taq (Clontech). Each control extension oligonucleotide was composed of a 5Ј sequence complementary to each of the EraCode sequences, a three-carbon spacer, and a 3Ј sequence complementary to the template oligonucleotide (5Ј-CACAGC-GAGGTGAGCCCA-3Ј). Reactions were subjected to the following thermal cycling protocol: 30 s at 95°C and 1 min at 65°C. Each 10-L extension reaction was then hybridized to the EraCode bead mixture according to the described hybridization protocol. In addition, to mimic MultiCode conditions, unextended TSEs to the other EraCodes were also added.
Hybridization of extension products in each 10-L reaction was performed by mixing 40 L of EraGen MC Hyb Solution A (prod. no. PN1237; EraGen Biosciences) and 0.25 L of EraCoded bead mixture. To each hybridization reaction, 0.5 L of 2 g/L streptavidin-R-phycoerythrin conjugate (Prozyme) plus 39.5 L of sheath fluid (Luminex Corp.) were added, and 65 L of the final reaction was injected into the Luminex 100 . pcr PCR primers were designed using Primer 3 (http:// www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi) or Hyther (http://ozone2.chem.wayne.edu). Primer pairs were designed to flank the polymorphism(s) of interest and have a melting temperature of 53-57°C (see Table 2 in the online Data Supplement). A single iC was added to the 5Ј end of the PCR primers.
PCR reactions were performed in a final volume of 8.0 L; each reaction contained EraGen MC PCR solution 35 (prod. no. PN1235 or PN1305 for the 579T system; Clinical Chemistry 50, No. 11, 2004 EraGen), 0.4 L of 20ϫ EraGen CFTR PCR primer mixture, 1ϫ Titanium Taq, and 1 L of diluted genomic target DNA. Reactions were cycled according to the following profile: 1 cycle of 30 s at 95°C, and 40 cycles of 1 s at 95°C, 10 s at 55°C, and 30 s at 72°C. Each of the primer mixtures contained forward and reverse primers for a subset of the mutations at concentrations of 100 -200 nM.
tse TSE was used to determine the exact mutations present. TSE requires TiTaq, diGTP-biotin, and extenders that contain 3Ј target-specific bases and 5Ј codes specific for the EraCode beads described above. During TSE, TiTaq incorporates labels on extenders only when specific targets having 5Ј iCs are present. The nonnatural base pair formed by iG and iC has been successfully used for both molecular recognition (13) (14) (15) and for site-specific enzymatic incorporation (16 -18 ) . To date, we have covalently coupled a series of different reporter groups to diGTP, using succinimidyl ester chemistry and confirmed enzymatic incorporation into nucleic acids by use of several commercially available polymerases (data beyond the scope of this report). In many cases, single amplicons spanning regions that incorporated multiple mutations were used as templates for multiple extenders. After TSE, EraCode-labeled beads and streptavidin-phycoerythrin were added, and the reactions were analyzed on the Luminex 100 . Data are reported in median fluorescence intensity (MFI).
For each polymorphism, pairs of TSE oligonucleotide primers were designed by use of Primer 3 and Hyther (see Table 3 in the online Data Supplement). Pairs were designed to have balanced melting temperatures ranging from 63 to 67°C. Each TSE oligonucleotide was composed of three components: a 5Ј sequence tag complementary to one of the EraCodes; a three-carbon spacer; and a 3Ј target-specific sequence fully complementary to the allele of interest. The polymorphic sequence for each pair of TSE oligonucleotides was the 3Ј base.
Extension reactions were performed by bringing the 8-L PCR reactions to 10 L with addition of 2.0 L of EraGen MC TSE solution 35 (prod. no. PN1236; EraGen Biosciences) including primers for the 579T system (prod. no. PN1310), and 0.4 L of 20ϫ EraGen CFTR TSE primer mixture; final concentrations of TSEs were 25-100 nmol/L (see Table 3 in the online Data Supplement). Reactions were cycled according to the following profile: 1 cycle of 30 s at 95°C; 5 cycles of 1 s at 95°C and 2 min at 65°C.
For this report, an assignment is defined as a genotype for a given target within the CFTR gene made for any given sample. Each sample tested will have 30 possible assignments because 30 targets for each sample are analyzed. To rapidly analyze the data obtained, we developed software to present the raw data from the instrument. The software directly imports the Luminex 100 data and organizes them by target mutation. Fractions of wild-type to wild-typeplus-mutant and raw MFI signals can be visualized graphically (Fig. 2) . The program sets default fraction windows at 1.0 -0.8 for wild-type samples, 0.6 -0.4 for heterozygous samples, and 0.0 -0.2 for homozygous mutant samples. Default windows can be changed by the end user. The area between the windows is designated as the no-call window. The program also sets no-amplification windows where wild-type and mutant MFIs are Ͻ200.
Template set-up within the Luminex LabMap software was required, and importation to analysis software was achieved by dragging the Luminex file folder into the Analysis Software. Data files were parsed, and the resulting raw MFI values were organized by target and sample. After data acquisition from all 225 clinical samples tested, default cutoff windows for each target were empirically determined and set in a blinded fashion. Once determinations were made, reports were generated for offline analysis. The analysis software was written in Java and is compatible with Windows, Mac OS X, and Linux operating systems. Data importation is achievable with Luminex LabMap 100 (software versions 1.7, 2.1, and 2.2) and Bio-Rad BioPlex 3.0. The software displays scatter plots and fraction plots of each given target for all samples.
automation Automation was performed on a Tecan Genesis Model 200 robotic workstation (TRP) starting from either the PCR amplicon formation or from isolated genomic DNA and continuing through data analysis. Samples used were a subset of the 225 used in the initial manual testing. Instructions for the automation program were written with Gemini software (Tecan). Initialization and launch of the bead-reading protocol on the Luminex 100 was performed automatically by use of a custom image-recognition and command-scripting program written in Java. After the read, the results were remotely analyzed by MultiCode Analysis software. Genomic DNA was purified manually before automation as described above. The TRP was fitted with the low-volume eight-channel dispense pipette system, TeStack, to supply disposable tips, a MJ Research thermocycler equipped with remote Alpha docks with Power Bonnet, and a robotic manipulator arm. Liquid reagents and disposable tips were also placed on the deck before runs were started. Reactions and additions for the automated process were the same as those described for the manual method unless noted. Reactions were performed in low-profile hard-skirted polypropylene well microplates, which allowed the robotic arm to grip and move the plates as needed. Each reaction was overlaid with 15 L of light mineral oil (Sigma) to prevent evaporation during thermal cycling. The robotic manipulator was used to transfer the plates to and from the thermocycler block and the Luminex 100 mounted off deck. Initialization and launch of the bead-reading protocol on the Luminex 100 were performed automatically by use of a custom image-recognition and command-scripting pro-gram written in Java. After bead-reading of each reaction, the results were analyzed by the MultiCode Analysis software as described above.
A schematic diagram of the MultiCode system is shown in Fig. 3 . The MultiCode process has three steps. For the first step, regions of the target of interest are amplified by PCR using primers that contain a single 5Ј-iC. In the second step, the amplicons from the first step are used as templates for the TSEs, and iC directs specific diGTP-biotin incorporation. The TSEs are coded on their 5Ј ends with sequences that specifically recognized short complementary sequences (EraCodes) attached to an addressed solid matrix. The molecular recognition of any given code with its EraCode is specific enough so that increased temperatures, incubation steps, or washings are not required (typical requirements for hybridization capture using only naturally occurring base chemistries). During this second step, labels are placed site specifically on the TSEs. This process requires the presence of iC-containing amplicons that are complementary to the TSE. When the correct target is available, the extension reaction continues to the 5Ј end of the amplicon, where the diGTP-biotin is used to insert the biotin into the TSE. Specificity and signal consistency arise when coded TSEs are covalently attached to a single biotin reporter in the presence of the correct target. In the final step, the TSEs are captured on the solid matrix and detected. The presence of the target is then determined by where the reporters are located spatially on the liquid matrix (created by the differentially colored microspheres).
Because each EraCode should be specific only for its complementary tag, only the bead that contains the specific EraCode should report a high MFI. Results show that for the correct complementary code, specific signal was between 2130 and 4357 MFI, with a mean (SD) MFI of 3040 (532; see Table 4 in the online Supplemental Data). Signals generated on microspheres that contained Era-Codes that were not completely complementary varied from 0 to 320 MFI with a mean (SD) MFI of 92 (28) . This set gave signal-to-noise ratios that ranged from 32:1 and 10:1 for the worst cases (i.e., where the specific signal was compared with the nonspecific signal from the microsphere presenting the highest noise). To our understanding, there have been no reports of a genetic-based molecular recognition system that was successfully implemented at room temperature without washing. mutation analysis using the manual cf multicode system PCR amplicons were generated for 17 regions within the CFTR gene, and 63 tagged extenders were used to analyze 27 mutations and 4 reflex targets. To improve the robustness of the overall results, the test was divided into four reactions with one additional test targeting the 5T7T9T reflex target. Two additional targets were included late in the development phase to include 394delTT and 3199del6. The test was transferred to the Wisconsin State Laboratory of Hygiene, where newborn CFTR genetic screening has been ongoing since 1994 and multimutation testing used since 2002.
For the CFTR-specific PLx assay, positive signal intensities varied between 2000 and 5000 MFI and background signal varied from 50 to 1500 MFI. The variation in signal depended strictly on the target sequence. We observed that determination windows varied from 0.99 to 0.7 for wild-type samples, from 0.4 to 0.6 for heterozygous samples, and from 0.0 to 0.2 for homozygous mutant samples.
The calling results are summarized in Table 1 [also see Table 4 in the online Data Supplement for additional details]. The 225 samples included 65 wild-type samples; 93 heterozygous samples representing 22 types; 36 compound heterozygous samples for 9 types; and 31 homozygous mutant samples for 6 types. Of the 225 samples tested manually, 203 (90.2%) samples reported all correct assignments. The failure rate of 10% was most likely a Step 2), tagged target-specific extenders are site-specifically labeled with diGTP-label when the correct amplicon is present. For this study, the label was biotin, and streptavidin-phycoerythrin was used for signaling purposes. ( Step 3), the tags are captured on EraCode-modified Luminex microspheres. The captured reactions are analyzed with the Luminex 100 system. product of DNA quality and not assay sensitivity. As stated in the Materials and Methods, the DNA samples were not fresh, and signal intensities indicated gross failure consistent with PCR failure. There were no discrepancies for genotype classification. For these 225 samples, there were a total of 6750 potential assignments (225 ϫ 27 mutations ϩ 3 reflex tests; 5T7T9T was not included). Correct assignments were made 99.3% (6704 of 6750) of the time. For the separate 5T7T9T-specific reflex test, 194 samples were tested and included at least 10 separate samples from each of the six possibilities (see Table 4 and Fig. 1 in the online Data Supplement). Test results showed that there were zero incorrect determinations and one no amplification failure.
mutation analysis using the automated cf multicode system
The final automated processes for the CFTR gene were conducted on a total of 86 samples. The results are reported from two separate processes: (1) PCR through data acquisition (20 samples) and (2) TSE through data acquisition (66 samples; Table 1 ; also see Table 5 in the online Data Supplement for further sample and testing information). Because workstation space is limited on the Tecan Genesis Model 200, tip usage is high when disposable tips are implemented, and amplicon carryover may be a concern. Process 1 may be applied for higher throughput scenarios where the PCR step could be implemented on yet another workstation or manually. For process 1, 20 (100%) samples reported calls correct. For process 2, 64 (97%) samples reported all correct assignments. There were no incorrect determinations.
After assay validation, which included the testing of various mutations and sample types, the PLx assay for CFTR gene analysis was directly compared with the CF Gold linear array method. Using freshly obtained blood cards from newborns that had immunoreactive trypsinogen scores in the upper 4%, we used both tests to analyze CFTR for the recommended core panel of mutations and reflex targets. The testing was performed on 419 samples, and 100% concordance was recorded (no incorrect assignments; Table 1 , column B). Of the samples tested, there were 21 samples (5%) in which the complete lists of calls could not be obtained with the PLx system. The failure rate of 5% was documented as errors resulting from pipetting errors. These samples were retested, and correct assignments were made. Of the possible 12 570 potential assignments, 12 521 (99.6%) were correct. Run time after sample preparation for the CF Gold test was ϳ7 h compared with Ͻ3 h for the PLx system. Total hands-on time for the CF Gold test was ϳ3.5 h compared with 0.5 h for the PLx system. The data output for the CF Gold test was visual inspection of the nitrocellulose strips, whereas the PLx system has a computerized calling system as described.
We have described a novel high-throughput platform that incorporates nucleic acid chemistries that simplify genetic analysis. PLx uses one additional base pair not found in nature in combination with microsphere flow cytometry technology to eliminate the most cumbersome steps commonly found in established multiplexed systems. The EraCode molecular recognition system described is particularly suited for the Luminex 100 instrumentation because the solid support can be dispensed into the complex reaction and decoding can be done at room temperature without washing, creating a true "Liquid Chip" type of system. Our study indicates that the chemistry is transferable to a clinical setting. In addition, although not required, the process can be performed with a robotic workstation for higher throughput.
We demonstrated the principles of the chemistry by use of an essential target and selected mutations found within the CFTR gene recommended by leading medical organizations. Data from the Wisconsin State Laboratory of Hygiene confirmed that the chemistry is accurate. For example, there were no incorrect determinations, and the percentage of correct determinations was high. The data presented here were tabulated by use of a broad sample set that included every mutation possible within the panel targeted with the exception of the 3199del6. For 24 samples, the entire manual process takes ϳ3 h to obtain final results. An upgraded prototype version of the CFTR test where all mutations are analyzed in a single well takes ϳ2.5 h to complete analysis of 96 patient samples (validation data not yet available). To demonstrate the system in a fully automated mode, we elected to use the Tecan genesis liquid-handling system. The entire automated process also takes ϳ3 h from prepared sample to final read for an entire 96-well plate or 24 samples. We presented data from two possibilities (when automation started at the PCR step and when it started at the TSE step) because there may be a need to perform PCR offline.
We believe that the expanded genetic alphabet enabled us to eliminate many of the steps that are required in technologies based on just the two base pairs provided by nature. The steps eliminated include solid support washings, transfers, aspirations, and high-temperature hybridizations. Specifically, we believe that there are many reasons that the extra base pair is beneficial. For example, we used short code sequences in the capture step to decode the complex extension mixtures. This may be possible with code sets constructed from only A, G, C, or T, but optimization would surely be more difficult because of cross-hybridization with sequence complements or close complements found in nature. The additional informational content that iC:iG builds into DNA also allows for greater diversity when compared with their all-natural counterparts. Specifically, if the four naturally occurring bases are used to make a library of 10mers, there are 4 10 , or 10 6 possibilities. In contrast, a six-letter system generates 6 10 , or 10 8 unique 10mers. As demon-Clinical Chemistry 50, No. 11, 2004 strated here, specific assembly can occur at conditions such as room temperature, which is in effect more amenable to and simplifies molecular diagnostics.
We also demonstrated for the first time that biotin labels attached to diGTP could be site specifically incorporated into extension primers. More generally, we demonstrated that labeled diGTP can be incorporated enzymatically into DNA. This could allow for broad use of such compounds in molecular biology. Whenever a reporter group is required at a specific site within an amplicon, transcript, or extension product, the use of an additional base pair may be helpful.
Recently we reported that site-specific enzymatic incorporation of a quencher attached to diGTP can be used to create a new and highly sensitive real-time PCR system (26 ) . Polymerase incorporation of labels only at sites where expanded genetic alphabet bases are positioned will also have other uses, such as 3Ј end labeling. Polymerase incorporation of EraCodes within PCR amplicons has now also been reported (27 ) . The EraCode system that uses additional nonnatural base pairing may have advantages over other DNA-based molecular recognition systems (28 -30 ) . These short DNA codes are simple to produce, specifically hybridize at room temperature, do not require wash steps, can be amplified with standard polymerases, and do not cross-hybridize with natural DNA. Taken together, we believe that the expansion of the genetic alphabet is a true paradigm shift to the ways molecular diagnostics will be developed in the future. Antisense-induced ribosomal frameshifting Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels. The standard triplet readout of the genetic code can be reprogrammed by signals in the mRNA to induce ribosomal frameshifting [reviewed in (1) (2) (3) ]. Generally, the resulting trans-frame protein product is functional and may in some cases be expressed in equal amounts to the product of standard translation. This elaboration of the genetic code (4, 5) demonstrates versatility in decoding.
Requirements for eukaryotic ribosomal frameshifting include a shift-prone sequence at the decoding site and often a downstream secondary structure in mRNA. The majority of À1 programmed frameshift sites consist of a heptanucleotide sequence X XXY YYZ [where X can be A, G, C or U; Y can be A or U; and Z can be any nucleotide (6) ].
In this configuration, the P-and A-site tRNAs can re-pair with at least 2 out of 3 nt when shifted 1 nt towards the 5 0 end of the mRNA. Similarly, for +1 frameshift sites, the identity of the codons in the P-and A-sites of the ribosome is critical for efficient frameshifting. One factor affecting +1 frameshift efficiency is the initial stability of the P-site tRNA-mRNA interaction in the 0 frame (7) . High-efficiency frameshifting occurs when the P-site tRNA does not form standard codon-anticodon interactions (8) . In some studies, a correlation between +1 frameshift efficiency and the final stability of the P-site tRNA-mRNA interaction in the +1 frame has been shown previously (9, 10) . However, in other systems there appears to be little correlation (11) . In addition, competition between decoding of the 0 frame and +1 frame codons in the A-site may affect frameshifting efficiency (7) . Slow to decode 0 frame codons such as stop codons or those decoded by low abundance tRNAs favor frameshifting, as do +1 frame codons with high levels of corresponding cognate tRNAs (12) (13) (14) (15) (16) .
High levels of frameshifting are often achieved by the stimulatory action of a cis-acting element located downstream of the shift site. A wide variety of structures, most commonly H-type pseudoknots (17) , have been identified which stimulate À1 frameshifting in eukaryotes [for reviews see (18, 19) ]. Mutagenic and structural data for several of the frameshift stimulators have demonstrated that each pseudoknot has key structural features required for frameshift stimulation (20) (21) (22) (23) (24) (25) (26) (27) (28) . However, unifying structural feature essential for frameshifting has not yet been identified. This observation combined with recent reports that simple antisense oligonucleotides can functionally mimic cis-acting 3 0 stimulators of À1 frameshifting (29, 30) demonstrates that many different structures can stimulate frameshifting. Although it should be noted that not all structures of equal thermodynamic stability can stimulate frameshifting (Discussion). RNA pseudoknots have also been shown to stimulate programmed +1 frameshifting in many eukaryotic antizyme genes (31, 32) . Antizyme is a negative regulator of cellular polyamine levels through its ability to target ornithine decarboxylase (the rate-limiting enzyme in polyamine biosynthesis) for degradation (33) (34) (35) , inhibits polyamine import (36, 37) and stimulates export (38) . Antizyme expression is induced by high-intracellular polyamine levels, and decreased with lowered levels. The polyamine sensor is a programmed +1 frameshift event that is required for antizyme synthesis. *To whom correspondence should be addressed. Tel: +1 801 585 1927; Fax: +1 801 585 3910; Email: mhoward@genetics.utah.edu Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
At low polyamine levels, termination at the end of open reading frame 1 (ORF1) is efficient, whereas at high levels of polyamines, a substantial proportion of ribosomes shift to the +1 reading frame and then resume standard decoding to synthesize the full-length and active antizyme protein.
Frameshifting at the mammalian antizyme mRNA shift site, UCC UGA, is stimulated by two cis-acting signals (39, 40) . One of these, the 5 0 element, encompasses $50 bases upstream of shift site and is important for the polyamine effect (39) (40) (41) . The other cis-acting element is a pseudoknot located 3 0 of the shift site. The mammalian antizyme pseudoknot and a structurally distinct counterpart in a subset of invertebrate antizyme mRNAs (31) are the only pseudoknots known to act as stimulators for +1 frameshifting in eukaryotes.
Although it is unknown if pseudoknots stimulate À1 frameshifting and +1 frameshifting by different mechanisms, one notable difference is found in positioning of the downstream structure relative to the shift site. Naturally occurring pseudoknots or stem-loop stimulators of À1 frameshifting typically begin $6-9 nt downstream of the A-site codon of the shift site (18) , whereas +1 frameshift pseudoknots are located closer with only a 2-3 nt separation from the A-site codon (31) . Mutagenic studies have revealed that altering the size of the spacer affects frameshifting and, in general, reduces efficiency (27, 31, (42) (43) (44) .
Here we have tested the ability of antisense oligonucleotides, annealed downstream of the shift-prone site, UCC UGA, to induce shifting of the ribosome to the +1 reading frame. The directionality of frameshifting (either into the +1 or À1 reading frame) is shown to be dependent upon the position of the duplex region relative to the shift site, and the efficiency of frameshifting is responsive to polyamine levels and enhanced by the inclusion of stimulatory sequences found upstream of the human antizyme +1 programmed frameshift site.
Complementary oligonucleotides, to construct the sequences described in this paper, were synthesized at the University of Utah DNA/Peptide Core Facility such that when annealed they would have appropriate ends to ligate into the SalI/ BamHI sites of the dual luciferase vector, p2luc (45) . Dual luciferase constructs were prepared and their sequence was verified as described previously (46) .
Insert sequences with shift site in boldface is given as follows:
P2lucAZ1wt: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-CCCCTCACCCACCCCTGAAGATCCCAGGTGGGCGAG-GGAATAGTCAGAGGGATCACAACGGATC;
P2lucAZ10sp: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGAC-CCTCACCCACCCCTGAAGATCCCAGGTGGGCGAGG-GAATAGTCAGAGGGATCACAACGGATC;
P2lucAZ1hp: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-
P2lucAZ1PKdel: TCGACGGTCTCCCTCCACTGCTG-TAGTAACCCGGGTCCGGGGCCTCGGTGGTGCTCCT-GATGCCCCTGGATC;
P2lucAZ1PKm1: TCGACGGTCTCCCTCCACTGCTGT-AGTAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTG-ATGCCCCTCACCCACCGGGATCACAAGGATC;
P2lucAZ1sl: TCGACGGTCTCCCTCCACTGCTGTAGT-AACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-CCCCTCACCCACCCGGATC;
P2lucAZ1FS: TCGACGTGCTCCTGATGCCCCTG-GATC;
P2lucAZ1FSUGG: TCGACGTGCTCCTGGTGCCCCTG-GATC. 
The dual luciferase constructs (0.1 mg) described above were added directly to TNT coupled reticulocyte lysate reactions (Promega) with 35 S-labeled methionine in a volume of 10 ml. Reactions were incubated at 30 C for 1 h. Radiolabeled proteins were separated by SDS-PAGE and the gels were fixed with 7.5% acetic acid and methanol for 20 min. After drying under vacuum, the gels were visualized using a Storm 860 PhosphorImager (Molecular Dynamics) and radioactive bands quantified using ImageQuant software. Percent frameshifting was calculated as the percentage of full-length (frameshift) product relative to the termination product and the full-length product combined. The value of each product was corrected for the number of methionine codons present in the coding sequence. The reported values are the average and standard deviations obtained from at least three independent measurements. Tables showing percent frameshifting and standard deviations can be found in Supplementary Data.
Plasmid p2lucAZ1PKdel was co-transfected into CV-1 cells with varying concentrations of AZ1B 2 0 -O-Methyl antisense oligonucleotides under the following conditions. CV-1 cells (1.5 · 10 4 ) in 50 ml of DMEM + 5% fetal bovine serum were added to wells (1/2 area 96-well tissue culture treated plates) containing 25 ng of DNA, varying amounts of AZ1B antisense oligonucleotides and 0.4 ml Lipofectamine 2000 (Invitrogen) in 25 ml of Optimem. Cells were incubated at 37 C (5% CO 2 ) for 20 h. Media were then removed from the cells and the transfected cells were lysed in 12.5 ml lysis buffer and luciferase activity determined by measuring light emission following injection of 25 ml of luminescence reagent (Promega). Percent frameshifting was calculated by comparing firefly/Renilla luciferase ratios of experimental constructs with those of control constructs: (firefly experimental RLUs/Renilla experimental RLUs)/(firefly control RLUs/Renilla control RLUs) · 100.
The ability of cis-acting RNA structures or trans-acting 2 0 -O-Methyl antisense oligonucleotides to induce ribosomal frameshifting was determined by in vitro transcription and translation of a dual luciferase reporter vector, p2Luc. p2Luc contains the Renilla and firefly luciferase genes on either side of a multiple cloning site, and can be transcribed using the T7 promoter located upstream of the Renilla luciferase gene (45) . Sequences containing shift-prone sites were cloned between the two reporter genes such that the downstream firefly luciferase gene is in the +1 reading frame. The resulting constructs were then transcribed and translated in vitro with or without complementary cis-Acting stimulators of frameshifting at the antizyme shift site Initially, three dual luciferase reporter vectors were generated containing the human antizyme 1 frameshift cassette (p2luc-AZ1wt) with the 5 0 and 3 0 stimulators of frameshifting, with the pseudoknot deleted (p2luc-AZ1PKdel), or replaced with a stem-loop (p2luc-AZ1hp) (Figure 1 ). Each constructs was then subjected to coupled transcription and translation reactions in the presence of increasing amounts of spermidine, and the 35 S-labeled products separated by SDS-PAGE. Table 2 ). Maximal levels of frameshifting were found to occur when 2-4 mM of antisense oligonucleotide was added to the transcription/translation reactions (Supplementary Table 3 ). In the presence of 0.4 mM exogenous spermidine, highly efficient shifting of ribosomes into the +1 reading frame (higher than that observed in the wild-type antizyme frameshift cassette) was observed with the addition of AZ1A (26.1%), AZ1B (51.8%) and AZ1C (31.8%) (Supplementary Table 2 ). The most efficient frameshifting is observed with the antisense oligonucleotide AZ1B which anneals such that spacing between the shift site and the beginning of the duplex region is the same as that observed between the shift site and the beginning of stem 1 of the natural antizyme 3 0 pseudoknot structure (i.e. each has a 3 nt spacer). To verify that the antisense oligonucleotide was activating ribosomal frameshifting and not transcription slippage, RNA was transcribed from p2luc-AZ1PKdel in the absence of oligonucleotide and added to reticulocyte lysate translations in the presence of increasing amounts of 2 0 -O-Methyl AZ1B oligonucleotide. Frameshifting levels were increased to the same level as that observed in coupled transcription and translation reactions demonstrating that the oligonucleotide acts to induce frameshifting during translation (Supplementary Figure) .
Surprisingly, the addition of AZ1A (0 spacer) also induced high-level frameshifting into the À1 reading frame in a manner which was modestly inhibited by the addition of spermidine (19% in the absence and 10% in the presence of 0.4 mM exogenous spermidine) ( Figure 3A and Supplementary Table 2 ). No À1 frameshift product was observed when the wild-type antizyme cassette was examined in the absence of antisense oligonucleotide addition (Figure 2 ; AZwt). As the AZ1A antisense oligonucleotide was designed to anneal directly adjacent to the UGA codon of the shift site, it was of interest to determine whether the wild-type antizyme pseudoknot could induce À1 frameshifting when located in the equivalent position. To address this, a new construct p2luc-AZ1-0sp ( Figure 1A ) was made by deleting the 3 nt spacer between the pseudoknot and the shift site of p2luc-AZ1wt. In this case, the wild-type pseudoknot is directly 3 0 adjacent to the shift site. The products of in vitro transcription and translation were separated by SDS-PAGE. No À1 frameshift product was observed and levels of the +1 frameshift product were significantly reduced to $3% ( Figure 3D and Supplementary Table 2) .
AZ1A, AZ1B and AZ1C were designed to complement RNA sequences encoded by the originating vector. To determine if duplexes formed between the antisense oligonucleotide and 3 0 adjacent antizyme sequences would result in more efficient frameshift stimulation, reporter vectors were designed to contain a portion of the antizyme 3 0 stimulator. Construct p2luc-AZ1PKm1 contains sequences from the 5 0 half of the axis formed by the stacking of stem 1 and stem 2 of the pseudoknot ( Figure 1A) . Two complementary 2 0 -O-Methyl antisense oligonucleotides were designed. First, PKm1 has perfect complimentarity to the region starting 3 nt and ending 21 nt downstream of the UGA shift site codon. Second, PKm2 is the same except that a mispaired C and bulged A were located at positions 9 and 16, respectively. These two alterations were included to more closely mimic the natural pseudoknot which also contains a mispaired C and bulged A at equivalent positions along the extended stem formed by the stacking of pseudoknot stems 1 and 2 ( Figure 1 ; compare p2luc-AZ1wt with the duplex formed between p2luc-PKm1 and antisense oligonucleotide PKm2). PKm1 and PKm2 induced 30 and 22% frameshifting, respectively, when added to coupled transcription and translation reactions of p2luc-AZ1PKm1 in the presence spermidine ( Figure 4A and B, and Supplementary Table 4 ). Neither PKm1 nor Pkm2 induced frameshifting to the same levels seen with AZ1B, suggesting that the sequence content of the duplex region can affect the efficiency of frameshift stimulation and that native antizyme sequences are not required.
A second construct, p2luc-AZ1sl, was designed to contain only the 5 0 half of stem 1 of the antizyme pseudoknot downstream from the UCC UGA shift site ( Figure 1A ). 2 0 -O-Methyl antisense oligonucleotides were designed to anneal between 3 and 15 nt (SL1) or 3 and 22 nt (SL2) downstream from the UGA codon of the shift site. Frameshift efficiency induced by these two antisense oligonucleotides, 8 and 22% respectively, was somewhat lower than that observed with PKm1 and PKm2 ( Figure 4C and D and Supplementary Table 4 ). In these cases frameshift efficiency was higher for the longer antisense oligonucleotide (SL2), suggesting that frameshift efficiency most probably correlates with stability of the duplex. As was seen with AZ1A, AZ1B and AZ1C, frameshifting efficiency stimulated by antisense oligonucleotides PKm1, PKm2, SL1 and SL2 was also strongly correlated with the concentration of exogenously added spermidine (Supplementary Table 5) .
The importance of the antizyme 5 0 sequence context to antisense oligonucleotide induced ribosome frameshifting was examined by testing the frameshift site, UCC UGA, without the 5 0 and 3 0 stimulatory antizyme sequences. To this end, the 5 0 antizyme stimulatory sequences were deleted from p2luc-AZ1PKdel to make p2luc-AZ1FS. Each of the antisense oligonucleotides AZ1A, AZ1B or AZ1C was added to coupled transcription and translation reactions with p2luc-AZ1FS in the presence or absence of spermidine. Frameshift efficiency was measured at 11, 8 and 4%, in the presence of spermidine and 3, 0.4 and 0.2% in its absence for AZ1A, AZ1B and AZ1C, respectively ( Figure 5A and B) .
To determine whether the stop codon of the shift site is essential for frameshifting, the UGA codon of p2luc-AZ1FS was altered to UGG such that the shift site was UCC UGG (p2luc-AZ1-UGG). Frameshift efficiency was significant, but reduced, compared to the shift site UCC UGA, and shows little stimulation by the addition of spermidine; AZ1A, AZ1B and AZ1C induced 3, 1 and 1.4% frameshifting in the presence of spermidine, and 1.9, 0.8 and 1.7% frameshifting in its absence, respectively ( Figure 5C and D) .
The ability of antisense oligonucleotides to induce frameshifting in cultured mammalian cells was examined by co-transfection of CV-1 cells with p2lucAZ1PKdel and increasing amounts of 2 0 -O-Methyl antisense oligonucleotides AZ1B as described in Materials and Methods. In the absence of antisense oligonucleotide frameshifting levels were determined to be 1.1%, whereas a graded increase in frameshift levels was observed upon the addition of AZ1B ( Figure 6 ). Maximal frameshifting levels were 13% in the presence of 2 mM AZ1B in the transfection media.
Several models attempting to explain pseudoknot stimulation of programmed À1 frameshifting have been proposed [for reviews see (18, 19) ]. Most models invoke a pausing mechanism whereby the ribosome is paused over the shift site such that time is allowed for the tRNAs to reposition in the new reading frame. This explanation is clearly too simplistic as stem-loops and pseudoknots of similar thermodynamic stability that cause ribosome pausing are not necessarily effective frameshift stimulators (47) (48) (49) . In addition, variations of the IBV pseudoknot have demonstrated a lack of correlation between the extent of pausing and the efficiency of frameshifting (47) . A recent publication by Brierley and co-workers (50) presents structural data demonstrating that the IBV frameshift stimulating pseudoknot blocks the mRNA entrance tunnel and leads to a structural deformation of the P-site tRNA. The resulting movement of the tRNA displaces the anticodon loop towards the 3 0 end of the mRNA. A model is presented in which this movement results in disruption of the codon-anticodon interactions, thus allowing for tRNA slippage relative to the mRNA. Similar tRNA movements were not observed with non-frameshift stimulating stem-loop structures. This model provides a feasible mechanistic explanation for the ability of some downstream structures to induce frameshifting.
The ability of antisense oligonucleotides to induce highlevel À1 frameshifting (29, 30) demonstrates that elaborate tertiary structures are not required, and that a duplex formed by complementary antisense oligonucleotides (with a variety of chemistries, including RNA, 2 0 -O-Methyl, morpholino) is sufficient to induce high-level frameshifting. Here we demonstrate for the first time that trans-acting antisense oligonucleotides may stimulate ribosome shifting to the +1 reading frame at surprisingly high levels, levels which are greater than those achieved by natural 3 0 cis-acting mRNA pseudoknot structures in programmed +1 frameshifting.
Structural studies indicating that the mRNA begins to enter the ribosome 7-9 nt downstream from the A-site codon is of direct relevance to this study (50, 51) . Our results indicate that maximal frameshifting is induced when the antisense-mRNA duplex begins 3 nt downstream of the UGA of the shift site, in agreement with the distance found between the UGA of the shift site and the beginning of stem 1 of the pseudoknot stimulator found in antizyme genes. Given this distance, the implication is that the stimulatory secondary structure would be encountered by the ribosome when the UCC codon enters the A-site of the ribosome. Perhaps as suggested by the structural studies of the IBV-1 frameshift inducing pseudoknot, the codon-anticodon interactions between the UCC codon and Ser-tRNA Ser are disrupted during translocation to the P-site. Given the importance of the UGA codon during frameshifting at the UCC UGA shift site, subsequent events following translocation of the UCC codon to the P-site and UGA to the A-site must influence frameshifting efficiency. This latter event most probably involves competition between termination and +1 frame decoding when the UGA codon is in the A-site. Various discussions have been presented for the importance of A-site and P-site events during ribosomal frameshifting (7, 52) and clearly, further investigations of this topic are warranted.
The observation presented here that the antisense oligonucleotide, AZ1A, which anneals directly adjacent to the UGA stop codon can induce ribosome frameshifts to either the +1 or À1 reading frame is surprising. In light of the above discussion of spacing for naturally occurring cis-acting frameshift stimulators, it is possible that frameshifting may occur at codons upstream of the known UCC UGA shift site. However, visual examination of upstream codons does not reveal an obvious À1 or +1 frameshift site.
The ability of spermidine to stimulate antisense oligonucleotide induced ribosome frameshifting to the +1 reading frame at the UCC UGA shift site in the absence of the natural 3 0 stimulator demonstrates that this cis-acting element is not required for polyamine responsiveness. Similarly, spermidine stimulation was observed in the absence of the 5 0 element but virtually eliminated by altering the UGA codon of the shift site to UGG. These observations are in agreement with previous studies examining the importance of cis-acting elements for polyamine induced frameshifting during expression of antizyme genes (39) (40) (41) .
Finally, the ability to direct ribosomes to the +1 reading frame in living cells ( Figure 6 ) suggests a potential therapeutic application for antisense oligonucleotides. Directed frameshifting to the +1 reading frame near a disease causing À1 frameshift mutation would cause some ribosomes to resume decoding in the wild-type ORF, thus restoring partial production of full-length protein from mutant alleles. The importance of the stop codon for efficient frameshifting suggests that the stop codon following the frameshift mutation presents a promising target for antisense induced phenotypic suppression, and that modulation of intracellular polyamine levels, although not essential, may increase the effectiveness of this approach. Further experiments are required to determine the therapeutic potential of this approach in vivo including the generality and efficiency of frameshift induction at non-programmed frameshift sites. The gene of an archaeal α-l-fucosidase is expressed by translational frameshifting The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a α-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a −1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed −1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea. Translation is optimally accurate and the correspondence between the nucleotide and the protein sequences are often considered as an immutable dogma. However, the genetic code is not quite universal: in certain organelles and in a small number of organisms the meaning of different codons has been reassigned and all the mRNAs are decoded accordingly. More surprisingly, the standard rules of genetic decoding are altered in specific genes by different events that are globally termed recoding (1) . In all cases, translational recoding occurs in competition with normal decoding, with a proportion of the ribosomes not obeying to the 'universal' rules. Translational recoding has been identified in both prokaryotes and eukaryotes. It has crucial roles in the regulation of gene expression and includes stop codon readthrough, ribosome hopping and ±1 programmed frameshifting [for reviews see (2) (3) (4) ].
In stop codon readthrough a stop codon is decoded by a tRNA carrying an unusual amino acid rather than a translational release factor. Specific stimulatory elements downstream to the stop codon regulate this process (5) . Hopping, in which the ribosome stops translation in a particular site of the mRNA and re-start few nucleotides downstream, is a rare event and it has been studied in detail only in the bacteriophage T4 (6) . In programmed frameshifting, ribosomes are induced to shift to an alternative, overlapping reading frame 1 nt 3 0 -wards (+1 frameshifting) or 5 0 -wards (À1 frameshifting) of the mRNA. This process is regulated and its frequency varies in different genes. The ±1 programmed frameshifting has been studied extensively in viruses, retrotransposons and insertion elements for which many cases are documented (7) (8) (9) . Instead, this phenomenon is by far less common in cellular genes. A single case of programmed +1 frameshifting is known in prokaryotes (10, 11) while in eukaryotes, including humans, several genes regulated by this recoding event have been described previously [(4) and references therein]. Compared to +1 frameshifting, À1 frameshifting is less widespread with only two examples in prokaryotes (12) (13) (14) and few others in eukaryotes (15) (16) (17) .
The programmed À1 frameshifting is triggered by several elements in the mRNA. The slippery sequence, showing the X-XXY-YYZ motif, in which X can be any base, Y is usually A or U, and Z is any base but G, has the function of favouring the tRNA misalignment and it is the site where the shift takes place (3, 18) . Frameshifting could be further stimulated by other elements flanking the slippery sequence: a codon for a low-abundance tRNA, a stop codon, a Shine-Dalgarno sequence and an mRNA secondary structure. It has been *To whom correspondence should be addressed. Tel: +39 081 6132271; Fax: +39 081 6132277; Email: m.moracci@ibp.cnr.it Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
reported that these elements, alone or in combination, enhance frameshifting by pausing the translating ribosome on the slippery sequence (4, 18) .
Noticeably, known cases of recoding in Archaea [recently reviewed in (19) ] are limited to termination codon readthrough events that regulate the incorporation of the 21st and 22nd amino acids selenocysteine and pyrrolysine, respectively (20) (21) (22) (23) .
No archaeal genes regulated by translational programmed frameshifting and ribosome hopping have been identified experimentally so far; therefore, if compared with the others domains of life, the study of translational recoding in Archaea is still at its dawn.
We showed that the a-L-fucosidase gene from the crenarchaeon Sulfolobus solfataricus is putatively expressed by programmed À1 frameshifting (24) . This gene, named fucA1, is organized in the open reading frames (ORFs) SSO11867 and SSO3060 of 81 and 426 amino acids, respectively, which are separated by a À1 frameshifting in a 40 base overlap ( Figure 1A ). We have reported previously that the region of overlap between the two ORFs had the characteristic features of the genes expressed by programmed À1 frameshifting including a slippery heptanucleotide A-AAA-AAT (codons are shown in the zero frame) flanked by a putative stem-loop and the rare codons CAC ( Figure 1A ) resembling the prokaryotic stem-loops/hairpins and the Shine-Dalgarnolike sites (24) . We showed that the frameshifting, obtained by mutating by site-directed mutagenesis the fucA1 gene exactly in the position predicted from the slippery site, produced a full-length gene, named fucA1 A , encoding for a polypeptide of 495 amino acids ( Figure 1B ). This mutant gene expressed in Escherichia coli a fully functional a-L-fucosidase, named Ssa-fuc, which was thermophilic, thermostable and had an unusual nonameric structure (24, 25) . More recently, we determined the reaction mechanism and the function of the residues of the active site of the mutant enzyme (26, 27) .
The functionality of the product of the mutant gene fucA1 A does not provide direct experimental evidence that programmed À1 frameshifting occurs in vivo and in S.solfataricus. To address these issues, we report here the study of the expression of the wild-type split gene fucA1 and of its mutants in the slippery sequence. We demonstrate here that fucA1 is expressed by programmed À1 frameshifting in both E.coli and S.solfataricus. This is the first experimental demonstration that this kind of recoding is present in the Archaea domain of life. The relevance of programmed À1 frameshifting in Archaea is also discussed. Figure 1 . The a-fucosidase gene. (A) Region of overlap in the wild-type split fucA1 gene. The N-terminal SSO11867 ORF is in the zero frame, the C-terminal SSO3060 ORF, for which only a fragment is shown, is in the À1 frame. The slippery heptameric sequence is underlined; the rare codons are boxed and the arrows indicate the stems of the putative mRNA secondary structure. The amino acids involved in the programmed À1 frameshifting and the first codon translated after this event in the À1 frame are shadowed. (B) Fragment of the full-length mutant fucA1 A gene. The small arrows indicate the mutated nucleotides.
Analysis of the a-fucosidase expression S.solfataricus cells were grown, and cell extracts obtained, as described previously (24, 28) .
The expression in the E.coli strain BL21(RB791) of the wild-type gene fucA1 and of the mutant genes fucA1 A [previously named FrameFuc in (24) ], fucA1 B , fucA1 sm and fucA1 tm as fusions of glutathione S-transferase (GST) and the purification of the recombinant proteins were performed as reported previously (23) . The nomenclature used in this paper for the different a-fucosidase genes is listed in Table 1 .
For the western blot studies, equal amounts of E.coli cultures expressing the wild-type and mutant fucA1 genes, normalized for the OD 600 , were resuspended in SDS-PAGE loading buffer containing 0.03 M Tris-HCl buffer, pH 6.8, 3% SDS (w/v), 6.7% glycerol (w/v), 6.7% 2-mercaptoethanol (w/v) and 0.002% blue bromophenol (w/v). The samples were incubated at 100 C for 5 min (unless otherwise indicated) and were directly loaded on to the gel. Western blot analyses were performed by blotting SDS-PAGEs of the concentrations indicated on Hybond-P polyvinylidenfluorid filters (Amersham Biosciences, Uppsala, Sweden); polyclonal anti-Ssa-fuc antibodies from rabbit (PRIMM, Milan, Italy) and anti-GST antibodies (Amersham Biosciences) were diluted 1:5000 and 1:40 000, respectively. The filters were washed and incubated with the ImmunoPure anti-rabbit IgG antibody conjugated with the horseradish peroxidase (HRP) from Pierce Biotechnology (Rockford, IL, USA). Filters were developed with the ECL-plus Western Blotting Detection system (Amersham Biosciences) by following the manufacturer's indications. The molecular weight markers used in the western blot analyses were the ECL streptavidin-HRP conjugate (Amersham Biosciences).
The protein concentration of the samples was measured with the method of Bradford (29) and the amounts of sample loaded on to the SDS-PAGEs are those indicated. The quantification of the bands identified by western blot was performed by using the program Quantity One 4.4.0 in a ChemiDoc EQ System (Bio-Rad, Hercules, CA, USA) with the volume analysis tool. The frameshifting efficiency was calculated as the ratio of the intensity of the bands of the frameshifted product/frameshifted product + termination product.
The mutants in the slippery sequence of the wild-type gene fucA1 were prepared by site-directed mutagenesis from the vector pGEX-11867/3060, described previously (24, 27) . The synthetic oligonucleotides used (PRIMM) were the following: FucA1sm-rev, 5 0 -TTTAGGTGATATTGGTGTT-CTGGTCTATCT-3 0 ; FucA1sm-fwd, 5 0 -GAACACCAATAT-CACCTAAAGAATTCGGCCCA-3 0 ; FucA1tm-rev, 5 0 -AGG-TGATATTGGTGTTCTGGTCTATCTGGC-3 0 ; FucA1tm-fwd, 5 0 -CCAGAACACCAATATCACCTCAAGAACTCGGCCCA-GT-3 0 , where the mismatched nucleotides in the mutagenic primers are underlined. Direct sequencing identified the plasmids containing the desired mutations and the mutant genes, named fucA1 sm and fucA1 tm , were completely re-sequenced.
The mutant Ssa-fuc B was prepared by site-directed mutagenesis from the vector pGEX-11867/3060, by using the same site-directed mutagenesis kit described above. The synthetic oligonucleotides used were FucA1sm-rev (described above) and the following mutagenic oligonucleotide: Fuc-B, 5 0 -GAACACCAATATCACCTAAAGAAGTTCGGCCC-AGT-3 0 , where the mismatched nucleotides are underlined. Direct sequencing identified the plasmid containing the desired mutations and the mutant gene, named fucA1 B , was completely re-sequenced. The enzymatic characterization of Ssa-fuc B was performed as described previously (24, 27) .
Samples of the proteins expressed in E.coli from the wildtype gene fucA1 and the mutants fucA1 A and fucA1 sm , purified as described, were fractionated on an SDS-PAGE. Protein bands were excised from the gel, washed in 50 mM ammonium bicarbonate, pH 8.0, in 50% acetonitrile, reduced with 10 mM DTT at 56 C for 45 min and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. The gel pieces were washed several times with the buffer, resuspended in 50 mM ammonium bicarbonate and incubated with 100 ng of trypsin for 2 h at 4 C and overnight at 37 C. The supernatant containing peptides was analysed by MALDIMS on an Applied Biosystem Voyager DE-PRO mass spectrometer using a-cyano-4-hydroxycynnamic acid as matrix. Mass calibration was performed by using the standard mixture provided by manufacturer.
Liquid chromatography online tandem mass spectrometry (LCMSMS) analyses were performed on a Q-TOF hybrid mass spectrometer (Micromass, Waters, Milford, MA, USA) coupled with a CapLC capillary chromatographic system (Waters). Peptide ions were selected in the collision cell and fragmented. Analysis of the daughter ion spectra led to the reconstruction of peptide sequences.
Genomic DNA from S.solfataricus P2 strain was prepared as described previously (24) . A DNA fragment of 1538 nt containing the complete fucA1 gene, was prepared by PCR, by using the following synthetic oligonucleotides (Genenco, Florence, Italy): FucA1-fwd, 5 0 -CTGGAGGCGCGCTAA-TACGACTCACTATAGGTCAGTTAAATGTCACAAAA-TTCT-3 0 ; FucA1-rev, 5 0 -GACTTGGCGCGCCTATCTAT-AATCTAGGATAACCCTTAT-3 0 , in which the sequence corresponding to the genome of S.solfataricus is underlined. In the FucA1-fwd primer, the sequence of the promoter of the T7 RNA polymerase is in boldface and the sequence of the BssHII site is shown in italics. The PCR amplification was performed as described previously (24) and the amplification products were cloned in the BssHII site of the plasmid pBluescript II KS+. The fucA1 gene was completely re-sequenced to check if undesired mutations were introduced by PCR and the recombinant vector obtained, named pBlu-FucA1, was used for translation in vitro experiments.
The plasmids expressing the mutant genes fucA1 A , fucA1 sm and fucA1 tm for experiments of translation in vitro were prepared by substituting the KpnI-NcoI wild-type fragment, containing the slippery site, with those isolated from the mutants. To check that the resulting plasmids had the correct sequence, the mutant genes were completely re-sequenced.
The mRNAs encoding wild-type fucA1 and its various mutants were obtained by in vitro run-off transcription. About 2 mg of each plasmid was linearized with BssHII and incubated with 50 U of T7 RNA polymerase for 1 h 30 min at 37 C. The transcription mixtures were then treated with 10 U of DNAseI (RNAse free) for 30 min. The transcribed RNAs were recovered by extracting the samples twice with phenol (pH 4.7) and once with phenol/chloroform 1:1 followed by precipitation with ethanol. The mRNAs were resuspended in DEPC-treated H 2 O at the approximate concentration of 0.6 pmol/ml.
In vitro translation assays were performed essentially as described by Condò et al. (28) . The samples (25 ml final volume) contained 5 ml of S.solfataricus cell extract, 10 mM KCl, 20 mM Tris-HCl, pH 7.0, 20 mM Mg acetate, 3 mM ATP, 1 mM GTP, 5 mg of bulk S.solfataricus tRNA, 2 ml of [ 35 S]methionine (1200 Ci/mmol at 10 mCi/ml) and $10 pmol of each mRNA. The mixtures were incubated at 70 C for 45 min. After this time, the synthesized proteins were resolved by electrophoresis 12.5% acrylamide-SDS gels and revealed by autoradiography of the dried gels on an Instant Imager apparatus.
Cells of S.solfataricus, strain P2, were grown in minimal salts culture media supplemented with yeast extract (0.1%), casamino acids (0.1%), plus glucose (0.1%) (YGM) or sucrose (0.1%) (YSM). The extraction of total RNA was performed as reported previously (24) . Total RNA was extensively digested with DNAse (Ambion, Austin, TX, USA) and the absence of DNA was assessed by the lack of PCR amplification with each sets of primers described below. The RT-PCR experiments were performed as reported previously (24) by using the primers described previously that allowed the amplification of a region of 833 nt (positions 1-833, in which the A of the first ATG codon is numbered as one) overlapping the ORFs SSO11867 and SSO3060 (24) .
For real-time PCR experiments total cDNA was obtained using the kit Quantitect RT (Qiagen GmbH, Hilden, Germany) from 500 ng of the same preparation of RNA described above. cDNA was then amplified in a Bio-Rad LightCycler using the DyNAmo HS Syber Green qPCR Kit (Finnzymes Oy, Espoo, Finland). Synthetic oligonucleotides (PRIMM) used for the amplification of a region at the 3 0 of the ORF SSO3060 were as follows: 5 0 -Real: 5 0 -TAAATGGC-GAAGCGATTTTC-3 0 ; 3 0 -Real: 5 0 -ATATGCCTTTGTCGC-GGATA-3 0 for the gene fucA1. 5 0 -GAATGGGGGTGATA-CTGTCG-3 0 and 5 0 -TTTACAGCCGGGACTACAGG-3 0 for the 16S rRNA gene.
For each amplification of the fucA1 gene was used $2500fold more cDNA than that used for the amplification of the 16S rRNA. Controls with no template cDNA were always included. PCR conditions were 15 min at 95 C for initial denaturation, followed by 40 cycles of 10 s at 95 C, 25 s at 56 C and 35 s at 72 C, and a final step of 10 min at 72 C. Product purity was controlled by melting point analysis of setpoints with 0.5 C temperature increase from 72 to 95 C. PCR products were analysed on 2% agarose gels and visualized by ethidium bromide staining.
The expression values of fucA1 gene were normalized to the values determined for the 16S rRNA gene. Absolute expression levels were calculated as fucA1/16S ratio in YSM and YGM cells, respectively. Relative mRNA expression levels (YSM/YGM ratio) were calculated as ( fucA1/ 16S ratio in YGM cells)/(fucA1/16S ratio in YSM). Each cDNA was used in triplicate for each amplification.
The wild-type fucA1 gene, expressed in E.coli as a GST-fused protein, produced trace amounts of a-fucosidase activity (2.3 · 10 À2 units mg À1 after removal of GST), suggesting that a programmed À1 frameshifting may occur in E.coli (24) . The enzyme was then purified by using the GST purification system and analysed by SDS-PAGE revealing a major protein band ( Figure 2A ). The sample and control bands were excised from the gel, digested in situ with trypsin and directly analysed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDIMS). As shown in Figure 2B and C, both spectra revealed the occurrence of an identical mass signal at m/z 1244.6 corresponding to a peptide (Peptide A) encompassing the overlapping region of the two ORFs. This result was confirmed by liquid chromatography online tandem mass spectrometry (LCMSMS) analysis of the peptide mixtures. The fragmentation spectra of the two signals showed the common sequence Asn-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys in which the amino acid from the ORF SSO11867 is underlined. These results unequivocally demonstrate that the protein containing the Peptide A is produced in E.coli by a frameshifting event that occurred exactly within the slippery heptamer predicted from the analysis of the DNA sequence in the region of overlap between the ORFs SSO11867 and SSO3060 ( Figure 1A) .
Remarkably, the MALDIMS analysis of the products of the wild-type fucA1 gene revealed the presence of a second Peptide B at m/z 1258.6 that is absent in the spectra of the Ssa-fuc control protein ( Figure 2B and C). The sequence of Peptide B obtained by LCMSMS ( Figure 2D ) was Lys-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys. This sequence differs only by one amino acid from Peptide A demonstrating that the interrupted gene fucA1 expresses in E.coli two fulllength proteins originated by different À1 frameshifting events. Polypeptide A results from a shift in a site A and it is identical to Ssa-fuc prepared by site-directed mutagenesis (24) , suggesting that the expression occurred with the simultaneous P-and A-site slippage. Instead, polypeptide B, named Ssa-fuc B , is generated by frameshifting in a second site B as the result of a single P-site slippage ( Figure 2E) .
To measure the global efficiency of frameshifting in the two sites of the wild-type gene fucA1 we analysed the total extracts of E.coli by western blot using anti-GST antibodies ( Figure 2F ). Two bands with marked different electrophoretic mobility were observed: the polypeptide of 78.7 ± 1.1 kDa migrated like GST-Ssa-fuc fusion and was identified as originated from frameshifting in either site A or B of fucA1. The protein of 38.1 ± 1.2 kDa, which is not expressed by the mutant gene fucA1 A (not shown), had an electrophoretic mobility compatible with GST fused to the polypeptide encoded by the ORF SSO11867 solely (27 and 9.6 kDa, respectively). This polypeptide originated from the translational termination of the ribosome at the OCH codon of the fucA1 N-terminal ORF ( Figure 1A) . The calculated ratio of frameshifting to the termination products was 5%.
To test if the full-length a-fucosidase produced by the À1 frameshifting event in site B (Ssa-fuc B ), resulting from the single P-site slippage has different properties from Ssa-fuc, whose sequence arises from the simultaneous P-and A-site slippage, we prepared the enzyme by site-directed mutagenesis. The slippery sequence in fucA1 A-AAA-AAT was mutated in A-AAG-AAG-T where mutations are underlined. The new mutant gene was named fucA1 B . The first G, producing the conservative mutation AAA!AAG, was made to disrupt the slippery sequence and hence reducing the shifting efficiency. The second G was inserted to produce the frameshifting that results in the amino acid sequence of Peptide B. Therefore, the sequence of the two full-length mutant genes fucA1 A and fucA1 B differs only in the region of the slippery sequence: A-AAG-AAT-TTC-GGC and A-AAG-AAG-TTC-GGC, respectively (the mutations are underlined, the nucleotides in boldface were originally in the À1 frame) ( Table 1) .
The recombinant Ssa-fuc B was purified up to $95% (Materials and Methods). Gel filtration chromatography demonstrated that in native conditions Ssa-fuc B had the same nonameric structure of Ssa-fuc with an identical molecular weight of 508 kDa (data not shown). In addition, Ssa-fuc B had the same high substrate selectivity of Ssa-fuc. The two enzymes have high affinity for 4-nitrophenyl-a-L-fucoside (4NP-Fuc) substrate at 65 C; the K M is identical within the experimental error (0.0287 ± 0.005 mM) while the k cat of Ssa-fuc B (137 ± 5.7 s À1 ) is $48% of that of Ssa-fuc (287 ± 11 s À1 ). In addition, 4-nitrophenyl-a-L-arabinoside, -rhamnoside, 4-nitrophenyl-a-D-glucoside, -xyloside, -galactoside and -mannoside were not substrates of Ssa-fuc B as shown previously for Ssa-fuc (24) . This suggests that the different amino acid sequence did not significantly affect the active site. Both enzymes showed an identical profile of specific activity versus temperature with an optimal temperature higher than 95 C (data not shown). The heat stability and the pH dependence of Ssa-fuc and Ssa-fuc B are reported in Figure 3 . At 80 C, the optimal growth temperature of S.solfataricus, the half-life of Ssa-fuc B is 45 min, almost 4-fold lower than that of Ssa-fuc ( Figure 3A ). The two enzymes showed different behaviour at pH <6.0 at which Ssa-fuc B is only barely active and stable ( Figure 3B) ; however, the two enzymes showed similar values of specific activity at pHs above 6.0, which is close to the intracellular pH of S.solfataricus (30) .
Characterization of the slippery sequence of fucA1 in E.coli
The experimental data reported above indicate that the predicted slippery heptanucleotide in the region of overlap between the ORFs SSO11867 and SSO3060 of the wildtype gene fucA1 could regulate in cis the frameshifting events observed in E.coli. To test this hypothesis, we mutated the sequence A-AAA-AAT into A-AAG-AAT and C-AAG-AAC (mutations are underlined) obtaining the fucA1 single mutant ( fucA1 sm ) and triple mutant ( fucA1 tm ) genes, respectively. It is worth noting that the mutations disrupt the slippery sequence, but they maintain the À1 frameshift between the two ORFs (Table 1) . Surprisingly, the expression of fucA1 sm in E.coli produced a full-length polypeptide that, after purification by affinity chromatography and removal of the GST protein, showed the same electrophoretic migration of Ssa-fuc and Ssa-fuc B ( Figure 4A ). This protein was then characterized by mass spectrometry analyses following in situ tryptic digestion. Interestingly, the MALDI spectra revealed the presence of a single peptide encompassing the overlapping region between the two ORFs with a mass value of 1259.7 Da (peptide C; Figure 4B ). The sequence of peptide C, determined from the fragmentation spectra obtained by LCMSMS analysis, was Glu-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys ( Figure 4C ). Remarkably, apart from the Glu residue, this sequence is identical to that of peptide B produced from fucA1, indicating that in the mutant gene fucA1 sm only one of the two frameshifting events observed in the wild-type fucA1 gene had occurred. The presence of a Glu instead of Lys was not unexpected. The mutation A-AAA-AAT!A-AAG-AAT in fucA1 sm was conservative in the zero frame of the ORF SSO11867 (AAA!AAG, both encoding Lys), but it produced the mutation AAA!GAA (Lys!Glu) in the À1 frame of the ORF SSO3060.
It is worth noting that the frameshifting efficiency of the gene fucA1 sm , calculated by western blot as described above, was 2-folds higher (10%) if compared to fucA1 (5%) ( Figure 4D ). This indicates that the mutation cancelled the frameshifting site A and, in the same time, enhanced the frameshifting efficiency of site B.
In contrast, the triple mutant fucA1 tm produced in E.coli only the low molecular weight band resulting from translational termination ( Figure 4D ). No full-length protein could be detected in western blots probed with either anti-GST ( Figure 4D ) or anti-Ssa-fuc antibodies ( Figure 4E ). These data show that the disruption of the heptameric slippery sequence completely abolished the frameshifting in E.coli confirming that this sequence has a direct role in controlling the frameshifting in vivo.
To test whether fucA1 is expressed in S.solfataricus we analysed the extracts of cells grown on yeast extract, sucrose and casaminoacids medium (YSM). Accurate assays showed that S.solfataricus extracts contained 3.4 · 10 À4 units mg À1 of a-fucosidase activity. These very low amounts hampered the purification of the enzyme. The extracts of S.solfataricus cells grown on YSM revealed by western blot a band of a molecular mass >97 kDa and no signals were detected with the pre-immune serum confirming the specificity of the anti-Ssa-fuc antibodies ( Figure 5A) . The different molecular mass may result from post-translational modifications occurred in the archaeon or from the incomplete denaturation of a protein complex. In particular, the latter event is not unusual among enzymes from hyperthermophilic archaea (31, 32) . To test which hypotheses were appropriate, cellular extracts of S.solfataricus were analysed by western blot extending the incubation at 100 C to 2 h. Interestingly, this treatment shifted the high-molecular mass band to 67.6 ± 1.2 kDa ( Figure 5B and C) , which still differs from that of the recombinant Ssa-fuc, 58.9 ± 1.2 kDa, leaving the question on the origin of this difference unsolved. To try to shed some light we immunoprecipitated extracts of S.solfataricus with anti-Ssa-fuc antibodies and we analysed the major protein band by MALDIMS. Unfortunately, we could not observe any peptide compatible with the fucosidase because the heavy IgG chain co-migrated with the band of the expected molecular weight (data not shown).
To test if the scarce amounts of the a-fucosidase in S.solfataricus extracts was the result of reduced expression at transcriptional level, we performed a northern blot analysis of total RNA extracted from cells grown either on YSM or YGM media. We could not observe any signal by using probes matching the 3 0 of the ORF SSO3060 (data not shown). These results suggest that fucA1 produced a rare transcript; therefore, we analysed the level of mRNA by RT-PCR and by real-time PCR. A band corresponding to the region of overlap between the ORFs SSO11867 and SSO3060 was observed in the RNA extracted from cells grown on YSM 2 mg) , the product of the gene fucA1 sm (2 mg), and Ssa-fuc (4 mg). The bands with faster electrophoretic mobility result from the proteolytic cleavage of the full-length protein (25) . (B) Partial MALDIMS spectrum of the tryptic digest from mutant fucA1 sm expressed in E.coli. The mass signal corresponding to peptide C encompassing the overlapping region is indicated. (C) LCMSMS analysis of peptide C. The amino acid sequence inferred from fragmentation spectra is reported. (D) Western blot of E.coli cellular extracts expressing fucA1 A , the wild-type fucA1, fucA1 sm and fucA1 tm genes (Materials and Methods). The blot was probed with anti-GST antibodies. (E) Western blot of partially purified protein samples expressed in E.coli fused to GST from wild-type and mutant fucA1 genes. Cellular extracts were loaded on GST-Sepharose matrix. After washing, equal amounts of slurries (30 ml of 300 ml) were denaturated and loaded on a 8% SDS-PAGE. Extracts of E.coli cells expressing the parental plasmid pGEX-2TK were used as the negative control (pGEX). The blot was probed with anti-Ssa-fuc antibodies. and YGM media, demonstrating that under these conditions the two ORFs were co-transcribed ( Figure 6A) .
The experiments of real-time PCR shown in Figure 6B demonstrated that rRNA16S was amplified after $17 cycles while the amplification of fucA1 mRNA was observed after 38 cycles, despite the fact that we used $2500-fold more cDNA for the amplification of fucA1. This indicates that the gene fucA1 is transcribed at very low level. No significant differences in the fucA1 mRNA level were observed in cells grown in YSM or YGM media. This is further confirmed by the analysis by western blot of the extracts of the same cells of S.solfataricus used to prepare the total RNAs, which revealed equal amounts of a-fucosidase in the two extracts ( Figure 6C) . Therefore, the low a-fucosidase activity observed under the conditions tested is the result of the poor transcription of the fucA1 gene.
To determine whether, and with what efficiency, the À1 frameshifting could be performed by S.solfataricus ribosomes, mRNAs obtained by in vitro transcription of the cloned wild-type fucA1 gene and the mutants thereof were used to program an in vitro translation system prepared as described by Condò et al. (28) . To this aim, a promoter of T7 polymerase was inserted ahead of the gene of interest to obtain RNA transcripts endowed with the short 5 0untranslated region of 9 nt observed for the natural fucA1 mRNA (24) . Autoradiography of an SDS-PAGE of the translation products (Figure 7 ) revealed that the wild-type fucA1 transcript produced a tiny but clear band whose molecular weight corresponded to that of the full-length Ssa-fuc obtained by site-directed mutagenesis (24); the latter was translated quite efficiently in the cell-free system in spite of being encoded by a quasi-leaderless mRNA. Judging from the relative intensity of the signals given by the translation products of the wild-type fucA1 and the full-length mutant fucA1 A , the efficiency of the À1 frameshifting in the homologous system was $10%. No signals corresponding to the polypeptides expected from the separated ORFs SSO11867 and SSO3060 (9.6 and 46.5 kDa, respectively) were observed. However, it should be noted that the product of SSO11867, even if synthesized, is too small to be detected in the gel system employed for this experiment. The larger product of ORF SSO3060, on the other hand, is certainly absent. These data unequivocally demonstrate that the ribosomes of S.solfataricus can decode the split fucA1 gene by programmed À1 frameshifting with considerable efficiency producing a full-length polypeptide from the two ORFs SSO11867 and SSO3060.
Remarkably, under the same conditions at which fucA1 drives the expression of the full-length protein, we could not observe any product from the fucA1 sm and fucA1 tm constructs. These data demonstrate that the integrity of the heptanucleotide is essential for the expression of the fucA1 gene in S.solfataricus, thus further confirming that the gene is decoded by programmed À1 frameshifting in this organism. In addition, the lack of expression of fucA1 sm by translation in vitro in S.solfataricus contrasts with the efficient expression of this mutant in E.coli, indicating that the two organisms recognize different sequences regulating the translational frameshifting.
The identification of genes whose expression is regulated by recoding events is often serendipitous. In the framework of our studies on glycosidases from hyperthermophiles, we identified in the genome of the archaeon S.solfataricus a split gene encoding a putative a-fucosidase, which could be expressed through programmed À1 frameshifting (24) . We tackled this issue by studying the expression of fucA1 in S.solfataricus and in E.coli to overcome the problems connected to the scarcity of expression of the a-fucosidase gene and to the manipulation of hyperthermophiles. As already reported by others, in fact, it is a common strategy to study recoding events from different organisms in E.coli (23, 33) .
The expression in E.coli of the wild-type split gene fucA1 led to the production by frameshifting of two full-length polypeptides with an efficiency of 5%. This is a value higher than that observed in other genes expressed by translational frameshifting in a heterologous system such as the proteins gpG and gpGT (0.3-3.5%) (33) .
The gene fucA1 is expressed in S.solfataricus at very low level under the conditions tested. In particular, the transcriptional analysis of the gene revealed that it is expressed at very low level in both YSM and YGM media. Similarly, no differences in the two media could be found by western blot probed with anti-Ssa-fuc antibodies, indicating that the low expression of the enzyme in S.solfataricus is the result of scarce transcription rather than suppressed translation.
Western blots allowed us to identify a specific band $8.7 kDa heavier than that of the recombinant Ssa-fuc and experiments of translation in vitro showed that the wild-type gene expresses a full-length polypeptide exhibiting the same molecular mass of the recombinant protein. This demonstrates that the translational machinery of S.solfataricus is fully competent to perform programmed frameshifting. It seems likely that the observed discrepancy in molecular mass might arise from post-translational modifications that cannot be produced by the translation in vitro. Further experiments are required to characterize the a-L-fucosidase identified in S.solfataricus.
MALDIMS and LCMSMS analyses of the products in E.coli of the wild-type split gene fucA1 demonstrated that two independent frameshifting events occurred in vivo in the proposed slippery site. In particular, the sequences obtained by LCMSMS demonstrate that peptide A results from a simultaneous backward slippage of both the P-and the A-site tRNAs ( Figure 8A) . Instead, the sequence of peptide B is the result of the re-positioning on the À1 frame of only the P-site tRNA; in fact, the next incorporated amino acid is specified by the codon in the new frame ( Figure 8B) . Therefore, the expression by À1 frameshifting of the wild-type gene fucA1 in E.coli follows the models proposed for ribosomal frameshifting (34) . We confirmed the significance of the slippery heptanucleotide in promoting the programmed frameshifting in vivo by mutating the putative regulatory sequence. The triple mutant fucA1 tm gave no full-length products; presumably, the mutations in both the P-and in the A-site of the slippery sequence dramatically reduced the efficiency of the À1 frameshifting as observed previously in metazoans (35) . This result confirms that the intact slippery sequence in the wild-type gene fucA1 is absolutely necessary for its expression in E.coli. In contrast, surprisingly, the single mutant fucA1 sm showed an even increased frequency of frameshifting (10%) if compared to the wild-type and produced only one polypeptide by shifting specifically in site B. We explained this result observing that the mutation in the P-site of the slippery sequence A-AAA-AAT!A-AAG-AAT created a novel slippery sequence A-AAG identical to that controlling the expression by programmed À1 frameshifting of a transposase gene in E.coli (36) . Therefore, apparently, the single mutation inactivated the simultaneous P-and A-site tRNA re-positioning and, in the same time, fostered the shifting efficiency of the tRNA in the P-site. It is worth noting that, instead, in S.solfataricus, only the simultaneous slippage is effective ( Figure 8B ) and even the single mutation in the slippery sequence of fucA1 sm completely annulled the expression of the gene. This indicates that this sequence is essential in the archaeon and that programmed frameshifting in S.solfataricus and E.coli exploits different mechanisms. Furthermore, since the only difference between the enzymes produced by the frameshifting sites A and B, Ssa-fuc and Ssa-fuc B , respectively, is the stability at 80 C, which is the S.solfataricus physiological temperature, the functionality of Ssa-fuc B in the archaeon appears questionable.
The reason why fucA1 is regulated by programmed À1 frameshifting is not known. However, the physiological significance of programmed frameshifting has been assigned to a minority of the cellular genes while for most of them it is still uncertain [see (4) and reference therein; (16) ]. This mechanism of recoding is exploited to set the ratio of two polypeptides such as the t and g subunits of the DNA polymerase III holoenzyme in E.coli (12) . Alternatively, programmed frameshifting balances the expression of a protein, as the bacterial translational release factor 2 and the eukaryotic ornithine decarboxylase antizyme [see (4) and (18) and references therein]. In the case of fucA1, the polypeptide encoded by the smaller ORF SSO11867 could never be detected by western blots analyses. In addition, the modelling of Ssa-fuc on the high-resolution crystal structure of the a-L-fucosidase from Thermotoga maritima (25, 37) showed that the fucA1 N-terminal polypeptide is not an independent domain. Moreover, we have shown recently that SSO11867 includes essential catalytic residues (27) , excluding the possibility that a functional a-fucosidase can be obtained from the ORF SSO3060 alone. Therefore, several lines of evidence allow us to exclude that programmed À1 frameshifting is used to set the ratio of two polypeptides of the a-fucosidase from S.solfataricus. More probably, this translational mechanism might be required to control the expression level of fucA1.
Noticeably, this is the only fucosidase gene expressed by programmed À1 frameshifting. Among carbohydrate active enzymes, the only example of expression through this recoding mechanism is that reported for a gene encoding for a a(1,2)-fucosyltransferase from Helicobacter pylori that is interrupted by a À1 frameshifting (38) . In this case, the expression by programmed frameshifting would lead to a functional enzyme synthesizing components of the surface lipopolysaccharides to evade the human immune defensive system. It is hard to parallel this model to fucA1. Nevertheless, the monosaccharide fucose is involved in a variety of biological functions (39) . Therefore, the a-L-fucosidase might play a role in the metabolism of fucosylated oligosaccharides; experiments are currently in progress to knockout the wild-type fucA1 gene and to insert constitutive functional mutants of this gene in S.solfataricus.
FucA1 is the only archaeal a-L-fucosidase gene identified so far; hence, it is probably the result of a horizontal gene transfer event in S.solfataricus. However, since there are no a-fucosidases genes regulated by programmed frameshifting in Bacteria and Eukarya, it is tempting to speculate that this sophisticated mechanism of translational regulation preexisted in S.solfataricus and it was applied to the fucosidase gene for physiological reasons. The identification of other genes interrupted by À1 frameshifts in S.solfataricus would open the possibility that they are regulated by programmed À1 frameshifting. Recently, the computational analysis of prokaryotic genomes revealed that seven Archaea harbour interrupted coding sequences, but S.solfataricus is not included in this study (40) . A computational analysis on several archaeal genomes revealed that 34 interrupted genes are present in the genome of S.solfataricus, 11 of these genes are composed by two ORFs separated by À1 frameshifting and could be expressed by recoding (B. Cobucci-Ponzano, M. Rossi and M. Moracci, manuscript in preparation).
We have experimentally shown here, for the first time, that programmed À1 frameshifting is present in the Archaea domain. This finding is the missing piece in the puzzle of the phylogenetic distribution of programmed frameshifting demonstrating that this mechanism is universally conserved. Role of RNA helicases in HIV-1 replication Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly >200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. Helicases are enzymes that separate in an energy-dependent manner stretches of duplexed DNA and/or RNA into singlestranded components. Currently, based on characteristic motifs and the sequence comparisons, three superfamilies (SF1 through 3) and two smaller families (F4, F5) of helicases have been identified (1) . Superfamily 1 and 2 contain helicases which share seven or more recognized signature amino acid motifs while SF3 and F4 and F5 helicases are characterized generally by three conserved motifs (2) ; the F4 and F5 proteins are largely bacterial and bacteriophage proteins. Currently, it should be cautioned that many 'helicases' are not bona fide helicases, but may only function as RNA translocases, perhaps to fulfill functions in the remodeling of ribonucleoprotein complexes (RNP).
DEAD-box and the related DEAH, DExH and DExD (3) helicases are the most numerous members of SF2 and are ubiquitously present in eukaryotic genomes. These helicases share eight conserved motifs and are commonly refered to as the DExH/D family of helicases. Humans, Arabidopsis and Saccharomyces have 38, 55 and 25 such entities, respectively (4) . Differing from DNA helicases and DExH proteins, DEAD helicases are poor in unwinding long nucleic acid duplexes and are best suited for separating short RNA hybrids. DEAD-box proteins bind with high-affinity RNAprotein complexes while exhibiting little RNA sequence preference. This suggests that the specificity determinants for DEAD helicases may be through the recognition of protein factors. In this regard, a better understanding of the roles for DEAD proteins depends on the clear characterization of their respective interacting proteins.
Although the precise substrate for most helicases awaits definition, DEAD helicases are generally thought to participate pleiotropically in many aspects of RNA metabolism including transcription, mRNA splicing, mRNA export, translation, RNA stability and mitochondrial gene expression (5) (6) (7) (8) . Some examples of helicases and their attributed functions include the following. UAP56, Brr2, Prp16, Prp22 and Prp43 play roles in RNA-splicing (4, 9) , while Dbp5 (10, 11) and DDX3 (12) chaperone RNAs from the nucleus into the cytoplasm. eIF4a and Ded1 serve for translation of mRNAs while Rh1B, Ski2, Dob1, Dhh1 helicases contribute to mRNA stability (4) . Other DEAD helicases act in ribosome biogenesis through regulation of small nucleolar RNAs and ribosomal RNAs (rRNAs) interactions (13, 14) . Finally, Neurospora and Trypanosoma DEAD proteins contribute to mitochondrial gene expression (15, 16) ; a Cryptococcus DEAD helicase is required for cryptococcosis pathogenesis (17) , and the dipteran Chironomus tentans uses a hrp84 DEAD helicase to regulate mRNA transport from the nucleus into the cytoplasm onto polyribosomes (18) .
Given that helicases significantly contribute to normal cellular metabolism, are they similarly essential to viruses?
The operational answer appears to be a qualified 'yes'. Indeed, when DEAD/DEAH-box helicase motif (InterPro IPR001410) was used to search the EMBL-EBI database, 1561 matches to individual viral sequence entries were found (http://www.ebi.ac.uk/interpro/DisplayIproEntry?ac¼ IPR001410), suggesting that many viruses have evolved to encode directly helicase or helicase-like proteins. The strongest biological evidence which supports the importance of a helicase in the virus life cycle comes from those viruses with an RNA genome. Hence, all positive-strand RNA viruses encode one or more helicase/helicase-like open reading frame (ORF) which, aside from the RNA-dependant RNA polymerase, is the most highly conserved viral sequence.
Although less ubiquitous, helicases are also found in other types of viruses (see some examples listed in Table 1 ). Direct mutagenesis studies have confirmed that a helicase function is biologically required for the replication of many viruses including vaccinia virus (19) , poliovirus (20) , alphaviruses (21) , brome mosaic virus (22) , nidoviruses (23, 24) and flaviviruses (25) (26) (27) .
In 1981, the first cases of acquired immunodeficiency syndrome (AIDS) were described in American homosexual men. Thereafter, within three short years, French and American scientists confirmed that the human immunodeficiency virus (HIV) is the causative agent for AIDS. In the ensuing 20 years, >20 million individuals have died from AIDS; and currently, in 2006, 50 million people worldwide are infected by HIV-1 with 3 million incremental AIDS deaths and 4-5 million new infections occurring annually. The magnitude of this burden casts urgency to medical research on HIV/AIDS.
HIV-1 is a retrovirus of the lentivirus genus with an RNA genome of 9 kilobases which encodes nine polypeptides. The major HIV-1 structural proteins are encoded by three genes, gag (group-specific antigen), pol (polymerase) and env (envelope), while the accessory proteins, Vif, Vpu, Vpr and Nef, and the regulatory proteins, Tat and Rev, are the primary translation products of multiply-spliced mRNA. HIV-1 infects CD4+ human T-cells and macrophages and integrates as a provirus into the host cell's DNA. Gene expression of HIV-1 is governed transcriptionally by a viral protein, Tat (28, 29) , via its binding to a nascent viral TAR RNA (30) , and post-transcriptionally by a second viral protein Rev (31,32) through its association with the viral RRE RNA. Both Tat and Rev interact with several host cell proteins in their transcriptional and post-transcriptional functions (33) . HIV-1 does not encode for any RNA helicase; however, findings suggest that host cell RNA helicases may be involved in the reverse transcription of HIV-1 RNA, in HIV-1 mRNA transcription and in the nucleus-to-cytoplasm transport of viral mRNA.
A recent unexpected finding revealed the possibility that an RNA helicase may potentially contribute roles in HIV-1 particle assembly and reverse transcription (34) . Using proteomic analyses, Roy et al. (34) reported that the DEAH protein RNA helicase A (RHA) was found associated with HIV-1 Gag and packaged into HIV-1 virions in an RNAdependent manner. When RHA was knocked down in cells, HIV-1 particles which were produced from these cells were significantly less infectious. This appears to be compatible with two possible explanations. First, it is conceivable that RHA participates in the formation of infectious virus particles either by shaping Gag-RNA interaction during viral particle assembly or by budding. Failure of RHA to properly restructure viral RNP could explain the observed reduced infectivity. Second, Roy et al. (34) reported evidence that HIV-1 particles that do not contain RHA showed reduced virionendogenous reverse transcriptase activity. In this respect, it may be that RHA assists HIV-1 reverse transcriptase to more efficiently copy RNA by unwinding RNA secondary structure or by promoting the interaction of viral RNA with the nucleocapsid protein in order to assemble a better reverse transcription complex.
Separate from reverse transcription, the unwinding of highly structured RNAs might also be reasoned to be important for transcription (35) . However, direct evidence for an RNA helicase role has been somewhat elusive. There are several examples which seemingly support an activity for RNA helicase in transcription. First, in vaccinia virus, it has been postulated that the NPH-II helicase assists transcription by strand-separating duplexed RNA structures to prevent R-loop formation behind the elongating RNA polymerase (36) . Second, RHA has been invoked to provide a factorrecruitment role, bridging at the promoter the CREB-binding protein (37) and RNA polymerase II (37) . Third, the p68 DEAD-box helicase was shown recently to be a novel transcriptional co-activator for p53's transcriptional function (38) . Interestingly, in the latter two instances, neither the ATPase nor the helicase activity of RHA and p68 is apparently required for their attributed transcriptional roles.
For HIV-1, two recent studies provide clues that RNA helicases may also serve co-factor function for transcription from the viral long terminal repeat (LTR). Fujii et al. (39) observed that RHA conserves in its N-terminus two double-stranded RNA-binding (dsRBD) domains characterized previously for the TAR RNA-binding protein, TRBP (40, 41) . These investigators found in both reporter and virus replication assays that RHA activated, in a TAR RNA-binding dependant manner, HIV-1 LTR-directed transcription (39 roles in transcription or indirectly influence the milieu of polymerase II initiation/elongation at the LTR. Downstream from transcription, the fate of HIV-1 encoded RNA is regulated at the step of export of unspliced/partially spliced moieties from the nucleus into the cytoplasm. Unspliced and partially spliced viral RNAs code for genomic RNAs that are packaged into progeny virions and structural proteins. Hence, the egress of these RNAs from the nucleus into the cytoplasm is critical to the life cycle of the virus. Exit of HIV RNAs from the nucleus is a significant issue because unspliced/partially spliced cellular mRNAs are routinely retained in and not permitted export from the nucleus (43) (44) (45) (46) . A large body of work has suggested an elegant solution to this conundrum. Thus, it was established that the HIV-1 encoded Rev protein binds a highly secondary structured element (Rev responsive element; RRE) present in all unspliced and partially spliced HIV transcripts (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) ; and this binding specifically distinguishes, for purposes of nuclear export, viral transcripts from cellular RNAs.
New evidence now suggests that RNA helicases are also co-factors for Rev-directed export of HIV-1 mRNAs (58) . In its role of transporting unspliced and incompletely spliced viral RNAs from the nucleus, Rev directly interacts with nuclear export receptor CRM1 (59, 60) , and CRM1 is required for Rev-mediated export of HIV RNAs (59, 61, 62) . A recent report provides data that an RNA helicase, DDX3, is an additional player in the Rev-CRM1-RRE complex (12) . Thus, it was shown that DDX3 over-expression enhanced Revdependent, but not other export, pathway; and that DDX3 is a nucleo-cytoplasmic shuttling protein which binds CRM1 and Rev. Moreover, DDX3's necessity for Rev/RRE/CRM1 function was demonstrated by knock-down of cell endogenous DDX3. Finally, because DDX3 locates to nuclear pore complexes (NPC), Yedavalli et al. (12) further proposed that this human helicase, like the analogous yeast Dbp5p (11), may function with Rev/CRM1 to remodel and 'thread' large unspliced HIV-1 RNAs through the nuclear pore, facilitating their final release to the cytoplasmic side of the NPC.
The above DDX3 results are consistent with two additional papers which described similar findings for a related RNA helicase, DDX1. Thus, Pomerantz and co-workers (63) showed that DDX1 binds directly to the N-terminus of Rev and to the RRE-RNA motif and participates in the export of unspliced HIV-1 RNA from the nucleus to the cytoplasm. Additionally, they illustrated that reduced expression of DDX1 in astrocytes explains the previously observed tissue restricted function of HIV-1 Rev (64). Fully spliced viral mRNAs encoding for viral Tat, Rev and Nef, proteins have been shown previously to exit the nucleus using the cellular mRNA export pathway. Export of these mRNA may require the RNA helicase Dbp5 (65, 66) . As yet, the involvement of Dbp5 in export of spliced HIV-1 viral RNA has not been fully clarified.
The story of HIV-1 and RNA helicases is, however, likely to be more complex than and unlikely to conclude simply with RHA, RH116, DDX1 and DDX3 (Figure 1 ). HIV-1 RNAs are extensively regulated through splicing. Splicing is a multiple-step process requiring the recognition of splice sites by spliceosomes. It is generally believed that remodeling of RNA-RNA and RNA-protein interactions within the spliceosome is catalyzed by a family of DEAD/DExH box RNA helicases. To date, seven mammalian proteins that are RNA helicases have been implicated in mRNA splicing (67, 68) . Whether there is specific preference by subclasses of RNA helicases for viral mRNA splicing remains to be clarified. Moreover, how cellular RNA helicases might contribute to the translation of viral mRNAs also require further investigation.
Recently both Van't Wout et al. (69) and Krishnan and Zeichner (70) have provided evidence that the expression of several cellular RNA helicases including DDX24, DDX21, DDX18, DDX11 and DDX9 is modulated during HIV-1 infection; however, the precise cellular role and significance of these helicases for HIV-1 pathogenesis have not be characterized. Interestingly, Krishnan and Zeichner reported microarray data which examined the transition of HIV-1 infection from latency to productive replication, and found that several cellular RNA helicases were upregulated (71) . For future understanding of functions, it will be important to design experiments which can segregate helicases which serve direct, although perhaps overlapping and redundant, roles on HIV-1 from those that might participate indirectly in the viral life cycle. Nevertheless, the convergence of evidence would support that several discrete cellular RNA helicases contribute importantly to the efficient execution of several steps in the HIV-1 replicative cycle.
Given that the HIV-1/AIDS disease burden has reached pandemic global proportions, new antiviral strategies that target molecularly delineated mechanisms used by this virus are urgently needed (72) . Is there a possibility that host cell helicases can be therapeutic targets for anti-HIV-1 chemotherapy? Implicit within this question is the concept that one could attack a host cell protein in order to treat an infecting pathogen. Although targeting a cellular protein involved in a viral pathway risks obvious cytotoxicity, this approach avoids the inherent problem posed by rapid HIV-1 mutation to all currently utilized chemotherapeutics targeted to virus-encoded proteins. We note that inhibition of cell-encoded enzymes in medical therapy is not an unprecedented strategy. Suppression of angiotensin-converting enzyme (ACE) is widely used to treat hypertension, congestive heart failure, myocardial infarction, endothelial dysfunction and renal disease (73) . Elsewhere, aromatase inhibitors have been used to treat hormone-dependent breast cancer (74) , and inhibitors of cellular secretory proteases are contemplated for Alzheimer's disease (75) . We recently inhibited the cellular polyprotein convertase, furin, at minimal toxicity to the cell in order to block HIV-1 replication (76). Thus, a priori exclusion of cellular helicase as an antiviral target is not warranted.
Guarded optimism that small molecule helicase inhibitors can be developed against viruses arises from encouraging progress in non-retroviral systems. Unlike HIV-1, human herpesviruses physically encode helicases. The herpes simplex virus UL5 and UL9 genes are helicases in superfamily 1 and 2, respectively (77) . HSV UL5 together with UL8 and UL52 form a heterotrimeric helicase-primase complex responsible for unwinding duplex viral DNA at replication forks. Two recent studies provide proof-of-concept that the HSV helicase-primase can be targeted at low host cell toxicity by two new classes of drugs, amino-thiazolyphenylmolecules (78) and thiazole amide derivatives (79) . In addition, other studies suggest that the NS3 protein, a RNA helicase encoded by Hepatitis C virus and related West Nile virus and Japanese Encephalitis virus can be targeted to inhibit viral replication (80) (81) (82) . This conceptual break through in drug development is important because it indicates that target discrimination between different helicases by small molecule inhibitors is possible. Of relevance to HIV-1, a synthetic immunomodulator Murabutide was shown recently to suppress HIV-1 replication in macrophages and T cells. Murabutide was shown to inhibit the activity of RNA helicase RH116, blocking its positive transcriptional activity for HIV-1 gene expression (42) .
If one looks beyond the signature motifs conserved amongst helicases, then it becomes clear that the different proteins are widely divergent in their coding sequences. In principle, this suggests that individual helicases can be abrogated with specificity in a knowledge-directed manner.
In theory, a helicase can be attacked by (i) inhibition of NTPase activity through direct competition for NTP binding, (ii) inhibition of substrate binding through direct competition at active site, (iii) allosteric mechanisms to affect NTPbinding/NTP hydrolysis and/or polynucleotide binding, and (iv) inhibition of unwinding activity by steric hinderance of helicase translocation along the polynucleotide substrate (83, 84) . Because the NTP-binding and substrate-binding pockets may be sufficiently similar between various helicases, specificity of inhibition through these sites will likely be extremely difficult, although perhaps not impossible. On the other hand, the tremendous variations in sequence and sizes of helicases, in their oligomerization states, in their discrete domains responsible for protein-protein interactions and/or for targeting to specific nucleic acids (85) , and in their differential localizations within cells (86) offer interventional possibilities outside of the NTP-or polynucleotidebinding sites.
We are in the preliminary stages of screening ringexpanded nucleoside analogs found previously to be successful NTPase/helicase inhibitors of West Nile virus, Hepatitis C virus and Japanese encephalitis virus (81, 82) . We have observed that a few of these candidate inhibitors have substantial anti-HIV-1 activity at doses that do not incur cytotoxicity to cells treated in tissue culture for 1 week. Further studies are needed before concluding that these compounds exert specific inhibition of DDX3, one of the other cellular helicases, or some other target altogether.
There is another area where a cellular helicase activity and HIV-1 are likely to intersect. An emerging research focus is the role of small interfering RNAs (87) and microRNAs (miRNA) as innate cell defenses against viruses including HIV-1 (87) (88) (89) (90) . In human cells, the precursor for miRNA (pre-miRNA) is processed by DICER (Figure 2A) which is a ribonuclease with a bona fide RNA helicase domain (91, 92) . A surprising recent finding revealed that the human TRBP, which has been shown to be a potent binder of the HIV-1 TAR RNA RNA (40, 41) , is an indispensable dsRNA-binding partner of DICER which allows the latter to associate with pre-miRNA (91, 92) . Without TRBP, DICER's miRNA processing activity is lost. Thus an intriguing scenario can potentially unfold. Accordingly, whereas the ribonuclease-helicase protein DICER requires TRBP to process duplex-structured miRNAs in order that the cell can use such matured miRNAs for antiviral defense, it could be speculated that HIV-1 has evolved to restrict this defense by the ability to transcribe viral TAR RNA to squelch TRBP away from DICER (Y. Bennasser, M. L. Yeung and K. T. Jeang, manuscript submitted) ( Figure 2B ). If this thinking is correct, then HIV-1 has developed mechanisms not only to co-opt the active functions of a virus-propitious cellular helicase (i.e. DDX3) but also to inactivate the role of a second virus-pernicious helicase (i.e. DICER) for purposes of selfish gain.
In a separate perspective, virus infection can trigger through double-stranded viral RNAs an innate antiviral immune response. Thus viral dsRNAs can be recognized by cellular proteins [pattern-recognition receptors (PRRs)] which initiate antiviral responses by inducing the production of a variety of cytokines including type I interferons (IFN-a and IFN-b) and initiating additional inflammatory and adaptive immune responses. Recently, DExD/H RNA helicases such as RIG-1 (retinoic acid inducible gene-1) (93) and Mda5 (melanoma differentiation-associated gene 5) (94) have been identified as suppressors of viral replication by binding to virus associated dsRNA and activating type I interferon-dependent antiviral immunity. Over-expression of RIG-1 and Mda5 was found to enhance dsRNA induced type I interferon antiviral response. Currently, it remains speculative whether helicases like RIG-1 and Mda5 may recognize HIV-1 dsRNA and trigger an innate immune response. Intriguingly, several reports exist in the literature that HIV-1 infection does induce activation of type 1 interferons (95, 96) .
In conclusion, by studying helicase proteins one can gain insights into normal cellular metabolic processes, abnormal inherited human diseases (e.g. Bloom syndrome, Werner syndrome, Cockayne's syndrome and xeroderma pigmentosum; all diseases with mutations in cellular helicases), and remarkably also the biology of viruses. MIMOX: a web tool for phage display based epitope mapping BACKGROUND: Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. RESULTS: We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. CONCLUSION: A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at . Since the pioneering work of Smith and co-workers [1] [2] [3] , phage display technology has been widely used in both basic research such as the exploration of protein-protein interaction sites and networks [2] [3] [4] [5] , and applied research such as the development of new drugs, diagnostics, and vaccines [6] [7] [8] . Phage display has also become a promising epitope mapping method, which has been applied in many fields such as allergology [9] and oncology [10] . The phage display based epitope mapping is usually accomplished through comparing the sequence of mimotopes (antibody-selected phage displayed peptides) to the anti-gen. In some cases, the mimotope sequence is identical or very similar to a sequence in the antigen [2] , there by indicating the location of the native epitope. These cases are rare however, and usually the mimotope sequence has little, if any, similarity with the antigen sequence. Compared with traditional epitope mapping methods such as solving the crystal structure of the antigen-antibody complex or scanning overlapping peptides of the antigen, phage display based epitope mapping is generally much cheaper and less arduous.
Though epitope mapping based on phage display can be done manually [11] , it is quite tedious and time-consuming to compare a set of mimotopes to the antigen without computational support. The low sequence similarity between the mimotope and the antigen often makes the mapping even harder. To solve these problems, several groups have researched algorithms and programs that assist and automate phage display based epitope mapping [12] [13] [14] [15] [16] [17] . According to their dependency on antigen structure, the existing programs for phage display based epitope mapping can be classified into three categories. Program in the first category such as FINDMAP, only work with sequence data from the mimotopes and antigen [13] . The second category needs both the sequence data and the antigen structure. SiteLight [12] , 3DEX [14] , and Mapitope [16, 17] belong to this category. A very recently published work: MIMOP [15] makes the third category, which integrates the two different approaches and can work with or without the antigen structure. Though implemented differently, all the existing programs have succeeded in given cases. However, most of the existing tools have not been implemented as a freely available online service until now, making it less convenient for the community to access, utilize, and evaluate them.
In the present study, we describe a web-based tool for phage display based epitope mapping named MIMOX. It was coded with Perl as a CGI program and can be used to align a set of mimotopes and derive a consensus sequence. The consensus sequence, or a single mimotope sequence, can then be mapped on to the antigen structure, and potential epitopes determined by spatial clustering of the mapped residues. The results mapped on to the antigen's 3D structure can then be viewed interactively. To validate this web-based tool, we compared the results from MIMOX with the results from other computational tools and experimentally identified native epitopes in several case studies.
Overall architecture of MIMOX MIMOX was coded with Perl using modules from the Bioperl [18] project. The whole online service provided by MIMOX is accomplished through a set of CGI scripts. The MIMOX service can be divided into two main sections. In the first section, MIMOX provides a simple interface for ClustalW [19] to align a set of mimotope sequences; this is implemented as the script mimosa.pl. The alignment can then be used to derive the consensus sequence through a simple statistical method; this is implemented as the script mimocs.pl. The alignment can also be viewed and managed through an embedded Java applet version of JalView [20] ; this is implemented as the script jalviews.pl. In the second section, MIMOX tries to map the user supplied sequence on to the given antigen structure. This is implemented as the script mimox.pl. The program NAC-CESS [21] is also wrapped into mimox.pl and used to calculate the surface accessibility of the mapping results. All mapping results are ranked based on their solvent accessible surface. Each mapping result has detailed information of the accessibility of each candidate residue, which is parsed through the script parsa.pl and displayed as a table in a new window. Each mapping result can also be viewed interactively on the antigen structure. This is implemented as the script jmol.pl, which wraps a Java applet version of Jmol [22] . The overall architecture of MIMOX is shown schematically in Figure 1 .
As described above, MIMOX wraps ClustalW to align a set of mimotope sequences and then allows the alignment to be viewed, edited, and analyzed through an embedded version of JalView. Based on the review by Smith et al [3] , we also implemented a simple statistical method in the script mimocs.pl to derive a consensus sequence from the alignment. Firstly, the script counts the appearance of each kind of amino acid at each position in the alignment and calculates the percentage frequency of each one. The frequency of a given amino acid X at the position i of the alignment (f xi ) is defined as where Xi means the times that the given amino acid X appears at the position i of the alignment and N is the number of sequences in the alignment. All frequencies are compared to a threshold value, which is 25% by default. If a frequency is more than the threshold, the corresponding residue is considered as a motif residue at that position. If the sum frequency of similar residue at the same position is above the threshold, the similar residues are also regarded as motif residues. In MIMOX, there are five similar residue groups (L, I, V; T, S; E, D; Q, N; K, R; F, W); other residues are considered unique. This classification scheme is the same as that used by Mapitope [16, 17] . If no motif residue is found at a given position, then X is used to stand for any amino acid residue. Motif residues at all positions of the alignment are then displayed in a table.
Overall architecture of MIMOX Figure 1 Overall architecture of MIMOX. MIMOX has two sections. The first section has 3 perl scripts. The script mimosa.pl aligns a set of mimotope sequences powered by ClustalW. The script jalviews.pl wraps JalView to view and manage the alignment. The script mimocs.pl derives a consensus sequence from the alignment. The second section also has 3 perl scripts. The script mimox.pl maps the user supplied sequence on to the given antigen structure and utilizes NACCESS to calculate the accessibility. The script parsa.pl displays the detailed accessibility information of each mapping result. The script jmol.pl wraps Jmol to view the mapping result interactively on the antigen structure. Thus a consensus sequence is suggested by the program. The script also creates a 3D bar figure based on the statistical analysis above, where the X axis represents the 20 amino acid types and gap type, Y axis is the frequency and Z axis stands for the position of the aligned sequences (Shown in Figure 2 ).
Since the mimotopes and the native epitope on the antigen bind to the same antibody, it is assumed that the mimotopes and the native epitope have similar physicochemical properties and similar spatial organization. This assumption is the basis of the MIMOX algorithm. The mapping process of MIMOX is based on the input sequence (such as the consensus sequence) and the uploaded antigen structure. A fragment of the sequence can also be used as input.
Firstly, for each position in the input sequence, MIMOX searches the uploaded PDB structure for matching residues and places them into an array of candidate residues for that position. Two matching modes are available at present. One is strict mode, which means the type of mimotope residue must match the antigen residue exactly. The other is called conservative mode, which means similar residues are also included in the candidate residue array. There are 5 groups of similar residues (L, I, V; T, S; E, D; Q, N; K, R; F, W) in MIMOX, which has been described in previous section.
Web interface of MIMOX section 1 Figure 2 Web interface of MIMOX section 1. Mimotopes selected out with trastuzumab [10] are input and aligned with wrapped Clus-talW. The frequency of a given amino acid at each position of the alignment is calculated and displayed in a table. A 3D bar figure is also created, where the X axis represents the 20 amino acid types and gap, Y axis is the frequency and Z axis stands for the position of the aligned sequences. A consensus sequence is then suggested, which can be used in further mapping with MIMOX. The alignment can also be managed with the embedded JalView [20] .
The array of candidate residues for each position is then added to an array of arrays. MIMOX finds all the residue neighbour pairs between consecutive candidate residue arrays in the array of arrays. Whether two residues are neighbours is determined by the distance between the two residues and the distance threshold value. If the distance between two residues is below the threshold, the two residues are taken as a neighbour pair. MIMOX provides three methods to calculate neighbour residue pairs. One method is to take the distance between the Cα atoms of the two amino acids as the distance between the two residues. Using Cα atoms may better reflect the backbone positions. The second method is to use the distance between the Cβ atoms, which may better reflect the side chain position (Cα atom is still used when it is a glycine because it does not have a Cβ atom). The third method described below, is based on the distances between all the heavy atoms of the two amino acids. All of the distances mentioned above are Euclidean distances, calculated as:
where D 21 means the distance between atom 2 and atom 1 and x 2 , y 2 , z 2 , x 1 , y 1 , z 1 are coordinates of atom 2 and atom 1. When the methods based on Cα or Cβ atom position are used, the default distance threshold is 7.0 angstroms, as it approximates the upper limit for noncovalent interactions in macromolecular structures [23] . When the third method is used, the distance threshold is calculated as
where DT is the distance threshold, DF is the Distance Factor (given by user), and vdwAtom is the Van der Waals radius of the atom. The default DF value is 1.11. If two residues have a pair of heavy atoms which are nearer than the distance threshold calculated from their Van der Waals radius, the two residues are taken as a neighbour pair.
MIMOX then recursively links the neighbour pairs until all possible ways of forming the input sequence are made. Each result is then ranked according to the sum of the absolute residue accessibility of each residue calculated from the NACCESS result file. In the end, the results are displayed in a table with hyperlinks to call the script parsa.pl which can parse and display the accessibility data in detail, and the script jmol.pl to view the result interactively mapped onto the antigen structure.
Web interface of MIMOX MIMOX has successfully been implemented as an online service, which has a simple web interface both for input and output. As described previously, MIMOX can be divided into two sections; we show here the input and output of the two sections in Figure 2 and Figure 3 respectively.
To test MIMOX, we have applied it to several cases taken from other similar research and literature. We compared the results from MIMOX with the results from other computational tools and the native epitope itself if the epitope is known in the CED database [24] . It should be pointed out that cases using monoclonal antibodies are most appropriate for testing [15] . However, in order to compare with previously published tools, some less appropriate cases (using polyclonal antibodies) taken from the corresponding literature are also used. More case studies [see Additional file 1] can also be found on the test dataset page of MIMOX[25].
The first case is taken from FINDMAP [13] . In 1999, Jesaitis and co-workers used an anti-actin polyclonal antibody to select a phage displayed random peptide library; VPHPTWMR was one of the consensus sequences they derived from the selected mimotopes. They manually mapped VPHPTWMR to the known structure of actin [PDB: 1ATN] and suggested that it might correspond to residues: V129, P130, H101, P102, T358, W356, M355, R372 [11] . In 2003, Mumey et al used FINDMAP to align VPHPTWMR to the actin sequence without utilizing information on the antigen structure. The result from FIND-MAP shows VPHPTWMR can be mapped to residues as V129, P130, H101, P102, T103, W356, M355, R372 [13] . FINDMAP mapped the input sequence to a slightly different set of residues (using T103 instead of T358). When running MIMOX with all parameters as defaults, we got no result. However, after the distance threshold is changed to 12 Å (the maximum distance allowed in MIMOX), we find that the two mappings above are returned as candidate cluster 5 and candidate cluster 17. As the side chain of some amino acids (such as arginine) can span a distance as great as 12 Å, MIMOX takes this value as the maximum allowable distance. This distance restriction is also used by Mapitope [16, 17] . In this case, the need for the higher distance threshold is due to R372 which lies some distance from the other mapped residues. MIMOX also suggested other possibilities such as cluster 1(V96, P102, H101, P130, T358, W356, M355, R372) which has a bigger solvent accessible surface, and cluster 26 (V96, P98, H101, P102, T103, W356, M355, R372), which clearly has 3 sequential segments, i.e. VPHPT, WM, and R.
The second case is taken from work by Enshell-Seijffers [16] . They used monoclonal antibody 17b, which is against HIV gp120 envelope glycoprotein, to select a phage displayed random peptide library and got a set of Figure 4 . As the latter two mapping results suggested by MIMOX are more exposed, they might be able to bind to the antibody more easily.
The last case is taken from MIMOP [15] . BO2C11 is a human monoclonal antibody against human coagulation factor VIII. Villard et al selected two phage displayed random peptide libraries with BO2C11 and got a set of 27 mimotopes [26] . Very recently, Moreau et al have applied their newly developed tool MIMOP to analyze these mimotopes. Combining the two methods MimAlign and Mim-Cons in MIMOP, the BO2C11 epitope is predicted be composed of a segment YFTNMF (2195-2200) and residues T2202, K2207, R2215, R2220, Q2222. The structure of human coagulation factor VIII in complex with
Comparison of three mapping results. MIMOX was used to map LLTTNKD to HIV gp120 with three different methods. Taking together, our initial case studies show that MIMOX can fully or partially repeat results from manual mapping, other existing tools, and also provide novel suggestions. MIMOX is designed to be a tool which is more interactive than automatic. We acknowledge that tuning the probe sequences and parameters are often required to get good results. This interactive process gives hints to users step by step and greatly decreases the load of the server and prevents the loss of some reasonable results. MIMOX lists all the matched results with no prediction threshold. This allows users to find the reasonable results by themselves based on their background knowledge on a given antibody, a given antigen and a given phage display experiment. Nevertheless, according to the test dataset page of MIMOX, the true epitope (or its segments) often falls in the top 5 (if the there are only a few result entries) or top 10% (if the there are many result entries) of the results.
Where the real epitope is unknown, we would suggest running MIMOX with a range of parameters and consensus sequence derived fragments to find overlapping or otherwise promising (high surface accessibility) candidate.
As we have mentioned previously, several groups have researched algorithms and programs that may assist and automate phage display based epitope mapping. Based on the dependency on antigen structure, the existing programs can be classified into three categories. FINDMAP belongs to the first category, which is independent of any structural information. FINDMAP has been implemented as a C++ program. It aligns a probe (e.g. a consensus sequence derived from a set of mimotopes) to the sequence of native antigen, allowing any permutation of the probe sequence. It uses a two-part scoring system to evaluate the quality of alignments and a branch-andbound algorithm to find an alignment with maximum score [13] .
The programs in the second category include SiteLight, 3DEX, Mapitope, and MIMOX. SiteLight was implemented in C++ and it has been tested on Red Hat Linux. First, the program divides native protein surface into overlapping patches based on geodesic distances between residues; then aligns each mimotope in the library with each patch and scores and sorts them; finally, high scoring matches are selected iteratively until 25% of the native protein is covered [12] . Another program 3DEX was implemented in Visual Basic and could only run on Windows. It divides a sequence into a set of overlapping subsequences with a user-defined length (3-maximum length of mimotope). Then, it searches for matching residues at each position of the above subsequences against the sequence or PDB structure of native protein and links the neighbours iteratively until the first subsequence is complete. This is repeated for the following subsequences to complete the mimotope and return the result [14] . Mapitope was also implemented in C++ and its algorithm was first described by Enshell-Seijffers in 2003. Briefly, Mapitope deconvolutes a set of mimotope sequences into a set of overlapping amino acids pairs (AAP). Then a set of major statistically significant pairs (SSP) are identified based on the AAP. Later, the SSP are mapped and clustered in the antigen structure. Finally, the most elaborate and diverse clusters on the antigen surface are identified and regarded as the predicted epitope candidates [16, 17] . [15] . It seems that MIMOP can work with or without the antigen structure from the published case studies. However, the sequence of the only case that is independent of antigen structure is just a continuous subsequence of the antigen sequence. Thus, more studies are still needed to prove that MimAlign can work without antigen structure information.
All the existing programs described above have succeeded in given cases. However, a systematic evaluation on these tools is absent. Moreover, as shown in the Table 1 , most of the existing tools have not been implemented as a publicly accessible online service until now, making it less convenient for the community to access, utilize, and evaluate them.
Like all software, bugs will have crept into MIMOX during the programming. We expect users will send their feedback to help us maintain and improve MIMOX in the future. A new version of MIMOX with more user definable options and supporting multiple-chain antigens will be implemented in the future, allowing epitopes formed by residues from different polypeptide chains to also be predicted. A systematic evaluation and comparison study with all available tools including MIMOX that assist phage display based epitope mapping is also under our consideration.
MIMOX, a web application for phage display based epitope mapping has been coded with Perl. It is helpful for molecular biologists to identify the native epitope of an antibody based on the antigen structure and a set of mimotope sequences they get through phage display technology. As a publicly accessible web tool in this area, MIMOX is very convenient for the community to access, utilize, and evaluate, complementing other existing programs.  Endogenous Cell Repair of Chronic Demyelination In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2(−/−)mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2(−/−)mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2(−/−)mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2(−/−)mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination. In central nervous system (CNS) demyelinating diseases such as multiple sclerosis (MS), myelin damage impairs impulse conduction along denuded axons. Limited remyelination occurs in MS lesions (1Y3) but is typically insufficient to prevent long-term neurologic disability. After a demyelinating event, improved remyelination could maximize functional recovery of viable axons and prevent associated axonal damage and degeneration.
In rodent models, extensive spontaneous remyelination and functional recovery is possible after an episode of acute transient demyelination (4, 5) . Proliferation of immature cells and differentiation into myelinating oligodendrocytes is required for this extensive remyelination (6, 7) . Similarly, immature oligodendrocyte lineage cells persist in the adult human CNS and may proliferate to increase in number within and near MS lesions (8, 9) . However, these immature cells often fail to differentiate sufficiently to remyelinate throughout the extent of MS lesions. Therefore, the repair capacity of endogenous cells may be limited by nonpermissive signals in chronic MS lesions.
Growth factors, cytokines, and cell adhesion molecules may regulate oligodendrocyte progenitor (OP) differentiation into remyelinating oligodendrocytes in the environment of a demyelinated lesion. Among these potential signaling molecules, we have examined the in vivo role of endogenous fibroblast growth factor 2 (FGF2), which is upregulated during postnatal development and in acute demyelination (10Y 12). During remyelination after acute demyelination, FGF2 null mice exhibit enhanced oligodendrocyte repopulation of demyelinated lesions (12) . This improved oligodendrocyte regeneration was further studied using in vivo retroviral lineage analysis in wild-type versus FGF2 null mice, which demonstrated that the predominant effect of FGF2 in vivo during remyelination is inhibition of OP differentiation (11) .
The current study examines whether this FGF2 effect on OP differentiation and oligodendrocyte regeneration has a significant impact on remyelination. Acute demyelination models in rodents typically exhibit spontaneous remyelination that may be so robust as to prevent appropriate evaluation of strategies for improving remyelination. Therefore, the current study challenges the capacity of endogenous cells to regenerate oligodendrocytes and remyelinate chronically demyelinated lesions.
We induced active demyelination in mice by adding 0.2% cuprizone to the diet so that return to normal chow would allow analysis of spontaneous remyelination without ongoing disease pathogenesis. Cuprizone reproducibly demyelinates the corpus callosum of mice (13) . Spontaneous remyelination is greatly reduced after chronic cuprizone demyelination in contrast to acute demyelination (14 Y 16) . Importantly, after chronic cuprizone demyelination, axons remain viable and can be remyelinated by transplanted OP cells (16) . In this chronic demyelination model, the current study demonstrates dramatically improved remyelination by endogenous cells in mice with genetic deletion of FGF2 as compared with wild-type mice. Thus, FGF2 expression in chronic lesions may limit remyelination and attenuation of FGF2 may generate a sufficiently permissive environment for spontaneous remyelination by endogenous cells.
Mice were bred and maintained in the Uniformed Services University of the Health Sciences (USUHS) animal housing facility and all procedures were performed in accordance with guidelines of the National Institutes of Health, the Society for Neuroscience, and the USUHS Institutional Animal Care and Use Committee. FGF2 knockout mice on the 129 Sv-Ev:Black Swiss genetic background were obtained from breeding heterozygous pairs (provided by Dr. Doetschman, University of Cincinnati). This FGF2 knockout was generated by a targeted deletion replacing a 0.5-kb portion of the FGF2 gene, including 121 bp of the promoter and the entire first exon with an Hprt minigene (17) .
Cuprizone ingestion results in a reproducible pattern of extensive corpus callosum demyelination (12, 13) . Cuprizone treatment was started at 8 weeks of age and only male mice were used. Cuprizone (0.2% (w/w), finely powdered oxalic bis(cyclohexylidenehydrazide) (Sigma-Aldrich, St. Louis, MO), was thoroughly mixed into chow (diet TD.01453; Harlan Teklad, Madison, WI), which was available ad libitum. Mice were maintained on the cuprizone diet until perfused for analysis or returned to normal chow after 6 weeks or 12 weeks of cuprizone ingestion.
Mice were perfused with 4% paraformaldehyde and then brains were dissected before overnight postfixation in 4% paraformaldehyde (18) . Brain tissue was cryoprotected overnight at 4-C in 30% sucrose and frozen in OCT compound for immunostaining and in situ hybridization.
In situ hybridization and preparation of digoxigeninlabeled riboprobes were performed as previously detailed (18, 19) . Antisense riboprobes were used to detect mRNA transcripts for proteolipid protein (PLP; gift from Dr. Lynn Hudson; National Institutes of Health [20] ) and PDGF>R (gift from Dr. Bill Richardson; University College London [21] ). The digoxigenin-labeled riboprobes were hybridized to 15-Km-thick coronal brain sections. Digoxigenin was detected with an alkaline phosphatase-conjugated sheep antidigoxigenin antibody (Boehringer Mannheim, Indianapolis, IN) followed by reaction with NBT/BCIP substrate (DAKO, Carpinteria, CA).
To identify OPs in situ, 15-Km coronal brain sections were immunostained for NG2 and PDGF>R (12, 19) . Primary antibodies used were rabbit polyclonal antiNG2 antibody (gift from Dr. William Stallcup, La Jolla, CA) and rat monoclonal antiPDGF>R antibody (APA5; Pharmingen, San Diego, CA). Donkey antirabbit IgG F(ab') 2 conjugated with Cy3 (Jackson Immunoresearch, West Grove, PA) was used to detect NG2, whereas the PDGF>R was detected with biotinylated donkey antirat IgG F(ab') 2 (Jackson Immunoresearch) followed by coumarin tyramide amplification (New England Nuclear, Boston, MA).
Myelin was immunostained with monoclonal antibody 8-18C5, which recognizes myelin oligodendrocyte glycoprotein (MOG). Hybridoma cells were provided by Dr. Minetta Gardinier, University of Iowa (22) . MOG immunolabeling was detected with donkey antimouse IgG F(ab') 2 conjugated with Cy3 (Jackson Immunoresearch).
Cell proliferation was estimated with immunostaining for Ki-67 antigen, which is expressed in the nuclei of actively dividing cells but absent at G 0 (23). Ki-67 was recognized with a rat monoclonal antibody to mouse Ki-67 antigen (DAKO) followed by detection with the ABC elite kit using 3,3-diaminobenzidine (DAB) as a substrate (Vector Labs, Burlingame, CA).
Images of in situ hybridization and immunostaining results were captured with a Spot 2 CCD digital camera using Spot Advanced image acquisition software (Diagnostic Instruments, Sterling Heights, MI) on an Olympus IX-70 microscope. Images were prepared as panels using Adobe Photoshop (San Jose, CA).
For comparing cell densities, all quantification was performed by an investigator blinded to the treatment condition. Cells expressing PLP mRNA were quantified using unbiased stereologic morphometric analysis (12) (Stereologer System from Systems Planning and Analysis, Inc., Alexandria, VA). Analysis was restricted to the corpus callosum region from the midline and extending laterally to below the cingulum in 15-Km-thick coronal sections. Using the Stereologer System, the specimen thickness contributes to the sampled volume so that measurements reflect cells/mm 3 . The unbiased stereologic method could not be used appropriately for conditions with relatively few cells of interest in any chosen category. Therefore, quantification of cells in the corpus callosum expressing PDGF>R or Ki-67 required counting all labeled cells and using the Spot 2 CCD camera and software to measure the area sampled, resulting in density units of cells/mm 2 (12) .
Quantification of corpus callosum myelination was estimated from MOG immunofluorescence detected with a Spot 2 CCD camera. Using Metamorph software, pixel intensity values were normalized between sections by thresholding to exclude values below the level of immunoreactivity in the dorsal fornix, which was selected as an adjacent white matter tract that is not demyelinated by cuprizone. The percent area of the corpus callosum (midline bilaterally to a point under the cingulum apex) with MOG immunoreactivity above the threshold level was then used as an estimate of the myelinated area.
Each category analyzed included three or more tissue sections per mouse and three or more mice per condition, except where larger sample sizes are noted in text and/or figure legends. One-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test was used to determine significant differences among stages of disease progression or treatment. Unpaired Student t-test was used to compare between FGF2 genotypes in nonlesioned mice. Significance of an FGF2 genotype effect across multiple treat-ment conditions was calculated using a two-way ANOVA. No statistical comparisons were made between mice with different genetic backgrounds (i.e. C57Bl/6 mice and FGF2 mice).
Chronic Cuprizone Demyelination Provided a Relevant Model of Insufficient Remyelination to Characterize the Repair Capacity of Endogenous Cells C57Bl/6 mice were used to establish parameters for analyzing, and later manipulating, the capacity of endogenous FIGURE 1. Spontaneous remyelination of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Corpus callosum myelination was estimated by immunofluorescence for myelin oligodendrocyte glycoprotein (MOG). Pixel intensity values were normalized between tissue sections by thresholding to exclude values below the immunoreactivity in the dorsal fornix, which was not demyelinated by cuprizone (see DF in [C]). The percent area of the corpus callosum with MOG immunoreactivity above the threshold level was then used as an estimate of myelinated area. Bar colors indicate treatment conditions: no cuprizone (white), acute cuprizone (up to 6 weeks, gray), or chronic cuprizone (9 weeks and above, black). At least three sections were quantified per mouse and at least four mice per condition. After acute cuprizone, myelination returned to near nonlesioned values (p > 0.05; no cuprizone, n = 4; 6 weeks cuprizone 6 weeks off, n = 5). Recovery to nonlesioned levels did not occur after chronic cuprizone (p G 0.001; 12 weeks cuprizone 6 weeks off; n = 6). Values for 12-week cuprizone three off were not significantly different than either 12-week cuprizone or 12-week cuprizone 6 off. Significant differences, p G 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone. oligodendrocyte lineage cells to repopulate and remyelinate chronically demyelinated lesions. We used the cuprizone model to take advantage of the ability to stop active demyelination simply by returning the mice to a normal diet. As characterized in C57Bl/6 mice, continuous cuprizone ingestion results in persistent demyelination with a variable degree of partial remyelination, indicating a regenerative potential of endogenous cells even while mice are still fed cuprizone (24) . However, after 12 weeks of continuous 0.2% cuprizone ingestion in C57Bl/6 mice, spontaneous remyelination remained limited through the 6-week recovery period (Fig. 1 ). This poor remyelination after chronic demyelination contrasted with the almost complete remyelination seen within the same recovery period after acute demyelination (i.e. 6 weeks on cuprizone followed by 6 weeks off cuprizone; Fig. 1 ).
To better evaluate the underlying causes of limited remyelination after chronic demyelination, we characterized the oligodendrocyte lineage cell responses relative to disease progression with a focus on the repair potential. Oligodendrocyte and OP populations were characterized during multiple stages of disease progression: nonlesioned (no cuprizone), acute demyelination (3Y5 weeks of continuous cuprizone), chronic demyelination (12 weeks of continuous cuprizone), and recovery after chronic demyelination (12 weeks of continuous cuprizone followed by normal chow for 3 or 6 weeks). In situ hybridization was used to identify oligodendrocytes expressing PLP mRNA transcripts (Fig. 2) and OP cells expressing FIGURE 2 . Oligodendroglial repopulation of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of C57Bl/6 mice. In situ hybridization for PLP mRNA, which identifies premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. Bar colors indicate no cuprizone (white), acute cuprizone (gray), or chronic cuprizone (black). The oligodendrocyte density after 12 weeks of cuprizone followed by 6 weeks for recovery was still significantly below nonlesioned values (p > 0.05; n = 5 for both conditions). Significant differences, p G 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone and with a carrot (^) for comparisons of recovery stages with 12 week cuprizone. platelet-derived growth factor > receptor (PDGF>R) mRNA transcripts (Fig. 3) .
Quantitative analysis of the oligodendrocyte population revealed ongoing partial regeneration during and after chronic demyelination (Fig. 2) . The oligodendrocyte density was lowest after the initial 3 weeks of cuprizone in the acute phase. After 12 weeks of continuous cuprizone, the oligodendrocyte density continued to be severely reduced relative to values from nontreated mice. Ongoing partial regeneration during chronic demyelination was indicated by the fact that the oligodendrocyte density after 12 weeks was higher than after the initial 3 weeks. After ending the chronic cuprizone treatment, this oligodendrocyte regeneration improved during the recovery period. Interestingly, as shown in Figure 1 , remyelination did not progress similarly during this 6-week recovery period. The PLP mRNA in situ hybridization used to identify oligodendrocytes should detect both premyelinating and myelinating oligodendrocytes because PLP transcription precedes myelin formation (25) . These results indicate that after chronic demyelination, a proportion of premyelinating oligodendrocytes that are generated during the recovery period may fail to differentiate into myelinating oligodendrocytes.
The OP population dynamics were dramatically different in response to acute versus chronic demyelination (Fig. 3) . In response to the initial episode of acute demyelination, the OP population was amplified almost fourfold (Fig. 3) . However, by the end of the chronic demyelination period, the OP density was greatly reduced and remained low during the recovery period. In the chronic lesions, similar results were observed with OP identification using PDGF>R (Fig. 3A ) and using NG2 (12 weeks cuprizone = 155 cells/mm 2 , n = 5; 12 weeks cuprizone plus 3 weeks recovery Figure 1 ) of corpus callosum myelination estimated by immunostaining for myelin oligodendrocyte glycoprotein (MOG) in cuprizone-treated FGF2 j/j mice (A) and FGF2 +/+ mice (B). The corpus callosum showed persistent demyelination with 0.2% cuprizone ingestion throughout 9 weeks and 12 weeks. After 12 weeks of cuprizone treatment, on return to normal chow, remyelination in FGF2 j/j mice significantly improved (p G 0.001; 12 weeks cuprizone, n = 4; 12 weeks cuprizone 6 weeks off, n = 4) and recovered to nonlesioned levels (p > 0.05; no cuprizone, n = 5; 12 weeks cuprizone 6 weeks off). In contrast, values in FGF2 +/+ mice did not increase after chronic demyelination (p > 0.05; 12 weeks cuprizone, n = 4 vs. 12 weeks cuprizone 6 weeks off, n = 3) and remained significantly below nonlesioned levels (p G 0.001; no cuprizone, n = 5 vs. 12 weeks cup). = 169 cells/mm 2 , n = 6; 12 weeks cuprizone plus 6 weeks recovery = 115 cells/mm 2 , n = 5). At 12 weeks of cuprizone treatment, very few cells in the corpus callosum were identified as actively undergoing mitosis using DAPI counterstaining to reveal mitotic chromatin figures, but each actively dividing cell was also immunostained for NG2 (inset, Fig. 3A ). Immunostaining for Ki-67 nuclear antigen was used to more broadly identify actively cycling cells within the corpus callosum (Fig. 3B) . Ki-67-labeled cycling cells were present at less than half the density of OP cells. This cycling population, although still relatively small, was significantly increased within the corpus callosum of mice fed cuprizone for 12 weeks as compared with age-matched controls or with mice that had recovered for 6 weeks after chronic demyelination. Together, these findings demonstrated compromised but continued OP cycling and regeneration of oligodendrocytes during chronic demyelination and the subsequent recovery period (Figs. 2, 3 ).
FGF2 expression is increased during acute cuprizone demyelination and can inhibit OP differentiation and oligodendrocyte regeneration during subsequent remyelination (11, 12) . Using in situ hybridization for FGF2 mRNA transcripts, we have shown that elevated FGF2 expression persists within chronic cuprizone lesions, as compared with nonlesioned corpus callosum (Fig. 4) . FGF2 mRNA transcript abundance related to changes in FGF2 protein detection in developing white matter and after acute demyelination (10, 19) . We predicted that in chronic lesions, FGF2 inhibition of differentiation could contribute to the limited capacity to generate remyelinating oligodendrocytes (Figs. 1, 2) , especially in the context of a reduce OP pool (Fig. 3A) .
Although the endogenous populations did not efficiently remyelinate after chronic cuprizone demyelination in C57Bl/6 mice (Fig. 1) , transplantation of OP cells has shown that the axons remain viable for remyelination (16) . Therefore, we used this chronic demyelination model in FGF2 FIGURE 6 . Oligodendroglial repopulation of the corpus callosum after chronic demyelination was dramatically improved in FGF2 j/j mice as compared with FGF2 +/+ mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of FGF2 mice. In situ hybridization for PLP mRNA, which identified premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. The oligodendrocyte density after 12 weeks of continuous cuprizone was significantly below nonlesioned values (noted by asterisks [*]; p G 0.01 for FGF2 +/+ ; p G 0.05 for FGF2 j/j mice; n = 4 for each condition). After 12 weeks of cuprizone and a subsequent 3-and 6-week period for recovery on normal chow, oligodendrocyte densities in FGF2 j/j mice were no longer significantly different as compared with nonlesion values (p > 0.05 at 3 weeks and at 6 weeks; n = 4 for each condition). Comparison of the FGF2 +/+ values with the matching conditions in FGF2 j/j mice demonstrated a significant effect of genotype (p = 0.0196). knockout mice to test whether repair from the endogenous OP population might be improved by removing endogenous FGF2 from the lesion environment. The extent of demyelination after 12 weeks of cuprizone ingestion was similar in C57Bl/6 mice and mice of both FGF2 wild-type (FGF2 +/+ ) and null (FGF2 j/j ) genotypes (Figs. 1, 5) . The percentage of the corpus callosum area with myelin immunolabeling was 45.8 T 14.1% in C57Bl/6 mice, 50.05 T 7.8% in FGF2 +/+ mice, and 46.9 T 4.3% in FGF2 j/j mice. After the chronic demyelination, only in FGF2 j/j mice did myelin immunostaining in the corpus callosum significantly increase during the recovery period (Fig. 5) . Surprisingly, the myelin immunostaining recovered to approximately nonlesion levels by 6 weeks after removal of cuprizone from the diet of FGF2 j/j mice. In contrast, in C57Bl/6 mice (Fig. 1) or FGF2 +/+ mice (Fig. 5) , the myelinated proportion of corpus callosum area did not significantly increase during the recovery period.
This striking improvement in remyelination corresponded with a dramatic increase in oligodendrocyte repopulation of chronic lesions in FGF2 j/j mice, which was not found in FGF2 +/+ mice (Fig. 6) . Before cuprizone treatment, nonlesioned FGF2 +/+ and FGF2 j/j mice had a similar density of oligodendrocytes in the corpus callosum at 26 weeks of age, which corresponded with the longest cuprizone treatment and recovery protocol. In addition, the cuprizone-induced loss of oligodendrocytes in FGF2 +/+ and FGF2 j/j mice was similar at the end of the chronic cuprizone treatment. In conjunction with the quantitative myelin immunostaining analysis, these findings indicate that FGF2 +/+ and FGF2 j/j mice experienced a similar chronic disease severity. Importantly, during the 6-week recovery period after chronic demyelination, the oligodendrocyte density returned to nonlesioned values in the FGF2 j/j mice, but not in FGF2 +/+ mice. Analysis of an additional intermediate recovery period in FGF2 j/j mice confirmed this increased oligodendrocyte regeneration and demonstrated that this repopulation could occur within 3 weeks after removal of cuprizone from the diet.
We previously showed that the absence of FGF2 did not significantly alter OP amplification in response to acute cuprizone demyelination (12) . A robust OP proliferative response occurred after 5 weeks of cuprizone in FGF2 null and wild-type mice that was similar to the previously mentioned data for C57Bl/6 mice (Fig. 3) . However, the OP population was depleted during chronic demyelination in contrast to the amplified OP response to acute demyelination in C57Bl/6 ( Fig. 3) . Therefore, we examined the effect of FGF2 genotype on the density of OP cells and cycling cells in the corpus callosum after chronic demyelination. FGF2 genotype did not appear to alter the OP population dynamics of chronically demyelinated mice or age-matched nonlesioned mice (Fig. 7) . The density of OP cells, identified by in situ hybridization for PDGF>R mRNA, was similar in FGF2 +/+ mice and FGF2 j/j mice (Fig. 7A) . Somewhat unexpectedly, mice of both FGF2 null and wild-type genotypes, which are on a 129 Sv-Ev:Black Swiss genetic background, exhibited an increase in OP cell density after 12 weeks of cuprizone that was not observed in C57Bl/6 mice (Fig. 3A ). This strain difference in OP accumulation after chronic demyelination corresponded with fewer oligodendrocytes observed during the recovery period in FGF2 +/+ mice (Fig. 6 ) as compared with C57Bl/6 mice (Fig. 2) . Therefore, the FGF2 background appears to be even less favorable than the C57Bl/6 background for OP differentiation and oligodendrocyte regeneration, yet the detrimental effects of the chronic lesion environment can still be overcome in the FGF2 j/j mice.
Ki-67 immunostaining (Fig. 7C ) indicated that the density of cycling cells in the corpus callosum was less than 20% of the density of OP cells, indicating a relatively low level of ongoing proliferation. Cells with nuclear Ki-67 immunoreactivity were often observed as doublets, as is appropriate for confirming detection of a cycling population. The density of cells immunolabeled for Ki-67 nuclear antigen increased in chronically lesioned corpus callosum with a return to nonlesioned levels during the recovery phase. The density of endogenous cells that were proliferating in the corpus callosum of FGF2 j/j mice was similar to the values for FGF2 +/+ mice (Fig. 7B ) and for C57Bl/6 mice (Fig. 3) . The similarity of FGF2 j/j mice and FGF2 +/+ mice in our analysis of OP cells and cycling cells indicates that the dramatic differences observed in oligodendrocytes and remyelination may result from a permissive effect of FGF2 removal on OP differentiation into remyelinating oligodendrocytes.
In MS lesions, an initial episode of transient demyelination may be followed by spontaneous remyelination. However, remyelination typically fails with recurring or chronic myelin damage. The current studies show the advantages of the cuprizone model of chronic demyelination for focusing on this compromised repair response. Importantly, our analysis in FGF2 j/j mice demonstrates that it is possible to overcome chronic lesion effects on endogenous oligodendrocyte lineage cells to increase remyelination. More specifically, taken together with our previous work (11, 12) , our findings in FGF2 j/j mice indicate that removing FGF2 inhibition of OP differentiation promotes spontaneous remyelination from a depleted pool of endogenous progenitors that persists after chronic demyelination.
The current findings in C57Bl/6 mice are consistent with previous reports of cuprizone treatment causing extensive demyelination of the corpus callosum in this mouse strain (13, 16, 24, 26) . To take further advantage of this model for examining mechanisms to promote remyelination from endogenous cells, we have further characterized the endogenous oligodendrocyte lineage population responses during the recovery period. We used C57Bl/6 mice for this part of our study to facilitate comparison with other studies, because differences in mouse strain can influence the response to cuprizone as well as overall cellular responses to CNS injury (13, 27) . After a transient episode of demyelination from 6 weeks of cuprizone ingestion, spontaneous remyelination is effective throughout the corpus callosum. In contrast, after a prolonged period of demyelination, from 12 weeks of continuous cuprizone, the remyelination remains limited and does not progress during a recovery interval that was examined out to 6 weeks. This limited remyelination corresponds with depletion of the OP pool ( [16] current study). However, the number of oligodendrocytes increases somewhat during the recovery period without a corresponding increase in remyelination of the corpus callosum. Taken together, these findings indicate that the depleted OP pool has a limited capacity to generate oligodendrocytes, and available OP cells may fail to fully differentiate into remyelinating oligodendrocytes in the environment of a chronic lesion. In lesions of some MS cases, immature oligodendrocyte progenitors and premyelinating oligodendrocytes may be present in relatively normal densities without efficiently remyelinating (9, 28) . Therefore, further studies will be important for better understanding regulation of the OP pool size and the transition of OP cells into premyelinating and then myelinating oligodendrocytes.
Robust OP proliferation in response to acute demyelination appears to be required for spontaneous remyelination (29) . During acute demyelination, the OP pool is dramatically amplified so that efficient differentiation into mature oligodendrocytes may not be required for extensive remyelination. PDGF-AA ligand, acting through PDGF>R activation, is an important mitogen for this OP amplification in response to acute cuprizone demyelination (11, 30) . In PDGF>R heterozygous mice, amplification of the OP pool in response to acute cuprizone demyelination is compromised and the corresponding generation of oligodendrocytes is reduced (11) . Crossing these PDGF>R heterozygous mice to FGF2 j/j mice actually increased oligodendroglial repopulation of lesions after acute cuprizone demyelination (11) . Therefore, in both acute and chronic lesion environments, FGF2 removal may promote the generation of oligodendrocytes from OP cells during remyelination.
Endogenous FGF2 has been predicted to contribute to proliferation of neonatal and adult OPs, especially when present in combination with PDGF-AA, based on in vitro studies (31Y33). In our current analysis, the densities of OP cells and Ki-67-immunolabeled cells were similar between FGF2 wild-type and null genotypes. These results are consistent with our previous BrdU incorporation studies indicating that endogenous FGF2 is not a predominant mitogen for OPs in the acute cuprizone model (11) . However, subtle changes in the proliferation rate among an asymmetrically dividing OP pool could be relatively difficult to detect in a small population of cells over a prolonged disease and recovery course. FGF2 is also predicted to stimulate neural stem cells in the subventricular zone (SVZ) to contribute to repopulation of corpus callosum lesions (34) . During the prolonged demyelination period of the current study, the contribution of cells derived from the SVZ should have been evident in the dynamics of the OP pool in the corpus callosum. The lack of a detrimental effect of FGF2 absence on the OP pool may indicate that FGF2 effects on the SVZ cells may not be a significant contribution to the remyelination of the overlying corpus callosum. However, more direct analysis of the response of cells in the SVZ is required to make this determination. In addition, endogenous elevation of FGF2 in lesions may elicit specific effects that are not replicated with in vivo methods of elevating exogenous FGF2 levels (34Y37).
FGF2 is among the most effective neuroprotective growth factors in diverse models of CNS injury, including stroke, trauma, excitotoxicity, and axotomy (38, 39) . In contrast to this expected protective effect of FGF2 on neurons, removal of endogenous FGF2 actually improved regenerative parameters in lesions that involved remyelination. Improved regeneration of oligodendrocytes was observed in FGF2 j/j mice after acute cuprizone demyelination of the corpus callosum and after murine hepatitis virus strain A59 demyelination of spinal cord ( [11, 12] current study). In an example from the peripheral nervous system, FGF2 knockout mice were used to prevent the normal upregulation of FGF2 and FGF receptor (FGFR) activation associated with Schwann cells and macrophages at a site of sciatic nerve crush injury (40) . Compared with wild-type mice, the FGF2 knockout mice exhibited improved myelination and increased axon diameter during regeneration from sciatic nerve crush (40) . These results indicate that a detrimental effect of endogenous FGF2 on remyelination may outweigh FGF2 protection from acute axon damage in these lesion models. Indeed, the improved remyelination in nerves of the FGF2 knockout mice may provide significant protection of axons from damage subsequent to the initial crush injury. Correlative support for a similar role of FGF2 in a chronic autoimmune model of MS can be found in a study of neural stem cell transplantation improved into mice with experimental allergic encephalomye-litis (EAE) (41) . The transplanted mice exhibited improved remyelination, with a major contribution from endogenous cells, and had less axonal loss in lesion areas. Neural stem cell transplantation was also associated with a significant decrease in FGF2 expression and reduced astroglial scar formation in the EAE lesions. FGF2 levels may modulate scar formation in demyelinated lesions because expression of FGF2 and FGFR1 corresponds with scarring, but not nonscarring, astroglial responses after other forms of CNS injury (42) .
It is not yet clear which FGFR type, or types, mediates the effect of endogenous FGF2 in the context of demyelination and remyelination. Differential effects of FGF2 at different stages of the oligodendrocyte lineage may occur through differential expression and activation of FGFR isoforms (43) . Oligodendrocyte lineage cell expression of high-affinity FGFRs and coreceptors varies with developmental stage and FGF2 exposure (44) . Furthermore, expression of multiple FGFR types is significantly increased in response to demyelination (19) . FGF2 may also have differential effects based on changing interactions with other signaling pathways that regulate oligodendrocyte lineage differentiation and myelination such as Notch1 (45, 46) and neuregulin (47, 48) . Interpreting a direct effect of FGF2 is complicated because neurons and glial cells express multiple FGFR types within and near demyelinated lesions (19) . Therefore, future studies will be required to identify the specific signaling components mediating endogenous FGF2 signaling in the in vivo context of demyelination and remyelination.
In MS lesions, the pathology is heterogeneous and the effect on the oligodendrocyte lineage population varies dramatically (49) . Oligodendrocyte density is severely compromised in demyelinated lesions in some patients with MS. Further studies are imperative to optimize regenerative responses from immature oligodendrocyte lineage cell populations that have been observed in some MS lesions (9, 28) . Targeting inhibitory signals that are upregulated in lesions should be a viable strategy for therapeutics to promote differentiation as needed near lesions. FGF2 expression has been reported in reactive astrocytes of acute and chronic MS plaques (50) . In addition, in cultures from adult human brain white matter, FGF2 inhibited the differentiation of preoligodendrocytes into oligodendrocytes (51) . Strategies to promote remyelination by attenuating FGF2 inhibition of OP differentiation may take advantage of reagents to modulate FGF2 signaling, which are currently being developed for angiogenesis and cancer treatments. In our in vivo analysis, absence of FGF2 was not detrimental in the normal adult or throughout the disease process. Therefore, inhibition of FGF2 signaling may be acceptable across demyelinating disease stages, which would improve treatment feasibility in diseases such as MS that have an unstable disease course. Treatments to promote remyelination should be a valuable complement to strategies that abrogate ongoing causes of demyelination such as immunomodulatory therapies. Automated identification of multiple micro-organisms from resequencing DNA microarrays There is an increasing recognition that detailed nucleic acid sequence information will be useful and even required in the diagnosis, treatment and surveillance of many significant pathogens. Because generating detailed information about pathogens leads to significantly larger amounts of data, it is necessary to develop automated analysis methods to reduce analysis time and to standardize identification criteria. This is especially important for multiple pathogen assays designed to reduce assay time and costs. In this paper, we present a successful algorithm for detecting pathogens and reporting the maximum level of detail possible using multi-pathogen resequencing microarrays. The algorithm filters the sequence of base calls from the microarray and finds entries in genetic databases that most closely match. Taxonomic databases are then used to relate these entries to each other so that the microorganism can be identified. Although developed using a resequencing microarray, the approach is applicable to any assay method that produces base call sequence information. The success and continued development of this approach means that a non-expert can now perform unassisted analysis of the results obtained from partial sequence data. For both surveillance and diagnostic applications, fine-scale pathogen identification and near-neighbor discrimination is important; therefore, an assay that monitors at this very specific level is desirable for many types of samples such as clinical and environmental (1) (2) (3) . To successfully use any method based on DNA or RNA detection, these assays must be coupled with large databases of nucleic acid sequence information for assay design to ensure that the desired information is provided and for the interpretation of raw data. Several well-established techniques use PCR to amplify individual target pieces of sequenced genomes to provide detection of organisms (4) . These methods can roughly be divided into approaches that target individual short sequence lengths or probes (<40 bp) and methods that examine longer probes. The advantage of using short probes is that when the uniqueness of the probe has been assured and unique primers are also selected, this method gives good specificity. This approach is capable of providing fine-scale identification of several genetically close organisms by selecting a sufficient number of probes. However, this can rapidly lead to a very large number of total probes being required to detect all organisms of interest. In addition these selected probes, which in the initial selection process were determined to be unique, are often later found to be less specific as more organisms are sequenced or are less specific under conditions that differ from the original conditions. This is particularly a problem for organisms belonging to a family with a high mutation rate and also for pathogens that have relatively few neighboring pathogens sequenced. In addition, PCR approaches focused on short unique probes are not capable of detecting the presence of new significant mutations nor can they easily resolve base sequence details. Approaches that use longer individual probes avoid many of these issues at the cost of being less specific. This issue means most of these approaches are not suitable for providing the information desired, providing impetus to this work.
High-density resequencing microarrays produce variable length segments, 10 2 -10 5 bp, of direct sequence information. This target sequence falls in the longer target regime of PCR approaches but rather than being hybridized to a longer lessspecific probe on the microarray, many shorter specific probes are placed on the microarray to allow more detailed determinations from the entire PCR amplicon. This also means that the specificity of the primers used can be relaxed. They have been successfully used to detect single nucleotide polymorphism (SNP) and genetic variants from viral, bacterial and eukaryotic genomes (5) (6) (7) (8) (9) (10) (11) (12) . Their use for SNP detection has clearly established their ability to provide reliable quality sequence information. In most cases, the microarrays were designed to study a limited number of genetically similar target pathogens and for many cases, the detection methods relied only on recognizing hybridization patterns for identification (6, 9, 10, 13, 14) . Taking advantage of the sequential base resolution capability of resequencing microarrays that is required for SNP detection, resequencing has recently been successfully adapted recently using a different approach for organism identification of multiple bacterial and viral pathogens while allowing for fine detailed discrimination of closely related organisms and tracking mutations within the targeted pathogen (15) (16) . The new methodology differed from earlier work by using the resolved bases as the query of a similarity search of DNA databases to identify the most likely species and variants that match the base calls from the hybridization observed. The system was capable of testing for 26 pathogens simultaneously and could detect the presence of multiple pathogens. A software program, resequencing pathogen identifier (REPI), was used to simplify data analysis by performing similarity searches of a genetic database using basic local alignment search tool (BLAST) (17) . The REPI program used BLAST default settings and would only return sequences that might represent the hybridization if the expect value, a quantity calculated by the BLAST program that indicates the likelihood that the sequence match found would have occurred by random chance in the database, was <10 À9 . This screened out all cases that had insufficient signal; however, the final determination of what pathogen(s) was detected and to what degree discrimination was possible required manual examination of the returned results. This method successfully allowed fine discrimination of various adenoviruses and strain identifications of Flu A and B samples in agreement with conventional sampling results (15, 16) . Two important advantages of this approach were that the information was always recovered at the most detailed level possible and that it was capable of still recognizing organisms with recent mutations. This approach also maintained specificity well, as it was not dependent on the uniqueness of a few individual short probes.
Although this analysis method has utility, there are several shortcomings: it is time consuming, not optimized to maximize sensitivity, has complicated results, is suitable only for an expert, and contains redundant or duplicate information. The process was time consuming because only the initial screening was handled automatically while the remaining steps required manual interpretation before the detection analysis was complete. Because a simple criterion (expect value cutoff of 10 À9 ) and non-optimized BLAST parameters were used to consider a pathogen detected, the REPI algorithm provided a list of candidate organisms but did not make a final simple conclusion or relate the results of one prototype sequence to another. Instead a manual process was used to make the final determination, but because the REPI program provided all similar results and the use of public nucleic acid databases containing redundant entries, a large amount of data was presented to a user that was not useful. In addition, with a manual process it was not possible to establish that the algorithm developed was generally applicable for any organism where nucleic acid base resolved sequence information has been provided.
In this paper, we describe a new software expert system, Computer-Implemented Biological Sequence Identifier system 2.0 (CIBSI 2.0), that successfully uses resolved base sequence information from custom designed Affymetrix resequencing microarrays to provide a simple list of organisms that are detected. This algorithm addresses the most important shortcoming of previous methods by incorporating new features to completely automate pathogen identification. We have demonstrated the effectiveness of this algorithm for identification via several examples. The single program is capable of making correct decisions for all 26 pathogens contained on the Respiratory Pathogen Microarray v.1 (RPM v.1), whether detected alone or in combinations, with improved sensitivity. Although the program is currently applied to resequencing microarrays, the methodologies developed remain generally applicable. Only the first portion of the algorithm handles issues specific to microarrays while the remainder deals with sequences that are suitable for use as a query by the BLAST algorithm. In developing the general identification algorithm, we have identified and resolved issues specific to resequencing microarrays that complicate their use. Because the entire decision process for what is detected has been automated, it is straightforward to test whether the rules used to make identifications are rigorous and applicable to any pathogen. With this efficient program, resequencing based assays can provide a competitive method to test simultaneously for many possible pathogens, providing output that can be interpreted by a nonexpert.
The details of the RPM v.1 design and the experimental methods have been discussed in previous work (15, 16, 18) (Lin et al., submitted for publication). Briefly, the RPM v.1 chip design includes 57 tiled regions allowing resequencing of 29.7 kb of sequences from 27 respiratory pathogens and biothreat agents. These were selected based upon clinical relevance for the population of immediate interest (United States military recruit in training) (19) (20) (21) . Partial sequences from the genes containing diagnostic regions were tiled for the detection of these pathogens. The experimental microarray data used in the present analysis were obtained using a variety of purified nucleic acid templates and clinical samples culture (throat swabs and nasal washes) using random and multiplexed RT-PCR amplification schemes (for more detail description of amplification methods see Supplementary Data). Resequencing microarrays provide base call resolution by comparing the intensities between a set of four 25mer probes that differ from each other at the same position (13th base). An amplicon or target sequence is represented by numerous overlapping probe sets. GCOSÔ software v1.3 (Affymetrix Inc., Santa Clara, CA) was used to align and scan hybridized microarrays to determine the intensity of each probe in every probe set. Base calls were made based on the intensity data of each probe set using GDAS v3.0.2.8 software (Affymetrix Inc.) which used an implementation of the ABACUS algorithm (5) . The sequences were represented in FASTA format for later analysis steps.
In this paper, target pathogens are the organisms the assay was specifically designed to detect. The sets of probes that represent reference sequence selected from target pathogen genomes are referred to as a Prototype Sequence or 'ProSeq' for brevity. The set of resolved bases that result from hybridization of genomic material to a ProSeq is referred to as the hybridized sequence or 'HybSeq'. The HybSeq is split into possible subsequences or 'SubSeqs'.
The CIBSI 2.0 program implemented in Perl described in this study handled a hierarchy of three tasks ( Figure 1 ): (I) Pro-Seq identification; (II) ProSeq grouping; and (III) pathogen determination. The most developed and important portion of the algorithm deals with ProSeq identification Task(I) and is handled in three important subtasks: initial filtering of individual HybSeqs into SubSeqs suitable for sequence similarity comparisons ( Figure 2 ), database querying of individual SubSeqs ( Figure 3 ) and taxonomic comparison of BLAST returns for each SubSeq (Figure 4 ). The NCBI BLAST and taxonomy databases were used for the queries and images were obtained on February 7, 2006. For the ProSeq grouping Task(II), ProSeqs were compared to determine if they supported the same identified organism. In the pathogen determination Task(III), detected organisms were compared to the list of target pathogens the assay was designed for in order to determine if any were positively detected or were possibly related close genetic near neighbors. The level of discrimination that a particular sample supported was automatically determined.
An initial filtering algorithm, REPI, was developed previously (16) and the general concepts with revisions were incorporated into the current (automated detection) algorithm used in the CIBSI 2.0 program. Filtering and subsequence selection were used to remove potential biasing caused by reference sequence choice and by other sources (i.e. primers). When PCR amplification was used, microarrays were hybridized in the presence of only primers to determine locations where they resulted in hybridization. Any portions of the ProSeqs that hybridized with the primers were masked as N calls so that the HybSeq did not contain biased information. Normally the primers are designed to be outside the Pro-Seq region to minimize the interference caused by primers, and so minimize the bases to be masked. There is still the chance that some bases require masking because with the large number of primers used in the multiplex, short stretches of a ProSeq not corresponding to primer locations may still hybridize with the primers. Such regions could be removed from the reference sequences and so not appear on the microarray. However, determining such locations are a difficult and time-consuming task that for most cases is not worth the effort. The first subtask of ProSeq identification Task(I) is noted in Figure 1 and shown schematically in detail in Figure 2 . This subtask uses a procedure to examine a Hyb-Seq to find the longest possible subsequence of base calls (SubSeq) that can be submitted as a query to BLAST.
It produces a group of SubSeq that contain all portions of a HybSeq that have a chance of producing a limited list of returns from a BLAST query. When a HybSeq has two regions separated by a long stretch of continuous N calls, the relational positioning of the two regions cannot be trusted and so must be sent as separate queries. In addition for shorter subsequences, the number of base calls that must be made is dependent on the length. It was also recognized that for very long sequences a longer WORD size in BLAST may be used. A detailed description of the criteria and process used for each step is contained in Supplementary Data. Upon completion, the algorithm returned to the Task(I) loop and performed the BLAST subtask.
The database query subtask performed a batch similarity search of a database using SubSeq as the queries. The BLAST program used was the NCBI Blastall -p blastn version 2.12 with a defined set of parameters. The masking of low complex regions was performed for the seeding phase to speed up the query; however, low complexity repeats were included in the actual scoring. The entire nucleotide database from NCBI acquired on February 7, 2006 was used as the reference database. (Note that earlier images of the database were used during development but all experiments were rerun with the algorithm as described with the Figure 3 . Detailed schematic representation of the second subtask of ProSeq identification Task(I), organism identification for an individual SubSeq. Each SubSeq sent to BLAST returned a list of possible matches contained in a Return array that was sorted through to find best bit score/expect value pair (MaxScore). If the MaxScore was greater than MIN (10 À6 ), all returns that had this best Score were sorted into a new array Rank1. Detailed SubSeqs requirement is described in Supplementary Data.
image of the database obtained on this date.) The default gap penalty and nucleotide match score were used. The nucleotide mismatch penalty, -q, parameter was set to À1 rather than the default. The results of any BLAST query with an expect value <0.0001 were returned in tabular format from the blastall program. The information about each return (bit score, expect value, mismatches, length of match) was placed in the Return{hash key}{info} hash using the SubSeq identity as the hash key for further analysis.
The next subtask of ProSeq identification Task(I) carried out was the determination of SubSeq() states and is shown in Figure 3 . The BLAST algorithm gives a ranking score which can be reported as accounting for the size of the database (expect value) or not (bit score). The full taxonomic classifications of every return for a SubSeq were retrieved from the NCBI taxonomy database obtained on February 7, 2006. Using the scores and taxonomy relationships it was possible to find a reduced number of returns that had the best match with the HybSeq. These results were summarized by identifying the taxonomic class to which all the returns belonged to, 'identified organism', and a parameter that indicated how they are related to each other, 'organism uniqueness'. A detailed description of the steps is contained in Supplementary Data. After each SubSeq was examined, the algorithm moved to the next subtask, which was to determine the identified organism of the ProSeq from the SubSeq (Figure 4 ). The subsequences from the same ProSeq were only allowed to support a single 'identified organism' determination. The procedure shown in Figure 4 demonstrates the decision method used to arrive at this determination (detailed description in Supplementary Data). After the subtask covered in Figure 4 was completed, the ProSeq identification Task(I) loop continued until all ProSeqs were examined. A list of ProSeqs that had detected organisms was built up in the Result1 array.
After the ProSeq identification Task(I) was completed, ProSeq grouping Task(II) (Figure 1 ) was used to examine the identified organism values listed in Result1 and grouped them together if they identified the same taxonomic class. Each entry in Result1 was examined and a new entry was created in Result2 if the identified organism did not appear in this list. The entries of Result2 represented the distinct individual organisms identified, but might still contain redundant information. When the ProSeqs were designed to detect the same organism and they all hybridized well, this grouping led to a reduction in redundant information being reported. But, when one ProSeq did not hybridize as well for a variety of possible reasons, multiple entries would appear in Result2 that actually represent hybridization from the same pathogen. This is because there is an alternative cause for the ProSeq hybridizing in this manner. This hybridization could be caused by two different but closely related organisms both being present in a sample and hybridizing to the microarray. Because we have not yet developed methods to distinguish these cases, no further reduction of the list of organisms is made for in ProSeq grouping Task(II) in cases where the level of identification varied on different ProSeq targeted for the same organism.
Although it was difficult to relate results from separate Pro-Seqs to each other, it was desired to have a simple final detection decision be made in pathogen determination Task(III). The first task was specifically implemented so that information about what a ProSeq was intended to detect was not considered and the second task only minimal consideration of this was taken into account. This allowed these initial tasks to be capable of recognizing not only just positive and negative identifications of target pathogens but also cases that were indeterminate. In the final task, the algorithm considered whether the identified organisms belonged to the list of organisms the ProSeqs were designed to detect. The task would group organisms from ProSeq grouping Task(II) together that belonged to or were child classes of the taxonomic class of a target organism. The taxonomic class reported was the common taxonomic group of all the organisms. When all the ProSeqs for a pathogen hybridized well, a fine level discrimination was reported. But if one or more ProSeqs hybridized less well, the reported positive target pathogen was only identified at the level of the less detailed level. This is conservative because methods have not yet been developed to clearly discriminate mixtures of very closely related organisms causing different ProSeqs to hybridize from variable hybridization of a single organism on several ProSeqs. The results of all When there was a single SubSeq or one scored much better than the other, the ProSeq inherited the properties of that SubSeq. Detailed SubSeqs requirement is described in Supplementary Data. three tasks were reported and a more experienced user can view ProSeq grouping Task(II) results to clarify some cases. Note that organisms identified in ProSeq grouping Task(II) that only belonged to target pathogens were reported as positives. Clear negative ProSeqs were not mentioned in the output. ProSeqs that were indeterminate or that detected close genetic organisms were never reported as positives. These organisms were instead reported as being detected.
A resequencing microarray (RPM v.1) was designed previously for detection and sequence typing of 20 common respiratory and 6 CDC category A biothreat pathogens known to cause febrile respiratory illness based on ProSeqs without relying on predetermined hybridization patterns (15, 16, 18 
Purified Chlamydia pneumoniae nucleic acid samples with 10-1000 genome copies (via method in Lin et al., submitted for publication) were chosen to illustrate how pathogen detection and identification were done when multiple ProSeqs were targeted for the same pathogen. RPM v.1 has three highly conserved ProSeqs selected from the genes encoding for the major outer membrane proteins VD2 and VD4, and the DNA-directed RNA polymerase (rpoB) gene. The HybSeqs from the different samples differed only in the number of unique base calls as shown in Table 1 . The percentage of the ProSeq called varied from 80 to 100% except for one case at a concentration of 10 that had only 11% of the rpoB ProSeq producing unique base calls. Because the samples at this concentration are not reproducibly generating the same percentage of base calls, this is probably the detection limit of this ProSeq of the assay. Table 1 listed the determinations made for the SubSeq and at the end of each task for the various samples. The ProSeq from the different cases produced the same number of SubSeqs. These SubSeqs from different samples reported different bit scores for the same top ranked returns from BLAST. In fact VD2 and VD4 produced exactly the same results. The NCBI taxonomy database classified the returns into four distinct groups, which represented the C.pneumoniae taxonomic group and three child strain groups. AE001652, AE002167, AE017159 and BA000008 appeared in the returns of all the ProSeqs for each sample, since they represented database entries of completely sequenced genomes. One rpoB Sub-Seq produced for its organism uniqueness, SeqUniqu. All other SubSeqs were TaxAmbig as multiple returns from different taxonomic classes were returned. Since the VD2 and VD4 ProSeq each have a single SubSeq, Task(I) assigned the Pro-Seq the state of the SubSeq. For the rpoB ProSeq, the bit score of one SubSeq was large enough that the algorithm assigned that SubSeq's identification to the ProSeq. Task II of the algorithm grouped all three ProSeqs together since they all had the same identified organism and TaxAmbig was assigned. The result of Task(III) was positive for target pathogen C.pneumoniae and this decision was straightforward as all the ProSeqs agreed with each other and belonged to the same target pathogen taxonomic class. Although the rpoB ProSeq was SeqUniqu, this was not the final conclusion for Task(II) as the ProSeq that was SeqUniqu was not the child taxonomic group and other ProSeq were TaxAmbig. The three recognized strains scored the same, which indicated that the sequence selected for the ProSeqs was very conserved and would not allow discrimination between the strains. Influenza and Human Adenovirus (HAdV) were the only pathogens that had ProSeq selected that would permit detailed strain level discrimination as discussed in previous work (15, 16) . This previous work using manual analysis found that the microarray results were in excellent agreement with the conventional sequencing results for clinical samples. A few of the results of running the CIBSI 2.0 program using the updated NCBI database on the raw microarray results are presented in Table 2 (the results for all samples used in the previous work are presented in Supplementary Table A) . The identified organisms were not identical to the original findings due to the difference in database used and because all ProSeqs were considered rather than only the Flu A and B hemagglutinin. In fact, the conventional sequencing results that were submitted to NCBI from that work were found for every sample to be among the returns with the best score for the hemagglutinin ProSeq (Supplementary Table B ). It should be noted that the previous work based its analysis upon only the results of the hemagglutinin ProSeq. For 8 of 13 Influenza A and 3 of 12 Influenza B cases, the results of ProSeq identification Task(I) and ProSeq grouping Task(II) found that the conventional sequencing was the single best return for the hemagglutinin ProSeq. Owing to the large number of isolate sequences in the database for the hemagglutinin gene it was not surprising that in some cases a single unique entry was not found. In each of the remaining five Influenza A samples, the other sequences returned differed by <0.2% from the conventional sequence. The fewer samples with unique isolate identifications for Influenza B were due to an older reference sequence used for the ProSeq, which allowed less Note: Within a row the first listing of a specific strain was followed by a two-letter abbreviation used in the remaining columns of that row. hybridization to occur (18) . This also meant that when multiple sequences were returned for a sample they represented greater genetic variation, up to 2%. As a result of the current method of making pathogen determination Task(III) level identification, the final organism reported was less specific (H3N2 or Flu B) for every sample than what was reported as possible in ProSeq grouping Task(II). For HAdV samples, the algorithm also reproduced the finer scale discriminations that had been made previously by manual methods (data not shown).
The next example of detection for the Mycoplasma pneumoniae pathogen demonstrated a case where there was only a single ProSeq for the target pathogen. A total of 48 test samples were performed using multiplex PCR (via method in Lin et al., submitted for publication) where for 46 of the samples M.pneumoniae organism was spiked into nasal wash with several other pathogens from 100 to 100 000 colony forming units per ml, the remaining 2 samples were purified with nucleic acid from culture stock at a concentration of 1000 genome copies per reaction volume. This ProSeq was also not optimal for fine discrimination because it was selected from a highly conserved region (345 bp) of the cytadhesin P1 gene. In every case taxonomic database entries for M.pneumoniae or its one recognized distinct strain tied for MaxScore (Supplementary Table 3 ). To better understand these returns, the database sequences were examined and subdivided into three groups of sequences, A, B and C, based on how well they matched the reference sequence used to make the ProSeq. The placement of the database entries into the three groups was determined from a CLUSTAL alignment of the sequences of this gene. This alignment confirmed that the database entries differed significantly more from each other in regions not represented by the ProSeq and contained sufficient variability that would have allowed finer discrimination. Members of Group A exactly matched the ProSeq and could not be distinguished between on the microarray. Similarly, members of group B matched the Pro-Seq except at the 199th position where the base called was C rather than T. Group C sequences contained a few database entries that were more variable and might be distinguished from other entries within the ProSeq. For the 48 experimental tests of M.pneumoniae, as much as 80% of the ProSeq hybridized for 19 samples, yet only 5 of these samples had an unambiguous base call at the 199th position. When it was unambiguous, it always matched group B sequences. In the cases where an N base call was made at the 199th location, both groups A and B sequences were returned with the same score. Regardless of this, the target pathogen positively identified was M.pneumoniae for every sample tested.
These examples showed how decisions were made independent of whether single or multiple ProSeqs were dedicated to a target pathogen. They also illustrated that the level of discrimination possible was strongly determined by the quality of the selected ProSeq. It is possible that for some pathogens fine level discrimination is not required and the currently tested selections on RPM v.1 would provide satisfactory information. The CIBSI 2.0 algorithm demonstrated its capability to automatically report the maximum level of discrimination that could be supported by the HybSeq information.
To demonstrate how the algorithm handled closely related genetic species, a sample of a non-targeted pathogen was considered using multiplex PCR (via method in Lin et al., submitted for publication). For Variola major virus, one of the biothreat pathogens on the RPMv.1, the validation runs demonstrated that Variola major virus purified DNA templates of plasmids were always positively identified when detected (Table 3) . Table 4 shows the results when purified Vaccinia genomic DNA was spiked into nasal washes and processed at various concentrations using multiplex PCR. The array has two ProSeqs from hemagglutinin (VMVHA, $500 bp) and cytokine response modifier B (VMVcrmB, $300 bp) genes for Variola major virus detection. The percentage of the ProSeq that hybridizes is sufficient that if hybridization patterns were only considered one might assume that this tile is identifying the presence of its target. This would indicate that reference sequence selected was not the best choice. However, when our algorithm was applied none of the samples is in fact identified as Variola major or minor virus. Vaccinia was always one of the Orthopoxvirus species listed with the highest scores for VMVcrmB ProSeq, but in only seven cases was it uniquely identified as the probable species detected. In only one sample at the lowest concentration and fraction of VMVcrmB hybridizing, did this ProSeq even identify Variola major and minor virus as one among the Orthopoxvirus species that could be the cause of the hybridization. The lower limit of detection for the amplification method used was between this concentration and the one above it for Variola major itself. Table 2 showed that HybSeqs split into multiple SubSeqs were capable of very specific identification.
To illustrate the other filtering performed, when multiplex strategies rather than generic were used for amplification Figure 5 also contains the raw and mask filtered results of this region for the A/Puerto Rico/8/34 sample. It was necessary to perform additional filtering to remove potential biasing caused by the specific primers as described in the methods. In the case shown in Figure 5 , a sequence of 18 bases present in the raw result are made N after filtering since they are in a location that interacts with the primers. If these base calls were included in the subsequences constructed, even though the HybSeq would still be split into the same number of SubSeq, the query for the ProSeq would favor an incorrect strain.
The algorithm we have developed successfully provided pathogen identification to the maximum level of detail possible (species or strain) depending on the quality of each Pro-Seq. This identification capability requires minimal input on the identity of the pathogens, making non-expert use feasible. The crucial feature incorporated that allowed complete automation was the use of taxonomic databases, which classify organisms into ordered groups and provide relationships between organism entries, allowing removal of redundancies, comparison of different related prototype sequences and simplification of data presentation. This allows databases, i.e. NCBI, that are redundant and subject to minimal curation but which constantly receive updated and new sequence information to be used with great success. Although we have demonstrated this using only the NCBI databases, other databases or custom made ones could have easily been used, which might improve performance. The algorithm is capable of providing accurate identifications at all analysis levels for pathogens that are less variable or are represented by highly conserved ProSeqs. For more variable or rapidly mutating pathogens, e.g. Influenza A virus, ProSeq identification Task(I) and ProSeq grouping Task(II) still provided accurate detailed identifications, but the pathogen determination Task(III) was unable to report fine scale discrimination. The comparison of the conventionally sequenced Influenza virus gene sequences illustrated that the algorithm is capable of automatically adjusting for updates in databases. The algorithm demonstrated its capability to properly distinguish hybridizations on a ProSeq caused by the specified pathogen from those caused by genetically close (near neighbor) strains and did not make incorrect identifications, eliminating one potential cause of false positives. Filtering the raw hybridization results served to reduce the computation time, accounted for potential primer interference and more importantly reduced potential biasing. This simple integrated algorithm provided sufficient and accurate identification, so that immediate use of the RPM v.1 or similar resequencing arrays and assay is possible. Although not discussed in this paper, the algorithm has successfully detected the presence of simulated multi-infections (Lin et al., submitted for publication). The algorithm as currently developed will detect mixtures when the organisms have sufficient variation; however, detection of a mixture of an organism and its mutation strain in a sample is uncertain in its present phase. In principle it may be possible to detect such mixtures as the resequencing microarray can detect and sequence diploid organisms.
Besides demonstrating the success of the CIBSI 2.0 program, the work involved in developing the algorithm allowed insight into the importance of proper ProSeq selection. The RPM v.1 was the first resequencing array designed specifically for multiple pathogen detection using database similarity searching and served as a prototype for this application. We have demonstrated that a single ProSeq with as few as 100 bp, when designed correctly, can be sufficient to unambiguously identify an organism. However, it is clearly indicated that several longer ProSeqs provide better confirmation and more detailed information of a pathogen. Although the algorithm provides accuracy equivalent to manual analysis for determinations of individual ProSeqs, the current algorithm is only partially successful in integrating information from multiple ProSeqs. The emphasis of the design to this point has been on capabilities that are generally applicable to any pathogen. We are incorporating these insights in our newer more comprehensive resequencing array designs. Improving on level of detail reported in pathogen determination Task(III) will require more information about an individual pathogen and may have to be developed for each specific pathogen or class of pathogens. This information is also required for the algorithm to identify which differences between a sample and database entries represent significant mutations. Future work will involve improving the use of the current taxonomic database or potentially developing a new relational database that is specific to our needs and then incorporating more specific information of target pathogens. The hierarchal design of the data analysis makes it easy to incorporate analysis that build upon the analysis already performed.
We have met with some success in the current version but want to have increased automated discrimination. We have a well-defined path to completing this aim. The use of properly designed resequencing microarrays and this automated detection algorithm provides a way forward to developing assays that can test for multiple organisms simultaneously while providing fine strain level discrimination giving access to information about detailed strain recognition, antibiotic resistance markers and pathogenicity. This is a capability that other approaches cannot currently provide. In addition, since the design of the original 30 kb RPM microarray, the possible sequence content of the current array has increased 10-fold to 300 kb and further increases in array density are still attainable. This, coupled with our identification algorithms, will allow the analysis of partial sequence information from even more organisms for applications such as differential diagnostics for illnesses with multiple potential causes (i.e. febrile respiratory illness), tracking of emergent pathogens, distinction of biological threats from harmless near genetic neighbors in surveillance applications and for tracking the impact of coinfections or super infections. The concept of categorizing and reporting different degrees of identification depending on the quality of samples and set of target sequences is not limited to resequencing microarrays but is more generally applicable to any platform that is capable of returning sequence level calls that can be used to query a reference DNA database. As the trend for assays that test for multiple pathogens increases, automated analysis tools, such as this one, become more crucial for rapid identification in simple formats useful to the non-expert on a day to day basis. The remaining hurdle to using resequencing microarrays as a routine assay method is now clearly the sample processing methods. Further automating these steps is an important area of future research and development.
The program can be obtained free of charge for research purposes by contacting the authors. Molecular dynamics simulations of human [Formula: see text]: the role of modified bases in mRNA recognition Accuracy in translation of the genetic code into proteins depends upon correct tRNA–mRNA recognition in the context of the ribosome. In human [Formula: see text] three modified bases are present in the anticodon stem–loop—2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms(2)t(6)A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm(5)s(2)U34) and pseudouridine (ψ) at position 39—two of which, ms(2)t(6)A37 and mcm(5)s(2)U34, are required to achieve wild-type binding activity of wild-type human [Formula: see text] [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390–13395]. Molecular dynamics simulations of nine tRNA anticodon stem–loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms(2)t(6) modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms(2)t(6)A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to ψ39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps. Ditchfield, R., Hehre  A novel endonuclease IV post-PCR genotyping system Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection. Nucleic acid assays utilizing cleavage enzymes to generate a fluorescent signal from dual-dye-labeled oligonucleotide probes have been extensively used in diagnostics (1) (2) (3) . In these assays, the ability of fluorescent dyes to transfer energy absorbed from light to nearby molecules forms the basis of homogeneous nucleic acid based assays (1, 4, 5) . In the unhybridized state the fluorophore and quencher are within close proximity, which allows the quencher to absorb energy from the fluorophore to affect quenching. The use of fluorogenic probes in a 5 0 -nuclease polymerase assay, where the probe is enzymatically cleaved to release the fluorophore (2) , is well known. The signal amplification reaction utilizes invasive cleavage with structure-specific 5 0 -nucleases (6) . The method requires annealing of two oligonucleotides, called the upstream oligonucleotide and the probe, to a target sequence, which results in the formation of a unique substrate for the 5 0 -nuclease. In the DNAzyme-PCR strategy the primer contains a target-specific sequence and harbors the antisense sequence of a 10-23 DNAzyme. During amplification, amplicons are produced containing active sense copies of DNAzymes that cleave a reporter substrate included in the reaction mixture (7) . A target nucleic acid sequence can be amplified exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H (RNA cleavage) and a DNA-dependent RNA polymerase (8) . Cycling probe technology represents a simple method for the detection of DNA target sequences, utilizing a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target (9) .
Apurinic/apyrimidinic (AP) sites in DNA arise via spontaneous or mutagen-induced hydrolysis of the N-glycosylic bond, or through the repair activity of DNA glycosylases (10) . If left unrepaired, AP sites are potentially lethal or mutagenic (11). To cope with the deleterious consequences of AP sites, organisms possess AP endonucleases that initiate the repair of these DNA lesions (10) . Class II AP endonucleases initiate repair by catalyzing the hydrolysis of the 5 0 -phosphodiester of an abasic site to generate a 3 0 -OH group and a 5 0 -abasic phosphate residue (10) . The substrate specificity of human apurinic endonuclease on synthetic substrate analogs has been reported previously (12) .
Here we describe a novel endonuclease IV (Endo IV) assay substrate ( Figure 1C ) that mimics the abasic lesions that normally occur in double-stranded DNA ( Figure 1A and B). The first lesion ( Figure 1A ) is a typical abasic (apurinic or apyrimidinic site generated by spontaneous or enzymatic loss of a nucleic acid base. The second lesion ( Figure 1B) is an atypical abasic site appearing as a result of inherent instability of the 3 0 -phosphodiester bond in abasic deoxyribose in lesion 1 or its cleavage by a Class I AP endonuclease. As shown by the arrow, AP endonucleases cleave the phosphodiester linkage at the abasic sites. In the novel Endo IV assay a short probe with a fluorophore linked through a phosphate at the 3 0 end and a short enhancer oligonucleotide generate an artificial, lesion 2 type Endo IV substrate. This arrangement allows the specific and efficient cleavage of the phosphodiester bond of the probe by Endo IV and release of fluorescent dye ( Figure 1C ). The addition of a quencher at the 5 0 end of the probe allows quenching of the fluorescence in the uncleaved probe. The specific cleavage of the phosphate bond and the generation of fluorescence is the basis of the new assay.
In this study we used endonuclease IV from Escherichia coli (Endo IV). The enzyme functions as a Class II AP endonuclease and as a 3 0 terminal phosphodiesterase (13, 14) . Endo IV is enzymatically similar to exonuclease III, the major AP endonuclease of E.coli, but it differs from the latter enzyme mainly in lacking the associated exonuclease activity (14) . It was also found that E.coli Endo IV is fairly heat stable to $70 C (15, 16) .
Full characterization for all the compounds described below can be found in the Supplementary Data.
A total of 102 unrelated Centre Etude Polymorphism Humaine DNA samples were obtained from the Coriell Institute of Medical Research (http://locus.umdnj.edu/) after specifying that the DNA samples should be used for research purpose only. The list of templates used is available at http:// snp500cancer.nci.nih.gov.
For the synthesis of the fluorophore-modified controlled pore glass(CPG) supports, please see the Supplementary Data.
Fluorogenic probes were synthesized on fluorophore-linkerbased CPG supports (Scheme 1) using standard 3 0 -DNAphosphoramidites. An Eclipse Dark Quencher was introduced at the 5 0 end using the corresponding phosphoramidite (www. glenresearch.com). Probes were purified by reverse-phase high-performance liquid chromatography (HPLC), dried and re-dissolved in 1· TE buffer. A nearest-neighbor model was applied to calculate extinction coefficient (e 260 ) of oligonucleotides (17) . A 260 measurements were made in PBS (pH 7.2) at ambient temperature and assumed to be a random coil DNA structure in solution. For each Eclipse Quencher, fluorophore 1 (FL1), fluorophore 2 (FL2) or fluorophore 3 (FL3) substitution an e 260 correction of +6600, +18 500, or +25 100 or 28 600 M À1 cm À1 was used, respectively.
Preparation of labeled probes with aminohexanoylextended hydroxyprolinol linker (FI 4, Table 1) 3 0 -Hydroxyprolinol modified probe precursor was synthesized using a modified CPG support (18) , and 3 0 -DNAphosphoramidites and Eclipse Quencher phosphoramidite on a 0.2 mmol scale. The probe was purified by reversephase HPLC using a gradient of acetonitrile in 0.1 M triethylammonium bicarbonate buffer (pH 8-9) followed by drying in a SpeedVac evaporator. The probe precursor was redissolved in 50 ml of dry DMSO and treated with 10 mmol of pentafluorophenyl N-FMOC-6-aminohexanoate (19) for 10 h. The FMOC-aminohexanoyl modified probe was isolated by reverse-phase HPLC and deprotected by treatment with concentrated ammonium hydroxide for 1 h at room temperature. The deprotected oligonucleotide was further purified by reverse-phase HPLC using the triethylammonium bicarbonate buffer as described above, dried and re-dissolved in 50 ml of dry DMSO. To this solution, 10 mmol compound 8c (Scheme 2) and 1 ml triethylamine were added. After being kept at room temperature for 10 h the reaction was combined with 1 ml of 2% solution of NaClO 4 in acetone. The precipitated crude conjugate was further purified by reverse-phase HPLC, dried and reconstituted in 1· TE buffer. An additional molar absorbance (e 260 ) of 28 500 M À1 cm À1 was used in the probe concentration calculation to correct for the presence of the dye.
The synthesis of special CPG supports for the preparation of required oligonucleotide probes are shown in Schemes 1-3. Methyl 3-(3-chloro-2,4-dihydroxyphenyl) propanoate (4a) (Scheme 1) was synthesized starting from 3-chloro-2,4dimethoxybenzaldehyde (1) (20) , which was converted to substituted cinnamic acid 2 by Knoevenagel condensation. Intermediate 2 was hydrogenated in the presence of Pd/C to yield substituted phenyl propionic acid 3, which was first demethylated with HBr/HOAc and then converted to the methyl ester 4a.
Synthesis of the pentafluorophenyl ester dye intermediates 11 required to synthesize the dye-modified CPG supports 12 is shown in Scheme 2. Compounds 4a and b were reacted with substituted phthalic anhydrides in the presence of AlCl 3 to give the benzophenones 5, which were reacted with the resorcinol analogs 6 (a-c) in trifluoroacetic acid/ methanesulfonic acid to yield the carboxyethyl substituted dyes 7. Treatment of compounds 7 with trifluoroacetic anhydride generated lactones 8, which were first reacted with the DMT-protected hydroxyprolinol 9 (21) and then with trimethylacetic anhydride to afford the protected dyes 10. Reaction of intermediates 10 first with succinic anhydride and then with pentafluorophenyl trifluoroacetate, yielded the PFP esters 11.
The reaction of the PFP ester 11 with the long chain aminoalkyl CPG (LCAA-CPG) yielded the desired CPG supports 12 as shown in Scheme 1.
Endo IV cleavage activity was measured in real-time as shown in Figure 2 . The model system in Figure 3A was used to determine optimum E.coli Endo IV (Trevigen, Gaithersburg, MD) concentration, magnesium concentration and pH; the latter two were determined to be at 20 mM Tris buffer, pH 8. 6 
The endo IV reaction was prepared by adding 5 ml Endo IV Master Mixture to 5 ml PCR mixture. Reaction was incubated at 50 C for 50-60 min. Fluorescent detection was performed on ABI 7700 or 7900 in the two channels typically used to detect FAM (for FL1) and VIC (for FL2, FL3 and FL4) utilizing the ABI software for Allelic Discrimination. Alternatively, the fluorescence could also be detected in a Figure 3B and D, respectively. The reaction mixture contained Endo IV at 0.04 U/ml concentration, probe and enhancer at 150 nM, and the target at 5 nM. The experiment was performed on LightCycler. LightCycler Ò LC24 Real-Time PCR System. All assays unless otherwise indicated were performed at least in triplicate.
Thermodynamic parameters of duplex formation were derived by the van't Hoff analysis methods. The shape of each melting curve was fitted to the two-state model with linear base lines (22) using a nonlinear least-square program (23) . Unless otherwise stated, T m of DNA duplexes were measured at a concentration of 5 · 10 À7 M in 1· PCR buffer containing 10 mM Tris-HCl (pH 8.5), 5 mM MgCl 2 and 40 mM NaCl, as described earlier (23) .
The MGB Eclipse Ò Design Software 3.0 (http://www. nanogen.com/products/customassays/mgb_ecliplse/) was used to design the primers for PCR. It was also used to calculate the T m of the enhancer and probe.
An example of the new Endo IV-based assay is shown in Figure 2 . A probe and an enhancer are hybridized to a complementary synthetic target with a one base gap between them.
The signal is substantially diminished in the absence of the enhancer. A rigid hydroxyprolinol linker ( Figure 3B ) between the oligonucleotide and fluorophore FL1, critical for sensitivity and specificity, was used in this experiment. The results in Figure 2 illustrate the importance of all components used in the assay. The cleavage of the fluorophore is highly target dependent and its rate is amplified in the presence of the enhancer oligonucleotide. Very low non-target-dependent cleavage is an important feature of the Endo IV assay.
Effects of pH and cations on E.coli Endo IV activity was determined using the probe, enhancer and target ( Figure 3A) . It was shown that Endo IV cleavage of the probe has a relatively flat pH optimum in the range between pH 8.5 and 9.5. For compatibility with post-PCR Endo IV amplification the assay buffer with pH 8.6 was chosen, similar to that of the PCR master mixture. Inclusion of the divalent magnesium cation in the buffer ranging from 0 to 20 mM, resulted in more than 2-fold increase in cleavage rate, the optimum range was $5-8 mM. A concentration of 5 mM was chosen for the Endo IV assay. It was determined that the monovalent cations, such as Li, Na, K and Rb, inhibit Endo IV cleavage activity for the concentrations studied in the range from 0 to 100 mM. An inhibition of $30% was observed at 20 mM monovalent cation concentration, which increased to $60-70% at 100 mM concentration. The optimum probe concentration was determined to be $600 nM, please see Supplementary Figures 1-3 . Figure 3A shows a model Endo IV assay that requires a target complementary to an enhancer and a probe labeled at the 5 0 end with an Eclipse Dark Quencher and a fluorescent dye at the 3 0 end. As shown, the probe (calculated T m ¼ 46.7 C) and enhancer (calculated T m ¼ 50.2 C) are separated by one base to mimic a natural abasic site. Four probes with different fluorescent dyes and linkers ( Figure 3B ) were evaluated for their performance in the new assay. Fluorophores FL1, FL2 and FL3 of the probes I, II and III (with similar quantum yields) contained a hydroxyprolinol linker between the 3 0 -phosphate of the probe sequence and the core of the dyes. Fluorophore FL4 of probe IV was analogous to FL3 of probe III, with an additional aminocaproic spacer introduced between the hydroxylprolinol moiety and the dye; the purpose of this spacer was to distance the fluorophore from the cleavage site.
The probe specificity was evaluated by comparing the cleavage rates in the presence and absence of target. Targetdependent (specific) and target-independent (non-specific) cleavage rates of the probe were measured as the percent target cleaved per minute. The results are summarized in Table 1 . Probes I-III demonstrated similar specific cleavage. A 2-to 3-fold decrease in the rate of non-specific cleavage for probes II and III versus probe I was observed. These low, non-specific cleavage rates translate into high-specific to non-specific cleavage ratios. It appears that chloro-or chloro/ methyl substitutions in the fluorophores of probes II and III, respectively, are beneficial for the reduction of non-specific cleavage. One possible explanation for this reduction may be an effect of increased hydrophobicity of FL2 and FL3 on enzyme activity. In the case of probe IV, with the extended hydroxyprolinol linker, the rate of specific cleavage almost doubled. However, the corresponding increase in the rate of non-specific cleavage was $20 times higher than that observed in probe III, substantially compromising the ratio of specific to non-specific cleavage rates. Once again, this result implies the existence of inhibiting interactions between Endo IV enzymatic activity and fluorophore which can be adjusted by moving the fluorophore away from the active center.
The rigid hydroxyprolinol linker with low non-specific cleavage rates utilized in probes I-III was identified as the linker of choice in the Endo IV assays.
The use of a flexible, straight chain linker (C6) instead of hydroxyprolinol-based linker increased the rates of nonspecific cleavage to even higher degree than the extended hydroxyprolinol linker and reduced the specific to nonspecific ratios to an unacceptable level (data not shown).
It appears that both the structure of the sterically constrained hydroxyprolinol linker and proximity of the bulky dye moiety to the cleavage site moderate cleavability of the 3 0 -terminal phosphodiester bond and practically eliminate non-template-dependent cleavage. Figure 4A shows the target, probe and the enhancer aligned with the target to indicate different gaps. The T m of the enhancer is typically chosen to be at least 5 C higher than that of the probe. The cleavage rates in the presence of enhancer are shown with 1-5 base gaps ( Figure 4B ). As expected, based on the natural abasic substrate requirement shown in Figure 1B , a one base gap gave the highest cleavage rate. The probe in the absence of enhancer showed a cleavage Table 1 . Effects of dyes and linkers on the rates and ratios of specific and non-specific cleavage Probe nos Dye Substitution on Figure 3B Cleavage rate Specific/non-specific rate ratios Target-specific %/min Target-non-specific %/min rate nearly equivalent to that of a 4 base gap in the presence of enhancer. As shown in Figure 4C , the maximum cleavage rate is achieved at a concentration of enhancer greater than $80 nM. In practice, the concentration of the enhancer is typically at least equivalent or larger than that of the probe. Some cleavage is observed with no enhancer present.
I FL1 R 1 -R 7 ¼ H; n ¼ 0 0.97 0.0031 313 II FL2 R 1 ¼ R 3 ¼ R 4 ¼ R 5 ¼ R 6 ¼ R 7 ¼ Cl; R 2 ¼ H; n ¼ 0 0.93 0.0012 775 III FL3 R 1 ¼ R 3 ¼ R 5 ¼ R 6 ¼ Cl; R 4 ¼ R 7 ¼ H; R 2 ¼ CH 3 ; n ¼ 0 1.04 0.0010 1040 IV FL4 R 1 ¼ R 3 ¼ R 5 ¼ R 6 ¼ Cl; R 4 ¼ R 7 ¼ H; R 2 ¼ CH 3 ; n ¼ 1 1.90 0.0260 73 - - - - - - - - - - - - - - - - -4 - - - - - - - - - - - - - - - -2 - - - - - - -3 - -5 3'-GCTGAGCCGGGAACGGGCGGTGTAACCGTGGCTGACACTGA -5' |||||||||| 5'-Q-CTCGGCCCTT Dye
Probe T m optimization Figure 5 shows how the cleavage rate of probes with different T m s changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in Figure 5 with calculated T m s of between 14 and 60 C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated T m s of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated T m or slightly above it. Performing the assay at a temperature slightly higher than the T m ( Figure 5 ) showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this assay.
The ability of the Endo IV assay to detect different target concentrations is shown in Figure 6 . It was observed that in the case of 125 nM target concentration, the probe-based fluorescence plateaued in $1 h, while in the presence of 25 nM target a fluorescence plateau was reached in $3 h. The limit of detection of this assay is 0.04 nM, clearly distinguishable from the stable background ( Figure 6 ). This stable background, a characteristic feature of the assay, is maintained over a period of 15 h. This property is also observed in the assay with other nucleic acid targets.
The ability of the Endo IV assay to discriminate mismatches at different positions of a 14mer probe is illustrated in Figure 7 . Mismatches were introduced in the probe one at a time, from positions 1 to 8. The Endo IV assay shows excellent specificity for the different mismatches from base 1 to base 6 with large match/mismatch signal ratios. As shown in Figure 7 , the mismatch signal is often about the same as that of the NTC or slightly higher. The exceptions are the difficult 4-G/T-and 8-T/G-mismatches (24) where match/mismatch ratios of $16 and 7 were observed, respectively. These ratios are quite adequate to differentiate matched and mismatched sequences. It appears (data not shown) that the discrimination in positions 1 and 2 is largely determined by substrate requirements while discrimination in other positions is determined by thermodynamic considerations. Experiments with a large number of assays have also indicated that, with 10mer to 12mer probes, satisfactory discrimination is observed with the mismatch position in all except the last three bases at the 5 0 end.
The exquisite specificity of the Endo IV assay appears to be the result of at least two factors. The first is the strict 
Relative Flourescence @ 60°C intrinsic substrate specificity requirement of the Endo IV enzyme which appears to require the rigid prolinol linker ( Figure 3B , n ¼ 0) used in the probes I-III. Second, the use of short probes (10mer to 12mer) gives the Endo IV a distinct advantage over most other assays where probes are typically much longer (1,2,6,25).
The application of the Endo IV cleavage as a post-PCR detection system is shown in Figures 8 and 9 . Following an asymmetric PCR using primers to produce predominantly single-stranded amplicon, two allele-specific probes, an enhancer and Endo IV enzyme are added to the amplification mixture and incubated isothermally for about an hour. The cleaved fluorescence from the two allele-specific probes, each labeled with a different fluorescent dye, is then plotted in a scatter diagram as shown in Figures 8 and 9 for the genotyping analysis of two single nucleotide polymorphisms (SNPs) which are of immediate importance to in cancer (http:// snp500cancer.nci.nih.gov). The scatter diagrams of two polymorphisms, namely agouti signal molecular epidemiology studies protein (ASIP-01) and adenomatous polyposis coli (APC-03) (http://snp500cancer.nci.nih.gov) are shown in Figures 8 and 9 , respectively. In both cases, a probe specific to the wild-type allele is labeled with FL1 and a probe specific for the mutant allele labeled with FL3 is used. The probe sets, designed to analyze ASIP-01 and APC-03 polymorphisms, were used to assay 102 unrelated human DNA samples obtained from the Coriell Institute. In the case of the ASIP-01 target the mismatch was situated two bases from the 3 0 end of the probe, while in the case of APC-03, the mismatch was six bases from the 3 0 end of the probe.
From our experience, the spacing of the allele clusters in Figures 8 and 9 are comparable or better than those scatter plots observed in other methods (2, 26, 27) . The exquisite specificity achieved with the Endo IV enzyme, combined with the shorter probes used and the high signal accumulation, gives this assay an advantage over other SNP assays. In comparison with other signal accumulating assays (2), the Endo IV assay has similar levels of high signal accumulation, but uses shorter probes with higher specificity than those assays. In the case of hybridization-based assays (5, 25) , the Endo IV assay has not only a signal accumulation advantage, but also uses shorter probes with a specificity advantage. In addition, the shorter probes allow much more flexibility in assay design compared to other methods with longer probes.
We disclosed a novel nucleic acid signal detection system based on the cleavage of a phosphodiester linkage in a dualdye-labeled oligonucleotide probe by the Endo IV enzyme from E.coli. The parameters influencing the activity of the Endo IV enzyme were optimized. This optimized Endo IV detection assay requires, in addition to an oligonucleotide probe substrate, a fluorophore attached through a relatively rigid linker to the oligonucleotide and a helper oligonucleotide. The rigid linker conveys the exquisite specificity of Endo IV cleavage of the phosphodiester linkage in the probe and shows high-specific/non-specific rate ratios. The synthesis of fluorophore analogs coupled to a rigid linker and solid support, used in the synthesis of labeled oligonucleotides, was also disclosed. Excellent mismatch discrimination is observed from positions 1 to 6 in a 12mer probe. The ability to put the mismatch in any one of positions 1-6, allows for flexibility in probe design for genotyping assays where sequence and secondary structural constraints are considered. This method allows the use of probes shorter than those typically used in other methods (28) contributing to the improved mismatch discrimination.
The combination of the Endo IV assay with a PCR as a post-amplification detection system has been demonstrated. Post-PCR Endo IV genotyping shows excellent spacing of scatter plot endpoint signals of no template controls, wildtype, mutant and heterozygous samples allowing clear allele differentiation. In contrast, other endpoint genotyping methods may suffer from overlapping endpoint signals due to PCR efficiency issues (25, 29) . This Endo IV post-PCR genotyping has been successfully used in an industry setting for >5000 genotype assays. Pandemic influenza preparedness: an ethical framework to guide decision-making BACKGROUND: Planning for the next pandemic influenza outbreak is underway in hospitals across the world. The global SARS experience has taught us that ethical frameworks to guide decision-making may help to reduce collateral damage and increase trust and solidarity within and between health care organisations. Good pandemic planning requires reflection on values because science alone cannot tell us how to prepare for a public health crisis. DISCUSSION: In this paper, we present an ethical framework for pandemic influenza planning. The ethical framework was developed with expertise from clinical, organisational and public health ethics and validated through a stakeholder engagement process. The ethical framework includes both substantive and procedural elements for ethical pandemic influenza planning. The incorporation of ethics into pandemic planning can be helped by senior hospital administrators sponsoring its use, by having stakeholders vet the framework, and by designing or identifying decision review processes. We discuss the merits and limits of an applied ethical framework for hospital decision-making, as well as the robustness of the framework. SUMMARY: The need for reflection on the ethical issues raised by the spectre of a pandemic influenza outbreak is great. Our efforts to address the normative aspects of pandemic planning in hospitals have generated interest from other hospitals and from the governmental sector. The framework will require re-evaluation and refinement and we hope that this paper will generate feedback on how to make it even more robust. In this paper, we present an ethical framework for pandemic influenza planning. The ethical framework was developed with expertise from clinical, organisational and public health ethics and validated through a stakeholder engagement process. The ethical framework includes both substantive and procedural elements for ethical pandemic influenza planning. The incorporation of ethics into pandemic planning can be helped by senior hospital administrators sponsoring its use, by having stakeholders vet the framework, and by designing or identifying decision review processes. We discuss the merits and limits of an applied ethical framework for hospital decision-making, as well as the robustness of the framework.
The need for reflection on the ethical issues raised by the spectre of a pandemic influenza outbreak is great. Our efforts to address the normative aspects of pandemic planning in hospitals have generated interest from other hospitals and from the governmental sector. The framework will require re-evaluation and refinement and we hope that this paper will generate feedback on how to make it even more robust.
As the world prepares for the emergence of a pandemic strain of influenza, trans-national, national and local organisations and agencies are designing plans to manage community outbreaks. In addition, the medical commu-nity is identifying scientific research priorities and needs related to the anticipated pandemic [1] [2] [3] [4] [5] . There is also a need to examine the ethical issues that arise from planning for a public health crisis of this magnitude. Who should get the limited supply of antivirals? Are health care workers duty-bound to care for the ill in a pandemic when they may have competing familial obligations? Who will be prioritized for scarce ventilated hospital beds? When should hospitals cancel elective surgeries or restrict hospital visitation? To date, the bioethics community has been slow to respond to public health issues in general [6, 7] , and pandemic influenza planning in particular [8, 9] . In this paper we discuss the need for ethics in pandemic influenza planning and discuss the ethical framework we developed to guide pandemic planning in hospitals.
In the only article we could find that has an in-depth analysis of the ethics of pandemic planning, Kotalik offers an ethical analysis of the pandemic plans of three countries. His arguments are primarily about the ethics of pandemic planning efforts, as opposed to the ethics in pandemic planning. For example, he argues persuasively that it is problematic that all three countries' plans accept particular conditions of resource scarcity as planning assumptions [10] . While Kotalik has raised important issues about the ethics of pandemic planning in his article, our ethical framework focuses specifically on providing guidance to decision-makers about ethical issues in pandemic planning. This includes providing guidance on how to design an ethical process for decision-making, and providing guiding ethical values for the consideration of substantive issues.
The framework here proposed is an example of practical ethics that attempts to provide decision-makers with an introduction to and articulation of generally accepted ethical principles or values. The significance of this ethical framework is a) in the unique collaborative approach taken to its development that involved ethicists with different areas of expertise and a variety of health care stakeholders, and b) that it fills an important need in pandemic planning for an ethical framework to guide decision-making that has been unmet in most pandemic planning processes world wide.
One of the characteristics of a public health crisis is that health needs overwhelm available human and material resources. Difficult decisions must be made about how, where and to whom resources should be allocated. Medical science provides valuable information to help make these decisions. However, science alone is insufficient. Now consider that resource allocation decisions are just one kind of decision decision-makers face in preparing for, and getting through an influenza pandemic [9] . As a few scholars have begun to point out, pandemic planning needs to take ethical considerations seriously, and not allow the urgency of logistical and scientific needs to sideline a discussion of ethical considerations [10, 11] .
Kotalik argues that as "every discourse about health care has not only a scientific but also a moral dimension, [pandemic influenza] plans also presuppose certain ethical values, principles, norms, interests and preferences" [10] . It is important to make these presuppositions explicit, because, as the SARS experience in Toronto taught health care organisations, the costs of not addressing the ethical concerns are severe: loss of public trust, low hospital staff morale, confusion about roles and responsibilities, stigmatization of vulnerable communities, and misinformation [12] [13] [14] . Another key insight from SARS that we overlook at our peril was that in times of crisis, "where guidance is incomplete, consequences uncertain, and information constantly changing, where hour-by-hour decisions involve life and death, fairness is more important, rather than less [emphasis added]" [14] . As we shall argue, fairness considerations are both procedurally and substantively important: there is a need for fair decision-making processes, as well as equitable distributions of scarce human and material resources.
Take the example of triaging ventilated beds in an ICU. In theory, decision-makers rely on scientific evidence to determine how best to maximise benefit in the allocation of ventilated beds, but science cannot tell us whether or not the initial decision to maximise benefit is just. Because the notion of maximising benefit is derived from a reflection on values, ethical analysis is required to determine why a utilitarian approach to triage though maximisation of benefit is preferable to the assignment of ventilated beds on a different basis, for example that of greatest need. Even if the utilitarian maximisation of benefit is thought to be ethically sound, how to implement a system based on this criterion is not ethically straightforward, and requires ethical reflection about what counts as good stewardship, and about the moral obligation to demonstrate transparency, accountability, fairness and trustworthiness in the allocation of scarce resources.
The importance of ethics to pandemic planning is in the "the application of value judgements to science" [15] , especially as they are embedded in planning assumptions, and within the practice of medicine itself. For example, while ethics might have little to contribute to understanding the mechanism of influenza virus transmission, it can make a significant contribution to debates such as what levels of harm the public are prepared to accept, how the burdens of negative outcomes should be distributed across the population and whether or not more resources should be invested in stockpiling antiviral medications.
The use of ethical frameworks to guide decision-making may help to mitigate some of the unintended and unavoidable collateral damage from an influenza pandemic. As Kotalik argues, the incorporation of ethics into pan-demic plans can help to make them "instruments for building mutual trust and solidarity at such time that will likely present a major challenge to our societies" [10] . Using ethical frameworks to help guide decisions can offer greater assurance that the values instantiated within them, such as accountability, transparency and trust, will be carefully thought about in decision-making and when reviewing decisions with stakeholders.
One of the key lessons from the Toronto SARS experience was that health care institutions and their staff could benefit from the development of ethical frameworks for decision-making [12] . The intention of this section is not to systematically derivate from normative theory the values and principles in the framework. This paper has a more narrow focus -it is an example of applied/practical ethics that attempts to introduce and articulate values that are already commonly accepted. It is not our intention to comprehensively defend the values in the framework, but rather to show from which areas of scholarship they were drawn, articulate their relevance to pandemic planning, and to demonstrate their discursive legitimacy through a process of stakeholder engagement and vetting. To our knowledge, no other pandemic planning process has attempted to a) develop an ethical framework to guide pandemic influenza planning and b) assess an ethical framework's robustness and resonance in the community of its intended users. Thus, the significance of the procedural elements of the development of the framework is not to be minimized, nor are the insights we have gleaned from implementing the framework in health care organisations and in a governmental setting.
Building on key lessons from SARS [12] [13] [14] and the "emergency ethics" literature and drawing on our expertise in clinical, organisational, and public health ethics, we identified key ethical processes and values that are relevant for health care organisations. These values were presented to and vetted by a variety of health care stakeholders. Thus, this framework is the product of an iterative and inclusive process.
In Ontario the need for guidance on the ethical issues pertaining to an influenza pandemic has been widely acknowledged. As word of our work on an ethical framework for Sunnybrook and Women's College Health Science Centre (S & W) became known, we were invited to join other hospitals' pandemic planning efforts. There was also broader sectoral interest in ethics, and we were invited to join the Ontario Ministry of Health and Long Term Care's (MOHLTC) efforts to design a pandemic plan.
Our working group was formed in response to the pandemic planning initiative that took place at S & W in early 2005. The hospital's Clinical Ethics Centre was invited to provide ethics support in this planning initiative. It soon became apparent that the scope of the issues went beyond the purview of clinical ethics to include organisational and public health ethics. Expertise in organisational and public health ethics was quickly procured through the University of Toronto Joint Centre for Bioethics which is a partnership between the University and sixteen affiliated healthcare organizations that includes S & W among its partners. S&W was subsequently de-amalgamated into Sunnybrook Health Sciences Centre and Women's College Hospital, thus the ethical framework is currently being implemented at Sunnybrook HSC.
As the framework took shape, we were invited to join the MOHLTC planning efforts. We began to work with the Vaccine and Antiviral working group at the MOHLTC, and we adapted our work to meet the related but distinct challenges facing government. While our work with the MOHLTC began with the Vaccine and Antiviral working group, the ethical framework we developed for the MOHLTC was eventually included in the Ontario Health Pandemic Influenza Plan [16] not as an annex to the section on vaccines and antivirals as we had originally anticipated, but as an ethical framework for the plan as a whole.
Expertise in clinical ethics was important to the development of this framework because of the knowledge, skills and experience clinical ethicists need to address dilemmas or challenges found in the daily clinical arena. An obvious challenge was how to integrate expertise in public health ethics into a framework designed to guide decision-making in clinical health care settings. A related challenge was to thoughtfully integrate generally accepted principles and values from clinical ethics with those in public health ethics. In order to meet this challenge, the authors turned not only to the respective ethics literature, but also to the SARS experiences of Toronto hospitals and health care providers. A review of the SARS literature, and that of public health ethics more generally, guided the integration of the public health and the clinical ethics perspectives [6, 9, 10, [12] [13] [14] [17] [18] [19] . The Toronto experience with SARS demonstrated that organisations faced unique ethical challenges when dealing with a public health crisis, and much of the ethics literature identified a need for greater forethought in how organisations can foster ethical decision-making in times of crisis [12] [13] [14] . We reasoned that the legitimacy of this framework would be enhanced by including insights from the analysis of a recent public health crisis like SARS.
Not surprisingly, the literature on clinical ethics has little to say about disaster preparedness and how to make decisions about such things as triage under extraordinary circumstances. The ethics literature on bioterrorism and battle-field triage informed our thinking and called our attention to important issues such as the duty to care, reciprocity, equity and good stewardship [20] [21] [22] [23] [24] [25] . The importance of having ethically robust criteria and policies developed in advance of a pandemic influenza outbreak is underscored in this literature, for "critical decisions like these should not be made on an individual case-by-case basis" and "physicians should never be placed in a position of individually deciding to deny treatment to patients without the guidance of policy or protocol" [22] . Robust disaster preparedness requires practising preventive ethics.
The ethical framework was vetted through S & W's Pandemic Planning Committee, the Joint Centre for Bioethics' Clinical Ethics Group (comprised of the affiliated health care organizations' clinical ethicists), the MOHLTC Vaccine and Antiviral Working Group, and the MOHLTC pandemic planning committee. Through this process, we refined the framework and we are grateful to these groups for their valuable insights.
The ethical framework is intended to inform decisionmaking, not replace it. It is intended to encourage reflection on important values, discussion and review of ethical concerns arising from a public health crisis. It is intended also as a means to improve accountability for decisionmaking and may require revision as feedback and circumstances require.
The framework is divided into two distinct parts, and begins with the premise that planning decisions for a pandemic influenza outbreak ought to be 1) guided by ethical decision-making processes and 2) informed by ethical values. Ethical processes can help to improve accountability and it is hoped that, to the extent that it is possible for ethical processes to produce ethical outcomes, the substantive ethical quality of decisions will be enhanced. Recognising, however, that ethical processes do not guarantee ethical outcomes, we have identified ten key ethical values to guide decision-making that address the substantive ethical dimensions of decision-making in this context.
In planning for and throughout a pandemic influenza crisis, difficult decisions will be made that are fraught with ethical challenges. Our framework around ethical proc-esses is based upon the "accountability for reasonableness" model developed by Daniels & Sabin [26] and adapted by Gibson, Martin & Singer [27] . This model provides a useful means of identifying the key elements of ethical decision-making processes. An extensive literature has developed around Daniels' and Sabin's accountability for reasonableness framework. The Daniels and Sabin framework has broad applicability across institutional settings and priority setting situations [28] [29] [30] [31] [32] [33] [34] [35] . Because the Daniels and Sabin framework applies deliberative theories of democratic justice to the specific problem of health care priority setting, and because it is unique in this regard, we felt it promoted the kind of deliberative approach to pandemic planning that this ethical framework is intended to support. Table 1 outlines the characteristics of an ethical decision-making process. Stakeholders will be more able to accept difficult decisions during a pandemic influenza crisis if the decisionmaking process has, and is perceived to have, ethical legitimacy.
The second part of the framework identifies ten key ethical values that should inform the pandemic influenza planning process and decision-making during an outbreak. These values are intended to provide guidance, and it is important to consider that more than one value may be relevant to a situation. Indeed, the hallmark of a challenging ethical decision is that one or more value(s) are in tension and that there is no clear answer about which one to privilege in making the decision. When values are in tension with one another, the importance of having ethical decision-making processes is reinforced (see above.)
The values identified in our ethical framework were based initially on previous research findings on ethics and SARS at the University of Toronto Joint Centre for Bioethics (JCB). This work was funded by a Canadian Institutes of Health Research grant in 2004 through 2006 and has led to several key publications on the ethical dimensions of SARS [14, [36] [37] [38] [39] . In particular, Singer et. al., in their seminal British Medical Journal article begin to identify key ethical values that were of relevance during the SARS epidemic in Toronto. These values were then further articulated by our working group and adapted for the pandemic influenza planning context. Through a discursive process of stakeholder consultation with public health specialists, ministry officials, S & W's pandemic influenza committee, and the Clinical Ethics Group at the JCB, we augmented the values to include two new values (stewardship and trust [40, 41] ) and refined the definitions of each value in light of the anticipated demands of a pandemic influenza crisis compared to a hospital-based epidemic such as SARS. The substantive values identified and articulated in the framework are not intended to be an exhaustive set, and they may underdetermine how best to achieve the overall goals of pandemic planning, which generally include the minimization of morbidity, mortality, and societal disruption. Nevertheless, this is not to say that that a procedural engagement about the overall goals of a pandemic response would not benefit from using the ethical framework to guide and shape debate. A description of the values that should guide decision-making can be found in Table 2 .
Included in the framework are "hot button" ethical issues that we identified through our work with Toronto hospitals and the MOHLTC. These issues were as follows: These "hot button" issues are not intended to be exhaustive, but rather they serve to illustrate how the values in the ethical framework can be used to identify key ethical aspects of decision-making.
Let us take the issue of targeting and prioritizing populations for vaccine and antivirals to illustrate how the values in the ethical framework can help guide decision-making. The values of solidarity and protecting the public from harm would require that priorities be set to maximize the capacity to help society ensure that the ill are cared for during a pandemic. Furthermore proportionality would require that decision-makers consider who within the community are most vulnerable to the contagion as well as who are most likely to benefit from immunization. A well-informed public conversant with the values in the ethical framework and aware of the expertise that informed the ranking of priorities for immunisation would be consistent with value of trust and the principle of transparency.
Lastly, while knowing how to use the framework to inform decision-making is vital, there is more to ensuring that the framework will be used or useful.
We have identified three necessary, if not exhaustive elements to the successful integration of ethics into hospital pandemic planning processes. These elements are 1) sponsorship of the ethical framework by senior hospital administration; 2) vetting of the framework by key stakeholders and; 3) decision review processes. 
There should be mechanisms in place to ensure that ethical decision-making is sustained throughout the crisis.
Decisions should be made explicitly with stakeholder views in mind and there should be opportunities for stakeholders to be engaged in the decision-making process. For example, decision-making related to staff deployment should include the input of affected staff.
Decisions should be publicly defensible. This means that the process by which decisions were made must be open to scrutiny and the basis upon which decisions are made should be publicly accessible to affected stakeholders. For example, there should be a communication plan developed in advance to ensure that information can be effectively disseminated to affected stakeholders and that stakeholders know where to go for needed information.
Decisions should be based on reasons (i.e., evidence, principles, values) that stakeholders can agree are relevant to meeting health needs in a pandemic influenza crisis and they should be made by people who are credible and accountable. For example, decision-makers should provide a rationale for prioritising particular groups for antiviral medication and for limiting access to elective surgeries and other services.
There should be opportunities to revisit and revise decisions as new information emerges throughout the crisis as well as mechanisms to address disputes and complaints. For example, if elective surgeries are cancelled or postponed, there should a formal mechanism for stakeholders to voice any concerns they may have with the decision. 
Duty to Provide Care The duty to provide care and to respond to suffering is inherent to all health care professionals' codes of ethics. In an influenza pandemic, demands on health care providers and the institutions in which they work will overwhelm resources. Health care providers will have to weigh demands from their professional role with other competing obligations to their own health, to family and friends. Health care workers will face significant challenges related to resource allocation, scope of practice, professional liability, and workplace conditions. Decision makers should:
• Work collaboratively with stakeholders and professional colleges in advance of an influenza pandemic to establish practice guidelines • Work collaboratively to develop fair and accountable processes to resolve disputes • Provide supports to ease this moral burden of those with the duty to care • Develop means through which institutions will handle appeals or complaints, especially with regards to work exemptions, or the vaccination/prophylaxis of staff Health care workers who are at increased risk because they are caring for patients with influenza must weigh familial obligations, and obligations to self with their professional duty to care. In addition, they may also have to comply with vaccination or antiviral regimens for prophylaxis which may conflict with their individual liberty.
The principle of equity holds that, all things being equal, all patients have an equal claim to receive needed health care. During influenza pandemic, however, tough decisions will need to be made about which health services to maintain and which to defer because of extraordinary circumstances. Measures taken to contain the spread of a deadly disease will inevitably cause considerable collateral damage. In an influenza pandemic, this will extend beyond the cessation of elective surgeries and may limit the provision of emergent or necessary services.
In allocating scarce resources, the value of equity could guide in developing fair criteria for allocation while consideration is given also to compensation for those who will not meet inclusion criteria yet are entitled to receive care.
Individual liberty is a value enshrined in health care practice under the principle of respect for autonomy. Under usual circumstances, health care providers balance respect for individual autonomy with a duty to protect individual patients from harm. In a public health crisis, however, restrictions to individual liberty may be necessary to protect the public from serious harm. Patients, staff, and members of the public may all be affected by such restrictions.
• Be proportional to the risk of public harm • Be necessary and relevant to protecting the public good • Employ the least restrictive means necessary to achieve public health goals • Be applied without discrimination Social distancing strategies that employ visitor restrictions in hospitals must be necessary for the protection of the public and must be proportionate to the threat being allayed.
Individuals have a right to privacy in health care. In a public health crisis, it may be necessary to override this right to protect the public from serious harm. A proportionate response to the need for private information requires that it be released only if there are no less intrusive means to protect public health.
• Disclose only private information that is relevant to achieve legitimate and necessary public health goals • Release private information only if there are no less intrusive means to protect public health • Determine whether the good that is intended is significant enough to justify the potential harm that can come from suspending privacy rights, (e.g. the harm from stigmatization of individuals or particular communities) • Provide public education to correct misconceptions about disease transmission and to offset misattribution of blame to particular communities
The need to conduct contact tracing of possibly infected people might require that particular groups or even individuals are identified publicly. The need to do so must be weighed against the potential harm of exposing communities and individuals to stigmatization.
Proportionality requires that restrictions to individual liberty and measures taken to protect the public from harm should not exceed what is necessary to address the actual level of risk to, or critical need of, the community. Decision makers should: • Use least restrictive or coercive measures in limiting or restricting liberties or entitlements • Use more coercive measures only in circumstances where less restrictive measures have failed to achieve appropriate public health ends.
The decision to close an emergency room must consider if the potential harm in keeping the emergency room open is significant enough to warrant its closure.
A foundational principle of public health ethics is the obligation to protect the public from serious harm. This principle requires that citizens comply with imposed restrictions in order to ensure public wellbeing or safety. To protect the public from harm, hospitals may be required to restrict public access to service areas (e.g. restricted visiting hours), to limit availability of some services (e.g. elective surgeries), or to impose infectious control practices (e.g. masks or quarantine).
• Weigh the medical and moral imperative for compliance • Ensure stakeholders are made aware of the medical and moral reasons for public health measures • Ensure stakeholders are aware of the benefits of compliance & the consequences of non-compliance • Establish mechanisms to review these decisions as the public health situation changes and to address stakeholders concerns or complaints When making the decision to quarantine individuals, protection of the public from harm must be weighed against individual liberty. Note that while the ethical value of individual liberty is often in tension with the protection of the public from harm, it is also in individuals' interests to minimize harm to others.
Reciprocity requires that society supports those who face a disproportionate burden in protecting the public good and takes steps to minimise their impact as far as possible. In an influenza pandemic, measures to protect the public good are likely to impose a disproportionate burden on health care workers, patients, and their families. Health care workers may face expanded duties, increased workplace risks, physical and emotional stress, isolation from peers and family, and in some cases, infection leading to hospitalization or even death. Similarly, quarantined individuals or families of ill patients may experience significant social, economic, and emotional burdens.
• Easing the burdens of health care workers, patients, and patient's families in their hospitals and in coordination with other health care organizations • Ensuring the safety of their workers, especially when redeploying staff in areas beyond the usual scope of practice
The provision of antiviral medication and/or vaccination to hospital staff for prophylaxis is one way hospitals can ensure the safety of their workers who may be exposed to greater than usual risks in discharging their duty to care.
SARS heightened the global awareness of the interdependence of health systems and the need for solidarity across systemic and institutional boundaries in stemming a serious contagious disease. An influenza pandemic will not only require global solidarity, it will require a vision of solidarity within and between health care institutions. Solidarity requires:
• Good, open and honest communication • Open collaboration, in a spirit of common purpose, within and between health care institutions • Sharing public health information • Coordinating health care delivery, transfer of patients, and deployment of human and material resources Territoriality between hospital departments and between health care institutions needs to be overcome with good communication and sense of common purpose in order to provide equitable care across jurisdictions
In our society, both institutions and individuals will be entrusted with governance over scarce resources, such as vaccines, antivirals, ventilators, hospital beds and even health care workers. During a pandemic influenza outbreak, difficult decisions about how to allocate material and human resources will have to be made, and there will be collateral damage as a result of these allocation decisions. Those entrusted with governance roles should be guided by the notion of stewardship. Inherent in stewardship are the notions of trust, ethical behaviour, and good decision-making. Decision makers have a responsibility to: • Avoid and/or reduce collateral damage that may result from resource allocation decisions • Maximize benefits when allocating resources • Protect and develop resources where possible • Consider good outcomes (i.e. benefits to the public good) and equity (i.e., fair distribution of benefits & burdens)
A hospital's decision to stock-pile antiviral medication must consider whether this is an effective way of protecting staff from infection, where the money for stockpiling will come from, and whether that money could be put to better use elsewhere.
Trust is an essential component in the relationships between clinician and patient, between staff and the organization, between the public and health care providers, and between organizations within a health system. In a public health crisis, stakeholders may perceive public health measures as a betrayal of trust (e.g. when access to needed care is denied) or as abandonment at a time of greatest need. Decision-makers will be confronted with the challenge of maintaining stakeholders' trust while at the same time stemming an influenza pandemic through various control measures. It takes time to build trust.
• Take steps to build trust with stakeholders before the crisis hits not while it is in full swing • Ensure decision making processes are ethical and transparent to those affected stakeholders Early engagement with stakeholders may go some distance to justify stakeholder confidence in decision-makers' trustworthiness. In part, the value of trust is respected and promoted by following the ethical processes outlined above. 
Whether or not an ethical framework is used to inform decision-making in a health care institution depends to a large extent on people in senior positions of an organisation seeing its relevance to the decision-making process.
In part, this is dependant on how robust the framework is, but it also requires the willingness to frame (at least some) pandemic planning issues as normative in nature.
Some may argue that the values in the framework are too stringent or impractical to implement under crisis conditions, especially those found in the Ethical Processes part of the framework (see Table 1 ). Certainly, crisis conditions may place constraints on the extent to which each principle can be acted upon. However, efforts should be made to put them into action to the fullest extent possible under the circumstances and in our experience this is only possible with the support of senior administrators.
The senior administration at S & W (many of whom were part of the Pandemic Planning Committee) had previous experience with the accountability for reasonableness framework for decision-making, and thus their pandemic influenza planning committee was already familiar with the Ethical Processes part of the framework, and they were receptive to the idea of being guided by an ethical framework. Senior administrators may also have been receptive to the ethical framework because, as they learned from SARS, organisations that did not have decision-making processes that honoured the values for ethical process during SARS have been dealing with a legacy of collateral damage to staff and patients in the form of distrust and low morale [12] . For these reasons, the senior administrators at S & W played an important role in vetting the ethical framework. Ensuring that institutional "sponsors" are in favour of adopting an ethical framework is important for gaining widespread support for using an ethical framework in decision-making, and for ensuring that the ethical framework does not become something that looks good but remains unused.
In order to obtain support for, or "buy in" to an ethical framework, it is important that key stakeholders in an institution vet the framework. This requires careful consideration of who the key stakeholders are in an institution. Not only should this include those with responsibility for decision-making, but also those who will be affected by decisions taken. For the vetting process is not just intended to create "buy in" but also to decrease the likelihood that interests and issues that are (morally) relevant to pandemic planning will be neglected or overlooked, thereby enhancing the moral legitimacy of the values in the framework. In addition, a process of stakeholder vetting increases the likelihood that the values instantiated in the framework resonate with the stakeholder community.
It has been our experience that the values in the framework did resonate with the pandemic planners with whom we have shared this ethical framework. The primarily pragmatic justification for the selection of the values in the framework means that the framework is provisional so it ought to be subject to revision in light of compelling argument, empirical evidence and further stakeholder feedback. It is important to note, however, that the iterative and inclusive process through which the values in the framework were deliberated amongst the various stakeholder groups lends them a form of discursive ethical legitimacy and helps to justify their inclusion in the ethical framework. We intend that the framework invite further dialogue about its legitimacy and its adequacy. We will return to this issue in the final section of this paper.
Ideally, the vetting process would include people who can represent the interests of patients, families and volunteers who are part of the hospital's constituency. Although patient relations, human resources and occupational health representatives from S & W provided guidance and feedback in the development of the framework, direct input from patients and family representatives was not obtained. One limitation of our framework is that is has yet to be vetted by these important stakeholders.
The importance of solidarity to the management of a public health crisis would also suggest that the public and other health care organisations be considered stakeholders in hospital pandemic planning. While it may not be pragmatic for hospitals to undertake broad public consultation and vetting processes for their pandemic plans in general, and their ethical frameworks in particular, solidarity and equity suggest that these broader stakeholder interests are relevant to pandemic planning. Consequently, opportunities for broader ethical dialogue about pandemic planning need to be encouraged.
In order to ensure that the support of key stakeholders is maintained through an outbreak, there need to be effective communication mechanisms in place. An important aspect of responsive decision-making processes is ensuring that there are formal opportunities to revisit and revise decisions as new information emerges. As part of our ethical framework, we formulated a template for decision review processes, (adapted from, Gibson, JL: Formal decision review process template. Unpublished; 2003) that aids organisations in identifying existing and establishing new mechanisms that can be used for the formal reviews of decisions. We believe decision review mechanisms are an essential part of ethical decision-making in a public health crisis, and are one way to put the values in the ethical framework in to action.
Formal mechanisms for reviewing decisions are needed in order to capture feedback from stakeholders on key decisions, and to resolve disputes and challenges. These processes are important for ensuring that decisions are the best possible under the circumstances given changing information and for engaging stakeholders constructively around the difficult decisions that must be made. Given the unpredictable nature of public health emergencies and the difficulty this poses for those in charge of planning and decision-making, it is reasonable to assume that decisions will be revised throughout the pandemic influenza crisis. Disputes or challenges may arise from the restrictions or requirements imposed on staff, patients and families during a pandemic influenza outbreak. Thus, decision review processes are essential. Again, while some may argue that this is too stringent a measure for a time of crisis, we argue that reviews of decisions will be taking place regardless (most likely in an ad hoc manner), and that to formalize this process is to increase its fairness and moral legitimacy. Indeed, there may be existing mechanisms which can handle these kinds of reviews.
It is important to distinguish between different types of ethical analyses in order to explain the approach that was taken to the development of the ethical framework discussed herein. Callahan and Jennings draw a useful distinction between applied ethics and critical ethics [7] . Our ethical framework is an example of applied ethics because the framework identifies and relies on "general principles that can be applied to real-world examples of professional conduct or decision-making" [7] and because it is "designed to give professionals guidance and to give clients and the general public standards to use in assessing professional conduct" [42] . While there is certainly a need for critical ethical analysis that pays attention to problems that are the "result of institutional arrangements and prevailing structures of cultural attitudes and social power" [7] , one would not expect a ethical framework designed to guide clinical decision-making to explicitly address these kinds of issues.
This is not to say that this ethical framework cannot address the kinds of issues that a critical ethical analysis might address. For example, the framework promotes values and processes that seek to redress the power disparities within institutions. The section of the framework that deals with ethical processes in particular is a challenge to how institutional decisions are typically made. For example, the value of "inclusiveness" as a process principle is essential for redressing power differences amongst key stakeholders [27] . Thus, while the ethical framework is the product of applied ethical analysis, and should be evaluated in light of this, one of its strengths is that it can also redress what Callahan and Jennings would characterize as "critical" ethics problem of power disparities within institutions.
Within pluralistic societies, there are many different ethical perspectives that exist simultaneously on issues about global, public and individual health. An ethical framework to guide decision-making is robust to the extent that it reflects the values and beliefs of the decision-makers who refer to it and the values and beliefs of those affected by the decisions being taken. Our framework relied heavily on the Toronto experience with SARS to surface and examine the ethical values that are important for a public health crisis. An influenza pandemic is likely to present us with particular ethical challenges that are different from SARS due to the predicted severity of the contagion and its spread to the community. It would therefore be important not to uncritically adopt such a framework but rather to use it as a basis for continued reflection and re-evaluation to ensure its relevance and responsiveness during the unfolding health crisis. It is also important to consider the extent to which an ethical framework is reflective of the community in which it is to be used. Lessons from SARS as it was experienced in China would likely surface some different ethical values, or emphasise different aspects of our framework. As Callahan and Jennings have argued:
We submit that a rich discourse on ethics and public health cannot be advanced without relating it to the background values of the general society, and the particular communities, in which it will be carried out. [7] Indeed, as previously maintained, there are many issues related to pandemic influenza planning -particularly those raised by a critical ethical analysis -that require broad public debate. While these kinds of issues require public debate that takes place at the societal level, ethical pandemic planning requires that organisations and agencies foster internal dialogue about the values instantiated in an ethical framework. For it is imperative that the values outlined in a framework resonate with the members of an organisation, and the community it serves. The procedural aspects of the framework provide a means to ensuring that the values of the community are reflected in decision-making through the procedural principles of inclusiveness and responsiveness.
It is important, too, to recognise that values are not static, and that circumstances will evolve rapidly during a pandemic influenza outbreak. Ethical frameworks will also require re-evaluation and revision. The challenge will be to continue to recognise the importance of moral reflection under circumstances that are not conducive to it and to encourage a process of re-evaluation that strives to assess whether resulting decisions are consistent with those values the framework is intended to promote. For this reason, it is imperative to start the ethical dialogue in advance, and to find ways to encourage consideration of ethical issues at all stages of decision-making. We hope that this paper will go some way towards advancing this objective, and that this paper stimulates discussion of the ethical issues and values that pervade pandemic planning.
We believe that this framework is unique in its blending of clinical, public health, and organizational ethics. One of its strengths is that it draws on lessons from the recent public health crisis of SARS in Toronto, and it is to some extent empirically grounded. Another strength is that it is the product of an inclusive process of development that included stakeholder vetting. It is also unique in its attempt to provide guidance to decision-makers facing a public health crisis. We hope that the framework's acceptance by hospitals and the provincial government in Ontario signals a change in the way that decisions are taken by institutions that are charged with making decisions that have life and death consequences for the public.
• Good pandemic planning requires reflection on values because scientific information alone cannot drive decision-making.
• The development of an ethical framework for hospital pandemic planning calls for expertise in clinical, organisational and public health ethics.
• Stakeholder engagement is essential for the ethical framework to be relevant and legitimate.
• The ethical framework contains procedural and substantive ethical values to guide decision-making.
• Three key elements of integration of ethics in to pandemic planning are 1) sponsorship from senior hospital administration; 2) vetting by stakeholders and; 3) decision review processes.
• An ethical framework is robust to the extent that pandemic influenza planning decisions are seen to be ethically legitimate by those affected by them.
• In order to increase the robustness of pandemic planning in general, timely public debate about the ethical issues is essential. Open lung biopsy in early-stage acute respiratory distress syndrome INTRODUCTION: Acute respiratory distress syndrome (ARDS) has heterogeneous etiologies, rapid progressive change and a high mortality rate. To improve the outcome of ARDS, accurate diagnosis is essential to the application of effective early treatment. The present study investigated the clinical effects and safety of open lung biopsy (OLB) in patients with early-stage ARDS of suspected non-infectious origin. METHODS: We undertook a retrospective study of 41 patients with early-stage ARDS (defined as one week or less after intubation) who underwent OLB in two medical intensive care units of a tertiary care hospital from 1999 to 2005. Data analyzed included baseline characteristics, complication rate, pathological diagnoses, treatment alterations, and hospital survival. RESULTS: The age of patients was 55 ± 17 years (mean ± SD). The average ratio of arterial partial pressure of oxygen (PaO(2)) to fraction of inspired oxygen (FiO(2)) was 116 ± 43 mmHg (mean ± SD) at biopsy. Seventeen patients (41%) were immunocompromised. Postoperative complications occurred in 20% of patients (8/41). All biopsies provided a pathological diagnosis with a diagnostic yield of 100%. Specific pathological diagnoses were made for 44% of patients (18/41). Biopsy findings led to an alteration of treatment modality in 73% of patients (30/41). The treatment alteration rate was higher in patients with nonspecific diagnoses than in patients with specific diagnoses (p = 0.0024). Overall mortality was 50% (21/41) and was not influenced by age, gender, pre-OLB oxygenation, complication rate, pathological results, and alteration of treatment. There was no surgery-related mortality. The survival rate for immunocompromised patients was better than that for immunocompetent patients (71% versus 33%; p = 0.0187) in this study. CONCLUSION: Our retrospective study suggests that OLB was a useful and acceptably safe diagnostic procedure in some selected patients with early-stage ARDS. The clinical definition of acute respiratory distress syndrome (ARDS) includes the acute onset of bilateral pulmonary infiltrates, a ratio of arterial partial pressure of oxygen (PaO 2 ) to fraction of inspired oxygen (FiO 2 ) of 200 mmHg or less, and no evidence of left atrial hypertension [1] . Many risk factors, such as pneumonia, sepsis, and aspiration, are associated with the development of ARDS. However, other diseases and conditions, such as bronchiolitis obliterans organizing pneumonia (BOOP), adverse reaction to drugs, diffuse alveolar hemorrhage (DAH), and hypersensitivity pneumonitis (HP), can also cause ARDS; despite similar clinical presentations, etiological diagnosis can be difficult especially for early-stage ARDS. Although the mortality rate of patients with ARDS ALI = acute lung injury; ARDS = acute respiratory distress syndrome; BAL = bronchoalveolar lavage; DAD = diffuse alveolar damage; FiO 2 = fraction of inspired oxygen; HRCT = high-resolution computed tomography; ICU = intensive care unit; OLB = open lung biopsy; PaO 2 = arterial partial pressure of oxygen; PEEP = positive end-expiratory pressure.
improves recently [2] , the rapid clinical deterioration of such patients, who often progress to multiple organ failure, remains a significant challenge for intensivists in the intensive care unit (ICU). To halt the disease progression of early-stage ARDS, accurate diagnosis is critical.
It can be difficult to differentiate between infectious and noninfectious etiology as the cause of ARDS in its early stages. Current microbiological sampling techniques are insufficiently sensitive to determine the causes of ARDS in all patients [3] [4] [5] . In patients with negative microbiological cultures, separating a true infection from an inflammatory response with clinical data remains problematic. Empiric broad-spectrum antibiotics are typically prescribed to these critically ill patients immediately after admission. However, unnecessary antibiotic therapy for non-infectious patients can enhance the occurrence of antibiotic-resistant strains of bacteria and increase the potential for subsequent nosocomial infections.
The therapeutic benefit of prolonged glucocorticoid therapy during the fibroproliferative stage of ARDS emphasizes the need for the elucidation of the underlying lung pathologies [6] . Additionally, the specific diseases such as BOOP, drug reaction, DAH and HP can cause an ARDS response to steroid therapy. However, inappropriate steroid therapy for patients with ARDS may be associated with complications such as gastrointestinal bleeding, hyperglycemia and increased susceptibility to infection.
Some previous studies have demonstrated that open lung biopsy (OLB) is a useful and acceptably safe diagnostic technique for patients with ARDS [7] [8] [9] . In the study by Papazian and colleagues [7] , the results of OLB directly altered the therapeutic management for 34 of 36 patients with ARDS (94%), and the OLB complication of an air leak occurred in five patients (14%). The OLB results obtained by Patel and colleagues [8] led to a change in management in the majority of 57 patients with ARDS, the addition of specific therapy for 34 patients (60%), and the withdrawal of unnecessary therapy in 24 patients (37%); major complications occurred in four patients (7%). However, in both studies the duration from intubation to OLB was long: in Papazian and colleagues' study [7] the range was 5 to 89 days, and in Patel and colleagues' study [8] it was 0 to 25 days. This retrospective study attempted to evaluate the utility and safety of OLB in patients with clinically suspected non-infectious early-stage ARDS.
The records of patients with ARDS who received OLB in two ICUs at a tertiary care referral center over a five year period between January 1999 and April 2005 were examined. Charts with a discharge diagnosis code 518.82 of the International Classification of Diseases, Ninth Revision, Clinical Modification, suggesting ARDS not related to surgery or trauma, were reviewed for possible inclusion in this study. A total of 819 patients with ARDS were identified and OLBs were performed in 68 patients (8.3%). Forty-one OLBs were performed during early-stage ARDS (one week or less after intubation). Patients supported with noninvasive positive-pressure ventilation or intubated for more than seven days at the time of biopsy were excluded.
All patients met ARDS criteria defined by the American-European consensus conference [1] . Decisions to perform OLB were made by senior intensivists in charge of the respective ICUs. OLB was indicated when ARDS was suspected to be noninfectious in origin, with no obvious etiology and with a possible indication for corticosteroid treatment based on clinical presentations with rapid progression, relative symmetric distribution on chest X-ray, and predominant ground-glass attenuation in high-resolution computed tomography (HRCT) of the chest. Informed consent for OLB was obtained from each patient's family.
Chest HRCT was performed before bronchoscopic sampling and OLB. The location for bronchoalveolar lavage (BAL) sampling was selected on the basis of HRCT findings, or on a chest X-ray when HRCT was unavailable. BAL was performed by introducing 200 ml of sterile warm (37°C) saline solution into a lung subsegment and aspirating it back in four 50-ml aliquots. The first aliquot returned (bronchial fraction) was discarded. Each specimen was sent for bacterial examination for Legionella, Mycoplasma pneumoniae, Pneumocystis carinii, and Mycobacteria, and for fungal and virological (cytomegalovirus, influenza virus, parainfluenza virus, adenovirus, herpes simplex virus, respiratory syncytial virus, and coxsackie virus) analyses. Specimens were also sent for cytology and iron stain analysis. BAL results were deemed positive when at minimum one microorganism grew to a concentration of more than 10 4 colony-forming units/ml. All procedures were performed within 24 hours of OLB.
OLB was performed in an operating room or at the bedside in an ICU by an experienced thoracic surgeon. Bedside OLB was indicated when the FiO 2 used reached 1 with an applied positive end-expiratory pressure (PEEP) of at least 12 cmH 2 O. With regard to mechanical ventilator settings to prevent air leakage, PEEP was immediately reduced 2 cmH 2 O from the baseline level after surgery. Pulmonary tissue was harvested from a site considered new or from a progressive lesion identified by chest HRCT or chest X-ray.
Each tissue specimen was cultured and examined by a pulmonary pathologist.
Medical records from these 41 patients were reviewed and analyzed for the following data: age; gender; Acute Physiology and Chronic Health Evaluation (APACHE) II scores at admission to the ICU; acute lung injury (ALI) scores, PEEP, and PaO 2 /FiO 2 ratio at ARDS diagnosis; dates of ARDS onset, respiratory failure, intubation, and biopsy; underlying diseases; diagnostic tests before biopsy; and medications at time of biopsy. Results regarding complications of biopsy, pathological diagnosis, and postoperative therapeutic changes (addition or removal of drugs) were also analyzed. Outcome parameters, including ICU and hospital survival rates and cause of death, were also evaluated.
For normally distributed data, values are reported as means ± SD. Student's t tests were used to compare normally distributed continuous variables. Differences between subgroups were compared by using the χ 2 test or Fisher's exact test when the expected number of events was less than five. The significance level (α) for all statistical tests was set at 0.05, and p < 0.05 was considered statistically significant.
Sixty-eight patients underwent OLB for ARDS evaluation during the study period, of whom 27 were excluded because the duration between intubation and OLB exceeded seven days. A total of 41 patients were enrolled. Table 1 lists the baseline characteristics of the patients studied. Twenty-four patients (59%) were immunocompetent and 17 patients (41%) were immunocompromised. Causes of immunocompromise status were hematological malignancy in 10 patients and solid tumors in four patients (three had bronchogenic cancers and one had breast cancer), HIV infection in two patients and renal transplantation in one. The duration from intubation to OLB for these 41 patients was 3.0 ± 1.9 days (mean ± SD; range 1 to 7).
BAL was performed 24 hours before OLB. Findings of BAL were compatible with pathological diagnosis for only four patients with diagnoses of bacterial pneumonia, mycobacterial tuberculosis, cytomegalovirus pneumonitis, and Pneumocystis carinii pneumonia. Twenty-two patients (54%) had chest HRCT before OLB to identify an appropriate biopsy site. For the remaining 19 patients who did not undergo chest HRCT, OLBs were performed from the right middle lobe in 12 patients and from the lingular lobe in seven patients.
Of the 41 patients, 26 (63%) underwent OLB in an operating room and 15 (37%) received bedside OLB in an ICU. Videoassisted thoracotomy was performed in eight patients, and the remaining patients underwent limited anterior thoracotomy. No intra-operative complication occurred, and eight patients (20%) had postoperative complications (less than seven days after the operation). Two patients developed transient hypotension after OLB and regained normal status after fluid resuscitation and vasopressor treatment for 12 hours. Two patients had pneumothorax diagnosed by chest X-ray and required a chest tube with low-pressure suction (10 cmH 2 O) drainage for 24 hours after OLB. Two patients had subcutaneous emphysema localized in the chest area after OLB, which resolved spontaneously in two days. Additionally, two patients had bronchopleural fistula with persistent air leaking from the operative chest tube for at least one day and did not need further surgery. Although six of these eight patients (two with transient hypotension, one with pneumothorax, one with subcutaneous emphysema and two with bronchopleural fistula) died, no surgical complication resulted directly in death. The incidence of postoperative complication was 15% (4/26) and 27% (4/15) for patients undergoing OLB in an operating room or at the bedside in an ICU, respectively. Complication rates were not significantly different between these two groups (p = 0.3799).
All biopsies provided sufficient data for pathological diagnosis (diagnostic yield 100%). The specimens obtained during OLB were sent for tissue culturing (for both bacteria and viruses); all culture results were negative. Pathological diagnoses were subdivided into specific and nonspecific categories. Eighteen patients (44%) had specific diagnoses established by OLB, and 23 (56%) had nonspecific diagnoses (Table 2) .
Overall, OLB findings led to alteration therapy for 30 of 41 patients (73%). After OLB, 18 patients were administrated high-dose corticosteroid therapy (1 g/day methylprednisolone in divided doses for three days) and seven patients were treated with low-dose corticosteroid therapy (2-3 mg/kg per day methylprednisolone in divided doses). Three patients received co-trimoxazole for Pneumocystis carinii pneumonia. Antibiotics were changed in one patient and discontinued in one patient on the basis of pathological findings. Treatment was not changed in 11 of 41 patients (27%). Table 3 presents comparative results for patient characteristics, complication rates, alterations in treatment, and survival rates of patients with specific and nonspecific pathological diagnoses by OLB. The rate of treatment alteration was higher in the nonspecific pathological diagnosis group than in that with a specific diagnosis (56% versus 87%; p = 0.0243). No other significant differences between these two groups were noted.
Twenty-one patients died in the ICU, resulting in an ICU survival rate of 49% (20/41). The hospital survival rate was the same as the ICU survival rate. Multiple organ dysfunction syndrome was the leading cause of death in 10 patients, followed by septic shock in nine patients, hypovolemic shock in one patient and acute myocardial infarction in one patient. Table 4 presents comparative results of patient characteristics and outcomes for survivors and nonsurvivors. No significant differences were observed between survivors and nonsurvivors for baseline data, such as age, gender, severity of illness, complication rate, and treatment alteration rate, between these two groups. Significantly more immunocompromised patients were in the survivor group than in the nonsurvivor group (60% vs 24%; p = 0.0187).
Comparisons between immunocompromised and immunocompetent patients (Table 5 ) showed that immunocompromised patients were younger (p = 0.0004) and had lower ALI scores (p = 0.0045). Furthermore, immunocompromised patients had better hospital survival rates than immunocompetent patients (71% versus 33%; p = 0.0187).
This study showed that OLB is an acceptably safe and useful procedure for some selected patients with early-stage ARDS. The treatment alteration rate was higher in patients with ARDS with nonspecific pathological diagnoses than in those with specific diagnoses.
In recent studies of patients with ARDS [7, 8] , OLB was employed relatively late, and the time from intubation to OLB was considerable (5 to 89 days in the study by Papazian and colleagues, and 0 to 25 days in the study by Patel). In the present study, OLB was performed within one week of intubation (3.0 ± 1.9 days), substantially earlier than in the previous two studies. Patel Specific diagnosis rates based on OLB findings vary among studies of patients with different disease entities. The specific diagnostic rates in a review by Cheson and colleagues were 21 to 68% in immunocompetent patients and 37 to 95% in immunocompromised patients [11] [12] [13] [14] . In this study, specific and nonspecific diagnostic rates were 44% (18/41) and 56% (23/41), respectively, and specific diagnostic rates for immunocompetent and immunocompromised patients were 33% (8/24) and 59% (10/17), respectively. Although not statistically significant (p = 0.1052), the specific diagnostic rate between immunocompetent and immunocompromised patients was similar to that in previous studies, indicating that OLB obtains a high percentage of specific pathological diagnoses for immunocompromised patients.
In this study, the rate of therapy alterations after OLB was 73% (30/41) and was not lower than those in previous reports (range 59 to 75%) [10, 14, 15] . For groups with nonspecific and specific pathological diagnoses, the rate of changed therapy was higher in the nonspecific group (87% versus 56%; p = 0.0243). This analytical finding resulted from a large number of patients with nonspecific pathological diagnoses undergoing corticosteroid treatment as a rescue or anti-inflammatory therapy after excluding potential active infection, such as the fibroproliferative stage of DAD [16] [17] [18] , interstitial pneumonitis, nonspecific interstitial pneumonitis, and organizing pneumonia. Early OLB can achieve diagnoses other than fibrosis that are potentially treatable with corticosteroid. Furthermore, the recent study by the ARDS Clinical Trials Network [19] did not support the routine use of methylprednisolone in patients with persistent ARDS (at least seven days after the onset) and
suggested that methylprednisolone therapy might be harmful when initiated more than two weeks after the onset of ARDS. The duration of ARDS before corticosteroid treatment interacted significantly with survival.
For immunocompromised patients, some studies [11, 20] suggested that OLB is advantageous for diagnosis and for treatment alteration but that its benefit to survival remains unclear.
McKenna and colleagues [21] found that for immunocompromised patients, early OLB (average 3.6 days after admission) benefited the histological diagnosis of interstitial pneumonitis treated with steroids; however, OLB did not improve clinical outcome for all patients. The overall mortality rate was 51% (21/41) in the present study, which is similar to that obtained in previous reports (range 47 to 50%) [7, 8] . More immunocompromised patients were in the survivors group and had a better survival rate than the immunocompetent patients (60% versus 24%; p = 0.0187); the young age and low ALI scores of immunocompromised patients probably accounted in part for their better outcome. Furthermore, the enhanced survival rate of immunocompromised patients might be attributed to more immunocompromised patients (9/13; 69%) than immunocompetent patients (9/17; 53%) receiving high-dose corticosteroid therapy after active infection had been excluded by OLB. Various pulmonary conditions such as infection, disease progression, therapeutic reaction, new and unrelated pathologies, or a combination of these can be present in immunocompromised patients [21, 22] . For diagnostic yield and adequate treatment, early OLB has been considered to be a reliable diagnostic modality, providing an early and accurate etiological diagnosis in immunocompromised patients. Operative complication rates reported for OLB in patients with ARDS have ranged from 17 to 39% [7, 8, 10] . In this study, the overall rate of OLB postoperative complications was 20% (8/ 41). In the late fibrotic stage, lung parenchyma is stiffer than in the earlier exudative or fibroproliferative stages of ARDS. Although operative complications are multifactorial, early OLB in non-stiff lungs (less fibrosis in the present study than in other reports) may account for the low surgical complication rate in this study. Of the 41 patients in the present study, 15 could not be transported to an operating room because they were being administered 100% O 2 and a high PEEP; consequently, OLB was performed at the bedside in the ICU. No intra-operative complications or exacerbation of oxygenation and hemodynamics occurred, even in patients with ARDS with severe hypoxemia. Of these 15 patients, four developed postoperative complications of hypotension, pneumothorax, subcutaneous emphysema, and bronchopleural fistula, respectively. No death was attributable to OLB. The risk for complications due to OLB in early-stage ARDS was therefore acceptable, even for the most critically ill patients with severe hypoxemia.
Several limitations of this study should be considered. First, because of its retrospective nature our study cannot directly address the question of whether early OLB has a survival benefit. However, understanding of a specific etiology would permit the initiation of specific therapy assuming that such a therapy is available. Many of the diagnoses found in this study (such as metastatic malignancy, infectious pneumonia and hypersensitivity pneumonitis) may have an established positive therapeutic effect on outcome. Second, the result of this study cannot be generally applied to all patients with ARDS. The decision to perform OLB was not made at random and the patients referred for OLB were unlikely to be a representative sample of our ARDS population. This selection bias of patients and intensivists would be expected to increase the possibility of an alternative intervention. A third limitation is that some specific diagnosis such as viral pneumonitis may be underdiagnosed because its identification depends on the availability of laboratory facilities. A standardized comprehensive microbiological examination of BAL before OLB should be established.
This retrospective study demonstrates that OLB had a high diagnostic yield rate and an acceptable complication rate for some selected patients with early-stage ARDS. The rate of treatment alteration was higher in patients with nonspecific pathological diagnoses than in those with specific pathologically diagnosed ARDS. Further prospective, randomized and control studies should investigate the appropriate indication and effect of OLB on outcome in patients with ARDS.
• Open lung biopsy is an acceptably safe diagnostic procedure for some selected early-stage patients with acute respiratory distress syndrome.
• In patients with early-stage acute respiratory distress syndrome of suspected non-infectious origin, open lung biopsy may have a high diagnostic yield rate.
• The role of open lung biopsy in patients with acute respiratory distress syndrome needs to be investigated in prospective, randomized and controlled clinical trials. Association and Host Selectivity in Multi-Host Pathogens The distribution of multi-host pathogens over their host range conditions their population dynamics and structure. Also, host co-infection by different pathogens may have important consequences for the evolution of hosts and pathogens, and host-pathogen co-evolution. Hence it is of interest to know if the distribution of pathogens over their host range is random, or if there are associations between hosts and pathogens, or between pathogens sharing a host. To analyse these issues we propose indices for the observed patterns of host infection by pathogens, and for the observed patterns of co-infection, and tests to analyse if these patterns conform to randomness or reflect associations. Applying these tests to the prevalence of five plant viruses on 21 wild plant species evidenced host-virus associations: most hosts and viruses were selective for viruses and hosts, respectively. Interestingly, the more host-selective viruses were the more prevalent ones, suggesting that host specialisation is a successful strategy for multi-host pathogens. Analyses also showed that viruses tended to associate positively in co-infected hosts. The developed indices and tests provide the tools to analyse how strong and common are these associations among different groups of pathogens, which will help to understand and model the population biology of multi-host pathogens. Pathogens have highly variable host ranges: in natural conditions some infect only one or a few related species (i.e., specialist pathogens) while other can infect a wide range of hosts belonging to different taxonomic groups (i.e., multi-host or generalist pathogens). A large fraction of described pathogens of humans, animals and plants are generalists [1] [2] [3] . The ability to infect different hosts conditions the epidemiology and pathogenicity of generalist pathogens and, therefore, is highly relevant for pathogen management and disease control [1, 4] . The distribution of multihost pathogens over their host range, i.e. the frequency of infection in the various host species within an ecosystem, may vary largely, which could determine the population dynamics and structure of the pathogen. The distribution of a pathogen species over its host range may also determine important aspects of its biology in hosts significant from an anthropocentric viewpoint (i.e. target hosts), such as reservoirs and inoculum sources, emergence and reemergence, population thresholds for disease invasion or critical community size for disease persistence [e.g., 1, [4] [5] [6] [7] .
Animal or plant species may be hosts for a range of pathogens, and most host populations encounter a large number of different pathogen species [8] . For significant host species, there is abundant evidence of differences in the infection frequency of the various pathogen species present in an ecosystem. The distribution of pathogens over their hosts, and the distribution of different pathogens within a host species, will affect the frequency of multiple infection of an individual host by different pathogens. Multiple infection may have important consequences for the infected hosts, for the pathogens, and for host-pathogen coevolution [8, 9] . In the host, frequent co-infections may lead to heterozygote superiority against multiple pathogens and contribute to the persistence in host populations of alleles conferring susceptibility to disease [10] . In multiple infected hosts, pathogens can cooperate or can compete for host resources, which will affect each other's fitness. Hence, multiple infections will be a factor in pathogen evolution. Theoretical analyses predict that the withinhost dynamics of microparasites in multiple infected hosts may have important consequences in the evolution of their virulence [11] [12] [13] [14] , and there is evidence that multiple infection may result in either increased or reduced virulence [e.g., [15] [16] [17] . Multiple infection of a host may also directly affect the genetic diversity of the pathogen population, as co-infection is a prerequisite for genetic exchange between different pathogen species or strains. Also, infection by one pathogen may result in an increased host susceptibility to a second pathogen, a common phenomenon named facilitation or predisposition by animal and plant pathologists, respectively [8, 18] .
In spite of its potential impact on pathogenicity, evolution, epidemiology and control, the distribution of pathogens over their host range and the occurrence of co-infections have been largely overlooked, and most research on pathogen ecology and epidemiology has dealt with specific pathogen-host interactions [8] . To our knowledge, it has not been analysed whether the distribution of pathogens over their host range is random or, alternatively, associations between pathogens and hosts occur, neither has been addressed whether host co-infection by different pathogens is random or associations between pathogens occur in particular hosts. Here we address these issues.
First, we propose indices for the observed patterns of host infection by different pathogens, and the observed patterns of coinfection, and tests to analyse if they conform to the null hypothesis of randomness or reflect associations. Second, we apply these tests to data on the prevalence of five insect-borne virus species in wild plant species within an agroecosystem in Central Spain. Results of these analyses uncover patterns that, if general, would be highly relevant to understand the ecology and evolution of pathogens. [19] . Except for TSWV, which has a single-stranded RNA genome of negative and ambisense polarity, all other viruses have single-stranded RNA genomes of messenger polarity. AMV, CMV and WMV are transmitted by aphids in a non-persistent manner, i.e. the virus is retained in the distal structures of the aphid mouth parts for short period of time. BWYV is transmitted in a circulative, non-propagative manner, i.e., the virus penetrates through the gut wall into the haemocoel of the insect vector, and circulates with the haemolymph to reach the salivary glands, from where it is inoculated into new plants. TSWV follows a similar path within the thrips body, but infects and multiplies in the insect cells [19] . All five viruses cause important diseases in vegetable crops worldwide, including the studied region in Central Spain, but infection in the analysed wild hosts was asymptomatic. Table 1 shows the number of samples analysed and the number of infected plants by each of these five virus species, in single or multiple infection, in the 21 most frequently found plant species in three monitored habitats (see Methods) for the analysed period.
To this data set tests for association between hosts and pathogens (see Methods) were applied. The index of selectivity of pathogen (ISP), and its significance, is shown in Table 2 for the five viruses. The distribution of three of five analysed viruses over their hosts was significantly non-random, i.e. some of the available hosts were preferentially infected. Fig. 1 shows the relationship between prevalence and the ISP for the five viruses. A positive correlation was found for both parameters (r = 0.9347, P = 0.0189 in a Spearman rank correlation test), i.e., the more host-selective viruses were those with a highest prevalence in the analysed ecosystem. Similarly, the index of selectivity of the host (ISH), and its significance, was calculated for the 21 host plant species in Table 1 , and values are shown in Table 3 . For about half (9/21) of the analysed hosts (Amaranthus spp., Cirsium arvense, Convolvulus arvensis, Diplotaxis erucoides, Lactuca serriola, Medicago sativa, Portulaca oleracea, Solanum nigrum and Taraxacum spp.) differences in the prevalence of the five viruses departed significantly from random. Fig. 2 shows the relationship between virus prevalence and the ISH for the 21 host species. Again, a positive correlation between both parameters was found (r = 0.5161, P = 0.0166, in a Spearman rank correlation test), i.e., the more virus-selective hosts were those with a higher prevalence of virus infection. The relationships between prevalence and selectivity for viruses and hosts were not due to a coincidence in the frequency of infection among hosts by different viruses, as shown by a contingency analysis of counts of infected hosts by the different viruses (P,10 24 ).
For 16 of the 21 plant species in Table 1 , co-infection with more than one of the five viruses occurred. For these 16 plant species, 102 plants were infected by at least one virus out of 1060 analysed plants ( Table 4 ). The above described test of association between pathogens was applied to this set. The data in Table 4 showed a tendency of the analysed viruses to associate positively: the distribution of the association index (AI) was skewed towards positive values (Fig. 3 ) so that out of 68 AIs computed for the five viruses in 16 plant species, 47/68 (more than two thirds) were positive and 21/68 were negative. Moreover, there was a conspicuous tendency of the positive AI values to have smaller probabilities (r = 20.6575, P,10 24 , in a Spearman rank correlation test). When the pooled sample from the sixteen plant species was considered, the AI was positive and significantly different from zero for each of the five viruses, i.e. each of the five viruses was found in co-infection with a frequency significantly higher than expected from the null hypothesis of independence of infection. However, this was not so when the data for each of the sixteen plant species were analyzed separately. Hence the association analysis uncovered two patterns that were not obvious: i) a general tendency of the analysed viruses to associate positively, ii) association depended on both the plant and the virus species.
Most efforts to understand the population biology of pathogens have focussed on specialist pathogens, and population biologists have successfully developed a formal understanding of the dynamics and evolution of single-host pathogens. However, most pathogens of humans, animals and plants are multi-host pathogens [1] [2] [3] 20] . As stated by Woolhouse et al. [1] ''understanding the more complex population biology of multi-host pathogens will be one major challenge in the 21st century ''. There is evidence that within an ecosystem the prevalence of multi-host pathogens may differ largely for the different species of their host range [e.g., [21] [22] [23] [24] [25] ]. Similarly, there is evidence of large differences in the prevalence on a host species of the various pathogens that are able to infect it [e.g., [26] [27] [28] [29] ]. However, no attempt has been made, to our knowledge, to analyse if differences in the distribution of multihost pathogens over their hosts are random or if there are associations between hosts and pathogens. The uncovering of associations between hosts and pathogens would be highly relevant to understand and model the population biology of multi-host pathogens, and for understanding the phenomenon of generalism itself.
We present here indices and tests to analyse if there is association between multi-host pathogens and their hosts. The proposed indices of selectivity for the pathogen and for the host measure the degree of association between hosts and pathogens.
The tests analyse the homogeneity of distribution of a pathogen over different host species or populations, and of different pathogens on a host, and analyse how significantly the values of the indices departs from zero (i.e. no association). The literature on pathogen ecology does not abound with data on the prevalence of various pathogens on various hosts. Hence, we have applied these indices to our unpublished data on the prevalence of five insectborne plant viruses on 21 species of wild plants in an agroecosystem in central Spain over a three year period.
The analysis of the prevalence of the different viruses in each host species by the homogeneity test that we propose, shows that half of the analysed plant species showed an index of selectivity of the host (ISH) significantly different from zero. The distribution of the host species showing virus selectivity was not related to taxonomy, habitat (fallow fields, edges or wastelands), seasonality or vegetative cycle (annual vs. perennial) (not shown). Interestingly, there was a positive correlation between the ISH and the average virus prevalence for these 21 host plant species, showing that the more selective hosts are more prone to be virus-infected, obviously by the virus(es) that better infects them. This phenomenon suggests that in spite that each host encounters a wide array of pathogens, mechanisms of escape and/or resistance [30] to some of them would operate, which could explain their selectivity. In fact, contingency analysis of counts of infected hosts by different viruses, suggest that different viruses specialise on different hosts. The analysis of the homogeneity of prevalence of a virus over its host species showed that for three of the five analysed viruses there was a significant host association, i.e., the value of the index of selectivity for the pathogen (ISP) significantly departed form zero. One major and unexpected finding of the analysis was that there was a positive and highly significant correlation between the value of the ISP and the prevalence of the viruses. The value of the ISP was not conditioned by the number of host plant species infected by each virus, as there was no correlation (r = 0.60, P = 0.173 in a Spearman rank correlation test) between ISP and the number of plant species that each virus infected in the analysed system i.e., the more selective viruses were not those infecting a smaller number of plant species. Thus, the more host-selective viruses were those that did best in the analysed ecosystem. This result could be highly relevant for understanding the evolution of generalism in pathogens. Although most described pathogens are generalists, the advantages of generalism are poorly understood. A generalist strategy provides the pathogen with more opportunities for transmission and survival, but it is predicted that evolution would favour specialism, because pathogen-host co-evolution could result in functional trade-offs that would limit the generalist fitness in any one host [1, [31] [32] [33] [34] . Our results are compatible with the hypothesis that specialism is advantageous for pathogens, as host selectivity is the rule for the analysed set of generalist viruses, and the more host selective is the virus, the more successful its strategy. Hence, our results could suggest that for generalist pathogens a degree of host specialisation, i.e. host-selectivity as defined here, is a successful strategy. Host specialisation in generalist pathogens would also be relevant for important issues of host and pathogen biology, as host specialisation will affect hostpathogen co-evolution and co-speciation, would reduce the opportunities for host switches and jumps, thus constraining the evolution of host expansion, and may result in spatial heterogeneity of hosts, thus favouring the stable maintenance of pathogen and host diversity [6, [35] [36] [37] . In addition, host specialisation may affect the opportunity for different pathogens of sharing a host and, thus, the consequences of multiple infection for pathogen and host evolution, as discussed below. We propose here also a simple procedure to estimate association among pathogens, which enables to compute an association index whose significance can be tested against the null assumption of independence of infections that follow a binomial distribution. The test was applied to the same data set as above, and the second major contribution of our analysis is the finding that co-infection was mostly non-random and that associations among the five analysed viruses were mostly positive. This result is relevant because co-infection of different pathogens may have important consequences for the pathogens, the infected hosts, and for hostpathogen co-evolution [8, 9, 14] . For viruses, co-infection of a host may result in the generation of new genotypes by recombination or by reassortment of genomic segments between different viral species or strains, often with dramatic changes in host range or pathogenicity. The classical example is the reassortment of avian and human strains of influenza A resulting in novel viruses with pandemic potential [38] [39] [40] [41] , but examples abound for both animal and plant viruses [e.g., [3, [42] [43] [44] [45] [46] [47] [48] ]. In the individual host, coinfection may lead to aggravated disease, often resulting from extracellular cooperativity of independently replicating viruses, by which one virus modulates the host response to infection to the benefit of the other [49, 50] . In addition, direct interactions of different viruses in co-infected cells may result in complementation of highly pathogenic defective genotypes, in increased virus replication or in modified cell and tissue tropisms [e.g., [51] [52] [53] [54] [55] [56] [57] ]. Alternatively, there is also evidence that mixed infections of pathogens result in reduced pathogenicity and less severe disease [17] . Examples from viruses include mixed infection with satellite or with defective interfering nucleic acids [58] . In our data set, association between viruses depended on each particular virus-host system. Hence, data suggest that in some hosts, but not in all, coinfection would be advantageous for some viruses, though the underlying mechanism remains to be analysed. The analysis here reported of plant virus infection on weeds has uncovered two major features that should be relevant to understand the population biology of viruses: i) the more hostselective viruses do better on the analysed ecosystem, ii) viruses tend to associate positively in co-infected hosts. It would be of high interest to know how general are these features and in which types of pathogens would they occur. The indices and tests that we propose here could be of general use in the analysis of the ecology of pathogens, and we hope that our results would prompt research on the ecology of pathogen-host and pathogen-pathogen associations, as these analyses might uncover pathogen properties relevant to the formal understanding of the population biology of multi-host pathogens.
We study two factors relative of the distribution of pathogens in different hosts (i.e. different host populations, genotypes, species etc): if there are associations between pathogens and their hosts and if there are associations among pathogens. To analyse these two factors we propose the following tests and indices:
Association between pathogens and their hosts Let us call N k the number of analysed individuals in host k (k~1, 2, :::: , n k ) and X ik the number of these individuals that are infected by pathogen i (i~1, 2, :::: , n i ). The prevalence of pathogen i in host k will be the ratio P ik = X ik /N k .
The average prevalence of pathogen i over hosts will be
Conversely, the average prevalence of the different pathogens in host k can be defined as
Homogeneity of the prevalence of a pathogen among hosts can be tested by means of a 2xn i contingency table with elements X ik and (N k 2X ik ) [59] . Different proportions (i.e. lack of homogeneity) will indicate a property of the pathogen that we will call selectivity. Selectivity will be measured by the Cramer's coefficient of contingency [59] of the contingency table. If x 2 i is the chisquared of the 2xn i table, the index of selectivity of the pathogen will be: Both of these indices range from zero to one, with zero meaning equal prevalence of the pathogen over hosts, or of pathogens over the same host, i.e. no selectivity for the pathogen or the host.
Association between different pathogens Let us call Xs ik and Xa ik the number of analysed individuals of host k that are infected only by pathogen i (single infections) and by pathogen i and at least another one (associated infections), respectively, (X ik = Xs ik +Xa ik ).
The frequency of pathogen i in host k can be estimated as:
which equals the above defined prevalence. Under the null hypothesis of independence of infection by different pathogens, the probability of a sampled host individual being infected only by pathogen i is:
The conditional probability of non-infection by any other pathogen given the presence of pathogen i is:
ps ik~P j=i (1{P jk ), and the conditional probability for the observed multiple infections given the presence of i is:
So, under the hypothesis of independence of infection by different pathogens (non-association between pathogens), Xs ik will be distributed as a binomial with X ik trials and probability ps ik . We define the association index (AI) for pathogen i in host k as the difference between the proportion of samples that being infected by pathogen i are infected also by at least another pathogen (Xa ik /X ik ), minus the expectation of this proportion under the null hypothesis (pa ik ). This index has a range from one to minus one and an expected value, under the null hypothesis, of zero. The significance of the observation can be estimated as a onetail test from the binomial above.
To test for association of different pathogens within a host, or for a given pathogen across different hosts, we follow the same process, as the expectation of a sum of observations will be equal to the sum of their expectations, and the corresponding sums of observations will be binomially distributed given the X ik .
To single out significant tests in a group, raw significance probabilities were corrected by the sequential Bonferroni method for multiple independent tests as indicated in [60] .
Plants were sampled monthly for three years in a horticultural area in central Spain within three habitats characterised by different degrees of human intervention: fallow fields, edges between fields, and wastelands. Plants were sampled systematically along fixed itineraries, with no consideration of symptom expression, as described in Sacristán et al. [21] . Infection by AMV, BWYV, CMV, WMV and TSWV in the sampled plants was analysed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using commercial antisera (Bio-Rad, Marnes-La-Coquette, France), according to the manufacturer's instructions.
The distribution of the host species showing virus selectivity according to taxonomy, habitat (fallow fields, edges or wastelands), seasonality or vegetative cycle (annual vs. perennial) was analysed by chi-squared tests of 2x N contingency tables, and their significances assessed, as in the rest of tests of this work, by simulation following model III. The Effectiveness of Contact Tracing in Emerging Epidemics BACKGROUND: Contact tracing plays an important role in the control of emerging infectious diseases, but little is known yet about its effectiveness. Here we deduce from a generic mathematical model how effectiveness of tracing relates to various aspects of time, such as the course of individual infectivity, the (variability in) time between infection and symptom-based detection, and delays in the tracing process. In addition, the possibility of iteratively tracing of yet asymptomatic infecteds is considered. With these insights we explain why contact tracing was and will be effective for control of smallpox and SARS, only partially effective for foot-and-mouth disease, and likely not effective for influenza. METHODS AND FINDINGS: We investigate contact tracing in a model of an emerging epidemic that is flexible enough to use for most infections. We consider isolation of symptomatic infecteds as the basic scenario, and express effectiveness as the proportion of contacts that need to be traced for a reproduction ratio smaller than 1. We obtain general results for special cases, which are interpreted with respect to the likely success of tracing for influenza, smallpox, SARS, and foot-and-mouth disease epidemics. CONCLUSIONS: We conclude that (1) there is no general predictive formula for the proportion to be traced as there is for the proportion to be vaccinated; (2) variability in time to detection is favourable for effective tracing; (3) tracing effectiveness need not be sensitive to the duration of the latent period and tracing delays; (4) iterative tracing primarily improves effectiveness when single-step tracing is on the brink of being effective. Control of epidemics of (emerging) infectious diseases, such as SARS, pandemic influenza, or foot-and-mouth disease, always faces the difficulty that some infectives are not yet observed. By concentrating control measures only on observed cases (treatment, isolation, culling), resources are used efficiently but control is often not effective enough. On the other hand, by directing control to the whole population (mass vaccination, prophylactic treatment, preventive culling), epidemics are most likely contained, but at high cost.
Contact tracing of symptomatic infecteds works on an intermediate level: treatment or quarantine of contactees (by contact we mean the possible transmission event, by contactee the individual that is contacted) may be effective because unidentified infecteds are most likely to be found among contactees, and efficient because the resources can be directed towards individuals at risk only. Tracing and quarantine has been successfully applied for smallpox control, where the term 'Leicester method' refers to exactly this strategy, with the establishment of specific smallpox hospitals [1] . It was also successful during the more recent SARS epidemic [2] , but not during the British foot-and-mouth epidemic [3] . Here we wish to investigate how these differences can be accounted for by the tracing process, thereby distinguishing tracing and quarantine as having separate effects which can be explained as follows.
Symptoms and detection divide the reproduction ratio R (the average number of secondary infecteds per primary infected in a susceptible population) into a part before detection and isolation and a part after [4] :
where c is the reduction factor due to isolation, R 0 is the basic reproduction ratio, when no efforts are made to isolate, and R 0 pre is that part of R 0 occurring prior to detection. Contact tracing will lead to earlier prevention of transmission due to quarantine of traced infecteds, thereby reducing the uncontrolled transmission R 0 pre to R pre , and the reproduction ratio R to R q :
The effect of decreasing R 0 pre to R pre is distinct from the effect of isolation and quarantine which reduce c. For tracing to be effective, R q should be smaller than 1, so R pre should be smaller than (12c)/(12cR 0 ), a threshold value determined by c and R 0 [4] [5] [6] . For comparison of contact tracing in different situations, we will choose c = 0 (as in [6] [7] [8] [9] [10] ), because it is easy and as arbitrary as any other value. The value c = 0 is valid for foot-and-mouth disease where traced herds are culled.
Several tracing studies have been published, some focussing on specific infections [10, 11] and some with the objective to obtain more general insight, e.g. in a general Markov-type SIR model [7] , in a model incorporating symptom development [4] , or in simulation models with specific network contact structures [8, 9] . Different assumptions were made with regard to the possibility of only tracing contacts of symptomatic infecteds (single-step tracing [4, 10, 11] ), or of iteratively tracing the contacts of traced infecteds (iterative tracing [6] [7] [8] [9] ). However, no general insight has yet been obtained in reducing R pre in relation to time-related characteristics of the infection and the tracing process, such as the latent and infectious periods, the time of symptom-based detection, and delays in the tracing process.
In this paper we consider detection and isolation as an autonomous process most likely governed by detection via clinical symptoms. This allows us to concentrate on contact tracing and quarantine, which are initiated by this autonomous process. Also we only regard transmission prior to control: we will consider tracing effective if R pre ,1.
We study an epidemic in its initial phase and use a branching process for its description, which means that the epidemic can be regarded as a growing tree. Each branch (contact) connects two nodes (contactees) with a hierarchical relation, the infector having infected the infectee. If one of the contactees is notified as being infected, by becoming symptomatic or by being traced via a secondary contact, the contact is traced with a probability p c (all parameters and functions of the model are given in Table 1 ). Each contact may be traced only once, either from infector to infectee (forwards tracing) or from infectee to infector (backwards tracing), so its traceability can be determined at the time of transmission. Thus, infection trees with traceable and untraceable contacts arise (Figure 1 ).
On these infection trees, two types of contact tracing are distinguished. The first type is single-step contact tracing, in which all traceable contactees of a symptomatic case are quarantined, but tracing only continues when an undiscovered infected, quarantined in this way, is detected itself ( Figure 1A ). The second type is iterative tracing, in which tracing of traceable contactees is continued, and a whole cluster of infecteds that is linked through traceable contacts is quarantined ( Figure 1B ). Such continuation would be possible if a test were available to determine the infection status of traced contactees.
The underlying model for infection dynamics is based on the framework of [4] . In our model, t measures time since infection of an individual, which starts with a latent period until t = t lat without transmission of the pathogen. During the infectious period, lasting from t lat to t inf , infecteds give rise to b new infecteds per unit of time, as long as they are not detected. As we are interested in the effectiveness of tracing only, we do not model possible transmission that might occur while being isolated, so transmission 'ceases' after detection of the infected. This leaves us with the basic reproduction ratio prior to detection, defined as the expected number of secondary infections per infected in a susceptible population before detection, R 0 pre . Here we have adjusted the interpretation of the model in [4] , where R 0 is divided in an asymptomatic and a symptomatic part, by adding a detection delay after becoming symptomatic. The part R 0 pre is determined by t lat , t inf , b, and the distribution of the detection time, a Gammadistribution with mean 1 (so time is measured relative to the mean detection time) and shape parameter a. Throughout our analyses, b will be scaled accordingly to achieve a desired value of R 0 pre . This model construction allows a flexible way of exploring different assumptions about the time to detection, the infectious period, and their overlap, and it enables us to evaluate tracing effectiveness for most infections.
For full understanding of contact tracing in our model, we analyzed four special cases regarding the infectious period and the detection time distribution. The infectious period was assumed to be either very short (all transmission occurs instantaneously, so t inf = t lat ) or very long (of infinite duration, so t inf = '). (In our model, infinite duration can be assumed because each infected will stop spreading the infection after detection. If some infecteds would never be detected, some large t inf ,' should be taken.) The detection time was assumed to be either fixed (a = ') or highly variable (a = 1, i.e. exponentially distributed). As a control we analyzed intermediate cases (results not shown) and four examples of real infections (influenza, SARS, smallpox, and foot-and-mouth disease), of which the parameter values are listed in Table 2 . These parameter values were obtained from literature [2, 3, [12] [13] [14] , assuming that the time to detection consists of the incubation period (time to symptom onset) plus a symptom-to-detection delay, which we assumed to be distributed as observed in the SARS epidemic (average 3.67 days, [2] ).
In our analyses tracing effectiveness will be expressed as the critical tracing probability p c *, defined as the proportion of contacts that need to be traced to achieve R pre = 1. If at least that many contacts are traced, epidemics will certainly die out if transmission during isolation or quarantine is prevented or limited to a small number of health-care workers that do not re-introduce the infection into the general community (see also [5] ). If such reintroductions cannot be excluded, a lower R pre may be aimed for. Because of this threshold of 1, the R 0 pre values (without tracing) were assumed to have some value larger than 1 (otherwise tracing would not be needed at all) and less than published R 0 values for the specific cases (Table 2) . First we study p c * as a function of R 0 pre and t lat (with tracing delay d = 0), and second as a function of t lat and d (with R 0 pre = 1.5).
In an epidemic where single-step tracing is applied, asymptomatic infecteds can spread the infection until they are detected and isolated. Detected infecteds are asked to name their contactees, and a proportion p c of all contactees will be reported and quarantined. Only when quarantined infecteds are detected themselves, tracing is continued ( Figure 1A) . We determined the critical tracing probability p c * as a function of the latent period for three values of R 0 pre (1.5, 2, and 3). The results are shown in Figure 2 . If the detection time is fixed (a = '), a too large latent period (larger than the detection time) results in a situation where every infected is detected before transmitting the infection, so tracing need not prevent any transmission; hence the maximum t lat of 1 in panels 2B and 2D. If we locate the approximate position of real infections (parameter values in Table 2 ) in Figure 2 , we observe that the long infectious period will be the best approximation for most infections, because we only regard pre-detection transmission and infecteds will often still be infectious at the time of detection. This does not entirely hold for influenza, which therefore fits best between the short and long infectious period. Because of the detection period distributions, smallpox and FMD are best described by panel 2D, whereas SARS and influenza fit best into panel 2C (influenza also into 2A).
In three special cases (panels 2A,B,D), with fixed incubation period and/or short infectious period, the proportion of contacts to be traced is at least 121/R 0 pre . This lower limit 121/R 0 pre is due to forwards tracing, when all infecteds that are traceable via their infector, are quarantined before the end of their latent period. Then, the effective reproduction ratio will be equal to the untraceable proportion of R 0 pre , i.e. R q = (12p c )R 0 pre , resulting in p c * = 121/R 0 pre . This is likely to be case with smallpox (panel 2D). However, if the latent period is short, as seen for influenza and possibly FMD, quarantine will occur too late to prevent all infections and more contacts need to be traced.
In the fourth panel (2C), with variable detection time and long infectious period, effective contact tracing requires a proportion of contacts smaller than 121/R 0 pre to be traced, if the latent period is large enough (like SARS). This can be explained as follows. If the detection time is variable and the latent and infectious periods are large, many infecteds will be detected before becoming infectious. If many infecteds are not infectious before being detected, the few that are should be very infectious (''superspreaders'') to attain a given R 0 pre (the average number of secondary infections before detection). Because many of the infectees of these superspreaders will be detected early (variable detection time), the superspreaders will be quarantined after backwards tracing, which adds to the effect of forwards tracing preventing infectees to reach their infectious period. Effectiveness may be very sensitive to the latent period, especially if there is little variation in the detection time. This is most apparent in the sharp transition in panel 2B, where tracing is only effective if all infectors are detected (at t = 12t lat ) before the infectious period (at t = t lat ), so t lat .0.5. The high sensitivity can be a problem for assessing the tracing effectiveness for a specific infection: the conclusion may largely depend on how correct the available estimates for the latent and incubation periods are, the incubation period determining the time to detection. Figure 2 indicates that this might be a problem for FMD.
In the previous section tracing was an instantaneous process: detection and isolation of a given case were followed by quarantine of traceable contactees of that case without any delay. The effect of a delay of duration d is that contactees of detected infecteds can continue transmitting the infection for an extra d time units, unless they are detected themselves during this interval.
The effect of a delay in each tracing step was studied by determining the critical tracing probability p c * for R 0 pre = 1.5, as a function of the latent period and tracing delay. Figure 3 shows the resulting contour plots for the four special cases with singlestep tracing, and it indicates the approximate positions of the four real infections. Along the t lat axis (d = 0) lie the R 0 pre = 1.5 curves of Figure 2 .
It appears that the iso-p c * contours in Figure 3 are linear in three of the four special cases (panel 3A,B,D), and approximately linear in the fourth case, with long infectious period and variable incubation time (panel 3C: t inf = '; a = 1). The slopes of all (approximate) lines are always between 0.5 and 1.5. This means that the effect of tracing delays is comparable to the sensitivity to the latent period as observed in Figure 2 , so plots of the critical tracing probability as a function of the delay will resemble the plots in Figure 2 , only mirrored (as in Figure 4 ).
In the contour plots in Figure 3 , tracing is ineffective in the dark grey areas (small latent period or large delay), so smallpox and SARS control are predicted to be able to cope with some delays, whereas it might be more of a problem with FMD and influenza. If forwards tracing is maximally effective and not affected by the delay, p c * = 121/R 0 pre = 0.33 as indicated by the light grey area (smallpox if d,0.5). If the detection time is variable, tracing may be effective even if a proportion less than 121/R 0 pre is traced (SARS if d,0.4, panel 3C). Because backwards tracing is needed to attain this result, it is only possible when the tracing delay is shorter than the infectious period (not shown in the Figures).
The contour plots show that for some combinations of latent period and delay, the sensitivity to small changes in the delay is large (contours lie close to one another). Then, small changes in the delay can have a major impact when the time of quarantine is shifted from just before to just after the end of the latent period, similar to the sensitivity to t lat as observed in Figure 2 . This effect is most dramatic if there is little variation in the detection time (smallpox: d<0.7, and FMD: d<0.2). Figure 4 shows the effectiveness of single-step tracing for SARS, smallpox, influenza, and FMD using the parameter values listed in Figure 2 . The effectiveness of single-step contact tracing without tracing delays. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *). The plots show p c * as a function of the latent period relative to the mean time to detection (t lat ). There are four special cases: A. Short infectious period and variable time to detection; B. Short infectious period and fixed detection time; C. Long infectious period and variable time to detection; and D. Long infectious period and fixed detection time. The three curves denote p c * for different values of the pre-isolation reproduction ratio R 0 pre . Indicated by dashed lines are the average t lat for four infections, in the panels with closest correspondence to the actual parameter values ( Table 2 ). Influenza appears in two panels with long and short infectious period, because it corresponds to both parameter sets equally. doi:10.1371/journal.pone.0000012.g002 Figure 3 . The effectiveness of single-step contact tracing with tracing delays, with the pre-detection reproduction ratio R 0 pre = 1.5. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *). The contour plots show p c * as a function of the tracing delay d and the latent period t lat , measured relative to the mean detection time, for four special cases: A. Short infectious period and variable incubation period; B. Short infectious period and fixed incubation period; C. Long infectious period and variable incubation period; and D. Long infectious period and fixed incubation period. Dark grey shadows indicate areas where tracing is ineffective, light grey shadows indicate areas where p c * = 0.33. Indicated by dashed lines are the average t lat for four infections, in the panels with closest correspondence to the actual parameter values ( Table 2 ). Influenza appears in two panels with long and short infectious period, because it corresponds to both parameter sets equally. doi:10.1371/journal.pone.0000012.g003 Figure 4 . The effectiveness of single-step and iterative contact tracing for control of influenza, smallpox, SARS, and foot-and-mouth disease. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *); p c * is plotted as a function of the relative delay (d, proportion of the incubation period) or the absolute delay (days). doi:10.1371/journal.pone.0000012.g004 Table 2 . Panel 4A shows the relation between p c * and the relative tracing delay d, from which it appears that the general cases shown in Figures 2 and 3 are good predictors for assessing tracing effectiveness. Influenza can be placed somewhere between long and short infectious periods (panels 2A,C, and 3A,C): it is hardly effective without delay, and ineffective already if d = 0.1. SARS control requires a tracing probability p c *,121/R 0 pre which is relatively insensitive to delays. Smallpox requires p c * = 121/R 0 pre , and is insensitive to delays up to some point (d<0.6) where tracing becomes quickly ineffective. Finally, FMD can be traced effectively only if d is small, but it is sensitive to delays already if d is small and requires a tracing probability p c *.121/R 0 pre . Measured in real time (panel 4B), effectiveness of tracing appears to be highly dependent on the actual generation time of the infection. Influenza control is hardly possible, FMD control will be difficult, whereas tracing is likely to be successful for smallpox and SARS.
Suppose a test is available to determine the infection status of traced contactees. We can then continue tracing iteratively before infected contactees are detected due to symptoms, until no further infecteds are found ( Figure 1B) .
We evaluated iterative tracing with and without delays for the same cases as single-step tracing, resulting in Figures similar to Figures 2 and 3 (see Supporting Information). As expected, the universal effect of iterative tracing is to lower p c *, although the lower limits 121/R 0 pre as observed in three of the four special cases remain intact. The largest difference between single-step and iterative tracing is observed when single-step tracing is on the brink of being effective, so that is the only situation where iterative tracing will make a difference (see also Figure 4 ). Without delays this difference is rather intuitive: single-step tracing is not effective if the latent period is small, but iterative tracing will always be effective, because if all contacts are traceable, the first detection is immediately followed by quarantine of every infected. This is the case with influenza, although the improvement will probably not be sufficient to making tracing effective for influenza control. On the other hand, when the latent period is large and single-step tracing is effective, iterative tracing only improves on this if there are significant delays, providing one or two more days to reach the required p c * (smallpox, SARS, and FMD).
We studied the effectiveness of contact tracing in a model for the start of an epidemic, that is flexible enough to use for most infections. Effectiveness of contact tracing was expressed as the minimum proportion of contacts that need to be traced to obtain a reproduction ratio prior to control of 1 (p c *). Other threshold values may be chosen if more is known about transmission of the infection to the general community while infectious individuals are being isolated or quarantined. For instance, if isolated infecteds still cause an average of 0.3 new cases in the general community after isolation or quarantine, p c * should be redefined such that R pre = 0.7.
The first conclusion from our model is that, for a given R 0 pre , the critical tracing probability can take any value depending on all infectious disease characteristics in the model: the latent period, the infectious period, and the detection time distribution. In contrast to some earlier publications on contact tracing [7, 9] , there exists no general expression for p c * as there is for the proportion to vaccinate for effective control in a well-mixed population. For smallpox the relation p c * = 121/R 0 pre holds reasonably well, but for SARS it is smaller, and for influenza and FMD it is larger.
The second conclusion is that a variable detection time improves tracing effectiveness, possibly even resulting in p c *,121/R 0 pre . This does not mean, of course, that one should aim at late detection of some infecteds (which would increase R 0 pre ), but that apparent variability is an argument to use tracing. The reason is that the few infecteds that are detected late (or not at all, which is essentially the same) will be discovered by backwards tracing which is an additional effect to forwards tracing that in itself may already result in p c * = 121/R 0 pre . It was earlier found that p c * can be smaller than 121/R 0 pre [9] , but by a different mechanism, namely the presence of shared contacts in a network.
The third conclusion is that the sensitivity of tracing effectiveness to the latent period and tracing delay may be large, especially in the case of single-step tracing. If this is the case already with a small delay (influenza and to a lesser extent FMD), reliability of parameter estimates will be crucial to establish whether tracing is advantageous. If it is only the case with larger delays (smallpox, SARS), tracing may be effective as long as it is done quick enough.
The fourth conclusion is that in most situations single-step and iterative tracing are almost equally effective. A considerable difference can only be expected if single-step tracing is not or hardly effective, which is also when the sensitivity to the latent period and tracing delay is largest (see above). Thus, influenza control will benefit from iterative tracing already without delays, whereas with the other infections one or two days may be gained ( Figure 4C,D) , thus having more time to achieve the required p c *. In the real world, the choice between single-step and iterative tracing will be based on what is possible. Capacity problems may reduce the effectiveness of iterative tracing if effort is directed towards secondary contactees prior to primary contactees; on the other hand, if not quarantine but vaccination is applied, it might be worthwhile to traced contactees of contactees even without diagnostic tests [6] .
For determining the cost-effectiveness of contact tracing, not only the critical tracing probability p c * is required, but also how easily that p c * can be achieved. A key aspect will be the possibility to distinguish relevant contacts: for control of sexually transmitted diseases relevant contacts are easily identified (that is, if people are willing to co-operate), but in case of respiratory pathogens like SARS or influenza the notion of relevant contacts is rather diffuse. Good insight will be obtained by not only regarding the sensitivity of tracing (p c ), but also the positive predictive value: what proportion of traced individuals is actually infected, and if we put more effort into increasing p c , how will it decrease the positive predictive value?
A relatively easy way to increase the proportion of traced contacts for respiratory pathogens like influenza might be quarantine of households. This can be effective if households are the prime location for spread of the infection; specific models taking into account the contact structures within and between households will be better-suited to study this strategy.
In our model we only regarded transmission of the infection before tracing or isolation, allowing us to focus on the characteristics of the contact tracing itself. Regarding the control of animal disease epidemics, e.g. foot-and-mouth disease or avian influenza, it is not unreasonable to assume that there will be no other transmission: all infected and suspected farms will be culled and they really will stop being infectious. However, with human infections it is very likely that isolation and quarantine will be less effective. This results in a complicated situation, because transmission will not only be reduced after detection, the contact structure will also change, with only a limited number of people (health-care workers) having contact with multiple isolated patients. For the case of SARS, a more detailed model on exactly this aspect was studied by Lloyd-Smith et al [5] , who indeed concluded that minimizing within-hospital transmission might be crucial, especially if tracing and isolation occur rather slowly. Further studies are required for this complex problem. The present paper provides a good understanding of the principles of contact tracing, and how infectious disease characteristics determine the effectiveness.
The effect of single-step tracing can be measured by the effective reproduction ratio R q , the average number of new infections per infected. Whereas the rate of being traced backwards at time t since infection (due to detection of any infectee) is equal for all infecteds, the rate of being traced forwards (due to detection of the infector) depends on the generation-time distribution, which in turn depends on the number of traceable generations backwards in the transmission tree (traceable ancestors). Hence, infectives need to be typed according to the number of traceable ancestors j, and R q is the largest eigenvalue of the next-generation matrix K with entries k ij being the expected number of type-i infecteds (with i traceable ancestors) produced per type-j infected [15] . Because type-j infecteds only produce type-0 and type-j+1 infecteds, by untraceable and traceable contacts respectively, all entries other than k 0j and k j+1,j are equal to 0.
Even though the matrix is infinitely large, we conjecture from all observed numerical calculations that the positive elements k j+1,j converge in the sense that |k j+1,j 2k j+2,j+1 |R0 as jR'. Therefore, for numerical evaluation we 'closed' the matrix to an (n+1) 2 matrix with k nn = k n+1,n . For the four special cases, R q could be calculated numerically in MathematicaH, but for other cases considered, the entries of the matrix had to be determined by numerical simulation. Analysis of single-step tracing with delay could be done by calculating R from the next-generation matrix, as for the model without delay (see Supporting Information for all details).
With iterative tracing, isolation of a single infected results in quarantine of a cluster of infecteds, all mutually linked by traceable contacts ( Figure 1B) . By recognizing these clusters of infectives, an epidemic of infected individuals can be regarded as an epidemic of traceable clusters. Each untraceable contact in the transmission tree results in infection of a new cluster index case, so the average number of untraceable contact infections caused by a single cluster, the cluster reproduction ratio R c , determines the effectiveness of iterative contact tracing. If Y(p c ) denotes the expected cumulative infectiousness of a cluster at the time of cluster quarantine, then
As with single-step tracing, effectiveness of iterative tracing is determined by considering the critical tracing probability (for achieving R c = 1):
If p c = 0, then each infective will be a separate cluster, and R c = Y(0) = R 0 pre by definition. For p c .0 the cluster size will be larger than one, but cluster infectiousness need not be larger, as backwards tracing can reduce the infectious period of superspreaders with large detection time as it does in single-step tracing.
For two of the four special cases (a = '), and for the case considered by Müller et al [7] (a = 1, t inf = ', t lat = 0), Y(p c ) can be calculated numerically in MathematicaH. For all other cases, including the real infections, stochastic simulations were needed.
Incorporating delays into iterative tracing does not generally change the concept of R c , but it makes the calculation of Y(p c ) very complicated, because there is no longer a single time of cluster quarantine. Two complications arise: first, delays may cause contactees in the chain emanating from a symptomatic infected to become symptomatic themselves before the tracing process reaches them. They then initiate a new tracing process of their own within the same cluster. Second, if the infection process is faster than the tracing process, the cluster size grows infinitely large and iterative tracing becomes ineffective. We determined p c * by stochastic simulation of clusters until quarantine of the final infected (see Supporting Information for all details).
Supporting Information Supporting information for the paper "The Effectiveness of Contact Tracing in Emerging Epidemics" Found at: doi:10.1371/journal.pone.0000012.s001 (0.60 MB PDF) The Waiting Time for Inter-Country Spread of Pandemic Influenza BACKGROUND: The time delay between the start of an influenza pandemic and its subsequent initiation in other countries is highly relevant to preparedness planning. We quantify the distribution of this random time in terms of the separate components of this delay, and assess how the delay may be extended by non-pharmaceutical interventions. METHODS AND FINDINGS: The model constructed for this time delay accounts for: (i) epidemic growth in the source region, (ii) the delay until an infected individual from the source region seeks to travel to an at-risk country, (iii) the chance that infected travelers are detected by screening at exit and entry borders, (iv) the possibility of in-flight transmission, (v) the chance that an infected arrival might not initiate an epidemic, and (vi) the delay until infection in the at-risk country gathers momentum. Efforts that reduce the disease reproduction number in the source region below two and severe travel restrictions are most effective for delaying a local epidemic, and under favourable circumstances, could add several months to the delay. On the other hand, the model predicts that border screening for symptomatic infection, wearing a protective mask during travel, promoting early presentation of cases arising among arriving passengers and moderate reduction in travel volumes increase the delay only by a matter of days or weeks. Elevated in-flight transmission reduces the delay only minimally. CONCLUSIONS: The delay until an epidemic of pandemic strain influenza is imported into an at-risk country is largely determined by the course of the epidemic in the source region and the number of travelers attempting to enter the at-risk country, and is little affected by non-pharmaceutical interventions targeting these travelers. Short of preventing international travel altogether, eradicating a nascent pandemic in the source region appears to be the only reliable method of preventing country-to-country spread of a pandemic strain of influenza. The emergence of a pandemic strain of influenza is considered inevitable [1] . Provided the emerged strain is not too virulent, it may be possible to eliminate a nascent influenza pandemic in the source region via various combinations of targeted antiviral prophylaxis, pre-vaccination, social distancing and quarantine [2, 3] . If early elimination in the source region is not achieved, then any delay in a local epidemic that a country can effect will be highly valued. To this end, countries may consider introducing non-pharmaceutical interventions such as border screening, promoting early presentation of cases among arriving passengers, requiring the use of personal protective equipment during travels (e.g. the wearing of masks), and reducing traveler numbers. While the case for believing that measures such as these can not stop the importation of an epidemic from overseas has been argued strongly, whether it be SARS or influenza [4] [5] [6] , the extent to which such interventions delay a local epidemic is currently not well quantified, and hence of considerable interest.
In this paper we demonstrate how the delay to importation of an epidemic of pandemic strain influenza may be quantified in terms of the growing infection incidence in the source region, traveler volumes, border screening measures, travel duration, inflight transmission and the delay until an infected arrival initiates a chain of transmission that gathers momentum. We also investigate how the delay is affected by the reproduction number of the emerged strain, early presentation of cases among arriving passengers, and reducing traveler numbers. As noted in previous simulation modeling [7] , many aspects of this delay have a significant chance component, making the delay a random variable. Therefore, the way to quantify the delay is to specify its probability distribution, which we call the delay-distribution. Some issues of the delay distribution, such as the natural delay arising in the absence of intervention and the effect that reducing traveler numbers has on this delay has been studied previously [6] [7] [8] . Specifically, if the originating source is not specified, and homogeneous mixing of the worlds population is assumed, then the most likely time to the initial cases arising in the United States is about 50 days assuming R 0 = 2.0 [7] . The additional delay arising from travel restrictions appears minimal until a.99% reduction in traveler numbers [6] [7] [8] .
This paper adds to previous work [5] [6] [7] [8] by simultaneously including a wider range of epidemiological factors and possible interventions, such as elevated in-flight transmission, flight duration, the effect of wearing of mask during flight, early presentation of cases among travelers, and quarantining all passengers from a flight with a detected case at arrival.
Consider a region in which a new pandemic strain of influenza has emerged, and a region currently free from the infection. We refer to these as the source region and the at-risk country, respectively. Travel between these countries is predominantly via commercial air travel and/or rapid transport which could potentially be subject to border screening and other interventions. We restrict our discussion to air travel. The aim is to assess the effects that a variety of non-pharmaceutical border control measures have, individually and in combination, on the time it takes before the epidemic takes off in the at-risk country. An epidemic is said to have ''taken off'' when it reaches 20 current infectious cases, after which its growth is highly predictable (i.e. nearly deterministic) and the probability of fade-out by chance is very low, if intervention is not enhanced. The source country of origin will undoubtedly have a large impact on the natural delay until importation of an epidemic, although this is difficult to quantify [7] . An alternative is to fix the originating city, for example a highly connected city such as Hong Kong [6] , with the obvious effect that results are highly dependent on the choice. We adopt no specific source region, but assume that the number of international travelers originating from it is reasonably small (see Methods), suggestive of a rural or semirural source region [2] . It is further assumed that the current heightened surveillance for pandemic influenza is continued and that a nascent pandemic with human-to-human transmission is identified and the pandemic is declared when there are 10 concurrent cases in the source region.
For an epidemic to take off in an at-risk country, a series of events need to occur. First, the epidemic needs to get underway in the source region. Second, an intending traveler needs to be infected shortly before departure. Third, the infected traveler must actually travel and successfully disembark in the at-risk country. Fourth, the infected traveler, or fellow travelers infected during the flight, must initiate an epidemic in the at-risk country with the infectiousness that remains upon arrival. Finally, the epidemic needs to reach a sufficient number of cases to begin predictable exponential growth.
International spread of the emerged pandemic strain of influenza may occur when a recently infected person travels. By 'recently infected' we mean that their travel is scheduled to occur within ten days of being infected. We assume that the number of individuals traveling from the source region to the at-risk country each day is known. The probability that a randomly selected traveler is a recently-infected person is taken to be equal to the prevalence of recently-infected people in the source region on that day. The incidence of infection in the source region is assumed to grow exponentially initially, with the rate of exponential growth determined by the disease reproduction number (the mean number of cases a single infective generates by direct contact) and the serial interval (the average interval from infection of one individual to when their contacts are infected) ( Figure 1A) .
The time since infection of a recently-infected traveler is a key component of the calculations, because it affects the chance of positive border screening, the chance of in-flight transmission and the infectivity remaining upon arrival in the at-risk country. The time since infection at the time of scheduled departure is random and the dependence of its probability distribution on the exponential growth rate of infection is illustrated by Figure 1B (see also Supporting Information). The higher the epidemic growth rate in the source region, the greater the probability than an infected traveler will have been infected more recently.
It is assumed that individuals detected by departure screening are prevented from traveling. To be detected by screening an infected traveler must be symptomatic and positively screened. An individual is assumed to become symptomatic 48 hours after being infected (cf. [3] who use 1.9 days). The probability of being symptomatic when presenting for departure screening is computed from the curve in Figure 1B . The distribution of the time since infection immediately after departure screening, given that the infected traveler was not detected, is given by the curve in Figure 1C . It contains an adjustment for the probability of being detected at departure.
The instantaneous rate at which susceptible contacts are infected depends on the time since infection, and is described by an infectiousness function ( [9] , page 45). We use a peaked infectiousness function, motivated by viral shedding and household transmission data [2] , which has a serial interval of 2.6 days. The basic reproduction number (R 0 ), namely the reproduction number when there is no intervention in place and every contacted individual is susceptible, is given by the area under the infectiousness function. However, our concern is with the effective reproduction number R that holds when various interventions are in place. We obtain any R by simply multiplying the infectiousness function by the appropriate constant (to make the area under the curve equal to R). This keeps the serial interval the same. In the absence of suitable data we assume for most scenarios that the aircrafts ventilation and filtration systems are functioning properly, and that infected travelers transmit the infection at the same rate during a flight as they would while mixing in the community. We examine the sensitivity of this assumption by increasing the inflight transmission by as much as 10-fold (as could potentially happen if air-circulation and filtration systems malfunction, e.g. see [10] ). The in-flight transmission rate is set to zero under the optimistic scenario that all travelers wear 100% effective masks during transit. In terms of a sensitivity analysis this illustrates what would be achievable in a best-case scenario. The number of offspring that an infected traveler infects during a flight is a random variable, taken to have a Poisson distribution with a mean equal to the area under the infectiousness function over to the flight duration.
Travelers infected during flights of less than 12 hours duration are asymptomatic at arrival and will not be detected by screening. The probability that an arriving traveler who was infected in the source region is detected on arrival is computed from the distribution of the time since infection on arrival. This distribution is obtained from the curve in Figure 1C by shifting it to the right by an amount equal to the duration of the flight. The distribution of the time since infection for an individual infected in the source region, who passes through arrival screening undetected has a further adjustment for the chance of being detected at arrival ( Figure 1D ). This curve shows that an infected traveler who escapes detection at departure and arrival is highly likely to enter the at-risk country with most, or all, of their infectious period remaining.
Authorities are assumed to implement one of two control options when detecting an infected traveler by arrival screening. Under option one (individual-based removal), all passengers who test negative are released immediately and only passengers who test positive are isolated. Under the second option (flight-based quarantining), authorities prevent all passengers from dispersing into the community until the last person has been screened from that flight. Should any one passenger be detected as infected then all passengers will be quarantined, as previously recommended [5] .
Transmission chains can be initiated in the at-risk country by infected travelers who mix within the community upon arrival. Suppose now that a flight arrives with one, or more, infected passengers who mix within the community. We classify these infected arrivals into those who are 'pre-symptomatic' and those who are 'symptomatic' at entry. It is assumed that the 'symptomatic' infected arrivals do not recognize their symptoms as pandemic influenza and will not present to medical authorities. In other words, they spend the remainder of their infectious period mixing in the community. On the other hand, the 'presymptomatic' infected arrivals, including all individuals infected during flight, are assumed to mix freely in the community only from entry until they present to medical authorities after some delay following the onset of symptoms.
Not all infected travelers entering the community initiate a 'major' epidemic, even when the reproduction number (R) exceeds one. Quite generally, the distribution of the size of an epidemic initiated by an infected arrival is bimodal, with distinct peaks corresponding to a major epidemic and a minor outbreak ( Figure 1E ). In the latter event the outbreak simply fades out by chance despite there being ample susceptibles in the population for ongoing trans- In (B), the step illustrates the probabilistic removal of travelers who have completed their incubation period. In (D), the distribution of time since infection in (C) will have shifted to the right by an amount equal to the flight duration, and cases incubated in-flight may be detected by symptomatic screening, as will those symptomatic cases that were not detected previously. Screening sensitivity for this illustration is 60% on both departure and arrival. (E) Upon entering the community undetected, an infected traveler may initiate a minor (inconsequential) or major epidemic, depending on the characteristics of the disease and public health policy. doi:10.1371/journal.pone.0000143.g001 mission [11] . The number of cases in an outbreak that fades out is typically very small compared to an epidemic.
The probability that a typical infective generates a local epidemic is computed by using a branching process approximation [12] for the initial stages of the epidemic, and equating 'epidemic' with the event that the branching process does not become extinct. This calculation is well known (e.g. [13] , page 473), but is modified here to allow for the fact that the process is initiated by a random number of infected arrivals and some of them have spent a random part of their infectious period before arriving in the at-risk country. The distribution for the random number of individuals infected by an infected individual when all their contacts are with susceptible individuals is needed for the calculation. The lack of data prevents a definitive conclusion for the most appropriate offspring distribution for influenza transmission [14] , and we use a Poisson distribution with a mean equal to R, discounted for individuals who spent only some of their infectious period mixing in the at-risk country. A Poisson offspring distribution is appropriate when the area under the infectiousness function is non-random (i.e. all individuals have the same infection 'potential'). We assume that R is the same in the source region and the at-risk country. For an undetected infected traveler and all their in-flight offspring to fail to initiate an epidemic on arrival, all of the chains of transmission they initiate must fail to become large epidemics (see Supporting Information).
We calculate the probability distribution of D, the total delay until an epidemic gathers momentum by noting that it is given by D = D 1 +D 2 , where D 1 is the time until an epidemic is first initiated and D 2 is the time from initiation until the local epidemic gathers momentum. For an epidemic to be first initiated in the at-risk country on day d, it must have not been initiated on all previous days. Hence the probability distribution of the time delay (D 1 ) until the epidemic is first initiated in the at-risk country following identification in the source region is described by:
Pr (D 1~d )~ p p 1 p p 2 p p 3 ::: p p d{1 p d where p d denotes the probability that the epidemic is initiated on day d , and p p d~1 {p d denotes the probability that the epidemic is not initiated on day d (see Supporting Information for calculation of p d ).
Once successfully initiated, an epidemic may initially hover around a handful of cases before reaching a sufficient number of cases for its growth to become essentially predictable. As mentioned, 20 concurrent cases is our criterion for an epidemic to have gathered momentum. We determine the distribution of D 2 , the time to this occurrence, from 10,000 stochastic simulations and approximate this empirical distribution by a shifted gamma distribution. Our criterion of 20 concurrent cases is conservatively high, as results from the theory of branching processes shows that the probability of a minor epidemic (and hence no take-off) starting from 20 concurrent cases is about 3610 28 when R = 1.5, and even smaller for higher values of R. Finally, the distribution of the total delay (D = D 1 +D 2 ) from the pandemic being identified in the source region until 20 cases in the at-risk country was calculated by the convolution of the distributions of D 1 and D 2 .
For the illustrative purposes, we chose values of 1.5, 2.5 and 3.5 for R, which encompass estimates proposed for previous pandemics [2, 3, 15] . The number of people within the infected source region was assumed reasonably small (5 million), and there was one flight per day traveling from the source region to the at-risk country carrying 400, 100 or 10 passengers. A higher number of travelers affects the delay only marginally, assuming the epidemic takes off in the source region (see Results). We assume a typical travel duration between attempted departure and possible arrival of 12 hours, but also examine the effect of varying this from 0-48 hours. The time to presentation following symptom onset is varied from 'immediately' to 'never presenting', with a time of 6 hours considered likely in the presence of an education campaign. The sensitivity of symptomatic screening is varied from 0-100%, with results presented for 0, 50 and 100% sensitivity.
The probability that a recently infected traveler evades screening is substantial even if screening reliably detects symptomatic travelers (Figure 2A) , because the typical travel duration is shorter than the 2-day incubation period. In addition, during the early stages of the epidemic a high R in the source region acts to increase the probability that an infected traveler has been infected quite recently and hence will escape detection due to being asymptomatic during their travels (Figure 2A ). For example, assuming 100% sensitivity for detecting symptomatic infection, we calculate that during the early stages of the epidemic the proportion of infected travelers that evade both departure and arrival screening after 12 hours of travel is 0.26, 0.45 and 0.59 for disease reproduction numbers 1.5, 2.5 and 3.5, respectively.
As the duration of travel approaches the disease incubation period, effective symptomatic screening substantially reduces the likelihood that a traveler evades screening and initiates an epidemic ( Figure 2B ). Reducing the time from the onset of symptoms to presentation (and subsequent isolation) for each infected arrival also reduces the probability that a major epidemic is initiated, however the best case scenario of infected travelers and all their in-flight offspring presenting immediately following the onset of symptoms still poses a substantial risk of epidemic initiation arising from pre-symptomatic transmission ( Figure 1C ).
The delay contains a fairly substantial natural component, primarily due to the time it takes to increase the number of infectives in the source region sufficiently to make the chance of a recently infected traveler appreciable ( Figure 3A ), and the time (D 2 ) it takes for a local epidemic in the at-risk country to gather momentum following successful seeding ( Figure 4A ). In the absence of any interventions, the number of infected individuals who successfully enter the community of the at-risk country initially increases exponentially ( Figure 3A ). With individual-based removal of infected travelers, the number of individuals entering the at-risk country undetected by screening is proportionately reduced over the course of the epidemic ( Figure 3A ). With flightbased quarantining, the number of infected individuals entering the at-risk country undetected is dramatically reduced over the course of the epidemic, even for relatively insensitive screening ( Figure 3A ). With flight-based quarantining, the number of infected passengers slipping through undetected is bimodal, with the first peak occurring when the number of infected travelers attempting to travel is still in single figures.
Without screening, the daily probability that an epidemic is initiated (p d ) increases, and becomes near certain once the number of infected travelers arriving undetected exceeds about 10 ( Figure 3B, solid line) . With screening and individual-based removal of infected individuals, p d follows a similar pattern only reduced somewhat. With screening in combination with flightbased quarantining, this probability is changed dramatically. After an initial rise it dips, to become essentially zero during the height of the epidemic in the source region ( Figure 3B , dotted line). This arises because once a flight has several infected travelers, the probability that at least one is detected approaches one (even if screening is imperfect), and all passengers on such a flight are quarantined. Once the epidemic starts to wane in the source region (assuming the unlikely event of the pandemic strain is restricted to the source region), the probability of initiation rises once again. The corresponding distribution of D 1 , the delay until The effects of R and the time from symptom onset to presentation on the probability that an infected traveler, having entered the wider community following arrival, will initiate an epidemic. There is no screening. doi:10.1371/journal.pone.0000143.g002 the epidemic is first initiated in the at-risk country, is bi-modal in the presence of screening ( Figure 3C) .
Although flight-based quarantining is effective in preventing the entry of infected travelers during the height of the epidemic, a substantial cumulative risk of initiation has already occurred before this from the handful of infectives that have slipped through undetected ( Figure 3B ). Hence, whilst the effect of border screening, particularly in conjunction with flight-based quarantining, on the daily probability of initiation is dramatic, its effect on the delay to initiation is much less pronounced ( Figure 3C ). Border screening, even with perfect sensitivity for detecting symptomatic cases, tends to increase D 1 , the time to an epidemic being initiated, by a matter of days to weeks. The time (D 2 ) from initiation (the arrival of the index case) to an epidemic reaching 20 concurrent cases within the at-risk country is adequately modeled using a shifted Gamma distribution ( Figure 4A ). The convolution of this right-skewed Gamma distribution with the left-skewed delaydistribution of D 1 (Figure 3C ) yields the distribution for D, the total delay until the epidemic reaches 20 cases in the at-risk country ( Figure 4B ). The distribution of D is approximately symmetrical. The effect of border screening on the total delay D is quite modest, though sensitive to how screening is implemented. For example, with R = 1.5 and 400 travelers per day, 100% sensitive screening with individual-based removal increases the median delay from 57 to 60 days ( Figure 4B ). Flight-based quarantining would extend the median delay to 70 days. In general, the added delay arising from flight-based quarantining is about four-fold that arising from individual-based removal.
The natural component of the delay is highly sensitive to the disease reproduction number ( Figure 5A ). For example, with 400 passengers per day departing the source country and in the absence of any interventions, the median delay ranges from a low of 17 days for R = 3.5 to 57 days for R = 1.5 ( Table 1 ). The delay is less sensitive to the number of intending travelers, with little appreciable increase in the median delay occurring until traveler numbers become very low ( Figure 5B ). For example, if R = 1.5, with no other border control measures, decreasing the number of intending travelers departing the source region from 400 to 100 per day increases the median total delay D from 57 to 66 days. A further decrease in the number of intending travelers to 10 per day increases the median delay to 83 days ( Table 1) .
The delay is quite insensitive to the rate of transmission in-flight. For example, with R = 1.5, a 12-hour flight, 400 travelers per day and no other interventions, preventing in-flight transmission altogether increases the median delay from 57 to 58 days. Conversely, doubling the rate of in-flight transmission reduces the median delay from 57 to 56 days. A 10-fold increase in the rate of transmission in-flight only decreases the median delay from 57 to 53 days. Encouraging the early presentation of cases among travelers following the onset of symptoms has a limited effect on the delay distribution ( Figure 5C ). For example, for R = 1.5, 400 intending travelers per day and no other interventions, reducing the time to presentation from 'never presenting' to 6 hours increases the median delay from 57 to 61 days. Immediate presentation at symptom onset only increases the median delay a further day in this scenario.
In general, the additional delay achieved by introducing nonpharmaceutical border control measures is generally small in comparison with the natural delay ( Figure 5D ). For the scenario with R = 1.5 and 400 intending travelers per day, a combination of 100% flight-based quarantining, 100% compliance with mask wearing during travel and immediate presentation at symptom onset extends the estimated median delay from 57 to 79 days ( Figure 5D ). This added delay diminishes in absolute terms as R increases. For example, if the same interventions are applied with R = 3.5, the median delay is extended from 17 to just 20 days ( Figure 5D ). The one exception to this generalisation is when travel numbers are reduced dramatically. The added delay achieved when a drastic reduction in travel numbers is combined with other border control measures appears to be greater than adding the delays each achieves on its own. For example, if R = 1.5, and we reduce the number of intending travelers from 400 to 10 per day, implement 100% flight-based quarantining, implement compulsory mask wearing during travel and presentation at 6 hours following symptom onset then there is a substantial probability (0.74) that the pandemic strain will never be imported (assuming the epidemic is confined to the source country). The estimated quartile delay (the median in this case is undefined) to the start of a major epidemic in an at-risk country is extended from 50 to 125 days. Again, the added delay decreases rapidly as R increases, and if the above interventions were applied with R = 3.5, the estimated median delay is extended from 17 to 26 days, and the importation of the epidemic is certain ( Figure 5D ).
We have formulated a model of the importation of an infectious disease from a source region to an at-risk country that permits a comprehensive analysis of the effect of border control measures. Our results are most relevant to the early stage of a pandemic when most cases are contained within a single source region. Once the pandemic has spread to several countries, models with greater complexity and ability to more realistically model global mixing patterns [6] [7] [8] are required. Our model is developed with a pandemic-strain of influenza in mind, but could apply to any emerging infectious disease that is transmitted from person to person. We have assumed a Poisson distribution for the number of secondary infections, which a natural choice when each infected individual has the same infectivity profile. A distribution with a larger variance is appropriate when individuals vary substantially in their infectiousness. Our results are conservative in the sense that they give an upper bound for the probability that an infected traveler manages to initiate an epidemic, compared to an offspring distribution with a greater variance but the same reproduction number [14] .
The nature of the next pandemic influenza virus, and particularly its reproduction number, is uncertain. If its reproduction number is low (R,2.0), our results indicate that at-risk countries receiving a reasonably small number of travelers (say 400 per day) from the infected source region can expect a natural delay until importing an epidemic of the order of 2 months. This is quite variable and under favourable conditions it could be 4 months. However, the natural delay decreases rapidly as R increases.
The additional delay from isolating individuals detected by border screening is merely a few days under most plausible scenarios, even if both departure and arrival screening is introduced and screening detects every symptomatic traveler. While the extra delay is more than quadrupled if flights with a detected case(s) are quarantined, the effect remains modest (weeks at most) and it is questionable whether the extra delay achieved warrants the disruption created by such a large number of quarantined passengers.
In-flight transmission is a commonly raised concern in discussions about the importation of an infection, so inclusion of in-flight transmission is an attractive feature of our model. Events of substantial in-flight transmission of influenza have been documented [10, 16] and modeling of indoor airborne infection risks in the absence of air filtration predicts that in-flight transmission risks are elevated [17] . However, it difficult to estimate the infectiousness of influenza in a confined cabin space, as there is undoubtedly substantial under-reporting of influenza cases who travel and fail to generate any offspring during flight. Provided the aircraft ventilation system (including filtration) is operational, it is considered that the actual risk of in-flight transmission is much lower than the perceived risk [18] . Our results indicate that the delay is relatively insensitive to the rate of in-flight transmission, making in-flight transmission less of an issue than commonly believed. A highly elevated transmission rate inflight will hasten the importation of an epidemic only marginally. Consistent with this, eliminating in-flight transmission by wearing protective masks increases the delay only marginally.
Early presentation by infected arrivals not detected at the borders was found to add only a few days to the delay. To some extent this arises due to our assumption that pre-symptomatic transmission can occur, for which there is some evidence. In contrast, Ferguson et al. [2] assume that the incubation and latent periods are equal, with a mean of 1.5 days. In their model presymptomatic transmission is excluded and infectiousness is estimated to spike dramatically immediately following symptom onset and declining rapidly soon afterwards. Under their model assumptions, immediate presentation at onset of symptoms would reduce transmission effectively. However, as presentation occurs some time after onset of symptoms and the bulk of infectivity occurs immediately after onset of symptoms the results on the effect of early presentation of cases are likely, in practical terms, to be similar to those found here. Given the variable nature of influenza symptoms, there is likely to be a difference between the onset of the first symptoms as measured in a clinical trial (e.g. [19] ) and the time that a person in the field first suspects that they may be infected with influenza virus. To fully resolve the issue of how effective very early presentation of infected travelers is in delaying a local epidemic we need better knowledge about the infectiousness of individuals before and just after the onset of symptoms. It is assumed that the pandemic is identified and declared when there are 10 concurrent cases in the source region attributed to human-to-human transmission, and that screening is applied at both departure and arrival. The time between screening events is assumed to be 12 hours and infected travelers are not isolated following the onset of symptoms. Of the border control measures available, reducing traveler numbers has the biggest effect on the delay and even then it is necessary to get the number of travelers down to a very low number. An equivalent control measure is to quarantine all arriving passengers with near perfect compliance.
Our results indicate that short of virtually eliminating international travel, border control measures add little to avoiding, or delaying, a local epidemic if an influenza pandemic takes off in a source region. All forms of border control are eventually overwhelmed by the cumulative number of infected travelers that attempt to enter the country. The only way to prevent a local epidemic is to rapidly implement local control measures that bring the effective reproduction number in the local area down below 1, or to achieve rapid elimination in the source region, in agreement with other recent studies [6] [7] [8] . Preventing the exponential growth phase of an epidemic in the source region appears to be the only method able to prevent a nascent influenza pandemic reaching atrisk countries.
Text S1 Estimating the daily probability of epidemic initiation Found at: doi:10.1371/journal.pone.0000143.s001 (0.08 MB PDF) Neutralizing Antibody Fails to Impact the Course of Ebola Virus Infection in Monkeys Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody. Editor's note: The potential efficacy of pre-and post-exposure prophylaxis against Ebola virus infection, as well as the fundamentally important question of whether neutralizing bodies are important for Ebola virus resistance, is addressed by a related manuscript in this issue of PLoS Pathogens. Please see doi: 10 .1371/journal. ppat.0030002 by Feldmann et al.
Passive transfer of relatively high concentrations of neutralizing antibodies can protect against challenge with a range of viruses in animal models and in humans [1] [2] [3] . Protection in some cases is in the form of sterilizing immunity, i.e., no viral replication is observed following challenge [2, 4, 5] . In other cases (e.g., [5, 6] ), some replication is observed but protection from disease is achieved, presumably because neutralizing antibody sufficiently blunts infection for T cell and innate immunity to resolve infection [7] . It might be expected that passive neutralizing antibody would be most effective against challenge with acute viruses. Many acute viral infections are resolved even in the absence of neutralizing antibody, and the blunting effect of passive antibody would provide more time for the development of effective cellular immune responses. In contrast, chronic viruses may present a greater challenge to passive antibody, since, in the absence of sterilizing immunity, there is a window of opportunity for the virus to establish a chronic infection before cellular immunity can be mobilized.
Ebola virus (EBOV) causes a severe acute infection in humans [8] . Infection with the Ebola Zaire strain, Zaire ebolavirus (ZEBOV), produces mortality in the range of 60%-90% [9] with death generally occurring around 7-11 d following the appearance of symptoms [8] . There is a single report describing the use of convalescent sera to treat EBOV infection [10] . However, the patients in this report may have already been through the worst stages of the disease, and it is not clear that serum antibodies were responsible for their recovery [10] . Further, neutralizing antibody titers in survivors of EBOV infection tend to be rather low, although we have isolated a neutralizing human monoclonal antibody (mAb), KZ52, of good potency from a convalescent individual [11] .
The ability of passive antibody to protect against EBOV has been investigated in a number of animal models. The guinea pig and mouse models use EBOVs that have been serially passaged to adapt to replication in the respective animals and are highly lethal. Protection has been demonstrated in the guinea pig model using neutralizing horse, sheep, and goat immunoglobulin G (IgG) against EBOV [12, 13] and the human anti-EBOV GP mAb, IgG KZ52. This antibody neutralizes ZEBOV (1995, Kikwit) with a 50% inhibitory concentration (IC 50 ) of 0.05-0.3 lg/ml and an IC 90 of 0.5-2.6 lg/ml in Vero cells [11, 14] and an IC 50 of approximately 0.05-1 lg/ml and a IC 90 of 0.5-2 lg/ml in primary human monocytes/macrophages [14] . We showed that when administered subcutaneously at a dosage of 25 mg/kg up to 1 h after challenge, the antibody protects against robust ZEBOV challenge (10,000 plaque-forming units [pfu] ) in the guinea pig model [6] .
Macaques provide a model of EBOV infection that is likely closer to human infection. The human virus can be used directly in macaques without need for adaptation and the course of disease mirrors that seen in humans [8] . In cynomolgus macaques (Macaca fascicularis), ZEBOV infection produces a mortality rate of 100% with death occurring 6-8 d following infection with 1,000 pfu [15] , while in rhesus macaques (Macaca mulatta) ZEBOV produces about 100% mortality with death occurring 7-10 d after infection with 1,000 pfu [16] . In contrast to the guinea pig experiments, the passively transferred polyclonal equine neutralizing IgG described above provided only some minor benefit in the form of a slight delay in the onset of viremia from day 5 to day 7 [13] following ZEBOV challenge of cynomolgus monkeys. No significant reduction in mortality was observed. However, protection against EBOV in primates has been observed in a low dose challenge model. Thus, neutralizing equine IgG protected baboons from ,30 LD 50 (50% lethal dose) ZEBOV challenge when the IgG was given up to 1 h after infection and the serum contained high neutralizing antibody titers (1:128 to 1:512) [17, 18] , and, similarly, neutralizing ovine serum protected baboons against 0.6 LD 50 ZEBOV challenge [19] .
Here, we studied the ability of passively transferred neutralizing human mAb KZ52 to protect against ZEBOV challenge in rhesus macaques. This passive transfer failed to protect the macaques against challenge with ZEBOV, and, furthermore, had a minimal effect on the explosive viral replication following infection. We showed by ELISA that antibody was present at high levels in serum of the monkeys and that neutralization escape was not responsible for the resistance of virus to antibody prophylaxis.
To evaluate whether IgGl KZ52 could protect against Ebola virus infection in a nonhuman primate animal model, antibody was passively transferred to rhesus macaques followed by challenge with the 1995 ZEBOV (Kikwit) isolate 24 h later. Protection against virus challenge by neutralizing antibodies in naive animals often requires high doses of antibody [2] . Therefore, we used a high dose of 50 mg/kg KZ52, which was close to the maximum practically achievable. In addition, we gave a second bolus of 50 mg/kg of KZ52 on day 4 following infection. The results for the four antibodytreated animals show a steady increase in plasma viremia up to 10 5 -10 7 pfu/ml on day 7 ( Figure 1 ). These levels of plasma viremia closely parallel those seen in the control animal and typically seen in historical controls [15] . The second bolus of antibody given on day 4 did not appear to have any impact upon the rate of increase of plasma virus ( Figure 1 ). Three of the treated animals were euthanized when moribund at day 9 or 10 post infection. The fourth treated animal showed a decrease in plasma viral load after the peak and survived to day 28 before becoming moribund when it too was euthanized. Although monkey CH46 had less severe symptoms than the other animals in the study, it was concluded that this animal, too, was suffering from disease due to ZEBOV, as evidenced, for example, by copious Ebola virus antigen in the lungs (see below).
Serum antibody (KZ52) loads were measured 1 d before virus challenge (day À1) and 4 d after challenge but before antibody boosting (day 4) by an ELISA designed to detect KZ52 as a human antibody that has bound to immobilized ZEBOV glycoprotein. The serum KZ52 antibody levels on day 4 were in the approximate range 200-400 lg/ml ( Table 1 ). The Figure 1 . Plasma Viremia in Macaques Challenged with ZEBOV Shown is the measured viremia, in log 10 pfu per ml, for four antibodytreated monkeys (CH46, CH56, CH57, and CH83) and one untreated control animal (EHD) at days 4, 7, 9, and 10 in plasma by plaque assay as described in Materials and Methods. 50 mg/kg of KZ52 IgG1 human antibody [11] was given intravenously to four rhesus macaques 1 d before and again 4 d after challenge with 1,000 pfu (intramusculary) of the 1995 ZEBOV (Kikwit) isolate. Ab, antibody. doi:10.1371/journal.ppat.0030009.g001
PLoS Pathogens | www.plospathogens.org January 2007 | Volume 3 | Issue 1 | e9 0063
Anti-Ebola Antibody Activity In Vivo
Ebola virus is one of the most feared of human pathogens with a mortality that can approach 90% and an extremely rapid disease course that can lead to death within days of infection. Antibodies able to inhibit viral infection in culture, neutralizing antibodies, can typically prevent viral infection in animals and humans when present prior to infection, at sufficient concentration. Such neutralizing antibodies may be provided through passive administration or induced by vaccination. We have previously shown that a human neutralizing antibody can protect guinea pigs against Ebola virus. However, here we show that this antibody does not protect monkeys against Ebola virus and surprisingly appears to have very little impact upon the rapid course of infection, despite being present at very high levels in the blood of the monkeys. We conclude that administering antibody prior to or immediately following exposure to Ebola virus, for example, after an accident in a research setting or a bioterrorist attack, is unlikely to be effective in preventing disease. Recent successes in protecting monkeys against Ebola virus through vaccination may be independent of antibody, or, more likely, critically dependent on the cooperation of antibody and cellular immunity.
two control monkeys, EHD (untreated and challenged), and a negative monkey (neither treated nor challenged), had very similar background levels of reactivity to the glycoprotein and anti-human antibody as each of the treated monkeys before treatment. A 50-mg/kg dose typically produces serum mAb concentrations in animals on the order of 500 lg/ml after injection [6] . Since the neutralization titer of KZ52 (IC 90 ) for ZEBOV is on the order of 0.5-2.5 lg/ml, depending upon the target cell and the presence of complement, the concentrations of KZ52 in the animals at the time of challenge and for the first few days were, as expected, greater than, or on the order of 100 3 IC 90 . These concentrations typically provide sterilizing immunity against challenge by a number of viruses [4, 20] .
One formal possibility is that the neutralizing antibody has little effect on the course of infection in the treated monkeys because of the rapid emergence of neutralization escape mutants. Accordingly, virus was isolated from a selection of plasma from day 4 (monkeys CH56, CH57, and CH83) and day 7 (monkeys CH56 and CH57). All of these viruses were sensitive to KZ52 so that essentially 100% neutralization was observed in vitro at 40 and 400 lg/ml KZ52 in a plaque assay (see Materials and Methods) using Vero E6 target cells.
In order to gain a better understanding of any differences in pathology between the control and antibody-treated animals, the levels of virus in different organs were surveyed postmortem. Viral levels in the liver, spleen, kidney, adrenal glands, lung, and mesenteric and inguinal lymph nodes were high (10 4 -10 6 pfu/g) in the control and three of the four treated animals (Table 2 ). However, monkey CH46, who survived much longer than the other animals (to day 28) showed some major histopathological differences from the other infected monkeys and from the norm for ZEBOV infection [15] . Relatively low viral levels were observed in most of the organs of CH46, and none in the liver, spleen, and adrenal glands. In addition, large immunoreactive monocytes were found in the blood of monkeys CH56, CH57, and CH83, but not in CH46 (Figure 2) . Typically, smaller immunoreactive monocytes are seen with ZEBOV infection [15] . The presence of large immunoreactive monocytes may simply reflect uptake of antibody-coated virions via Fc receptors and subsequent viral clearance. However, it is interesting to note that previous studies have implicated mononuclear phagocytes as vehicles for transport of filovirus particles to specific organs such as liver and spleen [21] [22] [23] [24] . If virus particles could remain infectious following Fc receptormediated uptake in a subset of cells (compare DC-SIGN mediated uptake of HIV-1 by dendritic cells [25] ), then the course of disease in monkeys CH56, CH57, and CH83 might represent the net result of inhibition by neutralization and enhancement by antibody-mediated cellular uptake. Interestingly, monkey CH46, who fared somewhat better than the others and lacked virus in the liver and spleen, did not show the presence of large immunoreactive monocytes. This is suggestive of lowered Fc receptor-mediated uptake of virus or reduced activity of the mononuclear phagocytic system.
Here, we describe the case of a potent neutralizing human monoclonal antibody, administered to give a high serum concentration, which is shown to be unable to protect macaques against challenge with a lethal dose of ZEBOV. The antibody appeared to have very little effect on the course of virus replication or disease in three of four treated animals. Neutralization escape does not appear to explain the lack of protection observed by the antibody. In one animal, a more limited infection was observed, but this macaque did also eventually succumb to the effects of viral disease.
The challenge dose of 1,000 pfu used corresponds to the amount of EBOV contained in a relatively small quantity of fluid (on the order of 1 ll) from an infected individual given the high titers of virus typically found in such individuals (on the order of 10 6 pfu/ml of blood, for example). Therefore, the challenge dose was not unreasonable in terms of a natural exposure to virus. The negative results with passive antibody contrast strongly with recent successes in preventing EBOV infection in macaques through vaccination [26, 27] . Does this mean that neutralizing antibody is unimportant in vaccine protection?
The answer to this question must await further studies. However, a plausible hypothesis to explain all the data would still allow for an important contribution of antibody to vaccine protection. This hypothesis would argue that passive antibody is unable to completely block all EBOV entry to cells, and once a few cells are infected, virus replication is so explosive that it cannot be contained by a de novo generated cellular immune response. Vaccination, on the other hand, will provide CD8þ memory T cells that can be rapidly recruited to become effector cells and limit infection. Certainly, although mAb KZ52 was able to provide protection from disease following ZEBOV challenge in the guinea pig model, immunity was not sterilizing and some viral replication was noted [6] . Since 1 pfu of EBOV is a lethal dose for primates [8] , a failure of passive antibody to achieve sterilizing immunity may be critical. Immunohistochemistry, as discussed above, gives some intriguing hints that uptake of antibody-coated virions by monocytes may possibly have a role to play in the course of infection following antibody treatment. We note, however, that previous in vitro studies using isolated human monocytes/macrophages did not find evidence of infectivity-enhancing antibodies [14] . More detailed in vitro and in vivo investigations will be required before any firm conclusions can be drawn. We also note that our experiments were carried out with a single neutralizing monoclonal antibody. It is possible that a more favorable outcome may have been apparent for a combination of neutralizing antibodies or even a combination of neutralizing and nonneutralizing antibodies [2] . However, these possibilities should be weighed against the very high concentrations of neutralizing monoclonal antibody used in the experiments and the efficacy of the antibody in the guinea pig model.
In summary, the inability of high concentrations of neutralizing antibody to even slow viral replication in infected macaques is remarkable and implies a mechanism of infection propagation that is virtually insensitive to antibody. Overall, the results suggest that monoclonal antibody prophylaxis or post-exposure prophylaxis alone are unlikely to be effective strategies in protecting against EBOV, for example, following a needle-stick accident in a research setting or a bioterrorist attack.
Passive transfer experiment. 50 mg/kg of KZ52 IgG1 human antibody [11] was given intravenously to rhesus macaques (weight, 3.9-4.4 kg) 1 d before challenge (day 0) with 1,000 pfu intramuscularly of the 1995 ZEBOV (Kikwit) isolate and again 4 d later (day þ4). One monkey was not given any antibody treatment. The animals were carefully monitored for signs of disease, and Ebola virus plasma viremia was determined at days 4, 7, 9, and 10 in serum by plaque assay as described below.
The investigators adhered to the Guide for the Care and Use of Laboratory Animals when conducting research, using animals [28] . The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) animal facilities and animal care and use program are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All infectious material and animals were handled in a maximum-containment biosafety level 4 facility at USAMRIID under standard operating conditions. Antibody purification. IgGl KZ52 was produced and purified as described by Parren et al. [29] and was .98% pure, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained ,1 IU of endotoxin/ml, as determined in a quantitative chromagenic Limulus amoebecyte lysate assay (BioWhittaker, Cambrex, http://www.cambrex.com).
Viremia determined by plaque assay. Plasma viremia and viral loads in organs was determined by virus titration in a conventional plaque assay on Vero E6 cells, as described elsewhere [13, 21] .
Neutralization assay. Samples were diluted into Eagle's minimal essential medium (EMEM; Invitrogen, http://www.invitrogen.com) with 5% heat-inactivated fetal bovine serum (FBS). In the presence and absence of a constant dilution of 1:10 or 1:100 of 4 mg/ml KZ52 (thus, 0.4 mg/ml or 0.04 mg/ml), plasma were titrated from 10 À1 to 10 À6 dilutions. Viremia was determined by counting pfu on Vero E6 cell monolayers. Cells grown to confluence in 6-well plates were given 0.2 ml of plasma with and without additional KZ52. The titrated samples were incubated in the presence of KZ52 for 1 h at 37 8C in 5% CO 2 . After absorption, the cells were overlaid with 2 ml of EMEM containing 5% FBS, 25 mM HEPES buffer, 50-lg gentamicin per ml, and 1% agarose. After 10 d, plaques were visible and the cells were removed from the humidified 37 8C incubator to visualize plaques with an inverted phase microscope. 2 ml of neutral red (1:6,000 final concentration; Sigma-Aldrich, http://www.sigmaaldrich.com) was added to each well, and after an additional 24-h incubation, the plaques were counted [11, 30] .
ELISA. Nunc-Immuno Maxisorp enzyme-linked immunosorbent assay (ELISA) plates (Nunc, http://nuncbrand.com) were coated with 100 ll/well of 10 lg/ml lectin from Galanthus Nivalis (Sigma-Aldrich) in PBS and incubated overnight at 4 8C. The plates were then blocked in phosphate-buffered saline (PBS) containing 10% FBS for 2 h. The wells were then washed twice with wash buffer, PBS containing 0.2% Tween 20 (Sigma-Aldrich). 293 cells were transfected with a mammalian expression plasmid coding for transmembrane domaindeleted Ebola glycoprotein. Supernatant (100 ll) from these cells, which contains 0.8-1.3 mg/ml total protein, was used to coat each well for 1 h at room temperature (RT) after the blocking solution was removed from the ELISA plates. Plates were then washed six times with wash buffer. Monkey sera were added in 10-fold dilutions from 1:10 to 1:10 5 in dilution buffer (PBS containing 1% BSA and 0.02% Tween) and incubated at RT for 1 h. The wells were then washed six times, and a secondary antibody alkaline phosphatase-conjugated goat anti-human immunoglobulin G (IgG) against the F(ab9) 2 portion of the antibody (Pierce, http://www.piercenet.com) diluted 1:500 was added, and this was incubated for 1 h. Finally, the plates were washed again six times and developed by one tablet of phosphatase substrate Typically smaller immunoreactive monocytes are seen with ZEBOV infection [15] . Immunohistochemistry was performed as described in Materials and Methods. doi:10.1371/journal.ppat.0030009.g002
(Sigma-Aldrich) in 5 ml of alkaline phosphatase stain buffer (pH 9.8) per plate. The assay was performed as per manufacturer's directions. The plates were read at an optical density of 405 nm on a microplate reader (Molecular Devices, http://www.moleculardevices.com) at 30 min after adding substrate. A panel of normal sera was run each time the assay was performed.
Immunohistochemistry. Sections were pretreated with Dako Ready to Use Proteinase K (Dako, http://www.dako.com) for 6 min at RT after deparaffinization and rehydration through a series of graded ethanols. Blocking was performed with Dako's Serum-Free Protein Block for 20 min pre-antibody exposure. The tissue sections were then incubated overnight at 4 8C in primary antibody using an equal mixture of mouse monoclonal antibodies to EBOV GP and VP40 (1:5,000). An alkaline phosphatase-labeled polymer (Dako Envision System, alkaline phosphatase) was incubated on the sections for 30 min, and then color was developed by exposing tissue to 6-bromo-2hydroxyl-3-naphtholic acid (HistoMark Red; Kikegaard and Perry Laboratories, http://www.kpl.com) substrate for 50 min in the dark. Counterstaining was done with hematoxylin. Positive controls included archived EBOV-infected cynomolgus tissue, and negative controls included replicate sections exposed to anti-Marburg virus antibodies and uninfected cynomolgus macaque tissue [15] . Public health preparedness in Alberta: a systems-level study BACKGROUND: Recent international and national events have brought critical attention to the Canadian public health system and how prepared the system is to respond to various types of contemporary public health threats. This article describes the study design and methods being used to conduct a systems-level analysis of public health preparedness in the province of Alberta, Canada. The project is being funded under the Health Research Fund, Alberta Heritage Foundation for Medical Research. METHODS/DESIGN: We use an embedded, multiple-case study design, integrating qualitative and quantitative methods to measure empirically the degree of inter-organizational coordination existing among public health agencies in Alberta, Canada. We situate our measures of inter-organizational network ties within a systems-level framework to assess the relative influence of inter-organizational ties, individual organizational attributes, and institutional environmental features on public health preparedness. The relative contribution of each component is examined for two potential public health threats: pandemic influenza and West Nile virus. DISCUSSION: The organizational dimensions of public health preparedness depend on a complex mix of individual organizational characteristics, inter-agency relationships, and institutional environmental factors. Our study is designed to discriminate among these different system components and assess the independent influence of each on the other, as well as the overall level of public health preparedness in Alberta. While all agree that competent organizations and functioning networks are important components of public health preparedness, this study is one of the first to use formal network analysis to study the role of inter-agency networks in the development of prepared public health systems. International and national events have brought critical attention to the Canadian public health system and how prepared the system is to respond to various types of con-temporary public health threats. Whether those threats result from emerging infectious diseases or bioterrorism, the public health system is responsible for protecting the health of the population. Outbreak response occurs first at the local or regional levels and thus the potential impact that a threat will have on the overall Canadian population can differ significantly depending on the capacity of public health systems to respond [1] . For example, following the SARS outbreak in Toronto, the Canadian National Advisory Committee (NAC) on SARS and Public Health held it as fortunate that the SARS outbreak struck primarily in Toronto and not in other parts of Canada where the capacity to combat public health threats is limited [2] . While a "general renewal of the public health infrastructure" and its capacity to respond are in order the effective coordination of all agencies at regional, provincial, and federal levels is necessary to assure a comprehensive surveillance and management of public health threats. As Ralph Klein, the Premier of Alberta, commented: "Albertans and all Canadians understand a disease outbreak like SARS or West Nile virus in one region affects other parts of the country. Coordinated approaches will help deal with public health threats."
Despite the commitment of federal and provincial agencies in Canada to improve public health system performance and develop overall public health preparedness, little is still known about the current systems-level state of public health preparedness in Canada. While coordination is seen as a necessary element for public health preparedness, few measures of inter-organizational collaboration relevant to public health preparedness have been developed and assessed in relation to other features of the organizational and inter-organizational environment. This study protocol presents the premises, conceptual model and methods used to measure and assess public health preparedness in Alberta, Canada. Although provincial public health agencies are themselves embedded within federal and global public health preparedness systems, we focus primarily on the organizational environment of public health preparedness in Alberta so as to develop contextually-relevant measures and evaluation techniques as well as address issues related to potential cross-Alberta variations in public health preparedness.
In Alberta, public health and emergency response procedures are primarily governed under the Disaster Services Act [3] and the Public Health Act [4] . Provincial response to a public health disaster event may thus call into action organizations and units under the jurisdiction of local municipalities, regional health authorities, emergency management districts, and/or the provincial government. There are a number of organizational actors that may be involved in preparation, response, or recovery phases of a public health disaster, including departments or portfolios within agencies. Each of which are potential actors in the provincial public health and emergency response network. While we consider the overall public health and emergency management network to consist of all poten-tial organizational actors involved in responding to public health threats, the precise set of organizations that come together around a particular public health threat depends on the nature of that threat. For example, pandemic influenza and the West Nile virus are both emerging infectious diseases but the nature of their transmission differs and distinctive sets of organizational actors must be in place to confront them. In the case of West Nile Virus, Alberta's Department of the Environment and Sustainable Resource Development would be integrally involved for mosquito surveillance and control whereas it would not be as integral in the event of a pandemic influenza outbreak [5] . To assess potential differences by public health threat in Alberta public health preparedness, we examine and compare two potential public health threats: pandemic influenza and West Nile Virus.
Using an embedded multiple-case study design[6] and integrated qualitative and quantitative methods, the project proposes to measure empirically the degree of seamless coordination existing among municipal, regional or district-level and provincial public health and emergency management agencies in Alberta. Although federal authorities and agencies play an important role in provincial-level responses to public health emergencies, for the purposes of this research, the focus will be on the inter-organizational linkages and organizational environments of provincial and sub-provincial actors; relevant federal-level organizations will be identified for future investigation. The seamless coordination of activities and tasks that should characterize the overall federal-provincial-regional linkages should also characterize the intraprovincial linkages. Fluid communication and resource flows among such diverse organizations and administrative authorities require an integrated and coordinated network of actors. Our research is premised on empirical research that has shown that effective and coordinated approaches to public health threats require a well-integrated and responsive inter-organizational system and that the most appropriate method in which to analyze inter-organizational relations and the degree of integration is one based on network analysis [7] . Yet, strong inter-organizational ties are not enough for effective preparedness if the organizations involved do not have the necessary individual capacity to respond to public health threats. In this regard, fluid communication and integrated organizational connections are but one element in an overall systems-level approach to assessing public health preparedness. As shown in Figure 1 , we view public health preparedness to be a product of several interrelated factors: 1) individual organizational attributes, 2) the inter-organizational networks existing among relevant organizational actors, 3) the institutional environments in which organizations and inter-organizational networks are located and in which threats first emerge, and 4) the integrated coordination of specific action-sets. An actionset refers, in this case, to a subgroup of organizations in the network that come together purposefully to address a specific public health threat. We see these four factors together influencing system-level public health preparedness.
Our target population for the research is all public health and emergency management-related organizations in Alberta. The construction of the project's sampling frame is based on a stratified, multistage cluster design. The strata are four administrative divisions: (1) provinciallevel agencies, (2) sub-provincial administrative agencies, i.e., emergency-management districts (disaster services) and regional health authorities, (3) metropolitan areas, and (4) towns and local areas. Within strata 1-3, the sampling frame includes all public health or emergency management organizations, i.e., agencies, departments, or units, within those strata. The fourth stratum will be substratified according to the Alberta Emergency Management district divisions into 6 geographical areas: (1) northwestern Alberta, (2) northeastern Alberta, (3) northcentral Alberta, (4) central Alberta, (5) south-central Alberta, and (6) southern Alberta. From each of the 6 geographical regions, we will draw a proportionate, random sub-sample of towns. For stratum 4, the sampling frame consists of all public health or emergency managementrelated organizations in those towns randomly selected. The project's frame population will thus consist of those organizations listed in the strata 1-3 sampling frame and those listed in the stratum 4 sampling frame.
This study will be conducted in 2 phases: phase 1 qualitative and phase 2 quantitative research. Phase 1 is used in part to help identify the organizations responsible for public health and emergency responses and the relevant relationships among them. Initial qualitative research will consist of the analysis of official documents and reports and interviews with key organizational representatives. Government documents and reports will be analyzed to 1) help identify agencies involved in the response system, 2) determine the formal tasks assigned to those agencies, and 3) initially assess the overall level of preparedness and responsiveness for the three case studies. Interviews with 
Attributes key informants and health officials will be used to determine 1) if there are additional organizations involved in the response system that were not noted in the official documents, 2) the types of relationships characterizing the inter-organizational ties within and among actionsets, and develop 3) relevant measures of the institutional environment and of public health preparedness and responsiveness. While laws mandate that certain organizations be involved in emergency responses to specific public health threats, key organizational representatives may identify agencies that play an important albeit informal role in local emergency response systems.
In addition to helping identify organizations that may be absent from official documents, the interviews will also investigate the perceived characteristics of inter-organizational relationships. Mandated relationships, for example, tend to be characterized by lower levels of perceived cooperation among organizations [8] . While such characteristics will be investigated using quantitative methods in phase 2, the qualitative research will reveal the points of conflict and cooperation that emerge in the course of public health and emergency responses and thus provide a basis for the questions that will be used in the organizational questionnaire. The final focus of the phase one interviews will centre on the development of measures of the institutional environment, network effectiveness, and public health preparedness. What are the resources that key agency representatives themselves identify as critical and important to their organization and the network? What processes do the respondents use to evaluate if their organization or the system was prepared?
In phase 1, we will draw a non-proportional sample from the project's sampling frame. We will interview 6 organizational representatives from the provincial level (stratum 1), 12 representatives from the sub-provincial level (strata 2 and 3), and 18 representatives from the local level (stratum 4). Phase 1 interview respondents will be organizational representatives who are randomly selected from each of these strata and choose to participate.
Interviews will be tape-recorded and transcribed so that content analysis, thematic, and inter-textual analyses can be conducted on the interviews. Transcripts will be coded for such indicators as (1) organizations involved in the public health and emergency management preparedness, (2) types of information-sharing among organizations, (3) types of resource flows existing among organizations, and (4) perceptions of local and provincial public health preparedness. Content analysis will be used to identify relevant organizations that were not identified in the documentary research [9, 10] . Thematic analyses will be used to identify the critical themes emerging in the 36 interviews; inter-textual analyses will be used to compare the responses of interviewees across the three strata dimensions of jurisdiction, domain, and geographical location.
Phase 2 involves the administration of an organizational/ inter-organizational questionnaire to Alberta-based public health and emergency management agencies. Information on organizations and inter-organizational networks will be collected by web-based or telephone questionnaires. For phase 2, we intend to administer questionnaires to representatives of all organizations listed in the project's sampling frame (described earlier). The questionnaire consists of three components: 1) an organizational attribute component, 2) an inter-organizational network component, and 3) an organizational environment and network assessment component. The organizational attribute component consists of questions on the general characteristics of organizations such as staff size, training background of specific occupational roles in the organization, sectoral operations, budget and more emergency-response related questions, such as whether an agency has formally assessed its epidemiology capacity.
The inter-organizational network component consists of items that ask representatives to identify those organizations with which their own organization has ties along specific dimensions. The specific content and dimensions of the network to be mapped will be determined following phase one analysis but candidate dimensions used in other organizational network studies include (i) information-sharing, (ii) resource-sharing, including staff and (iii) joint-planning. For each of the critical dimensions identified in phase one, the respondent will be presented with a list of organizations within the relevant action-set and asked, for each one, "who do you share information with?" or "who do you share resources with."
The third component of the questionnaire will consist of the respondent's subjective assessments of the institutional environment and inter-organizational network, and the impact that each has on perceived public health preparedness. For example, how do representatives assess the environment in which they operate, i.e., do they see resources as easily available or scarce? How prepared do they perceive their own organization and local system to be for a public health threat? Since mandated relations involve "sequential interdependence," an important question is whether organizations perceive the task transitions among organizations as functioning smoothly. The actual questions that will be used in this component of the questionnaire will be determined following phase one data analysis.
Ethical approval of the research has been granted by the Conjoint Health Research Ethics Board of the Calgary Health Region and University of Calgary, Faculty of Medicine (ID#17873) and of the Capital Health Region and University of Alberta (#B-251005).
The capacity of any organization to be prepared for a public health threat is influenced in part by the system in which the organization is located, just as the preparedness of the overall system can be influenced by the strengths and weaknesses of any single organization. The cross-case and intra-provincial comparative design allows our research to discriminate among different system elements and examine the independent influence of organizational attributes, institutional environments, and inter-organizational networks on public health preparedness. In addition, the mixed methods approach enables our research to analyse the role of both informal and formal relationships in the emergence of inter-organization networks and the development of prepared and responsive public health systems. As far as we are aware, our project is one of the first to assess public health preparedness at the systemslevel while accounting for the critical role that inter-organizational relationships play in the emergence of a cohesive and responsive system. Moreover, in deciphering the independent influence of inter-organizational ties within the overall system, the research will provide measures that will help researchers and policy-makers evaluate the degree of coordination among organizational actors.
Where are the gaps in system linkages? Which organizations need to be made more central to preparedness activities? These are just a few of the questions that our research is designed to answer. The immunoregulatory and allergy-associated cytokines in the aetiology of the otitis media with effusion. Inflammation in the middle ear mucosa, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions and reflux, is the crucial event in the pathogenesis of otitis media with effusion (OME). Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic inflammatory processes and stimulate the chronic condition of OME. Cytokines are the central molecular regulators of middle ear inflammation and can switch the acute phase of inflammation in the chronic stage and induce molecular-pathological processes leading to the histopathological changes accompanying OME. In this review we present cytokines identified in otitis media, immunoregulatory [interleukin (IL)-2, IL-10, transforming growth factor-beta]) and allergy associated (IL-4, IL-5, granulocyte-macrophage colony-stimulating factor), as crucial molecular regulators, responsible for chronic inflammation in the middle ear and the chronic condition of OME. The immunoregulatory cytokines [interleukin (IL)-2, IL-10, transforming growth factor (TGF)-b] and allergy-associated cytokines [IL-4, IL-5, granulocyte Á/ macrophage colony-stimulating factor (GM-CSF)] are mediators of the immune system, which are actively involved in regulation of molecular and cellular processes accompanying different types of inflammation.
IL-2 is the up-regulating cytokine, which stimulates primarily the cell-mediated inflammatory response by promoting growth, proliferation and differentiation of T cells, B cells, natural killer (NK) cells, monocytes and macrophages. It is secreted mainly by activated T cells. IL-2 induces activation and rapid clonal expansion of mature T cells, and cytokine production in T cells, including interferon (IFN)-g and IL-4; 1 growth of B cells and immunoglobulin (Ig) J-chain switching and secretion of IgM in B cells; 2 proliferation, production of IFN-g and cytolytic activity of NK cells; 3 proliferation and differentiation of macrophage precursors; 4 and cytolytic activity of monocytes. 5 In contrast to IL-2, IL-10 (known as the cytokine synthesis inhibitory and macrophage deactivating factor) down-regulates the immune reactions accompanying acute inflammation and limits the duration of inflammatory responses. IL-10 can also promote and regulate chronic inflammatory processes. IL-10 is produced by a variety of cell types, including CD4 ' T cells, activated CD8 ' T cells and activated B cells. The main anti-inflammatory activities of IL-10 */ namely, inhibition of cytokine production in macrophages, neutrophils, T cells and NK cells, 6 Á 9 and inhibition of the macrophage Á/monocyte activation and the antigen presentation abilities of these cells 10 */lead to the resolution of inflammation. However, if the acute inflammatory process has not been resolved, IL-10 can induce humoral inflammatory reactions such as the immunoglobulin isotype switching in B cells 11, 12 and differentiation of B cells into plasma cells, 13 and thus promote switching of inflammation in the chronic stage.
TGF-b, produced by T cells, platelets and monocytes, plays an important role in regulation of the inflammatory processes and in tissue reparation. The effects of TGF-b depend on the differentiation state of the responsive cells. 14 TGF-b, as well as IL-10, participates in resolution of acute inflammation by inhibiting antigen presentation and cytokine production in macrophages, 15 and suppressing proliferation of T cells and NK cells and cytokine production in activated T cells. 14 Simultaneously, TGF-b recruits monocytes to the site of inflammation and upregulates their pro-inflammatory activity; secretion of cytokines and growth factors, in particular. 14 The contradictory effects of TGF-b on monocyte/macrophage lineage, such as up-regulation of monocyte functions and deactivation of macrophages, demonstrate the highly coordinated role of TGF-b in regulation of inflammatory responses.
The immunoregulatory and allergy-associated cytokine IL-4, produced by CD4 ' T cells, mast cells and basophils, is actively involved in regulation of different types of inflammation. IL-4 up-regulates humoral inflammatory responses by inducing growth of B cells, isotype switching in activated B cells and differentiation of B cells into antibody producing plasma cells. 16 IL-4 also stimulates the cell-mediated inflammatory processes by promoting growth of activated T cells 17 and enhancing the development of virus-specific cytotoxic T cells. 18 Simultaneously, IL-4 possesses powerful anti-inflammatory effects. In particular, IL-4 controls numerous molecular processes in monocytes, macrophages and neutrophils, which down-regulate production and secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-a, IL-1 and IL-8, 19 Á 21 deactivate inflammatory macrophages and lead to suppression of acute inflammation. IL-4 can also induce switching of acute inflammation in the chronic stage owing to its ability to up-regulate expression of the mannose receptor on activated macrophages. 22 The mannose receptor promotes fusion of activated macrophages and formation of giant multinucleated cells in the zone of inflammation 23 that create the cellular background for manifestation of chronic inflammation.
IL-5 is an important regulator of the humoral immune response and is produced by CD4 ' T cells as well as NK cells. 24 Being a late regulating factor in differentiation of B cells, IL-5 plays an essential role in cytokine-induced production and secretion of immunoglobulins and supports humoral inflammatory reactions such as production and secretion of IgA. 25 IL-5 is also the allergic cytokine, which by stimulating production, activation, differentiation and migration of eosinophils creates a strong molecular background for eosinophilic inflammation. 26, 27 GM-CSF regulates proliferation and maturation of granulocyte and macrophage precursors and activates mature neutrophils, eosinophils and macrophages, 28 and thus participates in different types of inflammation. 29 Monocytes, T cells, fibroblasts and endothelial cells activated by macrophage cytokines IL-1b or TNF-a produce GM-CSF. The GM-CSF enhances neutrophil phagocytosis, enhances release of chemotactic factors by neutrophils, 30 induces production of the pro-inflammatory cytokines by macrophages and increases their function as antigen-presenting cells, 31 and in this way promotes acute inflammatory reactions. In the presence of IL-4, GM-CSF can initiate a specific differentiation of monocytes into osteoclast-like multinucleated giant cells 32 that create a background for chronic inflammation. GM-CSF can also contribute to allergic inflammation by promoting growth, prolonged survival and activation of eosinophils and basophils. 33 Both groups of cytokines, the immunoregulatory IL-2, IL-10 and TGF-b and the allergy-associated IL-4, IL-5 and GM-CSF, have been identified in otitis media and their presence is the evidence of their participation in regulation of local inflammatory processes. These cytokines can switch the acute phase of middle ear inflammation into the chronic stage and lead to the chronic condition of otitis media with effusion (OME). OME: a brief description OME, the commonest cause of childhood deafness in the developed world, is a chronic inflammatory condition of the middle ear cleft with repetitive recurrences of acute middle ear inflammation. The disease is characterized by a middle ear effusion that persists for months to years, cannot be cleared by the normal mucociliary transport mechanisms and is must usually removed by surgical operation.
A surgical procedure, which includes myringotomy, aspiration of fluid and the insertion of ventilation tubes (grommets) in the anterior tympanic membrane (Fig. 1) , is the most effective option in treatment of OME. The main problem with ventilation tubes is secondary infection and post-operative otorrhea, which can be managed using antibiotic eardrops. 34, 35 However, increasing antibiotic resistance of bacterial pathogens 36 and possible ototoxicity of topical antibiotics 37 can complicate the antimicrobial therapy in post-surgical management of otitis media. Other non-surgical approaches in treatment and prophylaxis of OME include traditional methods, such as combined steroid Á/antimicrobial therapy 38, 39 and polyvalent pneumococcal vaccination, 40, 41 and relatively new methods based on herbal medicine 42, 43 and homeopathy. 44, 45 The chronic condition of otitis media is associated with proliferative changes in the middle ear tissues, especially in the surface middle ear mucosa, which is presented in OME as a modified pseudostratified epithelium, 46 where basal cells are differentiating into goblet cells, 47 goblet cells are proliferating with enhanced secretory activity 48 and formation of mucus glands occurs 49, 50 (Fig. 1) .
Goblet cells produce and secrete mucins, which are important glycoproteins in the mucociliary transport system of the middle ear and are the main component of middle ear effusions, responsible for the viscous properties of effusions. 51, 52 However, under disease conditions, alterations that occur in the middle ear and eustachian tube mucin metabolism, 53 in the structure of mucin glycoproteins 54, 55 and in the glycoconjugate expression in cilia and goblet cells 56 promote the dysfunction of the normal mucociliary transport system and the formation of effusion in the middle ear cleft.
The expression of several mucins has been detected in OME. 53, 57 However, overproduction of only two mucins has been observed in otitis media; namely, the membrane-bound MUC4 53 and the secreted MUC5B. 53, 55, 58, 59 Another secreted mucin (MUC5AC) is always presented in effusions, but in varying amounts, 53, 55, 57, 60 and its levels maybe linked to levels of the pro-inflammatory cytokines TNF-a, IL-6 and IL-8, which can promote different levels of MUC5AC secretion. 61, 62 The initial step in the pathogenesis of OME is the inflammatory process in the middle ear mucosa. Bacteria, 63, 64 viruses, 65,66 allergic reactions 67,68 and even gastroesophageal reflux 69 Á 71 in tandem with predisposing factors such as eustachian tube dysfunction, 72 cleft palate 73 and obstructive adenoids 74 can stimulate the middle ear inflammation.
Different groups of inflammatory mediators were identified in the human middle ear mucosa, fluids and effusions. A lot of different mediators participate in initiation and early stages of the middle ear inflammation, including arachidonic acid metabolites (prostaglandin E 2 and leukotrienes LT-B 4 , LT-C 4 ), 75, 76 histamine, 77,78 platelet-activating factor, 79 surface cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial leukocyte adhesion molecule-1, platelet endothelial cell adhesion molecule), 80,81 soluble cell adhesion molecules (soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1), 82, 83 chemokine RANTES, 84 complement C3a anaphylatoxin 85 and interferon-g. 86 However, cytokines are the key mediators of the middle ear inflammation. Cytokines regulate different stages of inflammation, are responsible for resolution of inflammation and can initiate local molecular processes leading to histopathological changes in the middle ear mucosa and submucosa, and the chronic condition of otitis media. Different groups of cytokines have been identified in the middle ear effusions and mucosa: the pro-inflammatory TNF-a, IL-1b, IL-6 and IL-8; the immunoregulatory IL-2, IL-10 and TGF-b; and the allergy-associated IL-4, IL-5 and GM-CSF.
The pro-inflammatory cytokines and their participation in the pathogenesis of otitis media have been already discussed, 87 and are more related to the acute phase of middle ear inflammation. In the present review we have analysed the immunoregulatory and the allergy-associated cytokines in otitis media, which, by providing the cellular and molecular background for the chronic inflammation in middle ear, promote the chronic condition of OME.
The immunoregulatory cytokines IL-2, IL-10, TGF-b and their cellular and molecular networks in OME IL-2 in otitis media IL-2 has been detected in middle ear effusions from children with chronic OME at high concentration (greater than 300 pg/ml). 88 The analysis of peripheral blood immunologic pattern in patients with persistent middle ear effusions 89,90 showed the following: (1) the number of CD4 ' and CD8 ' T lymphocytes was decreased and the T-cell subset ratio (CD4 ' / CD8 ' ) was reduced; (2) proliferative responses of blood T cells were diminished; and (3) generation of IL-2 by peripheral blood lymphocytes was decreased. Another immunologic investigation of children with recurrent otitis media revealed a consistent inability of adenoidal T cells to turn on B cells to mature into immunoglobulin-secreting plasma cells that has been explained by defective production of IL-2 in adenoids. 91 This assumption was confirmed by cytokine assay of IL-2, which has been done simultaneously in nasopharyngeal lymphoid tissues and in peripheral blood of children with recurrent otitis. The adenoidal lymphocytes produced significantly less IL-2 compared with the patient's peripheral blood lymphocytes. 92 It has been assumed that poor production of IL-2 in adenoidal tissues is probably linked to a deficiency of IL-2 production in the middle ear tissues, and that could be associated with the persistence of OME. Thus, general and/or local deficiency of the IL-2 production could induce defective immunoregulation of the middle ear inflammation and promote the persistence of OME.
The investigation of IL-2 in experimental models of otitis media showed the involvement of IL-2 in regulation of acute inflammation because: (1) the IL-2 producing cells were predominant in acute OME compared with chronic OME, 93 and (2) recombinant IL-2 trans-tympanically injected into the middle ear of normal guinea pigs caused the inflammatory middle ear effusion within 24 h, which cleared by 72 h after inoculation. 94 However, differing degrees of responsiveness of effusion production were observed following the instillation of IL-2 in the middle ear, 95 varying from pronounced middle ear effusion causing rather severe mixed hearing loss to complete lack of effusion, and IL-2 was detected in all the middle ear effusions in experimental OME with signs of sensorineural hearing loss. 96 By analysis of clinical and experimental data we can propose the following conclusions about the role of IL-2 in OME:
1. IL-2 is involved in regulation of the middle ear inflammation and, as the main T-cell growth and differentiation factor, IL-2 can support acute and chronic inflammatory processes in the middle ear. 2. IL-2 can regulate local acute inflammatory response by inducing activation, differentiation of T cells and production of the proinflammatory (IL-1b, IL-6) and anti-inflammatory cytokines (IL-10, TGF-b) by activated T cells (Fig. 2 ). 3. The deficiency of local or general IL-2 production can suppress reactions of acute inflammation and promote persistence of OME, which can develop with time into the chronic OME (Fig. 3 ). 4. The excessive production of IL-2 can provoke chronic cell-mediated and (or) humoral inflammatory processes (proliferation of activated T cells, increased production of cytokines IL-4, IL-5 and GM-CSF by proliferated T cells, immunoglobulin-switching and IgM secretion in B cells), which will induce switching of the middle ear inflammation in the chronic stage with subsequent development of chronic OME (Fig. 3) . 
The clinical investigations demonstrated the involvement of IL-10 in chronic otitis media. 97,98 IL-10 was identified in middle ear effusions from children undergoing tympanostomy tube placements and its mean concentration was 569/58.7 pg/ml. 97 It has been assumed that the presence of IL-10 in middle ear effusions might be one of the causes of a lack of clinical features of acute inflammation and could lead to a chronic inflammatory state. Chronic otitis media has been also associated with dysregulated local production of IL-10. 98 Patients with chronic otitis media showed high levels of IL-10 ( /100 pg/ml) produced by sinus lavage cell culture and simultaneously low production of IL-10 by peripheral blood mononuclear cells. Investigations of cytokine profiles in different experimental models of OME showed a rapid appearance of IL-10 in the early stages of acute otitis media 99 Á 103 and that IL-10 in mucosa was produced predominantly by CD4 ' T cells. 104 Streptococcus pneumoniae induced expression of IL-10 mRNA in middle ear mucosa within 1Á/2 days after inoculation. 99, 100 Lipoteichoic acid, one of the components present within the cell wall layer of most Grampositive bacteria, 101 and non-typeable Haemophilus influenzae (NTHi) 102,103 induced the expression of IL-10 mRNA in middle ear mucosa even faster, within 6 h after inoculation. It is interesting to note that the expression of IL-10 mRNA followed the expression of TNF-a and IL-6 mRNAs, 99,100 and sometimes occurred simultaneously with the pro-inflammatory cytokines. 101, 102 Thus, clinical and experimental data about IL-10 in OME lead to the following conclusions:
1. IL-10 is involved in regulation of the middle ear inflammatory response induced by bacterial infection. 2. The expression of IL-10 in acute otitis media is up-regulated shortly after or even simul- taneously with the pro-inflammatory cytokines, TNF-a and IL-6. That is, the evidence of initiation of corrective immunoregulation in the middle ear mucosa, because IL-10 as the main anti-inflammatory cytokine, can down-regulate the local population of inflammatory cells, macrophages and neutrophils, and production of the proinflammatory cytokines TNF-a, IL-1b, IL-6, IL-8 and promote resolution of inflammation (Fig. 2 ). 3. If the acute inflammatory response is not resolved correctly, the prolonged presence of IL-10 in the zone of inflammation can provoke the amplification of chronic humoral inflammatory processes, such as increased secretion of immunoglobulins, especially IgA, by activated B cells and possible generation of plasma cells, in the presence of IL-4 ( Fig. 4) . In this way, IL-10 can contribute to switching of the disease into the chronic stage. 4. The deficiency of IL-10 in acute otitis media can also lead to chronic condition of OME, because shortage of IL-10 will promote overproduction of the inflammatory cytokines, especially IL-1b, TNF-a and IL-6, which can induce tissue damage and irre-versible changes in the middle ear mucosa (Fig. 4) .
General conclusions about the role of IL-2 and IL-10 in an aetiology of OME Both cytokines, IL-2 and IL-10, participate in balanced immunoregulation of the middle ear inflammatory response. IL-2 up-regulates cellular and molecular events, accompanying acute inflammation, whereas IL-10 down-regulates acute inflammatory reactions and promotes resolution of inflammation. Any imbalance in production of these cytokines will induce chronic inflammatory processes (cellmediated or humoral, or both) or stimulate uncontrolled inflammatory-related damage of the middle ear tissues (Figs 3 and 4) . Thus, IL-2 and IL-10 can be considered as the key cytokine mediators, regulating switching of the acute phase of middle ear inflammation in the chronic stage, which will lead to the chronic condition of OME.
Clinical investigations of TGF-b in OME showed its relation to chronic otitis media. 105, 106 The identification of TGF-b in serous and mucoid middle ear effusions was associated with a history of previous tympanostomy tube placements. 105 Investigation of growth factors in tympanic membrane perforations showed that TGF-b could promote development of the fibrotic scar at the perforation margin, explaining the deficient healing pattern of tympanic membranes in chronic otitis media. 106 However studies of cytokines in experimental OME demonstrated the presence of TGF-b also in acute otitis media. 100, 102, 103 The expression of TGF-b mRNA was up-regulated in acute otitis media caused by S. pneumoniae 100, 102 and NTHi infections, 102, 103 and was detected early during the course of acute inflammation, within 24 h after inoculation, together with cytokines IL-1, TNF-a and IL-10. However, the highest mRNA levels for TGF-b were recorded considerably later, on days 6 and 28, for the NTHiinfected and pneumococcus-infected animals, respectively, and its expression continued even after resolution of acute otitis media. 102 TGF-b regulates host response in infectious diseases through the TGF-b-activated kinase 1 107 and TGF-b-Smad signaling pathway. 108 It has been shown that NTHi, a major human bacterial pathogen of otitis media, strongly induced up-regulation of MUC5AC mucin in human epithelial cells. This induction occurred via activation of the Toll-like receptor 2-MyD88-dependent p38 mitogen-activated protein kinase (MAPK) pathway. However, activation of TGF-b-Smad signaling by TGF-b resulted in downregulation of p38 MAPK by inducing MAPK phophatase-1, and subsequent down-regulation of MUC5AC transcription. 108 TGF-b thereby acted as a negative regulator of MUC5AC mucin gene expression. This is a very significant fact in understanding the role of TGF-b in otitis media, because varying levels of the secreted mucin MUC5AC are presented in the middle ear mucosa and effusions in patients with OME. 53, 55, 57 By analysis of these data we can make the following conclusions about the role of TGF-b in OME:
1. TGF-b participates in regulation of the middle ear inflammation. 2. TGF-b is a negative regulator of acute inflammation in the middle ear. Early activation of TGF-b together with another antiinflammatory cytokine, IL-10, in the course of acute otitis media demonstrates the involvement of TGF-b in the correct resolution of acute inflammatory response. TGF-b as well as IL-10 can inactivate macrophages and inhibit pro-inflammatory cytokine production at the site of inflammation (Fig. 2 ). 3. TGF-b can be also involved in down-regulation of bacterial-induced MUC5AC mucin production, and that is the additional evi- Thus, TGF-b can contribute to the pathogenesis and persistence of chronic OME in many ways.
The allergy-associated cytokines IL-4, IL-5 and GM-CSF and their cellular and molecular networks in OME
IL-4 was identified in the middle ear effusions of children with persistent OME 109 and in atopic children with OME undergoing myringotomy and ventilation tube placement. 110 The cytokine analysis of effusions showed a higher mean level of IL-4 in the allergy-positive group compared with the allergynegative group 109 and a higher percentage of cells expressing IL-4 in atopic patients with OME compared with that seen in non-atopic patients. 110 A higher level of IL-4 in effusions correlated with predominance of T lymphocytes, which was the sign of chronic inflammation and was also related to the atopic background of patients with OME. 110 In effusions of non-atopic patients, the level of IL-4 was lower and the predominance of neutrophils 109 demonstrated signs of an acute inflammatory response.
Investigation of adenoids in patients with recurrent otitis media 92,111 resulted in the following important findings: (1) expression and secretion of IL-4 was detected in adenoids; (2) adenoidal lymphocytes produced the same level of IL-4, or even slightly more, compared with the patient's peripheral blood lymphocytes; 92 and (3) the epsilon germline transcripts for IgE were detected in the adenoids and the level of IgE epsilon germline transcript expression was dependent on the level of IL-4 mRNA expression. 111 This local IL-4-induced immunoglobulin class switching to IgE in the adenoids was considered the essential molecular process contributing to chronic inflammation in the middle ear and promoting the pathogenesis and persistence of OME.
Thus, IL-4 was presented in both groups of patients with OME, allergic and non-allergic. However, local overproduction of IL-4 was associated predominantly with an allergic background of patients.
Studies on IL-4 in experimental OME 93,112 demonstrated the involvement of IL-4 in regulation of different types of otitis media. In trans-tympanically challenged animals the expression of IL-4 was detected during acute and chronic otitis media, with the predominance of IL-4 ' cells in the acute form. 93 The trans-tympanical injection of soluble receptors for IL-4 prevented the allergic Eustachian tube dysfunction and formation of effusion in the middle ear cleft. 112 The involvement of IL-4 in regulation of MUC5AC and MUC5B mucin gene expression and secretion has been shown in human airways. 113 Á 115 The secreted mucins, MUC5AC and MUC5B, are presented in the middle ear mucosa and effusions in patients with chronic OME. 53,55,57 Á 60 Although relationships between IL-4 and production of MUC5AC and MUC5B mucins in the course of otitis media have not been investigated, the ability of a soluble receptor for IL-4 to prevent Eustachian tube dysfunction and formation of effusions in the middle ear cleft 112 demonstrated the potential involvement of IL-4 in the regulation of the middle ear mucin metabolism.
All these data allow us to make the following conclusions about the role of IL-4 in OME:
1. IL-4 is involved in regulation of different types of otitis media. 2. IL-4 is one of the cytokines responsible for an allergic type of inflammation owing its ability to induce local immunoglobulin class switching to IgE (Fig 6) . 3. IL-4 can support local humoral inflammatory responses by increasing production and secretion of immunoglobulins in activated B cells (Figs 4 and 6 ). 4. IL-4 can also support cell-mediated chronic inflammation in the middle ear by promoting generation of multinucleated giant cells (Figs 5 and 6 ), although it is necessary to note that giant cells in OME have not been investigated. 5. IL-4 can also participate in the regulation of mucin metabolism and the mucociliary transport system in middle ear inflammation.
Thus, IL-4 is the important regulator of cellular and molecular processes, accompanying different types and different stages of the middle ear inflammation, and an important factor contributing to chronic condition and persistence of OME.
IL-5 was detected in the middle ear mucosa and effusions in atopic patients with persistent OME. 110, 116, 117 The expression of IL-5 mRNA in mucosa was up-regulated and correlated with increased expression of major basic protein (cell marker for eosinophils) and CD3 (cell marker for activated T lymphocytes). 116 Examination of the cytokine profile in the middle-ear specimens showed an increased number of cells expressing IL-5 and its correlation with the percentage of eosinophils and T lymphocytes. 110 Correlation between the percentage of eosinophils and levels of IL-5 in effusions was particularly striking in OME patients with asthma, 117 suggesting that middle ear eosinophilia could be dependent not only on the local, but also on the general level of IL-5 production. The percentage of cells expressing IL-5 in effusions of atopic patients was the same or slightly higher compared with cells expressing IL-4; 110 this was evidence that both cytokines IL-5 and IL-4 contributed to development of allergic inflammation in the middle ear.
Thus, overproduction of IL-5 in OME is associated with the atopic background of patients, and IL-5 together with IL-4 is responsible for an allergic type of inflammation in the middle ear (Fig. 6) . This assumption was confirmed in an experimental model of OME, 112 where the late-phase allergic inflammatory response was prevented by pretreatment with IL-5 antibodies or soluble receptors for IL-4.
However, the involvement of IL-5 in regulation of the non-allergic inflammatory response in the middle ear should not be ignored. The production of IL-5 was detected in effusions of non-atopic patients with OME, 110, 117 although it was significantly less compared with atopic patients. Analysis of immunoregulatory cytokines in experimental models of OME 93 demonstrated the absence of IL-5 in acute otitis media and significant up-regulation of IL-5 production in chronic otitis media, where IL-5 ' cells were numerous. Thus, the presence of IL-5 in OME of nonallergic patients shows the involvement of IL-5 in regulation of non-allergic chronic inflammatory reactions in the middle ear, where IL-5 can support humoral processes such as increased production and secretion of immunoglobulins, IgA in particular (Fig. 6) .
Thus, IL-5 can be considered the late mediator of middle ear inflammatory response, regulating allergic inflammation and increasing humoral inflammatory processes, which contribute to the chronic condition of OME.
Our knowledge about GM-CSF in OME is insufficient. However, the involvement of GM-CSF in regulation of middle ear inflammation has been shown as GM-CSF was identified in effusions of acute purulent and chronic otitis media. 118 The level of GM-CSF in acute otitis media was higher compared with the chronictype otitis media.
Despite a lack of experimental data about GM-CSF in OME, this cytokine could participate in regulation of different stages of the middle ear inflammation. GM-CSF was identified in acute otitis media, where it could activate matured neutrophils and macrophages and support the acute inflammatory response (Fig. 2) .
GM-CSF in the presence of IL-4 can induce monocyte differentiation towards generation of mul-tinucleated giant cells, and in this way create the cellular background for chronic middle ear inflammation (Fig. 5) .
Finally, GM-CSF being the eosinophil survivalpromoting cytokine together with IL-5 can up-regulate allergic eosinophil-mediated inflammation in the middle ear (Fig. 6) . However, in the past year of investigations GM-CSF has been shown as a potential down-regulator of allergic inflammation, because GM-CSF induced apoptosis of human eosinophils through the eosinophil receptor Siglec-8 (sialic acid binding immunoglobulin-like lectin). 119 Thus, GM-CSF can be a very important regulator of the middle ear inflammation and additional experimental work needs to be done to clarify the exact role of this cytokine in the pathogenesis of chronic OME.
Different types of immunoglobulins, namely IgM, IgG, IgA, secretory IgA and IgE, have been identified in effusions and middle ear fluid of chronic OME. 120 Á 123 The effusion level of IgM in some patients with chronic otitis media was markedly elevated. 120 The mucoid type of effusions contained a high level of IgG, IgA 121 and IgE. 124 Comparison of the immunoglobulin levels measured in effusions and in sera showed that, in many cases, the effusion level of secretory IgA 122 and IgE 125 was significantly higher than the corresponding serum level, and this was the evidence of local overproduction of immunoglobulins in the middle ear.
The immunologic investigation of effusions detected the immune complexes of IgG (IgG Á/ICs) and FIG. 6. Cellular and molecular networks of the allergy-associated cytokines leading to the chronic condition of OME.
IgA (IgA Á/ICs) in both acute and chronic otitis media. 126, 127 The highest level of IgG Á/ICs was found in subacute cases, whereas IgA Á/ICs were predominant in chronic OME. The immunoglobulin immune complexes provided the prolongation of inflammatory process in the middle ear.
The presence of immunoglobulins in chronic OME was associated mainly with the bacterial infection. 128 Á 131 IgG and IgA antibodies specific to Hemophilus influenzae and S. pneumoniae , 128 IgG, IgM, IgA and secretory IgA antibodies specific to outer membrane antigens of Moraxella catarrhalis, 129 and Staphylococcus aureus -harboured bacteria, intensely coated with secretory IgA and IgG antibodies, 130, 131 were identified in chronic effusions. Only one type of immunoglobulins, the secretory IgA, was identified in effusions infected with respiratory viruses. 132 The presence of IgE in chronic OME has not been always associated with local allergic inflammation. 124, 133 However, local overproduction of IgE was usually accompanied by local allergic reactions, such as degranulation of mast cells found in the middle ear biopsy specimens 124 and expression of IgE on mast cells detected in nasal mucosa specimens from patients with OME. 134 Thus, the activity of immunoglobulins in chronic OME is evidence of chronic humoral inflammatory processes in the middle ear, which is obviously controlled by cytokines. Correlation between the levels and types of immunoglobulins and the immunoregulatory and allergy-associated cytokines in chronic OME has not been investigated. However, the presence of the main types of immunoglobulins, IgM, IgG, IgA, secretory IgA and IgE, in effusions is indirect evidence that cytokines IL-2, IL-10, TGF-b, IL-4 and IL-5, which are involved in regulation of immunoglobulin production and secretion in general, 2, 11, 12, 16, 25 participate also in local production and secretion of immunoglobulins and regulate humoral immune reactions during the course of middle ear inflammation (Figs 3 Á/6 ).
The immunoregulatory cytokines and the allergyassociated cytokines can be considered the key regulators of the middle ear inflammation responsible for the molecular and cellular background of chronic OME.
The immunoregulatory cytokines IL-2, IL-10 and TGF-b initiate and support molecular switching of the acute phase of inflammation in the chronic stage, whereas the allergy-associated cytokines IL-4, IL-5 and GM-CSF very probably provide the molecular and cellular background for chronic humoral, cell-mediated and allergic inflammatory processes in the middle ear, which lead to the chronic condition of OME.
However, it is necessary to note that additional experimental work needs to be done in order: (1) to elucidate the molecular mechanisms of cytokine regulation of the middle ear inflammation; and (2) to analyse the possibility of anti-cytokine therapy in clinical treatment of OME. Bench-to-bedside review: Critical illness-associated cognitive dysfunction – mechanisms, markers, and emerging therapeutics Cognitive dysfunction is common in critically ill patients, not only during the acute illness but also long after its resolution. A large number of pathophysiologic mechanisms are thought to underlie critical illness-associated cognitive dysfunction, including neuro-transmitter abnormalities and occult diffuse brain injury. Markers that could be used to evaluate the influence of specific mechanisms in individual patients include serum anticholinergic activity, certain brain proteins, and tissue sodium concentration determination via high-resolution three-dimensional magnetic resonance imaging. Although recent therapeutic advances in this area are exciting, they are still too immature to influence patient care. Additional research is needed if we are to understand better the relative contributions of specific mechanisms to the development of critical illness-associated cognitive dysfunction and to determine whether these mechanisms might be amenable to treatment or prevention. Since its advent more than 40 years ago, the specialty of critical care has made remarkable advances in the care of severely ill patients. Mortality rates for many commonly encountered critical illnesses such as severe sepsis [1] and acute respiratory distress syndrome (ARDS) [2] have declined sharply over the past 2 decades. As greater numbers of patients survive intensive care, it is becoming increasingly evident that quality of life after critical illness is not always optimal. For instance, nearly half of ARDS survivors manifest neurocognitive sequelae 2 years after their illness, falling to below the 6th percentile of the normal distribution of cognitive function [3] . Considering that 89% of Americans would not wish to be kept alive if they had severe, irreversible neurologic damage [4] , these findings are quite concerning.
Cognitive dysfunction (CD) is quite common in critically ill patients, not only during the acute illness but also long after the acute illness resolves [5] . Delirium, a form of acute CD that manifests as a fluctuating change in mental status, with inattention and altered level of consciousness, occurs in as many as 80% of mechanically ventilated intensive care unit (ICU) patients [6] . Most clinicians consider ICU delirium to be expected, iatrogenic, and without consequence. However, recent data associate delirium with increased duration of mechanical ventilation and ICU stay [7] , worse 6-month mortality [8] , and higher costs [9] . Chronically, critical illnessassociated CD manifests as difficulties with memory, attention, executive function, mental processing speed, spatial abilities, and general intelligence. Interestingly, patients who develop acute CD often go on to develop chronic CD after hospital discharge [10] [11] [12] [13] , suggesting that the two entities may share a common etiology.
Although there are clearly defined risk factors for critical illness-associated CD, there is little understanding of the underlying pathophysiology. The precise mechanisms are unknown and there are likely to be multiple mechanisms at work in any given patient ( Figure 1 ) [5, 14, 15] . We have chosen to focus on two mechanisms that appear to have the greatest merit: neurotransmitter abnormalities and occult diffuse brain injury. In this bench-to-bedside review, we discuss the evidence supporting these mechanisms, potential markers that could be used to evaluate each mechanism in individual patients, and emerging therapies that may prevent or mitigate critical illness-associated CD. relative dopamine excess in the central nervous system (CNS). Antipsychotics such as haloperidol, which antagonize central dopamine receptors, can counteract the cognitive effects of anticholinergic medications, further supporting the anticholinergic hypothesis.
Drugs with potent central anticholinergic effects, such as tricyclic antidepressants and antihistamines, are particularly likely to cause delirium. Many medications that are commonly used in the ICU yet not generally considered to be anticholinergic, such as H 2 blockers, opiates, furosemide, digoxin, glucocorticoids, and benzodiazepines, were recently shown to have central anticholinergic properties [16, 17] . Volatile anesthetics, such as sevoflurane, and intravenous anesthetics, such as propofol, also have anticholinergic effects and may be responsible not only for postoperative delirium but also for the more complex phenomena of postoperative cognitive dysfunction [18] . Acute illness itself may be associated with production of endogenous anticholinergic substances [19] . In one study, 8 out of 10 elderly medical inpatients had had detectable anticholinergic activity in their serum, even though no medication used by these individuals had anticholinergic activity. Characterization of such substances might improve our understanding of delirium and lead to useful intervention strategies. Considering that activation of specific cholinergic pathways can inhibit proinflammatory cytokine synthesis and protect against endotoxemia and ischemia-reperfusion injury [20] , it is tempting to speculate that inhibition of these pathways, whether exogenous or endogenous, might contribute not only to CD but also to other outcomes of critical illness.
In assessing the overall risk for developing CD posed by medications with central anticholinergic activity in a given patient, individual differences in drug pharmacokinetics make the dose received a poor estimate of a patient's overall anticholinergic burden [21, 22] . However, we can objectively measure anticholinergic burden in individual patients using an assay referred to as serum anticholinergic activity (SAA) [16] . First described by Tune and Coyle [23] , SAA measures the ability of a individual's serum to block central muscarinic receptors using a rat forebrain preparation. Elevated SAA levels are associated with cognitive impairment in studies of medical ward inpatients and community dwelling seniors [16, [24] [25] [26] [27] . Only a single, small study has used this assay to investigate CD in ICU patients. Golinger and colleagues [28] examined SAA levels in surgical ICU patients and found the mean SAA level drawn 4 hours after mental status change was significantly greater in delirious patients (n = 9) than in those without delirium (n = 16; 4.67 ng/ml versus 0.81 ng/ml; P = 0.007). Whether these results apply to all critically ill patients is uncertain because no study has examined SAA across a broad range of ICU admitting diagnoses or in medical ICU settings. Furthermore, because SAA measurement requires fresh rat brain preparations, its use is likely to remain limited to research settings for the foreseeable future. Pathophysiologic mechanisms and predisposing factors thought to underlie critical illness-associated cognitive dysfunction [5, 14, 15] . Apo, apolipoprotein; HIV, human immunodeficiiency virus; 5-HT, serotonin (5-hydroxytryptamine); GABA, γ-aminobutyric acid; NE, norepinephrine (noradrenaline).
Other neurotransmitter systems such as dopamine, serotonin, γ-aminobutyric acid (GABA), norepinephrine (noradrenaline), and glutamate are also thought to contribute to critical illnessassociated CD. Dopaminergic hyperfunction is thought to underlie the cognitive symptoms of schizophrenia, and dopamine administration itself may be a risk factor for delirium [29] . Serotonin syndrome, a consequence of excess serotonergic agonism, can be seen not only with selective serotonin reuptake inhibitors but also with a variety of drugs and drug combinations [30] . Even a single therapeutic dose of an selective serotonin reuptake inhibitor can cause the syndrome, which manifests as mental status changes, autonomic hyperactivity, and neuromuscular abnormalities.
GABA abnormalities are thought to contribute to hepatic encephalopathy, perhaps mediated by branched chain and aromatic amino acids acting as false neurotransmitters [31] . Excess GABA activity, such as that which occurs after withdrawal from chronic ethanol or benzodiazepine use, is a well known and quite dangerous cause of delirium [32] . Acutely, sedatives that stimulate GABA receptors, such as benzodiazepines and (probably) propofol, impair cognitive function and are deliriogenic [8, [33] [34] [35] . This raises the possibility that strategies to minimize sedative drug accumulation, such as daily interruption of sedative infusions [36] , which have been shown to reduce duration of mechanical ventilation, and ICU and hospital length of stay, might also reduce the incidence or duration of delirium. Whether these sedative drugs lead to neurocognitive deficits long after their use is unknown, but this has been suggested in certain high-risk groups, such as the very old (>75 years) and those with pre-existing cognitive impairment [37, 38] .
Noradrenergic hyperfunction, as part of the 'fight or flight' response, can lead to panic attacks and delusions. Glutamate has been implicated in the 'Chinese food syndrome', in which food with high amounts of monosodium glutamate interferes with normal neurotransmission causing confusion [39] . For a more complete review of the other neurotransmitter abnormalities that may underlie delirium, the reader is referred elsewhere [40, 41] .
If critical illness-associated CD were solely due to acute medication effects, it would probably resolve after the exposure has ended. However, a significant percentage of individuals developing delirium in the hospital continue to demonstrate symptoms of CD after discharge [10] [11] [12] [13] . These patients manifest decreased cerebral activity and increased cognitive deterioration, and are more likely to develop dementia than patients without delirium. Also, patients who develop delirium have a greater rate of decline on cognitive tests than do nondelirious patients [10] [11] [12] [13] . Taken together, these observations raise the possibility that some degree of occult diffuse brain injury, as a consequence of the local hypoxia, hypoperfusion, cytokine-mediated inflammation and microvascular thrombosis that characterize the multisystem organ dysfunction of critical illness, might have occurred in these patients [42] . Given that every other organ system can be damaged by these forces, it seems implausible that the brain would be uniquely spared.
Many of the data supporting occult diffuse brain injury as a cause of critical illness-associated CD come from studies of sepsis and septic encephalopathy, a form of delirium. In animal models of sepsis, oxidative damage occurs early in the hippocampus, cerebellum, and cortex [43] , and significant alterations in cerebral vascular hemodynamics and tissue acid-base balance indicate that cerebral ischemia and acidosis do occur [44] [45] [46] [47] [48] . Sharshar and colleagues completed several studies comparing brain pathology in small numbers of patients who died from septic shock with that in patients who died from other causes. Septic patients demonstrated diffuse severe ischemic and hemorrhagic CNS lesions [49] , which correlated with persistent hypotension and severe coagulation disorders. Multiple microscopic foci of necrosis involving the white matter of the pons [50] were seen, as well as ischemia and apoptosis within the cerebral autonomic centers [51] . The white matter lesions were associated with elevated levels of proinflammatory cytokines, suggesting a possible role of inflammation and microvascular thrombosis in the genesis of CNS injury [52] . Although those studies demonstrated that ischemic brain injury occurs in sepsis, they did not determine whether delirium occurred.
Two studies attempted to examine the relationship of ischemic brain injury to delirium. In one study of 84 patients with severe sepsis and multiple organ dysfunction [53] , severe hypotension was the only factor in multivariable analyses that was associated with delirium, suggesting that sepsis-related encephalopathy may be caused by ischemic damage rather than metabolic abnormalities. Another study examined cerebral blood flow and cerebral oxygen metabolic rates in patients with septic encephalopathy and multiple organ dysfunction [54] , and it found that both were significantly lower than those in normal awake individuals. Although these studies support the idea of occult brain injury as a cause of delirium, the authors did not use a standardized Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV based tool to diagnose delirium, such as the Confusion Assessment Method for the ICU [6] .
Lending support to the hypothesis that acute inflammation leads to brain injury and subsequent development of delirium, a recent study found that delirium in postoperative hipfractured patients was significantly associated with serum levels of C-reactive protein, an acute-phase protein that is a marker of acute inflammation [55] . Importantly, patients in the study were diagnosed with delirium using the Confusion Assessment Method (the ward-based predecessor to the Confusion Assessment Method for the ICU), providing the first DSM-IV based evidence that acute inflammation may be in the causative pathway of delirium. The brain is a target for free radical damage because of its large lipid content, high rate of metabolism, and low antioxidant capacity. Free radical induced oxidative stress may play a role in the delirium seen after cardiopulmonary bypass. Karlidag and colleagues [56] noted that patients with low preoperative levels of catalase, a erythrocyte-based antioxidant enzyme, were more susceptible to delirium postoperatively. They suggested that preoperative catalase levels might some day be used to identify at-risk patients who could then be put on antioxidant treatment preoperatively. Whether this would reduce the incidence of delirium remains speculative.
Regional cerebral blood flow appears to be reduced in delirium. Using xenon-enhanced computed tomography (CT), Yakota and colleagues [57] demonstrated significant focal and global brain hypoperfusion in 10 ICU patients with hypoactive delirium. After recovery from delirium cerebral blood flow returned to normal, implying that cerebral hypoperfusion may contribute to the development of delirium.
Studies of ARDS survivors suggest that a combination of acute hypoxia, hypoperfusion, and hyperglycemia plays an important role in the long-term cognitive sequelae of critical illness [3, 58, 59] . However, it has been difficult to demonstrate a clear relationship, given the lengthy interval between stimulus and effect and the great number of additional contributing variables that can obscure downstream effects. Among ARDS survivors, Hopkins and colleagues showed that the degree of CD at 1 year is significantly correlated with the durations of hypoxia [58] and mean arterial blood pressure less than 50 mmHg during the ICU stay [3] . In animals, hyperglycemia markedly enhances hypoxic-ischemic brain damage due to increased brain edema and disrupted cerebral metabolism [60] . In ARDS survivors, the duration of blood glucose greater than 180 mg/dl has been shown to correlate with worse visual spatial abilities, visual memory, processing speed, and executive function at 1 year [59] . Given the recent interest in maintaining tight glucose control during critical illness as a means of reducing mortality, it will be interesting to see whether patients managed using this technique have better cognitive outcomes. Clearly, such an approach will need to balance the benefits of tight glucose control with the known risks that hypoglycemia poses to the CNS.
One of the perceived difficulties with looking for evidence of occult brain injury in humans is the apparent need for CNS tissue specimens to prove that brain injury actually occurred. However, studies of stroke, trauma, and cardiopulmonary bypass-associated brain injury show that serum markers of brain injury correlate well with the extent of CNS damage. S-100β, neuron-specific enolase (NSE), and myelin basic protein (MBP) are three such markers that could be used to look for evidence of occult brain injury in critical illnessassociated CD.
S-100 is a dimeric calcium-binding protein consisting of two subunits (α and β) [61] . The β unit (S-100β) is highly brain specific, located mainly in astrocytes. Circulating levels of S-100β are elevated in patients with cerebral ischemia [62] , cardiopulmonary bypass-associated decline in explicit memory function [63, 64] , and traumatic brain injury (TBI) [65] [66] [67] . Even in mild head injury, serum levels of S-100β are correlated with clinical measures of injury severity, neuroradiologic findings, and outcomes, including postconcussion symptoms [68] . Elevated serum S-100β levels were recently demonstrated in critically ill patients with respiratory failure [69] and in porcine models of endotoxic shock [70] and acute lung injury [71] . In this latter group, elevated S-100β levels were associated with hippocampal histopathologic changes, including basophilic shrunken neurons in the pyramidal cell layer [71] . Interestingly, S-100β may have both beneficial and detrimental effects, in that lower levels may have protective neurotrophic effects, yet higher levels can lead to exacerbation of neuroinflammation and neuronal dysfunction [72] .
Whereas S-100β is a marker of astrocyte damage, NSE and MPB are markers of neuron and white matter (myelin) damage, respectively. NSE is protein-based enzyme that is found primarily in neurons. Serum levels of NSE are elevated after TBI, exhibiting a close relationship with outcome in severe head injury [73, 74] and with volume of contusion in minor head injuries [75] . Interestingly, elevated NSE levels were recently shown to predict death in one small study (n = 29) of patients with severe sepsis [76] , even though these patients had no acute CNS disorders, such as stroke or neurotrauma. MBP is the major protein component of myelin. Serum levels of MBP are elevated in diseases in which there is myelin breakdown. Studies of patients with TBI have shown that MBP levels correlate with clinical measures of severity and may allow early prediction of outcomes [74, 77, 78] .
New developments in neuroimaging, such as functional magnetic resonance imaging (MRI) and positron emission tomography, have revolutionized our understanding of abnormal brain function in many disease states, including schizophrenia, Parkinson's disease, and post-traumatic stress disorder. To study further whether critical illness-associated CD is associated with occult brain injury in humans, it would be useful to have an imaging test that can detect subtle evidence of brain injury. Unfortunately, traditional CT scans and MRI do not appear to be sensitive enough to pick up the microscopic cellular changes that may underlie CD [42] . Two small studies assessed brain CT findings in critically ill patients with sepsis [79, 80] . Neither study demonstrated any CT abnormalities, although brain pathology in nonsurvivors was consistent with the previously cited findings of Sharshar and colleagues [49] [50] [51] [52] . A recent study of ARDS survivors (n = 15) [81] found that many of these individuals exhibited signs of significant brain atrophy and ventricular enlargement on head CTs obtained during their acute illness, but there were no significant correlations between these abnormalities and subsequent neurocognitive scores.
A new MRI technique may prove useful for identifying occult brain injury in critically ill patients. Specifically, highresolution, three-dimensional MRI can be used to assess noninvasively differences in brain tissue sodium concentration, which is a highly sensitive marker of tissue viability that highlights areas that traditional MRI can miss [82] [83] [84] [85] [86] . The method is based on sodium ion homeostasis, which is tightly regulated in the body and is a major energy consuming process. Any event that perturbs the energy level of the cell enough to disrupt the sodium ion gradient, such as ischemia, has an important impact on cell viability. Although tissue sodium concentration MRI has been successfully used to evaluate the CNS, including nonhuman primate studies and clinical studies of stroke and reversible focal brain ischemia [87] [88] [89] , it has not been used to assess patients with either acute or chronic critical illnessassociated CD.
There are several recent developments that, although preliminary, are of interest because of their potential to prevent or mitigate critical illness-associated CD.
Haloperidol has been used for many years to manage agitation in mechanically ventilated ICU patients, and it is the recommended drug for treatment of ICU delirium [90] . Kalisvaart and colleagues [91] compared the effect of haloperidol prophylaxis (1.5 mg/day preoperatively and up to 3 days postoperatively) with that of placebo in 430 elderly hip surgery patients at risk for delirium. Although there was no difference in the incidence of postoperative delirium between treatment and control groups, those in the haloperidol group had significantly reduced severity and duration of delirium (5.4 days versus 11.8 days; P < 0.001). Haloperiodol also appeared to reduce the length of hospital stay among those who developed delirium (17.1 days versus 22.6 days; P < 0.001). A recent retrospective cohort study examined haloperidol use in 989 patients who were mechanically ventilated for longer than 48 hours [92] . Despite similar baseline characteristics, patients treated with haloperidol had significantly lower hospital mortality than did those who never received the drug (20.5% versus 36.1%; P = 0.004), an association that persisted after adjusting for potential confounders. Because of the observational nature of the study and the potential risks associated with haloperidol use, these findings require confirmation in a randomized, controlled trial before they may be applied to routine patient care.
Leung and colleagues [93] tested the hypothesis that using gabapentin as an add-on agent for treating postoperative pain reduces the occurrence of postoperative delirium. Patients aged 45 years or older undergoing spine surgery were randomly assigned to gabapentin 900 mg or placebo by mouth 1 to 2 hours before surgery and continued for the first 3 days postoperatively. Postoperative delirium occurred in 0% (0/9) of gabapentin-treated patients and 42% (5/12) of placebo patients (P = 0.045). Reduction in delirium appeared to be due to the opioid-sparing effect of gabapentin. Given the small size of the study, these results require confirmation.
Donepezil, a cholinesterase inhibitor that increases synaptic availability of acetylcholine, improves cognitive function in Alzheimer's disease. Sampson and colleagues [94] randomly assigned 33 elderly patients undergoing elective total hip replacement to donepezil 5 mg or placebo immediately following surgery and every 24 hours for 3 days. Donepezil was well tolerated with no serious adverse events. Although the drug did not significantly reduce the incidence of delirium (9.5% versus 35.7%; P = 0.08) or length of hospital stay (mean ± standard error: 9.9 ± 0.73 days versus 12.1 ± 1.09 days; P = 0.09), both outcomes showed a consistent trend suggesting possible benefit. The authors project that a sample size of 95 patients would be required for a definitive trial.
Dexmedetomidine's sedative effects are due to selective stimulation of α 2 -adrenoreceptors in the locus ceruleus of the CNS. Because it does not have anticholinergic or GABA-stimulating effects, it has the potential to be a delirium-sparing sedative. In preliminary results presented in abstract form [95] , cardiac surgery patients (n = 55) randomly assigned to dexmedetomidine for postoperative sedation had a nonsignificantly lower incidence of postoperative delirium as compared with those sedated with propofol or a combination of fentanyl and midazolam (5% versus 54% versus 46%). The authors of that report plan to enroll a total of 90 patient in the study; perhaps these impressive differences will be statistically significant with a greater number of patients.
Recombinant human erythropoietin (rHuEPO) has received considerable attention as a potential transfusion sparing strategy in the ICU. Interestingly, EPO and its receptor are both expressed by the nervous system, and systemically administered rHuEPO can reach sites within the brain. In preclinical studies, rHuEPO reduced neuronal injury produced by focal ischemia, TBI, spinal cord injury, and subarachnoid hemorrhage [96] [97] [98] . Enthusiasm regarding its use as a general neuroprotectant in the ICU has been tempered by potential risks such as thromboembolism and the considerable cost of the drug. Concerns over safety may be at least partially addressed by the recent finding of erythropoietin derivatives with tissue protective but not hematopoietic properties [99] .
Xenon is a chemically inert gas that has been used as an anesthetic agent and for contrast enhancement in CT scans.
In rats xenon appears to protect the brain from the neurologic damage associated with the use of cardiopulmonary bypass, an effect that is potentially related to N-methyl-D-aspartate receptor antagonism [100] . However, its tendency to expand gaseous bubbles, such as bypass-associated cerebral air emboli, could abolish any beneficial effect or even worsen cerebral outcome [101] .
In the setting of ischemic stroke or TBI, there are a variety of compounds with the potential to improve neurologic outcomes. For example, NXY-059, a free radical trapping agent, reduced disability at 90 days when given within 6 hours of stroke onset [102] . In a pilot randomized trial in 56 patients, simvastatin given up to 12 hours after stroke onset significantly improved neurologic functioning (National Institutes of Health Stroke Scale score) at 90 days [103] . Ethyl pyruvate, a pyruvate derivative that prevents mortality in murine sepsis models, reduced motor impairments, neurologic deficits, and infarct volume in a rat stroke model when given as late as 12 hours after middle cerebral artery occlusion [104] . In rodent models of TBI, cyclosporin A reduced acute motor deficits and improved cognitive performance, even when given after the traumatic insult [105] . A phase II dose escalation trial is currently underway in humans.
Mounting evidence suggests that mild-to-moderate hypothermia can mitigate neurologic injury. Shankaran and colleagues [106] found that whole-body hypothermia (33.5°C for 72 hours) reduced the risk for death or disability in infants with moderate or severe hypoxic-ischemic encephalopathy. In adults successfully resuscitated after cardiac arrest, moderate hypothermia (32-34°C for 12 to 24 hours) increased rates of favorable neurologic outcomes and reduced mortality [107, 108] . A practical limitation of therapeutic hypothermia is that reaching target temperatures takes at least 2 hours using the fastest currently available cooling techniques. However, Polderman and colleagues [109] demonstrated that hypothermia could be induced safely and quickly (about 60 min) by means of ice-cold intravenous fluid combined with icewater cooling blankets.
Cognitive rehabilitation involves the teaching of skills and strategies to target specific problems in perception, memory, thinking and problem solving, with the goal of improving function and compensating for deficits. The benefits of cognitive rehabilitation are well known to those that care for patients with stroke, anoxia, or TBI. Predicting who will benefit and how much has proven challenging, but even severely disabled patients sometimes make dramatic neurocognitive recoveries [110] . Although there are no studies evaluating the effectiveness of cognitive rehabilitation in patients recovering from non-neurologic critical illness, it stands to reason that such patients could benefit when they are found to be cognitively impaired. Because cognitive impairments in critically ill patients appear to be underrecognized by ICU and physical rehabilitation providers [111] , few patients are referred for cognitive rehabilitation therapy [3] . Education regarding the cognitive sequelae of critical illness is needed to enhance referrals for rehabilitation, not only for weakness and physical debilitation but also for cognitive impairments.
Cognitive function is an important and relatively understudied outcome of critical illness. Evidence suggests that neurotransmitter abnormalities and occult diffuse brain injury are important pathophysiologic mechanisms that underlie critical illness-associated CD. Markers that could be used to evaluate the influence of these mechanisms in individual patients include the following: SAA, certain brain proteins (S-100β, NSE, and MPB), and MRI tissue sodium concentration. Although recent advances in this area are exciting, they are still too immature to influence patient care. Additional research is needed if we are to understand better the relative contributions of specific mechanisms to the development of critical illness-associated cognitive dysfunction and to determine whether these mechanisms might be amenable to treatment or prevention.
This article is part of a thematic series on Translational research, edited by John Kellum.
Other articles in the series can be found online at http://ccforum.com/articles/ theme-series.asp?series=CC_Trans ProCAT: a data analysis approach for protein microarrays Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approach for analyzing many types of protein microarrays. DNA microarray technologies have proven to be extremely valuable for probing biological processes by measuring mRNA expression profiles. However, studies at the protein level have the potential to provide more direct information since most genes function through their protein products. Traditional investigations focus on individual proteins in a system and then combine such individual analyses to provide a more global perspective. Recently, technologies to analyze proteins in a high throughput and unbiased fashion have become feasible [1] . One particular powerful technology is protein microarrays, which contain a high density of proteins and allow a systematic probing of biochemical activities [2, 3] .
There are two types of protein microarrays [3] . A 'functional protein microarray' contains a set of proteins individually produced and positioned in an addressable format on a microarray surface. Functional protein microarrays are useful for identifying binding activities or targets of modification enzymes. The first version of a proteome microarray was reported in 2001 and contained 5,800 yeast proteins with amino-terminal glutathione S-transferase (GST) tags printed on the array [4] . A second version of yeast protein microarrays was generated recently and contained 5,600 proteins with carboxy-terminal 6His-HA-ZZ domain tags [5] . Proteins from both collections were overexpressed, purified and spotted onto the protein microarrays. Global proteome studies were performed on these chips to understand various biological mechanisms. For example, 87 yeast kinases were examined for their substrates using yeast protein microarrays and over 4,200 in vitro substrates representing 1,325 unique proteins were identified [6] . Compared with the approximately 150 known in vivo kinase-substrate interactions, this global study served as an important first step for dissecting yeast signaling networks. In addition to searching for kinase substrates, proteome chips can be probed with labeled proteins, DNA, lipids, antibodies and many other molecules to search for interacting proteins [4, 7, 8] . Large amounts of data have been generated using protein microarrays, presenting significant challenges in developing robust methods to process the raw data and building reasonable biological hypotheses from the datasets.
The second type of protein microarray, the 'analytical protein microarray' or 'antibody microarray', shares similarities with immunoassays and uses antibodies to detect specific probes. Studies have shown that these antibody arrays can recognize specific targets and generate dose-dependent signal intensities, indicating that they can be used to quantify levels of various targets in a crude mixture [9, 10] . Because of the crossreactivity of certain antibodies with a variety of proteins, only highly specific antibodies are suitable for this type of study. This remains a limiting factor in preparing antibody microarrays.
Both DNA and protein microarrays are prone to systematic errors that are usually generated from different sources, such as surface defects and spatial artifacts. Many studies have offered insight on noise subtraction in DNA microarrays [11] [12] [13] [14] , but little investigation has been done for protein microarrays. Functional protein microarrays differ in many respects from DNA microarrays. First, the goals of these two microarrays are different. DNA microarrays measure the relative DNA levels in a pool of probes, whereas functional protein arrays often aim at discovering global interactions of a single probe molecule. Second, a typical DNA microarray experiment measures signal ratios between two color channels, one for a tested mRNA sample and the other for a reference sample [15] . Signals in the second channel may serve as intrinsic controls that can help to decrease the effects of various amounts of reagent on the arrays and any local array nonuniformity. Furthermore, many current scaling methods are then based on the assumption that signal intensities should be balanced between the two color channels despite variation in slide location, intensity and other sources of systematic variation [16] [17] [18] . However, such controls are missing in onecolor-channel protein microarrays. Third, several scaling approaches in DNA microarrays are based on a set of 'housekeeping' genes that give constant signal intensities at different conditions [19, 20] . However, in protein microarrays, such a control group must be customized according to the type of activities that are assayed, and, therefore, a ubiquitous reference group does not exist. Fourth, unlike DNA microarrays, in which non-specific binding can often be addressed by signal comparison with mismatch probes [21] , cross-reactivities of protein microarrays can not be as directly corrected for. A separate slide is, therefore, often required to be probed in parallel as a negative control in protein microarray experiments. Finally, several protein-specific artifacts serve as common noise sources in protein microarrays. In the kinase assay, for example, the signal from strongly phosphorylated spots can bleed into neighboring spots, leading to incorrect background measurement. These differences are particularly applicable to functional protein microarrays in comparison to antibody arrays, and, therefore, the normalization techniques used for DNA microarrays are usually not directly applicable to functional protein microarrays.
We have developed a new protein chip analysis tool (ProCAT) to deal with various artifacts specific to functional protein microarrays. The work started from a careful survey and characterization of all potential sources of systematic errors in protein microarrays. Specific approaches were then designed to deal with each type of noise. A correction approach is applied to reduce measurement errors in the background signals. In addition, spatial variations can be reduced efficiently through a novel two-parameter signal normalization approach and calling positive spots locally. After generating a list of positives, negative control slides are analyzed in the same approach and spots are subtracted from the list if they appear in the control slide. Slide features with poor signal qualities are also removed. Finally, signal intensities of the positives are normalized according to their protein amounts. All modules that account for the challenges in data processing specific to protein microarrays are built into ProCAT and tested.
ProCAT contains a flexible modular design whose individual components can be adjusted according to the experimental designs and stringency level selected by the users. Six sequential modules are currently implemented in ProCAT before a final annotation report is assembled ( Figure 1 ). These modules carry out: background correction; signal normalization; positive spot identification; spot cross-reactivity filter; signal qualities inspection; and protein amount normalization. The performance of many of the steps was tested using several types of experiments as described below.
A fundamental issue in all microarray experiments is background correction, which aims at reducing noise in background quantification. Signal intensities are generally quantified by subtracting the foreground intensities with the local background intensities, which are measured as the background signals immediately surrounding the spot of interest (termed here the 'adjacent background'; Figure 2b ). However, in protein microarrays local background regions can be easily skewed by artifacts such as small speckles. In addition, strong positive signals from on-chip kinase assays tend to produce signal smears on both film and phosphoimagers that exceed the normal feature size (Figure 2a) . In both cases, the measurement for that spot will be inaccurate. First, the background intensity will be arbitrarily high, which will diminish the real signal intensity for that spot. Second, the intensities will be affected by the alignment of the grid and extent of the smear, and, therefore, the variance of the same protein at replicate experiments will be increased.
Two methods can reduce the artifacts in local background. The user can manually adjust the grid size to fit the circles to each individual spot. However, the aligning process requires considerable time and effort. The size of the smear may even prevent refitting the grid without adversely affecting neighboring spots. Additionally, a larger spot size can diminish the signal of the spot because the signal density decreases with increasing spot size. The second method for background correction, which is applied in ProCAT, replaces the background intensity of the central spot with the background from its local neighborhood. A three by three surrounding window is assigned to each protein spot, and the median background of the nine spots will be used as the 'neighborhood background' value for the central spot (see Materials and methods for more details). No additional time is needed for further alignment, yet this method will significantly reduce artifacts that can produce erroneous measurements on spots background.
In the analysis of the phosphorylome dataset [6] , we applied the neighborhood background correction and observed a high sensitivity in identifying positive targets. To further characterize the effects of neighborhood background correction, we performed a test kinase assay with 100 nM protein kinase A (PKA) spotted at 96 locations on one slide (Figure 2a) . Each of the 48 blocks on the slide contains two PKA pairs with random yeast proteins spotted elsewhere (approximately 12,000 spots). After incubating the slide with 33 P-γ-ATP, all of the PKA spots autophosphorylated and showed strong signals, and in many cases the signal went beyond the grid circle boundaries (Figure 2b ). We then applied the neighborhood background correction to the PKA spots. As expected, the median for PKA signal intensities was enhanced by 53%. Furthermore, the PKA signals from different positions are more similar to each other; the variance within them is decreased by 41% (p value = 0.006; Fig 2d) . Therefore, the neighborhood method for accessing background provides more robust measurements than that of the adjacent background method.
Spatial artifacts arise from uneven signal distribution across the slide, in part due to uneven probing conditions and smear artifacts [13] . Uneven probing can occur by several means, such as uneven mixing of the probe, exposure to the probe solution, or uneven washing and drying of the slides. Twocolor-channel experiments of DNA microarrays provide intrinsic controls that can be used to account for spatial artifacts. Functional protein microarrays often use only one color channel and, therefore, are especially prone to spatial artifacts. Spatial artifacts will cause inaccurate measurements of signal intensities and can hinder the identification of significant interactions. Adding more controls can help remove spatial artifacts since the signal of each spot can then be normalized according to its local controls. Due to the variable shape and size of spatial artifacts, ideally a large number of controls would be needed. However, space constraints of the protein chip and an inability to anticipate all the uses of the arrays usually prevent the necessary number of controls to fully account for spatial artifacts on the array.
A scaling method that reduces signal variations among spots of the same proteins at different array locations decreases spatial artifacts. We developed a new normalization method to deal with the spatial artifacts specific to functional protein microarrays. By assuming that signal distribution in large windows is consistent across the slide, the foreground signal of each spot can be normalized according to signal intensities in its surrounding neighborhood. This assumption is usually valid in protein microarray experiments in which proteins are randomly printed on the array ( Figure 3 ). Two parameters, the median and the median absolute deviation (MAD), are calculated to represent the signal distribution in the local window ( Figure 4 ). To perform the normalization, the median and MAD of all sliding windows are averaged. The average values are then used to correct the signal of the central spot to To test the performance of this two-parameter scaling approach for signal normalization within one slide, we designed a test microarray containing multiple positive controls printed at different positions on the slide. The test array was organized in the same format as the commercially available protein microarrays (Invitrogen). Each protein was printed in duplicate, and the array contained 24 blocks of 16 by 16 printed proteins (Figure 5a ). Two GST-fusion proteins, Sla2p and Myo4p, were purified separately and a 1:1, 1:5, and 1:25 dilution of each protein was prepared. Sla2p and Myo4p at each concentration were printed at eight random positions on the array. Other spots were occupied with bovine serum albumin (BSA) as negative controls. In order to visualize the two fusion proteins, anti-GST antibody was used to probe the slide, and one probing with typical spatial artifacts is shown in Figure 5 . The artifact-containing slide showed different signal levels between the edges and the middle portion of the array. This produced blocks that had a variable signal distribution that ranged from high to low from one edge of the slide to the opposite edge; the variability occurred across blocks and simple block normalization methods adopted in DNA microarray normalization approaches [17] would not be suitable for dealing with this problem.
We applied ProCAT to normalize the slide with several different parameters ( Figure 5) . Five window sizes were tested, termed windows 1, 3, 5, 7, and 9. These numbers correspond to the window size as a function of the number of spots on one edge of a block. For example, a block of 20 by 20 spots analyzed using window 1 would have a window size of 0.1 that of the block edge, or in this case 2 spots above, below, and to either side of the central spot, whereas a window size of 9 would contain a 37 by 37 area roughly as large as 4 blocks.
Three observations were made from the analysis of different window sizes. First, as the window size increases, the computational time used for the normalization also increases. Second, no obvious spatial artifacts were left after the normalization with any of the window sizes tested ( Figure  5b ). Third, a small window size diminishes any signal inequality that exists between positive signals and background noise. Indeed, a small scaling window tends to introduce extreme changes to the original signals and, therefore, increases the discrepancy between the duplicate spots of the same protein. The variance of the signals for the same protein after normalization with different window size was calculated. In five out of the six cases (three dilutions of two proteins) the scaling window 9 can successfully reduce the signal variance in a range from 31% to 90% (Figure 5c ). Decrease of signal variation suggests that a large scaling window will help to reduce spatial artifacts. Although larger window sizes are possible, 9 was used as the default number for ProCAT because the analysis can be done in a reasonable time and minimal improvement has been achieved after window size 7 (Additional data file 1).
In addition to providing accurate measurements of spot intensities, ProCAT has been developed to assign thresholds for identifying positive targets in one experiment. Traditionally, a global cutoff can be calculated from all spots and applied to the whole slide. Due to variable spatial artifacts, cutoffs were assigned locally in ProCAT. For each spot on the array the signal distribution within a nine by nine window was calculated and a cutoff defined as a number of standard deviations away from the mean; the default for ProCAT is two standard deviations. This cutoff corresponds to 5% significance level if the signal distribution within this local window is normal. When many spots with strong signals are included in the window, the cutoff will be arbitrarily high and thus decrease the sensitivity of detecting positive spots by the program. To avoid this loss in sensitivity, ProCAT has a built in function to identify possible outliers, to remove those outlier spots that have extremely strong signals, and then to calculate a cutoff for identifying positive spots using the remaining spots.
A receiver operating characteristic (ROC) curve was used to compare the performance of local window cutoffs versus a global cutoff on the test slide [22] . Area under ROC curve (AUC) is a performance indicator that ranges from 0 to 1, with 1 for the best performing method. Using GST-Sla2p and GST-Myo4p as positive controls and BSA as negative controls, the sensitivity and specificity for both local and global cutoff methods was estimated. Five window sizes were tested and compared with the global cutoff ( Figure 6 ). Prediction performance is increased significantly when using local windows with nine or more spots on one edge. Thus, a nine by nine window is used as the default in ProCAT since a larger A representative protein microarray with high-quality data Figure 3 A representative protein microarray with high-quality data. The slide image was reconstructed from a protein microarray experiment with minimal noise in the data. Density plots of signals in local 37 by 37 windows (window size 9) for all spots were computationally combined, and they showed high similarities. window size results in increased computing time with only minimal improvement in sensitivity. The AUC value is much larger in local cutoffs (0.992) compared to global cutoffs (0.916) and the improvement is unlikely to be due to random chance (p value = 0.002) [20] . Therefore, we can conclude that the local cutoff is significantly better in identifying positive spots than a global cutoff.
Scheme for the signal scaling method Figure 4 Scheme for the signal scaling method. The signal of one spot on the array is normalized according to the distribution in its local neighborhood. For each spot, a surrounding window is chosen and all spots in this window are defined as its neighborhood. The signal of a center spot will then be normalized by comparing the local median and MAD with the average values. Norm, normalized signals; Origin, original signals.
Average distribution 
Two layers of filters are implemented in ProCAT. First, all positive spots from negative control experiments are removed. For example, in on-chip kinase assays, kinase dead alleles were probed on separate arrays using the same experimental conditions as used with wild-type kinases. Spots that produce signals in the absence of active kinase were identified by ProCAT and removed from the target lists of kinase probings. When probing tagged protein to detect protein-protein interaction, testing the epitope tag in the absence of the protein of interest is also an essential control. If proper negative control experiments are available, ProCAT will analyze them in the same way as regular experiments to construct experimental positive spot lists void of proteins producing positive signals under control conditions.
The second filter checks the quality of each positive spot. All proteins are spotted in duplicates on protein microarrays, hence should have very similar signal intensities. ProCAT then uses the difference between duplicate signals as an indicator of the signal qualities. The difference between signals of two duplicate spots (s 1 , s 2 ) is calculated as (s 1 + s 2 )/(|s 1 | + |s 2 |) and then fitted to a normal distribution. Proteins with exceptionally large differences in their duplicate spots are more likely to be biased by certain artifacts, and thus are removed from the positive list. The default threshold for the duplicate spot difference in ProCAT is set at two standard deviations away from the mean.
One of the goals for protein microarray experiments is to identify the affinity of a binding interaction (in a protein-protein interaction assay) or the extent of phosphorylation (in a kinase assay) so that one can compare the relative strength of the reaction for each positive protein. Ideally, the spot intensity would directly correspond to the strength of interaction. However, a number of other factors contribute to the array signal intensities, including the systematic noise from various artifacts, as was already discussed, and the amount of protein printed on the chip. Nonetheless, semi-quantitative estimates can be obtained. After background correction and signal normalization, the raw signals can be standardized by relative protein amounts before they can be used to estimate the interaction strength.
Although proteins on the microarray can have very different amounts, they do share the same epitopes for the purpose of large-scale protein purification [4, 5] . Therefore, probing with anti-epitope antibodies will provide an estimate of the relative protein amounts in each spot on the array. After the protein amount is determined for one spot at row i and column j, ProCAT divides the raw signal intensities S i,j by the protein amount signals A i,j and uses the quotient as an approximation of the strengths of interactions:
This approximation generally works well across the slide except for the following two situations. Less abundant proteins will be biased because the A i,j values estimated in antiepitope probings are more susceptible to background noise and slide artifacts. On the other hand, overpowering spots can also be biased if they have saturated signal intensities. A saturated S i,j value is an underestimate to the real signal. For these two reasons, only proteins with amounts more than a minimal cutoff and signal intensities lower than a saturation threshold will be normalized with protein amounts. Proteins that do not conform to these two requirements will be recorded with unnormalized signals and flagged for further inspection. An additional caveat is that the relative protein amount assessed using antibodies includes both native and denatured protein at a given spot. Therefore, the estimation of interaction strength will be an underestimate since the amount of functional protein may be an overestimate.
ProCAT was designed as a flexible tool to analyze functional protein microarray data. The program was scripted in Perl (version 5.6.1) on top of a Tomcat (version 5.0.30) web server [23] . Each module discussed above was implemented independently and can be included or excluded depending on various experimental designs. To input a dataset, the user has to characterize the data in three aspects: experimental designs, data file formats and normalization parameters. Experimental design contains parameters such as the number of test arrays and negative control arrays for one particular assay. Data file format describes the layout in the uploaded dataset so that ProCAT can recognize and extract the useful information from it. Normalization parameters allow users to try different stringency levels. These three levels supply sufficient information to uniquely characterize an experiment while still allowing ample flexibility for the individual user to customize parameters to suit many different types of experimental designs.
After inputting all three descriptions and uploading the dataset, ProCAT takes five minutes on average to complete all analysis modules for each array. The time may vary depending on the selected analysis modules and the size of the protein microarrays. Each task is assigned a unique ID and results are organized into a database for future queries. Processed data including analysis parameters, a list of positive spots with protein annotations, and normalized signal intensities will be available for the users to download from the server.
Functional protein microarrays serve as an efficient platform for screening protein biochemical functions. Here we present ProCAT as a systematic approach to process and analyze data specific to functional protein microarrays. Calibrated by explicit test experiments, ProCAT has proven to be able to handle many types of functional protein microarray studies with three unique features. ProCAT includes novel scaling methods that provide robust and reproducible measurement for quantitative signals. This is crucial for protein microarrays as chip signal intensities often indicate strength of interactions. In addition, by calling positive candidates locally, ProCAT demonstrated excellent performance in identifying positives in comparison to global thresholds. Finally, each step has been integrated into a modular design to fit various experimental designs and stringency requirements.
A major challenge in designing any automated data processing method is thinking of and anticipating all possible situations that may arise. ProCAT uses a local three by three window to correct background containing signal smears or dust speckles. This method assumes the artifacts are sparse enough so that the majority of the nine spots in the local window still provide correct measurements of the background signals. Since the median value of nine spots is used to correct the background, a few biased spots within the window will not severely affect the corrected background value. This assumption is usually valid since the percentage of spots that are either positive or whose signal is contaminated by artifacts in protein microarray experiments is generally quite low. In extreme cases where such spots are likely to be very close to each other, a larger window (five by five for example) can be used. Large artifacts such as bright speckles and incubation bubbles may affect many spots in a particular region. Since the shapes of these artifacts are variable, it is necessary to manually flag these spots initially and then remove them from future analysis. Many commercially available software packages for microarray experiments have a built in flagging function, and ProCAT will automatically discard flagged spots.
A key aspect of ProCAT is the two-parameter approach for reducing spatial nonuniformity. Several factors can affect the performance of ProCAT's normalization. First, ProCAT normalizes the signal of a spot according to the signal distribution in its local neighborhood. It diminishes the signal intensity if the spot is located in a high signal neighborhood, while compensating the intensity if it is in a low signal neighborhood. This approach is based on the assumption that signal intensities across the slide share the same distribution, and it holds true if and only if the regional variations observed on the slide are due to technical artifacts and not from real biological differences. Since proteins are printed in a random order on most of the current protein microarrays, it is unlikely a particular region of the slide will gain high intensities as a result of biologically relevant reasons. Second, the size of the neighborhood window can also largely affect the performance of the normalization. Small window sizes tend to add biases to signals and diminish all local variations, whereas large window sizes increase the computational burden and tend to preserve local variations. We found that the optimal window size of ProCAT is 9 for protein-protein interactions; this figure corresponds to approximately four blocks on the chip and is used as the default. Other window sizes can also be chosen to fit various shapes of spatial artifacts.
ProCAT can be applied to many experiments using protein microarrays, such as kinase assays, protein-protein interactions and protein-DNA interactions. Thus far, the twoparameter scaling approach has only been used in single chip normalization; however, a similar strategy can be extended to rescale multiple slides by assuming signals in neighborhood windows on different slides are similarly distributed. Overall, ProCAT provides a powerful and flexible new approach for optimal processing and analysis of functional protein microarrays.
For the slide used for testing background correction, 100 nM PKA (Sigma, St. Louis, MO, USA) was spotted at 96 different places as positive control. The slide was incubated with 200 μl of kinase buffer (100 mM Tris pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 20 mM glutathione, 20% glycerol) plus 0.5 mg/ ml BSA, 0.1% Triton X-100, and 2 μl 33 P-γ-ATP in a humidified chamber at 30°C for 1 hour. The slide was then washed twice with 10 mM Tris pH 7.4, 0.5% SDS and once with double distilled H 2 O before being spun dry and exposed to X-ray film (Kodak, Rochester, NY, USA).
For the anti-GST probing, slides were printed with Sla2p and Myo4p as positive controls and 150 nM BSA as a negative control. The array surface was blocked using SuperBlock (Pierce, Rockford, IL, USA) at 4°C for 1 hour. Rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated with the slides at 1,000-fold dilution. The array was then washed with PBST (Sigma) and incubated with a 1:1,000 dilution of Cy5-conjugated anti-rabbit IgG antibody (Jackson Laboratories, Bar Harbor, ME, USA). Slides were then washed with PBST five times and scanned in an Axon
Testing experiment for the signal scaling approach Figure 5 (see previous page) Testing experiment for the signal scaling approach. (a) The design of the test slide with positive spots shown as red spots and the five tested normalization window, indicated by red squares, for a given spot on the array, shown in blue. (b) Comparison of signal intensity before and after normalization using window size 9 on the testing experiment. The two images were computationally reconstructed from the signal files, either without or with normalization. GenePix scanner (Molecular Devices, Sunnyvale, CA, USA). Raw signals were extracted with GenePix Pro 6.0 software (Molecular Devices).
For one spot, let i be the row and j the column on a protein microarray. Thus, B i,j represents the adjacent background intensity and F i,j denotes the foreground intensity. The raw signal intensity S i,j is calculated as:
In neighborhood background correction, we use neighborhood background to replace the adjacent background. A local three by three window around B i,j is chosen and the neighborhood background is defined as:
In a protein slide with N rows and M columns, a local window W i,j around one spot (i, j) is defined as signals of a set of spots S i,j that satisfy:
The size parameter k is dependent on window size factor f win and the block size f block :
in which f block represents the number of spots on one edge of the block, and f win is chosen by users from five options: 1, 3, 5, 7 and 9. Different windows can overlap with each other and go beyond the block edges. Let s denote signal intensities of spots within the local window; ProCAT uses two parameters to characterize the signal distribution of s: median (MED) and median absolute deviation (MAD):
After calculating MED i,j and MAD i,j for all the spots on the array, they are averaged to obtain the two parameters and for the reference distribution.
For one spot (i, j), ProCAT normalizes its raw signal S i,j by comparing MED i,j and MAD i,j with the average values:
For a given spot at row i and column j, its normalized signal is compared to surrounding spots in a nine by nine window W ij (4) . Signals within this window are fit to a normal distribution. The mean μ i,j and standard deviation σ i,j will be calculated and the default threshold is set at two standard deviations above the signal mean. A spot (i, j) will be called positive only if its signal is above the threshold:
When positive spots are likely to be close to each other, Pro-CAT uses box plots to examine and remove possible outliers from the surrounding window [24] . Let Q 1 be the lower quartile (25th percentile) and Q 2 be the upper quartile (75th percentile); the difference between Q 1 and Q 2 is termed interquartile range ΔQ. A spot (i', j') is then defined as an outlier if its signal:
ROC curve comparing the global cutoffs and local cutoffs in calling positive spots Figure 6 ROC curve comparing the global cutoffs and local cutoffs in calling positive spots. The test slide has six unique positive controls (Sla2p and Myo4p in three different titrations). The performance of identifying the positive controls is increased by using local cutoffs generated in relatively large surrounding windows. Five window sizes were tested and the best performance was achieved using nine by nine or larger windows. To obtain a robust threshold, the corrected mean and standard deviation are generated using the non-outlier spots.
The following additional data files are available with the online version of this paper. Additional data file 1 is a figure illustrating the variance reduction in positive controls using different normalization window sizes. Additional data file 2 is a table listing the raw signals generated in the autophosphorylation experiment for testing the background correction method. Additional data file 3 is a table listing the raw signals generated in the anti-GST probing experiment calibrating the signal scaling approach.
Additional File 1 Variance reduction in positive controls using different normaliza-tion window sizes Variance reduction in positive controls using different normaliza-tion window sizes. Click here for file Additional File 2 Raw signals generated in the autophosphorylation experiment for testing the background correction method Raw signals generated in the autophosphorylation experiment for testing the background correction method. Click here for file Additional File 3 Raw signals generated in the anti-GST probing experiment cali-brating the signal scaling approach Raw signals generated in the anti-GST probing experiment cali-brating the signal scaling approach. Click here for file Positional clustering improves computational binding site detection and identifies novel cis-regulatory sites in mammalian GABA(A) receptor subunit genes Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A γ-aminobutyric acid receptor (GABA(A)R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA(A)R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system. Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A g-aminobutyric acid receptor (GABA A R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA A R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system.
Co-regulation is a basic mechanism to coordinately control expression of genes in modules, biochemical pathways and protein complexes (1) (2) (3) . In eukaryotes, expression is most often mediated by transcription factors (TFs) that bind upstream of the transcription start site (TSS) and recruit the polymerase assembly (4) . TFs bind, with varying affinity, to a set of similar, short (6-20 nt) sequences (5) . Computational binding site discovery focuses on finding significantly overrepresented sequences in upstream regions of co-regulated genes (6) (7) (8) . Thus, computational TFBS prediction algorithms must begin with an input set of promoters from genes hypothetically co-regulated by a shared TF. The algorithms aim to predict the binding positions and consequently the nucleotide specificity of the TF (9) (10) (11) .
The first part of transcription factor binding site (TFBS) discovery, the input set, can be identified using either computational or experimental methods. Experimental techniques, such as chromatin immunoprecipitation (ChIP) (12) , have been successfully used to generate a genome scale mapping of approximate TF-binding positions (10, 13, 14) . Computational techniques, such as phylogenetic profiling (15, 16) and artificial neural networks, can also be used to identify sets of co-regulated genes. Both experimental and computational approaches, however, suffer from a significant false positive (FP) prediction rate. Inclusion of extraneous promoters in the input sets dilutes the enrichment of the shared TFBS sequences making computational TFBS discovery significantly more challenging (17) . We term such erroneously included promoters decoy sequences (DSs).
After receiving a set of upstream regions co-regulated by a shared TF as input, computational methods aim to predict the binding positions of that TF (6) (7) (8) 18) . Given a set of input promoters, motif detection algorithms identify a set of short, oligonucleotide segments hypothesized to bind to the TF of interest. The predicted sequences can be used to construct a position weight matrix (PWM) representing the average nucleotide frequencies for each position in the site (19) . Ideally, computational detection will return all sequences that bind to every TF with biologically relevant function in those upstream regions. However, since the source of binding specificity for TFs is not well understood (20) , heuristic approaches and ad hoc multiple alignment based scoring schemes are used to identify locally optimal solutions (17) . Each local optimum that exists in a given set of promoters may correspond to distinctly different motifs, and may score differently relative to each other according to different scoring schemes.
Binding site prediction algorithms are generally confounded by several factors: degeneracy in the binding site; the unknown length of the binding site; the relatively large length of promoters; and the inclusion of DSs in the input sets (17, 21, 22) . As a result as few as 10% of predicted positions correspond to biologically functional binding sites (23) . Due, in part, to the low accuracy rate, computational binding site identification has been of limited use (23) . Problems identifying binding sites are further exacerbated in mammalian genomes by larger promoter regions (24) and scarcity of reliable information on co-regulation of genes. Thus, the most demanding test of efficacy for TFBS identification approaches lies in their application to mammalian systems and subsequent validation of predictions.
Because of computational complexity of the problem, Gibbs sampling is often used to identify binding positions (18) . In this paper, we present a new strategy that clusters Gibbs sampling results at each input nucleotide-a technique we term positional clustering-to improve accuracy of predicted TF binding. We evaluate the efficacy of our approach using known examples of binding and regulation in yeast and experimentally testing predicted TF-binding sites upstream of the subunit genes coding for the heteromeric mammalian neurotransmitter receptor system, the type A g-aminobutyric acid receptor (GABA A R).
The GABA A R is the major inhibitory neurotransmitter receptor in the central nervous system (CNS) (25, 26) with important roles in development (27, 28) and disease (29) (30) (31) . The receptor is believed to be a pentamer made up of multiple subunits that come from at least four different subunit classes (a, b, g and d) (32) . At least 19 genes code for the various subunits that differentially combine to form numerous pharmacologically distinct GABA A receptor isoforms (29, 30) . Isoform utilization depends in part on the relative abundance of the subunits, which may change under various conditions (33) (34) (35) . Understanding subunit regulatory mechanisms may provide insight into GABA A receptor isoform usage and related phenotypes (36) .
In the current study, we test the ability of positional clustering to detect known TF-binding sites in a series of increasingly noisy sets of yeast promoters, and found marked improvement in the percentage of correct predictions over Gibbs sampling alone. We also present de novo predictions of TF-binding sites in promoter regions of GABA A receptor subunit genes (GABRs) whose expression is altered (either up-regulated or down-regulated) in an animal model of temporal lobe epilepsy (35) . Positional clustering identified a number of putative cis-regulatory sites, many of which correspond to known binding elements for TFs found in the CNS. Mobility shift assays showed several predicted GABR-binding sequences specifically bind nuclear proteins derived from primary neocortical neurons kept in culture. Furthermore, a particular non-consensus GABR putative regulatory sequence was shown to have a functional role in cultured cortical neurons demonstrating the efficacy of positional clustering in detecting functional regulatory elements in mammals.
We identified S.cerevisiae genes predicted at high confidence (P < 0.001) to be regulated by the TF STE12 in YPD growth media, according to whole-genome TF location data (14) . For the 51 identified genes, we collected upstream intergenic promoters. Intergenic regions were truncated at 1 kb upstream of the gene's TSS.
We selected for study a set of six GABRs: GABRA1, GABRA4, GABRB1, GABRB3, GABRD and GABRE. Promoters were extracted for each gene, including two alternative first exons of the GABRB3 (37), giving a set of seven promoters. The length of each promoter was: GABRA1, 3733 bp; GABRA4, 1546 bp; GABRB1, 1353 bp; GABRB3 (exon 1), 1310 bp; GABRB3 (exon 1A), 2080 bp; GABRD, 6625 bp; and GABRE, 5278 bp. We augmented the input set with orthologous promoters from rat, with the exception of GABRB3 for which an orthologous gene from mouse was used. In total, 14 promoters upstream of six GABRs were selected for analysis.
For a given input set of promoters, we ran the Gibbs sampler BioProspector (8) 400-550 times, evenly distributed across all motifs widths from 6-15 bp. We used a third-order background model derived from appropriate genomic promoters. We collected the best three results from each BioProspector run. We counted the number of times BioProspector identified each nucleotide in the input set. For each promoter, we identified the maximally occurring nucleotide, and extracted all positions identified by BioProspector >35% of the maximum. We clustered together neighboring positions into putative TFBS. As a dust filter, we removed all putative TFBSs <6 bp long ( Figure 1 ).
For sets of S.cerevisiae promoters, we used 1200 results from 400 BioProspector runs in our evaluation. For GABRps, we considered all 127 non-empty subsets of the seven promoters (orthologous sequences were always considered together). We used results from 70 000 BioProspector runs, evenly distributed across all promoter subsets, in our analysis. In addition to dust filtering, we required putative TFBSs to occur both in the human and in the orthologous rodent promoter.
We used positive predictive value,
to evaluate STE12-binding site predictions. We classified predictions as true positive (TP) or false positive (FP) by comparison to the STE12-binding motif, TGAAACA, as determined by (14) . For each sequence, we calculated distance from the known STE12 PWM using a modified local ungapped sequence alignment similar to that in (38) . Alignments were scored as the sum of Pearson's correlation coefficient,
between prediction X and the STE12 PWM across all aligned positions. Thus, scores ranged from zero, with no positions aligned, to seven, the length of the STE12 PWM. We observed a bimodal distribution of scores (Supplementary Figure S1) , and chose the alignment score corresponding to the minima of the distribution (alignment score ¼ 4.5) as the threshold to classify predictions as TP or FP.
We complemented the seed set of 51 STE12-bound promoters with 1-50 randomly chosen yeast promoters. We performed our motif detection procedure on each input set, and compared the PPV of putative TFBS with that of raw BioProspector results ( Figure 2 , solid lines).
To evaluate the background rate of STE12-binding site recovery, we created a seed set of 51 randomly chosen S.cerevisiae promoters. We evaluated the percentage of STE12-like binding sites identified in the random seed set, as well as in versions of the seed set augmented with 1-50 randomly chosen yeast promoters (Figure 2, dashed lines) .
For additional yeast evaluations (HAP4, TEC1, YAP1 and YDR026C), we substituted for BioProspector an in-house implementation of the BioProspector algorithm. Comparisons of results from each implementation show the two implementations to be approximately equivalent.
We ran MotifScanner (39) to search GABR promoters for all vertebrate TF-binding motifs found in TRANSFAC (40) . For each promoter analyzed, we used a prior probability of 0.1 and the corresponding organism specific third-order promoter background model from Eukaryotic Promoter Database (EPD) (41) .We considered positional overlap between Motif-Scanner predictions and putative TFBSs indicative of known binding motifs in our predictions.
Double-stranded oligonucleotides for EMSA contained the following sequences: Nuclear extracts were prepared (42) and used for gel shift analysis after concentration (Microcon no. 10 columns, Amicon, MA). Quantification was performed on EMSAs under conditions that yield a standard curve for band intensity.
Single-stranded sense and antisense phosphorothioate oligonucleotides for the predicted GGCGGCGTGCACACACACGC-CCACCGCGG binding site are annealed by boiling sense and antisense oligonucleotides for 5 min at equal molar ratios in dH 2 O. Oligos are then cooled to room temperature and placed on ice. Transfections using DOTAP (Roche)/HEPES solutions are performed with oligonucleotides corresponding to wildtype, mutant or with DOTAP (Roche)/HEPES solution lacking oligonucleotides (MOCK) as described in (29) . Effects of oligonucleotide application to neurons are assessed by real-time RT-PCR.
Since TFBS are predicted computationally by local optimization strategies, we evaluate the extent to which one of these strategies, Gibbs sampling, identifies the same set of segments in repeated runs using the same input data. Identifying stably recurring motifs requires clustering of related results which, in turn, requires definition of 'related'. Sequence similarity based clustering is impaired by the combination of sequence variation within motifs, the short length of TF-binding sites, and aligning motifs of different lengths. Instead of using sequence based clustering, we chose to cluster results by position, counting the number of times Gibbs sampling identifies each nucleotide in the promoter (Figure 1 ). We find that Gibbs sampling predictions, generated using BioProspector (8) are power-law distributed over nucleotide position (Supplementary Figure S2) . Gibbs sampling converges on the majority of nucleotides very infrequently, and a small number of nucleotides very frequently. Thus, the most frequently recurring nucleotides appear in as few as 10% of results. Moreover, we find the power-law distribution of results is robust to Gibbs sampling algorithm and scoring scheme (data not shown). We can hypothesize that the most frequently occurring positions are the most biologically significant. Thus, discarding the least frequent Gibbs sampling results may yield higher accuracy and robust identification of biologically insignificant positions.
As a preliminary test of the above hypothesis, we applied repeated runs of Gibbs sampling to a set of 51 S.cerevisiae promoters enriched in STE12 binding as identified by wholegenome ChIP-chip experiments (10) . We used positional clustering of 1200 results to identify the most frequently recurring positions (see Methods). Incorporation of additional results did not significantly alter the distribution of results (data not shown). We chose STE12 because it is one of the best studied TFs, with a well known, highly conserved and experimentally well-defined binding motif (10, 43) . The most frequently recurring positions were compared with the known STE12binding motif (40) . We classified predictions into two categories: true positive (TP) if they resemble the experimentally identified STE12-binding motif, and false positive (FP) otherwise (see Methods). Finally, we calculated the positive predictive value PPV as PPV ¼ TP/(TP + FP).
We find that positional clustering and subsequent selection of frequently recurring nucleotides improved the PPV of the STE12 binding site by at least 37% over Gibbs sampling alone (Figure 2 ). To validate that the above results were not specific to the number of input promoters, the STE12-binding motif, or the particular Gibbs sampling implementation, we repeated the above prediction process for promoters predicted to bind to YAP1, TEC1, HAP4 and YDR026C. We also repeated the analysis replacing the original Gibbs sampling procedure with our own implementation and MotifSampler (44) . In all cases, we found positional clustering significantly improves on results over local optimization procedures alone ( Figure 3 ).
Computational discovery of TFBS can have two types of FP predictions. One type is the identification of an incorrect motif from a set of upstream regions known to bind to a TF of interest as described above (see Methods). The second type of FP error is the background discovery rate of the correct motif using upstream regions that do not bind to the TF. To simulate this rate for STE12-like binding site recovery we repeated the analysis as described above starting with 51 randomly chosen yeast promoters. We find that positional clustering identifies STE12-like sites in <5% of results, compared with 10-15% for Gibbs sampling alone. Thus, using positional clustering, the performance of computational motif discovery is enhanced not only by improving the positive predictive value in promoters of genes co-regulated by STE12, but also by decreasing the false discovery of STE12-like sites by 10%.
Next, we evaluated the effect of adding DSs on the performance of Gibbs sampling with and without positional clustering. Addition of DSs dilutes enrichment of the TF-binding site in the input set, making motif detection more challenging (17, 22) . Modeling DSs, we repeated our estimate of PPV of TFBS detection with the addition of 1-50 random yeast promoters (DSs) to the original set of 51 STE12-bound promoters. We found that positional clustering improves the PPV of Gibbs sampling by >20% through the addition of up to 80% noise or 40 DSs (Figure 2, Supplementary Figure S3 ). Additionally, results of Gibbs sampling both with and without positional clustering decay linearly with the addition of decoys [R 2 ¼ 0.81 and 0.95, respectively (Supplementary Figure S4) ]. Extrapolating, we predict positional clustering will maintain an improved PPV through the addition of >100% noise or 70 DSs.
To address issues of generality, we repeated the procedure on additional sets of S.cerevisiae promoters (YAP1, TEC1, HAP4 and YDR026C). An added benefit is that we can evaluate the effect of information content of the binding motif and number of promoters on the improvement from positional clustering (22) . Repeating the analysis, we again find that independently of the set or sampling procedure, positional clustering improves accuracy through a broad range of random DSs (Figure 3 ). Improvement appears to be limited and unreliable only when sampling alone correctly identifies the binding site in fewer than 20% of results. This result is consistent with our analysis of STE12-bound promoters (Figure 2) , and may correspond to a lower limit for the efficacy of positional clustering.
Recently, researchers have noted that complementary motif detection approaches can be used together to predict binding sites more effectively than either method alone (23) . With this in mind, we evaluated positional clustering in terms of its ability to combine results from two different sampling implementations. For each dataset, an equal number of results from each approach were combined into a single dataset, and positional clustering was used to predict binding sites as described above ( Figure 3C ). We measured the average percent change in PPV for each TF on each dataset, and found positional clustering improved combined sampling by 94% compared with 25% and 27% improvement for BioProspector and MotifSampler, respectively. Additionally, clustering combined sampling improved 19 of the 22 datasets evaluated, whereas clustering of BioProspector and MotifSampler results improved 17 and 16 datasets, respectively. Thus, positional clustering is an effective mechanism to integrate results from multiple sampling procedures.
Identification of GABR cis-regulatory sequences As described above in Introduction, identifying functional TFBS in mammals is difficult due in part to inclusion of decoy sequence from long upstream regions and lack of information on co-regulation of genes. Positional clustering, as shown above, is more robust to noisy input than Gibbs sampling alone, and thus may be better suited to identify de novo cis-regulatory elements in mammalian promoters that are coordinately regulated. To test this possibility, we chose seven mammalian GABR promoters (GABRps) whose activity is potentially altered in response to status epilepticus as identified through change in mRNA levels of the gene products (10) . For each set, the initial promoters were analyzed using Gibbs sampling with positional clustering (solid triangles) and without (open triangles). Two Gibbs sampling approaches were applied to each dataset: a Gibbs sampler procedure similar to BioProspector (8) (row A), and MotifSampler (39) (row B). Row C shows the combination of both sampling procedures, along with positional clustering of the combined results. x-axis counts over addition of DSs. We evaluated the positive predictive value of each technique on each dataset, and found positional clustering generally improved the PPV through addition of 100% random DSs. (31, 35) . We also included orthologous rodent promoters in the input sets (45) . Orthologous promoters were included to provide more instances of binding sites in the input set than would be expected by random, allowing for easier detection of the sites. Inclusion of orthologous promoters has the additional effect of selectively amplifying evolutionarily conserved binding sites. Such binding sites are more likely to have major functional roles in the regulation of the GABR receptor. Thus, sensitivity to such sequences is improved at the expense of sensitivity to species-specific binding sites. With this effect in mind, we require all GABR-binding site predictions to exist in orthologous promoters. Since the mechanisms of co-regulation for the seven GABRs are unknown, hypothetical co-regulation models were evaluated by querying all 2 7 possible subsets of the seven GABRps. Clustering results on nucleotide positions and selecting the most frequently occurring positions, we predicted 13 functional TF-binding sites.
Predictions were compared with instances of known binding motifs from TRANSFAC (40) , and 8 of the 13 predictions (61.5%) resembled known binding sites for 10 TFs (Table 1) . Of the 10 TFs, 7 have been identified in the CNS of rodents: SP-1 (46); AP-2, TST-1 (POU3F1), OCT-1 (POU2F1), OLF-1 (47); CP-2 (48); and RREB-1 (49) . Furthermore, previous analyses of GABR promoter regions agree with our predictions that assign putative regulatory roles to SP-1, OCT-1, OLF-1 in the regulation of GABRs (29) . We chose to validate novel motif predictions with EMSAs and functional studies in primary cultured neurons.
EMSA (50) was performed with an excess of cold competitors to define specificity of protein binding in nuclear extracts derived from primary neocortical neurons and fibroblasts (FIBs) kept in culture. As shown in Figures 4-6 , out of six predicted binding sites found upstream of the (a, b, g and d) subunit genes, four (GABRA4, GABRB1, GABRB3 and GABRD) displayed specific binding. In addition to specific binding of neuronal extracts to novel GABRA4 motifs, we have evidence for specific binding using FIB extracts ( Figure 5A and B) , of especial interest given that the expression of GABRs is restricted to the nervous system and repressors such as the RE1-silencing transcription factor (REST) (51, 52) expressed in non-neuronal cells have been implicated in the neural specificity of gene expression.
Clearly, protein binding to DNA does not always necessitate regulatory function. To begin to address the functional Table 1 . Positional clustering based predictions of transcriptional regulatory sequences upstream of GABRs In total, we predict 15 orthologous pairs of regulatory sequences, representing 13 unique sequences. Comparing with known mammalian binding motifs, eight of the predictions show similarity to previously characterized TFBS, as indicated. Where no known binding motif exists, the corresponding in vitro EMSA and functional assay, if applicable, is indicated. Similar predictions are grouped together and aligned by hand. significance of our predicted regulatory motifs, we evaluated the effects of transfecting neurons with double-stranded oligonucleotides containing one of the GABRA4 novel binding motifs (dsA4O), as described above. GABRA4 is especially interesting given that it is regulated by brain derived neurotrophic factor (BDNF) after status epilepticus (31, 53) . Transfection with the dsA4O produced a significant downregulation of GABRA4 gene expression in neocortical neurons as monitored by quantitative real-time RT-PCR with no change after MOCK transfection or transfection with a dsO containing three copies of a cAMP regulatory element (CRE) (Figure 6 ).
How reliable are the binding site predictions returned by Gibbs sampling based TFBS identification algorithms? We began by evaluating the stability of binding site predictions via repeated runs of Gibbs sampling. To quantify the robustness of predictions, we counted the number of Gibbs sampling results at each nucleotide position in the input set ( Figure 1 ) over a large number of repeated trials. We find that the most frequently returned positions better predict TF binding sites than the maximally scoring motifs from Gibbs sampling (Figures 2 and 3 ). Since scoring functions are empirically derived and do not necessarily represent biological reality, the result is not altogether unexpected (17) . However, we find that selecting frequently recurring positions allows filtering of up to 90% of spurious sampling results caused by convergence on biologically uninformative local minima. Positional clustering allows unbiased aggregation of results from different motif widths, thus approximating the width of the binding site 'for free' (54) .
Next we show that positional clustering improves robustness to the addition of DSs (Figures 2 and 3) . Such sequences arise from inclusion of promoter regions in input sets without direct binding to the TF either due to experimental error or computational mis-annotation (17, 22) . In the STE12 example studied, linear regression models indicate our approach will maintain an advantage over traditional Gibbs sampling through addition of up to 150% noise to the original signal (Supplementary Figure S4) . Empirical data, however, show a sharp decrease in improvement close to the addition of 45 DSs, or roughly double the input set ( Figure 2 ). Moreover, evaluations using promoters co-regulated by other TFs Figure 6 . Double-stranded oligonucleotide functional assay for GABRA4 regulation. Primary cultures of rat neocortical neurons were treated with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) alone (Mock) or with DOTAP and phosphothioate oligonucleotides from either a cAMP response element (CRE Decoy) or a sequence from the GABA-A4 promoter predicted using positional clustering (GABA-A4 Decoy) (GTGCACACACACGCCCACCGCGGCTCGGG). mRNA was harvested after 24 h, and real-time RT-PCR was performed with GABA-A4 specific primers. Error bars refer to individual experiments; i.e. different platings of cells from different animals. Data was normalized to rRNA levels, and expressed as relative mRNA levels (GABA-A4/rRNA). Results are shown as mean ± SEM, N ¼ 3, asterisk indicates significantly different from control at the 95% confidence interval. Figure 5 . EMSA of three putative TF binding sites form DNA-protein complexes in neocortical and fibroblast nuclear extracts. Neocortical (NEO) and fibroblast (FIB) nuclear extracts from E18 rat embryos were incubated with three 32 P-radiolabeled probes from human A4 and D receptor subunits. Cold wild-type oligonucleotides were used to define specificity through competition. Cold oligonucleotides were added at 100-fold excess over probe. indicate positional clustering is less likely to improve predictions when Gibbs sampling identifies a correct site in <20% of repetitions ( Figure 3 ). Thus, it is possible the rather simplistic linear model overestimates improvement in robustness beyond what is practically achievable. Moreover, when multiple motifs exist in the input promoters, preliminary evidence suggests positional clustering will uniquely identify a single dominant motif (Supplementary Figure S5) . With further refinement, however, it may be possible to recover subordinate motifs, enabling identification of cis-regulatory modules. In spite of these limitations, using positional clustering of repeated runs, researchers can successfully apply sampling algorithms in identification of functional binding sites in datasets with a significant proportion of noise.
Computational prediction of TF binding in mammalian genomes poses just such a challenge due to increased decoy sequence in large upstream regions (24) . Thus, having established increased robustness to DSs in yeast, we applied our approach to identify potentially unknown GABA A receptor subunit gene regulatory sequences that may participate in the response of the genome to seizure activity. We reasoned that GABA A receptor subunit genes either up-regulated or down-regulated in the animal model of epilepsy would share common binding motifs. Using positional clustering, we predicted 13 TF-binding sites upstream of GABA A receptor subunit genes ( Table 1) . Twelve of our predictions were verified by either comparison to known binding sites or experimental verification using in vitro binding assays. Initially positive experimental results highlight the ability of computational techniques to direct research into transcriptional regulation in mammalian models. As such, our approach may be applicable in the study of other protein complexes in the mammalian proteome.
The reported predictions may enable pharmacologically important downstream research. For example the predicted sites can be used as a starting point for quantifying in vivo effect on downstream transcription; for identifying the TFs bound; and even for the more complex task of understanding the role of each site in determining the relative abundance of GABA A receptor isoforms. Research along these lines may dramatically improve our understanding of GABA A receptor regulation and its role in disease and development. Additionally, a more comprehensive evaluation of the remaining GABA A receptor subunit genes may reveal additional TFbinding sites that uncover the evolutionary significance of g-a-b GABR clusters in the human genome.
Supplementary Data are available at NAR Online.
Charles DeLisi is partially supported by NIH grants A08 POGM66401A and J50 01-130021. Daniel S. Roberts is supported by NIH training grant 2T32 GM00854. Shelley J Russek is supported by NIH/NINDS Grant NS050393. Funding to pay the Open Access publication charges for this article was provided by the Boston University Bioinformatics Program. The Transmissibility of Highly Pathogenic Avian Influenza in Commercial Poultry in Industrialised Countries BACKGROUND: With the increased occurrence of outbreaks of H5N1 worldwide there is concern that the virus could enter commercial poultry farms with severe economic consequences. METHODOLOGY/PRINCIPAL FINDINGS: We analyse data from four recent outbreaks of highly pathogenic avian influenza (HPAI) in commercial poultry to estimate the farm-to-farm reproductive number for HPAI. The reproductive number is a key measure of the transmissibility of HPAI at the farm level because it can be used to evaluate the effectiveness of the control measures. In these outbreaks the mean farm-to-farm reproductive number prior to controls ranged from 1.1 to 2.4, with the maximum farm-based reproductive number in the range 2.2 to 3.2. Enhanced bio-security, movement restrictions and prompt isolation of the infected farms in all four outbreaks substantially reduced the reproductive number, but it remained close to the threshold value 1 necessary to ensure the disease will be eradicated. CONCLUSIONS/SIGNIFICANCE: Our results show that depending on the particular situation in which an outbreak of avian influenza occurs, current controls might not be enough to eradicate the disease, and therefore a close monitoring of the outbreak is required. The method we used for estimating the reproductive number is straightforward to implement and can be used in real-time. It therefore can be a useful tool to inform policy decisions. A new highly pathogenic strain of avian influenza, H5N1, emerged in the poultry markets of Hong Kong in 1997 and subsequently re-emerged in Vietnam in 2003. From this time onwards it has rapidly spread across the globe and is likely to be endemic in poultry in many parts of the world. Although onward transmission to humans at present remains limited, the high case fatality rate in those people that are infected has raised concerns about the impact of a potential human pandemic [1, 2] . Whilst much research and planning is currently underway to contain any outbreak in humans, relatively little is known about the extent of infection in poultry and, in particular, the transmissibility of highly pathogenic avian influenzas between poultry farms. Such understanding is vital if we are to limit the potential for a human pandemic by reducing the extent of infection in poultry, either through movement restrictions, culling or vaccination.
Avian influenza occurs naturally in wild water fowl, usually in a low-pathogenic version (LPAI) causing no symptoms or only mild disease. However, in poultry some strains also occur in a highlypathogenic form (HPAI) and result in a devastating disease which can kill up to 100% of infected birds within 48 hours, and is highly transmissible between individual birds [3, 4] . Transmission between flocks kept at different farms is thought to occur via movement of infected birds, equipment or staff, with current evidence suggesting that air-borne transmission over long distances is rare [5] . There has been an increase in HPAI outbreaks over the past ten years [4] . In addition to their implications for human health, these outbreaks also have severe economic consequences for the affected countries. Typical control measures for HPAI in poultry comprise of swift isolation and culling of flocks on infected farms, the restriction of movements between farms, increased bio-security, and the culling of flocks in the vicinity of infected farms to deplete the susceptible poultry population. Vaccination, if coupled with a strict surveillance programme, has also been demonstrated to be effective in reducing the risk of further outbreaks [5, 6] .
The reproductive number for infected poultry farms, defined as the average number of farms that each original infected farm infects at the start of an outbreak (i.e., when most farms are susceptible), is an important measure of the overall transmissibility of the virus in a population. It determines whether a self-sustaining epidemic will occur and, more importantly, yields a tool to assess the effectiveness of control measures. If, on average, at any point in time, each infected farm infects more than one further farm, the epidemic will continue. However, if on average, each infected farm infects less than one further farm, the epidemic will decline and the intervention measures applied at that point can be interpreted as being sufficient to control the outbreak.
In this paper, we analyse published data from four outbreaks of HPAI in commercial poultry in industrialised countries to estimate the farm-to-farm reproductive number of HPAI to explore the extent to which different intervention measures implemented during these outbreaks reduce the reproductive number. The results from our analyses can be used to inform current planning for an outbreak of HPAI in similar commercial poultry sectors.
We analyse data from three different outbreaks of HPAI that occurred in the past 8 years in industrialised countries: an outbreak of H7N1 in Italy in 1999/2000, an outbreak of H7N7 in the Netherlands in 2003, that will be treated as two distinct outbreaks due to geographic separation, and an outbreak of H7N3 in Canada in 2004. Figure 1 shows the time course of these outbreaks. Brief details of these outbreaks are given below.
2.1.1. Outbreak of H7N1 in Italy in 1999/2000 Northern Italy has experienced a number of avian influenza outbreaks from 1997 onwards [6] [7] [8] [9] [10] . These all occurred in an extremely dense poultry production area (up to 70 000 birds/km 2 ) and involved a significant number of farms keeping turkeys, a species known from experimental studies to be highly susceptible to avian influenza [11] . Furthermore, in this region there are many wetlands and resting sites for migratory waterfowl in close proximity to the poultry industry, which likely lead to multiple introductions from the wild bird host.
In March 1999, H7N1 LPAI was detected in a farm keeping turkeys [6, 7, 9] . This outbreak was not controlled rigorously and so AI continued to circulate. In December, a case of H7N1 HPAI was found and strict control measures were implemented, including culling of affected flocks, movement restrictions and pre-emptive slaughter of flocks deemed at high risk. However, due to LPAI circulating at the time, the confirmation of HPAI was delayed, and so the disease had already infected a number of farms by the time control measures were enforced. This resulted in an HPAI epidemic affecting a total of 413 flocks. The LPAI/HPAI epidemic lasted until April 2000, and involved a total of over 13 million birds.
The H7N7 epidemic in the Netherlands in 2003 affected a total of 255 commercial flocks in two distinct geographical and temporal clusters. The outbreak was situated in the Gelderse Vallei, the densest poultry production area in the Netherlands, in which over 10 million birds are kept in 984 flocks with a density of 4 flocks/ km 2 [12, 13] . Two months into the outbreak the infection passed to Limburg, another very dense poultry production area, where it continued to spread.
In the Gelderse Vallei, HPAI was confirmed on 28 th February, 6 days after clinical signs appeared in the first infected farm, and between March and early April, a total of 212 farms were infected. In Limburg, a further 43 farms were infected between April and early May.
A number of control policies were enforced in several stages. From 1 st March all movement of poultry and poultry products was banned, the tracing of dangerous contacts was initiated and reinforcement of strict bio-security measures was implemented. Two days later, from 3 rd March, culling of infected farms was initiated. On 5 th March the additional pre-emptive culling of farms within a 1 km radius of any infected farms was put in place. This was further extended to a 10 km radius for turkey flocks and 3 km radius for all other flocks on 7 th April [14] . However, these control measures were insufficient and it is hypothesized that the epidemics in both areas finally came to a halt due to depletion of susceptible flocks, after the culling of 30 million birds in 1,255 commercial and 17,421 hobby flocks [15] . During this epidemic, 89 human infections were also reported, most of whom presented with conjunctivitis or mild influenza-like illness. One person died from their infection. There was also evidence of limited human-to-human transmission [16, 17] . Following detection of the index case, a broiler breeder farm, a surveillance program was initiated, which led to the detection of the second case on 11 th March. The Fraser Valley south of the River Fraser was declared a Control Area, restricting movements of birds, bird products and equipment. Furthermore, active surveillance was undertaken in a High Risk Region (HRR, 5 km around the index case) and in flocks deemed dangerous contacts in a Surveillance Region (SR, 10km around index case).
After the identification of 7 infected farms, all birds within the HRR were slaughtered from 24 th March onwards, but as this failed to stop transmission, on 5 th April it was decided to depopulate the whole Control Area, containing approximately 19 million birds.
Infected farms were located mainly in three distinct local clusters within the Control Area. It is hypothesized that long distance spread between these clusters was due to bird, equipment or people movement, whereas once a farm in a densely populated area became infected, where sheds are sometimes within a few hundred metres from each other, the virus spread via dust or feather debris.
Assuming homogeneous mixing, that all farms are equally infectious, and that the time-dependence of infectiousness from the point of infection is identical, we can estimate both the distribution of generation time intervals and the reproductive numbers of individual farms from the time-course of an epidemic using the following method [19] .
Suppose there are N infected farms, labelled i = 1,…,N, and ordered so that the first k farms are those that contracted their infection from outside sources. The infection times of these farms are t = (t 1 ,…,t N ) such that t 1 = … = t k = 0. Under the simplest model that neglects any differences between farms, spatial locations, etc., the probability that farm j[fkz1,:::,Ng was infected by farm i[f1,:::Ng is
where the generation time distribution has density w(T;h), which is defined to be 0 if T,0 and indexed by unknown parameter vector h. Under the above assumptions, the number of farms infected by farm i (i.e. the reproductive number of farm i) in the outbreak can be represented as an outcome from a random variable
that is, a sum of Bernoulli random variables, which has expected value
Now denote the 'infection tree' by v = (v k+1 ,…,v N ), defined such that v j = i if farm j was infected by farm i. Under (1), the likelihood for h when v and t are observed is
But as v is unobserved we sum over all possible infection trees to obtain the 'integrated likelihood'
where S j = {1,…,N}\{j} is the set of all indices other than j. The integrated likelihood is a genuine likelihood (up to a multiplicative constant) permitting valid inferences about h conditional on outbreak size N.
time The maximum likelihood (ML) estimateĥ is obtained by minimizing twice the negative log-likelihood
More details on how ML estimation is performed are given in Appendix S1. We assume the generation times T = t j 2t i are Weibull distributed, with density
and so h = (k, g). Minimization was performed using the Downhill Simplex method [20] , the code used for these calculations is given in Code S1 and Code S2; to ensure the global minimum is reached, the procedure was run from 10000 different starting points. We further investigated whether the generation time distribution changed after control measures were introduced. To do this, we extended the above model to allow for distinct parameters for the generation times before and after controls, h pre and h post , see Code S3 and Code S4. The improvement in fit compared to the original model was assessed using a likelihood ratio test.
2.2.2. Estimation of the reproductive number Givenĥ we can estimate the mean and variance of the generation time distribution. Moreover, we can estimate the reproductive number for each infected farm via equation (3), and the mean reproduction number for any subset of infected farms.
To calculate confidence intervals for the reproductive number we use an approximation of the parametric bootstrap percentile interval method [21] . To obtain proper parametric bootstrap intervals would involve generating infection times and trees according to the underlying epidemic model, which we do not wish to specify completely. Instead, the following two-step approximation is used, which we propose will be a good approximation for large N . These two steps approximate generating realisations from the underlying epidemic process. First, we take bootstrap samples of parameter values from the conventional approximation to the sampling distribution of the ML estimator,
that is, from a bivariate normal distribution with mean (k k,ĝ g) and
variance-covariance matrix V based on the inverse of the observed information matrix (see Appendix S1 for further details, the code used to generate the bootstrap sample is given in Code S5 and Code S6). This first stage can be loosely thought of as sampling the mean behaviour for a subgroup of possible outbreaks. To allow for variability within each subgroup, stage two involves fixing (k * ,g * )and independently generating reproductive numbers for each farm according to model (2) . Steps one and two together give R * = {R * i :i21,…,N}, an approximate bootstrap sample of the reproductive numbers for each farm. Here, 1000 samples of (k,g)pairs were drawn, and 500 sets of reproductive numbers generated for each. Finally, the approximate 95% CI for each R i is given by the 2.5 th and 97.5 th percentiles of the bootstrap distribution. The second stage of the calculation of the approximate CIs was done using Code S7 and Code S8.
The generation time is defined as the time between the infection of a farm and the time at which the farm passes on infection to another farm. We have assumed the generation time distribution is Weibull. While this is a biologically plausible choice, we cannot verify it empirically. As such, we assessed robustness to this choice using other plausible choices such as the gamma distribution (results not shown). However, the following results under these alternatives did not differ substantively from those shown below. Figure 2 shows the estimated generation time distribution for the four different outbreaks; the parameter estimates are detailed in Table 1 . The estimates, and hence the distribution, differs substantially between the outbreaks. It could be hypothesised that the generation time would shorten after measures were put in place to isolate the infected farms. However, allowing for different generation time distributions for the pre-and post-control time periods did not significantly improve the model fit. Table 2 ) with upper 95% bounds in the range 1.5-3.6.
The impact of control measures on the effective reproductive number can be clearly seen in all four outbreaks. For the outbreak in Italy (Figure 3a) , their introduction rapidly reduced the reproductive number, hovering around the threshold of 1 for the next few months before finally dying out. In British Columbia (Figure 3b ) controls were put in place after detection of the first IP. However our estimates of the reproductive number remain high until 24 th March when the decision was taken to cull the whole high risk region. Our estimates show that the control activities following this decision were effective in reducing the reproductive number to below one.
Our results show that the situation in the Gelderse Vallei, The Netherlands (Figure 3c ) differed in that the initial control measures failed to bring the reproductive number reliably below 1, and the epidemic only died out at the end of March after the depletion of susceptible flocks in the affected area [15] . The same controls were applied to the Limburg epidemic but our estimates show in this case the reproductive number was reduced to just below 1 (Figure 3d) , and so potentially effective in controlling the outbreak. However, the end of the epidemic in late April coincided here too with the depletion of susceptible flocks and therefore it is possible that the epidemic would have taken substantially longer to control had there been a larger pool of susceptible flocks in the area.
Our estimates of the farm-to-farm reproductive number prior to interventions for HPAI are in the range 1.1 to 2.4 and were remarkably consistent across the four datasets. However, these estimates are substantially lower than those previously reported for the Dutch epidemic. Prior to the implementation of control measures we obtained estimates of 1.1 (95% CI 0.9-1.5) in the Gelderse Vallei and 1.9, (95% CI 1.0-3.0) in Limburg which are significantly lower than those previously reported for the same outbreak prior to notification (6.5 (95% CI 3.1-9.9) for the epidemic in the Gelderse Vallei). However, as demonstrated in Figure 3c , there was substantial variation in our estimates of individual reproductive numbers prior to interventions. In addition, in the previous study, the generation time was not estimated directly from the data but based on observational and experimental data on the course of infection in the farms. Our estimate of the generation time for this region is of the order of 2 days, whereas the values previously assumed for the infectious period were defined per flock as the time between detection and culling plus an additional 4 days to cover the time before the infection was detected but during which birds were infectious. The previously published estimates therefore assumed a much longer mean generation time and this could also lead to a higher estimated reproductive number. Our results showed substantial differences between the estimated generation time distributions for the different outbreaks. Whilst much is known from experimental studies on the course of infection in individual birds [11, [22] [23] [24] , estimates of the generation time at the farm-level are more difficult to obtain. Although it is perhaps surprising that the generation time differs between the outbreaks, it is plausible that such differences could arise because of variation in farming practices or in the contact patterns between farms. In addition, the latent and infectious periods determining the generation time may differ by the strain of HPAI. Alternatively, the estimates may be biased because of assumptions made in the method. In particular, we assumed that the datasets were complete (and thus that all infected farms were detected) and that only the first farm in each outbreak was infected from an outside source. If, however, further undetected farms had played a role in transmission, this would substantially alter the estimates of the generation time and the reproductive number, particularly if these infections occurred towards the beginning or end of the epidemics where overall cases are sparser.
All of the outbreaks investigated here occurred within dense poultry farming areas and hence were difficult to control. The control policies implemented in the different outbreaks were similar, comprising strict bio-security measures for movement of poultry and poultry products, swift culling of infected flocks, and if these failed to control the epidemics, additional pre-emptive culling of flocks in the neighbourhood of any infected farms. Our results demonstrate that the bio-security measures, movement restrictions and culling of infected farms, all of which were initiated early on in the outbreaks, did have an effect but for all four outbreaks only reduced the reproductive number to close to the threshold value of 1. The additional pre-emptive culling of flocks and de-population of the areas was needed to fully control the outbreaks. Current contingency plans for HPAI outbreaks in Europe focus on the former set of control measures to contain any outbreak [25] . Whilst differences in farming practices between countries mean that it is difficult to predict whether these measures will be sufficient for a new outbreak, our analyses suggest that additional interventions may well be required. Close monitoring of outbreaks, coupled with quantitative estimation of the reproductive number, is therefore needed to ensure that such additional measures, if required, are promptly implemented.
The method used here to estimate the reproductive number and generation time parameters is an extension of that developed by Wallinga and Teunis [19] for the SARS-epidemic. This method requires only time-series data for an outbreak, and is therefore easily applied even in real-time. Technically, appropriate censoring terms should be added to the likelihood to account for infection times yet to occur, but a straightforward application of the method as described here will give estimates unbiased in an asymptotic sense. If data on the spatial location of infected farms are also available, this information can easily be incorporated to estimate the spatial transmission kernel and improve the estimation of the reproductive numbers. Such an approach was successfully applied to the Foot-and-Mouth epidemic in the UK in 2001 [26] . Further work is required to explicitly incorporate missing data, as this is likely to have a strong influence on the estimates of both the generation time and the reproductive number. Such methods are of particular importance to estimating the reproductive number for outbreaks of HPAI in Asia in which, with high general levels of poultry mortality, cases are likely to be less well documented.
Appendix S1 Code S1 Source code for the maximum likelihood estimation of the parameters of the generation time distribution, shown in Figure 2 and Table 1 Code S7 Source code for estimating R0 and the confidence intervals based on the uncertainties inherent in the estimation procedure and the uncertainty of the generation time distribution, shown in Figure 3 .  The influenza pandemic preparedness planning tool InfluSim BACKGROUND: Planning public health responses against pandemic influenza relies on predictive models by which the impact of different intervention strategies can be evaluated. Research has to date rather focused on producing predictions for certain localities or under specific conditions, than on designing a publicly available planning tool which can be applied by public health administrations. Here, we provide such a tool which is reproducible by an explicitly formulated structure and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. RESULTS: InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java, operates platform independent and can be executed on regular desktop computers. CONCLUSION: InfluSim is an online available software which efficiently assists public health planners in designing optimal interventions against pandemic influenza. It can reproduce the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability. Preparedness against pandemic influenza has become a high priority public health issue and many countries that have pandemic preparedness plans [1] . For the design of such plans, mathematical models and computer simulations play an essential role because they allow to predict and compare the effects of different intervention strategies [2] . The outstanding significance of the tools for purposes of intervention optimization is limited by the fact that they cannot maximize realism, generality and precision at the same time [3] . Public health planners, on the other hand, wish to have an optimal combination of these properties, because they need to formulate intervention strategies which can be generalized into recommendations, but are sufficiently realistic and precise to satisfy public health requirements.
Published influenza models which came into application, are represented by two extremes: generalized but oversimplified models without dynamic structure which are publicly available (e.g. [4] ), and complex computer simulations which are specifically adjusted to real conditions and/or are not publicly available (e.g. [5, 6] ). The complexity of the latter simulations, however, is not necessary for a reliable description of infection dynamics in large populations [7] . A minimum requirement for a pandemic influenza planning tool is a dynamic modelling structure which allows investigation of time-dependent variables like incidence, height of the epidemic peak, antiviral availability etc. The tool should, on the other hand, be adjustable to local conditions to adequately support the pandemic preparedness plans of different countries which involve considerably different assumptions (Table 1) .
Here we describe a publicly available influenza pandemic preparedness planning tool [8] which is designed to meet the requirements in preparedness planning. It is based on an explicitly formulated dynamic system which allows addressing time-dependent factors. It is sufficiently flexible to evaluate the impact of most candidate interventions and to consider local conditions like demographic and economic factors, contact patterns or constraints within the public health system. In subsequent papers we will also provide examples and applications of this model for various interventions, like antiviral treatment and social distancing measures.
The model is based on a system of 1,081 differential equations which extend the classic SEIR model. Demographic parameters reflect the situation in Germany in 2005, but can be adjusted to other countries. Epidemiologic and clinic values were taken from the literature (see Tables 1,  2 , 3, 4, 5, 6 and the sources quoted there). Pre-set values can be varied by sliders and input fields to make different assumptions on the transmissibility and clinical severity of a new pandemic strain, to change the costs connected to medical treatment or work loss, or to simply apply the simulation to different demographic settings. Model properties can be summarized as follows. The mathematical formulation of this model is presented in detail in the online supporting material. The corresponding source code, programmed in Java, and further information can be downloaded from [8] .
According to the German National Pandemic Preparedness Plan [9] , the total population is divided in age classes, each of which is subdivided into individuals of low and high risk ( Table 2) . Transmission between these age classes is based on a contact matrix (Table 3) which is scaled such that the model with standard parameter values yields a given basic reproduction number R0. Values for the R0 associated with an influenza strain with pandemic potential are suggested to lie between 2 and 3 [10] . This value is higher than the effective reproduction number which has been estimated to be slightly lower than 2 [11, 12] . As a standard parameter, we use R0 = 2.5 which means that cases infect on average 2.5 individuals if everybody is susceptible and if no interventions are performed.
Susceptible individuals who become infected, incubate the infection, then become fully contagious and finally develop protective immunity (Table 4) . A fraction of cases remains asymptomatic; others become moderately sick or clinically ill (i.e. they need medical help). Depending on the combination of age and risk group, a fraction of the clinically ill cases needs to be hospitalized, and an agedependent fraction of hospitalized cases may die from the disease ( Table 5 ). This partitioning of the cases into four categories allows combining the realistic description of the transmission dynamics with an easy calculation of the resources consumed during an outbreak. The degree and duration of contagiousness of a patient depend on the course of the disease; the latter furthermore depends on the age of the patient (Table 5) . Passing through the incubation and contagious period is modelled in several stages which allows for realistic distributions of the sojourn times ( Table 4 ). The last two stages of the incubation period are used as early infectious period during which the patient can already spread the disease. Infectiousness is highest after onset of symptoms and thereafter declines geometrically (Table 6 ). Clinically ill patients seek medical help on average one day after onset of symptoms. Very sick patients are advised to withdraw to their home until their disease is over, whereas extremely sick patients need to be hospitalized and may die from the disease (Table 4) . After the end of their contagious period, clinically ill patients go through a convalescent period before they can resume their ordinary life and go back to work (Table 4) .
We provide some examples of model output of InfluSim [8] , version 2.0, by means of four sensitivity analyses; further investigations will be presented elsewhere. Figure 1 shows the graphical user interface of the software which is divided into input and output windows. The user may set new values in the input fields or move sliders to almost simultaneously obtain new results for the course of an epidemic in a given population. Figures 2A and 2B show pandemic waves which result from varying the basic reproduction number from 1.5 to 4.0. Using the standard parameter values as given in Tables 2, 3 , 4, 5, 6 and omitting all interventions in a town of 100,000 inhabitants results in a pandemic wave which lasts for about ten weeks (Figure 2A , with R 0 = 2.5). The peak of the pandemic wave is reached after six to seven weeks, with a daily incidence of up to 2,340 influenza patients seeking medical help, with up to 280 hospital beds occupied by influenza cases and with up to 14,000 out of 60,000 working adults unable to go to work because of illness or convalescence. These results depend on the assumptions concerning the yet unknown contagiousness and pathogenicity of the virus. Figures 2C and 2D show how the shape of the curves depends on the course of contagiousness: the pandemic wave proceeds relative slowly if the contagiousness does not change during the infectious period (x 50 = 50%), but proceeds quickly if the contagiousness is highest after onset of symptoms and decreases thereafter (x 50 > 50%).
The influenza pandemic preparedness planning tool InfluSim stands between simple spreadsheet models and sophisticated stochastic computer simulations. It describes a pandemic wave within a homogeneously mixing population like a town or city, but surprisingly produces the same dynamics as individual-based simulations which explicitly consider geographic spread through the US (cf. [6] and [5] with Figure 2 using R 0 = 2). Similar observations were made with a simple deterministic compartmental model [7] . Stochastic models are known to behave quasi-deterministically when the simulated population becomes very large.
A further reason for the congruence of complex stochastic and simple deterministic models must lie in the incredi-bly quick way in which pandemic influenza spreads geographically. Unless being controlled at the place of origin [12, 13] , a pandemic starting in a far-off country will lead to multiple introductions [14] into the large industrialized nations where it can be expected to quickly spread to neighbouring towns and to rural areas. The large populations which have to be considered susceptible to a pandemic virus and the quick geographic spread tend to diminish the differences between the results of sophisticated individual-based and simple deterministic models.
However, a deterministic model like InfluSim cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities. Examples are: (i) a geographically limited spread and fairly effective control measures can imply that the epidemic affects only a small population and thus, may be strongly influenced by stochastic events [15] [16] [17] ; (ii) transmission which predominantly occurs in households or hospitals, or which is driven by other substantial features of the contact network is not in agreement with the assumption of homogeneous mixing in the deterministic model cannot reliably predict the spread of infection [18] [19] [20] [21] [22] [23] . In particular, (iii) super-spreading events can substantially change the course of an epidemic compared to the deterministic prediction [24] [25] [26] [27] . Apart from such factors, the predictability of intervention success is generally subject to uncertainties in the choice of parameter values, Assumed scenarios and outcomes of pandemic preparedness plans. * Gross attack rate (i.e. clinically ill and moderately ill cases). A population of N = 100,000 inhabitants of Germany is subdivided according to age a and risk category r. We assume that all age groups are fully susceptible at begin of the outbreak. A fraction of F a = 6% of all children (age < 20 years) are regarded as being under high risk (r = r 1 ) after an influenza infection whereby the remaining 94% are under low risk (r = r 2 ). The high risk fractions of working adults (ages 20-59) and elderly (ages 60+) are F a = 14% and F a = 47%, respectively. Source: [9] demanding additional efforts like Bayesian approaches [28] to evaluate the reliability of predictions [29] .
Pandemic preparedness plans must consider constraints and capacities of locally operating public health systems. The time-dependent solutions of InfluSim allow assessing peak values of the relevant variables, such as outpatients, hospitalizations and deaths. Various interventions may be combined to find optimal ways to reduce the total number of cases, to lower the peak values or to delay the peak, hoping that at least part of the population may benefit from a newly developed vaccine.
Special care was taken when implementing a variety of pharmaceutical and non-pharmaceutical interventions which will be discussed in subsequent papers. Despite its comprehensible structure, the model does not suffer from over-simplifications common to usual compartment models. Instead of implicitly using exponentially distributed sojourn times, we have implemented realistically distributed delays. For example, the model considers that individuals may transmit infection before onset of symptoms, and that some cases may remain asymptomatic, but still infecting others. Such features have serious implications for the success of targeted control measures.
InfluSim is freely accessible, runs on a regular desktop computer and produces results within a second after changing parameter values. The user-friendly interface and the ease at which results can be generated make this program a useful public health planning tool. Although we have taken care of providing a bug-free program, including the source code, the user is encouraged to treat results with due caution, to test it, and to participate in bug-reports and discussions on the open-source platform [30] which also provides regular updates of InfluSim. 
The author(s) declare that they have no competing interests.
ME developed the model, MS designed the software, HPD wrote the manuscript and SOB formulated the public The who-acquires-infection-from-whom matrix shows the frequency of contacts (per week per person) between different age classes.
Source: [38] . Distribution of sojourn times (the last two stages of the latent period are used as early infectious period with an average duration of D L = 0.5 days). Sources: A [11] , B [39, 40] , C assumed, D [41] health requirements of the software. All authors read and approved the final manuscript.
Susceptible individuals S a, r are infected at a rate λ a (t) which depends on their age a and on time t. Infected individuals, E a, r , incubate the infection for a mean duration D E . To obtain a realistic distribution of this duration, the incubation period is modelled in n stages so that progression from one stage to the next one occurs at rate δ = n/D E .
The last l incubation stages are regarded as early infectious period during which patients may already spread the infection (this accounts for an average time of lD E /n for the "early infectious period" which is about half a day for the standard set of parameters). After passing through the last incubation stage, infected individuals become fully contagious and a fraction of them develops clinical symptoms. The course of disease depends on the age a of the infected individual and on the risk category r to which he or she belongs: a fraction c a, r (A) becomes asymptomatic (A a ), a fraction c a, r (M) becomes moderately sick (M a ), a fraction c a, r (V) becomes very sick (V a ) and the remaining fraction c a, r (X) becomes extremely sick (X a ) and need hospitalization (i.e., c a, r (A) + c a, r (M) + c a, r (V) + c a, r (X) = 1 for each combination of a and r). ) . A fraction f V (t) of all severe and a fraction f X (t) of all extremely severe cases who visit the doctor within D T days after onset of symptoms are offered antiviral treatment, given that its supply has not yet been exhausted. As our model does not explicitly consider the age of the disease (which would demand partial differential equations), we use the contagious stages to measure time since onset and allow for treatment up to stage m a, T Sources: Contagiousness of asymptomatic cases: [11] ; degree of contagiousness during the early infectious period and equality of the contagiousness of moderately and severely sick cases: assumed. Independent of age a and risk group r, a fraction c a, r (A) = 33% of infections result in asymptomatic cases, a fraction c a, r (M) = 33.5% become moderately sick and the remaining fraction develops severe disease. An age-and risk-dependent fraction h a, r of untreated patients with severe disease needs hospitalization. An age-dependent fraction d a of hospitalized cases dies. Sources: fraction of asymptomatic cases: [11] ; 50% of symptomatic cases see a doctor: [9] ; hospitalizations per severe case: [9] ; case fatality of hospitalized, but untreated patients calculated from [4] .
(see below for details). This imposes some variability to the maximum time until which treatment can be given, which may even improve the realism of the model with respect to real-life scenarios. Antiviral treatment reduces the patients' contagiousness by f I percent and it reduces hospitalization and death by f H percent. Extremely sick patients, whose hospitalization is prevented by treatment, are sent home and join the group of treated very sick patients(W a, T ). The remaining duration of disease and contagiousness of treated cases is reduced by f D percent so that their rate of progressing from one stage to the next has To obtain a realistic distribution of this sojourn time, convalescence is modelled in j stages so that progression from one stage to the next occurs at rate ρ = j/D C . Fully recovered patients who have passed through their last stage of convalescence join the group of healthy immunes I; working adults will go back to work. Further interventions, describing the reduction of contacts, will be discussed after the presentation of the differential equations.
InfluSim user interface Figure 1 InfluSim user interface. x 50 = 95% means that 95% of the cumulative contagiousness is concentrated during the first half of the contagious period, see Table 6 ). D: Cumulative number of deaths for values of x 50 as in C. All other parameters as listed in Tables 2-6 . Hospitalized, but untreated cases
Contact matrix For the mixing of the age classes, we employ a whoacquires-infection-from whom matrix which gives the relative frequency of contacts of infective individuals of age a i with other people of age a s . In this paper, we assume bi-directional contacts (e.g. children have the same total number of contacts with adults as adults with children). Multiplication of this matrix with an appropriate constant scaling factor κ (see below)
results in the matrix of crude contact rates .
In the absence of interventions, we have to multiply these contact rates with the contagiousness factors b L , b A , b M and b V to obtain the effective contact rates:
during the early infectious period, of asymptomatic cases, of moderately sick cases, of (untreated) very sick cases.
To assess the effect of day care centre and school closing on the transmission of an infectious disease, we have to first make an assumption on what fraction r sch of the contacts among healthy children who are in the same age class occurs in day care centres and schools. The contact rates between very sick or hospitalized children (who do not attend day care centre or school) and other children need, therefore, be reduced to (contact rate between healthy and very sick children in the same age class, i.e. a i = a s ).
As very sick children have to be taken care of by adults at home or in hospital, their contact rate to adults increases by a factor F HC (contact rate between very sick children of age a i and adults of age a s ).
Contacts between very sick children and other children in a higher or lower age class remain unchanged: (contact rate between healthy children of age a s and very sick children of a different age a i ).
Closing day care centres and schools at time t will not necessarily prevent all the contacts that would have happened with other children. During the closing of schools and day care centres, the contact rates between susceptible children of age a s and infected children of age a i who are in their late incubation period ( ), who are asymptomatic ( ), or who are moderately sick ( ) are reduced by the factor r sch if the children are in the same age class:
where 1 sch (t) is a function which indicates when schools and day care centres are opened or closed: 
,..., While day care centres and schools are closed, children (age a i ) need adult supervision at home. Their contact with susceptible adults (age a s ) increases by the "child care factor" F CC :
Child care at home also increases the exposure of healthy children (age a s ) to contagious adults (age a i ):
Cancelling mass gathering events effects only the contacts of adults who are healthy enough to attend such events. Assuming that such an intervention at time t reduces contacts by a fraction r mass , we get for all contacts between susceptible adults of age a s and infectious adults of age a i the following contact rates:
where 1 mass (t) is a function which indicates when mass gathering events are possible or when they are closed:
As contacts with adults who are too sick to attend such mass gathering events cannot be prevented by this measure it is .
During some time in the epidemic, the general population may effectively reduce contacts which can be a result of wearing facial masks, increasing "social distance", adopting improved measures of "respiratory hygiene" or simply of a general change in behaviour. This will be implemented in the program by reducing the contacts of susceptible individuals at that time t by factor r gen (t 
while mass gathering events are forbidden while m mass gathering events are allowed. while the population reduces their contacts while the population behaves as usual.
The contact rates of cases in the late incubation period and that of asymptomatic cases remain unchanged:
for infected individuals in the late incubation period, for asymptomatic cases.
To allow for a contagiousness which changes over the course of disease, we multiply each contact rate with a weighting factor whereby k is the stage of contagiousness. This leads to the following contact rates:
for asymptomatic cases in For x = 1, contagiousness is equally high in all stages; for x = 0, only the first stage is contagious; for 0 <x < 1, the contagiousness decreases in a geometric procession. We make the simplifying assumption that contagiousness does not change during the late incubation period for cases in stage k = n -l,..,n of the incubation period.
At time t = 0 and in the absence of interventions, the next generation matrix has the following elements where is the fraction of untreated extremely severe cases who die from the disease (see below for details). The dominant eigenvalue of this matrix is called the basic reproduction number R 0 . If κ (which determines the value of the contact rates ) is given, the eigenvectors of this matrix can numerically be calculated. The user-specified value of R 0 is now used to determine numerically the scaling factor κ. Let be the eigenvector which has the largest eigenvalue R 0 . 
) ) − 
Using the user-specified numbers of people N a in the age classes and the fractions F a of people under high risk within each age class (Table 2) , we obtain the initial population sizes according to age and risk class: Using these initial values, the set of differential equations is solved numerically with a Runge-Kutta method with step-size control. 
if and in treatment window otherwise 0 ⎩ ⎩ Immune pathways and defence mechanisms in honey bees Apis mellifera Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens. While evident in social organisms ranging from humans to birds (Brown & Brown, 2004; Masuda et al ., 2004) , the impacts of sociality on disease are especially vivid within social insect colonies. Here, typically thousands of individuals interact in close quarters, at densities far exceeding those of even the most crowded vertebrate social groups (Wilson, 1971) . This density, coupled with a relatively homeostatic nest environment and the presence of stored resources, makes social insects attractive targets for disease agents (Schmid-Hempel, 1998) . As expected based on their parasite and pathogen pressures, social insects have evolved both individual and group strategies to combat disease. Grooming, nest hygiene and other behavioural traits found throughout the social insects can reduce the impacts of pathogenic bacteria, fungi and parasitic mites. For example, 'hygienic behaviour' first described for honey bees (Rothenbuhler, 1964) is now a classical example of a social defence, whereby workers identify and remove infected larvae from among the healthy brood (Spivak & Reuter, 2001) . Other defences enabled by sociality include the construction of nests from antimicrobial materials (Christe et al ., 2003) , the raising of offspring in sterile nurseries (Burgett, 1997) , social 'fever' in response to disease (Starks et al ., 2000) , transference of immune traits (Traniello et al ., 2002; Sadd et al ., 2005) , and heightened risk-taking by infected individuals (Schmid-Hempel, 2005) . Like most eukaryotes, colony members also possess individual defences, including immune responses toward disease agents (Casteels-Josson et al ., 1994; Evans, 2004) . The recent sequencing of the honey bee genome (Honey Bee Genome Sequencing Consortium, 2006) allows the first global analysis of immune components in honey bees, and the second opportunity (after humans) to use genomic insights to better understand disease resistance in a highly social organism.
Insects have diverse mechanisms to combat infection by pathogens. Many insects are protected by a layer of antimicrobial secretions on their exterior, and by a gut environment that is hostile to pathogens. When pathogens move beyond these defences, the epithelium is often sufficient to stop further progress. Should pathogens defeat the morphological defences of insects, they are often met by efficient cellular and humoral immune defences. Insect immunity shows many parallels to the innate immune responses of humans and other vertebrates, involving a diverse set of actions including the secretion of antimicrobial peptides, phagocytosis, melanization and the enzymatic degradation of pathogens (Hoffmann, 2003; Hultmark, 2003) . Further, insect immune pathways share both an overall architecture and specific orthologous components with the innate immune system of vertebrates (Beutler, 2004) . This suggests both a shared root for these immune pathways and selection to conserve many components over hundreds of millions of years.
In the first part of this paper, we propose honey bee models for four non-autonomous pathways implicated in inducible host defence, Toll, Imd, Janus kinase (JAK)/STAT and JNK (Boutros et al ., 2002) , based primarily on extensive searches for orthologues to well-studied fruit fly, mosquito and moth species. While these pathways engage in cross-talk and can direct some of the same immune effectors, they have well-defined structures and interaction sets, and are best tackled as individual entities. Most honey bee components for these pathways remain to be validated by functional tests, yet we feel that the presented models serve two important purposes. First, they point toward the most likely orthologues involved with all stages of the immune response, thus setting the stage for postgenomic functional work on honey bee immunity. Second, the models themselves show intriguing differences between species for these canonical immune pathways with respect to gene losses and duplications.
Next, we show that many immune-gene families in bees appear to be reduced in number, when compared to Drosophila and Anopheles . While genome-wide analyses of the honey bee have identified many gene families with reduced diversity (Honey Bee Genome Sequencing Consortium, 2006) , such reductions appear to be especially pervasive in the immune system. These reductions hold for each stage of immunity, from recognition and signalling to immune effectors. We couple gene-family data with data on specific orthologues to test five hypotheses:
(1) missing genes are not represented in the current draft honey bee genome assembly but are present in the genome; (2) immune-related genes in the bee have diverged especially quickly at the sequence level and, as such, have escaped annotation based on sequence similarity to other species; (3) honey bees enact immune responses using pathways and/or components not currently identified as immune players in other insects; (4) honey bees are targeted by a small set of coevolved pathogens and their immune systems are thereby tuned to these pathogens at the expense of being responsive to a wider range of threats; and (5) 'social' defences and barriers in honey bee colonies are effective in reducing pathogen pressure and, as such, bees are not as reliant as other insects on individual immune responses.
Honey bees possess apparent orthologues for the core members of each of the four pathways implicated in immunity (Figs 1 and 2, Supplementary Material Table S1 ) and precise 1 : 1 : 1 orthology between honey bees and the flies Drosophila melanogaster and Anopheles gambiae is evident for most pathway members, especially for the intracellular components. Of the dozens of described actors in four signalling pathways predicted to play a role in insect immunity, only one protein appears to be completely absent in the bee genome: the ligand unpaired from the JAK/STAT pathway. The presence of the JAK/STAT cytokine receptor domeless and all other members of this pathway (Fig. 2) suggest that JAK/STAT remains functional in honey bees and is triggered by a currently unrecognized ligand.
Insect Toll and the Toll-like receptors (TLRs) are transmembrane signal transducing proteins that play critical roles in both immunity and development. They are orthologous to mammalian TLRs, all of which have been implicated in immunity (Beutler, 2004) . In Drosophila , the Toll signalling pathway is enacted when the cytokine-like molecule Spaetzle binds to the extracellular domain of the transmembrane receptor Toll. The Drosophila genome encodes a family of six Spaetzle-related molecules, that are believed to function as ligands for the nine Drosophila Toll receptors (Parker et al ., 2001) . Two plausible Spaetzle orthologues are evident in the bee genome (GB15688 and GB13503; Fig. 1 ), and functional tests will be needed to determine which act as Toll-binding cytokines. Following conformational changes of the activated receptor, several intracellular death-domain (DD) containing proteins are recruited to form a receptor complex. Activation of this complex leads to the degradation of the NF kappa B inhibitor (I κ B) Cactus and subsequent nuclear translocation of the NF-κ B transcription factor Dorsal (or the Dorsal-related immune factor, Dif, in Drosophila (Royet et al ., 2005) . Two homologues of Dorsal were found in the honey bee genome (Fig. 1) , neither of which was orthologous with Dif. This lends support to the view that Dif is a highly derived branch found in brachyceran flies but absent from other insects. In mosquitoes (Shin et al ., 2005) , and arguably honey bees, Dorsal (called REL1 in mosquitoes) is a functional alternate for Dif. Functional tests can help determine which of the two dorsal paralogues is the key transcription factor for this pathway. The intracellular components Tollip, Pellino, Cactin and TNF receptor associated factor-2 (TRAF-2) are believed to aid the main players of this pathway, and all appear to be present in both fly species as well as the honey bee. Candidate effectors for the immune-related Toll pathways in honey bees include a compliment of antimicrobial peptides, the melanizing agent phenoloxidase and three lysozymes. While it has not been confirmed that these effectors are triggered by the Toll pathway as opposed to other pathways described below, it is evident that some of the bee effectors are responsive to pathogens and/or mechanical wounding of bees (Fig. 3) . Candidate honey bee members for the Toll pathway. Names are given for the Drosophila pathway components, along with vertebrate orthologues (in parentheses). Honey bee matches given as named during the genome project. Honey bee names in italics refer to genes with close paralogues which cannot readily be distinguished with respect to pathway components from Drosophila. Underlining indicates genes shown to be transcriptionally up-regulated after immune challenge.
Candidate honey bee members for the Imd, JNK and JAK/STAT pathways, below names for Drosophila pathway components along with vertebrate orthologues (in parentheses). Honey bee matches presented as named during the genome project. Honey bee names in italics refer to genes with close paralogues which cannot readily be distinguished with respect to pathway components from Drosophila. Underlining indicates genes shown to be transcriptionally up-regulated after immune challenge.
While Toll signalling in flies serves a dual purpose in development and immunity, the signalling process activated by peptidoglycan recognition protein (PGRP)-LC and Imd is specific for antimicrobial defence and is dispensable for normal development (Hultmark, 2003) . Via the NF-κ B-like transcription factor Relish, this signalling induces transcription of all major antimicrobial effector peptides in Drosophila . In Drosophila , Imd signalling is often said to be specific for Gram-negative bacteria, although Gram-positive bacteria with diaminopimelic acid-type peptidoglycans are at least as strong as elicitors. A weaker response is also seen to other types of peptidoglycan and even to fungi (Hultmark, 2003; Werner et al ., 2003; Stenbak et al ., 2004) . This broad specificity is caused by the three alternative splice forms of Drosophila PGRP-LC, which carry different peptidoglycan recognition domains (Werner et al ., 2003; Mellroth et al ., 2005 . Chang et al ., 2006 . Interestingly, the Imd signalling pathway is highly conserved in the honey bee, with plausible orthologues for all components (Fig. 2) . While this strongly suggests that Imd signalling is similar in flies and bees, it does not necessary imply similar biological roles.
Besides the activation of Relish, Imd signalling also leads to activation of components of the JNK signalling pathway (Boutros et al ., 2002) , and recent evidence indicates that this pathway can provide both positive and negative feedback for the expression of the antimicrobial peptides (Wojda et al ., 2004) . Plausible orthologues for each of the major components of the JNK signalling were also identified in the honey bee genome (Fig. 2) .
The JAK/STAT signalling pathway may also contribute to innate immunity by induction of complement-like factors and the overproliferation of haemocytes. JAK/STAT appears to be initiated via cytokine-like molecules in blood cells (Agaisse & Perrimon, 2004) . In flies, the extracellular glycosylated protein Upd acts as a ligand that activates the JAK/STAT pathway, which in turn promotes phagocytic activity of haemocytes. The JAK/STAT pathway has also recently been shown to participate in an antiviral response in Drosophila (Dostert et al ., 2005) . Honey bee homologues for the Drosophila JAK/STAT signalling pathway (Fig. 2 ) comprise the cytokine receptor domeless (Dom), JAK tyrosine kinase (Hopscotch), the STAT92E transcription factor 3. Transcript abundances for immune candidate genes in adult workers 24 h after injections of Escherichia coli (Ec), saline buffer, or the bee pathogen Paenibacillus larvae , and controls (left four columns). Two columns on right show transcript abundances in 2nd-instar larvae challenged orally with an infective dose of P. larvae or unchallenged controls. Cluster A = genes strongly up-regulated by adult injection or wounding, Cluster B = genes up-regulated in infected larvae, Cluster C = genes down-regulated or minimally changed in challenged bees. and two negative pathway regulators SOCS (suppressor of cytokine signalling) and PIAS (protein inhibitor of activated STAT). Orthologues of two recently identified components of this pathway (Baeg et al ., 2005; Muller et al ., 2005) , the tyrosine phosphatase Ptp61F (XP392429) and the WD40and bromo-domain-containing protein BRWD3 (XP395263), are also present in the honey bee. Although the key ligand (Upd) for the JAK/STAT pathway was not found in the honey bee genome, the presence of the gp130 cytokine receptor homologue Domeless and all other members of the signalling pathway indicates that this mechanism may be common across insects and is intact in honey bees as well as in flies. In addition to up-regulating the complement-like thiolestercontaining proteins (TEPs; Lagueux et al. , 2000; Boutros et al ., 2002) , the JAK/STAT pathway in Drosophila regulates expression of the Turandot (Tot) genes that encode humoral factors induced by severe stress Agaisse et al ., 2003) . None of the Tot factors (Tot A-Z) are apparent in honey bees.
While honey bees appear to have maintained each of the known insect immune-related pathways, they appear to do so with a reduced number of paralogous members. When comparing a set of 17 gene families and functional groups implicated in immune responsiveness (Christophides et al ., 2002) , honey bees have substantially lower paralogue counts than either Drosophila or Anopheles (Table 1 ). The 71 genes placed into these groups for honey bees are in sharp contrast to the 196 and 209 found in Drosophila or Anopheles , respectively. Bees have the lowest gene counts for 12 of the 17 families and are tied for the lowest count two more times. Drosophila and Anopheles were lowest for only one family each (defensins and dorsal, respectively). In contrast, Drosophila and Anopheles show the highest paralogue counts for this triad seven and eight times, respectively, versus once in bees (for the Toll-pathway candidate cactus, with three copies). These rankings are significantly different under an ordinal contingency-table analysis ( P < 1.0 × 10 -4 ), and reflect differences for genes involved with pathogen recognition and signalling, as well as effectors.
PGRPs, major players in pathogen recognition (Hultmark, 2003; Steiner, 2004; Royet et al ., 2005) are less diverse in honey bees versus flies and other insects for which genomic data exist (e.g. the moth Bombyx mori ). There are only four PGRPs in the honey bee genome, compared to 13 and seven in Drosophila and Anopheles , respectively (Fig. 4A , Table 1 ). Further, bees show no capacity for the splice variation that contributions to diversify peptidoglycan recognition specificity in flies and mosquitoes. Specifically, the single membrane-bound PGRP in bees (PGRP-LC, GB17188) is similar to fly and mosquito PGRP-LC but lacks the potential to insert alternative peptidoglycan recognition domains by alternative splicing, a factor in recognition breadth for flies. PGRP-LC and PGRP-S2 are both up-regulated in honey bees after disease challenge (as in flies), suggesting that the products of these genes indeed play a defensive role (Fig. 3) . As in Anopheles , there is only one Class C Scavenger Receptor (SR-C) in the honey bee. This group has diversified into four members in Drosophila , three of which show selective signs suggestive of an immune role (Lazzaro, 2005) . There are 10 Class B scavenger receptors in the bee, a number roughly similar to that in the fly and mosquito (Fig. 4B , Table 1 ). Several other recognition classes also seem to be reduced in honey bees, including β -glucan recognition proteins ( β GRPs), galectins and fibrinogen-related proteins (Table 1) . Of these, the fibrinogen-domain genes are especially striking, due to the absence in bees of high lineage-specific diversification found in mosquitoes and Drosophila [resulting in 57 and 13 domain-family members, respectively (Christophides et al ., 2004) ].
Along with PGRP-LC and SR-C , two other receptors were recently found to participate in the phagocytosis of infectious non-self in Drosophila , DSCAM and Eater. DSCAM, long implicated in neuronal development, was shown to have a likely role in the binding of bacteria by Drosophila haemocytes (Watson et al ., 2005) . This gene, which has > 12 000 potential splice variants in honey bees thanks to three sets of highly interchangeable exons (Graveley et al ., 2004) , is a very interesting candidate for determining the extent to which bees might better tune their immune response to specific pathogens. Another Table 1 . Gene counts for a subset of gene families implicated in insect immunity. Anopheles gambiae and Drosophila melanogaster counts based on Christophides et al. (2002 Christophides et al. ( , 2004 recently identified protein involved in Drosophila cellular immunity is Eater, a phagocytic receptor characterized by several repeats of an EGF motif in its extracellular domain (Kocks et al., 2005) . Many genes with EGF motifs are present in the bee genome (e.g. GB14654, Supplementary Material Table S1 ), as in flies, although their orthology with Eater is unclear. Bees are also likely to engage in the encapsulation of endoparasites and pathogens, and show typical integrins (Honey Bee Genome Sequencing Consortium, 2006) implicated in lamellocyte encapsulation . None of these cellular immunity components appear to be more diverse in bees than in fly species (Supplementary Material Table S1 ).
In addition to their function in digestion of food, serine proteases (SPs) in insects participate in regulatory cascade pathways in embryonic development and in immune responses (Kanost & Clarke, 2005) . Many haemolymph SPs and serine-protease homologues (SPHs) implicated in immunity contain one or more clip domains at their amino terminus, which may regulate or localize the immune responses stimulated by protease cascades (Jiang & Kanost, 2000) . Among the 57 SP-related proteins in the honey bee genome, 12 SPs and six SPHs contain at least one clip domain, significantly fewer than in Drosophila (24 SPs and 13 SPHs) (Ross et al., 2003) or Anopheles (26 SPs and 15 SPHs), and smaller than the number of clip domain SPs identified to date (n = 14) from expression data in Manduca sexta (Jiang et al., 2005) . Additional phylogenetic and functional relationships among insect SPs and SPHs are discussed in Zou et al. (2006) . Serine protease inhibitors from the serpin superfamily regulate protease cascades in mammals and in arthropods (Reichhart, 2005) . In insect haemolymph, serpins inhibit activated proteases to maintain homeostasis and prevent unregulated activation of immune responses such as melanization or Toll-mediated antimicrobial protein synthesis (Kanost & Clarke, 2005) . In the honey bee genome, there are seven annotated genes encoding five serpins and two proteins with serpin-like regions (GB10078 and GB15070). The number of serpin genes in the honey bee is much lower than in Drosophila (28) or Anopheles (14), mirroring the reduced size of the protease gene family in bees.
Nine Toll-related receptor genes are known from the Drosophila genome and 10 from Anopheles (Tauszig et al., 2000; Christophides et al., 2004) . In Drosophila, Toll is the primary family member implicated in immune-related function (Lemaitre et al., 1996) , although it appears that Drosophila Toll-5 and Toll-9 (the paralogue that is structurally most similar to mammalian TLRs), are also involved in immune-related signalling (Ooi et al., 2002; Bilak et al., 2003) . We have identified only five Toll-related genes in the honey bee: Toll1, Toll2/18w, Toll6, Toll8/Trex/Tollo and Toll10.
Additional Toll members in Drosophila (Toll-3, -4, -5, -7 and -9) apparently reflect gene duplication events in the fly lineage ( Fig. 4) or, in the case of Toll-9, arguably a loss in the honey bee and lepidopteran lineages. For instance, the ancestral Toll appears to have diverged into two different groups, Toll-1 and Toll-5, in flies. In contrast, Toll-1 remains the only member of this clade in honey bees. Similarly, the Toll-7/2 clade is represented only by a single honey bee homologue Am18w (Aronstein & Saldivar, 2005) . Presumably, the ancestral Toll 7/2 gene was duplicated in flies following their divergence 300 Mya from the lineage leading to honey bees. A protein named Apis mellifera toll7 (Kanzok et al., 2004) , seems more likely to be an orthologue of Toll-10. The five Toll receptors present in the A. mellifera genome (Toll-1, -6, -2/7, -8, -10) are also present in the sequenced genomes of other insects that belong to the orders Diptera, Lepidoptera and Coleoptera, with few exceptions. For example, whereas orthologues of Toll-6, -7 and -8 are found in D. melanogaster and A. gambiae (Diptera), Bombyx mori (Lepidoptera), and Tribolium castaneum (Coleoptera), Toll-1 appears to be absent from the genome of the lepidopteran insect B. mori, whereas Toll-10 appears to have been lost in the fruit fly. This suggests that these five genes encode the basic set of Toll receptors that was present in the common ancestor of these insects. These five receptors are highly expressed, in a dynamic and tissue-specific manner, during Drosophila embryogenesis (Kambris et al., 2002) . Of note, Toll-6 and Toll-8, are adjacent in both the D. melanogaster and A. mellifera genomes.
Among the immune effectors, the total of six honey bee antimicrobial peptides contrasts with the 20 and nine found in Drosophila and Anopheles, respectively (Table 1) . Two of these (abaecin, and apidaecin) are in the class of prolinerich antimicrobial peptides, two are conventional defensins and two (apisimin and hymenoptaecin) are distinct from all other recognized antimicrobial peptides. Genomic analysis reveals that the gene encoding apidaecin consists of a conserved N-terminus followed by several exons, each of which encodes a complete 28-amino acid peptide. Peptide and cDNA evidence for this gene was used to predict a mechanism for ratcheting up expression of apidaecin in response to bacterial challenge (Casteels-Josson et al., 1993) . The genomic structure of apidaecin raises the possibility of a mechanism for generating specific responses to pathogens by splice variation. As each exon is a functional and distinct antimicrobial peptide, it is conceivable that splice variation at this locus can further refine this gene as an immune effector. Apidaecin exons differ greatly across individual honey bees in both number and in their encoded amino acid sequences. The two bee haplotypes sequenced in this project differed in sequence and exon number and also differed from three previously described apidaecin cDNAs (Casteels et al., 1993) . Collectively, the two haplotypes sequenced here, and the three described by Casteels et al. encode a range of 4-11 secreted peptides each. While some peptides are shared between the various haplotypes for this gene, there is a surprisingly high level of sequence variation, such that the 35 peptides expressed by these five haplotypes reflect 23 different amino acid variants.
With the exception of defensin-2 (identified from genomic sequences generated during this project; Klaudiny et al., 2005) all of the honey bee antimicrobial peptides were first characterized by protein sequencing (Casteels-Josson et al., 1994) , a fact that belies the difficulty in discovering such genes by sequence similarity across the millions of years separating insect species. Still, there is no evidence for close paralogues for any of the honey bee antimicrobial peptides, in contrast to such patterns in other insects (e.g. the gene-rich cecropin family). Five of the six bee antimicrobial peptides are up-regulated across diverse immune challenges ( Fig. 3; Evans, 2004) . Honey bees possess only one prophenoloxidase (proPO) gene, versus three and nine in Drosophila and Anopheles, respectively. Like most proPO, the honey bee proPO lacks a signal peptide and has the consensus sequence of NRFG around the activation site. The gene encoding proPO is expressed more strongly in older honey bee larvae and pupae (Lourenço et al., 2005) . This gene was not up-regulated in our challenge experiments, but a gene identified as a proPO activator was up-regulated during natural infection (Fig. 3) . There are only three lysozymes in the honey bee genome, two c-(chicken) type and one i-(invertebrate) type. One of the c-class lysozymes is up-regulated by challenged honey bees (Fig. 3) .
There were fewer thiolester-containing proteins (TEPs) in the bee genome (four) than expected based on flies ( Table 1 ). The Anopheles genome encodes 15 TEPs, most of them originating from species-specific expansion (Christophides et al., 2004) versus six members of this gene family in Drosophila (Agaisse & Perrimon, 2004) . TEPs are induced after septic injury and promote phagocytosis in mosquitoes (Blandin & Levashina, 2004; Moita et al., 2005) . They also play a central role in vertebrate innate immunity as the complement factors. In Anopheles, TEP1 was found to promote phagocytosis of Gram-negative bacteria and is also a major player in the host response to plasmodium infection. Members of this group are implicated as both recognition proteins and effectors (opsonins) in insects (Levashina et al., 2001; Moita et al., 2005; Stroschein-Stevenson et al., 2006) .
As social animals, honey bees are at considerable risk from parasites and pathogens. Specifically, increased genetic relatedness and the high population densities that typify honey bee societies can strongly favour pathogen spread and epizootic outbreak. However, such social costs to honey bee immunity might be offset by social defences including mating strategy (e.g. multiple mating by queens: Tarpy, 2003) , mutual grooming, and the maintenance of a sheltered environment for colony members. Given their well-studied natural pathogens, immune pathway models from the current annotated draft genome and unique genetic traits, honey bees can join with several fly and moth species as important systems with which to understand the genetic causes of immunity and disease. They also join humans as organisms for which there is great interest in understanding the social drivers of disease, and in using this information to improve host survival.
Our analyses indicate that the basic set of molecules defining the insect host-defence system is present in honey bees, including intact pathways for the key processes implicated in immunity and development. Single orthologues can be assigned for many pathway members, while others show several potential bee genes for which further work is needed to confirm roles. Interestingly however, whereas in Drosophila and Anopheles the host appears to have diversified its molecular arsenal through species-biased gene duplication (Table 1) , we have not found examples of such gene expansions in honey bees. Thus, despite having wideranging parasites and pathogens, and tremendous losses to these pests at least in domesticated settings (Morse & Flottum, 1997) , bees appear to have relatively diminished capacities to respond to and defend against pathogens.
Our first hypothesis to help explain this observation, that bee gene families are systematically smaller than in other insects, is not supported because there is no evidence for a systematic downward bias in paralogue counts across the bee genome at the level seen for immune-gene families (Honey Bee Genome Sequencing Consortium, 2006) . Hypothesis two, that bee genes were simply missed due to sequence divergence, should apply particularly for immune genes that are short and subject only to limited sequence constraints, such as those encoding antimicrobial peptides. Indeed, few such peptides have been identified from bees (n = 6) and the means with which these few were discovered (directly at the protein level in all but one case) support the idea that sequence-level searches might have missed additional family members. Nevertheless, members of the remaining gene-poor families do not comprise especially short genes, nor genes divergent enough to be missed completely by comparisons at the level of insect orders. In fact, at least one significant bee:Drosophila:Anopheles orthologue is present in each of the remaining discussed families, indicating sufficient sequence-level conservation for identification of bee counterparts. Our third hypothesis, that bees simply have an undescribed mechanism for broadening their immune efficacy, is not testable at this point but would be very surprising given similarities in immune actors across the diverse insect orders studied to date. Two of our initial hypotheses, that bees face a less diverse set of successful parasites and pathogens, and that societal defences by bees lessen pathogen pressures, therefore seem best supported by the data in hand and can now be compared. On one level, bee pathogens are diverse, ranging from Gram-positive and Gram-negative bacteria to fungi, RNA viruses, microsporidia and amoebae (Morse & Flottum, 1997) . Bees are also parasitized by mites and other arthropods, raising risks of both pathogen infection (Kanbar & Engels, 2003; Chen et al., 2004) and lower abilities to combat disease (Gregory et al., 2005; Yang & Cox-Foster, 2005) . Still, despite having pests that range over several kingdoms, common disease agents in bees are in fact restricted to several pathogens, two of which (the bacterium Paenibacillus larvae and the fungus Ascosphaera apis) are predominant. While functional data on the specificity of responses toward these and other pathogens are not yet available, gene-expression changes after challenge do not appear to be especially precise with respect to honey bee pathogens vs. exotics (e.g. Escherichia coli) or stress generally (Fig. 3) . Thus, there is no compelling evidence at this point that the bee immune response is channelled just toward a small set of 'true' pathogens.
With respect to the final hypothesis, bees, like many social insects, are relentlessly hygienic, removing alien organisms from their nests, and secreting antimicrobial substances that can reduce the viability and growth of pathogens in the colonies. Bees also raise their young in individual cells using, as a food source, substances with strongly antimicrobial properties (e.g. royal jelly; Albert & Klaudiny, 2004) . A testament to this hygiene is the fact that, even when facing severe colony-level infections by bacterial pathogens such as Paenibacillus larvae (for which < 10 spores are normally fatal to young larvae (Brodsgaard et al., 1998) , the vast majority of larvae show no signs of exposure (Evans & Pettis, 2005) . 'Social' barriers might also reduce exposure to minor, opportunistic, pathogens or saprophytes that have been proposed as generalized targets of insect immune defences (Hultmark, 2003) . While bees do carry an assemblage of microbes, and bacteria in particular (Gilliam, 1997) , exposure to these microbes is arguably lower than in free-living Drosophila (decaying plant material as larvae and adults) or Anopheles (septic aqueous environments as larvae). Further, bacteria found in bee colonies have only rarely been associated with disease pathologies, despite extensive study. In fact, some resident bacteria in colonies appear to add to external defences through their inhibition of bee pathogens (Evans & Armstrong, 2006) .
Future genomic work can help reveal whether other species of highly social insects, including ants, wasps, bees and termites, also appear to have more simplistic innate defence systems. More generally, social and solitary insects with more 'exposed' life histories are predicted to have a greater number and higher functional diversity of immunepathway genes and end products, when compared to sister taxa that are more sheltered. Data on parasite and pathogen abundance across social (e.g. Boomsma et al., 2005) and solitary insects could be used as a surrogate for disease loads in different taxa, although field and epidemiological data are most needed to assess the relative fitness impacts of disease and the efficacies of different lines of defence.
Through this analysis, we present the first plausible models for immune pathways in a social insect, the honey bee. We show nearly complete conservation of candidate genes for these pathways yet show that bees have consistently undercut numbers of genes that embellish these pathways in other insects. Genome-wide expression studies newly available for bees, and the proven success of gene knockdown techniques such as RNA inactivation (Lourenço et al., 2005) , allow for more refinement of the roles played by pathway members as well as the discovery completely novel players in honey bee immunity. The latter discoveries, combined with analyses across more species of social and solitary bees, will help determine whether the observations described here are unique to the highly social honey bees. Longstanding agricultural interest has helped generate a wealth of data on honey bee pathologies (Morse & Flottum, 1997) , and it is now possible to connect these data with immune traits that help limit pathogen efficacy. Through these connections, honey bees will provide a valuable and tractable model for disease transmission, immunity, 'socialized medicine' and pathology.
Immune-gene candidates from other insects were used in several ways to query the honey bee genome, primarily using the BLAST family of search functions (www.ncbi.nlm.nih.gov). Most searches were initiated by BLASTP queries against the consensus protein list (GLEAN3, derived from HBGP assembly 2.0) using BLASTP and algorithms (BLOSUM and PAM variants) appropriate to gene size and structure. Honey bee orthologues were also identified by searching honey bee genome assemblies 2.0 and 3.0 directly using TBLASTN and either local databases or the BeeBase BLAST server (http://racerx00.tamu.edu/blast/blast.html). Searches for missing genes were also carried out a on smaller coverage set of honey bee contigs that were too short to be included in the assembly, as well as the unassembled reads from the project (http://www.hgsc.bcm.tmc.edu/projects/honeybee/). Given honey bee candidates, searches were repeated in the hope of identifying paralogues missed by interspecific comparisons. PSI-BLAST was used to identify honey bee genes on the basis of conserved domains, followed by RPS-BLAST to confirm the significance of these domain matches. Putative matches were aligned and, in the case of serine proteases, scavenger receptors and C-type lectins, screened for additional motifs using pfam categories (http://sanger.ac.uk/software/pfam). Tentative matches were aligned and checked for gene-prediction errors (in the case of genes from the official protein list) as part of the annotation of immune candidate genes for the Honey Bee Genome Project (Honey Bee Genome Sequencing Consortium, 2006). All protein matches were ported to Apis mellifera assembly 3.0 using the alignment program BLAT (Jim Kent, University California, Santa Cruz) to establish scaffold locations (Supplementary Material Table S1 ).
Best-match honey bee sequences were then aligned with counterparts from D. melanogaster and A. gambiae, along with other insects where sufficient genome-level data were available. Amino acid sequence alignments were carried out using GONNET series weight matrices, with the program CLUSTAL_X (Chenna et al., 2003) . Alignments were used to propose phylogenetic relationships using maximum-parsimony and neighbour-joining algorithms, with the programs PAUP* (Sinauer, Sunderland, MA) or PHYLIP (http:// evolution.genetics. washington.edu/phylip.html). The PGRP tree is based on the conserved domain region only. Other alignments were edited manually to reduce or remove ambiguous regions. For scavenger receptor class B proteins, human (NP_005497) and mouse (NP_031669 CD36) proteins were added to alignments. Honey bee scavenger receptor AmelSCRB8 was not included because its relatively short predicted length (apparently the result of an intercontig gap) precluded an unambiguous alignment. All alignments are available on request.
Two experiments were carried out to screen for immune-related transcript changes. In the first, adult worker bees from a single local A. mellifera ligustica colony were removed, then injected abdominally with either dilute phosphate-buffered saline or saline solution containing 103 live cells of E. coli or 103 vegetative spores of the honey bee bacterial pathogen Paenibacillus larvae. These bees, along with uninjected controls, were maintained for 24 h at high humidity and 34 °C and then were immediately frozen at −70 °C prior to RNA extraction. To assess immune responses following natural infection, eight 1st-instar larval bees from the same stock were given per os challenges of P. larvae in their food [(50 spores/µl as described in Evans (2004) ], then maintained 24 h at 34 °C and high humidity. Parallel control larvae were given the same food without bacterial spores. All samples were frozen at −80 °C following incubation.
RNA was extracted from whole abdomens of the adult bees using a standard TRIzol (Invitrogen, Carlsbad, CA, USA) procedure while RNA was extracted from individual larvae using the RNAqueous kit (Ambion, Austin, TX). RNAs were pooled by sample duration for the eight larvae challenged with the bacterial pathogen P. larvae, and the eight controls prior to cDNA synthesis, giving six RNA pools. DNA was removed from all extracts, then first-strand cDNA was synthesized as described by Evans (2004) . Transcript abundances for these cDNAs were assayed by quantitative real-time PCR with an Icycler real-time PCR machine (Bio-Rad, Hercules, CA, USA). Primer pairs were designed to amplify 120-300 bp sections of 39 honey bee immune-related genes derived from Supplementary Material Table S1 and ribosomal protein S5 as a control gene (primers in Supplementary Material Table S2 ). Primer sequences were modified, where necessary, to run in duplicate on 96-well plates using a fixed thermal protocol consisting of 5 min at 95 °C, then 40 cycles of a four-step protocol consisting of 94 °C for 20 s, 60 °C for 30 s, 72 °C for 1 min, and 78 °C for 20 s was used (Evans, 2006) . Reactions were carried out on 0.5-2 µg cDNA along with 1 U Taq, the provided PCR buffer (Roche Applied Sciences, Indianapolis, IN, USA), 1 mM dNTP mix, 2 mM added MgCl 2 , 0.2 µM each primer, 1× concentration SYBR-Green I dye (Applied Biosystems, Foster City, CA, USA), and 10 nM fluorescein in a 25 µl reaction volume. Amplification was followed by a meltcurve dissociation program in order to confirm expected product size. Thresholds were calculated individually for each target gene on the 96-well plate. For adult bee samples, data were pooled for the three replicates in each single-bee injection treatment (or controls). Results were screened for the appropriate dissociation (melt-curve) values, and by 1% agarose gels, in order to ensure against primer artefacts and the presence of DNA contamination (which would have been evident for numerous primers spanning two exons). Immune-gene transcripts were normalized relative to expression levels for the gene encoding ribosomal protein S5, a gene with consistent expression across honey bee life stages and disease status (Evans & Wheeler, 2000; Evans, 2004) . For display purposes, transcript abundance values (CTcontrol-CTtarget) for each gene were median-normalized across each panel of genes and clustered by average linkage clustering (using Cluster 3.0, M. Eisen, www.rana.lbl.gov/EisenSoftware.htm) and presented as relative grey-scale values (using Treeview, M. Eisen). ElaD, a Deubiquitinating Protease Expressed by E. coli BACKGROUND: Ubiquitin and ubiquitin-like proteins (Ubl) are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. FINDINGS: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs). Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. CONCLUSION: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan. Ubiquitin, as well as Ubls such as Nedd8 and SUMO, are proteins (almost) exclusively expressed by eukaryotes [1] . Ubiquitination controls many cellular processes, including degradation of proteins by the proteasome and intracellular trafficking. Nedd8 is a ubiquitin-like modifier that regulates the rate or extent of ubiquitination, whereas SUMO1 is involved mostly in regulation of transcription factors and in nuclear import. The attachment of ubiquitin or ubiquitin-like modifiers to substrate proteins is covalent, yet reversible [2] . A large family of eukaryotic cysteine proteases is involved not only in generation of ubiquitin(-like) proteins from their precursors, but also in their removal from modified substrates [3, 4] . Pathogens can tamper with the ubiquitin-proteasome system to cripple the cell's defenses [5] . For instance, ubiquitination and proteasomal degradation of p53, initiated by a Human Papillomavirus protein [6] , or stabilization of Ikb-a by Yersinia deubiquitinases [7] have been described. The continuing discovery of new deubiquitinating proteases in viruses broadly hints at how important it is for these pathogens to seize control of posttranslational modifications in host cells [8] [9] [10] . Here, we focus on cysteine proteases of the CE clan [11] , as defined by the MEROPS database [12] . CE peptidases are expressed by viruses, bacteria and eukaryotes. In eukaryotes, this clan represents the family of Ubl-specific proteases (ULP/SENP), which remove SUMO or Nedd8 from substrate proteins [13, 14] . Viral homologs of ULP/SENPs can act as deubiquitinases, but they also cleave unrelated proteins, as long as a glycine motif is present at the C-terminus of the substrate [15, 16] . Examples of bacterial deubiquitinases include YopJ and ChlaDUBs. First, YopJ is a protease that is secreted into host cells by Y. pestis [17] , and homologs to this peptidase can be found in other bacteria, too [18, 19] . Injection of YopJ eventually suppresses the inflammatory response in affected cells. The precise molecular mechanism of YopJ family peptidases has not yet been solved, as they lack a hallmark tryptophan following the active-site histidine [20] . Yet, in vitro and in vivo data strongly suggest that these proteins are indeed proteases with specificity for ubiquitin or SUMO [7, 18, 19] , although their effectiveness as virulence factors might depend on additional functions such as acetylation [21, 22] . A second example of bacterial CE peptidases can be found in pathogenic Chlamydiae. We have shown that pathogenic Chlamydiae, but not a nonpathogenic environmental strain, express proteases that specifically recognize ubiquitin and Nedd8. They do so presumably to remove both modifiers from target proteins of the host cell, as Chlamydiaelike most other bacteria-possess neither a ubiquitin nor a Nedd8 homolog [23] .
Intrigued by the finding of CE peptidases in bacteria and considering their functional similarity to eukaryotic ULP/SENP proteases, we explored whether additional bacterial homologs with deubiquitinating activity might exist.
To search the sequenced genomes of bacteria for new members of the CE protease clan, we employed PHI-BLAST [24] to find proteins with the typical catalytic triad of histidine, aspartate (or glutamate or asparagine), and the active-site cysteine [25] . Candidate proteins were subjected to a second round of analysis, in which we excluded proteins without the hallmark oxyanionstabilizing group, consisting of at least one glutamine (or asparagine) close to the active-site cysteine. Lastly, the predicted secondary structure of candidate proteases was compared to the solved structure of eukaryotic CE clan homologs [26, 27] . Apart from YopJ and the ULP/SENP homologs in Chlamydiae, we found potential CE peptidases in Alpha-, Beta-, and Gammaproteobacteria ( Figure 1 ), but not in bacteria from other branches. We also found a peptidase homolog in the giant Acanthamoeba Polyphaga Mimivirus [28] and in African Swine Fever Virus, the first virus in which a ubiquitin conjugating enzyme was discovered and for which deubiquitinating activity has been suggested [29] [30] [31] [32] . The bacteria we identified share a close (symbiotic or pathogenic) relationship with eukaryotes, and all viruses with CE proteases possess a dsDNA genome. The sequence variations among these peptidases are too extensive to allow for significant bootstrap values and it is therefore not possible to faithfully infer phylogeny from this dataset. Yet, this is also true for the eukaryotic ULP/SENPs, which fall into three functional classes, specific for either SUMO (ULP1 and ULP2 group) or Nedd8 (SENP8 group). While best reciprocal BLAST hits and consensus phylogram trees tend to correctly predict to which functional class a particular ULP/SENP homolog belongs, bootstrap support is generally not significant [33] .
We were especially interested in the protein elaD (belonging to ''group II'', see Figure 1 ), expressed by E. coli and with orthologs in Legionella pneumophila and in all currently sequenced strains of Salmonella ( Figure 2 ). E. coli is an abundant commensal in the human gut and also a relevant pathogen. Nonetheless, little is known about genes that define pathogenicity of various E. coli strains, apart from those that encode obvious toxins [34] [35] [36] . We furthermore chose elaD, because it represents one of the more distantly related hits in our bioinformatics screen and we aimed to test the robustness of our prediction by examining this protein's function. Figure 1 . Phylogram representation of CE clan proteases in viruses, bacteria, and eukaryotes. Eukaryotic peptidases (in blue) belong to the C48 subfamily and can be separated into three groups: ULP1 (including the mammalian proteases SENP1, 2, 3, and 5), ULP2 (including SENP6 and 7), and the SENP8 group with proposed specificity for SUMO (ULP1 and ULP2 group) and Nedd8 (SENP8 group), respectively. Bacterial proteins are indicated with a preceding ''B'', viral proteins with a ''V''. We have further divided microbial protease homologs by color: green for biochemically tested proteases, red indicating the absence of published data on the function of these putative proteases, and yellow for the group representing elaD and its orthologs. The C5 family contains Adenovirus proteases with deubiquitinating activity, C55 comprises the bacterial YopJ homologs, and C57 the Vacciniavirus I7 peptidases. Based on sequence similarity, two bacterial C48 family groups can be distinguished: a group of Proteobacteria (located at one o'clock) which appear to be closely related to fungal SENP8 homologs (common node indicated with a circle, bootstrap support.60%), and Chlamydiae, for which we had previously shown the presence of deubiquitinating and deneddylating activity. Three additional groups have not yet been assigned to specific CE clan subfamilies in the MEROPS database, including Mimivirus (''group I''), Gammaproteobacteria (''group II''), and Rickettsiae (''group III''). The African Swine Fever Virus protease and the I7 Vacciniavirus protease have not been tested for deubiquitinating or Ublspecific activity, but they both require a glycine-based motif at the C-terminus of the substrate, as found in ubiquitin or Ubls. The unrelated CD clan peptidase Clostripain is used as outgroup in this phylogram. For clarity, this tree does not contain all orthologs and paralogs of the different groups or families. Sequence information is provided in Table 2 . doi:10.1371/journal.pone.0000381.g001
The goal of our first experiment was to confirm protease activity and to determine the substrate specificity of elaD. Because of its relationship to ULP/SENPs, we hypothesized that elaD might recognize Ubls or ubiquitin. We expressed elaD by in vitro transcription/translation in rabbit reticulocyte lysate and incubated the metabolically labeled polypeptide with electrophilic probes, in which a Michael acceptor was added to the C-terminus of ubiquitin or the Ubls SUMO1, Nedd8, and ISG15 [14] . As shown in Figure 3 , elaD readily forms a covalent adduct with the ubiquitin probe and to a much lesser extent also with the Nedd8 probe, but not detectably with SUMO1 or ISG15. Moreover, when mutating the putative active-site cysteine at position 313 to serine, covalent binding to the electrophile is abolished. This indicates that the cysteine residue in elaD is essential for catalytic activity, as has been observed for YopJ [7] , for the Chlamydia protease CT868 [23] and for the eukaryotic ULP/SENPs [2] . To date, specific labeling of putative proteases with activity-based probes has shown excellent correlation with enzymatic activity [14, 37, 38] .
Next, we assessed enzyme kinetics by measuring hydrolysis of fluorogenic substrates derived from ubiquitin, SUMO1 and Nedd8. For these experiments, we expressed elaD in E. coli. The growth rate of the bacteria was unaffected when overexpressing elaD, but we only recovered about 50% of the wildtype protein when compared to the amounts of C313S mutant ( Figure 4A ). As demonstrated by release of the fluorophore 7-amino-4-methylcoumarin (AMC), elaD cleaves ubiquitin-AMC, but not SUMO1-AMC or Nedd8-AMC, and the C313S mutant of elaD fails to cleave either substrate ( Figure 4B and data not shown). The initial rate of hydrolysis with 100 nM ubiquitin-AMC and 50 nM elaD is in the order of 0.3-0.6 per minute, defining elaD as a moderately active deubiquitinase, compared to the rapid Isopeptidase T with a rate of ca. 8 per minute [39] or to the much slower ubiquitinprotease USP14 with a rate of,0.01 per minute (data not shown). It should be noted that we could not assess V max , because the enzymatic rate increased linearly with substrate concentration (we tested up to 20 mM ubiquitin-AMC; 50 nM elaD then hydrolyzed ubiquitin-AMC at an initial rate of 5 per minute). Similar observations have been made with the SARS virus deubiquitinase [8] . Overall, our functional analyses confirm the prediction of our bioinformatics screen and define elaD as a deubiquitinating protease.
Why do eukaryotic CE peptidases show specificity distinct from their prokaryotic counterparts? Could this indicate a profound discrepancy, hinting towards a separate origin of these two protease groups? We set out to challenge the presently held notion that ULP/ SENP proteases do not exhibit ubiquitin-specific activity [13] , while most tested bacterial homologs apparently do. To this end, we chose to biochemically define CE clan members of a more deeply branched class of eukaryotes. Pezizomycotina, a subgroup of fungi that includes A. fumigatus, A. nidulans, M. grisea, N. crassa, and G. zeae encode putative SENP8 proteases that are related in amino acid sequence to a group of yet uncharacterized bacterial C48 homologs ( Figure 1 ). In our phylogram, the common node between the respective prokaryote C48 group and SENP8 homologs of Pezizomycotina replicates with a bootstrap support of.60%. We cloned, expressed and tested the putative SENP8 protease of G. zeae, as a representative of Pezizomycotina. Unlike mammalian SENP8, the G. zeae ortholog displays dual activity towards Nedd8 and ubiquitin, similar to the previously defined CE clan protease CT868 Figure 2 . Sequence comparison between SENP8 and its homologs in human pathogenic bacteria. Multiple sequence alignment of the catalytic core region of human SENP8 (NCBI protein sequence identifier GI: 33942066, shown are residues 100-165) with the homologs in C. trachomatis (CT868, GI: 76789615, residues 199-284) [23] , E. coli (elaD, GI: 15832411, residues 228-319), L. pneumophila (GI: 52843101, residues 189-265), and S. typhi (sseL, GI: 29141091, residues 197-264) [42] . The arrows indicate active-site histidine, aspartate (or asparagine), the catalytic cysteine, and the oxyanion-stabilizing group. Predicted secondary structures are shown at the bottom and have been confirmed with the solved structure of SENP8 [27] . doi:10.1371/journal.pone.0000381.g002 Figure 3 . Biochemical assay for substrate specificity of elaD. 35 S-methionine-labeled in vitro translated wildtype elaD forms covalent adducts with suicide inhibitors based on ubiquitin (ubiquitin-vinylmethylester, VME) and Nedd8 (Nedd8-vinylsulfone, VS), but not with probes based on SUMO1 and ISG15. All probes were tested for activity with bona fide substrates (not shown) [14] . Mutation of the active-site cysteine at position 313 to serine abolishes adduct formation of elaD to electrophilic probes. Samples were resolved by reducing SDS-PAGE and visualized by fluorography. Indicated at the right is the molecular mass in kDa. doi:10.1371/journal.pone.0000381.g003 of the prokaryote C. trachomatis ( Figure 5 ) [23] . This result indicates that ubiquitin-specificity is not restricted to bacterial or viral CE peptidases, but also exists in some ancient eukaryotic members of this protease clan.
We have found previously undescribed CE peptidase homologs in several bacterial and in one viral species (Figure 1) . We have furthermore proven that one of the more distantly related homologs-the protein elaD in E. coli-can act as a deubiquitinating enzyme in vitro. Earlier, we had shown that related Chlamydia proteases also have deubiquitinating activity and similar observations had been made regarding the YopJ protease family. This suggests that the yet undefined bacterial CE peptidases could also display ubiquitin-or Ubl-specificity, especially considering their even closer sequence relationship to eukaryotic ULP/SENP proteases. These findings raise two important questions. First, are these proteases really specific for ubiquitin in vivo, or are we simply measuring an offtarget artifact, with the true substrates merely resembling ubiquitin? Second, why are these proteases-which are present in every eukaryote-so widely distributed in bacteria and viruses as well, and what can we learn about their genetic origin?
One might argue that the bacterial CE clan peptidases have distinct specificity for bacterial substrates. For instance, it has been proposed that the origin of the ubiquitin system predates the split between eukaryotes and prokaryotes [40, 41] . Factors that distantly resemble Ubls and their conjugating and deconjugating enzymes can be found in bacteria. In this manner, the cleavage of ubiquitin by elaD could just be an artificial byproduct of our assays, while the true substrate is of bacterial origin. Similarly, the Adenovirus CE protease has relaxed specificity for a consensus site that is present in ubiquitin, but also in certain viral substrates [15, 16] . However, we have extended our analysis of elaD's specificity to the ubiquitin homologs Nedd8 and ISG15. Both share significant sequence similarity to ubiquitin, and ISG15 is even identical at the critical C-terminal region. We observed no reactivity between elaD and ISG15-vinylsulfone, and the binding of elaD to Nedd8vinylsulfone was significantly weaker than to the ubiquitin probe. Furthermore, we detected hydrolysis of the C-terminal peptide bond in ubiquitin-AMC, but not in Nedd8-AMC. These features clearly distinguish elaD from the more promiscuous viral CE peptidases. Given the similarity of primary, secondary and tertiary structure among these Ubls, we conclude that hydrolysis of ubiquitin by elaD reflects a highly specific interaction. With the exception of A. avenae, no bacterial strain in our dataset encodes a homolog of ubiquitin, making it likely that the substrate of elaD is indeed eukaryotic ubiquitin. Moreover, the ortholog of elaD in Salmonella-sseL-has recently been shown to be a virulence factor and to display deubiquitinating activity in vitro and in vivo [42] . This enzyme is encoded by all currently sequenced Salmonellae, but only present as a pseudogene in Shigellae [43] . Likewise, elaD is not essential for E. coli under laboratory conditions [44] . A comparison of the genomes of all 16 sequenced E. coli strains reveals that elaD is present in the commensal E. coli strain K12, and in all intestinal pathogenic strains (EAEC, EHEC, EIEC, EPEC, ETEC), but absent from all ExPEC strains (APEC, NMEC, UPEC) ( Table 1 ).
To answer the second question raised above, the fact that all CE proteases show the same signature motifs at the catalytic domain and that they have substrate specificity for ubiquitin, Ubls, or related products, suggests a common genetic and functional origin of these proteases. The viral and bacterial organisms that express these CE proteases all share an intimate relationship with eukaryotes, either as commensals, symbionts or as pathogens. Additionally, lateral gene transfer has been proposed between eukaryotes and dsDNA viruses [28] , as well as between eukaryotes and bacteria such as Chlamydiae, Rickettsiae, and L. pneumophila [45, 46] . The notion of a dynamic horizontal gene transfer involving deubiquitinases is further underscored by the distribution of ubiquitin-specific proteases in Chlamydiae: all pathogenic strains express CE peptidases, except for C. pneumoniae, which instead encodes a homolog of the eukaryotic otubain-type deubiquitinating enzymes [47] . Also, one group of bacterial C48 peptidases in particular clusters close to the SENP8 homologs of Pezizomycotina ( Figure 1 ). The sequence relationship and the shared habitat of these organisms-plant symbionts and phytopathogens vs. environmental fungi-raises the possibility of gene transfer between them [48] . From a functional perspective, we could show that a homolog of SENP8 in Pezizomycotina does exert ubiquitin-specific hydrolase activity, like previously characterized bacterial CE proteases ( Figure 5 ). The dual specificity of SENP8 from the fungus G. zeae towards ubiquitin and Nedd8 is not trivial. Although Nedd8 is arguably the closest relative to ubiquitin, there are sequence differences that require distinct conjugating machineries and deconjugating proteases [27, 49] . In particular position 72-an arginine in ubiquitin, and an alanine in Nedd8-can act as a compatibility switch and a single replacement at this side chain can cause ubiquitin to mimic Nedd8 and vice versa [50, 51] . In this respect, the recognition of both ubiquitin and Nedd8 by the SENP8 homolog of G. zeae is in contrast to what has been observed in mammalian SENP8 [27] .
One possible explanation for these observations is that CE proteases originally derived from a deubiquitinase, a protease specific for this most conserved eukaryotic protein. As CE peptidases in eukaryotes structurally diversified to accommodate the evolving family of Ubls, protease counterparts in bacteria, viruses, and some deeply branched eukaryotes retained their specificity for the ''ur-substrate'' ubiquitin.
Together with the published literature, our data supports the notion that the clan of CE proteases was acquired by bacteria and viruses via horizontal gene transfer from eukaryotes. Why this family of enzymes forms such a particularly attractive substrate for genetic exchange is an intriguing question. The distribution of CE proteases in symbiotic and pathogenic prokaryotes and viruses is suggestive of a general role in host-microbe interactions, as exemplified by the Salmonella protease sseL [42] .
Protein and DNA sequence data were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), the Institute for Genomic Research (www.tigr.org), and the University of Wisconsin E. coli Genome Project (www.genome.wisc.edu). Protein sequence identifiers are listed in Table 2 . Sequences containing and surrounding the catalytic core of the proteases were aligned with the ClustalX algorithm (default parameters) (bips.u-strasbg.fr/fr/Documentation/ClustalX) [52] , manually edited with Genedoc (www.psc. [53] . Secondary structures were predicted with JPred (www.compbio.dundee.ac.uk/,www-jpred/) [54] . Phylograms were constructed with the MEGA software package [55] , using the Neighbor-Joining Method with Poisson correction (all substitutions, homogeneous pattern, c-distribution 2.0) and pairwise deletion of gaps. Figure 1 shows a consensus tree based on 100 bootstrap replications.
Cloning, expression, and biochemical analysis of elaD and G. zeae SENP8
The full-length elaD gene (NCBI protein sequence identifier GI: 16130204) was amplified by PCR from the K12 strain BL21 (Novagen) and cloned into pET28a (Novagen The putative proteases are separated by groups (as shown in Figure 1 ), by species, and by subdivision of Proteobacteria. This list is not complete in terms of orthologs/paralogs or bacterial species.  Mutational analysis of human CEACAM1: the potential of receptor polymorphism in increasing host susceptibility to bacterial infection A common overlapping site on the N-terminal IgV-like domain of human carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) is targeted by several important human respiratory pathogens. These include Neisseria meningitidis (Nm) and Haemophilus influenzae (Hi) that can cause disseminated or persistent localized infections. To define the precise structural features that determine the binding of distinct pathogens with CEACAMs, we have undertaken molecular modelling and mutation of the receptor molecules at previously implicated key target residues required for bacterial binding. These include Ser-32, Tyr-34, Val-39, Gln-44 and Gln-89, in addition to Ile-91, the primary docking site for the pathogens. Most, but not all, of these residues located adjacent to each other in a previous N-domain model of human CEACAM1, which was based on REI, CD2 and CD4. In the current studies, we have refined this model based on the mouse CEACAM1 crystal structure, and observe that all of the above residues form an exposed continuous binding region on the N-domain. Examination of the model also suggested that substitution of two of these residues 34 and 89 could affect the accessibility of Ile-91 for ligand binding. By introducing selected mutations at the positions 91, 34 and 89, we confirmed the primary importance of Ile-91 in all bacterial binding to CEACAM1 despite the inter- and intraspecies structural differences between the bacterial CEACAM-binding ligands. The studies further indicated that the efficiency of binding was significantly enhanced for specific strains by mutations such as Y34F and Q89N, which also altered the hierarchy of Nm versus Hi strain binding. These studies imply that distinct polymorphisms in human epithelial CEACAMs have the potential to decrease or increase the risk of infection by the receptor-targeting pathogens. The bacterial pathogens Neisseria meningitidis (Nm) and Haemophilus influenzae (Hi) are frequently found in the nasopharynx of a substantial proportion of the healthy population but are capable of causing serious infections in susceptible individuals (Turk, 1984; Foxwell et al., 1998) . Nm and typable Hi (THi) can invade the nasopharyngeal epithelial barrier to cause septicaemia and meningitis, which in the case of Nm, may rapidly become life threatening (van Deuren et al., 2000) . Non-typable Hi (NTHi), which lack a polysaccharide capsule, are associated with localized respiratory tract and conjunctival infections (Foxwell et al., 1998) . Strains belonging to Hi-biogroup aegyptius (Hi-aeg) are also associated with Brazilian purpuric fever (Foxwell et al., 1998) . The factors that determine susceptibility to infection by these frequent colonizers are not entirely clear.
For both colonization and pathogenesis, the first essential step is adherence to mucosal epithelial cells. Many investigations have shown bacterial targeting of specific human signalling molecules such as integrins, sialic acid binding Ig like lectins (Siglecs) and carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) can lead to cellular invasion (Virji et al., 1995; 1999; Hauck and Meyer, 2003; Jones et al., 2003) . Of these, CEACAMs have emerged as common targets of several respiratory mucosal pathogens and include Nm, Hi, Moraxella catarrhalis, as well as the urogenital pathogen Neisseria gonorrhoeae and enteric pathogens Escherichia coli and Salmonella (Leusch et al., 1991; Virji et al., 1996a; Chen et al., 1997; Gray-Owen et al., 1997; Hill and Virji, 2003) .
Carcinoembryonic antigen-related cell adhesion molecules belong to the immunoglobulin (Ig) superfamily. Several members of the CEACAM subgroup are expressed on human epithelial cells and include the widely expressed transmembrane CEACAM1 as well as GPI-anchored CEA and CEACAM6. All CEACAMs have an N-terminal IgV-like domain and variable numbers of IgC2-like A and B domains. CEACAM1 comprises up to four extracellular domains: N, A1, B and A2 and either a long or a short cytoplasmic tail (Tsutsumi et al., 1990; Prall et al., 1996; Hammarstrom, 1999) . Various functions have been attributed to CEACAM1 including cell-cell adhesion, insulin regulation and angiogenesis (Obrink, 1997; Hammarstrom, 1999; Wagener and Ergun, 2000; Najjar, 2002) . Targeting of CEACAMs by N. gonorrhoeae, as well as Nm and Hi leads to cellular invasion and passage across polarized monolayers (Virji et al., 1999; Gray-Owen, 2003; M. Soriani, K. Setchfield, D.J. Hill, and M. Virji, unpublished data) .
A wide range of bacterial adhesins are involved in targeting the CEACAM N-terminal domains and includes Opa proteins, a major adhesin family of pathogenic Neisseria and P5 proteins of Hi (Chen and Gotschlich, 1996; Virji et al., 1996a; Hill et al., 2001) . Nm and N. gonorrhoeae contain multiple copies of Opa genes that encode conserved domains which form b-barrel structures in bacterial membranes and variable domains that form surface exposed loops. In spite of the surface diversity afforded by the hyper-variable domains of the loops, the majority of the Opa proteins are capable of targeting CEACAMs (Virji et al., 1996a; Hauck and Meyer, 2003) . The P5 proteins of Hi are similar b-barrel forming proteins, also with surface variable loops (Webb and Cripps, 1998; Vandeputte-Rutten et al., 2003) . The interactions between these bacterial ligands and CEACAMs are complex and the binding domain appears to involve more than one variable loop of the bacterial adhesins (Virji et al., 1999; Bos et al., 2002; de Jonge et al., 2003) . Interestingly, antibody inhibition studies have shown that the diverse ligands of neisseria and haemophilus bind to an overlapping site on the N-domain. In addition, mutational analysis of the N-domain of CEACAM1 has identified several critical residues particularly Ile-91. Alanine substitutions at these sites abrogated binding of most Opa-and P5-expressing bacteria to CEACAM1. Additional residues such as Tyr-34, Ser-32, Val-39, Gln-44 and Gln-89, most of which located in the vicinity of Ile-91, appear to determine the efficiency of interactions of various Opa and P5 molecules (Virji et al., 1999; . The bacterial binding surface on the CEACAM1 N-domain is the protein face composed of the beta strands C′′, C′, C, F and G (CFG for brevity). Despite the extensive investigations in a number of laboratories particularly on neisserial Opa proteins, it remains unclear as to precisely how CEACAM-binding ligands engage with the receptors.
A three dimensional structural model of the human CEACAM1 N-domain has been previously generated based on other Ig family molecules (Virji et al., 1999) . In the current investigation, we have refined our previous model based on murine CEACAM1 crystal structure (Tan et al., 2002) . Examination of this model showed a better confluence of the above residues into a continuous binding site and suggested that substitutions at positions 34 and 89 at the core of the binding region may affect bacterial access to the implicated key binding residue Ile-91. By introducing conservative and non-conservative substitutions at positions 34, 89 and 91, we have examined the binding of a variety of bacterial strains to the receptor constructs. The data suggest that single nucleotide polymorphisms (SNPs) in individuals or populations that may introduce substitutions in CEACAM sequence particularly at the bacterial binding site, could not only decrease but also significantly increase the functional affinity of pathogen interactions. Increased binding affinity may result in increased cellular invasion and thus may lead to increased host susceptibility to infection by CEACAM-targeting bacteria.
A three-dimensional model of CEACAM1 N-domain was previously produced based on the Ig family molecules REI, CD2 and CD4 (Fig. 1A ) (Virji et al., 1999) . In this model, whilst most mutations affecting binding of Nm and Hi were located centrally on the CFG face of the protein, Val-39 involved in Opa binding, was located at a distance towards the bottom of the CFG face (Fig. 1A) . Subsequently, a crystal structure of murine soluble CEACAM1a domains 1 and 4 was reported (Tan et al., 2002) . Based on this, we have remodelled the human CEACAM1 Ndomain. The CC′ loop that contains Val-39 folds back against the CFG face of this model such that it lies in close proximity to the other critical residues involved in bacterial adhesion (Fig. 1B) . Further examination of this model suggested that the bacterial binding pocket forms a rather flat surface in the centre of the CFG face. It also appears that substituting neighbouring residues Tyr-34 and Gln-89 by Phe and Asn, respectively, could result in a further flattening of the bacterial binding surface (Fig. 1C-E) , providing increased access to the key Ile-91. These residue changes also increase the size of the hydrophobic patch centred on I91 (Fig. 1C-E) . Both of these effects might facilitate binding of some bacterial ligands to the target receptor.
In order to assess the importance of these residues in interactions with mucosal pathogens, substitutions were introduced by site-directed mutagenesis at three sites (91, 34 and 89) on the N-domain. The substitutions introduced in CEACAM1 [NA1B]-Fc are shown in Fig. 2 . Chimeric receptors proteins with the native sequence or with sub- (Virji et al., 1999) . (B) New model based on the murine CEACAM1a N-domain crystal structure (Tan et al., 2002) . The models are presented as Van der Waals surface representation. In the latter case V39 locates more centrally with the other critical residues for bacterial binding. (C-E, left) Stereo pairs presented are ribbon diagrams for CEACAM1 N-domain in the native form (C), with Y34F (D) or Q89N (E) substitutions show a flattening of the bacterial binding region on CEACAM1 and improving accessibility of the primary binding residue I91. The side-chain atom colouring (C-green, O-red, N-blue) show the increased hydrophobic nature of the binding site around I91 in the mutant structures. (C-E, right) Surface presentations of the binding site (views corresponding to a 90°rotation of the stereo images in the horizontal axis) coloured according to hydrophobicity. stitutions were produced by transient transfection of COS cells for functional studies described below.
The N-domain specific monoclonal antibody (mAb) YTH71.3 has been shown to require Gln-89 and Ile-91 for receptor recognition and substitution of these amino acids had no effect on the binding of the polyclonal anti-CEACAM antibody A0115 (Virji et al., 1999) . The novel receptor constructs produced in the current studies were first examined for their ability to bind to YTH71.3, A0115 as well as Kat4c. The latter mAb recognizes the A and B domains of the receptor (Jones et al., 1995) . AO115 and Kat4c bound to various receptor constructs and to the native NA1B-Fc molecule with equal efficiency. For YTH71.3, Leu but not Thr at position 91 supported binding to the same extent as Ile of the native molecule. Similarly at position 34, only Phe could be effectively substituted for Tyr. Finally Q89A and Q89N completely abrogated the antibody binding.
Opa-expressing phenotypes of two Nm strains (C751 and MC58) were used to investigate their binding to the modified NA1B-Fc receptors. Three C751 derivatives express-ing distinct Opa proteins (OpaA, OpaB and OpaD) and the MC58 derivative expressing an Opa protein designated OpaX (Virji et al., 1999) were assessed by receptor overlay experiments (Fig. 3) .
All the Nm isolates showed reduced binding to the soluble receptor with Y34A and I91A substitutions whilst Q89A affected OpaD binding most significantly confirming previous studies (Virji et al., 1999) . Introduction of a Leu or Thr residue at position 91 was generally less disruptive to Opa interactions. I91T substitution either did not affect binding (OpaB and OpaX) or reduced binding by 50-60% (OpaD and OpaA). I91L substitution had no effect on OpaD or OpaB whilst having opposite effects on OpaA (~50% reduction) and OpaX (twofold increase) binding.
Substitutions at position 34 other than Ala also supported binding of some but not all Opa proteins. Y34S substitution was unsuitable for the three Opa proteins of strain C751 but was tolerated by OpaX of MC58. Interestingly, Phe proved to be a more favoured residue in at least two cases with OpaX as well as OpaB binding to Y34F construct at threefold higher levels than the native receptor. Substitutions of Asn at Gln-89 revealed distinct patterns of interactions and binding reduced in the order OpaA > OpaX > OpaB/D (Fig. 3) .
Overall, the data suggest that Opa binding to the receptor requires an extended aliphatic chain at position 91 and certain arrangements of the chain may facilitate binding of most Opa proteins (Ile = leu > Thr). At position Α,Β D, E β−strand: (Virji et al., 1999) . Side chains of the residues introduced at positions 34, 89 and 91 in this study are shown in B for comparison 34, the removal of the hydroxyl residue (Y43F) is tolerated or preferred whereas the absence of aromatic ring (Y34S) has an overall deleterious effect on Opa binding. Finally, reduction of the side chain extension (Q89N) reduces binding of three of the four Opa proteins.
Comparison of C751 Opa-A, -B and -D in which the differences exist only between their surface exposed loop structures (HV1 and HV2, shown in Fig. 9 ), suggests further that a combination of the two loop structures must be involved in presenting the appropriate binding partners for the distinct residues of the receptor.
One non-typable (A950002) and two THi strains (Rd and Eagan) were used in receptor overlay experiments as above (Fig. 4) . Alanine substitutions at Ile-91 confirmed previous results (Virji et al., 2000) . Taken individually, substitutions of Ile-91 for Ala or Thr abrogated binding of Fig. 3 . Relative binding of CEACAM1-Fc constructs to N. meningitidis isolates expressing distinct Opa proteins. Bacterial lysates were dotted on to nitrocellulose and overlaid with NA1B-Fc constructs as indicated. Binding relative to the native receptor was determined by densitometric analysis of immno-blots using NIH Scion Image programme. One hundred per cent binding level is indicated by the horizontal line allowing comparison to native receptor binding. Mean values and SE of > 3 replicates are shown in each case, except Q89A (n = 1) for OpaA, B and X. However, alanine substitution at all three positions confirm previous observations (Virji et al., 1999) . Black inserts in C show the levels of binding of the various receptor constructs to Opaisolate of strain C751; experiments were conducted simultaneously with C751OpaD in the presence of the receptors shown. all three strains. However, substitution I91L reduced binding only of the THi strain Eagan, suggesting a requirement for the extended hydrophobic arm of Ile in Eagan binding. Alanine substitution of Tyr-34 abrogated the binding of Rd and Eagan, whereas no effect on NTHi A950002 was observed. In contrast, Y34F substitution led to an increased binding of all three strains varying from 45 to 80% above native receptor binding levels, whereas Y34S had a differential effect on the three strains tested, ranging from no binding of Rd to a slight increase in the binding of A950002. These data broadly reflect results with neisserial Opa binding. Finally substitutions at Gln-89 show that reduction or abrogation of the side chain (Q89N and Q89A) has no deleterious effect, rather its effect is often that of enhanced binding (Fig. 4) .
In summary, Hi strains primarily require Ile-91 to enable receptor targeting. Tyr-34 influences binding of THi and as for Nm, removal of the OH (Y34F) provides a more favourable environment. Gln-89 side chain also limits bacterial interaction and its substitution to shorter side chains (Q89A and Q89N) is more favourable especially for Rd-CEACAM binding. 
In previous studies, interactions of Hi-aeg strains were shown to be more analogous to Nm (Virji et al., 2000) . The current studies accordingly demonstrated a requirement both for Ile-91 and Tyr-34 for all Hi-aeg (Fig. 5) . In addition, as with some Nm strains, the requirement for hydrophobic and aliphatic chains was partly fulfilled by Leu and to a lesser extent by Thr. Substitutions of residue Tyr-34 with Phe or Ser produced a complex profile. Phe generally created a suitable or better environment but the loss of the aromatic ring was also tolerated (Y34S). At position 89, Q > N substitution enhanced binding dramatically for Ha3.
Overall, the three strains tested exhibited similar binding to all receptor constructs that had substitutions at residue
Ile-91 as well as those with alanine substitution at residue Tyr-34 (Fig. 5) . However, strains exhibited differences in binding to the receptors with substitutions Y34F and Q89N demonstrating the structural diversity of the CEACAM1-binding ligands of distinct Hi-aeg strains also.
From the above data, it is clear that receptor constructs with Q89N substitution have the capacity to increase the binding of some Hi over that of Nm. In previous investigations, we have shown that certain isolates of the two bacteria are capable of competing with the native receptor and that Nm C751 derivatives can displace Ha3 as well as Eagan from the receptor (Virji et al., 2000) . Here we investigated how changes in receptor structure at position 89 may influence this phenomenon. We used receptor dot blot overlay in the presence of varying amounts of the soluble receptor constructs to estimate the ability of C751OpaB and Ha3 to selectively adsorb the receptor. Consistent with the lower affinity of Ha3 compared with C751OpaB/D for CEACAM1 (Virji et al., 2000) , the proportion of the native CEACAM1 that interacted with Ha3 compared with C751OpaB was lower and declined further at limiting concentrations of the receptor (Fig. 6A ). In contrast, Ha3 and C751OpaB had similar affinities for the receptor when the Q89N construct was present in excess (> 0.13 mg ml -1 ; Fig. 6B ). Further, at limiting concentrations of Q89N, the greater affinity of Ha3 was apparent and bound this construct threefold more than the Nm derivative (Fig. 6B ). The data demonstrate the potential influence of structural modulations at the bacterial binding site in changing the colonization profile of the target tissue.
To assess whether CEACAM mutations that either significantly increase or decrease bacterial binding to the soluble constructs also affect bacterial binding to cellexpressed receptor constructs, we analysed COS cells transiently transfected with CEACAM1-4L containing Q89A, Q89N and I91A substitutions. Initially, binding of the anti-CEACAM antibodies to transfected CHO cells was examined and their binding was as observed for the soluble receptors (Fig. 7) . However, bacterially expressed ligands may overcome decreased affinity for specific mutant proteins due to multiple ligand-receptor engagement at the target cell surface. Examination of adherent bacteria and levels of receptor expression by microscopy revealed that bacteria bound to all transfectants other than sham-transfected cells (Fig. 8) . However, receptor expression levels had to be considerably high in the case of I91A for significant bacterial numbers to bind the transfectants. Whereas for Q89A and Q89N, bacteria could bind to cells with barely detectable levels of receptors (Fig. 8) .
Using CHO cells expressing full length CC1 or CC1(Q89N), the ability of the soluble native (CC1-Fc) or CC1(Q89N)-Fc to inhibit bacterial binding was assessed using C751 OpaB bacteria (Fig. 9 ). Data show the following: (i) Binding to CHO cells as reported previously (Virji et al., 1996b) was observed only with Opa+ and not Opa-bacteria and no binding was seen with untransfected cells (not shown), (ii) Despite the low levels of binding of the isolate to the soluble CC1 construct carrying the Q89N mutation in the dot immunoblot analysis (Fig. 3B) , bacterial binding to the receptor expressed on the cells was clearly visible (Fig. 9D) , (iii) As in the case of THi Rd described above, association was only significantly high with cells expressing high levels of the receptor (assessed in parallel experiments, not shown), (iv) CC1-Fc could compete with bacterial binding to the homologous native structure expressed on CHO cells (Fig. 9B) , confirming previous results (Virji et al., 1996b) , (v) In comparison, CC1-Fc could more efficiently compete out C751 OpaB binding to the CC1(Q89N) receptor expressed on CHO cells (Fig. 9E) , consistent with its higher affinity for the isolate than CC1(Q89N)-Fc (Fig. 3B) , (vi) CC1(Q89N)-Fc construct was largely ineffective in competing with either cell-expressed receptor ( Fig. 9C and F) ent bacterial numbers are thus also low (cf. Fig. 9D and F: lack of low level binding in F compared with D). The data emphasize the importance of cell presentation of receptor in addition to receptor structure in bacterial interactions.
The final outcome of this depends on the balance between the effects exerted by residue substitutions and receptor density. Both can affect functional affinity of bacteria-host interactions. 
To assess the relative importance and the degree of influence of each substitution on bacterial interactions, degrees of receptor recognition were assigned several categories as shown in Fig. 10A . The data show that substitution I91A has a profound effect on the interactions of all strains and whilst the side chain of Ile-91 is best suited to all, Leu can effectively substitute for Ile for several strains, whilst the polar Thr is less well tolerated. However, some Nm Opa proteins can also tolerate I91T substitution. Tyr-34 is also required in most cases. Interestingly, Phe-34 is preferred in general at this position over the native Tyr. Substitutions at Gln-89 have a delete- For the native receptor, expression levels and bacterial binding correlated more frequently than for the mutated molecules. In the case of substitutions at position 89, bacterial binding could be seen at very low levels of receptor expression (arrows in Q89A and Q89N panels). The reverse was the case with I91A, where even at high receptor levels (arrowhead, left panel), bacterial binding (arrowhead middle panel) was relatively low. In sampling of 100 cells, c. 50% exhibited this phenomenon. Note that the receptor recognition by Kat4C was not affected by mutations in the N-domain as determined by immunoblotting or by immunofluorescence microscopy (Fig. 7) .
rious effect on binding of some Nm Opa derivatives and the smaller Q89N apparently is better suited overall for binding by Hi and especially Hi-aeg strains. The data emphasize the extensive inter-and intraspecies ligand variations in this receptor targeting, perhaps with greater interstrain differences in Nm. Structural aspects of bacterial ligands that affect the receptor recognition can be considered for strain C751 Opa proteins whose structures are known (Fig. 9B ) (Hobbs et al., 1994) . The hypervariable loops HV1 and HV2 of Opa proteins have been implicated in the binding of CEACAMs (Virji et al., 1999; Bos et al., 2002; de Jonge et al., 2003) . Given that the combinations of HV regions of OpaA, B and D of strain C751 provide three distinct combinations (Fig. 10C) , this is consistent with the distinct patterns of targeting of the variant receptor molecules observed in the study. However, the proteins must all contain sufficient similarity to bind the hydrophobic region around I91.
The N-terminal domains of the cell-expressed CEA family of molecules are highly homologous and the majority of CEACAMs are targeted by one or more pathogenic neisserial adhesins belonging to the Opa family of proteins (Virji et al., 1996a; Chen et al., 1997; Gray-Owen et al., 1997) . Despite the structural variability, all Opa proteins target a common site on the receptors whose centre of binding appears to be Ile-91 on the CFG face of the N-domains. Ile-91 is conserved throughout the CEA members. Most of the other important residues on CEACAM1 identified by alanine scanning mutagenesis located in a close proximity of Ile-91 (Virji et al., 1999) . Precisely how variant Opa proteins can bind to a common receptor site is not entirely clear but a complementary set of sequences of more than one variable domains of Opa proteins may be involved. This variability of the ligands may determine the binding preference for distinct members of the CEA family which represents one mechanism that may determine tissue tropism (Virji et al., 1999; Bos et al., 2002; Gray-Owen, 2003; Hauck and Meyer, 2003; de Jonge et al., 2003) .
Diverse strains of typable and NTHi lineages including the biogroup aegyptius also bind to the CFG face of the N-domain of CEACAM1 (Virji et al., 2000) . In our previous analysis of receptor binding, THi isolates were shown to behave similarly: each strain tested having a primary requirement for Ile-91 and in addition, was affected by Y34A and Q44A substitutions. In the current study also, the THi strain Rd and Eagan demonstrate similar overall binding patterns. However, in all cases, intraspecies dif- Fig. 9 . Binding of N. meningitidis isolate C751OpaB to cell-expressed receptors: competition between cell-expressed and soluble receptors. Bacterial binding to cell-expressed CC1 or Q89N construct was detected using anti-Nm antisera and TRITC-conjugated secondary antibodies. The interactions of bacteria with the cell-expressed receptors were investigated in the absence (A, D) or presence of competing soluble receptors. Although binding to the soluble receptor Q89N is much lower than the native CC1 in dot-blots (Fig. 3B) , it is relatively high on certain cells (presumably, on those expressing high levels of the receptor) (D). In competitive experiments, the native CC1-Fc (B, E) and the CC1(Q89N)-Fc (C, F) were preincubated at 10 ug ml -1 with bacteria for 15 min, prior to infection of target cells. CC1-Fc inhibited bacterial binding to the homologous receptor significantly (B), and almost abrogated binding to cell-expressed Q89N (E); whereas the soluble Q89N was inefficient at inhibiting bacterial binding to CHO-CC1 (C). However, a level of homologous inhibition was apparent when examining the adhesion of bacteria to cells expressing low levels of the receptor (e.g. 'peppered' areas shown in D are less evident in F).
CHO-CC1(Q89N)
ferences in response to different amino acid substitutions were apparent. One consistency between all species and strains tested was the dramatic loss of binding following I91A substitution which, as observed previously, appears to be central in an overlapping bacterial binding footprint on CEACAM1 (Virji et al., 1999; . A similar situation occurs on the mouse CEACAM1a (MHVR1a) N-terminal domain, in which Ile-41 appears to engage with the mouse hepatitis virus spike protein. Replacement of Ile-41 by Thr in the MHVR1b allele reduces virus binding significantly (Tan et al., 2002) . Cell surface receptor interactions with their ligands often involve hydrophobic contact points which provides the major binding energy. Hydrophobic residues surrounding these contribute to the specificity of binding (Clackson and Wells, 1995; Kwong et al., 1998; Kim et al., 2001) . Thus in murine CEACAM1, the protruding Ile-41, which is surrounded by a number of surface exposed charged residues, e.g. Asp-42, Glu-44, Arg-47, Asp89, Glu-93 and Arg-97, might form such a binding area (Tan et al., 2002) . Accordingly from current studies also, mutations introduced at position 91 in the human receptor support the requirement for a hydrophobic pocket at this site. In addition, an extended aliphatic chain is preferred as Ala disrupted binding of all bacteria and the polar Thr reduced binding of all Hi and several Nm strains. Only I91L was more frequently tolerated. This binding pocket is flanked by several polar residues (Fig. 1 ) whose contribution to bacterial ligand binding is apparent and variable (Virji et al., 1999; . The importance of the Ile-91 and the surrounding residues on human CEACAM1 in bacterial binding is also supported by the observation that the mAb YTH71.3 directed against the N-domain also requires several residues in common with bacteria and blocks binding of all CEACAM1-binding bacteria we have investigated (Virji et al., 1999; Hill and Virji, 2003) .
The murine N-domain strand arrangement derived form crystal structure depicts the CC′ loop to assume a convoluted conformation. The previous model of human CEACAM1 contained a flat CC′ loop like the Ig-folds on Fig. 10 . Binding of bacterial ligands to CEACAM1-Fc constructs as determined in the current study. A. Receptor recognition categories are colour coded according to percent binding relative to the native molecule*. B and C. Diagrams showing CEACAM-binding meningococcal ligand structures. As only the Opa protein structure has been analysed in details so far with respect to CEACAM binding (de Jonge et al., 2003) , for clarity and ease of discussion, a 2D Opa protein structure (B) and relationship of the three variable domains of strain C751 Opa proteins are shown (C). SV, semivariable domains of strain C751 Opa-A, -B and -D are identical as are the hypervariable structures HV1 of OpaA and OpaB and the HV2 of OpaB and OpaD. which it was based. This resulted in Val-39 and Gly-41 being located at a distance from the binding focus Ile-91. Both Val-39 and Gly-41 have been implicated in bacterial binding from alanine scanning and homologue scanning mutagenesis (Bos et al., 1999; Virji et al., 1999) . The remodelling shown here of CEACAM1 produces the convoluted structure of the CC′ loop relocating Val-39 close to Ile-91. Further, the aromatic ring of Tyr-34 suggested to be required to maintain the convoluted structure of the CC′ loop (Tan et al., 2002) when substituted with Phe almost always supported bacterial binding. Indeed, Y34F provides a better environment for most bacterial strains tested. On the other hand, Y34A frequently abrogated receptor recognition. However and surprisingly, Y34S is tolerated by a substantial number of bacterial ligands. Whether this is due to the flexible variable loop domains of the bacterial ligands which may produce an induced fit around the receptor needs consideration. Interestingly, Tyr-34 is conserved in the majority of the human CEACAMs with the exception of CEACAM4 which contains His at this site. However, the importance of Tyr-34 in human CEACAM1 maintaining the three dimensional structure requires human receptor crystallographic data.
Substitutions Y34F and Q89N also produced interesting data from the point of view of pathogenesis. Whilst Q89A and Q89N appear to affect some Opa proteins by reducing receptor recognition, suggesting its potential contribution in determining tissue tropism (Virji et al., 1999) , Q89N substitution occasionally caused dramatic increase in bacterial adhesion, especially of Hi isolates. Y34F, as observed above, increased binding of strains within all species examined. As it is possible that multiple receptors presented on the target cells may overcome the reduced binding affinity of mutated receptors, we examined strain Rd binding to I91A and Q89A/N-substituted receptors expressed in transiently transfected COS cells. Whilst the latter receptors were targeted on cells with low levels of receptor expression, only a proportion (c. 50%) of cells with very high levels of I91A receptors had significant numbers of bacteria attached. Thus point mutations of CEACAMs can both decrease and increase bacterial load and additional factors that dictate bacterial binding include receptor levels on the target cells.
The ligands of Neisseria (i.e. Opa proteins) and of Hi so far identified (i.e. P5 proteins) share similar beta-barrel structure with surface-exposed variable loops. The regions of P5 that may engage with the receptor have not been identified but those of Opa proteins were studied by mutagenesis of strain H44/76 (de Jonge et al., 2003) . This strain is related to strain MC58, one of the strains used in the current study. The studies of de Jonge et al. implicated G ¥ (I/V/l) ¥ (S/E/Q) as the key motifs of HV2 regions (Fig. 9 ) of meningococcal Opa proteins in receptor targeting. Together with this, an 99 ELK motif of the Opa HV1 region might be involved in the three dimensional presentation of the receptor-engaging residues of the bacterial ligand (de Jonge et al., 2003) . Within the strain C751, OpaB and OpaD proteins contain the motif GxLxS at positions 172-176 and 167-171, respectively, whereas Opa A contains a 168 PxIxN motif. In the HV1 region, OpaA and B contain 99 DLK whereas OpaD contains EDK. Studies in our laboratories are in progress to assess the precise variant C751 Opa and receptor residue pairs involved in mutual recognition.
Polymorphisms that affect host susceptibility may be found at various sites in the genes encoding host receptors targeted by pathogens and may result in loss or gain of receptor-associated functions. Some SNPs may lead to multiple and diverse downstream effects, e.g. altered transcriptional response and manifestation of disease (Sakuntabhai et al., 2005) . Extracellular domain polymorphisms may have a more direct effect via altered binding of pathogen ligands to their receptors. SNPs of the innate immune system especially those affecting pathogen associated pattern recognition receptors and cytokines have been studied extensively. Changes such as Asp299Gly and Thr399Ile in the extracellular domains of LPS-binding Toll-like receptor 4 (TLR4) have been implicated in increased risk to bacterial infections (Schroder and Schumann, 2005) . These SNPs have also been associated with severe respiratory syncytial virus (RSV) bronchiolitis in infants. In this case, altered interaction of the viral fusion (F) protein, implicated as a ligand for TLR4, is regarded as the primary mechanism (Tal et al., 2004; Schroder and Schumann, 2005) . However, these TLR4 SNPs could not be correlated with meningococcal disease (Read et al., 2001) . In contrast, rare SNPs were found more commonly in the TLR4 genes of patients with meningococcal disease (Smirnova et al., 2003) . This supports the notion that rare rather than common variants of TLR4 may be associated with infectious disease susceptibility.
Studies presented here suggest some possible polymorphisms that can increase bacterial load. Several SNPs in CEACAMs have been identified and are listed in the NCBI SNP database and three have been identified in the N-domain of CEACAM1 including one at Gln-89. In this case, a Gln-89 to His substitution is observed. Such a residue difference occurs within the members of CEACAM family. For example, CEA contains H at position 89 (Fig. 2) . This is the only major difference between CC1 and CEA that could affect bacterial binding (Fig. 2) . As such, the mutation Q89H in CC1 would be expected to produce CEA-like binding pattern and could affect tropism of the bacteria as observed for CEA (Virji et al., 1999; de Jonge et al., 2003) . Further studies are required to assess whether other CEACAM polymorphisms, for example, substitutions such as Y34F and Q89N occur in human Molecular analysis of bacterial ligand-CEACAM1 interactions 341 populations and their frequencies in susceptible populations. SNP substitutions may also change the colonization profile of the nasopharynx, because in our competition studies we could demonstrate that increased binding afforded by Q89N to Hi-aeg isolate Ha3 increases the binding of Ha3 such that it out-competes Nm isolate C751OpaB in an in vitro competition assay for this receptor. The situation with the native receptor was the reverse. As shown in Figs 8 and 9, additional factors that affect bacterial binding to cell-expressed receptor include receptor density. Whilst certain residue substitutions, e.g. I91A, reduce functional affinity of bacterial interactions, high receptor densities increase such affinity. The final outcome must depend on the interplay between these two parameters. In recent studies, the role of receptor density on enhancement of bacterial attachment and invasion have been evaluated in detail (Bradley et al., 2005; Rowe et al., 2006) . It would be interesting to analyse bacterial invasion in cell lines expressing variant CEACAM1 carrying the above mutations by employing cell lines in which the receptor expression levels can be controlled, which are under development.
Besides receptor polymorphisms, several other scenarios may lead to increased bacterial ligand binding to CEACAMs. Both Nm and Hi CEACAM-binding ligands (Opa and P5) are known to undergo antigenic variation. Thus, in any population, antigenic/structural variants are present and may be selected for during the course of host colonization and subsequent pathogenesis (Virji et al., 1996a; Duim et al., 1997; Meyers et al., 2003) . The receptor repertoire and subtypes may select bacteria capable of binding with high affinity. As mentioned above, upregulation of receptor expression on target cells may also increase bacterial binding affinity. In such cases, high affinity interactions result in cellular invasion, whereas lower affinity or load of bacteria may not proceed beyond surface adhesion (Tran Van Nhieu and Isberg, 1993; Bradley et al., 2005) . The expression of CEACAMs on normal epithelia of the respiratory tract has been reported, which would allow bacterial attachment and possible subsequent penetration into these tissues (Tsutsumi et al., 1990; Virji, 2001) . Following exposure to cytokines such as IFN-g or TNF-a, CEACAM expression by colonic carcinoma cells has been shown to increase (Fahlgren et al., 2003) . In addition, certain viral infections have also been shown to upregulate CEACAM1 expression in several epithelial cell lines (Avadhanula et al., 2006) . Increased cytokine levels following viral infection could lead to increased CEACAM expression and bacterial association with respiratory epithelia and subsequent invasion of deeper tissue by these organisms. Such a situation may explain the epidemiological association of increased incidence of Nm and Hi infections following certain viral infections (Cartwright et al., 1991; Takala et al., 1993) .
In summary, little is known about why certain people are more susceptible to infection by some of the frequent colonizers of the human nasopharynx. Interestingly, opportunistic pathogens such as Nm and Hi as well as M. catarrhalis (not investigated here) target CEACAM1. As specific substitutions such as Y > F and Q > N produce more favourable targets for distinct mucosal isolates, it is possible that occurrence of such receptor polymorphisms in the human population could lead to greater bacterial binding thus increasing the chances of cellular invasion. Given the colonization rate of these organisms (generally > 10% of the population) and the frequency of invasive infection (up to 3:100 000 population), a combination of events may be required to increase host susceptibility. Inflammatory conditions that increase receptor density in populations carrying specific polymorphisms could provide the worst scenario.
The sequence of mature human CEACAM1 N-domain was aligned with the corresponding domain of murine CEACAM1, giving a gapless alignment for residues 1-109 with a residue identity of 42%. The crystal structure co-ordinates of mouse CEACAM1 (residues 1-109) were taken from the structure file containing domains 1 and 2 (PDB code 1L6Z) and a homology model of human CEACAM1 N-domain was built using standard methods. Final refinement of the model was performed by soaking it with a 5 Å thick layer of water and energy minimizing while constraining the backbone atoms to their original positions in the template structure. The final round of minimization was for 2000 conjugate-gradient steps, constraining the backbone heavy atoms with a force constant of 0.5 kcal/Å. A stereochemical analysis of the structure was performed using Procheck and found to be of similar quality to the template crystal structure.
Production of mutants was carried out by site-directed mutagenesis of the pIG construct containing the DNA encoding the CEACAM1 NA1B domains described previously (Watt et al., 1994) . The oligonucleotide primers used to create amino acid substitutions at positions 91, 34 and 89 of the N-domain are shown in Table 1 . Some primer sequences have been published previously (Watt et al., 1994) . For introducing mutations, CEACAM1 was amplified by polymerase chain reaction from the pIG-NA1B construct using either the common forward primer and a reverse primer containing the desired mutation, or a complementary forward primer containing the mutation and a common reverse primer. CEACAM1 with the appropriate mutation was amplified using the common forward and common reverse primers. The gene was then cloned into pIG using the restriction sites HindIII and EcoRI.
Chimeric soluble receptor proteins containing the appropriate amino acid substitutions were prepared as previously described by transient transfection of COS cells (Teixeira et al., 1994; Virji et al., 1999) . The CEACAM1-Q89A-Fc used in overlay experiments was kindly donated by Dr S. Watt (Virji et al., 1999; Watt et al., 2001) .
The strains used in this study have been described previously (Virji et al., 1999; . Nm strain C751 (serogroup A) variants used were C751OpaA, C751OpaB and C751OpaD. The strain MC58 (serogroup B) variant used expressed an Opa previously designated OpaX, which is encoded by the opaB locus. Opa -C751 isolate, which has been shown not to bind to CHO-CC1 (Virji, 1999) and RdCC-, a derivative of THi Rd, known not to bind to CHO-CC1 (M. Virji, unpublished) were used as controls. THi strain Rd is an acapsulate serotype d isolate, Eagan is serotype b isolate and A950002 is a NTHi strain. Hi-aeg strains Ha3, Ha30 and F2087 are all conjunctiva isolates. Nm was grown on brainheart infusion (BHI) agar supplemented with 10% heated horse blood (HBHI). Hi strains were grown on HBHI agar further supplemented with Levinthal base (10 mg ml -1 each of NAD and haemin). All strains were cultured at 37°C in 5% CO2.
COS-1 cells (African green monkey kidney cells) used for transient transfection were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2-10% heat-inactivated Foetal Calf Serum (FCS, Gibco™), 2 mM glutamine, 50 mg ml -1 penicillin and 50 mg ml -1 streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
Antibody binding to CEACAM1 constructs. NA1B-Fc proteins were dotted at 0.2 mg ml -1 on to nitrocellulose and non-specific binding sites blocked using 3% (w/v) BSA in Dulbecco's PBS containing 0.05% Tween-20 (PBST) for 1 h at room temperature. Receptor was detected using the following antibodies, rabbit polyclonal AO115, rat monoclonal YTH71.3 both directed against the N-domain and mouse monoclonal Kat4c which recognizes the A and B domains (Jones et al., 1995) . Bound antibody was subsequently detected using an appropriate secondary antibody conjugated to alkaline phosphatase and developed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.
Bacterial interactions with receptor constructs. Bacterial lysates (c. 2 ¥ 10 7 bacteria) were applied to nitrocellulose strips, air-dried and non-specific binding sites blocked using 3% BSA in PBST for 1 h at room temperature. Strips were overlaid with either native or mutated soluble NA1B-Fc diluted in 1% BSA in PBST at required concentrations for 1 h at room temperature. In most experiments, excess (1-3 mg ml -1 ) of the receptor was used. In competition studies, a range of concentrations (0.008-0.5 mg ml -1 ) was employed. Following washing to remove unbound NA1B-Fc, receptor binding was detected using anti-human-Fc alkaline phosphatase conjugate and substrates as described above. For quantification, densitometric analyses of the developed immunoblots were carried out using NIH Scion Image software. In most cases, multiple estimations were carried out and means and SE of each determination have been reported.
Site directed mutagenesis of CEACAM1-4L receptor gene was performed using the QuickChange® Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Primers (Table 1) were used to introduce the desired mutations into the pRc/CMV-CEACAM1-4L construct (kindly provided by Professor Wolfgang Zimmermann). Following sequencing to ensure the desired substitution had been obtained, the pRc/CMV-CEACAM1-4L construct was transiently transfected into COS-1 cells for functional analysis using DEAE dextran method described previously (Teixeira et al., 1994; Virji et al., 1996a) .  Immune reconstitution inflammatory syndrome (IRIS): review of common infectious manifestations and treatment options The immune reconstitution inflammatory syndrome (IRIS) in HIV-infected patients initiating antiretroviral therapy (ART) results from restored immunity to specific infectious or non-infectious antigens. A paradoxical clinical worsening of a known condition or the appearance of a new condition after initiating therapy characterizes the syndrome. Potential mechanisms for the syndrome include a partial recovery of the immune system or exuberant host immunological responses to antigenic stimuli. The overall incidence of IRIS is unknown, but is dependent on the population studied and its underlying opportunistic infectious burden. The infectious pathogens most frequently implicated in the syndrome are mycobacteria, varicella zoster, herpesviruses, and cytomegalovirus (CMV). No single treatment option exists and depends on the underlying infectious agent and its clinical presentation. Prospective cohort studies addressing the optimal screening and treatment of opportunistic infections in patients eligible for ART are currently being conducted. These studies will provide evidence for the development of treatment guidelines in order to reduce the burden of IRIS. We review the available literature on the pathogenesis and epidemiology of IRIS, and present treatment options for the more common infectious manifestations of this diverse syndrome and for manifestations associated with a high morbidity. Since its introduction, ART has led to significant declines in AIDS-associated morbidity and mortality [1] . These benefits are, in part, a result of partial recovery of the immune system, manifested by increases in CD4 + T-lymphocyte counts and decreases in plasma HIV-1 viral loads [2] . After initiation of ART, opportunistic infections (OI) and other HIV-related events still occur secondary to a delayed recovery of adequate immunity [3] .
Some patients initiating ART experience unique symptoms during immune system recovery. In these patients, clinical deterioration occurs despite increased CD4 + Tlymphocyte counts and decreased plasma HIV-1 viral loads [4] . This clinical deterioration is a result of an inflammatory response or "dysregulation" of the immune system to both intact subclinical pathogens and residual antigens [5] [6] [7] [8] [9] . Resulting clinical manifestations of this syndrome are diverse and depend on the infectious or noninfectious agent involved. These manifestations include mycobacterial-induced lymphadenitis [5] , paradoxical tuberculosis reactions [6, 7, 10, 11] , worsening of progressive multifocal leukoencephalopathy (PML) [12] , recurrence of cryptococcosis and Pneumocystis jirovecii pneumonia (PCP) [8, [13] [14] [15] [16] , Cytomegalovirus (CMV) retinitis [17] , shingles [18] , and viral hepatitis [19] , as well as noninfectious phenomena [20] .
Because clinical deterioration occurs during immune recovery, this phenomenon has been described as immune restoration disease (IRD), immune reconstitution syndrome (IRS), and paradoxical reactions. Given the role of the host inflammatory response in this syndrome, the term immune reconstitution inflammatory syndrome (IRIS) has been proposed [21] and has become the most widely used and accepted term to describe the clinical entity. Possible infectious and noninfectious etiologies of IRIS are summarized in Table 1 .
To date, no prospective therapeutic trials concerning the management of IRIS have been conducted. All evidence regarding the management of IRIS in the literature relates to case reports and small case series reporting on management practice. This does not provide reliable evidence regarding either the safety or efficacy of these approaches, but merely guidance regarding the practice of others in managing this difficult condition. In severe cases where the discontinuation of ART is a possibility, the potential disadvantages of therapy cessation, such as the development of viral resistance or AIDS progression, should be considered.
Despite numerous descriptions of the manifestations of IRIS, its pathogenesis remains largely speculative. Current theories concerning the pathogenesis of the syndrome involve a combination of underlying antigenic burden, the degree of immune restoration following HAART, and host genetic susceptibility. These pathogenic mechanisms may interact and likely depend on the underlying burden of infectious or noninfectious agent.
Whether elicited by an infectious or noninfectious agent, the presence of an antigenic stimulus for development of the syndrome appears necessary. This antigenic stimulus can be intact, "clinically silent" organisms or dead or dying organisms and their residual antigens. IRIS that occurs as a result of "unmasking" of clinically silent infection is characterized by atypical exuberant inflammation and/or an accelerated clinical presentation suggesting a restoration of antigen-specific immunity. These characteristics differentiate IRIS from incident opportunistic infections that occur on ART as a result of delayed adequate immunity.
Examples of IRIS in response to intact organisms include, but are not limited to, the unmasking of latent cryptococcal infection [22] and infection with Mycobacterium avium complex (MAC) [4, 5, 23, 24] . The most frequently reported IRIS symptoms in response to previously treated or partially treated infections include reports of clinical worsening and recurrence of clinical manifestations of Mycobacterium tuberculosis (TB) and cryptococcal meningitis following initiation of ART [6, 7, 10, 13, 16, [25] [26] [27] [28] . In noninfectious causes of IRIS, autoimmunity to innate antigens plays a likely role in the syndrome. Examples include exacerbation of rheumatoid arthritis and other autoimmune diseases [29] . Given the role of this antigenic stimulus, the frequency and manifestations of IRIS in a given population may be determined by the prevalence of opportunistic and non-opportunistic infections to initiation of ART.
The mechanism receiving the most attention involves the theory that the syndrome is precipitated by the degree of immune restoration following ART. In assessing this theory, investigators have examined the association between CD4 cell counts and viral loads and the risk of IRIS. Some studies suggest differences in the baseline CD4 profiles or quantitative viral load at ART initiation or their rate of change during HAART between IRIS and non-IRIS patients [4, [30] [31] [32] [33] [34] , while other studies demonstrate only trends or no significant difference between IRIS and non-IRIS patients [7, 35] . These immunological differences between groups have been difficult to verify due to small numbers of IRIS cases and lack of control groups. An alternative immunological mechanism may involve qualitative changes in lymphocyte function or lymphocyte phenotypic expression. For instance, following ART an increase in memory CD4 cell types is observed [36] possibly as a result of redistribution from peripheral lymphoid tissue [37] . This CD4 phenotype is primed to recognize previous antigenic stimuli, and thus may be responsible for manifestations of IRIS seen soon after ART initiation. After this redistribution, naïve T cells increase and are thought to be responsible for the later quantitative increase in CD4 cell counts [38] . These data suggest IRIS may be due to a combination of both quantitative restoration of immunity as well as qualitative function and phenotypic expression observed soon after the initiation of ART.
The third purported pathogenic mechanism for IRIS involves host genetic susceptibility to an exuberant immune response to the infectious or noninfectious anti-genic stimulus upon immune restoration. Although evidence is limited, carriage of specific HLA alleles suggest associations with the development of IRIS and specific pathogens [39] . Increased levels of interleukin-6 (IL-6) in IRIS patients may explain the exuberant Th1 response to mycobacterial antigens in subjects with clinical IRIS [9, 40] . Such genetic predispositions may partially explain why manifestations of IRIS differ in patients with similar antigenic burden and immunological responses to ART.
Despite numerous descriptions of the infectious and noninfectious causes of IRIS, the overall incidence of the syndrome itself remains largely unknown. Studies to date are often retrospective and focus on specific manifestations of IRIS, such as tuberculosis-associated IRIS (TB-IRIS). In a large retrospective analysis examining all forms of IRIS, 33/132 (25%) of patients exhibited one or more disease episodes after initiation of ART [4] . Other cohort analyses examining all manifestations of IRIS estimate that 17-23% of patients initiating ART will develop the syndrome [32] [33] [34] . Another large retrospective study reported 32% of patients with M. tuberculosis, M. avium complex, or Cryptococcus neoformans coinfection developed IRIS after initiating ART.
Risk factors identified for the development of IRIS in one cohort included male sex, a shorter interval between initiating treatment for OI and starting ART, a rapid fall in HIV-1 RNA after ART, and being ART-naïve at the time of OI diagnosis [31] . Other significant predictors have also included younger age, a lower baseline CD4 cell percentage, a lower CD4 cell count at ART initiation, and a lower CD4 to CD8 cell ratio at baseline [4, 32] . It should be noted cohorts differ substantially in study populations and the type of IRIS (i.e. TB-IRIS only) examined, making conclusions regarding risk factors for IRIS difficult. Clinical factors associated with the development of IRIS are presented in Table 2 .
Case reports describing different clinical manifestations of IRIS continue to appear, expanding the clinical spectrum of the syndrome. Because the definition of IRIS is one of clinical suspicion and disease-specific criteria have yet to be developed, determining the true incidence will be difficult. Taken together, these studies suggest IRIS may affect a substantial proportion of HIV patients initiating ART. Future epidemiologic and genetic studies conducted within diverse cohorts will be important in determining the importance of host susceptibility and underlying opportunistic infections on the risk of developing IRIS.
In order to aid clinicians in the management of IRIS, we review the epidemiology, clinical features, and treatment options for the common infectious manifestations of IRIS. Additionally, manifestations associated with significant morbidity and mortality, such as CMV-associated immune recovery vitritis (IRV) or immune recovery uveitis (IRU), are also reviewed. Treatment options and their evidence are presented. Until disease specific guidelines are developed for IRIS, therapy should be based on exist- [4, 6, 7, 10, 11, 26, 30-32, 41, 43, 45] Rheumatoid arthritis [29] Systemic lupus erythematosus (SLE) [91] Graves disease [92] , Autoimmune thyroid disease [93] Mycobacterium avium complex [4, 5, 23, 31, [94] [95] [96] Sarcoidosis & granulomatous reactions [20, 97] Other mycobacteria [4, 56, 57, 98, 99] Tattoo ink [100] Cytomegalovirus [4, 33, 61, 63] AIDS-related lymphoma [ [112] Molluscum contagiosum & genital warts [32] Sinusitis [113] Folliculitis [114, 115] ing evidence and individualized according to the severity of presentation.
Mycobacterium tuberculosis (TB) is among the most frequently reported pathogen associated with IRIS. Narita et al performed the first prospective study to evaluate the incidence of paradoxical responses in patients on TB therapy and subsequently initiated on ART. Of 33 HIV/TB coinfected patients undergoing dual therapy, 12 (36%) developed paradoxical symptoms [7] . The frequency of symptoms in this group were greater than those observed in HIV-infected controls receiving TB therapy alone, supporting the role of an exaggerated immune system response in the pathogenesis of the syndrome. Retrospective studies corroborate the finding that a significant proportion of HIV/TB coinfected patients undergoing HAART have symptoms consistent with IRIS, with estimates ranging from 7-45% [10,26, 30, 35, [41] [42] [43] .
The association between a shorter delay between TB treatment initiation and ART initiation is an area of debate. While some investigators have found no difference in time from TB therapy to initiation of ART between IRIS and non-IRIS subjects [30] , others have reported a significant differences between groups [31, 35] . In general, IRIS occurred in subjects initiated on ART within two months of TB therapy initiation [35] . Based on these and other data, a decision analysis on ART initiation timing in TB patients found the highest rates of IRIS occurred in patients initiated on ART within two months of TB therapy initiation [44] . However, withholding or deferring ART until two to six months of TB therapy was associated with higher mortality in scenarios where IRIS-related mortality was less than 4.6%. Future reports from large, prospective observational cohorts may aid in resolving this difficult issue.
Although consisting primarily of case reports [45, 46] , TB-IRIS affecting the central nervous system (CNS) poses a unique problem. As the availability of ART increases in endemic countries, the incidence of CNS TB-IRIS may increase. Thus, clinicians should be vigilant in its diagnosis.
The commonest clinical manifestations of TB-IRIS are fever, lymphadenopathy and worsening respiratory symptoms [47] . Pulmonary disorders, such as new pulmonary infiltrates, mediastinal lymphadenopathy, and pleural effusions are also common [7] . Extrapulmonary presentations are also possible, including disseminated tuberculosis with associated acute renal failure [6] , systemic inflammatory responses (SIRS) [48] , and intracranial tuberculomas [45] . Pulmonary TB-IRIS can be diagnosed by transient worsening of chest radiographs, especially if old radiographs are available for comparison. Other symptoms are nonspecific, and include persistent fever, weight loss, and worsening respiratory symptoms. Abdominal TB-IRIS can present with nonspecific abdominal pain and obstructive jaundice.
In most studies, TB-IRIS occurs within two months of ART initiation [6, 7, 10, 11, 25, 35, 45, 48] . Among 43 cases of MTB-associated IRIS, the median onset of IRIS was 12-15 days (range 2-114 days), with only four of these cases occurring more than four weeks after the initiation of antiretroviral therapy [7, 10, 25, 26, 30] . These studies suggest the onset of mycobacterial-associated IRIS is relatively soon after initiation of ART, and clinicians should maintain a high level of vigilance during this period.
Paradoxical CNS TB reactions are well described in HIVnegative patients, and include expanding intracranial tuberculomas, tuberculous meningitis, and spinal cord lesions [49] [50] [51] . TB-associated CNS IRIS has also been reported in HIV-positive patients [45, 46, 52] . Compared to non-CNS TB-IRIS, symptoms tend to occur later, usually 5-10 months after ART initiation [45, 50, 52] . Crump et al [45] described an HIV-seropositive patient in who developed cervical lymphadenopathy after five weeks of ART. Five months later, CNS symptoms associated with an expanding intracranial tuberculoma appeared after initiation of antituberculous therapy. The significant morbidity in this case illustrates the importance of maintaining a high clinical suspicion for the disease, particularly in endemic areas.
Treatment for mycobacterial-associated IRIS depends on the presentation and disease severity. Most patients present with non-life threatening presentations which respond to the institution of appropriate antituberculous therapy. However a range of life threatening presentations, such as acute renal failure [6] and acute respiratory distress syndrome (ARDS) [11] , are described and have significant morbidity and mortality. Morbidity and mortality might also be greater in resource-limited settings where limited management options exist. Since the pathogenesis of the syndrome is an inflammatory one, systemic corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDS) may alleviate symptoms. In studies where therapy for IRIS was mentioned, the use of corticosteroids was variable [7, 24, 25, 31, 41, 43] and anecdotally effective. Therapies ranged from intravenous methylprednisolone 40 mg every 12 hours to prednisone 20-70 mg/day for 5-12 weeks. These practices reflect the lack of evidence from controlled trials for the use of anti-inflammatory agents in IRIS. A randomized, placebo controlled trial examining doses of prednisone 1.5 mg/kg/day for two weeks followed by 0.75 mg/kg/day for two weeks in mild to moderate TB-IRIS is currently underway in South Africa. Until data become available, it is reasonable to administer corticosteroids for severe cases of IRIS such as tracheal compression due to lymphadenopathy, refractory or debilitating lymphadenitis, or severe respiratory symptoms, such as stridor and ARDS. Interruption of ART is rarely necessary but could be considered in life-threatening situations.
In HIV-negative patients, adjuvant corticosteroid use in tuberculous meningitis provides evidence of improved survival and decreased neurologic sequelae over standard therapy alone [53, 54] . Once other infectious etiologies, have been excluded, standard antituberculous therapy should be initiated or continued as the clinical situation dictates, and a course of corticosteroid therapy should be considered for CNS TB-IRIS. Continuation of ART is desirable, although its discontinuation may be necessary in unresponsive cases or in those presenting with advanced neurological symptoms.
In addition to TB, atypical mycobacteria are also frequently reported as causative pathogens in IRIS. Early observations involving atypical presentations of Mycobacterium avium-intracellulare (MAC) were first noted with zidovudine therapy [55] . Reports of atypical presentations of both Mycobacterium tuberculosis (MTB) and MAC increased in frequency with the introduction of protease inhibitors and ART. In larger cohorts, MAC remains the most frequently reported atypical mycobacterium [4, 5, 24] . Other atypical mycobacteria rarely associated with IRIS are referenced in Table 1 .
In general, MAC-associated IRIS typically presents with lymphadenitis, with or without abscess formation and suppuration [5] . Other less common presentations include respiratory failure secondary to acute respiratory distress syndrome (ARDS) [56] , leprosy [57] , pyomyositis with cutaneous abscesses [23], intra-abdominal disease [58] , and involvement of joints, skin, soft tissues, and spine [58, 59] .
Several studies have characterized the time of onset of Mycobacterium-associated IRIS. In one study of MAC lymphadenitis, the onset of a febrile illness was the first sign of IRIS and occurred between 6 and 20 days after initiation of antiretroviral therapy [5] . In another study, the median time interval from the start of antiretroviral therapy to the development of mycobacterial lymphadenitis was 17 days (range 7-85 days) [24].
As with TB-IRIS, evidence for treatment of IRIS due to atypical mycobacteria are scarce. Occasionally, surgical excision of profoundly enlarged nodes or debridement of necrotic areas is anecdotally reported [23, 59] . However, healing is often poor leaving large, persistent sinuses. Needle aspiration is another option for enlarged, fluctuant and symptomatic nodes. Otherwise, treatment is similar to TB-IRIS (see Mycobacterium tuberculosis IRIS -Treatment).
In the pre-ART era, CMV retinitis, a vision-threatening disease, carried a high annual incidence and was one of the most significant AIDS-associated morbidities [60] . After the introduction of HAART, Jacobson et al described five patients diagnosed with CMV retinitis 4-7 weeks after ART initiation. They speculated that an HAART-induced inflammatory response may be responsible for unmasking a subclinical infection [17] . In addition to classical CMV retinitis, ART led to new clinical manifestations of the infection, termed immune recovery vitritis (IRV) or immune recovery uveitis (IRU), in patients previously diagnosed with inactive AIDS-related CMV retinitis [61] . Distinct from the minimal intraocular inflammation of classic CMV retinitis, these manifestations exhibit significant posterior segment ocular inflammation thought to be due to the presence of residual CMV antigens or proteins which serve as the antigenic stimulus for the syndrome [62] . Clinical manifestations include vision impairment and floaters.
In a retrospective cohort, CMV-related IRIS was common (6/33 of IRIS cases, or 18%) [4] . In prospective cohorts, symptomatic vitritis occurred in 63% (incidence rate 83 per 100 p-yr) of ART responders who carried a previous diagnosis of CMV retinitis but had inactive disease at the onset of antiretroviral therapy. The median time from ART initiation to IRV was (43 weeks) [63] . Another large prospective surveillance study [64] identified 374 patients with a history of CMV retinitis involving 539 eyes. Thirtyone of 176 ART responders (17.6%) were diagnosed with IRU. Male gender, use of ART, higher CD4 cell counts, and involvement of the posterior retinal pole as factors associated with a reduced risk of developing IRU, whereas prior use of intravitreous injections of cidofovir, large retinal lesions, and adequate immune recovery on ART were associated with increased risk.
The diagnosis of ocular manifestations of IRIS requires a high level of suspicion. In addition to signs of retinitis, inflammatory symptoms include vitritis, papillitis, and macular edema, resulting in symptoms of loss of visual acuity and floaters in affected eyes. Treatment of IRIS associated CMV retinitis and IRV may involve anti-CMV therapy with gancyclovir or valgancyclovir [17, 65] . However, the occurrence of IRU in patients receiving anti-CMV therapy draws its use into question [64, 66, 67] . The use of systemic corticosteroids has been successful, and IRV may require periocular corticosteroid injections [61, [68] [69] [70] . Due to its significant morbidity and varying temporal presentations, clinicians should maintain a high level of vigilance for ocular manifestations of CMV-associated IRIS.
With the introduction of protease inhibitors, increasing rates of herpes zoster were noted in HIV-infected patients. Two studies comparing ART and non-ART patients reported increased incident cases of zoster and rates estimated at 6.2-9.0 cases per 100 person-years, three to five times higher than rates observed in the pre-HAART era [18, 71] . While another study [72] reported no difference in overall incidence between HAART eras (3.2 cases per 100 person-years), the use of HAART was associated with increased odds of developing an incident zoster outbreak (OR = 2.19, 95% confidence interval: 1.49 to 3.20). These studies suggest that ART may play a role in increasing the risk of zoster, which is reflected in large observational IRIS cohorts, where dermatomal varicella zoster comprises 9-40% of IRIS cases [4, 32, 33] . Mean onset of disease from ART initiation was 5 weeks (range 1-17 weeks) [71] , and no cases occurred before 4 weeks of therapy [18] . Both studies identified significant increases in CD8 T cells as a risk factor for developing dermatomal zoster.
Although complications such as encephalitis, myelitis, cranial and peripheral nerve palsies, and acute retinal necrosis can occur in immunocompromised HIV patients, the vast majority of patients exhibit typical or atypical dermatomal involvement without dissemination or systemic symptoms [18, 71, 73] .
A randomized, controlled trial demonstrated oral acyclovir to be effective for dermatomal zoster in HIV-infected patients, facilitating healing and shortening the time of zoster-associated pain [74] . Its use in cases of varicella zoster IRIS appears to be of clinical benefit [18] . The benefit of corticosteroids in combination with acyclovir in acute varicella zoster has been demonstrated in two large randomized, controlled trials. The combination of corticosteroids and acyclovir decreased healing times, improved acute pain, and quality of life, but did not affect the incidence or duration of postherpetic neuralgia [75, 76] . The incidence of postherpetic neuralgia in immunocompetent individuals does not differ significantly from HIV-infected patients, but increases with increasing patient age [77] . Successful symptomatic management involving opioids, tricyclic antidepressants, gabapentin, and topical lidocaine patches individually or in combination has been shown to be beneficial [78] [79] [80] [81] [82] and should be attempted in HIV patients with postherpetic neuralgia as a complication of herpes zoster IRIS.
Accurate incidence of C. neoformans-associated IRIS is unknown. It is infrequently reported in overall IRIS cohorts, and many cases appear as single case reports. 
A recent study [90] evaluated antifungal combination therapies in the treatment of C. neoformans meningitis in HIV patients. Although significant log reductions in colony forming units were observed with all combinations, substantial numbers of patients remained culture positive 2 weeks after therapy. It may be important to delay ART until CSF sterility can be achieved with effective antifungal combinations such as amphotericin B and flucytosine. However, the exact timing of ART and whether attaining CSF culture sterility is important in avoiding IRIS is unknown. This is illustrated by cases of reactivation cryptococcal meningitis described in four patients who had received at least four weeks of antifungal therapy prior to ART [13, 22, 83] . It is reasonable to administer systemic corticosteroids to alleviate unresponsive inflammatory effects, as anecdotal benefits have been observed in these patients [21, 84] . Furthermore, serial lumbar punctures may be required to manage persistent CSF pressure elevations in these patients [85, 86] . Although continuation of ART has been performed safely [13, 84] , interruption of antiviral therapy may be necessary in severe or unresponsive cases.
Other less common infectious etiologies, as well as noninfectious etiologies, are listed in Table 1 . Because these other infectious and non-infectious etiologies are rare, no recommendations exist for their management.
While exact estimates of incidence are not yet available, IRIS in patients initiating ART has been firmly established as a significant problem in both high and low income countries. Because of wide variation in clinical presentation and the still increasing spectrum of symptoms and etiologies reported, diagnosis remains problematic. Fur-thermore, no test is currently available to establish an IRIS diagnosis. Standardized disease-specific clinical criteria for common infectious manifestations of the disease should be developed to: 1) identify risk factors for developing the syndrome and 2) optimize the prevention, management of opportunistic infections. Results of trials addressing the optimal timing and duration of treatment of opportunistic infections will assist in developing guidelines for the prevention and management of IRIS. Treatment of IRIS will remain a clinical challenge due to the variety of clinical presentations and the presence of multiple pathogens capable of causing the syndrome. Until a greater understanding of the syndrome is achieved in different regions of the world, clinicians need to remain vigilant when initiating ART and individualize therapy according to known treatment options for the specific infectious agent.  Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry BACKGROUND: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. METHODS AND PRINCIPAL FINDINGS: Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. CONCLUSION/SIGNIFICANCE: Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance. Influenza viruses cause serious global economic and public health burdens. Annual influenza epidemics resulted in more than 30,000 deaths a year in the United States during 1990-1999 [1, 2] . Periodic pandemics result in significantly higher death tolls. Emergence of new influenza A virus strains can be caused by ''antigenic shift,'' resulting from reassortment of gene segments, including H and/or N types [3, 4] , ''antigenic drift'' resulting from the continuing accumulation of mutations in the H and N genes [5] , or a pathogenic virus jumping species and acquiring the ability to infect and be transmitted among humans, as in the 1918 pandemic [6] . The recent outbreak of highly pathogenic H5N1 avian influenza virus (HPAI), which originated in Southeast Asia and has since spread globally, has resulted in 166 deaths (272 confirmed human cases) as of February 6, 2007 (http://www.who. int/en/). The global emergence of this virus has brought renewed urgency to the effort to track the spread and the evolution of influenza viruses.
Currently, rapid methods for influenza virus diagnosis rely on antigen-specific antibody probes [7] , or real-time reverse transcription PCR (RT-PCR) analysis of the matrix (M) gene for identification of the viral species [8, 9] followed by H and N subtype specific RT-PCR assays for determination of the viral subtypes [10, 11] . Since there are many H and N subtypes with significant intra-and inter-subtype sequence variations, these methods do not identify all H and N subtypes, nor are they likely to identify reassortants or newly emerging genetic variants. Further, none of the current surveillance methods provide information relevant to tracking antigenically novel strains that emerge each year or distinguish amongst multiple lineages of influenza viruses that can co-circulate and persist in a popula-tion [12] . Secondary genome sequence comparisons and phylogenetic analyses are necessary to fully understand the multiple lineages of viruses, recognize newly emergent influenza variants, and monitor global spread of these viruses [12, 13] . For instance, analysis of human influenza virus H3N2 sequences from 1999-2004 revealed that at least three major clades of influenza viruses were in circulation after the 2002-2003 influenza season [12] . The differences were due to multiple reassortment events though all shared a common H-gene lineage. Several similar wholegenome studies with avian influenza viruses have revealed the presence of multiple, region-specific sub-lineages of the HPAI H5N1 virus in Southeast Asia that are spreading to Europe and Africa [14] [15] [16] [17] [18] .
We have developed a method based on broad-range RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ ESI-MS) for rapid and accurate detection of influenza virus, subspecies characterization, and early identification of genetic changes in circulating viruses. This method has previously been applied to detection of other pathogens in human clinical samples [19, 20, 21, 22] , but it has unique capabilities and advantages for influenza surveillance. Here, we show how a highthroughput assay incorporating eight parallel RT-PCR reactions followed by ESI-MS analysis can be used to simultaneously survey for all species of influenza viruses, provide clade-level resolution, identify mixed viral populations in the same sample, detect reassortants, and facilitate monitoring of viral evolution, all integral components of broad influenza surveillance.
To measure the breadth of coverage and resolution offered by the panel of primers described in Methods (details in Table S1 ), we tested 92 well-characterized influenza virus isolates collected from human, avian, and animal species. Despite the extensive genetic diversity of this sample set, the broad-range primers generated amplicons from all isolates, and base composition signatures distinguished the isolates (Figure 1 ). Most isolates showed base compositions consistent with expected signatures for the corresponding H/N sub-types based on bioinformatic analysis of existing sequence data. Two of the isolates, however, showed previously unknown base compositions at several primer loci suggesting these might be novel influenza virus types; these are noted as ''Unknown'' in Figure 1 .
Base composition signatures provide a multidimensional fingerprint of the genomes of the various viruses, which can be used to determine clusters of related species/sub-types. One such representation ( Figure 2 ) shows base composition data derived from the PA, PB1, and NP gene segments analyzed on individual axes. Importantly, only three of the six influenza A primer pairs from Figure 1 are visualized on this three-dimensional plot. There was strong agreement between the bioinformatic analysis of sequence data from GenBank and experimental measurements of base composition signatures from Figure 1 . Human H3N2 and H1N1 viruses clustered independently from each other and from the avian/human H5N1 and H1N1 viruses.
To assess the utility of the RT-PCR/ESI-MS assay for surveillance of influenza virus in human populations, we analyzed 656 blinded clinical samples collected over a seven-year period (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) . The results were compared with conventional analysis of the same samples by virus culture/serology and real-time RT-PCR methods. Two hundred forty-three samples were influenza positive both by RT-PCR/ESI-MS and conventional assays. Ten samples were positive only by RT-PCR/ESI-MS while eight samples were positive only by culture/real-time RT-PCR, corresponding to approximately 97% sensitivity and 98% specificity. Of the influenza-positive samples, RT-PCR/ESI-MS analysis identified 186 as influenza A virus and 67 as influenza B virus, in complete agreement with conventional typing methods.
Base composition analysis of multiple RNA segments enables further categorization of isolates into previously established clades determined by sequencing (details shown in Table S2 ). Of the 186 influenza A samples, we determined that 149 of were H3N2 subtype and 34 were H1N1. The subtype of three samples could not be distinguished between H3N2 and H1N2 because these viruses probably arose from a recent reassortment of the H gene from an H1N1 virus with gene segments from an H3N2 virus [12] . Importantly, our predictions agreed completely with serology and direct RT-PCR analysis of the H and N segments from these samples. Nonetheless, although base composition analysis of the PB1, NP, M1, PA, and NS gene segments can be highly predictive of the H and N types, direct analysis of the H and N segments is necessary for unambiguous subtyping due to the potential for viral reassortment.
In addition to identification and species typing, RT-PCR/ESI-MS provided a quantitative estimate of the number of viral genome copies in the original patient sample. This was achieved by including a fixed amount (300 copies/well) of an internal RNA calibration standard in each PCR reaction [19] [20] [21] [22] . The genome copy numbers in the influenza samples ranged from low (,100 genome copies per well) to intermediate (100-2,500 copies/well) to high (.3,000 copies per well). Based on the samples within the linear range of the calibration standard, we estimated an average genome load of 750 copies/PCR reaction, representing an average viral load of ,1.5610 4 genomes from material extracted from the original swab or 200 mL of a nasal aspirate. Six of the samples required serial dilutions to obtain viral concentrations in the quantifiable spectrum and ranged from 1.5610 6 to 8.0610 7 genomes/swab. Thus, for influenza-positive patients with respiratory symptoms, we observed a range of five orders of magnitude in viral RNA shedding.
To demonstrate the capabilities of RT-PCR/ESI-MS to track the evolution of circulating influenza viruses, we created a tree representation of the H3N2 influenza virus sequences from Genbank ( Figure 3 , black) as described in Methods. The 104 experimentally determined H3N2 base compositions were mapped onto this tree (Figure 3 , blue). Analysis revealed a distribution very similar to the sequence-derived clades of Holmes et al. [12] . The branch terminating in clade A represents the dominant branch between years 1999-2004, with a clade B branch that co-circulates in the same time period. Strikingly, however, clade-A isolates were not detected after this time and Table S3 ) and confirmed the findings described above. A visual display of the most likely relationships among the isolates is shown in Figure 4 . Collectively, these results demonstrate the richness of the genetic information provided by direct RT-PCR/ESI-MS analysis of human clinical materials. Figures 5A and B show the spectra from samples containing a mixture of the ''parent'' BCtype AADFAA and single-nucleotide variations AAHFAA and AADFBB, respectively, providing a snapshot of enduring ''fit'' quasispecies contributors and of potential viral evolution in action.
The dynamic range for mixed RT-PCR/ESI-MS detections has previously been determined to be approximately 100:1 [20] , which allows for detection of viral variants with as low as 1% abundance in a mixed population. Figures 5C and 5D show results obtained with a clinical sample containing a mixture of viruses at the limit of detection for mixed populations. To demonstrate that these peaks truly represented mixed viral populations, the PCR amplicons were cloned and 450 independent colonies were sequenced. Nine (2%) of these 450 clones had the predicted mutations, correlating well with the measured amplitude of the low-abundance peaks.
A total of 293 non-overlapping nucleotides, excluding the primer regions, were analyzed using the genetic loci targeted by the influenza A primers. This corresponds to 2.15% of the influenza A virus genome. Out of 174 human samples analyzed from the 2005-2006 season, only two showed evidence for mixed viral populations at one of the six loci, corresponding to 1.1% of the samples. Thus, assuming the same mutation rate for the broader viral genome as for the region analyzed by PCR/ESI-MS, about 50% of the human H3N2 virus samples would have a mixed population of viruses.
Choosing amongst the various molecular methods available for pandemic influenza surveillance requires consideration of both practical issues (e.g., broad availability, convenience, cost, and throughput) and scientific issues relevant to public health (e.g., sensitivity, breath of coverage, and the depth and value of the information provided). At one end of the spectrum, a conventional RT-PCR test with specific primers and probes provides a highlyspecific, sensitive, rapid, convenient, quantitative, relatively inexpensive, and high-throughput format that can provide valuable surveillance information. However, these tests are not optimal for surveillance when the exact nature of the pandemic virus is not known. Moreover, without supplemental nucleic acid sequencing, conventional RT-PCR-based tests are not capable of signaling the appearance of new genetic variants, except by potentially demonstrating a loss of sensitivity. Further, a single RT-PCR test can achieve only a single presence/absence analysis limited to the specific target for which it was designed. Discrimination of all known variants of influenza at the level of resolution described here would require hundreds of independent RT-PCR reactions. At the other end of the spectrum, virus isolation using culture methods followed by complete genome sequencing does not require prior knowledge of the virus' sequence and provides cladelevel resolution and highly detailed information regarding virus evolution. Unfortunately, this method is slow, labor intensive, expensive, and low throughput, rendering it ineffective in public health arenas requiring rapid response.
In this work we describe a novel method that employs some of the best properties of each of the discussed techniques, and also supplies additional valuable information not provided by those techniques. For example, our method may identify mixed Black font: types determined through sequence analysis; blue font: experimentally determined base composition types; red font: experimentally determined base composition types for season 2005-06. Ten rare sequence types (,1.5%) were not uniquely discernable by the base composition analysis of the eight amplicons used in this analysis, as more than one subtype produced the same BC-type. These BC-types are indicated by asterisks. doi:10.1371/journal.pone.0000489.g003 populations of viruses, either as viral quasispecies as previously illustrated (i.e., development of ''drift'' strains) or co-infections with circulating strains (i.e., potential for development of ''shift'' strains). Recent advances in ESI-MS using bench-top mass spectrometers have enabled analysis of PCR amplicons with sufficient mass accuracy that the nucleotide base composition (the A, G, C, and T count) of the PCR amplicon can be unambiguously determined [23] . The approach outlined here provides two important advantages for surveillance. First, broad-range primers targeted to highly-conserved sites within the influenza virus family that flank highly variable, information-rich regions can be used to amplify sequences from highly diverse viruses in the same assay. The measured base compositions allow identification of the viral species with a high degree of resolution. For this approach, we selected primer pairs targeting the core gene segments (PB1, PB2, PA, M, NS, and NP) that have conserved regions spanning all known influenza viruses and used the resultant base compositions to infer the influenza virus H/N types. In silico analysis of influenza virus sequences from the GenBank database showed that this approach would detect all known influenza viruses and distinguish .90% of all species and types. This overcomes the limitations of directly targeting the H and N segments, which requires specific primer pairs for each known H/N type. Further, since the H/N segments evolve rapidly, newly emerging strains might not be readily detected by the traditional approach.
Second, mixed viral populations in the same sample that differ only by a single mutation and is present in as low as 1-2% of the virus population can be identified, providing early insights into viral evolution as an integral component of surveillance. This information-rich result is provided with the same throughput and consumable costs as conventional, sequence-specific RT-PCR assays.
The results from 174 influenza A H3N2 samples from the 2005-2006 season in the northern hemisphere were particularly interesting because they revealed viral evolution during a single season. The viruses detected appeared to have been seeded from two of the more abundant BC types circulating during the previous season in the southern hemisphere. The majority (97) of the samples had identical BC types and probably arose from a single founder from the previous season in the southern hemisphere. Most of the remaining samples differed from this founder BC type by one or two additional point mutations within the target regions described here. Surprisingly, when mutations occurred, they became fixed rapidly in the viral population, since only two of the 174 samples from the 2005-2006 season showed evidence of mixed populations. Sequencing revealed that both mutations were silent transitions in third codon positions.
In summary, the RT-PCR/ESI-MS method has the capacity to provide rapid diagnosis of human influenza in individual patients with respiratory symptoms, as well as public health surveillance of emerging, potentially pandemic strains, including novel reassortants. The use of RT-PCR/ESI-MS for human as well as avian/ animal surveillance offers the potential for new insights into viral evolution on a scale and at a cost previously not possible. Benchtop mass spectrometers are capable of analyzing complex PCR products at a rate of approximately one reaction product/minute, making the RT-PCR/ESI-MS technology practical for large-scale analysis of clinical specimens or for animal surveillance. Further, as we have demonstrated with influenza viruses, this method provides sensitive detection directly from patient specimens with specificity approximating sequence-level resolution. The ability to quantitate virus shedding, detect low-abundance, mixed infections, and identify new genetic variants without prior knowledge of viral sequence also make this technology ideally suited for monitoring the emergence of drug-resistance mutations during therapy or for identifying newly emerging antigenic variants. Sample processing Viral stock samples consisting of cultured virus or stocks obtained from ATCC were prepared for analysis using the Qiagen QiaAmp Virus kit (Valencia, CA). Both manual (mini spin) kits and BioRobot kits were used. Robotic-based isolations were done on both the Qiagen MDx and Qiagen BioRobot 8000 platforms. Clinical swab samples were stored in Viral Transport Media (VTM). VTM solution (1 ml) was passed over a 0.2 micron filter, which was then subjected to bead beating in a small amount of lysis buffer. The resulting viral lysate was then used following the same protocol as above.
Based upon analysis of multiple influenza sequence alignments, pan-influenza virus RT-PCR primer sets were developed that were capable of amplifying all three influenza virus species (A, B, and C) and subtypes (HxNy) from different animal hosts (e.g., human, avian, and swine) and distinguishing essential molecular features using base composition signatures. Additional primer pairs were designed that broadly amplified all known members of a particular species, but that did not cross-amplify members of different species (e.g., pan-influenza A and pan-influenza B primers). A surveillance panel of eight primer pairs (Table S1 ) was selected comprising one pan-influenza primer pair targeting the PB1 segment, five pan-influenza A primer pairs targeting NP, M1, PA, and the NS segments, and two pan-influenza B primer pairs targeting NP and PB2 segments. All primers used in this study had a thymine nucleotide at the 59 end to minimize addition of non-templated adenosines during amplification using Taq polymerase [24] . The sensitivity of each RT-PCR primer pair was determined using known quantities of a synthetic calibrant RNA template as described previously [20] . Each of the primer pairs was sensitive to as few as twenty copies of the calibrant RNA and several primers were sensitive to five copies (Table S1 ).
One The following RT-PCR cycling conditions were used: 60uC for 5 min, 4uC for 10 min, 55uC for 45 min, 95uC for 10 min, followed by 8 cycles of 95uC for 30 seconds, 48uC for 30 seconds, and 72uC for 30 seconds, with the 48uC annealing temperature increasing 0.9uC each cycle. The PCR was then continued for 37 additional cycles of 95uC for 15 seconds, 56uC for 20 seconds, and 72uC for 20 seconds. The RT-PCR cycle ended with a final extension of 2 min at 72uC followed by a 4uC hold.
The RT-PCR products were analyzed using the Ibis T5000 universal biosensor platform (Ibis Biosciences, Inc., Carlsbad, CA; http://www.ibisbiosciences.com), which performs automated post-PCR desalting, ESI-MS signal acquisition, spectral analysis, and data reporting as described previously [23] . Briefly, the steps were as follows: 15 mL aliquots of each PCR reaction were desalted and purified using a weak anion exchange protocol as described elsewhere [25] . Accurate mass (61 ppm), high-resolution (M/dM.100,000 FWHM) mass spectra were acquired for each sample using high-throughput ESI-MS protocols described previously [20] . For each sample, approximately 1.5 mL of analyte solution was consumed during the 74-second spectral acquisition. Raw mass spectra were post-calibrated with an internal mass standard and deconvolved to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides [26] . Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 100 molecules [20] .
To demonstrate the capabilities of RT-PCR/ESI-MS to track the evolution of circulating influenza viruses, we used a bioinformatic approach to develop a framework on which to display the RT-PCR/ ESI-MS results obtained with H3N2 viruses. Complete genome sequences of all H3N2 human influenza viruses available in GenBank were analyzed. A total of 731 genomes were included, from which we inferred the phylogeny of H3N2 influenza virus since 1996. Using the 565-nucleotide concatenated sequence of the six loci that we analyzed by RT-PCR/ESI-MS, we constructed a nonredundant alignment of 105 sequence types. Base compositions were determined for each genome segment (i.e., locus) analyzed and each unique base composition at each of these loci was assigned a letter according to decreasing number of occurrences (therefore, the letter A represents the most common allele identified at each locus). The concatenation of the six base-composition letters from the PB1, NP, M1, PA, NS1, and NS2 loci from the sequences of 731 H3N2 viruses (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) available in GenBank yielded 95 six-letter codes referred to as base composition types (BC types). The predominant type is labeled AAAAAA. The topology of the tree was then deduced from the alignment of non-redundant sequences using the programs dnadist and neighbor from the Phylip package (http://evolution. genetics.washington.edu/phylip.html). Since the sequence types differ mostly by discrete single mutations, the original computergenerated tree was then extensively edited using graphics tools to place the labels of intermediate types within the tree itself in lieu of zero-length branches. Readability was further improved by sorting parallel branches chronologically. Table S1 Influenza virus primer pairs used in this study. Genbank reference sequence for each segment is indicated; however, the primer sequences are not identical to the reference sequence as described in the Methods. The limits of detection for each RT-PCR primer pair were determined using known quantities of a synthetic calibrant RNA template. Found at: doi:10.1371/journal.pone.0000489.s001 (0.06 MB PDF) Table S2 Distribution of BC-types observed in influenza A H3N2 positive human respiratory samples. Unique base compositions at each genome segment locus analyzed were assigned letter codes and concatenation of letter codes across the six loci analyzed yielded BC-types. H1N1 samples were not assigned a BC-type. Experimentally determined BC-types (marked RT-PCR/ESI-MS Analysis results) were compared to BC-type signature information of sequences currently available in GenBank and the closest matching strain is shown (right pane). Last column shows comparison to clade designation described in Holmes et al [12] . Found at: doi:10.1371/journal.pone.0000489.s002 (0.24 MB PDF)  Transmission Parameters of the 2001 Foot and Mouth Epidemic in Great Britain Despite intensive ongoing research, key aspects of the spatial-temporal evolution of the 2001 foot and mouth disease (FMD) epidemic in Great Britain (GB) remain unexplained. Here we develop a Markov Chain Monte Carlo (MCMC) method for estimating epidemiological parameters of the 2001 outbreak for a range of simple transmission models. We make the simplifying assumption that infectious farms were completely observed in 2001, equivalent to assuming that farms that were proactively culled but not diagnosed with FMD were not infectious, even if some were infected. We estimate how transmission parameters varied through time, highlighting the impact of the control measures on the progression of the epidemic. We demonstrate statistically significant evidence for assortative contact patterns between animals of the same species. Predictive risk maps of the transmission potential in different geographic areas of GB are presented for the fitted models. The 2001 FMD epidemic in the UK had a substantial cost in human, animal health and economic terms (Alexandersen et al. [1] , Kao [2] ). Understanding the risk factors underlying the transmission dynamics of that epidemic and evaluating the effectiveness of the control measures are essential to minimise the scale and cost of any future outbreak. Epidemic modelling [3, 4, [5] [6] [7] proved critical to decision making about control policies which were (in some cases controversially) adopted to control the 2001 epidemic [8] [9] [10] . Modelling now has a 'peace-time' contingency planning role.
One weakness of the modelling studies undertaken in 2001 was the relatively ad-hoc nature of the parameter estimation methods employed. In their first paper, Ferguson et al. [4] used maximum likelihood methods to fit to the observed incidence time series, but did not attempt to fit to the spatio-temporal pattern of spread. In their later work, the same authors developed a more robust method for estimating species-specific susceptibility and infectiousness parameters and spatial kernel parameters (see Supplementary Information to [3] ), but at the time the statistical basis for the methods developed was lacking. In retrospect, the methods developed turned out to be closely related to those developed during the SARS epidemic by Wallinga and Teunis [11] , although the earlier work incorporated population denominator data to allow for spatial-and species-based heterogeneity in disease transmission. Nevertheless, the methods employed had the limitation of not being fully parametric, meaning they could not be extended to fit arbitrary transmission models to the observed data. Keeling et al. [5] used maximum likelihood methods to estimate transmission parameters, but it was also supplemented by more ad hoc least-squares matching to regional incidence time series.
Therefore there remains a need to develop rigorous modern statistical approaches for parameter estimation of non-linear models for the 2001 FMD outbreak. Bayesian Markov Chain Monte Carlo (MCMC) techniques are the best established such methods and have been successfully employed in the analysis of a range of spatiotemporal outbreak data in the past [12] [13] [14] , as well as to purely temporal incidence data [15, 16] . Here we develop MCMC-based inference models for the 2001 FMD epidemic in GB. The models examine: the extent to which transmission was spatially localised and the temporal variation in transmission, species-specific variation in susceptibility, infectious-ness and heterogeneity in contact rates between and within species.
We take the farm as the unit of our study and ignore the possible impact of within-farm epidemic dynamics. Thus we implicitly assume disease spread within a farm is so rapid as to be practically instantaneous, with all animals on a farm becoming infectious at the same time.
Our data consists of information on all the farms in the UK listed in the 2000 agricultural census [see http://www.defra.gov. uk/footandmouth/cases/index.htm ]. There were a total of 134,986 farms listed in that dataset and uniquely identified by their County/Parish/Holding (CPH) number. Their spatial coordinates are provided together with the number of animals by species within each farm. A partition of all GB farms according to the animal types represented is shown in Figure 1a . Their geographical distribution is represented in Figure 1b as the number of farms per 565 km. Notice the high density areas in the North West (Cumbria), South West (Devon), Wales and Scotland where the main epidemic foci developed. There is also an area of high density in the Shetland Islands corresponding to very small crofter smallholdings. Figure 1c and d show the numbers of sheep and cattle kept per 565 km square.
During the 2001 FMD outbreak, a total of 2026 infected premises (IPs) were recorded -farms where FMD was diagnosed, and which were subsequently culled. The IP dataset contains, for each farm, the estimated date of infection (determined by a clinical evaluation of the age of lesions on affected animals), and the dates of disease reporting, confirmation and culling.
A total of 7457 other (non-IP) farms were also culled -mostly as contiguous premises (CPs, about 3103) or dangerous contacts (DCs, about 1287), but some under other local culling policies used in Cumbria and Scotland. For instance about 1846 (79%) out of a total of 2342 sheep farms in Cumbria had all sheep culled under the ''local 3 km radial sheep cull'' policy adopted there. Some of the farms (about 30) were recorded both as DCs and CPs. Multiple records per farm were often found in the disease control management system dataset, and it was often unclear whether this was due to data entry errors or as a result of sequential species-specific culls on the same farm. In our analysis we therefore considered the whole farm to be culled at the last recorded date of culling.
The most frequent species are cattle and sheep (see Figure 1a ). There are less than 3% farms with pigs only and only 10 farms with just pigs were diagnosed as IPs in 2001 (less than 1% of all the IPs). This indicates a-priori that pigs contributed far less to the 2001 outbreak than many other FMD outbreaks (despite their high levels of shedding [1, 17] ), and we therefore decided to discard pigs-only farms from the current study to simplify the analysis. The Sensitivity Analysis section shows that this simplification does not significantly affect estimates of other epidemiological parameters. We discarded another three IPs due to missing information or possible mistakes regarding their location or number of animals, leaving a total of 2013 IPs in our analysed dataset.
We model the epidemic as a space-time survival process [18] . The total observation time T is the 240 days between 7 th February and 5 th October 2001. Each farm i at the location (x i , y i ) is associated with an infection time t i (if infected), a removal time r i (if slaughtered) and two integers n c i and n s i representing, respectively, the number of cattle and sheep on the farm. S c and S s represent per-capita cattle and sheep susceptibility, respectively, while I c and I s represent per capita cattle and sheep infectivity. The susceptibility is a relative measure of animal sensitivity to the disease whereas infectivity represents the infectious risk posed by an animal to others. We use a continuous kernel to describe how the probability of contact between farms scaled with distance. Transmission is naturally assumed to decrease with the distance between farms according to the power law
where d ij represents the Euclidian distance between the infected farm i and the susceptible farm j. The parameters a (kernel offset) and c (kernel power) are to be estimated. The kernel captures all forms of movement and contact between farms and as such, the use of a simple 2 parameter function is inevitably a highly simplified representation of the true complexity of inter-farm contacts. We examined other functional forms for the kernel (such as those used in some other analyses [19] ) but the resulting model fits were much poorer than found using the power-law kernel above.
Given the susceptibility and infectiousness parameters and the kernel, the infection hazard from an infected farm j to a susceptible farm i is then quantified by 
This model is over-specified as stated, so we arbitrarily assume S s = 1 throughout, meaning S c represents the ratio of cattle-tosheep susceptibility. For a constant (distance-independent) kernel this is just a mass-action closed epidemic model with heterogeneous susceptibility and infectiousness. This model assumes susceptibility and infectiousness parameters scale linearly with the number of animals of different species on the farm, a relatively strong assumption imposed for model parsimony reasons. The mixing matrix embedded in (2) quantifies the 4 species-specific mixing rates between animals on different farms: cattle-to-cattle (S c I c ) sheep-to-cattle (S c I s ), cattle-to-sheep (S s I c ) and sheep-to-sheep (S s I s ). This model formulation is identical to that used by Keeling et al. [5] , except for the functional form of kernel used. The force of infection on a susceptible farm i at time t depends on the whole history of events and is just
where
, if the farm i is susceptible and the farm j is infectious at the time t 0, otherwise
By default, we assume a latent period of 1 day (latency is represented within the function L); i.e. farms are infectious the day after they are infected. However, we test the sensitivity of our estimates to the assumption by also examining latent periods of 2 and 3 days. The probability density function that farm i is infected at time t is then given by
Hence, the contribution that a farm i, observed to be infected at time t, makes to the log likelihood is just:
A farm which is not infected contributes to the overall likelihood the probability that it escapes infection during the observation period, i.e. until the time it is culled (r i ) or for the duration of the epidemic T, whichever is shorter. Its contribution to the log likelihood is therefore
The total log likelihood of the model can be written as
We then extend the simple model above by introducing an additional parameter to understand to what extent the transmission within species is altered by between species transmission. The parameter r quantifies the degree to which mixing between species is assortative -with r,1 representing assortative mixing and r.1 disassortative mixing. The interaction model still assumes constant parameters with respect to time along the whole observation period T. The mixing matrix defined in equation (2) becomes S c I c rS c I s rS s I c S s I s ð8Þ
where we again fix S s to be 1 to avoid model over specification. The force of infection (3) and model log likelihood equation (7) change accordingly. Assuming transmission parameters were constant in time throughout the epidemic is obviously a crude simplification. However, allowing infectivity to vary continuously in time results in an over-specified model and problems of parameter identification and confounding. We therefore examined two sets of models in which changes in transmission parameter were restricted to 2 significant points in time denoted by T cut , namely 23 rd February (when the national ban on animal movements was introduced) and 31 st March (when control measures were intensified and the so called 24/48 hour IP/CP culling policy was introduced). Models were respectively fitted to the individual case data from the start of the epidemic (conditioning on the first infection) or from after 23rd February (conditioning on the 54 farms that were already infected by that date). A detailed history of the epidemic is given by Kao [9] . We separately fitted model variants which assumed a discrete change in parameters on 23 rd February and on 31 st March. Confounding meant that only a very limited number of parameters could be varied in time, so we examined the effect of varying infectiousness and kernel parameters separately. We fitted four separate time-varying model variants: (i) varying the cattle infectivity by a factor and keeping sheep infectivity constant through time (Cattle Infectivity model); (ii) varying sheep infectivity by a factor but not cattle infectivity (Sheep Infectivity model); (iii) varying both cattle and sheep infectivity by the same ratio (Cattle & Sheep Infectivity model); (iv) varying the kernel parameters (Time Varying Kernel model). For the last model variant we also fitted a version which includes non-assortative mixing between species (see equation (8)). Hence the most general mathematical expression of the transmission model is:
where
The scripts pre and post are self-explanatory for time varying parameters. When fitting models with time varying infectivity parameters we actually fit I post and the ratio m = I pre /I post we called infectivity factor. This is a within species ratio, a parameter directly fitted by the models, unlike the between species infectivity ratio additionally calculated as explained later in the text (see Parameter estimates section). Note that all models above treat the epidemic as fully observed, i.e. infection times are assumed to be known (when in fact only estimated infection times are known -see Sensitivity Analysis section), and only IPs are assumed to be infectious.
We adopt a Bayesian framework for statistical inference and use MCMC methods for fitting the model to individual case data. This is not strictly necessary, given our simplifying assumption that the epidemic was completely observed, but it provides a more consistent and robust framework within which to relax that assumption in future work.
We obtained parameter estimates and equal-tailed 95% credible intervals from the marginal posterior distributions of the fitted parameters. For the basic model for instance we estimated the relative cattle susceptibility, S c , two infectivity parameters (I c (t);I c and I s (t);I s for all t) and two kernel parameters (c(t);c post ;c pre ;c and a(t);a post ;a pre ;a for all t).
We used the posterior mean deviance as a Bayesian measure of fit or model adequacy as defined by Spiegelhalter et al. [20] . The posterior density deviance is defined as:
where log{P(y|h)} is the log-likelihood function for the observed data vector y given the parameter vector h and C is a constant which does not need to be known for model-comparison purposes (being a function of the data alone). The smaller the mean posterior deviance, the better the corresponding model fits the data.
If the posterior deviance distributions for two different models overlap significantly, it is necessary to use additional criteria to compare model fit -namely a comparison of the relative complexity of the models. The Deviance Information Criterion (DIC) is perhaps the most general of such methods, being a generalisation of the Akaike information criterion for Bayesian hierarchical models [20] . We define the complexity of a model by its effective number of parameters, p D , defined as
where E[ ] represents taking expectations (the posterior average).
The DIC is then defined as
A lower value of DIC corresponds to a better model. This criterion offers flexibility for comparing non-nested models [20] and it is straightforwardly computed within an MCMC algorithm.
We applied the classic random walk Metropolis Hastings algorithm [21, 22] and a block-sampling of parameters due to the computationally expensive form of the likelihood [23, 24] . A log scale has been used for sampling as the parameters were all positive definite and were expected to potentially vary by orders of magnitude. However, linear scale sampling yielded similar results. The convergence of the chains was also very much improved (see Robert [25] for more on perfect sampling and reparameterization issues) compared with sampling on a linear scale. The model was coded in C and parallelized using OpenMP 2.0.
The MCMC sampler was allowed to equilibrate with convergence being evaluated visually from the likelihood and parameter traces. For the simpler models, 5,000 iterations were sufficient for equilibration, while this increased to 20,000 for the most complex models. Also, using log scale sampling, we verified that the chains were able to converge even if started with initial parameter values far from the final posterior mean values. Posterior distributions were estimated from 100,000 iterations. The rate of the acceptance varies from model to model. For the baseline model we achieved a 25% rate of acceptance and for the most complex model (8 parameters), a rate of approx 10%. These values compare well with the ''golden'' acceptance rate for Random Walk Metropolis Hastings of 23% (Roberts [26] ).
We did not encounter common problems in MCMC estimation like slow convergence and slow mixing (O'Neill [27] ). There were some correlations between parameters, mostly having biological explanations (cattle and sheep infectivity for instance), but a careful parameterization lowers them. We verified parameter estimates were not dependent on parameterization choices -e.g. no difference was seen whether we fitted species infectivity individually, or just fitted sheep infectivity and then the ratio of cattle-to-sheep infectivity. Table 1 lists the parameter estimates we obtained for a set of fitted models conditioned only on the first infection whereas Table 2 presents the estimates for models conditioned on infections occurring up to 23 rd February. The posterior deviances for each set of models are plotted in Figure 2a and Figure 2b , respectively. Figure 2a illustrates some clear conclusions. Of the two models without time variation in parameters, the interaction model fits significantly better than the baseline model without heterogeneous mixing between species. However, fitting the interaction model broadened the credible intervals of the infectivity parameter estimates (Table 1) , indicating (unsurprisingly) slight confounding between the 4 infectivity and susceptibility parameters.
Of the models which allowed infectivity to vary on 23 rd February, allowing only cattle infectivity variation gave a slightly better fit than varying sheep infectivity or both. However, of the models with parameters which vary on 23 rd February, the model variants which allow the 2 kernel parameters to vary at that time point fit substantially better (by both deviance and DIC criteria, see Table 1 ) than those which just allow a species-specific variation in infectivity. This is encouraging for the inference procedure, as the main control measure initiated on that date was the banning of (Figure 3a and Figure 3b ). The parameter estimates are less precise before 23 rd February (Table 1) due to the relatively small number of IPs (about 57) before that date. Looking at the most complex model (namely the interaction model with time varying kernel), cattle were estimated to be 5.7fold (4.6, 6.8) more susceptible than sheep (see Figure 3c and Table 1 ). Rather than mentioning animals' specific infectivity (see Figure 3d and Table 1 ), it is more informative to comment on the cattle:sheep infectivity ratio parameter for the most complex fit (this ratio does dot appear in the tables as it is not a model parameter). We calculated it within the MCMC algorithm as the ratio of the two species infectiousness for each sampled parameter point. The most complex model suggests that cattle are 5.95-fold (4.54, 7.63) more infectious than sheep (Figure 3e) .
The parameter quantifying assortativity in mixing was estimated at r = 0.45 (0.31, 0.61) -well below 1, the level at which mixing between species is random (Figure 3f ). By comparison with the model with a time varying kernel but random mixing between species, the effect of heterogeneous mixing between species modified the between-species transmission as given by matrix (1.9) as indicated below. 
Cattle-to-cattle and sheep-to-sheep transmission is higher (by 19% and 54% respectively) for the model with non-random mixing, whereas the sheep-to-cattle and cattle-to-sheep transmissions dropped by 41% and 37 % respectively. Conditioned on 23 rd February, 7 model variants have been considered (Table 2 and Figure 2b) . We examined the baseline and interaction models (no change in parameters over time), allowing cattle infectivity to vary on 31 st March and both cattle and sheep infectivity to vary by the same factor after 31 st March (with and without heterogeneity in mixing) and allowing both kernel parameters to vary on 31 st March.
Unsurprisingly, the kernel parameters were not significantly different if allowed to be different before and after 31 st March, neither did this model prove to be the best fit. Overall, while the variations in mean deviance (Figure 2b ) seen between model variants were much smaller than for the models conditioned on the first infection (Figure 2a ), the interaction model allowing for time varying cattle infectivity gave the most adequate fit (measured by both mean deviance and DIC, see Table 2 ).
We cannot statistically compare the two sets of models in Table 1 and Table 2 , as the data used are different for the two cases. However, the parameter estimates from the best-fitting models of each table are largely consistent. Each post-23 rd February estimated value from the best-fit model in Table 1 is included in the corresponding pre-31 st March 95% credible interval of the best fit model in Table 2 (and vice-versa).
The most important message from the second set of models is that all models with cattle time varying infectivity (best fit) indicated higher values of infectivity after 31 st March than before (m = 0.73 (0.63, 0.83)) ( Table 2 ). This may seem paradoxical but reflects the fact that while culling (the effect of which is explicitly included in the input data) dramatically reduced case incidence in April, from May to September 2001, case incidence maintained itself at a low level -but almost entirely within cattle farms. This increase in cattle infectivity may therefore really reflect the impact of reduced biosecurity and/or increased non-compliance with movement controls.
It is informative to examine what our parameter estimates imply in terms of geographic variation in transmission potential. Given the parameter estimates for each model, we can define the relative risk of transmission an infectious farm j would pose to all susceptible farms in the country r j : r j~Ic n c j zI s n s j P i=j Sc Ss n c i zn s
This quantity multiplied by the average duration of infectiousness of a farm (time from end of latency to culling) gives the reproduction number R 0j of the farm j. We divided the UK into 5 km squares and then calculated the average transmission risk of all farms in each square (local R 0 ). Figure 4 shows how geographic risk changed before and after 23 rd February for our best fit model conditioned on the first infection. The kernel shape has a major influence on the average risk distribution throughout the country. Figure 5 shows the corresponding risk maps for the estimates inferred from our best fit model conditioned on 23 rd February. A slightly higher risk is predicted after 31 st March by the model conditioned on 23 rd February due to the increase in the cattle infectivity after this date. The risk estimates after 23 rd February from the first set of models appear consistent with those obtained from the models conditioned on 23 rd February, though a rigorous statistical comparison is not appropriate. 
We have made the strong assumption for this study that the only infected farms during the 2001 epidemic were the reported IPs, and hence that any farms which were infected but culled before clinical diagnosis were not responsible for causing any infections. It is therefore interesting to calculate how many of the proactively culled farms our model predicts might have been infected (but, by definition, not diagnosed).
To calculate the probability p i that a particular proactively culled farm i was infected, we need to adjust the infection hazard by the probability that the farm would have not been reported as a clinical case before its culling date T i c . From the outbreak data, we calculate the probability density of the time from infection to report for reported IPs and hence the cumulative probability distribution of the time from infection to report, denoted by F. Then, with l i (t) being the force of infection on a proactively culled farm i at time t (from the best fit model conditioned on 23 rd February), the probability that that farm gets infected and escapes reporting between its potential infection time and culling time T i c is
We calculate the expected number of infections in different classes (e.g. DCs, CPs) of proactively culled farms culled within a particular time interval (T i c [ T 0 ,T 1 ½ ). For instance, the expected number of CPs culled at the time T i c [ T 0 ,T 1 ½ which are predicted to have been infected can be formally written as
This is a simplification, as in reality the delay from infection to report almost certainly depends on the size and species mix on a farm, but the result is nevertheless indicative of the expected level of infection in proactive culling. Also, at this stage, the calculations are made as if culling was a non-informative censoring process. This is a reasonable assumption for all proactively culled farms except for DCs (which by definition had been identified by veterinarian as having had a high risk of exposure) but our method may underestimate the infection rate. In calculating the infection to report delay distributions, we divided the epidemic after 23 rd February into 3 time periods: 23 rd February-31 st March, 31 st March-1 st May and 1 st May-5 th October. In these intervals a total of 1332, 4498 and 1627 farms were slaughtered, respectively. Our best fit model conditioned on 23 rd February predicts different infectivity regimes before and after 31 st March (see Parameter Estimates and Table 2 ) but we split further the second period of time due to different delays in reporting to culling. The infection to report delay is 8.6 and 8.8 days for the last two periods of time respectively but the infection to cull delay drops from 9.4 and 8.8 days respectively. Applying this approach to the interaction model with time varying cattle infectivity which conditioned on the 23 rd of February, we calculated the expected proportion of proactively culled farms which were infected. We estimate that approximately 1.3% (1%, 1.6%) of 7457 culled non-IP farms may have been infected -97 in total (Figure 6a ). Of the 1332 farms culled between 23 rd February and 31 st March, 1.7% (1%, 2.4%) may have been infected (23 farms). Of the 4498 farms culled between 31 st March and 1 st May, we estimate 0.7% (0.5%, 1%) were infected (34 farms). In the period 1 st May to 5 th October, we estimate that 1.6% (1%, 2.3%) of 1627 farms culled were infected (27 farms).
The proportion of CPs estimated to have been infected is 2% (1.5%, 2.5%), equating to 62 farms (Figure 6b ). Over the whole epidemic, we estimated 1.5% (0.8%, 2.1%) of farms designated as DCs were infected (19 farms). This estimate (Figure 6c ) does not allow for higher risk of infection implied by the veterinary judgement that led to those DCs being identified, which may mean that a higher proportion were in fact infected. If we assume that DCs were 3 times more likely to be infected due to their status than the model would predict, then the incidence of infection in DCs goes up accordingly, i.e. to 4.6% or 59 farms.
Farms culled neither as DCs or CPs (typically those culled under the 3 km and local sheep cull policies in the Cumbria, Dumfries and Galloway areas) had the lowest estimated rate of infectiona mere 0.5 % (0.2%, 0.8%) or 16 out of 3067 farms.
In this section we examine the sensitivity of our results to a number of factors: leaving pigs out of the analysis, possible errors in the estimated IP infection dates, and the assumed latent period.
To justify the simplification of the analysis by discarding the number of pigs in a farm, we present some more detailed statistics regarding this variable. We also fit the simplest model conditioned the last two farms are exclusively pig farms. We denote by n p i ,S p ,I p the number of pigs in farm i, pigs susceptibility and pigs infectivity respectively. The simplest model similar to (1.2) conditioned on the first infection has been fitted, reducing the number of parameters in the same manner.
In addition we estimated pig:sheep susceptibility ratio and pig infectivity, assuming all parameters constant through time. We found that cattle:sheep susceptibility ratio is 6. Table 1 shows parameter estimates for cattle and sheep are largely unaffected by ignoring the pig population, with none of the estimates from the two analyses being significantly different. We conclude that including pigs would not change the conclusions presented in Table 1 regarding cattle and sheep (given the very small number of IPs which had pigs) but it would decrease the power of the analysis and increase model complexity.
To understand to what extent our estimates are affected by the assumption that the infection dates have been accurately observed, we randomized the estimated infection dates by adding a Gaussian noise with zero mean and a standard deviation of 2 days. This is motivated by the substantial proportion in the observed standard deviation (73.5% less or equal than 2 days) of the distribution time from the estimated infection date to the report date of IPs. We then fitted the simplest model (conditioned on both first infection and 23 rd February) to 10 such randomised datasets. The average estimates across them are given in Table 3 . They lie well within the confidence intervals we predicted in Table 1 . The average cattle:sheep infectivity ratio is also very close to the values estimated using the original data.
The average estimates across 10 randomized datasets using the most appropriate model conditioned on 23 rd February (i.e. cattle infectivity and interaction model) are also in Table 3 . The values are within the 95%CI presented in Table 2 . We assessed a sensitivity analysis for the estimated proportion of infections in proactively culled farms (see the previous section) with respect to infection times. Using the predicted parameters for each dataset, we calculated the average proportions across all of them, for each category of proactively culled farms. The average proportion of infections between DC farms is 1.37% (2%, 0.78% and 0.72% for each period of time, respectively). For CP farms, the same quantities evaluate to 1.9% with 1.8%, 1.3% and 1.98%, respectively. Overall proactively culled farms, we obtained an average percentage of 1.25% with 1.64%, 0.81% and 1.6% for each considered period of time. All the values are well within the 95%CIs predicted by the original data (see the previous section and Figure 6 ).
All the results presented above assume a fixed latent period of 1 day. We tested the sensitivity of parameter estimates to this assumption by examining latent periods of 2 and 3 days. Overall, we would expect infectiousness parameters to increase to compensate for the shorter infectious period, and thus slightly increased generation time (namely the mean time from infection of one case and the time of infection of the cases that case generates). Interestingly, however, it is the kernel parameter estimates which are altered as the latent period is varied with the kernel becoming slightly less local with increasing latent period. For two and three days latent period, pre 23 rd February, the values of c dropped from 1.69 (Table 1) 
This paper has presented a statistical analysis of the spatiotemporal evolution of the 2001 foot and mouth outbreak in GB. Qualitatively, the results agree with those obtained by Keeling et al. [5] in identifying cattle as being the key species in the 2001 epidemic. Using the interaction model conditioned on 23 rd February with time varying cattle infectivity, we estimated that 88% of IPs between 23 rd Feb-31 st March were infected by cattle and only 12% by sheep. Sheep-to-sheep transmission only accounts for 3.1% of IPs in that period. After 31 st March (when we estimated that cattle infectivity increased slightly, see Table 2 ) Allowing for non-random mixing between species indicates contacts between farms are assortative on the basis of species composition of the farm; i.e. like species mix with like. This agrees with intuition about the nature of farming practices (e.g. sharing of personnel and equipment is likely to be more common if 2 farms have the same livestock species). The implications of the moderate degree of assortativity we found for control measures remains to be explored.
We did not use data collected during the epidemic on traced contacts between farms to fix the spatial kernel function in our analysis, since in the final version of the FMD epidemic data warehouse [http://www.defra.gov.uk/footandmouth/cases/index.htm] very few of the contacts apparently identified early in the epidemic remain confirmed. Also we shared the concern of earlier work that the distribution of contact distances in traced contacts may well be biased [3] . We therefore estimated the kernel function, using an offset power-law functional form. The higher value of the kernel power parameter we estimated after 23 rd February (2.67 vs. 1.70 before - Figure 3a) is consistent with the expected dramatic shortening in the typical contact distance following the national movement ban. This localized spread together with the higher estimated level of infectivity in cattle after 31 st March explains the long tail of the epidemic seen in 2001.
In estimating the transmission risk between farms, we assumed a dependence on the Euclidian distance between them. In reality, other metrics (e.g. the time required to travel between two farms) might be more reasonable, and should be examined in future work. We also did not include information on landscape (e.g. height above sea-level, location of rivers, trees etc).
The estimated risk maps (Figure 4 and Figure 5 ) match the areas of the country where highest case incidence rates were seen -with the notable exception of Wales. The discrepancy between the high predicted risk in Wales and the small number of cases observed may reflect inaccuracies in the input data set -Keeling et al. [5] reduced farm-level sheep population numbers by 30% in Wales and obtained a better geographic match to the data (Matt Keeling, personal communication). However, the discrepancy may also reflect model inadequacy. We have not here allowed for other farm-level risk factors, such as the farm fragmentation index considered by Ferguson et al. [3] . We have not explored more complex non-linear models of the dependence of susceptibility and infectiousness on the number of animals on a farm or relaxed our implicit assumption that contact rates between farms scale linearly with the local density of farms. All these assumptions are being relaxed in ongoing work.
The most important issue to be revised in future work is to allow for proactively culled farms which were not diagnosed as IPs to be potentially infected and infectious to other farms. This requires modification of the inference model used to allow for an arbitrary number of unobserved infections. The very low numbers of proactively culled farms we estimated as infected suggested that the effect of this model refinement may be limited. It should be noted though that these infection prevalence estimates are in part a result of the relatively non-local kernel estimated simultaneously. If kernel estimates change in a refined analysis -and if DCs were attributed a much higher risk of infection than estimated here due to their status -then it is possible that estimated infection rates in DCs and other proactively culled farms may increase somewhat.
However, even if these factors increased our estimated infection prevalence among proactively culled farms 5 fold (which seems unlikely from ongoing work), it would still mean that only a small proportion (,10%) of DCs and CPs culled were infected. This does not imply that proactive culling had no effect on the epidemic -as the largest expected effect of such culling is via the targeted depletion of susceptible animals. In this regard, proactive culling has the same epidemiological impact as vaccination. Future work will revisit past estimates of exactly how important such culling was for the control of the 2001 FMD epidemic. Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems. Pneumonia virus of mice (PVM) infection in mice was originally described by Horsfall and colleagues [1, 2] , but until relatively recently, the sole interest in this virus was as a pathogen of laboratory rodent colonies [3] [4] [5] . Over the past several years, we and others have built on Horsfall's early studies, and have developed and characterized an in vivo model of severe respiratory virus infection using PVM [reviewed in [6, 7] ]. Among our findings, we have shown that a minimal, physiologically relevant inoculum of PVM (typically <100 pfu) results in robust virus replication in lung tissue, accompanied by influx of granulocytes in response to local production of specific proinflammatory chemokines [8] . The pathophysiology of PVM bronchiolitis leading to pneumonia and acute respiratory distress syndrome (ARDS) is similar to that observed in response to severe respiratory syncytial virus (hRSV) infection in human infants [9] .
While PVM clearly replicates efficiently in mouse lung tissue, the in vitro propagation of this pathogen is significantly less straightforward. The primate BS-C-1 epithelial cell line supports minimal rates of PVM replication in vitro [10] . The BS-C-1 cell line has been used for traditional plaque assays, but PVM-induced plaques develop slowly, have relatively indistinct borders, and are difficult to eval-uate quantitatively [see Figure 1A ]. Furthermore, from an evolutionary perspective, one would prefer to perform molecular studies of virus pathogenesis in cells from a relevant species, i.e...a rodent cell type or cell line. We have demonstrated that PVM replicates in the mouse LA4 respiratory epithelial cell line [11] , but virus growth is similarly slow, even at temperatures permissive for virus propagation in vitro.
In this work, we explore PVM replication in several independent cell lines and identify the mouse macrophage 
The rodent L2, LA4, RAW 267.4, J774A.1, RLE and 3T3 and primate A549, BS-C-1, and HEp-2 cell lines obtained from American Type Culture Collection (Manassas, VA) were maintained in Iscove's Modified Dulbecco's medium with 10% heat-inactivated fetal calf serum, 2 mM glutamine and penicillin-streptomycin at 5% CO 2 and 32°C (permissive for virus growth in culture) unless otherwise indicated. Mouse-passaged PVM prepared as described was stored in liquid nitrogen at ~10 6 pfu/ml [12] . Virus replication in RAW 264.7 cells was determined by both Q-RT-PCR detection of the virus SH gene [see reference [13] ] for complete method] and by western blot [14] probed with a 1:200 dilution of polyclonal anti-PVM N peptide antibody prepared against sequence SQQLN-IVDDTPDDDI encoding amino acids 379 -393 of the PVM N protein. Proinflammatory cytokines in culture were evaluated by ELISA (R&D Systems, Minneapolis, MN). Q-RT-PCR detection of interferon-β was via standard methods using primer -probe set Mm00439546_s1 (ABI, Columbia, MD) normalized as described [13] on RNA prepared from infected and control uninfected cells in culture (RNazol B, Friendship, TX).
The cell lines evaluated for the ability to support virus replication included rat epithelial L2 and RLE, mouse epithelial LA4, mouse macrophage RAW 267.4 and J774A.1, and primate epithelial A549, BS-C-1, and HEp-2. All were inoculated with PVM on day 0 (MOI = 0.02, 10 4 pfu per 5 × 10 6 cells). On day 7, virus titer in the culture supernatants was determined by standard plaque assay [12] . Although pneumoviruses maintain strict host-pathogen specificity in vivo, we determined that PVM replicated to a limited extent in vitro (< 10 3 pfu/ml supernatant) in each of the aforementioned cell lines. The mouse monocyte/ macrophage RAW 264.7 cell line (established from a tumor induced by Abelson murine leukemia virus) generated the highest virus titers (10 4 pfu/ml) under culture conditions described. Cells of the RAW 264.7 line also support replication of other unrelated virus pathogens, including murine hepatitis virus and Japanese encephalitis virus [15] [16] [17] .
To evaluate the kinetics of virus replication and production of proinflammatory mediators in the RAW 264.7 cell line, cells at 50% confluence were inoculated with PVM (MOI 0.1) on day 0 and harvested on days 2 -5 thereafter. RAW 264.7 is a semi-adherent cell line, and is not well-suited for plaque assays. Here, virus replication was examined qualitatively on western blot of cellular homogenates probed with PVM-specific antisera [ Figure 1B ]. Virus was first detected in infected cultures on day 3 post-inoculation, and then in increasing amounts through day 5. No immunoreactive PVM N protein was detected in uninfected control cultures.
Virus replication was also examined quantitatively by Q-RT-PCR using the virus SH gene as a target sequence [13] , [ Figure 1C ]. PVM replication was readily detected in inoculated RAW 264.7 cells, reaching ~4 × 10 5 copies per microgram total RNA on day 5 of infection. No copies of the virus SH gene were detected in uninfected cells.
RAW 264.7 cells respond to infection with PVM by producing a variety of proinflammatory mediators. Transcription of interferon-β in response to virus infection was detected by Q-RT-PCR [ Figure 1D ]. Cytokines MIP-2, TNF-α, MIP-1α, and MIP-1β were detected in culture supernatants by ELISA [ Figure 1E ]. Interestingly, MIP-1α and MIP-2 are among the most prominent mediators detected in BAL fluid of infected mice; MIP-1α levels correlate directly with the severity of pneumovirus disease in both PVM and hRSV infection [18, 19] . In parallel to our findings, hRSV replicates in the human monocytic THP-1 cell line [20] , and several groups have provided evidence consistent with hRSV and bovine RSV (bRSV) replication in alveolar macrophages, although this point remains controversial [21] [22] [23] [24] [25] . Furthermore, hRSV infection of the human monocytic U937 cell line was associated with production of the proinflammatory mediator, platelet-activating factor (PAF) [26] .
In summary, PVM has recently emerged as a useful novel model for the study respiratory disease in mice [7, [27] [28] [29] [30] ; this has provided significant incentive toward identifying tissue culture systems for virus propagation. The mouse RAW 264.7 cell line supports efficient replication of PVM in vitro and responds to infection by augmenting production of cytokines implicated in the pathogenesis of respiratory disease. Use of this ex vivo model of PVM infection will permit further study of biological responses associated with virus infection and the cellular and molecular level.  Designing and conducting tabletop exercises to assess public health preparedness for manmade and naturally occurring biological threats BACKGROUND: Since 2001, state and local health departments in the United States (US) have accelerated efforts to prepare for high-impact public health emergencies. One component of these activities has been the development and conduct of exercise programs to assess capabilities, train staff and build relationships. This paper summarizes lessons learned from tabletop exercises about public health emergency preparedness and about the process of developing, conducting, and evaluating them. METHODS: We developed, conducted, and evaluated 31 tabletop exercises in partnership with state and local health departments throughout the US from 2003 to 2006. Participant self evaluations, after action reports, and tabletop exercise evaluation forms were used to identify aspects of the exercises themselves, as well as public health emergency responses that participants found more or less challenging, and to highlight lessons learned about tabletop exercise design. RESULTS: Designing the exercises involved substantial collaboration with representatives from participating health departments to assure that the scenarios were credible, focused attention on local preparedness needs and priorities, and were logistically feasible to implement. During execution of the exercises, nearly all health departments struggled with a common set of challenges relating to disease surveillance, epidemiologic investigations, communications, command and control, and health care surge capacity. In contrast, performance strengths were more varied across participating sites, reflecting specific attributes of individual health departments or communities, experience with actual public health emergencies, or the emphasis of prior preparedness efforts. CONCLUSION: The design, conduct, and evaluation of the tabletop exercises described in this report benefited from collaborative planning that involved stakeholders from participating health departments and exercise developers and facilitators from outside the participating agencies. While these exercises identified both strengths and vulnerabilities in emergency preparedness, additional work is needed to develop reliable metrics to gauge exercise performance, inform follow-up action steps, and to develop re-evaluation exercise designs that assess the impact of post-exercise interventions. Since 2001, state and local health departments in the US have accelerated efforts to prepare for bioterrorism and other high-impact public health emergencies. These activities have been spurred by federal funding and guidance from the US Centers for Disease Control and Prevention (CDC) and the Health Resources and Services Administration (HRSA) [1] [2] [3] . Over time, the emphasis of this guidance has expanded from bioterrorism to include "terrorism and non-terrorism events, including infectious disease, environmental and occupational related emergencies" [4] as well as pandemic influenza [5] .
For any locality, the rarity of major public health emergencies necessitates the use of practice-based exercises to simulate real life experiences in order to develop and improve skills and to assess response capabilities over time. The US Federal Emergency Management Agency (FEMA) describes six levels of exercises, increasing in complexity from informational seminars that minimally exercise response capacities to simulations that mimic reality and exercise participants' capacity to implement emergency response functions [6] .
Intermediate in this progression is the tabletop exercise, which FEMA describes as a "facilitated group analysis of an emergency situation." As practiced in public health, there is considerable variability in how tabletop exercises are designed and conducted. Tabletop exercises may be structured discussions of evolving events or unstructured reactions to short scenarios; participants may be limited to public health staff or involve representatives from partner agencies or organizations; scenarios may range from simple to complex; and facilitation may range from being minimally directive, allowing participants to assume responsibility for managing the discussion through "role play," to highly directive, enabling the facilitator to assure that specific questions are addressed.
Recognizing the need to exercise public health emergency response, and enabled by funding and directives from CDC and HRSA, health departments throughout the US have implemented exercise programs. These exercise programs have had varying goals, including building relationships among stakeholders [7, 8] , training staff [9] [10] [11] , and evaluating preparedness levels [12, 13] , and they have been used for a variety of purposes, including to identify gaps in preparedness [14] , make recommendations for improving preparedness [15] , and identifying variations in preparedness across health departments [16] . These exercises have involved diverse groups of stakeholders involved in public health preparedness, such as representatives from public health [17] , health care [18] , agriculture [19] , and emergency medical services [20] . Despite the commonality of preparedness and response compo-nents across a variety of biological threats, most exercises have been designed for single use and focus on single disease, such as smallpox [21] , pandemic influenza [22] , or a novel virus [23] .
These exercises have focused attention on the interaction between preparedness goals and exercise strategies, and have illuminated strengths and vulnerabilities in public health emergency decision making and response capacities. The increased utilization of tabletop exercises by health departments has not been accompanied by a parallel increase in knowledge sharing about lessons learned from them, either with regard to identifying common challenges that confront health departments or strategies for effective exercise design and management. Further, the literature dealing with tabletop exercises to date consists almost entirely of case studies and descriptions of a single exercise or a single disease. This paper describes lessons learned by public health researchers at RAND, and their collaborators, about the process of developing and conducting tabletop exercises in collaboration with state and local health departments in the US and their implications for public health emergency preparedness.
Data for this paper come from four related projects conducted from 2003-2006. Taken together, these projects involved developing, conducting, and evaluating 31 tabletop exercises with state and local health departments of different sizes and structures in 13 different states across the northeast, south, mid-west, and west regions of the country (Table 1) . Participating health departments did not incur any expenses through their involvement in these exercises other than the staff time required to participate.
Two of these projects, one in California and the other in Georgia, involved the conduct of exercises in multiple jurisdictions in the same state. In California, the Little Hoover Commission, a bipartisan, independent state body, asked RAND to assess California's public health infrastructure. A key component of the project, described in greater detail elsewhere [16] , was the development of a tabletop exercise that simulated a smallpox outbreak. This exercise was conducted in seven local health departments across California. In Georgia, RAND collaborated with the Georgia Division of Public Health and the Rollins School of Public Health at Emory University to develop, conduct, and evaluate a series of tabletop exercises focusing on different biologic agents in seven local health departments across Georgia, as well as one exercise focused at the state level.
The two remaining projects were funded by US Department of Health and Human Services (HHS) and involved the participation of multiple local health departments.
The first project involved developing ten different tabletop exercise templates and formats focusing on the local public health response to bioterrorist agents. These were tested in 13 local health departments in 12 different states. The second project involved developing a tabletop exercise to examine the interface between local health departments and health care systems in a hypothetical influenza pandemic. This exercise was tested in three local health departments in different states. Greater detail on the structure for these tabletop exercises as well as the tabletop exercise templates themselves can be found elsewhere [7, 17] .
All exercises focused on at least one of three related objectives: training, relationship-building, and evaluation. The structure and design of the tabletop exercises varied from project to project because their objectives were somewhat different. The key domains covered are outlined in Table   2 . The level of facilitator involvement varied with the exercise objectives. At one extreme, the facilitator's role was limited to introducing the exercise scenario and periodically interjecting updates. During these exercises, the participants were encouraged to lead the discussion themselves, based on their respective roles in their agency or organization. At the other extreme, the facilitator took a very active role by leading the discussion and interjecting questions or prompts. In between were exercises in which the facilitator turned the discussion over to participants but occasionally joined the discussion to request clarifications from the participants or assure that issues critical to the exercise objectives were discussed.
Despite these differences, all of the exercises shared common elements, including: evolving hypothetical scenarios, facilitated group discussions, and some level of collective decision making by participants emphasizing *< 100,000 = small, 100,000-1,000,000 = medium, > 1,000,000 = large; **Mild involvement-most of exercises was role played by participants, with very little intervention or direction from facilitators; Moderate involvement-most of exercises was role played or issue discussion, with the facilitator inserting additional probes and ensuring the discussion stayed on track; Active-most of the exercise was more discussion based, with facilitator asking questions or identifying issues that were subsequently discussed.
the role of local health departments in recognizing and initiating a response to an emergency. The scenarios typically began with a single case report or series of case reports that heralded a nascent disease outbreak and required a public health assessment. These situations exercised the internal communication and coordination across disciplines within health departments as well as the communication and coordination with partner agencies and organizations such as health care facilities and emergency medical service agencies. Several exercises extended beyond this initial response and included scenarios that progressed days or weeks into an outbreak, requiring greater interactions between local-and state-level authorities and attention to health care surge capacity.
Every exercise concluded with a "hot wash" in which participants discussed their collective performance, identified strengths and weaknesses, and when relevant, related their performance to experience with actual outbreaks or crises. In the latter exercises, participants were prompted to develop an initial 'action plan' that addressed key vulnerabilities identified in the exercise. The facilitators subsequently generated a written "After Action Report" (AAR) that summarized the exercise experience and highlighted the observed strengths and areas for improvement. In addition, participants completed exercise evaluation forms. These consisted of a series of structured and semistructured questions that asked participants to discuss what they learned during the exercise and to evaluate aspects of the exercise structure and conduct. For example, participants were asked to identify key gaps in preparedness that occurred during the exercise and to identify the most useful thing they learned during the tabletop exercise. The observations reported here are based on reviewing the after action reports, participant evaluations, as well as internal team discussions and consensus following the exercise debriefings.
The performance of health departments that participated in our tabletop exercises varied from agency to agency. However, there were consistent themes that emerged across the agencies, regardless of the structure or the biologic agent/disease discussed; nearly all agencies struggled with a common set of challenges. These challenges, summarized in Table 3 and described below, represent critical dimensions of an outbreak response.
Many local health departments did not have a structured process for notifying or soliciting case reports from health care providers in the community other than those in hospitals, largely because they did not have reliable contact information for private providers or a sure means to reach them rapidly. In most instances local health departments had good relations with staff in local hospitals (e.g., emergency department staff and infection control practitioners) but did not appear to have similar working relationships with non-hospital based practitioners.
Local public health officials were sometimes unsure about their direct role in following up with suspected ill patients and collecting and shipping clinical samples for laboratory testing. For example, there was frequently confusion around whether it was the responsibility of the local health department, the state health department, or the medical personnel at the hospital to collect laboratory samples. Once the samples were collected there was often confusion around whose responsibility it was to transport the samples, and in a few sites, local law enforcement were surprised to find out that they were the responsible party. A related issue was the ability of health departments to realistically generate enough surge capacity in their public health workforce to investigate or respond to a large event, especially one that encompassed multiple jurisdictions in the same state, thereby limiting the state health department's ability to shift manpower and resources from one jurisdiction to the next.
Few of the health departments in which we conducted exercises were proactive in their contacts with the media, and most waited until they were contacted by the media to begin communicating with the public. One consequence of this passive approach was that public health officials often responded defensively to early and sometimes unexpected media requests and in turn, had trouble quickly formulating an initial message to the public that was clear, informative, and alleviated anxiety.
Health departments consistently expressed uncertainty about how to effectively communicate with vulnerable or underrepresented population groups in their jurisdictions, and few had well established relationships with community leaders or organizations that could serve as messengers or communications channels to these groups.
In several sites, law enforcement and EMS personnel present in exercises had greater familiarity with these groups and could help identify trusted community messengers. Further, in some communities, health departments had limited language capacities or were not sufficiently familiar with community leaders to communicate effectively with these groups.
Communicating fully and effectively with response partners (e.g., law enforcement and EMS) about their occupational health risks and personal protection was also a challenge for local public health. In particular, while public health officials were usually quick to notify response partners soon after determining an event to be significant, response partners in many cases felt that public health officials were slow to provide them with critical information about the disease in question, what their risks might be, or what actions they should take to protect themselves. As a result, response partners frequently reported feeling either left out of the process or expressed concerns about continuing to work unless the risks to them were clarified and more was done to ensure their safety on the job.
The use of the National Incident Management System (NIMS) and its associated Incident Command Structure (ICS) structure is relatively new to public health. This was evident in the exercises, in that nearly all health departments had difficulties deciding if and when to implement the ICS process and in identifying the party who would serve as incident commander. Similar challenges were seen in the decision and processes related to opening an Emergency Operations Center (EOC). As a result, in many exercises, local public health officials delayed taking these steps and preferred maintaining a more informal management process. This approach was preferred even as the outbreak became progressively larger, thereby stressing these informal networks.
As outbreaks evolved, there was often a lack of clarity about whether and when local health departments should hand off control to the state health department, how responsibilities should be jointly shared between local and state authorities, and whether or when federal agencies, such as CDC, should become involved. In many of the exercises, state health departments were surprised by the level of assistance requested by their local health departments especially in the early stages of the outbreak; in other more rare examples, state health departments surprised local health departments by assuming roles and responsibilities local health departments regarded as their own. Regardless, the general consensus among local public health participants in most exercises was that CDC staff would be on the ground to help them fairly quickly, particularly in situations where bioterrorism was considered likely.
Most local health departments articulated some type of plan for increasing medical surge capacity by developing alternative care sites. In most instances however, these plans were unable to hold up to even a modest amount of scrutiny during the exercise because they were superficial and lacked sufficient detail necessary for rapid implementation. Related to this issue, local health departments frequently reported that there were not enough local health care workers to manage these sites even if they could create them. For example one participant noted, "We have pop-up tents and beds to increase capacity, we just don't have pop-up people to staff them."
Even obtaining a census of available staff members turned out to be challenging as many health care participants noted that some staff would likely be double-counted, particularly nurses and security officers who might work in several institutions. Increasing staff capacity through the use of community volunteers, including retired medical personnel, while often recognized as one potential solution to staffing shortages, proved to be extremely difficult to actually implement. Public health participants universally recognized the importance of volunteers, but learned that their plans to recruit, train, and mobilize large numbers of volunteers were too vague and lacked concrete actionable steps for realistic application during a real emergency.
In nearly all exercises, we also identified a number of strengths within participating health departments. However, there was far less commonality in these strengths than we observed with the areas for improvement. Universally, we observed public health leaders and staff who were committed and struggling to 'do the right thing.' The most commonly observed strengths were strong relationships between epidemiologists and hospital infection control practitioners and between public health workers and other emergency coordinators. In some instances, prior experience with emergency planning or response, such as involvement of health departments and emergency service agencies in coastal areas in preparing for or responding to hurricanes, was associated with stronger and more facile interactions between health department officials and partner agencies.
Our exercises were conducted over a period of several years. While we did not conduct exercises with any health department more than once and did not employ an experimental design to assess changes over time, we were struck by how the performance of health departments overall improved over time. First, compared to earlier exercises, local health departments appeared far more sophisticated about their early internal processes related to notification, enhanced surveillance and large outbreak investigations. In addition, by the end of the exercise period, health departments had considered plans for surge capacity, and participating hospitals had explicit plans for cancelling emergency surgery and discharging less severely ill patients. They also appeared more acutely aware of the challenges in assuring adequate numbers of staff to provide care.
The large number of tabletop exercises we conducted allowed us to test and compare different strategies for designing and conducting tabletop exercises. These comparisons enabled us to modify our exercises over time to build upon lessons learned from previous exercises. Below we briefly highlight five lessons we learned from this experience.
Exercises should be designed to achieve a specific objective When first developing the tabletop exercises, our assumption was that a single exercise could achieve multiple objectives, such as training, relationship building, and evaluation. While these objectives are interrelated and opportunities often exist to achieve them concomitantly in the same exercise, it is critical to define the priority objective for the exercise because different objectives have different implications for exercise design. For example, if exercise participants outlined a response that was flawed or problematic, in an exercise primarily focused around the objective of training it would be appropriate for the facilitator to pause and help the participants re-think their approach. On the other hand, if the objective of the exercise is evaluation, this type of facilitator involvement can lead the participants to choose a different course of action and therefore bias the overall outcome being evaluated. Taken further, in an exercise designed to build relationships and links across disciplines or agencies, a facilitator intervention implying that a participant had made a mistake could be embarrassing or diminish that person's credibility, depending on the level of trust among participants.
Exercises should be as realistic as possible while remaining logistically feasible Taken together, the optimal mix of design elements represents a balance between exercise objectives and logistic feasibility. The ideal balance is one that assures sufficient realism to provide a meaningful experience while minimizing distractions associated with the necessary artifice of exercise scenarios. Some departures from reality may be inadvertent if scenarios are developed with insufficient attention to local routines, forcing participants to sidestep usual procedures. Even seemingly minor design errors, such as using an outdated name for a hospital, or a time course for a disease that is inconsistent with its known epidemiology, can undermine the credibility of the exercise and can distract participants enough to take them out of their roles, thus disrupting the flow of the exercise.
The desire for a realistic exercise scenario can lead to the development of tabletop exercises around scenarios rather than issue areas based on local preparedness needs and priorities. This does not ensure that the participants will address the important issue areas. A broad mix of challenges related to a given scenario must be addressed, often simultaneously, ranging from conducting epidemiologic or environmental investigations, implementing and modifying interventions as information becomes available, communicating within and across agencies, and communicating with political leaders and the public. Introducing this full set of tasks into an exercise scenario in a way that meaningfully exercises relevant capacities is unlikely to align with the exercise's objective. Moreover, different stakeholders may want to address different issue areas and may become frustrated if their expectations are not met. It is therefore important for stakeholders to agree on a limited number of priority issue areas for the exercise and then to focus the design of the scenario around these areas.
For example, one set of exercises we designed focused on pandemic influenza preparedness in local health departments. Because it was infeasible to exercise the entire pandemic plan around a single scenario and in a single exercise, we developed the scenario and then the exercise by first meeting with local stakeholders to decide on the issue areas that would be covered in the exercise. These issue areas included disease surveillance, medical surge capacity, non-pharmacological disease control, and the use of antiviral medications. The scenario was then customized to unfold to deal with each of these issue areas.
Key decisions for each discussion point were then developed, as well as facilitator probes and instructions based on the specific objective of the exercise.
If not designed or facilitated properly, an exercise can lack focus and resolution, leaving participants to wonder what exactly was accomplished during the exercise. Therefore, it is paramount that exercises are designed to focus on issue areas that require concrete decisions over a limited period of time. For example, an exercise dealing with a simulated smallpox outbreak that unfolds over time might involve a discussion period dealing with movement restrictions in which participants are asked one or more questions such as, "Should schools be closed at this point?" Participants should then be given a limited amount of time to discuss this issue and make a decision. It is the facilitator's job to keep the discussion focused on the issue area and the specific question(s) at hand and to ensure that at the end of discussion, participants have collectively made the decision(s) they were tasked to make. Exercises can be designed to have multiple such issue area discussions as the scenario unfolds.
Depending on the goals and objectives of an exercise, an exercise can involve a narrow or wide range of potential participants. While broader inclusion would likely be more realistic, such inclusiveness needs to be weighed against the logistics of effectively managing a larger number of participants and the potential adverse effects of inclusion. For example, participants may be less comfortable discussing ideas, taking risks, or making mistakes, depending on who is in the room. Such constraints may impede the exercise process and undermine achievement of exercise objectives.
One solution to this problem would be to sequentially stage the involvement of different participants or to physically separate different groups in a way that more closely mimics actual situations. For example, some conversations that involve airing uncertainties or weighing difficult alternatives may normally involve a limited group of people, and members of that group may be more comfortable exercising such a conversation apart from colleagues from other agencies or organizations.
The disadvantage of this approach is that it is substantially more difficult logistically and it diminishes the opportunity for people from different groups to gain an understanding of one another's role and approach to problems. In those exercises where certain participants, notably law enforcement, joined the scenarios at different stages, the feedback was generally critical, and participants felt that staging participation diminished learning and teambuilding opportunities. Another solution is to split exercise participants into two or more groups that allow everyone to participate, often placing people at similar levels of responsibility in the same group, and to conclude the exercise with a session that brings everyone together to share what they learned.
The exercises described in this report represented collaborations between people familiar with local circumstances and people from outside the participating jurisdictions who had expertise in exercise design and facilitation. Because we did not test an alternative approach that exclusively involved local personnel, it is difficult to generalize from this experience about the value of engaging people from outside the participating agencies. Nonetheless, it was our impression that at certain points in the development, facilitation, and feedback steps, there was value in involving people who were not personally invested in local relationships or situations and who could offer seemingly independent advice or perspectives.
Tabletop exercises can provide useful insights into both strengths and vulnerabilities in public health preparedness. It is important to recognize, however, that exercise outcomes are influenced by the way they are designed and conducted. The exercises described in this report emphasized varying dimensions of public health preparedness, reflecting differences in state or local priorities for prioritizing exercise objectives. For example, some emphasized the early response to initial reports of suspect illness while others emphasized management of surges in demand for health care services that are likely to occur later emergency scenarios. Given the intellectual and emotional demands of participation in an exercise, participants (and facilitators) may be less energetic during later rather than earlier stages of an exercise scenario, affecting perceived capacity to execute different elements of a response. Potential gaps between observed and actual preparedness should be considered in interpreting after-action reports and evaluating exercises themselves.
The utility of tabletop exercises as tools to identify areas for improvement and make improvements on these problems is still evolving. Our ability to evaluate exercise performance is hampered by the lack of an evidence base about what constitutes optimal performance and by the lack of standards for assessing public health preparedness. There is a need to move beyond qualitative performance measures to ones that are quantifiable and can be measured over time. These quantifiable measures can range from simple checklists to Likert rating scales to scorecards. For example, in a series of our exercises we used checklists to assess the performance of health departments related to surveillance, risk communication and other functions.
One fairly consistent observation was that health departments identified gaps that had been identified in prior exercises or actual experience, but had not yet been addressed. Reasons for this included lack of time, and lack of knowledge about how to make change. We now conclude exercises by having health departments prioritize the challenges observed during the exercise and then have them develop initial action plans related to up to three priority items.
There are important limitations to our work and its interpretation that must be recognized. First, the nature of our exercises changed over time on a number of important dimensions, including the scenario, priority objectives, facilitation, exercise designers and facilitators, and attention to beginning an action plan after the hot wash. As a result of this variation, we are unable to provide a numerical tabulation of the numbers of health departments that struggled with each gap or displayed given strengths. Second, because we did not employ a methodology that could conclusively assess change over time, we cannot be certain that the improvements we identified were truly reflective of improvement, and not due to the inclusion of more sophisticated health departments in the latter part of our exercise period. We doubt this is the case, however, given the national emphasis on preparedness and planning and the ways in which health departments participating in later years qualitatively described their improvement. Furthermore, similar observations regarding improvements in public health preparedness during the same time period have recently been reported by others [24, 25] . We also cannot asses the potential influences that external events (e.g., hurricanes, outbreaks) may have had on health departments during the time period of our work, but it is noteworthy that all exercises were concluded before Hurricane Katrina struck. In addition, our exercises were not conducted in a random sample of health departments, and the findings may not be generalizable to all health departments. Finally, as discussed above, the evidence base for determining best practices in the design and conduct of exercises is extremely thin. We share our experience in the hope that it will help others, but do not propose that our recommendations constitute best, proven practices.
Developing, conducting, and evaluating tabletop exercises requires considerable planning and the perspectives of a variety of stakeholders. While these tabletop exercises identified both strengths and vulnerabilities in emergency preparedness, additional work is needed to develop reliable metrics to gauge exercise performance, inform followup action steps, and to develop re-evaluation exercise designs that assess the impact of post-exercise interventions. Transcript-level annotation of Affymetrix probesets improves the interpretation of gene expression data BACKGROUND: The wide use of Affymetrix microarray in broadened fields of biological research has made the probeset annotation an important issue. Standard Affymetrix probeset annotation is at gene level, i.e. a probeset is precisely linked to a gene, and probeset intensity is interpreted as gene expression. The increased knowledge that one gene may have multiple transcript variants clearly brings up the necessity of updating this gene-level annotation to a refined transcript-level. RESULTS: Through performing rigorous alignments of the Affymetrix probe sequences against a comprehensive pool of currently available transcript sequences, and further linking the probesets to the International Protein Index, we generated transcript-level or protein-level annotation tables for two popular Affymetrix expression arrays, Mouse Genome 430A 2.0 Array and Human Genome U133A Array. Application of our new annotations in re-examining existing expression data sets shows increased expression consistency among synonymous probesets and strengthened expression correlation between interacting proteins. CONCLUSION: By refining the standard Affymetrix annotation of microarray probesets from the gene level to the transcript level and protein level, one can achieve a more reliable interpretation of their experimental data, which may lead to discovery of more profound regulatory mechanism. Microarray technology was invented to rapidly profile the quantities of mRNA transcripts in a particular cellular context [1] [2] [3] . Its application has become universal in biomedical researches. Although it is mRNA that is actually detected by microarray experiments, and it is mRNA that has the direct relationship with protein, the methodology and algorithms for data analysis are commonly gene based. As evident in the probe annotation file provided by Affymetrix, gene-level annotation is widely accepted even though it fails to discriminate multiple mRNAs transcribed from the same gene. As a result, the analysis results are usually summarized at the gene level, such as differentially expressed genes [4] [5] [6] or gene-sets [7, 8] . Even in the recent works that integrate protein-protein interaction data and microarray data [9] [10] [11] , the analysis unit is reduced to the gene instead of mRNA. This practice could be attributed to the fact that most of the functional knowledge is at gene level instead of transcript level, and the functional differences between mRNA variants transcribed from the same gene are seldom clear. Although the gene-level analysis ignores the difference among mRNAs variants, this strategy is still biologically meaningful considering that the diversity of genes is much higher than that of transcripts encoded by the same gene.
It has been well established that alternative splicing increases mRNA diversity, and over 60% of human genes are involved in this mechanism [12] . In addition, other RNA processing events, such as RNA editing, also account for the increased diversity at the mRNA level [13] . Since these events enable one gene to encode multiple proteins which might be functionally heterogeneous, we feel it necessary to separate transcript-level synonymous probesets from gene-level synonymous ones. The probesets that hybridize to more than one transcript variant of the same gene are referred to as gene-level synonymous probesets; while the ones that hybridize to a single variant are named as transcript-level synonymous probesets. It has been noticed that transcript-level synonymous probesets tend to have similar expression profiles, while gene-level synonymous probesets may have distinct expression profiles [14, 15] . In fact, individual reports demonstrated that the expression of transcript variants could be totally different [16, 17] . These phenomena indicate that the gene-level strategy of microarray data analysis is imprecise enough that one may overlook the expressional inconsistencies among gene-level synonymous probesets.
As a matter of fact, Affymetrix suffixes their probeset ID according to the probeset's specificity. For example, probesets that recognize unique transcript variants are suffixed with _at, and probesets that recognize multiple alternative transcripts from a single gene are suffixed with _a_at or _s_at, and so on. This suffix system gives a hint on the varied specificities of the probesets, and could be considered as an endeavor trying to do away with customers' worry about the gene-level data analysis strategy. However, the correctness of the suffix system has been in doubt [14, 18] . Therefore it is not reliable to perform transcriptlevel analysis on the basis of this imperfect suffix system. As the standard annotation files and most of the analysis algorithms are gene-oriented, analysts often average out the expression heterogeneity of the same gene when dealing with probeset level data [19] .
In this paper, we linked the probesets of two widely used Affymetrix arrays with the International Protein Indexes (IPIs) [20, 21] through proper association and rigorous alignment procedures, and demonstrated the statistically significant advantage of interpreting microarray data at the transcript-level or protein-level. Our results can be viewed as a more precise annotation of Affymetrix array's probesets, with which one may achieve a more reliable interpretation of their experimental data. Moreover, the application of this new annotation substantially increased the expression correlation between interacting proteins.
Two Affymetrix arrays, MOE430A_2 and HG-U133A, were chosen for this study, in which 21,097 and 16,213 putative protein-coding probesets for the two arrays were subject to the alignment investigation (Table 1) . Candidate probeset-mRNA relationships were compiled based on the probeset-gene mapping information specified in the standard Affymetrix annotation files and gene-mRNA mapping relationships provided by separate mRNA transcript sources. Rigorous blast procedure similar to that described in [22] were performed to filter these candidate probeset-mRNA relationships. Finally, the mRNA targets passing the filtering criteria were linked to protein IDs in the IPI database.
Through the rigorous association and alignment, we obtained precise annotations for 18,894 and 15,288 probesets in Affymetrix arrays MOE430A_2 and HG-U133A respectively (see Additional file 1). These annotations discriminate alternative mRNA variants transcribed from a same gene, thus are at transcript level as opposed to the standard gene-level annotation files provided by Affymetrix. It is worth noting that since the transcript data we used were quite redundant, a conceptual transcript variant may be represented by multiple redundant transcript accessions in the transcript database. In our transcriptlevel annotation file, each conceptual transcript is identified with one IPI ID, as we only investigated the probesets associated with protein-coding transcripts.
Statistics on the non-control, investigated and annotated probesets, together with the number of involved genes and proteins, are shown in Table 1 . It is evident that the proportion of genes covered by our annotated probesets to those covered by all non-control probesets ('gene retaining percentage' in Table 1 ), 95.2% for MOE430A_2 and 85.4% for HG-U133A, are higher than the corresponding probeset retaining percentages, 83.5% and 68.8%, indicating that the gene coverage has only been slightly reduced by our filtering procedures. This observation is in support of our primary goal, that is to refine gene-level probeset annotations to transcript-level, but not to simply remove the poor-quality gene-level annotations.
Furthermore, we classified the probesets based on the way they linked to proteins. By checking the alignment results, the probesets in our annotation tables were divided into two groups, namely one-to-one and one-to-many. In our annotation table, a one-to-one probeset was linked to only a single protein, while a one-to-many probeset was linked to multiple proteins due to alternative splicing of one gene. The rest of the investigated probesets that were not linked to any protein were categorized into the third group of "one-to-null". The statistics of these three groups are shown in Table 2 . As we know, Affymetrix suffixes their probeset ID according to the probeset's specificity. The over ten types of probeset suffixes can be categorized into three groups: transcript-specific, with '_at', gene-specific, with '_a_at' or '_s_at', and other suboptimal probesets that may cross-hybridize with unrelated sequences. The first two groups are comparable to our one-to-one and one-to-many probesets respectively. However, we found that, out of a total of 21,097 and 16,213 investigated probesets in MOE430A_2 and HG-U133A respectively (Table 1) , our one-to-one type accounts for only 53.7% and 43.3%, which were significantly lower than that of _at probesets, 74.0% and 72.6%, in the respective arrays. This suggests that some of _at probesets are not really specific for a transcript.
To further clarify the issue of probeset specificity, we grouped the investigated probesets with the varied Affymetrix suffixes as well as our own categorizing system (Table 3 ). It is evident that overall one-to-one and one-tomany take up the majority of the _at group and the _a_at/ _s_at group respectively, but there are some disagreements between the two classifications. For example, the Affymetrix _at group has 30.1% one-to-many probesets and 11.7% one-to-null probesets, suggesting that these so-called 'transcript-specific' probesets do not have the expected high specificity, and that they should be treated with caution in data analysis. On the other hand, the Affymetrix _a_at/_s_at group contains 42.0% one-to-one probesets. We attributed this mainly to the trimming of Table 2 : One-to-one and one-to-many probesets in our annotation tables.
Annotated probesets One-to-one probesets 11327 (53.7% of investigated) 7014 (43.3% of investigated) One-to-many probesets 7567 8274
Sum 18894 15288
One-to-null probesets 2203 925
Total (Investigated probesets) 21097 16213 the originally false transcripts during the update of sequence information, as they may lead to the absence of the transcript targets of some probesets. Similarly, there are a large number of one-to-one probesets in the socalled Affy others group, 1,206 out of 2,619 for MOE430A_2 and 476 out of 1,555 for HG-U133A. These probesets, however, might not be really one-to-one mapping to the identified transcript targets, as our strategy was based on the premise that most probesets were specific for certain genes, and our blast was limited to the candidate transcripts associated with the probesets, but not the entire transcript pool.
These subgroups of the investigated probesets with different Affymetrix suffixes indicate the imperfection of the Affymetrix suffix system, thus affirming the necessity of our transcript-level or protein-level probeset annotations. In fact, several other research groups have addressed the misleading nature of the Affymetrix suffix system and the imperfection of its standard annotation file, including some re-annotation works for array HG-U133 [14, 18, 19, 23] . We will discuss these related works in detail in next section.
Since the probesets were linked to transcripts and proteins through rigorous association and alignment procedures, the expression profiles of transcript-level synonymous probesets were supposed to be more consistent than those of gene-level synonymous probesets [14, 15] . This was taken as the basis for the evaluation of our annotations.
We downloaded 30 expression datasets assayed with MOE430A_2 from the Gene Expression Omnibus database (GEO) [24, 25] , and calculated the Pearson correlation coefficients (PCCs) of expression profiles of synonymous probesets at gene level and transcript level. Since transcript-level synonymous probesets in our work recognized a single protein-coding transcript variant identified with a unique IPI ID, transcript-level synonymous probesets were also named as protein-level synonymous ones. Practically, we grouped gene-level synonymous probesets according to gene ID, and protein-level ones according to IPI ID. The probeset-protein mapping tables provided at NetAffx, the official protein-level annotation of Affymetrix probesets [26] , was used as comparison. Probesets corresponding to a single protein according to NetAffx were designated as 'Affy-protein' level synonymous probesets, which was a third level of synonymy.
Within each synonymous group, all pair-wise PCCs were calculated and then summarized to one value indicating the expression correlation of this group. The expression correlations of all synonymous groups at one level were then averaged into an overall value for a dataset (see Method section for details). The correlations of synonymous groups for 30 datasets at three different levels were depicted in parallel in Figure 1A . It is noticeable that the synonymous probesets at Affy-protein level show higher correlations than those at gene level, but the synonymous ones based on our protein level annotation show even higher correlations consistently over 30 datasets (p < 0.05 for 30 datasets under student's t-test, see Additional file 2 for details). The comparison proves the rationality and necessity of our protein-level probeset annotation.
These results support the argument that it is more reliable to interpret microarray data at transcript level than at gene level. The inferiority of the Affy-protein level annotation to our annotation could be attributed to the technical details in their alignment and association procedures [26] . First, they used the representative mRNA sequence ('consensus' or 'exemplar' sequence of each probeset), instead of the probe sequences themselves, as the query sequence in the alignment. Second, they aligned against the GenBank non-redundant protein database, rather than a comprehensive pool of mRNA sequences. In microarray experiment hybridization takes place between the probe sequences immobilized on the array and the cDNA sequences from the sample, so one can deduce that the alignment between the representative mRNA sequences and the protein sequences cannot precisely simulate the hybridization between probes and mRNAs. Finally, NetAffx filtered the blast results according to a cut- off of E-value, which indicates the likelihood of the observed alignment by chance [27] . Although frequently adopted for sequence homology analysis in closely related species, E-value is not sensitive enough to grade the many well aligned targets from the same species. In our practice, we took the probe sequences as the query and the mRNA sequences as the alignment targets, and adopted the matching nucleotide proportion as the filtering criterion (see Methods).
A similar comparison was conducted for HG-U133A array, involving another transcript-level annotation by Harbig et al. [14] . Across the whole 28 datasets, the annotation by Harbig et al. showed advantage over Affy-protein-level annotation, while our transcript-level annotation performed best (p < 0.05 for 18 datasets under Student's t-test, see Figure 1B and Additional file 2). As our work was done two years later than Harbig et al.'s, the updated mRNA sequences in the probe-mRNA alignment is one of factors contributing to the increased performance. The other contributing factor is different approach we used to identify the mapping of probes to mRNA targets. Harbig et al. performed a two-phase blast: first, blast probeset target sequences against mRNA sequence pool, and then blast probe sequences against the retrieved mRNA sequences. Efficient as it was, this two-phase-blast strategy reduced the alignment precision as compared to our direct probe-against-mRNA blast strategy. Moreover, Harbig et al. accepted the mRNA with the highest average probe matches as the target of a probeset, even if the highest value could be suboptimal.
According to our data, the puzzling fact that probesets for one gene may show variable expression profiles can be clarified when viewing the data at the protein level. Such genes are likely to involve alternative transcript variants which are constitutively different in expression levels. We looked at two genes as examples. Shown in Figure 2A , three probesets in GDS1277 dataset, 1448556_at, 1421382_at and 1451844_at, were mapped to the mouse Prlr gene (GeneID: 19116), with the first probeset correlating to protein IPI00321091, or PRL-R3, and the other two correlating to protein IPI00408593, or PRL-R2. The Prlr gene was reported to encode at least seven isoforms of prolactin receptor precursor in mouse [16] . The two probesets corresponding to the same protein IPI00408593, showed similar expression profiles as expected, with a PCC value of 0.6738 (P = 4.57e-6); while the probeset corresponding to another isoform, IPI00321091, showed a significantly different expression profile, with PCCs of 0.2530 and -0.0331 separately for 1421382_at and 1451844_at ( Figure 2A ). These data were consistent with the previous report that these two isoforms were predominantly expressed in liver and kidney, where PRL-R3 was highly expressed and PRL-R2 was weakly expressed [16] . We noticed that the shape of the expression curve of PRL-R3 seemed to be somewhat comparable to those of PRL-R2, although PRL-R3 probeset presented an overall higher expression level. These phenomena may suggest that PRL-R3 and PRL-R2 are partially co-regulated so that they show a comparable expression pattern, but they are expressed at different levels probably due to diverse roles of the regulatory elements.
Shown in Figure 2B , three probesets in GDS1076 dataset, 1419114_at, 1419115_at and 1419116_at, were mapped to the mouse gene Alg14 (GeneID: 66789), with the former two corresponding to IPI00132168 and the latter one corresponding to IPI00405947. Both proteins were indicated in IPI as homologs of yeast asparagine-linked glycosylation 14 without any further information. We notice two interesting phenomena in this case. First, the two probesets correlating to a same protein do not show similar expression profiles. Instead, they behave like the probesets correlating to different variants characterized by a PCC value of 0.4080 (P = 0.0415). This issue might be due to some factors causing microarray hybridization efficiency shift. Secondly, according to our calculation, the expressions of these two variants are negatively correlated with PCC values of -0.7740 (P = 5.04e-5) for 1419116_at and 1419115_at, and -0.4402 (P = 0.0296) for 1419116_at and 1419114_at. So far there are no reports on expression regulation of these two transcript variants of gene Alg14, and no function reports of the corresponding protein isoforms. This negative correlation suggested that these two proteins might perform different roles thus should be distinctly annotated.
In recent years, considerable efforts have been devoted to identifying and characterizing protein-protein interaction (PPI). Besides investigations on the molecular events involved in PPI, functional annotation of an unclassified protein according to its interacting partners is also an important topic [28] . Since it is too bold to infer protein functions according to the "majority rule" that utilizes only the PPI network structure [29, 30] , many studies integrate other data sources into the functional characterization of PPI, among which the gene expression data is the favorite [9, 31, 32] . All these works assumed that interacting protein pairs were characterized with higher expression correlation than random ones. However, previous investigations indicated that the relationship between expression correlation and PPI was weak on a genomic scale [33] [34] [35] although a recent work strengthened the association by integrating cross-species conservation information [10] .
We noticed that in these genome-scale studies PPI information was always first converted to gene pairs, after which the Pearson correlations of the probeset pairs corresponding to the gene pairs were evaluated. That is, the analysis targets were expanded from real interacting protein pairs to all possible cross-gene protein pairs for which interaction may not always exist. As illustrated in Figure 3 , suppose we have gene a (abbreviated to Ga) and gene b (Gb), with Ga encoding protein a1 (Pa1) and protein a2 (Pa2), Gb encoding protein b1 (Pb1) and protein b2 (Pb2). Among these protein variants, only proteins Pa2 and Pb1 interact with each other, while the other three possible cross-gene interactions, including Pa1-Pb1, Pa1-Pb2 and Pa2-Pb2, do not really happen. The four probesets, Pst_a1, Pst_a2, Pst_b1, and Pst_b2, recognize transcript variants Ta1, Ta2, Tb1 and Tb2 respectively, producing proteins Pa1, Pa2, Pb1 and Pb2. In the conventional genome-scale studies mentioned above, besides the probeset pair (Pst_a2, Pst_b1) corresponding to the real interacting protein pair, the other three cross-gene pairs, (Pst_a1, Pst_b1), (Pst_a1, Pst_b2) and (Pst_a2, Pst_b2), were also included, which would blunt the expression cor-Expression profiles of synonymous probesets Figure 2 Expression profiles of synonymous probesets. A) Three probesets on the array MOE430A_2 are associated with the mouse gene Prlr. Probeset 1448556_at correlates to protein IPI00321091, or PRL-R3; probesets 1421382_at and 1451844_at both point to protein IPI00408593, or PRL-R2. GEO dataset GDS1277 was analyzed. B) Three probesets on the array MOE430A_2 are associated with the mouse gene Alg14. Probesets 149114_at and 149115_at both point to protein IPI00132168; probeset 1419116_at correlates to protein IPI00405947. GEO dataset GDS1076 was analyzed.
relation between the real interacting entities according to our preceding observations. We propose that this might partly explain the weak coherency between PPI and expression correlation.
In order to validate our proposition, we investigated the relationship between protein interactions and expression correlation at both gene-level and protein-level perspectives, using 1,037 interacting protein pairs from the HPRD [36] and 28 microarray datasets assayed with HG-U133A from the GEO. As depicted in Figure 3 , the probeset pairs corresponding to all possible cross-gene protein pairs are termed as GGI pairs, out of which only the probeset pair corresponding to the real PPI, such as Pst_a2-Pst_b1, is a PPI pair. We calculated PCCs of both PPI pairs and GGI pairs for all available 1,037 interactions, evaluated the statistical significances of these PCC values under one-tailed t-test, and estimated the corresponding false discovery rates (FDR) using the SPLOSH FDR estimation method [37] .
For all datasets, we observed strengthened expression correlations between interacting proteins under the PPI schema relative to the GGI schema. Taking the GDS987 dataset including 41 arrays as an example ( Figure 4A 153, respectively, and the former was significantly smaller than the latter (Kolmogorov-Smirnov test p value is 9.9E-12). Summarizing the comparisons at the positive side and the negative side, we conclude that the expression correlation between interacting proteins is strengthened when the non-interacting protein pairs are excluded from the interacting gene pairs, that is, with the PPI PCC calculation in place of the conventional GGI PCC calculation.
In Figure 4A , we notice that the negative correlation is also strengthened by the PPI PCC calculation as well as the positive correlation. That is to say, PPI pairs seem to be either positively correlated or negatively correlated, but not exclusively 'co-expressed' as previous publications reported [10] . This phenomenon is more evident when we examine the PCC values for each coupled PPI pair and GGI pair. Figure 4B shows a scatter plot of the PPI PCC values versus the corresponding GGI PCC values. It is evident that most points fall into the 1st and the 3rd quadrants, indicating that each pair of PPI PCC value and GGI PCC value tends to have the same signs. The scatter plot suggests a linear relationship between the PPI PCCs and GGI PCCs, and indeed we get a linear regression formula, y = 0.5612x + 0.0046, at high confidence (p < 2e-16).
Since the estimated coefficient, 0.5612, is far less than 1, we may conclude that the absolute PCC values of PPI pairs are often larger than those of the corresponding GGI pairs. So the PPI PCC calculation preserves the original positive or negative correlation tendency revealed by the conventional GGI PCC calculation, and strengthens it with larger absolute correlation values. Such correlation tendencies between interacting proteins, especially those negative ones, would very likely be submerged under the Figure 3 Illustration of Entities and terminologies involved in protein-protein interaction. A) Relationship among genes, transcripts, proteins and probesets. B) Two types of pairs mentioned in the text. PPI pair indicates the probeset pair correlating to a real interacting protein pair; while GGI pairs are those probeset pairs correlating to all possible cross-gene pairs. PPI may not exist in all cross-gene pairs.
background correlations of random pairs if the non-interacting protein pairs are included in the analysis.
Similar observations were made over all datasets (see Additional file 4 and Additional file 5). Given the results from all 28 datasets, we were also able to compare the PPI PCC calculation and GGI PCC calculation at a higher level. Under a FDR threshold of 0.1, the list of significant PPI pairs or GGI pairs from each dataset was determined. Based on these lists, we counted the significantly correlated PPIs or GGIs (termed 'significant PPIs' or 'significant GGIs') over each dataset, and the datasets on which a PPI or GGI demonstrates significant correlation (termed 'PPIsignificant datasets' or 'GGI-significant datasets'). The former is a 'twenty-eight by two' table, shown in Table 4 ; the latter is a '1,037 by two' table, shown fully in Additional file 4 and partly in Table 5 . In both summary tables, we find that the statistics of PPI PCC calculation are mostly larger than the counterparts of GGI PCC calculation, indicating that PPI PCC calculation can detect more correlated PPI pairs in a certain experiment setting ( Table  4 ), and that it can detect the correlation tendency across more experiment settings ( Table 5 ).
The same experiments were also implemented with 274 PPI pairs extracted from the IntAct database [38] , and sim- Protein pairs with the false discovery rate of PCC value less than 0.1 were deemed as significantly correlated pairs. 'Significant PPIs' are pairs detected by the PPI calculation; 'Significant GGIs' are those detected by the GGI calculation. ilar conclusions were obtained. More details can be found in Additional file 6 and Additional file 7.
In this work, we re-annotated the probesets of two widely used Affymetrix arrays, MOE430A_2 and HG-U133A, via proper association and rigorous alignment procedures in a transcript perspective, and demonstrated the necessity and advantage of exploring microarray data at the transcript or protein level, instead of the conventional gene level.
Although Affymetrix utilized the most complete information available at the time of array design, tremendous progress in genome sequencing and annotation in recent years renders existing probeset designs and target identifications suboptimal. In recent years, there have been continuous reports on systematic false expression signals of Affymetrix probesets [39] , spurious expression correlation caused by cross hybridization [18] , and expressional inconsistency among different microarray platforms or even different generations of one platform [40] [41] [42] [43] . A few research groups performed probe-against-mRNA blast similar to ours [22, 42, 44] , but mostly they centered around UniGene [45] and therefore improved the accuracy of annotation only at gene level. A major trend among these efforts was to redefine probesets so that probes matching the same molecular target were placed into custom probesets, as proposed by [19, 23, 39, 42] , but as the authors of [19] pointed out, 'these transcript-targeted probesets are not transcript-specific, as probesets targeting transcripts from the same gene may share many or even all probes'. Thus the probe re-organization strategy may be used to make distinction at the level of genes, but not at the level of transcripts or splice variants [18] . Besides, this strategy takes the probe-level intensity file (the CEL file) as a prerequisite, however only around half of the expression datasets deposited in public databases like GEO were found with CEL files.
In order to make distinction precisely at the transcript level, we preserved the classical Affymetrix probesets, but distinguished them among their alternatively spliced transcript targets according to the consistent alignments of probes against up-to-date mRNA sequences. Our annotation table clearly divides the Affymetrix probesets into three groups with increased transcript-level specificity (reliability): one-to-null probesets that do not recognize any transcript, one-to-many probesets that hybridize to multiple alternative transcript variants of the intended gene, and one-to-one probesets that hybridize to unique alternative transcript variants of the intended gene. We IPI1  IPI2  Gene1  Gene2  PPI-significant datasets  GGI-significant datasets   IPI00003326  IPI00015161  ARL2  PDE6D  15  7  IPI00003894  IPI00019930  RNF11  UBE2D1  17  1  IPI00007411  IPI00021831  AKAP11  PRKAR1A  15  2  IPI00008529  IPI00008527  RPLP2  RPLP1  25  5  IPI00011118  IPI00026689  RRM2  CDC2  17  16  IPI00013871  IPI00011118  RRM1  RRM2  14  10  IPI00015952  IPI00029012  EIF4G2  EIF3S10  14  3  IPI00016910  IPI00290460  EIF3S8  EIF3S4  16  10  IPI00018350  IPI00184330  MCM5  MCM2  16  15  IPI00021700  IPI00026689  PCNA  CDC2  14  13  IPI00022865  IPI00026689  CCNA2  CDC2  16  15  IPI00026689  IPI00015105  CDC2  CKS2  15  13  IPI00027462  IPI00007047  S100A9  S100A8  23  17  IPI00028266  IPI00026689  CCNB2  CDC2  17  15  IPI00165506  IPI00025616  POLDIP2  POLD2  14  2  IPI00246058  IPI00025277  PDCD6IP  PDCD6  16  1  IPI00290461  IPI00029012  EIF3S1  EIF3S10  15  4  IPI00291006  IPI00018206  MDH2  GOT2  16  11  IPI00294696  IPI00026689  CCNB1  CDC2  17  14  IPI00306708  IPI00026689  PBK  CDC2  16  14  IPI00328118  IPI00026689  SPAG5  CDC2  15  10  IPI00604664  IPI00291006  NDUFS1  MDH2  18  10  IPI00647217  IPI00552920  SKIV2L2  EXOSC8  18  2 A PPI-significant dataset is a dataset where the false discovery rate of the PCC value for the PPI pair is less than 0.1, while a GGI-significant dataset is a dataset where the false discovery rate of the PCC value for the GGI pair is less than 0.1. Only PPI pairs with more than 14 'PPI-significant datasets' datasets are shown. The full table is provided in Additional file 4.
discriminate the intended alternative transcript variants of Affymetrix probesets based on the NetAffx's gene-level annotation for the first time. Given the fact that existing solutions are accompanied with imperfections and no consensus has been reached on an overwhelming strategy, our alternative solution to the problematic standard annotation points out a new way to improve the interpretation and exploitation of Affymetrix microarray data.
Although the transcript collections were not identical and the reannotation strategies differed more or less, we made out some similar discoveries to previous reports. For example, Harbig et al. found that a number of probesets did not detect any transcript and attributed this phenomenon to the elimination of the target sequence in the process of sequence update [14] . In our study, altogether 16.5% and 31.2% of non-control probesets in the MOE430A_2 and HG-U133A arrays were not found with any transcript targets in the pool of GenBank, RefSeq and Ensembl. Using newer and larger collection of transcript sequences, we even obtained a quite similar statistics of the percentage of 'multiple-targeting' probesets to that estimated in a foregoing study [18] , specifically 54.6% for MOE430A_2 and 54.1% HG-U133A (see Table 2 ). The significant mutual agreement among the related researches justifies the necessity to set up an improved annotation mechanism of the Affymetrix probes in the face of the continual growth of genomic and transcriptomic knowledge, ideally at transcript or protein level.
Over the past few years, the analysis of alternative splicing has emerged as an important new field in bioinformatics, and several recent large-scale studies have shown that alternative splicing can be analyzed in a high-throughput manner using DNA-microarray methods [46, 47] . Most of these studies used arrays particularly manufactured for analyzing alternative splicing, such as genomic tiling array and exon-exon junction array. Constructed without any priori knowledge of the possible exon content of a genomic sequence, the genomic tiling array [48, 49] is in principle capable of detecting novel alternative splicing events of diverse types, but it is in doubt whether their data will be readily interpretable as successful experiences remain insufficient [46] . On the other hand, although designed particularly to address the alternative splicing issue, exon and exon-exon junction arrays [49] were reported to be plagued by problematic probe specificity and unsatisfying hybridization efficiency because of the necessity of probe coverage across the full length of the gene (including 5' end) [5] . Many questions about the reproducibility of the amplification protocol, the quantitative accuracy, and the data analysis need to be addressed as a prerequisite to reliable quantitative analysis using these splicing arrays [50] . Given the current imperfection of splicing array techniques and inconvenience in deci-phering their generated data, it is an economic way to do large-scale investigations of alternative transcribing events with standard gene expression arrays, provided that the recognizing targets of the probes can be rigorously defined at the transcript level. Hu et al. proposed a primitive analysis method to explore alternative splicing with Affymetrix 3' gene expression arrays, though they regretted that only alternative splicing biased toward the 3'end of the gene can be detected in their way [51] . In the present paper, we conducted a large-scale alignment of the probe sequences in traditional gene expression arrays against the currently most comprehensive collection of transcript sequences, highlighting the probesets mapping to unique alternative transcripts unambiguously. For each of the two Affymetrix expression arrays tested in this study, we found over 40% of all probesets could be mapped to transcripts in a oneto-one manner, so our work strongly validate that it is feasible to analyze alternative splicing using traditional gene expression arrays. While the foregoing work contributed by Hu et al. remains as a qualitative analysis method aiming at detecting novel alternative splicing events, our work gives explicitly the relationship between the probesets and the currently known alternative transcript variants, which can be immediately exploited to facilitate quantitative analysis of alternative variants. As our mapping relationships are defined for the standard probesets of the traditional gene expression arrays, they can be conveniently exploited as the standard NetAffx annotation information, without any ad hoc influence on the widely applied experiment protocols or the routine data processing algorithms. In the demonstrative implementations of the novel annotation tables, we actually observed several examples of negatively correlated alternative variants (see Figure 2B for one of them), which will shed light on further studies of expression regulation of alternative transcript variants.
To sum up, we re-annotated two popular Affymetrix gene expression arrays, MOE430_2 and HG-U133A, in a transcript-level perspective, aiming at identifying probesets' detecting targets precisely at the transcript level. Although previous works addressed similar issues [14, 15, 18, 19, 22, 23] , we are the first to rigorously link existing Affymetrix probesets to their specific transcript targets and their corresponding proteins. Armed with this new annotation, we re-examined a number of previous studies, 30 datasets for MOE430_2 and 28 datasets for HG-U133A from GEO, and revealed increased expression consistency among synonymous probesets and closer expression correlation among interacting proteins. This transcript-level annotation of Affymetrix probesets allows for a more reliable gene expression data analysis and a more accessible protein-level correlation study.
The Affymetrix 3' eukaryotic gene expression analysis arrays MOE430A_2 and HG-U133A were selected for this study. Probe sequence files and corresponding annotation files, 'Mouse430A_2_annot_csv.zip' (annotated on 2005-12-19) and 'HG-U133A_annot_csv.zip' (annotated on 2006-04-11), were downloaded from Affymetrix website [52] . Also downloaded there were the NetAffx probesetprotein mapping files for MOE430A_2 (file 'Mouse430A_2_blast_csv', updated on 2005-12-18) and HG-U133A ('HG-U133A.na21.blast.csv.zip', updated 2006-04-11), which were the blast results of the representative mRNA sequence of probes against protein sequence databases [26] . Ensembl transcripts: 37,854 mouse transcript sequences were obtained from the Ensembl database (release 38) [56] . Mapping tables between Ensembl Gene ID, Ensembl Transcript ID, and Ensembl Peptide ID were obtained from Ensembl martview [57] .
IPI entries and their mappings to external protein accession numbers were acquired from the International Protein Index (IPI) database [21] (release 3.17). Also obtained there were the mapping relations between IPI numbers and transcript IDs (GenBank, RefSeq, and Ensembl). The counterpart file for human was downloaded there too (release 3.16).
Microarray datasets were downloaded from the Gene Expression Ominibus on April 15, 2006 . Array MOE430A_2, indexed as GPL339, was associated with 2,276 samples in GEO, ranking the second among all registered Affymetrix mouse arrays. All 31 GDS datasets profiled with MOE430A_2 were used in the analyses except for GDS1057, which contains only two samples. Array HG-U133A, indexed as GPL96, was associated with 8,698 samples in GEO, ranking the first among all registered Affymetrix human arrays. For our analysis, we downloaded 31 GDS datasets with largest sample sizes, and used 28 of them in our analyses, excluding GDS534, GDS1329, and GDS1324 as they are in a data format inconsistent with the others. Details about the used datasets can be found in our Additional file 8. 
Out of the total 22,690 and 22,283 probesets in arrays MOE430A_2 and HG-U133A, respectively, 64 and 68 control probesets were firstly removed. The remaining probesets were associated with genes according to the probeset-gene mapping information provided in Affymetrix's standard annotation file. The probeset-transcript mapping relationships were obtained based on the gene-mRNA mapping tables from GeneBank, RefSeq and Ensembl. In the process, we only included probesets that were identified with one Entrez Gene ID or one Ensembl gene ID. We ignored the probesets that were associated with multiple entities or no entity in the two gene-centric databases, since their gene-level specificity appears doubtful in the standard annotation file. This filtered out 3.2% and 5.2% of non-control probesets in MOE430A_2 and HG-U133A, respectively. For the rest of the probesets, we linked the candidate transcript targets to their corresponding protein entries in IPI database. IPI is currently the least redundant yet most complete protein database for featured species, with one protein sequence matching each transcript variant. Those probesets of which transcript tar-gets do not have any protein counterparts were also excluded from the following blast validation in order to focus our attention to the transcripts with well-characterized functions at protein level. The remaining probesets, 21,097 for MOE430A_2 and 16,213 for HG-U133A, were selected for the BLAST procedure.
We then filtered the candidate probeset-mRNA mapping relationships by aligning probe sequences in these probesets against their corresponding transcripts. Probes were blasted against their candidate mRNA targets using the bl2seq program [61] , and the probe to transcript matches were accepted if no more than one mismatch was found. At the level of probesets, the probeset to transcript matches were accepted only if more than 90% of all probes within a probeset (over 10 probes for the typical 11-probeset) were mapped to the transcript in the same orientation.
The probeset-transcript-protein links related to the above probesets passing BLAST filter were retrieved. After reducing the redundancy information of multiple transcripts corresponding to the same IPI, we finally obtained rigorous probeset annotation files for Affymetrix arrays MOE430A_2 and HG-U133A. There are two types of probesets in the new annotation file: one-to-one probesets, where one probeset maps to only one IPI ID; and one-to-many probesets, where one probeset maps to two or more IPI IDs. Only the one-to-one probesets were used in the subsequent analyses.
We grouped gene-level synonymous probesets according to gene ID (gene-level), and protein-level ones according to IPI ID (protein-level). Additionally, probesets corresponding to a single protein according to NetAffx probeset-protein mapping tables (see Materials) are grouped as 'Affy-protein' level synonymous probesets. In the case of the HG-U133A array, we included a fourth level, the 'Harbig-protein level', for comparison. The Harbig-protein level reflects the probeset-protein association transformed from a recent alternative annotation of the Affymetrix U133 plus 2.0 array [14] , also proposed in a transcript-level perspective. The downloaded annotation file mapped 33,579 probesets to 287,791 GenBank mRNA accessions, among which 21,669 were found on HG-U133A array, mapped to 186,085 GenBank mRNA accessions. The probeset-mRNA associations related to HG-U133A array were further linked to IPI IDs, finally giving rise to 26,960 probeset-IPI mapping relationships among which 12,146 were one-to-one. The following treatments were the same as those implemented to the standard gene-level annotation, the NetAffx Protein annotations, and our novel annotations. 30 and 28 expression datasets were selected from GEO respectively for MOE430A_2 and HG-U133A, and the original intensity data within each GEO dataset (GDS) were transformed to log 2 base and normalized to a constant median across all samples. For a synonymous group, if the expression values of all probesets in all samples were no larger than the constant median value, the probesets in this group were regarded not moderately expressed, and their expression profiles not informative enough. Therefore, we only kept the synonymous groups with at least one expression value above the constant median value, similar to the filtering procedure used by Tian et al. [62] . For the remaining synonymous groups, Pearson correlation coefficients (PCCs) were calculated for the expression profiles of each probeset pair. The minimum value of these PCCs was taken as a measurement of the expression coherence of this group. We used the minimum aggregation because the gene level synonymous probesets gave rise to within-protein PCCs (which are theoretically higher) and across-protein PCCs (which are theoretically lower), and the former was identical to the result of the protein-level synonymous group. In such a setting, the maximum did not result in any difference, and the average aggregation was not as sensitive as the minimum in terms of differentiating the two groups, so we adopted the minimum aggregation.
Finally, the mean of the expression coherences of all synonymous groups over a dataset was calculated. In this way we obtained an evaluation of expression consistencies within synonymous probesets for a microarray dataset, and may compare the expression consistencies at the three levels over different microarray datasets.
Given protein-protein interaction data from HPRD or IntAct, we first transformed the binary relations of protein accessions to IPI-IPI pairs, and also got the corresponding Gene-Gene pairs. For each PPI, we assembled the PPI probeset pairs and the GGI probeset pairs as illustrated in Figure 3 , where PPI pairs are those associated with the interacting IPI IDs while GGI pairs are those associated with the corresponding Gene IDs. For all probeset pairs associated with the IPI-IPI pair (PPI pairs) and those associated with the corresponding gene-gene pair (GGI pairs), the PCCs were calculated and averaged into a PPI PCC and GGI PCC, respectively. These PCCs of interacting pairs were further calculated to obtain the accompanying false discovery rates using the SPLOSH FDR estimation method.
The distributions of the PPI PCCs and the GGI PCCs were plotted in a same figure to show the contrast ( Figure 4A ). In addition, a background distribution of the PCCs of ran-dom probeset pairs was overlaid on the same figure. We let the number of random pairs equal to the number of PPI or GGI pairs, but repeated the process of calculating random PCC distribution 20 times and averaged over the 20 separate random distributions in order to cut down on random fluctuation. Within each run of calculating random PCC distribution, we randomly compiled 2 × n (n = 1037 or 274, for HPRD or IntAct, respectively) pairs of probesets, where each two probeset pairs formed a group. The two PCCs of each group were firstly averaged into a group-level PCC, and the distribution was calculated over the n group-level PCCs. The group-level averaging was devised to mimic the counterpart operation in PPI PCC or GGI PCC calculation. Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1 The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor–ligand signaling interactions can be selectively regulated by an extracellular redox catalyst. Disulfide bonds have long been recognized as structural elements stabilizing proteins in harsh extracellular environments. More recently, an additional concept has emerged: some disulfide bonds operate as dynamic scaffolds capable of regulated rearrangement into a variety of functional forms (Jordan and Gibbins, 2006) . Consistent with this notion, various cell surface processes have long been known to depend on catalyzed thiol-disulfide exchange including cell adhesion (Essex, 2004) , uptake of bacterial toxins (de Paiva et al, 1993) and viral fusion with the host membrane (Sanders, 2000) . Moreover, a variety of cell surface signaling receptors appear to exist in more than one thiol-disulfide configuration, for example CD28 (Greene et al, 1996) . However, in most cases, neither the catalyst driving thioldisulfide exchange nor the functional differences between the redox forms have been elucidated.
A number of thiol-disulfide oxidoreductases are known to be secreted and to act on the cell surface. One of these redox catalysts is protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily. Cell surface-PDI has been found to act on transmembrane and surface-associated proteins, including the envelope protein of HIV-1, to cause its fusogenic conformation (Markovic et al, 2004) and integrins, to mediate platelet adhesion (Lahav et al, 2003) . Another thiol-disulfide oxidoreductase associated with extracellular functions is Trx1. Best known for its intracellular roles, Trx1 reduces transiently formed disulfide bonds of cytosolic and nuclear target proteins and thereby participates in a multitude of fundamental processes, ranging from oxidant scavenging and DNA synthesis to regulation of apoptosis and cell proliferation (Powis and Montfort, 2001) . In addition, Trx1 is released to the extracellular environment by a variety of normal and neoplastic cells (Rubartelli et al, 1992) . Human Trx1 was first purified as a cytokine-like factor from supernatants of virally transformed lymphocytes and initially named adult T-cell leukemia-derived factor (Tagaya et al, 1988) , Tac-inducing factor (Tagaya et al, 1989) , B-cell stimulatory factor or 'B cell IL-1' (Wakasugi et al, 1990) . Extracellular Trx1 is present in the circulation of healthy subjects and its levels increase under inflammatory conditions, including viral infection (Nakamura et al, 2001a) . Circulatory Trx1 acts as a chemoattractant for monocytes, neutrophils and lymphocytes (Bertini et al, 1999) , and inhibits neutrophil migration into inflammatory sites both in vitro and in vivo (Nakamura et al, 2001b) . More recently, Trx1 was found to be secreted by dendritic cells upon cognate T-cell recognition and to contribute to subsequent T-cell activation (Angelini et al, 2002) .
At present, the mechanism(s) and pathway(s) by which extracellular Trx1 influences cellular behavior remain unknown. As many of its reported extracellular activities depend on a functional active site, it appears likely that Trx1 catalyzes thiol-disulfide exchange in one or more cell surface target proteins through its enzymatic activity. However, thiol-disulfide exchange reactions, even if highly specific, are too transient to be detected by conventional techniques. To date, only a single cell surface receptor, CD4, a member of the immunoglobulin superfamily, has been shown to be susceptible to Trx1 redox activity (Matthias et al, 2002) . Other cell surface proteins targeted by the enzymatic activity of Trx1 await identification.
In this study, we address the question as to which cell surface receptors expressed on lymphocytes specifically interact with extracellular Trx1 by way of disulfide bond exchange. Using a kinetic trapping technique that enables the detection and isolation of otherwise short-lived reaction intermediates on the surface of intact cells, we identify and validate the tumor necrosis factor receptor superfamily member CD30 (TNFRSF8) as the principal target molecule for Trx1 on infected and transformed lymphocytes. The cell surface activity of Trx1 is highly selective, discriminating between different members of the TNFR superfamily. Trx1-mediated thiol-disulfide exchange leads to a structural change in the CD30 ectodomain that can be detected with conformationsensitive antibodies. We demonstrate that disulfide exchange between Trx1 and CD30 interferes with binding of the CD30 ligand (CD30L) to its cognate receptor and that Trx1 affects CD30-dependent changes in lymphocyte effector function. As CD30 is implicated in both stimulatory and apoptotic signaling, our findings suggest that Trx1 interacts with CD30 to modulate lymphocyte behavior and survival under conditions of infection and inflammation.
To identify candidate Trx1 target proteins on the cell surface of lymphoid cells, we applied a trapping technique based on the reaction mechanism. This approach makes use of the finding that mutant thiol-dependent oxidoreductases lacking the C-terminal cysteine of the CXXC active site motif form long-lived mixed disulfide intermediates with target proteins. Thus, target proteins remain covalently linked to the mutant oxidoreductase and become amenable to isolation and analysis (principle shown in Figure 1A ). Kinetic trapping has been applied previously to identify interaction partners of Trx family members in plants (Motohashi et al, 2001) and in the secretory pathway of human lymphocytes . In these studies, the CXXC-based trapping technique identified both established and novel target proteins, subsequently confirmed by independent techniques, demonstrating the competence of this technique to identify bona fide interaction partners.
To determine whether kinetic trapping can be applied to human Trx1, we created recombinant wild-type and mutant Trx proteins, each equipped with a C-terminal dual affinity tag composed of a streptavidin-binding peptide (SBP) and a hexahistidine tag. To create a trapping mutant, the second cysteine of the 32 CXXC 35 motif was replaced by serine (C35S). Trx1 harbors three additional cysteine residues distal to the active site (cysteines 62, 69 and 73). As these residues are dispensable for catalytic activity but may cause oxidative inactivation by either intra-or intermolecular disulfide bond formation (Casagrande et al, 2002; Watson et al, 2003) , we also created mutants containing amino-acid substitutions for those additional cysteines ( Figure 1B ; CCCCC, CCAAA, CSCCC, CSAAA and SSAAA annotate the identity of residues 32, 35, 62, 69 and 73).
To test whether Trx1(C35S)-based trapping is capable of identifying known Trx1 target proteins, Trx1(CSAAA) was allowed to react with cytosolic proteins released from digitoninpermeabilized cells. Incubation led to the formation of a reproducible pattern of distinct mixed disulfide conjugates as visualized by silver staining of a SDS-PAGE gel under nonreducing conditions ( Figure 1C , lane 7). In accordance with the trapping mechanism, conjugation strictly depended on the availability of the N-terminal thiol (Cys-32) and the concurrent absence of the C-terminal thiol (Cys-35), as wild-type or cysteine-free Trx1 did not capture any proteins ( Figure 1C, lanes 3, 5 and 9 ). The pattern of trapped proteins was not significantly influenced by the presence or absence of the non-catalytic cysteines (data not shown). The main cytosolic interaction partner of Trx1(CSAAA) was identified as peroxiredoxin-1 (Prx1) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The Trx1-Prx1 association was further confirmed by immunoblotting (data not shown). The Trx1-Prx1 disulfide-linked conjugate is maintained under non-reducing conditions ( Figure 1C , lane 7) and cleaved into its monomer constituents under reducing conditions ( Figure 1C , lane 8).
To test whether Trx1(CSAAA) would also undergo authentic interactions under conditions more typical of an extracellular environment, we allowed Trx1(CSAAA) to react with human plasma proteins. To avoid nonspecific absorbance to high-abundance serum proteins, we applied Trx1(CSAAA) to a o30 kDa plasma ultrafiltrate, leading to the capture of a small number of proteins, as visualized by colloidal Coomassie staining ( Figure 1D ). Using LC-MS/MS, the principal interaction partner from the plasma ultrafiltrate was identified as peroxiredoxin-2 (Prx2), a well-established target protein of Trx1, recently found to be present in human plasma (Chen et al, 2004) . These experiments provided proof-of-principle evidence that kinetic trapping is capable of capturing and identifying proven target proteins of human Trx1 from both intra-and extracellular environments.
To further investigate whether the capture of proteins by Trx1(CSAAA) is Trx-specific, we directly compared Trx1(CSAAA) with the corresponding trapping mutant of another member of the Trx superfamily, glutaredoxin-1 Grx1(CSAAA) (CSAAA annotates the identity of residues 22, 25, 7, 78 and 82). Grx1, like Trx1, uses its active site thiol to act as a disulfide reductase in the cytosolic environment. However, in contrast to Trx1, Grx1 is specialized in the recognition and reduction of protein-glutathione mixed disulfide bonds and forms mixed disulfide intermediates with glutathione rather than with proteins (Yang et al, 1998; Peltoniemi et al, 2006) . As expected, we did not detect trapping of peroxiredoxins (or other Trx1-interacting proteins) by Grx1(CSAAA), neither on silver gels ( Figure 1E , lanes 3 and 4) nor by immunoblotting (data not shown). The activity and thiol reactivity of the Grx1 trapping mutant was confirmed in independent experiments (data not shown), thus demonstrating that the mere availability of an active site thiol does not explain the profile of proteins captured by the Trx1 trapping mutant. Instead, our results support the notion that Trx-mediated reducing activity is steered toward distinct target disulfide bonds by specific protein-protein interactions.
Having established the Trx1 kinetic trapping approach for soluble target proteins, we asked whether the kinetic trapping technique can also be applied to the surface of intact cells in culture. Given previous indications of disulfide bond exchange between CD4 and wild-type Trx1 (Matthias et al, 2002) , we asked whether kinetic trapping would enable us to detect this interaction on the surface of the CD4 positive promyelocytic cell line U937. In brief, we allowed mutant Trx1 to interact with the surface of live cells, removed unreacted oxidoreductase by washing and collected disulfide-linked Trx1 complexes from cellular lysates by and non-trapping (SSAAA) mutants of Trx1 were incubated with a o30 kDa fraction prepared from fresh human serum. Disulfide-linked complexes were analyzed by colloidal Coomassie staining under non-reducing and reducing conditions. The Trx1-Prx2 conjugate and the Trx1 dimer as well as monomeric Prx2 and Trx1 are indicated. (E) Kinetic trapping is mediated by specific protein-protein interactions. Cytosolic proteins from digitonin-permeabilized Jurkat cells were incubated with SAv sepharose, Grx1(CSAAA) or Trx1(CSAAA). Disulfide-linked complexes were analyzed by silver staining under nonreducing and reducing conditions. Trx1 conjugates formed with Prx1 and Prx2 are indicated. Other bands correspond to additional cytosolic proteins interacting with Trx1. streptavidin (SAv) affinity purification. We found that cell surface CD4 forms a mixed disulfide with exogenously added Trx1(CSAAA) (Supplementary Figure S1 , left panel), which could be dissociated by DTT treatment (Supplementary Figure S1 , right panel). This result confirmed that kinetic trapping can indeed be used to identify specific Trx1-reactive cell surface proteins and should therefore allow de novo identification of previously unknown cell surface target proteins.
Human Trx1 was first identified as an autocrine factor secreted by and acting on virally transformed lymphoid cell lines. We therefore applied cell surface trapping to a human EBV-transformed lymphoblastoid B-cell line (LCL-721.220) derived from the same parental cell line (LCL-721) as the 3B6 cell line, originally used in the description of the costimulatory factor '3B6-IL1', later identified as Trx1 (Wakasugi et al, 1990) . Mixed disulfide complexes, which formed on the surface of LCL-721.220 cells were isolated and analyzed by Trx1-specific immunoblotting to visualize overall mixed disulfide conjugates. Interestingly, we found that Trx1 predominantly engages a single protein on the lymphoblastoid surface, suggesting a highly selective interaction ( Figure 2A , lane 3). As expected, the trapping product, a mixed disulfide conjugate of about 160 kDa, was susceptible to reduction ( Figure 2A , lane 4). The 160 kDa conjugate did not form on the surface of the EBV-negative Burkitts lymphoma cell line BL-41 ( Figure 2B ). In contrast, a conjugate of the same size was found to be formed on the surface of CCRF-CEM T cells ( Figure 2C ) and YT large granular lymphoma cells (data not shown). Pretreatment of the cell surface with the alkylating agent iodoacetamide (IAA) did not interfere with the formation of the 160 kDa mixed disulfide conjugate, thus confirming that the conjugate was formed by the expected disulfide exchange mechanism (rather than by de novo disulfide bond formation between two thiol groups).
To identify the unknown Trx1 target protein, we performed cell surface trapping on a larger scale (5 Â10 9 LCL-721.220 cells), purified the Trx1-interacting surface protein by SAv affinity purification and visualized the protein by colloidal Coomassie staining ( Figure 3A , left panel). The 160 kDa band was absent in the control precipitation with Trx1(CCAAA). Corresponding bands from non-reducing and reducing lanes were subjected to tryptic digestion and LC-MS/MS analysis. From both samples the unknown protein was identified as TNFRSF8 (CD30), a member of the TNFR superfamily. To validate the direct covalent interaction between Trx1(CSAAA) and CD30, an aliquot of trapped complexes from the same experiment was separated under non-reducing and reducing conditions and subjected to immunoblotting analysis with anti-Trx1 ( Figure 3A , middle panel) and anti-CD30 antibodies ( Figure 3A , right panel), respectively. The observed mobility difference between non-reducing (NR) and reducing (R) lanes demonstrated the formation of a mixed disulfide conjugate ( Figure 3A , right panel). Additional immunoblotting experiments demonstrated that low nanomolar concentrations of Trx1(CSAAA) are sufficient to detect the interaction with CD30 ( Figure 3B ) and also confirmed that trapping of CD30 depends on the N-terminal cysteine of the CXXC motif ( Figure 3C, lanes 1-8) . Application of the Grx1 trapping mutant under the same conditions did not lead to its conjugation to CD30 ( Figure 
Ectodomains of TNFR superfamily proteins typically are composed of one to four cysteine-rich domains (CRDs), each normally harboring three disulfide bonds (Bodmer et al, 2002) . To exclude the possibility that Trx1 interacts with CRDs uniformly, we tested whether Trx1 discriminates between distinct members of the TNFR superfamily. As shown in Figure 4A , CD95 (TNFRSF6) did not form a mixed disulfide conjugate with Trx1(CSAAA) on the same cells under identical conditions. The same result was obtained for the epidermal growth factor receptor (EGFR), which contains a total of 25 ectodomain disulfide bonds, and is expressed at substantial levels on A431 cells (Gill and Lazar, 1981 ) ( Figure 4B ). These findings indicate that Trx1 reactivity of cell surface receptors is selective and is not determined by the presence of CRDs or the number of ectodomain disulfide bonds. To further exclude that Trx1 reactivity of receptors is determined or limited by surface expression levels, we ectopically overexpressed CD30 or other TNFR superfamily members under control of the same promoter in HeLa cells and analyzed Trx1 cell surface trapping by indirect immunofluorescence. While mock-transfected HeLa cells did not capture Trx1(CSAAA) on their surface ( Figure 4C , lower row), expression of CD30 led to a strong Trx1 surface association and colocalization of both proteins ( Figure 4C , upper row). In contrast, expression of CD95 ( Figure 4D ), TNFR1 or NGFR (data not shown) did not promote Trx1 interactions with the cell surface, further strengthening the notion that Trx1 reactivity is a specific property of CD30. The domain structure of human CD30 differs from other members of the TNFR superfamily and from its murine homologue by the presence of two additional CRDs, arising from the internal duplication of two exons (Burgess et al, 2004) . We asked whether this unusual feature might confer Trx1 reactivity to human CD30 and tested whether the shorter murine CD30 could also interact with Trx1. As shown in Figure 4E , murine CD30 expressed on the Rauscher murine leukemia virus-induced T-cell lymphoma line RMA is efficiently targeted by Trx1(CSAAA), thus demonstrating that the additional CRDs in human CD30 are not required for Trx1 reactivity. The result also suggests that the enzymatic affinity of Trx1 for CD30 has been conserved during mammalian evolution.
To demonstrate by an independent method that Trx1 attacks and breaks a disulfide bond in CD30, we used thiol-specific cell surface biotinylation to verify that Trx1 activity generates free thiols within the CD30 ectodomain (Supplementary Figure S3 ). Subsequent analysis of CD30 cell surface expression by flow cytometry and fluorescence microscopy revealed that a brief treatment of CD30 þ cells with wild-type Trx1 led to the complete loss of CD30 recognition by the Ki-1 antibody ( Figure 5A , upper panel and Figure 5B , second column). Similar results were obtained when another monoclonal antibody against CD30, MAB229 (R&D Systems; Clone 81337), was used to examine CD30 expression ( Figure 5A , middle panel). Under the same conditions, recognition by the Ber-H2 antibody was only slightly affected ( Figure 5A , lower panel and Figure 5B , third column), indicating that Trx1-mediated disulfide bond reduction (and possibly rearrangement) induces a structural alteration in the CD30 ectodomain, which disrupts or conceals the Ki-1 epitope.
Pursuant to the observation that recognition of CD30 by antibodies Ki-1 and MAB229 is affected by CD30 redox state, we used flow cytometry to analyze the response of CD30 to with Trx1(CSAAA) and complexes were analyzed by anti-EGFR immunoblotting. Cellular lysate was included as control. (C) HeLa cells were transiently transfected with an expression construct for human CD30 or empty vector incubated with Trx1(CSAAA) and analyzed by immunofluorescence microscopy using CD30-and Trx1-specific antibodies (scale bar, 20 mm). (D) HeLa cells were transiently transfected with expression constructs for human CD30 or CD95, treated as described in (C) and analyzed by immunofluorescence microscopy using CD30-, Trx1-and CD95-specific antibodies (scale bar, 20 mm). (E) RMA mouse lymphoma cells were incubated with Trx1(CSAAA) or Trx1(CCAAA). Disulfide-linked Trx1 complexes were analyzed by anti-mouse CD30 (mCD30) immunoblotting under non-reducing and reducing conditions. The disulfide-linked Trx1-mCD30 complex and monomeric mCD30 are indicated. The additional band of higher molecular weight has not been further characterized but might represent a conjugate between Trx1 and a dimer of mCD30. reduction in greater detail. Addition of oxidized Trx1 did not influence antibody reactivity of CD30 (data not shown). When reduced Trx1 was applied in the absence of a regenerating system, a conformational change in CD30 could be observed, but remained incomplete (data not shown). Complete and sustained loss of antibody reactivity required a source of reducing equivalents for the oxidoreductase. Both DTT and Trx reductase (TrxR)/NADPH were found to be effective as regenerating systems. Importantly, neither DTT nor TrxR/NADPH had an effect when applied in the absence of Trx1 ( Figure 5A , middle panel and data not shown). Wild-type Grx1 ( Figure 5C , left panel) and the redox-inactive mutant of Trx1 ( Figure 5C , middle panel) did not alter the redox-sensitive CD30 epitopes. Other cell surface receptors, for example CD28, analyzed in parallel on the same cells were unaffected by Trx1 treatment ( Figure 5C, right panel) . Loss of CD30 antibody recognition typically occurred within minutes ( Figure 5D , upper panel). Testing the influence of Trx1 concentration under the same conditions, consistent effects on CD30 conformation became evident at concentrations around 100 nM ( Figure 5D , lower panel).
The observation that antibody binding to CD30 is influenced by Trx1 suggested a redox-dependent conformational change within the CD30 ectodomain. To test whether Trx1-mediated reduction of CD30 influences binding of CD30 to its ligand (CD30L), we analyzed the interaction between CD30 and recombinant soluble CD30L (sCD30L) on the cell surface by flow cytometry. A brief incubation of CD30 þ Hodgkin's lymphoma HDLM-2 cells with wild-type Trx1 led to a substantial loss in CD30L binding to the cell surface ( Figure 6A) . A similar result was obtained for the large granular lymphoma Figure S4) . The same effect was evident in the absence of an exogenously added reducing system (Supplementary Figure S5) , thus demonstrating that under the given conditions Trx1-substrate interactions are not limited by oxidative inactivation. As shown by fluorescence microscopy, sCD30L binds to the surface of CD30-transfected HeLa cells and colocalizes with its receptor ( Figure 6B , upper row). Treatment of HeLa cells with Trx1(CCAAA) ( Figure 6B , middle row) but not a redox-inactive mutant ( Figure 6B , lower row) strongly interferes with CD30L binding and colocalization.
Given its influence on ligand binding, we reasoned that Trx1 might interfere with CD30-mediated signaling. To test whether Trx1-mediated conformational alteration of CD30 affects CD30-dependent cellular responses, we made use of the YT large granular lymphoma line that has previously been used to study signals emanating from CD30 and to define the genes regulated by such signals (Muta et al, 2000) . Consistent with previous results (Bowen et al, 1993) , we observed that stimulation of CD30 with either agonistic antibodies or sCD30L led to upregulation of the IL-2Ra chain (CD25) within 24 h ( Figure 7A , lower panel, compare columns 3, 4 and 7). Treatment of YT cells with Trx1(CCAAA) (but not with redox-inactive Trx1) prevented CD25 upregulation ( Figure 7A , lower panel, columns 5 and 8), concomitant with the redox change in CD30 ( Figure 7A , upper panel, column 4), thus demonstrating that the redox interaction between Trx1 and CD30 has a pronounced influence on CD30-mediated gene expression.
YT cells respond to CD30 signals by downregulating the expression of cytotoxic effector molecules, including FasL, thus decreasing their cytotoxicity against Fas-expressing target cells (Bowen et al, 1993; Muta et al, 2000) . To test if Trx1 influences CD30-mediated suppression of cytotoxicity, we treated YT cells with Trx1(CCAAA) or Trx1(SSAAA) before stimulation with agonistic anti-CD30 antibody and quantified cytotoxicity against Cr-labeled Raji cells. Upon CD30 stimulation, cytotoxicity was markedly reduced ( Figure 7B , compare columns 1 and 4). The decrease in cytotoxicity was completely reversed by Trx1(CCAAA) but not the catalytically inactive mutant (SSAAA) ( Figure 7B , columns 5 and 6). As judged by RT-PCR, changes in cytotoxicity correlated with changes in FasL mRNA expression ( Figure 7B, lower panel) . These results confirm that the catalytic activity of Trx1 modulates CD30-dependent changes in cellular behavior and function.
Accumulating evidence indicates that the reduction and rearrangement of disulfide bonds constitutes a mechanism controlling protein function on the cell surface (Hogg, 2003) . The idea that disulfide bonds can act as dynamical redox switches, specifically operated by secreted redox catalysts, represents a novel concept in signal transduction (Jordan and Gibbins, 2006) . However, technical difficulties in detecting and analyzing individual disulfide rearrangements on the cell surface have made progress slow.
Trx1 is recognized as one of the most important regulators of cellular and organismal redox homeostasis (Gromer et al, 2004) . In particular, intracellular Trx1 counteracts oxidative stress, promotes cell growth and inhibits apoptosis. Under conditions of oxidative stress, Trx1 is released by cells and accumulates at sites of inflammation (Nordberg and Arner, 2001) . Numerous studies have reported that secretory Trx1 influences effector functions and proliferation of lymphocytes (Nakamura et al, 1997) . However, proteins and pathways coupling extracellular Trx1 redox activity to defined cellular responses have remained unknown. In this study, we addressed the question regarding which lymphocyte surface receptors are targeted and regulated by the redox activity of extracellular Trx1. For this purpose, we made use of a mechanism-based kinetic trapping approach to capture mixed disulfide intermediates formed between exogenous Trx1 and its target proteins on the cell surface of living cells. Activity-based techniques offer the opportunity to identify interactions too short-lived to be detectable by conventional methods. To our knowledge, this is the first reported application of kinetic trapping to identify novel target proteins of mammalian Trx1 and the first application of this technique to the surface of intact cells. We demonstrate that Trx1 interacts with intraand extracellular target proteins in a highly selective manner, guided by specific protein-protein recognition rather than random encounters with disulfide bonds.
Applying the approach to the surface of cell lines representative of the lymphoid lineage, we observed that Trx1 basically targets a single cell surface protein, subsequently identified as TNF receptor superfamily member 8, also known as CD30. The pronounced preference of Trx1 for one particular target protein might seem surprising, but could be due to the fact that we assessed Trx1 reactivity of proteins as they are embedded in their natural microenvironment, namely the intact surface of the active plasma membrane of living cells. It is conceivable that protein disulfide exchange interactions are limited and controlled by their native context and location.
To scrutinize the specificity of the observed interaction, we asked if the preference of Trx1 for CD30 might be caused by an unusual density of disulfide bonds within CD30 and/or exceptional cell surface expression levels. Although the CD30 ectodomain harbors a significant number of predicted disulfide bridges within CRDs, it does not appear to be unusual in terms of disulfide bond composition/density when compared to other members of the superfamily. When tested experimentally, Trx1 failed to interact with other CRD-containing proteins, including the EGFR featuring a total of 25 ectodomain disulfide bonds. The preference for CD30 could not be explained by exceptional surface expression levels either. While Hodgkin's disease cell lines typically express high levels of CD30, other cell lines including LCL-721.220 or CCRF-CEM show at least 20-to 100-fold lower expression as determined by flow cytometry, yet the same selective targeting was observed. Conversely, ectopic overexpression of several related TNFR superfamily members in HeLa cells did not lead to their interaction with Trx1, yet CD30 strongly interacted on the same cells under the same conditions. Consistent with these findings, recent experiments demonstrate that Trx1 targets a particular site within the CD30 ectodomain (Y Balmer and TP Dick, unpublished data) .
To facilitate identification of low-abundance cell surface proteins, in vitro trapping experiments were typically performed using Trx1 concentrations of 1-3 mM. However, when disulfide exchange was subsequently tested at lower concentrations, Trx1(CSAAA) concentrations in the low nanomolar range (4-40 nM) were found to give rise to the formation of proportional amounts of Trx1-CD30 mixed disulfide intermediates ( Figure 3B ), thus demonstrating that the observed interaction is compatible with the expected physiological concentration range of secretory Trx1 (see below).
Wild-type Trx1 is known to act as a multiple-turnover catalyst if a suitable reducing system and electron source is provided for its regeneration. In agreement with these considerations, we observed that sustained reduction of CD30 in cell culture requires a Trx1 regenerating system. Using flow cytometry to monitor conformational changes in the CD30 ectodomain, CD30 was found to respond to Trx1 concentrations in the nanomolar range, starting at around 100 nM ( Figure 5E , lower panel). However, the minimal Trx1 concentration required for sustained CD30 reduction might be substantially lower in specific environments, which are efficient in delivering reducing equivalents and preventing oxidative inactivation of Trx1.
At present, it is not clear how extracellular Trx1 is regenerated in vivo. Despite the overall oxidizing character of the extracellular compartment, reductive processes are known to take place on the cell surface. On the one hand, there is longstanding evidence for the existence of transplasma membrane redox systems delivering electrons to the cell surface (Crane et al, 1985) . On the other hand, Trx1 may be regenerated by co-secreted reductants, as Trx1 secretion in DC-T co-culture is accompanied by the release of reduced cysteine and the creation of a reducing microenvironment between interacting cells (Angelini et al, 2002) . In addition, TrxR was found to be secreted by activated monocytes and might be part of an extracellular Trx1 reducing system (Soderberg et al, 2000) .
The concentration of Trx1 in human plasma is in the low nanomolar range (1-5 nM), and is found to be elevated several-fold under inflammatory conditions (Yoshida et al, 1999) . However, plasma Trx1 is oxidized and appears to represent systemic dilution of Trx1 previously released within tissues. Accordingly, local tissue concentrations of secretory Trx1, for example, within activated lymph nodes, are expected to be markedly higher than in plasma. Overall Trx1 concentrations in mammalian tissues can be as high as 20 mM (Gromer et al, 2004) . Certain Trx1-secreting cell types, including macrophages and dendritic cells, distinctly upregulate expression of Trx1 upon activation (Angelini et al, 2002) . In vitro studies of Trx1 secretion suggest that a substantial fraction of intracellular Trx1 can be released within a few hours (Rubartelli et al, 1992) . Although direct measurements of extracellular Trx1 within tissues are not available, physiologically relevant extracellular Trx1 concentrations may well reach into the upper nanomolar, if not lower micromolar range.
We found that Trx1-mediated disulfide reduction changes the conformation and functional properties of the CD30 ectodomain. In the reduced state, CD30 lost its ability to interact with its cognate ligand CD30L or agonistic antibodies. The presence of catalytically active Trx1 impeded CD30-dependent signaling in the YT lymphoma cell line, as demonstrated by its effect on CD25 and FasL expression, as well as its influence on cytotoxicity against Fas-expressing target cells.
The physiological role of the CD30-CD30L system has remained unclear. In vitro studies focusing on CD30 þ lymphoid malignancies showed that triggering of CD30 signaling can induce either proliferation, activation, growth arrest or apoptosis, depending on cell type and stimulatory conditions (Schneider and Hubinger, 2002) . In vivo, cell surface expression of CD30 appears to be tightly regulated and restricted to B and T lymphocytes undergoing activation in lymphoid tissues. It has been proposed that CD30 provides proliferation and/or survival signals during lymphocyte responses (Croft, 2003) .
Under inflammatory conditions, CD30 expression is markedly induced. In vivo activation of CD30 can be monitored by the release of sCD30, shed from the plasma membrane upon CD30L binding (Hansen et al, 2000) . Similar to serum Trx1, serum sCD30 is increased in infection, autoimmunity and allergy, for example systemic lupus erythematosus, rheumatoid arthritis and atopic dermatitis (Horie and Watanabe, 1998) . Both Trx1 and CD30 appear to play a role in the regulation of the antiviral inflammatory response. Both Trx1 secretion and CD30 expression have been associated with virally transformed lymphocytes. Elevated levels of sCD30 occur during viral infection. Likewise, viral infection leads to elevated Trx1 plasma levels and several studies indicate that secreted Trx1 modulates the antiviral inflammatory process (Nakamura et al, 2001b (Nakamura et al, , 2002 .
In this study, we have identified an enzyme-substrate relationship between Trx1 and CD30, a receptor of activated lymphocytes involved in the regulation of inflammation. As lymphocytes migrate between different microenvironments, it is conceivable that Trx1 catalyzes disulfide exchange dynamically, activating or inactivating the CD30 pathway in response to the redox environment. The interaction between Trx1 and CD30 might represent a regulatory link between oxidative stress and lymphocyte function. Understanding of this relationship in vivo awaits the generation of suitable experimental tools.
Cell culture BL-41, CCRF-CEM, HDLM-2, Jurkat, RMA and U937 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heatinactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco). LCL-721.220 and YT cells were cultured in IMDM (Gibco), HeLa and A431 cells in DMEM (Gibco) with the same supplements.
Depending on the type of experiment, recombinant trapping mutant was applied to cytosolic preparations, human serum ultrafiltrate or intact cells. A detailed description of the different substrate trapping protocols is provided as Supplementary information.
For reduction of cell surface CD30, 2.5 Â10 5 cells were incubated with 5 mM Trx1 together with 200 mM DTT or 100 nM human Trx reductase (TrxR)/500 mM NADPH for 30 min at 41C. To monitor reduction of cell surface CD30, cells were stained with anti-human CD30 monoclonal antibody MAB229 (R&D Systems), anti-human CD30 monoclonal antibody Ki-1 (Santa Cruz) or anti-human CD30 monoclonal antibody Ber-H2 (DakoCytomation) followed by incubation with R-PE-conjugated goat F(ab') 2 anti-mouse Ig's (Biosource). For control, cells were stained with R-PE-conjugated anti-human CD28 monoclonal antibody (BD Pharmingen). Cells were analyzed using a FACSCalibur (Becton Dickinson) and CellQuest software.
A total of 2.5 Â10 5 cells were incubated with 5 mM Trx1 (SBP-CCCCC) and 200 mM DTT for 30 min at 371C, washed three times and incubated with 250 ng/ml recombinant CD30L-His 10 (R&D Systems) for 10 min at RT. After washing, cells were stained with anti-polyHis monoclonal antibody (Sigma) followed by incubation with R-PE-conjugated goat F(ab') 2 anti-mouse Ig's (Biosource).
HeLa cells were seeded on coverslips and transfected with expression constructs using CaCl 2 precipitation. After 2 days, transfected cells were fixed with 3% formaldehyde and 2% sucrose in PBS for 7 min at RT. Fixed cells were washed three times with PBS and incubated with different Trx1 constructs or recombinant CD30L (R&D Systems). Proteins were visualized using appropriate primary antibodies (Anti-CD30L polyclonal antibody (R&D Systems), anti-CD30 monoclonal antibodies Ki-1 (Santa Cruz) or Ber-H2 (DakoCytomation), anti-CD95 monoclonal antibody (a kind gift from Dr P Krammer), anti-Trx1 polyclonal antibody (M Preuss and TP Dick, unpublished) followed by FITC-conjugated anti-goat IgG, FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG and analyzed with a Nikon C1Si confocal microscope.
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Screen for ISG15-crossreactive Deubiquitinases BACKGROUND: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. RESULTS: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. CONCLUSIONS: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. Posttranslational modification by ubiquitin regulates processes such as proteasomal degradation, intracellular trafficking, and transcription. Ubiquitin is attached to substrates in covalent isopeptide linkage or as an N-terminal fusion [1] [2] [3] . Ubiquitination, however, is reversible: the ubiquitin moiety can be released from substrates through the action of deubiquitinating proteases, which may rescue ubiquitinated substrates from their degradative fate [4] . In contrast, proteasome-associated DUBs enhance the rate of proteasomal degradation by removing bulky poly-ubiquitin chains from substrate proteins prior to proteolysis. Such DUBs enhance the processivity of the proteasome toward target proteins, and also recycle ubiquitin, a modifier that itself turns over slowly [5, 6] . DUBs are furthermore required to hydrolyze the ubiquitin precursor and generate the active ubiquitin monomer. Inspection of mammalian genomes shows the presence of more than 100 genes that encode putative DUBs, consistent with their specific and diverse regulatory functions. Ubiquitin-specific proteases (USPs) are the dominant family among DUBs [7] .
Ubiquitin-like molecules show sequence and structural similarity to ubiquitin. Unlike ubiquitination, modification by UbLs usually does not target proteins for destruction by the proteasome. A notable exception may be FAT10, a modifier that serves as a ubiquitin-independent signal for proteasomal degradation [8] . The conjugation of UbLs to target proteins follows reaction pathways similar to those involved in ubiquitination [9] . The enzymes that attach or cleave UbLs are generally distinct from the ligases or proteases of the ubiquitin pathway.
A closely related homolog of ubiquitin in vertebrates is the UbL polypeptide ISG15, an interferon-inducible gene product that is strongly upregulated following viral or bacterial infection [10] . However, the molecular and regulatory consequences of ISGylation remain unknown [11] . ISG15 consists of two ubiquitin domains in a tandem arrangement, similar to FAT10. Unlike other members of the UbL family, ISG15 co-opts at least one of ubiquitin's conjugating enzymes, Ubc8 [12, 13] and the ubiquitin ligase Herc5 [14] [15] [16] [17] . USP18 constitutes the only presently appreciated isopeptidase specific for ISG15, and its absence has profound effects on innate immunity, leading to increased resistance to certain viral infections [18, 19] . Notably, these effects appear not to be contingent upon proteolytic activity of USP18 [20, 21] . Apart from USP18, additional proteases for ISG15 must exist, since the ISG15 precursor protein is cleaved properly in USP18 knockout mice [19] .
The C-terminal six amino acids of ubiquitin and ISG15 are identical. This tail region is required for specific recognition of ubiquitin by conjugating enzymes, and also for recognition of ubiquitin adducts by isopeptidases [22, 23] . The overlap in conjugation between ubiquitin and ISG15, as well as their Cterminal similarity, imply the existence of promiscuous DUBs, capable of removing both ubiquitin and ISG15 from substrate proteins. Here, we report on the identification of new ISG15specific proteases measured by reactivity toward active-site directed probes and isopeptide-linked substrates [24] [25] [26] .
Activity-based profiling of DUBs Figure 1 shows a consensus phylogram based on the alignment of catalytic core sequences of DUBs, including the majority of known human USP homologs. In this tree, the ISG15-protease USP18 clusters close to USP5 (IsoT1) and its isoform USP13 (IsoT3). Previous work had identified USP5 as a protease with affinity for both ubiquitin [27] and ISG15, as shown by its reaction with an electrophilic ISG15 derivative, ISG15-vinyl sulfone (ISG15VS) [28] . To probe for additional ISG15-reactive proteases, we have cloned and expressed a total of 22 human DUB homologs from different clades of this phylogram (indicated with arrows), 17 of which reacted with a ubiquitin-based probe and/or an ISG15based probe (see below). The screen was based on in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SU-MO1VME and ISG15VS, respectively [28] . DUBs generated by IVT were incubated with each of these three probes (Figure 2 ), followed by direct analysis of the reaction mixture by SDS-PAGE. We have determined by X-ray crystallography that probes of this type form a covalent adduct with the catalytic cysteine residue of active DUBs to yield a thioether-linked adduct between enzyme and probe [26] . When unmodified IVT products are run adjacent to samples incubated with these activity-based probes, the adduct is readily detected through a shift in apparent molecular mass. USP2, USP5, USP13 and USP14 reacted with ISG15VS ( Figure 2B , C, D, E).
The following observations confirm the validity of our assay. First, the bona fide ISG15-isopeptidase USP18 displayed reactivity only towards ISG15VS (Figure 2A ), whereas most of the DUBs reacted only with UbVME. An example is shown with CGI-77 ( Figure 1 , 2F), a previously uncharacterized Otubain-homolog. Second, as a negative control, the SUMO protease SENP2 formed an adduct exclusively with SUMO1VME ( Figure 2G ). We found no evidence for any of the DUBs evaluated here to display reactivity toward the SUMO1 probe (not shown). The presence of the reactive group alone is clearly not sufficient for binding to the active-site cysteine of a protease and specificity of a DUB thus depends on the peptide moiety of the probe, containing either ubiquitin, SUMO1 or ISG15. Lastly, all covalent modifications of DUBs by active-site directed probes were blocked by pretreatment of the translated polypeptides with the sulfhydryl alkylating agent N-ethylmaleimide (NEM) (Figure 2 ), confirming the cysteinedependency of adduct formation.
In accordance with previous results [28] , we observed a non-linear decrease in electrophoretic mobility of the ISG15VS adducts. The ISG15 probe has a mass of 17.4 kDa, whereas the size increase observed for each of the DUBs investigated here is in the order of 25-110 kDa, when bound to ISG15VS. The correlation between the abnormal shift and the initial mass of the unmodified DUB suggests that the decrease in gel mobility is based on steric properties of the branched adduct, and is not caused by covalent modification of a single DUB by multiple ISG15VS molecules. In fact, in our sample set, the observed size increase of the ISG15VS-DUB adducts based on SDS-PAGE very closely matched a logarithmic equation ( Figure 3A and see Methods). To further verify that DUBs are modified by only a single ISG15VS probe per molecule, we replaced the catalytic cysteine at position 114 in USP14 with a serine residue. As expected, this mutation abolished all labeling ( Figure 3B ). Our assay was conducted in IVT lysate and the size increase of the ISG15VS adduct could potentially reflect modification of USP14 by additional factors. However, even USP14 that was recombinantly expressed in bacteria and .95% pure showed the same abnormal electrophoretic mobility for its ISG15VS adduct ( Figure 3C ). Mass spectrometry confirmed the monovalent modification of purified USP14 by ISG15VS and excluded covalent binding of additional factors to the complex ( Figure 3D ). Collectively, these experiments establish that the observed shift in apparent molecular mass of the ISG15VS-DUB adducts is solely a consequence of its unusual electrophoretic behavior. It also underscores the uncertainties in estimating the degree of modification of a target protein with UbLs by SDS-PAGE alone.
USP14 reacts more efficiently with ISG15VS in its proteasome-associated form USP14 and its yeast counterpart Ubp6 show significantly higher activity when bound to the 26S proteasome [29] . This activity may in fact be strictly dependent on association with the proteasome, as shown for Ubp6 [30] . As further evidence for a physiological role of the interaction of USP14 with ISG15, we investigated whether the allosteric activation of USP14 also influences its reactivity toward ISG15VS. We examined labeling of USP14 with the ubiquitin-and the ISG15-based probes as a function of the concentration of added purified proteasomes. As a negative control, we evaluated USP5, a DUB that is not a known interaction partner of the 26S complex in vivo. As anticipated, the inclusion of purified proteasomes had no effect on the ISG15VS-or UbVME-reactivity of USP5 ( Figure 4B ). In contrast, we observed a dose-dependent increase in ISG15VS adduct formation of USP14 with increasing proteasome concentration, indicating enhanced activity of this DUB. The effect was similar in magnitude to that seen for the ubiquitin probe ( Figure 4A , C).
While recombinant USP14 in its purified form bound to electrophilic probes ( Figure 3C ), we did not detect robust hydrolytic activity against ubiquitin-AMC or against ubiquitinor ISG15-linked isopeptide fusion proteins (data not shown). However, using sequential ultracentrifugation to obtain a cytosolic fraction that is enriched in 26S proteasomes [29] , we could show that proteases in this fraction efficiently and specifically cleave an ISG15-isopeptide linked substrate ( Figure 5A , B). The absence of proteolytic intermediates suggests specific cleavage of the isopeptide bond. In addition, the same bait peptide linked to SUMO1 was stable and not hydrolyzed, even upon prolonged incubation for over 24 hours with the proteasome fraction. Proteolysis of the ISG15-linked peptide substrate was inhibited by inclusion of NEM, indicating cleavage by cysteine proteases. Analysis by reaction with ISG15VS supports that USP14 is the only active Red arrows depict DUBs that bound to neither probe (UbVME, ISG15VS, or SUMO1VS), whereas black arrows indicate DUBs that formed covalent adducts with the indicated probes. Our screen represents the first biochemical proof for protease activity of USP13 and the Otubainhomolog CGI-77 (DUB homologs without publication record regarding biochemical function are marked with an asterisk). Otubain1 (OTU1) is an exception in that it binds to alkylhalide-or aldehyde-based probes, but not to the Michael acceptors employed in this study (data not shown). doi:10.1371/journal.pone.0000679.g001 ISG15-specific protease in the proteasome-enriched fraction ( Figure 5C ). While we cannot formally exclude the possibility of a yet undefined enzyme binding to ISG15VS, we consider this unlikely: such a protease would have to display a mass highly similar to that of USP14 and, furthermore, it would have to sediment after centrifugation for 5 hours at 100,000 g. However, only few deubiquitinating enzymes are sedimentable, none at a level comparable to USP14 [29, 31] .
The wealth of USPs found in the human proteome likely reflects substrate specificity, but potentially also complementation in terms of expression profiles and subcellular distribution. We therefore sought to analyze the intracellular distribution pattern of a subset of our crossreactive DUBs, using confocal microscopy. The analysis of a genome-wide set of C-terminal GFP fusion proteins for yeast had shown remarkably few with altered function or subcellular distribution (,5%), validating the choice of such Cterminal modifications [32] . Using anti-G/YFP antibodies, we also utilized this tag to assay for activity of DUBs in cell lysate. We cloned and transiently expressed five USP-EYFP constructs in 293T cells: USP5, USP13, USP14, USP3, and USP36 ( Figure 6A ). Lysate of USP14 EYFP transfected cells was incubated with the ubiquitin and the ISG15 probe, and assayed by anti-YFP immunoblot analysis ( Figure 6B ). Whereas the USP14 EYFP construct reacted with both probes, the respective C114S mutant Mutation of the catalytic cysteine residue to serine (C114S) in USP14 abolishes its reactivity toward UbVME and ISG15VS. When stored for longer periods at room temperature, the probes polymerize covalently, presumably by formation of secondary amine bonds between internal lysine residues and the reactive Michael acceptor at the C-terminus, thus resembling isopeptide-linked polyubiquitin. Such polymeric probes of UbVME likely caused the additional high-molecular mass adducts observed for USP14. Note that the smallest version of these adducts (a UbVME dimer) has a maximum electrophoretic mobility similar to that of the diubiquitin-like ISG15VS when complexed to USP14. The absence of any adducts in the C114S mutant of USP14 excludes the possibility of multiple binding sites for the probes. (C) Purified recombinant USP14 labels with UbVME and ISG15VS and results in the same abnormal mobility shift for ISG15VS-USP14 as seen in the IVT labeling experiments. (D) MALDI-TOF mass spectroscopic analysis of USP14 after incubation with ISG15VS. As described above for UbVME, ISG15VS also engages in internal polymerization. Molecular masses consistent with tri-and tetrameric ISG15VS are marked in this spectrogram by the numbers in superscript. Monovalently modified USP14 results in an adduct of predicted size, indicated with a red arrow. This complex is unique to the mixture containing both USP14 and ISG15VS, and is absent in the mass spectra of either component alone (data not shown). doi:10.1371/journal.pone.0000679.g003 did not, in agreement with the results of our IVT screen. With respect to subcellular distribution, we were particularly interested in the expression pattern of USP5 and USP13, given that these two isoforms displayed different specificity in UbVME and ISG15VS labeling experiments. USP5 EYFP was found throughout the cell ( Figure 6C, upper left panel) , similar to USP18 [33] . In contrast, its close relative USP13 EYFP was expressed mainly in the nucleus in a speckled pattern ( Figure 6C, upper right panel) . Consistent with the in vivo interaction between USP14 and the proteasome [5] , we observed USP14 EYFP predominantly in the cytoplasm, though we also noticed fluorescence in the nucleus ( Figure 6C, lower left panel) . To demonstrate that the presence of the C-terminal YFP fusion does not interfere with the endogenous distribution of these enzymes, we analyzed USP3 EYFP ( Figure 6C , lower right panel) and USP36 EYFP ( Figure 6C , lower right panel). USP3 is predicted to be a nuclear protein [34] and USP36 was . Reactivity of USP14 toward ISG15VS is augmented by proteasomal association. USP14 and USP5 were generated by IVT. Their activity toward ISG15VS and UbVME was analyzed in the presence of increasing concentrations of purified human 26S proteasomes. (A) Activity of USP14 toward UbVME and ISG15VS increases as a function of the concentration of added purified 26S proteasomes (in mg/ml). (B) The activity of USP5 remains unaffected. (C) Quantification of the radioactive signal of covalently modified USP5 and USP14. Binding affinity is depicted on the y-axis as percent in labeling intensity, determined by the ratio of labeled versus unlabeled USP5 or USP14. The ratio in the absence of exogenous proteasomes is defined as 100%. doi:10.1371/journal.pone.0000679.g004 Figure 5 . Proteasome-associated USP14 has ISG15-specific isopeptidase activity. (A) Scheme depicting the UbL-peptide conjugate used to assay isopeptidase activity. The biotinylated peptide heptamer is attached to either ISG15 or SUMO1 in isopeptide-linkage. Upon hydrolysis of the isopeptide bond by a specific DUB, the heptamer is released and the biotin signal lost. (B) Incubation of proteasomeenriched fraction (''5 hr pellet'') with UbL-peptide conjugate. After overnight incubation, the ISG15-peptide conjugate is completely cleaved, resulting in loss of the biotin-signal (significant proteolysis occurs already after one hour, data not shown). This activity is sensitive to NEM. Hydrolysis is not observed for the SUMO1-peptide conjugate. (C) Anti-HA immunoblot of HA-ISG15VS treated subcellular fractions. Based on previous identification [28] and on electrophoretic mobility, USP5 is the dominant ISG15-reactive DUB in the five-hour supernatant, which is enriched for uncomplexed proteins of light and moderate size (red asterisk). The five-hour pellet represents heavy cytosolic complexes, in particular the 26S proteasome, and contains USP14 as the only ISG15reactive DUB (blue asterisk). doi:10.1371/journal.pone.0000679.g005
identified as a nucleolar protein [35] . Both proteins were detected in the expected subcellular compartment. Combined, these results indicate that ISG15-specific proteases are expressed throughout the cell -a feature also proposed for DUBs. This observation supports the notion that unlike SUMOylation, which is believed to mostly occur in the nucleus [36] , ISG15-modification affects many cellular compartments.
Our data show the existence of multiple ISG15-reactive DUBs, a finding that further strengthens the similarities between ubiquitin and ISG15. USP2 is a highly active protease [37] and it represents one of only few mammalian DUBs with a known target. USP2 exhibits oncogenic potential in prostate cancer by stabilizing its substrate Fatty Acid Synthase (FAS) [38] , and FAS has indeed been identified as a target of ISG15 modification [39] . Furthermore, USP2 has been implicated in the regulation of the p53 pathway [40] . The recently solved structures of USP2 and USP14 [41, 42] show that both proteases accommodate the ubiquitin molecule in a shallow pan-like protrusion. Based on the orientation of the ubiquitin protein in both structures, ISG15 easily fits into the catalytic domain of USP2, USP14, and USP5 (data not shown) without apparent steric clashes (Figure 7 ) [23, 43] .
Stimulation by interferons alters the composition of the proteolytic proteasome core [44] , tailoring its activity toward generation of peptide-MHC complexes for inspection by the immune system. Interferon treatment also results in enhanced modification of proteins by ISG15 -a factor that evolved in the vertebrate lineage, and whose origin thus coincides with that of the adaptive immune system. Interestingly, inhibition of the proteasome leads to rampant accumulation of ISG15-modified substrates [45] . While nothing is known about the molecular functions of ISG15, its structural relative FAT10 is a modifier that destines proteins for degradation by the proteasome [8] . We now have demonstrated that USP14 exhibits proteasome-associated isopeptidase activity toward ISG15. Could therefore ISG15 be a (co-)modifier of proteins destined for proteasomal degradation? We have found no evidence that USP14 markedly changes the amount of ISG15-modified substrates in cells (data not shown). However, USP14 is not a vital protease [30, 46] and its low catalytic turnover does not affect overall ubiquitin conjugation either [47] . A recent study suggests that USP14 might have a more complex role, by inhibiting the proteasome in addition to acting as a deubiquitinase [48] .
The close sequence relationship between USP5 and USP13 (61.4% identity, 26.9% similarity) is contrasted by the functional differences and localization of these two proteases. These enzymes provide a unique opportunity to investigate the structural features that may contribute to ubiquitin versus ubiquitin-like specificity. A characteristic of USP5 and USP13 is the tandem occurrence of a UBA domain, which has been implicated in the binding of ubiquitin [49] . Our results raise the possibility that UBA domains in general interact not only with ubiquitin, but also with ISG15. Alternatively, ISG15 with its multiple lysines could act as a ubiquitination anchor, and USP5 may be a protease responsible for depolymerization of such chains [50, 51] . Moreover, the Cterminal hydrolase that processes the ISG15 precursor has not been identified yet, and any of the novel ISG15-specific proteases described here are potential candidates.
The ubiquitin gene is prone to duplications and insertions, leading to the formation of new fusion proteins [52, 53] . ISG15 likely emerged approximately 400-600 million years ago, when a nucleotide stretch from the polyubiquitin precursor gene, encoding a ubiquitin-dimer, was accidentally inserted in an area of the genome that was or that came under control of an interferon promoter. From an agnostic point of view, one could argue that ISG15 simply has no relevant function. The moderate or absent phenotype of the ISG15 knockout in mice [11] , the fact that ISG15 has not (yet) established its very own family of conjugating and deconjugating enzymes, and ISG15's relatively low degree of conservation between species would all support this view. Yet, ISG15's massive expression upon interferon challenge [54] likely reflects a role in anti-microbial or anti-viral defense [55] [56] [57] [58] [59] . And adaptation to the specific needs of host immunity often demands polymorphism. As a result, some genes most critical to the immune response are paradoxically least conserved. For example, cytokines and cytokine receptors substantially differ between species [60] and it is interesting to note that ISG15 and ubiquitin were initially reported to be cytokines [61, 62] . Similar observations were made for the ubiquitin-like modifier FUBI (also known as Fau or MNSFb) [63, 64] . If true, how do these factors gain access to the secretory pathway? It may pay to approach ISG15 from a less conventional perspective and from this vantage point, we might uncover new functions of ubiquitin as well.
USP2 and USP18 cDNAs were cloned from a human kidney cDNA library (BioChain Institute, Inc.). The cDNAs encoding the other human DUBs were obtained from ATCC. All cDNAs encoding fulllength DUBs were subcloned into pcDNA3. 
The protein sequences of human DUBs were obtained from the National Library of Medicine and the core domains were aligned with the CLUSTALW algorithm (EBI server) [65] and manually edited with Genedoc (http://www.psc.edu/biomed/genedoc/) by K.B. Nicholas & HB Nicholas Jr., using the putative active-site cysteine as an alignment anchor. The phylogram represents a consensus tree based on 100 bootstrap iterations, calculated by the Minimum Evolution method under default parameters [66] .
IVT was performed using the ''TNT-T7 Quick Reticulocyte Lysate System'' kit (Promega) for 30 to 45 min (0.25-1 mg DNA per reaction). Then, aliquots of the reaction mix were treated with RNase B (1 mg/ml, Sigma) for 10 min and incubated with the probes as described below. SDS-PAGE followed by fluorography was performed as described [67] .
The synthesis of human ubiquitin and UbL probes has been described [24, 28] . IVT products were incubated with saturating amounts of the individual probes (0.2-0.4 mg/10 ml IVT lysate). Preincubation with NEM was performed for 10 min at room temperature at a final concentration of 10 mM, after RNase B treatment. Autoradiograms were subjected to quantification of the optical density with NIH Image software (version 1.32j) as ratio of labeled versus unlabeled IVT products. Purified human 26S proteasomes for the experiments in Figure 4 were purchased from Biomol International and inhibited with MG132 (50 mM). E. coliexpressed human recombinant USP14 for the experiments shown in Figure 3C 
To 50 mL of conjugation buffer (100 mM Tris, pH 7.4, 5 mM MgCl 2 , 20 mM DTT, 40 mM ATP) was added ISG15 (Boston Biochem, 9.4 mM final) or GST-SUMO1 (Boston Biochem, 10.4 mM final) and biotinylated peptide 7-mer (biotin-VKAKIQD-OH, 250 mM final). The solution was mixed thoroughly and to this was added ISG15 activating enzyme (Boston Biochem, ISG15 E1, 50 nM final) and UbcH8 (Boston Biochem, 250 nM final), or SUMO activating enzyme (SAE1/SAE2 heterodimer, 50 nM final) and UbcH9 (250 nM final). The solution was mixed and incubated for 15 hours at 37uC. The reaction mixture was transferred to a microcentrifuge membrane filter (Vivascience, 5000 Da MWCO), diluted to 600 mL total volume with 50 mM Tris, pH 7.4 and concentrated at 4uC to 50 mL. This dilution/concentration procedure was repeated six times. The products were transferred to a clean tube and diluted with 50 mM Tris, pH 7.4 to a final volume of 100 mL. The SUMO1-and ISG15-linked biotinylated isopeptide was detected after transfer to a PVDF membrane (Perkin Elmer) with Streptavidin-HRP (Amersham).
EL4 cells were lysed with glassbeads and the proteasome-enriched fraction was retrieved by consecutive ultracentrifugation steps as previously described [29] . Proteasome activity was inhibited with MG132 (50 mM). 10 mg of fraction protein were incubated with 0.2 mg of N-terminally HA-tagged ISG15VS in a total volume of 10 ml for two hours at room temperature to detect ISG15VSreactive proteases by anti-HA immunoblotting. The cleavage assay for ISG15-or SUMO1-branched peptides was performed at 37uC using 20 mg of total protein from the proteasome-enriched fraction and 5 ml of branched peptides in a total volume of 10 ml. HA-ISG15VS treated subcellular fractions were analyzed with a monoclonal anti-HA antibody (12CA5). Immunoblotting was performed as published [67] .
293T cells were maintained in DME medium as described [67] . Various constructs were expressed by transient transfection, using a liposome-mediated transfection protocol (5-10 mg of DNA/ 20 ml of Lipofectamine-2000 per 10 cm dish; Invitrogen) as described [67] . Cells were analyzed between 24 and 48 h after transfection. C-terminal EYFP fusion proteins of DUBs were generated by subcloning from pcDNA3.1 into pEGFP-N1 (Clontech). NP40 lysates of USP14 EYFP and USP14 C114S-EYFP transfected 293T cells were prepared and incubated with activesite probes as described [24] . Due to the high similarity with GFP, EYFP fusion proteins can be detected with a polyclonal anti-GFP rabbit serum [68] . Immunoblotting was performed as published [67] .
Immunofluorescence experiments were performed as described [67] with minor modifications. Cells were allowed to attach to slides overnight. After fixation with 3.7% paraformaldehyde for 20 min at room temperature, subcellular localization of EYFP fusion proteins was analyzed with a Perkin Elmer spinning disk confocal microscope Ultraview RS system. The microscope used was a Nikon TE2000-U inverted unit with a Nikon 1006 1.4NA DIC lens. The imaging medium was Nikon type A immersion oil. s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine. Screening for genetic changes to unveil molecular attributes of human specimens is important for a variety of medical applications, including genotyping for inherited disorders, prediction of the pathologic behavior of malignancies, identification of cancer biomarkers and can affect treatment decisions for individual patients (1) (2) (3) . For example, mutations in genes like EGFR can profoundly influence chemotherapeutic response in lung cancer (2) (3) (4) (5) and the response is modulated by mutations in other genes of the same signaling pathway [e.g. K-ras, HER2, ErbB-3 (1, 6) ]. Therefore there is a need for efficient and high-throughput mutation screening of multiple genes along identified signal transduction pathways in tumor samples. Because a large portion of cancer-causing genetic changes remains unknown and can occur in numerous positions along tumor suppressor genes (e.g. p53, ATM, PTEN) mutation scanning rather than detection of specific mutations is frequently required for molecular cancer profiling.
Sequencing is often considered the gold standard for comprehensive mutation analysis. Multi-capillary electrophoresis, re-sequencing arrays or pyrosequencing provide platforms for highly parallel genetic analysis (7) (8) (9) (10) (11) (12) (13) . However, the expense associated with these techniques is currently high both for instrumentation and for runningcosts. Since somatic mutations for most genes are relatively rare events it can be inefficient to scan for mutations using expensive approaches that in several cases provide unnecessary data (14, 15) . Another issue with direct sequencing or re-sequencing arrays is the difficulty *To whom correspondence should be addressed. Tel: +1-617-525-7122; Fax: +1-617-587-6037; Email: mmakrigiorgos@partners.org ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
in detecting a small fraction of mutated alleles in the presence of a high excess normal alleles, which is frequently the case with clinical cancer samples (16) . As a less expensive alternative, rapid pre-screening methods such as SSCP, DGGE, dHPLC, CCM, CDCE or HR-melting are widely utilized to identify DNA fragments that contain mutations prior to performing full sequencing (14, (16) (17) (18) (19) (20) . Enzymatic mutation detection based on mismatch scanning enzymes like MutY, TDG or T4 endonuclease VII for mutation pre-screening has also been employed (21) (22) (23) (24) (25) , albeit with modest success since these enzymes cannot detect all possible mutations and deletions (22) and some of them have substantial activity on homoduplex DNA (16) . Recently an enzymatic mutation scanning method based on the Surveyor TM (CELI/II) nuclease (26, 27) combined with dHPLC or gel electrophoresis detection was introduced that shows satisfactory selectivity and reliability (1% mutant to wild-type alleles is detectable) while it also identifies all base substitutions and small deletions that are important to cancer (17, 28) or to biotechnology and plant genetic applications [TILLING method (29) (30) (31) (32) (33) (34) ]. While reliable, the use of dHPLC for examining Surveyor TM -generated DNA fragments is a slow endpoint detection method restricted to examining a single DNA fragment at a time and the resulting DNA fragments cannot be sequenced. This limits analysis of cancer specimens when numerous samples or genetic regions need to be screened.
We introduce a new approach that enables Surveyor TM to scan for mutations over one or several PCR products simultaneously and selectively amplify and isolate the mutation-containing DNA fragment(s) via linkermediated PCR. By selectively amplifying mutationcontaining DNA from wild-type fragments, the present approach de-couples enzymatic mutation scanning from the endpoint detection step. As a result, following enzymatic action on mismatches any chosen DNA detection method (real-time PCR, gel/capillary electrophoresis, microarray-based detection) can potentially be used to identify the mutated DNA fragments in a simplex or multiplex fashion. Here we utilize real-time PCR coupled with melting curve analysis (Surveyor TMmediated Real Time Melting, s-RT-MELT) to validate the new technology. We demonstrate that this approach increases the mutation scanning throughput by 1-2 orders of magnitude when several (4100) samples are to be pre-scanned for mutations, enables mutation scanning over several PCR fragments simultaneously and mutationpositive samples can be directly sequenced when somatic mutations are at a low-level ($1-10% mutantto-wild-type ratio) in surgical cancer specimens.
Genomic DNA from cell lines with defined mutations in p53 exons, DU145 (exon 6), SW480 (exons 8 and 9), DLD1 (exon 7) and BT483 (exon 7) was extracted from cell lines purchased from the American Type Culture Collection (ATCC), or purchased as purified DNA when available. Surgical colon and lung cancer tumor samples were obtained from the Massachusetts General Hospital Tumor Bank following Internal Review Board approval. DNA from the EGFR mutation-positive cell lines A549, HCC827, H1975 and LU011 and from formalin-fixedparaffin-embedded lung cancer samples were obtained from the Lowe Center for Thoracic Oncology, Dana Farber Cancer Institute following Internal Review Board approval. We isolated genomic DNA using DNeasy TM Tissue Kit (Qiagen).
PCR with primers containing 5 0 -GC-clamp and 5 0 -M13 Sequences for the 5 0 M13 and GC-clamp portion of the primers, as well as the gene-specific portion of the primers used in this investigation are listed in Supplementary  Table 1 . The M13f and GC-clamp sequence was added to the 5 0 end of forward and reverse gene-specific primers respectively, or vice versa. Twenty microliter PCR reactions were performed from genomic DNA with final concentrations of reagents as follows: 1X JumpStart TM buffer (Sigma), 0.2 mM each dNTP, 0.2 mM forward and reverse primer, 1X JumpStart TM Taq polymerase (Sigma). PCR cycling was done on a Perkin Elmer 9600 PCR machine. The cycling conditions were: 948C, 90 s; (948C, 20 s/658C, 20 s/688C, 1 min) Â 10 cycles, with annealing temperature decreasing 18C/cycle, touch-down PCR; (948C, 20 s/558C, 20 s/688C, 1 min) Â 30 cycles; 688C, 5 min. This PCR program was linked to a program for denaturation and re-annealing of the PCR product over 10 min.
Five-microliter PCR product (300-500 ng) was mixed with 0.5 ml Enhancer TM and 0.5 ml Surveyor TM (Transgenomic) and incubated at 428C for 20 min followed by adding 0.5 ml Stop-solution, as per manufacturer's protocol. The inactivated Surveyor TM -digested product was purified with PCR QiaQuick TM purification kit (Qiagen) and eluted in 35 ml water. In some experiments, the PCR product was mixed with an approximately equal amount of PCR product from wild-type DNA prior to forming cross-hybridized sequences, to facilitate detection of homozygous mutations.
Addition of polyA-tail on the 3 0 -end Following purification of the Surveyor TM -treated sample, Poly-adenine 'tail' was added to the 3 0 -ends of DNA fragments. For each reaction, we added 5 ml purified surveyor-digested PCR product to a final volume of 20 ml with final concentration of 1X reaction buffer-4, 1X CoCL 2 , 0.2 mM dATP, 4 U Terminal Transferase (New England Biolabs). The reaction was incubated at 378C for 10 min and inactivated by heating at 758C for 10 min.
The real-time PCR amplification was performed using Titanium-Taq TM polymerase (BD-Biosciences -Clontech) in a Smart Cycler (Cepheid) real-time PCR machine. For each reaction, we added 0.5 ml polyA-tailed DNA to a final volume of 20 ml with final concentration of 1X Titanium buffer, 0.2 mM each dNTP, 0.1 Â LCGreen (Idaho Technologies), 0.2 mM m13f primer, 0.2 mM oligodT-anchor mix GACCACGCGTATCGATGTCG ACTTTTTTTTTTTTTTTTV [V represents A, C and G each oligodT-anchor concentration is 0.067 mM, as per RACE protocol (35)], 1 Â Titanium TM polymerase (Clontech-BD Biosciences). The thermocycling program was as following: 1 cycle of 948C for 2 min, 25 cycles of 948C for 15 s, 558C for 20 s and 688C for 30 s for reading fluorescence. Temperature titration was performed using different denaturation temperatures, 94-828C to experimentally determine conditions that selectively enable mutation-containing fragments to amplify.
The real-time PCR step was immediately followed by real-time differential melting curve analysis using the SmartCycler TM machine. DNA melting was performed immediately following PCR on the Smart Cycler I machine. Samples were heated from 708C to 958C at 0.18C/s. Differential fluorescent intensity curves (ÀdF/dT) were smoothed and used for identification of melting peak (s).
Altenatively, real-time PCR products were examined via dHPLC chromatography on a WAVE TM system (Transgenomic). Mutation-positive PCR products were purified via PCR purification kit (Qiagen) and sequenced using the M13f primer. All experiments were repeated at least three times in independent runs from genomic DNA.
The OpenArray TM high-throughput, massively parallel real-time PCR platform (36) (BioTrove) was tested for compatibility with s-RT-MELT. Forty-eight samples of p53 exon 8 PCR products were generated from 48 different lung adenocarcinoma samples and mutation-containing cell lines and processed via the hybridization and enzymatic steps of s-RT-MELT. Real-time PCR in the OpenArray TM platform was performed with the LightCycler FastStart TM DNA Master SYBR Green TM I (Roche) using 0.2 mM M13f and 0.2 mM oligodT-anchor-mix as primers pre-positioned on the array through-holes (36) and polyA-tailed DNA as template. The cycling conditions were as follows: 1 cycle at 948C for 2 min, 25 cycles of 908C for 15 s, 558C for 20 s and 688C for 30 s for reading fluorescence using a high sensitivity imaging camera (36) . The real-time PCR step was immediately followed by real-time differential melting curve analysis. Raw data were exported in Excel software for further analysis. The OpenArray TM experiment was repeated twice at the company's headquarters.
To estimate T m,min, the PCR denaturation temperature below which PCR is not efficient it was assumed, as an initial approximation, that495% hypochromicity must be present for PCR to work (i.e. any given sequence must be completely denatured, otherwise it re-forms immediately when temperature is lowered in the reaction and inhibits primer binding). The percent melting (hypochromicity)versus-temperature relations for GC-clamp-containing PCR products and Surveyor TM activity-generated products were estimated using the POLAND algorithm (37) , and the thermodynamic parameters determined by Blake and Delcourt for 75 mM NaCl in the solution (38) were used. In order to force agreement at a single point, predicted and observed values for a p53 exon 8 sequence containing a short GC-clamp were normalized at 888C. This shift accounts for the influence on T m,min of NaCl and Mg++ content in the reaction, the presence of the SYBR-GREEN/LC-GREEN dyes and the proprietary composition of PCR buffers. The T m,min of all other PCR products was then estimated using these semi-empirically determined parameters.
The 'enriched PCR' method by Behn et al. (39) was used to sequence codon 273 mutation of p53 exon 8 from sample CT20 and wild-type samples. In addition, a second method [Amplification via Primer-Ligation At The Mutation (40, 41) ] was used to distinguish mutant and wild-type samples by virtue of the de novo Nla-III site generated in the mutant sample by the p53 codon 273 G4A mutation.
The s-RT-MELT assay converts PCR fragments generated at positions of mutations by the Surveyor TM enzyme to fully amplifiable sequences that enable selective PCR amplification in a subsequent quantitative PCR detection method. Following denaturation and re-annealing of PCR products that leads to formation of cross-hybridized sequences at the positions of mutations ( Figure 1A ) the sample is exposed to Surveyor TM endonuclease that recognizes base pair mismatches or small loops with high specificity (28) and generates a break on both DNA strands 3 0 to the mismatch. The resulting DNA fragments participate in a terminal transferase (TdT) reaction that leads to polynucleotide 'tailing' (sequential addition of adenine, poly-A-tail) at the 3 0 -ends. A real-time PCR reaction is subsequently performed using adjusted conditions that enable selective amplification of the mutantonly fragments, followed by real-time melting curve analysis for identification of mutations in the presence of SYBR-GREEN TM or LC-GREEN TM DNA dye.
To enable selective amplification of the mutationcontaining fragments in the real-time PCR step, modified primers are employed for the original amplification from genomic DNA ( Figure 1B ). The forward primer contains a region specific to the target gene and a high melting domain (GC-clamp), while the reverse primer contains a region specific to the target gene and an M13 tail (or vice versa). Following the TdT tailing reaction, the M13 primer is used for real-time PCR in conjunction with a primer that binds to the poly-A tail. The denaturation temperature of the real-time PCR reaction is lowered to enable PCR amplification only for fragments that do not contain GC-clamps. Because the PCR products that escape digestion by Surveyor TM contain GC-clamps ( Figure 1B ), these fragments do not amplify efficiently during PCR, thereby enabling selective amplification of Surveyor TM -selected fragments, i.e. an effective 'purification' of mutation-containing fragments. The subsequent closed-tube melting curve analysis enables clear separation of true mutant sequences from PCR dimers or other artifacts.
Because s-RT-MELT does not require size-separation for identification of enzymatically generated fragments, more than one sequence can be scanned in parallel for unknown mutations in a single-tube reaction of Surveyor TM . This simple procedure enables the specificity of the Surveyor TM enzyme to be combined with the throughput and convenience of real-time PCR for rapid mutation scanning. Finally, because the amplified mutated sequences contain defined primers at their ends, direct sequencing of enzymatically selected PCR products is readily possible following the real-time melting step, enabling sequencing of low-level mutations identified by Surveyor TM .
To provide initial proof of principle for unknown mutation scanning using s-RT-MELT we utilized cell lines and tumor samples containing sequencing-identified mutations at several positions of p53 exon 8. Figure 2A depicts dHPLC chromatograms of the products obtained using a sample containing a p53 exon 8 G4A mutation or a wild-type sample. The standard Surveyor TM -dHPLC approach (28) was first employed to identify the mutation following PCR amplification of exon 8 from genomic DNA. The resulting dHPLC traces contain a single product for the wild-type and two products for the mutation-containing sequences (Figure 2A , curves 1 and 2, respectively). Next, s-RT-MELT was used to screen the same p53 exon 8 sequence. Following PCR amplification Simplex or Multiplex PCR amplification of one or more exons
PCR PRODUCT(s) G C -C L A M P M 1 3 Self-hybridize or cross-hybridize with wild type DNA: generate mismatches at positions of mutations in one or more PCR fragments Scan for mismatches all fragments simultaneously using CEL I /Surveyor TM enzyme.
CEL I /Surveyor TM enzyme.
Use TdT enzyme to add oligonucleotide tail (e.g. oligo-dA) to 3′OH ends, to serve as primer anchor
Un-digested fragments Digested fragments
TdT tailing of 3′ DNA ends Amplify only mutated fragment(s) coupled w. real time melting analysis (see B)
Detect mutations via closed tube, high-throughput melting curve analysis.
IF positive, sequence the amplified mutated DNA fragment with GC/M13-modified primers we cross-hybridized PCR products and exposed them to Surveyor TM and TdT tailing. The subsequent real-time PCR was run at different denaturation temperatures and the products were examined either via dHPLC or via real-time melting-curve analysis. At the standard denaturation temperature of 948C the mutation-containing sample contains two peaks, corresponding to the anticipated amplification of both Surveyor TM -digested and un-digested fragments (Figure 2A, curve 3) . However, when the PCR denaturation temperature is lowered (e.g. 86-888C) a single PCR product is generated for the mutant sample, while the wild-type sample demonstrates no product (Figure 2A , curves 4-7). In Figure 2B , real-time differential melting curves for the PCR reaction run at 888C are depicted. A peak corresponding to the PCR product from the mutant sample is again clearly evident, which is absent in the wildtype sample. Finally, Figure 2C depicts sequencing of the s-RT-MELT-generated PCR fragment, as well as the direct sequencing from genomic DNA. The G4A mutation is evident in both samples. In the s-RT-MELT product the anticipated addition of the poly-A tail at the 3 0 -position next to the mutation is illustrated.
To examine the selectivity of s-RT-MELT, dilutions of mutant to wild-type DNA were performed using DNA from SW-480 cells that harbor a p53 exon 8 14487G4A homozygous mutation. The real-time PCR reaction was again performed at 888C and mutant-to wild-type ratios of $1-10% were distinguished from the wild-type using either dHPLC ( Figure 2D ) or melting curve analysis ( Figure 2E ). In these samples, direct di-deoxy-sequencing could not identify a mutation if the ratio of mutant-towild-type was 5$30-40% (data not shown). On the other hand, sequencing of s-RT-MELT products was possible including the lower dilutions ( Figure 2F ). sRT-MELT sequencing generated traces with poly-A tails depicting the presence and the position of the mutation, although the exact nucleotide change was less clear than the one in exon 5 (i.e. the position AE1 base from the mutation might also be confused to be a mutation). The reason for this AE1 base ambiguity of the exact position of the mutation can be probably understood. The PCR performed following poly-A tail addition contains an equimolar mixture of three reverse primers (3 0 ending in V = G, A or C). Depending on the exact nucleotide at the mutation, the correct primer should in theory be preferred, while the other two primers should not allow efficient polymerase extension due to the mismatched 3 0 -end. However, in practice this 'allele-specific PCR' step occasionally allows 3 0 -mismatched primer extension, enabling more than one version of the primer to amplify over the position of the mutation, or alternatively the incorporation of the poly-A tail may occur AE1 base from the exact position of the mutation. We conclude that in certain cases sRT-MELT indicates the position of the mutation to within 1 base, while in others (e.g. p53 exon 5) it indicates the position 'and' the actual nucleotide change.
Next, p53 exon 8 was amplified using DNA from a group of 48 surgical lung adenocarcinoma samples and s-RT-MELT was used for the screening of unknown mutations via melting curve analysis. Mutations at different positions along exon 8 were present in several of these clinical samples, as indicated by the shift in melting profiles obtained ( Figure 2H ) and subsequently verified via sequencing. In this set of samples, sRT-MELT-sequencing detected a low-level mutation on a colon cancer specimen (CT20) that direct sequencing failed to identify ( Figure 2I ). As with Figure 2F , sequencing of sample CT20 indicated the position of poly-A tail addition to within one base, but the actual nucleotide change was difficult to identify. To exclude the possibility for a false positive, two independent RFLP-based methods were used to verify the presence of the mutation. Thus, since the position of poly-A tail addition was known ( Figure 2I , codon 273 of p53 exon 8) the mismatched primer approach by Behn et al. (42) was used to introduce an MluI restriction site for the wild-type p53 sample but not for the codon 273 mutants. Subsequent restriction with MluI enzyme followed by PCR generated a product with a 14487G4A mutation for the CT20 sample but not for the wild-type sample (Supplementary Figure 1 , Frame A). As an additional verification for the low-level CT20 mutation, we observed that G4A mutation generates a de-novo Nla-III site at the position of the mutation. Accordingly, we applied 'Amplification via Primer-Ligation At The Mutation', a method that we described previously (40, 41) to ligate a primer at the Nla-III-digested site, and preferentially amplified the mutant fragment in a second PCR. The sequenced PCR product identified again the 14487G4A mutation (Supplementary Figure 1, Frame B) . In conclusion, sRT-MELT identified correctly a p53 codon 273 low-level mutation on CT20 that was missed by regular sequencing. This is very significant as p53 exon 8 mutations at codon 273 have been associated with bad prognosis in cancer (43, 44) . Table 2 of Supplementary Data depicts a good agreement between standard Surveyor screening, s-RT-MELT screening and di-deoxy-sequencing, except for the low-level mutation discovered on sample CT20 via s-RT-MELT. s-RT-MELT-sequencing traces for two samples with p53 exons 6 and 7 mutations are also depicted.
The data in Figures 2A-D and H indicate a lack of substantial PCR amplification at denaturation temperatures 4888C for fragments containing the GC-clamp and a selective amplification of the mutation-containing fragments for several different mutation positions on p53 exon 8. To estimate the influence of the GC-clamp length on PCR efficiency versus temperature and the PCR amplification of fragments generated for mutations lying at different positions along the sequence, a calculation based on the POLAND algorithm (37) was performed. The predicted minimum temperatures for substantial PCR amplification were then plotted versus the experimentally observed values. Three possibilities were simulated, no GC-clamp, 26 nucleotides (nt) GC-clamp and 117-nt GC-clamp. DNA fragments corresponding to mutations at several positions along exon 8 were also simulated and compared to the experimentally observed minimum temperatures for generating a PCR product for three samples that contained mutations at different positions along p53 exon 8 (SW480, CT5 and TL50). The results ( Figure 2G ) indicate agreement to within $1.08C between theoretical prediction and experimental observation. For denaturation temperatures in the region 85-888C in combination with a 26-nt GC-clamp all the available mutations on p53 exon 8 are predicted to result in selective amplification of the mutation-containing fragment and inhibition of the GC-clamp-containing fragment. This prediction is consistent with the experimental results obtained from PCR temperature-titration experiments ( Figure 2G ). The developed calculation algorithm can thus be used to predict the appropriate PCR denaturation temperature for additional PCR fragment/GC-clamp combinations.
As a further validation for s-RT-MELT, we utilized the method to identify mutations in additional p53 exons. Figure 3A depicts the chromatographs obtained when a 1:1 mixture of DNA from SW-480 cells (homozygous mutation at p53 14686 C4T exon 9) and from wild-type cells was screened. The real-time PCR reaction was performed at different denaturation temperatures and the products were examined both via dHPLC and via melting curve analysis for comparison. As also observed for p53 exon 8, at 948C denaturation temperature both the Surveyor TM -digested and the undigested PCR products are amplified during real-time PCR ( Figure 3A , curves 1 and 2, mutant and wild-type, respectively). By lowering denaturation temperature to 858C or 848C, a single PCR product is obtained from the mutant while no product, other than primer dimer, is obtained by the wild-type sample ( Figure 3A , curves 3-6). Figure 3B depicts the melting curves obtained following real-time PCR at 858C denaturation temperature for the mutant and wild-type samples. s-RT-MELT was subsequently applied in the same manner to screen for p53 mutations in exons 5-7 from cell lines and surgical colon samples harboring sequencing-identified mutations including a single-base frameshift mutation in exon 7 (listed in Supplementary Table 2 ). The melting curves from mutant and wild-type samples in p53 exons 5-7 are depicted in Figure 3C -E. The data indicate that results similar to those obtained for p53 exon 8 are also obtained for p53 exons 5, 6, 7 and 9.
Detection of mutations in EGFR exons 18-21 is of particular clinical interest as these alterations can modulate response to EGFR inhibitors in lung adenocarcinoma patients (2,3) . Figures 3F, G and H depict the application of s-RT-MELT for screening DNA from lung cancer cell lines that harbor dHPLC-identified alterations in EGFR exons 19-21, including a two-codon deletion (del L747-E749, exon 19). The ability of s-RT-MELT for detecting low-level EGFR mutations was evaluated by performing DNA dilutions of a heterozygous EGFR exon 20 into a homozygous sample. A 1-10% mutant-to-wildtype ratio was detectable in this dilution experiment ( Figure 3F) . Finally, the application of s-RT-MELT in detecting mutations in DNA from formalin-fixed paraffinembedded (FFPE) samples was examined by screening four clinical FFPE lung adenocarcinoma specimens. Two of these samples were known to harbor EGFR exon 21 mutations (L858R), while the other two were negative for mutations when independently evaluated via dHPLC (28) . Figure 3I demonstrates the identification of the mutational status of these samples via s-PCR-MELT.
Multiplex s-RT-MELT or OpenArray TM -based s-RT-MELT increases the throughput of mutation scanning A significant potential advantage of enzymatic mutation scanning is the ability to screen several sequences simultaneously for mutations. To demonstrate that s-RT-MELT can be used for parallel scanning of mutations in several PCR products, we mixed equimolar amounts of PCR products from p53 exons 5-9 containing mutations either in exon 8 or in exon 9. We then formed 'cross-hybridized sequences' and screened the mixture for mutations in p53 exons 5-9 in a single tube using s-RT-MELT, as depicted in Figure 1A . Following real-time PCR and melting curve analysis, the exon 8 or exon 9 mutants were clearly distinguished from the wild-type sample ( Figure 4A, curves 1-3) . Next, the mutant exon 8 DNA sample was first diluted 10-fold into wild-type exon 8 and the equimolar mixture of p53 exons 5-9 was prepared and screened again in a single tube via s-RT-MELT. The exon 8 mutation was again distinguished from the wild-type mixture of exons ( Figure 4B , curves 1-3). Since 480% of p53 mutations in human tumors are encountered in exons 5-9 (45), the multiplex single-tube s-RT-MELT reaction could be used to identify most p53 mutations encountered in clinical tumor samples. Combined with multiplex PCR directly from genomic DNA, this approach could result to a convenient, high-throughput method for mutation scanning.
By adopting a real-time PCR platform as endpoint detection for s-RT-MELT, the throughput for mutation scanning increases drastically over other mutation prescreening approaches that utilize dHPLC, or capillary and gel electrophoresis. To demonstrate better this point, a highly parallel nano-technology platform was adopted for the real-time PCR step of s-RT-MELT that enables an array of 3072 nl volume real-time PCR reactions (OpenArray TM system) to be carried-out simultaneously followed by differential melting curve analysis (36) . As a proof of principle of the compatibility of s-RT-MELT with OpenArray TM , p53 exon 8 PCR products were generated from 48 different lung adenocarcinoma samples and mutation-containing cell lines and processed via the hybridization and enzymatic steps of s-RT-MELT. The 48 samples were each dispensed in 10 replicate nano-liter volume reactions on OpenArray TM plates pre-fabricated to contain the appropriate primers and amplified in 3072 real-time PCR reactions using a denaturation temperature of 908C in the presence of SYBR-GREEN I dye. Melting curves were subsequently obtained using the OpenArray TM melting curve analysis mode. The PCR growth curves and smoothed differential melting curves obtained distinguish clearly the mutation-containing samples from wild-type samples ( Figure 4C and D, representative results from 3072 reactions). Furthermore, identification of mutation-containing samples is in good agreement between the conventional and the nano-technology platforms ( Figure 4D versus Figure 2H ).
These data indicate that s-RT-MELT is compatible with high-throughput nano-technology detection formats and reiterates the advantage of de-coupling enzymatic selection from the detection step. Comparison of the throughput using conventional pre-screening method (dHPLC or dHPLC/Surveyor TM ) to s-RT-MELT (Table 1) indicates that s-RT-MELT is 1-2 orders of magnitude faster when a large number of samples (4100) are screened for mutations. If the multiplex s-RT-MELT format is adopted, the throughput can increase further.
The intrinsic potential of enzymatic mutation scanning for parallel identification of mutations can, in principle, be very high since the enzyme operates on numerous distinct mismatch-containing sequences on a molecule-tomolecule basis thus providing highly parallel mutation scanning. However, in the past the selectivity of the enzymes used and the endpoint detection method has limited the realization of this potential. Here we enabled Surveyor TM , an endonuclease that recognizes selectively mismatches formed by mutations and small deletions following 'cross-hybridized sequence' formation, to generate mutation-specific DNA fragments that are amplified and screened via differential melting curve analysis. The replacement of size-separation methods (capillary/gel electrophoresis, dHPLC) by real-time PCR technology as the endpoint detection platforms and the ability to scan more than one sequences in parallel result in a highly increased throughput for s-RT-MELT while retaining the ability to detect diverse mutations at low-levels. Cel I/II endonucleases have also been known to have exonuclease activity on 5 0 DNA-ends (26, 27) . For this reason, s-RT-MELT was designed to attach an oligonucleotide linker to the 3 0 -DNA ends via terminal transferase (TdT) instead of using the 5 0 -DNA ends. The exonuclease activity also tends to degrade the attached 5 0 -GC-clamps in s-RT-MELT, thereby eliminating their influence in reducing amplification of un-digested fragments. We found that if exposure of DNA 'cross-hybridized sequences' to Surveyor TM is limited to 15-20 min, the substantial degradation of 5 0 -GC-clamps is avoided. For multiplexing mutation detection using several PCR products simultaneously, the size of the GC-clamp on each PCR amplicon may need to be individually adjusted to ensure that mutations along all sequence positions of the PCR products included in the mixture can be screened at a single real-time PCR temperature and that undigested fragments do not amplify. The calculational tools developed in this work can be used to guide the individual design of GC-clamps. s-RT-MELT detects heterozygous SNPs as well as mutations. As with other mutation prescreening techniques, the presence of a SNP concurrently with a mutation might be difficult to identify without performing sequencing. Because SNPs occur at fixed positions, melting peaks originating from SNPs have a reproducible pattern and melting temperatures (46, 47) thus in many cases they should be distinguishable from mutations. Finally, it is noteworthy that s-RT-MELT is a general methodology that may also be applied to isolate mutations using mismatch-cutting enzymes other than Surveyor TM when enzymes with satisfactory properties for mutation detection become available. Detection platforms other than real-time PCR/melting (e.g. DNA microarraybased) may also be envisioned following enzymatic mutation selection.
In summary, we developed a new method for rapid mutation scanning, s-RT-MELT that utilizes the Cel I/II (Surveyor TM ) and terminal deoxy-nucleotide transferase (TdT) enzymes to isolate and amplify mutation-containing DNA fragments without the requirement of DNA sizedependent techniques. Besides enabling highly increased throughput, multiplexed mutation screening and direct sequencing of the identified mutant DNA fragments, s-RT-MELT also retains the advantages of the Surveyor endonuclease over alternative pre-screening methods, such as reliability and identification of genetic alterations present at low (1-10%) fractions in the sample. s-RT-MELT provides a significant advancement in unknown mutation scanning in cancer research and diagnostics as well as for general medical, biological and biotechnology applications. Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. Human Plasmodium falciparum (Pf) infection is a dramatic public health problem. Today approximately forty percent of the world's population is at risk of malaria. Malaria causes more than 300 million acute clinical cases, and at least one million deaths annually. Ninety percent of malaria deaths occur in sub-Saharan African countries mostly among young children and pregnant women (http://rbm.who.int). Thus, there is an urgent need of a malaria vaccine. However, vaccine discovery, in general and particularly in malaria, is still a very empirical process. In fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. For protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. In addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. Hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [1, 2] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. Proper protein folding and solubility remain a limitation in numerous cases. Thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge.
We addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. We focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. LSA-1 [3] , LSA-3 [4] , MSP-3 [5] , and MSP-6 [6] and other pathogens [7] . MSP-1, another leading malaria vaccine [8] , also contains predicted a-helical coiled coil regions (unpublished results). Our choice was based on the following considerations. First, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. This motif can be easily identified by bioinformatic analysis. Secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [9] . Thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. In addition, these domains are short (about 40 residues) and can be rapidly produced by chemical synthesis. Furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. Here, we focus on the Pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis.
The screening of the Pf genome [10] using generalized sequence profiles [11] identified several hundred proteins containing putative a-helical coiled coil motifs. Through proteome and transcriptome data [12] [13] [14] we assessed which of these molecules are expressed in the Pf parasite erythrocytic stage. The combined analysis/assessment identified over 100 segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (Table S1) . Out of these a-helical coiled coil fragments, in general 30-40 amino acids long, present either in the same protein or in different ones, 95 were chemically synthesized and HPLC purified. Among them, longer peptides (up to 70 amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigenS 1, 12 and 83; Table  S1 ). The selected antigens were then tested in ELISA assays for reactivity with three panels of sera obtained from adult donors from Burkina Faso, Tanzania and Colombia, respectively. To our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (Tables 1 and  S1 ). In this manner, 71 proteins were identified whose lengths varied from 200 to 10,000 amino acids. Twenty-one peptides with the highest prevalence of responders and ELISA mean OD value were selected for further studies. Variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. The high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution.
Indeed, circular dichroism (CD) studies of selected peptides associated with biological activities (Tables 1 and 2) indicate that they predominantly assume an a-helical conformation in water. Peptides 14, 27 and 45 ( Figure S1A ) exhibit a CD pattern characteristic of a high a-helical content, whereas the remaining peptides show CD profiles similar to that shown for peptide 12 ( Figure S1B ) or intermediate between those shown in Figures S1A and S1B characteristic of a partial a-helical organization. When analyzed by size exclusion chromatography on FPLC columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (MW 24 and 13kDa, respectively). The CD and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by NMR and ultra-centrifugation studies.
To test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from Papua New Guinean adults. The 3 serum pools were first tested in ELISA assays against 21 peptides that were the most antigenic (Table 1) ; from these, 18 peptide-specific antibodies were purified from the most positive serum pool and tested again in ELISA. These 18 antibodies all reacted with parasite native proteins in infected red blood cells as shown by IFAT ( Figure 1A ; Table 2 ). Reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by IFAT competition assays with the corresponding peptide ( Figure 1A ).
The specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (Glu)-rich sequences which are known to generate cross reactivity among several malarial Glu-rich proteins [15] . Cross-reactions were systematically investigated using each of the 18 affinitypurified antibodies on each of the 18 peptides. Results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (Table S2) . To determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, ELISA competition assays were performed. To this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. Only homologous peptides competed best whereas peptides having sequence similarity did not (Figures 2A,  2B , and S2B), or at a much higher concentration ( Figure S2A ) including the shorter Glu-rich peptides derived from the Cterminus of peptide 27 and 45 (Figures 2A and 2B) . Finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide.
Antibodies corresponding to the 18 selected peptides were tested for direct and cell-mediated anti-parasite activity. Clinical experiments have shown that the Antibody-Dependent Cellmediated Inhibition (ADCI) of P. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [16] . Twelve peptide-specific antibodies proved able to induce a strong (more than 40%) and intermediate (lower than 40%) monocyte-dependent parasite killing (Table 1) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. The effects were in the range observed with antibodies from African adults who have the highest natural protection known against malaria. Therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. Thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria.
Sixteen peptides -twelve targeted by ADCI positive antibodies and four controls-were used to immunize CB6F1 mice ( Table 2) . Eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by IFAT ( Figure 1B ; Table 2 ). As seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in IFAT assays (data not shown) and by IFAT competition assays with the corresponding peptide ( Figure 1B ). Anti-peptide 27 mouse antibodies, which are positive in IFAT, are also specific for the homologous peptide 27 but not for the sequence related peptides 9, 12 and 45 (Table S2) . Thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. Improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the N-and C-terminal ends, b) stabilizing the a-helix as suggested by Cooper et al. and Lu and Hodges [17, 18] and/or c) use of other adjuvants.
Genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. However available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (PlasmoDB 5.2, and unpublished sequencing data). A few peptide DNA sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation.
With regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for ADCI assays ( Table 1) , 15 of the proteins contain a pentapeptide conforming to the PEXEL consensus [19, 20; 21, 22] , but that none of these have a position within the amino acid sequence that conforms to the location of known active PEXEL motifs (see Materials and Methods and membrane segments, and none of them has a GPI anchor. Only one protein contains a signal sequence. Fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (Table S3 ). The prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [23] . Further investigations will be required to determine the actual localization of the corresponding antigens. The predicted localization is a priori surprising for molecules able to trigger an ADCI activity. However, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering ADCI provided they defined at least two epitopes [24] , which is the case for a-helical coiled coil heptad repeats. Therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link Fc-c receptors on monocytes to achieve Pf parasite killing.
In conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. In fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. In principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens.
The Pf 3D7 genome [10] was used for the bioinformatics analysis. The generalized sequence profile method and the pftools package [11] were used to search for the short a-helical coiled coil domains. The coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. Two profiles containing four and five heptad repeats were used for the analysis. The cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known 3D structures. Subsequently, the coiled coil Figure 1 . Immunofluorescence microscopy analysis of Pf 3D7 parasites with peptide specific antibodies. Acetone/methanol-fixed schizonts and merozoites were reacted with A: human peptide specific, affinity purified antibodies obtained with peptides 12 and 14 (Table 1) and B: sera from mice immunized with peptide 27 (Table 1) . Grey: bright field images; blue staining: indicates DAPI nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by Cy3-conjugated anti-human or anti-mouse IgG specific antibody. Merge picture is an overlay of the blue and red fluorescence channel. doi:10.1371/journal.pone.0000645.g001 regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. These proteins were also analyzed by the COILS program [25] .
The selected a-helical coiled coil containing proteins were further tested on their possible surface location and GPI anchoring by using the following programs: identification of potential signal peptides, SecretomeP and SignalP (http://www. cbs.dtu.dk/services/) [26] ; transmembrane spanning regions (TMPRED http://www.ch.embnet.org/software/TMPRED_ form.html and TMHMM http://www.cbs.dtu.dk/services/ TMHMM; [27, 28] ), and GPI-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [29] ) and prediction of sub-cellular localization (pTARGET http://bioinformatics. albany.edu/,ptarget; [30] ). To identify the PEXEL-like motifs in sequences of the selected proteins we used the following pattern [ 
[DEQ] that represents a combination of the PEXEL patterns indicated in recent papers [21, 22] . The presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of P. falciparum (www.PlasmoDB.org; [31] ).
Peptides were synthesized on the Advanced ChemTech (Hatley St George, UK) AC T348 Omega multi channel synthesizer and the Applied Biosystem synthesizer 431A and 433A (Foster City, CA) using solid-phase Fmoc chemistry. Crude peptides were purified by RP-HPLC (C18 preparative column) and analyzed by mass spectrometry (MALDI-TOF; Applied Biosystem, Foster City, CA). Chemicals and solvents used for peptide synthesis were purchased from Fluka (Buchs, Switzerland) and Novabiochem (Laufelfinger, Switzerland).
Circular dichroism (CD) spectra of peptides were recorded on a JASCO J-810 spectrometer (JASCO corporation, Tokyo, Japan) equipped with a temperature controller and a 0.1 cm path length cuvette. The measurements were made in water at pH 7.3 and 22uC and at a peptide concentration of 0.2 mg/ml.
The sera from Burkina Faso were collected in the village of Goundry located in the central Mossi Plateau, between 15 and 50 km north of the capital Ouagadougou, in the province of Oubritenga. The climate is characteristic of areas of Sudanese savannah, with a dry season from November to May and a rainy season from June to October. Malaria transmission is very high during the rainy season and markedly seasonal. Ethical clearance was obtained from the Ministry of Health, Burkina Faso. After obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season 1998.
The Tanzanian sera came from a large-scale community based study undertaken in Kikwalila village, Kilombero District, Morogoro Region from 1982 to 1984. Blood samples from adults (.15 years) were taken by finger prick and the serum was kept at -70uC until use. Research and ethical clearance for the study was obtained by the Tanzanian Commission for Science & Technology.
The Colombian sera were collected in Buenaventura the main port on the Colombian Pacific Coast after human informed consent, during a cross sectional survey carried out from February to May 2002 within the framework of a project supported by the Colombian Research Council, COLCIENCIAS. The area has unstable transmission of both P. falciparum and P. vivax malaria. Ethical clearance to draw blood from human volunteers was Human purified antibodies were used at 5 mg/mL; IFAT was not performed on ring stages. 
Valle. Blood was taken by venipuncture into tubes containing EDTA and sera fractionated and stored frozen until use. The sera from adults from Papua New Guinea (PNG) pooled for affinity purification were collected in the Maprik district of the East Sepik Province, during a cross sectional survey in July 1992 within the framework of the Malaria vaccine Epidemiology and Evaluation Project (MVEEP) supported by the United States Agency for International Development [32] . The area is highly endemic for malaria. Ethical clearance for MVEEP was obtained from the PNG Medical Research Advisory Committee. Blood was taken by venipuncture into tubes containing EDTA.
The pool of immune African globulins (PIAG) used for ADCI was prepared from immune individuals living in endemic areas and negative control IgG (N-IgG) was obtained from a pool of more than 1000 French adult donors with no history of malaria. Briefly, the IgG fractions from both positive and negative controls were purified using a size exclusion TrisacrylH GF05M (Pall BioSepraH; Pall life Sciences, NY) column followed by an ionic exchange DEAE Ceramic HyperDH F column (Pall BioSepraH). Purified IgG were then extensively dialyzed against RPMI and kept at 4uC until use.
CB6F1 mice were injected 3 times with 20 mg of the indicated peptide in Montanide ISA 720 at the base of the tail on day 1, 22 and 78. Bleeding was performed 10 days after the second and third immunization.
ELISA was performed according to Lopez et al. [33] and anti human IgG-or anti mouse IgG conjugated to alkaline phosphatase was used (Sigma, St Louis, MO) as second antibody. Individual human sera from 37, 42 and 39 adults donors from Burkina Faso, Tanzania and Colombia respectively were used at 1:200 dilution. Serum was considered positive if the optical density (OD) reading was higher than the mean OD value+3 standard deviation (SD) of the negative controls (individual serum samples from 8 to 11 naïve Swiss donors) or if the OD ratio of the mean of duplicate experimental values to the mean OD of the negative control was higher than 2. For mouse sera, the end point value was determined as the last dilution of the mean OD value+3 standard deviation (SD) of the negative control (non immune sera).
ELISA competition assays were performed by incubating either each of the 18 selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the 18 antigens over the indicated range of concentrations for 30 minutes at room temperature prior to addition to the ELISA peptide-coated plate wells.
Antigen-Sepharose conjugate preparation: 5 mg of antigen was dissolved in 1 mL of coupling buffer (0.1 M NaHCO 3 containing 0.5 M NaCl, pH 8.0). The CNBr-sepharose 4B (Amersham Bioscience AB, Uppsala, Sweden) was activated by swelling in 1 mM HCl and then washed with coupling buffer. The antigen solution was added to the gel and the mixture was stirred for 1h at RT. After the coupling reaction, excess antigen was washed away with coupling buffer. The unreacted activated groups were blocked by treatment with ethanolamine (0.25 M; pH 8.0) for 30 min at RT. The gel was then washed with sodium acetate buffer (0.1 M; pH 4.0), followed by coupling buffer. The antigensepharose beads were either used or stored at 4uC in PBS (1x) containing 1 mM azide.
Isolation of specific antibody: Pooled human serum was diluted five times with PBS (1x) containing 0.5 M sodium chloride and mixed with antigen-sepharose conjugate. This mixture was then stirred gently on a wheel O/N at 4uC. After centrifugation the supernatant was collected and stored at 220uC for further use. The antigen-sepharose beads were then washed with 5 mL of trizma base TRIS (20 mM containing 0.5 M NaCl, pH 8.0) then with 5 mL of TRIS (20 mM, pH 8.0). The elution of bound antibody was achieved with glycine (0.1 M, pH 2.5). The fractions obtained were instantly neutralized with TRIS (1 M, pH 8.0), dialyzed against phosphate buffer (0.1M, pH 7.0) and the antibody concentration was determined by the absorbance of the solution at 280 nm.
Slides coated with Pf sporozoites were dried at RT for 30 minutes, fixed with 100% acetone at 4uC for 10 minutes, washed 2 times in PBS-0.05% Tween 20, dried carefully and blocked with 20 mL/ well of PBS-3% bovine serum albumin (BSA) for 30 minutes at RT. Slides coated with Pf merozoites were fixed with 100% acetone at 220uC for 15 minutes and dried O/N at RT. The appropriate antibody or serum dilutions prepared in PBS-3% BSA were distributed (10 mL/well) and incubated for 1h at RT in a humid chamber. After washing with PBS-0.05% Tween-20, goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to Cy3 (Molecular Probes) diluted 1/500 in PBS-3% BSA or anti-human IgG (Fc specific) FITC conjugate (Sigma) diluted 1/50 in Evans blue solution (1/50000) was added (400 mL/slide) and incubated for 1h at RT in a humid chamber in the dark. Slides were washed as above, covered with 50 % glycerol, sealed and read using a fluorescence microscope (Leica DMIRB DC200).
The Uganda Palo Alto strain (FUP/C) was cultured in RPMI-1640 supplemented with 0.5% albumax I (GibcoBRL-Invitrogen, San Diego, CA). For ADCI assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over 24 h, by floatation on 1% porcine skin gelatin type A (Sigma).
Blood monocytes (MN) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (Lecourbe Blood Bank, Paris, France). Peripheral blood mononuclear cells (PBMC) were separated on Ficoll density gradients J PREP (TechGen, Les Ulis, France) and washed in Ca 2+ and Mg 2+ free HBSS buffered with 10 mM HEPES (both from GibcoBRL-Invitrogen). Cells were then distributed on polystyrene 96-well flat-bottomed culture plates (TPP, Trasadingen, Switzerland) and adherent MN were selected by incubation for 2 h at 37uC, in a humidified 5% CO 2 atmosphere. More than 90% of the adherent cells obtained in this manner were MN as estimated by the non-specific esterase test (a-naphtyl acetate esterase; Sigma). MN from each donor were tested prior to ADCI assays and only those without direct inhibitory effect were used in assays.
To wells containing 2610 5 MN purified as described above, 50 ml of an asynchronous parasite culture at 0.5 % parasitemia and 4 % hematocrit were added. Wells were then supplemented with test or control antibodies (Ab) and the total volume adjusted to 100 ml with culture medium. After 48 h and 72 h, 50 ml of culture medium were added to each well and after 96 h the ADCI assay was stopped and the final parasitemia was determined by light microscopy on Giemsa-stained smears by counting $50,000 red blood cells. For each Ab tested, duplicate wells included the following controls 1) non-specific monocytic inhibition, both MN+parasite, and MN+N-IgG+parasites and 2) direct inhibition by control or test IgG, both N-IgG+parasites, and test Abs+parasites. PIAG and N-IgG were used at a final concentration of 1 mg/ml as positive and negative controls respectively. Immunopurified tests Abs were used at 15 mg/ml. The Specific Growth inhibitory Index (SGI) which considers the parasite growth inhibition due to the effect of test Abs cooperating with MN was calculated as follows: SGI = 1006[12(% parasitemia with MN and test Abs/% parasitemia test Abs)/(% parasitemia with MN and N-IgG/% parasitemia N-IgG)]. Figure S1 CD spectra of the peptides 45 (S1A) and 12 (S1B) Found at: doi:10.1371/journal.pone.0000645.s001 (12.32 MB TIF) Figure S2 ELISA inhibition assay using anti-human peptide specific antibodies. Binding of peptide specific antibodies to peptides 76 (S2A) and 9 (S2B) absorbed on ELISA plates was inhibited by incubating specific antibodies (1-2 mg/ml) with peptides 14, 76 and 81 (S2A) and peptides 8 and 9 (S2B), respectively (see Material and Methods). Peptides 14, 76 and 79 share NNM or MNN as sequence similarity while peptides 8 and 9 do not exhibit any apparent sequence similarity. Table S3 Structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in ADCI (Table 1) . Found at: doi:10.1371/journal.pone.0000645.s005 (0.05 MB DOC) FluGenome: a web tool for genotyping influenza A virus Influenza A viruses are hosted by numerous avian and mammalian species, which have shaped their evolution into distinct lineages worldwide. The viral genome consists of eight RNA segments that are frequently exchanged between different viruses via a process known as genetic reassortment. A complete genotype nomenclature is essential to describe gene segment reassortment. Specialized bioinformatic tools to analyze reassortment are not available, which hampers progress in understanding its role in host range, virulence and transmissibility of influenza viruses. To meet this need, we have developed a nomenclature to name influenza A genotypes and implemented a web server, FluGenome (http://www.flugenome.org/), for the assignment of lineages and genotypes. FluGenome provides functions for the user to interrogate the database in different modalities and get detailed reports on lineages and genotypes. These features make FluGenome unique in its ability to automatically detect genotype differences attributable to reassortment events in influenza A virus evolution. Infections with influenza A viruses continue to be a public health problem, causing seasonal epidemics and sporadic but devastating pandemics. Each year in the US, influenza epidemics cause more than 200 000 hospitalizations and result in over 30 000 influenza-related deaths (1) . Influenza pandemics are infrequent but they can result in high mortality. It is estimated that $20-100 million people were killed worldwide by the 1918-1919 influenza pandemic (2) (3) (4) . The current level of pandemic alert is at the highest level, phase 3, since the most recent pandemic of 1968 (5) .
Influenza viruses belong to the family Orthomyxoviridae and are classified into three types, A, B and C based on the identity of major internal protein antigens (6) . Influenza A and C viruses can infect multiple mammalian species, while influenza B virus is almost exclusively a human pathogen (7) . Influenza A viruses cause the greatest morbidity and mortality in humans. Interestingly, the largest pool of influenza A viruses is maintained by horizontal spread in wild aquatic birds, in which the virus does not normally cause any disease (6, 8) . Food and companion animal populations such as poultry, swine, horses and dogs support sustained replication of certain lineages of influenza A, with minimal to lethal disease depending on the virulence of the strain (6) . Influenza viruses have evolved in association with their various hosts in different continents for extended periods of time (9) . This co-evolution has resulted in extensive genetic divergence among the extant viruses currently available for analysis.
Influenza A viruses are classified into subtypes on the basis of antigenic analysis of hemagglutinin (HA) and neuraminidase (NA) glycoproteins. So far, 16 HA subtypes and 9 NA subtypes have been found (10) . In recent years, gene sequences have become available for a large number of viral strains creating a diverse pool of influenza A viruses from historical and current isolates collected in multiple geographic regions. Comparison of the deduced amino acid sequences of the HA and NA revealed an excellent agreement between the results of clustering viruses by the antigenic reactivity and sequence similarity. However, molecular genetic analysis allows a comprehensive analysis of the entire viral genome and is gaining popularity because it is more practical for most laboratories as a method for classification (11) . Most importantly, study of the influenza genomic structure, namely genotyping, could reveal mechanisms of virus evolution, spread and disease pathogenesis.
The influenza A genome consists of eight negativestranded RNA segments that encode at least 10 viral proteins (12) . The viral genome evolves through accumulation of mutation by the viral RNA-dependent RNA polymerase which lacks proofreading ability and through reassortment of entire gene segments (13) . Forces selecting viral variants such as the neutralizing antibody response of vertebrate hosts as well as species-related structural variation can also promote rapid evolution (14) . Each of the segments can evolve at a different rate if they are subject to differential selective pressures and functional constraints (15) (16) (17) (18) (19) . The segmented nature of the viral genome allows for segment exchange (termed reassortment) when two distinct viruses co-infect a cell and generate progeny with a mixed genome (20, 21) . Reassortment may theoretically yield 254 (2 8 -2) different combinations of gene segments from two parent viruses.
A comprehensive influenza genotype database that can be searched using a web tool for the genotyping viruses is not available. Unlike HIV and HCV, the influenza A virus has a segmented genome, so eight separate phylogenies must be analyzed to establish a genotype.
We approached the problem of genotyping influenza A viruses by analyzing each gene segment independently, segregating gene segments into subtypes and subsequently into lineages. The genotype of an influenza A viral strain is the sequential aggregate of the eight assigned gene segment lineages. A nomenclature for influenza A viral genotypes will allow researchers to unequivocally describe influenza A viral genotypes to analyze, compare and communicate the molecular epidemiology of the virus. In this report, we define a nomenclature for influenza A viral genotypes and describe a web tool developed for genotyping influenza A viruses from genome sequences. Our tool facilitates identification of reassortment events between divergent lineages.
Two nomenclature conventions are used routinely in influenza research: (i) the eight segments in the influenza A genome are numbered from 1 to 8 for PB2, PB1, PA, HA, NP, NA, M and NS, respectively; (ii) There are currently 16 alleles of the HA gene termed subtypes. Likewise, there are nine alleles for NA, and two alleles for non-structural (NS) proteins. Since influenza A viruses have an unusual genomic structure, we approached the genotyping problem by first analyzing each gene segment separately. According to the above conventions and considering that the evolutionary rate varies from segment to segment, we defined a genotype as a sequential combination of the lineages for each of the eight segments in a genome. A letter was assigned to each lineage of PB2, PB1, PA, NP and M, and a number followed by a letter was assigned to each lineage of HA, NA and NS with the number representing the subtype or allele. For example, [A,D,B,3A,A,2A,B,1A] is the genotype of a human seasonal subtype H3N2 virus with PB2 lineage A, PB1 lineage D, PA lineage B, HA subtype 3, lineage A and so on, following the convention for numbering of influenza genome segments. With this nomenclature, identifying genotypes and reassortment becomes an easy task accomplished by comparing the predicted genotype against all genomes that have been classified previously.
Genomic sequences of all influenza A viruses with 475% of the full segment length were downloaded from NCBI Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/ genomes/FLU/FLU.html). Alignments were performed for each individual gene segment using the ClustalW program (22) . The MEGA software was used to construct the phylogenetic trees with the neighbor-joining method and the HKY-85 model selected (23) . The goal of our genotype method is to determine when a reassortment event with a gene segment from a non-traditional host or location has occurred. The lineages of each viral gene were carefully determined as detailed subsequently: (i) using the phylogenetic trees constructed, significant clusters (which were segregated by $10% nucleotide difference by p-distance) were assigned lineages; (ii) bootstrap analysis was used on a smaller set of sequences with values 490% considered significant; (iii) the initial lineages were evaluated for nucleotide differences within and between other lineages and for strength of bootstrap support; (iv) approximately 10 sequences from each lineage were randomly selected for the maximum likelihood (ML) analysis for each gene segment, serotype (for HA, NA) or allele (for NS) on the MultiPhyl server (24) . The lineage assignment of each influenza gene available in the public databases was uploaded into the Segment Table in the database as described subsequently.
The FluGenome database contains three tables: Segment, Genome and Genotype. The Segment table contains information-related to sequences, including assigned lineage, strain name, segment, serotype, host, country, year, GenBank accession number, nucleotide sequence and sequence length. The Genome table contains the information for complete genomes, including assigned genotype and accession numbers of each gene segment. When more than one sequence was available for a gene segment, the longer of the two sequences was kept for the genome accession. Unique genotypes are stored in the Genotype table along with the total number of genomes that have that genotype. The Genotype table was created by querying the Genome table for distinct genotypes. Host categories were created to separate the genomes of each genotype, which include Human (Hu), Avian (Av), Swine (Sw), Equine (Eq), Canine (Ca) and Others (ONHM).
The FluGenome database is updated automatically every night. New sequences are downloaded from the NCBI Influenza Virus Resource (ftp://ftp.ncbi.nih.gov/ genomes/INFLUENZA/) and added into the FluGenome database. The lineage information predicted for new sequences is used to update Segment, Genome and Genotype tables if necessary. For sequences already in the database, the script checks to see what information needs to be updated, and the sequences entries are flagged for further validation.
The web interface and databases were implemented with the LAMP strategy. The server used Linux (L) for the operating system, along with Apache (A) as the web server. The genotyping database was built with the MySQL database management system (M). PHP and PERL (P) were used to code the two parts of the web tool: the back end program and the front end interface. JavaScript and HTML were used sparingly in the front end interface. A domain name, http://www.flugenome.org, was acquired to provide access to the database and the web tool.
The BLAST algorithm is used for sequence comparison, because of its advantages such as fast computation and accurate results in detecting local highly similar sequence regions. To overcome its inherent disadvantage (i.e. not a global alignment algorithm), we used a parameter called 'coverage' to detect gene-wide sequence similarity (25) . The default thresholds for identifying lineages were set to be 95% identity and 95% coverage. The user can reset the thresholds to any allowable value. The top BLAST results for a user-submitted query sequence are sorted by identity and coverage, and the best result is used to assign a lineage to the query sequence. If a result from BLAST falls below the thresholds, the lineage will be flagged with an asterisk ( Ã ).
To determine the genotype of a complete or partial influenza virus genome, a script is executed that first establishes the lineage of each viral gene segment. The genotype will be created by the sequential incorporation of the lineages for each of the eight segments, arranged per convention as shown in Table 1 . If a lineage does not meet the thresholds specified (95% default for both identity and coverage), the lineage will be assigned an asterisk ( Ã ) indicating the query sequence does not meet criteria and may be from a new lineage. If no BLAST results are found a blank lineage will be displayed. If all segments belong to known genotypes, the genotype of the query genomic sequence will be provided as output. The resulting genotype can be compared to previously identified genotypes in the Genotype database. This analysis can reveal reassortment events and host switching. If the genotype determined by FluGenome is not found in the Genotype database, the genome will be flagged as a virus with a potentially new genotype. Viral genotypes reported as new by FluGenome can simply result from identification of a gene from a novel phylogenetically defined lineage or the presence of genes from known lineages in novel combinations.
The online tool presents two query options to the user; entering gene segment sequence(s) or genotype sequence(s) (Figure 1) . The segment query 'Determine Individual Gene Segment Lineage' is used to identify the lineage of a viral gene segment of interest, for example PB2. In this case, the input FASTA file can contain one or many sequences, but all must correspond to the same gene segment. To analyze data sets from more than one gene simultaneously; e.g. both the PB1 and PB2, the user must first enter the number of different gene segments and then provide each sequence data set in a separate FASTA file.
The genotype query 'Determine Genotype' analyzes incomplete or complete genomes. Sequences from each genome must be in a separate FASTA file. Alternatively, the user can cut and paste sequences of one genome at a time. Multiple genomes can be analyzed simultaneously.
Nearly 30 000 sequences were collected from public databases and used for the lineage analyses, resulting in 184 lineages. The viral gene segments showed a wide range of diversity; HA was partitioned into 78 lineages whereas MP only into seven (Table 1) . Mining the aforementioned sequences resulted in $2300 complete genomes, which consists of 156 unique genotypes with 50 serotypes (http://www.flugenome.org/show_genotypes.php). Serotypes may comprise as many as 15, different genotypes; 
Step 1. Enter number of different gene segments to analyze
Step 2. Select which gene segment(s) to analyze
Step 3. Enter sequence(s) in FASTA format
Step 4. Results page with lineage(s)
Step 5. Show viruses with the same gene lineage(s)
Step 1. Enter number of influenza A genomes to compare
Step 2. Input genome sequence(s) in FASTA format
Step 3. Results page with genotype(s) 
We propose a nomenclature system for naming influenza A viral genotypes. This nomenclature was exploited to analyze $2000 complete viral genomes (nearly full-length or full-length segment sequences), revealing 156 unique genotypes. The FluGenome web server implementation also includes facilities for analysis and sorting of lineages and genotypes which allow the user to explore the evolutionary history of the viral strains. In particular, the FluGenome web server can provide genotype information that greatly facilitates the inference of genetic reassortment among influenza viruses. Influenza pandemic intervention planning using InfluSim: pharmaceutical and non- pharmaceutical interventions BACKGROUND: Influenza pandemic preparedness plans are currently developed and refined on national and international levels. Much attention has been given to the administration of antiviral drugs, but contact reduction can also be an effective part of mitigation strategies and has the advantage to be not limited per se. The effectiveness of these interventions depends on various factors which must be explored by sensitivity analyses, based on mathematical models. METHODS: We use the freely available planning tool InfluSim to investigate how pharmaceutical and non-pharmaceutical interventions can mitigate an influenza pandemic. In particular, we examine how intervention schedules, restricted stockpiles and contact reduction (social distancing measures and isolation of cases) determine the course of a pandemic wave and the success of interventions. RESULTS: A timely application of antiviral drugs combined with a quick implementation of contact reduction measures is required to substantially protract the peak of the epidemic and reduce its height. Delays in the initiation of antiviral treatment (e.g. because of parsimonious use of a limited stockpile) result in much more pessimistic outcomes and can even lead to the paradoxical effect that the stockpile is depleted earlier compared to early distribution of antiviral drugs. CONCLUSION: Pharmaceutical and non-pharmaceutical measures should not be used exclusively. The protraction of the pandemic wave is essential to win time while waiting for vaccine development and production. However, it is the height of the peak of an epidemic which can easily overtax general practitioners, hospitals or even whole public health systems, causing bottlenecks in basic and emergency medical care. The recent spread of highly pathogenic avian influenza from Asia to Europe and the transmission to humans has intensified concerns over the emergence of a novel strain of influenza with pandemic potential. While still being in an inter-pandemic stage, nations plan for pandemic contingency following recommendations of the WHO [1, 2] . National influenza preparedness plans are constantly being refined, aiming to mitigate the effects of pandemic influenza on a national, regional and local level. Even in the absence of a pandemic strain, seasonal influenza causes substantial morbidity and mortality [3] . Seasonal outbreaks put pressure on general practitioners and strain hospital resources, leading to bottlenecks in outpatient treatment and hospital admission capacities.
Various intervention strategies reduce the impact of influenza on individuals and public health systems. In interpandemic phases, vaccination is the most important tool to reduce morbidity and mortality, but a potent vaccine will probably not be generally available in the initial phase of a pandemic [4] . Other control strategies like pharmaceutical (antiviral) [5, 6] and non-pharmaceutical interventions (reduction of contact rates) [7, 8] will have to be implemented.
The use of antiviral drugs during a pandemic seems to be the treatment of choice at present [9] [10] [11] [12] , but not all countries can afford stockpiling enough drugs. Furthermore, concerns about the over-reliance of a "pharmaceutical solution" have been expressed [13] . An epidemic can also be mitigated by reducing contact rates in the general population and by decreasing the infectivity of cases [9] . Such reductions can be achieved by measures like quarantine and case isolation [14] , closing day care centres and schools, cancelling mass gathering events, voluntary self isolation and general behavioural changes in public and increasing social distance [8] .
The effectiveness of such interventions depends on various factors which must be prospectively explored by sensitivity analyses, based on mathematical models. Here, we use the freely available Java applet InfluSim [15] to investigate how effectively pharmaceutical and non-pharmaceutical interventions contribute to mitigate an influenza pandemic while vaccines are not available. In particular, we examine how intervention delays determine the course of a pandemic and constrict the success of interventions.
InfluSim is a deterministic compartment model based on a system of over thousand differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. Details of the simulation and a discussion of the standard parameter values have been described previously [15] ; a summarizing description of the model is provided in the Appendix. The program and its source code are publicly available [16] to offer transparency and reproducibility. The simulation produces time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses (neuraminidase inhibitors), hospitalizations, deaths and work days lost due to sickness, all of which may be associated with financial loss. The analyses presented here are based on InfluSim 2.0, using demographic and public health parameters which represent the situation in Germany in 2006. Interventions include antiviral treatment, isolation of patients, social distancing measures and the closing of day care centres and schools as well as cancelling mass gathering events.
Using the standard set of InfluSim parameters (freely accessible from [15] ), about one third of all infected individuals is expected to become severely ill and to seek medical help. Patients seeking medical help will be referred to as "outpatients" throughout this paper. An exponential distribution is used to model the delay between onset of symptoms and seeking medical help; on average, patients visit a doctor after 24 hours. If a patient seeks medical help within 48 hours after onset of symptoms, he or she is given antiviral treatment unless the stockpile of antivirals is exhausted. Antiviral treatment reduces the duration and degree of infectivity of the case and the number of hospitalizations (Table 1 ) [17] . For more detailed descriptions see [15] or the Appendix.
Non-pharmaceutical interventions examined in this paper are contact reduction measures and the isolation of cases. The latter effectively leads to reduced contact rates between individuals, too. In the scenarios presented below, we assume that everybody in the population avoids a given percentage of contacts (e.g. by improved hygiene, wearing masks, or behavioural changes) and that sick patients are isolated which reduces the contact rates of moderately sick, severely sick (but non-hospitalized) and hospitalized cases by 10%, 20% and 30%, respectively. Further interventions which comprise the closing of day care centres and schools, and the cancelling of mass gathering events will be examined in detail in a separate paper.
Assuming a basic reproduction number of R 0 = 2.5 and using the standard parameter set of InfluSim [15] , an epidemic in a population of 100,000 individuals reaches the peak about 40 days after introduction of the infection and is practically over three weeks thereafter if no interventions are performed (Figure 1 ). During the whole epidemic, 87% of the population become infected, 29% seek medical help, 0.7% are hospitalized and 0.2% die. Figure  1 shows how pharmaceutical and non-pharmaceutical interventions can mitigate this scenario. Contact reduction by isolation of cases alone (see Appendix), protracts the peak of the epidemic by about one week. Distribution of antivirals or additional contact reduction measures delay the epidemic by approximately 10 days and are hardly sufficient to provide a substantial delay. A combination of antiviral treatment, isolation of cases and social distancing in the general population seems to be necessary to delay the epidemic in the order of weeks. This example furthermore shows that an efficient mitigation of the epidemic is not necessarily associated with a significant reduction in the number of infections. For information on the proportions of infected people and outpatients see the legends to the Figures.
The mitigating effect of antivirals strongly depends on the onset of their distribution ( Figure 2 ). Antivirals can delay the epidemic if distributed very early while few cases exist in the population. Late distribution of antivirals (e.g. starting on day 30) leads to the paradoxical effect that the stockpile is exhausted even quicker compared to early distribution (shaded areas und the curves in Figure 2 ). Additionally, the mitigating effect of the intervention drastically diminishes and benefits are restricted to lowering the peak of the epidemic. Unrestricted availability of drugs (grey curves in Figure 2 ) still leads to an epidemic because (i) asymptomatic and moderately sick cases are not eligible for treatment, (ii) patients visit a doctor on average 24 hours after onset of symptoms while already being highly infectious and (iii) antivirals cannot fully prevent infectivity. Figure 3A ). In contrast, the mitigating effect becomes negligible, if antivirals are distributed with delay ( Figure 3B ). Independent of the delay in the distribution of antivirals, their quantitative availability affects only the height of the peak of the epidemic, but hardly the mitigation of the epidemic ( Figure 3A , B). For considerations into the final size of the epidemic see below. In summary, delaying the epidemic depends on early action, whereby lowering the peak depends on the quantitative availability of antivirals.
Contact reduction measures, comprising social distancing and the isolation of cases, can be an effective part of mitigation strategies; they have the advantage over antiviral treatment to be not limited per se, i.e. they can be continued for a sufficiently long period of time. Figure 4 examines the effect of isolation of cases and social distancing measures (see figure caption for details) in the absence of antiviral treatment. The peak of the epidemic is protracted by about 1 day for every percent of contact reduction if this intervention starts immediately after the introduction of the infection. Thus, a peak shift is not only possible by early action, but also by the degree of contact reduction. If contact reduction is initiated later, the peak shift diminishes, but the proportionality remains. For example, if the intervention starts three weeks after the introduction of infection, the peak of the epidemic is only mitigated by about half a day per 1% contact reduction ( Figure 4B ). Premature cessation of contact reduction measures restores the infection rates to the pre-intervention values which fuels the epidemic. It can lead to a delayed course and a higher total number of infections, involving a plateau or even a second peak of the epidemic ( Figure 4C ).
The preceding examples with interventions based on antivirals or contact reduction alone yielded peak delays only in the order of weeks, whereas months may be required for vaccine development and production, demanding for a combined intervention scheme ( Figure 5 ). We examine an optimistic scenario where antivirals are distributed immediately after the infection is introduced (dark bars in Figure 5 ), while varying the onset of social distancing measures. The antiviral stockpile lasts longer if social distancing measures are initiated earlier (pale bars in Figure  5 ). Immediate initiation of contact reduction can protract the epidemic by months, whereas a delayed initiation leads to a plateau in the epidemic curve at a time when antivirals are used up.
Without interventions, N i = 87% of the population become infected during the course of the epidemic and the cumulative number of outpatients reaches N o = 29%, reflecting the assumption that approximately one third of infected individuals becomes sufficiently sick to seek medical help. These outcomes remain surprisingly stable even for interventions assuming optimistic resources (cf. footnotes to Figures 1, 2 , 3, 4, 5). For instance, immediate and unlimited availability of antivirals reduces these fractions only to N i = 72% and N o = 24% ( Figure 2 ). This minor effect has three reasons: only about one third of cases seeks medical help and will receive antiviral treatment, many infections are passed on before cases seek medical help and antiviral treatment does not fully prevent further transmission. These disadvantages do not apply to contact reduction measures. For instance, a reduction of 20% of contacts reduces these fractions to N i = 68% and N o = 22% ( Figures 4A, B) . A combination of antiviral treatment and contact reduction can further reduce these values to N i = 53% and N o = 18% ( Figure 5 ).
In the preceding analyses it was assumed that parameter values are precisely known; in a real world scenario, however, uncertainty arises from biological variability, stochastic influences, heterogeneities, etc. We illustrate with a concluding example to which extent simulated epidemics are affected by uncertainty in the parameter values. As shown in Figure 6 , epidemics can be highly variable, although only four parameters have been varied within moderate ranges. Varying more parameters would further increase this variability.
For the interventions and parameter variations considered, the cumulative number of outpatients ranges from a few thousand to over twenty thousand (see inset in Figure  6 ). Among the four parameters, R 0 is the strongest predictor of the number of outpatients (analysis not shown) as it strongly determines how quickly antivirals become exhausted. In two out of 1,000 simulations the randomly chosen parameter combinations involved values for R 0 around 1.8 which led to very minor outbreaks given the intervention scheme. The cumulative number of outpatients escalates when antiviral stockpiles become exhausted while the proportion of susceptibles is still large enough to allow for further propagation of infectives. In this case, the epidemic curve proceeds with a second wave or a plateau.
With pandemic influenza, we have to "expect the unexpected" [18] . Historical reports frequently mention the surprising speed at which a pandemic wave travels through the population [19] [20] [21] . Predicting the course of a future pandemic which will be caused by a virus with unknown characteristics is based on substantial uncertainties and we must rely on sensitivity analyses, performed with mathematical models like InfluSim.
Because of the short serial interval of influenza, timely action is essential. Different control measures must be regarded as complementary and not as competing. Neither antiviral treatment nor non-pharmaceutical measures should be used exclusively to mitigate a pandemic influenza wave.
Infectious disease models have suggested that an upcoming influenza epidemic with a low basic reproduction number might be contained at the source through targeted use of antiviral drugs [9, 12] . The published scenarios concern WHO phases 4 and 5 (inter-pandemic alert period) and assume that an outbreak starts in a rural area with low population density. It can be expected that the pandemic virus will be introduced into Europe and the US after a local epidemic (i.e. in WHO phase 6). Communitybased prophylaxis, however, is of limited use for several reasons. Under a high prevalence of infection in phase 6, a wide distribution requires an enormous number of antiviral courses; with available stockpiles, it will be virtually impossible to locally contain the pandemic with targeted antiviral prophylaxis. Development of resistance, limited production capacities and extremely high costs are further limitations of this strategy, so that population-wide prophylaxis has not been recommended by the WHO for the final phase of the pandemic [1].
The discussion of pandemic influenza preparedness planning has frequently focussed on the amounts of drugs to be stockpiled and to whom and when they should be supplied [22] . Even if the currently stockpiled antiviral drugs will be fully effective against the pandemic strain, their use may not be able to sufficiently prevent the spread of influenza because (i) transmission of the infection may occur before the onset of clinical symptoms (as assumed in the InfluSim model) [23] , (ii) asymptomatic and moderately sick cases [6] are usually not treated despite contributing to transmission, and (iii) the occurrence of cases with influenza-like illness caused by other pathogens may lead to an accelerated depletion of the antiviral stockpile. Likewise, moderately sick cases or even healthy people may seek medical help and succeed in receiving antiviral treatment which would further deplete the stockpile. These factors reduce the efficacy of pharmaceutical control measures [24] , indicating the demand of extending this strategy by non-pharmaceutical intervention measures.
Especially if antivirals are limited, they should be supplied as early as possible. If their distribution is delayed, cases become so abundant that resources will quickly be exhausted without having much impact on the spread of the disease (Figures 2 and 3 ). This confirms that the amount of antivirals needed strongly depends on the number of infections that are present when the intervention is initiated [25] . If antiviral drugs are extremely limited, they should be used to preferably treat severe cases
Onset and sustainability of antiviral intervention Intervention with limited amounts of antivirals Figure 3 Intervention with limited amounts of antivirals. Number of outpatients expected during a pandemic wave, varied by the availability of antivirals. Parameter values are based on the InfluSim standard configuration [15] with R 0 = 2.5, except those listed at the end of this legend and indicated by superscripts 1 . Antiviral availability ranges from 0% (no antivirals available, dashed curves 2 ) to 10% (antivirals available for 10% of the population 3 Figure 4 Effects of contact reduction measures. Number of outpatients expected during a pandemic wave if contact reduction measures are implemented additionally to the isolation of cases. Parameter values are based on the InfluSim standard configuration [15] with R 0 = 2.5, except those listed at the end of this legend and indicated by superscripts 1 . The dashed curve shows the epidemic without intervention. Contact reduction involves social distancing 2 and isolation of cases 3 . The curves show the effects caused by social distancing, where contacts are reduced by 0% 4 (grey curve) up to 30% 5 in steps of 2% 6 (black curves, from left to right). Bars at the bottom of each graph illustrate the periods of contact reduction, which are in A: full, from day 0 to end, in B: delayed, from day 20 to the end, and in C: temporarily, from day 20 to day 50. 1 :Parameter modifications are given in the following and terms in italics refer to terms in the InfluSim user interface. InfluSim output: N i = cumulative proportion of the population infected, and N o = cumulative proportion of outpatients in the population. 2 that need hospitalization. Although this has practically no effect on the pandemic wave per se, it helps to reduce the death toll in the population (results not shown).
Rather than relying on a pharmaceutical solution, pandemic preparedness should also involve non-pharmaceu-tical measures (see above). Early self-isolation and social distancing measures can be highly effective, as shown for the SARS epidemic [26] : after the WHO's global alert and the implementation of massive infection control measures, the effective reproduction numbers in Hong Kong, Vietnam, Singapore and Canada fell below unity. Rigorous social distancing measures in the entire population, Parameter values are based on the InfluSim standard configuration [15] with R 0 = 2.5, except those listed at the end of this legend and indicated by superscripts 1 . The sensitivity analysis extends the scenario shown in Figure 5 , where antivirals are available for 10% of the population and are distributed from day zero 2 , and where contact reduction measures 3 , including the isolation of cases 4 , are initiated three weeks after the introduction of infection (scenario "day 21"). Right panel: parameter values for each realization are sampled independently from normal distributions as shown (means given in bold, 99% of the values lie within the range specified by dotted lines, except b A which is truncated). R 0 : basic reproduction number, x 50 : cumulative infectivity during the first half of the symptomatic period, b A : relative infectivity of asymptomatic cases, f c : antiviral treatment reduces infectivity by a factor of 1-f c . For each parameter, an increase of the value aggravates the epidemic. Large plot: from a hundred random realizations, we selected the two most extreme epidemics, and eight epidemics homogeneously placed between them. The epidemic with N 0 = 20800 is caused by parameter values drawn from the left tail of the corresponding distributions, and the epidemic with N 0 = 5000 is caused by parameter values drawn from the right tail of the corresponding distributions (see right panel). The epidemic curves show a plateau or a second wave when antiviral stockpiles are exhausted while the proportion of susceptibles is still large enough to allow for further propagation of infectives (thin curves in black); for optimistic parameter combinations (e.g. small R 0 ), the available stockpiles last over the whole period of the intervention and the epidemic curve proceeds without a plateau (bold curves in grey). Inset: distribution of cumulative number of outpatients obtained from 1,000 random realizations. 1 :Parameter modifications are given in the following and terms in italics refer to terms in the InfluSim user interface. 2 however, will tax the social and economic structure and the population may not be willing or able to reduce contacts during the whole course of a pandemic wave.
For Figure 5 , we assumed that contact reduction measures (e.g. improved hygiene, wearing masks, or behavioural changes) could add up to reduce contacts by 20%. Studies on the SARS outbreak suggest some preventative effect of wearing masks [27] [28] [29] , but compliance, availability of masks and their effectiveness against influenza infection remain unknown factors. Stockpiling surgical masks for the population results in exorbitant high numbers and may not be feasible [30] and individual stockpiling may be impossible due to economic limitations, especially in crisis situations. Since the specific effects of such behavio-ral changes remain uncertain, we modeled their contribution as a general reduction in contact rates.
In contrast to SARS, we will not be able to rely on isolating hospitalized cases when a new influenza pandemic emerges. Using the standard parameter settings of InfluSim, we expect only a total of 0.7% of the population to be hospitalized. Even for the worst case scenario of the US Pandemic Preparedness Plan, where this value may be up to ten times larger [31] , the wide majority of infected individuals is never hospitalized. With influenza, we have to rely on self-isolation of moderately sick cases and of bed-ridden patients who stay at home. As these cases form the majority of infections and exert the highest force of infection, even a moderate reduction of contacts between them and the general population can substantially change the pandemic wave.
Time is of the essence when controlling infectious diseases that spread at high speed and thus, interventions are most effective in the beginning when only few people are infected. Only a timely application of antiviral drugs (even with limited supplies) and a quick implementation of contact reduction measures will notably protract the peak of the epidemic and substantially reduce its height in a pandemic influenza wave. Whereby the protraction of the pandemic wave is essential to win time while waiting for vaccine development and production, it is the height of the peak of a pandemic wave which can easily overtax general practitioners as well as hospitals and whole public health systems, and can lead to dangerous bottlenecks in basic and emergency medical care. Vaccinating a small fraction of the population with a pre-pandemic vaccine would have a similar effect on the course of the epidemic as reducing the basic reproduction number by the percentage of immunized individuals (e.g. by 10%).
The sensitivity analyses at the end of the Results section shows that the planning of intervention strategies must not only be based on single parameter values, but must also address their variability. More detailed analyses into this will be presented in a subsequent publication. Mathematical models like InfluSim should not only be used to predict a specific outcome, but also to explore best and worst case scenarios.
The author(s) declare that they have no competing interests. 
InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java and open access [16] , it operates platform independent and can be executed on regular desktop computers.
The model structure of InfluSim is represented by Figure 7 , with descriptions given below.
Susceptible individuals (S) are infected at a rate which depends on their age and on the interventions applied at the current time. Infected individuals (E) incubate the infection for a mean duration of 1.9 days. To obtain a realistic distribution of this duration, the incubation period is modelled in 7 stages yielding a gamma distributed incubation period with a coefficient of variation of 37.8%. The last 2 incubation stages are regarded as early infectious period during which patients may already spread the infection. This accounts for an average time of about half a day for the standard set of parameters.
After passing through the last incubation stage, infected individuals become fully infective and a fraction of them develops clinical symptoms ( Figure 8A ). The course of disease depends on their age and risk group: one third remains asymptomatic (A), one third shows a moderate course of disease (M, "moderately sick") and the remaining third a severe course of disease (V, "very sick"); a small fraction of the latter third shows an extremely severe course of disease (X, "extremely sick") and needs hospitalization. The rationale for distinguishing extremely sick cases is that only these can die from the disease and need to be hospitalized; in all other aspects, both groups of severe cases are identical. The period of infectivity is gamma distributed and depends on the course of the disease and on the age of the case. To allow for an infectivity which changes over the course of disease, we apply weighting factors which depend on the stage of infectivity. Our standard value results in an infectivity which is highest immediately after onset of symptoms and which declines in a geometric progression over time ( Figure 8B ).
Severe cases seek medical help on average one day after onset of symptoms, whereby the waiting time until visiting a doctor is exponentially distributed. Very sick and extremely sick patients who visit a doctor may be offered antiviral treatment. Very sick patients are advised to withdraw to their home (W) until the disease is over whereas extremely sick cases are immediately hospitalized (H). Death rates of extremely sick and hospitalized cases are age-dependent. Whereas asymptomatic and moderately sick patients who have passed their duration of infectivity are considered healthy immunes, very sick and extremely sick patients first become convalescent before they resume their ordinary life (gamma distributed with a mean of 5 days and coefficient of variation of 33.3%). Fully recovered patients who have passed their period of convalescence join the group of healthy immunes; working adults will return to work, and children again visit day care centres or schools.
Antiviral treatment: Severe and extremely severe cases who visit the doctor within at most two days after onset of symptoms are offered antiviral treatment, given that its supply has not yet been exhausted. Antiviral treatment reduces the patients' infectivity by 80 percent, the duration of being diseased by 25%, and the risk of hospitalization by 50 percent. Extremely sick patients, whose hospitalization is prevented by treatment, are sent home and join the group of treated very sick patients.
Contact rates in the general population can be reduced by increasing "social distance", by closing schools and day care centres, by cancelling mass gathering events, or by behavioural changes.
Isolation of cases reduces their contact rates. Contacts are not necessarily reduced by 100%, but between 0 and 100%, as specified by the user. Our standard scenario considers reductions of 10%, 20% and 30% for moderately sick cases, very sick cases (at home) and extremely sick cases (hospitalized), respectively.
For the mixing of the age classes, we employ a "whoacquires-infection-from-whom matrix" (WAIFW matrix) which gives the relative frequency of contacts of infective individuals by age. InfluSim assumes bi-directional contacts (e.g. children have the same total number of contacts with adults as adults with children). In order to match the user-specified basic reproduction number R 0 , the diseasespecific infectivity and the durations of infectivity in this matrix must be incorporated, resulting in the next generation matrix. This matrix is multiplied with a scaling factor chosen such its largest eigenvalue is equal to the chosen value of R 0 . The force of infection is given as the product of the number of infective individuals and the corresponding age-dependent contact rates.
At the start of the simulation, one infection is introduced into the fully susceptible population. To avoid bias between simulations, the initial infection is distributed over all age and risk classes. DNA Vaccines against Protozoan Parasites: Advances and Challenges Over the past 15 years, DNA vaccines have gone from a scientific curiosity to one of the most dynamic research field and may offer new alternatives for the control of parasitic diseases such as leishmaniasis and Chagas disease. We review here some of the advances and challenges for the development of DNA vaccines against these diseases. Many studies have validated the concept of using DNA vaccines for both protection and therapy against these protozoan parasites in a variety of mouse models. The challenge now is to translate what has been achieved in these models into veterinary or human vaccines of comparable efficacy. Also, genome-mining and new antigen discovery strategies may provide new tools for a more rational search of novel vaccine candidates. In spite of the success of vaccines in public health, there are still numerous pathogens, and in particular protozoan parasites such as Plasmodium falciparum, Trypanosoma sp., or Leishmania sp. against which there are still no effective vaccine. However, the discovery that the direct injection of plasmid DNA encoding foreign proteins could lead to endogenous protein biosynthesis and a specific immune response against it opened new perspectives in vaccine development. Over 15 years later, DNA vaccines have gone from a scientific curiosity to one of the most dynamic fields of research and may offer new alternatives for the control of infectious diseases [1] . Indeed, the first two DNA vaccines have been licenced, in recent years, to protect horses from west nile virus and salmons from infectious hematopoietic necrosis virus, confirming the usefulness of this biotechnology. We review here some of the advances and challenges for the development of DNA vaccines against two well-studied protozoan parasites, Leishmania sp. and Trypanosoma cruzi. Both belong to the trypanosomatidae family and are ranked among the three major protozoan parasites affecting humans. Leishmaniasis is a complex disease caused by at least 18 species of parasites from the Leishmania genus and transmitted to humans by hematophagous sandflies. With an estimated 12 million cases, it has a major public health impact in several regions, and in particular in India, Sudan, and Brazil [2] .
Clinical manifestations range from self-healing cutaneous lesion to fatal visceral form, and this variety can be attributed in part to the respective parasite species, and each presents specific relationships with the host and diverse mechanisms of pathogenesis [3, 4] , which represents an additional difficulty for the development of treatments and vaccines. On the other hand, T. cruzi is the agent of Chagas disease, which is present from southern Argentina to the southern USA. An estimated 16-18 million persons are infected in the Americas and close to 100 million people are at risk of infection. After a short benign acute phase (a few weeks) and a very long (several years) asymtomatic phase, about 30-40% of infected patients develop chronic chagasic cardiomyopathy and eventualy die of heart failure. Current chemotherapy relies on nitrofurans (Nifurtimox), or nitroimidazoles (Benznidazole). However, the usefulness of these drugs is limited by their reduced efficacy (mostly during the early stages of the infection), serious side effects, and the emergence of drugresistant strains of parasites, and new treatments are slow to develop [5] .
DNA vaccines induce a complete immune response against the encoded antigen. The exact mechanisms involved in this proccess are still poorly understood, and particularly the type of CD4 + and CD8 + effector and memory cells activated, and some of these aspects have been reviewed in detail elsewhere [1] . Apart from their immunogenicity and efficacy that will be discussed below, there are several features of DNA vaccines that make them very advantageous against tropical diseases. First, they are extremely safe as they do not contain any pathogenic organism that may revert in virulence. The major concern of genomic integration of the plasmid DNA has also extensively been studied in safety studies and found to be rather unlikely [6] . Additional safety issues such as anti-DNA antibodies or autoimmunity have also been addressed in a growing number of preclinical and clinical studies [7] , which confirmed the high safety of these vaccines. With respect to manufacturing, storage, and distribution, they also present major benefits in that the production process is the same for any DNA vaccine, which is not the case for other types of biologicals and vaccines, for which a specific protocol has to be developed for each. This makes production easy and costs will likely go down as this type of vaccines become mainstream and future technological improvements are implemented. Also, plasmid DNA is a very stable molecule, specially compared to recombinant or live attenuated vaccines, which would greatly facilitate storage and distribution of DNA vaccine in tropical settings with limited health infrastructure as the huge costs associated with the cold chain may be offset. Administration is also easy as simple IM or ID injections can be sufficient, and multiple plasmids can be combined for the elaboration of multivalent vaccines [1] . Overall, DNA vaccines may thus represent an ideally affordable alternative for disease control, which explains in part the growing interest in their development for the control of tropical parasitic diseases such as malaria, leishmaniasis, or Chagas disease.
As mentioned above, leishmaniasis is caused by at least 18 species of parasites with diverse relationships with the host and mechanisms of pathogenesis [3, 4] . Early studies of cross-protection between Leishmania species clearly showed that it is a complex problem, with infection by one species protecting or not from subsequent infection by another species, depending on the species and the order of infections. Most vaccine studies have thus been focusing on homologous protection, although a single vaccine able to protect against all pathogenic species would be ideal.
The correlates for protection have been extensively studied in the case of L. major, and contributed considerably to the development of the Th1/Th2 paradigm [8] . Thus, there is a general agreement that a Th1-type immune response, characterized by a high IFNγ and low IL-4 and IL-10 production, leads to control of L. major infection, while a Th2type immune response does not [8] . Antibodies may have an exacerbatory role [9] , but may also contribute to T cell responses [10, 11] . Both IFNγ producing CD4 + and CD8 + T cells seem to contribute to protective immunity, and induction of NO production by macrophages is central to parasite elimiation [12, 13] . While it was assumed for a long time that this Th1/Th2 paradigm was applied to all Leishmania species, it has become clear in recent years that each species has a distinct relationship with the host, different mechanisms of pathogenesis, and possibly different correlates for protection [3, 4] . Nonetheless, IFNγ production seems to be a general requirement, although not necessarily sufficient, for protection against most if not all Leishmania species.
The earliest DNA vaccine experiments against Leishmania used L. major GP63 antigen, which has been extensively used as a recombinant or peptide vaccine. Immunization with a plasmid encoding GP63 was able to induce a Th1-type cytokine profile and a significant reduction of lesion size after challenge of the immunized mice with L. major [14] [15] [16] [17] . Subsequent studies investigated DNA vaccines encoding a large variety of Leishmania proteins ( Table 1 ) and showed that many different DNA vaccines were able to induce a Th1 immune response, and confer variable degrees of protection as assessed by reduction in skin lesion size and/or parasite burdens in mouse models. However, given the large variety in experimental models and designs, it is difficult to compare the effectiveness of the different vaccines to induce a protective immune response. Nonetheless, it is clear from studies comparing different DNA vaccines that the nature of the antigen encoded by the vaccine is a key parameter for efficacy.
Also, a few studies provided interesting comparisons of the same antigens administered as recombinant protein or DNA vaccines and showed that the latter were overall more effective than their recombinant protein counterparts. Indeed, DNA vaccines were able to induce a stronger Th1 bias in the immune response, a longer-lasting immunity, and/or a better protection against disease progression [19, 31, 32, 35, 40, 44] . While most of these studies have used a rather artificial infectious challenge based on the injection via nonnatural routes of high parasite doses, an experimental system criticized by some authors, the superior efficacy of DNA vaccines was also observed using a low-dose intradermal challenge in the ear, which was proposed to more closely mimick natural infection [45] . In these studies, both DNA and protein vaccination were able to induce very similar level of short-term (2 weeks postvaccination) protection against infection with L. major, but only DNA vaccine was able to induce long-term (12 weeks postvaccination) protection [45] .
These results thus confirmed the strong potential of DNA vaccines against Leishmania, but also indicated that in most cases only partial protection was achieved. Prime-boost immunization protocols have been tested with various antigens to increase vaccine potency (Table 1) . They are based on priming the immune response with a DNA vaccine and boosting with the corresponding recombinant vaccine based on recombinant virus or protein (Table 1) . In some studies, such immunization protocol resulted in increased immunogenicity of the vaccines and better protection levels [26, 27] , but in others, DNA only remained the best formulation for optimum efficacy [32] . Nonetheless, a major drawback of such vaccine formulation remains its complexity, which may limit their practical use.
An alternative way to broaden vaccine immunogenicity and increase its efficacy has been to use combination of plasmids encoding various antigens. For examples, cysteine proteinase (CP) a and b DNA vaccines are not protective when used individually, but immunization with a combination of both plasmids induces long-term protective immunity [34] . Alternatively, gene fusion has also been successively used to achieve expression of an antigenic fusion protein from a single plasmid construct [33] . Overall, expression of several antigens mostly resulted in increased efficacy, but this also depended on the antigen combination [13, 22, 23, 41, 45] .
Most authors thus argue that a successful Leishmania vaccine is likely to be based on multiple antigens.
Immunization with large number of plasmids is also the basis for expression library immunization, a powerful but laborintensive strategy for vaccine discovery [46] , which has been used with Leishmania. Immunization of mice with L. major genomic expression library fractions was able to induce significant protection, but these authors did not pursue library fractionation further [47] . In another study, the identification of protective library subsets from an L. donovani amastigote cDNA library and their successive fractionation into smaller protective libraries lead to the identification of novel protective antigens [48] . Interestingly, most of the antigens identified would not have been predicted to be good vaccine candidates. Indeed, they were not surface or secreted proteins, neither stage-specific, but were intracellular and some very conserved such as histones, or ribosomal proteins [48] . Vaccine discovery is also the next logical step following the recent completion of the L. major genome sequencing [49] . In one approach, the random screening of 100 genes upregulated in amastigotes tested as DNA vaccine allowed the identification of 14 novel protective and 7 exacerbating antigens [50, 51] . Again, function and cellular localization would have been poor predictors of the protective efficacy of these antigens, as most were not predicted to be localized on the surface, but shared similarity with ribosomal proteins, cytoskeleton, or metabolic enzymes [51] . It is thus becoming increasingly clear that there is little rationale to limit Leishmania vaccine discovery searches to surface or secreted antigens. Rather, new criteria need to be considered for the rational identification of vaccine candidates as strategies based on such random screening cannot be applied to large genomes such as that of Leishmania, with over 8000 annotated genes.
An additional advantage of DNA vaccines is their potential as therapeutic vaccines, aimed at reinforcing or redirecting the immune response of an infected host to control disease progression [58] . The major advantage of this strategy in addition to its efficacy is that it relies on short treatment regimens, and it is thus an attractive alternative to chemotherapy, particularly in the case of Leishmania with so few chemotherapeutic options. Thus, administration of as little as two doses of a DNA vaccine encoding PSA-2 can control an ongoing infection with L. major in mice [59] . The therapeutic effect is due to a shift of the immune response towards a Th1 immune response [59] . Similarly, a DNA vaccine encoding L. donovani nucleoside hydrolase NH36 has therapeutic activity against murine visceral leishmaniasis caused by L. chagasi [60] . The simplicity of such treatment makes them very advantageous compared to chemotherapy. In addition, the fact that the same DNA vaccine can be effective for both the prophylaxis [40] and the therapy of Leishmania infection is thus very promising as this would provide a versatile tool for the control of this parasite.
As mentioned above, an added challenge to Leishmania vaccine development is the large number of species, as well as the variability within species. Indeed, studies on the polymorphism of leading antigens such as GP63 quickly revealed that it was a very polymorphic [61, 62] . Such polymorphism has important implication for vaccine development as it may limit their efficacy against variant strains of parasites or novel escape mutants, and thus restrict vaccine protection to a single species [63, 64] . Antigen polymorphism between multiple strains and species is thus becoming a major issue in many vaccine development studies [65, 66] . In the case of Leishmania, few DNA vaccines have been tested against mul-tiple species. LACK antigen, initially identified in L. major, and found to be very conserved between Leishmania species, can protect mice against L. major [20] and L. amazonensis [67] , but not against L. mexicana [23] , L. donovani [25] , or L. chagasi [24] . On the other hand, L. amazonensis nuclease protein P4 can protect against both L. amazonensis and L. major, but cross-protection requires a different formulation (IL-12 or HSP70 as adjuvant, resp.) [38] . In other studies, antigens from one species were used to induce protection against another species [31] , but the extent of crossprotection against various species was not investigated. More recently, a single formulation of L. donovani NH36 DNA vaccine was found to induce a very good protection against both L. chagasi and L. mexicana, suggesting that this DNA vaccine may be able to provide broad protection against various Leishmania species [40] . Importantly, no DNA vaccine has yet been tested against L. braziliensis, in spite of this species being responsible of most cases of cutaneous leishmaniasis in South America.
While all the above DNA vaccines were based on Leishmania antigens, an alternative approach has used antigens derived from sand-fly saliva. Indeed, it has been shown that sand-fly saliva can exacerbate Leishmania infection [68, 69] , and preexposure of mice to saliva components may be sufficient to induce protection against infection [70] . Thus, a number of salivary antigens have been tested as vaccines against Leishmania. Maxadilan is a potent vasodilator from sand-fly saliva and was found to be responsible of most of the exacerbatory effects of whole saliva on Leishmania infection [71] . Immunization with this antigen (as a recombinant vaccine) protected mice against L. major infection [71] . Other salivary components, such as Phlebotomus papatasi SP15, have been tested as DNA vaccines and found to protect mice against L. major and while the vaccine induced both humoral and DTH responses, protection seemed to be mostly accounted for by the latter, as B-cell deficient mice remain protected [72] . Thus, characterization of sand-fly salivary proteins may lead to the identification of new vaccine candidates [73, 74] . However, as for Leishmania antigens, salivary protein polymorphism remains an important issue and may limit the usefulness of such antigens as vaccine candidates [75, 76] .
Based on the success of many of these DNA vaccine studies in mice, a few vaccine candidates have been tested in additional animal models, possibly more relevants for the development of a veterinary or human vaccine (Table 2) . PFR-2 and KMP11 antigens were tested as DNA vaccines in hamsters, a highly susceptible animal model. PFR-2 was tested as protein, DNA, or DNA-protein immunization, and protection levels against L. mexicana varied greatly depending on vaccine formulation, route of immunization, and sex of the animals [52] . Also, contrary to mouse studies, protein vaccination seemed more protective than DNA only vaccination. However, as in mouse studies, heterologous prime-boost vaccination with DNA and protein seemed better than DNA only [52] . Another DNA vaccine encoding PapLe22 was found to be immunogenic in hamsters and decreased parasitemia after infection with L. infantum, but further assessment of disease was not performed [54] . Immunization with KPM11 DNA induced a mixed Th1/Th2 response, but was able to protect hamsters against visceral leishmaniasis caused by L. donovani [53] . In dogs, while several protein vaccines have been tested and a purified protein vaccine has now been licenced for veterinary use [77] , very few DNA vaccine studies have been performed. A heterologous prime-boost strategy using CPa and CPb DNA and protein was reported as immunogenic and protective [57] , but the study was of limited power given the reduced number of animals. In another study, dogs were immunized with a mixture of DNA vaccines encoding 10 different antigens previously tested in mouse models, and this immunization induced a very good immune response, with a high production of IFNγ [56] . However, evaluation of protection was limited to an acute in vitro assay [56] and further studies will be required to assess the potential of this vaccine in dogs. In spite of their limitations, these studies clearly showed that several DNA vaccines can induce a potent immune response in nonmurine animal models, and it is likely that a good level of protection can be achieved in these as well, provided the correct antigens and vaccine formulation are used.
Vaccine development against Chagas disease has been dramatically limited because of extensive debate on the mechanisms involved in this pathology [78, 79] . Indeed, some studies suggested that tissue damage was associated with the presence and replication of intracellular amastigotes, while others proposed that autoimmunity induced by parasite antigens mimicking host proteins was responsible for it. It was thus unclear if the immune response needed to be inhibited, to reduce autoimmunity, or stimulated, to eliminate the parasite.
It is now accepted that the presence of parasites in cardiac tissue is necessary to initiate and maintain the inflammatory response, and that therapeutic treatments or vaccines aimed at eliminating T. cruzi would limit or prevent the progression towards chronic chagasic cardiomyopathy [80, 81] . There is a growing consensus that protection against T. cruzi relies on a Th1 immune response and the activation of cytotoxic CD8 + T cells [82] [83] [84] [85] .
The first DNA vaccines to be tested against T. cruzi encoded an antigen from the well characterized trans-sialidase family of proteins. There are over 1400 members in this family, making it one of the largest protein families of the parasite, and they are very abundant surface proteins. Several studies have used different members of this family, such as TS or TSA-1 (Table 3 ) [84, [86] [87] [88] . Immunization with TS was found to induce significant antibody titers able to inhibit trans-sialidase enzyme activity, a strong DTH, and lymphoproliferative response [86] . This immune response was protective as determined by an increase in survival and a decrease in parasitemia. Immunization with TSA-1 DNA was found to induce a specific CTL response which also lead to a lower parasitemia and increased survival in both BALB/c and C57BL/6 mice [88] . As in Leishmania vaccine studies, a few authors addressed the question of comparing protein and DNA vaccines encoding the same antigen [90, 98] . In A/Sn mice, immunization with recombinant TS induced a higher antibody titer than TS DNA, but a comparable decrease in parasitemia. However, the DNA vaccine was unable to increase survival, which the author attributed to the strain of the mice used, since this DNA vaccine was protective in BALB/c mice [90] . On the other hand, immunization with recombinant CRP or CRP DNA induced a comparable Th1 immune response, but only the DNA vaccine was protective against infection [98] .
A number of other studies showed that DNA vaccines encoding various antigens could induce significant protection against T. cruzi infection, as evidenced by decreased parasitemia and improved survival of vaccinated mice (Table 3 ). In addition, a few studies also presented evidence of a reduction in cardiac tissue damage and inflammation at the histopathologic level [87, 97] . Furthermore, T cell analysis confirmed that protection relied on CD8 + T cells [84, 91] and recent studies showed that these cells were very rapidly activated following infection of mice immunized with DNA vaccines [101] . DNA vaccines based on defined T cell epitopes from TS antigen have also been tested and it was found that 6 Journal of Biomedicine and Biotechnology IM: intramuscular; CTL: cytolytic activity; −: no protection; +: little protection; ++: fair protection; +++: very good protection.
both CD4 + and CD8 + T cell epitopes were necessary and sufficient to induce a protective immune response [102] . Taken together, these data clearly demonstrated that vaccination did not result in increased pathology, as initially feared, but allowed at least partial control of disease progression, thus confirming the central role of parasite persistence for Chagas disease pathogenesis and opening the way to further assessment of DNA vaccines against T. cruzi. However, it has to be noted that many of the antigens tested belonged to the trans-sialidase family of protein, so that there is still little diversity in terms of the antigens tested as vaccines against T. cruzi (Table 3) .
Because protection induced by single antigen DNA vaccine remained partial, a number of studies have evaluated strategies to increase vaccine efficacy. These include the use of cytokine/chemokine encoding plasmids to potentiate the immune response induced by the vaccine, and two of the most studied molecules have been IL-12 and GM-CSF, which both were generally able to potentiate protection ( Table 2) . Alternatively, mixtures of plasmids encoding distinct antigens were used for immunization, and as mentioned above for Leishmania vaccines. For example, immunization of mice with plasmids encoding TS and ASP-2 proteins resulted in a specific immune response against both antigens and an increased protection against infection [97] . On the other hand, an immunization with a mixture of DNA vaccines encoding up to 6 proteins from the mucin family resulted poorly protective, while a mixture of up to 7 proteins from the TS family was protective, but not as much as a single antigen vaccine encoding the TS-like antigen ASP-2 [96] . Similarly, a mixture of DNA vaccines encoding ASP-1, ASP-2, and TSA-1 had a similar protective activity as TSA-1 alone [87] . The lack of efficacy of these multivalent vaccines may be attributed to the presence of shared or immunodominant epitopes since they have significant sequence similarity that may not have resulted in a broader immune reponse.
Heterologous prime-boost approach has also been evaluated and immunization with some combinations of DNA and recombinant TS was found to enhance Th1 immune response, but protection was not significantly different from that obtained with DNA alone [103] . Taken together, these studies suggest that additional strategies need to be investigated to potentiate DNA vaccine efficacy against T. cruzi.
Therapeutic administration of DNA vaccines to control an ongoing infection with T. cruzi may represent an additional Eric Dumonteil 7 alternative for Chagas disease control. The concept was demonstrated in mice acutely or chronically infected, and in both cases the administration of only two doses of DNA vaccine encoding TSA-1 or Tc24 antigens was sufficient to limit disease progression, as treated mice presented increased survival and reduced cardiac tissue damage, as assessed by histopathologic analysis [104] . A comparative study of different DNA vaccines identified Tc52 antigen as another therapeutic vaccine candidate, while DNA vaccines encoding antigens from the TS family previously found to be protective had no signifiacnt therapeutic effect [105] . It was found that therapeutic vaccination rapidly induced spleen cell proliferation, including IFNγ-producing CD4 + and CD8 + T cells, while the effects on cardiac tissue inflammation and parasite burden take longer to be detectable [106] . Importantly, in all these studies, therapeutic vaccination of T. cruzi infected mice did not result in an increased inflammatory reaction in the heart, confirming that it is safe to stimulate the immune response of T. cruzi infected mice and that attacking the parasite can lead to a reduction of pathology. These studies thus open very attractive perspectives for the control of T. cruzi infection, and further studies on the efficacy of DNA vaccines encoding other antigens and on the immune mechanisms underlying their therapeutic effect should provide clues for the optimization of this strategy.
As for any vaccine, the nature of the antigen used remains a key factor for vaccine efficacy, and there is still little variety in terms of antigens evaluated as DNA vaccine candidates against T. cruzi. Thus, a number of studies have aimed at identifying novel antigens through various strategies. The most classical approach has been the screening of cDNA libraries using antibodies and screening an amastigote library allowed the identification of a novel antigen Tcβ3, and two previously characterized ones, LYT1 and FcaBP/Tc24 [96] . DNA vaccines encoding these antigens induced variable levels of protection, the best one being LYT [96] . Alternatively, expression-library immunization, described above for Leishmania, was also tested with T. cruzi, and found to be immunogenic, but there was no attempt at fractionating the library or identifying protective antigens [107] . A likely reason is that such strategy may be too labor-intensive for large genomes/libraries, and its usefulness may be limited to pathogens with small genomes. The availability of T. cruzi genome sequence also opens new possibilities for antigen discovery. In one of the first studies using such resource, a combination of bioinformatics analysis were used to identify GPI-anchored or secreted proteins, and most of the identified clones were immunogenic as DNA vaccines [108] . Further studies may confirm the usefulness of these new vaccines to protect against T. cruzi infection. Nonetheless, as discussed above for Leishmania, the rationale for limiting antigen searches to surface proteins may not be totally relevant, and additional strategies should also be used to include unbiaised genome-wide surveys for antigen discovery.
As detailed in this review, there have been considerable advances in DNA vaccines against Leishmania and T. cruzi in recent years. Taken together, these studies clearly validated the concept of using DNA vaccines for both protection and therapy against these protozoan parasites in a variety of mouse models. While sterile immunity seems to be an irrealistic goal for either Leishmania or T. cruzi, a reduction in disease severity and in the development of the pathology seems clearly within the reach of DNA vaccines. Nonetheless, the relevance of such mouse models for the development of veterinary or human vaccines against these parasites has been challenged by some authors. The few DNA vaccine studies in nonmurine models of leishmaniasis suggest that some extrapolation may be feasible, but certainly not completely. Additional advanced preclinical studies of DNA vaccine candidates in nonmurine animal models such as rats, hamsters, dogs, or monkeys are thus warranted in the next few years, to further explore the immunology and efficacy of DNA vaccines against these parasites. As already observed in such studies for other pathogens, this will lead to the challenge of achieving in these species an immunogenicity of comparable level and protective efficacy as that obtained in murine models. However, advances in adjuvants, DNA vaccine formulation, and delivery systems are likely to contribute to such results [1, 109] .
Another major issue is that of antigen discovery, and while a number of DNA vacines tested so far against Leishmania or T. cruzi have shown promise, we are still unsure if these are the best possible antigens, particularly since these parasites have relatively large genomes, and only a limited variety of antigens have been tested. The availability of the genome sequences of these parasites will without doubt be a key resource for genome-wide screenings for new protective antigens. A key lesson from the initial studies reviewed here [48, 51, 108] , together with other similar antigen discovery studies, seems to be that cellular localization and protein function are poor predictors of the antigenicity and protective efficacy of a protein. Alternative criteria should thus be used so that potent vaccine candidates are not missed, and the important development of genome-mining and bioinformatic tools is providing new tools for a more rational search of vaccine candidates [110] .
To conclude, those DNA vaccines represent a promising approach for the control of Leishmania sp. and T. cruzi, and such vaccines would have a major impact in developing endemic countries. Thus the question does not seem to be if DNA vaccines can control these parasites, since many studies have clearly showed that this is the case, but how to translate what has been achieved in mouse models into veterinary or human vaccines of comparable efficacy.
This work was funded by Grant SEP-2004-C01-47122 from the Consejo Nacional de Ciencia y Tecnología (CONACYT). Estimating Individual and Household Reproduction Numbers in an Emerging Epidemic Reproduction numbers, defined as averages of the number of people infected by a typical case, play a central role in tracking infectious disease outbreaks. The aim of this paper is to develop methods for estimating reproduction numbers which are simple enough that they could be applied with limited data or in real time during an outbreak. I present a new estimator for the individual reproduction number, which describes the state of the epidemic at a point in time rather than tracking individuals over time, and discuss some potential benefits. Then, to capture more of the detail that micro-simulations have shown is important in outbreak dynamics, I analyse a model of transmission within and between households, and develop a method to estimate the household reproduction number, defined as the number of households infected by each infected household. This method is validated by numerical simulations of the spread of influenza and measles using historical data, and estimates are obtained for would-be emerging epidemics of these viruses. I argue that the household reproduction number is useful in assessing the impact of measures that target the household for isolation, quarantine, vaccination or prophylactic treatment, and measures such as social distancing and school or workplace closures which limit between-household transmission, all of which play a key role in current thinking on future infectious disease mitigation. The household is a fundamental unit of transmission for many directly transmitted infections. In addition, the household provides a ''laboratory'' within which key measures of transmission such as infectiousness, generation time and the effect of immunity or vaccination can be studied [1] . In recent years considerable effort has gone into understanding the dynamics of transmission within populations organised into households using mathematical models [2, 3, 4, 5, 6] . Most effort has gone into analysing the asymptotic behaviour of these models, elucidating the threshold levels of transmission required for infection to be self-sustaining, calculating final epidemic sizes, or predicting the impact of generalised or targeted interventions designed to reduce or eliminate transmission. In parallel, methods have been derived to estimate the parameters which govern transmission within the household from detailed case reports [7, 8, 9, 10] . However, scant effort appears to have been paid to how to apply household structured models to the analysis of epidemics, either retrospectively or in real time.
Concurrently, mathematical models have played an ever greater role in interpreting and responding to emerging pathogens. These models have typically been either of the ''simple but tractable'' variety which ignore or average over demographic structure and social mixing patterns [11, 12] or the ''complex computer simulation'' variety that capture many details of demographic structure and dynamics, but of whom the behaviour can only be determined by intensive numerical analysis [13, 14, 15] . The aim of this study is to develop methods of a perhaps ''slightly less simple but still tractable'' variety that capture some of the detail that micro-simulations have shown is important, but which can be rapidly applied (say on a daily basis) in an emerging outbreak situation, to inform policy. More specifically, the aim is to arrive at a method to estimate the key transmission and control parameters for a model of transmission within and between households from as few detailed observations as are likely to be gathered in the heat of a major outbreak.
The resulting analysis will still be based on major simplifications in respect to all the spatial and other social constructs that govern disease transmission, but less so than those based on the very simplest assumption of free, homogeneous mixing. In this context, it should be stated that even in the best, most robustly parameterised microsimulations, gross approximations are made in describing the fabulously complex web of human behaviour, and even they are only attempts to characterise the statistical properties of the system as a whole. Extensive effort is, and should continue to be, spent on identifying the conditions where different types of simplification (household models, static network models, spatial metapopulation models…) can and can't be justified, and in developing analytical approximations to describe disease transmission within such simplified structures.
Individual based simulations of influenza and smallpox pandemic spread and control, incorporating detailed information on population density, age structure, commuting patterns, workplace sizes and long-distance travel have highlighted the particular importance of the household as a fundamental unit of transmission [13, 14, 16, 17, 18] (and reviewed in [19] ). Pure household models have been used fruitfully to explore detailed policy options in a city-wide response to an influenza pandemic [20] . It thus seems a priori that household models are a natural starting point in terms of extending theory previously developed for the simplest assumption of homogeneous mixing.
The analysis presented here will focus on deriving new estimators for individual and household reproduction numbers, denoted R (t ) and R * (t ) respectively. The individual reproduction number R (t ) is defined roughly as the average number of people someone infected at time t can infect over their entire infectious lifespan; as I will show below, there are several ways of defining this more precisely. The household reproduction number R * (t ) is defined here as the average number of households a household infected at time t can infect [3, 6] . The individual reproduction number R (t ) rightly plays a privileged role in epidemiology, as it is a meaningful measure within any contact network. However, of the possible summary measures of epidemic progress, it is not necessarily the most useful. For example, for an emerging directly transmitted pathogen, such as pandemic influenza virus, public health interventions may target the household rather than the individual, enforcing household quarantine as well as offering antivirals to the household to limit transmission within the household. In such a situation, the household reproduction number R * (t ) is more directly related to the parameters which characterize the intervention, and is thus a better measure of the effect of these interventions. These quantities (R (t ) and R * (t )) share the two essential properties of reproduction numbers, namely that they increase when infectiousness increases and decrease when infectiousness decreases (monotonicity), and that they mark a threshold that separates exponentially growing epidemics (when R (t ).1 or equivalently R * (t ).1) from exponentially declining epidemics (when R (t ),1 or equivalently R * (t ),1) [3, 6] .
The structure of the paper focuses first on deriving estimators for individual reproduction numbers, then on household reproduction numbers and finally on examples of pandemic influenza dynamics and measles.
Though less well known than their compartmental counterparts (SIR, SIS, etc…), time-since-infection models offer a more intuitive starting point for modelling infectious disease transmission, and importantly for this application, they provide two other major advantages. First, it is typically easier to identify their key parameters, and second they more readily adapt to describe multi-level transmission (by multi-level, I mean here withinhousehold and between household). A disadvantage is that it can be harder to include heterogeneities. Nomenclature is confusing, since both types of model have their origin in the same classic paper of Kermack and McKendrick [21] , and both the SIR model and the simplest time-since-infection model are known as ''the Kermack-McKendrick model''.
The model, in the formalism chosen here, predicts the changing incidence rate I (t) as a function of calendar time t in terms of the transmissibility, denoted b (t, t ), an arbitrary function of calendar time t and time since infection t. b (t, t) typically reflects pathogen load, or perhaps more precisely pathogen shedding. It is commonly a single peaked function reflecting pathogen growth followed by immune suppression, or host death, but can be more exotic such as the double peaked profile associated with early and late transmission of HIV [22] , or the repeated peaks of malaria [23] . b (t, t) also reflects the effective contact rate between infectious and susceptible individuals, which can change during the course of a single infection, increasing for example if a person coughs or sneezes due to respiratory disease, or decreasing if a person takes to bed with illness, and during the course of the epidemic as public health measures are implemented. More discussions of the components (infectiousness and contact) of b (t, t) can be found in [24] . Because I am interested in outbreaks of emerging infections, I will not describe explicitly reductions in the susceptible population caused by the epidemic. Formally this corresponds to working in the infinite population limit. This assumption is not essential for this section however, since b (t, t) could also be thought of as incorporating the proportion of cases that are susceptible; the assumption becomes more important in the later sections on household models.
Mathematically, transmission is defined by a Poisson infection process such that the probability that, between time t and t+d, someone infected a time t ago successfully infects someone else is b (t, t)d, where d is a very small time interval.
This assumption then results in a prediction that the mean incidence I (t ) at time t follows the so-called renewal equation
This equation states that the number of newly infected individuals is proportional to the number of prevalent cases multiplied by their infectiousness. It may often be convenient (and realistic) to truncate the function b (t, t) at a time t m such that b (t, t) = 0 for all t.t m .
The asymptotic behaviour of incidence I (t ) is determined by reproduction numbers [21, 25] . Two intuitively defined reproduction numbers are the case reproduction number, which I denote R c (t ), and the instantaneous reproduction number, which I denote R (t ). The case reproduction number R c (t ) is a property of individuals infected at time t, and is the average number of people someone infected at time t can expect to infect. For a person infected at time t it is the total infection hazard from time t onwards, i.e.
While the case reproduction number has been widely used, it may also be worth considering a quantity which I call the instantaneous reproduction number R (t ), a property of the epidemic at time t. It is the average number of people someone infected at time t could expect to infect should conditions remain unchanged. It is given by
To illustrate the distinction between R c (t ) and R (t ), consider a situation where the transmission rate is abruptly reduced at a time t = t I . The instantaneous reproduction number R (t ), which estimates how many people one case would infect if circumstances were to remain fixed, would abruptly switch from a high to a low value at time t I . The case reproduction number R c (t ), on the other hand, estimates how many people each case actually infects. It will thus account for the fact that someone infected at time t,t I may spend part of their infectious period before and after the reduction in transmission which occurs at time t I and thus R c (t ) will smoothly transition from higher to lower values.
To derive simple estimating equations for R (t ), I consider the case where this function is separable, which corresponds to saying that the relative progression of infectiousness as a function of time since infection is independent of calendar time. In this case b (t, t) can be written as the product of two functions w 1 (t ) and w 2 (t), i.e. b t,t ð Þ~w 1 t ð Þw 2 t ð Þ ð4Þ
A counter-example might be when reactive patient isolation is introduced and acts to reduce infectiousness in late stage infection, in which case b (t, t) can't be decomposed in this way. For this type of situation, it may be reasonable to assume the b (t, t) can be decomposed separately in different stages of the epidemic, pre-and post-implementation of isolation measures, for example.
Since b (t, t) is a product, I can arbitrary normalise one or other of the functions w 1 (t) and w 2 (t), so without loss of generality, I choose w 2 (t) to have total integral 1, i.e. Ð ? 0 w 2 t ð Þdt:1.
The function w 1 (t) is equal to the instantaneous reproduction number R (t). The function w 2 (t) is then the distribution of how these infection events are distributed as a function of time since infection t. This is an idealised definition of the generation time distribution, which I denote w (t). Thus, infectiousness can be decomposed as the product of the instantaneous reproduction number and the generation time distribution, i.e.
The relationship between the idealised generation time distribution w (t) and the distribution of observed generation times can be rather complex for a number of reasons. First, infections are rarely observed, and thus must be either backcalculated or the generation times must be based on a surrogate such as the appearance of symptoms [1, 12] . Second, right censoring can cause the observed generation times to be shorter or longer than expected for a growing or declining epidemic, respectively [26] . Third, as apparent here, if the reproduction number R (t) changes due to depletion of susceptibles, changes in contact rates or public health measures, then this will also change the observed generation times for infectious individuals during that period of change. Thus the distribution w (t) is really intended as a measure of infectiousness which will correspond to generation times for an index case in an ideal large closed setting where contact rates are constant. It can be inferred from data on the timing of cases, as in [10, 13] .
Inserting (6) into (1) yields a novel estimator for instantaneous reproduction number
By substituting the decomposition (6) into equation (2) , a relation between the instantaneous and case reproduction number is obtained:
i.e. the case reproduction number is a smoothed function of the instantaneous reproduction number. Usually, incidence is reported as a discrete time series of the form I i incident cases reported between time t i and time t i +1, in which case the generation time distribution should be appropriately discretised into a form w i such that P n i~0 w i~1 . The estimators for the reproduction numbers become
and
Equation (10) was proposed by [12, 27] as a real time estimator of the reproduction number, while equation (9) was first used for analysing polio transmission in India [28] (based on the work presented in this manuscript).
While the case reproduction number is an intuitively appealing quantity, the instantaneous reproduction number estimated by equation (9) should also be considered for practical applications as it may suffer fewer problems of right censoring in an incompletely observed epidemic. Right censoring is a real problem in using the case reproduction number to track an epidemic in real time, since the estimator for R c (t) at time t is seen in equation (10) to rely on knowing the incidence at future time-points. An algorithm to deal with this issue was proposed by [29] , but switching instead to the instantaneous reproduction number estimated by equation (9) may be a simpler solution. Right censoring is not however the only complication associated with estimating reproduction numbers in practice, and is not completely absent from (7) due to the delay in detecting infections. Left censoring may also arise due to not knowing the baseline number infected if an epidemic has been unfolding for some time before observations are recorded. Finally, estimating the generation time distribution may not be straightforward.
Several strategies are possible to deal with the fact that one never observes infections, but rather as a time series of cases of the form C i , where case definitions could be based on symptoms, hospitalisation or seroconversion. One strategy, used in [12] , is simply to ignore this and use cases as surrogates of infection for estimation of both the generation time and the reproduction numbers. Often though, it may be possible to characterise a distribution of the time from infection to becoming a case, say j i where P n i~0 j i~1 . If a case is defined by symptoms then this would be the incubation period distribution. One can then backcalculate incidence as follows
A drawback of this approach is that the estimated incidence time series Iˆi will tend to be over-smoothed relative to the original time series I i . It also makes clear that there is still a problem of right censoring in an incompletely observed epidemic in the estimator of equation (9), though less than in equation (10) .
Statistical properties of these estimators are straightforward [12, 28] . One previously noted point [12, 28] is that because these estimators are essentially ratios of incidences, they can be used in cases where only a fraction of cases are observed, such as for polio where only a tiny fraction of infections lead to disease (of the order of 1 in 200), though the confidence intervals will change.
A special case applicable to many cases where surveillance is poor is when only the epidemic growth/decline rate r is known. In this case the incidence takes the form I (t) = I (0)exp (rt) and both estimators (7) and (8) for the reproduction number become
where the reproduction numbers are now expressed as a function of the exponential rate of change r. This is likely a useful formula, presented and studied in detail in [30] , where the links to earlier ecological and demographic modelling were also highlighted.
Much of the subsequent analysis will concern itself with deriving an equation equivalent to (12) for the household reproduction number R * (r).
The model defined above assumes that the function b (t, t) describes the ''natural history'' of infection in each infected individual. Before specialising to the model of household transmission, it is first worth considering the case where different individuals experience different ''natural histories'', defined here by the susceptibility to infection, and infectiousness after infection. I denote a vector of random variables X = {X1, X2, …} to describe factors which influence susceptibility or infectiousness. For example for the standard SEIR model of infection the random variables would be the durations of the latent period (L) and the infectious period (D), i.e. X = {L,D}. Let f (X ) denote the probability distribution of these random variables amongst new infections (taking into account differences in susceptibility), defined such that
where the integral is taken over the domain of the random variables. In other words, f (X ) is the proportion of new infections that have state X. Let b (X, t, t) denote the infectiousness profile of an individual with state X.
Assuming that all individuals mix homogeneously, then the transmission model defined earlier by equation (1) is generalised to
where I (X , t) is the incidence of infections with state X. I define the function K(t) to denote the integral
which clearly depends only on time t and not state X. The total incidence at time t is defined by the integral
By substituting equation (14), which can be rewritten as I (X, t ) = f (X ) K (t ), into equation (16), I obtain that K (t ) = I tot (t ) and thus that
I can now substitute (17) into (14) to obtain
Dividing both sides of this equation by f(X)yields an equation for the total incidence
If I define the average infectiousness as follows
then equation (19) can now be seen to be the standard Kermack-McKendrick model of equation (1), i.e.
In other words, in this model of an emerging infectious disease epidemic with heterogeneities in susceptibility and infectiousness, the dynamics of mean total incidence of infection is exactly equivalent to the basic model where the infectiousness is appropriately averaged using equation (20) . Once an expression is derived for the average infectiousness b (t, t), the results such as equations (9) or (12) can be used without further consideration of the heterogeneities in infectiousness or susceptibility.
Heterogeneities which are transmitted or preserved from one infection to the next, for example due to non-random mixing between different risk groups, a situation not considered here, lead to a more complex result. Some public health interventions such as isolation and contact tracing can induce such heritability even if it is not a basic property of the transmission process [31, 32] .
A useful exercise in applying this formalism (not elaborated here) is the derivation of standard formulae for the basic reproduction number as a function of the exponential growth rate r for the SEIR model [30] .
One approach to estimating household reproduction numbers is simply to switch perspective from individual to household, directly estimate the generation time distribution (times taken for one household to infect another) and incidence of infection of households, and apply the results of equations (9) or (12) to estimate reproduction number as a function of time, R * (t), or exponential growth rate, R * (r). Because, as I have shown, the linearised Kermack-McKendrick model is applicable even when susceptibility and infectiousness are heterogeneous, this method is acceptable despite the fact that households may be quite heterogeneous in size and in the number of people infected. One analogous situation where this approach has been used is in estimating farm-to-farm reproduction numbers in the 2001 UK foot-and-mouth virus epidemic [27] . However, unless specifically tailored to this task, it is unlikely the data will be collected in the requisite form for this approach to be used in the human household situation. Thus, in this section I explore the alternative approach of explicitly modelling transmission within and between households.
Homogeneous transmission models can be interpreted as twolevel hierarchical models, where the processes which guide the natural history of infection within the host are considered separate from those which drive transmission between hosts. The link between the two can be thought of as the function b (t, t) which translates the impact of changing processes within the host into changing infectiousness as a function of time since infection. The approach taken here to modelling household transmission is to study a three-level hierarchical model of transmission. The three levels are within-host, within-household, and between households. The natural history of infection is described by the individual infectiousness function b (t, t). I assume in this section that individuals are homogenous in infectiousness and susceptibility. I then use this to predict the course of epidemics within households, and derive a function b * (t, t * ) which describes the average infectiousness of a household towards other households as a function of the time since the household was infected, t * (from here-on, I use the starred symbols to denote properties of households, and un-starred symbols to denote properties of individuals). The basic idea behind this analysis is illustrated in Fig 1. To simplify the notation, and because the main aim of this section is to study the case of an epidemic growing exponentially, I consider the situation where infectiousness is independent of calendar time t. This could be relaxed, though only if variation in time is somewhat slower than the typical duration of infection within a household.
More specifically, the model assumptions are that:
N individuals are distributed into households, and mix randomly and homogeneously outside of their household;
N within a small time interval d, an individual who has been infected a time t ago infects a person at random in the population with probability b G (t )d;
N within this same time interval he or she infects each susceptible individual in his or her household with probability b L (t, n)d (this is allowed to depend on the household size n, since empirical evidence suggests such variation may occur [10] ); N the population is large, and the disease has low prevalence, so that the probability of a household being repeatedly infected is negligible;
N the functions b G (t ) and b L (t, n ) are proportional to each other as functions of the time since infection t.
As a result of the last assumption and of the discussion around equation (6), the infectiousness functions can be decomposed as b G (t) = R G w (t) and b n (n, t) = r n w (t), where R G is the average number of people each infected individual infects through random (non-household) contacts, w (t) is the generation time distribution for between household transmission, and r n is a parameter describing infection within the household whose interpretation will be clarified below.
I start by analysing the process of transmission within a single infected household of size n in terms of the functions r n and w (t). Consider first a household of size 2, where one individual is infected at time t * = 0. Given the Poisson process described by the assumptions listed above, the probability that the second individual
The probability that the second person is never infected is
The distribution of times of infection of the second individual, conditional on infection, is then
where 2L q 2 (t * )/Lt * is the rate of change of the cumulative probability of not being infected, i.e. the probability density of being infected at time t * , and the normalising factor 1-Q 2 is the total probability of being infected. The difference between w 2 (t * ) and the standard generation time distribution w (t) is a saturation effect, so that the second case tends to get infected earlier as the infectiousness of the index case (r 2 ) is increased. The infectiousness of the second individual towards other nonhousehold members of the population, conditional on his or her infection, and described as a function of the time t * since the infection of the household is thus the convolution of w 2 (t * ) and b G (t), so that the total infectiousness of the household is
Generalising this exact result to larger households involves some complications. Consider for example a household of size 3, where one individual is infected at time t * = 0. The probability that neither of the other two individuals is infected by the first individual at time t * is q 3 t à ð Þ~exp {r 3 Ð tà 0 w s ð Þds À Á directly analogous to the situation for households of size 2. However this is somewhat greater than the actual probability that they are not infected at all, since once one of these two is infected, they can also infect the other, and thus the probability that they each escape infection is somewhat less than Q 3 :q 3 ?
ð Þ~exp {r 3 ð Þ. To progress further with analysing this system, I propose to approximate the process by assuming that infections within a household can be approximately described by a discrete generation Reed-Frost model, i.e. where the probability of not being infected in each generation is (Q n ) m where m individuals are infected in the previous generation and Q n u exp (2r n ). Q n is the escape probability of each infectious-susceptible pair of individuals considered in isolation. In the formalism proposed by Ludwig, this corresponds to using infectious rank as a surrogate for infectious generation [33] . Dynamics are recovered by assuming the times between generations are described by the standard generation time distribution w (t).
The ordering of infection events has no influence on the final number of individuals infected [33] , and therefore this approximation will produce exact results for the final number of people infected in each household. Because of the possibility of ''later'' generations preceding ''earlier'' ones, as noted in the case of households of size 3 above, and because of ignoring the saturation effect present in equation (22) in terms of the actual generation times within households, this approximation will overestimate the time taken for individuals to become infected in the household. Because of the general form of the relation between generation time and reproduction number seen in equation (12) , this will result in over-estimates of the household reproduction number R * (r). To provide a counter-balancing under-estimate of R * (r), I also consider an alternative approximation obtained by assuming the same total number of cases as predicted by this Reed-Frost model, but where all cases are assumed to be infected by the first index case. This is not a formal lower bound, since in the limit of infinite infectiousness within the household, all members of the household will be infected simultaneously upon introduction of the infection into the household. I find however that even for the example of highly infectious measles virus (below), the under-approximation is sufficient to provide a practical lower bound.
The probability of different chains of infection within households can easily be computed from the assumed Reed-Frost model [2] . I denote pr( {m 1 , m 2 , …, m n }|n) the probability of a chain of infection occurring in a household of size n where m 1 index cases infects m 2 , who in turn infect m 3 tertiary cases and so on, up to a maximum of n generations of infection. It is an assumption of the where pr m iz1 j m 1 , . . . ,m i f g ,n ð
ÞBinomial
The second approximation is that the time taken for one infected to infect the next is distributed according to the standard generation time distribution w (t). The time at which someone in the (i+1) th generation of infection is infected is as a result drawn from the i th auto-convolution of this distribution, denoted here w [i] (t * ) and defined by the recursive convolution equation
which satisfies
. Consider now an individual in the i th generation of infection in the household, and consider this household at a time t * after the first index case was infected. This individual must have been infected at some earlier time s ( t * distributed according to the distribution w [i-1] (s). His or her infectiousness to others outside of the household will be given by b G (t * -s). Thus, by averaging over all possible values of s, the average infectiousness of such an individual in the i th generation is
Thus having averaged over all possible times of infection in the chain of transmission events in the household, infectious households are stratified by their size and by the number of cases in each generation. Using the notation defined earlier, I define the state vector X = {n, m 1 , …, m n } of variables which define the infectiousness and susceptibility of the infected household, where n is the household size and m i is the number of infected individuals in the i th generation of infection in the household. The infectiousness of a household with this state X towards other households, mediated by random mixing of individuals between households, is the sum of the infectiousness of all the individuals each given by equation (27) 
Given that this infection process involves random mixing of individuals outside their household, the distribution of sizes of households which get infected is the so-called size-biased household distribution. This is the distribution of sizes one obtains by sampling individuals at random in the population and recording the size of their household, as opposed to the more commonly recorded household size distribution which is obtained by sampling households at random. If k n denotes the household size distribution, then
is the size-biased household size distribution. Given a household of size n gets infected, the probability of a chain of infections is given by the Reed-Frost probabilities pr ({m 1 , …, m n }|n). The distribution of the random variables X = {n, m 1 , …, m n } at infection is thus f X ð Þ~k n : pr m 1 , . . . ,m n f g j n ð Þ ð 30Þ
The mean infectiousness of a household is Let m = S i m i be the average total number of cases in an infected household. The household reproduction number takes an intuitive and well known form derived in [3, 6] , expressed in terms of the parameter R G as follows:
i.e. the household reproduction number is the product of the expected number of infections in a household multiplied by the number of people each individual infects out of their household. The mean household generation time distribution (time for one household infecting the next) is
The mean generation time for households, T g * , can be expressed in terms of the individual generation time T g as
The generation time distribution w * (t * ) can be used for the previously defined estimators of reproduction numbers (7)-(12) using household incidence data or just exponential growth rates. The exponential growth rate r for an exponentially growing epidemic is the same whether measured for individual or household incidence. For an exponentially growing or declining epidemic, one obtains the estimator
Now consider the integration
where the first equality uses the definition of the auto-convolution, the second is a re-ordering of integrals, the third involves changing variables to u = t * -s, the fourth is a factorisation and the fifth arises by induction. The sixth uses the definition of the individual reproduction number R (r ) one obtains ignoring household structure from equation (12) . The household reproduction number can be expressed in terms of the individual reproduction number R (r ) as
Examination of equation (33) immediately reveals that the estimate for the number of people each person infects out of the household is
I have thus derived a simple analytic relation between the individual and household reproduction numbers. Both are approximations, ignoring the effects of local saturation on the generation time, which will tend to produce overestimates of the reproduction number. An alternative approximating to the household reproduction number, which provides an underestimate, is found when all secondary household cases are assumed to arise in the second generation, i.e. using equation (38) 
There are two reasons for considering household structure in analysing the pandemic influenza situation. First, influenza transmission is known to be concentrated within the household, and thus parameter estimates which ignore this heterogeneity are likely to be frail. Second, many public health policies for future pandemics are likely to be organised around the household. The net effect of social distance measures such as school and workplace closures and cancellation of social gatherings is effectively to reduce transmission out of households (and perhaps inadvertently to increase transmission within them). Furthermore, antiviral treatment and prophylaxis and quarantine measures are likely to be targeted at whole households rather than individuals (though restricting families with one suspect case to stay together without any other support is possibly undesirable) [16, 17, 20] . A number of studies have identified the parameters needed to estimate the household reproduction number for influenza [8, 10, 11, 17] . It is important to bear in mind that these parameters could be quite different in future pandemics, and thus that robust methodology may be more useful in responding to new outbreaks than numerical estimates obtained for past outbreaks. While it would be straightforward to use demographic data and exponential growth rates from earlier pandemics combined with interpandemic data on the transmissibility of influenza within households to obtain estimates of R * for historical pandemics, it has not been shown that the within household transmission parameters for inter-pandemic influenza adequately describe the pandemic situation, so I focus instead on providing illustrative examples using current demographic data (on the household size distribution from the UK) [34], and recent data on the transmissibility of influenza in modern households [10] .
The household size data from 2001is truncated to size 6, and I assume that all households of size 6 or greater have size exactly 6. The data are k 1 = 29% (i.e. 29% of households are single person households), k 2 = 35%, k 3 = 16%, k 4 = 14%, k 5 = 5% and k 6 = 2%. The size of the mean household is thus 2.38 (average size of households where households are sampled at random), while the household of the mean individual has size 3.06 (average size of household to which individuals belong, where individuals are sampled at random).
From the French influenza study [10] , I obtain maximum likelihood estimates of the within household transmission parameter of r n = 1.35/n 1.0 (which is consistent with the best fit to the Tecumseh data [8] of r n = 1.27/n 0.97 ). The former study followed seronegative households for a two week winter outbreak of seasonal influenza. The corresponding escape probabilities are Q 2 = 50.9% (i.e. the probability of not being infected by the other household member in a household of size two is 50.9%), Q 3 = 63.8%, Q 4 = 71.4%, Q 5 = 76.4% and Q 6 = 79.9%. On the scale of other infections, this places influenza as being approximately as infectious as mumps, but a lot less infectious than either varicella-zoster or measles [1] . By applying the Reed-Frost model to these data with this distribution of households, I obtain estimates of the average number of infections in each generation of infection of m 1 u 1, m 2 = 0.64 (i.e. the first index case directly infects an average of 0.64 people in his or her household), m 3 = 0.19, m 4 = 0.036, m 5 = 0.0037 and m 6 = 0.00021, and thus the estimate for the total expected number of cases in an infected household is m = S 6 i = 1 m i = 1.87, to be compared to the mean size of 3.06. These calculations are performed in Microsoft Excel 2007 using equation (25) .
There is not yet a consensus on the generation time of influenza [13, 14, 16, 30, 35] , with estimates ranging from 2.6 days in [13] to 5.3 days in [14] . I use a Gamma distribution with mean T g = 2.85 days and standard deviation 0.93 days, as reported in [30] .
Based on these data, I compare the predicted and simulated infectiousness of households in Fig. 2 , which shows the average over all households sizes and compares this to the final analytical approximation given by equation (31) for b * (t * ), and also the alternate approximation which considers all secondary infections to arise in the second generation of infection ; the simulations and the first approximation are clearly in good agreement. Individual based stochastic simulations were programmed using Berkeley Madonna, and are described in Appendix S1.
For the case of an exponentially growing epidemic, the estimates of the individual and household reproduction numbers, R and R * respectively, are shown in Figure 3 , along with the estimate of the number of people one person infects outside their household, R G . For R * , both the under-and over-estimating approximations are shown, along with estimates obtained from the simulated generation time distribution. As expected for this low-infectiousness scenario, the simulated values are closer to the overestimating approximation. The range between these approximations which bracket the true value is rather narrow, indicating that the method is predictive.
For the 1918 ''Spanish Flu'' H1N1 pandemic, the median growth rate in large US cities was r = 0.20 per day [30, 35] , with comparable estimates in the UK [17] . This value also serves as an upper estimate for the spread of the H2N2 pandemic virus in 1957 [17] . Based on this growth rate, the estimated individual reproduction number is R = 1.74, while the estimated household reproduction number is R * = 2.26, and thus the out-of-household reproduction number is R G = 1.21. Of course, households were bigger in 1918 than now, so that the actual value of R * was likely higher than this. These estimates would imply that a proportion 121/R * = 56% of between household transmission would need to be blocked to prevent epidemic spread. Figure 3 could provide a rough guide to the likely values of R * and R G for a new influenza pandemic where the rate of exponential growth can reliably be determined.
Consider someone who the index case in their household; they would be expected to infect R G = 1.21 people out of their household and m 2 = 0.64 within their household. This validates assumed proportions of transmission within and between households from earlier simulation studies [17, 20] . The sum of these is greater than R since the reproduction number R is an average over different generations of infection within the household. For this value, the estimate of R which takes into account local saturation effects was determined numerically to be R = 1.79. Fig 3 shows that for all values of r, numerically estimated values for R (r ) are close to the curve estimated from application of equation (12) which ignores local saturation effects.
As a final check of the method, epidemics within a community of 2,000 households were simulated using an individual based stochastic model (see Appendix S1). I choose R G = 1.21 as inferred from an epidemic growth rate of r = 0.20 per day, and the other parameters as described above. The exponential rate of growth was then re-estimated directly from the simulated incidence timeseries to be r = 0.19 (Figure 4) , close to the predicted value of r = 0.20. This provides further support for the validity of this method, especially since no restrictions were placed on re-infection of households within this small simulated community.
As noted above, influenza is relatively uninfectious compared to other common viruses. For a contrasting application of the method, I now focus on measles which was the most infectious of the pathogens studied in [1] . Measles also has a more peaked generation time distribution, so that generations of infection are more distinct, and to make the contrast with the influenza estimates yet greater, I also use demographic data on household size chosen from the national census in 1961, when household sizes were greater than they are now. This analysis is perhaps a little artificial when applied to measles, since a large proportion of the population will have immunity either due to past infection or vaccination with the live MMR vaccine. The principal motivation is to further test and illustrate the methods in a case where good data on the transmission dynamics within households are available. Stratification by household of the recent outbreaks of measles caused by decreasing uptake of the MMR vaccine could reveal whether household heterogeneities should have be accounted for in estimating the changing reproduction number of measles [36] .
The household size data from 1961 is truncated to size 6, and I assume that all households of size 6 or greater have size exactly 6. The data are k 1 = 14% (i.e. 14% of households are single person households), k 2 = 30%, k 3 = 23%, k 4 = 18%, k 5 = 9% and k 6 = 7%. The size of the mean household is thus 2.99 (average size of households where households are sampled at random), while the household of the mean individual has size 3.66 (average size of household to which individuals belong, where individuals are sampled at random).
Hope-Simpson reported susceptible-infectious escape probabilities of Q = 69.9% for mumps, Q = 39% for varicella, and Q = 24.4% for measles in under 15s [1] . The results were reported independent of household size, and were regarded as unreliable in over-15s. Based on applying the Reed-Frost model to the measles (t) ) is shown, as is the infectiousness of the typical infected household (denoted b * (t * )). This latter curve is obtained by simulating over 10,000 epidemics of transmission within households starting from one infected case. The two analytical approximations described in the text are also shown. ''Approx 1'' is the main approximation described, while ''Approx 2'' is the one obtained by assuming that all infections occur in the second generation of infection within the household. Parameters are as described in the main text, and the curves are arbitrarily scaled such that each individual infects on average one person outside of the household (i.e. R G = 1). doi:10.1371/journal.pone.0000758.g002 estimate with this distribution of households, I obtain estimates of the average number of infections in each generation of infection of m 1 u 1, m 2 = 2.01 (i.e. the first index case directly infects an average of 2.01 people in his or her household), m 3 = 0.50, m 4 = 0.020, m 5 = 0.00036 and m 6 = 0.0000031, and thus the estimate for the total expected number of cases in an infected household is m = S 6 i = 1 m i = 3.54, to be compared to the mean size of 3.66. Hope-Simpson also reported the intervals between linked cases in households using different case definitions [1] ; the intervals for what he regarded as the most reliable case definition, ''maximum rash''. These data is well described by a Gamma distribution (not shown). The maximum likelihood estimate of the generation time is T g = 10.5 days with standard deviation 2.4 days.
Based on these data, I repeat the simulations of the previous section on influenza but with parameters for measles in Figs 2, 3 and 4. Figure 2B shows that, as expected, the average infectiousness of a household is less well approximated by either approximation than for the much less infectious case of influenza. In this case, multiple peaks of infectiousness corresponding to generations of infection within the household can be clearly distinguished, and there are more cases in the second generation of infection than in the first.
In terms of the predicting of the household reproduction number R * , the method is still found to be strongly predictive (as evidence by the small gap between upper and lower estimate) and reliable (compared to numerical estimates). While in influenza, the simulations were close to the upper approximation, here they are closer to the lower approximation, as expected for the more infectious situation of measles transmission. Simulations of transmission within a community of households were again found in Figure 4B to validate the approach. The difference in the shape of the epidemic curve with influenza reflects the different shape of the generation time distribution, though the exponential growth rate is the same.
New methods were presented to estimate both the individual and household reproduction number during an epidemic. The new method presented for estimating the individual reproduction number relates closely to earlier work [12, 27, 30] , but provides an alternative and possibly simpler solution to the problem of incomplete observations during an unfolding epidemic [29] . It also provides an alternative and perhaps more satisfying solution than the incidence-to-prevalence ratio method [37, 38] to the problem of long generation time distribution infections such as HIV, where epidemiological circumstances can change substantially within the course of a single infection, and thus the case reproduction number represents too much of an average to convey secular changes in behaviour and transmission.
Nothing in this study challenges the central role of the individual reproduction number as an epidemiological measure; because the empirical measures of reproduction number proposed here and in [12, 27, 29] use incident observed cases as the base, all of the complication in defining the 'typical' or 'eigen' case for structured models discussed most clearly in [24] are neatly sidestepped. What this study does highlight is that much complexity is hidden in effectively defining and estimating the generation time distribution for a structured population. In the case studied here, generation times between individuals are shorter for within household transmission than between household transmission, particularly for more infectious pathogens, and this resulted in systematic biases associated with estimating the reproduction number while ignoring this effect, which were quite substantial in the case of highly infectious measles virus.
The methods presented for the estimation of household reproduction numbers were not affected by this problem in the same way. Analytical approximation were derived which bracketed estimates between a lower and upper bound, and numerical simulations showed the range within these brackets to be narrow. These approximations were shown to be robust, but it is worth noting that assumptions are made about the population mixing randomly out of their households and results are only valid in the scenario of an emerging pathogen where overall prevalence is low.
The usefulness of these methods is likely to be found in predicting and understanding the impact of household targeted infection control measures in an emerging epidemic. This actually covers a wide class of interventions since the household is a central living and administrative unit in most populations. Decisions regarding isolation, quarantine, vaccination and prophylaxis may often be made for entire households. Similarly school and workplace closures as well as restrictions on leisure activity can be thought of as trying to reduce between household transmission. Analytical approaches are also invaluable in calibrating and providing independent checks on more detailed individual based micro-simulations, such as [13, 17, 20] . Some control interventions require more subtle analyses; for example it has been shown that vaccinating whole households is not the most effective strategy for a given vaccine coverage rate, and that alternative strategies such as preferentially vaccinating larger households could be considered [39] .
Further avenues of research include studying the statistical properties of these estimators for different situations. The assumption made here, that individuals mix nearly homogeneously out of their household may be an appropriate approximation for describing transmission within a neighbourhood or even a city [20] , but ultimately one should also consider developing the estimators for more complex demographic situations such as a hierarchy of organisations (household, to village, to region, to country, etc…) or a more complex overlap of households, workplaces and regular social spaces. Also of interest is the study of intervention measures, particularly those that respond to the presence of a symptomatic cases; the measures of pre-symptomatic transmission presented in [25] clearly generalise to a household, but analytical results on the efficacy of isolation and quarantine are not evidently obtainable.
The estimators of the household reproduction number have been shown here to be robust on their own terms, but I have not addressed the issue of model misspecification, for example to inaccurate determination of the generation time distribution or to individual heterogeneity in infectiousness or susceptibility within households. Further scenarios could be explored both to test the method with different infections and to address the issue of model misspecification.
There are many cases where it may be desirable to quantify household transmission, but where a degree of natural or vaccineinduced immunity may be present in the population, a problem not addressed here. In considering these more complex situations, while it may not be possible to obtain analytic forms for the infectiousness of a household, numerical forms can usually be obtained quickly and still offer benefits over full individual based micro-simulations in easily exploring a wide range of parameters.
Finally, the likely practical benefits of estimating household transmission parameters in an emerging epidemic need to be clearly established and communicated, and the most effective ways to enhance data collection protocols to allow their rapid estimation need to be identified.
Appendix S1 Description of the simulations Found at: doi:10.1371/journal.pone.0000758.s001 (0.08 MB PDF) Natural Killer Cells Promote Early CD8 T Cell Responses against Cytomegalovirus Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-α/β production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-α administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity. The development of antiviral immune responses involves the orchestration of a complex network of innate and adaptive immune cells to promote health over disease. Natural killer (NK) cells, plasmacytoid dendritic cells (pDCs), CD11b and CD8a conventional dendritic cells (cDCs), B cells, and CD8 T cells have all been demonstrated to be important for the generation of protective immunity to various viral infections [1] [2] [3] [4] [5] [6] . However, how the antiviral defense as a whole is coordinated, and in particular how the functions of different types of immune cells impact the shaping of the global immune response to viruses in vivo, is not thoroughly understood.
The importance of an efficient NK cell response for the promotion of a favorable outcome to viral infections has been demonstrated in both mice and humans [2, 7] , and the rapid activation of NK cells after infection is a hallmark of their potency as innate immune system effectors [2] . Increasing evidence supports the idea that optimal coordination of immune responses involves an intricate relationship between NK cells and other innate leukocytes. For example, several reports have documented the importance of NK/DC ''crosstalk'' for the reciprocal activation of these cell types and in the promotion of antitumor immunity as recently reviewed [3, 8] . Others have shown the involvement of macrophages as an intermediary in the activation of NK cells via Va14i NK T cells [9] . NK cells have also been shown to have the capacity to interact with neighboring NK cells as well as T cells to stimulate cellular proliferation [10, 11] .
pDCs were initially identified in humans and mice based on the unique ability of these cells to secrete enormous amounts of IFN-a/b early in response to viral challenge [12] . pDCs respond in this way as a consequence of their ability to recognize molecular signatures of viruses in a manner that is independent of pDC infection [12, 13] . Early during the course of murine cytomegalovirus (MCMV) infection, pDCs are the major producers of interferons a and b (IFN-a/b) [13, 14] , which are critical for host survival [15, 16] . IFN-a/b have direct antiviral effects because they can inhibit viral replication in infected cells as well as convey to noninfected cells defense mechanisms that protect them from viral infection. IFN-a/b also mediate a variety of immunoregulatory functions, either activating or inhibitory [17, 18] . They contribute to the proliferation of NK cells and induce them to express functional cytotoxic granules, promote cDC phenotypic and functional maturation, and can help to activate antiviral CD8 T cells. In contrast, depending on the context, IFN-a/b can also compromise the immune response of the host by inhibiting DC differentiation [19] or by directly leading to the attrition of CD8 T cells [20, 21] . Indeed, different viruses have recently been suggested to induce a profound immunosuppression in the host by inducing overwhelming levels of IFN-a/b [19] . The importance of the host genotypes in the efficiency of IFN-a/b antiviral functions in the context of the infection with specific viruses are well documented [22, 23] . In contrast, it is not known whether naturally occurring differences in the interactions between a virus and its hosts exist that may shape IFN-a/b responses by specifically dampening the immunosuppressive functions of these cytokines that are detrimental to the host and beneficial to the pathogen. The identification of such conditions will help to better understand the physiopathology of viral infection and may lead to the development of innovative treatments to fight these infections.
In the early phase of MCMV infection there is a tight temporal relationship between the activation and exertion of effector functions between NK cells and pDCs because the production of IFN-a/b by pDCs promotes NK cell proliferation and cytotoxicity [13, 24] . However, the impact of NK cell functions on the pDC response to MCMV remains unexplored. The goal of this study was therefore to evaluate the effect of efficient NK cell responses on pDC IFN-a/b production and on the development of the adaptive immune responses of the host. C57BL/6 mice are able to mount NK cell responses that can control MCMV replication very efficiently, due to their expression of the Ly49H activating receptor, which endows NK cells with the ability to recognize and kill viral-infected cells expressing the viral ligand m157 (reviewed in [1] ). Although the NK cells from BALB/c and other strains of mice lacking the Ly49H gene are strongly activated for the acquisition of the cytotoxic machinery and for the production of IFN-c early after MCMV infection, they fail to recognize and kill infected cells and can therefore be considered inefficient. Inbred mouse strains that are resistant or sensitive to MCMV infection in an NK cell-dependent manner differ in many other immune parameters, including major histocompatibility (MHC) haplotype as well as DC subset frequency and function [25] . Therefore, rigorous evaluation of the impact of NK cell responses on pDC and adaptive responses requires comparison between mice of the same genetic background differing only in the ability of their NK cells to control the infection. For this, we took advantage of the fact that the Ly49H/m157 activation axis is sufficient for NK cell control of MCMV when introduced into the BALB/c genome [26] . The response of pDCs and CD8 T cells to MCMV were therefore compared between BALB/c mice (Ly49H À ) and animals on the BALB/c background that are congenic for the C57BL/6 natural killer complex and in particular for Ly49H (C.B6-Klra8 Cmv1-r /UwaJ mice, hereafter named Klra8 [27] ). This system thus provides a unique model to study antiviral immune responses in vivo in the context of both efficient (Ly49H þ ) and inefficient (Ly49H À ) NK cell activation, in the absence of any broad defect in NK cell development or activity that may affect the homeostasis or functions of other immune cells. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identified the underlying mechanismthe ability of NK cells to limit pDC IFN-a/b production to levels not immunosuppressive to the host-which itself results from the early control of viral replication.
Ly49H triggering by m157 is required for the proliferation and accumulation of NK cells between days 3 and 7 after MCMV infection [28] . Therefore, we first used NK cell expansion as an indicator to show that Klra8 and BALB/c mice generate an NK cell response of different quality to MCMV infection ( Figure S1A ). We next compared the ability of Klra8 and BALB/c mice to control early viral load. Consistent with reported differences in viral load seen between mice lacking or possessing the Ly49H gene [26, 29, 30] , there was a substantially higher viral burden within the spleens of BALB/c mice when compared to those of congenic Klra8 animals ( Figure S1B ). In addition, NK cell depletion in Klra8 mice increased viral replication to levels similar to those observed in BALB/c animals ( Figure S1C ). Thus, these data confirm previous reports demonstrating that Klra8 mice mount a strong NK cell response that is able to efficiently control MCMV replication, in contrast to BALB/c animals.
To evaluate the impact of efficient antiviral NK cell activity on the induction of IFN-a/b, we first examined the levels of the cytokines present in the serum of Klra8 and BALB/c mice. The serum levels of IFN-a/b were dramatically lower (100fold) at the peak of the response (day 1.5), and decreased at all 
To fight viral infections, vertebrates have developed a battery of innate and adaptive immune responses aimed at inhibiting viral replication or at killing infected cells. These responses include the early production of innate antiviral cytokines, especially interferons a and b (IFN-a/b), and the activation of cytotoxic lymphocytes such as the innate natural killer (NK) cells and the adaptive CD8 T cells. While critical for antiviral defense, cytokine or CD8 T cell responses can be detrimental or even fatal to the host when deregulated. Therefore, we need to better understand how the different arms of antiviral immunity are regulated. In particular, NK cells are proposed to play a protective role in a variety of viral infection in humans, but the underlying mechanisms remain poorly understood. Here, in a mouse model of cytomegalovirus infection, we demonstrate that NK cells prevent an excessive production of IFN-a/b and promote more efficient antiviral CD8 T cell responses. We thus show that NK cells can help promote health over disease during viral infections by regulating both innate and adaptive immune responses. It will be important to examine in humans whether NK cells control innate cytokine production to prevent immunopathology and to promote adaptive immunity against herpesviruses, HIV-1, influenza, or SARS.
other times points examined, in Klra8 mice as compared to BALB/c mice ( Figure 1A) . The serum levels of other innate cytokines were also significantly lower in Klra8 mice, including IL-12p70 ( Figure 1A ) and TNF-a (unpublished). Similar results were observed when comparing serum cytokine levels between control-treated and NK cell-depleted Klra8 mice ( Figure S2 and unpublished data). Thus, while the systemic production of IFN-a/b and of other innate cytokines in response to MCMV infection is very high in susceptible animals, it is greatly reduced in the presence of an NK cell response that controls viral replication early and efficiently.
In MCMV-infected 129 or C57BL/6 immunocompetent animals, most of the systemic production of IFN-a/b and a significant proportion of that of IL-12p70 comes from pDCs [13, 14, 31] , which are not productively infected [13, 32] , and does not come from infected cells ( [13] and unpublished data). To our knowledge, the contribution of pDCs to IFN-a/b or other innate cytokine production has not been assessed in animals on a BALB/c genetic background, which are the most susceptible to MCMV infection. cDCs infected with MCMV in vitro have been reported to produce high levels of IFN-a/b [33, 34] , and more generally, any virus-infected cell could theoretically produce these cytokines. Therefore, the increase in the systemic levels of IFN-a/b observed in BALB/c animals could result either from a high activation of pDCs or from a more significant contribution to this function from other cell types or from the high numbers of infected cells in these animals [33, 34] . Therefore, we next compared the activation of pDCs for IFN-a/b or IL-12p70 production between BALB/c and Klra8 animals by intracellular staining for these cytokines on ex vivo isolated splenocytes ( Figure 1B ). Less than 1% of the pDCs were found to produce IFN-a/b or IL-12 in Klra8 as compared to 10%-12% in BALB/c mice. Consistent with previous reports on 129 or C57BL/6 animals [14, 35] , pDCs were the sole producers of IFN-a/b at all the time points examined in BALB/c and Klra8 mice (unpublished data). Both pDCs and cDCs contributed to the production of IL-12 in Klra8 mice. However, the high IL-12p70 serum levels observed in BALB/c mice could solely be attributed to pDCs ( Figure 1B and unpublished data). Therefore, a very high activation of pDCs for innate cytokine production was observed in BALB/c mice, which was drastically reduced in congenic animals endowed with efficient antiviral NK cell functions.
To further link the early viral load and the level of IFN-a/b production by pDCs, we performed infections of BALB/c and Klra8 mice with 5-fold serial dilutions of viral inoculums, leading to challenge doses from 10 3 to 1.25 3 10 5 pfu/mouse. Systemic IFN-a titers in these animals were measured by (B) Splenic leukocytes were isolated from Klra8 and BALB/c mice and analyzed for the frequency of IFN-a/b þ pDCs and IL-12 þ pDCs directly ex vivo. Numbers in dot plots represent the percent of IFN-a/b þ and percent of IL-12 þ cells within the total pDC population. One representative animal from groups of three mice for day 1.5 post-MCMV infection is shown. Graphs represent the total numbers of IFN-a/b þ pDCs and IL-12 þ pDCs present in the spleens of Klra8 and BALB/c mice on days 0, 1.5, 2, and 3 post-MCMV infection. Results are expressed as mean 6 SD of three mice per group and in ten thousands of cells. One experiment representative of three for (A, B) is shown. *p 0.05; **p 0.01; § ¼ not detected. doi:10.1371/journal.ppat.0030123.g001 ELISA on serum samples, and the numbers of pDCs producing IFN-a/b by flow cytometry ( Figure S2 ). The results clearly showed that the amount of IFN-a/b secreted by pDCs increases dramatically at high viral dose inoculums in Klra8 mice to levels similar to those observed in BALB/c animals. A 5-fold difference in the virus inoculums from 5 3 10 3 to 2.5 3 10 4 pfu/mouse is sufficient to switch on IFN-a/b production by pDCs in Klra8 mice from very low levels to nearly maximal levels similar to those observed in BALB/c animals. At low dose viral infection (10 3 pfu/mouse), BALB/c mice are still activated for nearly maximal pDC cytokine production. Thus, BALB/c and Klra8 animals are confirmed to exhibit dramatically different levels of pDC IFN-a/b production over more than a 10-fold range of viral inoculums. These data also show that the pDCs of Klra8 mice are able to produce high levels of IFN-a/b under conditions of stimulation with high viral inoculums. Moreover, NK cell depletion in Klra8 mice infected with low virus inoculums was also shown to lead to a dramatic increase in pDC IFN-a/b production, almost to the levels observed in BALB/c animals ( Figure S2 ). Altogether, these data thus show that the ability of the host to control MCMV replication early through NK cell responses limits the activation of pDCs and therefore prevents the induction of very high systemic levels of IFN-a/b and IL-12. These data also demonstrate that the pDCs of Klra8 mice are not intrinsically deficient for IFN-a/b production in response to MCMV infection.
High systemic levels of IFN-a/b production have been shown to lead to the ablation of cDCs [19] and to the attrition of both antigen-specific and bystander CD8 T cell populations during infection with lymphocytic choriomeningitis virus (LCMV) [20, 21] . We therefore hypothesized that the high systemic levels of IFN-a/b induced during MCMV infection in the context of inefficient NK cell activity could lead to the ablation of cDCs and may impinge on the development of antiviral CD8 T cell responses. Indeed, it has been previously reported that MCMV infection leads to a severe ablation of CD8a cDCs in BALB/c mice that is prevented by efficient antiviral NK cell functions in C57BL/ 6 animals [36] . However, the cause for this ablation of cDCs in BALB/c animals has not been identified, and the effect of this ablation on the development of antiviral CD8 T cells has not been evaluated. Thus, we next compared the numbers of DC subsets and the activation of antiviral CD8 T cells at different time points after infection between Klra8 and BALB/c mice.
We first confirmed that pDC numbers remained at homeostatic levels in BALB/c mice throughout the early phase of MCMV infection, while a severe ablation of cDCs occurred for both the CD11b and CD8a subsets ( Figure 2 ). In contrast, in the presence of an efficient NK cell response, in Klra8 mice, both the cDC and pDC compartments were preserved, consistent with the previous observations reported in C57BL/6 animals [36] .
We next compared the kinetics of CD8 T cell expansion between Klra8 and BALB/c mice ( Figure 3A ). We observed a significant increase in total CD8 T cells in the spleen at day 4 post-infection in Klra8 mice but only later in BALB/c animals. We investigated whether this accelerated expansion of CD8 T cells was a result of a faster activation of antiviral effector CD8 T cells. We first analyzed total splenic CD8 T cells for the expression of an O-glycosylated, activation-associated isoform of CD43. The 1B11 monoclonal antibody (mAb) specifically recognizes this isoform of CD43 on CD8 T lymphocytes [37] and identifies the subset of effector cells capable of IFN-c production and potent cytolytic activity ex vivo during the acute phase of a variety of viral infections [38] [39] [40] [41] [42] . We observed the appearance of CD43 hi effector CD8 T cells starting at day 4 post-infection in Klra8 mice but only later in BALB/c animals ( Figure 3B ). We then used MHC class I tetramers loaded with a peptide derived from the IE-1 protein of MCMV in order to quantify the subset of antiviral CD8 T cells specific to this epitope ( Figure 3C ). We observed a faster expansion of the anti-IE-1 CD8 T cells in Klra8 mice, similar to what was observed for total or effector CD8 T lymphocytes. Similar results were obtained when analyzing the CD8 T cell response against another viral peptide, derived from protein m164 (unpublished data). Conversely, and in Splenic leukocytes were isolated from Klra8 and BALB/c mice and analyzed for the frequency of pDCs (120G8 þ CD11c int ), CD11b cDCs (120G8 À CD11c hi CD8a À ), and CD8a cDCs (120G8 À CD11c hi CD8a þ ) within the DX5 À and TCRb À population. Numbers in dot plots represent percent pDCs, percent CD11b cDCs, and percent CD8a cDCs within the total splenocyte population for one representative animal from groups of three mice for days 0 and 2 post-MCMV infection. Graphs represent the total numbers (in millions) of pDCs, CD11b cDCs, and CD8a cDCs present in the spleens of Klra8 and BALB/c mice on days 0, 1.5, 2, and 3 post-MCMV infection. Results are expressed as mean 6 SD of three mice per group. One experiment representative of three is shown. *p 0.05; **p 0.01. doi:10.1371/journal.ppat.0030123.g002 accordance with what has been previously reported in C57BL/ 6 mice depleted of NK cells by antibody treatment [43] , we observed accumulation of effector CD8 T cells in BALB/c mice at later time points after challenge.
In order to investigate the effector potential of the MCMVspecific CD8 T cells observed at day 4 post-infection in Klra8 mice, we measured different parameters associated with the protective functions of antiviral CD8 T cells, namely their proliferation, by monitoring the expression of the marker Ki-67 ( Figure 4A ), their capacity to produce IFN-c in response to antigen-specific restimulation in vitro using intracellular staining ( Figure 4B ) and ELISPOT ( Figure S3 ), and their cytotoxic potential by measuring antigen-specific target cell killing in vivo ( Figure 4C ). For each of these parameters, we observed an accelerated acquisition of effector functions by CD8 T cells from Klra8 mice when compared to BALB/c animals.
As Klra8 and BALB/c mice differ for the whole NK cell locus, which includes genes expressed in several leukocyte subsets, we sought to ensure that the differences observed in the activation kinetics of CD8 T cells between these two mouse strains i) were not due to intrinsic differences in the reactivity of CD8 T cells and ii) were dependent on Ly49Hmediated NK cell activation. This was achieved by showing that no accelerated expansion of CD8 T cells occurs in Klra8 animals i) when the antibacterial responses of Klra8 and BALB/c mice are compared during Listeria monocytogenes infection ( Figure S4 ), or ii) in response to MCMV infection when Klra8 mice are depleted of NK cells ( Figure 5A ), or when the CD8 T cell responses of Klra8 and BALB/c mice are compared during infection with a Dm157 virus as opposed to infection with wild-type or revertant viruses ( Figure 5B ). Moreover, the impact of efficient NK cell activity on the development of antiviral CD8 T cell responses was confirmed in mice of another genetic background, as a delay in antiviral CD8 T cell activation was observed in B10.D2 animals deficient for Ly49H-mediated NK cell activation due to the inactivation of the associated adaptor molecule DAP12 (B10.D2.DAP128), as compared to wild-type controls ( Figure  5C ). Altogether, these data show that efficient NK cell responses to a viral infection accelerate the development of effector antiviral CD8 T cell responses.
To determine whether the dramatic reduction of pDC IFN-a/b production by NK cell functions in Klra8 mice could in part account for the ability of these mice to preserve an intact cDC compartment and to mount early CD8 T cell responses, we examined whether exogenous administration of the cytokines in these animals could directly impact the initiation of cDC and CD8 T cell responses despite efficient NK cell-mediated control of viral load. MCMV-infected Klra8 mice were treated with recombinant mouse IFN-a as described in Materials and Methods, and their DC or CD8 T cell compartments were compared to those of controltreated Klra8 and BALB/c mice ( Figure 6A ). The treatment of Klra8 mice with IFN-a indeed led to a drastic reduction in both CD11b and CD8a splenic cDCs, but not in pDCs, when compared to their control-treated Klra8 counterparts. Likewise, IFN-a treatment induced a delay in the expansion of total, CD43 hi , and IE-1 Tet þ CD8 T cell numbers ( Figure 6B ).
Of note, the IFN-a treatment administered to the Klra8 mice did not significantly change the level of viral replication in these animals. Very low but detectable levels of infectious viral particles were observed in both control-treated and IFN-a-injected Klra8 mice (1.9460.35 versus 1.9260.11 log pfu/spleen), which contrasted sharply with the high viral replication observed in BALB/c animals (5.260.04 log pfu/ spleen). These results demonstrate that exogenous injection of IFN-a in Klra8 mice is sufficient to decrease the numbers of cDCs and to ablate early antiviral CD8 T cell responses, and, therefore, that excessive levels of IFN-a can have a direct negative impact on antiviral immune cell responses in a manner that is independent of the level of viral replication in the host. The possibility remains that other innate cytokines, such as IL-12 and TNF-a, which are produced at much higher levels in BALB/c as compared to Klra8 mice, may also bear some contribution to this function, in a synergistic or redundant manner with IFN-a/b. In any case, our data strongly suggest that the ability of Klra8 mice to preserve an intact cDC compartment and to mount early CD8 T cell responses is in part due to their ability to control viral replication very early without the need for the host to produce high systemic levels of IFN-a/b. Altogether, our data thus identify how naturally occurring differences in the interactions between a virus and its host can tilt the balance between the various functions of IFN-a/b, and eventually other innate cytokines, towards conditions promoting the induction of early adaptive immunity, rather than the development of a state of transient immunosuppression.
We next aimed at further understanding the link between NK cell functions, IFN-a/b production, and downstream effects on DCs and CD8 T cells. As BALB/c mice have a much higher viral burden after infection with MCMV, it seemed plausible that the difference in viral load between Klra8 and BALB/c mice could have an impact on the intensity of the DC and CD8 T cell responses. In order to test the impact of the extent of viral replication in vivo on pDC cytokine production and on antiviral CD8 T cell activation, independently of the function of NK cells, we sought a strategy to control viral replication efficiently in BALB/c mice with a tightly controlled timing and magnitude. To achieve this, we utilized an MCMV (DN-SCP-MCMV) that is genetically engineered so that its in vivo replication can be effectively arrested by the administration of doxycycline [44] .
Using this system, we were able to design a doxycycline treatment protocol which, in BALB/c mice, closely mimics the extent and the kinetics of efficient NK cell-mediated control of viral replication observed in Klra8. Indeed, under the experimental conditions selected, the viral burdens in Klra8 mice and in DN-SCP-MCMV-infected, doxycycline-treated BALB/c mice were comparable at the different time points examined, and much lower than in untreated DN-SCP-MCMV-infected BALB/c animals ( Figure 7A ). The reduction in viral burden in doxycycline-treated DN-SCP-MCMVinfected BALB/c animals led to a significant, strong decrease in the serum levels of the pDC-derived cytokines IFN-a and IL-12p70 ( Figure 7B ) and allowed the maintenance of cDCs ( Figure 7C ). Moreover, we observed an accelerated activation Figure  7D ). The treatment of DN-SCP-MCMV-infected Klra8 mice with doxycycline had no effect on any of the parameters tested (unpublished data). Thus, our data indicate that, in Klra8 mice, the ability of NK cells to dampen innate cytokine production by pDCs and thus to promote optimal conditions for the initiation of antiviral cDC and CD8 T cell responses result from their exquisite capacity to control viral replication early and efficiently after infection.
The results from this study demonstrate that efficient NK cell responses promote the accelerated generation of effector antiviral CD8 T cells during infection in vivo, in part by preventing the generation of very high, immunosuppressive levels of antiviral cytokines. Thus, our study demonstrates that NK cells can serve not only as effector lymphocytes but also as a regulator of immune system function for defenses against viral infections. Therefore, our data bring important and original advances to the understanding of the contributions NK cells make to immunity against infectious disease, by demonstrating that i) they control the functions of another critical player in the innate antiviral responses, the pDC, and modulate the cytokine milieu induced early after challenge, and ii) they accelerate the generation of effector antiviral CD8 T cells. Other mechanisms through which NK cell activities can modulate immune responses to pathogens have been summarized in several recent reviews [8, 45, 46] and include in vivo i) the regulation of the homeostasis and of the maturation of DCs and ii) the prevention of a detrimental persistence of the activation of the CD8 T cells at later time points during the immune response. Very recently, it was also shown that perforin-mediated NK cell killing down-modulates the activation of macrophages and prevents the development of a hemophagocytic lymphohistiocytosis-like syndrome during MCMV infection [47] .
Our results demonstrate that NK cells can dramatically decrease the intensity and duration of pDC activation by controlling viral burden, which prevents the production of very high systemic levels of IFN-a/b, and eventually other innate cytokines, that can have detrimental effects for the host. We show that this mechanism also protects against the MCMV-mediated loss of splenic cDCs [36] . It has been reported that that both measles virus and LCMV can exploit the host's IFN-a/b response to inhibit cDC development and drive cDC loss in vivo [19] . Our results suggest that MCMV also can induce high production of IFN-a/b to promote its own survival by ablating cDCs and delaying the activation of antiviral effector CD8 T cells. In light of this observation, it is interesting to note that MCMV has developed strategies to actively counteract the antiviral responses to IFN-a/b or IFNc within infected cells [48] , as opposed to the mechanisms employed by negative-strand RNA viruses, which act to shut down the production of these cytokines by infected cells [49] or pDCs [50] . Indeed, even though complete deficiency in IFN-a/b responses is associated with a dramatic increase in the susceptibility of mice to MCMV infection [16] , it clearly appears that the benefit of high level IFN-a/b production for the host is less than that brought by an efficient NK cell response (since Klra8 mice show viral titers that are 1,000fold lower than those seen in BALB/c mice, even though Klra8 mice produce 100-fold less IFN-a/b). Thus, it is tempting to speculate that the efficient NK cell activity driven by the Ly49H activating receptor and its ability to dampen pDC IFN-a/b production and to promote adaptive immunity is a direct host countermeasure to the subversion of the IFN-a/b response by MCMV. Altogether, our results suggest that the NK cell response governs the balance between the positive and negative effects of IFN-a/b, and eventually other innate cytokines, for the optimal orchestration of the immune response to MCMV.
Our data indicate that efficient NK cell activity contributes to the adaptive arm of the immune response to MCMV by promoting the accelerated expansion of antigen-specific CD8 T cells. Like the contribution NK cells make to the maintenance of the cDC compartment, our data support a role for NK cell control of pDC IFN-a/b production for the promotion of antigen-specific CD8 T cell expansion. IFN-a/b can have both positive and negative effects on CD8 T cell responses [20, 21, [51] [52] [53] . For example, the optimal expansion of CD8 T cells in response to LCMV infection has been demonstrated to be dependent on the ability of the CD8 T cell compartment to receive IFN-a/b-mediated signals [53] . However, within the same viral system, IFN-a/b also drives the early attrition of both antigen-specific and bystander CD8 T cell populations [20, 21] . Here, we show that the delay in the expansion of antiviral effector CD8 T cells in the absence of an efficient NK cell response to MCMV occurs within the context of excessive production of IFN-a/b by pDCs. Moreover, we demonstrate that exogenous administration of IFN-a can override the ability of efficient NK cell activity to promote the accelerated expansion of functional antiviral CD8 T cells. The IFN-a-mediated effects on CD8 T cell expansion could be direct or a downstream consequence of the loss of cDCs. However, our results suggest a direct activity of IFN-a/b on CD8 T cells, as the administration of IFN-a leads to the complete disappearance of early antiviral CD8 T cells but only to an incomplete decrease in cDC numbers.
Although not the focus of this study, our data also show that delayed CD8 T cell responses reach higher levels and are sustained for longer periods of time in the absence of efficient antiviral NK cell activity, which is consistent with previous observations [43] . This extended activation of the T cell compartment is required for MCMV control under these conditions and likely results from the poor ability of the innate immune system to control the virus early. One could hypothesize that this could also lead to chronic inflammation and long-term detrimental effects for the host, including the increased susceptibility of BALB/c mice to MCMV-induced T cell-dependent autoimmune diseases such as myocarditis [54] . Thus, efficient NK cell responses could be beneficial to the host not only by early direct antiviral effects but also by reducing the degree of antiviral T cell activation required for later control of viral replication and therefore the indirect costs this may bear for health.
To our knowledge, this study is the first to demonstrate how naturally occurring, genetically determined differences in the interactions between a virus and its host can tilt the balance between the various functions of IFN-a/b towards conditions promoting the induction of early adaptive immunity rather than towards the development of a state of transient immunosuppression. Our data also confirm that NK cell antiviral functions prevent the generation of a chronic state of CD8 T cell activation later during the infection that may otherwise lead to immunopathology. A key question for future studies will be to determine how general this function of NK cells is for antiviral defense. Such mechanisms may also be in place during HIV infection where genetic [55] or functional [56] [57] [58] evidences suggest a role in the variation of NK cell functions for resistance or susceptibility to the development of AIDS, where detrimental effects of excessive levels of IFN-a/b [59] or of pro-inflammatory cytokines [60, 61] have been recently suggested, and where the initiation of antiviral CD8 T cell responses appears delayed [62] [63] [64] . This could also be the case for infections with highly pathogenic strains of influenza, as excessive production of pro-inflammatory cytokines have been shown to occur and proposed to play a crucial role in immunopathology [65, 66] , while NK cells can confer resistance through recognition of infected cells by the NKp46 receptor [67] . Very recently, researchers have demonstrated that human NK cells are able to recognize DCs infected with the influenza or Ebola viruses [68, 69] , while a correlation between unregulated IFN-a/b responses and a malfunction of the switch from innate to adaptive immunity has been reported during fatal SARS [70] , which further highlight the potential relevance of our observations to a variety of viral infections. Thus, it will be important to determine the impact of NK cell function on the modulation of innate and adaptive immunity and on the development of immunopathology in other models of viral infection. Moreover, our results imply that in individuals susceptible to such viral infections, pharmacological control of viral replication very early should benefit them not only by directly limiting viral cytopathogenicity, but also by establishing conditions better suited to the development of balanced, protective immunity, since this is the case in mice susceptible to MCMV infection when viral replication is controlled by drug treatment.
A role for NK cells in the generation of antitumor CD8 T cell responses has been linked to their ability to increase inflammation via secreting IFN-c and promoting IL-12 production by DCs [71, 72] . Collectively, these studies and ours reveal a role for NK cells as mediators of an ''innate cytokine balance'' for the optimal generation of the immune response, whereby NK cells are required to produce cytokines to increase inflammation when it is intrinsically low (during tumor development) and to prevent excessive production of innate cytokines when it can be intrinsically high (in the context of pathogenic encounters). Of note is the apparent requirement, in both instances, for NK cells to engage in cognate receptor-mediated interactions to naturally develop immunoregulatory functions. For example, during the response to MCMV, the systemic activation of NK cells by IL-12 to produce IFN-c in a Ly49H À environment is not sufficient to promote their control of viral replication and their associated immunoregulatory functions. These cognate interactions could also enable NK cells to act as direct regulators of antiviral immunity, as already illustrated for the generation of antitumor CD8 T cell responses. During the response to MCMV, Ly49H expression by NK cells dramatically increases the efficiency of recognition and killing of infected cells. Ly49H may also enable NK cells to deliver IFNc in a proper place and time to help the priming of antiviral CD8 T cells in infected mice through a tripartite interaction with MCMV-infected m157-expressing DCs. The importance of these cognate interactions for the promotion of efficient NK cell functions may differ depending on the tissues and on the target cell types, as exemplified by the heightened requirement for Ly49H-dependent mechanisms for MCMV control in the lungs [73] .
In conclusion, we demonstrate that efficient NK cell activity contributes to the optimal orchestration of innate and adaptive immunity during the course of a viral infection in vivo. The implications of these findings extend to the design of therapeutic strategies to fight human disease in two ways, as they emphasize i) that the benefits of innate cytokine production for immune cell recruitment and activation can be outweighed by detrimental effects that can directly reduce the capacity of the host to fight infection, as well as ii) the need to better understand and take into account the complex interactions of relatively rare cell types that occur in a physiological context for the purpose of exploiting the human immune system to promote health over disease.
Mice. BALB/c mice (Charles River Laboratories, http://www.criver. com/) were purchased for use in these studies. C.B6-Klra8 Cmv1-r /UwaJ, referred to as Klra8, (Jackson Laboratory, http://www.jax.org/), B10.D2, and B10.D2-DAP12-deficient (DAP128) animals were bred in pathogen-free breeding facilities at the Centre d'Immunologie de Marseille-Luminy (Marseille, France). Experiments were conducted in accordance with institutional guidelines for animal care and use. Protocols have been approved by the French Provence ethical committee (number 04/2005) and the US Office of Laboratory Animal Welfare (assurance A5665-01).
In vivo treatment protocols. Stocks of wild-type Smith Strain MCMV salivary gland extracts [14] and bacterial artificial chromosome (BAC)-derived wild-type, DN-SCP-MCMV [44] , Dm157-MCMV, and m157-revertant were prepared as previously described [73] . Infections were initiated on day 0 with the i.p. delivery of 5 3 10 3 PFU (for in vivo-derived virus) or 2 3 10 5 PFU (for in vitro-derived virus). These modest doses were chosen because they do not induce lymphopenia in infected animals who harbor leukocyte numbers equal to or greater than those of uninfected controls (not shown). In addition, these doses are likely to be closer to the physiologic doses reached upon natural exposure to the virus through contact with infected animals. For the in vivo arrest of DN-SCP-MCMV replication, 200 lg of doxycycline was delivered by i.p. injection 20 h postinfection followed immediately by the addition of 2 mg/ml doxycycline plus 5% sucrose in the drinking water. 50,000 units of recombinant mouse IFN-a (HyCult Biotech, http://www.hbt.nl/) was administered by i.p. injection at 30 and 48 h post-MCMV infection. This dose was titrated in ELISA and shown to correspond to 25 ng of the ELISA IFN-a standard. Thus, since the cytokine titers measured by ELISA in the serum of BALB/c mice at 36 h post-infection range between 5 and 10 ng/ml, and the volume of the lymph and blood of an adult mouse can roughly be estimated around 5 ml, the dose of rmIFN-a injected in Klra8 mice should be similar to the physiologic levels of the cytokine naturally induced in infected BALB/c mice. NK cells were depleted by delivery of 100 lg of purified anti-NK1.1 mAb (PK136) on days À1 pre-MCMV infection, and days 1 and 3 post-MCMV infection. Control mice were treated with 100 lg of mouse IgG (Jackson ImmunoResearch Laboratories, http://www. jacksonimmuno.com/). Uninfected mice were depleted of NK cells on the schedule of day 4 infected mice. To assess NK cell depletion, a combination of anti-DX5 and anti-TCRb staining was used in order to avoid the risk of underevaluating the numbers of remaining NK cells, as may have occurred due to a potential problem of epitope masking if the antibody used for immunophenotyping had been the same as the one used for depletion. NK cell depletions were greater than 98% ( Figure S5) .
Isolation of lymphocytes. For the analysis of CD8 T cell populations, spleens were minced, passed through nylon mesh and washed in PBS, 5 mM EDTA, and 3% FCS (PBS/EDTA/FCS). For the analysis of DC populations, spleens were digested by collagenase (liberase CI; Boehringer Mannheim, http://www.roche.com/) and teased apart by repeating pipeting in PBS/EDTA/FCS. In both protocols, erythrocytes were osmotically lysed by ammonium chloride treatment. Thereafter, cell suspensions were kept in PBS/ EDTA/FCS unless specified otherwise. Total live splenocytes were counted by trypan blue exclusion using a hemocytometer. Total numbers of specific leukocyte subsets were calculated for each individual mouse as (percent of these cells in the live gate of total splenocytes) 3 (total live splenocyte numbers).
CD8 T cell stimulation for IFN-c production. For intracellular IFNc detection, splenic lymphocytes were isolated and then incubated with IE-1 ( 168 YPHFMPTNL 176 ) or m164 peptide ( 257 AGPPRYSRI 265 ) (10 À7 M) pulsed P815.B7 cells at a 10:1 ratio for 6 h with brefeldin A (Sigma-Aldrich, http://www.sigmaaldrich.com/) added for the last 3 h of culture. Cells were then harvested and analyzed for CD8a, CD43, and intracellular IFN-c protein by three-color staining followed by flow cytometry.
In vivo cytotoxicity assay. Antigen-specific CD8 T cell-mediated cytotoxicity was assayed as described in [74] . Briefly, splenocytes from naive mice were costained with PKH26 (Sigma-Aldrich) and either 1 lM, 100 nM, or 1 nM CFSE (Molecular Probes, http://probes. invitrogen.com/). Labeled cells were then pulsed with the indicated peptides, mixed in equal ratios, then transferred i.v. (5 3 10 6 total cells) into the indicated groups of mice. Lymphocytes were isolated from the spleens of recipient mice 4 h post-transfer and analyzed for PKH26 (all transferred cells) and level of CFSE expression (unpulsed or peptide-pulsed cells). Percent killing within the PKH26 þ gate was calculated by: 100 À ([(% peptide-pulsed in infected/% unpulsed in infected)/(% peptide-pulsed in uninfected/% unpulsed in uninfected)] 3 100).
Quantification of viral titers and serum cytokine levels. Spleens were homogenized [14] , and viral titers were determined by plaque assay using mouse embryonic fibroblasts with centrifugal enhancement or NIH-3T3 cells [73] . Serum was collected at the indicated time points and cytokine levels were determined by ELISA for IFN-a (PBL Biomedical Laboratories, http://www.interferonsource.com/) and IL-12p70 (R&D Systems, http://www.rndsystems.com/) per the manufacturer's instructions.
Abs and reagents. DX5-PE, CD43-PE (clone 1B11), CD11c-PE (clone HL3), CD8a-PerCp (clone 53-6.7), TCR-b-allophycocyanin (clone H57-597), IFN-c-allophycocyanin (clone XMG1.2), IL-12allophycocyanin (clone C15.6), and streptavidin-PE were purchased from BD Pharmingen (http://www.bdbiosciences.com/). CD8a-FITC (clone CT-CD8a) was purchased from Caltag (http://www.caltag.com/). Ki-67-FITC (clone MM1) was purchased from Novocastra (http://www. vision-bio.com/). Purified rat anti-mouse IFN-a (clone F18 and RMMA-1) and anti-IFN-b (clone RMMB-1) were purchased from TEBU-Bio (http://www.tebu-bio.com/). 120G8 mAb was provided by Schering-Plough (http://www.schering-plough.com/) and conjugated to Alexa Fluor-488 using a kit from Molecular Probes. Isotype controls for each mAb were purchased from the appropriate manufacturer. The following tetramers conjugated to PE were obtained through the NIH Tetramer Facility (Atlanta, Georgia, United States): H-2L(d)/IE-1( 168 YPHFMPTNL 176 ) and H-2D(d)/ m164( 257 AGPPRYSRI 265 ). The above-mentioned reagents were used for FACS analysis in this study.
Flow cytometric analysis. Cells were first incubated with 2.4G2 mAb for 20 min. Cells were then stained with mAbs specific for cell surface markers or isotype controls for 30 min at 4 8C. Cells were then washed and fixed in 2% paraformaldehyde in PBS. Intracellular staining for Ki-67, IFN-c, and IL-12 was performed using the Cytofix/ Cytoperm kit (BD Pharmingen). Intracellular staining for IFN-a/b was performed as previously described [35] . Depending on the experiments, 2.5 3 10 5 to 2 3 10 7 events were collected on a FACSCalibur. The data were acquired and analyzed using CellQuest software (BD Biosciences). Isotype controls were used to set gates for the presented FACS analyses.
Statistical analyses. Statistical analyses were performed in Microsoft Excel 5.0 (Microsoft Corporation, http://www.microsoft.com/) using Student's two-tailed t tests. Mean 6 standard deviation (SD) was calculated for each graph. If visually absent, error bars are too small to be depicted based on the scale of the y-axis.  The Trojan Chicken Study, Minnesota We conducted a study in the summer of 2004 at county fairs in the Midwest to investigate the role poultry exhibits have in spreading avian pathogens to humans. A nearly invisible powder (pathogen surrogate) that fluoresces under UV light was surreptitiously sprinkled each day on 1 show bird at each of 2 fairs. A UV light box was used to daily examine the hands of 94 poultry-exhibit participants (blinded regarding UV box results) for up to 4 days during the poultry shows. Enrollment and end-of-study questionnaires collected data on pathogen risk factors. Eight (8.5%) of 94 participants had evidence of fluorescent powder contamination (95% confidence interval 2.76%–14.26%). This contamination and infrequent handwashing practices suggest that county fairs are a possible venue for animal-to-human pathogen transmission. We conducted a study in the summer of 2004 at county fairs in the Midwest to investigate the role poultry exhibits have in spreading avian pathogens to humans. A nearly invisible powder (pathogen surrogate) that fluoresces under UV light was surreptitiously sprinkled each day on 1 show bird at each of 2 fairs. A UV light box was used to daily examine the hands of 94 poultry-exhibit participants (blinded regarding UV box results) for up to 4 days during the poultry shows. Enrollment and end-of-study questionnaires collected data on pathogen risk factors. Eight (8.5%) of 94 participants had evidence of fluorescent powder contamination (95% confidence interval 2.76%-14.26%). This contamination and infrequent handwashing practices suggest that county fairs are a possible venue for animal-tohuman pathogen transmission. R ecently, the Centers for Disease Control and Prevention (CDC) declared avian influenza to be the world's number-1 health threat (1); in particular, the wide and rapid spread of the H5N1 strain has heightened concerns. All H5N1 cases to date have been associated with direct contact with poultry, but recently, human-to-human transmission has been purported in Thailand (2) . Previously healthy children and young adults seem to be especially susceptible to this illness (3) . As of February 27, 2006, a total of 173 confirmed human cases of avian influenza A (H5N1) and 93 deaths have been reported to the World Health Organization, for a case-fatality rate of 53.8% (4) .
Close contact with live poultry has been implicated in recent outbreaks of avian influenza in humans in Southeast Asia and elsewhere (2, (5) (6) (7) (8) . In the 1997 Hong Kong outbreak, live bird markets were implicated as the source of exposure to the virus (8) . In the United States, live bird markets are a known reservoir for avian influenza (9-11), but thus far they have not been associated with human avian influenza infection. Live bird markets involve a mixing of birds from diverse areas, crowded conditions for humans and livestock, mixing of different species of animal, and often a lack of proper sanitation, thus providing opportunity for outbreaks of disease. Transport of animals to market is a source of stress than can induce increased shedding of infectious agents. Stressed birds are also more susceptible to infections (12) .
While live bird markets are uncommon in the Midwest, animal exhibits such as those at county fairs are quite common. Such exhibits are similar to live bird markets in that they involve transport and mixing of animals from different locations, crowded conditions, and a general lack of sanitation. Approximately 125 million people visit agricultural fairs every year in the United States (13) . Fairs usually involve close proximity of food vendors to animal exhibits. Many animal exhibits encourage or allow visitors to touch animals. Small children are frequent visitors to county fairs and animal exhibits, and children also engage in behavior such as nail biting that may make them more likely to ingest infectious agents. Live animal exhibits such as petting zoos and open farms, which are in many ways similar to county fairs, have also been implicated in outbreaks of Escherichia coli O157:H7 and other bacterial diseases (13, 14) .
Proper handwashing is recommended to protect persons from infection (15) . However, animal exhibits often lack adequate handwashing facilities, and many persons may be unaware of the risk such exhibits pose. Direct contact with animals, indirect contact with contaminated objects, or inhalation of aerosolized virus could contribute to transmission of pathogens in such settings.
Because little is known about the possible spread of pathogens at county fairs, and because most cases of avian influenza have resulted from close contact with poultry, a study was undertaken to model interspecies transmission of pathogens at county fair poultry shows. The specific aims of this study were to determine the proportion of The Trojan Chicken Study, Minnesota human poultry show participants who demonstrate hand contamination by a surrogate marker for an avian pathogen and to determine possible risk factors associated with such contamination.
A feasibility study was conducted at a county fair in Iowa (county A) to evaluate study methods. After the feasibility study, human poultry fair participants were enrolled at a larger county fair in Minnesota (county B). Both fairs were held within small cities with populations of ≈100,000.
At county fairs, poultry judging often takes place in show areas that are open to the public. Birds are usually placed in cages that are stacked one upon another and set upon tables ( Figure) . Because poultry classes are judged separately and competitors may show their birds in several poultry classes, birds are frequently moved in and out of their cages for grooming and competition. During the competition, birds are moved to competition cages that have previously housed birds from other competition classes. Judges typically handle each bird individually; they take the bird from the exhibitor, examine it, and then hand it back to the exhibitor ( Figure) . Handwashing is not generally performed as the judge moves from bird to bird, nor is handwashing common before or after exhibitors handle their birds. After competition, birds often remain on exhibit for several days, and they may be touched by the general public.
This study was reviewed and approved by the University of Iowa's Institutional Review Board and Animal Use and Care Committee. The investigators participated in online human and animal subjects training. Informed consent was sought from participants before they were enrolled.
Anyone >7 years of age present in the poultry exhibit area at any time during the period when poultry were on active exhibit was eligible to enroll in the study. Recruitment focused on members of 4-H clubs and openclass exhibitors, their families, and 4-H club staff, but also included other visitors. Enrollment occurred continuously over a 4-day period (Monday through Thursday) while poultry were exhibited at the fairs. A special sign and an information table were used to promote the study. Study participants were recruited for enrollment as they walked through the poultry exhibit area. After providing informed consent, study participants were asked to complete a 1page questionnaire that gathered demographic and poultry exposure data. Participants were also asked to complete a 1-page end-of-study questionnaire after they completed their experience at the poultry exhibit (day 4). This instrument gathered data on handwashing and types of animals handled at the fair.
GloGerm (GloGerm Company, Moab, UT, USA), a benign, synthetic, organic colorant A-594-5 that fluoresces under a black light, was used as a surrogate marker for an avian pathogen. This powder (also found in liquid or gel form) is commonly used in handwashing training in hospitals and businesses (16) . Each day, the white powder was surreptitiously applied to the same single chicken at the fair to imitate a single source of pathogen. White broiler chickens were chosen as the exposure birds since the powder was not detectable on their feathers. Each "Trojan chicken" was otherwise treated the same as the other chickens in the poultry shows. While county fair authorities gave permission for the study, neither the judge nor the study participants were aware of neither the surrogate exposure nor which of the chickens were of particular hygienic concern. Instead several participants remarked that they thought the UV light box (see below) in which photographs were taken could somehow detect generic bacterial contamination on the hands. At county A, chicken powdering was conducted early in the mornings of the 3 days of competition, when competitors were not at the poultry exhibit. At county B, the same strategy was followed but the Trojan chicken was also surreptitiously powdered again in the early afternoon for 3 days of the show. During the powdering, approximately one-third cup powder was liberally sprinkled onto the underside of the chicken to imitate fecal shedding of pathogen. The chicken was then returned to its cage. The Trojan chickens each shared their cage with another, very similar, white broiler chicken, since these birds are normally shown in matched pairs.
To evaluate potential avian influenza transmission, a 2 × 2 × 2-foot wooden box was constructed from plywood. Three black 1-foot × 18-inch fluorescent lights (15 watts) and 1 white 1-foot × 18-inch fluorescent light (15 watts) were mounted under the lid of this isolation box. Study participants inserted their hands through hand holes in 1 side, and they were blinded as to the result of the fluorescence examination of their hands. From an opening in the box on the opposite site, digital photographs of the ventral and dorsal images of the hands were taken with a digital camera (Figure) . A log was kept to match the sequentially captured photograph numbers with the participants' names (data were later de-identified). Beginning on day 1 of each poultry show, daily photographs were taken of study participants' hands under the black lights (Figure) . Photographs continued to be taken through the afternoon of day 4 (last day of the shows).
Statistical analysis was performed with SAS version 8.0 (Cary, NC, USA). Chi-squared analysis and Fisher exact test were used to compare categorical variables with powder contamination. We used t tests to compare continuous variables. Logistic regression modeling was attempted, but the models did not converge. Odds ratios and confidence intervals were calculated by using EpiInfo (CDC, Atlanta, GA, USA) ( Table 1) .
Ninety-four persons participated in the study by having their hands photographed. Among these were 30 poultry exhibitors ( Table 2) . Of the study participants, 82 (87.2%) completed the enrollment questionnaire, and 44 (46.8%) completed the end-of-study questionnaire. Of all participants in county B, 29 (30.9%) were male.
The mean age of those who completed the enrollment questionnaire was 33 (range 7-79) years. Eighteen participants were poultry exhibitors, who showed 1-10 birds each (mean 3.4). Fifty-five participants (67.1%) of 82 were residents of farms.
Eight participants exhibited hand contamination ( Table  1) . Of these, all 8 completed the enrollment questionnaire, and 7 completed the end-of-study questionnaire.
Participant gender and hand contamination were not associated. Of participants whose hands were contaminated, 3 were male and 5 were female. None of the persons whose hands were contaminated were exhibitors: 3 were family members of exhibitors, 3 were visitors, and 1 was in the "other" category.
In the age group of 7 to 12 years, 1 (7.7%) participant had hand contamination ( Table 1) . None of the participants in the 13-to 21-year age group showed hand contamination. Four participants (10.8%) in the 22-to 50-year age group had contaminated hands, and 2 participants (14.3%) who were >51 years showed hand contamination. Contamination rates did not differ by age group.
Our study demonstrated that pathogen transmission is possible through poultry handling at county fairs. A contact transmission proportion of 8.5% (8 persons of the 94 participants had contaminated hands) is high, when one considers the insensitivity of the measure (gross fluorescence) and the number of persons possibly exposed at a county fairs. Both male and female participants were affected, as well as most age and role groups.
This study had some unique characteristics. Digital photography of a fluorescent powder on hands was a successful surrogate for contamination. However, this rather gross measure was likely insensitive when one considers how few bacterial or viral particles are needed to cause certain zoonotic diseases. The black light box was also successful in blinding participants to their contamination status, since they were unable to see inside the box, and few seemed to grasp the experimental nature of the study.
Some of our study findings were unanticipated. We expected contamination proportions to vary by age, gender, and role because we expected these factors to affect the amount of contact with birds and handwashing behavior. However the rates did not vary by these variables. This finding could be due to the study's limited power to detect such differences. If the differences between those exposed and those unexposed were statistically significant (e.g., also occurring in a similar study with a larger sample size), they might be consistent with studies that suggest that animal handlers (exhibitors) practice better hygiene compared to nonhandlers in the same environment. Alternatively, animal handlers may engage in other behavior that affects their contamination status, such as handling enough animals that the surrogate powder wears away more quickly than it would for someone who does not handle animals.
This theoretical model had limitations. Hand contamination with the fluorescent powder was considered a surrogate for pathogen transmission in this study; however, hand contamination of a pathogen does not necessarily lead to transmission. Transmission is dependent upon the amount of inoculated pathogen (dose), the ability of the pathogen to cause disease (virulence), and the ability of the host to defend against infection (host susceptibility) (17) . These variables are complex and difficult to measure in settings such as a county fair. Additionally, such variables often vary by pathogen and host; hence, we measured only surrogate markers for exposure because such exposure is a requirement for disease to occur.
GloGerm powder contamination may or may not be reflective of true pathogen transmission. The product is useful in handwashing training because it is generally not visible to the naked eye and persons are usually unaware that they have become contaminated. In our study, GloGerm was additionally useful because study participants were also unaware that a chicken was contaminated. Proper handwashing removes the powder, as it would pathogens. However, the amount of time the powder remains on a person's hands without handwashing varies and may be different from the amount of time that a pathogen would be viable on hands. In addition, dusting the chicken with powder is an attempt to model pathogen shedding, but this practice may not truly reflect the amount of pathogens an infected bird would shed. The undersides of the birds were dusted to model fecal shedding and dispersal of pathogens. However, the amount of powder used may be higher or lower than true pathogen shedding.
The study design was further limited in that we did not account for time after exposure when photographs were taken. Since participants could drop by any time of the day, the time after exposure and duration of exposure likely varied between participants. In both the feasibility and pilot studies, the return rate was low, and tracking down participants was difficult. If similar studies are conducted in the future, a reward system might be used to increase compliance.
Petting zoos and agricultural fairs are common in the Midwest and attract many thousands of people. While concern about viral and bacterial zoonotic disease transmission in these settings is growing, they are not usually thought of as a public health concern. The observations from this modest study, even with the limitations described above, suggest that live poultry exhibits may pose a disease transmission risk. Of particular concern is the relatively high proportion of powder transmission to poultry show visitors, who have casual and limited exposure to poultry. Larger future studies of similar design might help identify specific risk factors for zoonotic disease transmission and appropriate interventions for such settings. As a minimum contribution, these study data suggest that hygienic educational programs and disease prevention programs are warranted in poultry exhibits. The Restriction of Zoonotic PERV Transmission by Human APOBEC3G The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections. , bats [2] and chimpanzees [3] . Domesticated animals can also function as zoonotic intermediates (e.g., [4, 5] ). Additional and unprecedented opportunities for zoonoses occur when live cells, tissues or organs are transplanted from one species to another [6] . However, despite risks and technical and immunological challenges, several xenotransplantation procedures have shown preclinical promise for treating diabetes, heart, kidney and other human diseases (e.g., [7] [8] [9] ).
Pigs are favourable xenotransplantation sources because of their human-like physiology, large litters, short gestation period and genetic malleability [10] . However, pig to human virus transmission has been a concern since it was shown that porcine endogenous retroviruses (PERVs) could infect human cells in culture [6, [11] [12] [13] . Although PERV transmission has yet to be documented in xenotransplantation patients, significant concerns still exist regarding PERV and other potentially pathogenic viruses [14, 15] . Strategies to reduce the likelihood of PERV transmission have been proposed, such as selective breeding for lower levels of PERV, RNAi transgenesis to knock-down PERV expression or systematic deletion of active PERV copies (e.g., [15, 16] ). The first two are unlikely to be completely effective or risk-free and the third, albeit theoretically feasible, may be overly technical and prohibitively expensive. Therefore, alternative, robust and costeffective methods to reduce PERV transmission and possible xenozoonotic infections are desirable.
APOBEC3G is a single-strand DNA cytosine deaminase best understood as a potent inhibitor of HIV-1 replication [17] [18] [19] [20] [21] [22] [23] . It can however also inhibit a variety of other exogenous and endogenous retroviruses/elements (e.g., [17, 18, [24] [25] [26] [27] [28] [29] ). APO-BEC3G engages an assembling retrovirus particle, accesses the RNA genome-containing virus core and, upon reverse transcription, deaminates cDNA cytosines to uracils (C-to-U). Catastrophic levels of uracil either directly inactivate the coding capacity of the virus or trigger the degradation of the viral DNA. The former manifests as genomic strand-specific guanine to adenine (G-to-A) hypermutations (cDNA strand C-to-T transitions). However, in several instances, it is noteworthy that the deaminase activity of APOBEC3G or other APOBEC3 proteins is partly or even completely dispensable (e.g., HIV-1, hepatitis B virus, L1 and Alu [27] [28] [29] [30] [31] [32] ).
Interestingly, throughout evolution, the retroviruses of many mammals appear to have become largely immune to APOBEC3G or to the APOBEC3G-like proteins of their hosts. HIV-1 expresses an accessory protein, Vif, which neutralizes APOBEC3G through ubiquitination and degradation [33] [34] [35] [36] . Simian immunodeficiency virus (SIV) uses a similar Vif-dependent mechanism [17, 26] . Foamy viruses employ an unrelated viral protein called Bet, for which the precise neutralization mechanism is currently unclear [37, 38] . Murine leukaemia virus (MLV) and human T-lymphotrophic virus-1 (HTLV-1) may simply avoid APOBEC3 proteins by preventing encapsidation [26, 39, 40] .
However, cell-based studies have indicated that an APOBEC3 protein from a mammal to which the virus has not yet adapted may provide an effective strategy for thwarting species-specific viral counter-defenses. For instance, human APOBEC3G can potently inhibit the replication of SIV (except isolates such as SIV cpz which encode a Vif protein closely related to that of HIV-1), feline foamy virus and MLV (e.g., [17] [18] [19] [20] 25, 26, 39, 41, 42] ). Similarly, mouse APOBEC3 can potently block HIV-1 replication (regardless of Vif), although it is completely unable to impede the replication of MLV [26, 41] . One of the most dramatic examples to date used human and mouse APOBEC3 proteins to inhibit the mobilization of the yeast retrotransposons Ty1 and Ty2 [43, 44] . Thus, it is reasonable to hypothesize that cross-species expression of an APOBEC3 protein may be used to create a powerful barrier to impede or perhaps even block retrovirus infection. Here, this rationale is applied to the specific question of whether human APOBEC3G expression can inhibit the transmission of PERV from pig to human cells. The results demonstrate that PERV transmission can be strongly inhibited by APOBEC3G.
To determine whether expression of human APOBEC3G would inhibit the transfer of PERV from pig to human cells it was first necessary to establish a long-term co-culture system. A trans-well assay was set up to monitor PERV transmission from pig kidney PK-15 fibroblasts to recipient human embryonic kidney 293T cells ( Figure 1A ). These two cell types were used because transmission from PK-15 to 293 cells had been reported previously [11] . The trans-well system enabled co-cultures to be sustained for several weeks, and it facilitated the recovery of each cell type for downstream analyses. An additional benefit of this co-culture system (not provided by transient assays) is that it enables the simultaneous analysis of multiple, endogenous PERV elements, which are precisely the targets one would want to monitor and ideally inhibit in (xeno)transplantation procedures.
At each co-culture passage point, surplus human 293T cells were used to prepare genomic DNA. PERV transmission was monitored by subjecting these samples to quantitative (Q)-PCR. PERV-specific pol gene PCR products could be detected in the human genomic DNA samples after approximately two weeks of continuous co-culture ( Figure 1B) . From the point of first detection onward, the total number of PERV transmissions continued to increase, averaging 190 new events per day per 50,000 cells (10 5 beta-actin copies; SEM = 62; n = 5 experiments). Importantly, pig cells did not breach the 293T cell compartment because Q-PCR analyses of the same human genomic DNA samples failed to detect a concomitant transfer of pig genomic DNA ( Figure 1B ; also see Online Figure S1 for PERV pol gene Q-PCR standard curves, representative PERV-specific datasets and human beta-actin controls). Moreover, PERV copy number did not increase over a two week interval when infected 293T cells were grown in isolation, indicating that PERV was not replicating in the human cells and that the majority of the observed transmission events were derived from the PK-15 cells (i.e., new events). These results combine to indicate that the trans-well assay provided a robust system for monitoring bona fide zoonotic PERV transmissions.
The second key step in addressing our experimental question was isolating PK-15 clones that stably expressed human APOBEC3G. Clones expressing human APOBEC3G cDNA or an empty vector control were established in parallel ( Figure 2A ). Immunoblotting identified clones with APOBEC3G levels similar to those in known APOBEC3G-expressing human T cell lines CEM and H9, which are non-permissive for growth of Vif-deficient HIV-1 [19] . Although it was impossible to achieve a physiologic expression level, the comparative immunoblot at least ensured that the levels of APOBEC3G were equal or lower than those present in wellstudied, non-permissive human T cell lines.
Co-culture experiments were set up to compare PERV transmission from two independently derived PK-15 clones expressing human APOBEC3G and two vector expressing controls. Remarkably, the human APOBEC3G expressing PK-15 clones showed levels of PERV transmission that were lower than the Q-PCR detection threshold of approximately 10 copies ( Figure 2B ; . This graph summarizes data for two independently derived PK-15 clones, V1 (squares) and V2 (diamonds). All data points were calculated using results from duplicate Q-PCR reactions of genomic DNA from three parallel (but independent) co-cultures. The error bars indicate the SEM. See the Materials and Methods and Online Figure S1 for additional details, representative raw data and controls. doi:10.1371/journal.pone.0000893.g001
Online Figure S1 ). In contrast, the control clones showed high levels of PERV transfer by co-culture day 17 and transmission events continued to accumulate through the duration of the experiment. The kinetics of PERV transmission were similar to those reported in Figure 1B (these results also contributed to the aforementioned transfer rate calculations). These data were further corroborated by additional experiments where PERV transmission was monitored simultaneously by Q-PCR and by reverse-transcriptase ELISA assays (Online Figure S2 ).
An ultimate application of the technology described here raises the potential problem that human APOBEC3G may not be subjected to (proper) post-translational regulation in pig cells and it may therefore promote carcinogenesis. Expression of human APOBEC3G in a heterologous system has been shown to trigger elevated levels of genomic C/G-to-T/A transition mutation [44] . Therefore, to help mitigate this risk (in addition to establishing clones that expressed relatively modest APOBEC3G levels; above), we asked whether the predominantly cytoplasmic localization pattern of human APO-BEC3G would be maintained in PK-15 and in a swine testes cell line, ST-IOWA ( Figure 3 ; compare with other APOBEC3G reports [18, 35, 41] ). Unlike some other APOBEC3 proteins such as human APOBEC3B, which is mostly nuclear, both human APOBEC3G and pig APOBEC3F appeared predominantly cytoplasmic in either the human or the pig cell lines (Figure 3 ; [28, 31, 41] ). Both proteins also appeared to concentrate in cytoplasmic punctae, which varied in number and were apparent in some of the cells regardless of species of origin (described previously for APOBEC3G; e.g., [45, 46] ). Overall, these near-identical localization patterns suggested that human APOBEC3G is not aberrantly regulated in pig cells and, interestingly, that these proteins might be subjected to the same cellular regulatory mechanism(s).
During the course of these experiments, we reported some of the activities of pig APOBEC3F [41] . It could strongly inhibit the replication of HIV (regardless of Vif) and modestly inhibit the replication of MLV, a gamma-retrovirus phylogenetically related to PERV. Therefore, we wondered whether pig APOBEC3F was expressed in PK-15 and, if so, whether PERV resists this cellular defense. To begin to address this possibility, RT-PCR was used to test PK-15 cells for pig APOBEC3F expression. Pig APOBEC3F mRNA was detected readily (Online Figure S3A ). Full cDNA sequencing revealed that the predicted APOBEC3F protein of PK-15 cells was 98% identical to the variant we reported previously [41] . Eight amino acid differences were found, but both the PK15 and the previously reported APOBEC3F sequences were represented in pig genomic DNA sequences suggesting that these may be breed-specific polymorphisms (R.S.L. and R.S.H., manuscript in preparation). These observations indicated that either PERV resists the endogenous APOBEC3F protein of its host or that the level of suppression by pig APOBEC3F is not sufficient to inhibit PERV transmission.
To begin to distinguish between these two hypotheses, PK-15 clones over-expressing pig APOBEC3F were established and used . PK-15 and 293T cell lysates were used as negative controls. CEM and H9 were used as positive controls for APOBEC3G expression. A non-specific (but pan-species) band is shown as a protein loading control (marked by an asterisk). (B) Q-PCR data using genomic DNA prepared from 293T cells co-cultured with two independently derived APOBEC3G expressing PK-15 clones (G1 and G2, circles and triangles, respectively) or two vector control clones (V1 and V2, diamonds and squares, respectively). The experimental parameters are identical to those used in Figure 1B in transmission experiments. The former hypothesis was favored because pig APOBEC3F over-expression did not significantly interfere with PERV transmission (Online Figure S3B ). These results were further supported by PCR experiments showing that PERV could be amplified readily from 293T cells that had been co-cultured with PK-15 over-expressing pig APOBEC3F (unlike the APOBEC3G scenario; below).
We further noted that it is highly unlikely that another resident APOBEC3 protein contributes to PERV restriction, because genomic DNA sequencing showed that pigs have only one APOBEC3 gene, APOBEC3F (R.S.L. and R.S.H., manuscript in preparation). These observations combined to indicate that PERV is resistant to the endogenous APOBEC3 protein of its host. In hindsight, this was not particularly surprising given the emerging trend that (successful) retroviruses are selected in part by their ability to evade the APOBEC3 proteins of their host species (see Introduction).
The hallmark of APOBEC3G-dependent retrovirus restriction is plus-strand G-to-A hypermutation, which is caused by the deamination of minus-strand cDNA C-to-U during reverse transcription [17, 18, 20, 24, 25] . The deamination of cytosines within singlestrand DNA requires glutamate 259 (E259) of APOBEC3G [47] [48] [49] . Based on homology to structurally defined deaminases, E259 likely functions by helping position the water molecule that ultimately initiates the deamination reaction by attacking the cytosine ring (as a hydroxide; reviewed by [17, 23, 50, 51] ).
To determine whether DNA deamination is required the APOBEC3G-dependent inhibition of PERV transmission, we established a new set of PK-15 clones expressing APOBEC3G, APOBEC3G E259Q or a vector control (Figure 4, inset) . Surprisingly, both APOBEC3G and the E259Q derivative diminished PERV transmission to near background levels ( Figure 4) . These data demonstrated that the mechanism of inhibition does not require the DNA deaminase activity of APOBEC3G. These data were further supported by the fact that plus strand G-to-A hypermutations were not apparent in the DNA of the rare transmission events that occurred in the presence of human APOBEC3G (below).
To begin to genotype the infectious PERVs and to further probe the mechanism of PERV restriction by human APOBEC3G, the PERV pol gene DNA was amplified from human 293T cells, cloned and sequenced ( Figure 5A ; Online Figure S4 ). Twenty-nine and twentytwo sequences were analysed from APOBEC3G and control experiments, respectively. To minimize possible PCR biases, any sequence that was recovered multiple times was considered one event, unless it arose from independent experiments. These DNA sequence analyses revealed several important points.
First, in contrast to vector control and pig APOBEC3F overexpressing co-cultures, PERV pol gene DNA was difficult to amplify from the genomic DNA of 293T cells that had been cocultured with APOBEC3G-expressing PK-15 cells ( Figure 5B and every significant sampling point in our Q-PCR experiments). Taking this together with the observation that APOBEC3G does not effect PK-15 virus production (similar RT levels were observed in cell-free supernatants in the presence or absence of APO-BEC3G; data not shown), we infer that APOBEC3G restricts PERV transmission after virus production but before provirus integration (i.e., between entry and integration). APOBEC3G may restrict PERV at an early reverse transcription stage, possibly by interfering with primer binding, DNA synthesis and/or integration Is Deamination-Independent. PERV-specific Q-PCR data using genomic DNA prepared from 293T cells co-cultured with PK-15 clones expressing APOBEC3G (G; triangles), APOBEC3G-E259Q (GE259Q; circles) or empty vector (V; squares). Two datasets, each with an independent PK-15 clone in three replica co-culture wells, were collected in parallel and averaged for each data point. One standard error of the mean is shown. The experimental parameters are identical to those used in Figure 1B as shown recently for APOBEC3G and HIV-1 substrates (e.g., [30, [52] [53] [54] [55] ).
Second, control co-culture PERV transmission events were exceptionally diverse, as 11 unique pol sequences were detected and only 4 were found multiple times (Online Figure S4) . These data suggested that PK-15 cells have at least 11 active PERVs capable of infecting human 293T cells, a number consistent with previous studies that reported the existence of approximately 17-50 PERV copies in total (with only a fraction being replicationcompetent; [11, 12] ). In contrast, the rare PERV sequences derived from the APOBEC3G co-culture experiments showed a much lower genetic complexity. Only three unique sequences were recovered, each differing by a single nucleotide (Online Figure S4) . In parallel experiments with HIV-based viruses, this APOBEC3G expression construct caused approximately 30 G-to-A hypermutations per 1000 bases analyzed (e.g., [41] ). Thus, approximately 12 G-to-A transitions should have been recovered in these PERV DNA analyses (nearly 90 if multiply recovered sequences would have been considered). The absence of hypermutated PERV proviral DNA provided further support for a deaminase-independent mechanism of restriction, which may share features with other instances described previously (e.g., [27] [28] [29] [30] ).
We have established a quantitative assay to monitor the zoonotic transmission of PERV to human 293T cells. Expression of human APOBEC3G in the pig PK-15 cell line strongly inhibited PERV zoonoses, while the endogenous APOBEC3F protein of pigs appeared considerably less effective. These data are the first to show that human APOBEC3G can inhibit PERV and the first to demonstrate that APOBEC3 proteins can be used purposefully to reduce if not prevent zoonotic retroviral infections. These results were not anticipated because human APOBEC3G has a relatively weak effect against the PERV-related gamma-retrovirus MLV [26, 39] .
Our data indicate that the engineering of pigs to express human APOBEC3G (or an equally potent non-porcine APOBEC3) may result in animals whose cells and tissues are much less likely to disseminate functional PERV. The deamination-independence of the restriction mechanism suggests that a catalytically inert APOBEC3G protein, such as E259Q, may be equally potent and simultaneously reduce the risk of cancer-promoting mutagenesis. APOBEC3G or APOBEC3G-E295Q expressing pigs may therefore constitute safer source animals for pig-to-human xenotransplantation procedures. In contrast to knockdown, knockout (by gene targeting or selective breeding) or most chemical-based anti-viral approaches to neutralize PERV [15] , the APOBEC3 antiviral defense system has several advantages including a potentially broad neutralizing activity (effective against PERV and likely several other endogenous and exogenous viruses) and an applicability to situations where many copies of a virus are already present in a genome. Analogous transgenic applications can be envisaged, such as using cross-species APOBEC3 expression to purposefully impede known viruses (e.g., the AIDS virus HIV-1 or the Hepatitis B virus HBV). Moreover, for humans and other mammals with multiple APOBEC3 proteins, our data encourage the development of methods to induce/up-regulate endogenous APOBEC3 proteins, which have the capacity but may not normally restrict a particular virus (e.g., human APOBEC3B and HIV-1).
The porcine kidney PK-15 fibroblast cell line and the swine testes ST-IOWA cell line were obtained from the ATCC and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini), and 25 units/ml penicillin and 25 mg/ml streptomycin at 37uC and 5% CO 2 . Human embryonic kidney 293T and HeLa (A. Bielinsky, University of Minnesota) cell lines were grown under the same conditions. The T cell lines H9 and CEM (M. Malim, Kings College London) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gemini), and 25 units/ml penicillin and 25 mg/ml streptomycin at 37 uC and 5% CO 2 . Plasmids encoding human APOBEC3G, human APOBEC3G-E259Q and porcine APO-BEC3F were described previously [41] . The human APOBEC3G and porcine APOBEC3F cDNA sequences used here are identical to GenBank accession numbers, NM_021822 and NM_001097446, respectively.
Stable APOBEC3G-or vector control-expressing PK-15 cell lines were constructed by transfection using FuGENE6 according to the manufacturer's protocol (Roche) or by electroporation (BioRad, 250V, 950 mFa). Clones were selected using growth medium containing 1 mg/ml G418 (Roche), and APOBEC3G expressing clones were identified by immunoblotting using a polyclonal antibody toward human APOBEC3G (J. Lingappa, University of Washington). All PK-15 clones were maintained in growth medium supplemented with 250 mg/ml G418 to ensure stable expression.
Long-term co-culture assays were performed in 6 well tissue culture plates with inserts (TranswellH, Corning Inc.). This system uses a membrane with 0.4 mM diameter pores, which keeps the two cell types separated physically but simultaneously allows diffusion of nutrients and small molecules including virus particles of approximately 0.1 mM (including PERV). Each experiment was initiated with 75,000 PK-15 cells (insert) and 75,000 293T cells (well) as illustrated ( Figure 1A ). At 72 hr intervals, each cell type was washed with PBS, subjected to mild trypsinization and diluted into 4 parts fresh growth medium. Excess 293T cells were used to prepare genomic DNA (Qiagen DNeasy kit).
The rate of PERV transfer was calculated using the pol gene levels from the last two data points (usually spanning a 3 day period). The difference between these levels represents PERV pol gene DNA that has accumulated per 100,000 human beta-actin gene copies (50,000 cells assuming that the 293T cell line has two beta-actin copies) per time period. Individual rates from 5 independent experiments were averaged to determine the overall transmission rate (190+/262 events per day per 50,000 cells). Data from Figures 1B, 2B and S2A contributed to rate calculations.
Genomic DNA was isolated from human 293T cells using the DNeasy kit (Qiagen). Duplicate 25 ml PCR reactions consisting of 10 ng of 293T genomic DNA, 100 nM primers and 26 iQ SYBR Green super mix (BioRad) were run on an iCycler iQ Multicolor Real-Time PCR detection System (BioRad). The thermocycler conditions consisted of an initial denaturation of 95uC for 5 min and 50 cycles of denaturation (95uC for 15 sec) and annealing (58uC for 30 sec). After the 50 cycles, a melting curve analysis (55uC to 95uC) was performed to confirm product specificity. The cycle threshold (C T ) was generated using BioRad software and it was used to calculate the amount of target DNA (PERV pol or human beta-actin). A standard curve was generated using the method of Dorak [56] and a dilution series (10 to 10 7 copies) of a linearized plasmid containing the relevant 193 bp PERV pol gene fragment. The equation generated from the standard curve (slope and y intercept) was used to determine the efficiency of the PCR reaction and to quantify the number of PERV pol gene or human beta-actin copies in the Q-PCR reactions. PERV copy numbers were normalized to those of beta-actin using the method of [56] . The primer sets used in this study were: PERV pol (193 bp):  59-AAC CCT TTA CCC TTT ATG TGG AT and 59-AAA GTC  AAT TTG TCA GCG TCC TT; 
Standard reverse transcription (RT)-PCR reactions were performed using RNA prepared from PK-15 cells (TRIzol protocol, Invitrogen), M-MLV reverse transcriptase was used for cDNA synthesis using an oligo dT primer (Ambion) and Taq polymerase was used for PCR (Roche). The primers specific to pig APOBEC3F were 59-TGG TCA CAG AGC TGA AGC AG and 59-TTG TTT TGG AAG CAG CCT TT (175 bp). The semi-nested primer set used to detect plasmid-expressed pig APOBEC3F was 59-CCA AGG AGC TGG TTG ATT TC (exon 6, reaction 1), 59-CTG GAG CAA TAC AGC GAG AG (exon 7, reaction 2) and 59-TAG AAG GCA CAG TCG AGG, with the latter being vector specific (319 bp and 190 bp products, respectively). The mammalian beta-actin primers were 59-CCT TCA ATT CCA TCA TGA AGT G and 59-CCA CAT CTG CTG GAA GGT (236 bp). These primers amplify equally well a 236 bp beta-actin fragment from all mammals tested, including pigs and humans (e.g., Online Figure S3 ).
The human APOBEC3G-GFP, pig APOBEC3F-GFP and GFP expression constructs were described previously [28, 41] . The pig and human cell lines were maintained as above. One day prior to transfection, 5,000-20,000 cells were seeded onto LabTek chambered coverglasses (Nunc). After 24 hrs incubation, these cells were transfected with 250 ng of the relevant plasmid construct. After 24 hrs of additional incubation, images of the live cells were acquired using a Zeiss Axiovert 200 microscope at 4006 total magnification. Images were analyzed using Image J software (http://rsb.info.nih.gov/ij).
Whole cell protein extracts were prepared from 293T cells by suspending 500,000 cells in PBS, sonicating twice for 5 seconds and clarifying the lysates by centrifugation. Soluble protein levels were quantified using a BioRad Bradford assay. 10 mg of cell lysate was tested for reverse transcriptase activity using a C-type-RT activity assay (Cavidi Tech) following the manufacturers' instructions. Cell-free PK-15 supernatants (PERV-containing) were assayed directly using the Cavidi Tech ELISA assay.
Human 293T cell genomic DNA was prepared from terminal cocultures and 50 ng was used for high fidelity, PERV pol genespecific PCR reactions (Phusion polymerase; Finnzymes). 193 bp products were cloned using the Zero Blunt TOPO PCR Cloning kit (Invitrogen) and sequenced (University of Minnesota Advanced Genetic Analysis Facility). Sequence comparisons were performed using Sequencher software (Gene Codes Corp.) and publicly available Clustal W alignment algorithms (http://align.genome. jp/). Figure S1 Quantitative Real-time PCR Analyses. (A) Standard curves depicting Q-PCR data obtained using dilutions of a linearized PERV pol gene plasmid alone (squares) or diluted plasmid plus 10 ng of 293T cell genomic DNA (diamonds). Under both conditions, all template amounts (10 to 10 7 copies) amplified efficiently (the log-linear slope equations are shown). The correlation co-efficiency value (R 2 ), which reports the technical accuracy of the assay, is also indicated. The standard curve data points were the average of 2 independent reactions with deviations smaller than the symbols (i.e., C T errors for each point ranged from 0 to 0.4). (B) Two representative control Q-PCR datasets showing the amplification of PERV pol gene DNA from pig PK-15 cell genomic DNA (circles). Two additional control Q-PCR datasets showing that the PERV-specific primers fail to amplify product from uninfected human 293T cell genomic DNA (squares). The reaction threshold, 10 times the mean standard deviation of the background fluorescence level (BioRad), is indicated. (C) Representative co-culture Q-PCR amplification curves of PERV pol gene DNA. Template genomic DNA isolated from human 293T cells co-cultured with vector expressing PK-15 cells (diamonds) or human APOBEC3G-expressing PK-15 cells (triangles) was used. (D) Representative Q-PCR amplification curves of the 293T cell beta-actin gene, which served as an internal standard for quantifying the real-time PCR data. Raw Q-PCR data will be made available on request. Found at: doi:10.1371/journal.pone.0000893.s001 (9.93 MB TIF) Figure S2 APOBEC3G inhibits PERV transmission. (A) A graph showing the accumulation of PERV pol gene-specific PCR products in 293T cells co-cultured with a control cell line (V3) but not with an APOBEC3G-expressing cell line (G1). The data points were an average of two Q-PCR runs and the difference between each run was smaller than the plotted symbol. The experimental parameters were identical to those used in the experiments shown in Figures 1B and 2B. (B) Relative levels of reverse transcriptase(RT)-activity detected in soluble extracts of day 28 co-cultured 293T cells, which were used to generate the Q-PCR data shown in Figure S2A . Uninfected 293T cell lysates had a relatively high endogenous RT activity. Therefore, to help with the presentation of these data, this level was normalized to one and all of the other data were calculated relative to this value. The level of RT activity in PK-15 extracts was much higher than that of 293T cell extracts (+/2PERV) and it had reached saturation (out of range) when these data were collected. Found at: doi:10.1371/journal.pone.0000893.s002 (4.76 MB TIF) Figure S3 Pig APOBEC3F Is Expressed in PK-15 Cells and its Over-expression Does Not Markedly Inhibit PERV Transmission. (A) An image of an ethidium bromide-stained agarose gel showing the results of an RT-PCR amplification experiment using PK-15 cellular RNA and appropriate controls. The top panel shows that PK-15 and representative PK-15 derived clones all expressed pig APOBEC3F, as indicated by the specific 175 bp pig APOBEC3F PCR product (confirmed by DNA sequencing). 293T cell mRNA and a diluted pig APOBEC3F expression plasmid were used as negative and positive controls, respectively. A larger, non-specific band was apparent only in the 293T cell RT-PCR reactions. The bottom panel shows that a conserved, 236 bp beta-actin gene fragment could be amplified from both PK-15 cells and human 293T cells (but not from diluted plasmid DNA). Note that this primer set differs from the human-specific set used in the Q-PCR experiments. The sizes of the marker (M) DNA bands are shown. (B) An image of an ethidium bromide-stained agarose gel showing expression of plasmid-derived pig APOBEC3F in PK-15 cells after 26 days of continuous co-culture. Non-transfected (NT) cells and diluted APOBEC3F plasmid DNA (pDNA) provided negative and positive controls, respectively. The larger 319 bp (far right lane only) and smaller 190 bp bands are the specific PCR products of the first and second rounds of semi-nested PCR, respectively (confirmed by DNA sequencing). (C) A histogram summarizing the level of PERV transmission that was observed after 23 days of co-culturing human 293T cells with PK-15 cells expressing a vector control or over-expressing pig APOBEC3F. Two datasets, each with an independent PK-15 clone in three replica co-culture wells, were collected in parallel and averaged for each histogram bar. One standard error of the mean is shown. The experimental parameters are identical to those used in Figure 1B . Found at: doi:10.1371/journal.pone.0000893.s003 (8.52 MB TIF) Figure S4 Genetic Variation in Zoonosed PERV pol Gene Sequences. (A) Sequences of the PERV pol gene fragments cloned from 293T cells co-cultured with control vector-expressing PK-15 cells. The number of times that each sequence was recovered is shown (N). Experiments 1 and 2 used genomic DNA prepared from the 293T cells used to generate the data shown in Online Figure S2 (day 28 samples) and Figure 2B (day 23), respectively. The most frequently detected 147 bp PERV pol gene sequence is shown in its entirety (which together with PCR primers makes up the 193 bp product shown in Figure 5 ). Identical nucleotides in other sequences are represented by dashes and non-identical nucleotides by the indicated DNA bases. GenBank accession numbers are shown for pol gene fragments with 100% identity to previously reported sequences. (B) Sequences of the PERV pol gene fragments cloned from 293T cells co-cultured with control APOBEC3G-expressing PK-15 cells. Parameters are identical to those described above. Found at: doi:10.1371/journal.pone.0000893.s004 (0.05 MB DOC) Experimental infection of H5N1 HPAI in BALB/c mice BACKGROUND: In 2005 huge epizooty of H5N1 HPAI occurred in Russia. It had been clear that territory of Russia becoming endemic for H5N1 HPAI. In 2006 several outbreaks have occurred. To develop new vaccines and antiviral therapies, animal models had to be investigated. We choose highly pathogenic strain for these studies. RESULTS: A/duck/Tuva/01/06 belongs to Quinghai-like group viruses. Molecular markers – cleavage site, K627 in PB2 characterize this virus as highly pathogenic. This data was confirmed by direct pathogenic tests: IVPI = 3.0, MLD(50 )= 1,4Log10EID(50). Also molecular analysis showed sensivity of the virus to adamantanes and neuraminidase inhibitors. Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Mean time to death of infected animals was 8,19+/-0,18 days. First time acute delayed hemorrhagic syndrome was observed in mice lethal model. Hypercytokinemia was determined by elevated sera levels of IFN-gamma, IL-6, IL-10. CONCLUSION: Assuming all obtained data we can conclude that basic model parameters were characterized and virus A/duck/Tuva/01/06 can be used to evaluate anti-influenza vaccines and therapeutics. Influenza A (H5N1) virus now becomes a real threat for humans. Since 1997, when first human case of H5N1 HPAI had been reported, more than 317 people were infected and 191 died [1] . Before 2005 attention was attracted to Thailand, Vietnamese and Indonesian viruses. In the beginning of 2005 outbreak on Quinghai lake occurred [2] . Later "Quinghai-like" viruses spreaded to most part of Russia, European countries and Africa and caused numerous outbreaks. Only in Russia more than 1 million of different species and sorts of poultry died and been slaughtered [3] . Confirmed cases in Azerbaijan, Egypt, Iraq, and Turkey was caused by Quinghai-like viruses. Earlier HPAI viruses were investigated in mice [4, 5] and murine models were successively used for reverse genetics made influenza vaccines [6] . It was shown that H5N1 HPAI viruses could have different pathogenicity for mice [7] . Several molecular markers were choused to explain differences. Multibasic cleavage site with 627K in PB2 designate to highly pathogenic phenotype for mice. Also important role of pulmonary cytokines elevation was highlighted [8] . Combination of adaptation for wild waterfowl and high virulence for mammals makes Quinghai-like viruses presumably pandemic. Also, in future, because of ability for rapid spreading for long distances, this group of viruses can appear in North and South America and cause outbreaks.
Human disease caused by HPAI viruses can be characterized as acute viral pneumonia aggravated by ARDS, toxic shock and multiple organ failure. System dysfunction mediated by hypercytokinemia and high viral load [9] . To be ready for new influenza pandemy it is necessary to use animal models, in vaccine and antivirals studies, which most closely reflect human disease. Isolates from FRSI SRC VB "VECTOR" repository which were characterized previously were examined for MLD 50 , molecular markers of pathogenicity, sensitivity to amantadines and neuraminidase inhibitors, to be candidates for murine model. Among the investigated isolates A/duck/Tuva/01/06 has best features to be used.
Genes of A/duck/Tuva/01/06 were sequenced and analyzed for molecular markers of pathogenicity. Also phylogenetic analysis was performed. Results are presented in figure 1 . A/duck/Tuva/01/06 belongs to group of Qinghailike viruses. HA contains 5 polybasic aminoacids (PQGRRKKKR↓GL) in cleavege site of HA [15] . The receptor binding domen can be characterized as "avian" [16] . High pathogenicity to mammals in general correlates with presence of 627K in PB2 [17] .
The analysis of non-structural protein 1 (NS1) which also could be contributed for high virulence of H5N1 viruses revealed deletion of 5 amino acids similar to those in H5N1 viruses of genotype Z which could be contributed to increased expression of TNF-α and IP-10 protein in primary human macrophages [18] . A/duck/Tuva/01/06 con-tained Glu 92 in the NS1 and contained "avian-like" PDZdomain ligand ESEV [19] . It was shown that the most recent H5N1 strains isolated in Southeast Asia were resistant to amantadine and rimantadine; antiviral drugs targeted the M2 ion channels of influenza A viruses [20, 21] . It was also reported about Oseltamivir resistant H5N1 viruses isolation from humans [22, 23] . To determine the potential sensitivity of studied H5N1 viruses to these antivirals, amino acid sequences of the M2 and NA proteins were analyzed.
Variants of influenza A viruses resistant to amantadine possessed amino acid substitutions at one of 5 residues (26, 27, 30, 31, and 34) in the M2 protein [24, 25] . Sequence analysis did not reveal any mutations associated with resistance to amantadine. Thus all A/duck/Tuva/01/ 06 is potentially sensitive to this class of antiviral agents. Amino acid residues 119, 274, 292 and 294 in the NA protein (numbering according to the HA of H2 subtype) are crucial for the sensitivity of influenza A viruses to neuraminidase inhibitors [26] ; substitution H 274 →Y in the NA conferred resistance to Oseltamivir was observed in clinical H5N1 isolates [25, 26] . Sequence comparison of the NA protein of A/duck/Tuva/01/06 aligned with the NA of N2 subtype of A/Wuhan/359/95 (H3N2) influenza virus showed phenotype potentially sensitive to neuraminidase inhibitors.
A/duck/Tuva/01/06 showed wide cross-reactivity with sera against H5N1 HPAI viruses isolated earlier in South-Eastern Asia. HI results can be found in table 1. These features persuade to use this virus in studies of vaccines made from various H5N1 influenza viruses.
First MID 50 and MLD 50 for A/duck/Tuva/01/06 were determined (table 2) . To determine mean time to death (m.t.d) and 2C. In several cases (9 animals totally) the disease was complicated by severe intestine atony, which can independently lead to death or by pressuring on diaphragm can intensify respiratory failure.
We also determined virus titers in several organ tissues. As it was expected the highest titers was observed in lungs -5,3 log EID 50 . Brain titers were also high -3,4 log EID 50 . In spleen, liver and kidney tissues virus titers were lower then 1 logEID 50 and considered not significant.
We investigated the involvement of several cytokines in immunopathogenesis of experimental H5N1 HPAI infection in mice. Results of ELISA technique revealed alteration of expression both pro-inflammatory and antiinflammatory cytokines after the challenge (figure 3). In general, the most marked changes of cytokine levels were observed before the death of mice.
The minimal concentration of IFN-γ was detected on day 5 (14.3 ± 10.8 pg/ml), however, its levels enlarged about 8-fold (256 ± 27 pg/ml) during the course of the infection when compared with uninfected animals. On days 3 and 5 systemic production of TNF-α was below the detection limit of the assay. A peak was reached on day 7 by the cytokine (24 ± 3.2 pg/ml) and its levels remained elevated on day 8. Interestingly, concentrations of IL-1β in mice after the challenge were significantly lower in comparison with the constitutive expression of the mediator in intact animals. An abrupt decrease of IL-1β was detected on day 3 post infection, but was followed by step increase from day 5. After the 2.5-fold enlargement on day 3 the levels of IL-6 decreased dramatically on day 5, and the highest levels of the cytokine were determined at the end of observation period (133 ± 12 pg/ml). The constitutive production of IL-10 was undetectable. The dynamics of IL-10 showed a gradual growth with the maximum level (92.1 ± 6.0 pg/ml) reached before the death of mice. We observed statistically significant increase of IL-12 after the challenge. Concentrations of the cytokine retained constant in infected mice, except the unexpected decline occurred on day 7. The expression of IL-18 could not be detected throughout the entire period of observation.
Until 2005 avian influenza was regional problem of sev- [4] and question "why had only some wild waterfowl died?" is still unclear. Most of the outbreaks in Russia associated with wild birds. The same time viruses adapted to wild birds are extremely pathogenic for poultry and mice. This "competitive advantage" makes Quinghailike viruses most probable candidate to be precursor for new pandemic influenza virus. At the same time pathogenesis of different (phylogenetical clades) HPAI reveal common causes. The principal causes of rapid mice death after infecting with HPAI are primary viral pneumonia, ARDS, lesions of central nervous system and multiple organ failure. Our data suggest that A/duck/Tuva/01/06 strain of HPAI caused lethal pneumonia and spread systemically to the brain in BALB/c mice. Lesion of respiratory epithelium and following an activation of monocytes/macrophages results in a release of proinflammatory cytokines (TNF-α, IL-6) which are a hallmark of ARDS in murine model [27] . Despite powerful anti-influenza virus effects of TNF-α in lung tissue, as it was described previously [28] , we consider that elevated production of the cytokines seems to be crucial in the pathogenesis of HPAI infection. Moreover, it was shown that lethal H5N1 viruses are resistant to antiviral effects of interferons and TNF-α [29] . Virus-induced overexpression of TNF-α as well as high IFN-γ lead to activation of endothelium and imbalance in blood coagulation system [30] . This may explain the hemorrhagic syndrome as observed in some of animals. To pay attention that IL-12 is a potent inducer of IFN-γ synthesis by blood mononuclear cells [31] , we concluded the same cytokines hyperproduction reflects macrophage overactivation and subsequent hypercytokinemia. This cascade of events Phylogenetic tree based on full length sequencesof HA Figure 2 Phylogenetic tree based on full length sequencesof HA. Nucleotide sequences were analyzed by using the neighborjoining method with 500 bootstraps. The phylogenetic tree was rooted to the HA gene of A/goose/Guangdong/1/96 (H5N1) virus.
including inflammatory mediator production, changes in blood coagulation system and microvascular permeability was denoted as systemic inflammatory response syndrome (SIRS) [32] . On the other hand, we proposed that the prominent production of IL-10 from the early stages of the experimental HPAI infection was the compensatory response to overproduction of proinflammatory cytokines such as TNF-α, IL-6 and IL-12. However, the role of IL-10, which principle function seems to be containment and eventual termination of inflammation [33] , in HPAI pathogenesis is unclear. Also there is an uncertain discrepancy between undetectable expression of IL-18 and high levels of other Th1-cytokines (IFN-γ and IL-12).
Summing up, in our study BALB/c mice infected with HPAI, strain A/duck/Tuva/01/06, appeared to be able to produce the innate immune response, which culminated to the development of shock and subsequent multiple organ failure. The main characteristics of our model are comparable to the previously described fatal cases of H5N1 influenza in humans [10, 11] . Proposed model reflects lesions not only same organs but also mediating levels of some (IFN-γ, IL-6, IL-10) cytokines in terminal conditions.
The implication of different cytokines in immunopathogenesis of experimental HPAI is beyond question. But to understand exact mechanisms, which determine the disease outcome, further experiments remain to be done. 
All experiments were performed in BSL 3+ facilities of FSRI SRC VB "Vector" of Rospotrebnadzor licensed for working with highly pathogenic avian influenza viruses.
Stock of A/duck/Tuva/01/06 was produced in 9 days-old chicken embryos. Allantoic fluid was aliquoted and stored at -80°C. The infectivity of stock viruses was determined in 10 days-old embryonated chicken eggs; titers were calculated by the method of Reed and Muench [10] and were expressed as log 10 of 50% egg infective dose (EID 50 ) in 1 ml of allantoic fluid.
Viral RNA was isolated from virus-containing allantoic fluid with the RNeasy Mini kit (Qiagen, Valencia, CA) as specified by the manufacturer. Uni-12 primer was used for reverse transcription. PCR was performed with a set of primers specific for each gene segment of Influenza A virus [11] . PCR products were purified with the QIAquick PCR purification (Qiagen).
Sequencing was done with Beckman Coulter Genom-eLab™ Methods development kit Dye terminator Cycle Sequencing according instructions of manufacturer. Primers for sequence were obtained from E. Hoffman (SJCRH, Memphis, TN). Sequence products were analyzed on automatic sequence analyzer Beckman Coulter CEQ2000.
Phylogenetical analysis was done on HA full gene sequence DQ861291 using MEGA 2.1 software. Phylogenetical tree was built by Neighbor-Joining method; matrix of distances was counted with p-distance algorithm. Reliability of clades was checked with bootstrap analysis with 500 replications. Other genes in GenBank DQ861291-DQ861295.
Cross-reaction of A/duck/Tuva/01/06 was defined by hemagglutination inhibition test (HI) with 0.5% CRBC [12] with a panel of antisera against H5N1 HPAI. 
Six-week-old inbred male BALB/c mice (vivarium of FRSI SRC VB "Vector"). Animals were placed to individual cages with food and water available ad libitum. To determine the MLD 50 and MID 50 , mice were anaesthetized by diethyl ether inhalation and infected intranasally with 50 µl 10-fold serial dilutions of allantoic fluidin PBS (pH 7,2). Each group contained 10 animals. Animals were observed daily for 15 days for mortality (MLD 50 ) or sacrificed on day 5 after the challenge with following virus detection in the lungs by inoculation of 10 days-old embryonated chicken eggs (MID 50 ). MLD 50 and MID 50 were calculated by the method of Reed and Muench. Animals from group where 1MLD 50 had been observed were taken to determine virus titers in lung, spleen, kidneys, and liver and brain tissues. Mind time to death (m.t.d) was calculated as previously described [13] . Pathogenicity to chickens was determined by IVPI test [14] . All animal studies were performed according protocols approved by Animal Care & Use committee of FSRI SRC VB "Vector".
To determine IFN-γ, TNF-α, IL-6, IL-10, IL1-β, IL-12 we use ELISA R& D Systems kits (Minneapolis, MN, USA). Serum levels of IL-18 were measured using commercial Mouse IL-18 ELISA test kit (MBL, Nagoya, Japan). Detection limits were as follows: TNF-α, less then 5,1 pg/ml; IL1-β, 3,0 pg/ml; IL6, 3,1 pg/ml; IL10, 4,0 pg/ml; IL-18, 25 pg/ml. Sera was taken on 0,3,5,7,8 days and aliquots and stored -80°C upon usage. Day 8 was chosen because m.t.d defined earlier in the work was 8,19 ± 0,18 days. Statistics was performed with Student t-test. Values p < 0,05 considered to be reliable. Prediction of RNA Pseudoknots Using Heuristic Modeling with Mapping and Sequential Folding Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5′ to 3′ folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results. is a bracket graph developed by Hofacker et al. [1] , where the bracket is matched by an equal and opposite bracket on the 3' side. Figure S2d There are many examples of symmetric I-loops found in RNA structure databases. Some asymmetric I-loops ( n ) can also be found.
))).))))
((((....(((((....)))))))))
g h e f 5' 3'
3' ((((.....) )))..(((((.....))))).)))). (4) For pseudoknots: parts of the structure still satisfy cases 1 through 3.
However, in addition, in at least one part of a region between and l such that , there exists some base pairs that satisfy either or k , , ', '
Hence, many more possibilities can be generated once we start allowing pseudoknots.
Pseudoknots differ from real knots in the sense that the strand does not pass completely through the loop but only becomes potentially entangled with it. Anyone who has tried to untangle a pair of earphones or tried to untangle a recently, neatly wound up cord can realize that knots naturally occur on long flexible cords. Indeed, great effort seems to be required to avoid tangling a heap of cords. Figure S5a shows a common pseudoknot known as an H-type pseudoknot. This is also known as an ABAB pseudoknot. The structure is notated below with the standard parenthesis notation for the basic secondary structure (here shown in blue) and square brackets for the pseudoknot linkage (here shown in red). Green indicates the regions of free strand that are not forming base pairs (bps).
The color distinction of the stems in this example is not important because both stems are the same length and both stems are less than 10 bps. [[[[[[[[[[.) ))))))))))))) . [.) ))))))))))))) . and also known as an ABACBC pseudoknot. (f) The structure shown in bracket notation. (g) A pseudoknot. The difference between (c) and (g) is that the linkage stem is shorter than 9 contiguous bps. (h) The same structure in bracket notation. Figure S5c shows a knot and the corresponding bracket structure is shown below in Fig. S5d .
Both stems in Fig. S5c contain 14 base pairs (bp). Since the helical axis makes a rotation of 360 o every 10 bps, this means that the structure in Fig. S5c is tangled in a knot. There is no reason why such a structure cannot form. Indeed, knots are known to form in some rare proteins [2, 3] . However, it is not a pseudoknot and the current approach is not designed to estimate its existence or the likelihood of its formation. One important feature of a pseudoknot is, therefore, that the linkage stem (here shown in red) must be shorter than 10 contiguous bps. Figure S5e shows two stem-hairpins-loops (blue and purple) that are joined by a linkage stem (red). The stems are all short as in Fig. S5a . This is a pseudoknot, sometimes referred to as a kissing loop. It is also known as an ABACBC pseudoknot. It is also observed in a number of places, although less frequently than H-type pseudoknots. The corresponding bracket notation for this structure is shown below (Fig. S5f) . Belvedere knot secondary structure Supplement Figure S6 . A special type of knot (right hand side) that becomes entangled due to the way the structure folds up. The two dimensional nature of the RNA schematics tends to hide curious possibilities as this. The knot is seen in equilibrium between the unknotted structure (left hand side) and the "Belvedere knot" (right hand side).
Functional RNA structures that contain knots of the form shown in Figs. S5c or S6 are currently unknown or have not been reported. However, pseudoknots are often observed in functional RNA structures, particularly H-type pseudoknots (Fig. S5a) . Single strand RNA sequences such as messenger RNA with introns can have sequences with lengths that number in the tens of thousands of nucleotides. With such a propensity for a few simple cords to become tangled, and, since the cell can have many thousands of protein and RNA strands present within the cellular environment, this suggests that there is a fair amount of effort made within the cell to prevent or get rid of knots [2] .
In this work, we are concerned with the prediction of pseudoknots. These structures have the property that they can be evaluated as structures resulting from reversible folding. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-α amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP−PMOs. Insertion of 6-aminohexanoic acid (X) or β-alanine (B) residues into oligoarginine R(8) decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 μM. Up to 60 μM, only the conjugates with ⩾ 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP−PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues. Steric-blocking antisense oligonucleotides (AOs) are considered potential therapeutics for genetic diseases such as Duchenne muscular dystrophy (DMD) and b-thalassemia. For their potential to be realized, however, the AOs must be effectively delivered to cell nuclei.
Cationic lipoplex-or PEI-based transfection methods used to deliver charged AOs are not suitable for the delivery of uncharged AOs such as phosphorodiamidate morpholino oligomers (PMO, Figure 1 ) (1) and peptide nucleic acids (PNAs) (2) . Conjugation of PMO to short CPPs is a good method to enhance the cytoplasmic and nuclear delivery of PMO because the conjugates are simple to use and because the short peptides and their AO conjugates can be easily manufactured and characterized in a qualitycontrolled manner. Examples of well-studied CPPÀPMO conjugates include those with Tat and oligoarginine peptides (3, 4) Important considerations in the design of effective CPPs include the ability to deliver AO efficiently, stability in living systems and toxicity. We have reported that Tat and oligoarginine peptides are not stable in human serum (5) , and are therefore ill-suited for in vivo applications. Oligoarginine peptides incorporating non-a amino acids have been proven superior to oligoarginine alone. CPPs containing 6-aminohexanoic acid (X) and b-alanine (B) were more stable in human serum than Tat or oligoarginine peptides (5) . A CPPÀPMO conjugate, (RXR) 4 ÀPMO, has been shown to be more efficient in the correction of pre-mRNA mis-splicing (6) and in inhibition of the replication of mouse hepatitis virus in vivo (7) than an oligoarginine peptide. In addition, (RXR) 4 ÀPMO conjugates have been shown to cause effective exon skipping in muscle cells from DMD dogs (8) , in human muscle explants (9) and in mdx mice (10) , as well as inhibiting the replication of various viruses in cell cultures (7, (11) (12) (13) and in mice (7, 13) .
The above studies have helped make it clear that unnatural amino acids can confer enhanced stability and activity, and therefore improve the potential of CPPs to deliver therapeutic PMO. In pursuit of CPPs with improved characteristics, we have carried out a structure-activity relationship study to investigate the effects of unnatural amino acid insertions in oligoarginine peptides on cellular delivery, nuclear antisense activity, toxicity and serum-binding characteristics of the resulting CPPÀPMO conjugates. The unnatural amino acids studied here are X, B and D-arginine (r). We chose to study the X amino acid based on the successes of the (RXR) 4 CPP in several studies as shown in the previous paragraph. B and r amino acids were chosen because they have good enzymatic stability (5) . The CPPs are (i) the oligoarginine sequences, R 8 and R 9 , (ii) sequences with RXR, RX and RB repeats, as well as various combinations thereof, and (iii) sequences containing D-arginine, r 8 , (rX) 8 (rXR) 4 , (rXr) 4 and (rB) 8 . The CPPÀPMO conjugates were evaluated for their relative (a) cellular uptake, as determined by flow cytometry, (b) antisense activity, as determined by a splice correction assay (13) and (c) cellular toxicity, as determined by MTT cell viability, propidium iodide membrane integrity and hemolysis assays, as well as by microscopic imaging.
CPP nomenclature and sequences are listed in Table 1 . Chemical structures of PMO and (RX) 8 ÀPMO are shown in Figure 1 . The antisense PMO (CCT CTT ACC TCA GTT ACA) is designed to target a b-thalassemic mutant splice site present in the human b-globin intron 2 of a positive-readout antisense activity assay system (13) as described in the Results section. Synthesis of PMO, described previously (15, 16) , and the CPPs, using standard Fmoc chemistry (17) , were performed at AVI BioPharma, achieving purities of 490% as determined by HPLC and mass spectrometry analysis. Conjugation of a CPP to a PMO through an amide linker, described previously (6) , was followed with an additional purification step to remove nonconjugated peptide. Samples were loaded on source 30S resin (Amersham Biosciences, Pittsburgh, PA) in a 2 ml Biorad (Hercules, CA) MT2 column at 2 ml/min with running buffer A (20 mM Na 2 HPO 4 , 25% acetonitrile, pH 7.0) and purified into 45-s fractions with 0-35% buffer gradient (buffer B: 1.5M NaCl, 20 mM Na 2 HPO 4 , 25% acetonitrile, pH 7.0) over 60 min, using a Biorad BioLogic low pressure chromatography system. The desired faction was desalted by a method described previously (6) . HPLC and MS analyses revealed that the final product contained 490% CPP conjugated to fulllength PMO, with the balance composed of CPP conjugated to incomplete PMO sequence, nonconjugated full-length or incomplete PMO.
The HeLa pLuc705 (pLuc705) (14) cell line was obtained from Gene Tools, LLC (Philomath, OR). Human liver cell line HepG2 was from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 2 mM L-Glutamine, 100 U/ml penicillin and 10% fetal bovine serum (FBS) (HyClone, Ogden, UT) at 378C in a humidified atmosphere containing 5% CO 2 . All treatments were carried out in OptiMEM medium (Gibco, Inc., Carlsbad, CA.) with or without FBS.
Cell uptake assay pLuc705 cells were seeded 20 h prior to treatment in 12-well plates at 100 000 cells/well. Cells were treated with 2 mM fluorescein-tagged CPPÀPMO conjugates for 24 h. After treatment, cells were washed with 500 ml of 7 RXRXRXRXRXRXRXB 7 (RX) 5 RXRXRXRXRXB 5 (RX) 3 RXRXRXB 3 (RXR) 4 RXRRXRRXRRXRXB 5 (rXR) 4 rXRrXRrXRrXRXB 5 (rXr) 4 rXrrXrrXrrXrXB 5 (RB) 8 RBRBRBRBRBRBRBRBB 0 (rB) 8 rBrBrBrBrBrBrBrBB 0 (RB) 7 RBRBRBRBRBRBRBB 0 (RB) 5 RBRBRBRBRBB 0 (RB) 3 RBRBRBB 0 Ã The sequences of peptides are written from N to C terminus. R = arginine, r = D-arginine, X = 6-aminohexanoic acid, B = b-alanine. Each peptide had an acetyl group at the N-terminus and a carboxyl group at the C-terminus. Cell viability assay and microscopy pLuc705 cells were seeded 20 h before the treatment in 96-well plates at 9000 cell/well and then treated with the conjugates. The microscopic phase images of treated cells were visualized by a Nikon Diaphot inverted microscope (Melville, NY), captured by an Olympus digital camera and processed by the Magnafire software (Optronics, Goleta, CA). After imaging the cells, the cell viability was determined by the methylthiazoletetrazolium assay (MTT, Sigma, St. Louis, MO) assay. MTT solution (5 mg/ml) was added to the treatment medium to a final concentration of 0.5 mg/ml and incubated for 4 h at 378C. 85% of the media of each well was then replaced with DMSO containing 0.01M HCl and further incubated for 10 min at 378C and the absorbance measured at 540 nm. Percent cell viability was determined by normalizing the absorbance of each treated sample to the mean of untreated samples.
Propidium iodide membrane integrity assay pLuc705 cells were seeded 20 h before treatment in 12-well plates at 100 000 cells/well. Cells were treated by removing the medium, washing with 500 ml PBS and incubating with medium containing CPPÀPMO conjugates. Treatment medium was collected in tubes and cells were washed with PBS once, and then treated with 400 ml of 10% trypsin for 10 min at 378C. Trypsin was neutralized with 500 ml of the serum containing medium. Cells were transferred to the tubes containing previously collected treatment medium, pelleted by centrifugation at 1000g for 5 min, washed with PBS once, and re-suspended in 200 ml of 0.05 mg/ml propidium iodide (PI) in PBS. Cells were further incubated at 378C for 15 min and analyzed by the Beckman Coulter cytometer (30 000 events/sample collected).
The hemolytic activities of the conjugates were determined in fresh rat blood according to a method described elsewhere (18) .
Cellular uptake of CPPÀPMO conjugates was investigated using the 3 0 -carboxyfluorescein-tagged PMO (PMOF) and flow cytometry. We chose 2 mM as the treatment concentration because none of the conjugates caused any detectable cytotoxicity at this concentration, as demonstrated by the MTT and PI uptake assays. After treating with the conjugates, cells were treated with trypsin (19) to remove membrane-bound conjugates. We found that a heparin sulfate washing step prior to trypsin treatment did not remove additional membrane-bound conjugates but caused some cellular toxicity (data not shown); therefore, only the trypsin treatment step was used in this study.
To determine the effect of serum on cellular uptake of the various conjugates, uptake evaluation assays were carried out in the medium containing various concentrations of, or in the absence of, serum. Cellular uptake of CPPÀPMOF conjugates increased with the number of arginines and decreased with the X and/or B residue insertion (Figure 2A and B). The oligoarginine R 9 ÀPMOF had a mean fluorescence (MF) of 662, nearly 3-fold higher than the 234 produced by R 8 ÀPMOF, indicating that a difference of a single arginine can make a substantial difference in the biological properties of a CPP. Insertion of an X or B residue in the R 8 sequence reduced the MF from 234 of R 8 ÀPMO to 42, 70 and 60 of (RX) 8 À, (RXR) 4 À and (RB) 8 ÀPMOF, respectively ( Figure 2A ). The number of RX or RB repeats affected cellular uptake, with conjugates having fewer RX or RB repeats generating lower MF ( Figure 2B) .
While the addition of 10% serum to the medium caused a decrease in the uptake of the R 8 À or R 9 ÀPMOF conjugates, it increased the uptake of conjugates containing RX, RB or RXR motifs (Figure 2A and C) . Serum reduced the MF of R 9 À and R 8 ÀPMOF from 662 and 234 to 354 and 158, respectively, and increased the MF of (RX) 8 À, (RXR) 4 À and (RB) 8 ÀPMOF from 41, 70 and 60 to 92, 92 and 111, respectively. These differences were statistically significant (Figure 2A ). However, higher serum concentrations (30 and 60%) decreased the uptake of (RXR) 4 ÀPMOF and oligoarginineÀPMOF ( Figure 2C ).
Arginine stereochemistry (D versus L) had little effect on the uptake of CPPÀPMOF conjugates. We compared the MF of R 8 À, (RB) 8 À and (RX) 8 ÀPMOF with their respective D-isomer conjugates, r 8 À, (rB) 8 À and (rX) 8 ÀPMOF and found that there was no significant difference between each pair, as shown in Figure 2D for the (RX) 8 À and (rX) 8 ÀPMOF pair.
Nuclear antisense activity.
The effectiveness of each CPPÀPMO conjugate was determined in a previously described splicing correction assay (14) , considered a reliable method to assess nuclear antisense activity of a steric-blocking AO. This assay utilizes the ability of steric-blocking AOs to block a splice site created by a mutation in order to restore normal splicing. The luciferase coding sequence was interrupted by the human b-globin thalassemic intron 2 which carried a mutated splice site at nucleotide 705. HeLa cells were stably transfected with the plasmid therefore named as pLuc705 cell. In the pLuc705 system, steric-blocking AOs must be present in the cell nucleus for splicing correction to occur. Advantages of this system include the positive readout and high signal-to-noise ratio. With this system the relative efficiencies of various CPPs to deliver an AO with sequence appropriate for splice-correction to cell nuclei can be easily compared.
Oligoarginine, RX, RXR and RB panels. The CPP conjugates with the highest nuclear antisense activities were (RXR) 4 À and (RX) 8 ÀPMO. Figure 3A and B show luciferase activity normalized to protein of cells treated with various conjugates at 1 and 5 mM for 24 h. At both concentrations, (RX) 8 À and (RXR) 4 ÀPMO were more effective than the other conjugates tested, with the difference more prominent in serum-containing medium at 1 mM than at 5 mM. Cells treated with 1 mM of either conjugate had luciferase activity 10-15-fold over the background while the remaining conjugates yielded about a 2-4-fold over the background (Figure 3A ). At 5 mM, all conjugates generated higher luciferase activity Figure 2 . Cellular uptake of the CPPÀPMOF conjugates. pLuc705 cells were treated with carboxyfluorescein-tagged PMOs conjugated to CPPs in OptiMEM with or without 10% serum for 24 h, followed by flow cytometry analysis. Data are presented as mean fluorescence (MF) AE SD of six data points from two independent experiments. (A) Cells treated with 2 mM of R 8 2, R 9 2, (RXR) 4 2, (RX) 8 2 or (RB) 8 2PMOF conjugates. (B) Cells treated with 2 mM of (RX) 8 2, (RX) 7 2, (RX) 5 2, (RB) 8 2, (RB) 7 2 or (RB) 5 2PMOF conjugates in the absence of serum. (C) Cells treated with 2 mM of (RXR) 4 2 or R 9 2PMOF in media containing 0, 10, 30 or 60% serum. (D) Cells treated with (RX) 8 2 or (rX) 8 2PMOF in media containing 10% serum. than at 1 mM, with (RX) 8 ÀPMO and (RXR) 4 ÀPMO again the most effective, followed by (RB) 8 ÀPMO ( Figure 3B ). Figure 3C shows that at 10 mM, the activity of RX or RB conjugates decreased as the number of RX or RB repeats in the CPP decreased. The peptides with 5 or 3 RX or RB repeats, (RX) 5 , (RX) 3 (RB) 5 or (RB) 3 , generated much lower luciferase activity than those with 8 and 7 repeats.
Number and position of X residues. Having shown that (RB) 8 ÀPMO had less activity than (RXR) 4 ÀPMO or (RX) 8 ÀPMO, we further investigated the effect of the number and position of X residues on the activity of conjugates. Eleven CPPÀPMO conjugates containing 0, 2, 3, 4, 5 or 8 Xs were compared (Figure 4) . Generally, CPPs containing a higher number of Xs had higher activities. At 2 mM, (RX) 8 ÀPMO (8 X residues) had the highest activity followed by (RXR) 4 ÀPMO (5 X residues) and the conjugates with fewer Xs had lower activities. At 5 mM, three conjugates containing 3 (3d), 4 (4c) and 8 ((RX) 8 ) X residues had the highest activities, suggesting that the position of X residues affects activity.
L-Arginine versus D-arginine. Arginine stereochemistry had little effect on the nuclear activity of the R 8 À and (RB) 8 ÀPMO conjugates but affected the (RX) 8 ÀPMO ( Figure 5 ). Replacement of the eight L-arginine residues in R 8 À or (RB) 8 ÀPMO with D-arginine residues did not change the luciferase activity generated over the 1-5 mM ( Figure 5A and B) . However, the replacement did cause a small but statistically significant decrease in the activity for (RX) 8 ÀPMO at 1 mM (P = 0.03) and 2 mM (P = 0.01) ( Figure 5C ).
Serum effect on activity. The effect of serum on the antisense activity of the conjugates depended on the CPP sequences, as shown in Figure 3A -D. Addition of 10% serum to the medium decreased the activity of oligoarginineÀPMO conjugates (R 8 ÀPMO and R 9 ÀPMO) but increased activity of conjugates containing RXR, RX and RB repeats. The addition of 10% serum nearly doubled the luciferase activity of (RXR) 4 À, (RX) 8 À and (RB) 8 ÀPMO at 5 mM ( Figure 3B ). We further studied this effect for (RXR) 4 ÀPMO up to 60% serum. While the activity almost doubled as the serum concentration increased from 0 to 10%, it gradually decreased as the serum concentration increased to 60%, at which activity was similar to that in 0% serum which was still significantly above the background. This 'up and down' profile was also observed with the 1 mM (RXR) 4 ÀPMO treatment. Unlike (RXR) 4 ÀPMO, the luciferase activity of R 8 ÀPMO or R 9 ÀPMO (data not shown) only decreased as the serum concentration increased, with an approximately 30% reduction in 10% serum and no activity in 60% serum. R 8 ÀPMO or R 9 ÀPMO did not display any detectable activity at 1 mM, regardless of the serum concentration (data not shown).
The cellular toxicity of the various CPPÀPMO conjugates was determined by MTT-survival, propidium iodine (PI) exclusion and hemolysis assays and microscopic imaging. The MTT and PI exclusion assays measure metabolic activity and membrane integrity of cells, respectively. The hemolysis assay determines compatibility with blood. Microscopic images were used to verify the MTT results and observe the general health of the cells.
MTT assay. pLuc705 cells were treated at concentrations ranging from 2 to 60 mM for 24 h. As shown in Figure 6 , all conjugates, except (RX) 8 and (RXR) 4 , had no toxicity at up to 60 mM. Up to 10 mM, (RX) 8 and (RXR) 4 conjugates exhibited no toxicity, at higher concentrations they reduced cell viability in a concentration-dependent manner, with (RX) 8 being more toxic than (RXR) 4 ( Figure 6C and D) . Replacement of L-arginine with D-arginine in R 8 À, (RB) 8 À and (RXR) 4 ÀPMO did not change the viability profiles of these conjugates ( Figure 6A-C) . Surprisingly, the L!D replacement in (RX) 8 ÀPMO decreased the toxicity. Cell viability with 60 mM treatment was 40% for (RX) 8 ÀPMO, but 80% for (rX) 8 ÀPMO ( Figure 6D ). The eight conjugates containing fewer than 5 X residues did not inhibit cell proliferation up to 60 mM ( Figure 6E ). Monomers of arginine or X, individually or in combination, at 500 mM each, produced no inhibition of cell proliferation ( Figure 6F ). The toxicities of the CPPÀPMO conjugates, (RXR) 4 ÀPMO, 3dÀPMO and 4cÀPMO, were also evaluated in human liver HepG2 cells. We found that only (RXR) 4 ÀPMO caused dose-dependent inhibition of cell proliferation while other two conjugates had no toxicity up to 60 mM, the highest concentration tested in this study (data not shown).
Microscopic images. We sought to verify the MTT results by collecting microscopic images of cells treated with 60 mM of the conjugates. The images correlated well with the cell viability data. Images of (RX) 8 À, (rX) 8 À, (RXR) 3 RBRÀ (4c), (RXR) 4 ÀPMO and vehicle-treated cells are shown in Figure 7 . Cells treated with (RX) 8 ÀPMO and (RXR) 4 ÀPMO appeared rounded and detached from the culture well, and appeared to have fewer live cells. Interestingly, cells treated with (rX) 8 ÀPMO appeared to have normal morphology and cell density. The replacement of one X of (RXR) 4 ÀPMO . Nuclear activity of CPP-PMO conjugates: number and position of X residues. pluc705 cells were treated with the conjugates having 0, 2, 3 (3a, 3b, 3c and 3d), 4 (4a, 4b and 4c), 5 and 8 X residues (see sequences in Table 1 ) in OptiMEM medium with 10% serum for 24 h. Nuclear activity of a conjugate is indicated by relative luciferase activity (RLU) per microgram of protein. Data represent a mean AE SD of 9-12 data points from four independent experiments. Propidium iodine exclusion assay. The effect of the conjugates on integrity of cell membranes was investigated by a propidium iodine (PI) exclusion assay. PI can only permeate unhealthy/damaged membranes, so positive PI fluorescence indicates compromised cell membranes. Only (RXR) 4 À and (RX) 8 ÀPMO conjugates were found to significantly affect membrane integrity at higher concentrations (up to 60 mM tested). Figure 8A shows the histograms of pLuc705 cells treated with (RXR) 4 ÀPMO at 60 mM for 0.5, 5 and 24 h. The PI positive (PI+) region was defined by the cells permeabilized with ethanol (positive control) as indicated by the gate in the histogram. The PI histogram shifts from the PI-negative region to PI-positive region in the longer incubations, indicating the conjugate caused membrane leakage in a time-dependent manner. The 0.5-and 5-h-treatments caused a slight shift towards the PI+ region, while the 24-htreatment produced a distinct peak which corresponded to 57% of cells that were in the PI+ region. Figure 8B shows the histograms of cells treated with (RXR) 4 ÀPMO at concentrations of 2, 10, 20, 40 and 60 mM for 24 h. There was no significant PI uptake at concentrations up to 20 mM. At higher concentrations, the PI+ population appeared and the percentage of PI+ cells increased as the treatment concentration increased, indicating that there were more leaking cells at the higher treatment concentration. Similar concentrationand time-dependent PI uptake profiles were observed for (RX) 8 ÀPMO but not for (RB) 8 ÀPMO and the remaining conjugates (data not shown). Addition of 10% serum to the treatment medium significantly reduced membrane toxicity for the (RXR) 4 À ( Figure 8C ) and (RX) 8 ÀPMO conjugates (data not shown).
Hemolysis assay. The (RXR) 4 À and (RX) 8 ÀPMO conjugates were tested in a hemolysis assay and found to be compatible with red blood cells. Fresh rat red blood cells were treated with the conjugates at 60 mM, PBS (background) or 0.005% TX-100 (positive control). The supernatants of conjugate-and PBS-treated samples had small and similar amounts of free hemoglobin released, far lower than that of the TX-100-treated samples ( Figure 8D ).
The naturally occurring CPPs such as Tat peptide are not stable in blood and neither are oligolysine/oligoarginine (5) , rendering these CPPs unfavorable as transporters for therapeutic AOs. We reasoned that one approach to improve stability would be to use non-a amino acids or D-amino acids. In this study, we investigated whether incorporation of 6-aminohexanoic acid (X), b-alanine (B) and D-arginine (r) amino acids into the CPP would affect cellular delivery, antisense activity, toxicity and serum binding of the resulting CPPÀPMO conjugates.
We found that CPPÀPMOF conjugates containing X/B residues did not enter cells as efficiently as R 8 À and R 9 ÀPMO conjugates. This is consistent with our previous finding for the (RXR) 4 conjugate (6). We have found that cell surface proteoglycans were involved with binding of the TatÀ, R 9 F 2 À and (RXR) 4 ÀPMO conjugates with the (RXR) 4 conjugate having the lowest binding affinity. Insertion of X into an oligoarginine CPP reduces the charge density and may lead to decreased binding affinity for proteoglycans. Despite the lower cellular uptake of X/B-containing CPP-PMO, they generated higher antisense activities in the cell nucleus than oligoarginineÀPMO. We have found that endocytosis was the internalization mechanism (at least primarily) for oligoarginine-and (RXR) 4 ÀPMO conjugates. Indication of different uptake mechanisms was not found among these conjugates (6) . Therefore we hypothesize that X/B-containing conjugates have a greater ability to escape from endosomes/lysosomes than oligoarginine conjugates by a mechanism as yet to be studied.
The number of X residues affects both the nuclear antisense activity and the toxicity of conjugates. The CPPÀPMO conjugate with 8 X residues [(RX) 8 ÀPMO] had the highest activity followed by one with 5 Xs [(RXR) 4 ÀPMO] (Figures 1 and 4) . However, these conjugates were toxic to cells at higher concentrations, which may be a concern when considering potential applications for in vivo delivery of PMO. Replacement of all 8 Xs with Bs decreased both toxicity and antisense activity. The combination of 3-4 Xs with several B residues yielded CPPs with no detectable toxicity, and at some concentrations several of them had similar antisense activity as (RX) 8 ÀPMO. We think this type of CPP, having Bs and fewer than 5 Xs, will offer balanced activity and low toxicity as well as the stability, and have considerable potential for delivery of therapeutic AOs. Further investigation into the toxicity and activity versus dosing levels of these CPPs in vivo is warranted.
Surprisingly, the replacement of L-arginine with D-arginine enhanced neither uptake nor antisense activity for oligoarginine, or X-and B-containing conjugates. In the case of (RX) 8 ÀPMO, the replacement actually caused a small but statistically significant decrease in activity. Our observation is different from the results reported by others (20, 21) who found that D-CPPs had higher cellular uptake than L-CPPs, although no biological functional cargo was used in their study. The difference between results may be due to the type and size of cargos and the cell lines used for the assays. Whether the use of D-arginine-containing peptides results in superior CPPÀPMO functional activity in vivo remains to be tested.
We attempted to understand the nature of (RX) 8 À and (RXR) 4 ÀPMO toxicity. It is apparent that these two conjugates caused little immediate membrane damage with 0.5 or 5 h treatment at concentrations as high as 60 mM (Figure 8 ). However, these two conjugates had dose-dependent toxicity with 24 hr treatment as shown by the leaky cell membranes and fewer cells compared to controls ( Figure 6C&D, Figure 8 ). Interestingly, the replacement of Xs with Bs in (RX) 8 ÀPMO abolished the toxicity, and the replacement of L-arginine with D-arginine reduced the toxicity of (RX) 8 ÀPMO ( Figure 6 ). We have found that (rX) 8 ÀPMO was completely stable and the peptide portion of (RB) 8 ÀPMO was only partially degraded, whereas the peptide portion of (RX) 8 ÀPMO was completely degraded in cells (5) . We wondered whether the difference in toxicity among (RX) 8 , (RB) 8 and (rX) 8 ÀPMO conjugates was caused by differences in intracellular stability, resulting in the metabolized products of (RX) 8 ÀPMO producing toxicity. The identifiable metabolized products of (RX) 8 ÀPMO were XRXBÀPMO and XBÀPMO (5) but neither product had any detectable toxicity as measured by MTT assay (data not shown). It is possible that the CPP portion was degraded into free amino acids and/or smaller peptide fragments which were toxic. However, our investigation revealed that neither free R nor X, alone or in combination, caused cellular toxicity. Another possibility is that because of the high hydrophobicity of X compared to B, X in combination with positively charged arginine residues leads to toxicity not generated by B residue combinations. However, this explanation does not account for the difference in toxicity observed between (RX) 8 ÀPMO and (rX) 8 ÀPMO, which have the same hydrophobicity. Perhaps the toxicity of (RXR) 4 ÀPMO and (RX) 8 ÀPMO was caused by the peptide fragments that we could not identify by mass spectrometry.
Unlike the toxicity difference between (RX) 8 À and (rX) 8 ÀPMO, the L!D replacement did not change the toxicity of (RXR) 4 ÀPMO ( Figure 6 ). Substitution of either one R (rXR) or two R (rXr) from the RXR repeat neither reduced nor increased the toxicity profile of (RXR) 4 ÀPMO. At this point, we do not fully understand the mechanisms of (RXR) 4 À and (RX) 8 ÀPMO conjugate toxicity, but look forward to studying this topic further.
Serum effect on the activity of a CPPÀAO conjugates is an important issue when considering potential in vivo applications. X/B-containing conjugates were still active in 60% serum while oligoarginine conjugates were not. The greater stability of the X/B-containing conjugates to serum enzymes is likely a factor contributing to their high activity. The loss of activity in high serum concentrations makes oligoarginine CPPs undesirable as potential therapeutic AO carriers.
In summary, we have found that the X/B-containing CPPÀPMO conjugates are superior to oligo-arginineÀPMO conjugates for the following reasons: they display higher activity in cell nuclei, are less affected by serum and are more stable in blood (5) . The toxicity of the X/B-containing CPPs can be reduced by keeping the number of X residues below 5 while still maintaining a reasonable delivery efficacy and stability. This study provides a basis for further optimization of CPP sequence using R, r, X and B residues in the interest of further reducing toxicity and increasing antisense activity, which will likely lead to more effective AO transporters for potential therapeutic applications. Lost in the World of Functional Genomics, Systems Biology, and Translational Research: Is There Life after the Milstein Award? We've always wanted to save the world from the scourges of virus infection by developing better drugs and vaccines. But fully understanding the intricacies of virus-host interactions, the first step in achieving this goal, requires the ability to view the process on a grand scale. The advent of high-throughput technologies, such as DNA microarrays and mass spectrometry, provided the first opportunities to obtain such a view. Here we describe our efforts to use these tools to focus on the changes in cellular gene expression and protein abundance that occur in response to virus infection. By examining these changes in a comprehensive manner, we have been able to discover exciting new insights into innate immunity, interferon and cytokine signaling, and the strategies used by viruses to overcome these cellular defenses. Functional genomics may yet save the world from killer viruses. At the time I received the Milstein Award in September 1999, my laboratory was embarking on an exciting new venture. I had become fascinated by the novel opportunities that were becoming available through the sequencing of the human genome and the advent of highthroughput technologies such as DNA microarrays and mass spectrometry [1] . Because these technologies provided a way to obtain a nearly comprehensive view of a biological system, I was convinced they offered the best chance to learn everything there is to know about the complex interplay between viruses and the cells they infect. Over the past eight years, my laboratory has worked tirelessly, and even obsessively, to incorporate genomic, proteomic, and information technologies into our research. Although we certainly don't yet know everything there is to know about virus-host interactions, we've made tremendous progress, and I remain convinced that genomic and proteomic approaches will be a fundamental force for pushing the field of virology forward in the years to come. In this article, we provide a sampling of how we're using these technologies, the discoveries we've made, and how the technologies themselves have continued to evolve.
Our initial foray into the world of genomics began with cDNA microarrays that were spotted in-house using a commercially available collection of cDNA clones. Getting this new technology up and running required months of work. But after seemingly endless frustration, Gary Geiss was finally able to use these arrays, representing all of 1,500 human genes, to evaluate the changes in cellular gene expression that occurred in a CD4+ T-cell line infected with human immunodeficiency virus type 1 (HIV-1) [2] . This was a straightforward study in which we analyzed two time points after infection, and we were thrilled to observe the temporal regulation of genes involved in T-cell signaling, subcellular trafficking, and transcriptional regulation. Despite how simplistic this study now appears, it was the first ever to apply genomics to AIDS research and it was the cover article when published in Virology. With the technology now in place, we were eager to apply it to a broad range of experimental systems. The possibilities seemed endless (as indeed they are), and we rapidly began "doing arrays" on cells infected with a variety of different viruses or exposed to double-stranded RNA (dsRNA), interferon, or other treatments ( Figure 1 ).
Our next study was a quantum leap in sophistication. Our microarrays now contained over 4,600 cDNAs and our experimental system was more complex. This time, we profiled the cellular gene expression changes that occurred in HeLa cells infected with influenza virus, again analyzing two time points after infection [3] . In addition, we evaluated the cellular response to a heat-inactivated virus, allowing us to identify gene expression changes that were independent of viral replication. Genes involved in protein synthesis, transcriptional regulation, and cytokine signaling were induced by the replicating virus, and many of these changes suggested an active cellular antiviral response. Interestingly, viral replication also resulted in the down-regulation of many cellular genes, particularly at the later time point. The gene expression pattern induced by the inactivated virus included the induction of the cellular metallothionein genes, which may represent a protective response to virus-induced oxidative stress.
Our early use of microarrays also included the first systematic attempt to define the full repertoire of dsRNA-regulated genes [4] , and we used arrays, in conjunction with polysome fractionation, to identify cellular and viral mRNAs that increased in abundance and which were selectively recruited to polyribosomes following influenza virus infection [5] . Our dsRNA study identified 175 dsRNA-stimulated genes, which encoded proteins involved in a broad range of cellular functions and metabolic pathways. In addition, this study showed for the first time that many genes are also down-regulated by dsRNA. Our polysome fractionation studies, headed by John Kash, revealed that many of the cellular mRNAs that are recruited to polyribosomes during influenza virus infection are involved in the control of cell proliferation and survival, mitogenesis, or the inflammatory response. In addition, many of these mRNAs contain a putative binding site for GRSF-1, a cellular mRNA-binding protein that also specifically interacts with a discrete and conserved region of the viral 5′ untranslated region [6] .
As our use of microarrays extended to an increasing number of experimental systems, we quickly found ourselves having to cope with an avalanche of data. To manage the huge amounts of information generated from multiple array experiments, our bioinformatics group, then headed by Jeff Furlong, developed a custom gene expression database, called Expression Array Manager [7] . In addition to serving as a central data repository, Expression Array Manager functions as our laboratory information management system, provides a streamlined data analysis pipeline, and provides a mechanism for publishing our gene expression data to the scientific community (available at http://expression.viromics.washington.edu).
Most of our core genomic and proteomic data analysis functions are now performed using the Rosetta Biosoftware systems Resolver and Elucidator. These systems are designed for managing and analyzing large amounts of gene expression and mass spectrometry data and both contain a suite of higher-order analysis tools. To explore genomic and proteomic data in greater depth, we also use a variety of tools that provide a diversity of analytical choices. As an example, we use Spotfire DecisionSite to format data for further analysis using tools such as Ingenuity Pathway Analysis or MetaCore (GeneGo). These tools allow us to leverage proprietary biological content databases and examine gene expression (or protein abundance) data in the context of complex pathways to identify connections between differentially expressed genes. Sean Proll, Bryan Paeper, and Jon Rue provide the laboratory with expert guidance on the use of these tools and ensure that data moves efficiently through this complex pipeline.
We had earlier discovered that hepatitis C virus (HCV) uses its nonstructural 5A (NS5A) protein to repress the interferon-induced protein kinase, PKR, a primary mediator of the antiviral effects of interferon [8] . Our first application of gene expression profiling to HCV research was therefore to evaluate the effects of interferon treatment on cellular gene expression and to determine whether NS5A was able to alter this pattern of expression [9] . Gary Geiss again headed these studies in which we examined the effects of interferon treatment by using several types of human cells, including HeLa cells, liver cell lines, and primary fetal hepatocytes. In response to interferon, 50 of the approximately 4,600 genes examined were consistently induced in each of these cell types and another 60 were induced in a cell typespecific manner. A search for interferon-stimulated response elements (ISREs) in genomic DNA located upstream of interferon-stimulated genes revealed both previously identified and novel putative ISREs. The expression of NS5A partially blocked the interferon-mediated induction of interferon-stimulated genes and inhibited the induction of a reporter gene driven from an ISRE-containing promoter, further suggesting that NS5A may play a role in HCV resistance to interferon. This study also set the stage for our use of genomics to better understand the interferon response and the varied mechanisms used by viruses to evade the effects of interferon.
What really accelerated our HCV genomics program was being awarded a National Institute on Drug Abuse (NIDA) P30 Center to do translational and clinically relevant genomics studies (http://nida.viromics.washington.edu/). Maria Smith headed many of our early studies, the first of which looked for novel tumor markers in surgical liver samples from patients with hepatocellular carcinoma (HCC) [10] . This study, which by now used microarrays containing over 13,000 human cDNAs, revealed a set of 50 potential HCC marker genes that were upregulated in the majority of the tumors analyzed. This HCC marker set contained several cancer-related genes as well as a set of genes encoding secreted or plasma proteins that may provide potential serological markers for HCC.
We performed a similar set of analyses on surgical material and core biopsy specimens obtained from HCV-infected patients with liver cirrhosis [11] . Importantly, this study also included an analysis of normal liver samples to determine normal physiologic variation in gene expression. To identify markers associated with cirrhosis, genes that were differentially expressed in normal liver, or in HCC, were subtracted from the set of genes differentially expressed in cirrhotic livers. The resulting gene set included genes expressed in activated lymphocytes infiltrating the cirrhotic liver and genes involved in the remodeling of the extracellular matrix, cell-cell interactions associated with cytoskeleton rearrangements, the anti-apoptotic pathway of Bcl-2 signaling, and the interferon response. Together, this analysis identified several potential gene expression markers of HCV-associated liver disease and contributed to our rapidly expanding database of experiments describing HCV pathogenesis.
Perhaps one of our most promising applications of genomics to HCV-associated liver disease is our work with serial liver biopsy samples from HCV-infected liver transplant patients. Liver transplant recipients infected with HCV develop recurrent hepatitis soon after transplantation and, in some cases, progress to fibrosis within two years of the transplant. Our goals are to identify molecular processes influencing liver disease progression and to find potential gene expression markers of early fibrosis. To achieve these goals, we are working closely with clinicians in the liver transplant unit at the University of Washington, including Anne Larson, Robert Carithers, and James Perkins, to collect biopsy samples and to integrate the gene expression data obtained from these samples with clinical observations. Our initial analyses were performed on serial liver biopsy specimens obtained from 13 transplant recipients at 0, 3, 6, and 12 months after transplantation [12] . Gene expression data were compared with clinical observations and with gene expression data obtained from 55 nontransplant HCV-infected and uninfected liver samples. Our analyses revealed several specific gene expression patterns, the first of which was unique for the transplant recipients regardless of their infection status. The corresponding genes encoded stress response proteins and blood proteins involved in coagulation that were differentially expressed in response to post-transplantation graft recovery. The second pattern was specific to HCV-infected samples and included the increased expression of genes encoding components of the interferonmediated antiviral response and immune system. This pattern was absent or suppressed in the patients who developed early fibrosis, indicating that disease progression might result from an impaired liver response to infection. Finally, we identified gene expression patterns that were specific for the 12-month biopsy specimens of all four HCV-infected patients who developed early fibrosis after transplantation. The identified gene expression patterns may prove useful for diagnostic and prognostic applications in HCV-infected patients, including predicting early progression to fibrosis. We are continuing to collect and analyze biopsy samples as new patients are recruited into the study, which should enable us to obtain increasing levels of confidence in the predictive power of the markers that we identify.
In other recent studies, Kathie Walters and Sharon Lederer have examined the gene expression profiles that differentiate alcohol-and HCV-induced liver cirrhosis [13] and the gene expression patterns associated with HCV-induced pathogenesis in individuals co-infected with HIV-1 [14] . We found that global gene expression patterns vary significantly depending upon the etiology of liver disease and that stages of liver cirrhosis can be differentiated based on gene expression patterns in ethanol-induced, but not HCV-induced, disease. Many of the gene expression changes specifically observed in HCV-infected cirrhotic livers are associated with activation of the innate immune response. In contrast, we found that intrahepatic global gene expression profiles do not differ between HCV-and HCV/HIV-1 co-infected individuals. However, a specific gene expression pattern that may be associated with HCV-induced pathogenesis was identified. This pattern has similarities to the gene expression profiles in transplant patients who progress to fibrosis within one year of transplantation, suggesting it may also be relevant to predicting disease progression.
Although clinical samples can provide considerable insight into the changes in cellular gene expression that occur during liver disease progression, they are not necessarily ideal for studies investigating the molecular mechanisms responsible for these changes. These types of studies typically require an animal model that can be experimentally manipulated. In this regard, research in the HCV field has been significantly enhanced by the development of a mouse model of HCV infection. This model, developed by Lorne Tyrrell and Norman Kneteman at the University of Alberta, Canada, is based on the severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) transgenic mouse. Intrasplenic injection of freshly isolated human hepatocytes into these mice leads to the repopulation of the mouse liver with human hepatocytes. The result is a mouse containing a chimeric mouse-human liver and these animals can be persistently infected with a variety of HCV genotypes [15] . The chimeric mouse model provides a number of features that make it a useful system in which to study HCV-host interactions. Because groups of mice are transplanted with hepatocytes from different donors, we have the opportunity to analyze host-specific responses to HCV. In addition, the lack of an adaptive immune response in these animals makes it possible to distinguish between virus-mediated and immune-mediated effects on gene expression. Kathie Walters is using this model to characterize the host transcriptional response to HCV infection. Because liver samples from these animals typically have small percentages of contaminating mouse liver tissue, she first evaluated the level of cross-hybridization of mouse liver mRNA to the human probes present on the microarray [16] . The small set of genes identified (less than 2% of the genes present on the array) is either removed from subsequent data analysis or changes in the expression of these genes are validated using human-specific quantitative realtime PCR.
In an initial series of experiments, groups of mice transplanted with hepatocytes from different donors were inoculated with a single source of HCV and gene expression profiling was performed to characterize the host response to infection [17] . Although all HCV-infected animals showed evidence of an interferon response, the level of this response, both in the intensity and number of up-regulated interferon-induced genes, varied between animals depending upon the origin of the donor hepatocytes. These results indicate that host genetic factors contribute to the variation in the host response to HCV, including the activation of innate antiviral signaling pathways. Mice with weak interferon responses also tended to have high levels of intrahepatic HCV RNA, indicating that an ineffective interferon response may allow increased levels of viral replication. These animals also accumulated higher numbers of differentially expressed genes than did mice with strong interferon responses. Interestingly, we have also observed impaired interferon responses and the accumulation of increased numbers of differentially expressed genes in the intrahepatic gene expression profiles of transplant patients who develop fibrosis within one year of transplantation.
HCV research and the NIDA P30 Center also provided our entry point into proteomics. The ability to look at the protein content of a cell provides a strong complement to our genomic analyses, allowing us to evaluate how gene expression changes in response to virus infection translate into changes in the abundance of proteins, the molecules that directly carry out biological functions. Moreover, proteomic analysis of body fluids, such as blood, holds the promise of identifying candidate biomarkers for human diagnostic or prognostic applications.
Our initial venture into the realm of proteomics was led by Wei Yan in collaboration with Ruedi Aebersold at the Institute of Systems Biology. We began by using tandem mass spectrometry to analyze the global proteome of the Huh7 hepatoma cell line and cultured primary and immortalized human fetal hepatocytes [18] . We also used isotope coded affinity tag (ICAT) technology to measure the changes in protein abundance that occurred in human hepatocytes in response to interferon treatment [19] . These analyses led to the generation of a liver proteome dataset consisting of 2,159 unique proteins. Among the identified proteins were 78 interferon-regulated proteins that play roles in a multitude of cellular functions including antiviral defense, immune response, cell metabolism, and signal transduction. These data also contributed to the development of PeptideAtlas, a public resource for further annotating and validating the human genome by mapping identified peptide sequences to the human genome sequence [20] .
For our more recent proteomic work, we have teamed with Richard Smith at Battelle, Pacific Northwest National Laboratory (PNNL). In our first studies with the Smith group, we used high mass accuracy Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, coupled with the accurate mass and time (AMT) tag approach [21] , to perform global proteomic analyses on Huh-7.5 cells containing a full-length HCV replicon [22] . Using this more sensitive technology, we identified over 4,200 proteins. As a first-pass means to detect changes in protein abundance associated with HCV infection, we also performed a semi-quantitative comparison of total peptide identifications. This peptide-spectral count ("peptide-hits") approach revealed HCV-related perturbations in the abundance of a variety of proteins associated with lipid metabolism and oxidative stress.
We have also taken advantage of the nanoproteomic platforms available at PNNL to perform proteomic studies on human liver biopsy samples that yield only limited amounts of protein.
These studies were performed using a high-sensitivity 11.4-tesla FTICR mass spectrometer coupled with the AMT tag approach and our Huh-7.5 cell protein database. Using this method, we identified over 1,500 proteins from only 2 μg of a protein digest obtained from a liver biopsy sample [22] . The proteins identified included many of relevance to HCV infection and liver disease, including cellular proteins involved in lipid metabolism and the interferon response. The number of proteins identified by this approach is considerably larger than what has been reported in other published studies of the liver proteome, where less-sensitive methods, requiring milligram amounts of protein lysate, have identified only a few hundred proteins. Deborah Diamond leads the proteomics research in our laboratory and has recently authored a review that more fully discusses current developments in the application of proteomics to liver disease research [23] .
Influenza research has been a mainstay of the laboratory for over 20 years, and as mentioned earlier, some of our earliest genomic studies were performed on cells infected with influenza virus. Today, with worldwide attention focused on highly pathogenic avian influenza and the looming threat of a new pandemic, influenza research has taken on a renewed vigor. One of the priorities in this research is the development of improved animal models to better understand influenza pathogenesis and to develop new diagnostic, therapeutic, and vaccine strategies, all of which are important components of preparedness for the next influenza pandemic [24] .
Over the past two years, we have taken on this challenge by developing a macaque influenza infection model that features genomic and proteomic analyses as a key component of the experimental design. Carole Baskin has taken a leading role in this effort and was the first to report on the use of genomic technologies to characterize experimental influenza virus infection using a pig-tailed macaque model [25] . This study was performed using a mildly pathogenic strain of virus (A/Texas/36/91) with the goal of constructing a blueprint of an uncomplicated influenza infection. Gene expression profiling performed on lung tissue and tracheobronchial lymph nodes revealed that numerous genes associated with the interferon response were differentially expressed, particularly at day 4 after inoculation. We also observed an increase in the expression of genes encoding various mediators of chemotaxis, adhesion, and the transmigration of immune cells, suggesting the trafficking of these cells into the lungs and tracheobronchial lymph nodes, which was confirmed by histopathology. In addition, this study revealed gene expression changes relevant to the processing of antigens on MHC class I complexes, particularly in lung tissue, and many genes encoding proteins relevant to T-cell function were induced at one or more time points.
The gene expression profiling performed for this study was done using human cDNA microarrays, which until recently was the only option available for studies using nonhuman primates. However, because of the extensive use of macaques as models for a wide variety of human diseases (and in AIDS research in particular), we have been part of a push to develop genomic resources focused on macaque species [26] . This effort resulted the sequencing of the rhesus macaque genome [27] , and James Wallace headed an effort to use this extensive sequence information to develop an oligonucleotide microarray containing over 17,000 unique macaque sequences [28] . Additional information related to nonhuman primate genomics is also available at macaque.org, a Web site we have developed to disseminate macaque genomic and proteomic data and resources.
The macaque oligonucleotide array was used in a follow-up study in which we focused on the early innate immune response of macaques infected with influenza virus [29] . In this study, led by Tracey Baas and Carole Baskin, we also performed genomic analyses on whole blood samples from infected animals to determine whether we could detect gene expression signatures in the blood that would correlate with those detected in the lung. In general, genomic analyses of lung tissue showed an increase in the expression of many genes associated with interferon signaling early after infection, while other immune and cytokine responses were sustained throughout the course of the infection. We also observed a number of genes (many of which were involved in the interferon response) that were differentially expressed in a similar fashion in lung and blood samples, particularly early after infection. This finding suggests that microarray analyses of blood samples may hold promise for diagnostic applications. Finally, this study also included a first of its kind global proteomic analysis of macaque lung tissue. This analysis identified over 3,500 proteins, and consistent with the gene expression data, proteomic data also revealed an increase in the abundance of many proteins involved in the innate immune response.
What influenza virologist wouldn't be interested in understanding the molecular mechanisms underlying the extreme pathogenesis of the virus responsible for the 1918 influenza pandemic? Studies related to this virus, which killed and estimated 50 to 100 million people worldwide [30] , have been (and remain) a prominent fixture of the laboratory, and genomic analyses are providing important new insights into what made this virus so lethal. In our first experiments associated with the 1918 virus, we evaluated global gene expression patterns in cells infected with wild-type influenza, engineered viruses lacking all or part of the NS1 gene, or with a virus containing the NS1 gene of the 1918 pandemic virus [31] . Deletion of the NS1 gene increased the magnitude of expression of cellular genes implicated in the interferon, NF-κB, and other antiviral pathways, and a virus with a C-terminal deletion in its NS1 gene induced a cellular gene expression pattern intermediate between the patterns induced by wild-type and NS1 knockout viruses. In contrast, a virus containing the NS1 gene from the 1918 pandemic virus was more efficient at blocking the expression of interferon-regulated genes than its parental virus. Together, these results suggested that the cellular response to influenza virus is significantly influenced by the NS1 gene and that the 1918 NS1 is a particularly effective interferon antagonist. Of course, we have since learned that the story is much more complicated.
We next used a mouse infection model to assess the contribution of the 1918 HA and NA genes to viral pathogenesis [32] . These studies, led by John Kash, evaluated mouse-adapted A/WSN/ 33 viruses that were engineered to contain the HA and NA genes from the 1918 virus or from the nonlethal A/New Caledonia/20/99 virus. Microarray analyses performed on lung tissues isolated from all infected animals showed the activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes. However, consistent with histopathology analysis, the parental and 1918 HA/NA:WSN recombinant viruses showed increased expression of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in mice infected with the New Caledonia HA/NA:WSN virus. These studies documented clear differences in gene expression profiles that were correlated with pulmonary disease pathology and suggested that an intrinsic property of the 1918 HA and NA proteins may be the production of a longer and more severe immune response culminating in a more destructive viral infection.
The reconstruction of all eight gene segments of the pandemic virus provided the opportunity to find out what genomics could reveal about the high-virulence phenotype of the 1918 virus. The initial characterization of this virus by Terrence Tumpey and colleagues demonstrated that it highly virulent in mice and most animals died within five days after infection [33] . Genomic analyses, led by John Kash, showed that animals infected with the reconstructed virus showed an increased and accelerated activation of genes associated with pro-inflammatory and celldeath pathways by 24 h after infection [34] . Significantly, these genes remained activated until the death of the animal. This was in contrast to less dramatic and delayed host immune responses (and less severe disease pathology) in mice infected with influenza viruses containing only subsets of genes from the 1918 virus. These findings indicate a cooperative interaction between the 1918 influenza genes and suggest that enhanced inflammatory and celldeath responses may contribute to severe immunopathology.
Our most recent work with the reconstructed 1918 virus was done in collaboration with Yoshihiro Kawaoka. In these studies, we evaluated the host response to the reconstructed virus using a cynomolgus macaque infection model [35] . The 1918 virus replicated to high levels and spread rapidly throughout the respiratory tract of infected animals, causing severe damage and masses of infiltrating immune cells throughout the course of infection. Genomic analyses revealed that the 1918 virus triggered an aberrantly high and sustained expression of numerous genes involved in the innate immune response, including proinflammatory cytokines and chemokines. The early and sustained host response in macaques infected with the 1918 virus was similar to what we observed in mice, indicating that critical host responses that influence disease outcome may occur very early after infection. Interestingly, the 1918 virus also appeared to selectively attenuate the expression of specific innate immune response genes, including certain genes associated with the type 1 interferon response. We have not determined the mechanism for this attenuation, but it is possible that the viral NS1 gene may play a role in regulating this response. Although the details remain to be uncovered, the atypical innate immune response induced by the 1918 virus was insufficient for protection and may actually contribute to the lethality of the virus.
Our interests in combining genomics and macaque infection models have led to the establishment of the Division of Functional Genomics and Infectious Disease at the Washington National Primate Research Center. Our laboratory is the central component of this Division, which is devoted to developing genomic and proteomic technologies (along with Richard Smith's group at PNNL), immunologic resources (in collaboration with Edward Clark and Murali-Krishna Kaja), and advanced computing and bioinformatics applications to enhance the use of nonhuman primates as models for influenza virus infection and AIDS. Our goal is to develop and apply the tools needed to perform comprehensive and integrated analyses on nonhuman primates and to analyze the response to virus infection at multiple points along the flow of biological information: from the whole animal to DNA to RNA to protein to biological function (Figure 2 ). By integrating these diverse types of data, we have the opportunity to better understand the dynamics of the host response to infection and the molecular mechanisms underlying the progression to virus-mediated disease, immunopathology, or the development of protective immunity. We also have the opportunity to better understand how gene expression changes in response to infection translate into changes in protein abundance and function, and how these changes correlate with clinical outcome. Moreover, we have the opportunity to assess how changes in gene expression and protein abundance affect immune cell function, and how the innate immune response develops and its link to adaptive immunity. Ultimately, we believe this integrated approach will translate into molecular signatures that predict protective immunity or pathology, biomarkers for diagnostic or prognostic assays, and a rational base for improvements to antiviral therapies or vaccine strategies.
The studies outlined in this review provide just a glimpse of how we're using genomic and proteomic technologies to unravel the complexities of virus-host interactions. In addition to work with HCV and influenza, we're using genomic approaches to study a variety of other viruses. Tracey Baas heads collaborative research on SARS coraonavirus [36] and herpes simplex virus [37] . When not working on pandemic influenza, John Kash also leads efforts focused on Ebola virus [38] and vaccinia virus [39] , and Matthew Thomas and Michael Agy have explored gene expression changes associated with simian immunodeficiency virus infection [40] . We've also worked with Michael Gale on West Nile virus [41] , and in our work on P58 IPK (a cellular PKR inhibitor), we've even explored gene expression patterns associated with pancreatic β-cell depletion in diabetic mice [42] . Jamie Fornek is continuing the work on P58 IPK , including using P58 IPK knockout mice and genomics to examine the role of this PKR inhibitor during influenza virus infection. Gregory Zornetzer is developing a targeted proteomics approach to identifying protein-protein interactions associated with key viral proteins.
The technologies we use continue to improve and data is accumulating at an ever increasing rate. Today's microarrays represent over 18,000 genes (over ten-fold more than when we started) that together with assorted controls are arrayed as an impressive 44,000 oligonucleotides per slide. Barry Robinson is working to incorporate laser capture microdissection into our studies, which will give is our first opportunity to examine the gene expression changes that occur in specific cell types isolated from complex tissues. Bioinformatics capabilities are becoming evermore sophisticated and Jon Rue, Eric Flamoe, and Matthew Harding ensure that we stay atop the most recent advances in data management and analysis. Our Expression Array Manager database now contains information from over 7,000 expression profiles representing more than 50 million measurements of differential gene expression.
As we've discussed elsewhere [43] , the future of virology depends on making progress in understanding how things work at a systems level, and we must ensure that genomic and proteomic technologies do more than simply generate ever larger quantities of data without providing clues to underlying function. Nevertheless, we are confident that as technologies and experimental systems continue to progress, and data integration strategies become more mature [44] , functional genomics will make ever greater contributions to virology. As an example, we are continuing to take our use of genomics in new directions and Robert Palermo is working together with Marjorie Robert-Guroff (National Cancer Institute) to pursue genomic approaches to improving AIDS vaccine development. Genomic analyses during vaccine trials may reveal gene expression markers of protective immunity or gene expression changes that are indicative of a predisposition to a particular response to immunization and subsequent challenge. All told, the past eight years have been an exciting journey full of discoveries, and we look forward to new breakthroughs in the future. Indeed, life after the Milstein award hasn't been too bad. Representation of the range of viruses and experimental systems we have evaluated using genomic and proteomic technologies. Highlights of experiments related to the use of many of these experimental systems, particularly for hepatitis C virus and influenza, are summarized in this review. SARS-CoV: severe acute respiratory syndrome-associated coronavirus; SIV: simian immunodeficiency virus; WNV: West Nile virus; HIV-1: human immunodeficiency virus type 1; HSV-1: herpes simplex virus type 1; EAM: Expression Array Manager. The Division of Functional Genomics and Infectious Disease provides the virus infection models and the clinical, genomic, proteomic, and immunologic resources needed to perform comprehensive analyses on nonhuman primates. This allows us to analyze the response to virus infection at multiple points along the flow of biological information. EST: expressed sequence tag; AMT: accurate mass and time. Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens. Mass spectrometry has much to offer the field of genomic analysis, particularly in terms of multiplexed analysis and accurate quantification. To date, many mass spectrometry-based approaches for genomic analysis have been based on direct detection of nucleic acids particularly using matrix assisted laser desorption ionisation time-offlight (MALDI TOF) MS analysis. MALDI TOF is well suited to this approach due to the high mass range achievable by TOF analysis, however, MALDI TOF instrumentation is relatively expensive and sample preparation can be quite laborious. In addition, direct analysis of nucleic acids by mass spectrometry suffers from problems such as depurination leading to fragmentation (1) or cation adduct formation (2, 3) . These issues notwithstanding, MALDI TOF analysis of nucleic acids has been applied to DNA sequencing (4, 5) , RNA sequencing (6, 7) , analysis of DNA tandem repeats (8) . In particular, PNA probes, with and without noncleavable mass modifiers, have been used for characterization of genomic DNA libraries (9) , detection of DNA methylation (10) and detection of single nucleotide polymorphisms (SNPs) (11, 12) by MALDI TOF mass spectrometry.
Electrospray ionisation (ESI) mass spectrometry has also been used for direct detection of nucleic acids (13) (14) (15) . ESI-MS has some advantages over MALDI TOF MS particularly the availability of lower cost instrumentation. Moreover, sample handling can be simpler since most molecular biology assays are carried out in solution and such liquid samples are injected directly into the instrument. Furthermore, very high molecular weight species can be analysed due to the propensity for large molecules, such as PCR products, to form multiply charged ions that have relatively low overall mass-to-charge ratios under electrospray ionisation conditions. However, direct analysis of nucleic acids by ESI-MS still suffers from the same problems as MALDI TOF MS such as cation adduct formation (14) . In addition, the multiply charged ion spectra that are generated for large nucleic acid fragments can be very complicated reducing sensitivity and making multiplexing difficult (16) .
As an alternative to direct analysis of nucleic acids, mass spectrometry can also be used to detect nucleic acids indirectly through the use of cleavable mass tags, which avoids many of the limitations of direct analysis of nucleic acids while also offering numerous advantages such as ease of multiplexing, more robust detection of tag species and higher sensitivity and less demanding workup and sample preparation for analysis. Mass spectrometric analysis of mass tags, by ESI or MALDI also offers the possibility of accurate quantification through the use of isotopic tags. This capability of mass spectrometry has not really been exploited in genomic analysis but has been quite widely used in proteomic analysis (17) (18) (19) and is a key advantage of the mass spectrometric approach.
Again MALDI TOF analysis of nucleic acids with cleavable mass tags has been demonstrated by various groups (20) (21) (22) (23) (24) but it would be advantageous to be able to take advantage of lower cost ESI-MS/MS instruments and to avoid the laborious sample workup requirements of most MALDI approaches. A matrix-free laser desorption approach, which has reduced workup requirements has been demonstrated (21, 25) but this still requires that the sample be spotted onto a MALDI target or hybridized to an array. To our knowledge, only one mass tagging approach employing ESI-MS analysis has been demonstrated (26) . In this approach, mass tags are photocleavably linked to oligonucleotides and tag detection requires a photo-cleavage step and a tag isolation step outside of the mass spectrometer prior to tag detection, i.e. the workup is not much simpler than that required for MALDI TOF analysis.
Here, we describe the synthesis of novel ESI-cleavable Tandem Nucleic acid mass Tag-peptide nucleic acid conjugates and their analysis by ESI-MS/MS. We demonstrate a novel mass tag cleavage method in which source voltages in the electrospray ionisation source are used to cleave an ESI-cleavable linker, by a collision-based process, releasing the mass tag from the oligonucleotide during ionization. This method allows for direct analysis of assay solutions without requiring complex workups to cleave and isolate tags. In principle, this cleavage method would also allow in-line separation, by capillary electrophoresis for example, of labelled nucleic acids with direct spraying of the separated material into the ion source where tag cleavage would take place automatically.
In addition, we demonstrate a novel MS/MS-based tandem nucleic acid mass tag (TNT) design and detection process that allows highly specific detection of TNTs in a complex background. The Tandem Nucleic acid mass Tag design also allows easy synthesis of large sets of isotopic tags supporting the development of multiplexed and quantitative assays. We demonstrate the quantitative nature of the TNT approach. Furthermore, we evaluate ESI-cleavable TNT-PNA conjugates as hybridization probes for the detection of target DNA sequences via the use of ESI-MS/MS.
The TNTs described here are constructed using FMOC peptide synthesis chemistry. Synthesis of peptide nucleic acid-TNT peptide conjugates is relatively straightforward as PNA synthesis can be achieved using the same FMOC protection groups that are used in peptide synthesis (27) . This means that peptide TNT-PNA conjugates can be synthesized on the same resin in a continuous process. PNA is a useful analogue of DNA and is advantageous for this application due to its high specificity and its neutral backbone, which means that it does not require high concentrations of salt to hybridize making it highly compatible with mass-spectrometry-based detection methods (28) (29) (30) .
The outline of the general approach for the detection of DNA sequences via ESI-cleavable TNT-PNA conjugates is presented in Figure 1 . The ESI-cleavable TNT-PNA conjugates consist of a PNA probe portion, which interacts with the immobilized target sequence (DNA or RNA) and a peptide tandem nucleic acid mass tag portion, which is ultimately detected. Note that the TNT shown is merely a representation of the tag and not a real structure ( Figure 2 ). An ESI-cleavable linker connects the PNA probe portion of the conjugate to the tandem nucleic acid mass tag peptide. The complete TNT 'Parent Tag' comprises the red 'Tag Fragment' portion and the blue 'Mass Normalizer' portion shown in Figure 1 . The TNT marker is designed to have a unique combination of parent tag mass and tag fragment mass, released during collision induced dissociation (CID), and it is this pair of masses that serves as the sequence identifier. In a typical scenario (Figure 1 ), a set of PNA hybridization probes labelled with different TNTs is first hybridized to the captured target nucleic acids of interest (step (1)). After stringent washes to remove the non-hybridized probes (step (2)), the probes are denatured from the target (step (3)) and injected into an ESI-MS/MS instrument for detection (step (4)). In the mass spectrometer, the TNTs cleave from the PNAs during electrospray ionization (step (5)). The TNT parent tag ions are then selected from the background, fragmented by CID and finally, daughter, tag fragment ions from the fragmentation are detected (step (6)), confirming that the signals are indeed due to the presence of tagged probes, thereby detecting the presence of the target sequences.
The use of this MS/MS-based approach offers high specificity allowing TNT labels to be detected in a background of fragmentation noise. In addition, the MS/ MS detection means that tags can share the same mass as long their tag fragment ions are distinguishable from each other. This means that many TNTs can be detected in a compressed mass range. This feature combined with the relatively low overall mass required for TNTs, means that TNT technology will be able to exploit lower cost compact and portable ESI-MS/MS instrumentation that is currently under development (31) (32) (33) (34) (35) (36) .
FMOC-protected peptides were custom synthesized by PepSyn Ltd (Liverpool, UK) using commercially available FMOC-protected amino acids on a Beckman synthesizer.
4-FMOC-piperazin-1-ylacetic acid was obtained from Fluka (Sigma Aldrich, Dorset, UK). The amino acid isotope 13 C 3 , 15 N-FMOC-L-alanine was obtained from Cambridge Isotope Laboratories, Inc (Andover, MA). The sequences of the TNT peptides used for the preparation of ESI-cleavable TNT-PNA probes are shown in Table 1 .
Peptide nucleic acid oligonucleotide syntheses were carried out using a 2-mmol cycle on an Expedite 8900 synthesizer (Applied Biosystems, Foster City, CA). For the preparation of peptide-PNA conjugates, the FMOCprotected peptide TNT sequence was synthesized and left on the resin by PepSyn Ltd with the N-terminal FMOC left intact. The resin was extracted from the peptide synthesizer column and was then loaded into the Expedite synthesizer column (2 mmol per column). PNA synthesis was carried out as normal on the preloaded resin. The yield of each purified conjugate was in the range 4.3-32 OD 260 , which corresponds to a 1.5-21% yield based on the 2-mmol synthesis scale. The sequences and yields of the TNT-PNA probes are shown in Table 2 .
Biotinylated, fully complementary target 50-mer oligodeoxyribonucleotides for the hybridization experiments were synthesized by Yorkshire Bioscience (York, UK) on a 1-mmol scale. Target sequences are shown in Table 3 .
TNT-PNA stock solutions were made up at a concentration of 20 pmol/ml in water. Biotinylated target sequences were made up in stock solutions of 50 pmol/ml in water.
Six aliquots of 20 ml of stock solution of the first TNT-PNA probe was mixed with 20, 10, 4, 2, 1 and 0.5 ml of the second TNT-PNA, respectively. To these, water was added to make up the solution to a total of 40 ml. The aliquots were then made up to 80 ml with methanol and formic acid to give a final solution of the TNT-PNA probes in 50:50 water:methanol with 1% formic acid. These samples were then analysed by direct injection ESI-MS/MS.
Five aliquots of 50 ml of MyOne Streptavidin C1 Dynabeads (10 mg/ml suspension) were separated from their storage buffer and washed with twice with 50:50 methanol:water to remove potential mass spec contaminants. The beads were then washed with 1 Â Bind & Wash (B&W) buffer. B&W buffer for Dynabead incubation was made up according to the manufacturer's instructions: 20 mM Tris, pH 8.0, 2 mM EDTA, 2 M NaCl. Six aliquots of 20 ml (1 nmol) of stock solution of one biotinylated target was incubated with the Dynabeads. These aliquots were all made up to 40 ml with the addition of 20 ml of 2 Â B&W buffer. The biotinylated targets were then incubated at room temperature with the streptavidin beads for 1 h according to the manufacturer's instructions to immobilize the targets on the beads. The target solution was then removed from the beads and the beads were washed twice with hybridization buffer (20 mM Tris, pH 7.5, 10 mM MgCl 2 , 25 mM NaCl). A sixth aliquot of 100 ml of MyOne Streptavidin C1 Dynabeads was made up in the same way but this aliquot was incubated with 40 ml (2 nmol) of stock solution of the same biotinylated target and 40 ml of 2 Â B&W buffer.
Each aliquot of the first five aliquots of bead-captured target was then incubated with 50 ml of a TNT-PNA probe solution comprising 1 nmol of the TNT-PNA probe complementary to the target on the beads. Similarly, the sixth aliquot was then incubated with 100 ml of a second TNT-PNA probe solution comprising 2 nmol of the TNT-PNA probe complementary to the target but with the alternative TNT tag on the probe. Hybridization of all the aliquots was carried out at room temperature for two hours. After hybridization, the probe solution was removed from the beads and the beads were washed three times with ice-cold 70 mM aqueous ammonium citrate solution. After the wash step, the sixth aliquot was resuspended in 100 ml of ammonium citrate. Aliquots of the sixth aliquot were then pipetted into the first six aliquots: 20, 10, 4, 2, and 1 ml, respectively. The mixtures The TNT tags, outlined by the red broken line, are modular comprising different functional components that correspond to individual synthetic components in the automated synthesis of these reagents. Each TNT is composed of two parts. The first part is a tag fragment, drawn in red, that comprises a charge-carrying group, the piperazine moiety outlined by the black broken box, and an alanine mass modifier group, outlined by the green broken box. The tag fragment is linked to the second part of the tag, which is a mass normalization group, drawn in blue, that ensures that each tag in a pair of tags shares the same overall mass and atomic composition. Note that the tags shown above are not strictly isobaric due to an issue with the synthesis discussed in the text. The mass normalization group is essentially uncharged and comprises a second alanine mass modifier group to adjust the overall mass of the tag and a proline group that is part of the electrospray cleavable linker. The ESI-cleavable linker is outlined by another black broken box and comprises an aspartic acid proline linkage. Part (ii) of Figure 2a Tables 1 and 2. were then mixed thoroughly. The hybridized TNT-PNA probes were then denatured from their captured target by incubating the beads at 858C for 20 min in 50:50 water:methanol with 1% formic acid. The elution solutions were then analysed by direct injection ESI-MS/MS.
Six aliquots of 125 ml of MyOne Streptavidin C1 Dynabeads (10 mg/ml suspension) were separated from their storage buffer and washed with twice with 50:50 methanol:water to remove potential MS contaminants. The beads were then washed with 1 Â B&W buffer. Six aliquots of 20 ml (1 nmol) of stock solution of one biotinylated target was mixed with 20, 10, 4, 2, 1 and 0.5 ml, respectively of the other biotinylated target. To these, water was added to make up the solution to a total of 40 ml. The aliquots were then made up to a volume of 80 ml by addition of 40 ml of 2 Â B&W buffer. These aliquots were then added to the aliquots of washed beads. The biotinylated targets were then incubated at room temperature with the streptavidin beads for 1 h according to the manufacturer's instructions to immobilize the targets on the beads. The target solution was then removed from the beads and the beads were washed twice with hybridization buffer.
Each aliquot of captured target was then incubated with 100 ml of TNT-PNA probe solution comprising 1 nmol of each of two TNT-PNA probes of the same length in hybridization buffer, i.e. 1 nmol of ANTHRAX-12 would be mixed with 1 nmol MOMP-12. Hybridization was carried out at room temperature for two hours. After hybridization, the probe solution was removed from the beads and the beads were washed three times with ice-cold 70 mM aqueous ammonium citrate solution.
The hybridized TNT-PNA probes were then denatured from their captured target by incubating the beads at 858C for 20 min in 50:50 water:methanol with 1% formic acid. The elution solutions were then analysed by direct injection ESI-MS/MS.
ESI-MS/MS spectra were obtained on a Micromass Q-TOF Micro mass spectrometer (Micromass (Waters), Wythenshaw, UK). The TNT-PNA oligonucleotides were denatured from the Dynabeads into 50:50 Methanol:Water with 1% formic acid. Mass spectra were externally calibrated using the manufacturer's standards and calibration protocols.
Tandem nucleic acid mass tag-PNA oligonucleotide probe design and synthesis Two pairs of example TNT-PNA oligonucleotide probes are shown in part (i) of Figure 2a and b. The TNTs are peptides comprising two parts, the tag fragment portion, which carries a charge due to the presence of a tertiary amino-functionality and the mass normalization portion, which remains essentially uncharged. These two portions of the tag are linked by a cleavage enhancement group, a piperazine ring, which also carries the charge of the tag fragment on its tertiary amino-group. The two tag portions both comprise a mass modifier component, which are isotopes of alanine in this tag although a large number of different mass modifiers could be used. It can be seen from Figure 2 that the two tags shown employ the same mass modifier components but the order differs Beta Alanine -Aspartic Acid -Proline -13 C 3 , 15 N-Alanine -Piperazin-1-ylacetic acid -Alanine TNT2
Beta Alanine -Aspartic Acid -Proline -Alanine -Piperazin-1-ylacetic acid -13 C 3 , 15 N-Alanine between the tags. Thus, the overall masses of the tags are the same but the tag fragments have different masses, which are equalized by the mass of the mass normalization portion. These tags are designed so that on analysis by collision-induced dissociation (CID), the tag fragment is released to give rise to a uniquely resolvable ion. Thus, this pair of tags allows a pair of PNA probes to be distinguished by MS/MS analysis. Each tag is linked to a PNA oligonucleotide probe by a second linker, comprising aspartic acid and proline that is easily cleaved by CID (37) . As shown in Figure 2 , the aspartic acid/proline linker is used to cleave the tags from their oligonucleotides during electrospray ionization of the tagged oligonucleotides. The expected structures and mass-to-charge ratios of the cleaved parent tag ions generated by dissociation of the aspartic acid/proline linkage are shown in part (ii) of figure 2a and b (37) . Similarly, the expected structures and mass-to-charge ratios of the tag fragment ions and the structures of the neutral mass normalizer fragments, based on the predictions of the 'mobile proton' model of peptide fragmentation (38, 39) , are shown in part (iii) of Figure 2a and b. The use of FMOC peptide synthesis chemistry to synthesize the TNTs combined with FMOC PNA chemistry to synthesize the oligonucleotide probe sequence means the same resin can be used to synthesize both portions of the tagged probe in a single total synthesis approach. For the probes discussed here, the TNT portion of the probe was synthesized in a commercial peptide synthesizer using standard peptide synthesis resin cartridges. The cartridge was then opened and the resin within was loaded into a cartridge suitable for a commercial oligonucleotide synthesizer that supports PNA synthesis (Expedite DNA/PNA synthesizer), allowing the completion of the synthesis of the probe. The completed probe was then cleaved from the resin and deprotected in one step (TFA/cresol) and then purified. With this approach, only one purification step is required resulting in better yields of finished probe than multi-stage processes. Figure 3 illustrates the ease with which a substantial number of tags can be synthesized from a small set of mass modifier components: nine tags, shown in Figure 3b can be made from three mass modifiers, which are the three commercially available isotopes of alanine shown in Figure 3a . In fact, the number of tags that can be synthesized increases as the square of the number of mass modifier components, e.g. there are at least five isotopes of alanine with different masses that are commercially available which would actually allow the synthesis of 25 tags using the design presented here.
One issue that emerged from the experiments presented here was a loss of 13 C isotope from the carboxylic acid of alanine when the alanine isotope was present at the C-terminus of the peptide TNT, i.e. in TNT2 shown in Table 1 . This loss is consistent across every TNT-PNA synthesized with TNT2 and may be an effect of the resin used, which relied on a 4-HydroxyMethyl-Phenoxy Acetic acid (HMPA) linker to the carboxylic acid group of the first amino acid to allow the peptide to be cleaved from the resin at the end of the synthesis. Other resins will be tested in future to avoid the loss of isotope. This has meant that the TNTs synthesized were not completely isobaric as shown in Figure 2 . The mass of the singly charged parent tag ion of TNT1 is 388.2 while that of TNT2 is 387.2. For the MS/MS analysis of these tags, both tags could still be selected simultaneously by the first quadrupole of the Q-TOF instrument, as the mass range that is gated is actually about 3 daltons for the default setting of The TNT approach is similar in principle to other mass tagging techniques and enjoys the same features as other approaches, such as ease of multiplexing and the ability to design tag masses to suit applications, with some additional advantages. TNT tags can be made chemically identical, even sharing the same mass as long as the tag fragments are different, so they can act as more precise reciprocal internal standards, which leads to more accurate quantification and the same behaviour in analytical separations, hybridizations and labelling reactions thus avoiding 'dye effects' that plague fluorescent methods (40, 41) . The use of an MS/MS-based detection method allows TNTs to be selected from background noise thus improving signal to noise ratios. This allows untagged material to be ignored, greatly improving data quality.
To confirm that the TNT-PNA oligonucleotide conjugates cleave as they are expected to (see expected fragment structures and mass-to-charge ratios in Figure 2 ), the TNT-PNA oligonucleotides were analysed by ESI-MS/MS on a micromass quadrupole-time-of-flight (Q-TOF) mass spectrometer. The complete TNT-PNA probe molecules were initially ionized with the cone voltage (an accelerating voltage in the ESI source that can be varied on the micromass instrument to control the levels of collision induced dissociation) set to minimize fragmentation of the whole TNT-PNA probe conjugate ions. The whole molecular ions were selected using the first quadrupole of the instrument and were then subjected to collision induced dissociation at different collision energies. Fragmentation of the complete TNT-PNA conjugate ions was carried out as it allows both the cleavage of the TNT parent tag from the PNA and the cleavage of the tag fragment from the parent tag to be seen simultaneously in the TOF analyser of the Q-TOF instrument thus demonstrating both cleavage processes and their relative efficiencies. Typical results are shown in Figure 4 , in which it can be seen that, as the collision energy is increased, the whole TNT-PNA probe ion fragments to release the parent tag ion as the predominant fragmentation product. As the fragmentation energy is increased further, the parent tag ion undergoes subsequent consecutive fragmentation to give the desired daughter fragment ion. As the collision energy increases further, the intensity of the parent tag ion increases. Similarly, the ratio of the daughter ion to parent ion increases more. These results show that the TNT-PNA probe molecules fragment as anticipated with the aspartic acid/proline linkage cleaving more easily than the piperazine linkage. The higher collision energy spectra are quite noisy as these also contain other products from the fragmentation of the TNT-PNA probe molecule, which would not normally be present when the TNT parent tag ions are analysed by themselves.
The normal mode of analysis is shown in Figure 5 . Here the cone voltage in the electrospray ion source is increased to 25 V increasing the level of fragmentation during ionization thus releasing the parent tag ion from the TNT-PNA conjugate. The parent tag ion is then selected from background by the first quadrupole in the Q-TOF instrument. The parent tag ion is subsequently subjected to CID in the second quadrupole of the Q-TOF instrument. A collision energy of 25 V was used for CID. MS/MS spectra showing the detected tag fragment ions are shown in Figure 5 . These spectra show ratios of two tags and demonstrate the accuracy of quantification of the TNT technology, which is discussed in the next section.
A brief experiment to determine whether there were any obvious size dependent effects on the efficiency of the cleavage of the TNT from the TNT-PNA conjugate during electrospray ionisation was carried out. In this experiment, pairs of TNT-PNA oligonucleotides of different lengths were mixed together in 1:1 concentrations. It might be expected that the larger TNT-PNA probes would fragment somewhat less easily due to their greater size and the consequent ability to dissipate kinetic energy from collisions over many more different modes of vibration. This would mean that the larger probes should be detected with less sensitivity. It turned out that the larger sequences gave slightly more sensitivity with the 16-mer being almost twice as sensitive as the 8-mer in this experiment. This result may reflect the mechanisms of cleavage and detection, which are dependent on protonation of the aspartic acid and the piperazine groups in the tags. The larger probes tend to adopt higher charge states (not shown), i.e. they are more heavily protonated and the availability of more free protons on the larger probe ions may facilitate the cleavage of the tags, masking steric effects. However, only three different size molecules were evaluated, and this will be explored more fully in future.
A key feature of the Tandem Nucleic acid mass Tag design is the ease with which large numbers of chemically identical, isotopomeric tags can be made (Figure 3) . Sets of TNT isotopes should have almost identical behaviour in analytical separations (17) and during the ionization process. This means that it should be possible to use these tags to accurately quantify their associated oligonucleotide sequences as the ratios of the intensities of the TNT isotopomer fragments should reflect the ratios of the concentrations of the probes in solution or hybridized on their targets ( Figure 5 ). To demonstrate this feature of the TNT design, various experiments were conducted. In these experiments, pairs of TNT-PNA conjugates are analysed by ESI-MS/MS, where the parent tag ions are cleaved from their probes in the ESI ion source using a cone voltage of 25 V. The cleaved parent tag ions are subsequently selected from background by gating both 387.3 and 388.3 ions with the first quadrupole of the Q-TOF instrument. The gated parent tag ions are then subjected to CID in the second quadrupole of the Q-TOF instrument using a collision energy of 25 V followed by mass separation and detection in the TOF analyser to determine the ratios of the tag pairs. In the first set of experiments, pairs of TNT-PNA oligonucleotide probes of the same length and sequence, but with different tags were mixed in a predefined ratio and diluted to determine how well the ratios are conserved as the concentration of probes is decreased. Results are shown in Figure 6 . It can be seen that the ratios of the isotopic TNT-PNA probes are conserved over the range of concentrations investigated.
In a second experiment, pairs of TNT-PNA probes of the same length were mixed in various different ratios. The correlation between the expected and measured quantities of these different TNT-PNA ratios is shown in Figure 7 . It can be seen that there are simple linear correlations between the expected and measured ratios. The blue crosses in Figure 7 indicate the results of experiments where the two TNT-PNA probes with the same sequences but with different isotopomeric tags were mixed, i.e. the whole TNT-PNA probes were isotopes of each other. The measured ratios for these probes closely match the expected ratios.
The red squares in Figure 7 indicate the results of experiments where the two different probe sequences of the same length were mixed, i.e. although their TNT labels were different isotopes of each other, the complete TNT-PNA probes were not isotopes of each other. The red line represents a linear regression through these data points. Although the predicted and expected ratios do not match exactly, there is a good correlation between the results indicating that the measurements are quantitative. Since the TNT-PNA probes were actually different in these experiments, it is pleasing to see that there is correlation between the measured and expected quantities and the result suggests that, in future use, the measurements of the quantity of different targets in a sample could be calibrated against an internal control such as a housekeeping gene or, preferably, a known quantity of a spiked target sequence.
Quantification of hybridized probes was also evaluated. In the first experiment, external calibration of the quantities of hybridized probes was assessed, i.e. the amount of target in one sample was probed with a TNT-PNA whose abundance was then determined by comparison with a second reference sample comprising a predefined quantity of the duplex of the same target sequence and PNA probe sequence, but with a different TNT isotope conjugated to the probe, after the hybridization. In these studies, aliquots of a synthetic biotinylated 50-mer DNA oligonucleotide target was captured onto avidinated magnetic beads and hybridized with TNT-PNA probes with identical probe sequences but different tags. The hybridized beads were then mixed in different ratios. The target, arbitrarily selected, was a fragment of a sequence from the MOMP gene from Chlamydia pneumoniae. A fixed quantity of one TNT-PNA probe complementary to one of the targets was hybridized to the captured target sequences. The aliquots of beads were then washed extensively to remove probe that had not hybridized. The captured TNT-PNA probe mixture was then eluted into 50:50 water:methanol with 1% formic acid (a solvent suitable for ESI-MS/MS analysis) by thermal denaturation. The eluted TNT-PNA and the spike were then injected directly into the Q-TOF instrument for MS/MS analysis. The ratios of the intensities of the two tags derived from the TNT-PNAs should allow the amount of the target sequences in the pooled samples to be determined. Figure 8 shows the actual correlation between the expected and measured quantities of the target sequences. The results are very similar to the experiments where TNT-PNA probes with the same sequence but different tags are simply mixed together: the measured ratio matches very closely the expected ratio. Negative controls in which the target was absent do not show significant binding of TNT-PNA conjugates to the beads so the probe binding is sequence specific. This gives a clear indication that the probes behave quantitatively in hybridization assays and that TNT-PNA probes can be used for accurate quantification.
A further evaluation of the quantification of the TNT-PNA conjugates was carried out to determine whether accurate relative quantification can be derived from TNT-PNA pairs with different PNA sequences, i.e. can a reference sequence in a sample probed with one TNT-PNA be used to quantify a second sequence with a different PNA probe as long as the TNTs used in the probe pair are isotopes of each other. This would enable quantification by internal calibration using spiked sequences, housekeeping genes or similar controls in quantitative expression profiling or diagnostic assays. In these experiments, a pair of biotinylated 50-mer target oligonucleotides was used (MOMP-50 again and a sequence from B. anthracis, ANTHRAX-50; see Table 3 ). These were captured onto streptavidin-coated magnetic beads. The quantity of one target was fixed while the relative quantity of the second was varied. The captured targets were then hybridized at room temperature with a probe solution comprising equal quantities of MOMP-12-TNT1 and Anthrax-12-TNT2 probes (see Table 2 ). Probes of the same length were used together. After the hybridization, the magnetic beads were washed as before and the hybridized TNT-PNAs were eluted from their targets on the beads into 50:50 methanol:water with 1% formic acid and analysed as described earlier. Typical results are shown in Figure 8 . As observed in the simple mixture experiments, the measured TNT ratios show a linear relationship with the expected ratios but the measured quantities do not exactly match the expected ratios when the TNT-PNA probes being compared are not true isotopes but the linear relationship does mean that the measurements are quantitative. These data suggest that with appropriate choice of reference sequences, quantitative internal calibration should be achievable, which would be very useful in situations where suitable reference samples are not available for external calibration.
In this article the synthesis, characterization and application of ESI-cleavable TNT peptide-PNA conjugates to the quantitative detection of target DNA sequences by ESI-MS/MS has been described. The conjugates were prepared by first synthesizing the TNT tag peptide sequence in a peptide synthesizer, after which the peptide synthesis resin was transferred to a column compatible with a DNA synthesizer in which PNA can be prepared. The PNA sequence was extended directly from the peptide TNT. The use of PNA has several advantages for this application: (i) the oligonucleotide and the peptide TNT are generated in a single synthesis on the same resin, which means only a single purification step is required after the synthesis is completed; (ii) PNA is approximately 15% lower in mass than a corresponding DNA sequence, which Figure 8 . The relationship between the expected and measured ratio of mixtures of captured target sequences probed with 12-mer TNT-PNA probes. Experiments to quantify targets by external calibration where the PNA probes had identical sequences but different TNTs are shown with blue crosses and the blue regression line while experiments to quantify targets by internal calibration, i.e. using one sequence as a reference to quantify a different sequence are shown as red squares and the red regression line. enhances mass spectrometric sensitivity; (iii) PNA has enhanced binding affinity for its target compared with a corresponding DNA sequence, so a shorter probe can be used to achieve a corresponding level of specificity; (iv) PNA can hybridize under low salt conditions, which is more favourable for ESI analysis of samples as ESI-MS is susceptible to signal suppression by high salt concentrations.
We have shown that these TNT-PNA probes hybridize quantitatively and specifically to their targets and that the probes perform reliably over a wide dynamic range providing a new platform for multiplexed and quantitative genomic analysis.
The ESI-cleavable TNT mass markers described in this article have several properties that make them very useful for quantitative, multiplex assays, including the following: (i) The TNT portion can be elaborated into very large arrays of tags using only small numbers of starting components. The 20 standard amino acids as well as the large number of isotopic variants of these amino acids that are available provide the possibility of synthesis of numerous marker molecules that are easily resolved by the unique combination of parent and daughter ion massto-charge ratios; (ii) The ESI-cleavability allows direct analysis of solution phase assays without complex workup of the samples unlike MALDI, which requires that samples are spotted onto targets; (iii) The ESI-cleavable linker connecting the DNA and the TNT components cleaves virtually instantaneously during electrospray ionisation that will allow separations such as capillary electrophoresis to be performed in-line with the MS/MS analysis; (iv) In-line separation allows for a further level of multiplexing over and above the large numbers of available tags since the probes can be identified by their elution time as well as by their tag and in future work, we will explore the use of this feature to enable in-line coupling of analytical separations such as capillary electrophoresis; (v) sets of isotopic TNTs can be synthesized that behave identically during separations, hybridizations and labelling reactions enabling accurate measurements of quantities of target nucleic acids without 'dye effects' widening the range of applications for which mass tags can be employed.
The use of TNT-PNA oligonucleotide conjugates offer many of the advantages of fluorescent detection such as high specificity, ease and safety of handling and high sensitivity with the additional unmatched advantages that result from being able to generate large numbers of tags with predefined masses and from being able to construct these sets of tags with stable isotopes generating chemically identical entities that will behave the same in labelling reactions and in separation steps. This means that multiplexed analyses with accurate quantification are now enabled in a user-friendly format. Future experiments will be directed towards evaluation of the TNT-PNA probes for post-PCR amplicon detection. In addition, the development of TNT-DNA oligonucleotide conjugates and evaluation of these probes as primers for multiplexed PCR amplification and subsequent detection of PCR amplicons will also be pursued. The ability to employ in-line capillary electrophoresis with immediate cleavage and detection of tags will be of particular interest as many genomics assays, such as restriction fragment length polymorphisms, satellite marker analysis and multiplexed PCR employ size separations and the ability to perform such analyses with the higher levels of multiplexing enabled by this technology will be of great advantage. Hairpin structure within the 3′UTR of DNA polymerase β mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1 Aberrant expression of DNA polymerase β, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol β expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3′UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3′UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element—the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA–protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol β mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol β. In eukaryotic cells, DNA polymerase b (Pol b) is essential for base excision repair (BER) and involved in recombination and drug resistance (1, 2) . Pol b expression levels are important for the maintenance of genome integrity. On account of its low fidelity, the overexpression of Pol b, which has been reported in several cancer types, leads to a mutator phenotype associated with genetic instability and decreased sensitivity to anti-cancer chemotherapeutics (3) (4) (5) (6) . Conversely, cells deficient in DNA polymerase b display hypersensitvity to alkylating agent-induced apoptosis and chromosomal breakage (7) . Sugo et al. (8) have demonstrated that Pol b-deficient mice die immediately after birth, due to extensive apoptosis in the developing central and peripheral nervous systems. These data indicate that DNA polymerase b expression level is extremely important and must be tightly regulated.
Transcription of the Pol b gene is upregulated by the phosphorylated transcription factor CREB-1 in response to alkylating agent exposure. This upregulation requires the presence of the specific cAMP response element (CRE) in the promoter of the Pol b gene (9, 10) . It has also been shown that the Pol b promoter contains a binding site for the transcription factor SP1 (11) . Although regulation at the transcription level is apparent, expression of the Pol b transcripts might also be controlled at the posttranscriptional level. In rat cells there are two alternatively polyadenylated Pol b transcripts, with 3 0 UTRs of significantly different lengths (12) . The expression of the two transcripts is tissue specific. The short transcript is present in most tissues, with significantly higher expression in testis, while the long transcript is expressed mostly in the brain and lungs (13) . Motifs present in the 3 0 UTRs of the two transcripts are likely to be responsible for regulation of tissue-specific expression.
Sequence of the short 3 0 UTR is highly similar in the rat and human genes (up to 90% of homology in the conserved region), but in the further region of the long 3 0 UTR similarity abruptly decreases, which is consistent with the observation that there is only one (short) Pol b transcript in the human cells. Sequence similarity indicates that regulatory motifs present in the short 3 0 UTR of the rat Pol b transcript may act in the same manner and bind similar factors in human cells.
Structure prediction analysis (14) of the short 3 0 UTR revealed the presence of a putative hairpin element, $50 nt upstream of the polyadenylation sequence and 40-50 nt downstream of the termination codon (12) . There are several examples of transcripts with similar structures within the 3 0 UTR, constituting cis-acting regulatory elements, involved in mRNA localization. Structural motifs (hairpins) present within the 3 0 UTR of c-fos (15) , c-myc (16) , MT-1 [metallothionein-1, (17) ], slow troponin C (18) and vimentin (19) mRNAs have been shown to be involved in targeting these mRNAs to the perinuclear cytoplasm and, presumably, anchoring them in this location by binding to cytoskeletal elements (20) . Hairpin elements within the 3 0 UTRs were also reported to stabilize transcripts by binding factors that protect against nuclease cleavage [e.g. binding of IRP protein to the IRE sequence in the 3 0 UTR of transferrin receptor mRNA, (21) ] and to re-program translation as in the case of the SECIS element which directs an insertion of selenocysteine into in-frame UGA codons (22) .
In this report, we confirm the existence of the hairpin structure within the 3 0 UTR of the Pol b mRNA and demonstrate that this element influences the expression of a reporter gene. We describe the identification of a protein factor binding to this motif-Hax-1, an anti-apoptotic, cytoskeleton-related protein, which is known to bind a hairpin structure within the 3 0 UTR of vimentin mRNA. We demonstrate that binding occurs only for a Hax-1 dimer, though RNA binding is not a prerequisite for the dimerization itself. We confirm the importance of the hairpin structure for binding of Hax-1 by its mutagenic disruption, which impairs the RNA-protein interaction. We also report strong association of Hax-1 with the nuclear matrix, which is a novel finding, consistent with its transcript-binding properties. Taken together, these data suggest that the hairpin element within the Pol b 3 0 UTR represents a novel motif important for posttranscriptional regulation of expression.
The template for in vitro transcription encompassing the whole short 3 0 UTR of rat Polb (208 nt) was synthesized by PCR with a forward primer containing the T7 RNA polymerase promoter sequence (5 0 -TAATACGACTCAC TATAGGGCCTGCCCCACCCAGGCCT) and reverse primer (5 0 -AAACCATGGTACTGCGATC). The PCR was performed with the plasmid bearing the Pol b short 3 0 UTR sequence (pGEM-4Z/H), in the following conditions: 948C for 1 min followed by 35 cycles at 948C for 1 s, 608C for 1 s and 728C for 30 s.
The transcription reaction was carried out in 50 ml containing 20 pmol of PCR product, 500 mM rNTPs, 3.3 mM guanosine, 40 U of ribonuclease inhibitor RNase Out (Invitrogen) and 400 U of T7 RNA polymerase (Ambion). The reaction was carried out at 378C for 2 h and the transcript was purified from a denaturing 10% polyacrylamide gel, and 5 0 -end-labeled with T4 polynucleotide kinase and [g 32 P]ATP (4500 Ci/mmol; ICN). The labeled RNA was again purified by electrophoresis on a denaturing 10% polyacrylamide gel.
Prior to structure probing reactions, the labeled RNA was subjected to a denaturation and renaturation procedure in a buffer containing 2 mM MgCl 2 , 80 mM NaCl, 20 mM Tris-HCl pH 7.2 by heating the sample at 808C for 1 min. and then slowly cooling to reaction temperature. Limited RNA digestion was initiated by mixing 5 ml of the RNA sample (50 000 c.p.m.) with 5 ml of a probe solution containing lead ions, nuclease S1 or ribonucleases T1, T2 or Cl3. The reactions were performed at 378C for 10 min. and stopped by adding an equal volume of stop solution (7.5 M urea and 20 mM EDTA with dyes) and sample freezing.
To determine the cleavage sites, the products of the RNA fragmentation reaction along with the products of alkaline hydrolysis and limited T1 nuclease digestion of the same RNA molecule were separated on 10% polyacrylamide gels containing 7.5 M urea, 90 mM Trisborate buffer and 2 mM EDTA,. The alkaline hydrolysis ladder was generated by the incubation of the labeled RNA in formamide containing 0.5 mM MgCl 2 at 1008C for 10 min. The partial T1 ribonuclease digestion of RNAs was performed under semi-denaturing conditions (10 mM sodium citrate pH 5.0; 3.5 M urea) with 0.2 U/ml of the enzyme and incubation at 558C for 15 min. Electrophoresis was performed at 1500 V and was followed by autoradiography at À808C with an intensifying screen.
Site-directed mutagenesis SDM disrupting the hairpin structure was performed using the QuickChange Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. pGEM-3Z/H, containing 208 bp of the Pol b 3 0 UTR in the sense orientation, was used as a template for mutagenesis, resulting in the generation of pGEM-3Z/Hmut. Primers used for mutagenesis (changed nucleotides in bold):
Mutagenic primer #1 (forward):
Mutagenic primer #2 (reverse): 5 0 -GAAGAGGCAATCACCTAAAACACCCAAGGG TTACATAGCAAAGG-3 0
A fragment of 208 bp, containing the whole short Pol b 3 0 UTR sequence, generated as described in Structural analysis of RNA, was inserted downstream of Firefly luciferase in the pCMLuc vector (pCM2 derivative, a gift from Dr D.Weil, Institute Andre Lwoff, Villejuif, France) into the EcoRI site present in the polylinker, generating pCMLucH. The same fragment containing a mutation of the hairpin-forming region was cloned into the EcoRI site of pCMLuc, generating pCMLucHmut. The rat hepatoma FTO-2B cell line was grown on DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Transfection of FTO-2B was performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's guidelines. Two hundred nanogram of the appropriate plasmid was used for transfection, performed in 50 ml of medium in a 96-well plate. The activity of Firefly luciferase in lysates prepared from cells transfected with pCMLuc, pCMLucH and pCMLucHmut was measured using a luciferase reporter assay system (Dual-Glo, Promega). Assays were performed according to the manufacturer's instructions. Light emission was measured with LumiCount microplate luminometer (Packard). Transfection efficiencies were normalized by co-transfection with phRL-CMV vector (Promega) containing the Renilla luciferase.
Total RNA from transfected cells was isolated using the NucleoSpin RNA II kit (Macherey-Nagel). The first strand of cDNA was obtained with SuperScript Reverse Transcriptase (Invitrogen) from 1 mg of RNA, according to the manufacturer's instructions. Primers used in the experiment were designed to amplify 209 bp of the firefly luciferase transcript (forward: 5 0 -TCGTTGACCGCCT GAAGTCT-3 0 , reverse: 5 0 -GGCGACGTAATCCACGA TCT-3 0 ) and, as a reference, 232 bp of the Renilla luciferase transcript (forward: 5 0 -TGGAGCCATTCAA GGAGAAG-3 0 , reverse: 5 0 -TTCACGAACTCGGTGT TAGG-3 0 ). Quantitative PCR was performed using ABI Prism 7000 Sequence Detection System (Applied Biosystems). Power SYBR Green PCR Master Mix (Applied Biosystems) was used for detection. PCR was performed in the following conditions: precycling hold at 958C for 10 min, cycles: 958C, 30 s, 568C, 30 s, 728C, 30 s, up to 40. The ÁÁC T method was used for quantity calculations (23) . The slope of the validation curve was 50.1, which ensures, that target and reference efficiencies are approximately equal.
Yeast three-hybrid screen cDNA library construction. Total RNA from rat testis (Lewis) was isolated with Trizol reagent (Sigma) and mRNA was purified on Oligo(dT) cellulose columns (Molecular Research Center). Hundred microgram of mRNA was used for cDNA synthesis using the Gibco BRL cDNA Synthesis System, according to the manufacturer's instructions. EcoRI adapters (Gibco BRL) were ligated to the cDNA, followed by insertion into the EcoRI site of the pYESTrp1 vector (Invitrogen). The generated cDNA library contained roughly 1.5 Â 10 6 clones.
Generation of R40C-W. The yeast strain R40C (coat) (24) was disrupted in TRP1 gene in order to obtain tryptophane auxotrophy required for transformation with the pYESTrp1 vector. The disruption cassette was constructed in the YDp-W (25) vector by cloning KanMX resistance marker from pFA6akanMX4 (26) into XbaI-HindIII sites (blunted) within TRP1 gene. R40C was transformed with BamHI-BamHI fragment containing the disruption cassette and selected on geneticin (G418, 200 mg/ml) -containing medium.
Three-hybrid screen. A hybrid RNA construct (pRH5 0 H) containing the sequence of Pol b 3 0 UTR (from +3 to À208 downstream of the stop codon) was generated by inserting the Pol b sequence fragment into the unique SmaI site downstream of the phage MS2 sequence in the pRH5 0 vector (Invitrogen). The pRH5 0 H (H for hairpin) and cDNA library plasmids were sequentially transformed using the lithium acetate method (27) into the yeast R40C-W strain, which contains both HIS3 and b-galactosidase (b-gal) promoters integrated into its genome. Transformed yeast were plated on YC medium lacking tryptophan, uracil and histidine (YC-WUH) and containing 5 mM 3-aminotriazole (3-AT). Transformants were replicated on 25 mM 3-AT and after 3-6 days large colonies were picked for analysis. Selected transformants were screened out by the expression of beta-galactosidase using the colony lift assay with 5-bromo-4-chloro-3indolyl-beta-D-galactopyranoside (X-gal) as the substrate (CLONTECH Yeast Protocols Handbook). To eliminate RNA-independent false positives, plasmids were isolated (28) from selected blue colonies and used to transform electrocompetent E. coli KC8 cells. This bacterial strain enables complementation of yeast auxotrophy marker genes. Colonies with plasmids bearing the yeast TRP1 gene (pYESTrp1 cDNA clones) were isolated on M9 minimal medium lacking tryptophan. Plasmids were isolated, verified by restriction analysis and re-transformed into R40C-W, previously transformed with pRH5 0 H. Double transformants were checked again by the b-gal colony lift assay. This method of selection eliminated false-positive clones, leaving RNA-dependent positives. Positive clones were sequenced and analyzed by BLAST search.
Positive control vectors for the screen: pRH3 0 /IRE and pYESTrp1/IRP, were taken from RNA-Protein Hybrid Hunter Kit (Invitrogen).
Western blotting was performed with ECL Plus (Amersham) according to the manufacturer's instructions. Monoclonal anti-HAX-1 antibody (BD Bioscience) was used in 1:250 dilution. The secondary antibody, goat-antimouse (Pierce) was used in 1:2000 dilution. Anti-BclX L mouse antibody (Clone 2H12, Sigma) was used in dilution 1:80 and anti-proteasome 20S subunit alpha 1,2,3,5,6,7 (PW8195, Affiniti Research) mouse antibody was used in dilution 1:1000, both with secondary goat-anti-mouse antibody (Pierce) used in dilution 1:15 000. Anti-matrin 3 antibody (a kind gift from Ronald Berezney, State University of New York at Buffalo, Buffalo) was used in 1:1000 dilution with secondary rabbit-anti-chicken antibody (Pierce) used in dilution 1:15 000. Polyclonal rabbit anti-H2B antibody (UPSTATE) was used in dilution 1:5000 with secondary goat-anti-rabbit antibody (BioRad) used in dilution 1:5000.
Hax-1 cDNA was amplified with specific primers (forward: 5 0 -CCAGGATCCGAGCGTCTTTGATCTTTTC CGAGGCT-3 0 , reverse: 5 0 -GCTTGTCGACTCGGGAC CGAAACCAACGTCCTA-3 0 ) and the PCR product was cloned into pET201 (a gift from Csaba Koncz, Max Planck, Institute for Plant Breeding Research, Cologne). pET201 is a non-commercial vector representing a derivative of pET vector series used for expression of recombinant proteins fused to N-terminal bacterial thioredoxin and C-terminal 6xHis tags. pET201 vector encoding for thioredoxin was used concurrently to produce thioredoxin as a control protein. Bacteria were grown at 378C to an optical density of 0.5 (OD 600 ), followed by induction with 1 mM IPTG for 4 h. Recombinant proteins were purified under native conditions according to the Qiagen protocol for His-tag protein purification. Eluted proteins were analyzed by 12% SDS-PAGE and stained by Coomassie. Western blot analysis was carried out using monoclonal anti-Hax-1 antibody (BD Bioscience). Purified proteins were stored in 50% glycerol at À208C.
The 208 bp of Pol b 3 0 UTR, encompassing the whole short 3 0 UTR sequence with the hairpin encoding region, was cloned into pGEM-3Z and pGEM-4Z vectors (Promega) in both orientations, generating a set of templates for transcription of sense and antisense mRNAs. In vitro transcription was carried out with polymerases SP6 and T7 (Promega). [a-32 P]UTP (3000 Ci/mmol) was used for labeling. RNA integrity was controlled by polyacrylamide gel electrophoresis. Labeled transcripts were gel-purified (5% polyacrylamide, 8 M urea).
In vitro transcribed RNA with heparin (5000 U/ml, Sigma) and 160 U of RNAse inhibitor (RNase OUT, Invitrogen) was incubated on ice for 15 min in cross-link buffer (20 mM Tris, pH 7.9, 2.5 mM MgCl 2 ) with 0.5 mg of recombinant Hax-1 protein, BSA (Sigma) or thioredoxin (recombinant thioredoxin purified from bacteria using pET201 vector in the same conditions as Hax-1 protein). Cross-linking was performed on ice for 30 min in the UV Stratalinker 1800 (Stratagene). After cross-linking, the reaction was incubated in the presence of RNase A (final concentration 0.6 mg/ml) for 20 min. in room temperature. Laemmli loading buffer was added and the samples were heated to 958C for 5 min. Samples were analyzed on SDS-PAGE, with Prestained SDS-PAGE Standards, Kaleidoscope (Bio-Rad).
Purified recombinant Hax-1 protein (1 mg for the western blot, indicated amount for silver staining) was suspended in 100 ml of sample buffer (SB: 10 mM HEPES, pH 7.4, 120 mM potassium acetate, 2.5 mM MgCl 2 , 1.6 mM dithiotreitol), incubated for 15 min in room temperature to establish monomer-dimer equilibrium and then incubated for 20 min in 228C with 0.00125% glutaraldehyde (Sigma). The reaction was stopped by the addition of 50 ml of 3-fold concentrated loading buffer (150 mM Tris pH 6.8, 6 mM EDTA, 6% SDS, 30% glycerol, 1 M urea, 0.003% bromophenol blue). Samples were heated (908C) and separated on the SDS-PAGE. Proteins were detected by silver staining (29) and western blot. The lanes in the silver-stained gel were scanned and the areas under the peaks were quantified using Multi-analyst (Bio-Rad). The results were analyzed by nonlinear regression analysis to determine an approximate dimer dissociation constant, as described in (30) .
The filter-binding assay was performed by a modified method described by Wong et al. (31) . Briefly, radiolabeled, in vitro transcribed RNA at a concentration of $10 nM was heat denatured, allowed to refold and incubated for 15 min in room temperature in binding buffer (20 mM Tris, pH 7.9, 100 mM KCl, 2.5 mM MgCl 2 ) containing heparin (5000 U/ml, Sigma) and 160 U of RNAse inhibitor (RNase OUT, Invitrogen) with purified Hax-1 protein. The reaction mix was loaded onto a presoaked nitrocellulose membrane (0.45 mm, Osmonic Inc.) on top of a nylon membrane (Hybond-N, Amersham) and filtered under pressure in a slot-blot apparatus. Following filtration, each filter was dried and quantitated on a PhosphorImager (BioRad) using QuantityOne software. Dissociation constants (K d ) for the RNA-protein complexes were obtained by fitting the empirical data to a sigmoidal curve by nonlinear regression analysis, using Maxima 5.4. Fitting was performed in respect to dimer concentration calculated as a function of the total protein concentration, as in (30) .
Four hundred milligram of pulverized rat testes were homogenized in 1 ml of 2Â MEB buffer (100 mM Tris-HCl pH 7.5, 20% glycerol, 4 mM DTT, 10 ml/ml protease inhibitor cocktail [Roche]) in a Potter homogenizer, filtered on 50 mm Nitex membrane (Tetko) and centrifuged at 2000g, 48C. The pellet (nuclear fraction) was washed two times with 0.5 ml of 2Â MEB buffer. Nuclei were gently resuspended in 0.5 ml of 2Â MEB (small aliquot was analyzed by DAPI staining), passed through a needle and incubated 30 min on ice with 10 ml of DNase I (Warthington). The supernatant was centrifuged at 10 000g, 48C (mitochondrial fraction). After separation of the mitochondrial fraction, the supernatant was centrifuged at 100 000g in an ultracentrifuge and the supernatant (cytoplasmic fraction) was collected.
Procedures were adapted from Reyes et al. (32) . Highsalt method: isolated nuclei were resuspended in 1 ml of CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM surcose, 3 mM MgCl 2 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.5% TritonX-100 with protease inhibitor cocktail [Roche]-one tablet per 50 ml), incubated for 3 min at 48C and spun down at 5000g for 3 min to separate nuclei from soluble proteins. Next the chromatin was solubilized by RNase-free DNase I digestion (0.1 mg, Warthington) in 1 ml of CSK buffer for 15 min at 378C. Next, ammonium sulfate was added to a final concentration of 0.25 M and after a short incubation (5 min at 48C) the samples were centrifugated again. The resulting pellet was extracted three times with 2 M NaCl in CSK buffer in the following steps: resuspension, incubation at 48C for 5 min and centrifugation. 
In the first step of our study, we undertook the determination of the structural features of the entire short 3 0 UTR of the rat DNA polymerase b transcript (208 nt). For this purpose, we used limited fragmentation of the 5 0 -end-labeled RNA with six well-characterized biochemical structural probes: lead ions and five enzymes ( Figure 1A ). Nuclease S1 and lead ions have no documented nucleotide specificity, while ribonuclease T2 and A recognize all nucleotides but have a higher activity for adenosines and pyrimidines, respectively. RNase T1 exhibits specificity exclusively for guanosines and RNase Cl3 digests C-residues only. All the above structural probes recognize flexible and single-stranded regions in the RNA structure. Prior to the probing experiments, the 5 0 -end labeled transcript of the Pol b short 3 0 UTR was analyzed by non-denaturing gel electrophoresis. The result of this test suggests that only a single conformer is formed by the analyzed RNA molecule (migrated as a single band on a gel, not shown).
Probing data demonstrated that the short 3 0 UTR of Pol b transcript forms three separate structural modules, designated M1-M3 ( Figure 1B) . Module M1 represents a small hairpin structure with a C-rich terminal loop. The 3 0 -part of this structure is efficiently hydrolyzed with lead ions, even at a low concentration of the probe, which suggests that M1 hairpin has binding capacity for this metal ion. Module M3 contains the polyadenylation signal, followed by the cleavage and polyadenylation site, so only a part of it is present in the mature short Pol b transcript, which implies minor importance of the whole M3 module as a post-transcriptional regulatory element.
Module M2 is formed by nucleotides located between bases G29 and C96 of the analyzed transcript and is composed of three helical regions, two 6 bp and one 14 bp in length. Each of these helical regions is resistant to enzymatic digestion and lead ion hydrolysis. The three helixes are separated by two asymmetric, internal loops (b and c), which are mapped very well by all probes used. The longest helical region includes as many as six non-WC, U-G and G-U base pairs. Four of these exist as two tandems: U-G, G-U and U-G, U-G, which are known to be potential metal-or protein-binding sites. The hairpin structure contains a small 3-nt terminal loop (5 0 -UAU), which is also well recognized by both lead ions and enzymes ( Figure 1A and B) . The M2 hairpin structure is conserved among species (Figure 2) , which suggests its significant role in regulation of transcript fate.
In order to demonstrate the essential role of the evolutionarily conserved hairpin structure in the regulation of Pol b expression, we carried out in vitro mutagenesis to disrupt the M2 hairpin element. Mutagenic primers were designed based on an MFOLD (14) prediction of the potential effect of nucleotide change on hairpin structure ( Figure 3 ). Substitution of three C-residues for three G-residues in the stem-forming region (positions 67-69) changed the predicted free energy increment (dG) from À14.1 kcal/mol for the intact structure to À3.6 kcal/mol for the mutant. The mutated sequence (Hmut) was used in luciferase reporter assays and served as a template for in vitro transcription to generate mRNA for subsequent crosslink and filter-binding analysis.
The influence of the M2 hairpin structure within the short 3 0 UTR of Pol b mRNA on expression was analyzed utilizing a luciferase reporter system. The rat hepatoma cell line FTO-2B was transfected with reporter constructs bearing the Firefly luciferase gene appended by the Pol b 3 0 UTR sequence containing the M2 hairpin element, (pCMLucH) and by the same sequence containing a structure-disrupting mutation in the hairpin-forming region (pCMLucHmut). A vector bearing the unmodified luciferase gene served as a control (pCMLuc). To asses mRNA levels of the reporter, quantitative PCR was performed for cDNA preparations obtained from cells subjected to the same transfections as for luciferase assays. The results (Figure 4) show a significant decrease of mRNA levels for the construct with the hairpin structure (H), compared with almost unchanged mRNA levels for the mutated hairpin (Hmut). These differences, however, are not reflected at the protein level: both constructs (H and Hmut) caused an increase in luciferase expression, although only for the Hmut is this increase significant. The relative increase of protein levels in respect to mRNA levels is therefore more than 3-fold for the construct with the intact hairpin, while only a slight relative increase ($1.7-fold) was observed for the mutated construct. These changes in expression indicate a complex posttranscriptional regulation in which the hairpin element has a key function.
In order to identify proteins interacting with the M2 hairpin element present within the 3 0 UTR of the Pol b transcript, we performed a yeast three-hybrid screen of a rat cDNA library. The IRE-IRP interaction served as a positive control, while negative controls consisted of empty pRH5 0 and pYESTrp1 vectors and single transformants of pRH5 0 H 'bait' plasmid or of the protein-hybrid clone isolated from the library. Out of 63 positive clones obtained after the first selection, only 11 were RNA dependent, and these were sequenced and analyzed. Of these, only one was in the proper reading frame, had the correct in-frame orientation and represented a coding sequence-the terminal 622 bp of the rat Hax-1 mRNA(corresponding to the last 150 aa of the protein), ( Figure 5 ). This clone exhibited strong growth on 25 mM 3-AT medium without histidine and tested positive in the b-gal plate test (Table 1) .
UV-cross-linking confirms specific binding of Hax-1 to the mRNA region containing the M2 hairpin structure
In order to confirm the results of the 3-hybrid screen, analysis of in vitro binding was carried out using purified (14) . The free energy increment for the two structures varies considerably, and amounts to À14.1 for the M2 hairpin and À3.6 for the mutant. Presence of the hairpin-containing 3 0 UTR confers mRNA instability but also enhances expression at the protein level. Disruption of the hairpin prevents mRNA degradation, but a mutation-containing construct still slightly enhances expression at the protein level. Constructs used for transfections: control vector (pCMLuc), hairpin-containing construct (pCMLucH) and mutated hairpin (pCMLucHmut). mRNA levels were quantitated for each construct by RQ-PCR in cDNA isolated from transfected cells (8 independent transfections), in triplicate, normalized with respect to Renilla luciferase mRNA levels. Firefly luciferase levels were assessed for the same eight transfections (in six repeats) and normalized with respect to Renilla luciferase levels, which served as a control of transfection efficiency. Statistical significance is reported as P-value: Ã P50.01, ÃÃ non-significant. UV-cross-linking was performed using the purified recombinant Hax-1 and in vitro transcripts of the hairpin-containing region (H), a control antisense transcript of the same region (A), and the mutated transcript (Hmut). Bovine serum albumin and purified thioredoxin served as controls for interaction specificity. Hax-1 demonstrated a specific interaction only with the hairpin-containing RNA, and this transcript did not interact with either BSA or thioredoxin ( Figure 6) . The cross-linked band migrated at a molecular weight of $100 kDa, which corresponds to a recombinant Hax-1 dimer (the recombinant protein has a higher molecular mass than endogenous Hax-1 [35 kDa], due to its fusion with the thioredoxin sequence). The protein dimer is likely formed during the UV-cross-linking procedure, as has been previously documented (34) . No band corresponding in size to monomeric recombinant Hax-1 (ca. 50 kDa) was observed, which indicates that the protein interacts with RNA exclusively in the form of a dimer.
To confirm that Hax-1 forms a dimer, purified recombinant protein was used for chemical cross-linking with glutaraldehyde. As indicated in Figure 7 , monomers with a molecular mass of around 50 kDa were detected, as were dimers with a molecular mass around 100 kDa. Since dimer formation was detected in an in vitro experiment performed with purified protein, one can conclude that the RNA molecule is not necessary for dimerization. As deduced from the UV-cross-linking experiments, RNA is bound exclusively by dimeric Hax-1, hence the dimerization rate may represent an important factor for RNA-protein complex formation. Glutaraldehyde cross-linking with increasing protein amounts ( Figure 7A ) allowed for the estimation of the monomerdimer equilibrium dissociation constant (K ddim ) at 13.5 mM AE 5.5.
To corroborate the UV-cross-link results, filter-binding assays were performed with increasing amounts of Hax-1 protein and a constant amount of the same test RNAs (hairpin-containing region-H, antisense-A and mutant-Hmut). Data were analyzed on a Klotz plot ( Figure 8 ) and the apparent K d for each complex was calculated. The fit of a nonlinear binding curve to the experimental data points was best when calculations were performed in respect to dimer concentration, which indicates that the RNA interacts only with a dimeric form of the protein, thus confirming the UV-cross-link results. The approximate K ddim established from the titrated glutaraldehyde cross-linking experiments (13.5 mM AE 5.5) was in the same range as the K ddim predicted by curve-fitting (fitting performed simultaneously for the H and Hmut data point series in respect to variable K d and constant K ddim values yielded a K ddim of 10 mM AE 4). Transcript H showed the greatest affinity for Hax-1, with a K d of 28 nM AE 7, and significantly lower Table 1 . Three-hybrid screen of a rat testis cDNA library proteins interacting with the M2 hairpin-containing region of the Pol b mRNA 3 0 UTR Plasmids used for transformation of R40C-W Growth on 25 mM 3-AT b-gal plate test
The interaction for the single positive clone encoding for the terminal 622 bp of the rat Hax-1 gene (Hax-1-C) was tested for growth on 25 mM 3-AT medium without histidine and using the b-gal plate test. The IRE/IRP interaction served as positive control, while transformation with empty vectors as well as single transformants of tested constructs represented negative controls. Hax-1 localizes in mitochondria, but also in the nuclear matrix
While Hax-1 mitochondrial localization has been confirmed in many reports (35) (36) (37) , this is not obviously consistent with its transcript-binding properties. Several other reports have shown localization of the protein to the endoplasmic reticulum (35, 36, 38) , apical membrane of hepatocytes (39) and nuclear envelope (35) . In order to establish Hax-1 cellular localization in rat cells, we performed organellar fractionation and subsequent fractionation of nuclei, followed by SDS-PAGE and western blot. These experiments confirmed the presence of Hax-1 in mitochondria, but also indicated its localization in the nucleus ( Figure 9A ) with only traces of Hax-1 detectable in the cytoplasm. Subsequent nuclear fractionation performed with two different methods, revealed that Hax-1 is present in the fraction containing nuclear matrix proteins ( Figure 9B and C), while it was not detected in the other fractions containing soluble and chromatin-associated proteins.
To ensure proper quality of nuclear fractions, western blots with appropriate marker proteins were performed. Fraction 1 in both methods contains soluble, chromosomal proteins, released after DNaseI treatment, represented here by histone H2B. These proteins were washed out in subsequent steps of the preparation (fractions 2-3). The nuclear matrix fraction (fraction 4) was probed with a matrix-specific protein matrin 3 antibody (40) . Close association of Hax-1 with the nuclear matrix sheds new light on its mRNA-binding capacity and may indicate its role in regulation of transcript fate.
Stable secondary structures in 3 0 UTRs have been shown to play a role in mRNA sorting and localization (20) . Some of them have been also reported to influence mRNA stability (21) . The existence of a stable structural motif in the 3 0 UTR of the rat Pol b mRNA was predicted previously (12) and it was speculated that it may have a regulatory role. In the present work, secondary structure analysis by lead ion hydrolysis and enzymatic digestion revealed the existence of several motifs in the analyzed sequence, namely, a region of strong lead ion binding (M1), a hairpin-forming and highly evolutionarily conserved region (designated as M2) and the region containing polyadenylation sequence (M3). Only M2 is evolutionary conserved, the rest of the untranslated region has high interspecies sequence variation, which suggests lesser functional importance. The sequence in the conserved region exhibits 86.8% identity to the homologous human sequence (100% in the upper stem region), which may indicate a similar role of this element in human cells.
We show here that the hairpin structure within the 3 0 UTR influences the expression of a luciferase reporter gene. Lowering of luciferase mRNA levels for the construct with an intact hairpin structure in contrast with almost unchanged mRNA levels for the mutated structure indicate that the hairpin is in fact an RNA destabilizing element. However, at the protein level, expression for both constructs exceeds the expression of the control. This indicates the presence of at least two regulatory events in which the hairpin structure is involved: (i) mRNA degradation, (ii) enhanced mRNA transport (possibly coupled with mRNA stabilization) and/or enhanced translation. These effects may be the consequence of the competitive binding of trans-acting factors for the binding site within the hairpin. Thus, identification of the hairpin-binding factors is important for assessment of its physiological role.
A yeast three-hybrid screen identified Hax-1 as the binding partner for the hairpin structure of the Pol b 3 0 UTR. Hax-1 is an RNA-binding protein, known as an anti-apoptotic factor (36) associated with cytoskeletal proteins and involved in cell migration (38, 41) .
Hitherto only one RNA target of Hax-1 has been identified: vimentin mRNA (42) . Data from several reports suggest that Hax-1 binding to the hairpin element within the 3 0 UTR of the vimentin transcript plays a role in its localization to the perinuclear cytoplasm (19, 20, 42) . The importance of proper vimentin transcript localization is illustrated by the fact that its misdirection alters cell morphology and motility (43) . The identification of a second RNA target of Hax-1-the hairpin element present in the Pol b transcript-raises the question as to the identity of other mRNA targets of the protein. One may speculate that there is a pool of such mRNAs, especially because vimentin and Pol b are not functionally or evolutionarily related nor are they involved in the same pathway.
Comparison of the hairpin motifs in vimentin and Pol b mRNAs did not reveal any significant similarities, which could suggest substrate requirements for Hax-1 binding. The presence of U-rich single-stranded regions (vimentin: AGUUUU in the terminal loop, Pol b: AGUUAU in the internal loop) represents the only similarity between the two structures, but the helical regions adjacent to this U-rich sequence in the Pol b mRNA are not evolutionarily conserved. The lack of similarities suggests that Hax-1-binding mechanism and affinities might be different for these two structures.
It is an open question if Hax-1 is in fact a destabilizing factor or if its actions in respect to the Pol b transcript consist of mRNA stabilization, possibly coupled with its transport and/or localized translation. The fact that Hax-1 binds to the instability element (the hairpin) and does not bind to the stable mutated transcript suggests a role in mRNA destabilization. However, data concerning the role of Hax-1 in the regulation of vimentin transcript, indicating that it facilitates its transport to the perinuclear space, are rather contradictory to its potential functions in mRNA degradation. Another possible explanation is that Hax-1 may stabilize otherwise unstable mRNAs and facilitate their transport or enhance the translation rate, conferring elevated luciferase levels in respect to mRNA levels.
If this latter case is true, considering that Hax-1 has been identified as the same trans-acting factor for both vimentin and Pol b mRNAs, the hairpin element within the 3 0 UTR of the Pol b transcript may also represent a motif directing mRNA to the perinuclear space. Perinuclear localization of certain transcripts, and their subsequent translation at this site, could facilitate an efficient nuclear import of newly synthesized proteins (15, 16, 17) . DNA polymerase b as a nuclear protein could also benefit from such a mechanism.
To present a satisfactory explanation of the role of Hax-1 transcript binding in the cell, one has to resolve the question of the subcellular location of Hax-1. Hitherto, Hax-1 has been reported to localize predominantly in the mitochondria (35, 36, 37) but it has also been detected in the endoplasmic reticulum (35, 36, 38) , apical membrane (39), lammelipodia (38) and nuclear envelope (35) . In the last case, the presence of Hax-1 in the nuclear envelope was interpreted as a consequence of its association with intracellular membranes (by its putative transmembrane domain), as a continuum of endoplasmic reticulum localization. Our data reveal for the first time, that Hax-1 is associated with the nuclear matrix, which is coherent with its transcript-binding capacity and supports the notion of its role in post-transcriptional regulation.
Some new data support Hax-1 association with the nucleus. In a recent report, Kawaguchi (44) shows that Hax-1 is present in the nucleus of systemic sclerosis fibroblasts (but not in normal fibroblasts) and is involved in pre-IL-1a translocation into the nucleus-a process blocked by inhibition of Hax-1. This activity is contradictory to previously reported Hax-1 involvement in the cytoplasmic retention of IL-1a (45) and EBNA-LP (46) . The role of Hax-1 in protein import into the nucleus might suggest that it is shuttling between the nuclear matrix and perinuclear space, transporting different cargo molecules.
Participation of the hairpin element in the binding between the Pol b 3 0 UTR and Hax-1 was demonstrated by UV-cross-linking, in which the transcript with an intact hairpin structure bound to the protein, whereas a transcript with a disrupted hairpin did not. Cross-linking also revealed that Hax-1 binds to mRNA only in the form of a dimer. The presence of a band of a molecular weight of 100 kDa indicates that UV exposure cross-linked a complex consisting of RNA bound to a protein dimer. RNA-monomer complexes were not detected. Data from chemical cross-linking lead us to conclude that the RNA molecule is not necessary for dimerization. However, given that only a small percentage of protein dimerizes in vitro in the absence of mRNA, the possibility that RNA binding influences the dimerization rate is tempting, and remains to be assessed.
Filter-binding experiments, complementing the crosslinks, showed that the binding, though not completely abolished, is substantially weakened for a mutated sequence with a disrupted hairpin structure.
Only the C-terminal part of Hax-1 appears to be involved in mRNA binding, since a truncated peptide bearing only the last 150 aa of the protein suffices for binding the hairpin-containing element, as demonstrated in our experiments utilizing the yeast three-hybrid system ( Figure 5 ). Considering that an interaction with RNA occurs only for dimeric Hax-1, these findings suggest that the domain responsible for the dimerization is also located in the C-terminal part of the protein. From these results, we deduce that BH domains (BH1 and BH2) present in the N-terminal part of the protein do not take part in the dimerization. BH domains and a transmembrane domain (present at the C-terminus of Hax-1) represent the features of Bcl-2 family proteins, but there is no significant sequence homology between these proteins and Hax-1only a weak, partial homology to pro-apoptotic Nip3 (35) . Even though BH domains are known to be important for oligomerization of the proteins from the Bcl-2 family, data seem to exclude the possibility that they are responsible for dimerization of Hax-1.
We have demonstrated that the hairpin structure within the 3 0 UTR of the Pol b mRNA represents a posttranscriptional regulatory element. Hax-1 protein, which binds to this element, appears to be an important transacting factor, though the exact mechanism of Hax-1-mediated regulation remains to be elucidated, and the mechanisms implicating its role in control of mRNA stability, transport and/or localized translation must be verified by subsequent experiments. Hax-1 is a multifunctional protein, active in different cellular compartments and involved in various cellular processes. Attention has been focused on its functions in apoptosis and regulation of cell motility, but it seems that it has a more complex mode of action and plays a regulatory role in the context of its specific mRNA targets. Global public goods and the global health agenda: problems, priorities and potential The 'global public good' (GPG) concept has gained increasing attention, in health as well as development circles. However, it has suffered in finding currency as a general tool for global resource mobilisation, and is at risk of being attached to almost anything promoting development. This overstretches and devalues the validity and usefulness of the concept. This paper first defines GPGs and describes the policy challenge that they pose. Second, it identifies two key areas, health R&D and communicable disease control, in which the GPG concept is clearly relevant and considers the extent to which it has been applied. We point out that that, while there have been many new initiatives, it is not clear that additional resources from non-traditional sources have been forthcoming. Yet achieving this is, in effect, the entire purpose of applying the GPG concept in global health. Moreover, the proliferation of disease-specific programs associated with GPG reasoning has tended to promote vertical interventions at the expense of more general health sector strengthening. Third, we examine two major global health policy initiatives, the Global Fund against AIDS, Tuberculosis and Malaria (GFATM) and the bundling of long-standing international health goals in the form of Millennium Development Goals (MDG), asking how the GPG perspective has contributed to defining objectives and strategies. We conclude that both initiatives are best interpreted in the context of traditional development assistance and, one-world rhetoric aside, have little to do with the challenge posed by GPGs for health. The paper concludes by considering how the GPG concept can be more effectively used to promote global health. Although the health of the world's poor has been an apparent humanitarian concern of the world's rich for many years, results based on appeals to such 'humanity' have not been sufficient. Even recent high-profile engagements by the Global Fund Against AIDS, Tuberculosis, and Malaria (GFATM), the United States President's Emergency Plan for AIDS Relief (PEPFAR), the Bill and Melinda Gates Foundation, and WHO disease-targeted programs such as Stop TB and Roll Back Malaria have failed to bring us to the levels of assistance needed to achieve the healthrelated Millennium Development Goals (MDGs).
Beginning in the late 1990s, the suggestion emerged to address this situation by encouraging policy makers in rich countries to view health assistance not only as humanitarian but as a selfish investment in protecting the health of their own populations. The key concept underlying this new interpretation is that of 'global public goods' (GPGs) [1] . This paper briefly outlines and clarifies the GPG concept, and identifies two major health GPGs: health research and development (R&D), and communicable disease control. However, just because a problem is global and formidable, or just because the response is multilateral, does not necessarily mean that it has anything to do with the undersupply of GPGs. We show this by considering two major global health innovations, GFATM and the rebranding of traditional health objectives in the form of MDGs. Based on the review, in a concluding section we suggest three ways in which the GPG concept can be more effectively deployed to promote global health development.
The GPG concept is an extension of the economic tradition of classifying goods and services according to where they stand along two axes: one measuring rivalry in consumption; the other measuring excludability. Pure private goods are those that we are most used to dealing with in our day-to-day lives, and are defined as those goods (like a loaf of bread) that are diminished by use, and thus rival in consumption, and where individuals may be excluded from consuming them. At the opposite end of the spectrum are pure public goods, which are non-rival (not diminished by use) and non-excludable (if the good is produced, it is freely available to all). Public security is an often-cited example. In between these extremes are 'impure' goods, such as 'club goods', which have low rivalry but high excludability, and 'common pool goods', which have low excludability but high rivalry [2] . Clearly in this case, 'health' itself is a private good, as are the majority of goods and services used to produce health [3] .
One of the fundamentals of public economics is that the free market -the interplay of individual supply and demand decisions mediated through the price systemwill result in the provision of less than the collectively optimal level of public goods. Thus, the state has a role to play, either in producing the good directly (the traditional approach) or at least in arranging for its production by a private firm (the increasingly popular 'outsourcing' strategy). Examples of national public goods run from police protection to national security to financial regulation to museums and artistic ensembles. But some goods are quite clearly public at the global level. The classic case is greenhouse gas emission control.
A reasonable functional definition would be that a GPG is "a good which it is rational, from the perspective of a group of nations collectively, to produce for universal consumption, and for which it is irrational to exclude an individual nation from consuming, irrespective of whether that nation contributes to its financing" [[3], page 9]. The main issue facing non-national (global or regional) public good provision is how to ensure collective action in the absence of a 'government' to directly finance and/or provide the public good, the response in the case of national public goods [4] .
Given the reluctance of voters to support programs some of whose benefits are felt beyond the borders, an aspect deserving special attention is mobilizing non-traditional sources of finance [5] . Cutting across all aspects of the GPG concept is the key fact that collective action is in donor countries' self-interest.
The GPG concept thus has a specific meaning within economics. However, it has suffered as it has found currency as an advocacy tool for global resource mobilisation [6] [7] [8] .
Since a GPG calls for collective action, then, clearly, one's favourite program must be producing a GPG. This has given rise to "fuzziness" and "trendiness" [ [9] , page 2). The GPG 'tag' is at risk of being attached to anything of particular attraction and importance, to the point that, at the limit, anything promoting development could be considered a GPG. This is to be avoided, as overstretching the concept devalues the validity of the point that there really is a class of GPGs requiring public support or provision [10] .
Indeed, Smith et al [11] suggest that the GPG concept may perhaps be most usefully applied to just two aspects of health. The first is research and development (R&D) and the second is communicable disease control (epidemiological surveillance, immunization, and other preventive measures). In the next section, we ask how the GPG concept, particularly the need for collective action, has affected policies and programs in these areas.
Health R&D unquestionably has GPG aspects, and there is not enough of it in fields that would benefit poor countries. Historically, the public and the not-for profit sectors have carried out research resulting in new drugs and treatments, but the private for-profit sector now plays the largest role [12, 13] . An important policy question is therefore how to encourage private sector firms to engage in research benefiting poor countries and peoples: the ubiquitous '90-10 problem' (that 90% of global R&D spending in health is targeted at diseases affecting only 10% of the world's population) [14] . A related but distinct question is how to bolster the demand for drugs in low-income countries (and hence firms' willingness to engage in R&D); we touch on this in a section below in which we discuss Advance Purchase Commitments and other financial innovations.
A GPG perspective would argue that provision of adequate R&D related to diseases of the poor requires innovative collective action. This no-more-business-as-usual attitude has clearly motivated the explosion in the number of Global Public-Private-Partnerships (GPPPs) undertaking R&D related to diseases of the poor [15] [16] [17] . An order-of-magnitude estimate of GPPPs' annual spending might be US$1 billion [18] . This may be compared with total global health R&D spending on the order of US$100 billion [14] . This sounds small, but when the US$1 billion is compared to the estimated US$2.5 billion spent on health R&D by governments in low-and middleincome countries, the perspective is much more favourable for the important role played by GPPPs. In the area of R&D related to "neglected diseases" of the tropics, GPPPs occupy a decisive position.
The GPG perspective supports collective action in the area of infectious disease control when reduction in disease prevalence in Country A has a benefit for Country B as well. Areas in which this is particularly true are diseases for which eradication is feasible (polio) and diseases that are highly transmissible around the world, whether by human carriers (SARS), by trade in products (BSE), or by animal vectors (West Nile Virus, avian influenza). The control of antibiotic resistance is a closely related GPG problem.
As in the case of R&D, the GPG perspective has informed a number of major new initiatives to provide (not only develop new means of), communicable disease control. These include the GAVI Alliance (formerly the Global Alliance for Vaccines and Immunization), Stop TB, Roll Back Malaria, and others. The main question such initiatives face is the form that assistance should take.
There are basically three types of public health interventions: vertically targeted interventions (focused immunization or disease eradication campaigns, for example), horizontally targeted interventions (universal access to a basic medical care package including vaccination, for example), and sector-wide interventions such as capacity building for improved infrastructure and administration. For many years, donors and health officials in low-and middle-income countries gave emphasis to vertical interventions financed by 'earmarked' funds. Problems with this approach include duplication and lack of coordination among projects, 'recipient fatigue' in health ministries forced to administer multiple grants, distortions in local resource allocation such as poaching skilled personnel, and "crowding out" (of which more below) [19] . All donors and the partner countries have committed themselves, through the 2005 Paris Declaration, to pursue harmonisation of practices, standards, and criteria in foreign assistance. Yet the urge to compete rather than cooperate is strong, and well-entrenched donor practices and protocols disappear only stubbornly.
One of the ironies of the current health landscape is that, as many in the public health community moved away from vertical interventions towards broader approaches the GPG perspective has helped to fuel the proliferation of specific infectious disease-targeted programs. Yet the experiences of programs in immunization, malaria control, and tuberculosis control demonstrate that impacts are limited by sector-wide weaknesses such as lack of a cold chain, shortages of skilled personnel, insufficient resources for operating vehicles, etc. The plethora of new vertical initiatives may contain the seeds of its own failure if health systems are not generally strengthened (including the crucial human resources aspect) [20] . The danger is that the GPG agenda will promote focused interventions easy to "sell" to voters at home because they address an identifiable menace, at the expense of broader health system strengthening. One response is to identify the health system as a prime 'access good' -not a GPG itself, but a fundamental requirement for the provision of GPGs [11] .
The GPG perspective has contributed to a large number of new programs. However, some caveats are in order. Providing an adequate supply of a GPG requires spending more than would have been spent in the absence of collective action, i.e. "additionality." The additionality debate is complex, and involves at least three questions: -At the individual country level, we may ask whether international assistance for production of GPGs in Country X reduced (or, in development parlance "crowded out") Country X's government spending for production of GPGs. Since most of the countries in question are very poor and local needs take clear precedence over global ones, it is probably safe to answer this question in the negative.
-More pressing is the question whether international assistance for production of GPGs in Country X reduced international assistance for production of non-GPGs in Country X. A direct answer to this question is difficult to provide because data on foreign assistance at the level of destination countries are much worse than data by country of origin. Reisen et al [21] , looking at the latter, concluded that the average bilateral donor's allocation of US$1 to GPG production reduced its spending on non-GPG foreign assistance by US$0.25.
-Finally, additionality questions may be posed as between donors. The focus of this particular storm con-cerns the activities of the GFATM and PEPFAR. Many have complained that PEPFAR diverts U.S. resources away from GFATM, a multi-lateral agency, to a bilateral and highly politicised program. Questions of donor additionality are not confined to HIV/AIDS. Cohen [17] , in looking at the involvement of GPPPs in health R&D, reports that, in expert interviews, researchers complain that the new availability of philanthropic funds for medical research is "crowding out" funding that would have been received from government agencies such as the U.S. National Institutes for Health.
While the additionality of resources is ambiguous, we can answer a closely related issue definitively. Resources being used for the provision of GPGs in the area of health R&D and communicable disease control come from traditional, not innovative, sources. The typical new initiative depends on a philanthropic institution for its start-up, followed by infusions of government support channelled through bilateral aid organizations [15] . Yet, the GPG concept is firmly rooted in the self-interested use of domestic monies and as such sees funding as distinct from current aid and philanthropic flows. While there have been innovative fund raising suggestions ranging from a tax on airline travel to a global lottery, progress has been slow. In the health field, Advance Purchase Commitments and the "front-loading" of international assistance for immunization via the GAVI Alliance's International Finance Facility) represent steps forward [22] . In the first case, donors pre-commit to vaccine purchases if R&D is successful; in the second, future aid commitments are collateralized so that funds can be raised immediately in the international bond markets. These are welcome innovations, but they still tap the same source: international donor agency budgets.
Inverting the logic above, in this section we wish to clarify that a number of the major priorities in global health today do not represent GPGs. This does not diminish their importance, but it means that progress in these areas should not be equated with progress regarding the undersupply of GPGs. The two innovations we review are the GFATM and the re-framing of traditional health goals in the form of time-bound Millennium Development Goals.
Of the three scourges fought by the GFATM, only tuberculosis can be said with accuracy to represent a global public "bad." Malaria has significant cross-border aspects, but these make malaria control a regional public good (and one requiring collective action at the regional level), not a global one. Apart from R&D aspects, the public good problems associated with HIV/AIDS are regional at most, not global [23] . Despite inflammatory rhetoric heard at the beginning of the pandemic, HIV/AIDS has not proven to be a disease that spreads globally like SARS or pandemic influenza. Obvious cross-border aspects like transmission associated with long-haul truckers and migrant workers in Southern Africa call for a regional, not a global response.
Even if its transmission did qualify HIV/AIDS as a GPG, the GFATM response is not dealing with the disease using GPG logic. The main concern of GFATM (and PEPFAR, and the WHO's "Three by Five" program, and the G8 nations' commitment to universal access by 2010) is the provision of subsidized antiretroviral therapy (ART) to AIDS sufferers in low-income countries. ART is rival (therapy made available to one person or nation cannot be made available to another) and excludable (persons can be barred from receiving it). By contrast, AIDS prevention, in the form of media campaigns, condom distribution, voluntary counselling and testing, reduction of sexually transmitted infections, and encouragement of male circumcision, is non-rival (if A remains HIV-negative as a result of a prevention program, his sex partners B and C are protected equally) and non-excludable (no one can prevent C from enjoying the same protection as B).
Another argument runs that the destabilizing effect of HIV/AIDS in seriously affected countries gives rise to global impacts, but so do the destabilizing effects of everything else, like unemployment and hunger, that contribute to misery. And again, even it the disease's destabilizing potential did qualify it as a GPG, the international policy response does not prioritize GPG aspects. In a world of finite resources, the provision of ART in lowincome countries must come at the expense of prevention (if resources were not finite, of course, such tragic choices would not need to be made). The lopsided cost-ineffectiveness of ART means that each disability-adjusted life year (DALY) saved by treatment comes at the expense of many, many more DALYs lost in the future because the prevention measures needed to reduce transmission cannot be implemented [[24], p. 139]. If it were the looming scale of the AIDS catastrophe and its global spill over effects that were of greatest concern to donors, they would give priority to prevention, not treatment.
The need for collective action against AIDS was not the driving force behind the founding GFATM. It was born rather of frustration, especially on the part of AIDS activists, that good ideas from the field were not receiving deserved support because of donor red tape [25] . The response was to be a funding agency which would not assess proposals itself, relying rather on an independent panel, and would use local accounting firms to monitor implementation. Its comparative advantage would be focusing resources quickly on 'best shot' programs in countries with greatest need, as well as raising the profile of the disease. The hands-off approach to program formulation and implementation, it was argued, would mean that the GFATM would have no agenda of its own; aidrecipient countries would be able (through their representation on the review panel) to set their own priorities. The absence of a programmatic/operational agenda would allow the Fund to concentrate on mobilising and disbursing resources [26] . In sum, the rationale for the GFATM was not so much that a GPG was being undersupplied, but that assistance was not being provided efficiently or in a way consistent with needs.
Even if the GFATM is interpreted as a bold initiative -and not all do so, since most proposals still come through governments -we run into the caveats made above. The Fund may have generated additional resources or it may not have -the fact that it has struggled against resource constraints practically since its inception [26] gives some reason to suspect the latter. So do anecdotes making the rounds that the arrival of GFATM in some countries has led bilateral donors (apart from the US, through PEPFAR) to limit their own AIDS efforts. GFATM has not mobilized non-traditional sources of finance. As of mid-2007, out of the US$10.9 billion cumulatively pledged for all three diseases through 2008, only US$707 million comes from private firms, foundations, and individuals; this consists almost in its entirety of a US$650 million grant from the Bill and Melinda Gates Foundation. With its resources being pledged almost entirely from traditional donor countries' aid budgets, the GFATM is replicating the source-structure of existing aid flows.
A word on the U.S. PEPFAR is in order. PEPFAR promises US$15 billion (US$9 billion of which are claimed to represent new resources) for the global fight against AIDS but only allocates US$1 billion to GFATM [27] . Over half of the resources are targeted to providing AIDS treatment. Of funds allocated to prevention, a significant proportion is earmarked for faith-based programs encouraging teenaged sexual abstinence and discouraging multiple-partner sex. The ability of PEPFAR to finance activities benefiting key target populations -commercial sex workers and injecting drug users -is tightly constrained by law. It is hard to consider PEPFAR as collective provision of a GPG when the resources it makes available could have been channelled through a genuinely collective institution (GFATM) and when its prevention programs are designed to cater to a domestic political constituency.
The main focus of global health development at present is the health Millennium Development Goals (MDGs) (General Assembly, A/55/L.2, September 18, 2000) .
The targets associated with the health MDGs are to: (i) reduce child mortality by two-thirds between 1990 and 2015; (ii) reduce the maternal mortality ratio by three quarters between 1990 and 2015; (iii) halt and begin to reverse the spread of AIDS by 2015; and (iv) halt and begin to reverse the incidence of malaria and other major diseases by 2015. Developed countries and the development agencies will, in return for low-and middle-income countries' devoting effort to attaining the MDGs, take primary responsibility to establish a global partnership for development. In the area of health, the associated target is: (v) provide, in cooperation with pharmaceutical companies, access to affordable essential drugs in developing countries.
How do MDGs for health relate to the GPG perspective? Some of them, for example, those related to tuberculosis and access to drugs (or at least the R&D aspect of that problem), address GPG problems directly. Others, for example those related to maternal mortality, most child mortality, and HIV/AIDS, respond to humanitarian concerns, not GPG problems.
As in the case of the GFATM, when we look carefully at the origins of the MDG approach, we find that GPG logic is absent. The MDGs emerged from profound dissatisfaction with the effectiveness of aid to date and insistence on a "results focus" and improved monitoring and evaluation [28] . This process is the elaboration of a Poverty Reduction Strategy Paper or PRSP [29] ; the PRSP process, in turn is meant to encourage countries to adopt a long-term vision "by bringing out explicit awareness of poverty issues and promoting participation of stakeholders" [ [29] , page 11). PRSPs are meant to be country-driven ('ownership'), results-oriented, and participatory; reflect input of civil society and the private sector [30] . Countries are meant to prioritize the MDGs in accordance with their long-term vision of development needs. The PRSP process is also meant to force explicit linkages between fiscal resource allocation decisions and poverty reduction through the putting-in-place of Medium-term Expenditure Frameworks or MTEFs [31] .
"We will recognize country ownership and a partnership of equals," runs the donor governments' position, "if you will deliver results and ensure stakeholder participation." "We will deliver results and ensure stakeholder participation," runs beneficiary governments' position, "if you will acknowledge country ownership and a partnership of equals." This is a laudable win-win outcome -but it responds to a crisis in traditional development assistance, not to the need for collective action to supply GPGs. "Country ownership" and acceding to equal partnership are the last things that would be stressed in an approach built on GPG logic. Far from encouraging donor-country voters to support generous foreign aid programs because they are in their own interest, these discourage them from doing so [25] .
To conclude, the two initiatives examined show that massive mobilization of humanitarian assistance in pursuit of common goals should not be confused with collective action to ensure the adequate supply of GPGs. That observation does not lessen the importance of such actions, but it guards us against the fallacy of concluding that, just because there is a multiplication of high-profile innovations, fundamental GPG problems are being effectively addressed.
The GPG concept, discovered by the aid community in the late 1990s, can be a powerful tool in promoting global health because it marshals arguments of self-interest. It can be used to identify areas in which global collective action is needed, specify where the costs and benefits will rest and communicate to the public why spending to promote health thousands of kilometers around the world is not a waste of their tax dollars. Yet, we find that the GPG perspective has been a mixed blessing.
We looked at two acknowledged GPGs related to health, namely R&D and communicable disease control. While recognition of the need for global collective action has supported a large number of new initiatives, it remains to be determined what the result is in terms of additional funds. Those funds that have been generated have come from traditional philanthropic and public sources. The proliferation of infectious disease initiatives has promoted a vertical, "stovepipe" approach, to the detriment of broad health sector strengthening.
We then looked at two of the major global innovations in health, the GFATM (and, closely related, PEPFAR) and the re-packaging of traditional health concerns in the form of MDGs. We concluded that both can be more easily understood as addressing weaknesses in traditional humanitarian aid -red tape, lack of country ownerships, insufficient stakeholder involvement, need for results-based management, etc. -than as addressing problems of GPG provision.
All of the new initiatives we have discussed here, and many that we have not mentioned, are funding or doing valuable work. How might the GPG perspective strengthen them and lead to other efforts, as well?
First and foremost, within existing programs and when proposing new ones, the aid community should adhere to the strict economic definition and avoid the temptation to use the GPG 'tag' as a general-purpose fund-raiser. If we focus GPG logic on those goods and services where global collective action really is needed, that action is more likely to be achieved. Where humanitarian grounds, not rational self interest, are the main motivation for action -as in providing subsidized treatment for AIDS sufferers in poor countries -we should say so without equivocation. Where general health system strengthening is required to guarantee access to GPGs such as immunization or tuberculosis control, this should be stated explicitly, even if it means that budgets for GPG provision strictly defined may be reduced as a result.
Second, the aid community should stress to policy makers that, where the GPG label is appropriate, as in the case of communicable disease control, what is needed is not only new packaging/labeling of existing resources, but resources additional to those already being made available, which means mobilizing innovative sources of financing. The current elevated level of concern over emergent diseases, including pandemic influenza, is an ideal context in which to press for a more pro-active response. So is the rapid development of financial engineering tools related to aid, such as advance purchase commitments, collateralization future aid commitments in the bond market so as to "frontload" aid, etc.
Third, the relative ease of financing disease-specific actions, as opposed to broad sector strengthening, should not be allowed to distort health sector policy or dictate the structure of support. Where sector support serves an "access" function, the argument that it is a prerequisite for provision of GPGs (essentially, communicable disease control) can be used to strengthen its claim on resources.
The aim of this paper was to provide an introduction to the key concepts, and to consider some innovative developments in global health from the GPG perspective. Hopefully this has illustrated the potential and limitations of the concept, and provided a foundation for further discussion of these. Outcome of paediatric intensive care survivors The development of paediatric intensive care has contributed to the improved survival of critically ill children. Physical and psychological sequelae and consequences for quality of life (QoL) in survivors might be significant, as has been determined in adult intensive care unit (ICU) survivors. Awareness of sequelae due to the original illness and its treatment may result in changes in treatment and support during and after the acute phase. To determine the current knowledge on physical and psychological sequelae and the quality of life in survivors of paediatric intensive care, we undertook a computerised comprehensive search of online databases for studies reporting sequelae in survivors of paediatric intensive care. Studies reporting sequelae in paediatric survivors of cardiothoracic surgery and trauma were excluded, as were studies reporting only mortality. All other studies reporting aspects of physical and psychological sequelae were analysed. Twenty-seven studies consisting of 3,444 survivors met the selection criteria. Distinct physical and psychological sequelae in patients have been determined and seemed to interfere with quality of life. Psychological sequelae in parents seem to be common. Small numbers, methodological limitations and quantitative and qualitative heterogeneity hamper the interpretation of data. We conclude that paediatric intensive care survivors and their parents have physical and psychological sequelae affecting quality of life. Further well-designed prospective studies evaluating sequelae of the original illness and its treatment are warranted. the quality of life (QoL) in survivors and in their families are important outcome measures.
Historically, outcome research in paediatrics is either based on an age-specific approach, such as follow-up studies of premature infants [41, 72, 73] , or on a more disease-oriented approach, such as follow-up studies in survivors of cardiothoracic surgery or trauma [15, 55, 64, 70] . These studies have shown substantial physical, psychological and neurocognitive sequelae, interfering with daily life and normal development. In addition, effects on parents and siblings have been shown [26] . Evaluative research of adult intensive care survivors showed the effect of intensive care treatment per se. Irrespective of the underlying illnesses, sequelae on all domains with effects on QoL were found [2, 19, 58, 75] . In multi-disciplinary paediatric intensive care unit (PICU) populations, reports on outcome are scarce [24, 25] .
Based on these observations, we believe that follow-up research of paediatric intensive care survivors and their families is needed to evaluate: (1) physical sequelae and their impact during growth and development; (2) psychological sequelae in patients and their families and their impact on the QoL of patients and family members; and (3) the need for treatment and support after discharge.
The aim of this article is to provide an overview of the available literature concerning the different domains of QoL (i.e. physical, psychological and social functioning) in children surviving paediatric intensive care, including the effect on parents, and to suggest directions for future follow-up research.
To identify studies eligible for this review, we searched Medline , EMBASE (1974 EMBASE ( -2006 , CINAHL (1982 CINAHL ( -2006 , pre-CINAHL and the Cochrane Library (2006) in March 2006. In the search strategy, all terms mapped to the appropriate MeSH/EMTREE subject headings and "exploded" were used; among them were: paediatric intensive care unit (PICU), septic shock, respiratory insufficiency, meningococcal disease, central venous catheterisation, intubation, physical and psychological sequelae, post-traumatic stress disorder (PTSD), QoL, health status and long-term outcome.
Functional health is defined as an individual's ability to perform normal daily activities, to fulfil usual roles and to maintain health and well-being.
QoL is defined as an individual's perception of their position in life, in the context of the culture and value systems and in relation to their goals, expectations, standards and concerns [1] .
Health-related QoL (HRQoL) is defined as QoL in which a dimension of personal judgement over one's health and disease is added [21] .
Studies were selected for review if they met two inclusion criteria: (1) study of a representative population of PICU survivors (defined as a population consisting of medical and/or surgical PICU patients <18 years old) and (2) evaluation of physical sequelae, measurement of QoL or functional health >30 days after PICU discharge. Because of the limited number of studies, the measurement tools did not need to be standardised. Studies with a retrospective and prospective design were included.
Excluded were: (1) studies in homogeneous PICU populations (e.g. survivors of cardiothoracic surgery and trauma) reporting diagnosis-related outcome in particular but not intensive care treatment as such, and (2) studies evaluating mortality only.
Eligible studies and quality of the studies Twenty-seven studies were found in which one or more aspects of long-term sequelae in PICU survivors and/or their families were described. The patient characteristics, populations, measurement tools and outcomes are described in Tables 1 and 2 . The quality criteria are described in Table 3 . None of the studies met all of the quality criteria. In studies describing the same outcome aspect, differences in study population, follow-up time and measurement tools make the comparison and synthesis of results difficult.
Physical and neuro-cognitive sequelae (Table 1) In 12 studies that included in total 340 patients, aspects of physical and neuro-cognitive sequelae were evaluated.
Neurological evaluation was conducted in five studies including 275 survivors. The majority of the children were neurologically normal. In the remaining children, disabilities such as hearing loss, coordination, cognition and developmental problems turned out to be severe [23, 35, 43, 53, 59] .
Pulmonary evaluation was conducted in six studies including 65 patients [6, 14, 22, 30, 48, 74] . Restrictive and obstructive disease and hypoxaemia during exercise was found. Cardiac evaluation was conducted in two studies including 23 survivors [22, 74] . No abnormalities were found, except for left ventricular hypertrophy in one child.
Renal evaluation was conducted in one study including 12 survivors [62] . In two children, glomerular filtration was impaired, one had hypertension and one had proteinuria.
Psychological sequelae (Table 2) Various questionnaires were used. Cut-off points for the diagnosis of PTSD differed between studies but all of them showed high scores for PTSD in children and parents.
Psychological evaluation of children was conducted in five studies including 202 children [40, [50] [51] [52] 61] . Symptoms of PTSD were found in 11 of 74 evaluated children. In one study, a relation was found between invasive procedures and high scores [52] .
Psychological evaluation of parents was conducted in six studies including parents of 547 children [4, 8, 20, 40, 50, 61] . Symptoms of PTSD were found in 72 of 295 evaluated parents. In some studies, a relation was found between high scores and illness severity as perceived by parents [4, 50, 61] . In one study, these high scores decreased over time [8] .
Functional health and QoL (Tables 1 and 2) Evaluation of functional health was conducted in three studies including 821 children [9, 12, 44] . The majority of the children seemed to have normal functional health; the remainder was found to be seriously impaired.
Evaluation of QoL was conducted in four studies including 1,664 children [27, 38, 47, 65] . QoL was evaluated using three different questionnaires. In the majority of children, the QoL was normal or equal to the QoL before PICU admission. In all studies, some of the children had poor QoL.
Only 27 studies consisting of 3,444 PICU survivors met our inclusion criteria. The small numbers, heterogeneity of the 4  yes  yes  yes  yes  no  6  no  no  yes  yes  no  8  no  no  yes  yes  yes  9  no  yes  yes  no  no  12  yes  yes  yes  yes  no  14  yes  yes  yes  yes  no  20  yes  yes  yes  yes  no  22  no  no  no  yes  no  23  no  no  yes  yes  yes  27 yes studied populations and the used measurement tools, the frequent use of non-validated measurement tools and the various aspects of outcomes studied make aggregation of the data and, therefore, strong conclusive statements difficult.
The reviewed studies report distinct physical sequelae, including neurological abnormalities in PICU survivors. Standardised neurological examination of PICU survivors was validated in 1994 but very few studies have been carried out since [24, 25] . As neurological problems have a great impact on daily life, standardised evaluation and adequate support and rehabilitation seem to be relevant, similar to in NICU survivors [11, 46, 56] . Follow-up studies evaluating lung function in children are hampered by the small incidence of severe respiratory insufficiency in children [49] . In adult respiratory distress syndrome (ARDS), the recovery of lung function is shown during the first year and physical limitations seem to be partly dependent on lung function [34, 58] . In infants and children, post-natal lung growth may contribute to the improvement of lung function after critical illness. In addition to lung function, the long-term effect of small airway disease should be evaluated, for instance, in children with respiratory syncitial virus infection.
Data on the structured evaluation of cardiac and renal function in paediatric and adult ICU survivors is not available. In young children, septic shock and the need for vasoactive support of the circulation may interact with the developing myocardium and may have persistent effects on cardiac growth and function [10, 67, 77] .
Complications of intensive care procedures per se, (e.g. vascular complications due to intra-vascular catheters and side-effects of ototoxic drugs and sedatives) are not evaluated [5, 18, 32, 33, 45, 54, 57, 63] . One can assume the exact incidence of physical sequelae to be higher than has been reported so far.
In the reviewed studies, psychological sequelae have been established in 10-14% of survivors and their parents. The comparison of findings is hampered due to different measurement tools and cut-off points for the diagnosis of PTSD and various follow-up intervals. Risk factors accounting for hampered psychological outcome could be diverse (severity of illness, being removed from one's child, having been witness to the accident, mental health, family functioning, social support, coping strategies and lack of information from the medical team) [17, 26, 29, 31] . Psychological support to improve coping strategies and prevent over-protection might improve psychological out-come in children and parents [3, 28] . Further research is essential to establish the appropriate time and extent of the psychological support needed.
Cognitive sequelae have rarely been studied in the reviewed studies. Adequate neuro-cognitive evaluation is both expensive and time-consuming. Studies in neonatal ICU survivors show substantial cognitive dysfunction with great impact on daily life [7] . Consequently, early intervention, education and rehabilitation are expected to improve daily life [11, 46] .
A majority of PICU survivors seem to have unchanged functional health and good QoL. In the reviewed studies, functional health is evaluated by telephone interviews [27, 38, 47, 65] . In most of these studies, the physician rather than the child or its parents evaluates functional health. Ideal (HR)QoL questionnaires should measure all aspects of QoL and preferably be filled in by the children themselves. Proxy investigation of functional health and (HR) QoL (in children <6-8 years of age) is second best [36, 37, 39, 66] . Besides, the pre-morbid state is probably an important factor which is difficult to assess [16] .
The reviewed studies have a number of methodological limitations. Heterogeneity is the most important one. Consensus on all aspects of follow-up research is essential for well-founded conclusions. For example, structured and standardised evaluation of: (1) organ system function with a validated tool such as the Paediatric Logistic Organ Dysfunction (PELOD) score [13, 42, 60, 71] ; (2) neurocognitive function; (3) complications of PICU treatment; and (4) (HR)QoL are warranted. Multi-centre studies as proposed by the Collaborative Pediatric Critical Care Research Network (CPCCRN) with a uniform approach will provide answers either in general PICU cohorts or in disease-oriented study groups [76] .
In conclusion, this review indicates that PICU survivors and their parents may have substantial physical and psychological sequelae interacting with QoL. Because of longer life expectancy, longer follow-up time is warranted, emphasising the consequences for health care in children. We believe that paediatric intensivists and psychologists should be involved as core members of follow-up teams. An evaluation of Comparative Genome Sequencing (CGS) by comparing two previously-sequenced bacterial genomes BACKGROUND: With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. It is critical though to know the accuracy of the technique used, and to establish confidence that all of the mutations were detected. RESULTS: In order to evaluate the accuracy of genome resequencing using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc., we resequenced the E. coli strain W3110 Kohara using MG1655 as a reference, both of which have been completely sequenced using traditional sequencing methods. CGS detected 7 of 8 small sequence differences, one large deletion, and 9 of 12 IS element insertions present in W3110, but did not detect a large chromosomal inversion. In addition, we confirmed that CGS also detected 2 SNPs, one deletion and 7 IS element insertions that are not present in the genome sequence, which we attribute to changes that occurred after the creation of the W3110 lambda clone library. The false positive rate for SNPs was one per 244 Kb of genome sequence. CONCLUSION: CGS is an effective way to detect multiple mutations present in one bacterium relative to another, and while highly cost-effective, is prone to certain errors. Mutations occurring in repeated sequences or in sequences with a high degree of secondary structure may go undetected. It is also critical to follow up on regions of interest in which SNPs were not called because they often indicate deletions or IS element insertions. Genome resequencing is the determination of a genome sequence using an already established genome sequence as a reference. In hybridization-based resequencing, the reference is necessary for the generation of microarray probes and for signal normalization. In other types of resequencing the reference is used as a scaffold for the assembly of short sequence reads. The genome to be rese-quenced must be substantially similar to the reference; otherwise the reference looses its effectiveness.
Resequencing is useful for relating phenotype to genotype and for analyzing natural variation. It has been used to study the acquisition of antibiotic resistance [1, 2] , the analysis of variation in pathogenic bacteria and viruses [3] [4] [5] [6] [7] , and to study the experimental evolution of bacteria and yeast [8] [9] [10] [11] . Methods of resequencing utilize micro-arrays [2, 7, 10] , polonies [9] or sequencing-by-synthesis technology [12] . The Comparative Genome Sequencing service provided by Nimblegen Systems Inc. is a hybridization-based method and consists of two steps [2] . In the first step, an experimental and reference genomic DNA sample are labeled and hybridized to microarrays containing ~30-mer 'tiled' oligonucleotides spaced every ~7 bp of the genome on both strands. Probes cover every nucleotide of the genome and are designed to have isothermal hybridization characteristics. Probes showing differences in signal intensity are flagged as Regions Of Interest (ROIs) for further investigation. A second microarray is then designed to interrogate the ROIs at single base-pair resolution, with all four possible nucleotides synthesized for each interrogated position on both strands (8 probes per position). If the sequence difference is a single-base polymorphism, the pattern of probe intensities at that position will be discernibly different than expected. Other types of sequence differences such as insertions/deletions (indels) or differences of more than one bp will not be determined conclusively.
In order to attribute phenotype to a genotype it is important to know that all relevant sequence differences were detected. False negatives (i.e. failures to detect mutations that are actually present) can result in erroneous interpretation, especially considering that a single mutation can have a dramatic impact on phenotype. False positives on the other hand (i.e. the reporting of sequence differences that are not actually present) are not a big problem in most cases since even 100 false mutations can be checked and refuted with PCR amplification and Sanger sequencing for less money than a typical resequencing experiment.
In order to determine the false negative and false positive rates for CGS, we utilized two related strains of E. coli for which high-quality genome sequences exist. Strain W3110 was resequenced with strain MG1655 as the reference, but this setup could just as easily have been reversed. Our results demonstrate the accuracy of CGS resequencing but also raise caveats about CGS and the instability of bacterial strains.
The closely related and fully sequenced E. coli strains W3110 and MG1655 represent an excellent test for the accuracy of resequencing technology. Hayashi et al. [13] compared the genome sequences of these two strains and resolved all discrepancies, generating a pair of highly accurate genome sequences. The genome sequence of strain W3110 differs from MG1655 by seven single nucleotide polymorphisms (SNPs), one 2 bp insertion, 12 IS element insertions, one deletion of 6.6 kb, and an inversion of 783.1 kb [13] . A summary of all sequence differ-ences between strain MG1655 and W3110 is presented in Table 1 .
To test the accuracy of CGS, E. coli strains W3110 Kohara and MG1655 were obtained from the E. Coli Genetic Stock Collection (CGSC). DNA was extracted and submitted to Nimblegen Systems Inc. for CGS, using MG1655 as the reference. Nimblegen reported 25 SNPs in strain W3110, 4 of which corresponded to known sequence differences. Regions surrounding the other SNPs were PCR amplified and subjected to Sanger sequencing. In this way, 19 of the remaining putative SNPs were refuted, though 2 were confirmed. These two mutations are not present in the W3110 genome sequence, and must have been introduced into the strain after Kohara et al. [14] made the lambda phage library that was sequenced. In sum, there were 19 false positive SNPs out of 25 reported, or one false positive per 244 kb ( Table 2 ). Figure 1 shows a sample of hybridization signals surrounding three different sequence differences. It can be seen that the ratio of signal intensities near the mutations were distinctly elevated. When these regions were resequenced at single bp resolution, the sequence differences in ycdT and acnA were correctly determined. The intensity of hybridization signals in other areas where there were no sequence differences varied considerably.
In addition to SNPs, Nimblegen also provided the location of probes that showed hybridization differences, yet could not be resolved as SNPs ("non-called Regions Of Interest" or ROIs). These probes may indicate the presence of mutations other than SNPs, such as insertions or deletions. Nimblegen reported 1094 ROI probes located in 36 clusters, a cluster being a group of probes that are within 500 bp of each other. One of these clusters corresponded to the known 6.6 kb deletion of intZ thru yffS (b2442-2450) ( Table 1) . Three other clusters corresponded to known sequence differences that were not reported as SNPs -the substitutions in rpoS and crp and the 2 bp insertion in dcuA. Nine clusters corresponded to known IS-element insertions in W3110. Most of the IS-element insertions were evident as clusters consisting of multiple ROI probes, though three were only evident as singleprobe clusters. There were three IS element insertions that were not detected as either SNPs or ROIs. The large inversion in W3110 of all genes between ribosomal RNA genes rrlD and rrlE was not detected as either SNPs or ROIs.
One novel cluster of ROI probes was very large, indicating the possible deletion of three genes, ynaJ, uspE, and fnr (b1332-1334). Such a deletion is not present in the genome sequence of W3110. PCR amplification using primers located in adjacent genes confirmed the deletion; a 3.8 kb PCR product was obtained from MG1655 while a 1.4 kb product was obtained from W3110. Deletions of fnr have been noted previously in strains of MG1655 obtained from the CGSC [16] . To determine if the deletion occurred before or after the strain was deposited at CGSC, a culture was obtained from the original lyophil made when stock # 7167 was deposited in 1990 by Akira Ishihama. PCR amplification with the same primers showed two products, one at 1.4 kb and another at 5 kb.
Sequencing the ends of the 5 kb band revealed the known IS5 element b1331 to the right of ynaI on one side and a new IS5 element inserted to the left of ogt (b1335) on the other. This result seems to indicate that the strain of W3110 deposited with CGSC has two copies of IS5 on either side of ynaJ, uspE and fnr that undergo recombination with each other at high frequency leading to the deletion of the intervening genes. The remaining 22 clusters of ROI probes were PCR amplified and Sanger sequenced to see if they indicated additional mutations not present in the W3110 genome sequence. Indeed, seven IS-element insertions were discovered, while the other 15 clusters showed no mutations. We note that all but one of the false positive ROIs were single-probe clusters and that all of them contained inverted repeats of between 6 and 13 nt. These repeats may lead to hairpin structures and poor hybridization properties of those probes [15] . In total, 10 mutations (2 SNPs, 7 IS-insertions and 1 deletion) accumulated in strain W3110 in the time period between when Kohara et al. generated the lambda library and when we obtained it from CGSC. The history and handling of the strain before Ishihama deposited it with CGSC in 1990 is not known by the authors of this study, though we speculate that it may have been stored as a stab at room temperature.
In this study, we sought to evaluate the accuracy of Nimblegen's microarray-based CGS resequencing technology so that the results obtained from experimental samples can be interpreted judiciously. Our results indicate that the false positive rate for SNPs was one per 244,193 bp of sequence. The false positive rate for clusters of ROI probes was one per 309,312 bp of sequence. These rates have been shown to depend on the thresholds chosen for mutation-calling [17] . In theory, low thresholds should be used for mutation mapping, increasing the number of false positives but reducing the number of false negatives. The methods used by Nimblegen to identify ROI's and to determine sequence differences have been described elsewhere [2] [18] . ROIs are identified by discarding erroneous probes then picking probes for which the ratio on both strands exceeds 3.5 standard deviations of the 80 th percentile within a 1800 bp local window [2] . For sequence determination, Molla et al. [18] . describe a machine-learning algorithm that uses relative positions within "feature space" rather than a training set and describe the effects of changing thresholds on sensitivity.
In future work, false positive rates might be reduced by additional development of these algorithms and discard-ing single-probe clusters containing inverted repeats. Error analysis for development of improved algorithms or array designs should take into account whether errors occurred in the first "mapping" step or the second single bp resolution step of CGS.
A high false discovery rate is generally not a problem with resequencing projects. The cost of PCR amplifying and Sanger sequencing up to 100 candidate regions is small compared to the cost of resequencing itself. The false negative rate on the other hand is very important. In order to associate genotypic change to phenotypic change it is critical to know with some certainty that all of the important mutations were detected. Nimblegen estimates that the false negative rate for SNPs is less than 5% (Tom Albert, personal communication). We found that only half of the small sequence differences actually present were reported as SNPs. If we consider detection in ROI probes as well, then 7 out of 8 small sequence differences were detected, yielding a false negative rate of 12.5%. Given the small sample size, this rate may be consistent with Nimblegen's claims. CGS is meant for the detection of SNPs, but ROI data also reveals deletions and IS-element insertions. The false negative rate for detection of IS element insertions was double the rate for small sequence differences, possibly because CGS has not been optimized for detection of insertions. Conceptually, insertions and inversions could result in a more pronounced hybridization difference at the insertion site. If the insertion site/inversion breakpoint occurs in the middle of the region covered by a probe, then genomic DNA will only match one half of the probe or the other. Detection of insertions might be improved by manipulation of the hybridization/wash conditions or changes to the algorithm to specifically identify probe series indicative of insertions.
The single SNP not detected is a substitution in the gene rrlE, which is duplicated 6 other times in the E. coli genome. In previous work, we noted the failure of CGS to detect a 9 bp duplication, a 1 bp deletion and a 28 bp deletion near a transcriptional terminator [8] . It appears that CGS has trouble in regions of high local secondary a. This number only includes those differences reported in reference 13, and not the new ones discovered in this study. b. The number of non-called ROI's that did not contain mutations was taken as the number of false positives for both IS-element insertions and indels.
Sample mutation mapping data Figure 1 Sample mutation mapping data. The ratio of signal of W3110 vs. MG1655 is shown for probes spaced every ~7 bp surrounding an IS5 insertion in dcuC (top), a SNP in ycdT (middle), and a SNP in acnA (bottom). acnA region structure and with sequences repeated multiple times in the genome [15] [17] . In addition, detection of SNPs may be less accurate in areas with low overall intensity. Fortunately, such regions are relatively uncommon, and CGS appears to detect most mutations with a reasonable degree of confidence.
Site-directed mutagenesis can overcome uncertainty caused by the possibility of false negatives. In our previous work [8] , we introduced all of the mutations identified into the wild type strain and were able to reconstruct the observed change in growth phenotype in 4 of 5 cases, indicating that all of the important mutations were found in most cases. An additional precaution against false negatives is resequencing multiple replicates from the same experimental setup. Our previous work identified mutations in the gene glpK in 4 of the 5 replicates. Sequencing glpK from the one remaining replicate with other technology showed a 9 bp duplication undetected by CGS. This demonstrates the value of replicates and why genes containing mutations in some replicates should be screened for false negatives in the other replicates.
Resequencing technology allows a sequence to be determined at less than one tenth the cost of traditional Sanger sequencing, but it is less accurate with some types of sequences and requires a closely related reference sequence. Other methods of resequencing [9, 12] are likely to differ from CGS in cost and accuracy. The control experiment presented here comparing E. coli strains W3110 and MG1655 can now be performed with these other technologies to allow cross-comparison.
We determined the false positive rate for CGS to be one per 244 Kb for SNPs and one per 309 Kb for non-called ROIs. We observed a false negative rate of 12.5% for small sequence differences and 25% for IS-element insertions.
The one large deletion present in the genome sequence was easily detected, though the chromosomal inversion was not. We conclude that the accuracy of CGS is sufficient for effective resequencing studies, but with some precautionary notes. All clusters of non-called ROI probes should be PCR amplified and Sanger sequenced to detect IS-element insertions and small indels. Also, multiple replicates and/or site-directed mutagenesis should be used in cases where rare false negatives may affect the scientific interpretation.
We made the unexpected discovery of 10 mutations accumulated in strain W3110 after Kohara et al. generated the lambda clone library that was sequenced. This highlights the ability of CGS to reveal unexpected results and the rapid degeneracy of strains under some kinds of storage conditions. Strains with the same name (e.g. W3110, MG1655) often differ depending on their source and storage conditions, so sequenced strains should only be obtained from the laboratories that sequenced them or their designated depositories.
E. coli strain W3110 Kohara and MG1655 (seq) were obtained from the E. Coli Genetic Stock Collection (CGSC # 7167 and # 7740, respectively). Cells were grown in liquid LB medium and then DNA was extracted using DNAeasy Tissue Kit (Qiagen). The CGS results provided by Nimblegen are given in Additional File 1.
Regions containing putative mutations were PCR amplified using oligonucleotide primers listed in Additional File 2. They were then purified using Qiaquick (Qiagen) and sequenced using Sanger sequencing. Sequence differences were confirmed by manual examination of the trace data. Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions: A summary report from a Global HIV Vaccine Enterprise Working Group The authors discuss humoral immune responses to HIV and approaches to designing vaccines that induce viral neutralizing and other potentially protective antibodies. T he Global HIV Vaccine Enterprise convened a two-day workshop in May of 2007 to discuss humoral immune responses to HIV and approaches to design vaccines that induce viral neutralizing and other potentially protective antibody responses. The goals of this workshop were to identify key scientific issues, gaps, and opportunities that have emerged since the Enterprise Strategic Plan was first published in 2005 [1] , and to make recommendations that Enterprise stakeholders can use to plan new activities.
Most effective viral vaccines work, at least in part, by generating antibodies that inactivate or neutralize the invading virus, and the existing data strongly suggest that an optimally effective HIV-1 vaccine should elicit potent antiviral neutralizing antibodies. However, unlike acute viral pathogens, HIV-1 chronically replicates in the host and evades the antibody response. This immune evasion, along with the large genetic variation among HIV-1 strains worldwide, has posed major obstacles to vaccine development. Current HIV vaccine candidates do not elicit neutralizing antibodies against most circulating virus strains, and thus the induction of a protective antibody response remains a major priority for HIV-1 vaccine development. For an antibody-based HIV-1 vaccine, progress in vaccine design is generally gauged by in vitro assays that measure the ability of vaccine-induced antibodies to neutralize a broad spectrum of viral isolates representing the major genetic subtypes (clades) of HIV-1 [2] . Although it is not known what magnitude and breadth of neutralization will predict protection in vaccine recipients, it is clear that current vaccine immunogens elicit antibodies that neutralize only a minority of circulating isolates. Thus, much progress needs to be made in this area. Also, though virus neutralization is considered a critical benchmark for a vaccine, this may not be the only benchmark for predicting success with antibody-based HIV-1 vaccine immunogens.
The main targets for neutralizing antibodies to HIV-1 are the surface gp120 and trans-membrane gp41 envelope glycoproteins (Env) that mediate receptor and coreceptor binding and the subsequent membrane fusion events that allow the virus to gain entry into cells [3] . Antibodies neutralize the virus by binding these viral spikes and blocking virus entry into susceptible cells, such as CD4 + T cells [4, 5] . In order to chronically replicate in the host, the virus exploits several mechanisms to shield itself against antibody recognition, including a dense outer coating of sugar molecules (N-linked glycans) and the strategic positioning of cysteine-cysteine loop structures on the gp120 molecule [6] [7] [8] . These shielding mechanisms, although highly effective, have vulnerabilities imposed by fitness constraints. Information on the precise location and molecular structure of these vulnerable regions could be valuable for the rational design of improved vaccine immunogens.
Participants in the workshop identified four areas that, if given proper attention, could provide key information that would bring the field closer to an effective antibody-based HIV-1 vaccine: (1) structure-assisted immunogen design, (2) role of Fc receptors and complement, (3) assay standardization and validation, and (4) immunoregulation of B cell responses.
Clinical studies have demonstrated that immunization with the gp120 surface unit of the HIV-1 envelope protein does not lead to the induction of potent or broadly reactive neutralizing antibodies. In order to develop better immunogens, it is likely that we will need a more detailed understanding of the atomic level structure of epitopes on the native envelope glycoprotein. Data on the X-ray crystal structure of liganded and unliganded partial gp120 molecules have provided valuable information about the atomic level interaction of gp120 and neutralizing antibodies [9] [10] [11] [12] . The recent atomic level resolution of monoclonal antibody (MAb) b12 bound to the CD4 receptor binding site of the gp120 molecule provides new insights into how successful neutralizing antibodies access functionally conserved regions of the Env glycoprotein [13] . Crystal structures of complete monomeric gp120 and gp120-gp41 trimer complexes in their native unliganded form need to be elucidated, as these are the natural targets for neutralizing antibodies. This information is needed for multiple genetic subtypes of the virus and for transmitted strains of the virus. Coupled with this effort should be a program to make necessary improvements in electron tomography technology to gain a higher resolution of native Env spikes as they exist on virus particles [14] [15] [16] . An improved understanding of the structural basis of antibody binding to the HIV-1 Env glycoprotein will likely form the foundation for a rational program of novel vaccine design. Ongoing efforts to stabilize gp120 into more immunogenic forms or to scaffold conserved neutralization epitopes into foreign proteins may lead to more promising antibody responses.
Induction of an effective neutralizing antibody response will require that a vaccine deliver to the naïve B cell repertoire epitopes that are both immunogenic (i.e., possess favorable properties for B cell inductive pathways) and antigenic (i.e., available for high affinity antibody binding on functional Env spikes). Viral epitopes that are conserved among most viral strains are more likely to generate cross-reactive antibodies. In this regard, researchers have focused on a small number of human MAbs, from clade B HIV-1-infected individuals, that possess broadly cross-reactive neutralizing activity [17, 18] . The cognate viral epitopes for these MAbs have been well characterized and are being evaluated as vaccine immunogens. However, for reasons that are not completely understood, these conserved viral epitopes have either been poorly immunogenic or have elicited antibodies of restricted reactivity. Improvements are being sought by introducing specific structural alterations [19, 20] and by targeting autoreactive B cell pathways [21] . These and other efforts to improve the immunogenicity of conserved neutralization epitopes should remain a high priority. Workshop participants recognized the need to expand efforts to identify and characterize new MAbs, with special attention to MAbs from non-clade B HIV-1 infections. New technologies are now available that might afford an advantage for identifying novel antibody specificities that were previously undetected [22, 23] . In addition to this focus on MAbs, sera from selected HIV-1infected individuals that can broadly neutralize HIV-1 isolates should be studied in detail. New assays allow more precise mapping of the polyclonal antibody response in these sera to better understand the epitopes targeted [5, [24] [25] [26] . Such studies may reveal novel antibody specificities and their associated viral epitopes that could be useful for immunogen design.
While there has been considerable interest in conserved epitopes, less attention has been paid to more variable epitopes that might be useful if administered in the form of a polyvalent vaccine. Of particular interest are the epitopes that drive the autologous neutralizing antibody response in infected individuals. These epitopes may be quite variable, but recent evidence suggests that there are constraints on the extent of variation the virus can tolerate in these regions [27, 28] . Detailed molecular and immunologic studies of the autologous neutralization response would enhance our understanding of viral determinants that are vulnerable to antibody attack. Similarly, it is possible that combinations of antibodies will have desirable additive or synergistic effects on virus neutralization [29-32]. An example is seen in how soluble CD4 binding rearranges the structure of gp120 to expose the highly conserved coreceptor binding domain, which allows antibody binding and virus neutralization to occur [33, 34] . Such effects of antibodies might be discovered by applying high throughput screening methods to the plethora of existing MAbs as well as new MAbs that become available in the future.
Recent findings have generated renewed interest in so-called "non-neutralizing" antibodies that are unable to directly inhibit free virus entry into target cells, but nonetheless exhibit antiviral activity mediated by the Fc region of the antibody molecule. These antibody effector mechanisms include complement binding and viral lysis, phagocytosis of antibody-coated virions, and antibody-dependent cellular cytotoxicity [35] [36] [37] [38] . Recent studies have suggested examples of Fc-dependent antiviral effects of HIV-1-positive serum in cases where there was little or no detectable activity in conventional neutralization assays [39, 40] .
In addition, passive transfer studies in a relevant monkey model suggest that Fc receptor (FcR) binding capacity of a protective antibody makes a substantial contribution to the antibody-mediated protection [41] . Antibody effector functions that mediate complement activation and FcR engagement on macrophages, dendritic cells, natural killer cells, and other cell types need to be evaluated to determine their relevance to HIV-1 vaccines. Assays that measure these antiviral antibodies should be standardized and used to assess biologic relevance in passive protection experiments in animal models using antibodies that exhibit the different effector functions in vitro.
In order to adequately monitor neutralization breadth and potency and to compare and prioritize immunogens, assays are needed that are sensitive, quantitative, high throughput, and have correlative value. Substantial improvements have been made in the past several years in assay technology and in available reference reagents. Thus, cumbersome and expensive assays using peripheral blood mononuclear cells (PBMC) and uncloned viruses are being replaced with assays that utilize molecularly cloned Env-pseudotyped viruses and genetically engineered target cells lines [2, [42] [43] [44] [45] . This new technology affords greater sensitivity, reproducibility, high throughput, and cost-effectiveness compared to PBMC assays, and as a result, it has been responsible for an explosion of new data. Steps are being taken by the Collaboration for AIDS Vaccine Discovery to transfer this new technology to multiple laboratories around the world and to implement a validated proficiency testing program to assure inter-laboratory equivalency in assay performance.
Recently, several cases were identified where neutralization was considerably more potent or only detected in the older PBMC assay compared to the newer assay technology [43, 46, 47] . This raises important questions about current plans to employ a single assay for routine use, and it points to the need for a better understanding of the mechanisms of neutralization. Thus, it may be necessary to use more than one assay to assure that all neutralizing antibodies are detected. There is a need to standardize and compare neutralizing antibody assays and to decide which assay or combination of assays should be used for standardized assessments of vaccine-elicited neutralizing antibody responses. A major priority is to strengthen the standardization of the PBMC assay, given that it is the only assay that has been at least partially validated in passive antibody experiments in animal challenge models.
Important decisions need to be made about the types of antibodies and assays that have greatest relevance to HIV-1 vaccines. Validation experiments in animals models are needed to determine the potential correlative value of new assay technologies that rely on the use of genetically engineered cells lines and Envpseudotyped viruses. Ideally, this would be done by employing several different assays to study the antibody response in a clinical efficacy trial in which the vaccine was at least partially protective. Because no such vaccine is currently available for HIV-1, studies in animal models are the next best choice. In this regard, two animal models are widely used for HIV vaccine development: simian immunodeficiency virus (SIV) and chimeric simian-human immunodeficiency virus (SHIV) infection in monkeys [48] . Quantitative passive transfer experiments in either model with antibodies that exhibit different effector functions could be used to address the biological relevance of in vitro assays. Unfortunately, very few SHIVs are currently available and, among these, most are derived from a single genetic subtype (clade B) and exhibit properties that may not be well suited to assay validation [49] . The creation of new and better SHIVs from non-clade B viruses would facilitate assay standardization as well as vaccine challenge models.
This workshop identified several critical gaps in the current understanding of B cell regulatory pathways that impede a more rational development of an effective antibody-based HIV-1 vaccine. For example, broadly neutralizing antibodies in patient serum bind epitopes that are present on monomeric gp120 [25] , yet this is a poor immunogen for neutralizing antibody induction in vaccine recipients. Moreover, as mentioned above, viral epitopes for the known broadly neutralizing MAbs appear to be poorly immunogenic in infected individuals and as vaccine candidates. Insights into the immunoregulation of some of these latter epitopes (e.g., epitopes defined by MAbs 2F5 and 4E10) was provided by recent studies in which the MAbs were discovered to bind one or more self antigens [50, 51] , raising the possibility that these antibody specificities are subjected to negative regulation mechanisms, such as receptor editing or deletion. Thus, Env as an immunogen may bypass key steps in the B cell inductive pathway, or may actively induce negative production or downregulation of production of some broadly neutralizing antibodies [52] [53] [54] .
The receptor-ligand interactions and intracellular signaling pathways that govern the production of antibodyproducing plasma cells and the persistence of plasma and memory B cells are poorly understood. Additional information on the mechanisms responsible for B cell migration, selection, and differentiation within and between specialized anatomical sites, particularly within lymphoid follicles, might be used to target suitable Env epitopes to appropriate B cell inductive pathways. An example would be to provide necessary signals to generate long-lived and high affinity memory in the marginal zone B cell compartment. Another example would be to discover ways to modify germinal center formation, positive and negative selection, and B cell differentiation to drive long-lived high affinity antibody responses against key epitopes that tend to be poorly immunogenic.
In parallel to these efforts, genetic studies at the population level could provide critical information on the most promising paths to follow. In particular, the recent completion of the International HapMap Project now permits whole genome associated studies to be conducted with a minimum number of single nucleotide polymorphism tags [55, 56] . This powerful new technology could be used to identify genes that are associated with the wide variation in neutralizing antibody responses in HIV-1-infected individuals and in vaccine recipients. A critical question to ask is whether the potent neutralizing antibody response in a small subset of infected individuals is due to unique viral epitopes or to host genetic polymorphisms. Current evidence suggests that both might make a substantial contribution in the context of combined epitope and allelic representations [28, 47, 57] .
To date most studies of the humoral responses in HIV infections have investigated immunoglobulins, the final product of B cell responses. Relatively few studies have examined B cell immunopathogenesis. A number of basic questions are still unanswered (e.g., extent and reason for perturbation of B cell subset changes, including memory B cells and plasma cells, in peripheral blood and tissues). Questions also remain about other potential functional contributions of B cells to HIV infections (e.g., role as antigen-presenting cells). In vivo studies should be performed in the nonhuman primate animal model to determine the emergence of pathologic events in the B cell compartment, in particular in lymphatic and gastrointestinal tissues of naïve and vaccinated animals that are challenged with pathogenic SIV or SHIV. These investigations should be done in parallel to detailed analyses of the magnitude and function of HIV-specific immunoglobulin responses determined in plasma and tissue secretions, and of HIV-specific B cells on a single cell basis.
The establishment of a research consortium to study fundamental B cell biology as it relates to HIV-1 vaccines is recommended. This program should be structured in a way that asks key scientific questions about B cell regulatory pathways that modulate Env immunogenicity. Studies could address B cell receptor-ligand interactions and intracellular signaling pathways that govern the production of antibodyproducing plasma cells, the persistence of plasma and memory B cells, the mechanism of action of adjuvants, and host genetic associations with immune responses. immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Eng Design  Host Gene Expression Profiling of Dengue Virus Infection in Cell Lines and Patients BACKGROUND: Despite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication. CONCLUSION/SIGNIFICANCE: Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy. Although dengue-related disease results in an estimated 50-100 million cases of dengue fever and 250,000 to 500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year [1, 2] , a clear understanding of dengue pathogenesis remains elusive. Dengue virus is an enveloped, positive-stranded RNA virus of the Flaviviridae family transmitted by the mosquito Aedes aegypti and Aedes albopictus. Four serotypes of dengue virus (DENV1-4) circulate in endemic areas. Although infection with one serotype of dengue virus confers life-long protective immunity to that serotype, it does not protect the host from infection with other serotypes [3] .
The initial target cells during dengue infection are believed to be Langerhans cells [4] . Through means not yet fully understood, Langerhans cells spread the virus, via the lymphatic system, to other tissues such as liver, spleen, kidney and blood, whereas monocytes, macrophages and endothelial cells are the major cell types in which the virus replicates [5] . In the majority of symptomatic dengue infections, a fever of 5-7 days duration develops together with bone and joint pain, retro-orbital pain, nausea and fatigue, this is called dengue fever (DF). While the majority of DF patients recover without intervention, 2-5% develop a more severe form of the disease, called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), characterized by thrombocytopenia and vascular leakage, causing hypervolemic shock and death if not promptly treated [6] . The cause of DHF/DSS is not clear. Antibody dependent enhancement (ADE) is the most widely supported theory explaining the higher risk of DHF/DSS associated with a heterologous secondary infection [7] . The phenomenon of T cell ''original antigenic sin'' has also been described [8] . Like many viruses, dengue inhibits IFNa and IFNb signaling by suppressing Jak-Stat activation, resulting in reduced host antiviral response [9] . The combination of a reduced host defense, increased uptake of the virus and delayed viral clearance likely synergizes to produce higher viremia resulting in a more severe outcome.
Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology has allowed researchers to study gene expression changes on a much larger scale. A summary of published microarray data from infection of different host cell types with bacteria, viruses, yeast, protozoa and helminthes revealed a common host-transcriptional-response consisting of a cluster of IFN-stimulated and immune mediating genes [10] . One particularly successful application of microarray technology was the identification of a novel drug target, c-kit, in endothelial cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) [11] .
With regard to dengue infection, gene expression studies have been carried out in infected human umbilical vein endothelial cells (HUVECs) by differential display reverse transcription (DD-RTPCR) and Affymetrix oligonucleotide microarrays [12] . Genes having a role in the IFN antiviral response and immune defense such as 29-59 oligoadenylate synthetase (OAS), myxovirus protein A (MxA), TNFa, galectin-9, phospholipid scramblase 1 and human inhibitor of apoptosis-1 (IAP1) were shown to be upregulated upon infection [12] . Infection with dengue of a more transformed HUVEC-like cell line, ECV304, was analyzed by a different microarray system containing 7600 cDNA oligonucleotides [13] . In this study, the expression of 15 genes involved in cell cycle, apoptosis, membrane trafficking and cytoskeleton was found to be altered after infection [13] . Using a different gene expression approach, primary macrophages infected with a clinical isolate of dengue were analyzed by a cytokine array containing 375 human cytokine-related genes with approximately 20 genes observed to be either up-or down-regulated [14] . However, the functional importance of these changes, if any, was not studied [14] .
We have used Compugen human 19K oligonucleotide arrays to study the host response to dengue infection, initially in a human hepatocytic cell line (HepG2). We identified the upregulation of three major clusters of genes; NF-kB-mediated cytokine/chemokine responses, type I IFN response, and the ubiquitin-proteasome system. Increased expression of selected genes, identified by Compugen array, was confirmed using a taqman low density array (TLDA) and results extended to include an additional, readily infectable, cell line (A549) and PBMC derived from adult dengue fever patients from a Singapore dengue prospective cohort study. Next we sought to confirm the functional effects of these upregulated genes. We confirmed the high levels of two novel chemokines, IP-10 and I-TAC, in dengue infection, in both cell lines and in dengue patients. We also determined that overexpression of viperin, an upregulated gene in the IFN pathway, and proteasome inhibition, with MG-132 or ALLN, reduced virus replication. In summary, we have used microarray analysis to identify new host genes associated with dengue infection, and to show that components of the host response can control dengue viral replication and therefore represent potential targets for drug therapy.
Cell lines, A549, BHK-21, C6/36, HeLa, HepG2, HUV-EC-C, K562, SK-Hep1 and THP-1, were obtained from ATCC and maintained as instructed. The type 2 dengue virus strain TSV01 was obtained from a dengue outbreak in Townsville, Australia [15] (GenBank, accession number AY037116) and was propagated in the C6/36 cell line. Heat-inactivated virus was prepared by incubating virus samples in a 55uC water bath for 1 hr. Each cell line was infected with dengue virus at a multiplicity of infection (MOI) of 1 and 10 for 3, 6, 12, 24, 48, and 72 hrs before being evaluated for replication efficiency by plaque assay. Infection of cell lines was not continued beyond 72 hrs as after this point a significant degree of cell death became apparent.
BHK-21 cells were cultured overnight in 24 well plates before media was removed and serial dilutions (10-fold) of virus culture supernatants added to individual wells. Plates were incubated for 1 hr before media was aspirated and replaced with 0.5 ml of 0.8% methyl-cellulose medium (with 2% FBS). Plates were then incubated for 5 days before the media was removed and cells fixed in 4% formaldehyde for 20 min, rinsed in water, stained with crystal violet for 20 min then rinsed again. Plaques were counted manually and concentrations of plaque forming units per ml (pfu/ ml) in the cell culture supernatant calculated. For drug intervention studies, HepG2 cells were treated with compound, or DMSO, at the indicated concentration and time. While the culture supernatants were collected for plaque assay, cytotoxicity in HepG2 cells was monitored using fluorescein diacetate (FDA, Acros Organics, Belgium), 10 mg/ml added to cells for 25 min at room temperature, and measured using a fluorescent plate reader (485 nm excitation/535 nm emission).
Dengue E-protein was assessed in suspension HepG2 cells by intracellular staining and fluorescence activated cell sorter (Becton Dickinson, USA) and an Alexa-647 conjugated (AlexaFluor conjugation kit, Invitrogen, USA) monoclonal antibody (4G2, ATCC). Cells were scraped and permeabilized using BD FACS Perm/Wash solution (Becton Dickinson, USA) and acquisition and analysis was performed using CellQuest software (Becton Dickinson, USA).
Dengue is the most prevalent mosquito-born viral disease affecting humans, yet there is, at present, no drug treatment for the disease nor are there any validated host targets for therapeutic intervention. Using microarray technology to monitor the response of virtually every human gene, we aimed to identify the ways in which humans interact with dengue virus during infection in order to discover new therapeutic targets that could be exploited to control viral replication. From the activated genes, we identified three pathways common to in vitro and in vivo infection; the NF-kB initiated immune pathway, the type I interferon pathway, and the ubiquitin proteasome pathway. We next found that inhibiting the ubiquitin proteasome pathway, or activating the type I interferon pathway, resulted in significant inhibition of viral replication. However, inhibiting the NF-kB initiated immune pathway had no effect on viral replication. We suggest that drugs that target the ubiquitin proteasome pathway may prove effective at killing the dengue virus, and, if used therapeutically, improve clinical outcome in dengue disease.
Virus quantification by real time RT-PCR RNA was extracted from infected cells using the RNeasy Mini Kit (Qiagen, Netherland). Dengue serogroup 2 virus TSV01 detection by TaqMan PCR was adapted to the ABI7900 real time instrument using previously published primers and probes [16] . Standard ABI conditions were used, incorporating primers at 900nM. Quantification was achieved by relating viral Ct value to the Ct value on a standard curve of a measured number of copies of a 750 bp section of the virus (forward: 59-AAAGATCAGTGG-CACTCGTTCC-39 and reverse: 59-GCAGGTCTAAGAAC-CATTGCCT-39), cloned into pCR2.1-TOPO (Invitrogen, USA).
Human arrays of 19,800 60mer oligonucleotide probes (representing 18861 genes), designed by compugen and manufactured by Sigma-Genosys were used according to the standard protocol described for cDNA microarray [17] . Dye swap was performed for each sample, at every time point and a rigorous quality check was performed before an array was used for downstream analysis [18] . 60 mer oligonucleotide probes were spotted onto poly-L-lysinecoated microscope slides using GeneMachines OmniGrid Microarray Spotter (USA). For fluorescence labeling of target cDNAs, 20 mg of total RNA from universal human reference (Strategene, USA) and experiment samples (RNA extracted from infected cells using Qiagen RNeasy Mini Kit ) were reverse transcribed in the presence of Cy3-dUTP and Cy5-dUTP (Amersham Biosciences, UK) using the Superscript reverse transcription kit (Invitrogen, USA). Labeled cDNA were pooled, concentrated, re-suspended in DIG EasyHyb (Roche, Switzerland) buffer and hybridized overnight (14-16h) in the MAUI Hybridization chamber (BioMicro, USA). The arrays were scanned using a GenePix 4000B Scanner (Axon Instruments, USA) to generate Tiff images. The images were analyzed by GenePix Pro 4.0 software (Axon Instruments, USA) to measure Cy3 and Cy5 fluorescence signals intensity and format data for data base deposition. The array data then underwent lowess normalization [19] available in an R package aroma to remove channel specific biases (R Development Core Team).
Differentially expressed genes were selected using a procedure known as Significance Analysis of Microarrays (SAM) [20] , described in brief below. The statistic used in SAM is given as
where; the numerator is the group mean difference, s the standard error, and s 0 a regularizing constant. Setting s 0 = 0 will yield a t-statistic. This value, called the fudge constant, is found by removing the trend in d as a function of s in moving windows across the data to reduce false positive results. As the statistic is not t-distributed, significance is computed using a permutation test. Genes with a computed statistic larger than the threshold were considered significant. The false discovery rate (FDR) associated with the given threshold can also be calculated from the permutation data.
Quantitative PCR (qPCR) by TaqMan Low Density Array (TLDA)
RNA was extracted using RNase Easy kit (Qiagen, Netherland) from infected cells. For patients blood samples (see Patient Samples), 2.5 ml of blood was collected in PAXgene tubes, RNA was extracted using PAXgene Blood RNA Kit (PreAnalytiX, Qiagne, Netherland). RNA was subjected to DNase treatment using an RNase-Free DNase Set (Qiagen, Netherland). 100 ng of total RNA was reverse transcribed using the High-Capacity cDNA Archive Kit (ABI, USA) and processed for TaqMan Micro Fluidic Cards (3M Company, ABI, USA) according to manufacturers instructions, together with data analysis using SDS2.2 software (ABI, USA). Differentially expressed genes were detected as above, using SAM. We analyzed SAM gene lists using the Applied Biosystem online program PANTHER [21] (http://www. pantherdb.org/). Pathway, interaction and Gene Onotology analysis was performed using MetaCore, version 3.2.0 (GeneGo, Inc, USA).
Subjects were enrolled from the Early DENgue (EDEN) study, a dengue investigation conducted at a primary healthcare clinic in Singapore, for which local ethical approval had been granted, and informed consent from patients obtained. Participation required a PCR diagnosis of fever with duration less than 3 days (for the details of the PCR diagnosis, see [22] ). The enrolled dengue cases were either of serotype 1 or 3. Ten dengue positive subjects (n = 10, 3 males) were selected to represent the most severe cases of dengue by the criteria of a platelet counts below 30 (x 10 3 /ml; range 8-30, mean 20.5). Their mean age was 43.2 years (range 24-67). The mean fever was 38.3uC (range 37.7-39.1) at the first visit (Time Point 1), with a mean duration of 43 hours (range 14-72) from the onset of the fever. The second visit (Time Point 2) occurred at 80 to 96 hours after the first visit, mean fever was 37.1uC (range 36.2-38.7). Dengue negative patients were enrolled with the same criteria (fever with no respiratory infection symptoms), but were PCR negative for dengue. Ten subjects were taken with (n = 10, 4 males) a mean age of 43.2 years old (range 24-67). Their mean fever was 38.4uC (range 37.6-39.4) with a mean duration 27 hrs (range 8-60) at the first visit (Time Point 1). The convalescence sample was collected at the third visit which was 3 to 4 weeks later (Time Point 3). At each time point, a 2.5 ml blood sample was collected in PAXgene tubes for RNA analysis. A 10 ml blood sample was collected, serum was separated within 5 hrs and stored at 280uC. Serum IP-10 and I-TAC concentrations were measured using ELISA kits from R&D Systems as per manufacturers instructions.
A549 cells were transfected with an expression construct encoding viperin using lipofectamine 2000 (Invitrogen, USA). Cells were selected using 500 mg/ml G418 and screened for viperin expression by immunoblotting. Cells were cultured overnight in 6 well plates before IFNb (Glycoferon, Singapore), at a final concentration of 500 U/ml, was added to each well while control wells remained untreated. Twelve hours post IFNb treatment, cells were infected with dengue virus (TSV01; MOI 1) for 48 hrs and the plaque assay was used to determine virus production.
While a number of different cell lines have been used as models for dengue infection it is not clear which of these represents the most appropriate model for the analysis of host response by microarray, which requires a high rate of infection. As such, we screened seven human cell lines for their ability to support replication of dengue virus. We used the clinical, dengue serotype 2 isolate TSV01 (Accession number: AY037116) strain for infection. Human cell lines were ranked by maximum plaque forming units (pfu)/ml titer produced with A549.HepG2.SK-Hep1.K562.HUV-EC-C.THP-1.HeLa (data not shown). The same results were obtained with the widely used NGC strain (data not shown). The highest yielding cell lines A549 (a lung carcinoma) and HepG2 (a hepatoma cell line) were used in further studies, with HepG2 as the primary focus because of evidence of liver injury in DHF/DSS and the detection of dengue antigens in hepatocytes in liver [23, 24] .
Microarray identification of host responses to dengue virus replication using the HepG2 infection model Viral replication in HepG2 cells infected with dengue virus TSV01 for 3, 6, 12, 24, 48 and 72 hours, compared to heat inactivated virus, was determined by plaque assay of the virus released in the cell culture medium ( Figure 1A ), FACS analysis of infected cells labeled intracellularly with an Alexa 674-conjugated antibody against dengue E protein (4G2) ( Figure 1B ) and real-time PCR analysis of viral RNA ( Figure 1C ). All three methods showed that new viral replication began after 24 hours and reached a plateau at 72 hrs, with FACS analysis showing 28% of cells infected at this point. After 72 hrs a degree of cell death become apparent (data not shown) and the experiment was not continued. Analysis of microarrays, performed in duplicate (dye-swapped) on three biological replicates at each time point, comparing infectious with heat inactivated virus; using a SAM q value (false discovery threshold) of 25%, revealed no significantly differentially expressed genes at 3, 6, 12 or 24 hrs post infection. However, there were 24 transcripts identified at 48 hrs and 124 at 72 hrs (a total of 132 transcripts representing 124 genes; Table S1 ) that were differentially expressed. At both 48 hrs and 72 hrs, clustering of transcripts using PANTHER analysis identified the IFN-mediated immunity pathway as the most significant with a P value of 10 215 (at 72 hrs). Genes that are typically induced after type I IFN stimulation, including OAS1, OAS2, OAS3, OASL, STAT1, STAT2, MX1, IFIH and IFNb, featured prominently in this cluster ( Figure 2 and Table S1 ). Further analysis of the 124 genes suggested the involvement of NF-kB-mediated cytokine/chemokine responses (NFKBIB, NFKB1A, TNFA1P, CCL4, CCL5, IP-10 and I-TAC amongst others) and ubiquitin related genes (HERC5, HERC6, UBE2L6, USP15 and others) ( Figure 2 ). Although not completely overlapping, a core host response to pathogen involving the IFN response and the NF-kB-mediated immune defense response [10] was observed in our array results. Other genes that were significantly changed included those associated with cell signaling, lipid metabolism, cell cycle and vesicular transport (Table S1 ). We did not detect any significantly down regulated genes in our system. All the microarray data were deposited in a public database accessible at http://www.ncbi.nlm. nih.gov/projects/geo (accession number is GSE6048).
With the evidence of upregulation of three major pathways (NF-kB, IFN and ubiquitin) by microarray, we decided to pursue these pathways with further confirmation and validation. Firstly, we chose 59 genes from the three major pathways that were upregulated in the HepG2 cell line as shown by microarray. Secondly we selected another 36 genes which had a functional association with these pathways or might otherwise be associated with dengue infection. A Taqman Low Density Array (LDA) was then constructed for these 95 genes in order to confirm their (Table S2) , indicating a high rate of validation of the genes detected by microarray.
In order to exclude gene responses that were specific to the HepG2 cell line alone, we decided to test the expression level of these genes in the A549 cell line using the same LDA. In the A549 infection model, plaque assay revealed that peak viral production occurred earlier than in the HepG2 model (1.1610 5 61.4610 4 pfu/ ml at 48 hrs) but was still substantial at 72 hrs post-infection (7.3610 4 63.8610 4 pfu/ml). The quantitative PCR also revealed a higher number of differentially expressed genes, with 63 genes at the 48 hr time point and 82 genes at the 72 hr time point (Table S2 ).
Seeking confirmation that these genes were relevant in a physiological setting, we sought to test these genes in dengue patients. Whole blood RNA samples were obtained from ten adult fever patients (age .21 years old) enrolled in the Singapore Early Dengue (EDEN) cohort study in 2005 [22] . Each was PCR diagnosed positive for either dengue serotype 1 or 3 and all showed typical dengue fever symptoms. At the second visit (,5 days after onset of fever, onset of fever considered day 1), their platelet count had dropped below 30 (x 10 3 /ml; range 8-30, mean 20.5), and they were admitted to hospital. When these patient samples were tested for expression of the 95 selected genes by LDA, 67 genes were shown to be differentially expressed comparing blood samples taken at acute fever stage (first visit, 1-3 days after onset of fever) to convalescence (third visit, 3-4 weeks after first visit) (Table S2) .
After confirmation of the expression of certain genes in two different dengue-infected cell lines and in dengue patients, we Table S1 . Colour intensity is derived from mean relative expression fold changes of dye swap results in comparison to universal reference (Strategene USA), red for upregulated, green for down regulated, black for no change, and grey for missing data. Time course (3, 6, 12, 24, 48, selected those that were upregulated in at least one time point in HepG2 cells and in at least one time point in A549 cells and in dengue patient samples (comparing acute to convalescent blood samples). Fifty genes fulfilled these criteria (Table S2 ) and, we felt, represented common genes involved in dengue virus response, or virus replication, while excluding responses that were cell type specific. These 50 common genes were mapped by direct interactions using the MetaCore program which illustrated the close clustering and interconnectedness of a network of 29 of genes around NF-kB, TNF-a and STAT1. (Figure 3A) . The NF-kB gene alone was added to the network, despite not being from our common list, to illustrate the connections between those induced by it. For example, the upregulation of IP-10, I-TAC, VEGF, PAI1, B2M, TNFAIP3 and RIG-1 could all be linked to the activation of NF-kB, while NFKB1B and NFKB1A, two feedback control genes for NF-kB activation, were also upregulated reflecting the self containment of this activation ( Figure 3A ). The degree of upregulation varied between the genes in this pathway with I-TAC and IP-10 being the most highly up-regulated (average expression in the two cell types and patients) genes ( Figure 3B) . A number of genes clustered around STAT1 were associated with the IFN pathway, and examination of the common list revealed another four genes (viperin, IFI44, IFIH1, G1P3) related to the IFN pathway that were upregulated but not mapped by the MetaCore program ( Figure 3C ). Finally, a number of genes related to the ubiquitin-proteasome pathway also appeared on the MetaCore map (HERC5, USP18 and Hdm2) with several more (unmapped) genes appearing on the common list ( Figure 3D )
The two most highly upregulated common genes from the NF-kB pathway were IP-10 (or CXCL10/IFN-inducible protein 10) and I-TAC (or CXCL11/IFN-inducible I cell a chemoattractant) ( Figure 3B) . In order to determine if this up-regulation of gene transcription lead to translation and protein release, concentrations of IP-10 and I-TAC protein in cell culture supernatant following dengue infection were determined by ELISA. In both the A549 and HepG2 infection models, dengue infected cells produced moderate, but significant (compared to heat-inactivated virus treated cells), concentrations of IP-10 ( Figure 4A ) and I-TAC ( Figure 4B ) at 72 hrs, but not at earlier time points (data not shown). We next investigated if these, or other, NF-kB induced proteins were influencing dengue virus replication. Adding dexamethasone to the HepG2 infection model to inhibit NF-kB activation [25] prevented IP-10 and I-TAC production, but had no effect on viral replication (data not shown). These results suggest that NF-kB activation, and IP-10 and I-TAC production, do not have a direct effect on viral replication and are, rather, simply part of the immune response to infection. Serum concentrations of IP-10 and I-TAC in the ten dengue fever patients described above, together with ten fever patients who did not have dengue (viral PCR negative) were also determined by ELISA. High concentrations of IP-10 ( Figure 4C ) and I-TAC ( Figure 4D) were present in the serum of dengue fever patients. There was significantly more IP-10 in the serum of the patients during the first (1-2 days after fever onset) and second (4-5 days after fever onset) visits, compared to the convalescent serum (P = 10 215 and P = 10 211 , respectively) as well as to non-dengue fever patient serum (P = 10 29 and P = 10 27 , respectively). I-TAC level was also significantly higher in first visit dengue patient serum comparing to both the convalescent (P = 10 27 ) and non-dengue fever (P = 10 26 ) patients. I-TAC levels in dengue fever patients at the second visit were significantly lower than at first visit (Fig 4D) .
We detected a large number of IFN response genes induced in both cell line infections and in dengue patients (see Figure 3C ). In our study, viperin was one of the most highly upregulated genes in the type I IFN response pathway. Viperin has previously been identified as an IFN-induced anti-viral protein in HCMV and HCV infection [26, 27] . In order to investigate the role of viperin in dengue infection, we used an established A549 cell line stably overexpressing viperin (Vip) [26] . Comparing to infection in wild type A549 cells (WT), viperin overexpressing cells were significantly resistant to viral replication, as shown by plaque assay two days after infection ( Figure 5A ), with and without pre-treatment with IFNb (+IFN; 500 U/ml). Although pre-treatment with IFNb had the greater anti-viral effect, viperin over expression alone resulted in a small, but significant, reduction in virus production both with (P = 0.038) and without (P = 0.0004) IFNb pretreatment. These results suggest that viperin is an functional component of the IFN-mediated response to dengue, and demonstrate, for the first time, that viperin could be part of the anti-dengue response.
Ubiquitination, another pathway identified in our list of common genes, is a key component of the immune system, the conjugation of single, or multiple, ubiquitin molecules to a protein targets it for defined subcellular localization, or destruction in the proteasome [28] . Components of the ubiquitin-proteasome system have been shown to be required for the maturation and release of a number of viruses (see review [29] ). In order to investigate the role of ubiquitin-proteasome system in dengue infection, we introduced proteasome inhibitors, MG-132 and ALLN, to the HepG2 dengue infection model [30] . At lower concentrations, both MG-132 and ALLN significantly reduced virus replication in the cell lines by over 50% (Figure 5B ). An examination of the effects of these compounds on the integrity and viability of inhibitor-treated cells using fluorescein diacetate revealed no cytotoxicity at these concentrations ( Figure 5C ). Higher concentrations of the inhibitors had an even greater effect (.90% reduction in pfu/ml) but with a degree of cytotoxicity ( Figures 5B, 5C ).
Despite the fact that dengue is a major disease affecting the tropical world, little is known of its pathogenesis due, partly, to the lack of a suitable animal model and the complex cell interactions in infected individuals. Using microarray analysis of gene expression and high throughput quantitative real-time PCR, we investigated the host response to dengue infection in cell lines and in DF patients. We further validated our microarray results by functional study of the identified genes. Although dendritic cells, Langerhans cells and monocytes have been proposed as the principle reservoirs of viral infection [4] , it is not clear which other tissues, if any, are targeted. Immunohistochemistry and in-situ hybridisation studies of biopsies from DHF patients have indicated that a range of tissues are infected by the virus, including cells in the liver, spleen, lung, kidney and peripheral blood [5] . We chose the HepG2 cell line as the primary cell model for this study as it readily supported viral replication, and it is derived from the liver, which might be of some clinical relevance. The A549 cell line was included as it was also an excellent supporter of viral replication even though there is, presumably, little or no clinical relevance. The final clinical relevance came from the validation of the genes in 10 DF patients from the Singapore EDEN cohort. The 10 patients were selected based on low platelet count, because of the lack of WHO DHF/DSS manifestation in Singapore adult patients. Despite the limited sample number, and the wide range of collection times, variation between individuals at the same time point was ruled out by statistical analysis. We believe that with more patients, more genes that are differentially expressed would be identified.
The overlap of upregulated genes, determined by quantitative PCR, in the two cell systems and in patients removed any responses that were unique to the cell type. In fact, we observed cell type specific gene changes such as IL-8, PAI-1 and RANTES that were upregulated in the cell lines but not the patient samples while the anti-inflammatory cytokine IL-10 was upregulated in the patient samples but not the cell lines, indicative of the effects of multiple cell types in an in vivo system. Similarly, IFNb was upregulated in the cell lines while IFNc was in patient samples. However, it is the large overlap between the in vitro infection and patient samples that warrants most attention.
The two most highly upregulated chemokines were IP-10 and I-TAC. IP-10 and I-TAC are both ligands for the CXCR3 chemokine receptor and the production of these chemokines leads to the recruitment of CXCR3 expressing T cells and NK cells [31, 32] . Increased IP-10 and I-TAC expression has been seen in various viral infections, especially viral meningitis [33] . In SARS patients, elevated IP-10 early in infection was shown to be a predictor for a more severe outcome [34] . Neuronal IP-10 was shown to be involved in the recruitment of T cells in West Nile virus encephalitis [35] . Elevated I-TAC mRNA and protein has also been found in the liver of chronic Hepatitis C patients [36] . In dengue infection, various chemokines have previously been found to be induced in dengue patients, including IL-8, MIP-1a, MIP-1b, RANTES and MCP-1, but IP-10 and I-TAC have not been examined (see review [37] ).
In a dengue intracerebral mouse infection models, CXCR3 2/2 and IP10 2/2 mice both had higher mortality rate than wild type mice after infection, indicating that IP-10 and CXCR3 receptors are part of the host defense mechanism, most likely in recruitment of T cells to the infection site [38] . IP-10 was also proposed to compete with virus binding on the receptor in vitro [39] . However, the relevance of this study to human patients is not clear. Although IFNc, a/b and NF-kB could all induce the production of IP-10 and I-TAC, prevention of IP-10 and I-TAC via NF-kB inhibition clearly reduced the IP-10 level in HepG2 cells. Furthermore, IFNb and IFNc were not consistently upregulated in the two cell lines or in DF patients in our study (data not shown). Therefore, we believe that NF-kB activation rather than IFN induction played the major role in elevated IP-10 and I-TAC in dengue infection. The inhibition of NF-kB, and consequent IP-10 and I-TAC inhibition, had no demonstrable effect on dengue replication in cell lines, suggesting the more complex role for these chemokines in a multi-cellular in vivo setting. Concentrations of each chemokine were significantly higher during the early stages of dengue fever, while I-TAC levels were reduced at the second visit, IP-10 levels remained high. The persistently high level of IP-10 might also contribute to the immunopathogenesis of dengue although this would require further investigation. It may be that concentrations of IP-10 and I-TAC, in combination with a viral antigen, could be used as an early marker for dengue fever. It is interesting to note that the level of IP-10 and I-TAC were independent of previous history of dengue infection, as half of the ten tested patients were suffering from a secondary infection. The common clinical feature of the ten selected patients was the low platelet count at second visit. Because of the lack of severe dengue cases (by WHO definition) in Singapore, the low platelet count was used as the measure of severity by which the ten patients from the cohort study were classified as having more severe dengue fever. We cannot at this point make a link between the level of IP-10, I-TAC and disease severity because of the small number of cases used in this study, a more detailed clinical study, currently underway, aims to determine if the concentrations of IP-10 and I-TAC during early stages of infection are linked to the progression to more severe forms of disease.
The ability of IFN pre-treatment to inhibit subsequent dengue replication has been previously reported [9, 40] , as has the importance of IFN in the anti-viral response [41] . Our results indicated that viperin was one of the most highly upregulated genes in all models following dengue infection. Viperin encodes for an IFN-inducible antiviral protein shown to be associated with the endoplasmic reticulum and redistributed to the Golgi apparatus and cytoplasmic vacuoles following human cytomegalovirus (HCMV) infection [26] . The induction of viperin has been suggested to be anti-viral in both HCMV [26] and hepatitis C (HCV) [27] infections, but this is its first association with dengue infection. The use of viperin overexpressing A549 cells demonstrated that, in addition to being significantly upregulated during infection, viperin is directly involved in the anti-viral response to dengue, as shown by the suppression of dengue replication in the presence of increased expression of viperin. This effect was not as significant as the effect of IFNb alone suggesting that viperin is only a part of the IFN-mediated response to dengue. The molecular mechanism of the action of viperin to counter dengue infection will need to be further studied.
The ubiquitin-proteasome system is the cellular machinery involved in the conjugation of single or multiple ubiquitin molecules to direct protein trafficking or degradation (reviewed in [28] ). In HCV infection, E6AP ubiquitin ligase was reported to mediate the ubiquitination and degradation of the virus core protein [42] . HCV polymerase has also been shown to interact with a ubiquitin-like protein leading to its degradation [43] . In dengue, ubiquitin-proteasome genes have not been reported to be involved in the dengue virus life cycle although a recent publication has shown that dengue envelope protein interacts with SUMO-1 conjugating enzyme 9 (Ubc9) and that overexpression of Ubc9 reduces virus production in a cell line [44] .
Our TLDA results indicated that there was significant upregulation of a number of ubiquitin-proteasome system related genes during dengue virus replication but it was unclear if this response was anti-viral or if the dengue virus utilised components of the ubiquitin-proteasome system for replication. Use of proteasome inhibitors, MG-132 and ALLN, significantly reduced the release of dengue virus following infection of the HepG2 cell line. Previously, it has been shown that the effect of proteasome inhibitors may be virus specific with these compounds less (or non-) effective against virus such as influenza [45] and equine infectious anemia virus [46] . The effect of proteasome inhibition on viral replication appears to be mediated via a number of different processes including the involvement of ubiquitin or the proteasome in virus assembly [45] , budding [47] and release and maturation [48] . Further studies may elucidate the exact role the ubiquitin-proteasome plays in dengue virus replication which may involve trafficking of the virus to the plasma membrane or the maturation and fusion of the virus upon release [49] . It is instructive to compare our results with those of a published microarray study investigating host responses in patient blood [50] . That study identified genes whose protein products are expressed in the ER to be the most significantly enriched, which directly overlaps with our identification of the ubiquitin pathway; specifically including UBE2 and the PSMB genes. This may represent a process fundamental to viral replication in any system. In addition, Simmons et al. described host responses associated with dengue shock syndrome that clearly overlap with our interferon related gene list. In particular, G1P genes, OAS genes, IFI genes and Mx genes were found in both studies, suggesting that the amount of viral replication may be directly related to clinical outcome.
Gene arrays of a number of cell systems during dengue infection revealed many genes, and host response pathways, that were upregulated during dengue infection. Further functional analyses distinguished between host response pathways involved in initiating an innate signaling response (NF-kB mediated genes and IFN pathway) and those involved in virus replication (ubiquitin-proteasome system). Specific components of the response to virus, such as viperin and IP-10 and I-TAC have been implicated in dengue infection for the first time. Further investigation of these components, together with the precise role of the ubiquitin-proteasome system in virus replication, may lead to drug targets for dengue. The use of gene array in multiple cell systems to investigate genes involved in virus replication, used in concert with functional studies, has proved to be a valid approach for discovery of novel markers and genes for understanding the host response to dengue and, ultimately, therapeutics against dengue.
Table S1 HepG2 Microarray Gene List. List of HepG2 transcripts (n = 132), identified as differentially expressed by SAM analysis, in response to dengue virus TSV01 compared to heat inactivated virus 48 and 72 hours post infection. Transcripts are placed in groups according to biological processes. qV is the SAM calculated q value for each gene (following SAM significance selection based on a Delta value calculated from the variance between the sample sets, see Material and Methods). F.C. indicates fold change. ''-'' represents no significant change. Genes selected for real-time PCR validation through a TaqMan low density array (TLDA) platform are indicated by a tick. Found at: doi:10.1371/journal.pntd.0000086.s001 (0.28 MB DOC) Table S2 Quantitative PCR by Taqman based low density array in HepG2, A549 and Singapore dengue fever patients. Fold increase in gene expression as determined by quantitative PCR. For HepG2 and A549, the fold change was calculated based on dengue virus infection over heat-inactivated virus infection. For dengue fever patients (Patients), the fold change was calculated using patient blood samples collected at the first visit (,1-2 days after onset of fever) over the convalescence (3-4 weeks after the acute fever). Upregulation is shown in black and down-regulation in red. ''1.0'' represents no significant change (significance determined by q value ,5, see Material and Method). Genes that were significantly up-regulated in at least one time point in HepG2 and in at least one time point in A549 and in Patients are indicated with P-values, calculated by standard student T test and selected based on a cut off at P,0.05. Found at: doi:10.1371/journal.pntd.0000086.s002 (0.19 MB DOC) Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-related immune receptors in the dog BACKGROUND: Although the impact of pathogens on the evolution of the mammalian immune system is still under debate, proteins, which both regulate immune responses and serve as cellular receptors for pathogens should be at the forefront of pathogen-driven host evolution. The CEA (carcinoembryonic antigen) gene family codes for such proteins and indeed shows tremendous species-specific variation between human and rodents. Since little is known about the CEA gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging CEA family by analyzing the CEA family of the dog. RESULTS: Here we describe the complete CEA gene family in the dog. We found that the gene coding for the ITIM-bearing immunoregulatory molecule CEACAM1 gave rise to a recent expansion of the canine CEA gene family by gene duplication, similar to that previously found in humans and mice. However, while the murine and human CEACAMs (carcinoembryonic antigen-related cell adhesion molecules) are predominantly secreted and GPI-anchored, respectively, in the dog, most of the CEACAMs represent ITAM-bearing transmembrane proteins. One of these proteins, CEACAM28, exhibits nearly complete sequence identity with the ligand-binding N domain of CEACAM1, but antagonizing signaling motifs in the cytoplasmic tail. Comparison of nonsynonymous and synonymous substitutions indicates that the CEACAM28 N domain is under the strongest purifying selection of all canine CEACAM1-related CEACAMs. In addition, CEACAM28 shows a similar expression pattern in resting immune cells and tissues as CEACAM1. However, upon activation CEACAM28 mRNA and CEACAM1 mRNA are differentially regulated. CONCLUSION: Thus, CEACAM1 and CEACAM28 are the first paired immune receptors identified within the CEA gene family, which are expressed on T cells and are most likely involved in the fine-tuning of T cell responses. The direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that CEACAM28 evolved in response to selective pressure imposed by species-specific pathogens. The evolution of immunoglobulin superfamily (IgSF) members is largely influenced by the nature of their ligands. The extracellular part of IgSF members which are predominantly expressed in the nervous system are well conserved, while the extracellular domains of members expressed by immune cells or cells involved in reproduction tend to diversify much more rapidly [1] . The CEA gene family, which belongs to the IgSF, is a tandemly clustered multigene family. Such gene families are subject to rapid evolution due to ongoing gene duplication, deletion and mutational events [2] . At present, the CEA family is subdivided into two main subgroups: the CEA-related cell adhesion molecule (CEACAM) and the pregnancy-specific glycoprotein (PSG) subgroups. The CEACAM subgroup in humans consists of 12 members composed of a single immunoglobulin variable (IgV)-like N-terminal (N) domain followed by zero to six Ig constant (IgC)-like domains of A and B subtypes and one member which consists of two IgC-like domains and two IgV-like domains, one at each end of the molecule. CEACAM1, CEACAM3, CEACAM4, CEACAM18, CEACAM19, CEACAM20 and CEACAM21 are transmembrane molecules while CEA/ CEACAM5, CEACAM6, CEACAM7, and CEACAM8 are linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors and CEACAM16 is most likely a secreted molecule. For CEACAM1 (also known as CD66a, BGP and C-CAM105) and the recently discovered CEACAM16, CEACAM18, CEACAM19 and CEACAM20, but not for other family members have orthologs been identified in rodents [3] . In addition to these orthologs, the transmembrane-bound CEACAM2 (in mice), CEACAM17 (in mice and rats) and the secreted members CEACAM9, CEACAM10, CEACAM11 CEACAM12, CEACAM13, CEACAM14, CEACAM15 (in mice and rats) exist in rodents [3, 4] . In mice, rats and cattle, at least two CEACAM1 alleles each have been identified, which differ considerably in their N domain sequences [5] . No such allelic variation has been observed in humans. The PSG, which are specifically expressed in the trophoblast, are the most abundant fetal proteins in the maternal bloodstream during late human pregnancy. The PSGs are thought to play a pivotal role in the regulation of the maternal immune response to the fetal semi-allograft [6] . The diversity of the CEACAM/PSG family of proteins is further enhanced by species-specific differential splicing of several family members [7] . As expected from the variable structure of the CEA family members, these molecules have diverse functions. It is well established that various CEA family members play crucial roles in cell-cell adhesion, tumor development, angiogenesis, insulin metabolism, reproduction and, as more recently realized, in immunity [8] . CEACAM1 functions as a natural killer cell inhibitory receptor [9, 10] , regulates T and B cell proliferation [11, 12] , induces dendritic cell maturation [13] and facilitates granulocyte and monocyte survival [14, 15] . In addition, CEACAM3, CEACAM6 and CEACAM8 were found to be pivotal for the regulation of granulocyte activation in humans [16] . While human CEACAM1 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic domain, one CEACAM1 ITIM is replaced by an immunoreceptor tyrosine-based switch motif (ITSM) in mice and rats. Human CEACAM3 and possibly CEACAM4, CEACAM19 and CEACAM20 harbor functional immunoreceptor tyrosine-based activation motifs (ITAM) in their cytoplasmic tails [17, 18] . In contrast, except for CEACAM19 and CEACAM20 no such ITAM-containing CEA family members exist in rodents. Remarkably, several pathogens namely, Neisseria sp., Haemophilus influenzae, and Moraxella catarrhalis in humans [19] [20] [21] and mouse hepatitis virus (MHV) [22] use CEACAMs to anchor themselves to or to invade host cells. In addition, it has been suggested that bacterial pathogens can down-regulate immune functions by binding to CEACAM1 on CD4+ T lymphocytes and signaling via the CEACAM1 ITIM motif [23] . It was recently proposed that the human granulocyte-specific CEACAM3 functions as a specifically adapted single-chain ITAM-dependent phagocytic receptor involved in the clearance of CEACAM-binding bacteria by human granulocytes [24] . This indicates that fixation of genes in the human genome coding for ITAM-bearing CEACAMs can be the result of a selective pressure mediated by CEACAM-binding pathogenic microbes. In contrast, one of the two allelic variants of murine CEACAM1 exhibits a significant lower virus receptor activity suggesting that, in mice, mutations in the CEACAM1 gene rather than the fixation of an activating virus receptor were acquired to evade pathogen attack. The availability of a high-quality draft genome sequence of the domestic dog (Canis familiaris) [25] provided the possibility to analyze the complete CEA gene family in a third mammalian order. Here we show that in the dog multiple ITAM-bearing CEACAM1-related CEACAMs evolved and that CEACAM28 and CEACAM1 have undergone concerted evolution by gene conversion. Based on sequence analyses and comparison of expression profiles we suggest that CEACAM28 and CEACAM1 are paired activating and inhibitory immune receptors involved in the regulation of T cell responses. The possibility that sporadic recurrence of selective pressure mediated by species-specific pathogens was the driving force for the evolution of multiple ITAM-bearing CEACAM1-related CEACAMs in the dog is discussed.
In an effort to identify all CEA gene family members in the dog genome we employed the BLAST program to search the GenBank and Ensemble databases (CanFam 1.0, July 2004) using cDNA nucleotide sequences from all known human and mouse CEA family members as query sequences. The human and mouse CEA gene families are located within the expanded leukocyte receptor complex on chromosome 19q13 ( Figure 1A ) and chromosome 7, respectively. This approach yielded the sequences of the CEACAM1, CEACAM16, CEACAM18, CEACAM19 and CEACAM20 orthologs. Their predicted full length mRNA sequences (Additional file 1) encode proteins with domain organizations identical with their human and murine counterparts: CEACAM16 (N1-A-B-N2); CEACAM18 (N-A-B-TM-Cyt); CEACAM19 (N-TM-Cyt); CEACAM20 (truncated N-A1-B1-A2-B2-TM-Cyt) [3] . Like their mouse and human counterparts, both CEACAM19 and CEACAM20 contain presumed ITAM consensus sequences in their cytoplasmic domains. However, like in mouse, no CEACAM19 cytoplasmic domain exon 1 present in human CEACAM19 was found [3] . As for human CEACAM18, no cytoplasmic domain exon could be identified due to its small size and lack of corresponding EST sequences. In a second round of search we used the BLAST program and cDNA nucleotide sequences from the identified orthologous canine genes to screen the Gen-Bank and Ensemble databases. This strategy was employed in order to identify dog-specific genes which have evolved after the separation of the dog and primate/ rodent lineage. This approach led to the identification of 7 additional CEACAM genes (CEACAM23-CEACAM29) using CEACAM1 nucleotide sequences as query sequences. CEACAM23, CEACAM24, CEACAM25, CEACAM29, CEACAM28 and CEACAM1 are arranged between XRCC1 and LIPE, while CEACAM26 and CEACAM27 are located between CD79A and TGFβ1 on the opposite strand ( Figure 1) . Surprisingly, no genes corresponding to the PSG genes were found. The murine Psg genes are located between Hif3a and Pglyrp1 spanning nearly 1.8 Mbp. In dog, however, the distance between HIF3A and PGLYRP1 comprises only 0.1 Mbp which rules out the presence of a larger set of genes between these marker genes. Similarly, there is not enough space between XRCC1 and CEACAM23 (0.2 Mbp) to accommodate an expanded PSG gene family as found in humans at the corresponding chromosomal locations location where the distance is approximately 1 Mbp ( Figure 1B) . The canine CEACAM gene loci on dog chromosome 1 exhibit a similar arrangement to that found for the human CEA gene family on chromosome 19 but it is partially different from that of the murine CEA gene family on chromosome 7 ( Figure 1B) .
In order to identify the very short cytoplasmic exons of the CEACAM1-like genes which cannot be localized by direct search strategies we first localized the transmembrane exons of all CEACAM genes using transmembrane exon sequences of the human and mouse CEACAM genes as query sequences. Surprisingly, the 6 predicted transmembrane domain exons of the CEACAM1-related canine genes clustered with the sequences of the transmembrane domain exons of human CEACAM3, CEACAM4 and CEACAM21 which are connected to cytoplasmic domain exons encoding a C-terminal ITAM motif (CEACAM3 and CEACAM4) or a homologous truncated cytoplasmic domain with a premature stop (CEACAM21). The sequence of the transmembrane exon of canine CEACAM1 clustered with transmembrane sequences from murine Ceacam1 and Ceacam2 and with a group of exons coding for hydrophobic GPI signal peptides from human CEACAM1, CEACAM5, CEACAM6, CEACAM7, CEACAM8 (Figure 2A and data not shown). CEACAM5-CEACAM8 GPI signal peptide exons are thought to have evolved from the transmembrane exon of an ancestral CEACAM1 by introduction of a stop codon [26] . CEACAM1/Ceacam1 and Ceacam2 encode cytoplasmic domains which contain ITIM motifs. Taken together, this indicates that TM and cytoplasmic domain exons are exchanged together during evolution and predicts the presence of ITAM signaling motifs rather than ITIM in the cytoplasmic tails of the dog CEACAM23-CEACAM25, CEACAM28 and CEACAM29 molecules. Subsequently, we screened about 2,000 bp of nucleotide sequence downstream of the predicted canine transmembrane domain exons for the presence of the short cytoplasmic exons. Indeed, small cytoplasmic domain exons similar to the ones found in human CEACAM3 and CEACAM4 could be identified, which encode ITAM motifs close to the C-terminal end ( Figure 2B ). Although homologous cytoplasmic domain exons 1-3 are present in CEACAM29 the loss of the splice donor site in the cytoplasmic domain exon 3 leads to read through into the adjacent intron and truncation by a stop codon following after five codons. CEACAM26 and CEACAM27 probably represent pseudogenes due to multiple deletions in their extracellular (in particular N exons) and cytoplasmic domain exons (Figure 3) .
To determine the degree of relatedness between the primordial canine CEACAM genes with the orthologous genes in mice and man, the nucleotide and encoded amino acid sequences of the leader, the IgV-type N domain, the IgC-type A and B domains, the transmembrane domain and the cytoplasmic domain exons were compared ( Table 1 ). The degree of sequence identity between corresponding extracellular Ig-type domains in dog, humans and mice varies greatly at the amino acid sequence level. The lowest value is observed for CEACAM1 N domains (56% and 42% for human and mouse, respectively), the highest for the CEACAM16 N2 domain (92 and 96% for human and mouse, respectively; Table 1 ).
Based on the genomic sequence information we designed gene-specific primers for the amplification of full length CEACAM1-related cDNAs from total RNA, isolated either from liver or spleen. In the published genomic dog sequence, one nucleotide is missing from the A1 exon of canine CEACAM1. Cloning and sequencing of full length CEACAM1 clones from a mix-breed dog revealed a complete 279 bp CEACAM1 A1 exon. In total, we could iden- Expansion of ITAM-bearing CEACAM1-related CEA family members in dog No leader exon could be identified for CEACAM23 because of a sequence gap (indicated by brackets) in the publicly available genomic sequences. The genes are arranged in the order and orientation as found on dog chromosome 1.
and 4. The cloned CEACAM25 splice variants also code for proteins with only one N domain followed by a transmembrane domain. Three out of four clones encode cytoplasmic domains which contain the predicted ITAM motif (GenBank accession no. EF137908). In one clone, the absence of the 53 nucleotide cytoplasmic domain exon 1 leads to a frame shift and the usage of an alternative stop codon located in cytoplasmic domain exon3 (GenBank accession no. EF137909). Using supposedly CEACAM28specific primers, two products were amplified which differ in their length by 276 bp. Cloning and sequencing revealed that the CEACAM28 gene codes for a protein with one N domain, two A domains, a transmembrane domain and a cytoplasmic tail which contain an ITAM motif (GenBank accession no. EF137910). The sequence of the cDNAs cloned from the smaller PCR product was different from all genomic sequences published either for the boxer or the poodle breed and, therefore, was named CEACAM30. The differences are more or less randomly distributed within the whole coding region which consists of an N, A, transmembrane and cytoplasmic domain region. Two CEACAM30 splice variants were identified, both containing an ITAM motif in the cytoplasmic tail. They differ, however, in the usage of alternative splice acceptor sites of the cytoplasmic domain exon 1 (Gen-Bank accession nos. EF137911 and EF137912) ( Figure 4 ).
To analyze the evolutionary relationship of the newly discovered CEACAM genes, multiple nucleotide sequence alignments of Ig domain exons of human and dog CEACAM genes were performed and phylogenetic trees constructed. As expected, the N domain sequences of the orthologous genes (CEACAM16, CEACAM18-CEACAM20) clustered pairwise, while all new canine N domain exon sequences (CEACAM23-CEACAM30) clustered with that from dog CEACAM1 ( Figure 5A ). Similarly, the primate-specific CEACAM1-related N domain nucleotide sequences (CEACAM3-CEACAM8) clustered with the human CEACAM1 N domain exon sequence except for CEACAM21 N. The close relationship between the new dog CEACAM genes and dog CEACAM1 could also be demonstrated when the A and B domain exon nucleotide sequences were compared ( Figure 5B ). Nine of the 17 IgC-like domains of the new CEACAM1-related canine molecules are of the CEACAM1 A2 type and five each were of the CEACAM1 A1 and B type ( Figure  5B ).CEACAM23 appears to be the only CEACAM1-related gene which contains all extracellular domain exons present in CEACAM1, in particular three IgC-like domain exons (A1, A2 and B). All other members of this subgroup either lack one of these exons or contain exons corrupted by nonsense, frame shift or splice site mutations leading to smaller predicted proteins (Figure 3, 4) . Assuming that the N domain is the most relevant domain for ligand interaction, the ligand specificity of CEACAM28 seems to be most closely related to that of CEACAM1. The N domain amino acid sequence of CEACAM28 differs from that of CEACAM1 in only two positions. These amino acid changes do not affect the center of the CFG face, a topological N domain region of CEACAM members essential for ligand and pathogen interactions ( Figure 6A,B) . Interestingly, CEACAM28 lies next to CEACAM1 in the CEACAM1 gene cluster which hints to an evolutionary recent duplication event ( Figure 1B) . CEACAM25 is the most distant CEACAM1 relative within the group of CEACAM1-related genes, sharing only 66% of its N domain exon encoded amino acid sequence (Figure 5A, 6A; Table 2 ). Most of the amino acid sequence differences cluster to the CFG face of the N domains, indicating a positive selection for ligand diversification. To test this possibility, we calculated the ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitution rates on the branches of an N exon phylogenetic tree. The lowest Ka/ Ks ratio (0.1) is observed for CEACAM28. This indicates that the CEACAM28 N domain is under purifying selection. For all other canine CEACAM1-related CEACAMs, the N domain amino acid changes appear to evolve neu- (51) 56 (40) The trally or to be positively selected for during evolution since the corresponding Ka/Ks values are close to 1 or higher ( Figure 6C ).
To identify the CEACAM gene expression pattern and splice variants, their nucleotide sequences were aligned, and regions of the transmembrane domain-encoding exons with sequence differences were selected to design primers that are specific for each of the CEACAM1-related cDNAs except for CEACAM28 and CEACAM30 which were amplified with a common primer pair ( Table 3 ). The primers were used to amplify cDNAs from individual CEACAM by RT-PCR from RNA isolated from liver, bone marrow, spleen, purified peripheral blood mononuclear cells (PBMC), and granulocytes ( Figure 7) . Except CEACAM29, all analyzed CEACAM1-related genes, (CEACAM1, CEACAM23, CEACAM24, CEACAM25, CEACAM28, and CEACAM30) were found to be expressed in spleen. CEACAM1, CEACAM24, CEACAM25, CEACAM28 and CEACAM30 transcripts were detected in bone marrow and CEACAM1, CEACAM24, CEACAM28 and CEACAM30 mRNAs in PBMC. Granulocytes express in addition to different CEACAM1 splice variants (preferentially with the long cytoplasmic tail) all the ITAM-bearing molecules except CEACAM23. In liver, the presence of mainly CEACAM1 and CEACAM23 transcripts could be demonstrated. CEACAM26 and CEACAM27 are not expected to be expressed because the sequences of essential exons appear to be corrupted (see above).
To further analyze the expression of CEACAM1-related CEACAMs by immune cells, we isolated T cells from PBMC by MACS sorting using CD4-and CD8-specific mAbs. This procedure resulted in a purity of the T cell populations of >97%, as determined by flow cytometry (data not shown). Activation of T cells by the various stimuli was controlled by analysis of blast formation using flow cytometry ( Figure 8A) . RT-PCR analysis demonstrated that resting T cells express CEACAM1, CEACAM28 and CEACAM30 mRNA simultaneously. In addition, expression of these CEACAMs was also found in T cell-depleted PBMC, which are mainly composed of Bcells ( Figure 8B ). Stimulation of PBMC with anti-CD3 mAb or IL-2 had only a minor effect on CEACAM1 expression in T celldepleted PBMC (data not shown) and T cells. In contrast, the content of CEACAM28 mRNA seem to be reduced in T cells upon activation ( Figure 8B ). In unstimulated T cells the CEACAM1/CEACAM28 cDNA ratio was < 3 whereas the ratio was > 7 and > 11 in T cells stimulated with IL-2 and anti-CD3 mAb, respectively ( Figure 8C ). The content
Domain organization of dog CEACAM1-related protein splice variants The amino acid (A; below the diagonal) and nucleotide sequences (N; above the diagonal) of IgV-like domain exons were compared pairwise using the ClustalW software (Weight Matrix BLOSOM). The degree of identity is indicated in %. Figure 8B ).
The near identity of the N domains between CEACAM1 and CEACAM28 is most likely due to a recent gene conversion event. Indeed, we could identify a 2,332 bp genomic region in CEACAM1 which shows 99% identity with the corresponding region of CEACAM28. The high similarity of this segment starts at position -984 of the 5'flanking region and ends at position 114 of the intron between the Ndomain and the A1 domain exons ( Figure   9A ). Upstream of this segment, the sequence similarity between homologous regions of CEACAM28 and CEACAM1 (~1,200 bp) drops to 80%. The downstream homologous region of ~1,700 bp demarcated by a SINE element in CEACAM28 exhibits a similarity of 92%. The occurrence of such a regionally restricted gene conversion event is also supported by the high degree of amino acid sequence similarity of the leader (100%) and N domain (98%). The A1 and A2 domains of CEACAM28 encoded outside of the conserved genomic region share only 88% and 86% of their amino acid sequence with that of the homologous CEACAM1 A2 domain ( Figure 9B ). These data suggests that the similarity between CEACAM1 and
Relationships of the N-domains of dog CEA gene family members the CEACAM28 ancestor was ~92% before the gene conversion event occurred.
Recent molecular studies have established strong evidence for four primary, superordinal mammalian clades: Afrotheria, Xenarthra, Euarchontoglires and Laurasiatheria [27, 28] . The CEA gene family is well known in primates (humans) and rodents (mice and rats). However, both orders belong to the same principal lineage of placental mammals, the Euarchontoglires. Since carnivores belong to the Laurasiatheria clade, we expected to gain new insights into the evolution of the rapidly diverging CEA family by analysis of the CEA family of the dog. First of all, identification of CEACAM1, CEACAM16, CEACAM18, CEACAM19 and CEACAM20 orthologs in the dog genome at corresponding chromosomal locations, strongly indicates that the CEA gene family of the common ancestor of Euarchontoglires and Laurasiatheria consisted of at least five genes. It remains to be seen whether species which represent older mammalian lineages like Afrotheria, marsupials and monotremes contain the same set of primordial genes. Furthermore, the findings of this work supports our previous hypothesis that CEACAM3-CEACAM8, CEACAM21 and CEACAM9-CEACAM15, CEACAM17, none of which is present in the dog genome ( Figure 2A and data not shown), represent primate and rodent-specific genes, respectively [3] . The ancestral members of the CEA gene family are located in three different loci (Figure 1 ). While CEACAM16, CEACAM19 and CEACAM20 are close together (CEACAM16 and CEACAM19 are next to each other), CEACAM1 is separated from this locus by more than 1 Mbp and CEACAM18 is displaced into the SIGLEC gene cluster in humans, mice and dogs.
Out of these ancestral genes, probably only CEACAM1 ancestors gave rise to massive gene expansions in a orderspecific manner [3] . The CEACAM1-related genes form compact clusters next to CEACAM1 and near the CD79A gene. In rodents even a third locus (around the Hif3a gene) has been encroached by amplified CEACAM1related genes. With analysis of the CEA family in a third order (carnivores), the inversion event observed for the chromosomal region flanked by Cd79a and Lipe can now be decided to have happened in the rodent rather than in the primate lineage, since the order of genes in this locus is conserved between humans and dogs ( Figure 1 ).
CEACAM1-related genes can be subdivided into the PSG and the CEACAM subgroup genes. The birth of the ancestral PSG has been estimated to have occurred some 90 Myr ago [29] , approximately the time of rodent-primate divergence [30] . The rapid, independent expansion of the human and mouse PSG gene families occurred through further gene duplication and exon shuffling events [31] [32] [33] . Surprisingly, we did not find any PSG gene in the dog genome indicating that the PSG function exerted in primates and rodents is dispensable for the dog, possibly because dogs have an endotheliochorial placenta type which is different from the hemochorial placentae of primates and rodents [34] . Semi-allogeneic fetal trophoblast
Expression pattern of dog CEACAM1-related genes Figure 7 Expression pattern of dog CEACAM1-related genes. CEACAM1-related transcripts were identified by RT-PCR using gene-specific primers which are located in the N domain and transmembrane exons. For the detection of CEACAM1 transcripts, primers in the N domain and cytoplasmic domain exon 3 were used. The products were separated by agarose gel electrophoresis in the presence of ethidium bromide and visualized by UV illumination. One-kb and 100-bp DNA fragment ladders were used as markers.
The possible domain organization of the proteins encoded by the splice variants (number of Ig domains) is indicated in the right margin. Sequence determination of the CEACAM28 PCR products revealed simultaneous detection of CEACAM28 and CEACAM30 cDNAs (till then unknown). C, CEACAM. 
Expression of the paired receptors CEACAM1 and CEACAM28 in stimulated T cells forward scatter sideward scatter cells are less invasive and concomitantly less prone to encounter the maternal immune system in endotheliochorial placentae in comparison to the hemochorial placentae where the trophoblast cells are bathed in maternal blood [35] . Therefore, PSG could be involved in the regulation of trophoblast invasion and/or modulation of the maternal immune system as has been suggest before [3, 36] . Alternatively, the function of the PSG could have been taken over by an unrelated gene family due to functional convergence at the molecular level, similar to that found for natural killer (NK) cell receptors, where killer cell immunoglobulin-like receptors (KIR) and Ctype lectin-like receptors (Ly49) are used in primates and rodents, respectively.
In contrast to the PSG, the CEACAM subgroup of canine CEACAM1-related genes underwent a recent expansion similar to that found in primates and rodents. In general, IgSF members which are expressed by leucocytes are diverging rapidly within their extracellular domains. This results in an average amino acid identity of about 54% between human and mouse orthologs [1] . The conservation of the CEACAM1 N domain, most relevant for pathogen and cell-cell interactions [37] , lies within this range, with an amino acid identity of 56% (dog versus human) and 42% (dog versus mouse). This is consistent with the notion that CEACAM1 plays a key role in various functions of the immune system [8] . In contrast, between mouse and human CEACAM16, CEACAM18 and CEACAM19 orthologs, the N domain amino acid identities are higher ranging from 60% to 91%, which argues for non-immunological functions, especially for CEACAM16, which exhibits an overall amino acid sequence identity of about 90% ( Table 1 ).
The dog CEACAM1-related genes are arranged head to tail, like the KIR genes in the human KIR locus [38, 39] . Such an arrangement was found to facilitate unequal crossing over leading to gene expansion and generation of multigene families [40] . Furthermore, the KIR gene family is subject to birth-and-death evolution, domain shuffling and mutational changes [41] . Our analyses indicate that the CEA gene family evolved similarly. One characteristic for this kind of evolution is the presence of multiple pseudogenes, which also exist within the canine CEACAM1like gene locus (CEACAM26, CEACAM27).
This type of evolution also facilitates a mechanism for qualitatively changing a receptor's signaling potential. A number of type I transmembrane IgSF members including CEACAMs and KIRs are composed of an amino-terminal extracellular "environmental sensor" and a carboxy-terminal cytoplasmic tail which can contain either inhibitory (ITIM) or activating motifs (ITAM or a positively charged amino acid in the transmembrane region permitting coupling with activating adaptors such as DAP12 [42] ). Unequal crossing-over at such gene loci encompassing the corresponding exons could result, in a single step, in genes encoding receptors with a drastically changed signaling potential by combining the same extracellular domain with a cytoplasmic tail of opposite function. This allows rapid adaptation to environmental changes and the creation of so-called paired immune receptors. An example for such a mechanism is found for the KIR gene KIR3DL/S, where either an inhibitory variant or an activating variant encoding KIR gene is found in different human individuals at the same locus [43] . In addition, a change in the receptor's signaling potential which may be the result of an arms race between pathogens and the host immune system has been reported recently for mice. The murine cytomegalovirus (MCMV) induces in infected cells expression of the virus-encoded GPI-anchored m157 protein, which binds to the activating, DAP12-associating killer receptor Ly49H in MCMV-resistant mice leading to NKcell-mediated cytotoxicity. MCMV-susceptible mouse CEACAM1 and CEACAM28 genomic regions involved in gene conversion Figure 9 CEACAM1 and CEACAM28 genomic regions involved in gene conversion. The 2332 bp genomic region of CEACAM1 which covers nearly 1 kb of the 5'-flanking region (5'-FR), the leader exon (L), intron 1 (IntI), the N domain exon (N) and part of intron 2 (Int II) is highly conserved in CEACAM28 (99%) and, therefore, probably participated in a recent gene conversion event. The homologous sequences upstream and downstream from the conserved region are less conserved (80% and 92%, respectively) (A). This can also be deduced from the degree of conservation of the amino acid sequences encoded by the leader, N and A domains which is much less outside of the gene conversion region. Note that CEACAM28 contains two A2 type domains (B). [44] . It was suggested that the activating receptor gene was derived from the gene encoding the inhibitory receptor to counteract a virus that exploits the inhibitory receptor during infection and that this mechanism may apply to many other activating receptors that have inhibitory counterparts [45, 46] . However, recently it was shown for the human SIGLEC5 and SIGLEC14 genes that the direction of gene conversion that generates paired immune receptors is not always the same. This indicates that also other needs drive the evolution of paired receptors like the fine tuning of immune responses [45] . Host-pathogen interactions involving both proteins with ITIM or ITAM signaling motifs have also been reported for humans. The primate-specific CEACAM1-related protein CEACAM3 which contains an N domain closely related to that of CEACAM1 (88% amino acid sequence identity) and a cytoplasmic tail with an ITAM is exclusively expressed in granulocytes. Due to its close relatedness with the ITIM-bearing gonococcal receptor CEACAM1 it can act as decoy receptor for Neisserial pathogens and is able to induce clearance of gonococcal infections by granulocytes. Mutational analyses demonstrated that the cytoplasmic ITAM is instrumental for this process [24] .
To our knowledge, CEACAM1 and CEACAM28 represent the first paired coexpressed immune receptors which contain antagonizing ITIM and ITAM (and not just a transmembrane domain with a positively charged amino acid which serves as a docking site for ITAM-containing adaptor molecules, like DAP12) in their respective cytoplasmic tails. CEACAM28 was probably formed from a CEACAM29 ancestor by a recent gene conversion encompassing the leader and the N exon from CEACAM1. The latter exon encodes the region most instrumental for ligand and pathogen binding in CEACAM molecules. Several findings, presented here, argue for a role of CEACAM28 as a CEACAM1 decoy receptor in the dog. First, the direction of the gene conversion was most likely from the inhibitory receptor to the activating receptor, second, the Ka/Ks ratio of the N domain exons of these family members is indicative for a purifying selection, whereby the specificity of the putative CEACAM1 ligand is probably conserved for CEACAM28, third, the amino acid changes observed for the CEACAM28 N domain most likely have no effect on the structure of that part of the N domain shown to be the pathogen binding site in human and mouse CEACAM1 and fourth, is expressed by immune cells (T, probably B and/or NK cells and granulocytes), a prerequisite for a defense function against a putative pathogen. Based on a highly similar amino acid sequence and expression pattern CEACAM30 can be envisioned to also function, like CEACAM28, as a decoy receptor.
However, this hypothesis raises questions about the driving force behind the fixation of the other ITAM-bearing CEACAM1-related genes in the dog. Most likely, selective pressure by pathogens changes with time. In times where the selective pressure exerted by pathogens on a receptor system is low, Darwinian selection will act and change the specificity of a receptor now possibly recognizing an endogenous ligand. Once the receptor has gained a new function it also can change the signaling properties which may explain why older i.e. more diverged ITAM-bearing receptors e.g. like CEACAM24 and CEACAM29 have lost the ITAM or have gained new splice variants which do not contain an ITAM.
Taken together, the presence of multiple CEACAM1related dog CEACAMs with ITAMs could reflect a repeated arms race between species-specific pathogens and the dog immune system. Fixation and expression by effector cells of ITAM-containing CEACAMs would result from either bacterial pathogens binding to CEACAM1 or viruses which induce the expression of CEACAM1 ligands by infected cells as found for MCMV. This putatively pathogen-driven evolution of the CEA gene family led to the development of the CEACAM1/CEACAM28 receptor pair which could also be involved in the fine tuning of T cell responses.
Different canine tissue samples were collected from healthy mix-bread dogs and flash-frozen in liquid nitrogen. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood of healthy mixbread or beagle dogs by density-gradient centrifugation through Ficoll-Paque (GE Healthcare, Freiburg, Germany). Granulocytes located on top of erythrocytes were harvested and remaining erythrocytes were lysed (ammonium chloride buffer).
Stimulation of PBMC with PHA (2 µg/ml for 72 hr), CD3 (CA17-2A12; [47] ) (0,5 µg/ml for 96 hr) and human IL-2 (200 U/ml for 7 days) was performed at a concentration of 5 × 10 5 cells/ml in RPMI-1640 supplemented with 10% fetal calf serum (FCS "Gold"; PAA Laboratories, Coelbe, Germany), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, non-essential amino acids and 1 mM sodium pyruvate (GIBCO/Invitrogen, Karlsruhe, Germany). T cells were isolated from either stimulated or unstimulated PBMC by positive MACS sorting following the manufacturer's instructions (Miltenyi Biotec, Ber-gisch-Gladbach, Germany). Stimulated and unstimulated PBMC were incubated with mouse anti-canine CD4 (CA13-1) and rat anti-canine CD8 mAbs (Dog 10-8) [48] and subsequently with bead-coupled anti-mouse Fc mAb which also reacts with rat IgG.
For surface staining, cells were suspended in PBS/0.3% (w/v) BSA supplemented with 0.1% (w/v) sodium azide. Cells were incubated with 0.5 µg/10 6 cells of the relevant mAb for 30 min at 4°C. Cells were washed twice and analyzed with a FACScan (BD Biosciences, Mountain View, CA). Dead cells were excluded by propidium iodide staining. The following reagents and mAbs from Serotec were used: FITC-conjugated anti-CD3, FITC-conjugated anti-CD4 and PE-conjugated anti-CD8α.
Total RNA extraction was performed using either the TRIzol ® reagent (Invitrogen Life Technologies, Karlsruhe, Germany) or the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufactures' protocols. One µg of total RNA was used for cDNA synthesis by reverse transcription (RT) in a total volume of 20 µl using either the Reverse Transcription System ® (Promega, Mannheim, Germany) or the First-Strand cDNA Synthesis ® Kit (MBI Fermentas, St. Leon-Rot, Germany). The RT product (1 µl) was amplified by polymerase chain reaction (PCR) with Taq polymerase (Qiagen). After an initial denaturation step at 95°C for 45 s, 35 PCR cycles (denaturation: 95°C, 30 s; annealing: 60°C, 1 min; extension: 72°C, 1.5 min) and a final extension step at 72°C for 15 min were performed. The primers were designed using the Primer3 software. Gene-specific primers were selected that exhibited > 2 mismatches at the 3'-end of the oligonucleotide to all other members of the canine CEACAM family, except for CEACAM28 and CEACAM30 (see below). The primers were validated by eye using multiple nucleotide sequence alignments of the aforementioned CEA family members. The primer sequence and the predicted sizes of the amplified products are summarized in Table 3 . Canine GAPDH cDNA was amplified using GAPDH.dogforward 5'-GCCAAAAGGGTCATCATCTC and GAPDH.dog reverse 5'-GCCCATCCACAGTCTTCT primers at a annealing temperature of 56°C and 30 cycles. Eight µl of each PCR were analyzed by electrophoresis on a 1.8 % agarose gel and visualized by ethidium bromide staining. The amount of cDNA was quantified by endpoint determination with the Quantity 1 ® software (Bio-Rad Laboratories, Munich, Germany).
Based on published genomic sequences and comparison with human CEACAM1 the 5'-and the 3'-untranslated regions of canine CEACAM1 were predicted and used to design primers for amplification of full length cDNA (CEACAM1cf-5i and CEACAM1cf-3i; Table 3 ). These primers introduced HindIII and XbaI restriction sites at the 5'-and 3'-ends of the PCR products, respectively. PCR was performed with cDNA from dog liver using Pfu DNA polymerase according to the protocol supplied by the manufacturer. After electrophoretic separation, 6 distinct ethidium bromide-stained DNA fragments were excised from the agarose gel and purified using the Perfectprep ® Gel Cleanup Kit (Eppendorf, Hamburg, Germany). The full length cDNAs were digested with HindIII and XbaI and cloned into the pRc/CMV expression vector. Plasmid DNA isolated from 12 clones were analyzed by PCR and sequencing. Full length cDNAs of CEACAM23, CEACAM24, CEACAM25, CEACAM28 and CEACAM30 were cloned using the TOPO TA Cloning ® Kit (Invitrogen Life Technologies). PCR was performed with cDNA from dog spleen using Taq polymerase (Qiagen) and the CfCEACAM1-5i-f forward primer for all CEACAMs combined with gene-specific reverse primers from the 3'untranslated regions (UTR; Table 3 ).
Sequence similarity searches were performed using the NCBI BLAST tools [49] and Ensembl BLAST/SSAHA search programs [50] . For the identification of CEACAM genes not yet annotated in the dog genome, sequences from human and mouse CEACAM and PSG cDNAs were run against the WGS databases or whole genome sequences. Multiple sequence alignments were performed and phylogenetic trees were constructed with Clus-talW [51] using default parameters.
The three dimensional structures of CEACAM N domains were modeled using the Geno3D-release 2 software based on the published crystal structure of the murine CEACAM1 N-A2 fragment [37, 52] . Leader and transmembrane domains were identified with the aid of programs SignalP and TMHMM, respectively. The NetPhos 2.0 server was used to produce neural network predictions for serine, threonine and tyrosine phosphorylation sites in the cytoplasmic domains of canine CEACAMs [53] . Comparison of the number of nonsynonymous substitutions per nonsynonymous site (Ka) with the number of synonymous substitutions per synonymous site (Ks) was performed with the Ka/Ks Calculation Tool [54] . The substitution rate ratio Ka/Ks measures the molecular selective pressure. If Ka/Ks = 1, the amino acid changes are neutral and will be fixed at the same rate as silent mutations. If Ka/Ks < 1, the amino acid changes are deleterious and purifying selection will reduce the fixation rate. If Ka/ Ks > 1, the amino acid changes are evolutionarily advantageous and positive selection will increase the fixation rate. Nucleolus: the fascinating nuclear body Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed. Brief history of the nucleolus An ovoid body visible in the nucleus was probably the Wrst observation of the nucleolus more than two centuries ago by F. Fontana. Since that time, the nucleolus has been the object of intense investigation and interestingly our vision of the nucleolus has evolved with technical progress. During the nineteenth century, using light microscopy, numerous cytologists described the variability of nucleolar morphology with great precision (Montgomery 1898) . In 1934, McClintock proposed that the "nucleolus is organized in the telophase through the activity of ... the nucleolar-organizing body" (McClintock 1934) . Since the nucleolar-organizing body corresponds to a speciWc region of chromosome 6 in Zea mays, this was the Wrst time the nucleolus was related to gene activity. In the 1950's the presence of RNAs in the nucleolus was demonstrated, and in the 1960's in situ hybridization techniques made it possible to identify ribosomal genes (rDNAs) in the nucleolar organizer region (NOR) (Caspersson 1950; Perry 1962; Ritossa and Spiegelman 1965) . During the same period, mass isolation of nucleoli became possible leading to the biochemical characterization of nucleolar components. Based on these results it was proposed that ribosome biogenesis occurs in nucleoli. Given that the nucleolus became a subject of great interest, the "International symposium on the nucleolus-its structure and function" was organized in Montevideo in 1965 and the contributions published in Natl Cancer Inst Monogr no 23 (USA) in 1966. Since 1969, at the initiative of W. Bernhard and H. Busch, "Nucleolar Workshops" on nucleolar organization, the biochemistry of nucleolar proteins, rRNA processing as well as variability in cancer cells were regularly organized. Several books on nucleoli were published; among them, the famous "The nucleolus and ribosome biogenesis" is still a very useful source of information (Hadjiolov 1985) . Between 1980 and 2000, the functional organization of the nucleolus was deciphered in large part due to the improvement of labeling by the electron microscopy (EM).
Recently a new Weld of investigation was opened when molecules not involved in ribosome biogenesis were detected in the nucleolus (Carmo-Fonseca et al. 2000; Pederson 1998; Politz et al. 2002; Visintin and Amon 2000) . In accordance with these nucleolar localizations, nucleolar mass spectrometry analyses identiWed »700 nucleolar proteins, some of them not related to ribosome biogenesis (Andersen et al. 2005) . The area of plurifunctional nucleolus was opened. Consequently "The nucleolus", a book that presents the state of the art was published (Olson 2004) as well as several reviews on the multiple functions of the nucleolus (Boisvert et al. 2007; Hernandez-Verdun 2006; Hiscox 2007; Raska et al. 2006 ).
General information "The nucleolus: an organelle formed by the act of building a ribosome" (Mélèse and Xue 1995) reveals by its size and organization the eYciency of ribosome biogenesis. For example the nucleolus is a prominent nuclear structure in cycling cells but of limited size in the terminal stages of diVerentiation such as in lymphocytes or chick erythrocytes. If ribosome biogenesis is blocked, reorganization of the nucleolar components is visible in segregated nucleoli. In mammalian cells, the nucleolus is disorganized in prophase and reassembled at the end of mitosis using the nucleolar machineries from the previous cell cycle. On the contrary, in yeast the nucleolus is present and active throughout the cell cycle even though condensation of the rDNAs is necessary for transmission of the nucleolus in anaphase (D'Amours et al. 2004; Sullivan et al. 2004; Torres-Rosell et al. 2004) .
The nucleolus is the ribosome factory of the cell. In the nucleolus rDNAs are transcribed, the 47S precursor ribosomal RNAs (pre-rRNAs) are cleaved, processed and assembled with the 80 ribosomal proteins and the 5S RNA to form the 40S and 60S ribosomal subunits (selected reviews Gébrane-Younès et al. 2005; Hernandez-Verdun and Junéra 1995; Scheer et al. 1993; Scheer and Hock 1999; Shaw and Jordan 1995; Thiry and Goessens 1996) . This complex series of maturation and processing events, presently better characterized in yeast than in higher eukaryotes is under the control of about 150 small nucleolar RNAs (snoRNAs) and 2 large RNP complexes: (1) the small subunit (SSU) processome containing the U3 snoR-NAs and 40 proteins or Utps (U three proteins) required for the 40S ribosomal subunit, and (2) the large subunit (LSU) processome required for the 60S ribosomal subunit (de la Cruz et al. 2004; Fatica and Tollervey 2002; Fromont-Racine et al. 2003; Sollner-Webb et al. 1996; Tollervey 1996) . The snoRNAs associated with proteins, function in the maturation of rRNAs creating two types of modiWed nucleotides (2Ј-O-methylation and pseudouridylation) and mediating endonucleolytic cleavages of pre-rRNAs (Gerbi and Borovjagin 2004) .
Our objective is to focus this review on the ribosome biogenesis processes occurring in the nucleoli that might help to decipher the global organization of nuclear functions. We describe nucleolar organization and dynamics, propose our view on nucleolar targeting, report the relationship between the nucleolus and the cell cycle, review particular relationships between nucleolus and virus, and nucleolus related to cancer.
Three main components in the active nucleolus
The nucleolus has been proposed as the paradigm of nuclear functional compartmentalization (Strouboulis and WolVe 1996) . It is the site of ribosome biogenesis and in addition the nucleolar machineries are distributed in diVerent compartments. When observed by EM, three main nucleolar components (compartments) can be discerned in mammalian cells: the Wbrillar centers (FCs), the dense Wbrillar component (DFC) and the granular component (GC) (Fig. 1a) . The FCs are clear areas, partly or entirely surrounded by a highly contrasted region (Goessens et al. 1987) , the DFC. The FCs and the DFC are embedded in the GC, mainly composed of granules of 15-20 nm in diameter. The most contrasted structures in the EM sections stained with uranyl and lead correspond to high concentrations of nucleic acids. The condensed chromatin surrounding part of the nucleolus is visible using standard or preferential staining methods and also as a network within the nucleolus (Fig. 1b) . The global amount of intra-nucleolar chromatin is probably low since by light microscopy, DNA staining by DAPI excludes the nucleolus.
It has become apparent that nucleoli of diVerent cell types exhibit a variable number of FCs of diVerent sizes, with an inverse proportion between size and number (Hozak et al. 1989; Pébusque and Seïte 1981) . Generally cells with a high rate of ribosome biogenesis possess numerous small FCs. On the contrary, cells with greatly reduced metabolic and transcription activities, present small nucleoli with one large-sized FC such as in lymphocytes and in inactive mammalian neurons (Hozàk et al. 1994; Lafarga et al. 1989 ). In the more active neurons, one giant FC (GFC) of 1-2 m is observed together with small FCs (Fig. 1c, d) . It was demonstrated that the GFC is enriched in the upstream binding factor, the UBF transcription factor, in a small ubiquitin-like modiWer (SUMO)-1 and Ubc9 but lack ubiquitin-proteasome and 20S proteasome (Casafont et al. 2007 ). However, the possibility that only one FC might play a role in storage and become a GFC during intense nucleolar activity is still an open question.
It is also remarkable that the tripartite nucleolar organization is not general since the nucleoli of Drosophila and insects lack FCs (Knibiehler et al. 1982; Knibiehler et al. 1984) . It has been proposed that this diVerence in organization could be linked to the evolution of the rDNAs, in particular to the size of the intergenic sequences (Thiry and Lafontaine 2005) .
The localization of the nucleolar machineries is related to their function in the production of the small and large ribosome subunits. These Wndings have led to assigning speciWc functions to speciWc compartments of the nucleolus. Nascent transcripts appear at the junction between the FCs and DFC and accumulate in the DFC (Cmarko et al. 2000; Guillot et al. 2005; Hozàk et al. 1994; Puvion-Dutilleul et al. 1997; Shaw and Jordan 1995) . This was recently conWrmed in the GFC since no transcripts can be detected in these large structures (Casafont et al. 2007 ). Processing of the 47S pre-rRNA starts at the site of transcription in the DFC (Cmarko et al. 2000) and continues during the intranucleolar migration of the RNA towards the GC. The nucleolar proteins that participate in the early stages of rRNA processing, localize in the DFC, such as Wbrillarin and nucleolin along with the U3 snoRNAs (Biggiogera et al. 1989; Ginisty et al. 1998; Ochs et al. 1985b; Puvion-Dutilleul et al. 1991) , whereas proteins B23/NPM (nucleo- Fig. 1 The nucleolus of mammalian cells as seen by electron microscopy. a In the human HeLa cell, the three main nucleolar components are visible in a section of material Wxed in glutaraldehyde and osmium tetroxyde, embedded in Epon and the section contrasted with uranyl acetate and lead citrate. FCs of diVerent sizes are visible and the largest is indicated by an asterisk. The FCs are surrounded by the DFC and are embedded in the GC. b Preferential contrast of DNA using NAMA-Ur staining in a PtK1 cell (courtesy J. Gébrane-Younès). The nucleolus is the gray structure surrounded by highly contrasted chromatin (arrow). Some chromatin Wlaments are also visible inside of the nucleolus (Nu). c, d Nucleolus of rat neurones (courtesy M. J. Pébusque) in the day (c), and during the night (d) which is the active period for the nucleolus of the rat. In the nonactive period (c), the nucleolus is reticulated with small FCs (asterisk). In the active period, one giant FC is visible (d, asterisk) . Bar in a: 0.5 m and bars in b, c and d: 1 m phosmin) and PM-Scl 100 (rrp6 in yeast) that are involved in intermediate or later stages of processing have been localized to the GC (Biggiogera et al. 1989; Gautier et al. 1994) . Recent advances in the isolation of large RNP complexes by tandem aYnity puriWcation and the characterization of their constituents demonstrated that two largely independent processing machineries exist in yeast nucleoli, the SSU processome (Dragon et al. 2002; Grandi et al. 2002) and the LSU processing/assembly factors (Raué 2004) . The SSU/90S processome is localized in the DFC and most of the 60S processing occurs in the GC. There is no particular domain characterized in the GC corresponding to the 43S subunit. This is most probably due to the limited events of 40S processing in the GC since the last step of processing occurs in the cytoplasm. In conclusion it seems that in the nucleoli, the vectorial distribution of the machineries successively involved in ribosome biogenesis correlates with the diVerent processing steps of the biogenesis of the ribosome subunits.
When ribosome biogenesis is active, the conWnement of certain machineries in the FCs, DFC or GC makes it possible to reveal these subnucleolar constituents by immunoXuorescence as illustrated for FCs ( Fig. 2A) , DFC (Fig. 2Ba , b), and GC (Fig. 2Bc, d) . The factors associated with the rDNA transcription machinery are distributed in several foci, most frequently inside the nucleolar volume as illus-trated for UBF. These foci correspond to FCs. A distribution within the network inside the nucleolus is typical of the DFC as demonstrated for Wbrillarin. Labeling of the nucleolar volume excluding small areas contained within the volume is typical of the GC as illustrated for B23/NPM. These labeling patterns (FCs, DFC, GC) in the nucleoli provide a good indication of the step of ribosome biogenesis concerned and also reveal the blockage of ribosome biogenesis when this organization is disturbed (see below).
Transient association of functionally related components appears necessary to generate a morphologically deWned nucleolus with its three distinct components, thereby maintaining the nucleolus in its usual organization. This suggests that such an organization results from the activity of ribosome biogenesis. Indeed nucleolar reorganization is induced when ribosome biogenesis is impaired either by inhibiting rDNA transcription, or inhibiting rRNA processing and/or transport.
Nucleolar segregation is observed upon rDNA transcriptional arrest either in physiological conditions or induced Fig. 2 The subnucleolar constituents revealed by Xuorescence microscopy. (A) The rDNA transcription machinery, illustrated by UBF labeling, is localized in several foci corresponding to FCs in HeLa cells (a). rDNA transcription sites detected by in situ BrUTP incorporation (b), mainly colocalize with UBF as seen by the merge (c). The nucleus is visualized by Dapi staining (d). (B) HeLa cells expressing either Wbrillarin-GFP fusion or DsRed-B23 fusion. Fibrillarin decorates the DFC (a, b) whereas B23 decorates the GC (c, d). In ActDtreated cells nucleoli are segregated, Wbrillarin localizes in caps (e, f) contrary to B23 that localizes in the central body and outside the caps (g, h). Arrowheads point the caps. Bars: 10 m by low doses of actinomycin D (ActD). The segregation of nucleoli is characterized by the separation of the nucleolar components that remain close to each other but no longer intermingle ( Fig. 2Be -h) (for reviews see Hadjiolov 1985; Hernandez-Verdun and Junéra 1995; Scheer and Benavente 1990) . The eVect of ActD on nucleolar organization follows sequential changes: Wrst the Wbrillar components (FCs and DFC) condense and migrate towards the periphery of the nucleolus, after which the nucleolar components segregate to Wnally form a central body associated with caps (Hadjiolova et al. 1995) . In the caps are several proteins related to the RNA polymerase (pol) I transcription machinery such as UBF, close to Wbrillarin-containing caps. In the central body are proteins derived from the GC, some of which are progressively released, such as PM-Scl 100. It was recently demonstrated that certain nucleolar caps of segregated nucleoli could recruit factors involved in mRNA splicing. In this case, localization is induced by inhibition of both RNA pol I (rRNA transcription arrest) and RNA pol II (mRNA transcription arrest) (Shav-Tal et al. 2005 ). This is not observed when only RNA pol I is inhibited indicating that the composition of a segregated nucleolus can be more complex when induced by general transcription inhibition.
One question that remains unanswered is how nucleolar components continue to be maintained in segregated nucleoli in spite of the absence of transcription or pre-rRNA processing. Nucleolar proteins may still be capable of forming complexes during inhibition of transcription, but why these complexes remain juxtaposed is presently unknown. Recently, it was reported that re-localization of proteins in speciWc caps of segregated nucleoli (after inhibition of RNA pol I and II transcription) is an energy-dependent repositioning process that requires active metabolism of the cells (Shav-Tal et al. 2005 ) most probably also ATP and GTP.
A clue to the question of rRNA degradation in the nucleolus was recently proposed in yeast: a surveillance pathway that eliminates defective 60S pre-ribosomal subunits after addition of poly(A) tails was described (LaCava et al. 2005) . RNA degradation appears to occur preferentially within a subnucleolar structure, the No-body, and is mediated by the exosome (Dez et al. 2006) . Similarly, when the nuclear protein of the exosome rrp6 was deleted, poly(A) rRNAs and poly(A) U14 snoRNAs colocalized in one focus with Nop1 (Wbrillarin in human), most probably the Nobody (Carneiro et al. 2007 ). This body is distinct from the nucleolar body that functions in snoRNA maturation in yeast and could be a compartment where polyadenylation and degradation of nucleolar RNAs take place. This compartmentation would promote eYcient recognition of rRNAs in view of degradation by the exosome (Carneiro et al. 2007 ). During nucleolar segregation induced by ActD in human cells, rRNAs are degraded. However, the formation of one focus containing PM-Scl 100 has not been described; it could be either an early event that was not carefully examined, or rRNA degradation could be diVerent in yeast and mammalian cells.
Disconnection between rDNA transcription sites and the late rRNA processing proteins can be induced either by kinase inhibitors or by modiWcations of snoRNA domains (Chan et al. 1996; Colau et al. 2004; David-Pfeuty et al. 2001; Rubbi and Milner 2003; Sirri et al. 2002) . The separation of the DFC and GC can be reversed by removal of a CK2 inhibitor, restoring nucleolar organization. The CK2 kinase is known to phosphorylate several nucleolar proteins (Meggio and Pinna 2003) . We postulate that the connection between DFC and GC is controlled at least in part by phosphorylation of these proteins. This hypothesis was veriWed for B23 by mutation of the major site of CK2 phosphorylation (Louvet et al. 2006 ). In conclusion, the rRNA processing proteins can be disconnected from the rRNA transcription sites indicating that rRNA transcripts are not suYcient to attract the processing proteins in these conditions. The dynamics of nucleolar reformation and the connection between DFC and GC is ATP/GTP dependent, sensitive to temperature, and is CK2-driven.
The analysis in living cells of intranuclear dynamics has recently become possible using Xuorescent fusion proteins. Time-lapse videomicroscopy can track the movement of large Xuorescent complexes in the cell, and Xuorescent recovery after photobleaching (FRAP) can measure the intracellular mobility or the residency time of Xuorescent proteins (Lippincott-Schwartz et al. 2001) . Inverse FRAP (iFRAP) quantiWes the loss of Xuorescence of the region of interest (ROI) after complete bleaching outside this region (Dundr et al. 2004 ). This constitutes a direct evaluation of the residency time of the proteins in the ROI. Another approach is the use of photoactivatable GFP (PA-GFP) to follow the traYc of the activated proteins (Patterson and Lippincott-Schwartz 2002) . This process is similar to pulsechase experiments since it makes it possible to follow a pool of labeled proteins starting at time zero. These technologies applied to nuclear dynamics have introduced new dimensions and unexpected concepts concerning nuclear functional compartmentation. The mobility of several GFPtagged nuclear proteins (nucleolar proteins, histones, DNA binding proteins, transcription factors, splicing factors, nuclear receptors) has been estimated by FRAP and the recovery of Xuorescence was slower than predicted for isolated diVusing proteins of similar size. FRAP recovery rates change with inhibition of transcription, decreased temperature and depletion of ATP indicating that recovery is correlated with nuclear activity.
It was demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm (Phair and Misteli 2000) . The recovery curve of GFP-Wbrillarin (DFC marker) in the nucleolus reached a plateau, 60 s after bleaching and the plateau indicated an immobile fraction of »15%. The diVusion coeYcient of Wbrillarin (estimated between 0.02 and 0.046 m 2 s ¡1 ) was 10 times lower in the nucleolus than in the nucleoplasm (Chen and Huang 2001; Phair and Misteli 2000; Snaar et al. 2000) . This value is proposed to reXect the time of residency of Wbrillarin engaged in nucleolar activity, and could explain the fact that the time of residency of Wbrillarin is shorter in the Cajal body than in the nucleolus (Dundr et al. 2004 ). The nucleolar proteins engaged in rRNA transcription and processing (respectively UBF, B23, Nop52, nucleolin and Rpp29) also move with rapid recovery rates in the nucleolus as does Wbrillarin (Chen and Huang 2001; Louvet et al. 2005) . Conversely the recovery rates of ribosomal proteins are slow (»3 times slower than nucleolar proteins). This could reXect a slower mechanism for ribosome protein assembly compared with transcription and processing (Chen and Huang 2001) , or alternatively, more stable associations of ribosomal proteins with the pre-rRNAs.
B23 (also designated NPM) is a multifunctional protein, abundant in the GC of the nucleolus that undergoes diVerent phosphorylation events during the cell cycle. It was recently demonstrated by FRAP that the kinetics of B23 depends on its phosphorylation status (Negi and Olson 2006) . During interphase, the half-time (t 1/2 ) recovery of B23 is 22 s in nucleoli but when the CK2 phosphorylation site is mutated (S125A) the t 1/2 increases to 44 s, and when a mutant mimicking phosphorylation charges of the four sites of mitotic CDK1 phosphorylation, the t 1/2 decreases to 12 s. This could indicate that the S125A-B23 protein has a higher aYnity for the nucleolar components (Negi and Olson 2006) . Alternatively this could correspond to a decreased turner-over in the nucleolar complexes in correlation with the disconnection of the DFC and GC occurring by overexpression of S125A-B23 (Louvet et al. 2006) . Overexpression during interphase of B23 mimicking four sites of mitotic phosphorylation increased the mobility of B23. It is tempting to propose that this results from a defect of aYnity for rRNAs of these B23s as demonstrated for mitotic phosphorylation of B23 (Okuwaki et al. 2002) .
Inhibition of pol I transcription by ActD does not prevent traYc of nucleolar proteins. However, if the diVusion coeYcient of nucleolar proteins in the nucleoplasm is similar for active and repressed pol I transcription, the traYc in segregated nucleoli changes diVerently for diVerent nucleolar components. TraYc of UBF in the nucleolus is decreased by ActD, whereas it is similar for nucleolin or increased for ribosomal proteins (Chen and Huang 2001) .
In contrast to the well-deWned nucleolar structures visible by EM, all the nucleolar proteins involved in ribosome biogenesis that have been examined, cycle between the nucleolus and the nucleoplasm in interphase cells. To summarize, it is now established that rapid diVusion of nucleolar proteins occurs in the nucleoplasm and recruitment to the nucleolus is permanent. Moreover, the diVerence in kinetics of several proteins shared between the nucleolus and the Cajal body suggests the existence of compartment-speciWc retention (Dundr et al. 2004 ).
To be localized or retained within the nucleolus In eukaryotic cells, once imported or diVused into the nucleus, some proteins distribute throughout the nucleoplasm and others are targeted to speciWc nuclear compartments such as nucleoli. Proteomic analyses revealed that at least 700 proteins are localized in nucleoli (Andersen et al. 2002 (Andersen et al. , 2005 Leung et al. 2003) . Whereas the rules and signals that govern the nuclear localization and nuclear export of proteins are now well deWned, those concerning nucleolar localization are still debated.
Contrary to the nuclear localization signals (NLSs), nucleolar localization signals or sequences (NoLSs) are not well characterized. Although several NoLSs have been described, no obvious consensus sequence has emerged. Nevertheless all NoLSs reported for nucleolus localizing virus proteins, such as HIV-1 Rev (Kubota et al. 1989 ), HIV-1 Tat (Dang and Lee 1989) and human T-cell leukaemia virus type 1 Rex (HTLV-1 Rex) (Siomi et al. 1988) , and for cellular proteins such as the nucleolar protein p120 , Survivin-deltaEx3 (Song and Wu 2005) and HSP70 (Dang and Lee 1989) are rich in basic residues. The capacity of numerous proteins to adopt nucleolar localization has been correlated with interaction of these proteins with B23. Owing to the ability of numerous nucleolar proteins to interact with B23 and because this major nucleolar protein shuttles constantly between the nucleus and the cytoplasm (Borer et al. 1989) , it was frequently suggested that B23 might be a transporter for nucleolar proteins possessing a NoLS (Fankhauser et al. 1991; Li 1997; Valdez et al. 1994) . Even if this tempting hypothesis has never been demonstrated, recent results obtained using stable U2OS-derived cell lines with reduced B23 expression levels showed that the nucleolar localization of ARF is linked to B23 (Korgaonkar et al. 2005) . Indeed, reduced expression of B23 induced a partial delocalization of ARF from nucleoli to nucleoplasm. The authors therefore concluded that B23 targets ARF to nucleoli in a dose-dependent manner. Nevertheless, this result does not allow discriminating between a role for B23 in the transport of ARF from nucleoplasm to nucleoli and/or in the retention of ARF in nucleoli.
A NoLS, i.e. a sequence essential for nucleolar localization, is most probably a sequence involved in nucleolar retention by interacting with a nucleolar molecule such as B23 (Lechertier et al. 2007 ). Indeed, recent analyses of the intranuclear dynamics of proteins in living cells revealed that nuclear proteins could diVuse within the nucleoplasm (Phair and Misteli 2000; Sprague and McNally 2005) . As for the nucleolus, it was demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in a continuous exchange with the nucleoplasm (Chen and Huang 2001; Dundr et al. 2004; Phair and Misteli 2000; Snaar et al. 2000) . There probably exist compartment-speciWc retention mechanisms for proteins in nuclear bodies, implying that the residency time of a particular molecule in a given nuclear body depends on its speciWc interactions (Misteli 2001) . In support of this possibility, we have recently shown that the fusion of a B23-interacting sequence with Wbrillarin makes it possible to re-localize Wbrillarin from the DFC to the GC of nucleoli where B23 is mainly localized (Lechertier et al. 2007) . Similarly, by fusing the B23-interacting sequence to MafG (part of the nuclear transcription factor NF-E2 composed of both MafG and p45), NF-E2 is redirected from the nucleoplasm to the GC. Therefore, interactions most probably govern the nuclear distribution of proteins and a NoLS is very likely a nucleolar molecule-interacting sequence.
However, nucleolar localization of a protein is most probably governed by several factors and the presence of a NoLS in its sequence is not suYcient to predict nucleolar localization of the protein. In particular, a nucleolar protein must Wrst be localized in the nucleus, and consequently all mechanisms that interfere with nuclear import and/or nuclear export of a nucleolar protein will modify its localization at the steady state. A good illustration is provided by the major nucleolar protein, B23. This multifunctional protein is normally mainly located in the GC of nucleoli but exhibits an aberrant cytoplasmic localization in one-third of acute myeloid leukemias due to mutations in its C-terminal coding exon that causes a frameshift and the formation of an additional CRM1-dependent nuclear export signal (NES) (Mariano et al. 2006) . Another example showing the diYculty encountered in predicting nucleolar localization is provided by the box C/D snoRNPs: it seems clear that the nucleolar localization of box C/D snoRNPs is linked to their biogenesis (Verheggen et al. 2001; Watkins et al. 2002) . Indeed, by modifying the conserved stem II of the box C/D motif present in the U14 snoRNA, both the spe-ciWc assembly of the box C/D snoRNP and nucleolar localization are lost (Watkins et al. 2002) . Moreover, genetic depletion of one of the four core proteins, namely 15.5kD, Nop56, Nop58 and Wbrillarin, also inhibits the nucleolar localization of box C/D snoRNPs (Verheggen et al. 2001 ). However, targeting of box C/D snoRNPs to nucleoli is not yet fully understood. Indeed, unexpectedly two nuclear export factors, PHAX and CRM1 appear to be stably associated with the U3 pre-snoRNPs (Boulon et al. 2004; Watkins et al. 2004 ). Boulon and coworkers proposed that U3 precursors bind PHAX, which targets the complex to the Cajal body, and that subsequently CRM1 further targets the U3 complexes to the nucleolus. Even if PHAX and CRM1 play an important role in the transport of box C/D snoRNPs to the nucleus, the possibility that these proteins may also function in the nuclear export of snoRNPs cannot be excluded (Watkins et al. 2004 ). This possibility is reinforced by a recent study showing that in addition to nuclear export factors, the nuclear import factor Snurportin 1 is involved in U8 box C/D snoRNP biogenesis (Watkins et al. 2007) . Nucleolar localization of the components of the box C/D snoRNPs would therefore depend on the biogenesis of the box C/D snoRNP complexes, which would imply nuclear export.
rDNA transcription machinery rDNAs are found in multiple, tandem, head-to-tail arrayed copies in the nucleoli of eukaryotic cells (Hadjiolov 1985) . In mitotic human cells, rDNA clusters are localized on the short arm of the Wve pairs of chromosomes 13, 14, 15, 21 and 22 and are termed NORs. Each rDNA unit consists of a transcribed sequence and an external non-transcribed spacer (Hadjiolov 1985; Liau and Perry 1969) in which all the sequences necessary for proper RNA pol I transcription such as proximal promoters, spacer promoters and terminators are located (Hadjiolov 1985) . In the rDNA promoter two important elements have been described, a CORE element and an upstream control element (UCE) (Haltiner et al. 1986; Windle and Sollner-Webb 1986; Xie et al. 1992 ) that function synergistically to recruit a transcriptionally competent RNA pol I complex. This complex contains in addition to RNA pol I, the upstream binding factor (UBF) (Pikaard et al. 1989; Voit et al. 1992) , the selectivity factor protein complex SL1 (Learned et al. 1985 ) also called TIF-1B in mouse cells (Clos et al. 1986 ), consisting of the TATA-binding protein (TBP) and four transcription activating factors [TAF I s110, 63, 48 and 41 (Comai et al. 1994; Gorski et al. 2007; Zomerdijk et al. 1994) ], the transcription initiation factor TIF-IA, the mouse homolog of Rrn3p (Bodem et al. 2000; MooreWeld et al. 2000) and the transcription termination factor TTF-1 (Bartsch et al. 1988 ). The UBF containing HMG boxes (Bachvarov and Moss 1991; Jantzen et al. 1990 ) that confer a high aYnity for DNA structures plays an architectural role on the rDNA promoter (Mais et al. 2005) . It was proposed that UBF activates rDNA transcription because it stabilizes binding of SL1 required to recruit the initiation-competent subfraction of RNA pol I. This recruitment is achieved by interaction of UBF with the RNA pol I-associated factor PAF53 (Schnapp et al. 1994) , and by interaction of SL1 with TIF-1A/ Rrn3p (Miller et al. 2001) . TIF-1A/Rrn3p interacts also with the RPA43 subunit of RNA pol I and thus facilitates linking between RNA pol I and SL1 complexes (Peyroche et al. 2000; Yuan et al. 2002) . Following initiation, TIF-1A/ Rrn3p is released and can associate with another preinitiation complex. Recycling of TIF-1A/Rrn3p requires a posttranslational phosphorylation event that appears to play a role in its initiation activity (Cavanaugh et al. 2002; Zhao et al. 2003) . Moreover, it was proposed that TTF-1 is not only involved in termination of transcription in cooperation with the release factor PTRF (Jansa and Grummt 1999), but also in the remodeling of ribosomal chromatin by recruiting ATP-dependent remodeling factors to the rDNA promoter (Längst et al. 1997 ). The nucleolar remodeling complex (NoRC) (Strohner et al. 2001) , which acts in repression at the rDNA promoter level (Li et al. 2006; Santoro et al. 2002) , and the transcription activator CSB (Cockayne syndrome group B protein), a DNA-dependent ATPase, interact with TTF-1 (Yuan et al. 2007 ). The Wnding that TTF-1 interacts with both CSB and NoRC suggests that competitive recruitment of CSB and NoRC may determine the epigenetic state of the rDNA.
It is now established that the presence of a fully active nucleolus depends on cell cycle regulators. rDNA transcription is maximum in the S and G2 phases, silent in mitosis, and slowly recovers in G1. Post-translational mod-iWcations of the RNA pol I machinery are required for the formation of a productive preinitiation complex. The phosphorylation status of several components of the RNA pol I machinery can modify the activity and interactions of these proteins and thus can modulate rDNA transcription during the cell cycle. Concerning silencing of rDNA transcription during mitosis, it is well established that some components of the rDNA transcription machinery such as SL1 (Heix et al. 1998 ) and TTF-1 (Sirri et al. 1999) , are mitotically phosphorylated by CDK1-cyclin B. As shown in vitro, CDK1-cyclin B-mediated phosphorylation of SL1 abrogates its transcriptional activity (Heix et al. 1998 ). Moreover CDK1-cyclin B is necessary not only to establish repression but also to maintain it from prophase to telophase. Indeed, in vivo inhibition of CDK1-cyclin B leads to dephosphorylation of the mitotically phosphorylated forms of components of the RNA pol I machinery and restores rDNA transcription in mitotic cells (Sirri et al. 2000) . On the other hand, rDNA transcription also appears regulated by CDK(s) during interphase: the increase of rDNA transcription during G1 progression depends on phosphorylation of UBF by G1-speciWc CDK-cyclin complexes (Voit et al. 1999) , and CDK inhibitor treatments partially inhibit rDNA transcription in interphase cells (Sirri et al. 2000) . ModiWcations of the phosphorylation status of UBF and/or TAF I 110 aVect the interactions between UBF and SL1 necessary for recruitment of RNA pol I (Zhai and Comai 1999) . In addition to phosphorylation, it has been speculated that acetyltransferases might also regulate the activity of RNA pol I transcription factors. Indeed, two studies have demonstrated that UBF and one of the SL1 subunits are acetylated in vivo (Muth et al. 2001; Pelletier et al. 2000) . Functional studies indicated that acetylated UBF is transcriptionally more active than deacetylated UBF. However, acetylation of UBF does not aVect its DNA binding activity as shown for other transcription factors, and it is unclear how this post-transcriptional modiWcation modulates UBF activity. The TAF I 68 subunit of SL1 is speciWcally acetylated by recruitment of PCAF (p300/CBP associated factor) to the rDNA promoter. In vitro analyses indicate that acetylation of TAF I 68 is likely to increase the activity of SL1 facilitating interaction of the complex with DNA. Sirtuins, the human homologues of the yeast Sir2 (silent information regulator) with NAD-dependent deacetylase and ADP-ribosyltranferase activity, have recently been implicated in the regulation of the RNA pol I machinery. In particular, nuclear sirtuin1 deacetylates TAF I 68 and represses RNA pol I transcription in vitro (Muth et al. 2001) . Conversely, the nucleolar sir-tuin7 is described as activator of rDNA transcription by increasing RNA pol I recruitment to the rDNA, but no substrates of such activity have as yet been identiWed (Ford et al. 2006) . Additional in vivo approaches are necessary to better understand the role of sirtuins in the regulation of rDNA transcription.
SUMO modiWcation is reported to inXuence the assembly of transcription factors on promoters and the recruitment of chromatin-modifying enzymes, and is often associated with transcriptional repression (Gill 2004) . Recently, the colocalization of SUMO-1 and UBF in the GFC (Casafont et al. 2007 ) of neuronal cells and the nucle-olar localization of the sentrin/SUMO-speciWc proteases, SENP3 and SENP5 (Gong and Yeh 2006; Nishida et al. 2000) suggest a potential role of sumoylation on the regulation of rDNA transcription. Further studies of the identiWcation of sumoylated nucleolar transcription factors will be necessary to verify this possibility.
In higher eukaryotic cells at the beginning of mitosis when rDNA transcription is repressed, the nucleoli disassemble and are no longer observed throughout mitosis. Conversely nucleoli assemble at the exit from mitosis concomitantly with restoration of rDNA transcription and are functionally active throughout interphase.
In late prophase when mitotic repression of rDNA transcription occurs, the rDNA transcription machinery remains associated with rDNAs in the NORs as revealed by the analysis of diVerent components at the steady state (Roussel et al. , 1996 Sirri et al. 1999 ). Nevertheless, more recent quantitative kinetic analyses have revealed that some RNA pol I subunits, including RPA39, RPA16 and RPA194, transiently dissociate from the NORs during metaphase and reappear in anaphase (Chen et al. 2005; Leung et al. 2004) . As for the mechanism that governs disassembly of nucleoli in prophase, it may be assumed that it is linked to repression of rDNA transcription, most probably caused by CDK1-cyclin B-directed phosphorylation of components of the rDNA transcription machinery (Heix et al. 1998; Sirri et al. 1999) .
At the beginning of prophase, the components of the pre-rRNA processing machinery do not remain in the vicinity of the rDNAs (Gautier et al. 1992 ) but become partially distributed over the surface of all the chromosomes (reviewed in Hernandez-Verdun et al. 1993) . The nucleolar proteins that relocate to the chromosome periphery are components of the DFC and GC of the active nucleolus. In living cells, nucleolar proteins tagged with GFP are concentrated around the chromosomes during mitosis and migrate with the chromosomes (Savino et al. 2001) . However, the mechanisms maintaining interactions of nucleolar processing proteins with chromosomes during mitosis have not been characterized. The colocalization of the diVerent factors involved in pre-rRNA processing suggests that processing complexes are at least to some extent maintained during mitosis. It is as yet unknown whether migration of the nucleolar processing proteins occurring at the onset of mitosis (Fan and Penman 1971) takes place as a consequence of the arrest of pre-rRNA synthesis or whether it is also regulated. Indeed, it is noticeable that (1) during prophase, the components of the rRNA processing machinery appear to be delocalized before total repression of rDNA transcription occurs, and (2) the most recently synthesized pre-rRNAs accumulate as partially processed 45S pre-rRNAs (Dousset et al. 2000) suggesting that total repression of pre-rRNA processing could occur prior to total repression of rDNA transcription. These observations therefore raise the possibility that rDNA transcription and pre-rRNA processing are both repressed during prophase by distinct mechanisms.
Nucleoli assemble at the exit from mitosis concomitantly with restoration of rDNA transcription at the level of competent NORs (Roussel et al. 1996) . Until recently it was admitted that transcriptionally active rDNAs, serving as nucleation sites, possessed by themselves the ability to organize the nucleoli (Scheer and Weisenberger 1994) . Results obtained in the laboratory showed that (1) reactivation of rDNA transcription in mitotic cells does not lead to the formation of nucleoli (Sirri et al. 2000) , (2) initiation of nucleolar assembly occurs independently of rDNA transcription (Dousset et al. 2000) , and (3) at the exit from mitosis nucleologenesis is impaired in the presence of a CDK inhibitor even if rDNAs are actively transcribed (Sirri et al. 2002) . Consequently, the formation of functional nucleoli at the exit from mitosis is not governed solely by the resumption of rDNA transcription. Based on previous studies (Sirri et al. 2000 (Sirri et al. , 2002 , we propose that the formation of nucleoli is a process regulated by CDK(s) at two levels: resumption of rDNA transcription but also restoration of rRNA processing.
In anaphase, early and late processing proteins (respectively Wbrillarin, and Bop1, B23, Nop52) are homogeneously distributed around the chromosomes. During telophase and early G1, along the translocation pathway between chromosome periphery and transcription sites, processing proteins concentrate in foci designated prenucleolar bodies (PNBs), Wrst described in plant cells (Stevens 1965) . PNB formation is a general phenomenon occurring during the recruitment of the nucleolar processing proteins at exit from mitosis (Angelier et al. 2005; Azum-Gélade et al. 1994; Dundr et al. 2000; Jiménez-Garcia et al. 1994; Ochs et al. 1985a; Savino et al. 2001 ). This appears to be a cell cycle regulated process since when the nucleolar function is established during interphase, recruitment of processing proteins is not associated with PNB formation. Inactivation of CDK1-cyclin B occurring at the end of mitosis induces the Wrst events of nucleologenesis. Strikingly, Wbrillarin concentrates in PNBs and rDNA clusters when decrease in CDK1-cyclin B activity overcomes the mitotic repression of RNA pol I transcription (Clute and Pines 1999) , while Nop52 and other GC proteins are recruited later on transcription sites. This late recruitment is under the control of cyclin-dependent kinases since CDK inhibitors block this process (Sirri et al. 2002) . Thus, it seems that recruitment of the processing machinery at the time of nucleolar assembly is a regulated process most probably dependent on cell cycle progression. This provides a physiological situation to investigate the formation, control and dynamics of nuclear bodies.
The dynamics of the processing nucleolar proteins was analyzed at the transition mitosis/interphase using rapid time-lapse video microscopy (Fig. 3) . The Wrst detectable assembly of proteins in foci occurred on the surface of the chromosome during telophase (Savino et al. 2001 ), followed by the progressive delivery of proteins to nucleoli ensured by progressive and sequential release of proteins from PNBs (Dundr et al. 2000) . Based on the observations of diVerent Wxed cells, it was concluded that the early processing proteins are recruited Wrst on transcription sites while the majority of the late processing proteins are still in PNBs (Fomproix et al. 1998; Savino et al. 1999 ). This sequence of events was conWrmed in living HeLa cells. Fibrillarin resides brieXy in PNBs (»15 min) before recruitment to the nucleolus, while Nop52 is maintained longer in PNBs (»80 min) (Savino et al. 2001) . The relative dynamics of early and late rRNA processing proteins at the time of PNB formation was examined using coexpression of GFP-Wbrillarin and DsRed-B23 (Angelier et al. 2005) . Once near the poles, 1-2 min after the onset of telophase, numerous bright Xuorescent foci containing both GFP-Wbrillarin and DsRed-B23 appeared almost simultaneously. For about 10 min, the relative amount of B23 in foci was Wve to six times higher than that of the dispersed proteins whereas the amount of Wbrillarin in the same foci was three to four times higher than that of dispersed proteins. Subsequently, Wbrillarin was released while B23 was still present in the foci. This clearly illustrates the presence of the two types of nucleolar processing proteins in the same PNBs and suggests diVerential sorting of these proteins. Conversely in the same conditions, simi-lar dynamics and Xows of GFP-Nop52 and DsRed-B23 were observed. Thus the processing proteins passed through the same PNBs and were released simultaneously suggesting that these proteins could form complexes in PNBs.
Time-lapse analysis of Xuorescence resonance energy transfer (FRET) was chosen to determine whether nucleolar processing proteins interact along the recruitment pathway. The apparatus used to determine FRET performs tdFLIM (time domain Xuorescence lifetime imaging microscopy) by the time and space-correlated single-photon counting method (Emiliani et al. 2003) . This technique directly yields the picosecond time-resolved Xuorescence decay for every pixel by counting and sampling single emitted photons. Positive FRET between GFP-Nop52 and DsRed-B23 in nucleoli indicates that the distance and most probably the interactions between the proteins can be evaluated by this approach (Angelier et al. 2005) . Since it is possible to detect FRET between B23 and Nop52 in nucleoli, FRET was tracked during the recruitment of these proteins into nucleoli from anaphase to early G1. FRET was never detected during anaphase at the periphery of the chromosomes whereas it was registered in 20% of the PNBs at the beginning of telophase, in about 40% at the end of telophase, and in 55% in early G1. Thus, interaction between GFP-Nop52 and DsRed-B23 was established progressively in PNBs, as the number of PNBs exhibiting FRET increased. Such data indicate that Nop52 and B23 did not interact until they were recruited in PNBs. It is noteworthy that a given PNB can alternatively present or not present FRET. Based on the behavior of these two proteins, one possibility is that late rRNA processing proteins already interact in PNBs. Were this to be conWrmed for other rRNA processing complexes, PNBs could be proposed as assembly platforms of processing complexes at this step of the cell cycle. It would be very interesting to establish whether this role can be extended to the early rRNA processing machinery (Angelier et al. 2005) .
In conclusion, assembly of the nucleolus requires reactivation of the rDNA transcription machinery, and also recruitment and reactivation of the pre-rRNA processing Fig. 3 At the exit from mitosis, the dynamics of DsRed-B23 is followed in living cell. In telophase (0 min), the B23 signal is visible in small foci. These foci corresponding to PNBs are clearly visible 20 min later. The B23-containing PNBs are distributed in the nucleoplasm and B23 is progressively recruited in the incipient nucleolus (40 min). Nu nucleolus machinery. Indeed cells exiting from mitosis in the presence of a CDK inhibitor exhibit neither relocalization of the late pre-rRNA processing components from PNBs to rDNA transcription sites, resumption of proper rRNA processing, nor formation of functional nucleoli.
The link between cell proliferation, cancer and nucleolar activity has been well established during the past several decades (more than 5,000 references). Half of the studies related to the nucleolus and cancer are dedicated to the prognostic value of AgNOR staining, a technique revealing the amount of nucleolar proteins. The aim of this technique is to evaluate the proliferation potential of cancer cells by measuring nucleolar activity. B23, nucleolin, UBF and subunits of RNA pol I were found to be the argyrophilic proteins responsible for the silver-staining properties of nucleolar structures (Roussel et al. 1992; Roussel and Hernandez-Verdun 1994) . In interphase cells, the amount of major AgNOR proteins, B23 and nucleolin, is high in S-G 2 and low in G 1 phases and thus a higher value of AgNOR corresponds to actively cycling cells (Sirri et al. 1997) . Standardization of the AgNOR staining method permits routine application of this technique for clinical purposes. The size of the nucleolus is generally enlarged in cancer cells, and this has been correlated with cell proliferation.
A new Weld of research was recently opened by the discovery that several tumor suppressors and proto-oncogenes aVect the production of ribosomes (Ruggero and PandolW 2003) . rRNA synthesis is enhanced by c-Myc (Arabi et al. 2005) and it was proposed that this stimulation is a key pathway driving cell growth and tumorigenesis (Grandori et al. 2005) . On the contrary, the decrease of ribosome production induces apoptosis in a p53-dependent or independent manner (David-Pfeuty et al. 2001; Pestov et al. 2001) and the disruption of the nucleolus mediates p53 stabilization (Rubbi and Milner 2003) . The cross talk between the p53 pathway and the nucleolus is at least in part mediated by localization of Mdm2 in the nucleolus, an E3 ubiquitin ligase involved in p53 degradation. Nucleostemin, a nucleolar protein discovered in stem cells and in cancer cells interacts with p53 McKay 2005, 2002) . It was proposed that nucleostemin might regulate p53 function through shuttling between the nucleolus and the nucleoplasm. The major nucleolar protein B23 is directly implicated in cancer pathogenesis as demonstrated by mutation of the gene in a number of hematological disorders (Grisendi et al. 2005) . Importantly, in acute promyelocytic leukemia, the fusion protein NPM/RAR localizes in the nucleolus indicating a role of this nucleolar protein in this disease (Rego et al. 2006 ).
Within the last few years, increasing evidence has revealed that viruses require the nucleus and in particular the nucleolus to target proteins indispensable for their replication. An increasing number of key proteins from both DNA-and RNA-containing viruses are localized in the nucleolus: viruses of the family Herpesviridae, Adenoviridae, Hepadnaviridae, Retroviridae, Rhabdoviridae, Orthomyxoviridae, Potyviridae, Coronaviridae and Flaviviridae, encode such proteins. Viruses have developed diVerent strategies to facilitate targeting of their proteins to the nucleolus: (1) it was reported that the sequences of certain viral proteins harbor NoLS and NES (Harris and Hope 2000; Hiscox 2007; Kann et al. 2007) . Recently it was demonstrated by mutagenesis that the nucleocapsid (N) protein of infectious bronchitis virus (IBV), presents an 8 amino acid-long motif that functions as NoLS, and is necessary and suYcient for nucleolar retention of the N protein and colocalization with nucleolin and Wbrillarin; the NoLS is required for interaction with cell factors.
(2) Other viral proteins present sequences rich in arginine-lysine (Ghorbel et al. 2006; Reed et al. 2006) known to be nucleolar retention signals; generally, these sequences overlap the NLS.
(3) Some viral proteins that target the nucleolus present motifs with aYnity for double-stranded RNA (dsRNA), for RNA binding or for DNA binding (Melen et al. 2007) . (4) Other studies showed that nucleolar localization of viral proteins, is cell cycle-dependent (Cawood et al. 2007 ); using synchronization studies coupled to live cell confocal microscopy, the authors demonstrated that the concentration of N protein in the nucleolus was higher in the G2/M phase than in other phases, and that in this phase the protein was more mobile in the nucleoplasm. In all the cases examined, the viral proteins depend on cell factors to successfully shuttle between the nucleolus and the cytoplasm.
Why must viral proteins target to the nucleolus?
The answer to this question is not clear; however, diVerent authors had reported that such viral proteins are involved both in replication of the viral genome, and in transcriptional and post-transcriptional regulation of viral genome expression (Dang and Lee 1989; Pyper et al. 1998) . For example, some plants viruses are known to encode a protein designated movement protein, responsible for long-distance movement of the viral RNA through the phloem (Ryabov et al. 1999) . Movement strictly depends on the interaction of the viral movement protein with the nucleolus and the Cajal bodies, which contain snRNPs and snoR-NPs (Kim et al. 2007a, b) . The open reading frame (ORF) 3 of Groundnut rosette virus is one such protein; it is Wrst localized in Cajal bodies and forms Cajal body-like struc-tures, it is then localized in the nucleolus when the Cajal body-like structures fuse with the nucleolus, and Wnally it exits to the cytoplasm (Kim et al. 2007b) . Another study showed that this shuttling is indispensable to form the RNPs essential for systemic virus infection (Kim et al. 2007a) . In this process, the interaction of the viral ORF3 with Wbrillarin is absolutely required. Interestingly, silencing of the Wbrillarin gene blocks long-distance movement of the virus but does not aVect virus replication and movement via plasmodesmata. Because the mobility of nucleolar components depends on the interactions and functions of the components (Olson and Dundr 2005) , we suggest that targeting of viral proteins to the nucleolus could help viral protein traYc and diVusion of viral infection.
The activity of the human immunodeWciency virus (HIV)-1 Rev protein is essential for virus replication. Its subcellular localization is nucleolar, but it has the ability to shuttle continuously between the nucleus and the cytoplasm (Felber et al. 1989; Kalland et al. 1994) . Rev possesses both an NES and an NLS; the NLS is associated with importin-as well as with B23 (Fankhauser et al. 1991; Henderson and Percipalle 1997) . Rev-GFP movement in the nucleolus is very slow, implying that it is attached to aYnity binding sites in this subcellular compartment (Daelemans et al. 2004 ). In addition, the transport of Rev from the nucleolus to the cytoplasm can be aVected negatively by NF90, a cellular protein that colocalizes with Rev in the nucleolus (Urcuqui-Inchima et al. 2006) (Fig. 4) . This indicates that the transport of HIV transcripts by Rev to the cytoplasm is a regulated process. Because Rev is concentrated in the nucleolus, it was suggested that the passage of Rev to the nucleolus is an indispensable step for Rev function, and hence for HIV-1 replication. Indeed, based on HIV-1 RNA traYcking through the nucleolus, this organelle is an essential participant of HIV-1 RNA export (Michienzi et al. 2000) .
As discussed above for Rev, it has been shown that the Herpes virus saimiri ORF57 protein is required for nuclear export of viral intronless mRNAs (Boyne and Whitehouse 2006) . In addition, the expression of ORF57 induces nuclear traYcking, which is essential for nuclear export of such RNAs; the human transcription/export protein involved in mRNA export, is redistributed to the nucleolus in the presence of the ORF57 protein. Based on these Wndings, the authors concluded that the nucleolus is required for nuclear export of the viral mRNAs.
What are the consequences for the cells of the passage of viral proteins via the nucleolus?
It is known that all viruses whether with DNA or RNA genomes interfere with the cell cycle, aVecting host-cell functions and increasing the eYciency of virus replication.
The data obtained suggest that targeting of virus proteins to the nucleolus not only facilitates virus replication, but may also be required for pathogenic processes. Recent studies following infection by IBV, revealed a change in the morphology and protein content of the nucleolus . This included an enlarged FC and an increase in protein content; interestingly, the tumor suppressor protein p53, normally localized in the nucleus in virus infected cells, was redistributed mainly in the cytoplasm. The Hepatitis B virus (HBV) core antigen (HBcAg) is responsible for export of the virus with a mature genome (Yuan et al. 1999a, b) . Indeed a change from isoleucine to leucine in position 97 (I97L) of HBcAg causes the cell to release virus particles with immature genomes. HBcAg with a mutation Fig. 4 HIV Rev-GFP and NF90-RFP fusions were expressed in HeLa cells. Both proteins colocalize in nucleoli as seen by the merge. The nucleus is visualized by Dapi staining. Bars: 10 m in position 97 (I97E or I97W) has been detected in the nucleolus colocalizing with nucleolin and B23, and this colocalization was often related with binucleated cells or apoptosis (Ning and Shih 2004) , suggesting that the localization of HBcAg in the nucleolus could perturb cytokinesis. The authors propose that this event may be associated with liver pathogenesis. Some factors expressed by west nile virus (WNV) such as NS2B and NS3 and the WNV capsid (WNVCp) participate in WNV-mediated apoptosis (Oh et al. 2006; Ramanathan et al. 2006) . It is well known that p53 is activated in response to oncogenic or DNA damaging stresses, inducing cell cycle arrest and apoptosis (Harris and Levine 2005) . In normal conditions, HDM2 targets p53 and blocks abnormal accumulation of p53 by HDM2-mediated ubiquitinylation, followed by 26S proteasome-dependent degradation of p53 (Haupt et al. 1997; Kubbutat et al. 1997) . Recently it was demonstrated that WNVCp could bind to and sequester HDM2 in the nucleolus, blocking p53-HDM2 complex formation (Yang et al. 2007 ). This phenomenon causes stabilization of p53 and Bax activation and thereafter apoptosis. In addition, the authors show that WNVCp is able to induce apoptosis-dependent processes, suggesting that the viral protein mediates apoptosis through p53-dependent mechanisms by retention of HDM2 in the nucleolus.
The conclusions are based on the perspectives and the tendency that can be anticipated from the present research in the Weld of the nucleolus.
We propose that in the future, a better understanding of the complexity and variability of ribosome biogenesis will need to be established. For example, the diVerence between the information available in yeast and mammalian cells is of major importance. The diVerent steps of ribosome biogenesis and protein complexes are well characterized in yeast due to easy access of mutants. Similarly, Miller chromatin spreading for electron microscopy in yeast strains carrying mutations reveals the coupling of RNA pol I transcription with rRNA processing (Schneider et al. 2007 ). Additionally, the compaction into SSU processomes of pre-18S ribosomal RNA before cleavage was observed on Miller spreads (Osheim et al. 2004 ). There is presently no comparable information for the mammalian genes. Yet the tendency is to generalize and suppose that the information is similar in the two models. In the future, diVerences will most probably be revealed as well as the complexity of the regulation in diVerentiated cells. Along this line, it was demonstrated that basonuclin, a cell-type-speciWc rDNA regulator transcribes only one subset of rDNAs of a cell (Zhang et al. 2007b ). In such a diVerentiated cell, it remains to be established how the subset of rDNA repeats is selected.
The nucleolus was proposed to be a domain of the sequestration of molecules that normally operate outside this organelle, mainly in the nucleoplasm. Sequestration in the yeast nucleolus of the phosphatase cdc14 and its release into the cytoplasm at anaphase was demonstrated to be a key event in cell cycle progression (for a review see Cockell and Gasser 1999; Guarente 2000; Visintin and Amon 2000) . However, it is important to recall that there is no nucleolus during mitosis in mammalian cells. In mammalian interphase cells, the nucleolus is a domain of retention of molecules related to cell cycle, life span, and apoptosis, and is in particular an actor of the p53-dependent pathway. Recently nucleolar retention of the Hand1 transcription factor was observed in trophoblast stem cells (Martindill et al. 2007 ). Phosphorylation of Hand1 induced nucleolar to nucleoplasm translocation of Hand1 and commitment of stem cells to diVerentiate into giant cells. Hand1 translocation to the nucleoplasm might regulate a crucial step of stem cell diVerentiation into polyploid giant cells but the targets of Hand1 in the nucleoplasm are still undeWned.
The nucleolus is generally surrounded by highly condensed chromatin Wrst described in rat hepatocytes and presently known as heterochromatin. By following the movements of chromosome sequences introduced in diVerent sites in chromosomes of living cells, it was demonstrated that loci at nucleoli periphery and nuclear periphery are less mobile than in other sites. Disruption of the nucleoli by a CK2 inhibitor increases the mobility of the perinucleolar loci. It was proposed that the nucleolus and nuclear periphery could maintain the three-dimensional organization of chromatin in the nucleus (Chubb et al. 2002) . Recently the perinucleolar ring of chromatin was brought to the fore when its role in the maintenance of inactive X (Xi) was demonstrated (Zhang et al. 2007a) . During middle and late S phase, Xi contacts the nucleolar periphery when it is replicated during the cell cycle. It was discovered that the perinucleolar chromatin is enriched in Snf2h, the catalytic subunit of a remodeling complex required for replication of heterochromatin. These observations demonstrate the role of the perinucleolar compartment in maintaining the epigenetic state of Xi (Zhang et al. 2007a) . The presence of inactive rDNA repeats in perinucleolar heterochromatin is known in many plant cells and in Drosophila. It was recently demonstrated that disruption in Drosophila of histone H3K9 methylation, a marker of heterochromatin, induced nucleolar disorganization and decondensation, and disorganization of rDNA repeats (Peng and Karpen 2007) . The authors suggest that condensation of a part of the rDNA copies into heterochromatin could be a general strategy against recombination of these highly repeated genes.
For long, interest concerning the nucleolus was to establish how eYcient ribosome biogenesis occurs and the link of this function with the cell cycle. More recently the eVect of the disruption of ribosome biogenesis appeared very important when it was proposed that the nucleolus is a sensor of stress (Rubbi and Milner 2003) . Indeed disruption of ribosome biogenesis releases ribosomal proteins from the nucleolus that bind to MDM2 and inhibit p53 degradation (Lindstrom et al. 2007) . A connection between ribosomal stress and p53-dependent cell cycle arrest is now proposed (Gilkes et al. 2006) .
Considering the diversity of the recent information gathered on the nucleolus, it is clear that this is a very dynamic and rapidly progressing research area. The most promising aspect is the contribution of new models (pseudo-NORs, Prieto and McStay 2007) , new species (not only yeast), new approaches (Miller spreads using mutants, proteomics) and new questions (for instance the role of siRNAs or antisens RNAs in the activity of the nucleolus). Antidiabetes and Anti-obesity Activity of Lagerstroemia speciosa The leaves of Lagerstroemia speciosa (Lythraceae), a Southeast Asian tree more commonly known as banaba, have been traditionally consumed in various forms by Philippinos for treatment of diabetes and kidney related diseases. In the 1990s, the popularity of this herbal medicine began to attract the attention of scientists worldwide. Since then, researchers have conducted numerous in vitro and in vivo studies that consistently confirmed the antidiabetic activity of banaba. Scientists have identified different components of banaba to be responsible for its activity. Using tumor cells as a cell model, corosolic acid was isolated from the methanol extract of banaba and shown to be an active compound. More recently, a different cell model and the focus on the water soluble fraction of the extract led to the discovery of other compounds. The ellagitannin Lagerstroemin was identified as an effective component of the banaba extract responsible for the activity. In a different approach, using 3T3-L1 adipocytes as a cell model and a glucose uptake assay as the functional screening method, Chen et al. showed that the banaba water extract exhibited an insulin-like glucose transport inducing activity. Coupling HPLC fractionation with a glucose uptake assay, gallotannins were identified in the banaba extract as components responsible for the activity, not corosolic acid. Penta-O-galloyl-glucopyranose (PGG) was identified as the most potent gallotannin. A comparison of published data with results obtained for PGG indicates that PGG has a significantly higher glucose transport stimulatory activity than Lagerstroemin. Chen et al. have also shown that PGG exhibits anti-adipogenic properties in addition to stimulating the glucose uptake in adipocytes. The combination of glucose uptake and anti-adipogenesis activity is not found in the current insulin mimetic drugs and may indicate a great therapeutic potential of PGG. Type 2 diabetes has developed into a worldwide epidemic (1) . Ironically, the dramatic increase in the prevalence of type 2 diabetes can be attributed to the rapid economic development and correlated to changes in lifestyle within the last 50 years. Type 2 diabetes is closely associated with obesity. Up to 90% of the patients in the US with type 2 diabetes are either overweight or obese (2, 3) . It seems likely that the readily available high calorie food and a sedentary life style are major causes for obesity. Obesity contributes to insulin resistance and type 2 diabetes. Reducing obesity and stopping weight gain constitutes a way to slow down the rate of occurrence of type 2 diabetes. Type 2 diabetes is caused by insulin resistance, which is defined as defective insulin signaling and a decreased insulin efficiency to induce glucose transport from the blood into key target cells such as muscle and fat (adipocyte) cells (3) . In general, obesity leads to hyperglycemia, which in turn leads to and exacerbates insulin resistance. Insulin resistance, if not treated, results in hyperinsulinemia and eventually leads to full blown type 2 diabetes (3, 4) . Obesity or excessive adiposity, particularly visceral adiposity, contributes to and worsens insulin resistance (2, 5) . Most antidiabetic drugs are hypoglycemic or anti-hyperglycemic (blood glucose level reducing). However, most of these drugs are, to different extents, weight gain promoting (adipogenic) (6, 7) . Thus, these drugs treat one of the key symptoms of type 2 diabetes, hyperglycemia, but exacerbate the condition of being overweight or obese, one of the leading causes of type 2 diabetes. Therefore, while these drugs are beneficial over the short term, they are not optimal for long term health of type 2 diabetic patient. The most desirable situation would be the development of new types of antidiabetic drugs that are either hypoglycemic or anti-hyperglycemic without the side effect of promoting weight gain (adiposity). Herbal medicines known to be useful in diabetes treatment may be able to lead to compounds with such a combination of ideal therapeutic properties (8) (9) (10) (11) (12) (13) .
Lagerstroemia speciosa (Fig. 1) , also called banaba in the Tagalog language of the Philippines, is a tropical plant found in many parts of Southeast Asia including the Philippines, Vietnam, Malaysia and southern China. It is a tree that can grow as tall as 20 m. Despite growing in several countries, only in the Philippines are the dried and shredded banaba leaves known to be used as a treatment for diabetes and kidney disease. It is not clear if banaba plants grown in different countries are equally effective in the treatment of diabetes.
Garcia published the first research on banaba's insulinlike, hypoglycemic effect as early as 1940 (14) (15) (16) (17) (18) . Later, the popular use of banaba in the Philippines was noticed and led to its introduction in Japan. However, it was not until 50 years after Garcia's first publication that scientific interest in banaba's potential for the treatment of diabetes resurfaced. Scientists from countries including Japan, the Philippines, Korea and the United States are currently studying banaba. Lagerstroemia speciosa (banaba) has become relatively popular in the form of health-promoting tea products in Eastern Asia and the United States.
In 1996, Kakuda et al. (19) studied banaba's antidiabetic activity by preparing water and methanol extracts of the plant. After feeding the extracts to hereditary type 2 diabetic KK-Ay/Ta Jcl mice, they found that food containing either 5% of water extract (BE) or 3% of methanol extract was effective in reducing blood glucose and insulin levels (P50.05) (19) . It is interesting to note that the total cholesterol in treated mice was also significantly reduced, but the plasma triglyceride level remained unchanged (19) .
In a second study by Kakuda's research group, food containing 5% banaba water extract was used to feed female obese KK-Ay/Ta Jcl mice. Obese mice treated with banaba extract had a significantly reduced body weight (10%) compared with control mice fed with a regular diet (20) . No change in food intake was observed (20) . Interestingly, it was also discovered that liver triglyceride content was reduced by more than 40% in the banaba extract-treated mice. In addition, the parametrial adipose tissue was 10% lighter (P50.01) (20) . However, as in the previous antidiabetic activity study of BE in animals (19) , both the identity of the effective component(s) and the mechanism for the activity were not studied. Nevertheless, these two studies clearly demonstrated the in vivo antidiabetic and anti-weight gaining efficacy of the extract.
In 1993, a group of scientists from Hiroshima University used an Ehrlich ascites tumor cell line coupled with a bioassay guided fractionation to screen compounds isolated by HPLC from banaba extract in order to identify the effective antidiabetic component (21) . Corosolic acid (2a-hydroxyursoloic acid) was identified as the effective compound in the methanol extract of banaba leaves in a glucose uptake assay ( Fig. 2A, 21) . However, this result should be considered with caution since the tumor cell line used in this study is a very unusual and unconventional cell line for diabetes studies or antidiabetic compound screening. Furthermore, the result could not explain the discrepancy that both the banaba water extract and the methanol extract were active in antidiabetic and anti-obesity animal studies since corosolic acid only exists in the methanol extract (19) (20) (21) . Realizing the potential problems associated with the cell line selection and assay method, the researchers acknowledged in a later publication that corosolic acid 'could not represent the whole activity of the banaba extract' (22) . Consequently, the researchers switched their cell model from the original tumor cells to a natural cell target of insulin, adipocytes, in order to allow for the 'isolation and the identification of more active compounds using the improved methodology' (22) . After HPLC purification, ellagitannins were identified in the water extract of banaba as the activators of glucose transport in fat cells with a glucose uptake assay (22) . One of the most potent ellagitannins was named 'Lagerstroemin' (Fig. 2, 22) .
In a recent study, the same group reported the activation of the insulin receptor (IR) by Lagerstroemin (Fig. 2, 23) . In this study, Lagerstroemin was able to induce phosphorylation of the b-subunit of IR at 150 mM (23) but the mechanism responsible for IR activation was not found. The researchers speculated that Lagerstroemin could act intracellularly or bind to the insulin receptor (IR) extracellularly.
In 2004, a separate group of researchers from several universities found that glucose transporter 4 (GLUT4) translocation from the intracellular microsomal membrane to the plasma membrane was significantly increased in the muscle cells of mice treated orally with corosolic acid (P50.05, 24). This result is both interesting and puzzling. GLUT4 is the major glucose transporter protein in both muscle and adipocytes and GLUT4 is insulin-responsive (4, 25) . However, since corosolic acid does not possess insulin-like glucose transport stimulatory activity, the process that leads to GLUT4 translocation is not known. The GLUT4 membrane translocation mechanism initiated by corosolic acid as described (24) must be independent of the IR mediated signaling pathway since corosolic acid does not use this pathway for its activity. In 2006, the same group of researchers found that corosolic acid significantly reduces blood glucose levels and plasma insulin levels in KK-Ay diabetic mice (26) . Additionally, this group of researchers published another article in 2006 in which they showed that corosolic acid significantly lowered blood glucose levels at the 90 min mark in an oral glucose tolerance test done on type 2 diabetic patients (27) . However, no statistical difference between the treated group and the control group was found for any other time point during the test (27) .
Our interest in the isolation and identification of antidiabetic compounds from natural sources initiated our investigation of banaba extracts with 3T3-L1 adipocytes as a cell model and a radioactive glucose uptake assay as a screening method for the identification of potential antidiabetic compounds. In our study, we confirmed that the water and methanol extracts of banaba leaves exhibit glucose transport stimulatory activity (28) . In addition, we showed that the activity induced by the extract had a concentration-activity profile similar to that of insulin, suggesting that the activity of banaba extract (BE) might be triggered via an insulin-like mechanism (28) . Furthermore, we demonstrated that the same BE also inhibits adipocyte differentiation (28) . 3T3-L1 preadipocytes treated with methylisobutylxanthine, dexamethasone, and insulin (MDI) and BE did not differentiate into adipocytes as they normally do under the sole influence of MDI. This result indicated that BE inhibits the adipocyte differentiation activity induced by MDI. Unlike the glucose transport stimulation, the anti-differentiation activity is anti-insulin like, since insulin plays an important role in adipocyte differentiation and is a component of MDI ('I' in MDI stands for Insulin). Thus, BE differs from insulin in that it is anti-adipogenic whereas insulin is adipogenic, which is considered to be a negative side effect of insulin.
In order to identify the component(s) in BE responsible for the antidiabetic activity, our first goal was to confirm that corosolic acid was responsible for the glucose transport stimulatory activity. Since tannins comprise up to 40% of the material in the extract, we were interested in separating it from corosolic acid first. To our surprise, after removing tannins by either gelatin or bovine serum albumin tannin precipitations (29), the remaining extract was not able to induce glucose transport. We concluded that the glucose transport activity was caused by the tannin component of the extract, and not corosolic acid (29) . Furthermore, we tested pure corosolic acid and found that it was not able to stimulate glucose transport in our cell model (Fig. 3) . Although we cannot exclude the possibility that corosolic acid may have some antidiabetic activity, we can eliminate corosolic acid from having the insulin-like glucose transport stimulatory activity found in adipocytes. It should be noted that banaba extracts prepared from banaba leaves from different sources may have different chemical compositions, which in turn may lead to different experimental results (our lab used dried banaba leaves from the Philippines).
After testing corosolic acid, we also tested the corosolic acid-based banaba extract product glucosol (30) . The glucose uptake assay revealed that glucosol was activating glucose transport. Its activity was dose-dependent (Fig. 4) . However, since corosolic acid itself does not have any insulin receptor mediated glucose transport activity, the effect of glucosol is likely to originate from other chemical compounds in the banaba extract and not from corosolic acid.
Tannins comprise a large and diverse class of polyphenolic compounds (31, 32) . Our search for the active components in the tannin fraction of BE was made much easier after we discovered that commercially available tannic acid (TA) shows similar glucose transport stimulatory activity to BE (29) . The main components of TA are gallotannins, a subclass of the tannins usually consisting of a glucose core connected to a variable number of galloyl groups via ester bonds (Fig. 5) .
TA is known to exhibit various health-beneficial activities (33) (34) (35) (36) . As a constituent of red wine, it has been shown to effectively reduce blood glucose levels in type 2 diabetes patients (37) and the production of endothelin-1 (38), a key protein factor intimately involved in the development of cardiovascular disease (38) . In our study, TA was found to be much more potent and efficacious than the ellagitannin Lagerstroemin (29) . Therefore, TA was chosen as the focus of our research for isolating active compounds from the tannin fraction Figure 3 . Absence of glucose transport stimulatory activity of corosolic acid in adipocytes. Pure corosolic acid in aqueous solution was added to 3T3-L1 adipocytes grown in wells of 6-well cell culture plates to induce glucose transport by a commonly used procedure (28, 29) . Cells treated with either 1 mM insulin or 30 mM penta-galloyl-glucose (PGG) were used as positive controls. Cells treated with water vehicle served as negative controls. Samples were in triplicate in the experiment, and the experiment was repeated three times. No difference was found by a one-way ANOVA statistical analysis between the negative (vehicle) control and corosolic acid samples at any concentration.
of BE. Components of TA were separated by HPLC, and active fractions were identified with a glucose uptake assay in 3T3-L1 adipocytes (29) . The study led to the discovery of penta-O-galloyl-D-glucopyranose (PGG) as the most effective compound in TA (Fig. 5, 39 ). Both anomers of PGG occur in nature (40) . The a-anomer was found to be slightly more active than the b-form in its glucose transport stimulatory activity (39) .
Corosolic Acid and Lagerstroemin versus Tannic Acid and PGG: Which are More Bioactive?
In the ellagitannin study mentioned earlier, Lagerstroemin exhibits glucose transport stimulation at 40 mM with an EC 50 of 80 mM (22) . In comparison, aand b-PGG exhibit activity at a concentration as low as 10 mM with EC 50 of 17 and 18 mM (39) . In other words, aand b-PGG are about five times more potent than Lagerstroemin in stimulating glucose transport. We would like to emphasize that both aand b-PGG possess the adipogenesis inhibitory activity exhibited by the banaba extract (28) and by TA (29) . This suggests that these two activities are associated with the same molecules. It also indicates the functional differences between insulin (glucose transport inducing and adipogenic) and PGG (glucose transport inducing but anti-adipogenic). Both compounds may have the potential to reduce hyperglycemia without increasing adiposity, a very desirable combination of properties that insulin lacks.
The Lagerstroemin study also showed that its glucose transport inducing activity is about 54% of that of insulin (22) . In comparison, aand b-PGG showed 60-70% of insulin's glucose transport inducing activity (39) . . Glucose transport stimulatory activity of glucosol as compared with banana extract. Banaba extract (BE) was prepared in house (28) . Glucosol (30) was purchased. Glucosol was compared with BE in a regular glucose uptake assay (28, 29) . Samples were assayed in triplicates, and the assay was repeated three times. The result of the assays was analyzed with a one-way ANOVA. *P50.05, **P50.01, ***P50.001. All samples were compared with the negative (vehicle) control. Thus, aand b-PGG are at least 30% more effective than Lagerstroemin (Table 1 ). It is interesting that PGG possesses many other health-beneficial bioactivities, such as anticancer (41, 42) , anti-inflammation (43, 44) , antivirus (anti-HIV, 45; anti-SARS, 46) and antioxidant activity (47, 48) .
From the known studies, we conclude that tannin molecules are responsible for the insulin-like glucose transport stimulatory activity of the banaba extract. Gallotannins such as PGG appear to be more potent and efficacious than ellagitanins such as Lagerstroemin in IR binding, IR activation and glucose transport induction. Corosolic acid does not possess any insulin-like glucose transport stimulatory activity. If its antidiabetic activity can be confirmed, it is likely to be induced via a noninsulin like, indirect mechanism. Although tannins were identified as the effective component for the insulin like glucose transport inducing activity in banaba extract, the most effective tannin molecule in the extract has not been identified. A well designed bioassay (glucose uptake assay) guided isolation should be able to complete this task.
In the past 10 years, other studies regarding banaba extracts or chemicals derived from banaba extracts were reported (49) (50) (51) (52) . These studies indicate that banaba extracts contain interesting biomedical substances that have attracted significant scientific attention. More detailed studies at molecular and cellular levels as well as in animal models are required to elucidate banaba extract's antidiabetic activity and other health beneficial activities such as its anti-adipogenesis activity. 
Corosolic acid -0
Insulin 1 nM 156 *Activity of b-PGG was arbitrarily assigned as 100. Transmissibility of the Influenza Virus in the 1918 Pandemic BACKGROUND: With a heightened increase in concern for an influenza pandemic we sought to better understand the 1918 Influenza pandemic, the most devastating epidemic of the previous century. METHODOLOGY/PRINCIPAL FINDINGS: We use data from several communities in Maryland, USA as well as two ships that experienced well-documented outbreaks of influenza in 1918. Using a likelihood-based method and a nonparametric method, we estimate the serial interval and reproductive number throughout the course of each outbreak. This analysis shows the basic reproductive number to be slightly lower in the Maryland communities (between 1.34 and 3.21) than for the enclosed populations on the ships (R(0) = 4.97, SE = 3.31). Additionally the effective reproductive number declined to sub epidemic levels more quickly on the ships (within around 10 days) than in the communities (within 30–40 days). The mean serial interval for the ships was consistent (3.33, SE = 5.96 and 3.81, SE = 3.69), while the serial intervals in the communities varied substantially (between 2.83, SE = 0.53 and 8.28, SE = 951.95). CONCLUSIONS/SIGNIFICANCE: These results illustrate the importance of considering the population dynamics when making statements about the epidemiological parameters of Influenza. The methods that we employ for estimation of the reproductive numbers and the serial interval can be easily replicated in other populations and with other diseases. The emergence of the highly pathogenic avian influenza strain H5N1 has raised concerns of an imminent influenza pandemic. Public health workers, government officials and disaster planners have an increasing interest in better understanding the potential impact of an influenza pandemic and possible strategies for containment. Crucial in this planning is an understanding of the basic epidemiology of the disease in various settings. This has led to a growing interest in the analysis and understanding of past epidemics, particularly that of 1918, the most virulent and deadly influenza epidemic of the 20th century. Mortality has been estimated at 50-100 million people worldwide as a result of influenza in the 1918 pandemic [1] . It is reasonable to suppose that by better understanding the transmission dynamics of the highly pathogenic virus in 1918, we can gain greater insight into the dynamics, and thus potential methods of control, for a future pandemic [2] . Important parameters for understanding disease transmission are the reproductive number and the serial interval [3] . The basic reproductive number is defined as the average number of secondary infections created from a primary infection in an entirely susceptible population [4, see also 5] . A more complex, but perhaps meaningful parameter is the effective reproductive number which defines the average number of secondary infections an infected will create at a given point in the epidemic. This parameter takes into account that not all contacts of an infected individual are with susceptible persons, as well as the impact of public health control measures. Control strategies are typically targeted to drive this number below one and maintain it there, as this will lead to eventual extinction of the epidemic. An example of this is herd immunity, or immunity to a disease that is incurred from a sufficiently large proportion of the population being immune to a disease. Modeling techniques are often used to determine the proportion of the population that should be vaccinated in order to have the reproductive number low enough to avoid outbreaks of disease [6] .
The serial interval can be defined as the time interval between a primary case presenting with symptoms and its infectee developing symptoms [7, 8] . Thus this quantity is completely observable. This is a mixture of the incubation period and the infectious period, both of which are useful to understand, but difficult to measure. The SARS outbreak of 2003 had a relatively long serial interval, estimated to be between 8 and 10 days on average and following a Weibull distribution [9] making case isolation extremely effective in containing the epidemic.
Methods for the estimation of basic epidemiological parameters are still in development phase. [10] provides a thoughtful summary of methods for estimating the reproductive number. One particularly interesting and useful method has been previously described by [7] for estimating the daily reproductive number, R t , or the average number of cases an infected individual on day t would cause. One interesting feature of this method is that for days where no cases are observed, the estimated effective reproductive number is zero. Another observation is that this method essentially estimates a curve for the effective reproductive number that traces the epidemic curve, lagged by the average serial interval length. This nonparametric method presupposes information on the serial interval distribution. This is typical as most methods for estimating the reproductive number rely on knowledge of the serial interval.
Few have described analytical methods for estimating the serial interval, making most methodologies dependent on contact tracing data, which is often difficult and expensive to attain. [11] describe a method to estimate the reproductive number that relies on limited contact tracing information but not a full estimate of the serial interval. [12] have recently described a method to estimate the serial interval and then used this estimate with the estimator proposed in [7] of the daily reproductive number and have applied their method to data from outbreaks of avian influenza in poultry farms in Europe.
Several researchers have studied the 1918 pandemic and estimated some of these key epidemiological parameters. Estimates have ranged from 2-3 for the basic reproductive number, R 0 , when using an SEIR model with a mean latent period of 1.9 days and infectious period of 4.1 days [13, 14] . Using an exponential model and assuming the serial interval to be four days (somewhat based on the assumptions of [13] ), [15] estimated R 0 to be 2.6-10.6 for confined settings (such as prison and ships) and 2.4-4.3 for community settings. The estimates for the mean latent and infectious periods come from [16] and were used again by [17] and [18] . It appears that the original estimates were derived from epidemic data, although their source is not well documented.
In what follows, we introduce new methodology for the estimation of both the daily reproductive number and the serial interval. We apply this method to data from two outbreaks on military ships in the 1918 influenza outbreak, as well as welldocumented outbreaks in five Maryland communities. The results from this method are compared to that of [12] . The results illustrate the differences in infectious disease dynamics between outbreaks in a closed population and a dynamic community.
We analyze data from several well-documented influenza outbreaks in 1918. First we consider data from two troop ships that embarked in the late fall of 1918 [19] . The Medic reported two initial cases on November 11. Out of 989 passengers (156 crew members, 829 soldiers, 4 civilians) 313 became sick with influenza over a 40 day period (Attack Rate, AR, = 0.32), though most of the cases occurred within the first fourteen days. The Boonah left Durban and in five days, on November 29, reported the first three definitive cases of influenza. Those who collected the data note that there were likely some initial cases that were not identified. Out of 1095 on board (164 crew members and 931 troops), 470 cases were reported (AR = 0.43) in the 40 days of the epidemic.
The United States Public Health Service created special surveys of 18 localities during the pandemic [20] . Reported results from six communities in Maryland are derived from house-to-house surveys requesting the date of onset of influenza for all infected, and the sex and age of each case of pneumonia and influenza. A summary of these populations is provided in Table 1 .
We describe a likelihood based methodology for estimating the reproductive number at each day in the epidemic as well as the serial interval. The method builds on that described by [21] . We assume that the population is closed, that all cases are observed, and use daily case counts only (i.e. number of new cases each day).
Let N = {N 0 , N 1 , N 2 ,…, N T } represent the daily cases counts of influenza for the T days of the epidemic and X ij represent the number of cases that appear on day j that are infected by individuals that appeared sick on day i. Following is a representation of the disease transmission model in the population.
. .
We assume that the total number of cases produced by those on day i, X i? , are Poisson distributed with parameter N i R i , where R i is the reproductive number for cases on day i. We further assume that X i = {X i,i+1 , X i,i+2 ,…,X i,i+k } follows a multinomial distribution with parameters X i? , p, k, where p = {p 1, p 2,…, p k } represent the distribution of the serial interval. Using these assumptions we can construct a likelihood function (see details in the Supplemental Information), which, when simplified, yields the following convenient form
where m i~Ri ( X k j~1 p j N i{j ) [21] . Maximization of this likelihood with respect to R i and p yields estimates of these parameters. To further simplify this process and create a more parsimonious model, we parameterize p by allowing it to follow a traditional parametric form for a serial interval (for instance a Weibull, Gamma, Log Normal, or Exponential distribution). Then the p j are functions of the parameters of the density (for instance in the case of the Gamma distribution, the p j only depend on the shape and rate parameters of the Gamma).
Similarly R i can be modeled parametrically as a function of time. One example of a reasonable model for this is the four parameter logistic curve [22] [23] [24] given by The parameters of this curve describe the initial height of the curve (approximately a+b), the point of inflection (d), the curvature over the inflection (c) and the final height of the curve (a). These parameters have biological meaning in this setting where the initial height corresponds to the values of R i prior to intervention and significant depletion of the susceptible population. The inflection point and its steepness would describe the timing of intervention and the rapidity with which it impacts transmission. The final height would describe the ultimate value of R i, which typically is less than one, indicating that disease transmission is in a sub epidemic state.
In our analysis, we also implement the method described by [12] (hereafter referred to as the Garske et al. method) and compare the results of the two methodologies. This method first estimates the generation time distribution using a likelihood based method. Then the effective reproductive number is estimated using the method described by [7] (hereafter referred to as the WT method). We fit the likelihood for both methods using a Nelder-Mead maximization procedure and use 576 starting values in order to ensure that we reach the global maximum. All analyses were done using R 2.4.1.
Both methods assume homogenous mixing in the population, no missing data (clearly violated with the data from the Maryland communities), that a primary case experiences symptom onset prior to any cases that it infects and a completely closed system where all cases are infected by a case that has been observed. In the case of the Maryland data, where only a sample of the total number of cases was surveyed, we can observe the efficacy and robustness of these methods with sample data. Certainly results should be interpreted with caution, however, as we will show, the results that are obtained are consistent with previous estimates for influenza.
Standard errors were calculated for the MLE method using a parametric bootstrap. One thousand epidemics were simulated using the parameter estimates and estimates were obtained from each of these simulated epidemics. The standard deviation of the 1000 estimates was used as the standard error estimates. We used the method described in [12] to estimate the standard error for their estimates, however our simulations based on their assumption of asymptotic normality yielded a large number of negative estimates for the parameters. It is possible that this is due to the non-independence in the data and lack of theoretical underpinnings for the method that they propose. These results make their standard error estimates infeasible to estimate in this case. Therefore we do not present standard error estimates for the results obtained using their methodology.
In order to determine the accuracy and relative merit of the estimates obtained from each methodology, we compute one-stepahead residuals and implement a cross validation approach to analyze the generalizeability of the estimates obtained. The onestep-ahead residuals were calculated by first using the estimates from a particular location along with the data to predict the next days' number of cases,Ñ i as follows:
EachÑ i is calculated using N 0 , N 1 , …, N i21 . Then the one-stepahead residuals are calculated as
We present these residuals averaged over the T days observed.
Generalizeability of the results was studied using an ad hoc cross validation (CV) technique. This is done by using the estimates obtained from one location to calculate the one step ahead residuals for another location. Specifically we use the Boonah ship estimates to calculate residuals with the Medic data and then use the Medic estimates to calculate the residuals for the Boonah data. For the Maryland communities, we report the average of the residuals obtained using the estimates from one community to predict the epidemics in each of the other four communities, creating five CV estimates (one for each community). Table 2 gives the results for the serial interval distribution estimates. Notable in these results is the striking consistency in the estimates of the first moment, with the exception of Cumberland. The second moments vary much more, however. In general they tend to be much larger for the ships when using the Garske et al. method compared to the MLE method. For the communities, we observe that they are consistently around 10 for the Garske et al. method and vary much more for the MLE method. Also of interest in these results are the large error estimates, particularly for Cumberland, but also to a smaller extent for Frederick. This is perhaps indicative of the model not fitting the data as well, for instance the logistic model may not be the best fit in this scenario, or that the lack of census data on cases might be more problematic here.
In Table 3 and Figure 1 , we present the results for estimation of the effective reproductive number. Evident in these results, is the large initial reproductive number for the Boonah ship. This is likely due to some of the missing data at the beginning of the epidemic and thus the model attributing the large number of cases that rapidly develop to the few individuals who were initially reported. The logistic model fits this as accurately as possible, but perhaps the important message is the qualitative result, indicating that initial transmission in this susceptible, non-quarantined population was very high and rapidly decreased as many became infected. The result is similar for Medic though the initial value is not high. We also note that the reproductive number dropped to sub epidemic levels rapidly (around 10 days for both ships).
In the Maryland communities the initial reproductive number tended to be slightly lower (ranging from 1.34 in Salisbury to 3.21 
In Table 4 , we present the results of the residual analysis. We notice here that the Garske et al. method often does better than the MLE method. It is important to point out that the WT method of fitting the effective reproductive model over fits the model and suffers from generalizeability. This method essentially traces the epidemic curve, lagged by the mean of the generation time distribution. Thus, according to the residuals, it appears that the WT method outperforms the MLE. However, considering the importance of external validation and reproducibility, the model suffers somewhat as evidenced by the CV measures. The exceptions to this are in the case of the Boonah where the CV measure is impacted by the large initial MLE estimate of the reproductive number and in Cumberland where it appears that either the parametric model chosen may not represent the best fit to the data or there were sensitivities to the survey data. 
We have presented results that are informative with regard to the dynamics of the 1918 influenza pandemic in different populations and provide insight into two methodologies for estimating basic epidemiological parameters. Both methods assume that the population is closed, there are no missing cases and no migration to or from the population. The second of these assumptions is clearly violated with the data from Maryland; however the results appear to be reasonably robust to this discrepancy, except in the case of Cumberland. The purpose of this exercise determines to some extent which methodological approach we might favor. If the intent is to simply estimate the parameters for a specific epidemic and better understand what exactly was occurring in that setting, then the method presented by [12] (Garske et al.) appears to provide good fit. The caveat that we see in this method is that by estimating the effective reproductive number with the methodology of [7] (WT) there is an over fit of this parameter and it essentially traces the epidemic curve, lagged by the mean of the serial interval. It is not clear if this is a desirable or informative property. The MLE method has greater promise for generalizeability. While it can be argued that adhering to a parametric definition of the shape of the effective reproductive number leads to a greater chance of lack of fit, it can also lead to a result that can be interpretable for other settings that are similar to that being studied.
One can choose any reasonable parametric form for modeling the effective reproductive number. Here we have only shown the four parameter logistic model, and feel that it is suitable in most cases where the epidemic curve has a single peak. It is feasible that this model may not apply well in all situations. Another approach might be to analyze the data using the Garske et al. method and then smooth the plot of the effective reproductive number and from this determine a parametric form that closely approximates the smoothed curve. Multiple models could be implemented, then the residual analysis that we have shown provides a valuable tool for model assessment and comparison.
The results of these models can be sensitive to underreporting initially in the epidemic. We see this clearly in Boonah, where it was acknowledged that there was underreporting early on and this led to us getting very high estimates for the initial reproductive number. Similarly, in Cumberland, if we remove the first five days of data (three cases on the first day, six cases on the second and then no cases the following three days) we get much more reasonable estimates (m~6:00,ŝ 2~1 0:32) with smaller residuals (6.00). Therefore, it is important to note that unusual observations in the first few days can impact the estimates and one should pay careful attention to this possibility.
Overall both methodologies presented are valuable tools that can be used in tandem for understanding the dynamics of infectious disease epidemics. These methods are easy to implement and interpret.
The results that we have presented suggest that the average serial interval for pandemic influenza in 1918 was consistently between three and four, regardless of the setting. The standard deviation for the serial interval distribution varied much more for the MLE method depending on the location. Garske et al. estimates indicate that the value was consistently smaller in the communities than in the ships. It is not clear exactly how to interpret this result. Further, we consistently see a large initial value for the reproductive number. In the ships, this value is higher and rapidly drops off, perhaps due to the close quarters and extremely rapid transmission that could take place in these very vulnerable populations. In the communities, the reproductive number tended to drop off later, typically around day thirty. This could be due to a larger initial susceptible population and more complicated dynamics for the disease to spread, leaving large pockets of susceptible individuals unexposed for a longer period of time than in the ships.  Surfactant therapy for acute respiratory failure in children: a systematic review and meta-analysis INTRODUCTION: Exogenous surfactant is used to treat acute respiratory failure in children, although the benefits and harms in this setting are not clear. The objective of the present systematic review is to assess the effect of exogenous pulmonary surfactant on all-cause mortality in children mechanically ventilated for acute respiratory failure. METHODS: We searched the MEDLINE, EMBASE, CINAHL and Ovid Healthstar databases, the bibliographies of included trials and review articles, conference proceedings and trial registries. We included prospective, randomized, controlled trials of pulmonary surfactant that enrolled intubated and mechanically ventilated children with acute respiratory failure. We excluded trials that exclusively enrolled neonates or patients with asthma. Two reviewers independently rated trials for inclusion, extracted data and assessed the methodologic quality. We quantitatively pooled the results of trials, where suitable, using a random effects model. RESULTS: Six trials randomizing 314 patients were included. Surfactant use reduced mortality (relative risk = 0.7, 95% confidence interval = 0.4 to 0.97, P = 0.04), was associated with increased ventilator-free days (weighted mean difference = 2.5 days, 95% confidence interval = 0.3 to 4.6 days, P = 0.02) and reduced the duration of ventilation (weighted mean difference = 2.3 days, 95% confidence interval = 0.1 to 4.4 days, P = 0.04). CONCLUSION: Surfactant use decreased mortality, was associated with more ventilator-free days and reduced the duration of ventilation. No serious adverse events were reported. Acute respiratory failure remains the primary indication for admission to North American paediatric intensive care units (PICUs) and accounts for significant mortality, morbidity and resource utilization [1] . Respiratory infections, in particular pneumonia and severe bronchiolitis, are the most common causes of respiratory failure requiring mechanical ventilation in children [1] .
Alterations in endogenous surfactant play a role in the pathogenesis of many causes of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [2] . Surfactant dysfunction, destruction and inactivation have also been demonstrated in children with acute respiratory insufficiency due to bronchiolitis [3, 4] . The administration of exogenous surfactant may reduce the need for mechanical ventilation and its associated sequelae by restoring surfactant levels and function. Inspired by the success of surfactants in reducing mortality and the need for mechanical ventilation in neonatal respiratory distress syndrome [5] , investigators have studied exogenous surfactant in other populations with various causes of respiratory failure. Trials of surfactant in adults with ALI and ARDS have not demonstrated a mortality benefit [6] [7] [8] [9] , perhaps due to inherent differences in the aetiology of lung injury in adults, the design features of the trials, the mode and timing of surfactant administration or the type and dose of surfactant used. In children with respiratory failure, the efficacy of exogenous surfactant has been suggested in uncontrolled studies ALI = acute lung injury; ARDS = acute respiratory distress syndrome; FiO 2 = fractional inspired oxygen; PaO 2 = arterial oxygen tension; PICU = paediatric intensive care unit; RSV = respiratory syncytial virus.
(page number not for citation purposes) [10, 11] . The relatively low mortality rate, the diversity of the study populations and the shorter duration of mechanical ventilation are factors that make large-scale randomized controlled trials in this population challenging to conduct. Two of the largest trials were stopped early due to slower than expected enrolment [12, 13] . While the use of surfactant in ARDS/ALI has not been previously systematically reviewed, its use in children with bronchiolitis has been [14] .
We anticipated that including trials enrolling children with acute respiratory failure from a variety of causes would result in a heterogeneous population and would increase the generalizability of the results. Our confidence in the results of the present review would also be increased if a consistent effect is shown in subgroups and across a spectrum of disease severity.
The primary objective of the systematic review is to assess the effect of the administration of pulmonary surfactant compared with no therapy or with placebo on all-cause mortality (at or before hospital discharge) in mechanically ventilated children with acute respiratory failure.
We included trials that were prospective, that were randomized, that enrolled children intubated and mechanically ventilated for acute respiratory failure and that compared the intratracheal administration or nebulization of at least one dose of natural or artificial pulmonary surfactant with a placebo or no intervention. We excluded trials exclusively enrolling neonates or patients with asthma. We used the trial authors' definitions of paediatric.
The primary outcome measure was all-cause mortality at or before hospital discharge. Secondary outcomes were ventilator-free days to day 28 (a composite of mortality and duration of ventilation, defined as days alive and free from mechanical ventilation) [15] , the duration of mechanical ventilation (from intubation to extubation, death or trial withdrawal), the duration of PICU stay, the use of rescue therapy (such as extracorporeal membrane oxygenation, high-frequency oscillatory ventilation, open label surfactant and nitric oxide), and complications and adverse effects as reported by the trial authors.
One of us searched for published and unpublished trials, examining trial registries, conference proceedings and the bibliographies of any identified trials and relevant reviews (the search strategy is available upon request). We polled paediatric intensivists and pharmacists at our institution for additional trials. We selected search terms from the keywords and MESH terms of previous surfactant trials and from the generic and brand names of commercially available surfactants. We imposed no language restrictions.
One of us screened the title (and abstract if required) of all citations retrieved. We selected citations for further evaluation if they reported the administration of at least one dose of surfactant to at least one child or if the title or abstract did not give enough information to make an assessment. Two reviewers independently reviewed all citations meeting criteria for further review and applied the inclusion criteria. Disagreements between reviewers were resolved by consensus in consultation with a third reviewer. We considered agreement between reviewers to be acceptable if the kappa value was greater than 0.8.
We used the following characteristics to assess the methodologic quality: allocation concealment (sealed envelopes or central randomization were considered adequate), blinding (which of the trial personnel and caregivers were blinded, and the methods used to ensure blinding), completeness of followup (assessed by the number of patients randomized for whom there were no outcomes), similarity of the groups at baseline (with respect to known prognostic factors: age, aetiology, severity of illness as measured by the Pediatric Risk of Mortality score, and immunosuppression), whether a standard or recommended strategy for mechanical ventilation was used, and whether a priori criteria for the use of co-interventions were used. Effective blinding of surfactant is challenging because of the large volumes of milky fluid administered, which can often be seen by caregivers in the patients' ventilator tubing or endotracheal tube, particularly during suctioning.
We pretested and refined the developed forms on two trials of surfactant therapy for adults, and clarified definitions based on feedback from the reviewers. Two reviewers then independently used these forms to abstract trial quality, blinded to the authors, the journal, the country of origin and the results. We resolved any disagreements by consensus in consultation with a third reviewer if needed.
After pretesting and refining the forms on two trials of surfactant therapy in adults and clarifying definitions based on feedback from the reviewers, two reviewers then independently abstracted the data. Reviewers were only provided with a full-text version of the trials from which the introduction, conclusions and discussion were omitted and from which the author, journal and country of origin were deleted. We thereafter examined these sections of the reports for any missing data. We resolved any disagreements between reviewers by consensus in consultation with a third reviewer if needed. We asked the authors to supply data not included in the published reports. Two reviewers performed data entry in duplicate.
We quantitatively pooled the results of individual trials when possible. We expressed the treatment effect as a relative risk for dichotomous outcomes and as a weighted mean difference for continuous outcomes with 95% confidence intervals. We considered effects statistically significant if P < 0.05. A z test was used to statistically test the estimates of treatment effect between groups [16] . We assessed heterogeneity among trials using the I 2 statistic, and considered an I 2 value greater than 50% to indicate substantial heterogeneity [17] . RevMan 4.2 software and a random effects model were used to perform the analyses [18] . We chose the random effects model because it gives a more conservative estimate of the precision of the treatment effects and because the true effect of the intervention probably varies given the different populations enrolled in these trials [19] . A subgroup analysis was planned based on the aetiology of respiratory failure (trials enrolling exclusively patients with respiratory syncytial virus (RSV)/ severe bronchiolitis compared with all other trials) if sufficient data were available, because these trials were likely to enrol a younger, more homogeneous, population with a lower predicted risk of mortality. We also planned sensitivity analysis based on methodological features of the included trials (trials reporting adequate allocation concealment compared with all other trials).
We identified 742 unique citations, six of which met our inclusion criteria ( Figure 1 outlines the reasons for exclusion). Most reports excluded enrolled neonates or were retrospective or uncontrolled in design. Chance corrected agreement was excellent (kappa = 0.91, 95% confidence interval = 0.73-1.1). Table 1 presents a complete description of our quality assessment. Only one trial did not report allocation concealment [20] . Although effective blinding of surfactant is challenging, two trials reported blinding of the PICU team [12, 20] . The two Flow diagram of included trials Flow diagram of included trials. RCTs, randomized controlled trials. groups were generally well matched in terms of baseline characteristics in most trials. The most significant imbalance was the numerically higher number of immunosuppressed patients in the placebo group. These patients had higher mortality (56%) than the immunocompetent group (13%). The authors attempted to adjust for this imbalance with logistic regression, which suggested that the treatment effect seemed to be relatively consistent between the two groups [12] . Only one trial reported a priori criteria for rescue therapy [13] . Table 2 describes the included trials. Three trials enrolled exclusively infants with RSV-induced respiratory failure [20, 21] or with severe bronchiolitis [22] . The remaining three trials enrolled a heterogeneous group of patients with ARDS or ALI [12, 23, 24] . While the individual treatment protocols varied, all trials used comparable doses (50-100 mg/kg phospholipids) of natural or modified natural surfactants and each patient typically received one or two doses. A variety of interventions were used in the control groups: no intervention, air placebo or similar sedation and ventilation manoeuvres without a placebo. Although one study [20] used a modified natural surfactant, all the products used contained surfactant proteins B and C. All studies administered surfactant early in the course of respiratory failure; most patients were treated within 12-48 hours of requiring mechanical ventilation.
The baseline characteristics of the patients are presented in Table 3 . While there was significant heterogeneity among and within trials with respect to age and cause of respiratory failure, we considered the initial Pediatric Risk of Mortality scores and the initial PaO 2 /FiO 2 ratios to be clinically comparable.
Mortality data were available for all six trials, randomizing 311 patients and reporting data for 305 patients. There were no deaths reported in the three RSV/severe bronchiolitis trials; thus our estimate is based on three trials randomizing 232 patients, 64 of whom died. In the pooled analysis, surfactant was associated with significantly lower mortality (relative risk = 0.7, 95% confidence interval = 0.4-0.97, P = 0.04). There was no evidence of heterogeneity (I 2 = 0%) ( Figure 2 ).
Ventilator-free days to day 28
The number of ventilator-free days to day 28 was available for six trials randomizing 311 patients and reporting data for 305 patients. In the pooled analysis, surfactant was associated with significantly more ventilator-free days (weighted mean dif- (Figure 3 ).
The duration of mechanical ventilation was available for six trials randomizing 311 patients and reporting data for 305 patients. In the pooled analysis, surfactant was associated with a significantly shorter duration of mechanical ventilation (weighted mean difference = 2.3 days, 95% confidence interval = 0.1-4.4 days, P = 0.04) (Figure 4) .
The duration of PICU stay was available for five trials randomizing 273 patients and reporting data for 272 patients. In the pooled analysis, surfactant was associated with a shortened duration of PICU stay (weighted mean difference = 2.6 days, 95% confidence interval = 0.02-5.2 days, P = 0.05), but this difference was not statistically significant ( Figure 5 ).
Data on the use of rescue therapy were available for six trials randomizing 311 patients and reporting data for 305 patients.
In the pooled analysis, the surfactant was associated with a significantly lower use of rescue therapy (relative risk = 0.4, 95% confidence interval = 0.3-0.7, P < 0.0001). There was no evidence of heterogeneity (I 2 = 0%). This summary estimate should be interpreted with caution as only one trial reported a protocol for initiating rescue therapy. The decision to use a rescue therapy, particularly an open-label surfactant, may be influenced by knowledge of the patient's allocation; furthermore, only two trials reported blinded caregivers and the methods used to ensure blinding may not be adequate.
Surfactant therapy was well tolerated (see Table 4 ), but only three of the trials reported any definitions or a priori criteria or of collecting adverse events [12, 21, 23] . Transient hypotension and transient hypoxia were the most commonly reported adverse events in the largest trial. These responded to a brief adjustment in ventilation, to a slowing of the rate of surfactant administration or to fluid administration. There was no difference in the incidence of air leaks in the two trials that reported this outcome. No patient was withdrawn from any of the trials because of adverse events. We did not pool the data on adverse events associated with the trial interventions from the six trials because of the inconsistent manner in which the events were documented and reported.
The effect of surfactant on ventilator-free days, the duration of mechanical ventilation and the duration of PICU stay was not significantly different when we compared the three trials that enrolled exclusively patients with RSV/severe bronchiolitis with the three other trials (Table 5) . A 100% survival in the bronchiolitis trials subgroup precludes formal subgroup analysis for the primary outcome of mortality.
All but one of the included trials reported adequate allocation concealment (defined as sealed envelopes or central telephone randomization). Since there were no deaths in this trial we could not assess the effect of inadequate allocation concealment on mortality. Pooling the five remaining trials did not change the direction of the effect and did not significantly Meta-analysis of trials of surfactant in children with acute respiratory failure: Mortality Meta-analysis of trials of surfactant in children with acute respiratory failure: Mortality. ALI, acute lung injury; ARDS, acute respiratory distress syndrome; 95% CI, 95% confidence interval; RR, relative risk; RSV, respiratory syncytial virus.
Meta-analysis of trials of surfactant in children with acute respiratory failure: Ventilator-free days Meta-analysis of trials of surfactant in children with acute respiratory failure: Ventilator-free days. ALI, acute lung injury; ARDS, acute respiratory distress syndrome; 95% CI, 95% confidence interval; RSV, respiratory syncytial virus; SD, standard deviation; WMD, weighted mean difference.
change the point estimates for the secondary outcomes of ventilator-free days, duration of ventilation or duration of PICU stay (Table 6 ).
In the present systematic review and meta-analysis of the effect of surfactant for critically ill children with acute respiratory failure we found that surfactant therapy significantly reduced our primary outcome of mortality. Surfactant was associated with more ventilator-free days, with decreased duration of ventilation and with less use of rescue therapy as compared with standard therapy. There was no significant difference in the duration of PICU stay. Surfactant therapy was well tolerated; while transient hypoxia and hypotension were reported during surfactant administration, no study reported any serious adverse events. The patients enrolled in these trials are representative of the heterogeneous group of children with early, severe acute respiratory failure that is seen in clinical practice. These patients had similar severity of illness scores and a similar degree of respiratory failure (as measured by Pediatric Risk of Mortality scores and PaO 2 :FiO 2 ratios).
The heterogeneity of results for our primary outcome of mortality was low. The presence of significant heterogeneity reduces the strength of inferences we can make regarding the effect of surfactant on the secondary outcomes of ventilator-free days,
Meta-analysis of trials of surfactant in children with acute respiratory failure: Duration of mechanical ventilation Meta-analysis of trials of surfactant in children with acute respiratory failure: Duration of mechanical ventilation. ALI, acute lung injury; ARDS, acute respiratory distress syndrome; 95% CI, 95% confidence interval; RSV, respiratory syncytial virus; SD, standard deviation; WMD, weighted mean difference.
Meta-analysis of trials of surfactant in children with acute respiratory failure: Duration of PICU stay Meta-analysis of trials of surfactant in children with acute respiratory failure: Duration of PICU stay. ALI, acute lung injury; ARDS, acute respiratory distress syndrome; 95% CI, 95% confidence interval; PICU, paediatric intensive care unit; RSV, respiratory syncytial virus; SD, standard deviation; WMD, weighted mean difference.
duration of ventilation and duration of PICU stay. Separately pooling the trials that exclusively enrolled patients with RSV/ severe bronchiolitis and those enrolling patients with ARDS/ ALI from a variety of causes did not significantly reduce the heterogeneity. Changing ventilation strategies and the use of a variety of natural and modified natural surfactants may have increased the heterogeneity of our results. Ventilation strategies, such as the use of lower tidal volumes and earlier use of high-frequency oscillatory ventilation, have evolved significantly in the 10-year span over which the included trials were conducted [25] [26] [27] . The surfactants used in the included trials were all natural or modified natural surfactants; however, these surfactants may have slightly different effects on oxygenation and compliance due to the differences in phospholipid and surfactant protein composition, which may have influenced individual study results. The strengths of the present review include a comprehensive search strategy, broad inclusion criteria (resulting in a representative, heterogeneous population) and abstraction of clinically important outcomes in duplicate, independently blinded to information that may bias evaluation. The strength of the inference we can make from our subgroup analysis is limited because we were unable to extract all subgroup data from these trials. Access to individual patient data would allow better examination of the treatment effect in subgroups of patients and would facilitate further exploration of possible causes of heterogeneity.
We found that mortality was very different between the trials that exclusively enrolled patients with RSV/severe bronchiolitis and those that enrolled patients with ARDS/ALI from a variety of causes. We pooled the results because both conditions result in abnormal surfactant function and because of the substantial overlap between the two groups; up to 17% of children in the ARDS/ALI trials had RSV and up to 50% of the children in some bronchiolitis studies also had pneumonia.
The reduction in mortality and the increased ventilator-free days have important implications as very few trials in paediatric critical care suggest a favourable impact on mortality [28] . The present review suggests that surfactant could be an important adjunct in the management of paediatric respiratory failure. Uncertainty exists, however, about the reproducibility of treatment effects generated from relatively small unblinded trials; questions remain about adverse affects, which may be undetected or under-reported in this literature. Also, a large proportion of patients and events are reported in one trial [12] . Furthermore, issues of the optimal dose and the timing of administration, and which patients are most likely to derive benefit, should be studied in further adequately powered multicentre trials. The Pediatric Acute Lung Injury and Sepsis Investigators network is planning a large rigorous randomized trial enrolling children with acute hypoxemic respiratory failure to address these issues.
Surfactant use decreased mortality, was associated with more ventilator-free days and reduced the duration of ventilation. No serious adverse events were reported. Most trials enrolled small numbers of children, and further well-designed and adequately powered multicentre trials are therefore required. 
• Surfactant decreased mortality in a heterogeneous population of children with acute respiratory failure.
• Surfactant was associated with more ventilator-free days and a reduced duration of ventilation.
• No serious adverse events were reported.
• Further well-designed and adequately powered multicentre trials are required. Clinical review: Update of avian influenza A infections in humans Influenza A viruses have a wide host range for infection, from wild waterfowl to poultry to humans. Recently, the cross-species transmission of avian influenza A, particularly subtype H5N1, has highlighted the importance of the non-human subtypes and their incidence in the human population has increased over the past decade. During cross-species transmission, human disease can range from the asymptomatic to mild conjunctivitis to fulminant pneumonia and death. With these cases, however, the risk for genetic change and development of a novel virus increases, heightening the need for public health and hospital measures. This review discusses the epidemiology, host range, human disease, outcome, treatment, and prevention of cross-transmission of avian influenza A into humans. Human influenza pandemics over the last 100 years have been caused by H1, H2, and H3 subtypes of influenza A viruses. More recently, avian influenza virus subtypes (that is, H5, H7) have been found to directly infect humans from their avian hosts. The recent emergence, host expansion, and spread of a highly pathogenic avian influenza (HPAI) H5N1 subtype in Asia have heightened concerns globally, both in regards to mortality from HPAI H5N1 infection in humans and the potential of a new pandemic. This paper will review the current human infections with avian influenza and their public health and medical implications.
Influenza A, B and C are the most important genera of the Orthomyxoviridae family, casusing both pandemic and seasonal disease in humans. Influenza A viruses are enveloped, single-stranded RNA viruses with a segmented genome (Table 1 ) [1] . They are classified into subtypes on the basis of the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins expressed on the surface of the virus [1, 2] . Influenza A viruses are characterized by their pathogenicity, with highly pathogenic avian influenza (HPAI) causing severe disease or death in domestic poultry [3] . Molecular changes in the RNA genome occur through two main mechanisms: point mutation (antigenic drift) and RNA segment reassortment (antigenic shift) [4, 5] . Point mutations cause minor changes in the antigenic character of viruses and are the primary reason a vaccination for influenza A is given yearly. Reassortment occurs when a host cell is infected with two or more influenza A viruses, leading to the creation of a novel subtype. The influenza subtypes of the 1957 (H2N2) and 1968 (H3N2) pandemics occurred through reassortment, while the origins of the 1918 (H1N1) pandemic are unclear.
The HA glycoprotein mediates attachment and entry of the virus by binding to sialic acid receptors on the cell surface. The binding affinity of the HA to the host sialic acid allows for the host specificity of influenza A [6, 7] . Avian influenza subtypes prefer to bind to sialic acid linked to galactose by α-2,3 linkages, which are found in avian intestinal and respiratory epithelium ( Table 2 ) [8] . Human virus subtypes bind to α-2,6 linkages found in human respiratory epithelium [8, 9] . Swine contain both α-2,3 and α-2,6 linkages in their respiratory epithelium, allowing for easy co-infection with both human and avian subtypes (thus acting as a 'mixing vessel' for new strains) [10] . Humans have been found to contain both α-2,3 and α-2,6 linkages in their lower respiratory tract and conjunctivae, which allows for human infections by avian subtypes [9, 11, 12] . The HA glycoprotein is the main target for immunity by neutralizing antibodies.
The NA glycoprotein allows the spread of the virus by cleaving the glycosidic linkages to sialic acid on host cells and the surface of the virus. The virus is then spread in secretions or other bodily fluids. The NA glycoprotein is not the major target site for neutralization of the virus by antibodies. 
Influenza A viruses infect a wide range of hosts, including many avian species, and various mammalian species, such as swine, ferrets, felids, mink, whales, horses, seals, dogs, civets, and humans [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . Wild birds (ducks, geese, swans, and shorebirds) are important natural reservoirs of these viruses, and all of the known 16 HA and 9 NA subtypes have been found in these birds [32] [33] [34] [35] . In most cases, these subtypes are found within the gastrointestinal tract of the birds, are shed in their feces, and rarely cause disease [32] . Since 2002, however, HPAI H5N1 viruses originating in Asia have been reported from approximately 960 wild bird species, causing disease in some instances and asymptomatic shedding in others [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . The virus has now spread across Asia, Europe, the Middle East, and some African countries. Additional species, such as tigers, leopards, cats, stone martens, and humans have also become infected with HPAI H5N1 [49] . This spread of H5N1 into a wide range of animal and avian species may enhance the spread of the virus into the human population as it interacts with animals in a number of ways (increased land use, markets, consumption) [44] . Thus, the potential contact, transmission, and mutability of HPAI H5N1 worldwide will increase as the number of species and their interactions increase, complicating prevention, surveillance and treatment possibilities.
The incidence of avian influenza infections in humans has increased over the past decade (Table 3) . Initially, cases of avian influenza (H7N7) in humans occurred in association with poultry outbreaks, manifesting as self-limiting conjunctivitis [30, [50] [51] [52] [53] . Then, in 1997, a large scale HPAI H5N1 outbreak occurred among poultry in Hong Kong, with 18 documented human cases [29, 31, 54, 55] . Two subsequent poultry outbreaks in Hong Kong in 1999 and 2003 with HPAI H5N1 occurred without human cases until 2003 when two members of a family in Hong Kong contracted HPAI H5N1 [56] . In December of 2003, HPAI H5N1 surfaced in poultry in Korea and China, and from 2003 to 2006 the outbreak stretched worldwide in the largest outbreak in poultry history. Human cases of HPAI H5N1 followed the poultry outbreak, with a total of 256 cases and 151 fatalities thus far [57] . Other limited outbreaks have occurred, causing variable human disease (Table 3 ) [52, 58] . However, HPAI H5N1 remains the largest and most significant poultry and human avian influenza outbreak.
Epidemiological investigations of human cases of avian influenza show that the virus was acquired by direct contact with infected birds [29] [30] [31] [50] [51] [52] [53] [54] [55] [56] . Influenza A is transmitted through the fecal-oral and respiratory routes among wild birds and poultry [32] . Human interaction with these infected secretions and birds was the major mode of transmission, with contact including consumption of undercooked or raw poultry products, handling of sick or dead birds without protection, or food processing at bird cleaning sites. All birds were domesticated (chicken, duck, goose) and no transmission from birds in the wild (migrating) or contaminated waterways has been documented. In a few cases, limited human to human transmission has been reported among health care workers and family members (Table 4 ) [59] [60] [61] [62] [63] . In each of these cases, no personal protective equipment was used, which is the major factor in transmission between humans [60] .
The clinical manifestations of avian influenza in humans has ranged from mild conjunctivitis to severe pneumonia with multi-organ system failure ( [53] . However, with HPAI in Hong Kong in 1997 and in Southeast Asia currently, pneumonia progressing to multiorgan failure, acute respiratory distress syndrome (ARDS), and death are the predominant findings [17, 55, [65] [66] [67] [68] . Rye syndrome, pulmonary hemorrhage, and predominant nausea, vomiting, and diarrhea complicate these cases [68] . Laboratory findings include both thrombocytopenia and lymphopenia [65, 66] . Chest radiographic findings include interstitial infiltrates, lobar consolidation, and air bronchograms. The clinical course of patients with HPAI H5N1 is rapid, with 68% percent of patients developing ARDS and multiorgan failure within 6 days of disease onset [69] . The case fatality rate ranges form 67% to 80%, depending on the case series [17, 55, 65, 66] . Once the patients reached the critical care unit, however, the mortality rate was 90% [69] . The average time of death from disease onset was nine to ten days.
Avian influenza A infections in humans differ from seasonal influenza in several ways. The presence of conjunctivitis is Available online http://ccforum.com/content/11/2/209 Number of fatalities (percent) 0 (0) 6 (33) 0 (0) 1 (1) 0 (0) 151 (59) H, hemagglutinin; ILI, influenza like illness; N, neuroaminidase. more common with avian influenza A infections than with seasonal influenza. Gastrointestinal symptoms, as seen with HPAI H5N1, and reports of primary influenza pneumonia and development of ARDS are also more common with avian influenza A infections [65, 67, 69] . Finally, the rapid progression to multi-organ failure and eventually death occurs at a much higher rate with avian influenza A infections [69] .
Post-mortem studies have illustrated findings consistent with an overwhelming systemic inflammatory response syndrome, including diffuse alveolar damage, acute tubular necrosis and atrophy, disseminated intravascular coagulation, and multiorgan damage [70, 71] . Interestingly, the virus has been isolated from the lungs, intestine, spleen, and brain, suggesting viremia, but active replication of the virus has been limited to the lungs [71] . This overwhelming inflammatory response, with acute lung injury and ARDS as the predominant features, coincides with the findings of preferential binding of the avian influenza A viruses to α-2,3 linkages in type II pneumocytes of the lower respiratory tract of humans and a vigorous cytokine response, including increased interleukin-6, interleukin-10, and interferon beta release [11, 12, 70, 71] .
The clinical diagnosis of avian influenza infection in humans is difficult and relies on the epidemiological link to endemic areas, contact with sick or dead poultry, or contact with a confirmed case of avian influenza (Table 6 ). Since many infectious diseases present with similar symptoms, the only feature significant to the clinician may be contact in an endemic area, through travel or infected poultry, and the clinician should always elicit a detailed patient history.
The definitive diagnosis is made from isolation of the virus in culture from clinical specimens. This method not only provides the definitive diagnosis, but the viral isolate is now available for further testing, including pathogenicity, antiviral resistance, and DNA sequencing and analysis. Alternatively, antibody testing can be performed, with a standard four-fold titer increase to the specific subtype of avian influenza virus. Neutralizing antibody titer assays for H5, H7 and H9 are performed by the micorneutralization technique [72] . Western blot analysis with recombinant H5 is the confirmatory test for any positive microneutralization assay [59, 60, 72] . More recently, rapid diagnosis can be performed with reverse transcription-PCR on clinical samples with primers specific for the viral subtype [73] [74] [75] . This test should be performed only on patients meeting the case definition of possible avian influenza A infection.
Any suspected case of avian influenza in a human should be investigated by the public health officials in the province or country of origin [39, 76] . Additionally, governmental labs are often equipped with the appropriate biolevel safety 3 laboratories, primer libraries, and associated expertise to confirm the diagnosis quickly and efficiently. Any clinical specimens should be submitted with the assistance of the public health experts.
Treatment of avian influenza infections in humans includes antiviral therapy and supportive care. Controlled clinical trials on the efficacy of antivirals (NA inhibitors), supportive therapy, or adjuvant care have never been performed, so current recommendations stem from the experiences of past avian influenza outbreaks and animal models. The adamantanes (rimantadine and amantadine) and NA inhibitors (oseltamivir and zanamivir) are the antivirals used for treatment and prophylaxis of influenza infections in humans. In avian influenza virus infections, adamantanes have no role due to widespread resistance through a M2 protein alteration. In addition, over 90% of isolates of H1 and H3 human subtypes during seasonal influenza have had resistance to the adamantanes [77] . Their role has now been limited to prophylaxis in the community when the circulation strain is know to be susceptible to the adamantanes [78] [79] [80] .
NA inhibitors (oseltamivir and zanamivir) have been studied for both treatment and prophylaxis with the human influenza A subtypes H1, H2, and H3 as well as influenza B (Table 7 ) [80] [81] [82] . In animal models with HPAI H5N1, their efficacy has been well documented, with improved survival rates seen after infection [83] [84] [85] . Oseltamivir has been used in avian influenza outbreaks involving H7N7 and HPAI H5N1, and therapy with oseltamivir has been shown to decrease the viral load in nasal secretions in patients infected with HPAI H5N1 [11, 86, 87] . Resistance to oseltamivir has been documented in a HPAI H5N1 subtype in a Vietnamese girl treated with 75 mg daily for 4 days as post-exposure prophylaxis [68] . The NA glycoprotein had a histidine to tyrosine substitution at position 274, conveying a markedly higher IC50 for oseltamivir [68, 88] . In one study, the viral count of HPAI H5N1 in nasal secretions did not decrease with the administration of oseltamivir when the H5N1 isolate carried this resistance mutation [68] . However, resistance produced by this change may be overcome with higher doses of oseltamivir in vitro, and this change has not been documented to confer resistance to zanamivir [88] .
The timing of treatment with NA inhibitors is paramount, as early therapy is directly related to improved survival [66, [83] [84] [85] . The greatest level of protection was seen if the NA inhibitors were started within 48 hours of infection, and protection rapidly dropped after 60 hours [78, 79] . These initial studies, however, were performed with seasonal human influenza A and B, where the period of viral shedding is approximately 48 to 72 hours. In HPAI H5N1 cases from Southeast Asia, survival appeared to be improved in patients who received oseltamavir earlier (4.5 days versus 9 days after onset of symptoms) [66] . Both of these time periods are much longer than documented in animal models, so the window of optimal therapy is still unknown, particularly if viral shedding exceeds the average 48 to 72 hour period seen in seasonal influenza A and B infections.
Combination therapy with influenza A viruses has not been studied [84] . Ribaviron by inhalation has been evaluated in vitro with some avian influenza A subtypes and has been found to reduce mortality from influenza B in a mouse model [89] . Further animal model studies are indicated to determine if there is a role for ribaviron or combination therapy with avian influenza A viruses.
Supportive care with intravenous rehydration, mechanical ventilation, vasopressor therapy, and renal replacement therapy are required if multiorgan failure and ARDS are a feature of disease [69, 90] . Due to the progression of pneumonia to ARDS, non-invasive ventilation is not recommended, and early intubation may be beneficial before overt respiratory failure ensues. Corticosteroids have been used in some patients with HPAI H5N1, but no definitive role for steroids has been determined. Other immunomodulatory therapy has not been reported [91] .
Human vaccination for avian influenza viruses has not been widely used, although multiple vaccination trials are underway. Prior avian vaccines in humans have been poorly immunogenic and thus have limited use. An inactivated H5N3 has been tested and was tolerated but with limited immunogenicity [91, 92] . Other H5 vaccines have resulted in the development of neutralizing antibodies, but to a limited degree [93, 94] . Recently, a large randomized trial looked at an H5N1 attenuated vaccine from the Vietnam strain [95] . Only a modest immune response was seen, with microneutralization antibodies being developed at 12 times the dose used in the seasonal influenza vaccine. The side effects were minimal. A number of other industry trials with adjuvant vaccines are currently ongoing. Although promising, human vaccination against avian influenza viruses is still under development. Underscoring this development is the uncertainty of a pandemic strain, which may have vastly different antigenic properties from any developed H5 vaccine.
Health care infection control is a crucial component in the management of avian influenza infection or a new pandemic strain. Experience from the severe ARDS outbreak in 2002 has illustrated that appropriate infection control measures are paramount to reduce spread to health care workers and, possibly, the community [96] [97] [98] . Therefore, the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC) recommend contact and airborne precautions for any initial suspected case of avian influenza in a human [99] . In late October 2006, the CDC released updated interim guidance on the use of masks and respirators in the health care setting (Table 8 ) [99] . In certain high risk procedures, additional protection may be considered given the likelihood of generating aerosol particles that may enhance transmission (Table 9 ) [99] . Respiratory protection should be worn along with an impermeable gown, face shield, and gloves. Initial cases should be placed in a negative pressure isolation room with 6 to 12 air changes per hour. Hand hygiene with antibacterial soap or alcohol based washless gel should be standard, with appropriate basins at each patient room. Seasonal vaccination of all health care workers should be preformed and further emphasized in order to reduce the likelihood of co-infection with two stains of influenza. Visitors and family members should be strictly monitored and their access to the patient limited to reduce the likelihood of spread. Finally, antiviral chemoprophylaxis should be available to any health care workers exposed to an infected individual. Any symptomatic worker should be taken off duty and workplace surveillance should occur. With these aggressive measures, risk to health care workers, patients, and family members will be reduced.
Avian influenza viruses have occurred with increased incidence within the human population, reflecting the delicate and tangled interaction between wildlife, domesticated animals, and humans. Disease in humans can be limited to conjunctivitis or an influenza-like illness, but HPAI H5N1 causes mainly severe pneumonia, respiratory failure, and death. Most cases have occurred through direct transmission from infected poultry or waterfowl, with only a few limited cases of human to human transmission. Treatment has been successful with the NA inhibitors if started early, and vaccine development is underway with a more immunogenic attenuated H5N1 virus preparation. Infection control measures are the mainstay for prevention and disease reduction. Avian influenza viruses may constitute part of the next pandemic, so appropriate knowledge, prevention, and treatment will reduce the likelihood of this occurrence. Table 9 High risk aerosol procedures in avian influenza
Non-invasive mechanical ventilation Bronchoscopy Humidified oxygen delivery Non-rebreather mask without expiratory filter This article is part of a thematic series on Disaster management edited by J Christopher Farmer.
Other articles in this series can be found online at http://ccforum.com/articles/ theme-series.asp?series=CC_Disaster Clinical review: Mass casualty triage – pandemic influenza and critical care Worst case scenarios for pandemic influenza planning in the US involve over 700,000 patients requiring mechanical ventilation. UK planning predicts a 231% occupancy of current level 3 (intensive care unit) bed capacity. Critical care planners need to recognise that mortality is likely to be high and the risk to healthcare workers significant. Contingency planning should, therefore, be multi-faceted, involving a robust health command structure, the facility to expand critical care provision in terms of space, equipment and staff and cohorting of affected patients in the early stages. It should also be recognised that despite this expansion of critical care, demand will exceed supply and a process for triage needs to be developed that is valid, reproducible, transparent and consistent with distributive justice. We advocate the development and validation of physiological scores for use as a triage tool, coupled with candid public discussion of the process. It is widely accepted that conditions exist for the evolution of a new strain of influenza virus with the potential to cause a human pandemic [1] . The biggest challenge in planning for an influenza pandemic is the range of unknown factors; its nature and impact cannot be fully predicted until the pandemic virus actually emerges. Those planning for a pandemic must, therefore, work from a number of assumptions based on knowledge gained from previous pandemics and scientific modelling of a range of potential scenarios. The UK Pandemic Influenza Plan [2] sets out a range of possible scenarios for clinical attack rates and case fatality rates during a pandemic, including the potential for more than one wave. The base scenario assumes a clinical attack rate of 25% and a case fatality rate of 0.37%, giving rise to 53,700 excess deaths in the UK. A reasonable worst case scenario involves a cumulative clinical attack rate of 50% with 2.5% case fatality, causing 709,300 excess deaths. Similarly, the US Department of Health and Human Services predicts that in a "moderate" scenario based on a virus with 1968-like pathogenicity, 865,000 will require hospitalisation and 65,000 (7.5%) will require ventilation. They also outline a "severe" 1918-like scenario with 9.9 million hospitalisations and 743,000 patients requiring ventilation [3] .
An influenza pandemic will undoubtedly create a major increase in demand for critical care services. The majority of UK hospital intensive care units (ICUs) are already operating at > 98% bed occupancy. Integral to the success of any emergency planning strategy is 'surge capability', incorporating the ability to scale up the delivery of appropriate specialist care to those that require it [4] . Modelling of the impact of an influenza pandemic on UK critical care services has been carried out using the FluSurge 1.0 programme developed at the US Centers for Disease Control [5] . With simulation of an 8-week epidemic and 25% attack rate the demand for critical care beds from patients with influenza would represent 208% of current combined level 2 (highdependency unit) and level 3 (ICU) bed capacity, and 231% of current level 3 capacity [6] . Even allowing for optimistic estimates of other modulating factors (50% reduction in ICU demand with use of neuraminidase inhibitors and 50% upgrade of level 2 to level 3 beds), level 3 bed occupancy due to the pandemic would remain at 75%. Furthermore, occupancy of level 3 beds by 'flu patients' was unsustainable at approximately 50% in terms of care for other patients even in the most optimistic conditions. SARS outbreak, up to 32% of cases were admitted to ICU, 25% were mechanically ventilated and 28 day mortality for ventilated patients was 45% [13] . In Singaporean SARS patients admitted to ICU, 98% developed ARDS [13] .
Properly constructed plans for the delivery of critical care during an influenza pandemic must include the ability to deal with excessive demand, high and possibly extreme mortality, and the risk to the health of critical care staff.
The consequences of a pandemic, both in terms of numbers of patients and the effect on the healthcare system, are likely to precipitate a 'major incident' where special arrangements are needed to manage the system while it is under extreme pressure. It is anticipated that there will be an overwhelming demand for critical care services, not only for respiratory support through mechanical ventilation but also for a full range of care to manage multi-organ failure. Assuming that the next pandemic derives from the H5N1 strain, the epidemiological evidence to date suggests extremely high mortality and, although not precisely quantifiable, a significant risk to health care workers. Both of these will undermine the ability to deliver critical care to influenza patients even before consideration is given to the duty of care to other critically ill patients.
Coherent incident response requires a robust command and control structure, with the ability to make rapid informed decisions across an organisation and also across a health economy. In the UK, health incident management is based on a 'medallion' structure, with gold, silver and bronze corresponding to strategic, tactical and operational command levels [14] . North American and Asian health institutions tend to use the Hospital Emergency Incident Command System [15] . The common theme in both systems is a clear command and control structure with which healthcare staff should be familiar [4, 14, [16] [17] [18] [19] . Their generic hierarchical structure allows application to a wide range of incidents whilst retaining familiarity gained from training and exercises. The importance of familiarity with the command and control structure was highlighted in a recent Delphi study [20] and European survey [21] .
Critical care contingency planning guidance from the UK Department of Health places an expectation on providers to expand their level 3 bed capacity by a factor of 3 but no more. Provision of full multiorgan level 3 support is recognised to be unrealistic, but principally respiratory support is felt to be achievable. Cancellation of elective surgery to minimise alternative sources of demand for critical care, upgrading level 2 to level 3 facilities and recruitment of theatre recovery areas and even operating theatres may allow expansion of ICU-like care capacity. Staff in these areas already have the competencies to manage sedated patients and those receiving respiratory support. Escalating their clinical role should require relatively limited focussed training [22] . Other staff may need to be redeployed and receive training in the management of critical care patients to support fully trained staff, permitting a dilution of the standard critical care nurse to patient ratio [23] . Flexibility around dependency level and staff experience will be required [24] . The expansion of ICU capacity to provide critical care in other areas will require the pre-emptive identification, tracing and maintenance of all usable equipment and potentially the stockpiling of key items to allow for rapid up-scaling of activity in response to demand.
It is likely that there will be some variability in the prevalence of influenza across the country during a pandemic wave, with peaks in demand staggered across geographical areas. It may be possible to disperse some of the patient load by interfacility transfer if this occurs to any significant extent.
The expansion of ICU facilities during the SARS epidemic in Hong Kong and Singapore was recently described [25] . Infection control is recognised as an overriding priority for the delivery of critical care, including the ability, in the early stages, to cohort cases. This should ideally include the use of separate entrances and exits, isolation rooms with negative pressure ventilation and dedicated separate healthcare staff. The Toronto experience identified 21 secondary cases of nosocomial transmission of SARS in ICU from an initial index case before infection control measures were introduced. Even following the introduction of extensive protective equipment, nine healthcare workers developed SARS as a result of being present in the room during the intubation of a single patient. In terms of personal protection, planning and practice in the donning of protective equipment (PPE) and prior fit testing is essential [26] . The practicalities of being able to manage patients when fully attired must be understood and consideration given to the fact that any procedure or task will take longer. This will impact on care efficiency and the staff to patient ratio.
While beds can be scaled up and extra areas recruited to provide critical care, without trained staff the planning will be ineffective. Staff illness rates and the risk to staff must be factored into the planning process. In the UK, staff illness has been estimated at 30% with work absences of up to 8 days [2] . Normal working patterns may need to be revised and facilities provided for staff to stay on site rather than go home to their families. Staff absence tends to be greater the longer special circumstances apply and the greater the impact on the lives of the staff [27] . The preventive effectiveness of neuraminidase inhibitors may make focussed chemoprophylaxis a strategy for reducing staff illness in critical care areas [28] .
The evolution of a new pandemic strain of influenza will inevitably result in a major increase in demand for critical care services. It is likely that these services will rapidly reach their capacity and even their contingency arrangements for extended facilities will be overwhelmed. Excessive demand where resources are finite creates an ethical dilemma and many emergency plans apply a utilitarian approach of 'best care for the greatest number' [29] . There is a legitimate debate about how limited capacity can best be utilised, but a number of themes are recurrent. There needs to be a legal and ethical framework for the process decided in advance, the rationale for triage should be fair and transparent and it should meet the principles of distributive justice [30] [31] [32] . Triage can conflict with human rights legislation and even humanitarian laws but 'accountability for reasonableness' can temper the disagreements about priority setting [33] .
The decision making process needs to be valid and reproducible. Although there are a number of triage systems available for mass casualty incidents, there has been little validation of any of them in the field [34] , and what there has been relates to 'big bang' single incidents and the apparent unreliability of triage [35, 36] . While it does not need to be explicit ahead of time, the decision thresholds should be based on both the cumulative evidence about the disease process and prognosis, and the number of patients and severity of illness making the demands on the service [31] . In effect, triage may result in a gradual degradation of care with the increasing scale of the incident and become a 'societally mandated Do Not Resuscitate order'. On these grounds the process needs to be carefully considered at an appropriately senior level and applied consistently [32] .
Allowing for the utilitarian approach, it is recognised that in mass casualty incidents, the standard of care for all patients, including those not immediately related to the incident, may need to be adjusted and reduced. While this may infringe individual rights, the higher ethical principle of 'wellness of society as a whole' allows for the direction of resources to those where it is felt most effective. It may also allow for an expansion in the scope of practice of non-physicians [37] .
It may be unrealistic and impractical to expect that senior medical intensive care staff will make all decisions regarding instituting critical care and there will be a need to empower more referring general clinicians to do so. This is at odds with the need for decision making by the most senior person [32] and will require a change in practice for many clinicians; it is not current practice in the UK. The use of track and triage protocols will be essential to direct this decision making and ensure its consistency. Ardagh [38] has developed a set of pragmatic questions for the clinician facing acute problems of resource allocation; the only point lacking in his assessment process is a tool for the 'ranking' of patients in terms of likelihood of benefit from the limited resources.
We believe that the basic criteria for a system for triage to critical care in a pandemic are fourfold; it should identify patients sick enough to require higher level care at some stage in their illness, it should be able to recognise those patients who are too acutely or chronically unwell to benefit from critical care, it should be consistently applicable by healthcare professionals and support workers from a variety of backgrounds within the constraints of the pandemic and should ideally also be scalable to reflect any mismatch between need and capacity. In order to fairly allocate resources across both flu and non-flu patients it should also be disease non-specific and allow prognostic comparisons across disease categories.
A number of scoring systems have been advocated for use in a pandemic. The UK Department of Health currently recommends a six-point pneumonia severity score [2] . Although US guidelines emphasise the importance of triage in primary influenza, specific tools are only recommended for assessment of post-influenza bacterial pneumonia [39] .
The majority of available potential scores were developed as mortality indicators and perform less well for predicting critical care usage. Amongst ICU admissions with community-acquired pneumonia in Massachusetts in 1996 to 1997, 10/32 scored CURB-65 1 or 2 (that is, low risk) and 5/32 were classified as PSI (Pneumonia Severity Index) class III (intermediate risk) [40] . Even amongst patients with pneumonia included in the PROWESS study, only 90.5% were PSI class IV or V, and only 70.3% had a CURB-65 score of 3 or above [41] .
There is no guarantee that pandemic influenza will be primarily pneumonic in its presentation; case reports have documented H5N1 influenza presenting with diarrhoea [42, 43] and coma [43] and a World Health Organisation summary has described absence of respiratory symptoms in a number of cases [44] . The utility of disease-specific pneumonia scores may also be limited by mortality from comorbidities such as cardiovascular disease.
A number of intensive care scoring systems have demonstrated their power in using physiological derangement to predict mortality or higher resource requirements, whatever the presenting diagnosis [45] [46] [47] [48] [49] . Physiological scores have also been demonstrated to be good predictors of requirement for higher level care on hospital wards [50] , in medical assessment units [51, 52] and in the Emergency Department [53] . We have demonstrated that a purely clinical score incorporating acute physiological derangement and chronic health and performance status can reliably predict requirement for critical care [54] .
It is inevitable that if an influenza pandemic reaches the scale of some predictions, some patients who, in normal circumstances, would benefit from critical care will not be offered it. Critical care triage will need to evolve from a process of identifying cases who need high level care to one that determines those patients most likely to benefit from the limited resources available and distinguishes them from those where care is likely to be futile. This is recognised by the Emergency Medicine community and the US administration in terms of disaster triage [37, 55] . The American Thoracic Society adopted the utilitarian principle a decade ago, stating that "the duty of health providers to benefit an individual patient has limits when doing so unfairly compromises the availability of resources needed by others" [56] . The problem now facing policymakers and clinicians is defining a process for resource allocation that meets the requirements of distributive justice and accountability for reasonableness [33] . As the Working Group on Emergency Mass Critical Care of the Society for Critical Care Medicine recognised, "an ideal triage system is based on data collected at hospital admission, requires little or no laboratory testing, and has been proven to predict hospital survival" [57] .
The Ontario Ministry of Health Long-term Care working group have courageously taken the first steps in defining a triage protocol for critical care [58] and their use of serial Sequential Organ Failure Assessment (SOFA) scores to place a ceiling on care provided to non-responding patients is to be supported. However, it is unlikely to be feasible for all patients to have a trial of inotropes and/or ventilation and some way of screening out the sicker patients at ward/floor level will be required.
We are not aware of the use of objective prognostic scores to allocate or refuse critical care resources at present and indeed most research demonstrates the ad hoc nature of admission decision-making [59] . However, if, as is likely, review by experienced critical care physicians is impractical, decision support will be required for the non-critical care specialist. Emergency physicians, for example, had a positive predictive value (PPV) of only 73% in identifying those with a low chance of survival, as opposed to critical care fellows (PPV 83%) and the Mortality Probability Model (MPM 0 ; PPV 86%) [60] .
SOFA scoring has previously been demonstrated on a multinational basis to predict high risk of mortality (a SOFA score of over 15 was 98.9% specific for mortality) [61] . Other critical care scoring systems show comparable performance in mortality prediction; discrimination as measured by area under Receiver Operator Characteristic (ROC) curve was 0.825 to 0.901 for Acute Physiology and Chronic Health Evaluation III (APACHE III) [62] [63] [64] [65] , 0.79 to 0.846 for Simplified Acute Physiology Score II (SAPS II) [62, 64, 66] , and 0.928 for the Multiple Organ Dysfunction Score [67] . However, calibration of these scores to give absolute risks of mortality has not always been reliable [65] and has required customisation for international use [68, 69] .
Concentrated work is clearly required to amend and validate existing scoring systems so that they are suitable for use as triage tools. We suggest that this should be done on two levels. While disease specific scoring systems are valuable and should continue to be refined, there is a need to develop an appropriately generalisable scoring system for as unselected a group of patients as possible. To have the discriminating power, it will need to take place on a multicentre or, preferably, on a multi-national basis.
It is a general principle of major incident planning that procedures should not be changed at precisely the moment when the system or institution is under its greatest stress, so planning for pandemic flu needs to make use as much as possible of systems and procedures already in place. Development of a triage system and tool needs to be accompanied by planning for hospital command and control (to dictate scalability as related to available resources) and by training for staff whose roles may change.
Researchers, clinicians and policymakers in the field need to analyse systems and scores already in existence and improve and validate them as triage tools (though this may not be the purpose for which they were originally developed). At the same time ethical principles require transparency and consistency in the decision-making process, and involvement of public in its development.
In reality, perhaps the question we need to address is the action required when critical care services are overwhelmed. The scalability of triage tools may aid in decision making by objectively altering the threshold for admission to critical care. However, the time may come when we need realistically to evaluate the effectiveness of critical care in influenza. If survival with the benefit of critical care is marginal (for example, <10%) and there is a significant cross-infection risk, perhaps critical care should then close and concentrate its efforts on outreach to other areas, including wards. Direction and support from professional bodies and health departments will be required to support the medical staff with such difficult decisions possibly against a ground swell of media-driven public opinion.
DW is a member of the UK Department of Health Critical Care Contingency Planning Working Group. The other authors declare that they have no competing interests. Avian influenza outbreak in Turkey through health personnel's views: a qualitative study BACKGROUND: Avian influenza threatens public health worldwide because it is usually associated with severe illness and, consequently, a higher risk of death. During the first months of 2006, Turkey experienced its first human avian influenza epidemic. A total of 21 human cases were identified, 12 of which were confirmed by the National Institute for Medical Research. Nine of the cases, including the four fatal ones, were from the Dogubeyazit-Van region. This study aims to evaluate the efforts at the avian influenza outbreak control in the Van-Dogubeyazit region in 2006 through the experiences of health personnel. METHODS: We conducted in-depth interviews with seventeen key informants who took active roles during the avian influenza outbreak in East Turkey during the first months of 2006. We gathered information about the initial responses, the progress and management of the outbreak control, and the reactions of the health professionals and the public. The findings of the study are reported according to the topics that appeared through thematic analysis of the interview transcripts. RESULTS: Following the first suspected avian influenza cases, a Van Crisis Coordination Committee was formed as the coordinating and decision-making body and played an important role in the appropriate timing of decisions. The health and agriculture services could not be well coordinated owing to the lack of integrated planning in preparation for outbreak and of integrated surveillance programs. Traditional poultry practice together with the low socio-economic status of the people and the lack of health care access in the region seemed to be a major risk for animal to animal and animal to human transmission. The strengths and weaknesses of the present health system – primary health care services, national surveillance and notification systems, human resource and management – affected the inter organizational coordination during the outbreak. Open communication between the government and the public played an important part in overcoming difficulties. CONCLUSION: Although there were problems during the avian influenza outbreak in Turkey, the rapid responses of the central and regional health authorities and the performance of the health workers were the key points in controlling the epidemic. The lessons from this outbreak should provide an opportunity for integrating the preparation plans of the health and agricultural organizations, and for revising the surveillance system and enhancing the role of the primary health care services in controlling epidemic disease. Developing successful strategies based on knowledge and experience may play a valuable role in delaying an avian influenza pandemic. The highly pathogenic Avian Influenza A (H5N1) virus has caused more than ten outbreaks worldwide and many related human fatalities have occurred since 1997. A total of 258 laboratory-confirmed cases have been reported in South East Asia, North Africa and Europe by the World Health Organization (WHO) and 154 people with confirmed avian influenza died between 2003 and 2006 [1,2] . Most patients have similar features; they are children, they have a history of close contact with poultry or wild birds, and they live in some of the poorest areas of the world. The human cases in Turkey and Azerbaijan were the first confirmed reports of human avian influenza infection outside Asia and Africa. This was alarming not only for Turkey but also for the European Union and other countries. After considering the epidemiological and laboratory evidence, WHO has maintained its pandemic alert at Phase 3 (of 6), indicating that the new influenza strain causes human infections with no or very limited human to human transmission [3] .
Effective surveillance, early warning systems and containment measures based upon a general capacity for health care have been recognized by WHO as the essential action strategies [4, 5] . In May 2002, "The Global Agenda for Influenza", which was issued by WHO, stressed the necessity of expanding animal influenza surveillance, and noted that studies at the domestic-wild bird and humandomestic bird interfaces are part of this activity [6] .
The H5N1 type of avian influenza virus outbreak was lived in Turkey on January 2006; a total of 21 human cases were identified [3] , 12 of which were confirmed by the National Institute for Medical Research [3, 7] . Nine of the cases, including the 4 fatal ones, were from the Dogubeyazit-Van region. The confirmed cases were people aged 3-16 years who had close contact with ill poultry in the rural area in Dogubeyazit or in migrant wards in Van [3, 7, 8] . The timeline of events was summarized in Table 1 .
In this period, more than one hundred suspected human cases (21% of total suspected patient in YYU Hospital) underwent prophylactic therapy in the outpatient clinic and twenty five percent of patients who had a history of contact and/or clinical findings were hospitalized in YYU Hospital [7] . Tens of thousands of chickens were culled [8] .
Avian influenza infections in Turkey provide an example of concurrent animal and human avian influenza epidemics. These were the first occurrences of human cases in the country, and the impact of the epidemic was quite strong. It was the main agenda for the local and national institutions of health, agriculture and other sectors, the community and the media during the first months of 2006.
This study aims to understand the course of the avian influenza outbreak control in Turkey through the views of health care providers. It will also highlight what local health personnel in various positions experienced during that period.
This qualitative study was carried out in Dogubeyazit and Van in May 2006. We interviewed seventeen key informants who took active roles during the outbreak of avian influenza in East Turkey during the first months of 2006. The twelve of interviewees were medical doctors (directors, specialists and general practitioners), three were allied health personnel (one director and two health officers), and two were midwife and nurse (primary health care provider and director). Most of the informants were senior staff and had primary responsibilities for the management of the outbreak control.
Five informants were from the Van Provincial Health Directorate (PHD). These informants were the Director and the Deputy Director of the PHD, who were the main coordinators of the avian influenza outbreak intervention, two department chiefs who were involved in implementing the intervention and one health officer who is responsible for transportation of the samples and record keeping.
Four informants were health personnel in primary health care centers; three general practitioners and one midwife, who have active roles in surveillance in Van and Dogubeyazit. One of the general practitioners made the initial diagnosis of the avian case at a primary health care center in Van. Two general practitioners were staff of the primary health care center in Dogubeyazit. One of the doctors were the chief of the primary health care center and worked as a coordinator of avian influenza outbreak intervention and the other one was assigned to the avian influenza surveillance.
Three informants were staff of the State Hospitals in Van and Dogubeyazit. The first was an infectious disease specialist working in Van State Hospital. The second was the Director of Dogubeyazit State Hospital during the outbreak. The third was a pediatrician working in Dogubeyazit State Hospital who pre-diagnosed the avian cases in Dogubeyazit. They worked as if gate-keepers between the primary health care center and the YYU Hospital during the outbreak.
Five informants were Van YYU hospital staff. They were the Director of the University Hospital, the chief of nursing staff, two specialists and one health officer. The infectious disease specialist is on duty in the infectious disease control committee of YYU Hospital, who was a counselor in the Van Crisis Coordination Committee (CCC) and communicated with WHO representatives for the outbreak control. One internal medicine specialist who was also a native of Dogubeyazit helped the Provincial Health Directorates and WHO representatives to communicate with families from Dogubeyazit. The health officer was assigned to the emergency services during the outbreak and he was also the representative of the Health Workers Union in Van.
We used a semi structured guide consisting of open ended questions during in-depth interviews. All interviews were audio-taped and field notes were taken afterwards from each interview for data-gathering. The appointments with the informants were arranged by the second author of this study, who worked in Van Yuzuncu Yil University, and the interviews were conducted by the first author, who worked outside the region. The authors clarified the purposes of the study and the interviews were conducted with the informants' consent for audio-taping and for the use of their words in the report. In the institutions where the authors were affiliated, interview studies are not subject to permissions from ethical committees. Thus, the authors did not apply for approval of these institutions.
Following the interview guide, we asked the participants to inform us about the events chronologically: the first responses of the organizations, the management of outbreak control, the reactions of the health personnel and the resident population, the inter-organizational coordination, and interviewees' roles during the avian influenza outbreak in Turkey. We also asked them to evaluate the strength and weakness of the outbreak control, and the lessons from the avian influenza outbreak.
Records were transcribed verbatim. The authors read and identified codes for major themes according to their interest for the study. These codes were then marked to lines of text and the relevant codes were collected one under the other for preliminary analysis. The second analysis [7] involved recovering the relevant codes from the text. The results of the study are reported according to the topics revealed by the analysis.
In the avian influenza outbreak, the studies by health organizations were defined by two features as suggested by the interviewees: it was perceived as a new and unknown disease; and it was encountered as a regional, national and international crisis by the health authorities. The preparedness of animal and human health organizations for outbreak-human resource, management, surveillance approach and notification systems-affected inter organizational coordination during the outbreak. The comments related to follow up show us that there is an inconsistency between the pandemic influenza action plan and avian influenza surveillance approach. Although a circular of Turkish MoH was announced on October 2005 including a flow chart to evaluate the suspected cases, the exclusion of primary health care centers and hospitals in the sentinel surveillance and the lack of regional preparedness might have caused health care workers to miss some cases in the referral and reporting procedures. The cost of the culling animals was partially paid to the owners to compensate their economic loss. But in practice the official records couldn't be made exactly, therefore most of poultry owners could not receive the compensations and they had been subject to high rates of financial loss. The situation was expressed by the general practitioner from the primary health care center in Dogubeyazit:"Now about 90-100 thousand chickens were slaughtered. Five Turkish liras were to be paid for each but only 30% of this price was paid because 70% of them were taken without any official record so people couldn't claim their rights."
After the declaration of avian influenza outbreak by the Turkish MoH, people's reactions to the disaster and patient applications increased to health care centers. The fear and panic were experienced among health care providers. This situation was explained by a health officer from YYU hospital: "When cases arrived in Van According to the health care providers' experiences, to deal with an outbreak, the health system must be strengthened, health services should be coordinated, surveillance system and the notification for communicable diseases must be operated efficiently, human and animal health care should be integrated.
Health personnel suggested that the preparedness of the health organizations is very important for an outbreak in terms of equipment, human resources and information gathering The preparation studies should not only be on the management level, but also throughout the health organization and the experience of public health workers must be taken into account: "My opinion is that continuing education of health care providers should be provided, so the practitioner who notices a suspected case can take the details of the patient's history. If it is done like this, the system will work better, a recording system is formed".
According to the experiences of health personnel, sectors and institutions related to the outbreak control must work in collaboration and be coordinated: "(...) avian influenza control absolutely a multi-sector activity. No one should try to be a hero. Every health organization continues with its task; if the organizations of agriculture, municipality, gendarme, police, mufti, education are not included, your success will be overshadowed, and your aim is not achieved."
The subjects that most stressed by the health personnel were the prevention of transmission from animal to animal and from animal to human. The health personnel agreed that besides culling animals, studies on preserving animal health must be developed and sustained. In place of traditional poultry farming, alternatives suggested included changes to modern poultry farming at nearby settlements:
"At this time our advice to the Van PAD was that since all of them were culling, new projects must be developed and information should be gathered on poultry farming. Of course, this should be done with government support."
"The origin oriented control should be. Firstly, a surveillance system at our borders must work well, especially in terms of animal surveillance, but also mainly before disaster happens, not only when a pandemic or epidemic occurs (...)."
As the health personnel, communication and information gathering is very important for the outbreak control. The public's psychology and needs must be taken into account to enable them to cooperate with the health teams. The general practitioner who has a close relationship with the local people emphasized the importance of people's psychology and environmental factors. "I mean, why wasn't 'a child playing with chicken heads like puppets' put on the agenda? When she was discharged from the hospital, she came to visit me and I bought her a doll and advised her not to play with chickens, which are microbial things. She slept with the doll for many nights. Why didn't anyone underline this occasion? Why was the people's psychological status not taken into account? Why wasn't attention paid to the problems related to the housing infrastructure or the lack of clean water? I still can't accept this; it still affects me."
The Turkish Avian Influenza outbreak is an example for other countries in respect of the experiences and lessons achieved by the health organization managers and health care providers who actively worked in the process.
The health care providers who participated in our study think that the avian outbreak reached national and regional crisis level because they faced an unknown emerging infectious disease and the threat to health that it was not predicted.
According to the health care providers, the most critical issue was the coordination between the agriculture and health organizations during crisis control. The main reason for this problem was the lack of organizational preparation compatible with central or regional plans. To be prepared for an outbreak, it is vital to define the central, regional and local organizational framework, to maintain close cooperation and collaboration between the health and agriculture sectors and to share information on surveillance, evaluating the risk to humans and planning interventions at the time when a case occurs [9] [10] [11] [12] . WHO indicated that Turkey's preparedness plan provided a framework for action, even if not yet fully developed [3] .
Indeed the Turkish MoH published the country's first national pandemic influenza action plan in October 2005 and a circular was sent to health organizations about possible avian influenza case descriptions and precautions [3, 8] . On the other hand, the results of this study indicates that, the circulation of the avian influenza action plan was insufficient, and regional and national activities related the outbreak control only started after following the verification of H5N1 virus in poultry. As preventive human and animal health care, organizational preparations were incomplete. WHO states that there was no active surveillance in neighbouring provinces after a domestic animal outbreak was confirmed in Igdir in December 2005 [3] .
According to the health care providers who participated in our study, a reference laboratory could not meet the demand for the animal samples and after the human cases arose, the Turkish MoA decided to cull all the poultry without searching for possible or confirmed cases. Because there were not enough agriculture personnel for the culling at Van, the Turkish MoA purchased support from private veterinary clinics. This shows us the size of crisis and the lack of preparedness such as reviewing organizational facilities and operational plans in pre-outbreak phase. As a result, the work of outer-organizational teams, which proceeded in their own region, was not integrated with that of other regional health teams that continued surveillance. The evaluation of animal health services at that period is limited in this study because we did not interview agriculture personnel. Future research should focus on the experiences of the animal health service personnel.
Evidently, chaos is unavoidable in primary health care services that do not include central and organizational level intervention plans for emerging avian influenza-like infectious diseases, as in the Turkey example. The health care providers think that the strengths and weaknesses of the health organizational structure before the outbreak affected the success of the intervention and problems were encountered in coordination between health institutions. While a failure is expected even in the health services of developed countries following a possible avian influenza pandemic [13, 14] , the chaos can be bigger in developing countries due to the weaknesses of health organization. Therefore, strengthening the healthsystem in developing countries shouldbe considered as a factor in delaying the worldwide spread of avian influenza. National and international network and partnerships have to maintain current public health and animal health infrastructures and resources for construction, modernization, enhancement and recruitment for unprecedented emerging and reemerging disease [15] .
The most critical issue related to the health system is the surveillance system. The notification of communicable diseases in Turkey was changed in recent years. In the new system, influenza group diseases are subject to sentinel surveillance. According to the sentinel surveillance, primary health care centers have no obligation to notify influenza group diseases including the possible avian influenza cases. Only the training and research hospitals in some selected provinces have the duty for notification of confirmed influenza cases. During the Turkish avian influenza outbreak, Van and Dogubeyazit (Agri) were not included among the selected provinces.
The health care providers who participated in our study emphasized that the widespread of the outbreak requires follow-up and assessment of all influenza cases who applied to health care centers. As the interviewees stated, trace-back investigations were not performed because of the current surveillance system mentioned above. The need for an active surveillance system is clear for an epidemic disease such as avian influenza, which is seasonal, regional and closely related to epidemic diseases related to agricultural practice. In the WHO Report dealing with lessons from the avian influenza outbreak in Turkey, the need for active surveillance of animal and human avian influenza outbreaks was underlined [3] . A well integrated effort such as lining up a network between animal and public health laboratory system is needed to define an epidemic earlier, ensure more effective control measures [7, 15] . This will possibly make us gain valuable time by delaying a pandemic.
Practitioners, midwives and nurses, who are responsible for population-based health care services in the primary health care system, became the most important human resource for surveillance during the avian influenza outbreak in Turkey. This experience clearly shows that primary health care centers should be included in the surveillance system. For a efficient surveillance system, upgrading the sentinel physician network by enlisting and retraining more participants and [16] the coordination among public health workers, clinicians and managers is most necessary. It is accepted that public health workers will play an integral role in an influenza pandemic [17] . The health managers and providers whom we interviewed believe that their experiences must be reflected in national avian influenza preparedness plans.
Another problem encountered during the avian influenza outbreak in Turkey was the fear of contamination risk among the health care providers who have no protective equipment against H5N1 when the outbreak started. It is as important to train the health personnel to be prepared for an outbreak as it is to stock protective equipment for them at the sites of preparation studies [14, 15, 18] . Interviewees believe that the delay in supplying protective equipment and the lack of orderly planning in the distribution of it caused concerns among health care providers about contamination risk, which affected their work output. Health personnel must be accepted as the primary group in prevention, and mortality and morbidity among them must be minimized to allow for efficient intervention by the maximum number of personnel during an avian influenza pandemic. Even if epidemiological evidence about the spread of the H5N1 virus from patients to health personnel is limited [19] , studies indicate that nurses who work in hospitals and public health workers touching the suspected avian influenza cases may experience fear and anxiety for their own and their families' health and can face ethical dilemmas when deciding between continuing their work and maintaining their families' health [20, 21] .
Several problems were experienced during intervention in the outbreak by health care providers who participated in the study. They may be classified as problems related to the socio-economic conditions of people living in the region, and those related to the health care system, including the management and surveillance systems:
The health care providers emphasized that people who lived in the rural part of Dogubeyazit and the suburban areas of Van that gather migrants lacked basic needs such as health, education and infrastructure. According to the interviewees, the outbreak in Turkey was influenced by factors closely related to traditional poultry farming and poverty-line economics, as was the case in Asian countries [22] . According to the health personnel who participated in our study, the infection spread rapidly because domestic birds belonging to neighbourhood backyards were able to walk around freely. It is possible that wild migrant birds may be in contact with poultry in the area for food or water. Family-based small-scale poultry farming is a major source of income and nutrition for the region.
All our interviewees thought that the most important factor in the transmission of the H5N1 virus from animal to human was the sharing of shelter according to the low socioeconomic status of the family. Especially during fall and winter, family members share their one sleeping and dining room with poultry. From the year 2004, the contact histories of children and young adults who are described as a risk group for avian influenza cases throughout the world resemble the ones in Turkey [23] . Health care providers who worked in Van and Dogubeyazit stated that people had eaten ill chickens before death and young adults had taken part in the cleaning, cooking and slaughtering of the chickens. Children had played with sick animals and corpses.
The low socio-economic status of the people was accompanied by insufficient primary health care services in the region. The first avian influenza cases were diagnosed after lower respiratory tract infection symptoms appeared. All the fatalities were among those who had recently applied to a health center and been diagnosed as avian influenza. Informants have differing explanations for the late diagnosis of the patients. According to some, the first admission of the families was late. Others claim that the family of the first cases was sent back home during the first admission, and avian flu was diagnosed only after their second admission.
The community was affected psychologically and economically from the disaster. Avian influenza created the fear of a mysterious disease on the people. Most of the people were subject to a financial loss because of the slaughtering of their poultries and were impoverished relative to previous status. In this study we found that the communication between community and health personnel is important for establishing public support for outbreak control and to overcome obstacles such as lack of confidence in governmental organizations. After the avian cases were verified in Turkey, the declaration by the Turkish MoH was evaluated affirmative by the health care providers. It is very important that managers be clear about the known and unknown issues by the first stage of the avian influenza crisis, inform the people about health risks and precautions as early as possible, and be sensitive the worries of people [24, 25] .
According to the health personnel interviewed, the reason why poultry were kept as a supply of food and living in Van and Dogubeyazit at the time the culling started was the lack of public education about preventable health problems in the region, as much as economic security. The experiences of health personnel indicate that the people participated more after the crisis centers were established by the health and agriculture boards, phone counselling services were made available, good communication was established between health personnel and the people during surveillance, and information studies were applied by media channels.
Turkey experience in avian influenza outbreak shows that preparation planning and surveillance systems should be rational and sustainable. The lack of organizational emergency disease plans delineating the tasks and responsibilities of health care providers in the event of a possible avian influenza outbreak, and the lack of training about preventive care, caused health personnel to be caught unprepared by the outbreak. The problems in supplying and distributing protective equipment, reflecting another dimension of the lack of organizational preparedness, influenced the efficiency of the health care providers' work because they were anxious about their own and their families' health. During the preparation and updating of national epidemic and pandemic plans, and during outbreak management, public health workers should participate effectively in decision-making and risk communication. A well designed communication strategy people's participation in control and may relieve from the psychological effects of the outbreak.
Animal and human health care services could not be well coordinated owing to the lack of integrated preparation and planning. Because the sentinel surveillance of influenza infections in Turkey was based on the confirmation of the cases diagnosed at the training and research hospitals in selected provinces, detection of possible human avian influenza cases at the primary health care level was hindered and trace-back investigation was precluded. To evaluate the spread of the avian influenza outbreak accurately, evaluation and follow-up of influenza cases needs to be done in all health care centers. Such evaluation also necessitates integrated human and animal surveillance and control measures.
The causes of transmission of the H5N1 virus from wild to domestic animals in Turkey are closely related to socioeconomic conditions and traditional poultry farming. Poverty in the region played a predisposing role in the outbreak and the outbreak also increased poverty. Limited access to education, shelter, water supply and waste removal formed the basis for virus transmission from animals to animals and animals to humans. To reduce the risks rising from these predisposing conditions, infrastructure needs to be built specially at the village level.
Although the limited access to health care services and the high contact rate with ill chickens were among the characteristics of the vulnerable group, the rapid organization of the health authorities during intervention studies prevented an increase in the mortality rate. Despite the above-mentioned problems in preparedness about coordination during the avian influenza outbreak control, the rapid response and performance of the health workers played an important role in controlling epidemic.
the manuscript. TE arranged appointments with the informants during the course of data gathering and contributed to analyzing the data. Both authors read drafts of the manuscript, made comments and approved the final version. Influenza activity in Europe during eight seasons (1999–2007): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (spread) across Europe BACKGROUND: The European Influenza Surveillance Scheme (EISS) has collected clinical and virological data on influenza since 1996 in an increasing number of countries. The EISS dataset was used to characterise important epidemiological features of influenza activity in Europe during eight winters (1999–2007). The following questions were addressed: 1) are the sentinel clinical reports a good measure of influenza activity? 2) how long is a typical influenza season in Europe? 3) is there a west-east and/or south-north course of peak activity ('spread') of influenza in Europe? METHODS: Influenza activity was measured by collecting data from sentinel general practitioners (GPs) and reports by national reference laboratories. The sentinel reports were first evaluated by comparing them to the laboratory reports and were then used to assess the timing and spread of influenza activity across Europe during eight seasons. RESULTS: We found a good match between the clinical sentinel data and laboratory reports of influenza collected by sentinel physicians (overall match of 72% for +/- 1 week difference). We also found a moderate to good match between the clinical sentinel data and laboratory reports of influenza from non-sentinel sources (overall match of 60% for +/- 1 week). There were no statistically significant differences between countries using ILI (influenza-like illness) or ARI (acute respiratory disease) as case definition. When looking at the peak-weeks of clinical activity, the average length of an influenza season in Europe was 15.6 weeks (median 15 weeks; range 12–19 weeks). Plotting the peak weeks of clinical influenza activity reported by sentinel GPs against the longitude or latitude of each country indicated that there was a west-east spread of peak activity (spread) of influenza across Europe in four winters (2001–2002, 2002–2003, 2003–2004 and 2004–2005) and a south-north spread in three winters (2001–2002, 2004–2005 and 2006–2007). CONCLUSION: We found that: 1) the clinical data reported by sentinel physicians is a valid indicator of influenza activity; 2) the length of influenza activity across the whole of Europe was surprisingly long, ranging from 12–19 weeks; 3) in 4 out of the 8 seasons, there was a west-east spread of influenza, in 3 seasons a south-north spread; not associated with type of dominant virus in those seasons. Influenza has an important impact on societies each season. Surveillance data not only provide valuable information on the burden of disease in the population [1, 2] , but also enables an assessment of whether the vaccine is a good match with the circulating virus [3, 4] . Surveillance may help to plan and allocate health care resources and is important for pandemic preparedness [5] . In addition, the surveillance infrastructure can be used to monitor new emerging respiratory diseases, like SARS or avian influenza in humans [6] . Countries in Europe have shared detailed clinical and virological data via the European Influenza Surveillance Scheme (EISS) since 1996 [7] . This collaborative project is partially funded by the European Commission through the European Centre of Disease Prevention and Control (ECDC) and currently includes 30 countries. The scheme covers a total population of about 450 million inhabitants and an area of roughly 12 million square kilometres. EISS collects two types of data on influenza activity each season: 1) clinical and virological data collected by sentinel GPs and 2) virological data from non-sentinel sources. In the present study the EISS dataset was used to characterise important epidemiological features of influenza activity in Europe during eight winters (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) .
In recent seasons there have been indications that influenza activity first appeared in the west of Europe and then moved east across Europe. As an example, during the 2003-2004 season, activity began in Ireland, the United Kingdom, Portugal and Spain in early August and reached Poland in February 2004 [8] . These observations led us to assess three related questions:
1. Are the sentinel clinical reports a good measure of influenza activity? 2. How long is a typical influenza season in Europe?
3. Is there a west-east spread and/or south-north spread of influenza in Europe?
Data from a median of 17 (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) . Countries were included in the analysis if they were at least 5 years active member of EISS and if weekly data were available for the full season. The assessment of influenza activity presented in this paper is largely based on data reported by sentinel GPs. The GPs report clinical cases of influenza-like illness (ILI) and/or acute respiratory infection (ARI) to a central registry and take respiratory specimens that are sent to a national reference laboratory for testing. This ensures that the clinical data reported by the sentinel physicians are validated by virological data on influenza. The national reference laboratories also report laboratory test results on non-sentinel respiratory specimens e.g. specimens from hospitals or non-sentinel physicians. These data were collected to have an additional indicator of influenza activity and to validate the sentinel virological data. The national reference laboratories participate in the 'Community Network of Reference Laboratories for Human Influenza in Europe' (CNRL), which is coordinated by EISS [9] . CNRL works closely with the WHO through its network of National Influenza Centres and collaborates with the Centre for Reference and Research on Influenza at Mill Hill, London, UK.
In the current study only the time points of highest clinical and virological activity were used, because only peak levels represent undisputable markers of activity in a given country. The reason not to take whole incidence curves into account is that incidence rates in Europe vary considerably, because: 1) case definitions are not yet harmonised across Europe; most countries report cases of ILI, but some report cases of ARI, 2) the denominator calculations vary by country and, 3) consultation rates for ILI and ARI vary among countries. They not only depend on cultural factors, but also on the delivery of health care. For example, in some European countries a doctor's certificate is required for a single day of absence from work (leading to a higher consultation rate), whilst in others a certificate is only required after absence of 5 days or more, leading to a lower consultation rate.
The weeks of peak activity were selected by plotting the clinical and virological data available for each country. If the clinical and virological activity was very low during a season (e.g. below or around the baseline level; defined as level of influenza activity in the period when no influenza virus was detected), it was difficult to identify the peak week and no peak was selected. Most countries reported cases of ILI to EISS (13 out of the 17 countries); four countries used the less restrictive case definition of ARI (Czech Republic, France, Germany and Romania). Since 2004 the Czech Republic and Romania also report cases of ILI [10] . The case definitions of ILI and ARI have been described by EISS and discussed by Aguilera et al. [11, 12] . Briefly; the general criteria for ILI are: sudden onset of fever > 38°C, with respiratory (i.e. cough, sore throat) and systemic symptoms (headache, muscular pain); the criteria for ARI are: sudden onset of respiratory symptoms, accompanied by fever and headache in the absence of other diagnosis.
For the validity analysis of sentinel reports, we defined a good match as a situation where the sentinel and virological peaks occurred in the same week, or when there was a difference of only one week. For example the peak of the incidence of ILI consultations in the Netherlands during the 2002-2003 season was week 10 and the peak of positive laboratory reports of the dominant influenza virus was week 9. This difference of 1 week was considered to be a good match. Therefore, a time difference between peaks of 1 week or less was taken as a measure for a good match (irrespective of which peak presented first); a difference of 2 weeks was taken as a reasonable match and a longer period as a poor match. The analysis was based on the percentage of countries fulfilling the criteria of a good or moderate match during 8 influenza seasons. In a second validity analysis, using similar criteria, the sentinel clinical incidences were compared with non-sentinel laboratory reports. Because the case definitions of ILI and ARI differ considerably, the validity analyses were performed separately for countries using ILI (n = 13) and countries using ARI criteria (n = 4). Differences in matching percentages between ILI and ARI were statistically evaluated using the Chi-square test or Fisher exact test if the expected value in one of the cells was less than five.
The length of an influenza season was roughly calculated by subtracting the earliest and latest week of peak clinical activity across Europe for each season. Per season the aggregated data of participating countries were used. Knowing that using peak weeks as indicator for activity would lead to underestimation of the length of the epidemic, because periods of high activity at the beginning and the end were not taken into account, 4 weeks were added: 2 weeks before the earliest peak and 2 weeks after the last peak. These periods still represent a rather conservative estimate of the slopes of increased activity around the incidence peaks. Eight countries participated throughout the 8 seasons (their longitudes ranging from -4 to 15.3); 6 countries during 7 seasons (longitudes -8 to 19.3) and 14 countries were included during less than 7 seasons (longitudes -3 to 25).
The sequence of peak activity of influenza in the various European countries was taken as a measure for the spread of influenza across Europe. We are well aware that the use of the word 'spread' is based on the liberal assumption that the sequence of peak activity across Europe parallels the actual spread of influenza. Therefore, as we have no clear insight into the dynamics of influenza between countries, in the present study 'spread' should be appreciated with some caution and in a very general context. In order to assess a possible west-east spread or a south-north spread of influenza activity in Europe, the peak week data of influenza activity in EISS countries were plotted against the longitude and latitude of the central point in each country. For the purpose of finding the appropriate geographic centre of a country, rounded longitude and latitude figures were used, based on The Gazetteer of Conventional Names, third Edition, August 1988 [13] . For Northern Ireland, Scotland, Wales and England such central points were not available. Therefore we took the capital cities of these regions: Belfast, Edinburgh, Cardiff and London as best substitutes. Considering it was difficult to identify a peak during seasons of low influenza activity, the sentinel virological data, if available, were used to select the peak. If no sentinel virological data were available, no peak was selected.
Regression analysis and analysis of significance was performed using SPSS 11.5. The variance was expressed as squared correlation coefficients (R 2 ), interpreted as follows: < 0.1 very weak correlation; 0.1-0.25 weak; 0.25-0.50 moderate; 0.5-0.75 strong; 0.75-0.9 very strong; > 0.9 exceptionally strong correlation. Table 1 presents the clinical data in 17 countries during eight seasons. When using the norm of +/-1 week, the mean total overlap of sentinel clinical and virological data was 72% (median 71%; range 25%-100%) for countries using ILI as case definition, and 71% (median 73%; range 57%-83%) for countries using ARI as case definition. When using the norm of +/-2 weeks, the mean overlap for ILI-countries rose to 84% (median 86%; range 50%-100%) and rose to 85% (median 86%; range 71%-100%) for countries using ARI as case definition. Table 1 also compares the sentinel clinical data with the non-sentinel virological data. When using a match of +/-1 week, the mean overlap of sentinel clinical and non-sentinel virological data for countries using ILI was 63% (median 67%; range 0%-100%), and 46% (median 50%; range 43%-50%) for countries using ARI. When accepting a match of +/-2 weeks, the overlap rose to 84% for ILI (median 86%; range 50%-100%), and to 68% for ARI (median 60%; range 50%-100%). The differences between ILI and ARI were statistically not significant (1week match, p = 0.26; 2-week match, p = 0.19). Table 2 No correlation was found between length of the epidemic and type of virus or virus combinations. Table 2 assesses the west-east and south-north spread of influenza activity in Europe after respectively plotting the longitude and latitude of each country to the peak clinical level of activity for each season. As an example the plot of the 2003-2004 season is shown in Figure 1 . The assessment was based on calculation of the squared correlation coefficient. In four consecutive seasons (2001) (2002) (2003) (2004) (2005) , there was a moderate to strong correlation between longitude and peak influenza activity, indicating a west-east spread of influenza. It is noteworthy that during these four seasons A(H3) was the dominant virus. However, this was also the case during the 1999-2000 and 2006-2007 seasons, in which there was no indication for a west-east spread. We repeated the analysis for the latitudes of each country and found a moderate correlation in three season, favouring a south-north spread. During two of these seasons (2001-2002 and 2004-2005) there was also a clear west-east spread of influenza. Taking all eight seasons into account the overall indication for a west-east spread was twice as strong than for a south-north spread (mean R 2 west-east: 0.277; mean R 2 south-north: 0.137).
In the present study routinely collected surveillance data were used to assess the timing, length and spread of influenza activity in Europe during eight winter seasons. It first tried to establish the validity of the sentinel reports and then used these data to assess the length of an average season in Europe and whether or not there is a general westeast spread of influenza activity.
The analysis in this paper is largely based on clinical sentinel reports and it was therefore important to validate this S: Sentinel, information provided by sentinel general practitioners NS: Non-sentinel, data obtained from non-sentinel physicians, hospitals and institutions ILI: influenza-like illness used as case definition by 13 countries ARI: acute respiratory disease used as case definition by 4 countries 1 week overlap: occurrence of peak incidences differ 1 week or less (good match) 2 week overlap: occurrence of peak incidences differ 2 weeks or less (moderate match) Percentages represent the mean overall match aggregated from data of most participating countries obtained during 8 influenza seasons data source, first with the virological sentinel data and then with non-sentinel laboratory data. We found that the data were valid, irrespective of whether ILI or ARI was used as case definition. There was an overall match of 72% (+/-1 week) between the clinical and virological sentinel data and a 60% match (+/-1 week) between sentinel clinical data and the non-sentinel virological data. Allowing a larger overlap (+/-2 weeks) provided a match of 84% and 80%, respectively. The results indicate that the sentinel system is very adequate in estimating influenza activity in a continent, irrespective of case definition. The strength of the system is that it combines communitybased clinical and virological data and also can provide age-specific data. The sentinel approach has already been implemented in other continents, our results imply that it should be considered in other parts of the world [14] .
It is not surprising that there was a close relationship between the clinical and virological sentinel data. This relationship should be close, because the diagnosis of ILI or ARI and the subsequent collection of respiratory specimens is done by the same person (sentinel GP). Sampling of specimens is also usually at its highest during the period of increased influenza activity, which will lead to increased numbers of positive specimens. Some of the non-matches of sentinel data occurred during the Christmas/New Year period when the clinical and virological surveillance systems are affected by holidays.
The second comparison we performed was between the sentinel clinical and the non-sentinel virological data. This was an important validity check because two independent surveillance systems were compared. Many countries (e.g. the U.S.) do not collect sentinel virological data, but base the virological assessment of influenza activity solely on data from non-sentinel sources [15] . Our study found a good match between sentinel clinical and nonsentinel virological sources. Again, the clinical rates can sometimes be unreliable during seasons when activity is very low, which may lead to mismatches. On the other hand, a large outbreak in a major hospital or region may be another important factor that can affect the non-sentinel data. Such localized outbreaks may lead to increased non-sentinel reports that are not picked up by the sentinel clinical reports, resulting in a mismatch. Taken together, we do favour the sentinel collection of virological data because the approach is more systematic, less prone to pre-selection and differs less among countries than clinical sampling.
The mean length of a typical influenza season in Europe based on the peak activity levels of ILI/ARI was 15.6 weeks. It is a conservative estimate, because it does not include the period of increased influenza activity. If this period is also taken into account the average European influenza season lasts about 4.5 months. This is a important finding as it highlights the fact that influenza activity occurs for a long period of time in Europe each season. It also highlights the need to present country-specific data, in order to get insight into the diversity of activity [16] .
Because the spread of influenza depends of many factors one might not expect a particular pattern. Still, four out of eight seasons showed a clear west-east spread of influenza. In three seasons there was a south-north spread. The overall indication for a west-east spread was stronger than for a south-north spread, which means that a west-east spread of influenza is a more common, but far from consistent phenomenon in Europe. It is noteworthy that the westeast spread occurred in 4 consecutive seasons during which the more virulent A(H3) was the dominant virus. It should be noted that the data for 1999-2000 and 2000-2001 were not complete: a number of important countries to the east of Europe (Latvia, Lithuania, the Slovak Republic and Poland) were not included in the analysis because data were not available. This might have affected our westeast analysis.
In an analysis of the spread of influenza in the US over 30 years, Viboud et al. observed a consistent early onset of the epidemic in California, which is the most populous state in the U.S. [17] . Interestingly, the onset in California was earlier than in 3 populous Eastern states, suggesting that in addition to population factors also geographical and climate factors, such as:, mountain ranges, plains, lakes, and predominant wind direction may drive early epidemic activity. Some of these factors may be involved in a west-east spread in Europe: the western of Europe is the most populated part and factors that contribute to spread, such as commuting and airline travel consequently are more intense in the western than in the eastern part of Europe [18] .
We have used a rather crude method to assess the geographic spread of influenza activity in Europe. A more refined method would be to collect doctor specific data as routinely is done in France [19] . As these data are not available yet within EISS we had to use the methodology of analysing peak levels of activity in each country. In 2005 EISS initiated a European Mapping Project, in which data from Germany and the Netherlands were brought together to map the spread of influenza on a weekly basis, using data from about 500 physicians in Germany and 84 in the Netherlands [20] . This project has now been extended to 7 countries and hopefully the upcoming data will allow us to better assess the spread of influenza [21] . These results have important consequences for public health: it allows better planning of health care resources at a local level, and at a European level better recommendations can be made about the timing of vaccination.
Our analysis has demonstrated that the sentinel clinical data, the main indicator used to measure influenza activity in Europe, is a valid indicator for influenza activity. We also found that the length of influenza activity in Europe was surprisingly long. Finally, during 4 out of 8 seasons there was a clear indication of a west-east spread and in 3 seasons a moderate indication of a south-north spread of influenza activity across Europe. Diagnosis and treatment of severe sepsis The burden of infection in industrialized countries has prompted considerable effort to improve the outcomes of patients with sepsis. This has been formalized through the Surviving Sepsis Campaign 'bundles', derived from the recommendations of 11 professional societies, which have promoted global improvement in those practices whose primary goal it is to reduce sepsis-related death. However, difficulties remain in implementing all of the procedures recommended by the experts, despite the apparent pragmatism of those procedures. We summarize the main proposals made by the Surviving Sepsis Campaign and focus on the difficulties associated with making a proper diagnosis and supplying adequate treatment promptly to septic patients. Severe sepsis and septic shock are currently among the most common causes of morbidity and mortality in intensive care, and their incidences have increased during the past decade as the population has aged [1, 2] . The emergency department (ED), where patients are treated for community-acquired infection, many of whom require intensive care unit (ICU) management [3] , has been identified as a setting in which these syndromes and their outcomes may readily be observed.
Despite dramatic improvements in diagnostic and treatment procedures, mortality rates among patients with sepsis remained unchanged from the 1960s through to the late 1990s. Diagnostic algorithms have therefore been developed to identify at-risk populations, and professional societies have worked to implement treatment procedures that focus efforts on early intervention. The Surviving Sepsis Campaign proposed management procedures that differentiate between 'resuscitation bundles' for the first 6 hours and 'management bundles' to be applied until the end of the 24th hour [4] . These procedure recommendations have been disseminated worldwide and are focused on global improvement in practices whose primary goal it is to reduce sepsis-related death. As a consequence of the recommendations, a trend toward decreasing mortality has been observed during the past few years.
Difficulties remain, however, in applying all of the procedures recommended by the experts. This article summarizes the main proposals raised by the Surviving Sepsis Campaign and focuses on the difficulties associated with applying these guidelines in an appropriate time frame.
Definitions of sepsis, severe sepsis, and septic shock were proposed 15 years ago. They were based on expert advice and used criteria that identify progression of the infection along with appropriate responses [5] . However, these criteria are clearly inadequate in terms of allowing detection of severe infections in routine daily practice. A study of a large multicenter cohort of ICU patients with infection [6] concluded that simply categorizing an infectious process as 'sepsis' or 'severe sepsis' did not predict prognosis. A high score indicating a septic condition did not necessarily predict a patient's outcome, even though that outcome might be affected by sepsis-related organ dysfunction.
With regard to patients presenting at the ED because of community-acquired pneumonia (CAP), a recent report from Dremsizov and coworkers [7] illustrated the limited value of the well established criteria for 'systemic inflammatory response syndrome' (SIRS) in predicting outcome. That work emphasizes the inability of the SIRS designation to identify which infected patients were at risk for developing severe sepsis or shock. These findings prompted experts to propose new scoring systems aimed at identifying patients who are at [9] outcomes in patients presenting at an ED with infection, and calculation of this score requires data that are immediately available in the ED. Despite its ability to predict all-cause death in the study population, the accuracy of the MEDS score has not been tested at the individual patient level; its use at the bedside has not been evaluated, and therefore this tool should not be used in decisions regarding triage and ICU referral [10] . Most of the other newly developed scoring systems appear to have only marginal utility in daily routine practice because they require microbiologic identification and 24-hour clinical evaluation; hence, they lack the immediacy that is required for decision making in emergency medicine [6] . To date, the Pneumonia Severity Index is the only scoring system that is considered to help physicians to assess severity of illness in the ED [11] . Using this score at the bedside allows better triage of lowrisk patients [12] [13] [14] , but it does not alter outcomes in more severe pneumonia [15] , in which it is only slightly more effective than the inadequate SIRS classification [7] .
Evaluation of biologic factors also may help in determining the severity of illness. Cady and coworkers [16] proposed use of the arterial blood lactate level to identify patients with severe illness and to assess the severity of sepsis. The Surviving Sepsis Campaign Management Guidelines Committee [4] , and the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference Committee [17] have also proposed guidelines that help to identify those patients who are at greater risk for sepsis. Recent reports from Shapiro [18] and Nguyen [19] and their colleagues have emphasized the importance of lactate clearance in identifying those patients who will respond to treatment and have a favorable outcome. Lactate clearance was shown to be a better prognostic factor than a single lactate determination performed on ED admission [18, 19] . However, a single venous lactate measurement above 4 mmol/l predicted short-term and in-hospital risk for death in patients presenting at the ED with suspected infection [20] , even in those with normal arterial blood pressure [21] . A single lactate dosage is thus a valuable tool that may facilitate early detection of at-risk patients. Plasma procalcitonin may also be valuable in this setting. Procalcitonin is a more specific test than C-reactive protein [22] and interleukin-6, and can help the physician to detect sepsis [23] . Higher levels of procalcitonin are sufficiently specific to identify those septic patients who will develop severe sepsis, but it is not sensitive enough for routine use in ED triage [24] .
It is clear that the site of infection should be managed promptly in patients with severe infection, including emergency surgery when applicable. However, efforts should also focus on early and carefully controlled antimicrobial therapy.
Minimizing the delay between admission and beginning antimicrobial treatment is key to achieving a successful outcome.
The potential influence of delayed antibiotic therapy was first evaluated in patients with CAP. In a series of 18,209 Medicare patients older than 65 years admitted because of CAP [25], the antibiotic regimen used saved lives when the first dose was administered before hour 4 after admission. Of note, fewer than 50% of patients received antibiotics during the first 4 hours in this study and as many as 17% received antimicrobial treatment after hour 6. Those patients in whom administration of antimicrobial agents was delayed were elderly people with an atypical CAP presentation, or they exhibited clinical features inconsistent with a diagnosis of sepsis, such as the absence of fever and altered mental status [26] (specifically, patients in whom the diagnosis of infection was not obvious). Such a lack of aggressive and early antimicrobial therapy has been identified in various settings in which patients were being treated for such conditions as meningitis, cancer, CAP, and nosocomial pneumonia [27] [28] [29] [30] [31] [32] [33] . A recent retrospective analysis quantified the impact of delayed antimicrobial treatment in patients with severe sepsis. Kumar and coworkers [34] demonstrated that every additional hour without antibiotics increased the risk for death in hypotensive septic patients by 7.6% during the first 6 hours. Early antibiotic therapy has been incorporated into the Surviving Sepsis Campaign recommendations [35] , and we expect compliance with this component of the guidelines to increase from its current low level [36] .
The focus of infection is sometimes difficult to ascertain, but treatment must effectively target the responsible pathogen, from among a wide range of potentially etiologic agents [37] . Initial selection of an antimicrobial agent with good activity against the causative organism is crucial for survival. A prospective evaluation of sepsis [38] emphasized that, other than comorbidity, the factor most strongly associated with death was ineffectiveness of antimicrobial treatment against the micro-organism identified in blood cultures. Several large reports corroborated the relation between ineffective antibiotic treatment and poor prognosis. Consequently, broadspectrum antibiotics have been recommended, and the agent selected should provide coverage against the microorganisms that are usually involved in the suspected focus of infection [35] . Supportive clinical evidence for use of broadspectrum antibiotics will probably remain sparse [36] , but effective antimicrobial management requires good microbiologic sense.
Adherence to such guidelines regarding use of antibiotics may positively influence prognosis [39] , but efforts to improve detection of pathogens should continue because enhanced specificity allows one to focus treatment on the responsible micro-organism and so limit the spectrum of coverage. The usual microbiologic techniques of detection may lack effectiveness. The use of urine antigens to Streptococcus pneumoniae and Legionella pneumophila type 1 can help in patients with pneumonia. Apart from their good sensitivity, the presence of these antigens can be detected long after an infection and, in the case of pneumoccocal related infection, may reflect carriage of the micro-organism in the upper respiratory tract [40] . Sensitive genomics tools are now available to detect both bacteria and viruses, and multiplex platforms allow screening of a wide range of micro-organisms [41] . The position of these techniques in the diagnostic armamentarium is yet to be defined, but efforts to improve antimicrobial therapy must continue so that our practices and therefore outcomes may be improved in the future.
Among the symptomatic treatments, need for hemodynamic management is the most apparent, but modalities continue to be discussed and the scientific literature abounds with studies in this area. Efficient restoration of circulating blood volume is the primary goal of resuscitation in septic patients [42] . Albumin was the first product to be broadly used for intravenous fluid loading, but a meta-analysis comparing albumin with other fluid loading agents [43] identified an increased risk for death among patients who received albumin for supportive treatment during shock. However, subgroup analysis (septic patients with hypoalbuminemia) [44] revealed a trend toward greater efficiency of fluid loading with albumin. The cost-benefit balance is another factor that has restricted use of albumin, but in their recent report Guidet and colleagues [45] indicated that albumin infusion was potentially cost-effective in patients with sepsis. Thus, use of albumin should be considered with caution; it currently lacks the support needed for it to be recommended for use in patients with septic shock.
Transfusion of packed red cells may also be considered in septic patients because transfused hemoglobin may contribute to improved oxygen transport and delivery. Few controlled studies have tested this option, however, and it has been reported that liberal transfusion is potentially ineffective [46, 47] . Since the publication of the findings of Rivers and coworkers [48] , use of packed red cells has been regarded as a valuable approach to improving tissue oxygenation, but the specific indications for transfusion of packed red cells in this setting remain unclear.
Although controversy persists in this area, preferential use of crystalloids rather than colloids is supported by the available literature. For the same amount of volume expansion, there is no difference between these two treatments in terms of ejection stroke volume or oxygen delivery [49] . Systematic reviews and meta-analyses that included patients with sepsis and other types of patients concluded that crystalloids and colloids were generally similar in effect; an exception was one study that identified an advantage for crystalloids [50] . This finding received support from a randomized trial [51] that found that patients with septic shock receiving colloids had greater renal impairment. A recent study [52] was conducted to compare colloid with crystalloid volume resuscitation, with the aim being to identify the safest choice for use in patients with sepsis. This study, which employed a prospective randomized multicenter design, compared the influence on outcome of Ringer's lactate versus hydroxyethyl starch and of intensive versus conventional insulin therapy in patients with severe sepsis and septic shock. Experts have already criticized this study on the grounds that its design confounds applicability of the findings to routine daily care [53] . To summarize, although infusing fluids is a cornerstone of supportive care during sepsis, the optimal modalities and volume are difficult to determine and choices should be driven by objectives in the individual patient [48] .
A solid rationale explains the utilization of vasopressors in daily practice [54] , but the few comparative studies and the combination of different molecules account for their practical selection. Combining norepinephrine (noradrenaline) and dobutamine improved hemodynamic parameters of hepatosplanchnic circulation [55] but required invasive monitoring procedures, without clinical benefit. Dopamine and epinephrine are vasoconstrictors that also increase cardiac output, but their metabolic effects may be harmful [56, 57] . In addition, use of vasopressors has been associated with poorer outcomes in septic patients, but their influence on mortality was unclear [58] .
To assist physicians in their use of vasoactive drugs, professional associations have proposed guidelines that allow an opportunity to administer epinephrine or a combination of norepinephrine and dobutamine to more severely ill patients [4] . A recently reported study [59] indicated that these two strategies were equivalent in terms of both efficacy and safety. Interest in vasopressin is reflected in a growing number of publications, but the available evidence does not allow its integration into a global therapeutic scheme. However, recent data [60] may justify reconsideration of vasopressin in severe sepsis management guidelines in the near future. The VAsopressin in Septic Shock Trial (VASST) study [61] is currently comparing vasopressin with norepinephrine as initial vasopressor in septic shock patients. Because the study is not yet completed, no analysis or definite conclusions can yet be drawn from this trial.
Whichever drug is selected, introduction of vasopressors should be considered after optimal fluid loading; these agents may allow therapies to be applied earlier and more aggressively in order to improve physiological parameters and ultimately outcomes [48, 62] .
In the initial management of patients with sepsis, improving physiological parameters such as blood pressure and tissue oxygen delivery is a clear goal, as has been emphasized by experts since the late 1990s [63] . Previous studies underscored that applying an early goal-directed therapy (EGDT) approach could improve survival. The landmark study conducted by Rivers and coworkers [48] emphasized this concept in the field of sepsis. Its publication in 2001 prompted a debate in basic medical practice centered on the question, is it possible to improve outcomes in septic patients by increasing tissue oxygenation parameters during the first 6 hours of management?
The protocol proposed by Rivers and coworkers involves attainment of physiological levels of hemodynamic parameters (arterial blood pressure and central venous oxygen saturation [ScvO 2 ], by using fluid loading, vasopressors, packed red cells, and early initiation of mechanical ventilation) as rapidly as possible. The overwhelmingly positive results of this EGDT study prompted a number of ED and ICU teams to change their daily care in accordance with the study protocol. Some papers [62, [64] [65] [66] reported partial or absolute adherence to the procedures evaluated by Rivers and coworkers. Others proposed adapting the procedure to their medical system with either less aggressive therapy or by forming 'sepsis teams' specifically tasked with managing patients with severe infection [67] [68] [69] [70] [71] [72] . The overall result of these reports was a trend toward improved outcomes in septic patients [73] .
However, these findings have been tempered by a number of barriers. Not all EDs have access to the same equipment, and ability to monitor hemodynamic parameters invasively varies widely [74] . Another unresolved issue is that not all ED physicians have the necessary resuscitation skills to administer optimal treatment, as observed in ICUs [75] . Additionally, a number of recent reports have identified the fact that EDs are increasingly overburdened. This can compromise the quality of care delivered to patients, especially those who require highly technical care that many ED physicians do not have time to practice because of everincreasing numbers of patients [76] [77] [78] .
Finally, studies are now emerging that indicate how few of the recommendations have been implemented. Early administration of antimicrobial therapy was poorly adhered to, even in recent reports. In these, although the Surviving Sepsis Campaign proposals were implemented, the mean delay to first infusion of antibiotics remained in excess of 3 hours [62] , and as many as 68% of patients did not receive their first dose within this period [79] . Only a few EGDT validation studies have been conducted in EDs applying aggressive treatment outside the ICU. However, even in those EDs, mortality sometimes remained at 31% before and after the institution of procedures to improve coordination between ED and ICU [80] . In addition, effort should be maintained after the initiation of an EGDT strategy because performance dramatically decreased after initial implementation [81, 82] .
In addition to the pragmatism of this therapeutic approach, the optimal tools with which to evaluate attainment of physiological goals have also been subject to debate. Although ScvO 2 is a valuable parameter when it is abnormal, it may be in the normal range even in severely septic patients [73] . The hemodynamic presentation, of which there are many, depends on comorbidities and stage of sepsis [83] . In addition to ScvO 2 , central venous pressure may also provide useful information. A low central venous pressure indicates hypovolemia, and a high central nervous pressure with a low ScvO 2 indicates myocardial suppression or mismatch of supply and demand. In any clinical situation, the findings must be interpreted alongside other clinical data. Other indicators may help, and systolic volume and pulse pressure variation of 10% or above also provide valuable information regarding blood volume [84] . Relatively liberal use of packed red cells to improve ScvO 2 may be offset by its potential harm [46, 47] but in the setting of severe sepsis and septic shock the theoretical risks appear balanced by the benefits in terms of tissue oxygenation [85] . Although use of central venous pressure and ScvO 2 to evaluate attainment of physiologic goals can be debated [73] , it is clear that defining reasonable goals to treat sepsis is important whatever the local organization and the available means to achieve those objectives are [86] .
For the past two decades therapeutic trials attempting to elicit a change in the host response to infection have failed to improve patients' conditions despite positive preclinical data [87] [88] [89] [90] [91] . However, the results of two recent studies have led to a more promising approach to this problem with recombinant human activated protein C (rhAPC) and low-dose steroids. The hemodynamic effects of steroids have been widely discussed since their use was found to allow early withdrawal of vasopressor treatment in a prospective doubleblinded, multicenter study [92] . The positive effects of steroids on adrenergic receptor cycling and sodium and water balance have been proposed as explanations for this efficacy. Their anti-inflammatory role as well as their anticoagulant effect, caused by limiting membrane expression of tissue factor, may contribute to the clinical benefit. A major difficulty lies in defining adrenal deficiency in septic shock patients, and a number of definitions have thus far been used. A recent retrospective multicenter cohort study conducted by the Corticus study group [93] emphasized the importance of cortisol variation after corticotropin stimulation. That study additionally raised the possibility of a deleterious effect of etomidate on hormonal response and outcome, a concern that was previously reported by others [94] . This specific point is still subject to debate [95] . Efforts are currently being made to define the best strategy for use of steroids during sepsis.
The efficacy of rhAPC has been tested in a large multicenter study, the results of which have been widely debated. This compound was initially designed to compensate for a deficit in the natural anticoagulant protein C during sepsis, and thus it limited organ failures and improved the survival of septic shock patients [96] . Since then a number of studies have demonstrated that it has additional beneficial effects on complex interactions with inflammation, innate immunity, and apoptosis [97, 98] . rhAPC also protected animals and healthy volunteers from hypotension after lipopolysaccharide challenge. A similar finding was also reported in the PROWESS (Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis) study, with more rapid improvement in hypotension and vasopressor withdrawal [99] . These clinical effects could be related to endocrine modulation (adrenomedullin was implicated in this regard) and vasoactive capacity. Mechanisms, efficacy, and safety of rhAPC are discussed in other reviews included in this supplement.
Despite the strong evidence base, use of adjunctive therapies has remained sparse in the setting of sepsis. Questionnaire surveys have attested to the under-use of such adjunctive therapies. Once again, the need for medical adherence to new therapies must be promoted by implementation of local guidelines that are inspired by the recommendations of the Surviving Sepsis Campaign (Figure 1 ).
New standards of care, such as low tidal volume mechanical ventilation and tight blood glucose control, have recently emerged and are now a cornerstone of treatment for critically ill patients. Low tidal volume (≤ 6 ml/kg) as compared with 'standard' mechanical ventilation (12 ml/kg) has improved survival in patients with acute respiratory distress syndrome in independent studies [100, 101] . Two landmark studies by van den Berghe and colleagues [102, 103] suggested that aggressive insulin therapy improved 30-day survival in critically ill surgical patients, and reduced morbidity indicators such as weaning from mechanical ventilation and hospital days in medical ICU patients. Whereas occurrence and management of hypoglycemia appeared irrelevant in the main papers and additional data, hypoglycemia has been identified as potentially causing harm by others [104] . Even if these standards are still discussed and do not specifically impact on sepsis, they may also contribute to quality-of-care improvement and finally to patients' successful outcome [83, 84] .
Guidelines that were proposed through the Surviving Sepsis Campaign to improve outcome in septic patients are difficult to apply routinely in most EDs. Attempts to apply these procedures fully have varied widely; diagnosis may be problematic because of atypical or unspecific presentations, biomarkers are of little help at the start of treatment and are unspecific, supportive treatment often depends on local supply of resources, and specific devices are often absent in EDs for initial therapy and monitoring. Even adherence to early administration of antibiotic therapy is poor, with delays being common. Our goal is now to improve the level of care by applying evidence-based procedures.
J-FD was an investigator in the PROWESS study and is a consultant for Eli Lilly and Company. Y-EC declares that he has no competing interests.  Simian virus 40 vectors for pulmonary gene therapy BACKGROUND: Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy. METHODS: Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/luc) were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. RESULTS: Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. CONCLUSION: In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool. Sepsis is the leading cause of death in critically ill patients [1] . Despite advances in treating the sepsis syndrome, the incidence and mortality of sepsis remains high (35-45%) [2] . Lung is the organ most often involved, with lung injury taking the form of Acute Respiratory Distress Syndrome (ARDS) [3] [4] [5] , carrying 40% mortality [6] . The pathological hallmark of ARDS is airspace flooding with proteinaceous fluid, basement membrane disruption, hyaline membrane deposition, surfactant depletion, interstitial swelling, and interstitial neutrophilic infiltration [7] . Histological sections of the lungs from patients dying of ARDS and from animal models of the disease reveal: interstitial edema, followed by extensive necrosis of alveolar epithelial cells [8] .
Alveolar epithelium in the adult lung consists of two cell types: Type I, differentiated cells that facilitate gas exchange and Type II, metabolically active cells involved in surfactant secretion and epithelial repair, serving as progenitors for injured type I cells [9] . Injury to type II cells impairs gas exchange by reducing the surfactant supply and limits type I cells regeneration, their preservation being essential for recovery from ARDS [10] .
Nowadays therapy for ARDS remains mainly supportive, designed to prevent secondary injury [11] . However, recent studies demonstrated that organ damage in sepsis involves an alteration in gene expression. Decreased transcription of surfactant proteins [12] as well as profound pulmonary epithelial dysregulation together with altered levels of Hsp70 were found in animal models of ARDS [13] . We also showed in our previous studies that severe sepsis induced by cecal ligation and puncture (2CLP) precipitates ARDS [13, 14] . Furthermore, we demonstrated that enhanced Hsp70 expression in pneumocytes using an adenoviral vector (AdHSP) not only decreased histological abnormalities in the lung but also improved shortterm outcome [15] . Specifically, we showed that AdHSP limited sepsis-induced acute inflammation by suppressing NF-κB activation [50] .
One approach to correct deficient protein expression is to use viral mediated gene transfer [16] . An optimal vector for gene therapy should be safe, efficient, nonimmunogenic, and available in high titers [17] . There is currently no single vector with all these advantages. SV40 based vectors are efficient gene delivery vehicles for a wide spectrum of ex vivo and in vivo targets, including hematopoetic, liver and kidney cells [18, 19] . The vector was shown to be potentially effective in a number of clinical models, including Crigler Najjar syndrome [20] , HIV, [21] SV40 evades host immune response most likely by caveolar endocytosis, followed by vesicular transport to the endoplasmic reticulum [27], thus avoiding the more common endosomal-lysosomal pathway used by most viruses [28] . SV40 vectors were found to be nonimmunogenic, allowing repeated administrations and long survival of transduced cells [29, 30] . Replication incompetent SV40 vectors in which the viral T-antigen is replaced by the gene of interest are produced in vivo in cells that supply T-anti-gen in trans-, such as COS or COT cells [31] , and high vector titers may be readily prepared [32] . Cloning capacity of these vectors is limited; nevertheless, many potentially therapeutic genes may be accommodated, as shown by the wide spectrum of applications already investigated.
The use of SV40 vectors for gene transfer to the lung has not been explored. In this work we found that SV40 vectors may be used for lung cell transduction. After in vivo vector delivery, we established and measured expression of the reporter gene in rats with sepsis induced ARDS. Our results lead the way for studies regarding gene delivery to the lung, in order to modulate pulmonary disease processes.
SV/luc is a T-antigen replacement vector carrying the firefly luciferase (luc) reporter gene [33] . Preparation of the SV/ luc vector was performed as previously described [34] . Recombinant E1, E3-deleted adenoviral vectors (Ad/luc) were propagated in HEK293T cells, by commonly used methods [35] .
Animal procedures were approved by the Institutional Animal Care Ethical Committee. Under Ketamine/Xylasine/Isoflurane anesthesia, severe sepsis was induced in Sprague-Dawley rats using cecal ligation double puncture (2CLP) [14] . 1.25 × 10 8 IU/ml (infectious units) of SV/luc in 300 μl PBS were administered via a tracheal catheter to 2CLP and sham operated (SO) rats immediately after the procedure and to unoperated (UO) rats. In the positive control group, 10 9 IU/Ad/luc in 300 μl PBS were administered in the same way to 2CLP animals [15] . Negative control animals received PBS only.
The reporter gene used encodes the luciferase protein, an enzyme that converts luciferin in presence of oxygen and ATP to a bioluminescent substance. In vivo light detection was performed using the Roper Chemiluminescence Imaging System, (CCCD-cooled coupled charged camera) model LN/CCD-1300EB equipped with ST-133 controller and a 50 mm Nikon lens (Roper Scientific, Princeton Instrument, Trenton, NJ). The system enables detection of an internal light signal emerging from mammalian tissue. The measurement is the sum of the integrated light signal subtracted the background light emission of an area of equal size [36] . A pseudo color image represents light intensity spectrum (from blue -least intense, to redmost intense).
Forty eight hours after vector administration, rats were reanesthetized. 125 mg/kg Beetle Luciferin (Promega, Madison, WI) was administered intraperitoneally, and luciferase activity was measured by photographing the animals first in light (to obtain the animal's image) and then in the dark (to measure light emission). A composite photograph was obtained by superimposing the two images. The average amount of fluorescence measured in light units per area, as detected by the CCCD camera represents signal intensity.
After the light emission measurement, at 48 hrs, the animals were sacrificed and internal organs harvested. One lung was removed, preserved in formalin, embedded in paraffin, cut at 5 μm thickness and stained with hematoxylin and eosin. H&E sections were evaluated for lung injury, degree of injury and its distribution within the lungs [14] . Part of the spleen, liver, heart and one kidney were also harvested and prepared in the same way. The remaining part of the spleen, heart, liver, the second kidney and lung were frozen in fluid nitrogen and preserved at -80°C for RNA.
Immunostaining was performed using the procedure described by Lavon et al [37] . For luciferase we used a primary rabbit anti-mouse polyclonal antibody (Cortex, San Leandro, CA) at 1:50 dilution, followed by a secondary goat anti-rabbit IgG antibody (biotin conjugated, Zymed, San Francisco, CA). VP1 capsid protein detection was performed using a rabbit anti-SV40 polyclonal primary antiserum [38] followed by FITC-conjugated goat antirabbit IgG secondary antiserum (Zymed). DAPI (blue) was used as nuclear counterstain. Double immunostaining for surfactant protein C (ProSP-C) and VP1 was performed in order to detect internalization of VP1 viral capsid protein into the alveolar cells. Serial lung sections were immunostained for VP1 using the same rabbit polyclonal antibody as above followed by goat anti-rabbit Cyte 5, and for ProSP-C using rabbit anti-ProSP-C polyclonal primary antiserum (Alomone Inc, Israel) followed by FITC-conjugated goat anti-mouse IgG. Immunohistochemical detection of Lymphocyte (CD3+ T cells) infiltration was performed using the immunoperoxidase avidin biotin methodology. A primary CD3+ T cell antibody (monoclonal mouse anti-rabbit, affinity purified, Biosource, Carlsbad, California) at 1:100 dilution was used, followed by a secondary goat anti-rabbit IgG antibody (biotin conjugated, Zymed).
Total RNAs from the frozen tissues were extracted using Trireagent (Sigma, Saint Louis, MO) according to the manufacture's instructions. A quantity of 2.5 μg of extracted RNA (for each sample) was subjected to reverse transcription using 400 u/μl RNAse, 0.5 μg/μl oligo dT, 200 u/μl M-MLV RT and 2.5 mM dNTPs. 2 μl of the reacting cDNA products were used as template for PCR. PCR was performed using PCR primers specific for Luciferase: 5'-TGGTCTGCCTAAAGGTGTCG-3' (forward) and 5'-ATGTAGTCTCAGTGAGCCC-3' (reverse), and carried out for 35 cycles. pGL3 Luciferase reporter vector (Promega) served as a positive control. GAPDH was used as a house keeping control gene, using PCR primers specific for GAPDH: 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'.
In the present experiment, ARDS was induced secondary to intra-abdominal sepsis (2CLP). Starting 12 hours after 2CLP, and more prominently at 24 and 48 hours, 2CLP rats displayed the typical signs of sepsis: intense pallor/ cyanosis of the mucous membranes, dirty fur with erected hairs, distended abdomen, diarrhea, tachypnea (40-60 breath per minute). Animal behavior was grossly abnormal: periods of agitation followed by periods of sleepiness along with limited movements and inability to feed.
Histological examination of the lung sections from 2CLP animals showed changes consistent with ARDS. Macroscopically: lungs were less aerated, covered with white fibrin patches and pleural fluid was found in different quantities. H&E stained sections depicted: alveoli filled with proteinaceous fluid, septal thickening, and interstitial neutrophilic infiltration ( Figure 1 ). Mortality was present only in the septic (2CLP) animals. The 48 hrs mortality rate following 2CLP was similar to previously published numbers in sepsis induced ARDS [13] [14] [15] .
SV/luc or Ad/luc vectors were directly administered into the trachea of 2CLP and SO rats, immediately following the procedure. Using this route we achieved maximal vector concentration and distribution to the lung, limiting the systemic spread. Histopathologically there was no difference between the lungs of the septic rats given PBS or SV/luc ( Figure 1 ). Mortality rate was similar in 2CLP animals given either PBS or SV/luc, suggesting that the vector itself did not add to the sepsis/ARDS induced mortality.
At 24 and 48 hours, all the animals were reanesthetized and photographed with the CCCD camera to detect light emission. At 48 hours all animals were sacrificed and the lungs were harvested.
At 24 hours, Luciferase activity was not detected in any animal groups. We explain this finding by the time needed for the reporter gene to express. However, at 48 hours, luc activity (detected as luminescence by the CCCD camera) was seen over the lung areas in 2CLP animals given SV/luc (Figure 2a) . Low levels of luc activity were detected in the tracheostomy region, confirming a degree of vector affinity for airway epithelium (Figure 2b ). No luc activity was detected in rats given PBS. No significant in vivo luc activity was detected in the heart, liver, spleen or kidneys of the animals given SV/luc.
Fluorescence over the lungs of SO rats was significantly lower than that seen in 2CLP animals (Figure 2a ). Intratracheal administration results in little vector uptake in the normal lungs of SO rats, while changes in the intracellular structure occurring during ARDS may be responsible for enhanced viral expression. Similar findings were observed with adenoviruses, and explained by an ARDS-induced exposure/expression of the CAR and integrin receptors that mediate adenoviral entry into the alveolar cells [14] . This mechanism may also apply to the SV40 vector, although different receptors or increased endocytosis may be involved. [39] [40] [41] .
Based on our previous findings [14, 15] , a comparison of luc activity between SV/luc and Ad/luc vectors was performed. As expected, luc expression was lower in the 2CLP-SV/luc than in the 2CLP-Ad/luc group (Figure 2c,  2d) , because of the natural tropism of adenoviral vectors for the pulmonary epithelium [14] . Another factor may have been the higher titer of the adenoviruses used, a total of 10 9 IU/ml adenoviral vector as compared to 1,25 × 10 8 IU/ml of the SV40 vector.
Luc immunostaining was nonspecific, involving both alveolar type I type II cells. It was moderate in septic animals and low in SO animals ( Figure 3 ). This may be due to moderate infectivity of the SV40 vector in the lungs or weak activity of the SV40 promoter in alveolar cells. This finding is also consistent with the literature, describing less transgene expression of some non -mammalian, non -vertebrate encoded proteins (e.g. luciferase, GFP, lac Z) commonly used as markers for transduction in SV40 vectors, than is usually seen with other vector systems (e.g., adenovirus) [18] .
Immunostaining for luciferase in the liver, kidney, spleen and the heart was negative in both 2CLP and SO animals groups given SV/luc, suggesting that direct intratracheal administration of the vector results in negligible systemic spread despite alterations in membrane permeability and capillary leak seen in ARDS.
In order to test if the low level of Luc immunostaining in lung tissue is due to a low vector penetration into the alveolar epithelium or to low expression of the reporter gene, we tested for the presence of the VP1 capsid protein by fluorescent immunostaining. A significantly higher number of alveolar cells contained viral capsid proteins compared with the number of luc positive cells (Figure 4a , 4b, and
Lung pathology Figure 1 Lung pathology. H&E stained lung tissue, shown at ×20 and ×100 magnifications. Untreated control rats show normal lung histology; SO rats given intratracheal SV/luc also show normal histological appearance; 2CLP rats given intratracheal PBS or SV/ luc show distinct ARDS pathology: alveoli filled with proteinaceous fluid, septal thickening, interstitial lymphocytic and neutrophilic infiltration. compare with Figure 3 ). This suggests that vector penetration into lung epithelial cells is efficient, but transgene expression in many of the cells is below detection level. It is possible that the SV40 promoter used in the present vector is weak and therefore gene expression is lower than gene delivery. A stronger, lung specific promoter may significantly improve expression of a therapeutic gene in alveolar cells.
Alveolar type II cells participate in the regenerative process of the lung. Therefore their transduction is critical for successful ARDS gene therapy.
To examine this we performed co-immunostaining of lung tissue for surfactant protein C (Pro SP-C) a marker for alveolar type II cells and VP1 viral capsid protein (Figure 5 ). The figure depicts co-localization of Pro SP-C and VP1. These findings support our conclusion that the vector is internalized into alveolar type II cells. The ability to transduce these cells is essential because of their impor- tance in alveolar epithelial repair processes and lung recovery in ARDS [10] .
RT-PCR was performed on frozen tissues of the lung, kidney, liver, spleen and heart. High levels of mRNA were detected in the lungs of 2CLP animals, similar to those found in the plasmid pGL3 used as positive control for luciferase. Minimal levels were present in the liver, kidney spleen and none at all in the heart, confirming minimal systemic spread of the vector after direct intratracheal instillation. Interestingly, in T0 and SO controls, minimal Detection of the VP1 capsid protein in lung tissue Figure 4 Detection of the VP1 capsid protein in lung tissue. 4a: Positive staining for VP1 protein appears as red intracytoplasmatic coloration: moderate for SO rats (above) and high for 2CLP rats (below). DAPI (blue) was used as nuclear counter-stain. 4b: Negative control for VP1 immunostaining. Above: lung tissue from control septic rats given intratracheal PBS, and below: normal lung tissue from a control T0 rats. The minimal staining seen represents the background. DAPI (blue) was used as nuclear counter-stain. Shown at ×40 magnification. Figure 3 Luciferase immunostaining. Was performed on the lung tissue harvested from 2CLP and SO rats given intratracheal SV/luc. Positive immunostaining appears as brown intracytoplasmatic coloration (Black arrows). Shown at ×100 magnification.
expression was noticed in the lungs, in spite of the same route of vector administration, suggesting that the pathological processes taking place in the sepsis-induced ARDS lungs (as in 2CLP animals) enhances vector penetration and expression by an unknown mechanism. Regarding the kidney, liver, spleen and heart of T0 and SO animals, the same minimal expression was observed as in the septic animals ( Figure 6 ).
Direct intratracheal administration of SV/luc induced no immunological response as measured by lymphocytic and neutrophilic infiltration of the H&E stained lung sections compared to PBS treated septic lung (Figure 1 ). To further confirm this we performed immunohistochemical detection for lymphocytic (CD3+ T cells) infiltration ( Figure  7a, 7b) . As expected, inflammatory cell infiltration was higher in the ARDS compared to the SO lungs. However lymphocytic infiltration in the SV/luc and PBS treated septic rats was similar, indicating that the SV40 vector does not elicite an excessive cellular immune response. These findings support our previous results which demonstrated that there was no cellular immune response against the vector or the transgene following liver transduction by the SV/luc vector, measured by lymphocyte proliferation assay at 84-110 days following vector administration [30] .
Moreover, literature data reported that neutralizing antibody activity against an SV40 vector was not detected in the serum of treated mice, even after 8 consecutive intraperitoneal or subcutaneous inoculations [29] . In our previous study we detected marginal humoral immune response against the vector only at very high vector concentration [30] . Two factors appear responsible of this behavior: the uncommon cell entry route (SV40 vectors evade immune surveillance most likely by entering cells via caveolar endocytosis, followed by vesicular transport directly into the endoplasmic reticulum, bypassing the endosomal-lysosomal pathway) [27] , and deletion of T antigen of the viral genome (renders the vector nonimmunogenic) [32].
The results presented in this article are only preliminary data. Currently, the most common vectors for gene transfer to the lung are replication-deficient adenoviruses. The major advantage of adenovectors is their excellent efficiency in gene transfer. However, gene expression is transient and the immunogenicity prevents repeated administrations. Moreover, the preparation of a gutless adenovirus with a more extended expression is technically demanding [42] . Figure 5 VP1 detection in type II cells. The cells were double-stained for VP1 and Pro-SP C as described in Materials and Methods. An SO lung is shown at the top row and ARDS lung in the bottom row. VP1 was detected by Cyte5 (red fluorescence, left panels) and Pro-SP C by FITC (green fluorescence, middle panels). The upper and lower right images show co-localization of both stainings inside alveolar type II cells (yellow). Shown at ×100 magnification After a detailed search of the literature data we can confirm that this is the first experiment in which an SV40 based vector was used for transduction of lung cells. The goal of our research was to provide a basis for the use of these vectors in face of their advantages over adenoviral vectors, as well as their limitations.
In spite of the two major barriers met by the viruses after intratracheal administration: the mucociliary clearance system and the glycocalix [42] , in the present study we have demonstrated efficient uptake of an SV40 vector in the lungs of animals with sepsis-induced ARDS. Although nonspecific, these vectors appear to be capable of in vivo transduction of alveolar type II cells, key cells involved in regenerative process of the lung [10] .
Acute respiratory disorders like ARDS are the result of a variety of endogenous and exogenous influences, arising rather as a misbalance between protective and destructive mechanisms. Transient gene therapy may thus help restore the homeostatic balance by short term expression of protective genes or suppression of damaging genes [43] . In the near future we plan taking this research a step further by replacing the reporter gene with a therapeutical one (encoding one of the surfactant proteins, Hsp70 or interleukin 6) and trying to alter the course of the disease. SV40 vectors are generally considered non-integrating, transiently expressing vectors [44] . Although others reported a prolonged expression of rSV40 vectors (1 month -1 year) [30], we did not estimate the duration of transgene expression, as our study was limited to 48 hours. Short term or transient gene expression may be sufficient and even advantageous for the treatment of an acute condition like ARDS, where a therapeutic protein or enzyme may be only needed during the acute state. However, as these vectors elicit marginal immune response, repeated administrations, if required, would be possible.
Our results show moderate expression of the luciferase reporter gene. Use of a CMV promoter in the construction of the SV40 vector may improve transgene expression. However, low level of proteins expression may not be entirely disadvantageous. Supra-physiological production of specific proteins may lead to unexpected side effects. In contrast, low expression may be augmented by repeated administrations of the vector [40] . With the SV40 vector RT-PCR for luciferase mRNA detection Figure 6 RT-PCR for luciferase mRNA detection: High levels of mRNA were detected in the lungs of 2CLP animals (plasmid pGL3 used as positive control for luciferase). Minimal levels were present in the liver, kidney spleen and none in the heart. Minimal expression in the lungs, kidney, liver, spleen, and heart of T0 and SO animals. GAPDH was used as a house keeping control gene, using PCR primers specific for GAPDH.
repeated administrations are possible due to its low immunogenicity.
Safety considerations require that the vectors do not contain replication associated T antigen sequences. For this reason our vector is T antigen deleted. Vector propagation is achieved in COT18 cells, cell lines with minimal sequence identity with the vector. Reacquisition by rSV40 of T antigen DNA and reemergence of wtSV40 virions remains extremely unlikely [30].
Another major advantage with SV40 vectors is that they may be constructed in vitro by packaging DNA of choice in recombinant capsids [45] . These vectors, which combine efficient gene delivery of viral vectors with safety and flexibility of non-viral vectors, were shown to have similar tropism as standard SV40 vectors such as the SV/luc described here [30] . Furthermore, these vectors accommodate plasmids as large as 17 kb, significantly larger than SV40 DNA, and may be therefore used to deliver a variety of genes with complex regulatory signals [46] . Furthermore, these vectors do not require any SV40 sequences, providing additional safety margin [47] [48] [49] .
The present study suggests that in vitro constructed SV40 vectors may be tailored to deliver genes of choice with lung-specific promoters for treatment of acute respiratory diseases like ARDS. 
Immunostaining for CD3+ T cell Figure 7 Immunostaining for CD3+ T cell. 7a: Spleen tissue from 2CLP rats serving as positive control for CD3+ T cell immunostainin 7b: left: minimal lymphocytic infiltration of the lung tissue in SO rat. Middle: moderate infiltration in a 2CLP lung rat after SV/luc administration. Right: moderate infiltration in a 2CLP lung rat not treated with SV/luc. Note that the degree of lymphocyte infiltration is similar in the middle and right panels, suggesting that it is part of the disease process rather than being induced by the vector. Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses BACKGROUND: We previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range. RESULTS: We expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry. CONCLUSION: Virus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific. The CAT-1 receptor mediates the entry of ecotropic gammaretroviruses into rodent cells. Virus properties that rely on receptor recognition such as host range or pathogenicity could potentially be affected by polymorphisms that alter the receptor or the receptor binding domain (RBD) of the virus. In previous studies we identified two unusual ecotropic mouse leukemia virus (MLV) variants [1, 2] . Both of these viruses have altered host range, both are cytopathic, and both have amino acid substitutions at the same site in their RBDs. Spl574 is a Moloney MLV (MoMLV) variant with the substitution S82F, and F-S MLV is a Friend MLV (FrMLV) variant with the substitution S84A. Both viruses cause the formation of large multinucleated syncytia on cells derived from the wild mouse species M. dunni two days after infection, and syncytium formation is accompanied by the accumulation of large amounts of unintegrated viral DNA [2] . These two viruses also differ from each other and from their respective parental MLVs in host range. Spl574 replicates efficiently only in M. dunni cells and very inefficiently in other mouse cells such as NIH 3T3 and SC-1 cells. F-S MLV shows no unusual pattern of infectivity in mouse cells, but is capable of infecting hamster cells that are normally resistant to ecotropic MLVs.
The fact that these two viruses are only cytopathic in M. dunni cells suggests involvement of the receptor-virus interaction for two reasons. First, the amino acid residue that is modified in both viruses has been identified as one of the critical amino acids forming the receptor binding site [3, 4] . Second, M. dunni cells differ from other mouse cells in their resistance to MoMLV [5] , and these cells are known to carry a modified CAT-1 receptor (dCAT-1). The dCAT-1 gene of M. dunni cells differs from the prototypical CAT-1 gene of the laboratory mouse (mCAT-1) in that the third extracellular loop that contains the virus binding region has a substitution (I214V) as well as an inserted glycine after Y235, a residue critical for receptor function [6] (Fig. 1A) .
In this study, we examined the role of the dCAT-1 receptor in syncytium formation and susceptibility to infection by different ecotropic MLVs. We generated an expression vector containing dCAT-1 and transfected either this clone or the mCAT-1 gene into cells of non-rodent species that are not normally infectible by ecotropic virus. The transfected cells were then evaluated for susceptibility to infection by ecotropic MLVs and for virus induced syncytia. While virus induced syncytia were only seen in the dCAT-1 transfectants, a different panel of virus isolates was capable of efficiently infecting and/or inducing syncytia in these transfectants suggesting that virus-cell fusion and cell-cell fusion are distinct receptor mediated phenomena. The possible contribution of differential glycosylation to these phenotypic differences was evaluated using Western analysis, treatment by glycosylation inhibitors and mutagenesis to remove glycosylation sites.
HA-tagged mCAT-1 and dCAT-1 clones were transfected into three cell lines that are not naturally susceptible to infection by ecotropic mouse gammaretroviruses: MA139 (ferret), Tb-1-Lu (bat lung), and MDCK (canine kidney) cells. As a control, mCAT-1 was transfected into M. dunni cells. Pools of stably transfected cells were used for analysis along with single cell derived clones of transfected MA139 cells. mCAT-1 and dCAT-1 expression in transfected cells was confirmed by Western analysis (Fig. 1B) . Consistent with previous observations [7] , CAT-1 was detected as a heterogeneously glycosylated protein in each cell line. The size range distribution for the mCAT-1 and dCAT-1 proteins was similar for each cell line, but the size range and band patterns were variable between cell lines suggesting cell specific differences in glycosylation. Thus, for example, the molecular weight range of CAT-1 was lower in MDCK cells (not shown) and Tb-1-Lu cells than in MA139 cells and M. dunni cells (Fig. 1B) .
These stable transfectants of MA139, Tb-1-Lu and MDCK cells were infected with a panel of ecotropic gammaretroviruses including two, Spl574 and F-S MLV that induce multinucleated syncytia in M. dunni cells but not in other mouse cell lines. The infected cells were examined for cytopathicity over a period of 2-5 days. Transfectants of all 3 cell lines expressing mCAT-1 showed no signs of cytopathicity following virus infection as shown for the MA139 and MDCK transfectants in Fig. 2 . In contrast, dCAT-1 expressing MA139 and MDCK cells (Fig. 2 ) as well as Tb-1-Lu cells (not shown) formed syncytia within two days of infection with Friend virus isolate F-S MLV.
Several separate pools of MA139 transfected cells were generated and tested. Virus-induced syncytia were observed in two independently derived pools of dCAT-1 transfected MA139 cells as well as three independently isolated clonal lines (FerrD2, N65FerrC2 and N65FerrB6), but not in 3 independently derived pools of mCAT-1 transfected MA139 cells.
Among the ecotropic isolates tested, F-S MLV was most efficient in inducing syncytia in all dCAT-1 transfected cells (Fig. 2 ), but syncytium formation was also observed following infection with the Friend MLV isolates FBLV and FrMLV57. Infection with MoMLV or Spl574 occasionally resulted in syncytium formation in these transfectants, but these syncytia were smaller and fewer in number, and, appeared 1-2 days after the appearance of syncytia in parallel cultures infected with the most cytopathic isolate, F-S MLV. Thus, cells expressing the dCAT-1 receptor can, like M. dunni, produce syncytia in response to virus infection, but these transfectants differ from M. dunni in their relative insensitivity to Spl574 and their sensitivity to syncytium formation by virus isolates that are not typically cytopathic in M. dunni cells.
Virus induced syncytium formation in M. dunni cells was previously shown to be accompanied by the appearance of high levels of unintegrated viral DNA [2] , a phenomenon also observed for other pathogenic retroviruses [8] .
To determine if the transfected cells show this same response to cytopathic virus, we extracted Hirt DNA from CAT-1 transfected MA139 cells 3 days after infection with F-S MLV (Fig. 3A ).
At the time of DNA extraction, FerrM cells, expressing mCAT-1, showed no cytopathic response and the observed level of unintegrated viral DNA was low (4.0% of M. dunni, Fig. 3A ,B). In contrast, virus-induced syncytia were observed in all 3 dCAT-1 transfectants. 2 of these 3 transfectants had large multinucleated syncytia that involved >50% of the cells in infected cultures (Fig. 3C) ; levels of unintegrated linear DNA in these cells were high (53% and 68% of M. dunni) (Fig. 3A,B) . The third dCAT-1 transfectant showed fewer and smaller syncytia ( dCAT-1-g : : : : V : : : : : : : : E : : : : : V : : : : : : G : : : :
Thus, viral DNA accumulation is observed in dCAT-1 but not mCAT-1 transfectants, increased viral DNA is associated with virus-induced cytopathicity in the transfected cells, and the amount of viral DNA varies with the severity of the cytopathic response.
To define the relationship between syncytium formation and productive virus infection, transfected cell lines carrying either mCAT-1 or dCAT-1 were tested for susceptibility to a panel of ecotropic MLVs using the XC plaque overlay test (Table 1 ). In this assay, clusters of infected cells expressing ecotropic Env glycoprotein are identified by plaques of syncytia formed by overlaid rat XC cells [9] . For the cytopathic viruses Spl574 and F-S MLV, the number of syncytia induced directly by these viruses in susceptible cells is approximately equivalent to the titer determined by this XC overlay assay; for example, parallel cultures of infected M. dunni cells produced an XC titer of 10 5.1 (Table  1 ) compared to Spl574 syncytium titer of 10 4.6 .
Transfected M. dunni cells expressing mCAT-1 in addition to the endogenous dCAT-1 gene were significantly more susceptible to MoMLV infection than untransfected M. dunni cells (Table 1) , consistent with a previous study indicating that the dCAT-1 sequence variation is responsible for M. dunni resistance to MoMLV [6] . No difference was noted in the XC plaque titer of Spl574 in M. dunni cells expressing mCAT-1 in addition to the endogenous dCAT-1, and no viruses other than Spl574 and F-S MLV were cytopathic in the mCAT-1 transfected M. dunni cells.
The differences between M. dunni and NIH 3T3 cells in susceptibility to ecotropic viruses were not reproduced in MA139 cells expressing dCAT-1 (FerrD2) or mCAT-1 (FerrM). In fact, there were no significant differences in the XC titers of different MLVs in FerrD2 and FerrM (Table  1) . FBLV, F-S MLV, and, surprisingly, MoMLV efficiently infected both FerrM and FerrD2 with slightly higher XC titers for all viruses in FerrD2. Also, even though Spl574 efficiently replicates in M. dunni, Spl574 produced comparably low XC titers in both FerrD2 and FerrM. Thus, FerrD2 does not resemble M. dunni cells in its susceptibility to infection by MoMLV and Spl574; this difference suggests the involvement of additional factors independent of the CAT-1 receptor sequence.
The cytopathicity of different virus isolates did not always correlate with the efficiency of virus replication in FerrD2 as determined by XC virus titer. While on the one hand, Spl574 produced low XC titers on FerrD2 (Table 1) and was also poorly cytopathic, high XC titer viruses did not all produce syncytia in these cells. Thus, the most cytopathic virus in FerrD2 cells, F-S MLV, produced an XC titer comparable to that of the rarely cytopathic MoMLV. Efficient virus replication is thus not sufficient to generate a cytopathic response.
To further investigate the observed differences in XC titers for cells expressing different CAT-1 genes, we assessed infectivity using viral pseudotypes in a single round infectivity assay ( The Spl574 pseudotype is restricted in NIH 3T3 cells as is the Spl574 virus (Tables 1, 2) suggesting that this restriction is entry related. In contrast, the Spl574 pseudotype was not restricted in FerrD2 or FerrM cells although Spl574 virus produces low XC titers in both of these transfectants. This shows that the failure of Spl574 to replicate efficiently in the transfected cells is not entry related and suggests the involvement of factor(s) restricting post-entry stages of Spl574 virus replication in ferret cells.
M. dunni and FerrD2 cells express the same dCAT-1 receptor, but these cells differ in their relative infectivity by MoMLV and Spl574, and they produce syncytia in response to different virus isolates. One possible explana- tion for these differences is that CAT-1 may undergo different post-translational modification in the two cell lines. It has been shown that resistance of M. dunni cells to MoMLV infection is reduced by treatment with the inhibitor of glycosylation, tunicamycin (Tu) [10] . The involvement of glycosylation is also suggested by the observation that the CAT-1 glycosylation patterns differ in transfected MA139 and M. dunni cells ( Fig. 1B ; lanes e,f).
To determine if glycosylation contributes to the observed differences, we generated a dCAT-1 clone from which the N-glycosylation sites had been removed.
The CAT-1 protein has two glycosylation sites, and both carry N-glycans [7] . Both sites are in the third extracellular loop which also contains the residues implicated in virus binding and entry [11, 12] . Both glycosylation sites were removed by PCR mediated site-specific mutagenesis from the dCAT-1 variant (Fig. 1) , and the resulting clone, dCAT-1-g, was transfected into MLV, nor did it result in syncytium formation by viruses not cytopathic in M. dunni cells. The transfectants, however, showed increased susceptibility to MoMLV compared to untransfected M. dunni cells (Table 3 , Exp.1), as also shown above for M. dunni(mCAT-1) ( Table 1) ; the transfectants showed no increase in their susceptibility to other ecotropic viruses. This is consistent with the conclusion that glycosylation of dCAT-1 is associated with MoMLV resistance.
MA139 cells expressing dCAT-1-g resembled FerrD2 in their susceptibility to virus infection ( Table 3 , Exp. 2 and Table 1 ) and sensitivity to F-S MLV-induced syncytia (Fig.  4B ). The cells with the unglycosylated receptor were, like FerrD2, efficiently infected by MoMLV. Syncytia were produced in these transfectants with the same viruses that are cytopathic in FerrD2, and no cytopathic response was observed with viruses that are also noncytopathic in FerrD2. Thus, the complete absence of N-glycans on dCAT-1 did not alter the ability of the dCAT-1 receptor to mediate virus induced syncytium formation in MA139 cells, nor did it alter the panel of viruses that were cytopathic and/or infectious in the transfectants.
The glycosylation inhibitor tunicamycin (Tu) was previously shown to reduce resistance to MoMLV in M. dunni cells [10] . We tested the ability of multiple glycosylation inhibitors to alter infectivity of ecotropic MLVs in mouse cells expressing the two functional CAT-1 variants: mCAT-1 (NIH 3T3 cells) and dCAT-1 (M. dunni cells). The 6 inhibitors included Tu which blocks generation of the carbohydrate-dolichol precursor needed for N-linked glycosylation, the sugar analog 2-deoxy-D-glucose (2DG), and 4 inhibitors which inhibit different enzymes involved in oligosaccharide trimming: castanospermine (CST), deoxymannojirimycin (DMM), deoxynojirimycin (DNM) and swainsonine (Sw). Western analysis of M. dunni cells transfected with HA-tagged mCAT-1 (Fig. 5A) showed that none of the inhibitors had a significant effect on expression levels, although all inhibitors reduced the size range of the mCAT-1 glycoprotein.
Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells Because the resistance of NIH 3T3 cells to Spl574 infection is comparable to the resistance of M. dunni cells to MoMLV, we treated NIH 3T3 cells with 5 different glycosylation inhibitors before Spl574 infection (Table 4 ). All 5 inhibitors significantly reduced resistance to Spl574 replication, but inhibition of N-glycosylation did not affect the XC titer of other ecotropic viruses in NIH 3T3 cells, as shown for MoMLV. Resistance of SC-1 cells to Spl574 [1] is similarly relieved by glycosylation inhibitors (data not shown).
M. dunni cells were also treated with the same set of glycosylation inhibitors prior to virus infection ( Table 4 ). All inhibitors reduced the resistance of M. dunni cells to infection with MoMLV, but no comparable increase in titer was noted with Spl574. To confirm that this effect is on entry, DMM-treated M. dunni cells were infected with LacZ pseudotypes of MoMLV; pseudotype titer was 10 3.6 on DMMtreated cells compared to no detectable LacZ expressing cells in untreated M. dunni.
To determine if altered infectivity results from inhibitormediated changes in cell surface receptor levels, we measured biotinylated CAT-1 in M. dunni cells transfected with HA-tagged mCAT-1. As shown in Figure 5B , surface mCAT-1 in DMM-treated cells shows the expected reduction in size because of the predominance of smaller highmannose N-glycans, but quantitation of this expression by densitometric scanning shows that the level in DMM treated cells is not significantly different from the untreated control.
These results, taken together, indicate that N-glycans can impede ecotropic MLV entry in cells expressing mCAT-1 as well as cells expressing dCAT-1, and that these N-glycans obstruct different ecotropic isolates in NIH 3T3 and M. dunni cells. Also, the fact that the effect on entry is seen with inhibitors other than Tu suggests that inhibition may be due to N-glycan type or size.
Three factors contribute to the observed variations in host range and/or cytopathicity of mouse ecotropic gammaretroviruses: specific sequence differences in the viral env, differences in the CAT-1 receptor, and glycosylation of cellular proteins. The role of specific env sequence variations in virus-induced syncytium formation was previously suggested by our identification of two MLV isolates that are uniquely cytopathic in M. dunni cells. Both isolates have amino acid substitutions at the same RBD residue that is critical for receptor binding: S82F in Spl574 and S84A in F-S MLV. That mutations in the viral receptor binding site contribute to cytopathicity is also supported by the observation that a third MLV variant, TR1.3, is cytopathic in SC-1 cells and brain endothelial cells because of a single substitution, W102G [13] , at a site that together with S82 and D84 forms the receptor binding site [3, 4] .
The involvement of CAT-1 in the cytopathic response in M. dunni cells was suggested by the specific sequence differences that distinguish the dCAT-1 receptor variant from mCAT-1. These 2 receptors differ by 4 amino acids of which two are within the third extracellular CAT-1 loop that contains the virus binding site: I214V, and a glycine insertion within the YGE virus binding site [6] . As shown in the present paper, all cells expressing the dCAT-1 variant and none expressing mCAT-1 are susceptible to virusinduced syncytium formation. This indicates that one or both of these two amino acid changes, I214V and Δ236G, are responsible for the cytopathic response mediated by this receptor variant.
Previous studies with cytopathic retroviruses such as HIV have identified the accumulation of unintegrated DNA as a hallmark of cytopathicity [8] . Analysis of MA139 cells expressing the naturally occurring mouse receptor types, mCAT-1 and dCAT-1, shows that receptor type also correlates with this aspect of cytopathicity, and that in different dCAT-1 transfected lines the amount of unintegrated DNA corresponds to the extent of syncytium formation. This cell-virus system may thus be useful in further studies on the mechanisms thought to be involved in this cell killing such as endoplasmic reticulum stress induced apoptosis [14] .
It is known that the glycans on various cell surface receptors can modulate virus entry (for example, [15] ). The CAT-1 receptor is glycosylated at two sites, and previous studies have shown that glycosylation inhibitors reduce resistance to ecotropic MLV infection in rat and hamster cells expressing the rCAT-1 and haCAT-1 receptor variants [16] [17] [18] [19] , as well as resistance to MoMLV in M. dunni cells with dCAT-1 [10] . It has also been shown that in mink cells expressing mCAT-1, glycosylation affects SU binding and the down-modulation of receptor by virus infection [20]. Our results show that glycosylation modulates virus entry mediated by the laboratory mouse CAT-1 receptor, mCAT-1, in NIH 3T3 cells. This resistance is specific to Spl574 and is not seen in heterologous cells expressing mCAT-1. The control of this differential sensitivity of mCAT-1 to a specific ecotropic isolate by cell specific glycosylation has not been previously described.
The present study also considered whether altered glycosylation could explain why two cells expressing the same dCAT-1 receptor, M. dunni and FerrD2, produce syncytia in response to different viruses. As shown by the inhibitor results, however, while N-glycans contribute to the restriction of MoMLV entry into M. dunni cells, comparisons of ferret transfectants expressing the dCAT-1 or dCAT-1-g receptor variants produced no evidence that N-glycans modulate virus infectivity or virus-induced cytopathicity in the MA139 cells.
N-glycans can have high mannose, complex or hybrid structures. The various glycosylation inhibitors target different steps in protein glycosylation and can be used to manipulate the carbohydrate composition of glycoproteins. The inhibitor CST blocks glucose trimming, and DMM and SW inhibit successive steps in mannose trimming. The fact that all of these inhibitors along with the sugar analog 2DG and glycosylation inhibitor Tu relieved the resistance of M. dunni cells to MoMLV and of NIH 3T3 to Spl574 suggests that these viruses are most effectively blocked by the large complex oligosaccharides produced in the terminal stages of glycosylation. These results, taken together, suggest roles for N-glycans in virus entry that are virus-specific and cell-specific, and also indicate that this regulation may be sensitive to small sequence changes in both virus and receptor. These results indicate that N-glycans broadly regulate ecotropic gammaretrovirus interactions with the CAT-1 receptor in cells of their natural host [21] , although it is possible that glycosylated proteins other than CAT-1 may contribute to this resistance.
Our demonstration that not all infectious viruses are cytopathic in M. dunni and FerrD2 cells supports the idea that virus-cell fusion and cell-cell fusion are distinct receptormediated phenomena. A similar lack of correlation between infectivity and syncytium formation has been reported, for example, in a mouse cell line that is unusual in its resistance to HTLV Env-mediated syncytium formation although it is highly susceptible to virus infection [22] . It has also been shown that, for a transformed NIH 3T3 cell line subject to MoMLV-induced syncytium formation, chloroquine treatment blocks MoMLV entry but does not also block syncytium formation [23] . Our results further distinguish cell fusion and virus entry as separate receptor functions.
Finally, these studies also identify differences between M. dunni and FerrD2 cells that are clearly not receptor mediated. Use of LacZ pseudotypes shows that Spl574 Envs efficiently mediate entry into FerrD2 cells, but XC titers in Spl574 virus infected FerrD2 cells are clearly reduced as is virus-induced syncytium formation. This indicates a postentry block to virus replication leading to reduced surface Env, and the nature of this block is under investigation.
The CAT1 receptor mediates ecotropic gammaretrovirus entry and the cytopathic response to virus infection. Use of virus env variants, receptor mutations, and inhibitors of glycosylation demonstrate that both of these virus-receptor interactions are modulated by a small number of critical amino acid residues in virus and receptor, and that Nlinked glycans can modulate entry for specific virus-cell combinations. Virus stocks were made by collecting culture fluids from infected or transfected cells. These stocks were titered by the XC overlay test [9] following infection of NIH 3T3, SC-1 [24] , or M. dunni [5] and cells transfected with CAT-1 receptor. Cells were plated at 1-2 × 10 5 cells/60 mm dish and infected with 0.2 ml of appropriate dilutions of virus stocks in the presence of polybrene (4 ug/ml; Aldrich, Milwaukee, WI). Cells were irradiated 4 days after virus infection with ultraviolet light from germicidal bulbs (30 sec at 60 ergs/mm 2 ) to kill the cells but not the virus, and were then overlaid with 10 6 XC cells/plate. XC cells produce plaques containing syncytia in response to focal areas of virus infected cells. Plates were fixed and stained 3 days later and examined for plaques of syncytia.
To screen for the formation of multinucleated syncytium in virus infected cells, 2 × 10 4 cells in six-well tissue culture plates or 10 5 cells in 60 mm plates were infected with virus-containing medium in the presence of 4 ug/ml polybrene. After 2-4 days, the cells were examined by light microscopy using objective lenses of 4×-20× and photo- The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5′ and 3′ end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal. The precursor of HIV-1 structural proteins, Gag, and the precursor of the viral enzymes, Pol, are translated from the full-length viral messenger RNA (mRNA). Gag is produced by conventional translation whereas Pol requires a programmed -1 ribosomal frameshift during the elongation step of translation, which generates the fusion protein Gag-Pol (1, reviewed in 2, 3) . Previous studies showed that a 2-to 20-fold increase in the Gag-Pol to Gag ratio prevents viral infectivity (4-7) and our group showed that a decrease in the frameshift efficiency as low as 30% severely impairs the replication of the virus in cultured cells (8) . The Gag-Pol to Gag ratio is therefore critical for viral infectivity and the programmed -1 frameshift that determines this ratio represents an interesting target for the development of novel antiretroviral agents against HIV-1.
The HIV-1 frameshift event requires two cis-acting elements in the viral mRNA: a slippery sequence, UUUUUUA, where the frameshift occurs (1, reviewed in 2,3), followed by an irregular stem-loop (9) (10) (11) , the frameshift stimulatory signal, that makes the ribosomes pause over the slippery sequence and controls the frameshift efficiency. Only a fraction of the ribosomes that encounter the stimulatory signal make a frameshift. After the pause, the ribosomes unfold the signal, which can reform after their passage.
HIV-1 can use a cap-dependent mechanism to initiate translation of its mRNAs, like most eukaryotic mRNAs (for a review on translation initiation, see [12] [13] [14] [15] . There are two major control steps in eukaryotic cap-dependent translation initiation (see details in Figure 1A ). One is the binding of the initiator tRNA, Met-tRNA i Met , to the 40S ribosomal subunit, which requires the participation of the initiation factor 2 (eIF2) associated to GTP. The other one is the binding of the 40S subunit bearing the ternary complex to the 5 0 cap structure of the mRNA, which is controlled by the eIF4F complex. Double-stranded RNA (dsRNA), such as the TAR RNA structure, can modify the rate of translation initiation. TAR, the transactivation response element, is a 59-nt stem-bulge-loop structure present at the 5 0 and 3 0 end of all HIV-1 mRNAs in the nucleus and the cytoplasm (reviewed in 16) . It is also present under a free form of 58-66 nt in the cytoplasm of cells infected with the virus (17, 18) . In the nucleus, TAR mediates transcription activation by binding to the viral Tat protein and the cellular cyclinT protein (19, 20) . In the cytoplasm, a low concentration of TAR activates PKR, the dsRNA-dependent protein kinase, whereas a higher concentration of TAR inhibits this kinase by blocking its dimerization, which is essential for its activity (reviewed in 21) . When PKR is activated, it phosphorylates the a subunit of eIF2, interfering with translation initiation, whereas, when it is inhibited, the amount of eIF2 phosphorylated decreases and the rate of translation initiation increases.
In this study, we investigated whether the presence of TAR affects HIV-1 frameshift efficiency in relationship with the changes it causes in the rate of cap-dependent translation initiation. To this end, we used a dualluciferase construct (8) which expresses the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) separated by HIV-1 frameshift region as a fusion protein. Rluc is expressed following conventional rules of translation whereas Fluc expression requires a -1 frameshift in the HIV-1 frameshift region. This type of construct is adapted from Grentzmann et al. (22) , who pioneered the use of a dual-luciferase reporter for studying recoding signals. CD4+ T cells (Jurkat) or 293T cells were transfected with the dual-luciferase plasmid and TAR was added either in cis or in trans of the reporter mRNA. Several conditions were assayed to characterize the effect of TAR on frameshift efficiency and the involvement of PKR in this effect, such as the introduction of a small or a large amount of TAR in the cells, the use of mutants of TAR that cannot perturb PKR activity and the silencing of PKR expression with short interfering RNA (siRNA).
Our results show that HIV-1 frameshift efficiency increases at a low concentration of TAR, when capdependent translation initiation is slowed down, whereas it decreases at a high concentration of TAR, when translation initiation is stimulated. These effects were shown to be dependent on PKR. A model is presented which relates the effects of TAR on frameshift efficiency to changes in the spacing between the elongating ribosomes on the mRNA caused by changes in the rate of translation initiation. Such changes affect the frequency of encounter between the ribosomes and the frameshift stimulatory signal.
To measure HIV-1 frameshift efficiency, we used the dualluciferase reporters pDual-HIV(-1) and (0) (8) . These plasmids are derived from pcDNA3.1Hygro+ (Invitrogen) and contain the HIV-1 frameshift region inserted between the coding sequences of the Renilla (15) . The figure is adapted from Gebauer and Hentze (14) . Only the factors we refer to in the text are named. The 40S ribosomal subunit associates with the ternary complex [initiation factor 2 (eIF2) plus GTP plus the initiator tRNA, Met-tRNA i Met ] and with other factors, and binds to the 5 0 cap structure of the mRNA. This binding requires the eIF4F complex formed by three initiation factors: eIF4E, the cap-binding protein, eIF4G, a scaffold protein and eIF4A, a RNA helicase that unfolds secondary structures. After each round of initiation, eIF2 is released from the ribosome in association with GDP. Phosphorylation of the a subunit of eIF2 (eIF2-a) prevents the recycling of eIF2-GDP in eIF2-GTP, blocking translation initiation. Thapsigargin induces endoplasmic reticulum stress, which stimulates the PERK kinase that phosphorylates eIF2-a, reducing the level of functional eIF2 (55) (56) (57) . Rapamycin shuts down the mammalian target of rapamycin (mTOR) pathway, which blocks the phosphorylation of the translation repressor 4E-BP, and hypophosphorylated 4E-BP sequesters the initiation factor eIF4E (58, 59) . Hippuristanol is a selective inhibitor of eIF4A (60) , which interferes with the binding of the 40S subunit to the mRNA. luciferase (Rluc) and the firefly luciferase (Fluc). Expression of these genes is under control of a CMV promoter, which is followed by a T7 promoter. Plasmid pDual-HIV(0) differs from pDual-HIV(-1) by the addition of an adenine after the slippery sequence in the frameshift region. Derivatives of pDual-HIV(-1) and (0) were constructed where the TAR sequence was inserted after the CMV and T7 promoters. A TAR-containing fragment flanked with HindIII sites obtained from pcDNA3-RSV-TAR-Rluc plasmid (23), a kind gift from L. DesGroseillers (Universite´de Montre´al), was cloned in the HindIII site of pDual-HIV to produce pDual-HIV-TAR(-1) and (0), where the TAR sequence is located at a distance of about 40 nt from the 5 0 end of the reporter mRNA. To produce pDual-HIV-50TAR(-1) and (0), where the TAR sequence is at a larger distance from the 5 0 end of the reporter mRNA, a cassette of a 50-nt noncoding sequence was inserted in the AflII site of pDual-HIV, followed by the insertion of TAR immediately after these 50 nt, in the HindIII site. The oligonucleotides for the cassette were cass50nt-fwd and cass50nt-rev (see the sequence of all the oligonucleotides used in this study in Table 1 of the Supplementary Data). Plasmid pTAR, which expresses the free TAR sequence in trans from the reporter mRNA, was made by inserting the TARcontaining fragment flanked with HindIII sites into the HindIII restriction site of pcDNA3.1Hygro+. Derivatives of pTAR, pTARuucg à and pTARibulge à , which express mutants of TAR, were constructed by cloning oligonucleotide cassettes (cass_TAR-uucg à fwd and cass_TAR-uucg à rev or cass_TAR-bulge à fwd and cass_TAR-bulge à rev) between the two NheI restriction sites present in the TAR sequence of pTAR. In the first mutant, the upper loop, CUGGGA, is replaced with UUCG and, in the second mutant, the bulge UCU preceding the upper loop is deleted. Plasmid pCGNiC [a generous gift from N. Hernandez, Cold Spring Harbor Laboratory (24) ] expresses a mutant of the TAR-binding protein Tat (Tat à ), named TatC30,31A.
Jurkat cells (CD4+ T cells) were maintained in RPMI 1640 medium (Wisent) supplemented with 10% (v/v) FBS (Wisent) and HEK 293T cells (human embryonic kidney cells transformed with adenovirus and simian virus 40 large-T) were maintained in DMEM (Gibco) supplemented with 10% (v/v) FBS. Transfections were performed with polyethylenimine (PEI) (Polysciences, Inc.) in six-well plates containing Jurkat cells (1.2 Â 10 6 ), 293T cells (4.0 Â 10 5 ) or 293T stable transfectants (6.0 Â 10 5 cells) expressing a dual-luciferase HIV reporter (see subsequently). PEI was added drop-wise to serum-free medium and incubated 10 min at room temperature. In parallel, serum-free medium was added to DNA. The diluted PEI was added to the DNA solution (PEI to DNA ratio of 2:1) and incubated at least 15 min at room temperature. An empty plasmid, pcDNA3.1Hygro+, was added, when required, to maintain an equivalent DNA input.
Translation inhibitors were added as follows: rapamycin (Fisher), 16 h post-transfection (final concentration: 25 nM), hippuristanol (a generous gift from J. Pelletier, McGill University), 24 h before harvest (final concentration: 400 nM) and thapsigargin (Sigma), 4 h before harvest (final concentration: 300 nM). Transfected cells were harvested 48 h post-transfection. Non-adherent cells were centrifuged at 3000 g for 5 min, washed with PBS and lysed in 100 ml of Cell Passive Lysis Buffer (Promega). Adherent cells were washed with PBS and lysed in 400 ml of Cell Passive Lysis Buffer. Cell lysates were centrifuged 2 min at 13 000 g at 48C to remove cell debris, before luciferase assays.
Plasmids pcDNA5-Dual-HIV(-1) and (0) were made by inserting the HindIII-ApaI fragment from pDual-HIV(-1) or (0), respectively, into pcDNA5-FRT (Invitrogen), which contains a resistance gene to hygromycin B. An in-frame construct without the HIV-1 frameshift region was generated by cloning an oligonucleotide cassette (inframe-fwd and inframe-rev) into the KpnI and BamHI restriction sites of linearized pDual-HIV. In pDual-in-frame, the luciferase coding sequences are in the same reading frame and separated by a short linker. The HindIII-ApaI fragment from pDual-in-frame was cloned into pcDNA5-FRT.
Cell lines stably expressing the (-1) or (0) dual-luciferase HIV reporter, or the in-frame construct, were generated following the manufacturer's instructions, using 293T Flp-in TM cells (Invitrogen). Individual clones that stably incorporated the plasmids were selected on the basis of their resistance to hygromycin B (Wisent) (250 mg/ml) and maintained in hygromycin B.
293T transfectants (6.0 Â 10 5 cells) stably expressing the (-1) and (0) dual-luciferase HIV reporter were transfected with 150 ng of the PKR ShortCut Õ siRNA Mix or the eGFP ShortCut Õ siRNA Mix (New England BioLabs), using PEI. The TAR-expressing plasmids were transfected 24 h after the transfection with a siRNA mix. Cells were harvested 48 h after this second transfection and luciferase assays were performed.
293T transfectants, transfected with a siRNA mix, as described above, were harvested 48 h after the transfection, washed in PBS and lysed in 100 ml of Ripa-Doc (final concentration: 140 mM NaCl, 8 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.05% sodium dodecyl sulphate), containing a cocktail of protease and phosphatase inhibitors. Equal amounts of proteins (15 mg) were separated on a 10% SDS-PAGE gel, transferred on a nitrocellulose membrane and immunoblotted with a mouse anti-PKR hybridoma supernatant (clone F9) (a generous gift from A. Koromilas, McGill University) and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Amersham) diluted 1/1500. After detection of the antigen-antibody complexes, the membrane was washed with 25 ml of stripping buffer (final concentration: 0.08 M b-mercaptoethanol, 2% sodium dodecyl sulphate and 0.06 M Tris-HCl, pH 6.9) for 30 min at 508C, and immunoblotted with a mouse antia-tubulin monoclonal antibody (clone B-5-1-2 Sigma) diluted 1/5000 and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody diluted 1/1500. Antigen-antibody complexes were detected with an enhanced chemiluminescence (ECL) system.
The Fluc versus the Rluc activities of the (-1) and (0) constructs were measured as relative light units with a Berthold Lumat LB 9507 luminometer, as previously described (8) . A Dual-Luciferase Reporter Assay System kit (Promega) was used for Jurkat cells and home-made reagents (25) were used for 293T cells. The Rluc activity is used to normalize the Fluc activity (Fluc/Rluc). The frameshift efficiency is equal to: ½FlucðÀ1Þ=RlucðÀ1Þ= ½Flucð0Þ=Rlucð0Þ þ FlucðÀ1Þ=RlucðÀ1Þ:
Our aim was to investigate whether the presence of TAR affects HIV-1 frameshift efficiency in relationship with its effect on cap-dependent translation initiation. To this end, we used a dual-luciferase construct, pDual-HIV(-1), which contains the Rluc and the Fluc reporter genes separated by the HIV-1 frameshift region ( Figure 1B ). In this construct, the Fluc is produced only by ribosomes that make a -1 frameshift when translating the HIV-1 frameshift region. To assess the frameshift efficiency, we used a control construct, pDual-HIV(0), in which an adenine is added after the slippery sequence in the frameshift region, so that the Fluc coding sequence is in-frame with the Rluc coding sequence. The Rluc is synthesized by conventional translation in both (-1) and (0) constructs. Before investigating the effect of TAR, we verified that changes in cap-dependent translation initiation affect HIV-1 frameshift efficiency. Jurkat cells, a CD4+ T-cell line, were transfected with pDual-HIV(-1) or (0) plasmids and treated with thapsigargin, rapamycin or hippuristanol, three inhibitors perturbing a different step of capdependent translation initiation ( Figure 1A ). The frameshift efficiency, which is 5.1 AE 0.4% in the absence of inhibitors, was increased about twofold in the presence of either one of these three inhibitors ( Figure 1C) .
The presence of a high amount of TAR decreases HIV-1 frameshift efficiency
We next assessed the effect of TAR on the frameshift efficiency. TAR (Figure 2A ) was inserted at about 40 nt from the 5 0 end of the mRNA in pDual-HIV, generating pDual-HIV-TAR(-1) and (0) ( Figure 2B ). We avoided placing TAR at the very end of the mRNA, since such a position could interfere with the binding of the 40S subunit to the messenger (23,26,27 and references therein). We first examined the effect of a high amount of TAR that inhibits PKR and stimulates translation initiation (21) . The frameshift efficiency was assessed in Jurkat and 293T cells. When 2 mg of pDual-HIV-TAR were delivered into the cells, the frameshift efficiency was decreased to 70% of its value in absence of TAR in either Jurkat or 293T cells ( Figure 2C and D) . Under the conditions of these assays, the frameshift efficiency in absence of TAR was 6.1 AE 0.2% in Jurkat cells and 11.3 AE 0.9% in 293T cells. These values, and the value of 5.1 AE 0.4% observed in the experiment described in the preceding section with Jurkat cells that were transfected under slightly different conditions (see details in 'Materials and Methods' section), are comparable to the values obtained with different heterologous systems containing the HIV-1 frameshift region, which were shown to range between 2 and 10% in mammalian cultured cells (8, 22, 28, 29) . It can be recalled here that several groups observed that the absolute value of the frameshift efficiencies changes, depending upon various parameters such as the conditions used for the assay and the type of cultured cells (30) . We then investigated whether the decrease in frameshift efficiency observed with pDual-HIV-TAR was influenced by the position of TAR in cis or in trans from the reporter mRNA. Two other constructs were used, pDual-HIV-50TAR, where the distance between TAR and the 5 0 end of the reporter mRNA was increased by 50 nt compared to pDual-HIV-TAR, and pTAR, that provides TAR in trans from the reporter mRNA expressed from pDual-HIV ( Figure 2B ). The frameshift efficiency was decreased to 75 and 60%, respectively, in Jurkat cells and 293T cells transfected with pDual-HIV-50TAR compared to the value in absence of TAR. When Jurkat and 293T cells were co-transfected with 2 mg of pDual-HIV and 2 mg of pTAR, the frameshift efficiency was reduced to 70% of its value in absence of TAR, a decrease similar to that observed when TAR was present in cis of the reporter mRNA ( Figure 2C and D) . These results indicate that it is the presence of TAR in the cells and not its presence in the reporter mRNA that decreases HIV-1 frameshift efficiency. The effect of TAR on the frameshift efficiency was confirmed when using an infection system to deliver the reporters into the cells (see Figure 1 in the Supplementary Data).
To verify that PKR was involved in the changes in HIV-1 frameshift efficiency observed with a high amount of TAR, we created two constructs, pTARibulge à and pTARuucg à , expressing mutants of TAR that cannot bind PKR (31) ( Figure 3A ). When Jurkat cells were co-transfected with pDual-HIV and plasmids generating these TAR mutants, the frameshift efficiencies were similar to that obtained in absence of TAR and significantly higher than the value obtained in the presence of wild-type TAR ( Figure 3B ). This result supports that PKR is involved in the changes of frameshift efficiency observed in the presence of TAR.
To further confirm that inhibiting PKR decreases HIV-1 frameshift efficiency, a plasmid expressing Tat, a HIV-1 viral protein, was co-transfected with the dualluciferase plasmids. In addition to its well-characterized transactivation effect on transcription of the viral mRNAs by binding to TAR, Tat influences translation by inhibiting PKR, either directly by binding this kinase or indirectly by blocking the binding of TAR to PKR (32, 33) . We used a Tat mutant (Tat à ) that can bind TAR and inhibit PKR but cannot transactivate transcription, and, thereby, that does not affect mRNA levels (24) . Jurkat cells were co-transfected with the plasmid coding for this Tat mutant and with pDual-HIV, pDual-HIV-TAR or pDual-HIV-50TAR. In the presence of Tat à , the frameshift efficiency was decreased to approximately 60% of its value in absence of Tat à ( Figure 3C ). The decrease with Tat à was the same, whether TAR was present or not, which suggests that Tat à and TAR both act via the same mechanism, the inhibition of PKR.
Next, we investigated the effect of a small amount of TAR, which activates PKR and thus interferes with translation initiation (21) . We used stable 293T transfectants expressing a dual-luciferase HIV reporter. Stable transfectants expressing a (-1) or (0) dual-luciferase HIV reporter were transfected with pTAR, pTARibulge à or pTARuucg à in amounts ranging from 0 to 2.3 mg. Figure 4A shows the effect of wild-type TAR. In the presence of a small quantity of TAR, the frameshift efficiency increases to about 140% of its value in absence of TAR but with a larger quantity of TAR, the frameshift efficiency decreases to about 80%, a decrease comparable to that observed with a transient transfection of pDual-HIV ( Figure 2 ). As a control, we used stable 293T transfectants expressing Rluc and Fluc in-frame, separated by a linker instead of the HIV-1 frameshift region. The ratio of Fluc activity to Rluc activity in lysates from these transfectants was unchanged in the presence of pTAR (data not shown), confirming that changes in the Fluc to Rluc ratio observed with stable transfectants expressing the dual-luciferase HIV reporter are due to variations in the frameshift efficiency. When the stable 293T transfectants expressing the dual-luciferase HIV reporter were transfected with plasmids producing TAR mutants that cannot bind PKR, the frameshift efficiency was unaltered ( Figure 4B) . The effect of a low amount of TAR was also assessed by transient co-transfection of Jurkat cells with pDual-HIV and different quantities of pTAR, ranging from 0 to 2 mg, the ratio of pTAR to pDual-HIV being equal or inferior to 1:1. The frameshift efficiency also increases under the conditions corresponding to low amounts of TAR, the highest increase being $140% of the frameshift efficiency without TAR (data not shown).
We investigated the involvement of PKR in the changes in frameshift efficiency observed with a low amount of TAR. To this end, PKR expression was silenced by transfecting a PKR siRNA mix into stable 293T transfectants expressing a dual-luciferase HIV reporter. After 24 h, cells were transfected with pTAR in different amounts and harvested 48 h later. As a negative control, an eGFP siRNA mix targeting GFP was used. In the presence of the eGFP siRNA, the frameshift efficiency increases when TAR is present. However, when PKR expression is silenced, this effect disappears, supporting that it is related to PKR activation ( Figure 5A ). Effective silencing of PKR is achieved under the conditions of the assay as shown in Figure 5B . It can be noted that the response of the cells to the increase in the amount of TAR appears to differ from that in Figure 4 . This is due to a difference in the experimental protocol resulting in a lower ratio of the quantity of transfected pTAR to the number of cells (see 'Materials and Methods' section).
Using a dual-luciferase reporter system in Jurkat and 293T cells, we showed that the presence of TAR alters HIV-1 frameshift efficiency. The addition of a high amount of TAR, in cis or in trans of the reporter mRNA, decreases the frameshift efficiency. This effect is related to an inhibition of PKR. Conversely, a low amount of TAR increases the frameshift efficiency, by activating PKR.
Activation or inhibition of PKR is well-known to affect translation initiation via changes in eIF2 phosphorylation (reviewed in 21). However, it is also known that transformed cells, such as those we used in this study, tolerate a certain degree of endoplasmic reticulum stress leading to a certain level of phosphorylation of eIF2 via PERK, a kinase functionally homologous to PKR (34) . Our experimental conditions do not drastically affect the expression of our reporters, implying that the changes in the translation initiation rate caused by activation or inhibition of PKR are small and that the changes in eIF2 phosphorylation should be modest. Using western blotting, we could not detect significant variations in the phosphorylation level of eIF2 in 293T or Jurkat cells transfected with different quantities of TAR (data not shown). We nevertheless suggest that the effect of PKR on HIV-1 frameshift efficiency results from changes in eIF2 phosphorylation that are too small to be detected in presence of the endogenous signal for phosphorylated eIF2 in these cells. However, we cannot exclude that PKR could also influence HIV-1 frameshift efficiency via another yet undiscovered mechanism.
Contradictory effects were seen in previous observations on the influence of the translation initiation rate on the frameshift efficiency. The frameshift efficiency of a plant virus, the beet western yellow virus (BWYV), was higher in a reticulocyte lysate than in a wheat germ extract, which has a lower rate of translation initiation (35) . Also, the frameshift efficiency of the human T-cell leukemia virus type II (HTLV-2), when measured in a reticulocyte lysate, was higher with capped than with uncapped mRNAs, which have a lower rate of translation initiation (36) . These observations disagree with our results that show a negative relationship between the rate of translation initiation and the frameshift efficiency. However, Paul et al. (37) , when comparing the frameshift efficiency of the barley yellow dwarf virus (BYDV) with capped and uncapped mRNAs in a yeast extract, found that increasing the translation initiation rate decreased the frameshift efficiency. Furthermore, Lopinski et al. (38) , who investigated in vivo the effect of a reduced translation initiation rate on the frameshift efficiency of the L-A virus of S. cerevisiae, found that this efficiency was increased under these conditions. The results of Paul et al. (37) and Lopinski et al.(38) are in perfect agreement with our findings, and, in line with them, we present the following model that explains our results ( Figure 6 ).
When a ribosome translates the HIV-1 frameshift region, it encounters the frameshift stimulatory signal and makes a pause, its decoding center covering the slippery sequence (39) (40) (41) . During the pause, the ribosome can shift or not the reading frame, and, after the pause, the ribosome unfolds the frameshift stimulatory signal and translation continues. If the upstream ribosome reaches the frameshift region before the signal has refolded, the probability that the frameshift occurs is extremely weak. The spacing between ribosomes translating the HIV-1 frameshift region, which is determined by the rate of translation initiation [basal rate estimated to about one initiation event every 6.5 s (42)], could thus affect the frameshift efficiency. Therefore, if we assume an average elongation speed of five amino acids per second per ribosome, corresponding to a displacement of 15 nt per second on the mRNA (42) , the minimal distance between the decoding centers of two ribosomes translating a mRNA would be of about 100 nt. A ribosome covers about 32 nt on the mRNA and heel-printing studies showed that the first base of the P-site codon is at a distance of 12 nt from the 5 0 edge of the ribosome and of 20 nt from the 3 0 edge (43) . From these calculations, there would be about 70 exposed nt between two elongating ribosomes. Thus, the HIV-1 frameshift region, including the 43-nt frameshift stimulatory signal, would be exposed after the passage of the first ribosome. The signal would then re-form, which takes only a few microseconds (44), before the upstream ribosome reaches the region of the mRNA containing the sequence of this signal. However, the pause made by the first ribosome when encountering the signal decreases the distance with the following ribosome, which has continued to progress during the pause of the first ribosome. This second ribosome could reach the region corresponding to the stimulatory signal before this signal could refold, being still partially covered by the first ribosome. A pause of about three seconds for the first ribosome is sufficient to prevent the refolding of the stimulatory signal. The second ribosome would thus avoid frameshifting and the spacing between this ribosome and the third ribosome would not be altered. As a consequence, the third ribosome would encounter the stimulatory signal and pause, and frameshifting would be possible. This analysis shows that the signal affects every other ribosome under basal conditions. According to this model, an increase in the rate of translation initiation would decrease the frameshift efficiency, since ribosomes would be closer to each other and a smaller proportion of ribosomes would encounter the folded frameshift stimulatory signal. Conversely, a decrease in translation initiation would increase the frameshift efficiency since ribosomes would be further apart and it is very likely that each ribosome would encounter the folded signal. Interestingly, Lopinski et al. (38) , when studying the effect of a reduced translation initiation rate with the L-A virus in yeast cells, observed that the frameshift efficiency doubled, independently of the severity of the initiation defect. Their interpretation was that every other ribosome encounters the signal under basal conditions and that, with a reduced initiation rate, every ribosome encounters this signal. Our analysis fully supports this interpretation. Figure 6 . Changes in the rate of translation initiation influence the frameshift efficiency by modifying the spacing between elongating ribosomes. This model shows elongating ribosomes that reach the frameshift region and explains how the rate of translation initiation, which determines the spacing between these ribosomes, affects the frameshift efficiency (see the text). Note that a ribosome must encounter a folded frameshift stimulatory signal to make a frameshift, but this encounter does not ensure that frameshifting will occur.
Although HIV-1 does not induce a rapid and dramatic global shutdown of host cell translation following infection, in contrast to other viruses such as poliovirus, cap-dependent translation initiation is decreased due to cellular stress following infection by this virus (25, 45) and this decrease can be related to PKR activation (46) . Our results suggest that a change in cap-dependent translation initiation could affect HIV-1 frameshift efficiency in infected cells. As mentioned in the 'Introduction' section, the virus replication appears to be exquisitely sensitive to changes in frameshift efficiency. Given the detrimental effect of such changes, the virus likely uses various strategies to counteract this effect. One strategy is inhibition of PKR (reviewed in 25, 47) to stimulate translation initiation. HIV-1 uses two major ways to inhibit PKR: its Tat protein inhibits PKR and its TAR RNA structure blocks PKR dimerization when present in large quantities. TAR is located at the 5 0 and 3 0 end of all HIV-1 mRNAs and is also present under a free cytoplasmic form of 58-66 nt (17, 18) . All these forms of TAR can participate in the inhibition of PKR.
However, inhibition of cap-dependent translation initiation can occur independent of PKR activation. Indeed, the HIV-1 Vpr protein is capable of inducing G2 arrest in cultured CD4+ T cells (48, 49 and references therein), and, during such arrest, cap-dependent translation initiation is severely impaired (50) . Another possible strategy to circumvent the problem caused by this situation is the use of a cap-independent mechanism by HIV-1 to initiate the translation of its full-length mRNA (25, 45) . The virus would thus continue to express Gag and Gag-Pol and would maintain a frameshift efficiency that is optimal for its replication. An internal ribosomal entry site (IRES) was identified in the 5 0 UTR region of HIV-1 full-length mRNA (51) and another IRES was found in the beginning of the gag coding sequence (52) . IRES have also been found in HIV type 2 (53) and in simian immunodeficiency virus (54) , two viruses related to HIV-1. However, the use of an IRES by HIV-1 in the context of replication-competent viruses remains to be proven (45) . The two strategies that are described above are not mutually exclusive. HIV-1 could first counteract changes in cap-dependent translation initiation by inhibiting PKR, until a larger stress in the cellular environment severely perturbs cap-dependent initiation. The virus would then switch to an IRES-driven mode to translate its full-length mRNA.
This scheme is deduced from studies in cultured cells and it will now be important to investigate the frameshift efficiency in the context of a viral infection. A detailed understanding of the mechanisms used by HIV-1 to control its frameshift efficiency will provide valuable information for the design of drugs targeting the frameshift event. Establishing a nationwide emergency department-based syndromic surveillance system for better public health responses in Taiwan BACKGROUND: With international concern over emerging infectious diseases (EID) and bioterrorist attacks, public health is being required to have early outbreak detection systems. A disease surveillance team was organized to establish a hospital emergency department-based syndromic surveillance system (ED-SSS) capable of automatically transmitting patient data electronically from the hospitals responsible for emergency care throughout the country to the Centers for Disease Control in Taiwan (Taiwan-CDC) starting March, 2004. This report describes the challenges and steps involved in developing ED-SSS and the timely information it provides to improve in public health decision-making. METHODS: Between June 2003 and March 2004, after comparing various surveillance systems used around the world and consulting with ED physicians, pediatricians and internal medicine physicians involved in infectious disease control, the Syndromic Surveillance Research Team in Taiwan worked with the Real-time Outbreak and Disease Surveillance (RODS) Laboratory at the University of Pittsburgh to create Taiwan's ED-SSS. The system was evaluated by analyzing daily electronic ED data received in real-time from the 189 hospitals participating in this system between April 1, 2004 and March 31, 2005. RESULTS: Taiwan's ED-SSS identified winter and summer spikes in two syndrome groups: influenza-like illnesses and respiratory syndrome illnesses, while total numbers of ED visits were significantly higher on weekends, national holidays and the days of Chinese lunar new year than weekdays (p < 0.001). It also identified increases in the upper, lower, and total gastrointestinal (GI) syndrome groups starting in November 2004 and two clear spikes in enterovirus-like infections coinciding with the two school semesters. Using ED-SSS for surveillance of influenza-like illnesses and enteroviruses-related infections has improved Taiwan's pandemic flu preparedness and disease control capabilities. CONCLUSION: Taiwan's ED-SSS represents the first nationwide real-time syndromic surveillance system ever established in Asia. The experiences reported herein can encourage other countries to develop their own surveillance systems. The system can be adapted to other cultural and language environments for better global surveillance of infectious diseases and international collaboration. groups starting in November 2004 and two clear spikes in enterovirus-like infections coinciding with the two school semesters. Using ED-SSS for surveillance of influenza-like illnesses and enteroviruses-related infections has improved Taiwan's pandemic flu preparedness and disease control capabilities.
Taiwan's ED-SSS represents the first nationwide real-time syndromic surveillance system ever established in Asia. The experiences reported herein can encourage other countries to develop their own surveillance systems. The system can be adapted to other cultural and language environments for better global surveillance of infectious diseases and international collaboration.
With the recent global concern over emerging infectious diseases (EID) and the challenges of the 2003 SARS epidemics, government health officials in SARS-affected countries have begun to consider various measures of improving their infectious disease surveillance systems [1] [2] [3] [4] . Infectious disease epidemiologists and several leading public health administrators at the Centers for Disease Control in Taiwan (Taiwan-CDC) becoming aware of the importance of early detection of EID or bioterrorism, started developing an automatic alert system. Therefore, the Automatic Syndromic Surveillance Planning Task Force Committee was created and recruited infection physicians, epidemiologists, biostatisticians, and information technology (IT) experts in July 2003 to oversee the initiation and development of Taiwan's first medical informatics-based emergency department syndromic surveillance system (ED-SSS).
To prepare for this project, we reviewed the syndromic surveillance systems of other countries and officials of health informatics at Taiwan-CDC started collaborating with the Real-time Outbreak and Disease Surveillance (RODS) Laboratory at the University of Pittsburgh to develop a real-time syndromic surveillance system for Taiwan in August 2003 [1, [4] [5] [6] [7] [8] . RODS, used during the 2002 Olympic Winter Games, is the first commonly used syndromic surveillance system in the United States and has been found to efficiently process and analyze data in a timely manner [9] [10] [11] . Together, the task force and the RODS group aimed to establish a nationwide syndromic surveillance system within six months to meet the challenges of potential avian flu outbreaks for up-coming winter seasons and other future EIDs. To gain more operational level experiences, we also visited the Department of Health in New York City, where syndromic surveillance system was established and has been in daily operation since 2001 [12] . There, the task force members observed routine workflow processes and became familiar with other practical concerns of operating an ED-SSS on a daily basis. Based on these experiences and high population density in Taiwan, we decided to create a nationwide surveillance system. To this nationwide ED-SSS, we added geographical information system (GIS) technology, meant to facilitate epidemiological investigation and feedback between data providers and decision-makers [13] .
Using the electronic data from the health information systems already in place in about eighty percent of the hospitals in Taiwan required by the National Health Insurance Payment Program and the technical support of the RODS Laboratory at the University of Pittsburgh, Taiwan's ED-SSS has been in operation since March, 2004 [14] . It is the first time in Taiwan that information technology and timely data directly from hospitals has been used with systematic approaches to facilitate public health surveillance. This report shares our experience of establishing an ED-SSS in a non-English-speaking country. It covers the process of taking into account the various needs at different levels of hospitals, discusses the stages of developing the system, and highlights the characteristics of ED-SSS data collected during the first year. The experiences reported here may benefit other countries seeking to establish or improve their own surveillance systems for infectious disease.
Initially, two Taipei City Municipal Hospitals that kept electronic files of their emergency department patients' medical information were selected as pilot sites. From these two pilot hospitals, we had learned work flow involved in the process of data transfer, format of Chinese chief complaints, practical concern of ED (such as heavy workload etc.) during and after the 2003 outbreak of SARS, and available electronic ED information from nationwide emergency care hospitals. To obtain more representative data from various geographical areas, we gradually enlisted the cooperation of 189 hospitals nationwide, all offering emergency healthcare. Because many outbreaks of EIDs require emergency health care, these emergency care-designated hospitals were required transmit their ED triage and patient data to the Taiwan-CDC electronically on a daily basis ( Figure 1 ).
Nurses at the triage stations at all participating hospitals generated the bulk of the information needed for syndromic surveillance. That information included time and date of admission, date of birth, gender, home zip-code, body temperature, triage categories and chief complaints for patients admitted to their EDs. Because the National Health Insurance requires hospitals to keep clinical data written in ICD-9 codes based on the criteria of the international classification of diseases, 9th revision, clinical modification (ICD-9-CM) for billing purposes, we were also able to collect clinical data from initial assessment of each case by an ED physician. These fundamental data were provided in either the health level-seven protocol (HL-7) format or extensible Markup Language (XML) format, if HL-7 was not used by a hospital during the time of the study. Thus, ED-SSS is capable of accepting these data of above-mentioned variables (Table 1) , including the patients' clinical and demographical information, and hospital identification numbers, in either format.
All the sentinel hospitals recruited into our system had independent MySQL servers on which their data were saved, plus a remote connecting program for automatic transfer of data. Data files generated by the 189 hospitals, including data from their triage classification systems, hospital information systems (HIS) and clinical information systems (CIS), were firstly de-identified and then transferred hourly to a Microsoft SQL Server 2000 at the Taiwan-CDC. Hyper text transfer protocol over secured sockets layer (HTTPS) or secured file transfer protocol (SFTP) was used in this process. All communication histories were recorded in a log file in the SQL server at the Taiwan-CDC and monitored daily by health informatics personnel. A program was written into the system so that each transfer attempt to the Taiwan-CDC would automatically generate an e-mail to the hospital notifying them whether the transfer had been successful or not.
Three different data storage tables were designed to process the data in the Taiwan-CDC's syndromic surveillance database. All information received is initially fed into the first table, with a serial number generated in an additional column for each case. The system picks up the data from the first table every five minutes and moves it into a second temporary table for a logic check and data cleansing. At this point, the system checks for unambiguously erroneous data, e.g., a birth date later than the admission date or other variables such as body temperatures that fall outside of reasonable ranges. The data cleansing work is accomplished through a system algorithm written with SQL commands. The cleaned-up data are transferred to the third table for further epidemiological analysis, aberration detection, and then sent them to related local public health agencies.
Although only a few variables were collected from each hospital on consecutive days, one major difficulty we had was the data presented by discontinuous data, i.e. data that sometimes be there and sometimes not. Sometimes data were repeated. To handle this problem, specific criteria of data cleansing were used for different variables, including the logic checks described above and double checks for possible presence of duplicate patient records. If data in the chief complaint field was written as "test" or the field was left empty or if the ICD-9 field was written as "test" or left empty, they were deleted before data analysis. Hospital ID, date of birth, admission time, gender, and home zip-code were used as key indicators of whether a listing is a duplicate listing and be deleted as repeated data. The system was capable of performing frequent and rapid checks of any subject of hospital identification code and time format of all time fields. It was capable of moving erroneous data to an "error table" for storage. Incorrectly formatted ICD-9-CM data were also moved to the error table. All deletion and removal operations were recorded in the log file for monitoring. In certain situa-tions in case possible systematic errors were found (i.e., aberrant number of ED visits on certain days or occasionally inconsistent formats of ICD-9 codes), the data examiner would contact the medical informatics officers of those specific hospitals to discuss improving data entry.
After the data cleansing, we categorized ED visits into 11 different syndromic groups important in Taiwan. There were: (1) fever, (2) respiratory, (3) skin, (4) neurological, (5) upper gastrointestinal (GI), (6) lower GI, (7) haemorrhagic, (8) influenza-like illness (ILI), (9) asthma, (10) enterovirus-related infection (EVI) syndrome, and (11) syndrome for severe illness or death. Since only about 25% of all chief complaints were written fully in English and the grouping of syndromes by chief-complaints due to Chinese language barriers would have effects on the outbreak detection ability, we first analyzed our data according to the ICD-9 coded syndrome groups [15] . Definitions of clinical syndromes were based on two different sources: (1) those associated with bioterrorism-related agents as announced by the Centers for Disease Control and Prevention (CDC) in the U.S.; and (2) those identified as important by the ED-SSS Advisory Committee in Taiwan, whose members include infectious disease physicians, emergency doctors, pediatricians, and epidemiologists [16] . For example, because the epidemics of enterovirus 71 caused severe fatal cases in Taiwan in the years of 1998, 2000 and 2001, the EVI syndrome group was considered as an important syndrome group locally (ICD-9 codes listed in Table 2 ) [17] . All patient information was de-identified and only aggregated data was used for data analysis. The protocol for this study was approved by the Research Ethical Committee (Institutional Review Board) of National Taiwan University. 20 Coxsackie carditis, unspecified 074. 21 Coxsackie pericarditis 074. 22 Coxsackie endocarditis 074. 23 Coxsackie myocarditis, Aseptic myocarditis of newborn 074. 3 Hand, foot, and mouth disease Vesicular stomatitis and exanthem 074. 8 Other specified diseases due to Coxsackie virus, Acute lymphonodular pharyngitis Although the ED-SSS data started transferring on March 10, 2004, we confined our analysis to data collected between April 1, 2004 (when the data became more stabilized) and March 31, 2005 . Data were organized using statistical programs to perform a descriptive analysis of the daily and weekly plots of different syndrome cases and obtain a baseline pattern for each syndrome in Taiwan. We initially generated the SQL commands for data querying and data grouping into the 11 different syndromic groups. To increase the sensitivity of this ED-SSS in monitoring regional patterns of these 11 syndrome groups, we categorized ED-SSS data by four different geographical areas (northern, central, southern and eastern Taiwan), based on major regional variations in the types of infec-tious diseases. In analyzing the seasonal patterns of ED visits, the correlation between the ILI syndrome and respiratory or asthma syndrome was assessed by the value of Pearson's coefficient (R). the Taiwan-CDC on a daily basis. The greatest challenge as began to develop this system was communication with different hospitals. Because different hospitals were using different information systems and inputting different data with various formats, it took long time to agree which variables and their data format should be collected. At the very beginning to build the ED-SSS, we had at first intended to collect information on a large set of epidemiologically useful variables, including occupation, travel history, family clustering, other exposure-related factors, and address for each patient to help detect possible zoonosis. However, such data were not collected during routine medical examination and care. Finally, we decided to capture only parameters usually collected by the hospitals during examination, intake and care.
During the planning phase, it was necessary to gain a full understanding of what not only just public health personnel expected but also what the medical staff at participating hospitals expected from the ED-SSS. Public health officials tended to prefer a timely and sensitive surveillance system able to detect all possible outbreaks of emerging or known infectious diseases. Mostly concerned over the limited public health resources, they wanted more evidence to prove the cost-effectiveness of ED-SSS and fewer false positive signals from pilot studies before integrating the system into routine public health surveillance workflow. On the other hand, the hospitals and their medical staff had three major expectations. First, the hospitals expected an easily operated feedback mechanism and quick feedback of useful information for better decision-making. Those who had experienced nosocomial infection of SARS during the 2003 SARS outbreak were particularly interested having analyzed information, based on their own hospital or regional/national hospital data, quickly fed back them. They believed that this would provide incentive for them to share hospital data and routinely maintain the high quality of their data for public health usage. Second, the hospitals anticipated two-way communication with public health agencies, as they frequently been requested or even forced to send data on short notice when they were too busy or too involved in emergency care. What made matters worse, despite their compliance; they had difficulty in obtaining useful feedback information from the public health agencies so that they could improve their care of patients at the time of an outbreak crisis. Third, the hospital decision-makers wanted immediate firsthand feedback, particularly with regard to control of nosocomial infection and hospital management in order that their health-care workers could be protected during regional outbreaks. Considering the expectations of both public health agencies and hospitals, we learned that the syndromic surveillance system should provide efficient means of feedback and effective two-way communication.
From April 1, 2004 to March 31, 2005 , data transmitted from the 189 hospitals on 2,692,325 ED visits were collected and stored in the Taiwan-CDC database. Initially, we appointed two computer engineers to cleanse the data by checking the log files and inform the hospitals by telephone on weekdays to correct errors or provide missing data. Then, these cleaned data on daily counts of ED visits collected from ED-SSS were analyzed. The time series plot of rough data on daily numbers of ED visits in our nationwide ED-SSS during the study period is shown in Figure 3 .
The computer system shut down twice during this period. The first time occurred from August 8 th to August 9 th , when no daily procedure was installed to monitor the quality of uploaded data. To help both hospitals and public health agencies perform routine data quality checks, we installed a computer program having check-up procedures of data quality after each data transfer from the hospital to the Taiwan-CDC for automatic quality control of data. This program records all the logs of each data sending from the participated hospitals. If Taiwan-CDC doesn't receive data from hospitals, program will send e-mail automatically to inform the personnel of health informatics in that hospital about failure sending. System maintenance personnel need to check the log daily and make a phone call to the hospital to verify successful data transfer and quality of data if there are no data transfers in two consecutive days.
Only 5.04% of hospitals failed to send ICD-9-CM data, but almost half (47.4%) failed to send chief complaints. For example, of the 239,617 sets of cleaned data received in July 2005, about 7.1% of ICD-9-CM information was lacking or filled out as 'null' and 54.82% of cases did not include chief complaints. Additionally, certain hospitals transmitted the patients' chief complaints in Chinese, further complicating the analysis. Because of these difficulties, this report focuses on the data based on ICD-9-CM diagnostic codes only. The time-series plots of the 11 syndrome groups (Figure 4 , 5) that may correlate to infectious diseases and important health problems in Taiwan (e.g., asthma has become an important pediatric problem in recent years) were analyzed.
Understanding the characteristics and patterns of numbers of ED visits over time from our established ED-SSS in Taiwan is very crucial before we set up appropriate threshold levels of different syndrome groups for outbreak detection, There was a significant difference in daily counts between weekdays and weekends, which occurred on a weekly basis. ED visits were 1.288-fold-higher on weekends than on weekdays (p < 0.001), while national During this first year study, the ED-SSS found patients throughout Taiwan were more likely to seek emergency medical services at medical centers than at district or local hospitals. Most patients visiting ED were 60 years old or older (21.46%) or below the age of 10 years old (18.55%). These two groups were also at greatest risk for various infectious diseases, especially influenza (Table 3) . Young adults between 20 to 39 years old ranked the third most frequent visitors to ED (17.43%), though traffic accidents, not infectious diseases, were the main reasons for their visits. These age distributions suggest that the newly established ED-SSS was capable of providing information for the age groups most at risk for severe cases of infectious diseases in Taiwan. Male ED patients slightly outnumbered female ED patients (male : female = 1.12:1), which approximates the general distribution of gender in Taiwan (male/female ratio = 1.10). For elderly ED patients (age > 65 years old), the male/female ratio in our sample was the same as general population (male: female = 1.10:1).
To understand the epidemiological characteristics of the eleven important syndrome groups and asthma syndrome in ED-SSS, data of their daily and weekly counts were plotted and shown in Figure 4 and 5, respectively. As can been seen in time series plots in Figure 6 , the seasonal patterns for ED visits due to respiratory syndrome and ILI syndrome were quite similar with high correlation (R = 0.98), while that for asthma syndrome, which had distinct peaks, was nonetheless not highly correlated with ILI syndrome (R = 0.78) nor respiratory syndrome (R = 0.77). Importantly, the ED-SSS was able to detect peaks of these respiratory-related syndromes even occurred in or around the summer season (July to September), though their most noticeable peaks were found during the Chinese new year holidays ( Figure 5B, 5C, 5D ). Like respiratory infection syndrome, visits due to the fever syndrome also showed another peak during the summer (July to September) ( Figure 4A ). ED visits for respiratory syndrome peaked earlier (mid-September) than those visits for ILI (mid-October) and asthma syndrome (end of September, e.g. the transition period between summer and autumn). Visits for pediatric asthma syndrome for children 12 years old and below peaked in mid-autumn, around mid-October (data not shown). Later during winter season (between November and February), there was an increase 
Respiratory Syndrom Asthma Visits In gastrointestinal (GI) syndromes, visits due to upper GI or lower GI or total GI started increasing November 2004 and peaked during the Chinese New Year holidays ( Figure  4F, 4G, 4H) . Interestingly, cases of hemorrhagic syndrome also increased slightly in the winter season ( Figure 4I ).
For those syndrome groups with severe symptoms, including skin rash, neurological symptoms and death/ coma that might be related to bioterrorism attacks, there was no significantly cyclic or seasonal patterns ( Figure 4J , 4K and 4L). One spike of syndrome cases with clinical severity (severe syndrome) appeared in mid-May, but that occurred as a result of one hospital sending duplicate data that escaped from our check algorithm ( Figure 4L ).
Taiwan has very high population density (Taiwan, 632.23 persons/km 2 ; Taipei, 9662.53 persons/km 2 ), which increases the spread of many human-to-human infectious diseases [19] . This makes the surveillance of infectious diseases very important for this island. Taiwan's ED-SSS is the first syndromic surveillance system to be implemented in Asia. It also represents the first time that Taiwan's public health agencies have attempted active nationwide surveillance. Its automatic data collection mechanism is capable of capturing comprehensive population-based health information and providing important details on current disease epidemics at the community level. The information it provides can also be used as community baseline data for further infectious disease modeling and can also improve the detection of emerging infectious diseases.
In addition to the information that the ED-SSS can provide for disease control, it can open avenues for further investigation. For example, in addition to the neurological syndrome, asthma syndrome, and syndrome for severe symptoms, there were clear and consistent weekend and holiday increases in the visits of other nine syndrome groups. Because cost of ED visits in Taiwan is not as expensive as it is in other countries, especially the United States, it is very likely that many patients seek ED medical care when local clinics are closed on weekends and holidays. It is also possible that the gathering of people on the holidays would increase the transmission of certain pathogens, particularly on cold days in closed spaces where respiratory viruses including influenza virus are easily transmitted. Therefore, future research might want to investigate the effect of holidays on the aberration detection of outbreaks and prediction of number of cases for certain infectious diseases using Taiwan's ED-SSS.
We also found differences in seasonal trends in visits due to symptoms/signs related to respiratory, influenza-like illness and asthma syndromes. Our ED-SSS found a sum-mer peak in visits for cases with influenza-like illnesses in 2004. This has seldom been found by the previous passive surveillance systems used in Taiwan. These summer cases of influenza-like illness occurred before annual vaccinations usually done in October or November. Therefore, a further longitudinal analysis of influenza-like syndrome patterns is needed to formulate the best vaccination policy on human influenza.
Another epidemiological finding from our ED-SSS was an increasing trend in visits due to gastrointestinal syndrome starting late autumn 2004. Such trends have not been detected by other infectious disease surveillance systems in Taiwan. There are two possible explanations for this finding. One reason for the increases might be related to the increased activity of certain pathogens, including rotavirus or norovirus, during winter season, as was found during the winter of 2006 in both Japan and Taiwan [20] [21] [22] [23] [24] [25] [26] [27] . Another reason might be the social habits of Taiwanese who like to dip raw meats and seafood into boiling water fondues and eat from the chafing pot during the winter season. This would increase the change that inexperienced or careless diners would consume undercook seafood or use chopsticks contaminated by raw seafood.
The findings of our ED-SSS, the first time in Taiwan to use daily rather than weekly data, suggest further directions for research into GI syndrome and many other diseases of significant interest to public health. For example, during the 1998, 2000 and 2001 enterovirus 71 epidemics, children aged 3 years and younger who were at higher risk of severe or fatal cases of the disease were identified for more effective prevention only after the occurrence of several cases of sudden deaths from weekly sentinel physician surveillance and later retrospective epidemiological data analysis on those cases when sample size became larger. Therefore, prospective monitoring of daily ongoing data of EVI syndrome in this high risk age group and early ED-SSS detection of enterovirus activities by local public health personnel might help minimize social panic among parents. Furthermore, the results on seasonal pattern of enterovirus-like infection in our ED-SSS was consistent with the previous epidemic patterns in Taiwan, again demonstrating the usefulness of ED-SSS to avoid future large-scale or severe epidemics caused by enteroviruses [18] . In summary, these initial findings suggest that it is necessary to develop algorithms capable of detecting aberrations for different syndrome groups from patients in different geographical areas of Taiwan, taking into account variations in the levels of medical care and the effect of weekends and holidays on ER visit.
The ED-SSS did not, however, reveal obvious trends in all syndrome groups. For example, it was hard to find seasonal patterns or secular trends in cases of coma/death, skin rash, or neurological symptoms -the three syndrome groups that might be useful in the detection of severe outbreaks caused by bioterrorism, e.g., anthrax, during the study period without bioterrorism attacks [28, 29] . Certainly, continuous monitoring for these syndrome groups at both local and national levels will be very helpful in detecting possible bioterrorism or EIDs in future years. Using those trends in coma/death and other syndrome groups of clinical severity or unexpected symptoms/signs, our ED-SSS data have provided directions for further research in the areas of pathogen detection, epidemiological clues, and improvement in public health policies. Therefore, future investigations have to control the weekend and holiday effects of ED visits for better aberration detection even during long holidays.
In daily public health practice to monitor the data of ED-SSS, careful verification and systematic management is needed once the aberration signals are detected. The server needs an automatic error feedback system function instead of the original use of engineers to double check for data errors would increase the efficiency and completeness of surveillance. Future efforts require closer collaboration between computer-science professionals and medical informatics personnel at the Taiwan-CDC to establish a system with the standard operating procedures (SOP) for database maintenance and to provide more continuous on-job training for both hospital users and local and central public health agencies [30] .
The major difficulty in developing our ED-SSS was diverse formats for different types of data, including categories of chief complaints, the ways to fill out ICD-9-CM codes, and even the different number of digits used in home zip codes in different participated hospitals. For example, most Taiwan hospital ED physicians/nurses EDs only write down one chief complaint, which is very different from the ED reports made by most U.S. hospitals which list all possible complaints (with text format) in English. Several participating hospitals only had a paper system for recording triage chief complaint data. A standard format for select syndromes and variables needs to be established and continuously reevaluated to improve data quality and stability of data transmission. There are needs to have more research into the chief complaints with Chinese styles, the suitability of chief complaints vs. ICD-9 codes, how to combine symptoms/signs and link data to improve sensitivity.
With regard to the current epidemics of avian influenza H5N1 in China and many other southeast Asian countries, an ED-SSS like the one we developed in Taiwan may play an important role in detecting an outbreak possibly caused by human-to-human transmission even when cluster size is small [31] [32] [33] [34] . Through early detection, ED-SSS may help minimize the adaptation of avian influenza virus to human populations. Because of the large volume of business traffic, international travelers, and workers from Southeast Asia coming to Taiwan, it has previously been difficult to do real-time surveillance for imported infectious diseases, including dengue, malaria, acquired immunodeficiency syndrome (AIDS) and SARS. However, using the ED-SSS to monitor health status at the community level may help public health decision-makers handle unexpected health threats. Because countries are so interconnected today, it is imperative that we share our health information and experiences with other countries if international health is to be guarded. Our ED-SSS has equipped Taiwan the ability to closely monitor avian influenza and other potential EIDs in Asia and worldwide. We hope that by sharing our experiences developing ED-SSS, other countries can be encouraged to develop and improve their own surveillance systems for infectious disease.
The author(s) declare that they have no competing interests.
TSJW was in charge of epidemiological data analysis, improvement of the ED -Syndromic Surveillance System, and manuscript writing. FYFS initiated the thoughts on Syndromic Surveillance for detecting emerging infectious diseases in 2003 and contributed to syndrome groupings, selection of variables for ED-SSS, and system improvement based on clinical data analysis. MYY contributed to syndrome groupings, initiating the standard format for collecting Chinese Chief-Complaints in our ED-SSS, and system improvement with regard to clinical aspects. JSJW initiated the thoughts on Syndromic Surveillance for Emerging Infectious Disease in 2003. SWL worked on computer programming on ED-SSS and health informatics for surveillance systems of infectious disease at Taiwan-CDC. KCMC was a leader and coordinator on health informatics for surveillance systems of infectious diseases at Taiwan-CDC and gave the most administrative support on systematic improvement in health informatics. CH provided statistical consultation and chose the best statistical modeling method for outbreak detection in the initiation stage of establishing the ED-SSS. JHC was the Deputy Director at Taiwan-CDC in charge of improving surveillance of infectious diseases, coordinated the 189 hospitals designated for emergency health care to participate the ED-SSS, and provided strong administrative support on ED-SSS. YTC was a research assistant of ED-SSS in charge of the data analysis and project administrative assistance. HC was the Director of Department of Health in Taipei City and participated in the task force meetings from planning to implementation of ED-SSS in the perspective of the local government. CHC was the Section Chief of Department of Health in Taipei City in charge of surveillance, prevention and control of infectious diseases in Taipei City and gave suggestions health informatics and practical concerns from the viewpoints of local government. FCRT helped set up the Real-time Outbreak and Disease Surveillance (RODS) system at Taiwan-CDC. MMW introduced our public health officials and scholars in Taiwan to the practical applications of RODS in the USA and informed us of recent progress of RODS. IJS, the former director of Taiwan-CDC, had the vision to invite scholars to discuss the improvement of infectious surveillance system in Taiwan right after the 2003 outbreak of SARS. CCK, involved in the improvement of infectious disease surveillance in Taiwan for more than twelve years, initiated research on syndromic surveillance, coordinated each trouble-shooting step as the ED-SSS was developed and implemented, and was involved the revision of the manuscript. All authors read and approved the final manuscript.
Publish with Bio Med Central and every scientist can read your work free of charge  Amino Acid Similarity Accounts for T Cell Cross-Reactivity and for “Holes” in the T Cell Repertoire BACKGROUND: Cytotoxic T cell (CTL) cross-reactivity is believed to play a pivotal role in generating immune responses but the extent and mechanisms of CTL cross-reactivity remain largely unknown. Several studies suggest that CTL clones can recognize highly diverse peptides, some sharing no obvious sequence identity. The emerging realization in the field is that T cell receptors (TcR) recognize multiple distinct ligands. PRINCIPAL FINDINGS: First, we analyzed peptide scans of the HIV epitope SLFNTVATL (SFL9) and found that TCR specificity is position dependent and that biochemically similar amino acid substitutions do not drastically affect recognition. Inspired by this, we developed a general model of TCR peptide recognition using amino acid similarity matrices and found that such a model was able to predict the cross-reactivity of a diverse set of CTL epitopes. With this model, we were able to demonstrate that seemingly distinct T cell epitopes, i.e., ones with low sequence identity, are in fact more biochemically similar than expected. Additionally, an analysis of HIV immunogenicity data with our model showed that CTLs have the tendency to respond mostly to peptides that do not resemble self-antigens. CONCLUSIONS: T cell cross-reactivity can thus, to an extent greater than earlier appreciated, be explained by amino acid similarity. The results presented in this paper will help resolving some of the long-lasting discussions in the field of T cell cross-reactivity. Each T cell expresses thousands of T cell receptors (TCR) of a single specificity that allows inspection of peptide fragments bound by major histocompatibility complex molecules (MHC) on the surface of other cells. Peptides originate as the product of intracellular protein turnover, and both foreign and self-peptides are able to form peptide:MHC complexes (pMHC). Presentation of peptides for which the inspecting CTLs have not been tolerized, triggers a cytotoxic response. Although much has been learned about peptide processing and MHC presentation [1, 2] it is still largely unknown why roughly half of all natural foreign pMHC are ignored [3, 4] . The processing and MHC binding of naturally processed foreign peptides is a primary requirement for the initiation of a cellular immune response. However, the availability of a suitable TCR further determines if a peptide is immunogenic. The structural mechanism of T cell recognition is a highly debated subject in the immunological literature and a consensus view of the promiscuous peptide recognition has not yet been reached (see e.g., [5] ). The core problem is that T cells seem to combine high specificity with the ability to recognize a surprisingly large number of dissimilar antigens. Two terms are often used to describe this nature of T cell recognition. Poly-specificity is used to emphasize TCR's ability to recognize multiple distinct/unrelated pMHC ligands with high specificity (with little or no tolerance to substitutions of the ligands) [6, 7] . Cross-reactivity is a term that was originally used to indicate unexpected reactivity to targets that differed from those used to initially define the T cell clone [8] . Several studies suggest that T cells can recognize seemingly dissimilar epitopes (for a summary see [6] ), while other studies have established that substitutions affect peptide recognition in a predictable and additive manner [9] suggesting that the majority of cross-reactive pMHC complexes share structural similarities. One outstanding question in T cell biology is therefore whether T cell cross-reactivity is mostly a stochastic phenomenon induced by unpredictable structural constraints or, whether we can predict which peptides should be cross-reactive. Previous studies of crossreactivity have focused on limited data covering a single or a few T cell clones. Here, we investigate a simple model of T cell crossreactivity and perform a large-scale analysis spanning both a broad set of experimental settings, heterogeneous pathogens, MHC molecules and T cell clones. We use this benchmark to investigate whether cross-reactivity is either generally predictable or mostly random. Finally, we test whether the degree of host mimicry is negatively correlated with immunogenicity. By analyzing a large set of known HLA-A2 restricted HIV epitopes, we investigate if potential HIV epitopes with high similarity to self are able to trigger detectable immune responses. Our results suggest that amino acid similarity, rather than identity, is a predictive measure of cross-reactivity.
We analyzed public data on CTL sensitivity and created a visualization of how CTLs react to single amino acid substitutions. Lee et al. [10] analyzed the specificity of CTL responses against the immunodominant HLA-A2 restricted HIV Gag epitope SLFNTVATL (SFL9). IFNc production was measured in response against all 171 single mutant variants of SFL9. Abrogated TCR responses were mostly due to loss of TCR binding as the majority of SFL9 variants retained binding to MHC. The cross reactivity data for the three data sets: G10, T4 and PBMC were converted into a position-specific-scoring-matrix (PSSM) as described in Materials and Methods. The recognition motif of the T4 clone (the PSSM matrix) is visualized in Fig. 1A as a Logo plot [11] . The plot shows a stack of the possible amino acid mutations on each position in SLFNTVATL (x-axis). The height of the stack is reciprocal to the number of tolerated mutations (i.e., it indicates the degree of T cell recognition specificity at this position, see Materials and Methods). Few tolerated mutations translate into tall stacks while many tolerated mutations show up as short stacks (bars). For example, in position five, only one variant is tolerated (T5S) and is shown as a tall bar. The remaining variants on position 5 were unable to bind the TCR even though the binding to MHC was mostly preserved. On the contrary, in position one, 18 out of 19 variants preserved the TCR recognition. The logo plot for CTL clone T4 given in Fig. 1A suggests that the central peptide positions are most important for peptide-TCR binding, which is in agreement with earlier data [12, 13] . The average Shannon information [14] plot for T4, G10 clones and PBMC is shown in Fig. 1C . This figure also indicates that positions 2-6 and 8-9 are most important for peptide recognition whereas position one is consistently of little importance. Position 2 and 9 are the main positions determining peptide binding to the HLA-A*0201 molecule, see Fig. 1B . Thus, positions 3-6 and 8 were consistently involved in the primary TCR recognition motif. Moreover, the sequence motif for T4 clone suggests that tolerated substitutions tend to be conservative with respect to the original epitope sequence, SLFNTVATL. Examples are F3Y (both non-polar and aromatic), T5S (both polar), and V6I (both aliphatic). Similar observations on tolerated substitutions were made for the other two CTL clones (data not shown). Taken together, these data suggest that amino acid similarity could be a major component of T cell recognition.
Using the information in the TCR amino acid position specific scoring matrices, we estimate the number of ligands recognized by a given T cell clone by assuming that recognizable peptides contain only those amino acids giving a detectable ELISPOT response in the Lee et al. [10] study. Non-recognized peptides are the ones containing at least one prohibited amino acid for which no response was detected. The number of recognizable peptides was computed by the following procedure. The degeneracy of a TCR on a single position was measured as the diversity of amino acids present at that position defined in terms of the Simpson index (see Materials and Methods). This diversity measure yields a value between 1 and 20. Here, 20 means that all amino acids are used with equal frequency at a position, and 1 means that only a single amino acid is found. The higher the diversity the more degenerate the TCR is at this position. In the binding motif of T4 clone (Fig. 1A ) the first position diversity is very high, 13.26, as [14] which is a measure of how conserved a position is. Rigid positions have few but tall letters, while very degenerate positions have many but very short letters. For example, position 1 was mutated 19 times of which 18 variants preserved TCR binding, only the S1R variant compromised TCR binding while the MHC binding was preserved (see [38] ). The frequency of amino acids occurring in this TCR motif can also be used to estimate the number of distinct ligands this T cell clone can recognize (see text for details). (B) Sequence motif of HLA-A2 binding peptides (277 HLA-A2 restricted peptides were extracted from the SYFPEITHI database [15] ). (C) The average Shannon information at each position, for the CTL clones: G10 and T4, and PBMC. doi:10.1371/journal.pone.0001831.g001 expected, because this position is highly degenerate. In the conserved position five, the Simpson diversity drops to 1.29. The product of the tolerated amino acid diversity at each position can provide an estimate of the number of ligands a T cell clone can recognize. For T4, we estimate a total of 5.6?10 5 ligands in this way and this value is in good agreement with previous estimates [8] . For the G10 clone, we estimate 3.2?10 6 ligands, suggesting that this clone is more degenerate. Similarly, one can estimate the number of ligands that can bind to a MHC molecule. For example, the HLA-A*0201 molecule (see Fig. 1B for the binding motif) can bind 4.8?10 9 distinct peptides [15] . Thus, measured in this way the CTL binding event is three orders of magnitudes more specific than that of the MHC.
The above calculation suggests that a single T cell receptor can recognize as many as 10 6 ligands. How related are these ligands, and is the cross-reactivity of a T cell clone predictable? A few studies suggest that cross-reactivity is not completely random [9, 16] , while others argue that T cells can recognize unrelated ligands (see e.g. [17] ). Here, we investigate whether TCR peptide cross-recognition can be predicted by a quantitative model of peptide similarity using amino acid similarity matrices (SM) as explained in detail in Materials and Methods. The peptide similarity score is unity for two identical peptides, and 0 for peptides of maximum dissimilarity, as defined by the SM. Note, that this simple model does not differentiate between positions. Below, the predictions made from this model are tested on several independent data sets, and compared against the performance of random predictors. [10] ). The Pearson correlation coefficients between their relative SFU and our peptide similarity score were: 0.40, 0.39, and 0.35 for G10, T4, and PBMC data, respectively (p,0.0001, Monte Carlo randomization exact estimate). Since PBMC consist of two clones, where one clone is dominant [10] , the prediction performance on this data set is similar to the performance on the single clonal data. These significant correlation coefficients suggest that peptide crossreactivity can, to some degree, be estimated from peptide similarities. Thus the proposed model of peptide similarity is capable of producing significant predictions of the loss of recognition due to single amino acid substitutions. Iversen et al. [18] measured IFNc secretion by T cells specific for SLYNT-VATL (SYL9), when they are stimulated with naturally occurring (i.e, patient derived) variants of SYL9. Data consisted of 21 variants of SYL9. Each variant peptide had between 1 and 3 mutations with respect to SYL9. Fig. 2B presents a scatter plot of the data from Iversen et al. [18] for the T4 clone, where the peptide similarity is plotted on the y-axis against the relative IFNc secretion (x-axis). Using the BLOSUM35 matrix to calculate the peptide similarity score (see Materials and Methods) the Pearson correlation was 0.65. Similar results were obtained using BLOSUM matrices 35-90 (data not shown). For the remaining CTL clones (G10, C-3, C-4, C-22 and C-32) tested by Iversen et al. [18] correlations were 0.49, 0.47, 0.55, 0.60 and 0.57 respectively (all values are significantly different from zero with p,0.02 Monte Carlo randomization exact estimate). This model of peptide similarity (or cross-reactivity) was thus able to explain around 20240% of the IFNc secretion. Still, a number of SYL9 variants, for which we predict rather high peptide similarity to SYL9, hardly induce an IFNc response, e.g., A7S, A7V, T5A mutants given in the upper left corner of Fig. 2 . Part of this discrepancy is due to the fact that our model is not position specific, and thus underestimates the effect of mutations in the central positions, which are crucial for T cell recognition (see Fig. 1A ). When more data becomes available, the peptide similarity model can be extended with a weighting accounting for the relative importance of the peptide positions.
We were able to achieve similar performances while testing the model on other peptide scanning data, e.g. La Rosa et al. [19] (HLA-A2 restricted CMV epitope, data not shown). Thus, our . The x-axis shows the relative IFNc secretion measured for 171 single mutants of SLFNTVATL (SFL9). Immunogenicity was grouped in four bins with average ELISPOT responses of 0, 0.15, 0.50 and 0.85 of maximal ELISPOT for SFL9. In both figures the y-axis shows the predicted CTL recognition in terms of BLOSUM35 similarity scores (see Eq. 2). Unfavorable (non-conservative) substitutions (low x) are associated with a low similarity score (low y) whereas conservative substitutions (high x) in general are associated with higher similarity scores (high y). (B) Observed and predicted recognition of patient derived SLYNTVATL (SYL9) variants with 0-3 mutations. The axis shows the relative IFNc and peptide similarity scores. Note, that the IFNc response falls to a half when peptide similarity is around 0.85. doi:10.1371/journal.pone.0001831.g002 model was able to predict cross-reactivity of T cell clones measured in at least two different peptide-scanning library studies.
Striking examples of T cell cross-reactivity have been reported for CTL responses to viruses [17, 20] . It was shown that CTLs that were elicited during a primary viral infection might also respond when the same mice are re-infected with unrelated viruses. By mapping the different viral epitopes to which a particular T cell clone can respond, it was demonstrated that these cross-reactive epitopes can share very little sequence identity [17, 20] leading to the conclusion that CTLs are extremely non-specific [8, 17, 20] . Reviewing the literature, we compiled a set of 19 cross-reactive epitopes in Table 1 . These epitopes are restricted to the K b , K d , D b , HLA-A1, HLA-A2, and HLA-B62 MHC alleles. Some of the epitopes share only a few amino acids; one is even different on all positions, while others share the majority of the amino acids. We assumed that the first epitope (x) in a cross-reactive pair (x,y) is the original epitope for which the cross-reactive CTL clone was first raised, and that it was observed to respond to y later (see Table 1 ). To test whether these cross-reactive epitopes that differ markedly in their sequence could nevertheless have structurally similar amino acids on the non-identical positions, we did the following. First we computed the similarity of the cross-reactive epitopes S O . Then we constructed an ensemble of random peptides that have the same identical positions as the cross-reactive epitope pair but otherwise consist of random amino acids (see Materials and Methods for details). We then computed the baseline (or the expected) peptide-similarity as the average random similarity denoted S E . In 16 out of 19 pairs the observed similarity S O exceeded the expected baseline similarity S E (see Table 1 and Fig. 3A, p,0 .02, Fisher's exact test). Fig. 3A shows the observed (S O ) versus the baseline expected similarity (S E ) and the solid line presents the case where S O = S E . This plot demonstrates that crossreactive epitopes are significantly more similar than unrelated peptides with the same level of sequence identity. Thus, in crossreacting T cell ligands non-identical positions are significantly more conservative than random. Fig. 3B shows this more explicitly. The 19 epitope pairs were split in two groups according to the level of sequence identity; less than 50% and larger than or equal to 50% identity. For both groups we compute the percent excess observed similarity of the cross-reactive constituents defined as 100?(S O 2S E )/S E From Fig. 3 , we clearly see that for ''seemingly'' unrelated sequences (identity,50%) the excess observed similarity (y-axis) is on average 25.8% +/2 10.8%, i.e., when sequence identity is low, the observed similarity is much higher than the expected similarity. Conversely, for epitopes sharing more than half the amino acids, excess similarity drops markedly (2.0% +/2 4.3%) probably because cross-reactivity is maintained by the more numerous identical positions. The difference in excess observed similarity between the groups is highly significant (p,0.001, rank test), which suggests that amino acid identity is a poor measure for estimating physicochemical similarity, and thus T cell crossreactivity. In summary, the above results demonstrate that biochemical similarity plays a large role in defining CTL crossreactivity when sequence identity is low. In such cases, crossreactivity is observed for non-identical, but conservative, substitutions preserving structural and/or physiochemical properties satisfying the idiosyncratic binding constrains of the responding TCR. The columns are as follows: 1) MHC restriction, 2) source pathogen and protein for initial infection, 3 
Another open question in T cell response is why roughly half of all foreign cell surface-presented antigens fail to raise a T cell response [3, 4, 21] . Tolerance to self-antigens could explain this lack of immunogenicity, in which case the degree of similarity to self-antigens should predict which foreign antigens are likely to be non-immunogenic. We examined this effect of self-tolerance on immunogenecity using our cross-reactivity model. First, a large set of self-antigens was defined, and secondly, a list of non-self (e.g., HIV) antigens was built, labeled as either immunogenic or nonimmunogenic according to experimental evidence (data obtained from the Los Alamos HIV database, see Materials and Methods). The expectation was that T cell clones, with high affinity for HIV peptides similar to self peptide(s), have been tolerized during thymic education via negative selection [22, 23] . Such TCRs should therefore not be present in the functional T cell repertoire thus causing tolerance to molecular mimics of self-peptides. We define a score of cross-reactivity to self as the maximum peptide similarity between the non-self antigen and the set of all selfantigens (see Materials and Methods) and test whether nonimmunogenic peptides have a higher cross-reactivity score to self when compared to immunogenic ones. We downloaded the human proteome from the NCBI website and identified a set of 230,460 potential HLA-A2 self-antigens (see Materials and Methods). Next, we downloaded the HIV proteome from the Los Alamos HIV database and predicted a set of potential HLA-A2 epitopes. 33 of the 91 predicted HIV candidate epitopes were annotated as A2 supertype restricted epitopes in the Los Alamos database of CTL HIV epitopes, while the remaining 54 of the HIV peptides were never identified as epitopes. Another four peptides were found to be immunogenic for other HLA alleles than HLA-A2. Since it would be wrong to tag these epitopes as ''non-immunogenic'', they were excluded from the data set. The 33 confirmed HLA-A2 epitopes were labeled: confirmed HIV epitopes and the remaining 54 possible non-immunogenic peptides were given the label: putative, non-immunogenic HIV peptides. It is possible that future studies reveal that a number of the putative nonimmunogenic HIV peptides do in fact elicit CTL responses in HLA-A2 + patients. Nevertheless, this set of HIV peptides should be enriched in HIV peptides that fail to generate CTL responses. Maximal similarity scores were computed between all 87 HIV peptides (33 immunogens and 54 putative non-immunogens) and the set of 230,460 predicted HLA-A2 self-epitopes. Fig. 4 shows a scatter plot of the 33 HIV immunogens (black diamonds) and 54 putative non-immunogens (open circles). The x-axis shows the predicted antigen presentation score (NetCTL) while the y-axis shows the estimated maximum similarity to the self-antigens S SELF (x,y) (see Materials and Methods). Immunogenic peptides tend to be less self-like, although the difference between immunogens and non-immunogens is not significant (p = 0.2, Mann-Whitney). Drawing a horizontal line at y = 0.85 separates the most self-similar HIV antigens from the rest (the results presented in Fig. 2B suggest that IFNc response would fall to a half when the similarity drops to 85%.) For the antigens that have a self-similarity score above 0.85, most (14/16) are classified as nonimmunogenic HIV antigens i.e. predicted epitopes not confirmed by experimental evidence (p-value,0.05, Fisher's exact test). Note, that the NetCTL score does not correlate with the maximal selfsimilarity score (p-value = 0.42, exact estimate) and the above difference between the immunogenic and non-immunogenic antigens is therefore not explained by the difference in the NetCTL scores. Repeating our analysis for HLA-A3 and HLA-B7, we found similar tendency of more-self-likeness among nonimmunogenic HIV-1 peptides (p,0.3 and p,0.45 respectively). Summarizing, these results suggest that similarity to self-antigens plays a role in discriminating immunodominant from cryptic peptides. 
Many studies have suggested that T cells can recognize totally unrelated peptides and a new term, poly-specificity was coined to express the high specificity of T cell receptors to unrelated peptides [6] . The ''unrelatedness'' of the peptides was defined as low sequence identity, however, sequence identity might not be able to account for the total amount of structural similarity that drives TCR recognition. Here, we demonstrate that this is indeed the case: T cell receptors recognize biochemically and structurally related peptides and cross-reactivity is, up to a degree, predictable. The loss of recognition simply depends on the number and similarity of non-identical amino acid between cross-reactive constituents. We find that the majority of the seemingly ''unrelated'' cross-reactive peptides have a significantly higher biochemical similarity to each other than what would be expected from truly ''unrelated'' peptides. This is especially true for peptides with very limited identity. To our knowledge this is the first study that analyzes a large set of cross-reactive peptide-MHC combinations and demonstrates that the cross-reactivity can, up to a certain extent, be predicted.
Because negative selection of immature thymocytes remove high affinity TCR specific for self-antigens [7] , we expected that this should leave a ''hole'' in the T cell repertoire around negative selecting self-antigens. Hence, if an infected cell presents a nonself antigen that is highly similar to a negative selecting self-antigen, then this foreign antigen might not be matched by any available TCR which could provide an explanation for why around half of foreign pMHC do not generate a T cell response [3, 4, 21] . We tested this hypothesis for HLA-A2 restricted HIV-1 response and showed that the absence of T cell response to part of the non-confirmed (i.e. putative non-immunogenic) HLA-A2 restricted HIV-1 peptides can be explained by their similarity to self antigens. These results are in agreement with a recent study by Rolland et al. [24] , who showed a trend of more-selflikeness (measured in terms of number of shared amino acids) among HIV peptides with no detectable CTL responses in a large study group. We predict that the correlation found by Rolland et al. would be stronger if amino acid similarity is taken into account.
If peptide similarity can be used to describe T cell reactivity, what would then be the best model to describe similar peptides? We have chosen for the simplest model (given by Eq. (2) in Materials and Methods) because systemic data on cross-reactive peptides is very limited. An obvious extension of this model would be to add position dependence, i.e., account for the fact that central positions play a larger role.
The number of potential antigens exceeds the number of T cells in the immune system and the ability to recognize multiple ligands is required to mount at least a few responses to all potential pathogens [8] . Here, we demonstrate that the number of expected T cell ligands is not necessarily reduced by restricting T cell recognition to cover only similar peptides: it is still possible for a T cell to recognize 10 5 210 6 peptides. These estimates of the complexity of CTL recognition are well within the bounds of earlier estimates [8] . In summary, the results presented here quantify, to our knowledge for the first time, that the basis of T cell recognition is amino acid similarity, defined in terms of biochemical properties of amino acid side chains.
Lee et al. [10] analyzed the specificity of CTL responses against the immunodominant HLA-A2 restricted HIV Gag epitope SLFNTVATL (SFL9). IFNc production was measured in response against all 171 single mutant variants of SFL9 for two T cell clones (G10 and T4), and for purified Peripheral Blood Mononuclear Cells (PMBCs) using an ELISPOT assay. Purified PBMCs consisted of just two clones where one was dominant. CTL responses were reported as the percentage of maximal IFNc (I) obtained for the reference ELISPOT of SFL9 and discretized in Peptide similarity score
The un-normalized peptide similarity score A(x,y) between reference epitope: x = {x 1 , L, x N } and peptide: y = {y 1 , L, y N } of the same length N is defined as the sum of substitution scores along the sequences expressed by the relation:
W(xi, yi) is the amino acid substitution matrix, e.g., BLOSUM35, providing a measure of how conservative substitutions are. The peptide-similarity score for the reference peptide x spans the interval:
where the length of the interval |I| = A max 2A min depends on references peptide x and matrix W. Two different intervals (I x , I y?x ) are not comparable per se. Thus, we define the normalized peptide similarity using the relation
This equation constitutes the model of peptide similarity used throughout subsequent analysis. S(x,y) measures how much peptide y resembles x in terms of the number and magnitude of conservative substitutions. A x max is the auto-peptide-similarity score of x. Thus, A x max = A(x,x). If peptide y is a mimic of x then S(x,y) should be close to 1. The other extreme value (A x min ) is found by comparing x with a peptide x, where on each position the amino acid in x corresponds to the substitution in x with the smallest value i.e. the least likely substitution. In this way 0#S(x,y)#1 for all peptides y. The peptide-similarity score is asymmetric i.e. S(a,b)?S(b,a) despite W being symmetric. The reason is that the extreme values (A min ,A max ) cannot be guarantied to be identical for any pair of peptides (a,b). Reference peptides x which are dominated by amino acids like tryptophan that hardly ever substitute, will have few highly similar peptides (y) which satisfy the condition: S(x,y) < 1. In contrast, reference peptides which are enriched in amino acids that are more likely to substitute (given the matrix W) have a greater number of highly similar peptides. This property is captured by the asymmetry of S.
The observed similarity S O of pairs of experimentally verified cross-reactive epitopes (x,y) is to be compared to unrelated peptides (z i ) which retain the sequence identity of (x,y) but have an otherwise random amino acid on non-identical positions. The procedure to compute the ''unrelated'' or ''baseline'' expected similarity is best illustrated by an example: The HLA-A2 epitopes x = GLCTLVAML and y = GILGFVFTL from EBV and influenza-A share 3 identical positions: G1, V6 and L9. We first compute the observed similarity S O = S(x,y) between epitopes x and y using Eq. (2). Then we generate a set of N = 10.000 random peptides, z 1 , z 2 , L, z N , with the same identical positions, i.e. we have z i = G......V...L where a dot can be any amino acids avoiding identity with x at that position. The expected similarity between the primary (original) epitope (x) and the unrelated but semi-identical artificial peptides z is then defined as the average similarity to the ensemble of unrelated peptides as: S E~1 N P N i~1 S x,z i ð Þ.
The HIV-1 HXB2 sequence for Env, Pol, Vpu, Rev, Tat, Vif, Vpr, p17, p24 and p2p7p1p6 and the HIV-1 clade B consensus sequence for Nef (due to a stop codon in HXB2-Nef) were downloaded from the Los Alamos database (www.hiv.lanl.gov). There was also one stop codon in HXB2 sequence for TAT, however, no TAT peptides were predicted to be HLA-A2 epitopes and thus the stop codon did not interfere with our results. Out of 3,063 HIV nonamers, 91 were predicted to be HLA-A2 epitopes using NetCTL version 1.2 [25, 26] and default selection threshold (0.75). NetCTL predicts the level of antigen presentation by combining three separate predictions of: proteasomal cleavage, TAP affinity and MHC biding. Four out of the 91 predicted HIV epitopes were found to be immunogenic for other supertypes than HLA-A2 and were filtered out. These were: QLQARILAV, RILAVERYL (class II, DPW4.2), TLYCVHQRI (HLA-A11) and SINNETPGI (HLA-A25). Of the remaining 91-4 = 87 peptides, 33 were confirmed HLA-A2 epitopes by cross-referencing the records of the LANL CTL epitope summary table (downloaded December 2006). Thus, the epitope prediction resulted in the identification of 87 possible HLA-A2 restricted HIV epitopes where 33 (38%) were confirmed and 54 (62%) were not.
The human proteome was downloaded from the NCBI website (www.ncbi.nlm.nih.gov/Genomes/date: 29 march 2006) and contained 34.460 protein sequences. The removal of proteins containing the words: predicted, hypothetic or isoform in the protein description label lead to a final core human proteome of 14,034 human protein sequences. We predicted A2 self-antigens using NetCTL version 1.2 [25, 26] for all these protein sequences (default epitope selection threshold). Repeats were removed, along with a small set of self-peptides, which contained the unknown amino acid (X). The final set consisted of 230,460 predicted human HLA-A2 restricted self-antigens each of length 9.
HIV self-similarity (HLA-A2)
The maximal similarity between predicted HIV antigens (x) and the set of human 230,460 self-antigens (y) was defined as the selfsimilarity score S self (x) = max(S(x,y)) for HIV peptide x. Self-similarity scores were obtained for all confirmed HIV epitopes and putative HIV antigens. Because no identical matches were found between HIV epitopes and self-antigens, self-similarity scores were always S(x,y),1. Confirmed HIV epitopes and putative non-immunogenic HIV peptides were ranked on maximum self-similarity, and the combined ranking was split in two parts: a) The peptides with a selfsimilarity score greater than 0.85 and b) and peptide with a selfsimilarity score below 0.85. We used Fisher's exact test to compute the significance of the difference in the frequency of putative HIV epitopes in the top versus the bottom. Distinguishing Molecular Features and Clinical Characteristics of a Putative New Rhinovirus Species, Human Rhinovirus C (HRV C) BACKGROUND: Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected. METHODOLOGY/PRINCIPLE FINDINGS: Genomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe. CONCLUSIONS: The divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission. Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs). HRVs have been associated with lower respiratory tract (LRT) illness and more serious clinical outcomes within pediatric and other vulnerable populations [1] . Despite this, HRV strains continue to be commonly defined, en masse, by their most prolific and currently most well-defined role in causing the 'common cold' [2, 3] .
Classified within the family Picornaviridae, the genus Rhinovirus consists of 100 serotyped strains divided into two species, Human rhinovirus A (HRV A; n = 75) and Human rhinovirus B (HRV B; n = 25) [4] . Species classification was initially based on the susceptibility of strains to capsid binding antivirals [5, 6] and subsequently confirmed by phylogenetic studies [4, 7] . HRVs are also subdivided by receptor usage; major group HRVs use an intercellular adhesion molecule (ICAM-1; n = 88 strains), which interacts within a depression of the viral surface known as the canyon [8] . Minor group HRVs (n = 12) receive a molecule from the low density lipoprotein receptor family (LDLR) at protrusions along the 5-fold axis of the capsid surface [9] [10] [11] [12] . Recently, heparan sulphate was identified as a pH dependent, low efficiency receptor for HRV-54 [13] . A relationship between receptor usage and species identity is only evident among minor group HRVs; all are HRV As. Major group HRVs reside in both species. Studies have identified four neutralizing immunogenic sites on the surface of a major group HRV (HRV-14; NImIA, NImIB, NImII and NImIII) [14] and three sites on a minor group HRV (HRV-2; site A, B and C) [15] , with some overlap [16] . These sites consist of discontinuous amino acid sequence within VP1, VP2 and VP3.
The human enteroviruses (HEVs) are the closest genetic relatives of the HRVs but they belong to the genus Enterovirus. HEV strains are classified into four species (A-D) and some have been implicated in ARTI [17] . Similar to the major group HRVs, some HEVs have been shown to use ICAM-1 as their primary cellular receptor [18] . Other host molecules employed by HEVs include the decay accelerating factor (DAF) [19, 20] , poliovirus receptor (PVR) [21] and coxsackie-adenovirus receptor (CAR) [22] . Under a proposal currently before the International Committee on Taxonomy of Viruses (ICTV), HRV and HEV strains will be combined into the genus Enterovirus, at the expense of Rhinovirus, an issue of contention for decades [23] .
Picornavirus genomes typically consist of a 7.2 to 7.5 kb positive-sense, single-stranded RNA molecule located within a non-enveloped icosahedral capsid. The genomes contain a single open reading frame flanked by 59and 39untranslated regions (UTR). The polyprotein is first cleaved into three precursor polyproteins, P1, P2 and P3 ( Figure 1 ). P1 is further cleaved into the structural proteins VP4, VP2, VP3 and VP1. Union of a single copy of each structural protein constitutes a viral protomer; the HRV capsid consists of 60 protomers arranged in a T = 1, pseudo T = 3 (i.e. T = p3) conformation. P2 and P3 are cleaved into seven non-structural proteins including two proteases (2A pro and 3C pro ) and an RNA-dependant RNA polymerase (3D pol ) [24] .
Recent studies have generated 27 additional HRV A and 12 additional HRV B genomes [25, 26] also resulting in the proposal that individual HRV genomes are under purifying selective pressure and that significant intra-strain variation occurs only at the antigenic sites [26] . This is in stark contrast to the HEVs for which recombination in the non-structural regions is a significant contributor to viral diversification [27, 28] . Untypeable rhinoviruses have been described using PCR-based tools, since 1994 [29] but it was only in 2007 that the complete coding sequences of a number of novel and apparently divergent HRV strains were reported, permitting a for identification of the first new strains in two decades [30] . The initial strain described, HRV-QPM, was identified from children with presenting symptoms often suggesting bronchiolitis [31] . Subsequently, 'HRV-Xs' were found in an adult asthma study in the United States [32] and 'HRV-Cs' were described from a study of pediatric infection by human bocavirus in Hong Kong [33] . Some notable features associated with these six putative viruses include the reliance upon molecular methods for their detection, their apparent endeminicity, their failure to produce cytopathic effects in culture, their frequent detection in subjects with expiratory wheezing and their limited sequence identity with existing HRV strains but high shared sequence identity. Studies employing subgenomic sequences proposed that there were many of these putative viruses occupying a distinct position within the genus Rhinovirus, which we collectively called HRV A2 [34] . It has been proposed that these strains may constitute a new picornavirus species [33] . Data have not yet been presented which appropriately address classification using current ICTV criteria and the scope of the clinical impact of these divergent strains has not yet been studied in detail. Because it appears that the members of this clade are frequently associated with LRT illness we sought to better characterise them in the hope of finding features that might prove useful in predicting the outcome of HRV A2 infection.
Intensive genomic analysis and computer-based modelling resulted in comprehensive characterisation of all HRV A2 polyprotein sequences and, by superimposing the sites of known receptor contacts, we were able to propose a more robust taxonomic placement for these newly identified viruses (NIVs). Detailed medical chart reviews on HRV-QPM-positive (prototype HRV A2) individuals were augmented by a severity scoring system which quantified the outcome of infection by this apparently uncharacteristically pathogenic clade of HRV strains.
We undertook a comparative analysis of all HRV A2 complete coding regions (n = 6; HRV-QPM, -X1, H2, -C024, -C025 and C026) against all HRV A (n = 34), HRV B (n = 13), most HEV sequences (n = 71, including species A-D) and two non-human enteroviruses (simian, SEV and porcine, PEV).
HRV A2 sequences had a higher G+C content (average, 42.4%) than all other HRVs (average 38.7%) or HEV Ds and a shorter coding region (average, 2144 aa) than any other picornavirus investigated. The final residue of the HRV A2 genomes was an isoleucine, distinguishing them from all other picornavirus genomes we investigated, which ended at the preceding phenylalanine; the biological implications of this feature are unknown.
We identified 10 protease sites (data not shown) dividing the HRV A2 polyproteins into four structural and seven nonstructural proteins ( Figure 1A ). However, unlike previous studies that relied on amino acid alignments to determine cleavage sites [33] , we obtained additional evidence that these sites were recognised by 2A pro and 3C pro by using a neural network protease cleavage-site prediction tool. A single cleavage by 2A pro was predicted between an A or L and a G residue at the VP1/2A junction. The remaining cleavages, predominantly occurring between Q and G residues, were mediated by 3C pro . Three motifs crucial for RNA polymerase binding (YGDD, TFLKR and SIRWT) were identified in all HRV A2 sequences [35, 36] .
Using SimPlotß [37] , the HRV A2 sequences were found to be most similar to those from HRV A strains (54% identity) whereas identity with HEV species ranged from 46%-49% ( Figure 1B ). Dips in identity occurred across all regions but only the structural region was investigated further because its components contain antigenic sites and receptor contact residues, or 'footprints'; the latter being significant domains for defining the major and minor group HRVs [38] and other picornaviruses.
Although identifiable using amino acid alignments [33] , the scope and impact of the many HRV A2 sequence deletions could not be fully qualified or quantified in this form. However, the inability to isolate the HRV A2 strains in vitro will hinder crystallography studies seeking to identify structural features, so we created structure-based alignments of the VP1-4 sequences from the prototype HRV A2 strain, HRV-QPM and used them to predict the presence of a-helices and b-sheets. These were identified in the eight, anti-parallel b-sheet 'jellyroll' conformation common among picornavirus proteins VP1, VP2 (including the EF 'puff') and VP3 [39] (Figure 2 ). Deletions in the VP1 protein imparted the most dramatic changes, reducing the protrusion of the BC, DE and HI loops compared with the other HRVs and introducing an additional C-terminal sheet-loop-sheet structure (arrow, Figure 2B ). Similar deletions were noted in all HRV A2 strains but only HRV-C026 was predicted to encode a similar, Cterminal structure in VP1. We next assembled the HRV-QPM structural proteins into a protomer by associating them with the closest sequence match (HRV-16) and then replicated this structure in silico into a viral pentamer. RMSD values were derived from the VP1, VP2 and VP3 structures of all HRVs with crystallography-derived data available (HRV-1A, -2, -3, -14 and -16) and compared to the predicted HRV-QPM structure. HRV-QPM and HRV-16 shared the highest conformational similarity (0.145, 0.142 and 0.147 Å for the VP1, VP2 and VP3 structures respectively), reflecting the use of HRV-16 in predicting the structure HRV-QPM. The poorest agreements were between HRV-1A and HRV-3 in VP1 (0.793 Å ) and VP3 (0.740 Å ) and between HRV-2 and HRV-14 in VP2 (0.697 Å ). At the ICAM-1 footprint, HRV-QPM values ranged from 0.064-0.106 Å and 0.709-0.779 Å in comparison to the same regions on HRV-16 and -14, respectively. Values could not be determined for HRV-2 because of the impact of deletions. We also examined the conformational agreement between an HEV footprint e.g. the CAR site employed by CV-B3, and the predicted HRV-QPM structure. A space filling model of the pentamer was next used to display SimPlot data in order to visualise regions of sequence conservation and diversification with a focus on the major (ICAM-1; Figure 3A ) and minor (VLDL-R; Figure 3C ) group domains. Overall, greater sequence identity was apparent between HRV-2 than HRV-14 reflecting the genomic similarity to HRV A strains, however within the domains defining HRV groups, HRV-QPM was most similar in its antigenic sites to the major group representative, HRV-14. Most sequence diversity was found in the VP2 protein while the most conserved protein in these comparisons was VP3.
To study these regions in greater detail, we mapped known HRV antigenic sites and receptor footprints onto ribbon depictions of pentamers derived from empirically determined structural data (major group, HRV-14, Figure 3B ; minor group, HRV-2, Figure 3D ) and compared their locations and structures to the predicted HRV-QPM pentamer. HRV-QPM and HRV-14 appeared to share a number of similar structures in the region of the ICAM-1 footprint [6, 33, 40] ( Figure 3B ). The noteworthy structural disparities were two small helices present in the HRV-QPM sequences; one in the VP1 and the other in the VP3 ICAM-1 footprint ( Figure 3B ; VP1 and VP3, ICAM-1 arrows). Similar sequences were infrequent among the other HRV strains we examined. The HRV-QPM NImIA and IB sites appeared to differ structurally due to the shortening of the BC and DE loops in VP1 while the NImII and NImIII sites appeared structurally similar.
Comparison to HRV-2 ( Figure 3D ) also revealed the impact of VP1 deletions. The resulting loops were shorter, which ablated any analogue of antigenic site A and may hamper binding of the VLDL-R molecule, the only LDLR family member with a welldefined footprint. The area in VP1 forming site B was altered due to the presence of more hydrophilic amino acids at the equivalent positions in HRV-QPM ( Figure 3D ; VP1 site B, arrow). Deletions were apparent but did not confer obvious structural changes to the VP2 portion of site B. Furthermore, in antigenic site C (VP2), a single deletion and a hydrophobic to hydrophilic amino acid change contributed to a protrusion in HRV-QPM compared to the same region in HRV-2.
Given the comparative differences we observed when locating and comparing the HRV domains, we also investigated the HRV- The deletions in the VP1 BC loop had a less obvious impact on HEV receptor footprints which were mostly located on the bsheets rather than the loop (Figure 4 ). The EF loop in HRV-QPM VP1 provided the greatest observable identity with any HEV footprint while the C-terminal sheet-loop-sheet structure may interfere with binding to the HEV receptors we investigated. All the HEV VP2 receptor footprints occured in the EF 'puff' but in this region of HRV-QPM a number of deletions and residue substitutions were apparent ( Figure 4 ) that affected the appearance of predicted structural overlap. HEV footprints in VP3 differed from HRV-QPM at the N-terminus and the CD loop due to amino acid substitutions and loop-shortening deletions but they overlapped in the GH loop.
HRV-QPM protomers were replicated and mapped onto an icosahedral lattice (T = 1 configuration; Figure 5A ), rendered in 3D and depth cued in order to predict capsid structures. Since HRV-QPM protein and protomer structures were inferred by homology to their closest available relatives, these capsids (HRV-16 and HRV-14) were produced from the available empirical data, using the same approach. Visual comparisons revealed that the deletions in the HRV-QPM VP1 most obviously reduced the size of protrusions around the 5-fold axis of the capsid.
Previously, phylogenies had been estimated using subgenomic sequences from HRV A2-like strains [31] [32] [33] . To test whether this approach accurately represented inter-strain relationships, we [10] . Attachment of the VLDL-R involves adjacent VP1 molecules. Magnified VP1 area represents one half of a VLDL-R footprint [42] . Amino acid substitutions (arrowed) contributed to the differences between minor group sites B and C. doi:10.1371/journal.pone.0001847.g003 compared the entire polyprotein sequences from 120 previously described and, for the first time, all six newly-identified picornavirus strains ( Figure 6 ). We confirmed that the newlyidentified HRVs occupy a distinct phylogenetic position with the genus Rhinovirus [34] and are most closely related to the HRV A strains (also supported by SimPlot mapping; Figure 3 ). We also found support for our earlier data [31] that this clade is divided into two distinct subgroups. In the 2C and 3CD regions, these subgroups share less than 70% amino acid identity, which also occurs among members of the existing HRV species.
Previously, preliminary and potentially subjective notes made by physicians upon first contact with ill individuals had been used for an approximate determination of the clinical impact of HRV-QPM [31] . To better address clinical impact, a comprehensive review of available patient charts was undertaken and two objective severity scoring tools were applied (Table 1) . Most HRV-QPM-positive individuals (76.5%) were admitted to hospital ( Table 2 ). Five patients were admitted for 96 hours or longer; two with severe illness and only HRV-QPM detected (variants 005 and 012) and one with moderate illness positive for HRV-QPM (variant 017) and HCoV-NL63. The remaining two had underlying medical conditions and multiple infections (HRV-QPM variant 009 and HCoV-229E with cystic fibrosis deterioration and HRV-QPM variant 008, HBoV and HCoV-NL63 with a viral URTI during admission for cardiac surgery). Oxygen therapy was required by almost half of all HRV-QPM-positive individuals studied. In addition to those described previously [31] , a new co-detection was identified with the newly identified polyomavirus, WUPyV [41] in a patient exhibiting a persistent cough, already positive for HRV-QPM (variant 013) and HBoV.
In silico data obtained from this study permitted an enhanced analysis of HRV A2 strains using the ICTV criteria which assign a member to the genus Rhinovirus or Enterovirus [42] (Table 3) . The requirement common to both genera was for .70% amino acid identity in the P1 and 2C+3CD regions; this was not met by the HRV A2 genomes ( Figure 1B ). The remaining Rhinovirus species criterion was classification based on capsid-binding antiviral susceptibility and we found here, building upon earlier data [31] , that all HRV A2 strains contain a Thr 191 . This residue reportedly contributes to conveying resistance to the capsid-binding antiviral Pleconaril [4, 43] , placing the divergent HRVs into antiviral Group A [5] .
Comparison to the Enterovirus species criteria revealed several features that may also be applicable for classifying HRV A2 strains. Firstly, although we could not identify a specific receptor using these in silico methods, the HRV-QPM models most closely accommodated interaction with an ICAM-1-like molecule, similar to some HEV C viruses [44] . Secondly, HRV A2 genomes exhibited #2.5% variation in G+C composition compared to HEV C and HEV D and ,5% compared to HEV A and HEV B. Thirdly, HRV-and HEV-like protease cleavage sites and locations suggested similar proteolytic processing ( Figure 1A ). Although we have been unable to find any indication of recombination in HRV-QPM [31] , the differences in amino acid identity within the novel HRVs, particularly in the 2C and 3CD region of HRV-X2, -C024 and -C025 compared to HRV-QPM, -X1 and -C026 (data not shown), may indicate a relevant event requiring further study. In sum, these data indicate that the HRV A2 strains cannot be assigned to an existing HRV or HEV species and should be assigned to a new species, tentatively called Human rhinovirus C.
In 2007, the sequences of a number of divergent HRV strains were reported as a result of worldwide molecular investigations into cases of suspected ARTI [26, 31, 33] . Because these strains formed a distinct clade within the existing HRV As, we previously assigned to all Australian and similar New York strains, the title of HRV A2 [31] . Kistler et al. identified similarly divergent strains [32] and Lau et al. used sequence-based criteria to propose that some HRV A2-like strains could be classified as a new species [33] . Our ongoing efforts to characterise additional HRVs led us to catalogue the distinguishing molecular and clinical features of these HRV A2-like strains and to better address their taxonomic placement. We undertook in silico analyses, for the first time using all complete HRV A2 coding sequences and performed a retrospective review of the medical records of cases from which variants of the prototype strain, HRV-QPM, had been previously detected. To date the studies reported herein are the first of their kind to have been conducted on a single HRV strain.
During our genomic analysis we found HRV A2 strains shared only 50%-53% average amino acid sequence identity with other HRV strains ( Figure 1) ; less with any strain from an HEV species. Average HRV A2 G+C content (42.4%) fell between the rhinovirus (38.7%) and enterovirus (45.6%) genera, suggesting an intermediate evolutionary path for HRV A2 strains. Nonetheless, a large number of HRV motifs, including those recognized by the RNA-dependent RNA polymerase, were retained by HRV A2 strains. This supported the proposal that some HRV genomic regions are under purifying selection [26] , even among the newly identified and divergent strains. HRV A2 strains were most similar to the HRV As overall, but amino acid sequences were as different from any traditional HRV or HEV species as the species in those genera were from each other. Previous subgenomic HRV A2 nucleotide phylogenies [31] [32] [33] were confirmed and expanded herein reinforcing that these NIVs reside within a distinct clade of the genus Rhinovirus, branching from the existing HRV A species ( Figure 6 ). The inability to propagate an HRV A2 virus could be due to many factors including the age, storage, site of origin and amount of the inoculum, its handling during processing, how long after symptoms appear before sampling occurs and the culture conditions used. It is also possible that an unknown primary receptor is employed or that binding of a secondary receptor is required for successful viral attachment and entry; or a mix of both. We sought to approach receptor-related issues using homology models derived crystallography data which already exist for a small number of HRV strains. Models of the component structural proteins from the prototypical HRV A2 strain, HRV-QPM, predicted that the characteristic 'jellyroll' conformations were retained (Figure 2 ) despite frequent and distinguishing deletions and inter-and intra-genus diversity at the sites of receptor contact and antigenicity (Figure 3 ). This also supported findings that HRV variability is mostly localized to receptor and antigenic sites [24, 33, 45] and begins to extend the data to include the more divergent strains.
The VP1 BC and HI loops receive the VLDL-R molecule among some minor group HRV strains [12, 46, 47] . The comparative reduction in size of protrusions along the 5-fold axis, resulting from deletions in the BC, DE and HI loops of the HRV-QPM VP1, together with structure-altering deletions and substitutions affecting some antigenic sites previously identified from a minor group strain (HRV-2), particularly site A, lead us to propose that HRV A2 viruses are unlikely to behave as members of the minor group. Mapping the known ICAM-1 contact sites onto the predicted HRV-QPM structure revealed that most of the footprint was retained in structure by HRV-QPM, although it differed in sequence compared to other major group strains. SimPlot data revealed that sequence variation was not uncommon among strains within the major groups. Two small a-helices predicted to form in the HRV-QPM VP1 and VP3 footprints may disrupt receptor binding but since ICAM-1 is predicted to attach at differing angles in different HRV strains [8] , it may be suitably flexible to overcome such obstacles. Although NImIA and NImIB, first identified in a major group virus (HRV-14), were affected by the deletions in VP1, additional support for HRV-QPM belonging to the major HRV group came from structural similarities in the NImII (VP2) and NImIII (VP3). Amino acid substitutions occurred at these sites (ERG and GRT respectively) rather than insertions or deletions. These variations may result in distinct immunogenicity.
Localisation of four known HEV receptor footprints to the predicted HRV-QPM structure identified at least one major sequence and/or structural divergence in each ( Figure 4 ). However, like the ICAM-1 interactions with HRV-14 and -16, HEV receptors could still bind at the same locations but utilize different amino acids on the capsid of HRV A2 strains [20] .
Recent findings by Bartlett et al have shown that once past the receptor, HRV replication can occur within non-human tissues [48] exemplifying the importance of receptors in moderating the potential for more widespread HRV replication and illness. The high detection frequency of HRV A2 strains from patients with acute LRT illness may be another indication that these divergent viruses employ a tissue-specific receptor or it may be that all HRV strains cause similar illness, they simply have not been subjected to sufficient individual study. The LDLR family of molecules are located on many tissues in many species [10] and yet all these locations do not, to our knowledge, host HRV replication. Identifying an HRV A2 receptor in vitro cell culture system will be important for empirically addressing receptor-and replicationrelated issues and the outcomes may have broad implications for what is currently known of the attachment, entry and replication of HRV strains. Based on our predictive in silico studies of HRV-QPM, an ICAM-like molecule may be an early candidate for the role of receptor. Future in silico studies could examine all available HRV polyprotein sequences to predict the extent of flexibility in receptor interactions 'normally' occurring in the major and minor groups. This would provide a context for the structural differences we observed in HRV-QPM. The sequence of most HRV genomes is unknown and our understanding of HRV structure is mostly due to crystallography data derived from less than 5% of HRV strains. There are limited structural data to support the wholesale extrapolation of receptor binding behaviour and capsid structure to the other HRV strains. Without data to the contrary, in silico analyses appear to provide a suitable surrogate to address this issue. By employing RMSD calculations to examine the conformational similarity between predicted HRV-QPM structures and those from HRV structures determined empirically, we could be confident that our predictions approximated experimental data. All comparative values (0.064-0.766 Å ) were well below the maximum resolution used to originally determine HRV crystal structures (2-3 Å [14,15,49] ). Comparison to an HEV footprint also returned values below this threshold.
While the inability to cultivate divergent HRV strains is a defining feature of the HRV A2s it is also a hindrance to classifying them. In our experience (data not shown), the earliest PCR-based methods [50] are already efficient at detecting these divergent strains. By extrapolating from published subgenomic sequence data, this clade is populated by a great number of divergent strains which are distributed both geographically and temporally, suggesting an endemic aspect to these previously uncharacterised viruses. No evidence exists to suggest that the HRV A2 strains are emerging viruses, rather it appears that they are newly identified strains that have been contributing to respiratory illness, in the absence of detection using culture-based diagnostic methods, for many years. There has now arisen a need for reliable protocols to characterise these putative viruses but in the absence of an HRV A2 isolate, neutralization, acid sensitivity and antiviral susceptibility studies, important for taxonomic placement, cannot be obtained. We have catalogued a series of features using methods which, while requiring further validation, contribute to our understanding of the biology and pathogenesis of HRV A2 strains. Additional molecular approaches will also be useful to classify other recently described divergent HRV strains [51] . Our data demonstrated that the newly identified HRVs, residing within the HRV A2 clade, do not satisfy the criteria for assignment to any existing HRV or HEV species and most likely constitute one or more novel species in the current genus Rhinovirus, tentatively called Human rhinovirus C (HRV C).
Until recently, the genus Rhinovirus has been frequently neglected from clinical laboratory diagnosis and to date its members are not sought independently. Improved diagnostic methods, the broader application of existing PCR technology, new sequence data and many new insights have greatly enhanced our understanding of human rhinoviruses and the selective pressures impacting on their evolution [25, 26] . Similarly, the identification of a putative new HRV species, HRV C, the characterisation of HRV-QPM's molecular features and epidemiology and the identification of similar viruses around the world [31, 34, 52] have potential to reinvigorate HRV research. We found that the variants of one strain of the HRV Cs, HRV-QPM, sought in a well characterised population of specimens collected over all months of a single year, were more often associated with LRT illness than is commonly reported. The illness frequently presented as exacerbation of expiratory wheezing or persistent cough, with a requirement for supplemental oxygen, steroids and bronchodilator treatments. Despite exhaustive PCR-based investigation for other respiratory viral causes in our previous retrospective studies [31, 41] , HRV-QPM detection, in the absence of any other pathogen, occurred among infants with mild, moderate and severe (in both instances) LRT illness and most cases (88.2%) were admitted to hospital. Associated illness was usually (92.3% of the time) scored as mild to moderate and 58.8% of the HRV-QPM positive cases were children aged 12 months or less. The same age group represented 43.2% of the total study population originally screened for HRV-QPM (n = 1,244). While it is to be expected that a paediatric hospital-based population would overestimate the severity of HRV C clinical impact compared to a communitybased study, our study may in fact better represent the impact of first infection with HRV strains, the major pathogens detected during the first year of life [53] .
It will be important to further characterize these NIVs and determine whether their differences confer distinctive biological behaviours, such as unique growth properties, antiviral resistance and discrete clinical outcomes among infected individuals. Strainfocussed studies could also identify how many distinct HRVs circulate in a single respiratory season, how often a given strain recurs in a population and just how many HRV strains there are.
Nucleotide and amino acid compositions were determined using BioEdit Sequence Alignment Editorß v7.0.5.3. Picornavirus coding sequences (obtained from GenBank or picornaviridae.com) were determined by multiple alignments and subsequent truncations of the 59and 39UTRs. Discrepant nucleotides were substituted with the most frequently employed nucleotides, as determined by consensus alignment. Alignments available upon request. Predicted picornavirus cleavage sites were identified by amino acid sequence submission to the NetPicoRNA World Wide Web server [54] .
Amino acid similarity was determined using SimPlotß v3.5 [37] employing the Hamming method on a 50% consensus of each group, a sliding window of 100 bp and a 1 bp step. Similarity data were mapped to the HRV-QPM capsid residues on the original sequence alignment and visualized using Chimera [55] .
Picornavirus polyprotein sequences were compiled, translated, and aligned with the program BioEdit [56] . A neighbour-joining tree was generated using the Kimura two-parameter estimation in MEGA 3.1 [57] . Nodal confidence values (%), noted at the relevant nodes, indicate the results of bootstrap resampling (n = 1000). Because of the sequence diversity among the viruses analyzed, we undertook an additional analysis on a subgroup of strains whereby we gap-stripped the alignment, removing all gaps/ highly divergent regions and re-estimated the phylogeny (data not shown). This confirmed the phylogenetic position determined in the first instance.
Secondary structures in the HRV A2 structural proteins were predicted using amino acid sequences submitted to the Jpred web server (http://www.compbio.dundee.ac.uk/,www-jpred/ submit.html). The location of a-helices and b-sheets were revealed by comparison to other picornavirus sequences using Cn3D 4.1 (http://130.14.29.110/Structure/CN3D/cn3d.shtml) (data not shown).
To determine the predicted protomer structure of HRV-QPM, DeepView/Swiss-Pdb Viewer v3.7, [58] were used to locate and individually thread HRV-QPM amino acid sequences into HRV reference structures. Of the five HRV strains with empirically determined structural data available, HRV-14 [14] and -16 [49] shared the highest sequence identities with HRV-QPM in the regions chosen. Proteins VP1 to VP3 were aligned and threaded through HRV-16 (1ayn1, 1ayn2, 1ayn3 respectively) and VP4 through HRV-14 (1na1). Models were submitted to the SWISS-MODEL server for final threading analysis. The resulting HRV-QPM protein data bank files (PDB) were then matched and structurally aligned to a template HRV protomer (HRV-16, 1AYN) . Other picornavirus reference PDB files used in this study: HRV-2 (1FBN), HRV-3 (1RHI), HRV-14 (4RHV), CV-A21 (1Z7S), E-11 (1H8T), PV-1 (1HXS) and CV-B3 (1COV). Structural matching, alignments, ribbon figures, capsid predictions and root mean square deviation (RMSD) data were produced using Chimera [55] . Epithelial Cell Apoptosis and Neutrophil Recruitment in Acute Lung Injury—A Unifying Hypothesis? What We Have Learned from Small Interfering RNAs In spite of protective ventilatory strategies, Acute Lung Injury (ALI) remains associated with high morbidity and mortality. One reason for the lack of therapeutic options might be that ALI is a co-morbid event associated with a diverse family of diseases and, thus, may be the result of distinct pathological processes. Among them, activated neutrophil- (PMN-) induced tissue injury and epithelial cell apoptosis mediated lung damage represent two potentially important candidate pathomechanisms that have been put forward. Several approaches have been undertaken to test these hypotheses, with substantial success in the treatment of experimental forms of ALI. With this in mind, we will summarize these two current hypotheses of ALI briefly, emphasizing the role of apoptosis in regulating PMN and/or lung epithelial cell responses. In addition, the contribution that Fas-mediated inflammation may play as a potential biological link between lung cell apoptosis and PMN recruitment will be considered, as well as the in vivo application of small interfering RNA (siRNA) as a novel approach to the inhibition of ALI and its therapeutic implications. The aim of this review is, therefore, to elucidate mechanisms of ALI pathogenesis, focusing on two main theories. The first, that neutrophils (PMN) can play a central role in driving ALI and the second, that lung epithelial cell apoptosis represents an important pathological aspect inducing lung injury. In this regard, we discuss the data in the field as well as some of our own findings using a clinically relevant double-hit mouse model of indirect ALI induced by hemorrhagic shock and subsequent polymicrobial sepsis. In addition, a novel strategy, i.e. the use of siRNA in mouse lungs in vivo, has served us in broadening our understanding of the pathology of ALI.
The pathophysiologic mechanisms of ALI are insufficiently understood. In the common finale of this heterogeneous entity, the alveolo-capillary barrier is compromised allowing consecutive edema formation in the interstitium as well as alveoli, thus compromising gas exchange leading to organ dysfunction and respiratory failure. Histological evaluation of lungs from ALI patients indicated substantial accumulation of activated PMNs, diffuse alveolar damage, loss of epithelial integrity, and increased pulmonary edema (16, 17) . There are several proposed mechanisms which are believed to account for this phenomenon.
Among leukocytes, PMNs are considered the first line of defense against microorganisms. Rapidly recruited to the inflammatory site/organ, they exert a variety of primarily beneficial functions (phagocytosis, production of reactive oxygen species [ROS] , and nitric oxide species [NOS] , degranulation of lytic enzymes, etc.), that, when well orchestrated, enable clearance of the invading pathogen. However, it is also hypothesized that activated PMN may possess harmful potential when these same functions are directed at otherwise normal host tissue, culminating in injury and organ damage (Figure 1 ). There is sub- Figure 1 . Proposed mechanisms of acute lung injury through hemorrhagic shock (HEM) "priming" for inflammation (Infl./red color)/apoptosis (Ao/ gray color)/injury, and "triggered" by a subsequent infectious insult: The resting lung (A) is primed by divergent inflammatory mediators released during an initial event (for example, shock, inflammation, etc.) that acts on a number of cells in the blood (MΦ: monocytes, PMN: neutrophils) and lung (EP: epithelial cells, EC: endothelial cells, AMΦ: alveolar macrophages) (B). These cells, in turn, stimulate, either separately or concomitantly, the pro-inflammatory response and/or the Ao of a small number of EP, both through Fas-FasL activation. The release of chemokines like MCP-1 then primes the AMΦ. When, at a later time, a subsequent inflammatory/infectious ('trigger') event takes place (C) the local EC, AMΦ, and/or EP become activated, release chemokines and activating agents that recruit the primed and now activated leukocytes (PMN, MΦ) to the lung (D). These activated leukocytes then transmigrate into the interstitium and alveoli where they perform their effector roles (in the absence of infection, the effector response may be solely injurious). In addition, they may propel the inflammatory/apoptotic response into a vicious cycle by further activating Fas through FasL on their cell surface (E). stantial evidence that the lung is particularly susceptible for PMN accumulation. In contrast to most other organs, where PMN sequestration occurs at post-capillary venules, pulmonary PMN retention takes place within the pulmonary capillaries, representing a complex interconnecting network of short capillary segments where the course from arteriole to venule crosses numerous alveolar walls and often includes more than 50 capillary segments. The blood in this complex network contains 50 times more PMN compared with most other vascular beds (reviewed in 18) .
PMN accumulation has been observed early in lung tissue (19, 20) as well as in bronchoalveolar lavage fluids (BALF) of ARDS patients (16) . When developed during neutropenia, ALI rapidly progressed once PMN counts were restored (21, 22) . Furthermore, the degree of neutrophilia in BALF has been correlated with poor prognosis in septic ARDS (23) . In an experimental setting of indirect ALI stemming from hemorrhagic shock (HEM) followed by polymicrobial sepsis, we found that, in response to HEM, circulating PMNs exhibited an ex vivo increase in respiratory burst capacity and a decrease in apoptosis. This is consistent with the concept that shock/injury can produce in vivo "priming," the significance of which could be seen when HEM then was followed by sepsis, as recruitment of these PMNs into the lung occurred, along with the development of ALI (24) . Also, when these HEM-primed PMNs were injected intravenously into PMN-depleted animals, which subsequently underwent cecal ligation and puncture (CLP) to induce sepsis, ALI again resulted (24) . Upon this background, using different models of ALI, we (25) and others (26) (27) (28) (29) (30) (31) (32) have found that depletion of PMNs actually may serve to decrease injury associated with ALI. Thus, depletion of PMNs prior to HEM and sepsis markedly reduced the extent of lung inflammation and ameliorated lung protein influx and the severity of ALI (25) , which is in line with findings during transfusion-induced ALI (29) .
Inflammation is closely linked to the pathogenesis of ALI. Levels of Interleukin (IL)-8 and IL-1 have been found increased in the lungs of ALI patients (33, 34) and the persistent increase of inflammatory cytokines in the lung correlated with poor outcome in ALI (35) (36) (37) . Inflammatory mediators exert several effects on PMNs. For example, complement protein C5a activates PMNs, thus contributing to their pulmonary sequestration and mediating lung tissue injury (38, 39) . Several inflammatory mediators, including complement, cytokines, chemokines, and lipid mediators also are able to induce the expression of surface molecules on the endothelium, which further promote PMN recruitment (40, 41) . Tumor necrosis factor (TNF)-α and IL-1β are two important cytokines that are found regularly in the BALF of ARDS patients (36, 42, 43) at higher concentrations than in their plasma, thus supporting their local origin (35, 44) . However, anti-TNF and anti-IL-1 therapies have failed to protect septic patients from ALI (44, 45) . Proinflammatory cytokines also are regularly expressed by activated PMNs traveling to the lung (32) . They appear to be a relevant source of IL-1β, favoring the subsequent release of other mediators, such as TNF-α, MIP-2, and IL-8 (46) (47) (48) . However, an early anti-inflammatory response driven by IL-1-, TNF-and IL-6-receptors, with early peaking IL-10 levels, was also shown in ARDS patients (41) . In addition, PMN stimulation with lipopolysaccharide (LPS) or TNF-α showed an activation of nuclear factor kappa B (NF-κB), p38, and AKT (49, 50) . The degree of nuclear translocation of NF-κB in peripheral PMNs from patients with septic ALI was linked to ventilator time and survival (50) .
PMNs on Their Way to Lung-Sequestration, Adhesion, Migration, Activation, and Tissue Injury. First, in response to inflammatory mediators, PMNs migrate to the pulmonary capillaries in preparation for extravasation ( Figure 1 ). Initial changes in the cyto-skeleton prevent PMNs from deforming, making it less likely for them to pass through the pulmonary capillaries (51) . In this regard, activated PMNs from ARDS patients appeared even more rigid than those from septic patients (52) . Second, emigration of PMNs and passage through the endothelium can be regulated through adhesion molecules (51) . While their initial sequestration appears to be independent from adhesion molecules, the durability of this process could rely on them. Thus, while L-selectin-deficient mice showed a normal pool of marginated PMNs and cleavage of L-selectin from the PMN surface did not alter this margination (53, 54) , L-selectin deficient mice exhibited only a very short and transient leukopenia in response to complement activation (53, 55) . PMNs can adhere to endothelial cells using CD11/CD18 interacting with intercellular adhesion molecule-1 (ICAM-1) on the endothelial side. Thus, PMN emigration in response to Escherichia coli (E.coli), E.coli lipopolysaccharide, Pseudomonas aeruginosa, immunoglobulin (Ig) G immune complexes, and IL-1 was mediated through CD11/CD18, but this did not appear to be the case in response to Streptococcus pneumoniae, Group B Streptococcus, Staphylococcus aureus, hyperoxia, or C5a (56) . Even in CD11/CD18-dependent migration, blocking of CD18 reduced PMN emigration only by 60% to 80%, suggesting that other redundant mechanisms mediated these effects (56) . Cytokines and chemokines such as IL-8 also have been shown to be involved in increasing β2-integrin avidity (57) . It is important to understand that activation of PMNs is associated closely with the endothelial interaction mediated by the adhesion molecules (57, 58) . Rolling PMNs might well integrate the sum total of inputs received while scanning the endothelium. If an activation threshold is reached, β2-integrins switch to the high-affinity conformation, redistribute on the cell surface, and trigger arrest and adhesion (59, 60) . However, integrins also are involved in PMN migra-tion, phagocytosis, respiratory burst, and even cytokine production (57, 61, 62) .
As eluded to above, chemokines are critically involved in the activation and recruitment of PMNs to the lung, potentially contributing to their harmful effects on an organ level. In this regard, associations between chemokines, lung neutrophil influx, and alveolo-capillary dysfunction have been reported (63, 64) . In our experiments, we observed that during hemorrhage-induced septic ALI (24) , as well as after experimental blunt chest trauma (65) , chemokines are markedly upregulated locally in the lung of the animals, as well as systemically. Blockade of the CXCR (chemokine [CXC motif] receptor) 2 using antileukinate, a hexapeptide inhibitor, following HEM, reduced lung PMN influx in response to subsequent sepsis and additionally decreased lung inflammation and lung protein leak, while pulmonary IL-10 levels were markedly increased (66) . We also administered anti-KC or anti-MIP-2 antibody into mice immediately following HEM. The adoptive transfer of PMN isolated from the anti-MIP, but not from the anti-KC-treated donors, exhibited a reduction of lung inflammation and lung PMN influx in the recipients in response to sepsis when compared with control Ig-treated donors (67) . These data demonstrated that hemorrhage-induced priming of PMNs not only mediated experimental ALI, but also that this process was differentially effected by MIP-2 and KC, although both signal through CXCR2 in mice. However, as humans utilize IL-8 in place of the two mouse α-chemokines and because receptors for IL-8, i.e. CXCR1 and CXCR2, also are utilized differently, these murine chemokine data may not translate directly to the patient setting of ALI/ARDS (68) .
Once activated PMNs have reached the lungs, they possess the potential to induce tissue injury. The release of ROS/NOS has long been thought to be a central effector mechanism of PMNmediated lung injury (69) . In this regard, while the inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase attenuated sepsisassociated ALI (70) , surprisingly, NADPH oxidase-deficient animals were not protected from complement-induced ALI (71) . On the other hand, inhibition/ deficiency of the nitric oxide synthetase showed protection in complement-/ endotoxin-induced ALI (71, 72) . However, ROS and NOS also play additional roles as signaling molecules modulating kinases and phosphatases, receptors and transcription factors (reviewed in 73) complicating the interpretation as to whether their net pulmonary effect is one of harm or help.
Proteolytic enzymes also are a part of the PMN pathogen-clearing arsenal. While neutrophil elastase, cathepsin G, and metalloproteinases all have important roles in clearing invading pathogens and function in aspects of tissue remodeling/wound healing, they also are able to mediate neutrophil-induced tissue damage and can degrade extracellular matrix efficiently. Thus, while inhibition of neutrophil elastase improved respiratory function during septic ALI (74), its deficiency also rendered mice more susceptible to gram negative, but not gram positive sepsis (75) . In addition, deficiency of elastase and/or cathepsin G was accompanied by increased susceptibility to fungal infections, but decreased susceptibility to endotoxin-mediated inflammatory ALI (76) . Activated gelatinase A correlated with protein accumulation in the epithelial lining fluid in ARDS patients and might, therefore, be a potential marker for ARDS (77) . The inhibition of matrix metalloproteinases and elastase prevented lung dysfunction and reduced septic ALI in pigs (78) . The detailed mechanisms by which PMN mediate ALI via the involvement of proteolytic enzymes are reviewed in detail elsewhere (79, 80) .
Regulation of PMN Apoptosis. Neutrophils, once mature, exhibit a constitutive form of programmed cell death with a life span between 6 to 12 h in circulation. Under normal circumstances, activated PMNs are eliminated fairly quickly once the invading pathogen has been cleared. Several inflammatory agents, such as LPS, TNF, IL-8, IL-6, IL-1, Granulocyte [macrophage] colony-stimulating factor (G [M] -CSF), etc., can inhibit PMN apoptosis (81) (82) (83) (84) . The delayed apoptotic response provides PMNs with a longer life span, which, in turn, allows them to accumulate at local tissue sites of inflammation/infection. In this respect, PMNs obtained from patients following major trauma (85, 86) , burn injury (87), sepsis (88) , and ARDS (89) all showed evidence of decreased apoptosis. The anti-apoptotic effect of ARDS plasma on PMNs appears to be mediated through the GM-CSF receptor (90) . In sepsis, the delay in apoptosis was associated with a decrease in caspase-3 and -9 activity and a prolonged maintenance of the mitochondrial membrane potential (88) . The delay of PMN apoptosis also involved the active regulation of CXC receptors by PMNs themselves (91) . However, controversy persists as to whether this sustained activation actually contributes to organ injury. Activated PMNs have been shown to exert damaging effects in the lungs (68, 92) . However, following G-CSF treatment in pneumonia patients, no differences in outcome or time of recovery were noted, while it appeared that complications such as ARDS were decreased (93) . These data were complemented by studies that indicated that GM-CSF in the air spaces was associated with improved survival in patients with ARDS (94) .
In our model of hemorrhage-induced septic ALI, it was evident that prolonging the lifespan of activated PMNs by using transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in a myeloid-restricted fashion (95) did not exacerbate acute lung injury, although a prolonged presence of activated PMNs in the lung was evident (96) . In contrast, we observed an initial survival benefit when the life span of PMNs was further prolonged, likely due to an enhanced capacity to clear bacteria as evidenced by the lower bacteria counts in the lung (96) . In addition, in an inflammatory/ non-infectious environment, such as SIRS, mimicked by intraperitoneal injection of E. coli-LPS, prolonging the lifespan of activated PMNs was detrimental for the animals' survival and was associated with an exacerbation of lung injury (96) . Additionally, during non-infectious/ inflammatory ALI, failure to clear PMNs from the lungs contributed to increased inflammation and mortality (97) . Together, these data imply that the tissue environment (infectious versus inflammatory) that the neutrophil encounters plays an important role in determining whether PMNs mediate organ damage or not. However, the mechanisms which determine whether PMN turn bad or good are not understood well and require further investigation.
The above information makes a strong case for PMNs playing a significant role in ALI. However, it is interesting to note that ARDS has been described as developing in patients with neutropenia (98) (99) (100) . In addition, following G-CSF therapy in pneumonia and sepsis patients, the incidence of ARDS was not increased (93, 101) and, during experimental ALI or pneumonia, PMNs have been shown to migrate to the lungs without exerting deleterious effects (102, 103) . Thus, other mechanisms also are likely to be involved in the pathogenesis of ALI.
In the development of ALI/ARDS, the loss of epithelial cells in the lungs with consecutive impairment of the integrity of the alveolo-capillary barrier is commonly noted (19, 104) . Type I epithelial cells make up approximately 90% of the pulmonary surface, and, while type II epithelial cells account for only 10%, they are critical because they produce surfactant and may differentiate into type I cells (17) . Impairment of the alveolar epithelial barrier is, in many ways, important in the development of ALI. On the one hand, the epithelial barrier is, under physiologic conditions, less permeable when compared with the endothelial barrier, thus, destruction of its integrity prompts a progressive influx of proteinrich fluid into the alveoli (17, 102) . On the other hand, the loss of epithelial integrity represents an impairment of the physiologic trans-epithelial fluid transport and further inhibits the re-absorption of the alveolar edema (105, 106) . Thus, while an isolated injury to the endothelium still leaves intact the capacity of the epithelium to counteract pulmonary edema formation, the fluid balance becomes rapidly disturbed upon injury of the lung epithelium (107, 108) . Therefore, it is not surprising that the extent of epithelial damage and impaired edema re-absorption was linked clinically to outcome in ALI (109) . Additionally, the accompanying reduction of surfactant production led, via an increase in the surface tension, to an augmented respiratory effort affecting gas exchange (110, 111) .
Programmed cell death/apoptosis of lung epithelial cells represents a potentially important mechanism contributing to the loss of this cell type in the development of ALI (Figure 1 ). Bachhofen et al. reported that patients who died from ARDS exhibited excessive apoptotic alterations in the chromatin of their alveolar type II cells (19) . Subsequent studies then confirmed these DNA fragmentations (112) as well as an increased expression of the pro-apoptotic protein Bax (Bcl-2 associated X protein) (113) . More recent studies also have suggested a role for lung epithelial cell apoptosis in pediatric ARDS (114) . Importantly, epithelial cell and PMN apoptosis is differentially regulated during ARDS. While, as mentioned earlier, PMNs show a decreased rate of apoptosis, it is increased markedly in epithelial cells (107) . Local mediators in the lungs of these patients may regulate these opposing effects (115, 116) . Interestingly, the induction of lung epithelial cell apoptosis appears to lead to the development of ALI, which is further aggravated, when apoptosis is additionally induced in macrophages (117) . Furthermore, inhibition of apoptosis, using a caspase inhibitor, protected mice from the lethal consequences of endotoxin-associated ALI (118) . In addition, Bcl-x(L) treatment reduced albumin leakage and lung tissue damage in LPS-mediated ALI (119).
In the induction of lung apoptosis during ALI, Fas-(an apoptotic death receptor pathway) mediated cell death also appears to play a major role (Figure 2 ). In this regard, during endotoxin-induced ALI, an increase of Fas expression on epithelial cells, as well as an immigration of Fas-ligand (FasL) expressing cells in(to) the lung, was observed (120). Human lung epithelial cells also expressed Fas and were, particularly in the distal airways, sensitive to Fas-mediated apoptosis (121) . Caspase-cleaved cytokeratin-18, a marker for epithelial cell apoptosis, also was increased in BALF during ARDS (122) . During ALI and ARDS, an increased concentration of Fas and FasL in patients' BALF and lung tissue was detected (122, 123) and BALF from ARDS patients was shown to induce apoptosis in healthy lung epithelial cells (116) . Furthermore, ARDS non-survivors exhibited markedly higher FasL concentrations when compared with survivors (116) . Particularly during septic ALI, an infection-severity dependent activation of the Fas-FasL system in the lung was observed (124) . FasL concentrations have been reported to be much higher locally than systemically, suggesting its origin is pulmonary (122, 123) . In this respect, there appear to be several potential sources of FasL: infiltrating monocytes have been implicated (but not alveolar macrophages) (125), PMNs have been implicated (126) , and FasL may be cleaved from cell membranes by the activation of matrix metalloproteinases 3 and 7 (127, 128) . However, anti-apoptotic proteins such as soluble Fas (129) also appear to be increased during ARDS (122, 123) and have been described to correlate with clinical features such as the PaO 2 /FiO 2 ratio (122) . This may point toward a simultaneous activation of protective anti-apoptotic mechanisms during ALI.
Under experimental conditions, the instillation of Fas-activating antibody into murine lungs induced apoptosis of lung epithelial cells, PMN recruitment and impairment of the alveolo-capillary barrier (130) . This development of Fas-dependent lung injury was linked to Fas-expression on non-myeloid but not myeloid cells (131) . Furthermore, blocking of the Fas-FasL system reduced the development of endotoxin-mediated ALI (120) . Our experiments further demonstrated the relevance of Fas-mediated apoptosis in the lung during HEMinduced septic ALI (132) . Fas and FasL mutant animals exhibited less pulmonary epithelial cell apoptosis in response to the insult when compared with background animals. In addition, the extent of ALI as assessed histologically and by protein influx was diminished significantly. This was associated with a survival benefit for Fas mutant mice when compared with background animals (132) . It also has been reported that Fas-FasL deficiency is associated with a reduction in the degree of injury seen during direct ALI in E. coli, S. aureus, or S. pneumoniae sepsis models (133) . Similar results were described for sepsis induced by Legionella pneumonia (134) . In contrast, data from pulmonary sepsis fol-lowing lung infection with P. aeruginosa suggest a reduction in lung apoptosis in the absence of Fas signaling as well, but a decreased survival rate was observed, associated with an increased dissemination of the bacteria (135) . It should be noted that Fas-mediated apoptosis in the lung appears to be modulated by several factors such as surfactant protein A, Angiotensin II, transforming growth factor (TGF)-beta, decoy receptor 3, etc. (reviewed in 108) , and that other apoptotic pathways, for example, mediated by TNF-α (136) or mitochondria (137) , appear to play a role under certain circumstances, too.
Recent studies suggest that activation of Fas serves not only to induce apoptosis, but also to induce the secretion of cytokines and chemokines by a variety of cell types (138) (Figure 2 ). In murine lungs, activation of Fas initiated a pro-found inflammatory response with an early generation of chemokines and subsequent recruitment of neutrophils (130, 132, 139) . This response could be reduced by antagonizing Fas ligand (139) . Triggering of Fas also led to increased concentrations of TNF-α, MIP-1 α, MIP-2, monocyte chemotactic protein (MCP)-1, and IL-6, and compromised the alveolo-capillary barrier (133) . LPSmediated pulmonary inflammation also appears to be regulated through Fas (140) . Mice, genetically altered to express Fas either on myeloid or on nonmyeloid cells in the lung, presented no marked increase of inflammatory mediators following Fas activation (131) , in contrast to non altered mice lungs (130, 132, 139) . With respect to cell types involved, it has been indicated that activation of Fas on monocyte cell lines induced production of MIP-2 (141) . The activation of an inflammatory program by Fas also was shown for peritoneal macrophages (142) , monocytes and macrophages (143) , endothelial cells (144) , and others (145) (146) (147) (148) (149) (150) (151) .
Our own experiments with the Fas and FasL mutant mice showed a marked decrease in inflammation in the lungs of these animals in response to hemorrhageinduced septic ALI, when compared to background animals (132) . This phenomenon also was evident following Fas silencing in the mouse lungs using siRNA (152) and was associated, in both cases, with a reduction of PMN immigration into the lungs (132, 152) . Further experiments revealed that lung epithelial cells were capable of secreting MIP-2, KC, and MCP-1 in vitro in response to Fas activation, through mechanisms involving ERK and, potentially, FLIP (132), complementing the findings of other researchers who demonstrated that a NF-κB dependent mechanism appears to underlie this response (151) . Instillation of a Fasactivating antibody into transgenic murine lungs in which lung macrophage numbers are markedly reduced displayed a similar inflammatory response as seen in background animals, further supporting a role for Fas-mediated, ep- ithelial cell-induced pulmonary inflammation in vivo (132) . The fact, that inhibition of Fas activation modulated both PMN recruitment into the lung as well as pulmonary epithelial apoptosis, intriguingly points at its value as a potential therapeutic target in ALI.
The use of silencing RNA (siRNA) represents not only a potentially powerful experimental approach to allow us to better understand the evolving pathology of ALI, but may possibly represent a novel therapeutic approach to the treatment of this condition. The history of siRNA, its discovery, development, the mechanisms involved, as well as its successful initial uses in mammals in vivo are described elsewhere (153) (154) (155) (156) . With respect to its application in vivo, the lung appears to be a good candidate, as it can be accessed straightforwardly intranasally (i.n.) or intratracheally (i.t.). Nevertheless, nucleic acid transfer efficiency can be diminished substantially by phospholipids and proteins, which are major components of the airway surface liquid (157) . Interestingly, unlike systemically, delivery of naked siRNA into the lungs has proven very efficient (152, 158, 159) , thus potentially complex and costly approaches using vector systems or chemical siRNA modifications may not be necessary. In this regard, the delivery of siRNA inhibitors of SARS coronavirus in 5% glucose was superior, even in reducing the severity of the disease, than when given in modified calf pulmonary surface active material (160) . As early as 2004, the feasibility of a surfactantbased or naked siRNA approach in the mouse lung targeting glyceraldehyde-3phosphate dehydrogenase (GAPDH) or heme oxygenase-1 during ischemiareperfusion, respectively, has been demonstrated (161, 162) . Phase I/II trials for Respiratory Syncytial Virus have been conducted and are now on their way to evaluate the safety, tolerability, and antiviral activity of siRNA treatment in human lungs (155, 158, 163) .
With respect to the application of siRNA in the in vivo setting of the lung, initially we attempted to extend our observation that blockade of MIP-2 or KC with conventional antibodies effected the development of ALI (67) by using an in vivo approach silencing these same chemokines locally in the lung (164) . Subsequently, we initiated studies with antiapoptotic siRNA against Fas and caspase-8 assessing their capacity to protect the lung from the detrimental effects of hemorrhage-induced septic ALI (152) .
However, to establish the initial feasibility of in vivo siRNA administration, mice overexpressing green fluorescent protein (GFP) were chosen to receive a single intratracheal instillation of a GFPsilencing RNA. What we found was that green fluorescence in the lungs of these animals at 18 h post-instillation was reduced by at least 65% when compared with vehicle-treated GFP mice, while no decrease in fluorescence was seen in other organs outside the lungs (152, 164) . The use of siRNA at these concentrations neither induced substantial activation of type I interferons (165, 166) via activation of TLR-3 (167), TLR-7 (168), TLR-8, TLR-9, and protein kinase-R(PKR) pathways (169) (170) (171) nor via more classic proinflammatory processes (169, 171) . This is in line with reports demonstrating that intravenous delivery of naked siRNA did not induce an interferon response (170) . However, the induction of STAT (signal transducers and activator of transcription)-1 in this context appears to be dose dependent (165) , and thus it cannot be ruled out that different dosages, delivery, or timing of administration might have provided dissimilar results. To gain some insight into the cellular localization of siRNA in the lung, we followed the intrapulmonary deposition of Cy-5 fluorochrome labeled siRNA uptake by confocal immunofluoresence microscopy. Interestingly, labeled siRNA was found to co-localize only with lung epithelial cells counterstained with anti-cytokeratin-18 at 24 h post instillation, but not alveolar macrophages (152) . However, negative results for alveolar macrophages may not preclude the uptake of siRNA by this cell type as one can imagine different kinetics in the various cell types with respect to siRNA uptake and processing. Relative to this, the feasibility of gene silencing in macrophages using siRNA has been described in vitro (172) (173) (174) , however, silencing of typically macrophagederived molecules such as TNF-α and IL-6 during indirect murine lung injury remained unsuccessful in our hands (unpublished observations). Whether this might be attributable to the underlying cell type or perhaps to the degree of upregulation of a certain gene during pathological conditions remains to be elucidated. Irrespective these and the experiments below represent the first studies to demonstrate the feasibility of in vivo lung siRNA as treatment for developing ALI.
To finally assess the therapeutic capacity of such a siRNA approach, experiments were designed to modulate PMN immigration based on the neutrophil hypothesis using in vivo siRNA constructs against the murine chemokines KC and MIP-2 during the development of indirect septic ALI. To this end, suitable siRNA constructs were instilled into the lungs of animals 2 h following HEM and prior to CLP. ALI was assessed 24 h after the induction of sepsis. Silencing of MIP-2 markedly reduced tissue and plasma IL-6 concentrations, tissue MIP-2, as well as lung PMN influx, interstitial edema, alveolar congestion, and disruption of lung tissue architecture (164) . In contrast, KC-siRNA treatment, while reducing plasma KC, tissue KC, and tissue IL-6, produced neither a significant reduction in plasma IL-6 nor lung PMN influx nor lung damage (164) . Alternatively, based on the epithelial cell hypothesis, siRNA sequences against Fas and caspase-8 were intratracheally instilled during septic ALI. Interestingly, while these sequences markedly diminished lung Fas and caspase-8 expression in a gene specific fashion, when lung apoptosis was assessed, pulmonary tissue caspase-3 activity was reduced only in response to Fas but not caspase-8 silencing. As silencing here, as commonly observed, did not result in a total depletion of the target protein, but rather in a diminished expression, it is possible that sufficient active caspase-8 was still present to mediate the apoptotic effects either through direct activation of caspase-3 or indirectly through cleavage of the proapoptotic protein Bid (152, 175) . Our data also indicated that silencing of Fas on lung epithelial cells was associated with a reduction in lung inflammation, PMN influx, and a diminished extent of lung injury (118) , thus emphasizing the relevance of epithelial cell integrity during indirect septic ALI.
Keeping in mind the diversity of clinical scenarios, we use the inclusive acronym ALI, one could think that it might be exactly this imprecise global view that prohibits the development of novel therapeutic approaches for ARDS. The diversity of underlying pathologic mechanisms prohibits the formulation of a unified pathophysiology of this clinical entity. Whether different forms of ALI (for example, direct versus indirect) are more of a response to a certain patho-mechanism than another remains to be determined. However, while there may be numerous diverse stimuli that can initiate the pathogenesis of this clinical entity, the final steps in ALI/ARDS, such as compromise of the alveolocapillary barrier function, appear to be somewhat common. Here we have reviewed the data which support the role of the activated PMN in lung injury as well as the contribution of epithelial cell death as independent entities contributing to ALI. Further, we have shown how recent studies looking at epithelial cells and the silencing of Fas-induced apoptosis and/or inflammation in these cells may serve as a lynchpin linking these two pathological processes. Upon early Fas activation, alveolar macrophages and lung epithelial cells might produce chemokines in the lung, thus attracting activated and potentially harmful neutrophils, monocytes, or even T-lymphocytes to the site of injury and potentiating the degree of injury. Finally, we trust we have illustrated how the application of siRNA-induced gene knockdown in the lung not only has produced novel insights into pathogenesis of ALI, but may represent a potential exciting therapeutic approach for its treatment. The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection RNA–RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2–RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis. RNA-RNA recombination is one of the most important pathways in virus evolution. It was first discovered in the early 1960s in poliovirus (1, 2) and has been documented in various genera of animal viruses, plant viruses and bacterial viruses group. Studies conducted on viruses during the last 40 years indicated that RNA-RNA recombination could occur between RNAs of the same or different strains of one species (3, 4) , of different species (5) or between viral and host cellular RNAs (6, 7) . RNA hairpins or mutations in a replicase domain have been implicated to play an important role in promoting RNA-RNA recombination (8) (9) (10) (11) . While none of those studies demonstrated a role for nonreplicase proteins in viral RNA-RNA recombination or selection of recombinants, recombination without replication has been described (12, 13) .
RNA recombination has been studied extensively in the plant virus genus Cucumovirus. Those studies have involved two of the three recognized viral species in this genus, Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). These viruses both contain a single-stranded and positive-sense RNA genome divided into three species designated RNAs 1, 2 and 3 (14, 15) . RNA 1 encodes the 1a protein involved in virus replication (16, 17) . RNA 2 encodes two proteins: the 2a, also involved in virus replication (16, 17) and the 2b protein, involved in virus movement and the suppression of RNA silencing (18) (19) (20) (21) . RNA 3 also encodes two proteins, the 3a movement protein and the capsid protein, both of which are also involved in virus movement (22) (23) (24) . Protein 2b is translated from a subgenomic RNA of RNA 2, RNA 4A (25, 26) , while the capsid protein is translated from a subgenomic RNA of RNA 3, RNA 4 (27) . The 3 0 terminal 305-310-nt non-translated region (NTR) is nearly identical in each of the three genomic RNAs of each virus and represents promoters for (À) viral RNA synthesis during viral replication (23, 28, 29) . The location of most of the recombinants described involving CMV and TAV is within these 3 0 NTR sequences.
RNA-RNA recombination was first described in cucumoviruses isolated from tobacco plants after multiple passage of a pseudorecombinant (reassortant) virus containing RNAs 1 and 2 from CMV, and RNA 3 from TAV (30) . The progeny RNA 3 consisted mostly of TAV RNA 3 but with the 3 0 terminal sequence from CMV RNA 2. In addition, in an earlier passage, there was an intermediate RNA 3 present containing a duplicated 21-nt sequence at the crossover site (30) . Subsequently, several other cucumoviral recombinant RNAs were also discovered in plants infected with a so-called quadripartite hybrid virus having RNAs 2 and 3 from CMV, and RNAs 1 and 2 from TAV (31) , with a mixture of wild-type (wt) CMV and wt TAV (32, 33) , with mixed CMV isolates (34) (35) (36) , or within a single wt CMV (37) . These characterized recombinant RNAs had non-templatederived nucleotides at the crossover site (31) and had CMV sequences at one end of the crossover site and TAV sequence at the other end (32) or had the same 3 0 -terminal NTR sequence for all three genomic RNAs (37) . Some of the recombinants had crossover sites localized within a short region of high sequence similarity (32) . Others were predicted to occur in regions of high secondary structure (33, 34, 38) . TAV RNA 3 itself has a direct repeat of 163 nt (differing by one nucleotide) in the 3 0 NTR, which is not essential for infection (39) and in some cases, was deleted during long-term incubation (40) . While some of the above studies examined recombinants formed in the directly inoculated leaves and thus avoided detecting only those replicable recombinants that might be selected (32, 33) , other studies examined those recombinants detected after slow selection (31, 38) and found that some recombinants could co-exist in the same plants (38) . However, what factors may have led to the selection of particular recombinants has not previously been examined.
Here we show that infection and passage of a chimeric virus having RNAs 1 and 2 from CMV, with RNA 3 and the 2b gene from TAV consistently resulted in the slow selection of recombinant viruses. We demonstrate that the 2b gene plays a key role in promoting this selection.
Plasmid cDNA clones, with transcription in planta controlled by the cauliflower mosaic virus (CaMV) 35S promoter and terminator, were used for plant inoculation. A pGEX2T-based plasmid (Amersham Pharmacia Biotech), pGEX-T2b, in which the gene encoding the TAV 2b protein was fused with sequences encoding GST, was used to express GST/TAV2b for RNA-binding assay. Various plasmids used in this study are shown in Figure 1 . Plasmid cDNA clones C1 (previously named pQCD1), C2 (pQCD2), C2 T2B (pQCD2qt), C2 T2BC2a (pQCD2qt1), C2 W2B (pQCD2qw), C3 (pQCD3), T1 (pCass1T1), T2 (pCass1T2), T3 (pCass1T3) and T3 Á163(A) (pCass1T3 Á163(A) ) have all been previously described (26, 39, (41) (42) (43) . The other plasmid cDNA clones were constructed as follows.
Plasmid cDNA clones C1 Á23 , C2 T2BÁ23 and T3 Á163(A)Á23 were constructed through three steps: first, amplifying two fragments with appropriate primer pairs (see Supplementary Table 1 Figure 1 . Schematic representation of constructs used in this article. The constructs regulated from the 35S promoter and 35S terminator are indicated. The open reading frames encoding various cucumoviral proteins on the constructs are shown. The 23 nt of sequence identity between all genomic RNAs is indicated by a short thick bar. The tandem repeat of 163 nt is indicated by an arrow. The crosshatch lines represent the amino acid sequences different from the native sequence for either 2a or 2b. T3 Á163 (A)Á23 ; and finally, by ligating the two digested  fragments into the appropriate sites created by digestion  with the same enzymes. Plasmid cDNA clones C1 3081-3390 ,  C2 W2B2757-3065 ,  T3 Á163(A)Á141 ,  T3 1-2060 ,  T3 1-2223 ,  T3 1902-2386 and T3 2065-2386 were constructed via two  steps: first amplifying appropriate fragments, and then ligating these fragments into the pCass1 vector (43) . Plasmid cDNA clones C2 W2BC2a and T3 Á163(G) were constructed with a QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instruction. All the amplified fragments as well as their joined regions in the plasmids were sequenced to ensure whether they were correct.
Two plant species, Nicotiana clevelandii and Nicotiana glutinosa, were chosen for inoculation of the plasmid cDNA clones. Unless specified otherwise, all inocula used were a mixture of the three genomic cDNA clones (corresponding to RNAs 1, 2 and 3), each at an equal concentration of 10 mg/10 ml per plant. For each test, at least five, four-leaf plants of each of the above two species were inoculated mechanically after a 24-h dark treatment. Each test was repeated at least once.
Total plant RNAs were extracted from infected plants and analysed by northern blot hybridization as described previously (26) . The nylon membranes were hybridized with a 32 P-labelled transcript probe, complementary to the 3 0 terminal sequences of either TAV RNA 3 (nucleotide 2287-2386) or RNA 2 of the Q-strain of CMV (Q-CMV; nucleotide 2871-3035). These two probes would hybridize to all the genomic and subgenomic RNAs of the corresponding viruses (39) . An additional probe complementary to nucleotide 1-1884 of TAV RNA 3 was specifically used to hybridize to the 5 0 -terminal region of TAV RNA 3.
Viral particles and virion RNAs were purified according to the method of Peden and Symons (44) . Individual viral RNAs were purified from the virion RNAs first via an agarose gel and then via a polyacrylamide gel (45) . The purified, single, viral RNAs were polyadenylated with Escherichia coli poly(A) polymerase and then RT-PCR was done using an oligo dT primer as described previously (43) . The amplified products were cloned into the pBluescript SK + vector and sequenced from both orientations.
The purified, single, recombinant RNAs, total RNAs extracted from plants and total virion RNAs were also reverse transcribed using primers T3 0 and C3 0 , respectively. T3 0 is complementary to the 3 0 -terminal sequence of TAV, while C3 0 is complementary to the 3 0 -terminal sequence of Q-CMV (see Supplementary Table 1 for details). The synthesized first-strand cDNAs from the templates were amplified with different pairs of primers, C3 0 /T3-1187, C3 0 /C2-980, C3 0 /C1-2032, T3 0 /T3-1187, T3 0 /C2-980 and T3 0 /C1-2032 (see Supplementary Table 1 for details), using the thermocycler program: 1 min at 948C, 1 min at 528C and 2 min at 728C for 30 cycles.
The amplified products were also cloned into the pBluescript SK + vector and sequenced from both orientations. At least five clones were sequenced from each PCR product.
DNA sequencing was carried out using 4 pmol of a specific primer and 0.1 mg of a cDNA clone. The sequence reaction was incubated at 378C for 10 min and then analysed via a manual sequencing gel system. RNA sequencing was carried out under the same conditions as the DNA sequencing except that 10 mg of the total plant RNAs, 2 mg of the virion RNAs or 1 mg of a single RNA were used as a template and that the sequence reaction was incubated at 428C for 30 min.
The plasmid pGEX-T2b was constructed via two steps: amplifying the PCR fragments first and then cloning the PCR fragments into appropriate vectors (as above). PGEX-T2b was transformed into E. coli BL21 (DE3). Protein expression was induced with 0.1 mM IPTG (isopropyl-b-D-thiogalactopyranoside). Purification of the protein was carried out using glutathione beads. The cleavage of GST/TAV2b protein was carried out using thrombin (Novagen) as recommended by the manufacturer. The purity of the purified protein was analysed by SDS-PAGE. The amount of purified protein was measured using the Bio-Rad protein assay.
Preparation of full-length TAV RNA 3 transcripts DNA templates representing a full-length RNA 3 of TAV and including T7 or T3 promoter were generated by PCR using the infectious cDNA clone of TAV RNA 3 (T3) as a template and primer pairs T3 0 and T7-TR3(+) (a primer used for transcription of the sense RNA), or T5 0 (nucleotides 1-27 of TAV RNA 3) and T3-TR3(À) (a primer used for transcription of the anti-sense RNA; see Supplementary Table 1 for the primer sequences). Labelled transcripts from the DNA templates were obtained using [ 32 P]-a-UTP and T7 or T3 DNA-dependent RNA polymerase. The free nucleotides in the transcription reaction were removed using P-30 micro Bio-Spin columns (Bio-Rad), while the DNA templates were removed by incubation with DNase I. The labelled TAV RNA 3 transcripts were purified through a 4% denaturing polyacrylamide gel and quantified by UV spectrophotometry (Beckman).
Three different amounts (150, 300 and 450 ng) of the TAV 2b protein purified from E. coli were each incubated with 4 ng of each of the [ 32 P]-labelled TAV RNA 3 transcripts in a binding buffer [50 mM Tris-HCl (pH 8.2), 10 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 200 ng of yeast tRNA (Sigma), and 2 U of RNase inhibitor (Promega)] at 258C for 30 min (46) . After incubation, potential protein-RNA complexes were analysed via electrophoresis on a 4% non-denaturing polyacrylamide gel and detected by autoradiography.
Pseudorecombinant viruses formed between RNAs 1 and 2 of CMV and RNA 3 of TAV have been shown gradually to yield stable, recombinant viruses after multiple passage (30, 31, 38) . However, the effects of the various viral genes on the selection of these recombinant viruses have not been examined. Previously, using interspecies hybrid viruses, we examined the roles of different 2b genes on virulence and virus accumulation and showed that while the nature of the 2b gene influenced virulence, the RNA 3 (and therefore the two genes encoded by RNA 3; the 3a movement protein and the capsid protein) did not (21, 47) . Therefore, the hybrid viruses generated previously, C1C2 T2B C3 and C1C2 T2B T3, where C indicates Q-CMV, T indicates TAV, 1, 2 and 3 indicate RNAs 1, 2 and 3, and the subscript T2B indicates that the 2b gene of Q-CMV was replaced precisely with that of TAV ( Figure 1 ), were subjected to long-term, multiple passages in N. glutinosa to determine whether the 2b gene had any effect on the selection of recombinant viruses. The progeny viral RNAs were examined by northern blot hybridization, with probes specific to the 3 0 terminal NTR sequences of either the three Q-CMV RNAs or the three TAV RNAs (Figure 1 ). This analysis indicated that in the multiple passage plants TAV RNA 3 derived from C1C2 T2B T3 had recombined with CMV RNA, as the progeny RNA 3 was detected by both the Q-CMV-specific and TAV-specific probes (Figure 2A and B, compare lanes 9 and 20). By contrast, RNA 3 derived from the same virus maintained for several weeks in the plasmidinoculated plants was not a recombinant RNA, as it was only detected by the TAV-specific probe, but not by the Q-CMV-specific probe (Figure 2A and B, compare lanes 5 and 16). The specificity of the CMV-specific and TAV-specific probes was demonstrated in that both probes only detected their own specific viral RNAs ( Figure 2A and B, compare lanes 2 and 3, and lanes 13 and 14) . The other RNAs of C1C2 T2B T3 were still detected by their specific probes in both the inoculated plants and the multiple passage plants (Figure 2A and B) .
To confirm that RNA 3 was a recombinant, the RNA 3 derived from C1C2 T2B T3 was gel-purified, reverse transcribed with primers C3 0 and T3 0 , respectively, and then amplified with different pairs of primers as described in the Materials and Methods section. Only the primer pair C3 0 /T3-1187 was able to generate a specific RT-PCR product (data not shown). T3-1187 corresponded to sequences ending with nucleotide position 1187 of TAV RNA 3, while C3 0 was complementary to the 3 0 -terminal sequence of Q-CMV. This indicates that the RNA 3 was indeed a recombinant, having a TAV RNA 3 sequence in the capsid protein region and a CMV RNA sequence at the 3 0 terminus.
To determine the nature of the recombinant RNAs derived from C1C2 T2B T3, the recombinant RNAs derived from this virus were purified from infected plants, subjected to RT-PCR and the RT-PCR products were then cloned into the pBluescript SK + vector for sequencing. The sequencing results from five independent clones showed that there was one type of RNA 3 recombinant derived from C1C2 T2B T3, containing 2370 nt, of which the 5 0 terminal 2060 nt was derived from the 5 0 terminal 2060 nt of TAV RNA 3 ( Figure 3A right, open bar), while the 3 0 terminal 310 nt was identical to the 3 0 terminal 310 nt of Q-CMV RNA 1 ( Figure 3A right, stippled bar). The crossover site on the TAV RNA 3 sequence was 7 nt 5 0 of a homologous sequence of 23 nt conserved in all three genomic RNAs, and 3 nt 5 0 of this conserved 23 nt sequence in the Q-CMV RNA 1 sequence.
The TAV RNA 3 sequence in the recombinant RNA 3 retained the first repeat [in order of 5 0 to 3 0 on the (+) sense strand] of two 163-nt tandem repeats (39) , but lacked the second one, which originally was present in the TAV RNA 3 inoculum. The difference between the two 163-nt repeats is an A at position 1966 in the first repeat, with a G at the corresponding position (2129) in the second 163-nt repeat ( Figure 3A left) (39) . The repeated sequences showed no difference in viral pathogenicity when only one or the other was present (39) . The 3 0 terminal 310 nt of the recombinant RNA 3 was not only the same as the 310 terminal nucleotides of Q-CMV RNA 1, but was also the same length as Q-CMV RNA 5. Q-CMV RNA 5 is a mixture of subgenomic RNAs derived from the 3 0 terminal NTR of RNAs 1, 2 and 3 [(48); our unpublished data]. The level of RNA 5 has a strong effect on the severity of symptoms; the presence of more RNA 5 in the plants results in less severe symptoms (our unpublished data).
Repeated multiple passages of virus generated from the plasmids comprising C1C2 T2B T3 in 10 N. glutinosa plants or 10 N. clevelandii plants resulted in the appearance of the same recombinant RNA 3s with the same border sequences among the sequenced clones. These recombinant RNAs were not detected by northern blot hybridization in the inoculated parental plants, even if the plasmid-inoculum was increased 5-fold (50 mg/10 ml) or decreased 20-fold (0.5 mg/10 ml), although the same recombinant RNAs were detected again in the multiple passage plants, when the initial inoculum concentration was changed (data not shown).
The observation that recombinant RNAs were detected when the 2b gene of Q-CMV was substituted by that of TAV (Figure 2A , lane 9), and not when the 2b gene of Q-CMV was present in the interspecific hybrid virus C1C2T3 (Figure 2A , lane 11) suggested that the 2b gene has some role in the generation and/or selection of the recombinant viruses. However, as the replacement of the 2b sequence also changed the sequences encoding the C-terminal 41 amino acids of the 2a protein, it cannot be ruled out that the C-terminal 41 amino acids of the 2a protein might play a role in the recombination and/or selection. Therefore, to determine whether the C-terminal 41 amino acids of the 2a protein encoded by the overlapping TAV 2a gene sequence had such a role, we introduced a stop codon upstream of the TAV 2b gene in C2 T2B to prevent the expression of the C-terminal 41 amino acids of the 2a protein (see C2 T2BC2a in Figure 1 ). Bioassay of C1C2 T2BC2a T3 onto N. glutinosa showed that the virus induced symptoms similar to those induced by C1C2 T2B T3 and northern blot hybridization and RT-PCR analyses showed that recombinant RNA 3 still accumulated in the multiple passage plants, but not in the inoculated plants (data not shown). Moreover, the recombinant RNA 3 generated was the same as that derived from C1C2 T2B T3 (data not shown). Therefore, the C-terminal 41 amino acids of the 2a protein encoded by the TAV 2b sequence had no role in the recombination and/or selection, indicating that the TAV 2b gene was required for the recombination and/or selection.
That the 2b protein rather than the 2b RNA sequence itself affected the generation and/or selection of recombinant RNA 3s could not be established conclusively, since expression of the 2b gene was shown to be necessary for the cell-to-cell movement, but not the replication of the pseudorecombinant virus C1C2T3 (21) . However, if the 2b protein was actually involved in the recombination/ selection process, then at a minimum the 2b protein should be capable of binding viral RNA. To determine 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 C1C2T3 whether there was some interaction between TAV RNA 3 and the 2b protein, we performed an RNA-proteinbinding assay. The TAV 2b protein ( Figure 4B , lane 3) purified from E. coli ( Figure 4A ) was able to bind to the T7-transcribed (+) sense TAV RNA 3 ( Figure 4C , lanes 2-4) as well as to the T3-transcribed (À) sense TAV RNA 3 ( Figure 4D , lane 3). The binding ability increased with an increasing amount of the 2b protein ( Figure 4C , compare lanes 1-4). By contrast, comparable amounts of GST alone, which was expressed and purified under the same conditions as the TAV 2b protein ( Figure 4B , lane 2), did not bind to TAV RNA 3 (data not shown). This demonstrated that RNA-binding activity was not due to a co-purified, contaminating protein from E. coli. The significance of the more efficient binding of the 2b protein to (+) versus (À) sense RNA is unknown. The different binding abilities probably are due to different sequences and/or structures between RNA 3(+) and RNA 3(À). Since the 2b protein was designated a movement protein (19) and was shown to be required for the movement of pseudorecombinant viruses containing C1C2T3 (21), the 2b protein probably also binds to TAV RNA 3 in vivo.
Another approach to determine the role of the 2b protein in the generation and/or selection of the recombinant RNAs, was to use a different 2b gene in the heterologous interspecific hybrid virus. To this end, we replaced the whole 2b gene of (the subgroup II strain) Q-CMV with that from a subgroup IA strain of CMV, WAII-CMV, generating the hybrid virus C1C2 W2B T3 (see C2 W2B in Figure 1 ). The WAII-CMV 2b gene and the Q-CMV 2b gene are only 53.4% identical at nucleotide level and the WAII-2b protein is 10 amino acids longer than the Q-2b protein.
Co-inoculation of C2 W2B , C1 and T3 onto N. glutinosa plants followed by multiple passages at 1-2 weekly intervals showed that C1C2 W2B T3 induced symptoms as severe as those induced by C1C2 T2B T3, which were much more severe than those induced by either of the parental viruses (21) . Northern blot hybridization showed that RNA 3 derived from C1C2 W2B T3 also had recombined with a CMV sequence in the passage plants, but not in the inoculated plants ( Figure 2C and D, compare lanes 27 and 34, and lanes 29 and 36). This was supported by RT-PCR analysis (data not shown). These data strongly indicate that the 2b protein was required for recombination and/or selection or the recombinant RNA 3s, and that this process was not TAV 2b protein-specific.
To determine the nature of the recombinant RNAs derived from C1C2 W2B T3, the recombinant RNA 3 was subjected to RT-PCR, cloning and sequence analysis. Surprisingly, the RNA 3 recombinants derived from C1C2 W2B T3 differed from those derived from C1C2 T2B T3 or C1C2 T2BC2a T3 in the following respects: First, the recombinant RNA 3 derived from C1C2 W2B T3 was 22 nt longer than that derived from C1C2 T2B T3 or C1C2 T2BC2a T3 (2392 versus 2370). Second, the 3 0 terminal NTR of the recombinant RNA 3 derived from C1C2 W2B T3 contained a Q-CMV RNA 2 NTR sequence, rather than the Q-CMV RNA 1 NTR sequence (compare Figure 3A right and B right) . Third, the crossover sites on RNA 2 and on RNA 3 were different in the recombinant RNA 3 derived from C1C2 W2B T3 versus either C1C2 T2B T3 or C1C2 T2BC2a T3 (compare Figure 3A and B). And fourth, the recombinant RNA 3 derived from C1C2 W2B T3 contained an imperfect repeat of 19 nt near the crossover site preceded by an A between the TAV RNA 3 sequence and the 3 0 CMV RNA sequence ( Figure 3B) . Moreover, the recombinant RNA 3 derived from C1C2 W2B T3 also was missing the second 163-nt repeat. These similarities on one hand and differences on the other suggested that the recombinant RNA 3s derived from C1C2 T2B T3 and C1C2 T2BC2a T3 versus C1C2 W2B T3 might be generated using different templates, but occurred via a similar mechanism. Taken together, the above results indicate that the 2b protein determined either the RNA species participating in the recombination and the crossover site, or selected for particular recombinant RNAs generated at random that have the above characteristics.
The 2b protein and a 163-nt tandem repeat on RNA 3 both affect the rate of recombinant selection while a single nucleotide within the repeat determines the crossover site on RNA 3
Both the TAV 2b protein and WAII-CMV 2b protein were able to promote the recombination and/or selection of RNA 3 in the passage plants, but not in the inoculated plants. To determine when the recombinant RNA 3s could first be detected, a time-course experiment was done involving three viruses: C1C2 T2B T3, C1C2 T2BC2a T3 and C1C2 W2B T3. The appropriate plasmid combinations were inoculated onto 10 N. glutinosa plants and sap was then passaged onto healthy N. glutinosa plants for 10 passages, at 6-day intervals. The inoculated leaves of the passage plants were detached 3 days after inoculation, total RNAs were extracted and the RNAs were analysed by northern blot hybridization. These analyses showed that the recombinant RNAs were only detected from the fourth passage and onwards in plants infected with C1C2 T2B T3 or C1C2 T2BC2a T3, or from the sixth passage and onwards in plants infected with C1C2 W2B T3 (Supplementary Figure 1A, data not shown) . These results indicated that selection of the recombinant RNA 3s is a slow process and therefore it seems more likely that the 2b proteins were selecting for specific recombinants, rather than being involved in the generation of the recombinant RNA 3s. This is also consistent with the different subcellular localization of the replication proteins and the 2b protein (49) (50) (51) and the lack of detectable interaction of the replicase proteins with the 2b protein (52) . In addition, virus containing the TAV 2b protein showed selection of the recombinant RNA 3s two passages earlier than did virus containing the WAII-CMV 2b protein. Therefore, it seems likely that the source of the 2b protein also affected the rate of selection.
To examine the role of the two 163-nt repeats in the recombination and/or selection, we deleted each repeat independently in C1C2 T2B T3. The resultant deletion mutants were designated C1C2 T2B T3 Á163(A) and C1C2 T2B T3 Á163(G) (Figure 1) , where the subscripts Á163(A) and Á163(G) represent the first 163-nt repeat deletion and the second 163-nt repeat deletion, respectively. Bioassay of C1C2 T2B T3 Á163(A) and C1C2 T2B T3 Á163(G) onto N. glutinosa plants showed that both deletion mutants induced similar symptoms to those induced by C1C2 T2B T3 (data not shown). Northern blot hybridization analysis showed that neither deletion affected the presence of recombinant RNA 3s in the multiple passage plants (Figure 2A, lanes 8 and 19 , data not shown). However, both deletion mutants yielded recombinant RNA 3s in both the inoculated plants and the passage plants (Figure 2A and B, compare lanes 4 and 15, and lanes 8 and 19, data not shown). Repeated inoculation and northern blot hybridization analysis gave the same results as earlier. This suggests that the two 163-nt repeats affected the rate of the recombination and/or selection of recombinants.
To confirm the role of the two 163-nt repeats in affecting the rate of recombination and/or selection, we generated two other deletion mutants, C1C2 W2B T3 Á163(A) and C1C2 W2B T3 Á163(G) . Bioassay of C1C2 W2B T3 Á163(A) and C1C2 W2B T3 Á163(G) showed that these two deletion mutants also induced symptoms similar to those induced by C1C2 W2B T3 (data not shown). Northern blot hybridization analysis showed that the deletions also resulted in the generation of the recombinant RNA 3s in both the inoculated and passage plants ( Figure 2C and D, compare lanes 26 and 33, and lanes 28 and 35, data not shown). These results showed that the two 163-nt repeats indeed affected the rate of the recombination and/or selection.
To determine the nature of the recombinant RNA 3s derived from the 163-nt repeat-deletion mutants, we again purified the recombinant RNA 3s of C1C2 T2B T3 Á163(A) , C1C2 W2B T3 Á163(A) , C1C2 T2B T3 Á163(G) and C1C2 W2B T3 Á163(G) , used RT-PCR to amplify the recombinant sequences, cloned the RT-PCR products and sequenced them. The sequences of the recombinant RNA 3 derived from C1C2 T2B T3 Á163(A) contained the 5 0 -proximal 2082 nt from TAV RNA 3 and the 3 0 proximal 310-nt 3 0 NTR of CMV RNA 1 ( Figure 5A ). The CMV RNA 1 NTR sequence was identical to that present in the recombinant RNA 3 derived from C1C2 T2B T3. However, the TAV RNA 3 sequence was 22 nt longer than that of the recombinant RNA 3 derived from C1C2 T2B T3 (compare Figures 3A  and 5A) , suggesting that the first 163-nt repeat might have a role in determining the crossover site on one of the participating RNAs, despite not affecting the crossover site on the other participating RNA. By contrast, the sequence of the recombinant RNA 3 derived from C1C2 T2B T3 Á163(G) was exactly the same as that derived from C1C2 T2B T3 (compare Figures 3A and 5B) , indicating that the second 163-nt repeat had no role in determining the crossover sites on either RNA.
The recombinant RNA 3 derived from C1C2 W2B T3 Á163(A) consisted of the 5 0 proximal 2082 nt from TAV RNA 3 and the 3 0 proximal 309-nt 3 0 NTR of CMV RNA 2 ( Figure 5C ). Although the 2082 nt sequence from TAV RNA 3 was exactly the same as that in the recombinant RNA 3 derived from C1C2 T2B T3 Á163(A) , it was 18 nt longer than the one seen in the recombinant RNA 3 derived from C1C2 W2B T3 (compare Figures 3B  and 5C ). This result supports the conclusion that the first 163-nt repeat might indeed affect the left border of the crossover site. The CMV RNA 2 sequence present in the recombinant RNA 3 derived from C1C2 W2B T3 Á163(A) was 19 nt shorter than the CMV RNA 2 present in the recombinant RNA 3 derived from C1C2 W2B T3 (due to the absence of the additional A and the first 19-nt repeat shown in Figure 3B , but with an additional U residue, nucleotide 2757), suggesting that the first 163-nt repeat might also affect the right border of the crossover site. In the recombinant RNA 3 derived from C1C2 W2B T3 Á163(G) , the sequence was exactly the same as that of the recombinant RNA 3 derived from C1C2 W2B T3 (compare Figures 3B and 5D) , confirming that the second 163-nt repeat had no role in determining the position of the crossover sites on either RNA. Since only one nucleotide differed in the two 163-nt repeats (A versus G), the above results indicate that the position of the crossover sites is actually affected by this single nucleotide difference within each repeat.
To determine whether the recombinant RNA 3s derived from C1C2 W2B T3 or C1C2 W2B T3 Á163(G) , both of which contained a 19-nt repeat sequence, might represent an intermediate, buffered extracts from plants infected by viruses containing these recombinant RNA 3s were passaged onto five healthy plants for more than 20 passages done at 1-week intervals. RT-PCR and sequence analyses of the recombinant RNA 3s from the passage plants showed no sequence difference from the original recombinant RNA 3s described above, indicating that the 19-nt repeat was still present in the same position as before (data not shown). These results indicate that recombinant RNA 3 derived from C1C2 W2B T3 or C1C2 W2B T3 Á163(G) containing the 19-nt repeat was not an intermediate, but the stable recombination end-product.
To examine how soon after infection this 19-nt repeat was generated and whether there was an intermediate generated before this 19-nt repeat appeared, a timecourse experiment was done, in which two viruses, C1C2 T2B T3 Á163(G) and C1C2 W2B T3 Á163(G) , were inoculated onto N. clevelandii. Nicotiana clevelandii was chosen as the host because the symptoms are more readily seen on this host than on N. glutinosa (unpublished data) and C1C2 T2B T3 Á163(G) and C1C2 W2B T3 Á163(G) were chosen because both viruses showed more rapid appearance of recombinant RNA 3 (see above). Moreover, the recombinant progeny virus of C1C2 W2B T3 Á163(G) contained the 19-nt repeat, which could be used as a marker to determine whether there was an intermediate before this final product. After inoculation of each virus to 200 plants, leaves were detached from groups of five inoculated plants at 1-day intervals for up to 40 days. Northern blot hybridization of the total RNAs extracted from the detached leaves showed that recombinant RNA 3s derived from any of the above viruses did not accumulate to a detectable level before day 35; the earliest time when a recombinant RNA 3 could be detected by northern blot hybridization was at day 35 in the C1C2 T2B T3 Á163(G)infected plants (Supplementary Figure 1B) . The recombinant RNA 3 derived from C1C2 W2B T3 Á163(G) was not detected during the whole time-course of the experiment (data not shown). However, by using RT-PCR with the primer pair C3 0 /T3-1187, the recombinant RNA 3 derived from C1C2 T2B T3 Á163(G) was detected as early as day 22 (Figure 6 upper panel) , while the RNA 3 recombinant derived from C1C2 W2B T3 Á163(G) could be detected at day 35 (data not shown). These results also confirmed that the TAV 2b protein promoted the recombination/selection faster than the WAII-CMV 2b protein.
To characterize the RT-PCR products further, Southern blot hybridization was done using CMV-specific and TAV-specific probes. All the RT-PCR products on the gel were the same size ( Figure 6 middle and lower panels) and the size was also the same as for the RT-PCR products obtained from the passage plants (data not shown). In addition, all the RT-PCR products were able to hybridize with both the Q-CMV-specific probe and the TAV-specific probe ( Figure 6 middle and lower panels). Sequencing of these RT-PCR products showed that the RT-PCR products from the C1C2 T2B T3 Á163(G) -inoculated plants contained 874 nt derived from TAV RNA 3 (up to nucleotide 2060 of TAV RNA 3) and 310 nt from CMV RNA 1. This sequence was exactly the same as that derived from the same virus in the passage plants and also the same sequence as that derived from C1C2 T2B T3, either in the inoculated plants or in the passage plants ( Figures 3A and 5B ; data not shown). All of the RT-PCR products derived from C1C2 W2B T3 Á163(G) also were exactly the same in sequence, having 878 nt from TAV RNA 3 and 326 nt from CMV RNA 2 including the 19-nt repeat ( Figure 5D ; data not shown). The sequence of the RT-PCR products also were the same as those from the passage plants, or those derived from C1C2 W2B T3 in both the inoculated plants and passage plants ( Figure 3B ; data not shown). These results indicate that the recombinant RNA 3s accumulating in the infected plants did not contain a detectable intermediate, and that the crossover sites on both donor and acceptor RNAs occurred at the same position.
The conserved 23-nt sequence and the 3' NTR of RNA 3 are critical for infection
The conserved 23-nt sequence at the beginning of the 3 0 NTR has been implicated in the recombination and nucleotides 1-20 of the 23-nt sequence also have been suggested to be an internal subgenomic promoter site (33, 38) . This sequence also was always near the crossover sites in the recombinant RNA 3s observed here, and the 19-nt imperfect repeat also comes from this sequence. Therefore, we examined the requirement for this sequence in infection. Three deletion mutants were constructed: C1 Á23 , C2 T2BÁ23 and T3 Á163(A)Á23 , where the conserved 23-nt sequence was deleted from each of C1, C2 T2B and T3 Á163(A) , respectively. Co-inoculation of each deletion mutant in the combinations C1 Á23 with C2 T2BÁ23 and T3 Á163(A)Á23 , C1 Á23 with C2 T2B and T3 Á163(A) , C1 with C2 T2BÁ23 and T3 Á163(A) , or C1 with C2 T2B and T3 Á163(A)Á23 , onto N. glutinosa and N. clevelandii plants, showed that none of these combinations was able to infect the plants (Table 1) . However, when either C1 Á23 was combined with C2 and C3, or T3 Á163(A)Á23 was combined with T1 and T2, the mixtures were infectious (Table 1) , indicating that the presence of this 23-nt sequence is essential for infection of the pseudorecombinant viruses, but not the parental viruses. Since the pseudorecombinant viruses containing the 23-nt sequence were viable without detectable recombination for some time (Figure 2) , these results indicate that the 23-nt sequence is not essential for replication of the pseudorecombinant virus, but may be essential for maintaining infection of the pseudorecombinant viruses through the plants, just as the 2b protein was required for this task, but not for the maintenance of infection of the parental viruses (19, 21) .
The above results showed that the CMV sequence in the recombinant RNA 3 was similar or identical to the CMV RNA 5 sequence. Correspondingly, the TAV RNA 3 sequence in the same recombinant RNA 3s lacked the second 163-nt repeat and the rest of the 3 0 NTR (which together corresponds to the TAV RNA 5 sequence). To determine whether CMV RNA 5 itself or the 3 0 NTR of CMV RNAs 1 and/or 2 could be used as a template for the synthesis of the recombinant RNA 3, we prepared the appropriate templates that would be expressed transiently in inoculated plants and assessed these for the presence of recombinant virus. In the templates, we deleted 326 nt from the 3 0 NTR of T3 to make T3 , and deleted the first 163-nt repeat plus 141 nt from the 3 0 NTR of T3 to make T3 Á163(A)Á141 (Figure 1 ). These two deletions had the same length of the TAV RNA 3 sequence as in the recombinant RNAs. In addition, we also made four RNA 5-like cDNA clones, C1 3081-3390 , C2 W2B2757-3065 , T3 1902-2386 and T3 2065-2386 . The former two were based on the CMV RNA 1 and C2 W2B sequences in the recombinant viruses, respectively, while the latter two were based on the sequence of TAV RNAs 3B and 5, respectively (39) . All of these clones were placed into the pCass1 vector, under transcriptional control of the CaMV 35S RNA promoter and terminator sequences. The infectivity results of the various plasmid mixtures tested on N. glutinosa and N. clevelandii plants are shown in Table 1 (Table 1 ). These results indicate that the sequences containing the 3 0 NTR are essential for the viral infection and could not be separated functionally from the rest of the TAV RNA 3 sequence. In addition, the results also indicate that the recombination observed in other experiments did not occur during the synthesis of (À) sense RNA from (+) sense templates, since there was no repair of the 3 0 NTR from synthesis initiated on either genomic RNAs 1 or 2, or subgenomic RNAs 3B or 5 (Table 1) and template switching to TAV RNA 3 lacking the 3 0 NTR. Such repair recombination is common among tombusviruses (8, 53) and occurs with the related brome mosaic virus (54) (55) (56) . Therefore, an alternative explanation is that the recombination occurred during (+) sense RNA synthesis on the (À) sense RNA templates, with recombination occurring as a result of template switching of the viral replicase from the donor (TAV RNA 3) strand to the acceptor (CMV RNA) strand.
Based on putative secondary structures for the 3 0 NTRs of CMV and TAV, models have been proposed to account for the generation of some of the other recombination products previously observed to occur between CMV and TAV (33, 38) . These models are based on template switching of the viral replicase on a stem-loop structure present in the (À) RNA 3 (L1/L2 in Figure 7 ) that may be the promoter for the synthesis of subgenomic RNAs 3B and 5 (33, 38) . Similar structures can be used to explain the various recombinant products obtained here and the effect of the 163-nt repeats and the single G to A substitution on the recombination event.
This model assumes that intramolecular recombination is a more frequent event than intermolecular 1-2060 T3 1902-2386 À À C1C2 T2B T3 1-2060 T3 2065-2386 À À '+' indicates infection, while 'À' indicates no infection. The nature of the plasmid constructs is described in Figure 1 . recombination. Therefore, generation of TAV RNA 3 progeny with deletion of one of the 163-nt repeats is more likely to occur than recombination between TAV RNA 3 and another viral genomic RNA. That is, if one repeat is missing, then only intermolecular recombination can occur, which may explain why the recombinants were seen to accumulate more rapidly in the plants directly inoculated with virus containing only one repeat ( Figure 2 ). On the other hand, loss of a repeat was not a prerequisite for intermolecular recombination, since intramolecular recombination would involve loss of the first repeat [163(A)], while the recombinants formed from the full-length T3 all retained the first repeat. Intramolecular recombination would be expected to occur during (+) TAV RNA 3 synthesis, if the polymerase jumped from the beginning of the first repeat in stem-loop L2 ( Figure 7A ) to the beginning of the second repeat in stemloop L1 ( Figure 7B ). These sequences also contain the putative RNA 5 subgenomic promoter sequence (33, 38) that would facilitate this recombination and those described previously that involved this 'hot spot' for intermolecular recombination between TAV and CMV (33, 38) . The presence of another stem-loop structure (K1/K2 in Figure 7 ) immediately adjacent to the putative subgenomic promoter may slow the polymerase down sufficiently to stutter and in some cases switch templates. In the case of the first TAV-CMV RNA 3 recombinant described (30) , this occurred just before another stem-loop structure in the first repeat (J2 in Figure 7A ; see arrow), but in that instance, the polymerase jumped to the putative RNA 5 subgenomic promoter of (À) CMV RNA 2, leading to an initial recombinant that had a duplication of the two, putative subgenomic RNA promoters, which then led to a second recombination event, with the intervening TAV RNA 3 sequences deleted, giving the final, stable recombinant RNA 3 described (30) . The full-length TAV RNA 3, as well as the T3 Á163(G) variant gave rise to similar recombination products, with the exact switchover position on the donor RNA of the recombinant determined by the 2b gene present (T2B versus W2B), while the switchover position on the receptor RNA is the beginning of the RNA 5 sequence in either CMV RNAs 1 or 2, again determined by the specific 2b gene present. In the case of C1C2 T2B T3 and C1C2 T2B T3 Á163(G) , the recombination occurred immediately after nucleotide 2060. For C1C2 T2B T3, switchover would have had to occur at the base of stem-loop L1 ( Figure 7B ), while for C1C2 T2B T3 Á163(G) , switchover would have had to occur within stem-loop G ( Figure 7B and C), since the sequences comprising stem-loop L1 (plus stem-loops K1 to H1) would not exist in C1C2 T2B T3 Á163(G) . Since stem-loops L1 and G are quite different in most of their sequence and structure, as well as stability (ÁG = -1.3 kcal/mol for stem-loop G and À6.9 kcal/mol for stem-loop L1), it seems likely that the common structures preceding these stem-loops (J2-I2-H2 versus J1-I1-H1; ÁG = À3.8, À4.8 and À2.9 kcal/mol, for each stem-loop structure) may have slowed the polymerase sufficiently to allow it to pause at nucleotide 2060, in either stem-loop L1 or G ( Figure 7B ).
In the case of C1C2W 2B T3 Á163(G) , switchover occurred following nucleotide 2064 in stem-loop G, but did not immediately proceed to the (À) CMV RNA 2 as the receptor RNA. Rather, the polymerase jumped back to the subgenomic promoter stem-loop (L2) and copied this through the A residue in the gap between stem-loops L2 and K2 ( Figure 7A) , before pausing and jumping to the same subgenomic promoter sequence on (À) CMV RNA 2. The small difference in ÁG between the 6 bp stem-loop K2 in (À) TAV RNA 3 (ÁG = À2.7 kcal/mol) and a weaker 3 bp stem-loop (K 0 ) structure adjacent to the subgenomic promoter in (À) CMV RNA 2 (ÁG = À2.1 kcal/mol) may be sufficient to prevent the polymerase from pausing at the base of stem-loop K 0 in (À) CMV RNA 2. This explains the duplication of the 19 nt seen in Figure 5D , following the A residue (from copying the first nucleotide, U-1902 of the RNA 5/3B promoter). In the case of C1C2 W2B T3, the same recombinant progeny RNA 3 is seen, but it must have been generated from a different starting point, since the second repeat is present in the template but missing in the recombinant progeny RNA 3. Here, template switching occurred either immediately after nucleotide 2084 (the A residue in the gap between stem-loops L1 and K1), again leading to a partially duplicated subgenomic promoter with the 19 nt repeat, or it occurred at nucleotide 2064, in the lower region of stem-loop L1 ( Figure 7B ), and then proceeded as described above for C1C2 W2B T3 Á163(G) .
In contrast to C1C2 T2B T3 Á163(G) , C1C2 T2B T3 Á163(A) and C1C2W 2B T3 Á163(A) underwent recombination 22 nt further along the TAV RNA 3 molecule after nucleotide 2245, adjacent to stem-loop E (in Figure 7C) , regardless of the nature of the 2b protein. Therefore, while the nature of the receptor RNA and the apparently slow selection of the recombinants (Figures 2 and 6) were determined by the 2b gene, the generation of recombinants at position 2245 was determined by the absence of the first repeat. Since the first and second repeats differ by only one nucleotide (underlined blue letters in Figure 7A and B, at positions 1966 and 2129), we propose that there must be a difference in structure between the virus containing only one repeat, when this is the second repeat versus the first repeat, and that this difference in structure affects pausing by the polymerase, leading to switchover to another template. This difference could be the presence (or absence) of a weak pseudoknot structure formed between the three A residues in the loop of stem-loop G and the three U residues in the non-base-paired region between stem-loops K2 and J2 (see blue letters in Figures 7) . This pseudoknot would not be stable in T3 Á163(A) , since it would only contain two A:U base pairs. This weak pseudoknot structure may be sufficient to affect pausing by the polymerase in the T3 Á163(G) variant, if it can re-form after the polymerase has gone through it the first time and then transcribed through the various, following stem-loop structures (stem-loops J, I and H in Figure 7 ), leading to template switching in stem-loop G. However, as this pseudoknot is absent in T3 Á163(A) , the polymerase would continue through stem-loop G until it pauses at the base of stem-loop E ( Figure 7C ; ÁG = À6.8 kcal/mol), before switching to the receptor RNA.
It would seem likely that the polymerase is as equally able to use RNA 1 as RNA 2 for the receptor RNA, since the polymerase is essentially the same in all cases, and the presence or absence of the 2a protein C-terminal sequences (where the recombinant polymerases showed some differences in sequence) did not affect the nature of the recombinant RNAs. However, since only one type of recombinant progeny was observed in each case, this demonstrates that there must have been selection for a particular progeny RNA, which depended on the source of the 2b gene and its encoded protein.
How could the nature of the 2b protein affect the selection of recombinant RNAs? It is conceivable that the selection of recombinant RNAs is a manifestation of the RNA silencing suppression-related functions of the 2b proteins, since some of these also vary between strains of CMV (57, 58) . However, based on the spatial separation of the 2b protein from the replication machinery within the cell (49) (50) (51) , it is difficult to envision how different 2b proteins provide the selection observed through their RNA silencing suppressor functions. In the case of the tombusvirus cucumber necrosis virus, inhibition of expression of the 20-kDa protein now known to be the RNA silencing suppressor of this virus, led to the rapid appearance of defective-interfering RNAs (59) . Thus, in that, the absence of the silencing suppressor led to enhanced generation or selection of recombinant RNAs, while in the case of CMV, the source of the 2b protein determined the nature of the recombinant RNAs selected and pseudorecombinant viruses could not be selected in its absence (21) . Moreover, in the case of influenza virus, where the NS1 gene encodes an RNA silencing suppressor (60) , mutation in a different gene (NS2) led to the rapid appearance of defective interfering particles (61) . At the time it was thought that the influenza NS2 protein might be involved in replication. In fact, the NS2 protein is involved in selecting ribonucleoprotein complexes for transport from the nucleus to the cytoplasm and is now called the nuclear export protein (62) .
Based on previous observations that the 2b protein is also a movement protein (19, 63) , that the presence of the 2b protein was necessary for the cell-to-cell movement of pseudorecombinants viruses (21) , and that the 2b protein is a viral RNA-binding protein (Figure 4) , it seems likely that the 2b binds to and selects specific recombinant viral RNAs for cell-to-cell movement. The different forms of recombinant RNAs generated may have different affinities for the various 2b proteins, leading to selection of one recombinant form over another by a particular 2b protein, which leads eventually to selection of the recombinant RNA over the parental RNA. Since selection to reach detectable levels was only observed after several weeks, the recombinant viral RNAs do not appear to be under strong selection pressure, although there is clearly a gradual selection for the recombinant RNAs over the parental RNA 3. This was seen in the slow appearance of the recombinant RNAs in both sets of time course experiments ( Figure 6 and Supplementary Figure 1 ). The rate of selection could be tested further by direct competition experiments. The Q-CMV 2b protein did not select for any recombinants, while the TAV and WAII-CMV 2b proteins selected for different recombinants, possibly reflecting differences in their RNA-binding properties. It would also be interesting to determine whether various 2b mutants would show differences in their selection of different recombinants. Epigrass: a tool to study disease spread in complex networks BACKGROUND: The construction of complex spatial simulation models such as those used in network epidemiology, is a daunting task due to the large amount of data involved in their parameterization. Such data, which frequently resides on large geo-referenced databases, has to be processed and assigned to the various components of the model. All this just to construct the model, then it still has to be simulated and analyzed under different epidemiological scenarios. This workflow can only be achieved efficiently by computational tools that can automate most, if not all, these time-consuming tasks. In this paper, we present a simulation software, Epigrass, aimed to help designing and simulating network-epidemic models with any kind of node behavior. RESULTS: A Network epidemiological model representing the spread of a directly transmitted disease through a bus-transportation network connecting mid-size cities in Brazil. Results show that the topological context of the starting point of the epidemic is of great importance from both control and preventive perspectives. CONCLUSION: Epigrass is shown to facilitate greatly the construction, simulation and analysis of complex network models. The output of model results in standard GIS file formats facilitate the post-processing and analysis of results by means of sophisticated GIS software. Epidemic models describe the spread of infectious diseases in populations. More and more, these models are being used for predicting, understanding and developing control strategies. To be used in specific contexts, modeling approaches have shifted from "strategic models" (where a caricature of real processes is modeled in order to emphasize first principles) to "tactical models" (detailed representations of real situations). Tactical models are useful for cost-benefit and scenario analyses. Good examples are the foot-and-mouth epidemic models for UK, triggered by the need of a response to the 2001 epidemic [1, 2] and the simulation of pandemic flu in differ-ent scenarios helping authorities to choose among alternative intervention strategies [3, 4] .
In realistic epidemic models, a key issue to consider is the representation of the contact process through which a disease is spread, and network models have arisen as good candidates [5] . This has led to the development of "network epidemic models". Network is a flexible concept that can be used to describe, for example, a collection of individuals linked by sexual partnerships [6] , a collection of families linked by sharing workplaces/schools [7] , a collection of cities linked by air routes [8] . Any of these scales may be relevant to the study and control of disease spread [9] .
Networks are made of nodes and their connections. One may classify network epidemic models according to node behavior. One example would be a classification based on the states assumed by the nodes: networks with discretestate nodes have nodes characterized by a discrete variable representing its epidemiological status (for example, susceptible, infected, recovered). The state of a node changes in response to the state of neighbor nodes, as defined by the network topology and a set of transmission rules. Networks with continuous-state nodes, on the other hand, have node' state described by a quantitative variable (number of susceptibles, density of infected individuals, for example), modelled as a function of the history of the node and its neighbors. The importance of the concept of neighborhood on any kind of network epidemic model stems from its large overlap with the concept of transmission. In network epidemic models, transmission either defines or is defined/constrained by the neighborhood structure. In the latter case, a neighborhood structure is given a priori which will influence transmissibility between nodes.
The construction of complex simulation models such as those used in network epidemic models, is a daunting task due to the large amount of data involved in their parameterization. Such data frequently resides on large geo-referenced databases. This data has to be processed and assigned to the various components of the model. All this just to construct the model, then it still has to be simulated, analyzed under different epidemiological scenarios. This workflow can only be achieved efficiently by computational tools that can automate most if not all of these time-consuming tasks.
In this paper, we present a simulation software, Epigrass, aimed to help designing and simulating network-epidemic models with any kind of node behavior. Without such a tool, implementing network epidemic models is not a simple task, requiring a reasonably good knowledge of programming. We expect that this software will stimulate the use and development of networks models for epidemiological purposes. The paper is organized as following: first we describe the software and how it is organized with a brief overview of its functionality. Then we demonstrate its use with an example. The example simulates the spread of a directly transmitted infectious disease in Brazil through its transportation network. The velocity of spread of new diseases in a network of susceptible populations depends on their spatial distribution, size, susceptibility and patterns of contact. In a spatial scale, climate and environment may also impact the dynamics of geographical spread as it introduces temporal and spatial heterogeneity. Understanding and predicting the direction and velocity of an invasion wave is key for emergency preparedness.
Epigrass is a platform for network epidemiological simulation and analysis. It enables researchers to perform comprehensive spatio-temporal simulations incorporating epidemiological data and models for disease transmission and control in order to create complex scenario analyses. Epigrass is designed towards facilitating the construction and simulation of large scale metapopulational models. Each component population of such a metapopulational model is assumed to be connected through a contact network which determines migration flows between populations. This connectivity model can be easily adapted to represent any type of adjacency structure.
Epigrass is entirely written in the Python language, which contributes greatly to the flexibility of the whole system due to the dynamical nature of the language. The geo-referenced networks over which epidemiological processes take place can be very straightforwardly represented in a object-oriented framework. Consequently, the nodes and edges of the geographical networks are objects with their own attributes and methods (figure 1).
Once the archetypal node and edge objects are defined with appropriate attributes and methods, then a code representation of the real system can be constructed, where nodes (representing people or localities) and contact routes are instances of node and edge objects, respectively. The whole network is also an object with its own set of attributes and methods. In fact, Epigrass also allows for multiple edge sets in order to represent multiple contact networks in a single model. Figure 1 Architecture of an Epigrass simulation model. A simulation object contains the whole model and all other objects representing the graph, sites and edges. Site object contaim model objects, which can be one of the built-in epidemiological models or a custom model written by the user.
These features leads to a compact and hierarchical computational model consisting of a network object containing a variable number of node and edge objects. It also does not pose limitations to encapsulation, potentially allowing for networks within networks, if desirable. This representation can also be easily distributed over a computational grid or cluster, if the dependency structure of the whole model does not prevent it (this feature is currently being implemented and will be available on a future release of Epigrass). For the end-user, this hierarchical, object-oriented representation is not an obstacle since it reflects the natural structure of the real system. Even after the model is converted into a code object, all of its component objects remain accessible to one another, facilitating the exchange of information between all levels of the model, a feature the user can easily include in his/her custom models.
Nodes and edges are dynamical objects in the sense that they can be modified at runtime altering their behavior in response to user defined events. In Epigrass it is very easy to simulate any dynamical system embedded in a network. However, it was designed with epidemiological models in mind. This goal led to the inclusion of a collection of built-in epidemic models which can be readily used for the intra-node dynamics (SIR model family). Epigrass users are not limited to basing their simulations on the built-in models. User-defined models can be developed in just a few lines of Python code. All simulations in Epigrass are done in discrete-time. However, custom models may implement finer dynamics within each time step, by implementing ODE models at the nodes, for instance.
The Epigrass system is driven by a graphical user interface(GUI), which handles several input files required for model definition and manages the simulation and output generation (figure 2).
At the core of the system lies the simulator. It parses the model specification files, contained in a text file (.epg file), and builds the network from site and edge description files (comma separated values text files, CSV). The simulator then builds a code representation of the entire model, simulates it, and stores the results in the database or in a couple of CSV files. This output will contain the full time series of the variables in the model. Additionally, a map layer (in shapefile and KML format) is also generated with summary statitics for the model (figure 3).
The results of an Epigrass simulation can be visualized in different ways. A map with an animation of the resulting timeseries is available directly through the GUI (figure 4). Other types of static visualizations can be generated through GIS software from the shapefiles generated. The KML file can also be viewed in Google Earth™ or Google Maps™ (figure 5).
Epigrass also includes a report generator module which is controlled through a parameter in the ".epg" file.
Epigrass is capable of generating PDF reports with summary statistics from the simulation. This module requires a LATEX installation to work. Reports are most useful for general verification of expected model behavior and network structure. However, the LATEX source files generated Workflow for a typical Epigrass simulation Figure 3 Workflow for a typical Epigrass simulation. This diagram shows all inputs and outputs typical of an Epigrass simulation session.
Epigrass graphical user interface Figure 2 Epigrass graphical user interface.
by the module may serve as templates that the user can edit to generate a more complete document.
Building a model in Epigrass is very simple, especially if the user chooses to use one of the built-in models. Epigrass includes 20 different epidemic models ready to be used (See manual for built-in models description).
To run a network epidemic model in Epigrass, the user is required to provide three separate text files (Optionally, also a shapefile with the map layer):
1. Node-specification file: This file can be edited on a spreadsheet and saved as a csv file. Each row is a node and the columns are variables describing the node.
2. Edge-specification file: This is also a spreadsheet-like file with an edge per row. Columns contain flow variables.
3. Model-specification file: Also referred to as the ".epg" file. This file specifies the epidemiological model to be run at the nodes, its parameters, flow model for the edges, and general parameters of the simulation.
The ".epg" file is normally modified from templates included with Epigrass. Nodes and edges files on the other hand, have to be built from scratch for every new network. Details of how to construct these files, as well as examples, can be found in the documentation accompanying the software, which is available at at the project's website [10] 
In the example application, the spread of a respiratory disease through a network of cities connected by bus transportation routes is analyzed.
The epidemiological scenario is one of the invasion of a new influenza-like virus. One may want to simulate the spread of this disease through the country by the transportation network to evaluate alternative intervention strategies (e.g. different vaccination strategies). In this problem, a network can be defined as a set of nodes and links where nodes represent cities and links represents transportation routes. Some examples of this kind of model are available in the literature [8, 11] .
One possible objective of this model is to understand how the spread of such a disease may be affected by the pointof-entry of the disease in the network. To that end, we may look at variables such as the speed of the epidemic, number of cases after a fixed amount of time, the distribution of cases in time and the path taken by the spread.
The example network was built from 76 of largest cities of Brazil (>= 100 k habs). The bus routes between those cities formed the connections between the nodes of the networks. The number of edges in the network, derived from Epigrass output visualized on Google-Earth Figure 5 Epigrass output visualized on Google-Earth. Figure 4 Epigrass animation output. Sites are color coded (from red to blue) according to infection times. Bright red is the seed site (on the NE).
the bus routes, is 850. These bus routes are registered with the National Agency of Terrestrial Transportation (ANTT) which provided the data used to parameterize the edges of the network.
The epidemiological model used consisted of a metapopulation system with a discrete-time SEIR model (Eq. 1). For each city, S t is the number of susceptibles in the city at time t, E t is the number of infected but not yet infectious individuals, I t is the number of infectious individuals resident in the locality, N is the population residing in the locality (assumed constant throughout the simulation), and n t is the number of individuals visiting the locality, Θ t is the number of visitors who are infectious. The parameters used were taken from Lipsitch et al. (2003) [12] to represent a disease like SARS with an estimated basic reproduction number (R 0 ) of 2.2 to 3.6 ( Table 1) .
To simulate the spread of infection between cities, we used the concept of a "forest fire" model [13] . An infected individual, traveling to another city, acts as a spark that may trigger an epidemic in the new locality. This approach is based on the assumption that individuals commute between localities and contribute temporarily to the number of infected in the new locality, but not to its demography. Implications of this approach are discussed in Grenfell et al (2001) [13] .
The number of individuals arriving in a city (n t ) is based on annual total number of passengers arriving trough all bus routes leading to that city as provided by the ANTT (Brazilian National Agency for Terrestrial Transportation). The annual number of passengers is used to derive an average daily number of passengers simply by dividing it by 365.
Stochasticity is introduced in the model at two points: the number of new cases is draw from a Poisson distribution with intensity and the number of infected individuals visiting i is modelled as binomial process:
where n is the total number of passengers arriving from a given neighboring city; I k, t and N k are the current number of infectious individuals and the total population size of city k, respectively. δ is the delay associated with the duration of each bus trip. The delay δ was calculated as the number of days (rounded down) that a bus, traveling at an average speed of 60 km/h, would take to complete a given trip. The lengths in kilometers of all bus routes were also obtained from the ANTT.
Vaccination campaigns in specific (or all) cities can be easily attained in Epigrass, with individual coverages for each campaign on each city. We use this feature to explore Vaccination scenarios in this model (figures 6 and 7).
The files with this model's definition(the sites, edges and ".epg" files) are available as part of the Additional files 1, 2 and 3 for this article.
To determine the importance of the point of entry in the outcome of the epidemic, the model was run 500 times, randomizing the point of entry of the virus. The seeding site was chosen with a probability proportional to the log 10 of their population size. These replicates were run using Epigrass' built-in support for repeated runs with the option of randomizing seeding site. For every simulation, statistics about each site such as the time it got infected and time series of incidence were saved.
The time required for the epidemic to infect 50% of the cities was chosen as a global index to network susceptibility to invasion. To compare the relative exposure of cities to disease invasion, we also calculated the inverse of time 
, for all k neighbors elapsed from the beginning of the epidemic until the city registered its first indigenous case as a local measure of exposure.
Except for population size, all other epidemiological parameters were the same for all cities, that is, disease transmissibility and recovery rate. Some positional features of each node were also derived: Centrality, which is is a measure derived from the average distance of a given site to every other site in the network; Betweeness, which is the number of times a node figures in the the shortest path between any other pair of nodes; and Degree, which is the number of edges connected to a node.
In order to analyze the path of the epidemic spread, we also recorded which cities provided the infectious cases which were responsible for the infection of each other city. If more than one source of infection exists, Epigrass selects the city which contributed with the largest number
Cost in vaccines applied vs. benefit in cases avoided, for a simulated epidemic starting at the highest degree city (São Paulo) Figure 6 Cost in vaccines applied vs. benefit in cases avoided, for a simulated epidemic starting at the highest degree city (São Paulo).
Cost in vaccines applied vs. benefit in cases avoided, for a simulated epidemic starting at a relatively low degree city(Salvador) Figure 7 Cost in vaccines applied vs. benefit in cases avoided, for a simulated epidemic starting at a relatively low degree city(Salvador).
of infectious individuals at that time-step, as the most likely infector. At the end of the simulation Epigrass generates a file with the dispersion tree in graphML format, which can be read by a variety of graph plotting programs to generate the graphic seen on figure 8.
The computational cost of running a single time step in an epigrass model, is mainly determined by the cost of calculating the epidemiological models on each site(node). Therefore, time required to run models based on larger networks should scale linearly with the size of the network (order of the graph), for simulations of the same duration. The model presented here, took 2.6 seconds for a 100 days run, on a 2.1 GHz cpu. A somewhat larger model with 343 sites and 8735 edges took 28 seconds for a 100 days simulation. Very large networks may be limited by the ammount of RAM available. The authors are working on adapting Epigrass to distribute processing among multiple cpus(in SMP systems), or multiple computers in a cluster system. The memory demands can also be addressed by keeping the simulation objects on an objectoriented database during the simulation. Steps in this direction are also being taken by the development team.
The model presented here served maily the purpose of illustrating the capabilities of Epigrass for simulating and analyzing reasonably complex epidemic scenarios. It should not be taken as a careful and complete analysis of a real epidemic. Despite that, some features of the simulated epidemic are worth discussing. For example: The spread speed of the epidemic, measured as the time taken to infect 50% of the cities, was found to be influenced by the centrality and degree of the entry node (figures 9 and 10). The dispersion tree corresponding to the epidemic, is greatly influenced by the degree of the point of entry of Spread of the epidemic starting at the city of Salvador, a city with relatively small degree (that is, small number of neigh-bors) Figure 8 Spread of the epidemic starting at the city of Salvador, a city with relatively small degree (that is, small number of neighbors). The number next to the boxes indicated the day when each city developed its first indigenous case.
Effect of degree(a) and betweeness(b) of entry node to the speed of the epidemic Figure 9 Effect of degree(a) and betweeness(b) of entry node to the speed of the epidemic.
Effect of betweeness of entry node on the speed of the epi-demic Figure 10 Effect of betweeness of entry node on the speed of the epidemic.
the disease in the network. Figure 8 shows the tree for the dispersion from the city of Salvador.
Vaccination strategies must take into consideration network topology. Figures 6 and 7 show cost benefit plots for three vaccination strategies investigated: Uniform vaccination, top-3 degree sites only and top-10 degree sites only. Vaccination of higher order sites offer cost/benefit advantages only in scenarios where the disease enter the network through one of these sites.
Epigrass facilitates greatly the simulation and analysis of complex network models. The output of model results in standard GIS file formats facilitates the post-processing and analysis of results by means of sophisticated GIS software. The non-trivial task of specifying the network over which the model will be run, is left to the user. But epigrass allows this structure to be provided as a simple list of sites and edges on text files, which can easily be contructed by the user using a spreadsheet, with no need for special software tools.
Besides invasion, network epidemiological models can also be used to understand patterns of geographical spread of endemic diseases [14] [15] [16] [17] . Many infectious diseases can only be maintained in a endemic state in cities with population size above a threshold, or under appropriate environmental conditions(climate, availability of a reservoir, vectors, etc). The variables and the magnitudes associated with endemicity threshold depends on the natural history of the disease [18] . Theses magnitudes may vary from place to place as it depends on the contact structure of the individuals. Predicting which cities are sources for the endemicity and understanding the path of recurrent traveling waves may help us to design optimal surveillance and control strategies. Autoimmune Cholangitis in the SJL/J Mouse is Antigen Non-specific Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by intrahepatic bile duct destruction and the production of anti-mitochondrial antibodies (AMA). The absence of an animal model has been a striking impedance in defining the molecular basis of disease. Previous work has suggested that SJL/J mice immunize with the pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen of PBC, leads to the development of lymphoid cell infiltration in portal tracts and a model system coined autoimmune cholangitis. We hypothesized that this pathology would be augmented if immunization occurred in the presence of IFN-γ injections. Accordingly, SJL/J mice were immunized with PDC-E2 and, for purpose of control, α-casein. Subgroups of mice were also treated with exogenous IFN-γ. As expected, mice immunized with PDC-E2, with or without IFN-γ, developed high titer AMAs. In contrast, mice immunized with α-casein, develop antinuclear antibodies. More importantly, the livers from mice immunized with PDC-E2 and/or those immunized with α-casein all displayed lymphoid cell infiltration to the portal tracts, irrespective of bile duct size. Indeed, there was no significant difference between the experimental and the control groups by histologic analysis. Thus, autoimmune cholangitis in these mice is antigen non-specific. Primary biliary cirrhosis (PBC) is a chronic inflammatory, organ specific autoimmune disease of the liver characterized by high titer anti-mitochondrial antibodies (AMA) and intrahepatic bile duct destruction (Nakanuma and Ohta, 1979; Scheuer, 1994; Kaplan, 1996) . The etiology and pathogenesis of PBC are unknown. High titer AMA react with a series of highly conserved intramitochondrial proteins including the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen of PBC. Whether liver injury is caused by AMA (Malmborg et al., 1998; Feehally and Allen, 1999) , PDC-E2 specific T cells (Van de Water et al., 1997; Shimoda et al., 1998; Inada et al., 2000) , NK cells (Tsuneyama et al., 1998; Iwata et al., 2000) or pathological alterations of the bile ducts themselves (Yasoshima et al., 1995; Kimura et al., 1996; Leon et al., 1997) are all being investigated. However, it is important to bear in mind that PBC is an autoimmune disease that most likely develops as a multi-hit disease, not just one insult. It has been theorized that PBC, in a genetically susceptible host, may be initiated by a molecular mimic (Coppel and Gershwin, 1995; Shimoda et al., 2000) or a xenobiotic (Uibo and Salupere, 1999; Long et al., 2001) . However, to this end, no definitive causative agent has, as yet, been identified.
The first reported animal model for PBC was observed in an inbred rabbit strain with spontaneous nonsuppurative cholangitis (Tison et al., 1982) . However, the specificity and repertoire of AMA in these rabbits failed to mimic human disease. More recently, PBC epithelial cells were injected intravenously into neonatally thymectomized A/J mice on the premise that altered epithelial cells may initiate disease (Kobashi et al., 1994; Masanaga et al., 1998) . Also, injection of C57BL/6 mice with purified PDH in LPS or injection of recombinant polypeptides of PDC-E2 (Ide et al., 1996; Kimura et al., 1996) have been attempted. However, although AMAs were induced in all of these proposed models, pathological changes in the liver similar to human PBC were not induced. In contrast, a GVHD model for PBC has also been developed which induces histological pathology but serological responses are not specific for mitochondrial antigens (Tanaka et al., 1999; Zeniya, 2000) . As a whole, all animal models developed to date fail to accurately mimic human PBC by the induction of both AMA and intrahepatic liver damage. With these observations in mind, Jones et al. (2000) reported the appearance of autoimmune cholangitis in SJL/J mice following the immunization of bovine PDC-E2, emulsified in CFA. The SJL/J mouse was chosen because it has been successfully used as models of other autoimmune disease, including experimental allergic encephalomyelitis, systemic lupus erythematosus and mercury induced immunopathology (Vidal et al., 1994; Abedi-Valugerdi and Moller, 2000; Winer et al., 2001) . Further, T cells isolated from affected mice displayed a mixed Th1/Th2 response when stimulated with autoantigen in vitro (Jones et al., 1999) . IFN-g is a Th1 cytokine involved in the generation of antigen-specific T cells and has already been shown to be involved in induction of experimental myasthenia gravis (Wang et al., 2000) . Therefore, we hypothesized that injection of exogenous IFN-g would enhance the development of a PBC-like disease in SJL/J mice immunized with bovine PDC-E2. However, we report herein that while SJL/J mice do demonstrate evidence of significant cholangitis, that such cholangitis is antigen non-specific regardless of IFN-g injection and thus not reflective of PBC.
A mitochondrial fraction isolated from bovine heart tissue was prepared as described previously (Fregeau et al., 1990) ; native PDC-E2 was thence purified (Stanley and Perham, 1980) . As a negative control, a-casein, was purchased from Sigma Inc. (St Louis, MO). A lipoated, huPDC-E2 peptide (p163) spanning amino acids 163-176 (GDLLAEIETDKATI), previously shown to be a major T cell epitope (6 -8), was synthesized by Alpha Diagnostic International (San Antonio, TX). Peptide purity was confirmed by reversed phase HPLC.
Female SJL/J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and all procedures conducted according to the principles outlined in the Guidelines of the Committee on Care and Use of Laboratory Animals. In the first set of experiments, mice were immunized at 12 weeks of age with a single 200 ml intraperitoneal injection of 500 mg of either bovine PDC-E2 or a-casein emulsified in complete Freund's adjuvant as described (Jones et al., 2000) ; the complete Freund's adjuvant (CFA) used contained 10 mg/ml Mycobacterium tuberculosis strain H37Ra (Difco) in incomplete Freund's adjuvant purchased from Sigma Inc. (St Louis, MO). In subgroups of mice, 3000 units of murine recombinant IFN-g was administered intraperitoneally twice a week for four weeks (Kyuwa et al., 1998) . The mice were divided into the following groups: (A) PDC-E2; (B) PDC-E2 and IFNg; (C) a-casein; and (D) a-casein and IFN-g in groups of 8 -12 animals (Table I) . Mice were serially observed and by 30 weeks of age, mice were sacrificed. This time period was chosen based on earlier observations by Jones et al. (2000) . Sera were assayed for autoantibodies. In addition, the liver, salivary gland, thymus, lung, heart, kidney, spleen, stomach, small and large intestine, were all removed for evaluation. Tissue specimens were fixed with 10% neutral buffered formalin and embedded in paraffin. Deparaffinized thin sections from each paraffin block were stained with hematoxylin and eosin for histology. Because some pilot data suggested the possibility of amyloid deposition, a Direct Fast Scarlet stain was performed and tissues viewed using polarized microscopy.
In the second set of experiments, two groups of five mice were immunized at 8 weeks of age with a single 200 ul intraperitoneal injection of the following: (E) 50 ug of p163 in incomplete Freund's adjuvant (IFA); or (F) IFA alone. Mice were serially observed and at 36 weeks of age, they were sacrificed. Sera were assayed for autoantibodies. The livers were removed for evaluation as described above.
Antimitochondrial antibodies were assayed using a combination of ELISA, immunoblotting and immunohistochemical staining of HEp-2 cells. For ELISA, microtiter plates were coated with 10 mg/ml PDC-E2 suspended in carbonate buffer (pH 9.6) and incubated overnight at 48C. After washing three times with 0.05% Tween-20 in PBS (PBS/Tween), plates were blocked with 3% milk in PBS for 1 h at room temperature (RT). Diluted mouse sera (1:200) was distributed into individual wells and incubated for 1 h at RT. After washing, the plates were incubated with 0.1 ml of HRP-conjugated goat anti-mouse immunoglobulin (Zymed, South San Francisco, CA) for 1 h. Reactivity was visualized by addition of 40 mM 2,2 0azinobis (3-ethylbenzthiazoline-6-sulfonic acid) dissolved in 0.05 M citric acid buffer (pH 5) containing 0.5 M H 2 O 2 .
The reaction was stopped after 15 min by the addition of 5% SDS. Reactivity was measured using a Wallac spectrophotometer at a wavelength of 405 nm. Known positive and negative sera were included with each assay. For immunoblotting, samples were suspended in 250 ml of sample buffer (125 M Tris -HCl (pH6.8) containing 4% SDS, 20% glycerol and 5% 2-ME), boiled for 5 min, and resolved by SDS-PAGE using 1.5 mm-thick slab gels with a 4.75% stacking gel and a 10% separating gel. Separated proteins were transferred electrophoretically to nitrocellulose filters purchased from Micron Separations (Westboro, MA). After transfer, nitrocellulose filters were cut into strips, blocked with 3% milk in PBS for 1 h at RT and probed by incubation for 1 h with mouse sera diluted at 1:200. After washing with PBS/Tween, strips were incubated for an additional hour with HRP-labeled goat anti-mouse or anti-human polyvalent immunoglobulin (1: 2000). Strips were washed and visualized with enhanced chemiluminescent substrate purchased from Pierce (Rockford, IL). Known AMA positive PBC sera and control negative sera were used throughout these studies as controls. Immunohistochemical staining of Hep-2 cells were performed using Hep-2 cell slides purchased from Antibodies Inc. (Davis, CA). Slides were incubated at RT for 1 h with SJL/J mice sera, human PBC sera or control sera diluted at 1:40. After incubation, slides were washed in PBS for 10 min. The reaction was followed by a 30 min incubation with FITC-conjugated goat anti-mouse or anti-human IgG diluted in PBS. Once again, known positive and negative sera were used throughout.
One defining serologic characteristic of PBC is the development of high titer AMA specific for the E2 subunit of PDC-E2 . Mice were bled and screened for the development of AMA. All sera collected at 7 weeks from SJL/J mice immunized with PDC-E2 (group A) showed positive reactivity against PDC-E2 by immunoblot (Fig. 1, lanes 1 and 2) . In contrast, sera isolated at 7 weeks from mice immunized with a-casein (group C) failed to react with PDC-E2 (Fig. 1, lane 3) . All responses from mice immunized with PDC-E2 (group A) were similar to responses of mice immunized with PDC-E2 plus IFN-g (group B), including reactivity with the conserved inner lipoyl domain of PDC-E2 (data not shown). At subsequent time points, through sacrifice (30 weeks), sera reactivity was very much similar to that determined at 7 weeks post-immunization ( Fig. 1, lanes 4 and 5). In contrast, sera isolated at 30 weeks from mice immunized with a-casein (group C) failed to react with PDC-E2 (Fig. 1, lane 6) . Results performed by immunoblot were similar to those obtained by ELISA. Further, sera from mice primed with PDC-E2 (Groups A and B) displayed a typical speckled (punctate) antimitochondrial staining pattern (Fig. 2a) . However, a single mouse primed with PDC-E2 and who received IFN-g (group B) demonstrated an anti-nuclear antibody pattern at a 1:40 dilution. Sera isolated from mice immunized with a-casein (groups C and D) also displayed weak antinuclear antibody staining patterns (Fig. 2b) .
At 7 and 30 weeks post-immunization, chronic portal inflammation and granulomas were observed in all mice, irrespective of the antigen used (Table I, Fig. 3a and b) . Mild bile duct injuries were prevalent as demonstrated by the irregular arrangement of the biliary epithelium (Fig. 3c) . Moreover, significant lymphoid cell infiltration into the biliary epithelium and isolated apoptotic biliary FIGURE 1 Immunization of SJL/J mice with PDC-E2 induces AMA. Sera isolated from SJL/J mice immunized with PDC-E2 as well as PBC patient sera recognized PDC-E2 (74 kD) and OGDC (48 kD). Lanes 1 and 2 represent sera isolated at 7 weeks from mice immunized with PDC-E2. Lane 3 represents sera isolated at 7 weeks from a mouse immunized with a-casein. Lanes 4 and 5 represent sera isolated at 30 weeks from mice immunized with PDC-E2. Lane 6 represents sera isolated at 30 weeks from mice immunized with a-casein. Lane 7 is serum from a PBC patient. epithelial cells were observed in all groups. Of note, this pathology was observed in both small and large bile ducts (data not shown). Loss of bile ducts and destruction of the portal tracts leading to cirrhosis failed to develop in any mice.
Interestingly, focally globular amyloid depositions developed along the walls of hepatic vessels in mice immunized with bovine PDC-E2 regardless of IFN-g treatment (Fig. 3d) . These amyloid depositions were characterized as AA type as the amyloid was sensitive to KMnO 4 treatment. In contrast, amyloid deposition was not detected in mice injected with the a-casein and there was no significant difference in histology between a-casein groups treated with or without IFN-g administration (data not shown).
Although PDC-E2 is a ubiquitous antigen found in every cell in the body, pathology is localized to small bile ducts and salivary glands. Thus, it was important to confirm the specificity of the immune response induced by injection of bovine PDC-E2 by not only analyzing liver tissue, but also selected control tissues. It was interesting that there was lymphoid cell infiltration associated with mild duct injuries in the salivary glands of mice in all groups (Fig. 3e) . Moreover, after 30 weeks, lymph node swelling was evident in all mice. However, in contrast to the liver and salivary glands, there were no indications of significant inflammatory responses in other organs. Thus, immune responses to bovine PDC-E2 and a-casein with or without IFN-g were localized to the ducts of the liver and salivary glands and appeared to be due to CFA or impurities in our antigen preparations.
The initial study by Jones et al. (23) indicated that immunization of SJL/J mice with an immunogenic huPDC-E2 peptide (p163) was as effective as whole, bovine PDC in inducing hepatic histologic changes characteristic of PBC. To eliminate differences in antigen and CFA preparation as a reason for the difference between our results and those of Jones et al., studies were conducted using synthesized p163 and IFA. At 36 weeks post-immunization, we observed significant chronic portal inflammation with bile duct invasion and damage in 2 of 5 mice injected with lipoated p163 þ IFA (Table II) . No granulomas were detected in any of these livers. Low titers of anti-PDC-E2 antibodies were detected by immunoblotting against purified human PDC-E2 only in the sera of the two mice with histologic changes (data not shown). Pre-incubation of sera with p163 inhibited immunoblotting of huPDC-E2. However, portal inflammation with bile duct invasion and damage, though less severe, was also observed in 3 of 5 mice injected with IFA alone. Granulomas were observed in two of these mice. Anti-PDC-E2 antibodies were not detected in the sera of these mice. Serum alkaline phosphatase levels were elevated only in the mouse (E4) with the most severe portal tract damage (data not shown). Portal inflammation with bile duct damage occurred as frequently in SJL/J mice injected with IFA alone as compared to mice injected with p163 and IFA. 
PBC is an autoimmune disease associated with an inflammatory response localized to the small bile duct cells of the liver Ludwig, 2000) . While attempts to establish an animal model for PBC in a number of animal species, including several strains of mice, have had with little success (Krams et al., 1989; Zeniya, 2000) , Jones et al. have proposed an animal model for PBC termed "experimental autoimmune cholangitis" in which SJL/J mice are immunized with purified bovine PDC-E2 (Jones et al., 2000) . Both the use of SJL/J mice and bovine PDC-E2 may have been important factors in establishing this proposed model of PBC. Unclear susceptibility factors in SJL/J mice have made these mice useful for the establishment of models of other autoimmune diseases (Vidal et al., 1994; Abedi-Valugerdi and Moller, 2000; Winer et al., 2001) . Additionally, purified bovine PDC-E2 may be more efficient at inducing an immune response for several reasons. In our hands, native PDC-E2 isolated from bovine heart tissue induces a stronger response than E. coli-produced recombinant protein when injected into mice (unpublished data). First, it has been suggested that lipoic acid is essential for optimal antigenicity (Fregeau et al., 1990; Bassendine et al., 1998; Koike et al., 1998) . Native protein is lipoated while synthetic peptides and recombinant proteins may not be lipoated. Second, it is important to note that bovine PDC-E2 differs from murine PDC-E2 by 28% at the amino acid level as determined by a protein blast. However, the antigenic region of bovine PDC-E2, including lipoic acid, has high homology with the murine protein and thus select regions may be recognized as a foreign antigen or altered self by the murine immune system. It thus may induce a stronger immune response against the autoantigen than a recombinant self protein or peptide (Coppel and Gershwin, 1995; Mackay, 2000) .
In the study herein, we conducted a thorough serologic and histologic review to evaluate the premise that the SJL/J mouse is a suitable animal model for PBC. We also injected purified bovine PDC-E2 as the antigen to initiate the immune response in SJL/J mice. Our control group was not injected with CFA alone, as in the study by Jones et al., but with a-Casein and CFA. Mild bile duct pathology was induced by immunization with bovine PDC-E2 emulsified in CFA. However, similar pathology was also noted in the mice immunized with the control protein (a-Casein) emulsified in the same adjuvant. Antigen specific AMA were successfully induced in experimental SJL/J mice but not control mice. However, the characteristic bile duct injury typical of PBC failed to develop even after 30 weeks, although mild bile duct pathology and portal inflammation persisted. Furthermore, inflammation and diffuse bile duct pathology were observed, irrespective of bile duct size. In contrast, only small bile ducts are damaged in human PBC and affected bile ducts are distributed sparsely, not diffusely (Sasaki et al., 2000) .
To magnify the inflammatory environment in SJL/J mice, we injected exogenous IFN-g but such injections failed to alter the pathology of PBC. The addition of IFN-g did not change the specificity or intensity of histologic or serologic effects. These results are not surprising since the pathology observed herein is antigen non-specific. Hence, this system is different from the disease associated features of IFN-g when NOD mice are injected with exogenous cytokines (Xiang and Zaccone, 1999) . Alternatively, treatment of IFN-g may cause classswitching of antigen specific AMA to IgG1. It is possible that IgG1 is not pathogenic. In fact, recently our lab has demonstrated that IgA may significantly contribute to the pathology of PBC (Reynoso-Paz et al., 2000) . In this respect, we should note that mice do not have IgA receptors on their bile duct cells and, in fact, have a significantly different mucosal immune system than that of humans .
To eliminate non-specific effects caused by impurities in our antigen or adjuvant preparations, SJL/J mice were also injected with a lipoated, immunogenic huPDC-E2 peptide (aa 163 -176) emulsified in IFA or with IFA alone. Portal inflammation with bile duct invasion and damage occurred with similar frequency in both groups. There is no indication that the development of portal inflammation was specific to a loss of tolerance to PDC-E2, though the severity of inflammation was greater in mice expressing anti-PDC-E2 autoantibodies. Thus, both the development of autoantibodies and portal tract inflammation may simply reflect the severity of the inflammatory response following ip injection. SJL/J mice appear to be predisposed to the development of portal inflammation with minimal provocation.
It is of interest that systemic amyloidosis of AA type was induced in most of the SJL/J mice immunized with PDC-E2 but not in controls with a-casein. Generally, AA type amyloidosis occurs secondary to systemic chronic inflammatory changes such as rheumatoid arthritis and tuberculosis. Heretofore, AA amyloidosis in mice has been previously induced in a number of strains of mice by repeated injections of a-casein, chemicals and endotoxin (Skinner et al., 1977; Baumal, 1979; Rokita et al., 1989; Kisilevsky and Young, 1994; Sipe, 1994) , and in a transgenic mouse model carrying human IL-6 gene with mouse metallothionein-I promoter (Solomon et al., 1999) . Spontaneous amyloid deposits have also been noted in aged SJL/J mice. In fact, the injection of amyloid enhancing factor (AEF) together with a-casein in CFA induced AA amyloidosis in SJL/J mice, while a-casein alone did not induce amyloidosis (Rokita et al., 1989) . Our findings are in agreement with these findings but not those of Jones et al. (1999) .
The SJL/J mouse is an interesting model for a number of diseases and it remains possible that with further study, significant information may be forthcoming on the inflammatory environment within the liver and salivary glands. However, it appears that such responses are antigen non-specific. La Crosse virus infectivity, pathogenesis, and immunogenicity in mice and monkeys BACKGROUND: La Crosse virus (LACV), family Bunyaviridae, was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl with fatal encephalitis in La Crosse, Wisconsin. LACV is a major cause of pediatric encephalitis in North America and infects up to 300,000 persons each year of which 70–130 result in severe disease of the central nervous system (CNS). As an initial step in the establishment of useful animal models to support vaccine development, we examined LACV infectivity, pathogenesis, and immunogenicity in both weanling mice and rhesus monkeys. RESULTS: Following intraperitoneal inoculation of mice, LACV replicated in various organs before reaching the CNS where it replicates to high titer causing death from neurological disease. The peripheral site where LACV replicates to highest titer is the nasal turbinates, and, presumably, LACV can enter the CNS via the olfactory neurons from nasal olfactory epithelium. The mouse infectious dose(50 )and lethal dose(50 )was similar for LACV administered either intranasally or intraperitoneally. LACV was highly infectious for rhesus monkeys and infected 100% of the animals at 10 PFU. However, the infection was asymptomatic, and the monkeys developed a strong neutralizing antibody response. CONCLUSION: In mice, LACV likely gains access to the CNS via the blood stream or via olfactory neurons. The ability to efficiently infect mice intranasally raises the possibility that LACV might use this route to infect its natural hosts. Rhesus monkeys are susceptible to LACV infection and develop strong neutralizing antibody responses after inoculation with as little as 10 PFU. Mice and rhesus monkeys are useful animal models for LACV vaccine immunologic testing although the rhesus monkey model is not optimal. proteins in overlapping reading frames: the nucleoprotein (N) and a non-structural protein (NS S ) which suppresses type 1 interferon (IFN) in the mammal host. The M segment encodes a single polyprotein (M polyprotein) that is post-translationally processed into two glycoproteins (G N and G C ), and a non-structural protein (NS M ) [3] . G N and G C are the major proteins to which neutralizing antibodies are directed [4, 5] . The L segment encodes a single open reading frame for the RNA-dependent RNA polymerase (L) [6] .
The virus is transmitted by hardwood forest dwelling mosquitoes, Aedes triseriatus, which breed in tree holes and outdoor containers. Ae. triseriatus feed on Eastern chipmunks (Tamias striatus grinseus) and Eastern gray squirrels (Sciurus carolinensis) which serve as amplifying hosts for LACV [7] [8] [9] . Interestingly, the virus can be maintained in the mosquito population in the absence of vertebrate hosts by transovarial (vertical) transmission, thus allowing the virus to over-winter in mosquito eggs [9] . More recently, LACV has been isolated from naturally infected, non-native Aedes albopictus mosquito [10] . The infection of Ae. albopictus may represent a shift in virus ecology and increases the possibility for generation of new reassortants [11] .
LACV was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl from Minnesota who suffered meningoencephalitis and later died in La Crosse, Wisconsin [12] . In humans, the majority of infections are mild and attributed to the "flu" or "summer cold" with an estimated 300,000 infections annually, of which there are only 70-130 serious cases reported [1, 2, 13, 14] . Isolation of virus is rare and has been made from post-mortem brain tissue collected in 1960, 1978, and 1993 [15-18] . Two isolates from non-fatal LACV cases were collected in 1995, one from a brain biopsy of a child and one from cerebrospinal fluid [16, 19] .
Histopathologic changes in two human cases, one from 1960 and one from 1978, were characteristic of viral encephalitis. Inflammatory lesions consisted of infiltration of mononuclear leukocytes either diffusely or as microglial nodules. The largest inflammatory foci were observed in the cerebral cortex, including the frontal, parietal, and temporal lobes, and foci could also be found in the basal ganglion and pons. In these two cases, there was a lack of inflammatory lesions in the posterior occipital cortex, cerebral white matter, cerebellum, medulla, and spinal cord [17] . Brain biopsy from a non-fatal LACV infection contained areas of perivascular mononuclear cuffing and focal aggregates of mononuclear and microglia cells [16] . RT-PCR analysis of neural tissues from the 1978 case could only detect viral RNA in the cerebral cor-tex and not in the medulla, cerebellum, spinal cord, basal ganglion, or temporal lobe [20] .
In children and adults, severe LACV encephalitis clinically mimics herpes simplex virus encephalitis with fever, focal signs, and possible progression to coma [16, 21, 22] . Confirmatory diagnosis has been made by RT-PCR of cerebrospinal fluid to exclude herpes simplex virus and by fluorescent staining for LACV antigen in brain biopsy sections [16] . Children who recover from severe La Crosse encephalitis may have significantly lower IQ scores than expected and a high prevalence (60% of those tested) of attention-deficit-hyperactivity disorder [13] . Seizure disorders are also common in survivors [23] . Projected lifelong economic costs associated with neurologic sequelae range from $48,775 -3,090,398 per case [24] . Currently, a vaccine or specific antiviral treatment is not available, but could serve to reduce the clinical and economic impact of this common infection.
Although evidence of LACV infection has been reported for several species, only limited research has been done to understand LACV pathogenesis in its natural host or experimental rodents [8, [25] [26] [27] . LACV administered subcutaneously to suckling mice first replicates in muscle, and viremia develops with virus invading the brain across vascular endothelial cells [28] [29] [30] [31] . Virus replication in muscle was confirmed by immunohistochemical (IHC) staining, and the predominant cell type infected in the CNS is the neuron [28, 32] . The virulence of LACV for mice decreases with increasing age, similar to humans in which it causes CNS disease predominantly in pediatric populations [13, 28] . As an initial step in the establishment of animal models useful for vaccine development for humans, we sought to better characterize the tissue tropism of LACV in mice by identifying the tissues that support LACV replication after peripheral inoculation. We have previously described Swiss Webster mice as suitable for characterization of LACV infection at birth and at 3weeks of age [6] . Here we inoculated 3-week old Swiss Webster mice with either 1 or 100 LD 50 of virus intraperitoneally. Twenty tissues were individually collected for six consecutive days and processed for virus titration, immunohistochemical staining, and histopathology studies.
Since experimental infection of non-human primates with LACV has not been reported, we also sought to determine if rhesus monkeys were susceptible to LACV infection. Rhesus monkeys were chosen since they are susceptible to a variety of neurotropic arboviruses, including flaviviruses [33] . In this study, rhesus monkeys were infected intramuscularly or subcutaneously with a mosquito or human isolate of LACV. These two isolates were used since preliminary genomic sequence analysis indicate that there are host specific differences between LACV isolated from humans and mosquitos [6] . LACV was found to be highly infectious for rhesus monkeys, but infection did not result in viremia, disease, or significant changes in blood chemistries or cell counts. However, high titers of neutralizing antibodies developed in all monkeys indicating that rhesus monkeys, although not optimal, will be useful for studying the infectivity and immunogenicity of LACV vaccine candidates.
Previous evaluation of LACV in suckling mice revealed that it first replicated in striated muscle cells from which it seeded the blood and next invaded the CNS, where it replicated in neurons [28, 32] . In developing a rodent model for our live attenuated LACV vaccine development program, we sought to characterize LACV infection in outbred weanling mice, rather than suckling mice, since older mice can be used to study both the level of attenuation and the immunogenicity of our LACV vaccine candidates.
To identify key steps in pathogenesis of LACV in weanling mice, we evaluated LACV tissue tropism after peripheral inoculation of wild-type virus and sought to identify tissues in which virus replicated efficiently. Weanling Swiss Webster mice (21-23 days-old) were inoculated intraperitoneally with 1 or 100 LD 50 (2.5 or 4.5 log 10 PFU) of LACV/human/1960, and the tissues indicated in Figures 1  and 2 were collected from 3 mice per day on days 1-6 post inoculation. Following inoculation of either dose, virus could first be detected in tissue near the inoculation site such as inguinal lymph nodes, spleen, and ovaries/uterus. Virus was detected in serum on days 1-3, but rarely on subsequent days even in moribund mice. By day three, virus distribution was widespread and could be found in the majority of tissues sampled, albeit at very low levels with titers rarely exceeding those found in serum. The highest virus titers detected were present in nasal turbinates, brain, and spinal cord. Respiratory tissue, including lung and nasal turbinates, contained virus following inoculation with 100 or 1 LD 50 beginning on days 1 and 2, respectively. CNS infection appeared to follow respira-Tissue distribution of La Crosse virus following intraperitoneal (IP) inoculation of Swiss Webster mice with 10 2.5 PFU Figure 1 Tissue distribution of La Crosse virus following intraperitoneal (IP) inoculation of Swiss Webster mice with 10 2.5 PFU. a Percent of mice positive by plaque assay represented by shading: 100% black, 66% dark gray, 33% light gray, 0% no data entry. Mean virus titer calculated only for virus positive tissues. Areas left blank indicate virus titer below detection limit of 0.7 log10 PFU/tissue. tory tissue infection, with virus being present in the brain 5 days after infection with 1 LD 50 and on day 2 after infection with 100 LD 50 . At either virus dose, infection appears early in the lymph nodes and major organs, with subsequent infection of the upper respiratory tract (nasal turbinates) followed by infection of the brain and eventually the spinal cord. By day six, mice began to succumb to infection in the high dose group showing signs of paralysis whereas mice in the low dose group failed to show clinical signs at this time, but would have succumbed later in infection. These results indicate that LACV replicates to low to moderate levels in peripheral tissues in weanling mice, with the nasal turbinates rather than striated muscle being the major site of replication.
Since LACV replicated to high titers in the nasal turbinates, we sought to determine if intranasal inoculation of mice with LACV could lead to infection. Three-weekold Swiss Webster weanling mice (n = 6/dose) were inoculated intranasally (IN) (10 µl volume) or intraperitoneally (IP) (100 µl volume) with serial dilutions of LACV/ human/1960, and the LD 50 and 50% infectious dose (ID 50 ) were determined. Clinical disease served as a surrogate for lethality and mice were promptly euthanized prior to succumbing to LACV disease. In both groups, clinical disease was first noted on day 6 ( Figure 3 ). Twenty days post-inoculation, the LD 50 was determined. All surviving mice were tested for the development of a neutralizing antibody response. respectively) were slightly lower than the LD 50 titers, indicating LACV can cause a subclincal infection in weanling mice, but only at low doses.
To further characterize the LACV infection in weanling mice, an additional group was inoculated intraperitoneally with 100 LD 50 of LACV/human/1960 and selected tissues (serum, muscle, nasal turbinate, brain, and spinal cord) were collected for virus quantitation (n = 5, daily for six days) to confirm titers found in Figure 2 and for histopathological and immunohistochemical (IHC) examination (n = 3, daily for six days), and the data is summarized in Table 1 and Figures 4 and 5. Virus titers for nasal turbinates, brain, spinal cord, muscle, and serum were in agreement with findings in Figure 2 (data not shown). Histopathologic lesions in the brain (including areas of the olfactory bulb, cerebral cortex, thalamus, hippocampus, and medulla oblongata) and spinal cord included perivascular cuffing ( Figure 4A ), neuronal degeneration ( Figure 4B -C), necrosis (either single cell or small foci), and apoptosis (4F). There was an infiltration of CD3+ lymphocytes and macrophages ( Figure 4D -E).
The most extensive lesions occurred in the medulla oblongata and were associated with perivascular cuffing.
Histopathological changes were minimal outside the CNS. In the spleen, lymphoid atrophy was only observed on days 3-4 post-infection. Plasmacytosis in the spleen was observed on days 4-6 and areas of necrosis were observed on days 4-5 with myeloid hyperplasia on day 5.
Histopathological changes were not observed in respiratory nasal epithelium or muscles of the upper rear limb.
To determine the location of cells expressing viral antigen, tissues were immunostained for La Crosse virus antigens. Viral antigens were not observed in the nasal turbinates, muscle, spleen, or pancreas. However, viral antigen was detected in the olfactory bulb of the brain, thalamus, cerebrum, medulla oblongata, and spinal cord.
On day 5, viral antigen could be detected in all sampled brain regions with a greater number of cells and regions positive for viral antigen than for overt histologic lesions. Mild perivascular cuffing was seen in a few areas and single cell necrosis was seen in areas stained positive for viral antigen. At this time and at later days, the olfactory bulb was more extensively involved ( Figure 5A ), although viral antigen was not detected in sections of the underlying nasal epithelium. Viral antigen could be detected in the brain tissues of all mice on day 5, but only small foci of antigen were seen in the spinal cord of one mouse suggesting brain infection proceeds spinal cord infection.
By day 6, viral infection in the CNS was widespread and extensive throughout all regions of the brain and spinal cord ( Figure 5B ). Neurons were the main cell expressing viral antigens, but supporting glia also appeared positive ( Figures 5D-F) . Single cell necrosis, focal necrosis (more than one cell), and apoptotic bodies were prominent throughout the lesions ( Figure 4C ) and apoptotic bodies stained positive by TUNEL staining ( Figure 4F ). Degenerative neuronal changes were also commonly seen, includ- 
To develop a non-human primate model of LACV infection for pathogenesis studies and for testing of future vaccine candidates, rhesus monkeys were inoculated with 10 5 PFU of biologically cloned (terminally diluted) human or mosquito LACV (LACV/human/1978-clone, LACV/mosquito/1978-clone). Illness was not observed following virus administration, and virus was not detected at any time in serum samples ( Table 2 ). Virus was present at a low titer in lymph nodes on days 6, 8, and 12, however, virus replication in these tissues could not be identified by IHC staining. Despite the low level (or absence) of viremias and highly restricted replication in the tissues sampled, all monkeys developed neutralizing antibody responses that were first detected on days 6-8 indicating that the each monkey was infected. Twenty-eight days after inoculation, neutralizing antibody titers (PRNT 60 ) for each group were in the range of 1:560 -1:2186 (Table  2) . Low-level cross-reactive antibodies were present in two monkeys (CL6E and DBOH) at the start of the experi-ment. A boost in antibody titer in these monkeys was not observed compared to other monkeys, suggesting that this experimental LACV infection was a primary infection.
Complete blood counts (CBC) with differential and blood chemistries were analyzed at each blood collection. Since LACV infection in monkeys was asymptomatic and also since differences between the four virus groups indicated in Table 2 were not observed, the CBC and blood chemistry data were averaged for the 16 animals to detect changes in blood values during the course of infection (Table 3) . Days in which specific parameter values for a significant number of monkeys were greater than 1 standard deviation from normal appear boxed in Table 3 with mean values for each test shown. After day 6, the majority of monkeys experienced a slight anemia, which may in part be associated with the overnight fast in preparation for anesthesia prior to each blood collection. This analysis suggests that infection of major organs such as liver was minimal or absent.
To estimate the minimum dose required to infect a monkey, rhesus monkeys were inoculated with LACV/mosquito/1978-cl at 10 1 or 10 3 PFU subcutaneously. Blood was collected on days 0, 21, 28, and 42, and serum neutralizing antibody titers were determined. Mean neutralizing antibody titers were 1:355 and 1:82 for the 10 1 or 10 3 PFU dose groups, respectively, and all monkeys seroconverted by day 28 (PRNT 60 ≥ 40) (Table 4 ). Thus, LACV is highly infectious for rhesus monkeys even at a dose of 10 1 PFU and results in the induction of a high level of neutralizing antibody. However, LACV infection did not result in identifiable clinical abnormalities in this group of 24 monkeys. 
As an initial step in development of a live attenuated LACV vaccine, we sought to better characterize LACV infection in weanling mice because at this age mice can be used to evaluate both the level of attenuation and immunogenicity of candidate vaccine viruses. Previous LACV pathogenesis studies in suckling mice inoculated subcutaneously with a mosquito isolate of LACV demonstrated that viral antigen was detected in the cytoplasm of striated muscles, the interscapular brown fat, and the endothelial and smooth muscle cells of small arteries and veins [28] . When virus was first detected in the brain, it was confined to cerebral vascular endothelial cells but later spread to neurons. The authors suggest that the late infection of the dorsal route ganglion indicates that the virus does not penetrate the CNS via nerves but rather by vascular endothelial cells [28] . This previous model therefore suggests that virus first replicates in muscle cells leading to the development of viremia with subsequent hematogenous spread to the CNS, and we sought in the present study to examine if this pattern of infection also occurred in weanling mice. In weanling mice inoculated intraperitoneally with 1 or 100 LD 50 of LACV, virus was first detected on days 1 -3 in tissues near the inoculation site. At either dose, virus was no longer detectable by days 4 and 5 in these tissues, suggesting that it was rapidly cleared by the innate immune system. Interestingly, the virus was not detected in muscle tissue until day 3 post inoculation and clearly did not preferentially infect this tissue. Rather, outside the CNS, the virus replicated to highest titers in the nasal turbinates and appears to spread from this site into the brain. Immunohistochemical staining of the nasal turbinate tissue was not sensitive enough to identify the LACV infected cells, but is thought LACV may gain access to the CNS via cells in the nasal turbinates. This suggestion is offered with the caveat that respiratory epithelial cells could also have been infected, but the magnitude of the infection could not be detected with the IHC staining. To travel from the nasal olfactory epithelium to the olfactory bulb, the virus a Days with significant number of monkeys 1 SD from the pre-inoculation mean (day -7 and 0 combined) are boxed (chi square, p < 0.05, n = 16), mean values displayed. b Abbreviations: MCV-mean corpuscular volume, MCH-mean corpuscular hemoglobin, MCHC-mean corpuscular hemoglobin concentrations, ASTaspartate aminotransferase, ALT-alanine aminotransferase, ALP-alkaline phosphatase, BUN-blood urea nitrogen. c Pre-inoculation mean determined from samples collected on days -7 and 0 post inoculation.
would follow olfactory neurons into the brain, as infection is first detected in the rostral section of the brain.
Although virus replication in nasal turbinate tissue was detected, we were unable to identify the cells that were infected. Viruses such as vesicular stomatitis virus, rabies virus, mouse hepatitis virus, Borna disease virus, pseudorabies virus, and herpes simplex virus have all been demonstrated to enter the mouse CNS via olfactory neurons [34] [35] [36] [37] [38] [39] [40] . It is important to note that 2 of 62 mice tested had detectable virus within the brain without detectable virus in the nasal cavity (individual data not shown) suggesting that more than one route might be used to gain access to the CNS. We were surprised by the ability of the virus to infect intranasally, and found that the LD 50 and ID 50 were almost identical by either route by of inoculation. The kinetics of the development of clinical disease that occurred following intranasal administration of virus was similar for virus given IP or IN.
The finding that LACV is able to infect very efficiently via the nasal route has possible implications for the ecology of the virus. It is possible that infectious virus is present in water collections containing Aedes mosquito larvae, e.g., tree holes, since the virus has been shown to be transmitted from infected mosquitoes to larvae via eggs. A mammalian host that drinks the water would intake both fluid, which might contain free virus from lysed larvae, and infected larvae, either of which could initiate an infection in the mammalian host. Thus, exposure to such water could lead to an alternate route of infection for the natural hosts, i.e., the oral/nasal route in addition to the vectorbourne route. This hypothesis needs to be confirmed experimentally but remains an interesting possibility.
In the CNS of the weanling mouse, LACV infects predominantly neurons (some microglial cells are also infected) with spread in a rostral to caudal direction eventually reaching the lumbar spinal region. In both mice and humans, virus has been detected in the cerebral cortex, however infection appears more widespread in the mouse CNS with virus detection in the medulla oblongata, cerebellum, thalamus, olfactory bulb, and all regions of the spinal cord. The virus used in this study, LACV/human/ 1960, was isolated and passaged twice in C6/36 mosquito cells and was not previously adapted to growth in mouse neural tissue. One surprising difference between human and mouse infection was the detection of virus replication by IHC and the development of lesions in the mouse spinal cord [17] .
Taken together, these data suggest that in weanling mice the virus first replicates in the periphery near the inoculation site. If the infection is not quickly controlled, the virus disseminates, most likely via blood, to the nasal turbinates. The detection of virus and lesions first in the rostral brain suggest the virus may utilize olfactory neurons to gain access to the CNS. The differences in findings between our study and previous work may be the result of differences in virus strain (mosquito vs. neurovirulent human isolates), mouse strain (outbred white vs. Swiss Webster), mouse age (suckling vs. weanling), inoculation route (subcutaneous vs. intraperitoneal) and dose (1000 TCID 50 vs. 1 or 100 LD 50 ). Although sequence data is not available for the strain used in the previous mouse pathogenesis work, it is known that the virus was a mosquito isolate and not directly linked to human disease [28] .
In humans the majority of infections are asymptomatic, but children hospitalized with severe disease present with fever (86%), headache (83%), vomiting (70%), disorien- [13] . Like the majority of human infections, rhesus monkeys in the current study experienced a subclinical infection without the development of systemic disease or neurologic symptoms. A much greater number of monkeys would probably need to be tested to detect neurologic symptoms after peripheral inoculation. Nevertheless, all monkeys were infected with LACV and developed neutralizing antibody responses, even after inoculation with as little as 10 PFU. Future work will include the intracerebral inoculation of rhesus monkeys to determine if LACV is neurovirulent in this species, but this will wait until vaccine candidates have been identified.
It is still unclear why so many human LACV infections remain asymptomatic. In our mouse model, infection with 1 LD 50 of virus resulted in delayed CNS infection compared to mice receiving 100 LD 50 . Mice were able to control virus infection at doses at or below the LD 50, and developed strong neutralizing antibody responses. The LACV ID 50 for humans is unknown, but if human exposure is limited to a small dose, the virus may be effectively controlled by the immune system and CNS infection may be averted. If, like our mouse model, an individual is exposed to a greater dose of virus, virus growth may outpace control mechanisms leading to CNS infection. To further support the role of immune control affecting human disease outcome, it has been shown that individuals residing in endemic areas with major histocompatibility complex molecule B49 on CD8+ cytotoxic T lymphocytes (HLA-B49) had a greater relative risk of developing encephalitis after infection (relative risk 17.65, χ 2 = 7.3, P < 0.1). Infected individuals with HLA-DR5 had a lower relative risk of developing seizures (relative risk 0.22, χ 2 = 5.10, P < 0.025) [41] .
In weanling Swiss Webster mice, the LD 50 and ID 50 are similar, indicating that most infections lead to a lethal outcome at this age. LACV first replicates in tissues near the inoculation site, enters the blood stream, infects the nasal turbinates, and gains access to the CNS, presumably via olfactory neurons. This model will be useful to identify attenuated vaccine candidates that are deficient in the ability to disseminate from the site of inoculation, to replicate to high titers in the nasal turbinates, or to establish infection in mouse CNS. The CNS infection of mice appears more widespread than described for human infection, both in the brain and spinal cord. LACV is highly infectious both by the IP and IN routes suggesting that infection of natural mammalian hosts such as the chipmunk or squirrel might occur by the oral/intranasal route in addition to the bite of an infected mosquito. In rhesus monkeys, LACV is highly infectious with as little as 10 PFU resulting in the development of neutralizing antibodies, but clinical disease is not observed at any dose tested, suggesting that rhesus monkeys will be useful for studying the infectivity and immunogenicity of live attenuated virus vaccine candidates, but will be of limited usefulness for the evaluation of their level of attenuation.
C6/36 cells (Aedes albopictus mosquito larvae) were maintained in Earle's MEM supplemented with 10% fetal bovine serum (HyClone), 2 mM L-glutamine (Invitrogen), and 1 mM non-essential amino acids. Vero cells (African green monkey kidney) were maintained in Opti-PRO™SFM medium (Invitrogen) supplemented with 4 mM L-glutamine (Invitrogen).
LACV/human/1960 was isolated from post-mortem brain tissue collected from a Minnesota patient hospitalized in La Crosse, Wisconsin and passaged two times in C6/36 cells. LACV/mosquito/1978 was isolated from mosquitoes collected in North Carolina and passaged once in mouse brain and three times in Vero cells. LACV/human/ 1978 was isolated from post-mortem brain tissue collected in Wisconsin and passaged once in mouse brain, twice in BHK-21 cells, and once in Vero cells. Biological clones were generated by terminal dilution in Vero cell cultures. Passage histories and complete genomic sequences for all stocks used in this paper have been previously published (EF485030-EF48538) [6] .
Weanling Swiss Webster mice (Taconic Farms, Germantown, NY) were inoculated with LACV/human/1960 diluted in L15 media (Invitrogen) intraperitoneally (100 µl volume) or intranasally (10 µl volume) while under isofluorane anesthesia. Mice were observed daily for 20 days for clinical disease including tremors, seizures, and limb paralysis. Moribund mice were promptly euthanized. Serum was collected 20 days after inoculation for determination of the presence and titer of neutralizing antibodies.
The replication of LACV virus was evaluated in 3-week-old weanling female Swiss Webster mice (Taconic Farms, Germantown, NY). All animal experiments were carried out in accordance with the regulations and guidelines of the National Institutes of Health. The Swiss Webster mice, were inoculated IP with 1 or 100 LD 50 in a volume of 100 µl [6] . The tissues indicated in Table 1 were collected individually (3 mice per day for 6 days at each dose of virus), weighed, homogenized in L15 with SPG buffer (final concentration 218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM dibasic potassium phosphate, pH 7.2). Homogenates were centrifuged for 10 minutes at 3000 rpm to remove cell debris and aliquots were frozen at -80°C until virus quantitation was performed. All mice were carefully observed twice daily for clinical disease including tremors and limb paralysis. Mice exhibiting clinical disease were promptly euthanized.
Monolayer cultures of Vero cells grown on 24-well plates were infected in duplicate with ten-fold serial dilutions of tissue homogenate, and the cells were overlayed with OptiMEM (Invitrogen) supplemented with 1% methylcellulose, 5% FBS, 2.5 µg/ml amphotericin B, and 20 µg/ml ciprofloxicin. Five days after infection the overlay was removed and cells were washed twice with PBS. The cells were fixed and stained for 10 minutes with crystal violet solution, virus plaques were enumerated, and tissue titers were expressed as mean log 10 PFU/g tissue.
Weanling Swiss Webster mice were inoculated intraperitoneally with 100 LD 50 of LACV/human/1960, and tissues were collected for six consecutive days for virus titration (n = 5) and pathology (n = 3) per day. Virus titrations were performed to confirm previous virus kinetics. Tissues collected for pathology were fixed in 10% neutral buffered formalin (NBF) for a minimum of 72 hours, embedded in paraffin and sections were prepared at 4-5 (µm). When bone was present in the tissue, as with muscle, nasal cavity, and spinal cord, tissues were decalcified using Immunocal (Decal Chemical Corp. Tallman, NY). Sections were stained with hematoxylin and eosin (H&E). A serial section was saved for immunohistochemical staining (see below).
For immunohistochemical analysis, fixed serial sections of mouse tissues were mounted onto slides and deparaffinized with xylene, rehydrated in a series of ethanol solutions (100%, 95%, 70%, 50%), and washed with deionized water. TUNEL Staining to detect apoptosis was preformed using the "DeadEnd™ Colorimetric TUNEL System" (Promega USA, Madison, WI). Sections were pretreated with Pro K enzyme (provided in the kit, diluted 1:500 with PBS). Strepavidin (also provided in the kit) was diluted at 1:500 in PBS.
To detect CD3+cells, slides were steam hydrated and pretreated with Diva Solution (Biocare Medical Concord, CA) for 20 minutes. The primary antibody, rabbit antihuman CD3, (A-0452, Dako Corporation, Carpentaria, CA) was used at a dilution of 1:300. The detection system was the Standard ABC kit (Vector Laboratories, Burlingame, CA), with 3,3'-diaminobenzidine (DAB) as the chromogen and a modified Harris hematoxylin counterstain.
To detect macrophage antigen 2 (MAC-2) expressing cells, slides were stream hydrated with citrate buffer for 20 minutes. The primary antibody, rat anti-Mac-2 (TIB-166, ATCC, Mannasas, VA) was used at a 1:200 dilution followed by a biotinylated goat-anti-rat IgG secondary antibody and developed with Streptavidin HRP (Biocare, Concord, CA).
Rhesus monkeys were inoculated with 10 5 PFU of biologically cloned human or mosquito LACV (LACV/human/ 1978-clone, LACV/mosquito/1978-clone). Each virus was inoculated intramuscularly (n = 4) or subcutaneously (n = 4). Blood samples were collected on days -7, 0, 2, 4, 6, 8, 10, 12, 14, 21, 28 post inoculation for blood chemistries, virus titration, and neutralizing antibody titration. Axillary or inguinal lymph nodes were surgically excised on days 4, 6, 8, and 12 post inoculation for viral titer or fixed in buffered formalin for histopathology and immunohistochemical analysis. Monkeys were observed daily for clinical disease. To determine LACV infectivity at low doses, rhesus monkeys (n = 4) were inoculated with 10 1 or 10 3 PFU LACV/mosquito/1978-clone subcutaneously. Blood samples were collected on days 0, 21, 28, and 42 post inoculation for neutralizing antibody titration.
Neutralizing antibody in mouse and monkey serum was quantitated by a plaque reduction neutralization assay. Test sera were heat inactivated (56°C for 30 minutes) and serial 2-fold dilutions beginning at 1:5 or 1:10 were prepared in OptiMEM (Invitrogen) supplemented with 2% FBS, 50 µg/ml gentamicin, and 0.5% human albumin (Talecris Biotherapeutics, Inc., Research Triangle Park, NC). The homologous LACV was diluted to a final titer of 500 PFU/ml in the same diluent and 10% guinea pig complement (Cambrex Bioscience Walkersville, Inc., Walkersville, MD) was added to equal volumes of the serum dilutions and mixed well. Serum/virus mixture was incubated at 37°C for 30 minutes, added to confluent monolayers of Vero cells, and incubated for 1 hour to allow virus attachment. Cells were overlayed with 1% methylcellulose and incubated for 5 days at 37°C. After incubation, the overlay was removed, and the monolayers were washed twice with PBS and stained with crystal violet to allow for the enumeration of virus plaques. A 60% plaque-reduction neutralization titer was calculated. The intrinsically disordered C‐terminal domain of the measles virus nucleoprotein interacts with the C‐terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, N(TAIL) (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in N(TAIL), an α‐helical molecular recognition element (α‐MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small‐angle X‐ray scattering, we have derived a low‐resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P‐induced folding within the α‐MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N‐terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517–525) involved in binding to XD but not in the unstructured‐to‐structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure. Nucleoproteins of Paramyxoviridae are divided into two regions: a structured N-terminal moiety, N CORE (aa 1-400 in MV), which contains all the regions necessary for self-assembly and RNA-binding Curran et al. 1993; Bankamp et al. 1996; Liston et al. 1997; Myers et al. 1997b Myers et al. , 1999 Karlin et al. 2002a; Kingston et al. 2004b) , and a C-terminal domain, N TAIL . N TAIL is intrinsically unstructured (i.e., it lacks any stable secondary and tertiary structure in physiological conditions) ) and is exposed at the surface of the viral nucleocapsid (Heggeness et al. 1980 (Heggeness et al. , 1981 . The presence of a flexible region protruding from the viral nucleocapsid allows the establishment of interactions with numerous different viral partners and with several cellular proteins (Moyer et al. 1990 ; De and Banerjee 1999; tenOever et al. 2002; Zhang et al. 2002; Laine et al. 2003) . In Morbilliviruses and Respiroviruses, N TAIL is responsible for binding to P (Curran et al. 1993; Harty and Palese 1995; Bankamp et al. 1996; Liston et al. 1997; Longhi et al. 2003; Kingston et al. 2004b) , to the polymerase complex P-L, and to the matrix protein (Coronel et al. 2001) . Beyond viral partners, N TAIL also interacts with several cellular proteins, including the interferon regulatory factor 3 (tenOever et al. 2002) and the heat-shock protein Hsp72 (Zhang et al. 2002) . Moreover, N TAIL within viral nucleocapsids released from infected cells also binds to a yet unidentified protein receptor expressed at the surface of human thymic epithelial cells (Laine et al. 2003 (Laine et al. , 2005 .
The P protein of Paramyxovirinae plays multiple roles in both transcription and replication: It is an essential subunit of the viral polymerase complex and acts as a bridge between the nucleocapsid template (N NUC ) and the polymerase complex L-P. P is a modular protein, consisting of an intrinsically unstructured N-terminal moiety (PNT) (Karlin et al. 2002b , and of a well conserved C-terminal moiety (PCT) which contains all the regions required for transcription (Curran 1996) . Paramyxovirinae PCT have a modular organization, consisting of alternating disordered and structured regions . In particular, they possess a coiled-coil domain (referred to as PMD, for P multimerization domain) responsible for both oligomerization and binding to L (Smallwood et al. 1994; Liston et al. 1995) , and a C-terminal globular region (referred to as XD), that is involved in binding to both monomeric and assembled forms of N (Curran et al. 1994 (Curran et al. , 1995a Harty and Palese 1995; Tuckis et al. 2002; Johansson et al. 2003; Kingston et al. 2004b) . Figure 1 shows a schematic representation of the MV N NUC -P complex, which highlights the role of N TAIL in the recruitment of P via the binding to XD.
We have previously reported the crystal structure of the C-terminal globular domain of MV P (XD, aa 459-507): It consists of a monomeric protein composed of three a-helices, forming an anti-parallel three-helix bundle . We have also shown that N TAIL undergoes an induced folding in the presence of XD and that this unstructured-to-structured transition implies a gain of a-helicity . Using a combination of computational and biochemical approaches, we have identified within N TAIL an a-helical molecular recognition element (a-MoRE, aa 488-499) involved in binding to P and in the induced folding of N TAIL (Bourhis et al. 2004) . The a-MoRE has been modeled in the crystal structure of XD, in the long hydrophobic cleft delimited by helices a2 and a3 ). In this model, the contact of N TAIL with a hydrophobic patch at the surface of XD would be the driving force in the induced folding of the a-MoRE of N TAIL by burying apolar residues at the protein-protein interface. Very recently, Kingston et al. (2004a) have reported the crystal structure of a chimeric protein composed of MV XD and a peptide corresponding to residues 486-505 of MV N. The structure of the complex is a four-helix bundle in which the ahelix of N is bound in the reverse orientation with respect to the model proposed by Johansson et al. (2003) . Using NMR and crystallographic studies, Kingston et al. (2004a) deduced that the induced folding of N TAIL is restricted to only 18 residues (out of 125), although the analysis was restricted to the N region encompassing residues 477-505. (B) . The encapsidated RNA is shown as a dotted line. PMD is represented with a dumbbell shape according to Tarbouriech et al. (2000) . The tetrameric P (Rahaman et al. 2004 ) is shown bound to N NUC through three of its four C-terminal XD ''arms,'' as in the model of Curran and Kolakofsky (1999) . The L protein is shown as a rectangle contacting P through PMD by analogy with SeV (Smallwood et al. 1994) .
In this paper we examine this localized induced folding event in the context of the entire N TAIL domain. A low-resolution structural model of the complex between XD and N TAIL shows the presence of a bulky globular region and of an extended and elongated shape. The model shows that the N-terminal region of N TAIL (residues 401-488) remains predominantly unfolded, and the 489-525 region is packed against XD, thus suggesting that beyond the a-MoRE the C terminus may play a role in the interaction with XD. Indeed, we present several lines of experimental evidence confirming that beyond the region encompassing the a-MoRE, an additional N TAIL region encompassing residues 517-525 also contributes to binding to XD, although without undergoing any gain of regular secondary structure.
Small angle X-ray scattering (SAXS) is a valuable technique for the study of flexible, low compactness macromolecules in solution, which has already been successfully used to characterize N TAIL ). Therefore, we used SAXS to study the N TAIL -XD complex. To this endeavor, we cloned, expressed, and purified from the soluble fraction of Escherichia coli an N-terminally histidine-tagged form of N TAIL (i.e., N TAILHN ) (data not shown). The identity of the recombinant products was confirmed by immunoprecipitation (IP) studies using anti-N Cl25 and anti-hexahistindine tag monoclonal antibodies (mAbs) (data not shown).
Beforehand, we checked whether XD possesses the same structure in solution as in the crystal. SAXS experiments performed on XD showed that it has a globular shape with a radius of gyration (R g ), extrapolated at a nil concentration, of 12.1 6 0.8 Å and a maximum diameter D max of 41 6 1 Å (data not shown). Comparison with the crystal structure using the program CRYSOL (Svergun et al. 1995) indicates that the scattering profile calculated for the crystal structure (PDB code 1OKS) is identical to the experimental one, fitting the data with a 2 of 1.4 and giving a theoretical R g of 12.3 Å (data not shown). These results suggest that the overall conformation of XD in solution is similar to that observed in the crystal.
The scattering profile of the N TAILHN -XD complex was obtained as described in Materials and Methods. Analysis of the curve in the low q region with the Guinier approximation gave an R g of 32.7 6 0.7 Å . The molecular mass (MM) calculated from the forward scattering intensity I(0) is 23.5 6 2 kDa, in agreement with the value expected for a 1:1 stoichiometric complex (21.3 kDa), thus suggesting that the complex did form in solution. The high value of R g indicated that the overall structure of the N TAILHN -XD complex is not compact. The distances distribution function inferred from the scattering curve of the N TAILHN -XD complex exhibits a maximum at 20 Å , with a shoulder at about 30 Å and a long tail up to 146 Å , typical of an elongated object (see Fig. 2A ). The bump most probably corresponds to the intramolecular distances within the globular portion of the complex (see below), while the tail indicates that N TAIL possesses regions with an extended conformation.
The overall envelope of the complex was restored ab initio from its scattering profile using the program GASBOR ). Several independent runs yielded different shapes with recurrent features: they were all elongated, with a globular cluster of always the same size at one extremity and an elongated protuberance with varying bends and cross-sections. The quality of the fit to the experimental data was similar in all cases, with a 2 of 1.3 to 1.6. The globular part most probably corresponds to XD packed against the folded region of N TAIL , while the outgrowth corresponds to unfolded regions of the latter. The crystal structure of the XD-N TAIL486-505 complex (PDB code 1T6O), which encompasses the a-MoRE (residues 488-499), was inserted in the shape using the program SUPCOMB (Kozin and Svergun 2001) (Fig. 2B ). Interestingly, only one protuberance was observed, corresponding to the N-terminal area of N TAIL , and no other protuberance was restored from the shape determination in the opposite region of the globular cluster, thus suggesting that the C-terminal region of N TAIL (aa 506-525), although not visible per se, is packed inside the bulky portion of the complex (Fig. 2B ). In order to get further insights into the conformation that the regions of N TAIL encompassing residues 401-485 and 506-525 adopt in the complex with XD, we used the program package CREDO, which is an extension of GASBOR and calculates the position of missing loops in crystal structures. The results obtained with several independent runs using the crystal structure of XD-N TAIL486-505 as the template, revealed at one side a peptide chain in an extended conformation and at the other side a chain packed against the bulky portion of the XD-N TAIL486-505 complex. The extended conformation, which protrudes from the globular cluster of the complex and points to the solvent, corresponds to the 92-residue-long N-terminal region of the N TAILHN construct. In contrast, the more compact peptide chain, corresponding to the 20-residue-long C-terminal region of N TAIL (aa 506-525), always packs against the XD-N TAIL486-505 complex but at varying distances and positions. An example of one of the calculated conformations is shown in Figure 2C . The experimental data were fitted by these models with similar 2 of about 0.8-1.0 (see Fig. 2C , inset). The various possible conformations provided by the program CREDO together with the extended structural properties of the complex (as indicated by the distance distribution function, the R g and the restored shape, with respect to the number of amino acids of the complex) are typical of disordered polypeptide chains lacking a stable regular structure. Accordingly, the structure shown in Figure 2C represents only one possible conformation found by CREDO among many others. Indeed, CREDO restores only one conformation per run, and therefore cannot account for multiple conformations due to disorder when fitting the data. Therefore, although a unique conformation of the N TAIL region encompassing residues 506-525 cannot be derived, all the solutions provided by CREDO consistently revealed a packing of the Cterminal region of N TAIL against XD. The recurrence of these packed conformations suggests that the Cterminal region of N TAIL interacts with XD.
Moreover, we checked the influence of the Box3 conformation on the scattering curve by calculating the scattering profile of pseudomodels of the complex. In these pseudomodels, the N-terminal region of N TAIL is in the same conformation as in the credo model shown in Figure  2C . The only difference concerns the orientation of Box3, which points out of the complex, in different solventexposed conformations. Noteworthy, the theoretical scattering curves calculated from these pseudomodels using CRYSOL (Svergun et al. 1995) poorly fit with the experimental curve, with an average 2 of $ 7.0 (data not shown). This confirms that the C terminus of N TAIL does contribute to the observed scattering profile, thus attesting the reliability of the model provided by CREDO.
In order to further explore the possible contribution of N TAIL regions other than the a-MoRE to XD binding, we have looked at the regions of homology conserved amongst members of the Morbillivirus genus (Diallo et al. 1994) . These regions of homology are herein referred to as Box1, Box2, and Box3 and span N residues 401-420, 489-506, and 517-525, respectively (see Fig. 3A ), with the a-MoRE being located within Box2. We have then designed three deletion constructs bearing different combinations of these homology boxes (Fig. 3A) .
The gene fragments encoding the different N TAIL deletion proteins were cloned into the pDest14 vector (Invitrogen) to yield N-terminally histidine-tagged and C-terminally flag-tagged recombinant products, the expression of which is under the control of the T7 promoter. In all cases, most recombinant protein was recovered from the soluble fraction of bacterial lysates (Fig. 3B , lanes SN). The N TAIL deletion proteins were purified to showing the location of the a-MoRE, is given below. The inset shows the experimental scattering curve (black circles) and fit (red line) obtained by CREDO with the low-resolution model. homogeneity (>95%) in two steps: Immobilized Metal Affinity Chromatography (IMAC), and gel filtration (Fig.  3B ). The identity of the recombinant products was confirmed by IP studies using anti-N Cl25, anti-flag, and antihexahistindine tag mAbs (data not shown). As shown in Figure 3B , the three N TAIL deletion proteins migrate in SDS-PAGE with an apparent MM of either 20 kDa (N TAILD3 and N TAILD1 ) or 18 kDa (N TAILD2,3 ) (expected MMs are 14.5, 13.4, and 11.5 kDa, respectively). This abnormal migratory behavior has already been documented for N TAIL , where mass spectrometry analysis and N-terminal sequencing gave the expected results . The anomalous electrophoretic mobility is therefore due to a rather high content of acidic residues, as frequently observed in intrinsically disordered proteins (Tompa 2002) . Likewise, the behavior of the truncated N TAIL proteins can probably be accounted for by this sequence bias composition.
The number of different conformations of the N TAIL deletion proteins is limited, as indicated by the sharpness of the peaks observed in gel filtration (data not shown). As expected for intrinsically disordered protein subdomains, the Stokes radius (R S ) values, as inferred by gel filtration (27 6 3 Å , 22 6 3 Å , and 27 6 3 Å for N TAILD3 , N TAILD2,3 , and N TAILD1 , respectively), are consistent with extended conformations (see Materials and Methods). Thus, these deletion proteins share similar hydrodynamic properties with full-length N TAIL , all possessing an elongated shape.
In order to directly measure the contribution of the different N TAIL boxes to binding, we have studied binding reactions between XD and N TAIL deletion proteins. Changes in surface plasmon resonance were monitored in real time as the N TAIL proteins passed over sensor chips to which XD was covalently coupled. This analytical approach is ideally suited to study reversible, lowaffinity protein-protein interactions that typify interactions involving intrinsically disordered proteins (Wright and Dyson 1999; Dunker and Obradovic 2001; Uversky 2002) . Moreover, reaction rate and equilibrium constants were calculated for reactions with different protein substrates, allowing differences in XD binding affinities to be readily quantified.
Binding affinities between XD and N TAIL constructs were established using 180-225 RU of immobilized XD and N TAIL concentrations ranging from 0.1 to 10 mM (see Materials and Methods). Dosage-dependent binding was observed in this range. Reactions conformed to a 1:1 ligand-substrate (Langmuir) binding model, exhibiting an excellent fit (i.e., a 2 value <1 and residuals within the range of 62) following global analysis of sensorgrams. Binding reactions between XD and N TAILHNFC exhibit an equilibrium dissociation constant of 80 nM (see Table 1 ). The XD binding affinity for N TAILD1 (50 nM) is similar to that of N TAILHNFC indicating that Box1 does not participate in binding. In contrast, removal of either Box3 alone or Box2 plus Box3 results in a strong decrease (three orders of magnitude) in the equilibrium dissociation constant, where N TAILD3 and N TAILD2,3 display similar binding affinities (see Table 1 ). The strong decrease in the affinity resulting from removal of Box3 clearly indicates that Box2 is not the sole region involved in binding to XD, and attests that Box3 also plays a role in the interaction with XD, as already suggested by SAXS studies.
The far-UV circular dichroism (CD) spectra of N TAIL deletion proteins at neutral pH are typical of unstructured proteins, as seen by their large negative ellipticity at 198 nm and very low ellipticity at 185 nm ( Fig. 4A -C). The solvent 2,2,2-trifluoroethanol (TFE) mimics the hydrophobic environment experienced by proteins in protein-protein interactions, and is therefore widely used as a probe to unveil disordered regions having a propensity to undergo an induced folding (Hua et al. 1998 ). Thus, we have recorded CD spectra of N TAIL deletion proteins in the presence of increasing concentrations of TFE ( Fig. 4A -C). All proteins show an increasing gain of a-helicity upon addition of TFE, as indicated by the characteristic maximum at 190 nm and minima at 208 nm and 222 nm ( Fig. 4A -C). In the case of N TAILD3 and N TAILD1 , most unstructured-to-structured transitions take place in the presence of 20% TFE, a concentration at which the a-helical content is estimated to be about 13% (using the ellipticity at 222 nm) (Fig.  4D ). On the other hand, in the case of N TAILD2,3 , TFE concentrations as high as 30% are required for most pronounced unstructured-to-structured transitions to take place (see Fig. 4B ). Moreover, in the presence of 30% TFE, the a-helical content of N TAILD2,3 is not only lower (11%) than that (16%) of N TAILD1 and N TAILD3 , but also lower than the a-helical content observed at 20% TFE for the two other N TAIL deletion proteins (Fig. 4D) . Therefore, N TAILD2,3 displays the lowest a-helical potential, while N TAILD1 and N TAILD3 exhibit a folding propensity similar to that observed for N TAIL Bourhis et al. 2004 ). These results, beyond confirming that the region spanning residues 489-516 affects the folding potential of N TAIL (Bourhis et al. 2004) , suggest that neither Box1 nor Box3 contribute to the a-helical propensity of N TAIL , in agreement with the secondary structure prediction provided by PSI-PRED (McGuffin et al. 2000) and PHD (Rost 1996) , which both predict an a-helix (residues 489-504, see 
In order to investigate whether the N TAIL deletion proteins retained the ability to undergo induced folding in the presence of XD, we have used far-UV CD spectroscopy. The N TAILHNFC protein, bearing an N-terminal hexahistidine tag and a C-terminal flag was purified from the soluble fraction of E. coli (data not shown) and used as the reference in these experiments to allow direct comparison with the N TAIL truncated proteins. Noticeably, the CD spectrum of N TAILHNFC is fully superimposable on that of N TAILHN (data not shown), thus ruling out the possibility that the flag sequence might affect the folding properties of the protein.
The far-UV CD spectrum of XD ( Fig. 5A -D, gray line) is typical of a structured protein with a predominant ahelical content, as indicated by the positive ellipticity between 185 nm and 200 nm, and by the two minima at 208 nm and 222 nm. After mixing N TAILHNFC with different molar excesses of XD, the observed CD spectra differed from the corresponding theoretical average curves calculated from the individual spectra. Since the theoretical average curves correspond to the spectra that would be expected if no structural variations occur, deviations from these curves indicate structural transitions. The results obtained in the presence of a threefold molar excess of XD indicate a random coil to a-helix transition, as judged by the much more pronounced minima at 208 nm and 222 nm, and by the higher ellipticity at 190 nm of the experimentally observed spectrum compared to the corresponding theoretical average curve (see Fig. 5A ). Although even more dramatic structural transitions of full-length N TAIL have been previously observed with a twofold molar excess of XD , the results obtained with a threefold molar excess have been selected for presentation in order to allow direct comparison with the N TAIL deletion proteins (see below). Figure 5 , panels B-D, show the results obtained for the three N TAIL deletion proteins in the presence of a threefold molar excess of XD, a condition leading to the most dramatic structural transitions. Removal of Box3 significantly reduces, but does not abrogate, the folding ability of N TAIL . Indeed, the experimental CD spectrum does not significantly deviate from the average curve in the 200-260 nm region, but it considerably deviates from the average curve in the 185-195 region (58% mean increase of ellipticity) (Fig. 5B ), thus supporting partial folding ability of N TAILD3 in the presence of XD. Further removal of Box2 results in a truncated N TAIL form, which has completely lost its ability to fold in the presence of XD, as indicated by the good superimposition between the experimental and the average spectra (Fig. 5C) . Conversely, removal of Box1 does not affect the folding ability of N TAIL , as the deviations from the average spectrum are similar to those observed with the full-length form (Fig. 5, cf. D and A) . The mean increase in ellipticity in the 185-195 nm region (77%) and the decrease in the ellipticity value at 220 nm (46%) observed with N TAILD1 , are comparable to the corresponding values observed with N TAILHNFC (70% and 46%, respectively).
As a control, we recorded CD spectra of N TAILHNFC in the presence of lysozyme (data not shown). The absence of significant structural variations even with molar excesses as high as 5, confirms the specificity of the deviations observed upon addition of XD to the N TAIL proteins.
A two-and threefold molar excess of XD is required to induce the most pronounced structural transitions of fulllength and truncated forms of N TAIL , respectively. In contrast, an equimolar amount of PCT is sufficient to produce the same effect Bourhis et al. 2004) . This difference can be accounted for by a lower affinity of N TAIL towards XD, compared to PCT, rather than to the formation of a 1:2 or 1:3 stoichiometric complex. Indeed, formation of a 1:1 stoichiometric complex between XD and a peptide corresponding to residues 477-505 of N, has been documented using isothermal titration calorimetry (Kingston et al. 2004b ). The higher affinity of N TAIL for PCT compared to XD could be ascribed either to cooperativity phenomena among the different XDs within the PCT tetramer (Rahaman et al. 2004) or to the possible contribution of other PCT regions to binding. This latter possibility can be ruled out based on recent data pointing out XD as the sole PCT region contributing to binding (Kingston et al. 2004b; S. Longhi and M.J. Oglesbee, unpubl.) .
In conclusion, these results indicate that (1) Box1 is fully dispensable for binding to XD and induced folding, (2) Box2 is strictly required for induced folding to take place, and (3) Box3 contributes to binding to XD, as pointed out by the reduced ability of N TAILD3 to undergo induced folding. Moreover, the fact that N TAILD3 , N TAILD1 , and N TAIL Bourhis et al. 2004) share similar folding propensities suggests that the contribution of Box3 to the interaction can be accounted for more in terms of binding rather than of induced folding.
In order to further characterize the contribution of Box3 to binding, we have used fluorescence spectroscopy. Accordingly, we have designed an N-terminally hexahistidine-tagged N TAIL variant form bearing a tyrosine to tryptophan substitution at position 518 (see also Fig. 3A ). Introduction of a tryptophan residue in Box3 allowed binding events to be followed by fluorescence spectroscopy, while maximizing the conservative nature of the substitution (note that neither N TAIL nor XD contain any tryptophan residue).
Most recombinant product was purified from the soluble fraction of the bacterial lysate (Fig. 6A) . The identity of the recombinant product was confirmed by IP studies using anti-N Cl25, and anti-hexahistindine tag mAbs (data not shown). As already observed in the case of full-length N TAIL and N TAIL deletion proteins, N TAILW518 migrates in SDS-PAGE with an apparent MM higher than expected (Fig. 6A) . The mutated protein, displays the same gel filtration elution profile as the wt form (data not shown), leading to an estimated R S of 27 6 3 Å .
As shown in Figure 6B , the far-UV CD spectrum of N TAILW518 is almost perfectly superimposable on that of N TAILHN , thus indicating that the introduction of the tryptophan residue does not affect the overall secondary structure content of the protein. In order to investigate whether the variant form retained the ability to undergo induced folding in the presence of XD, we have added various molar excesses (ranging from 1 to 4) of XD to N TAILW518 , and recorded the corresponding far-UV CD spectra (data not shown). These latter studies indicate that the tyrosine to tryptophan substitution does not affect the ability of the protein to undergo induced folding in the presence of the partner, thus supporting the biochemical relevance of this variant form.
Fluorescence spectroscopy studies showed that N TAILW518 has a maximum of emission at 356 nm, indicating that Trp 518 is fully exposed to the solvent (data not shown). Addition of gradually increasing XD concentrations triggers an increase in the fluorescence intensity in a dose-dependent manner, which indicates a modification in the pattern of interactions with neighboring groups. At the same time, addition of XD causes a progressive shift in the emission maximum from 356 nm to 352 nm (data not shown), thus indicating that Trp 518 becomes only slightly less exposed to the solvent.
No significant variations are observed in the fluorescence spectrum obtained after addition to N TAILW518 of a 2 mM solution of an irrelevant protein (SARS virus, unclassified protein 5) of similar size and devoid of tryptophan residues (data not shown). After plotting the relative fluorescence intensity increase as a function of the XD concentration (Fig. 6C) , an apparent constant equilibrium dissociation (K Dapp ) value of 133 6 32 mM is derived. This value is in good agreement with the value obtained by surface plasmon resonance studies with wt N TAIL . Although the environment of Trp 518 remains mostly polar upon binding to XD, the observed increase in the fluorescence intensity indicates that the chemical environment of Trp 518 is affected, thus further suggesting that the C terminus of N TAIL interacts with XD.
In order to further explore the nature of the interaction established between Box3 and XD, we have used NMR spectroscopy. To this endeavor, we have recorded a HSQC spectrum of 15 N uniformly labeled N TAILHN either alone or in the presence of a twofold molar excess of XD, as well as of 15 N uniformly labeled N TAILD3 in the same conditions. This analysis allowed a quantitative estimation of the number of residues involved in the interaction with XD by following chemical shift changes in the backbone amide and proton resonances upon addition of unlabeled XD. Upon addition of XD to N TAILHN , 16 correlation peaks are displaced. Among them, 11 undergo an upfield shift (see Fig. 7 , stars), which indicates a random coil to a-helix transition, and two correspond to the side chain of either a Gln or an Asn. Additionally, at least seven additional peaks undergo a less dramatic displacement (see Fig. 7 , diamonds). Because of its small amplitude, this shift most likely does not reflect a conformational change. Nevertheless, it is indicative of a change in the chemical environment of these residues resulting either by direct interaction with the partner or by local magnetic perturbations of residues spatially close to the newly formed a-helix. Based on this experiment, however, the determination of N TAIL residues involved in these two types of shifts is not possible. The precise identification of these residues would require the full assignment of the N TAIL HSQC spectrum, which is complicated by the strong signal overlapping inherent in the predominantly unfolded nature of N TAIL .
Comparison of present results to those of the NMR spectroscopy studies of Kingston et al. (2004a) suggest that the 11 residues undergoing the random coil to a-helix transition are most likely located within the 486-503 region. In order to assess whether the additional N TAIL residues undergoing a less pronounced displacement upon binding to XD are located within Box3, we recorded a HSQC spectrum of 15 N uniformly labeled N TAILD3 either alone or with a twofold molar excess of XD. As shown in Figure 7 , the same peaks undergoing the large displacement in the N TAILHN -XD complex are also observed in the N TAILD3 -XD complex. In particular, among these peaks, the occurrence in both complexes of the 11 peaks that undergo the random coil to a-helix transition (see Fig. 7 , stars), provides further support that the helical folding occurs within Box2. On the other hand, the seven peaks that undergo a less dramatic displacement upon complex formation with XD (see Fig. 7 , diamonds) are not present in the spectrum recorded on the N TAILD3 -XD complex. This latter observation indicates that these seven peaks correspond to residues that are located within Box3.
In conclusion, these experiments reveal that complex formation between N TAIL and XD implies two types of interaction: one, mediated by residues belonging to Box2, involves a significant gain of a-helicity, while the other, attributable to Box3 residues, is not accompanied by a significant structural transition.
In this paper we show that N TAIL remains predominantly unfolded after binding to XD, with two distinct sites being involved in the interaction. Although the XDinduced gain of regular secondary is restricted to Box2 (aa 489-504), the region encompassing residues 489-504 is not the only N TAIL region involved in binding to XD. In particular, we present several lines of evidence indicating that the extreme C terminus of N TAIL (Box3, aa 517-525) also contributes to binding, without however gaining any regular secondary structure.
The C terminus of N TAIL is an additional XD binding site SAXS studies suggest that the C terminus of N TAIL interacts with XD. In particular, the calculated overall envelope of the complex shows the presence of a globular cluster of invariant size at one extremity and of an elongated protuberance with varying shapes. The elongated protuberance corresponds to the 92-residue-long N-terminal region of N TAIL , while the more compact region accommodates the structure of the XD-N TAIL486-505 complex as well as the 20 C-terminal residues of N TAIL . Data from overall shape calculations thus clearly indicate that the C terminus is not protruding towards the solvent, and remains close to XD. On the other hand, attempts to more precisely model the conformation adopted by the C-terminal region of N TAIL within the complex led to an ensemble of solutions. In all these solutions, the C-terminal region of N TAIL always packs against the XD-N TAIL486-505 complex, rather than being extended and exposed to the solvent. Identification of the possible gain of regular secondary structure within the C-terminal region of N TAIL is beyond the resolution limits of SAXS. However, results provided by CD, fluorescence spectroscopy, and heteronuclear NMR all converge to suggest that the C-terminal region of N TAIL does not gain any regular secondary structure (see below). Besides supporting a role for the C-terminal region of N TAIL in the interaction with XD, these data also indicate that most of N TAIL remains disordered within the complex. The prevalent disorder of N TAIL within the complex is in agreement with other data reported in the literature where an intrinsically disordered protein largely preserves its overall extended conformation even after interaction with a binding partner (see Tompa 2002 , and references cited therein; Permyakov et al. 2003) . Finally, it is noteworthy that in the SAXS model the N terminus of XD is exposed to the solvent (see Fig. 2B ), a position that would accommodate the remaining part of P.
The involvement of additional N TAIL regions other than the a-MoRE in binding to XD is confirmed by surface plasmon resonance studies, where the contribution of Box3 to XD binding can be quantitatively estimated. Removal of either Box3 alone or Box2 plus Box3 leads to a strong decrease (three orders of magnitude) in the affinity as compared to full-length N TAIL , thus indicating that Box3 contribution to binding is similar to that of Box2.
Spectroscopy studies showed that Box3 contributes to binding to XD but does not undergo any gain of regular secondary structure. Although Box3 does not affect the folding potential of N TAIL , as indicated by CD studies in the presence of TFE, the removal of Box3 significantly reduces the ability of N TAIL to undergo induced folding in the presence of XD. This supports a role for Box3 in binding to XD. Further removal of Box2 results in a truncated form that has a significantly decreased folding potential and has lost the ability to undergo induced folding in the presence of XD. These results are consistent with the unique a-helical forming potential of Box2 and its role as primary binding site for XD.
Contribution of Box3 to binding without dramatic structural change is supported by fluorescence spectroscopy data, which show an increase in the fluorescence intensity of N TAILW518 upon addition of XD. Increases in the fluorescence intensity upon binding to a partner/ ligand have been already documented, for both folded (Bette et al. 2002) and intrinsically unstructured proteins (Raggett et al. 1998) , and indicate that the chemical environment of the Trp, although remaining mostly polar, is changed as a result of the addition of the partner. However, the fluorescence data do not enable us to discriminate between a direct Trp 518-XD interaction and secondary effects resulting from local perturbations triggered by a direct Box2-XD interaction.
That complex formation with XD involves both Box2 and Box3, and that only Box2 undergoes a gain of a-helicity, is confirmed by NMR studies. Heteronuclear NMR experiments show that upon complex formation with XD, 11 N TAIL residues, belonging to Box2, undergo a random coil to a-helix transition, while at least seven additional residues undergo a shift in their chemical environment not accompanied by the gain of regular secondary structure elements. Our data show that these latter peak displacements correspond to residues belonging to Box3, as indicated by the absence of such peaks in both the spectra of N TAILD3 alone and in complex with XD. Thus, the NMR studies, beyond confirming the role of Box3 in the interaction with XD, also highlight that binding of N TAIL to XD implies two types of interaction, where gain of regular secondary structure is restricted to only one of the binding determinants, Box2.
The accommodation of the 486-505 region of N within XD (Kingston et al. 2004a ) triggers some minor rearrangements at the surface of the latter, compared to the crystal structure of the uncomplexed form . In particular, the largest movements are observed for the side chains of residues Arg15 and Glu17 of XD (corresponding to residues 472 and 474 of P). The Arg15 NH2 atom moves 9.7 Å away in the chimeric structure compared to the structure of XD alone, while the Glu17 C atom undergoes a shift of 2.5 Å . These two residues are both located within the loop connecting a1 and a2 helices, and occur at least 9 Å away from Box2, thus ruling out the possibility that the observed spatial rearrangements they undergo could be ascribed to Box2 embedding. The significant displacement they undergo in the complex therefore indicates that binding of Box2 induces local conformation changes in XD that could favor interaction with Box3. The surface displaced residues of XD could, in fact, be part of a Box3 binding site.
We tentatively propose that binding to XD might take place through a sequential mechanism involving successive binding of disordered domains, as it has been recently reported for p27 upon binding to the CdK2-cyclin A complex (Lacy et al. 2004 ).
The K D value between N TAIL and XD, as measured by both fluorescence spectroscopy and surface plasmon resonance, is in the 100 nM range. Surprisingly, this value is considerably lower than that reported by Kingston et al. (2004b) (13 mM) and derived from isothermal titration calorimetry studies. A weak binding affinity, associated with a fast association rate, would ideally fulfill the requirements of a polymerase complex, which has to cartwheel on the nucleocapsid template during both transcription and replication. However, a K D in the mM range would not seem to be physiologi-cally relevant considering the low intracellular concentrations of P in the early phases of infection. Moreover, such a weak affinity is not consistent with the ability to readily purify nucleocapsid-P complexes using rather stringent techniques such as CsCl isopycnic density centrifugation (Robbins and Bussell 1979; Stallcup et al. 1979; Robbins et al. 1980; Oglesbee et al. 1989) . Our data support a higher affinity between P and N, resulting in a stable P-N TAIL complex that would be predicted to hinder processive movement of P along the nucleocapsid template. In agreement with this prediction, Box3 has been shown to have an inhibitory role upon transcription and replication, as indicated by previous minireplicon experiments, where deletion of Box3 enhanced basal reporter gene expression (Zhang et al. 2002) . We can speculate that the transient nature of the N TAIL -XD interaction might be ensured by the possible intervention of cellular and/or viral cofactors. Indeed, the requirement for cellular or viral cofactors in both transcription and replication has been already documented in the case of measles (Vincent et al. 2002) , respiratory syncytial (Fearns and Collins 1999) , and Ebola viruses (Hartlieb et al. 2003) . These cofactors may serve as processivity or transcription elongation factors and could act by modulating the strength of the interaction between the polymerase complex and the nucleocapsid template. Such a mechanism may explain the stimulatory effect of hsp72 on MV transcription and genome replication, an effect mediated by Box3-hsp72 interaction (Zhang 2002) . In this capacity, hsp72 may neutralize the contribution of Box3 to a more stable complex between P and N TAIL , thereby promoting successive cycles of P binding and release that are essential to polymerase processivity.
Using different physico-chemical approaches we have shown that the interaction of N TAIL with XD involves an additional, previously unreported site located at the extreme C terminus of N TAIL . While the primary site (i.e., Box2) gains a-helical structure upon binding to XD, this additional site does not gain any regular secondary structure elements. Nevertheless, it plays a crucial role in stabilizing the complex.
We have previously shown that N TAIL belongs to the premolten globule subfamily, e.g., it possesses a certain extent of residual secondary and/or tertiary structure Bourhis et al. 2004 ). In agreement with these findings, NMR studies showed that the N TAIL region encompassing residues 486-503 is not a statistical random coil (Kingston et al. 2004a ). These results, supporting a restricted conformational freedom of this N TAIL region, are in agreement with the speculation that residual structure within N TAIL may play a role for efficient binding to P Bourhis et al. 2004) . That residual structure within intrinsically disordered proteins may favor the folding process triggered by binding to a partner has already been reported (Bienkiewicz et al. 2002; Fuxreiter et al. 2004; Csizmok et al. 2005) .
N TAIL provides an interesting model system for the study of the interaction of an intrinsically disordered protein and its partners. Its multiple-site mode of interaction well illustrates the complexity of the contacts established by intrinsically disordered proteins and emphasizes the need for thorough investigations of the molecular mechanisms underlying recognition of the partner. Indeed, binding of disordered regions to their targets involves different types of interactions, such as binding coupled to folding, binding with no gain of regular secondary structure, or coexistence of both interaction mechanisms (for a review, see Dyson and Wright 2005) .
Binding coupled to folding has been reported for the phosphorylated kinase-inducible domain (pKID) of CREB, which undergoes a coil to a-helix folding transition upon binding to the KIX domain of the transcription coactivator CBP (CREB Binding Protein) (Radhakrishnan et al. 1997) . The amphipathic helix aB of pKID interacts with a hydrophobic groove defined by helices a1 and a3 of the partner. The other pKID helix, aA, contacts a different face of the a3 helix. This mode of interaction is reminiscent of that occurring between N TAIL and XD. Similarities concern the involvement of two distinct sites within the disordered domain (where Box2 and Box3 resemble helices aB and aA, respectively) and embedding of an a-helix within a hydrophobic cleft delimited by a-helices from the structured partner. The difference concerns the mode of interaction of the second site, where Box3 of N TAIL does not gain any regular secondary structure element, contrary to aA of pKID. Nevertheless, complexes with dual interaction have already been described in the literature. Among them, we mention the case of the binding of p27 to the cyclinA-Cdk2 complex (Lacy et al. 2004) , and that of the activation domain of CITED2 to the TAZ1 domain of CBP (De Guzman et al. 2004 ).
On the other hand, binding without any concomitant gain of regular secondary structure, has been also reported. This is the case of the unfolded proteins 4E-BP1 (4E binding protein 1), an inhibitor of translation. Its binding to eIF4E does not involve any transition to stable regular secondary structure (Fletcher et al. 1998) . Interestingly, when eIF4E binds to another partner, namely eIF4G, it induces a coil-to-helix transition in the latter (Marcotrigiano et al. 1999) , highlighting the diversity of the structural transitions that can be triggered by a structured partner.
Finally, there are a few examples of proteins that undergo different structural transitions as a function of the partner they bind. Notably, HIFa (hypoxia-inducible factor a ) possesses an intrinsically disordered domain, which can interact with two different partners. This unstructured domain adopts an a-helical structure when bound to the TAZ1 domain of CBP. However, binding of the same region of HIFa to an asparagine hydroxylase results in a highly extended conformation, which is required for the enzymatic activity of the latter. These findings provide an elegant example of the plasticity that intrinsically disordered proteins display for different partners involved in different functions. We can speculate on a similar behavior in the case of N TAIL . In particular, Box2 and Box3 of N TAIL interact both with at least two distinct partners, P and Hsp72, and competition between XD and Hsp72 for binding to N TAIL has been recently shown (Zhang et al. 2005) . It is conceivable that binding of N TAIL to P or to Hsp72 may involve a different structural transition, as in the case of HIFa.
Last, but not least, our findings, beyond contributing to elucidate the dynamics of the interactions established by intrinsically disordered proteins, provide an interesting target site for mutational studies aimed at exploring further the mode of interaction between N TAIL and XD.
The E. coli strains DH5a (Stratagene) was used for selection and amplification of DNA constructs. The E. coli strains Rosetta [DE3] pLysS (Novagen) and C41 [DE3] (Avidis) were used for expression of recombinant proteins. E. coli was grown either in Luria-Bertani (LB) medium, or in minimal M9 medium supplemented with 15 NH 4 Cl.
Pfu polymerase was from Promega. Primers were purchased from Invitrogen. The anti-hexahistidine tag mAb was purchased from Qiagen. The anti-flag mAb was purchased from Sigma. The anti-N Cl 25 (Giraudon et al. 1988; Buckland et al. 1989 ) mAb was kindly provided by D. Gerlier.
The XD gene construct, encoding residues 459-507 of the MV P protein (strain Edmonston B) with an hexahistidine tag fused to its C terminus, has already been described .
All N TAIL constructs were obtained by PCR using the MV N gene, strain Edmonston B, as template. The N TAILHN gene construct, encoding residues 401-525 of the MV N protein with a hexahistidine tag fused to its N terminus, was obtained using the plasmid pET21a/N (encoding the MV N protein; Karlin et al. 2002a ) as template. Forward primer (5 0 -gatagaac catgCATCATCATCATCATCATactactgaggacaagatcagtaga-3 0 ) was designed to introduce a hexahistidine tag encoding sequence (upper case) at the N terminus of N TAIL , while reverse primer (5 0 -ggggaccactttgtacaagaaagctgggtcttagtctagaa gatttctgtcattgta-3 0 ) was designed to introduce an AttB2 site (bold). The PCR amplification product was further used as template in a second PCR step, using forward primer (5 0 -ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgCAT CATCATCAT-3 0 ), designed to introduce an AttB1 site (bold), and reverse primer as above. After purification (PCR Purification Kit; Qiagen), the PCR product was cloned into the pDest14 vector (Invitrogen) using the Gateway recombination system (Invitrogen). The final construct is referred to as pDest14/N TAILHN .
The N TAILHNFC gene construct, encoding residues 401-525 of the MV N protein with an N-terminal hexahistidine tag and a C-terminal Flag sequence (dykddddk) (Brizzard et al. 1994) , was obtained using the plasmid pDest14/N TAILHN as template. Forward primer (5 0 -ggggacaagtttgtacaaaaaagcaggcttcgaaggagata gaaccatgCATCATCATCAT-3 0 ) was designed to introduce an AttB1 site (bold), and reverse primer (5 0 -gtcttaTTTGTCGTCAT CGTCTTTATAATCgtctagaagatttctgtcattgta-3 0 ) was designed to introduce a Flag encoding sequence (upper case). The PCR amplification product was further used as template in a second PCR step, using the same forward primer as above, and reverse primer (5 0 -ggggaccactttgtacaagaaagctgggtcttaTTTGTCGTCAT CGTCTTT-3 0 ), which was designed to introduce an AttB2 site (bold). After purification, the PCR product was cloned into the pDest14 vector to yield pDest14/N TAILHNFC .
The N TAILD3 gene construct, encoding residues 401-516 of the MV N protein with an N-terminal hexahistidine tag plus a C-terminal Flag sequence, was obtained using the plasmid pet21a/N as template. Forward primer (5 0 -gatagaaccatgCA TCATCATCATCATCATactactgaggacaagatcagtaga-3 0 ) was designed to introduce a hexahistidine tag encoding sequence (upper case) at the N terminus of N TAIL , and reverse primer (5 0 -gtcttaTTTGTCGTCATCGTCTTTATAATCtataggggtgtcc gtgtctgagcc-3 0 ) was designed to introduce a Flag encoding sequence. The PCR amplification product was further used as template in a second PCR step, using forward primer (5 0 -ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgCATCA TCATCAT-3 0 ) and reverse primer (5 0 -ggggaccactttgtacaa gaaagctgggtcttaTTTGTCGTCATCGTCTTT-3 0 ), which were designed to introduce an AttB1 and an AttB2 site (bold), respectively. After purification, the PCR product was cloned into the pDest14 vector to yield pDest14/N TAILD3 .
The N TAILD2,3 gene construct, encoding residues 401-488 of the MV N protein with an N-terminal hexahistidine tag plus a C-terminal Flag sequence, previously referred to as N TAIL2 (Bourhis et al. 2004) , was obtained using the plasmid pet21a/ N as template. Forward primer (5 0 -gatagaaccatgCATCATCA TCATCATCATactactgaggacaagatcagtaga-3 0 ) was designed to introduce a hexahistidine tag encoding sequence (upper case) at the N terminus of N TAIL , and reverse primer (5 0 -gtctta TTTGTCGTCATCGTCTTTATAATCactgtcctgcggatcttggctg ga-3 0 ) was designed to introduce a Flag encoding sequence (upper case). The PCR amplification product was further used as template in a second PCR step, using the same pair of primers as in the second PCR step, which yielded the N TAILD3 amplification product. After purification, the PCR product was cloned into the pDest14 vector to yield pDest14/ N TAILD2,3 .
The N TAILD1 gene construct, encoding residues 421-525 of the MV N protein with an N-terminal hexahistidine tag plus a C-terminal Flag sequence, was obtained using the plasmid pet21a/N as template. Forward primer (5 0 -gatagaaccatgCA TCATCATCATCATCATcacggtgatcaaagtgagaatgag-3 0 ) was designed to introduce a hexahistidine tag encoding sequence (upper case) at the N terminus of N TAIL , and reverse primer (5 0 -gtcttaTTTGTCGTCATCGTCTTTATAATCgtctagaagattt ctgtcattgta-3 0 ) was designed to introduce a Flag-encoding sequence (upper case). The PCR amplification product was further used as template in a second PCR step, using the same pair of primers as in the second PCR step, which yielded both N TAILD3 and N TAILD2,3 amplification products. After purification, the PCR product was cloned into the pDest14 vector to yield pDest14/N TAILD1 .
The N TAILW518 gene construct, encoding residues 401-525 of the MV N protein with a Tyr ! Trp substitution at position 518 and with a hexahistidine tag fused to its N terminus, was obtained by PCR, using the plasmid pET21a/N as template. Two separate PCR steps were carried out in parallel, yielding amplification products A and B. Product A was obtained using forward primer (5 0 -gatagaaccatgCATCAT CATCATCATCATactactgaggacaagatcagtaga-3 0 ), designed to introduce a hexahistidine tag-encoding sequence (upper case) at the N terminus of N TAIL , while reverse primer (5 0 -aaga tttctgtcattCCAcactatTggggtgtc-3 0 ) was designed to introduce a Trp at position 518 (bold and upper case) and a silent mutation at nucleotide position 1545 (upper case) thus resulting in the introduction of a BstXI site (underlined). Product B was obtained using forward primer (5 0 -gacaccccAatagtgTGG aatgacagaaatctt-3 0 ) designed to introduce a Trp at position 518 (bold and upper case) and a silent mutation at nucleotide position 1545 (upper case) thus resulting in the introduction of a BstXI site (underlined), and reverse primer (5 0 -ccgggcatgc atccggatatagttcctcctt-3 0 ) designed to anneal with nucleotide positions 1755-1775 of pet21a/N (where the A of the ATG codon of the N ORF was set as nucleotide position 1). A further PCR step was carried out using amplification products A plus B as template, to yield the N TAIL W518 amplification product. Forward primer (5 0 -ggggacaagtttgtacaaaaaagcaggct tcgaaggagatagaaccatgCATCATCATCAT-3 0 ) was designed to introduce an AttB1 site (bold), while reverse primer (5 0 -ggggac cacttttgtacaagaaagctgggtcttagtctagaagatttctgtcattCCA-3 0 ) was designed to introduce an AttB2 site (bold) and a Trp at position 518 (upper case). After purification, the PCR product was cloned into the pDest14 vector using the Gateway recombination system. The final construct is referred to as pDest14/ N TAILW518 . Candidate clones bearing the desired mutation were selected on the basis of the ability of their recombinant plasmids to be restricted by BstXI, a unique restriction site introduced by PCR together with the Tyr to Trp substitution.
The sequence of the coding region of all expression plasmids was verified by sequencing (MilleGen).
E. coli strain Rosetta [DE3] (Novagen) was used for the expression of N TAIL constructs. Since the MV N gene contains several rare codons that are used with a very low frequency in E. coli, coexpression of N TAIL constructs with the plasmid pLysS (Novagen) was carried out. This plasmid, which supplies six rare tRNAs, carries also the lysozyme gene, thus allowing a tight regulation of the expression of the recombinant gene, as well as a facilitated lysis. Cultures were grown overnight to saturation in LB medium containing 100 mg/mL ampicilin and 17 mg/mL chloramphenicol. An aliquot of the overnight culture was diluted 1/25 in LB medium and grown at 37 C. At OD 600 of 0.7, isopropyl b-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the cells were grown at 37 C for 3 h. The induced cells were harvested, washed, and collected by centrifugation. The resulting pellets were frozen at À20 C.
Isotopically substituted ( 15 N) N TAILHN and N TAILD3 were prepared by growing transformed bacteria in minimal M9 medium supplemented with 15 NH 4 Cl (0.8 g/L). A 50-mL preculture grown overnight to saturation in LB medium containing 100 mg/mL ampicillin and 17 mg/mL chloramphenicol, was harvested, washed in minimal M9 medium, and inoculated into 1 L of minimal M9 medium supplemented with ampicillin and chloramphenicol. The culture was grown at 37 C. At OD 600 of 0.5, IPTG was added to a final concentration of 0.2 mM and the cells were grown first at 37 C for 3 h, and then over night at 28 C. The induced cells were harvested, washed, and collected by centrifugation. The resulting pellets were frozen at À20 C.
Expression of tagged XD was carried out as described in Johansson et al. (2003) .
Cellular pellets from bacteria transformed with the different N TAIL expression plasmids were resuspended in 5 volumes (v/ w) buffer A (50 mM sodium phosphate at pH 8, 300 mM NaCl, 10 mM Imidazole, 1 mM phenyl-methyl-sulphonyl-fluoride [PMSF]) supplemented with lysozyme 0.1 mg/mL, DNase I 10 mg/mL, protease inhibitor cocktail (Sigma) (50 mL/g cells). After a 20-min incubation with gentle agitation, the cells were disrupted by sonication (using a 750 W sonicator and four cycles of 30 sec each at 60% power output). The lysate was clarified by centrifugation at 30,000g for 30 min. Starting from a 1-L culture, the clarified supernatant was incubated for 1 h with gentle shaking with 4 mL Chelating Sepharose Fast Flow Resin preloaded with Ni 2+ ions (Amersham Pharmacia Biotech), previously equilibrated in buffer A. The resin was washed with buffer A, and the N TAIL proteins were eluted in buffer A containing 250 mM imidazole. Eluates were analyzed by SDS-PAGE for the presence of the desired product. The fractions containing the recombinant product were combined, and concentrated using Centricon Plus-20 (molecular cutoff, 5000 Da) (Millipore). The proteins were then loaded onto a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech) and eluted in either 10 mM sodium phosphate at pH 7 or 10 mM Tris/HCl at pH 8. The proteins were stored at À20 C.
Purification of histidine-tagged XD was carried out as described in Johansson et al. (2003) .
All purification steps, except for gel filtrations, were carried out at 4 C.
Apparent molecular mass of proteins eluted from gel filtration columns was deduced from a calibration carried out with LMW and HMW calibration kits (Amersham Pharmacia Biotech). The theoretical Stokes radii (R S ) of a native (R S N) and fully unfolded (R S U) protein with a MM (in Daltons) were calculated according to (Uversky 1993) : log(R S N) = 0.369 Á log(MM) À 0.254 and log(R S U) = 0.533 Á log(MM) À 0.682.
Protein concentrations were calculated either using the theoretical absorption coefficients e (mg/mL Á cm) at 280 nm as obtained using the program ProtParam at the EXPASY server (http://www.expasy.ch/tools), or the Biorad protein assay reagent (Bio-Rad).
IP experiments were carried out using the anti-N Cl 25, the anti-flag, and the anti-hexahistidine tag mAbs, and bacterial lysates expressing N TAIL proteins as described in Longhi et al. (2003) .
Small angle X-ray scattering All protein samples were prepared by dilution of the purified N TAILHN and XD solutions in buffer 10 mM Tris/HCl at pH 8, 40 mM NaCl, with 1 mM DTT as radiation scavenger. The complex N TAILHN -XD was prepared by mixing XD and N TAILHN with a molar ratio of 2:1 in the same buffer and at a final protein concentration of 10 mg/mL. The samples were filtered prior to each measurement (Millex syringe filters 0.22 mm, Millipore) to eliminate possibly existing large aggregates.
SAXS experiments were carried out on beamline ID02 (Narayanan et al. 2001 ) at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The wavelength was 1.0 Å and the sample-to-detector distance was 3.0 m and 1.0 m, leading to scattering vectors q ranging from 0.02 to 0.20 Å À1 and 0.05 to 0.40 Å À1 , respectively. The scattering vector is defined as q = 4 p/l sin, where 2 is the scattering angle. The detector was an X-ray image intensified optically coupled to an ESRF developed FReLoN CCD camera. Forty successive frames of 1.0 sec with a 5-sec pause between each frame were recorded for each sample. The protein solution was circulated through an evacuated quartz capillary between each frame. Thus, no protein solution was irradiated longer than 1.0 sec. Each frame was then carefully inspected to check for possible bubble formation or radiation-induced aggregation. No such effect was observed, and individual frames could then be averaged. Absolute calibration was made with a Lupolen sample. A series of measurements at different protein concentrations ranging from 1.8 to 10 mg/mL were performed for every protein (XD, N TAILHN , and the mixture N TAILHN -XD) to check for interparticle interaction. Background scattering was measured before or after each protein sample using the buffer solution and then subtracted from the protein scattering patterns after proper normalization and correction from detector response. All the experiments were carried out at 20 C. The data acquired at both sample-to-detector distances of 3 m and 1 m were merged and extrapolated to zero concentration for the calculations using the entire scattering spectrum.
The scattering pattern of the N TAILHN -XD complex was obtained following this process: to avoid any possible bias on the absolute intensities due to the concentration, the experimental merged scattering curves obtained as described above were normalized by their theoretical I(0). The scattering pattern of XD was then subtracted twice from the scattering pattern of the mixture N TAILHN -XD (molar ratio 1:2). Given the rather low K D between N TAIL and XD ($100 nM; see Results section), we assumed that the concentration of uncomplexed N TAILHN in solution is negligible at the concentration used in the SAXS experiments (2 to 10 mg/mL). The scattering pattern of the complex thus obtained was then used for further calculations.
The value of the R g was derived from the Guinier approximation (Guinier and Fournet 1955) : I(q) = I(0) exp(Àq 2 R g 2 /3), where I(q) is the scattered intensity and I(0) is the forward scattered intensity. The R g and I(0) are inferred, respectively, from the slope and the intercept of the linear fit of Ln[I(q)] versus q 2 at low q values (q R g < 1.0). The distance distribution function P(r) is the histogram of all the interatomic distances within a molecule. This function also provides the maximum dimension D max of the molecule, which is defined as the point where P(r) becomes zero. The P(r) function was calculated by the Fourier inversion of the scattering intensity I(q) using GNOM (Svergun 1992) and GIFT (Bergmann et al. 2000) on the entire scattering spectra.
The low-resolution shape of the N TAILHN -XD complex was determined ab initio from the scattering curve using the program GASBOR ). This program restores lowresolution shapes of protein and calculates a volume filled with densely packed spheres (dummy residues) fitting the experimental scattering curve by a simulated annealing minimization procedure and considering the protein as an assembly of dummy residues centered on the C a positions (spheres of 3.8 Å diameter) with a nearest-neighbour distribution constraint. Several independent fits were run with no symmetry restriction with an input of 188 dummy residues, corresponding to the 56 residues of X D and 132 residues of N TAILHN . The program package CREDO (CHADD and GLOOPY) was used to restore the low resolution model of N TAILHN in complex with XD, with the crystal structure of the chimeric protein between XD and the region of N TAIL encompassing residues 486-505 (XD-N TAIL486-505 ) (Kingston et al. 2004a ) as template. CREDO is an extension of the original program GASBOR. It calculates the structure of missing domains or loops of crystal structures from the experimental scattering curve of the entire particle and represents them by an ensemble of dummy residues forming a chain-compatible model.
Binding between purified XD and purified N TAIL proteins was analyzed by using BIAcore 3000 (Amersham Pharmacia Biotech), a system for real-time biomolecular interaction analysis that is based upon surface plasmon resonance technology. Purified XD (1.4 mg/mL in acetate buffer at pH 5.5) was covalently bound to carboxy-methyl groups of CM5 sensor chips using amine-coupling chemistry (Biosensor AB, Amersham Pharmacia Biotech). The levels of immobilized XD were comprised between 180 and 225 RU (1000 RU equal a change in mass of 1 ng/mm 2 on the sensor surface) (Zhang et al. 2002) . Kinetic and equilibrium constants were calculated from global analysis of reactions with multiple analyte concentrations (0.1, 0.2, 0.5, 1, 2, 5, and 10 mM). Reactions were performed at 25 C. Remaining flow channels on the sensor chip included a control for a nonspecific interaction with the sensor chip (i.e., activated/ blocked flow channel) and a control for nonspecific interactions with irrelevant protein targets (i.e., lactoferrin conjugated flow channel). Protein analytes were passed over the sensor chip in HBS-P buffer (0.01 M HEPES at pH 7.4, 0.15 M NaCl, 0.005% surfactant P-20) containing 2.5 mM magnesium acetate and 2.5 mM ATP. Sensorgrams plotted changes in surface plasmon resonance (measured in RU) as a function of time. Multiple sensorgrams representing various analyte concentrations were analyzed by using BIAevaluation 3.1 software. Background interaction of N TAIL proteins (i.e., analytes) with the sensor surfaces were measured on flow channels that were activated and subsequently blocked under buffer conditions used to immobilize XD. This background was subtracted from all binding curves (i.e., sensorgrams) prior to global analyses. Immobilized lactoferrin was used as a specificity control, and the resultant sensorgrams ruled out high affinity interactions between N TAIL constructs or peptides and this irrelevant protein ligand (data not shown). Global fitting of experimental data to well-characterized binding reactions was used to define reaction rate and equilibrium constants. Signal changes on the activated/ blocked control channel were subtracted from the peptide-XD and N TAIL protein-XD interactions using in-line reference and the subtracted sensorgrams were analyzed. Curves generated with serial analyte concentrations were applied globally to the 1:1 Langmuir binding model with or without correction for baseline drifting depending on baseline status. 2 and residual values were used to evaluate the quality of fit between experimental data and individual binding models. Plots of residuals indicate the difference between the experimental and reference data for each point in the fit. The 2 value represents the sum of squared differences between the experimental data and reference data at each point. A good fit between experimental and reference data has small residuals in the À2 to +2 range that randomly distribute about the X-axis, and 2 values are less than 10.
The CD spectra were recorded on a Jasco 810 dichrograph using 1-mm thick quartz cells in 10 mM sodium phosphate (pH 7) at 20 C. Structural variations of N TAIL proteins were measured as a function of changes in the initial CD spectrum upon addition of either increasing concentrations of TFE (Fluka), or different amounts of XD or lysozyme (Sigma). CD spectra were measured between 185 and 260 nm, at 0.2 nm/min and were averaged from three independent acquisitions. Mean ellipticity values per residue ([Q]) were calculated as [Q] = 3300 m DA/(l c n), where l (path length) = 0.1 cm, n = number of residues, m = molecular mass in daltons, and c = protein concentration expressed in mg/mL. Number of residues (n) are 140 for N TAILHNFC , 131 for N TAILD3 , 103 for N TAILD2,3 , 120 for N TAILD1 , 132 for N TAIL W518, 56 for XD, and 129 for lysozyme, while m values are 15,626 Da for N TAILHNFC , 14,523 Da for N TAILD3 , 11,539 Da for N TAILD2,3 , 13,440 Da for N TAILD1 , 6690 Da for XD, and 14,300 Da for lysozyme. Protein concentrations of 0.1 mg/mL were used when recording spectra of both individual and protein mixtures. In the case of protein mixtures, mean ellipticity values per residue ([Q]) were calculated as [Q] = 3300 DA/{[(c 1 n 1 / m 1 ) + (c 2 n 2 /m 2 )] l}, where l (path length) = 0.1 cm, n 1 or n 2 = number of residues, m 1 or m 2 = molecular mass in daltons, and c 1 or c 2 = protein concentration expressed in mg/mL for each of the two proteins in the mixture. The theoretical average ellipticity values per residue ([Q] Ave ), assuming that neither unstructured-to-structured transitions nor secondary structure rearrangements occur, were calculated as follows: [Q] Ave = [([Q] 1 n 1 ) + ([Q] 2 n 2 R)]/(n 1 + n 2 R), where [Q] 1 and [Q] 2 correspond to the measured mean ellipticity values per residue, n 1 and n 2 to the number of residues for each of the two proteins, and R to the excess molar ratio of protein 2. The a-helical content was derived from the ellipticity at 220 nm as described in Morris et al. (1999) .
Fluorescence intensity variations of the single tryptophan in N TAILW518 was measured by using a Cary Eclipse (Varian) equipped with a front-face fluorescence accessory at 20 C, by using 2.5 nm excitation and 10 nm emission bandwidths. The excitation wavelength was 290 nm and the emission spectra were recorded between 300 nm and 540 nm. Titrations were performed in a 1-mL quartz fluorescence cuvette containing 1 mM N TAILW518 in 10 mM sodium phosphate buffer at pH 7, and by gradually increasing the XD concentration from 0.05 mM to 3 mM. Experimental fluorescence intensities were corrected by subtracting the spectrum obtained with XD alone (note that XD is devoid of tryptophan residues). Data were analyzed by plotting the relative fluorescence intensities at the maximum of emission at increasing XD concentrations. The dissociation equilibrium constant (K Dapp ) value was determined from data fitted to a single exponential equation, by using the PRISM 3.02 nonlinear regression tool (GraphPad).
2D-HSQC spectra (Mori et al. 1995) were recorded on a 500-MHz DRX Bruker spectrometer either on 0.5 mM uniformly 15 N-labeled N TAILHN or on 0.125 mM uniformly 15 N-labeled N TAILD3 in 10 mM sodium phosphate buffer at pH 7.0 containing 10% D 2 O (v/v) alone or after addition of a twofold molar excess of XD. The temperature was set to 283 K and the spectra were recorded with 2048 complex points in the directly acquired dimension and 256 points in the indirectly detected dimension, for 6 h each. Solvent suppression was achieved by the WATERGATE 3-9-19 pulse (Piotto et al. 1992) . The data were processed using the UXNMR software; they were multiplied by a sine-squared bell and zero-filled to 1 K in first dimension prior to Fourier transformation. For the spectra recorded after addition of XD, the number of scans was multiplied by 4.
The accession number of MV N is P04851. Secondary structure predictions were carried out using both PSI-PRED (McGuffin et al. 2000) and PHD (Rost 1996) . L. Kingston for kindly providing us with the PDB file of the chimeric construct between XD and the N TAIL peptide (PDB code 1T6O). This study has been carried out with financial support from the Commission of the European Communities, specific RTD program ''Quality of Life and Management of Living Resources,'' QLK2-CT2001-01225, ''Towards the design of new potent antiviral drugs: Structure-function analysis of Paramyxoviridae RNA polymerase.'' It does not necessarily reflect its views, and in no way anticipates the Commission's future policy in this area. This work was also in part supported by the CNRS and by funds from the National Institute of Neurological Disorders and Stroke (R01 NS31693). A survey of knowledge, attitudes and practices towards avian influenza in an adult population of Italy BACKGROUND: Several public health strategic interventions are required for effective prevention and control of avian influenza (AI) and it is necessary to create a communication plan to keep families adequately informed on how to avoid or reduce exposure. This investigation determined the knowledge, attitudes, and behaviors relating to AI among an adult population in Italy. METHODS: From December 2005 to February 2006 a random sample of 1020 adults received a questionnaire about socio-demographic characteristics, knowledge of transmission and prevention about AI, attitudes towards AI, behaviors regarding use of preventive measures and food-handling practices, and sources of information about AI. RESULTS: A response rate of 67% was achieved. Those in higher socioeconomic classes were more likely to identify the modes of transmission and the animals' vehicles for AI. Those older, who knew the modes of transmission and the animals' vehicles for AI, and who still need information, were more likely to know that washing hands soap before and after touching raw poultry meat and using gloves is recommended to avoid spreading of AI through food. The risk of being infected was significantly higher in those from lower socioeconomic classes, if they did not know the definition of AI, if they knew that AI could be transmitted by eating and touching raw eggs and poultry foods, and if they did not need information. Compliance with the hygienic practices during handling of raw poultry meat was more likely in those who perceived to be at higher risk, who knew the hygienic practices, who knew the modes of transmission and the animals' vehicles for AI, and who received information from health professionals and scientific journals. CONCLUSION: Respondents demonstrate no detailed understanding of AI, a greater perceived risk, and a lower compliance with precautions behaviors and health educational strategies are strongly needed. The first known direct avian to human transmission of influenza A (subtype H5N1) viruses was reported during an outbreak in Hong Kong in 1997 and exposure to infected poultry was identified as the probable route of transmission [1] [2] [3] .
Since then, outbreaks of the H5N1 highly pathogenic avian influenza strain have been identified in birds, wild and domestic poultry, in several countries, particularly in Vietnam, Indonesia, Thailand, China, Cambodia and, more recently, in Turkey and Iraq. In Italy, no human cases have been reported and two epidemics occurred in poultry causes by avian influenza virus H5 and H7 subtypes. Alongside these massive avian outbreaks, the World Health Organization (WHO) reported more than 300 confirmed human cases of avian influenza A (H5N1), approximately two thirds of whom have subsequently died [4] . Nearly all of these cases are traceable to exposure to infected poultry or birds, but there has not yet been a mutation allowing the H5N1 and H7N7 viruses to spread efficiently in human [5] .
However, concern is widespread that the current situation favors the emergence of a highly pathogenic influenza virus with the ability for efficient transmission from person to person, particularly in the presence of a mutation in the viral genome leading eventually another pandemic human influenza.
Several public health strategic interventions are required for effective disease prevention and control of the multifaceted issues posed by avian influenza [6] . Of these interventions, it is necessary to create a communication plan to keep the population fully and adequately informed on how to avoid or reduce exposure. A similar approach has been applied during the SARS epidemic [7] and the implementation of appropriate infection control measures was a key aspect for its control. In the past years a limited number of studies have been published investigating knowledge, attitudes, and practice about avian influenza among target groups [8, 9] and general population [10] [11] [12] [13] . This area of investigation seems to be an important one because members of the public often misinterpret their risk of health problems. Therefore, the objectives of the present investigation were to assess the knowledge, attitudes, and behaviors relating to avian influenza and to evaluate the effect of several potential predictors on such outcomes of interest in an adult population in Italy.
This cross-sectional survey was conducted from December 2005 to February 2006 in the geographic area of Naples (Italy). A two-stage cluster sampling technique was employed to draw the required sample. In the area surveyed there were 40 schools and each school was considered a cluster. The first stage consisted of selecting four clusters through random sampling. The second stage consisted of randomly select 255 adults from the parents' files of each sampled school that contained 500 students.
The questionnaires used for this study were handed out, in sealed envelopes, to the Referent of the health education activities in each school with instructions to distribute one to each family. Families received an information sheet which explained the purpose of the project and requested that the survey be completed by one parent only, a self-administered anonymous questionnaire, and an envelope to facilitate the return of the completed questionnaire. In addition, the letter assured parents about the anonymity and confidentiality of all responses. Participants were asked to return the completed questionnaires to the school personnel anonymously via the envelope enclosed with each questionnaire. Participation was on a voluntary basis and all participants had the right to comply with or refuse participation. The response to questionnaire constituted the participants' informed consent. The study was approved by the Institutional Ethics Review Board.
The survey, that was a modification of an instrument previously used [8] , was arranged in different sections enquiring about participants' demographic and socioeconomic characteristics, knowledge of the definition, modes and vehicles of transmission, risk groups, and preventive measures about avian influenza, attitudes towards avian influenza, behaviors regarding use of preventive measures and food-handling practices, whether they had ever received advice and information about avian influenza and, if so, the sources. The response choices for all knowledge questions were given on a three-point Likert-type scale using "yes", "no", "do not know" options for the modes and vehicles of transmission, and risk groups and "agree", "uncertain", and "disagree" for the measures concerning the preventive measures; whereas, the response choice for the question about the knowledge of the definition was open. The responses for all statements relating to attitudes towards avian influenza, to ascertain level of agreement or disagreement were given on a three-point Likert-type scale (from 1 to 3, 1 = agree, 2 = uncertain, 3 = disagree); in two questions respondents were also asked to use a ten-point Likert-type scale to assess perceived degree of risk for contracting avian influenza for him/her and for friends/familiars with responses ranged from 1 (low risk) to 10 (high risk). Five possible categories of responses were allowed, ranging from never to always, to measure compliance with recommendations of the WHO to avoid spread of avian influenza through food [6] . We have also asked whether the respondent and/or member(s) in the household, in the three months preceding the survey, have modified the habits in buying foods or in dietary habits for fear of contracting avian influenza.
The original version of the survey instrument underwent a pilot study among a convenient group of 50 subjects to ensure practicability, validity, and interpretation of answers. On the basis of the comments obtained, the questionnaire was revised in item, wording, and format before distribution to the study sample.
Multivariate stepwise logistic and linear regression analyses investigated the independent contribution of potential predictors to the following primary outcomes of interest: knowledge about the main modes of transmission (animal-to-animal and animal-to-human) and the animals classified as common vehicles (poultry and birds) for avian influenza (model 1); knowledge that wash hands with soap before and after touching raw poultry meat and use of gloves is a hygienic practices to avoid spreading of the avian influenza virus through food (model 2); wash hands with soap before and after touching raw poultry meat and use of gloves (model 3); perception of risk of contraction avian influenza for him/her (model 4). For the purposes of analysis, the outcome variables originally consisting of multiple categories in the logistic analysis were collapsed into two levels. In Model 1, respondents were divided into those who knew the main modes of transmission (animal-to-animal and animal-to-human) and the animals classified as common vehicles (poultry and birds) for avian influenza versus all others; in Model 2, those who knew that wash hands with soap before and after touching raw poultry meat and use of gloves is a hygienic practices to avoid spreading of the avian influenza virus through food versus all others; in Model 3, those that wash hands with soap before and after touching raw poultry meat and use gloves versus all others. In all models the following independent explanatory variables were included: age, gender, marital status, educational level, number of children, employment status, health professionals and reading scientific journals as sources of information, and need of additional information. Others independent explanatory variables were also included in the different models: perception of risk of contraction avian influenza (models 1-3); knowledge about the modes of transmission of avian influenza (models 2 and 3); correct definition of avian influenza and know that avian influenza could be transmitted by eating and touching raw eggs and poultry foods (model 4); knowledge that wash their hands with soap before and after touching raw poultry meat and use of gloves is a hygienic practices to avoid spreading of the avian influenza virus through food (model 3). Before testing multivariable logistic regression models assessing predictors of the outcomes of interest, we examined correlations to assess collinearity among the independent variables and bivariate relations between the independent variables and the dependent variable. The criterion to be met before any independent variable was considered for entry into an initial multivariable logistic regression model was a p-value ≤ 0.25 obtained for each outcome variable in the univari-ate analysis and noncollinear with other predictors. Furthermore, the significance level for variables entering the logistic regression models was set at 0.2 and for removing from the model at 0.4. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated in the model for the independent variables. When building linear regression model, we have first included only one possible variable at a time. Then, using the variables that were significant at p-value ≤ 0.25, we constructed a stepwise multivariate linear regression model and the significance level for variables entry the model was set at 0.2 and for removal at 0.4. Statistical significance level was defined as a two-tailed p-value ≤ 0.05. Stata version 8.1 software program was used for all statistical analyses [14] .
The sample consisted of 683 individuals for a participation rate, defined as the number of completed questionnaires divided by the number of those randomly selected, of 67%. Socio-demographic characteristics of the respondents are reported in Table 1 . The average age was 40.7 years, two thirds were female, almost all were married, the majority had not reached college level education, more than half is inactive or housewife, and one-third has three or more children. Table 3 ).
Respondents did not recognize the major risk groups, since a large percentage agreed that poultry workers (88%) were at risk, but lower values were reported for butchers (55.1%), hunters (30.7%), and veterinarians (23.6%). Moreover, 34.6% knew that washing their hands with soap before and after touching raw poultry meat and using gloves is a hygienic practice to avoid spreading of the avian influenza virus through food. Those older (OR = 1.03; 95% CI 1.01-1.05), who knew the modes of transmission and the common vehicles for avian influenza (OR = 1.63; 95% CI 1.11-2.39), and who still need additional information about avian influenza (OR = 1.52; 95% CI 1.09-2.12) were more likely to know this practice (Model 2 in Table 3 ).
More than half of the respondents thought that avian influenza was a serious disease (61.9%) and that it was possible to prevent (53.3%). The respondents' level of perceived risk of contracting avian influenza for them and for friends/familiars resulted in a mean total score respectively of 5.9 ± 2.9 and 6.2 ± 2.8, indicating a high risk perception with respectively 19.3% and 20.4% of the respondents having reported feeling "very much" at risk by answering "10". A stepwise multivariate linear regression model was constructed to search for associations with the attitude, aiming at understanding which variable had stronger associations with the perception of risk of contraction avian influenza by the respondent. Respondents considered the risk for them of being infected significantly higher if they were from lower socioeconomic classes, had lower educational level, if they did not know the definition of avian influenza, if they knew that avian influenza could be transmitted by eating and touching raw eggs and poultry foods, and if they believed that they did not need additional information about the disease (Model 4 in Table 3 ).
Participants were asked about their behaviors regarding the use of preventive measures and food-handling practices. Approximately two-thirds reported that, in the three months preceding the survey, at least one member in the household had modified the habits in buying foods (63.3%) or in dietary (59.9%) for fear of contracting avian influenza. As regards hygienic practices to avoid spreading of the virus through food, among those who prepared food, only 26.9% reported washing their hands with soap before and after touching raw poultry meat and using gloves. Multivariable logistic regression analysis indicated that compliance with the hygienic practices was more likely by those who perceived a higher risk of contracting avian influenza (OR = 1.13; 95% CI 1.06-1.19), by those Table 3 ).
Almost all respondents recalled receiving some information about avian influenza (97.9%) mostly through mass media (85.8%), health professionals (26.5%), and scientific journals (8.4%). A vast majority (65%) reported interest in receiving further information on avian influenza.
The results of the present survey depict a mosaic of opinions outlining the stated knowledge, attitudes, and selfreported behavior patterns concerning avian influenza among a large cross-section of a random sample of an adult population in one region of Italy. Guidelines and recommendations have been developed to prevent and control the spread of avian influenza at source and in responding to the pandemic threat [6, [15] [16] [17] [18] [19] . These attempts to provide public health related measures in the community, in workers involved in outbreak disease control and eradication activities, in people involved in producing, marketing, and living with poultry, and in travellers who are visiting countries experiencing outbreaks. The main recommended measures which need to be used in concert, are: 1) intensify collaboration between the animal and public health sectors; 2) appropriate personal protective equipment for medical workers that transport/treat avian flu patients and for workers involved in the culling, transport or disposal of avian influenzainfected poultry; 3) effective disease surveillance for early detection and reporting of outbreaks; 4) food safety of poultry products; 5) control of movement of birds and products that may contain virus; 6) risk communication; 7) rapid, humane destruction of infected poultry and poultry at high risk of infection, and 8) proper use of vaccination.
Data gathered showed that a high number of respondents had no detailed understanding of avian influenza. Specifically, less than half answered correctly the questions on the modes and vehicles of transmission and on the recommended hygienic practices to avoid spreading of the avian influenza virus through food. Moreover, it was disturbing to note that detailed questioning revealed gaps in knowledge about the risk groups. That this happened, despite the fact that almost all received information about avian influenza from different sources, is troubling. In rural Thailand a community cluster survey on 200 people has shown widespread knowledge regarding avian influenza and the effective means of protection with, for example, 76% recognizing that people could get the disease from chicken or other poultry [11] . In our study, the investigation of correlates in multivariate comparison, which allowed us to control for different risk factors, yielded several interesting findings such as that older respondents with a higher educational level and from higher socioeconomic class were more likely to be knowledgeable. The inclusion of measures such as participants' level of schooling and employment status greatly reduces the size differences between groups. Failure to account for these socioeconomic factors would result in biased estimates and artificially high differences across groups. Finally, most respondents recognized that their knowledge on preventive measures was fair and indicated the need for increasing that knowledge. This finding is important because it has already been reported that public health education campaigns and general media reports about avian influenza appear to have been effective in reaching those who were at greatest risk of acquiring the disease through contact with backyard poultry [11] .
This study revealed a relatively high degree to which respondents themselves perceived themselves to be at risk of avian influenza virus as a health threat and demonstrated a readiness on the part of respondents to be educated. In our sample respectively 19.3% and 20.4% said that they were "very much" concerned about the risk of contracting avian influenza for them and for friends/ familiars in the future. In a previous telephone study, that examined the perceived risk of avian influenza from live chicken sales involving Hong Kong households, it was documented that one third of those surveyed perceived some risks and almost 50% indicated that their friends had expressed anxieties [10] . It has been well established that worried individuals were significantly more likely to have received advice or instruction about the disease by health professionals and by reading scientific journals and to feel the need for more information. Our findings do suggest that the perception of risk was a significant determinant of greater compliance with recommended precautionary procedures. Indeed, respondents worried about their own risk were more likely to use gloves and wash hands with soap before and after touching raw poultry meat. Such findings suggest that the population would both welcome and benefit from tools and strategies that would help them to reduce their fear because it is important in whether or not they adhere to these procedures.
The respondents to our questionnaire exhibited higher compliance with recommendations of the WHO to avoid spread of avian influenza through food [6] , such as hand washing and using protective gloves, when compared with the findings reported in two previous surveys about food-borne diseases. In one study only 20.8% of respondents claimed that they used protective gloves, 53.9% reported washing hands before and after touching raw and unwrapped food, and 50.4% reported using soap to wash hands [20] . In the other one respectively 68.7% and 66.2% of food handlers routinely washed their hands before and after handling any food [21] . As we hypothesized, in accordance with a previous study, knowledge influences behavior [8] . Our survey indicates a significant association between those who fail to wash hands and to use gloves and the lack of knowledge that these are standard hygienic practices to avoid spreading of the virus through food. In addition, it is notable that the main source of information was the media and not qualified healthcare representatives. It has been observed that our results confirmed the hypothesis that those who received information from health professionals and scientific journals had higher relevant compliance than those who did not receive information from these sources. These findings support the importance for media campaigns to implement educational and policy strategies and to increase patient education by medical professionals in the context of routine medical care.
Despite the novelty and significance level of these findings, some methodological considerations ought to be highlighted when interpreting our results. First, the analyses were based on cross-sectional data and the findings of the analysis of factors associated with the outcomes of interest should be interpreted with caution given the nature of the associations that limited us from drawing definitive causal conclusions or direction of causality about the observed relationships between among independent characteristics and the outcomes measured. However, we feel that our study design was adequate to assess the knowledge, attitudes, and behaviors relating to avian influenza and identify what percentage of these could be explained by several potential predictors. Second, the study was limited to those parents of children in randomly selected schools, which may have implications for the generalizability of the results. However, because in our country the education is mandatory until the age of 16 irrespective of the characteristics of the parents, we believe our results are generalizable to all population. Third, all variables used in this analysis were gathered using entirely respondents' self-reports and self-perceptions, and biases in perception and reporting cannot be ruled out. The problem with self-reporting is that participants' responses may reflect intentionally or unintentionally perceived desirable responses or an attempt to inflate or minimize reports of behaviors. However, to gain a true reflection of knowledge and behavior, respondents were assured that their responses would not be shared, since the survey was delivered with the responses that were anonymous to encourage accurate recording. Fourth, it was not possible to identify characteristics of those who failed to return the questionnaire, so it was not possible to establish whether they were in any way different from those who did return it. However, there is no obvious reason to suspect that non-responders were substantially different from responders. Despite the potential limitations, the main advantage of the current study is that we were able to achieve a relatively large sample and the high response rate excludes one major potential source of bias in the results. We believe that this high response rate was made possible through the extreme importance of the topic surveyed.
In conclusion, the results of this study illustrates that, despite being given information, respondents had no detailed understanding of avian influenza, had a great perceived risk of experiencing avian influenza, and had a low compliance with precautions behaviors. These observations raise concerns about a clear need to find the optimal way of correcting these deficiencies by developing and implementing public health policy regarding priorities for tailored educational and promotion strategies and in particular more attention should be given on using preventive approaches in these population. Nevertheless, it is important to consider that dissemination and widespread adoption of preventive measures require education. Encouragingly, respondent's interest in learning more about avian influenza was high in our survey. Therefore, designing and implementing avian influenza educational programs and measuring their effectiveness should be priorities to incentive the population to take a more active role. The evolution of human influenza A viruses from 1999 to 2006: A complete genome study BACKGROUND: Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. Few complete genome sequences of influenza A viruses from Europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza A viruses. We have studied the evolution of circulating human H3N2, H1N1 and H1N2 influenza A viruses from 1999 to 2006, we analysed 234 Danish human influenza A viruses and characterised 24 complete genomes. RESULTS: H3N2 was the prevalent strain in Denmark during the study period, but H1N1 dominated the 2000–2001 season. H1N2 viruses were first observed in Denmark in 2002–2003. After years of little genetic change in the H1N1 viruses the 2005–2006 season presented H1N1 of greater variability than before. This indicates that H1N1 viruses are evolving and that H1N1 soon is likely to be the prevalent strain again. Generally, the influenza A haemagglutinin (HA) of H3N2 viruses formed seasonal phylogenetic clusters. Different lineages co-circulating within the same season were also observed. The evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the Fujian-like viruses in 2002–2003. Also evolutionary stasis-periods were observed which might indicate well fit viruses. The evolution of H3N2 viruses have also been influenced by gene reassortments between lineages from different seasons. None of the influenza genes were influenced by strong positive selection pressure. The antigenic site B in H3N2 HA was the preferred site for genetic change during the study period probably because the site A has been masked by glycosylations. Substitutions at CTL-epitopes in the genes coding for the neuraminidase (NA), polymerase acidic protein (PA), matrix protein 1 (M1), non-structural protein 1 (NS1) and especially the nucleoprotein (NP) were observed. The N-linked glycosylation pattern varied during the study period and the H3N2 isolates from 2004 to 2006 were highly glycosylated with ten predicted sequons in HA, the highest amount of glycosylations observed in this study period. CONCLUSION: The present study is the first to our knowledge to characterise the evolution of complete genomes of influenza A H3N2, H1N1 and H1N2 isolates from Europe over a time period of seven years from 1999 to 2006. More precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition. Every year the influenza A virus causes human infection with varying severity depending on the host acquired immunity against the particular virus strain. Three to five million people experience severe illness and 0.25 to 0.5 million people die of influenza yearly worldwide (WHO EB111/10). The influenza virus evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA) or by reassortment of segments from different viruses co-infecting the same cell leading to a new stain with a HA (and NA) not seen in the population before (shift). In the worst case, shifts may cause pandemics. There have been three pandemics the last hundred years, the Spanish flu in 1918 (H1N1), the Asian flu in 1957 (H2N2) and the Hong Kong flu in 1968 (H3N2). It is believed that new pandemics emerge through shifts with strains from the avian reservoir, as was the case of the pandemics of 1957 and 1968, or by direct introduction of an avian strain into the human population as suggested for the 1918 pandemic [1] . At present only two of the 16 possible HA subtypes (H1 and H3), and two of the nine possible NA subtypes (N1 and N2) are circulating in man. H3N2 and H1N1 influenza A viruses have co-circulated in the human population since the re-emergence of H1N1 in 1977, increasing the possibility for genetic reassortments. The prevalence of the different subtype combinations may vary from season to season. The H3N2 has been the predominant influenza A strain during the last 20 years, with the exception of the 1988-1989 and 2000-2001 seasons where H1N1 infections dominated [2] . In the 2000-2001 season a new reassorted human strain, H1N2, emerged in Europe and became established in the autumn 2001 [3, 4] . The new H1N2 subtype was covered by the 2002-2003 H1 and N2 trivalent vaccine components and because both H1 and N2 viruses had circulated the previous years some degree of herd immunity against the new strain was expected. The H1N2 viruses were not associated with severe influenza illness that season. In 2002, a new lineage A/Fujian/411/02(H3N2)-like emerged in Asia and caused significant outbreaks on every continent [5, 6] .
For the northern hemisphere the WHO issues the recommendation for strains to be included in the trivalent vaccine for the next season based on epidemiological data and antigenic and genetic analyses of circulating strains.
Until the recent release of over 1,800 complete influenza A genome sequences from the Influenza Genome Sequencing Project managed by US National Institute of Allergy and Infectious Diseases [7, 8] 
The relative prevalence of influenza virus varies from season to season. Influenza A H3N2 was the dominating strain in Denmark during the last seven years, with the exception of the 2000-2001 season where the H1N1 viruses dominated, as can be seen in Figure 1 . 
Based on phylogenetic analysis of the HA and NA nucleotide sequences from 1999 to 2006 (Figure 2 ), ten isolates representative for the phylogenetic clustering of sequences from each subtype in each season, as far as possible, were included in the final HA and NA tree ( Figure 2 ) and representatives were chosen for complete genome sequencing. Generally the H3N2 HA and NA genes formed seasonal phylogenetic clusters ( Figure 2 ). However, we observed that strains of different lineages and clusters cocirculated within the same season and that viruses had reassorted with viruses from previous seasons ( Figure 2 ).
The HA gene of the influenza H3N2 strains from the 1999-2000 season formed a phylogenetic subclade to A/ Moscow/10/99(H3N2) and A/Sydney/5/97(H3N2) (represented by A/Memphis/31/98) (Figure 2 ), located between A/Moscow/10/99 and A/Panama/2007/99 (not shown). The antigenicity of these strains was A/Moscow/ 10/99(H3N2)-like in a haemagglutination inhibition assay, and will therefore be referred to as Moscow-like throughout this report. The NA and the internal genes were all A/Moscow/10/99(H3N2)-like, with the exception of the matrix (M) gene that clustered as a subclade to the A/New York/55/01-like strains ( Figure 3 ). Figure 1 ). Thirteen isolates from this season were available for sequencing, and all were of the H1N1 subtype. These sequences represented two different co-circulating lineages (Figure 4 ). Lineage I is A/Bayern/7/ 95(H1N1)-like and lineage II include the H1N1 strains of today and the A/New Caledonia/20/99(H1N1) vaccine reference strain (Figure 4 ). The phylogenetic trees of NA and the internal genes showed the same topology ( Figure  3 and 4) . The lineage II strains are characterised by a deletion K130 in HA (K134 in H3 numbering) ( Table 1) 
* Amino acids in brackets indicate less than half but more than two substitutions at the given amino acid position within a season. A single amino acid change in one position is not shown. Amino acids separated by '/' indicate equal substitutions of either amino acid at the given position. Letters in upper case above an amino acid indicate the antigenic site location of the residue. In N1 the upper case letter ' P ' stands for phylogenetically important region (PIR) and the following letters indicate the actual PIR. Based on the ratio of non-synonymous versus synonymous substitutions none of the influenza A genes were directly influenced by positive selection (dN/dS<1) (Table  3) . However, as expected the HA1 region of both H3 and H1 viruses were more influenced by evolutionary pressure (Table 3) . We applied FEL and SLAC maximum-likelihood methods to estimate individual positively selected sites in H3N2 HA and NA and added REL for smaller datasets in all genes (se methods section). The FEL method found one site in the H3 protein (n = 204), position 199 (p = 0.046) to be positively selected, while the more conservative SLAC analysis found none. No positive selected sites were predicted for the N2 genes (n = 166) and none in the internal genes (n = 15) estimated by FEL and SLAC. The REL analysis retrieved four sites in the M1 gene to be selected namely positions 208, 211, 218 and 219. No sites in HA (n = 27) and NA (n = 30) of the H1N1 viruses were directly positively selected with any of the three methods of analysis. [26] and it was anticipated that there would be some extent of herd immunity in the population against this new reassortant.
The phylogenetic trees of H3N2 HA and NA showed seasonal clusters but also co-circulating lineages within seasons. The introduction of the A/Fujian/411/02(H3N2) strains in 2002-2003 caused a "jump" in the evolution of both HA and NA genes. Many of the substitutions in HA introduced with the A/Fujian/411/02(H3N2)-like viruses have become fixed, probably reflecting a very fit virus. The genetic variation before the 2003-2004 season may have been more influenced by introduction of new viruses through viral migration than adaptive evolution of the genes. A constant rate of drift was not observed for HA but instead periods of change followed by stasis periods. The low dN/dS ratios (0.232 for HA and 0.247 for NA) also indicated that the influenza genes were not directly influenced by positive selection. Reassortments between co-circulating strains and viruses from previous seasons, and introduction of viruses from other parts of the world might play a larger role than natural selection for some seasons, as also observed by others [27, 28] . [2, 4, 25, 26] . The reassorted H1N2 viruses possessed only the HA from A/New Caldonia/20/ 99(H1N1)-like viruses and the rest of the genome from A/ Moscow/10/99(H3N2)-like viruses as also reported by others using partial sequences [25] . The H1N2 viruses have been introduced to Denmark from elsewhere and are not a local reassortant. (Table 1) suggesting they may be important for viral escape from the host immune system and the overall fit of the virus.
The A/Fujian/411/02-like(H3N2) viruses did not antigenically match the A/Moscow/10/99(H3N2) strain included in the 2002-2003 vaccine [29, 36] . The HA substitution D144N was however not responsible for the antigenic drift of the Fujian-like viruses. It has been shown that only two amino acid changes, H155T and Q156H, specified the antigenic difference from Moscow-like to Fujian-like [37] , both are located at antigenic site B. The T155 and H156 amino acids have been maintained in all Danish isolates after the introduction of the Fujian-like viruses. With the A/California/7/04(H3N2)-like viruses in 2004-2005, site 145 has changed from K to S in some isolates and to N in others. S145 has been found at this position before 1999 and N145K was the only cluster-difference substitution between isolates from the seasons 1987/ 1989 and 1992/1995 [38] . It was therefore suggested that substitutions at this site alone could have large antigenic effect. Antigenic site A, located in a loop, makes few contacts with the rest of the structure, therefore 144 and 145 may change drastically without influencing on the overall shape of the HA molecule.
Antigenic site A is supposed to be ideal for antibody binding and for amino acid replacements [33] . The preferred antigenic site in the change from A/Moscow/10/ 99(H3N2) to A/Fujian/411/02(H3N2)-like viruses was site B. This observation is in accordance with previously published data [39] . One region in HA, position 225 to 227, that influence antigenic site D, has changed drastically during the study period from GVS → DVS → DIP → NIP. The influence of antigenic site D was first apparent in the antigenic change from Fujian-like viruses to California-like viruses; however, the antigenic site B was still the preferred site for antigenic change. It has been proposed that a minimum of four substitutions in two or more antibody binding sites are required for an epidemically important strain [40] . 
Gulati et al., [41] [22, [42] [43] [44] [45] .
The substitution H274Y is the only neuraminidase resistance mutation identified in H1N1 viruses to date [46, 47] . Danish isolates did not possess any resistance mutations in the NAs. Neuraminidase inhibitory drugs are rarely used for influenza prophylaxis or treatment in Denmark.
T-cell epitopes are more conserved than antibody epitopes. Fifteen per cent of the T-cell epitopes are conserved while only 2.7% of the antibody epitopes [48] . The reason for this higher degree of conservation is that 80% of the antibody epitopes are located in the variable glycoproteins HA and NA, while only 40% of the T-cell epitopes are found in these proteins [48] . Recent research has shown some degree of escape from CTL-mediated immunity in addition to escape from neutralizing antibodies [28] . In our dataset we found several substitutions in regions involved in protective T-cell response [48] in NA, PA, M1, NS1 and most in the NP protein. This is not unexpected because most T-cell epitopes are defined for the NP protein and this protein is the main target for the cytotoxic host immune response [49, 50] . The extensive variations in the T-cell epitopes during 1999 to 2006 suggest that these regions and the antibody epitopes are working together for efficient escape from the host defence responses.
The M2 proteins from the Danish Wisconsin-like viruses in 2005-2006 possessed the substitution S31N, associated with resistance to matrix inhibitory drugs, like amantadine [22, 44, 45, 51] . These types of drugs are not used for prophylaxis or treatment in Denmark. The S31N substitution is therefore not a local introduced resistance mutation. We cannot exclude that this substitution has arisen by chance, but it is more likely that the mutation has emerged as a resistance mutation in other countries like the USA [52] and Australia [53] where the prevalence of amantadine resistance is high. The resistance may also be related to the increased use of this drug in Asia during the SARS epidemic [21] .
The A/Fujian/411/02(H3N2)-like clinical Danish viruses had several substitutions in HA at sites that might influence the virus' capability for egg growth [10, 37] . These include; A131T, I144N, H155T, Q156H, W222R and G225D. The A/California/20/99(H3N2)-like viruses had further changes at positions K145S/N, Y159F, S193F and V226I and A/Wisconsin/67/05(H3N2) possessed in addition S193F and D225N. All isolates after the 1999-2000 A/Moscow/10/99(H3N2)-like viruses possess S186G. In recent years H3N2 viruses have had poor replication efficiency in eggs [54, 55] . It has been shown that positions 186, 226 and 196 are critical determinants for egg growth.
The changes G186V and V226I increased egg viral replica-tion of A/Fujian/411/02(H3N2) viruses so did the changes G186V and A196T for A/California/20/ 99(H3N2) viruses [56, 57] . On the contrary, others have stated that the V226I change in combination with T155 and H156 do not result in viral recovery in eggs [37, 54] . This might explain the delay in the 2006-2007 vaccine production for the northern hemisphere due to egg propagation difficulties with the A/Wisconsin/67/05(H3N2) strain. The influence on replication efficiency by the other substitutions observed at receptor binding sites should be investigated further.
We did not observe amino acids in the N2 NA protein that would decrease virus replication in eggs. The amino acids known to give good replication in eggs (Q119, K136 and Y347) [56] were all present in this dataset.
Oligosaccharides at the surface proteins HA and NA might have greater impact on viral escape from the immune system than single amino acid changes in the antigenic sites. Oligosaccharides which are recognised as "self" by the host immune system may pose conformational changes in the molecule and mask antigenic sites, thereby prevent binding of host antibodies. The number of N-linked glycosylation sites in the H3 HA protein has increased during the years from only three attachment sites in 1968 [58, 59] to ten predicted sites in the Danish isolates after 2004.
The A/Fujian/411/02(H3N2)-like stains from the 2002-2003 season gained a potential glycosylation site at position 144, thereby masking the supposed "key" site for antigenic change [33] . Substitutions at antigenic site B and the predicted N-glycosylation at position 144 in HA antigenic site A together with a stronger NA might have contributed to the increased infectivity of the reassorted Fujian-like viruses of the 2003-2004 season, causing an epidemic in Denmark. We have shown that the preferred antibody epitope for genetic change is antigenic site B for the Danish dataset also reflecting that site A is camouflaged by glycosylation. Thus the antigenic changes at a glycosylated site A may not play a major role in escape from the immune system as long as the glycosylation is present.
Six potential N-glycosylation sites have been conserved in the N2 NA Danish dataset from 1999 to 2003. The majority of isolates from 2003 to 2006 have lost the site at position 93 which is located in a CTL epitope (HLA-A*0201) region of NA [12] . The recent NAs may therefore be more easily recognised by the cytotoxic immune system. We found two predicted N-glycosylation sites (61 and 70) in the N2 NA stalk region in sequences from 1999-2006. Greater density of carbohydrate in the stalk region of NA might reflect a need for proteolytic protection. The two observed carbohydrates in the stalk have been reported for other isolates in other time periods and other continents [58, 60] . The stalk region has therefore stayed unchanged and the two sequons seem to be conserved.
As expected a higher dN/dS ratio was observed for the surface glycoproteins, although none were directly influenced by positive selection. We observed that the HA1 region and the M2 protein have a slightly higher global dN/dS ratio than the other genes (Table 3 ). This is consistent with the findings of others [11, 61] . The M2 protein is a membrane ion channel protein on the surface of the virus molecule, a higher dN/dS ratio for this protein compared to the internal proteins is expected. The ratio might, however, be biased because the M2 protein is spliced from M and the dS is suppressed for overlapping regions giving a higher dN/dS ratio [11] .
Earlier findings support positive selection at sites involved in receptor and antigen binding [11, 62] . Five of the 18 codons in HA proposed by Bush et al., [63] to be under positive selection were found to have changed in our HA dataset of H3N2 since 1999, namely: pos 145, 156, 186, 193 and 226. In our H3 dataset (n = 204) site 199 was influenced by positive selection as calculated by FEL analysis, but no positively selected sites were found applying the more conservative SLAC method. Position 199 may be involved in receptor binding and influence on the virus ability to grow in eggs [10] . In a similar study on a slightly larger dataset (n = 284) positions 220 and 229 were found to be positively selected [11] . Another study found positions 13 and 236 [62] ; however, suggested positively selected sites may vary by the dataset applied, method used and the significance level selected for a site to be classified as positively selected. REL analysis identified four sites (208, 211, 218 and 219) in M1 under positive selection pressure. REL analysis tends to give better estimates on small datasets than SLAC and FEL. Sites 211, 218, and 219 were still selected when the bayes factor cut-off was increased from 50 to 200. Further analysis would be needed to determine if these sites actually are positively selected.
There is a need for complete genome analysis of European human influenza A viruses in order to gather a comprehensive picture of the evolution and migration of viruses.
Our results support the suggestion that the evolution of influenza A viruses is more complex than originally believed [28, 62] . Local short term evolution of influenza virus is a stochastic process, also involving gene reassortments. It will be interesting to further investigate how viruses from other parts of Europe influence on the evolu-tion of Danish isolates when more full length sequences from Europe are made public. 
Viral RNA was extracted from 140 µl of human nasal swab suspension or nasopharyngeal aspirate by QIAamp ® Viral RNA Mini Kit (QIAGEN, Germany) as described by the manufacturer or by an automated MagNA Pure LC Instrument applying the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Basel, Switzerland). The different gene segments were amplified by OneStep RT-PCR Kit (QIAGEN) as previously described [64] , applying a two minute elongation step for all genes. The primers for RT-PCR were segment specific but subtype universal targeting the highly conserved noncoding RNA regions at the 5'-and 3'-end of each segment [65] . PCR products were purified using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Germany) prior to sequencing.
Purified PCR products were sequenced directly. Primer sequences are available upon request. The sequencing reaction was performed by ABI PRISM ® BigDye™ Terminators v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) as described previously [66] . The sequences were developed on an automatic ABI PRISM ® 3130 genetic analyzer (Applied Biosystems) with 80 cm capillaries. Consensus sequences were generated in SeqScape ® Software v2.5 (Applied Biosystems). Sequence assembly, multiple alignment and alignment trimming were performed with the BioEdit software v.7.0.5 [67] . Distance based neighbor joining (NJ) phylogenetic trees and character based maximum parsimony (MP) trees were generated using the Molecular Evolutionary Genetics Analysis (MEGA) software v.3.1 [68] . Maximum likelihood trees were generated by the Phylogenetic Analysis Using Parsimony (PAUP 4.0) Software (Sinauer Associates, Inc.) [69] applying the HKY85 nucleotide model, allowing transitions and transversions to occur at different rates.
The between-seasons nucleotide distance means were computed as the arithmetic mean of all pair wise distances between two seasons in the inter-season comparisons using the MEGA v.3.1 software [68] . The global rate between dN and dS substitutions and the individual sitespecific selection pressure were measured by the likelihood based single likelihood ancestor counting (SLAC) method in Datamonkey (modified Suzuki-Gojobori method) [70, 71] . For datasets over 100 sequences (H3 n = 204 and N2 n = 166) the HyPhy package [72] was applied. The estimations are likelihood-based, employing a codon model cross between HKY85 and MG94. To elucidate single, positively selected amino acids, the HA and NA datasets were analysed with SLAC and a two rate fixed effects likelihood (FEL) [73] using a likelihood approach with neighbor joining phylogenetic trees (HyPhy). Sites with dN>dS with a <0.05 significance level for likelihood ratio test (LRT) were implied as positively selected for the large HA and NA datasets. For the small datasets (genes coding for the internal proteins <15 and the H1N1 viruses <30) we additionally ran a random effects likelihood (REL) analysis using an empirical Bayes approach with NJ phylogenetic trees in Datamonkey [70] . This method is expected to calculate positively selected sites more accurately in small datasets. The accepted significance level for a positively selected site was set at <0.1 (two-tailed binominal distribution) for SLAC and FEL analyses and >50 bayes factor for REL.
Potential N-linked glycosylation sites were predicted using nine artificial neural networks with the NetNGlyc 1.0 Server [74] . A threshold value of >0.5 average potential score was set to predict glycosylated sites. The N-Glycosite prediction tool at Los Alamos [75] was used to visualise the fraction of isolates possessing certain glycosylated sites along the sequence alignment.
The specific measure of antigenic distance between two strains of influenza were calculated as P epitope values by the method suggested by Muñoz, et al., [39] . The P epitope value was calculated as the number of mutations within an antibody antigenic site divided by the number of amino acids defining the site. It is assumed that an antigenic epitope which has the greatest percentage of mutations is dominant, because that epitope is influenced by the greatest selective pressure from the immune system. The P epitope distance is defined as the fractional change between the dominant antigenic epitopes of one strain compared to another. The P epitope Calculator [76, 77] was applied for H3 sequences. Residues in antigenic epitopes were collected from several references [9, 13, 39, 40, 48, [78] [79] [80] [81] [82] [83] 83, 84] . Can "presumed consent" justify the duty to treat infectious diseases? An analysis BACKGROUND: AIDS, SARS, and the recent epidemics of the avian-flu have all served to remind us the debate over the limits of the moral duty to care. It is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints. The purpose of this study was to investigate the opinions and beliefs held by both physicians and dentists regarding the occupational risks of infectious diseases, and to analyze the argument that the notion of "presumed consent" on the part of professionals may be grounds for supporting the duty to treat. METHODS: For this cross-sectional survey, the study population was selected from among physicians and dentists in Ankara. All of the 373 participants were given a self-administered questionnaire. RESULTS: In total, 79.6% of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. Of the participants, 5.2% said that they would not have chosen this profession if they had been informed of the risks. It was found that 57% of the participants believed that there is a standard level of risk, and 52% of the participants stated that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented. CONCLUSION: If we use the presumed consent argument to establish the duty of the HCW to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. Our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. Furthermore, in terms of the moral duty to provide care, it seems that most HCWs are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. For all these reasons, the presumed consent argument is not persuasive enough, and cannot be used to justify the duty to provide care. It is therefore more useful to emphasize justifications other than presumed consent when defining the duty of HCWs to provide care, such as the social contract between society and the medical profession and the fact that HCWs have a greater ability to provide medical aid. In the course of providing health care service, health care workers (HCWs) are continually exposed to many workrelated health risks. One of these risks is the exposure to infectious diseases. These diseases can include the flu, AIDS, tuberculosis, and hepatitis, and can be transmitted through physical contact, exposure to contaminated blood, or via the respiratory system. And, needless to say, such risks do indeed at times prove fatal. The consequences of occupational exposure to pathogens are not limited solely to bodily infections. Each year, thousands of HCWs are adversely affected by psychological trauma stemming from months of anxiously awaiting the results of serological tests, tests made necessary due to potential infection incidents. The anxiety experienced by HCWs is related to the perception of risk from the incident and the resulting infection that may occur, and by the worry of what the reactions of others might be, such as colleagues, family, and friends, all who have to be informed. During this uncertain waiting period HCWs will frequently experience intrusive thoughts, problems concentrating, difficulty sleeping, frequent loss of temper, and a decrease in sexual desire, which can act as a catalyst to exacerbate any pre-existing and unresolved emotional issues [1] . And if it turns out that the health care worker has indeed been infected by one of these contagions, the serious personal consequences to that health care worker can include the postponement of childbearing, damaged personal relationships, having to alter sexual practices, experiencing the side effects of prophylactic drugs, chronic disabilities, loss of employment, denial of worker compensation claims, possible need for a liver transplant, and premature death [2] . AIDS, SARS, and the recent epidemics of the avian-flu have all served to remind us of the occupational risks faced everyday by HCWs; the result being that the recent appearance of these diseases has forced this issue onto the common agenda and helped to spark renewed interest in the debate over the limits of the moral duty to treat. In seeking an answer to this question it is useful to have an understanding of the occupational risks faced by HCWs as well as an understanding of the attitudes of HCWs to these risks. For example, studies conducted in various countries have shown that, especially when there was a risk of being infected with AIDS, HCWs may refuse to treat a patient on the grounds that there is a risk of being infected by this patient [3] [4] [5] [6] [7] [8] [9] [10] [11] . And despite the fact that the hepatitis viruses are transmitted more easily than HIV, it is the fear of being infected with HIV that causes many HCWs to experience the greatest amount of stress and anxiety [12] . In a study which compared the relative risks of transmission of both HBV and HIV, the reasons for physicians' underlying fears of particular contagions were also inves-tigated and described. [13] . According to the study, people initially percieve the risk to be greater when there is a high likelihood of death involved with infection (as with HIV) even though there may be less risk of infection, as opposed to when there is a higher risk of infection but a lower risk of death involved with that infection (as with HBV). Additionally, since the likelihood of sexually transmitting HBV between heterosexual partners is less than that of transmitting HIV, the consequences of HBV infection are again percieved to be less severe than the consequences of HIV infection. In this way, the hazards posed by HBV infection conflict less with the obligation to protect family members from harm. It was also found to be important that there is less of a stigma attached to having HBV than there is to having HIV. And, finally, the fact that there is a vaccine for HBV infection, which is more than 90 percent effective (for vaccinated HCWs the risk of death from infection is reduced by a factor of nearly twenty), also was found to greatly influence the perceptions of the physicians.
Additionally, factors other than a fear of the contagion can contribute to the reluctance to treat a particular patient. Some physicians and dentists express concern that if it is discovered that they treat patients with AIDS, then those patients who don't have HIV may shun their practice. Still, other physicians insist they do not know enough about HIV infection and are too busy to learn [14] . Another reason for which a physician may refuse to treat HIV-positive patients is that the physician feels they have a duty to protect their other patients, basing their reasoning on the principle of "First do not harm". By treating HIV-positive patients they claim that they may potentially be putting their other patients at risk for infection [15] . Furthermore, as has been reported, there is always the possibility that when a HCW is able to reject the patient based on a more benign excuse, for example if the patient does not have enough money, it is even easier, and all the more likely, for treatment to be refused, even though this refusal was done in the interest of protecting the physical health of the individual health care provider [14] .
In the literature, most studies have concentrated primarily on the attitudes and rationale behind the refusal to treat. Before one can set out to effectively explore the attitudes of HCWs however, it is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints, such as providing health care to a patient suffering from an infectious disease which may be particularly contagious or for which adequate treatment measures may not yet be available.
Clark, in his article about physicians' duty to treat, claimed that there are three reasons in which such a duty is grounded [16] : "... since the ability to render aid is greater, the obligation to assist is (...) elevated. Second, by consideration of Daniels' argument that by freely joining a profession designed to combat disease, one consents to some standard of risk, and third, by realizing that the profession has flourished due to socially negotiated promises to be available in such times of duress."
In his article "Duty to treat or right to refuse", Daniels argues that when a person chooses a career in a particular profession, it must be understood by all parties that this individual has both accepted and is willing to take the risks that are inherent to that profession [14] :
"Consent is crucial where obligations to take risks exist in various occupations or professions. For example, we assume that in choosing their careers, undergoing the training involved, and agreeing to follow the codes and practices regulating their work, firefighters and police have given consent to facing the significant risks they are obliged to take. There are strong parallels to medicine. People who enter medical fields clearly had alternatives. There is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training." Daniels proposes, however, that some situations can exceed the standard level of risk (SLR) [14] :
"For example, it is common to screen new house staff and nurses in medical centers to determine whether any individuals face special risks of contagion, such as immunosuppression or pregnancy. Those at high risk may then be asked to avoid certain treatment situations, materials, or hospital areas.(...) Protecting immunosuppressed providers is reasonable "risk management", a measure taken to reduce bad outcomes. But such special protection supports the claim that only standard risks are included in the duty to treat.(...) Some nosocomial risks clearly take us beyond what duty requires."
It is perhaps more illustrative if this argument (from this point on, this statement will simply be referred to as the "presumed consent") is written in classic form:
Health care services should be provided to patients who have a contagious disease.
Contracting an infectious disease while providing health care services to a patient with a contagious disease is an occupational risk.
It is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion. This assumption is based on the following facts:
a. There is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training.
b. People who enter medical fields clearly had alternatives.
Some nosocomial risks clearly take us beyond what duty requires.
There is a moral duty to treat patients who have a contagious disease so long as the risk to the HCW is below the SLR.
If we are to accept this argument, then the pressing question becomes how to determine and define the risks which are deemed to be standard and acceptable versus those which are believed to exceed and, indeed, outweigh the duty of the health care provider to treat. In order to begin to answer this question, it will be useful to investigate the nature of the choice (and all that goes along with making it) that an individual makes when they decide to enter a particular profession. For instance, how wise is it to assume that at the time of choosing their future profession the HCW was fully aware of the risks involved with such work? Perhaps they were not made aware of the risks until their education and training. Furthermore, if they were aware of, and fully appreciated, the risks prior to deciding on a particular profession, would they have even chosen that profession in the first place? And, finally, how is the SLR to be determined, and which of the infectious diseases would then exceed this SLR? In order to effectively analyze the presumed consent argument it is necessary to have an awareness of the diverse opinions and beliefs of HCWs and, also, to understand their different motives and backgrounds. Additionally, knowledge of what HCWs feel about the risk concept and of how they feel about their duty to treat patients with contagious diseases can also be of great value to educators as they plan their curricula and it can be used by the authorities in charge of health care systems in order to better organize their services. The purpose of this study was to analyze whether or not the third premise grounds the duty to treat, namely, "it is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion". In order to carry out this analysis, the opinions and beliefs of physicians and dentists regarding the occupational risks of infectious diseases were investigated; and, by extension, the argument that the notion of "presumed consent" may be grounds for supporting the HCWs' duty to treat was also analyzed.
For this cross-sectional survey, the study population was selected from among physicians and dentists in Ankara, the capital of Turkey. A self-administered questionnaire designed to assess the beliefs and opinions of the participants regarding the occupational risks of infectious diseases was used. This questionnaire was also used to obtain the socio-demographic information of the participants. The 17 items on the questionnaire were developed by reviewing previous studies in the literature [1, [3] [4] [5] [6] [7] [8] [9] [10] . A draft of the questionnaire was distributed to experienced health care professionals and later revised based on their criticism and suggestions.
In both of the universities in which this study was conducted there are ethics committees which had been established for the purpose of determining the ethical appropriateness of pharmaceutical trials using humans; since our study involved only the use of a questionnaire and not an experimental drug, we did not apply for approval from either of these ethics committees. Instead, written permission to carry out the study was granted by the dean of the faculty of medicine and by the chief manager of university hospitals. In addition, all of the potential participants were fully informed about the aim and structure of the study. Furthermore, potential volunteers were all made aware that participation was strictly voluntary and that all of the answers they provide would be done so anonymously.
The questionnaire was administered to a total of 373 health care workers: all of the 236 physicians who work in surgical specialties at the Ankara University Ibn-i Sina Hospital and to all of the 137 dentists in the Gazi University Faculty of Dentistry. Dentists were included in this study because, aside from being HCWs themselves, there are a number of studies in the literature which show that dentists, citing various reasons, may also refuse to treat patients with contagious diseases. And, in order to better assess the fact on which the third premise of presumed consent is based, we decided to include only professional health care workers, instead of students and others who might still be in the process of deciding whether or not to currently enter the field.
In total there were 230 participants, 101 physicians and 129 dentists, who completed the questionnaire, for an overall response rate of 61.7%. The questionnaire was later sent back to the non-respondents one month after the first survey, and 28 of these were completed and returned to us. The mean age of the participants was 33.8 ± 9.6 years, while 56.5% were male and 43.5% were female. Additionally, the average amount of time that they had been working in the medical profession was found to be 8.5 years (min. 0, max. 40). All of the data was collected anonymously. The difference between the two groups, physicians and dentists, was compared using the chi-square test, with a p-value of <0.05 accepted as statistically significant. All analyses were carried out using SPSS 11.5.
Of the HCWs surveyed in this study, roughly half stated that they understood that by choosing their profession they would be exposing themselves to an increased risk of contracting contagious disease (55.2%). And at the time of entering the faculty, 24.4% of the participants expressed that they were unaware of any increased risks; however, they later learned of these risks during their education and training. In other words, 79.6% of the participants stated that they had known about the risks either at the time they chose their profession or that they had later learned of the risks during their training and education. Additionally, 6.5% of the participants answered that they had only come to realize the kinds of risks they would face after starting to work. The percentage of participants who claimed that if they had been aware of the risks earlier they would not have chosen to enter or continue in the medical profession was 5.2%. Table 1 are statements which physicians and dentists chose as best reflecting their personal opinions regarding the occupational risks of infectious disease. In general, the physicians, prior to their education and training, were significantly more aware of the potential risks associated with their profession than were the dentists (p < 0.05). A significantly higher percentage of the dentists however, stated that they only learned of the occupational risks of dentistry during their education and training (p < 0.05). There was no statistically significant difference between the two groups in terms of the other opinions questioned.
The participants were also asked whether or not they agree with the argument "When people choose, and continue to practice, the medical or dentistry profession, they are then required to accept all of the occupational risks resulting from the infectious diseases they might confront". The aim of this question was to determine whether or not the HCWs each have their own individual working-definition for the SLR. Of the participants, 57.4% believed that there is such a level. 52.2% felt that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented, and 5.2% said that some diseases were always beyond the SLR, no matter what precautions might be taken. No statistically significant difference was found between the physicians and the dentists.
Listed in Table 2 are the diseases which, under certain circumstances, were cited as potentially exceeding the SLR. Among the participants who stated that there would be a SLR for providing health care to the patients of specific diseases unless protective measures were implemented, AIDS and Hepatitis C and B were the most frequently cited of these diseases (71.7%, 64.2%, and 56.7%, respectively). The participants who felt that some diseases would always exceed a SLR expressed, that Hepatitis B, Tuberculosis, and Bacterial meningitis always would go beyond the SLR (41.7%, same for all). According to these participants, the occupational risk of potentially being infected with HIV is paramount to all other risks. Percentage-wise, AIDS was the most frequently mentioned disease that would exceed the SLR, more so than SARS.
All of the participants who answered that some diseases would be beyond the SLR were then asked what criteria they used to make their determination. The most com-monly expressed criteria, in order, regarding the diseases, were the likelihood of transmission, whether or not protective measures are available, and whether or not immunization is possible (66.7%, 65.2%, and 58.3%, respectively). The distribution of these criteria among the physicians and dentists can be seen in Table 3 . Physicians expressed significantly more often than dentists that if there was no immunization or treatment available for a particular disease, then that disease would exceed the SLR (p < 0.01). In terms of other criteria, there were no significant differences observed between the two groups.
The primary aim of this paper is to evaluate the claim that presumed consent may constitute grounds for the moral duty to treat. The presumed consent argument is valid, because its conclusion should logically be accepted if its premises are taken into account. To analyze the soundness of the argument we carried out a survey investigating the opinions of HCWs about the occupational risks of infectious diseases. In total, 79.6% of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. In other words, one fifth of the participants either lacked adequate knowledge about the occupational risks when they chose their profession or were not sufficiently informed of these risks during their faculty education and training. This means that the assumption stated in Premise 3 may be wrong for an important proportion of health care workers. It seems reasonable to suggest that the words "there is a general understanding" would be misleading if used to characterize a social concept of which the applicability and, indeed, the very existence, are yet to be established by sociological studies.
It is also useful to discuss the other problems associated with presumed consent; in particular, the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the nearimpossibility of being able to evaluate, in retrospect, whether or not every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases.
If we are to use the presumed consent argument, then the findings of this study indicate that when a new epidemic of a contagious disease occurs, or when the medical profession is confronted with a disease for which no immunization or treatment options are available, some HCWs are not bound by the duty to treat according to the presumed consent argument. This seems potentially problematic and demands serious consideration. How appropriate is it to describe the healthcare provider's responsibility and duty as stemming from their 'consent'? To address this question, it is helpful to reflect on the conditions required for an individual to be able to give consent that is well-informed.
For an HCW's consent to be informed, the following should first be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the SLR, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. If not necessarily when they choose their profession, then at least after being given the relevant knowledge during education and training, the person's choice should be regarded as informed. It should therefore be ensured that HCWs are acquainted with each new and emerging risk, and with any methods of prevention developed during or after their education and training. If a person's choice is to be confidently regarded as informed, it is imperative that these conditions be met. Of course, the question now becomes: how possible is it to satisfy all these conditions?
It is quite easy to imagine more than one answer to this question, but one thing is for sure: any thoughtful answer would acknowledge that choice is determined both by factors that are under the control of the individual and by factors that are not. Personal factors such as educational status, perception of the world and ambitions all influence an individual's choice of profession strongly. Nevertheless, factors outside the individual's control also play a large role in determining that choice. The environment in which the person grew up -their family life, the jobs of their parents, their community, social class and cultureall contribute to forming that individual's background, which (needless to say) has a very large influence on the opportunities and choices available to them.
Even though a person may not have been sufficiently informed when they chose their profession, it can be argued that during their education and training they will learn all relevant knowledge about the occupational risks associated with working in the medical profession. If so, it is fair to assume that when this individual begins to work after graduation they will be willing to confront any of those risks. In theory at least, it can be presumed that every student who passes their exams and goes on to graduate from the faculty is informed of the risks; so it can be argued that all HCWs who are currently active in their profession have consented to accept the risks posed by all the contagious diseases known at the time of their graduation. Of course, the diverse factors that determine the quality of education, such as the particular educational methods used, the course content, the abilities and knowledge of the instructors, role-models, and the personal features and motivations of students, are all potential sources of variation. But for the sake of argument, let us assume that a standardized education program is implemented throughout all medical schools. If that were the situation, then the argument that the individual has been made aware of the occupational risks during education would be true to the extent that the education program addressed those risks sufficiently. Nevertheless, it would be hard to claim that presumed consent is valid for every individual. For many people, a degree in medicine is very costly, both financially and in terms of time and energy. Under such circumstances, it is difficult for a person to quit their schooling despite the awareness they gain of the occupational risks involved. Individuals might feel pressured and confused by the two options confronting them: on the one hand, dropping out of medicine and forfeiting all the time, effort and money spent on schooling towards that aim; and on the other, reluctantly accepting the occupational risks, which may look frightening to the individual at that moment. Of course, it is important to remember that the person chose the medical profession in the first place, and numerous positive and beneficial elements are associated with working within it, which may ultimately serve to temper and override the individual's fear of the risks. An additional source of pressure may be that, for whatever reasons, switching educational tracks is too difficult; it may appear too daunting or be financially untenable. It seems likely that in the end the individual will choose to continue with their education and embark on a career in medicine, despite hesitation and fear of the risks. It is difficult to describe a decision made under conditions of such uncertainty and stress as 'informed'.
As can be seen, we do not choose our profession from among a wide array of possibilities spread out in front of us by thoroughly researching each one so that we are fully informed of its nature; everybody's options are different and it is a large and difficult task to make oneself sufficiently informed of them. Moreover, as described above, the decision to quit medical school can be quite difficult: on one side of the dilemma there are occupational risks that must be accepted regardless of misgivings on the part of the individual; on the other side, very influential factors pressure the individual to continue their medical education. Thus, the claim that "People who enter medical fields clearly had alternatives" is debatable and sometimes even doubtful. In theory it sounds right; nobody has to be a physician. But in practice, having alternatives does not mean that all our decisions are made freely or autonomously.
Theoretically, the fundamental problem with the presumed consent argument is that it cannot explain why there is always some degree of uncertainty about the occupational risks of working in the medical profession, particularly stemming from new and emerging diseases. Quite simply, if there was little or no knowledge of a risk at the time the individual became informed and gave their (tacit) consent, then this individual never accepted the risk, implicitly or otherwise, because it was unknown at the time the individual was informed. From a historical perspective, it is possible to see that while the medical profession was once concerned only with treating diseases, its vocational responsibility came in time to include preventative, promotive and rehabilitative healthcare services. As the notions of human rights and patient rights have developed and become widespread, perceptions about the health profession have changed at the community level. Diseases that once killed millions of people can now be treated with a simple medicament, but today we are faced with new and challenging diseases unheard of in the past. As a result, the continuous cycle of changespurred on by greater knowledge and technological advances and confrontations with new and untreatable diseases -serves to alter the identity and nature of the medical profession, and out of all this arises a constant degree of uncertainty. This characteristic uncertainty is present both when the individual chooses the profession and throughout their education and training periodand, indeed, for the entirety of their professional career. It is therefore not possible for someone to be fully informed when they choose their profession, nor is it possible for them to become fully informed during their education and training; nevertheless, the person should be informed about the uncertainty involved with working in the medical profession. In the light of this uncertainty, answers such as "if I had known I would not have chosen it" are not very meaningful, because there is no way to anticipate all the potential risks one might encounter in the course of a professional life. The only sure thing amid the uncertainty is that diseases such as SARS and avian 'flu will always continue to emerge.
So far in the discussion, the difficulties of satisfying the conditions needed to validate the presumed consent argument have been described. It seems virtually impossible to fulfill all these conditions satisfactorily. At this point it is important to discuss two particular problems regarding the argument itself.
First, it seems nearly impossible to evaluate individually whether the choice made by every HCW to enter the medical profession was informed. The only way to do that would be laboriously to ask each HCW whether they were informed when they decided on the health care profession. Even then, irrespective of whether the person's answer to this question reflects the truth, the only thing that could be learned from such a broad and extensive interrogation would be the individual's perception, not their actual knowledge. By extension, we could not question the participants' level of knowledge in this study, but rather how they perceive their level of knowledge. Because it is futile to seek objectivity in people's perceptions, it would not be sound to use those perceptions to determine whether the HCW's choice was informed, and thus whether they have taken on the duty to treat. And if these perceptions are regarded as subjective, as they should be, then it would be very difficult to develop a set of standard criteria that could be used to establish whether a HCW has been informed. This would also complicate efforts to reach a consensus on forming criteria by which various levels of risk could be defined universally. Unfortunately, such difficulties can only hamper efforts to protect the right of every patient to receive the best treatment available.
The second problem is that there is very likely to be more discrimination against patients with certain diseases, as HCWs use this argument to justify their refusal to treat those diseases. The World Medical Association's Declaration of Geneva, which states the basic moral values of the medical profession, specifies that there should be no discrimination, regardless of the circumstances: "I will not permit considerations of age, disease or disability, creed, ethnic origin, gender, nationality, political affiliation, race, sexual orientation, or social standing to intervene between my duty and my patient" [17] . However, the physicians' right to choose their patients is described by the same organization in another policy called "Twelve Principles of Provision of Health Care in any National Health Care System" [18] : "Any health care system should allow the patient to consult the physician of his choice, and the physician to treat only patients of his choice, without the rights of either being affected in any way." For regulations at the national level, it is generally held that if there is an urgent need for medical care [19, 20] , or if no other physician is available to whom the patient can apply [21, 22] , this right would be negated and the available physician must treat the patient. If we look at these obligations in reverse, we can see that the physician might refuse to provide health services to the patient if there is no urgent need for medical care and/or if another physician is around to whom the patient can be referred. Besides, the physician might also refuse the patient on the grounds that their prejudices may adversely affect the advice or treatment that they provide [20] . In actuality, this flexibility was written into the regulations in order to ensure that the patient receives the highest quality care possible; but as can be seen, it could also be abused. And if the presence or absence of consent is used as a criterion to define the duty to treat, in addition the existing flexibilities, then a mechanism would be created by which HCWs may freely, and perhaps excessively, discriminate among patients.
To summarize, considering all the points discussed above, the soundness of the presumed consent argument can be doubted. Therefore, it should not be claimed that there is a duty to treat on the basis of the presumed consent argument, as the argument itself is not persuasive.
It is also important to note that 5.2% of our participants said that they would not have chosen this profession if they had been informed of the risks. In other words, most of those HCWs who claimed that they were uninformed of the occupational risks when they entered the faculty, or were not fully informed of them during their education period, stated that they still would have chosen the medical profession even if they had been more aware of the risks. This finding tells us that, generally speaking, HCWs place relatively little importance on being informed beforehand. Further support for these findings comes from the answers given by the participants to the other questions. Nearly half said that there is no SLR, and the other half felt that the diseases they evaluate would not surpass SLR if the appropriate protective measures are available. Also, Table 2 indicates that at least 28.3% of the participants thought that none of the diseases listed in that table would exceed the SLR regardless of circumstances. It can therefore be concluded that a large majority of the HCWs place more emphasis on their working conditions than on being informed beforehand. In addition, the criteria most commonly stated by the participants for determining the SLR were the likelihood of transmission of a disease, whether protective measures are available and whether immunization is possible. Each of these criteria is related to protecting the HCW from infection, not to the treatment or the effects of a particular disease. This means that as long as protective measures are available, the HCWs would regard a given disease as below the SLR, so it has nothing to do with being informed beforehand. Besides, the only disease used as an example in this study that could be claimed to exceed the SLR was SARS; AIDS, hepatitis C, hepatitis B, tuberculosis and bacterial meningitis all fell below the SLR according to these criteria. Nevertheless, only 30.9% of the participants suggested that SARS would surpass the SLR. To put this into perspective, SARS was not even observed until 2003, and the research in the present study was conducted in 2004 and 2005. Therefore, most participants in this study were not aware of SARS when they chose the medical profession, nor were they ever informed of it during education or training. They nonetheless felt that the duty to treat pertained even to patients with SARS. All of this suggests that factors more useful and relevant than presumed consent influence the decision of HCWs to choose and continue in the medical profession; these factors may include the social contract between society and the medical profession, and the greater ability of HCWs to provide medical care [16] . It is these factors that should be investigated and emphasized when defining a moral duty to treat.
This study could be limited by several factors. The first limitation could be due to a socially desirable response bias; some participants might have given what they perceived as the 'right' answers to the questions rather than the answers that reflect their opinion or belief. In order to address this concern, future studies could benefit by using qualitative methods, which provide more reliable results about the motives and opinions of participants. Also, this study was not prospective, so recall bias might have affected the responses of the participants. Furthermore, the extent to which the results of this study are applicable to HCWs such as nurses or physicians who work in internal specialties is uncertain. Future studies that include other HCWs as participants may broaden our understanding of the beliefs and opinions of HCWs, thereby allowing us to state our claims and shape our arguments more precisely. Finally, it should be mentioned that the response rate for this study (61.7%) was slightly lower than is generally expected for a survey. Nevertheless, despite all these methodological limitations, we believe that our findings support our conclusion about the persuasiveness of the presumed consent argument.
If we use the presumed consent argument to establish the duty of the HCW to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. Our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. As we stated above, in order for a candidate HCW to be informed literally, three items should be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the SLR, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. In this study it has been shown that at least some HCWs may not be informed of (a). Also, it is not currently possible to inform HCWs of (b) since there are no widely-agreed criteria and definitions to allow for a universally accepted SLR; and there is currently no standard education for all HCWs to ensure that (c) is satisfied. Considering this in addition to the problems mentioned above, the third premise of the presumed consent argument appears implausible and, consequently, the duty to treat cannot be grounded persuasively on the consent assumption. It is therefore more useful to emphasize justifications other than presumed consent when defining the duty of HCWs to provide care, such as the social contract between society and the medical profession and the fact that HCWs have a greater ability to provide medical aid.
Furthermore, in terms of the moral duty to provide care, it seems that most HCWs are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. It seems important that further research be carried out to improve understanding of the opinions and perceptions of HCWs and the basis of their definitions, as this information could prove very helpful in defining a duty to treat that can be effectively put into practice. It is also important that a well-organized ongoing educational program that is needs-based and easily accessible be provided to HCWs at both the graduate and postgraduate levels. In particular, this program must be continuously updated regarding AIDS and other diseases that may cause the HCWs to behave discriminatively towards patients, even though these diseases are below the SLR. Such continuing medical education is the best answer to the justification "When I chose the profession/when I graduated, this disease did not exist!" for refusing treatment. Emphasizing the social role of HCWs, and educating them about the professional obligations derived from the social contract betweeen the profession and the wider social order, would further reduce that kind of reasoning. In addition, stricter standards for the duty to provide care should established by determining the criteria for a SLR and identifying the situations and conditions that would exceed this SLR. Each of these measures could serve to remind HCWs that they have a moral responsibility, as individual HCWs, to be aware of professional obligations and to act as responsible members of the profession. Moreover, the working environment of HCWs should be provided with preventative measures that can be applied both generally and specifically and should emphasize their use. For a circumstance in which a preventative measure has been developed for a disease but is not available for treating a particular case, it would not be easy to justify the claim that there is an undeniable duty to provide care at that moment. Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system BACKGROUND: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control. RESULTS: By applying on-line pO(2 )monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels. CONCLUSION: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems. The methylotrophic yeast Pichia pastoris is a favored yeast species as a host for heterologous protein production (for reviews see [1] [2] [3] [4] [5] [6] ). P. pastoris has the potential for high expression levels, efficient secretion of target proteins, posttranslational modifications, and is easily grown to high cell densities on mineral salt medium in bioreactors. It has been demonstrated that P. pastoris is an efficient production system also for very large and complex proteins, such as collagens, which besides the recombinant gene(s) needed for the collagen polypeptide chain(s) needs the parallel expression of two different genes coding for collagen prolyl 4-hydroxylase (C-P4H), an enzyme required for the thermal stability of collagens [7] [8] [9] .
Although different promoter systems exist for a controlled or continuous expression of heterologous proteins in P. pastoris, frequently the strong and tightly controlled promoter of alcohol oxidase 1 (AOX1) is applied. The AOX promoter is induced by methanol [6] . Methanol, aside from being the inducer of the promoter is also a carbon/ energy substrate. Generally, in such P. pastoris processes, methanol is the only carbon substrate during the production phase of the AOX1-promoter controlled protein. A drawback is the toxicity of methanol. Generally, methanol concentrations above 3.6% inhibit the yeast growth and lead to death [2] .
In previous studies for shake flask cultures, the commonly used protocol recommended by Invitrogen [10] for methanol feeding has been modified individually depending on the product. For the expression of proteins in shake flask cultures it is proposed that methanol is added twice per day [11, 12] . However, due to the small window in which the methanol concentration must be kept for an optimal process, pulse-based methanol addition for optimization of processes in shake flasks seems not to be the best way to provide data for the development of a fermentation process.
Optimization work for recombinant processes with P. pastoris is generally performed in bioreactors by the fed-batch strategy. The basic scheme follows in principle the original protocol of Brierley and coworkers [13] . A glycerol batch phase is followed by a transition phase and by a methanol induction phase [14] . Thereafter, methanol is added continuously. Different variations of this strategy have been proposed, mostly evaluated as being advantageous for a specific protein (comprehensively reviewed by [2] ).
The requirement of bioreactors is a limitation for parallel fast optimization of processes. Principally parallel bioreactor systems exist on the market, but they are generally expensive and non-scalable. Here we describe a solution, which allows the application of the fed-batch principle in shake flasks. We apply a recently described monitoring system [15] which is connected to a radio-modem controlled feeding device, which can be placed directly on shakers.
The recombinant protein produced in the present investigation is human type II collagen which is the major collagenous component in the cartilage [16] . Purified recombinant human type II collagen is regarded very useful in applications for cartilage repair [17] . The formation of stable triple helical collagen at physiological temperature requires C-P4H activity [16, 18] . Consequently, the coexpression of C-P4H, an α 2 β 2 tetramer located within the lumen of the endoplasmic reticulum, has been proven essential for the production of temperature-stable recombinant collagens [7, 8] . Recombinant type II collagen has been produced successfully in the baculovirus expression system [19, 20] and in P. pastoris [8, 20] .
In this study, application of semicontinuous feeding of methanol to shake flask cultures improved significantly the production of stable human type II collagen, which is documented by protein and mRNA analyses of the collagen and C-P4H.
We have shown previously by applying on-line monitoring sensors in shake flasks that the commonly used methanol feeding protocol for shake flask cultures of P. pastoris (two manual pulses of methanol per day) leads to long starvation phases between feeding pulses [15] . Starvation is obvious by a pO 2 peak after exhaustion of methanol, related to declining respiratory activity ( Figure 1A ). We concluded from this data that the methanol feeding in this shake flask process is not optimal. In the first step of optimization, the rapidly increasing pO 2 level after consumption of methanol was used as a signal to manually add a new methanol pulse. Each single methanol dose was the same as in the standard protocol and consequently much more methanol was added during the course of the cultivation. Although the procedure was laborious and difficult to reproduce manually, a 30% higher cell density was obtained and the product related mRNAs showed higher expression, such as the mRNAs encoding alcohol oxidase 1 (AOX), formaldehyde dehydrogenase, yeast PDI, type II procollagen chain and the C-P4H α(I) and PDI/β subunits ( Figure 1B) . Unexpectedly, the amount of collagen II was not increased, however (not shown).
Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for produc-tion of recombinant human collagen II in shake flasks Figure 1 Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for production of recombinant human collagen II in shake flasks. Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO 2 -dependent manual feeding of methanol (red filled circles, red continuous line) were compared.
Although the results from the manual feeding experiment were promising, due to the long cultivation time a real optimization of the feeding procedure would be possible only by a computer-controlled feed, which allows the addition of methanol in a quasi-continuous way, similar as in bioreactor fed-batch cultivations.
Therefore, a computer-controlled feeding device was developed, consisting of an encapsulated microcomputer which was connected to feeding valves. The microcomputer is controlled by a normal desktop computer over a radio-module working at 868 MHz as shown schematically in Figure 2 . If needed, the feeding device can be powered by a rechargeable lead battery.
Methanol was fed from a sterile 50 ml syringe through a silicon tube connection (1 mm diameter) and a steel needle connected to one of the three side necks of the shake flask. The feeding pulse was given by a miniature LFV valve (The Lee Company, U.S.A.) which was integrated into the wireless feeding device. In the performed experiments a flow through the valve was simply created by gravitation over an approximately 1 m height difference, but we could also generate a constant flow of solutions with low viscosity by applying an air overpressure container or a micropump (data not shown).
With the developed device and the programmed backchannel software, different feeding protocols could be applied to shake flasks (not shown). Opening of the feeding valves in short time intervals yielded a quasi-continuous flow.
The effect of quasi-continuous methanol feeding into the shake flasks was tested by repeatedly performing series of two parallel 200 mL cultures with different computer controlled feeding schemes.
In each experiment for one of the shake flasks (reference culture) the feeding strategy was based on the commonly used pulse-feeding method [10, 12] in which a 5% methanol solution was fed twice a day to a final methanol concentration of 0.5% (v/v). Into the other flasks methanol was fed in a quasi-continuous mode. Within the first 2 hours a methanol pulse was given every 12 min and after that the feeding interval was 30 min. Each methanol pulse was 0.8 ml of the 5% methanol solution. The total feeding volume and total amount of added methanol was the same in both parallel flasks.
In the reference cultures the addition of a methanol pulse was followed by an immediate drop of the pO 2 level. However, within 5 to 10 h after a methanol pulse the pO 2 raised, indicating exhaustion of the substrate (methanol). The pO 2 dropped to zero only when the shaker was Schematic presentation of the wireless data collection and control system for shake flask cultivations Figure 2 Schematic presentation of the wireless data collection and control system for shake flask cultivations.
P. pastoris cultivation in shake flasks with methanol feeding without (A, B) or with (C, D) manual pH adjustment Figure 3 P. pastoris cultivation in shake flasks with methanol feeding without (A, B) or with (C, D) manual pH adjustment. Cultivations were performed in two phases: initial batch phase in BMG-medium and fed-batch in BMM-medium. Dissolved oxygen (pO 2 ) and pH were measured with a wireless measuring system, cell growth was followed by measurement of the OD 600 . Vertical lines represent methanol feeding points in A and C or start of methanol feed in B and D.
stopped for sampling (see pO 2 spikes in Figures 3A and 3B), but remained otherwise above 50%.
In the flasks with quasi-continuous feeding the first methanol pulse caused a decrease of the pO 2 . Later, when the feeding intervals were longer, the pO 2 level was stabilized to about 90% ( Figure 3B ). Only after 50 h of cultivation, when the methanol started to accumulate, the pO 2 level increased indicating a higher maintenance or toxification.
The methanol concentration in the culture medium of the reference culture was close to the detection limit almost during the whole incubation time. Only towards the end of the cultivation a low concentration of residual methanol could be detected in the medium. It should be remarked that the samples for methanol analysis were always collected before the next methanol pulse. Therefore we concluded that methanol had been fully consumed before the next pulse. This was also obvious from the pO 2 profile. In contrast, small amounts of methanol were present in the culture with quasi-continuous methanol addition. Until 50 h of cultivation the methanol concentration was below 1 g L -1 . Only towards the end of the cultivation an increase of the residual methanol concentration was detected.
As the pO 2 level was high in the culture with quasi-continuous feeding it was tested whether also a higher feeding rate would be possible. Feeding with a double amount of methanol was not beneficial, but instead led to the accumulation of the methanol concentration above 5 g L -1 already within 20 h after the start of the feeding (data not shown).
Without pH control, the pH value declined during the whole cultivation. The decrease occurred stepwise in the reference culture and was clearly related to the feed pulses (see Figure 3A ). At the end of the cultivation the final pH was very low (close to 2), which surely is not beneficial for the cell growth and production. As the pH in fermenter cultivations is usually kept between 4 and 6, we decided to adjust the pH during the experiment to 5.5 by intermittent addition of 10% (v/v) ammonia.
The control of the pH did not result in major changes in the general culture parameters ( Figure 3C,D) . The variations, especially the higher methanol concentration in the culture with quasi-continuous feed ( Figure 3D ) may be mainly attributed to the larger amount of samples collected during the cultivation for analysis of the protein product and mRNA levels.
All cultures were followed by analysis of collagen II, C-P4H enzymatic activity and product-related mRNAs. The amount of type II collagen chains (139 kD) derived from correctly assembled protease-resistant triple-helical collagen II was analyzed by reducing SDS-PAGE after HCl extraction with pepsin-treatment according to Myllyharju et al. [8] . Stable and correctly assembled collagens are resistant against pepsin. Collagen II showed a clear band ( Figure 4 ) which was significantly stronger in the cultivations with the quasi-continuous feeding profile, compared to the standard pulse feeding profile. The results were reproducible (see Table 1 ) despite experiment to experiment variations. Generally, more correctly assembled triple-helical collagen was produced in the experiments with quasi-continuous feeding of methanol.
These findings were supported also by the higher amount of C-P4H activity (see Figures 3C and 3D , cf. Table 1 ). C-P4H activity was measured from several time points and found to be 2 to 8 times higher in different cultures with quasi-continuous methanol feeding compared to the parallel reference cultures in each experiment (see Figure 5 ). Doubling the methanol concentration in the feeding solution (cf. experiment 4 A in Table 1 , and grey bar in Figure  5 ) led to higher activities compared to the reference, but the C-P4H activity was slightly lower than in the experiment with the lower feed rate. Surprisingly, also the cell density was not increased in this cultivation compared to the culture with quasi-continuous feed of more diluted feed.
SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1 ) after 21, 46 and 72 hours cultivation Figure 4 SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1 ) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.
The effect of the feeding procedure on the formation of the product was investigated in more detail at the mRNA level. A quantitative sandwich hybridization assay which was developed earlier in our laboratory was applied [21, 22] . Figure 6 shows the levels of mRNAs encoding the procollagen II chain, AOX1, EF3 (translational factor), and C-P4H. The level of procollagen II mRNA was already high during the first 12 h when the cultures were grown in a batch mode on methanol in both types of cultivations. In the reference culture with pulse feeding, the procollagen II mRNA level decreased to a very low level after the start of the feeding. In contrast, the mRNA level stayed high during the whole cultivation in the culture with quasi-continuous feeding of methanol, being approximately 10 times higher when compared to the pulse feed method.
The level of AOX1 mRNA, encoding the alcohol oxidase enzyme and therefore indicating the induction of methanol as well as an active methanol metabolism, was much lower than the level of procollagen II mRNA. Interestingly, the procollagen II mRNA decreased fast in the reference culture with pulse feeding strongly during the shift from the methanol batch to the pulse feeding, similarly to all other mRNAs analyzed. Possibly this is a result of growth inhibition due to carbon/energy source starvation as shown above. In contrast, the AOX1 mRNA level was approximately the same over the whole cultivation when the quasi continuous feeding mode was applied. The same behavior was detected for the EF3 mRNA, a translation factor, and for the level of the C-P4Hα(I) mRNA. The higher level of the C-P4Hα(I) mRNA correlates well with the higher amount of C-P4H activity obtained.
Experiments at the scale of shake flasks are generally batch processes. This restricts the applicability of the results for the further development of fermentation processes, which normally apply the fed-batch principle. Despite of this limitation shake flask cultivations are commonly used due to their simplicity and high flexibility.
In many cases it might be useful to apply the fed-batch principle already in the earliest process optimization phase, not primarily for obtaining higher cell densities, but for applying similar physiological conditions as in the later process. As such tools are not commonly available we developed a new feeding system which works wireless and therefore can be easily applied on any shaker. If powered by a lead accumulator, the feeding unit can be C-P4H activity in three different experiments, each with a quasi-continuous feeding culture (black bars) and a reference culture (manual feeding twice a day, white bars) Figure 5 C-P4H activity in three different experiments, each with a quasi-continuous feeding culture (black bars) and a reference culture (manual feeding twice a day, white bars). Additionally, in experiment no. 4 double concentrated methanol was fed (grey bar). mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask culti-vation of P. pastoris Figure 6 mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of P. pastoris. Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h) . Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1 .
directly placed on the shaking table without the need for any direct electrical contact. This unit works together with the earlier described SENBIT wireless measurement system [15] . Control functions and set-points can be easily programmed in the controlling software (BackChannel) which communicates with the measuring software (SEN-BIT).
Recombinant P. pastoris cultures were an interesting test case to apply the feeding system in shake flasks. Especially with the widely used AOX1 promoter system, the results from shake flask experiments may be of low practical value, because the methanol concentration has to be controlled within a narrow concentration window which is not possible in a batch system.
The starting point for our optimization approach was the generally applied recommended protocol for pulse feeding of methanol twice a day [11, 12] . This procedure is very different from the various cultivation techniques applied in fermentation processes which all are based on continuous addition of methanol. As model system we used the expression of recombinant human collagen II in P. pastoris, which has been described earlier [8] . The assembly of the triple-helical collagen is directly correlated with the hydroxylation of prolyl residues by C-P4H, which is coexpressed together with the collagen polypeptide chains.
It is very obvious that the proposed pulse feeding protocol is unfavorable for collagen production. During cultivation, long phases of carbon/energy starvation between the feeding pulses were observed by measurement of oxygen levels in shake flasks. Presence of these phases was confirmed by analysis of process-related marker mRNAs and analysis of the products. Overall, lower expression of these mRNAs was observed compared to a constant feeding protocol.
We could prove that a quasi-continuous feeding in the shake flask scale can significantly improve the conditions for collagen production and leads to increased expression of triple-helical stable collagen by simultaneous enhancement of C-P4H expression and activity. The corresponding mRNAs were also expressed at higher level than in the culture with pulse feeding.
Almost 10-fold higher amount of procollagen II mRNA was detected in the cultures with quasi-continuous feeding of methanol compared to the pulse method. Also the expression of the translation factor EF3, which is an indicator of protein synthesis, was higher with constant feeding. Generally, in the case of such optimization approaches, and not limited to shake flasks studies, the quantitative analysis of mRNAs, especially for C-P4H and collagen in our case, seems to be a good indicator for the quality of the cultivation.
Although we applied here mainly a predetermined feeding strategy, also other feeding schemes and control principles are applicable with the newly developed feeding system, such as the pO 2 -dependent feeding. Although the DO-Stat control principle did not improve the product yield in our case (data not shown), such extended studies are principally interesting and will widen the usefulness of shake flask cultures.
As discussed above, it is generally not the aim of shake flask studies to reach the high cell densities which are commonly reached in bioreactors. In the experiments described here cell concentrations were rather moderate which is due to the lower oxygen transfer rate in shake flasks compared to bioreactors. It was the intention in our experiments to keep the oxygen level high as it is a necessary cofactor in the C-P4H reaction, although for other proteins good production may be achieved also by running the culture into oxygen limitation which has been recently shown for the production of an scFv antibody fragment [23] . Furthermore, the moderate cell densities in our experiments are also due to the use of diluted methanol feed solutions. To avoid long time intervals without any feed and to ensure a feed which is metabolically seen as continuous methanol was fed as a 5% solution, which correspondingly dilutes the medium and results in a lower final cell yield. The use of low-power micropumps instead of microvalves is currently tested, and may provide an improved continuous supply of small liquid amounts.
Simple controlled bioreactors can be obtained by applying feeding to shake flask cultivations. Although this will not generally change specific characteristics for shake flask cultures such as the problem to establish specific pO 2 levels and poorly controllable aeration of the cultures, the proposed solution is a way to control the physiological state of the cultures in early screening phases in a similar way as during later process development. Furthermore, the described system is advantageous in terms of flexibility and investment costs compared to parallel bioreactor systems.
The comparison of the commonly used pulse feeding of methanol to P. pastoris cultures in shake flasks with the quasi continuous feeding clearly showed clearly that the commonly applied standard protocol is not favorable to optimize recombinant processes, mainly due to the longterm starvation phases. Commonly such optimization is performed in bioreactors, which however are not available for many of the molecular biology laboratories. We show here a simple solution by applying a quasi-continuous feed profile, which is a method commonly used in bioreactors. However, the developed feeding device is not limited to such simple applications, but pre-determined feed functions and even feed-back control of the feed rate on the basis of measured parameters can be easily programmed and principally turn the shake flask into a bioreactor.
In our case the quasi-continuous feeding profile increased both the amount of C-P4H and stable collagen II. This clearly demonstrates in the case of one complicated large recombinant protein the power of monitoring and control in shaken cultures. The tools applied here provide a valuable system for parallel optimization at small scale also for other kind of cultivations.
The P. pastoris strain sCII7 (Mut + phenotype) contained the pICZB expression vector with the cDNAs coding for the human procollagen II chain and the two C-P4H subunits (kindly provided from Fibrogen Europe Ltd., Helsinki, Finland). The cells were stored as glycerol stocks at -70°C.
Cultivation medium and cultivation conditions P. pastoris was cultivated in BMG or BMM culture medium containing 0.1 M phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg L -1 biotin, and 1% glycerol (BMG) or 0.5% methanol (BMM) respectively [10] .
Cultivations were performed in 1 L Erlenmeyer flasks with three baffles and three side necks for the sensors and sampling (Glasgerätebau Ochs GmbH, Bovenden, Germany). The flasks contained 200 mL of BMG and were incubated at 30°C on a rotary shaker (Certomat H, B. Braun Int.) at 250 rpm. When the OD 600 reached the value between 2 and 6, the cells were centrifuged at 3500 rpm for 5 min, and washed with BMM by further centrifugation. The supernatant was decanted and the cell pellet was resuspended in BMM to obtain an initial OD 600 of about 1.5. The baffled shake flasks, containing 200 mL of BMM and the pH-and pO 2 -electrodes, were incubated at 30°C on a rotary shaker at 250 rpm. To maintain the expression of the product, 100% of methanol was added to a final concentration of 0.5% (v/v) twice a day or manually at the time when the pO 2 level increased (shake flask experiment with optimized feeding, cf. Figure 1) . Alternatively, the feeding was performed with the wireless feeding device with a 5% or 10% methanol solution as explained in the results section by a quasi-continuous predetermined feed rate, or on the base of the pO 2 signal (pO-stat principle). In some experiments ammonia (10%) was added manually to keep the pH between 4 and 6, as described in detail in the results section.
The SENBIT wireless system (teleBITcom GmbH, Teltow, Germany) was used in the shake flask cultures to follow the pO 2 and pH as described earlier [15] . The feeding module was constructed as described in the results section.
The samples were taken with a sterile needle (0.9 × 120 mm) connected to three-way sterile plastic valve with Luer-lock (Oriplast GmbH) inserted into a side neck of the flask and tightened with a rubber seal into 10 ml syringes. After immediate cooling on ice the samples were portioned for the further analyses, with exception for the mRNA samples which were directly chilled in the inhibition solution (see below).
Growth was monitored by measuring the turbidity (OD 600 ) at 600 nm.
Samples were centrifuged (2 min, 13000 rpm, 4°C) and the cell-free supernatant was analyzed by gas chromatography.
The analysis was performed as recently described [24] with the following modifications for P. pastoris. For immediate chilling of metabolic activities P. pastoris samples (4 × 2 mL) were immediately mixed with 200 μL inhibition solution (95:5 v/v ethanol/phenol, pre-cooled at -20°C). After centrifugation (2 min, 13000 rpm, 4°C) the supernatant was removed and the pellet was dispensed in 100 μl of RNA Later (Ambion) and stored at -70°C until analysis. Total RNA extraction was performed with the RNeasy Mini Kit and mechanical cell disruption according to the manufacturers instructions (Qiagen).
Oligonucleotide probes ( Table 2) were designed using the CloneManager5 program with following submission of the sequences a NCBI BLAST search [25] to exclude alignments with other genes. HPLC-purified unlabelled and biotin-labeled oligonucleotide capture probes were purchased from Metabion GmbH (Martinsried, Germany). Dig-tail labeling of the detection probes was performed according to manufacturer's instructions using the Roche Dig-tailing kit (2 nd generation, Roche Diagnostics GmbH, Mannheim, Germany).
In vitro RNA standards were designed for the quantitative analysis of each gene as described before [22, 24] . Therefore, primers as indicated in Table 3 were used for in vitro transcription (purchased from Sigma-Genosys, Cambridge, UK).
Protein analysis P. pastoris cell pellets were broken with zirconia beads both in acidic conditions followed by pepsin digestion for collagen II analysis. Protein samples were analyzed on SDS-page.
C-P4H activity was analyzed from P. pastoris cell pellets that were broken with zirconia beads in basic conditions. The activity assay is based on the hydroxylation-coupled decarboxylation of 2-oxo [1-14 C] glutarate with (Pro-Pro-Gly) 10 as the peptide substrate [26] . The total protein concentrations were determined with the RC DC Protein assay Kit (Biorad Laboratories).
probes and primers and performed together with MK the mRNA analyses. MR, AN and ERH performed the protein analyses. JM and ERH contributed with their experiences in the collagen production process. MR wrote the initial manuscript, which was read and approved by all authors. PN initiated and practically supervised the scientific work. HMGB1: Endogenous Danger Signaling While foreign pathogens and their products have long been known to activate the innate immune system, the recent recognition of a group of endogenous molecules that serve a similar function has provided a framework for understanding the overlap between the inflammatory responses activated by pathogens and injury. These endogenous molecules, termed alarmins, are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. One nuclear protein, High mobility group box-1 (HMGB1), has received particular attention as fulfilling the functions of an alarmin by being involved in both infectious and non-infectious inflammatory conditions. Once released, HMGB1 signals through various receptors to activate immune cells involved in the immune process. Although initial studies demonstrated HMGB1 as a late mediator of sepsis, recent findings indicate HMGB1 to have an important role in models of non-infectious inflammation, such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. Furthermore, in contrast to its pro-inflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration. The complex functions of HMGB1 as an archetypical alarmin are outlined here to review our current understanding of a molecule that holds the potential for treatment in many important human conditions. Activation of the innate immune system is a necessary step in mounting an anti-microbial response to pathogens. Clinicians have long appreciated that infectious insults and tissue injury from sterile inflammatory states produce a similar inflammatory response. Recent advances in understanding the mechanisms of innate immune system activation have pointed to certain pattern recognition receptors, such as the family of Toll-like receptors, as a common pathway for immune recognition of both microbial invasion and tissue injury. By recognizing either pathogens or endogenous danger signals released upon cellular stress or damage, these pattern recognition receptors are capable of alerting the host of danger by activating the innate immune system. In this review, we will describe one such endogenous danger signal, High mobility group box-1 (HMGB1), and its role in the pathogenesis of many disease states.
Pathogens have long been known to cause both local and systemic inflammation via activation of the immune system. Classically, descriptions of immune regulation arose from the idea that immune cells can discriminate self from non-self, and activate innate immunity when there is foreign invasion. This model, which stemmed from work done in the 1960s by Burnet and Medawar, has been modified through the years to account for new discoveries in immune cell function (1, 2) . Although the self/nonself model is suitable in explaining immune activation and inflammation that occurs as a consequence of clinical scenarios, such as invasion by foreign pathogens or transplant rejection, it fails to account for the inflammation which occurs in settings such as trauma or autoimmunity, which are void of non-self stimuli. In an attempt to better describe these phenomena, Matzinger has proposed the concept of danger signaling (3) . In this model, initiation of the inflammatory response occurs in response to molecular patterns that are associated with both pathogens and some normal cellular components that are released by damaged cells during both infectious and sterile processes (4) . The concept of cellular communication by "danger signals," whether exogenous or endogenous, reconciled the paradox of immune activation in both infection and sterile injury.
In the danger model, immune activation is the result of recognition of molecular patterns by cellular receptors. Molecular elements from pathogens that elicit an immune response (i.e. lipopolysaccharide [LPS], bacterial DNA, viral RNA) are specific patterns termed Pathogen Associated Molecular Patterns (PAMPs). These PAMPs are generally invariant and
it is their recognition by the immune system that triggers the inflammatory response (5, 6) . The cellular receptors that recognize these patterns are evolutionarily conserved and called Pattern Recognition Receptors (PRRs) (6) .
Inflammatory responses following sterile injury closely mimic those seen during infection, with similar cytokine and chemokine production patterns (7, 8) . Several endogenous molecules that are released during both infectious and sterile inflammation, such as HMGB1, heat shock proteins (HSPs), S100s, and hyaluronan, have been implicated as possessing the capacity to trigger the immune system through PRRs, much like PAMPs (9) (10) (11) . These signals are normal cell constituents and are released either passively by necrotic cells or actively secreted by stressed cells in response to cellular injury. While the term PAMP is restricted to patterns located on pathogens, these endogenous analogues are termed alarmins (12) . The exogenous PAMPs and endogenous alarmins are subgroups of the larger category of danger signals termed Damage Associated Molecular Patterns (DAMPs) (13, 14) .
Alarmins are of particular interest because of their role in both infectious and sterile inflammation. They are present either locally or systemically in severe sepsis, burns, infection, arthritis, and cancer (10, (15) (16) (17) . Alarmins are found in a variety of organelles in all cell types studied and maintain functions in normal cellular homeostasis. They are found in the nucleus as transcription factors (HMGB1), in the cytoplasm as calcium regulators (S100s), in exosomes as chaperones (HSPs), or as components of the cell matrix (hyaluronan). Although diverse in their locations during homeostatic conditions, alarmins have many common functional characteristics. In addition to immune activation, an alarmin is released rapidly during necrosis, sequestered in apoptosis, has potential for active secretion by immune cells, and ultimately promotes homeostasis (13) . Because alarmins are a diverse group of ubiquitous molecules impli-cated in nearly all inflammatory states, understanding and ultimately modulating their activity may allow us to control the inflammatory processes.
The high mobility group (HMG) nuclear proteins were discovered in 1973 in an effort to better define the specific regulators of gene expression (18) . This group of non-histone, chromatin-associated proteins has since been characterized to be involved in DNA organization and regulation of transcription. This family of proteins shares structural characteristics which make them unique from other chromosomal proteins. These characteristics include transcripts with long AT-rich 3′ untranslated regions as well as carboxy-terminal regions which are highly negatively charged (19) . HMG proteins are constitutively expressed in the nucleus of eukaryotic cells. Collectively, they share functional motifs that bind specific DNA structures and induce conformational changes without specificity for target sequences.
HMGB1 is a member of a subfamily of the HMG proteins. On average, HMGB1 is found at concentrations of 10 6 molecules per cell and non-specifically binds to the minor grooves in DNA (19) . This binding is regulated by two 80 amino acid HMG-1 domains (or boxes), each structurally represented as three α-helices in a characteristic L-shaped fold (20) . The nuclear localization of HMGB1 and its affinity for DNA is regulated through phosphorylation and acetylation, and has been found to have a dynamic relationship with chromatin (21) . Like the other members of this protein family, HMGB1 plays an important role in DNA architecture and transcriptional regulation.
HMGB1 first was implicated as an important endogenous signaling molecule in 1999 when Wang et al. described the cytokine activity of HMGB1 by identifying it as a late mediator of endotoxinrelated lethality in mice (10) . This study reported increased serum levels of HMGB1 from 8 to 32 h after endotoxin exposure, attenuation of lethality with delayed administration of an HMGB1 neutralizing antibody, and lethality with direct administration of HMGB1 (10) . This groundbreaking work sparked renewed interest in HMGB1 as an important component of the immune response.
The specific interactions and functions of the HMGB1 DNA binding domains have been described. As noted above, the structure of HMGB1 contains two separate "boxes," the A-and B-boxes, each containing ~80 amino acids in an L-shaped fold, along with an acidic C-terminal tail (20) . These separate structural motifs appear to function differently when isolated from HMGB1. Several studies have identified the B-box domain as important for many of the proinflammatory properties of HMGB1 including cytokine release (22, 23) . In comparison, the A-box does not possess the pro-inflammatory properties of the B-box and instead competes with HMGB1 for binding sites leading to attenuation of the inflammatory cascade (24) .
HMGB1 is released passively during cellular necrosis by almost all cells which have a nucleus and signals neighboring cells of ongoing damage (25) . However, HMGB1 also is secreted actively by immune cells such as monocytes, macrophages, and dendritic cells (10, 16, 26) . Active secretion of HMGB1 is generally through non-traditional, leaderless pathways which are not routed through the endoplasmic reticulum or Golgi apparatus, similar to IL-1 (27) . During normal cellular homeostasis the dynamic relationship of HMGB1 with the nucleus and cytoplasm heavily favors the nucleus. However, HMGB1 localizes in secretory lysosomes when hyper-acetylated on lysine residues and then is released upon appropriate signaling stimuli (21, 28) . When HMGB1 is not acetylated, it remains localized to the nucleus and is not secreted or released, such as during apoptosis (25) . How the acetylation of HMGB1 is regulated is yet to be determined but is surmised to in-volve deacetylases in the nucleus (16, 21, 29) . In other studies, specifically with TNFα stimulated macrophages, the secretion of HMGB1 is dependent upon phosphorylation (30) . While these modifications are clearly important in the release of HMGB1, it currently is unclear how these specific modifications differ.
Stimuli for secretion of HMGB1 from immune cells are diverse and include PAMPs, cytokines, and certain states of cellular stress. Macrophages release HMGB1 starting approximately 8 h following exposure to LPS (10,31-33) but do not demonstrate the same response to CpG DNA (34) . It is notable that release due to LPS is dependent, at least partially, on TNFα (31) . HMGB1 also can be released in response to polyinosinicpolycytidylic acid (a model of viral infection) (33, 35) , while in vitro viral stimulation has demonstrated only passive release of HMGB1 due to cytokines or necrotic cells (36) . In addition to these PAMPs that result in the release of HMGB1, endogenous molecules such as cytokines released during other states of injury can result in secretion of HMGB1. While first demonstrated with IFNγ, macrophages also release HMGB1 in response to stimulation with TNFα, IL-1, and oxidative stress (35, (37) (38) (39) (40) . Interestingly, the PAMPs and cytokine stimuli for HMGB1 secretion from macrophages occurs through distinct pathways (35) . For example, while LPS regulates HMGB1 release by hyper-acetylation, TNFα-induced secretion is mediated through phosphorylation, which directs it to the cytoplasm for release (30) .
While it was thought initially that HMGB1 was released only from cells of the immune system, other cells have since been demonstrated to actively secrete alarmins. The first non-immune cell to be studied for active HMGB1 secretion was the pituicyte, which was found to release HMGB1 in response to IL-1 or TNFα stimulation (41) . Enterocytes release HMGB1 following stimulation with a cytokine mixture (42) . Hepatocytes also can release HMGB1 in response to hypoxic conditions or oxida-tive stress; this release appears to be mediated by calcium signaling changes within the cell (43) .
The inhibition of HMGB1 also has been an important topic for those seeking to ameliorate injury by decreasing the level of HMGB1 release. A variety of pharmacologic agents have been studied for their potential to inhibit release of HMGB1; however, a full discussion of pharmacologic inhibition of HMGB1 is beyond the scope of this paper. It is worth noting, though, that HSP72, an endogenous molecule that has itself been indicted as an alarmin, has been demonstrated to inhibit HMGB1 release. Originally, it was demonstrated that brief heat shock resulted in decreased HMGB1 release from LPS-stimulated macrophages, but no specific pathway was identified (32) . Recently, HSP72 overexpression has been shown to inhibit HMGB1 release from macrophages in response to LPS, TNFα, or oxidative stress (hydrogen peroxide). This inhibition appears to be due to intranuclear interactions between HSP72 and HMGB1 (44, 45) .
As noted above, PRRs are a group of molecules that recognize the molecular patterns of DAMPs. These receptors may be activated by PAMPs, alarmins, or both, to activate the immune system. Several important receptors have been implicated in HMGB1 signaling, including the receptor for advanced glycation end products (RAGE) and members of the Toll-like family of receptors (TLRs). RAGE is a transmembrane protein expressed at low levels in normal tissues that is upregulated at sites where its ligands accumulate (46) . The receptor first was identified to bind advanced glycation end products in diabetes, but has since been identified to bind other ligands and is involved in multiple inflammatory states (46, 47) . RAGE was also the first receptor demonstrated to bind HMGB1 (48) . At the time, the consequences of HMGB1 interaction with RAGE were unknown, but it was discovered later that HMGB-1 signaling through RAGE promotes chemotaxis and the production of cytokines in a process that involves the activation of the transcription factor nuclear factor-κB (NF-κB) (49, 50) . Other RAGE-dependent effects of HMGB1 appear to involve the maturation (23, (51) (52) (53) and migration (38, (53) (54) (55) (56) of immune cells as well as the upregulation of cell surface receptors (57) (58) (59) .
In addition to RAGE, the Toll-like family of receptors has been demonstrated to be important in HMGB1 signaling. Members of the TLR family share many structural similarities, both extracellularly and intracellularly, but they differ from each other in ligand specificity, expression patterns, and, in some instances, the signaling pathways they activate. Generally, TLRs can recognize both DAMPs and PAMPs and hence are involved in immune response to both infection and injury. Members of the Toll family have specific ligands, and TLR4 might be the most well known and for its role in the bacterial endotoxin (LPS) recognition complex (60) . TLR4, TLR2, and TLR9 have all been implicated as HMGB1 receptors. HMGB1 binding of TLR2 and TLR4 results in NF-κB upregulation (61) (62) (63) , thus making it likely that HMGB1 stimulation of these receptors can lead to cytokine release. Interestingly, HMGB1-mediated TLR4 activation is different from that resulting from LPS stimulation (50, 62) . For example, when applied to cell cultures, HMGB1 activates both IKKα and IKKβ, whereas LPS only activates IKKβ. Additionally, the MAPK protein activation and cytokine production profiles differ between HMGB1-and LPS-treated cells.
There is much speculation that HMGB1 does not act alone in the triggering of receptor activation. Proof of this concept was provided by Tian et al. who demonstrated that HMGB1-DNA complexes activate TLR9 signaling (64) . HMGB1 involvement in TLR9 activation appears to be mediated by HMGB1-DNA complexes rather than HMGB1 alone. While HMGB1-DNA complex stimulation of TLR9 is involved in maturation of immune cells and cytokine secretion (64, 65) , there is some evidence that these complexes may also suppress the immune response in some cell types (66) . In addition to the HMGB1-DNA interactions that activate TLR9, HMGB1 interactions with other cytokines such as IL-1β, IFNγ, and TNFα lead to an increased pro-inflammatory response compared with HMGB1 stimulation alone (67) . Ultimately, whether other interactions or modifications of HMGB1 are required for pattern recognition receptor binding or activation is uncertain.
As HMGB1 has multiple downstream signaling responses due to activation of different receptors, it also induces cell specific responses when it stimulates cells of the immune system ( Figure 1 ). HMGB1 induces DC maturation as measured by the increased expression of many cell surface markers as well as the secretion of inflammatory cytokines (23, (51) (52) (53) . Monocytes stimulated with HMGB1 have an increased capacity for adhesion (38) and release numerous cytokines and inflammatory mediators (61, (68) (69) (70) . This effect is augmented when administered concomitantly with other cytokines (67, 71) . Neutrophil stimulation with HMGB1 increases the interaction of MAC-1 and RAGE, thus activating the adhesive and migratory function of these cells (56) . Furthermore, HMGB1 stimulates the production of reactive oxygen species by neutrophils through a TLR4 dependent activation of NAD(P)H oxidase (72) , as well as increases the activation of NF-κB which results in increased production and release of cytokines (50, 62) . T cells stimulated with HMGB1 release cytokines and appear to have increased proliferation, survival, and Th1 functional polarization (23, 51) .
The response of endothelial cells to exogenous HMGB1 has been studied in relation to its possible pro-inflammatory role in vascular disease. Endothelial cells release TNFα, IL-8, and MCP-1, all of which enhance the local inflammatory en-vironment (57, 58) . Additionally, HMGB1 stimulation appears to increase the expression of ICAM-1 and VCAM-1 on the surface of endothelial cells, thus increasing the adhesion of inflammatory cells (57, 58) . These effects appear to be at least partially mediated by RAGE, which also appears to be upregulated in HMGB1stimulated endothelial cells (57, 58) .
In contrast to their role in promoting inflammation, the ability of alarmins to promote tissue repair and regeneration is of increasing interest (73) . Importantly, HMGB1 induces migration of stem cells toward inflamed regions to promote repair and regeneration (74) . Furthermore, it results in increased mesangioblast and endothelial proliferation and migration to sites of inflammation and induces transit of these cells across the endothelial layer (75, 76) . Myoblasts stimulated by HMGB1 migrate toward damaged regions and stimulate repair (77, 78) . These effects on specific cell types are reflected in increased regeneration at the tissue level. In smooth muscle, HMGB1 induces proliferation and rapid changes in cellular architec-ture leading to cell migration (79, 80) . In skeletal muscle, HMGB1 promotes increased myogenesis and angiogenesis (49, 76, 77) . Further confirmation of this is reflected in the finding that inhibition of HMGB1 signaling leads to decreased vessel density and tissue regeneration (77) . Additionally, direct injection of exogenous HMGB1 to peri-infarcted cardiomyocytes in mice results in increased myocytes within the infarcted area and ultimately improved outcomes by both structural and functional measures (81) . In an animal model of wound healing, topically applied HMGB1 accelerates the process in diabetic mice while inhibition of HMGB1 signaling slows it in normal mice, thus implicating functional HMGB1 signaling as an important component of diabetic wound healing (82) . The ability of HMGB1 to stimulate angiogenesis has been demonstrated independently in studies demonstrating that exogenous HMGB1 stimulates endothelial proliferation and migration (75, 83) . Interestingly, many of these restorative effects are mediated through the same receptors (i.e. RAGE) that mediate the pro-inflammatory properties of the molecule (75, (77) (78) (79) 84) . Taken together, the regenerative properties of HMGB1 represent a potentially important manner by which the functions of an alarmin can be manipulated to promote healing as opposed to injury.
While early work on HMGB1 demonstrated its role as a late mediator of sepsis (10), more recently HMGB1 has been implicated as a putative danger signal involved in the pathogenesis of a variety of non-infectious inflammatory conditions including autoimmunity, cancer, trauma, and hemorrhagic shock, and ischemia-reperfusion injury. Furthermore, it has been studied in a number of organ systems including liver, heart, pancreas, brain, bone, and kidney. In addition to these studies highlighting the pro-inflammatory effects of HMGB1 in in vivo models of multiple diseases, there is emerging evidence to suggest that HMGB-1 can participate in tissue repair and remodeling as well as preconditioning; all of which are increasingly being recognized as important capacities of danger signals.
Considerable evidence exists implicating extracellular HMGB1 in the pathogenesis of a variety of autoimmune diseases. Anti-HMGB1 antibodies are present in the serum of patients with rheumatoid arthritis and drug-induced systemic lupus erythematosus (85, 86) . Furthermore, HMGB1 has been found to be overexpressed in the extracellular milieu of synovial biopsy specimens in rheumatoid and experimental arthritis (87, 88) . When given via direct intraarticular injection, HMGB1 induces arthritis in mice, while treatment with HMGB1 antagonists ameliorates collagen-induced arthritis in both rats and mice (89, 90) . Increased extracellular HMGB1 also is detectable in the dermis and epidermis of skin lesions in patients with cutaneous lupus erythematosus and in biopsy specimens of the minor salivary glands of patients with Sjogren's Syndrome (91, 92) .
HMGB1 was initially identified as a nuclear DNA-binding protein. As such, it plays a role in the transcription of several genes, some of which include those that have been implicated in cancer development including E-selectin, TNFα, insulin receptor, and BRCA (93) (94) (95) (96) . Furthermore, cancer cells that have undergone necrotic cell death can release HMGB1 into the local microenvironment. Extracellular HMGB1 can lead to chronic inflammatory/reparative responses that, in the setting of cancer, may lead to tumor cell survival, expansion, and metastases (97) . Interestingly, numerous studies suggest that HMGB-1 plays a role in metastasis development, and thus links it to poor prognosis in a variety of cancers including prostate, breast, pancreas, and colon (98) (99) (100) (101) (102) (103) (104) (105) (106) .
In contrast to its potential role in tumor growth and spread, it also has been shown that HMGB1-induced TLR4 signaling is required for effective responses to chemo-radiation in established tumors in animal models (107) . These findings are validated further by the observation that node positive breast cancer patients carrying a loss of function TLR4 allele tend to relapse more quickly after treatment with chemo-radiation as compared to patients with wild-type TLR4 alleles (107, 108) . More recently, HMGB1 has been recognized as a tumor derived DAMP capable of recruiting and activating eosinophils. The role of tumor infiltrating eosinophils is still being elucidated as they can either limit tumor growth through destructive effector functions or promote tumor growth through immunoregulation and tissue repair/remodeling (109, 110) .
In addition to the evidence linking HMGB1 to established cancers, it has also been linked to the pathogenesis of premalignant conditions. Elevated serum levels of HMGB1 are present in mice with chemically induced colitis. Furthermore, neutralizing antibodies to HMGB1 decrease tumor incidence and size in colitis-associated cancer models (111) .
The sum of these findings suggests that HMGB1 plays a role in tumor development, growth, and spread. Additionally, immune responses to established cancers prior to, as well as during, systemic treatment appear dependent upon HMGB1/TLR-4 mediated signaling cascades. Consequently, HMGB1 warrants further investigation as a possible therapeutic target in malignant processes.
End organ dysfunction in trauma and hemorrhagic shock results from systemic inflammatory responses (112) . Barsness et al. provided indirect evidence that HMGB1 may be involved in the pathogenesis of end organ injury following hemorrhagic shock by linking TLR4 activation to the development of acute lung injury (113) . Subsequently, it has been shown that pulmonary HMGB1 levels are increased as early as 4 h after the initiation of hemorrhagic shock (114) . Furthermore, HMGB1 neutralizing antibodies ameliorate hemorrhage-induced acute lung injury (114) as well as gut barrier dysfunction and ultimately lead to improved survival (115) . These findings are even more relevant given the clinical observation that circulating HMGB1 levels are elevated in trauma patients with hemorrhagic shock as compared with normal volunteers (115) .
Additionally, HMGB1 has been shown to play a central role in the initiation and propagation of the inflammatory response following traumatic injury. In a mouse femur fracture model, administration of neutralizing HMGB1 antibodies results in decreased serum IL-6 and IL-10 levels. This blunted systemic response corresponds with decreased hepatic and gut barrier dysfunction as measured by serum transaminase levels and NF-κB activation, respectively. Interestingly, the contribution of HMGB1 to end organ injury in this model appears once again to be TLR4 dependent, as mutants are protected when compared with wild-types. No additional protec-tion is seen when antibodies are administered to TLR4 mutants (116) .
The role of HMGB1 in the pathogenesis of acute or chronic viral illnesses remains elusive. As mentioned, HMGB1 can escape extracellularly upon necrotic cell death. West Nile encephalitis and acute hepatitis are viral-induced pathologies associated with necrotic cell death either via direct cytotoxic effects of the virus itself or the inflammatory response to viral infection (36) . In a recent study utilizing hepatitis B virus (HBV) transgenic mice injected with virus specific cytotoxic T lymphocytes (CTLs), HMGB1 translocates from the nucleus to the cytoplasm of hepatocytes surrounding CTLcontaining necroinflammatory foci. Furthermore, treatment with HMGB1 inhibitors in this model significantly decreases the recruitment of inflammatory cells (117) .
In addition to passive release from necrotic cells, HMGB1 can be secreted actively by inflammatory cells (monocytes, macrophages, dendritic cells, etc.). A number of viral illnesses including SARS and influenza result in the elevation of proinflammatory cytokines (TNF, IL-1, IL-6, types I and II interferon) that could be responsible for inducing release of HMGB1 from inflammatory cells, thus leading to the propagation of the immune response (36) .
A preponderance of evidence exists implicating HMGB1 in the pathogenesis of ischemia/reperfusion injury (IRI) in multiple organ systems including kidney, brain, heart, and liver. Much of the seminal work identifying extracellular HMGB1 as an alarmin that initiates the inflammatory response resulting from IRI was performed using a model of partial warm hepatic IRI in mice. In this model, tissue levels of HMGB1 are elevated as early as 1 h following reperfusion (118) and continue to increase for up to 24 h following the insult. Furthermore, in this same model, neutralizing antibodies to HMGB1 ameliorate the damage resulting from IRI in a TLR4-dependent manner.
The importance of HMGB1 signaling through TLR4 in hepatic IRI was further clarified in subsequent in vivo experiments by the generation of chimeric mice in which TLR4-derived bone marrow cells were shown to be largely responsible for the initiation and propagation of the inflammatory response. In this set of experiments, mice expressing TLR4 mutant bone marrow-derived cells were protected from liver damage as compared to those expressing TLR4 wild type bone marrow-derived cells. This occurred independently of the TLR4 phenotype expressed on hepatocytes (119). Subsequently, it was shown that increasing the number of hepatic dendritic cells worsened liver damage in TLR4 wildtype but not TLR4 mutant animals (59) , thus suggesting that dendritic cells are involved in the recognition of and response to DAMPs (HMGB1 included) released after acute injury in this model. The molecular mechanisms of HMGB1/ TLR4-dependent IRI involve the production of reactive oxygen species in a process that appears to be dependent on calcium signaling via the calcium/ calmodulin-dependent kinases (CaMK) whose activation is ultimately involved in the release of HMGB1 (43) .
While the significant body of literature described above elucidates the role of HMGB1 in hepatic IRI, similar work in other organ systems including the heart (120,121), kidney (122) , and brain (123) (124) (125) has provided confirmatory evidence that HMGB1 is involved in the initiation of the inflammatory response following IRI.
A well characterized effect of pathogen-associated molecular patterns (PAMPs), such as LPS, is their ability to confer protection when administered in small doses prior to more significant insults in a process that has come to be known known as preconditioning.
Therefore, alarmins ought to be able to precondition against later insults as well. Proof of principle of this concept has been demonstrated by HMGB1's ability to provide protection when administered as a precondition agent in hepatic IRI (126) . HMGB1 administration 1 h prior to the onset of injury results in a dose-dependent protection as evidenced by decreased circulating biochemical markers of liver damage as well as decreased serum TNFα and IL-6 levels.
This preconditioning effect appears to be mediated through an inhibition of TLR4 signaling (126) .
There is a growing body of literature surrounding the specific functions of HMGB1 on cells of the immune system as well as its role in important disease states. As the mechanisms promoting the release of HMGB1 and the signaling pathways it activates remain to be completely elucidated, evidence that suggests its potential as a therapeutic target/agent in various models of inflammation continues to accumulate. Importantly, human-subject studies which demonstrate its elevation in several disease states further indicate its importance. Interestingly, in addition to its proinflammatory roles, HMGB1 also appears to have several regenerative effects that lead to tissue repair. Understanding HMGB1 and its complex effects on the immune system may lead to the development of novel strategies to attenuate inflammation and/or promote tissue repair and regeneration in various clinical states. Quantitative measurement of thyroglobulin mRNA in peripheral blood of patients after total thyroidectomy Previous studies have reported the clinical usefulness of reverse transcription-polymerase chain reaction (RT-PCR) detection of thyroglobulin (TG) mRNA in the peripheral blood of patients with differentiated thyroid carcinoma. To evaluate this usefulness, we measured TG mRNA in the peripheral blood of patients diagnosed with thyroid carcinoma after total thyroidectomy by real-time quantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Surprisingly, we detected TG mRNA in all samples obtained after total thyroidectomy, including those from 4 medullary carcinomas. Further, there was no statistical difference in expression levels of TG mRNA in the patients with or without metastasis, and no significant correlation was found between serum TG concentrations and the expression levels of TG mRNA. These results give rise to a question regarding the clinical applications of not only RT-PCR detection but also quantitative measurement of TG mRNA in peripheral blood. © 2001 Cancer Research Campaign http://www.bjcancer.com Thyroid carcinomas often recur many years after surgery (Loh et al, 1997) . The monitoring of serum thyroglobulin (TG) by immunoassay can be used in detecting residual or recurrent differentiated thyroid carcinoma (DTC) after total thyroidectomy. However, the usefulness of this method is limited by both the requirement for thyroid hormone withdrawal to attain optimal sensitivity and interference by antithyroglobulin antibodies (Singer et al, 1996) . Recent reports have demonstrated that the reverse transcription-polymerase chain reaction (RT-PCR) can be used to detect circulating cancer cells in the peripheral blood of patients with malignancies such as prostate cancer (Ghossein et al, 1995) and neuroblastoma (Mattano et al, 1992) .
A sensitive RT-PCR assay amplifying thyroid-specific mRNAs such as TG or thyroid peroxidase (TPO) may be utilized for the early detection of DTC recurrence and thus may have important therapeutic and prognostic implications. In fact, 2 recent reports have shown the clinical usefulness of the RT-PCR detection of TG, TPO, and ret /PTC in the follow-up of DTC (Ringel et al, 1998; Tallini et al, 1998) . For example, TG mRNA in peripheral blood became detectable earlier than serum TG in a case of recurrent thyroid papillary carcinoma. Recently, Ringel et al have reported the clinical usefulness of the real-time quantitative measurement of TG mRNA in the peripheral blood of the patients with DTC. In their report, however, they did not use an appropriated internal reference such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA .
Encouraged by these optimistic reports, we applied this method to our patients for the follow-up of DTC. However, we found that TG and TPO mRNAs were detectable in the peripheral blood of all the patients tested, a result that differs from those reported previously, and we therefore felt a need to re-evaluate TG mRNA detection. One of the problems of previous studies has been that the expressions levels of TG mRNA have not been calculated in conjunction with the use of an appropriate internal control, as the results of simple RT-PCR detection can vary in response to subtle changes in conditions.
Patients who have undergone total thyroidectomy constitute good model cases for evaluating this problem. As such, we measured TG mRNA in the peripheral blood of patients after total thyroidectomy and determined the expression levels of TG mRNA by real time-quantitative RT-PCR using GAPDH mRNA as an internal control.
We evaluated 57 patients (3 males and 54 females, between 15 and 77 years of age, 49 papillary, 4 follicular, and 4 medullary carcinomas) who underwent total thyroidectomy. Blood was drawn at least 6 months after surgery or the administration of radioactive iodine. Of the 53 DTC patients, 21 had evident metastases (16 lung, 1 bone, 2 mediastinum, and 2 lung and bone) detected by either computed tomography or 131 I scintigram. All the patients with metastasis and 21 of 32 patients without metastasis received 5 to 150 (mean: 55.7 mCi) and 5 to 100 (mean: 17.0 mCi) 131 I, respectively. All the patients after total thyroidectomy, except those with medullary carcinoma, received a suppressive dose of thyroxine so that their serum thyroid stimulating hormone (TSH) levels were undetectable. 17 healthy subjects (5 males and 12 females, between 29 and 59 years of age) with no evidence of thyroid disease were also examined as controls. The protocol was approved by the institutional review boards at the participating institutions, and informed consent was obtained.
Peripheral blood was collected in heparinized tubes in 10 ml samples and placed immediately on ice. Next, 10 ml of 3% dextran 200 000 (WAKO, Osaka, Japan) was added to the tubes, which were than placed in ice for 30 min. The supernatant was collected and centrifuged. The precipitant was dispersed in distilled water to lyse the remaining erythrocytes, and an equal volume of 1.8% NaCl was then immediately added. The isolated cells were washed, then centrifuged. Total RNA was extracted following standard procedures (Chomczynski and Sacchi, 1987) and resolved in 20 µl of distilled water, then stored at -70˚C.
1 µl of total RNA was reverse transcribed in an RT mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl 2 , 0.5 mM deoxynucleotide triphosphates (dNTPs), 200 U M-MLV reverse transcriptase (Gibco, Gaithersburg, MD), 2 U µl -1 RNase inhibitor (Takara, Shiga, Japan), and 2.5 µM random hexamer (Takara) in a total volume of 20 µl at 42˚C for 60 min.
1 µl of first-strand cDNA was used as a template for the PCR reaction with specific primers for TG cDNA as previously described (Tallini et al, 1998) . Each reaction mixture consisted of 1 µl of cDNA, 0.5 µM of each primer, 1 µl of 10 × Ex Taq Buffer, 0.8 µl of 2.5 mM dNTP mix, 0.5 U of Ex Taq polymerase, and nucleasefree water to a final volume of 10 µl. 10 × Ex Taq Buffer, dNTP mix, and Ex Taq polymerase were obtained from Takara. The reaction mixture was then subjected to 35 cycles of denaturation (94˚C, 1 min), annealing (55˚C, 1 min) and extension (72˚C, 1 min). After PCR amplification, the reaction mixture was run on a 2% SeaKem GTG agarose gel (Takara). The gel was then stained with ethidium bromide. Samples without reverse transcription were used as negative controls.
Real-time quantitative RT-PCR (TaqMan PCR) of TG and GAPDH mRNAs using an ABI PRISM 7700 Sequence Detection System and a TaqMan PCR Core Reagent Kit (PE Biosystems, Foster City, CA) was performed as previously described (Takano et al, 1999) . The primers used for amplification of TG cDNA were changed to the same set of primers used in the report by Tallini et al. The probe for TG cDNA used for the TaqMan PCR was: (5′-FAM-CACTTCGAGTTCCAGGAATGGCCTGACCCT-TAMRA-3′).
1 µl of each cDNA was used for measurement of the copy number. The conditions for the TaqMan PCR were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 seconds and 60˚C for 1 min. Recombinant pGEM T-vectors (Promega, Tokyo, Japan) containing TG or GAPDH cDNA were constructed by PCR cloning with the same set of primers used in the TaqMan PCR and were used as standard samples. The expression levels of TG mRNA were calculated by dividing the copy number of TG mRNA by that of GAPDH mRNA. Intraassay variabilities were 35% and 24% at 10 and 100 ng l -1 thyroid total RNA, respectively. Interassay variabilities across a 1-month period were 38% and 28% at 10 and 100 ng/L thyroid total RNA, respectively.
A portion of normal thyroid tissue from the opposite lobe of a thyroid papillary carcinoma was obtained by surgery. Total RNA was extracted following standard procedures.
Serum TG was measured using a commercial radioimmunoassay kit (Ab-Beads Thyroglobulin, Eiken, Tokyo, Japan). Intrassay variability was 8.6% at 5 µg l -1 . Interassay variabilities across a 1month period were 11.8%, 7.4%, 3.1% at 15, 15, 50 µg l -1 , respectively. Assay sensitivity as determined from the 20% interassay coefficient of variation (CV) was 0.5 µg l -1 . The cut off level of this kit was 28.4 µg l -1 . Serum anti-TG antibody was detected with a semi-quantitative microtitre particle agglutination test for the in vitro diagnostic detection and titration of anti-TG antibodies in human serum, SERODIA-ATG (Fujirebio, Tokyo, Japan).
Statistical analysis of differences between the groups was carried out using the Mann-Whitney U test. Linear correlation analysis was used to examine the correlation between serum TG and expression levels of TG mRNA in the peripheral blood. Ps of <0.05 were considered significant.
TG mRNA was detected in all the samples tested, including the samples from 4 patients diagnosed with medullary carcinoma. Representative data are shown in Figure 1 .
The reliability of the quantitative RT-PCR was estimated by adding total RNA from a normal thyroid to that from 10 ml of blood of a healthy subject. The TG/GAPDH mRNA ratio in the sample without the addition of thyroid RNA was 6.75 × 10 -5 . As the thyroid RNA was increased from 10 ng per 1 l blood to 10 µg per 1 l blood, a linear increase in the TG/GAPDH mRNA ratio was observed, indicating the reliability of this quantitative measurement ( Figure 2) . These results were used in the construction of the standard curve in the following experiments.
Using this method, the expression levels of TG mRNA in the peripheral blood of 57 patients and 17 healthy subjects were calculated. All the samples were successfully measured by real-time quantitative RT-PCR, which confirmed the expression of TG mRNA in all the samples. There was no statistical difference between the patients with and without metastasis (Figure 3 ). The serum TG was measured in the 49 DTC patients without serum anti-TG antibody, and the set of results were compared (Figure 4) . No statistical differences in the TG mRNA levels were observed. While serum TG was detectable in all the patients with metastasis, it was undetectable in 14 of 29 patients without metastasis. Further, serum TG was undetectable in all 4 patients diagnosed with medullary carcinoma, while quantitative measurement of TG mRNA was possible (Figure 3) . These results suggest that serum TG is a superior marker of distant metastasis of DTC than TG mRNA in peripheral blood. Further, the correlation between TG mRNA and serum TG was estimated in these 49 patients and no correlation was observed between the two ( Figure 5 ).
The vast majority of thyroid carcinomas are differentiated tumours that originate from the follicular epithelium and show Total RNA from a normal thyroid was added to that from 10 ml of blood from a healthy subject, after which real-time quantitative RT-PCR, as described in Methods was carried out. The copy number of GAPDH mRNA was simultaneously measured, and the expression levels of TG mRNA were calculated as the ratio of TG and GAPDH mRNA. The results are shown with mean ±SD for triplicate determinations either follicular or papillary structure. Serum TG immunodetection measurements are currently the best available indicator of tumour metastases, even though their diagnostic usefulness has several limitations. Circulating tumour cells can be detected by sensitive PCR amplification of tumour-specific abnormal gene sequences or through the detection of tissue-specific abnormal gene transcripts normally absent in the peripheral blood. Thyroid tumours represent the ideal target for such studies because they actively express tissue-specific markers such as TG and TPO. Despite promising reports in 1998 on the detection of TG mRNA in peripheral blood, our preliminary study showed that TG transcripts were detectable in all samples tested, including the ones from healthy subjects. Recently, 2 studies suggesting limitations in the clinical applications of this method have been reported. Wingo et al have detected TG transcripts in the peripheral blood of healthy subjects that can be measured by real-time quantitative RT-PCR (Wingo et al, 1999) . Bojunga et al have found that TG mRNA expression is not specific to thyroid tissue and is not correlated with a diagnosis of thyroid cancer in patients, and they have recommended further examination of this method by quantitative measurement (Bojunga et al, 2000) . We therefore decided to re-evaluate this method using samples obtained from patients after total thyroidectomy, in which TG transcripts in the circulating thyroid cells from the normal thyroid may be negligible. We detected TG transcripts in all the samples tested, even in that of the patient diagnosed with medullary carcinomas, in whom the circulating of thyroid follicular cells was not likely to occur. Real-time quantitative RT-PCR assay confirmed the expression of TG mRNA in these samples, in which an increase in the fluorescent signal from hybridized TG cDNA-specific probe was observed.
By real-time quantitative RT-PCR, high levels of TG mRNA were expressed in some patients without evident metastasis. For example, in one patient, the copy number of TG mRNA in 1 ml of peripheral blood was comparable to that in over 300 pg total RNA from normal thyroid tissue, which is approximately equal to 30 thyroid cells. It is hard to believe that this many thyroid cells still circulated in the peripheral blood of a patient after thyroidectomy without any evidence of a distant metastasis, which could be a source of circulating cells. TSH receptor mRNA, which has previously been considered to be expressed only in the thyroid, was found to be expressed in adipocytes and lymphocytes (Francis et al, 1991; Endo et al, 1995) . The TG mRNA detected by our study thus might not have been derived from thyroid cells; instead, it is most likely that lymphocytes express small quantities of TG mRNA, as they are known to express various kinds of mRNAs such as alpha-fetoprotein (AFP) or carcinoembryonic antigen (CEA) (Lafarge-Frayssinet et al, 1989; Coutelier et al, 1994) , which used to be considered tumour-specific. Further, the expression of TG mRNA is not likely to be limited to thyroid follicular cells, as we have recently found it to be expressed in cell lines derived from lung or gastric carcinomas (data not shown). Possibly, the regulation of TG mRNA expression in lymphocytes is controlled by some unknown factors. If so, the detection of TG mRNA is not likely to become an alternative means of achieving early detection of recurrent thyroid carcinomas because sensitive detection of recurrence would be interfered with by basal expression of TG mRNA in peripheral blood.
It therefore seems likely that the diagnostic methods presented by Tallini and Ringel may be useful only in limited populations of patients in whom a considerable number of thyroid tumour cells expressing a high copy number of TG mRNA are circulating. In fact, our results showed no statistical difference between the samples from patients with or without distant metastasis. Further, we obtained no evidence that this method is superior to the conventional measurement of serum TG, as it was hard to utilize the TG mRNA data to differentiate the patients with metastasis from those without, whereas serum TG measurements showed more than 5 µg l -1 in all patients with distant metastasis, and the high values of interassay CV in the quantitative measurement of TG mRNA can be a disadvantage in the follow-up of a patient for a long period. Moreover, TG mRNA and serum TG measured in the same samples showed no significant correlation. These results suggest that the sources of TG mRNA and serum TG might be different, and, as discussed above, the former might not be derived from thyroid follicular cells.
The use of TPO mRNA in the detection of thyroid carcinomas has also been reported in conjunction with some studies. However, our preliminary data showed that TPO mRNA was detectable by RT-PCR in all the samples tested, including those from healthy subjects and patients after total thyroidectomy without distant metastasis (data not shown). Considering that TPO mRNA are usually less abundant than TG mRNA in thyroid cells, it is not likely that the use of TPO mRNA instead of TG would improve the clinical usefulness of this method.
In summary, we have found no advantages in diagnosing DTC by the quantitative measurement of TG mRNA in peripheral blood. We consider that an intensive re-evaluation, including a determination of what percentage of TG mRNA derives from thyroid follicular or cancer cells, is necessary with regard to this method before considering the clinical applications.
This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (to TT; No. 12771474) from the Ministry of Education, Science, Sports and Culture of Japan.  Functional Analysis of the 5′ Genomic Sequence of a Bovine Norovirus BACKGROUND: Jena Virus (JV), a bovine Norovirus, causes enteric disease in cattle and represents a potential model for the study of enteric norovirus infection and pathogenesis. The positive sense RNA genome of JV is organised into ORF1 (non-structural proteins), ORF2 (major capsid protein) and ORF3 (minor capsid protein). The lack of a cell culture system for studying JV replication has meant that work to date has relied upon in vitro systems to study non-structural protein synthesis and processing. PRINCIPAL FINDINGS: Only two of the three major ORF1 proteins were identified (p110 and 2C) following in vitro translation of JV RNA, the N-term protein was not detected. The N-term encoding genomic sequence (5′GS) was tested for IRES-like function in a bi-cistronic system and displayed no evidence of IRES-like activity. The site of translation initiation in JV was determined to be at the predicted nucleotide 22. Following the insertion of an epitope within the 5′GS the JV N-term protein was identified in vitro and within RNA transfected cells. CONCLUSIONS: The in vitro transcription/translation system is currently the best system for analysing protein synthesis and processing in JV. Unlike similarly studied human noroviruses JV initially did not appear to express the N-terminal protein, presenting the possibility that the encoding RNA sequence had a regulatory function, most likely involved in translation initiation in an IRES-like manner. This was not the case and, following determination of the site of translation initiation the N-term protein was detected using an epitope tag, both in vitro and in vivo. Although slightly larger than predicted the N-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the ORF1 polyprotein but also activity of the viral protease. These findings indicate that the block to noroviral replication in cultured cells lies elsewhere. Jena virus, a bovine norovirus, is a member of the Caliciviridae family of positive sense RNA viruses and was first isolated from the diarrhoeic stools of newborn calves [1, 2] . JV is a type I genogroup III (GIII) norovirus which is closely related to the type II GIII bovine noroviruses Newbury agent 2 and Dumfries [3, 4] . The GIII noroviruses are responsible for causing enteric disease in cattle [2, 5] and, thus, likely share a similar tissue tropism to the human-associated enteric noroviruses. Like human noroviruses [6] bovine noroviruses have a high seroprevalence [4] . JV is therefore a potentially useful model for studying the molecular biology of enteric norovirus pathogenesis and replication. The 7.3 kb polyadenylated RNA genome of JV has been characterised previously [7] and, like other noroviruses, is organised into 3 open reading frames (ORFs). ORF1 encodes the non-structural proteins in the form of a large 185 kDa polyprotein, which is subsequently cleaved into functional replication proteins by the viral encoded 3C-like protease. ORF2 encodes the structural capsid protein (56 kDa) and ORF3 encodes a small basic protein, which has been shown to function as a minor capsid component [8] . JV ORF1 is consistent with other caliciviruses in that it encodes a 39 kDa 2C-like nucleoside triphosphatase (NTPase), a 3C-like protease and a 56 kDa 3D-like RNA-dependent RNA polymerase [7, [9] [10] [11] [12] [13] . However, the genomic sequence within the 59 region of JV ORF1 (59GS) displays a high level of divergence. This divergence is mainly attributed to the presence of several proline-encoding polypyrimidine tracts within the region predicted to encode a 35 kDa Nterminal protein [7] . The predicted size of N-terminal proteins relative to the size of the respective 2C proteins differs within the norovirus genus. Within the GI noroviruses, such as Southampton virus, the N-terminal protein (44.8 kDa) is larger in size compared to the 2C protein (39.6 kDa). This is in contrast to the GII noroviruses, such as Lordsdale virus and Camberwell virus, in that the N-terminal protein is smaller in size compared to the 2C protein [11, 14] . This is also the case for Jena virus in which the predicted JV N-terminal protein (35 kDa) is smaller than the JV 2C protein (39 kDa) [7] .
The norovirus N-terminal protein varies in relative size across the genus, and the encoding sequence bears no similarity to other cellular or viral proteins. Alignment of the N-term protein sequences of various noroviruses indicates little similarity between genogroups within the first 180 residues, however towards the Cterminal end of the protein similarity between the amino acid residues increases. Recent studies investigating the functions of the Norwalk virus N-terminal protein have successfully demonstrated association with the Golgi apparatus in transfected cells [15] . In addition this study also identified a picornaviral 2B like region within the N-terminal protein, suggesting that the protein is involved with host cell membrane interactions, reinforcing other findings that have suggested that the Norwalk virus N-terminal protein disrupts intracellular protein trafficking, including proteins destined for the host cell membrane [16] . A 3C protease-mediated cleavage event within the N-terminal protein (37 kDa) was described for Camberwell virus, a genogroup 2 norovirus, yielding proteins of 22 kDa and 15 kDa [17] . Based on these observations and location within the genome it was hypothesised that the Nterminal protein of noroviruses corresponds to the 2AB region in picornaviruses.
Another possibility is that the N-term encoding RNA itself serves to function as a translational enhancer by interacting with cellular proteins involved in translation. Indeed, this phenomenon has been previously reported for Norwalk virus, within which a double stem loop structure has been predicted at the 59 end of the genomic RNA [18] . It was subsequently demonstrated that elements within the 59 end of Norwalk virus bind specifically with cellular proteins such as La, PTB and PCBP2 [19] which have all been implicated in IRES-mediated cap-independent translation in the closely related picornaviruses [20] [21] [22] [23] . In this study the role of the JV 59GS was investigated, including its potential to direct capindependent translation initiation. The precise location of translation initiation in JV was also investigated.
Previous studies of norovirus polyprotein processing have yielded three major products following in vitro transcription and translation, representing the uncleaved 3ABCD, N-term and 2C proteins. However, initial analysis of JV polyprotein processing indicated that only two major proteins are synthesised initially which, based on molecular weight predictions, are the 3ABCD (110 kDa) and the 2C (39 kDa). The lack of an N-terminal protein encoded by the JV 59GS, predicted to be 35.3 kDa, is unique among the noroviruses that have been studied in this way. The in vitro transcription and translation profile for JV was therefore studied in more detail. As initial experiments had analysed TNTH reactions following a 1 hr incubation, reaction aliquots were harvested at time points before and after the recommended 1 hr incubation.
The results in Figure 1 show that there are no major reaction products synthesised prior to the 1 hr time point, at which time the 3ABCD/p110 and 2C/p39 proteins are clearly visible. Extended incubation past the 1 hr point resulted in further proteolytic cleavage of p110 that coincided with the appearance of proteins of the following sizes: 86 kDa, 55 kDa, and 51 kDa. In addition proteins of 29 kDa, 22 kDa and 20 kDa were also visible at the 24 hr time point (Figure 1, lane 7) . The only protein that was consistently visible following the 1 hr time point was the 2C/p39 protein. Despite prolonged incubation there was no indication that the N-terminal/p35 protein was synthesised.
A comprehensive study of polyprotein processing within the murine norovirus (MNV) suggests likely identities for the equivalent proteins in the similar profile for JV [12] . Using region specific antisera the authors were able to identify p110 as the 3ABCD uncleaved precursor, p90 as the 3BCD, p57.5 as the 3D-like polymerase, p52 as a 3ABC precursor and p40 as the 2Clike NTPase, which was determined by mutagenesis and microsequencing experiments. The 19 kDa protein was identified as the 3C-like protease. The antisera used to detect the MNV Nterm protein recognised 3 products; one was the predicted molecular weight at 39 kDa and the other two bands migrated as a 45 kDa doublet.
The 59GS region of JV is highly divergent compared to other noroviruses, mainly due to the relatively high cytosine content (32%), which contributes to an overall G/C content of 58%. There are many polypyrimidine tracts within the sequence, potentially yielding a relatively high degree of RNA secondary structure. Previous studies have described potential secondary RNA structure and interaction with proteins involved with IRESmediated translation within the 59 genomic region of Norwalk virus [18, 24] . It was of interest therefore, based on these findings, to ascertain whether or not the 59GS of JV possessed IRES-like properties within the context of a 'Bi-cistronic' expression system, independently of other viral proteins, including the VPg which, in other caliciviruses, has been shown to be associated with translation initiation factors [25, 26] .
Traditionally the bi-cistronic vector system has been used to define potential IRES-like sequences from a variety of viral and cellular mRNAs, and is recognized as being the standard test for this function [27] . A bi-cistronic vector is comprised of a 59 and 39 cistron; translation of the 59 cistron being cap-dependent and translation of the 39 cistron regulated by the putative IRES-like sequence. Thus, if the 39 cistron is translated in addition to the 59 cistron then the sequence of interest is said to have IRES-like properties, as translation is initiating internally.
To test for IRES-like function in JV, bi-cistronic constructs were made with a cap dependent 59 EGFP cistron and a 39 lacZ cistron under the translational control of either the JV 59GS (pEGFP-C1/ JV59GS/lacZ) or an authentic EMCV IRES (pEGFP-C1/IRES/ lacZ). CRFK cells were transfected with the bi-cistronic constructs and, following incubation, were assayed for EGFP and lacZ expression.
Both constructs were able to direct translation of the EGFP cistron effectively as expected (Figure 2a and Figure2d). The use of an authentic EMCV IRES to direct translation of the lacZ cistron was also effective (Figure 2e), with levels of b-galactosidase activity comparable to those of the b-galactosidase reporter ( Figure 2f ). However, no b-galactosidase activity was detected from cells transfected with the pEGFP-C1/JV59GS/lacZ construct (Figure 2b ), demonstrating that the JV 59GS was unable to initiate translation, and therefore, in this context, did not possess any IRES-like functions.
As it was clear that the JV 59GS did not posses any IRES-like functions it was necessary to determine the location of translation initiation within ORF1. This was predicted be the ATG encoding methionine at nucleotide position 22, as it is situated in a favourable context for translation initiation [7] . To investigate this multiple translation termination codons (polySTOP) were inserted into the JV genome within the 3B-encoding region, downstream of the 59GS, to halt translation at a defined point. In vitro transcription and translation of this construct would, in theory, yield a product whose size would relate to the initiation codon used within the 59GS (Figure 3 ). To address the unlikely event of translation read-through or re-initiation downstream of the polySTOP, which would result in subsequent translation of the 3C protease and cleavage of the truncated ORF1 polyprotein, a mutation was made within the active site encoding region of the 3C protease within JV ORF1, to prevent any viral mediated cleavage of ORF1 translation products (JV 3C mut /polySTOP). A point mutation of the critical cysteine residue within the highly conserved GDCG motif to a glycine residue was performed, and this approach has been described for the successful inactivation of other norovirus' 3C activity [28] . In vitro transcription and translation analysis was performed on JV wild type ( Figure 4 within the 3C region of JV successfully inactivated the 3C protease, thus a large, .200kDa uncleaved polyprotein is yielded following TNTH. The major product generated by JV 3C mut / polySTOP was calculated to be 103kDa in size. Based on computer predictions this is in agreement with the initiation of translation occurring at nucleotide 22, which demonstrates that the JV N-term protein is translated in full in vitro. At this time it is not possible to determine whether translation of intracellular VPgbound viral RNA initiates at nucleotide 22, although it is likely given the favourable context in which the initiation codon is situated.
As the JV N-term was found to be translated in vitro attempts were made to express and purify the protein in bacteria for immunisation so that the protein could be identified by radioimmune precipitation assay (RIPA), as it was possible that the Nterm protein was migrating on gels aberrantly and possibly comigrating with 2C. Attempts to express the protein in bacteria were unsuccessful due to toxicity. Therefore, the 14aa V5 epitope encoding sequence was cloned in frame into the JV cDNA construct at nucleotide position 123 (JV V5). The V5 epitope originates from the P and V proteins of the SV5 paramyxovirus [29] , for which a commercially available monoclonal antibody is used for detection.
Following in vitro transcription and translation of JV V5 a new product, approximately 42 kDa in size, was visible ( Figure 5 , lane 2). This product was not observed in any prior analyses of JV. To confirm that this protein was V5/N-term associated the TNTH reaction was subjected to RIPA using the anti-V5 antibody ( Figure 5, lane 3) . This confirmed expression of the N-term protein in vitro.
To confirm expression of the V5/N-term protein in cell culture capped RNA was synthesised from the JV V5 T7 cDNA construct, which was used to transfect CRFK cells. As there is currently no host cell line in which to propagate JV the CRFK cell line was used as it has been shown to support the replication of feline calicivirus [30] . Confocal immunofluorescence of transfected cells using the anti-V5 antibody demonstrated expression of the V5/Nterm protein in cultured cells ( Figure 6 ). Expression of the V5/Nterm protein was diffuse and did not co-localise with the Golgi/ ER/plasma membrane marker wheat germ agglutinin (WGA) and therefore displays a different pattern of cellular expression compared to Norwalk virus [15] . Cells transfected with the wild type full length JV RNA were negative for fluorescence (data not shown).
Lysates of cells transfected with wild type JV and JV/V5 RNA were subjected to western blot using the anti-V5 antibody ( Figure 7) . No product was present for cells transfected with wild type JV RNA, but a protein of approximately 42 kDa in size was visible in cells that had been transfected with JV/V5 RNA, confirming N-term expression and size as seen in the in vitro system. In addition, this important observation also confirms for the first time that the JV 3C protease was active in cells transfected with capped RNA as the size of the V5/N-term indicated successful cleavage of the protein from the ORF1 polyprotein.
To address the issue of potential rapid degradation of the JV Nterm protein CRFK cells were transfected with JV V5 RNA and were harvested at designated time points following the addition of the protein synthesis inhibitor cycloheximide. Cell lysates were analysed by Western blot using the anti-V5 antibody (Figure 8 ). The consistent appearance of the N-term/V5 protein suggested that it is stable and insensitive to degradation by viral and host cell proteases.
The predicted molecular weight of the JV N-term is 35.3 kDa, based on the site of initiation of translation and location of conserved cleavage sites. The appearance, therefore, of a previously unseen 42 kDa protein in the in vitro transcription and translation profile was unexpected but this protein does represent a translation product for the JV 59GS. To date, it has not been possible to explain the difference in the predicted and observed sizes for the JV N-term, and the addition of the 14 amino acid V5 epitope within JV N-term does not account for this apparent large shift in molecular weight. However, a recent study described a similar anomaly when investigating proteolytic processing in the murine norovirus MNV-1 [12] . The predicted molecular weight for the MNV-1 N-term protein was 38.3 kDa. The authors successfully generated antisera against the MNV-1 N-term and used it to immunoprecipitate the protein from in vitro transcription and translation reactions and observed that the N-term existed as a 45 kDa doublet, in addition to the predicted size of 38 kDa. However, when MNV-1 N-term antisera was used to probe MNV-1-infected cell lysates only the 43-45 kDa doublet and a large 115 kDa precursor could be detected, suggesting that the predicted 38 kDa form of the N-term is not generated in cell culture. Again, it was not possible to conclusively determine the cause of this discrepancy, but it was speculated that the N-term protein may migrate abnormally in SDS-PAGE, or may be proteolytically processed at a previously unknown cleavage site downstream of the protein's predicted C-terminus. It is also possible that the N-term protein might be modified in some way leading to a shift in observed molecular weight. At this time the same conclusions would seem appropriate for the JV N-term. In addition, it is not known why the JV N-term was previously not detected in in vitro transcription and translation studies prior to the insertion of the V5 epitope. It cannot be ruled out, however, that the wild type JV N-term aberrantly co-migrates with the 39 kDa JV 2C protein in SDS-PAGE. Indeed, the appearance of the V5/ N-term product from transfected cell lysates would appear to be one of a doublet (Figure 8) , also analogous to the observed appearance of the MNV N-term protein in infected cells, suggesting the likelihood of a further cleavage site within the JV N-term protein which has yet to be elucidated. Nevertheless, these studies clearly demonstrate that a protein representative of the 59GS of JV is translated both in vitro and in vivo and is proteolytically processed from the ORF1 polyprotein following translation initiation at nucleotide 22.
Human norovirus infection has been shown to be the leading cause of non-bacterial gastroenteritis [31] , however there is currently no cell culture system available to facilitate viral replication and ethical considerations have hindered progress in establishing a permissive human organ culture system. The study of Jena virus offers a potential animal model of enteric noroviral infection. However, until a permissive bovine cell and/or organ culture systems is established analysis of the molecular mechanisms underpinning viral replication and pathogenesis rely upon in vitro systems, most notably polyprotein synthesis and processing.
Unlike similarly studied human noroviruses JV initially did not appear to express the N-terminal protein, presenting the possibility that the encoding RNA sequence had a regulatory function itself, most likely involved in translation initiation in an IRES-like manner. This was shown not to be the case and, following determination of the site of translation initiation at the predicted nucleotide 22 the N-term protein was detected following the insertion of an epitope tag, both in vitro and in vivo. Although slightly larger than predicted the N-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the ORF1 polyprotein but also activity of the viral encoded protease. These important findings indicate that the block to replication of enteric norovirus in cultured cells cannot be attributed to a failure to synthesise and process the non-structural proteins. The detection of processed and active ORF1 proteins in transfected cultured cells, however, highlights the potential for the development of cell and bovine organ based systems to facilitate the replication of Jena virus.
The pEGFP-C1 vector (Clontech) comprises of an EGFP coding sequence under the control of a CMV promoter and a Kozak translation initiation site. Downstream of the EGFP sequence is the multiple cloning site containing unique BglII, SacI, HindIII and ApaI restriction sites. Contruction of pEGFP-C1/JV 59 GS/lacZ was as follows; The JV 59 GS sequence was amplified from the JV full length cDNA clone [7] using Bio-X-Act DNA polymerase (Bioline) with the primers 59 GS F (59-AACTGCA-GATCTTAATAAGTGAATGAAGACTTTGACGAT-39), containing the BglII restriction site (bold) and two in-frame translation termination codons (underlined) to ensure that translation of the EGFP sequence did not carry over to the 59 GS, and 59 GS R (59-AACTGCAAGCTTCTGCAGGACACAATGAGG-39), containing theHindIII restriction site. The JV 59 GS amplicon was ligated to the pEGFP-C1 vector, following restriction enzyme digestion of both amplicon and vector with BglII and HindIII restriction enzymes, and the ligated DNA used to transform E.coli Top10 (Invitrogen). This intermediate construct was named pEGFP-C1/ JV 59 GS. The lacZ coding sequence was amplified from the pSVb-Gal reporter vector (Promega) using Bio-X-Act DNA polymerase and the primers lacZ F (59-AACTGCAAGCTTGA-TATGGGGGATCCCGTCGTTTTACAACG-39), containing the HindIII restriction site (bold) and a kozak translation initiation site (underlined), and lacZ R (59-AACTGCGGGCCCTTAT-TATTTTTGACACCAGACCA-39) containing the ApaI restriction site (bold) and translation termination codons (underlined). The lacZ amplicon was ligated to the pEGFP-C1/JV 59 GS vector following restriction enzyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The construct was verified by sequencing. Construction of pEGFP-C1/IRES/lacZ was as follows; The EMCV IRES sequence was amplified from the pIRES2-EGFP vector (Clontech) using Bio-X-Act DNA polymerase and the primers IRES Bgl F (59-ACTCGAAGATCTTAA-TAGAGCTTCGAATTCTGCAGTCGA-39), containing the BglII restriction site (bold) and translation termination codons (underlined) to prevent carry over translation as before, and IRES Sac R (59-ACTCGAGAGCTCTGTGGCCATATTATCATC-GTG-39), containing the SacI restriction site (bold). The IRES amplicon was ligated to the pEGFP-C1 vector follwing restriction Whole cell lysate was collected at the following time points following CHX treatment: 0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr. Bradford analysis was performed on the lysates to ensure equal loading. Following Western analysis the ECL treated membrane was exposed to film for 1 min. Molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g008 enzyme digest of both amplicon and vector with BgIII and SacI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The lacZ amplicon described previously was ligated to the intermediate pEGFP-C1/IRES vector following restriction enzyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10.
The JV 3C protease mutant was created by point mutation of the critical TGT encoded cysteine residue, within the GDCG active site motif, to a GGT encoded glycine residue by mutagenic overlap PCR using Bio-X-Act DNA polymerase. Three rounds of amplification using the JV full length cDNA clone as template were used to generate the final mutant protease cassette. Round 1 used the primers JV F1 (59-CGTCTCAGGGTTGATACT-39) and JV Mut 1 (59-GCAACCACCGTCACCAG-39), yielding a 222 bp amplicon (point mutation nucleotide shown in bold). Round 2 used the primers JV Mut 2 (59-CTGGTGACGGT-GGTTGC-39) and JV R2 (59-TTCCTGGGAGGAACAAGTT-39), yielding a 651 bp amplicon. Amplicons generated in rounds 1 and 2 were pooled to serve as template for round 3 using the primers JV NF (59-ATGTCAACCACCACCAGC -39) and JV NR (59-AAGGGCTCCGGTGAAGG-39). This cassette contained two BclI restriction sites flanking the 3Cprotease active site, as also found in the wild-type full length clone. Restriction digest using BclI was used to remove the appropriate wild-type cassette from the JV full length clone. The mutant cassette was also digested with BclI prior to ligation to the BclI-digested JV full length clone. The ligated DNA was used to transform E.coli Top10, and was designated JV 3C mut .
Construction of JV 3C mut /polySTOP was as follows: complementary oligonucleotides with three translation termination codons (underlined) in each reading frame in sense and anti-sense orientations were desgined in such a way that upon annealing the duplex would contain blunt termini. The oligonucleotides were termed pSTOP Top (59-CTAGGTAAGTAAACGCGTCTACT-CACTCAC-39) and pSTOP Comp (59-GTGAGTGAGTA-GACGCGTTTACTTCAATAG-39). Each oligo (1 mg) was incubated with T4 polynucleotide kinase and ATP to phosphorylate the 59 termini, pooled and heated to 75uC for 15 min, and left to cool to room temperature to anneal the oligos. Following purification the polySTOP duplex was ligated to Eco47III digested JV 3C mut , and ligated DNA was used to transform E.coli Top10. The duplex contained the unique restriction site MluI (shown in bold) to assist screening of recombinant clones.
The V5 epitope (N-Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-C) is recognized by the anti-V5 monoclonal antibody (Invitrogen). Complementary oligonucleotides encoding the V5 epitope were designed in such a way as to generate SacII compatible termini following annealing (bold), and to preserve the reading frame when inserted into the SacII restriction site at nucleotide 123 within the 59 GS of the JV genome (underlined). The oligos were termed V5 Top (59-GGTAAGCCTATCCC-TAACCCTCTCCTCGGTCTCGATTCTACGAGC-39) and V5 Comp (59-TCGTAGAATCGAGACCGAGGAGAGGGT-TAGGGATAGGCTTACCGC-39). The oligos were phosphorylated and annealed as described previously and the duplex ligated to the SacII digested JV full length clone. Ligated DNA was used to transform E.coli Top10.
In vitro coupled transcription and translation was performed using the TNTH Coupled Reticulocyte Lysate System (Promega) as per the manufacturer's instructions. Reactions were incubated at 30uC for 1-2 hr. For non-radiolabelled reactions the 35 S-Methionine was replaced with 1 mM unlabelled Methionine (2 ml). Reaction products (1-2 ml) were analysed by SDS-PAGE. Gels were stained and prepared for autoradiography by incubating for 30 min in a solution containing 32 g sodium salicylate, 100 ml methanol and 100 ml dH 2 O. Gels were dried under vacuum and the reaction products were detected by exposure to Kodak X-Omat scientific imaging film (Sigma) at 270uC for 16 hr followed by developing using a Kodak automated developer.
Specific V5-tagged proteins synthesised by TNTH were precipitated from 5-10 ml of reaction product using the anti-V5 monoclonal antibody (Invitrogen) at the recommended dilution in 600 ml of 16 RIPA buffer (diluted from 10x stock: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.15 mM NaCl, 0.1% SDS, 0.5% Empigen BB, 0.1 mM phenylmethylsulphonylfluoride) for 1 hr at 37uC. This was followed by a second incubation of tube for 2 hr rotating at room temperature with goat anti-mouse immunoglobulin G agarose beads (Sigma) to absorb the immune complexes. The beads were washed three times with 500 ml 16 RIPA buffer and once with 500 ml PBS. The beads were resuspended in sample buffer for analysis by SDS-PAGE and autoradiography as before.
Endotoxin-free preparations of plasmid DNA were prepared using the GenElute TM Endotoxin free plasmid midi prep kit (Sigma). Crandall-Reese Feline Kidney cells (CRFKs) were seeded into a 12 well tray at approximately 40-50% confluence. CRFK cells were transfected with no DNA (negative control), pSV-b-Gal (control for b-galactosidase activity), pEGFP-C1/JV 59 GS/lacZ and pEGFP-C1/IRES/lacZ (control for IRES activity) using the Superfect TM transfection reagent (Qiagen) as per the manufacturer's recommendations. Following a 16 hour incubation the cells were observed for EGFP expression using a Leica Leitz DMRB fluorescence microscope. The cells were washed in PBS and fixed using a 0.5% solution of glutaraldehyde for 30 min at room temperature. The cells were incubated with an X-Gal stain solution: 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 , 1x X-Gal (Sigma) for 4 hours at 37uC and were observed for b-glactosidase activity by light microscopy. The experiment was performed more than once to confirm the results.
JV V5 and JV FLC T7 cDNA plasmid constructs were linearised using NdeI (Invitrogen). Capped RNA was synthesised using the mMessage mMachineH Capped RNA Transcription kit (Ambion) according to the manufacturer's instructions. CRFK cells were seeded into 6 well trays at approximately 50% confluence and were transfected with 2 mg purified RNA per well using Transmessenger transfection reagent (Qiagen) according to the manufacturer's instructions.
For immunofluorescence CRFK cells were seeded onto 19 mm coverslips in 6 well trays and were transfected with RNA as described. Following a 24 hr incubation the coverslips were washed with PBS and fixed in 4% formaldehyde for 15 min at room temperature. Cells were permeabilised and blocked in saponin buffer, also used as staining buffer, (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hr at 4uC. Cells were stained using an anti-V5 monoclonal antibody (Invitrogen) followed by an anti-mouse Alexafluor 488 conjugated secondary antibody (Molecular Probes) at the recommended dilution in staining buffer for 30 min in the dark. Cells were then stained for 30 min in the dark with a Wheat Germ Agglutinin Alexafluor 594 nm conjugate (Molcular Probes) to allow identification of plasma and Golgi membranes. Coverslips were washed and mounted onto slides using Vectashield containing DAPI (Vector Labs). Microscopy was performed using an inverted Leica TCS-NT confocal laser scanning microscope.
The anti-V5 antibody was also used to detect V5-tagged protein by Western blot. Cell lysates were prepared following transfection using lysis buffer (0.15 M sodium chloride, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-Cl pH 8.0) and protease inhibitor cocktail (Sigma). Lysates were incubated for 15 min on ice followed by sonication to shear genomic DNA. Following Bradford analysis equal protein content from JV V5 and JV FLC lysates were run on a 10% SDS-PAGE gel and subsequently transferred onto Immobilon-P PVDF membrane (Millipore) according to the manufacturer's recommendations.
The membrane was probed using the anti-V5 monoclonal antibody at the manufacturer's recommended dilution, followed by an anti-mouse HRP-copnjugated secondary antibody (Santa Cruz) at the recommended diltution. The ECL Western blotting reagents kit (G.E. Healthcare) was used to detect antibody bound protein, which was visualised by exposure to BioMax Light film (Kodak).
CRFK cells were seeded into 6 well trays and transfected with capped JV V5 RNA as described. Following a 24 hr incubation cycloheximide (Sigma) was added to the cells at a final concentration of 50 mg/ml. Cells were harvested at indicated times for the preparation of lysates for V5 Western analysis as described above. Bradford reagent (Sigma) was used to ensure equal loading of lysates according to the manufacturer's recommendations. Preliminary Findings of a Randomized Trial of Non-Pharmaceutical Interventions to Prevent Influenza Transmission in Households BACKGROUND: There are sparse data on whether non-pharmaceutical interventions can reduce the spread of influenza. We implemented a study of the feasibility and efficacy of face masks and hand hygiene to reduce influenza transmission among Hong Kong household members. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cluster randomized controlled trial of households (composed of at least 3 members) where an index subject presented with influenza-like-illness of <48 hours duration. After influenza was confirmed in an index case by the QuickVue Influenza A+B rapid test, the household of the index subject was randomized to 1) control or 2) surgical face masks or 3) hand hygiene. Households were visited within 36 hours, and 3, 6 and 9 days later. Nose and throat swabs were collected from index subjects and all household contacts at each home visit and tested by viral culture. The primary outcome measure was laboratory culture confirmed influenza in a household contact; the secondary outcome was clinically diagnosed influenza (by self-reported symptoms). We randomized 198 households and completed follow up home visits in 128; the index cases in 122 of those households had laboratory-confirmed influenza. There were 21 household contacts with laboratory confirmed influenza corresponding to a secondary attack ratio of 6%. Clinical secondary attack ratios varied from 5% to 18% depending on case definitions. The laboratory-based or clinical secondary attack ratios did not significantly differ across the intervention arms. Adherence to interventions was variable. CONCLUSIONS/SIGNIFICANCE: The secondary attack ratios were lower than anticipated, and lower than reported in other countries, perhaps due to differing patterns of susceptibility, lack of significant antigenic drift in circulating influenza virus strains recently, and/or issues related to the symptomatic recruitment design. Lessons learnt from this pilot have informed changes for the main study in 2008. TRIAL REGISTRATION: ClinicalTrials.gov NCT00425893 HKClinicalTrials.com HKCTR-365 A randomised controlled trial of face masks and hand hygiene in reducing influenza transmission in households.
Relevant definitions: Index case: the first subject to be infected with influenza in a household. Household contact: any person living in the same household as the index case. Secondary attack ratio (SAR): the proportion of household contacts of an index case who subsequently become infected with influenza. Hand washing: a process for the removal of soil and transient microorganisms from the hands [1] . Hand antisepsis: a process for the removal or destruction of transient microorganisms [1] .
The investigational products include surgical face masks, liquid hand soap and hypoallergenic waterless alcohol-based hand cleanser with emollient. The interventions will incorporate distribution of these investigational products and education on their proper use.
There is currently concern about the possibility of an impending emerging influenza pandemic. In the event of such, a limited number of interventions would be available to reduce and control the spread of the disease and thus the resulting morbidity and mortality [2, 3] . Antiviral drugs could be used subject to availability, although their effectiveness against the novel pandemic strain is uncertain and resistance could develop quickly with large-scale use. Furthermore a vaccine specific to the pandemic strain would take optimistically, given current technology and production capacity, at least 6 months to develop and mass produce [4] . In addition to vaccination and targeted antiviral prophylaxis, other population-level social distancing measures such as school and workplace closures and travel restrictions are likely to be somewhat effective in reducing influenza transmission in the community [5, 6] , but implementation on a prolonged basis and with repeated waves of the pandemic could be difficult. Household-based quarantine and isolation will likely be effective in mitigating the impact of a pandemic [5] [6] [7] . There is however considerable uncertainty about the efficacy of some non-pharmaceutical interventions at the personal level including face masks and hand hygiene. Our proposed study, to assess the efficacy of masks and hand-hygiene for influenza control, is a direct response to the World Health Organization's recent call for urgent research on the efficacy of nonpharmaceutical public health interventions [3] .
In addition to pandemic preparedness, knowledge about the efficacy of masks and hand hygiene would also be important for inter-pandemic influenza control. In western temperate and regional subtropical countries, influenza is a major source of morbidity and mortality during the seasonal periods of epidemic circulation [8, 9] , and a number of measures are typically taken to try to reduce transmission in hospitals and elderly care homes [1] . However there are few data on the efficacy of such measures in the household, although household transmission is thought to be one of the most important settings for the community transmission of influenza [10] .
Previous studies have tentatively suggested that prophylaxis with amantadine, rimantadine, and the neuraminidase inhibitors oseltamivir (Tamiflu ® ) and zanamivir (Relenza ® ) are effective in reducing influenza transmission in households [11, 12] . Vaccination is known to be an effective preventative measure [13] provided that the at least one of the vaccine strains closely matches the circulating strain [14] . Protective interventions at the personal level to reduce influenza transmission such as wearing masks and improving hand hygiene are often recommended [15] but few studies have investigated the efficacy of these measures outside nosocomial settings in a rigorous manner. Influenza is thought to be mainly transmitted through airborne droplet nuclei [16] but to a significant extent also spread by hand and surface transfer [17] . Some studies have suggested that transmission of upper respiratory tract infections was reduced after household-based hygiene interventions [18] [19] [20] , while a recent case-control study has suggested that masks and hand-washing may have been effective in reducing the transmission of SARS in a hospital setting [21] . A recent population study has suggested that improved hygiene measures and decreased community mixing during the SARS outbreak in Hong Kong resulted in reduced incidence of respiratory viral infections [22] .
The results of this study will have important implications for influenza prevention both in a pandemic and in interpandemic periods. Quantitative estimates of the efficacy of non-pharmaceutical interventions will inform resource allocation under pandemic preparedness plans.
Wearing masks may diminish the rate of influenza transmission by reducing the amount of virus-containing droplet nuclei entering the surrounding area after leaving the mouth and nose of an infected subject. When worn by a non-infected subject in the presence of infected airborne droplets, surgical masks may reduce the amount of infected droplets inhaled and thereby reduce the chances of infection. However the degree to which a surgical mask can reduce airborne transmission is difficult to quantify given the lack of prior research in this area, and the expected benefit is uncertain. There are few apparent risks of wearing a mask, perhaps the greatest risk being that the mask engenders a feeling of overconfidence in the ability of the mask to prevent infection, leading to riskier activity (e.g. sitting closer to family members at mealtimes) than might have taken place if the mask were not worn, and thus an increased rather than decreased risk of influenza transmission.
Proper hand hygiene is thought to reduce community transmission of some viral infections including rhinoviruses and RSV [10] , but the specific effect of hand hygiene on influenza has not been quantified. Again there are few apparent risks of proper hand hygiene, perhaps the greatest being the detrimental effects on skin of frequent hand washing, particularly with alcohol-based products although most of these contain emollients to buffer against excessive drying. To try to mitigate these effects as much as possible we will only supply an alcohol-based hand cleanser which includes emollients, and encourage study participants to be wary of skin irritation. The other very rare potential adverse consequence, as with all topical applications, is allergic reaction. We will use a hypoallergenic product and exclude participants who have a known allergy to alcohol or additive components of the alcohol handrub deployed.
The face masks and hand washing interventions will include an intensive counselling session to describe and demonstrate proper use of the respective hygiene aids.
Given that the index case could be symptomatic for a further 5 days, and asymptomatic incubation of the disease in household contact could take 1-2 days before symptoms appear, we propose that the hygiene measures should be maintained for at least 7 days.
The trial will be conducted in compliance with this protocol, GCP, and the applicable regulatory requirements.
The population studied are households in Hong Kong containing three or more individuals where at least one individual is suspected to be infected with influenza (ascertained either by meeting specific symptom criteria or by a positive result on a rapid diagnostic test, see 4(d)) and where no other household members have experienced symptoms of influenza-like-illness in the preceding two weeks.
To quantify the efficacy of face masks and/or hand hygiene in reducing household transmission of influenza.
The primary outcome measure is the SAR i.e. the proportion of household contacts with laboratory-confirmed influenza during the study period (follow-up period of 10/7 days in pilot/main study). Inference will be made at the individual rather than household level, adjusting for the potential within-household correlation.
The main study will follow a parallel design with three intervention arms (i.e. routine health education only, hand hygiene only, masks and hand hygiene). Households will be randomly assigned to one of the three interventions although intention-to-treat analysis will be at the individual level; therefore this is a cluster-randomised trial design. The pilot study will have three arms (mask only, hand hygiene only, routine health education only).
Subject recruitment will take place at selected government general outpatient clinics, group/managed practices, public hospital emergency rooms, private hospital outpatient departments, and private primary care clinics throughout Hong Kong, Kowloon and the New Territories.
For the pilot study we propose to recruit 500 individuals with ILI symptoms and apply the QuickVue rapid diagnostic test, so that we can follow-up a maximum of 200 index cases with a positive test result, and their households. We will stop recruiting as soon as we have 200 influenza-positive index cases, and we will stop recruiting after we have used 1,000 rapid diagnostic tests even if we have fewer than 200 influenzapositive index cases. Each household will be randomized to receive one of the three interventions, and all household contacts will be followed up. Details of the power calculation to justify this sample size are given below. Given an average household size of 3.8, a study of 200 households will involve the enrolment of a total of 760 individuals (200 index cases and 560 household contacts).
For the main study we propose to recruit approximately 6,000 individuals with symptoms of influenza-like illness (ILI) and follow-up an anticipated 1,200 index cases who meet specific criteria (in most cases a positive rapid diagnostic test result, or in some cases those meeting symptom based criteria -further described in 4(d)) and their households. Each household will be randomized to receive one of three interventions, and all household contacts will be followed up. Given an average household size of 3.8, a study of 900 households (from 1,200 randomized households after an anticipated 25% dropout) will involve the enrolment of a total of 3,420 individuals (900 index cases and 2,520 household contacts). Details of the power calculation to justify this sample size are given in 9(c) below.
A cluster-randomisation design has been chosen because simple individual randomisation of household contacts would almost certainly result in crosscontamination between family members who are assigned to different intervention arms. This cluster randomisation design requires a larger sample size to allow for potential within-household variability in SAR.
Given the nature of the interventions, participants will be un-blinded to the intervention received although they will be blinded to the nature of the other interventions. The same will apply to our research nurses who will be educating participating households on uses of assigned interventions and prevention of influenza transmission. Nurses will initially be randomly assigned to administering one of the three interventions and will receive training relevant to their assigned intervention only, hence avoiding potential cross-contamination by nurses.
When the details of a new index case are uploaded to the online database by fax to the trial manager, a unique identifier will be assigned. A pre-specified table of random numbers will be used to assign one of the three interventions to the household of the index case. Therefore the randomised intervention will be unknown to the doctor at or after the time of recruitment to minimise allocation and ascertainment biases respectively.
In the majority of recruiting sites, the criteria for further household follow-up will be a positive result for influenza A or B using the QuickVue rapid diagnostic test on a nose and throat swab (Quidel Corp, San Diego, CA) which has a reported sensitivity of 79% and specificity of 92% for influenza A or B [23] . If the QuickVue test is positive and informed consent is obtained, the index case and their household will be further followed up with home visits as described above. We will calculate the sensitivity and specificity of the rapid diagnostic test in our study setting, using viral culture or PCR of a nose and throat swab as the gold standard.
In a small number of recruiting sites at defined periods during the pilot and main studies we will use a symptom-based criteria to determine eligibility for further follow-up; specifically we will enrol subjects and their households for further followup if they present with at least two of the following symptoms: fever≥37.8°C (≥38°C in the pilot study); cough; headache; sore throat; pain in muscles or joints [24] .
In a small number of recruiting sites at defined periods during the main study we will use an alternative rapid diagnostic test, namely the HX Diagnostics Influenza A+B test (HX Diagnostics, San Francisco, CA) which is based on 3rd generation lateral flow immunoassay technology and may be more sensitive and specific than the QuickVue test. In this case, subjects with a positive result on the HX Diagnostics Influenza A+B test would be eligible for further follow up as described above. We will calculate the sensitivity and specificity of the rapid diagnostic test in our study setting, using viral culture or PCR of a nose and throat swab as the gold standard. See also 7(c).
See 6(a).
Following randomization, an immediate home visit will be scheduled (to take place within at most 36 hours, and ideally within 12 hours) to implement the intervention. During the home visit by a trained nurse, the purpose of the study will be explained to all household contacts and their consent obtained. Consent for children aged 17 years or younger will be obtained from their parents. Assent will also be obtained for children aged between 7 through 17. The nurse will then collect details on household composition, risk perceptions, attitudes and beliefs on influenza (questionnaire Q2, appendix), and take a nose swab and a throat swab from each household contact, except for asymptomatic children under the age of 2. Due to concerns about difficulties of taking nose and throat swabs from infants, for household members who are under 2 years of age only information on clinical symptoms will therefore be collected unless they are symptomatic. For participants who are symptomatic, a nose and a throat swab will be collected regardless of their age. The swabs will later be tested to confirm the absence of influenza in any household contact at baseline. The nurse will provide and describe proper use of a free tympanic thermometer, and the daily symptom diaries. Finally, the nurse will administer the standardized intervention through intensive counselling, and demonstration of proper wearing of masks or hand washing.
Three and six days (±1 day) after the initial home visit, a trained nurse will revisit each household. During the visit, the nurse will take nose swabs and throat swabs from the index case and all household contacts, except for children under 2 years of age who are asymptomatic, and collect the symptom diaries (questionnaire Q3, appendix) from each household contact. During the final visit (day 6), the nurse will ask the household members to complete a final questionnaire (Q4, appendix) and assess their adherence to the interventions (questionnaire Q5, appendix).
The 8-week pilot study will take place from January to April 2007. The exact starting and stopping dates of patient recruitment will not be fixed in advance. Recruitment will begin after the start of the annual influenza peak season (typically Feb/Mar) has been confirmed by the Department of Microbiology, HKU, and will continue until the prespecified sample size is reached. The 39-week main study will take place from January to September 2008.
Individuals may at any time decide to stop participating if they wish, without prejudice or any adverse consequences. There are no formal rules for stopping the trial early.
When the details of a new index case are uploaded to the online database, a unique identifier will be assigned. A table of random numbers will be generated by the trial statistician prior to the start of the trial, and this will be used to assign one of the three interventions to the household of the index case. Randomisation codes will not be used since for the first home visit it is necessary for the nurse to know which of the interventions has been assigned.
Randomisation codes will be masked from those assessing the outcomes.
k) The identification of any data to be recorded directly on the case record forms (i.e. no prior written or electronic record of data) and to be considered source data.
See questionnaires Q1-Q5 (appendix) for the source data that will be recorded in this study. Note that we will not access subjects' medical records.
Nurses will be offered influenza vaccination prior to the study.
We will offer each participating household a HK$200 (US$25) supermarket voucher (HK$150 / US$20 in the pilot study).
Specimens collected in recruiting clinics will be stored in a 2-8°C refrigerator (overnight, if required). Specimens collected during home visits will be stored at room temperature or if necessary (i.e. in hot weather) in a cool box with at least 2 icepacks immediately after collection. Before the end of the day of a home visit, the study nurse will take any collected samples to the nearest recruitment clinic for storage in a 4°C refrigerator (overnight, if required) or directly to the Department of Microbiology, HKU. Samples stored at 2-8°C in recruiting clinics will be delivered to the Department of Microbiology, HKU as soon as possible, via courier and maintained at 2-8°C en route. Samples will be eluted and cryopreserved at minus 70°C at the destination.
The criteria for further follow-up are discussed in 4(d), in the majority of recruiting sites the criteria will be a positive result on the QuickVue Influenza A+B rapid diagnostic test.
During the final home visit in the main study we will request buccal swabs from all household members. The samples will be anonymised and then processed by a biotech company specialising in DNA extraction from human samples. These data will be studied at a later date to investigate possible genetic factors in influenza susceptibility or transmission.
Inclusion criteria for index cases are as follows: (1) a Hong Kong resident; (2) reporting ILI symptoms including at least two of fever (recorded fever ≥38°C), cough; nasal congestion; sore throat; headache; runny nose and pains in muscles or joints [24] (3) onset of symptoms within the preceding 48 hours. If these conditions are satisfied, the subject will be approached to determine household eligibility to enrol in the study as below prior to further follow-up as described in 4(d) and 4(f).
Inclusion criteria for households are as follows: The household must contain at least three people including the index case and any domestic helpers, and where no household contacts have had ILI symptoms in the preceding two weeks.
In the main study (January to September 2008), if we are ahead of our recruitment target at any time in March or later, we will consider revising the inclusion criteria to include only subjects with onset of symptoms in the preceding 36 (or 24) hours, since we believe the interventions will be most effective if applied sooner after symptom onset. If implemented, this protocol change would be taken into account in the final analyses.
There are no exclusion criteria.
The entire household will be withdrawn from the study if there is failure to obtain proper informed consent from any one or more individual household contacts for whatever reason. Informed consent will be sought from each individual household contact, or proxy consent from the parents of any individual under the age of 18. Assent will also be obtained for children age between 7 through 17. Households will be withdrawn from the trial if no home visit can be scheduled within 36 hours. Withdrawn households will be replaced to maintain the specified sample size. If one or more household contacts are not present during one of the home visits, we will attempt to reschedule a supplementary home visit to collect clinical specimens, and in the case where the initial home visit was missed we will request signed informed consent and apply interventions as necessary during the supplementary home visit.
In the main study, if a randomized household refuses to allow any home visits, we will request permission to contact them by telephone after 7 days to enquire how long the index case experience symptoms for, and whether any household members reported clinical influenza; if available, these data will allow a simple comparison of households who dropout with those who are successfully followed up.
We will randomize households among three study arms, each of which will include intensive counselling during the first household visit. All recruited households will receive educational pamphlets specific to their assigned intervention arms in English or Chinese or both, as appropriate. The intervention arms are as follows:
This group will receive education about the importance of a healthy diet and lifestyle for boosting the immune system against influenza and other directly transmitted respiratory pathogens leading to ILI symptoms, both in terms of illness prevention and symptom alleviation.
This group will receive the control intervention (health education) plus education about the potential reduction in transmission of respiratory infections to household contacts if all parties maintain proper hand hygiene, and demonstration of proper hand washing and hand antisepsis. Each household member (including the index case) will receive a uniquely labelled 100ml bottle of hypoallergenic waterless alcohol-based hand rub for individual use only, and households will be provided with one 220ml bottle of antimicrobial Ivory liquid hand soap (Proctor & Gamble, Cincinnati, OH) for each washroom and kitchen sink.
This group will receive the control intervention plus the hand-hygiene intervention plus education about the potential reduction in transmission of acute directly transmitted respiratory infections to household contacts if all parties wear masks, distribution of 50 (75) surgical masks for each adult (child aged 3-7) household member, and demonstration of proper face-mask wearing.
The pilot study will include the first two arms and a third mask-only arm (i.e. the mask intervention but not the hand hygiene intervention). For the first 100 households, we will apply an unbalanced randomisation of 2:1:1 among arms (1), (2) and (3). For the subsequent households (up to a further 100 households) we will apply an unbalanced randomisation of 8:1:1 among arms (1), (2) and (3) to allow us to extract maximum information about the transmission dynamics of influenza in the absence of non-pharmaceutical control measures. If the maximum sample size of 200 households is reached, there will be approximately 130, 35 and 35 households in arms (1), (2) and (3) respectively. Following completion of the pilot study, information derived about the characteristics of influenza transmission will be invaluable not least in validating our sample size calculation for the main study.
The main study will randomise households equally among the three arms above, using a block randomisation structure with randomly permuted block sizes of 18, 24 and 30.
We will use separate randomisation tables for subjects recruited with different criteria (as described in 4(d)) to ensure the intervention groups are balanced; this is because the QuickVue test is likely to capture subjects with on average higher viral shedding than a symptom-based criteria.
There are no restrictions on the use of other medications during the trial period. However the use of antivirals, antibiotics and other Western and Chinese medicine to relieve ILI symptoms during the study period will be recorded by the visiting nurse in the nurse's assessment sheet at the first and last home visits (ie. Questionnaire Q2 and Q4 respectively, appendix).
Self-reported use of hygiene measures including mask wearing and hand washing will be recorded in the symptom diaries (Q3) by each household contact and the index case. Overall use will be reported at the final home visit, and quantities of masks, alcohol and liquid hand soap used will be measured by the visiting nurse during the final household visit.
The primary outcome measure is the secondary attack ratio (SAR) which is the proportion of household contacts with laboratory-confirmed influenza during the study period (10/7 days after recruitment in the pilot/main studies). We will preferentially use the laboratory definition of influenza rather than the clinical definition, when available.
Household contacts will be confirmed to be influenza-free at the first household visit, within 36 hours of recruitment of the index case.
In the main study, clinical influenza is defined as the presence of at least two of the following symptoms: feverishness (we will strongly encourage household members to use the supplied thermometer to assess whether a fever is ≥38°C); cough; headache; sore throat; pain in muscles or joints (following [24] ). In the pilot study (as per the initial protocol), clinical influenza is defined as the presence of feverishness, or at least two of the following symptoms: cough; sore throat; nasal congestion, rhinorrhoea, or sneezing; fatigue; headache; stiffness; myalgias.
In the main study during the follow-up period of 7 days after recruitment of the index case, the index case and all household contacts will be asked to maintain a daily record of their symptoms (questionnaire Q3, appendix) and their tympanic temperature. A nurse will visit the household on three occasions during follow-up, namely 3 and 6 days after the initial home visit (day 0) with a window period of ±1 day. During each visit the nurse will collect nose swabs and throat swabs from the index case and all household contacts. These will be cryopreserved at the Department of Microbiology HKU as soon as possible as decribed in 4(n). The nose swabs and throat swabs taken during the follow-up visits will provide independent confirmation of the presence or absence of influenza virus in all household contacts, and the duration of viral shedding in the index case. In the pilot study the follow-up period will be 10 days, with visits on days 0, 3, 6 and 9.
The primary endpoint of our study will compare the SAR in each of the intervention groups with the control intervention. We will use χ 2 tests and odds ratios adjusting for potential within-household correlation, with a 5% type I error rate.
The criteria for further follow-up of the index subject and their household were described in 4(d) above. In the majority of cases, the criteria for further follow-up will be a positive result on a rapid diagnostic case. In some recruiting clinics the criteria will be presentation with at least two of the following symptoms: fever≥37.8°C (≥38°C in the pilot study); cough; headache; sore throat; pain in muscles or joints. However we will only include households in the final analyses if the index subject is laboratory confirmed to have influenza infection. This laboratory confirmation will require a positive result for influenza A or B by viral culture or standard PCR of a nose and throat swab collected from the index case at the recruitment site, and/or during the first home visit.
During the first household visit, a responsible adult (usually the household head or a parent) will be asked to provide an overview of the composition of the household, and details on past illness history and influenza vaccinations (Q2, appendix). At the final household visit, the nurse will collect information (questionnaire Q4, appendix) on the overall self/proxy-reported compliance with the intervention, and on any medication taken during the follow-up period, by asking household members and also by personally checking how many masks remain unused, or how much soap or alcohol is left in the bottles and dispensers.
There are no safety parameters in this trial.
The characteristics of households, index cases and household contacts in the three intervention groups will be compared and assessed for similarity with χ 2 tests, adjusting the comparison of household contacts for potential within-household correlation. In the primary analyses, households will only be included if the index case has laboratory-confirmed influenza infection -all other households will be dropped from the primary analysis. Furthermore, in the main study households will be dropped from the primary analysis if any of the household contacts are found to have laboratory-confirmed influenza infection at baseline although this will not be incorporated in analyses for the pilot study as per the original protocol. Therefore our results will not be biased by the potentially different transmission dynamics of other respiratory diseases compared to influenza A or B, or by the potential for more than one index case in a household when the interventions are applied.
The primary endpoint of our study will compare the SAR in each of the intervention groups with the placebo intervention (1). We will use χ 2 tests and odds ratios adjusting for potential within-household correlation [25] , with a 5% type I error rate.
We will investigate the efficacy of the interventions on the SAR in multivariable logistic regression models with a generalized estimating equations approach to allow for potential within-household correlation [26] . Analysis will first be performed including only the effects of masks and hand hygiene. Further analyses will allow for the effects of potential confounders on the SAR. Confounders of the SAR that we will assess for each household contact include the age, gender, smoking status, chronic disease status, prior vaccination status, and additionally the age and gender of the corresponding index case. We will investigate the intervention effect in age/gender subgroups, although the statistical power for these analyses is unlikely to be high. We will further investigate the intervention effect in households where the intervention was applied sooner (with 36 hours) after symptom onset in the index case.
We will further investigate the intervention effects for influenza A and influenza B separately, although with likely lower incidence the statistical power for the latter may be low.
We will also assess the adherence of the index case and household contacts to the interventions, and conduct as-treated analyses of the primary outcome measure. We will conduct sensitivity analyses excluding households where the index case was prescribed antiviral medication, since onward transmission may be less likely in this scenario.
In further analyses, we will investigate the effect of the interventions on secondary outcomes listed below, and further adjust for the effect of possible confounders in multivariable logistic and proportional hazards regression models where appropriate.
1. The proportion of household contacts with clinical influenza, adjusting for the potential within-household correlation. 2. The proportion of households with one or more secondary case of influenza (laboratory definition used in preference to clinical definition where available). 3. The proportion of households with one or more secondary case of clinical influenza.
We will investigate the predictors of influenza infection and the factors affecting duration of symptoms. We will further examine the effect of environmental and lifestyle factors, and measures of risk perception on the disease course and onward transmission using regression models. We will examine the factors affecting adherence to interventions using regression models.
We will develop and apply novel modelling approaches to the analysis of the household infection data to estimate specific transmission parameters, building on previous research [27] .
We will investigate the performance of the QuickVue Influenza A+B rapid diagnostic test (and the HX Diagnostics Influenza A+B test) by comparison with the gold standard of laboratory confirmed influenza by viral culture or PCR. We will further investigate the factors potentially affecting rapid diagnostic test performance, including age, gender, and time since symptom onset.
If funding is available, we will conduct further laboratory tests of collected samples to allow us to investigate the incidence and transmission dynamics of non-influenza respiratory viruses. Subjects' consent for these additional respiratory virus tests are provided on the current version and all previous versions of the informed consent forms.
If funding is available, we will apply quantitative PCR tests to nose and throat swabs collected from home visits, to investigate potential correlation between the degree of viral shedding and onward transmission, as well as the degree of viral shedding in any resulting secondary cases.
Finally, if funding is available, we will sequence the genome of influenza viruses detected in index cases and secondary cases to investigate genetic variability in the virus as well as the evolution rate between successive cases (and indeed whether secondary cases were truly infected by their corresponding household index, or from some other source).
Pilot study: A simple calculation of the SAR is given by dividing the number of household contacts with influenza by the total number of household contacts. To estimate the anticipated SAR of 0.241 to within ±5% would require 283 household contacts. Given an average household size of 3.8 (i.e. 2.8 contacts per household) we would require at least 101 households in the placebo group; we propose to recruit 130 households in this arm to allow for some households being lost to follow-up. To test the randomization and intervention procedures we will recruit a further 35 households to the second and third study arms (masks and hand-hygiene). This will also enable a preliminary estimate of the efficacy of hand hygiene and masks although the power of the pilot study will be low to detect small or medium effect sizes with statistical significance. More importantly, the pilot study will allow an idea of the feasibility of these interventions, and coherence to them.
Main study: For the sample size calculation we require an estimate of the anticipated SAR (P), the degree of within-household correlation (ρ) in the SAR, the relative risk (r) that we would like to detect, and the relevant critical values of the standard normal distribution Z for a specified power (1-β) and type I error rate (α). For average household size m, the required number of individuals n in each intervention arm is given approximately by
and thus the number of required households is given by n/m [25] .
A recent study of influenza transmission in household contacts in France found a SAR of 24.1%, and a within-household correlation of ρ=0.29 [28] . We will assume a reduced SAR of 20% to allow for the possibility of some transmission occurring prior to randomization and the likely inclusion of some index cases without influenza. A relative risk reduction of at least 30% is generally accepted to be clinically and epidemiologically important. We note that the efficacy of masks and hand-washing were estimated to give relative risk reductions of 90% and 75% respectively during a nosocomial outbreak of SARS, and while we doubt such high efficacy in the household setting we anticipate relative risk reductions of perhaps around 30%-50%, although there is no literature to guide us on such estimates (and hence the need for this trial!). The average household size in Hong Kong excluding houses with single or double occupancy is 3.8 (source: Hong Kong Thematic Household Survey 2002), therefore the average number of household contacts per index case would be m=2.8.
We would like to have at least 80% power to detect a 30% reduction (i.e. r=0.7) in the relative risk between intervention 2 (or intervention 3) and intervention 1 (anticipated P=0.2), with a 5% type I error rate. Using the formula given above we calculate that we would require the randomization of 840 household contacts into each arm of the study. Allowing for a 25% dropout rate following randomization, we would require the randomization of 840 household contacts into each study arm corresponding to a total study requirement of 2,520 household contacts in 900 households. Thus we will recruit a total of 3,420 individuals including 900 index cases with positive results on the QuickVue rapid diagnostic test, and 2,520 household contacts. The specified sample size would also have high statistical power to detect larger relative risk reductions if the observed secondary attack ratio were lower (Table 1) . To achieve this sample size, we would need to recruit approximately 6,000 suspected influenza cases and follow-up those who meet the specific criteria (in the majority of recruited subjects this would be a positive result on the QuickVue Influenza A+B rapid diagnostic test), and our reasoning is as follows. During the peak season we would conservatively anticipate that 50% of subjects with ILI symptoms, as we have defined these above, are infected with influenza rather than another virus (3,000 of the 6,000 tested). Recent international oseltamivir trials found that during the influenza peak season the proportion of subjects with influenza-like-illness who had confirmed influenza was 60% [29] and 66% [30] . Allowing for a sensitivity of 79% for the QuickVue test, only 2,370 index cases would be correctly identified (the remainder would be misclassified as false negatives). Given the estimated specificity of the QuickVue test of 92%, testing the 3,000 non-influenza index cases would result in misclassification of 240 subjects without influenza (false positives), and their households would also be visited. While the specificity of the symptom-based definition in 4(d) is likely to be lower, the sensitivity may be higher and with symptom-based recruitment only occurring in a small number of sites the approximate calculation above is appropriate. Thus we anticipate that we would need to recruit approximately 6,000 index cases with ILI symptoms and apply the specific criteria (typically the rapid diagnostic test), of who 1,477 (2,370+240) would meet our criteria and be subject to randomization and follow-up. Given the intention-to-treat approach, randomization and follow-up of some non-influenza cases is an expected consequence of the speed required by this study.
d) The level of significance to be used We will use a significance level of α=0.05.
Given the short duration of the trial, we do not plan to conduct any interim analyses or specify any early-stopping rules.
The laboratory definition of influenza will be preferentially used as the primary outcome measure, but when this is unavailable we will use the clinical definition. In the pilot study, index cases and household contacts with missing data on important predictors will be excluded from analyses. In the main study, we will use multiple imputation [31] with 10 imputed datasets to replace missing values on outcome and predictor variables. If 10 imputed datasets are not sufficient to ensure stability of estimates we will use 20 imputed datasets. Multiple imputation makes maximum use of available data and maximises statistical power while requiring less strict theoretical assumptions than to a complete case analysis, or single imputation of mean values. We note that this is now one of the preferred (and standard) methods for analysing clinical trials data [32] .
Any deviations from the original statistical plan will be described and justified in the final report.
We will follow an intention-to-treat approach in the analyses. Households without a laboratory confirmed index case, and additionally in the main study those households where a household contact is laboratory confirmed to have influenza infection at baseline, will be excluded from the primary analysis.
We will permit direct access to source data and documents for the purposes of trialrelated monitoring, audits, IRB/IEC review and regulatory inspections. As required by the NIH/CDC funding agency, the anonymised individual participant data will be made publicly available after publication of our results in peer-reviewed journals and no later than 24 months after the conclusion of our study.
An important ethical consideration is that households randomised to the control intervention might be considered to have less benefit from the trial than those assigned to the mask or hand washing interventions. However we note that there is little evidence that masks or hand washing can reduce influenza transmission, whereas this study will provide that evidence. Further, the participation of a control arm is essential to allow estimation of the effect of the interventions, given the lack of localspecific data on the SAR in typical circumstances. Thus we believe that those in the control arm, as the whole of society, will still benefit indirectly from this research.
Socio-demographic and epidemiological data; confidential patient details (name address) will be collected from all subjects via the four questionnaires included in the appendix. All data will be anonymized when entered into an electronic database (double entry) and stored in the Department of Community Medicine, HKU. Original identities will be kept in a separate file accessible only to the trial manager. Original documents will be destroyed at the conclusion of the pilot study.
This study is financed by a grant from the Centers for Disease Control and Prevention (Appendix A).
The results will be published in international peer-reviewed journals. Real Time Bayesian Estimation of the Epidemic Potential of Emerging Infectious Diseases BACKGROUND: Fast changes in human demographics worldwide, coupled with increased mobility, and modified land uses make the threat of emerging infectious diseases increasingly important. Currently there is worldwide alert for H5N1 avian influenza becoming as transmissible in humans as seasonal influenza, and potentially causing a pandemic of unprecedented proportions. Here we show how epidemiological surveillance data for emerging infectious diseases can be interpreted in real time to assess changes in transmissibility with quantified uncertainty, and to perform running time predictions of new cases and guide logistics allocations. METHODOLOGY/PRINCIPAL FINDINGS: We develop an extension of standard epidemiological models, appropriate for emerging infectious diseases, that describes the probabilistic progression of case numbers due to the concurrent effects of (incipient) human transmission and multiple introductions from a reservoir. The model is cast in terms of surveillance observables and immediately suggests a simple graphical estimation procedure for the effective reproductive number R (mean number of cases generated by an infectious individual) of standard epidemics. For emerging infectious diseases, which typically show large relative case number fluctuations over time, we develop a Bayesian scheme for real time estimation of the probability distribution of the effective reproduction number and show how to use such inferences to formulate significance tests on future epidemiological observations. CONCLUSIONS/SIGNIFICANCE: Violations of these significance tests define statistical anomalies that may signal changes in the epidemiology of emerging diseases and should trigger further field investigation. We apply the methodology to case data from World Health Organization reports to place bounds on the current transmissibility of H5N1 influenza in humans and establish a statistical basis for monitoring its evolution in real time. A pandemic of H5N1 influenza in birds is presently unfolding, with over 50 countries around the world affected, resulting in hundreds of millions of dead animals through infection or culling [1] [2] [3] . This emergency and the associated risk of a devastating new human pandemic [4] [5] [6] stress the need for new approaches targeted specifically at detecting and monitoring the evolution of emerging infectious diseases [7] [8] [9] . Assessing the risk of emergence of a human epidemic at the genetic level requires accounting for rare stochastic events, associated with genetic mutation and recombination, over vast pathogen and host populations [4, 8, 10] . This makes prediction of pathogenic evolution at the molecular level typically still very difficult. Consequently, the first indications of disease emergence are usually observed as infected cases in human and animal populations. Thus, for early assessments of the epidemic potential of a new outbreak, it is essential to assign quantitative meaning to existing epidemiological surveillance data in real time, with quantified uncertainty, and to use this knowledge to enable primary prevention strategies targeted at reducing chances of pathogenic evolution.
The quantity that measures the epidemic potential of a pathogen is the basic reproduction number R 0 [11, 12] . R 0 is defined as the average number of new infections created by an infectious individual in an entirely susceptible population. For established human pathogens, leading to standard epidemics, R 0 .1, as is the case of seasonal or pandemic influenza [13] [14] [15] [16] [17] [18] [19] . In practice, epidemiological data typically permit only the estimation of the effective reproduction number R, which may differ from R 0 due to acquired immunity and other factors. For an emerging infectious disease, when transmission is only incipient [20] and the pathogen is adapting to the population, it becomes crucial to monitor quantitative changes of the effective reproduction number over time. Thus, the detection and tracking of an emerging disease can be formalized in terms of monitoring R, as it evolves and approaches the critical threshold RR1. This is likely the current state of H5N1 avian influenza in humans, where complete absence of human to human transmission would imply R = 0, but likely R is very small, as a few cases of possible human contagion suggest [21] [22] [23] .
Notwithstanding a marked recent increase in systematic surveillance by national and international organizations, and the advent of real time reporting of many public health indicators (syndromics) [24] , the epidemiological regime of incipient but evolving transmission has received little attention in terms of quantitative modelling [22, [25] [26] [27] [28] . The main difficulty is that data in these circumstances tend to be very stochastic, involve small case numbers and may be plagued by uncertainties and inconsistent reporting. As an example, we contrast in Figure 1 the time series of confirmed new human cases of H5N1 avian influenza in Vietnam, reported by the World Health Organization (WHO), with weekly isolate numbers for seasonal H3N2 influenza in the USA during 2004-2005 (see Methods for ''Data Sources''). The ultimate objective of this paper is to propose a methodology to extract quantitative inferences and generate epidemiological outlook in real time from time series like that of Figure 1a .
Recently the problem of real time monitoring of (emerging) communicable diseases has gained growing attention, with a few new methods proposed to estimate R. One method proposes the analysis of the distribution of the sizes of case clusters to provide indications of changes in R. Specifically, increases in R(,1) translate on average into larger case cluster sizes [21, [27] [28] [29] . Another approach [30] relies on the inference of probable chains of transmission among observed cases from knowledge of the statistical distribution of the infectious period. From an ensemble of such chains and their associate compounded probability, R can be estimated. This method has recently been applied to ''real time'' monitoring of SARS [31, 32] , via a Bayesian inference scheme. The strength of this class of methods is that they allow insights into heterogeneities in the population. This demands the consideration of all pairs of possible transmissions, which may become computationally intense as case numbers rise and can be sensitive to under reporting, competing risk and to the details of the distribution of infectious periods. Moreover those studies considered the efficacy of control measures for a disease with an initial R.1 and no new cases introduced during the epidemic, whereas it is typical of emerging communicable diseases that adaptation of the pathogen's tropism to the host population is the result of numerous such introductions [22] .
Here we propose an alternative approach, which addresses the issue of new introductions, requires in general smaller computational overhead and results in the estimation of the full probability distribution for R. The method is based on the probabilistic formulation of standard SIR disease transmission models analogous to the time-series SIR (TSIR) approach [33] , which simplifies the need to reconstruct transmission chains by aggregating all infectious and susceptible individuals into classes that are assumed to mix homogeneously. A Bayesian procedure is then employed to translate the time series of case numbers into a probability distribution for epidemiological parameters. The method adopts the standard assumptions made in epidemiological compartment models with homogeneously mixing classes, and benefits from their simpler computational structure allowing efficient estimation with available sparse empirical data. The estimation method developed here has been applied once before [34] to 1918 influenza pandemic death notifications time series for San Francisco, with the purpose of comparing its performance with other conventional methods for estimating R. Here we present its full derivation, provide more details and examples, include introductions from an animal reservoir and show how the method can be used to provide statistical expectations for new case predictions. We also show how case predictions with quantified uncertainty do, in turn, define possible statistical anomalies for future case numbers, which can be used to inform surveillance and logistical management in the event of a new or continuing outbreak. As an example, we apply the method to human case data of H5N1 influenza in Vietnam and Indonesia, to produce bounds on its effective reproduction number, R, and establish a basis for its continued monitoring in real time.
Time series of H5N1 influenza cases in humans were assembled from World Health Organization (WHO) reports of confirmed cases (http://www.who.int/csr/don/en/), from January 2004 to June 1, 2006 (see Supplementary Material S1, including Figure S1 , for more information). Data for H3N2 seasonal influenza was obtained from the Centre for Disease Control (CDC) Surveillance Weekly Reports in the United States (http://www.cdc.gov/flu/ weekly/fluactivity.htm).
New human cases of avian influenza may result from two alternative processes: i) infection of humans from animal sources [4] , or ii) human to human transmission [23] . For a standard epidemic, explicit consideration of multiple introductions is not important as each case produces many secondary infections. For emerging infectious diseases multiple introductions from a reservoir [22, 25] may constitute an important fraction of all observed cases, and the progression of secondary cases must be carefully assessed and monitored.
Our objective is to cast standard SIR-class models in a form that directly relates to time series data of emerging infectious diseases by i) accounting for cases from reservoir sources, ii) casting the model variables in terms of observable quantities reported from field surveillance, iii) formulating the model in a discrete probabilistic form, and iv) quantifying uncertainty in the estimation of epidemiological parameters and future cases, and assimilate new data to reduce it.
We consider a standard epidemic susceptible-infected (SIR) model
where S(t) is the average number of susceptibles at time t, I(t) is the average number of infectious, N is the size of the population, which decreases due to disease-induced deaths (taken as a fraction a of progressing infections), b is the contact rate, and c 21 is the infectious period. After an average residence time c 21 , infectious individuals recover or die (not shown in [1] ). The total number of cases up to time t, T(t) obeys the equation dT/dt = b S/N I. Epidemic reports most commonly state the occurrence of new infected cases, which over the period t, are given by
To find the expression accounting for the evolution of new cases DT(t+t) we integrate Eq. [1] , for I(t) between t and t+t, to obtain
reproduction number) is a function of time; the last expression is exact if S(t)/N(t) is constant in the period [t, t+t] . This simplifying assumption is generally excellent for emerging infectious diseases, which result in few cases within a much larger population. Generally the validity of the assumption can be assessed through consideration, from [1] , of its evolution equation
This condition is usually satisfied as the fraction of infectious at a given time, I(t)/N(t), is typically less than a few percent (even for seasonal influenza), while other quantities in the product are of order unity. The quantity, in expression
, accounting for the number of new cases resulting from infections over time t [35] .
To obtain the disease progression in terms of epidemiological observables, we discretize the differential equation for the change in total number of cases between t and t+t as
where we used [2] and the assumption that S(t)/N(t) is piecewise constant over [t, t+t], but does vary between intervals contributing to changes in R t . At time t, the total number of cases is also
Substituting expression [5] into [4] , we obtain:
We see that the well known multiplicative progression between new cases at successive times due to contagion appears, on average, as a linear relation between DT(t+t) and DT(t) in an epidemic time delay diagram, Figures 2a-d. Expression [6] generalizes similar relations in the TSIR literature by casting them in terms of new cases over arbitrarily chosen observation intervals t, not necessarily coinciding with the average generation time c 21 . Expression [6] also shows how the initial R t can be estimated geometrically (without the need for parameter search or numerical optimization) from an epidemic time delay plot of surveillance data: b(R t ) is the slope of the tangent at the origin of case trajectories (dashed line in Fig. 2a, b) . For emerging infectious diseases relative fluctuations in case numbers are large, see e.g. Figure 2d , and this simple geometric approach is not valid, thus making more robust estimation methods, as the one presented here, necessary.
For emerging infectious diseases, many introductions from a reservoir may occur before the pathogen adapts its tropism to the new host population and produces epidemic outbreaks [22, 25] . As a result epidemiological models for the time evolution of new cases must account for two processes: (incipient) human transmission and infections from the reservoir.
We introduce a new source of infected individuals, through contact with the reservoir (birds). The evolution of I is now given by dI dt~b
The first term on the right accounts for the human-to-human infectious process. The last term is a source, creating new I through contact with a reservoir of infectious agents of size K(t), with contact rate b bh .
We denote the number of new infections from the reservoir per unit time as dB/dt = b bh S(t) K(t). As a result the number of humans infectious, I, and the total number of cases evolve as
The evolution of I(t) between t and t+t, accounting for the effects of the inhomogeneous source term, is
where Y(t, t ,B) denotes the integral. We use this expression to solve for the number of new cases (Eq. [8] ), giving
Probabilistic models of contagion A probabilistic description is crucial for realistic modelling of new cases of emerging infectious diseases, which are typically characterized by large coefficients of variation. This probabilistic description is achieved, as in [33] , by defining the number of new cases, DT(t+t) as a stochastic discrete variable generated by a probability distribution with average number of cases given by [10] , i.e.
where P{l} denotes a discrete probability distribution with mean l. If the only information on case evolution is their average number then the maximum entropy distribution for P{} is Poisson, which we adopt throughout this paper. If additional information on the magnitude of fluctuations were also known, a generalized distribution should be employed, such as a negative binomial [33] , which can account for clumping effects (we reproduce all estimations from the main paper using a negative binomial in Supplementary Material S1, Table S2 ). To use expression [11] in practice, we need to evaluate the integral Y. Assuming the introductions from the reservoir per unit time to be approximately constant between t and t+t, the integral can be calculated to first order as Y(t,t,B) = t dB/dt and, [11] is written as
where we replaced tdB/dt by its discrete approximation DB(t). This is the expression used in practice in all quantitative estimations presented.
Parameter estimation with quantified uncertainty can be achieved using a Bayesian approach in the context of probabilistic epidemiological models. Bayes' theorem expresses the full probability distribution for model parameters, such as the effective reproduction number, R, in terms of the probabilistic epidemiological model [12] , given the time series for new cases. Specifically, the probability distribution of R, compatible with the observed temporal data stream is given by
P[R] is the prior, which captures given knowledge of the distribution of R. The distribution P[DT(t+t)rDT(t)] is independent of R, and corresponds to a trivial normalization. From successive applications of Bayes' theorem, a sequential estimation scheme, that uses streaming epidemiological observations performed in real time, can be constructed using the posterior distribution for R, at time t as the prior in the next estimation step at time t+t, leading to an update scheme via iteration of Eq. [13] . The resulting probability distribution for R includes information on all observations up to time t, and contrasts with the ''instantaneous'' R t , used above, which only considers the data at times t and t+t. Thus, R is a robust estimator of the effective reproduction number assumed to be constant for the whole epidemic up to time t. Any changes in R over time result from the assimilation of each new data point, leading to an updated estimate of R. This in turn allows the use of our estimation procedure as an anomaly detection tool (see below).
Ideally, field case tracing should provide a measure of the likelihood that a new case resulted from contact with the (animal) reservoir, DB in Eq. [12] , or was due instead to human contagion. Another possibility is to explicitly model the introductions from the reservoir, K(t) in Eq. [7] . Although some empirical studies start to address this possibility [27, [36] [37] [38] , it is still difficult to calibrate such models and uncertainties remain large. Thus, for the calculations in this study, we choose to use a minimal statistical approach. Formally, assuming statistical independence between different cases, we model each new introduction as a Bernoulli trial with probability h. h is defined as the average probability that a case is attributed to human to human contagion, and 1-h that it is the result of an infection from the reservoir. Note that for emerging infectious diseases this probabilistic model is more appropriate and generalizes (see Supplementary Material S1, Figure S2 ) the more typical modelling of introductions as homogeneous Poisson processes [33] .
We can provide an upper bound for h by considering observed clusters of cases. If we take all confirmed cases in clusters, except the index case, to be due to human contagion, then an estimate for h is given by the proportion of such cases divided by the total number observed over the same period. This gives an upper bound on h, because it is unlikely that all cluster cases arise from human infection, rather some could have a common reservoir source. Two epidemiological studies of H5N1 influenza, one from January 2004 to July 2005 [39] and another from July 2005 to June 2006 [40] , found that 26 of 109 cases and 15 of 54 cases, respectively, occurred in family clusters Attributing those cases to human infection gives h = 0.24-0.28. This estimate of h is consistent with an independent statistical analysis of a case cluster in Indonesia, which found that the secondary attack rate for household transmission of H5N1 influenza was 0.29 [28] .
In the remaining of this paper we treat h as a constant parameter common to all reported cases. The sensitivity of R estimates is then assessed as a function of h (Table 1 ). In addition to tests of the estimation procedure on epidemic data [34] , we also verified the precision of our methodology on an extensive number of simulated case time series, based on a standard SIR model, with introductions from a reservoir and different R 0 .
We used the observed time series of new cases of human H5N1 influenza DT(t) to compute the probability distribution for R using programs implemented both in Matlab and Fortran. We used an unbiased uniform distribution for R between 0 and 3 as the initial prior. For each subsequent weekly iteration, we computed the full posterior distribution from [13] using the posterior at the previous week as the new prior. The product of the two probabilities on the right-hand side of [13] was evaluated as a non-parametric function defined in terms of 1000 discrete bins in R between 0 and 3, as shown in Figure S3 in the Supplementary Material S1. Parameters choices used in the calculations in the main text are: t = 1 week, c = 1 week 21 , h variable as in Table 1 . We also explored other parameter choices reported in the literature for seasonal influenza and corresponding results are given in Table S1 of Supplementary Material S1.
Here we show how the method performs at estimating R from single realization time series, produced by simulation with a known value of R 0 . In all instances the time series for human H5N1 cases in Vietnam (Figure 1a ) was used as introductions into the human population. For a choice of R 0 .1 in the simulation, any introduction readily develops into an epidemic. For R 0 ,1 each introduction leads to small outbreaks that eventually become extinguished.
The effective reproduction number, R, calculated by our method changes over time, because of decreases in the fraction of susceptibles, S(t)/N(t), and the availability of more information, as more cases are observed. Thus, we use the values obtained for R at early times, when S(t)/N(t) approximates its initial value, to estimate R 0 of the simulation by assuming that max(R) = R 0 . As shown in Figure 3 , in all circumstances, the method gives an excellent estimation of R 0 as outbreaks unfold, usually making accurate predictions when supplied with a mere two or three observation points. Uncertainty, measured by the width of the 95% credible interval, is reduced by larger case numbers, so that it typically remains higher the smaller the R 0 . In all instances uncertainty is reduced as more cases are reported over time.
We next applied our method to the time series of cases of H5N1 influenza in humans. We produce estimates and credible bounds for R, under different scenarios for the expected fraction of new observed human cases that is attributable to human contagion h. Summary results for Vietnam (and Indonesia) are given in Table 1 . Even in the worst case scenario, where all observed cases are attributed to human transmission (h = 1), the most likely (as of June 2006) R is 0.53 (0.56), with an upper 95% bound of R,0.77 (0.89). For the estimated h = 0.29 (see Methods), the most likely R for both Vietnam and Indonesia is 0, although the estimated upper 95% bound in Vietnam gives the bound R,0.42. For Indonesia, the corresponding estimate gives an R entirely consistent with zero at the 95% credible level.
For less than 20% of the cases attributable to human transmission, R is entirely consistent with zero, even when accounting for the uncertainty in the duration of the infectious period (c 21 ). Reported information does not allow at present a precise determination of c 21 for H5N1 influenza in humans, so that different scenarios are possible, which we explore in detail in Supplementary Material S1 ( Figure S4 , Table S1 ). Data permitting, a hierarchical Bayesian estimation method for the distribution of c can also be envisaged [32] . Figure 4 shows the evolution of R and of its corresponding 95% credible interval. The computation of successive probability distributions for R gives a basis for assessing the evolution of transmissibility over time, including the approach to the epidemic threshold RR1. At present we conclude that, even in the unrealistic worst case scenario, where cases are aggregated at the national level and all cases are attributed to human transmission, R remains below unity.
The emergence of a new epidemic in humans often requires shifts in pathogen biology and/or changes in the human population structure. The methodology developed here can signal these events as anomalies in the expected number of new cases. Assuming no change of epidemiological conditions, knowledge of the distribution of R, accumulated until time t, provides expectations for future case numbers DT(t+t) , with quantified credible intervals, via
where P[R] is taken as the posterior in [13] at time t, and P[DT(t+t)rDT(t)|R] is the statistical epidemic model. Failure to predict future observed cases at time t+t, can then be formulated as a p-value significance test at any chosen level of credibility. A statistical anomaly, i.e., future cases falling outside the credible interval defined by previous observations, may signal changes in epidemiological parameters, specifically in transmissibility (either by pathogen evolution or host population changes) as measured by R. We provide an example in Figure 5 , for simulated data with R 0 = 0.8 changing to R 0 = 1.3, where we show the predicted 95% credible interval for new cases vs. the number of cases actually observed (see also Figure S7 in Supplementary Material S1).
Emerging and re-emerging infectious diseases pose some of the greatest health risks to human populations worldwide. Increasingly they are a feature of our time, stoked by changes in human demographics, mobility, land use and climate, and compounded by poor standards of public health in parts of the world [26, 41] . Importantly, new surveillance and intervention strategies are now becoming possible, guided by quantitative interpretation of epidemiological data, potentially strengthening the hand of primary prevention efforts.
The modelling and prediction approaches developed here (see also Chowell et al. [34] for a comparison to other methods) provide tools for real time estimation of epidemiological parameters that are appropriate for emerging infectious diseases. The method is intentionally simple, relying on standard epidemiological population models, in order to be commensurate with the paucity of epidemiological data typically available for emerging infectious diseases. These features are illustrated by the application of the method to H5N1 influenza infection time series in humans. Clearly, the SIR class of models, even when cast in probabilistic terms, relies on several general assumptions, which are simplistic in specific situations. First, these models do not account for contact heterogeneities, resulting from spatial effects, age, and/or the structure of social networks. These effects can be partially addressed by structuring the population compartments in terms of spatial and risk classes [42] . For example, different regions in an affected country may be taken as separate compartments, provided that there are data pertaining to each region. Indeed the method would then allow estimation of correlations between R at different spatial locations. Second, in its present form, the model does not include independent estimation of infectious or incubation periods. It is straightforward to include an incubation period [43] and, given data on the duration of these periods on a case by case basis, these issues can be addressed by including additional Bayesian estimation steps (see [32] ). Notwithstanding these limiting features, the SIR structure allows reliable real time parameter estimation with quantified uncertainty at very low computational overhead, as verified extensively via simulations at varying known input R, and applications to past pandemic outbreaks (Supplementary Material S1 - Fig. S5 and S6 -and Ref [34] ). One feature of the bounds on R derived here is their dependency on the fraction of cases attributed to human transmission, h. Although h is judged to be small from present surveillance [23, 27, 28] , it remains hard to quantify with certainty. Given the paucity of data, we chose in practice to assume independence and use a binomial probability to attribute cases to human transmission vs. infection from the reservoir. However, other procedures to determine h are possible. If enough data were available, an explicit dynamical model of the (animal) reservoir could be built, or an empirical function correlating introductions through time could be used. Indeed, in the optimal scenario, the actual cases of introduction from the reservoir would be known from field work, and the method proposed here could incorporate that information directly. We note that in the most relevant case, when R becomes larger than 1, the effect of h,1 quickly vanishes, as cases multiply exponentially. For R,1, even a choice of h = 1 will lead to estimates of R,1, but different values of h may lead to credible intervals that include the critical threshold. In general, we believe that a suspicion of a possible R<1 should be followed up with careful field investigations.
We presented a general methodology capable of interpreting quantitatively emerging disease surveillance data in real time with quantified uncertainty that complements other methods proposed recently [21, 29, 30, 32] . Although we illustrated the method with data for H5N1 influenza, these inference strategies are general and can be applied to time series from other communicable diseases. We verified that the model developed here also applies to standard epidemics, yielding agreement with previous estimates of R for the 1918 influenza pandemic in US cities [14, 44] (Figures S5, S6 in Supporting Information) and with other estimation methods [34] . We have also shown how to construct p-value statistical significance tests suitable for automatically monitoring changes in transmissibility of (emerging) communicable diseases [43] .
While still in their infancy, we believe that the current emerging trend in mathematical epidemiology towards real time predictive methods will enable a shift towards more quantitative surveillance and primary prevention, resulting in more consistent and extensive monitoring of emerging infectious diseases and improved designs for health interventions and logistic allocations as epidemics unfold.
Material S1 In Supplementary Material S1, we present further details of the method, including extensions and additional examples. Found at: doi:10.1371/journal.pone.0002185.s001 (0.28 MB PDF)
We thank Miles Davenport, Mac Hyman, Alan Perelson, Timothy Reluga, our PLoS One Editor and anonymous referees for comments that substantially improved the manuscript. Indonesia, under the pessimistic assumption that 29% of reported cases are due to human-to-human transmission (see Table 1 ); and (c) for seasonal H3N2 human influenza isolates in the USA during the 2004-2005 season. (Note that isolates represent only a small fraction of total cases, and may contain reporting biases.) The estimate of the effective reproduction number for an epidemic outbreak asymptotes to unity at late times because initial growth and long-term decay in new case numbers (due to depletion of susceptibles) average out over the history of the outbreak. doi:10.1371/journal.pone.0002185.g004 Between weeks 74 and 75, the reproduction number is shifted R 0 = 0.8R1.3 to create an epidemic. Although we continued to iterate the R distributions via the Bayesian procedure described in the text, note that the shift in R upwards leads to many statistical anomalies (indicated by black arrows). The anomaly is detected immediately, on weeks 75 and 76. Anomalies here are defined as observed numbers of new cases that fall outside the expected 95% credible interval. These anomalies indicate a violation of the hypothesis that R is unchanged, and could be used to trigger alerts in surveillance. doi:10.1371/journal.pone.0002185.g005 DetectiV: visualization, normalization and significance testing for pathogen-detection microarray data DNA microarrays offer the possibility of testing for the presence of thousands of micro-organisms in a single experiment. However, there is a lack of reliable bioinformatics tools for the analysis of such data. We have developed DetectiV, a package for the statistical software R. DetectiV offers powerful yet simple visualization, normalization and significance testing tools. We show that DetectiV performs better than previously published software on a large, publicly available dataset. One of the key applications of metagenomics is the identification and quantification of species within a clinical or environmental sample. Microarrays are particularly attractive for the recognition of pathogens in clinical material since current diagnostic assays are typically restricted to the detection of single targets by real-time PCR or immunological assays. Furthermore, molecular characterization and phylogenetic analysis of these signatures can require downstream sequencing of genomic regions. Many microarrays have already been produced with the aim of characterizing the spectrum of microorganisms present in a sample, including detection of known viruses [1] [2] [3] [4] [5] , assessment of bioterrorism [6, 7] and monitoring food quality [8] .
However, the use of DNA microarrays for routine applications produces many challenges for bioinformatics. Firstly, probe selection is a difficult and time consuming process. There are a huge number of diverse species in nature, of which we have sequence information for only a tiny fraction. This makes it difficult to find oligonucleotides, either alone or in combination, that uniquely identify species of interest. Oligos may have homology to multiple species, which results in a complex and noisy hybridization pattern. Secondly, each nucleic acid sample tested will typically contain a mixture of DNA and RNA from the organism of interest, the host and from a variety of contaminants, which may all contribute to the resulting microarray profile. Furthermore, this may be complicated by the presence of multiple, possibly related, pathogen species, making it difficult to separate patterns due to cross-hybridization from a true positive result.
Urisman et al. [9] have previously reported E-Predict, a computational strategy for species identification based on observed microarray hybridization patterns. E-Predict uses a matrix of theoretical hybridization energy profiles calculated by BLAST-ing completely sequenced viral genomes against the oligos on their array, and calculating a free energy of hybridization. Observed hybridization profiles are then compared to the theoretical profiles using a similarity metric, and a p value calculated using a set of experimentally obtained null probability distributions. E-Predict has been shown to produce useful results in a number of situations. However, at present, E-Predict does not contain any tools for visualization, and requires extensive customization and calculation before it is applicable to new arrays. Also, E-Predict is only available as a CGI script for Unix/Linux platforms.
We present DetectiV, a package for R [10] containing functions for visualization, normalization and significance testing of pathogen detection microarray data. R is a freely available statistical software package available for Windows, Unix/ Linux and MacOS, meaning DetectiV is a platform independent solution. DetectiV uses simple and established methods for visualization, normalization and significance testing. When applied to a publicly available microarray dataset, DetectiV produces the correct result in 55 out of 56 arrays tested, an improvement on previously published methods. When applied to a second dataset, DetectiV produces the correct result in 12 out of 12 arrays.
DetectiV is implemented as a package for R, a powerful, opensource software package for statistical programming [10] . Many packages for R already exist for the analysis of biological datasets, including microarray data, and the bioconductor project [11] is just one example of a group of such packages. As it is implemented in R, DetectiV easily integrates with many of the packages available for microarray analysis, including limma [12] , marray [11] and affy [13] .
DetectiV is written in the native R language and uses standard functions within R. As R is available on Microsoft Windows, Unix (including linux) and MacOS, DetectiV represents a platform independent solution for the analysis of pathogendetection microarray data.
The flow of information through DetectiV is shown in Figure  1 . The basic dataset required is a matrix of data, with rows representing probes on the array, and columns representing measurements from individual microarrays. This dataset is easily produced from data structures created by limma [12] , which includes functions for reading in many common microarray scanner output formats, and affy [13] , which provides functions for reading in affymetrix data. Commonly, researchers will have an additional file of information giving details about each probe. In the case of pathogen detection arrays, this file will most often contain the type, species, genus and other classification data for the pathogen to which each probe is designed. It should be noted that there may be more than one entry in this file for each probe; for example, if a given probe is thought to hybridize to multiple pathogens. In text format, these may be read in using the native read.table command, or in excel format using the RODBC library.
Once these two datasets are in R, DetectiV prepares them for analysis using the prepare.data function. This function joins the array data to the probe information data based on a unique ID. The researcher may choose to subtract local background if appropriate. The default at this stage is to average over replicate probes, again based on a unique ID. This will result in a single value for each unique probe for each array. The data will have one or more columns of extra information from the annotation file, and these columns will be used to group the data for further analysis.
Researchers will wish to visualize their data in order to compare the hybridization signals for the probes recognizing the different pathogen signatures. DetectiV provides a function called show.barplot for this. The output from prepare.data is passed to the function, along with the name of the column containing the variable by which the data will be grouped, referred to here as group. An example in pathogen detection data may be species, genus, family, and so on. The data are sorted into unique groups as defined by the unique values of group. A barplot is drawn, with one bar per unique probe. Probes from the same group are drawn together. Each group is represented by a unique background color, enabling the user to easily visualize the different groups. An example output is shown in Figure 2 . This sample comes from Urisman et al [9] and represents data from a virus detection microarray hybridized with amplified RNA from nasal lavage, positive for respiratory syncytial virus by direct fluorescent antibody (DFA) test. The group chosen here is virus family. It is quite clear from this image that there is a virus from the family Paramyxoviridae present in the sample, demonstrated by the high bars associated with that family.
These images are often very large, and so DetectiV offers the ability to subset the data before plotting by using the get.subset function. Figure 3 shows a similar barplot using a subset of the data: only those oligos representing species that belong to the Paramyxoviridae family. It is clear from this image that those oligos representing different groups/species of respiratory syncytial virus have the highest intensity, as we would expect, although there is cross-hybridization with oligos for human metapneumovirus (another paramyxovirus in the same sub-family: Pneumovirinae).
DetectiV may also carry out normalization and significance testing. For this, there is the function normalise. Here, the aim of normalization is to represent the data in relation to a negative control. The idea is that if the values for each probe are divided by the negative control and then the log2 taken, then the data should be normally distributed, and each group should have a mean of zero (providing a pathogen is not present). Traditional statistical tests can then be used to test if any group of probes is significantly different from zero. DetectiV offers three methods of normalization, each using a different 'type' of negative control, and these are summarized in Table 1 .
Flow of information, and steps taken, when analyzing pathogen detection microarray data using DetectiV Figure 1 Flow of information, and steps taken, when analyzing pathogen detection microarray data using DetectiV. 
The median method calculates the global median value for each array. It should be noted that this method assumes that most probes will not hybridize to anything. If this assumption is false then this method should not be used. However, if the assumption holds, then the median is a good representation of that value we would expect to see from probes that have not hybridized to anything.
The control method relies on specific negative controls having been spotted on the array. The researcher may then choose one of these controls, and the mean value is calculated for that control for each of the arrays. The mean control value for each array is then used as a divisor for each probe on their respective arrays.
Finally, the array method utilizes an entire control array or channel. In this instance, an entire array is chosen to be the negative control, and all probe values are divided by their respective elements from the control array. An obvious example for a control array may be RNA from a known uninfected animal. The control array therefore has a value for each specific probe representing that value we would expect to see if that specific probe has not hybridized to anything.
In all instances, after taking the log2, groups of probes that have not hybridized to anything should be normally distributed and have mean zero. We can therefore split the probes into groups and perform a t-test for each one. DetectiV does this using the do.t.test function. The normalized (or raw) data are split into groups as defined by the unique values of a user defined annotation column. Providing each group has more than two probes, a t-test is performed to test the difference of the observations from zero. The average value is also calculated. The output is a table, sorted by p value.
The data used were downloaded from the Gene Expression Omnibus (GEO) [14] , accession number GSE2228. The array platform for this data is GEO accession GPL1834, and includes over 11,000 oligos representing over 1,000 viral and bacterial species [4] .
The dataset itself consists of 56 arrays including 15 independent HeLa RNA hybridizations, 10 independent nasal lavage samples positive for respiratory syncytial virus, 7 independent nasal lavage samples positive for influenza A virus, a serum sample positive for hepatitis B virus, a nasal lavage sample positive for both influenza A virus and respiratory syncytial virus, and culture samples of 11 distinct human rhinovirus serotypes.
Both DetectiV and E-Predict [9] have been used to analyze the data. For DetectiV, the data were not corrected for local background. Missing, negative and zero values were set to a nominal value of 0.5. Intensities were averaged across replicate probes. Median normalization was then carried out, followed by a t-test grouping the data by virus species. Probes representing actin, GAPDH and Line_Sine were filtered from Table 1 DetectiV normalization methods
Median Where is the value for probe i on array j and is the median value for all probes on array j
Where is the value for probe i on array j and is the mean value for control oligo c on array j Array Where is the value for probe i on array j and is the value for probe i on control array/channel c Explanation of the three normalized statistics offered by DetectiV. the results. Results were first filtered such that groups had a normalized log2 ratio greater than or equal to 1 (a ratio of two to the control) and then sorted by p value. This method will be referred to as DetectiV.
For E-Predict, default values for all parameters were used, and are shown in Table 2 . Data points were corrected for local background, as per the examples in Urisman et al. [9] . E-Predict filters out 266 oligos by default, and this setting was kept. In all cases, E-Predict carried out two iterations, although only results from the first iteration are shown here. The best performing method of interpreting the results was to take those species with a p value ≤ 0.05 and sort by distance (termed E-Predict.dist). Note that this is the method cited in [9] , example 3, used to demonstrate E-Predict's ability to detect SARS.
Pathogen detection arrays have also been implicated in the discovery of SARS. Urisman et al. [9] reported that although their original platform did not contain oligos designed to SARS, once the SARS genome had been published, it was possible to recalculate the energy matrix for E-Predict and find that the energy profile for SARS was the top hit (after taking those viruses with low p values and sorting by distance). We have applied DetectiV to the same dataset (GEO accession GSE546). To include oligos for SARS, we searched a database of oligo sequences on the array with sequence NC_004718 from RefSeq using NCBI blast. There were 61 oligos on the array that hit the SARS genome with greater than 80% identity across an alignment of 20 bp or more. In the analysis, these oligos were assigned as representative of two viruses: their original virus and SARS. The data were median normalized and a t-test carried out using DetectiV.
Finally, having established that DetectiV compares favorably with previously published software, we have validated the DetectiV software by applying it to a second dataset. The data used were downloaded from the GEO [14] , accession number GSE8746. The array platform for this data is GEO accession GPL5725, and consists of 5,824 oligos representing over 100 viral families, species and subtypes. The dataset itself consists of 12 arrays, 4 hybridized with RNA from cell cultured footand-mouth disease virus (FMDV) type O, 3 hybridized with RNA from FMDV type A, 1 hybridized with RNA from a sheep infected with FMDV type O, and 4 hybridized with cell-cultured avian infectious bronchitis virus (IBV). Analysis using DetectiV was carried out as described above.
We present here results from two methods of analysis, termed DetectiV and E-Predict.dist, as described above. There are 56 arrays in the dataset, the expected results of which are known.
Each array was hybridized with RNA containing a single virus, except GSM40845, which was infected with both influenza A and respiratory syncytial virus. We assigned a correct result for each method if the top hit from the analysis was the same as the known infectious agent or, if that agent was not represented on the array, the top hit was a very closely related virus. In the case of GSM40845, we report a correct result if both viruses were at the top of the reported hits, to the exclusion of other virus species (but not closely related strains).
Additional data file 1 gives the top hit for both analysis methods in all 56 arrays. As can be seen, DetectiV generated a correct result in 55 out of the 56 arrays. In comparison, the E-Predict.dist method gave a correct result in 53 out of the 56 arrays. These results are discussed in greater detail below.
Full results for each of the arrays can be found on the DetectiV website [15] . Within the 55 correct results, there are three classes that require slightly different interpretation, examples of which are GSM40806, GSM40810 and GSM40817. Results for these arrays are given in Table 3 .
Array GSM40806 was hybridized with amplified HeLa RNA, and the top hit from DetectiV is human papillomavirus type 18, as expected. This virus has both the smallest p value and largest mean normalized log ratio. There is also clear GSM40810 was hybridized with RNA containing human rhinovirus 28. There are 24 distinct groups of human rhinoviruses represented on the array, including a group of oligos for all members ('human rhinovirus sp.), one each for human rhinovirus A and B, and several groups for distinct serotypes. Human rhinovirus 28 is not one of those serotypes specifically targeted by the array; however, as a serotype of the human rhinovirus A species, we would expect the groups for human rhinovirus sp. and human rhinovirus A to be prevalent amongst the results. As can be seen from Table 3 , the top hit from DetectiV is human rhinovirus sp., closely followed by human rhinovirus A, the expected result. The reason we have highlighted this array, however, is that the result for Enterobacteria phage M13 shows a higher mean normalized intensity than any of the rhinovirus groups. This is representative of a class of result from DetectiV whereby a virus group has a higher mean normalized log ratio, but a larger p value, than the top hit. Here, as in GSM40806, we see orders of magnitude between the p value for the top hit and that for Enterobacteria phage M13, which identifies human rhinovirus as being the infectious agent, but in this case we cannot rely on the mean normalized intensity. In this particular instance, Enterobacteria phage M13 is represented by 10 oligos, all of which have intensities far greater than the global median, but which vary considerably between 982 and 18,864. These high values may be due to hybridization with a cloning vector.
Finally, array GSM40817 was hybridized with respiratory syncytial virus. The results are again shown in Table 3 , but for this array only, they have not been filtered on mean normalized intensity. Human herpesvirus 5 has by far the smallest p value of any of the virus groups; however, it also has a very small mean normalized log ratio. The correct hit, respiratory syncytial virus, has the second smallest p value, but has a much larger mean normalized log ratio. This represents the final class of result seen by DetectiV, where the correct virus group does not have the smallest p value, but does have a much larger mean normalized log ratio than those groups that have smaller p values. The small p value of respiratory syncytial virus combined with the large mean normalized log ratio identifies respiratory syncytial virus as the only infectious agent. In this instance, human herpesvirus 5 is represented by 241 oligos, 167 of which are greater than the global median, but all of which have intensities less than 1,000. This could be due to the oligos for human herpesvirus 5 having distant homology with the infectious agent or host cell.
These three types of result are typical of DetectiV, and explain why both the p value and the mean normalized log ratio must be taken into account when interpreting the results. Thus, if the results from DetectiV are filtered such that only viruses whose mean normalized log ratio is ≥ 1, and then sorted by p value, the three scenarios described here are accounted for, and we obtain the correct result in 55 out of the 56 arrays.
The single incorrect result for DetectiV comes from GSM40816, which reports human herpesvirus 7 as the top hit, whereas the infectious agent was in fact respiratory syncytial virus. The top five hits for this array using the DetectiV method are shown in Table 4 . As can be seen, bovine respiratory syncytial virus and respiratory syncytial virus are second and third, respectively. Both respiratory syncytial virus and bovine respiratory syncytial virus have higher mean values than human herpesvirus 7, although the latter has a smaller p value and a mean value that is above the cut-off of 1. Had the results been filtered for p value ≤ 0.5 and then ordered by average value, then the top hit would have been respiratory syncytial virus; similarly, if a cut-off of 2 had been applied instead of 1, a correct result would have been reported. However, across the entire dataset these methods of interpreting the results perform worse than the DetectiV method described above. It is worth noting here that for this array, E-Predict gives the correct top hit.
The results from E-Predict follow similar patterns to those of DetectiV. In most cases it is obvious which virus is the infectious agent, either by examining the p value, the similarity or both together. Full results can be seen on the DetectiV website [15] . However, there are certain results reported by E-Predict where it is impossible to obtain the correct result no matter which combination of p value and similarity is used. These arrays are arrays are GSM40809, GSM40821 and GSM40847, and the top five results for these arrays can be seen in Table 5 .
GSM40809 was hybridized with RNA containing human rhinovirus 26. Again, this is a serotype not specifically targeted by the array; however, as a serotype of human rhinovirus B we Array GSM40821 was infected with hepatitis B virus but E-Predict reports orangutan hednavirus as having both a smaller p value and a higher similarity. This is not that surprising given that hepatitis B and orangutan hepadnavirus are closely related; however, the fact remains that with no a priori knowledge, the only logical conclusion from this result would be that the infectious agent was orangutan hepadnavirus. Again, DetectiV calls this array correctly.
Finally, array GSM40847 was hybridized with RNA containing human rhinovirus 87. Again, this is a serotype not specifically targeted by the array, and is not present in the NCBI taxonomy database [16] at the time of writing. We can therefore expect the 'human rhinovirus sp.' group to be high amongst the results (in fact, it is the top result for DetectiV). E-Predict reports human enterovirus B as having the smallest p value and human echovirus 1 as having the largest similarity. In fact, E-Predict does not report any rhinovirus oligos in the first iteration at all, and it is only in the second iteration that the group human rhinovirus A is reported as significant.
In the three cases outlined above, there is no clear way of distinguishing the incorrect virus from the correct one. There is also no consistent method of sorting or filtering the results that would give the correct results. In these three cases, E-Predict is unable to distinguish closely related virus species and serotypes. We have reported here the best performing method of interpreting E-Predict results, whereby virus groups with a p value ≤ 0.05 are sorted by distance. This results in a success rate of 53 out of 56 arrays.
The top five hits from the analysis of the SARS dataset can be found in Table 6 . As can be seen, the top hit is SARS, with the lowest p value and the highest mean normalized log ratio. SARS is distinct from the other viruses, having a p value three orders of magnitude lower than the second top hit.
Full results can be found on the DetectiV website [17] . The top hit from DetectiV for each of the 12 arrays from GSE8746 can be found in Table 7 . As can be seen, DetectiV clearly identifies the infectious agent in all 12 cases. DetectiV works for both the cell-cultured samples and the infected sheep, and shows the ability of the array to distinguish between different subtypes of FMDV.
Developing a quick and reliable test for the presence/absence of thousands of bacterial and viral species in a single experiment is an attractive proposition, and a function that DNA microarrays are ideally suited to. Microarrays are extremely high-throughput and relatively cheap. In the case of pathogen detection, the aim must be to quickly and clearly identify those pathogens present in a sample with high confidence, keeping false positives and false negatives to a minimum.
However, the data from such microarrays pose many problems. Firstly, oligos may not be unique to the species they are designed to. For certain species it is impossible to find a large number of oligos that are unique only to that virus that meet the criteria for oligo selection. This is particularly problematic for closely related species and strains. In such cases, the 'best' oligos are added to the array, in the knowledge that multiple viruses may hybridize to them. This leads to noisy signals across multiple virus families, species and serotypes. Secondly, infected biological samples may contain many different virus species and strains, making interpretation difficult. Thirdly, it is known that certain oligos simply do not work, even when the array is hybridized with the species that those oligos were designed to. Without testing the array with each virus, we are incapable at present of predicting which oligos will work and which will not. With thousands of species per array, many of which cannot be cultured in vitro, it is unfeasible to challenge arrays with every species. Finally, we of course do not know, nor can we ever know, the complete genome sequence of every virus we may encounter. Therefore, though we think we have oligos unique to a species or strain, that is only ever in the context of our knowledge at the time of design, and they may not in fact be unique.
Despite these problems, many species detection arrays have been developed [1] [2] [3] [4] [5] . However, reliable methods of data analysis have been rare. Initial methods included visual inspection of the array [4] and clustering [18] , both of which are subjective and time-consuming. To combat this, Urisman et al. [9] have proposed a more robust method, E-Predict. E-Predict utilizes a pre-calculated energy matrix for each oligo on the array and uses a variety of normalization and similarity metrics to calculate a p value and similarity for each virus. The advantages of E-Predict are that it is quantitative, produces good results and is extensible, through the extension of the energy matrix. The disadvantages of the software are a lack of visualization tools, the need to customize parameters for different array platforms and hybridization conditions, and the availability of the software only as a CGI script on the Unix/Linux platform.
We have developed DetectiV, a package for R containing visualization, normalization and significance testing functions for pathogen detection data. DetectiV uses simple and well established visualization and statistical techniques to analyze data from pathogen detection microarrays. DetectiV offers a powerful visualization option in the form of a barplot, enabling researchers to quickly and easily identify possible infectious agents. Data can then normalized to a negative control (be that a specific probe, array or the global median), transformed by taking the log2 and then subjected to a t-test for each species on the array. Oligos are allowed to represent any number of viruses, and thus any analysis is easily extensible by simply updating the list of which oligos represent which species.
DetectiV requires minimal set up and configuration, requiring only an additional file detailing which species each oligo represents. In the majority of cases, these files will already exist. It is then possible to apply DetectiV 'out of the box' to any array data that is readable by R or bioconductor. DetectiV requires no training, configuration or customization specific to each array. DetectiV is available as a package for R on both Windows and Linux/Unix, and as such may be considered platform-independent.
In this study, DetectiV produced the correct result in 55 out of 56 arrays, by filtering for viruses with a mean normalized log ratio greater than 1 and then sorting by p value. We make the distinction here between biological and statistical significance. A statistically significant result may be obtained by a group of oligos that display intensities only marginally larger than the negative control (in this case the global median intensity). This is demonstrated by human herpesvirus 5 on array GSM40820 (Table 3) . However, we know that from a biological perspective, we would expect to see intensities far higher than the negative control, and that intensities only marginally higher result from low homology between the probe and the sample. We can therefore use the statistical significance (p value) in combination with our idea of biological significance (the mean normalized log ratio) to successfully call the correct result in over 98% of the arrays.
In the majority of cases there is a clear difference in the p value, the mean normalized log ratio, or both, between the correct hit and subsequent hits, allowing for both automatic and manual detection of true and false positives. However, this does require careful interpretation. Both DetectiV and E-Predict predict multiple, significant matches on all of the arrays. When using DetectiV, it is only when looking for major changes between the top hit and subsequent hits, in terms of p value or mean log ratio, that it is possible to separate the true positives from the false positives. In many cases, using automatic rules will result in the correct result; however, there will inevitably be borderline cases where human inspection of the results is required. This is all the more important when considering the possible economic impacts of a false positive for certain species. At present, the safest way to employ such arrays, and their analysis methods, may be simply as a first step towards identifying infectious agents, informing researchers about which viruses they should test for using more conventional methods.
The results from the application of DetectiV to the SARS dataset are encouraging. Here, oligos designed to SARS were not present on the array. However, using a simple NCBI blast search, it was possible to extend the range of viruses covered by the array to include SARS -61 existing oligos showing significant homology to the SARS genome. On application of DetectiV to the updated data, SARS was the top hit. Not only does this offer the promise of being able to extend the coverage of the array without adding further oligos, it also suggests that it is possible to detect viruses without having any unique oligos. This may inform the oligo selection process -it may be equally desirable to have multiple, non-unique oligos to represent a species as it is to have a few that are unique.
The results from the application of DetectiV to a second dataset are also encouraging, with the correct result being the top hit in all 12 cases. Of particular interest is the ability of the array, and DetectiV, to distinguish not only between separate viral species, but also between different subtypes of FMDV. It should be noted that in order to apply DetectiV to a second dataset from a completely different array to the first dataset, the user only has to change the GEO accession number and the number of arrays within that dataset. This compares favorably with E-Predict, which would require a separate training dataset from the second array, the calculation of a large and complex sequence similarity matrix and the optimization of several parameters.
There are a number of ways in which DetectiV may be developed. In terms of visualization, better browsing capabilities of the barplots would be desirable, perhaps using a web-interface. In terms of the analysis, we may borrow ideas from gene expression arrays. For example, limma uses an empirical Bayes method to shrink each gene's standard error towards a common value, and has been shown to perform better than standard statistical methods [12] . It may be that we can apply a similar method here to shrink the standard error for each virus species towards a common value, thus increasing sensitivity. It may also be possible to apply multiple-testing procedures to the resulting p values. The Bonferroni correction may be appropriate, in which the p values are multiplied by the number of comparisons, or a more conservative approach may be needed, such as that suggested by Benjamini and Hochberg [19] , in order to control the false discovery rate.
In conclusion, DetectiV is a highly accurate tool for the analysis of pathogen detection microarray data, offering simple but powerful visualization, normalization and significance testing functions. DetectiV performs better than previously published software on a publicly available microarray dataset. DetectiV is available as a package for R, a platform-independent statistical software package, and requires little configuration or customization. It is released under the GNU General Public License and may be downloaded from the DetectiV website [20] . Expression of Foot-and-Mouth Disease Virus Capsid Proteins in Silkworm-Baculovirus Expression System and Its Utilization as a Subunit Vaccine BACKGROUND: Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD(50) (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD(50) per dose. CONCLUSION: The results suggest that this strategy might be used to develop the new subunit FMDV vaccine. Foot-and-mouth disease (FMD) is an economically important disease of domestic and wild cloven-hoof animals including cattle, swine, goat, sheep and buffalo. It can result in great reduction of productivity in adult animals and death in young animals. FMD is endemic in parts of Asia, Africa, the Middle East and South America (with sporadic outbreaks in other ''free areas''). In countries affected by the disease, livestock trade and animal products have been impacted. Even in developed countries and areas, outbreak of FMD would greatly affect the economy. In 2001, the outbreak of FMD in England brought a loss of 8 billion dollars [1] , and the consequent occurrence in Holland killed 0.2 million animals [2] . In addition, the 1997 outbreak in Taiwan brought a loss of about 3.6 billion dollars to its exports [3] . Therefore, controlling bath endemic and epidemic FMD has become a global concern in livestock raising.
At present, vaccination is a major means of FMD control in most endemic areas. The present method of FMDV vaccine production is this: The virus is propagated in BHK-21 cell line, concentrated, and chemically inactivated. Although the inactivated vaccine has been shown to be effective, it may lead to new outbreaks of FMD because of either the incomplete inactivation of FMDV in large-scale production or the escape of the live virus from vaccine production workshops [4] . Therefore several expression systems such as E.coli [5] , transgenic plant [6] , yeast [7] , adenovirus vector [8] [9] [10] [11] [12] , vaccinia virus vector [13] , and DNA vaccine [14] , have been used for expression of FMDV antigen to prepare subunit vaccines. But such methods have problems such as poor immunogenic capability or low efficiency. Adenovirus based vaccine known for its best protective effects can protect 5 of 5 vaccinated cattle, but this vaccine is still unacceptable because of safety problem and preservation difficulty. The baculovirus expression system, a valuable expression system to produce virus-like particles, has successfully produced many kinds of empty viral capsids [15, 16] , such as rabbit hemorrhagic disease virus, Norwalk-like viruses,SARS and so on [17, 18, 19] . Compared to the baculovirus expression system (AcNPV-Sf cell), silkworm-baculovirus expression system has distinct advantages [20, 21] . First, expression levels in silkworm are 50-1000 times higher than that in insect cell lines. Second, silkworm don not have any pathogens that can cross infect with vertebrates and animal serum is not needed to produce foreign proteins in this expression system, so that the expressed antigens are safer to vertebrates. In view of all these advantages, the silkworm-baculovirus expression system was employed for expression of intact P1-2A, 3C coding regions of FMDV Asia I/HNK/ CHA/05. All five cattle that were vaccinated with diluted expression antigen were induced specific antibody, four of which were considered completely protected. Furthermore, the PD 50 (50% bovine protective dose) value of the subunit vaccine was 6.34 in bovine potency test.
Intact P1-2A and 3C protease coding regions from FMDV Asia I/HNK/CHA/05 strain were amplified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-P12A3C. The plasmid was digested with BamH I/EcoR I to generate fragments of 2.3 kb and 10 kb, while fragments of 0.7 kb and12 kb were generated with EcoR I/Bgl II. Based on the length of these fragments, it was confirmed that the full target fragment (P12A3C) was correctly incorporated into the transfer vector and that the expression cassette was located downstream of polyhedrin promoter. Sequence analysis indicated that the P12A3C was 3,024 bp containing full P1-2A and 3C genes and partial 2B,3B genes.
Bm-N cell line was co-transfected with baculoviral transfer plasmid pVL-P12A3C and linearized BmBacPAK-6 DNA. The supernatant was collected 4 days post transfection as the viral stock for screening of recombinant virus. Twenty four isolated viral plaques from the plaque assays were cultivated in a 24-well plate and were inoculated into silkworms. The plaque of recombinant virus expressed at maximal activity was selected to purify. The pure recombinant virus from the last round was used as stock virus and confirmed to contain the full expression cassette. The recombinant virus Bm-P12A3C was used to express FMDV protein in cells or silkworm.
The expression of polyprotein in Bm-N cells was analyzed by IFAT and sandwich-ELISA. IFAT pictures demonstrated that Bm-N cells infected with Bm-P12A3C produced specific fluorescence, while only very weak background fluorescence appeared in the control cells (Fig. 1 ). This indicated that polyprotein was indeed expressed in Bm-N cell. The sandwich-ELISA results indicated that the FMDV antigen in Bm-P12A3C infected cells was expressed at levels about equivalent to the positive control, but was not detected in BmBacPAK-6 infected cell lysate.
The Bm-P12A3C virus was inoculated 1,000 silkworms. The dying silkworm's haemolymph was collected (about 4-5 d post infection, corresponding with rearing mean temperature about 25-27uC). Sandwich-ELISA was conducted to evaluate the expressed antigen in silkworm. The results indicated that OD value of the harvested haemolymph from silkworm infected by Bm-P12A3C decreased as the dilution rate increased, which was in good agreement with variation of positive control of FMDV antigen. The expression yield was about 100 fold (positive antigen: 1/32,0.964; expressed antigen in haemolymph: 1/4096, 0.995) more than the positive control (BHK-21 cell vaccine which had a PD 50 value of 3.6), but was not detectable in the negative control (BmBacPAK-6 infected silkworm's haemolymph) (Fig. 2) .
In order to determine the time course of expressed antigen in silkworm and the optimum acquisition time for large scale's production, haemolymph from 10 silkworms was harvested every 12 h beginning at 60 hpi and stored at 220uC. Subsequently, the haemolymph was diluted to 1000 folds for detection of expression products (Fig. 3 ). There was a little at 60 hpi, and the accumulation of recombinant products were dramatically increased from 84 hpi and kept at the high levels during the late phase of infection. So, expressed antigen could be harvested at 108-120 hpi (at the condition about the mean rearing temperature of 25uC).
Cattle were vaccinated by the vaccine prepared from 1/30 diluted expressed antigens (the quantity of antigen was about 3 folds of the BHK-21 cell vaccine). LPBE-antibody titer was determined at 7, 14, 21 and 28 dpv following the LPBE method. It was found that all five cattle vaccinated with Bm-P12A3C antigen developed a detectable FMDV-antibody response at 7 dpv, and dramatically reached to high level at 14 dpv. By 21and 28 dpv, the antibody level was maintained at the same level or higher, and reached to a titer of 360 in cattle No33 and No50. In contrast, antibody level in the two control cattle (vaccinated with vaccine prepared from BmBacPAK-6) was not boosted (Table 1) .
Furthermore, sera were analyzed for neutralizing antibodies against FMDV (Table 2) . While the two control cattle could not develop any neutralization antibodies against FMDV, all five cattle vaccinated with Bm-P12A3C antigen developed a FMDV-specific neutralizing antibody response in 14pv and maintained at the same level or higher in 28 dpv. The result was in agreement with the LPBE-antibody titer.
All vaccinated cattle were challenged with 10,000 BID 50 of Asia I/HNK/CHA/05 at 28 dpv. Body temperature, mouth and feet were observed consecutively for ten days to evaluate the incidence of disease (Table 3) . Four of the five cattle were considered completely protected. Only one vaccinated cattle, No45, developed lesions. The lesions were detectable by 6 dpc and the clinical signs were less severe compared to control group. By contrast, vesicles developed in the control animals by 2 dpc at the sites of all feet and mouth. This indicated that antigen produced in silkworm could be effectively protective.
The PD 50 test was performed to assess the subunit vaccine potency by following the bovine potency test protocol described by the OIE to test the traditional inactivated FMD vaccines. In this research, the result showed the vaccine potency of the batch immunized with the expressed antigens reached 6.34 PD 50 per dose (Table 4 ). Discussion FMD still threatens world livestock production. Seven distinct serotypes of FMDV have been identified, named A, O, C, Asia I, SAT I, SAT and SAT with none cross immune protection occurring between them. The FMDV serotype Asia which was first isolated in Pakistan is epidemic within Southeast Asia and Indian peninsula, disseminating among Near East, Middle East and Far East [22] . In March 2005, FMDV serotype Asia was found in HongKong (Asia I/HNK/CHA/05 strain). Subsequently, this type of the virus was reported from mainland of China in April 2005 [23] . The P1 sequence of Asia 1/HNK/CHA/05 isolate was aligned and compared with 9 reference sequences. The result confirmed that Asia 1/HNK/CHA/05 has a high identity with nine Asia I reference sequences from 85.9 to 92.6% [24] .
Expression products of baculovirus expressing system are generally considered to be well immunogenic and possess the ability to assemble empty viral capsid. When the same FMDV expression cassette were expressed in E.coli and baculovirus expression system, the expression products from baculovirus excels that from E.coli in terms of the immunogenic [25] and protective effects [26] . Empty capsid comes into being only when capsid precursor P1-2A, protease L and 3C coding region from FMDV O1K serotype were all expressed in baculovirus expressing system(AcMNPV-Sf cell). Truncated protease L can not be selfcleaved from VP0. But, the expressed protease L is harmful to host cell growth, reducing the expression efficiency [27] . In adenovirus expression system, P12A3C expression cassette, including full structure of P1-2A,3C and portion of 2B and 3B, can be expressed and assembled into empty capsids [8] . Myristoylation of the animo terminus of P1-2A is of great importance to the assembly of viral particles [28] . It has been reported that the expression products of AcMNPV-Sf cell can be myristoylated well [29] . Based on the above studies, and using the design previously published by Mayr et al [8] , the P12A3C expression cassette of FMDV serotype Asia I was constructed. After two sorting rounds of recombinant virus and measurements of expression efficiency for more than 20 viral clones, the over-expressed recombinant virus Bm-P12A3C was obtained. It can express with very high efficiency in the hyperexpression variety of silkworm (JY1). The specific antigen produced per milliliter in silkworm haemolymph at least 100 folds more than the BHK-21 cell vaccine which had a PD 50 value of 3.6.
Because cattle are the most important economic and susceptible cloven-hoof animal, we designed an experiment to verify whether this produced antigen can be used for preparing a cattle FMD vaccine. We followed the bovine potency test protocol described by the OIE to test this subunit vaccine potency. We used 1/30 diluted dosage to vaccinate five cattle and two controls were vaccinated with vaccine prepared from BmBacPAK-6's. By two weeks post vaccination, the antibody level of the five vaccinated cattle reached a high titer. The antibody level has some ascension but maintained thereafter two weeks, while the control group maintained lower than titer 8. After virulent homologous virus challenge, four of the five were considered protected, and one delayed the disease and ease the clinical symptom, but two unvaccinated cattle developed lesions on all the feet and in the inside of mouth on the second day. Cell-mediated -immune response was probably involved in the protection: that would explain why animal 45 has the same neutralizing antibody titers as 122 but is not protected. This demonstrated that the expression products from silkworm-baculovirus expression system were immunogenic as well. Based on above result, we did the PD50 test to assess the bovine potency of the subunit vaccine. When employed for routine prophylactic use, the vaccine should contain at least 3 PD 50 per dose for cattle by OIE recommended. The result showed the subunit vaccine potency could get 6.34 PD 50 a dose for cattle. This leads to a conclusion that it is feasible to use the silkworm-baculovirus expression system for FMD vaccine production. 
Genomic RNA was extracted from the viral supernatant with RNeasy (Qiagen) and used immediately for cDNA synthesis. cDNA synthesis was performed with Superscript II reverse transcriptase (Invitrogen). PCR was used to amplify the Intact P1-2A and 3C protease coding regions from the cDNA using two pairs of specific primer:
Forward primer for P1-2A: 59-ATAGGATCCACCATGG-GAGCCGGGCAATCCAGCC-39 (BamH I site was introduced)
Reverse primer for P1-2A 59-CGCGAATTCTGACATGT CCTCCTGCATCTGGTTG-39 (EcoR I site was introduced)
Forward primer for 3C:59-GCGGAATTCAAGAAACCT GTCGCTTTGAAAGT-39 (EcoR I site was introduced)
Reverse primer for 3C:59ATAAGATCTCTACTCGTGG TGTGGTTCGGGAT-39 (Bgl II site was introduced)
The PCR products were separated by 1% Agarose gel electrophoresis. The target fragments of P1-2A and 3C were inserted into baculoviral transfer vector pVL1393 using BamH I/ EcoR I and EcoR I/Bgl II sites following the routine protocols to generate a transfer plasmid. The whole P1-2A and 3C coding regions in transfer plasmid was sequenced and named pVL-P12A3C.
The baculoviral transfer plasmid pVL-P12A3C was co-transfected with linearized Bm-BacPAK6 DNA into Bm-N cells by liposome-mediated method using transfection reagent lipofectamin 2000 (Invitrogen) [30] . The co-transfection supernatant was subject to plaque assays to screen the individual viral plaques. PCR amplification was conducted to confirm that the P1-2A and 3C genes had been incorporated into the baculoviral genome.
Primers were designed as follows: Sense: 59-ACTGTTTTCG-TAACAGTTTTGTAA-39 and Reverse: 59-CTACTCGTGGT GTGGTTCGGGAT-39. Another two rounds of screen were performed and the pure recombinant virus was used to generate high titer viral stocks for expression.
The expression of FMDV polyprotein in Bm-N cells infected with Bm-P12A3C was analyzed by immunofluorescence test (IFAT) and sandwich-ELISA. The Bm-P12A3C was multiplied in Bm-N cells. Bm-N cells (2.0610 5 ) were cultured on cover slips and inoculated at a MOI of 10 pfu with Bm-P12A3C. After 48 hours post infection (hpi), IFAT was conducted to analyze the expression of FMDV proteins. Cells were then rinsed with PBS for 1 or 2 times and fixed in 100% cold acetone (220uC for 30 min). Samples were incubated with rabbit serum against FMDV (37uC for 30 min) in humid box, washed with PBS for five times, and then stained with fluorescein-conjugated goat anti-rabbit serum at 37uC for 30 min. The cover slips were coated with glycerin and observed on an Olympus fluorescence microscope. Bm-N cells infected with BmBacPAK-6 were used as control.
When Bm-N cells infected with Bm-P12A3C were partial floating, they were detached and collected (about 72 hpi), the cells pellet was freezed and thawed at -70/37uC in PBS for three times and centrifuged at 10,000g for 5 min at 4uC. The supernatant was tested using the sandwich-ELISA method. 96-well plat-bottomed plates (Costar) were coated with the rabbit serum against FMDV overnight at 4uC and blocked with defatted milk powder for 1h. Then the plates were washed five times. FMDV antigen (from the vaccine which had a PD 50 value of 3.6), lysates of Bm-N cells with Bm-P12A3C and BmBacPAK-6 infected were diluted in a twofold series and incubated at 37uC for 1h. Subsequently, the plates were washed thoroughly and guinea pig sera against FMDV was added to each well. The plates were incubated at 37uC for 1 h, and then rabbit anti-guinea pig IgG peroxidase conjugate (Sigma) at 1:10000 dilution was added and reacted at 37uC for 1 h. Substrate (0.05% H 2 O 2 plus orthophenylene diamine) was added, reacted for 15 minutes and stopped by the addition of 1M sulphuric acid. Absorbance was determined at 492 nm.
Early fifth-instar silkworms were infected with the recombinant virus at about 10 5 pfu per larva. The dying silkworm's haemolymph was collected on ice and stored at 220uC for sandwich-ELISA.
The infected silkworm's haemolymph was collected every 12 h starting at 60 hpi for determining the expression course of target antigen.
Silkworm haemolymph was lysed ultrasonically and cell debris was removed by centrifugation. The diluted supernatant was used to produce vaccine. Liquid-phase blocking ELISA(LPBE)(http:// www.oie.int/eng/normes/MMANUAL/A_00024.htm) was performed to determine the antibody titer for screening of candidate cattle according to the standard method of World Organization for Animal Health, Office International desEpizooties (OIE) before vaccination. Candidates with a potency lower than 8 were housed in disease-secure isolation facilities in Lanzhou Veterinary Research Institute. Detection kit was prepared by Lanzhou Veterinary Research Institute. Seven cattle (6-8 months old) were immunized by intramuscular inoculation at the site in the neck.
Five cattle were vaccinated with 3ml/animal of vaccine with Bm-P12A3C's, while two control cattle were vaccinated with the same dose of vaccine with BmBacPAK-6's.
Cattle serum were collected at 7, 14, 21and 28 days postvaccination (dpv). Antibody against FMDV was detected by LPBE method (as above) and SNT.
For SNT, the sera was diluted in DMEM as two-fold in a 96well flat-bottomed tissue culture plates (Castor, USA). Virus suspension with a titer of 100 TCID 50 in 50 ul was added to each sera well and the mixture was incubated for 1 h at 37uC and 5% CO2. 50 ul of BHK-21 cell suspension (1.5610 6 ml 21 ) was added to each well and incubated for 4-5 days. Appropriate serum, virus and cell control were included in this test. The plates were observed via microscope for cytopathic effect.
According to the descriptions by standard protocol of OIE (http://www.oie.int/eng/normes/MMANUAL/A_00024.htm), all animals were challenged by intradermal inoculation at two sites in the tongue with 10,000 bovine infectious doses (BID 50 ) of Asia I/HNK/CHA/05 at 28 dpv. The body temperature of the animals was monitored daily. The restrained animals were carefully examined in the mouth, and feet every day for the first 10 days after challenge.
We followed the bovine potency test protocol described by the OIE to test this subunit vaccine potency. Three groups of five cattle per group and a control group of two non-vaccinated animals were vaccinated. The vaccinated groups were administered different doses (1 dose, 1/3 dose, 1/9 dose)of the subunit vaccine prepared from diluted expression products and all animals were challenged 3 weeks after vaccination with 10,000 BID 50 of Asia I/HNK/CHA/05 to the vaccine under test by intradermal inoculation into two sites on the upper surface of the tongue. The animals were observed daily for 10 days after challenge for clinical signs of FMD. Control animals developed lesions on at least three feet. Unprotected animals showed lesions at sites other than the tongue. From each animal protected in each group, the PD 50 (50% bovine protective dose) content of the vaccine was calculated based on the Reed-Muench method. The effect of network mixing patterns on epidemic dynamics and the efficacy of disease contact tracing In networks, nodes may preferentially contact other nodes with similar (assortatively mixed) or dissimilar (disassortatively mixed) numbers of contacts. Different patterns of contact support different epidemic dynamics, potentially affecting the efficacy of control measures such as contact tracing, which aims to identify and isolate nodes with infectious contacts. We used stochastic simulations to investigate the effects of mixing patterns on epidemic dynamics and contact-tracing efficacy. For uncontrolled epidemics, outbreaks occur at lower infection rates for more assortatively mixed networks, with faster initial epidemic growth rate and shorter epidemic duration than for disassortatively mixed networks. Contact tracing performs better for assortative mixing where epidemic size is large and tracing rate low, but it performs better for disassortative mixing at higher contact rates. For assortatively mixed networks, disease spreads first to highly connected nodes, but this is balanced by contact tracing quickly identifying these same nodes. The converse is true for disassortative mixing, where both disease and tracing are less likely to target highly connected nodes. For small epidemics, contact tracing is more effective on disassortative networks due to the greater resilience of assortative networks to link removal. Multi-step contact tracing is more effective than single-step tracing for assortative mixing, but this effect is smaller for disassortatively mixed networks. For a wide range of epidemic and epizootic diseases, individuals can be usefully modelled as nodes in a network, where the network links represent potentially infectious contacts between individuals. This network representation applies to many complex systems such as the Internet, the World Wide Web (Albert & Barabási 2002) , social and transportation networks (Liljeros et al. 2001; Jones & Handcock 2003; Hufnagel et al. 2004) and livestock movement networks Robinson et al. 2007) . Models that use explicit contact structures between individuals, households, cities, regions, countries or farms have been used to analyse the spread of human diseases, such as SARS (Hufnagel et al. 2004; Meyers et al. 2005) and pandemic influenza (Eubank et al. 2004; Ferguson et al. 2005) , and animal diseases, such as foot-and-mouth disease (FMD; Green et al. 2006a; Kao et al. 2006; Kiss et al. 2006a ) and avian influenza (Le Menach et al. 2006) . Much attention is now focused on analysing network properties and the dynamic processes they support (for reviews, see Newman (2003b) and Keeling & Eames (2005) ), as well as how to effectively control these processes.
Contact tracing is commonly used to identify individuals that have been in contact with infectious individuals, to reduce the number of potential onward infections from traced individuals. Contact tracing was successful in the 2003 SARS epidemic (Lipsitch et al. 2003) , but unsuccessful in the 2001 FMD epidemic in the UK (Ferguson et al. 2001; Keeling et al. 2001; Kao 2003) . For simple unstructured populations, contact tracing has been modelled using a combination of branching process analysis complemented by stochastic simulation models (Müller et al. 2000) . However, in more highly structured contact networks, the properties of the network markedly influence the efficacy of contact tracing. For example, contact tracing is more effective on clustered networks than on random networks (Eames & Keeling 2003; Kiss et al. 2005) , and on scale-free networks a higher tracing effort is needed to control an epidemic than on random Poisson networks .
In this paper, we examine the impact of departure from the common assumption of proportionate (random) mixing upon the efficacy of contact tracing. Proportionate mixing assumes that the probability of any two nodes being connected is proportional to the product of their number of contacts. This is unlikely in many cases where 'like attaches to like, assortatively mixed' (e.g. STI transmission; Anderson et al. 1990; Catania et al. 1992) or where 'opposites attract, disassortatively mixed' (e.g. livestock trading among farms and markets in the UK ; correlation properties of the Internet (Pastor-Satorras et al. 2001) ). The empirical evidence and importance of connectivity correlations have led to numerous network-and differential equation-based models (Boguñá et al. 2003a; Newman 2003a; Barthélemy et al. 2005) . Many of these models focus on the effect of connectivity correlations on the epidemic threshold, initial growth rate and hierarchical spread. For example, it has been shown that epidemics on networks characterized by high node degree variance grow rapidly, and in the limiting case of infinite variance, instantaneously, independently of the mixing pattern (Boguñá et al. 2003b) . The initial growth of the epidemic changes from exponential to power law if non-random mixing is combined with small-world properties, with the powerlaw exponent determined by the average node distance on the network ( Vazquez 2006) . While in the limit of infinite populations, the implications of preferential mixing for disease invasion are well known, here we focus on disease control and consider the efficacy of contact tracing for different mixing patterns. For networks that are poorly characterized, the efficacy of contact tracing is difficult to determine without exact knowledge of the underlying contact network, i.e. who is connected to whom? By analysing disease transmission on theoretical networks with different mixing patterns, we aim to identify the implications of non-random mixing for epidemic dynamics and control strategies.
The network-based disease transmission and contacttracing model is based on models considered by Huerta & Tsimring (2002) , Eames & Keeling (2003) and Kiss et al. (2005 Kiss et al. ( , 2006a . Each node in the network is classified according to one of four states of disease progression: susceptible nodes (S ); nodes at once infected and infectious (I ); nodes 'triggering' contact tracing (T ), which are identified as being infectious, are immediately isolated and initiate tracing; and, finally, removed nodes (R), which are no longer infectious and do not initiate tracing. The transitions between states are depicted in figure 1.
Infection S/I. The epidemic is seeded with one or more infected nodes. Thereafter, infection progresses via a contact network; the probability of a node becoming infected depends on the state of the nodes directly linked to it. The probability p of a susceptible node with k infectious contacts becoming infected in a small time interval Dt is pZ1Kexp(KtkDt). Here, t is the rate of infection spreading through a single contact between an infectious and a susceptible node.
Contact-independent identification of infectious nodes I/T. Disease is detected at an infectious node (e.g. via clinical signs or screening) at rate a. This results in the isolation of the node and triggers the tracing of its contacts.
The removal of triggering nodes T/R. Triggering nodes are removed at rate d.
Multi-step contact tracing I/T. The infectious neighbours of triggering nodes (T ) can themselves become triggering nodes through contact tracing if they are found to be infected, creating a multi-step contacttracing chain that tracks the paths of disease transmission. Traced I nodes are not directly removed, but instead enter the T state at rate 4. Unless otherwise stated, we consider multi-step tracing.
Single-step contact tracing of infectious nodes I/R. Diagnostic tests are often necessary to determine the status of traced nodes (individuals); these may be imprecise or slow and the isolation and observation of traced individuals may not be a viable option. In such cases, multiple-step contact tracing is less likely. In an alternative single-step contact-tracing model, the infectious neighbours of triggering nodes are traced at rate 4 per contact. These are then directly removed and do not initiate further tracing. Control is always modelled through either multi-step or single-step tracing.
Undirected networks with different mixing patterns (assortative and disassortative) are generated using a method proposed by Newman (2003a) . The mixing pattern here is based on node degree (i.e. the number of links of each node). The level of mixing is given by the correlation coefficient of the 'excess' degrees (see below) calculated on all pairs of connected nodes. The generation of networks with different mixing properties is based on a Monte Carlo sampling scheme with repeated link switching at a probability determined by the values of the connectivity matrix EZe ij . Here, e ij is the probability that a randomly chosen link connects a node with i connections to a node with j connections where the link under consideration is itself not counted. Defining the distribution of the 'excess' degree (i.e. degree minus one) of vertices at the end of links as q k Z P j e jk , the level of mixing by vertex S T R I Figure 1 . Transitions among the four disease progression states. Contact tracing is represented by either the I/T transition (multi-step, dashed) or the I/R transition (singlestep, dotted), both with rate 4. Tracing occurs through either multi-step or single-step tracing. degree is given by
where s 2 q is the variance of the distribution q k . For disassortatively mixed networks K1%r!0, for random networks rz0 and for assortatively mixed networks 0!r%1.
Many real networks display a wide distribution of node degrees (Newman et al. 2001; Albert & Barabási 2002) . To reflect such systems, we consider networks that are generated according to an exponentially truncated power-law degree distribution
The function Li n ðxÞ is the nth polylogarithm of x and acts as a normalizing constant. The exponential cut-off of the scale-free distribution is determined by K. Following Newman (2003a), a value of gZ2.5 is used for the power-law exponent. The network analysis that illustrates the effect of preferential mixing is performed for KZ100. For this value of K, the average number of connections per node in the networks is hkiz1.7. To explore a range of parameter values that support meaningful epidemics (i.e. where the epidemic is large enough to be of a significant public health concern), hki must be sufficiently large to allow for a range of new infections in the first generation well above 1. Therefore, the networks are generated by only accepting nodes with kR3 (see Kiss et al. 2006b ). Moreno et al. (2003) proposed a numerical method for epidemic models that can account for connectivity correlations. While this method works without explicitly generating the network, it offers less flexibility when considering disassortatively mixed networks. Here, we use epidemic simulations on networks with NZ10 000 nodes and consider values of rZK0.2, K0.10, K0.05 for disassortatively mixed networks and rZ0.05, 0.10, 0.2 for assortatively mixed networks. The limits are chosen as the values over which the algorithm appears to be robust (M. E. J. Newman 2006, personal communication) ; this range of r values is sufficient to give marked differences in the epidemic threshold for the transmission rate, number of nodes traced, final epidemic size and to illustrate important trends in contact-tracing efficacy. A further check on the mixing pattern is illustrated in the electronic supplementary material by a plot of the average connectivity of a neighbour as a function of node connectivity for different values of r.
All simulated epidemics were seeded with 10 index cases chosen at random, in order to avoid early stochastic extinction. Averages of 10 000 simulations are presented, consisting of 100 epidemic runs on each of 100 different network realizations. Proportions across all plots are relative to the total network size NZ10 000. The simulation time step used is DtZ0.04. Smaller time steps produced effectively identical results (not shown). The s.e. values of the averages of simulation outputs are at least three to four orders of magnitude smaller than the measurement itself and therefore are not discussed further.
The giant component (GC) is the largest subset of nodes such that any two nodes from this subset can be connected by a series of links. For undirected networks, the GC size represents the upper limit for the potential size of an epidemic. In figure 2 , the structural differences of preferentially mixed networks are demonstrated by the size of the GC as a function of the cut-off parameter K for three different values of the assortativity coefficient r. As the cut-off parameter increases, the number of links in the network increases and the network becomes denser. A K-value exists where the GC size is equal on disassortatively and assortatively mixed networks. Consistent with previous results ( Newman 2002 ( Newman , 2003a , below this value, GC size is larger on assortatively mixed networks and above it, the GC is larger on disassortatively mixed networks, with GC size generally intermediate on random networks.
The two different regimes are a direct consequence of the mixing pattern. In assortatively mixed networks, nodes of high degree preferentially connect to each other and form a highly connected core group. Therefore, for low link density, a larger GC size is found than for either disassortatively mixed or random networks. Link density within the GC is higher than in the network as a whole. In contrast, in disassortatively mixed networks at low link density, the links are more dispersed, forming many isolated components of small size (figure 3). As link density increases, the probability that a link with one node in the GC will only connect to another node already in the GC also increases; this is a finite size effect that is exacerbated by assortative mixing since high-degree nodes are relatively few. Thus, at higher link densities, assortatively mixed networks have a smaller GC size compared with disassortatively mixed networks, as in the latter, added links are more likely to result in smaller components being absorbed into the GC (figure 2). 
The analysis of the GC growth and component distribution was performed on networks generated using gZ2.5 and kR1. For KZ100, the average number of connections per node is hkiz1.7. For random networks, the percolation transition occurs at hkiZ1, above which the network will support large epidemics, i.e. epidemics that scale with total population size (e.g. Moore & Newman 2000; Kao et al. 2006) . Thus, hkiz1.7 supports only a narrow range of epidemiological parameters over which large epidemics can occur. To circumvent this problem, the networks used below were generated using the same parameter values, but only nodes with kR3 were accepted during the network generation process , resulting in networks with hkiz6.
We define two variables T p and F for ease of reporting and comparing results. With per-contact transmission rate t and detection rate a, the transmission probability per link over the period before the detection of the infectious node is given by T p Zt/(tCa) (Keeling & Grenfell 2000) . Constant hkiT p provides a constant number of secondary infections caused by the introduction of infection at a node when the remainder of the network is susceptible (Keeling & Grenfell 2000) , at the cost of having different epidemic dynamics as hki varies ). In a parallel manner, with per-contact-tracing rate 4 and removal rate of triggering nodes d, the tracing probability per traceable link over the whole tracing triggering period is given by FZ4/(4Cd).
In figure 4 , in the absence of tracing, the final epidemic size R(N) is plotted against the transmission probability for r2{K0.2, K0.1, K0.05, 0, 0.05, 0.1, 0.2}. In this case, compartments T and R are equivalent and the current SITR model is equivalent to the well-known SIR model with an effective infectious period of 1/a. The relationship between R(N) and T p is qualitatively similar to the relationship between GC size and link density in figure 2. The epidemic threshold for assortatively mixed networks occurs at a lower transmission probability than for the other two mixing patterns. However, the final epidemic size approaches its asymptote, total network size, faster for disassortatively mixed networks. The two network types with rZK0.2 and 0.2 produce approximately equal epidemic sizes at tz0.0528 (transmission probability T p z0.15), when R(N)z0.225 on both types.
Epidemics on assortatively mixed networks have a faster initial growth rate and a shorter duration than those on disassortatively mixed networks (figure 5a). This is mainly due to the GC containing a 'core' group of high-degree nodes that are highly connected. The differences in the initial epidemic growth rate across the different network types are directly related to the basic reproduction number R 0 (Anderson & May 1991) . The value of R 0 can be estimated as the lead eigenvalue of the next-generation matrix CZ(c ij ) (Diekmann & Heesterbeek 2000) , and, in this case, it can be approximated by the product of the contact matrix and the per-link probability of transmission c ij ZT p M ij . A non-zero entry M ij Z1 denotes that an infectious node j can transmit the infection to a susceptible node i; the magnitude of c ij is given by the per-link transmission probability T p . Estimates of R 0 averaged over 100 generated networks of each type are as follows: R 0 Z12.12T p (s.e. 0.11) for disassortatively mixed networks; R 0 Z15.0T p (s.e. 0.11) for random networks; and R 0 Z17.81T p (s.e. 0.14) for assortatively mixed networks. As expected, the threshold for epidemic outbreaks occurs at lower infection probabilities for assortatively mixed networks than for either disassortatively mixed or random networks.
The differences in the initial epidemic growth rate and epidemic duration are likely to have consequences for the efficacy of contact tracing on the different networks. For example, on assortatively mixed networks with fast epidemic turnover, efficient contact tracing has to be comparably fast. The prevalence of traced nodes (dashed line) for assortatively and disassortatively mixed networks is illustrated in figure 5a . The average degree of newly infected (Barthélemy et al. 2004 ) and contact-traced nodes is plotted in figure 5b . While assortatively mixed networks sustain epidemics with fast turnover and quick initial growth rate, they also allow contact tracing to remove a larger number of highly connected nodes early on in the epidemic. In contrast, on disassortatively mixed networks, disease spread is slower, but contact tracing is also less efficient. Contact tracing can be viewed as an exploration of the local network structure (Cohen et al. 2003) , and thus we would expect its efficacy to depend on the mixing patterns of the network. This is investigated by varying 4, while keeping d fixed. For comparison purposes, we contrast the cases where R(N) is the same on both assortatively and disassortatively mixed networks, but possibly with differing transmission rates, later concentrating on the unique transmission rate that results in the same R(N) on both networks.
The final epidemic size R(N), the proportion of nodes that become triggering nodes via clinical signs or screening and the proportion of nodes that have been contact traced during the epidemic are plotted in figure 6 as a function of tracing probability F for disassortatively (rZK0.2) and assortatively (rZ0.2) mixed networks. First, transmission rates are chosen such that R(N)z0.73 on both networks (tZ0.125 and 0.175 on disassortatively and assortatively mixed networks, respectively; figure 6a ) and, second, the transmission rates are the same (tZ0.0528) with R(N)z0.225 on both networks (figure 6b). For the first case, the effect of contact tracing is similar on both networks with comparable R(N); however, we identify two distinct regimes above and below FZ0.61, above which contact tracing becomes more effective on disassortatively mixed networks (figure 6a).
In figure 6a , if F%0.61, R(N) is smaller on assortatively mixed networks. Contact tracing on assortatively mixed networks removes nodes of higher degree (figure 5b) than on disassortatively mixed networks. For small F values, the final epidemic size is still high and the population of highly connected nodes is depleted well before the epidemic ends, as shown by the crossover between the average degree of newly infected nodes on the two network types (figure 5b). Over the epidemic, susceptible nodes are on average of lower degree and are more difficult to reach. On disassortatively mixed networks, the depletion of highly connected nodes is less marked and epidemics persist, with disease spread alternating between highly and less well-connected nodes producing an average degree of newly infected nodes which is more even in time (figure 5b). In the latter stages of the epidemic, on disassortatively mixed networks, highly connected nodes are not completely depleted and can become infected. Thus, on the assortatively mixed networks, the epidemic ends earlier with a smaller final epidemic size. On assortatively mixed networks, when global depletion of susceptible nodes is important, contact tracing acts to enhance the early depletion of nodes of high degree (figure 5b). At higher tracing probability, the proportion of nodes removed through contact tracing (figure 6a) decreases with increasing tracing probability. This is indicative of effective control with a limited proportion of nodes becoming infectious and, hence, fewer targets for tracing. In the regime of more effective control (FR0.61), the epidemics die out early on and susceptible depletion becomes less important. This is illustrated in figure 7 , showing that the depletion of susceptibles is only found at low F values. This is also reflected in the very rapid reduction of R g (i.e. average reproduction ratio in generation g defined as the ratio between the number of nodes infected in consecutive generations) below 1 when F is high. This is a more general effect corroborated by examining contacttracing efficacy in a different parameter regime. In figure 6b , the scenario where tZ0.0528 on both networks is considered. For this value of t and in the absence of tracing, the final epidemic size is the same on both networks (R(N)z0.225). Here, the final epidemic size is similar to the point at which contact tracing starts to perform better on disassortatively mixed networks in figure 6a. In this case, for all F values, contact tracing always performs better on disassortatively mixed networks. The behaviour in this regime can be explained by interpreting contact tracing as a mechanism acting to reduce the effective transmission probability T p past the first generation of infection. Contact tracing achieves this by shortening the average infectious period of traced nodes. In figure 4 , lower transmission probabilities correspond to lower values of the transmission rate t. While we do not have exact analytic relationships between the implications of reducing the transmission probability through the different routes, the effect of contact tracing can be approximated by following the trend of the final epidemic size (figure 4) as the transmission probability decreases. The steeper curve of the final epidemic size on disassortatively mixed networks suggests that a small decrease in the transmission probability has a more marked effect on final epidemic size than on assortatively mixed networks. This effect is especially dominant since in the absence of tracing the same R(N) is observed on both networks. This supports the higher contact-tracing efficacy observed on disassortatively mixed networks.
Multiple-step contact tracing is not always logistically feasible. It is therefore important to determine the benefit provided by it. For both network types (figure 8), there are considerable differences between the final epidemic sizes for single-and multiple-step contact tracing at high tracing rates, particularly for assortatively mixed networks (figure 8b). In multiple-step tracing, a triggering node can generate other triggering nodes, potentially creating a cascade of triggering nodes throughout the infected portion of the network. This leads to tracing a higher number of infectious nodes and reducing the number of links that successfully transmit the disease. For high tracing rates, the epidemics are short-lived, and, on assortatively mixed networks, as a result of the faster initial epidemic growth rate, the extra proportion of untraced infectious nodes in the case of single-step tracing generates a higher number of infections and hence there is a marked difference between single-and multistep tracing. In figure 8 , the faster decrease in the proportion of traced nodes with increasing tracing probability indicates earlier and more effective control in the case of multiple-step contact tracing compared with the single-step case.
Contact tracing performs comparably well on both assortatively and disassortatively mixed networks. This is mainly explained by a balance that is reached between the epidemic time scale (i.e. slow for disassortatively mixed and fast for assortatively mixed; figure 5a) and the hierarchy of spread (figure 5b) on assortatively mixed networks (and the lack of it on disassortatively mixed networks) on the one hand, and the contact-tracing mechanism. On assortatively mixed networks, the epidemic spread is faster and the disease typically spreads to nodes with high degree. This is counterbalanced by contact tracing that removes infectious nodes with high degree. On disassortatively mixed networks, the epidemic spread is slower; however, owing to the connectivity pattern, contact tracing also alternates between removing poorly and highly connected nodes and therefore is comparably less effective. The higher average degree of traced nodes is a reflection of the average degree of nodes becoming infected earlier on in the epidemic when highly connected nodes are more abundant. This combined with the depletion of highly connected nodes, accentuated by finite size effects, leads to producing a higher average degree of traced nodes when compared with the average degree of infected nodes.
In the case of large epidemics and small values of the contact-tracing rate, contact tracing is more effective on assortatively mixed networks than on disassortative mixed networks, although the difference is small. Here, on the assortatively mixed networks, the early, global depletion of highly connected nodes results in a rapid increase in the proportion of susceptible nodes that are poorly connected nodes, and thus are less likely to become infected. For smaller values of the final epidemic size, the epidemics die out earlier and the depletion of susceptible nodes is less important. The efficacy of contact tracing in this case is determined by the more resilient nature of the assortatively mixed networks to the removal of potentially infectious links through tracing. In the case of singlestep versus multi-step contact tracing, the differences are more marked for assortatively mixed networks and small epidemics.
The algorithm used to generate networks with different mixing patterns is robust for mixing values in the range of K0.2 to 0.2. This range covers many of the values measured from networks based on real data (Newman 2002) . For more marked differences in the mixing pattern, we expect similar qualitative conclusions with possibly more significant quantitative differences. For the models presented above, differences in contact-tracing efficacy were investigated for various parameter values in addition to those presented. The results agreed qualitatively across the range of parameter values studied. However, further investigation is needed to determine the relative contributions of the different determinants of contact-tracing efficacy. The model presented here does not incorporate time or resource constraints for contact tracing. The implementation of epidemic control strategies often involves qualified personnel and costly or time-consuming diagnostic tests. Although contact tracing performs comparably well on both network types, the faster time course of the epidemic on assortatively mixed networks is more likely to stretch resources in real situations, since it requires a greater and more timely concentration of resources.
As in previous studies investigating the effects of contact clustering (Kiss et al. 2005 ) and degree distribution ) on contact tracing, we show that, unless contact tracing is very good, the mixing patterns have little effect on the course of the epidemic and the number of nodes removed. These results would suggest that it is difficult to exploit network structure to achieve better control via tracing. This may seem somewhat surprising, as previous studies have shown that control strategies such as acquaintance sampling (Cohen et al. 2003 ) that is based on the local exploration of the population contact structure provide an efficient epidemic control strategy, compared with random removal of nodes. However, such studies considered only networks that are randomly mixed and compare random removal with targeted removal (a form of contact tracing). Here, however, we compare targeted removal but where the networks themselves differ. Like the disease itself, contact tracing exploits the local network structure and, for assortatively mixed networks, identifies and removes early the highly connected nodes. This beneficial effect, however, is counterbalanced by the fast initial disease spread to such highly important nodes. Therefore, in the present case, the properties of the network can at the same time enhance disease spread and also increase control efficacy, highlighting the non-trivial interactions between the network structure and the dynamics on networks, showing that added attention is needed when evaluating the efficacy of epidemic control strategies. General Practice and Pandemic Influenza: A Framework for Planning and Comparison of Plans in Five Countries BACKGROUND: Although primary health care, and in particular, general practice will be at the frontline in the response to pandemic influenza, there are no frameworks to guide systematic planning for this task or to appraise available plans for their relevance to general practice. We aimed to develop a framework that will facilitate planning for general practice, and used it to appraise pandemic plans from Australia, England, USA, New Zealand and Canada. METHODOLOGY/PRINCIPAL FINDINGS: We adapted the Haddon matrix to develop the framework, populating its cells through a multi-method study that incorporated the peer-reviewed and grey literature, interviews with general practitioners, practice nurses and senior decision-makers, and desktop simulation exercises. We used the framework to analyse 89 publicly-available jurisdictional plans at similar managerial levels in the five countries. The framework identifies four functional domains: clinical care for influenza and other needs, public health responsibilities, the internal environment and the macro-environment of general practice. No plan addressed all four domains. Most plans either ignored or were sketchy about non-influenza clinical needs, and about the contribution of general practice to public health beyond surveillance. Collaborations between general practices were addressed in few plans, and inter-relationships with the broader health system, even less frequently. CONCLUSIONS: This is the first study to provide a framework to guide general practice planning for pandemic influenza. The framework helped identify critical shortcomings in available plans. Engaging general practice effectively in planning is challenging, particularly where governance structures for primary health care are weak. We identify implications for practice and for research. Primary health care, and in particular general practice, will be at the frontline in the response to pandemic influenza. Preparedness planning for this sector has lagged behind public health planning, despite evidence from SARS [1, 2] and influenza epidemics [3] of the important role played by general practice. Preparedness may be defined as the capacity to respond to a range of public health threats including natural disasters and infectious disease outbreaks, human-caused accidents and intentional attacks [4] . There is an increasing recognition of the need for an 'allhazards' approach to planning that integrates acute clinical care, public health, and emergency management systems [4] . Since September 2001, the US government has invested about $5 billion to upgrade preparedness plans for emergency management systems [5, 6] .
There are three challenges for pandemic planning by general practice. First, there is no systematic framework for planning this sector's response. Preparing for health threats and emergencies is an essential function of public health, but is not core business for general practice. Second, the way in which ambulatory health services will interact with each other and with the broader health system response to a pandemic is unclear. General practitioners (GPs) in Canada [7] , Australia [8] and the UK [9] have expressed uncertainty about how to participate in such a response. Third, planning and implementing changes for pandemic influenza across the health system is complex. Although there is little evidence linking specific preparedness activities to effective system-wide responses to pandemic influenza [5, 6] , change management theories point to a need for dynamic partnerships between general practices and other ambulatory care services, hospitals and public health departments [10] . The strength and structure of these linkages vary around the world, depending on decentralisation processes, the regulatory and legal system, and financing within health systems [11, 12] . Although general practice, or family medicine, is organised differently in different countries, there is considerable potential for transferable learning at the meso-level of management planning [11] .
We aimed to develop a framework that will facilitate systematic planning for the general practice response to pandemic influenza and used it to appraise coverage of key elements in publicly available pandemic plans from Australia, England, USA, New Zealand and Canada.
To guide planning and to appraise available plans, we adapted the Haddon Matrix, a planning tool developed in the field of injury research and intervention [13] , and more recently applied to the public health response to bioterrorism, SARS [14] , and pandemic influenza [15] . The matrix consists of a grid of columns of four factors (human, agent, and physical and organisational environment) impacting upon the event [15] . Pandemic influenza may be perceived as a form of injury on a mass scale and the matrix helps us understand the multi-dimensional nature of epidemics and of the associated challenges that could be expected by general practice. The framework can be readily shared with public health units and other parts of the health system, as it identifies the general practice contributions to primary health care services and to public health surveillance and control. Because all disasters are local, the matrix is flexible enough to allow a focused analysis of the smallest unit of study, such as an individual, or group of general practitioners.
The methods used to construct the cells of the modified Haddon matrix have been detailed elsewhere [16] . In brief, a team with expertise in social science, public health and general practice reviewed objectives and strategies in WHO guidelines for preparing and responding to a pandemic [17] to define the context and potential contributions of general practice. Next, we undertook a narrative review of the peer-reviewed and grey literature on pandemic influenza to identify papers that elaborated strategies relevant for general practice. A search of the peerreviewed literature through PubMed using the terms 'general practice', 'family physician', 'family medicine' and various combinations of the terms 'influenza', 'epidemic', 'preparedness' and 'pandemic' yielded 24 eligible papers from 157 search results . The process of constructing the framework and populating the cells was informed by organisational theories that emphasise multilevel approaches to change from the individual to the broader health system [10, 18] , and by methods for measuring [5] and improving the quality [6] of public health emergency preparedness.
We tested our framework through interviews with a purposive sample of health professionals engaged in pandemic planning. Nineteen general practitioners and practice nurses with expertise in pandemic planning were nominated by the two participating Divisions of General Practice, each of which was a national leader in disaster preparedness and response. Eight general practice policy leaders were identified by representative organisations (Australian Medical Association, Royal Australian College of General Practitioners, Australian General Practice Network). Group interviews were held with 14 state and territory public health leaders attending a national pandemic preparedness meeting. We held two workshops, attended by representatives of state and territory health services, Commonwealth policymakers, non-government organisations, and general practice organisations. In addition, we conducted two focus groups of GPs and nurses working in aged care in two cities. Finally, we undertook four desktop exercises [19] attended by 25 GPs, 11 practice nurses and 10 administrative staff.
The five countries in this study had national response plans. Contextualised detail about health-sector responses is contained in plans at the level of administrative decentralisation where decisions are made about patient-service groupings including general practice. In practice, this level was the state or provincial health departments in Federal systems where those jurisdictions have responsibility for health service management and planning (USA, Canada, and Australia). In England, the managerial level for health services is located at the Primary Care Trust (PCT), while in New Zealand it occurs at the level of the District Health Board. Although these are not identical loci of health service governance, they were sufficiently similar in the planning aims for comparisons to be drawn.
Plans were obtained from websites of health departments of states or provinces (USA, Australia, Canada), District Health Boards (New Zealand) and PCTs (England) ( Figure S1 ). For New Zealand and England, publicly available records of Board Meetings were also examined. Consumer information and isolated sub plans (e.g. for infection control) were excluded. Plans for 95 jurisdictions were identified; six were excluded as they addressed isolated aspects such as only the distribution of medications, or communication with the public, leaving 89 plans suitable for analysis.
Of the five countries, Canada exhibits the most variation between provinces in health system coordination. We examined the websites of Canada's 84 provincial regional health authorities (RHAS, 14 plans identified) and Ontario's 36 public health units (26 plans identified) and 14 Local Health Integration Networks (no pandemic plans identified). We excluded the RHA and public health unit plans from inter-country quantitative analysis, as their level of devolution and/or responsibilities for health management differed from those examined in the other four countries, but have included descriptive details from some of the RHA plans where they illustrate innovative approaches.
All plans were examined by two clinicians, and searched for the following terms: primary care, primary health, ambulatory, general practice, general practitioner, GP, family practice, family physician. The roles of general practice/family practice in the plans were assessed across the four domains of general practice identified in the first part of this project. No attempt was made to quantify the extent of coverage of general practice in the plans as this rarely extended beyond a few sentences. Where there was detailed coverage of an issue, we analysed the text and the health system context.
The study was approved by the Australian National University Human Research Ethics Committee and the National Research and Evaluation Ethics Committee of the Royal Australian College of General Practitioners. Written informed consent was obtained from participants.
A conceptual framework of the general practice response to pandemic influenza is shown in Table 1 .
The framework identifies four domains of practice: clinical services, public health responsibilities of general practice, internal (physical and organisational) environment of the general practice unit, and the macro-environment of general practice. In each domain, we list the key challenges to be anticipated by general practice during an influenza pandemic, and the type of responses that need to be addressed in the plan. Table 2 summarises the organisational levels in the five countries, the proportion of jurisdictions with accessible pandemic plans, and coverage of general practice in these plans. While almost all plans from US jurisdictions were accessible, three quarters of Australian states/territories and one third of New Zealand's District Health Boards had accessible plans. Only 13% (20/152) of England's PCTs had pandemic plans available in the public domain. Figure S1 shows the jurisdictions and health management systems whose plans were included in this study; they comprise 49 jurisdictions from the USA, 20 from England, 8 from Canada, and 6 each from Australia and New Zealand. Table 3 shows the number and rates of coverage of each of the four domains of the general practice response in jurisdictional plans of the five countries. The domain covered most frequently was influenza-related clinical care (in all plans from England and Canada). Overall less than half the plans mentioned non-influenza clinical care, with the exception being England, where 90% of PCT plans mentioned non-influenza clinical care. Public health surveillance was addressed in all plans from Canada and New Zealand and infection control in general practice in almost all plans from England and Canada. Functional linkages of general practice with other parts of the health system were addressed in almost all the English plans, but a smaller proportion of other plans. Clinical care
Essential planning elements. This domain includes two sets of clinical care needs. The first, prevention and treatment of influenza, includes care for the surge in patients with acute respiratory illness, and for people at high risk of exposure to, or complications from, influenza. These aspects are discussed extensively in the literature [20] [21] [22] [23] . Most people with influenza can be managed in the community, protecting hospitals by delaying or avoiding admission and facilitating early discharge.
The second clinical care need is for non-influenza-related care. General practitioners provide most chronic disease care, though there is inter-country variation in their capacities to do this efficiently [24, 25] . While activities like cervical screening may cease in a pandemic, chronic illnesses like diabetes or cardiac disease will still need management. Some acute care usually undertaken in hospitals, like acute asthma or injuries, may be transferred to the community. In an earlier paper, we advanced a range of models of practice to balance clinical services for influenza and non-influenza care [16] .
In the recovery phase, the clinical needs of patients are for psychological care and chronic illness management. If the pandemic occurs in waves, as in 1918-19, recovery activities may need to be tempered by preparations for the next wave.
Coverage of essential elements in plans. All Canadian and English plans outlined a role for general practice in clinical care for influenza. While only 41% of plans from the USA addressed clinical care for influenza by primary care practitioners (Table 3) , every US plan included guidelines on influenza management by hospital physicians. Some plans articulated a surge in demand for influenza care as a threat to general practice's survival, and proposed assessment and treatment clinics as a way of protecting them [26, 27] . In other plans [28] [29] [30] the response to a surge was to support general practices to become more resilient by collaborating and changing their work practices. In two US state plans, the failure of the ambulatory care sector in the face of a surge was assumed. The planning challenge became to find ways to redeploy workers into other health care sectors [31, 32] .
Most plans were sketchy on systems to maintain non-influenzarelated clinical care, with the exception of some PCT plans, which included activities like triage, extended prescribing, identifying deferrable reasons for presentation, and management of more acute problems to protect hospitals [29, [33] [34] [35] [36] . The main non-influenza clinical area was mental health care, mentioned in six plans from the USA [37] [38] [39] [40] [41] [42] (reflecting a focus in the national plan [43] ) and one Canadian plan [44] . Coverage of the needs of vulnerable populations-the elderly, homeless, prisoners and the psychologically unwell -was most detailed in plans from Canada and England.
Essential planning elements. This domain includes surveillance of influenza-like illness and influenza virology, and control of influenza in the general practice and the community. Surveillance includes early diagnosis and notification, and specimen collection to confirm clinical diagnosis and to monitor viral characteristics and resistance to antiviral drugs. GPs and private specialists are currently central to surveillance activities [45] [46] [47] [48] . In the early stages of the pandemic, it is likely that public health authorities will undertake contact tracing to facilitate containment, but their capacity to sustain this approach as the epidemic continues will be limited. General practice may then be expected to include contact tracing, and monitoring and support of people in quarantine or home isolation. Other responsibilities may include prescribing and dispensing antiviral drugs and participating in mass immunisations against the pandemic strain of the virus.
Coverage of essential elements in plans. Surveillance in general practice was mentioned in 53% of US plans and in only 33% of English plans, in all Canadian and New Zealand plans, and all but one Australian plan ( Table 3 ). The low rates of coverage of surveillance in PCT plans are not in accord with the UK plan which imputes to general practice a role in surveillance, and recommends that PCTs operationalise this recommendation [49] . The College of Family Physicians in Canada is a partner in FluWatch, recruiting sentinel physicians to undertake surveillance, so this role is well understood within the Canadian health sector.
The role of general practice in contact tracing, in monitoring people in home isolation, and in distributing antiviral drugs is unclear in most plans. Home care by GPs for people in quarantine is mentioned in two US Plans [50, 51] , and one English plan [36] , though the recently released guidelines for PCTs anticipate a role for general practices in home care [52] . In all country plans, dispensing antiviral medications was generally performed by public health units. Only 22% of PCT plans and 40% of US plans mention a role for primary care in dispensing antiviral medications. None of the Canadian plans, and only one NZ and two Australian state plans, mentioned antiviral dispensing by primary care. The only plan to set out contingencies when decisions about dispensing may change was one Canadian RHA plan [27] . Although immunisation was mentioned most frequently after surveillance as a public health activity by general practices, in most plans the immunisations were against pneumococcal disease and seasonal influenza, but not mass immunisations against pandemic influenza.
Essential planning elements. This domain includes the physical environment of the general practice and its practice-level organisation. The risk of transmission of infections within the surgery could be minimised through separate waiting rooms and entrances, triage and personal protective equipment and handwashing facilities. Hogg has outlined infections control procedures in the practice and the associated financial costs [53] . Some general practices (for example, those with small waiting rooms, or only one consulting room) may be deemed too much of a transmission risk to continue providing face-to-face services.
The practice needs to develop strategies to maintain reliable and efficient access to essential drugs and equipment and influenza and pneumococcal vaccines. It also needs to strengthen the capacity of its communication technologies with patients and the broader health system, including telephones, faxes, internet, work-from-home technologies for staff, compatible software for sharing electronic medical records, and recall and reminder systems for patients.
Preparation at the organisational level relates mainly to business continuity plans. These plans should include leadership delegations, staffing contingencies, safe and flexible working hours and family care plans for staff, criteria for considering clinic closure, recruiting and training ancillary staff, early psycho-social support, support for making difficult clinical decisions, record keeping to ensure accountability for actions and 'inactions', use of antiviral medications, and plans for simulation exercises to complement training, and to evaluate and refine local practice plans. Tools [54, 55] and desktop simulation exercises [19] are available to help GPs plan for continuity.
Coverage of essential elements in plans. Infection control strategies were well covered in plans from Canada and England, but were mentioned in only 39% of US plans ( Table 3) . None of the plans provided an inventory of fixed features, such as size and layout of waiting room, or a single entrance, which could compromise infection control.
Business continuity was a focus of the English plans, which frequently referenced resources available on the UK Resilience website [56] . This aspect of preparedness was enhanced after the Exercise Winter Willow simulation in February 2007, and new PCT guidelines addressing workforce planning [52] . Some PCT plans addressed the need for general practice resilience in the face of workforce sicknesses [33] , increased aggression from patients, and threatened loss of capacity in single doctor practices [57] . Few plans from other countries discussed business continuity for primary care in such detail. This may be because such issues are felt to be outside the normal purview of state or provinces, and to be the responsibilities of the businesses themselves or corporate interests.
Essential planning elements. This domain includes the overall organisation of, and interactions with, the health system that will facilitate or impede effective functioning of general practice services during a pandemic, including adaptation of relevant regulatory and financing systems.
The health system requires a plan that adopts the 'all-hazards approach' and integrates roles, responsibilities and actions for acute clinical care, public health, and emergency management systems [4] . This calls for coordination across general practices and other ambulatory care services to ensure primary health care needs within the community are effectively monitored and addressed; with hospitals to avoid/delay hospitalisation and facilitate early discharge; and with public health units to share responsibilities for contact tracing, monitoring and treating people in home isolation or quarantine, dispensing of anti-viral medications, and participation in mass immunisations against pandemic strains of the virus (when these become available).
Neighbouring general practices and other ambulatory care services will need local leadership with strategic approaches to collaborate and maintain services through a pandemic. England's PCTs and New Zealand's Primary Health Organisations (PHOs) represent two ways of linking general practices under the governance of regional boards. These networks are consolidated by financial relationships between the PCT or the PHO and general practices. The links between Australia's Divisions of General Practices and GPs are purely voluntary. In the USA, managed care systems function as another way of linking ambulatory and hospital services. Communication infrastructure between Canada's family practitioners, 25% of whom are solo practitioners [58], is still being developed, as is the incorporation of general practice into Canada's Pan-Canadian Public Health Network [59] .
The regulatory environment includes accreditation of retired medical practitioners and allied health professionals, laws and regulations which support or hinder the flow of qualified personnel across a jurisdiction's health facilities [48] , and ensuring an appropriate medicolegal framework to support clinical decisions on prioritising medical care during a pandemic, for example, modifying clinical standards, deferring treatment, and restricting access to certain treatments.
Funding mechanisms for general practice may impact upon the capacity to provide extra services. In countries with fee-for-service payment systems, general practices may profit from a surge in attendances, but may equally run into business difficulties if they are short-staffed for prolonged periods. GPs funded through a capitated system may have more freedom to alter their practice to provide different service mixes.
In the post-event phase, patients and GPs may require support for psychological recovery. It may be necessary to provide some formal relief through a system of locum GPs from areas less affected by the pandemic. Organisational partnerships at this stage may need to be with social services and mental health support services.
Coverage of essential elements in plans. Countries with mechanisms for linking general practices with other sectors were more likely to address networking in their plans. Ninety five per cent of English plans addressed systems to support collaboration between general practices (Table 3) . These plans addressed buddy systems, practice networks, and contingency plans for communities of practice. Four of the six New Zealand plans also addressed collaboration, though only one in significant detail; this plan outlined a distinction between key practices, and other practices which might decide to partner one another [55] . Of the three Canadian provincial plans that addressed collaboration, the most comprehensive was from Quebec, which identified a need to bridge the gap between salaried practitioners and independent physicians. The plan of the Montreal Regional Authority [60] operationalises this by setting up a system of active and sustained outreach by the public health department to independent physicians.
The absence of plans for networking between general practice and public health is most marked in the USA. With the exception of Louisiana [61] , US plans which mentioned networking did so in one line, generally advocating partnership between private and public services without indicating how this might occur. Louisiana's strategic approach built a participatory structure for rural practitioners through a partnership between the state public health department and the Bureau of Primary Rural Health Care.
The Canadian national pandemic plan [62] is framed around a set of ethical precepts incorporated into pandemic planning at the provincial and regional health level. The UK has recently released an ethical framework for policy and planning, though this has not yet been incorporated into planning documents [63] . The regulatory framework most mentioned was in relation to credentialing for retired GPs and other volunteers [33, 64, 65] , and less frequently, indemnity [36] . Although most plans include coverage of the relevant public health legislation, no country's plan included an inventory of legislation relevant to general practice that might need to be amended.
Only one plan [66] and the PCT guidelines [52] , canvas the potential of recompense for financial loss to a general practice. The only country in which the planning level coincided with the level that made decisions about funding of health care was Canada. One regional health authority plan provided an outline of specific issues likely to affect physicians, and raised the possibility of reviewing funding mechanisms in a pandemic [67] . There appear to be no ancillary plans addressing principles of altered funding for private physicians in a pandemic.
This is the first study to provide a framework that brings together multiple functions, structural relationships and the responsiveness of general practice to prepare for pandemic influenza. The framework provides clarity of purpose and a structure to guide planning through four functional domains: clinical care, public health responsibilities, and the internal and macro environments of general practice. The domains have been structured as integral components of a complex system that can respond to uncertainty [68] and be adapted for a given local setting and health system context.
We draw three conclusions regarding general practice from our analysis. First, none of the 89 jurisdictional plans addressed all domains of the general practice response during a pandemic. Second, while many aspects of the first three domains are included in plans for general practice, there are critical gaps and inconsistencies in the fourth domain (macro-environment) that render some elements of the jurisdictional plan ungrounded or unrealistic. Third, few plans addressed the broader ambulatory care context, including the need to engage private specialists and other allied health professionals [48] .
Planning and implementing change across the health system is complex. Targeting individual sectors for change (e.g. public health departments, hospitals or general practices) without securing reciprocal changes and strengthening inter-relationships across the health system, is unlikely to succeed [10, 18] . Planners must consider how connectivity across the health system might be strengthened to enable optimal use of general practice resources for planning [68] . While this may be challenging, particularly in countries with weak governance structures for primary health care, omitting general practice input into the planning process may be considered unethical [69] and counterproductive.
Limitations of the study: Our findings are exploratory rather than definitive, and indicate directions for further planning and research. Like any new tool, the framework and its application in a given context needs testing and refinement through simulation exercises targeting ambulatory care services as well as the broader health system.
Planning is an evolving activity that reflects a 'map' rather than a 'destination', and our findings provide a snapshot of the plans accessible in late 2007. The scope and content of the plans will change over time, as seen in two countries that adjusted their plans after simulation exercises, Exercise Cumpston in Australia [70] and Winter Willow in the UK [71] . Interestingly, the former identified specific weaknesses in the involvement of the primary health care sector and made recommendations to better integrate primary health care providers into planning at the national and jurisdictional levels [70] . National and sub-national pandemic plans may be intended to provide a strategic focus and not to elaborate on operational activities; it is possible the latter may have been addressed, but were not accessible at the time of our study.
Another potential limitation of our study is that the gaps we identified in many plans were grounded in theories about the ways to enhance the quality and outcomes of clinical care [10, 18] or of public health preparedness planning [6] . The science of preparedness planning is still maturing [4] [5] [6] and there is relatively little systematic evidence for linking specific preparedness structures to the ability to implement efficient and effective responses [5, 6] .
Two important limitations to the implementation of preparedness activities are uncertainties in knowing how much preparedness is enough [5] and in having a measurable assessment of the outcomes of preparedness activities. It may be more meaningful to perceive of the activities as a 'preparedness production system' in which a variety of processes and activities have been completed to prepare for an optimal response [6] . We are unable to comment on the extent to which these preparedness plans have been implemented, except in the case of those jurisdictions which have held pandemic exercises [70, 71] . General practice response is rarely tested in pandemic exercises, which tend to focus on hospital and public health responses. A notable exception is Operation Sparrowhawk in Singapore, where the feasibility of general practice influenza clinics was tested [72] The Haddon matrix is not a final check-list for preparedness planning but a problem-solving tool used as a starting framework for planning. The contents of each cell of the matrix help identify a particular problem or challenge that needs to be addressed. We recognise that the challenges will be neither static over time, nor uniform across general practices; responses will have to be modified in the context of the general practice setting as the pandemic evolves and as other parts of health system, particularly hospitals and public health units respond to the epidemic.
Implications of our study for primary health care in developing countries: Endemic and epidemic infectious diseases inflict high levels of morbidity and mortality in developing countries because of a combination of poor living conditions, effects of multiple concurrent illnesses particularly in children, fragile national health systems, overburdened and overstressed health workers, and negative work environments [73] . Although our study targeted general practice in developed countries, the conceptual framework we developed (Table 1) can be used by primary health care services in developing countries to deconstruct the multidimensional challenges posed by pandemic influenza. Identifying possible solutions and apportioning responsibilities across components of the health system is more complex. Operational guidelines have been developed for the detection and rapid containment of a potentially pandemic strain of influenza to the epicentre of the outbreak [74] , for example, if this were to occur in a South East Asian country. However, because of the immense global implications of such an event, this intensive strategy will need to be supported by extraordinary resources from the global community, an action not sustainable once the pandemic strain spreads beyond the initial epicentre.
In an analysis of pandemic influenza plans in Asia-Pacific countries in 2006, Coker found that although all countries recognised the importance of pandemic planning, operational responsibility particularly at the local level, remained unclear; most plans relied on specialised flu hospitals, while few developed the possibility of caring for patients at home [75] . (The study made no reference to primary health care or the private practice sector). In his analysis of public health emergencies in developing countries, Quarantelli identified relatively poor adaptive capabilities to be the key barrier to effective responses at the central and local levels [76] . Possible reasons included poorer public health infrastructures and human and financial resources, organisational structures that functioned mainly in a top-down manner with a strong emphasis on structures more than functions, and lack of planning initiatives the further away one moved from central level [76] .
Many poor countries already have a health crisis, and need massive international investments, including mobilisation and strengthening of human resources to build sustainable health systems, strong leadership and political commitment [73] . In the face of the pandemic threat, primary health care in developing countries will need resources to develop a suite of policies, including: clarification of what essential primary health care will continue through a pandemic, developing health workforce plans that may entail diverting clinicians from other areas of the health workforce, establishing non-hierarchical links between primary health care, hospitals and public health, and injecting funds into hospital and primary care preparedness simultaneously.
It may be argued that the absence of general practice elements from pandemic plans is not problematic, that it is outside the responsibility of public health departments that do not have a governance role for general practice. We argue instead that the general practice sector, which is characterised by loose networks between ambulatory care services, and often lacks the appropriate organisational structure and mandate, cannot spearhead many elements of planning for primary care. This calls for actions by health departments as well as by general practices.
Actions by health departments. Ensuring that the community receives appropriate health care during public health emergencies is a government responsibility. Consequently, health departments must emphasise in national and sub-national plans, the critical need for all levels of the health system to integrate the general practice sector in the planning process. This should include appropriate general practice representation in high level planning and decision-making committees, in incident-commandcontrol structures and in the management of community-based specialised clinics such as 'fever clinics' or 'community information and assessment centres'.
Good planning must focus on the planning process rather than the production of a written document [76] . The process includes collaborative activities such as meetings, drills, exercises, simulations, developing techniques for training, knowledge transfer, identifying and obtaining resource materials, and continually updating materials and strategies. These planning activities are important not only because they inform, but because they also foster collaborative learning and problem-solving, and generate an atmosphere of mutual trust and solidarity among people who will be affected by a pandemic and whose collaboration will be essential in the response.
The willing general practitioner sector [7, 8] is an essential resource for extending the surge capacity of health departments. Health departments should harness and support interactions and networking among general practices, and between them and ambulatory health care providers, hospitals and public health units. The role of general practice in contact tracing, monitoring and treating people in home isolation or quarantine, dispensing antiviral drugs and participating in mass vaccinations -omitted in most plans -needs to be clarified. In addition, health departments should modify or adopt where appropriate, legislation and financing mechanisms to enable general practices to function optimally during the pandemic.
Action to support planning by general practice. While the diversity of the general practice sector means that there will not be guidelines to cover all scenarios and contexts, a coherent approach would enable multi-actor accountability and more efficient, contextual planning by jurisdictions. The guidelines for PCTs [52] are an example of such an approach, designed for a particular health system. They could act as a useful point of departure for planning integrated general practice plans by other health systems.
There is a need for a system of sharing innovations and exemplary solutions to challenges for pandemic planning by general practice, analogous to those targeting mainly hospitals and public health departments [77] . Given the diversity in organisation of general practice systems, a web presence comparing exemplary approaches from different health systems would be a useful resource for planners.
An important challenge will be ensuring collaboration and coordination across the health sector during a pandemic. Research is needed to identify the prevailing barriers and facilitators to effective collaboration across the health sector, how these may change under the stressor of a pandemic, and how this information could be used to optimise the response.
The regulatory environment is founded on a set of ethical principles, often unarticulated. Since there is likely to be some dispute between utilitarian philosophical approaches used in public health and deontological or virtue ethical approaches used in clinical medicine [78] , there is a need for some preparatory work with general practitioners clarifying ethics of clinical behaviour, restriction of liberty under quarantine orders, and resource allocation and distribution.
In an established pandemic, it is likely that there will be shortfalls in the GP workforce, due to illness among GPs, caring duties or closure of small practices. Non-hospital clinical specialists, retired general practitioners, allied health professionals and medical students could be trained to fill the gap in services. Research is needed to define the clinical work that can be done by other health personnel in general practice, eligibility criteria and accreditation processes for this cadre of workers, and optimal training processes.
All public health problems have a clinical dimension, and all clinical problems have a public health dimension. At present, the plans in the five countries provide more detail on the public health dimension of the pandemic. There are intercountry differences in the emphases provided to different domains of the general practice response. Some of this reflects the emphasis on particular elements contained within the relevant national plan. Some of the differences are due to the ways in which general practice is structured in a country, and the strengths of its linkages to other components of the health sector. There is an urgent need to incorporate general practice and the broader primary care sector into pandemic planning activities, and to undertake the preparedness activities that would make this sector, which provides the majority of health care work, a true partner in pandemic response. Figure S1 Jurisdictions or health management organizations whose plans were included in the study. Found at: doi:10.1371/journal.pone.0002269.s001 (0.04 MB DOC) Apoptotic signals induce specific degradation of ribosomal RNA in yeast Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast. Gene expression in all organisms is regulated at multiple levels, including transcription initiation, mRNA stability and turnover, translation and protein degradation. Not surprisingly, rapid changes in cell metabolism and most responses to environmental stimuli involve significant flux of many cellular RNAs, of which mRNA transcriptome profiles have been most extensively studied to date (1, 2) . However, also stable RNAs, such as ribosomal, transfer, nuclear and nucleolar RNAs (rRNAs, tRNAs, snRNAs and snoRNAs), are likely to undergo specific transformations in altered conditions. It has been demonstrated that certain pathways of cell death are accompanied by the destruction of nucleic acids. For example, in metazoans the programmed cell death (PCD) called apoptosis, in addition to irreversible DNA damage, which is considered an apoptotic hallmark (3) , also involves specific cleavage of several RNA species, including 28S rRNA, U1 snRNA or Ro RNP-associated Y RNAs (4) . It was proposed that rRNA degradation could contribute to cell autodestruction, whereas degradation of anti-apoptotic factors mRNAs would accelerate apoptosis. In higher eukaryotes, RNA cleavage is probably carried out by RNase L, a 2 0 5 0 -oligoadenylate-dependent endoribonuclease, which functions in RNA decay during the interferon-induced response to viral infection, and whose activation in animal cells causes apoptosis (5) (6) (7) . However, RNase L-independent cleavage of 28S rRNA in virus infected cells has also been reported (8) .
The occurrence of apoptosis was assumed to be limited to metazoans, where elimination of single cells does not kill the whole organism. Nevertheless, recent studies revealed the existence of cell death pathway in yeast, Saccharomyces cerevisiae, and other unicellular eukaryotes, with typical hallmarks of apoptosis: DNA fragmentation, externalization of phosphatidyl serine and chromatin condensation. PCD in yeast is triggered by several different stimuli, including ageing, expression of mammalian pro-apoptotic proteins, exposure to low doses of H 2 O 2 , acetic acid, hyperosmotic stress and mating-type a-factor pheromone (9) (10) (11) . Unicellular organisms are believed to undergo PCD for a variety of reasons, including elimination of old, infected and damaged cells in growth-limiting conditions for better survival of the remaining population, and adaptation of the more fit subpopulation to the ever changing and challenging environment (12) . Orthologues of core regulators of mammalian apoptosis, such as the caspase-related protease Yca1, a homologue of mammalian pro-apoptotic mitochondrial serine protease HtrA2 (Nma111), a yeast EndoG nuclease Nuc1, apoptosis inducing factor (Aif1) involved in chromatin condensation, AIF-homologous mitochondrionassociated inducer of death (AMID) Ndi1 and an inhibitor of apoptosis (IAP) Bir1, are conserved in yeast (13) (14) (15) (16) (17) (18) . In addition, in both human and yeast cells, histone modifications (histone H2B phosphorylation at serine 14 in human and at serine 10 in yeast and H2B deacetylation at lysine 11 in yeast) play an important role in apoptotic chromatin condensation and cell death (19) (20) (21) . However, several apoptotic factors are missing in yeast, including the Bcl-2/Bax family and the apoptosis protease activator factor Apaf-1. Also, there is no good homologue of RNase L to execute possible RNA degradation. Nevertheless, yeast apoptosis has recently been shown to be activated in mRNA decay mutants (dcp and lsm) (22, 23) , supporting the notion that RNA metabolism and apoptosis are linked.
PCD occurs via a number of different mechanisms, e.g. caspase-dependent or independent, however, in all eukaryotes it is thought to be correlated with high levels of reactive oxygen species (ROS). ROS can either be generated exogenously through respiration or originate from exogenous sources such as exposure to hydrogen peroxide (H 2 O 2 ), superoxide anions or hydroxyl radicals. Excessive ROS results in damage of cellular components (DNA, lipids and proteins), cell cycle arrest, ageing and finally cell death (24) . Cells have developed a complex network of defence mechanisms, both enzymatic and non-enzymatic, against adverse consequences of oxidative stress (25) . Non-enzymatic system comprises a set of small molecules acting as ROS scavengers (e.g. glutathione, thioredoxin, glutaredoxin and ascorbic acid), whereas enzymatic system eliminates oxygen radicals by the action of specialized cytosolic or mitochondrial enzymes (e.g. catalases, superoxide dismutases, glutathione peroxidases and thioredoxin peroxidases) (25) . Most genes encoding components of these systems are induced in response to oxidative stress and are under transcriptional control of specific factors, for example Yap1, Msn2/Msn4 and Skn7 in budding yeast (26) . However, it appears that there is no general oxidative stress response. In S. cerevisiae, different response pathways are triggered by specific oxidants and different genes are involved in maintaining efficient cellular resistance to various sources of ROS (1, 2) . Interestingly, recent genomic approaches to identify these genes showed that strains lacking proteins which function in RNA metabolism were oversensitive to oxidative stress (1,2). These included genes encoding rRNA helicases (Dbp3, Dbp7), rRNA processing factors (Nop12, Nsr1), mRNA deadenylases (Ccr4, Pop2) and several mitochondrial RNA splicing components (2, 27) . This indicates that RNA processing and degradation may have a role in cellular response to ROS.
In this study, we examined the effects of elevated ROS levels generated by oxidative stress, ageing and other apoptotic-inducing treatments on the status of ribosomal RNA in yeast S. cerevisiae and we have shown that mature rRNAs become specifically fragmented as a result of the cell response to these conditions. RNA degradation coincides with fragmentation of chromosomal DNA but occurs considerably earlier and most likely upstream of the activation of major apoptotic regulators, Yca1 and Aif1. The existence of this mechanism underscores the role of gene expression, namely rRNA turnover, in regulating certain pathways of cell death, in this case most likely through destruction of ribosomes and subsequent inhibition of translation in the early stages of apoptosis.
Yeast strains and plasmids used in this work are listed in Supplementary Table S1 . The transformation procedure was as described (28) . Strains were grown at 308C either in YPD, YPGal or YPGly medium (1% yeast extract, 2% Bacto-peptone, 2% glucose or 2% galactose or 2% glycerol, respectively) or in synthetic complete medium (SC, 0.67% yeast nitrogen base, 2% glucose or 2% galactose, supplemented with required amount of amino acids and nucleotide bases). Strains W303-1A-Bax, W303-1A-Bcl-x L , W303-rDNA were grown in SC media without leucine, tryptophan or uracil, respectively.
Yeast cultures in early logarithmic phase (OD 600 $ 0.2) were stressed with H 2 O 2 (0.12-180 mM), menadione (0.05-0.6 mM), cumene hydroperoxide (CHP, 0.1-0.2 mM), tert-butyl hydroperoxide (t-BHP, 1-3 mM), paraquat (0.1-10 mM), diamide (0.2-2 mM) and linoleic acid hydroperoxide (LoaOOH, 0.05-0.2 mM) for 200 min (all reagents from Sigma). Treatment with acetic acid stress was performed as described (29, 30) . Cells were grown in SC media to early exponential stage, shifted to SC media, pH = 3, and treated with 175 mM acetic acid (Sigma) for 200 min. Hyperosmotic shock was achieved by growth of exponential cells in SC complete media containing 60% (wt/wt) glucose (Fluka) or 30% (wt/wt) sorbitol and 2% glucose (Sigma) for 2-8 h (31). Chronological ageing was performed by constant growth of yeast cultures in SC complete medium for 2-16 days (32) . For expression of murine Bax or Bcl-x L proteins, cells were grown to early exponential phase in SC-Leu or SC-Trp, respectively. Expression of murine Bax was induced by shifting the cells grown to early exponential phase in SC-Leu medium containing glucose to SC-Leu medium containing galactose for 6 h. Pre-treatment with ascorbic acid (20 mM, Fluka) and respiratory chain inhibitors oligomycin A (7.5 mg/ml, Sigma) and sodium azide (1.5 mM, Sigma) was performed for 30 or 60 min prior to treatment with H 2 O 2 . For inhibition of protein synthesis, cells were treated with 50 mg/ml or 200 mg/ml of cycloheximide (Sigma) for 30 min. Cell fixation was achieved by addition of formaldehyde (final concentration 1%) or EtOH (final concentration 70%) to exponentially growing yeasts and incubation in room temperature for 10 min or 60 min, respectively. Formaldehyde was quenched by addition of glycine to the final concentration of 0.4 M for 5 min. Following the removal of fixation agents, cells were exposed to H 2 O 2 for 200 min as described earlier.
Preparation of samples and analysis of chromosomal DNA fragmentation by pulsed field gel electrophoresis (PFGE) was performed exactly as described (30) . PFGE was conducted in a CHEF-DRIII Chiller System (Bio-Rad). One percent agarose gels were run in 0.5% Tris borate-EDTA buffer at 148C with an angle of 1208 with a voltage of 6 V/cm and switch times of 60-90 s for 24 h. Gels stained in ethidium bromide were analysed after destaining using Syngene Gene Genius Bioimaging System.
RNA extraction, northern hybridization and primer extension were essentially as described (33, 34) . Lowmolecular weight RNAs were separated on 6% acrylamide gels containing 7M urea and transferred to a Hybond N+ membrane by electrotransfer. High-molecular-weight RNAs were analysed on 1.2% agarose gels and transferred by capillary elution. Oligonucletides used for RNA hybridization and primer extension (W235 and W237) are listed in Supplementary Table S2 . Quantification of northern blots was performed using a Storm 860 PhosohorImager and ImageQuant software (Molecular Dynamics). Dideoxy-DNA sequencing was performed on PCR-templates prepared from genomic yeast DNA using the same primers as for primer extension (W235 and W237) and a fMolSeq kit (Promega) according to manufacturer's instructions.
The 3 0 RACE assay was carried out on total RNA (20 mg) isolated from untreated cells and treated with 1mM H 2 O 2 . DNA 'adaptor' oligonucleotide (W242) carrying aminolinker at the 3 0 -end was ligated with the 3 0 -end of total RNA using 20U of T4 RNA ligase (NEB). Ligation was performed in the presence of 25% PEG1000 (Sigma) at 378C. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.
RNase H cleavage was performed essentially as described (35) . Samples of 10 mg of total RNA were annealed with 40 ng of oligonucleotide complementary to the specific regions within rRNA at 688C for 10 min and digested with 1.5 U RNase H at 308C for 1 h. For detection, samples were separated on polyacrylamide gels and analysed by northern hybridization using probes located upstream of RHase H cleavage.
To examine the existence of the RNA degradation pathway in yeast under oxidative stress, we have performed treatments with low doses of oxidative agents generating different ROS. These included the inorganic H 2 O 2 (concentrations 0.4-3 mM), superoxide-generating menadione (concentrations 0.05-0.6 mM), paraquat (concentrations 0.1-10 mM), thiol oxidant diamide (concentrations 0.2-2 mM), organic CHP (concentrations 0.1-0.2 mM), t-BHP (concentrations 1-3 mM) and a LoaOOH (concentrations 0.05-0.2 mM). Such concentrations of oxidants result in 30-90% of cell death (2, 36, 37) .
Total RNA from wild-type W303 or BY4741 cells grown to early exponential phase (OD 600 = 0.25) in YPD media and treated with chemical compounds for 200 min was separated on 1.2% denaturing agarose/formaldehyde gels and analysed by northern hybridization using a probe complementary to the 5 0 -end of mature 25S rRNA (starting at position +40). This RNA species was chosen in the first place, since effects on 28S rRNA have been observed in apoptotic mammalian cells (8, 38, 39) . On treatment with two oxidants, H 2 O 2 and menadione, extensive decay of the mature 25S and accumulation of specific degradation products was observed, whereas little or no degradation occurred for other chemicals tested ( Figure 1A , data shown only for W303 strain and treatment with H 2 O 2 , menadione, CHP and t-BHP). This indicates that RNA cleavage accompanies some oxidative stress pathways, as it is known that different oxidants elicit specific cellular responses that, though partly overlapping, induce different groups of genes and require individual sets of specialized defence functions to maintain resistance (1, 2, 24) .
In addition to the 25S rRNA, other rRNA species were also probed for undergoing specific decay. After treatment with H 2 O 2 , RNA damage with accumulation of characteristic breakdown products occurred for 5.8S and, to a much lesser extent, for 18S, but not for 5S, (Figure 1B and C; data not shown). In the case of 18S, hardly any degradation intermediates were detected, there was some decay of the mature RNA, however, it was approximately 2.5 to 3-fold weaker than for the mature 25S. Therefore, we conclude that mainly the two components of the large ribosomal subunit, 25S and 5.8S, undergo specific apoptotic degradation.
The oxidants utilized, except for H 2 O 2 , had not been tested for apoptotic effects in yeast. One of the most recognized apoptotic markers is fragmentation of chromosomal DNA. Internucleosomal DNA laddering, typical for mammalian apoptosis, has not been detected during PCD in yeast; nevertheless, a higher order chromatin fragmentation to segments of several hundred kilobases also occurs in yeast (30, 40) . This DNA breakdown can be monitored either by the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay or by using PFGE of genomic DNA. The latter approach was applied to verify which oxidative agents lead to apoptotic phenotypes. Chromosomal DNA from cells treated with H 2 O 2 (1 mM), menadione (0.5 mM), CHP (0.2 mM), paraquat (5 mM), diamid (2 mM) and t-BHP (1 mM) for 200 min was analysed using PFGE ( Figure 1D ). Clear DNA degradation was observed only for cells exposed to H 2 O 2 and menadione, other treatments did not result in a visible apoptotic fragmentation. This is in a striking agreement with rRNA degradation that occurred only in H 2 O 2 -and menadione-treated cells. This strongly indicates that rRNA decay phenotype can be related to apoptosis.
To confirm this, we have examined other conditions known to provoke apoptosis in yeast, i.e. acetic acid, ageing and hyperosmotic shock ( Figure 1E -H). For treatment with acetic acid, cells were grown in SC complete medium (pH 3) to exponential phase and exposed to 175 mM acetic acid for up to 200 min (29,30). Hybridizations with probes against 25S (probe 007, position +40, lanes 1-6; probe W234, position +344, lanes 7-12; probe W236, position +600, lanes 13-18; probe W238, position +843, lanes 19-24; probe W239, position +2168, lanes 25-30 and probe W240, position +3323, lanes 31-36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3 0 product of the major cleavage, 5 0 product is marked with one asterisk. (B-C) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5 0 end of the mature 25S. DNA sequencing on a PCR product encompassing the 5 0 end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1-4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/. (D-E) 3 0 ends of cleaved-off products for the major cleavage at positions +610-611 were mapped by 3 0 RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated Hyperosmotic shock was achieved by growth of exponential cells in SC complete media supplemented with 60% (wt/wt) glucose or 30% (wt/wt) sorbitol (31) . And finally, chronological ageing was performed by constant growth of yeast cultures in SC complete medium for 2-16 days (32) . This analysis revealed that all apoptotic stimuli tested resulted in the 25S and 5.8 rRNA fragmentation with the degradation pattern specific for each condition (shown in Figure 1E -G for 25S in all apoptotic conditions and in Figure 1H for 5.8S during ageing). The accumulating intermediates generated by some factors were comparable (see for example, cleavages mediated by 60% glucose and 30% sorbitol, acetic acid and H 2 O 2 , Figure 1E and F); however, the general outcome of each treatment indicated differences in the course of events during each response.
The occurrence of RNA degradation triggered by H 2 O 2 was monitored during a time course between 5 and 60 min and over a broad range of concentrations (0.12-180mM for 200 min) ( Figure 1E , lanes 6-9 and Figure 2A ). Degradation was initiated relatively fast, since it was apparent at 5 min for 0.6 mM H 2 O 2 ( Figure 1E , lanes 6-9), 15-30 min for 175 mM acetic acid ( Figure 1E , lanes 1-5), 2 h for 60% glucose and 30% sorbitol ( Figure 1F ) and 3 days for ageing ( Figure 1G ) following the treatment. This onset of rRNA degradation distinctly precedes the timing of DNA damage characterized in apoptotic yeast exposed to the same stimuli (30) , indicating that RNA decay process is activated early during the response. Also, in the case of H 2 O 2 , low doses of the oxidant, starting with 0.12 mM and optimal at 0.4-3 mM, were sufficient to initiate rRNA degradation with the appearance of specific bands. When high concentration of H 2 O 2 (180 mM), believed to result in cell necrosis, was used, these specific degradation products were absent; however, the level of mature 25S and 18S rRNAs was also significantly reduced ( Figure 2A ; data not shown).
These data show that different ROS-generating treatments that lead to yeast apoptosis, namely H 2 O 2 and acetic acid, ageing and hyperosmotic shock, induce RNA fragmentation that most likely precedes the DNA damage and, as in higher eukaryotes, can be considered a hallmark of the induction of PCD in yeast.
Cleavages in the 25S rRNA are endonucleolytic and require cellular machinery Specific cleavages within the 25S rRNA generated in the presence of hydrogen peroxide were monitored by northern hybridization with probes located along the molecule to narrow down the regions to be further analysed ( Figure 2A ). This analysis showed the accumulation of diverse degradation products, some of which extended from the 5 0 -end of the molecule (Figure 2A, lanes 1-6) , whereas others were also truncated at their 5 0 ends ( Figure 2A, lanes 25-30) . The striking decrease in the level of the mature 25S rRNA at higher doses of the oxidant (1-3 mM) indicates that following specific cleavages the majority of rRNA becomes degraded, possibly by the exosome complex of 3 0 !5 0 exonucleases that participates in the decay of rRNA precursors and excised transcribed spacers (41) . The emergence of the characteristic cut-off in the signal at the fragment size corresponding to the position of the probe indicated that major cleavage sites are located around positions +400, +600 and +900 with respect to the 5 0 -end of the molecule. Two of these cleavages, at positions +610-611 and +398-403, were mapped for treatment with 1 mM H 2 O 2 by primer extension using primers W237 and W235 situated downstream of the expected cleavage sites ( Figure 2B ). Similarly, major cleavage sites were analysed in 16-day old chronologically aged cells using the same primers ( Figure 2C ) and mapped at positions +601-602 and +478-501. According to the secondary structure of the 25S rRNA taken from (42), the regions where mapped cleavages occur (shown in Figure 2B and C besides corresponding primer extension reactions) are located at unpaired nucleotides in loops or bulges. This points to the action of single-stranded RNA nucleases.
To establish the nature of the observed RNA fragmentation, 3 0 -ends of the products generated by the H 2 O 2mediated cleavage in the 25S at positions +610-611 and +398-403 were determined by the 3 0 RACE. To this end, DNA 'anchor' oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243). This served as a template to amplify products containing required fragments using the same 3 0 primer and a 5 0 primer that covers the 5 0 -end of 25S RNA starting at position +50 (W241). The ensuing PCR fragments ( Figure 2D ) were cloned into pGEM-Teasy and sequenced. The results of 10 sequenced clones for the cleavage at +610-611 and 19 clones for the cleavage +398-403 are shown in Figure 2E . In the case of the major site (cuts at positions +610-611), this analysis confirms that the 5 0 and 3 0 ends of this degradation product overlap ( Figure 2E , lower panel), which is consistent with the endonucleolytic mechanism of the cleavage. Mapping the 5 0 and 3 0 ends at site +398-403 by primer extension and 3 0 RACE produced a different pattern: these ends do not match ideally but the products with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an 'anchor' oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3 0 primer and the 5 0 primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398-404 (lower) and +610-611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3 0 RACE analysis for fragments cleaved at site +398-403 (19 independent clones) and site +610-611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. (F) Mapping 3 0 ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7D cells treated with 1mM H 2 O 2 (lanes, 2-4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3 0 ends of fragments cleaved at +398-403 (F-I) and at +610-611 (F-II), respectively. Arrows show more defined 3 0 ends of products cleaved at +610-611 for all strains and at +398-403 in the mutants; vertical bar in F-I indicates heterogenous 3 0 ends of products cleaved at +398-403 in wild-type cells. get progressively shorter pointing at the action of 3 0 !5 0 exonucleases ( Figure 2E, upper panel) . The most likely candidate is the exosome, a large complex with a 3 0 !5 0 exonucleolytic activity involved in the processing and degradation of mRNA, rRNA and other RNA substrates (43) . Mutants in the exosome core component Rrp41, the nuclear subunit Rrp6 and the cytoplasmic cofactor Ski7, were used to assess the status of the product 3 0 ends by a specific RNase H cleavage. This cleavage, directed by a DNA-RNA hybrid between oligonucleotide W244 and a complementary region in the 25S starting at residue +271, allows higher resolution of analysed RNAs. This analysis shows that products generated at site +398-403 in the exosome mutants rrp41-1 and ski7D, but not in rrp6D, are 3 0 extended and less heterogenous than corresponding fragments in the wild-type strain (Figure 2F-I; data not shown). In contrast, positions of cleavages at site +610-611 are not affected by mutations in the exosome (Fig. 2F-II) . This suggests that the cytoplasmic exosome may contribute to rRNA decay by digesting 3 0 ends of at least some cleavage products.
To ascertain that RNA degradation process is enzymatic and not chemically induced by various reactive compounds, yeast cells were fixed with 1% formaldehyde for 10 min or with 70% ethanol for 30 min prior to exposure to increasing concentrations of hydrogen peroxide ( Figure 3A and B) . Both fixation procedures preserve cellular structures, however, it is known that most fixatives have harmful consequences, e.g. cause some loss of cellular components, including ribosomes. Nevertheless, a similar approach had been used to demonstrate that DNA damage in apoptotic yeast cells was an enzymatic process (30) . Also, in the case of RNA, the appearance of specific H 2 O 2 -induced degradation products was prevented by fixation, although the overall level of rRNA was reduced. Some faster migrating RNA species were detected in formaldehyde or ethanol fixed cells, however, these were generated also in the absence of the oxidant and did not intensify after treatment ( Figure 3A and B, lane 4) .
Finally, to check whether rRNA destruction during apoptotic response is not due to cessation of translation in dying cells, cells were treated for 200 min with the translation elongation inhibitor cycloheximide (200 mg/ml) and this did not lead to an apparent rRNA degradation (Supplementary Figure S1A) . This is also supported by our earlier observations that exposure of yeast cells to many oxidative agents that cause cell death does not result in rRNA decay ( Figure 1A) .
Together, this strongly suggests that rRNA degradation observed in apoptotic and oxidative stress conditions is not simply a result of cell death but is produced in the process that requires enzymatic activity and functional cellular machinery. rRNA is most likely cleaved endonucleolytically and in some cases, dictated probably by the RNA structure, this is followed by exonucleolytic digestion by the exosome.
rRNA degradation is strongly correlated with ROS levels and is connected with oxidative stress response and apoptosis pathways Treatment with oxidative agents and apoptotic stimuli generate elevated levels of ROS in the cell. To test whether there is a direct link between RNA fragmentation and ROS, a potent ROS scavenger, L-ascorbic acid (vitamin C), was used (44) . The presence of 10mM ascorbic acid prior to treatment with standard doses of H 2 O 2 almost totally abrogated degradation of the 25S rRNA ( Figure 4A ). In addition, the ectopic expression of murine Bcl-x L protein of the anti-apoptotic mammalian Bcl-2 family, known to have a protective effect against ROS in yeast (45, 46) , also strongly safeguarded the 25S rRNA from rapid degradation by H 2 O 2 ( Figure 4B ). In contrast, expression of the mammalian pro-apoptotic Bax protein that increases ROS level (36, 47) , additionally enhanced the degradation phenotype ( Figure 4C ). This confirms the direct correlation between the production of ROS and the fate of cellular nucleic acids, leading not only to DNA but also rRNA damage and destruction of ribosomes.
From the data presented so far, it appears that the observed rRNA fragmentation may possibly represent a part of the cellular oxidative stress and apoptotic responses. This was assessed by testing the extent of the 25S rRNA degradation in different mutants defective in these pathways. In the first place, yca1D strain, lacking the only identified apoptotic metacaspase Yca1 in yeast, and aif1D cells not expressing the yeast apoptosis inducing factor Aif1 (13,15), were assayed for H 2 O 2 -induced RNA decay, however, no significant differences were observed (Supplementary Figure S1B and C) . Similarly, addition of a broad-range caspase inhibitor z-VAD-FMK (20 mM) that prevents Yca1-dependent cell death in yeast (13, 48) had no effect on rRNA fragmentation (data not shown). This indicates that rRNA degradation detected in all apoptotic conditions tested is independent of the two major apoptosis mediators, Yca1 and Aif1, and of other potential yeast caspases. Likewise, treatment with translation inhibitor cycloheximide, that has been shown to prevent apoptotic cell death induced by H 2 O 2 and acetic acid (29, 36) , had little or no effect on H 2 O 2 -mediated rRNA degradation (Supplementary Figure S1A) . However, it appears that events in the course of apoptosis that require protein synthesis are rather late, for example DNA fragmentation and chromatin condensation, whereas rRNA decay is initiated relatively fast. In contrast, different outcome was observed for mutants inhibiting chromatin condensation during H 2 O 2 -induced apoptosis. Phosphorylation of Serine 10 and deacetylation of Lysine 11, both in histone H2B, were reported to have an essential role for the progress of cell death in yeast (20, 21) . In agreement, S10A or K11Q mutations in histone H2B that prevent these modifications and abrogate apoptosis resulted in the significant reduction of the 25S rRNA degradation, both the decay of the mature rRNA and the amount of degradation products ( Figure 5A ). Together, these data show that the destruction of ribosomal RNA in cells treated with H 2 O 2 , and possibly with other apoptotic stimuli, is a part of a yeast cell death pathway that involves histone modification and not the caspase-dependent pathway. Remarkably, Nuc1-mediated apoptosis resulting from over-expression of yeast EndoG homologue Nuc1, a major mitochondrial nuclease, was also reported to be Yca1-and Aif1-independent and related to histone modifications (18) . Alternatively, it can be envisaged that damage of ribosomes triggered by apoptotic stimuli is an early event during the response, does not require protein synthesis, precedes caspase activation and acts as an upstream signal in the apoptotic pathway. This scenario is consistent with most observations so far.
As the oxidative stress in yeast proceeds through multiple pathways that involve different response mechanisms (1,2), we tested several known enzymes and factors that regulate these responses. These included two major transcription factors, Yap1 and Skn7, that control expression of several genes induced by oxidative stress and in this way participate in ROS sensing (1, 26, (49) (50) (51) , as well as components of antioxidant pathways, e.g. superoxide dismutases Sod1-2, glutathione peroxidases Gpx1-3, glutaredoxins Grx1-2, peroxiredoxins Tsa1-2, Prx1, Dot5 and Ahp1, thioredoxins Trx1-2 and thioredoxin reductases Trr1-2 (24,52). These enzymes are required for protection against ROS either by catalysing the breakdown of oxidative compounds or by restoring natural intracellular redox equilibrium. In addition, as glutathion protects cells against ROS, we also used a gsh1D strain lacking a g-glutamylcysteine synthetase, which, when grown on glutathion-free synthetic medium, leads to glutathion depletion and cell death (36, 53) . Strains lacking these proteins are more sensitive to several oxidants than their isogenic wild-types (2), and following treatment with H 2 O 2 they showed a marked increase in the 25S rRNA degradation, however, to different degrees depending on the mutant. In Figure 5B -D, yap1D, skn7D, gpx1/2/3D, sod1/2D, grx1/2-D, prxD (tsa1/2D/prx1D/ ahp1D/dot5D) and gsh1D strains are shown, which gave the most evident effects in comparison with their respective isogenic wild-types, particularly when considering the decay rate of the mature 25S rRNA. These data indicate that properly functioning oxidative stress response also protects cellular components such as nucleic acids from the attack by ROS and that defects at any step of this defence result in a more severe and faster breakdown. It is noteworthy that multiple anti-oxidant mutants lacking all components of each enzymatic pathway exhibit a stronger effect on the 25S degradation than single mutants, pointing to the additive protection actions of these systems. Striking effects on rRNA stability in strains lacking stress response transcription factors Yap1 and Skn7 indicate that the synthesis of new anti-oxidant proteins that are induced by oxidative stress might be required for protection of ribosomes. Consistently, blocking protein synthesis by pre-treatment with cycloheximide resulted in somehow stronger rRNA degradation (Supplementary Figure S1A, lanes 1-5 and 11-15 ).
Taken together, this indicates that targeting rRNA degradation during oxidative stress may directly contribute to cell death.
To test whether the level of rRNA, which reflects the amount of cellular ribosomes, may be somehow linked with cell survival under oxidative stress, we attempted to create a situation where the steady-state level of mature rRNAs will be increased or decreased. However, additional copies of rDNA present on a multicopy pNOY102 plasmid under control of the inducible GAL7 promoter (54) did not affect the amount of any mature rRNA species, possibly due to mechanisms that regulate ribosome abundance (data not shown). In contrast, the level of total genomic and plasmid-derived 18S and 25S rRNAs was, to our surprise, reduced to 70% in a strain transformed with the multicopy pJV12 plasmid expressing a tagged rDNA gene under the control of the constitutive PGK1 promoter (55) , when compared to a strain transformed with vector alone ( Figure 6A ). The basis of this effect is unclear, particularly that it was seen even though the tagged rRNA versions were expressed as confirmed by northern blots using probes specific for plasmid borne 25S rRNA ( Figure 6A ). Nevertheless, the strain carrying pJV12 showed a decreased viability already in the absence of oxidant (1.6-fold) and even more strikingly reduced following treatment with different concentrations of H 2 O 2 (4.6-fold for 0.6 mM and 6.24 for for 1 mM, respectively) relative to the strain with vector alone ( Figure 6B ). In another approach, we used the NOY504 strain, which carries a temperature sensitive (ts) RNA polymerase I (56) . At 378C, this strain ceases to grow but it sustains slow growth at 308C due to reduced levels of mature rRNA. Expression of additional copies of rDNA from pNOY102 or pJV12 plasmids improves growth at all temperatures and rescues the ts-lethal phenotype (55, 56) . Growth of NOY504 expressing additional rDNA under the control of GAL7 (pNOY102) or PGK1 (pJV12) promoters resulted in total rRNA levels lower by 15% in the latter case ( Figure 6C ). This relatively modest difference in rRNA abundance led to a 10% decrease in survival of cells exposed to oxidative stress ( Figure 6D ).
This indicates that there may exist a correlation between the quantity of ribosomal subunits and the capacity of the cell to elicit functional defence mechanisms and prevent cell death. It is possible that there is a feedback mechanism that controls this relationship: a healthy cell that contains an adequate number of ribosomes is able to respond more efficiently to stress stimuli to protect cell components from damage, including ribosomes themselves. Therefore, provided that the level of ribosomal RNA monitors cell fitness, its sudden reduction may act as one of the signals to initiate cell death mechanisms, including apoptosis.
rRNA degradation depends on the mitochondrial activity in the cell Mitochondria are the major source of endogenous ROS generated by oxidative phosphorylation. The extent to which mitochondria are involved in mammalian or yeast apoptosis is still questionable, although it appears that mitochondrial ROS could be important in some signalling pathways (57,58) and have a central role in some apoptotic pathways and less crucial in others (59) . For example, apoptotic cell death in yeast induced by acetic acid, pheromone and Bax expression was shown to be mediated by mitochondria (31, 60) .
The correlation between mitochondria and rRNA stability was assessed, in the first place, by checking rRNA level in respiratory-deficient rho0 cells lacking mtDNA in conditions inducing apoptosis, i.e. exposed to H 2 O 2 ( Figure 7A ), 175 mM acetic acid ( Figure 7B ), hyperosmotic stress (60% glucose, Figure 7C ) and during chronological ageing ( Figure 7D ). All treatments resulted in a remarkably robust degradation of the 25S and 5.8S rRNAs in rho0 strains when compared to the parental W303 and BY4741 strains (Figure 7 , Supplementary Figure S1E ; and data not shown). This points to the importance of the functioning mitochondria in the stressinduced rRNA degradation. To test the contribution of the oxidative phosphorylation, two mutants in these pathways were used, op1 with a point mutation in a major ADP/ATP carrier AAC2 (Arg96!His96), and a triple aac1/2/3Á deletion mutant (61) ( Figure 7E ). Both mutants behaved in a similar manner as rho0 cells and exhibited more pronounced rRNA degradation in the presence of H 2 O 2 than the isogenic wild-type; however, the phenotype was stronger for op1 than upon deletion of the three carrier proteins, possibly due to a dominant negative effect in the point mutant. In addition, treatment with F 0 -F 1 ATPase proton-pump inhibitors, oligomycin A (7.5 mg/ml) and sodium azide (NaN 3 , 1.5 mM) that block electron transfer and the synthesis of mitochondrial ATP (62-64), resulted in a moderate increase in the rRNA cleavage ( Figure 7F and Supplementary Figure S1D ). All these experiments indicate that the process of respiration, though generating the endogenous ROS, is also vital for counteracting its adverse effects. This was further supported by the degree of rRNA protection against oxidative damage caused by H 2 O 2 observed for yeast cells grown on different carbon sources, which are known to affect the level of respiration (65) . The most extensive RNA decay was observed in glucose, where a process called glucose repression discourages respiration. It was less pronounced in galactose and least of all in the nonfermentable source, glycerol, where mitochondrial respiration is forced ( Figure 7G) . These experiments directly correlate functional mitochondria and the process of respiration with the defence against oxidative stress triggered by H 2 O 2 , acetic acid, hyperosmosis and ageing that, among others, prevents destruction of cellular components, including rRNA.
Several cellular responses are regulated at the translational level, particularly by selective translation of specific mRNAs or by inhibition of the ribosome and protein synthesis, as these processes consume a large amount of energy. Such inhibition can follow various stimuli, including endoplasmic reticulum stress and unfolded protein response (UPR), transition into quiescence and different stress-related and cell death-related signals (66). The most straightforward and fastest way to achieve translation inhibition is to target ribosomal RNA. Interestingly, it has been proposed that repression of protein synthesis during UPR in human cells is due to the cleavage of 28S rRNA by hIRE1b, a second homologue of IRE1 (67) . Also, during apoptosis in some mammalian cells degradation of 28S rRNA by RNaseL, but also of other RNAs such as Y RNA or some mRNAs, has been suggested to block protein synthesis that contributes to, but could even initiate, cell death. Furthermore, damage to the 28S rRNA may act as a ribotoxic stress and induce an early death-committing signal through activation of SAP and MAP kinases (68, 69) . These possibilities were not examined in yeast PCD pathways. We have analysed the behaviour of ribosomal RNAs during oxidative stress and in apoptotic pathways that are induced by different stimuli. We have observed that cells exposed to all apoptotic conditions tested, such as H 2 O 2 , acetic acid, hyperosmotic stress (60% glucose) and ageing, reveal a significant degradation of the 25S and some of 5.8S rRNAs, with a much lesser effect on the 18S rRNA. The decay of mature rRNAs was accompanied by the accumulation of treatment-specific, yet partly overlapping degradation intermediates.
Although there is no evidence so far that rRNA damage during apoptotic conditions in yeast can directly initiate cell death by activating signalling pathways, it is tempting to speculate that this might be the case. Such signalling could be conveyed either by a critical decrease of the mature 25S rRNA or, alternatively, by the accumulation of degradation intermediates/products, which can function as signal molecules triggering a specific PCD pathway. In most cases, when ribosomal RNAs are depleted (e.g. in pre-rRNA processing mutants), rRNA degradation is conducted rapidly with no or little rRNA fragments detectable; however, lack of Lsm proteins has been reported to result in degradation of ribosomal RNAs with accumulation of unusual intermediates (70) . It is noteworthy that one apoptotic pathway that is linked with RNA metabolism is triggered by the defect in mRNA turnover caused by mutations in enzymes involved in 5 0 decapping, including components of Dcp1-2 and Lsm1-7 complexes (22) .
Each of the applied apoptosis-inducing conditions resulted in a clear-cut pattern of the 25S degradation intermediates or products. This points to the endonucleolytic nature of the reactions, though exonucleolytic destruction, with certain RNA fragments temporarily protected by compact structures or tight interactions with proteins, cannot be excluded. However, mapping 5 0 and 3 0 boundaries of the major H 2 O 2 -induced cleavage product by primer extension and 3 0 RACE, respectively, confirmed that both ends strictly overlap and thus result from the endonucleolytic cut.
It has been shown that apoptotic stimuli in yeast generate ROS that are closely correlated with the onset or progression of PCD (71) . We saw that, also the degree of rRNA decay corresponded to the cellular level of ROS, which was modified by using ROS scavenger (ascorbic acid) or ectopic expression of pro-or anti-apoptotic proteins (Bax and Bcl-x L , respectively) known to affect ROS generation. Also, rRNA degradation was more robust in oxidative stress defence mutants, both enzymatic and non-enzymatic, where intracellular ROS is not properly neutralized. All these observations argue that there is a direct link between ROS production and rRNA fragmentation. It can be envisaged that various reactive species themselves are able to produce endonucleolytic nicks in RNA molecules that will lead to breakdown, particularly as all mapped cleavages occur in singlestranded regions that constitute loops and bulges and are more accessible to chemical compounds in the solvent. However, this is not the case, given that specific RNA fragmentation did not occur in cells fixed with formaldehyde or ethanol prior to treatment with H 2 O 2 . In addition, oxidative agents used in this work generate different forms of ROS such as H 2 O 2 , hydroperoxide (LoaOOH, CHP and t-BHP), superoxide anion (menadione and paraquat) and hydroxyl free radical (produced from H 2 O 2 or superoxide anion). Although all were applied at toxic doses, only two of them, H 2 O 2 and menadione, mediated rRNA degradation, supporting the notion that oxidative compounds as such do not provoke cuts in RNA molecules within the cell. Taken together, this strongly suggests that active cellular machinery, such as signalling factors and RNA degrading enzyme(s), is required for this process. Nevertheless, these enzymatic activities are still to be identified. The closest yeast homologue of mammalian RNase L, Ire1, a sensor of the unfolded protein response, functions in the unconventional splicing of Hac1 pre-mRNA and contains protein kinase and endoribonuclease domains similar to those in RNase L (72) . Although our unpublished data show that deletion of Ire1 has no effect on the H 2 O 2 -induced rRNA degradation (M.S. and J.K.), it does not exclude its participation in other apoptotic pathways, for example those related to endoplasmic reticulum stress and unfolded protein response. We are currently testing several known yeast endo-an exonucleases for participation in apoptosis-related rRNA degradation. Preliminary observations suggest that, contrary to expectations, this function may involve not one but a number of unspecific nucleases, including mitochondrial Nuc1p, that act in a redundant fashion (M.S. and J.K., unpublished data).
The reason why treatment with some oxidative agents but not others produce fragmented RNA is not entirely clear, however, it is known that different chemicals induce specific responses leading to expression of distinct sets of genes (1, 2) , and possibly only a few activate pathways that involve RNA destruction. Our results suggest that rRNA degradation phenotype most likely accompanies apoptosis, and only apoptosis-inducing oxidants result in rRNA decay, whereas cell death caused by others probably occurs via a different mechanism. Another question is why each apoptotic stimuli resulted in slightly different set of cleavage products. Those which were mapped for treatment with H 2 O 2 and in aging cells illustrate that cleavages often occur in loops and bulges in closely located regions within the 25S rRNA, or rather within the accessible RNA elements in the compact RNP structures. The difference in cleavage patterns could be due to stress-induced subtle or severe alterations in ribosome particles that change the local accessibility of the rRNA components presented for cleavage. Alternatively, if rRNA decay is indeed carried out by more than one nuclease, these differences may reflect varying enzyme specificities in each death-inducing condition. It is also possible that somehow different set of nucleases is activated or recruited to rRNA substrates during apoptosis triggered by oxidative stress, acetic acid treatment and ageing.
Mitochondrial respiration protects rRNA against deleterious consequences of ROS Certain aspects of apoptosis, such as the change in mitochondrial membrane potential, fragmentation of mitochondria and the requirement of cyt c and AIF release to the cytoplasm, are strongly conserved among different organisms and point to the pivotal role of mitochondria. In yeast, PCD pathways triggered by acetic acid, Bax expression and pheromone, are strictly correlated with these events and do not proceed in cells devoid of mtDNA (rho0) (31, 60) . During chronological ageing and hyperosmotic shock, rho0 strains were reported to have somehow higher survival, which can be attributed to their long doubling time (1.5 to 2-fold), but they still die apoptotically (23, 73) . However, mitochondrial function is required for resistance to oxidative stress by way of detoxification or repair of the oxidative damage and, consequently, rho0 cells are more sensitive to several oxidants, have higher level of endogenous ROS and undergo apoptosis caused by H 2 O 2 or amino-acid starvation (61, 71, (74) (75) (76) (77) . Also, mammalian rho0 cell lines undergo apoptosis in response to some but not all cell death-activating stimuli. It has been postulated that these differences may arise from a distinct mechanism by which rho0 cells maintain membrane potential by way of ATP consumption (78) .
Our results show that, in all conditions tested, rRNA degradation is tightly connected with mitochondrial function and the active process of respiration. Rho0 cells that are less resistant to oxidative stress suffer severe rRNA degradation in the presence of H 2 O 2 , acetic acid and 60% glucose and during chronological ageing. Also, impediment of oxidative phosphorylation by protonpump inhibitors or by mutations in ATP/ADP carriers gives a similar outcome. In contrast, rRNA is markedly more stable when mitochondrial respiration is enhanced (e.g. by growth on glycerol versus glucose). This is consistent with the protective role of active mitochondria against the damaging effects of ROS on cellular components, rRNA destruction included. To begin with, several anti-oxidant enzymes localize to mitochondria and are more abundant in respiring cells. In addition, it has been proposed that some anti-oxidant activities may require energy (75) .
As some apoptotic pathways depend on the release of mitochondrial factors to the cytoplasm and are not induced in rho0 cells, this poses an important question regarding the link between rRNA degradation and apoptosis. rRNA degradation-apoptotic or not?
Although several apoptotic mediators (Yca1, Aif1, Nma111, Bir1 and Ndi1) have been identified in yeast, their networking in regulation of apoptosis is not yet fully understood. They may interact and function in a similar fashion as their mammalian counterparts, however, in contrast to mitochondrial mammalian proteins, Nma111 and Bir1 are located in the nucleus, so in yeast there might be some deviations from the mammalian model (13, 14, 71) . Nevertheless, apoptotic cell-death pathways in yeast induced by H 2 O 2 , acetic acid, hyperosmotic shock, ageing and increased mRNA stability were reported to require metacaspase Yca1, nominating them as caspasedependent pathways (13, 23, 31) . Still, deletion of Yca1 in the mRNA turnover mutant lsm1D does not attenuate mRNA decay, placing Yca1 action downstream of the signal rising from mRNA level (23) . In contrast, histone phosphorylation at Ser10, which requires prior deacetylation at Lys11, has been shown to mediate H 2 O 2 -induced apoptosis independently of Yca1 (20, 21) . Also, the activity of the major mitochondrial nuclease, Nuc1, in the celldeath pathway does not require either apoptotic mediators, Yca1 or Aif1, but is affected by H2B modifications (18) . Moreover, two PCD pathways, namely induced by defects in protein N-glycosylation and triggered by ammonia in multicellular yeast colonies, do not rely on Yca1 but on as yet unknown caspase-like activity (79, 80) .
Degradation of the 25S/5.8S rRNAs is observed in all conditions inducing apoptosis, however, this process is not dependent on Yca1 and Aif1. On the other hand, mutations in histone H2B that inhibit phosphorylation at Ser10 and block the progress of H 2 O 2 -induced apoptosis also severely affect rRNA degradation. More importantly, rRNA degradation coincides with apoptotic DNA fragmentation; from several compounds that lead to oxidative stress and cell death, only those that provoked DNA destruction also triggered rRNA decay. Furthermore, at least some apoptotic pathways strictly require the involvement of mitochondria and do not occur in yeast lacking mtDNA, whereas rRNA degradation in all stress conditions tested is more powerful in rho0 cells. To sum up: rRNA decay induced by apoptotic stimuli occurs during cell death pathway that involves ROS generation, mitochondrial activity, fragmentation of chromosomes and histone modification but not apoptotic regulators, Yca1 and Aif1. This could be due to the existence of numerous different but partly overlapping PCD mechanisms, whether caspase-and mitochondria-dependent or independent. Ever increasing numbers of such pathways has been described in the literature in recent years. However, we favour a different model, where all these elements function in concert at different steps of the whole scenario. Stress stimuli induce signals, possibly via ROS, affecting different levels of gene expression, namely chromatin modifications, transcription and translation, which as a result activate defence response. At this level, mitochondrial respiration helps to protect cellular components via adaptive mechanisms with anti-oxidant functions. Even so, when the attack is not successfully pacified, generated ROS molecules initiate the destruction of cellular machineries, targeting in the first place crucial elements such as protein synthesis (i.e. ribosomes). Now, the progression of the response comes to the crossroadsif the conditions are appropriate, the cascade of events leading to apoptosis is triggered (e.g. release of CytC and other mitochondrial factors to the cytoplasm) resulting in cell death with characteristic apoptotic markers. Alternatively, when apoptotic prerequisites are not met, cells do not enter this pathway. The more fit cells escape death, whereas others die anyway, maybe less rapidly and through a different pathway. In this scenario, certain events, including generation of ROS, histone modifications and possibly also RNA fragmentation, occur upstream of subsequent steps, such as activation of caspases and other apoptotic regulators with resulting apoptotic phenotypes. Virus Adaptation by Manipulation of Host's Gene Expression Viruses adapt to their hosts by evading defense mechanisms and taking over cellular metabolism for their own benefit. Alterations in cell metabolism as well as side-effects of antiviral responses contribute to symptoms development and virulence. Sometimes, a virus may spill over from its usual host species into a novel one, where usually will fail to successfully infect and further transmit to new host. However, in some cases, the virus transmits and persists after fixing beneficial mutations that allow for a better exploitation of the new host. This situation would represent a case for a new emerging virus. Here we report results from an evolution experiment in which a plant virus was allowed to infect and evolve on a naïve host. After 17 serial passages, the viral genome has accumulated only five changes, three of which were non-synonymous. An amino acid substitution in the viral VPg protein was responsible for the appearance of symptoms, whereas one substitution in the viral P3 protein the epistatically contributed to exacerbate severity. DNA microarray analyses show that the evolved and ancestral viruses affect the global patterns of host gene expression in radically different ways. A major difference is that genes involved in stress and pathogen response are not activated upon infection with the evolved virus, suggesting that selection has favored viral strategies to escape from host defenses. One of the first consequences of organisms' adaptation to new environments is the manipulation of resources [1] [2] [3] [4] . In this sense, the interaction between intracellular parasites and their hosts represents a paradigm of resource manipulation. In general, a virulent relationship results in the alteration of many aspects of cellular metabolism and development, which are taken over in the parasite's own benefit [5] [6] [7] . Whether the relationship between a host and a parasite evolves towards a more or less virulent or benign situation depends on several genetic and ecological factors that may affect virus accumulation and transmission between hosts [5] . Of particular interest in the context of emerging infectious diseases is the characterization of changes in the pathogen's genome that are responsible for adaptation to a new host after spilling over from the original one and to understand how these changes may alter host's metabolic and regulatory interactions.
High-density DNA microarrays offer an unparalleled view of the transcriptional events that underlie the host response to pathogens, providing a quantitative description of the behavior of tens of thousands of genes. In recent years, microarrays have been widely used to analyze the alteration of gene expression in host cells after infection with both animal [e.g., [8] [9] [10] [11] [12] [13] and plant [e.g., [14] [15] [16] [17] [18] viruses. However, a common drawback of these previous studies is that experiments were either done in cell cultures [8] [9] [10] [11] [12] [13] , which always represent an artificial and oversimplified environment, or using host-virus pairs whose previous evolutionary history of association is unknown and the degree of impact of abiotic environmental factor uncontrolled [14, 17] . Therefore, the relevance of these studies and, more importantly, their evolutionary implications for the problem of emergent infectious diseases, are rather limited. In the following, the results from an experiment simulating the emergence of a plant virus that crossed the species barrier and is horizontally spreading on a population of partially-susceptible hosts are reported. Evolutionary changes in viral genome and phenotypic properties and, more importantly, in the way it interacts with its host's transcriptome are the focus of the study.
The pathosystem Tobacco etch potyvirus (TEV)-Arabidopsis thaliana ecotype Ler has been chosen for the present study. TEV genome is composed of a 9.5 kb positive polarity single-strand RNA that encodes a large ORF whose translation generates a polyprotein that is subsequently self-processed by virus-encoded proteases into 10 mature peptides [19, 20] . TEV has a moderately wide host range infecting around 149 species from 19 families [21] , although most of its natural hosts belong to the family Solanaceae. In these plants TEV induces stunting and mottling, necrotic etching and malformation in leafs [21] . A. thaliana ecotypes vary in their susceptibility to TEV. Some ecotypes (e.g., C24 and Ler) are fully susceptible [22, 23] whereas many other (e.g., Col-0 and Ws-2) do not allow for systemic movement but support replication and cellto-cell spread in inoculated leafs [22, 23] . Arabidopsis is a member of the family Brassicaceae, which belongs to a different order than the Solanaceae within the class Magnoliopsida [24] . Therefore, adaptation of TEV to A. thaliana represents a jump in host species at the taxonomic level of orders.
TEV adaptation to A. thaliana: phenotypic changes
The ancestral TEV was poorly adapted to A. thaliana Ler and infection concurred with the development of very mild symptoms ( Figure 1 ). Furthermore, 21 days post-inoculation (dpi), the viral load in infected plants, measured as the number of lesion-forming units (LFU) produced per milligram of tissue, was low, 48.3362.95 LFU/mg (6SEM), and the infectivity of the newly produced viral particles (i.e., the efficiency of initiating a new infection using a normalized amount of viral particles) was as low as 17.95% [95% confidence interval (CI): 7.54-33.53%].
Viral particles obtained from a single tobacco plant were used to initiate an evolution experiment in A. thaliana Ler plants. Seven independent lineages were founded. Each lineage consisted on 10 plants. Twenty-one dpi, positive infections were confirmed by Western blot hybridization using an anti-coat protein antibody (data not shown). One of the infected plants from each lineage was randomly chosen to be the source of viral particles for infecting the next batch of plants. This basic transfer protocol was serially repeated every three weeks. In six out of seven cases, lineages went to extinction as a consequence of decreases in viral loads beyond the threshold value that ensures efficient transmission. The only surviving lineage was maintained for 17 serial passages (hereafter TEV-At17). The viral load reached by TEV-At17 21 dpi, was 2138.386134.08 LFU/mg. In other words, TEV-At17 accumulation was ,44-fold larger than the value estimated for the ancestral TEV (two-sample t-test, t 43 = 15.58, P,0.0001). Not only more viral particles were produced per gram of infected tissue, but also the infectivity of TEV-At17 was 100% (95% CI: 77.91-100%) and significantly larger than for the ancestral TEV (Binomial test, 1-tailed P,0.0001). Furthermore, symptoms induced by TEV-At17 were more severe (Figure 1 ), including stunting, vein clearing and leaf deformation.
TEV adaptation to A. thaliana: genotypic changes
The above phenotypic changes have a correlate at the genetic level. Full-genome sequencing of TEV-At17 indicates that six changes have occurred during adaptation (first six rows in Table 1 ); three of them were non-synonymous. The first non-synonymous change, A1047V, affected the P3 protein. P3 localizes in nucleus and nucleoli in association with the NIa protein and participates in virus amplification through its interaction with the CI protein [20] . In other potyviruses, P3 is also involved in systemic movement [25, 26] . The second mutation is a T1210M replacement in the 6K1 peptide. This short peptide has been implicated in plant pathogenicity since its deletion results in symptomless infections [20] . Finally, the third amino acid replacement observed is L2013F in the VPg domain of the NIa protein. VPg is covalently attached to the 59 end of the viral RNA and has essential functions in the viral replication and, relevant for the problem in hand, it has been reported as a key determinant in host-genotype specificity for systemic movement or replication [20] and it has been recently demonstrated that the proper interaction between the translation initiation factor eI4B and VPg is necessary for TEV infection [27] . In conclusion, these three mutations may explain the observed improvement in virus amplification and pathogenicity. The relevance of the three synonymous substitutions observed is not as clear, although their adaptive value cannot be ruled out.
To further characterize the relationship between these changes and symptoms severity, we introduced them by site-directed mutagenesis in the ancestral TEV clone. In addition, all three possible pairs of non-synonymous mutations and the triple non- synonymous mutant were also created. A. thaliana Ler plants were inoculated with these nine mutant clones and maintained in the same growth conditions for three weeks. The results of this experiment are summarized in Table 1 . All mutant genotypes were viable and replicated and accumulated in the plants, as confirmed by Western blot analysis (data not shown). Among the three single mutants, the only clone that produced visible symptoms was the one containing the L2013F allele in VPg. These symptoms were, nonetheless, qualitatively milder than those produced by TEV-At17 ( Figure S1 ). Concerning the three double mutants, only the combination of VPg and P3 substitutions induced symptoms that were qualitatively more severe than those produced by the single VPg L2013F mutant (Table 1 ) and almost as severe as those observed for TEV-At17. By contrast, mutation 6K1 T1210M does not have any effect on aggravating the symptoms associated with VPg L2013F. The combination of substitutions in P3 and 6K1 did not produce any symptom. Finally, the triple mutant recreated the strong symptoms characteristic of TEV-At17 (Table 1 and Figure S1 ). All together, these results suggest that the presence of substitution L2013F in the VPg protein is enough for triggering symptoms and that the severity of these symptoms is enhanced by the presence of substitution A1047V in P3, suggesting an epistatic interaction between these two mutations. The role of substitution T1210M in the 6K1 peptide remains unclear. It has been recently reported that the correct interaction between potyvirus' VPg and host's eIF4E is required to initiate a successful infection [27] . Recessive resistance of peppers to potyvirus depends on the substitution of relevant amino acid residues in eIF4E that disrupt the normal interaction between this translation factor and VPg. Resistance-breaking viral strains restore the normal interaction [27] . Therefore, we can hypothesize that TEV-AT17 has enhanced its ability to infect A. thaliana Ler by improving the interaction of its VPg with the host's translation initiation factor eIF4E.
Differential effect of evolved and ancestral viruses on the overall pattern of host gene expression Next, we sought to unravel what component of the plant gene interaction networks and metabolic pathways have been targeted by the virus during its adaptation to A. thaliana Ler. Our goal is not to identify single genes but rather global transcriptomic changes. Long-oligonucleotide microarrays representing almost all genes in A. thaliana genome have been used to this end. Five replicates were analyzed per experimental treatment (control mock-inoculated plants, and plants infected with TEV and TEV-At17) using a global reference experimental design. After quality analysis, a total of 13,722 spots, corresponding to 12,180 genes, were considered as valid for further analyses (Table S1 ). Data were normalized to the median expression of non-infected plants, and thus they reflect biological differences in gene expression in each sample analyzed. Statistical analysis allowed identification of genes whose expression responded differentially upon infection with either TEV or TEV-At17 ( Figure 2 ). When comparing global patterns of gene expression in plants infected with ancestral and adapted viruses, 496 genes showed higher expression and 1,322 genes lower expression in TEV-At17 infections than in TEV infections ( Figure 2 and Tables S2 and S3); which represents 2.7 times more down-regulated than up-regulated genes (Binomial test, P,0.0001).
Differentially expressed genes were grouped according to selforganizing maps (SOM) (Figure 3 and Table S4 ). Three global patterns of gene expression were observed among genes that were up-regulated by TEV-At17 infection ( Figure 3A ). The first pattern (SOMs A1 plus A2) corresponds to 130 genes whose expression was activated by both viruses but the magnitude of expression was magnified by TEV-At17. Genes belonging to this category include the pathogenesis-related protein PR1, which is well known to be a marker for the activation of salicylic acid-dependent defenses, such as the systemic acquired resistance (SAR) pathway [28, 29] . The second pattern (SOM A3) corresponds with 141 genes that were down-regulated after infection with TEV but showed expression levels similar to uninfected plants when infected with TEV-At17. The third pattern (SOM A4) corresponds to 234 genes whose expression was not significantly affected by TEV infection but show increased expression after infection with TEV-At17.
Three distinct patterns were also observed among genes downregulated after infection with TEV-At17 relative to the infection with TEV ( Figure 3B ). The first pattern (SOMs B1 plus B2) represents 683 genes that were over-expressed by plants infected with TEV but infection with TEV-At17 resulted in expression levels similar to those observed in uninfected plants. Interestingly, proteins related with disease response such as PR5 and several other PR-like proteins as well as four proteins of the TIR-NBS-LRR class [29, 30] belong to this category. The second pattern (SOM B3) includes 196 genes that were down-regulated after infection with both ancestral and evolved viruses, although the magnitude of down-regulation was larger for TEV-At17. Finally, the third pattern (SOM B4) corresponds to 456 genes whose expression was not affected by TEV but showed lower expression when TEV-At17 infected the plants.
The expression of transcription factors (TF) was also differentially affected by TEV and TEV-At17. Table S5 shows the list of differentially up-and down-regulated TF in plants infected with each type of virus. Fifty-one TFs, belonging to 20 families, were upregulated whereas 84 TFs, from 27 families, were down-regulated, including 13 ethylene-responsive binding factors (ERF), after infection with TEV-At. ERFs are linked to stress responses [31] and delays in ERF induction had been described in A. thaliana plants infected with virulent strains of the bacteria Pseudomonas syringae when compared with avirulent strains of the same bacteria [31] . Viral adaptation by avoidance of plant defenses Next, we examined the distribution of genes involved in related biological processes that are differentially affected by TEV and TEV-At17 (i.e., gene ontologies (GO) categories [32] ). The algorithm implemented in FatiGO [33] was applied to the nonredundant gene list grouped in each SOM (results are shown in Table S6 ). Only a significant differential category, response to salt stress, was identified for the SOM A3 of up-regulated genes shown in Figure 3A . By contrast, a large number of GO terms show significant over-and under-representation in the differentially down-regulated genes ( Figure 3B ). Table 2 shows the nonredundant functional categories that correspond to SOMs B1 plus B2 (i.e., genes over-expressed after infection by TEV but not differing from uninfected plants when infected with TEV-At17). Interestingly, significantly over-represented genes belong to functional categories which are related to plant responses to different abiotic (wounding, light intensity, temperature, salinity) and biotic stresses. Furthermore, genes involved in the SAR and in the activation of innate immune responses [29] were not expressed on plants infected with TEV-At17 while they were over-expressed on plants infected with the ancestral TEV, suggesting that the evolved virus acquired the ability to evade certain plant defense mechanisms, perhaps explaining the observed increase of viral load. Genes involved in basic cellular processes such as nucleic acid metabolism, translation and proteolysis were under-represented among down-regulated genes in SOMs B1 plus B2 ( Table 2 ), suggesting that the plant may be compensating for the consumption of these resources by an increased viral replication.
A single significant GO category was also found in SOM B3 of down-regulated genes ( Figure 3B ), that is, gene expression was repressed in presence of both viruses but to a larger extent when TEV-At17 was infecting plants. Genes involved in response to auxin were under-expressed to a larger extent by plants infected with TEV-At17 than with TEV.
We have shown that adaptation of a virus to a new host occurs by few changes in viral genome. The increase in viral fitness correlates with deep changes in the patterns of host's gene expression, illustrating that the subtle but dynamic interplay between the pathogen and the plant shifts as the virus adapts to its host. Under the experimental conditions imposed, it may be speculated that natural selection may had favored viral genomes that avoided plant defense mechanisms as suggested by the observation of stress-related genes not being activated after infection with the evolved virus (Table 2 ). Therefore, perhaps as a consequence, increases in the strength of symptoms, virus accumulation and transmissibility have been observed. These phenotypic changes are associated to a few genomic changes fixed in the viral genome. In particular, the development of symptoms is associated to a single substitution in the viral VPg protein, whereas ulterior mutations in other viral components simply magnify symptoms. Our starting hypothesis was that viral adaptation occurs throughout the integration of viral replication processes within host physiology and circuitry of genetic and metabolic interactions. Necessarily, this integration has to affect the patterns of host's gene expression. Our experiments directly test this hypothesis, supporting its validity and, furthermore, pinpointing some physiological processes that may be targeted by the virus as it improves its fitness. The obvious follow-up of this study is to dissect the physiological processes and identify, whenever possible, the precise steps and proteins that are getting targeted by the virus during its adaptation.
Serial-passage experiments simulating horizontal transmission are well known to produce increases on parasite's virulence due to enhanced within-host competition among pathogenic strains, the decoupling between intra-host growth rate and transmission rate, and the lack of evolutionary innovation in the host [34] . The outcome of a different experimental design in which transmission would be vertical, and hence making high virulence detrimental, or in which virus and host are engaged in a coevolutionary armsrace may produce different results; perhaps with the evolution of a less severe virus and different alterations in plant gene expression.
Finally, the findings here reported call for extra precaution when analyzing data from microarray experiments seeking for the effect of pathogen's infection on host gene expression: the pathogen effect on host's transcriptomic profiles would depend on the degree of adaptation of the pathogen to the host and to environmental conditions. Therefore, the only fully meaningful studies would be those in which pathogens and their experimental hosts would have an evolutionary history of association in the experimental growth conditions, whereas results from studies in which hosts are infected with naïve pathogens or the effect of environmental variables on pathogen's growth remain uncontrolled would be of very limited interest.
An infectious clone pTEV-7DA [35] (GeneBank DQ986288), kindly provided by Prof. J.C. Carrington (Oregon State Univ.) was used as ancestor virus. This infectious clone contains a full-length cDNA of TEV and a 44 nt long poly-T tail followed by a BglII restriction site cloned into the pGEM-4 vector downstream of the SP6 promoter. 59 capped infectious RNA was obtained upon transcription of BglII-digested pTEV-7DA using SP6 mMES-SAGE mMACHINE kit (Ambion). A stock of ancestral TEV viral particles was generated as follows. Five mg of RNA transcripts were rub-inoculated into the third true leaf of four-week old Nicotiana tabacum var Xanthi plants. Afterwards, plants were maintained in the green house at 25uC and 16 h light photoperiod. Seven dpi, virions were purified as described elsewhere [36] , aliquoted and stored at 280uC.
The viral load reached by replicating TEV populations in A. thaliana was estimated by the dilution-inoculation assay method on the local-lesion host Chenopodium quinoa [37] . In short, 2 g of tissue was ground in 1 mL of 0.5 M phosphate buffer. Four different leafs from each one of three different 4-week-old C. quinoa plants were rub-inoculated with 5 mL of undiluted, 5-and 10-fold diluted virus, respectively; 100 mg/mL carborundum were added to facilitate inoculation. Nine dpi, the number of local lesions was recorded and transformed into viral infectious loads (LFU/mg) by estimating the intercept of the regression line of the observed number of lesions on the dilution factor.
A. thaliana Ler seeds were obtained from Lehle Seeds (cat. # WT-04 18 01).
Seven independent evolution lineages of TEV were maintained by serial passages until extinction or up to 17 passages. All evolving lineages were initiated from the ancestral TEV stock population. Therefore, initial viral genetic variation among inoculated A. thaliana plants was minimal. To maximize transmission success, 10 plants were inoculated per lineage. Plants were inoculated between growth stages 3.5 and 3.7 [38] . Plants were maintained at 25uC and 16 h light photoperiod. Successful infections were confirmed by Western blot hybridization analysis 21 dpi using commercial antibodies anti-coat protein conjugated with horseradish peroxi-dase (Agdia). One gram of leaf tissue from a randomly-chosen infected plant per lineage were carefully ground in 1 mL 0.5 M phosphate buffer (pH = 8.0) and used to inoculate the next batch of 10 plants. Plants were always inoculated with similar viral doses.
The consensus full-genome sequence of TEV-At17 was obtained following standard methods. In short, RNA was extracted using the RNeasyH Plant Mini kit (Quiagen), it was reverse-transcribed using MMuLV polymerase (Fermentas) and PCR amplified with Taq polymerase (Roche). The ABI Prism Big Dye Terminator Cycle Sequencing Kit 3.1 (Applied Biosystems) was used for cycle sequencing with fluorescently labeled dideoxynucleotides. Cycle sequencing reactions were carried out on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). Labeled products were resolved in an ABI 3100 Genetic Analyzer (Applied Biosystems). Seven pairs of specific primers were used to amplify the 9.5 kb of TEV genome. The resulting fragments were overlapping, facilitating the task of fragment sequence assembly. Sequences were processed and analyzed with the STADEN 1.4b1. The 59-and 39-ends were sequenced by the RACE-PCR method [39] .
The seven mutant genotypes created in this study were generated by site-directed mutagenesis using the QuikchangeH II XL kit (Stratagene) and following the indications of the manufacturer. Mutagenic primers were also designed according to Stratagene recommendations. To minimize unwanted errors during the mutagenesis process, the kit incorporates the PfuUltra TM high fidelity DNA polymerase. The presence of the desired mutation was confirmed by sequencing. To assess the presence of undesired mutations on each clone, the Surveyor TM Mutation Detection Kit Standard Gel Electrophoresis (Transgenomic) was employed. All six mutant genotypes presented the expected genome-wide band pattern.
Total RNA was extracted from control and infected plants and used in an amplification reaction with the MessageAmp II aRNA Amplification kit (Ambion) following manufacturer's instructions.
Five replicates for each sample category were generated, and compared with a global reference, generated from an equimolar mix of amplified RNAs from each of the 15 plants. RNA from each individual sample, plus the reference, were amplified, and used for labeling. For each category, three samples were labeled with Cy5 and two with Cy3, and compared with the corresponding reversed-labeled reference mix. Long 70-mers oligonucleotide microarrays, provided by Dr. D. Galbraith (Univ. Arizona), contain 29,110 probes from the Qiagen-Operon Arabidopsis Genome Array Ready Oligo Set (AROS) Version 3.0. This oligo set represents 26,173 protein-coding genes, 28,964 protein-coding gene transcripts and 87 miRNAs and is based on the ATH1 release 5.0 of the TIGR Arabidopsis genome annotation database (www.tigr.org/tdb/e2k1/ath1/) and release 4.0 of the miRNA Registry at the Sanger Institute (www.sanger.ac.uk/Software/ Rfam/mirna/index.shtml). Further information can be found at the Operon website (omad.operon.com/download/index.php). Oligos were rehydrated and immobilized by UV irradiation. Slides were then washed twice in 0.1% SDS, 4 times in water, dipped in 96% ethanol for 1 min, and dried by centrifugation. Slides were prehybridized 30 min at 42uC with 100 mL of 66 SSC, 1% BSA and 0.5% SDS, under a 60622 mm coverslip LifterSlip (Erie Scientific) in an ArrayIt microarray hybridization cassette (TeleChem). Slides were then rinsed five times in H 2 O and dried by centrifugation. Slides were hybridized immediately. Labeled RNA was used to hybridize the slides basically as described in [40] . After hybridization and wash, slides were scanned at 532 nm for the Cy3 and 635 nm for the Cy5 dyes, with a GenePix 4000B scanner (Axon Molecular Devices), at 10 nm resolution and 100% laser power. Photomultiplier tube voltages were adjusted to equal the overall signal intensity for each channel, to increase signal-to-noise ratio, and to reduce number of spots with saturated pixels. Spot intensities were quantified using GenePix Pro 6.0 (Axon Molecular Devices).
Microarray raw data were deposited in the NCBI's GEO database under accession GSE11088.
Spots with a net intensity in both channels lower than the median signal background plus twice standard deviations were removed as low signal spots. Data were normalized by median global intensity and with LOWESS correction [41] using the Acuity 4.0 software (Axon Molecular Devices). Finally, only probes for which a valid data was obtained in at least 13 out of the 15 slides were considered for further analysis (13,722 spots; Table S1 ). Median, mean and standard deviations were calculated from each treatment (control, TEV-and TEV-At17-infected plants), and all data were normalized to the median of the expression in control samples. To detect differentially expressed genes in plants infected with TEV-At17 compared to TEV, data were analyzed with the SAM package [42] , using two-class comparison (TEV versus TEV-At17) with a false discovery rate (FDR) of 5.38% with no fold-change cut-off. Differentially over-and under-expressed genes were grouped in 262 self-organizing maps (SOMs) [43] using Acuity with Euclidean squared similarity metrics. Gene lists were further analyzed with FatiGO [33] to find differential distributions of gene ontology (GO) terms between statistically differential genes in each SOM and the rest of genes in the microarray, with P values adjusted after correcting for multiple testing [33] . SAM analysis at 1% FDR gave qualitatively identical results, confirming their robustness to changes in arbitrarily-chosen statistical thresholds. Figure S1 Representative plants showing the symptoms induced by several of the viral genotypes described in Table 1 Table S4 SOM clustering of significant genes, both up-and down-regulated, between TEV-and TEV-At17-infected plants. Genes belonging to each of the eight SOMs in Figure 3 are listed on different spreadsheets, along with their annotation and mean expression data in control and in TEV-and TEV-At17-infected plants. Found at: doi:10.1371/journal.pone.0002397.s005 (0.48 MB XLS) Table S5 Transcription factors differentially expressed after infection with TEV and TEV-At17. A. thaliana transcription factors and other transcription regulators were mainly downloaded from arabidopsis.med.ohio-state.edu/AtTFDB/index.jsp, and collapsed with the significantly up-regulated (Table S2 ) and downregulated (Table S3) Table S6 Differential GO categories among differential genes grouped by SOM. FatiGO analysis was carried out for each SOM in Figure 3 . Differential categories were identified for downregulated genes in SOMs B1 plus B2 and B3 ( Figure 3B ) and in up-regulated genes in SOM A3 ( Figure 3A ). List1 includes the differential genes (gene name, number and percentage) belonging to each GO category, while List2 include the rest of genes in the same GO category represented in the microarray. Unadjusted and adjusted P values after correcting for multiple-tests are also indicated.
Found at: doi:10.1371/journal.pone.0002397.s007 (0.07 MB XLS) Deletion of human metapneumovirus M2-2 increases mutation frequency and attenuates growth in hamsters BACKGROUND: Human metapneumovirus (hMPV) infection can cause acute lower respiratory tract illness in infants, the immunocompromised, and the elderly. Currently there are no licensed preventative measures for hMPV infections. Using a variant of hMPV/NL/1/00 that does not require trypsin supplementation for growth in tissue culture, we deleted the M2-2 gene and evaluated the replication of rhMPV/ΔM2-2 virus in vitro and in vivo. RESULTS: In vitro studies showed that the ablation of M2-2 increased the propensity for insertion of U nucleotides in poly-U tracts of the genomic RNA. In addition, viral transcription was up-regulated although the level of genomic RNA remained comparable to rhMPV. Thus, deletion of M2-2 alters the ratio between hMPV genome copies and transcripts. In vivo, rhMPV/ΔM2-2 was attenuated compared to rhMPV in the lungs and nasal turbinates of hamsters. Hamsters immunized with one dose of rhMPV/ΔM2-2 were protected from challenge with 10(6 )PFU of wild type (wt) hMPV/NL/1/00. CONCLUSION: Our results suggest that hMPV M2-2 alters regulation of transcription and influences the fidelity of the polymerase complex during viral genome replication. In the hamster model, rhMPVΔM2-2 is attenuated and protective suggesting that deletion of M2-2 may result in a potential live vaccine candidate. A more thorough knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication are being studied as we develop a potential live hMPV vaccine candidate that lacks M2-2 expression. Human metapneumovirus (hMPV) infection can cause acute respiratory illness in young infants, the immunocompromised, and the elderly [1] [2] [3] . HMPV infection has been detected in 4 to 15% of pediatric patients hospitalized with acute lower respiratory infections [4] [5] [6] [7] [8] [9] [10] . Cur-rently there are no licensed measures to prevent hMPV disease.
Based on analyses of genomic sequences hMPV has been assigned to the metapneumovirus genus of the pneumovirus subfamily within the paramyxovirus family [11, 12] .
The genome contains 8 transcription units with at least 9 open reading frames (ORFs) that encode a nucleocapsid protein (N), a matrix protein (M), a phosphoprotein (P) that likely associates with the polymerase complex, a fusion glycoprotein (F), an attachment glycoprotein (G), a large polymerase protein (L), a small hydrophobic protein (SH), and two proteins (M2-1 and M2-2) encoded by overlapping ORFs in the M2 gene. Among paramyxoviruses, SH is found in rubulaviruses and pneumoviruses, while M2 is found only in pneumoviruses. The functions of M2 proteins have not been studied extensively.
Mutants of hMPV have been constructed by deleting M2-1, M2-2, SH or G, either individually or in combination, using the CAN97-83 isolate of hMPV, which requires trypsin for growth in cell culture [13, 14] . Recombinant hMPV lacking either M2-2 or G were attenuated and immunogenic in African green monkeys and have been proposed as promising vaccine candidates [15] . Such a suitably attenuated live hMPV is desirable because it would deliver the nearly complete set of viral antigens and closely mimic a natural hMPV infection.
To construct a rhMPVΔM2-2 virus that can replicate efficiently in Vero cells without trypsin supplementation, we engineered the M2-2 deletion in a different subtype A hMPV strain. This recombinant strain is derived from hMPV/NL/1/00, and contains F 2 /F 1 cleavage-enhancing mutations in the F gene, a property that could facilitate the testing and manufacture of potential live hMPV vaccine candidates [16, 17] . The impact of the physical deletion of M2-2 on hMPV replication, and genetic stability in tissue culture were evaluated. rhMPV/ΔM2-2 exhibited somewhat restricted replication in Vero cells, but was significantly attenuated in hamsters. Hamsters immunized with rhMPV/ΔM2-2 were protected from experimental challenge with wthMPV/NL/1/00. The deletion of M2-2 resulted in higher levels of viral mRNA transcripts in tissue culture, giving rise to aberrant ratios of genomic RNA to viral transcripts. In addition, previously unreported genetic instability was observed, resulting in a higher frequency of point mutations and random insertions of U nucleotides in poly-U tracts of the rhMPV/ΔM2-2 genomic RNA.
Recombinant hMPV harboring a deletion in M2-2 gene was recovered from rhMPV/ΔM2-2 cDNA. The M2-2 deletion was designed to preserve the native ORF of M2-1, which overlaps the M2-2 ORF by 51 nucleotides. The first 21 amino acids of the putative M2-2 protein and the entire M2/SH non-coding region (NCR) were maintained ( Figure 1A ). Recombinant rhMPV/ΔM2-2 was efficiently recovered. RT-PCR was performed on the recovered rhMVP/ΔM2-2 virus to confirm the presence of the M2-2 deletion.
In Vero cells, rhMPV/ΔM2-2 plaques were less than 50% the size of rhMPV plaques (Figure 2A) . A 4-day multi-cycle growth curve was performed in Vero cells, a cell-line used for production of live vaccines, to compare the replication kinetics of rhMPV/ΔM2-2 and rhMPV. Data for the replication curves of these viruses were collected from three independently performed infections. The peak titer of rhMPV/ΔM2-2 in Vero cells was 7.22 +/-0.16 log 10 PFU/ ml, which was not significantly lower than the 7.52 +/-0.29 log 10 PFU/ml titer achieved by rhMPV ( Figure 2B ). However, the plaque size of rhMPV/ΔM2-2 was markedly diminished compared to rhMPV. Thus, hMPV M2-2 is dispensable for replication in Vero cells.
During the preparation of viral stocks, we noted several mutations in rhMPV/ΔM2-2. To further assess the genetic stability of rhMPV/ΔM2-2, one-step RT-PCR was performed on a virus stock that was serially passaged 4 times in Vero cells. Sequence analysis of an RT-PCR product spanning the M2 and SH genes (nt4536 to nt6205) revealed nucleotide polymorphisms in several poly A tracts (sense direction) in the M2-1 and SH genes. Figure  3A shows a representative chromatogram of the sequence of an RT-PCR product generated from a rhMPV/ΔM2-2 virus stock. The wild-type sequence AGAGAAACTGA 6 TT is shown overlapping another sequence containing an inserted A in the poly A 6 tract. Three independently derived virus stocks of rhMPV/ΔM2-2 had major subpopulations with inserted A nucleotides (nts) at nt5060, nt5166 or nt5222 in M2-1, each of which would cause a premature translation termination in the M2-1 ORF. (See figure 3D for numbering of A insertions). Subpopulations with inserted A's were also detected at nt5551 or nt5572 in SH that would result in premature translation termination in the SH ORF.
To compare the frequency of inserted A nucleotides in rhMPV/ΔM2-2 to that in rhMPV, RT-PCR products spanning nt4536 in F to nt5623 in SH were obtained from a passage 4 virus stock of rhMPV/ΔM2-2 or rhMPV. For this study, both positive sense and negative sense RNA were amplified using a one-step RT-PCR reaction. 1 kb RT-PCR fragments were inserted into pCR2.1 plasmids and 15 independent plasmids were sequenced. Surprisingly, 14 of the 15 (93%) cloned RT-PCR products of rhMPV/ΔM2-2 had an inserted A nucleotide at nt5060, nt5213 or nt5222 in the M2-1 gene ( Figure 3B ): there were 6 clones with insertion of A at nt5060, 2 with insertion at nt5213 and 6 with insertion at nt5222. Insertions of U, C or G Construction of cDNA for rhMPV/ΔM2-2, rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA Figure 1 Construction of cDNA for rhMPV/ΔM2-2, rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA. A) rhMPV/ΔM2-2 has a deletion in the M2-2 gene adjacent to a SwaI site. Nucleotides that were modified to introduce the SwaI site are underlined. Translational stop codons are bold and the intergenic (IG) sequence is bold italics. B) To construct rhMPV/GFPpolyA, an NheI site was introduced at the M2-2 stop codon of rhMPV and an NheI-N/P-GFP-polyA-NheI cassette was inserted. The modified nucleotides are underlined, the stop codon is bold and the IG sequence is bold italics. C) To construct rhMPV/ΔM2-2/GFPpolyA, an NheI site was introduced between the stop codon of M2-1 (bold) and the SwaI site (italics) in rhMPV/ΔM2-2 and an NheI-N/ P-GFP-polyA-NheI cassette was inserted. The modified nucleotides are underlined and the IG sequence is in bold italics. D) The reading frame of GFP is aligned with that of GFPpolyA to show the stop codon and frame shift resulting from the 11 nt insertion. were not observed. In sharp contrast, no A nucleotide insertions were detected in 15 cloned RT-PCR products derived from the identical region of rhMPV. Transitions, transversions, and deletions were also observed for rhMPV/ΔM2-2 in addition to insertions of A. For rhMPV/ ΔM2-2, 14 of 15 cloned RT-PCR sequences exhibited a total of 79 transition mutations, 2 transversions, and 1 deletion. Only 1 cloned sequence from rhMPV/ΔM2-2 had no nucleotide changes. In comparison, 7 of 15 cloned RT-PCR products of rhMPV showed a total of 17 transitions, 2 transversions, and 1 deletion. Eight cloned RT-PCR sequences from rhMPV had no nucleotide changes ( Figure 3B ). Thus, by passage 4, both rhMPV and rhMPV/ ΔM2-2 contained heterogeneous subpopulations and rhMPV/ΔM2-2 had a higher frequency of transition mutations and a propensity for insertion of A nucleotides in poly A tracts, compared to rhMPV.
To determine whether U insertions were present in the antisense genome, a two step RT-PCR was performed to specifically amplify only genomic RNA. Again, total RNA was extracted from a passage 4 stock of rhMPV/ΔM2-2 and the region from nt4536 in F to nt5623 in SH was amplified. 1 kb RT-PCR products were inserted in pCR2.1 and 15 individual plasmids were sequenced. All 15 cloned RT-PCR products contained an insertion of 1 or 2 T nts (antisense) in either the F gene (non-coding region), the M2-1 gene or the SH gene ( Figure 3C ). One sequence had an A inserted at nt4964 in the M2-1 gene. However, no insertions of C or G were observed. The 15 cloned RT-PCR products also contained 18 transitions and 4 transversions. Thus, there is a high frequency of U insertions in the genomic RNA suggesting that insertions were propagated in the viral genome. Whether the insertion events occurred during synthesis of the genomic or antigenomic RNA cannot be determined from these data.
We next examined the frequency of poly A and poly U tracts in the hMPV sequence spanning nt4536 to nt5623, to determine whether there is a bias between insertions of A or U. This region contains 14 poly A tracts and 3 poly U tracts with 4 or more contiguous A or U residues, respectively. Among the 15 cloned RT-PCR products amplified from the genomic RNA, 26 incidences of inserted A and 1 of inserted U were observed ( Figure 3C ). Thus, the data suggest a strong bias for insertions of A.
We also looked for insertions outside of the region that encoded the non-essential genes M2-1 and SH. RT-PCR was performed on rhMPV/ΔM2-2 and rhMPV total RNA to amplify the N/P, P/M, F/M2, SH/G and G/L non-coding sequences. There was a total of 23 poly A tracts and 2 poly U tracts with 4 or more contiguous A or U residues, respectively, among these sequences. However, no insertions of A were observed in the any of these non-coding sequences, showing that the high frequency of A insertions was predominantly confined to the region encoding the non-essential genes M2-1 and SH.
To investigate whether these insertions also occur in non-hMPV sequences, a GFP gene was inserted into the sixth gene position between the M2 and SH transcription units of rhMPV and rhMPV/ΔM2-2. An assay was developed to detect insertions, by designing a GFP ORF with an 11-nt sequence, CGA 6 TTA, positioned downstream of the first two GFP codons. This resulted in a frame shift in the downstream reading frame and a premature translational stop codon at the 6 th GFP codon, abrogating expression of GFP ( Figure 1B ,C and 1D). The modified GFP ORF is labeled GFPpolyA ( Figure 1D ). Insertion of a single nucleotide (or 4, 7, 10, etc.) in the A 6 tract of the CGA 6 TTA sequence would restore the translationally silenced GFP ORF, resulting in a fluorescent hMPV infectious focus. Four full-length cDNA's were engineered to recover
Growth of rhMPV and rhMPV/ΔM2 in Vero cells Titer (log 10 PFU/ml) rhMPV/GFP, rhMPV/GFPpolyA, rhMPV/ΔM2-2/GFP, and rhMPV/ΔM2-2/GFPpolyA viruses. Titers ranged from 6.6 log 10 PFU/mL for rhMPV/ΔM2-2/GFP to 7.3 log 10 PFU/ml for rhMPV/GFPpolyA and plaque sizes between all four viruses were similar ( Figure 4A ). However, rhMPV/ΔM2-2 and rhMPV/GFP plaques were both smaller than rhMPV plaques.
Vero cells were inoculated at MOI of 0.1 with rhMPV/GFP, rhMPV/GFPpolyA, rhMPV/ΔM2-2/GFP or rhMPV/ΔM2-2/ GFPpolyA as well as the control viruses rhMPV and rhMPV/ΔM2-2. Viruses were harvested on day 4 for Western blot analysis. The Western blot was probed for expression of hMPV F and GFP. Actin was also probed as a loading control ( Figure 4C ). The levels of hMPV F as detected by Western blot were considered equivalent among the GFP-viruses ( Figure 4B ). As expected GFP protein was detected by Western blot only in rhMPV/GFP and rhMPV/ΔM2-2/GFP, and not in rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA ( Figure 4D ). These data indi-Chromatogram and frequency of A insertions and point mutations in rhMPV/ΔM2-2 compared to rhMPV Figure 3 Chromatogram and frequency of A insertions and point mutations in rhMPV/ΔM2-2 compared to rhMPV. A) A chromatogram of the RT-PCR product derived from P4 of rhMPV/ΔM2-2, spanning nt4536 in F to nt6205 in NCR of SH, contained this sequence showing two subpopulations. One population is the correctly cloned sequence; the second population has one inserted A nt (sense direction) at nt5222 in the M2-1 gene. B) To assess the relative frequency of mutations, RT-PCR fragments spanning nt4536 in F to nt5623 in SH were obtained from rhMPV/ΔM2-2 or rhMPV using one-step RT-PCR, and were cloned into pCR2.1 plasmids. Among 15 independent plasmids the number of inserted As, single nt deletions, and point mutations (transition or transversion) for each virus were tabulated. 14 of the 15 (93%) rhMPV/ΔM2-2RT-PCR products had an inserted A (sense direction) nucleotide. No fragments containing A nucleotide insertions were detected in any of the 15 RT-PCR fragments spanning the identical region in P4 of rhMPV. C) To study frequency of mutations in genomic RNA, RT-PCR fragments spanning nt4536 to nt5623 were obtained from rhMPV/ΔM2-2 using two-step RT-PCR, and were cloned into pCR2.1 plasmids. Nucleotide insertions were predominantly T (genomic antisense direction), with one A, and were distributed among 8 locations in the fragments. D) To describe the position where insertion of an A was observed, the nt number of the last A in the poly A tract is used, though it is not known which A residue in the poly A tract is the inserted residue. The example shown is A inserted at nt5166. Gene: cate that insertion of the GFP cassette at this genome position was well tolerated by hMPV in vitro and insertion of the CGA 6 TTA sequences in the N terminus of the GFP ORF effectively silenced GFP expression.
To indirectly monitor A nucleotide insertions in GFP-polyA, Vero cells were inoculated with rhMPV/ΔM2-2/ GFPpolyA or one of the control viruses rhMPV/GFP, rhMPV/GFPpolyA or rhMPV/ΔM2-2/GFP, at MOI of 0.1, and viewed by fluorescence microscopy for 6 days. Fluorescence was readily observed throughout the monolayers Functional GFP expression in rhMPV/ΔM2-2/GFP6 poly A by A nucleotide insertion Figure 4 Functional GFP expression in rhMPV/ΔM2-2/GFP6 poly A by A nucleotide insertion. A) rhMPV and rhMPV/ΔM2-2 viruses containing the native GFP ORF or GFP-polyA sequences, harboring an engineered poly A tract that silenced the translation of GFP, formed comparable plaques in Vero cells. B) Western blots indicated F expression was comparable between viruses. C) A duplicate Western blot was probed with antibody directed to actin to serve as a loading control. D) GFP was detected by Western blot in viruses that contained native GFP ORFs. E) Fluorescence was robustly detected in viruses that contained native GFP ORFs, was readily detectable in some fluorescent foci in rhMPV/ΔM2-2/GFPpolyA, and was not detected in rhMPV/GFPpolyA. F) Nucleotide insertion of one A restored function of GFPpolyA ORF. Nucleotide insertion of 3 As would not restore functional GFPpolyA, but indicated heterogeneity at this polyA locus. of Vero cells infected with rhMPV/GFP or rhMPV/ΔM2-2/ GFP, but not in cells infected with rhMPV/GFPpolyA (Figure 4E ). Initially, no fluorescence was observed in cells infected with rhMPV/ΔM2-2/GFPpolyA. However, after two days, a few foci of fluorescent cells were observed in monolayers infected with rhMPV/ΔM2-2/GFPpolyA, suggesting that some cells were infected with GFP-expressing hMPV. One focus containing approximately a hundred infected fluorescent cells is shown ( Figure 4E ). The expression of GFP indicated that the reading frame of the GFP gene had been restored in some virions, and cell-to-cell spread within the focus of infection suggested that the restored GFP gene sequences were present in progeny virions. The low level of GFP expressed was only observable by fluorescence microscopy and not by Western blotting ( Figure 4D ).
To assess the frequency of insertions that restored expression of GFP, Vero cells in 96-well plates were inoculated with P2 stocks of rhMPV/ΔM2-2/GFPpolyA or rhMPV/ GFPpoly A. GFP expression was monitored by fluorescence microscopy 4 days post infection. Plates were inoculated with 1, 10, 100 or 1000 PFU/well (Table 1) . No GFP-expressing foci were observed in wells inoculated with either 100 or 1000 PFU/well of rhMPV/GFPpoly A (Table 1 ). In contrast, cells inoculated with 10, 100, or 1000 PFU/well of rhMPV/ΔM2-2/GFPpoly A developed fluorescent foci. Fluorescent multicellular foci were observed in 25 out of 384 wells (6%) inoculated with 10 PFU/well of rhMPV/ΔM2-2/GFPpolyA (Table 1) . At 100 PFU/well of rhMPV/ΔM2-2/GFPpolyA, fluorescence was observed in 65% of the infected wells (Table 1) . Finally, fluorescent multicellular foci were observed in 100% of wells inoculated with 1000 PFU/well of rhMPV/ΔM2-2/ GFPpolyA. Thus, this assay shows that at least one insertion occurs out of approximately every 17 infections at 10 PFU/infection and the frequency of insertions was significantly elevated in the absence of M2-2.
Viruses from 24 of the wells that exhibited fluorescence and that had been inoculated at a MOI of 0.1 were passaged once in Vero cells and each of the 24 viruses retained GFP expression. Total RNA was extracted from a mixture of cells plus supernatant and RT-PCR was performed to amplify a 1.5 kb fragment encompassing the GFPpoly A gene. The RT-PCR product was cloned into pCR2.1 and 8 individual clones were sequenced. 4 cloned GFP fragments contained the 11-nt CGA 6 TTA insert as constructed, 3 contained 1 inserted A that restored the reading frame of GFP, and 1 contained 3 inserted A nucleotides in the A 6 tract ( Figure 4F ). Thus, insertions of A nucleotides occurred frequently in non-hMPV sequences as well during rhMPV/ΔM2-2/GFPpolyA replication, suggesting that misincorporation of A nucleotides is not hMPV sequence-specific.
To further investigate the role of hMPV M2-2, we compared the transcription and genome replication of rhMPV/ΔM2-2 with rhMPV in Vero cells. First, we compared the amounts of rhMPV/ΔM2-2 viral transcripts with that of rhMPV by Northern blotting. Northern blot analysis was performed using hMPV-specific anti-sense DIGlabeled riboprobes to detect M2, SH, N, F, or G mRNA. At 24-hr intervals, RNA was extracted from Vero cells inoculated with rhMPV or rhMPV/ΔM2-2 at an MOI of 0.1, and Northern blot analysis was performed in 6 replicates. The M2 and SH riboprobes each detected two RNA species from rhMPV-infected cells (Lanes 1, 3, 5 and 7 of Figure  5A and 5B). The size of the minor species is consistent with the monocistronic transcript while the size of the major species coincided with the predicted size of the M2/ SH read-through product. No monocistronic M2 transcripts were observed at 24 or 48 hours post rhMPV/ΔM2-2 infection in Vero cells. The predicted M2/SH readthrough product showed a reduction in size in rhMPV/ ΔM2-2 infected cells consistent with the deletion of M2-2 (compare lanes 1 and 2 of Figure 5A ). The levels of bicistronic compared to monocistronic SH transcripts were higher in both rhMPV and rhMVP/ΔM2-2 infected cells, but the difference was more pronounced in rhMPV/ΔM2-2 infected cells ( Figure 5B ). This increased level of readthrough was unexpected since we had sought to preserve the native M2/SH noncoding sequences. One explanation could be that transcription termination at the M2 gene end sequences required nucleotides in the coding region of M2-2 that had been inadvertently removed and/or the M2 termination signal was altered by the introduction of the Swa I site. Next we compared the amounts of M2 transcripts in cells infected with rhMPV or rhMPV/ΔM2-2 at days 1 to 4 postinfection (p.i.). At day 1 p.i., the levels of transcripts were equivalent between both viruses (lanes 1 and 2 in Figure  5A ). By day 2 p.i., the relative levels had changed markedly. The amount of transcripts in cells infected with rhMPV/ΔM2-2 was several-fold higher compared to cells infected with rhMPV (lanes 3 and 4 in Figure 5A ). The upregulation was maintained up to day 4, when peak titers were observed (lanes 7 and 8 in Figure 5 ). More SH, N, F, and G transcripts were also observed in cells infected with rhMPV/ΔM2-2 compared to rhMPV ( Figure 5B ,C,D and 5E). Therefore, M2-2 deletion resulted in up-regulation of viral transcripts of genes upstream (N, F) and downstream (SH, G) of the M2 gene. However, the increased levels of viral transcripts produced by the rhMPV/ΔM2-2 mutant were not accompanied by an increase in virus titer. On days 2 and 3, rhMPV/ΔM2-2 had higher levels of transcripts but equivalent or lower titers compared to rhMPV ( Figure 5H) . Neither was there a concomitant increase in protein expression, at least for the F gene ( Figure 4B , lanes 1 and 2). Thus, the higher levels of viral transcripts produced by the M2-2 deletion mutant did not yield a greater number of infectious rhMPV/ΔM2-2 virions compared to rhMPV. We noted that the levels of rhMPV transcripts peaked at day 3 (lanes 1, 3, 5 and 7 in Figure 5 ), while the levels of rhMPV/ΔM2-2 transcripts remained the same on days 3 and 4 (lanes 6 and 8 of Figure 5 ).
RNA samples from day 4 were also probed for genomic (anti-sense) RNA using a mixture of three riboprobes directed to P, M and F genes. No significant differences were observed between the amount of genomic RNA in cells infected with rhMPV/ΔM2-2 and rhMPV (lanes 7 and 8, Figure 5F ). Thus, deletion of M2-2 altered the ratio between hMPV genomic RNA and mRNA.
Syrian Golden hamsters are highly permissive for hMPV replication and were used to assess the attenuation of rhMPV/ΔM2-2 [14, 18] . Groups of 8 hamsters were inocu-lated on day 0 with 10 6 PFU of wthMPV/NL/1/00, rhMPV or rhMPV/ΔM2-2. Both the recombinant viruses were P3 stocks. On day 4, titers of virus in the nasal turbinates and lungs were compared. As expected, the titers of wthMPV/ NL/1/00 and rhMPV in nasal turbinates and lungs were comparable (Table 2) . However, the titers of rhMPV/ΔM2-2 were 3.7 log 10 PFU/gm lower in the URT and 1.8 log 10 PFU/gm lower in the LRT, relative to rhMPV titers. Therefore, rhMPV/ΔM2-2 was approximately 10,000-fold and 100-fold more restricted in the URT and LRT, respectively, compared to rhMPV.
To determine if the lower level of replication in lungs and nasal turbinates of hamsters was sufficient to protect the hamsters from subsequent infection with hMPV, 4 hamsters were challenged with 10 6 PFU of wthMPV/NL/1/00 4 weeks post immunization. Four days post administration of the challenge, no virus was detected in either lungs or nasal turbinates of the immunized hamsters while unvaccinated animals had 5.6 +/-0.6 PFU/gm in URT and 4.5 +/-1.5 PFU/gm in the LRT (Table 2) . Therefore, replication of rhMPV/ΔM2-2 was restricted in hamsters and animals were protected from challenge with wthMPV/NL/1/ 00.
Using reverse genetics, we engineered rhMPV lacking the M2-2 gene with the aim of generating a potential vaccine candidate. rhMPV/ΔM2-2 grew to high titer in Vero cells, was attenuated in the respiratory tract of hamsters, and protected immunized hamsters from challenge with wthMPV/NL/1/00. These results agree with a similar study reported by Buchholz et al. in which a different subtype A hMPV strain, CAN97-83, with a deletion of M2-2 was proposed as a potential vaccine candidate [14, 15] . Our studies utilized the rhMPV/NL/1/00/E93K/S101P backbone which contained engineered mutations in the hMPV F gene that allows this virus to replicate efficiently in Vero cells without trypsin supplementation [17] . This property is expected to facilitate the testing and manufacture of potential live hMPV vaccine candidates. To assess the genetic stability of our M2-2 deletion mutant, sequence analyses were performed on P4 stocks of rhMPV/ΔM2-2. These analyses revealed major subpopulations (as high as 50%) that contained insertions of A nucleotides (sense direction) in the M2-1 and SH ORFs. These insertions appeared predominantly in A tracts and were also observed in non-hMPV sequences. Nucleotide insertions were also readily detected in an A tract introduced in the GFP ORF. Interestingly, insertions of A were not observed outside the region encompassing the nonessential genes M2 and SH. Transcriptional editing, whereby alternative reading frames of viral genes are accessed, has been observed in the P gene of several paramyxoviruses [19] [20] [21] [22] [23] . Therefore it is possible that an inserted A could occur frequently during transcriptional editing of paramyxovirus RNA. The nucleotide insertions observed in rhMPV M2-2 deletion mutants differ somewhat from transcriptional editing in that (i) the positions of inserted A nucleotides did not appear to be sequence biased beyond selecting for A tracts and is not hMPV sequence specific, and (ii) the nucleotide insertions were incorporated into the viral genome and could be propagated, as shown by passaging of fluorescent rhMPV/ΔM2-2/GFPpolyA viruses. Interestingly, these insertions did not appear to confer growth advantages in Vero cells because further passaging of rhMPV/ΔM2-2 promoted new A insertions and did not increase the subpopulations of earlier insertions. Many of the A nucleotide insertions caused premature translation terminations in the non-essential M2-1 and/or SH ORFs. These observations argue mechanistically against transcriptional editing and suggest that the insertions observed when M2-2 was deleted may be caused by an alteration in the fidelity of the replication complex directly or indirectly.
Removal of the hMPV M2-2 gene resulted in up-regulation of viral transcription, although there was no alteration in the level of genomic RNA. This had been observed previously for the respiratory syncytial virus (RSV) M2-2 gene as well as for hMPV [14] . Deletion of RSV M2-2 resulted in higher levels of viral transcripts compared to wt RSV. Based on these observations it was postulated that the RSV M2-2 is involved in regulating the balance between transcription and genome replication [24, 25] . Our observation that the levels of rhMPV transcripts peaked at day 3 p.i., while the levels of rhMPV/ΔM2-2 transcripts remained high through day 4 p.i. is also consistent with a higher level of viral transcripts in rhMPV/ ΔM2-2 infected cells. Thus, deletion of the hMPV M2-2, like its RSV counterpart, appears to cause aberrant regulation of viral transcription.
Comparison of monocistronic and polycistronic viral transcripts showed differences in the frequency of readthrough transcription at the M2 gene end sequences between rhMPV and rhMPV/ΔM2-2 infected cells. In RNA from cells infected with rhMPV, the M2 riboprobes detected a minor monocistronic M2 transcript and a major polycistronic M2/SH readthrough transcript. While transcription readthrough is not unique to the M2/SH intergenic region, the polycistronic readthrough transcripts at other noncoding regions such as N/P and F/M2 were less pronounced and monocistronic transcripts predominated. The genes immediately upstream and downstream of the M2 and SH transcription units also existed predominantly as monocistronic transcripts indicating that the M2 gene-end sequences are particularly prone to high frequency of readthrough transcription. The frequency of readthrough transcription at the M2 gene stop sequences appeared to be accentuated by the removal of the M2-2 ORF. This may in part be attributed to the sequences that were removed and/or altered by the introduction of a Swa I site at the proximity of the M2 gene end sequences. Nonetheless, the increased frequency of readthrough at this gene junction may perturb the expression of downstream genes such as SH, G and L. In rhMPV/ ΔM2-2 infected cells, there are major populations of M2-1 transcripts that contained premature termination codons introduced by the high point mutation frequency. Therefore, it is possible that M2-1 expression was significantly reduced during rhMPV/ΔM2-2 infection and this reduction in M2-1 expression may also contribute to the up-regulation of transcription and increased frequency of read-through observed.
Our results differ somewhat from that reported for the recombinant CAN97-83 strain of hMPV. Growth of recombinant rΔM2-2 CAN97-83 is trypsin-dependent and peak titer was not observed until 11 days post infection [14] . In contrast, our rhMPV/ΔM2-2 achieved peak titers at 4 days post-infection, a significant savings in production time. Interestingly, both ΔM2-2 viruses showed dramatic up-regulation of transcription at 48 hours post infection despite very different growth kinetics. No increase in the frequency of read-through transcription was observed for rΔM2-2 CAN97-83 whereas we observed increased polycistronic M2/SH transcripts in rhMPV/ ΔM2-2 infected cells. This may stem from differences in the construction of the M2-2 deletion. rΔM2-2CAN97-83 had a deletion of 152 nt in the M2-2 ORF whereas our construct had a deletion of 142 nt and a SwaI site introduced adjacent to the polyA tract of the M2 gene stop sequences. However, the ratio of polycistronic M2/SH transcripts to monocistronic M2 transcripts was significantly different even between the two wild-type hMPV strains, with the Netherlands strain exhibiting a higher frequency of readthrough at the M2/SH noncoding region than the Canadian strain.
Sequence analysis of rhMPV CAN 97-83, showed that mutations do develop in SH, G, L and non-coding regions (NCR), with a particularly high frequency in the SH gene [26] . While mutations and insertions were reported for rhMPV and rhMPVΔ G of the CAN 97-83 strain, the sequence analysis did not include viruses that lacked the M2-2 gene [26] . In a separate evaluation of rΔM2-2 CAN97-83, no increase in the frequency of point mutations was reported [14] . While the M2-2 proteins of both strains are completely identical, the SH protein of the CAN97-83 strain is only 83% identical to the NL/1/00 strain. There are also 26 amino acid differences in the L gene between the two strains. Finally, although deletion of the hMPV M2-2 ORF succeeded in attenuating both hMPV strains, the CAN97-83ΔM2-2 virus was more attenuated in hamsters than rhMPV/ΔM2-2 [14] . Clearly there are differences between the CAN97-83 and hMPV/NL/1/ 00 strains. Further study will be required to elucidate the differences in phenotype.
In summary, M2-2 plays an important role in the genetic stability of the hMPV genome. Silencing of M2-2 expression resulted in a greater frequency of hMPV subpopulations harboring insertions and point mutations.
Stabilizing the sequence of the rhMPV/ΔM2-2 genome by re-engineering all poly A tracts will only be partially effective because this does not address the increased frequency of point mutations. More studies are needed to gain detailed knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication. Ablation of the M2-2 ORF also resulted in up-regulation of viral transcription but not genomic RNA and increased the frequency of readthrough transcription at the M2/SH noncoding region. Aberrant transcription regulation and increased genetic instability could both contribute to the attenuation phenotype rhMPV/ΔM2-2. Further studies are being conducted to develop a potential live hMPV vaccine candidate. [2, 17, 27] . The following recombinant viruses were generated by reverse genetics from full-length cDNA plasmids using the rhMPV/NL/1/00/ E93K/S101P backbone: rhMPV/ΔM2-2, rhMPV/GFP, rhMPV/GFPpolyA, rhMPV/ΔM2-2/GFP, and rhMPV/ ΔM2-2/GFPpolyA.
The cDNA for rhMPV/NL/1/00/E93K/S101P was constructed as previously described [17] . To generate rhMPV/ ΔM2-2 cDNA, two Swa I restriction sites were introduced into the SphI/ClaI subclone, which contained the SphI (nt 100) to Cla I (nt8678) fragment derived from rhMPV/NL/ 1/00/E93K/S101P, using a Quik change mutagenesis kit (Stratagene). The first SwaI site was positioned at nt5326, down stream of the M2-1 stop codon, and was generated with the primer 5' CAGTGAGCATGGTCCAATTTAAAT-TACTATAGAGG and its complement. The stop codon of M2-1 is in bold and the Swa I restriction site is underlined. The second SwaI site was positioned at nt5468, upstream of the native stop codon of M2-2, and was generated using the primer 5' CATAGAAATTATATATGTCAAGGCTTATT-TAAATTAG and its complement. Digestion with Swa I resulted in the removal of 142 nts of the M2-2 gene. The Stu I (nt4495) to Cla I (nt8693) fragment in the subclone, containing M2-1, SH, and G genes, was transferred into the full-length rhMPV/NL/1/00/E93K/S101P cDNA to form rhMPV/ΔM2-2 as depicted in Figure 1A . rhMPV/ ΔM2-2 cDNA plasmid used to recover virus was sequenced from nt4495 to nt8693 to confirm that no unexpected changes were generated by PCR during cloning.
To construct the cDNA for rhMPV/GFP, a NheI-N/P-GFP-NheI cassette was constructed with the N/P noncoding region (NCR) upstream of the GFP gene. An NheI site was introduced into the hMPV SphI/ClaI subclone at nt 5474, located at the stop codon of M2-2, using the primer 5'GCTTACTTAAGCTAGCTAAAAACACATCAGAGTGG (NheI site underlined) and its complement. Following NheI digestion, the NheI-N/P-GFP-NheI cassette was ligated into the subclone. A StuI (nt4495) to ClaI (nt8693) fragment, containing M2, GFP, SH and G genes, was isolated from the hMPV SphI/ClaI subclone and inserted into the full-length cDNA to form rhMPV/GFP.
To construct rhMPV/GFPpolyA, an 11 nt Poly A insert was cloned into the NheI-N/P-GFP-NheI cassette using the primer 5' TGAGCTAGCTTAAAAAAGTGGGACAAGTCAAAATGGT-GCGAAAAAATTAAGCAAGGGCGAGG (hMPV P gene start sequences is in bold, the GFP sequences are italicized, and the 11 nt poly A insert is underlined) to generate the cassette NheI-N/P-GFP-PolyA-NheI. The NheI-N/P-GFP-PolyA-NheI cassette was ligated into the hMPV SphI/Cla I subclone at the Nhe I site located at nt5474. Again the StuI (nt4495) to ClaI (nt8693) fragment of the hMPV SphI/ Cla I subclone was inserted into the full-length cDNA to generate rhMPV/GFPpolyA ( Figure 1B) .
To construct rhMPV/ΔM2-2/GFP and rhMPV/ΔM2-2/ GFPpolyA, an Nhe I site was introduced into the SwaIΔM2-2 subclone at nt5316, using the primer 5'GCACTAATCAAGTGCAGTGAGCTAGCATTTAAATTAG and its complement (the stop codon of M2-1 is in bold and the Nhe I site is underlined). The NheI-digested GFPcontaining NheI-N/P-GFP-NheI or NheI-N/P-GFP-PolyA-NheI cassette was inserted at nt5316 to generate rhMPV/ ΔM2-2/GFP or rhMPV/ΔM2-2/GFPpolyA respectively ( Figure 1C ).
Recombinant viruses were generated from cDNA as described previously [27] . Briefly, 1.2 ug of pCITE hMPV N, 1.2 ug of pCITE hMPV P, 0.9 ug of pCITE hMPV M2, 0.6 ug pCITE hMPV L, and 5 ug of full-length hMPV cDNA plasmid in 500 uL optiMEM containing 10 uL lipofectamine 2000 (Invitrogen), was applied to a monolayer of 10 6 BSR/T7 cells. The medium was replaced with fresh optiMEM 15 hr post transfection and incubated at 35°C for 2 to 3 days. Cells and supernatant from the transfection were harvested together and used to infect Vero cells. Virus recovery was verified by positive immunostaining 6 days post inoculation with ferret polyclonal Ab directed to hMPV. Recovered viruses were further amplified in Vero cells by inoculating at a multiplicity of infection (MOI) of 0.1 PFU/cell.
Virus titers (plaque forming units (PFU)/ml) were determined by plaque assay in Vero cells. Monolayers of Vero cells in TC6-well plates were inoculated with 10-fold serial dilutions of virus. After 1 hour adsorption, the inoculum was aspirated, the monolayer was overlaid with optiMEM diluted 1:1 with 2% methylcellulose, and the plates were incubated for 7 days at 35°C. Plaques were immunostained with ferret polyclonal antisera directed to hMPV diluted 1:500 in PBS containing 5% powdered milk (w/v) (PBS-milk). The cells were then incubated with horseradish peroxidase-conjugated goat anti-ferret antibody (Dako) diluted 1:1000, followed by incubation with 3-amino-9-ethylcarbazole (AEC) (Dako) to visualize plaques. Ferret polyclonal antisera were generated by collecting blood 4 weeks after infection of 8-10 week old ferrets with 6.0 log 10 wthMPV/NL/1/00 administered intranasally.
Subconfluent monolayers of Vero cells in TC6-well plates were inoculated at a MOI of 0.1 PFU/cell with rhMPV or rhMPV/ΔM2-2 diluted in optiMEM. After 1 hr adsorption at 35°C, the virus inoculum was replaced with 2 ml opti-MEM. Combined cells and supernatant were collected at 24 hr intervals for 4 or 6 days, stabilized with 1× SPG and frozen at -70°C. Virus titers were determined by plaque assay in Vero cells.
Five-week-old Syrian golden hamsters (8 animals per group) were infected intranasally with 10 6 PFU/animal of virus or placebo medium in 100 uL. Four days post infection, the nasal turbinates and lungs were harvested from 4 animals per group, homogenized and titered by plaque assay in Vero cells. On day 28 post infection, immunized hamsters were challenged with an intranasal dose of 10 6 PFU/animal of wthMPV/NL/1/00. Four days post-challenge, the nasal turbinates and lungs were harvested and assayed for challenge virus replication by plaque assay.
Total RNA was extracted from hMPV-infected cells using TRizol (Invitrogen) reagent according to the manufacturer's instructions followed by phenol/chloroform (Amresco) extraction and ethanol precipitation. RT-PCR products of total hMPV RNAs were generated using a one step Ultrasense RT-PCR kit (Invitrogen) with sense and anti-sense primers designed to generate 1 to 2 kb fragments from total RNA. Genomic RNA was amplified with a two-step Super Script III platinum RT-PCR kit (Invitrogen). To ensure that only genomic RNA was amplified in the two-step process, only the sense primer was present during the RT step, the RT was deactivated by treatment of the product at 94°C for 15 minutes, and phenol/chloroform extraction was performed on the RT product to remove any residual RT enzyme prior to the PCR reaction. Sequence analysis was performed only on DNA fragments of the expected size isolated from agarose gels using a gel extraction kit (Qiagen).
Vero cells were inoculated at MOI = 0.1 and incubated at 35°C for 4 days. Cells and supernatant were scraped together and total RNA was extracted using Trizol reagent (Invitrogen) followed by an additional phenol-chloro-form extraction and ethanol precipitation. RNA was separated on 1% agarose gel in the presence of 0.44 M formaldehyde and transferred to a positively charged nylon membrane (Amersham Biosciences). The RNA was hybridized with gene-specific riboprobes labeled with digoxigenin using a DIG RNA labeling kit (Roche). The hybridized bands were visualized with a DIG luminescent detection kit (Roche).
Western blot analysis was performed as described previously [17] . Briefly, in duplicate, cell lysates of hMPVinfected Vero cells were separated on a 12% polyacrylamide Tris-HCl Gel (Bio-Rad), transferred to a Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences) and immunostained with either hamster Ab # 121-1017-133 (MedImmune) directed to hMPV F, mouse Ab directed to GFP (Roche Molecular Biochemicals), or mouse Ab directed to actin (Chemicon MAB #1501). Bands were visualized by incubation with horseradish peroxidase-conjugated anti-hamster Mab or anti-mouse Mab, developed with chemiluminescence substrate (Amersham Biosciences), and exposed to Biomax MR film (Kodak). No Longer an Innocent Bystander: Epithelial Toll-Like Receptor Signaling in the Development of Mucosal Inflammation Diseases of mucosal inflammation represent important causes of morbidity and mortality, and have led to intense research efforts to understand the factors that lead to their development. It is well accepted that a breakdown of the normally impermeant epithelial barrier of the intestine, the lung, and the kidney is associated with the development of inflammatory disease in these organs, yet significant controversy exists as to how this breakdown actually occurs, and how such a breakdown may lead to inflammation. In this regard, much work has focused upon the role of the epithelium as an “innocent bystander,” a target of a leukocyte-mediated inflammatory cascade that leads to its destruction in the mucosal inflammatory process. However, recent evidence from a variety of laboratories indicates that the epithelium is not merely a passive component in the steps that lead to mucosal inflammation, but is a central participant in the process. In addressing this controversy, we and others have determined that epithelial cells express Toll-like receptors (TLRs) of the innate immune system, and that activation of TLRs by endogenous and exogenous ligands may play a central role in determining the balance between a state of “mucosal homeostasis,” as is required for optimal organ function, and “mucosal injury,” leading to mucosal inflammation and barrier breakdown. In particular, activation of TLRs within intestinal epithelial cells leads to the development of cellular injury and impairment in mucosal repair in the pathogenesis of intestinal inflammation, while activation of TLRs in the lung and kidney may participate in the development of pneumonitis and nephritis respectively. Recent work in support of these concepts is extensively reviewed, while essential areas of further study that are required to determine the significance of epithelial TLR signaling during states of health and disease are outlined. Standing at the interface between the host and the environment, mucosal-lined surfaces represent the first line of defense against potential pathogens. This defensive role is particularly relevant to the mucosa of the gastrointestinal tract, the pulmonary system, and the urinary tract, each of which is particularly susceptible to the development of inflammatory diseases due to their role as a barrier that must not only protect, but also serve the physiological function of each of the organ systems. In the case of the gastrointestinal tract, mucosal inflammation is manifest as inflammatory bowel disease (including Crohn's disease and ulcerative colitis) (1) (2) (3) or necrotizing enterocolitis (NEC), a leading cause of death in preterm infants (4) . In the case of the pulmonary system, mucosal inflammation may be manifest as pneumonitis, pneumonia, or asthma (5-7), acute and chronic pulmonary conditions that have a high degree of morbidity and potential mortality. And in the case of the urinary tract, mucosal inflammation may be manifest as interstitial nephritis, cystitis, and urethritis (8) (9) (10) , causes of significant morbidity in patients of all ages. To elucidate the pathogenesis of mucosal inflammatory diseases, research over the past several decades has focused on the role of the immune system in their developmentin particular the relationship between mucosal lymphocytes, macrophages, and neutrophils, and the effects of their cellular by-products on mucosal integrity and function (11) (12) (13) . However, recent work has shed light upon the important role that the epithelia itself may play as a primary regulator of the immune response in the development of mucosal inflammation. No longer an innocent bystander, the epithelial-lined mucosa at each of these sites has been shown to possess all of the required armamentarium to allow an effective response to invading challenges, and to lead the battle to neutralize potential microbial threats (14) (15) (16) . Not only is the epithelium able to respond to potentially dangerous microbial products, it also may sense endogenous molecules that are released during conditions of stress, hypoxia, or injury-so-called danger molecules that may play a critical role in the development of mucosal inflammation (17) (18) (19) . In order, therefore, to understand the pathogenesis of mucosal inflammation and to assist in the rational design of anti-inflammatory strategies, it is necessary to define the receptors and signaling pathways that mediate the inflam-matory response with respect to the epithelium itself. The innate immune system consists of a series of receptors and their associated signaling molecules that is present both on leukocytes and epithelial cells through the body, and which initiates an immune response by responding directly to pre-formed ligands ( [20] provides a recent review). The innate immune system lies in contradistinction to the adaptive immune system, a set of cellular and molecular interactions that must first "learn" how to deal with a potential pathogen, and then respond through the release of antibodies or other cellular derived products. The innate immune system includes pattern recognition receptors such as the TLRs, the NOD-like receptors (NLRs), the RIG-like receptors (RLRs), and C-type lectins, and their role in inflammatory signaling in leukocytes has been extensively reviewed (21) (22) (23) 186) . Relatively few reports have focused on the ability of the innate immune system to signal within the epithelium, although emerging evidence from a variety of laboratories including our own indicates that innate immune signaling within the epithelium plays a critical role in the pathogenesis of mucosal inflammation. The current review will focus on the TLR family, which has been shown to play a critical role in the response of epithelial cells to bacterial and endogenous ligands in the pathogenesis of various mucosal inflammatory diseases (Table 1) .
A central controversy in the field of mucosal inflammation may be stated as follows (Figure 1 provides a pictorial representation of this): Is mucosal inflammation a reflection of a leukocyte-driven immune response that has gone awry, resulting in tissue injury and the loss of mucosal barrier function ( Figure 1A )? Or is it the mucosa itself, long known to play a role as a primary immune organ that is capable of producing a large number of pro-inflammatory molecules, that somehow has developed an exaggerated response that then leads to mu- Flagellin (58) 6 Diacyl Lipopeptides (144) Lipoteichoic acid (131) Zymosan (131) 7 Single-stranded RNA (145, 146) Imidazoquinoline (59) 8 Single-stranded RNA (146) Imidazoquinoline (147) 9 Bacterial (demethylated CpG) DNA (148) 10 Unknown 11 Profilin (149) Uropathogenic Bacteria (114) Figure 1 . Mechanisms by which TLR signaling leads to mucosal inflammation. As stated in the text, there are two potential mechanisms by which TLR signaling can lead to the development of mucosal inflammation. (A) TLR activation in response to DAMPs (damage associated molecular patterns) and PAMPs (pathogen associated molecular patterns) on leukocytes leads to the release of pro-inflammatory cytokines, resulting in epithelial destruction. (B) TLR activation by DAMPs and PAMPs on the epithelium itself directly impairs epithelial function and initiates the release of cytokines, leading to the development of mucosal inflammation. The development of mucosal inflammation likely arises from a combination of these mechanisms.
cosal injury ( Figure 1B) ? Mucosal surfaces, such as the intestinal mucosa and the upper respiratory tract, are constantly exposed to environmental stimuli, such as commensal luminal bacteria in the intestine, as well as endogenous stimuli, or, as is the case in the lower respiratory tract and urinary tract mucosa, may encounter and respond to endogenous and exogenous stimuli in disease states. In either case, the mucosa must be able to mount an effective immune response, resist barrier failure, and coordinate this response with both mucosal and sub-mucosal leukocytes, while avoiding initiation and propagation of an exaggerated inflammatory response. But where does the answer lie in terms of what is initiating the mucosal inflammatory response?
In seeking to answer this question, we and others have focused on the innate immune receptors that are present on the mucosa, and have examined the response of these receptors to known ligands in the development of mucosal inflammatory disorders. Such ligands may be broadly grouped into two categories: the so called "danger signals," a term used by Matzinger (24) , also called damage-associated molecular patterns (DAMPs) ( [25, 26] provide recent reviews); and those ligands on the surface or interior of pathogen-associated molecular patterns (PAMPs) ( Table 2) . Not surprisingly, there is a great deal of interest in identifying the important receptors for DAMPs and PAMPs expressed by various cell types, so as to accurately define their relative role in the development of inflammation. In this regard, several investigators have established that DAMPs and PAMPs are recognized by TLRs in many cells, including epithelial cells: a list of the TLRs and their cognate ligands appears in Table 1 . Although current dogma suggests that circulating leukocytes play a central role in the coordination of the immune response, emerging evidence suggests that the epithelium also plays a key role in the recognition and response to various "danger molecules" (27) (28) (29) (30) (31) . This review will examine in detail the various roles of epithelial signaling via TLRs in the development of common, and often devastating, mucosal inflammatory conditions. Much of the focus will be on the TLR-initiated signaling in response to PAMPs, as the majority of work has been performed in this area.
Several recent investigators have shed light upon the important role of TLR signaling in the development of mucosal inflammation (31) (32) (33) (34) . To understand how TLRs may signal within the epithelium, information may be gained by analyzing TLR signaling in other systems, primarily within leukocytes. A full description of the molecular mechanisms by which TLR signaling occurs is beyond the scope of this review; currently accepted concepts with respect to TLR signaling are described below (21, 35 have recent reviews).
The structure of each member of the TLR family of receptors provides important clues to how they function. All currently recognized TLRs are homologous with the interleukin-1 (IL-1) receptor, sharing an intracellular signaling domain, known as the Toll/IL-1R (TIR) domain (36) . A model that depicts the currently accepted mode of TLR signaling in leuko-cytes is shown in Figure 2 , in which the interaction with TLR4 and its cognate ligand lipopolysaccharide (LPS, endotoxin) is shown. The interaction of TLR4 with LPS leads to the activation of myeloid differentiation primary response protein 88 (MyD88)-dependent signaling, resulting in the induction of pro-inflammatory genes such as TNF-α, IL-1β, IL-6, and IL-10 (37-39) and MyD88-independent signaling cascades leading to activation of type-1 interferon (40 has a recent review) (38, 41) . MyD88-depedent signaling occurs as TIR domain-containing adapter protein (TIRAP/Mal) (42, 43) and MyD88 (44) (45) (46) interact with TLR4 and recruit IL-1 receptor-associated kinase (IRAK) family members IRAK1 and IRAK4 to the signaling heterocomplex consisting of TLR4, MyD88, and TIRAP (46) . Subsequent signaling occurs through tumor necrosis factor receptor-associated factor 6 (TRAF6)mediated (47) activation of transforming growth factor-β-activated protein kinase 1 (TAK1) (48) . TAK1 forms a complex with TAK1 binding proteins (TAB), TAB1 (49), TAB2 (50) , and TAB3 (51) . The TAK1/ TAB1/TAB2/TAB3 complex formation leads to the phosphorylation of IκB by IκB kinase (IκK) (52), initiating nuclear factorkappa B (NF-κB) signaling pathways and parallel activation of several mitogen-activated protein kinases (MAP-kinases) including c-Jun N-terminal kinase (JNK) (154) Viral proteins (106,143) Self DNA (155) Flagellin (58) Uric Acid (156) Bacterial DNA (148) Profilin (149) (53), p38 MAP-kinase (54) , and extracellular signal-regulated kinase (ERK) (55), ultimately resulting in pro-inflammatory gene induction (56, 57) . It is noteworthy that MyD88-dependent signaling is thought to be the predominant signaling pathway for TLR2 (41) , TLR5 (58), TLR7 (59) , and TLR9 (60,61). Figure 2 , TLR4 also may signal in the absence of MyD88. Evidence for this was demonstrated by Kawai et al., who described that the activation of NF-κB and MAP-kinases was reduced significantly, but not abolished, in MyD88-deficient mice (38) . The MyD88-independent signaling pathway proximally involves the activation of TRAM, a TIR-domain containing adapter molecule. TRAM associates with and activates TRIF, another TIR-domain containing adapter protein (62, 63) . TRIF then interacts with and activates TANKbinding kinase 1 (TBK1) and IKKε, two IκK homologs, which leads to the phosphorylation of IRF3 (64, 65) and translocation of IRF3 to the nucleus where it regulates the expression of various genes, including the type I IFN family of genes (66) . TRIF also interacts with TRAF6 and receptor interacting protein 1 (RIP), leading to the activation of NF-κB (67, 68) . Importantly, TLR3 signals mainly through the MyD88-independent, TRIFdependent pathway (62) .
There is a wide and diverse spectrum of diseases that involves the development of inflammation of the intestinal mucosa. Such diseases include inflammatory bowel disease that is, Crohn's disease and ulcerative colitis, necrotizing enterocolitis (which is a leading cause of death and disability in newborn infants) and a variety of infectious causes of intestinal dysfunction, due to enteroinvasive organisms such as Salmonella and Escherichia coli. As shown in Figure 1 , signaling via TLRs could lead to the development of intestinal inflammation through direct interaction of TLRs with the intestinal epithelium, or through effects on sub-epithelial and circulating leukocytes whose activation then leads to the initiation and propagation of mucosal inflammation. Although evidence exists to support this latter possibility, the expression of various TLRs in enterocytes (Table 3) suggests the possibility that direct interaction of intestinal TLRs with cognate ligands (see Table 1 ) may occur. Enteric bacteria in general, and LPS in particular, have been shown to play a critical role in the development of many diseases of intestinal inflammation (69) (70) (71) (72) , further suggesting the possibility that enterocyte TLR signaling may contribute directly to the development of these diseases.
To address the role(s), if any, of intestinal epithelial TLR signaling in the pathogenesis of intestinal inflammation, we, and others, have focused on TLR4, the receptor for LPS. Multiple enterocyte cell lines, including (IEC-6) rat enterocytes (27, 73) , primary and cultured (HT-29 and T84) colonocytes (74) (75) (76) and (CMT93) mouse rectal cells (75) express TLR4, the TLR4 adapter protein MD-2, and MyD88. In these cell lines, activation by LPS leads to pro-inflammatory signaling (74-76) as well as changes in cellular processes including proliferation (73) and intracellular TLR4 trafficking (77) . These findings provide evidence that enterocytes may respond directly to LPS via TLR4, yet by no means prove the physiological relevance of such a response. However, clinical significance for TLR signaling in the pathogenesis of intestinal mucosa is suggested as patients with inflammatory bowel disease demonstrate an increase in the expression of TLR4 and TLR2 in the intestinal mucosa (75, 78) , and we have found that TLR4 expression is increased in experimental and human necrotizing enterocolitis (32) . Sensitization of the intestinal mucosa through upregulation of TLRs also occurs in other diseases of intestinal inflammation, including inflammatory bowel disease and intestinal celiac disease (75, (79) (80) (81) , suggesting a potential role in the injury response.
In seeking to further understand the role of enterocyte TLR4 in the pathogenesis of intestinal inflammation, our laboratory recently has examined the role of enterocyte TLR4 activation in the pathogenesis of necrotizing enterocolitis (NEC) (32) . NEC is the leading cause of death from gastrointestinal disease in preterm infants (71) , and, currently, is one of the leading causes of death of newborns in the United States overall with a mortality rate of nearly 15% (82) . We have established recently that enterocyte TLR4 activation plays a critical role in the pathogenesis of NEC (32) . Specifically, we found that NEC in both mice and humans is associated with increased expression of TLR4 in the intestinal mucosa, and that physiological stressors associated with NEC development, namely exposure to LPS and hypoxia, sensitize the murine intestinal epithe-lium to LPS through upregulation of TLR4 (32) . In support of a critical role for TLR4 in the development of NEC, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates (32), a finding consistent with previous work by Caplan et al. (33) . TLR4 activation in vitro led to increased enterocyte injury by induction of enterocyte apoptosis and reduced epithelial healing, due to an inhibition of enterocyte migration and proliferation. This latter finding suggests a role for enterocyte TLR4 in the regulation of intestinal mucosal repair. In support of this possibility, increased NEC severity in wildtype C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation compared with TLR4-mutant mice. TLR4 signaling also led to increased serine-phosphorylation of intestinal focal adhesion kinase (FAK), a molecule necessary for efficient enterocyte migration. Surprisingly, TLR4 coimmunoprecipitated with FAK in enterocytes, and siRNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. Taken together, these findings demonstrate a critical role for TLR4 signaling in the intestinal epithelium in the development of NEC through effects on enterocyte injury and repair (32) .
In addition to the effects of enterocyte TLR4 activation on the regulation of intestinal injury and repair, our group also has demonstrated a surprising role for enterocyte TLR4 in the regulation of bacterial translocation across the intestinal barrier (see Figure 1A ). Translocation of bacteria across the intestinal barrier is important in the pathogenesis of not only intestinal inflammation, but also systemic sepsis, and may be a critical determinant of the development of multisystem organ dysfunction. We recently have shown that enterocyte TLR4 plays a key role in regulating the ability of enterocytes to internalize Gram-negative bacteria into membrane-bound phagosomes. Further evidence that TLR4 signaling is both necessary and sufficient for phagocytosis by epithelial cells was found as cultured enterocytes were able to internalize LPS-coated but not uncoated latex particles, and MD2/TLR4-transfected HEK-293 cells acquired the capacity to internalize E. coli, whereas non-transfected HEK-293 and HEK-293 transfected with dominant negative TLR4 bearing a P712H mutation did not. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice with mutations in TLR4 (27) . These data suggest a novel mechanism by which bacterial translocation occurs, and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.
The work reviewed above indicates that activation of TLR4 within the intestine is deleterious to the host, through effects on intestinal barrier injury, repair, and bacterial translocation. The overriding concept that enterocyte TLR4 activation has negative effects on intestinal homeostasis is supported by work demonstrating that TLR4 plays an important role in protecting the host from the development of chemical-induced colonic inflammation through the maintenance of intestinal homeostasis and the production of cytoprotective factors (83) (84) (85) . However, subsequent studies have demonstrated that TLR4 may play a permissive role in the development of spontaneous colonic inflammation (86) , suggesting either that the net effects of TLR4 on intestinal inflammation are dependent on the specific disease process examined, the anatomic location of the disease process, or that the interaction with various downstream effectors influences the extent of intestinal inflammation that develops. It is noteworthy that the inflammation observed in NEC is predominantly localized to the small intestine as opposed to the colon (4,87), implying that the effects of TLR4 activation within small intestinal epithelial cells may lead to different effects than its role on the colonic epithelia. In support of this concept, it has been demonstrated previously that small intestinal enterocytes are more responsive to LPS than colonic enterocytes, due in part to differences in TLR4 expression and/or activity (88, 89) . Moreover, the increase in expression of TLR4 within the ileum that we have observed after exposure to hypoxia and endotoxin suggests that TLR4dependent signaling within the small bowel mucosa may be increased after exposure to these stressors. The combined effects of the enhanced baseline sensitivity of the small intestine to LPS, and the upregulation of TLR4 expression in the intestine may partially explain the observed effects of enterocyte TLR4 in the induction of NEC. In support of this possibility, Caplan et al. have recently demonstrated that TLR4 expressing mice are more susceptible to the development of NEC in a model of formula feeding and cold asphyxia through a mechanism involving the enhanced interaction with luminal bacteria (33) .
In addition to TLR4, other TLRs have been shown to play a role in the pathogenesis of intestinal inflammation, poten-tially via TLR-dependent signaling of the enterocytes themselves. For instance, both TLR2-/-and TLR9-/-mice were found recently to develop more severe intestinal inflammation compared with wild-type counterparts (90, 91) . Moreover, TLR5-/-mice have been found to develop spontaneous colitis (92) and the TLR5 ligand flagellin has been found to protect against enterocyte apoptosis (93) . These findings indicate that TLR2, TLR5, and TLR9 may exert protective roles in the pathogenesis of intestinal inflammation, or indeed may provide support for the maintenance of intestinal homeostasis. Since TLR2, TLR5, and TLR9 share the downstream mediator MyD88, it is possible that these studies provide mechanistic insights into the protective role of MyD88 in the maintenance of intestinal homeostasis as identified by Medzhitov et al. (83) . Once again, though the story is more complicated than appears on first glance, as activation of TLR3, the only TLR family member that does not require MyD88 to signal, with the specific ligand polyinosinic:polycytidylic acid (poly I:C) protected against the severity of DSS-induced colitis (94) .
How do we reconcile the apparently contradictory roles of TLRs in the development of intestinal inflammation? It is possible that there may be cross talk between various TLR family members in the maintenance of intestinal inflammation, and the balance between intestinal homeostasis versus intestinal injury may be a reflection of the relative balance between TLRs and their associated signaling molecules. Alternatively, different TLRs within the intestine may be more or less susceptible to upregulation by different physiological stressors. We and others also have shown that intestinal mucosal TLR expression varies in different parts of the GI tract (SC Gribar and DJ Hackam, unpublished report) (95), which could explain in part the regional effects of TLR signaling on intestinal inflammation that is observed. It also may be possible that unique, epithelialspecific, intracellular signaling networks are activated by specific TLR ligation in enterocytes. Additional studies designed to delineate the precise interaction between the various enterocyte TLRs and their downstream receptors are required to resolve these possibilities.
Further insights regarding a potential role for TLR signaling in the pathogenesis of intestinal inflammation may be learned from studying genetic polymorphisms in humans with diseases of intestinal inflammation and sepsis. The TLR4 Asp299Gly mutation is known to render TLR4 hyporesponsive to endotoxin (99) . This mutation has been associated with an increased incidence of inflammatory bowel disease (ulcerative colitis and Crohn's disease) (100, 101) . Furthermore, pancolitis, the most severe manifestation of ulcerative colitis, is more common in patients with the TLR1 Arg80Thr polymorphism and the TLR2 Arg753Gly polymorphism (102) . In patients with Crohn's disease, the TLR1 Ser602Ile polymorphism is associated with a reduced risk of developing ileal disease (102) . While no genetic polymorphisms have been associated with NEC, further study is necessary as the current observations were made on small cohorts of patients (103) .
Pulmonary inflammatory diseases represent a broad spectrum of conditions that include allergic asthma, acute lung injury and acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and infectious pneumonia (96 has a recent review). Although these diseases traditionally have been considered to reflect the combined effects of activation of the adaptive immune system with the release of antibodies and mobilization of host immune cells, recent evidence has demonstrated an important role for the TLR family members of the innate immune system in their pathogenesis. And, in parallel with the mechanisms leading to the development of mucosal inflammation in the intestine, an emerging body of literature now provides evidence that epithelial TLR signaling plays a central role (15) . Previous authors have shown that the pulmonary epithelium expresses a variety of TLRs (see Table 3 ), suggesting their role in the pathogenesis of pulmonary inflammation. In support of a role for TLR signaling in the pulmonary epithelium in the development of pulmonary inflammation, Noulin et al. have demonstrated that TLR4 and MyD88dependent signaling are required for the bronchoconstriction, cytokine response, protein leak, and neutrophil recruitment observed in response to inhaled endotoxin (28) . Furthermore, using MyD88-/bone marrow chimeras, Noulin et al. demonstrated that both resident and hematopoietic cells are necessary for the mucosal inflammatory response to inhaled endotoxin (28). Hajjar et al. demonstrated that MyD88-deficient mice transplanted with bone marrow from MyD88-expressing mice showed reduced chemokine production compared with MyD88 expressing mice that were transplanted with MyD88-expressing bone marrow in a model of experimental Pseudomonas aerogeninosa pneumonia, indicating a requirement for resident pulmonary parenchymal cells in the response to experimental pneumonia. The local pulmonary cytokine response was predominately dependent on competent MyD88 signaling in bone-marrow derived cells, suggesting that collaboration between local parenchymal cells, including epithelial cells, and bone-marrow derived cells is required (29) . In a model of bacterial pneumonia that utilizes inhaled LPS, the uptake of LPS was observed in bronchial epithelial cells and was associated with increased TLR2 and TLR4 expression in the bronchial epithelium (97) . Similarly, in an equine model of recurrent airway obstruction associated with inhaled endotoxin-rich stable dust, increased epithelial expression of TLR4 was observed and was associated with increased IL-8 expression by the airway epithelium (98) . Taken together, these reports provide supportive evidence for an important role for epithelial TLR signaling in the pathogenesis of mucosal inflammation in the pulmonary system.
Several groups have shown that airway epithelial cells (AEC) express TLRs and secrete cytokines in response to TLR activation. AECs have been shown to express TLR2 and TLR4 and release IL-8 in response to Streptococcus pneumoniae, lipoteichoic acid, and lipopolysaccharide (99, 100) . Furthermore, TLR9 activation in bronchial epithelial cells has been shown to potentiate IL-8 release from bronchial epithelial cells (101) . Although it has been shown that multiple TLRs may signal in the airway epithelium (15) , microarray analysis of the lung has revealed that TLR4 signaling accounts for 74% of the pulmonary response to experimental Klebsiella pneumoniae pneumonia by comparing the pulmonary response in wildtype mice to C3H/HeJ TLR4 mutant mice. The particular TLR4-dependent responses included genes that are involved in cytokine and chemokine induction, neutrophil activation and recruitment, growth factor receptors, and TLR adaptor molecules (102) .
In addition to the evidence for TLR signaling in pulmonary epithelial cells in vitro, a variety of studies have shown that TLR activation may lead to the development of pulmonary inflammation in vivo. For instance, the TLR4 mutant strains C3H/HeJ and C57BL/10ScCr showed reduced clearance of pulmonary H. influenzae and E. coli (103, 104) . In an experimental Chlamydia pneumoniae pulmonary infection model, both TLR4 and TLR2 were found to be required for survival (105), while TLR4 and CD14 were found to play an important role in the response to respiratory syncytial virus (RSV) infection (106) . In response to pulmonary Streptococcus pneumoniae infection, TLR2-deficient mice revealed only modestly reduced inflammatory response and unchanged bacterial clearance (107) compared with wild-type counterparts. TLR3-deficient mice developed a survival advantage compared with wild-type mice, as well as reduced expression of IL-6 in the bronchoalveolar fluid in a murine model of influenza A virus infection (108) .
Additional evidence implicating a role for TLR signaling in the development of pulmonary inflammation may be found in studies examining the development of pulmonary inflammation in human patients with TLR polymorphisms. For instance, polymorphisms in TLR4 (Ala299Gly and Thr399Ile), which are known to lead to hyporesponsiveness to LPS (187), lead to a marked resistance to infection with Legionella pneumophila (188) . These TLR4 mutations have been correlated with the development of severe RSV infection in infants (120) . An inactivating polymorphism in TLR5 (TLR5392STOP) that encodes a stop codon in the ligand binding domain of TLR5 is associated with an increased susceptibility to infection with Legionella pneumophila causing Legionnaire's disease (188) . Taken in aggregate, the results of these in vitro and in vivo studies provide evidence for a role for TLR signaling in the pathogenesis of pulmonary inflammation. Additional studies are required utilizing pulmonary-specific TLR deletions to further delineate the relative contributions of pulmonary epithelial cell versus infiltrating leukocytes in the development of mucosal inflammation in the lung.
Akin to the gastrointestinal and pulmonary tracts, dysregulated epithelial signaling in the genitourinary system may lead to marked organ dysfunction. The expression of multiple TLRs within the urinary epithelium has now been established, suggesting the possibility that TLR signaling may regulate the interaction of the urinary epithelium with potential pathogens (see Table 3 ). TLR signaling within urinary tract epithelial cells leads to pro-inflammatory signaling in response to uropathogenic E. coli (UPEC) and LPS (109, 110) . Furthermore, modulation of uroepithelial inflamma-tion may be mediated by sensitization of the uroepithelium through regulation of epithelial TLR expression in response to infection or injury. An increase in TLR4 expression in the urinary epithelium has been observed during systemic sepsis in a murine model of cecal ligation and puncture (111) , and an increase in renal epithelial TLR2 and TLR4 expression has been observed in a murine model of local renal inflammation induced by ischemia (112) . Further demonstrating a role for TLR activation in uroepithelial inflammation, TLR4 mutant C3H/Hej mice failed to clear uropathogenic E. coli (UPEC) and showed reduced inflammatory mediator production compared with wild-type controls (109) . TLR4 mutant C3H/Hej mice were resistant to LPSinduced renal failure, had less renal neutrophilic infiltrate, and less renal cell apoptosis compared with wild-type controls (113) . In addition to TLR4, other TLRs may participate in the development of uroepithelial inflammation. For instance, TLR5-deficient mice were found to be more susceptible to experimental UPEC urinary tract infection compared with wild-type counterparts (16) , while mice with null mutations in TLR11, which is normally found to be strongly expressed in the bladder and kidney epithelium, developed markedly less severe kidney inflammation compared with wild-type counterparts (114) . TLR2-deficient mice were protected from tubular injury and renal function deterioration in a model of kidney ischemiareperfusion (115) . The clinical significance of a role for TLR signaling in the pathogenesis of genitourinary inflammation is found in clinical studies in which the incidence of acute rejection after kidney transplantation is reduced in patients who received a graft heterozygous for either the TLR4 Asp299Gly or Thr399Ile polymorphism compared with grafts without these mutations (189) , although conflicting results have been reported (190) . Taken together, these studies suggest an important role for TLR signaling in the development of urinary tract inflammation in a variety of models.
Which cells are required for the development of TLR-induced inflammation in the urinary tract? Evidence suggests that both epithelial and non-epithelial cell types may play a role. For instance, when TLR4 mutant C3H/Hej mice were transplanted with wild-type hematopoietic cells, the mice were unable to mount the necessary response to UPEC (30) . By contrast, in a model of cisplatin-induced renal injury, the development of inflammation was dependent on competent TLR4 signaling in resident renal parenchymal cells, as demonstrated in the study of TLR4-/-bone marrow chimeras (31) . Additional work is required to define more accurately the relative roles of TLR signaling within the epithelium versus the leukocytes in the development of mucosal inflammation in the epithelial tract.
The information reviewed above highlights the important roles that TLRs play in the regulation of the inflammatory response at mucosal surfaces. However, it is well known that these mucosal surfaces are constantly bathed in bacteria, and yet appear to mount little, if any, inflammatory response. These observations lead to the question, "What controls the activation of TLRs during basal states, and what leads to their activation during inflammatory conditions?" While a complete answer to this question remains lacking, current evidence suggests that the regulation of TLR activity occurs through altering TLR or co-receptor expression, TLR localization, TLR polarity, or signaling intermediate or negative regulatory protein expression, as described below.
Regulation of epithelial TLR expression has been suggested as a mechanism for the regulation of epithelial cell responsiveness in the setting of commensal bacterial exposure and during disease states. For instance, low expression of TLR4, as has been observed in colonic biopsies from humans (75) , has been suggested as a mechanism for colonocyte LPS hyporesponsiveness. Similar findings of low TLR4 expression have been observed in colonocyte cell lines (HT-29, SW480, colo205) and increased TLR4 expression after IFN-γ or TNF-α priming, as may occur during inflammatory states, has been shown to enable LPS responsiveness (88) . Similarly, low expression of TLR2 has been implicated in bronchial epithelial cell hyporesponsiveness to Gram-positive bacteria (15) .
Expression of TLR co-receptors in the epithelial cells also may play a role in epithelial TLR responsiveness. Hyporesponsiveness to LPS in colonic epithelial cell lines (Caco-2, T84, SW837, and in the basal state is associated with low or absent expression of the TLR4 coreceptor MD-2 (88, 191) and priming of cultured colonocytes (HT-29) with IFN-γ or TNF-α enabled LPS responsiveness in a mechanism that involved increased MD-2 expression (192) . Recently, we also have demonstrated a transient increase in the expression of the LPS co-receptor CD14 in enterocytes after exposure to LPS (193) . In the pulmonary system, absent expression of the TLR2 coreceptor CD36 has been implicated in the hyporesponsiveness of bronchial epithelial cell to Gram-positive bacteria (194) .
Changes in the subcellular localization of TLRs also may play a role in their responsiveness. Dissimilar to plasma membrane localized TLR4 in macrophages, TLR4 has been shown to be localized predominately in the Golgi apparatus in enterocytes (174) , and TLR4 activation in enterocytes has been shown to require intracellular recognition of LPS in the Golgi apparatus and recruitment of IRAK-1 and MyD88 to the Golgi apparatus (195) . Furthermore, colonic HT-29 and colo205 cells express TLR4 predominately in cytoplasmic fractions and are hyporesponsive to LPS in basal states (89) . Intracellular TLR redistribution has been suggested as a mechanism for flagellin tolerance, as prolonged flagellin exposure resulted in redistribution of TLR5 to an intracellular location in T84 colonocytes. Increased cell surface TLR expression also has been suggested as a mechanism of increased TLR sensitivity. Colonic SW480 cells are LPS-responsive and express TLR4 on the cell surface, where LPS internalization is not necessary for TLR4-LPS interaction (89) . Also, increased TLR9 surface expression was noted in response to DNA from pathogenic bacteria in HT-29 colonocytes (196) .
Epithelial cell polarity and differential localization of TLRs on the apical and basolateral cell surface also has been shown to play a role in TLR sensitivity as the apical surface of epithelial cells is more likely to encounter bacteria in the normal state, whereas the basolateral surface of epithelial cells may be more likely to encounter TLR ligands only in states of disease. In support of this concept, TLR4 and TLR2 are expressed at the apical pole of T84 cells and redistribute to a cytoplasmic compartment near the basal pole with activation (77) . Furthermore, differential TLR9 signaling has been shown in colonic HCA-7 epithelial cells. Apical TLR9 activation leads to attenuation of activation of NF-κB pathways, whereas basolateral TLR9 activation leads to activation of NF-κB signaling (91) . In the pulmonary system, TLR2 was located at the apical pole of airway epithelial cells, and increased surface expression was observed in response to bacteria, whereas TLR4 was noted predominately in a basolateral location (160) . In a recent finding by Soong et al., TLR2 became enriched in lipid rafts on the apical surface after bacterial infection in airway epithelial cells, suggesting a role in the regulation of TLR sensitivity (197) .
The regulation of signaling intermediate molecules also may affect TLR sensitivity. For instance, although fetal intestinal cells are known to be responsive to LPS, postnatal endotoxin hyporesponsiveness of enterocytes has been observed, and recently shown to be due to a decrease in the expression of the TLR4 signaling intermediate, IRAK1 (198) .
Negative regulatory molecules may play a role in regulating epithelial TLR signaling, including peroxisome proliferatoractivated receptor-γ; the cytoplasmic zinc finger protein, A20; and the negative regulator of TLR signaling, IRAK-M, as has been reviewed recently (199) . The relevance of these molecules to signaling within epithelial cells remains to be definitely demonstrated.
Given the importance of TLR signaling to the development of mucosal inflammation, it is understandable that a great deal of interest exists in the development of agents that can interfere with TLRsignaling pathways. Such an antiinflammatory approach may have particular relevance in the case of epithelial inflammation, due to ready access of the gastrointestinal, pulmonary, and urinary mucosa through ingestion, inhalation, or instillation via catheter delivery methods. Considerable attention has been placed on developing agents that are capable of modulation of the TLR4mediated response, in particular through manipulation of the lipid A moiety of LPS. Such lipid A mimetics, termed aminoalkyl glucosaminide phosphates (AGPs), have been demonstrated to reduce inflammation in experimental models of systemic sepsis induced by intravenous injection of Listeria monocytogenes (116) , pulmonary infection after intranasal administration of influenza virus (116) , murine models of colitis including DSS-induced colitis (117) , and spontaneous colitis in multidrug resistance gene 1a-deficient mice (117) . In parallel studies, soluble TLRs may reduce TLR signaling by binding to circulating ligands, rendering them unable to initiate pro-inflammatory signaling. Brandl et al. demonstrated that the synthetic molecule "LPS-Trap" was capable of blocking LPS-mediated macrophage activation in vitro by fusing MD-2 to the C-terminus of a soluble form of TLR4 (118) . Iwami et al. cloned an alternatively spliced soluble murine TLR4 (smTLR4) that, when transfected into murine macrophages, was secreted and inhibited LPS-mediated macrophage NF-κB activation and TNF-α release in vitro (119) . In addition, TLR-signaling intermediates have been targeted chemically to minimize the host inflammatory response. The synthetic peptide-mimetic compound ST2825 prevents MyD88 homodimer formation leading to an inhibition in MyD88dependent signaling, and prevents TLR9dependent inflammation in vivo (120) . A cyclohexene derivative, TAK-242, prevents TLR4 activation and was found to reduce the cytokine response to endotoxemic shock in mice (121, 122) . The Vaccinia virus protein A52R also reduces the TLR-mediated response by interacting with IRAK2 and TRAF6, and was found to reduce the cytokine response in an animal model of infectious otitis media (123, 124) and to increase survival in models of endotoxemia (125) .
As the effects of TLR signaling in hematopoietic cells, as well as epithelial cells, are more clearly defined, manipulation of TLR signaling may play a larger role in the treatment of patients with diseases of inflammation, and, as evidence continues to mount suggesting a protective role for particular TLR signaling, directed and specific TLR activation may hold therapeutic promise.
Mucosal surfaces and the epithelial cells that line them are constantly exposed to potential pathogens. The evidence reviewed above suggests that the innate immune system, comprised of TLRs and their associated molecules, plays a pivotal role in the regulation of mucosal inflammation in response to invading pathogens. However, the very fact that these mucosal surfaces are bathed in potential pathogens as part of their daily existence and yet don't develop inflammation under normal conditions raises an important scientific ques-tion: When does epithelial TLR signaling within mucosal surfaces become pathological? Or stated differently, when is the balance tipped between "physiological" signaling and "pathological" signaling in favor of a pathological response? A definitive answer to this important question not only is necessary to fully elucidate the steps required for the development of mucosal inflammatory diseases, but is central for the design of effective anti-inflammatory strategies.
Our current thinking in this area based upon our work and the work of others is shown in Figure 3 , in which the intensity of TLR signaling within the epithelium varies depending upon the prevailing degree of systemic stress. Under basal conditions, epithelial-bacterial interactions that may occur via TLRs are likely to play roles in the regulation of processes that regulate barrier integrity, such as epithelial migration, proliferation, and apoptosis ( Figure 3A ). However, during states of systemic stress, such as hypoxia or remote infection, we submit that the extent of TLR signaling within the epithelium becomes exaggerated in response to PAMPS and DAMPS that are encountered. This "tips the balance" in favor of mucosal barrier disruption, and adversely affects mucosal repair while worsening mucosal injury ( Figure 3B ). The extent of inflammation that develops within the local microenvironment likely is compounded further by the contribution of TLR activation on leukocytes, and the release of proinflammatory molecules. Under conditions in which the balance of TLR signaling within the epithelium can be "tipped back" to a homeostatic state, mucosal inflammation may not develop. By contrast, when the extent of TLR signaling is persistent, we propose that a "feedforward" loop develops within the mucosa, resulting in persistent TLR signaling, cytokine release, and mucosal inflammation. The evaluation of the factors that maintain the degree of TLR signaling within the mucosa in the maintenance of homeostasis and the pathogenesis of disease is a topic of intensive investigation. 
The importance of mucosal inflammation as a clinical problem is well accepted; however, the molecular and cellular signaling pathways that lead to its development remain incompletely understood. Although much attention has been placed on the role of the epithelium as a target in the mucosal inflammatory cascade, recent evidence has shed light upon the critical role that the epithelium itself, signaling in part through Toll-like receptors, may play in the initiation of a pro-inflammatory cascade in response to external stimuli. The field of mucosal inflammation research is likely to be advanced significantly through success in the following areas of study: 1) What are the relative roles of TLR signaling within the epithelium versus circulating leukocytes in the pathogenesis of mucosal inflammation? 2) What is the precise trigger for TLR signaling within the epithelium that adversely affects the host, and what are the essential roles played by mucosal TLRs in the maintenance of mucosal homeostasis? 3) Are there TLR-signaling molecular intermediates that differ between epithelial cells and leukocytes, and do such molecules confer epithelial-specific responses in the development of mucosal inflammation? 4) What regulates the interplay between the epithelium and the other cellular constituents of the mucosa, including neurons, endothelial cells, and endocrine cells during TLR activation? It is our belief that by addressing these important questions, one can be optimistic for the development of novel classes of anti-inflammatory strategies aimed specifically at the treatment of these devastating diseases of mucosal inflammation.
DJH is supported by grant R01GM078238-01 from the National Institutes of Health and the State of Pennsylvania Tobacco Settlement Fund. SCG is supported in part by the Loan Repayment Program for Pediatric Research of the National Institutes of Health and a Resident Research Award from the American College of Surgeons. WMR is supported in part by the Loan Repayment Program for Pediatric Research of the National Institutes of Health. Ubiquitination Is Required for Effective Replication of Coxsackievirus B3 BACKGROUND: Protein ubiquitination and/or degradation by the ubiquitin/proteasome system (UPS) have been recognized as critical mechanisms in the regulation of numerous essential cellular functions. The importance of the UPS in viral pathogenesis has become increasingly apparent. Using murine cardiomyocytes, we have previously demonstrated that the UPS plays a key role in the replication of coxsackievirus B3 (CVB3), an important human pathogen associated with various diseases. To further elucidate the underlying mechanisms, we examined the interplay between the UPS and CVB3, focusing on the role of ubiquitination in viral lifecycle. METHODOLOGY/PRINCIPAL FINDINGS: As assessed by in situ hybridization, Western blot, and plaque assay, we showed that proteasome inhibition decreased CVB3 RNA replication, protein synthesis, and viral titers in HeLa cells. There were no apparent changes in 20S proteasome activities following CVB3 infection. However, we found viral infection led to an accumulation of protein-ubiquitin conjugates, accompanied by a decreased protein expression of free ubiquitin, implicating an important role of ubiquitination in the UPS-mediated viral replication. Using small-interfering RNA, we demonstrated that gene-silencing of ubiquitin significantly reduced viral titers, possibly through downregulation of protein ubiquitination and subsequent alteration of protein function and/or degradation. Inhibition of deubiquitinating enzymes apparently enhances the inhibitory effects of proteasome inhibitors on CVB3 replication. Finally, by immunoprecipitation, we showed that coxsackieviral polymerase 3D was post-translationally modified by ubiquitination and such modification might be a prerequisite for its function in transcriptional regulation of viral genome. CONCLUSION: Coxsackievirus infection promotes protein ubiquitination, contributing to effective viral replication, probably through ubiquitin modification of viral polymerase. Coxsackievirus B3 (CVB3), a small RNA virus in the picornaviridae family, is an important human pathogen associated with various diseases, including myocarditis, aseptic meningitis, pancreatitis and possibly insulin-dependent diabetes. We and others have shown that CVB3 infection leads to activation of several intracellular signaling pathways [1] [2] [3] [4] [5] [6] [7] , and downregulation of host proteins likely through the ubiquitin/proteasome system (UPS) [7] [8] [9] .
It is well-established that the UPS is the major intracellular proteolytic system of all eukaryotic cells [10, 11] . The ATPdependent system begins with covalent attachment of ubiquitin to the ubiquitin-activating enzyme (E1). Then the ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2). Finally, ubiquitin ligase (E3) transfers the ubiquitin to the substrate protein. After several cycles of ubiquitination, multiple ubiquitin molecules are attached to the substrate which is then quickly recognized and subsequently degraded by the 26S proteasome. Ubiquitin is recycled through the actions of deubiquitinating enzymes (DUBs) [12, 13] . There are at least two classes of deubiquitinating enzymes, the ubiquitin C-terminal hydrolases (UCHs) and ubiquitin-specific processing proteases family.
In addition to the degradation of mutant, damaged and misfolded proteins, this system is responsible for the modulation of many regulatory proteins such as cyclins [14] , inhibitors of cyclin-dependent kinases (p21, p27) [15] , tumor suppressors (p53) [16] , and inhibitor of NFkB (IkB) [17] , which are essential for a variety of cellular functions, including cell-cycle regulation, apoptosis and host immune responses [18] . Unlike polyubiquitination in the regulation of protein degradation, monoubiquitination of cellular proteins, such as histones, calmodulins, actin, proliferating cell nuclear antigen and receptor tyrosine kinases, plays more diversified roles involving in the regulation of chromatin remodeling, DNA repair, transcriptional regulation and endocytosis [19] .
Since the first discovery that human papillomavirus protein E6 targets the cellular tumor suppressor protein p53 for the UPSmediated degradation [16] , increasing studies, including those from our laboratory, have suggested that various viruses evolve different mechanisms to utilize or manipulate the host UPS for their own benefits [9, [20] [21] [22] [23] [24] [25] . We have previously shown that CVB3 infection results in downregulation of several host proteins [7, 9] , such as cell-cycle protein cyclin D1, tumor suppressor p53, and transcription activator b-catenin in infected HeLa cells. The downregulation of host proteins following CVB3 infection is most likely through the UPS. Specific inhibitors to 26S proteasome reverse the degradation of proteins in HeLa cells [7, 9] and reduce CVB3 replication in murine cardiomyocytes [26] .
In this study, we investigated the possible underlying mechanisms by which the UPS regulates CVB3 replication. We demonstrated that protein ubiquitination was enhanced after coxsackievirus infection. We further showed that knockdown of ubiquitin expression by small-interfering RNA (siRNA) decreased CVB3 infection, likely through the downregulation of ubiquitination and subsequent alteration of protein function and/or degradation. In addition, we showed that inhibition of deubiquitinating enzyme increased the inhibitory effects of proteasome inhibitors on CVB3 replication. We also found that CVB3 RNA-dependent RNA polymerase 3D (3D pol ) was modified by ubiquitination. Taken together, our study suggests an important role of ubiquitination in the regulation of coxsackieviral replication.
To uncover the underlying mechanisms of the antiviral activities of proteasome inhibitors, we chose to use the well-characterized HeLa cells to further our study. We first examined the role of proteasome inhibition in CVB3 replication. As shown in Fig. 1 , we found that proteasome inhibitor, MG132, significantly reduced CVB3 viral RNA synthesis (Fig. 1A) . Both proteasome inhibitors used in the study, MG132 and lactacystin, decreased the synthesis of CVB3 capsid protein, VP1, in a dose-dependent manner (Fig. 1B ). In addition, two inhibitors inhibited CVB3 viral titers by up to fifteen folds (Fig. 1C) . Although MG132 and lactacystin significantly inhibited cellular 20S proteasome activities, we have previously demonstrated there was no apparent difference in proteasome activities between CVB3-infected and sham-infected HeLa cells [9] . Together, these results suggest that efficient replication of CVB3 requires the intact UPS function rather than the core proteasome activity alone.
We also performed cell viability assay and morphological examination to determine whether inhibiting viral replication by proteasome inhibitors is due to the toxicity. We found that there was no measurable cell death throughout the incubation period for all doses of proteasome inhibitors used in this study (Fig. 1D) . On the contrary, virus-induced cell death was markedly inhibited after the treatment of proteasome inhibitors as a result of decreased viral replication (Fig. 1D ).
As alluded to earlier, two successive steps are involved in protein degradation: (1) covalent attachment of ubiquitins to the target protein substrate, and (2) degradation of the polyubiquitinated protein by the 26S proteasome with the release of ubiquitin for recycling. To dissect out the role of ubiquitination and degradation in CVB3 infection, we next decided to investigate the protein ubiquitination after CVB3 infection. As shown in Fig. 2A , protein ubiquitination was gradually increased along the time-course of CVB3 infection, which was accompanied by a decrease of free ubiquitin levels. Densitometric analysis further demonstrated that the increases in protein ubiquitination at 3 h, 5 h, and 7 h post-infection were statistically significant as compared to sham infection (Fig. 2B) . We have previously demonstrated that 26S proteasome activities were unchanged during CVB3 infection [9] . Thus, the finding of increased accumulation of ubiquitinated proteins is likely due to enhanced protein ubiquitination as opposed to reduced proteasome activity. Decreased levels of free ubiquitin could be a direct consequence of the increased protein ubiquitination. These results suggest that enhanced ubiquitin conjugation may be a prerequisite for efficient synthesis of CVB3 viral RNA and continuation of its lifecycle.
In addition to blocking proteasome proteolytic activities, proteasome inhibitors are known to reduce free ubiquitin levels in treated cells [27] . It has been suggested that proteasome inhibition negatively affects the budding of retroviruses through reducing free ubiquitin level and subsequently interfering with ubiquitination of viral Gag proteins [22, 24, 25] . Ubiquitin is generated in the cell by proteolysis of polyubiquitinated proteins or ubiquitin fused to carboxyl extension proteins (CEPs) [28] . To investigate whether protein ubiquitination is beneficial to CVB3 replication in HeLa cells, we used the ubiquitin-specific siRNA to gene-silence the expression of human ubiquitin-CEP Uba80, which codes for ubiquitin fused to ribosomal protein S27a [29] . As shown in Fig. 3A , both ubiquitin conjugates and free ubiquitin levels were markedly knocked down after the treatment of ubiquitin siRNA. We further showed that viral titers were significantly reduced in the ubiquitin siRNA-transfected cells as compared to scramble siRNA control (Fig. 3B ), suggesting that protein ubiquitination is a critical process adopted by coxsackievirus for the successful completion of its lifecycle.
It has been demonstrated that protein ubiquitination can also be regulated by deubiquitinating enzymes that specifically cleave ubiquitin from ubiquitin-conjugated protein substrates [12, 13, 30] . To further explore the role of protein ubiquitination in viral replication, we examined the influence of DUB inhibition on viral protein expression. Two commercially available ubiquitin cterminal hydrolase inhibitors, UCH L1 and UCH L3 inhibitors, were used for this study. As shown in Fig. 4 , specific inhibition of UCH L1 or UCH L3 further reduced CVB3 protein expression and virus titers in proteasome inhibitor-treated cells, suggesting that these enzymes may be involved in the lifecycle of CVB3. Nevertheless, it was found that inhibition of the UCH L1 and L3 activities alone was not sufficient to block coxsackievirus replication since no significant changes in viral protein expression and CVB3 titers were observed in cells treated with two UCH inhibitors either separately or in combination (Fig. 4) . As discussed earlier, stabilization of short-lived host proteins and prevention of protein ubiquitination by reducing recycled ubiquitin likely contribute to the inhibitory effect of proteasome inhibition on viral replication. Thus, it is speculated that DUB inhibition by UCHL1/L3 inhibitors alone, in the absence of apparent inhibition of protein degradation, is not sufficient enough to block viral replication. However, additional reduction of recycled free ubiquitin by DUB inhibition can further enhance the inhibitory effect of proteasome inhibitor.
CVB3 RNA-dependent RNA polymerase 3D is ubiquitinated Some virus RNA-dependent RNA polymerases including the sindbis virus and the turnip yellow mosaic virus RNA polymerases have been demonstrated to be phosphorylated and ubiquitinated [31] . Although the role of ubiquitination of these RNA polymerases in the regulation of virus replication remains to be determined, such observation raises the interesting possibility that the ubiquitin/proteasome system may regulate CVB3 replication through ubiquitinating viral polymerase 3D, which is essential for initiating viral RNA replication. To examine whether coxsackieviral proteins are subjected to ubiquitination during viral infection, we performed immunoprecipitation with anti-ubiquitin antibody, followed by immunoblots using antibodies against 3D pol and viral capsid protein VP1, respectively. As shown in Fig. 5 , immunoreactive bands of around 60 kDa were detected in CVB3infected cells. Non-modified 3D pol has a molecular weight of about 53 kDa, thus this observation suggests that 3D pol likely undergoes post-translational modification by monoubiquitination. No protein ubiquitination was observed for VP1 (data not shown). Our results implicate that the ubiquitination process of CVB3 viral proteins might be required for successful replication of the virus.
In trying to understand the mechanisms by which CVB3 manipulates the UPS, we examined the protein expression of several key enzymes involved in the process of protein ubiquitination and deubiquitination. We measured expression levels of ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme Ubc H7, ubiquitin C-terminal hydrolase and two p53related E3 ligases, human papillomavirus E6-associated protein and mouse double minute 2 homolog. However, no apparent changes were observed during the time-course of CVB3 infection (data not shown). These results indicate that the manipulation of the UPS by CVB3 is unlikely regulated by the above-examined ubiquitin-related key enzymes or molecules. Future studies will determine whether CVB3 infection targets on specific ubiquitin ligases or deubiquitinating enzymes.
In the present study, we have provided further evidence that CVB3 manipulates the UPS for its infection. CVB3 infection results in increased protein polyubiquitination and a subsequent decrease in free ubiquitin levels. Knockdown of ubiquitin and ubiquitinmediated protein modification and/or degradation by siRNA It is increasingly apparent that viruses can evolve various strategies to utilize the host UPS for their own benefits. The UPS has been suggested to play a critical role in the different steps of viral lifecycle, including viral entry, viral replication, maturation, viral progeny release, and latent virus reactivation [32] [33] [34] . The mechanisms that the UPS regulates viral infection involve degrading intracellular proteins or excessive viral proteins that are against efficient viral replication and modulating viral protein function through ubiquitin-mediated modification or by directly encoding ubiquitin-related enzymes [35] . The finding in this study that CVB3 infection stimulates protein ubiquitination without inhibition of the core 20S proteasome activity highlights the possibility that CVB3 manipulates the UPS to destabilize or modulate the host and viral proteins. Polyubiquitination and degradation of host antiviral proteins has been suggested to be a mechanism of HIV-1 replication [36] . We have previously identified several proteins, such as cyclin D1, p53 and b-catenin, which are downregulated through the UPS after CVB3 infection [7, 9] . Destabilization of these short-lived host proteins is likely required for CVB3 viral RNA and protein synthesis in its lifecycle. Moreover, it is speculated that nonstructural viral proteins of CVB3 could also be potential targets of the UPS for degradation. Previous studies on picornavirus have shown that several viral proteins, such as encephalomyocarditis virus (EMCV) 3C protease and hepatitis A virus (HAV) 3C protease, are ubiquitinated and present in low concentrations in infected cells [37] [38] [39] . Several E3 ubiquitin ligases, such as human E3a ubiquitin ligase, have been shown to catalyze the ubiquitination of these viral proteins [38, 39] . Although the exact role of ubiquitination and subsequent degradation of nonstructural viral proteins of EMCV and HAV in infected cells remains elusive, such rapid turnover may be required for efficient viral RNA replication, viral protein synthesis and virus maturation.
As alluded to earlier, DUBs are a large family of cysteine protease responsible for the removal of ubiquitin from substrate proteins [40] . It is estimated that the human genome encodes more than 100 DUBs. Although UCHL1 is identified as an important DUB, inhibition of UCHL1 alone has been shown to only partially block the activities of DUBs [41] . Thus, the finding in this study that UCHL1/L3 inhibition is not as efficient in blocking viral replication as general inhibition of proteasome function or knockdown of ubiquitin is likely attributed to incomplete inhibition of DUBs by UCHL1/L3 inhibitors.
In addition to protein degradation, ubiquitin-modification has been suggested to be involved in the regulation of protein function. It was reported that monoubiquitination of the Gag protein of retroviruses is required for virus budding [22, 24, 25] . Depletion of free ubiquitin by proteasome inhibitors prevents Gag ubiquitination, subsequently blocks virus progeny release/budding. In addition, ubiquitination of human immunodeficiency virus type 1 Tat protein and human T-cell leukemia virus type 1 Tax protein has been shown to modulate their transactivation activities [20, 23] . We speculate that monoubiquitination is also an important machinery for post-translational modification and activation of CVB3 viral proteins. In the current study, we have shown that CVB3 RNA-dependent RNA polymerase 3D is posttranslationally modified by ubiquitination, suggesting a critical role of protein ubiquitination in the regulation of viral protein functions.
Based on the results in the manuscript, in combination of our previous findings that CVB3 infection promotes host protein degradation, including cyclin D1, p53 and b-catenin, a model system on the role of the UPS in CVB3 replication is proposed in Fig. 6 . Coxsackievirus infection facilitates host protein polyubiquitination, which subsequently increases intracellular protein degradation by the proteasome and/or viral protein modification, such as 3D pol , by monoubiquitination. Degradation of host antiviral proteins provides a favorable environment for virus to achieve successful replication. Knockdown of ubiquitin decreases host protein degradation and viral protein ubiquitination. Proteasome inhibition blocks host protein degradation and viral protein ubiquitination by reducing recycled ubiquitin. DUB inhibitors further decreases the viral replication when used together with proteasome inhibitors through the additional reduction of recycled free ubiquitin.
In conclusion, we have demonstrated for the first time that CVB3 infection results in increased protein ubiquitination and consequent decreases in free ubiquitin levels. We further demonstrate that protein ubiquitination is required for the completion of viral lifecycle, likely through ubiquitin modification of viral polymerase.
HeLa cells (American Type Culture Collection) were grown and maintained in complete medium [Dulbecco's modified Eagle's media (DMEM) supplemented with 10% heat-inactivated newborn calf serum (NCS) (Invitrogen)]. CVB3 (Kandolf strain) was propagated in HeLa cells and stored at 280uC. Virus titer was routinely determined by a plaque assay prior to infection as described below.
The monoclonal anti-b-actin and anti-ubiquitin antibodies were purchased from Sigma-Aldrich. The monoclonal anti-VP1 antibody was obtained from DakoCytomation. The ubiquitin siRNA, scramble control siRNA, and horseradish peroxidaseconjugated secondary antibodies were obtained from Santa Cruz Biotechnology. The proteasome inhibitors, MG132 and lactacystin, the UCH L1 inhibitor (LDN-57444) and the UCH L3 inhibitor (4,5,6,7-Tetrachloroindan-1,3-dione), and the polyclonal anti-ubiquitin antibody were obtained from Calbiochem. The polyclonal anti-3D pol antibody was a generous gift from Dr. Karin Klingel (University Hospital Tuebingen, Germany).
HeLa cells were grown in complete medium to 70-80% confluence, and then infected at a multiplicity of infection (MOI) of 10 with CVB3 or sham-infected with phosphate-buffered saline (PBS) for 1 h in serum-free DMEM. Cells were then washed with PBS and cultured in serum-free medium. For inhibition experiments, HeLa cells were infected with CVB3 for 1 h, washed with PBS, and then incubated with DMEM containing various concentrations of inhibitors. Immunoprecipitation and immunoblot analysis Cell lysates were prepared using lysis buffer (50 mM pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 mM Na 3 VO 4 , 10 mM HEPES (pH 7.4), 0.1% Triton X-100, and the protease inhibitor cocktail) as described previously [2] . For immunoblot analysis, equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked for 1 h with nonfat dry milk solution (5% in PBS) containing 0.1% Tween 20. Blots were then incubated for 1 h with the primary antibody followed by incubation for 1 h with the secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). When protein ubiquitination was examined, membrane was heat-activated by autoclaving at 121uC for 35 min prior to blocking with nonfat dry milk solution to enhance antigenic site recognition.
For immunoprecipitation, cells were lysed using the abovedescribed lysis buffer with freshly added 20 mM iodoacetamide. A total of 500 mg of cell lysates were incubated with a monoclonal anti-ubiquitin antibody (1:100) at 4uC overnight, followed by 2 h incubation with protein G-agorose beads (Amersham). Immunocomplexes were washed five times with the lysis buffer containing 20 mM iodoacetamide, and then boiled for 5 min in the 26 nonreducing sample buffer which lacks both b-mercaptoethanol and DTT, but with addition of 20 mM iodoacetamide. After centrifugation, the precipitated proteins were separated by SDS-PAGE. Ubiquitin conjugates were analyzed by immunoblot using polyclonal anti-3D pol antibody.
HeLa cells were grown and maintained on two-chamber culture slides (Becton Dickinson Labware). Subconfluent cells were infected with either PBS or CVB3 (MOI = 10). Following 1 h of incubation at 37uC, cells were washed with PBS and replenished with complete medium in the absence and presence of MG132. HeLa cells were incubated for an additional 6 h. The culture slides were then washed gently with PBS, fixed with formalin buffer for 15 min, and then air-dried at room temperature. Culture slides were then subjected to in situ hybridization assays to detect the sense-strand of CVB3 genomic RNA as previously described [26] .
Plaque assay CVB3 titer in cell supernatant was determined on monolayers of HeLa cells by an agar overlay plaque assay in triplicate as described previously [2] . Briefly, samples were serially diluted and overlaid on monolayer of HeLa cells. After 1 h incubation, medium was replaced with complete medium containing 0.75% agar. Cells were incubated for 72 h, then fixed with Carnoy's fixative (75% ethanol-25% acetic acid), and stained with 1% crystal violet. Plaques were counted and viral titer was calculated as plaque forming unit (PFU) per milliliter.
MTS (3, 4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt, Promega) assay was performed to determine cell viability as previously described [7] . Briefly, cells were incubated with MTS solution for 2 h prior to collection. Absorbance was measured at a wave length of 490 nm using an ELISA reader.
HeLa cells were grown to 50% confluency and then transiently transfected with ubiquitin-specific siRNA (200 nM) using oligofectamine according to the manufacturer's suggestion (Invitrogen). A scramble siRNA (200 nM) was used as a control. The silencing efficiency was detected by immunoblot analyses using the antiubiquitin antibody. After 24 h of transfection, cells were infected with CVB3 as indicated.
Statistical analysis was performed using the paired Student's t test. A p value of less than or equal to 0.05 was considered statistically significant.  Professional and Home-Made Face Masks Reduce Exposure to Respiratory Infections among the General Population BACKGROUND: Governments are preparing for a potential influenza pandemic. Therefore they need data to assess the possible impact of interventions. Face-masks worn by the general population could be an accessible and affordable intervention, if effective when worn under routine circumstances. METHODOLOGY: We assessed transmission reduction potential provided by personal respirators, surgical masks and home-made masks when worn during a variety of activities by healthy volunteers and a simulated patient. PRINCIPAL FINDINGS: All types of masks reduced aerosol exposure, relatively stable over time, unaffected by duration of wear or type of activity, but with a high degree of individual variation. Personal respirators were more efficient than surgical masks, which were more efficient than home-made masks. Regardless of mask type, children were less well protected. Outward protection (mask wearing by a mechanical head) was less effective than inward protection (mask wearing by healthy volunteers). CONCLUSIONS/SIGNIFICANCE: Any type of general mask use is likely to decrease viral exposure and infection risk on a population level, in spite of imperfect fit and imperfect adherence, personal respirators providing most protection. Masks worn by patients may not offer as great a degree of protection against aerosol transmission. With a potential influenza pandemic looming, governments need to decide how they can best use available resources to protect their people against severe illness and death, and to mitigate health and social effects for society as a whole. Much research is being devoted to develop optimal strategies for the use of (pre)pandemic vaccines and of anti-virals. There are only limited data to assess the potential effectiveness of non-pharmaceutical interventions to reduce the risk of transmission, including the effect of different kinds of face-masks worn by the general public or by patients.
Respiratory infections such as influenza are transmitted through infectious particles, small enough to be suspended in air [1] . Influenza transmission can occur via large droplets, which only remain suspended in the air for a short period of time thus requiring close contact, and can occur via small airborne particles, which remain suspended in air for considerable longer periods of time, and can thus be transmitted over larger distances [2] . Furthermore, some transmission may occur via direct contact with respiratory secretions such as on hands and surfaces [2] .
Interruption of transmission may allow containment of major outbreaks, like pandemic influenza. Opportunistic data collected during the SARS epidemic in Asia suggested that population-wide use of face masks may significantly decrease transmission of not only SARS but also influenza [3, 4, 5, 6, 7] . As part of pandemic preparedness, many are contemplating the contribution wide-spread use of masks could have [8, 9] . As this has major implications for resource allocation and for communication, there is great need for data to guide such decisions and make them evidence-based.
Protective effects of face masks have been studied extensively, but usually this involved personal respirators for professionals under idealized conditions, because of specific applications, for instance in military or occupational uses, involving protection of specifically trained personnel. This is different from deployment of masks in the general population during an outbreak of an infectious disease, where anyone may encounter the infectious micro-organism, implying much greater heterogeneity, in training levels (experience and understanding), goodness of fit of a mask, and activities interfering with mask use and thus reducing potential reduction of transmission. The protective effect of masks is created through a combined effect of the transmission blocking potential of the material, the fit and related air leakage of the mask, and the degree of adherence to proper wearing and disposal of masks. Personal respirators such as those worn by staff attending TB patients, are used primarily to protect the wearer, and are designed to fit to the face with as tight a seal as possible. Their efficiency is graded on the degree of protection the material offers, assuming a perfect fit and optimal compliance. In contrast, surgical masks, as commonly worn in the operating theatre, are primarily used to protect the environment from the respiratory droplets produced by the wearer. With these masks, facial fit is much looser. The fit of home made masks, which could be e.g. made of a tea cloth or other comparable material available in the home, is likely to be even looser. Thus personal respirators confer a higher degree of protection than surgical masks, and these are again likely to give a higher degree of protection than home-made masks. In professional situations, ample time might be available prior to use to ensure a perfect fit and to give extensive counselling on adherence, but it is unlikely this will apply to the general population in case of a pandemic. It is possible that the discomfort in wearing associated with a certain type of masks will lead to reduced adherence and thus to a loss in overall protectiveness [10, 11] . Indeed a review among health care workers could not determine whether personal respirators conferred better protection for the health care workers than surgical masks [10] .
To investigate the levels of protection, and their variation, wearing of face masks could convey to untrained subjects we designed a study in which healthy volunteers would be wearing different types of professional and home-made masks during a selection of activities, in different conditions (inward protection). We also assessed the protection different types of masks could convey when worn by a simulated infectious patient (outward protection). Resulting quantitative descriptions of distributions of protection factors may be used for assessing the importance of mask use in respiratory disease transmission.
Three different experiments were undertaken to assess 1) shortterm protection for different types of masks worn during 10-15 minutes by the same volunteer following a standardized protocol, 2) long-term protection of a specific mask worn continuously by a volunteer for 3 hours during regular activities, and 3) effectiveness of different types of mask in preventing outgoing transmission by a simulated infectious subject. Inward protection was defined as the effect of mask wearing to protect the wearer from the environment; outward protection was defined as the effect of a mask on protecting the environment from the generation of airborne particles by a patient (or in this case a mechanical head).
In the first short-term experiment, 28 healthy adult volunteers were recruited, as well as 11 children between 5 and 11 years of age. Each volunteer followed the same protocol wearing a Filtering Facepiece against Particles (FFP)-2 mask 1872VH (3M); which is the European equivalent of a N95 mask, a surgical mask (1818 Tie-OnH, 3M; with a filtering efficiency of around 95% for particles of sizes between 0.02 mm to 1 mm; http://jada.ada.org/ cgi/content/full/136/7/877) and a home-made mask (made of TD Cerise MultiH teacloths, Blokker). In this standard protocol, the volunteer was asked to perform five successive tasks in a fixed sequence 1.5 minute of duration each: no activity-sit still, nod head (''yes''), shake head (''no''), read aloud a standard text, stationary walk. In this sequence of activities, the respiratory rate is gradually increased. Throughout this exercise, the concentration of particles was measured on both sides of the mask through a receptor fixed on the facial and on the external side. These were connected to a portable counter of all free floating particles in the air via an electrostatic particle classifier and counter, the PortacountH. The PortacountH can register particles floating in the air with sizes between 0.02 mm to 1 mm, covering most of the size range of infectious respiratory aerosols [12] . Total inward leakage (TIL) percentage was calculated by dividing the concentrations on the outside and on the inside (TIL = (concentration inside/concentration outside)6100); the calculated quantitative protection factor was the inverse of the leakage (PF = (TIL/ 100) 21 ). To ensure small numbers of particles produced by the volunteers would not affect measurements, we checked that at least 10,000 particles per cm 3 particles of this size class (0.02 mm-1 mm) were present in the room which were produced by a number of lit candles. (Figure 1 )
In the second long-term experiment, 22 volunteers, all adults, 10 men, 12 women, were divided into 3 groups. Each group wore a single type of mask for a period of three hours, being either a FFP2 mask (4 males, 4 females), a surgical mask (3 males, 4 females) or a home-made mask (3 males, 4 females), similar to the masks used in the short-term experiment described above. At the beginning and end of each three-hour period, full series of measurements were taken using the standardised protocol as described for the short-term experiment, and during the three hour period while wearing the masks, participants reported back at regular intervals for a short measurement during rest (absence of activity). For the remainder of the period, participants carried on with their usual daily activities. During regular activities in between measurements, the probes of the masks were plugged which did not involve dislodging of the masks. In the final experiment, we assessed the effectiveness of different types of masks in reducing outgoing transmission from an infectious subject shedding aerosolised particles. This was simulated by fitting the different types of masks to an artificial test head, which was connected to PC-driven respirator (BacouH LAMA AMP, Modelref 1520307). Breathing frequency was varied to mimic different respiratory rates (15, 25 and 40/minute). Only expiration was simulated; twice for each mask at each respiratory rate. The breathing flow was defined as (respiratory rate/minute x volume per breath (2 litres)) resulting in a breathing flow of 30, 50 and 80 litres per minute, which correlates with light (walking), medium (marching with backpack) and strenuous (running) activities [13] . Concentrations of particles were measured as described above by a TSI Portacount Respirator Fit tester, model 8020, measuring outward protection, rather than inward protection.
All volunteers received written information prior to the experiments and gave oral informed consent. For the children also a parent gave oral informed consent, and a parent remained present during the experiments. The Dutch Central Committee on Research Involving Human Subjects (CCMO) informed us in writing that this project did not need to be assessed by an Ethics committee.
Protection factors (PF) calculated from measurements of particle concentration by PortacountH devices were reported as the ratio of particle concentrations outside and inside the mask. This is a similar concept to the fit factor as used by the US Occupational Safety and Health Administration (http://www.osha.gov/pls/ oshaweb/owadisp.show_document). Therefore, a higher PF is better and PF = 1 means complete absence of protection. For statistical analysis, the following transformation was used:
The inverse of the PF (1/PF) can be interpreted as a probability (that any particle succeeds in moving through the barrier the mask provides). The logit transformation is a standard transformation to transform the probability scale (0,1) to the real axis (-infinity, +infinity) to allow standard regression techniques (including ANOVA) to test the effects of co-variables (mask type, age class, sex, activity, duration of use) on transformed PFs in a linear model, using the statistical application R (version 2.5.0). The p-values are based on testing the ratio of mean squares for a factor (like 'mask') and the mean square of errors (random fluctuations), assuming that ratio is F-distributed. Whenever the p-value (the probability of a greater value of the tested ratio) is greater than 0.05, the ratio is considered significantly different from 1 ( = indifference) at the 95% level.
All masks provided protection against transmission by reducing exposure during all types of activities, for both children and adults (Table 1) . Within each category of masks, the degree of protection varied by age category and to a lesser extent by activity. We observed no difference between men and women. Surgical masks provided about twice as much protection as home made masks, the difference a bit more marked among adults. FFP2 masks provided adults with about 50 times as much protection as home made masks, and 25 times as much protection as surgical masks. The increase in protection for children was less marked, about 10 times as much protection by FFP2 versus home-made masks and 6 times as much protection as surgical masks.
In these short term experiments, adjusting for covariates, face mask type had a strongly significant independent effect on protection (p,0.001). Children were significantly less protected than adults (p,0.001). There was no significant impact of activity on protection.
As in the short term experiment, mask type was a strong determinant of protection (Table 2 ). Protection factors for each type of mask were similar to the protection factors measured in the short term experiments for adults. There was considerable variability between volunteers. The median protection factors measured over a 3 hour period increased for those wearing homemade masks, decreased for those wearing FFP2 masks, and did not show a consistent pattern for those wearing a surgical mask (Figure 2 ), but overall protection factors calculated per type of mask were stable over time, and did not change statistically significant with prolonged wearing. Overall, protection factors were relatively stable over time for each individual (ANOVA p = 0.4). Males and females did not have significantly different protection factors (ANOVA p = 0.9). As in the short term experiment, protection conferred by surgical masks was higher than protection given by a home-made mask, and protection provided by a FFP2 masks was again markedly higher than protection provided by a surgical mask. As in the short term experiment, more strenuous activities (reading and walking) tended to increase the protection of the home-made mask and to a lesser extent of the surgical mask, and decreased the protection by the FFP2 mask, but there was no overall significant effect of type of activity on PF (ANOVA p = 0.1). Outward protection experiment
In a final experiment, retention of particles expelled inside the masks was studied. Here again, mask type was strongly correlated with (transformed) protection factors. Protection factors for all type of masks were considerably lower than those observed for inward protection. The home-made masks only provided marginal protection, while protection offered by a surgical mask and an FFP2 mask did not differ ( figure 3) .
The simulated breathing frequency did not significantly affect the measured protection factors. Adjusting for covariates, mask type and particle concentration, but not flow rate, were significant factors for protection in the reverse flow experiment.
In our experiments, the main determinant of the magnitude of protection factors measured by masks was the type of mask, which can be seen as a proxy for potential reduction in infectious disease transmission. The duration of wear and the type of activity did not have a significant impact on exposure reduction. Thus, the expected superior protection conferred by a professional FFP2 mask compared to a surgical mask or a home-made mask was maintained when these FFP2 masks were worn by healthy lay people in spite of the increased risk of a poor fit and significant behavioural leakage.
Children were significantly less protected from exposure than adults, which might be related to an inferior fit of the masks on their smaller faces. Although we observed a high degree of individual variability in the degree of protection conferred as reflected in the wide interquartile ranges of the measured PFs, no systematic difference was found between men and women, suggesting a poorer fit only has a noticeable impact on protection when the mismatch between face and mask is considerable. All types of masks provided a much higher degree of exposure protection against inward transmission of particles, then in preventing outward transmission by a mechanical head as a proxy for an infected patient exposing the environment.
Data from professional users suggest a decrease in protection over time due to a reduction in fibre charges [13] . In our data, this effect was not significantly present, although a tendency towards reduced protection over time was seen for the FFP2 masks. Also, our study showed a high degree of individual variation in exposure protection. This is important as it reflects the presence of many different sources of variation, behavioural as well as anatomical, which can also be expected to be present if the general population would be requested to wear face masks in case of a pandemic. Furthermore, we do not know from these experiments whether reduced exposure has a linear or non-linear relationship to the reduction of infection risk.
Although this could imply that individual subjects may not always be optimally protected, from a public health point of view, any type of general face mask usage can still decrease viral transmission. Also, it is important not to focus on a single intervention in case of a pandemic, but to integrate all effective interventions for optimal protection. Surprisingly, the protection conferred by each of the masks appeared stable over time and was not dependent on activity. This suggests that leakage associated with suboptimal fit and compliance was stable over time. The tendency towards improved protection of the poorer fitting masks with increased activities such as reading, might be attributable to reduced leakage when breathing through the mouth rather than the nose, which could give some overpressure and thus reduce inward leakage. We had assumed that compliance would decrease during the three hours of continuous wearing, in particular with more strenuous activities. Indeed, among professionals like cullers, there have been some anecdotal reports that FFP3 masks were associated with poorer compliance than FFP2 masks in wearing. Where a reduction in protection was found with the FFP2 mask, the reverse was seen for the home-made mask. It is possible that the experimental situation, sufficient motivation to endure a relatively limited time of discomfort, and the absence of physically challenging activities, has provided more stable protection than might be found in reallife situations. However, overall these experiments show that significant protection against influenza transmission upon exposure can be conveyed also for lay people, including children, in spite of imperfect fit and imperfect adherence.
It is also clear that home-made masks such as teacloths may still confer a significant degree of protection, albeit less strong than surgical masks or FFP2 masks. Home made masks however would not suffer from limited supplies, and would not need additional resources to provide at large scale. Home made masks, and to a lesser degree surgical masks, are unlikely to confer much protection against transmission of small particles like droplet nuclei, but as the reproduction number of influenza may not be very high [14] a small reduction in transmissibility of the virus may be sufficient for reducing the reproduction number to a value smaller than 1 and thus extinguishing the epidemic [15] . Greater reduction in transmissibility may be achieved if transmission is predominantly carried by larger droplets. In a typical human cough half of the droplets may be small (,10 mm), but these comprise only a small fraction (2.5*10 26 ) of the expelled volume [12] . Smaller droplets may however more easily penetrate the smaller bronchi and be more effective in transmission [1] . A more detailed analysis of aerosol and droplet inoculation and infectivity may provide better insight into the impact of either transmission mode on population spread.
The difference in measured protection against inward and outward protection is remarkable, and cannot be explained from the available data as we only measured the overall effect. A differential effect on the amount of leakage seems most plausible. At the same time, we cannot exclude that wearing of face masks, even FFP2 or surgical masks by patients might still significantly reduce transmission. However, the observed limited particle retention in our experiments may still be an overestimate of protection, as it may for instance be challenging to enforce adherence to mask wearing by a patient who is short of breath. Wearing of masks by caregivers might be more feasible and more effective, in particular where additional preventive measures are in place as well for caregivers. Furthermore, we should bear in mind that this is an experimental study, with relatively small numbers of volunteers, which limits the generalisability of some of our findings. E.g., for masks to have any impact during an actual pandemic, people may need to be wearing masks during several weeks with many shorter or longer mask-free periods. Furthermore, the PFs may be an over-or underestimation of the actual protection conferred. And although our simulated patient varied its breathing frequency, we have not assessed the impact of e.g. coughing or sneezing on outward transmission through a mask.
A recent analysis of the 1918 epidemic, noted that cities where strict interventions were implemented early on to prevent transmission, were overall worse-off than cities where some degree of transmission occurred early on [16] . Given the need for the population to acquire sufficient natural immunity over time, it can not be excluded that the amount of protection conferred by home made masks might sufficiently reduce viral exposure to impact on transmission during the early waves, while allowing people enough exposure to start mounting an efficient immune response. Further field studies are needed to assess acceptability and effectiveness of masks worn by people from the general population. Also, experimental data are needed to develop dose-response models which may improve understanding of determinants of transmission. A cost-effectiveness analysis might give further insights in the relative benefits of home made masks.  Oct-4 Expression Maintained Cancer Stem-Like Properties in Lung Cancer-Derived CD133-Positive Cells CD133 (prominin-1), a 5-transmembrane glycoprotein, has recently been considered to be an important marker that represents the subset population of cancer stem-like cells. Herein we report the isolation of CD133-positive cells (LC-CD133(+)) and CD133-negative cells (LC-CD133(−)) from tissue samples of ten patients with non-small cell lung cancer (LC) and five LC cell lines. LC-CD133(+) displayed higher Oct-4 expressions with the ability to self-renew and may represent a reservoir with proliferative potential for generating lung cancer cells. Furthermore, LC-CD133(+), unlike LC-CD133(−), highly co-expressed the multiple drug-resistant marker ABCG2 and showed significant resistance to chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and paclitaxel) and radiotherapy. The treatment of Oct-4 siRNA with lentiviral vector can specifically block the capability of LC-CD133(+) to form spheres and can further facilitate LC-CD133(+) to differentiate into LC-CD133(−). In addition, knock-down of Oct-4 expression in LC-CD133(+) can significantly inhibit the abilities of tumor invasion and colony formation, and increase apoptotic activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). Finally, in vitro and in vivo studies further confirm that the treatment effect of chemoradiotherapy for LC-CD133(+) can be improved by the treatment of Oct-4 siRNA. In conclusion, we demonstrated that Oct-4 expression plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of LC-CD133(+). Future research is warranted regarding the up-regulated expression of Oct-4 in LC-CD133(+) and malignant lung cancer. Lung cancer is one of the leading causes of cancer-related deaths in industrialized countries [1, 2] . Radiotherapy and chemotherapy play significant and crucial roles in clinical antilung cancer treatment to achieve prolonged patient survival [3, 4] . However, a high failure rate and low median survival rate are observed in patients undergoing chemoradiotherapy with recurrent, intractable lung cancer [5] . To improve the patient survival rate, investigation to elucidate the mechanism of tumorigenesis of lung cancer is needed [5] . Recent data have demonstrated that tumors contain a small subpopulation of cells, i.e., cancer stem-like cells (CSCs) or cancer-initiating cells (CICs), which exhibit a selfrenewing capacity and are responsible for tumor maintenance and metastasis [6] . Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ''side population (SP)'' [7] . Ho and colleagues isolated and characterized SP cells from six human lung cancer cell lines and showed that an elevated expression of ABCG2 as well as other ATP-binding cassette transporters were positively correlated with resistance to multiple chemotherapeutic drugs [8] . In addition, Gutova and colleagues have purified uPAR-positive CSCs from three lung cancer cell lines. These uPAR-positive cells co-expressed with CD44 and MDR1, and had the ability to promote advanced malignancy and chemoresistance [9] .
CD133 (prominin-1), a 5-transmembrane glycoprotein, was first recognized in CD34 + progenitor populations from adult blood, bone marrow, and fetal liver cells [10] . Recently, CD133 has been considered an important marker to represent the subset population of CSCs in leukemia, brain tumors, retinoblastoma, renal tumors, pancreatic tumors, colon carcinoma, prostate carcinoma, and hepatocellular carcinoma [11] [12] [13] [14] [15] [16] [17] [18] [19] . Based on immunohistochemical findings, Hilbe and colleagues suggested that CD133-positive (CD133 + ) progenitor cells play a role in the development of tumor vasculature in patients with non-small-cell lung cancer (NSCLC) [20] . More recently, a well-designed study by Eramo and colleagues showed that lung cancer contains a population of CD133 + CSCs able to self-renew and generate an unlimited progeny of non-tumorigenic cells. These CD133 + cells are also resistant to conventional chemotherapy [21] . However, the gene regulation mechanisms in maintaining the self-renewal and drugresistant properties in putative cancer stem-like cells of lung tumors are still unclear.
Oct-4, a member of the family of POU-domain transcription factors, is expressed in pluripotent embryonic stem (ES) and germ cells [22] [23] . Oct-4 mRNA is normally found in totipotent and pluripotent stem cells of pregastrulation embryos [24] . Knocking out the Oct-4 gene in mice causes early lethality due to the lack of ICM formation, indicating that Oct-4 has a critical function for self-renewal of ES cells [25] . Oct-4 activates transcription via octamer motifs, and Oct-4 binding sites have been found in various genes, including fgf 4 (fibroblast growth factor 4) and pdgfar (platelet-derived growth factor a receptor) [26, 27] . This suggests that Oct-4 functions as a master switch during differentiation by regulating the pluripotent potentials of the stem cell, and Oct-4 plays a pivotal role in mammalian development [24, 25] .
In this study, the CD133-positive cells (LC-CD133 + ) and CD133-negative cells (LC-CD133 2 ) were isolated from tissue samples of lung cancer (LC) patients and LC cell lines. These LC-CD133 + cells possessed both the characteristics of stem-like cells and malignant tumors. Our data further demonstrated that Oct-4 expression in LC-CD133 + is involved in tumor malignancy of lung cancers and exhibits refractory properties for chemoradiotherapy in cancer stem-like cells. These results suggested that the expression of Oct-4 plays a crucial role in maintaining cancer stem-like and chemoradioresistant properties in lung cancerderived CD133 + cells.
This research followed the tenets of the Declaration of Helsinki and all samples were obtained after patients provided informed consent. The study was approved by the Institutional Ethics Committee/Institutional Review Board of Taipei Veterans General Hospital. The dissociated cells from the samples of nonsmall cell lung cancer patients (Table 1 ) and the lung cancer (LC) cell lines were labeled with 1 mL CD133/l micromagnetic beads per 1 million cells using the CD133 cell isolation kit (Miltenyi Biotech, Auburn, CA). CD133 + cells were cultured in a medium consisting of serum-free DMEM/F12 (Gibco-BRL, Gaithersburg, MD), N2 supplement (R&D Systems Inc., Minneapolis), 10 ng/ml human recombinant bFGF (R&D Systems) and 10 ng/ml EGF (R&D Systems) [28] .
The isolated CD133 + and CD133 2 cells were cultured in a 96well cell culture cluster (Corning Costar, Acton, MA) at a density of 3610 3 cells/well with 100 ml culture medium. At specific time points during cultivation, the medium was discarded and replaced with an equal volume (100 ml) of fresh medium containing 0.2 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, WI) and 0.038 mg/ml of phenazine methosulfate (PMS; Promega) and incubated for additional 1.5 hours in 37uC 5% CO 2 . Cell viability proportionate to optical density (OD) was measured by colorimetric assay in terms of mitochondria activity to convert tetrazolium salt into a colored soluble formazan product in the medium. The OD values were measured at the wavelength of 490 nm with a 1420 multilabel counter VICTOR from Wallac (PerkinElmer Wallac, Turku, Finland).
For real-time RT-PCR analysis, the total RNA of cells was extracted by using the RNA easy kit (Qiagen, Valencia, CA). Briefly, the total RNA (1 mg) of each sample was reversely transcribed in 20 mL using 0.5 mg of oligo dT and 200 U Superscript II RT (Invitrogen, Carlsbad, CA). The amplification was carried out in a total volume of 20 ml containing 0.5 mM of each primer, 4 mM 
An avidin-biotin complex method was used for the immunofluorescence staining in the differentiated spheroid and neuronallike cell. In brief, cells were plated onto poly-L-ornithine-coated glass coverslips and fixed with 4% paraformaldehyde for 15 to 20 minutes at room temperature, and then were washed twice (10 minutes each) with 16 PBS. Cells were permeabilized with 0.1% Triton X-100/PBS for 10 minutes at room temperature, and then twice (10 minutes each) with 16 PBS. The cells were then blocked with blocking solution for 30 minutes and were incubated with primary antibodies (Oct-4, Chemicon, Temecula, CA) for 1 hour at room temperature. We then washed the cells three times (10 minutes each) with 16 PBS. Immunoreactive signals were detected with a mixture of biotinylated rabbit antimouse IgG and Fluorsave (Calbiochem, San Diego, CA). Cells were further probed with fluorescein isothiocyanate (FITC)tagged secondary antibodies. Fluorescence images were visualized with a fluorescence microscope. To quantitatively analyze the fluorescence intensity, we recorded images with an inverted fluorescence microscope equipped with a CCD camera. The percentage of signal fluorescence per photographed field was analyzed by image processing software (Image Pro-Plus, Media-Cybernetics, Inc., Silver Spring, MD).
For cell surface marker identification, a single cell suspension of sixth-to eighth-passage cells from trypsinized spheres was stained with anti-CD133, CD117 (c-Kit), or ABCG2 and secondary fluorescein (FITC)-or phycoerythrin (PE)-coupled antibodies (Dako, Carpinteria). Cells were fixed with 2% paraformaldehyde and were analyzed with a BD FACSCalibur apparatus (Becton, Dickinson and Company, Franklin Lakes, NJ).
The gamma radiation (ionizing irradiation; IR) was delivered by a Theratronic cobalt unit T-1000 (Theratronic International, Inc., Ottawa, Canada) at a dose rate of 1.1Gy/min (SSD = 57.5cm). To evaluate the cell proliferation rate we seeded cells on 24-well plates at a density of 2610 4 cells/well. Cells were seeded 24 hours after IR and then they were analyzed by methyle thiazol tetrazolium assay (MTT assay, Sigma-Aldrich, St. Louis, MN). The amount of MTT formazon product was determined by using a microplate reader and the absorbance was measured at 560 nm (SpectraMax 250, Molecular Devices, Sunnyvale, CA).
Cisplatin, etoposide (VP16), and paclitaxel were obtained from Sigma-Aldrich and were dissolved in DMSO (Sigma-Aldrich) at 100 mM of stock solution.
The 24-well plate Transwell system with an 8-mm pore size polycarbonate filter membrane (Corning Costar, Corning, NY) was used. The filter membrane was coated with Matrigel (BD Biosciences, San Diego) diluted with serum-free medium and incubated overnight at 37uC. The cell suspensions were seeded to the upper compartment of the Transwell chamber at the cell density of 1610 5 in 100 ml serum free medium. After 24 hours, the medium was removed and the filter membrane was fixed with 4% formalin for 1 hour. The opposite surface of the filter membrane facing the lower chamber was stained with Hoechst 33342 (Sigma-Aldrich) for 3 minutes and the migrated cells were then visualized under an inverted microscope. The protocol of soft agar colony assay is described as follows. Each well (35 mm) of a six-well culture dish was coated with 2 ml bottom agar mixture (DMEM, 10% (v/v) FCS, 0.6% (w/v) agar). After the bottom layer solidified, 2 ml top agarmedium mixture (DMEM, 10% (v/v) FCS, 0.3% (w/v) agar) containing 2610 4 cells was added, and the dishes were incubated at 37uC for 4 weeks. Plates were stained with 0.5 ml of 0.005% crystal violet for 1 hour and then a dissecting microscope was used to count the number of colonies [29] .
The pLVRNAi vector and pCDH-MCS1-EF1-copGFP vector were purchased from Biosettia Inc. (Biosettia, San Diego, CA). The method of cloning the double-stranded shRNA sequence is described in the manufacturer's protocol. The siRNA oligonucleotide 59-CCGGCCCTCACTTCACTGCACTGTACTCGAGTA-CAGTGC AGTGAAGTGAGGGTTTTT-39 targeting human Oct-4 (NM_002701, nt 1035-1055) was synthesized and cloned into pLVRNAi to generate a lentiviral expression vector. The Oct-4 cDNA was amplified and purified by RT-PCR and cloned into a pCDH-MCS1-EF1-copGFP vector. Lentiviral production was done by transfection of 293T cells using Lipofectamine 2000 (LF2000, Invitrogen). Supernatants were collected 48 hours after transfection and then were filtered; the viral titers were then determined by FACS at 48 hours post-transduction. Subconfluent cells were infected with lentivirus at a multiplicity of infection of 5 in the presence of 8 ìg/ml polybrene (Sigma-Aldrich).
All procedures involving animals were in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. 1000, 3000, and 10 4 cells were injected into the tail vein of SCID mice and/or nude mice (BALB/c strain) each aged 8 weeks. In vivo GFP imaging was visualized and measured by an illuminating device (LT-9500 Illumatool TLS equipped with excitation illuminating source [470 nm] and filter plate [515 nm]). The tumor size was measured with calipers and the tumor volume was calculated according to the formula (Length6Width 2 )/2. The integrated optical density of green fluorescence intensity was captured and then analyzed by Image Pro-plus software [29] .
Statistical Package of Social Sciences software (version 13.0) (SPSS, Inc., Chicago, IL) was used for statistical analysis. The independent Student's t-test or ANOVA was used to compare the continuous variables between groups, whereas the x 2 test was applied for comparison of dichotomous variables. The Kaplan-Meier estimate was used for survival analysis, and the log-rank test was used to compare the cumulative survival durations in different patient groups. The level of statistical significance was set at 0.05 for all tests.
Using the magnetic bead method, we isolated CD133 + cells (Fig. 1A) from tissue samples of ten non-small cell lung cancer (NSCLC) patients (Table 1 ) and five lung cancer (LC) cell lines ( Table S1 ). The high percentage (97%) of CD133 + (LC-CD133 + ) subset was isolated in the LC tissues and parental LC cell line (Fig. 1A) . It has been reported that cancer stem-like cells can be cultured in suspension to generate floating spheroid-like bodies (SB) under serum-free medium with bFGF & EGF [30] . We found that LC-CD133 + isolated from these ten patients (Table 1 ) and five LC cell lines (Table S1 ) can form SB in DF-12 serum-free medium with bFGF and EGF ( Fig. 1B 1C ) and proliferation rate (Fig. 1D ) in LC-CD133 + were significantly higher than that in LC-CD133 2 (p,0.05). In addition, to determine the in vivo tumorigenic activity of LC-CD133 + and LC-CD133 2 , we injected respective amounts of 1000, 3000, and 10 4 cells into the tail veins of SCID mice. The results showed that 10 4 LC-CD133 2 did not induce tumor formation but 3,000 LC-CD133 + from the lung cancer tissues of ten patients and five LC cell lines in xenotransplanted mice can all generate visible tumors 4 weeks after injection (Table 1 and Table S1 ).
To characterize our isolated LC-CD133 + , FACS analysis was used to detect the expression profile of cells surface markers. As shown in Figure 2A , the majority of isolated LC-CD133 + were stained with higher expression levels of CD133, CD117 (c-Kit), and ABCG2 compared with LC-CD133 2 . This result demonstrated that isolated LC-CD133 + were almost ABCG2-positive cells (Fig. 2B) . To further evaluate the enhancement of tumorigenicity of LC-CD133 + , we examined in vitro Matrigel-combined Transwell invasion and soft agar colony formation assays. Compared with LC-CD133 2 , LC-CD133 + derived from NSCLC Patients No.1 (PLC) and No. 2 (LLC) showed higher invasion activity through Matrigel Transwell invasion assay (p,0.001; Fig. 2C ). Similarly, the foci formation ability of LC-CD133 + from PLC (No.1) and LLC (No.2) was enhanced when compared with the LC-CD133 2 of those two patients (p,0.001; Fig. 2D ).
Increased In Vivo Tumor-restoration and Proliferative Ability in LC-CD133 + We further evaluated the in vivo tumor-restoration and proliferative ability of LC-CD133 + and LC-CD133 2 by xenotransplanted tumorigenicity analysis (Fig. 3A) . Four weeks after 10 4 cells were injected into the tail veins of SCID mice, a significant increase in the multiple nodules of tumor formation on lung surface was noted in the LC-CD133 + -injected group (Figs. 3A4 & 3A7) but not in the LC-CD133 2 group (Fig. 3A1) . Diffuse infiltrations of LC-CD133 + from the lung parenchyma to the alveolar cavity were observed (Figs. 3A5, 3A6, and 3A8) . The histological examination demonstrated that the prominent neovascularization and thrombus formation were detected in the pulmonary parenchyma of LC-CD133 + -injected SCID mice (Fig. 3A9) . In contrast, no significant tumor foci or neovascular formation was found in the lungs of LC-CD133 2 -injected SCID mice (Figs. 3A2 & 3A3) . We further investigated the in vivo tumor growth rate in 10 4 LC-CD133 + cells, 10 6 LC-CD133 2 cells, and 5610 6 parent tumor cells from the same patient. The finding demonstrated that the tumor growth rate of the 10 4 LC-CD133 + group (from Patients No. 1, 2, 4, and 7; Table 1 ) was significantly higher than that of the 10 6 LC-CD133 2 group and 5610 6 parental tumor cell group (Fig. 3B) . Furthermore, 10 4 LC-CD133 + isolated from secondary tumors can further generate new (second) tumors from transplanted SCID mice. Results of flow cytometry showed that a high percentage (60%) of CD133-positive cells was detected in the second tumor (Fig. 3C ). In addition, one thousand LC-CD133 + isolated from the second tumor can also generate a new (third) tumor in transplanted SCID mice (Fig. 3C) . To sum, our data indicated that LC-CD133 + present self-renewing and repopulation capabilities both in vitro and in vivo.
Enhanced Chemo-and Radiation-resistance in LC-CD133 + We evaluated the multidrug (chemotherapy)-resistant abilities of LC-CD133 + and LC-CD133 2 . We further tested four common chemotherapeutic agents including cisplatin, VP16 (etoposide), doxorubicin, paclitaxel. Compared with LC-CD133 2, LC-CD133 + are significantly resistant to the four tested chemotherapeutic agents (p,0.01; Fig. 4A ). To further determine the radiation effect on the rate of tumor growth, we used an ionizing radiation (IR) dose from 0 to 10 Gy to treat both LC-CD133 + and LC-CD133 2 . As shown in Fig. 3B , after IR treatment, the survival rate and number of LC-CD133 + were significantly higher than those of LC-CD133 2 (p,0.01). We further found that the LC-CD133 + cells possess a higher degree of radioresistance (p,0.01; Fig. 4B ). Moreover, we investigated the combined treatment effect of radiochemotherapy in LC-CD133 + . Experiments were conducted with cisplatin (10 mM) alone, VP-16 (10 mM) alone, or combined cisplatin and VP-16 on IR (2 Gy)-treated LC-CD133 + . As shown in Figure 3C , the data revealed that the cell survival rate in IR-treated LC-CD133 + was not significantly decreased by the IR treatment combined with cisplatin, with or without VP-16 (p.0.05). On the contrary, cell survival significantly declined after chemotherapy with cisplatin combined with VP-16 in IR-treated LC-CD133 2 (p,0.01; Fig. 4C ). These results suggest that LC-CD133 + may play a vital role in the tumor's ability to resist radiation and chemotherapies. To investigate whether Oct-4 expression plays a role in maintaining self-renewal or cancer stem-like properties in LC-CD133 + , we used the siRNA method with lentiviral vector for knockdown of Oct-4 expression in LC-CD133 + . We found it important that the treatment of Oct-4 siRNA in LC-CD133 + can significantly interfere with the capabilities of spheroid-like bodies (SB) formation (p,0.001; Fig. 5C ). After 7 days of the Oct-4 siRNA treatment, the SB of LC-CD133 + could not maintain floating spheres but differentiated into attached epithelial-like cells (Fig. 5C ). In contrast, the treatment of scramble control siRNA did not influence the SB formation capability in LC-CD133 + (Fig. 5C) . The SB of LC-CD133 + endogenously expressed strong positive signals for Oct-4 and CD133 (Fig. 5D) . Furthermore, the immunofluorescent results demonstrated that both the CD133 and Oct-4 expressions in LC-CD133 + were significantly blocked after 7 days of Oct-4 siRNA treatment (Fig. 5D) . FASC assay confirmed that the amount of CD133 was dramatically decreased in Oct-4 siRNA-treated LC-CD133 + and the percentages of LC-CD133 2 were significantly increased in LC-CD133 + after 7 days of Oct-4 siRNA treatment (p,0.001; Fig. 5E ). These data suggested that Oct-4 may maintain the properties of primitive stem cells and inhibit the tendency for differentiation in LC-CD133 + .
To further study the role of Oct-4 in tumor malignancy of LC-CD133 + in vitro, the migration/invasive and soft agar colony assay were used. The results showed that the abilities of the in vitro migratory invasion and colony formation in LC-CD133 + treated by Oct-4 siRNA were significantly decreased compared with nontreated LC-CD133 + , or LC-CD133 + treated with scramble-siRNA (control; p,0.001; Fig. 6A ). Furthermore, the treatment effect of chemoradiotherapy for the LC-CD133 + group can be significantly improved by the treatment of Oct-4 siRNA compared with nontreated LC-CD133 + or LC-CD133 + treated by scramble-siRNA ( Fig. 6B; p,0.001 ). In addition, we found that the apoptotic activities of annexin V (Fig. 6C) and caspase 3 (Fig. 6D; upper part) were quickly and effectively induced in LC-CD133 + treated by Oct-4 siRNA after 72 hours. In accordance with the result of cell survival and treatment effects in Oct-4 siRNA-treated LC-CD133 + (Fig. 6B) , the western blot data further demonstrated that the amounts of activated (cleaved) form of PARP were consistently elevated in LC-CD133 + treated by Oct-4 siRNA with IR alone or combined with chemotherapy ( Fig. 6D; lower part) . Thus, knockdown of Oct-4 expression in LC-CD133 + can effectively enhance chemoradiosensitivities and apoptotic activities in response to IR and chemotherapy, suggesting that Oct-4 could be a key factor enables LC-CD133 + to resist radiochemotherapeutic stress.
To investigate the treatment effects of chemoradiotherapy on Oct-4 siRNA-treated LC-CD133 + , LC-CD133 + was first transfected by lentivector combined with green fluorescent protein gene (GFP), and then in vivo GFP imaging and histological study were used to monitor the tumor-growth effect. We first injected 10 4 LC-CD133 + -GFP cells into the subcutaneous sites of nude mice with different treatment protocols. The tumor volumes were significantly decreased in Oct-4 siRNA-treated LC-CD133 + when exposed to IR alone, cisplatin alone, or IR combined with cisplatin (p,0.01; Fig. 7A ). To further evaluate the capabilities of tumor invasion and metastasis of LC-CD133 + treated by different regimens, we injected 10 4 LC-CD133 + -GFP cells from each treatment groups into the tail vein of SCID mice. The results of in vivo GFP imaging showed that the tumor foci of lung and metastatic lesions in the Oct-4-siRNA-treated LC-CD133 + groups were significantly lower than those of the LC-CD133 + without Oct-4-siRNA-treated groups (p,0.01; Fig. 7B ). In addition, to investigate the treatment effects of Oct-4 expression in LC-CD133 + in vivo, we injected the seven groups with different regimens individually into the tail vein of SCID mice for xenotransplanted tumorigenicity analysis (Fig. 7C ). Immunohistochemistry (IHC) showed that the expression levels of Oct-4 in the lung tumors of LC-CD133 + -injected SCID mice were highly expressed in comparison with the other treated-groups (Fig. 7C) . Oct-4-IHC levels were significantly decreased in Oct-4 siRNAtreated LC-CD133 + when exposed to IR alone, cisplatin alone, or IR combined with cisplatin (p,0.01; Fig. 7C ). Moreover, using combined chemoradiotherapy with the treatment of Oct-4 siRNA, the mean survival rate of the LC-CD133 + group was significantly prolonged compared with the control or other treated groups (p,0.05; Fig. 7D ). This in vivo study also confirmed that the treatment effect of chemoradiotherapy for the LC-CD133 + group can be improved by the treatment of Oct-4 siRNA.
Self-renewal and pluripotency are the central features in the definition of embryonic stem cells (ESC), and Oct-4 is a key regulator in this process [24] [25] [26] . Oct-4 has been suggested as one of four major factors that render the reprogramming capability of adult cells into germline-competent-induced pluripotent stem cells [31] [32] [33] . Previous studies also showed that mouse pulmonary stem cells endogenously express Oct-4 [34] . Recently, Oct-4 transcript can be consistently detected in human embryonal carcinomas, testicular germ cell tumors, seminomas, and bladder carcinomas [35] [36] [37] [38] . The expression of Oct-4 has further been shown in human breast cancer stem-like cells, suggesting that its expression may be implicated in self-renewal and tumorigenesis via activating its downstream target genes [39] . Herein we reported the isolation of CD133-positive cells (LC-CD133 + ) from clinical tissue samples and lung cancer cell lines. LC-CD133 + showed strong proliferative and invasive capabilities in vitro and in vivo (Figs. 1, 2, and 3) . LC-CD133 + also displayed significant resistance to chemotherapeutic agents (Fig. 4) . We also demonstrated that Oct-4 expression was transcriptionally and translationally up-regulated in LC-CD133 + (Fig. 5) . Indeed, Oct-4 functions as a master switch during differentiation by regulating the pluripotent potential in stem cells [31] [32] [33] . Using the siRNA method with lentiviral vector for knockdown of Oct-4 expression in LC-CD133 + , our data showed that the treatment of Oct-4 siRNA can block the sphere formation of LC-CD133 + and further facilitate LC-CD133 + to differentiate into LC-CD133 2 (Fig. 5) . Furthermore, in vivo animal studies demonstrated the IHC of Oct-4 in the lung tumors of LC-CD133 + -injected SCID mice were prominently up-regulated, and the total lung tumor volume as well as Oct-4 IHC levels can be significantly decreased in LC-CD133 + -injected mice by the treatment of Oct-4 siRNA with or without chemoradiotherapy (Fig. 7) . In addition, we showed that increased incidence of Oct-4 expression correlated positively with the advanced stage of 78 lung cancers (Figs. S1A-C). To our knowledge, this is the first study to show that Oct-4 expression plays a crucial role in maintaining selfrenewal and cancer stem-like properties in LC-CD133 + .
The property of resistance to chemotherapy and irradiation treatment is the major clinical criterion to characterize ''cancer stem-like cells (CSCs)'' [6] . The existence of cancer stem-like cells may explain why conventional anti-cancer therapies are able only to suppress or shrink a tumor but often cannot completely eradicate it, resulting in eventual recurrence [6, 40, 41] . Consistent with these hypotheses, LC-CD133 + were significantly resistant to cisplatin, VP16 (eptoposide), doxorubicin, and paclitaxel than LC-CD133 2 (p,0.001; Fig. 4 ). Even IR alone or a single chemodrug can effectively inhibit cell growth of LC-CD133 2 (Fig. 4) ; however, IR treatment combined with cisplatin and VP-16 still failed to cause cell death in treated LC-CD133 + (Fig. 4D) . To overcome resistance to radiotherapy and chemotherapy in LC-CD133 + , treatment of Oct-4 siRNA was used and results showed that the knockdown Oct-4 in LC-CD133 + can significantly improve the anti-cancer effect in single-or combination-treated LC-CD133 + in vitro and in vivo (Figs. 6 and 7) . Moreover, the mean survival rate of the LC-CD133 + group can be significantly prolonged after the treatment of Oct-4 siRNA with IR and chemotherapy (Fig. 7) . Recently, Oct-4 has been suggested to be a protector for survival of ES cells from apoptosis induced by etoposide, UV, or heat shock through the Stat3/Survivin pathway [42] . Consistent with this important finding, our results suggest that knockdown of Oct-4 expression can effectively enhance the chemoradiosensitivity of LC-CD133 + through activating the apoptotic activities of caspase 3 and PARP (Fig. 6) . Importantly, our in vivo animal study and clinical data provide the evidence that the amount of Oct-4 in LC-CD133 + (Fig. 7C ) and in patients with high-grade lung cancer (Fig. S1 ) is positively correlated with the degree of resistance to chemoradiation therapy. Taken together, these results indicate that the up-regulated expression of Oct-4 in LC-CD133 + may contribute to the development of chemoradioresistance in patients with lung cancer.
Recent studies have revealed that the human ABCG2 transporter is a P-glycoprotein that causes multidrug resistance (MDR) including mitoxantrone, doxorubicin, and topoisomerase I inhibitors of irinotecan, topotecan, and 7-ethyl-10-hydroxycamptothecin (topoisomerase inhibitor) and gefitinib (an inhibitor of EGF receptor) in patients with lung cancer [41, 43] . Hirschmann-Jax and colleagues were the first to observe that ''side population'' cancer stem-like cells isolated from cell lines and patients with neuroblastoma expressed high levels of ABCG2 and ABCG3 transporter genes as well as a greater capacity to expel cytotoxic drugs [44] . Monzani and colleagues further showed that cancer stem-like cells derived from the melanoma cell line highly coexpressed CD133 and ABCG2 markers with enhanced tumorigenic potential [45] . In this study, we found that LC-CD133 + are highly co-expressed with ABCG2 transporter and are significantly resistant to conventional treatment methods compared with LC-CD133 2 (Figs. 2 & 4) . Interestingly, a significant down-regulating of ABCG2 expression and an increase in the chemosensitivity of LC-CD133 + were observed when the Oct-4 siRNA treatment was given (Data not shown). Thus, more studies are needed to investigate whether over-expression of Oct-4, CD133, and/or ABCG2 play a role in the development of MDR in LC-CD133 + or surrogate markers of therapeutic response in patients with lung cancer.
In conclusion, we demonstrated that LC-CD133 + display a higher Oct-4 expression with the ability to self-renew and may represent a reservoir with unlimited proliferative potentials for generating lung cancer cells. The resistance of LC-CD133 + to in vitro and in vivo chemoradiotherapy is partially due to preferential activation of Oct-4 gene expression. In addition, these data support that the up-regulated expressions of the Oct-4, selfrenewing gene of embryonic stem cells, play an important role in the tumorigenicity of patients with lung cancer.
Table S1
Found at: doi:10.1371/journal.pone.0002637.s001 (0.05 MB DOC)  Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-γ induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213–1220, 2005). This protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users. With an estimated death rate of more than 400,000 per year, mainly affecting children less than 5 years of age in developing countries, rotavirus is recognized as one of the major infectious diseases of the gastrointestinal tract [38] . Rotaviruses are members of the family Reoviridae, viruses with segmented double-stranded RNA genomes [17] .
In the small intestine, mature enterocytes near the top of the villi are the primary target cells for virus replication [29] . Replication in these cells provokes numerous intraand extracellular pathological changes that inevitably lead to disruption of the absorptive and digestive functions of the small intestine, and consequently, to malabsorption and diarrhea. These changes include destruction of enterocyte brush borders, enterocyte vacuolization, loss and destruction of enterocytes, villus blunting and atrophy, thinning of the intestinal wall, and crypt hyperplasia (for comprehensive reviews, see [29, 39] ). However, the nature and severity of histopathological alterations in vivo can be quite different depending on the species and virulence of the rotavirus strain. There is no clear correlation between these alterations and manifestation of clinical symptoms. A systemic inflammatory response can be absent, and rotavirus infections can be asymptomatic [29, 39] . This suggests that the interplay between host and viral factors is important for determining the course of this disease. For Electronic supplementary material The online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users.
instance, rotavirus NSP4 acts as an enterotoxin that induces diarrhea in mice in the absence of rotavirus replication [5] . NSP4 affects Ca 2+ and electrolyte homeostasis in an auto-and paracrine fashion in both rotavirus-infected and uninfected intestinal cells [41] . NSP4 increases Ca 2+ permeability of the ER and plasma membrane, resulting in an increased Ca 2+ concentration in the cytosol ([Ca 2+ ] cyt ), causing derailment of numerous Ca 2+ -dependent cellular processes [40] . In uninfected enterocytes and crypt cells this rise in [Ca 2+ ] cyt is induced by binding of exogenous NSP4 to an apical receptor that modulates the PLC-IP 3 pathway [16] . The higher [Ca 2+ ] cyt triggers laminal secretion of peptides and amines by uninfected enterocytes, and luminal Cland H 2 O secretion by crypt cells [28, 39] . In infected enterocytes, the rise in [Ca 2+ ] cyt is believed to be independent of PLC modulation [41] , and this rise perturbs cytoskeleton and tight junction integrity, which ultimately leads to cell lysis [9, 10, 26, 37] .
In vitro studies with cell lines, mainly derived from colon, have contributed significantly toward understanding the pathogenesis of rotavirus on a molecular level. However, the intestinal mucosa consists of a diversity of specialized cell types in different states of differentiation. Presumably, all these different types of cells respond differently to environmental changes, and accordingly to changes in their neighboring cells. Therefore, the regulation of genes responsible for these complex phenotypic responses in vivo may not be detected by challenging single types of cultured cells with rotavirus. To address this issue, we studied the early transcriptional response in jejunal mucosa of 3-week-old, just-weaned piglets after oral infection with virulent group A rotavirus. To assign measured responses exclusively to rotavirus, we performed these experiments in germ-free piglets. Differential expression patterns of uninfected versus infected jejunum were recorded 12 and 6 h before severe diarrhea was expected, using a homemade pig intestinal cDNA microarray [34] . The biological significance of elevated or reduced expression of these genes for rotavirus pathogenesis is discussed.
Seven germ-free piglets (Groot Yorkshire 9 [Cofok 9 Large White]) were obtained by caesarean section and housed in isolators, fed with sterilized condensed milk till the age of 14 days and thereafter with pelleted feed (sterilized by X-ray radiation) and water ad lib. On day 21, three of the seven piglets were transported to the necropsy room and served as uninfected control piglets.
The four remaining pigs were orally infected with virus suspension diluted in a total volume of 5 ml PBS and containing 2 9 10 7 rotavirus particles (as determined by negative-stain semi-quantitative electron microscopy) of strain RV277 [45] . The virus suspension was prepared from the contents of the small and large intestine of a rotavirus-infected gnotobiotic piglet [32] . The above applied oral dose caused severe diarrhea from 24 hpi (hours post infection) in 3-week-old gnotobiotic piglets [32] . Infected piglets were housed in their isolators under the same conditions as described above for another period of 12 (two piglets) or 18 h (two piglets) before they were transported to the necropsy room. Immediately after arrival in the necropsy room, 10 ml of EDTA blood for hematological analysis was collected from the jugular vein. Subsequently, animals were killed by barbiturate overdose and their intestines were taken out. The jejunum was opened and rinsed with cold saline, and 10 cm of mucosa in the middle of the jejunum was scraped off with a glass slide, frozen in liquid nitrogen, and kept at -70°C until RNA and DNA extraction. An adjacent part of the collected jejunum was fixed in 4% formaldehyde and used to determine the villus height and crypt depth. Villus and crypt dimensions were determined on hematoxylin-eosinstained 5-lm tissue sections [34] . During the experiment, fecal samples were collected at 0, 12 and 18 hpi from the rectum for determination of the percent dry matter [18] . Fecal samples were tested for the presence or absence of rotavirus by ELISA [33] . The germ-free status of each piglet was confirmed by analyzing throat saliva and feces samples, collected on days 6, 12 and 19, and on the day of slaughter, for the presence of microorganisms.
Isolation of RNA and DNA From 1 g of frozen mucosal scrapings, total RNA (DNasefree) was isolated using TRIzol Ò reagent (Invitrogen) as described recently [34] . The yield per gram of tissue and the purity of the RNA were calculated from measurement of the extinction at 260 and 280 nm. The integrity of all RNA samples was checked by analyzing 5 lg of RNA on a denaturizing 1% (w/v) agarose gel. After ethidium bromide staining, the gel was scanned to calculate the 28S/18S peak ratio (volume 28S over volume 18S) for each preparation. RNA with a ratio [2 was considered of adequate quality to be used for real-time PCR and microarray analysis. A part of the isolated RNA was used to prepare RNA pools for microarray analysis. A control pool was prepared by mixing equal amounts of RNA isolated from the jejunum of the three uninfected piglets (n = 3). The same was done for the two infected piglets slaughtered at 12 h and for the two piglets slaughtered at 18 h. After gentle homogenization in lysis buffer, DNA was extracted from 0.5 g of frozen mucosal scrapings, and 4 lg of purified DNA was analyzed on a 0.9% agarose gel [22] .
The relative concentrations of interferon-gamma (IFN-c) and ornithine decarboxylase antizyme 1 (OAZ1) mRNA in all RNA samples was determined by real-time PCR. Two hundred ng of total RNA was reverse transcribed in a standard RT reaction using Superscript II reverse transcriptase (Invitrogen) and pd(N) 6 primers. IFN-c cDNA in these RT reactions was quantified using labeled Light-Cycler probes (Roche Diagnostics) as described [15] and expressed as pg/ll control plasmid. A 20-mer forward primer (5 0 -GACCCGACGCTTGCTTCATG-3 0 ) and a 19mer reverse primer (5 0 -GAGTGAGCGTTTATTTGCAC-3 0 ), generating a cDNA fragment homolog to nucleotide 702-895 of the human OAZ1 mRNA reference sequence (gi:34486089), were used to quantify OAZ1 cDNA using Cybergreen as label in a standard LightCycler reaction. The relative concentration of OAZ1 mRNA was calculated by extrapolation on a standard curve prepared from dilutions of an RT reaction prepared from a reference RNA sample [34] . The quantity of 18S rRNA in each RNA sample was determined using the above described RT reactions by real-time PCR [15] and used to normalize the IFN-c and OAZ1 concentrations. The quantity of 18S rRNA showed no essential differences among all individual RNA samples (average concentration ± SD; 4.95 ± 0.82 lg/ll of control plasmid).
The same collection of pig probes (ESTs) used in earlier studies [34, 35] were spotted in triplicate on Corning Ul-traGAPS slides. Briefly, this collection consisted of 2,928 probes prepared from jejunal mucosal scrapings collected from 4-week-(672) and 12-week-(2,256) old pigs, probes coding for porcine cytokines (IFN-c, TNF-a, GMCSF, IL-2, 4, 6, 8, and 10) and lung surfactant proteins SFTPA and SFTPD, and 110 Marc1 and Marc2 probes (porcine ESTs) homolog to trefoils, collectins, defensins, and glycosyltransferases [34] . A list of the probes already sequenced/ annotated is accessible on the website of Arch Virol (gene list Hulst et al. pdf).
Dual-color (Cy3-Cy5) hybridization of slides was performed using the RNA MICROMAX TSA labeling and detection kit (PerkinElmer) as described earlier [34] . Messenger RNA levels in both infected pools (12 and 18 hpi) were independently compared to the expression levels in the control (uninfected) pool. For each comparison, a dye swap was performed. In addition, a control hybridization experiment was performed in which a microarray slide was simultaneously hybridized with Cy3labeled control RNA and Cy5-labeled control RNA. Scanning of slides, processing of raw images, creation of data reports, data-normalization and statistical analysis were performed as described by Niewold et al. [34] with minor modifications. Briefly, probes were considered to be differentially expressed when at least four of the six data points (spots) on both dye swap slides hybridized with a ratio of 3.6-fold (M = [log 2 (Cy3/Cy5)] \ -1.85 or [1.85) or more (3.6 is considered significant according to the manufacturer of the TSA kit) and were identified by significant analysis of microarrays (SAM) [43] with a median false discovery rate (FDR or q value) of \5%.
Equal amounts of total RNA (5 lg) were separated on a denaturizing 1% (w/v) agarose gel. After several washes with RNase-free water, the gel was blotted on Hybond-N membranes (Amersham), and blots were hybridized with 32 P-labeled DNA fragments homolog to the mRNA in question, in the same manner as was described in an earlier study [34] . After post-hybridization washes, the blots were scanned using a Storm phosphor-imager (Molecular Dynamics, Sunnyvale, California, USA).
Four 3-week-old germ-free piglets were orally infected with a dose of rotavirus that caused severe diarrhea from 24 hpi in 3-week-old gnotobiotic piglets [32] . For practical reasons, three uninfected germ-free piglets were slaughtered at the zero time point (mock, see Table 1 ). In order to isolate highquality RNA from jejunal mucosal scrapings, infected piglets were slaughtered 12 and 18 hpi. Thus, 12 and 6 h before severe diarrhea would have been induced. In three of the four infected animals, rotavirus was detected in their feces. Determination of the percent dry matter showed that only the fecal samples collected 18 hpi (piglets 65 and 67) had a significantly lower (pasty) consistency [18] . This indicated that not all of the piglets developed diarrhea before 12 hpi, and that the two piglets slaughtered 18 hpi did not develop the severe form of diarrhea normally observed at 24 hpi [32] . In jejunal tissue sections prepared from these two 18-h piglets, villus length was decreased to two-thirds of the average length measured in corresponding sections prepared from the three control piglets. No significant differences in crypt depths were observed between infected and control animals. For both piglets that were slaughtered at 18 h, these results indicated that the orally applied rotavirus reached the Transcriptional response to rotavirus 1313 jejunum and induced the desired limited (not severe) pathological symptoms. In addition, the lower concentration of lymphocytes in the blood indicated that the animals were effectively infected with rotavirus [47] .
In the feces of piglet 63, slaughtered at 12 h, no rotavirus could be detected. Moreover, only a small decrease in villus length (20%) was observed for this piglet and its 12-h replicate. To find additional evidence whether the jejunum of piglet 63 was effectively challenged with rotavirus, the level of IFN-c mRNA in jejunal mucosal scrapings was measured by real-time PCR (Fig. 1 ). In RNA samples isolated from the three uninfected pigs, hardly any IFN-c mRNA could be detected. In contrast, the IFN-c mRNA levels in scrapings of all infected piglets were up to 50-fold higher than in uninfected piglets, whereas OAZ1 mRNA levels were nearly equal for all RNA samples. This indicated that the jejunum of all piglets, including piglet 63, responded to the orally applied rotavirus challenge.
Despite the loss of cells from the tip of the villi, the amount of RNA (Table 1 ) isolated from all infected piglets was comparable to that of uninfected piglets. On an ethidium-bromide-stained agarose gel, no degradation of RNA was visible for any of the extracted RNA samples (see also Fig. 2a ). In addition, 28S/18S peak ratios were [2 for all these samples (Table 1 ). These results showed that scrapings collected from all piglets yielded high-quality RNA suitable for real-time PCR and microarray analysis. No random (necrotic) and/or fragmentized (apoptotic) DNA was visible after gel electrophoreses of DNA samples extracted from any of the piglets, indicating that the majority of cells imbedded in the epithelial layers of all the piglets were not apoptotic or necrotic (results not shown).
Mid-jejunal mucosal gene expression analysis was performed using a homemade pig cDNA small intestinal microarray [34] . In two separate hybridization experiments, mRNA expression levels in an uninfected RNA pool (n = 3) were compared to expression levels in RNA pools prepared from 12-and 18-h infected piglets (both n = 2). For both comparisons, dye swaps were performed. Probes that hybridized differentially with a ratio (FC; infected over uninfected) of \0.28 or [3.6 in both slides of the dye-swap and that were identified with the lowest possible false-discovery rate (FDR; based on SAM, [43] ), i.e., 0.35% for the 18-h comparison and 4.6% for the 12-h comparison, were selected for further analysis. Raising of the FDR to a maximum of 10% did not identify additional probes with a FC \ 0.28 or [3.6 in either comparison. Selected probes were sequenced and annotated after blastn or blastx analysis (when not yet annotated). For each differential expressed probe, the mean FC calculated from the two dye-swap slides is presented in Table 2 . When Cy3-and Cy5-labeled cDNA was prepared from the same uninfected RNA pool and simultaneously hybridized on the array, none of the probes that hybridized differentially in the 12-and 18-h comparisons hybridized differentially (results not shown). Six out of the nine probes that hybridized with a FC of 3.6-fold or more in the 12-h comparison (panel ''higher in infected'') also hybridized significantly more strongly in the 18-h comparison. For three of these probes (R14-R16), the ratio of differential expression further increased with time. In contrast, only one probe (R1) hybridized significantly less strongly at both time points. Based on literature search and data mining, a tentative function was assigned for the genes identified by blast analysis (see Table 2 ). In addition, the FC (infected over uninfected) of genes which were also found to be regulated in a previous study by Cuadras et al. [13] , i.e., 16 h after infection of human intestinal epithelial Caco-2 cells with rotavirus live virus vaccine RVV, is provided in parentheses in Table 2 after the annotations. Probes coding for IFN-c, TNF-a, GM-CSF, and IL-2, 4, 6, 8, and 10 were spotted on the array. However, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mRNA concentrations of these cytokines (including IFN-c) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [6] . 
Northern blots (NB) loaded with equal amounts of RNA from each of the piglets were hybridized with P 32 -labeled cDNA probes homologous to six differentially expressed mRNAs and to three mRNAs that were not identified as differentially expressed. For all nine mRNAs, the length of the transcript(s) detected on blots were comparable to the length of porcine or human mRNA reference sequences posted in the NCBI databank or reported in the literature.
In accordance with array data, NB analysis showed that expression levels of GBP-2, KRT20, SAT, MGAM, and CASP3 mRNAs were significantly higher in both 18-hinfected piglets than in uninfected piglets. For all of these mRNAs, hybridization intensities for piglet 65 were nearly equal to that of piglet 67. GBP-2, KRT20, SAT, and MGAM mRNA expression was also up-regulated in infected piglet 63, slaughtered at 12 hpi. This indicated that the response to rotavirus infection in this 12-h piglet was comparable to the response observed in both 18-h piglets. However, no significant up-regulation of these mRNAs was observed in the other piglet slaughtered at 12 h (64), indicating that this piglet responded differently to the rotavirus infection than its 12-h replicate and the two piglets slaughtered at 18 h. In accordance with array data, NB analysis showed that the expression level of IFABp2 mRNA was significantly lower in 18-h-infected piglets than in uninfected piglets. For mRNAs that showed no significant differential expression on the arrays (glutathione-S-transferase, calbindin-D, and aldolase-B), no large differences in hybridization intensities were observed between uninfected piglets and the two piglets slaughtered 12 hpi and one of the piglets slaughtered 18 hpi (65). However, significantly lower hybridization intensities were observed for calbindin-D and aldolase-B mRNAs for piglet 67 than for its 18-h replicate.
Using microarray analysis, we detected a set of genes that are differently expressed in rotavirus-infected jejunal mucosa compared to uninfected mucosa. For nine mRNAs, expression levels in individual piglets were analyzed by NB. These analysis confirmed the array data. In addition, NB analysis showed that one piglet slaughtered at 12 hpi responded quite similarly to rotavirus infection as both 18h piglets did, whereas its 12-h replicate did not, despite the fact that this latter piglet also showed an IFN-c mRNA response. In addition, 7 out of the 12 genes differentially expressed at 12 hpi also reacted at 18 hpi. These results indicated that three out of four infected piglets responded quite analogously. Because we used a limited number of germ-free piglets per time point and measured responses in a mixed population of cells, we imposed stringent criteria for selection of genes ([3.6-fold up-or down-regulation and a false-discovery ratio of less than 5%). Using this approach, we minimized the chance of selecting genes hybridizing differentially solely due to inter-animal variation in gene expression and/or cell composition. However, such stringent selection criteria could have excluded the detection of more rotavirus-regulated genes, especially of genes regulated exclusively in specific types of cells that are present in low quantities in the jejunal mucosa. The different responsiveness of one of the 12-h piglets, however, obliged us to interpret our overall results carefully, especially, concerning the five genes that reacted solely at 12 hpi (TXN, FRK, DppC-2, IF144, and ACTB; see Table 2 ). Nevertheless, data mining revealed relevant relationships between these five genes, 18 h response genes, and processes known to be important for rotavirus pathogenesis.
Cuardras et al. [13] measured the transcriptional response in the human enterocyte cell line Caco-2, 1, 6, 12 and 16 hpi with Rhesus rotavirus live vaccine. Four genes up-regulated in our experiments (GBP-2, SAT, MGAM, PPA1) were also up-regulated 16 hpi in Caco-2 cells. Recently, Aich et al. [1] profiled the transcriptional response in surgically prepared jejunal loops from 1-dayold colostrum-deprived calves after 18 h of perfusion with bovine rotavirus (BRV). Several genes for which we detected more than 3.6-fold up-or down-regulation (TXN, NADH5, SGLT1, ACTB, SAT, CASP3, and PPA1) were also present on the cDNA array they used (NCBI GEO acc. number GPL325). None of these genes showed a differential expression of twofold or more in their study. The different route of administration and virulence of the strain used, the digestive differences between the jejunum of omnivores and herbivores, and, in the case of the study of Cuardras et al., the various specialized cell-types present in the jejunum of living animals versus cultured colon-derived Caco-2 cells are probably responsible for the poor correspondence between these three studies.
Based on relevant literature and functional information in databanks, we assigned a function and a possible type(s) of cell(s) responsible for expression for most of the genes on our list (Fig. 3) . In this hypothetical model, information from existing models dealing with the pathogenesis of rotavirus infection [29, 39, 40] and the development and maintenance of the small intestinal epithelium [20] were used to fit in our data. Possible functions of these genes in relation to processes and pathways known to be important for rotavirus pathogenesis are discussed below.
Measurements of villus length indicated that considerable numbers of epithelial cells were lost from the tip of the villi, including (infected) mature enterocytes. In part, down-regulation of genes involved in transport of ions and nutrients over the membranes of mature enterocytes, like meprin A, SGLT1, and IFABp2, may be a direct result of this loss. In another part, replication of rotavirus in enterocytes imbedded in the epithelial layer could have down-regulated transcription of these genes. This may also be the case for other down-regulated genes from our list, especially for genes detected only at 18 hpi (R4-R13, Table 2 ). In addition to down-regulation of SGLT1, IFABp2, and meprin A, we observed up-regulation of two other genes that may affect the absorptive and digestive function of the intestine: a gene coding for a protein carrying a Ca 2+ -permeable cation channel CD20/IgE Fc receptor subunit b domain (MS4A2) and a gene homolog to a bacterial oligo/dipeptide permease (DppC-2). It is tempting to link up-regulation of MS4A2 directly to NSP4induced enhancement of Ca 2+ permeability of the plasma and ER membranes in intestinal epithelial cells [29, 40] . Likewise, up-regulation of the DppC-2 homolog may be related to enhanced laminal secretion of peptides and amines by uninfected epithelial cells, a process believed to be triggered by raised [Ca 2+ ] cyt [40] . Characterization of these porcine transcripts/proteins is needed to provide further insight in the role of these genes in rotavirus pathogenesis. The same applies for the TMEM106B gene. This gene showed the highest level of up-regulation. So far, only a DUF1356 protein domain with unknown function has been predicted in TMEM106B.
Recently, it was reported that rotavirus infection in infant mice induced apoptosis in vivo [7] . Although DNA analysis showed that the majority of cells present in infected mucosal scrapings were not apoptotic, we observed upregulation of the apoptosis effector protein CASP3. This suggests that programmed cell death in the epithelial layer was stimulated by rotavirus infection. NB analysis detected a considerable level of CASP3 mRNA expression in uninfected mucosal scrapings. This constitutive expression of CASP3 is, most likely, related to the process of maintenance of the absorptive status of the intestinal epithelial layer. A process in which mature enterocytes continually die due to apoptosis and are replaced by differentiating cells migrating from surrounding crypts to the tip of the villi [20] . In contrast to mature enterocytes, an in vivo study in mice showed that goblet cells are largely spared from apoptosis in rotavirus-infected mice [8] . Moreover, migration of goblet cells from the crypt to the tips of the villi was stimulated in these mice. We found up-regulation of the goblet cell marker gene KRT20 [51] and the lower and mid-villus immature enterocyte marker gene MGAM [42] . This could indicate that transcriptional activity in both of these cell types was promoted. Stimulation of apoptosis in rotavirus-infected enterocytes and higher proliferation/differentiating activity in goblet cells and immature enterocytes could be a coordinated response of the jejunum to remove infected enterocytes and overlay villus tips with fresh enterocytes, goblet cells, and mucus layer. We did not detect genes on our array that were directly associated with cell-cycle progression/arrest. However, down-regulation of the nuclear kinase FRK (an antagonist of cell proliferation) and TMS4F20 may be associated indirectly with this process. In humans, TMS4F20 is strongly homologous to TM4SF4, a protein that reduced the ability of the crypt cell line HT29 to proliferate [48] .
Several other genes that may play a role in cell death and repair were regulated. SAT was up-regulated, and TXN and PRR13 were down-regulated. For this later protein, reduced expression in cells was correlated with increased sensitivity to taxane-induced cell death, a caspase-independent process characterized by the polymerization of tubulins to extraordinarily stable microtubules [27, 30] . TXN is the major carrier of redox potential in cells, and it is crucial for the defence of cells against oxidative-stressmediated apoptosis. TXN also regulates gene expression by increasing binding of redox-sensitive transcription factors like p53 [44] , NF-jB [25] , and the Nrf-2/polyamine- Table 2 . Information about the Paneth cell marker THOC4 is provided in reference [24] Transcriptional response to rotavirus 1319 inhibited SAT expression [23] . Therefore, up-regulation of SAT gene expression at 18 hpi may be directly related to down-regulation of TXN at 12 hpi. SAT is a rate-limiting enzyme in spermine/spermidine metabolism. Acetylation of these polyamines by SAT promoted their degradation and excretion [46] . Recently, it was reported that depletion of polyamines suppresses apoptosis in normal intestinal epithelial cells by AKT-kinase-mediated inhibition of CASP3 activity [50] . NB analysis showed that the increase in SAT mRNA expression coincided with the goblet cell marker KRT20. Therefore, it would be interesting to determine whether SAT expression in goblet cells can be stimulated by rotavirus infection and whether it plays a role in protecting these cells from apoptosis [8] . Interestingly, it was recently demonstrated that RNA viruses can directly modulate polyamine metabolism by regulation of SAT transcription and splicing [36] .
A most interesting gene that we found more than tenfold up-regulated codes for an as yet not-well-characterized hypothetical human protein (LOC646627) carrying a phospholipase A2 inhibitor domain (PLA2-inh). The magnitude and kinetics of up-regulation of this gene corresponded exactly with GCNT3, suggesting that this gene was expressed in the same types of cells as GCNT3, most likely mucus-producing goblet cells and/or differentiating (immature) enterocytes. The amino acid sequence translated from our PLA2-inh EST showed an overall amino acid identity of 56%, and all cysteines aligned perfectly with cysteines of the human LOC646627 protein and of other proteins that bear a typical PLA2-inh domain. PLA2s comprise a diverse family of cytosolic and secreted enzymes that hydrolyze membrane phospholipids to free fatty acids. They play an important role in many exogenous and intracellular processes, ranging from fatty acid metabolism and lysis of membranes to the synthesis of arachidonic acid (A-acid), an essential precursor for the production of inflammatory mediators such as eicosanoids. Secreted PLA2s are calcium-dependent enzymes. Cytosolic PLA2s (cPLA2) can also be calcium-independent. A moderate increase in [Ca 2+ ] cyt mediated translocation of calcium-dependent cPLA2 to intracellular membranes where it hydrolyses phospholipids to A-acid [12] . A similar effect was observed after activation of the MS4A2 calcium-permeable cation channel (up-regulated here at 18 hpi, see above) on the surface of mast cells [14] . Perhaps, enhanced expression of a PLA2-inh in our study may be a countermeasure of specific intestinal epithelial cells to normalize and/or inhibit PLA2 enzyme activity in response to extra-and intracellular changes in [Ca 2+ ] evoked by rotavirus replication, either to protect specific cells from extra-and intracellular membrane-damage or to negatively regulate A-acid production. With respect to the latter process, we observed down-regulation of the enzymes CYP2C39 and ACSL3, which both utilize A-acid acid as substrate. Interestingly, the capsid protein of parvovirus possesses PLA2 activity [49] , and HMCV particles carry a cell-derived PLA2 activity [2] . For both viruses, PLA2 activity appeared to be essential for infectivity.
Our results showed that IFN-c mRNA expression in the jejunum of infected piglets peaked around 12 hpi and tended to decline beyond this time point (see Fig. 1 ). This suggests that IFN-c was produced for a short period.
Recently, Aich et al. [1] measured the mRNA expression levels of several cytokines in jejunal loops perfused for 18 h with BRV. However, they observed no IFN-c mRNA response. Because our orally administered rotavirus needed time to reach the jejunum, our 18 h infection period represents a shorter period than 18 h of perfusion. After 18 h of perfusion, expression of IFN-c mRNA may have dropped to normal levels. Interestingly, they did detect a rotavirus-induced IL-6 (alias IFN-b 2) mRNA response at 18 hpi [1] . Recent studies showed that the interplay between IFN-gamma and IL-6 controls the influx and clearance of neutrophils and, subsequently, the transition to a more sustainable influx of mononuclear cells during acute inflammation [21, 31] . Therefore, it would be interesting to study which immune cells produce IFN-c and IL-6 and whether an orchestrated action of these cells regulates an influx of vital immune cells in the jejunum after rotavirus infection.
The IFN-c-inducible GBP-2 gene was up-regulated 18 hpi. Overexpression of GBP-1 and GBP-2 in HeLa cells and NIH 3T3 cells abrogated the cytopathogenic effect mediated by VSV and EMCV, respectively, by an unknown mechanism [3, 11] . Furthermore, it was shown that expression of murine GBP-2 in NIH 3T3 cells neutralized the cytotoxic effect of the taxane drug Paclitaxel [4] . This drug specifically stimulates polymerization of tubulins to extraordinarily stable microtubules. These stable microtubules interfere with the function of normal microtubule filaments and inevitably induce cell death [4] . The reduced expression of PRR13 we observed (discussed above) may be an indication that the formation of extraordinarily stable microtubules in intestinal epithelial cells actually takes place in response to rotavirus infection. In fact, several studies have shown that rotavirus infection induces disorganization of the cytoskeleton network and microtubule filaments in enterocytes [9, 10, 26] . Moreover, we also observed the up-regulation of the IFN-a/b-inducible gene IFI44, a cytosolic protein associated with microtubular structures, and the cytoskeleton gene ACTB. Therefore, enhanced expression of GBP-2 in specific intestinal epithelial cells could contribute to a cellular mechanism(s) that impairs and/or prevents disorganization of the microtubule filaments.
Further in vivo studies are needed to determine whether the genes identified in this study are representative for an intestine with a normal microflora. If so, more focussed studies involving in situ hybridization and immuno-histology may specify where along the crypt-villus axis and in which type of epithelial (or immune) cells elevated or reduced expression of these genes is induced. Gatekeepers of health: A qualitative assessment of child care centre staff's perspectives, practices and challenges to enteric illness prevention and management in child care centres BACKGROUND: Enteric outbreaks associated with child care centres (CCC) have been well documented internationally and in Canada. The current literature focuses on identifying potential risk factors for introduction and transmission of enteric disease, but does not examine why these risk factors happen, how the risk is understood and managed by the staff of CCCs, or what challenges they experience responding to enteric illness. The purpose of this study was to explore the understanding, knowledge and actions of CCC staff regarding enteric illness and outbreaks, and to identify challenges that staff encounter while managing them. METHODS: Focus groups were conducted with staff of regulated CCCs in Southern Ontario. Five focus groups were held with 40 participants. An open ended style of interviewing was used. Data were analyzed using content analysis. RESULTS: CCC staff play an important role in preventing and managing enteric illness. Staff used in-depth knowledge of the children, the centre and their personal experiences to assist in making decisions related to enteric illness. The decisions and actions may differ from guidance provided by public health officials, particularly when faced with challenges related to time, money, staffing and parents. CONCLUSION: CCC staff relied on experience and judgment in coordination with public health information to assist decision-making in the management of enteric illness and outbreaks. Advice and guidance from public health officials to CCC staff needs to be consistent yet flexible so that it may be adapted in a variety of situations and meet regulatory and public health requirements. In 2003, over 2 million Canadian children from the ages of 0-5 years were in some form of non-parental care and 28% of these children were in a daycare [3] . The cost of child care ranged from $300 to over $700/week and varied between provinces, territories and age in 2005 [4] . Two types of child care, regulated and unregulated, are present in Canada. There are approximately 11, 000 regulated child care facilities in Ontario which include nursery schools, preschools, centre-based full-day child care and regulated home child care. These centres must meet legislated requirements for operation of services as set out by provincial/territorial regulations. Unregulated arrangements may consist of care provided by a relative, by an unregulated family child care provider, or in-home caregiver [5] .
Canadian data from 1998 showed that over 98% of child care centre (CCC) staff were female and 45% were under the age of 30. Staff had a high level of experience; 60% had worked in the field over 5 years and 71% of teaching staff held an early childhood education (ECE) credential. Hourly wages varied between provinces and territories. The national average hourly rate was $11.62 for a teacher and $9.59 for an assistant. Only 74% of staff reported paid sick days, which averaged 7.6 days/year. Even with these challenges, a high proportion of staff reported experiencing enjoyment and reward from their work. Twenty percent reported feeling disrespected by some other professionals [5] .
Numerous enteric illness outbreaks in CCCs have been reported, although investigations rarely identified a common source. Reports commonly cited person-to-person transmission or risk factors that increased the potential for transmission and illness [6] [7] [8] . These factors included inadequate hygiene related to diapering, toileting and hand-washing [9, 10] ; poor cleaning of environmental surfaces [6] ; improper food preparation [6] ; inadequate exclusion of ill children and staff [9, 10] ; and working or being cared for in a younger age group [7, 11] .
Acts and regulations related to child care and health are issued by the provinces and territories in Canada. Additionally, public health officials at the local level have a high level of responsibility for performing inspections, responding to outbreaks and providing guidance or developing resources for CCCs according to these Acts and regulations. Staff of CCCs are most likely made aware of these Acts and regulations through their formal training, but also through regular interaction with public health officials. The variation between provincial/territorial or local health regions can make consistent public health response to enteric outbreaks challenging. Currently, knowledge is limited on how staff working in CCCs interpret and implement the guidance they are provided from public health officials. A previous study identified that staff may have inconsistent definitions of what constitutes diarrhea, and that their actions during an outbreak may differ from those recommended by public health officials [11] . Furthermore, reports from three recent Canadian investigations recommended the development of consistent guidance for CCCs related to outbreak management, prevention and control of illness [9, 11, 12] .
Resources or programs can be more successful when developed in collaboration with the groups or people who may be impacted by them [13] . Staff of CCCs were identified as an important group to consult with regarding the development of consistent guidance. The exploration of perspectives, understanding and opinions were important aspects, and thus qualitative research approaches were selected as the most suitable tool to gather this information. The qualitative approach allowed for data to be collected in the form of the participant's own words which provided a more detailed and deeper understanding than would be possible through quantitative studies. Use of qualitative methods required an inductive approach to explore the participant's experiences and perspectives [14] . Focus groups are a mechanism for collecting data. This form of group interview sets up an environment for the participants to interact, and interaction can lead to a wide range of information on experiences and perspectives [15] .
The purpose of this study was to explore the perspectives, knowledge, self-described practices and challenges CCC staff working in regulated centres in southern Ontario have related to enteric illness and outbreaks. It was also intended to investigate how staff use current public health guidance. The final objective was to gather information to feed into the development of consistent recommendations for the prevention and management of enteric illness in child care settings.
Between November 2005 and January 2006, five focus groups were conducted with staff from regulated CCCs in Southern Ontario. Four groups were conducted at different CCCs. Each of the four groups consisted of staff from the respective CCCs. The fifth group was mixed and consisted of staff from different centres and who did not work together. Ethics approval was obtained from the Research Ethics Board of the University of Guelph. All participants provided written, informed consent
A purposive sampling approach was used to recruit participants from regulated CCCs in southern Ontario. Centres were selected by the researchers and contacted by phone.
Study information and requirements were sent to each centre for their further consideration and permission to conduct the focus groups. This sampling approach sought participants who worked in a variety of settings and who could contribute their experiences on managing enteric outbreaks. Centres and staff members were defined as those who provided daytime care for an entire day to a group of children (e.g. did not include those who provided only after-school care), and whose staff's first language was English. Participating staff had daily direct contact with the children and had worked in a centre for more than six months.
Preliminary research was conducted with public health officials and CCC staff to develop appropriate questions for use in the focus groups. One-on-one interviews were conducted with public health officials to gather information related to the types of centres that they worked with, how enteric illness outbreaks were typically managed, what support public health officials provided, and their experiences while working with CCC staff. Observations of daily operations were also conducted in five centres to better understand the working environment and how staff interacted with their coworkers and supervisors. Time spent observing in the centres ranged from two hours to a full day.
Open ended inquiry was used in each focus group. Sixteen questions were developed and used per session. Questions related to staff priorities, knowledge of enteric illness in the CCC, definitions and practices, how they used information from public health officials, challenges they faced when managing enteric illness and recommendations for improvement of these situations.
Public health officials assisted in the verification of the question set prior to the focus groups to ensure clarity of the order, timing and flow of discussion. The inductive process used during and between groups with the CCC staff allowed the questions to be altered or refined. For example, follow-up or probing questions were added to allow for experiences and details not in the original question set to be captured as directed by the participants. This process of modifying questions ensured that new situations and recurring trends were explored thoroughly. Focus groups were conducted in a CCC or public location and lasted between 45 and 60 minutes. The principal investigator moderated all groups and a co-facilitator assisted by taking detailed notes. All groups were audiotaped using a digital recorder and transcribed verbatim by the principal investigator. Additionally, detailed memos were written by the researcher following each group. Notes pertained to group participation, how questions were responded to, interesting features of the discussion, and considerations for future groups.
Content analysis was used to analyze the data. This involved examining the transcripts and use of descriptive codes to compare and look for common patterns and themes among the groups [14] . All transcripts were examined by the principal investigator for common patterns within groups. Next, transcripts were reviewed and coded using descriptive codes such as: "communication with parents", "description of diarrhea" and "acknowledgement". Further content and theme analysis was done using NVivo 7. NVivo is a software program often used by qualitative researchers to handle text based data. It assists with the categorization, analysis and displaying of data [16] . Descriptive codes were later grouped into larger categories. Through continuous comparison of the transcripts, categories that were common among the groups were refined. This constant comparison allowed for the identification of themes based on common categories among the groups to be identified.
A total of 40 staff participated in one of five focus groups. The number of participants per group ranged from 3-12. All participants were female and the median age was 35 years (range 20-60 years). Eighty-three percent of the participants had an ECE Diploma (college), and 15 % had a University degree. The median number of years working in a child care centre was 11 (range 2-30 years). The centres represented a variety of settings from large, public centres with multiple classes for infants, toddlers, preschool and school-aged children to smaller centres which only had two rooms. The centres also differed on geographic location and the type of population they served.
Five major themes emerged from the data. The following is a description of the themes and text based examples to illustrate them.
Among the groups, the health and safety of the children was the primary concern and responsibility of the staff. Staff ensured health and safety through observation and the creation of an informal surveillance system for any illness among the children. Staff reported an intimate knowledge of the behaviour and health status of each child they cared for, including indicators of enteric illness such as bowel movements.
Their intimate understanding of the children was used to monitor and look for cues that indicated a change in the child's health. These cues included changes in behavior, eating or sleeping patterns, additional symptoms as well as a change in the frequency, colour and consistency of a child's bowel movement.
Staff: You get to know the smell, like the regular smell of somebody's bowel movement, the colour, you get to know the timing, like something is different or not right.
Staff recognized that bowel movements, and the causes of irregular bowel movements, vary between children and took these factors into account when making decisions and taking actions. This knowledge allowed them to consider additional factors such as the use of antibiotics and medication, teething, nutritional intake as well as the period of time that would most likely indicate an infectious disease as the cause of illness.
Staff:...and that's the hardest thing..., is this really diarrhea from a virus or is it just they're on antibiotics or they're teething.
Staff worked in a dynamic and continually changing environment. Based on previous experience, staff knew when enteric illness appeared infectious, for example, if they observed multiple children ill throughout the centre with similar symptoms in a short period of time. They also noted that illness was often first identified in younger ages and spread throughout the centre rapidly.
Staff: The number of cases of anything, you know when you start to see so many children with similar symptoms-quickly in succession that's when you start wondering if something is happening.
Staff monitored the health and safety of the children by collecting information on various forms (attendance, toileting, health, etc.) and in personal journals. Staff described collecting a large amount of information, however, the number and types of forms being used, the detail collected and how the information was recorded and shared varied between centres. There was no standardardization of forms, although in some jurisdictions standard forms may have been recommended by public health officials. Information collected by staff often remained specific to the room or the group of children that they worked with. The supervisor or director of the entire centre was responsible to collect it centrally and track the information. Staff relied on the supervisor to identify centre-wide health concerns and inform all staff, as well as act as a liaison with public health.
Monitoring and observation was reduced when a child was at home or in school, wherein the staff were unable to monitor hand-washing or health status and had to rely on parents and teachers for information. The staff had less ability to monitor older children (closer to kindergartenage), as the child's independence was much higher. For older children, staff were less involved in diapering and toileting and less in depth records were kept.
The staff role as "eyes and ears" created an informal surveillance system for enteric illness within the centre and was also the first stage in a decision-making process. Staff demonstrated knowledge of the children they worked with and the potential causes and symptoms of enteric illness through various cues. The system relied on the staff's recognition of symptoms and their ability to report this information for tracking and communication purposes.
In order to maintain the health and safety of the children, staff acted practically and responses were action-oriented. When a child was ill, their first actions were to care for the child. Staff 2: Get out the gloves, the pads, the wet cloths, the concentrated cleaning, clean that off.
These practical actions ensured a safe environment for the children through thorough cleaning, hand-washing, and restricting play areas (communal water and sand play) where the potential for transmission of illness from child to child existed.
During an outbreak, staff remained action-oriented but began a more intensive cleaning procedure. Responses to outbreaks were described: Staff spoke easily and frequently about they type of cleaning they did during an outbreak, how often they did it, and how it became a habit that they integrated into their day.
Staff: I mean you end up putting it into your program, you end up putting it into the transitions of your day. Like in the infants [group] for example, once one staff goes outside with the children that are awake, the other staff stays inside with the children that are sleeping and they're washing toys, it just becomes a daily part, an everyday practice.
Staff had a high level of comfort with this response and it was equivalent to practices that would be recommended by public health officials. From their experience working in the centre, staff felt that cleaning stopped further transmission of illness. This assisted them in meeting their goals of a healthy and safe environment for the children.
When staff defined diarrhea, they often used visual and sensory based descriptions like: "uncontained", "out of the diaper", "running down their legs". Staff used these cues to take action. However, staff definitions or descriptions of diarrhea were not the same; there was a level of ambiguity and uncertainty and they experienced a dilemma in determining whether a child was ill or not. The staff described two reasons for this uncertainty:
1) Diarrhea could be child specific. One definition may not apply to everyone.
2) The definition of diarrhea could vary between staff members and often was considered a "judgment call".
Staff experienced a similar difficulty in clearly defining an "outbreak". In public health, an outbreak is often defined as a sudden or unexpected increase of disease within a population.
[17] Staff discussed that there were a number of ways in which they determined when an outbreak was occurring. These included: increased number of children who were ill, comparison to a set baseline, more illness when compared to previous years, or situations where there were multiple illnesses.
Staff: But there's centres that are confused about that, you know I thought it was always 10% and you might talk to one public health person and she may say, oh, it's 10%, you might talk to another one who might say go on past history.
Staff stated very clearly their need for standard definitions for both diarrhea and outbreak to assist them when managing enteric illness. They felt that if the definitions were clear, they would know which actions to take and when.
Staff: It's diarrhea, even to recognize diarrhea versus a loose bowel movement, cause they're two different things, that would help everybody, this is where we need experts to tell us what is the true definition of diarrhea.
In these situations staff used their experience working in the centre, knowledge of the children and personal judgment to enable them to develop definitions that assisted them, but expressed that further guidance from public health officials would be welcomed.
Staff expressed that at times they felt overloaded with demands and/or had limited resources to respond appropriately to unpredictable situations of enteric illness. In these situations, they required flexibility in how they provided care and what actions they took. Staff discussed their use of judgment and previous experience in consideration with guidance provided by public health officials to make appropriate decisions. Staff had different forms of education and varying experiences. This attribute influenced how staff worked with children and managed enteric illness and outbreaks. Although experience could assist in making judgments, experienced staff indicated their personal judgments of diarrhea and how they responded to situations varied. This was in part due to the uncertainty associated with how to define diarrhea. New or inexperienced staff required training by more experienced staff to assist with decision-making. Although policies and guidance related to management of enteric illness are provided to the centre as a whole, staff reported that modification took place on a situation specific basis. This was reported by staff when they felt that the procedures and guidelines were inflexible or differed from a situation that they experienced. In some cases, staff actions varied from what they knew, leaving the individual staff member with a great deal of responsibility in deciding what to respond to and when.
Staff: We have to say that this is our policy, like there are certain things that we can bend on, like if the fever is 99 degrees and our policy is whatever, then yes-bring your child in. But when it's something like that [excluding a child with diarrhea], it's "this is our 24 hour policy".
Guidance provided to staff from public health officials and CCC management in this dynamic environment needed to be flexible and pragmatic. Staff used the recommended information provided by public health officials, but also felt that they had adequate ability to modify it to unique situations. This created new practices at the frontline level that were often more specific to the situation and the centre. This responsibility, and the decisions staff made, could significantly influence the management of enteric illness and the potential for outbreaks.
In certain situations, staff experienced conflict in the care they provided due to a specific challenge. These challenges prevented them from taking required actions, and fell into four areas: money, time, staffing and parents.
Most centres identified that on a regular basis they used bleach and water to clean and disinfect. During outbreaks or increased illness one of the first precautions was to change to a more powerful cleaning product. Use of these products was considered important for making the environment safe for the children.
Staff: We tend to be a little proactive, well just yesterday we had one case of diarrhea and today we sent a child home with vomiting, so that room has now been totally disinfected with Virox.
Many staff reported that when the outbreak was considered to be over, centres returned to normal bleach and water solution due to the higher cost associated with using alternate cleaning products on a regular basis even though they felt the use of the product was beneficial.
Staff: I was going to say that's truly the reason why, the cost, bleach is a lot cheaper than Virox.
Staff described that they were always pressed for time, and in certain circumstances, additional responsibilities could require changes to routines in child care that may not be feasible. The expectations to keep the environment clean, especially during an outbreak, are very high and staff felt increased pressure to find ways to incorporate this demand into an already busy schedule and maintain a high level of care.
Staff: I mean it's a lot, on top of all the stuff with parents and the kids and keeping everyone healthy ... during outbreaks we are disinfecting every surface, every toy, like every half day to every day ... cause not only are you trying to run your usual program and everything else but you're also trying to take time within those hours to care for sick kids and do this mass disinfecting...it's just added stress and workload.
Staff worked hard to provide the cleanest and safest environment possible, but recognized they were limited by time and resources. Cleaning products which are fast acting and can be used around the children throughout the day were deemed as necessary. Staff also often suggested that having additional staff support just to clean would be high on their wish list.
Record keeping was cited as an important component of routine monitoring, and especially so during an outbreak. Staff used several forms related to the group they cared for (e.g. attendance) and each individual child (e.g. bowel movement records). This record-keeping required a large amount of time and may prevent staff from performing other actions such as cleaning, speaking with a parent, planning other programs and activities, or looking after themselves. Forms related to enteric illness were only some of the records staff kept. Staff were also responsible for documenting information related to diet, allergens, what activities the child participated in, or behaviour they exhibited.
Staff said that it was difficult for them to take time off from work when they were sick. Reasons included not having compensated sick days, concern over loss of pay, and the inability to find a substitute staff member. Staff knew that they should, and were expected to take time off when experiencing symptoms of enteric illness. For most of the staff, this could be a challenge due to a limited incentive to remain at home while ill, and they admitted not always excluding themselves.
When staff were ill, they relied on a substitute staff member to replace them. However, if a substitute staff member worked in a centre during an outbreak they were not permitted to work in another centre concurrently. This policy is in place to prevent disease transmission. Staff reported that when they are unable to find a substitute staff, they may choose to work.
Staff: If she happens to come to my centre and then the next day we were officially in an outbreak, I would have to call that supply teacher and say "you know what, you were here yesterday and we're in an outbreak". She can come back to my centre but she can't go to [another] centre, so that's the frustrating part.
Appropriate ratios between staff and children are regulated and must be maintained in a centre at all times. When staff became ill and were unable to work, or when an ill child needed to be segregated from the larger group until a parent could come and pick them up, the maintenance of ratios between staff and children could be compromised. In these circumstances, there may not be enough staff to segregate the child from the rest of the group. Staff balanced variables such as how ill the child was, how long they had already been in the centre, and the number of available staff, before proceeding to make an informed decision regarding how to appropriately exclude children while maintaining required ratios. Staff 2: sometimes we just don't have the staff or ability to segregate a child.
The relationship between staff and parents was described as positive. During an outbreak, or when a child needed to be excluded, it could become strained. Lack of understanding and level of parental awareness about the transmission of enteric illness created a barrier with staff. Some parents did not understand the importance of keeping their children at home when ill. Staff felt that experience working in the CCC gave them a greater understanding of the severity of symptoms, potential causes for illness, and when a child was truly ill due to infectious diseases.
Staff: That is probably one of the biggest reasons as well as being inconvenient, perhaps inconvenience is the first one...just the financial end of it is another reason why parents are perhaps tentative to withdraw their child from daycare..."we paid for that".
Staff recognized their inability to monitor the child and ensure proper hygiene when they were sent home.
They relied on the parent's honesty regarding their child's health status.
Staff 1: I mean the hard part with it is we are relying on parents to be completely honest. A number of reasons why they felt parents found it difficult to come and pick up an ill child or keep them at home was discussed. These reasons included a lack of access to alternate care providers or the inability to take time off for financial reasons or the nature of the work place. During the focus groups, a number of the staff expressed empathy for the parents' situations, and staff often felt conflicted between balancing the needs of the parents and what they deemed best for the centre. They expressed that overcoming the challenges associated with parents would be the most helpful to assist them when dealing with enteric illness. 
This study illustrated that CCC staff had an intimate knowledge of symptoms and potential causes of illness in the children they cared for. This knowledge was used in addition to experience related to enteric illness to guide their decisions and actions. An informal surveillance system to identify illness and take appropriate actions was created using the knowledge gained from daily interaction with the children and the observations and records staff kept.
Staff described a high level of comfort in their ability to thoroughly clean, and thereby to help prevent and manage enteric illness within the centre. When staff felt uncertain, they relied on their own judgment and experience to assist them. This was apparent when staff described their difficulty in defining both "diarrhea" and "outbreak". In situations where challenges related to money, time, staffing and parents were identified, or staff required flexibility in their response, staff adapted their actions to ensure appropriate care, even if it meant modifying recommendations provided by public health officials. Staff gave examples including: adapting cleaning schedules, exclusion guidance, and record-keeping. Public health officials provide guidance based on legislated regulations. The purpose of this guidance is to ensure that staff consistently achieve outcomes which protect the safety and health of the children. This includes the prompt identification of cases and outbreaks of enteric illness so that appropriate public health preventative measures can be put in place. Findings demonstrated the health and safety of the children was a priority for CCC staff but how objectives related to this priority were achieved and how guidance from public health officials was used may vary by staff and facility.
To the best of our knowledge, a study of this nature has not been done. However, the findings support other work that demonstrated staff may have inconsistent definitions and actions related to enteric illness [11] . The challenges identified, such as time, understanding and financial or logistical needs have also been documented in other studies that have examined health care practitioners and their challenges with hygiene [18] [19] [20] . Additionally, the findings help to support the need for consistent forms of management for enteric illness and outbreaks in CCCs. Consistent guidance was supported by the staff who discussed their needs related to standard definitions and actions to take based on the definitions.
The use of focus groups with staff of CCCs allowed for the collection of in-depth data about a wide range of experiences and opinions related to enteric illness and outbreaks. Development of the original question set using information collected from public health officials and staff of CCCs ensured thorough exploration of themes and experiences related to the management of enteric illness. The inductive process allowed for new questions and details to be explored, and led to a range of discussion about the perspectives, experiences and challenges of CCC staff that would not have been possible through the use of a standard, closed questionnaire. Using a purposive sampling approach ensured that participating centres represented a range of care settings available in Southern Ontario. Centres varied in the number and age of children cared for, and setting type (teaching facility, private, public, etc). Although there were some differences between staff and centres, the major themes, experiences and perspectives that the staff spoke of were the same regardless of what type of centre they worked in. Non-regulated facilities were not approached due to the challenges associated with identifying them, size and physical setting. The insight and understanding of CCC staff can be used in further development and implementation of practical guidance.
The process of developing, following and adapting policies at the frontline level has been examined in other public service workers. "Street Level Bureaucrats" are defined as public service workers who interact directly with citizens in the course of their job, and who have substantial discretion in the application of policies in the execution of their work [21] . This level of discretion and ability of the frontline worker to modify set government policies for the individual situation has also been described among police officers, social workers and others who work within the public sector [21] . Additionally, studies of nurses have demonstrated similar reliance on previous experience and visual cues such as touching, observing, listening, feeling or sensing, and "knowing" in decision-making [22, 23] . Conflict in care has also been reported in health care workers who demonstrated balancing the risks and demands of caring for patients [20] . The results of our study demonstrated that the decision making process and demands placed on CCC staff are similar to those experienced by other professionals, who work in high stress environments and care for others on a daily basis.
The decision-making process that CCC staff used could best be described as Naturalistic. Naturalistic decisionmaking is most often associated with proficient decisionmakers who have extensive experience [24] . The process is informal and relies on the intuition and judgment of the decision-maker. The environments where this decisionmaking process is most frequently used are those with shifting goals due to dynamic and changing conditions, time constraints and high stakes. Typically identified in firefighters or military personnel, this framework also appears to apply to CCC staff. Their decision-making relies heavily on using their judgment and experience to make decisions and modify plans to meet the needs of the situation in a workable and timely fashion [24, 25] .
Previous research has concluded that control measures in the form of standard guidance, education and hygiene are necessary to assist with the prevention and control of infectious diseases [26] . Other studies in areas of infection control and hygiene have highlighted issues related to compliance with guidance. To minimize these issues and increase compliance, they recommend guidance should be easy to follow, accessible and that identified challenges should be considered during their development. Strategies for changing practices should address needs at the individual and group level [27] [28] [29] . The staff in this study also highlighted these as considerations, and based on the findings in this study a number of factors were identified that could strengthen and be considered when developing further guidance to ensure optimal compliance.
The process of identifying, managing and preventing enteric illness in children in CCC settings is inherently variable due to the number of factors, but continuing to enhance consistent decision-making tools and resources for all CCC staff is important. For example, consistently updating public health manuals and onsite visits from public health officials would be beneficial. A visual framework to assist decision-making, which could be used by CCC staff and parents, could be designed to include the variables identified by CCC staff in this study, as well as additional factors considered important by public health officials (e.g., blood in the stool). This framework could be designed to allow CCC staff to incorporate their experience and knowledge into it.
Decisions and actions rely on a clear and consistent understanding of "diarrhea" and "outbreak" and CCC staff indicated a need for this guidance. Developing one definition may not be possible when there are a number of factors to consider, but inclusion of these factors in a clear decision making framework could be of assistance to staff. It is important for public health officials and management of CCCs to work together while continuing to develop and strengthen definitions of diarrhea and outbreak. As well, it remains an important task to continue to clarify procedures and activities with all CCC staff to ensure consistency.
In CCCs, staff require resources that they can use and adapt as needed. For example, improved record-keeping forms could be developed that are visual and easy for all staff to complete and which could improve challenges associated with time. The information collected on the forms is invaluable in illness surveillance for the entire centre. Staff were very aware of the children they work with but enhancing awareness and communication among staff could ensure the rapid implementation of preventative measures such as increased cleaning throughout the entire centre. This could be accomplished by exchanging information on a regular basis with staff in a consistent manner, through a regularly scheduled briefing session or standard communication log. Staff overwhelmingly expressed the desire for tools that would make the process of cleaning easier. This information is useful for public health officials and CCC management to consider when developing guidelines or providing guidance on cleaning methods and products that staff could use which are of high quality and efficiency but not seen as a challenge due to cost or time.
Consultation with public health officials indicated that public health training for CCC staff would be the most important tool to assist staff. In contrast, although continuing education offered by public health officials was important, CCC staff felt that education and information should also be made available to parents. This training would increase the parent's understanding of enteric disease and provide information on topics such as symptoms, the importance of exclusion and proper prevention and control. Outbreaks may provide educational opportunities to bring staff and parents together for education and information by public health officials. Therefore, it is suggested that educational material be directed to parents, as well as CCC staff.
Staff providing care to children on a daily basis were proud of the impact they had on children's development and education. The intrinsic value placed on child care is significant, but staff often felt that their work was undervalued. Staff need to be acknowledged for the work they do. Basic personal needs such as salaries, and paid sick days, should reflect the level of work and responsibility. Staff should not be penalized financially for taking time off when sick. Many of the staff relied on their wages to support themselves and their families and if not paid while ill, they might need to continue to work. Likewise, parents who keep their children home while ill receive no compensation for doing so, and in most cases they still pay for the days of care, even when their child is not there. Accommodations and incentives also need to be considered for families and parents, especially those in situations of financial need. In addition to staff specific needs, centres may require assistance to ensure additional staff for proper ratios, enhanced cleaning and ensuring substitute staff are available.
The relationship between CCC staff in this study and local public health officials was very positive. During an outbreak, staff looked to public health officials to provide assistance and to reassure them that they took appropriate actions, particularly when dealing with parents. Staff stated a number of times that they referred parents to public health for further support and regarded public health officials as an authority figure when further assistance was required. Guidance should be supported by all groups involved. The relationship between staff, parents and public health is essential to ensuring proper response and management of enteric illness.
As with all qualitative research, questions regarding representativeness and generalizability must be addressed. This study was restricted to a small geographic area in one province in Canada. Although the data and recommendations appear applicable to other jurisdictions, it would be useful to hold similar groups with staff in other jurisdictions to explore and confirm these themes further, taking potential regional differences into account. The one group that was mixed with staff from different centres demonstrated the highest level of interaction from the participants and also gave the greatest degree of contrast. Further research should consider maximizing the number of mixed groups to gain further interaction and insight which would allow for a deeper comparison between groups in analysis due to the potential for contrasting discussion. The relationship between staff and parents is important as it relates to monitoring and preventing enteric illness in CCCs. Recommendations from this study will have impact on parents and therefore further research with parents is required before any recommendations should be implemented. An intervention study could be conducted to test the effectiveness of the recommendations in reducing the amount of illness or improving the response and understanding of staff. Further work with public health officials to gain their perspectives regarding strategies to implement recommendations would also be of value.
This qualitative assessment provides an enhanced understanding and appreciation of the perspective, practices and challenges that staff of CCCs experience in responding to enteric illness and outbreaks. In general, it was found that CCC staff are dedicated to and well informed about the children they work with and have a tremendous responsibility. Results from this study will be useful to public health officials responsible for developing tools and resources to further support or better inform current knowledge and practices for preventing and managing enteric disease. The recommendations from this study were made based on data directly from staff of CCCs and are designed to be practical and developed in further collaboration with them. The experience and knowledge CCC staff use to identify and take action for prevention and management of enteric illness clearly demonstrates their responsibility as gatekeepers of health among the children they care for. Dating the time of viral subtype divergence Precise dating of viral subtype divergence enables researchers to correlate divergence with geographic and demographic occurrences. When historical data are absent (that is, the overwhelming majority), viral sequence sampling on a time scale commensurate with the rate of substitution permits the inference of the times of subtype divergence. Currently, researchers use two strategies to approach this task, both requiring strong conditions on the molecular clock assumption of substitution rate. As the underlying structure of the substitution rate process at the time of subtype divergence is not understood and likely highly variable, we present a simple method that estimates rates of substitution, and from there, times of divergence, without use of an assumed molecular clock. We accomplish this by blending estimates of the substitution rate for triplets of dated sequences where each sequence draws from a distinct viral subtype, providing a zeroth-order approximation for the rate between subtypes. As an example, we calculate the time of divergence for three genes among influenza subtypes A-H3N2 and B using subtype C as an outgroup. We show a time of divergence approximately 100 years ago, substantially more recent than previous estimates which range from 250 to 3800 years ago. Precise estimates are sorely lacking for dating the emergence and divergence of viral subtypes. Improved estimates equip epidemiologists and virologists to begin to correlate these important establishing events with historical demographic changes, geographical invasions and zoonoses, the transferring of a virus from one host species to another [7, 1, 25] . For example, archeological sequence data can furnish accurate dates and show that substantial genomic changes associate with geographical invasion and zoonosis [14, 17] . Further, the recent availability of viral gene sequences sampled at a pace commensurate with their rate of nucleotide substitution vastly augments the ability to rigorously infer the time scale of phylogenies and hence determine the time of the most recent common ancestor (TMRCA) for different viral types [18, 26, 6] .
Systematic studies characterize the substitution process and substitution rate process of several classes of viral subtypes in, for example, Dengue, influenza subtype A, human immunodeficiency virus (HIV) and the virus responsible for sudden acute respiratory syndrome (SARS). For the last three viruses, a unique zoonotic transfer appears to co-occur with substantial changes in both the composition of nucleotides and amino acids as well as alterations in the rate of nucleotide substitution [15, 14, 1] . In Dengue, where a single subtype simultaneously inhabits two hosts (humans and Aedes aegypti) in a persistent zoonotic process, the introduction of the virus to new geographical environments associates with a dramatic increase in sequence diversity [25] . Unfortunately, no studies thus far analyze the rate of nucleotide substitution during either geographical invasion or zoonosis. Consequently, studies of the date of origins of viral subtypes must use strong a priori assumptions on the rate structure of nucleotide substitution.
Two primary methods find use to date the time of viral subtype divergence. The most commonly employed approach determines the divergence time of subtypes using a molecular clock assumption (MCA) over an entire phylogeny [18, 21, 5, 26] . In its strict formulation, the MCA posits a proportional relation between the number of substitutions and the intervening time period over the entire phylogeny. Looser forms of MCAs require only that the proportionality hold along individual branches, with the rates across branches drawn from a pre-specified distribution [5] . Committed to some variant of the MCA, current algorithms then estimate the rate of nucleotide substitution over all taxa in a given set. Consequently, these methods provide inference most suitable for situations where sequence evolution follows a MCA (e.g. influenza A-H3N2 in human hosts, as in [9] ) or deviates from the MCA homogenously in time (e.g. perhaps influenza A in wild fowl, see [3] ). In considering divergence events between viral subtypes, even when the MCA well-approximates nucleotide substitution within a given subtype, the above methods may incorrectly infer the time of divergence across subtypes. By either assuming that a single rate of nucleotide substitution holds for the region preceding the common ancestor of each subtype or by smoothing the rate of nucleotide substitution over clades with different numbers of taxa, the adherence to a MCA prevents direct inference of the rate during subtype divergence. Suzuki and Nei (2002) propose an alternative, more heuristic method of estimation to counteract the problem of differing rates of substitution before and after zoonotic events [23, 25] . In these studies, the evolutionary models draw a distinction between the rate of substitution within a given subtype and the rate of substitution between subtypes. However, trouble arises since there are no methods for estimating the latter quantity. Consequently, the models assume that the rate of substitution for portions of the phylogeny between the subtypes equals the mean rate in the initial host species population. For instance, in dating the time of divergence between influenza B hemagglutinin and influenza C hemagglutinin-esterase, Suzuki and Nei use the rate of amino acid substitution for water fowl for the portions of the phylogeny previous to the TMRCA of these two proteins [23] . While this method may accurately reflect the rate within avian and human hosts, it neglects whatever additional changes in the rate of substitution are due to the process of zoonotic adaptation, likely leading to a substantial underestimation of the date of the TMRCA.
The study here focuses on influenza, although the techniques are readily applied to other rapidly evolving organisms. Influenza has three types, A, B and C, classified based on serological analysis. To date, only type A sequences have been demonstrably associated with global pandemics [4] . Since modern surveillance began in the 1930s, type B has only been responsible for mild epidemics while type C has been nearly asymptomatic in human infection. Several subtypes of A, notably H1N1 and H3N2, are currently co-circulating in the human population. As the H1N1 and H3N2 subtypes may be as divergent from each other as they are from types B and C, we will refer to all types and subtypes simply as subtypes for the remainder of this paper. We select for this study three genes, coding for hemagglutinin (HA), the matrix protein (MP) and the non-structural protein (NS) responsible for interfering with host immune response. Subtype C has a hemagglutinin-esterase gene that is analogous to the hemagglutin gene in other subtypes [1] . We hence refer to the hemagglutinin gene generally and the hemagglutininesterase gene when referring specifically to the subtype C sequences.
We present a simple estimation tool to determine the date of divergence among viral subtypes that overcomes the difficulties encountered with use of the MCA by measuring the pairwise rate of substitution between taxa. Our estimator derives from the triplet statistic developed in [26, 22, 13] , where each sequence member of the triplet draws from a different subtype. In this manner, we generate from each triplet an estimate of the rate of nucleotide substitution between the most recently diverged subtypes, and consequently provide an estimate of the TMRCA. This circumvents the problems posed by earlier methods by directly estimating the pairwise rate of nucleotide substitution over the set of pairs of sequences straddling the subtype divergence without any further rate assumptions other than the existence of a mean. However, this method is only capable of determining the rate between two subtypes where a third, more distantly related, subtype functions as an outgroup. This method thus trades the ad hoc rate assumptions of the previous methods with two implicit conditions: (i) that subtypes have a unique divergence and (ii) a third, comparable subtype is available to serve as an outgroup. In exchange, we arrive at a precise statistical measure of the TMRCA that converges as the number of taxa increases and is robust to the balancing of the numbers of taxa between different subtypes. We show that applying this method to dating the divergence of influenza subtypes A-H3N2 and B gives a time of diver-gence approximately 100 years before present, substantially more recent than previous estimates.
To calculate the rate of nucleotide substitution, we require a measurement of the number of nucleotide substitutions occurring in a given time interval. Starting from a given set of aligned sequences {s 1 , ..., s n } for n taxa, we define the pairwise distance in number of substitutions to be the estimates {K ij } under a given model of nucleotide substitution. Naturally the unobservable true values {D ij } of the pairwise distances differ from their estimates {K ij }. To understand this difference, we associate each D ij with an error ε ij and assume that ε ij tends to zero as sequence lengths increase without bound. We further assume that the covariance between errors, cov(ε ij ; ε mn ), is bounded and known. For time measurements, we assume that each sequence is labeled by a sampling time t i given in consistent units. Since we know only the sampling time of a given sample up to the unit of time reported (day, month, year) we posit an uniform error ν i ~ U [0, 1] underlying each t i over the unit sampling interval. To complete the error structure specification we force the two forms of error (ν i and ε ij ) to be independent. Finally, for a set of three sequences (s i , s j , s k ) and their associated pairwise distances, we enforce a fixed topology among sequences, as shown in Figure 1 , via methods outlined in [26] . We augment the topology with the observed sampling times of the three sequences, α, the divergence time between the two sequences of interest and β, the divergence time of all sequences. When necessary for clarity, we write α ij to indicate the true time of divergence between sequences i and j.
Under our triplet method, we aim to estimate the true rate of nucleotide substitution, p ij , between sequences s i and s j with an unobserved error δ ij . With respect to outgroup sequence k, an unbiased estimate is where the factor corrects for bias resulting from the time sampling error structure (see Appendix for derivation). We superscript to denote its weak dependence on outgroup sequence k. Dependence is weak as the path of evolution from t k to α is shared between the paths from sequence k to both sequence i and sequence j and hence largely cancels out in Equation 1. We make this transparent in the following derivation. For brevity, we consider only unobservable true values, ignoring error terms. Let u be the location on the triplet in Figure 1 corresponding to time α and let p xy be the true rate along the path connecting locations x and y. Then, as distance is rate multiplied by time, we have
Subtracting the first equation from the second equation
, which is equivalent to Equation 1. This derivation makes clear that the estimator (1) measures the rate along the path from sequence i to sequence j, with only incidental dependence on sequence k.
The variance for the estimator (1) is well approximated by Further, we can estimate the time of subtype divergence α ( Figure 1 ) between sequences via
We note that the term t i + t j -1 is used rather than t i + t j to account for the expected error coming from the uniformly
The phylogenetic relationships between three sequences s i , s j and s k , sampled on dates t i , t j and t k respectively As nucleotide data increases without bound, K ij → D ij and → p ij , ensuring that → α ij . For finite sequence lengths, this relation ensures that . To gain an understanding of this estimator, we note that with a standard model of substitution (e.g. JC69, HKY85), a rate of substitution of 10 -4 (s/s/yr) and a sequence of 2000 nucleotides, the above estimator yields a standard error of approximately 23 years [20] .
The above derivations express our rate and time estimates for a single triplet of sequences. We now consider estimates that combine information across multiple representative sequences from each subtype. For discussion, we label subtypes A, B and C (which are only incidentally the same as the labels for influenza) and we assume the topology in Figure 1 for these groups. We let n r , where r ∈ {A, where P α is the sum of the inverse variance of each estimate, . Having found , we estimate its variance by a bootstrap resampling of sequences from each subtype [8] .
The computational efficiency of this estimator is on the order O(n 3 ) for a tree of n taxa. This is natural as each of the initial rate estimates is composed of information concerning three taxa. While the growth of computational expense in the number of taxa may appear unpleasant, in practice this algorithm is both fast and stable, owing to the absence of costly optimization procedures for parameter inference, and is able to handle data sets of thousands of taxa. The authors detail the computational efficiency of a similar statistic in [26]. As an example, for the data presented below all computations required only a few seconds on a desktop computer.
We demonstrate the advantage of our triplet estimator through analysis of influenza A-H3N2/B subtype divergence using the hemagglutinin (HA), matrix protein (MP) and non-structural (NS) genes. Each analysis is performed on 60 gene sequences constructed from 20 genomes each drawn from influenza subtypes A-H3N2, B and C. We download these data along with their dates of sampling from the Los Alamos Influenza Database [16] . We perform sequence alignment using ClustalX [24, version 1.8].
For consistency with previous studies of A-H3N2 HA evolution, we use the HKY model of nucleotide substitution [10] . We use the TREBLE algorithm, which implements a MCA, on sets of sequences solely drawn from a single subtype to derive within-subtype rates. The phylogenetic tree, generated by TREBLE, for the HA gene is depicted in Figure 2 (± 0.48) × 10 -3 s/s/yr and the subtype C rate is 1.31 (± 0.33) × 10 -3 s/s/yr. Lastly, for the NS gene, the rates are similar to those of the MP gene. The subtype A-H3N2 rate is 2.14 (± 0.25) × 10 -3 s/s/yr, the subtype B rate is 1.92 (± 0.20) × 10 -3 s/s/yr, and the subtype C rate is 1.68 (± 0.51) × 10 -3 s/s/yr. Table 1 presents these results. Figure 3 provides histograms of the bootstrap distributions for all three genes and subtypes.
Assuming a molecular clock within a subtype and with the rates above, we generated the corresponding dates of the TMCRA. Figure 3 shows histograms of the TMRCA estimates for different genes and subtypes. All genes are similar in dating the TMRCA for A-H3N2 to approximately 1965 (1964, 1965, and 1962 for HA, MP and NS genes, respectively). These dates are consistent with the emergence of the A-H3N2 subtype into global circulation dur-ing the 1968 pandemic [1] . Both the MP and NS genes date the TMRCA of subtype B to 1943, while the HA rate places the TMRCA at 1953. This latter value is inconsistent with the influenza B sub-epidemics of 1950-51 but is consistent with the emergence of the more lethal Victoria strain of influenza B in 1953 [11] . Each of these estimates has a standard error of approximately 2 years and so these discrepancies may be accounted by measurement uncertainty. The 10 year gap between the TMCRA suggested by the different genes can be explained by a reassortment event. Finally, the TMRCA of subtype C is calculated as 1952 and 1953 by the MP and NS genes, respectively, while the HA gene places the TMCRA at 1906. This nearly half century discrepancy suggests that the subtype C HA gene experienced a markedly different evolutionary history than either the MP or the NS gene. A biologically plausible explanation would be a reassortment event. Another possible explanation is that non-MCA rate behavior has lead to substantial bias in dating the TMRCA.
We now compare the results from pairwise rate estimates across subtypes A-H3N2 and B with those from application of the MCA to the same data. These results are summarized in Table 2 and Histograms of the time of most recent common ancestor for subtypes A-H3N2, B and C, respectively, derived from molecular clock estimates on hemagglutinin (HA), matrix (MP) and nonstructural (NS) gene sequences This discrepancy between the two sets of estimates of the TMRCA likely owes to the inability of the MCA to integrate information from the period of evolution between the two subtypes, leading to a substantial underestimate of the rate of substitution, and consequent underestimation of the date of the TMRCA.
We present a new method for ascertaining the rate of nucleotide substitution between subtypes and apply this method together with traditional MCA methods to date the divergence of influenza subtypes A-H3N2, B, and C. We use three genes, HA, MP and NS, to date two types of divergence events: the time of the most recent common of each subtype and the time of divergence between two subtypes, A-H3N2 and B. For the former event type, we show that the three genes are loosely consistent in their dating of the TMRCA of the subtypes, with the notable exception of the HA-derived estimate of subtype C's TMRCA approximately 50 years before the MP-and NS-derived estimates. This discrepancy may indicate either that subtype C's hemagglutinin-esterase gene engaged in a biologically significant event, such as reassortment, or that MCA estimation does not adequately model the evolution of the gene.
For the divergence between subtypes A-H3N2 and B, previous studies using the MCA generally place a time of divergence of several hundred years ago, ranging from the 16th to early 19th centuries. Other analysis have yielded estimates of 3600 years ago [23] . In the current study, application of the MCA yielded estimates in the last half of the 18th century. However, applying the pairwise rate estimate developed above we find uniformly, across genes, that the divergence likely occurred in the very early 20th century. The discrepancy between these two measures is likely due to the increased modeling flexibility of the pairwise rate estimate relative to the MCA.
This discrepancy between the rates and corresponding TMCRA estimates has important biological consequence. The phylogenetic divergence between subtype A-H3N2 and B corresponds to a subspeciation event for the virus. The results in this study indicate that the process of speciation is not neutral but instead a period of rapid and intense genetic change. The three genes studied here consistently show large acceleration in the rate of nucleotide substitution for the divergence period relative to the rates observed within a stable subtype. This study gives strong evidence that, at least for influenza viral subtype divergence, the process of subspeciation is associated not just with large genomic changes but also with an accelerated, finite process of adaption.
Histograms of the time of most recent common ancestor of subtypes A-H3N2 and B, derived from molecular clock estimates (light grey) and pairwise estimates (dark grey) on hemagglutinin (HA), matrix (MP) and nonstructural (NS) gene sequences Assuming that the more recent estimate is correct, a subsequent question is whether or not a pandemic or epidemic associates with subtype A-H3N2/B divergence. In the twentieth century, all influenza pandemics associate with the emergence or reemergence of subtypes (A-H1N1 in 1918, A-H2N2 in 1957 and A-H3N2 in 1968). Serological analysis indicates that the 1897 pandemic was likely due to subtype A-H2N2. However, the pandemic of 1900 is of uncertain type, although it is commonly reported in the literature as being due to A-H3N2 [4] . The above analysis suggests that it is possible to postulate that the cause of this pandemic is due to the emergence of subtype A-H3N2 or B.
As noted above, we condition the results presented here on a specific sequence alignment. As the question under consideration concerns the divergence of specific genes and proteins over a (presumably) long time scale, the capacity to generate reasonable alignments diminishes with increasing time of divergence between types, conditional on the rate of substitution. We find that for the hemagglutinin gene, a proportion of sequence alignments support the split of subtype B from subtype C after the split between subtypes A-H3N2 and B, in opposition to the topology enforced in our analysis. Hence, to some unknown degree, our analysis is necessarily biased by the choice of alignment. This suggests that improved dating can be found by integrating estimation procedures over an ensemble of alignments [19] .
The pairwise estimate method presented above is accurate in the scale where is the total time over the phylogeny and p is mean rate over the phylogeny [26] . This relation dictates that as divergence events become more remote the ability of the triplet method to resolve the time of divergence diminishes. While this limit prohibits the calculation of remote divergence events, the example presented above lies within the appropriate scale.
In place of a specific MCA, the estimates presented here directly calculate the rate of substitutions between taxa from different viral subtypes. As such estimates span paths between subtypes, they simultaneously capture the rate evolution along branches both within and between subtypes. From these estimates, we are able to directly infer the time of divergence between subtypes. As a trade-off for limited MCAs, the method requires an outgroup subtype to function as an origin relative to the subtypes under consideration. We feel that the triplet method provides a simple and widely applicable way to calculate the dates of divergence of rapidly evolving organisms without the pitfalls of the MCA.
We present a simple method for calculating the time of viral subtype divergence that does not assume a molecular clock over the entire phylogeny. Additionally, the estimator of this method, a weighted sum of pairwise estimates, furnishes a defined variance for the time of the most common ancestor between subtypes. As a tradeoff for this increased precision, the structure of the triplet statistic requires an outgroup set of sequences, usually a closely related subtype. We apply this estimator to the case of influenza subtype divergence, considering three genes. We show that the estimated divergence time of subtypes A-H3N2 and B is more than a century later than those calculated with a molecular clock.
Since we assume that the ν and ε structures are independent, the right side of the equation can be further reduced, yielding Let Δt = t i -t j . The final expectation on the right hand side resolves by direct integration, We note that as the sampling time is independent of the rate of nucleotide substitution, the error increases in proportion to the magnitude of the initial statistic. We can then create a new, unbiased statistic by counterbalancing the original statistic with this factor, making a new statistic Discovery and Development of Toll-Like Receptor 4 (TLR4) Antagonists: A New Paradigm for Treating Sepsis and Other Diseases Sepsis remains the most common cause of death in intensive care units in the USA, with a current estimate of at least 750,000 cases per year, and 215,000 deaths annually. Despite extensive research still we do not quite understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. Endotoxin (lipopolysaccharide from Gram-negative bacteria, or LPS) has been implicated as a major cause of this syndrome. Inflammatory shock as a consequence of LPS release remains a serious clinical concern. In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure and death. A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by Gram-negative bacteria, including blockage of one or more of the cytokines induced by LPS. Recently several novel amphipathic compounds have been developed as direct LPS antagonists at the LPS receptor, TLR4. This review article will outline the current knowledge on the TLR4-LPS synthesis and discuss the signaling, in vitro pre-clinical and in vivo clinical evaluation of TLR4 antagonists and their potential use in sepsis and a variety of diseases such as atherosclerosis as well as hepatic and renal malfunction. Bacteria are classified into two groups based on a staining procedure (1) . This staining response is a consequence of the composition of their membranes. Gram-positive bacteria present a multi-layered, cross-linked polymer of peptidoglycan surrounding their plasma membrane, whereas Gramnegative bacteria have essentially a monolayer (1) . The Gram-negative outer membrane is an asymmetric lipid bilayer interspersed with proteins. The lipid of this outer leaflet is almost exclusively constituted by LPS molecules.
Bacterial infection can be life threatening, requiring the host organism to develop a system to respond to this insult. The innate immune response is the first line of defense against infectious agents and is devoted to recognize highly conserved pathogen motifs in lipopeptides, DNA, dsRNA, ssRNA, specific proteins and LPS. These motifs are known as pathogen-associated molecular patterns (PAMPs) (2) .
Lipopolysaccharide is composed of three distinct domains, lipid A, a short core of oligosaccharide and the O-antigen polysaccharide (Fig. 1) . The lipid A domain is the bioactive component and is recognized during human infection. The composition of the O-antigen varies between different Gram-negative bacterial strains. The presence or absence of O chains determines whether LPS is considered rough or smooth (3) . Full length O chains would render the LPS smooth while the absence or reduction of O-chains would make the LPS rough (3, 4) .
Lipopolysaccharide is a potential drug target since its presence is critical in membrane stability and also it plays a prominent role in raising an immune response (2) . LPS triggers the release of many inflammatory cytokines, in particular TNFα, interleukin-1β and IL-6, and it has been implicated as the etiological agent of a variety of pathologies ranging from mild (fever) to lethal (septic shock, organ failure and death) (5) . Thus the structure, function and biosynthesis of LPS have been areas of intense research in the last decade (6) .
The receptors capable of recognizing the pathogenassociated molecular patterns are Toll-like receptors (TLR) and scavenger receptors. Ten members of the TLR family have been identified in humans (7) . The Toll was originally described as a type I transmembrane receptor that controls the embryonic dorsal-ventral pattern of Drosophila (8) . In fact this pioneering work identified a group of ten different genes which when deleted produced qualitatively similar phenotypes. Null mutations on any of these genes lead to a failure to differentiate patterns on the dorsoventral axis and resulted on embryonic lethality. The identification of the sequence of Toll led to the recognition that its carboxyl terminal domain was significantly related to that of the vertebrate interleukin-1 receptor (IL-1R) (8) . IL-1R activation is part of a cascade of events linked to an acute phase response to infection. This suggested that TLRs could not only be involved in development but also in the initial responses to infection in vertebrates. This hypothesis received further support from the work of Lemaitre et al., who found that Toll and other genes from the dorsal group played a role in innate immune responses to pathogenic fungi and bacteria (9) .
The TLRs belong to a cluster of molecules called the IL-1R/TLR super-family characterized by the presence of cytoplasmic Toll/IL-1R (TIR) domains (10) . The three subgroups are: the IL-1R (which present extracellular immunoglobulin domains), the adapter subgroup (cytoplasmic proteins without extracellular region) and the TLRs (9) . TLRs are type I transmembrane proteins with extracellular amino terminus and a carboxy terminal intracellular domain. The extracellular domain of the TLR4 contains over 600 amino acids and is highly polymorphic compared with the transmembrane and cytosolic domains (6) . The TIR domain, composed of three highly conserved regions, contains 150 amino acids and modulates protein-protein interactions between the TLRs and the adaptor proteins involved in the signal transduction cascade (10) . Unlike other receptors, TLRs do not have an enzymatic activity (6) . Researchers have identified at least fifteen different negative regulators of the TLRs, including MyD88s (a short form of MyD88), IRAKM, suppressor of cell signaling-1 (SOCS1), nucleotidebinding oligomerization domain 2 (NOD2), phosphatidylinositol-3-kinase (PI3K) and Toll-interacting protein (TOLLIP) (11) . The TLR activation leads to responses that involve the induction of new genes via transduction pathways such as NFκB and AP-1 (9) . The discovery of TLR lead to the understanding that an adaptive response mediated by antibody responses and T cell activation is tightly coupled to a second unknown process that requires the presence of microbial extracts (2, 7) .
Toll-like receptor 4 (TLR4) is the central signaling receptor for LPS in mammals (12) . The current knowledge on the structure and function of the TLR4 has opened the possibility to develop new drug targets to fight sepsis and other diseases associated with this signaling molecule. TLR4 was identified as the first human homologue of the Drosophila Toll (13) . TLR4 not only engages LPS but it recognizes an envelope glycoprotein encoded by mouse mammary tumor virus (MMTV) (14) . In addition, TLR4 recognizes ligands such as heat shock proteins and EDA (extracellular domain A) in fibronectin (15, 16) .
TLRs activate a potent immunostimulatory response which needs to be tightly controlled. TLRs homo o heterodimerize upon ligand binding whereas TLR4 and TLR9 homodimerize (6) . TLR signaling involves a family of adaptor proteins which recruit downstream protein kinases which activate transcription factors such as nuclear factor-kB (NF-κB) and members of the interferon (IFN)-regulatory factor (IRF) family (10) . LPS signaling involves the binding of the LPS-binding protein (LBP) to LPS; this interaction leads to a disruption of LPS aggregates (10) (Fig. 2 LPS signaling, modified from (10) with permission). Upon ligand binding there is the formation of a TLR4 complex with CD14. CD14 was the first molecule shown to enhance LPS signals (17) . Interestingly TLR4 does not require CD14 to trigger epithelial signaling to uropathogenic E. coli since bladder cells do not express CD14 (18) . In addition a small molecule, myeloid differentiation 2 receptor (MD-2), participates in this complex by associating with the TLR4 extracellular domain (19) .
MD-2 binds to the LPS monomer and is sensitive to the acylation pattern of the lipid A moiety. Association of the MD-2:LPS complex to the ectodomain of the TLR4 finally transduces the signal through the association of intracellular TIR domain, recruiting the adapter proteins triggering the signaling cascade (20) . In a similar way to TLR2, TLR4 uses the myeloid differentiation primary-response gene 88 adapter like protein (MAL) as a bridging adaptor to recruit the myeloid differentiation primary-response gene 88 (MyD88) to activate the NF-κB, p38 and JNK/MAPK pathways via TRAF6 (9) . MAL is recruited to plasma membrane microdomains containing the phospholipid PtdIns (4,5)P 2 (phosphatidylinositol-4,5-bisphosphate). MAL subsequently recruits MyD88 (20) . Another pathway activated by TLR4 involves TRIF-related adaptor molecule (TRAM). Similar to MAL, TRAM is also membrane proximal and requires myristoylation to lodge into the membrane. TRAM recruits the Toll/interleukin-1 receptor (TIR)-domain-containing adaptor protein inducing interferonβ (TRIF) which activates the tumor-necrosis factor-receptorassociated factor 3 (TRAF3), TRAF6 and receptor interacting protein 1(RIP1). Recent work with CD14 knockout mice suggested that TRL4 can function in two ways: one where full signaling occurs in the presence of CD14 and one limited to MyD88-dependent signaling (21) .
In addition to blocking the intracellular LPS signaling there are other means to modulate the endotoxin response. Approaches to alleviate the morbidity and mortality of patients associated with severe sepsis and septic shock include: (a) neutralizing LPS or blocking initial LPS-signaling events by preventing the generation of cell-surface signals, (b) blocking the intracellular signals induced by endotoxin or the synthesis of cytokines and other cellular mediators, (c) inhibiting the release of cytokines (Il-1, IL-6 and IL-8) and cellular mediators, (d) blocking the TNF-α and IL-1 receptors to the cellular mediators on their responsive target cell and (e) inhibiting downstream pathophysiological events such as acute respiratory distress or aberrant blood clotting (22) .
TLR mediated innate and/or adaptive immune responses play an important role in a variety of diseases, including sepsis, infectious disease, atherosclerosis, kidney failure, liver disease, pulmonary disease and myocardial ischemia/reperfusion injury (5, (23) (24) (25) (26) (27) (28) . TLRs are expressed in a variety of cell types including immune and non-immune cells. In addition, the capability of these receptors to recognize PAMPs is indicative of their distinct roles in infection, inflammation and tissue damage (29) (Fig. 3 ).
According to the definition made by ACCP/SCCM Consensus Conference in 1992, sepsis is known to be an early syndrome that may progress to a pathologic state manifested by hypotension and hypoperfusion known as septic shock. LPS has been associated with sepsis and the high mortality rate seen in septic shock (5) . However, it is the exaggerated host response to the systemic release of endotoxin that accounts for septic shock from Gram-negative bacteria (23) .
TLR4 up-regulation in non-immune cells after initial TLR mediated immune response may trigger secondary responses such as activation of endothelial cells that promotes the production of adhesion molecules, followed by macrophage infiltration and vascular permeability during infection (30) . This cascade may result in a systemic septic syndrome including tissue perfusions, an imbalanced coagulation cascade and organ failure (31) .
Atherosclerosis is an inflammatory disease where activated cells are involved in its initiation and progression. Guha and Mackman (32) have shown that activated TLR4 elicits the production of inflammatory cytokines and chemokines. Edfeldt et al. (33) have also found that TLR4 is prominently expressed in endothelial cells of human atherosclerotic lesion, but poorly expressed in normal human arteries. In the early atherosclerotic lesion, LPS and other ligands can stimulate the TLR4 expression on macrophages. The activated receptors can then initiate the signaling cascade that induces the expression of inflammatory cytokines, proteases, and cytotoxic oxygen and nitrogen radicals. These entities further speed up the progression of the atherosclerotic lesion (34) . In advanced atherosclerotic lesion, LPS can induce the proliferation of vascular smooth muscle cells, as well as the expression of elastin-degrading enzyme via TLR4 (35) . Besides that, in response to chemokines, more smooth muscle cells will also migrate to the sites of the lesions (36). These predominant changes cause the accumulation of cells, extracellular matrix components, thickening of the intima, as well as the deformity of the arterial wall.
Furthermore, TLR4 signaling might also be involved in atherosclerotic plaque destabilization. Grenier and Grignon have demonstrated that LPS induces the expression of matrix metalloproteinase-9 (MMP-9) by TLR4 in macrophages; MMP-9 has been shown to degrade collagen fibrous cap, thus predisposing plaque to rupture (37) .
In many forms of liver diseases such as alcoholic or nonalcoholic liver disease, liver failure and inflammation are the result of a cascade of insults which result in hyper-activation of inflammatory pathways and liver injury (26, 27) . Velayudham and colleagues (26) have shown that there is an up-regulation of TLR4 receptors in liver granulomas and LPS induced liver injury. Pathogen-induced TLR4 activation also activates reac- tive oxygen species (ROS), which is a major source of acute hepatocyte injury and death in the liver. Up-regulation of peripheral blood monocyte expression of TLR4 also occurs in patients with chronic hepatitis C (38) . In addition, endogenous gut-derived bacterial LPS have also been implicated as important cofactors in the pathogenesis of liver injury.
Within the liver, LPS binds to LPS-binding protein (LBP), which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells in the liver (39) . Moreover, TLR4 can also interact with a protein ligand released from damaged hepatocytes to extend an existing injury in the liver (40) .
In other studies, there is evidence that high-mobility group box 1 (HMGB1) can interact with both TLR2 and TLR4 to induce an inflammatory response during liver ischemia/reperfusion (IR) injury similar to that initiated by LPS (41, 42) . HMGB1 is an intracellular protein present in many species that functions in regulation and modulation of gene transcription (42) . HMGB1 is released readily from necrotic or damaged cells, which may signal through TLR4 the presence of advancing tissue injury, initiating an inflammatory response that further damages viable cells (42) .
The restoration of blood flow to the ischemic heart has often caused myocardial ischemic/reperfusion (MI/R) injury. An inflammatory response triggered by MI/R injury can irreversibly cause damage to the viable tissue surrounding the infarct, thereby further extending the injury. It is still unclear how innate immune signaling pathways are initiated during MI/R injury (43) . However, TLR4, which is also present in cardiomyocytes, has been thought to play a role in mediating MI/R injury. Schuster and Nelson (28) have shown that TLR4 receptor is up-regulated in response to myocardial injury. Furthermore, Shimamoto and colleagues have shown that TLR4 activates NF-κB-dependent transcription of inflammatory cytokine genes in MI/R injury. The TLR4-mediated injury appears to occur through activation of c-Jun NH 2terminal kinase (JNK) and translocation of NF-κB (41) . It is also believed that TLR4 recognizes endogenous molecules that are exposed during cellular injury and extracellular matrix remodeling, independent of pathogen invasion (44) . Thus, inhibition of TLR4 signaling pathway may be a potential therapeutic target to treat the myocardium damage in the ischemia/reperfusion setting.
Acute renal failure (ARF) occurs in close to 5% of hospital admissions, and is a leading cause of morbidity and mortality. A common cause of ARF is sepsis, which results from overwhelming infection (25) . Cunningham and colleagues (25) have shown that LPS insult leads to renal cell apoptosis and renal neutrophil infiltration.
Tubular epithelial cells of the kidney are among the nonimmune cells that express TLR1, TLR1-2, TLR1-3, TLR1-4, and TLR1-6, suggesting that these TLR might contribute to the activation of immune responses in tubulointerstitial injury (45) . In addition, receptors such as TLR4, TLR2, CD91 and the receptor for the advanced glycation end-products (RAGE), allow leukocytes and renal cells to recognize molecules released by injured cells. These receptors are sentinels for tissue necrosis (46) . Upon stimulation with LPS in renal infection or other endogenous ligands from necrotic tubular cells, the activated TLR4 has been shown to specifically stimulate the NF-κB pathway in response to oxidative stress (47) . Furthermore, TLR4 activation on tubular epithelial cells and circulating immune cells leads to secretion of cytokines and chemokines that either directly or indirectly contributes to renal injury.
Inflammatory bowel disease (IBD) is a medical condition that predominantly affects the gastrointestinal tract (48) . De Jager et al. showed that TLR4 and its signaling molecule TIRAP affect susceptibility to IBD (49) . Recent studies have shown that TLR4 −/− and MyD88 −/− knockout mice tend to be more prone to severe dextran sulfate sodium-induced colitis than their wild-type littermates (50) . Interestingly, CRX-526 a TLR4 antagonist has been shown to prevent an inflammatory disease in the dextran sulfate sodium and mdr1a −/− /1b −/− deficient mice models (51) . To explain these contradictory results we have to consider that constitutive signaling through TLR4 may result in the production of tissue protective factors such as IL-6 and TNF-α (49) . This is the scenario in the MyD88 −/− knockout mice, while in the case of the CRX-526 we may have selective downregulation of one of the TLR-4/ LPS signaling pathways.
Simpson and colleagues observed an increased expression of TLR2, TLR4 and CD14, as well as the proinflammatory cytokines IL-8 and IL-1β in neutrophilic asthma and bronchiectasis patients compared to controls. These groups also had higher airway endotoxin levels than the control group (23) . In another study, there was also an increased pulmonary expression of inflammatory cytokines occurring in the lung during experimental endotoxemia. The cytokine production further contributes to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (52, 53) . Baumgarten and colleagues showed that LPS induces pro-inflammatory cytokines in the lung via the TLR4/CD14 signaling cascade, suggesting a role of the innate immune response in the pathogenesis of ALI/ARDS (52) .
LPS biosynthesis occurs by two distinct, yet convergent pathways: one for the lipid A core and another for the polysaccharide O antigen. After independent synthesis, the two parts are ligated together to complete the LPS molecule (4) . Amongst the most important TLR4 antagonists developed so far we have CRX-526, E5531 and E5564.
Aminoalkyl glucosaminide 4-phosphates or AGPs are a class of lipid A mimetics in which the reducing sugar of lipid A has been replaced with an N-acylated aminoalkyl aglycon unit (54) . The AGPs contain an L-serine-based aglycon unit as well as three (R)-3-n-alkanoyloxytetradecanoyl residues comprised of even-numbered normal fatty acyl chains between 6 and 14 carbon atoms in length. All members of this family have 14 carbon atoms in the "primary" fatty acid, which is the -hydroxy fatty acid attached directly to the AGP backbone. -Hydroxymyristic acid is the most common of the primary fatty acids present in lipid A. These compounds were used in a variety of cell-based and an in vivo model to determine structure-activity relationships related to AGP acyl chain length and stimulation via TLR4 (54) . Figure 4 shows the chemical structure of two AGPs: MPL and CRX-526. The structure of CRX-526 differs significantly from monophosphoryl lipid A (MPL) and other TLR4-agonist AGP in the length of its secondary fatty acyl chains (SAC): for instance CRX-526 contains 3 SAC of 6 carbons in length, whereas MPL and other AGP, which signal through the TLR4 complex, contain SAC of >10 carbons in length (55) .
The synthesis of AGPs has been described elsewhere (55) . Briefly, the AGPs were prepared by a highly convergent method, which allowed chemical differentiation of the hydroxyl and amino groups and sequential introduction of the (R)-3-n-alkanoyloxytetrahexanoyl residues. The AGPs were purified by flash chromatography on silica gel (to >95% purity) and analyzed as a triethylammonium salt by standard analytical methods. For stimulation in vitro, the AGPs were formulated in water containing 216 μg/ml dipalmitoylphosphatidyl choline [aqueous formulation (AF)], 0.2% triethanolamine (pH 7.4), or in 2% glycerol (i.v. formulation). (53) . The structure of E5531, an analog of the lipid A of Rhodobacter capsulatus, is presented elsewhere (56) . The structure of E5564 is depicted in Fig. 5 . The crystal structure of the TLR4-MD2 complex with bound E5564 was recently published (57) suggesting that the mechanism of action of E5564 is binding through a large internal pocket in MD-2.
An important consideration in the development of assays is to determine the efficacy and toxicity of the compounds and the potency required to obtain a biological effect. A high potency is a key factor that would limit the high cost of synthesis and purification of such a compound. To illustrate the challenges in developing a synthetic endotoxin receptor antagonist we present the case of E5564.
In vitro, E5564 dose-dependently inhibited LPS-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood at nanomolar concentrations as measured by production of tumor TNF-and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and had no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 μM. E5564 blocked LPS-mediated activation of nuclear factor-B in TLR4/MD-2transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to E5531, another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose-and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for a period of time likely sufficient to permit clinical application. These results indicate that E5564 is a potent LPS antagonist and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by LPS. Compared with E5531, E5564 is structurally and synthetically less complex, yet seems to possess superior activity and pharmacological characteristics. Although E5531 demonstrated potent inhibition of LPS when added to blood in vitro and in vivo, activity decreased as a function of time. This reaction has been shown to be due to interaction of E5531 with plasma lipoproteins (58) (59) (60) . E5564 is an inhibitor of LPS-mediated stimulation of responsive cells in vitro and in vivo as measured by production of cytokines, as well as morbidity and mortality associated with LPS poisoning in animal models. Because E5564 is a structural analog of the lipid A portion of LPS, it is logical to hypothesize that the antagonist interacts with the same signaling components that bind to LPS such as the soluble serum proteins LBP and sCD14, as well as membrane-associated CD14 and perhaps the TLR4/MD-2 receptor complex. E5564 blocked LPS/ sCD14-induced reporter activity in TLR4/MD-2-expressing HEK293 (61), but not TLR2-mediated signaling by heatkilled S. aureus. These findings indicate that E5564 selectively inhibits LPS signaling via TLR4/MD-2. However, a limitation to this model system is that LPS requires the presence of sCD14 for cellular activation, making it difficult to determine whether E5564 blocks LPS binding to sCD14 or TLR4/MD-2.
Results from experiments indicated that serum components did not affect the potency of E5564, indicating that they are not critical to E5564 antagonistic activity (61) .
Further support of the hypothesis that interaction of E5564 at CD14 does not play a key role in its activity comes from a previous study by Lien et al. describing the activity of novel synthetic acyclic lipid A-like agonists that activate TLR4/MD-2 in the absence of CD14 (62) . E5564 inhibited the actions of these agonists under serum-free conditions. Taken together, these lines of evidence make it tempting to speculate that E5564 binds to TLR4/MD-2 complex, thereby blocking LPS binding or transmembrane signaling. The downstream effect of inhibiting the initial signaling by LPS seems to be an inhibition of all LPS-induced cytokines measured, including TNF-, IL-1, IL-6, IL-8, IL-10, and nitric oxide, which was measured in cultured cells, whole blood, and in vivo. Recently the crystal structure of the TLR4/MD-2 and E5564 has been described confirming the physical interaction of these molecules (57) .
Comparisons of antagonistic potency in cells cultured in 10% serum versus whole blood allow us to determine whether the high concentration of proteins/lipoproteins present in serum inhibit E5564 activity. In all systems but the rat, antagonistic activity of E5564 in cultured cells was within fourfold that measured in high serum (blood) compared with assays done in low-serum conditions (cultured cells or monocytes) (60, 63) . This indicates that serum has little or no inhibitory effect on antagonistic activity under these in vitro conditions. However, extended incubations in whole blood demonstrated that activity of E5564 was measurably reduced.
Other studies indicate that like E5531, E5564 is not rapidly metabolized, but binds to lipoproteins, and time dependently loses antagonistic activity (64) . The observation that lipoproteins reduce drug activity may explain the poor activity of E5564 in rat blood that has relatively high lipoprotein content.
During extended incubation in whole blood, E5564 retained activity better than similar concentrations of the first-generation antagonist E5531. Based on the proposed mechanism of action as a cell surface antagonist, it is likely that E5564 can completely block cellular activation by LPS. This block is achieved by concentrations of E5564 as low as 10 nM (14 ng/ml) in vitro, and at doses of 1 mg/kg or less in animal models challenged with lethal LPS doses.
Both LPS-challenge model and infection model use animals that have been sensitized or primed to LPS by previous infection with BCG, increasing cytokine response and lowering the threshold lethal dose of endotoxin (61) . All animal models of sepsis and infection have been criticized for their inability to closely mimic human sepsis. The primed model is the most relevant to the study of endotoxin antagonists such as E5564. It is well known that compared with humans, unprimed rodents such as rats and mice and primates demonstrate a profound insensitivity to endotoxin, requiring endotoxin doses as high as milligrams per kilogram, whereas humans demonstrate reproducible response to endotoxin at doses as low as 2 ng/kg. This argues that either LPS contributes only weakly to the inflammatory process in animal models, or that response to infection occurs only after the level of infection is very high, representing a process different from that in more LPSsensitive species such as humans (61) .
Even in primed animal models, lethal doses of LPS are high, approximately 100 μg/kg, generating estimated plasma concentrations of ∼1 μg/ml. These plasma levels are still >100-fold that found in even the most extreme cases of human sepsis (65) . Because the dose of E5564 required to protect against LPS is proportional to the LPS challenge dose, studying E5564 in these animal models indicates that E5564 can be a safe and effective antagonist even under these extraordinary conditions. E5564 is approximately tenfold better in human blood than mouse blood (IC 50 =1.6 nM in human whole blood; Table I versus ∼20 nM in mouse whole blood; Table II) .
Complete block of cytokine response by 10 nM E5564 in blood extrapolates to a human dose of approximately 100 μg in a 70-kg individual. Recent studies have supported this extrapolation by finding that a dose of 100 μg of E5564 given to normal volunteers over 30 min completely blocks response (signs, symptoms, and cytokines) to a dose of 4 ng/kg endotoxin administered at the midpoint of the E5564 infusion (66) .
In vitro and ex vivo assays have found that low concentrations of E5564 time dependently lose ability to inhibit LPS response. In light of these observations, it is perhaps not surprising that low doses of E5564 demonstrate a time-dependent loss of activity after administration into normal volunteers. This loss in activity is overcome when E5564 doses are increased (67) . Phase I clinical safety and tolerability assays indicate that E5564 is safe and except for the occurrence of phlebitis, well tolerated at doses up to 252 mg administered over 72 h. At this dose, in vivo antagonistic activity is retained for at least an additional 72 h after discontinuing infusion. This leads us to believe that sufficient therapeutic activity can readily be administered to patients (67) . The safety and efficacy of E5564 are currently been analyzed in a phase III randomized controlled study.
TAK-242 has been demonstrated to suppress LPSinduced inflammation (68, 69) . Recently, TAK-242 has been shown to almost completely suppress production of nitric oxide or TNFα induced by LPS in mouse RAW264.7, human U937 and P31/FUJ cells (70) . In a HEK293 cell model where TLR4, MD-2 and CD14 were co-expressed, this antagonist showed specificity to TLR4 as other TLRs, TLR1/2, TLR2/6, TLR3, TLR5, TLR7 and TLT9 were not affected by this drug (70) .
Sepsis is a major cause of high mortality rate in intensive care units in the USA (71) . Severe sepsis usually leads to organ failure. Currently, over 30 pharmaceutical products have been in the development stage to treat this condition, yet only few have reached the market (72) . Many of these target specific inflammatory mediators have been unsuccessful because of the complex nature of sepsis. For the treatment of sepsis, there are a few products that are being investigated in clinical studies via blocking different mechanisms of the body's innate immune system. Eli Lilly's Xigris ® was one of the few drugs currently available on the market to treat sepsis. Xigris ® is a recombinant human activated protein C that has anti-inflammatory, anti-thrombotic and pro-fibrinolytic properties to block the coagulation cascade which plays a critical role in the development of organ failure due to sepsis (73) . In addition, simvastatin and atorvastatin had also shown to have some non-specific anti-inflammatory effects contributing to their clinical benefits in treating sepsis (63) . However, statins are currently not been considered as a treatment for sepsis. To find a more specific target, scientists have identified TLR4 as one of the candidates in blocking the innate immune system. Only two TLR4 antagonists, E5564 and TAK-242, have made far into the clinical phase (Table III) .
In Wong, et al., determined the safety and tolerability of E5564 following a 30-min intravenous infusion in healthy male volunteers (74) . This was a single-center, randomized, double-blind, placebo-controlled, sequential-group, singledose study of E5564. The drug dose levels used were 350, 1,000, 2,000 or 3,500 μg. All doses of E5564 presented a long pharmacokinetic half-life and short in vivo pharmacodynamic half life which generally less than several hours when it is coadministered with LPS in healthy volunteers (74) . The C max and AUC (area under the curve) of E5564 increased in a dose-dependent manner. E5564 pharmacokinetics was characterized by a slow clearance (0.67-0.95 ml h −1 kg −1 ), a small volume of distribution (41-54 ml/kg), and a relatively long elimination half life (42-51 h) in healthy male volunteers. Thus, to overcome this low PD, the doses of E5564 given to the volunteers needed to be adjusted. In summary, all doses were demonstrated to be safe and well tolerated. Safety and tolerability assessments included monitoring and questioning of the subjects about adverse events, physical examinations, clinical laboratory tests (including hematology, blood chemistry, and urinalysis), and vital sign measurements (including supine and standing pulse rate and blood pressure), and 12-lead electrocardiograms (ECGs) and cytokine concentration testing. In this study, E5564 inhibited LPS-induced tumor necrosis factor-α in a dose-dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 h post-infusion. E5564 lacked LPS-like agonist activity at doses up to 3.5 mg (74).
In another study of healthy volunteers with experimental endotoxemia, Lynn et al. (66) found phlebitis was only associated with 72 h continuous intravenous infusion of E5564 but not with four hour infusion of E5564 into a peripheral vein. In this study the authors explored the possibility of extended pharmacokinetic activity of E5564. The infusion period was changed from bulk dosing to a 4-and 72-h infusions of E5564 into normal volunteers. They observed that at 4 h infusion of E5564, 3 mg/h completely blocked endotoxin administered 8 h post-dosing. Additionally, they observed that administration of 3.4 mg of E5564/hX72 h completely blocked the effects of endotoxin challenge at the end of dosing (72 h), and at 48 and 72 h post dosing. A lower dose of E5564 of 2 mg was also studied, and they found that 0.5 mg/h×4 h, ameliorated but did not block most effects of endotoxin 8 h post-dosing. This work also studied the effect of varying plasma lipoprotein content on E5564 activity in subjects who have high or low cholesterol levels (>180 or <140 mg/dl) after a 72 h infusion of 252 mg of E5564. The distribution of E5564 into the lipoprotein fractions was not significantly different between the low-and highcholesterol groups (66) .
In another study by Rossignol et al., a 72 h intravenous infusion and higher doses (500, 2,000 or 3,500 μg/h) of E5564 were administered into healthy volunteers (67) . E5564 has a slow plasma clearance (0.679 to 0.930 ml h −1 kg −1 of body weight), a small volume of distribution (45.6 to 49.8 ml/kg), and a relatively long half-life (50.4 to 62.7 h). All these pharmacokinetic parameters obtained are comparable to the study done by Wong et al. (74) . The association of E5564 with plasma lipoproteins was also investigated and it was found that the majority (∼55%) of the drug was bound specifically to high-density lipoprotein (HDL), but not low-density lipoproteins, very-low-density lipoproteins, or albumin (67) .
A Phase II multi-site, double-blind, randomized, ascendingdose, placebo-controlled safety study on E5564 was conducted in cardiac surgery patients (75) . Patients undergoing coronary artery bypass graft and/or cardiac valvular surgery with cardiopulmonary bypass were enrolled. Patients received a four hour infusion of 2, 12 or 28 mg of E5564 before cardiopulmonary bypass. No significant safety concerns were identified. No significant difference was observed in most variables related to systemic inflammation or organ dysfunction/injury. This phase II safety study suggests that the administration of E5564 is not associated with toxicity in cardiac surgical patients. However, the relatively small sample size used in this study limits the conclusion regarding rare adverse events or the potential clinical benefits of this drug (75) .
The potential of E5564 as a sepsis treatment was addressed by Kaneko et al. (76) , Surface Plasmon resonance (SPR) analysis indicated that E5564 binds to LPS binding protein (LBP), in a manner similar to LPS. Blood withdrawn from healthy volunteers was treated with heparin to prevent clotting. At doses of E5564 relevant to its clinical use (i.e. 6 μg/ml), antibodies against LBP did not influence either the distribution of E5564 to non-HDL lipoprotein fractions or the transfer of E5564 from non-HDLs to HDL. LBP binds E5564 in a manner similar to LPS, but does not play a role in E5564 redistribution/binding to lipoprotein and plasma clearance.
Czeslick et al. (77) carried out an ex vivo study on the effect of E5564 on production of LPS-induced pro-inflammatory cytokines, particularly IL-6 and TNF-α, in LPS-induced human monocytes. In this study, they recruited 10 healthy volunteers and obtained their whole blood samples and preincubated with 0.001, 0.003, 0.01, 0.03, 0.1, 1 and 10 ng/ml E5564 for 45 min and after stimulated with 0.2 ng/ml of LPS. They found that E5564 (0.003 up to 10 ng/ml) caused a dosedependent inhibitory effect on IL-6 and TNF-α production in LPS-stimulated human monocytes. They concluded that E5564 has a significant LPS inhibitory effect via down regulation of the intracellular generation of pro-inflammatory cytokines IL-6 and TNF-α in human monocytes (77) .
The association of E5564 with plasma protein and lipoprotein was studied in plasma obtained from fasted human subjects with various lipid concentrations (64) . It was reported that the majority of E5564 was recovered in the high-density lipoprotein (HDL) fraction. Additionally, they had shown increasing levels of TG-rich lipoprotein (TRL) lipid (TC and TG) concentrations resulted in a significant increase in the percentage of E5564 recovered in the TRL fraction. Furthermore, their findings had suggested that E5564 does not influence CETP-mediated transfer activity (64) .
Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood obtained from healthy human volunteers by density gradient centrifugation (68) . TAK-242 was effective in human cells and inhibited the production of TNF-α, IL-6, and IL-1b from PBMCs stimulated with LPS and IFN-gamma, with IC 50 values of TAK-242 ranging from 5.3 to 58 nM. There were four donors used for this study. There was no marked difference in the IC 50 values of TAK-242 amongst them. TAK-242 showed suppressive effects on the production of various inflammatory mediators from human monocytes and macrophages stimulated with LPS. TAK-242 also suppressed the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC 50 values from 11 to 33 nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, TLR3, and TLR9. TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling (68) .
The manipulation or intervention of TLR-mediated immune responses is a potential approach to treat and prevent the septic shock and variety of associated diseases. However, blocking TLR may lead to 'inappropriate' immune responses such allergic Th2 responses, or immunological tolerance (78) . Thus, it seems clear that the risks and benefits of manipulation of TLR mediated immune responses need to be balanced and require further investigation. Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity BACKGROUND: Dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. During the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. METHODS: Seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. GLM models using bimodal or Gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. RESULTS: IL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7 and GM-CSF were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). In contrast, increased MIP-1β levels were observed in patients with mild dengue. MIP-1β was also associated with CD56+NK cell circulating rates. IL-1β, IL-8, TNF-α and MCP-1 were associated with marked thrombocytopenia. Increased MCP-1 and GM-CSF levels correlated with hypotension. Moreover, MIP-1β and IFN-γ were independently associated with both dengue severity and disease outcome. CONCLUSION: Our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. MIP-β is indicated for the first time as a good prognostic marker in contrast to IFN-γ that was associated with disease severity. During the last decades dengue became the most important arthropod-borne emerging viral disease in tropical countries [1] . It is estimated that about 2.5% notified cases are classified as dengue haemorrhagic fever (DHF) and about 2.5-20% of DHF cases are lethal [2] [3] [4] . In the last two decades, Latin America saw a dramatic increase in frequency and in geographic extension of dengue fever. Specifically, the situation in Brazil has worsened during the last decade since the introduction of the Dengue-3 serotype. In the past years Brazil had dengue outbreaks with at least 1 million cases (2001) (2002) and within the last 18 months 900 thousand cases were reported [5] . In addition, severe disease forms are occurring with increased frequency and mortality rates.
Dengue pathogenesis is not completely understood, and the main determinants of the development of severe forms are not yet well established. Increase in capillary permeability associated with endothelial activation and haemorrhagic phenomena are landmarks of severe clinical manifestations, strongly suggesting an alteration in immunoregulation [6] .
Cytokines are proteins secreted during innate and adaptive immunological responses, acting as inflammatory mediators or modulatory molecules during several haemorrhagic fevers [7] . Clinical studies support a key role for cytokines in the DHF pathogenesis [8] [9] [10] [11] [12] [13] . During Dengue virus infections, cytokines are involved in the disease onset and homeostatic regulation. Specifically, TNF-α, IL-1β and IL-6 have been associated with both coagulation (F1+2 and TATc) and fibrinolysis (t-PA, PAPc, and D-dimmer) activation markers [14] . This activation is more striking in patients with severe clinical manifestations, although it can be found at lower degrees in patients with mild disease [15, 16] .
Despite the fact that cytokine network and their multiple regulatory pathways are highly complex and not fully elucidated during dengue fever, these molecules seem to represent interesting markers for patient stratification or prognosis. An emerging interest has appeared in order to define biomarkers that may have pathophysiological roles during disease and that may be used as future therapeutic targets. New technologies have been developed in order to detect multiple biomarkers within a single and small blood sample. Such approaches may lead to the development of specific marker panels for dengue fever. Accordingly, cytokine patterns have been indicated as serum biomarkers during infectious diseases such as Hepatitis C [17] , ARDS [18] and sepsis [19] .
In this study, we prospectively evaluated the potential use of plasma cytokine concentrations for severity stratifica-tion of patients with dengue, using a 17 cytokine-multiplex assay. Among tested cytokines, we were able to recognize ten significantly altered circulating factors and to characterise cytokine patterns related to determined clinical manifestations and disease severity.
The Ethics Committee of the Oswaldo Cruz Foundation approved this study protocol and written informed consent was obtained from all patients or their guardians prior to blood collection.
We included prospectively 59 dengue-infected patients ( A detailed history and physical examination was performed to detect hemorrhagic manifestations (positive tourniquet test for capillary fragility, skin haemorrhages, epistaxis, gingival, gastrointestinal, or urinary tract haemorrhage), signs of plasma leakage (pleural or pericardial effusion, ascites), signs of circulatory failure (cold extremities, cyanosis, hypotension, tachycardia, shock), and hepatomegaly.
In addition to the suggestive clinical diagnosis, all patients had the Dengue virus infection confirmed either by antidengue enzyme-linked immunoabsorbent assay (ELISA)-IgM, serotype specific reverse transcription-polymerase chain reaction (RT-PCR) or by virus isolation [20] [21] [22] . Dengue immune response was considered as primary or secondary by IgG ELISA according to previously established criteria [23] .
As previously reported [24] [25] [26] , we also were often unable to characterize the severe disease forms based on WHO criteria [3]. In Nicaragua, Harris et al. [24, 27] described four key severe clinical manifestations associated with dengue -shock, plasma leakage, marked thrombocytopenia or internal haemorrhage -that do not fit DHF/DSS classification as single parameters. According to these criteria, we considered:
• Severe dengue -Dengue confirmed cases plus severe thrombocytopenia (<50,000 platelets/mm 3 ) and/or hypotension (postural hypotension with decrease in systolic arterial pressure in 20 mm Hg in supine position or systolic arterial pressure < 90 mm Hg) and/or plasma leakage (either haemoconcentration fluctuation of packed cell volume ≥ 20% during illness course and recovery or clinical signs of plasma leakage, such as pleural effusion) and/or severe haemorrhagic manifestations.
• Mild dengue -Dengue confirmed cases in absence of severe thrombocytopenia, hypotension, plasma leakage signs or haemorrhagic manifestations.
Blood samples were collected from a peripheral vein and kept on ice. Plasma was collected by centrifugation at 800 g for 15 min at 4°C, aliquoted, and stored at -70°C until the analysis day. A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA). Cytokines measured were: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, CXCL8 (IL-8), IL-10, IL-12 (p70), IL-13, IL-17, granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), monocyte chemoattractive protein (MCP-1/CCL2), macrophage inflammatory protein (MIP-1β/CCL4), and TNF-α. Briefly, 20 μl plasma samples were diluted 1:4 and incubated with antibodycoupled beads. Complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentration titres. Concentrated human recombinant cytokine was provided by the vendor (Bio-Rad Laboratories). A range of 1.95-32,000 pg/ml recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. Cytokine levels were determined using a multiplex array reader from Luminex™ Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories). The analyte concentration was calculated using software provided by the manufacturer (Bio-Plex Manager Software).
Liquid nitrogen cryopreserved peripheral blood mononuclear leukocytes were isolated by Histopaque-1077 (Sigma Chemical Co., Saint Louis, MO, USA) from 35 out of 59 dengue patients. Cells were stained for CD56 surface marker using anti-CD56-Cy5 (IgG1, clone B159) from Pharmingen (San Diego, CA, USA) and positive cells were detected by flow cytometry as described before [22] using FACScalibur (Becton-Dickinson). Events (10,000-20,000) were acquired and analyses were carried out with FlowJo (TreeStar, version 4.3) software.
The nonparametric Mann-Whitney U test was used to evaluate differences between cytokine ratios from severe and mild dengue patients.
GLM models were used to evaluate factors independently associated with quantitative variables. Analysis of factors independently associated with dengue severity and other clinical manifestations was performed with GLM with logistic regression or Gaussian family. Results from the logistic regressions are given as odds ratio (OR). The confidence interval (CI) was established at 95%. Alternatively, for a GLM Gaussian family t values were recorded. A probability value of P<0.05 was considered to be significant. The statistical programs R [28] and Prism 4 (Graph-Pad Software, San Diego, CA, USA) were employed.
The Fisher's exact test was applied to determine the significance of positive samples from patients when comparing different virus serotypes or sequential infections. Correlation between platelet counts and cytokine production in blood samples was estimated by Spearman's correlation.
From the 59 patients included, 39 were classified as severe dengue and 20 as mild dengue. Detailed demographic, clinical, and laboratorial data from dengue patients are summarized in Table 1 . Blood collection was performed between 3 and 10 days after disease onset. In order to avoid effects due to differences in the blood collection time, we compared mild and severe dengue patient groups using Mann-Whitney U Test, which showed no differences in the disease onset time at the moment of sample collection [see Additional file 1]. The original data used to perform this analysis is shown at Figure 1 .
Patients with mild and severe dengue were investigated for prior incidence of infection, detected by serologic immune response (IgG antibodies for DENV). Patients with severe dengue (42%; 14 out 33) were more likely to be experiencing a secondary Dengue virus infection than patients with mild dengue (28%; 6 out 20), although no statistical significance was found in Fisher's exact test (P = 0.3989). Among 22 patients with DENV-1, 5 were classified as secondary infection, whereas among 35 patients with DENV-3, 12 were classified as secondary infection (P = 0.3912).
Dengue fever is characterised by a high fever phase and an abrupt drop in body temperature that has been called defervescence phase. Characteristically the disease outcome is defined during this phase, when patients can either recover rapidly or progress to a severe life-threatening stage. Cytokines and immunoactivation markers such as IFN-γ, IL-2, soluble CD8 and receptors for TNF-α [12, 29] are associated with the defervescence phase and with disease severity. IFN-α levels are higher in DHF than in DF during defervescence [30] .
During the febrile phase significant increase in cytokine circulating levels was detected including IL-4, IL-6, IL-10, MCP-1 and MIP-1β levels, which were maintained also elevated in defervescence (data not shown, analysed by non parametric Kruskal-Wallis test and Dunn's Multiple Comparison Test when compared with controls, P < 0.05); no significantly altered febrile levels were found when compared to defervescence. During the febrile Cytokine levels in plasma from patients with mild and severe dengue Figure 1 Cytokine levels in plasma from patients with mild and severe dengue. Box-and-whiskers graph. The box extends from the 25 th to the 75 th percentile and the line at the middle is the median. The error bars, or whiskers extend down to the lowest value and up to the highest. Factors were sorted according to their functional groups. Mann-Whitney U test was used to evaluate differences between cytokine concentration from severe and mild dengue patients. * P < 0.05, ** P < 0.01 and ** P < 0.001.
phase IL-7 was significantly higher than in defervescence. IL-1β, IL-13, IFN-γ were significantly increased during defervescence as compared to control samples. Significant levels of IL-5, IL-12, and IL-17 were not detected during dengue disease in our patients. IL-2 was detected both in healthy individuals and in dengue patients but no difference between these two groups was detected [see Additional file 2].
We studied the cytokine profile from Brazilian patients in order to compare severe and mild dengue cases during the acute phase of the disease. Figure 1 shows data from patients with regard to their plasma cytokine contents, which were sorted in four groups according to their reported function. We observed that cytokine concentrations of IL-1β, IFN-γ, IL-4, IL-6, IL-7, IL-13 and GM-CSF were significantly increased during severe dengue as compared to mild dengue, while MIP-1β levels are higher in mild dengue.
MIP-1β and IFN-γ were independent variables associated disease outcome as determined by a logistic regression model (Table 2 and Figure 2 ). While MIP-1β was increased during mild dengue with odds ratio (OR) of 0.181 and confidence interval (CI) 0.045-0.72, IFN-γ was associated with severity with OR of 1.138 (CI, 1.0541-1.245).
To assess relationships between cytokine levels and several clinical manifestations, the patient cohort with severe dengue was divided into distinct subgroups: those with hypotension, thrombocytopenia (≤50.000 counts/mm 3 ) and/or haemorrhagic manifestations. A logistic regression model was used for binomial response subgroups and GLM models using Gaussian family were employed for subgroups with continuous response in order to determine cytokine profiles.
IL-1β was associated with marked thrombocytopenia with OR = 1.058 (CI, 1.012-1.106) in dengue patients. TNF-α was inversely related to thrombocytopenia with OR = 0.978 (CI, 0.962-0.995) (Table 2, Figure 2 ). Considering platelet counts as a continuous variable for statistical analysis with a Gaussian family, it was possible to determine that IL-8 (P = 0.0434) and MCP-1 (P = 0.0146) levels are inversely related to their counts, displaying therefore an association with thrombocytopenia, while MIP-1β (P = 0.0114) confirms its association with higher counts -normal or tending to normal (Table 3) .
GM-CSF (OR = 1.004; CI, 1.001-1.007) was related with hypotension, whereas IL-1β had a negative predictive Table 2 and Figure 2 .
Natural Killer (NK) cells have been earlier related to mild cases of dengue [22] . Forty-eight PBMC samples from thirty-five patients had their CD56+ rates determined by flow cytometry and a good correlation was observed with
Cytokines detected in plasma as independent factors in predicting severity Table 2 . their respective MIP-1β plasmatic levels (r = 0.4668; P = 0.0008).
Considering that different cytokines act in the immunological network as stimulating/up regulating factors and also in a feedback loop as down regulators, the cytokine balance might play a role in the immune response outcome. Therefore we calculated MIP-1β/IFN-γ ratios for every patient and compared those from mild dengue with those from severe dengue. Ratio averages ± SEM were respectively 3032 ± 514 and 864 ± 240 (P = 0.0003; Mann Whitney U test), confirming our earlier data that these cytokines are acting as opposing factors. The different models built here using clinical manifestations as independent variables each exhibit specific cytokine profiles.
The cytokine profile identified in patients with dengue may represent a valuable tool for the characterisation of immunological response patterns and may assist the identification of patient groups at risk for developing severe disease. In the present study, the use of a multiplex analysis for cytokine plasma detection in patients with dengue could identify cytokine profiles associated with the disease severity.
Early identification and management of severe dengue disease are essential to prevent death. It has been increasingly recognized that the inflammatory response and deregulated cytokine production play key roles in the development of severe clinical manifestations [31] . However, cytokine profiles associated with dengue evolution and prognosis are not well established. New technologies for cytokine quantification were developed including the multiplex immunofluorescent bead array analysis system, allowing multiple biomarkers to be tested simultaneously in a small volume from one single plasma aliquot.
Recently, this methodology has been used for cytokine profile evaluation during several infectious diseases including viral infections [17, 32, 33] and sepsis [19] , among others.
We were able to identify models for cytokine circulation during dengue acute phase that may vary with clinical manifestations. MIP-1β was for the first time associated with a good prognostic and was identified in the different disease models presented here. MIP-1β has been earlier detected after Dengue virus cell stimuli in vitro [34, 35] but preliminary studies in vivo did not report their role in severity. In accordance with a protective role for MIP-1β, changes in MIP-1β levels were significantly correlated with decreases in viral titre after Hepatitis C treatment [17] . In addition, MIP-1β was up regulated in acute infection in chimpanzees only when viral clearance took place, but not in those animals which failed to eradicate the virus [36] .
MIP-1β is produced by human monocytes and dendritic cells upon different stimuli [37] as well as by activated NK cells [38] and lymphocytes [39, 40] . Activated NK cells release granzyme A, which displays cytolytic functions and MIP-1β is chemoattractant for NK cells, recruiting them to inflammatory sites. NK cells have been associated with mild dengue [22] . Here a good correlation between MIP-1β plasma levels and NK cells was observed, reinforcing the relevance of these pathways and strongly suggest- ing their role in dengue protective mechanisms. An early and efficient virus clearance by direct or indirect NK functions is likely controlling virus replication, restricting intense immunological activation and the dengue immunopathology and therefore favouring a mild dengue disease.
In previous studies, TNF-α has been reported to be associated with severity, mainly during DHF in Brazilian patients [11, 13, 41] . In the present study, however, this cytokine was not strongly associated with severity. Indeed, other authors also found inconsistency or no difference in TNF-α levels in severe vs. mild disease forms [10, 42] . We may hypothesize that differences in Dengue virus serotypes or in host immune response such as different TNF-α genetic polymorphisms may explain the disease outcome. In our study from 2001 (Braga et al., 2001) , patients were Dengue-2 infected, while in the present study, patients were Dengue-1 and -3 infected. A recent report [43] describes non-significant TNF-α serum levels in adult patients and suggests that the discrepancy may have been caused by a transient TNF-α peak which was not detected at a later time point.
In the present study we observed an association of IFN-γ with disease severity. Indeed, increased IFN-γ concentrations have been detected in dengue patients in a variety of studies [29, [44] [45] [46] [47] . DHF induced by Dengue-3 was associated with higher viremia early in illness and earlier peak plasma IFN-γ levels; maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia [45] . However, in a previous study from our group we failed to observe association of IFN-γ with disease severity [44] , probably due to the small number of severe patients analyzed or to the Dengue-1 incidence.
IFN-γ is produced during a T-lymphocyte helper response type 1 and may reflect CD8+ T cell activation with production of inflammatory cytokines. High levels of IFN-γ were observed in patients with dengue from Asian and Latin America and were associated with severity [9] . IFN-γ produced by T cells may also activate mononuclear phagocytes (monocytes and dendritic cells), which would produce factors such as TNF-α, tissue factor, and plateletactivating factor, among other mediators. These factors may all participate in platelet and endothelial cell activation, leading to platelet consumption, increase in endothelial permeability, hypotension and ultimately to shock. IFN-γ has also been associated with secondary heterologous Dengue virus infections [47, 48] inducing a strong antigenically cross-reactive inflammatory response, probably inefficient in terms of antibody and T-cell specific response. Indeed, we observed earlier in several patients a CD8+T cell activation with HLA-DR+ subset increase that was associated with severity [9] .
GM-CSF acts at early differentiation processes at myeloid progenitors or resting monocytes [49] . An additional stimulus may be required to activate monocytes or dendritic cells in order to produce proinflammatory cytokines [50] . GM-CSF was associated with hypotension as well as MCP-1, likely acting both in concert, contrasting with MIP-1β, once more associated with good prognostics. MCP-1 was earlier detected in DHF patients [51] but for the first time we reported clinical and laboratory findings associated with severity.
IL-8 and MCP-1, here associated with thrombocytopenia, are chemokines and may contribute to platelet activation, either by their chemoattractant properties or by their effect on endothelial permeability. Both factors were detected in patients with DHF [51, 52] . These cytokines are produced by monocytes after various activation stimuli, such as virus infection, and increase the endothelial permeability by disrupting tight junctions among cells [53] .
Despite the fact that our study could identify cytokines with good accuracy for the stratification and/or prognosis of dengue, it has potential limitations. Here we identified cytokines related to dengue severity, but the small sample size represents a shortcoming regarding the generalization of our results. In addition, only one time point was used for the measurement of cytokines, not allowing further insights provided by sequential measurements. Moreover, classification of disease severity has been a matter of debate, especially for adult patients' management and classification. Indeed, the WHO criteria for DHF has failed to identify severe disease, including fatal cases, in adult Latin America population [24, 54] (S.M.O. Zagne, R.M.O. Nogueira, unpublished) and clearly do not satisfy the stratification of our studied population. Accordingly, in the present study severe disease forms were classified following other proposed criteria [24] . While a direct correlation of cytokine concentrations and the pathophysiology of severe dengue is tempting, we believe that the full burden of disease severity cannot be attributed to a single cytokine. Cytokines may be increased simply as one of the several steps in the network loops without necessarily playing a direct harmful role and most likely more than one factor may be involved, including others not tested here such as IL-18, TGF-β, and MIF among others [9] .
We can suggest a mechanism explaining our cytokine models for dengue fever (Figure 3) . MIP-1β would be associated with a protective pathway for its chemoattractive and activating effect on NK cells, which in turn are efficient cells in early virus clearance, by their antiviral cytokine production and cytotoxic activity against infected target cells. IFN-γ has a deleterious effect for the host regarding its action in activating T cells for virus anti-genic cross-reactive response and monocyte/dendritic cell activation. Mononuclear monocytes are activated by IFNγ and GM-CSF among other cytokines and in turn produce several factors such as IL-1β and MCP-1 that may act on vascular permeability leading to plasma leakage and haemoconcentration. As suggested by other authors, it is likely that viral replication in antigen presenting cells, cytokine liberation and circulation, and T cell activation may not be a linear process [55] , but in fact a complex interaction network, with positive and negative feedbacks, where viral clearance and pathologic events take place, such as increased vascular permeability and circulatory collapse, and their balance may determine the disease outcome.
Our study demonstrated the plasma cytokine profile in dengue fever from a Brazilian population detected by a multiplex bead immunoassay. MIP-β is indicated for the first time as a good prognostic marker and in contrast to IFN-γ that was associated with the disease severity. Both cytokines can discriminate mild from severe cases. Moreover, we show here for the first time that during the dengue course different cytokine profiles may be present and vary according to determined clinical manifestations. The cytokine profiles identified herein by bead array multiplex system may favour an early identification of patients with the worst prognosis and may contribute to the establishment of more directed therapeutic procedures than the present ones.
Hypothetic mechanism to explain cytokine models during dengue fever Figure 3 Hypothetic mechanism to explain cytokine models during dengue fever. MIP-1β is associated with a good prognostic and IFN-γ has a predictive value for severity. GM-CSF, MCP-1, IL-1β, IL-6, IL-8, IL-12, IL-13 are also playing important roles during dengue pathogenesis (see text for detailed description).  Temporal trends in the discovery of human viruses On average, more than two new species of human virus are reported every year. We constructed the cumulative species discovery curve for human viruses going back to 1901. We fitted a statistical model to these data; the shape of the curve strongly suggests that the process of virus discovery is far from complete. We generated a 95% credible interval for the pool of as yet undiscovered virus species of 38–562. We extrapolated the curve and generated an estimate of 10–40 new species to be discovered by 2020. Although we cannot predict the level of health threat that these new viruses will present, we conclude that novel virus species must be anticipated in public health planning. More systematic virus discovery programmes, covering both humans and potential animal reservoirs of human viruses, should be considered. Despite long-standing interest in global biodiversity (May 1988) , only recently has the diversity of human pathogens been catalogued . Approximately 1400 pathogen species are currently recognized ( Woolhouse & Gaunt 2007) . Fewer than 200 of these are viruses, but novel virus species are being reported in humans at a rate of over two per year, much faster than for other kinds of pathogen ( Woolhouse & Gaunt 2007) . Novel viruses are a major public health concern, whether causing disease on the massive scale of HIV/AIDS, more transient events such as the SARS epidemic or potential future threats such as pandemic influenza. An analysis of temporal patterns of virus discovery is therefore of considerable interest.
Our analysis is based on the rate of accumulation of new human virus species: the 'discovery curve'. Discovery curves have previously been used to estimate the total diversity of various plant and animal taxa (Dove & Cribb 2006; Bebber et al. 2007) . However, to our knowledge, the discovery curves have not previously been compiled for any category of human pathogen. Having compiled the discovery curve, we proceed to develop a simple statistical model which we use to estimate the size of the pool of human virus species, N, and the expected rate of discovery of new species to 2020.
A standard method for estimating numbers of species is to extrapolate the cumulative species discovery curve (Bebber et al. 2007) . We gathered data for this curve by systematically searching the primary literature for first reports of human infection with each of the currently recognized virus species, using species as defined by the International Committee on Taxonomy of Viruses (ICTV; http://www.ictvonline.org/). We note that the set of viruses we are interested in-those that can infect humans-is a small subset of the total (over 1500 species according to ICTV ) and, as is discussed below, not a closed set because many of these viruses can also infect other hosts . We regard this as analogous to constructing species discovery curves for any subdivision of geographical range or habitat. As we demonstrate below, this approach yields an excellent description of the discovery curve.
We used piecewise linear regression to test for changes in the slope of the discovery curve. The results suggested upswings in 1930 (95% CI, 1929 -1933 ) and 1954 (1953 -1956 . We therefore restricted detailed analysis to the period 1954-2006.
We modelled discovery since 1954 assuming a total number of species available to be discovered (the species pool) of N virus species, each discovered in any given year with probability p. The model was fitted to the data and assessed using Markov chain Monte Carlo (MCMC)-based Bayesian inference, generating distributions and credible intervals for the parameters. The model defines the expected number of discovered viruses in year t as l t ðN; pÞ Z Npð1K pÞ tK1 ;
ð2:1Þ
where year tZ1 corresponds to 1954. The binomial distribution B(N, p) can be accurately approximated by a Poisson distribution with parameter Np for the range of values of N and p of interest. We considered fitting a distribution for values of p; however, provided individual p-values are low there is minimal improvement in model fit. Thus, for a set of model parameters, the likelihood of observing data, XZ{x i }, the number of viruses discovered for years 1 to k, is given by
Parameter distributions for N and p were calculated using MCMC simulation using a standard Metropolis algorithm with flat prior information. It was necessary to compute a correlation matrix to define a joint proposal since N and p are closely correlated. We monitored convergence using two chains. Once they had converged, we had a burn in period of 10 5 samples.
We compared the model with the observed data by calculating the mean, trend in the mean and variance for the number of virus species discovered per year (based on five million simulations using best-fit parameter values). The model was extrapolated to year 2020 by calculating the expected number of viruses discovered using the best-fit model. The 95% posterior prediction intervals were calculated using two million model simulations taking into account parameter uncertainty (as given by data from 1954 to 2006) and natural model simulation stochasticity.
As a validation exercise, the model was also fitted to the curve for accumulated virus families from 1954 using the same methods, except that the Poisson approximation no longer holds, so a binomial distribution was used. A family (based on current ICTV classifications) was added to the total when the first post-1954 species was allocated to that family. We tested the assumption that species can be randomly assigned to families (weighted by the size of the families) by noting the number of years in which 0, 1, 2, etc. virus families were discovered. This was done one million times to obtain a distribution for comparison with the observed values.
From a comprehensive search of the primary literature, we found 188 virus species that have been reported to infect humans, going back to yellow fever virus in 1901 (table 1) . Since then, the number of human virus species discovered in any given year has ranged from zero to six. As is typical (Bebber et al. 2007) , the cumulative species discovery curve increases slowly initially and then more rapidly (figure 1). Piecewise linear regression suggests no further upswings since 1954, roughly corresponding to the advent of tissue culture techniques for virus detection (figure 1).
We confirmed that our model reproduced the observed slight downward trend in the rate of discovery since 1954 (figure 1) and the observed variance in the data from 1954 to 2006 (figure 2). The distribution of the number of virus species discovered per year shows slight overdispersion (meanZ2.69; varianceZ3.07; varianceto-mean ratio greater than 1) which falls within the predicted range (meanZ2.70 with 95% credible interval 2.41-3.00; varianceZ3.03 with interval 1.99-4.49). Together, these results support our choice of model, even though we do not explicitly consider heterogeneity in the probability of discovering a given species in any one year ( p) or temporal variation in sampling effort, detection techniques and reporting.
Noting that p and N are highly correlated (figure 3), our best estimate for p is 0.015 (95% credible interval, 0.004-0.026) with 117 (38-562) so far undiscovered virus species. Extrapolating the discovery curve, allowing for parameter uncertainty and stochastic discovery, we obtain a best estimate of 22 new species (10-40) by 2020 ( figure 1) .
Data on the cumulative discovery of new virus families are also reproducible (figure 4). The predicted distribution of the number of virus families discovered per year (assuming random allocation of species to families) compares favourably with the observed distribution ( figure 5 ). This provides further support for the appropriateness of our model. Virus discovery curve M. E. J. Woolhouse et al. 2113
We conclude that it is extremely probable that new human viruses will continue to be discovered in the immediate future; we are not yet close to the end of the virus discovery curve. As a direct result of this, it is not possible to estimate the size of the species pool for human viruses with precision. However, in contrast to the negative assessment by Bebber et al. (2007) of the use of incomplete species accumulation curves, we consider that the upper and lower limits to our estimate of the size of the species pool are of interest and also have practical implications. Current trends are consistent with a pool of at least 38 undiscovered species that will be reported at an average rate of at least approximately one per year to 2020. In this context, it is worth noting that three new species were reported in 2007: two polyoma viruses, Ki and Wu, and a reovirus, Melaka (Allander et al. 2007; Chua et al. 2007; Gaynor et al. 2007) . Other viruses may have been reported but not yet classified. In practice, future rates of discovery will, of course, be affected by any major advances in virus detection technology or by any major shifts (upwards or downwards) in the effort expended on virus discovery programmes. Tissue culture was regarded as the 'gold standard' for virus detection up until a few years ago when molecular methods came to the fore (Storch 2007) , although there has not been a detectable increase in discovery rates as a result. Indeed, it is striking that there have been no dramatic changes in the pattern of virus discovery for over 50 years; extrapolations from our data should therefore provide a useful benchmark for probable future discovery rates.
The upper limit for N is finite but large; we cannot rule out hundreds of novel human viruses to be reported in the future. There are two (not mutually exclusive) possible explanations for such a high level of diversity. First, it could reflect the largely unknown extant diversity of viruses in the non-human animal reservoirs that constitute the major source of emerging human pathogens Woolhouse & Gaunt 2007) . The majority of human viruses are known to be capable of infecting nonhuman hosts (almost exclusively mammals and birds), and the animal origin of many apparently novel human viruses (e.g. HIV1 and HIV2, SARS CoV, Nipah virus) has been frequently remarked upon (Morse 1995; Woolhouse & Gowtage-Sequeria 2005; Wolfe et al. 2007 ); indeed, recently discovered viruses are even more likely to be associated with a non-human reservoir ( Woolhouse & Gaunt 2007) . All these observations are consistent with the idea that a significant fraction of viruses discovered in the last few decades is ecological 'spillover' from animal populations rather than newly evolved specialist human viruses. We have very limited knowledge of the diversity of viruses present in most mammal and bird species (with most attention having been paid to viruses of domestic animals; Cleaveland et al. 2001) , so it is unclear for how long this process might continue. An alternative explanation for a large pool of human viruses is that this reflects a high rate of evolution (within a reservoir population) of truly novel species capable of infecting humans. This hypothesis is difficult to test directly without much more comprehensive sequence data from both human and non-human virus populations. We note that the finite upper limit for the current estimate of N does not necessarily imply that the process of virus discovery is not open-ended (as a result of the evolution of new species) since there could be a low background rate of virus evolution, which will remain once extant diversity has been fully revealed. The balance between revealing extant diversity and the continual evolution of new species could be explored using a more complex model than equation (2.1); however, the available data are insufficient to yield useful estimates of the additional parameters required.
Although we cannot know in advance how big a threat they will pose, novel human viruses must be anticipated in public health planning and surveillance programmes for emerging infectious diseases (King et al. 2006; Jones et al. 2008 ). However, current approaches to virus discovery are largely passive, usually relying on investigation of reports of human disease with unfamiliar clinical symptoms and uncertain aetiology. Recently, there have been calls for more active discovery programmes for viruses and other pathogens involving 'systematic sampling and phylogeographic analysis of related pathogens in diverse animal species' ( Wolfe et al. 2007) . We consider that such calls are supported by the results reported here. Virus discovery curve M. E. J. Woolhouse et al. 2115  Investigating selection on viruses: a statistical alignment approach BACKGROUND: Two problems complicate the study of selection in viral genomes: Firstly, the presence of genes in overlapping reading frames implies that selection in one reading frame can bias our estimates of neutral mutation rates in another reading frame. Secondly, the high mutation rates we are likely to encounter complicate the inference of a reliable alignment of genomes. To address these issues, we develop a model that explicitly models selection in overlapping reading frames. We then integrate this model into a statistical alignment framework, enabling us to estimate selection while explicitly dealing with the uncertainty of individual alignments. We show that in this way we obtain un-biased selection parameters for different genomic regions of interest, and can improve in accuracy compared to using a fixed alignment. RESULTS: We run a series of simulation studies to gauge how well we do in selection estimation, especially in comparison to the use of a fixed alignment. We show that the standard practice of using a ClustalW alignment can lead to considerable biases and that estimation accuracy increases substantially when explicitly integrating over the uncertainty in inferred alignments. We even manage to compete favourably for general evolutionary distances with an alignment produced by GenAl. We subsequently run our method on HIV2 and Hepatitis B sequences. CONCLUSION: We propose that marginalizing over all alignments, as opposed to using a fixed one, should be considered in any parametric inference from divergent sequence data for which the alignments are not known with certainty. Moreover, we discover in HIV2 that double coding regions appear to be under less stringent selection than single coding ones. Additionally, there appears to be evidence for differential selection, where one overlapping reading frame is under positive and the other under negative selection. In the past few years we have witnessed an explosion in the viral genomic data available. GenBank alone holds over 80,000 close to complete viral genomes, and numbers are rising fast. For example, since the submission of the first SARS genome in May 2003, over 140 more have been published. With this genomic data at hand we hope to finally be able to tackle our understanding of viruses. Mechanisms of selection, that is to say the rate f at which a mutation resulting in a change in amino acid is accepted, and evolution on viruses are still strongly debated, and a methodology which is trimmed towards answering these questions is required. A step towards this is our attempt to develop a method which can deal with the vast amount of viral data, as well as the complexity of viral genomes and their high divergence and subsequent unreliability of alignment.
Several papers [1, 4, 5, 7, [13] [14] [15] [16] 18] have been dedicated towards the study of selection on viral genomes, in particular focusing attention on the evolutionary behaviour of overlapping reading frames. These are a feature common to viruses, where due to the three periodicity of the genetic code, up to three genes may be encoded simultaneously. The constraints placed on a nucleotide involved in such a multiple coding region will naturally have an effect on its mutational pattern, and as a result the concept of selection is complicated further. Another complication is the uncertainty of alignments when dealing with genomes of reasonable evolutionary distance. Recent papers have shown that parameter estimation can be greatly biased by the use of a fixed alignment [10] .
It is often thought that overlapping regions tend to be more constrained in their evolution than single coding ones, since a mutation may cause a non-synonymous substitution in up to three genes simultaneously. Some methods rely on these assumptions for the de novo detection of overlapping genes [19, 22] .
Various researchers have attempted to measure selection acting on overlapping reading frames, by investigating the K a /K s ratio within these regions for seperate reading frames [4, 7, [14] [15] [16] . Comparing non-synonymous to synonymous substitution rates only makes sense when the synonymous substitutions are unconstrained. In the case of coding for multiple genes, however, a synonymous substitution in one gene may well be non-synonymous in the other and thus constrained. This biases the analysis towards an under-estimation of the 'true' synonymous substitution rate and thus can lead to the false inference of positive selection.
An attempt to resolve this problem has been made, for example by focusing on synonymous substitutions in one reading frame which indeed are unconstrained in the other [18] . Hein & Støvlbaek [5] developed an evolutionary model particular to multiple coding regions, and used this for a study of selection on these. de Groot et al. [1] used this model of varying selection to comparatively annotate two viral genomes with evolved gene structure.
McCauley et al. [13] incorporated a slightly extended version into their multiple sequence annotation method, which additionally provides a selection annotation of the genome. However, their method looks at selection on an individual nucleotide level, and does not make assumptions about the modelling of selection on specific regions.
Our method presented here looks at selection on genomic segments as opposed to nucleotides, and thus in overlapping coding regions can discern selection for different reading frames. We may therefore attempt to draw conclusions about the nature of not only selection but also the interaction of selection on two different genes. Also, to study the imprint of evolution on viral genomes, it is necessary for the samples to have a reasonably high level of divergence. A benchmark herefore in our experience would be an evolutionary distance a + 2b of at least 0.4. Since more divergent genomes are harder to align, this brings uncertainty about the alignment into the inference. We decide to circumvent this problem by considering the set of all possible alignments -and their corresponding likelihood under our model -, as opposed to a fixed 'optimal' alignment. This method has previously been used for similar purposes, to minimize variability in parameter estimation due to uncertain alignments [10, 12] .
We work with a simple indel model, together with our evolutionary model, to generate a pairwise statistical alignment. For two sequences x and y, a set of seed parameters then gives us the probability p ij of each i th position x i being aligned with each j th position y j . We subsequently work with expected observations as opposed to actual ones. We iteratively calculate the alignment probabilities and the maximum likelihood estimates of evolutionary parameters, until we reach a given level of convergence. We also extend our methodology to a multiple pairwise method.
The work presented in this paper thus improves on both the above methods [1,13] by our ability to pry apart selection for two genes on overlapping segments and us not having to rely on a fixed alignment anymore.
We run a simulation study to gauge the improvement made by considering all possible alignments as opposed to a single fixed one. Even though viruses containing a large number of multiple coding sites might be expected to be easy to align, our simulation results suggest that this is not necessarily the case. The improvement in parameter estimation made by getting rid of uncertainty in alignments appears to be non-negligible, even for viruses with overlapping reading frames.
We run our method on a set of 5 HIV2 sequences, as well as a set of 3 Hepatitis B genomes. These are good candidates for analysis of overlapping reading frames, with 11% of the HIV2 genome being double coding and an average overlapping segment being of length 171 nucleotides. Hepatitis B is even more compact with 49% of the genome being double coding and an average overlapping segment length of 532 nucleotides. We subsequently investigate various questions relating to overlapping read-ing frames and the selectional mechanism underlying these.
We test our method on simulated data, to see whether summing over all alignments does actually improve results notably. All the results in this section, unless stated otherwise, are obtained using the 'worst-case-scenario' of only two sequences.
By taking a 600 nucleotide sequence chunk out of a double coding region of the Hepatitis B NC00397 sequence, we construct a long double coding region, flanked by 300 nucleotides on either side of background sequence. We let this evolve according to the TKF91 model [21] into a descendent sequence, where the Match-Match state emits a descendant according to the Hein & Støvlbaek [5] model with specified evolutionary parameters. We use a gap opening probability of 0.02 and a gap extension probability of 0.4 -these being values similar to the ones encountered in the real sequences we wish to analyse. We also only allow gaps of length 3 within coding regions, so as not to cause a frame shift in coding. We fix all selection parameters to 0.5 and test a variety of evolutionary distances, with transition rate a ranging from 0.2 to 0.7 and transversion rate b = a/2.
We annotate using our statistical alignment method described above, as well as performing parameter optimization on a fixed alignment produced by both GenAl [6] and ClustalW [20] . As we can see from Figure 1 , ClustalW gives consistently rather bad results, since it is not designed to deal with overlapping coding regions. Our method achieves better results than GenAl on sequences of evolutionary distance less than 0.8, but cannot quite compete with GenAl on sequences further apart. Here our estimation error is shown as the fraction between the average absolute deviation of our estimated parameters to the true parameter value and the true value itself.
The statistical alignment method performs, when applied to evolutionary distances we are realistically going to encounter, within 10% of the true value. Similar results hold for a number of other tested scenarios, including cases where one reading frame is under much stronger selection than the other and both are under positive or both under strong negative selection.
We wish to find out what effect the length of a double coding region has on our estimation accuracy. Letting the length of the double coding region in our above simulation vary from 600 down to 25, with transition and transversion rate 0.4 and 0.2 respectively, we obtain Figure 2 . As to be expected, the shorter the region, the worse our prediction results, since our data set decreases. However, above a length of 50 nucleotides we start picking up selection within a distance of ± 0.15, and above 200 nucleotides we are within the ± 0.1 mark. This is reassuring, since as mentioned above the average double coding region in HIV2 and Hepatitis B is 171 and 532 respectively.
We test the confidence levels of our predictions, trying to create as 'realistic' simulated data as possible. In the light of our real data analysis, we take the Hepatitis B genome NC00397 and split it into 7 different regions, a new one starting whenever there is a change in gene structure. We evolve the sequence according to our indel model with varying transition and transversion rate of a = 0.2 -0.8 and b = a/2 respectively, and fixed selection strength of 0.5 for each of the different regions. Depending on the evolutionary distance and closely related to our results in Figure  1 , we achieve an accuracy of approximately 70 -94% with both the statistical alignment method as well as the fixed alignment method using GenAl, versus 20 -72% for the fixed alignment method using ClustalW. In contrast using the true alignment gives us an accuracy of 78 -96%. Here our estimate is counted as correct if the true value lies within the error bars around the estimated value. This is naturally highly dependent on the width of our error bars, which in some cases are indeed large, simply due to lack of data. However, the error bars for the parameter estimates of both the fixed and the summed alignment are close to identical, and thus the measure is valid if only for the sake of direct comparison.
One of the reasons for the comparatively low performance on ClustalW alignments might be that those alignments often do not conserve the reading frame. As we wish to make as fair a comparison as possible, we therefore additionally manually adjust alignments to be more 'reasonable' by adjusting gap placement to conserve the reading frame. This does indeed result in considerable inprovement, thus demonstrating the volatility of results when dependent on one particular alignment. However, even when improving the fixed ClustalW alignment, the resulting accuracy after manual adjustment still falls short of that achieved by the statistical alignment method, reaching only 40 -70%.
Finally, we compare our results on the last setup using simulated descendants of the Hepatitis B genome in a pairwise versus a multiple sequence scenario. When adding up to four sequences, we observe the error bars getting notably tighter and simultaneously our estimation error decreasing by about 0.01 per added sequence. This implies, as desired, a more precise estimation of selection factors for multiple sequences.
We run our method on the Hepatitis B strand NC003977 and 'descendants' Woodchuck Hepatitis B strand J02442 and Ground Squirrel Hepatitis K02715, with sequences and gene structure downloaded from GenBank. As seed parameters we have all values set to 0.5 and wait between iterations for a difference in our loglikelihood of <1. Our method takes ~40 seconds to reach convergence and results are shown in Figure 3 .
To see how a region acts when viewed as a whole, we also calculate the average selection acting on double coding regions, by weighting the expected counts for each mutation by the appropriate selection coefficient -in the case of a single non-synonymous change in gene A or B by the factor f A and f B respectively, and in the case of two nonsynonymous changes by the joint factor f AB . Table 1 shows the values obtained for the different regions, both single and double coding. We can see that when viewed like this, the double coding regions are on average under 0.41 selection, and thus not greatly different to the single coding ones at an average of 0.39.
Due to more than 1500 sites in the Hepatitis B genome being multiple coding, we may reasonably test whether the simpler multiplicative model is an equally good fit to the full one used above. Setting f AB = f A ·f B we may perform a likelihood ratio test between the full and the restricted model, where selection acting on two different genes simultaneously gets multiplied up. With -2log Λ = 18 for 3 added parameters, the full model fits the date significantly better than the restricted multiplicative one (P = 0.0004).
We apply our method to the HIV2 genomes J04542 with reasonably diverged 'descendants' U27200, M15390, DQ00835 and M30502, by splitting the genome into different regions whenever there is a change in gene structure. Setting all our initial parameters to 0.5, as above, we Simulation Results: Varying Evolutionary Distance Figure 1 Simulation Results: Varying Evolutionary Distance. Simulation results between two sequences for a double coding region of length 600 of varying evolutionary distance. The figure plots the average estimation error of the statistical method and the fixed alignment method using both ClustalW and GenAl, versus the evolutionary distance between the two sequences. The estimation error is measured as the fraction of the average absolute deviation to the true parameter value and the true value itself. The evolutionary distance is measured as a + 2b, where a and b are transition and transversion rates respectively. obtain a selection annotation for the different regions.
The results of our parameter estimates are given in Figure  4 .
As we can see, there is a marked difference between the estimated selection strengths underlying the different regions, with selection ranging from 0.21 -1.50. Our results seem to suggest that genes encoded by double-coding regions often show contrasting modes of evolution, where one gene is highly conserved, whereas the other is less so. Naturally all these estimates are made on relatively small regions, and thus have relatively large error bars, but tendencies towards a distinction between fast and slow evolving overlaps are nonetheless demonstrated. On the other hand, the selection on the overlap between the fifth and sixth gene in line -the vpr and the tat gene -is close to equal in both reading frames, thus indicating that the otherwise observed high and low selection values are not mere inevitable artefacts of our model. We return to this in the discussion.
One of the most remarkable observations is that within each reading frame, selection on single coding regions appears to be more constrained than in double coding ones. As before, we calculate the selection acting on each region as a whole, as shown in Table 2 is in line with the results shown by de Oliveira et al. [2] and more recently by McCauley et al. [13] , but somewhat contrary to general belief [19, 22] . Clearly within the HIV2 genome there is much less data than with Hepatitis B, so it is harder to assign a true significance to these figures. However, our results do appear to suggest less stringent selection on overlapping regions than on single coding ones, thus maybe indicating the overlapping regions to be a relatively young feature in the virus.
We have introduced a novel method for estimation of selection strengths that explicitly incorporates uncertainty in estimated alignments. By integrating a statistical alignment procedure into our parameter estimation, we do not rely on a fixed alignment input. Instead, we calculate the expected number of observations, and are thus weighting our parameter estimates by the probability of each possible alignment. We naturally can not compete with knowing the true alignment, something which sufficient and extremely time consuming manual work can get close to. We do however offer a fast, automatic and efficient alternative to the use of a fixed alignment, which provides a quick and easy way for producing selection factors for different regions in a viral genome. We outperform alignments given by ClustalW consistently. We even beat GenAl for sequences of evolutionary distance below 0.8 and only do slightly worse for ones further apart. It is however additionally worth noting that the sequences we have and generally will be dealing with, will generally The average selection acting on each of the seven regions of the Hepatitis B genome, measured by weighing each expected mutation by its appropriate selection coefficient. Selection on double coding regions appears to tend to be more lenient have an evolutionary distance of 0.4 -0.9. We are therefore encouraged to see that our method is competetive compared to the slightly more refined GenAl and hope that this is amplified once also extended to include protein alignment. More importantly our method is statistical, which means it can be more readily incorporated into a maximum likelihood estimation framework, whereas GenAl works on a count-basis.
We test our method in a number of different simulation studies against the use of a fixed alignment, which we obtain using ClustalW. We show that on average our statistical approach has up to 30% higher absolute sensitivity, and that both evolutionary distance and the length of a double coding region have a lesser effect on our results than when using a fixed alignment.
Our study focuses on trying to understand the selection mechanism underlying overlapping reading frames. On the Hepatitis B genome, which boasts over 1500 multiple coding sites, we investigate several questions such as the selection a mutation is under, when it causes a non-synonymous mutation in two genes simultaneously. That is to say, if gene A and gene B are under selection f A and f B respectively, will a mutation affecting both necessarily be [22] .
Another feature which is particular to our method, is that we may seperate selection acting on the different reading frames in an overlapping region. We find especially in HIV2 a certain division of selection occurring, similar to that observed in Potato Leafroll Virus [4] and in Microviridae [16] . Essentially, it appears as though in an overlapping region one gene can take over the fastly evolving function, whilst the other behaves more conserved. Since this is not something we observed in our simulation studies, it seems to be no artefact of our model.
One possible evolutionary scenario that could explain this observation is the following: when an overlapping region is 'created'for example by the elongation of one of the genes involved, then it is likely to initially be under nonnegative selection. Since the organism survived both with and without the overlap, it might be expected to be able to evolve without detrimental effects. A thus possible behaviour would be for the newly coding region to be encouraged to evolve quickly, whilst the other gene remains under negative selection as before. The estimated selection strengths may subsequently help deduce which overlaps are the 'newer' regions -for example our study suggests that the pol gene extended itself both onto the gag and the vif gene. The effect would essentially be similar to that noted on selection occurring on duplicated genes, where the duplicated gene reaches fixation in the population due to initially being under positive selection [24] .
Up till now, other methods dealing with related issues have made use of the concept of K a /K s ratio, which however creates problems when applied to overlapping reading frames [4, 7, [14] [15] [16] . For this reason McCauley et al. [13] decided on a different HMM based approach and estimated selection as acting on a single nucleotide basis, but at the cost of not being able to pry apart selection acting on different reading frames. Most importantly however, all of the above methods use a fixed alignment and are thus prone to a great variability in their estimated parameters, dependent on the alignment. Our method manages to circumvent this problem by using a statistical approach, and thus we account for the uncertainty inherent in the alignment by considering all, rather than picking a single "best" alignment. The improvement we observe by doing this, makes us suggest that our approach of marginalizing over alignments may benefit other sequence-based inferential methods, such as for example methods for identifying conserved binding motifs.
One drawback to our method is the fact that for each descendent sequence we model transition and transversion rates as constant along the genome. This is a gross simplification, and something that should be dealt with in future work. As mentioned above, we would also like to superimpose protein alignment on our existent statistical alignment method, in accordance with the idea behind [6] . Another point is our fixing a partition prior to analysis. It would be even more interesting to be able to incorporate a hidden Markov model approach, in which breakpoints between regions would be chosen organically from the data. We could then truly start questioning which parts of the genome behave in different ways, as opposed to being restricted to the 'trial and error' approach that is the essence of our method now.
We describe the type of problem we are confronted with according to a specific example, shown in Figure 5 . Due to the 3-periodicity of the genetic code, there are three global reading frames in which a sequence may code in the forward direction, henceforth referred to as GRF1, GRF2 and GRF3. In viruses these reading frames tend to encode simultaneously for up to three different overlapping genes on each strand, resulting in multiple coding regions. We will be looking at single stranded RNA viruses, which predominantly code in the forward reading direction only. Amendments to our model would have to be made to include reverse reading frame encoded genes.
We are given two sequences S 1 and S 2 , descended from a common ancestor, together with the gene structure G of S 1 -in the case of Figure 5 this is a genome with two genes which overlap. Say these genes code in GRFs 1 and 2 respectively. Let us first assume we already have an alignment between our two sequences, and we wish to understand the way selection works on different regions of the genome. An initial question to ask would be, whether single and double coding regions behave in the same way. We thus, as shown, partition the genome into five segments, making a split wherever a gene starts or stops. These five segments we then assign to be of one of three region-types: non-, single-and double coding. When considering the effect a mutation of the indicated nucleotide C in the overlapping region of S 1 might have, we must consider its coding role in both reading frames. In GRF1 it is in the third position of the codon AGC and in GRF2 in the second position of the codon GCT. In the genetic code the codon AGx codes for serine or argenine, depending on whether x is a purine or a pyrimidine, respectively. On the other hand GxT codes for four different amino acids, depending on the nature of x. Therefore a transition in the nucleotide C will have no effect on the amino acid encoded by GRF1, whereas a transversion will. In GRF2 on the other hand, both will result in a non-synonymous substitution. Additionally, the selection strengths acting on either gene might be different, due to one of them evolving faster than the other.
Since we wish to analyse selection happening over a reasonable evolutionary distance, our aim is to be able to draw conclusions without relying on a prior alignment. Instead of estimating evolutionary parameters using observed substitution counts froma fixed alignment, we will therefore use an alignment model to generate expected substitution counts and from these use a maximum likeli-hood method to estimate all evolutionary parameters. In this manner we may sum over the uncertainty of the alignment -an uncertainty that will be high for distantly related viruses. Since our alignment model includes a substitution model, we iteratively switch between both it and our ML-procedure. Figure 6 depicts the basic outline of our program.
To be able to calculate the probability of a certain alignment between S 1 and S 2 , we need to devise a model for the evolution of a sequence. We will be working with a simple 3-state HMM indel model, using a more complex nucleotide substitution model, given by an emission matrix E, for the emission probabilities. We wish to investigate region-specific selectional behaviour along the genome of S 1 . We may thus apply a partition P to our sequence S 1 , given by a sequence of partition points {p 0 , p 1 , ..., p |P| }, where clearly p 0 = 0 and p |P| = |S 1 |. Because we are interested in certain global features, we may wish to group particular partition segments together into regions of a Sequence Annotation Figure 5 Sequence Annotation. An example of our input data and annotation. We see here the 'ancestral' sequence S 1 , whose genes structure G is given by coding regions in two reading frames GRF1 and GRF2. We apply a partition P to the sequence, where a breakpoint occurs whenever there is a change in gene structure. We annotate this partition with R = 3 different types of region for non-, single and double-coding respectively. Finally we have the descendent sequence S 2 .
particular type. Say we have R regions, then each partition segment [p k , p k+1 ] with (0 ≤ k < |P|) gets assigned to a certain 'region-type' r, with (r ≤ R), where regions of the same type are assumed to evolve in a similar way.
As stated above, since we are interested in investigating the evolutionary behaviour of viruses in particular, we wish to work with a substitution model, which specifically accounts for the presence of multiple coding regions. For our evolutionary model E we use a model very similar to the one in de Groot et al. [1] . For the convenience of those not familiar with our earlier work, we include a description of it in the following. Most amino acids are encoded by several different codons, meaning that a mutation may often result in no change in amino acid. Regarding a certain nucleotide in a codon, depending on its context, we may generally divide it into the following degeneracy classes, where a substitution would result in • four times the same amino acid. Figure 6 Model Setup. A graphic representation of our method. As input, we give the 'ancestral' sequence S 1 , its gene structure G, our desired partition P and our region annotation R of the partition segments. We also input the 'descendent' sequence S 2 , as well as our seed parameters for , a, and b. From this we may generate both our seed emission matrix E and the typeannotation-array t = [t 1 , t 2 , t 3 ] belonging to each locus along the sequence S 1 . These then get input into our alignment procedure, which subsequently over the sum of all possible alignments, calculates the expected counts C of a certain substitution of a certain type in a certain region. This information gets transferred to our maximum-likelihood (ML) method, which generates our new parameter values, maximizing the expected observations C. The resulting emission matrix E gets fed back into our alignment procedure, and the loop continues until a change in parameters is below some given threshold.
• two different amino acids, depending on whether a transition or transversion has occurred.
• four different amino acids, regardless of the type of substitution.
We shorthand these as being of type 4, 2 and 1 respectively. A few sites are not classifiable into one of the above three classes -ATx codes for three isoleucines and one methionine and CGG and GGG are synonymous although one results from the other by a transversion. Treating, for example, ATG as a type 1 site and ATA, ATC and ATT as type 4 sites, means however, that the approximations made by us are most likely to be minor.
We model the evolution of our sequences according to the Hein & Støvlbaek [5] model. When looking at a nucleotide in the ancestral sequence, for each reading frame we assign a certain state-dependent 'degeneracy-type' t to it, depending on its context. This will, in a coding region in a particular reading frame, be either of degeneracy 1, 2 or 4 and for non-coding will always be designated as 0. Since we are considering overlapping reading frames, we thus obtain for each nucleotide in the ancestral sequence a certain state-dependent 'degeneracy-type-array' t = [t1, t2, t3] -an array consisting of the degeneracy annotation of a nucleotide for each of the three reading frames. In our example in Figure 5 we can see an overlap between two genes, say genes A and B. This results in an annotation of [2, 1, 0] for our nucleotide C in the overlap, meaning that we have a degeneracy annotation of 2 and 1 with respect to gene A and B respectively.
Using this degeneracy annotation we incorporate the concept of selection factors into our framework: transitions and transversions occur according to the Kimura [8] model, and non-synonymous substitutions get accepted by a selection factor specified in the following.
Consider a nucleotide x in a region of type r in S 1 with degeneracy-type array t. Then our factors will be given by where with F determined as explained above. We thus are able to construct an emission matrix E, where E(r, t1, t2, t3, x, y) is the probability of in region r, nucleotide x of type [t 1 , t 2 , t 3 ] mutating into nucleotide y.
We would like to note that even though our alignment model does assume independence of sites, we model a local dependency in our evolutionary model by conditioning our emission probabilities on the nucleotide context in the ancestral sequence. An undoubtable improvement would be to model the dependency as continuous throughout the evolutionary process [17] . However, as noted by the authors themselves, the elaborate MCMC method developed in order to do this makes it a computationally intractable option.
To compute the probability of an alignment we use a simple indel model with Match, Insert and Delete states. We have as alignment parameters a gap-opening, a gap-extension and a transition probability from any state to the end state. All other state transition probabilities may be derived from these. The Insert and Delete states emit a nucleotide from a uniform distribution, aligned to a gap. 
In the Match state nucleotide pairs are emitted according to our above model.
We wish to eliminate the bias in parameter estimation created by the use of a fixed alignment. For this, we work with a probabilistic alignment, which instead of producing an 'optimal' alignment, handles the set of all possible alignments and their likelihood. It computes posterior probabilities for each state at every nucleotide position. We thus are considering all possible sequence alignments and weighing them appropriately (see [23] ), according to our indel model. This method has been previously used and described in further detail in [9, 10] . Note, that when referring to the insertion and deletion states, the related posteriors are added together so that we obtain the posterior probability of a certain nucleotide not being aligned, as opposed to belonging to a particular gap.
During the alignment procedure, our alignment parameters are estimated in a few iterations of the Baum-Welch algorithm [3] . The implementation of the algorithm, including banding to cut computational demands, was generated automatically by the HMM compiler program HMMoC [11] .
As shown in Figure 6 , we initially have as an input all the sequence and genome structure data, as well as a set of seed parameters. We subsequently use our alignment model to generate the posterior probabilities of every nucleotide position being in each state. This is done by using the forward and backward algorithms applied to our alignment indel model, as is standard HMM procedure. From these posteriors we may calculate, for each degeneracy in each region, the expected number of times an identity, transition and transversion is used. For a site of degeneracy t = [t 1 , t 2 , t 3 ] in region r, let this be , and respectively. Since , and were the probabilities for a site of degeneracy t in region r of an identity, transition or transversion occurring (see equations 1, 2, 3), we may rewrite the emission term of the log likelihood log L as follows:
For this function of the 3R + 2 emission parameters , a and b we now find the maximum likelihood estimates using the Newton-Raphson iteration method. We may do this by taking the explicit derivatives of the likelihood function, possible because of the simple substitution model used. If we had opted for a more complicated model, we would need optimization methods that did not rely on derivatives and would subsequently be slower, though the estimation would still be possible. Once the change in log-likelihood between two iterations has fallen below some given threshold, we take the likelihood to have converged. We then generate a new emission matrix E to be fed back into our alignment procedure in order to generate new posterior probabilities.
Once the likelihood function has converged below some set threshold, we output the final set of estimated selection parameters. We may also, if desired, construct an alignment, either using the Viterbi path or posterior decoding.
We would like to be able to apply our method to multiple sequences, thus extrapolating more information where possible. We could of course devise a multiple alignment indel model, and develop a new likelihood function from which to maximize over all tree branches simultaneously. This however would be of computational much higher demand, runtime increasing exponentially with the addition of each new sequence. Instead, we therefore opt to work with a multiple pairwise alignment under the assumption of a star shaped tree, with the reference sequence as the root in the star topology. This implies viewing the evolution of the pairwise sequences as independent, which is an approximation which we wish to address in future work. This merely requires per additional sequence an extra transition and transversion parameter, since selection is acting on the gene in the ancestor and we assume this to be constant over all branches. The modification to our program is thus trivial, with only a linear increase in runtime.
As an input we have, for lack of better terminology, the ancestral sequence A and its N descendants D 1 , ..., D N , together with the seed parameters for the selection factors (f 1 , f 2 , f 3 ) R on each region R as well as n transition and transversion parameters (a, b) n respectively.
We then build a set of N pairwise alignments between the ancestor A and its N descendants. Each one of these obtains a likelihood function log L as given in equation 6. Now we create a new likelihood function log L* which is the sum of the N log likelihoods. If is the number of expected identities of type t in region r between the ancestral sequence A and its n th descendant, then our assumption of independence implies that
x ts t r , x id t n r , , is the full likelihood of observing all N sequences under our model. Note here, that the probabilities P are dependent on the sequence-dependent transition and transversion rates (a, b) n and the selection factors (f 1 , f 2 , f 3 ) R which in turn are not dependent on n, since we are assuming selection to occur on the gene in the ancestral sequence.
Maximizing this new log likelihood function, we proceed as above and estimate a new set of selection factors and a set of sequence specific transition and transversion rates, from which we may generate a new set of pairwise statistical alignments.
log log log log , , ,  Seasonality of Influenza A(H3N2) Virus: A Hong Kong Perspective (1997–2006) BACKGROUND: The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: 281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997–2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations. CONCLUSIONS/SIGNIFICANCE: The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population. Despite many hypotheses and studies, the underlying basis for the annual recurrence of seasonal influenza still remains a mystery [1] . Hammond et al. [2] postulated a rapid, global dispersion of 'airborne aerosolized influenza virus' via the atmosphere, to account for the persistence and spread of the disease. Recent reviews have discussed the various approaches to resolving this question, and identified various factors that may be involved, including: properties of the virus itself (mutation rates and immune escape), properties of the host (seasonal variation in host health and behavior, e.g. crowding and air travel, production and dissemination of bioaerosols through sneezing and coughing), and properties of the environment (temperature, humidity and weather variations, e.g. El Nino) [3] [4] [5] [6] [7] .
Some of these factors have been incorporated into mathematical models to attempt to understand the driving forces behind influenza seasonality [6, [8] [9] [10] [11] [12] [13] .
Sequence-based analyses have become very popular recently and have shed some interesting insights into possible underlying mechanisms of influenza seasonality. Many of these also urge for (or at least hint at) the need for more sequences from tropical regions to be made publicly available to increase the accuracy of such analyses [14] [15] [16] [17] [18] [19] [20] [21] . Other studies have analyzed genetic data together with the even more scarcely available antigenic data, in attempts to understand and even predict the most likely emerging strains [22] [23] [24] [25] . Even the application of mass spectrometry has been applied to influenza surveillance [26] .
Hong Kong is a subtropical region of almost 7 million people, 95% of whom are ethnic Chinese, with a mean temperature of 24uC and mean relative humidity of 79% [27] . It lies geographically in the Northern hemisphere, and its influenza season occurs during February-April, sometimes with a second peak during June-August, each year. In contrast, other Northern hemisphere countries usually have a more extended influenza season from November to March/April, whereas the influenza season of Southern hemisphere countries usually occur from May to September [7, 28] . Hence, Hong Kong may be unique in that its biphasic influenza seasonality seems to straddle those of the Northern and Southern hemisphere countries, making the molecular epidemiology of its circulating influenza viruses of great interest. In addition, Hong Kong and Southern China have been referred to as the 'epicenter' for new influenza A viruses with pandemic potential for over 25 years now [29] . For all of these reasons, any investigation of the underlying basis for influenza seasonality may benefit greatly from a study of influenza viruses isolated from Hong Kong.
In this study, an analysis is presented of 281 Hong Kong influenza A(H3N2) hemagglutinin (HA) and neuraminidase (NA) full-length, dated sequences collected over 10 years (1997-2006) to assist the ongoing efforts to elucidate the underlying basis for the seasonality of influenza A(H3N2).
The HA and NA ML phylogenetic trees (with and without the additional, down-loaded contemporary sequences from publicly available archives) produced in this study are too large to include as separate figures in this paper and have been published as online Supporting Information in a scrollable PDF format for further inspection on the PLoS ONE journal website (http://www. plosone.org/home.action).
For each of these trees, certain clusters of interest have been highlighted using annotated red boxes or ellipses, and will be specifically referred to, in the following text for further description and discussion.
All of these 281 Hong Kong influenza A(H3N2) HA and NA sequences have been deposited on GenBank (Accession nos.: EU856814-EU857094 for HA, and EU857095-EU857375 for NA sequences).
Figures S1 and S2 show the relationship between the 281 HA and NA sequences for the Hong Kong influenza A(H3N2) samples and 4 World Health Organization (WHO) influenza A(H3N2) vaccine strain HA sequences (Syd/5/97, Mos/10/99, Fuj/411/02 and Cal/7/04).
These Hong Kong HA and NA sequences were inspected to determine if there were any sequences from consecutive influenza seasons occurring on the same branch, indicating that viruses with the same or very similar HA and NA gene sequences were occurring in adjacent influenza seasons. This would suggest that that particular virus carrying this gene may have remained 'latent' in that population, to re-emerge in the same population the following season.
One example of such possible viral persistence between influenza seasons was found, with HA and NA sequences from the same viruses (5251Jan02 and 5267Jan03, as indicated in Figures S1 and S2 for the HA and NA phylogenetic trees, respectively), showing a similar clustering pattern for both these genes, separated by at least one year.
Interestingly, the HA sequence from sample 5250Jan02 clusters closely with those from samples 5251Jan02 and 5267Jan03 (Figures S1 and S3), but its NA sequence lies some distance away on a separate branch (Figures S2 and S4 ). This may suggest a possible reassortment event, either with its HA or NA gene segment. Further full genome sequencing and analysis may resolve this issue.
The The relationship between the WHO vaccine HA and NA sequences and those from Hong Kong and elsewhere can be seen even more clearly in Figures S3 and S4 when the 315 JCVI sequences are added to each tree. Although these contemporary JCVI sequences are mainly drawn from just three additional locations, they still represent the Northern hemisphere (New York, USA) and the Southern hemisphere (Western Australia and various locations in New Zealand). Again, for reference, the January Hong Kong HA and NA sequences from each year are again highlighted in red boxes.
In addition, in Figures S3 and S4 , red ellipses have been added to show where similar HA and NA sequences from other, non-Hong Kong locations have clustered with Hong Kong sequences on the same branch. The dates of such sequences may be the same (within the limit of the one month temporal resolution used in this study), relatively similar, or very different. These highlighted clusters serve to demonstrate the mobility and ubiquity of this influenza A(H3N2) virus, worldwide, during this 10-year period, i.e. genetically similar viruses can appear in different parts of the world at similar and also different times. These examples are not meant to be exhaustive and other such examples may be found in these trees.
These number and position of the clusters indicated by the red ellipses differ between Figure S3 (HA sequences) and Figure S4 (NA sequences) probably because there are different selection pressures acting on these two genes as they have quite different functions (i.e. the HA protein is used by the virus to bind to the host cell for entry, whereas the NA protein is an enzyme that enables new progeny viruses to leave the host cell). Also, in Figures 3 and 4 , for both the HA and NA gene sequences, respectively, there is a large region of transition between Mos/10/99-like and Fuj/411/02-like viruses, containing sequences collected during 1999-2005.
Inspection of the protein-coding alignment for NA sequences showed no evidence of any of the known N2 neuraminidase inhibitor (NAI) resistance-associated mutations, R292K and E119V, in any of these Hong Kong influenza A(H3N2) isolates collected between 1997-2006.
This comparative analysis of dated HA and NA sequences from influenza A(H3N2) viruses from 4 geographical regions of the world (New York, Western Australia, New Zealand and Hong Kong) attempts to elucidate more clearly the behavior of influenza A(H3N2). This study contributes an additional 10 years of influenza A(H3N2) HA and NA sequences, from Hong Kong, to the publicly available sequence database (GenBank), which should aid other researchers investigating an underlying basis for influenza A(H3N2) seasonality.
The approach of the analysis in this study has been to compare accurately dated HA and NA sequences using established phylogenetic techniques to examine which sets of sequences cluster together, and by examining the dates and locations from which they were collected, to infer the movements of the virus within those dated periods.
A similar analysis was recently performed using dated whole genome influenza A(H3N2) sequences from New York, New Zealand and Australia, downloaded from publicly available databases, in an attempt to test two competing hypotheses: whether seasonal influenza A(H3N2) viruses continuously 'migrate' around the world, particularly between Northern and Southern hemispheres; or whether the virus remains 'latent' in one location and reactivates each year to produce the familiar pattern of influenza seasonality [19] . As these authors stated, ideally, whole genomes should be used for more accurate phylogenetic analyses of influenza virus as has been reported previously [14, 15] , with at least one good reason for this being the potentially misleading conclusions caused by influenza viral HA and NA gene reassortments [19] . However, many laboratories worldwide do not have the resources to perform whole genome sequencing, which is expensive -particularly those in subtropical and tropical regions from where such influenza sequence data is significantly lacking.
In addition, with any phylogenetic study such as this, there is always a limitation on the number of sequences that are available (i.e. the number of respiratory samples containing influenza viruses that have been collected and sequenced in any one influenza season), and the number that can be comfortably analyzed within a given time-frame (i.e. the limitations on computing power, which again may be more of a problem in tropical and subtropical countries that are more resource-limited).
In this particular study, there is also a problem of sample bias as these sequences were obtained from only hospitalized children (rather than from those who remained in the community), and may therefore reflect the influenza virus population isolated from the more clinically severely ill patients (or those with more concerned, anxious parents). However, there was a rationale for deliberately selecting children's samples for this study. The reason for this is that, especially in Hong Kong, unlike adults, children of school age (1-10 years old) are more likely to stay close to home and not travel far from home, which would minimize any importation of influenza viruses from overseas. This would therefore reduce this confounding factor when assessing the migration vs latency hypotheses as an explanation for the underlying mechanism of influenza seasonality in Hong Kong.
Accepting all of these shortcomings, some of which are inevitable for such phylogenetic studies (since not all samples can be collected and sequenced from all infected individuals from all over the world for any particular virus), there are still some useful conclusions that can be gained from this study: i) from Figures S1 and S2, there is at least one example of a virus that reappears, relatively unchanged between consecutive influenza seasons, and which can be seen as some evidence to support the 'latency' hypothesis [19] . Since the same viruses show this same pattern of clustering in both their HA and NA genes, this reduces the likelihood that this was due to a reassortment event in one of these genes, i.e. it is less likely for the same viruses to have reassorted both the HA and NA genes during the same years -though of course this possibility cannot be ruled out. Whole genome sequencing would be useful to confirm this if resources are available in the future. ii) from Figures S3 and S4 , it is difficult to say whether viruses from Hong Kong preceded (or gave rise to) those from elsewhere, since viruses from outside Hong Kong can be found to both pre-date and post-date those isolated from Hong Kong. However, there are several examples of similar viruses being isolated in different parts of the world at about the same time (as shown in some of the red ellipses in Figures S3 and S4 Figures S3 and S4) , though this may not necessarily mean that the Fuj/411/02-like viruses originated from there.
Hence, these results suggest that the seasonality of influenza A(H3N2) may, in fact, result from a combination of these 2 models postulated by Nelson et al [19] , i.e. mainly migration, but with occasional examples of latency. Again, due to the unavoidable, incomplete sampling and sequencing of influenza A(H3N2) viruses worldwide, this support for these hypotheses ('migration' and 'latency'), as presented here, is admittedly, only patchy at best. However, the fact that we can find at least one example supporting the latency hypothesis, in these 281 HA and NA sequences spanning 10 years (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) , suggests that they may both play a part in the underlying mechanism governing influenza A(H3N2) seasonality.
The large variations in both the HA and NA sequences during the transitional period between Mos/10/99 and Fuj/411/02 ( Figure S3 and S4) may have reduced the protective efficacy of any pre-existing influenza antibodies (from prior infection or immunization by Mos/ 10/99-like viruses) before the inclusion of the Fuj/411/02 strain in the World Health Organization (WHO) recommendations for the seasonal influenza vaccine in 2004. Such a reduction in protective efficacy from the contemporary WHO influenza vaccine has indeed been suggested in several reports [14, 30, 31] . Similarly, although, the NA antigen is not usually specified in seasonal influenza vaccine, and its protective effect is unknown, its sequence variation in NA during this transitional period, may have also contributed to the reduced protection provided by the seasonal influenza A(H3N2) vaccine component during this period, when the Fuj/411/02-like viruses were emerging.
In summary, this study has provided additional data from Hong Kong to support a mainly migratory mechanism to explain the underlying seasonality of influenza A(H3N2) viruses. However, there may be small localities of so-called 'latency' where viruses remain circulating at low levels within that local population, to reemerge during the influenza season of the following year, with relatively little genetic change. This may affect only a minority of these populations, with the majority being infected by newly imported influenza viruses from elsewhere. These concepts are summarized in Figure 1 .
Two recent papers [37, 38] have suggested that the existing evidence tends to support a migration rather than a latency mechanism to explain the annual seasonality of influenza A. However, they did not have access to large numbers of influenza sequences from Southeast Asia. Thus, in view of the new data presented in this study, it is hoped that such hypotheses may be revised, to include a contribution from the latency mechanism. In some populations (perhaps those more localized in Southeast Asia), this latency mechanism may contribute more significantly to the underlying basis for the seasonality of influenza A -a possibility not entirely ruled out by one of these studies [38] . More data is required to explore this hypothesis in those populations. Admittedly, the fact that in this 10-year (1997-2006 ) data set of almost 300 influenza A(H3N2) HA and NA sequences from Hong Kong, we have only found one particular example supporting the latency mechanism, does suggest that this is a relative rarity, and that the migration mechanism is probably responsible for the majority of influenza seasonality patterns seen worldwide.
Finally, it is interesting, and to a certain extent, reassuring, to note that during this 10-year period (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) , no influenza A(H3N2) viruses isolated from this cohort of NAI-naïve children from Hong Kong, showed any evidence of naturally occurring, known NAI resistance-associated amino acid mutations (R292K and E119V).
There are many parts of the puzzle remaining, such as exactly how do some of these influenza viruses migrate so widely, yet still remain relatively similar over several years. How is the low level circulation of 'latent' influenza viruses accomplished between seasons? Is this a property of the population's host immune responses, the virus, the environment or some combination of each of these factors?
In a collaborative effort to answer these questions and perhaps to improve the accuracy of influenza strain forecasting and vaccine composition [38] , it is also hoped that this study will encourage more researchers in Southeast Asia to make their influenza sequences publicly available for analysis -especially whole genome sequences where resources permit, and particularly sequences from more tropical countries where influenza is prevalent all year round, with less well-defined seasonal peaks.
Approximately 30 influenza A(H3N2) isolates for each year of 1997-2006 obtained from the nasopharyngeal aspirates (NPAs) of children aged 1-10 years, admitted to the Prince of Wales Hospital (PWH) in Hong Kong, were retrieved from long-term archives (stored either at 270uC or in liquid nitrogen) for hemagglutinin (HA) and neuraminidase (NA) gene sequencing and analysis. These children presented with acute respiratory illness and did not receive anti-influenza therapy before or during their illness. As influenza A(H1N1) was the predominant circulating virus during 2006, very few H3N2 isolates were obtained for that year. Table 1 shows the age and sex distribution of these children.
Verbal consent for the initial diagnostic testing of these samples for respiratory viruses, including influenza, was obtained from the parents of these children. Such verbal (rather than written) consent is routine for such standard diagnostic tests in Hong Kong. The local Joint Chinese University of Hong Kong-New Territories East Cluster (Joint CUHK-NTEC) Research Ethics Committee institutional review board awarded ethics approval for this retrospective sequencing and molecular epidemiological study (study reference number: CRE-2005.390) without the need to obtain further, explicit, written consent. This is also in agreement with the American College of Epidemiology guidelines [33] for such retrospective epidemiological/surveillance studies.
The samples retrieved from the deep-freeze archive were first generation isolates, i.e. the original clinical samples (NPAs) had been inoculated, for routine diagnostic testing, into Madin-Darby Canine Kidney (MDCK) cells. These MDCK-cultured viral isolates were originally confirmed to be influenza A(H3N2) before being frozen and archived. For this study, these frozen isolates were retrieved from deep-freeze, thawed and then used directly for sequencing. If any of these newly-thawed, archived samples failed to amplify at this stage, as determined by ethidium bromide staining and gel electrophoresis, it was inoculated into MDCK cells and re-cultured. After this additional step, most isolates were successfully sequenced. If this step still failed, then an alternative isolate was retrieved from deep-freeze for sequencing.
Total RNA was extracted using the PureLink TM Viral RNA/ DNA Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions, and resuspended in 50 mL of RNase-free water. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out with SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase kit (Invitrogen, Carlsbad, USA) according to the manufacturer's protocols. In brief, a 25-mL reaction mix containing 0.5 mM of each forward and reverse primers and 10 mL of extracted RNA template were used for the RT-PCR. Sets of primers was designed to amplify the complete influenza A(H3N2) HA and NA genes ( Table 2) .
The RT reaction (55uC for 30 min) was followed by 94uC for 2 min and 40 cycles of PCR (94uC for 30 sec, 50uC for 30 sec, and 68uC for 1 min 45 sec, for each cycle) and a final extension at 68uC for 10 min.
The PCR products were purified by MicroSpin Sephacryl S-400 HR columns (Amersham Biosciences, UK). Sequencing reactions were performed using BigDyeH Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, USA) with 2.5 mL of template cDNA. For sequencing the HA and NA genes, the primers used are shown in Table 3 .
Sequencing reactions were performed on an Applied Biosystems 3130 ABI sequencer (ABI, Foster City, USA) and in both directions to cross-check the results. Alignments of nucleotides sequences were carried out using SeqScape v2.5 (Applied Biosystems, Foster City, USA).
These Hong Kong influenza A(H3N2) HA and NA gene sequences were aligned and edited in BIOEDIT v.7.0.9.0 [32] .
After alignment and manual editing, to enable all sequences (i.e. both the Hong Kong and JCVI reference sequences) to have the same final length for the construction for each of the phylogenetic trees shown in Figures S1 to S4 , the sequence lengths were: This was due to different HA and NA sequence lengths being available in the respective JCVI and WHO sequence data bases.
Phylogenetic tree construction was performed with PAUP* version 4.0b10 [34] by using a maximum likelihood (ML) approach, under an optimum model of evolution as determined by MODELTEST v3.7 [35] . Due to the large dataset and to reduce the time required for computation, optimal trees were searched for by using a nearest neighbor interchange (NNI) heuristic search strategy. Bootstrapping was performed within PAUP* and displayed using exported PDF files created using Figtree v1.0 (previously available from: http://tree.bio.ed.ac.uk/ software/figtree/), which were subsequently annotated using Adobe Acrobat Professional 6.0.
The phylogenetic trees for these influenza A(H3N2) HA and NA sequences for the period 1997-2006 were rooted against the reference HA and NA sequences obtained from the influenza vaccine strain A/Syd/5/97(H3N2) downloaded from the Los Alamos National Laboratory (LANL) database (http://www.flu. lanl.gov/vaccine/) [36] . This strain was used as it was representative of the influenza A(H3N2) viruses circulating in 1997, i.e. at the start of this Hong Kong influenza A(H3N2) archive. Other available HA and NA sequences for other vaccine strains (Mos/ 10/99, Fuj/411/02, Cal/7/04) were also downloaded and included in all the phylogenetic trees.
For comparison with other contemporary influenza A(H3N2) sequences worldwide between 1997-2006, all publicly available (at the time of this analysis) dated HA and NA sequences from children of similar ages (0-16 years old -the upper range was extended to include more sequences), spanning this period were downloaded from the then TIGR (The Institute for Genomics Presence/absence of established N2 neuraminidase inhibitor (NAI) resistance-associated mutations, R292K and E119V
Once the NA nucleic acid sequences were obtained, they were aligned, in-frame for protein coding, and converted to amino acids using the built-in function in BIOEDIT. The presence or absence of the established NAI resistance-associated mutations, R292K and E119V, was then determined by inspection of the resulting amino acid alignment, with reference to the influenza A/Syd/5/ 97 (H3N2) NA sequence, which was isolated before the licensing and widespread use of neuraminidase inhibitors that began after 1999/2000. Table S1 The 315 downloaded TIGR(JCVI) influenza A(H3N2) HA and NA sequences used to construct the multi-country HA and NA phylogenetic trees shown in Figures S3 and S4 . MS Excel file containing the GenBank Accession numbers of the 315 downloaded TIGR(JCVI) influenza A(H3N2) HA and NA sequences which were used to construct the multi-country HA and NA phylogenetic trees shown in Figures S3 and S4 Author Contributions H5N1 and 1918 Pandemic Influenza Virus Infection Results in Early and Excessive Infiltration of Macrophages and Neutrophils in the Lungs of Mice Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. These results together show that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection. In addition, primary macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro and in infected mouse lung tissue. The influenza pandemic from 1918 to 1919 was the most devastating infectious disease pandemic ever documented in such a short period of time, killing nearly 50 million people worldwide [1] . Unlike the epidemiological profiles of most influenza infections, young adults aged 18-35 yrs old had the highest mortality rate, so much so that the average life expectancy during those years was lowered by 10 years [2] . In 1918, severe destruction of lung tissue observed by pathologists at autopsy was unlike that typically seen in cases of pneumonia [3] and histopathological analysis of lung tissue showed severe tissue consolidation with unique destruction of the lung architecture [3, 4] . Human infections with highly pathogenic avian influenza (HPAI) strains of subtype H5N1 since the first outbreak in 1997 have also been particularly severe for children and young adults [5] [6] [7] . Assessing pulmonary infiltrates in response to influenza H5N1 virus infection has been difficult due to the lack of autopsy material. The basis for the high morbidity and mortality associated with the 1918 virus and recent H5N1 viruses remains inconclusive based on viral genetic analysis alone and accounts of patient lung pathology provide only qualitative information about the host factors contributing to disease [4, 8, 9] . Great concern about a pandemic caused by a novel avian H5 subtype virus warrants comparative studies to better understand the cellular pathology caused by a pandemic virus and potentially pandemic viruses. Identification and quantification of the inflammatory cell types associated with highly pathogenic respiratory infections represent prospective targets for modulation of host innate immune responses.
Recent studies using animal models to investigate the mechanism(s) of severe influenza virulence have implicated the innate immune system in complicating lung tissue recovery [10] [11] [12] [13] . Mouse models of highly pathogenic (HP) H5N1 [14] [15] [16] [17] [18] and 1918 [19, 20] influenza virus infection confirm histological observations of severe lung pathology in human patients, however, the types of immune cells present during the peak of lung pathology have not been fully elucidated. Excessive immune cell infiltration during an acute lung injury may impair tissue restoration directly by interfering with gas exchange, or indirectly through the release of soluble immune mediators. In the present study, we determined key immune cellular components in the murine lung following infection with matched H5N1 and H1N1 virus pairs that represent high and low virulence infections of each influenza subtype as previously determined in the mouse model [18, 21] . The two H5N1 viruses used in this study (A/Thailand/16/2004 and A/Thailand/SP/83/2004) were isolated in 2004 from fatal human cases in Thailand but have a differential pathogenic outcome in mice, specifically a low and high mouse lethal does 50 (LD 50 = 1.7 and 5.6 log 10 PFU respectively) [18] . For relevant comparison, we also used a contemporary (non lethal) seasonal H1N1 human isolate from 1991 (A/TX/36/91) and the reconstructed 1918 pandemic virus [21] . A detailed flow cytometry evaluation of lung cells demonstrated that macrophages and neutrophils are the prominent cell types associated with and potentially mediating the severe lung pathology following infection with the highly virulent H5N1 and 1918 viruses. Moreover, inoculation of macrophages and dendritic cells with the HP viruses in vitro or ex vivo reveals that some innate immune cells can themselves serve as targets of viral infection.
Highly pathogenic H1N1 and H5N1 viruses exhibit early and sustained replication in murine lung tissue following intranasal infection Female BALB/c mice were infected intranasally with either highly pathogenic (HP) or low-pathogenic (LP) influenza viruses ( Table 1 , Methods) based on known LD 50 's and phenotypes of disease in mouse [21] and ferret [18, 22] models. As shown in Figure 1 Increased cellularity in lungs of mice infected with highly pathogenic H1N1 and H5N1 influenza viruses Whole lungs collected (without perfusion, therefore including the bronchoalveolar lavage contents (BAL)) from both 1918 and Thai/16 virus-infected mice showed an increase in overall tissue cellularity as early as 3 days p.i. (Figure 2A ). By day 3 p.i., lungs infected with the HP influenza viruses (containing between 4.3-5.1610 7 cells) had nearly twice as many total lung cells than were measured in the HP infection groups just 24 hours previously at day 2 p.i. (2.1-2.4610 7 cells). Significant differences (*p,0.05) were observed between HP and LP infection groups in total lung cell number at every time after day 2 p.i. Total lung cell numbers doubled in HP infection groups between days 5 and 7 p.i. and by day 7 p.i., when viral titers were high for all four viruses, total cell numbers in the lungs of mice infected with either HP H1N1 or H5N1 viruses were 6-fold higher than those in PBS-inoculated mice, and at least 3-fold higher than those found in LP virusinfected lungs (Figures 1 and 2A) . On day 7 p.i., there were as many as 1.3610 8 cells in HP-infected lungs compared with 4.0-8.0610 7 cells in LP-infected and 1.8610 7 cells in PBS-inoculated lungs.
To quantify the immune cell sub-populations responding to viral infection, we next determined the total cell numbers of specific inflammatory cell populations in the infected lungs using flow cytometry ( Figure 2B ). Compared with PBS-inoculated
Patients who succumbed to influenza during the 1918 pandemic had severe lung pathology marked by extensive inflammatory infiltrate, indicating a robust immune response in the lung. Similar findings have been reported from H5N1-infected patients, raising the question as to why people expire in the presence of a strong immune response. We addressed this question by characterizing the immune cell populations in the mouse lung following infection with the 1918 pandemic virus and two H5N1 viruses isolated from fatal cases. Our data shows excessive immune cell infiltration in the lungs contributing to severe consolidation and tissue architecture destruction in mice infected with highly pathogenic (HP) influenza viruses, supporting the histopathological observations of lung tissue from 1918 and H5N1 fatalities. We found that certain cells of the innate immune system, specifically macrophages and neutrophils, increase significantly into the mouse lung shortly following HP virus infection. Interestingly, lung macrophages and dendritic cells were shown to be susceptible to 1918 and H5N1 virus infection in vitro or ex vivo, suggesting a possible mechanism of immunopathogenesis. Identification of the precise inflammatory cells associated with lung inflammation will be important for the development of treatments that could potentially enhance or modulate host innate immune responses. [18, 21, 22] . Generated by reverse genetics [21] . doi:10.1371/journal.ppat.1000115.t001 animals, mice infected with each of the viruses exhibited an increase in the numbers of macrophages (CD11b + , CD11c 2 , Ly6G/c 2 , CD4 2 , CD8 2 ) beginning 3 days p.i. and continuing through day 9 p.i. Strikingly, there were significantly (*p,0.05) more macrophages in HP virus-infected lungs than in LP virusinfected lungs from days 2 through 9 p.i. (Figure 2B , a). As early as 2 days p.i, there was 1-2 million more macrophages in the lungs of HP-infected lungs compared to LP infected lungs. 1918 and Thai/ 16 virus-infected lungs had twice as many and nearly 4 times as many macrophages compared to LP viruses at days 3 and 5, respectively. Numbers of lung macrophages peaked in 1918 and Thai/16 infected mice (1.5 and 1.2610 7 cells, respectively) at day 7 p.i. before waning as demonstrated at day 9 p.i.
An increase of at least twice as many neutrophils (CD11b + , CD11c 2 , Ly6G/c + , CD4 2 , CD8-) was observed as early as 1 day p.i. in all infection groups, compared with neutrophils numbers in PBS-inoculated mice. On day 2 p.i. there was on average (n = 3 mice) over four hundred thousand more neutrophils in the lungs of 1918 virus inoculated mice than TX/91 inoculated mice. Significant differences in neutrophil populations (*p,0.05) between the HP and the LP virus groups emerged on day 3 p.i. and were sustained at each subsequent time point measured ( Figure 2B , b). On day 3 p.i., more than twice as many neutrophils were found in HP compared to LP infected lungs. Between days 3 and 7 p.i., lungs infected with HP viruses displayed a three fold increase in neutrophil numbers. At the peak of neutrophil infiltration on day 7 p.i, there was 4-8 million more neutrophils in the lungs of HP-infected animals compared to LP virus-infected mice. Although numbers of dendritic cells (CD11b 2 , CD11c + , Ly6G/c 2 , CD4 2 , CD8 2 ) ( Figure 2B , c) and CD4 + T (CD11b 2 , CD11c + , Ly6G/c 2 , CD8 2 ) and CD8 + T (CD11b 2 , CD11c + , Ly6G/c 2 , CD4 2 ) cells ( Figure 2B , d and e) in all groups of infected mice increased slightly compared with numbers in PBSinoculated animals during the course of infection, no significant differences were found between HP and LP infection groups, suggesting that these cell populations are not major contributors to the histopathological consolidation observed in lungs during HP H5N1 virus infections in mice [16] .
Macrophages and neutrophils account for the highest percentage of the total lung leukocytes following infection with highly pathogenic influenza viruses We next determined specific immune cell sub-populations given by percent of the total lung leukocyte population in order to reveal differential population dynamics amongst the immune cell populations measured in these studies (Table 2) . Strikingly, macrophage populations by day 3 pi, represented 24% and 24.4% (nearly one-quarter) of the total gated lung leukocytes of mice infected with 1918 and Thai/16 (HP) viruses, respectively, which was significantly higher than the frequency of macrophages detected in TX/91 and SP/83 virus-infected mice from days 2 though 9 p.i. (^p,0.005). Neutrophil populations were elevated in all infection groups compared to mock levels beginning 1 day p.i ( Figure 2 , panel B) however percentages of neutrophils in HP virus infected mice were significantly higher (* p,0.05) than those in LP virus-infected mice beginning day 2 p.i. and levels remained elevated at each subsequent time point measured ( Table 2 ).
In contrast, dendritic cell populations as percent of the total leukocyte population declined over the course of infection with HP viruses while percentages increased in LP infections, peaking at days 3 and 5 p.i. (Table 2 ). Significant differences in percent dendritic cells between HP and LP infection groups were observed day 3 p.i. and at all other subsequent time points (* p,0.05). Percentages of CD4 + T cells in the lungs decreased in all infection groups but no significant differences were observed in CD4 + or CD8 + T cell population dynamics between HP and LP infection groups. Together, these results indicate that macrophages and neutrophils are responsible for the majority increase in total lung cell numbers following infection with the 1918 and HP H5N1 influenza viruses.
To better understand potential influences on immune cell population dynamics in the lung following HP influenza virus infection as demonstrated in Figure 2B and Table 2 , we analyzed 17 chemokines and cytokines in the lungs of mice on days 1 and 4 p.i and report data here on 6 of the analytes that revealed significant differences among the viruses tested ( Figure 3 ). These earlier time points were chosen in an effort to understand the temporal relationship with lung immune cell infiltration in 1918 and Thai/16 infections ( Figure 2 ) and day 4 p.i. is a time when significant differences were observed in lung virus titers between HP and LP infection groups ( Figure 1 ). As shown in Figure 3 , on day 1 p.i. TX/91 infected lungs exhibited higher titers of IL-1a, IFN-c, and KC compared to 1918 virus-infected lungs though levels of MIP-1a, MCP-1, and IL-6 were higher in 1918 virus infected lungs. Cytokine levels were similar between the H5N1 viruses at day 1 p.i.. In contrast, lung tissue cytokine and chemokine levels at day 4 p.i. were higher among 1918 and Thai/ 16 virus-infected mice compared to those infected with the subtype-matched LP (TX/91 and SP/83) counterpart viruses for all cytokines and chemokines measured. Protein levels of the potent monocyte chemoattractant MCP-1 were 10-fold higher in 1918 and H5N1 virus-infected lungs than TX/91 virus-infected lungs, whereas levels of the MIP-1a chemokine were notably elevated in Thai/16 virus-infected lungs. KC (the mouse equivalent of human IL-8) [20, 23] levels were 10-fold higher in Thai/16 and 3-fold higher in 1918 virus infected lungs compared to TX/91 virus-infected lungs with SP/83 levels similar to 1918 infected lungs. The HP viruses were also potent inducers of IL-1a and IFNc. IL-6, (a generally pro-inflammatory cytokine) was also 
Due to the increased presence of macrophages in the lungs following HP influenza virus infection, replication of paired H1N1 and H5N1 viruses (Table 1 ) was assayed over time in primary human PBMC-derived macrophages and mouse lung macrophages to address whether these cells are specific targets of viral infection and can productively replicate HP viruses. Mouse lung macrophages were harvested from naive BALB/c mice and infected in vitro (MOI = 0.1, Figure 4A ) as described. While the HP 1918 and Thai/16 viruses exhibited a slight increase in log titer very early after inoculation (13-24 hrs p.i.), overall these viruses did not replicate efficiently compared to the growth kinetics of these viruses observed in lung epithelial cells [22, 24] . At titers and these differences were statistically significant (^p,0.05) at 72 hrs p.i.. The lack of prolific replication of these four viruses in primary mouse lung macrophages was confirmed further when a higher MOI (1.0) was utilized (data not shown). Human macrophages also supported replication of all four viruses ( Figure 4B , MOI = 0.1). At 48 hrs p.i. 1918 and Thai/16 infected macrophages exhibited 180 times higher virus titers than TX/91 and SP/83 infected cultures. Interestingly, 1918 virus-infected macrophages exhibited a higher baseline titer soon following infection that was found to be statistically significant when compared to the other infection groups ( { p,0.05). In summary, while human macrophages are a target of viral replication and support replication well, mouse lung macrophages support low levels of 1918 and Thai/16 virus production early following infection.
Additional experimentation with primary human macrophages revealed that levels of pro-inflammatory cytokines were higher for H5N1-infected cells than either 1918 or TX/91 virus infected cells (48 hrs p.i., MOI = 0.1, Figure 5 ). Significant differences in cytokine levels were observed between H5N1 and H1N1 virus Figure 4B ). Thai/16 infected cultures elicited at least a 2 fold greater cytokine response than the SP/83 virus infected cultures in every cytokine measured except IL-8 and MCP-1 chemokines where Thai/16 levels were only slightly higher than SP/83 levels.
Because of their important role in antigen presentation to T cells, we also assessed the ability of primary dendritic cells to productively replicate pandemic H1N1 and human H5N1 viruses ( Figure 6A To determine if innate immune cells are being productively infected in vivo, macrophages and dendritic cells were purified from lungs of infected mice and cultured for infectious virus. Lungs from infected (3 days p.i.) mice were harvested and ex vivo cultures containing either lung macrophages or dendritic cells were sampled for infectious virus over a 65 hour time period. While the seasonal influenza isolate, TX/91 virus was not produced from either macrophages (CD11b+) or dendritic (CD11c+) cells, the 1918 pandemic virus as well as the two human H5N1 isolates were released into the culture supernatant (Figure 7) , indicating that these cells are being productively infected in the mouse lung. In CD11b+ (macrophages) cell cultures ( Figure 7A Figure 7B ). These data further demonstrate that mouse lung macrophages and dendritic cells are susceptible to highly pathogenic influenza virus infection in the lung tissue.
Lung consolidation has been described as a pathological feature of severe influenza virus infection caused by the 1918 pandemic virus and H5N1 viruses in humans [9, 25, 26] as well as in animal models [11, 12, 18] . Using a detailed flow cytometry evaluation, the current study set out to characterize differences in the cellular innate immune response in the mouse lung following highly pathogenic (HP) or low pathogenicity (LP) influenza virus infections. Lungs from mice infected with the HP 1918 H1N1 virus and a recent H5N1 human isolate (A/Thailand/16/04 (Thai/16)) exhibited a significant increase in cellularity in comparison to the LP seasonal H1N1 isolate, A/Texas/36/91 (TX/91). Significant differences in titer between HP and LP virusinfected mice were observed as early as day 1 post-inoculation (p.i.), likely forecasting the dramatic increase in immune cell infiltration in the lungs of these virus-infected mice. Interestingly at day 7 p.i. when peak lung cellularity was observed in HP virus infection groups, differences in virus titers between paired subtype viruses (1918 compared to TX/91 and Thai/16 compared to SP/ 83) were minimal and were limited to a maximum difference of 1 log (Figure 1 ), indicating a failure by the immune system to clear the viral infection. Lung cellularity was further investigated by characterizing the comprising immune cell sub-populations. We observed a significant increase in macrophages and neutrophils early following infection with the 1918 and Thai/16 viruses, and their sustained presence in the lung tissue mark a distinction between HP and LP influenza virus infection. These data show that virus replication in the lungs of HP influenza infections are sustained at high levels the first week of infection regardless of the high numbers of immune cells present in the tissue. Although the current study did not determine whether these cells are playing an antiviral role against the HP viruses, it has been previously shown that neutrophils and macrophages assist in the clearance of influenza virus early during infection; these cells appear to be capable of only partially reducing the virus load in the lung despite their presence at high numbers [20] . We also observed a decrease in the percentage of lung-associated dendritic cells and T cell lymphocytes during HP influenza virus infection. A decrease in the number of circulating lymphocyte populations has also been previously observed in the peripheral blood of humans and mice infected with H5N1 viruses [5, 9, 10, 15, 20, 27] . The precise mechanism of leukocyte depletion during H5N1 infections is not well understood, but evidence of apoptosis in the spleen and lungs of HP H5N1-infected mice detected in situ suggests a mechanism for cell loss [16] .
Influenza virus growth in the respiratory epithelium and the subsequent release of chemotactic proteins from those cells may encourage the increased presence of macrophages and neutrophils [28, 29] . Macrophages and neutrophils can secrete chemokines and cytokines that can act in an autocrine fashion which in turn can promote the increased migration of those cells and other leukocytes into the lung tissue [30] . Elevated levels of certain chemokines and cytokines have been associated with high viral load and severe disease in H5N1 virus infected patients [26, 31] . These studies show that infection with HP 1918 and Thai/16 H5N1 viruses result in elevated amounts of pro-inflammatory chemokines and cytokines in the lungs of mice day 4 post-infection compared with TX/91 and SP/83 infected mice, a time point that correlates with rising but significantly different lung virus titers between infection groups. Elevated levels of the chemokines MCP-1 [32] and MIP1-a were observed among H5N1 and 1918 virus infected mouse lungs. Although, MIP-1a does not appear to be critical for virus replication and spread in the mouse model [10] , this chemokine exhibits a variety of pro-inflammatory activities including macrophage and neutrophil recruitment and has been associated with fatal outcomes in human H5N1 virus infections [31] . Lungs infected with the 1918 pandemic and Thai/16 H5N1 viruses also exhibited significantly higher levels of IFN-c on day 4 p.i compared to their subtype-paired LP virus counterparts. IFN-c is known to mediate the increased production of nitric oxide [33] which can subsequently result in the recruitment of more neutrophils and macrophages. Higher levels of IL-6 were measured in 1918 and Thai/16 virus infected lungs, supporting observations obtained with the 1997 H5N1 viruses [10] and has been correlated with systemic illness symptoms and fever in experimental human TX/91 infections [34] . By directly measuring cytokine protein levels, these data provide confirming evidence of a heightened lung cytokine response to 1918 and H5N1 infection in mice [13] . Interestingly, while we reveal marked similarities between the HP 1918 and Thai/16 viruses in overall lung cellularity, virus growth and patterns of immune cell sub-population dynamics over time, Thai/16 virus infection consistently resulted in higher levels of chemokines and cytokines both in mouse lungs and human macrophages. Although it has been shown recently by two independent research groups that the lack of key cytokines (through the use of single cytokine gene knockout mice) had no effect on the overall disease outcome or virus replication among H5N1 virus-inoculated mice [10, 35] , the present results along with data from others continue to indicate that pro-inflammatory cytokines correlate with disease outcome [36] . It is thought that these immune mediators do not act singularly in vivo and it will be critical to reveal these concerted interactions (both locally in the lung and systemically) to further our understanding of the pathogenesis of H5N1 infection in animal models and in human patients.
Macrophages and dendritic cells play a fundamental role in the lung at all stages of influenza virus infection [20, 37, 38] . We have provided evidence regarding the higher replication efficiency of HP influenza viruses in primary human macrophages and dendritic cells, a property that has also been demonstrated previously in other primary cells [24, 39] . Mouse macrophages were also susceptible to virus infection in vitro, however they did not support productive replication to the level observed in primary human monocyte-derived macrophages ( Figure 4B ) or lung epithelial cells [21, 24] . However, the cytopathic effects (estimated by visual examination of monolayers) among HP virus-infected mouse and human macrophages was observed in a shorter period of time compared to LP virus infected cells. Interestingly, the 1918 virus exhibited higher baseline titers in human and mouse macrophages as early as 2 hrs p.i. compared to the TX/91 H1N1 virus or the H5N1 viruses, indicating a curious property of this pandemic virus. While the importance of the binding properties of the HA molecule has been demonstrated elsewhere extensively [22, 40, 41] , further resolution of this interesting finding and its immunological importance deserves further experimentation. The role of other cell surface molecules on innate immune cells such as the mannose receptor in HP influenza infection should be investigated [42, 43] . The higher viral replication of HP viruses in dendritic cells also correlated with the severe pulmonary disease observed in mice. We also demonstrated definitively that these cells are targets of infection ex vivo by highly pathogenic influenza viruses like the 1918 pandemic virus and recent H5N1 isolates (Figure 7) . Thus, it appears that macrophages and dendritic cells may contribute to the pathogenesis of HP virus infection due to their susceptibility to influenza virus infection. An inability to mount an adaptive immune response due to direct infection of important innate immune cells such as macrophages and dendritic cells may be a critical difference in host outcome during influenza virus infection [44] . This coupled with the phenomenon of T cell depletion in infected mice [17] and human patients [9, 31] may allow for uncontrolled viral replication. While reducing viral load through anti-viral intervention remains the best treatment option for H5N1 patients, therapies that moderate immunopathology may help to reduce the high case fatality rate currently associated with virus infection [45] . (Table 1) . Low pathogenicity in this manuscript refers to the non-lethal phenotype of the seasonal H1N1 TX/91 virus and the low virulence SP/83 H5N1 isolate [18, 21] . The Thai/16 and SP/83 viruses differ from each other in 13 amino acids in 7 proteins and this sequence comparison has been published previously [18] . The A/Texas/36/91 and H5N1 viruses were grown in 10 day old embryonated hen's eggs and the 1918 viruses grown in MDCK cells. All virus stocks were titered by plaque assay on MDCK cells prior to mouse infections.
Human peripheral blood monocytes (PBMC's) were obtained by Histopaque (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation of whole blood donated by healthy donors aged 20-40 yrs old without history of influenza vaccination in the past year. Whole blood was obtained through an approved protocol by both the Emory University Institutional Review Board (IRB) and CDC IRB (Emory University Hospital Blood Bank is an FDA-accredited Blood Bank). Human monocytes were obtained by negative selection column enrichment (Miltenyi Biotech, Auburn, CA) yielding approximately 90% CD14+ purity as determined by FACS analysis. For development of macrophages, monocytes were cultured at 37uC in 6 well plates in Macrophage SFM media (Gibco, Grand Island, NY) with 20% heat inactivated autologous serum for 7 days in the presence of GM-CSF (1000 U) before infection [46] . Human macrophages cultured in this manner typically displayed classical morphology with the phenotype: CD11b low , CD11c low , HLA-DR low , CD14 low , CD40 low , CD80 low , CD83 low , CD86-( Figure 4C and D). For the development of dendritic cells (DC's), monocytes were grown in RPMI (Gibco) with 20% heat inactivated autologous serum for 10 days in the presence of IL-4 (1000U) as previously described [46] . Dendritic cells developed in this manner typically displayed a classical morphology with the presence of dendritic processes with the phenotype: CD11b low , CD11c high , HLA-DR high , CD14 low , CD40 high , CD80 high , CD83 high , CD86 high ( Figure 6B) .
To obtain primary mouse lung macrophages and dendritic cells, lungs from naïve mice were removed and tissue disrupted as described above through the use of collagenase digestion and cell suspensions prepared. Macrophages (CD11b+) and dendritic cells (CD11c+) were extracted from contaminating cells by selection on magnetic columns (Miltenyi Biotech, Auburn, CA). Cells were washed twice with media containing 20% FCS (Macrophage SFM for macrophages (Gibco) or RPMI (Gibco) for dendritic cells) and cultured for 24 hours before in vitro infection. Primary mouse lung macrophages typically displayed the phenotype: CD11b high , CD11c-, MHCII high , CD40 high , CD80 high , CD83 high ( Figure 4A and B). Primary mouse lung dendritic cells typically displayed typical morphology with dendritic extensions and the phenotype: CD11b-, CD11c high , MHCII high , CD40 high , CD80 high , CD83 high ( Figure 6A ).
Primary human and mouse cells were washed 36 with serum free growth media and infected for 1 hour with viruses (Table 1) at a multiplicity of infection (MOI) of 0.1. Following infection, cells were washed 36 with serum free growth media and 1 ml SFM media, containing 1 mg/ml of TPCK-treated trypsin (Sigma-Aldrich), was placed into the wells. Virus growth was measured over time in triplicate wells for each experiment and titered in duplicate by standard plaque assay on MDCK cells. All macrophage and dendritic cell data reflects at least three independent experiments (Figures 4 and 6) . Cytokine levels produced from infected human macrophages (MOI = 0.1) were quantitated 48 hrs p.i. by BioPlex assay (Figure 5 ). Escherichia coli lipopolysaccharide (LPS, 100 ng, Sigma-Aldrich) and Poly I/C (100 ng, Sigma-Aldrich) were used as positive control stimulants.
All animal research was conducted under the guidance of CDC's Institutional Animal Care and Use Committee and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. 8-10 week old female BALB/c mice (Harlan, Indianapolis, IN) were anesthetized with Avertin [20] (Sigma-Aldrich) and infected intranasally (i.n.) with 50 ml of 10 2 PFU of influenza viruses prepared in phosphate buffered saline (PBS). Avertin was chosen as the anesthetic because it provides consistent mouse infections with the viruses used in these studies. Using the sublethal (10 2 PFU) inoculum, 1918 and Thai/16 virus infected mice survive a prolonged disease course allowing for the measurement of the influx of inflammatory cells into the lung tissue during a full course (,7-9 days) of influenza virus infection. At indicated times post-infection (n = 3 mice per virus group) mice were euthanatized and exsanguinated. Lungs were removed from individual mice without PBS perfusion and included total lung cell counts included cells located in the bronchoalveolar airways. Perfusion was not possible in many cases of HP influenza virus infection due to the presence of microvascular hemorrhage. We obtained similar results when performing these lung cell quantitation assays with or without lung perfusion in LP virus infected mice and therefore did not introduce this variable in our high containment laboratory. Whole lung cell suspensions were prepared in Dulbecco's minimal essential media (DMEM) with 20% fetal calf serum following collagenase-DNase treatment and manual disruption [47] . Red blood cells were removed by lysis buffer treatment (Sigma-Aldrich). Total viable lung cell number was determined for each mouse by trypan blue exclusion on a hemocytometer.
For the ex vivo experiment, macrophages and dendritic cells were isolated from the lungs of infected BALB/c mice. Two or three mice were infected i.n. with 10 2 PFU of each of the four viruses described in this study (Table 1) . Three days post-inoculation, lungs were removed without perfusion and cell suspensions were prepared as described above. Lungs were pooled from mice in each virus infection group. Macrophages and dendritic cells were isolated by positive selection on CD11b+ or CD11c+ MACS columns. Columns containing bound immune cells were washed extensively (5x) and CD11b+ or CD11c+ cells were eluted off the magnetic columns and cultured in 6-well plates in 5 ml of RPMI containing 5% BSA. Supernatants were collected at the indicated times and virus content was determined in a standard plaque assay on MDCK cells.
Lung cell suspensions were incubated with anti-Fc block (antimouse CD16/CD32) to reduce non-specific antibody binding for 10 min. prior to staining for 1 hr with fluorophore-conjugated antibodies (BD Biosciences, San Diego, CA) specific for immune cell populations according to standard protocols [48] and included: CD11b-PE (pan-macrophage), CD11c-APC (pan-dendritic cell), Ly6G/C-FITC (neutrophil), CD4-PE and CD8-APC T cell markers (Table 2, Figure 2 ). Cells were washed twice with PBS and fixed overnight at 4uC with 2% paraformaldehyde. Samples were safety tested for infectious virus and removed from the BSL3+ laboratory. Flow cytometry was performed on a FACSAria flow cytometer (BD Biosciences). To further characterize primary mouse and human macrophage and dendritic cells we utilized the following fluorescently conjugated antibodies for flow cytometric analysis: MHC II (I-A/I-E)-PE, HLA-DR-PE, CD14-FITC, CD40-FITC, CD80-FITC, CD83-APC, and CD86-APC (BD Biosciences) (Figures 4 and 6 ).
At various times post-infection (n = 3 mice per virus group) lungs were removed and stored at 270uC until virus and cytokine levels could be quantified. Lungs were homogenized individually in 1 ml PBS. Virus was titered from clarified lung homogenates by standard plaque assay on MDCK cells in duplicate and titers are reported as plaque forming units per ml PBS (PFU/ml, Figure 1 ). Cytokine protein levels were measured (day 4 post-infection ( p.i.)) by the Bioplex Protein Array system [49] (Bio-Rad, Hercules, CA) using beads specific for mouse G-CSF, IL-1a , IL-1b, IL-3, IL-6, IL-9, IL-12 (p40), IL-12 (p70), IL-13, Eotaxin, TNFa, RANTES, KC, MIP1-a, MIP-1b, MCP-1, and IFN-c. Cytokine protein levels were measured according to the manufacturers instructions by fluorescently conjugated monoclonal antibodies in duplicate against a standard curve (Figures 3 and 5 ).
Statistical significance of differences between experimental groups was determined through the use of the unpaired, nonparametric Student's t test. Values of p,0.05 were considered significant. Patterns of Positive Selection in Six Mammalian Genomes Genome-wide scans for positively selected genes (PSGs) in mammals have provided insight into the dynamics of genome evolution, the genetic basis of differences between species, and the functions of individual genes. However, previous scans have been limited in power and accuracy owing to small numbers of available genomes. Here we present the most comprehensive examination of mammalian PSGs to date, using the six high-coverage genome assemblies now available for eutherian mammals. The increased phylogenetic depth of this dataset results in substantially improved statistical power, and permits several new lineage- and clade-specific tests to be applied. Of ∼16,500 human genes with high-confidence orthologs in at least two other species, 400 genes showed significant evidence of positive selection (FDR<0.05), according to a standard likelihood ratio test. An additional 144 genes showed evidence of positive selection on particular lineages or clades. As in previous studies, the identified PSGs were enriched for roles in defense/immunity, chemosensory perception, and reproduction, but enrichments were also evident for more specific functions, such as complement-mediated immunity and taste perception. Several pathways were strongly enriched for PSGs, suggesting possible co-evolution of interacting genes. A novel Bayesian analysis of the possible “selection histories” of each gene indicated that most PSGs have switched multiple times between positive selection and nonselection, suggesting that positive selection is often episodic. A detailed analysis of Affymetrix exon array data indicated that PSGs are expressed at significantly lower levels, and in a more tissue-specific manner, than non-PSGs. Genes that are specifically expressed in the spleen, testes, liver, and breast are significantly enriched for PSGs, but no evidence was found for an enrichment for PSGs among brain-specific genes. This study provides additional evidence for widespread positive selection in mammalian evolution and new genome-wide insights into the functional implications of positive selection. Positive darwinian selection is an important source of evolutionary innovation and a major force behind the divergence of species. The Neutralist-Selectionist debate of the past 30 years has gradually given way to a general consensus that both neutral drift and positive selection play major roles in evolutionary change. Interest has therefore shifted to questions of which genes positive selection has affected, how strong was the effect, when did it occur, and what were its functional consequences. Heightening interest in these questions is a growing appreciation that methods for detecting positive selection can also be valuable tools for gaining insight into gene function [1] . Consequently, a wide variety of methods for detecting positively selected genes (PSGs) have been introduced, including comparative or phylogenetic methods, which make use of patterns of substitutions between species, and population genetic methods, which primarily rely on patterns of intraspecies polymorphism [2, 3] . Using these techniques, strong evidence of positive selection has been found for various genes in various organisms, including many genes involved in sensory perception, immunity, host-pathogen interactions, and reproduction (reviewed in [1, 3] ).
Phylogenetic and population genetic methods for detecting positive selection serve as complementary tools for functional and evolutionary analysis. These methods operate at different time scales, with phylogenetic methods being best suited for detecting selection that operates over relatively long periods in evolutionary history, and population genetic methods being best suited for detecting more recent selection. Population genetic methods can potentially detect selection operating at individual sites, through the effects of linkage with flanking alleles, while phylogenetic methods generally require multiple sites to have been affected in a sequence of interest. At the same time, decay of linkage disequilibrium at longer evolutionary time scales can allow phylogenetic methods to more accurately pinpoint the specific locations of functionally important substitutions. In some cases, phylogenetic methods also allow such substitutions to be mapped to particular branches of a phylogenetic tree, thereby providing useful insights about the evolutionary histories of the sequences in question.
With the availability of multiple complete genome sequences, it has become possible to apply phylogenetic methods for the detection of positive selection at a genome-wide scale. Within mammals, several genome-wide scans for positive selection on protein-coding genes have been conducted, using both phylogenetic [4, 5, 6, 7, 8, 9] and population genetic [10, 11, 12, 13, 14, 15] methods (reviewed in [16] ). These analyses have identified many new genes showing strong evidence of positive selection and have revealed striking differences in the prevalence of positive selection on different lineages and among different classes of genes. For example, it has been reported that PSGs are enriched for roles in sensory perception, immunity and defense, tumor suppression, apoptosis, and spermatogenesis [4, 5] ; that PSGs are associated with known Mendelian disorders [4] ; that PSGs often coincide with segmental duplications [8] ; and that more genes have undergone positive selection in chimpanzee evolution than in human evolution [9] . Genome-wide scans for PSGs have also helped to stimulate interest in detecting positive selection on noncoding sequences and on gene expression [17, 18, 19, 20] .
Nevertheless, much remains to be learned about positive selection in mammalian genomes, even within protein-coding regions. The most comprehensive scans for PSGs so far [4, 5, 7, 8, 9] have been based on at most three genome sequences-typically the highly similar human, chimpanzee, and/or rhesus macaque genomes (.97% average identity in orthologous coding regions [8] ). As a result, the power for detection of PSGs has been relatively weak [5, 8] . In addition, in several of these studies, at least one genome was of draft quality, which reduced the number of genes that could be examined and required additional care in avoiding false positive predictions.
Here we present a phylogenetic analysis of positive selection in the six eutherian mammalian genomes for which high-coverage, high-quality sequence assemblies are now available: the human [21] , chimpanzee [6] , macaque [8] , mouse [22] , rat [23] , and dog [24] genomes. The phylogenetic depth of this data set helps considerably in addressing the problem of weak power. Indeed, these genomes have a near-optimal degree of divergence for PSG detection, being distant enough to produce a strong phylogenetic signal, yet close enough that gene structures are well conserved, alignment is fairly straightforward, and synonymous substitutions are not saturated (e.g., [25] ). In addition, our data set for the first time allows positive selection of mammalian genes to be examined genome-wide on a nontrivial phylogeny, so that insight can be gained into the particular ''selection histories'' of individual genes-that is, the branches of the phylogeny on which they experienced positive selection. In our analysis, we employ models of codon substitution that account for variation of selective pressure over branches on the tree and across sites in a sequence, which can capture signatures of molecular adaptation that affect small numbers of sites [26, 27] . Using a series of likelihood ratio tests (LRTs) based on these models, we identify more than four hundred genes that show strong signatures of positive selection during mammalian evolution. Our detailed analysis of the functional roles, selection histories, and expression patterns of these genes follows.
Using the latest human, chimpanzee, macaque, mouse, rat, and dog genome assemblies, we identified 17,489 human genes with high-confidence orthologs in at least two of the remaining five species. These ortholog sets (human genes and non-human orthologs) were identified by an automatic pipeline that made use of syntenic whole-genome alignments, sequence quality scores, and other data (see Methods). Briefly, the pipeline began with 21,115 human genes drawn from the RefSeq [28] , UCSC Known Genes [29] , and VEGA [30] gene sets. These genes were mapped to the other genomes via syntenic pairwise alignments, then passed through a series of rigorous filters to ensure correct mapping, high sequence quality, and only minimal changes between species in gene structure. This approach exploits the fact that gene structures are generally well-conserved between mammalian species [22] and avoids any dependency on the non-human gene annotations, which-with the exception of mouse-are significantly less accurate and complete than those for human. Because low-quality sequence can produce a spurious signal for positive selection (e.g., [8] ), all bases with low quality scores (Phred quality ,20) were masked out for subsequent analyses. Masking (or truncation at the 59 or 39 end) was also used to exclude regions of genes in which minor differences in gene structure were apparent. Genes that showed signs of substantial disruptions to their exon-intron structures or open reading frames in one or more species (perhaps indicating pseudogenization) were masked out completely in those species. All masked bases were treated as missing data in the subsequent analysis of positive selection. This masking approach allowed the number of genes to be maximized while ensuring that the analyzed alignments were of high quality (Table 1) .
For this study, we chose to avoid recently duplicated gene families and to focus on 1:1 orthologs. This simplified the analysis, allowed for parameter sharing across genes (see Methods), and eliminated an important source of error by avoiding the need for a separate tree reconstruction for each gene family. (All ortholog sets were assumed to obey the species tree shown in Figure 1 ; because only an unrooted tree is needed, the topology is well accepted.) It was therefore necessary to discard any genes that showed evidence of recent duplication. This was accomplished in a pairwise fashion, by examining each human gene and orthologous non-human gene, and determining-based on BLAST matches to other genes and gene predictions in the same genome-whether either gene had a paralog that was more similar to it than the two orthologs
Populations evolve as mutations arise in individual organisms and, through hereditary transmission, gradually become ''fixed'' (shared by all individuals) in the population. Many mutations have essentially no effect on organismal fitness and can become fixed only by the stochastic process of neutral drift. However, some mutations produce a selective advantage that boosts their chances of reaching fixation. Genes in which new mutations tend to be beneficial, rather than neutral or deleterious, tend to evolve rapidly and are said to be under positive selection. Genes involved in immunity and defense are a well-known example; rapid evolution in these genes presumably occurs because new mutations help organisms to prevail in evolutionary ''arms races'' with pathogens. Many mammalian genes show evidence of positive selection, but open questions remain about the overall impact of positive selection in mammals. For example, which key differences between species can be attributed to positive selection? How have patterns of selection changed across the mammalian phylogeny? What are the effects of population size and gene expression patterns on positive selection? Here we attempt to shed light on these and other questions in a comprehensive study of ,16,500 genes in six mammalian genomes.
were to each other (see Methods). Requiring that each human gene had a high-confidence 1:1 ortholog in at least two other species reduced the total number of ortholog sets to 16,529. These sets contain a human gene and either five (42% of cases), four (28%), three (15%) or two (15%) non-human orthologs.
We performed a series of nine different LRTs to identify genes under positive selection on particular branches or clades of interest in the six-species phylogeny. In particular, we tested for selection on any branch of the tree ( Figure 1A ); on the branch leading to, and on any branch within, the primate clade ( Figure 1B ,C); on the branch leading to, and on any branch within, the rodent clade ( Figure 1D ,E); and on each of the four individual branches within the primate clade ( Figure 1F -I). These LRTs were all based on widely used site or branch-site models of codon evolution [31, 26, 27] (see Methods). The test for all branches was applied to all 16,529 ortholog sets. For the branch-and clade-specific tests, ortholog sets were discarded if they did not contain adequate ingroup or out-group data for the test in question, which somewhat reduced the number of tests (Text S1, Table S1 ).
The PSGs identified by each test ranged in number from only seven (the hominid branch) to 400 (the test for all branches; FDR,0.05 in all cases). As in previous studies, the numbers of genes identified by the tests for individual primate branches were small, primarily due to weak power caused by low levels of interspecies divergence. The inclusion of additional non-primate mammals does not appear to have improved the power of these tests substantially, but it does allow a distinction to be made between selection on the branches to the hominids and to macaque. The tests for selection on the branch to the primates and in the primate clade also yielded fairly small numbers of PSGs, but the tests for selection in, or on the branch to, the rodents identified somewhat (nearly three-fold) larger numbers. In general, even with the larger data set, our power to detect selection on individual lineages and clades is still fairly weak, and differences in numbers of identified PSGs almost certainly reflect differences in power more than differences in the prevalence of selection. Nevertheless, these LRTs together produced a fairly large set of high-confidence PSGs, permitting a more detailed and thorough functional analysis than has previously been possible in mammals (see below).
The identified PSGs are significantly enriched for a large number of functional categories, according to the Gene Ontology (GO) [32] and Protein Analysis Through Evolutionary Relationships (PANTHER) databases (Tables 2, S2, and S3). If these overrepresented categories are clustered by the PSGs that are assigned to them, major groups corresponding to sensory perception, immunity, and defense emerge (Figure 2 ), in agreement with previous genome-wide scans [4, 5] . However, the increased power of our analysis allows biological processes and functions associated with positive selection to be identified at much finer resolution than in previous analyses, as discussed below. The increased power also seems to diminish the dependency of functional enrichments on the database or statistical methodology selected for the analysis. In particular, better agreement was observed between functional categories over-represented among the identified PSGs, as determined by Fisher's exact test (FET), and categories whose genes displayed significant shift toward smaller LRT P-values (whether or not they met the significance threshold for PSGs), as determined by the Mann-Whitney U (MWU) test (see Methods). Better agreement was also observed between analyses based on the GO and PANTHER databases (see Tables S2 and S3 ). The observed enrichments do not appear to be an artifact of differences between categories in gene length or alignment depth per gene (Text S1). In the discussion below, we focus on GO categories and nominal P -values based on the MWU test, as applied to P-values from the LRT for selection on any branch of the tree (except when otherwise indicated); full results are shown in Table 2 and Text S1.
The PSGs are enriched for a wide variety of functions related to immunity and defense. Several over-represented categories describe activation in response to external or environmental stresses, such as from bacteria (P = 4.2610 28 ), viruses (P = 3.0610 28 ), wounding (P = 3.2610 28 ), and acute inflammation (P = 4.7610 211 ). In some cases, different categories reflect the same or very similar sets of genes (e.g., ''response to wounding'' and ''acute inflammatory response,'' or ''response to virus'' and ''response to bacterium''), while in others they reflect quite distinct gene sets (''response to wounding'' and ''response to virus'') ( Figure 2 ). Genes involved in both innate (P = 1.9610 29 ) and adaptive (P = 1.5610 25 ) immunity are over-represented, with many PSGs contributing to both classes. The conventional division of adaptive immunity into humoral (P = 1.6610 27 ) and cellular (P = 3.5610 27 ) responses is reflected in the enriched GO categories. Various mechanisms of immune response are represented, including previously identified categories for natural killer cell (P = 1.6610 28 ), B-cell (P = 4.8610 27 ), and T-cell (P = 1.2610 28 ) mediated immunity [5, 8] , and new categories such as cytokine/chemokine-mediated (7.6610 28 ) and complement-mediated immunity (P = 6.0610 26 ; see Table S3 ).
Some of the enriched categories point to particular pathways with large numbers of PSGs. A striking example is the complement immunity system, a biochemical cascade responsible for the elimination of pathogens. This system consists of several small proteins found in the blood that cooperate to kill target cells by disrupting their plasma membranes. Of 28 genes associated with this pathway in KEGG [33] , nine are identified as PSGs (FDR,0.05), and five others have nominal P,0.05 ( Figure S1 ). Most of these PSGs are inhibitors (DAF, CFH, CFI) and receptors (C5AR1, CR2), but some are part of the membrane attack complex (C7, C9, C8A), which punctures cell membranes to initiate cell lysis. Many of these PSGs are known to interact with one another, suggesting possible co-evolution. Two of three biochemical pathways known to activate the complement system are also enriched for PSGs (the classical complement pathway [P = 6.1610 27 ] and the alternative complement pathway [P = 1.5610 26 ]), as is the coagulation cascade that interacts with the complement system (''blood clotting,'' MWU P = 2.2610 27 ; Table S3 ). Other pathways that contain multiple interacting PSGs include those for apoptosis, taste transduction, antigen processing and presentation, and cytokine-and chemokine-mediated signaling (e.g., Figures. S4, S5) .
Several gene families of the immunoglobulin superfamily (''immunoglobulin mediated immune response,'' P = 1.1610 27 ) show particularly strong enrichments for PSGs. For example, five of the six SIGLEC genes included in our analysis are under positive selection (see [34] ). A detailed examination of one immunoglobulin gene for which structural information was available-a cellsurface receptor for hepatitis A and other viruses called HAVCR1 (LRT P = 6.9610 29 )-revealed several sites under positive selection in its N-terminal V-like immunoglobulin (IgV) domain. Three of these sites correspond to regions of the protein believed to play critical roles in binding to viruses or in regulating the immune function of the gene (Figure 3 ). In addition to its role in viral defense, HAVCR1 is a key player in the hygiene hypothesis explaining the increase in allergies and asthma [35] . It also interacts with IgA (CD79A; P = 5.4610 29 ), whose deficiency is associated with increased susceptibility to autoimmune and allergic diseases [36] .
The hierarchical clustering of GO categories ( Figure 2 ) reveals an unexpected similarity between the sets of PSGs involved in fertilization and cytolysis, and some similarity of both sets with immune-related PSGs. This association of immunity, fertilization, and cytolysis is driven by a group of genes that participate in sperm-egg interaction, but also have immune-related functions and destroy pathogens by cytolysis. Interestingly, PSGs with roles in both reproduction and immunity are often also related to cancer, and it has been hypothesized that most cancer genes under positive selection have been subject to antagonistic co-evolution, with lineage-specific variations in dynamics and strength [5, 37] . Several PSGs identified here are associated with both FAS/p53 apoptosis and cancer (Da Fonseca et al., in prep.), such as the protein p53, which also regulates maternal reproduction [38] ; the cell adhesion gene ADAM2 (P = 2.9610 26 ), which is integral to fertilization [39] ; and the related genes ADAM15 (P = 5.4610 24 ) and ADAM29 (P = 3.4610 24 ), which are strong candidates for cancer evolution driven by sexual conflict. In addition, the testes development-related gene CCDC54 (P = 3.3610 24 ) is currently a target of cancer immunotherapy research [40] .
A smaller and somewhat less diverse group of enriched categories is associated with sensory perception. Among the most inclusive categories of this type are ''sensory perception of chemical stimulus'' (24 PSGs; P = 4.3610 239 ) and ''G-protein coupled receptor protein signaling pathway'' (39 PSGs; P = 1.4610 27 ). Previously, enrichments for such categories have been attributed primarily to olfactory receptors [4, 5] . Indeed, 15 PSGs are labeled as having ''olfactory receptor activity'' (P = 6.9610 236 ). However, eight PSGs are involved in ''sensory perception of taste,'' including five taste receptors (P = 1.4610 210 ). Interestingly, several of these are bitter taste receptors. The sense of bitter taste is critical in allowing organisms to avoid toxic and harmful substances, and extensive gene expansion of bitter taste receptors is known to have occurred during mammalian evolution [41] , possibly driven by (or helping to drive) positive selection. Bitter taste receptors under positive selection include TAS2R1, TAS2R5, and a recently expanded cluster of genes at chr12p13 (TAS2R13, TAS2R14, TAS2R42, and TAS2R49). Another PSG, TAS1R2, is a receptor of sweet and umami taste, and the PSG RTP3 is a transmembrane protein that is involved in the transport of taste receptors and apparently influences their expression. The PSGs in the ''neurological processes'' category (P = 7.5610 27 ) are dominated by olfactory and taste receptors, but they also include other types of genes. For example, TMC2 (P = 1.1610 24 ) is expressed in the inner ear and is important for balance and hearing [42] . The acid-sensing ion channel gene ACCN4 (P = 1.0610 26 ) has been implicated in synaptic transmission, pain perception, and mechanoperception [43] . SLC6A5 (P = 3.0610 24 ) is associated with hyperekplexia, a neurological disorder characterized by an excessive startle response [44] . The neuromedin receptor NMUR (P = 6.1610 24 ) is involved in the mammalian circadian oscillator system [45, 46] . Finally, the neurotensin receptor NTSR1 (P = 8.1610 24 ) mediates hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion [47] .
Similarly, the PSGs associated with diet include but are not limited to taste and olfactory receptors. For example, MGAM (P = 2.4610 28 ) is essential for the small intestinal digestion of starch, giving it a critical role in human metabolism, as starches of plant origin make up two-thirds of most human diets [48] (see also [49] ). MAN2B1 (P = 1610 26 ) is involved in the cleavage of the alpha form of mannose, a sugar monomer. Defects in this gene cause lysosomal alpha-mannosidosis, a lysosomal storage disease characterized by the accumulation of unbranched oligosaccharide chains [50] . TCN1 (P = 2.9610 231 ) is a major constituent of secondary granules in neutrophils and facilitates the transport of vitamin B12 into cells, which is important for the normal functioning of the brain and nervous system, and for the formation of blood [51] . In addition, several PSGs participate in ''steroid hormone metabolism'' (P = 8.3610 24 ) including genes that metabolize xenobiotics and drugs (e.g., SULT1C3, UGT2B7, and CYP2C8). Positive selection in these and other genes is likely to Figure 2 . Hierarchical clustering of 27 over-represented GO categories identified by the Mann-Whitney U test (''biological process'' group only), based on the genes assigned to each category. This dendrogram is derived from a dissimilarity matrix defined such that any two GO categories, X and Y, have dissimilarity 0 when all genes assigned to X are also assigned to Y (or vice-versa), and dissimilarity 1 when the sets of genes assigned to X and Y do not overlap. Specifically, X and Y have dissimilarity have been influenced by changes in food preferences during mammalian evolution.
Few functional enrichments were evident for the PSGs identified by the branch-and clade-specific LRTs, primarily because these sets were quite small in size. However, the more powerful LRTs, such as those for the primate and rodent clades ( Figure 1C ,E), did produce significantly lower P-values for genes of certain functional categories than for others. Interestingly, these categories were dramatically different for the primate-and rodentclade LRTs, with nearly all of the primate categories relating to sensory perception, and nearly all of the rodent categories relating to immunity and defense (Table 4) . Indeed, the PSGs identified by the primate-clade test include several taste and olfactory receptors, as well as receptors for the sensation of pain (e.g., MRGPRE, NPFF2) and color vision (e.g., OPN1SW), and receptors involved in immunity (e.g., CCR1). The PSGs identified by the rodent-clade test include few such genes, but they include many genes involved in responses to wounding, inflammation, and stress, as well as genes involved in complement activation and innate immunity. Thus, we find little evidence that genes directly involved in brain development and function have (as a group) been driven by positive selection in primates, but many genes that provide sensory showing the interaction between two receptors that have been implicated in the regulation of HAVCR1's immune function. It is thought that clustering of receptors within the same cell surface might facilitate phosphorylation of the cytoplasmic tail, and that interaction between receptors from different cells might be a mechanism for B-T cell adhesion [91] . Predicted residue 39 falls within the region of these receptors, very near residue 37, which directly interacts with the opposite receptor (according to the available mouse structure). In addition, predicted residues 54 and 56 are adjacent to the virus-binding surface (shown in pink), as defined by a polymorphism in macaque [91] . Interestingly, the residue that falls between them (55) appears to be critical for virus-binding at the homologous loop in the CEA coronavirus receptor [91] . Residue 75 in the IgV domain also shows evidence of positive selection (PP.0.90, shown in orange) but its function is unknown. doi:10.1371/journal.pgen.1000144.g003 information to the brain do appear to have experienced positive selection. These changes in sensory perception could conceivably have been brought on by, or could have contributed to, increased brain size and complexity in primates.
To gain further insight into the patterns of positive selection that have shaped present-day mammalian genes, we devised a model that allows for probabilistic inferences about the selection histories of individual genes. A selection history is defined as an assignment to each branch of the phylogeny of one of two evolutionary modes: positive selection (each site evolves with v 0 ,1, v 0 = 1, or v 2 .1) or absence of positive selection (each site evolves with v 0 ,1 or v 0 = 1). The model allows a posterior distribution over selection histories to be inferred for each gene, and it allows for estimates of the number of genes under positive selection on individual branches and clades that consider uncertainty about selection histories. Unlike the branch-and clade-specific LRTs-which are simple one-sided hypothesis tests and are necessarily conservative about rejection of the null hypothesis-this model considers all candidate histories symmetrically, and allows for ''soft'' (probabilistic), rather than absolute, choices of history at each gene.
Briefly, the model is defined in terms of a simple switching process along the branches of the phylogeny. It has separate parameters for the rates of gain and loss of positive selection at several switch points on the tree, with two switch points per internal branch and one per external branch (see Figure 4A and Methods). The joint posterior distribution of these parameters and of all selection histories is inferred from the data by a Gibbs sampling algorithm (see Methods and Text S1). The inference procedure is computationally intensive, so it was applied only to the 544 genes identified by one or more LRTs as showing significant evidence of positive selection. Because in these cases the null model of no positive selection had already been rejected by a conservative test, the history without selection on any branch was excluded, leaving 2 9 21 = 511 possible histories for the nine-branch (unrooted) phylogeny. To reduce computational cost, the inference of selection histories was conditioned on the maximum likelihood estimates of the parameters of the codon models (see Methods).
The inferred rates of gain and loss are quite variable ( Figure 4A and Figure S2 ), with posterior means ranging from about 0.01 to 0.53. These rates are sharply reduced for the external branches of the tree, probably in large part because of diminished power to detect changes in selective mode on these branches. The number of genes inferred to be under selection also varies by branch, but not as dramatically, with expected values ranging between 207.9 and 393.9 and many 95% credible intervals overlapping ( Figure 4B ). Despite differences at individual branches, gains and losses appear to be roughly in equilibrium overall, with 61% of genes estimated to have been under selection at the root, and between 38% and 62% (averaging 50%) under selection at the leaves. The slight tendency to lose selection over time could reflect an ascertainment bias for genes that experienced selection early in mammalian evolution, which will tend to display signatures of selection on multiple long branches of the tree and therefore will be more easily detectable by the LRTs. The branches with the most genes under selection (such as those leading to the rodent and primate ancestors, and to dog and macaque) are generally long (see Figure 1A ), suggesting power may influence these estimates. Nevertheless, the unusually high rate of gain on the branch to the rodents, and the comparatively low rate of loss on that branch (both having fairly low posterior variance; Figure S2 ), suggest not just differences in power but a real tendency for a net gain of selection on this branch, perhaps due to larger population sizes in the rodents. Whether because of power or a genuine increase in selection, the rodent branch appears to play a major role in the identification of PSGs. An expected 72% of the 544 candidate PSGs are under selection on this branch.
The posterior distributions over histories suggest that few genes have experienced positive selection specific to individual branches or clades ( Figure 4B) . Instead, most genes appear to have switched between evolutionary modes multiple times. The estimated number of mode switches per gene (averaging across genes but considering the joint posterior distribution for all selection histories) is 1.6 (95% CI: 1.5-1.7), with 0.6 gains (0.5-0.7) and 1.0 losses (0.9-1.1). An expected 91% of PSGs have experienced at least one mode switch, and an expected 53% have experienced two or more switches. 54% of PSGs have 95% CIs excluding zero switches (i.e., with high confidence, these genes have switched modes at least once), and 10% have 95% CIs also excluding one switch (with high confidence, they have switched modes at least twice). Thus, this analysis suggests that positive selection tends to be gained and lost relatively frequently in mammalian genes. Episodic positive selection has been observed and analyzed in detail at individual loci (e.g., [52, 53] ) but to our knowledge genome-wide evidence of this phenomenon in mammalian phylogenies has not previously been reported. Interestingly, our observations are qualitatively compatible with Gillespie's theoretical model of an episodic molecular clock [54] , although our model differs from his in detail.
By pooling information across genes and allowing for uncertainty in selection histories, this method estimates much larger numbers of genes under positive selection on each branch of the tree than do the more conservative LRTs (Figure 1 ). For example, the expected number of genes under selection on the branch to the primates is 360.5 (95% CI 338-382), compared with 21 genes identified by the corresponding LRT, and the expected number under selection on the branch to the rodents is 393.9 (357-426), compared with 56 identified by the corresponding LRT. In this analysis, the estimated numbers of genes that have experienced positive selection on the various primate and rodent lineages are not dramatically different, suggesting that the sharp differences from the LRTs in large part reflect inequalities in power. They also suggest that the numbers of genes under selection in recent human and chimpanzee evolution are not as different as they appear from LRTs, which will identify only the most extreme cases [9] . Indeed, the 95% CIs for the human and chimpanzee estimates heavily overlap.
In addition to being useful in a bulk statistical analysis of all PSGs, the Bayesian framework can be used to identify the single most likely selection history for each gene. In some cases, these histories are consistent with known functional differences between species, and help to shed light on the evolutionary basis of these differences. For example, the sweet receptor TAS1R2 has been shown in knock-out experiments to be responsible for differences between species in preferences for sweet tastes [55] . (Humans can taste several natural and artificial sweeteners that mice cannot, such as monellin, thaumatin, aspartame, and neohesperidin dihydrochalcone.) This gene is predicted to have experienced selection on the primate clade and on the branches leading to the primate and rodent clades (posterior probability [PP] = 0.20), suggesting that positive selection on TAS1R2 in both primates and rodents could have contributed to differences in sweet taste preferences. Another example is the integral membrane glycoprotein GYPC, which plays an important role in regulating the mechanical stability of red blood cells. In humans, GYPC has been associated with malaria susceptibility, and predicted to have undergone recent positive selection [56] . However, we find evidence that GYPC has experienced positive selection on all branches of the primate clade (PP = 0.66), suggesting longer-term selective pressure that have also affected nonhuman primates. A third example is CGA, which encodes the alpha subunit of the four human glycoprotein hormones (chorionic gonadotropin, luteiniz-ing hormone, follicle stimulating hormone, and thyroid stimulating hormone). This gene shows strong evidence of positive selection specific to the primate clade (PP = 0.82), consistent with the proposal that relatively recent adaptations in pregnancy and development have played a critical role in the evolution of the human endocrine system [57] . Interestingly, the closely related genes CGB1 and CGB2 (which encode two of the six beta subunits of chorionic gonadotropin) are thought to have originated by gene duplication in the common ancestor of humans and great apes [58] , and these events could have contributed to positive selection on CGA. Finally, the complement components C7 and C8B, which encode proteases in the membrane attack complex, are predicted with high probability to be under selection in rodents only (C7: PP = 0.98 for selection in mouse; C8B: PP = 0.93 for selection in mouse and rat). Differences in complement proteases are thought to explain certain differences in the immune responses of humans and rodents [59] .
We examined the human mRNA expression levels of PSGs non-PSGs using public data from the Affymetrix Human Exon 1.0 ST Array, which contains probes for nearly all of our genes and permits accurate estimation of expression levels [60] . Our most striking finding was that PSGs show reduced expression levels in all of the 11 available tissues (breast, cerebellum, heart, kidney, liver, muscle, pancreas, prostate, spleen, testes, and thyroid; see Methods). In particular, a significantly smaller fraction of PSGs than of non-PSGs produce a hybridization signal above the background level for the array (P,4610 24 in all tissues for PSGs defined by the all-branch test, one-sided FET). Moreover, among genes expressed above background, expression levels are significantly lower for PSGs than for non-PSGs (P,7610 25 in all tissues, one-sided MWU test; Figures 5A-C) . PSGs also show significantly greater tissue bias than non-PSGs, as measured by the statistic t [61] ( Figure 5D ) or by an alternative statistic here denoted c [17] (Methods). The differences in expression level and tissue bias between the two sets of genes do not appear to be explained by differences in false negative or false positive rates in the detection of positive selection, and the differences in expression level do not appear to be a consequence of the differences in tissue bias (Text S1). In addition, the observed differences remain if the genes that belong to strongly enriched GO categories (Table 2) are excluded, indicating they cannot be attributed to particular classes of PSGs known to have tissue-specific expression patterns, such as those involved in immunity or spermatogenesis. That expression levels are reduced in all tissues further suggests the existence of a general relationship between expression patterns and the likelihood of positive selection.
Consistent with previous observations (e.g., [62] ), we found a significant negative correlation of v with expression level in all 11 tissues (Spearman's rank correlation coefficient r ranged from 20.25 to 20.43). In addition, we observed a positive correlation of v with tissue bias, as measured by t (r = 0.24) [63, 64] . (Similar correlations were observed when the log likelihood ratio in the test for positive selection on any branch-which increases with increasing evidence for selection-was used in place of v.) Unlike in previous studies, however, we were able to examine these correlations separately for positively and non-positively selected genes, using the set of PSGs identified by the all-branches LRT. Interestingly, the correlations of v with expression level t are much stronger within the non-PSGs than within the PSGs, indicating that the observed correlations are primarily driven by negative rather than positive selection (see also [65] ). Thus, while genes expressed at low levels and/or in a tissue-specific manner show an increased tendency to have experienced positive selection, the strength of positive selection does not appear to be strongly correlated with their expression patterns (see Discussion).
Of the 15,823 genes that were tested for positive selection and had detectable expression in at least one tissue, 1,509 showed a strong preference for one tissue and were designated as tissue specific (c t .0.25 for some tissue t and c t .0.25 for all t9 ? t; see Methods). Based on this designation, spleen-and testes-specific genes were strongly enriched for PSGs: 22 of 174 (12.6%) spleenspecific genes were PSGs, compared with only 2.2% of other genes (P = 8.7610 211 , one-sided FET); and 45 of 715 (6.3%) testesspecific genes were PSGs, compared with 2.1% of other genes (P = 8.2610 210 ). There were also significant, but weaker, enrichments for PSGs among liver-specific (P = 9.1610 23 ) and breastspecific (P = 1.0610 22 ) genes. Not surprisingly, the spleen-specific PSGs generally appear to be immune-related, and many of the testes-specific PSGs are involved in spermatogenesis or sperm adhesion (they include ADAM2 and SPAM1; Table 3 ). The liver and breast specific genes are more heterogeneous. In contrast, only 2 of 254 (0.7%) cerebellum-specific genes were PSGs, compared with 2.3% of other genes (P = 0.066, one-sided FET). Only a few tissue-specific genes were identified by the clade tests, so it was not possible to compare the relationships between tissue-specific expression and positive selection in primates versus rodents. However, there were significant enrichments for primate PSGs among spleen-specific genes, and for rodent PSGs among testesspecific genes.
Despite our large data set, we found no indication of a correlation between expression in the primate brain and recent positive selection in protein-coding regions [66] (see [67, 68] ). Indeed, we found some evidence to the contrary: PSGs identified by the primate-clade test show more sharply reduced expression levels (compared with non-PSGs) in the cerebellum than in any other tissue; cerebellum-specific genes are depleted, not enriched, for PSGs; and none of the primate PSGs show tissue-specific expression in the cerebellum. These findings, of course, do not rule out positive selection in individual genes of great importance in brain development, nor do they rule out positive selection on gene expression.
While positive selection was our primary focus, our data set also provides an opportunity to compare the average rates of protein evolution in various mammalian lineages. We estimated a separate nonsynonymous-synonymous rate ratio v for each branch of the six-species phylogeny, pooling data from all ortholog sets ( Figure 1A) . Consistent with previous findings [6, 8] , we observe that protein-coding genes, on average, have experienced moderately strong purifying selection (v « 1) on all branches of the phylogeny, but that estimates of v vary considerably within the mammals. These estimates are largest for the hominids (v<0.25), smallest for the non-primate mammals (0.12,v#0.14), and intermediate for non-hominid primates (0.17,v,0.21). It is thought that increased estimates of v in hominids primarily result from weakened purifying selection, owing to reduced effective population sizes [69, 5] . The intermediate values for non-hominid primates may also be influenced by population size.
To examine the relationship between v and population size further, we made use of a theoretical relationship between v and the scaled selection coefficient c (see [70, 71] ), which holds if nonsynonymous substitutions have equal (and small) selection coefficients, if synonymous substitutions are neutral, and if population sizes are sufficiently large (Methods). This relationship allows ratios of population sizes to be estimated from ratios of v estimates, under the assumption of constant selection coefficients across species. Here we further assumed that the ancestral population sizes of humans and the chimpanzee subspecies Pan troglodytes versus (to which the sequenced animal belonged) were roughly the same (N h = N c ) [5] , and estimated the ratio of v m in macaque to v h in human/chimpanzee from our 10,980 humanchimpanzee-macaque ortholog trios. Our estimate of v m / v h = 0.732 implies an estimate for the ratio of the macaque to human ancestral population sizes of N m / N h = 1.41 [bootstrapping 95% CI (1.15, 1.64)]. In comparison, the ancestral macaque population size has been estimated at ,73,000 [72] and ancestral human and chimpanzee population sizes at 40,000-70,000 [73, 74] , which would imply a ratio of 1.04-1.82, in reasonable agreement with our estimate. We used the same theoretical relationship to devise a LRT indicating whether or not each gene deviated significantly from the assumed model with N m / N h = 1.41 (Methods). For the vast majority (96%) of the 10,980 genes examined, no significant deviation was observed, indicating that the differences in selection pressure in macaque and the hominids are generally well-explained by differences in population size.
To compare the power of our LRTs with the power of previous tests based on hominid or primate genomes, we simulated data sets under a range of parameter values and measured the fraction of cases in which positive selection was predicted ( Figure 6) . These experiments show that power increases substantially when the set of species under consideration is expanded from the two hominid species to the three primates then to all six mammals. With hominid species only, power is poor even when selection is quite strong (e.g., ,20% with a constant v = 2 and ,40% with v = 4), suggesting that a genome-wide scan will tend to identify only the most extreme cases of positive selection. If a rigorous correction for multiple testing is applied, a test based on hominids only has essentially no power, even for fairly long genes under strong selection ( Figure S3 ; see also [5] ). The situation is considerably improved by the addition of the macaque genome, but power remains poor when controlling for multiple testing unless genes are long and selection is strong. When all six mammals are considered, however, power increases substantially. With the full data set, power is reasonably good ($70%) even when genes are short and selection is moderate in strength; it remains good when multiple comparisons are considered ( Figure S3 ). The absolute estimates of power from these experiments depend on the simplifying assumptions used in the simulations (including the unrealistic assumption of constant v among lineages and among sites), and they must be interpreted cautiously. However, estimates of relative power-which will be less sensitive to these simplifying assumptions-indicate a substantial improvement is achieved by the addition of the three non-primate mammals.
Since it first became possible to compare the sequences of complete mammalian genomes about five years ago, a number of genome-wide scans for positively selected genes (PSGs) have been conducted using phylogenetic methods [4, 5, 6, 7, 8, 9] . These studies have provided a valuable initial assessement of the genome-wide landscape of positive selection in mammals, but they have left many important questions unanswered. The analysis presented here, by incorporating non-primate mammalian genomes into a genome-wide scan for positive selection, represents a significant step forward. The larger, more divergent group of species improves power significantly, and the use of a nontrivial phylogeny provides insight into the particular patterns of positive selection that have helped to shape present-day genes. To our knowledge this is the largest and most detailed genome-wide analysis of positive selection to date, not only in mammals but in any group of organisms (although extensive analyses, similar in some respects, have been performed recently in Drosophila [75, 76] ).
One finding of particular interest was that several whole pathways are especially rich in PSGs. Examples include the classical and alternative pathways for complement-mediated immunity and the FAS/p53 apoptotic pathway ( Figures S1, S4 and S5). These findings suggest that positive selection may frequently act directly on whole protein complexes or pathways (see [77, 78] ). Alternatively, adaptive changes in one protein may sometimes have a cascade effect, leading to changes in other genes that bring a system back into equilibrium. Whether or not all changes affecting a pathway are driven by positive selection, one might expect to see similarities in the selection histories of gene with closely related functions. Indeed, we have found that genes with similar selection histories on average have substantially greater similarity in their GO categories than do genes with more divergent histories ( Figure S6 ). The observations that multiple interacting genes often show evidence of positive selection and that positive selection is frequently episodic may well be connected. For example, in some cases a transient external force could induce a burst of changes in multiple genes that participate in the same pathway, either separately or by triggering a cascade of interdependent events. Further unraveling the (co-)evolutionary histories of interacting PSGs promises to be a fertile area for future Table 3 . Summary of individual PSGs discussed in this article.
Cytokine/chemokine C-C motif: CCL1 (P = 5.2610 24 ), CCL20 (P = 7.6610 24 );
C-X-C and C-X3-C motifs: CXCL5 (P = 8.1610 24 work. Care will be required to distinguish between true coevolution and correlations that can be explained by dependencies on expression levels or other covariates of evolutionary rate [79] . Our finding that PSGs are expressed at lower levels and in a more tissue-specific manner than non-PSGs is consistent with a well-known negative correlation v with expression level, and a positive correlation of v with tissue bias (t or c). Various explanations have been proposed for the observed decrease in v among genes expressed at high levels and/or expressed broadly across tissues, including selection for translational efficiency, selection against misfolding, or increased selection due to pleiotropy [62, 68, 65] . In any case, these genes do appear to experience a reduction in their evolutionary ''flexibility'' compared with genes expressed at low levels and/or nonuniformly across tissues. Our observation of decreased rates of positive selection among these genes-and increased rates among lowexpression/high-tissue-bias genes-is consistent with this characterization. Interestingly, however, we observe that correlations of v with expression level and t hold strongly within non-PSGs, but are much less pronounced within PSGs. Thus, expression levels and patterns are strongly correlated with both the strength of negative selection and the likelihood of positive selection, but they are only weakly correlated with the strength of positive selection. It appears that genes may be more likely to come under positive selection if they are in a state of evolutionary flexibility brought on by reduced or tissue-specific expression, but once positive selection has taken hold their subsequent evolutionary course is not strongly dependent on their expression patterns.
As additional mammalian genomes become available, the statistical power to detect positive selection will improve. However, most forthcoming genomes are being sequenced at low coverage, and will inevitably exhibit increased levels of error in base calls, genome assemblies, ortholog identification (due to short contigs), and alignment-all of which can lead to spurious signals for positive selection. (The same errors tend to produce false negatives, rather than false positives, in the identification of conserved elements.) Thus, careful data quality controls will be needed to take advantage of these data. In addition, when considering the impact of additional sequences on statistical power, it is useful to distinguish between positive selection that acts continuously (or in recurrent episodes) over a long evolutionary period, and positive selection that acts transiently or in a lineagespecific manner. Deep phylogenetic sequencing should generally improve detection power for continuous or recurrent positive selection, but power for transient selection depends strongly on the sequenced species and the lineages of interest. For example, the genome sequences of a dozen non-primate mammals will likely have little effect on the power to detect human-specific selection, while the gorilla and neanderthal genomes could help considerably. There are fundamental limitations in the detection of weak, transient, or highly localized positive selection that will not be overcome by any amount of genome sequencing. Nevertheless, the availability of several new primate genomes, including those of the orangutan, marmoset, and gorilla, may significantly improve power for PSGs in primates.
Our ability to connect positive selection with function remains rudimentary, but gradual progress is being made. As additional sequence data becomes available, it will become possible to associate selection with individual residues of proteins with greater accuracy. At the same time, more data is becoming available on the specific functional roles of individual amino acids, for example, from structural or mutagenesis studies. As a result, it will increasingly become possible to find direct links between selection and function. Often these links will initially be tentative, as in our site-specific analysis of the HAVCR1 gene. Nevertheless, they provide a valuable starting point for experimental follow-up. At the same time, more can be done to incorporate non-sequence data-such as structural and expression data-into computational methods for detecting positive selection. Thus, improvements in both computational and experimental methods will be needed to establish deeper and more informative connections between evolutionary dynamics and molecular function.
The latest human (hg18), chimpanzee (panTro2), rhesus macaque (rheMac2), mouse (mm8), rat (rn4), and dog (canFam2) genome assemblies were obtained from the University of California, Santa Cruz (UCSC) Genome Browser. Humanreferenced whole-genome alignments were constructed from syntenic pairwise alignments with human (the ''syntenic nets'') using the UCSC/MULTIZ alignment pipeline [80, 81] . Low quality bases (Phred score ,20) from the chimpanzee, macaque, rat, and dog genomes were converted to 'N's in these alignments.
A starting gene set was composed from of the human RefSeq [28] , UCSC Known Genes [29] , and VEGA [30] annotations (downloaded from UCSC Feb. 19, 2007) . Transcripts that lacked annotated coding regions (CDSs), that had CDSs of ,100 bp, or that had CDSs whose lengths were not multiples of three were discarded, leaving 88,879 nonredundant transcripts. These transcripts were grouped by same-stranded CDS overlap into 21,115 genes (transcript clusters). All transcripts were mapped from human to each of the other five mammalian species via the syntenic alignments, then subjected to a series of filters designed to minimize the impact of annotation errors, sequence quality, and changes in gene structure on subsequent analyses. Briefly, each human transcript was required (1) to map to the non-human genome via a single chain of sequence alignments including $80% of its CDS; (2) after mapping to a non-human species, to have #10% of its CDS in sequencing gaps or low quality sequence; (3) to have no frame-shift indels, unless they were compensated for within 15 bases; (4) to have no in-frame stop codons and to have all splice sites conserved. To allow for genes that are mostly conserved but whose start or stop codons have shifted, incomplete transcripts-with ,10% of bases removed from the 59 and 39 ends of the CDS-were also considered. The final collection of ortholog sets was obtained by selecting, for each gene, the (complete or incomplete) transcript that successfully mapped to the largest number of non-human species. In the case of a tie, the transcript with the greatest total CDS length was selected. This procedure resulted in 17,489 genes with $2 non-human orthologs, averaging ,5 species per gene (including human; see Table 1 ).
To establish 1:1 orthology, each human gene and putative nonhuman ortholog was examined for evidence of an inparalog (a Figure S3 .) When v#1, these fractions are estimates of the false positive rate. Each data point is based on 1000 data sets simulated with evolver [84] under the assumption of a constant v among lineages and among sites (model M0). All other parameters (including the transition-transversion ratio k, the codon frequencies, and the branch lengths) were fixed at values estimated from the real data. Results are shown for short (200-codon) and long (500-codon) genes and three sets of species: hominids (human and chimpanzee), primates (human, chimpanzee, and macaque), and all six mammals. Details on the computation of P-values are given in Text S1. Note the logarithmic scale on the x-axis. doi:10.1371/journal.pgen.1000144.g006 paralog arising from a recent duplication [82] ) with respect to the other species. Specifically, if either gene had a BLASTN match within the same species (with $80% CDS alignment) that was more similar than the two orthologs were to each other, then that gene was considered recently duplicated and was excluded from the analyses of positive selection. The removal of a duplicated gene did not require an ortholog set to be discarded entirely, provided a human gene and $2 nonhuman orthologs still remained. A collection of genes and gene predictions from the UCSC Genome Browser were used in the identification of inparalogs. When comparing rodent vs. non-rodent and rodent vs. rodent distances, a simple correction for unequal rates of evolution was applied. Further details are given in Text S1.
The LRT for selection on any branch of the phylogeny is essentially Nielsen and Yang's [31] test of site models 2a versus 1a, and the lineage-and clade-specific LRTs are essentially instances of Yang and Nielsen's [26] test 2 (see also [83, 27] ). However, to reduce the number of parameters estimated per gene, the complete set of 17,489 genes was divided into eight equally sized classes by G+C content in third codon positions. The branch lengths and the transition-transversion rate ratio k were estimated for each class under the null model, and these estimates were subsequently held fixed, in a G+C dependent way, for the LRTs. Instead of a complete set of branch lengths, a single scale parameter m was estimated per gene. Thus, only the parameters m, v 0 ,1 and p 0 for the null model and the additional parameters v 2 .1 and p 1 for the alternative model, were estimated per gene (see [31, 26] ). This parameterization speeds up calculations substantially compared to estimating k and a set of branch length per gene, while its sensitivity, specificity and power to detect positive selection are comparable (Text S1). We developed our own software for likelihood computation and parameter estimation to support this parameterization.
For the LRT for selection on any branch, P-values were computed empirically, based on simulation experiments. 10,000 alignments were simulated under the 'nearly neutral model' (allowing for a fraction p 0 of sites to evolve with v 0 ,1 and a fraction 12p 0 to evolve with v 1 = 1) for each G+C class using evolver [84] . Alignment lengths and values of m, v 0 and p 0 were drawn from the empirical distribution defined by the real alignments (using estimates obtained under the null model), and the remaining parameters were fixed at global estimates for each G+C class. Log likelihood ratios (LLRs) were then computed exactly as for the real data. The nominal P -value for a LLR of r was defined as the fraction of all simulated alignments with LLR$r, unless the number of such alignments was ,10, in which case we assumed 2r*x 2 df~1 (an adequate approximation for small P-values, according to the simulation experiments). The method of Benjamini and Hochberg [85] was used to estimate the appropriate P-value threshold for a false discovery rate of ,0.05. For the lineage-and clade-specific LRTs, P-values were computed assuming the null distribution was a 50:50 mixture of a x 2 df~1 distribution and a point mass at zero (see [27] and discussion in Text S1).
Let X = (X 1 ,…, X N ) be the alignment data, with X i denoting the alignment for the i th gene (1#i#N; here N = 544), and let Z = (Z 1 ,…, Z N )be the set of selection histories, with Z i denoting the selection history for the i th gene (1#Z i #M; here M = 511). Recall that a selection history is defined as a pattern of presence or absence of positive selection on the branches of the unrooted phylogeny. Let Z ib M {0,1} indicate the selective mode (with 1 representing positive selection) for branch b M {1,…,B} (here B = 9) under history Z i . The parameters of the switching model, denoted h, are defined below. The model assumes independence of genes and independence of histories, and conditional independence of X and h given Z. Thus, the complete data likelihood is given by:
The probability of a history, P(Z i |h), is a function of the set of switches in selective mode required to explain the history parsimoniously. For each history to be explained parsimoniously, switches must be allowed to occur early (near the ancestor) or late (near the descendant) on each internal branch, as well as (early) on each external branch ( Figure 4A ; see Text S1 for a justification of the model). Thus, there are twelve possible switch points, with three of them adjoining each of the four internal nodes of the tree. It is convenient to denote these points P nb : n[N ,b[B n f g where N is the set of internal nodes and B n represents the branches adjoining node n. Let V nb M {0,1} and W nb M {0,1} indicate the selective states before and after point P nb , respectively. For a given history Z i , these variables are uniquely determined by parsimony according to a simple algorithm (see Text S1). The four possible values of (V nb , W nb ) correspond to four possible scenarios at P nbgain of selection (0,1), loss of selection (1,0), absence of gain (0,0), or absence of loss (1,1). The probabilities of these scenarios (i.e., the conditional probability of each W nb given V nb ) are defined by a parameter for gains (h nbG ) and a parameter for losses (h nbL ) at each point. In addition, the prior probability of selection at the root of the tree is given by a parameter h 0 . (For this analysis, the most recent common ancestor of the primates and rodents is treated as the root of the tree; see Text S1.) The set of parameters can thus be described as h~h nbe :
The prior probability of a history Z i is simply a product of the prior and the relevant switching probabilities:
where U 0 represents the selective state at the root. The switching model effectively defines a prior distribution over histories, which tends to favor simpler histories over more complex ones (typically h nbe ,0.5). The prior probability for each element of h is defined by a (conjugate) Beta distribution with parameters a and b (here, a = 1, b = 9). Because these elements are independent in the prior,
The term P (X i | Z i ) in equation 1 is simply the likelihood at gene i of a branch-site codon model that assumes selection history Z i . A full Bayesian approach would integrate over the parameters of these codon models, but this would be computationally prohibitive. Instead, we make the Empirical Bayes simplification of conditioning the analysis on maximum likelihood estimates of the parameters of the codon models. The maximized log likelihoods L ij for all genes i and histories j are precomputed using existing software (in parallel, on a large computer cluster) and stored in an N6M matrix, which is then used in the inference of selection histories.
The variables Z and h are unobserved, and the goal is to infer their joint posterior distribution,
This inference was accomplished by a Gibbs sampling algorithm that alternates between sampling each Z i conditional on X i and a previously sampled h, and sampling each element of h conditional on a previously sampled Z. It is straightforward to derive the required conditional distributions and to sample from them (Text S1). The Gibbs sampler converges rapidly and mixes well. Notice that, because the history without selection on any branch is excluded, all of the histories are described by codon models with the same number of parameters. Therefore, no penalty for parameter number is needed when comparing histories.
After an appropriate burn-in period, each iteration of the Gibbs sampler produces a sample (Z (t) , h (t) ) from P(Z, h|X). Estimated posterior expected values of interest were obtained by averaging these samples or functions of these samples, and Bayesian 95% confidence intervals were obtained by taking the 0.025 and 0.975 quantiles of the sampled values. For example, the posterior expected number of genes under selection on branch k (see Figure 4 ) was estimated as 1
, where T is the number of samples and the function f k (Z) counts the number of genes under selection on branch k in a set of histories Z.
Each gene was assigned categories from the GO [32] and PANTHER [86] databases (downloaded on June 26, 2007) , based on the Uniprot identifiers of associated transcripts. At least one GO category was identified for 14,137 (86%) genes, and at least one PANTHER category for 13,753 (83%) genes. To account for the hierarchical nature of these databases, each gene was also considered to belong to all parent categories of the ones to which it was directly assigned. For each category C and set of PSGs S, a 262 contingency table was constructed for the numbers of genes assigned or not assigned to C, and within and outsideS, then a (one-sided) P-value for independence of rows and columns was computed by Fisher's exact test. In addition, the distributions of LRT P -values among the genes assigned to C and not assigned to C were compared by a (one-sided) Mann-Whitney U (MWU) test. (Notice that S is not considered in this case.) Nominal P -values computed by the FET and MWU tests were corrected for multiple comparisons using the method of Holm [87] .
The analysis of gene expression was based on the publicly available ''Tissues+Mixtures'' sample data set for the Affymetrix GeneChip Human Exon 1.0 ST Array (http://www.affymetrix. com/support/technical/sample_data/exon_array_data.affx). The RMA-based probeset summaries [88] and DABG (detected above background) -values were used. Each probeset was assigned genomic coordinates using the ''Affy All Exon'' track in the UCSC browser (hg17), then was associated with any human gene from our set having an exon on the same strand that completely contained the probeset. Nearly every gene (98%) had at least one probeset.
To calculate a P-value for each gene6tissue, the DABG Pvalues of all associated probesets (pooling the three replicates per probeset6tissue) were combined using Fisher's method [89] . A gene was considered to be significantly expressed above background if it had (nominal) P,0.001. Similarly, an estimated expression intensity for each gene6tissue was calculated by first taking the median over the three replicates of each RMA-based probeset summary, then taking the median of these values over all probesets associated with the gene. The analysis of expression intensities was restricted to genes significantly expressed above background so that genes expressed at or near the background level did not drive the results.
To measure tissue bias, we used: (1) the statistic t [61] , which represents the average difference in normalized expression intensity from that of the tissue of maximal expression, and (2) a statistic, here denoted c, defined as c = max t c t , where c t is the squared cosine of the angle between the expression vector and the coordinate axis associated with t (see [17] ). In defining genes as tissue specific for tissue t we required c t .0.25 and c t9 ,0.125 for all t9 ? t. Further details are given in Text S1.
Maximum likelihood estimates of v for each branch were obtained using the codeml program in the PAML software package [84] , with F364 codon frequencies, estimation of k (fix_kappa = 0) and a single v across sites per branch (model = 1, NSsites = 0). The tree topology shown in Figure 1 was assumed. The alignments for all genes were concatenated for this analysis.
Assuming all non-synonymous mutations at a given gene have the same selection coefficient and all synonymous mutations are neutral, population genetic theory says that v should be given by [70, 90] :
where c = 2Ns. Therefore, c can be estimated as f 21 (v), where v = f(c) denotes the function above. (Values of c can be obtained numerically; see Text S1.) Ratios of population sizes can therefore be estimated from ratios of v estimates: N1
The LRT to test whether differences in population size can explain the differences in v in human and macaque was constructed as follows. The null model assumes v h = v c and v m = 0.732v h (see Results). The alternative model also assumes v h = v c but leaves v m as a free parameter to be estimated from the data. Because the models are nested, a x 2 df~1 distribution is used for significance testing. This test was applied separately to each gene.
A website is available at http://compgen.bscb.cornell.edu/ projects/mammal-psg/ with definitions of the candidate genes (accession numbers, genomic coordinates, and descriptions), multiple alignments of orthologous gene sets, GO and PANTHER category assignments, detailed results of the LRTs and the Bayesian analysis, and other resources. In addition, the candidate genes and predicted PSGs are displayed as a track in the UCSC Genome Browser (http://genome.ucsc.edu; assembly hg18).  Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval and is based on results from probability theory and statistics. We also provide formulas which can be used as close approximations. Digital PCR conventionally utilizes sequential limiting dilutions of target DNA, followed by amplification using the polymerase chain reaction (PCR) [1, 2] . As a result, it is possible to quantitate single DNA target molecules. We utilize the digital array, which is a novel nanofluidic biochip [2, 3] where digital PCR reactions can be performed ( Figure 1 ) by partitioning DNA molecules, instead of diluting them. This chip utilizes integrated channels and valves that partition mixtures of sample and reagents into 765 nanolitre volume reaction chambers. DNA molecules in each mixture are randomly partitioned into the 765 chambers of each panel (the total volume of the PCR mix in each panel: 6 nl6765 = 4.59 ml). The chip is then thermocycled and imaged on Fluidigm's BioMark real-time PCR system and the positive chambers that originally contained 1 or more molecules can be counted by the digital array analysis software ( Figure 2 ).
Copy number variations (CNVs) are the gains or losses of genomic regions which range from 500 bases on upwards in size. Whole genome studies have revealed the presence of large numbers of CNV regions in human and a broad range of genetic diversity among the general population [4, 5, 6] . CNVs have been the focus of many recent studies because of their roles in human genetic disorders [7, 8, 9] .
Current whole-genome scanning technologies use array-based platforms (array-CGH and high-density SNP microarrays) to study CNVs. They are high throughput but lack resolution and sensitivity. Real-time PCR is a sequence-specific technique which is easy to perform, but is limited in its discriminating power beyond a 2-fold difference [11, 12] .
CNV determination on the digital array is based upon its ability to partition DNA sequences. Given the number of molecules per panel and the dilution factor, the concentration of the target sequence in a DNA sample can be accurately calculated. In a multiplex PCR reaction with 2 or more assays, multiple genes can be quantitated simultaneously and independently, effectively eliminating any pipetting errors if separate reactions have to be set up for different genes. When a single copy reference gene (RNase P in this study, [10] ) is used in the reaction, the ratio of the target gene to the reference gene would reflect the copy number per haploid genome of the target gene. Primary contribution of this paper In this paper we will show that the digital array provides a robust and easy-to-use platform to study CNVs. We have derived a mathematical framework to calculate the true concentration of molecules from the observed positive reactions in a panel. We also show how to perform statistical analysis to find the 95% confidence intervals of the true concentrations and the ratio of two concentrations in a CNV experiment using the digital array with multiplex PCR.
The copy number variation problem can be stated as follows. Given two counts h 1 and h 2 of positive chambers for two genes in a digital array panel, how can one estimate a ratio of true concentrations r = l 1 /l 2 of the two genes and a confidence interval [r Low , r High ] on the estimation?
Our approach is built on well-known tools and techniques from statistics. It decomposes the problem into two parts. 2. Given estimated true concentrations l 1 and l 2 of the reference gene and the target gene, respectively, in the DNA sample and their respective confidence intervals, how can one estimate the ratio r = l 1 /l 2 and a confidence interval [r Low , r High ] on this estimation?
It turns out that the first question can be answered by applying sampling and estimation theories from statistics and probability, and the second question can be answered by a numerical algorithm based on generalization of a mathematical theorem.
For related work on answering the first question, using Bayesian approach, see unpublished preprint by Warren et al. titled ''The Digital Array Response Curve'' dated March 2007 at http:// thebigone.stanford.edu/papers.htm. Warren et al. assumed a uniform probability distribution of number of molecules, with maximum number assumed to be 4000, and using Bayesian and combinatorial methods, presented a solution. The confidence interval obtained using Bayesian probabilistic framework, is often referred to as credible interval or Bayesian confidence interval which requires one to incorporate problem-specific contextual information from the prior distribution.
This paper differs from this prior work by Warren et al. in two different ways. First, we consider the parameter l to be a fixed constant, unlike having a probability distribution as in Bayesian approach. Second, in addition to providing the answer to the first question, we are interested in estimating the confidence interval of the ratio of two concentrations which is new work. For difference between credible interval and confidence interval, see [13] . Both approaches give good results depending upon the question one is trying to answer.
We will prove mathematical correctness of our results in this paper and present simulation results to help the reader build useful insight. Finally, we present actual CNV experiments on the digital array with known ratios and show the results using the techniques developed in this paper.
DNA quantitation in the digital array is based on the partitioning of a PCR reaction into an array of several hundreds or even few thousands of chambers or wells. One panel of the digital array consists of 765 chambers and one can use up to 12 panels at a time. If the concentration of the target molecules is low in the DNA sample, most of the chambers capture either one or no molecules and the number of positive chambers at the end point of the PCR yields close approximation to the true concentration of the target. However, if the number of molecules is large, then there is greater probability of several molecules being in the same chamber, and therefore the number of positive chambers would be significantly lower compared with the number of molecules in the chambers.
We are interested in estimating the true concentration of the molecules in the DNA sample from which we extracted 6 nl6765 = 4.59 ml of sample for each panel.
Consider the universe of infinite number of the digital array chambers filled with an infinite amount of the DNA sample where the true concentration of the target molecules is l per chamber (per 6 nl). The true concentration is an unknown population parameter of this infinite DNA sample. If a chamber gets no molecule then it constitutes failure in the sense of Bernoulli experiment. If it gets one or more molecules, that is, if it gets a ''hit'' and is therefore positive, then it constitutes success. Let the probability of success be p. Note that p is an unknown population parameter. We will use the standard hat notation to denote sample estimators of population parameters. For example, p andl l will denote the estimators of p and l, respectively.
One can model K, the number of molecules in each chamber as a Poisson process, and this gives the relationship between p and l as follows
Alternatively, consider M molecules randomly distributed in C chambers. The probability of any given molecule being in any given chamber is 1 C . So the probability p of a given chamber having at least one molecule is
As number of chambers becomes arbitrarily large, the above approaches e 2l . Therefore, l~{ln 1{p ð Þ which establishes the relationship between l and p.
Confidence Intervals for estimation of p and l A chamber getting a hit or no hit is a binomial process, same as toss of a coin, with success probability p. Let the number of positive chambers in the panel be H. Considerp p~H C as an estimator of p. It is well known that p is an unbiased estimator of p and has expectation p and standard deviation ffiffiffiffiffiffiffiffiffiffiffi
and its sampling distribution f(p) is approximately normal for large C. See Figure 3 for illustration of the above ideas.
See [13, 14, 15] for extensive literature on obtaining confidence interval for the estimation of binomial probability. It is referred to as binomial sign test when the test statistic can be approximated with the chi-square distribution, specifically through the use of the chi-square goodness-of-fit. An alternative and equivalent approximation is obtained by using the normal distribution and then the test is referred to as the population proportion test, see [15] .
If C is large enough, then the confidence limits are approximately given byp
For 95% confidence interval, z c = 1.96. For the digital array, C is an integral multiple of 765 and is comfortably large enough for the above approximation.
Define the estimator of l aŝ l l~{ln 1{p p ð Þ
Since the probabilities in any given differential area of a probability density function are preserved under change of variables, the 95% confidence interval [l l Low ,l l High ] is directly given as followŝ p p Low,High~p p+1:96
See Figure 4 for illustration.
Let a random variable X have probability density function f X (x). If h(x) is either increasing or decreasing in x, then U = h(X) has density function given by
which follows from the fact that probabilities in any given differential area have to be invariant under change of variables, see [16] . Furthermore,
which can be expanded using Taylor series expansion of h(x) around the mean j = E(X), as follows
Since in our case, we have the followinĝ l l~{ln 1{p p ð Þ therefore, in above, we have x = p, u~l l and h(x) = 2ln(12x). Sincê l l is a monotonically increasing function of p, one can get the sampling distribution ofl l from the sampling distribution of p as
Note that due to nonlinear relationship betweenl l and p, one can not make assumptions about g. In general, g is not normal and
Now we derive an approximation for El l from the Taylor series expansion shown above. Higher order central moments of Gaussian function f(p) with mean p are
For proof see [17] . Since f(p) has very small s due to very large number of chambers, the higher order terms for all n.0 in the Taylor expansion are small,
and therefore the only contributing term is when n = 0, which 
It is informative and useful to run a simulation experiment on the computer to see how the real-world matches with the theory developed above. For this purpose, one can use a random number generator and a computer program to simulate the universe of the digital array chambers.
If a panel has C chambers, consider a universe of C6K many chambers where K is a large number chosen for simulation. Choose some value of l as the true concentration of molecules in one chamber. Therefore, in total, there will be l6C6K molecules. Assign each of these molecules randomly to one of the chambers.
Extract K panels out of this universe and for each of the panels, computep p~H C as an estimator of p and plot its histogram over all the K panels. The mean should be p = 12e 2l and standard deviation should be ffiffiffiffiffiffiffiffiffiffiffi
. For each of these panels, estimate l and compute the 95% confidence interval. In 95% of the K panels, the true value of l should lie within the confidence interval. For our simulation experiments we chose M = 400, that is, l~4 00 765 . We chose K = 70000. In Figure 5 we show the histogram of H which is really same as distribution of P scaled by a factor of 765. In Table 1 we show how the predicted values match with the actual simulation values. In the same way, the sampling distribution of number of molecules matched with what is predicted by theory. Though the results of the simulation follow from elementary probability, we conducted these simulations in order to build more advanced simulations for ratios of concentrations later. They also illustrate the meaning of the confidence interval.
In previous section, we established a method for estimating the true concentration of the target molecules in the DNA sample from the count of positive chambers as well as the 95% confidence interval for this estimation. We also showed how the sampling distribution gl l is related to the sampling distribution f(p). In CNV, the goal is to determine ratio of true concentrations of two genes, one being reference gene and the other being test gene, and associated confidence interval, which we now accomplish in next subsections. Table 1 . Comparison of the metrics of histogram, shown in Figure 5 , of number of positive chambers obtained in simulation with those predicted by the theory. 
Let the sampling distributions of the test gene and the reference gene be g 1l l 1 and g 2l l 2 , respectively. If these distributions were normal, then one can make use of Fieller's Theorem [18, 19] .
However, as mentioned in previous section, one can not make this assumption in general. It is useful to go through the geometric interpretation of Fieller's theorem so that one can solve the problem for arbitrary sampling distributions. See Figure 6 for geometric interpretation of Fieller's Theorem [20, 21] .
Assume g 1l l 1 and g 2l l 2 are normal. Forl l 1 andl l 2 , the ratior r~l l 1 .l l 2 can be shown as the slope of the line in the twodimensional plane which passes through the origin and the 2-D point (l l 2 ,l l 1 ). Luxburg et al. show in [20, 21] how a confidence ellipse in the two-dimensional plane can be constructed. Consider the two lines which pass through the origin and are tangents to this ellipse. The intersection of these lines with the vertical line at l l 2~1 gives the desired confidence interval.
In this paper we have presented data in a controlled experimental system, where a synthetic DNA construct was spiked into human cell line DNA at different concentrations. In this case, the synthetic construct, which was to the RPP30 gene, was used as the target, and the RNase P gene which was endogenous to the human cell line, was used as the reference gene. The two genes were identified using two separate PCR reactions, using separate PCR primers and probes. Since there is no reason to assume that the amplification and detection of the target and reference genes are linked,l l 1 andl l 2 are independent variables.
It is easy to see from the proof of Fieller's theorem and its geometric interpretation that one can compute sampling distribution q of the ratio estimatorr r~l l 1 .l l 2 as follows: This can be interpreted as cutting out thin wedges in the joint distribution of g 1l l 1 and g 2l l 2 and accumulating the probabilities inside the wedge to compute the function q in the corresponding thin interval of the ratio. This is the basis of our numerical algorithm which implements integration in order to compute q(r):
1. Build histograms of sampling distributions g 1l l 1 and g 2l l 2 . The tails of the histograms where probabilities become very small are approximated by zero. 2. Build a histogram of sampling distribution q(r) ofr r~l l 1 .l l 2 by considering each bin [r 1 , r 2 ] and by adding all the joint probabilities of different values of concentrations which give a ratio rM[r 1 , r 2 ]. 3. Compute the mean and the 95% confidence interval from the ratio histogram.
See Figure 7 for illustration of the above algorithm. One can still use direct formulas, as an approximation, to compute confidence interval as follows.
The means of g 1l l 1 and g 2l l 2 are l 1 and l 2 respectively. Let the standard deviations be s x and s y respectively. For given estimationsl l 1 andl l 2 , assuming that distributions are normal, it follows from the analysis in [20, 21] that the boundary of the confidence ellipse for a given confidence level z c would be defined by
It is easy to generalize this to the case, under a reasonably close approximation, when we have asymmetric distributions which are assumed to be normal in each of the four quadrants of the coordinate system centered at (l l 2 ,l l 1 ). Then the confidence region is made of union of four quadrant-wise elliptic regions. Let the asymmetric confidence intervals for specified z c and the two concentrations be [l l 1 {H B ,l l 1 zH T ] and [l l 2 {W L , l l 2 zW R ].
If W R = W L = z c s x and H T = H B = z c s y , it is symmetric case [20, 21] . Using simple algebraic manipulations, it can be shown, as in symmetric case, that the slopes of lines that will be tangents to this union of four quadrant-wise ellipses will bê r r Low~l
The above equations can be used as an approximation though numerical algorithm will give more accurate results as the algorithm does not make any assumptions and works with arbitrary sampling distributions. One detail has to be mentioned. Special care has to be taken if the confidence region gets too close tol l 1 axis whenl l 2 is small. If it touchesl l 1 axis, then r High = '. If eitherl l 1 orl l 2 is too small, one can build respective histogram with smaller bin size to get more accurate results.
See Table 2 for summary of equations derived in order to solve the copy number variation problem. Though the numerical approach based on histograms is recommended as it does not make assumptions, these direct formulas can be used as close approximation.
We conducted simulation studies, using a random number generator and a computer program as in previous section, by choosing a ratio of 2 of concentrations of two genes, which are independent of each other, and building a distribution of estimated ratios over 50 thousand panels. In 94.9% of the panels, the true chosen ratio did lie in the computed confidence intervals thereby showing the correctness of our mathematical analysis.
The copy number variation results for known ratios of 1, 1.5, 2, 2.5, 3 and 3.5 are shown in Figure 8 . Materials and methods for this experiment are discussed in next section. As the number of panels P increases, then the number of chambers C = 765 Pincreases and therefore the estimation of the ratio becomes more accurate as well as the confidence interval shrinks. When only 1 panel is used, there is significant overlap between 95% confidence intervals of certain ratios e.g. between ratio 2 and 2.5. There is no overlap when 3 or more panels are used. In all cases the known ratio lies within the computed 95% confidence interval. Note that using mathematical analysis one can find optimal numbers of positive chambers for each ratio which give smallest confidence intervals and which will therefore improve the results.
In summary, Fluidigm's digital array is capable of accurately quantitating DNA samples and is a valuable platform for studying copy number variation. It is a robust technology that is sequencespecific, easy-to-use, and extremely flexible. We have presented mathematical and algorithmic solutions to analyze CNV on a digital array. The solution is an elegant application of statistical sampling and estimation theories to such an important real-world Table 2 . Given number of chambers C and counts H 1 and H 2 of the positive chambers in a digital array for the target gene and the reference gene, respectively, list of formulas needed to analyze copy number variation. problem. We have shown how one can compute the true concentration of a target sequence in a DNA sample and the associated confidence interval on this estimation, and how one can compute the ratio of true concentrations of multiple sequences and the associated confidence interval on the estimation of this ratio.
A 10-ml reaction mix is normally prepared for each panel. It contains 16 TaqMan Universal master mix (Applied Biosystems, Foster City, CA), 16 RNase P-VIC TaqMan assay, 16 TaqMan assay for the target gene (900 nM primers and 200 nM FAMlabeled probe), 16 sample loading reagent (Fluidigm, South San Francisco, CA) and DNA with about 1,100-1,300 copies of the RNase P gene. 4.59 ml of the 10-ml reaction mix was uniformly partitioned into the 765 reaction chambers of each panel and the digital array was thermocycled on the BioMark system. Thermocycling conditions included a 95uC, 10 minute hot start followed by 40 cycles of two-step PCR: 15 seconds at 95uC for denaturing and 1 minute at 60uC for annealing and extension. Molecules of the two genes were independently amplified. FAM and VIC signals of all chambers were recorded at the end of each PCR cycle. After the reaction was completed, Digital PCR Analysis software (Fluidigm, South San Francisco, CA) was used to process the data and count the numbers of both FAM-positive chambers (target gene) and VIC-positive chambers (RNase P) in each panel.
A spike-in experiment was performed using a synthetic construct to explore the digital array's feasibility as a robust platform for the CNV study. A 65-base oligonucleotide was ordered from Integrated DNA Technologies (Coralville, IA) that is identical to a fragment of the human RPP30 gene. The sequences of the primers and FAM-BHQ probe used to amplify this construct are from Emery et al [22] . The primers and probe were ordered from Biosearch Technologies (Novato, CA).
Both RPP30 synthetic construct and human genomic DNA NA10860 (Coriell Cell Repositories Camden, NJ) were quantitated using the RPP30 assay on a digital array. Different amounts of RPP30 synthetic construct was then added into the genomic DNA so that mixtures with ratios of RPP30 to RNase P of 1:1 (no spikein), 1:1.5, 1:2, 1:2.5, 1:3, and 1:3.5 were made simulating DNA samples containing 2 to 7 copies of the RPP30 gene per diploid cell.
These DNA mixtures were analyzed on the digital arrays as described above. Five panels were used for each mixture and 400-500 RNase P molecules were present in each panel. The ratios of RPP30/RNase P of all samples were calculated using the techniques developed in this paper. For each ratio, we did pooled analysis by adding the numbers of positive chambers in the first P = 1,2,3,4,5 panels. The results are summarized in the previous section and in Figure 8 .  The geosimulation of West Nile virus propagation: a multi-agent and climate sensitive tool for risk management in public health BACKGROUND: Since 1999, the expansion of the West Nile virus (WNV) epizooty has led public health authorities to build and operate surveillance systems in North America. These systems are very useful to collect data, but cannot be used to forecast the probable spread of the virus in coming years. Such forecasts, if proven reliable, would permit preventive measures to be put into place at the appropriate level of expected risk and at the appropriate time. It is within this context that the Multi-Agent GeoSimulation approach has been selected to develop a system that simulates the interactions of populations of mosquitoes and birds over space and time in relation to the spread and transmission of WNV. This simulation takes place in a virtual mapping environment representing a large administrative territory (e.g. province, state) and carried out under various climate scenarios in order to simulate the effects of vector control measures such as larviciding at scales of 1/20 000 or smaller. RESULTS: After setting some hypotheses, a conceptual model and system architecture were developed to describe the population dynamics and interactions of mosquitoes (genus Culex) and American crows, which were chosen as the main actors in the simulation. Based on a mathematical compartment model used to simulate the population dynamics, an operational prototype was developed for the Southern part of Quebec (Canada). The system allows users to modify the parameters of the model, to select various climate and larviciding scenarios, to visualize on a digital map the progression (on a weekly or daily basis) of the infection in and around the crows' roosts and to generate graphs showing the evolution of the populations. The basic units for visualisation are municipalities. CONCLUSION: In all likelihood this system might be used to support short term decision-making related to WNV vector control measures, including the use of larvicides, according to climatic scenarios. Once fully calibrated in several real-life contexts, this promising approach opens the door to the study and management of other zoonotic diseases such as Lyme disease. The WNV is a flavivirus which was isolated for the first time in 1937. Its name comes from the district of West Nile in Uganda. It was detected in human, birds and mosquitoes in Egypt at the beginning of the fifties, and has then been found in various European countries [1] . It is however only with the important 1996 human epidemic in Bucharest, Romania, that WNV became a concern for public health. Moreover, there is no specific treatment of the disease and no vaccine is yet available for humans. The WNV was detected on the American continent in 1999 and more specifically in New York [2] . In Canada, WNV reached southern Ontario in 2001, while the first human cases were detected in August 2002 [3] .
WNV made its appearance in Quebec in July 2002. The virus was then propagated, like everywhere else, by the intermediary of mosquitoes and birds. The expansion of this epizooty forced the Government of Quebec to adopt an intervention plan which included in 2003 the implementation of a multi-faceted surveillance system [4] . This system brought together field data on human, avian and entomological infection and deaths.
While these monitoring activities were undertaken to better understand the epidemiology of WNV and the level of risk it can represent for the human population, they do not allow for forecasts of the probable propagation of the virus on the territory. Such a forecast, if it proved to be reliable, would allow public health authorities to initiate preventative actions at the right time and places and at the appropriate level of expected risk. Currently, one main control activity is larvicide spraying in urban and rural settings in order to reduce the population of mosquitoes infected with WNV. However, it remains difficult to determine the at-risk zones on a scientific basis and the efficacy of such measures has been challenged [5] , not to mention their high cost and environmental impacts. The identification of vulnerable zones and risk levels in due time remains a significant challenge for public health management due to the complexity of the phenomena related to the virus transmission.
Multi-agent geosimulation is an artificial intelligence modeling approach which might be used to develop public health management tools in order to anticipate the progression of the disease and to assess various intervention scenarios. This approach makes it possible to simulate the behaviours of thousands of agents in georeferenced virtual spaces. The MAGS System (Multi-Agent GeoSimulation) recently developed by Dr. Moulin's Groupe de Recherche en Informatique Cognitive at Laval University, can be used to create such simulations in virtual environments generated with georeferenced data obtained from geographic information systems (GIS). These agents are characterized by cognitive capacities such as perception of the environment and its content, autonomous navigation and reasoning [6] . Although one of the first applications of MAGS was related to the simulation of crowd behaviours in urban environments, MAGS is a generic platform allowing the simulation of several types of behaviours in various geo-referenced virtual environments. For example, it has already been used to simulate the behaviour of consumers visiting shopping centers and firemen intervention plans to contain the propagation of forest fires [7] .
The main objective of the WNV-MAGS Project reported in this paper, was to develop a system to simulate the behaviours and interactions of populations of indicator birds and of mosquitoes involved in the propagation and transmission of the WNV, taking into account the characteristics of the geographic environment. This simulation takes place in a virtual cartographic world representing a large territory (southern part of the province of Quebec, Canada). The simulation also takes into account various climatic scenarios and regimens of larvicide treatments.
In Section 2, we present an overview of the phenomena which are linked to the spread of WNV. Then, we present the conceptual model which was developed after setting some carefully chosen hypotheses. Next, we present the geosimulation of the populations of interest, using agents' roosts to represent the dynamics of the bird populations and an intelligent density map to represent the populations of mosquitoes. Some short-term climate scenarios and the calibration of the system are also presented in this section. In Section 3, we present a conclusion and some new work currently underway. In Section 4 we briefly present the design method used to develop the system, including the conceptual architecture and an overview of the mathematical model formalizing the evolution of relevant populations. We also comment upon the quality and availability of data used to feed the system. Finally, we briefly present the implementation context of the system.
Overview Figure 1 presents a synthetic view of the phenomena which are involved in the spread of WNV, as adapted for the Quebec context [8] . Indeed, there are mainly two populations involved in the transmission of the WNV: the population of mosquitoes (Culex sp.) and the population of birds. In this paper, we mainly consider the Corvidae family and more specifically crows which have been chosen by public health authorities as indicator birds for the WNV. Mosquitoes spawn eggs in sumps and other shelters. The larvae hatch from eggs and evolve into nymphs that emerge to become adult mosquitoes. This cycle mainly depends on temperature and humidity [9] . Besides, human intervention can reduce the population of mosquitoes through larvicide treatments (e.g. Methoprene) in order to kill larvae. The transmission of the WNV occurs mainly mosquitoes biting birds. An infected mosquito can infect a bird, which can in turn infect healthy mosquitoes that will subsequently bite the infected bird before its death [10] .
Regarding the populations of Corvidae, their spatial and temporal characteristics depend on geographic areas and on the periodicity of displacements and grouping. During early spring bird couples spread over the whole territory and remain for few months around their nesting areas. By the end of July, which happens to be the very beginning of human infections in Quebec [3] , Corvidae change their social behaviour and regroup in roosts at night. During the day, the birds fly to surrounding areas in search of food, but they go back to the roosts at night [11] . At the end of fall, many of them migrate to warmer areas south of the province [12] . Furthermore, the transmission of WNV to the populations of Corvidae can occur either by mechanical infection (an infection after a direct contact between birds) or through the biting of a healthy Corvidae by an infected mosquito ( Figure 1 ).
Since we wanted to simulate the progression of the WNV infection involving a large number of individuals of two main species interacting in a particular geographic region, we selected a geosimulation approach which allows for the study of the spatial and temporal characteristics of the populations' interactions in a virtual geographical environment [6] . However, given the enormous complexity involved in representing such phenomena and the lack of detailed data, we had to raise a number of reasonable simplifying hypotheses with regard to the species of mosquitoes and Corvidae of interest, to the factors influencing the evolution of these populations, the geographical region selected for the analysis, the period of simulation and the space-time scale. These hypotheses led us to identify a set of key parameters to carry out the simulations, based on the epidemiologic and surveillance experience with WNV in North America and more precisely in the province of Quebec [4] . For example, considering the availability of surveillance data, we selected the American crow as the main indicator bird species; and the Culex pipiens and Culex restuans as the main mosquito's species susceptible to bite crows (and possibly humans). Another example is the period of simulation for the WNV propagation: July 1st to October 1st was selected as the critical time window during which human cases have appeared in Quebec so far (Table 1) .
The objective of the conceptual model is to introduce a synthetic view of the phenomena of interest while taking into account the above mentioned simplifying hypotheses ( Figure 2 ). Let us briefly comment upon this model which represents the evolution and interactions of Culex sp. (pipiens/restuans) and crows. Moreover, we simplified the biological cycle of Culex to only consider the change from a larval state to an adult one. From a public health management's point of view, these two states are the most important ones since the virus is spread by adult females and treatments against the progression of the WNV are carried out using larvicides. This simplification has been validated by domain experts (see below). In addition, considering the spatial dynamics of crow populations, we selected the period of the year when Corvidae regroup in roosts. In our model, a roost is considered as the spatial extension of an aggregate of crows (a sub-population of crows which gathers in this roost for the period of the year of interest). During the day, crows fly a variable distance from the roost in search of food, and return at night. Hence, the spatial phenomenon of gathering and dispersion of this sub-population of crows can be represented in a synthetic way in the form of an expansion and a contraction of the area occupied by this sub-population. The surface over which the birds spread during the day ("roost expansion") depends on the roost size. Consequently, we can take into account the variable density of crows in this dynamically changing area. Another fact to consider is that the Culex mosquito has a mostly nocturnal activity [13] . Therefore, the crows located in roosts at night will be good targets for them. Moreover, preset variables have been used in order to compute parameters such as the infection probabilities and the mortality proportions. This conceptual model has been validated by domain experts (from the GDG Company, Université de Sherbrooke and Université du Québec à Trois Rivières -UQTR) and was used to orient the development of the geosimulation tool.
According to our conceptual model, the progression of the WNV infection involves a large number of individuals of two main species and their interactions depend on the probabilities of finding sub-populations of these species within the same geographic areas at specific times. We already mentioned the interest of using a multi-agent geosimulation approach in such a context. However, we had to adapt it to take into account the large geographic area of interest and the very large size of the involved populations, especially for Culex.
To create the virtual geographic environment representing the studied region ( Figure 3 ), we first collected geo-referenced data and generated the various spatial data layers needed by the MAGS platform. Then, we modelled the two populations involved in the transmission of the virus as well as their locations in this virtual environment. Indeed, the population of Culex represents an extremely large number of individuals and cannot be represented using individual agents. Instead, we decided to model the mosquito population as an 'intelligent density map' which is characterized by population data being attached to reference areas (municipalities) in the virtual space. The idea is to associate to each reference area a list of variables corresponding to the numbers of the different categories of mosquitoes (larvae, healthy and infected adults) located in this place. These numbers evolve during the simulation as a consequence of various parameter changes (temperature, degree of humidity resulting from 
Representative species of Corvidae populations: Corvus brachyrynchos (American crow). Other birds are also considered. Representative species of mosquito's populations: Culex pipiens and Culex restuans. Main factors influencing the behaviours Culex Climatic conditions (primarily the temperature and precipitations).
crows Zones and periodicity of displacements and grouping.
The Ecumene zone of the following administrative areas: Québec, Chaudière-Appalaches, Mauricie-Centre du Québec, Montérégie, Estrie, Montréal-centre, Laval, Laurentides, Lanaudière et Outaouais.
We decided to simulate the WNV propagation from July 1 until October 1.
An interval of a daily step with a weekly assessment.
The micro-space scale specifies the size of a pixel (4 km 2 ). The macro-space scale give an idea on the spread of the WNV propagation in order to bring a help to the decision-making concerning the larvicides treatments (interpolated by aggregation of pixels between 1 and 50 km 2 ).
rainfall, etc.) as well as encounters with crows. For the population of crows, we used agents to model groups of crows associated with specific areas where roosts have been observed in the field. The interactions of the two populations have also been modeled thanks to the geosimulation which enables the system to automatically determine the places and times where groups of crows (pertaining to roosts) will cross areas in which the Culex sub-populations are located.
The populations of Culex do not move much and they are present practically everywhere in the selected territory [3, 14] . Because of the extremely large number of individual mosquitoes, we represent sub-populations of mosquitoes as characteristics of the virtual geographic environment and we use what we call an "intelligent density map" that represents the distribution of Culex subpopulations over the different reference areas depending on the geographic characteristics and the locations of favourable habitats for mosquitoes. This intelligent density map is a kind of cellular automaton associated with rules that enable the system to simulate the evolution of the different categories of mosquitoes (larvae, adults, healthy, infected, etc.) in each reference area under the conditions that influence the mosquitoes' life cycle (temperature and precipitations). The system gets these conditions either from actual meteorological data (from specific databases: see Section 4.3) or from the parameters set in scenarios that the user wants to explore. This map contains the polygons representing all the municipalities of the Quebec province (the reference areas). On the user's screen, the color of each municipality changes according to the relative densities of the Culex populations (ratios of healthy, and infected adults) computed by the simulator (Figure 4 ).
A roost synthesizes the behaviour of a group of crows. It is modeled by an agent having some initial characteristics such as the number of individuals, the position of the roost on the map, and the maximum radius of its expansion area. These characteristics are computed using various field data as presented in Section 4.3. Moreover, this agent inherits from all the functionalities of MAGS agents. For example, it uses some behaviour rules in order to model how crows scatter around the roost. In addition, an operating range parameter is computed for each roost in order to estimate the maximum distances covered by the crows when they search for food during the day.
Each roost agent is implemented as a particle system [15] which simulates the way crows spread around a roost during the day. Hence, such particle systems behave as agents, as described above. Each particle represents one or several crows, depending on the number of individuals attached to the roost. In Figure 5 we can see a snapshot of a simulation in which roosts are displayed as "clouds of blue particles". Each particle has different characteristics (velocity, direction of movement) that enable it to travel at a distance from the roost location during a number of simulation steps representing a day. Hence, the set of particles associated with a given roost covers a circular area with a maximal radius set by the operating range parameter. We calibrated the parameters of the particle system by computing the density of crows in the area covered by the expansion of the roost and comparing it to observed field data and other estimated data.
The interaction between the two main populations is a very important functionality of our system, since it is the way of representing the evolution of the infection. Indeed, while traveling in the geographic space, one or several crows represented by a particle can cross areas in which Culex mosquitoes are located. Consequently, there is a probability that some of these crows will be bitten. Technically, in order to determine the probabilities of encounters between mosquitoes and crows, the corresponding particle takes into account the characteristics of the Culex population associated with each reference area of the 'intelligent density map' over which it travels. Therefore, the system can estimate the number of infected individuals, based on the likelihood that a number of individual crows be bitten by mosquitoes and be infected with WNV (using the equations of the mathematical model described in Section 4.2). Moreover, the user can visualize the extent of the spread of WNV on the map in different ways. The system can either change the color of the particles representing the infected crows or the color of the polygon representing a municipality containing a high density of infected Culex.
Our initial simulations involved the two main species of American crows and Culex pipiens/restuans that we selected and that enabled us to apply Wonham's mathematical model [16] (see Figure 12a in section 4.2). We quickly found out some limits with this model, since it does not take into account the influence of temperature on the evolution of mosquito's populations. When we applied this model, it led, after a number of iterations, to the complete "extinction of crows". This, obviously, does not conform to reality, although a dramatic decrease of Corvidae popu-
The geographic area of interest which contains all the municipalities belonging to the ecumene (Southern Quebec, Canada) Figure 3 The geographic area of interest which contains all the municipalities belonging to the ecumene (Southern Quebec, Canada).
lations have been observed in recent years due to the spread of WNV [17] .
Hence, we proposed an extension of this model which enables us to model several species of birds and to take into account the impact of the temperature in terms of cumulated degree-days which influence some parameters of the model (see Figure 12b in section 4.2). We cannot discuss here the details of such a model and its implementation (for more details, see [18] ). In the current experiments, we modeled the interactions between crows and mosquitoes as described in the previous section. Since surveillance systems provide data about crows as indicator birds, we used this species to set the simulation parameters and to calibrate the system. However, we added other bird species in the simulation to increase "the biting opportunities" for mosquitoes, so that the "crow popula-tion" does not become extinct by the end of the simulation period. Indeed, this is a plausible hypothesis: mosquitoes bite other birds as well as crows.
We thus introduced in the WNV-MAGS system another 'global' population of birds, that we called "generic birds" (Common Raven, Blue Jay, American Robin, House Sparrow, European starling and Mourning Dove) which are resident in the municipalities and known to carry WNV [17] . This population of generic birds appears in the mathematical model with similar equations as those used for the crows (each bird population is represented by a different index j in the equations: see Figure 12b in section 4.2). However, the parameters for each bird family may be different. Due to lack of data, we currently set some average parameters to the equations of the "generic birds". Getting more accurate parameters will require further
Using an intelligent density map to represent the population of Culex Figure 4 Using an intelligent density map to represent the population of Culex. research from bird specialists. In the simulation, one distinction that we established between crows and "generic birds" is that we assumed that birds do not move outside the municipality (as crows may do while flying away from the roosts). Hence, generic birds stay in contact with the same mosquito population during the simulation. Indeed, this is a simplification. Since our system is parameterised, we will be able to introduce parameters for other bird species as soon more precise data will be available with respect to the ecology and epidemiology of other birds affected by WNV.
Using various short-term climate scenarios In our system, multi-agent geosimulation is at the heart of a decision support tool. Hence, our approach is somewhat different from more traditional simulations used for prediction purposes [19] . The WNV-MAGS System simulates the WNV epidemics and enables a user to specify scenarios in order to explore various situations including climate change and different intervention strategies. The user may choose one among four different scenarios which influence the dynamics of the Culex population ( Figure 6 ). The first scenario is the default scenario which can be set in order to use average conditions of temperature and precipitations (using in this case the Canadian Climate Normals [20] ). In order to estimate the number of mosquitoes located in each municipality, we computed the number of sumps that are along the municipality's roads (see section 4.3). Sumps offer ideal locations for the maturation of larvae and the emergence of adult mosquitoes. They are also the main targets of larvicide spraying. But abundant rains may flush sumps, killing a large proportion of larvae. In a second type of scenario, the user can choose a date during which abundant rains may flush
Using roosts to represent the populations of crows Figure 5 Using roosts to represent the populations of crows.
sumps in some municipalities (Figure 7 ). In the same way, the third scenario is used to simulate the use of larvicides in a certain area (municipality). The last scenario is a combination of the second and third scenarios. Hence, it is possible to choose a date for the flushing of sumps and another date for the application of larvicides. Most larvae are supposed to die after the flushing of a sump, although the dynamics of the larval populations starts all over again since there are always Culex adults in the vicinity of the sump that will spawn new eggs. Moreover, the WNV-MAGS System offers a variety of functionalities to the user in order to modify the parameters of the mathematical model, to visualize the progress of the infection in and around the crows' roosts, to extract data from the simulation and to generate graphs showing the evolution of the involved populations.
The qualitative results of the model which represent the distribution of the populations were satisfactory. Indeed, the resulting curves reflect the biological behaviours of the studied species according to the opinion of the consulted domain experts (from GDG and UQTR). However, the quantitative data needed to be calibrated in order to be used in real-life situations. In fact, we calibrated the model by comparing simulation results and field observations (ISPHM-WNV data [4] ). We evaluated the ratio between the real populations of mosquitoes and the samples of mosquitoes captured in traps (absolute densities) as well between crows and the collected dead crows.
Regarding the populations of Culex, we used Reisen's work [21, 22] to estimate the mosquito density ratio. A Using management scenarios Figure 6 Using management scenarios.
The dynamics of the larval populations before (a) and after (b) the flushing of sumps in Laval Municipality on August 15 th (hypo-thetical scenario defined by user) Figure 7 The dynamics of the larval populations before (a) and after (b) the flushing of sumps in Laval Municipality on August 15 th (hypothetical scenario defined by user).
captured mosquito was considered to represent a population of 300 Culex over one km 2 . Since we did not have data for all regions, we only calibrated simulation results for some key municipalities where human infections had occurred. It appeared thereafter that there was a significant difference between the data generated by the model and those obtained from the field. Hence, we tuned up the initial settings of the simulation (e.g. the initial percentage of infected Culex or infected crows, distance between sumps, emerged Culex per sump, percentage of sumps containing larvae, etc) as well as some parameters of the mathematical model (e.g. mosquitoes biting rate of crows per capita, WNV transmission probabilities from Culex to crows of from crows to Culex, etc). These changes have helped us to quantitatively calibrate the model for the processed municipalities. Figure 8a presents the evolution of the total number of mosquitoes for the municipality of Laval between July 1 st and October 1 st . The smooth blue curve represents the data generated by the simulation while the rugged red curve represents averages of real data over four years (2003 to 2006) . We had to consider these averages because we do not have sufficient data from the field (trap measurements are sparse and not carried out regularly in Quebec municipalities). Moreover, these data averages enabled us to adjust our initial data in the simulation (mainly the initial number of mosquitoes) as it can be observed in Figure 8a . The simulation curve and the real data curve fit nicely between July 1 st and August 15 th . The two big drops that are observed in the real data curve are difficult to explain at this point since we consider the average measures over four years. This may be the result of systematic larvicide applications in July and August (3 applications in some municipalities during a WNV season), but we have no sufficient data to confirm this conjecture. In figure 8b , we also observe a similarity between the curves representing the infected mosquitoes. Again, the rugged red curve represents averages of real data over 2003-2006. All drops in the curve result from lack of sufficient field data.
Regarding the populations of crows, we used the results presented by David and colleagues [23] in order to determine whether the numbers of dead birds sighted and tested for WNV are representative of the true bird mortality. We also used the index trend obtained from the ÉPOQ database [24] and from the North American Breeding Bird Survey [25] to adjust the population of crows as well as the population of generic birds. Moreover, changes in the population of crows have been calibrated using field data collection of dead birds and their analyses in the laboratory, as it was done for the population of Culex.
Model calibration using the average total mosquitoes cap-tured in traps (a) and those among them which are infected with the WNV (b) during the considered simulation period represents a comparison of the simulated data (smooth blue curve) and real data (red rugged curve) for the collected dead crows. The general shapes of the curves are similar. This is encouraging since data available for dead birds are even sparser than for mosquitoes.
Then, we looked at real data for Laval Municipality for 2003, the year for which we have the most complete data set. We created the temperature scenario for 2003 and launched the simulation. Figure 10a presents the difference between the simulated data (blue smooth curve) and the real data. In order to explain this difference, we checked with the SOPFIM Company if larvicides had been sprayed in Laval Municipality in 2003. It was indeed the case, with interventions on June 18, July 17, and August 13. We created a new scenario using these three dates for larvicide spraying and we got the curve displayed in Figure  10b . The curve of simulated data now approximates the real data fairly well (the rugged curve of real data being again explained by missing data). This is an encouraging result showing that the parameters adjusted for calibration provide reasonable results.
In order to improve precision and validate our models and the simulation parameters, we will carry out the simulation on a different data set. We are currently collecting data (for mosquitoes and crows) for the city of Ottawa. We expect to get a more complete data set, since measurements have been more frequent and regular in the Ottawa region (Canada) over the past 6 years. This work is in progress.
In this paper, we presented a system that simulates the interactions of the populations of mosquitoes and birds which are involved in the propagation and transmission of the WNV. Moreover, we used a multi-agent geosimulation approach which takes into account the influence of the geographic characteristics of the various regions, thanks to the use of GIS data. For example, we determined the geo-referenced co-ordinates of crows' roosts in order to locate them on the map and we were able to develop rules which control the expansion/contraction of roosts over space and time. We also pre-processed climate data in a GIS in order to feed it to the simulation. We also used the geographic characteristics and the location of favourable habitats for mosquitoes in order to represent the populations of Culex using an intelligent density map. Consequently, we were able to implement the interactions between the mosquitoes' and birds' populations which can cause an outbreak of the virus and epidemic propagation of the disease. Even if other works [26, 27] also used GIS data to simulate the spread of the WNV, they did not offer a decision support system as we do. In contrast, our system enables users to simulate the propagation of WNV under various short-term climate scenarios and allows for local parameterization. This approach may be useful for practical decision-making. For instance, it has been shown [28] that the number of degree-days below -5°C in the winter and the number of degree-days greater than 25°C in the summer may contribute to a highly epidemic emergence of the virus during the summer under specific climatic conditions. Consequently, our system may be used to predict such an epidemic if we simulate the propagation of the WNV using a scenario in which seasonal forecasts of climatic data are favourable for the emergence of the virus [29] . By assessing the simulation results and comparing the outcomes of different intervention scenarios, the users of the WNV-MAGS System can make more informed decisions about the actions to be taken such as the application of larvicides or the stepping up of personal protection measures.
An important limit of this kind of approach is the lack of field data. As we have already shown in this paper, a good calibration and validation of the models depends on the availability of a large variety of data sets (related to mosquitoes and to different species of birds). There is also a difficulty in estimating the parameters needed in the mathematical model, which would require in some cases that additional field studies be carried out by entomologists and ornithologists. In addition, the potential effects of changing resistance and immunity in wild birds remain unknown and need to be studied by domain experts. Obviously, they have not been included in our models yet.
If we were able to collect sufficient data about the WNV spread in different regions during the past years, we could develop scenarios and simulations whose results could be compared to recorded field data. Consequently, we would be able to further validate the system and adjust the various parameters that are used for the simulations, taking into account the specificities of the considered regions and species.
Nonetheless, the system can already be used to compare different scenarios involving variations of the climatic data in relation to the potential spread of WNV in particular regions. As we have shown, the system can also be used to estimate the influence of human intervention based on larvicide application. However, since it still remains difficult to get accurate weather forecasts over long periods (several months), public health authorities will have to take into account this inherent limitation of meteorological science when developing intervention plans using such a tool.
Our MAGS approach and tool can be used not only to simulate the propagation of the WNV, but they can also be adapted to various other vector-borne diseases. We are currently working on the simulation of Lyme disease in Quebec. Moreover, the tool and approach can be extended to take into account the specificities of other similar diseases (e.g. SARS) in other geographic areas.
In order to develop the WNW-MAGS System, we applied an 'Agile' [30] analysis and design method which favours the collaboration with domain specialists and users, as well as quick adaptations of the software under development. We also applied classical knowledge engineering techniques [31] in order to acquire domain knowledge from the specialized literature and from domain experts (entomologists and ornithologists) after many work sessions. We then went through an exploration phase of the field by gathering the maximum useful information in order to understand all the phenomena which are related to the spread of WNV. We present in this section the conceptual architecture which is used as a basis of the simula-tion system. We also present the mathematical model which was chosen and adapted in order to compute the dynamics of the populations involved in the transmission of the WNV. We also present a subset of the relevant data which is used to feed the system. Finally, we present the implantation of the system.
Based on the requirement specifications and using the conceptual model of the phenomena (see Figure 2 in section 2.2), we designed a conceptual architecture of the WNV-MAGS system which includes all the needed system components and their relationships [32] . While constructing this architecture, we identified all the processes to be developed (represented as rectangles numbered as Pi in Figure 11 ) and all the data stores (represented as ovals numbered as Ai in Figure 11 ) that gather data and feed the system processes. Indeed, most of the necessary data are obtained from external databases (EPOQ [24] , Weather data, etc.) and GIS. They are represented as 'cylinders' at the bottom of Figure 11 .
The architecture is divided into four parts. The first part (processes P1 to P4) deals with data preparation, including the extraction of data from all the required databases. The second part (processes P5 and P6) computes the evolution of populations using the mathematical model presented in Section 4.2. The third part (processes P7 and P8) deals with the interactions between the sub-populations of crows and the sub-populations of Culex. It reflects the interactions between the agents' roosts and the intelligent density map. The last part (processes P9 to P12) is responsible for the management of scenarios, as well as for the display, analyses and calibration of the results. We used different databases (as presented in Section 4.3) in order to initialize the populations of Culex and crows at time t 0 (beginning of the simulation). We used these populations to compute the dynamics of crows, the dynamics of Culex and the interactions between the two populations while increasing the time by one step, at time t. Then, we get the new populations at time t+1 (taking into account the state changes of the sub-populations of mosquitoes and crows reflecting the infection spread and deaths) and the system triggers again the same processes to simulate the joint evolution of the two populations. The simulation results can then be displayed on the map. Domain specialists can also calibrate the simulation using the WNV surveillance data (that we have pre-processed). The user can manipulate the results and create various scenarios. Then, the simulation results can be assessed and compared.
We need to compute the evolution of the populations of Culex and the populations of crows in order to simulate their interactions using the geosimulation system. To this
Conceptual architecture of the system (based on EPAS method [32] ) Figure 11 Conceptual architecture of the system (based onEPASmethod [32] ).
end, we selected the model proposed by Wonham and colleagues [16] to compute the dynamics of the two populations. This model is based on 8 differential equations (Figure 12a ) which can compute over time the evolution of the different categories of individuals (called 'compartments'): susceptible,infected, recovered and dead birds, the larvae of mosquitoes and the susceptible, exposed and infected adult mosquitoes. We proposed some modifications in order to correct some discrepancies that we found in the model. We also included climate effects in the model using the work of Madder and colleagues [33] as a starting point. This was a difficult task because the model was no longer in equilibrium and this required several modifications to the differential equations [18] . Figure  12b presents an overview of the new equations of the proposed model. We notice that the birds equations (dS BJ /dt, dI BJ /dt, dR BJ /dt, dX BJ /dt) have an index j which represents a different bird species that we want to include in the simulation. Climate effects are computed using another set of equations that is not presented in this paper. These equations modify certain parameters in the differential equations of Figure 12b . For example v xL in the dL M /dt equation represents the rate of progress of larvae toward the state of nymphae, this rate depends on temperature conditions defined in a different set of equations. The adjusted model gives satisfactory results in terms of quality (e.g. distribution of the mosquitoes' generations). Indeed, the pace of the established curves reflects the biological behaviours of the studied species if we refer to the specialized literature. However, the quantitative results (e.g. the number of larvae, eggs, emerged Culex, dead crows, etc.) had not been conclusive with the first results of the simulation. We corrected this problem with the calibration of the system as presented in Section 2.4.
We already mentioned that our approach takes advantage of GIS data in order to properly locate the agents' roost in space. Indeed, we used the Geomedia GIS software in order to handle the geo-referenced data of the DMTI Spatial (CanMap Streetfiles) and the digital maps of INSPQ.
Using this data, we created the bitmap from which the MAGS platform generates the simulation environment. This bitmap contains polygons representing a list of 945 municipalities (out of a total of 1476 for the whole province of Quebec) being part of the ecumene (inhabited part of the studied region) of the geographical area of interest. Moreover, this bitmap is also used by the intelligent density map presented earlier (see Figure 3 in section 2.3).
In addition, we had to pre-process all the data needed to feed the system (see processes P1 to P4 of the conceptual architecture in Figure 11 , section 4.1). We estimated the initial populations of Culex and crows at the beginning of the simulation. For the population of crows, we used the SAS statistical software and the MapInfo GIS to compute a specific density of birds per region (number of individuals by square kilometer). This done by estimating an average of the sighting mentions provided by professional or amateur ornithologists (from 1997 to 2005 and located inside the ecumene) using the ÉPOQ database [24] (Table 2) . We also processed the data relative to crows' roosts (obtained through an email survey involving expert birders or extracted from the ÉPOQ database [24] ). This data included the co-ordinates (latitude/longitude) as well as an approximate number of individuals for each mentioned roost. We computed an average of the number of individuals in the case of several records of the same roost (same latitude and same longitude). This data is used to characterise the roost agents (Table 3) . Moreover, we studied the literature and discussed with domain experts in order to collect relevant information about crows' behaviours. We used this information in order to specify some behavioural rules for the roost agent such as the way that particles spread around the roost to simulate the birds' daily search for food.
Considering the Culex population, we computed the number of individuals of the initial population, estimat- ing the number of adults that emerge from the larvae laid down in sumps (which we supposed to be the main reservoirs of mosquitoes in urban and sub-urban areas). To this end, we developed a Visual Basic application in order to query the geo-referenced databases in Geomedia and to compute the total length of roads for each municipality of interest. We then computed the number of sumps in each municipality by using the total length of roads (Table 4 ). We assumed that there is an average of one sump for each 30 linear meters of road. This average distance conforms to the standards used by the Ministère des transports du Québec (MTQ) when they install sumps. However, the user of the WNV-MAGS system can modify this value, since it can vary in relation to the considered regions such as urban and rural areas. We also assumed that there are only 20% of sumps containing larvae. This default value can also be modified by the user as well as the average number of Culex adults which emerge from a sump at the beginning of a simulation. These numbers were obtained by consulting data provided by the SOPFIM Company [34] in relation to the monitoring of larval and adult Culex in certain Quebec regions during the summer of 2005.
Furthermore, we used a DLL which enables us to integrate the climate data into the system. This DLL represents one of the functionalities of the BIOSIM software [35] . We used it to interpolate values for temperature and precipitations at certain precise locations on the territory, taking into account the data of the four neighbouring weather stations and the elevation data. This computation can be done using either real data or the Canadian Climate Normals [20] which are produced over several years.
After the data preparation, we implemented the system using the MAGS platform which is developed in C++. This simulator includes several modules performing various tasks. It contains a controller to manage the threads of application (Processes Module), a user Interface and a module managing the simulation environment (Environment Module). In addition to these modules, MAGS contains a module in charge of data display, modules to specify agents' populations and agents' behaviours, and a module simulating particle systems. In order to simulate the propagation of the WNV, we added a module which simulates an epizooty (Epizootic Manager) which has been developed in a generic way in order to be easily extended to simulate epizooties different from WNV (Figure 13 ). 
The authors declare that they have no competing interests. Various components of the technical system architecture (the epizootic module was added to the pre-existing MAGS components) Figure 13 Various components of the technical system architecture (the epizootic module was added to the preexisting MAGS components). Alternative medicines for AIDS in resource-poor settings: Insights from exploratory anthropological studies in Asia and Africa The emergence of alternative medicines for AIDS in Asia and Africa was discussed at a satellite symposium and the parallel session on alternative and traditional treatments of the AIDSImpact meeting, held in Marseille, in July 2007. These medicines are heterogeneous, both in their presentation and in their geographic and cultural origin. The sessions focused on the role of these medications in selected resource poor settings in Africa and Asia now that access to anti-retroviral therapy is increasing. The aims of the sessions were to (1) identify the actors involved in the diffusion of these alternative medicines for HIV/AIDS, (2) explore uses and forms, and the way these medicines are given legitimacy, (3) reflect on underlying processes of globalisation and cultural differentiation, and (4) define priority questions for future research in this area. This article presents the insights generated at the meeting, illustrated with some findings from the case studies (Uganda, Senegal, Benin, Burkina Faso, China and Indonesia) that were presented. These case studies reveal the wide range of actors who are involved in the marketing and supply of alternative medicines. Regulatory mechanisms are weak. The efficacy claims of alternative medicines often reinforce a biomedical paradigm for HIV/AIDS, and fit with a healthy living ideology promoted by AIDS care programs and support groups. The AIDSImpact session concluded that more interdisciplinary research is needed on the experience of people living with HIV/AIDS with these alternative medicines, and on the ways in which these products interact (or not) with anti-retroviral therapy at pharmacological as well as psychosocial levels. A large number of new treatments offered to people living with HIV/AIDS (PLWA) have appeared over the last fifteen years in the therapeutic domain of AIDS. These med-icines are particularly heterogeneous, both in their presentation and in their geographic and cultural origin. They constitute a group of products with a therapeutic aim that occupies a space between the customary traditional, popular and biomedical sectors of health care [1] . These products often mix reference to biomedicine and science with notions of traditional health culture and nature in a syncretic way. They consist mainly of herbs and nutritional substances and are packaged as 'modern' pharmaceuticals: capsules, tablets, and solutions. The names of these alternative treatments reflect their reference to biomedicine: Immunocomplex, Viralgic, Virjint, etc. Their accompanying leaflets provide detailed information on substance, as well as dosage, indications, and biomedical efficacy claims. Their diffusion follows contemporary paths in the global economy and makes use of new information technologies. In this paper, we will use the term "alternative" to consider a generic category including medicines that recently appeared for AIDS which have not been authorised by drug regulatory authorities, nor recommended by WHO. Other terms, such as neo-traditional or neo-phytotherapeutic, may be discussed for the characterization of some of these treatments, related to their local meanings or their social status.
The emergence of alternative medicines for AIDS in Asia and Africa was discussed at a satellite symposium and the parallel session on alternative and traditional treatments of the AIDSImpact meeting, held in Marseille, in July 2007. We were especially interested in the role of these medications since the introduction and rapid scale-up of highly active anti-retroviral therapy (HAART) in resource poor settings.
Twenty anthropologists and health researchers attended the satellite session and presented exploratory findings from Asia and Africa (Uganda, Senegal, Benin, Burkina Faso, China and Indonesia). The aims of the satellite, the results of which were presented at the parallel session [2] , were to (1) identify the actors involved in the diffusion of these alternative medicines for HIV/AIDS, (2) explore uses and forms of these medicines, and the way they are given legitimacy, (3) reflect on underlying processes of globalisation and cultural differentiation, and (4) define priority questions for future research in this area. We present here the insights generated at the meeting, illustrated with some findings from the studies that were discussed.
There has been an increased professionalisation and commercialisation of traditional medicine in response to the development of biomedicine. This trend is not specific to AIDS and not necessarily a recent development. Social scientists first noted this trend in the late 1980s: Charles Leslie [3] for example has shown how, in India, in response to an increased authority of biomedicine and the globalisation of health markets, Unani and Ayurvedic medicine production changed; and Afdhal and Welsch [4] described the rise of 'modern' jamu in Indonesia. Jamu is the traditional term for Indonesian indigenous medicines usually prepared from herbal medicines such as leaves, bark, roots and flowers. Nowadays a multimillion dollar industry is involved in the production of Ayurvedic and Unani medicines in India, and of jamu in Indonesia. A rapidly expanding assortment of powders, creams, pills, capsules and cosmetics has been manufactured both in small cottage industries as in large factories with increasingly sophisticated technologies [3, 4] . The modernization of the manufacturing of these drugs has been accompanied with more modern biomedical modes of presenting their efficacy [5] . Under globalization, similar trends occurred in other regions and these products diffused more rapidly.
At the seminars in Marseille, we discussed the ways in which such alternative remedies operate in the therapeutic domain of AIDS care. In the first decade of the AIDS epidemic there was no effective treatment for HIV/AIDS and patients were faced with nearly certain premature death. At that time, there were regular hypes offering hope for life. But with the introduction of ART, alternative treatments are now marketed for many additional purposes too: to prevent AIDS, to kill viruses, to delay the need for ART, to restore and enhance health while on ART, to treat opportunistic infections, and to alleviate adverse side effects of other treatments. Biomedical practitioners generally discourage the use of alternative medicines, fearing interactions with ART and also through the concern that patients may stop using ART.
At the AIDSImpact sessions Egrot and colleagues [6] presented findings on the supply of what they label "neo-traditional medicines" to refer to the boundary-crossing nature of these treatments in West Africa. The "designers" of the inventoried products are extremely heterogeneous. In some cases these people are nationals of African countries who present themselves as healers. Some say they have undertaken "research" on the basis of therapeutic products that were already known locally. Others refer to a dream revelation (classic in the universe of healers in Africa) of a plant composition that is "efficacious" against AIDS, while yet others speak of a divine revelation. Physicians, scientists and academics are solicitated, brought into involvement or spontaneously engage themselves in the exploitation of neo-traditional products. The case studies in West Africa show that other treatments, such as Immunicomplex or Aloe Vera, originate in Europe and the USA. Alternative medicines from Europe and the USA occupy the same shelves in ordinary pharmacies as those originating from Africa and China, often along with a few 'immune-boosting' food products (honey, olive oil). Specialized "bio", "natural health" and "health food" shops make these products available to the more affluent. The distributors and marketing men of these products also target health workers and clinics directly. The West Africa case studies noted that health workers also have started to prescribe alternative products such as Immuboost (NHi2T) or Viralgic (Pharma Concept) (see Figure 1) .
A case study from Uganda showed how health workers operating an anti-retroviral treatment program adopted a locally available traditional ointment as an alternative medication for skins problems of people living with HIV and AIDS. The skin problems result from adverse effects of ART or symptoms of opportunistic infections. The health workers obtained the recipe from local traditional healers (patients had told them that the cream works well), and the patients help collect the ingredients. They 'repackage' this traditional remedy into what is now called 'mobile cream' (to make clear it is produced by the so called 'Mobile' ART program). One of the nurses reports:
"The mobile cream, which we ourselves prepare either at our chief nurse's home or here at the office depending on how busy we are at the office, is very efficacious for many kinds of skin related conditions. We are quick to prescribe it to the patients because we know it works and it is popular among patients too because it works for them [7] ." Content analysis of drug information leaflets, advertisements, product catalogues, and brochures distributed by medical representatives in the West Africa case studies [6, 8] casts light on the range of effects that are attributed to these drugs. Most commonly cited (biomedical) properties are immune-stimulation and antioxidant. Some manufacturers suggest that the products have antiviral properties as well. The antiviral dimension refers either to the opportunistic infections such as herpes (mentioned for example in the product information for Immuboost) or eventually to the immunodeficiency syndrome itself.
Indeed, some products boldly claim anti-HIV activity as well, and are marketed as natural ART (see Figure 2 ). However, such efficacy claims are not static. The producer of Virusinest (Nesto-Pharma) recently withdrew the antiviral claim, stating in its information leaflet: "the analyses carried out among patients do not allow the anti-HIV assertion to be upheld". There may be also inconsistencies between various information sources. The brochure for Viralgic (Pharma Concept) says that this is a product which renders the virus undetectable, but the website of the manufacturer presents the drug as immunostimulant (result of trials published on the web site), and present the product as treatment for opportunistic infections: "antiherpes...for healthy persons".
A case study on Indonesia [9] dealt with the demand for alternative medicines among PLWA. As Afdhal and Welsch noted two decades ago, Indonesia has a thriving market for jamu. Jamu are sold for a wide range of indications: common colds, influenza, headaches, aches and pains, high blood pressure, beauty, improvements in sexual performance, and recently to treat and prevent HIVrelated health problems. AIDS prevalence is below 1% (i.e. this is a low prevalence area), but the disease is stigmatised, because of its association with intravenous drug use and prostitution. Hardon and her colleagues conducted interviews with women and men who live with HIV and use anti-retroviral therapy, mainly intravenous drug users and their partners. All of them had better health since taking these modern drugs. Nonetheless, all of them see the need to take jamu as well. They do so in Tobacoak's, West Africa part out of their intention to live positively (i.e. eating and sleeping well, and keeping a positive outlook on life), as promoted by many of the support groups in which PLWA participate. The respondents do not make distinctions between modern medicines and jamu in these health maintenance and restoring practices. Rather they distinguish the drugs by their effects. They use popular jamu to treat side effects of HAART, such as itchiness. These jamus are not specifically promoted for HIV and AIDS in Indonesia, perhaps partly because the disease is so stigmatised.
However one neo-traditional preparation stood out in the narratives of our respondents as a product which can treat HIV/AIDS: virgin coconut oil. Ceri, for example, started using coconut oil shortly after she found out she was HIVpositive. She says:
Actually, the effect is not only for your immune system. So, I feel better, don't feel tired, and have more energy. I think what influences most is self-suggestion. It's self-suggestion that matters...
Mia (a 28 year old woman from Jakarta) was given virgin coconut oil by a friend from Yogjakarta: I got 70 boxes. A box contains 60 capsules. It took it every day until I felt sick, but there was no effect. My CD4 level did not increase. Three months, three months made me look like a coconut you just needed to squeeze (laughing). I became very oily. The good effect when you take VCO is that your skin is silk smooth, your face is fairer and if you take a shower, you don't need any lotion, because your skin is naturally oily. That is the positive effect. Your hair is also stronger.
But Buli, a 29-year-old ex-drug user from Karawang, one of the most active members of the support group in Karawang says:
In Indonesia, the drug sellers were not very willing to discuss the effects of VCO. They would acknowledge that indeed these drugs are used by PLWA, or they would deny knowing anything about the drugs. But their pharmacies are full of advertisements for the products and they have prominent positions on their shelves (see Figure 3 ).
Content analysis of the package information for VCO in Indonesia revealed that they are marketed as real 'curealls', i.e. to kill viruses and bacteria and/or strengthen the immune system, efficacy claims that we also found in West Africa. For example the package leaflet for Vicofit (manufactured by Sumber Dinamis in Bogor) states that the drug has "a high content of lauric acid which has antivirus, anti-bacterials and anti-protozoa properties." And that it is "believed to help improve the health condition of those with cholesterol, diabetes, coronary heart disease, hepatitis C, HIV positive, cancer, prostate, uric acid, osteoporosis, influenza and weight problems". The package for Virjint (produced by PT Vermindo International) states that the medicine is safe for daily use and without side effects. It lists two dosages: one for prevention (2 × 2 capsules per day) and another for treatment (2 × 3 capsules per day). The leaflet stipulates that the indications are:
-"to increase energy and body stamina -to increase body resistance (Meningkatkan daya tahan tubuh) against bacterial, viral and fungal pathogens -to reduce weight -anti-oxidant, anticancer, and anti-HIV -to overcome uric acid, hypertension, stroke, heart disease, atherosclerosis, osteoporosis, influenza, hepatitis, chickenpox, herpes, TB, diabetes, epilepsy, eczema, liver, haemorrhoids, kidney, peradangan (burning sensation), infection, degenerative disease."
The packages cite clinical research conducted elsewhere (Philippines, USA) to give legitimacy to the products. For example the leaflet of Holistic Virgin Coconut Oil states: "Based on research conducted in the Philippines, Holistic Virgin Coconut Oil is very effective to fight against SARS and AIDS". 
One of the key characteristics of alternative medicines in Asia and Africa is that they move from one cultural and geographic space to another, apparently without being constrained by trade-barriers, or regulatory mechanisms. Some governments promote the production and diffusion of neo-traditional medicines. They do so for economic reasons: alternative medicines are big business, but they also do so for ideological reasons: neo-traditional medicines reflect an attractive hybrid of modernity and national heritage, providing a sense of national identity in the globalized health economy [10] . The governments of India, China, Indonesia, and some African countries support research programs to further advance these neo-traditional products, and facilitate market diffusion. While registered pharmaceuticals are regulated heavily upon market entry (proof of efficacy is assessed by national drug regulatory authorities), this is not the case for alternative medicines. ART programs, which are sponsored by the same governments, usually discourage the use of alternative medicines, fearing the toxicity of the drugs, or that these medicines will interact with anti-retroviral medication and lead to discontinuation of ART therapy [11] . Governmental agencies may have contradictory attitudes towards the use of alternative medicines for AIDS, discouraging it within ART programs and supporting it within divisions of traditional medicine. An exception is the Chinese government, which officially supports a complementary medicine program for AIDS care and research [12] .
Mass-produced alternative medicines meet an increasing demand for health products, a trend which has been labelled the "commodification of health" [13, 14] : from the slums of Djakarta to rural settings in Burkina Faso, people believe more and more that they need pharmaceutical 'things' to protect their health and to treat illness symptoms. People living with HIV and AIDS are particularly uncertain about their health and their future: ART may be accessible and improve health now, but they wonder if this will be the case in the future. This uncertainty makes them an attractive market for the 'best of both worlds', alternative medicines, which come with assertions of 'natural' safety and 'biomedical' efficacy [15] . However the case studies presented in Marseille suggest that people especially want to use alternative medicines to delay onset of ART, treat opportunistic infections, restore health and alleviate adverse effects once on ART. Immuneboosters are popular, though our case studies suggest that PLWA are often ambivalent about alternative medicines that claim anti-HIV efficacy.
The case studies make clear that the market of alternative medicines for HIV/AIDS is dynamic. It adapts to progress in biomedicine, which has produced potent anti-retrovi-ral medications. In some cases, the efficacy claims for alternative medicines reinforce a biomedical paradigm for HIV/AIDS, and fit with a healthy living ideology promoted by AIDS care programs and support groups. More interdisciplinary research is needed on the experience of people living with HIV/AIDS with these alternative medicines, the ways in which the products and their representations move from one cultural setting to another, and on the ways in which these products interact (or not) with anti-retroviral therapy at pharmacological as well as psychosocial levels. More research is also needed to assess the economic impact of these therapies, since people seem to be spending much on these 'other' medicines while ART is provided for free. A blanket denial of the relevance of these products for the quality of life of PLWA does not make sense for patients, who need precise information that make clear which products are likely to have negative interactions with ART, and which ones could be beneficial. Unfortunately research on the interactions between alternative medicine and antiretroviral drugs is sparse [11] . To be able to inform patients better, more clinical research is needed on the benefits and risks of those alternative medicines that are perceived to be beneficial by people living with HIV and AIDS. Evolutionary and Transmission Dynamics of Reassortant H5N1 Influenza Virus in Indonesia H5N1 highly pathogenic avian influenza (HPAI) viruses have seriously affected the Asian poultry industry since their recurrence in 2003. The viruses pose a threat of emergence of a global pandemic influenza through point mutation or reassortment leading to a strain that can effectively transmit among humans. In this study, we present phylogenetic evidences for the interlineage reassortment among H5N1 HPAI viruses isolated from humans, cats, and birds in Indonesia, and identify the potential genetic parents of the reassorted genome segments. Parsimony analyses of viral phylogeography suggest that the reassortant viruses may have originated from greater Jakarta and surroundings, and subsequently spread to other regions in the West Java province. In addition, Bayesian methods were used to elucidate the genetic diversity dynamics of the reassortant strain and one of its genetic parents, which revealed a more rapid initial growth of genetic diversity in the reassortant viruses relative to their genetic parent. These results demonstrate that interlineage exchange of genetic information may play a pivotal role in determining viral genetic diversity in a focal population. Moreover, our study also revealed significantly stronger diversifying selection on the M1 and PB2 genes in the lineages preceding and subsequent to the emergence of the reassortant viruses, respectively. We discuss how the corresponding mutations might drive the adaptation and onward transmission of the newly formed reassortant viruses. The H5N1 highly pathogenic avian influenza (HPAI) virus was originally isolated from a farmed goose in Guangdong province of China in 1996 [1] , and soon spread to live-poultry markets in Hong Kong [2] , resulting in 18 cases of human infection in 1997, 6 of which were fatal [3, 4] . The first wave of H5N1 infection ceased after the depopulation of all poultry in Hong Kong, although the H5N1 virus was later found to circulate continuously in Southern China without causing apparent disease symptoms among infected poultry [5] . H5N1 outbreaks recurred in 2003, persistently affecting poultry farms in many Southeast Asia countries, such as China, Thailand, Vietnam, Indonesia and Cambodia. The viruses also spread outside Asia, including to some European countries. More importantly, occasional zoonotic transmissions to humans occurred in most of the affected Asian countries and the virus continued to pose a serious threat to global public health [6] .
H5N1 outbreaks in Indonesia were initially detected in poultry farms in December 2003 [7] . It was suggested that the H5N1 virus was first introduced to Java and subsequently spread to other parts of the country [8] . The virus rapidly became endemic in Indonesia [9, 10] , and continued to cause sporadic zoonotic transmissions to humans beginning in July 2005 [9] . Three clusters of H5N1 transmission among family members were identified in 2005, raising concerns of possible human-to-human transmission of the virus [11, 12] . As of April 8, 2008 , Indonesia had 132 confirmed human cases with 107 deaths [13] , the largest number of deaths among all affected countries.
Previous studies have shown that several H5N1 genotypes have emerged in Asia through reassortment between H5N1 viruses and other subtypes [14, 15] . One of these genotypes, Z, predominated the H5N1 outbreaks throughout 2003-2007, causing most H5N1 outbreaks in Asian countries, including Indonesia [16] . Moreover, a variety of antigenically distinct sublineages of Z genotype virus have been established [16] . Unlike Vietnam and Thailand, Indonesia was invaded by only a single sublineage of genotype Z virus. Previous phylogenetic analyses suggested that Hunan province of China may be the source of the initial H5N1 outbreak in Indonesia [17] , and classified the Indonesian H5N1 HPAI viruses into three groups [10] ; however, further statistical analysis is necessary to characterize and compare different aspects of their evolutionary histories. In this study, we examined molecular phylogeny of the most recent Indonesian H5N1 viruses isolated from avian and mammal hosts. A group of putative reassortant viruses was discovered and their genetic parents were identified. In addition, we investigated the evolutionary behaviors (including spatial migration, growth of genetic diversity, and evolutionary drift and selection) of the reassortant viruses and compare with those of the parental strain, thereby providing insights into the nature and impact of this emerging reassortant strain.
Phylogenetic trees of Indonesian H5N1 viruses were reconstructed from 12 separate gene datasets (Table S6) , using a maximum likelihood (ML) approach with bootstrapping analyses to assess clade robustness (Figures 1, S1-S3; computer files of dendrogram are available as Dataset S1). In all the phylogenies, viruses sampled from avian species during earlier years of outbreaks (predominantly 2003-2004) tended to cluster near the root as expected, but with a poorly resolved branching structure that is likely due to relatively low sequence divergence. In contrast, viruses sampled from recent infections (2005) (2006) (2007) from avian and mammalian hosts formed three well-supported lineages with bootstrap support (or posterior probabilities) over 90 (or 0.9) under neighbor-joining (NJ), ML and Bayesian Markov Chain Monte Carlo (BMCMC) methods. We denote these lineages as groups 1, 2, and 3 in the hemagglutinin (HA) and neuraminidase (NA) phylogenies ( Figures 1A and S2C ). This structure was preserved in the phylogenies of other genes for which sufficient sequence data were available (viruses from group 3 were missing sequence data for the NP, NS, NS1, NS2, and PB2 genes). The group 3 lineage in the MP, M1, M2, and PB1 phylogenies was only represented by the A/Indonesia/6/05 strain.
It is important to recognize that our phylogenetic groupings (groups 1, 2, and 3) of Indonesian H5N1 viruses ( Figure 1 and Table S6 ) are slightly different to those by Smith and coworkers [10] who did not require the same level of clustering support for each group, leading to the inclusion of earlier viruses (predominantly 2003-2004) . We chose to be conservative, and did not include poorly supported branches (e.g., earlier viruses) in our viral group definition. Therefore, we did not define a group corresponding to the group B of Smith et al., because most of group B taxa are earlier viruses. Groups 1 and 2 in this study correspond to groups C and A defined by Smith et al. respectively, plus some more recent viruses. Smith did not report group 3, because the sequences were unavailable at that time.
We found previously unrecognized phylogenetic discordance between gene trees involving human and cat isolates (n = 25, denoted in red in Figures 1, S1-S3)-the main focus of our study-suggesting that they are reassortant viruses descending from group 2 and 3 lineages. In addition, the placement of two avian viruses isolates from Java (Ck/IDN/Semerang1631-62/07 and Ck/IDN/Magelang1631-57/07, shown in blue in Figures 1A  and S2C ) differed between HA and NA phylogenies, suggesting another reassortment event.
To further investigate the putative reassortant human and cat viruses, a selected dataset (n = 24) of manually concatenated full genomes (Figure 2A ; see Methods) of Indonesian H5N1 HPAI viruses were analyzed using more sophisticated analysis methods, including similarity plots, bootscan analyses and GARD analyses (genetic algorithm for recombination detection). In the similarity and bootscan plots ( Figure 2B and 2C), the putative reassortants (represented by a consensus sequence) showed a high degree of sequence similarity and phylogenetic clustering with the group 3 strain A/Indonesia/6/05 in the MP and PB1 segments, but not in other genomic regions, where they were more similar to the consensus sequence of group 2 viruses ( Figure 2B and 2C). Moreover, GARD detected two well-supported breakpoints near the boundaries of MP and PB1 segments in the concatenated genomes ( Figure 2D ), suggesting that the phylogenetic incongruence was significant between the three regions. In summary, all three analyses agreed that the newly reassortant strains had arisen from acquiring PB1 and MP genome segments from the group 3 lineage and the remaining segments from the group 2 lineage.
Based on the HA phylogeny ( Figure 1A ), we further classified the reassortant viruses into three subgroups (R1, R2, and R3) with bootstrap support of 80% or better, as shown in the phylogenies containing only reassortant viruses ( Figure S4 ). Similar groupings were observed in the NA phylogeny ( Figures S2C and S4B ), although here subgroup R3 clustered with subgroup R1, and two reassortant viruses isolated in 2007 (IDN/CDC1046/07, IDN/ CDC1047/07) moved to a different subgroup. These inferred clustering patterns can be explained by multiple reassortment events, or by a single reassortment followed by divergence due to mutation and selection in different populations. We note that some group 2 viruses also cluster inside the reassortant subgroups ( Figures 1A and  S2C ) and may indicate more reassortment events; however, most of them formed polytomies close to the most recent common ancestor (MRCA) of the reassortant subgroups and had poor bootstrap support for their exact placement. As the divergence between the reassortant subgroups and other intercalating group 2 viruses are low, the three subgroups may actually be linked uninterruptedly, implying a single origin. Therefore, the times and number of reassortment events that generated the putative mosaic reassortant viruses remains elusive. We examine both the single and multiple origin hypotheses in subsequent analyses, excluding the intercalating group 2 viruses from the reassortant group.
To estimate the times of the reassortment events that generated the putative reassortant viruses, the times of the MRCA (tMRCA) of the three reassortant subgroups were estimated using BMCMC methods [18, 19] . Bayes Factors (BF) [20] were used to select Author Summary H5N1 highly pathogenic avian influenza (HPAI) virus emerged in China in 1996, and has spread beyond Asia since 2003. Following the first outbreak reported in Indonesian poultry farms in December 2003, the virus spilled over to 27 Indonesian provinces by June 2006, and became endemic in the country. In the following years, repeated sporadic human infections in Indonesia had been attributed to H5N1 HPAI viruses. Nonetheless, the viral evolution and transmission have not been fully understood. Here, we report phylogenetic evidence of a group of interlineage reassortant viruses isolated from human and cats in Java. Our comparative study of the reassortant viruses and one group of genetic parents found that although their rates of evolution were similar and both of their phylogenies were not geographically structured within mainland Java, the growths of genetic diversity were different. We also detected significant positive selection on the viral matrix and polymerase genes preceding and subsequent to the emergence of the reassortant viruses, which might correspond to viral adaptation. Based on our findings, we discuss the possibility of host switching in facilitating the emergence of the reassortant strain, and call for more extensive viral surveillances in the non-avian population in Indonesia. among strict and relaxed clock models of evolution [21] . The uncorrelated exponentially-distributed clock model (UCED) significantly outperformed the other models (lnBF.3) for most datasets, except for the NA gene of the reassortant viruses, for which the strict clock model was not rejected (lnBF,1; Table S4 ).
The results of tMRCA estimation are summarized in Figure 3C and 3D. In addition, sequence isolation dates were plotted against their genetic distance (units of substitutions/site) to their MRCA, to graphically show the accumulation of mutations through time ( Figure 3A and 3B). The tMRCA of all reassortant viruses (All- were isolated from central and east Jakarta (Table 1) . Parsimony reconstruction (see Methods) of binary ancestral geographical states (either Greater Jakarta or West Java) upon the HA and NA ML phylogenies suggested that the MRCA of all reassortants (and the MRCAs of each reassortant subgroup) likely originated from Greater Jakarta and surroundings (Table S2 ; result robust to random resolution of polytomies; see Methods).
The mean numbers of observed geographical state changes (GSC) of the reassortant and of the group 2 parental strains were estimated independently and compared with the null distribution of GSC values under the null hypothesis of completely unrestricted migration (i.e. panmixis; Figure S5 ) [22] . For the reassortant strain, the observed GSC value was not significantly lower than the GSC value expected under panmixis (Slatkin-Maddison test: p.0.2). Therefore the observed geographic structure is not significantly different to that expected by chance alone. For group 2 viruses, the observed GSC value for all geographical state pairs is significantly (p,0.0002) lower than the null value. However, the observed value of GSC within Java (i.e., migrations between Greater Jakarta and the rest of Java) and between the three islands (i.e. migrations between mainland Java, Sumatera and Sulawesi Selatan including Papua) are insignificantly (p.0.2) and significantly (p,0.0002) lower than the corresponding null values respectively, suggesting that the phylogeny of group 2 viruses is not geographically structured within Java, but is subdivided by island-to-island migrations. However, we could not address whether the viral migrations inside Sumatera and Sulawesi Selatan including Papua are panmictic or structured due to limited operative localities in our dataset to distinguish between different regions inside these islands. We also found that the migration of group 2 viruses from Greater Jakarta and surroundings to Sumatera and Sulawesi Selatan including Papua was more frequent than expected under the null hypothesis, and there is relatively little viral migration from the rest of Java to Sumatera and Sulawesi Selatan including Papua (Table S3 ). This observation suggests Greater Jakarta played a more salient role in dispersing group 2 viruses to other Indonesian islands than other parts of Java did. There is another family member (brother) who was confirmed with H5N1 infection (index #45, fatal); however, virus sequences are not available. f Numbers in parentheses are the unique references to the localities shown in Figure 4 . Numbers 1-6 were assigned to Greater Jakarta and surroundings; numbers 7-12 were assigned to West Java. n/a = Information not available. doi:10.1371/journal.ppat.1000130.t001
We used the Bayesian skyline plot (BSP) [23] to estimate the change of relative genetic diversity of the reassortant viruses and of the group 2 parental strain over time, as shown in Figure 3E -3H. For both the HA and NA datasets, the group 2 viruses consistently show a slow growth in relative genetic diversity over time which appears to follow a constant size or exponential growth model, whereas the reassortant viruses initially exhibited an abrupt rise in relative genetic diversity followed by stabilization, which visually resembles a logistic growth curve with two phases [24, 25] ( Figure 3E and 3F) . When the BSPs are superimposed upon the demographic results obtained under parametric growth models (i.e., constant, exponential and logistic growth; Figure S8 ), then a similar observation can also be made. However, BF tests (Table  S4) indicate there is insufficient statistical power to discriminate between the three parametric growth models (lnBF,2.99), suggesting a lack of strong demographic signal in these data. When the parametric demographic models were fitted to the data, the median estimates of growth rates for the reassortant datasets are generally higher than those estimated for the datasets of group 2 viruses (Table S1 ). However, the confidence intervals of some growth rate estimates are fairly large and overlapped among the reassortant and group 2 viral datasets.
Diversifying selection in the PB2 and M1 genes Using the Random Effects Likelihood (REL) method [26] we found sites under positive selection in the PB2 gene (codons 76, 534, 627, 677 and 740) and the PA gene (codon 409) of the reassortant viruses. The Fixed Effects Likelihood (FEL) method [26] was more conservative and only identified PB2 codon 534 as being positively selected. For the group 2 viruses, HA codon 129 (starting from HA1) and M1 gene codon 205 were the only selected sites identified by the FEL and REL methods, respectively. Using a lineage-specific selection model (see Methods), we identified elevated rates of diversifying selection, measured by the ratio of non-synonymous to synonymous substitutions (dN/dS), on the M1 gene in the lineage leading to the MRCA of the group 3 viruses and preceding the emergence of the reassortant viruses (highlighted in Figure 1B ). The dN/dS values for the M1 gene in this lineage (which we call the preemergence lineage) was estimated to be 1.514 (95% CI: 0.447-3.814; see Table S5 ), significantly higher (LRT p,0.002) than the mean estimates for other lineages (dN/dS = 0.077) in the Indonesian clade and for lineages in other H5N1 HPAI clades (e.g., Fujian, Qinghai, Thailand and Vietnam clades which have dN/dS ranging from 0.05 to 0.09). This lineage-specific elevation of dN/dS was not significant (LRT p.0.1) for other genes (i.e. HA, NA, M2, PB1; see Table S5 ). Four amino acid changes in M1 occurred along the pre-emergence lineage, including threonine to alanine at reside 37, arginine to lysine at reside 95, threonine to alanine at reside 137, glutamine to histidine at reside 249. Three (residue 37, 95, and 137) of them are located close to the electrostatic positive surface of the N-terminal domain of the M1 protein molecule ( Figure S7 ), and one (residue 249) is located in the remaining C-terminal fragment.
This study classified H5N1 HPAI viruses in Indonesia into three distinct viral lineages (groups 1, 2, and 3) and discovered a group of naturally occurring reassortant viruses that represent a newly emergent H5N1 HPAI strain in Java in 2006. Several phylogenetic methods concurred that two (MP and PB1) of the reassortant viruses' genome segments descended from the group 3 ancestral viruses, and the remaining six (PB2, PA, HA, NP, NA, NS) segments descended from the group 2 ancestral viruses. Although the majority of reassortant viruses (24/25) are human isolates, few of the associated human infections are epidemiologically linked (Table 1) , suggesting multiple sporadic zoonotic transmissions from birds. The phylogeographic results indicate that the parental viruses of the reassortants have been co-circulating in Java since 2005. Despite the identification of parental lineages, the exact number of reassortment events remains difficult to assess. Although the three fairly consistent phylogenetic subgroups (subgroups R1, R2, and R3 in Figure S4 ) formed by the reassortant viruses suggest three independent reassortments, the underlying uncertainty in our estimated phylogenies means that we cannot rule out the possibility of a single origin.
The hypothesis of three reassortments implies that the viruses have acquired exactly the same genome segments from the same group of parental viruses, which seems unlikely to occur by chance (probability = 0.0089, assuming panmixis and that exactly two genomic segments are swapped out). This probability might be increased if reassortments confer a selective advantage. We did detect a significantly stronger selection pressure on the M1 protein in the pre-emergence lineage of group 3 parental strain that led to the reassortant viruses (Table S5) . Previous reports suggested a few amino acid changes in M1 of influenza A and B viruses can confer a growth advantage in mouse lungs [27] [28] [29] . Although the M1 mutations identified in this pre-emergence lineage have not been functional characterized elsewhere in the authors' knowledge, one (residue 137, TRA) of them is close to a previously characterized mutation (residue 139, TRA) which controls the virulence in mouse model [27, 29] . Three of the inferred residue changes are located close to the electropositive surface of N-terminal domain of M1 protein ( Figure S7 ) that acts to bind viral RNA [30, 31] . The M1 matrix protein mediates encapsulation of viral RNAnucleoproteins into membrane envelope during packaging [31] , and has close contact with other viral proteins inside the viral particle. It seems possible that some of these changes may be involved in the adaptation of reassortant viruses, through promotion of structural interactions among viral proteins.
According to our analyses, the common ancestor of the reassortant viruses is dated to July 2005 (HPD: April-October), approximately 5 months prior to the first case of human infection caused by the reassortant virus (index case #15 defined by WHO; see Table 1 ). Our analysis of virus phylogeography suggests the ancestors of these reassortant viruses first arose in Greater Jakarta and surroundings, which agrees with the observation that the first two cases of human infection by the reassortant viruses occurred in Central and East Jakarta (index cases #15 and #16). The molecular dating and phylogenetic analyses suggest that nascent reassortant viruses might take several months to spread and expand their diversity in the local bird population, eventually leading to the exposure of human population. The subsequent spread of the reassortant strain seems to become more rapid and extensive, as human cases were reported outside Greater Jakarta one month later, and the reassortant virus spread to as far as the south and east of West Java in the following six months (Table 1 and Figure 4) . Commercial poultry transportation, as well as carriage by migratory birds, may facilitate the viral migration, but their tangible contributions need further studies. Our results suggest that the circulations of reassortant viruses and their genetic parent (group 2) were not restricted by geography within Java. The viral migration back to Greater Jakarta could be driven by the inter-province transfer of infected poultry, in particular the importation of live poultry or fresh poultry products to the densely human populated Jakarta from the remote provinces engaged in poultry-farming. Future studies on economic and social geography (e.g., addressing the modes of inter-provincial poultry transport) in Indonesia might help to further elucidate the effect on the viral dispersal by human, agricultural and industrial activities. In this study, we opted for a lower geographical resolution (i.e., four widely ranged geographical states instead of distinct geographical coordinate for each viral isolate) in our phylogeographic analyses because of the varying precision of the geographical data we have. Therefore, more complex hypotheses of viral origin and migration trajectory cannot be investigated here, but can be explored when more high-quality geographical data of Indonesian H5N1 viral samples is available.
The BSP analyses ( Figure 3E-3H) indicate that the reassortant viruses follow a logistic-like growth curve, which is typical for virus invasion and maintenance, especially in a structured population [24, 25] . In contrast, the group 2 viruses followed a more continuous and relatively slow growth in diversity. There was insufficient data in our samples to definitively discriminate between alternative population growth models and provide narrow confidence intervals for parameter estimates, but our results are suggestive and future sequencing will add to the needed statistical power.
What factors have contributed to the apparent difference in the growth of genetic diversity? Rates of molecular evolution between the two groups were similar ( Figure S6 ) and therefore are not likely to be the cause. Since our analyses could not resolve the temporal dynamics of population subdivision by geography, we cannot directly investigate how viral genetic variation is affected by the population structure. We expect future development of analysis methods will help to shed more light on the interaction between viral migration and genetic diversity.
Analysis of clinical records (Table 1) found that the mean duration from onset to death in those fatal human cases caused by Indonesian reassortant H5N1 viruses is 9.1 days (standard deviation [SD] = 3.9; n = 23) and those caused by other Indonesian H5N1 viruses is 7.7 days (SD = 2.7; n = 10), and their means are not significantly different (student t-test, p.0.25, two-tails). Therefore, based on the clinical records, the reassortant viruses did not kill human faster than other Indonesian H5N1 viruses did. However, we would recommend more experimental studies addressing the virulence, pathogenicity and immunogenicity of the reassortant viruses and the parent strains to verify this claim in the future.
Mechanisms of viral transmissions are sometimes correlated with genetic diversity dynamics. For example, hepatitis C viruses transmitted by drug injection or blood products have a faster rate of spread than endemic strains circulating in Asia and Africa [32] . It has also been suggested that mosquito susceptibility may affect the growth of dengue viruses [33] . Therefore, it is possible that a change of host species could generate the difference in the viral dynamics we observe. In our study, the majority of the reassortant viruses (24/25) were isolated from humans, whereas only a minority of the group 2 viruses were isolated from humans (10/57 and 10/41 in the HA and NA datasets, respectively).
It has been previously shown that the receptor binding specificity of hemagglutinin [34] and mutations in the viral polymerase (e.g., lysine at residue 627 of PB2) [35] [36] [37] can determine viral transmissibility and replication in different host species. None of the aforementioned HA mutations which confer recognition to human-type host cell receptors [34, 38] were found in the Indonesian reassortant viruses; however, our detection of positively selected sites in the PB2 gene of the reassortant viruses could potentially reflect adaptation to mammalian hosts, and requires further investigation. In particular, amino acid changes on two positively selected sites (threonine to methionine at reside 76, glutamic acid to glycine and alanine at reside 677) were found on the internal branches of the reassortant lineage, corresponding to molecular changes during sustainable transmissions. However, some of these positively selected changes may also result from the compensatory evolution as the mix of genome segments from different strains might alter their epistatic physiochemistry [39] . Although most of the human isolates in our datasets were epidemiologically unlinked, such linkage is theoretically possible if many asymptotic or mildly manifested human infections are not reported. Recently, some evidence of subclinical or asymptotic H5N1 infection in humans has been put forward [40, 41] ; however, the ability of the viruses to transmit from these infected individuals to other susceptible individuals remains unknown.
The possible role of other animal host species in the transmissions of reassortant viruses in Indonesia should not be neglected. In particular, one of the reassortant viruses was isolated from a dead cat in Jakarta, where H5N1 outbreaks in poultry and sporadic human infections have been reported [42] . Moreover, unusual high mortality of cats in the vicinity of H5N1 HPAI outbreaks has been reported [43] . An unofficial report also detected H5N1 HPAI sero-positivity in around 20% of 500 blood samples taken from stray cats near poultry markets in Java and Sumatera [44] . In addition to small cats in Germany [45] , Iran [46] , and Indonesia [42] , dogs and zoo tigers were also found infected with H5N1 HPAI viruses in Thailand [47, 48] . Furthermore, previous experimental studies have demonstrated that cats can be infected with H5N1 HPAI virus [49, 50] , and that cat-to-cat transmission is possible [49, 51] . Could cats, or other non-avian species, have played a role in spreading the reassortant viruses in Java? Similarly, could cats act as amplifying hosts facilitating viral expansion and cross-species transmission, as civets did in the SARS outbreaks [52] ? Future experimental studies on these reassortant viruses, that assess viral transmissibility between species, together with epidemiological studies, such as viral monitoring within Indonesian animal populations using serological tests and PCR detection, would give more clues to these questions. H5N1 HPAI viruses have been endemic and evolved into different genetic lineages that have spread across Indonesia. Areas where more than one lineage of virus is co-circulating, such as Jakarta, are most likely to generate novel viruses by inter-lineage reassortment. These reassortant viruses have distinctive evolutionary and transmission dynamics, as shown in this study. We suggest that more intensive and timely field surveillance and analysis of influenza viruses, including H5N1 HPAI and human H3N2, H1N1, and H1N2 epidemic strains, should be employed, so that bio-security can be undertaken promptly and appropriate strains can be selected for vaccine production whenever a novel reassortant strain emerges. The reassortant viruses reported in this study should be also added to the watch list for the future epidemiological surveillance.
Sequence data collection and alignment H5N1 influenza viruses isolated from avian and mammalian hosts in Indonesia during 2003-2007 were studied. Their genomic sequences (n = 807) were extracted from the Influenza Virus Resource [53] and the Influenza Sequence Database [54] in September 2007, and aligned using MUSCLE version 3.6 [55] . Columns with gaps were removed from the alignments, and sequences from the same virus strain (duplicated submission in the two databases) were filtered such that one copy was retained. Eight genome segment alignment datasets (PB2, PB1, PA, HA, NP, NA, MP, and NS), as well as four coding sequences (M1, M2, NS1, and NS2), were generated. Full details of our datasets can be found in Table S6 and S7.
Phylogenetic trees of 12 alignment datasets were reconstructed using the ML approach implemented in PhyML 3.412 [56] . The robustness of the ML tree topology was assessed by comparing the ML topology with the topologies sampled in the BMCMC analysis performed in MrBayes version 3.1.2 [57] , and with bootstrapping analyses of 1,000 pseudo-replicate datasets. ML and NJ trees were estimated from the bootstrap datasets using PhyML [56] and PAUP* version 4beta10 [58] , respectively. A general-time-reversal (GTR) substitution model with gamma distributed rate heterogeneity of 4 rate categories (C 4 ) and a proportion of invariable sites were used in all tree reconstruction methods. Phylogenies were rooted with the H5N1 HPAI strain A/Ck/HK/YU324/2003, which is genetically close to the newly reported Hunan strains [17] , and shares comparable genetic proximity to Indonesian clade.
Homologous recombination within each gene segment among Indonesian H5N1 isolates was extensively searched using Recombination Detection Program version 2 (RDP2) [59] , and the datasets are found to be free of homologous recombination. Putative reassortant viruses were preliminarily identified by their topological incongruity across the phylogenies of different gene segments. This was further investigated using a smaller set of Indonesian H5N1 virus isolates with full genome sequences, which included sequences of early viruses (n = 2), group 1-3 lineages (n = 12) and putative reassortant viruses (n = 10). The eight gene segment alignments were manually concatenated in the order of their length to generate a single alignment of complete genome sequences, and was further analyzed using 1) similarity plots and 2) bootscan analyses [60] implemented in SIMPLOT version 3.5.1 [61] , and 3) GARD [62] available via the Datamonkey website [63] . The hypothesis of reassortment was supported if the recombinant breakpoints were detected near the junctions where the genome segments were manually concatenated.
The geographic locations of virus isolation were either obtained from the sequence databases, or obtained through personal communication with Catherine Smith (from Disease Control and Prevention, Atlanta, USA), or inferred from their strain names (Tables 1 and S6 ). The locations of isolates were indicated on the map of main island of Java in Indonesia (Figure 4 ). Due to the limit of our geographical data, the localities of the isolates shown in the map (Figure 4 ) should be regarded as arbitrary within the province which is the highest precision level shared by all viral samples. Each of the reassortant viruses was assigned with a state of either Greater Jakarta (surroundings included) or West Java depending on its place of origin ( Table 1 ). The migratory history of the reassortant viruses (n = 25) between these geographical states were inferred based on the refined ML phylogeny of HA and NA ( Figure  S4 ) independently using two parsimony optimization methods, called ACCTRAN (accelerated transformation) and DELTRAN (delayed transformation) implemented in PAUP* software. The geographical states of all ancestral nodes in the tree were estimated to achieve minimum state changes in overall, and therefore the number of state changes and state of the MRCA of the reassortant was obtained. Polytomies were randomly resolved 1,000 times, and state changes were estimated separately for each resolution. The mean number of state changes was then calculated. To test against the null hypothesis of completely unrestricted migration between geographical states (panmixis), the mean number of observed state changes was compared with the frequency distribution of the mean number of expected state changes under the null hypotheses. The null distribution and critical values were generated by randomly shuffling the states of isolates 5,000 times (the Slatkin-Maddison test [22, 24, 64] ). The migratory history of group 2 viruses was also studied using the HA gene in a similar manner, while each group 2 virus was assigned to either of four widely ranged geographical states: Greater Jakarta and surroundings, the rest of Java, Sumatra, and Sulawesi Selatan, including Papua. This assignment scheme is comparable to that of reassortant viruses, as West Java is part of Java.
Parameters of codon-partitioned substitution rates, demographic functions, tMRCA and tree topologies were co-estimated from HA and NA gene datasets of reassortant and group 2 viruses separately in a BMCMC framework [18] using BEAST version 1.4.6 [65] . Substitution model HKY+C 4 with invariable site portion was used. Isolation dates were used to calibrate the molecular clock. Three clock models including strict clock, UCEN and UCLN relaxed clocks [21] were attempted independently, and the best-fit clock model was selected by comparing the BF calculated from their posterior distributions [20] . The Bayesian skyline plot [23] was used to estimate population dynamics, in terms of relative genetic diversity. Less complex parametric demographic models (constant size, exponential growth and logistic growth) were applied independently, and the best-fit models selected by BF tests were used to quantitatively estimate the growth rate and other demographic parameters. The BMCMC analyses contained 2610 8 states, with sampling every 1,000 states, and the first 10% of each chain was discarded as burn-in. Convergences and effective sample sizes of the estimates were checked using Tracer v1.4 [66] .
Positively selected sites were detected using random effect likelihood (REL) and fixed effect likelihood (FEL) methods [26] via the Datamonkey website [63] . Bayes factors larger than 50 and pvalues smaller than 0.1 were used as thresholds for strong evidence of selection in REL and FEL, respectively. To test lineage-specific positive selection, the two-ratio branch model was used, which pre-specifies a single rate of synonymous substitution (dS) for the whole phylogeny and two rates of non-synonymous substitution (dN 1 and dN 2 ). The dN 1 was specified for the pre-emergence lineage (indicated as the ancestral branch connecting the group 3 MRCA; see Figure 1B ) for the group 3 viruses (including the reassortant viruses for M1, M2, and PB1 genes). The dN 2 was specified for other lineages across the phylogenies. The ML estimates of these rate parameters were performed in HYPHY version 0.99 [67] . The resulting likelihood score of the two-ratio model was then compared with that of the one-ratio model, which assumes the same dN and dS across the phylogeny, using the likelihood ratio test (LRT, with degree of freedom = 1). The substitution model MG94XGTR+C 4 was used.
The ancestral nucleotide sequences of all internal nodes were reconstructed using joint ML method [68] implemented in HYPHY. Amino acid changes along the pre-emergence lineage were determined, and were then mapped onto the three-dimensional structure of the N-terminal domain of M1 matrix protein molecule [30] available (PDB-ID: 1EA3) in RCSB Protein Data Bank. Figure S6 Substitution rates of HA and NA genes from reassortant and group 2 parental strains. 95% higher probability densities (HPDs) are indicated by the error bars. 1st, 2nd, 3rd, and C denote the rate for the 1st codon position, 2nd codon position, 3rd codon position, and whole sequence (non-partitioned), respectively. Substitution rate units for codon partitioned and non-partitioned sequences are substitution/codon/year and substitution/site/year, respectively. Table S5 Estimations of dN/dS using 1-ratio and 2-ratio lineage-specific selection models. These estimations were performed in HYPHY software. Gene datasets other than PB1, HA, NA, M1, and M2 were not analyzed because group 3 is represented by the single virus IDN/6/05. Found at: doi:10.1371/journal.ppat.1000130.s014 (0.03 MB DOC)
Information and phylogenetic groupings of sequences used in this study. 1, 2, 3, and X denotes groups 1, 2, 3, and unclassified (early viruses; see main text for explanation). Empty entries indicate the unavailability (e.g., no sequence found, too short, too many ambiguous codes, and too many gaps) of the sequence.  Endothelial Cells Support Persistent Gammaherpesvirus 68 Infection A variety of human diseases are associated with gammaherpesviruses, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infections usually result in either a productive lytic infection, characterized by expression of all viral genes and rapid cell lysis, or latent infection, characterized by limited viral gene expression and no cell lysis. Here, we report characterization of endothelial cell infection with murine gammaherpesvirus 68 (γHV68), a virus phylogenetically related and biologically similar to the human gammaherpesviruses. Endothelial cells supported γHV68 replication in vitro, but were unique in that a significant proportion of the cells escaped lysis, proliferated, and remained viable in culture for an extended time. Upon infection, endothelial cells became non-adherent and altered in size, complexity, and cell-surface protein expression. These cells were uniformly infected and expressed the lytic transcription program based on detection of abundant viral gene transcripts, GFP fluorescence from the viral genome, and viral surface protein expression. Additionally, endothelial cells continued to produce new infectious virions as late as 30 days post-infection. The outcome of this long-term infection was promoted by the γHV68 v-cyclin, because in the absence of the v-cyclin, viability was significantly reduced following infection. Importantly, infected primary endothelial cells also demonstrated increased viability relative to infected primary fibroblasts, and this increased viability was dependent on the v-cyclin. Finally, we provide evidence for infection of endothelial cells in vivo in immune-deficient mice. The extended viability and virus production of infected endothelial cells indicated that endothelial cells provided a source of prolonged virus production and identify a cell-type specific adaptation of gammaherpesvirus replication. While infected endothelial cells would likely be cleared in a healthy individual, persistently infected endothelial cells could provide a source of continued virus replication in immune-compromised individuals, a context in which gammaherpesvirus-associated pathology frequently occurs. Endothelial cells create a physical barrier on the luminal surface of blood and lymphatic vessels. This barrier must be traversed by blood-borne pathogens and immune cells trafficking between tissues and the bloodstream. Many herpesviruses require systemic spread for persistence within a host, and therefore must cross such an endothelial cell barrier. To date, herpesviruses have been implicated as potential initiators of arterial injury, endothelial dysfunction, and local inflammation, possibly contributing to the pathogenesis of atherosclerosis [1] [2] [3] [4] . Human cytomegalovirus (HCMV), a betaherpesvirus, infects endothelial cells in vivo. Studies have shown that infected endothelial cells play a role in HCMV dissemination and pathogenesis [5] . Endothelial cells exhibit regional specialization in gene expression and morphology depending on the local physiologic demands of their respective organs and tissues [6] . In light of this diversity, it is not surprising that endothelial cells from different tissues differ in their susceptibility to HCMV infection [7] . Kaposi's sarcoma-associated herpesvirus (KSHV), a human gammaherpesvirus, also infects endothelial cells in vivo. More importantly, KSHV is the causative agent of the endothelial cell neoplasm, Kaposi's sarcoma (KS). The murine gammaherpesvirus, cHV68, has been detected in aortic endothelium after infection of apoE deficient mice, as well as on the luminal surface of explanted aortas infected in vitro [8] . While a relationship between gammaherpesviruses and endothelial cells has been noted, the role of endothelial cells in chronic gammaherpesvirus infection and pathogenesis is ill-defined.
Gammaherpesviruses are a lymphotropic family of viruses associated with a broad spectrum of malignancies and lymphoproliferative diseases. These oncogenic viruses persist for the life of the host by establishing and maintaining a latent infection. Because gammaherpesviruses are extremely host-specific, studying the human viruses Epstein-Barr virus (EBV) and KSHV in nonhuman systems does not mimic natural infection. cHV68 is a natural pathogen of wild murid rodents and therefore provides a valuable small animal model of gammaherpesvirus infection [9] [10] [11] [12] [13] . This virus has important biological and genetic similarities to the human gammaherpesviruses, and results in a variety of pathologies in defined mouse models. Primary cHV68 infection is characterized by virus replication in lung epithelial cells, and the establishment of latency in B cells, dendritic cells, and macro-phages [12] [13] [14] [15] [16] . Persistent cHV68 infection also occurs in lung epithelial cells [17] . The role of endothelial cells in cHV68 infection has not been explored to date.
At the cellular level, gammaherpesvirus infection encompasses two broadly defined outcomes: productive, lytic replication and non-productive, latent infection. During lytic replication, viral DNA is amplified and host cell machinery is utilized for the production of viral progeny. Viral genes are actively transcribed and translated, contributing to new virus production. Ultimately, the cell is lysed and infectious virus released. In light of the fact that this process occurs quickly (24 to 48 hours in vitro) our understanding of virus-host interactions during primary infection is quite limited. During latency, viral DNA is not amplified, but instead is maintained as a nuclear episome in latently infected cells, and little viral gene transcription occurs. Latently infected cells remain intact and do not produce new infectious virus. Furthermore, it is thought that these long-lived latent cells may serve as the major mechanism by which gammaherpesviruses promote life-long infection of their hosts.
Here we characterize cHV68 infection of endothelial cells. To date, in vitro infection of most cells with cHV68 supports significant viral replication and results in complete cell lysis (data not shown, van Dyk). We have infected both primary endothelial cells and endothelial cell lines and demonstrated that they produce comparable amounts of virus as fibroblasts. However, whereas fibroblasts were mostly lysed by 36-48 hours post-infection, a significant percentage of infected endothelial cells remained intact. Analysis of endothelial cell lines revealed that these intact cells 1) were actively infected and undergoing the lytic transcriptional program, 2) continued to proliferate for a prolonged time after infection, 3) were altered in morphology and cell-surface protein expression, compared to uninfected endothelial cells, and 4) continued to release infectious virions as far as 30 days postinfection. In the absence of the cHV68 viral cyclin, endothelial cell viability was significantly decreased after infection, indicating that an active viral process was responsible for promoting the outcome of infection in endothelial cells. Of major significance, we also provided evidence of endothelial cell infection in primary cells and in vivo in immune-deficient mice. These data demonstrated that endothelial cells supported persistent, productive gammaherpesvirus infection, an outcome not previously reported for cHV68 or other gammaherpesviruses. Together, these data indicated the potential of the endothelial cell as a critical cell type in cHV68 pathogenesis, and open a new avenue of research into herpesvirus manipulation of endothelial cell biology.
Endothelial cells are heterogeneous in nature, express unique surface antigens, and differ in their susceptibility and response to infection by various pathogens [6] . In light of this diversity, we investigated endothelial cell lines from various anatomic locations, and from BALB/c and C57BL/6 mice, for their ability to support cHV68 replication. We analyzed the outcome of cHV68 infection in endothelial cell lines from lung (CD3), brain capillary (MB114), and lymph node (SVEC) by single-step and multi-step growth assays. Single step growth proceeded with kinetics similar to previously published infection of the fibroblast cell line, NIH 3T12 cells [18] in each of the three endothelial cell lines analyzed (Fig. 1A , top panel). Though equivalent numbers of cells were plated per well, the differences amongst the endothelial cell lines in titer at time zero were not surprising given that cell lines grow at varying rates and to varying densities. A comparison of viral titers at 36 hours revealed that the three endothelial cells lines produced titers comparable to those achieved in 3T3 fibroblast cells. Additionally, multi-step growth in MB114 and CD3 endothelial cells was similar to growth in 3T3 fibroblasts (Fig. 1A, bottom  panel) . These data demonstrated that cHV68 is capable of replication in endothelial cells infected in vitro.
Using a GFP-labeled virus, we next sought to determine if the percentage of infected endothelial cells was comparable to percentage of infected fibroblasts. Following infection of MB114 endothelial cells and 3T12 fibroblasts, cells were analyzed for expression of GFP from the cHV68 genome by flow cytometry. At 24 and 48 hours post-infection the majority of endothelial cells and fibroblasts expressed GFP (Fig. 1B) . These data indicated that, like fibroblasts, endothelial cells were uniformly infected and supported the cHV68 lytic transcription program.
A population of endothelial cells remained intact following cHV68 infection and uniformly expressed lytic antigens Uninfected endothelial cells and fibroblasts grew as adherent monolayers ( Fig. 2A, top panel) . However, in infected endothelial cell cultures, we observed a population of intact, phase-bright, and non-adherent cells as early as 96 hours post-infection, whereas only cellular debris remained in the infected fibroblast cultures (data not shown). At six days post-infection, we collected non-adherent cells and cellular remains by centrifugation. After washing, we resuspended this material in complete media for culture and analysis. The cells harvested from the infected endothelial cultures appeared as individual, intact cells suspended in culture, whereas the material harvested from the infected fibroblast cultures appeared as clumps of cellular debris ( Fig. 2A, bottom panel) . Prior to centrifugation, we measured cell viability by trypan blue exclusion. Very few infected fibroblasts remained as viable, nonadherent cells at six days post-infection (Fig. 2B ). However, a significant proportion of endothelial cells remained as viable, nonadherent cells at six days post-infection. When MB114 endothelial cells were treated with a non-toxic dose of phosphonoacetic acid
Various human diseases are associated with gammaherpesvirus infections, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infection of cells usually results in either productive infection that is characterized by new virus production and rapid destruction of the host cell, or latent infection that is characterized by long-term carriage of viral DNA in intact cells that do not produce virus. Here, we characterize endothelial cell infection using a small animal model of gammaherpesvirus infection and disease. While infection of endothelial cells resulted in virus production as most cells do, infection of this cell type was unique in that cells remained intact and continued to proliferate. These intact, infected endothelial cells were significantly altered in appearance and gene expression compared to uninfected cells, changes predicted to impact endothelial cell growth and function. Endothelial cells were unique in their ability to support this type of persistent infection. These data demonstrate an additional mechanism, beyond latency, by which gammaherpesviruses may achieve long-term propagation, particularly in conditions of immune suppression. Our results suggest persistently infected cells as a therapeutic target for prevention/ treatment of chronic disease, and may provide a mouse model for future testing.
(PAA), an inhibitor of viral DNA replication and late gene synthesis, most cells remained adherent, and there were significantly fewer detached, viable cells (Fig. 2B) . PAA alone had no effect on the viability, adherence, or phenotype of uninfected MB114 cells in culture (data not shown). These data revealed that a population of endothelial cells became non-adherent and remained viable at six days post-infection, whereas fibroblasts were destroyed by cHV68 infection. Additionally, this outcome of infection in endothelial cells was influenced by late viral gene expression, as most cells remained adherent in the presence of PAA.
Next we determined the status of virus replication in the viable and non-adherent cells. Cells collected at six days post-infection were analyzed by flow cytometry for expression of the cHV68 glycoprotein, gp150, which is expressed during the lytic transcription program on the surface of infected cells [19] . Cell viability was also determined using propidium iodide (PI), a cell impermeant dye that is excluded from intact cell membranes. In these experiments we also included the S11E cell line, a mouse B cell lymphoma line which harbors latent cHV68, and therefore is negative for gp150 surface expression [20] . The majority of MB114 endothelial cells and 3T12 fibroblasts were positive for surface gp150 expression (Fig. 2C ). While 3T12 fibroblasts were mostly PI positive, MB114 endothelial cells excluded PI. These data indicated that endothelial cells which remained viable at six days post-infection were undergoing the lytic transcription program and were not latent or uninfected.
At 12 days post-infection we determined the viability of cells collected and cultured at six days post-infection. PI staining of S11 cells, after six days in culture, revealed that 51.7% (62.5 SEM) of cells were PI negative and thus viable. PI staining of post-infection fibroblasts, treated in parallel with infected endothelial cells, revealed that only 14.5% (61.5 SEM) of the cells harvested remained intact. In contrast, PI staining of post-infection endothelial cells revealed that 92.5% (60.3 SEM) of the cells harvested were intact and viable (Fig. 2D) . Analysis of the viral replication program in MB114 endothelial cells at 12 days postinfection revealed that the majority of cells continued to express gp150 (Fig. 3C) . Therefore, infection of endothelial cells with cHV68 resulted in a population of cells that escaped lysis and remained intact as far as 12 days post-infection, while undergoing the lytic transcription program.
The observed viability of endothelial cells after cHV68 infection could be the result of escape from infection or latent infection of these cells. To determine the infection status of the intact endothelial cells, we performed RT-PCR analysis of viral gene transcripts. The cHV68 M2 gene transcript is synthesized during both lytic and latent infection, as are the polymerase III (pol III) transcripts encoded at the left end of the viral genome [21, 22] . Latently infected S11 cells contained both pol III-1 and M2 transcripts (Fig. 3A) . We detected these transcripts in 3T3 fibroblasts at 24 and 36 hours post-infection, as well as from the 3T3 cellular debris harvested at six days post-infection, while no cellular b-actin transcript was detected from infected 3T3 cells at 36 hours post-infection. No RNA could be detected from infected 3T3 debris at 12 days post-infection, whereas we recovered RNA from infected MB114 cells as far as 12 days post-infection. Pol III-1 and M2 transcripts were detected in infected MB114 cells at 24 and 36 hours post-infection and from the intact, cultured cells at six and 12 days post-infection. We detected b-actin RNA in infected MB114 cells at 24 and 36 hours post-infection, but not at six and 12 days post-infection. However, we detected another cellular mRNA transcript, cyclophilin A, at six days post-infection in 3T3 fibroblasts and MB114 cells (Fig. S2) , and comparable detection of the cellular 18S rRNA transcript was observed in all conditions. Gradual loss of the cellular b-actin transcript in viable cells was not surprising given that b-actin has been documented to vary significantly in the setting of virus infection [23] , and selective degradation of certain mRNAs frequently occurs during virus infection [24] [25] [26] [27] [28] . Analysis of viral gene transcripts indicated that those endothelial cells which remained intact after cHV68 infection contained viral gene transcripts as far as 12 days postinfection.
To determine whether the intact, infected endothelial cells were undergoing active viral replication, we analyzed infected cells for the expression of early and late lytic replication-associated M3 and gB. As predicted, 3T3 fibroblasts, which support lytic replication, expressed both M3 and gB (Fig. 3B ). In contrast, S11 B cells, which contain latent cHV68, did not express either M3 or gB. Intact, infected MB114 endothelial cells not only expressed M3 and gB early (36 hours), but also at six and 12 days post-infection. A faster migrating gB-specific band was also detected at six days post-infection, and may indicate differential glycosylation or degradation. Additionally, infected MB114 endothelial cells were positive for surface protein expression of gp150 at both 12 and 24 days post-infection (Fig. 3C ). Our analysis of lytic protein expression indicated that endothelial cells which remained intact after cHV68 infection expressed early and late lytic proteins, indicating that not only did cHV68 infected endothelial cells still contain virus, these cells also expressed viral proteins indicative of active viral replication, and not latency.
To further test the status of viral replication in endothelial cells, we infected MB114 endothelial cells with GFP-cHV68 and collected the intact, non-adherent cells at six days post-infection. GFP expression is driven by the CMV immediate early promoter and has been demonstrated to be expressed only during lytic infection [29] . At the time of harvest, these cells uniformly expressed GFP (Fig. 3D histogram) and continued to do so at 20 days post-infection, (Fig. 3D micrographs) . Therefore, the nonadherent endothelial cells which remained intact after cHV68 infection did not escape infection and were not latently infected.
After establishing that the intact endothelial cells harvested at six days post-infection were indeed infected, we determined whether these cells produced mature virions. Transmission electron microscopy (TEM) of these cells revealed virions at various stages of maturation throughout the nucleus and cytoplasm of 100% of the 20 cells imaged at 12 days post- The culture conditions we established for maintaining the nonadherent cells included centrifugation and resuspension in fresh media every six days (Fig. S1 ). To test the contribution of nonadherent cells to virus production, aliquots of the supernatants were collected for measurement of cell-free virus. A culture of latently infected S11 cells did not yield any detectable cell-free virus after six days in culture by plaque assay (Fig. 4B ). In contrast, after six days in culture (12 days post-infection), cell-free supernatant from infected MB114 cells yielded significant viral titer, and this titer was significantly higher than that of supernatant taken from infected 3T3 cultures. Therefore, endothelial cells continued to release new infectious virions for at least 12 days post-infection. Infected endothelial cell supernatant contained 100-fold more virus than that of a lysed, infected, fibroblast culture.
After the initial observation that cHV68 infection of several endothelial cell lines from different anatomic locations resulted in a population of cells that escaped lysis, we focused our analysis on this outcome in MB114 endothelial cells. Beginning at the time of collection (day six post-infection), we counted intact, trypan blue excluding cells every three days. Infected MB114 cells remained viable for at least 30 days post-infection (Fig. 5A) .
During this course of infection, the number of intact cells in the culture increased from 7.08610 5 (60.16 SEM) to 8.51610 6 (60.28 SEM) during the first six days of culture, a ten-fold increase. A corresponding increase in the percentage of membrane viable cells also occurred during this time. Subsequent to day 12 post-infection, the total number of intact cells began to decrease, as did the overall viability of the culture (Fig. 5A ). The observed increase in both intact cell number and percent viability during the first six days of culture, concurrent with positive gp150 staining ( Fig. 2C and Fig. 3C ), indicated that endothelial cells were intact and proliferating in culture while maintaining the lytic transcription program.
To further examine the proliferation observed during the first six days of culture, we stained MB114 cells with carboxyfluoroscein (CFSE) prior to infection. CFSE is a fluorescent molecule used to measure cell proliferation, in that each time a cell divides, the two daughter cells contain half the CFSE of the parent cell. S11 cells demonstrated a drop in CFSE signal intensity after six days in culture, consistent with cell division. At the time of harvest (day six post-infection), MB114 cells also demonstrated a drop in CFSE signal intensity consistent with multiple cell divisions (Fig. 5B ). After six more days in culture (12 days post-infection) CFSE signal intensity dropped further, consistent with continued cellular proliferation. Thus, endothelial cells which remained intact after cHV68 underwent multiple rounds of proliferation.
Note that by 30 days post-infection, however, the viability of the culture was quite low in comparison to earlier time points (Fig. 5A ). To further test the fate of the surviving endothelial cells, we examined viability of infected cells by staining with annexin V and PI (Fig. 5C ). MB114 cells were 92.4% (60.6 SEM) viable (annexin V negative, PI negative) at two days post-infection and 72.5% (614.5 SEM) viable at four days post-infection. At six days postinfection all MB114 cells were non-adherent and 31.0% (610.74 SEM) of these non-adherent cells were viable. In contrast to MB114 cells, dying cells dominated infected 3T12 cultures at six days post-infection These data indicate that many MB114 cells died early during infection, and in agreement with our proliferation data ( Fig. 5A and 5B ) the population of viable cells increased to 86.4% (60.6 SEM) of the culture by 12 days postinfection. However, by 30 days post-infection dying cells finally dominated the infected culture. These data support that the subset of cells surviving and proliferating at six days post-infection were unique their extended survival, but did not remain viable indefinitely. Of significance, during the course of this infection most of the cells expressed the lytic glycoprotein gp150 on their surface at both early and late time points (Fig. 2C and Fig. 3C) . Moreover, the percentage of cells expressing gp150 did not change markedly with time ( Fig. 2C and Fig. 3C ). These data indicate that the vast majority of viable, infected endothelial cells were undergoing active viral replication, and that these cells were neither latently infected nor had escaped infection.
Intact endothelial cells were altered in size, shape, and surface marker expression after cHV68 infection To investigate the morphologic differences between uninfected endothelial cells and the endothelial cells harvested at six days post-infection, we quantified cell size and internal granularity by flow cytometric determination of forward versus side scatter. Uninfected MB114 cells exhibited a broad range of light scatter, indicative of a cell population diverse in size and internal granularity. In contrast, infected cells were very uniform in forward and side scatter (Fig. 6A) . PAA treatment immediately following MB114 infection resulted in significantly fewer cells becoming non-adherent (Fig. 2B) . Like the uninfected MB114 cells, MB114 cells treated with PAA following infection exhibited a broad range of forward and side scatter (Fig. 6A) . Thus, cHV68 infection of endothelial cells resulted in a uniform population of cells with altered morphology, and this outcome was dependent on productive viral infection and late viral gene expression.
To further investigate the differences between uninfected endothelial cells and the intact cells harvested six days after infection, we compared the expression of cell-surface proteins on these two cell populations by flow cytometry. Because the cells harvested at six days post-infection persisted in culture as nonadherent cells, we chose to investigate surface expression of the adhesion markers intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Additionally, we analyzed cells for surface expression of Thy1, a cell surface protein which is upregulated on the surface of activated endothelial cells and functions in cell-cell interactions [30] [31] [32] . Uninfected MB114 cells expressed ICAM-1, VCAM-1, and Thy1 (Fig. 6B) . In contrast, infected endothelial cells expressed neither ICAM-1 nor VCAM-1, but did express Thy1. Based on these data, cHV68 infected endothelial cells downregulate cell surface expression of two adhesion molecules, while maintaining surface expression of an activation marker.
In the absence of the viral cyclin, endothelial cell viability after cHV68 infection is reduced
We demonstrated that cHV68 infection of endothelial cells resulted in a population of intact cells which persisted in culture as non-adherent cells and continued to produce new virus. Our analysis of endothelial cell infection in the presence of PAA indicated that this outcome is influenced by viral DNA replication and/or late gene synthesis ( Fig. 2B and Fig. 4A ). To begin dissecting the mechanism of persistent cHV68 in endothelial cells infected in vitro, we tested the role of the cHV68 viral cyclin (v-cyclin) in this system. We measured expression of the cHV68 vcyclin in infected MB114 endothelial cells and infected fibroblasts by RT-PCR and western analysis. Notably, the cHV68 v-cyclin gene transcript and protein were detectable in infected endothelial cells and fibroblasts ( Fig. 7A and Fig. S3A) .
To determine the role of the v-cyclin in endothelial cell infection, we infected MB114 cells with either wildtype cHV68 or a v-cyclin deficient cHV68 (v-cyclin.STOP cHV68) [18] . Cells were harvested at six days post-infection and cultured as described previously. As per previously published reports in fibroblasts, we determined that the v-cyclin was dispensable for cHV68 growth in endothelial cell lines by multi-step growth assays (data not shown Because viability of MB114 cells after infection was impaired in the absence of the v-cyclin, we next determined the effect of this viral gene on persistent viral replication in viable endothelial cells. Cell free supernatants from infected MB114 cells were titered by plaque assay (Fig. 7C) . Infections with both wildtype and vcyclin.STOP virus resulted in persistent viral production up to 30 days post-infection. Total viral titers from v-cyclin.STOP infections were significantly less than wildtype infections at 12 and 24 days, however, an equivalent amount of virus was produced per cell as in wildtype cHV68 infections. Cell surface protein expression of ICAM-1, VCAM-1, and Thy1 was the same following infection with either wildtype or v-cyclin.STOP virus ( Fig. 6B and Fig. S3B ), thus the v-cyclin was not required for the surface phenotype of infected endothelial cells and the surface phenotype of infected endothelial cells did not predict survival. Therefore, upon infection, the primary function of the v-cyclin was to promote the viability of endothelial cells.
Primary endothelial cells were infected in vivo, and ex vivo demonstrated prolonged viability while supporting cHV68 growth
Given that immortalized endothelial cell lines are inherently different from endothelial cells in vivo, we next determined the outcome of cHV68 infection in primary endothelial cells. Primary endothelial cells were isolated from C57/BL6 mouse lungs and characterized as per previously published methods (Fig. S4) .
First, we determined if primary lung endothelial cells could support growth of wildtype cHV68 by multi-step growth assay (Fig. 8A) . Because of the apparent role for the v-cyclin in infection of endothelial cell lines, we also analyzed growth of v-cyclin.STOP virus in primary endothelial cells. Infection proceeded with kinetics comparable to previously published infection of NIH 3T12 fibroblasts [18] . Growth curves did not differ between wildtype and v-cyclin.STOP virus. These data revealed that primary endothelial cells supported growth of cHV68, irrespective of the vcyclin.
Second, we analyzed the percent of cells that remained viable after cHV68 infection at a low MOI. Immediately following removal of the viral inoculum (t = 0), mouse embryonic fibroblasts (MEFs) and primary lung endothelial cells did not differ significantly in viability (p.0.05) (Fig. 8B) . However, at 48 hours post-infection with wildtype cHV68, primary lung endothelial cells were significantly more viable than MEFs (p,0.001), and remained significantly more viable at 96 (p,0.05) and 120 hours post-infection (p,0.001). Notably, endothelial cells infected with the v-cyclin deficient cHV68 had reduced viability at 48, 96, and 120 hours post-infection. Given that cells were infected at a very low MOI, it was not surprising that a proportion of MEFs (28.5%611.1 SEM) were viable at 96 hours post-infection. However, unlike endothelial cells, MEFs were mostly lysed by 120 hours post-infection with wildtype virus. These data demonstrate that primary endothelial cells surpass MEFs in viability following cHV68 infection, and that this outcome is promoted by the v-cyclin.
Lastly, we examined lung endothelial cells following acute cHV68 infection in vivo. Our in vitro studies revealed that viable, infected endothelial cells were not latently infected, but uniformly supported a lytic viral program (Fig. 2C, Fig. 3C and 3D ). Because persistent viral infection and lytic viral antigen expression is likely to be cleared by an intact immune response, we analyzed endothelial cell infection in lung tissues of immune-deficient CD8-alpha null mice. At six days post-intranasal infection, lung cells enriched for those bearing the endothelial cell marker CD31 were analyzed alongside the remaining CD31 depleted lung cells. Given the possibility that a small percentage of non-endothelial cells can transiently express CD31 (i.e. macrophages and neutrophils), we depleted the CD31 positive cell population of these potential contaminating cells and analyzed resultant cell populations by flow cytometry (Fig. S5B ). PCR analysis with single copy sensitivity for cHV68 gene50 (Rta) was performed to determine the frequency of viral genome positive cells in the CD31-enriched and remaining CD31-depleted lung cell populations (Fig. 8C) . Data from this analysis revealed that approximately 1/102 CD31-enriched cells were viral genome positive, whereas 1/525 remaining CD31depleted lung cells contained viral DNA. Thus, these data support that a surprisingly large proportion of lung endothelial cells were viral genome positive following in vivo infection.
To further demonstrate that these viral genome positive cells are actively infected, and to exclude the possibility of endocytic uptake of virus or abortive infection, we performed RT-PCR analysis on endothelial enriched and depleted lung cells (Fig. 8D) . The transcript for the endothelial cell marker CD31 was robustly detected in the endothelial enriched lung cells from each of the four mice analyzed, whereas a very low level of this transcript was detected from the CD31 depleted lung cells of only one out of the four mice analyzed. In combination with flow cytometric analysis (Fig. S5B) , these data support that our enrichment strategy was effective in isolating CD31+ cells from total lung cells. Additionally, the viral pol III-1 transcript was detected from both endothelial cell enriched and depleted lung cells of cHV68 infected mice, and was absent from lungs of mock infected mice. Detection of viral gene transcripts from lung cells indicates virus infection of these cells. Notably, within each infected mouse, pol III-1 detection was comparable between endothelial enriched and CD31-depleted lung cell populations, suggesting that detection of this viral transcript in the endothelial cell population was not due a few contaminating infected non-endothelial cells. Taken together, these data demonstrate cHV68 infection of endothelial cells in vivo.
Herpesviruses have been implicated as potential initiators of a variety of endothelial pathologies [1] [2] [3] [4] . Given the intimate interactions observed between herpesviruses and endothelial cells, and the systemic spread of cHV68 during infection, we characterized the outcome of endothelial cell infection with cHV68, a small animal model for the human gammaherpesviruses. While cHV68 replicated comparably in endothelial cells and fibroblasts up to 36 hours post-infection, infected cultures of endothelial cells had a high percentage of viable, non-adherent cells that remained following infection. Of significance, these viable, non-adherent cells had not simply escaped infection, but instead were actively infected, as determined by the presence of multiple markers of the lytic replication program. While the absolute number of viable, infected endothelial cells varied among different cell lines and primary cells, the prolonged viability of endothelial cells in comparison to fibroblasts was remarkably consistent in endothelial cell lines from distinct anatomic locations, as well as in primary endothelial cells. Furthermore, optimal survival of both endothelial cell lines and primary endothelial cells was dependent on the presence of the cHV68 v-cyclin, indicating that this outcome was a process actively promoted by virus infection.
This conserved outcome of endothelial cell infection is particularly striking given that endothelial cells display phenotypic heterogeneity in structure and function depending on anatomic location [33] [34] [35] . Based on the persistent infection observed in diverse endothelial cell lines, and heightened viability of primary endothelial cells following infection, we propose that cHV68 may have evolved machinery to specifically promote persistent infection in endothelial cells.
Endothelial cells serve as a natural site of infection and possible viral reservoir of HCMV [36] [37] [38] [39] [40] , suggesting a role for HCMVinfected endothelial cells in viral spread and persistence. Additionally, recent reports implicate circulating endothelial progenitor cells as potential reservoirs of KSHV and possible precursors of KS spindle cells [41, 42] . However, the specific mechanisms by which infected endothelial cells contribute to the pathogenesis of these human viruses remains unclear. Murine cHV68 pathogenesis involves dissemination from the lung to lymph nodes, spleen, and peritoneum [12, 15] . In light of this systemic spread, cHV68 likely encounters an endothelial cell barrier.
The human gammaherpesvirus KSHV causes a serious endothelial cell malignancy, KS, which predominantly occurs in immunecompromised individuals (e.g. AIDS patients). KS tumors are comprised of distinctive spindle cells of endothelial origin and a variable inflammatory infiltrate [43] [44] [45] [46] . KSHV is detected primarily in the endothelial component of the lesion, and though most of these cells harbor latent KSHV, a subset of them enter the lytic cycle [47, 48] . While there is precedent for a mixed infection type within KS tumors (i.e. both lytic and latent infection), the history of the lytically infected cells remains elusive, though recent reports point to circulating endothelial progenitor cells [41, 42] . In vitro, endothelial cell infection with KSHV is predominantly latent [43, [49] [50] [51] . However, when these cells are transferred into mice, they show evidence of lytic gene expression and virus production [52] .
Because viral DNA replication and/or late gene synthesis were important for endothelial cell outcome of cHV68 infection (Fig. 2B and Fig. 6A ), our findings indicate that an active viral process occurred within the cells to yield these changes. Consistent with this hypothesis, we identified that the cHV68 v-cyclin is required for optimal endothelial cell viability after cHV68 infection in vitro. The cHV68 v-cyclin promotes cell cycle progression in primary lymphocytes and can function as an oncogene in transgenic mice [18] . While the v-cyclin is critical for reactivation from latency, to date the v-cyclin has been dispensable in all assays of lytic replication in vitro [53, 54] . Given the lytic nature of endothelial cell infection, the contribution of the v-cyclin to optimal endothelial cell viability after cHV68 infection indicates a role for the v-cyclin outside of its requirement in reactivation from latency. Additionally, the apparent role of the v-cyclin in this prolonged infection of both endothelial cell lines and primary lung endothelial cells may explain the slight decrease in lung titers that resulted in mice infected with low doses of v-cyclin.STOP cHV68 and other vcyclin mutant viruses compared to wildtype cHV68 [55] . Although the precise mechanism by which the v-cyclin promotes endothelial cell viability is unknown at this time, it is possible that the v-cyclin provides growth cues that allow for anchorageindependent growth of endothelial cells after cHV68 infection.
While our initial analysis focused on the role of the v-cyclin, it is very likely that additional viral genes facilitate persistent endothelial cell infection. Lead candidates include the anti-apoptotic viral bcl-2 gene M11 and the viral GPCR (ORF 74), whose homologs in KSHV have been implicated in endothelial cell survival and transformation [56] [57] [58] [59] . Additional candidates for optimal endothelial cell infection are the ribonucleotide reductase homologs, ORF 60 and 61 of cHV68. Although the role of these genes in cHV68 infection of endothelial cells is currently untested, the MCMV ribonucleotide reductase homolog is required for in vivo replication and pathogenesis of this endothelial cell-tropic virus [60] .
One of the most noticeable alterations of the persistently infected endothelial cells described here is the extent of change in their cellular morphology and properties. Though we did not test the oncogenic potential of these cells, endothelial cells achieved anchorage-independent growth, a property frequently associated with oncogenic transformation. These cells also underwent significant changes in protein expression on the cell surface, with down-regulation of the cellular adhesion proteins ICAM-1 (CD54) and VCAM-1 (CD106). While there was decreased expression of ICAM-1 and VCAM-1, changes in cell surface expression were not global, since Thy1 expression remained positive on these cells (Fig. 6B) . It is worth noting that infection of endothelial cells with KSHV results in down-regulation of MHC class I, PE-CAM (CD31), and ICAM-1 (CD54), but not LFA-3 (CD58) or Fas (CD95), and the viral genes K3 and K5 have been demonstrated to regulate this outcome [61] [62] [63] [64] [65] [66] . The contribution of the mK3 gene of cHV68 to persistent endothelial cell infection remains untested. Down-regulation of certain surface markers but not others during viral infection indicates a specific phenomenon, rather than global down-regulation, and provides further evidence that an active process was responsible for the observed endothelial cell outcome of cHV68 infection.
While cHV68 may have evolved mechanisms that promote persistent infection of endothelial cells (e.g. the v-cyclin), it is also possible that endothelial cells have a cellular program that limits the cellular lysis typically observed during productive infection. This putative cellular adaptation may be particularly important in limiting destruction of blood vessel integrity during viral infection. A recent report of host cell response to cHV68 in three different cell types (fibroblasts, endothelial precursor cells and macrophages) identified 148 genes whose expression was altered in endothelial precursor cells, but not macrophages or fibroblasts [67] . Taken together with the unique endothelial cell outcome of cHV68 infection reported here, these data make a compelling argument for cell type specific responses to productive cHV68 infection.
We have provided evidence for cHV68 infection of endothelial cells in vivo at early times post-infection. Interestingly, infected cells (including endothelial cells) were not abundant in acutely infected lung tissue, as little to no GFP was detected by flow cytometric analysis of unfractionated lung cells after infection with a GFPmarked virus (data not shown), but required sensitive PCR methods for detection. Our in vitro analysis demonstrated a change in surface phenotype of infected endothelial cells which could correspond to substrate detachment and release into circulation. This is an intriguing idea in light of published reports of circulating endothelial cells and progenitor endothelial cells in virus infection. Our in vitro studies implicated endothelial cells as a persistent source of virus production, however, the extent to which endothelial cells contribute to cHV68 persistence in vivo, and to what degree it might be influenced by host immune status, are important issues that remains to be addressed. While it is unlikely that an intact immune response would permit such long-term expression of viral antigens in vivo, we hypothesize that persistently infected endothelial cells could provide a significant source of continued virus replication in immune-compromised individuals (i.e. AIDS patients), a context in which gammaherpesvirusassociated pathology frequently occurs.
Given the unusual properties of these viable and infected endothelial cells (e.g. anchorage-independent growth and altered cell surface protein expression), it will be important to critically address the potential role of endothelial cells as a reservoir for infection in vivo in both immune-competent and immune-deficient individuals.
In conclusion, our data provide evidence for prolonged gammaherpesvirus infection in endothelial cells. This outcome appears to be the result of a specific interaction between cHV68 and endothelial cells, as it is promoted by a viral gene (the cHV68 v-cyclin) and, to date, is unique to endothelial cells. These data further refine the concept of gammaherpesvirus infection and demonstrate that a gammaherpesvirus can undergo robust productive replication in the context of prolonged host cell viability. Identification of intermediate outcomes of gammaherpesvirus infection, such as the one we have described here, has major implications for our understanding of the nature of gammaherpesvirus infection as it relates to specific cell types. Such a course of infection provides an additional mechanism, beyond latency, by which gammaherpesviruses can achieve longterm propagation.
Mouse fibroblast cell lines 3T3 (ATCC CRL-1658) and 3T12 (ATCC CCL-164) and mouse endothelial cell lines MB114 [68] , SVEC4-10 (ATCC CRL-2181), and CD3 [69] were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 5% FBS (Hyclone, Logan, UT), 2 mM L-glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin sulfate. S11 [20] and S11E tumor cells [22] were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 50 mM b-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin sulfate (complete RPMI). Mouse embryonic fibroblasts were isolated from C57/BL6 mice as previously described [70] and cultured in DMEM supplemented with 10% FBS, 2 mM Lglutamine, 10 U/mL penicillin, 10 mg/mL streptomycin sulfay7te, and 250 ng/mL fungizone. Isolation, characterization, and culture of primary endothelial cells from C57/BL6 mice was done according to previously published methods [71] and is described in Protocol S1, Text S1 and Figure S3A .
cHV68 WUMS (ATCC VR-1465) and all recombinant viruses were grown and titered as previously described [72] . DK3TET 2 cHV68 (cHV68-GFP) containing a GFP cassette under the control of an immediate early CMV promoter was generated and characterized by Dr. Phillip Stevenson [73] . cHV68 containing a stop codon within ORF 72 (v-cyclin.STOP. cHV68) was previously described [18] .
All infections were carried out at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Inoculum was removed after one hour of infection at 37uC, cell monolayers rinsed three times with sterile phosphate buffered saline (PBS), and complete media added. Intact and non-adherent cells were collected at six days post-infection, at which time cells and media were collected, counted to determine post-infection viability by trypan blue exclusion, and then centrifuged for 10 minutes at 2086g. Cell pellets were washed twice in sterile PBS and then resuspended in complete RPMI at a concentration of 5610 5 viable cells/mL for continued culture. Cells were counted every three days of culture, and adjusted to a concentration of 5610 5 viable cells/mL. Every six days of culture, cells were counted and centrifuged for 10 minutes at 2086g to remove cell-free cHV68 and cellular debris. Pellets were washed twice in sterile PBS and resuspended in RPMI at a concentration of 5610 5 viable cells/ mL. Culture scheme is depicted in supplement (Fig. S1 ).
To measure virus replication, infected samples were analyzed by plaque assay at various times post-infection. To measure cell-free virus titer from infected cultures, supernatants were collected every six days of culture and analyzed by plaque assay. Samples were thawed, serially diluted, and plated onto NIH 3T12 cells in 12 well plates in triplicate. Infection was performed at 37uC for one hour. Cells were overlaid with a 1:1 mix of DMEM and carboxymethylcellulose plus fungizone (final concentration 250 ng/mL). Plates were incubated for seven days at 37uC. On day seven, carboxymethylcellulose was removed, and plates were stained with 0.35% methylene blue in 70% methanol and rotated for 15-20 minutes, before rinsing with water and counting on a light box. All titers were determined in parallel with a laboratory standard.
For propidium iodine (PI) viability studies, cells were washed twice in PBS (five minutes, 10006g), resuspended in a 0.5 mg/mL PI solution, and incubated for 15 minutes. Following incubation, cells were centrifuged (five minutes, 10006g) and washed in a solution of PBS, 2% fetal calf serum, and 0.1% NaN 3 (buffer A). Cells were fixed in 1% paraformaldehyde, and analyzed by flow cytometry. For two parameter viability analysis, cells were washed in 1X annexin V binding buffer (BD Bioscience, San Jose, CA), resuspended in 0.5 mg/mL PI and 5 mL annexin V-FITC (BD Bioscience), incubated for 15 minutes, washed in binding buffer, fixed in 1% paraformaldehyde, and analyzed by flow cytometry.
For carboxyfluorescein (CFSE) proliferation studies, MB114 cells and Sll cells were washed twice in PBS (five minutes, 10006g) and resuspended in a solution of PBS and 2% fetal calf serum (buffer B) at a concentration of 1610 6 cells per mL. An equal volume of 4 mM CFSE in buffer B was added to the cell suspension (2 mM final concentration) and pipet mixed. After three minutes the labeling reaction was quenched with an equal volume of fetal calf serum for 30 seconds, and buffer B was added for a total volume of 50 mL. Cells were centrifuged (five minutes, 10006g), resuspended in buffer B, and aliquots collected for day 0 analysis. Remaining, labeled cells were resuspended in complete media and cultured. Labeled MB114 cells were infected at an MOI = 5 PFU/ cell. Infected cells were harvested as described at day six postinfection and analyzed by flow cytometry at six and 12 days post-infection. Stained and unstained S11 cells were analyzed at the same time as a positive control.
For surface marker staining, cells were washed twice in buffer A (five minutes, 10006g) and resuspended in primary antibody (1:200 in buffer A, 25% 24G2 [74] ). The following primary antibodies were used: CD106-biotin (Rat, IgG2a k, clone 429 (MVCAM.A)), CD54-FITC (Armenian hamster, IgG1 k, clone 3E2), Thy1.2-APC (Rat, IgG2a k, clone 53-2.1) (BD Bioscience), monoclonal mouse anti-cHV68 gp150 (mouse IgG2a, a kind gift from Dr. Phillip Stevenson) [75] . Cells were then incubated for 45 minutes at room temperature. Following incubation, cells were centrifuged twice in buffer A (five minutes, 10006g), resuspended in either buffer A or secondary staining reagent in buffer A (1:500), and incubated for 20-30 minutes at room temperature. Secondary staining reagents were either streptavidin-APC (BD Bioscience) or anti-mouse IgG2a-FITC (Rat IgG1 k, clone R19-15) (BD Bioscience). Cells were centrifuged twice in buffer A (five minutes, 10006g) and analyzed by flow cytometry.
For forward versus side scatter analysis, MB114 cells harvested at six days post-infection were analyzed. Uninfected MB114 cells were detached from flasks with 0.5 mM EDTA and analyzed in parallel with infected cells. In certain experiments, to determine the effect of viral DNA replication and/or late gene synthesis on forward and side scatter properties, MB114 cells were treated with 200 mg/mL phosphonoacetic acid (PAA) after one hour of infection. At six days post-infection, non-adherent cells were harvested and counted by trypan blue exclusion to determine the percent of cells infected that were viable and non-adherent at time of harvest. Cells which remained adherent to the flask were then detached with 0.5 mM EDTA, combined with the harvested nonadherent cells, and analyzed by flow cytometry for forward and side scatter properties. Effective block of late gene synthesis was confirmed by plaque assay titer of treated cells. Viral titer was reduced 96.2% after 24 hours and 99.9% after six days as compared to untreated cells. 10 micron control beads were run in parallel with each experiment (Beckman Coulter, Fullerton, CA).
Cells were collected and boiled in Laemmli buffer for 10 minutes. Total protein concentration of each cell extract was determined by Lowry assay (DC protein assay kit, Bio-Rad, Hercules, CA). 10-20 mg of total protein per extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electrotransferred (ThermoFisher Scientific, Portsmouth, NH) to PVDF membranes (Millipore, Billerica, MA) and blocked in PBS with 0.05% Tween-20 and 5% nonfat milk for one hour at room temperature. Western blots for cHV68 protein expression were incubated with 10 mg/mL monoclonal mouse anti-cHV68 gB [76] (kind gift of Dr. Phillip Stevenson), monoclonal mouse anti-cHV68 M3 at 1:50, polyclonal rabbit anti-cHV68 v-cyclin at 1:2000 (kind gifts of Dr. Herbert Virgin), and monoclonal antibody to mouse b-actin at 1:1000 (Sigma Chemical, St. Louis, MO). Blots were washed for 45 minutes in PBS with 0.05% Tween-20, then incubated with donkey antimouse or donkey anti-rabbit antibodies at 1:6000 for one hour. Blots were then washed in PBS containing 0.1% Tween-20 for 45 minutes. Proteins were visualized using an ECL Plus Western blotting detection kit (Amersham Pharmacia Biotech).
Total RNA was extracted from S11 cells, infected NIH 3T3 cells, infected MB114 cells, and murine lung cell fractions using mirVANA TM miRNA Isolation kit (Ambion, Austin, TX), per manufacturer's instructions. Amplifications were conducted using the following primer sets: cHV68 polIII-1 forward 59 CAA CAG GTC ACC GAT CC 39, cHV68 polIII -1 reverse 59 GGA AGT  ACG GCC ATT TC 39, cHV68 M2 forward 59 TAA GGA CCT  CGT AGA GAT TGG C 39, cHV68 M2 reverse 59 ACG TTA  AAG TCC CCA TGG AAG C 39, cHV68 v-cyclin forward 59  ATT AGC ACT GGG CGT TTC ATG 39, cHV68 v-cyclin  reverse 59 GAC CTC CGT CAG GAT AAC AAC 
Cells were pelleted (five minutes, 2086g), resuspended in 1 mL PBS, and pelleted again (five minutes, 2086g). Supernatant was removed and 2.5% glutaraldahyde solution (adjusted to pH 7.4 using HCl and to 400 mOsm using CaCl 2 ) added. Samples were processed and imaged by Dr. Gary Mierau, The Children's Hospital Department of Pathology/Laboratory Services, Aurora, CO. Briefly, samples were post-fixed in 2% cacodylate buffered osmium tetroxide (pH 7.4), dehydrated in a graded series of alcohols, and embedded in epoxy resin. Sections, approximately 80 nm in thickness, were stained with uranyl acetate and lead citrate prior to examination at 60 kV with a Zeiss EM-10 transmission electron microscope (Carl Zeiss Inc, Thornwood, NY).
Lung tissues were removed from CD8-alpha knock-out mice six days post-intranasal inoculation of 1610 6 PFU cHV68. Tissues were enzymatically digested and endothelial cells purified using the endothelial cell marker CD31 (PECAM-1) (Fig. S4A) . Briefly, cells were stained with the following antibodies: anti-CD31-biotin (PECAM-1, clone MEC 13.3, rat IgG2a k) (BD Bioscience), anti-CD45-PE (B220, clone RA3-6B2, Rat IgG2a k) (BD Bioscience), anti-F4/80-PE (clone BM8, Rat IgG2a k) (eBioscience, San Diego, CA), anti-CD4-PE (L3T4, clone RM4-5, Rat IgG2a k) (eBioscience), anti-CD8a-PE (Ly-2, clone 53-6.7, Rat IgG2a k) (BD Bioscience), and anti-Ly-6G and Ly-6c-PE (Gr-1, clone RB6-8C5, Rat IgG2b k) (BD Bioscience). Cells were magnetically labeled with Anti-Biotin MultiSort beads, separated with Octo-MACS separation unit, and released from the MultiSort beads as per manufacturer's instructions (Miltenyi Biotec Inc., Auburn, CA). Released cells were incubated with Anti-PE MultiSort beads and depleted of contaminating PE positive cells by magnetic separation. Flow cytometric analysis was performed to confirm flow through fraction from PE column as enriched in CD31 positive cells and depleted of PE positive cells (Fig. S5B) .
Limiting dilution nested PCR detection of cHV68 genome-positive cells
The frequency of lung cells from CD8-alpha knock-out mice containing cHV68 genome was determined using a previously described nested PCR assay (LD-PCR) with single-copy sensitivity to detect gene 50 of cHV68 [77] . Briefly, freshly isolated cells were counted, resupsended in isotonic solution, and then diluted in 10 4 uninfected NIH 3T12 cells prior to serial dilution plating. Plated cells were lysed overnight in proteinase K, followed by two rounds of nested PCR. Reactions were performed on 12 replicates per dilution per sample and products resolved on a 2% agarose gel and identified by ethidium bromide staining. PCR sensitivity was quantitated using 10, 1, or 0.5 copies of a gene 50 containing plasmid (pBamH I N) diluted in 10 4 uninfected NIH 3T12 cells.
Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). Data were analyzed using the paired Student's t test to determine statistical significance. Limiting dilution data were subjected to nonlinear regression analysis, and the frequency of genome-positive cells was calculated using the Poisson distribution to assume that the cell number at which 63.2% of events were detected corresponded to the occurrence of a single event. 
Text S1 Endothelial cell specific markers included CD31 and CD54. CD80 and CD86 were included as non-endothelial cell specific markers, though a previous report has identified low level CD80 expression on primary murine lung endothelial cells [71] . Fluorescence was determined relative to unstained cells (grey). Cell morphology and surface expression was similar to previously characterized primary endothelial cells [71] . Found at: doi:10.1371/journal.ppat.1000152.s006 (0.89 MB TIF) Figure S5 Isolation and characterization of murine lung endothelial cells infected in vivo. (A) Primary murine lung endothelial cells were isolated for analysis of infection in vivo as described in Materials and Methods. Lung cells were stained for the endothelial cell marker CD31, as well as with a cocktail of PE labeleled antibodies specific to the following cell types (which includes potential contaminating infected cells): macrophages, granulocytes, CD8 + T lymphocytes, CD4 + T lymphocytes, and B lymphocytes. CD31 positive cells were enriched from total lung cells and then finally depleted of cells stained with the PE cocktail. (B) Flow cytometric analysis of lung cell separation following in vivo infection was performed on total lung cells and PE depleted/ CD31+ enriched cells. Left and middle panels show representative data (1610 6 cells per stain) from an infected mouse (Fig. 8D ). Gates were set on unstained cells, and data are representative of the four mice analyzed in Fig. 8D . Splenocytes (right panel) were included as a staining control. Found at: doi:10.1371/journal.ppat.1000152.s007 (0.20 MB TIF) Analysis of synonymous codon usage and evolution of begomoviruses Begomoviruses are single-stranded DNA viruses and cause severe diseases in major crop plants worldwide. Based on current genome sequence analyses, we found that synonymous codon usage variations in the protein-coding genes of begomoviruses are mainly influenced by mutation bias. Base composition analysis suggested that the codon usage bias of AV1 and BV1 genes is significant and their expressions are high. Fourteen codons were determined as translational optimal ones according to the comparison of codon usage patterns between highly and lowly expressed genes. Interestingly the codon usages between begomoviruses from the Old and the New Worlds are apparently different, which supports the idea that the bipartite begomoviruses of the New World might originate from bipartite ones of the Old World, whereas the latter evolve from the Old World monopartite begomoviruses. Synonymous codon usage bias has been investigated in many organisms, as the genetic code is degenerate. The synonymous codons are also non-randomly used in viruses infecting living organisms. Several factors such as mutational bias (Jenkins and Holmes, 2003; Gu et al., 2004; Zhou et al., 2005) , translational selection (Sau et al., 2005a; 2005b; 2005c) , gene function (Wang et al., 2002; Gu et al., 2004; Zhou et al., 2005) , gene length (Sau et al., 2005a) , and CpG island (Shackelton et al., 2006) were found to influence codon usage in animal viruses and phages, and mutational bias was found as the major determinant factor. Adams and Antoniw (2004) also suggested that mutational bias rather than translational selection was the major determinant of codon usage variation amongst plant viruses.
Geminiviruses (family Geminiviridae) are single-stranded DNA (ssDNA) viruses that cause severe disease in major crop plants worldwide. Most geminiviruses belong to the genus Begomovirus, which are transmitted exclusively by the whitefly Bemisia tabaci (Harrison and Robinson, 1999) . Many begomoviruses have bipartite genomes known as DNA A and DNA B. DNA A contains the AV1 (coat protein) and AV2 ORFs (open reading frames) in the virus strand and, on the complementary strand, four ORFs: AC1 (replication initiation protein), AC2 (transcriptional activator protein), AC3 (replication enhancer protein) and AC4. The virus and complementary strands of DNA B contain two ORFs: BV1 (nuclear shuttle protein) and BC1 (movement protein). Some begomoviruses have a monopartite genome and lack DNA B. Phylogenetic analysis shows that begomoviruses can be generally divided into two groups, the Old World begomoviruses (eastern hemisphere, Asia, Africa, Europe, the Mediterranean areas) and the New World begomoviruses (western hemisphere, the Americas). All the New World begomoviruses are evolved to bipartite with lack of AV2 ORF in DNA A, whereas both bipartite and monopartite begomoviruses in the Old World encode AV2 ORF (Harrison and Robinson, 1999) .
Because of their destructive effect on cash crops (Moffat, 1999; Moriones and Navas-Castillo, 2000; Mansoor et al., 2006) , numerous studies on begomoviruses have been conducted to understand their symptoms, host range, distribution, genome structure, gene function, and so on Zhou et al., 2003) . In this paper, we report the analysis of codon usage bias in begomoviruses and also perform an evolutionary analysis based on their codon usage pattern.
The complete genomic sequences of 147 begomovirus species were downloaded from the GenBank database, from which a total of 932 variants of the 8 known protein-coding genes were extracted. To minimize sampling errors, 915 variants were selected for further analysis by the following sifting standard: (1) the selected genes should be complete coding DNA sequences (CDS) with correct initial and terminal codons; (2) only those CDS including at least 80 codons were selected in the dataset; (3) those CDS with uncertain annotation or annotated as hypothetical protein-coding genes were excluded from this study.
GC content is the frequency of G+C in a coding gene. GC1, GC2 and GC3 contents are the frequencies of G+C at the first, second and third positions of codons, respectively. A3, T3, G3 and C3 contents are the frequencies of A, T, G and C at the synonymous third position of codons, respectively. Effective number of codons (N c ), ranging from 20 to 61, is generally used to measure the bias of synonymous codons. When N c value approaches 20, only one codon is used with extreme bias for one amino acid and, if the value is up to 61, the anonymous codons are used equally with no bias (Wright, 1990) .
Relative synonymous codon usage (RSCU) is defined as the ratio of the observed frequency of codons to the expected frequency given that all the synonymous codons for the same amino acids are used equally. RSCU values have no relation to the amino acids usage and the abundance ratio of synonymous codons, which can directly reflect the bias of synonymous codon usage (Sharp and Li, 1986) . The codon adaptation index (CAI) was used to estimate the extent of bias towards codons that were known to be preferred in highly expressed genes (Sharp and Li, 1987) . It is now proved that CAI values mostly approach the theoretical values to reflect the expression level of a gene. Thus it has been widely utilized to measure the gene expression level (Naya et al., 2001; Gupta et al., 2004) . A CAI value ranges from 0 to 1.0, and a higher value indicates a stronger codon usage bias and a higher expression level.
CodonW version 1.4.2 (John Peden, available at http://sourceforge.net/projects/codonw/), an integrated program, was utilized for calculating GC, GC3 contents and N c values and then carrying out correspondence analysis (CA), while GC, GC1, GC2 and GC3 contents were calculated by practical extraction and report language (PERL) scripts which were written by us. A3, T3, G3 and C3 contents, as well as RSCU and CAI values, were also calculated by using PERL scripts.
CA is the most commonly used multivariate statistical analysis at present (Greenacre, 1984) . This method can successfully present variation trends among genes, and then distribute them along the continuous axis by using RSCU value as variable data. In CA, all genes were plotted in a 59-dimensional hyperspace, according to the usage of the 59 sense codons. Major variation trends can be determined using these RSCU values and genes ordered according to their positions along the major axis, which can also be used to distinguish the major factors influencing the codon usage of a gene.
The set of reference sequences used to calculate CAI values in this study were the genes coding for coat proteins. According to the calculated CAI values, 5% of the total genes with extremely high and low CAI values were regarded as the high and low dataset, respectively. Then we calculated the average RSCU values of the two gene samples and subtracted them subsequently in each dataset group (ΔRSCU). If the ΔRSCU values are larger than 0.08, then this codon will be defined as the optimal codon (Duret and Mouchiroud, 1999) .
In order to examine the base composition variation among different genes, the base composition of different protein-coding genes was calculated. Table  1 shows that with the exception of the AC3 gene that has a lower average GC content (0.391), no obvious difference in GC content was found among other tested genes. However, differences in GC content at the different synonymous positions of codons were apparent. For example, GC content at the synonymous first position of codons for the AV2 gene is 0.563, while that for the AC4 gene is only 0.412. GC content at the synonymous third position of codons for the AC4 gene is 0.534, while that for the BV1 gene is only 0.399.
It was also observed that the average percentage of GC content was generally higher at the first than at the second codon position (Table 1) , except for the AC4 gene whose GC2 content was larger than GC1 content. This result suggested that the AC4 gene might have a special codon usage pattern.
In addition we found that AV1 and BV1 genes had a tendency to usage bias at the synonymous third codon position. The AV1 gene does not tend to use A(T)-ending or G(C)-ending codons but tends to use T-ending codons relative to A-ending codons. However, BV1 gene apparently uses T-ending codons and seldom used C-ending codons. Therefore, AV1 and BV1 genes should show a stronger codon usage bias among the begomovirus genes.
An N c plot (a plot of N c vs GC3 content) was widely used to investigate the determinants of the codon usage variations among genes in different organisms. It was suggested that if GC3 content was the only determinant of the codon usage variation among the genes, then the N c value would fall on the continuous curve between N c value and GC3 content (Wright, 1990 ). In general, if genes are distributed in N c plots approaching the expected continuous curve with no selection, then codon usage bias of genes is mainly influenced by compositional constraints. Otherwise the codon usage bias of genes is more affected by other factors such as translational selection, etc. The N c plot for genes of the 147 begomoviruses showed that although a small number of genes were located on the expected curve, most points lay far below the expected curve ( Fig.1) , suggesting that apart from mutation bias, other factors might also play a role in shaping the codon usage bias of begomoviruses (Guo et al., 2007) . Table 1 The GC content at the different codon positions and the A, T, G and C contents at the third position of begomovirus genes Base composition analysis suggested that the codon usage of the AC4 gene, which is always embedded in the AC1 gene, might differentiate from other genes. In addition, strong codon bias was observed in AV1 gene. Because of the special characteristics of the abovementioned genes, we selected them for examining the influence factors in shaping the codon usage (Fig.2a) . The N c plots of AV1, AC1 and AC4 genes suggested that apart from compositional constraints, other factors might play important roles in shaping their codon usage, although high translational selection seems to lay stress on the AC4 gene because of the wide range of N c values for the same GC3 content.
To examine the reason for N c variation under the same GC3 content, the relationship between the GC1, GC2 and GC3 contents for AV1, AC1 and AC4 genes was further examined (Fig.2b) . It was observed that the GC1 content was always higher than GC2 content for the AV1 and AC1 genes, whereas the GC1 content of the AC4 gene was generally lower than the GC2 content. This result was coincident with the base composition analysis (Table 1) . Thus the variations of the synonymous first and second codon positions for the AC4 gene might be caused by the translational selection utilizing G or C at the synonymous second position.
As the AC4 gene is always embedded in the AC1 gene, the codon usage pattern of the AC4 gene might be influenced by the AC1 gene. The sequences of AC1 and AC4 genes were aligned. It was found that the synonymous third codon position of the AC1 gene corresponded to the second codon position of the AC4 gene for all the begomoviruses. Thus the base compositional environment of the AC1 gene might also influence the codon usage pattern of the AC4 gene. It could be postulated that the second codon position of the AC4 gene that tended to use G or C should be influenced by compositional constraints other than translational selection.
According to the variation analysis of base composition for the genes of begomoviruses, the AV1 gene was selected as a reference dataset to calculate the CAI values of all genes. The correlation analysis between CAI value and the positions of the genes along the first two major axes (generated by correspondence analysis), as well as other indices, was then calculated. It was observed that the CAI values were negatively correlated with the GC3 and GC contents (r=−0.234 and r=−0.535 respectively, P<0.01), while significantly positively correlated with axis 1 (r=0.587, P<0.01). Moreover the CAI value was also significantly negatively correlated with the N c value, indicating a tendency to a higher CAI value or a lower N c value and a higher expression level for begomovirus genes. Thus it is feasible to use the AV1 gene as a reference dataset for our estimation of the CAI value of begomovirus genes.
Based on the calculated CAI values, 5% of the total genes with extremely high and low CAI values were regarded as the high and the low datasets, respectively. Then we compared the codon usage of the high dataset to the low dataset. Table 2 shows that 14 codons that code 13 amino acids were apparently used at a high level, and can be determined as translational optimal codons. Out of the 14 optimal codons, 5 were ended with G, 1 with C and 8 with T.
CA was performed on the RSCU value based on the concatenated genes for each begomovirus genome. Fig.3 shows the positions of all tested begomoviruses along the first two major axes. The first major axis accounted for 15.2% of the total variations, while the second major axis accounted for 9.9% of the total variations.
In order to detect the codon usage variation of different genomes, the begomoviruses were divided into three groups including the Old World begomoviruses with monopartite genomes (OM), the Old World begomoviruses with bipartite genomes (OB), and the New World begomoviruses with bipartite genomes (NB). The distribution of the three groups along the first two major axes showed that the Old World monopartite begomoviruses and the New World bipartite ones were located in two independent fields, indicating that the two groups of begomoviruses exhibit a different codon usage pattern. Because the species with a close genetic relationship always present a similar codon usage pattern (Sharp et al., 1988) , the genetic relationship of the two groups of begomoviruses should be far removed from each other. As to the Old World begomoviruses with bipartite genomes, we found that the majority of them exhibited a similar codon usage pattern with the Old World monopartite begomoviruses, and a few of them showed a similar codon usage to the New World bipartite begomoviruses. An explication of this result might be that a number of Old World bipartite begomoviruses evolved to adopt the codon usage pattern of some Old World monopartite begomoviruses.
Moreover, the New World bipartite begomoviruses were closely related to a small number of the Old World bipartite ones and far removed from the Old World monopartite ones.
The results of the N c -plot and base composition analysis indicated that the codon usage pattern of begomoviruses was influenced by mutation bias as well as other factors such as translational selection. Comparative analysis of AC1 and AC4 genes showed that the compositional environment of the former genes might play a role in dictating the codon usage of the latter gene. Thus, although it seems that strong translational selection might have an influence on shaping the codon usage of AC4 genes, the compositional constraints derived from AC1 genes might be the major determinant in determining codon usage.
Consequently it can be speculated that mutation bias might play a major role in shaping the codon usage pattern of begomoviruses.
As to the gene itself, the selection pressures from the external environment always act as effective factors in promoting the gene to adapt to the change in the external environment. On the other hand, direct changes to a gene will interfere with or be harmful to the gene. Therefore, the base mutation at the synonymous third codon position may not affect the protein expression for AC1 gene because of the degeneration of genetic codons. But for the embedded AC4 gene, the corresponding base mutation occurs at the second codon position, which probably results in the loss of function of the translated protein. Thus we suggested that AC4 gene might degenerate step by step during the long period of evolution, which might be an important reason for explaining the loss of function for the AC4 gene in the bipartite begomoviruses with DNA B.
The variation analysis of the base composition for begomovirus genes showed that AV1 and BV1 genes exhibit a stronger codon usage bias and a higher gene expression level. Thus CAI values of different gene samples were calculated using the AV1 gene as a reference set. The results of correlation analysis indicated the reliability of choosing AV1 gene as a high expression gene sample for begomoviruses. Then 14 codons were determined as the major optimal codons for begomoviruses. That will be very important during the design of degenerate primers, the introduction of point mutation, the modification of the virus genes and the investigation of the evolution mechanism of species at the molecule level.
It was speculated that monopartite begomoviruses emerged approximately 130 million years ago (Rybicki, 1994; Mansoor et al., 2006) , suggesting that the begomoviruses should have evolved from the original monopartite viruses. Rybicki (1994) suggested that the significant expansion of the New World begomoviruses might have occurred after the transmission of the Old World begomoviruses to those of the New World by whiteflies. Based on codon usage pattern analysis, it could be inferred that there was no direct relationship between the Old World monopartite begomoviruses and the New World bipartite ones. Interestingly, a small number of the Old World bipartite begomoviruses exhibited a similar codon usage pattern to the New World bipartite ones, which suggested that the ancestor of begomoviruses could have evolved from monopartite to bipartite ones before they were transferred to the New World areas. Subsequently it was not the Old World monopartite begomoviruses but the Old World bipartite ones that transmitted to the New World in a certain way and finally evolved into the New World bipartite begomoviruses. In other words, the New World bipartite begomoviruses probably resulted directly from the Old World bipartite ones, while the latter evolved from the Old World monopartite begomoviruses.
It still remains unclear whether the New World begomoviruses directly evolved from the Old World bipartite begomoviruses because of the sharp environmental change after their transfer to the New World, or whether the Old World bipartite begomoviruses had evolved into the New World bipartite ones before their transmission to the New World. Rybicki (1994) suggested that the absence of the AV2 gene in all the New World begomoviruses could be attributed to its earlier loss after the New World begomoviruses arriving in the New World. These results suggest that the current New World begomoviruses evolved from ancient viruses after transferring to the New World. Ha et al.(2006) speculated that the new identified begomovirus termed Corchorus yellow vein virus (CoYVV) might belong to a New World begomovirus group that previously existed in the Old World, which suggested that the common ancestor of the New World begomovirus might originate from the Old World begomovirus. However, both the New World and Old World begomoviruses had began to evolve and coexisted in this area for a long time before the separation of the continents. In other words, the present New World begomoviruses might have evolved in the Old World, and then moved to the New World by some unknown means (Ha et al., 2006) . Animal models of acute lung injury Acute lung injury in humans is characterized histopathologically by neutrophilic alveolitis, injury of the alveolar epithelium and endothelium, hyaline membrane formation, and microvascular thrombi. Different animal models of experimental lung injury have been used to investigate mechanisms of lung injury. Most are based on reproducing in animals known risk factors for ARDS, such as sepsis, lipid embolism secondary to bone fracture, acid aspiration, ischemia-reperfusion of pulmonary or distal vascular beds, and other clinical risks. However, none of these models fully reproduces the features of human lung injury. The goal of this review is to summarize the strengths and weaknesses of existing models of lung injury. We review the specific features of human ARDS that should be modeled in experimental lung injury and then discuss specific characteristics of animal species that may affect the pulmonary host response to noxious stimuli. We emphasize those models of lung injury that are based on reproducing risk factors for human ARDS in animals and discuss the advantages and disadvantages of each model and the extent to which each model reproduces human ARDS. The present review will help guide investigators in the design and interpretation of animal studies of acute lung injury.  Intrapulmonary administration of recombinant activated factor VII in diffuse alveolar haemorrhage: a report of two case stories BACKGROUND: Diffuse alveolar haemorrhage (DAH) is a serious pulmonary complication characterised by a high mortality rate and the absence of specific treatment. The intrapulmonary administration of activated recombinant factor VII (rFVIIa) in DAH was recently published in six patients by Heslet et al with an efficient hemostatic effect. We describe two cases of DAH treated with intrapulmonary rFVIIa. METHODS: Two cases of DAH were admitted to the ICU after presenting abrupt desaturation, tachypnea, cough and haemoptysis, requiring orotracheal intubation and mechanical ventilation. The diagnosis was achieved by the bloody return during the bronchoalveolar lavage, during the procedure rFVIIa (50 μg/Kg in 50 ml of isotonic saline) was administered via the bronchoscope. RESULTS: Immediate cessation of bleeding was observed. Prior to intrapulmonary administration of rFVIIa, the FiO(2 )was 1, which was reduced to 0.4 24 hours later. Following the procedure, the haemostatic effect made blood transfusion superfluous. No thrombotic complications associated with administration of the drug were observed. After the intervention both cases progressed fast and was discharged from the ICU with no further episodes of bleeding. CONCLUSION: 1. Local intrabronchial deposition of DAH with rFVIIa has been shown to be effective in controlling life-threatening DAH. 2. In the case described above, no thrombotic complications were observed following the intrapulmonary administration of rFVIIa. Conclusion: 1. Local intrabronchial deposition of DAH with rFVIIa has been shown to be effective in controlling life-threatening DAH. 2. In the case described above, no thrombotic complications were observed following the intrapulmonary administration of rFVIIa.
Diffuse alveolar haemorrhage (DAH) is a serious pulmonary complication, characterised by the presence of haemoptysis, dyspnea, hypoxemia and anaemia, with a high mortality rate of over 50% of patients requiring mechanical ventilation [1] . Diffuse opacities found on X rays in patients with DAH, however, are unspecific [2] .
DAH is a complication of systemic diseases, and frequently manifests as an initial sign of these [3] . Bronchoalveolar lavage (BAL) is the most useful procedure for confirming initial clinical suspicion, BAL with a bloody return is the only way to confirm the diagnosis and, at times, fiberoptic bronchoscopy provides the treatment. In a new development, its use in intrapulmonary administra-tion of recombinant activated factor VII (rFVIIa) has been reported in six patients [4] .
The drug (rFVIIa) promotes local formation of thrombin when it combines with tissue factor exposed at the level of the endothelium. It was initially indicated to treat coagulopathies in patients suffering from haemophilia, although its use has extended to other haematological conditions. Beyond these indications, it is administrated on a more compassionate level. The requirements for its effective use in controlling serious haemorrhages are fibrinogen and platelet readings of over a 50 mg/dl and with 50.000/μl respectively, in addition to a pH > 7.20.
We present two cases of massive haemoptysis in which local administration of rFVIIa (Novoseven ® from Novonordisk) via BAL was used as an emergency measure.
Two cases of life-threatening DAH were admitted to the ICU after presenting abrupt desaturation, tachypnea, cough and haemoptysis, requiring orotracheal intubation and mechanical ventilation. Both cases were diagnosed with fiberoptic bronchoscopy and treated with local rFVIIa.
A 39 year old woman, with a personal history of acute promyelocytic leukaemia treated with chemotherapy and renal failure with recent arteriovenous fistula intervention. The patient was admitted to the ICU with hypoxemic respiratory failure with cough and haemoptysis, requiring orotracheal intubation and mechanical ventilation. The thorax X-ray showed a "patchy" infiltrate affecting bases and middle fields. Values of haemoglobin with 11,4 decreased to 7,9 g/dl concomitant with reduced platelet count from 100 × 10[3]/μL to 40 × 10 [3] /μL during the first 24 hours. Suddenly, the patient deteriorated with an abrupt desaturation and frank haemoptysis through the orotracheal tube. In order to achieve platelet readings of over 50,000/μL a transfusion of 8 units of platelets was necessary before the drug could be administered. An emergency fibrobronchoscopy confirmed DAH. Systemic administration of rFVIIa was considered, but the potential thrombogenic effect of the drug and the risk of obstruction of the recent arteriovenous fistula prompted us to decide to administer 50 μg/Kg of rFVIIa in 50 ml of isotonic saline via the bronchoscopy channel, 25 ml in each main bronchus; following this, immediate cessation of bleeding was observed. Prior to this, the families had been informed and their written consent obtained.
The inspired fraction of oxygen (FiO 2 ) which, before intrapulmonary administration of rFVIIa had been 1, was reduced to 0.6 during the first three hours subsequent to the administration of rFVIIa, and to 0.4 over the following 24 hours (figure 1). Haemoglobin readings remained unchanged without the need for blood transfusion. Although there were no new episodes of active DAH, weaning from ventilator was retarded due to muscular weakness. The patient was extubated at day 16 in the ICU and discharged to stationary ward without recurrent bleeding.
Changes in the FiO 2 following rFVIIa administration in two cases treated with intrapulmonary rFVIIa Figure 1 Changes in the FiO 2 following rFVIIa administration in two cases treated with intrapulmonary rFVIIa.
A 46 year old man, with a personal history of smoking, ex parenteral drug abuse, hepatitis B and C infection, hepatic cirrhosis evolving for years and HIV infection diagnosed in 1988; currently receiving antiretroviral treatment. He was admitted to the ICU with acute inferoposterior myocardial infarction. Treatment was commenced with low molecular weight heparin and double anti-aggregation therapy with aspirin and clopidogrel. After 24 hours, he suddenly developed haemoptysis, acute hypoxemic respiratory failure and bilateral crackles, requiring orotracheal intubation and mechanical ventilation with FiO 2 of 1 to maintain a SpO 2 of 85-90%. The BAL return was increasingly bloody from both lungs. Due to the potential thrombogenic effect of systemic rFVIIa administration in acute myocardial infarction, and after informing and obtaining consent from the family, we decided on intrapulmonary administration of the drug at a dosage of 50 μg/Kg in 50 ml of isotonic saline via the bronchoscopy channel, observing the immediate cessation of bleeding. The FiO 2 was reduced to 0.5 over the first three hours and to 0.35 after 24 hours (figure 1). After 12 days on mechanical ventilation the patient was extubated and transferred to a stationary ward.
Systemic administration of rFVIIa to patients with lifethreatening conditions due to active haemorrhage has increased in clinical practise, based more on presumed expectation than on scientific evidence supported by controlled and randomised studies [5] .
Edward et al carried out a survey in the American College of Chest Physicians (ACCP) in 1998 on the treatment of acute haemoptysis: 85% of specialists answered that intubation and connection to mechanical ventilation must be performed at an early stage and 64% considered it mandatory to carry out a fibrobronchoscopy during the first 24 hours [6] . Consequently, treatment undertaken in both clinical cases presented -mechanical ventilation support and fibrobronchoscopy -meets these recommendations and was performed as an emergency measure to treat a massive haemoptysis episode. In middle-sized hospital, where selective embolization of the bronchial artery techniques and/or thoracic surgery are not available, systemic rFVIIa has been used in massive haemoptysis cases, with good results [7] . DAH in haematological patients requiring mechanical ventilation has a high mortality rate of over 70% in series described [8, 9] . This is due to the absence of specific treatment for DAH of haematological origin. The results of a multicentre, randomised study on the efficacy and safety of three different dosages of systemic rFVIIa compared with a placebo in treating haemorrhagic complications in 100 bone-marrow transplant patients (seven with DAH) were inconclusive, 8% of thromboembolic events were observed in the group treated with rFVIIa [10] .
Pulmonary haemorrhage associated with myocardial infarction thrombolysis is an unusual complication; in 1996, Chang YC et al published a retrospective study, finding an incidence of 0.4% [11] . The same is true of platelet antiaggregation treatment, where isolated cases of DAH have been described [12] . In spite of its low incidence, DAH secondary to acute coronary syndrome treatment is a complication which maybe undetected, due to the common radiological findings in both DAH and ALI/ARDS and acute pulmonary oedema [13] . Bearing in mind the thrombogenic risk of a systemic administration of local rFVIIa, and with Heslet et al's publication as a reference, in which 6 consecutive critically ill patients with acute DAH are treated with local rFVIIa, intrapulmonary administration of the drug was chosen in the cases here presented with a view to avoiding the risk of thrombosis of the arteriovenous fistula in the first case, and of reinfarction in the second [4, 10] .
DAH is a life-threatening disease characterised by the lack of specific treatment and a high mortality of patients requiring mechanical ventilation. Bronchoscopy BAL with increasingly bloody return is the only diagnostic procedure for the diagnosis of DAH, and at times, provides the treatment. Local administration of rFVIIa via the fibrobronchoscope channel was used as an emergency measure in two cases of massive haemoptysis with an excellent hemostatic effect and without adverse effects.
ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome; BAL: Bronchoalveolar lavage; DAH: Diffuse alveolar haemorrhage; FiO 2 : Inspired fraction of oxygen; rFVIIa: Recombinant activated factor VII; HIV: Human inmunodeficiency virus; SpO 2 : Pulse oxygen saturation. Activation of the Unfolded Protein Response Is Required for Defenses against Bacterial Pore-Forming Toxin In Vivo Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack. Pore-forming toxins (PFTs) are the single most prevalent protein virulence factor made by disease-causing bacteria and are important for the virulence of many important human pathogens including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and Aeromonas hydrophilia [1, 2] . Crystal (Cry) toxins produced by the invertebrate pathogen Bacillus thuringiensis (Bt) are a large family of PFTs that target the intestinal cells of insects and nematodes [3, 4, 5] . The fact that some Cry proteins target nematodes, in particular C. elegans, has been exploited to provide the only in vivo genetic model for studying PFTs. This system led to the discovery of the first signal transduction pathway that protects cells against PFTs, the p38 mitogen-activated protein kinase (MAPK) pathway, which has been confirmed in mammalian cells [6, 7] . There is growing evidence that the response of cells to PFTs is, however, complex and there is a great deal yet to learn [8] .
The unfolded protein response (UPR) of the endoplasmic reticulum (ER) is a fundamental stress response used by eukaryotic cells to match protein synthesis demand to its capability to fold proteins within the ER to maintain cellular homeostasis [9] . In C. elegans and other animals there are three transducers that signal from the ER to activate this response. These three distinct arms of the UPR are mediated by IREI, ATF6, and PERK in mammals [10] , which correspond to the genes ire-1, atf-6, and pek-1 in C. elegans [11, 12, 13] . All three pathways are regulated by the ER chaperone BiP in response to an increase in unfolded proteins [9] .
Here we demonstrate that the ER stress response, in particular the ire-1 arm, is activated upon exposure of C. elegans and mammalian cells to PFTs. We demonstrate for the first time that the ire-1 -xbp-1 arm of the UPR (and to a lesser extent the atf-6 arm) is functionally important for defense against a pathogenic attack since loss of this pathway leads to animals hypersensitive to PFT, but not to other toxic insults. Furthermore, we demonstrate that activation of the ire-1-xbp-1 pathway by PFT requires p38 MAPK and its associated MAPK kinase and that the in vivo response of the UPR to a PFT can be separated from its response to unfolded proteins. These results indicate that activation of the UPR plays an important role in cellular defenses against pathogens.
In a genetic screen for genes involved in the cellular response of C. elegans to the PFT Cry5B, we found a mutant predicted to be defective in protein N-glycosylation in the ER (L.J.B. and R.V.A., manuscript in preparation). Since defects in protein glycosylation induce the UPR, this result suggested that perhaps the UPR might play a role in protection against PFTs. To test this hypothesis, we first investigated whether or not the UPR was activated by a PFT. The xbp-1 gene is spliced upon activation of the IRE-1 branch of the UPR, and its splicing is one marker for IRE-1 (and UPR) activation [13] . In C. elegans, the xbp-1 intron spliced by IRE-1 is 23 nucleotides and the induction of this splicing event can be detected by RT-PCR [14] . To analyze xbp-1 mRNA transcript splicing, animals were fed Escherichia coli expressing Cry5B and compared to worms fed control E. coli ( Figure 1A ). While there is abundant unspliced xbp-1 mRNA transcript in both samples, there is an increase in the spliced xbp-1 transcript from worms ingesting Cry5B, indicating activation of the IRE-1 pathway. Quantitative analyses indicate that the xbp-1 spliced transcript increases 2.3, 3.0, and 3.0 fold at the 7, 8, and 9 h time points respectively.
To independently test this result, we analyzed the in vivo expression of an ire-1 regulated gene, hsp-4, a BiP homolog. In vivo analysis of the hsp-4 promoter coupled to green fluorescent protein (GFP) demonstrated expression of this gene requires ire-1 and xbp-1 [13] . A C. elegans strain containing hsp-4::GFP was fed either control E. coli or Cry5B expressing E. coli for 8 hours at 20uC. As shown, a strong and specific increase in GFP expression in the intestine can be seen in the presence of the PFT ( Figure 1B , middle panel), consistent with activation of the ire-1-xbp-1 pathway by Cry5B. Heat shock of this strain in the absence of Cry5B confirms GFP could be induced in other cell types in addition to the intestine ( Figure 1B, right panel) , as was demonstrated with the N-glycosylation inhibitor tunicamycin [13] . The fact that Cry5B only induced expression in intestinal cells suggests the PFT is only targeting these cells (see below).
To address whether the ire-1-xbp-1 pathway is also activated in mammalian cells in response to a PFT, activation of the pathway was ascertained in HeLa cells exposed to the Aeromonas hydrophila PFT, aerolysin. As detected by the presence of the spliced protein isoform of XBP-1, treatment of mammalian cells with a PFT also results in robust activation of the ire-1-xbp-1 pathway ( Figure 1C ).
The ER stress response is required for defense of C. elegans against Cry5B
To determine whether the ER stress response played a role in the defense of C. elegans against the PFT, C. elegans mutants in the ER stress response pathway were qualitatively compared to wildtype N2 animals in their susceptibilities to Cry5B. The mutants that were tested included those encoding the three ER stress transducer genes, atf-6(ok551), pek-1(ok275), and ire-1(v33), as well as xbp-1(zc12); these mutations are predicted or known to be loss of function mutations in their respective genes [11, 12, 13] . In the absence of Cry5B, the wild type and mutant worms are healthy adults with similar appearance, except ire-1(v33), which is clearly smaller than the other strains ( Figure 2A ). In the presence of lowmoderate levels of the PFT Cry5B, wild-type worms are slightly intoxicated compared to those found on control no-toxin plates, as evidenced by their smaller sizes and paler appearances (Figure 2A ). To the same extent seen with wild-type worms, atf-6(ok551) and pek-1(ok275) mutant animals are also slightly intoxicated on lowmoderate levels of the PFT Cry5B, indicating lack of either of these genes does not result in overt hypersensitivity or hyperresistance to Cry5B (Figure 2A) . However under the same conditions, the ire-1(v33) and xbp-1(zc12) mutant worms are more severely intoxicated than wild-type worms as they are relatively smaller and considerably paler compared to their corresponding no toxin controls. The hypersensitivity to Cry5B resulting from lack of ire-1 and xbp-1 was also seen using RNA interference (RNAi; data not shown), confirming the phenotype is caused by loss of function in these genes. We call this hypersensitivity phenotype ''Hpo'' for hypersensitive to pore-forming toxin.
The sensitivity to Cry5B of animals mutant for the three ER stress response pathways was quantitatively assessed using a dosedependent lethality assay ( Figure 2B ). From these data, an LC 5s s (lethal concentrations at which 50% of the animals die) were obtained (Table 1) . Our quantitative results confirm that ire-1(v33) and xbp-1(zc12) mutant animals are statistically more sensitive to PFT than wild type animals (Table 1 ) and thus are Hpo relative to wild type (caution is called for in interpreting the ire-1(v33) data since many of these animals also have significant overt defects, e.g., developmental delays which prevents them from being as well synchronized at the start of the assay compared to the other strains [11] ). Our results indicate that atf-6(ok551) mutant animals are also Hpo, albeit to a lesser extent (2.8 vs. 5.8 fold increase in sensitivity for atf-6 vs. xbp-1). Although atf-6(ok551) hypersensitivity was not discerned with the plate assay, it is likely that the quantitative lethality assay is a more sensitive test for Cry5B hypersensitivity than the qualitative plate assay. In contrast to xbp-1 and atf-6 mutant animals, the sensitivity of pek-1(ok275) mutant animals is not statistically different from that of wild-type animals ( Table 1) .
To independently confirm these results, we used a developmental assay to assess the relative sensitivity of the four ER stress response mutants to Cry5B. This experiment was performed by placing newly hatched L1 stage worms on plates containing different percentages of Cry5B expressing E. coli and then counting the worms that developed to either the L4 stage or adulthood ( Figure 2C ). In the absence of Cry5B, nearly all worms developed to the L4 stage or adulthood for all strains with the exception of ire-1(v33). This result confirms developmental defects previously seen with this mutant [11] , and it was therefore excluded from subsequent analyses. Wild type N2 and pek-1(ok275) were both similarly inhibited in their development by increasing percentages of Cry5B. Compared to N2 and pek-1(ok275) animals, though, both atf-6(ok551) and xbp-1(zc12) were Hpo, i.e., each is more developmentally inhibited by Cry5B than wild-type animals ( Figure 2C ). Because the ire-1-xbp-1 pathway has a more discernible effect on protection against Cry5B than atf-6, further experiments were focused on this arm of the ER stress response.
Pore-forming toxins (PFTs) are bacterial toxins that form holes at the plasma membrane of cells and play an important role in the pathogenesis of many important human pathogens. Although PFTs comprise an important and the single largest class of bacterial protein virulence factors, how cells respond to these toxins has been understudied. We describe here the surprising discovery that a fundamental pathway of eukaryotic cell biology, the endoplasmic reticulum unfolded protein response (UPR), is activated by pore-forming toxins in Caenorhabditis elegans and mammalian cells. We find that this activation is functionally important since loss of either of two of the three arms of UPR leads to hypersensitivity of the nematode to attack by PFTs. The response of the UPR to PFTs can be separated from its response to unfolded proteins both at the level of activation and functional relevance. The response of the UPR to PFTs is dependent on a central pathway of cellular immunity, the p38 MAPK pathway. Our data show that the response of cells to bacterial attack can reveal unanticipated uses and connections between fundamental cell biological pathways.
Taken together, the above results suggest that the ire-1-xbp-1 pathway functions to protect the host against the PFT Cry5B. However, an alternative explanation for our results is that animals mutant in this pathway (e.g., xbp-1 mutant animals) are sickly and have compromised health and therefore would respond poorly to any toxic insult. To address this alternative hypothesis, we tested whether xbp-1(zc12) animals are hypersensitive to two toxic chemical compounds, the heavy metal CuSO 4 (a toxic insult that kills with kinetics similar to Cry5B) and the oxidative stress agent H 2 O 2 (a toxic insult that kills rapidly). The mutant xbp-1(zc12) has the same sensitivity as wild type to killing by either CuSO4 or H 2 O 2 ( Figure 2D and 2E; Table 1 ). These data argue against the supposition that this mutant is hypersensitive to the PFT merely because it is generally unhealthy. Rather, the protective response is somewhat specific against the PFT. These conclusions are strengthened by the finding that C. elegans lacking the UPR respond normally to attack by the pathogenic bacteria Pseudomonas aeruginosa, which does not make a PFT ( Figure 2F and Table 1 ).
The xbp-1 pathway functions in the intestine to protect against Cry5B PFT Mosaic and expression analyses have shown that the targeting of intestinal cells by the PFT Cry5B is both necessary and sufficient to intoxicate worms [15, 16] . If the ire-1-xbp-1 pathway is functioning directly to protect against the effects of the PFT, then we would predict that the ire-1-xbp-1 pathway should function in the target cells of the toxin, the intestinal epithelial cells. Alternatively, the pathway might be functioning indirectly to protect against the effects of the PFT (e.g., it might hypothetically function in neurons that then sends protective signals to the intestine). Consistent with the first hypothesis, that the pathway is functioning directly in the target cells to protect against the PFT, we previously noted that a marker for downstream activation of the pathway, hsp-4, is turned on exclusively in intestinal cells ( Figure 1B , middle panel), although the pathway is capable of being activated throughout the worm by a more general stress, such as heat shock ( Figure 1B, right panel) . elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20uC for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30uC for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 mg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane. doi:10.1371/journal.ppat.1000176.g001 Figure 2 . Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria. (A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO 4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO 4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H 2 O 2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H 2 O 2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments. doi:10.1371/journal.ppat.1000176.g002
To directly demonstrate the role of xbp-1 in protecting intestinal cells against Cry5B, the intestinal specific app-1 promoter [17] was used to drive expression of xbp-1 in xbp-1(zc12) mutant animals to determine if expression in the intestine is sufficient to rescue the Hpo phenotype. As a negative control, GFP was similarly expressed under control of the app-1 promoter in xbp-1(zc12) mutant animals. In control animals, expression of the GFP solely in intestinal cells was confirmed (data not shown). As expected, the majority of wild-type N2 animals showed only a low-modest degree of intoxication upon exposure to 25% Cry5B-expressing E. coli ( Figure 3A , B; they were smaller and somewhat paler than the wild-type worms on control plates but were still quite active). Also as predicted, both xbp-1(zc12) mutant animals and xbp-1(zc12) mutant animals transformed with app-1::GFP were Hpo and intoxicated to similar extents ( Figure 3A , B; most animals were very pale, small, and inactive). In contrast, xbp-1(zc12) worms expressing xbp-1 under the app-1 promoter were significantly healthier than either untransformed or app-1::GFP transformed xbp-1(zc12) animals fed with Cry5B ( Figure 3A, B) . However, these app-1::xbp-1-transformed xbp-1(z12) worms were not as healthy as wild-type N2 under the same conditions. This partial rescue could indicate the expression of the artificial xbp-1 transgenes did not fully recapitulate wild-type xbp-1 expression levels and/or that there is some role for the ire-1 -xbp-1 pathway in other cell types.
Nonetheless, our results support a significant protective function for xbp-1 within the cells targeted by Cry5B.
Induction of ire-1-xbp-1 pathway's role in response to PFT but not unfolded proteins is regulated by the p38 MAPK pathway ER stress responses have been studied extensively for their role in protecting cells against unfolded proteins [10, 18] . One way to assess the role of the ER stress pathways in protecting against unfolded proteins is with the drug tunicamycin (a natural compound that leads to the accumulation of unfolded proteins in the ER due to its inhibitory effect on N-linked protein glycosylation [19] ). Previous data in C. elegans have indicated different sensitivities of the three ER stress response pathways for tunicamycin [11, 12] . Using a different toxicity assay, we have confirmed these observations: atf-6(ok551) mutant animals have a similar sensitivity to tunicamycin as wild-type animals whereas both xbp-1(zc12) and pek-1(ok275) mutant animals are more readily killed by tunicamycin (Figure 4) . These results are in contrast to the response of these different ER stress pathways to Cry5B, to which atf-6 mutant animals are more sensitive than pek-1 mutant animals. These data suggest that there are differences in how ER stress pathways are activated in response to unfolded proteins and to the PFT Cry5B. It is known that PFTs trigger the activation of p38 MAPK, which promotes cell survival and cellular defenses and which seems to play a central role in cellular responses to PFTs [6, 7, 20] . We therefore investigated whether PFT-mediated activation of the UPR and the p38 MAPK pathway might be connected.
We first investigated whether the ire-1-xbp-1 pathway plays a role in the PFT-induced activation of p38 by comparing the activation of the p38 MAPK in wild-type and xbp1(zc12) animals. We find that addition of Cry5B to wild-type C. elegans results in an increase in phosphorylated p38, indicating the p38 pathway is activated by a PFT in C. elegans just as it is in mammalian cells [20] ( Figure 5A ). We find that p38 activation occurs normally in xbp-1(zc12) mutant animals ( Figure 5A ), indicating that the UPR is not required for activation of p38 MAPK pathway in response to PFT. We extended this result using ttm-2, a downstream transcriptional target of the p38 MAPK pathway in response to Cry5B and a gene required for normal defense against Cry5B PFT [6] . Upregulation of ttm-2 mRNA was dependent on the p38 MAPK pathway but not dependent on xbp-1 ( Figure 5F ).
We next analyzed the reverse relationship between the ire-1-xbp-1 and the p38 MAPK pathways, namely whether the p38 MAPK pathway is required for PFT-induced activation of the ire-1-xbp-1 pathway. We find that activation of the ire-1-xbp-1 pathway in response to PFT is dependent on the p38 MAPK pathway, namely on sek-1, the MAPK kinase (MAPKK) gene upstream of p38, and on pmk-1, the p38 MAPK downstream of sek-1 ( Figure 5 ). We find that increased splicing (activation) of xbp-1 in response to Cry5B does not occur in sek-1(km4) MAPKK mutant animals ( Figure 5B) . Quantitatively, at the 3 h time point the spliced form of xbp-1 is induced 1.9 fold in animals with an intact p38 MAPK pathway and depressed 1.8 fold in sek-1(km4) MAPKK mutant animals relative to untreated controls. However, sek-1 is not absolutely required for splicing of xbp-1 since, in response to tunicamycin, splicing of xbp-1 is normal in sek-1(km4) mutant animals ( Figure 5C ). In agreement with these results, we find that in vivo activation of the downstream target of the ire-1-xbp-1 pathway, hsp-4::GFP, by Cry5B within intestinal cells does not occur in pmk-1(km25) p38 MAPK mutant animals ( Figure 5D ), whereas activation of hsp-4::GFP by tunicamycin does occur normally in pmk-1(km25) mutant animals ( Figure 5E ).
To independently confirm and extend these results, we analyzed a different downstream target of the ire-1-xbp-1 pathway. Using proteomics, we identified a protein, Y41C4A.11 (a homolog of the beta-prime subunit of the coatomer complex), that increased 4.6 fold in C. elegans animals exposed to Cry5B and whose increase was completely dependent on xbp-1 (see Materials and Methods and Protocol S1). The gene encoding this protein was previously demonstrated to be transcriptionally regulated by tunicamycin in an ire-1 and xbp-1 dependent manner [12] . Using real time PCR, we find that both hsp-4 mRNA and Y41C4A.11 mRNA are induced by either Cry5B or tunicamycin ( Figure 5F ). Consistent with activation of the ire-1-xbp-1 pathway by p38 MAPK in response to PFT but not unfolded proteins, the full induction of both mRNAs by Cry5B, but not tunicamycin, is dependent on sek-1 MAPKK. Interestingly, whereas induction of both mRNAs by Cry5B is lacking in xbp-1(zc12) mutant animals (confirming that activation of hsp-4 and Y41C4A.11 by PFT is via the UPR), both mRNAs are still somewhat induced by Cry5B in a sek-1(km4) mutant, albeit at lower levels than in wild-type animals. These data suggest that some of the UPR-mediated transcriptional response is p38 pathway independent.
Based on these data, we predicted that animals mutant in the p38 pathway should be more sensitive to PFT than animals mutant in the UPR pathway. This hypothesis is based on the fact that the p38 pathway is upstream of the UPR, is required for full activation of the UPR in response to PFT, and is involved in UPRindependent PFT defense pathways (e.g., ttm-2). Comparison of sek-1(km4) and xbp-1(zc12) mutant animals on Cry5B indicates sek-1(km4) animals are more severely intoxicated than xbp-1(zc12) animals at the same dose of Cry5B ( Figure 5G ). This conclusion was quantitatively confirmed by performing LC 50 experiments on N2 and sek-1(km4) animals (Table 1) . Whereas the LC 50 of xbp-1(zc12) animals on Cry5B is 5.8 fold lower than N2, the LC 50 of sek-1(km4) animals on Cry5B is 170 fold lower than N2.
Here we demonstrate that ER stress response pathways play a central but heretofore unknown role in innate defenses in vivo. Specifically, we find that bacterial pore-forming toxins (PFTs) activate the ire-1-xbp-1 branch of the ER Unfolded Protein Response (UPR) in C. elegans and mammalian cells and that the ire-1-xbp-1 and atf-6, but not the pek-1, branches of the UPR are important for C. elegans cellular defenses against a PFT since elimination of either of these two branches leads to hypersensitivity to the PFT Cry5B.
The ER stress response has been previously associated with pathogenic attack, mostly in the opposite direction shown here, e.g., aiding viral replication and pathogenesis ( [21] and references therein). In a few cases, the ER stress response has been linked with innate immunity since induction of ER stress can activate CREB-H, which in turn promotes the acute inflammatory response [22] . It has also been suggested that IRE-1 could influence immunity via its association with TRAF-2, which in turn can regulate NF-kB [23] . Data from studies in plants suggest that in response to pathogens, signals can be produced that lead to an ''anticipatory'' UPR to handle the massive synthesis of new secretory proteins required [24] .
Here we definitively demonstrate a functional role of the UPR in defense against a pathogen in vivo. Loss of xbp-1 leads to animals nearly 6 fold more susceptible to PFT whereas loss of atf-6 leads to animals nearly 3 fold more susceptible.
Our data suggest that cells have adapted the UPR pathway for a specific response to PFTs in order to promote cellular defense against this common form of pathogenic attack. First, we found that loss of the xbp-1 arm of the UPR does not lead to hypersensitivity to a heavy metal or hydrogen peroxide nor does loss of either xbp-1 or atf-6 lead to decreased protection against a bacterial pathogen that lacks a PFT. Second, the ire-1-xbp-1 and atf-6 arms of the UPR are involved in the defense but the pek-1 arm is not. Third, the activation and function of the UPR in PFT defenses can be separated from the role of the UPR in dealing with unfolded proteins (here tested using the drug tunicamycin) in two ways: 1) the relative importance of the various arms of the UPR for defense against PFT is different than their importance for protection against unfolded proteins and 2) the activation of the ire-1-xbp-1 pathway by PFT, but not unfolded proteins, requires p38 MAPK (see below).
A link between the p38 and UPR pathways has been shown in previous studies, although not with the level of functional relevance demonstrated here. Various arms of the UPR have been shown as both upstream or downstream of the p38 pathway, depending on the circumstances [25, 26, 27, 28, 29, 30] . The p38 pathway itself is implicated extensively in innate immune protection of many organisms against pathogens [31] and against PFTs in worms and mammals [6, 7] . Our data presented here for the first time functionally link the UPR to this major innate immune signal transduction pathway. Our findings on the activation and role of the UPR and p38 pathways in defense against PFT are summarized in Figure 6 .
Why would induction of the ER stress response play a protective role against PFTs? It is possible that PFTs somehow lead to the accumulation of unfolded proteins in a cell. For example, PFTs are known to perturb calcium homeostasis and changes in calcium homeostasis are known to affect protein folding [32, 33] . In this model, cells would respond to the toxin via p38 MAPK and turn on the UPR to anticipate and ameliorate the detrimental effects of unfolded proteins. Arguing against this model, however, is our data showing that sensitivity of the three arms of the UPR to Cry5B is different than their sensitivity to a global unfolder of ER proteins, tunicamycin. A second model is that activation of the ER stress response by Cry5B in a p38 MAPK dependent manner may prepare the cell to handle an altered biosynthetic load in the ER to defend against a toxin. For example, transcriptional array analysis indicate that over 1000 genes are differentially regulated in C. elegans by Cry5B ingestion [6] , which could in turn lead to significant changes in the protein load of the ER. A third model is 4(bn2) and glp-4(bn2);sek-1(km4) after 3 hours of exposure to either control (DMSO) or tunicamycin (2 mg/mL). This is a representative experiment of three independent experiments. (D) In vivo induction of hsp-4::GFP by Cry5B requires pmk-1 (p38 MAPK). The strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25) were fed either control E. coli or E. coli expressing Cry5B for 8 hours and the expression of GFP was then analyzed. Cry5B induces GFP within the intestinal cells of the strain hsp-4::GFP but not in the strain containing the pmk-1(km25) mutant. The experiment was performed three times and representative worms are shown. Scale bar is 0.2 mm. (E) In vivo induction of hsp-4::GFP by tunicamycin does not require pmk-1 (p38 MAPK). The strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25) were exposed to either control (DMSO) or tunicamycin (2 mg/mL) for 8 hours and the expression of GFP was then analyzed. Tunicamycin induces GFP throughout both the strains hsp-4::GFP and hsp-4::GFP;pmk-1(km25), including within the intestinal cells. The experiment was performed three times and representative worms are shown. Scale bar is 0.2 mm. (F) Downstream targets of the UPR require the p38 MAPK pathway for induction by PFT but not unfolded proteins. The fold change in the levels of hsp-4 and Y41C4A.11 mRNA transcripts by Cry5B and tunicamycin were determined for glp-4(bn2), glp-4(bn2);xbp-1(zc12) and glp-4(bn2);sek-1(km4) using real-time PCR. In addition, the fold change in ttm-2 transcripts was determined in response to Cry5B. Data are mean and standard deviation of three independent experiments. (G) Animals lacking sek-1 MAPKK are more sensitive to Cry5B than animals lacking xbp-1. Wild-type N2, sek-1(km4), and xbp-1(zc12) animals were placed on plates spread with E. coli transformed with empty vector (0%) or spread with empty vector E. coli diluted 9:1 (10%) or 3:1 (25%) with Cry5B-expressing E. coli (% thus gives toxin dose on a plate relative to undiluted Cry5B-expressing E. coli). The assay was initiated with L4 stage worms and photographs were taken 48 hours later. In the absence of Cry5B, the worms developed into dark, gravid, active, healthy adults. On 10% Cry5B-expressing E. coli, xbp-1(zc12) were slightly smaller than N2 but healthier than sek-1(km4), which were as small, pale, inactive, and severely intoxicated. On 25% Cry5B-expressing E. coli, xbp-1(zc12) was more intoxicated than N2 but not as intoxicated as sek-1(km4) animals. Scale bar is 0.2 mm. doi:10.1371/journal.ppat.1000176.g005
based on the fact that activation of the ire-1-xbp-1 pathway leads to increased phospholipid biogenesis [34] . It is possible that the defensive role of the ire-1-xbp-1 pathway is to produce phospholipids that play a protective role against PFTs. Consistent with this, it has been shown that inhibiting the activation of SREBPs, the central regulators of membrane biogenesis, leads to hypersensitivity of mammalian cells to the PFT aerolysin [35] .
In summary, we have identified specifically the ire-1-xbp-1 and atf-6 ER stress transducer pathways as components of cellular defenses against a PFT. While p38 MAPK was previously demonstrated to function in this regard [6] , we have discovered a major and unexpected downstream target of this pathway for PFT defenses, namely the UPR. These results demonstrate the fundamental requirement for specific cell responses to bacterial PFTs and support the notion of intrinsic cellular defenses (or INCED, formerly, cellular non-immune defenses), a budding concept in immunity that emphasizes the intrinsic ability of epithelial cells to defend against bacterial toxins and the importance of these defenses as a supplement to the innate immune and adaptive immune systems [36] . Additionally, the differential importance of the three ER stress transducer pathways in response to Cry5B versus tunicamycin, the differential activation of ire-1-xbp-1 by p38 MAPK in response to Cry5B versus tunicamycin, and the divergent pathways regulated by p38 MAPK in protective responses reveal how studying pathogenesis can uncover a wonderful complexity and new connections among intracellular pathways.
C. elegans strains were maintained at 20uC on NG plates using Escherichia coli strain OP50 as the food source [37] . Strains used in this study were wild-type Bristol strain N2 [37] , atf-6(ok551), glp-4(bn2), ire-1(v33), pek-1(ok275), pmk-1(km25), sek-1(km4), SJ4005 (zcIs4 [hsp-4::GFP]) and xbp-1(zc12). atf-6(ok551) and pek-1(ok275) were each outcrossed a total of 6 times. SJ4005 was outcrossed an additional 4 times as it had been outcrossed twice upon receipt from the Caenorhabditis Genetics Center. xbp-1(zc12) was created by outcrossing strain SJ17 (xbp-1(zc12); zcIs4 [hsp-4::GFP]) four times and removing the integrated hsp-4::GFP during the outcrosses. Images were acquired with an Olympus BX60 microscope with the 106 objective linked to a 0.56 camera mount and a DVC camera. Worms were placed on 2% agarose pads containing 0.1% sodium azide for photography.
All assays were performed at 20uC unless indicated elsewhere. Qualitative toxicity assays based on visual comparison of worm intoxication were performed on plates with E. coli-expressed Cry5B as described [6, 38] . Beginning with the 4 th larval (L4) stage worms, worms were fed for 48 hours either on control plates with E. coli JM103 that did not express Cry5B (empty vector) or plates prepared with E. coli JM103 expressing Cry5B diluted 1:3 with empty vector transformed JM103. This amount of Cry5B (25%) mildly intoxicates wild-type C. elegans, which allows for identification of strains that are hypersensitive to Cry5B as these strains will be more severely intoxicated than wild type. Quantitative lethal concentration assays were performed as described [38] except the worms were scored after 8 days for Cry5B, CuSO 4 , and tunicamycin. Lethal concentration assays with H 2 O 2 did not include E. coli or 5-fluoro-29-deoxy-uridine, and worms were scored after 4 hours. Concentrations of each toxin were set-up in triplicate for each assay, and each assay was performed independently three times. Purified Cry5B was prepared as described [39] and dissolved in 20 mM HEPES (pH 8.0) prior to use. Approximately 1500 worms were scored for each strain in the calculation of the LC 50 values for each toxin. For tunicamycin assays, the set up was identical to the lethality assay with Cry5B. For the developmental inhibition assay, Cry5B plates were prepared as described [6, 38] . Approximately 100 L1 stage worms (from bleached embryos hatched off overnight) were placed on each plate (60 mm) and the number of worms at the L4 or adult stage 3 days later was determined. This assay was performed independently three times. The P. aeruginosa lifespan assay was performed on slow-killing plates as described [40] , with the following modifications: PA14 was cultured overnight in tryptic soy broth instead of King's broth and then spread on slow-killing plates complemented with 75 uM mM 5-fluoro-29-deoxy-uridine. The experiment was performed three times with approximately 100-150 worms total per strain, at 20uC. To determine if there was rescue of the hypersensitivity phenotype in the intestinal-specific promoter studies, 25% E. coli-expressing Cry5B plates were used to compare Cry5B sensitivities of wild-type N2, xbp-1(zc12), and xbp-1(zc12) that were transformed with constructs to express either green fluorescent protein (GFP) or xbp-1 mRNA within intestinal cells using the app-1 promoter (plasmids are described in Protocol S1). Transgenic L4 stage worms were placed on the 25% E. coli expressing Cry5B plates and their health status was assessed 72 hours later. Specifically, the relative health of each worm was determined qualitatively by comparing body size, darkness of the intestine as an indicator of feeding, and activity, including whether the worm demonstrated spontaneous movement. For scoring of the transgenic worms, comparisons were made using both N2 as a reference for healthy worms, as they demonstrated dark intestines and continuous spontaneous movement, and xbp-1 (zc12) as a reference for intoxicated worms that had pale intestines and demonstrated rare or no spontaneous movement.
The glp-4(bn2) strain was used for these experiments (including the double mutants glp-4(bn2);xbp-1(zc12) an glp-4(bn2); sek-1(km4)) since it has a greatly reduced number of germ cells when grown at Figure 6 . Schematic illustrating relationship between p38 MAPK, ire-1-xbp-1, and PFT defense pathways. PFTs at the cell surface of epithelial cells activate p38 MAPK that activates IRE-1 that induces splicing of xbp-1, which then turns on defense against PFTs. Residual activation of xbp-1 targets in the absence of the p38 MAPK pathway suggests there might be p38-independent activation of the ire-1-xbp-1 pathway in response to PFT as well (not shown). Independent of IRE-1 activation, p38 MAPK can also activate TTM-2 and other PFT defenses. Tunicamycin, which causes the accumulation of unfolded proteins in the ER, activates IRE-1 via a mechanism independent of the PFT and p38 MAPK. doi:10.1371/journal.ppat.1000176.g006
20uC. This helps remove the background of macromolecules not isolated from the intestine. The response to Cry5B is not altered in this strain compared to wild type [6] . Primers used for these experiments are described in Protocol S1. Approximately 15,000 L4 stage worms were used per 100 mm dish for each treatment group. Worms were exposed to Cry5B for the indicated period of time on either E. coli JM103 containing empty vector or E. coli JM103 expressing Cry5B as described [6, 38] . After exposure to each treatment, worms were rinsed from plates with water, centrifuged at 500 g for 45 seconds, and washed two additional times with water. RNA was prepared from worms using TRIZOL (Invitrogen) and further purified with RNeasy columns (Qiagen). cDNA was prepared by reverse transcription using oligo-dT. Standard PCR was used to detect xbp-1 splicing, and products were analyzed on 2% agarose gel. Unspliced xbp-1 transcript is 220 nucleotides and spliced transcript is 197 nucleotides. To quantitate the amount of xbp-1 splicing, loading was normalized by quantitating cDNA levels using real time PCR and eft-2 primers [6] . Equal amounts of cDNA were used for the xbp-1 splicing PCR experiments and 10 microliters of each reaction were loaded onto a 2% agaose gel and stained with ethidium bromide. NIH ImageJ was then used to quantitate the intensities of xbp-1 spliced forms in Cry5B treated samples relative to untreated samples at the same time point.
Real time PCR was performed on an ABI 7000 Instrument using SYBR Green detection (Applied Biosystems). eft-2 was used as the real time PCR normalization control [6] . Experiments with Cry5B used either a control plate (E. coli not expressing Cry5B) or a Cry5B plate on which 100% of the E. coli expressed Cry5B. Tunicamycin experiments used E. coli OP50 as a food source and either DMSO as the control or tunicamycin at 2 mg/mL incorporated into the plates. Three independent experiments for the splicing and real time PCR were performed for each treatment.
HeLa cells were cultured in MEM media supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% glutamine and 1% non-essential amino acids, in a humidified incubator with 5% CO 2 at 37uC. Aerolysin was purified as described [41] . Cells were continuously treated with 2 ng/mL (0.02 nM) of proaerolysin. At different time points, cells were washed with PBS and lysed at 4uC in 0.25 M sucrose supplemented with proteases inhibitor (Roche, Germany) using a needle. The whole cell extracts were subjected to SDS-PAGE and Western blotting. XBP1 (R-14) antibody was from Santa Cruz Inc. Band intensities were quantified, after background removal, using ImageJ software (NIH). The loading in each lane was normalized relative to the intensity of a nonspecific antibody-reacting band on the blot.
Approximately 750 L1 stage worms were grown in a single well of a 48 well plate containing 150 mL S media [42] and E. coli OP50. When worms had reached the L4 to young adult stage, glucose was added to 100 mM and either 20 mM HEPES (pH 8.0) or Cry5B dissolved in 20 mM HEPES (pH 8.0) to give a final concentration of 100 mg/mL was added. After one hour, worms were removed, centrifuged, and 175 mL of media was removed. Twenty five mL of 26 sodium dodecyl sulfate loading buffer was added, and worms were boiled for 5 minutes. Ten microliters of lysate were used for immunoblotting. Monoclonal antibody to phospho P38 MAPK (Cell Signaling Technology cat. no. 9215) was used at 1:300 and monoclonal antibody to a-tubulin (Sigma-Aldrich cat. no. T6199) was used at 1:4000.
L4 stage glp-4(bn2) and glp-4(bn2);xbp-1(zc12) worms were used for this experiment. Approximately 80,000 worms of each strain were used for both control and Cry5B treatments. Control plates consisted of 100 mm plates spread with E. coli that did not express Cry5B, while Cry5B treatments consisted of plates in which 100% of the E. coli expressed Cry5B. Approximately 20,000 worms were used per plate. Worms were fed on the bacteria for 6 hours at 20uC. For details of mass spectrometry, please see Protocol S1.
All experiments were performed a minimum of three times. LC 50 values were determined by PROBIT analysis [43] . The lethal concentration assays are represented graphically using nonlinear regression performed with the software GraphPad Prism. Statistical analysis between two values was compared with a paired t-test. Statistical analysis among three or more values was compared with matched one way ANOVA using the Tukey post test. Lifespan data was analyzed with Kaplan-Meier survival curves. Statistical significance was set at p,0.05.
Protocol S1
Found at: doi:10.1371/journal.ppat.1000176.s001 (0.03 MB DOC) Composition and Function of Haemolymphatic Tissues in the European Common Shrew BACKGROUND: Studies of wild animals responding to their native parasites are essential if we are to understand how the immune system functions in the natural environment. While immune defence may bring increased survival, this may come at a resource cost to other physiological traits, including reproduction. Here, we tested the hypothesis that wild common shrews (Sorex araneus), which produce large numbers of offspring during the one breeding season of their short life span, forgo investment in immunity and immune system maintenance, as increased longevity is unlikely to bring further opportunities for mating. In particular, we predicted that adult shrews, with shorter expected lifespans, would not respond as effectively as young animals to infection. METHODOLOGY/PRINCIPAL FINDINGS: We examined haemolymphatic tissues from wild-caught common shrews using light and transmission electron microscopy, applied in conjunction with immunohistology. We compared composition and function of these tissues in shrews of different ages, and the extent and type of inflammatory reactions observed in response to natural parasitic infections. All ages seemed able to mount systemic, specific immune responses, but adult shrews showed some signs of lymphatic tissue exhaustion: lymphatic follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; Wald = 11.1, p<0.05) and exhibited greater levels of depletion (Wald = 13.3, p<0.05). CONCLUSIONS/SIGNIFICANCE: Contrary to our expectations, shrews respond effectively to their natural parasites, and show little indication of immunosenescence as adults. The pancreas of Aselli, a unique lymphoid organ, may aid in providing efficient immune responses through the storage of large numbers of plasma cells. This may allow older animals to react effectively to previously encountered parasites, but infection by novel agents, and eventual depletion of plasma cell reserves, could both still be factors in the near-synchronous mortality of adult shrews observed shortly after breeding. The immune system is the primary mechanism through which animals defend against parasites and pathogenic organisms. Immunity is believed to be a major factor in regulating host survival, as under natural conditions, even a mild parasitic infection may weaken an animal sufficiently to increase the chances of mortality through starvation or predation [1] . However, maintenance and up-regulation of the immune system requires energetic and nutritional resources [1] , resulting in a trade-off between investment in immunity and other physiological processes, including growth and reproduction [2] [3] [4] . An appreciation of the extent to which hosts invest in immunity is therefore critical to understanding the strategies through which animals maximise fitness, and how trade-offs are mediated between current offspring production, longevity and future reproductive success [3, 4] .
While there have been a number of studies of ecological immunology in birds and insects [4] [5] [6] , there has been little effort to understand immune function of small mammals in this context outside of the laboratory. Here, we examined the unique haemolymphatic system of the European common shrew (Sorex araneus) to investigate the capacity of this short-lived mammal, restricted by a fast metabolism and extremely limited fat reserves, to defend against its unusually diverse parasite fauna, both as a young animal and an adult.
Common shrews have attracted considerable attention from both ecologists and parasitologists, and have a life history strategy characterized by a high investment in reproduction and a short life span [7] [8] [9] . Their life cycle takes 14 to 16 months to complete, with the first young born in mid-May [10] [11] [12] , and only one breeding season in the spring of the second year of life [9, 12, 13] . Both sexes can mate with multiple partners, and females are extremely promiscuous [14, 15] . Females can produce up to three litters of around seven offspring [10, 11, 16] , with energy intake during lactation increasing to around three times the non-reproductive level [8] . Both males and females die shortly after breeding, such that there is little overlap between generations [12, 13, [17] [18] [19] .
S. araneus has an unusually diverse parasite fauna, which includes ectoparasites [20] [21] [22] , Bartonella and trypanosome infections [23] [24] [25] [26] [27] , Anaplasma phagocytophilum [27, 28] , and Pneumocystis carinii [29] , as well as over 20 helminth species [30] . The extent to which shrews are able to mount immune responses to these parasites is unknown: commons shrews have a fast metabolic rate even for their small body size [31] but store very little energy as fat [32] . Lack of resources may therefore limit the capacity of S. araneus to mount immunological responses, particularly as reproductive adults, and parasitism has been suggested as one of the causes of mortality of adults after breeding [12, 13, 18] . Common shrews possess a large lymphatic organ, known as the Pancreas of Aselli, which may function in defence against parasites, though its exact role is unknown, and remains the subject of discussion [33] [34] [35] . To date, there have been no studies of spleen or bone marrow function in S. araneus.
We hypothesised that common shrews, which are not expected to survive beyond the first breeding season, would gain little benefit from investing their limited resources in immunity and immune system maintenance, at the expense of reproduction. Instead, we predicted that wild shrews would demonstrate only limited responses to parasites, and that their immune system would show signs of deterioration with age. The aim of the study was therefore to evaluate the capacity of sub-adult and adult shrews to mount immunological responses. We examined and compared the structure, composition and function of relevant haemolymphatic tissues including the pancreas of Aselli, in wild-caught common shrews of different ages pre and post maturation, and the extent and type of inflammatory reactions produced in response to naturally occurring parasitic infections. Light and electron microscopy were applied in conjunction with immunohistological characterisation of leukocyte populations. Contrary to our predictions, our results indicated that shrews are capable of mounting immune/inflammatory responses throughout their entire life span. While some degree of lymphatic exhaustion was obvious in adult animals (perhaps as a result of age-related changes, or reduced investment in immunity as a consequence of breeding effort), there was also evidence of some degree of compensation, in the form of storage of plasma cells particularly in the pancreas of Aselli, possibly as a defence against previously encountered parasites.
Forty-three common shrews (19 male, 24 female) were live-caught in Cheshire, England between September 2001 and June 2003. The work was performed with approval of and under a licence from English nature (licence number 20030767) held by PS. Shrews were classified into three age categories: 18 sub-adults, showing no sign of sexual development (11 female, 7 male), 3 animals undergoing sexual maturation (2 female, 1 male) and 22 sexually mature animals (11 female and 11 male) caught during or after the breeding season. Adult females all exhibited signs of mating, pregnancy and/or lactation. All animals appeared healthy when captured, and were killed humanely by overdose of inhalation anaesthetic (Fluothane, Schering-Plough Animal Health, UK). Animals were inspected for ectoparasites (data not presented) before full necropsy was performed and body mass (minus gastrointestinal tract) recorded. Bladder and oesophagus were removed and dissected in Hanks saline (406 magnification), with nematode and digenean parasites in both tissues counted and identified using keys [30] . Stomachs and guts were removed, weighed, stored in 10% formalin and later dissected in Hanks saline (406 magnification). Recovered helminths were identified as nematodes, cestodes or digeneans [30] and counted.
Tissue samples from all major organs from all animals were fixed in 4% buffered paraformaldehyde for 24-48 h prior to routine embedding in paraffin wax. Sections (5 mm) were stained with haematoxylin-eosin for histological evaluation. Sections from haemolymphatic tissues (spleen, pancreas of Aselli, bone marrow (sternum) and in selected cases mesenteric or mediastinal lymph nodes and thymus) were prepared for immunohistological examinations and the TUNEL method.
Samples from the pancreas of Aselli of one sub-adult common shrew were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in cacodylate buffer (pH 7.4) and subsequently embedded in epoxy resin. Semi-thin (1 mm) and thin sections were prepared and the latter examined using transmission electron microscopy.
Labelling of leukocytes, proliferating and apoptotic cells by immunohistology and the TUNEL method Leukocytes, proliferating and apoptotic cells in haemolymphatic tissue samples from 21 common shrews were identified using immunohistology and the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling of DNA fragmentation sites) method respectively. For immunohistology, monoclonal and polyclonal antibodies (cross) reacting in other species were used in conjunction with peroxidase anti-peroxidase and avidin biotin peroxidase complex methods as previously described [36] [37] [38] [39] . Antibodies, their sources and detection methods are listed in Table 1 .
Cellular turnover in haemolymphatic tissues was assessed by counting proliferating and apoptotic cells. Proliferating cells were identified by their expression of the proliferating cell nuclear antigen (PCNA; [38] [39] [40] ), while apoptotic cells were demonstrated in situ by the TUNEL method ( [39, 41] ) using a commercially available kit according to the manufacturer's instructions (ApopTag TM In Situ Apoptosis Detection Kit; Chemicon, California, USA).
Consecutive tissue sections, incubated with normal rabbit or rat serum or a non-reacting mouse monoclonal antibody, were used as negative controls for polyclonal and monoclonal antibodies respectively. For TUNEL, terminal deoxynucleotidyl transferase (TdT) was replaced by distilled water on negative control slides.
All antibodies used cross-reacted with shrew leukocytes. The B cell markers CD45R and CD79a were expressed by B cells, CD45R being strongly expressed in follicular germinal centres, but relatively faintly in well-differentiated B cells within follicular mantle zones, whereas CD79a was mainly expressed by mature B cells (weak expression in germinal centres (dark zone), strong expression in the periphery of secondary follicles, positive reaction of all cells in primary follicles). Plasma cells were negative or exhibited faint staining for both B cell markers. CD3 acted as a pan T cell marker, being expressed by entire T cell zones. Both the myeloid/histiocyte antigen and lysozyme were markers for mature monocytes/macrophages and their precursors, myelomonocytic cells, as they were expressed by monocytes and macrophages and a high percentage of cells in the bone marrow. However, they seemed not to be a marker for all neutrophils, as in both the splenic red pulp and sinuses of the pancreas of Aselli, a proportion of cells with the morphology of neutrophils were negative for both antigens. PCNA-positive, proliferating cells were present in follicle germinal centres, T cell zones, bone marrow (including megakaryocytes) and splenic red pulp, the sites in the haemolymphatic tissue expected to contain proliferating cells. The TUNEL method identified cells with the morphology of apoptotic cells [39, 41] as well as apoptotic bodies (both free and within tingible body macrophages), located predominantly in follicular germinal centres.
Assessment of lymphatic follicle and bone marrow activity, and the severity of inflammatory infiltration in the liver Spleen and lymph node exhibited a composition very similar to laboratory mice, which allowed direct comparison for evaluation.
Lymphatic follicles in the spleen and lymph nodes of all animals were classified as primary and/or secondary follicles and as small, medium or large. The presence and degree of follicle depletion (none, mild, moderate or severe) was assessed on the basis of the cellularity of the germinal centres. Large, undepleted secondary follicles were interpreted as evidence of high activity, whereas small, depleted primary follicles indicated lowest activity. Similarly, bone marrow activity was classified as low, moderate or high based on the ratio of haematopoietic cells to adipose tissue in a cross section of the marrow in the sternum.
The degree of inflammatory infiltration in the liver was assessed semi-quantitatively as mild, moderate or severe, based on the number of cells and cell layers in the portal areas or between hepatic cords.
Statistical analyses were restricted to sub-adult and adult animals as only 3 pubescent shrews were caught. Ordinal logistic regression examined whether bone marrow activity, lymphatic follicle activity (primary/secondary follicles, presence and degree of follicular depletion) in the spleen and pancreas of Aselli and severity of inflammatory infiltration in the liver (mild, moderate or severe) varied with age class or sex. Sex and age category were entered simultaneously as independent variables into models for each dependent variable listed above, and the significance of both terms assessed using Wald tests. Mann-Whitney U tests were used to test for differences in helminth abundances between sub-adult and adult shrews.
The spleen in adult shrews exhibits lesser activity in the white pulp but higher cellularity in the red pulp compared to the spleen in sub-adults Red and white pulp of shrews of all age groups were examined for their composition and cellular turnover. The white pulp (lymphatic follicles and T cell zones) was generally confined to the organ's centre, where follicles were arranged singly or in groups ( Fig. 1 ). Germinal centres exhibited numerous proliferating, PCNA-positive cells (up to 50%) and few (#5%) apoptotic cells. T cell zones were arranged around medium-sized arteries, forming periarterial lymphatic sheaths similar in size and cell density in all animals, exhibiting up to 10% proliferating, PCNA-positive cells and generally few apoptotic cells. The generally cell-rich red pulp contained neutrophils, erythrocytes and smaller numbers of lymphocytes (each ,5% up to 30% T cells and mature B cells) and macrophages as well as numerous evenly distributed megakaryocytes (Fig. 1B) ; the red pulp exhibited some degree of cellular turnover, with approximately 10% proliferating, PCNApositive cells and scattered apoptotic cells. Follicles and follicle groups were often delineated by a variably distinct rim of macrophages and neutrophils (Fig. 1C) , together with variable numbers of mature, strongly CD79a-positive B cells, T cells and erythrocytes. In general, 30-50% of cells in the red pulp were positive for myeloid/histiocyte antigen (Fig. 1D ) and lysozyme and had the morphology of monocytes/macrophages or neutrophils.
Age groups differed in both the composition and functional state of the spleen. Within the red pulp, the amount of neutrophils and Table 1 . Antibodies and detection methods used to identify leukocytes and proliferating cells in the common shrew with references to suppliers and use in other species. [36, 53] . f [37] . g [53] . h [38] [39] [40] . megakaryocytes often appeared greater in adult animals than in sub-adults. The white pulp of sub-adults was exclusively comprised of secondary follicles, which often appeared interconnected and formed large groups (Fig. 1A) . These follicles were for the most part large and without signs of depletion and included numerous apoptotic cells and tingible body macrophages, as well as several mitotic cells. In contrast, the majority of adults exhibited fewer follicles which were a mixture of primary and secondary follicles (Figs. 1B, 2) and generally smaller than those of sub-adults (n = 17, Wald = 17.17, p,0.05, Figs. 1A, 2). Follicles in adults also differed from those in sub-adults, in that they were only partially surrounded by distinct perifollicular rims. Where present, germinal centres in adults contained both mitotic as well as apoptotic cells. Follicle centres in four adults exhibited collagen deposition, and while there was a tendency for greater follicle depletion in older animals (Fig. 2) , the difference between adults and sub-adults was not statistically significant (Wald = 2.10, not significant (ns)). The white pulp of the three pubescent animals exhibited primary and/ or secondary follicles, the latter with features similar to those found in sub-adult shrews. Males (n = 18) and females (n = 21) did not differ with respect to either size (Wald = 0.67, ns) or depletion (Wald = 0.13, ns) of follicles in the spleen.
Lymph node composition is similar in all age groups, with a relatively high proportion of plasma cells, particularly in adults Mesenteric or mediastinal lymph nodes were examined from selected sub-adult and adult animals. All features normally associated with mammalian lymph nodes were represented: a cortex containing primary and secondary follicles, paracortex, lymphatic cords, medulla and both marginal and medullary sinuses. Compared to lymph nodes in other mammalian species [39, 42] , the medulla often appeared to contain a high number of plasma cells, particularly in adults. No other differences were observed between the age groups.
The pancreas of Aselli represents a specialised abdominal lymph node that appears to function as a plasma cell store, in particular in adult shrews
In the past, there has been some controversy as to the composition and function of the pancreas of Aselli (lymph nodelike or equivalent to the avian bursa of Fabricius [35] ). The aim of this study was therefore to clarify this matter with up-to-date methodology. In general, the composition of the pancreas of Aselli was very similar to that of a lymph node. Beneath the capsule were marginal sinuses of variable width, containing disseminated lymphocytes (mostly T and B cells in equal proportions), macrophages and neutrophils, the latter either disseminated or as small accumulations. In four adult shrews, the marginal sinuses exhibited focal to extensive fibrosis. Beneath the sinuses lay a cortex containing exclusively secondary follicles ( Fig. 3A-C) , with the exception of one adult male where both primary and secondary follicles were present (Fig. 4) . Germinal centres generally exhibited a high cellular turnover, with variable but high numbers of PCNA-positive, proliferating cells (often more than 50% of cells; Fig. 3E ) and often numerous apoptotic cells (Fig. 3F ). T cell zones formed a paracortex located immediately beneath the follicles (Fig. 3D ) and were generally similar in size and cell density in animals of all age groups. The organ's centre (medulla) contained loosely arranged sinuses with only low numbers of macrophages. The remainder of the medulla was made up almost entirely of plasma cells (Fig. 3G, H) .
In sub-adult animals the cortex generally appeared tightly packed with large follicles that exhibited no, mild or moderate depletion (Figs. 3A, 4) . In adult shrews, the cortex often contained only a small number of follicles (9/20 animals; 45%), frequently with large areas of cortex devoid of follicles (5/20; 25%; Fig. 3B ). Follicles occasionally seemed to extend outwards into marginal sinuses and in two animals exhibited central collagen deposition. Follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; Wald = 11.06, p,0.05, Fig. 4 ) and exhibited greater levels of depletion (Wald = 13.28, p,0.05, Fig. 4) . No difference was found between males (n = 17) and females (n = 22) with respect to follicle size (Wald = 0.30, NS) or depletion (Wald = 0.42, NS).
Cortical areas devoid of follicles were also devoid of T cell zones. As a consequence, overall numbers of T cells in the pancreas of Aselli often seemed lower in adult than sub-adult animals. Where the cortex was devoid of follicles, plasma cells extended from the medulla to the marginal sinuses or beyond to the capsule, such that the medulla often occupied most of the organ in adult animals (Fig. 3B) . Accordingly, in adult animals, the whole organ frequently appeared as an accumulation of plasma cells, surrounded by a fragmentary cortex and paracortex.
Extramedullary haematopoiesis was observed in 9/21 (43%) adult animals, as represented by scattered megakaryocytes within the outer medulla. This was not seen in sub-adult animals.
Thymic tissue was recovered from seven animals across all age groups. This generally consisted of a variable number of lymphocyte layers which were arranged around blood vessels and encased by a thin capsule of fibrous connective tissue. Taking the histological features of a mouse thymus in account, the thymus appeared to exhibit a variable, but generally low degree of involution in all examined shrews, regardless of age. The bone marrow generally exhibits moderate to high activity, regardless of age The bone marrow generally exhibited moderate to high activity. All haematopoietic cell types known from other mammalian species were present. Approximately 10% of cells were identified as T cells, 10% as B cells (CD79a-positive, CD45R-negative; interpreted as circulating mature B cells) and 30% to 50% were myeloid/histiocyte antigen-and/or lysozyme-positive cells. At least 30% to 40% of all cells were PCNA-positive, representing relatively high levels of proliferation. No difference in bone marrow activity was found between sub-adult (n = 15) and adult (n = 16) shrews (Wald = 1.91, ns) or between males (n = 12) and females (n = 19; Wald = 0.74, ns).
All animals harboured helminths ( Table 2 ). The abundance of infection was greater in adults than sub-adults (Table 3) . Nematodes (Liniscus incrassatus; Roots, 1992) recovered from the lumen and between epithelial cells of the urinary bladder were associated with mild to moderate acute to chronic lymphocytedominated cystitis and/or mild degeneration and sloughing of epithelial cells. Infestation of the gall bladder by the digenean Dicrocoelium soricis resulted in only a mild lymphocytic submucosal infiltration in one of two animals infected. Neither helminths within the gastrointestinal tract nor Porrocaecum sp. larvae within interscapular adipose tissue were associated with inflammatory reactions or other histological changes.
A high proportion of shrews (14/18 sub-adults; 78%, 2/3 pubescent animals; 67%, 12/22 adults; 55%) exhibited protozoan parasites within vessel walls that could not be further identified ( Table 2 ). These occurred predominantly in kidneys (21/43 shrews; 49%) and myocardium (12/43 shrews; 28%), but also occasionally in the liver, the splenic red pulp, the medulla of the pancreas of Aselli and the wall of the urinary bladder. In general, these cysts did not induce any alterations apart from an occasional slight thickening of the affected vessel wall, or a mild granulomatous inflammatory infiltration. Protozoan cysts with features of Sarcocystis sp. were found within skeletal muscle myocytes of one adult female, without any associated reaction.
The liver of all animals exhibited a variable degree of mixed cellular (neutrophils, lymphocytes and macrophages) portal inflammatory infiltration, where T cells and B cells were present in equal amounts. No difference in the severity of the inflammatory infiltration was apparent between sub-adults (n = 18) and adults (n = 22; Wald = 0.03, ns), or between males (n = 22) and females (n = 18; Wald,0.01, ns). Infiltrates often occurred together with follicle-like accumulations of lymphocytes, which occasionally exhibited germinal centres.
Additional findings in the liver included a granuloma with central necrosis in one sub-adult, focal necrosuppurative hepatitis in two adults and variably intense (multi)focal hepatic necrosis with haemorrhage and pyogranulomatous inflammation in another three adults. In one pubescent shrew helminth parasites were observed within and outside multifocal suppurative hepatitis and haemorrhage. Five of the 43 animals exhibited granulomas within the pancreas of Aselli, with a central area of necrosis and/or mineralization (four shrews) or an embedded nematode (one animal). Focal pyogranulomatous (two adults) or suppurative inflammation (two adults), the latter in one case surrounding a nematode, were also observed. The marginal sinuses of one subadult shrew contained extensive focal accumulations of neutrophils, occasionally surrounding areas of necrosis.
None of the animals exhibited other major gross or histological changes. In particular there were no findings suggestive of a systemic non-infectious disease and/or a neoplastic disease.
While studies of laboratory animals allow aspects of immunity to be studied in controlled, repeatable environments, they may not reflect how wild animals, constrained by limited resources and at threat from a variety of infectious agents, respond to parasites and disease. We predicted that common shrews, which are short lived, store little energy as fat, and invest heavily in reproduction, would show limited responses to parasites, and their haemolymphatic tissues would deteriorate with age as expected survival decreased.
Our study is the first to examine haemolymphatic tissue structure, compoosition and function in shrews using modern techniques, and one of only a small number to explore immune responses of wild animals in their natural environment. With regards to both morphology and composition, the spleen, lymph nodes, thymus and bone marrow in S. araneus were found to be very similar to their equivalents in other mammalian species [39, 42] . In common with a number of species, including the musk shrew Suncus murinus, both bone marrow and spleen were identified as sites of haematopoiesis in S. araneus [43] . The results of our study also confirm that the pancreas of Aselli, which is specific to shrews, can be considered as a large, specialised lymph node [33] . The presence of a cortex with both follicles and paracortical T cell zones renders previous controversial assumptions regarding the organ's function incorrect: the pancreas of Aselli is neither a specific site of exclusive B cell production nor a functional analogue of the bursa of Fabricius in birds [35] . However, it differs from normal lymph nodes in that the centre (medulla) contains a very high proportion of plasma cells. In adulthood, the number of plasma cells and the relative size of the medulla seem to increase, until almost the entire organ is composed of plasma cells. Such a feature has not been described under physiological circumstances in any other species, and suggests that the pancreas of Aselli in S. araneus functions as a storage site for plasma cells, particularly in older animals. Lymph nodes in S. araneus were also found to contain a higher number of plasma cells than normally observed in other species [39, 42] , which may emphasise a general tendency towards progressive plasma cell storage in common shrews.
In comparing the lymphatic tissues of sub-adult and adult shrews, young animals were generally found to have an activated immune system, as represented by a predominance of large, active, secondary follicles in the spleen and pancreas of Aselli. This suggests sub-adults were responding effectively to a diverse array of infectious agents, including the helminth and protozoan parasites detected in a number of tissues. Post-reproductive animals, however, exhibited characteristics indicative of immune system exhaustion: follicles were generally smaller and were often depleted, with a smaller proportion of secondary follicles, particularly in the spleen. This could indicate decreased follicular activity in adult animals, with impaired germinal centre reactions resulting in reduced B cell production [44] . Impairment of germinal centre reactions is a known feature of immunosenescence in vertebrates and has been studied extensively: in humans it has been shown to be a product of defective T cell-dependent B cell activation [44, 45] . Reduced lymphocyte production as a consequence of follicular and T cell impairment could explain why significantly lower numbers of white blood cells, and specifically lymphocytes, have been reported in old common shrews [46] .
Differences between sub-adults and adults were also evident in the so-called ''marginal'' or ''intermediate zone'' of the spleen, the variably distinct rim of macrophages, lymphocytes, neutrophils and erythrocytes surrounding follicles and follicle groups seen in S. araneus and previously described in other species, including the musk shrew [43, 47] . This zone is considered to be the site of most intensive blood filtration in the spleen [43, 47] , and its loss of integrity/intensity in older animals may indicate a reduction in filtration capacity. This might however be counterbalanced by an increase in the capacity of the peripheral phagocytic response, as represented by an increase in neutrophil numbers within both the spleen (as observed here) and the peripheral blood [46] .
Both in bone marrow and splenic red pulp, the degree of haematopoiesis was similar in animals from all age groups. This concurs with similar findings in the musk shrew [43] , where both the splenic red pulp and bone marrow have been identified as physiological sites of erythropoiesis, leukocytopoiesis and platelet production over the animal's lifespan. In this aspect, shrews are similar to some reptiles, whereas in other mammals the haematopoietic capacity of the spleen seems to cease after birth [43] . We also found evidence of haematopoiesis in the pancreas of Aselli in adult S. araneus, as demonstrated by the presence of megakaryocytes in the medulla of some individuals. Interestingly, we found no evidence of major thymic involution in S. araneus, even in older animals. The rate of thymic involution is known to vary between species and breeds and with intraspecific factors such as sex and diet [48, 49] . Perhaps in shrews thymic involution is delayed to maintain production of T cells into adulthood.
It has been suggested that short-lived species should limit their investment in immunity to immediate, innate responses, as the energetic costs associated with mounting specific immune reactions are unlikely to be outweighed by the benefits of increased long-term survival [1] . The dependence on innate responses may be greater for species with limited energetic reserves (such as S. araneus), as even a mild immune challenge is likely to result in starvation if allowed to persist for more than a short time [1] . Here, however, the presence of numerous active secondary follicles in the spleen and pancreas of Aselli, the development of small lymphatic follicles in portal areas in the liver and the generally high number of plasma cells in the pancreas of Aselli all indicate that common shrews remain consistently able to mount systemic, specific immune responses. We also observed macro- phage-dominated (granulomatous) inflammatory reactions with lymphocyte involvement in both sub-adult and adult shrews, which included reactions to helminths in tissues. The increasing number of plasma cells in the medulla of the pancreas of Aselli and in lymph nodes with advancing age might even suggest a 'refocusing' of the immune system, from reacting to novel antigens in follicles as a young animal, to combating previously experienced parasites or pathogens with appropriate antibody responses as an adult. Plasma cells are long-lived and can survive for weeks after immunisation, particularly when not too tightly packed [50] ; perhaps young common shrews invest in long term immunity by producing and storing plasma cells in the pancreas of Aselli, which can then be used to mount efficient responses against previously encountered parasites in adulthood, when reproduction places greater demands on internal resources [8] . While this strategy may allow older animals to react effectively to previously encountered parasites, infection by novel agents or eventual depletion of plasma cell reserves, could still be factors in the near-synchronous mortality of adult shrews observed shortly after the breeding season [12, 13, 18]  Prevention of Cytotoxic T Cell Escape Using a Heteroclitic Subdominant Viral T Cell Determinant High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity. Despite the antigenic complexity of microbes, primary pathogen-specific cytotoxic CD8+ T lymphocyte (CTL) responses are commonly directed to just one or a few determinants. Furthermore, even when multiple epitopes are targeted, distinct patterns of epitope hierarchy often emerge. Such immunodominant epitopes commonly elicit high-magnitude CTL responses characterized by potent cytolytic function, whereas subdominant determinants generate responses that are relatively lower in magnitude and often less efficacious. In general, potent anti-viral CTL strongly correlate with control of infection and less clinical disease. Viral progeny selected on the basis of CTL surveillance can evolve to evade T cell responses. This selective pressure results in mutations in immunodominant CTL determinants that abrogate recognition. CTL escape virus is commonly observed in humans and nonhuman primates infected with HIV-1, hepatitis C virus (HCV) or simian immunodeficiency virus (SIV) and its selection often correlates with disease progression [1] [2] [3] [4] . Escape mutations may diminish binding to the restricting MHC class I molecule, interfere with T cell receptor (TcR) recognition or interfere with antigen processing [5] [6] [7] . Escape mutations are usually detected in epitopes targeted by CTL that exhibit high functional avidity because the corresponding potent CTL response exerts high selective pressure on the virus [2] . In some HIVinfected patients, CTL escape occurs without an associated enhancement of virus replication, suggesting that the mutations compromised virus fitness or, alternatively, the variant determinant elicited a de novo CTL response [8, 9] . Virus fitness is sometimes restored, with concomitant increased virus replication, when a second, compensatory mutation is selected [10, 11] . Collectively these results suggest that given the importance of virus diversification (CTL escape) in disease progression, suppression of selection or outgrowth of CTL escape variants should improve outcomes in persistently infected animals and humans.
Modulating the immunogenicity of subdominant CTL determinants could potentially lead to the development of more efficacious vaccines that are more broadly protective and prevent or minimize the appearance of variant viruses that have mutated in dominant epitopes targeted by high-avidity CTL responses. Enhancement strategies, which result in augmented responses to the native, subdominant epitope, have been described for both MHC class I and class II-restricted determinants, whereby the most common approaches involve generating a series of conserved and non-conserved mutations at MHC anchor residues, followed by an empiric determination of whether each individual substitution augments T cell effector function [12] . Furthermore, evaluating the effect of epitope enhancement in vivo has generally been achieved via heterologous infection systems [13] [14] [15] . Thus, while these results demonstrate proof of principle, direct evidence for enhanced protection against autologous microbial infection in vivo is lacking. The design of heteroclitic determinants in which non-MHC anchor residues are targeted for substitution are also usually determined empirically. Studies of heteroclitic tumor epitopes have demonstrated the clinical utility of such determinants [16, 17] . Notably, however, there are no well defined examples of viral epitopes which demonstrate enhanced immunity and the molecular basis for the enhanced immunogenicity is not well understood.
Potential interventions to directly manipulate host-pathogen interactions and thereby diminish CTL escape variant selection are often difficult to evaluate since most examples of CTL escape occur in infected humans or non-human primates. By contrast, mice persistently infected with mouse hepatitis virus (MHV) strain JHM (JHMV) serve as a useful system for investigating anti-viral CTL responses and CTL escape [18] [19] [20] . Two JHMV-derived CTL epitopes are recognized in C57BL/6 (B6) mice. The immunodominant H-2D b -restricted CTL epitope (S510, CSLWNGPHL, spanning residues 510-518 of the Spike (S) glycoprotein) elicits a highmagnitude, high-avidity CTL response that drives virus diversification during persistent infection [18] [19] [20] [21] [22] . A second subdominant CTL epitope, S598 (H-2K b -restricted, RCQIFANI, spanning residues 598-605 of the S glycoprotein) also elicits an appreciable CTL response [21] ; however, S598-specific CTL exhibit ,100-fold lower functional avidity and do not protect from CTL escape in S510. The presence of a readily mutable dominant and a subdominant epitope with high and low functional avidity, respectively, in JHMV-infected mice is useful for investigating both epitope enhancement and approaches to diminishing CTL escape. Consistent with this notion, we have previously shown that the introduction of a second dominant CD8 T cell epitope into the JHMV genome (GP33 from lymphocytic choriomeningitis virus (LCMV)) protected mice from the development of CTL escape in S510 and enhanced virus clearance [22] .
Here we determined whether the CD8 T cell response to S598 could be enhanced so that it now elicited a more potent T cell response that protected mice against the development of CTL escape at S510 and subsequent clinical disease. We modified epitope S598 (S598 Q600Y ) such that it elicited a high-avidity CTL response and using the crystal structures of the H-2K b /S598 and H-2K b /S598 Q600Y complexes, determined the basis of this enhanced immune response. We then introduced this more immunogenic S598 epitope into a recombinant version of JHMV and showed that these high-avidity S598-specific CTLs protected against escape variants in the immunodominant S510 epitope. Immunization with the modified peptide resulted in an improved response to the native S598 epitope, demonstrating a true heteroclitic effect and suggesting that this strategy may have clinical applications for reducing viral titer and preventing CTL escape during chronic infections.
As we have previously observed with other MHC/Cyscontaining peptide complexes [19, 23] , complexes did not readily form unless the cysteine of the S598 peptide (RCQIFANI) was modified with L-a-aminobutyric acid (Aba, an isostereomer of cysteine). The Aba-modified peptides maintained immunogenicity since a higher frequency of splenic CD8+ T cells from JHMVimmune mice reacted to Aba-modified S598 peptide, relative to unmodified S598 peptide ( Figure S1A ).
Next, we used the consensus H-2K b binding motif [24] to engineer a novel, high-avidity S598 CTL epitope. Importantly, Gln-3 diverges from the consensus H-2K b -restricted ligand binding motif, in which a tyrosine is often present at position 3 [24, 25] . Therefore, we substituted a glutamine residue for tyrosine (Q600Y, CAA to TAT, RCYIFANI) at position 3 of the determinant with the aim of creating a peptide that bound more tightly to H-2K b . Stability of the H-2K b /S598 and H-2K b /S598 Q600Y complexes was assessed by circular dichroism (CD). As shown in Figure 1 , H-2K b /S598 Q600Y was considerably more thermostable than the native complex (Tm 54uC vs 64uC). To probe the biological properties of the Q600Y substitution, we used reverse genetics to engineer a recombinant version of JHMV expressing the S598 Q600Y epitope (Figure 2A) . Recombinant viruses encoding this substitution replicated as efficiently as wild type JHMV (rJ) in vitro during one-step growth kinetics analyses and in vivo virus competition assays ( Figure 2B , C). The immunogenicity of S598 Q600Y was assessed by intracellular expression of IFN-c by central nervous system (CNS)-derived lymphocytes. Since Cys-containing peptides are often diminished in
Enhancing the immune responses to pathogens is a chief goal of vaccine development. Here, we describe the development of an engineered CD8+ T cell epitope that elicits an immune response to the native epitope that is more potent than the one that occurs during the natural infection. We showed that this ''improved'' (heteroclitic) epitope protects against clinical disease and against cytotoxic T cell escape that frequently occurs in the immunodominant epitope expressed by the virus. We also performed structural analyses and showed that enhanced immunogenicity was associated with changes in the conformations of both the peptide and the region of the MHC class I molecule that is in close association with the peptide. These studies provide a model for designing T cell epitopes with enhanced immunogenicity that will be useful in vaccine development, with particular emphasis on diseases, such as HIV and hepatitis C, in which epitope mutation and escape is common. Seven days p.i., total RNA was harvested from the brains of mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products. The relative proportion of animals in which only rJ, only rJ.S Q600Y , or a mixture of the two viruses is shown. (D) High-ability to elicit a CTL response, we included a reducing agent (TCEP, Tris [2-carboxyethyl] phosphine) in the cultures. TCEP enhanced the stimulatory capacity of the S598 peptides ( Figure  S1B ), indicating that a proportion of the unmodified S598 peptide stock had undergone oxidation. Thus, we stimulated CNS-and spleen-derived CTL ex vivo in the presence of 500 mM TCEP, a concentration consistent with other work with Cys containing peptides [23, 26] .
The S598 Q600Y epitope elicited a CTL response in the CNS of rJ.S Q600Y infected mice, with nearly 30% of all CD8 T cells recognizing the determinant ( Figure 2D ). In addition, CTL primed by S598 Q600Y cross-reacted with the native S598 determinant. The converse was not true, however, as cells primed by the native epitope failed to produce IFN-c when stimulated with S598 Q600Y peptide ( Figure 2D ). Of note, the CTL response to the dominant D b -restricted S510 epitope in rJ.S Q600Y -infected mice was diminished relative to responses in mice infected with wildtype rJ virus ( Figure 2D , E).
Next, we assessed the relative functional avidity of CTL populations primed by native S598 and S598 Q600Y determinants, as a surrogate measure of the potency of the anti-virus CTL response in vivo. CNS-derived mononuclear cells were harvested from mice infected with rJ or rJ.S Q600Y and examined for IFN-c expression after stimulation ex vivo in the presence of 10-fold dilutions of the appropriate peptide. Cells primed to the native S598 epitope (cells harvested from rJ-infected mice) required approximately 100-fold more peptide than did S598 Q600Y -primed cells to elicit a half maximal response (100 nM vs. 1 nM, Figure 3A ,B).
Because we observed that a subpopulation of S598 Q600Y -primed cells cross-reacted with the native determinant, we next determined whether this subpopulation also exhibited high functional avidity. For this purpose, we isolated cells from the CNS of rJ-and rJ.S Q600Y -infected adult B6 mice and stimulated them with 10-fold dilutions of native S598 peptide. S598 Q600Y -primed cross-reacting cells exhibited 7-fold higher functional avidity, relative to those primed by the native determinant ( Figure 3C , D) and are therefore distinct from CTL primed by the native S598 determinant. Consistent with the presence of a distinct population of cells, there were modest differences in Vb chain utilization when total populations from rJ and rJ.S Q600Y -infected mice and when total and cross-reacting cell populations from rJ.S Q600Y -infected mice ( Figure S2 ) were compared. In all instances, Vb5.1/5.2 expression was relatively over-represented, but some Vb elements were preferentially utilized by specific responding populations (Vb11, Vb13 and Vb14). Also consistent with this observation, alanine scanning mutagenesis of the two determinants revealed that the CTL response to each cognate peptide following infection was also subtly different reflecting the altered repertoire. The CTL response to each cognate peptide was very sensitive to mutation at every position except 2 and 9 although mutations in the S598 Q600Y determinant were tolerated slightly better than changes in the S598 epitope ( Figure S2C -E).
If CTL recognizing S598 Q600Y exhibit high functional avidity in vivo, they should protect from CTL escape in S510 and might select for S598 Q600Y CTL escape variants. Diversification at S510 is observed in pups infected with neurovirulent JHMV at 10 days of age and nursed by JHMV-immune dams [27] . These mice are largely protected from developing acute lethal encephalitis, but a variable percentage (30-90%) later develop a demyelinating encephalomyelitis. Infectious virus isolated from these mice with late onset clinical disease is mutated in S510, resulting in enhanced virus replication [20] , with demyelination occurring during the process of virus clearance [28] . Thus, we next infected maternal antibody-protected suckling mice with rJ or rJ.S Q600Y viruses and monitored persistently infected mice for the development of clinical signs for 60 days post infection (p.i.). The presence of the highly immunogenic S598 Q600Y epitope did not protect mice from acute encephalitis ( Figure 3E ), perhaps because the presence of the improved S598 epitope was accompanied by a diminished response to S510 ( Figure 2D ). However, we found that among survivors (defined as survival past day 14 p.i.) there was a significant reduction in the incidence of clinical disease as well as in the development of CTL escape in S510 (Table 1) . Additionally, S598 Q600Y did not undergo CTL escape in mice persistently infected with rJ.S Q600Y , even though single nucleotide changes in the region of the S glycoprotein gene encoding the S598 determinant could potentially result in fifty-one CTL escape mutations. Thus, as expected, the ''improved'' S598 Q600Y epitope was protective in vivo in infected mice, likely because S598 Q600Yspecific CTL are present in higher numbers and exhibit higher functional avidity than the native S598-specific response.
Since this lack of mutation at S598 Q600Y might reflect enhanced suppression of virus replication mediated by co-dominant CTL responses directed against S510 and S598 Q600Y , we developed a recombinant virus encoding S598 Q600Y in the context of a common S510 CTL escape mutation, S510 W513R (rJ.S W513R+Q600Y , Figure 2A ). The CTL response is predicted to be largely directed at S598 Q600Y in mice infected with this virus. The W513R mutation (position 4 substitution in S510 epitope, CSLRNGPHL) occurs in 13% of all CTL escape variants [19, 20, 22, 29, 30] , and completely abrogates native S510 CTL recognition [31] . We verified that virusspecific CTL responses were focused on S598 Q600Y in adult B6 mice infected with rJ.S W513R+Q600Y ( Figure 3F ). To examine the phenotype of the S598 Q600Y /S510 W513R double mutant, we infected antibody-protected suckling B6 mice with this virus and appropriate controls and monitored mice for survival ( Figure 3G ). As expected, 93.3% of mice infected with rJ survived the acute infection (day 0-14 p.i.). All mice infected with rJ.S W513R developed fatal encephalitis but, in marked contrast, 66.6% of mice infected with rJ.S W513R+Q600Y survived. We also found that survival correlated with virus clearance ( Figure 3H ). Relative to rJ-infected mice, replication was suppressed in mice infected with virus encoding the S598 Q600Y epitope and greatly elevated in mice infected with rJ.S W513R . In mice infected with rJ.S W513R+Q600Y , virus titers were intermediate between rJ and rJ.S W513R . Thus, the presence of the heteroclitic S598 Q600Y determinant contributed to suppression of virus replication and to increased survival, even when the highmagnitude, high-avidity CTL response to S510 was largely abrogated.
Surprisingly, S598 Q600Y still did not undergo sequence diversification in mice that survived the rJ.S W513R+Q600Y infection ( Table 2) . This result was unexpected, as the majority of CTL in the rJ.S W513R+Q600Y -infected CNS specifically target the magnitude, unidirectional cross-reactivity. Representative dot plots demonstrating the frequency of epitope-specific CD8 T cells in a mouse infected with rJ (top panels) or rJ.S Q600Y (bottom panels). Numbers represent the frequency of epitope-specific CD8 T cells among total CD8 T cells recovered from the brains of mice 7 days p.i. (E) Summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific CD8 T cells recovered from the brains of rJ and rJ.S Q600Y -infected mice 7 days p.i. Data shown in D represent mean6SEM for 4 independent experiments. doi:10.1371/journal.ppat.1000186.g002 S598 Q600Y determinant and exhibit high functional avidity ( Figure 3A-C) . One possible explanation for this result is that S598 is not as plastic as S510, even though both determinants are derived from a region of the spike gene that is hypervariable and even deleted in some strains of MHV [32] .
To determine whether cells primed to S598 Q600Y that crossreact with the native S598 determinant were more protective in vivo than S598-primed cells, we vaccinated mice with bone marrow-derived dendritic cells (BMDC) alone, or BMDC pulsed with peptides corresponding to S598 or S598 Q600Y (Figure 4) .
Seven days later, mice were challenged via intranasal inoculation of 4610 4 PFU of wild type, non-recombinant JHMV. Similar to results observed following rJ.S Q600Y infection (Figure 3) , CTL primed via DC-S598 Q600Y vaccination exhibited higher functional avidity when reacted against the native S598 determinant when compared to those arising after DC-S598 vaccination ( Figure 4A ). In other experiments, we examined the survival of mice vaccinated with each determinant, but we observed no significant differences between groups (data not shown), probably because mortality is largely CD4 T cell-mediated in adult mice with acute encephalitis [33, 34] . In terms of virus titers, vaccination with either the native or enhanced S598 determinants resulted in ,70-80% reduction in virus burden compared to mice that received un-pulsed BMDC ( Figure 4B ). When we examined the One-fourth of the pups in an individual litter were infected with each virus. At the indicated day p.i., brains were aseptically harvested, homogenized in sterile PBS and clarified by centrifugation. Supernatants were collected and infectious virus was titered on Hela-MHVR (Hela cells transfected with CEACAM1, the JHMV receptor [59] ) as previously described [27] . Symbols on graph represent individual mice assayed from multiple independent litters. The limit of detection (LOD) for the assay is 80 PFU/brain. frequency and numbers of virus-specific CTL in the CNS of these same mice, we detected markedly fewer S598-specific CTL in the CNS of mice that received BMDC pulsed with S598 Q600Y peptides ( Figure 4C) . Calculation of the product of virus titers and CTL numbers within individual mice, as an approximate measure of CTL potency, indicated that the S598-specific CTL in S598 Q600Ycoated BMDC vaccinees were ,6-7-fold more efficacious on a per cell basis ( Figure 4D ). Thus, S598-specific CTL induced by the S598 Q600Y determinant show similar enhancement in function compared to S598-primed cells, whether measured in vitro ( Figure 3C ,D) or in vivo ( Figure 4D ).
While these studies clearly demonstrated that S598 Q600Y is heteroclitic, they did not provide a mechanism for the immune enhancement. To address this, we determined the crystal structures of the H-2K b /S598 (PDBid 2ZSV, Protein Data Bank Japan (http://www.pdbj.org/)) and H-2K b /S598 Q600Y (PDBid 2ZSW) complexes to 1.8 Å and 2.8 Å resolution respectively. The structure of H-2K b /S598 consists of two heterodimers in the asymmetric unit (r.m.s.d. of 0.18 Å for Ca atoms), with the S598 peptide clearly bound in the antigen binding cleft of the heavy chains (HC, Figure S3A ). The two peptide copies display a virtually identical configuration with root mean square deviation (rmsd) values of only 0.09 Å for all peptide atoms (0.05 Å for Ca atoms). The mode of S598 and S598 Q600Y binding within the Agbinding cleft is unambiguous, with the exception of Arg-1, whose side chain is partially disordered (Figure 5A,D; Figure S3C ). The S598 peptide adopts an extended conformation, with the side chains of Arg-1, Ile-4 and Asn-7 extending prominently out of the cleft ( Figure 5C ). Ala-6 is also largely solvent exposed with its side chain pointing towards the a2 helix, while the side chains of Cys (Aba)-2, Gln-3, Phe-5 and Ile-8 are buried within the cleft. While the cysteine analogue's side chain is not involved in any hydrophilic interactions, there are a number of suitably positioned hydrogen bonding partners (Glu-24, Tyr-45 and Asn-70), with which the original thiol side chain could potentially interact ( Figure S3B ; Table S2 ).
In addition to main chain interactions across the length of the peptide, S598 is anchored to the MHC via the side chains of Gln-3, Phe-5 and Ile-8. Gln-3 forms hydrogen bonds with the Ser-99 and Gln-114 of the HC (Figure 5B ), while Phe-5 and Ile-8 are buried within hydrophobic pockets. In addition, the side chains of Phe-5 and Gln-3 pack against each other and between the aromatic rings of HC residues Tyr-159 and Phe-74, constraining the peptide's backbone conformation at those positions.
The structure of H-2K b /S598 Q600Y consists of four heterodimers in the asymmetric unit (rmsd of 0.09 Å for heavy chain (HC) Ca's), the four copies of the peptide adopting virtually identical conformations (rmsd of 0.12 Å for all peptide atoms and 0.05 Å for Ca atoms). S598 Q600Y displays the same conformation as the S598 determinant (the average rmsd for peptide Ca atoms between the two structures is 0.24 Å ) and forms equivalent interactions with the MHC. The only prominent structural difference is observed at the mutated position (Q3RY3) ( Figure 5B,E, 6A,B) . In contrast to Gln-3, the Tyr-3 side chain is oriented towards the a-2 helix rather Figure 5E ). The side chain of Tyr-3 also forms a number of close contacts with the residues in its immediate environment, specifically HC residues Gln-114, Arg-155, Leu-156 and Tyr-159, as well as intramolecular interactions with Phe-5 (Table S2 ). In addition to Tyr-3, deviations of potential significance (.0.5 Å ) in the peptide structures that are attributable to the Q660Y mutation are observed at Ile-4 and Phe-5.
Overall the S598 determinant displays greater complementarity for the antigen binding cleft of H-2K b in its N-terminal region, with few stabilizing interactions observed between the HC and positions 6 and 7. (Figure S3B, D; Table S2 ). Nevertheless, a pocket is observed between the a-2 helix and the peptide near position 3 in the index structure that is filled by the steric bulk of Tyr-3 in the Q600Y structure ( Figure 5C,F) . This increase in surface complementarity and the greater number of observed interactions resulting from the Q3RY3 mutation would account for the enhanced thermostability (,10uC) measured for the H-2K b /S598 Q600Y -Aba complex by circular dichroism (Figure 1 ). This increase in complementarity of the MHC for S598 Q600Y and the greater stability of the resulting complex were predicted from comparisons of the WT determinant complex with existing structures of H-2K b bound with peptides similar to S598 and possessing the consensus tyrosine anchor residue at position 3, as well as another aromatic residue at position 5.
With respect to the HC, the a-1 and a-2 domains of the two structures superimpose well with an rmsd of 0.37 Å for Ca atoms (residues 1-176). Nevertheless, significant deviations (.0.5 Å ) are observed between the two structures at a number of positions in the region of the antigen binding cleft. In particular, changes in the conformation of Ser-99, Gln-114, and Gly-151-Glu-154 are associated with the bound peptides. Changes in the side chain conformations of Ser-99 and Gln-114 in the Q600Y complex structure are consistent with the loss of hydrogen bonding interactions with position 3. In the wild type peptide structure, Glu-152 forms a salt bridge with Arg-155, the guanadinium group of which also forms a hydrogen bond with the main chain carbonyl group of the peptide's Ile-4. While these interactions are maintained in the Q600Y complex, the side chain of Glu-152 displays a conformational shift consistent with the formation of a hydrogen bond with the peptide's Tyr-3 ( Figure 6 ). Interestingly the region of the a-2 helix around Glu-152 (Gly-151 -Glu-154) also displays significant main chain and side chain deviations between the two structures (rmsd of 0.77 Å for Ca atoms; Figure 6 ). Thus, consistent with the functional analyses, the structure of the heteroclitic variant of the S598 epitope displays relatively small changes in conformation yet the combination of these subtle changes in the TcR accessible residues and the structural landscape of the MHCp in addition to the enhanced stability of the complex lead to more efficacious CTL responses.
We describe the identification of a heteroclitic determinant that enhances recognition by virus-specific CD8 T cells, and use the crystal structure of the new determinant (S598 Q600Y ) to provide a basis why it elicits an enhanced CTL response. Comparison of S598 to the consensus binding motif suggested a suboptimal interaction with the H-2K b molecule at the secondary anchor position (Gln-3) and, consequently, an approach to enhance the immunogenicity of the determinant. Replacement of the Gln-3 with Tyr-3 (Q600Y) did, indeed, result in an determinant with increased thermostability without diminishing the CD8 T cell response. Most strikingly, the Q600Y change resulted in subtle changes in the conformation of the a-2 helix locally in the vicinity of Glu-152. These subtle changes are likely critical for the enhanced TcR recognition that we detected. S598 Q600Y elicited a response with higher functional avidity to both the cognate and native determinants than S598, and this was not reflected in differential Vb usage. The T cell response to S598 in rJ-infected mice is very diverse [35] . As assessed by Vb usage, the response to S598 and S598 Q600Y in rJ.S598 Q600Y was similarly diverse with only modest differences noted when cells from mice primed by S598 and S598 Q600Y were compared. While we cannot exclude the possibility that cross-reacting S598-specific cells primed by S598 Q600Y are biased for Vb chains not analyzed in this study, it is more likely that the fine specificity of the complementarity-determining region 3 (CDR3) determines their greater affinity for H-2K b /S598. Although an increase in stability of the MHC class I/peptide complex is not generally expected to enhance TcR affinity for the complex, similar results have been observed in mice immunized with analogues to a common tumor antigen [36] .
One unexpected result was that S598 exhibited both low MHC class I and low TcR avidity. Previous studies showed that this determinant exhibited low functional avidity [21] , but it was not known whether this reflected low binding to the MHC class I or to the TcR. Assuming that low affinity for MHC class I results in a low effective concentration of H-2K b /S598 complex on the cell surface, the responding T cells should be high avidity, based, primarily, on in vitro studies [37] . While the relationship between level of surface peptide and avidity of the responding T cells generated in vivo is not as clearcut [38] , there is no obvious explanation for how a peptide with low MHC class I binding also elicits a low avidity T cell response. This selection of only a subset of CD8 T cells capable of responding to S598 may partly explain why S598-primed cells do not recognize the Q600Y determinant.
The biological significance of the heteroclitic Q600Y determinant was shown by its ability to protect JHMV-infected mice from CTL escape at S510 and to diminish clinical disease. This was important to demonstrate because other studies, using tumor models, have shown that immunogenicity and tumor recognition are not necessarily concordant [39] . Mutations resulting in CTL escape occur most commonly in determinants that are exposed to high selective pressure [40] [41] [42] and outgrowth of CTL escape variants is efficiently suppressed by effective and rapid virus clearance [43] [44] [45] [46] , as occurs in mice infected with rJ.S Q600Y . Thus, even though CTL escape is not detected in normal mice infected with LCMV or influenza, escape does occur when mice transgenic for a single LCMV-specific TcR are infected with high amounts of virus [44, 47] . Under these conditions, the immune response is highly focused on a single CD8 T cell determinant and virus replication continues for extended periods of time, facilitating mutation at the targeted determinant.
In mice infected with wild type JHMV, the CTL response is functionally focused on S510 [21] ; the Q600Y substitution effectively prevents CTL escape at either S510 or S598 Q600Y by the induction of a second high avidity CTL response. Mutations in S598 Q600Y were not detected even when the CTL response was directed primarily at this determinant (e.g. mice infected with rJ.S W513R + Q600Y ). Consistent with this inability to readily tolerate mutations, we were unable to generate recombinant virus mutated in position Ile-4 (I601D,E,K,R,T) and recombinant virus mutated at Phe-5 (F602A) was highly attenuated (data not shown). The combination of induction of high avidity CTL and inability to tolerate mutation without adversely affecting virus fitness make S598 Q600Y an ideal target for the anti-JHMV CTL response. Further, the ala scanning results suggest that S598 Q600Y -specific CTLs may more readily tolerate changes in the H-2K b /peptide complex, and this plasticity would also minimize the likelihood of CTL escape. In contrast, we have previously shown that introduction of the LCMV-specific GP33 determinant, which also elicits CTL with high functional avidity, into JHMV greatly diminished clinical disease but did not prevent CTL escape [22] . The GP33 determinant was introduced at a site in the genome that tolerated mutation and deletion and intact determinant was no longer present in virus by day 20 p.i. Collectively these results suggest that a response to a second determinant that elicits CTL exhibiting high functional avidity at early times p.i. results in enhanced suppression of virus replication, but its presence throughout the infection is required to protect against CTL escape.
In conclusion, we have demonstrated that crystal structures are useful in gaining an understanding of the basis of heteroclitic epitopes and can also prove valuable in guiding the rational design of ''better'' CTL epitopes. In our mouse system, immunization with the heteroclitic determinant resulted in the generation of unique populations of CTL that respond with high functional avidity to an otherwise modestly immunogenic viral epitope. The generation of unique populations of CTL that respond with high functional avidity to weakly immunogenic epitopes will be useful for the treatment and prevention of human infectious diseases. Our proposed structure-guided approach has direct application to HIV, HCV and other chronic infections in which virus persistence and CTL escape occurs. By modulating T cell immunity through prophylactic or therapeutic peptide-based vaccination, virus titers may be reduced and CTL escape and other consequences of viral persistence circumvented.
Specific pathogen-free B6 and BALB/c mice were obtained from National Cancer Institute (Bethesda, MD). To obtain infected mice in which CTL escape at S510 was detected, suckling B6 mice were infected intranasally with 2-4610 4 PFU of recombinant JHMV at 10 days of age and nursed by dams that were immunized with JHMV, as described previously [27] . For experiments comparing multiple JHMV variants, each litter served as an internal control: equal numbers of pups were infected with rJ and one to three recombinant variant viruses, depending on litter size. All animal studies were approved by the University of Iowa Animal Care and Use Committee.
Mononuclear cells were harvested from the brains of acutely ill mice 7 days p.i. and analyzed for expression of IFN-c by an intracellular cytokine staining protocol as previously described [28] . Unless otherwise noted, peptides corresponding to epitopes were used at a final concentration of 1 mM and cells were stimulated in the presence of 500 mM TCEP (Sigma, St. Louis, MO). Cells were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Data sets were analyzed using FlowJo software (Tree Star, Inc, Ashland, OR). All antibodies and reagents were purchased from BD Pharmingen (San Diego, CA).
Recombinant wild-type and S510 and S598 variants of JHMV were generated as previously described [48, 49] . Briefly, overlapping extension polymerase chain reaction (PCR) was used to generate the S598 Q600Y and S510 W513R variants. Primers that overlapped the glutamine at residue 600 of the spike glycoprotein were (59 to 39) Q600Y fwd, ATGATCGCTGCTATATTTTTGCTAACATAT-TG; Q600Y rev, AATATGTTAGCAAAAATATAGCAGCGAT-CAT. Primers that overlapped the tryptophan at residue 513 were (59 to 39) W513R fwd, GTGAGTGTTCTCTTCGGAATGGGC-CCCATTTGCGCTCGGC; W513R rev, AGCGCAAATGG-GGCCCATTCCGAAGAGAACACTCAC. The outer primers for each targeted change were fwd, TGTTGATTGCGCCAG-CAGCTACATTAG; and rev, ACCTACGGATTGAACGCTAT-CATTGAC. Underlined nucleotides correspond to the nucleotides encoding the Gln to Tyr and Trp to Arg substitutions within S598 and S510, respectively. Recombinant viruses encoding the variant epitope(s) were selected, propagated and titered as previously described [49] . At least two independent isolates of each recombinant virus were analyzed.
Virus was inoculated onto confluent 17Cl-1 monolayers in a 12well plate at a multiplicity of infection (MOI) of 1.0. Groups of cells were harvested at the indicated time points and total virus (cellassociated and cell-free) was titered as previously described [27] .
Equal PFU (2-4610 4 ) of rJ and rJ.S Q600Y were combined and inoculated intranasally into 5-week-old B6 and BALB/c mice. Total RNA was harvested from the brains of mice 7 days p.i. and the relative representation of WT (wild type) versus variant template was determined by RT-PCR and sequencing. This assay can specifically detect a given species of template when that species comprises at least 20% of a heterogeneous pool [50] . Primers used were : forward, AACCCCTCGTCTTGGAATAGGAGG-TATGG; and reverse, CCTACGGATTGAACGCTATCATT-GACTAAC. PCR products were sequenced directly by the University of Iowa DNA Core.
For alanine scanning, cells were stimulated ex vivo with the indicated concentration of native or variant peptide and stained for CD8 and IFN-c as described above. Data were normalized to the frequency of cells that reacted to the unmodified S598 or S598 Q600Y peptides. For functional avidity determination, mononuclear cells were harvested from the brains of rJ or rJ.S598 Q600Y -infected mice 7 days p.i. and stimulated ex vivo in the presence of EL-4 cells pulsed with tenfold dilutions of peptide corresponding to native S598 or S598 Q600Y epitopes. After 5.5 hours, cells were stained for intracellular IFN-c as described above. For each epitope-specific population, data were normalized to the frequency of antigenspecific CTL detected using the highest titration of peptide (1 mM).
CD spectra were measured on a Jasco 810 spectropolarimeter using a thermostatically controlled cuvette at temperatures between 30 and 90uC. Far-UV spectra were collected and analyzed as described [23] .
Cells were harvested from the CNS of mice 7 days p.i. and stimulated ex vivo with S598 or S598 Q600Y peptides. Cell aliquots were subsequently stained for CD8 (PE-Cy7-anti-CD8a) and Vb (FITC-anti-Vb2, 3, 4, 5.1/5.2, 6, 7, 8, 9, 10 b , 11, 12, 13 or 14, BD-Pharmingen) followed by intracellular staining for IFN-c (PEanti-IFN-c). Data were collected using a Becton Dickinson LSR II instrument at the University of Iowa Flow Cytometry Facility. Data are expressed as the proportion of antigen-specific CD8 T cells that express each Vb chain.
Total RNA was purified with TRIzol (Invitrogen, Carlsbad, CA) from the CNS of mice. The 1055 base pair region of the spike glycoprotein encompassing both S510 and S598 was amplified by RT-PCR and sequenced directly as previously described [19] .
Bone marrow-derived DC were prepared, pulsed with peptides and injected into mice as previously described [51] . Briefly, 5610 5 LPS-matured DC were left uncoated, coated with S598 or S598 Q600Y peptides and injected via tail vein into groups of 4week-old mice. Seven days following DC-vaccination, mice were infected intranasally with 4610 4 PFU of JHMV. Seven days following virus infection, brains were harvested from mice and the frequencies of epitope-specific CD8 T cells were determined by ex vivo stimulation and intracellular cytokine staining as described above. In independent studies, DC-S598 and DC-S598 Q600Y priming was verified by harvesting spleens from several mice seven days following DC-vaccination.
Crystal structure of H-2K b /S598 and H-2K b /S598 Q600Y complexes Crystals of H-2K b S598-Aba were grown at 21uC in 0.1 M cacodylate pH 6.6, 16% PEG (polyethylene glycol) 8,000, 0.2 M Ca(OAc) 2 , using a protein concentration of 9 mg/ml. Crystals were cryoprotected by stepwise equilibration against mother liquor containing 5, 10 and 15% glycerol and flash frozen by placing in a nitrogen stream. A 1.8 Å resolution dataset was collected on an inhouse X-ray source. Crystals of H-2K b S598 Q600Y -Aba were grown at 21uC in 0.1 M cacodylate pH 6.5, 13% PEG 8,000, 0.2 M Ca(OAc) 2 , using a protein concentration of 6 mg/ml. Crystals were cryoprotected by gradual equilibration against mother liquor containing 20% PEG 8,000 and 5% glycerol before flash freezing. A 2.8 Å resolution dataset was collected on an in-house source. The WT data were integrated in MOSFLM [52] and scaled/merged using SCALA [53] . The Q600Y variant data were processed using HKL2000. Both structures were solved by molecular replacement in PHASER [54] , against previously solved H-2K b complexes (PDBid's: 1G7Q and 1RJY, respectively). The resultant models underwent iterative cycles of refinement in PHENIX [55] and, REFMAC5 [56] (restrained and TLS refinement) followed by model building/ correction in COOT [57] . The solvent structures were built using ARP/wARP [58] and COOT. A summary of the processing and refinement statistics is presented in Table S1 .
Statistical significance was determined by nonpaired, two-sided Student's t test or chi-squared test, where indicated. (A) Total mononuclear cells were harvested from mice infected with rJ or rJ.S Q600Y and stimulated ex vivo with S598 and S598 Q600Y peptides, respectively. Following stimulation, aliquots of cells were surface stained for CD8 and the indicated Vb chain followed by intracellular cytokine staining for IFN-c. (B) CNSderived cells from rJ.S Q600Y -infected mice were stimulated ex vivo with peptides corresponding to native S598 or S598 Q600Y epitopes. Following stimulation, cells were analyzed as described for (A). Data represent the fraction of IFN-c+CD8+ T cells expressing each Vb chain and are derived from cells pooled from 2-3 individual mice. (C) Alanine scanning of the native S598 determinant. CNS-derived mononuclear cells were recovered from rJ-infected mice 7 days p.i. and stimulated ex vivo in the presence of 500 mM TCEP and 1 mM of the indicated peptide then stained for CD8 and intracellular IFN-c. Data are normalized to the frequency of epitope-specific cells detected when stimulated with the native S598 determinant. (D) Alanine scanning of the S598 Q600Y determinant. Cells were harvested and tested as described for B except in this case the cells originated from the rJ.S Q600Y -infected CNS and were stimulated with 10 nM S598 Q600Y peptide. (E) Alanine scanning of the S598 determinant recognized by S598 Q600Y -primed, cross-reactive CTL. As in C, but cells were stimulated with 150 nM S598 peptide. For B-D, concentrations of peptide equivalent to 106 that required for half maximal stimulation were used; data are mean6SEM from four independent experiments. Note that the differential responses to RCAIFANI in panels C and D reflect the differing amounts of peptide used in the two assays. Found at: doi:10.1371/journal.ppat.1000186.s002 (0.76 MB TIF) Figure S3 Refined structures of WT and Q600Y S598-Aba bound to H-2K b . (A) View of the H-2K b antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The unbiased F o -F c map density for the peptide contoured at 2.5 s is shown as a magenta mesh. (B) The same view as in A displaying key interactions (dashed lines) between H-2K b and S598-Aba. Selected residues of the HC are drawn in stick format (slate carbon atoms) and ordered water molecules are shown as red spheres. Peptide residues are labelled in italics. C and D, Equivalencies to A and B, respectively, for the H-2K b / S598 Q600Y -Aba structure. In these panels the HC is drawn in green and the peptide in cyan.  HIV-Specific T-Cells Accumulate in the Liver in HCV/HIV Co-Infection BACKGROUND AND AIMS: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses. METHODS: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry. RESULTS: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4(+) T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8(+) and CD4(+) T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8(+) T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4(+) T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses. CONCLUSION: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease. Approximately 25% of Human Immunodeficiency Virus-1 (HIV) infected individuals are also infected with Hepatitis C Virus (HCV) [1] . HIV adversely affects each stage of the natural history of HCV infection. Fewer individuals recover spontaneously from HCV infection when also infected with HIV [2] . Among those with persistent HCV infection, HIV co-infection is associated with higher HCV viremia and more rapid progression to cirrhosis and hepatocellular carcinoma [3] . A recent meta-analysis showed that HIV co-infection increased the risk of histological hepatic cirrhosis by two-fold and clinically decompensated liver disease by six-fold [4] . In addition, HCV co-infection is associated with increased incidence of HAART (highly active antiretroviral therapy) related liver injury [5] .
The mechanisms for hepatic damage in HCV/HIV co-infection are poorly defined. Although intra-hepatic T-cell immune responses are necessary for HCV clearance, they have also been shown to play a central role in mediating hepatocellular injury by direct cytotoxicity or indirectly by releasing cytokines. In this regard, IFN-c has been shown to be anti-fibrogenic, whereas, TNF-a activates hepatic stellate cells, which induce fibrosis, and likely contributes to progression to cirrhosis [6, 7] .
Potent and broad CD4 + and CD8 + T-cell immunity are important for virologic control in both HCV and HIV viral infections. Ex-vivo HCV-specific CD8 + T-cell responses in peripheral blood mono-nuclear cells (PBMCs) from mono-infected individuals are generally weak [8] . Although, peripheral HCVspecific CD4 + and CD8 + T-cell responses are somewhat weaker in HCV/HIV co-infected individuals [9] , similar frequencies of intra-hepatic HCV-specific responses appear to be obtained in HCV versus HCV/HIV co-infection [10, 11] . However, ex-vivo HIV-specific CD8 + T-cell responses in PBMCs from HIV monoinfected individuals are about one log higher than ex-vivo HCVspecific responses in HCV mono-infection. In addition, impairment in cellular immune responses to HCV compared to HIV has been shown in HCV/HIV co-infection [12] . HIV-specific CD8 + T-cells are easily detectable in blood of untreated HIV infected individuals [13] . Such high frequencies of HIV-specific T-cells circulating in peripheral blood led us to question whether these cells could also migrate to the liver in HCV/HIV co-infection and through bystander responses add to the inflammation induced by HCV-specific T-cells.
HCV mono-infected and HCV/HIV co-infected individuals who required liver biopsies for work up of liver disease were recruited for the study (see Results and Table 1 ). All study participants provided informed, written consent and the study protocol was approved by the research ethics board at the University of Toronto and St. Michael's Hospital. Both blood and liver biopsy samples were received from each participant.
Liver biopsy samples were washed in RPMI-1640 to remove contaminating blood lymphocytes, manually homogenized with a plastic plunger, and treated with DNase (0.002%, Sigma) and collagenase IV (0.02%, Sigma) for 30 minutes, stirring at 37uC. The digested cell suspension was filtered through a 70 mm strainer, washed and re-suspended in R-10 medium (10% fetal calf serum).
In order to identify candidate epitope-specific responses to be detected in ex-vivo liver samples, we first mapped antigen-specific T-cell responses in blood against the entire HIV-1 clade-B and HCV-1a proteome using the matrix approach by IFN-c ELI-SPOT assay as described previously [14] . Mapped peptides were then pooled to evaluate hepatic responses. In order to address the possibility that differing epitopes were only targeted in the liver, we also used four peptide pools that previously were shown to target a majority of responses. These pools spanned HIV-Gag and HCV-NS3, HCV-NS4 and HCV-Core protein (2 mg/peptide/ml, from National Institute of Health Reagent Program). Of the HCV pools, the pool that gave the strongest ELISPOT response in PBMCs was used for hepatic cell stimulation (see below).
All the extracted cells from each liver biopsy were split in three wells and stimulated on the same day as PBMCs. 1610 6 PBMCs and liver-isolated cells were stimulated with either DMSO, HIV or HCV peptide pools as described previously [14] . HIV pools consisted of peptides that were screened by the matrix approach in that individual plus the HIV-Gag pool. Likewise, HCV pools consisted of mapped peptides plus an HCV pool that gave the strongest response in PBMCs. CD107a antibody (PE-Cy5, BD Pharmingen) was added at the time of stimulation. The following antibodies were used for staining: CD8-PE Texas-Red (Beckman Coulter), CD4-Pacific Blue (e-Bioscience), CD3-APCCY7, IFNc-FITC, TNFa-PECY7, IL2-APC, MIP-1b-PE (BD Bioscience), PD-1 FITC (Biolegend) and dead cell stain Aqua (Invitrogen, Molecular Probes).
Cells were analyzed on a multi-color FACSAria flow cytometer (BD Biosciences). For Blood samples between 500,000 to 1,000,000 total events and for liver biopsy samples between 50,000 to 200,000 total events were collected. Data analysis was performed using FlowJo version 8.6 (Treestar Inc., San Carlos, CA). Polychromatic FlowJo data were analyzed with PESTLE software, and pie-chart graphs were generated using SPICE software (obtained from M. Roederer, National Institutes of Health, Bethesda MD).
Multi-parameter analysis of HIV-Gag: 77-85 (SLYNTVATL: SL9) specific CD8 + T-cells was conducted in both blood and liver of co-infected individuals initially identified with a positive ELISPOT response to the 15-mer HIV peptide including SL9 epitope, using the corresponding tetramer (iTAg MHC Class-I tetramer, Beckman Coulter). Tetramer staining was performed prior to peptide stimulation, at room temperature for 20 minutes. Tetramer stained cells were then washed and stimulated with 10 mg/ml of SL9 peptide followed by ICS staining as mentioned above. Additional HIV, HCV and CMV-specific pentamer staining (Pro5 MHC class I Pentamers -Proimmune) was conducted followed by PD-1 staining. 
Data were analyzed by performing two-tailed non-parametric Mann-Whitney test using GraphPad Prism version 4.00. P-values#0.05 were considered significant.
Three groups of individuals were studied as depicted in Table 1 ; HCV mono-infected (n = 6), HCV/HIV co-infected who were not receiving HAART (n = 8) and HCV/HIV co-infected who were receiving HAART for greater than one year at the time of evaluation (n = 12). All individuals never received prior treatment for HCV and underwent liver biopsies for staging and evaluation for pegylated-interferon/ribavirin treatment. HCV/HIV co-infected individuals had higher HCV viral loads. On average, CD4 T-cell counts of HIV infected individuals were .400/ml in both groups. Of note, the mean hepatic fibrosis scores were higher in the HAART treated and mono-infected groups in this cohort, indicating that individuals in these groups had more advanced disease at the time of biopsy in this study.
Although similar frequencies of intra-hepatic lymphocytes were obtained in dual versus mono-infection, HAART-treated individuals showed a trend towards greater percentages of lymphocytes in their biopsies ( Figure 1a ). The percentage of intra-hepatic CD4 + T-cells was significantly reduced in dual infection [31.6%613.8 for HCV vs 6.5%62.9, for HCV/HIV therapy naïve, p,0.01], and was not associated with any improvement in HAART-treated individuals, as previously shown in the gut [15] (Figure 1b) . However, compared to HCV mono-infected individuals the percentage of intra-hepatic CD8 + T-cells was higher in both co-infected groups [33.8%65.5% for HCV vs 67.3%615.5% for HCV/HIV therapy naïve vs 59.5615.1% for HCV/HIV on HAART, p,0.01] (Figure 1b ).
To determine the presence of intra-hepatic viral specific T-cell responses, liver isolated cells were stimulated with HCV and HIV peptide pools. Summary data of viral specific responses are depicted in Figure 2 . In response to stimulation with HIV peptide pool, untreated co-infected individuals showed significantly higher frequencies of intra-hepatic CD4 + T-cells producing IFN-c, compared to HCV mono-infected [0.1660.05% vs 0.0260.01%, p,0.05], and HAART-treated co-infected individuals [0.1660.05% vs 0.0360.05%, p,0.05] (Figure 2a ). Untreated co-infected individuals showed a trend towards lower frequencies of intra-hepatic IFN-c producing CD4 + T-cells in response to HCV peptides. Surprisingly, HAART-treated co-infected individuals had significantly reduced HCV-specific IFN-c producing CD4 + T-cells when compared to untreated co-infected individuals [0.0260.01% vs 0.4660.11%, respectively, p,0.01] (figure 2a).
Therapy naïve co-infected subjects had greater IFN-c producing CD8 + T-cells in response to HIV peptides compared to HCV mono-infected individuals [1.3960.37% vs 0.0260.0%, p,0.05], and HAART was associated with a significant reduction in the frequencies of these cells [1.3960.37% vs 0.3060.26%, p,0.05] (figure 2b). Although there was a trend for enhanced intra-hepatic CD8 + T-cells producing IFN-c in response to HCV peptides in therapy-naive co-infection compared to HCV mono-infection, this was not found to be statistically significant. HAART on the other hand, was associated with a significant reduction in HCV-specific, intra-hepatic CD8 + T-cells producing IFN-c [1.360.37% vs 0.0360.01%, p,0.05] (figure 2b).
Similarly, co-infected individuals had significantly greater intrahepatic TNF-a expressing CD4 + T-cells after HIV peptide stimulation compared to HCV mono-infected [0.260.05 vs 0.0260.01, p,0.01], although HAART had no significant effect on their frequencies (Figure 2c ). HCV mono-infected individuals showed significantly higher frequencies of HCV-specific TNF-a producing CD4 + T-cells compared to HAART-treated co-infected individuals [0.9160.25% for HCV vs 0.2360.20 for HCV/HIV on HAART, p,0.01] (figure 2c), but did not show significant differences with the untreated co-infected group.
The therapy-naïve co-infected group showed significantly higher frequencies of intra-hepatic TNF-a producing CD8 + Tcells in response to both HIV-1 and HCV antigens. Both types of 
To study the functional profile of virus-specific T-cells in HCV/ HIV co-infection, simultaneous expression of 5 distinct CD8 + T-cell markers were analyzed in 3 individuals within each cohort using a previously developed multicolor flow cytometry method [16] . Expression levels of degranulation marker CD107a and cytokines IFN-c, TNF-a and IL-2, as well as the chemokine MIP-1b were simultaneously measured in response to HCV or HIV peptides in both blood and liver of each individual. Figure 3 depicts a representative multi-parameter analysis of CD8 + T-cell responses in liver and blood of a therapy-naïve, co-infected subject in response to HIV and HCV peptide pools. These data indicate that both HCV and HIV specific CD8 + T-cells expressing one or more functions are detectable in the liver and blood. Compared to blood, the frequency of HIV-specific T-cells producing CD107a and IL-2 was shown to be significantly higher in the liver of therapy-naïve, co-infected individuals. Consistent with previously reported data [10] , HCV-specific responses were compartmentalized to the liver and stronger than peripheral HCV-specific responses (Figure 3c ).
Recognition of functional CTL, specific for the HLA-A0201-restricted HIV-SL9 epitope in HCV/HIV co-infected liver
To determine if T-cells specific for an HIV immuno-dominant epitope are present in HCV/HIV co-infected liver, we quantified CD8 + T-cells specific for HLA-A*0201-restricted SLYNTVATL (SL9) epitope in the liver of individuals with positive SL9 responses in their blood. We identified 3 co-infected HLA-A*0201 individuals, among them one showed a response to the SL9 epitope of HIV-Gag antigen. Figure 4 shows the multi-parameter analysis of tetramer positive CD8 + T-cells in blood and liver of this therapy-naïve, co-infected individual. The tetramer cytokine response pattern was shown to be different in the liver compared to blood of the same individual, with diminished intra-hepatic tetramer-specific IFN-c responses and an increase in both CD107a and TNF-a responses, with the majority of SL9 tetramer positive cells expressing these two markers.
We also included CMV as a non-hepatotropic control virus in our liver analysis. Using a pool of HLA-A*0201-and HLA-A*2402-restricted, CMV-specific pentamers, we did not detect any CMV-specific CD8 + T-cells in HCV/HIV co-infected liver, although we could readily detect them in blood (Figure 4c ).
Using the panel of markers TNF-a, IFN-c, MIP-1b, IL-2, and CD107a, we characterized the ability of CD8 + T-cells to simultaneously exert these 'functions' in response to both HCV and HIV peptides. Figure 5 depicts a representative functional profile of virus specific CD8 + T-cells in blood and liver during HCV/HIV co-infection (Fig. 5a) and HCV mono-infection (Fig. 5b) . Analysis of co-infected subjects demonstrates a very limited functional hierarchy of HCV-specific T cells in the blood, with majority of T-cells producing one function. HIV-specific Tcells from blood had a more expanded functional hierarchy. In accordance with previous reports on HIV mono-infected individuals [16] , cells expressing all 5 functions were absent in the blood of co-infected subjects, mainly due to lack of IL-2 production. In co-infection, intra-hepatic CD8 + T-cells responding to HCV peptides were within the single-responding and 2+ populations. Intra-hepatic CD8 + T-cell responses to HIV peptides produced a larger spectrum of responses. CD107a responding cells were represented in nearly all of the different HIV-specific populations in the liver of co-infected individuals.
As expected, HCV-specific responses in the blood of HCV mono-infected subjects were mainly single functional. The profile of HCV-specific responses in the liver of HCV mono-infected individuals showed the appearance of a very small population of 4+ responding cells in the liver.
We and others have considered a cutoff point of more than 2 simultaneously expressed markers to demonstrate poly-functional characteristic of responding T-cells [16, 17] . Figure 5c shows a comparison between average frequency of intra-hepatic viralspecific responses within the pool of CD8 + T-cell populations expressing 2 markers or less, and those within the pool of populations expressing more than 2 markers simultaneously. For both HCV and HIV-specific CD8 + T-cells the majority of responses had two or less functions. However, intra-hepatic HIV-specific responses demonstrated more poly-functionality, compared to HCV-specific responses either within co-infected or mono-infected individuals [0.0560.01 vs 0.00760.00, p,0.05; HIV-specific responses vs HCV-specific responses in HCV/HIV co-infected group]; [0.0560.01 vs 0.0160.00, p,0.05; HIVspecific responses in HCV/HIV co-infected group vs HCVspecific responses in HCV mono-infected group]. In summary, although viral-specific T-cells, simultaneously expressing all 5 measured markers were rarely found in the liver, intra-hepatic HIV-specific T-cells showed greater functional capacity when compared to those being HCV-specific.
Based on the recently highlighted role of PD-1 contributing to the dysfunction of T-cells in chronic viral infections, we also determined whether HIV and HCV-specific intra-hepatic T-cells differ in the degree of PD-1 expression. In an HCV/HIV coinfected liver, we found that 100% of intra-hepatic HCV-specific CD8 + T-cells were PD-1 positive, compared to 48.8% of those cells that were HIV-specific (Figure 5d ).
This is the first study to demonstrate the presence of HIVspecific T-cells within the liver of HCV/HIV co-infected individuals. The finding of HIV-specific T-cells within liver of co-infected individuals may not altogether be surprising, given the high frequencies of HIV-specific CD8 + T-cells found in the peripheral blood in untreated HIV infection. Nevertheless, it is surprising to find functional T-cells of such viral specificities to be accumulating in liver. In contrast, we could not detect CMVspecific T-cells in co-infected liver despite their abundance in blood indicating that different viruses target T-cells to the liver. Recent studies have demonstrated that systemic viral infections may recruit viral-specific T-cells to the liver. The significance of non-hepatotropic viral-specific T-cells that are found in liver is unclear. It has been postulated that the liver can non-selectively trap activated T-cells during any infection, and thus act as a 'sink' or 'graveyard' [18] , however it is unclear whether these cells are rendered anergic while traveling in the liver or contribute to inflammation and damage as a result of bystander activation. Of note, is that hepatitis has been observed in measles [19] , SARS [20] and in 20% of individuals with acute HIV infection [21] . Polakos et. al. [22] found that some individuals infected with influenza-A develop transaminitis and showed in a murine influenza model that influenza-specific CD8 + T-cells migrate to Figure 3 . Polychromatic FACS analysis of viral-specific T cells in HCV/HIV co-infection. Shown are representative FACS data of the HIV and HCV specific multi-parameter CD8 + T-cell responses from (a) liver and (b) blood of subject OM 405, a therapy-naïve HCV/HIV co-infected individual, after in vitro stimulation using pool of HIV and HCV peptides. Initial gating on forward scatter area (FSC-A) versus height (FSC-H) was used to remove doublets. The events were further gated on forward scatter (FSC) versus the dead cell marker to remove dead cells. Lymphocytes were gated on the remaining live cells on a FSC versus SSC plot. Gates on CD3 + /CD8 + cells were then generated. All responses are gated on a CD3 + /CD8 + population and presented against TNF-a on the x-axis. Figure (c) shows a comparison of the frequency of HIV and HCV-specific CD8 + T-cells in the liver and blood of therapy-naïve, co-infected individuals. All intra-hepatic HCV-specific responses are significantly stronger than peripheral HCV-specific responses. doi:10.1371/journal.pone.0003454.g003 Non-hepatotropic viruses such as HIV, CMV and EBV, in general do not induce chronic hepatitis, thus, it is possible that the coexistence of hepatotropic viruses may alter the hepatic environment to allow recruitment of activated T-cells non-specifically. This could be due to an up-regulation of integrins such as ICAM-1 and VCAM-1 in hepatic sinusoids as previously shown during HCV infection [23] that could enhance T-cell recruitment. In this regard, Spangenberg et. al. [24] demonstrated the presence of influenza-specific T-cells in about 50% of liver biopsies from HCV mono-infected individuals.
There are several lines of evidence demonstrating that the liver efficiently clears many foreign pathogens, including RNA viruses. It is shown that liver is a major organ for clearing Simian Immunodeficiency Virus in rhesus monkeys [25] . There is also evidence for the detection of HIV RNA in the liver of HIV infected individuals [26] . These findings support the identification of HIV-specific T-cells in the liver. In HCV/HIV co-infection, it is possible that intra-hepatic HCV-specific CD4 + T-cells become infected with HIV and recruit HIV-specific immune responses to this site. Evidence for these potential mechanisms will need further analysis on liver biopsies of co-infected individuals.
Our analysis of liver biopsies from HCV/HIV co-infected individuals not only demonstrate that HIV-specific T-cells producing IFN-c and TNF-a are detected in the liver, but also exhibit comparable frequencies of responses to those that are HCV-specific. This observation may explain the added contribution of HIV-specific immune responses to the ongoing intrahepatic damage induced by HCV-specific T-cell responses that are inefficient in clearing the virus. Therapy naïve co-infected individuals demonstrated a higher frequency of intra-hepatic CD8 + T-cells that produce TNF-a in response to both HCV and HIV antigen stimulation compared to HCV mono-infected individuals. In addition, we identified CD8 + T-cells specific for an immunodominant HIV epitope in co-infected liver, demonstrating high frequency of TNF-a expression. Intra-hepatic TNF-a has been previously associated with liver fibrosis, and the accumulation of cells expressing this marker may explain in part the faster rate of liver disease progression found in HCV/HIV coinfection. Further comparisons of TNF-a responses between immunodominant HCV and other HIV epitopes in a larger cohort of individuals are warranted. Contrary to our expectation, viral-specific, intra-hepatic levels of IFN-c were also higher in the therapy-naïve co-infected group, which would be against the expected protective role of IFN-c. However, we interpret this observation as a potential effect of the fibrogenic TNF-a to mask IFN-c protection. On the other hand, viral-specific T-cells are composed of several major populations with unique functional patterns. Therefore, measurement of only one or two T-cell functions may not provide a comprehensive picture of the quality of T-cell responses.
Recent lines of evidence demonstrate the importance of the qualitative rather than quantitative characteristics of CD8 + T-cell responses to efficient viral control [13, 27] . The significance of Tcell populations simultaneously representing 5 different functions has been discussed as a hierarchical functional model in viral infections such as CMV and EBV which are effectively controlled by respective CD8 + T-cells [16] . HCV-specific CD8 + T-cells were not poly-functional which is consistent with the notion that although HCV-specific T-cells are found in hepatic tissue, their loss of poly-functionality may be associated with inefficient control of HCV replication. HIV-specific T-cells in the liver of co-infected individuals however, simultaneously could express 4 and 5 of the measured markers. Recently, T-cell exhaustion has been related to signaling pathways through PD-1 [28, 29] . Our analysis of PD-1 levels of antigen-specific CD8 + T-cells from co-infected liver demonstrates higher expression of PD-1 on HCV-specific T-cells, compared to those specific for HIV, supporting the notion that the former are less functional. The observed poly-functionality of intra-hepatic HIV-specific T-cells, should have little effect on HCV replication but would further enhance the cytokine milieu induced from bystander activation, and contribute to liver damage during co-infection with HCV. In this regard, we found that the degranulation marker CD107a dominates the HIV-specific CD8 + T-cell responses in the liver, with the majority of the responding cells expressing CD107a, a surrogate marker for the cytotoxic function of CD8 + T-cells. Activated HIV-specific CD8 + T-cells with the potential to degranulate could induce bystander damage. In addition, the release of chemokines such as MIP-1b by the same cells could also attract further lymphocytes without HCV specificity to the liver. Bystander function of these non-specific T-cells could expand the tissue damage triggered by HCV infection and ultimately activate fibrogenesis.
We found that the frequency of CD4 + T-cells within livers of coinfected individuals was reduced compared to HCV monoinfection. Surprisingly, HAART did not appear to reconstitute the CD4 + T-cell population within liver. Despite this defect of CD4 + T-cell help, comparable frequencies of HCV-specific-CD8 + T-cells were found in co-infected livers. HAART-treated biopsies showed further reduced frequencies of HCV-specific responses. These data support previous findings that show HAART induces CD4 + T-cell recovery but not any restoration of HCV-specific Tcell responses peripherally [30] . Further investigation is needed to clarify the role of CD4 + T-cell help in affecting the frequencies of HCV-specific CD8 + T-cells in HCV/HIV-1 co-infection. HAART was also associated with a reduction in frequencies of HIV-specific T-cell responses within liver, indicating that removing the HIV antigenic load may also reduce the opportunity for such cells to accumulate within hepatic tissue.
Here, we propose a novel mechanism for enhanced HCVrelated liver disease progression in HIV co-infection; that of bystander activation and induced inflammation from HIV-specific T-cells accumulated in the liver. Our data however are limited in the cross-sectional nature of our cohort, the low number of analyzed liver biopsies and the narrow range of CD4 + T-cell counts among the studied individuals. We should also acknowledge that ex-vivo functional T-cell capacity may not exactly reflect the situation in-vivo. Further studies, particularly, those which are prospective are warranted in order to understand the role that HIV-specific T-cells play in contributing to fibrosis and in particular how HAART modulates these responses. frequency of CD8 + T-cells specific for HCV, HIV and CMV in HCV/HIV co-infected liver (OM 385). Liver isolated mononuclear cells were stained with pools of HLA-A*0201 and HLA-A*2402-restricted pentamers (Pro5 MHC class I Pentamers, Proimmune), followed by staining for cell surface markers CD3 and CD8. The following pentamers were used for each group: HCV pentamers: NS3-CINGVCWTV and NS3-KLVALGINAV; HIV pentamers: Pol-ILKEPVHGV and Gag p24-TLNAWVKVV; CMV Pentamers: pp65-NLVPMVATV and pp65-QYDPVAALF. No CD8 + T-cells specific for CMV were detected in this co-infected liver sample. Similar findings were found in another individual (data not shown). doi:10.1371/journal.pone.0003454.g004 background is shown to become extremely low when examining combinations of functions, nearly reaching 0 events for multiple functions simultaneously. This permits a very low threshold for detection of positive responses from multiple combinations. Consequently, for multi-parameter analysis, the results were thresholded based on a minimum criterion of positivity, as calculated by SPICE software and presented as the 90 th percentile of negative values for each analysis. Each pie chart generated by SPICE software, represents the hierarchy of responses to either HCV or HIV antigen stimulation. For simplicity, responses are grouped by number of functions and matched to the colored bars, with black bars representing the percentage of responding cells to HIV peptides and gray bars representing the percentage of responses to HCV peptide stimulation. In all pie charts, color red represents the 5+ responding population and the colors blue, green, turquoise, and yellow representing the 4+, 3+, 2+, and 1+ populations respectively. Color-coded arcs represent the dominant marker within each pie slice, with color blue representing TNF-a, red for CD107-a, green for IFN-c and black for MIP-1b. Although IL-2 is included in the presentation and demonstrated by bar graphs, the software would not allow for arc colors for more than 4 responses. As a result there is no arc representative for IL-2. Figure (c) represents the average frequency of intra-hepatic viral specific responses within the pool of CD8 + T-cell populations simultaneously expressing 2 functions or less, compared with those within the pool of populations expressing more than 2 functions simultaneously; as analyzed in 3 subjects within each cohort of HCV mono and HCV/HIV co-infected individuals. The cutoff point of simultaneous expression of more than 2 measured markers is considered to show CD8 + T-cell poly-functionality.  Multiorgan failure due to hemophagocytic syndrome: A case report INTRODUCTION: Hemophagocytic syndrome (HFS) is a potentially lethal disorder due to an uncontrolled immune response to a triggering agent. Our objective is to raise the importance of HFS early diagnosis by presenting a representative case. CASE PRESENTATION: A sixteen-year-old girl with Still disease diagnosis developed a progressive multiorgan failure including acute respiratory distress (ARDS), anemia and thrombopenia, elevated liver enzymes, renal failure, coagulopathy with hypofibrinogenemia, and acute phase reactants elevation despite broad-spectrum antibiotics. A bone marrow puncture-biopsy was performed, and hemophagocytosis was found. Prolonged fever, splenomegaly, bicytopenia, hypofibrinogenemia, hyperferritinemia and hypertriglyceridemia confirmed HFS diagnosis. She received intensive care support therapy including mechanical ventilation and specific therapy according to HLH 2004 protocol, with a very good response. CONCLUSION: Our case shows complexity of HFS diagnosis, due to septic shock-like manifestations. Early diagnosis is essential to start appropriate treatment achieving a better outcome. Hemophagocytic syndrome (HFS) is a rare disorder characterized by prolonged fever, cytopenias, hepatosplenomegaly, hypertriglyceridemia, disseminated intravascular coagulation (DIC)-like coagulopathy and bone marrow, spleen, liver or lymphatic nodes histiocytosis [1] [2] [3] . A sudden presentation, like a septic shock is possible making its early recognition a challenging diagnosis [4] . It is well known that HFS could be a severe complication in some infections (mainly virals) or in some underlying diseases, such as chronic juvenile arthritis (CJA) [3, 5, 6] . Moreover, it is one of the differential diagnosis in fever of unknown origin [7] .
Taking into account that a better outcome has been related to an early treatment [3, 8] , presentation of a difficult diagnosis case in a young lady could be helpful and interesting.
A sixteen-year-old girl presented with rash, elevated fever and joints swelling. She was admitted to the hospital to make further examinations as the symptoms persisted for 15 days. A diagnosis of probable Still disease was made. In the following days, her clinical state worsened, with persistence of elevated fever. Serologic tests were negative for Bartonella, Parvovirus B19, Rickettsia, viral hepatitis (A, B and C), HIV, Toxoplasma, Salmonella and Yersinia. Blood cultures were also negative. No abnormalities were found in abdominal and cardiac ultrasonography and in cranial computed tomography (CT). A bone gammagraphy, showed enhanced captation in right tibial malleolus and in proximal interphalangeal joints of fingers 2 and 4 of the right hand, and finger 4 of the left hand. An abdominal CT showed a biliary bladder wall enlargement and a laparotomy was performed, but no signs of cholecystitis were found. A broad spectrum antibiotherapy was started with ciprofloxacin and imipenem. The patient was transferred to pediatric intensive care unit (PICU) as a consequence of progressive multiorgan failure including acute respiratory distress syndrome, liver failure, anemia, thrombopenia and increasing acute phase reactants (APR). On admission to PICU, the patient was on mechanical ventilation. Examination revealed generalized oedema, hypoventilation in both lung bases, abdominal distension and hepatoesplenomegaly. There was also active bleeding around puncture points and through nasogastric tube. Blood analysis on admission is shown in table 1.
A high positive end-expiratory pressure (PEEP) was set due to hypoxemia (up to 14 cmH 2 O), with a PO 2 /FiO 2 of 141. Thorax radiography showed bilateral diffuse infiltrates, and slight cardiomegaly. Inotropic support was needed (dopamine at 15 μg/kg/minute) and a perfusion with furosemide (0.5 mg/kg/hour) was started. Laboratory analysis showed abnormal values for haemoglobin (7.7 g/dL), platelets (29,000/mm 3 ) and coagulation times including hypofibrinogenemia (85 mg/dL). She received red blood cell concentrates, platelets and fresh frozen plasma. The same antibiotherapy was maintained and acute liver failure support treatment was started.
A bone marrow puncture-biopsy was performed. Activated macrophages with hemophagocytosis were found (figure 1), which together with the clinical and analytical data (bicytopenia, and coagulopathy with hypofibrinogenemia, and afterwards a ferritin of 190594 μg/L and triglycerides of 677 mg/dL) confirmed the HFS diagnosis. Specific treatment for this syndrome was started, following Hemophagocytic Lymphohistiocytosis (HLH) 2004 guidelines. She had a very good response, and at forth day from admission she was extubated to non invasive ventilation using a full-face mask. A progressive analytical normalization was observed and she was discharged after 12 days in PICU, without any sequelae.
She is currently being followed as an outpatient. She had two reactivations of her rheumatoid disorder, with a good response to corticoids.
HFS is an activation of mononuclear phagocyte system cells, with hemophagocytosis in bone marrow and the rest of reticuloendothelial system. This syndrome can be either primary/familial (familial hemophagocytic lymphohistiocytosis -FHL) or reactive/secondary. FHL has a recessive autosomal inheritance and it develops in chil- Bone marrow smear (optic microscope) Figure 1 Bone marrow smear (optic microscope). Activated macrophages and hemophagocytosis from bone marrow puncture-biopsy.
dren younger than 2 years, even though in rare cases it can feature later on [9] . It is rapidly lethal and it is sometimes related to some immunological diseases (X-linked lympho-proliferative, Chediak Higashi and Griscelli syndromes).
Secondary HFS has a better outcome than primary HFS. It is triggered mainly by viral infections (especially Ebstein-Barr virus) [10] , and also by bacterial, parasitic and fungal infections. It can also develop during malignancies and rheumatoid disorders (kwown in this case as macrophagic activation syndrome), as in our patient [6] .
The activation of mononuclear system cells occurs due to a hypersecretion of proinflammatory cytokines (IFNγ, TNFα, IL6, IL10, M-CSF), as a consequence of a triggering agent, which is often a viral infection [11] . The underlying problem is a T and Natural Killers cells dysfunction, which leads to an uncontrolled immunological response [12] due to inability to eliminate the triggering agent. All viral, bacterial, parasitic and fungal cultures performed in our case were negative. Impaired perforine function due to gene mutations seems to play an important role in HFS pathogenesis, as reported in literature [12] . They are implicated in cytotoxicity by forming a death-inducing pore in target cell [13] .
HFS diagnosis is made basing on clinical and histological criteria. Five out of 8 criteria must be fulfilled. Absence of hemophagocytosis does not exclude the diagnosis [2] . Multiorgan failure is the most severe presentation of HFS. In pediatrics, multiorgan failure is usually caused by sepsis. In the present case, the initial diagnosis was septic shock. Therefore, HFS has to be included between the causes of multiorgan failure in pediatrics to permit an early diagnosis and treatment. Central nervous system (CNS) is often involved, which has been linked with a poor prognosis [14] . Even though our patient developed a very severe form of HFS, there seemed to be no CNS involvement, and this agrees with the good outcome.
Treatment is nowadays applied according to HLH 2004 protocol, which is designed for primary HFS and also used in severe secondary HFS cases. Aggressive immunochemotherapy is given (dexamethasone, cyclosporine A, etoposide and in patients with CNS symptoms or abnormal CSF, also intrathecal methotrexate and corticoids) [2] . After initial treatment, bone marrow transplantation is indicated in primary disease and in severe and persistent secondary HFS [1] . 
Our patient had a very good outcome, without any sequelae. Early recognition of this syndrome to apply specific therapy as well as multiorganic failure treatment in PICU, are management key factors. HFS is probably underdiagnosed, as multiorgan failure is usually explained by other more common causes like septic shock. [4] Abbreviations APR: acute phase reactants; CJA: chronic juvenile arthritisl CNS:  Studying copy number variations using a nanofluidic platform Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies. Variation in the human genome occurs on multiple levels, from single nucleotide polymorphisms (SNPs) to duplications or deletions of contiguous blocks of DNA sequences (1) (2) (3) (4) (5) . Copy number variation (CNV) is an important polymorphism of DNA segments across a wide range of sizes and one of the primary sources of variation in the human genome (6) . Recently, CNV has been studied extensively because of its close association with large numbers of human disorders (7, 8) . An understanding of this variation is important not only to understand the full spectrum of human genetic variation but also to assess the significance of such variation in disease-association studies. The first human CNV map was constructed from a study of 270 normal individuals with a total of 1447 CNV regions in the whole genome (9) ; more than 15 000 CNVs have been found in the human genome (http://projects. tcag.ca/variation). A recent paper demonstrated the presence of 525 novel insertion sequences across the genomes of eight unrelated individuals, which were not present in the human reference genome, and showed that many of these have different copy numbers (10) . However, the current CNV analysis is mainly dependent upon microarray-based SNP and comparative genomic hybridization (CGH) platforms, or DNA sequencing, and is therefore subject to low sensitivity and low resolution. These techniques are high throughput but lack the flexibility of analyzing individual genes or sequences of interest. Other existing technologies, such as quantitative polymerase chain reaction (PCR), are limited because of their inability to reliably distinguish less than a twofold difference in copy number of a particular gene in DNA samples (11) (12) (13) .
In this study we demonstrate the use of a unique integrated nanofluidic system, the digital array, in the study of CNVs. The digital array (14, 15) is able to accurately quantitate DNA samples based on the fact that single DNA molecules are randomly distributed in more than 9000 reaction chambers and then PCR amplified. The concentration of any sequence in a DNA sample (copies/ml) can be calculated using the numbers of positive chambers that contain at least one copy of that sequence. In order to ensure that the apparent difference in gene copy numbers in different samples are real, and not distorted by differences in sample amounts, we use the expression 'relative copy number'. The relative copy number of a gene is the number of copies of that gene per haploid genome. It can be easily expressed as the ratio of the copy number of a target gene to the copy number of a single copy reference gene (two copies per cell) in a DNA sample, which is always 1 per haploid genome. By using two assays for the two genes (the gene of interest and the reference gene) with two fluorescent dyes on the same digital array, we are able to simultaneously quantitate both genes in the same DNA sample. The ratio of the numbers of molecules of these two genes is the relative copy number of the gene of interest in a DNA sample. A single copy gene should have a relative copy number of 1. A relative copy number greater than 1 indicates the presence of duplication of the target gene while a number smaller than 1 implies deletion of this gene.
Our data show that the digital array is able to distinguish less than twofold differences in gene copy number and differentiate between 1, 2, 3, 4, 5, 6 and 7 copies of a gene with great accuracy. It provides a reliable and robust platform to study copy number variations and has great advantages over conventional techniques.
The sequence of the RPP30 synthetic construct and the sequences of the primers and probe used to amplify this construct are shown in Supplementary The TaqMan assay for the RNase P gene (VIC) was ordered from Applied Biosystems (Foster City, CA).
The feasibility of digital PCR has previously been demonstrated by performing PCR on a single DNA sample obtained by a serial dilution process (16, 17) . Target molecules in a DNA sample could be quantitated by counting the number of positive reactions. We utilize the principle of partitioning instead of dilution in order to identify and quantitate individual DNA molecules.
The Fluidigm digital array is a novel nanofluidic biochip where digital PCR reactions can be performed (14, 15) . Utilizing nanoscale valves and pumps, the digital array delivers up to 12 mixtures of sample and PCR reagents into 12 individual panels. Each panel contains 765 independent 6-nl chambers. This nanofluidic platform utilizes soft lithography and silicone rubber to create nanoscale valves and pumps that can be used in serial or parallel applications. The digital array is composed of a PDMS (silicone rubber) Integrated Fluidic Circuit, an Integrated Heat Spreader to ensure rapid heat transfer and temperature uniformity within the array and an SBS-formatted carrier with inputs and pressure accumulator to act as an interface between the user and the PDMS chip. There are 12 carrier inputs corresponding to 12 separate sample inputs to the chip. Individual samples of a minimum volume of 8 ml each are delivered into 765 6-nl preprogrammed partitioning chambers in the chip by pressure-driven 'blind filling' in the PDMS. Control lines are primed with control fluid and are pressurized to actuate valves between the reaction chambers. The valves partition individual chambers that are kept closed during the PCR experiment.
One of the important applications of the digital array is absolute quantitation (14, 15) . The DNA molecules in each mixture are randomly partitioned into the 765 chambers of each panel. The chip is then thermocycled on Fluidigm's BioMark system and the positive chambers that originally contained one or more molecules will generate fluorescent signals and can be counted by the Digital PCR Analysis software. Since the volumes and dilution factors of the DNA samples are known prior to loading into the digital array, the DNA concentrations can be accurately calculated. The precision of this test is only dependent upon the sampling randomness and, like any biological experiments, will improve with multiple tests (panels). Digital array has been routinely used by us to quantitate DNA samples of unknown concentration and, especially, cDNA samples whose concentrations of the sequences of interest are hard to determine otherwise.
When duplication occurs, multiple copies of a gene might be closely linked on the same chromosome and therefore might not be separated from each other, even on the digital array. As a result, multiple copies might behave as a single molecule and the total number of copies of the gene would be underestimated. When two copies are separated by a large genomic distance, some of them might be separated when DNA molecules are fragmented during purification. However, in most cases this would not be sufficient (see Table 2 , sample NA11994 genomic DNA data). Specific target amplification (STA) is a good solution to this problem. STA is a simple PCR reaction with primers for both the reference gene and the gene of interest. It is typically performed for a limited number of thermal cycles (five in this study). The copy numbers of both genes are proportionally increased. Using this process, multiple copies of the gene of interest will be amplified separately and later randomly partitioned into chambers in the digital array. Since the newly generated molecules of both genes reflect the original ratio and they are not linked any more, a digital chip analysis can quantitate the molecules of the two genes and measure their ratio, and therefore the copy number of the gene of interest, very accurately ( Figure 3 ). It is very important that the amplification efficiencies of the two pairs of primers be approximately equal in order not to introduce any bias in the ratio of the two gene copy numbers in the limited number of STA thermal cycles, although this is likely to have an insignificant effect on our results since we utilized only five cycles of preamplification. The amplification efficiency of any pair of primers can be easily measured using real-time PCR (18) .
STA was performed on a GeneAmp PCR 9700 system (Applied Biosystems, Foster City, CA) in a 5 ml reaction containing 1 Â TaqMan PreAmp master mix (Applied Biosystems, Foster City, CA), 225 nM of primers for both RNase P and the target gene and 10-50 ng DNA. Thermocycling conditions were 958C, 10 min hot start and five cycles of 958C for 15 s and 608C for 2 min. The products were diluted prior to the copy number analysis on the digital array based on their initial concentrations so that there would be about 500-600 RNase P molecules per panel.
Copy number analysis using the digital array on the BioMark system Each panel of a digital array contains a total of 4.59 ml (6 nl  765 chambers) PCR reaction mix. However, 10 ml reaction mixes were normally prepared for each panel, containing 1  TaqMan gene expression master mix (Applied Biosystems, Foster City, CA), 1  RNase P-VIC TaqMan assay, 1  TaqMan assay (900 nM primers and 200 nM probe) for the target gene, 1  sample loading reagent (Fluidigm, South San Francisco, CA) and DNA with about 1100-1300 copies of the RNase P gene. The reaction mix was uniformly partitioned into the 765 reaction chambers of each panel and the digital array was thermocycled on the BioMark system (http:// www.fluidigm.com/products/biomark-main.html). Thermocycling conditions included a 958C, 10 min hot start followed by 40 cycles of two-step PCR: 15 s at 958C for denaturing and 1 min at 608C for annealing and extension. Molecules of the two genes were independently amplified. FAM and VIC signals of all chambers were recorded at the end of each PCR cycle. After the reaction was completed, Digital PCR Analysis software (Fluidigm, South San Francisco, CA) was used to process the data and count the numbers of both FAM-positive chambers (target gene) and VIC-positive chambers (RNase P) in each panel.
There are 765 chambers in each of the 12 panels in a digital array. When single DNA molecules are randomly partitioned into these chambers, it is possible that multiple molecules could partition into the same chamber. As a result there could be more molecules in each panel than positive chambers. The true number of molecules per chamber can be estimated using a simple Poisson distribution equation as described by Sindelka et al. (15) . We have developed a more robust computational algorithm to analyze CNV data obtained from the digital array. This algorithm has been integrated into the Digital PCR Analysis software and is detailed in (19) .
A proof-of-principle spike-in experiment was performed using a synthetic construct to explore the digital array's feasibility as a robust platform for the CNV study. A 65-base oligonucleotide that is identical to a fragment of the human RPP30 was ordered from Integrated DNA Technologies (Coralville, IA, USA). RNase P, a single copy gene, is used as reference in this study (20, 21) .
Both RPP30 synthetic construct and human genomic DNA NA10860 from the Coriell Cell Repositories (Camden, NJ, USA) were quantitated using the RPP30 assay on a digital array. Different amounts of RPP30 synthetic construct were then spiked into the genomic DNA so that mixtures with ratios of RPP30 and RNase P of 1 : 1 (no spike-in), 1 : 1.5, 1 : 2, 1 : 2.5, 1 : 3 and 1 : 3.5 were made, simulating DNA samples containing two to seven copies of the RPP30 gene.
These spike-in mixtures were analyzed on the digital arrays. Five panels were used for each mixture and 400-500 RNase P molecules were present in each panel. The ratios of RPP30/RNase P of all samples were calculated and are plotted against the expected ratios in Figure 1 . A good linear relationship can be observed. Also shown in Figure 2 is an example of a typical digital array experiment.
CNVs of the CYP2D6 gene CYP2D6 belongs to the cytochrome P450 system responsible for the metabolism of many commonly prescribed medications (22, 23) . The CYP2D6 gene is highly polymorphic and this can significantly influence the metabolic activity of the enzyme it codes for (debrisoquine 4-hydroxylase) and the therapeutic efficacy of the drugs. Therefore, the pharmacogenetic polymorphism information of this gene would be of great clinical importance in therapeutic decision-making (24) (25) (26) (27) . More than 100 alleles of the CYP2D6 gene have been identified (http://www.cypalleles. ki.se/cyp2d6.htm). Allele-associated variations in the activity of the CYP2D6 enzyme have been observed and individuals carrying these alleles are classified into poor, intermediate, extensive and ultrarapid metabolizers (28, 29) .
Genotyping patients would be able to identify those who are at risk of severe toxic responses (poor metabolizer) or in need of more than standard level of drugs (ultra rapid metabolizer). It has been shown that some poor metabolizers and ultra rapid metabolizers are caused by the deletion or duplication of the entire CYP2D6 gene (30, 31) .
These large structural changes can be detected using conventional technologies such as Southern blot and longrange PCR. However, it is believed that real-time PCR is Figure 1 . Quantitation of the RPP30 copy number in spike-in samples that contain two to seven copies of the RPP30 molecules per two haploid genomes. The x-axis shows the expected ratio of the numbers of RPP30 molecules to RNase P molecules. The y-axis shows the observed ratios. Each value is calculated using five panels of the same sample mix and the error bars represent standard errors. A good linear correlation can be seen with a coefficient of determination (R 2 ) of 0.996. currently the only promising technique that is able to provide information about the exact copy number of the CYP2D6 gene in a routine clinical setting (32) (33) (34) .
We used the digital array to measure the CYP2D6 copy numbers of three DNA samples from ParagonDX (Morrisville, NC). The CYP2D6 genotypes of these DNA samples had been characterized (Table 1 ). The samples were STA-treated (see Figure 3 and Materials and methods section) and the products were analyzed using five panels each on the digital arrays. The relative copy numbers of these three samples are 0, 0.49 and 0.98, respectively, highly consistent with their assumed CYP2D6 diploid copy numbers (0, 1 and 2) based upon their genotypes.
We also studied five cell line DNA samples from Coriell Cell Repositories (Camden, NJ). First, we measured their relative copy numbers using genomic DNA. The results showed that two of them have a single copy and two have two copies of the CYP2D6 gene per cell ( Table 2) . One sample had a relative copy number of about 1.17, equal to a diploid copy number of 2.34. We then STA-treated these five samples and ran the products on digital arrays. The relative copy numbers of the 1-and 2-copy samples remained the same and the fifth sample showed a relative copy number of about 1.5 or a diploid copy number of 3. Apparently this sample had a duplication of the CYP2D6 gene on one of the two chromosomes (35) . It has been previously demonstrated (31, 36) that when CYP2D6 duplication occurs, the two copies are separated by 12.1 kb. Therefore, the diploid copy number of 2.34 obtained when genomic DNA was used is likely the result of DNA breakage in this 12.1 kb genomic region in some DNA molecules that separated the two CYP2D6 copies. To confirm this, we ran a long range PCR [see (31) CYP2D6 duplication was observed only in the sample with a relative copy number of 1.5 ( Figure 4) .
ERBB2 (also known as HER2) is a receptor tyrosine kinase gene overexpressed in up to 30% of invasive breast cancer, resulting in a loss of normal cellular growth control. Most of these cases (97%) are caused by the amplification of this gene and the number of extra copies is closely related to the protein expression level (37) (38) (39) (40) .
ERBB2 amplification is well correlated with an aggressive phenotype characterized by reduced response to chemotherapy, high recurrence rate and short survival time and serves as a significant prognostic predictor for breast cancer patients (37, 41) . Trastuzumab (Herceptin), an FDA-approved monoclonal antibody against the ERBB2 protein, has been shown to dramatically increase response rate and extend survival in breast cancer patients with ERBB2 amplification. Given Trastuzumab's proven efficacy and substantial benefit in multiple clinical trials, detection of ERBB2 amplification has become critical (42) (43) (44) (45) .
There are different methodologies of determining the ERBB2 status in breast cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are two FDA-approved technologies for the detection of ERBB2 amplification. The former detects overexpression of the ERBB2 receptor on the cell membrane while the latter detects the copy number of the gene itself relative to the chromosome 17 centromere.
IHC is less expensive and easy to perform but is prone to a high rate of inaccuracies due to variations in tissue preparation, protein stability, antibody sensitivity and scoring subjectivity. On the other hand, FISH is accurate with good clinical correlation but it is expensive, time consuming, and labor intensive and requires very experienced personnel. Therefore, suggestions have been made to use a combination of IHC and FISH, where IHC is used as a screening procedure followed by a FISH confirmation if necessary (46, 47) .
We used digital arrays to analyze the ERBB2 copy numbers of 40 breast cancer and 8 normal breast tissue DNA samples from BioChain (Hayward, CA). All DNA samples were from Asian individuals except one normal sample that was from a Caucasian. Of the 40 breast cancer samples, 3 are adenocarcinoma, 1 is fibroadenoma, 2 are invasive lobular carcinoma, 1 is infiltrative ductal carcinoma and 33 are invasive ductal carcinoma. The samples were STA-treated and, for screening purpose, the products were analyzed using only two panels for each sample on digital arrays. The results are shown in Figure 5 . Fourteen breast cancer samples (35%) had a diploid ERBB2 copy number of more than five while all control samples were below five copies [an absolute number of ERBB2 copies greater than 4.0 per cell is considered amplification in FISH analysis (47) . Here we use five as the threshold]. The copy numbers shown are not all integers due to (i) heterogeneity of the cancer cells and (ii) sampling variations as only two panels were used for each sample.
A real-time PCR reaction was also performed on these 48 samples. Twenty-four replicates were used for each sample. Although the average copy numbers were close to the digital array data, large fluctuations (SDs of up to 0.5) were observed in the 24 reactions of each sample. Studies on other genes (for example, CYP2D6) showed that real-time PCR does not always produce accurate results (data not shown).
Genomewide analyses have shown the existence of large numbers of CNVs in the entire human genome with large interindividual diversity (48) (49) (50) (51) (52) (53) . Many of these CNVs colocalize with genes involved in a variety of diseases or disease susceptibility and are believed to play some role in pathogenesis (54) (55) (56) (57) . The first Mendelian disorder associated with the amplification of a 750 kb DNA fragment was reported recently (54) . It appears to only be a The results of both genomic DNA and STA products are shown. The ratios of the CYP2D6 gene to the RNase P gene should be close to multiples of 0.5. The genomic ratio of 1.17 for sample NA11994 (corresponding to a diploid copy number of 2.34) reflects the partial separation of the duplication alleles in the genomic DNA. A ratio of 1.51 (diploid copy number of 3) was obtained when the sample was subjected to STA prior to the digital PCR analysis. question of time before more genetic conditions related to CNV are identified. Two standard genomewide scanning methods for CNV detection are array-based CGH and high-density SNP genotyping arrays and both were employed in the construction of the first human CNV map (58) . These microarray techniques are able to generate whole-genome CNV data and are important in CNV discovery. Their resolution is also improving with the development of new probes. However, since they are both based on hybridization, the detection of copy number changes largely depends on signal-to-noise ratio, which is sensitive to reagent and manufacturing variability. Therefore, false positive and false negative results are sometimes inevitable (59) . Additionally, the lack of standard reference genomes in the studies using these technologies further complicates the interpretation of the results (60).
On many occasions, gene-or locus-specific (other than the whole genome) copy number information is required. This is especially true in the cases of CYP2D6 and ERBB2 described above in which therapeutic decision needs to be made based upon the copy numbers of these genes. In addition to other conventional methods (Southern blot, long-range PCR and FISH), the possibility of using quantitative PCR in the CNV study of these two genes has been previously explored (61) (62) (63) (64) (65) .
Quantitative PCR is simple and easy to perform. However, since the copy number of the target gene is derived from the Ct difference between the target gene and a reference gene, the results are very sensitive to the efficiency of the amplification reaction. Even if one compensates for the amplification efficiency, it is considered difficult to obtain a discrimination power of better than twofold (66) .
The digital array has the ability to absolutely quantitate any type of DNA sample. In a multiplex PCR reaction with two assays, the quantitation of two or more genes/sequences in a single sample becomes possible, effectively eliminating pipetting variations inherently occurring in any quantitation experiment. The accuracy of the results is only subject to the random distribution of the molecules and, like any biological experiments, can improve with the use of multiple replicates for each sample. STA can efficiently separate the linked copies of a gene on the same chromosome when duplication occurs while other methods, such as restriction digestion are also valid (data not shown).
We performed three experiments to test the feasibility of the digital array in the CNV study. First we measured the copy numbers of the RPP30 gene of a series of mixtures made of a human genomic DNA and a synthetic RPP30 construct. We observed a very good correlation between the results and the expected outcome. We then studied the CYP2D6 copy numbers of some DNA samples that were either genotyped elsewhere or characterized by us using conventional techniques. The results were also consistent. Lastly, we screened 40 breast cancer samples for the amplification of the ERBB2 gene. Although the clinical data (other than pathological classification) of these samples were lacking, about 35% of the samples had an increased number of this gene above 5, very close to the ERBB2 amplification frequency reported in the literature (67) .
In conclusion, this study shows that the digital array provides a new and robust technology to study geneand sequence-specific CNV and is able to detect gene copy numbers with great accuracy. Digital arrays provide a much greater discrimination power than quantitative PCR. CNV studies on the digital array are easy to perform, fast and the data obtained is easy to interpret. Furthermore, the platform is very flexible and can be tailored to any gene/sequence. It can also serve as an independent measure to verify results from the whole-genome scans using array technologies. The digital array is an excellent CNV platform for both basic research and clinical investigation.
Supplementary Data are available at NAR Online.
Funding for Open Access charge: Fluidigm Corporation.
Conflict of interest statement. The authors declare competing financial interests. All are employees of Fluidigm Corporation. (19) . An analysis of hospital preparedness capacity for public health emergency in four regions of China: Beijing, Shandong, Guangxi, and Hainan BACKGROUND: Hospital preparedness is critical for the early detection and management of public health emergency (PHE). Understanding the current status of PHE preparedness is the first step in planning to enhance hospitals' capacities for emergency response. The objective of this study is to understand the current status of hospital PHE preparedness in China. METHODS: Four hundred hospitals in four city and provinces of China were surveyed using a standardized questionnaire. Data related to hospital demographic data; PHE preparation; response to PHE in community; stockpiles of drugs and materials; detection and identification of PHE; procedures for medical treatment; laboratory diagnosis and management; staff training; and risk communication were collected and analyzed. RESULTS: Valid responses were received from 318 (79.5%) of the 400 hospitals surveyed. Of the valid responses, 264 (85.2%) hospitals had emergency plans; 93.3% had command centres and personnel for PHE; 22.9% included community organisations during the training for PHE; 97.4% could transport needed medical staff to a PHE; 53.1% had evaluated stockpiles of drugs; 61.5% had evaluated their supply systems; 55.5% had developed surveillance systems; and 74.6% could monitor the abnormity(See in appendix). Physicians in 80.2% of the analyzed hospitals reported up-to-date knowledge of their institution's PHE protocol. Of the 318 respondents, 97.4% followed strict laboratory regulations, however, only about 33.5% had protocols for suspected samples. Furthermore, only 59.0% could isolate and identify salmonella and staphylococcus and less than 5% could isolate and identify human H5N1 avian flu and SARS. Staff training or drill programs were reported in 94.5% of the institutions; 50.3% periodically assessed the efficacy of staff training; 45% had experts to provide psychological counselling; 12.1% had provided training for their medical staff to assess PHE-related stress. All of the above capacities related to the demographic characteristics of hospitals and will be discussed in-depth in this paper. CONCLUSION: Our survey suggested that, at the time of the survey, hospital preparedness for PHE in China was at an early stage of development. Comprehensive measures should be taken to enhance hospital capacity in the prevention and management of PHE. Public health emergency (PHE) is an event or events that cause or may cause harm to the health of a community or nation [1] . To prevent and/or minimize the harm caused by PHE, early detection and management are necessary. As hospitals are the main location for PHE surveillance and treatment, their preparedness is critical for PHE's early detection and management [2] . Evaluating the current status of PHE preparedness within the hospital system is the first step in improving a nation's preparedness for a PHE. Yet, there is no national data on China's hospital PHE preparedness capacity aside from two studies that addressed the issues at local level [3, 4] . To understand the current status of hospital PHE preparedness in China, a sample survey of hospitals in four representative city/ provinces were conducted between November 2004 and March 2005.
The survey used a cross-sectional study design to survey hospitals in different regions of China. Respondents were all secondary and tertiary hospitals(the detail of hospital classification see in appendix) in the city of Beijing and provinces of Shandong, Guangxi, and Hainan. The selection of hospitals in these four regions is intended to represent a variety of regional economic status. Broadly speaking, Beijing and Shandong are economically well developed, Hainan moderately developed, and Guangxi developing [5] . According to the Hospital Classification Method issued by the National Bureau of Statistics of China, the surveyed hospitals included general hospitals, hospitals of traditional Chinese medicine (TCM), hospitals of integrated traditional Chinese medicine and western medicine (TCM-WM), specialized hospitals, community health center, and medical emergency center (the definition of community health center and medical emergency center see in appendix) [6] . Four hundred secondary and tertiary hospitals were surveyed. The study was approved by the Institutional Review Board (IRB) of the School of Basic Medicine, Peking Union Medical College in Beijing, China.
Based on a literature and government document review, a detailed methodological approach for research framework and questionnaire development was followed to inform the development of this study [3] . An indicator system framework was created and questionnaire designed based on the framework. The questionnaire consists of 17 sections and 192 items. The questionnaire and the survey protocol (including field work manual and quality control procedures) were tested by a pilot study. For the purpose of this study, we analyzed the data focused on the following nine areas of interest: (1) hospi-tal's demographic data (including region, SARS crisis experience, teaching function, hospital type, and number of medical staff in related departments); (2) hospital PHE preparation (emergency plans, response initiating time, accessibility, and revision and implementation of emergency plan); (3) response to a community PHE (cooperation with local organizations, relationship with the community PHE network, medical treatment, and rescue work in the community); (4) stockpiles of drugs and materials (stockpiles of drugs and other resources and personal protective equipment); (5)PHE detection and identification (syndrome surveillance); (6) procedures for medical treatment (protocol for diagnosis, treatment, and transfer of PHE victims); (7) laboratory diagnosis and management (laboratory regulation and management system, sample disposal and evaluation system, collection and disposal of suspected samples, and diagnosis of pathogen/etiology); (8) staff training (organization of PHE training, current training of medical staff, curriculum development and training effectiveness assessment); and (9) risk communication (organization for communication of risk psychological counseling to victim and medical staff, and communication with public). Excluding aspect 1, items 2-9 (covering 88 survey questions) represent 8 types of PHE preparedness capacities. Each answered item was scored 1 for "yes" and 0 for "no" or "unknown". Item scores were calculated by adding together "yes" answers. Items scores were used as a proxy for measuring PHE preparedness in an institution. A total item score was measured by calculating the score across all 8 items. The higher the total item score, the better the hospital PHE preparedness capacity. Further analyses were conducted to understand the correlation between preparedness capacity and demographic information. The distribution of the related preparedness capacities across 10 categories of PHE [1] and 15 types of etiology was also assessed.
A computerized questionnaire stored in a CD was sent to the targeted hospitals accompanied by an official letter from each of the four city and provincial health departments stating the importance of the survey and requiring that each hospital designates a department director to be responsible for coordinating the completion of the questionnaire. Each returned questionnaire was carefully reviewed for its completeness and consistency. For those questionnaires with incomplete and/or inconsistent responses, one or two follow-up telephone calls were made to ensure completeness and consistency. The data from returned questionnaires were then transferred into a database for analysis.
A database was set up using Microsoft Excel 2003. Data was checked, cleaned, and analyzed using SPSS software version 11.5. Ninety-five percent confidence interval of means (95% CI) was used to describe PHE preparedness capacities. Categorical variables were analyzed with frequency and percentage. Comparisons of mean score of each of eight PHE preparedness capacities among different types of hospitals were performed with P < 0.05 as statistical significance using parameter test (Independent-Samples T Test (two-tailed) or One-way Analysis of Variance) and/or non-parameter test (Mann-Whitney Test or Kruskal-Wallis Test) based on data distribution characteristics and homogeneity.
Four hundred hospitals responded, with a response rate of 100%. However, seventy-seven questionnaires were excluded from analysis due to one of the following reasons: (1) if less than 50% of items in the questionnaire were not answered, or (2) hospital did not meet secondary and/or tertiary hospital standard according to the hos-pital classification system. Therefore, the valid response rate was 79.5%.
Of analyzed hospitals (318), 29.9% were in Beijing, 24.5% in Shandong, 40.6% in Guangxi and 5.0% in Hainan. In terms of hospital type, 72.4% were teaching hospitals. The mean number of physicians and nurses per hospital was 174.5, and the mean number of total medical staff per hospital was 206.1. The mean number of physicians and nurses in emergency department and infectious-disease department were 24.3 and 12.0, respectively. Table 1 shows the demographic characteristics of the analyzed hospitals.
Of 318 hospitals, 264(85.2%) had an emergency plan. Among the 264 hospitals that had an emergency plan, 92.6% reported that the institution possessed a protocol to initiate the emergency plan, 75.5% had a classification system for different PHE events, 55.3% had evaluated and revised their emergency plan at least once, and 79.6% reported that their emergency plan was accessible to all 2 and table 3 .
Of all analyzed respondents, 64.2% were designated as the local emergency hospital for PHE patient admissions and 53.0% of them were the designated hospitals to provide medical rescue services during a national disaster. Of all analyzed respondents, 97.4% could promptly transport needed medical staff to the PHE field, 84.5% reported that they were prepared to respond to the needs of vulnerable people (including women, children, pregnant women and the disabled) during a PHE, however, only 49.8% had evaluated their ability to increase beds and equipment for PHE. When performing a PHE preparedness drill, 22.9% of respondents reported that they would invite relevant community organizations to participate. With regard to capacity comparison, the statistics test showed: the total item score of hospitals in Beijing(95% CI:5.9,6.9) was lower than that of hospitals in Shandong (95% CI:7.0,7.9) and Guangxi(95% CI:6.7,7.4); the score of teaching hospitals(95% CI:7.0,7.5) was higher than that of non-teaching hospitals(95% CI:5.7,6.6); and the score of tertiary grade A (95% CI:6.8,8.0) and B (95% CI:6.7,8.4) hospitals was higher than that of secondary grade B ones(95% CI:5.4,6.9), respectively. Among all types of hospitals, community health center scored highest on this aspect.
Our results revealed that 53.1% of respondents had evaluated their stockpiles of drugs, and 61.5% had established a relationship with suppliers to provide emergency drug- supplies, however, only 43.2% had signed written contracts with suppliers. Of all analyzed respondents, 47.8% had drug-distribution plans, and 21.5% knew where the national or local pharmacy distribution centers were located. In regards to other medical materials, 80.1% had stockpiles of materials for responding to PHE. As for the stockpiles of drugs for infectious diseases, about 93.2%, 91.9% and 43.5% of responding hospitals had drug stockpiles for treating infectious diarrhea, influenza and botulismo toxin, respectively. When hospitals were compared on this item, statistical analysis showed that institutions in Beijing (95% CI:5.8,6.9) had a higher score than that of Shandong (95% CI:6.6,7.9). Tertiary hospitals generally had a higher score than secondary ones.
Among all the respondents, 55.5% reported that they had developed syndromic surveillance systems for certain diseases and 84.4% required that physicians on duty should report any abnormity to the hospital's presidents (the definition of abnormity see in appendix). Abnormity in admission diagnosis, routine microbiological tests, emergency room patients, and death with unknown causes were systematically monitored by 74.6% of institutions and 47.4% of hospitals shared their surveillance information with the local health authority. There were statistically significant differences between tertiary grade hospitals (Grade A 95% CI: 5.6,6.6; Grade B 95% CI: 5.6,7.3) and secondary grade B hospitals (95% CI:4.1,5.9) for this capacity, with tertiary hospitals scoring higher on their ability to detect and identify a PHE.
Physicians in 80.2% of the responding institutions reported being familiarized with the latest treatment protocol for a PHE, 92.8% could transfer PHE victims to corresponding medical agencies for appropriate treatment, and 98.0% could provide training on the protocol system. However, only 69.0% had specific procedures for patient transfer in a PHE. As for infectious disease treatment protocol, 80.1% had protocols for SARS, but only 37.3% for brucellosis. With regard to the capacity comparison 
Among all the respondents, 94.5% reported that they had a training program for the following medical staff: infection managers (56.3%); emergency department physicians and nurses (92.2%); and infectious disease ward physicians and nurses (71.8%). Staff training was supervised by a designated person in 82.3% of institutions and 65.8% had training curriculums, 66.5% of which was updated regularly. Effectiveness of PHE training was periodically assessed in 50.3% of respondents. For this capacity, statistical significance indicated that respondents in Shandong (95% CI:5.9,7.0) scored higher than participating institutions in Guangxi (95% CI:5.1,6.0). 
Serious PHE concerns were raised in China during the 2003 SARS crisis when it became apparent that hospitals possessed poor emergency preparedness [7] . Even the upcoming 2008 Olympics Game in Beijing and the 5.12 Earthquake Disaster in China have dramatically evoked the awareness of PHE preparedness capacity for hospital. Based on the experience of the SARS pandemic, all hospitals should possess fundamental PHE programs, including preparedness of drugs, equipment, staff, emergency education and staff training [3, 8, 9] , coordination with relevant community bodies [10], medical treatment [11] , early detection and warning [12] , laboratory diagnosis [13] [14] [15] and psychological intervention [8] . Since the SARS crisis, the central Chinese government has become more active in the construction of public health system, especially in regards to the medical emergency response system [16] . One major effort involved a 11.4 billion RMB investment in local governments to initiate the construction of regional PHE medical treatment systems [17] . In order to offer some insight into the development of hospital PHE preparedness capacity, this study examined the current status of hospital preparedness in Beijing, Shandong, Guangxi, and Hainan.
Emergency preparedness refers to the processes involved in ensuring an institution: (1) has complied with the preventive measures; (2) is in a state of readiness to contain the effects of a forecasted disastrous event in order to minimize loss of life, injury, and damage to property; (3) can provide rescue, relief, rehabilitation, and other services in the aftermath of the disaster; and (4) holds the capability and resources to continue to sustain its essential functions during a PHE [18] . An emergency preparedness systems primarily composed of emergency plans and organizational structures and lays the foundation for dealing with PHE [19] . Emergency plans establish the protocol for operation under a PHE [16] . For a hospital to mobilize all PHE resources in a short period of time, contingency plans must be issued in advance [9] . In addition, periodic review and updating of emergency plans enhance an institution's emergency response capacity [3] . Our study showed that most hospitals had emergency plans and that these plans focused on infectious diseases control with less attention to preparedness for biological, nuclear radiation and other terrorism attacks. Most of the hospitals had PHE command departments and emergency response teams, however, only 55.3% of hospitals with emergency plans reported they had evaluated and revised their PHE systems. Overall, tertiary hospitals performed better in PHE preparation than secondary hospitals. Meanwhile, no statistical significance was found between hospitals that had admitted SARS patients and those that had not, suggesting that after the SARS crisis, all hospitals raised awareness of emergency plans and implementation.
No hospital or medical system can manage a public health emergency without community networks and public involvement. Therefore, hospitals need to communicate and cooperate with other local health agencies, functioning as a networked public health provider. Problems like lack of communication and coordination between hospital departments and inter-agency networks hinder the availability of resources in a community and limit timely forecasting, public communication and effective regulation of a PHE [10]. Our survey revealed that if a PHE occurred, most of hospitals reported that they could take responsibility for PHE rescue service, transport the medical staff in a timely manner, and provide priority health services to vulnerable populations. Yet, less than one third of respondents attended regulation and revision workshops for emergency plans for infectious epidemic control held by local agencies. This lack of cross-institutional interaction indicated that the ability of hospitals to coordinate with community agencies in preparation for, or in the event of a PHE was generally poor. The survey showed that among all the types of respondents community health center were best able to respond to PHE and the respondents with multiple functions performed better suggesting that communication and coordination between hospitals and community agencies should be strengthened.
Characteristics of a PHE include suddenness and unpredictability [9] . For most hospitals, medicine storage may be in great demand when faced with a sudden increase in patients. Therefore, hospitals must have programs to ensure appropriate levels of emergency supplies including drugs, medical equipment, electricity, water and oxygen, disinfectant, etc. Our survey suggested that most of the hospitals could establish an emergency-drug-supply system for most of the infectious diseases we addressed in the questionnaire except anthrax, brucellosis, botulism toxin poisoning and tetramine poisoning. For most of surveyed hospitals possessed emergency resource reserves, but less than half of them had corresponding drug distribution programs. In addition, hospital capacity was affected by economic level and classification of the hospital, suggesting that the importance of local economic development strengthens hospital ability to provide PHE.
Early detection and identification of a PHE are amongst the most important objectives for prompt and effective public health response to a PHE [12] as well as an essential precondition for selecting appropriate prevention and treatment measures. This study showed that most of the hospitals could regularly train medical staff on how to report and identify suspicious PHE and that the institutions possessed surveillance systems to monitor various aspects of abnormity. Approximately half of the respondents could share surveillance information with the local health authorities. There were statistically significant differences among various classification of the respondents, which demonstrated that after the SARS crisis, hospitals at all levels attached high importance to PHE monitoring and early warning system, however, the capacity was affected by the comprehensive strength of hospital.
PHE happens suddenly and its incidence rate is relatively low, which leaves most medical staff inexperienced and unprepared [11] . Therefore, it is important that hospitals develop emergency plans for PHE treatment programs. In this survey, more than half of respondents showed that their physicians were aware of current PHE protocols. Most hospitals had transfer and treating procedures for infectious diseases, including SARS, influenza, and infectious diarrhea, but less held these procedures for biochemical incidents, leakage of nuclear, and terrorist attacks. Because they are easily used as biological terrorist attacks materials [20] , therefore, the prevention and control of these emergencies become very important. Our statistical analyses showed that tertiary-grade, teaching and TCM-WM hospitals performed better on medical treatment procedures preparedness, which might reflect the fact that different types of hospitals have different functions and mission in the community, however, for this capacity, the statistical significance among different regions showed the important role that economic factor plays.
Hospital laboratories not only have the task of clinical diagnosis, but take some responsibility in the surveillance of public health [13, 14] . Therefore, laboratory informa-tion plays an important role in detection of the PHE [13, 15] . Detecting PHE related pathogen/etiology can not only confirm clinical diagnosis, but also identify newly emerging infectious diseases [15, 21] . The presence of SARS in China in 2003, and the slow response to its emergence, revealed that China's public health laboratory systems were weak [13] . This survey indicated that many of the hospitals did not report adequate laboratory diagnostic capacities. Although hospital laboratory regulations seemed relatively good, only one-third of hospital laboratories had programs for dealing with suspicious samples collecting, disposal and delivery. [23] . When PHE occurs, hospital medical staff are usually the first responders and information providers, therefore, education and training are key measures to enhance PHE response [24] . Our survey suggested that after SARS crisis, most hospitals re-evaluated the importance of medical staff training for PHE. The majority of respondents offered training programs to their related medical staff. However, the effectiveness of these training programs needs to be periodically evaluated.
PHE can cause psychological as well as physical problems for the public and medical staff attending to victims [9, 25] . In a public health crisis or emergency, effective risk communication can help people cope, make decisions, and return their lives to normal. Crisis communication, as an important part of a PHE response [8] , is key to ensuring complete, transparent and prompt information exchange, and to help hospitals make timely responses and reduce the serious consequences [26] . The results of this survey revealed that medical staff in 12.1% of the hospitals underwent training for evaluation of PHE-related stress and only one-third of respondents had specific programs and spokespersons for communicating critical messages and information to the media, public, governments and stakeholders. These results indicated that most of the surveyed hospitals do not understand the importance of psychological care in a PHE emergency, do not have the resources to deal with it, or presume that it is not their place to do so. Indeed, this capacity evaluation revealed that when a PHE occurred, most hospitals' response plans focused on physiological medical treatment, but health education, psychological counseling, and crisis communication plans were rare. However, for this capacity, the statistical significance among different regions and levels showed the important role that economic factor and comprehensive level play.
The study has several limitations. First of all, the surveyed hospitals were restricted to four city and provinces, even some types of hospitals were rare (the number of the surveyed community health center and emergency center was just one, respectively), therefore, the results may not fully represent the PHE capacity of all hospitals in China. Secondly, because of self-report method there may be a respondent reporting bias. The inclusion of official documents from respective Health Bureaus, for example, may have encouraged respondents to complete survey but have also been interpreted as an official assessment of capacity leading some hospital representatives to overestimate PHE capacity. Thirdly, only quantitative data were collected to measure certain capacities of PHE preparedness. Most questions required a "yes" "no" or "unknown" answer which restricts the collated data to these three categories. Finally, this data set is not complete as some hospitals did not respond and others had to be excluded on the basis of incomplete answers or for ineligibility for hospital classification. To a certain extent, this loss of respondents caused a loss of information.
After several years of construction and development, the capability of hospitals in China to deal with PHE, in particular infectious diseases control, has improved greatly [3, 4] . Nevertheless, this research suggests that China has more progress to make before PHE preparedness is satisfactory. To enhance hospital preparation for dealing with PHE, governments at all levels should increase investment in the construction of infrastructure to create and sustain appropriate PHE capacity. On the other hand, hospitals at all levels should enhance their management, including updating and revising of emergency plans; strengthening communication and cooperation with other local agencies; enhancing the capacity of abnormity monitoring and laboratory diagnostic capability for infectious diseases; improving the treatment program for various PHE scenarios; and strengthening psychological intervention and risk communication capabilities. Finally PHE preparedness in relation to terrorism caused by nuclear radiation and biochemical substance was low in this study and should be further assessed for areas of need and improvement. Molecular evidence for the evolution of ichnoviruses from ascoviruses by symbiogenesis BACKGROUND: Female endoparasitic ichneumonid wasps inject virus-like particles into their caterpillar hosts to suppress immunity. These particles are classified as ichnovirus virions and resemble ascovirus virions, which are also transmitted by parasitic wasps and attack caterpillars. Ascoviruses replicate DNA and produce virions. Polydnavirus DNA consists of wasp DNA replicated by the wasp from its genome, which also directs particle synthesis. Structural similarities between ascovirus and ichnovirus particles and the biology of their transmission suggest that ichnoviruses evolved from ascoviruses, although molecular evidence for this hypothesis is lacking. RESULTS: Here we show that a family of unique pox-D5 NTPase proteins in the Glypta fumiferanae ichnovirus are related to three Diadromus pulchellus ascovirus proteins encoded by ORFs 90, 91 and 93. A new alignment technique also shows that two proteins from a related ichnovirus are orthologs of other ascovirus virion proteins. CONCLUSION: Our results provide molecular evidence supporting the origin of ichnoviruses from ascoviruses by lateral transfer of ascoviral genes into ichneumonid wasp genomes, perhaps the first example of symbiogenesis between large DNA viruses and eukaryotic organisms. We also discuss the limits of this evidence through complementary studies, which revealed that passive lateral transfer of viral genes among polydnaviral, bacterial, and wasp genomes may have occurred repeatedly through an intimate coupling of both recombination and replication of viral genomes during evolution. The impact of passive lateral transfers on evolutionary relationships between polydnaviruses and viruses with large double-stranded genomes is considered in the context of the theory of symbiogenesis. Approximately two-thirds of these wasps are endoparasites, meaning that the larval stages develop within the body cavity of their hosts, typically other insects. Among the most successful of these endoparasitic wasps are those that use lepidopteran larvae as hosts. Owing to the economic importance of these insects and the utility of their wasp parasites as biological control agents, the ability of these parasites to develop within lepidopteran hosts without triggering an intense immune response has been the subject of numerous studies over the past forty years.
Early studies of the Mediterranean flour moth, Ephestia kuhniella, parasitized by the ichnemonid, Venturia canescens, showed that eggs of this species are coated with particles that resemble virions [2] [3] [4] and contain surface proteins that mimic host proteins, thus keeping the eggs and larvae from being recognized as foreign material by their host. These particles lack DNA, and thus are not considered virions [5] . With respect to both species number and mechanisms that lead to successful parasitism, endoparasitic wasps are known to inject secretions at oviposition, but only a few lineages use viruses or virus-like particles (VLPs) to evade or to suppress host defences. In the family Ichneumonidae, for example, four types of host defence suppression mediated by the injection of fluids or suspensions are known that lead to successful parasitism. 1) Fluid injected with eggs bypasses host defences without the aid of viruses or VLPs [6] .
2) Wasps inject a virus that replicates in both the wasp and lepidopteran host. One example is the wasp Diadromus pulchellus, which injects an ascovirus, DpAV4 [7] into host pupae to circumvent host defence response.
3) The wasp injects VLPs capable of molecular mimicry and/or direct defence suppression.
4) The wasp injects polydnavirus particles that contain genes coding for proteins that interfere with host defence responses.
The last mechanism is by far the best-studied type of direct immune suppression by ichneumonid wasps, and occurs in many species belonging to genera Campoletis, Hyposoter and Tranosema (Ichneumonidae, Campopleginae), and Glypta (Ichneumonidae, Banchinae) [8] . In these cases, female wasps inject eggs along with ichnovirus particles into their hosts. Similarly, in certain lineages of endoparasitic braconid wasps, other types of immunosuppressive particles containing DNA occur in the fluid injected along with eggs [ [9] ; for a review, [10] ]. Once in the host, ichneumonid and brachonid particles enter host nuclei and their DNA is transcribed, producing proteins that selectively suppress various steps in the host defence response.
As a result of this unusual biology, these particles were described as symbiotic viruses belonging to new viral family, Polydnaviridae [10] [11] [12] Since the 1970's, it was assumed that the DNA in the polydnavirus particles, as with all other viruses, encoded typical enzymes and proteins for viral replication and virion assembly and structure. However, several recent genomic studies have shown that only a small number of the genes vectored into lepidopteran hosts, less than 2%, have homologs in other viruses. Most viral DNA is noncoding, except that which codes for wasp proteins involved in suppression of immune pathways, such as phenoloxidase activation and the toll pathways [8, 13, 14] . Even before these genomic studies, it was suggested that these particles were more similar to organelles than viruses [15] . The similarities between particle structure and virions of known types of complex DNA insect viruses are striking, and suggest these immunosuppressive particles originated by symbiogenesis between viruses and endoparasitic wasps, the same evolutionary process by which mitochondria and plastids originated from symbiotic bacteria [16] . For example, most braconid wasps produce enveloped bacilliform particles classified as bracoviruses, and these resemble baculovirus and nudivirus virions [10, 15] . Similarly, ichneumonid wasps produce enveloped spindle-shaped particles classified as ichnoviruses that resemble virions of ascoviruses, viruses lethal to lepidopterans, which, interestingly, are vectored by endoparasitic wasps [15] . It must also be noted that ichnoviruses resemble other true virus particles that are structurally very similar to virions of ascoviruses, but which remain unclassified because the lack of information about their genomes [17] [18] [19] [20] [21] . However, ascoviruses and ichnoviruses display very different genome properties; similar genomic differences occur between bracoviruses and baculoviruses or nudiviruses, suggesting that convergent evolution led to the origin the different polydnavirus types from at least two different types of viruses.
In ascoviruses, the genome consists of a single circular DNA molecule ranging from 119-to 180-kpb in size [7] . Phylogenetic analyses of several viral genes have revealed that ascoviruses are closely related to iridoviruses [22] , and likely evolved from them. In contrast, the genome of ichnoviruses is composed of multiple circular DNA molecules (25 to 105) representing a total size of 250 to 300kbp, all of which are replicated from the wasp chromosomes. The ichnovirus proviral genome is specifically excised and amplified in several segments in the female calyx cells, the only wasp tissue in which ichnovirus virogenesis occurs. After assembly, these particles are secreted into the female genital tract. Once injected into the host, the ichnovirus genome does not replicate, and does not lead to the production of a new virus generation. The third characteristic of ichnoviruses is that most of the genes borne by the particles are not related to viral genes. Among the 7 annotated ichnovirus gene families, there are four (rep, PRRP, N, and TrV) for which no homology with known eukaryotic (or prokaryotic) proteins has been detected and for which no function has been proposed. Among the remaining three (cys, ank and inx), cys-motif proteins have no clear homologs among eukaryotic (or prokaryotic) proteins, although the "cysteine knot" that they form is a folding domain found in many proteins, but not one that is necessarily related to eukaryotic host immune systems [10, 14] . However, some protein domains and their putative functions suggest that they might be related to regulatory components of eukaryotic host defence systems that are not sufficiently elucidated.
Although the resemblance of the polydnavirus virions to those of conventional insect viruses suggests that the former evolved from the latter, to date no molecular evidence supports this hypothesis. In the case of ascoviruses and ichnoviruses, well-conserved genes found among the three ascoviruses sequenced so far (SfAV1a [23] , TnAV2c [24] , and HvAV3e [25] ) are not found in ichnovirus genomes. As noted above, the principal reason for this is that the genomes of the latter viruses appear to contain mainly wasp genes, not viral genes. This highlights the need for new and alternative types of sequence data obtained from pertinent biological systems. In this regard, DpAV4 has features that could provide important insights. Indeed, it is the only ascovirus known to replicate in both its wasp and caterpillar hosts. It is transmitted vertically from wasp to caterpillars to suppress the defence response of the latter host, thereby enabling parasite development [26, 27] . Moreover, in males and females of D. pulchellus, the DpAV4 genome resides in the nuclei of all hosts cells, providing a possible example of what may have been an intermediate stage in the symbiogenesis that led to the evolutionary origin of ichnoviruses.
We recently sequenced the DpAV4 genome, and a combination of our analysis of this genome and recent data from new types of ichnoviruses, as well as new software programs that elucidate protein relationships based on structural analysis, have enabled us to detect phylogenetic relationships between proteins encoded by open reading frames of DpAV4 and the Glypta fumiferanae (GfIV) and Campolitis sonorensis (CsIV) ichnoviruses. In support of the symbiogenesis hypothesis for the origin of ichnoviruses, data and analyses suggest two independent symbiogenic events, in agreement with what was previously proposed [28] . The first led to the ichnoviruses in Banchinae lineage. This hypothesis is based on the occurrence of a gene cluster present in GfIV and DpAV4. The second symbiogenic event led to ichnoviruses in the Campopleginae wasp lineage. This hypothesis is based on relationships of the major capsid proteins among CsIV, ascoviruses and iridoviruses. Extending our investigations to proteins encoded by open reading frames of certain ascoviruses and bracoviruses, hosts and bacteria, in the light of recent analyses about the involvement of the replication machinery of virus groups related to ascoviruses in lateral gene transfer [29] , we discuss the robustness and the limits of the molecular evidence supporting an ascovirus origin for ichnovirus lineages.
The DpAV4 genome sequenced by Genoscope (France) is 119,334-bp in length. Its organization, gene content and evolutionary characteristics will be detailed in a separate publication (manuscript in preparation; Additional file 1). However, BLAST results obtained with several ORFs in the DpAV4 genome provide evidence that certain ichnovirus ORFs have their closest relatives in an ascovirus genome. Specifically, we identified a 13-kbp region that contains a cluster of three genes ( Fig. 1 , ORF90, 91 and 93; Additional files 1 and 2) that have close homologs in a GfIV gene family composed of seven members [28] . All contain a domain similar to a conserved domain found in the pox-D5 family of NTPases. To date, this pox-D5 domain has been identified as a NTP binding domain of about 250 amino acid residues found only in viral proteins encoded by poxvirus, iridovirus, ascovirus and mimivirus genomes. These genes seem to be specific to GfIV, as they are absent in the three sequenced genomes of other ichnoviruses, namely CsIV, Tranosema rostrales ichnovirus (TrIV), and Hyposoter fugitivus ichnovirus (HfIV).
More specifically, in DpAV4, ORF90 encodes a protein of 925 amino acid residues that is 40% similar from position 140 to 925 to a protein of 972 amino acid residues encoded by the ORF1 contained in the segment C20 in the GfIV genome (Fig. 2) . These two proteins can therefore be considered putative orthologs. The 480 C-terminal residues of this DpAV4 protein are also 42% similar to the Cterminal domain of the protein homologs encoded by the ORF1 of the D1 and D4 GfIV segments, 36% similar to the N-terminal and the C-terminal domains of the protein encoded by the ORFs 184R and 128L of the iridovirus CIV and LCDV, and 30% similar with those encoded by ORFs 119, 99 and 78 in the ascovirus genomes of HvAV3e, SfAV1a and TnAV2c, respectively. Overall, this indicates that this DpAV4 protein is more closely related to that of GfIV than to those found in other ascovirus and iridovirus genomes currently available in databases. ORF091 encodes a protein of 161 amino acid residues similar only with the C-terminal domain of three proteins encoded by the ORFs 1, 1 and 3, contained, respectively, in GfIV segments D1, D4 and D3. In contrast, ORF93 is closer to iridovirus and ascovirus genes than to GfIV genes. This protein of 849 amino acid residues is 43% similar over all its length to CIV ORF184R orthologs in all iridoviral and ascoviral genomes and is only 36% similar over 350 amino acid residues to the C-terminal domain of the GfIV protein homologs encoded by the ORF1, 2, 1, 1, 1 and 1 in, respectively, the C20, C21, D1, D2, D3 and D4 segments of this virus.
Analysis of the genes surrounding the DpAV4 ORF-90-91-93 cluster confirms that this virus has an ascovirus origin since this region contains ORFs that are close homologs of genes in iridovirus and ascovirus genomes. Upstream from the ORF-90-91-93 cluster, an ORF encoding the DNA-dependent RNA polymerase 1 subunit C is present, which is an ortholog of the iridoviral CIV ORF176R and the ascoviral SfAV1a ORF008. Downstream from this cluster, there are two genes, absent in known ascoviral genomes, but similar to the iridoviral CIV ORF115L and CIV ORF132L. These two genes encode, respectively, a chromosomal replication initiation protein and zinc finger protein. In between them, a gene encoding a small protein is present that is similar to that encoded by the ORF069L of the iridovirus CIV, and which corresponds to the ALI-like protein also found in entomopoxviruses [30] .
Since the three DpAV4 genes have relatives in all ascovirus and iridovirus genomes sequenced so far, their presence in the DpAV4 genome cannot result from a lateral transfer that occurred from an ichnovirus genome related GfIV to DpAV4. Thus, as these DpAV4 genes are the closest relatives of the pox-D5 gene family present in GfIV identified so far, they could be considered a landmark of the symbiogenic ascovirus origin of the ichnovirus lineage to which this polydnavirus belongs. An alternative explanation is that the presence of DpAV4-like genes in the genome of GfIV resulted from a lateral transfer from viral genomes closely related to those of GfIV and DpAV4.
Indeed, this might have happened when a Glypta wasp was infected by an ancestral virus related to DpAV4. Nevertheless, the symbiogenic origin of GfIV from ascoviruses is also supported by morphological features of its virions [28] , which, aside from similarities in shape, also show reticulations on their surface in negatively stained preparations, a characteristic of the virions of all ascovirus species examined to date [7] .
Because ascovirus virions and ichnovirus particles display structural similarities, we developed an approach to search for homologs of virion structural proteins in ichnoviruses. These approaches were initiated in 2000 and recently finalized, but some of the conclusions have been published [14] . To date, only two virion proteins from the Campoletis sonorensis ichnovirus (CsIV) have been characterized [31, 32] . The first is the P44 (Acc N° AAD01199), a structural protein that appears to be located as a layer between the out envelope and nucleocapsid, and the second, P12, a capsid protein (Acc N° AF004367). Presently, there are more than one hundred ascoviral or iridoviral MCP sequences in databases. BLAST searches using these sequences failed to detect any similarities between CsIV virion proteins and ascoviral or iridoviral MCPs, or any other proteins [33] . To evaluate the possibility that homology between ichnovirus and ascovirus virion proteins may simply not be detectable by conventional Blastp searches, we used a different method, WAPAM (weighted automata pattern matching; [34] ). The models were designed on the basis of a previous study [22] demonstrating that MCP encoded by ascovirus, iridovirus, phycodnavirus and asfarvirus genomes are related, and all contain 7 conserved domains separated by hinges of very variable size. We investigated these conserved domains further using hydrophobic cluster analysis (HCA, [35] ). This
Map of the 13-kbp region of the DpAV4 genome (EMBL Acc. N° CU469068 and CU467486) that contains the gene cluster with direct homologs in the genome of the Glypta fumiferanae ichnovirus 
Amino acid sequence comparison resulting from a BLAST search done with the DpAV4 ORF90 as a query, and the best hit corresponding to the protein encoded by the ORF1 of the ichnovirus segment GfV-C20 (Subject; Genbank Acc. N° YP_001029423) Figure 2 Amino acid sequence comparison resulting from a BLAST search done with the DpAV4 ORF90 as a query, and the best hit corresponding to the protein encoded by the ORF1 of the ichnovirus segment GfV-C20 (Subject; Genbank Acc. N° YP_001029423). analysis revealed that most conservation occurred at the level of hydrophobic residues, as expected for structural proteins (Additional file 3a and 3b). The size variability of the hinges between conserved domains and the conservation of hydrophobic residues might explain why BLAST searches using iridoviral and ascoviral MCP sequences have limited ability to detect MCP orthologs in phycodnavirus and asfarvirus genomes. We designed two syntactic models (see Materials and Methods), which together were able to specifically align all MCP sequences of the four virus families. Importantly, WAPAM aligned the CsIV ichnovirus P44 structural protein with both models. Complementary structural and HCA confirmed the presence of the seven conserved domains in this CsIV structural protein ( Fig. 3a and Additional file 3c).
In addition to the above analysis, ten syntactic models were developed using proteins conserved in the three sequenced ascovirus species (SfAV1a, TnAV2c, and HvAV3a) and twelve iridoviruses [36] . None of these 1 and 4, typed in black) , DpAV4 (lanes 2 and 5, typed in blue) and SfAV1a (lanes 3 and 6, typed in purple) . Conserved positions among the amino acid sequence of CsIV and those of DpAV4 and SfAV1a are highlighted in grey. Secondary structures in the three SfAV1a ORF061 orthologs were calculated with the Network Protein Sequence Analysis at http://npsa-pbil.ibcp.fr/ and the statistical relevance of the secondary structures were evaluated with Psipred at http://bioinf.cs.ucl.ac.uk/psipred/. C, E and H in lanes 4 to 6 respectively indicated for each amino acid that it is involved in a coiled, b sheet or a helix structure. Using default parameters of Psipred, upper case letters indicate that the predicted secondary structure is statically significant in Psipred results. Significant secondary structures are highlighted in yellow. In (a), the comparisons were limited to three of the seven conserved domains (Additional file 3a, b and 3c), the 2, 5 and 7. Indeed, classical in silico methods appeared to be inappropriate to predict statistically significant secondary structures in conserved structural protein rich in b strand such as iridovirus and ascovirus MCP. In contrast, a complete and coherent domain comparison was obtained by HCA profiles (fig. S3b, c) . , developed from small proteins encoded by the DpAV4 ORF041, SfAV1a ORF061, HvAV3a ORF74, and TnAV2c ORF118 in the ascovirus genomes, and iridovirus CIV ORF347L and mimivirus MIV ORF096R genomes, respectively. Importantly, these proteins have orthologs in vertebrate iridoviruses, phycodnaviruses, and asfarvirus. In SfAV1a, the peptide encoded by ORF061 is one of the virion components. In ascoviruses, iridoviruses, phycodnaviruses, and the asfarvirus, they have been annotated as thioredoxines, proteins that play a role in initiating viral infection [37] [38] [39] . Database mining with our model revealed four hits with CsIV sequences (Acc N°. M80623, S47226, AF236017, AF362508) each a homolog ORF of SfAV1a ORF061. In fact, these sequences correspond to several variants of a single region contained in the B segment of the CsIV genome. To date, these have not been annotated in the final CsIV genome, probably because they overlap a recombination site. HCA analyses confirmed that the hydrophobic cores were conserved ( Fig. 3b and Additional file 3d and 3e).
The chromosomal locations of genes encoding these two CsIV proteins, i.e., P44 and P12, were also consistent with the symbiogenesis hypothesis. In fact, the ORF encoding P44 is not found in proviral DNA. It is notable that no ORFs encoding orthologs of P44 or other structural proteins such as MCPs are found in any of the other three ichnovirus genomes sequenced -TrIV, GfIV, HfIV [8, 14] . Therefore, this indicates that the orthologs of ichnovirus MCPs and other virion structural proteins are also probably located in the genomes of these wasps, i.e., not in proviral DNA. In contrast to this, we found that the gene encoding the CsIV ortholog of SfAV1a ORF061 is located within the proviral DNA. Whether ortholog proteins are similarly involved in the TrIV, GfIV and HfIV biology, their genes are not found in proviral DNA, since no matches were detected in their viral genomes. The phylogenetic analysis performed previously on P44 and the SfAV1a ORF061 orthologs [15] indicated that they have an ancestor close to that of the ascoviruses and iridoviruses.
As in the case of genes encoding pox-D5 family of NTPases in all ascoviruses, iridoviruses, and GfIV, genes encoding virion proteins cannot result from a horizontal transfer from a Campoplegine or Banchine ichnovirus genome to all ascovirus, iridovirus, phycodnaviruses and asfarvirus genomes. As the ascovirus genes encoding the two virion proteins investigated here are the closest relatives of virion proteins in CsIV, they can be considered a landmark reflecting the symbiogenic origin of the two ichnovirus lineages from ascoviruses closely related to DpAV4. In fact, the difficulty encountered in elucidating their sequence relationships can be explained by a combination of the marked transition from ascovirus to ichnovirus, and the significant selection constraints that resulted as the latter virus type evolved from the former.
Analysis of available ascovirus, iridovirus and ichnovirus genomes provides some of the first molecular support for the hypothesis that ichnoviruses evolved from ascoviruses by symbiogenesis. However, examining genes shared only by ascovirus, iridovirus and ichnovirus genomes likely limits the sources of genes that contributed to the evolution and complexity of these viruses, especially of the role of lateral gene transfer. Relevant to this is the recent finding that an important part of the mimivirus and phycodnavirus genomes had a bacterial origin [28] . Obviously, this did not lead to the conclusion that these viruses had a bacterial origin. The cytoplasmic environment in which these viruses replicate is rich in bacterial DNA because their amobae and unicellular algae hosts feed on bacteria that they digest in their cytoplasm. Thus, it has been proposed [28] that lateral transfers of bacterial DNA within these viral genomes were driven by intimate coupling of recombination and viral genome replication. Indeed, replication of these viruses is similar to that of bacteriophage T4. This mode of replication has been called recombination-primed replication. It permits integration of DNA molecules with sequence homology as short as 12-bp [28, 40] . The replication machinery used by ascoviruses, iridoviruses, mimiviruses, phycodnaviruses, and other nucleocytoplasmic large DNA viruses (NCLDV) [41, 42] is common to all of them, despite differences in the specifics of replication in each virus family. It can therefore be expected that recombination-primed replication occurred repeatedly during evolution of both these viruses and the genome of their eukaryotic hosts. In an eukaryotic cellular environment in which bacteria, chromosomes, NCLDV viruses and non-NCLDVs (such as baculoviruses) intimately cohabit temporarily or permanently, recombination-primed replication is able to allow reciprocal passive lateral transfers between viral genomes, host chromosomes, and bacterial DNA. Under these conditions, lateral transfers are considered passive since they just result from the intimate environment and not from an active mechanism dedicated to genetic exchanges. In ascoviruses and iridoviruses, the occurrence of such lateral transfers is supported by BLASTp searches that detected the presence of ORFs whose closest relatives have their origin within eukaryotic genomes (e.g., for DpAV4, in Additional data 1, ORFs 029, 049, 077, 080, 083, 118), bacterial genomes (e.g., for DpAV4, in Additional data 1, ORFs 056, 057, 059, 112, 115 and119) or viruses belonging to other NCLDV and non-NCLDV families (e.g., for DpAV4, in Additional data 1, ORFs 007, 037, 062, 068).
The transmission of ascoviruses is unusual in that they are poorly infectious per os and appear to be transmitted among lepidopteran hosts by parasite wasp vectors at oviposition [7, 43] . The genome of the ascoviruses can be replicated in presence of polydnavirus DNA either within the reproductive tissues of female wasps or within the body of the parasitized hosts infected by both polydnavirus and ascovirus. Consequently, integrated sequences of ascovirus origin can be expected within wasp and polydnavirus genomes. Reciprocally, sequences of polydnavirus origin may have been integrated in ascovirus genomes, whatever the wasp origin, ichneumonid or braconid. One gene family related to a bacterial family of N-acetyl-L-glutamate 5-phosphotransferase (Acc. N° of the closest bacterial relatives YP_001354925, CAM32558, ZP_00944224, ZP_02006449), identified only within the SfAV1a, HvAV3e and TnAV2c genomes, supports this conclusion. It has been found in the genome of a bracovirus, Cotesia congregata BracoVirus (CcBV [13] ; Fig. 4 ). Since this gene is absent in the genome of Microplitis demolitor BV, a related bracovirus [8] , it is difficult to infer the direction of the lateral transfer between the common ancestors of the three ascoviruses and of the wasp C. congregata. However, they unambiguously indicate that there was at least one lateral transfer for this gene between the common ancestor of ascoviruses and the parasitic wasp.
Since iridoviruses, like ascoviruses and other virus species [44, 45] , are, in some cases, vectored by parasitic wasps, databases were mined using all the available ichnovirus virus proteins as queries. We found no significant relationships between CsIV, HfIV and TrIV genomes and genomes of their putative closest relatives NCLDV and non-NCLDV relatives. This indicates that passive lateral gene transfers from virus to eukaryotes that are successfully spread and maintained in ichnovirus genomes remain rare events. One case of such lateral transfer was described in the CcBV genome. In this genome, aside from the presence of cardinal endogenous eukaryotic retrotranposon and Polintons that transposed in the chromosomal DNA of the proviral form of CcBV [46] [47] [48] , two genes encoding AcMNPV P94-related proteins, which have their closest relatives among granuloviruses (XcGV), were found. This suggests that CcBV contained at least two cases of lateral transfers between non-NCLDV and a bracovirus.
Our results provide another source of evidence that passive lateral gene transfers have occurred regularly during evolution from bacteria to viruses and eukaryotes, and between viruses and eukaryotes [49] [50] [51] [52] . Even if the pox-D5 NTPase genes in the GfIV genome, and the MCP and SfAV061-like genes in the CsIV genome, indicate that they have an ascovirus origin, they provide only limited evidence supporting an ascovirus origin of ichnoviruses. Indeed, their sequence conservation and biological characteristics suggest that there were repeated lateral transfers during evolution between ascoviruses and wasp genomes, including the proviral ichnovirus loci. This raises an important issue about the role of lateral transfers during co-evolution of the NCLDVs and non-NCLDVs, ichnovirus, wasp and parasitized host. Indeed, genetic materials of various origins have been exchanged and maintained during co-evolution. This therefore suggests that ichnoviruses might be chimeric entities partly resulting from sev- Symbiogenesis was first proposed as an evolutionary mechanism when it became widely recognized that mitochondria and plastids originated from free-living prokaryotes [7] . The genomes of the endosymbiotic cyanobacteria and proteobacteria, respectively, at the origin of chloroplasts and mirochondria have evolved by reduction of several orders of magnitude to the approximate size of plasmids. Concurrently, nuclear genomes have been the recipients of plastid genomes. This relocation of the genes encoding most proteins of the endosymbiotic bacteria to the host nucleus is the ultimate step of this evolutionary process, so-called endosymbiogenesis [7, 53] . Recent studies of plants have revealed a constant deluge of DNA from organelles to the nucleus since the origin of organelles [54] . This allows the host cell to have the genetic control on its organelles, in a relationship that is closer to enslavement or domestication than to a symbiosis or a mutualism in which the organelles would recover benefits from their contribution to the eukaryotic cell well-being. To date, this deluge of DNA is considered to correspond to passive lateral transfers that result from the interactions between the life cycle of the organelle and nuclear replication.
Numerous cases of symbiogenesis between endocellular bacteria and a wide variety of eukaryotic hosts have been characterized. However, recent work has demonstrated that this evolutionary process was not restricted to bacteria. It also occurred between endocellular eukaryotes such as unicellular algae and fungal endophyte in plants [55, 56] . Endosymbiogenesis was also proposed as the evolutionary mechanism that allowed some invertebrate viruses with a large double-stranded DNA genome related to the nudiviruses and the ascoviruses [22] , to have led, respectively, to the origin of bracoviruses and ichnoviruses, which are currently recognized as forming two genera within the family Polydnaviridae. Although presently there is no definitive evidence ruling out the hypothesis that the resemblance between ichnovirus and ascovirus virions is only an evolutionary convergence, the genomic differences between ascovirus and ichnoviruses are in good agreement with the symbiogenetic hypothesis. Indeed, they match an evolutionary scenario of endosymbiogenesis during which, from a single integration event of symbiotic virus genome, viral genes were lost and/or translocated from the provirus to other chromosomal regions (Fig. 5 ). In parallel, host genes of interest for the wasp parasitoid were integrated and diversified by selection and gene duplication in the proviral DNA. In this scenario, the more ancient symbiogenesis, the rarer the traces of genes from viral origin in the ichnovirus genome would be. This constitutes a constraint that dramatically limits the possibility to investigate the evolutionary links between ascovirus and ichnovirus. Results of our analyses demonstrate that the situation is also complicated by the fact that lateral gene transfers unrelated to the origin of ichnoviruses cause important misleading background noise. Moreover, the scenario in Figure 5 is close to a previously proposed version [57] , but is not consistent with results presented here, nor with recently accumulated knowledge on DNA transfer from organelles into the nucleus. Since endocellular environments favour lateral transfers between virus and wasp nucleus, it can be proposed that genes of virus origin that are involved in the ichnovirus biology were passively integrated in one or several loci, step by step over time, alone or through transfers of gene clusters, or even the entire viral genome. Since parasitoid wasps are able to vector different viruses [44, 45] , this second scenario opens the exciting possibility that virus genes involved in the ichnovirus biology might correspond to a gene patchwork resulting from transfers from viruses belonging to different NCLDV and non-NCLVD families. Because of the background noise due to lateral gene transfers found in these systems, elucidating the origins of ichnoviruses will be very time-consuming, requiring new accurate experimental approaches to generate more robust evidence. Sequencing wasp genomes to identify proteins of viral origin that are components of virions and involved in the assembly of these may well contribute to our understanding of how ichnoviruses and bracoviruses evolved from other insect DNA viruses.
Searches for similarities were mainly developed using facilities of BLAST programs at two websites http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi and http:genoweb.univ-rennes1.fr/Serveur-GPO/out ils.php3?id_rubrique=47. For DpAV4 genes having their origin within eukaryotic, bacterial or virus genomes belonging to NCLDV and non-NCLDV families, the closest gene was located using the distance trees supplied with each BLAST search at the NCBI website.
Construction of syntactic models: Conserved amino acid blocks and positions described previously [15, 22] and with new data sets were verified or determined using MEME at http://meme.sdsc.edu/meme/meme.html. In the first step, we used motifs resulting from MEME to make MAST minings in databases at http:// meme.sdsc.edu/meme/mast.html. Since MEME motifs depend significantly on the data set use to calculate them, this approach did not enable an exhaustive detection of homologs among ascoviruses, iridoviruses, phycodnaviruses, mimiviruses and asfarviruses, and the detection sensitivity was ultimately very similar to that obtained with BLAST. To reach our detection objectives, we therefore constructed syntactic models that only included the most conserved positions and their variable spacing using WAPAM at the website. http://genoweb.univ-rennes1.fr/ Serveur-GPO/ outils_acces.php3?id_syndic=185&lang=en.
Defining these models was obtained empirically until they allowed an exhaustive detection in refseq-protein and Genbank databases of the homologs among ascoviruses, iridoviruses, phycodnaviruses, mimiviruses and asfarviruses. The procedures were done until we were only able to detect exact match with the syntactic model. Whatever obtained with WAPAM, they required a confirmation with other approaches. Here, we used Psipred result comparison for regions with scores over 7 and HCA analyses for regions having scores lower than 7 with Psipred. This simplified the statistical treatment of the result obtained with WAPAM, since all exact matches have significance or a score of 100%.
Syntactic Hypothetical mechanism for the integration and evolution of ascovirus genomes in endoparasitic wasps Figure 5 Hypothetical mechanism for the integration and evolution of ascovirus genomes in endoparasitic wasps. Schematic representation of the three-step process of symbiogenesis, and DNA rearrangements that putatively occurred in the germ line of the wasp ancestors in the Banchinae and Campopleginae lineages, from the integration of an ascoviral genome to the proviral ichnoviral genome. Sequences that originate from the ascovirus are in blue, those of the wasp host and its chromosomes are in pink. Genes of ascoviral origin are surrounded by a thin black or white line, depending on their final chromosomal location. Two solutions can account for the final chromosomal organisation of the proviral ichnovirus genome, monolocus or multilocus, since this question is not fully understood in either wasp lineage. More complex alternatives to this three-step process might also be proposed and would involve, for example, the complete de novo creation of a mono or multi locus proviral genome from the recruitment by recombination or transposition of ascoviral and host genes located elsewhere in the wasp chromosomes. This model for the chromosomal organization of proviral DNA in polydnaviruses is consistent with data recently published [58] .  Nasal Delivery of an Adenovirus-Based Vaccine Bypasses Pre-Existing Immunity to the Vaccine Carrier and Improves the Immune Response in Mice Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-γ+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-γ+ CD8+ T cells (3.9±1% naïve vs. 3.6±1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40±10 reciprocal dilution, both groups). The number of INF-γ+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146±14, naïve vs. 120±16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by ∼25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-γ+ CD8+ T cells 10 days after administration (0.3±0.3% PEG vs. 1.7±0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine. The ability of human adenoviruses to induce strong innate and adaptive immune responses makes them powerful adjuvants that facilitate the immune response against an encoded antigen. Recombinant adenoviruses have been shown to elicit significant immune responses to bacterial (anthrax, plague), viral (Hepatitis C, Rabies, SARS) and tumour-associated antigens [1] [2] [3] . While these results are encouraging, immunity eventually develops against virus capsid proteins. This severely reduces the immunogenicity of adenovirus-based vaccines in mice, [4] [5] [6] [7] [8] , primates [9] and humans [10] . This problem is also significant since a large portion of the Western world has marked levels of anti-adenovirus serotype 5 (Ad5) antibodies and is also prominent in regions of sub-Saharan Africa and Southeast Asia, where many of these vaccines are needed [11, 12] . Thus, assessment of the impact of pre-existing immunity on immune protection and alternative vaccination strategies may be needed for successful use of many adenovirusbased vaccines.
Several strategies have been developed to address the prevalence of pre-existing immunity to Ad5 in the general population. Increasing the vector dose or adopting a prime-boost regimen in order to overcome pre-existing immunity to the virus is a common approach [2] . There is mixed enthusiasm for this plan, however, due to the documented toxicity associated with high doses of adenovirus and the length of time required for primeboost regimens when only a prime could be sufficient [13, 14] . 'Seroswitching', using recombinant adenoviruses constructed from chimpanzees or rare human serotypes with limited exposure rates such as adenoviruses 35 and 11 can elicit potent immune responses that are minimally affected by pre-existing immunity [7, 8, [15] [16] [17] [18] [19] [20] [21] . Hexon-chimeric adenoviruses can also avoid neutralization [22, 23] . Both approaches offer promise in the context of addressing pre-existing immunity, but require further investigation in response to concerns regarding safety and feasibility of largescale production. Covalent attachment of polyethylene glycol or incorporation of the virus into polymer matricies can also effectively protect Ad5 from neutralization [24] [25] [26] [27] [28] [29] [30] [31] . Delivery of Ad5-based vaccines by mucosal routes can also circumvent the effect of pre-existing immunity and induce a significant immune response against an encoded antigen [32] .
Recombinant adenoviruses are one of the few well-studied vectors currently under development for vaccination against Ebola virus infection. The first protocol for an Ebola vaccine employed a prime-boost regimen consisting of naked DNA expressing either Ebola glycoprotein (GP) or nucleoprotein (NP) and recombinant Ad5 expressing Ebola GP to successfully protect non-human primates against a lethal challenge of Ebola [33] . This has since led to several Phase I clinical trials [34, 35] in which each component of the vaccine is administered by intramuscular injection. To date, there have been only two reports describing mucosal administration of an Ebola vaccine [36, 37] . Nasal administration of recombinant human parainfluenza virus type 3 (HPIV3) vectors expressing Ebola GP and/or NP to Guinea pigs and rhesus monkeys conferred complete protection against a lethal challenge with Ebola.
We have previously found that a single dose of a recombinant adenovirus expressing Ebola Zaire GP given by either the oral or the nasal route is capable of affording protection against lethal challenge in naïve mice and that mucosal immunization can stimulate a broad, prolonged T cell-mediated immune response in both the systemic and mucosal compartments [37] . The primary objective of this study was to test the hypothesis that administration of an Ad5-based Ebola vaccine by either the nasal or oral route can circumvent pre-existing immunity and confer full protection upon challenge. Systemic and mucosal T and B cell responses to Ebola GP were assessed in both naïve mice and those with pre-existing immunity. The influence of PEGylation of the vaccine carrier on the immune response after oral immunization is also described.
The E1/E3-deleted adenovirus vector expressing the Ebola Zaire glycoprotein was created by cloning the open reading frame (ORF) sequence of the glycoprotein in the plasmid pShuttle (Adeno-X Expression system I, BD Clonetech, Palo Alto, CA) for subsequent insertion in the E1 region of the human adenovirus serotype 5 genome. The human cytomegalovirus (CMV) promoter included in the Adeno-X expression system was used to drive the expression of the Ebola Zaire glycoprotein in the final recombinant adenovirus serotype 5 construct. Authenticity of the final product was confirmed by sequencing of the recombinant virus rescued by transfecting the linearized DNA into 293 cells. Virus was sequentially amplified to large-scale infections (5610 8 cells) and purified on an affinity column (Adeno-X virus purification mega kit, BD Clonetech, Mountain View, CA) according to the manufacturer's instructions. Genome structures of vectors were analyzed by restriction digestion of isolated viral DNA and compared with those of the original molecular clones. Particle number and infectivity of vectors were determined by standard optical density reading and immunodetection of the hexon protein, respectively, following infection of 293 cells with limiting dilutions of each vector preparation according to the recommendations by the manufacturer (Adeno-X rapid titer kit, Clontech, Mountain View, CA). Purified virus was administered in sterile phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less.
First generation recombinant adenovirus expressing Ebola Zaire glycoprotein was prepared and purified as described above. The protein content of the virus preparation was determined using BioRad DC Protein Assay reagents (BioRad, Hercules, CA) and bovine serum albumin as a standard in a microplate format. According to established protocols, 10 mg of monomethoxypoly(ethylene) glycol, activated by tresyl chloride (Sigma Aldrich, St. Louis, MO), was added for each microgram of protein present [38] . The coupling reaction was performed at 25uC with gentle agitation. The reaction was stopped by the addition of L-lysine, in a 10-fold excess with respect to the amount of PEG added. Unreacted PEG, excess L-lysine, and reaction byproducts were removed by buffer exchange over a second Econo-Pac 10DG disposable chromatography column equilibrated with 100 mM potassium phosphate-buffered saline (pH 7.4). PEGylated preparations were administered in sterile potassium phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less as determined by the Adeno-X rapid titer kit. Characterization of these preparations revealed significant changes in biophysical properties of the virus once the reaction was complete such as the PEG-Dextran partition coefficient and peak elution times during capillary electrophoresis (as described previously [30, 38] ). Approximately 13,000 PEG molecules were associated with each virus particle in the studies outlined here as determined by a PEG-biotin assay [30] .
B10.BR mice were immunized with 1610 10 particles of recombinant virus per mouse either by intramuscular injection (50 ml) in the right hindlimb, or by oral gavage (100 ml) using oral feeding needles (18G, 2.25 mm dia., Popper & Sons, Inc, New Hyde Park, NY). For nasal immunization, mice were anesthetized with isoflurane. Once anesthesia was achieved, 1610 10 particles of virus slowly delivered as a bolus into the nostrils using a standard micropipette (Gilson, Middleton, WI) as previously described [39] .
Pre-existing immunity to adenovirus serotype 5 was established by injecting 5610 10 particles of adenovirus expressing betagalactosidase (AdlacZ) by intramuscular injection in the right hindlimb 30 days prior to vaccination with Ad5-ZGP. This protocol has been documented to activate T and B cells against virus capsid proteins and elicit humoral immunity [8, 11] . At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1:320, which falls within lower range of average values reported in humans after natural infection [11] . Mice were challenged by intraperitoneal injection of 2006LD 50 of mouse-adapted Ebola virus, Zaire strain (MA-ZEBOV) in 200 ml sterile saline [40] . After challenge, the animals were weighed daily for 13 days and monitored for clinical signs of Ebola infection using an approved scoring sheet. All procedures and the scoring method were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) of the Public Health Agency of Canada (PHAC) according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the 'Biosafety Level 4' (BSL4) facility at NML, PHAC.
A) Anti-ebola neutralizing antibody (serum). Sera collected from immunized mice were inactivated at 56uC for 45 minutes. Serial dilutions of each sample (1:10, 1:20, 1:40, etc, in 50 ml of DMEM) were mixed with equal volumes of recombinant Ebola Zaire expressing the enhanced green fluorescent protein (EGFP) reporter gene (ZEBOV-EGFP, 100 transducing units/well, according to EGFP expression) and incubated at 37uC for 90 minutes [41] . The mixture was then added to subconfluent VeroE6 cells in 96-well flat-bottomed plates and incubated for 5-10 minutes at room temperature. Control wells were infected with equal amounts of either ZEBO-EGFP with media without serum or that containing non-immune serum. 100 ml of DMEM supplemented with 20% FBS was then added to each well and plates were incubated at 37uC in 5% CO 2 for 48 hr. Cells were subsequently fixed with 10% buffered formalin for 24 h and examined under a fluorescent microscope. Sample dilutions which showed .50% reduction in the number of green cells compared to controls scored positive for neutralizing antibody.
B) Anti-ebola neutralizing antibody (mucosal). For the evaluation of the specific levels of IgG and IgA antibodies, bronchoalveolar lavage (BAL) fluid was collected in situ with a 20gauge catheter inserted into the proximal trachea, flushing the lower airways three times with 1 milliliter of L15 media (Sigma). BAL from each animal was incubated at 56uC for 45 minutes. Two-fold serial dilutions were added to 96 well plates pre-coated overnight with 30 ng of ZEBOV-like particles per well and incubated at 37uC for 1 hr. Goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) was then added and the plate was incubated for one additional hour at 37uC. The ABTS Peroxidase Substrate System (KPL) was used for detection and data collected using an ASYS UVM 340 ELISA plate reader (Isogen Life Science) at OD405. All infectious in vitro work was performed in the BSL4 laboratory at the NML, PHAC.
C) Anti-adenovirus serotype 5 neutralizing antibody (serum). Pre-existing immunity against recombinant adenovirus 5 was assessed by determining the amount of neutralizing antibody present in serum according to established methods [30] . In brief, serum was incubated at 56uC for 30 minutes and then diluted in DMEM in twofold increments starting from a 1:20 dilution. Each dilution (100 ml) was mixed with an aliquot of a standard stock of adenovirus type 5 expressing E. coli beta-galactosidase (10 6 pfu), incubated for 1 hour at 37uC, and applied to HeLa cells in 96-well plates (2610 4 cells/well). One hundred microliters of DMEM supplemented with 20% FBS was then added to each well. Cells were incubated at 37uC for 24 hours. Neutralizing antibody titers were calculated as the highest dilution at which 50% of the cells stained blue by visual inspection.
For the evaluation of INF-c positive CD8+ T cells 10 days postimmunization, splenocytes were harvested and cultured (1610 6 / sample) for 5 hours at 37uC in 96-well round bottom microtiter plates in DMEM supplemented with 10% FBS, 2-beta-mercaptoethanol (10 26 M) and GolgiStop (1 ml/ml, BD PharMingen, San Diego, CA). The TELRTFSI peptide which carries the Ebola Zaire GP immunodominant MHC class I epitope for mice of the H-2 k haplotype (B10.BR) was used for stimulation at a concentration of 1 mg/ml [42] . Control cells were treated with either an unrelated peptide or no peptide. After washing, cells were stained with 100 ml of a FITC-anti mouse CD8a antibody (1:100 dilution, PharMingen) at 4uC for 30 minutes. Cells were washed again, permeabilized in 16Cytofix/Cytoperm (PharMingen) for 20 minutes at 4uC, washed with 16Perm/Wash (PharMingen) and stained with 100 ml of a PEanti mouse IFN-c antibody (1:100 dilution, PharMingen) in the same buffer at 4uC for 30 minutes.
Quantitation of INF-c positive CD8+ T cells isolated from splenocytes or mononuclear cells from bronchioalveolar lavage (BAL), mesenteric lymph nodes (MLN) and Peyer's patches (PP) 45 days after vaccination was performed using an ELISPOT assay (ELISPOT Mouse Set, BD PharMingen, San Diego, CA) according to the manufacturer's instructions. Briefly, a 96-well ELISPOT plate was coated with 5 mg/ml anti-mouse IFN-c capture antibody. Cells pooled from 4 B10.BR mice per experimental group were added to microwells along with the TELRTFSI peptide (2 mg/well). Control cells were incubated either without peptide or with the non-specific stimulator, SEB (200 ng/ml). After incubation with a biotinylated anti-mouse IFNc detection antibody and Streptavidin-horseradish peroxidase antibody, wells were counted using an ELISPOT reader (AID EliSpot reader system, Cell Technology, Colombia, MD).
Data were analyzed for statistical significance by performing unpaired T tests (two-tailed p value) or one-way analysis of variance (ANOVA) when appropriate. The differences in the mean or raw values among treatment groups were considered significant when p,0.05.
In an effort to correlate markers of immunity with protection against Ebola infection after mucosal immunization, T and B cell specific immune responses against Ebola glycoprotein were analyzed in mice in the presence or absence of pre-existing immunity (PEI) to adenovirus 10 days after vaccination with a first generation adenovirus serotype 5 vector expressing the Zaire Ebola glycoprotein (Ad5-ZGP). Intracellular staining and flow cytometry (FACS) revealed that Ebola glycoprotein peptide-specific activation of CD8+ T cells, as measured by production of IFN-c, occurred in naïve animals immunized by the nasal route at a frequency of 3.961% (I.N., Figure 1A ). Pre-existing immunity was induced by intramuscular administration of recombinant Ad5 expressing a nonrelevant antigen, beta-galactosidase (AdlacZ), 30 days prior to vaccination with Ad5-ZGP. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1:320. Pre-existing immunity did not significantly alter activation of CD8+ T cells when the vaccine was given intranasally (I.N.+PEI, 3.661%, p = 0.07). Oral immunization lowered the response against Ebola glycoprotein (P.O., 260.5%). Samples obtained from animals with pre-existing immunity and vaccinated in the same manner were barely above the positive threshold (three times the average background level obtained from animals treated with the irrelevant virus, AdlacZ). Samples obtained from naïve animals immunized by the intramuscular route (I.M.) contained the largest population of IFN-c positive CD8+ T cells (1062%). Animals treated with the AdlacZ vector alone (AdlacZ-control) served as negative controls and produced 0.5% CD8+, IFN-c+ T cells in response to the Ebola glycoproteinspecific peptide.
The B cell response against Ebola glycoprotein achieved by administration of the vaccine was determined by incubating a recombinant Ebola virus (Zaire strain) expressing green fluorescent protein (ZEBOV-EGFP) with serum collected 25 days after vaccination [8, 37] . Neutralizing antibody (NAB) levels equivalent to 40610 reciprocal dilution were detected in both naïve animals and those with pre-existing immunity after intranasal immunization ( Figure 1B) . Samples from naïve animals immunized by the oral route contained NAB levels equivalent to 2065 reciprocal dilution whereas anti-Ebola NAB could not be detected in samples obtained from animals immunized by the same route with preexisting immunity (P.O.+PEI). NAB levels of 80610 were detected in samples obtained from naïve mice immunized by the intramuscular route. Anti-Ebola NAB could not be detected in samples from mice given the AdlacZ vector alone (AdlacZ Control).
Since the mucosa is often the primary sight of exposure, longterm, localized immune responses against Ebola virus are desirable for providing optimal protection against infection. In this context, T cell mediated immune responses were assessed by ELISPOT from various tissue compartments specific to the route of immunization 45 days after vaccination. The number of activated IFN-c secreting mononuclear cells harvested from splenocytes was significantly reduced by 38 and 59% in mice vaccinated nasally or orally when compared to those immunized by intramuscular injection (p#0.05, Figure 2A ). In contrast, samples obtained from the spleen of animals with pre-existing immunity that were immunized by the I.N. route contained the highest number of activated IFN-c secreting cells (8156190 spot-forming cells (SFC)/ million mononuclear cells (MNCs), Figure 2C ) although this was approximately 30% lower than that seen in naive animals (1,3406146 SFC/million MNCs, Figure 2A ). Pre-existing immunity also significantly reduced the number of these cells produced by animals immunized by the I.M. (210668 SFC/million MNCs) and P.O. routes (71612 SFC/million MNCs) with respect to those seen in naïve animals (2,145612 (I.M.) and 8856168 (P.O.)), p#0.05, Figure 2A) . A very limited number of activated IFN-c secreting mononuclear cells were detected in splenocytes of naïve animals immunized with the AdlacZ vector (50620 SFC/million MNCs, Figure 2A ) and those with pre-existing immunity to the same virus (1261, AdlacZ, Figure 2C ). Significant levels of INF-c positive cells were detected in bronchioalveolar lavage fluid (BAL) after intranasal immunization (146614 SFC/million MNCs, p#0.05, Figure 2B ). This was not significantly compromised by pre-existing immunity to adenovirus (120616 SFC/million MNCs, p#0.06, Figure 2D) . A limited number of INF-c secreting cells were detected in BAL from naïve animals immunized by the I.M. or P.O. routes (462 and 1165 SFC/million MNCs respectively, Figure 2B ) and were similar to that seen in samples obtained from animals given the AdlacZ vector (1261, negative control). Pre-existing immunity to adenovirus reduced these cell populations further to values that were not statistically significant (161 (I.M.), 562 (P.O.) and 261 (AdlacZ) SFC/million MNCs (p = 0.08, Figure 2D) ). INF-c positive cells were found in mesenteric lymph nodes (MLN) and Peyer's Patches (PP) (146611 and 2965 SFC/million MNCs, respectively) only after oral vaccination with Ad-ZGP ( Figure 2B ). Pre-existing immunity, however, reduced production of these cells to 18612 SFC/million MNCs (MLN) and 1662 SFC/million MNCs (PP) ( Figure 2D ).
The NAB response was also monitored from bronchioalveolar lavage fluid 45 days post-vaccination. NAB to ZEBOV-EGFP was undetectable in samples obtained from control (AdlacZ) or I.M. immunized mice, whereas levels of 40610 and 1065 reciprocal dilution were detected in the BAL of nasally and orally vaccinated animals, respectively ( Figure 3 ). Those with pre-existing immunity and immunized by the nasal route had NAB levels of 30610. NAB was not detected in mice with pre-existing immunity to Ad5 and immunized by either the intramuscular or oral route.
Further characterization of Ebola glycoprotein-specific immunoglobulin isotypes obtained from bronchioalveolar lavage fluid revealed that pre-existing immunity correlated with a marked decrease in the production of both IgG and IgA antibodies in mice immunized by intramuscular injection (Figure 4A and 4B ). IgG and IgA levels were the lowest in that were vaccinated orally regardless of whether they had pre-existing immunity ( Figure 4A and 4B). Immunization by the nasal route induced a strong IgG response that was reduced by an average of 25% in the presence of pre-existing immunity. The strongest IgA response was detected in samples obtained from animals given the vaccine by the nasal route. IgA levels, however, were also reduced by 25% in mice with pre-existing immunity to Ad5.
The most direct means of evaluating vaccine efficacy in mice is to assess protection by monitoring weight loss and death rates after a lethal challenge of Ebola [43] . Therefore, mice were immunized with 1610 10 pre-existing immunity. At the time of vaccination, animals had an average anti-adenovirus neutralizing antibody titer of 1:320 reciprocal dilution. Naïve mice vaccinated by intramuscular injection with saline (vehicle) served as controls for complete lethality following challenge with mouse-adapted Ebola virus. Twenty-eight days after vaccination, animals were challenged with 200 LD 50 of mouse-adapted (MA)-ZEBOV. Only mice immunized by the nasal route survived the lethal challenge ( Figure 5A ). This was evident as early as 5 days after challenge when controls and mice immunized by the other routes began to lose weight ( Figure 5B ). All eventually expired 7 days post-challenge ( Figure 5A , some data not shown for clarity). In contrast, naïve mice given the vaccine survived challenge regardless of the route of administration.
The studies outlined above strongly suggest that only intranasal immunization can successfully afford protection against Ebola virus in the absence and presence of pre-existing immunity. It was also clear that additional strategies were needed to improve vaccination by either the oral or intramuscular route in the presence of preexisting immunity. Covalent attachment of activated monomethoxypolyethylene glycol to the protein capsid of the Ad5-ZGP vector was evaluated as a potential way to improve the immune response after oral vaccination. Based upon previous observations, we hypothesized that this modification would protect the virus from neutralization by immune sera and improve survival in gastrointestinal tract [26, 27, 30, 44] . The frequency of Ebola glycoprotein peptide-specific activation of IFN-c positive CD8+ T cells was not significant in naïve animals given the PEGylated virus (0.360.3%) with respect to those given the unmodified virus (2.060.5%, p = 0.09, Figure 6A ). Similar results were also seen in the presence of pre-existing immunity in either treatment group (0.460.2% PEGylated vaccine, 1.760.5%, unmodified vaccine).
In contrast, NAB levels equivalent to 30610 reciprocal dilution were detected in naïve animals given the PEGylated vaccine ( Figure 6B ). Samples obtained from animals given the unmodified virus had levels of 2065 reciprocal dilution. Animals with preexisting immunity against adenovirus and treated with the PEGylated preparation were also able to produce detectable levels of NAB (1065 reciprocal dilution) whereas NAB was not found in sera from mice given the unmodified vector orally. Further characterization of Ebola glycoprotein-specific Ig isotypes revealed that the PEGylated vector could possibly stimulate production of IgG in the presence or absence of pre-existing immunity with respect to levels found in animals given the unmodified vector ( Figure 6C ). Modification of the virus induced a slight increase of IgA in the presence of pre-existing immunity with respect to that seen in naïve animals given unmodified virus (Vaccine, Figure 6D ) and those with pre-existing immunity (Vaccine+PEI). Despite this, animals with pre-existing immunity to adenovirus and immunized with the PEGylated vaccine orally did not survive after challenge with 200 LD 50 of (MA)-ZEBOV.
With mortality rates as high as 95%, Ebola infection occurs largely by direct contact with blood, tissues or skin of patients, and through mucosal exposure [45, 46] . To date, few efforts have been made to focus on the mucosa as the primary sight of exposure to Ebola virus and priming it for participation in the host defense by vaccination [47] . This is surprising given that the pulmonary, nasal and oral immune systems which comprise the mucosal-associated lymphoid tissues (MALT) are responsible for the production of approximately 80% of all immunocytes [48, 49] and mucosal immunity is often the first line of defense against pathogens coming in contact with susceptible hosts. Many studies in rodents indicate that systemic immunization produces strong anti-viral systemic responses while mucosal vaccination can stimulate both the mucosal and systemic immune systems and can confer long-term immunological memory against a given pathogen despite the fact that the magnitude of these responses are often reported to be somewhat reduced [37, 48, 50] . We have found this to be the case in the studies outlined here since the systemic cellular response and neutralizing antibody levels against Ebola Zaire GP were consistently lower following nasal and oral vaccination. Mucosal vaccination did, however, generate significant cellular and antibody responses in the periphery (BAL, MLN or Peyer's patches) and a single intranasal immunization with Ad5-ZGP conferred 100% protection even in the presence of pre-existing immunity.
Administration of recombinant adenovirus-based vaccines to the mucosa has also conferred sufficient protection against challenge with a variety of pathogens in the presence of preexisting immunity to the vaccine carrier in mice and other preclinical models of disease [5, 19, 32, 51, 52] . This served as a basis for the present study. A single intranasal dose of a recombinant Ad5 vaccine expressing the Zaire Ebola glycoprotein conferred 100% protection in both naïve mice and those with pre-existing immunity despite the fact that the strength of the immune response generated by this route of administration was quantitatively lower than that seen in animals vaccinated by intramuscular injection. It is also important to note that pre-existing immunity induced by intramuscular injection did not severely compromise the T cell-mediated response at either the systemic or mucosal levels in these animals. The level of anti-Ebola GP antibodies in the circulation and in the lung was also not significantly compromised by pre-existing immunity to adenovirus. While these results suggest that intranasal vaccination with an Ad-based vaccine is indeed a promising strategy to overcome pre-existing immunity, one might wonder how accurately our results translate to natural exposure to the wild type virus via the respiratory tract. This is somewhat difficult to establish in the mouse model since the ability of the wild-type adenovirus to replicate is limited [53] . In addition, the amount of neutralizing antibody present in the nasal cavity of those in the general population with pre-existing immunity to adenovirus 5 has not been assessed to the degree that serum neutralizing antibodies have, making it difficult to set a relative parameter for one to target in pre-clinical animal models. Lack of this data may be due to the invasive nature of the technique for acquiring samples and/or the fact that antibody levels in the mucosa of individuals with established pre-existing immunity to adenovirus type 5 are quite low and transient in contrast to systemic levels of anti-adenovirus neutralizing antibodies which are quite robust and persist over time (unreported observations). Thus, for these studies, we decided to induce pre-existing immunity against adenovirus by intramuscular injection at a dose that has been shown to induce production of systemic neutralizing antibodies at 1:320, a level mostly below what was reported in humans with documented preexisting immunity [8, 11, 12] . Additional studies designed to assess the amount of neutralizing antibody to AdHu5 in the lung over time after intranasal administration of varying doses of AdHu5 are currently underway in an effort to further define stringent conditions under which pre-existing immunity can be established for experimental testing.
Data obtained from animals vaccinated by the oral route provided several insights about the immunological requirements for protection against Ebola in a mouse model. Immunization by this route induced quantitatively lower T and B cell mediated immune responses against Ebola glycoprotein with respect to that achieved by either intramuscular or intranasal immunization. Despite this, every naïve animal immunized with a single oral dose of Ad-ZGP survived challenge, giving better precision on the minimal threshold of immunity required to achieve protection. As seen with intranasal immunization, the T cell-mediated response was not compromised by pre-existing immunity 10 days after oral vaccination. The number of IFN-c secreting T cells in both mucosal and systemic compartments of these animals at day 45, however, was significantly reduced by pre-existing immunity. It is possible that the peak T cell response at day 10 does not reflect the extent by which pre-existing immunity decreases the Ad5-ZGP-induced T cell response. Alternatively, it is also possible that the ELISPOT assay could detect subtle variations that flow cytometery could not monitor accurately due to differences in the sensitivity of each assay. Neutralizing antibody was not detected in the serum of animals with pre-existing immunity given a single oral dose of the vaccine. More importantly, none of these animals survived challenge with mouse adapted Ebola.
We attempted to improve the immunogenicity of the adenovirus-based vaccine by protecting it from the harsh environment of the gastrointestinal tract and from neutralization by antiadenovirus antibodies by PEGylation. Interestingly, the T-cell mediated immune response was significantly reduced and antibody levels increased in naïve animals given a single oral dose of the PEGylated vaccine with respect to that seen in animals given the same dose of unmodified Ad5-ZGP. It has been shown that modification of virus capsids by PEGylation can significantly dampen the T-cell mediated immune response against the virus and stimulate the antibody response against a secreted antigen [26, 27, 30, 54] . Taken together, these data support the notion that the exposition of the virus capsid proteins facilitates the immune response against the encoded antigen. Optimization of PEGylation chemistries and/or densities on adenovirus-based vaccine that promote and strengthen protective immune responses following oral immunization is currently underway.
Delivery of recombinant adenoviral vaccines to either the nasal or intestinal mucosa is an attractive vaccination strategy for many reasons. Vaccines administered in this manner will offer improved safety with respect to disease transmission and needle-stick injuries among health care workers, significant issues of concern in developing countries where the demand for many vaccines is high [55] . Mucosal administration of vaccines reduces the pain associated with vaccination, eliminates the need for specialized training programs for large vaccination campaigns and makes selfadministration of the vaccine possible. This route of administration may also significantly reduce systemic toxicity associated with recombinant adenovirus despite the fact that it has been shown that nasal immunization with recombinant adenovirus-based vaccines can facilitate translocation of the virus to the central nervous system [52] . Even though testing of other virus-based vaccines such as influenza have reported similar findings and are currently used in the clinic [56] , studies designed to fully assess the toxicological profile of adenovirus vaccine after nasal administration are also currently underway.
We have shown that nasal immunization with an Ad5-based vaccine can induce a long-term protective immune response against Ebola virus in a mouse model which is not impeded by preexisting immunity to adenovirus serotype 5. While these results are extremely encouraging, further characterization of the immune response against both the encoded antigen and the adenovirus vector in larger, clinically relevant animal models is vital for both understanding the biology of Ad vaccines and for the development of an effective Ebola vaccine suitable to populations with different requirements [57, 58] . The issue of pre-existing immunity must also be adequately addressed in order to develop efficient recombinant adenovirus-based vaccines. While the majority of the literature suggests that pre-existing immunity significantly hamper the effective use of AdHu5 vaccine carriers, other investigators have reported that pre-existing immunity did not interfere with the potency of recombinant Ad5-based vaccines in both pre-clinical models of disease and in humans [59, 60] . Thus, additional studies identifying clinically relevant conditions under which to test Ad-based vaccine candidates are necessary to assess the full impact of pre-existing immunity on vaccine potency, including in different compartments. Better define the role of preexisting immunity on vaccine-induced immunity will further the understanding of how individuals previously exposed to adenovirus will respond to these immunization regimens. Screening Pneumonia Patients for Mimivirus Acanthamoeba polyphaga mimivirus (APM), a virus of free-living amebae, has reportedly caused human respiratory disease. Using 2 newly developed real-time PCR assays, we screened 496 respiratory specimens from 9 pneumonia-patient populations for APM. This virus was not detected in any specimen, which suggests it is not a common respiratory pathogen. I nvestigation of a suspected Legionnaire's pneumonia outbreak in 1992 led to the isolation of a new microorganism from a water cooling tower in Bradford, England. This pathogen was thought to be a bacterium because it resembled small gram-positive cocci; however, in 2003 it was correctly identifi ed as a virus (1) . Acanthamoeba polyphaga mimivirus (APM), named for its ameba host and bacteria-mimicking characteristics, is a double-stranded DNA virus with the largest viral genome described to date (1.2 Mb) (2) . Mimiviridae is the newest member of the nucleocytoplasmic large DNA virus (NCLDV) group, which also contains Poxviridae, Iridoviridae, Asfarviridae, and Phycodnaviridae (1) . APM encodes specifi c translation proteins that are more commonly associated with cellular organisms than with viruses (2) .
Other ameba-associated microorganisms from environmental sources, such as Legionella pneumophila, are known to cause outbreaks of acute pneumonia in immunosuppressed and elderly persons, although person-to-person transmission is uncommon. Whether APM is similarly responsible for individual cases or outbreaks of respiratory disease has yet to be conclusively determined. Previous studies have reported serologic evidence of APM infection in 7.1% to 9.7% of patients with community-or nosocomially acquired pneumonia (3, 4) . APM DNA was also amplifi ed by a nested PCR assay from a bronchoalveolar lavage specimen of a 60-year-old patient receiving intensive care for hospital-acquired pneumonia (3) . In this study, we used newly developed real-time PCR assays to screen pneumonia patients from a variety of epidemiologic settings for APM infections.
Real-time PCR assays for APM were developed from multiple primers and probes designed for conserved re-gions of class I NCLDV genes L396 and R596, class III NCLDV gene L65, as well as the R656 gene, from the published APM genome sequence (GenBank accession no. NC_006450) by using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). All probes were labeled at the 5′ end with 6-carboxy-fl uorescein and quenched at the 3′ end with Black Hole Quencher-1 (Biosearch Technologies, Novato, CA, USA). Different primer and probe combinations were evaluated, and the 2 PCR assays that gave the best performance were selected for further studies (Table 1 ). Assays were performed by using the iQSupermix Kit (Bio-Rad, Hercules, CA, USA) in 25-μL reaction volumes. Amplifi cation was performed on an iCycler iQReal-Time Detection System (Bio-Rad) by using the following cycling conditions: 95ºC for 3 min for 1 cycle; 95ºC for 15 s and 55ºC for 1 min for 45 cycles each. Total nucleic acid was extracted from all specimens by using either the NucliSens Automated Extractor (bioMérieux, Boxtel, the Netherlands) or the automated BioRobot MDx (QIAGEN, Valencia, CA, USA) according to the manufacturers' instructions. Each clinical specimen was also tested for the human ribonuclease P gene to measure nucleic acid integrity as previously described (5) .
For PCR-positive controls, recombinant plasmids containing APM DNA (kindly provided by Didier Raoult, Unite des Rickettsies, Universite de la Mediterranee, Marseille, France) were constructed. Primer pairs bracketing the L396 and R596 genes were used to amplify 1,560bp and 879-bp full gene regions, respectively, using 300 nmol/L of forward primers 396 F (5′-TTA ATC ATC TTC CAA AAA ATT TAA TTC-3′) and 596 F (5′-ATG TCG TTA TCA AAA CAA GTA GTT CC-3′), and 300 nmol/L of reverse primers 396 R (5′-ATG GCG AAC AAT ATT AAA ACT AAA A-3′) and 596 R (5′-CTA ATT TTC AAT ATA GTG CGT AGA TTC TA-3′). These PCR products were purifi ed by using the QIAquick Gel Extraction Kit (QIAGEN) and then cloned into a pCR-II TOPO vector by using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). Recombinant plasmids were then isolated by using the QIAprep Spin Miniprep Kit (QIAGEN) and quantifi ed by UV spectroscopy. Standard curves were prepared from serial 10-fold dilutions of the quantifi ed plasmid in nuclease-free water containing 100 μg/mL of herring sperm DNA (Promega, Madison, WI, USA).
The L396 and R596 real-time PCR assays could detect as few as 10 copies of plasmid DNA per reaction with amplifi cation effi ciencies of 99.6% [slope -3.33 and r 2 = 0.99] (Figure, left panels) and 99.2% [slope -3.34 and r 2 = 1.00] (Figure, right panels) , respectively. No amplifi cation was obtained by either assay with pooled total nucleic acid extracts from respiratory samples from healthy humans or from other common DNA respiratory viruses, including adenovirus, human bocavirus, or herpesviruses.
The real-time PCR assays were used to test respiratory specimens from 496 pneumonia cases representing 9 distinct patient populations, which consisted of hospitalized pneumonia patients from population-based pneumonia surveillance studies in Thailand and the United States, transplant recipients with pneumonia, and isolated pneumonia outbreaks in either retirement homes for the elderly or familial clusters ( Table 2) . Of the 496 specimens tested, no positive results were obtained for APM DNA by either assay.
We developed a rapid method of screening samples for APM DNA by using 2 sensitive and specifi c realtime PCR assays designed to target conserved NCLDV class I genes. With only 1 APM sequence published (NC_006450) (2), little is known of APM strain variation; therefore, use of assays that target different genes increases the likelihood that genetic variants of APM will not be missed. A suicide-nested PCR method for APM detection has been reported (3); however, the quicker turnaround time and lower risk for amplicon contamination makes the real-time PCR method more attractive for screening large numbers of samples. ACC TGA TCC ACA TCC CAT AAC TAA A  Reverse  Helicase  GGC CTC ATC AAC AAA TGG TTT A seroprevalence study of APM among Canadian patients with community-acquired pneumonia identifi ed APM antibodies in 9.7% of 376 patients compared with 2.3% of 511 healthy controls (3). However, seropositivity may refl ect exposure to APM antigen rather than active infection, and the potential for nonspecifi c cross-reactions with the serologic assays used may have infl ated the true prevalence of APM infection (6) . In a separate report, a laboratoryacquired APM infection was linked to acute pneumonia by seroconversion in a technician in Marseille, France, thus providing evidence that this virus can occasionally cause clinical disease (7) . However, using sensitive real-time PCR assays, we failed to detect APM DNA in 496 respiratory specimens from 9 epidemiologically varied pneumonia patient populations.
If we assume an APM prevalence of 0.2% (1 case in the study sample), the estimated probability of obtaining our results by chance, based on binomial analysis, would be 0.37. Most of the specimens we tested were from the upper respiratory tract, whereas the only reported APM PCR-positive sample was from a lower respiratory bronchoalveolar lavage specimen (3) . Moreover, the patient populations sampled may not represent those at highest risk for APM infection. Nevertheless, our study supports the fi ndings of an Austrian study that failed to detect APM in 214 nasopharyngeal specimens from hospitalized children with respiratory symptoms (8) .
Our study did not detect APM in a large collection of specimens from patients with pneumonia, which indicates that this virus is not a common cause of severe acute respiratory disease. Because APM is an ameba-associated pathogen like Legionella, exposures to APM are most likely to occur from environmental sources. Further studies of more epidemiologically appropriate populations may be necessary to adequately access the importance of APM as a potential human respiratory pathogen. The real-time PCR assays described here will help facilitate these studies.
Mr Dare is a microbiologist in the Division of Viral and Rickettsial Diseases at the Centers for Disease Control and Prevention. His research focuses on developing molecular diagnostic assays for respiratory viruses.  Resource Allocation during an Influenza Pandemic Resource Allocation during an Influenza Pandemic To the Editor: Planning for pandemic infl uenza is accepted as an essential healthcare service and has included creation of national and international antiviral drug stockpiles and novel approaches to emergency vaccine development (1) . The effectiveness of these strategies in a pandemic may be substantial but is unknown. More certain is that effective management of severe and complicated infl uenza will reduce deaths and that demand will exceed available treatment resources (2) . Appropriate allocation of treatment resources is therefore essential, perhaps more important than any specifi c treatment such as administering antiviral medication to symptomatic patients. Re- (4) . Even more important for most severely ill patients, however, will be deciding whether to admit them to the hospital at all. The UK pandemic-planning criteria currently recommend a scoring system for hospital admission based on an assessment of poor outcome rather than on capacity to benefi t (2). Indeed, age >85 years and severe underlying cognitive impairment, which would rule out admission to critical care in Canada, would strongly favor admission to hospital care in the United Kingdom, the opposite of the situation for a younger cognitively intact person with similar disease severity. If tools are to be developed to support triage at all stages of the patient pathway in a pandemic, societies must consider the ethical issues raised (4,5), debate them, and take a position on the values that should underpin decision making in a pandemic. Even when clear societal goals are established, much work remains to ensure that the healthcare community is equipped to steer healthcare resources to deliver these effectively (6) . Community-acquired pneumonia has been used as a surrogate for infl uenza to test predictive scoring systems for assessing severity and assisting triage decisions (7) . Seasonal infl uenza epidemics would provide the most realistic setting available, in particular, if protocols were in place to test criteria when a relatively severe infl uenza season occurs. In addition to identifying criteria for setting priorities within infl uenza management, such testing will need to consider the balance of resources between infl uenza treatment and treatment of other usual noninfl uenza conditions that will require emergency care during the pandemic. Decisions that must be made during a pandemic are complex, varying from when to stop major elective surgery so critical care capacity can be opened up, to how to triage those who have experienced major trauma and those with infl uenza. These decisions could differ from those same decisions made outside a pandemic, and an adequate evidence base is needed if they are to be of good quality.
The third component of our preparation for optimally deploying standard care in a pandemic is being able to change our approach quickly as new knowledge emerges. In the so-called Spanish infl uenza pandemic of 1918-19, the unfamiliar clinical course meant that infl uenza was not even considered when the fi rst cases appeared (8) , and expectations had to be revised concerning who was most vulnerable and at what stage in their clinical course they were most at risk. Therefore, healthcare professionals must develop and test the public health infrastructure to capture patient factors associated with outcome and treatment response during a pandemic and feed this information back into clinical practice rapidly and reliably, as occurred during the epidemic of severe acute respiratory syndrome (9) . International collaboration will be important for sharing this work (10) and developing useful tools early in a pandemic. Having recognized the risk for pandemic infl uenza, we must now complement the research into novel infl uenza treatments by addressing our knowledge gap on how best to use our resources to deliver optimal clinical care in the management of infl uenza guided by effective clinical surveillance. 
To the Editor: Tick-borne relapsing fever in western North America is a zoonosis caused by spirochetes in the genus Borrelia that are transmitted by argasid ticks of the genus Ornithodoros (1) . Human disease occurs in many focal areas and is associated with infections of Borrelia hermsii, B. turicatae, and possibly B. parkeri (2, 3) . Although the ecologic parameters that maintain B. hermsii and B. turicatae differ, human infections usually occur in rustic cabins (B. hermsii) and caves (B. turicatae) inhabited by ticks and their terrestrial vertebrate hosts (1) . Recently, Gill et al. (4) provided evidence that the argasid bat tick, Carios kelleyi, feeds upon humans. Subsequently, Loftis et al. (5) used PCR analysis and DNA sequencing to detect in C. kelleyi an unidentifi ed Borrelia species that was closely related to B. turicatae and B. parkeri. We report the partial molecular char-acterization of another novel tickborne relapsing fever spirochete in C. kelleyi, which expands our knowledge for this group of pathogenic spirochetes and their potential vertebrate hosts and tick vectors.
C. kelleyi were collected August 18, 2005, from a house in Jones County, Iowa, built in 1857. Bats had been excluded from the attic since 1992. Nine months before ticks were collected, bats were prevented from roosting under the eaves. DNA was extracted from 31 nymphal C. kelleyi, as described previously (6) . For each tick, regions of the glpQ, fl aB, and 16S rRNA genes were amplifi ed and sequenced as described (3, 7, 8) . Sequences were assembled by using the SeqMan program in the Lasergene software package (DNASTAR, Madison, WI, USA).
Fourteen (45.1%) of 31 ticks were positive by PCR for >1 of the genes tested. Partial DNA sequences were determined from tick no. 16, for which amplicons for all 3 genes were obtained. The partial fl aB sequence had 4 bases different from the 300-base sequence (98.66% identity) reported previously (GenBank accession no. AY763104) for another Borrelia sp. found in C. kelleyi (5) . We constructed a 1,992-bp concatenated sequence that contained 1,273 bp of the 16S rRNA, 351 bp of fl aB, and 368 bp of glpQ. This concatenated sequence was aligned with homologous, trimmed DNA sequences of the same length obtained from representative full-length sequences determined previously for B. hermsii, B. turicatae, and B. parkeri (3, 9) (Figure) . This C. kelleyi spirochete was more closely related to B. turicatae and B. parkeri than to B. hermsii but was clearly distinct from all 3 species (DNA sequence identities of 98.89%, 98.75%, and 95.98% to B. turicatae, B. parkeri, and B. hermsii, respectively).
A glpQ amplicon from another nymphal tick (no. 3) was sequenced (GenBank accession no. EF688578) and was unique in the database; it was also considerably different from the glpQ sequence determined from tick 16, with 325 of 368 bases matching (88.3% identity). The Borrelia glpQ sequence from tick 3 had 85.1%-89.1% identity compared with glpQ sequences from B. hermsii, B. turicatae, and B. parkeri. This fi nding suggests the presence of at least 2 relapsing fever group spirochetes in C. kelleyi that await further characterization.
We found a novel Borrelia in bat ticks that is closely related to, but distinct from, the other known species of tick-borne relapsing fever spirochetes in North America. The human health implications of the new relapsing fever group spirochete are not yet known. The willingness of C. kelleyi to feed on humans and the fact that infection with bacteria closely related to true relapsing fever spirochetes occurs in  WU Polyomavirus Infection in Children, Germany  U of HotStarTaq polymerase. The cycling conditions were 50 cycles (94°C for 30 s, 53°C for 40 s, and 72°C for 1 min) after a preheating step of 10 min at 95°C. All PCR products of positive reactions by agarose gel electrophoresis with ethidium bromide staining were sequenced completely in both directions for confi rmation of sequence specifi city. One negative control was extracted and amplifi ed for every 5 NPA samples. A plasmid containing the cloned PCR product was used as positive control. The sensitivity of the WUPyV PCR was 8.8 copies per reaction as determined by probit analysis, which corresponds to 440 copies per mL of sample. The study was approved by the ethics committee of the medical faculty at the University of Würzburg.
During the study period, 1,326 NPA of hospitalized children with febrile respiratory tract diseases were received for viral diagnostic evaluation. The median age of the patients was 1.6 years (mean age 3.2 years; range 7 days-22 years), and 58.4% were boys. DNA of 1,277 NPA from 1,085 children was available for retrospective testing. Of these, 62 (4.9%) samples from 59 children were positive by WUPyV PCR and subsequent sequencing. The median age of the WUPyVpositive children was 3.0 years (mean 2.9 years; range 4 months-6.3 years) ( Figure) , and 57% were boys. Of the children with WUPyV-positive NPA, 3.2% were >6 years of age, although children in this age group constituted 15.7% of the total population. Infections with WUPyV were found year round, but most occurred in the winter months. Yearly frequencies (July-June) of WUPyV-positive results varied from 3.2% to 8.5% during the observation period. These variations were not statistically signifi cant. In 34 (54.8%) of the WUPyV-positive samples, co-infections with other respiratory viruses were detected, most frequently with adenovirus (n = 10) and fl uA (n = 10), followed by hBoV (n = 9) and RSV (n = 5). The co-infections bronchitis, wheezing bronchitis, and pneumonia.
In the context of the previous reports of WUPyV detection in Australia and North America (3), our data suggest a worldwide distribution of WUPyV. Most of the WUPyV-positive children were <4 years of age, and WUPyV DNA was rarely found in children >6 years of age. This age distribution is compatible with WUPyV infection occurring in day nurseries and kindergartens. In keeping with the fi ndings of Gaynor et al. (3), we observed a high number of co-infections. The true number of co-infections in our study is probably higher than the reported 53.2% because we did not test for several respiratory pathogens, such as coronaviruses, rhinoviruses, enteroviruses, and the human metapneumovirus. Hypotheses to account for the detection of WUPyV in respiratory samples include the following: WUPyV is a persisting asymptomatic virus that is detected by chance, WUPyV is a persisting virus that is reactivated by an infl ammatory process, or WUPyV is a predisposing or aggravating factor of respiratory diseases. Further studies are necessary to determine whether WUPyV is a human pathogen.
To the Editor: Outbreaks of hepatitis E virus (HEV) have been documented in many geographic regions and nonindustrialized countries (1-3); they have been primarily associated with fecal contamination of drinking water (4). In the Central African Republic (CAR), economic indicators (CAR ranks 172/177 countries on the 2006 United Nations Development Program Human Development Index), political instability, geographic situation, a deteriorating health network, and a very poor epidemiologic surveillance system all contribute to the country's epidemic susceptibility.
In July 2002, Ministry of Health (MoH) and Médecins sans Frontières (MSF) teams working in the Begoua Commune Health Center, north of CAR's capital Bangui, reported an increased number of patients from the Yembi I neighborhood who were showing signs of jaundice and extreme fatigue.
Patients suspected of having hepatitis E were defi ned as those with clinical jaundice (yellow discoloration of the sclera) and symptoms of malaise, anorexia, abdominal pain, arthralgia, and fever. Confi rmed cases were those in which patients' serum samples were positive for HEV immunoglobulin (Ig) M or IgG.
Initially, 16 pairs of serum and stool samples were collected from jaundiced patients. Fecal samples were stored at -20°C and sent to the National Reference Center of Enterically Transmitted Hepatitis, Hospital Val de Grâce (Paris, France) for HEV marker testing; serum samples were tested at the Bangui Pasteur Institute for yellow fewer (YF) IgM by MAC-ELISA.
The HEV epidemic was confi rmed by the detection of HEV markers: HEV IgG (Enzyme Immuno Assay, HEV, Abbott Laboratories, Abbott Park, IL, USA), HEV IgM (Abbott Laboratories), amplifi cation of RNA (5), and the absence of YF IgM. The HEV genome was detected in 4 of the fecal samples. Genotyping and sequencing showed that one of these was genotype 1, prevalent in Africa; the others were related to genotype 2 (Mexico-like) (GenBank accession nos. DQ151640, DQ151640) (5,6).
Data suggest that the epidemic began in the Yembi I neighborhood, then spread to the rest of the Begoua commune and fi nally to Bangui or surrounding areas (Figure) . Of 715 suspected HEV case-patients recorded in the MSF hospital between July 22 and October 25, 2002, 552 (77%) lived in the Begoua commune (271 in the Yembi I neighborhood). The attack rate for the Begoua commune (20,080 inhabitants) was 2.7%. Of 351 suspected case-patients serologically tested for IgG and IgM anti-HEV antibodies, 222 (63%) had IgM antibodies, including 5/16 pregnant women (2.3% of all confi rmed cases). Most patients reported jaundice (97.5%) and choluria (95.1%);other reported symptoms Factors influencing psychological distress during a disease epidemic: Data from Australia's first outbreak of equine influenza BACKGROUND: In 2007 Australia experienced its first outbreak of highly infectious equine influenza. Government disease control measures were put in place to control, contain, and eradicate the disease; these measures included movement restrictions and quarantining of properties. This study was conducted to assess the psycho-social impacts of this disease, and this paper reports the prevalence of, and factors influencing, psychological distress during this outbreak. METHODS: Data were collected using an online survey, with a link directed to the affected population via a number of industry groups. Psychological distress, as determined by the Kessler 10 Psychological Distress Scale, was the main outcome measure. RESULTS: In total, 2760 people participated in this study. Extremely high levels of non-specific psychological distress were reported by respondents in this study, with 34% reporting high psychological distress (K10 > 22), compared to levels of around 12% in the Australian general population. Analysis, using backward stepwise binary logistic regression analysis, revealed that those living in high risk infection (red) zones (OR = 2.00; 95% CI: 1.57–2.55; p < 0.001) and disease buffer (amber) zones (OR = 1.83; 95% CI: 1.36–2.46; p < 0.001) were at much greater risk of high psychological distress than those living in uninfected (white zones). Although prevalence of high psychological distress was greater in infected EI zones and States, elevated levels of psychological distress were experienced in horse-owners nationally. Statistical analysis indicated that certain groups were more vulnerable to high psychological distress; specifically younger people, and those with lower levels of formal educational qualifications. Respondents whose principal source of income was from horse-related industry were more than twice as likely to have high psychological distress than those whose primary source of income was not linked to horse-related industry (OR = 2.23; 95% CI: 1.82–2.73; p < 0.001). CONCLUSION: Although, methodologically, this study had good internal validity, it has limited generalisability because it was not possible to identify, bound, or sample the target population accurately. However, this study is the first to collect psychological distress data from an affected population during such a disease outbreak and has potential to inform those involved in assessing the potential psychological impacts of human infectious diseases, such as pandemic influenza. Equine influenza (EI) is an acute, highly contagious viral disease which can cause rapidly spreading outbreaks of respiratory disease in horses and other equine species. It does not infect humans, but the virus can be physically carried on skin, hair, clothing, shoes, vehicles and equipment and through these means can be transferred to other horses. In addition, the windborne virus can be spread for distances up to eight kilometres [1] .
Australia's first outbreak of EI was confirmed on August 24 th 2007. It spread quickly, but was successfully contained within areas of South East Queensland (Qld) and New South Wales (NSW). Although EI was not detected in other States and Territories, stringent disease control procedures were put in place across all States; which included an initial stand-still of all horse movements and subsequent controls, movement restrictions, and biosecurity requirements for many months. Colour-coded EI control zones were established within four weeks of the outbreak based on the level of disease/disease risk in Local Government Areas in NSW and Qld; these were adjusted as the disease spread, and each zone was subject to specific controls and restrictions. Controls were reviewed, revised and expanded as the disease spread, subsequent disease containment and control progressed, and policies were revised. These zones are summarized in Table 1 . Further details of the outbreak, restrictions and zoning are available via the NSW Department of Primary Industries (NSW DPI) and Qld. Department of Primary Industries and Fisheries (DPI&F) websites [2, 3] . Throughout the outbreak movement restrictions and biosecurity requirements remained in place, and no (or very limited) horse movement was ever allowed from higher risk zones to lower risk zones.
The disease outbreak peaked in late September/early October 2007, and then declined as successful containment and eradication strategies were progressed. The last new infections of EI were reported in NSW and Qld in December 2007. In total approximately 6,000 properties and 47,000 horses were infected in NSW and at least 3,000 properties were infected in Qld. Current data from disease surveillance and monitoring indicates that no active infection is present in Australia and the expectation is that Australia will be declared EI-free by the end of June 2008; if successful, Australia will be the only EI infected country in the world to have eradicated the disease.
The effects of EI and the disease containment strategy, like the horse industry itself, were varied and wide-ranging; impacting differentially on horse owners and those involved with the horse industry nationally. In terms of support to those affected, a range of government financial support and assistance was available to many of those affected within a short time of outbreak onset and financial and economic impact surveys were undertaken to provide feedback information to government [4, 5] . The current study was conducted to gain additional complementary data to assess the impacts of EI on the social and emotional health and well-being of those affected. This paper reports data collected on non-specific psychological distress; however the full study covered many other aspects, such as adherence to biosecurity requirements, effects of social isolation due to quarantine and the consequences of restricted horse movement and related activities, and sources of support and coping during the EI outbreak.
Although EI is endemic in Europe and North America, and has occurred as an epidemic in many other countries, e.g. Japan, South Africa, Hong Kong, there does not appear to be any published studies of the human response or impacts to EI or the containment strategies used to control this disease. The best reported and documented research with respect to the impacts of infectious animal disease on people is the outbreaks of foot and mouth disease (FMD) in Europe in 2001, specifically in the UK and The Netherlands.
Like EI FMD is highly contagious, however, FMD is considerably more serious as it spreads to cloven-hoofed animals including cattle, sheep, pigs, and goats. During these FMD outbreaks an estimated 4 million livestock were slaughtered on 9,000 farms in the UK (including many healthy animals as part of 'contiguous' or preventative culling on farms neighbouring infected farms) and 270,000 were culled in The Netherlands. The impacts on people were both economic, through financial/business/ tourism-related losses, and psychological, through the exposure to loss of livestock, culling, and massive funeral pyres; the latter affecting not just farmers and their families, but also the wider population through media images on the television and in newspapers [6] [7] [8] . In the UK higher 'caseness' as indicated by the GHQ(G) was found in farmers from 'badly infected' areas, although higher psychological morbidity generally, was reported in farmers from both badly infected and unaffected areas [9] .
In a study of Dutch dairy farmers [10] around half of those whose animals were culled suffered from severe post-traumatic distress, (identified as a clinical level of distress (> 25) using the 15-item Impact of Events Scale), with this reducing to one in five for those where severe restrictions were imposed (but where no culling took place). Higher levels of symptoms were reported for older respondents and those with lower levels of education. In this same study differences in stress, psychological marginalization, and depression were reported for different disease control areas, i.e. culled-area, buffer-area, FMD-free area [11] .
Within Australia, the psycho-social impacts of Ovine Johne's disease have been reported [12, 8] in which grief, depression, and anxiety were profound in affected farming families, and the perceptions of the management control process were the cause of much of the distress. Government policies on quarantining and de-stocking farms were suspended due to mounting reports of severe emotional and social distress in farmers, rural families, and government employees implementing those policies. Further discussion of stress in emergency responders managing agricultural emergencies is considered in an Australian context in a recent paper by Jenner [13] .
The role of the animal-human bond on disaster preparedness and response is key feature in human response to animal disease, and has been review by Hall et al. [8] . These authors report several aspects of relevance to the current study, including the increasing role of horses as companion animals as opposed to livestock or economic investments, and hence an increasing emotional attachment to horses; the complex and dynamic emotional relationship between farmers and their livestock; the emotional and practical implications of the animal-human relationship in disaster management, e.g. compliance with disaster management behaviours; and the impacts on veterinarians as first responders in disasters. These authors conclude that recognizing the mental health aspects of the animalhuman bond is an important factor in public health approaches to disaster and can be critical in promoting the resilience of individuals and communities. Therefore, it follows that in an animal-centred disease outbreak, such as EI affecting horses, the potential disruption of the animal-human bond, and the impact of policies restricting animal-human activities could have significant implications for the mental health and resilience of those affected.
The main outcome measure in this study is non-specific psychological distress, as measured by the Kessler 10 (K10) [14] . The K10 was selected because it is a well-established and validated measure that is used widely in population research in Australia, it has been used in population health surveys in NSW [15] , Victoria [16] , South Australia [17] , and Western Australia [18] , as well as in National surveys conducted by the Australian Bureau of Statistics [19] , and therefore State and National prevalence data are available as benchmarks for the current study. Scores from the K10 can be related to levels of intervention, with 'very high' psychological distress scores (> 30) equating to 'caseness' for a mental disorder, and high scores are strongly associated with current diagnosis of anxiety and depression using the Composite International Diagnostics Interview (CIDI) [20] . The K10 is also able to discriminate between DSM-IV cases and non-cases, and is felt to be an appropriate screening instrument for identifying likely cases of anxiety and depression in the population providing a strong marker for a possible mental health disorder [21] [22] [23] .
In the most recent (2007) data from the NSW Adult Population Health Survey the combined proportion of the population reporting 'high' or above psychological distress (22-50) is 12.1% [24] . In addition, recent data collected in rural communities suggests that these figures may be slightly higher in rural-dwellers with 'very high' psychological distress of 5% reported in one study [25] and 13.4-13.8% for combined 'high'/'very high' psychological distress in another [26] . These findings are of relevance in the current study as it would be expected that horse-ownership would be linked to rural and peri-urban residency.
The questionnaire was designed for online completion to expedite data gathering whilst the EI outbreak was occurring. Questionnaire content was reviewed by subject matter experts, including a small group of public health professionals in NSW Health, some of whom had been involved in aiding the NSW DPI in disease control management, a NSW DPI Local District Control Centre Controller who was responsible for leading control activities, and representatives of the Australian Horse Industry Council (AHIC). Ethics approval for the study was obtained through the University of Western Sydney ethics committee.
Horse owners, and those involved in the horse industry were invited to take part in the study via an e-mail alerting service administered by AHIC; using the national Horse Emergency Contact Database (HECD). The HECD had been established before the EI outbreak and was used as a network to contact and inform horse-owners during emergencies, such as bushfires, and disease outbreaks, and had been used previously by AHIC for collecting financial impacts information relating to EI earlier in the outbreak. This alerting service was used regularly during the EI crisis to update registrants with government support agency communications and general industry news and support information. Approximately 8,000 addressees were registered on the HECD; most were individuals, but also included were industry associations, pony clubs, and horse groups that would forward information to their own memberships nationally. Horse owners in NSW were encouraged by the NSW DPI to register on the HECD to receive up to date information.
The initial invitation to participate was sent to those registered on the HECD on 14 November 2007 (Week 12 of the outbreak). The survey remained open until 7 January 2008 (Week 21 of the outbreak) and date of completion was recorded with each respondent's data.
The full survey comprised 166 questions, covering a wide range of subject areas; those reported here include demographic information, i.e. gender, age category, number of children, highest level of educational qualification, and State/Territory of residence. In addition, respondents were asked about the nature of their current main involvement with horses (i.e. their industry sector), for example breeding, equestrian, recreational; whether their primary source of income was linked to a horse-related industry, and their current colour-coded EI control zone.
The main outcome measure reported in this paper is nonspecific psychological distress as measured by the K10. This measure comprises 10 questions that ask respondents how often they have experienced certain symptoms during the preceding four weeks and responses are scored on a scale of 1 to 5 depending on how frequently each symptom is experienced, where 1 = 'none of the time', and 5 = 'all of the time'. Thus, a minimum score is 10, indicating no psychological distress, and a maximum score is 50, indicating the most severe level of psychological distress. Scores on the K10 are subsequently categorized into four levels: low (scores of 10-15); moderate (scores of [16] [17] [18] [19] [20] [21] ; 'high' (scores of [22] [23] [24] [25] [26] [27] [28] [29] and 'very high' (scores of 30-50) [27] .
Statistical analyses were undertaken using STATA, version 9.2 (2004; Stata Corporation, College Station, TX, USA). Exploratory data analysis was conducted using frequency distributions for categorical variables. In the logistic model, a binary coding of psychological distress was used in which high psychological distress was a combination of 'high' + 'very high' levels of psychological distress = 1 (i.e. K10 scores of 22 or greater) and low psychological distress was a combination of 'low' + 'moderate' levels of psychological distress = 0 (i.e. K10 scores of 21 or less). Simple binary logistic regression and backward stepwise multiple logistic analyses were performed to identify factors influencing high psychological distress. All variables were entered into the model initially, with the least significant variables removed one at a time until only significant variables associated with values of p ≤ 0.05 remained. All statistical tests were two-tailed.
Details of the study sample are presented in Table 2 . In total, 2,760 respondents completed the online survey, and of these 15% were male and 84% were female. More than a half of the sample (58.9%) had no children. A total of 40.2% of the respondents had a tertiary level educational qualification.
Just under half the sample (47%) was from NSW and respondents from Qld. and Victoria (Vic) comprised a further 40% of the sample (20% from each State). Thirty percent of respondents were in uninfected white zones in States other than NSW and Qld, and 22% were from the restricted high EI risk red zones in NSW and Qld.
Around three quarters of the sample (73%) were from three industry sectors; recreational, equestrian, and breeding/stud sectors (30%, 27%, and 16%, respectively). The majority of respondents (76%) reported that their main source of income was not linked to a horse-related industry.
The prevalence of the four levels of psychological distress for the whole sample during the equine influenza outbreak; were 39% of respondents reporting 'low', 27% reporting 'moderate' 20% reporting 'high' and 14% reporting 'very high' levels of psychological distress. Table  3 shows the proportion of respondents reporting each level of psychological distress for the main socio-demographic survey variables.
The greatest prevalence of 'very high' psychological distress was reported for those respondents in the 16-24 age group (21.2%), and the lowest prevalence was reported by those in the 55-64 age group and those under 16 (8.6% and 7.4%, respectively). With regard to the remaining socio-demographic variables the highest prevalence of 'very high' psychological distress were recorded for those respondents who were female, those with one child, and those with no formal educational qualifications.
The prevalence of 'very high' psychological distress was greater for respondents from Qld. (19.4%) with prevalence figures being slightly lower for respondents from NSW (14.5%) and lower again for respondents from Vic. (10.8%). The highest prevalence of 'very high' psychological distress was found for respondents in the red zones (18.2%) and lowest for those in the white zones (9.3%).
Those whose incomes were linked to horse-related industry had a higher prevalence of 'very high' psychological distress as compared to those whose main income was not linked to a horse-related industry (20.7% and 11.8%, respectively).
The four levels of psychological distress were combined in pairs ('low'/'moderate', and 'high'/'very high') to form a binary variable for subsequent statistical modelling. Figure 1 shows the prevalence of this binary high/low psychological distress variable by EI disease zones. Respondents in the red and amber zones reported higher prevalence of high psychological distress (41% and 39%, respectively) than those in the purple, green, and white zones (36%, 34%, and 26% respectively).
Univariate analysis Table 4 shows the unadjusted and adjusted odds ratios (ORs) for the associations between high psychological distress (≥ 22) and socio-demographic variables. Total count = 2760 unless otherwise given in brackets Respondents whose main source of income was from horse-related industry (unadjusted: OR = 2.19, 95% CI: 1.80-2.67; p < 0.001) were at a greater risk of high psychological distress than those whose main income was not linked to horse-related industry. chological distress as compared to those whose income was not linked to horse-related industry.
The most salient finding was the extremely high prevalence of high psychological distress in horse owners and those involved in the horse industry during a serious horse disease epidemic; with just over one third (34%) reporting levels of psychological distress that might require some form of external intervention, and 40% of these (14% of the sample) reaching levels that may be considered indicative of 'caseness' for a DSM-IV disorder. The prevalence of 'very high' psychological distress in this sample was approaching five times the level reported in recent population health data for NSW [24] . Although this prevalence is very high, and there are some methodological reasons why this may be distorted (see study limitations section) it is certainly true that many of those impacted by EI, or the threat of EI, were subject to a wide range of acute stressors over a prolonged period, in a country where EI and such rigorous disease containment and control measures were previously unknown.
Analysis of psychological distress prevalence within the sample indicated that EI control zone was associated with psychological distress. Those in the areas where EI was present had higher risk of high psychological distress, furthermore, risks were higher in areas where EI was more active or threatening and the tightest levels of disease control were in place (i.e. red and amber/buffer zones). This finding suggests high levels of anticipatory anxiety. Interestingly, the risks of higher psychological distress in the purple zone (the region in NSW with the highest infection rate and earliest infections) were lower than in the red and amber areas. It is probable that during the timing of the study EI was more of a 'known' threat to those in the purple zone and there would have been some habituation to this risk; with many properties already infected or recovering, and restrictions eased due to the decision to let EI 'run its course' in this area at that time.
As disease control (and zoning) was controlled at a State level there is geographical overlap and co-linearity of the Australian State/Territory and EI control zone variables in the analysis; in the backward stepwise multiple logistic analyses excluding one made the other a significant factor. With regard to analysis by State, it is interesting to note the high levels of psychological distress reported in Victoria.
Although Victoria remained EI-free throughout the crisis, those in Victoria were 1.57 times more likely to experience high psychological distress that those in the other uninfected States. There are probably a number of reasons for this effect: Victoria has a very extensive horse industry and is geographical closer to the infected States and diseaseaffected areas of NSW and Qld; there is also a high level of business interaction and physical movement of horses between Victoria and NSW and Qld; hence the level of proximal threat and the degree of disruption caused by disease control measures was probably experienced more widely in Victoria and may explain some of this effect. It should also be noted that although the remaining States were similarly uninfected, the overall prevalence of high psychological distress in horse owners from these States was still far higher than in the general population; those uninfected were not unaffected.
One of the other primary factors associated with high psychological distress was age. Those in the 16-24 year age category reported the highest levels of high psychological distress and analysis indicated that although prevalence and comparative risks of high psychological distress reduced from age 24 onwards, these reduced risks only became reliably statistically significant from age 45 onwards, and high psychological distress was certainly still a risk to those in the 35-44 year age category. This is interesting because in the general population psychological distress is generally found to peak around middle age (40s-50s). The study findings would suggest that younger people were particularly vulnerable and were coping less well with the consequences of EI. The reasons for this finding are not known, however, research literature suggests that younger people form stronger emotional attachments to animals [28] , and they are also less likely to be resilient or practised, generally, when it comes to coping with adversity. From the general perspective of mental and physical health of younger people, it is interesting to consider the longer term consequences and potential burden of disease if these effects are enduring.
It is also interesting to note here the association of psychological distress with having children. Data in this study indicated that those with one child had a 1.2 times higher risk of high psychological distress than those with no children; and having three or more children appeared somewhat protective against high psychological distress. National statistics would support the suggestion that those with one child are generally younger adults and/or are 'young families' with a single younger child. In this study, 17.8% of respondents with one child reported 'very high' levels of psychological distress (K10 score = 30-50). Given these family circumstances such a finding may be a cause for concern.
The final main factor associated with high psychological distress was having an income linked to horse-related industry. Unsurprisingly, those with financial dependence on an industry facing such a crisis are likely to be significantly predisposed to high psychological distress. Nothing has been mentioned in this paper on the industry sector from which respondents had their main involvement with horses. These data were reported as part of the sample description to illustrate the wide range of industries affected by EI and the complexity of the affected population, and to provide information to aid interpretation of the findings. The nature of the potential psychological impacts of EI on those in different sectors is extremely diverse; from purely economic impacts, to loss of leisure pursuits and disruption of social networks, to loss of futures and missed opportunities in time, and many other possible impacts. Time, money and support will help most recover but it is possible that some people's mental and physical health will be permanently affected by EI and some will take many years to recover professionally if they choose to stay in these professions.
Given the level of psychological distress noted in the current study, it is interesting to consider the distress that might result from other epizootics, such as foot and mouth disease or avian influenza, and how this, in turn, might compare to the levels of distress resulting from human epidemics, such as SARS and H5N1/pandemic influenza. As mentioned earlier, foot and mouth disease in Europe resulted in high distress and PTSD in farmers.
In relation to avian influenza, most research has focussed on risk perception and compliance with protective behaviours. A large European Union project on risk perception to avian influenza in Europe and Asia found moderate levels of risk perception generally, with higher levels of risk perception noted in Europe, and in females in most countries [29] . Considering distress and risk perception in relation to human epidemics; it is likely that psychological distress would be far greater, since these present a threat to human health and possibly death. Certainly data collected during and after SARS in Hong Kong found high levels of fear and PTSD in health care workers and hospital workers [30] , and high levels of emotional disturbance in the general population [31] . Research in Canada found enduring psychological distress, up to two years following SARS, among health care workers in a hospital that treated SARS patients [32] .
This study had a number of limitations that should be considered when interpreting the data. Firstly, the target population; those affected by EI, is a complex, disparate, and unknown population and therefore it is difficult to comment accurately on the representativeness of the sample. All horse owners in Australia are not registered on a centralized database, or otherwise controlled, and as a result, it is not possible to know how extensive the database used to access horse owners (the HECD) was. However, at a national cross-industry sector level it is believed that this was the most extensive and efficient online route to access the target population, and the use of the database as a central communication facility during the EI crisis meant that this was likely to have been a focus for those affected during the epidemic.
Due to demographic bias in the sample, in particular, a greater proportion of women, and those with higher levels of education it is possible that there will be response bias in the data. The high proportion of women in the sample may be due to greater interest and participation in studies of this nature, but may also be indicative of higher levels of females in the target population, in particular in the main industry sectors represented in the data, i.e. recreational and equestrian. There are no official statistics on gender breakdown across horse industry sub-populations in Australia, but data indicate that the equestrian sector in the United States may comprise 80% women [33] , so the gender bias may reflect a gender bias in the main industry sub-populations in our data. Research data often report higher levels of psychological distress in women in the general population, and therefore, the gender demographic bias in our study might have led to an elevation in the levels of psychological distress reported in this study. However, the absence of a significant gender effect in this study, and the close matching of relative levels of psychological distress in men and women with data from the Australian general population, suggests that EI, as an adversity, was exerting similar impacts on males and females. It is not possible to explain why there was an absence of a gender difference in the data. One possible explanation is that the timing of the study; around the height of the EI epidemic, and the high levels of psycho-logical distress generally, reflected peak, acute levels in which gender differences were minimised and insignificant.
As with gender bias, it is hard to define the impacts of education level in the data. Unlike the (female) gender bias in the data, higher levels of education offer a protective effect (as identified in the univariate analysis). Therefore, this source of bias may have led to an under-reporting of high psychological distress. Again, it is not possible to define or quantify the extent of this.
Finally, the use of an online survey imposes potential limitations. It is probable that the study findings under-represent the responses of those in certain demographics, e.g. those who are less educated (as noted), those less affluent, and older respondents. Not all horse owners would have access to the internet, and online survey methodology is relatively uncontrolled, e.g. the sample was self-selected and therefore may be more prone to response bias than a sample that was randomly selected or otherwise controlled. Also, those experiencing higher distress may have been more motivated to respond. The extent of this response bias on the data cannot be accurately estimated, however, in anticipation of potential response bias, actions were taken to ensure that the study was presented to potential respondents in a way that would minimize such effects; e.g. the study was presented as independent of any industry group or government organization and it was clearly identified as a university research study. It was hoped that such presentation of the study would reduce political or self-interest motivation for completing the study.
Despite some methodological limitations, this study was able to determine the psychological impact of Australia's first outbreak of equine influenza on a substantial sample of horse owners and those involved in horse-related industry. Study findings indicated that this affected population had highly elevated levels of psychological distress and that, although prevalence of high psychological distress was greater in infected EI control zones and States, elevated levels of psychological distress were experienced in horse-owners nationally, and not just in areas where equine influenza was present. Statistical analysis indicated that certain groups were more vulnerable to high psychological distress; specifically younger people, those with no formal educational qualifications, and those whose main income was linked to a horse-related industry.
Findings from this study generate further questions: What were the determinants of elevated psychological distress? Was it the risk of the disease itself, e.g. fear of the disease, or concern for horses? Was it the social and emotional impacts of disease control measures and restrictions, e.g. social isolation, quarantine, loss of freedom or control, stigma of being 'infected'? Was it loss of income or sporting aspirations? More importantly, how enduring is this elevated psychological distress, and what are the longer term mental or physical health consequences for those affected? The latter is of critical importance given the increased prevalence of high psychological distress reported in young people in this study. Some of these questions can be addressed using additional data collected in the wider study; however, the issue of enduring psychological distress will require further assessment.
Publish with Bio Med Central and every scientist can read your work free of charge  Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response. Influenza A viruses are negative-stranded RNA viruses that belong to the family of orthomyxoviruses. The segmented genome of influenza A virus encodes for up to 11 viral proteins. As many other viruses, influenza viruses have evolved strategies to counteract cellular antiviral responses, especially to circumvent the type I IFN system as a first line of defense against the pathogenic invader.
Among the influenza viral proteins, the NS1 has been identified as the main type I IFN antagonistic factor. So far two major mechanisms have been described by which NS1 suppresses the initial expression of IFNb. On the one hand NS1 inhibits vRNAmediated induction of the transcription factors interferon regulatory factor-3 (IRF-3), activating protein-1 (AP-1) and NF-kB that target the IFNb promoter. This most likely occurs via binding to the RNA-sensor retinoic acid inducible gene (RIG-I) and inhibition of RIG-I-mediated signaling in response to viral RNA [1, 2] . On the other hand NS1 inhibits maturation [3, 4] and nuclear export of host mRNAs [5] . Other functions of the multifunctional protein include block of activation of the dsRNAactivated protein kinase PKR by direct interaction [6] or activation of the phosphatidylinositol-3 kinase PI3K/Akt pathway to prevent premature apoptosis induction [7, 8] .
While the NS1-mediated antagonistic activities of influenza viruses mainly affect the induction of genes such as IFNb, so far no viral suppression of IFN signaling has been described.
IFN are among the first molecules synthesized in response to viral infections [9] . The IFN family includes three classes. Type I comprises the well known IFNa and IFNb. The only member of type II IFN is IFNc. Type III IFN comprises IFNl1, -l2, and -l3. All classes of IFN bind to different receptors and are structurally not related [10, 11] . Type I IFN belong to the key cytokines produced by influenza A virus-infected epithelial cells [12, 13] . The antiviral activity of type I IFN is mediated by a set of IFN-induced genes (ISGs).
Binding of IFNa/b to its receptor is the initial step in this signaling process, followed by activation of the JAK family and subsequent activation of STAT proteins [14] .
Ligand binding leads to dimerisation of the type I IFN receptor subunits IFNAR1 and IFNAR2 and causes their conformational change. The JAK kinase Tyk2, which is constitutively bound to IFNAR1, phosphorylates the receptor at tyrosine residues and creates a docking site for STAT2. Subsequently, Tyk2 phosphor-ylates STAT2 at Y690. At the same time the receptor-bound JAK1 phosphorylates STAT1 at Y701 [15, 16] .
The phosphorylated transcription factors dimerise and bind to IRF-9 [17] . The newly formed heterotrimer, called IFNstimulated gene factor 3 (ISGF3), translocates into the nucleus and binds to IFN-stimulated response elements (ISRE), to initiate gene transcription of ISGs. Treatment of cells with type I IFN upregulates expression of an array of genes including SP110, IRF-1 and many others [18] . Among these ISGs the 29, 59oligoadenylate synthetase 1 (OAS1), the Mx proteins and the dsRNA-activated protein kinase (PKR) are described to directly interfere with viral replication [19] . Both, PKR and the OAS1/ RNaseL system are capable of inhibiting cellular and viral translation.
IFN-induced JAK/STAT signaling can be inhibited at different levels by several viral and cellular factors through various mechanisms. The large T-antigen of murine polyomavirus (MPyV) binds to JAK1 and inhibits downstream signaling [20] , whereas the VP24 of Ebola virus (EBOV) binds to karyopherina-1 thereby blocking nuclear accumulation of STAT1 [21] .
Endogenous cellular key regulators, capable of negatively regulating JAK/STAT-mediated signal transduction, include suppressor of cytokine signaling (SOCS) proteins, protein tyrosine phosphatases (PTP) and protein inhibitor of activated STATs (PIAS). The family of SOCS proteins comprises eight members (cytokine-inducible SH2 domain-containing protein (CIS) and SOCS1-7). All members contain a central SH2 domain, an Nterminus of variable length and sequence and a C-terminal 40 amino-acid module called SOCS box [22] . The SOCS box is necessary for recruitment of the ubiquitin transferase system and for stabilization and/or degradation of SOCS proteins [23] [24] [25] . The N-terminus contains a kinase inhibitory region (KIR), which functions as pseudo substrate for the JAK [26] . SOCS-1 and SOCS-3 differ in their mode of action. For inhibition of the kinase activity of JAKs, SOCS-1 binds directly to the activation loop of JAKs [26] [27] [28] . In contrast, SOCS-3 first binds to the receptor [29, 30] .
Induction of SOCS-3 gene transcription by viruses was reported for HSV-1, HCV [31] [32] [33] and for respiratory viruses, such as SARS and RSV [34, 35] . The level of induction of SOCS-3 by HSV-1 seems to determine whether infection turns to acute or persistent progression [31] . For HCV it has been suggested that upregulation of SOCS-3 may contribute to the non-responsiveness of HCV patients to IFN therapy [33, [36] [37] [38] . Elevated SOCS-3 mRNA levels during RSV infection were linked to Th2 cell-mediated immune disease as atopic dermatitis and asthma [39, 40] .
In the present study we show that influenza A virus can be added to the list of viruses that induce SOCS-3 expression. The protein functionally interferes with viral replication by providing a virus-supportive IFN-antagonistic activity on the level of type I IFN-signaling that has not been described so far.
Phosphorylation of STAT1 and STAT2 by members of the JAK tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type I IFN-induced gene expression. Therefore, we analyzed whether STAT phosphorylation patterns are altered in influenza A virus infected cells that were stimulated with IFN at different time points post infection (p.i.). The human alveolar epithelial cell line A549 was infected with the influenza A virus strain A/Puerto-Rico/8/34 (H1N1) (PR8) ( Figure 1A ). Cells were subsequently stimulated with IFNb at given time-points p.i. and STAT phosphorylation was assessed in Western blots. Both STAT1 and STAT2 were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to 4 h p.i. ( Figure 1A ). Furthermore, virus infection alone resulted in a significant induction of STAT phosphorylation 4-6 h p.i., presumably caused by virus-induced IFN expression. However, at later time points (6-10 h p.i.), in A549 cells both virus-and IFN-induced STAT1 and STAT2 phosphorylation was markedly reduced ( Figure 1A ). Similar patterns were observed upon stimulation of cells with IFNa or upon infection with other viruses, such as the human influenza virus A/Victora/3/75 (H3N2) (data not shown). In addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line HEK293 ( Figure 2E ) or the human umbilical vein endothelial cells (HUVEC) ( Figure S1B ). Inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since IFNc-induced STAT1 phosphorylation was not affected at all ( Figure 1C ). Finally, involvement of any auto-or paracrine action of virus-induced type I IFN could be ruled out, as the inhibitory effect was also observed in Vero cells lacking functional type I IFN genes ( Figure 1E ).
With regard to the molecular basis of impaired IFNa/b-induced STAT phosphorylation in infected cells it was striking that the inhibitory effect correlated with the accumulation of viral proteins, as monitored in PB1 Western blots ( Figures 1A and 1E) . Thus, the question arose whether individual expression of viral proteins may result in the interference with STAT1 phosphorylation. Out of the 11 viral proteins of PR8 we choose the nucleoprotein (NP), the NS1 protein, the matrix protein (M1) (Figure 2A ) and the subunits of the viral polymerase, PA, PB1 and PB2 ( Figure 2C ), for a representative experiment. These proteins are known to bind to vRNA/RNPs or to interfere with the RNA-mediated innate immune response. For efficient transfection of the expression
The type I interferon (IFN) system is one of the most powerful innate defenses against viral pathogens. Most RNA viruses are sensitive to the action of type I IFN. Therefore, these pathogens have evolved strategies to evade this response. For example, influenza viruses express a viral protein, the non-structural protein 1 (NS1), that suppresses production of IFNb by lowering cellular sensitivity to viral nucleic acid as a pathogen pattern.
Here we present data indicating that influenza A viruses are not only capable of suppressing production of the IFNb gene but also inhibit action of this antiviral cytokine on cells. This occurs by viral induction of a cellular protein, the suppressor of cytokine signaling (SOCS)-3, a potent endogenous inhibitor of IFN signaling. This is a novel mechanism by which influenza viruses inhibit the antiviral response of the host and paves the path to efficient virus replication. This may be especially relevant for influenza viruses that induce high cytokine responses (cytokine burst), such as highly pathogenic avian influenza viruses of the H5N1 subtype. Induction of SOCS-3 expression would allow efficient replication despite high IFN and cytokine levels.
constructs we used the highly susceptible cell line HEK293 that also exhibits impaired IFNb-induced STAT1 phosphorylation at later stages of infection ( Figure 2E ). 24 h post transfection cells were stimulated with IFNb and STAT phosphorylation was monitored in Western blots (Figures 2A and C) . Expression of none of the viral proteins resulted in a significant decrease of IFNb-induced STAT1 or STAT2 phosphorylation (Figures 2A  and 2C ). Similar results were obtained in the human bronchial epithelial cell line H1299 when expressing M1, NS1 or NP alone or in different combinations (data not shown). Thus, we concluded that viral proteins most likely do not play a prominent role as blockers of IFNa/b-induced JAK/STAT signaling.
Decrease of STAT phosphorylation might also be due to the action of virus-induced phosphatases. On the one hand these enzymes may cause direct dephosphorylation of STAT proteins. On the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of JAKs resulting in an attenuated phosphorylation of STATs. Several protein tyrosine phosphatases (PTPs) are known to mediate dephosphorylation of both, JAKs and STATs [41] . In order to investigate whether influenza A virus activates phosphatases that subsequently target JAKs or STATs, we treated infected or uninfected A549 cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [42, 43] . Uninfected cells or cells infected with PR8 for 10 h were incubated with increasing amounts of this compound 10 min prior to stimulation with IFNb. This time point of infection was chosen since we observed considerable inhibition of IFN-induced STAT1 phosphorylation in the course of infection ( Figures 1A and 1E ). Increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. Accordingly, a gradual increase of STAT1 phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-STAT1 ( Figures 3A and B ). This is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the IFNb stimulated samples ( Figure 3B ). If the blockade of IFNb-induced STAT1 phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. However, the result in Figure 3B indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virusactivated phosphatase appears to be involved. In support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. This is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates ( Figure 3C ). Thus, involvement of phosphatases in influenza virus-induced alteration of STAT1 phosphorylation can be greatly ruled out.
Phosphorylation of STATs in the IFNb signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (SOCS) family. Action of these proteins is mainly controlled on the level of transcriptional activation. SOCS proteins are described to have high affinity for JAK and STAT proteins and to inhibit the transmission of IFNa and IFNb induced signaling [44, 45] . To examine whether expression of SOCS genes is induced in influenza virus infected cells, A549 cells were infected with PR8 for different time points. Subsequently total RNA was analyzed for the amount of SOCS-1 and SOCS-3 mRNA by means of quantitative real time-PCR (qRT-PCR). The mRNA Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNb (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNb at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji). doi:10.1371/journal.ppat.1000196.g002 levels of SOCS-1 and SOCS-3 differed notably in the time course ( Figure 4A ). While SOCS-3 mRNA is strongly and transiently elevated in the early phases of infection, SOCS-1 gene transcription is only significantly induced 15 h p.i.. Elevated SOCS-3 mRNA levels were also observed in other host cell types, such as HUVEC starting 3 h p.i. ( Figure S1A ). Although elevation of SOCS-3 mRNA levels in infected cells was rather transient, there appears to be a robust induction on protein level ( Figure 4C ). First detected at 4 h p.i., SOCS-3 protein levels increased and stayed on a high level throughout the observation period. Strikingly, expression kinetics of the SOCS-3 protein perfectly matched the kinetics of virus-induced inhibition of STAT1 phosphorylation ( Figure 4C ), indicating that both processes are functionally linked.
Virus mediated SOCS-3 gene induction at early stages of infection ( Figures 4A, 4C and S1A) appeared to occur concomitant with an immediate and strong induction of IFNb ( Figures 4B and S1D) . This prompted us to analyze whether SOCS-3 transcription might be induced due to an auto-or paracrine action of IFNb expressed during infection. A549 cells were stimulated with IFNb for different time points and SOCS-3 gene induction was measured by qRT-PCR ( Figure 4D ). As a control we monitored expression of 29, 59oligoadenylate synthetase (OAS1) and MxA, genes that are typically induced by IFNb. While OAS1 and MxA mRNAs were readily upregulated upon IFNb treatment SOCS-3 mRNA was not significantly elevated ( Figure 4D ). Similar results were obtained from HUVEC stimulated with IFNb ( Figure S1E ). To further confirm these results we knocked down the IFNAR in A549-cells by an siRNA approach. Although the knock down was efficient and leads to more than 60% inhibition of IFNb induced STAT1 phosphorylation ( Figure 4E ), the induction of SOCS-3 expression was not impaired ( Figure 4F ). SOCS-3 levels in the knock down cells were similar compared to wild type cells and even higher than in the vector control ( Figure 4F ). These results are consistent with data gained from previous experiments in Vero cells ( Figure 1E ) and indicate that neither induction of SOCS-3 mRNA nor inhibition of STAT phosphorylation is dependent on virus-induced type I IFN expression.
Since accumulation of viral RNA in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce SOCS-3 gene transcription. As a source for viral RNA, A549 cells were infected with influenza A virus for 10 h and total RNA from these cells was isolated. RNA from uninfected A549 cells served as a negative control. Different amounts of these RNAs were used for stimulation of A549 cells for 3 h (Figures 5A, 5B and 5C). Transfection of RNA from uninfected cells did not result in an increase of SOCS-1 or SOCS-3 gene transcription ( Figure 5A ) or IFNb induction as a control ( Figure 5B ). However, transfection of RNA from virally infected cells led to strongly elevated SOCS-3 mRNA amounts while SOCS-1 mRNA is only induced weakly ( Figure 5A ). This dose dependent induction of SOCS-3 by stimulation with increasing amounts of RNA from infected cells corresponds with a gradual decrease in the ability of this RNA to induce or potentiate STAT1/2 phosphorylation ( Figure 5C ).
In contrast to cellular RNA, influenza viral RNA carries a triphosphate group at its 59 terminus that was previously shown to be a major pathogen pattern that triggers cellular signaling [46] . To verify that indeed the viral 59 triphosphate RNA in the pool of RNAs from infected cells is the major trigger for induction of SOCS-3 expression, RNA from infected or uninfected cells was treated with phosphatase to remove the 59 triphosphate termini prior to stimulation of A549 cells ( Figure 5E and 5F). The dephosphorylated viral RNA was only poorly capable to induce SOCS-3 ( Figure 5E ) or IFNb ( Figure 5F ) mRNA expression. In addition, poly(I:C) was transfected to mimic action of double-stranded (ds) RNA ( Figure 5E and 5F, right bars). However, the dsRNA analog showed surprisingly little effects on SOCS-3 and IFNb mRNA induction.
Since viral RNA is able to induce IFNb gene transcription ( Figure 5B and 5F) we again wanted to rule out that induction of SOCS-3 by viral 59 triphosphate RNA is mediated by auto-or paracrine action of de novo synthesized IFNb. In order to do so, cells were stimulated with viral RNA after treatment with the protein synthesis inhibitor anisomycin at two different concentrations ( Figure 5G ). SOCS-3 mRNA was still induced to the same extent in the presence of the protein synthesis inhibitor, providing the ultimate proof that de novo protein synthesis is not required for SOCS-3 induction.
So far, our data suggest that influenza virus-induced transcriptional upregulation of the SOCS-3 gene is not mediated by the . Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNb (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified. doi:10.1371/journal.ppat.1000196.g004 autoregulatory action of type I IFNs ( Figure 4D and 4F) but is directly induced through accumulation of viral RNA during infection. This raises the question, which RNA-induced signaling pathways are responsible for SOCS-3 expression. The MKK/p38 mitogen activated protein kinase (MAPK) pathway [47] [48] [49] as well as the IkB kinase (IKK)/nuclear factor of kB (NF-kB) cascade [50] [51] [52] are both known to be activated by RNA or influenza virus infection and to be involved in the control of SOCS-3 expression. To assess whether the MKK6/p38-or the IKK/NF-kB-module is required for SOCS-3 gene induction, we generated A549 cell lines expressing dominant negative forms of either MKK6 (MKK6Ala) or IKK2 (IKK2KD) (Figure 6A to 6D). These mutants have been previously shown to efficiently block p38 or NF-kB signaling, respectively [52] [53] [54] . To monitor SOCS-3 gene induction, wild type, vector or mutant expressing cell lines were infected with PR8 ( Figure 6A ) or stimulated with RNA from virally infected or uninfected A549 cells ( Figure 6C ). Induction of IFNb mRNA was monitored as a control ( Figure 6B and 6D) . While MKK6Ala expression did not result in significant reduction of SOCS-3 in either infected ( Figure 6A ) or RNA-stimulated cells ( Figure 6C ), transcription is markedly reduced in IKK2KD expressing cell lines. To obtain independent evidence for NF-kB dependence of SOCS-3 gene transcription, A549 wild type cells were incubated with the NF-kB specific inhibitor BAY 11-7085 prior to stimulation with RNA from virally infected or uninfected A549 cells ( Figure 6E ). Again, IFNb mRNA levels were assessed for control purposes ( Figure 6F ). Both, SOCS-3 and IFNb mRNA levels were strongly reduced in BAY 11-7085 treated cells. This indicates that virus-induced SOCS-3 expression strongly depends on IKK2 and NF-kB activation, while the MKK6/p38 appears not to play a prominent role.
To further verify that influenza virus induces SOCS-3 via an RNA sensory pathway and in an NF-kB dependent manner we infected cells with the influenza A virus mutant deficient for NS1 (DNS1) (Figure 6G and 6H) . The NS1 protein is known to block RNA dependent signaling and NFkB activation [55] . Accordingly, infection of cells with the mutant virus resulted in a more pronounced and sustained, albeit delayed induction of SOCS-3 ( Figure 6G ) if compared to infection with the isogenic wild type, that is a very poor inducer of SOCS-3 but still reasonably well induces IFNb. Noteworthy, this isogenic wild type strain differs from the PR8 wild type virus used in the other experiments shown here (see Materials and Methods for details).
To analyze whether NF-kB activation is sufficient for SOCS-3 gene induction we stimulated cells with IL-1b ( Figure S2A ) or TNFa ( Figure S2B ) that are both strong activators of the transcription factor. While mRNA levels of IL-6, a strictly NF-kB dependent cytokine, are strongly elevated, SOCS-3 gene transcription is not significantly induced. Under the assumption that these cytokines do not additionally induce counteracting processes one can conclude that NF-kB is required, yet not sufficient for the induction of SOCS-3. Thus viral induction of SOCS-3 may require additional factors that are only active in virus-infected cells. Furthermore, these results rule out a potential role of virus-induced IL-1b or TNFa in the induction of SOCS-3. This is supported by the observation that neither expression of IL- 
To further assess a functional role of SOCS-3 in virus-induced suppression of STAT1 phosphorylation we analyzed mouse cells with a targeted deletion of the SOCS-3 gene [56] . Wild type and SOCS-3 deficient mouse embryonic fibroblasts (MEF) were infected for different time points with PR8. The time of infection was prolonged in comparison to the infection of A549 cells because the human PR8 replicates less efficiently in mouse than in human cells. Following infection lysates of these cells were assessed for STAT1 phosphorylation ( Figure 7A ). Both cell types showed no phosphorylation of STAT1 in the uninfected state. In contrast, infection of SOCS-3 knock out cells resulted in strongly elevated phosphorylation of STAT1 in a sustained fashion. To rule out that this STAT1 phosphorylation is due to altered secretion of IFNb or Figure 7 . Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNb-stimulated genes. Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNb for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described. doi:10.1371/journal.ppat.1000196.g007 other STAT1-activating cytokines in SOCS-3 deficient cells, we performed conditioned medium experiments ( Figure 7C ). MEF wild type and MEF SOCS-3 deficient cells were infected for 6 h and supernatants were subsequently harvested. Stimulation of MEF wild type cells with these different supernatants for 15 min. revealed no differences in STAT1 phosphorylation, indicating that both infected cell types secrete similar amounts of IFNb and other STAT1 activating cytokines. This is a strong indication that the observed differences in virus-induced STAT phosphorylation are directly due to the presence or absence of SOCS-3 in wild type and knock out MEF, respectively. To answer the question whether enhanced STAT phosphorylation in SOCS-3 deficient cells would also lead to enhanced expression of ISGs, total RNA was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of SP110, IRF-1 and OAS1 ( Figure 7E, 7F and 7G ). These genes are described as type I IFN-induced genes [18] . Indeed mRNA levels of all three representative ISGs were elevated in SOCS-3 knock out versus wild type cells at almost every time point during the course of infection. This indicates that enhanced STAT1 phosphorylation and activation in SOCS-3 deficient cells results in elevated expression of ISGs.
The remaining question was, whether the elevated IFN-induced gene response in knock out cells might also affect propagation of influenza A viruses. Thus, both wild type and knock out cells were infected with PR8 ( Figure 8A ) or the strain A/Victoria/3/75 (H3N2) ( Figure 8B ). Virus titers were assessed at different time points post infection. Progeny virus titers from SOCS-3 knock out cells were significantly reduced compared to titers from infected wild type cells. To independently confirm these results and to verify that the observed effects are really due to the lack of SOCS-3, we used an siRNA approach to specifically knock down SOCS-3 mRNA in A549 cells. Cells were transfected with 150 nM siRNA for 48 h and SOCS-3 protein levels were compared to control transfected samples ( Figure 8C, right) . Subsequently, cells were infected and progeny virus titers were determined by plaque assay (Figure 8C, left) . Similar to the results gained from infected knock out cells, knock down of SOCS-3 resulted in decreased virus titers.
On the contrary, over-expression of SOCS-3 resulted in elevated virus titers ( Figure 8D ) concomitant with an inhibition of IFNb-or virus-induced STAT1 phosphorylation ( Figure 8E) .
Taken together the data indicate that in the absence of SOCS-3, infection leads to a stronger activation of STAT1, resulting in enhanced expression of ISGs and reduced virus titers. Vice versa, over-expression of SOCS-3 leads to an inhibition of STAT1 activation and elevated virus titers, probably due to inhibited expression of ISGs.
This highlights the important role of virus induced SOCS-3 to limit the type I IFN-induced antiviral response program.
The type I interferon (IFN) system is one of the most powerful innate defenses of vertebrate cells, which limits replication and spread of viral pathogens including avian and human influenza viruses. Influenza virus propagation is sensitive to IFN activities and therefore, like other viral pathogens, these viruses do not only induce type I IFN but also antagonize the production and effects of these cytokines at the same time [55] . For influenza A and B viruses, this is accomplished through their non-structural NS1 proteins that are structurally related polypeptides of 26 kDa (A/ NS1) and 32 kDa (B/NS1), which are abundantly expressed in infected cells [55] . NS1 proteins predominantly act on the level of IFN gene induction in infected cells by obstructing RIG-Idependent signaling through interaction with cellular factor(s) and/or sequestration of RNAs generated during virus replication [1, 2, 57] . Some NS1 proteins were also described to inhibit the maturation of cellular pre-mRNAs raising the possibility that this activity additionally reduces production of IFNa/b in infected cells [58, 59] . While NS1 also interferes with the activity of some ISGs, such as the dsRNA dependent kinase PKR [5, 60] , so far no type I IFN antagonistic mechanism was described for influenza viruses that act on the level of IFN signaling rather than gene induction. Here we present data, showing that RNA-induced expression of SOCS-3 in early phases of infection leads to a functional inhibition of IFN-induced STAT activation and gene expression. This is a novel mechanism by which influenza virus suppresses the antiviral response of the host and paves the path to efficient virus replication.
While it was reported in the literature that expression of SOCS proteins can be induced upon stimulation with IFN [61] we could not detect any significant gene induction by IFNb in A549 cells. Instead we observed a significant up-regulation of SOCS-3 by viral 59 triphosphate RNA, indicating that gene induction occurs via accumulation of vRNA during infection. This appears to occur through the RNA-mediated activation of the IKK/NF-kB pathway, most likely activated through engagement of the RNA sensor RIG-I. At a first sight, this might appear controversial since NF-kB activation is among the RIG-I-induced signaling responses and NS1 was reported to inhibit this signaling pathway. However, despite the action of NS1 it is well known that NF-kB is still significantly activated upon influenza virus infection and many NF-kB and IFNb dependent genes are still expressed. We hypothesized previously that the incomplete inhibition conferred by NS1 is an indication that the virus exploits the remaining signaling activities for efficient replication [52, 62, 63] . The findings described here, namely NF-kB dependent induction of SOCS-3 and limitation of type I IFN signaling responses, provide yet another example how influenza viruses take advantage of NF-kB activity.
While the data show that NF-kB is required for viral SOCS-3 induction, the factor appears not to be sufficient, since prototype inducers of NF-kB, such as IL-1b or TNF-a would not induce SOCS-3. Thus there seems to be the need of additional virus or RNA-induced transcription factors. The most likely candidate would be the constitutively expressed interferon regulatory factor 3 (IRF-3), that is known to be simultaneously activated with NF-kB upon virus infection directly via the RIG-I RNA sensing pathway without the need of type I IFN. Furthermore IRF-3 is a factor suppressed by the NS1 protein.
Recently it was reported that IFN-induced gene expression responses are potentiated in cells, which lack the NF-kB factors p50 or p65 [64] . Although these authors described an inhibitory binding of NF-kB transcription factors to some IFN-induced gene, this mechanism might be cell type dependent since we could not observe similar effects in the cell types used here (data not shown). Thus, the underlying molecular mechanisms appear to be not fully clear. It is striking that the effects described for p50 and p65 knock out cells in these studies fully correlate with our observations in SOCS-3 deficient cells. While in the latter case cells lack the IFNb signaling inhibitor SOCS-3, the p50 and p65 knock out cells are deficient for the factors required for SOCS-3 induction. Thus, given the NF-kB dependent induction of SOCS-3 described in the present manuscript, we provide an additional molecular mechanism that may explain the phenomenon described by Wei et al. [64] .
First indications for beneficial effects of SOCS-3 gene expression on viral replication came from studies using the HCV core protein as a replacement for the influenza A viral NS1 in the context of infections with a NS1 deficient influenza virus [33] . One of the hallmark responses of HCV core expression is a rapid induction of SOCS-3 expression. Given the role of SOCS-3 described here, it was not surprising that HCV core could partially rescue growth of the NS1 deficient virus [33] .
While this manuscript was in preparation it was demonstrated by Pothlichet et al. that influenza A virus-induced SOCS-1 and SOCS-3 upregulation requires a TLR-3-independent, RIG-I/ MAVS-dependent pathway [65] . Moreover, over-expression of SOCS-1 and SOCS-3 in infected cells revealed that both molecules inhibit antiviral responses. These studies are perfectly complemented by our findings. Here we confirm involvement of RIG-I/MAVS by showing that 59 triphosphate RNA, the ligand for RIG-I, is a major inducer of SOCS-3. Furthermore, the finding that dsRNA is only a weak inducer of SOCS-3 is also consistent with the independence from the dsRNA sensor TLR-3. The only discrepancy of this work and the study of Pothlichet et al. is that they show a dependence on the type I IFN receptor. This may be due to the different virus-strains and cell types used. It is well known that the capability of type I IFNs to induce SOCS proteins is strongly cell type specific [31] . While in some cell types SOCS-3 expression appears to be type I IFN dependent (e.g. fetal liver cells) [31] it is clearly independent of IFN in other cell types [66] . Recently it was shown that SOCS-3 is not significantly induced by IFNa in A549 cells [18] , the major cell type used in our study. Evidence that cell type specificities may be the cause of discrepancy is additionally provided by the fact that Pothlichet et al. show identical induction kinetics of SOCS-1 and SOCS-3. In contrast the kinetics of the two proteins differ clearly in the cells we used, with SOCS-3 being induced much earlier than SOCS-1 on mRNA and protein level.
Finally, it should be stated that regardless whether SOCS-3 is additionally induced by type I IFNs at a later stage of infection, it is important that it can be induced earlier and in parallel to IFNb directly by vRNA accumulation. This is supported by the finding that IFNb and SOCS-3 induction occurs in parallel kinetics ( Figure 4A and 4B) while IFN-induced genes such as OAS1 and MxA are only up-regulated later in a delayed and more sustained fashion ( Figure 4D ). This makes a qualitative difference since the blocking effect of SOCS-3 on IFNb signaling already kicks-in during the first wave of IFNb action.
Taken together we describe here for the first time that at least some influenza A virus strains are able to suppress type I IFN signaling by a mechanism involving NF-kB dependent activation of SOCS-3 expression, which negatively affects STAT phosphorylation. This adds a new aspect to our knowledge of the strategies used by influenza A virus to antagonize type I IFN responses.
Human influenza A/Puerto-Rico/8/34 (H1N1) (PR8) (Giessen variant) and A/Victora/3/75 (H3N2) (Victoria) were originally taken from the strain collection of the Institute of Virology, Giessen, Germany. The human NS1 deficient influenza virus mutant DNS1 and its isogenic wild type variant were propagated and used as described earlier [7, 67] . It should be noted that this isogenic wild type strain as described by Garcia-Sastre et al. [68] is different from the PR8 (Giessen variant) used in the other experiments and in many previous studies [52, 67] . The supernatant was aspirated and cells were incubated with specific medium containing 0.2% BSA and antibiotics. To score for production of viral plaques the overlay was stained for 1 h using 1 ml neutral red in PBS per well [69] .
To trigger JAK/STAT signaling cells were stimulated using human IFNa/b or c as well as mouse IFNb. For stimulation of A549 cells or HUVECs 100 U/ml human IFNa or human IFNb was used. For stimulation of the green monkey epithelial cell line Vero or HEK 293 cells 500 U/ml human IFNb was applied. IFNc was always used in the concentration of 500 U/ml. Mouse embryonic fibroblasts (MEF) were incubated with 100 U/ml mouse IFNb. The different IFN were diluted in infection medium. For stimulation after infection, viral supernatants were aspirated and diluted cytokine was incubated for 15 minutes at 37uC. To investigate the potential of other cytokines to induced SOCS-3 gene expression A549 cells were stimulated with 100 U/ml IL1b or 20 ng/ml TNFa at 37uC for times indicated. After stimulation cells were lysed and subjected to immune blotting.
To block the activity of phosphatases after infection with influenza virus, sodium vanadate was used. Dilutions were prepared using infection medium. Sodium vanadate was added to the virus-containing infection medium at the time points indicated. After 10 minutes of incubation IFNb, diluted in infection medium, was added to the medium containing virus and sodium vanadate. The cells were stimulated with IFNb for 15 minutes. Incubation with sodium vanadate started 25 min before cells were lysed and subjected to Western blotting as described.
For conditioned medium experiments wild type and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for 10 h or left uninfected. Supernatants were used for stimulation of MEF wild type for 15 minutes. Cell lysates were subjected to Western blot analysis.
To investigate the induction of SOCS-3 expression by viral RNA, RNA isolated from infected or uninfected cells (control) was used. A549 cells were infected with PR8 (MOI = 5) or left mock infected. 10 h post infection RNA was isolated using the RNeasy mini Kit from Qiagen according to manufacturer's instructions.
To dephosphorylate viral 59 triphosphate RNA, calf intestine alkaline phosphatase (CIAP) (Fermentas) was used. Briefly, RNA was isolated using Trizol according to manufacturer's instructions. For dephosphorylation the reaction mix was set up in a 50 ml volume with 50 mg RNA, 25 U CIAP and 80 U RiboLock RNase inhibitor (Fermentas) and was incubated for 3 h at 42uC. Thereafter the RNA was isolated using the RNeasy mini Kit from Qiagen. RNAs used as control were mock-treated replacing CIAP by glycerol.
For stimulation, the different RNA species and analogues were transfected using Lipofectamine 2000 (L2000) according to manufacturer's instruction (Invitrogen). In brief, L2000 was incubated with OPTI-MEM for 5 minutes at room temperature; different amounts of RNA were added and incubated for additional 15 minutes. For stimulation of cells with cellular or viral RNA 400 ml RNA-L2000 mix were added to 2 ml serum-free medium. Cells were stimulated for 3 hours and subjected to either Western blot analysis or quantitative real time PCR.
For silencing SOCS-3 mRNA, A549 cells were transfected with 150 nM human SOCS-3 siRNA 48 h before infection using Hiperfect (Qiagen) according to manufacturer's instructions. In brief, 150 nM siRNA was added to a mixture of D-MEM without FCS/antibiotics and Hiperfect and incubated for 10 min at room temperature. For transfection 400 ml of this mixture were added to the cells. Subsequently cells were subjected to plaque assay analysis or Western blot analysis. Control siRNA was purchased from Qiagen. The sequences for the human SOCS-3 siRNA in use are: human SOCS-3 siRNA sense 59-CCA AGA ACC UGC GCA UCC AdTdT-39, human SOCS-3 siRNA anti-sense 59 -UGG AUG CGC AGG UUC UUG GdTdT-39 ) (see Table 1 for accession number of the human SOCS-3 gene).
To determine whether tyrosine phosphatases become activated upon infection with influenza virus a phosphatase assay using the Tyrosine Phosphatase Assay System (Promega) was performed. A459 cells were infected for 10 h (MOI = 5) or left uninfected. Cells were harvested in assay buffer (100 mM tris-HCl pH 5.2, 100 mM CaCl 2 , 100 mM MgCl 2 , 0.02% b-mercapto ethanol), cracked by a single freeze/thaw step at 280uC and disrupted by ultrasonic pulsing. Lysates were precleared from cell debris and residual free phosphates according to the manufacturer's instruction. Tyrosine phosphatase activity was measured by enzymatic release of free phosphate of two given pseudosubstrates (phosphorylated peptides representing target sequences for the most common tyrosine phosphatases). Quantification was performed in comparison to a given standard according to the manufacturer's instruction.
For Western blot analysis cells were lysed with RIPA [25 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% Glycerol, 0.1% SDS, 0.5% NaDOC, 1% IgePal, 2 mM EDTA, pH 8.0, pyrophosphate 5 mg ml 21 aprotinin; 5 mg ml 21 leupeptin; 1 mM sodium vanadate and 5 mM benzamidine] on ice for a minimum of 30 minutes. Supernatants were cleared by centrifugation in a standard tabletop centrifuge (Eppendorf) at maximum speed. Protein concentration was determined by Bradford assay.
The phosphorylated and unphosphorylated forms of STAT1 were detected using anti-STAT1 (Y701) antibody and anti-STAT1 (BD Bioscience). An antibody directed against Y690 of STAT2 was used for detection of the phosphorylated form of STAT2 (Upstate). Antibodies to detect influenza viral proteins were purchased from Serotec (NP, M1), Santa Cruz (PB1, PB2). The anti-PA antibody was kindly provided by J. Ortin (Madrid/ Spain). A monoclonal antibody directed against the viral NS1 was generated at the IMV, Muenster, Germany [70] . A monoclonal anti-Myc-tag antibody to detect Myc-M1 was kindly provided by Viktor Wixler. IMV, Muenster, Germany. All secondary antibodies were purchased from Amersham and diluted 1:2500 in TBS-T. Secondary antibodies were incubated for a minimum of 60 minutes at room temperature.
To synthesize cDNA from cells, RNA was isolated using Qiagen RNeasy mini kit according to manufacturer's instruction. In brief, cells were lysed in the presence of b-mercaptoethanol and lysates were loaded to a column, washed and eluted in RNase-free water. For reverse transcription 3 mg total RNA, 0.5 mg oligo dT primer in a total volume of 12 ml were heated for 10 minutes at 70uC. Enzyme mix was prepared (56 Enzyme Buffer (Fermentas), water and 500 mM dNTPs) and pre-warmed at 42uC for 2 minutes before adding 535 U/100 ml RevertAid H 2 M-MuLV (Fermentas).
Reverse transcription was performed at 42uC for 1 hour. The enzyme was inactivated at 70uC for 10 minutes. Samples were stored at 220uC or directly used in quantitative real-time PCR.
For analysis of gene expression relative quantification of the DNA amount was applied. In order to do that gene expression of the housekeeping gene GAPDH was determined. To ascertain changes in expression of the gene of interest the differences between expression of GAPDH and the gene of interest was calculated using the 2 2DDCT method [71] . For quantitative real time Brilliant QPCR SYBR Green Mastermix (Stratagene) was used according to manufacturer's instructions. The fragment of interest was amplified in 40 cycles. The following primers were used (see Table 1 
The pCFG5-EGZ retroviral vector used for transfection [72] as well as the constructs to express dominant negative MKK6 (MKK6Ala) or IKK2 (IKK2KD) have been described earlier [52, 73] . The Phoenix amphotropic retroviral producer cells (a gift from G. Nolan, Stanford, CA) [74] were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. Generation of MKK6Ala or IKKKD expressing producer cells as well as transduction of A549 cells to stably express these transgenes was performed as previously described [52, 53] . Figure S1 Infection of HUVEC results in inhibition of STAT1 phosphorylation and IFNb independent SOCS-3 gene transcription. HUVEC were infected with PR8 (MOI = 5) (A, B, D) or stimulated with 100 U/ml IFNb (E) for time points indicated. To assess the mRNA levels of SOCS-3 (A, E), IFNb (B) and MxA (E) RNA was reverse transcribed and cDNA was subjected to quantitative real time PCR. Equivalent mRNA amounts were normalized to endogenous GAPDH and calculated as n-fold of untreated cells that were arbitrarily set as 1. To assess the amount of phosphorylated STAT1 (B) A549 cells were infected with PR8 (MOI = 5) for time points indicated. Total cells lysate was subjected to Western Blot analysis using anti-phospho-STAT1, anti-STAT1 antibodies. To assess effective viral replication viral NS1 was detected using an anti-NS1 antibody. (C) Quantification of relative band intensities of (B) using AIDA software and 2D densitometry (Fuji). Figure S2 IL1b and TNFa do not affect induction of SOCS-3 gene transcription. A549 wt cells were stimulated with 100 U/ml IL1b (A), 20 ng/ml TNFa (B) or infected with PR8 (MOI = 5) (C) for time points indicated. Cells were lysed, and RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of SOCS-3 and IL6 (A and B) or IL1b (C). Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1.
Found at: doi:10.1371/journal.ppat.1000196.s002 (4.87 MB TIF) Biodefense versus bioterrorism Genomics was essential for identifying the source of the deadly anthrax strain released after the September 11 terrorist attacks in the US. The same research that is needed to combat low-probability bioterror attacks is needed to combat high-probability natural infectious agents.  Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR(−/−) mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease. Adenoviruses (Ads) cause mild disease in humans, but are hazardous pathogens in immuno-compromised individuals [1] . Human Ads are dsDNA viruses grouped into six species. Species A, C, D, E, and F and species B Ads use different infectious entry pathways [2] . Human Ads enter mouse cells and express their early genes; however, the virus genome is not replicated and no viral progeny is made during the infection of mouse cells in vitro or in vivo [3, 4] . Furthermore, early viral gene expression can be abolished by UV-inactivation and well-defined mutants with defects of viral early genes or viral entry are available [3, 5] . Thus, the effects elicited by different components of the virus-host interaction preceding viral replication can be accurately evaluated.
Ads transduce many different cell types and can be produced in vitro in sufficiently high amounts for in vivo administration. While these properties make them attractive for gene therapy applications, they can also trigger a severe systemic toxic reaction [6, 7] . Upregulation of inflammatory mediators, including cytokines and chemokines such as IL-1, IL-6, IL-8, IL-12, macrophage inhibitory protein-1/2, tumor necrosis factor-a (TNF) and recently also type I IFN has been observed in experimental and clinical infections with wt as well as with recombinant Ads [6, 7, 8, 9, 10] .
Type I IFNs represent one of the host's most important antiviral defense mechanisms. The type I IFN family comprises different IFN-a subtypes, a single IFN-b and other less well characterized proteins [11] . All IFN-a species and IFN-b interact with the same IFN-ab cellular receptor, the activation of which mediates a wide range of direct and indirect innate antiviral or antimicrobial effects and modulates the antiviral adaptive immune response [12, 13, 14] .
At present, two main mechanisms of type I IFN induction by viruses resulting from the extracytoplasmic or cytoplasmic virus recognition, respectively, are known [12, 13, 14, 15] . The extracytoplasmic induction is initiated by triggering the surfaceexpressed transmembrane protein toll-like receptor (TLR) 4 with certain non-nucleic viral constituents [16, 17, 18] or upon recognition of viral nucleic acids in the endosomes of specialized cells (dendritic cells and macrophages) via different members of the TLR family. These include TLR3, TLR7/TLR8 and TLR9, sensing dsRNA, ssRNA and CpG DNA, respectively. For IFN-ab induction TLR3 and TLR4 signal through the adaptor molecule TIR domain-containing adaptor protein inducing interferon b (TRIF). This results in the activation of interferon regulatory factor (IRF)-3. TLR7, 8 and 9 signal through the adaptor molecule Myeloid differentiation factor 88 (MyD88). An important result of the MyD88-mediated pathway is the activation of IRF-7 (but not of IRF-3), which together with the transcription factors NF-kB and AP-1 initiates the induction of both the IFN-a and IFN-b genes [13, 15] . This induction pathway is responsible for the strong, early IFN-ab response to several replicating and inactivated viruses in pDCs, which express preferentially TLR7 and TLR9 [19, 20, 21] .
The ''classical'' cytosolic pathway is the major IFN-ab producing mechanism in cells other than pDCs [22, 23] . Signal transduction leading to type I IFN gene induction is initiated by the recognition of intracellular virus-associated molecular patterns. dsRNA and 59-triphosphate RNA produced during viral replication are sensed by the RNA helicases RIG-I and MDA-5 [23, 24, 25, 26, 27, 28] . This pathway has been extensively studied, mainly in virus-infected fibroblasts. Triggering of the aforementioned RNA helicases leads to the activation of the transcription factors NF-kB, IRF-3 and IRF-7 that are important for the induction of IFN-ab and proinflammatory cytokines, including IL-6. In the cytosolic pathway, type I IFN gene induction is a sequential event and both IRF-3 and IRF-7 were shown to be important in the early phase when mostly IFN-b is produced. The late phase of the IFN-ab response is regulated by positive feedback via the increased levels of IRF-7 elicited by IFN-b production during the early phase [29, 30, 31] . In addition to fibroblasts, the potential of mDCs and macrophages to produce significant amounts of type I IFNs in response to viral replication has been demonstrated in vitro [32, 33] ; however, in vivo the specific contribution of these cells to systemic levels of IFN-ab is not well documented.
Recently, detection of bacterial DNA in cells infected with L. monocytogenes and recognition of transfected B-DNA has been shown to trigger IFN-b production. This type of response strictly requires IRF-3 [15, 34, 35, 36, 37, 38] . Such a sensing system has been suggested to represent a further mechanism of cytosolic DNA virus recognition [14, 15] and the Z-DNA binding protein 1 (Zbp1, also referred as DNA-dependent activator of IFN regulatory factors, DAI) was shown to be a candidate DNA sensor in this pathway [39] . Notably however, in a follow-up study the same group found a critical role for DAI in L-929 cells but not in mouse embryonic fibroblasts (MEFs) [40] . Furthermore, recent experiments with Zbp1/DAI knockout mice did not show the essential role of Zbp1/DAI in the induction of innate and adaptive responses to B-DNA in vivo and in macrophages, dendritic cells and MEFs in vitro [41] .
The induction of type I IFN in Ad-infected mice has been recently studied [10, 42] and associated with both the extracytoplasmic and intracytoplasmic pathways. It was claimed that a part of the IFN-ab response is initiated by TLR9 and MyD88 signaling in pDCs and another part by cytosolic DNA recognition in non-pDCs. However, the identification of IFN-ab producing cell types directly in infected mice was not carried out. In the present study we investigated the IFN-ab responses of Ad-infected mice and showed that the bulk of the in vivo induced IFN-ab is produced by splenic mDCs. Furthermore, we found that TLRs, including TLR9 play no major role. The Ad-elicited IFN-ab response required viral endosomal escape, suggesting a cytosolic induction pathway. Surprisingly however, the induction was independent of IRF-3 and dependent on stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity, which is in contrast to the known induction mechanism initiated through cytosolic DNA recognition. Instead, the induction required IRF-7, and a positive feedback regulation via the type I IFN receptor. Although this does not exclude a role for cytosolic nucleic acid sensors, our data do not support the involvement of MDA-5, RIG-I and Zbp1/DAI in the induction of the IFN-ab response to Ad. Furthermore, our results reveal distinct mechanisms in the induction of IFN-ab and IL-6 by Ad. Finally, we show that Ad-induced IFN-ab is a key mediator of hypersensitivity to bacterial lipopolysaccharides in infected mice. Enhanced susceptibility to LPS and to other microbial inducers of inflammation may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases.
In order to characterize the induction of type I IFNs by Ad in vivo, we first examined how two types of human Ad, Ad3 (species B) and Ad R700 (species C) [2] known to use distinct infectious entry routes, elicit an IFN-ab response in vivo. The results summarized in Fig. 1A and supplementary Fig. S1A show that all mice infected with either of the two viruses exhibited similar IFN-ab responses. IFN-ab was first detectable in plasma at 4 h, peaking at 8 h and declining to low levels 18 h after infection. We then investigated, whether the expression of viral genes is required and/or regulate the induction of IFN-ab by Ads. To this end, we injected mice with an UV-inactivated Ad3 (Fig. S1B ) or Ad R700, incapable of viral gene expression, or with a recombinant Ad5 (species C) that contains a deletion of the E1 and E3 Ad early regions and expresses GFP (Ad5-GFP) (Fig. S1C ). As shown in Fig. 1A , UV-inactivated Ads also induced a strong IFN-ab response which, however, peaked at 6 h after injection, i.e. 2 h earlier than the response to intact Ads. A similar early-peaking IFN-ab response was obtained in mice injected with UV inactivated Ad R700 (Fig. S2A ) and with the recombinant Ad5-GFP (Fig. S2B ).
Adenoviruses (Ads) are important pathogens and promising vectors for gene therapy applications. In the course of adenoviral infections innate immune responses are activated, which can be beneficial for the antiviral host defense but also detrimental if activated in a deregulated manner. Type I IFNs are crucial for the innate immune control of various viral infections in the mammalian host. So far, the early, systemic release of IFN-ab during viral infections has been attributed to specialized immune cells, the plasmacytoid dendritic cells. Here, in a mouse infection model, we show that wild type Ads, as well as adenoviral vectors, elicit rapid IFN-ab production almost exclusively in another cell population, the splenic myeloid dendritic cells. This IFN-ab storm depends on viral escape from endosomes to the cytosol and the requirements of the response are suggestive of a novel viral induction pathway. Furthermore, we show that virus induced IFN-ab is the key mediator of Ad-induced hypersensitivity to the cytokine-inducing and toxic activity of lipopolysaccharide, a common constituent of Gram-negative bacteria. Since these bacteria comprise several commensals and pathogens, enhanced susceptibility to lipopolysaccharide may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases. Titration of the viral preparations in mice revealed that a positive correlation between the viral dose and the height of the IFN-ab response existed only when relatively small doses of intact Ads were used. Higher doses of the Ads either did not elicit a further increase of the IFN-ab response (Ad3, Fig. 1B ) or led to a decrease of the response (Ad R700, Fig. S2C ). In contrast, injection of the corresponding UV-inactivated Ads always led to a gradual increase of the IFN-ab response and, at higher viral concentrations, even exceeded the response obtained with intact viruses. Thus, the expression of Ad genes is not required for the induction of IFN-ab in mice. The data also indicate that the expression of early adenoviral genes negatively regulates type I IFN production. Notably, the Ad E1A gene has been shown previously to suppress Newcastle Disease Virus and IRF-3 induced IFN-a4 promoter induction in transient expression assays in fibroblasts [43] .
Interestingly, however, viral gene expression did not inhibit the production of IL-6 in either Ad3-or Ad R700-infected mice ( Fig. 1C and Fig. S2D ). Further experiments revealed that, in contrast to the UV-inactivated Ad, heat-inactivated Ad did not elicit IFN-ab in mice (data not shown). Since heat inactivation prevents the entry of Ad into cells ( [44] and Fig. S2E ), we conclude that signal transduction leading to IFN-ab production is activated during Ad entry.
Previous studies have shown that, depending on the inducing virus, IFN-ab is produced ubiquitously or in a cell type specific manner [13, 14] . Here we stimulated different primary mouse cells, including bone marrow derived mDCs (BMDC), bone marrow derived macrophages (BMM), bone marrow derived pDCs and mouse embryonic fibroblasts (MEFs), with Ad2 (species C) or Ad3 (species B). Six hours later, the IFN-ab content of culture supernatants and the expression of the viral E1A gene in the cells were determined. BMDC and BMM, but not MEFs, produced IFN-ab (Fig. 1D ), although all cell types were successfully infected as shown by RT-PCR ( Fig. 1E and not shown). Very similar results were obtained when, instead of Ad2, Ad3 or the recombinant Ad5-GFP were used for infection of the three cell types (not shown). In addition, pDCs also produced IFN-ab in response to Ad infection in vitro (Fig. S3A) ; however, only at high multiplicities of infection. Finally, like MEFs, L-929 cells infected with Ad2 produced no IFNab either (Fig. S3B ). In agreement with previous studies [36, 37] , MEFs and L-929 cells produced IFN-ab following transfection with purified DNA (Fig. 1D and Fig. S10C ). In addition to mouse DCs and macrophages we also found that human monocyte-derived DCs produced IFN-ab upon infection with the adenoviral vector Ad5-GFP (Fig. S3C) . The present data confirm that Ads can trigger IFN-ab production in various immune cells in vitro [10, 45, 46] . However, it furthermore indicates that production does not proceed ubiquitously in all types of infected cells.
In order to identify the organ site of viral uptake and IFN-ab production in vivo, we analyzed the expression of the early viral gene E1A and of type I IFN mRNAs, respectively, in the spleen, liver, lung and kidney of Ad3-infected mice. We found expression of the early viral E1A gene in spleen and liver (Fig. S1B ), but not in lung and kidney (not shown), which agrees with earlier findings on in vivo Ad tropism [47, 48] . Expression of IFN-a and IFN-b mRNA was below the level of detection in the organs of non-infected controls. We also found that between 4 and 18 h after infection IFN-a and IFN-b mRNAs were expressed at high levels in the spleen (Fig. 1F ), but surprisingly not in the liver, despite the presence of Ad in both organs. As expected, IFN-ab was not expressed in the virus-free lung or kidney of infected mice (Fig. 1F) .
In order to identify the cell type(s) producing IFN-ab in vivo, we isolated splenocytes from Ad infected animals 8 h after virus treatment and separated them into CD11c + (DC-containing) and CD11c 2 (non-DC) populations. Both CD11c + and CD11c 2 populations contained viral DNA (not shown). This finding is in accordance with the report of Morelli et al describing that both splenic DC and non-DC contain the virus in Ad-infected mice [49] . However, quantitative RT-PCR determination of IFN-a and IFN-b mRNA in both populations revealed the presence of IFNab mRNAs predominantly in the DC-containing CD11c + fraction ( Fig. 2A, B ), but not in the macrophage containing CD11c 2 fraction. In contrast to IFN-a and IFN-b, the mRNA levels of IL-6, another cytokine known to be induced by Ad, were comparably upregulated in both the CD11c + and CD11c 2 population (Fig. 2C ). Since the latter non-DC population comprises more than 95% of all mouse splenocytes [50] , we conclude that most of the IL-6 made in the Ad-infected spleen is of non-DC origin.
According to published data, the CD11c + cell population contains different types of DCs, as well as other cells such as lymphocytes, macrophages, granulocytes and NK cells [51, 52, 53] . We therefore further separated the purified CD11c + cells into mDCs (CD11c + CD11b + Gr1 2 ), pDCs (CD11c + CD11b 2 GR1 + B220 + ) and a CD11c + CD11b-F4/80 + subpopulation ( Fig. S4A -D) and measured the expression of IFN-a, IFN-b and b-actin with real-time RT-PCR. After normalization to b-actin expression, we found that on a per cell basis mDCs expressed significantly more IFN-b than pDCs (Fig. 2D ), but both mDCs and pDCs expressed similar amounts of IFN-a (Fig. 2E ). In contrast, CD11c + , CD11b 2 cells carrying the macrophage marker F4/80 + did not express detectable amounts of IFN-a or IFN-b. Since mDCs comprise approximately 60% of all analyzed CD11c + splenocytes and their numbers are approximately 10-times higher than those of pDCs (Fig. S5A , B and [50] ), these results suggested that the vast majority of IFN-ab in Ad-infected mice was produced by splenic mDCs. To verify this assumption, we analyzed the Ad-elicited cytokine responses in mice depleted of CD11c high MHC II + myeloid DCs. To ablate these cells, we injected diphtheria toxin into the CD11c-diphtheria toxin receptor CD11cDTR/GFP transgenic mice [54] 24 h prior to infection with Ad. In agreement with previous reports [55, 56] , DT pre-treatment of DTR/GFP transgenic mice resulted in the ablation of CD11c high MHC II + CD11b + splenic mDCs, whereas the CD11c int Siglec H + CD11b 2 plasmacytoid DCs remained unaffected (Fig. S5A, B) . When DT pre-treated CD11cDTR/GFP transgenic mice were challenged with Ad3 and examined for IFN-ab in plasma 4 and 8 h after infection, only marginal IFN responses were found at both timepoints, in contrast to the strong responses of similarly infected transgenic control mice that had not received DT (Fig. 3A) . The same pretreatment with DT had no effect on the Ad elicited IFN-ab response of wild-type mice (Fig. 3A) .
Interestingly, the determination of IL-6 levels in plasma 8 h after infection revealed that the ablation of mDCs in CD11cDTR/GFP transgenic mice affected only moderately the induction of IL-6 ( Fig. 3B) , confirming that non-DCs contribute significantly to the Ad-elicited IL-6 response in vivo. The fact that different cell types are responsible for IFN-ab and IL-6 response to Ad may explain at least in part why viral gene expression did not inhibit the production of IL-6 in Ad3-or Ad R700-infected mice (see Fig. 1C and Fig. S2D ). In order to functionally evaluate the possible participation of pDCs to the systemic production of IFNab to Ad we also checked this response in mice depleted of pDCs. As shown in Fig. S6A and B, the injection of anti PDCA-1 antibody led to the substantial decrease of the number of splenic pDCs. Nevertheless, the production of IFN-ab was not changed in response to Ad infection (Fig. 3C) . The data collectively show that the vast majority of IFN-ab but not of IL-6 in Ad infected-mice is produced by splenic mDCs.
Recent studies have shown the involvement of different TLRs including TLR 2, 3, 4, 7 and 9 in the innate recognition of different viruses [16, 17, 18, 57, 58, 59, 60, 61] . Signaling via TLR9 was shown to be responsible for the strong type I IFN response of pDCs to Ad in vitro [10, 45, 46 ]. Here we investigated the possible involvement of TLR9 in the induction of type I IFNs by measuring IFN-ab in Ad infected wt and TLR9 2/2 mice. Fig. 4A shows that TLR9 2/2 mice produced normal levels of IFN-ab. upon infection with Ad3. Comparable results were obtained with the recombinant Ad5-GFP (Fig. S7A) . Moreover, Unc93B mice, deficient in signalling by intracellular TLRs showed normal IFN-ab responses to Ad5-GFP (Fig. S7A ). We further checked the possible involvement of the TLR system using mice deficient for TLR2, TLR4 or for the TLR adaptor proteins TRIF and MyD88. Fig. 4A shows that the Ad3 induced IFN-ab levels in all strains of mice were as high as in the respective wt controls. Similarly, comparable IFN-ab responses were also found in TLR-, MyD88-or TRIFdeficient mice and in the corresponding wt animals after infection with UV-inactivated Ad3, AdR700 and Ad5-GFP (not shown). Furthermore, comparable IFN-ab responses were found in cultures of Ad-infected BMDCs from wt, MyD88-and TRIFdeficient mice (Fig. 4B ). The various TLR deficient mice showed impaired responses to the corresponding ligands in control experiments (Fig. S7B ). Collectively, our data show that the TLR system plays no major role in the systemic IFN-ab responses to Ads in mice.
The type of virus, the IFN-ab-producing target cell, and the activation mechanism determines whether positive feedback signaling is involved in the induction of the IFN-ab response or not [13, 20] . Here, we studied the possible involvement of IFN feedback signaling in the IFN-ab and IL-6 response to Ad3 by using wt and IFN-abR-deficient mice. The absence of the IFN-ab receptor resulted in dramatically decreased levels of IFN-a and IFN-b protein in the plasma as well as of IFN-a and IFN-b mRNAs in the spleen (Fig. 5A , B) 4 and 8 h after Ad infection. The difference between the protein or mRNA levels of IFN-a and IFN-b in wt versus mutant animals was approximately 100 and 20fold, respectively. Furthermore, in contrast to wt mice, the characteristic rise in IFN-ab levels between 4 and 8 h after infection was absent in IFN-abR 2/2 mice. Thus, in Ad-infected mice, production of both IFN-a and IFN-b is strictly dependent on positive IFN-ab feedback. The determination of IL-6 protein and mRNA levels in the same plasma and splenic tissue samples revealed that the induction of IL-6 is also positively regulated by IFN-ab signaling (Fig. 5C ), which is in agreement with a previous study [10] .
We also tested whether IFN-abR-dependent signaling is involved in the IFN-ab response of Ad-infected BMDCs in vitro. As with the in vivo results, we found that cells from IFN-abR 2/2 mice infected with Ad3 produced significantly less type I IFN than similarly infected cells from wt mice (Fig. S8 ). The loss of IFN-ab signaling also resulted in a strong inhibition of the Ad induced IL-6 production in BMDCs (Fig. 5D ) and BMMs (Fig. 5E ). Furthermore, as shown using Ad-infected BMMs, it resulted also in a strong reduction of inducibility of IL-6 mRNA expression (Fig. 5F ). Because transcriptional changes are often determined by epigenetic factors [62] we checked the levels of hyperacetylated histone H4 (acH4), a permissive factor for transcription at the IL-6 promoter in control and Ad-infected BMMs from wt and IFNabR 2/2 mice. Chromatin immunoprecipitation (ChIP) assays showed a significant enrichment of acH4 at the IL-6 promoter in infected wt, but not IFNabR 2/2 BMMs (Fig. 5G) . In a control experiment, as expected, an enrichment of acH4 was observed at the promoter of the constitutively active Topoisomerase 3b but not of the l5 (not expressing, active only in early B-cells) gene in both cells types. From these results we conclude that IFN-ab exerts a positive regulatory effect on the Ad-induced IL-6 transcription and that its loss is at least partly responsible for the strong reduction of the IL-6 response in Ad-infected IFN-abR knockout mice.
Using real-time RT-PCR we then analyzed the spectrum of IFN-ab genes in wt mice as well as the impact of IFNabR deficiency on their induction. Included were IFN-a2, 4, 5, 6, 9, 11, 12, 13, 14 and IFN-b. All of them were induced in the spleen by Ad3 in vivo and in BMDC in vitro. IFN-ab subtypes were not detectable in the spleen of unstimulated mice or BMDCs (not shown). Fig. 6 shows the patterns of IFN-ab genes induced in vivo and in vitro in the presence or in the absence of IFN-ab feedback signaling. In wt mice and cells IFN-a5 and IFN-b were the most strongly expressed genes and IFN-a13 was the least activated IFNa subtype. IFN-a11 was not induced at all. IFNabR deficiency resulted in an inhibition of IFN-ab gene expression, the strength of which was subtype dependent. In some cases the inhibition was weak (IFN-a2, 4, 5 and IFN-b), in others strong or complete (IFN-a12, 13, and 14) , showing that the expression of different subtypes of IFN-ab are differentially affected by IFN-ab feedback signaling. Collectively, these data show that the adenovirus triggered production of IFN-ab and IL-6 in BMDCs in vitro and in mice in vivo is strongly dependent on intact IFN-ab signaling.
The critical role of IRF-7 but not IRF-3 in Ad-induced type I IFN production
The transcription factors IRF-3 and IRF-7 have distinct and important roles in IFN-ab production induced by viruses or other pathogens and their involvement can be characteristic for the induction mechanisms involved [13] . Specifically, IRF-3 has been shown to be critically involved in cytoplasmic DNA sensing and in the Ad-induced IFN-ab production in BMMs in vitro [63] . We show here that IRF-3 is critical for the induction of IFN-ab by isolated adenoviral DNA, but not by infection with whole virions in BMDCs (Fig. 7A ). In order to analyze the individual contribution of IRF-3 and IRF-7 to the Ad-induced IFN-ab response in vivo, we infected mice deficient for these transcription factors with Ad3. We also compared these responses to those triggered by poly I:C, a known activator of the cytosolic IFN-ab producing pathway in vivo. As shown in Fig. 7B , the lack of IRF-3 did not significantly influence the plasma levels of IFNab 4 or 8 h after infection in response to Ad. In contrast, Ad-infected IRF-7deficient mice did not exhibit detectable amounts of IFN-ab in the plasma. Very similar data were obtained with Ad5GFP (Fig. S9) . Thus, IRF-7, but not IRF-3, is essential for the induction of the IFN-ab response during Ad infection. Compared to wt mice, poly I:C injected IRF-7 deficient mice produced significantly less, but still well detectable amounts of IFN-ab.
The nucleic acid sensors MDA-5, RIG-I and DAI/Zbp1 are not critical for Ad-induced type I IFN production Next, we investigated whether the cytoplasmic RNA sensors MDA-5 or RIG-I, or the putative DNA sensor DAI/Zbp1 may play a major role in the induction of the IFN-ab response to Ad. The possible involvement of MDA-5 was tested using BMDCs and BMMs from MDA-5 deficient mice. Normal IFN-ab responses to Ad were obtained in MDA-5-deficient BMDCs cells (Fig. 8A ) and also in BMMs (not shown), whereas the responses to the known MDA-5 ligand Poly I:C were abrogated (Fig. 8A) . The possible involvement of RIG-I was checked using a dominant negative form of RIG-I (RIG-IC) [64] . BMDCs from IRF-3 2/2 mice were transfected with a GFP-expressing plasmid (transfection control) with or without RIG-IC and subsequently stimulated with Ad or control leader RNA. We used IRF-3 2/2 cells to avoid any induction of IFN-ab by the plasmid itself, which is, contrary to that by Ad, strictly IRF-3 dependent. The induction of IFN-b mRNA was analyzed in sorted GFP-positive cells. As shown in Fig. 8B , left, the transfection of RIG-IC prevented induction of IFN-b mRNA by the leader RNA, but not by Ad. The role of Zbp1/DAI was analyzed using siRNA-mediated knockdown of DAI/Zbp1 in BMDCs. For this purpose, cells from IRF-32/2 mice were co-transfected with DAI/Zbp1 targeting siRNAs and a GFP expressing plasmid and subsequently stimulated with Ad. As shown in Fig. 8B , right, the transfection of BMDCs resulted in strongly reduced DAI/Zbp1 expression but not in a reduced IFNb mRNA induction by Ad in GFP-positive cells. Control experiments with L-929 cells showed that transfection of the gene-specific siRNA downregulated the expression of DAI/Zbp1 on both the mRNA and protein levels and efficiently inhibited the IFN-b response to B-DNA ( Fig. S10A-C) . Collectively, our results indicate that known nucleic acid sensors such as MDA-5, RIG-I and Zbp1/DAI are not involved in the Ad-induced type I IFN production.
MAPKs have been previously shown to be activated by Ad in vitro, in different non-immune cell types and to be important for the induction of chemokines in response to Ad [65, 66, 67, 68] . Here we investigated whether members of the MAPK family play a role in the Ad-induced IFN-ab and IL-6 response. We infected BMDCs with Ad3 in the presence or absence of MAPK inhibitors and determined the levels of IFN-ab and IL-6 produced. Fig. 9A and Fig. S11 show that the pharmacological blockade of the SAPK/JNK MAPK almost completely inhibited the Ad3-induced production of both IFN-ab and IL-6. In contrast, the inhibition of the p38 MAPK pathway partially inhibited the production of IL-6, but had no effect on the production of IFN-ab. Finally, the blockade of ERK1/2 had no effect on the production of either IL-6 or IFN-ab. Very similar data on the effects of MAPK inhibitors were obtained using Ad2 and mutant Ad5-GFP to stimulate BMDC (data not shown).
Next, we analyzed the levels of activated SAPK/JNK MAPK proteins in BMDCs and found their robust phosphorylation 2 h after either Ad3 or Ad5-GFP infection (Fig. 9B) . We also tested the importance of SAPK/JNK signaling on Ad-induced IFN-ab production in vivo. Fig. 9C shows that the blockade of the SAPK/ JNK signaling pathway in mice completely inhibited the production of IFN-ab at 4 and partially at 8 h after Ad infection. Taken together, these data strongly indicate that the Ad-activated SAPK/JNK MAPK pathway plays an important role in the virusinduced production of type I IFNs and IL-6.
Endosomal escape triggers the Ad-induced production of IFN-ab and IL-6, but prevents TLR9-dependent innate recognition During the course of our adenovirus preparations, we regularly found ''empty capsids'' which we separated and purified in addition to the mature virions. We tested the IFN-ab stimulating activity of these preparations in vivo and observed that they were not active (Fig. S12A ). Since empty capsids lack viral DNA and exhibit an altered protein composition [69] , the absence of an inducing viral constituent(s) from these capsids could explain their inability to provoke an IFN-ab response. Another possible explanation could be that endosomal escape is required for IFNab induction, since empty capsids cannot escape from the endosome [69] . To test the latter possibility, we infected mice with 3.6610 10 viral particles of wt Ad2 and Ad2Ts1, a viral mutant deficient in endosomal escape [5] . As shown in Fig. 10A , in contrast to the wt virus, Ad2Ts1 did not induce detectable levels of type I IFN at 4 and 8 h after infection. Furthermore, the IL-6 response was also severely reduced in these animals (Fig. S12B) . In a control experiment, mice infected with Ad2Ts1 grown at permissive temperature (32uC) and thus capable of endosomal escape, exhibited normal IFN-ab responses (Fig. 10B) . The inability of Ad2Ts1 to escape from endosomes of mDCs was confirmed by electron microscopy (Fig. 10E-H) and in vivo by the lack of Ad early E1A gene expression in the spleen of mice infected with Ad2Ts1 (Fig. 10I) . Thus, escape from the endosome is critical for the induction of IFN-ab and IL-6 by adenoviruses.
It should be noted that a further increase in the Ad2Ts1 dose (2.16610 11 particles) resulted in detectable, albeit very low levels of plasma IFN-ab that were released with different kinetics (Fig. 10C) . In this case, IFN-ab was detectable as early as 2 h after infection and, in contrast to the results we obtained with wt viruses ( Figures 1A, 10A and S1A), the levels of IFN-ab did not increase significantly at the later time-points. This already suggests a mechanism for type I IFN induction by Ad2Ts1 that is fundamentally different from the IFN-ab induction seen with wt Ad2. Since Ads are DNA viruses, they can possibly be detected by TLR9. In fact, the innate immune recognition of Ad in pDCs is TLR9 dependent. We therefore repeated the experiment with a high dose of Ad2Ts1 using TLR9 2/2 mice. As shown in Fig. 10C , TLR9 2/2 mice did not produce IFN-ab in response to the mutant Ad2Ts1, quite in contrast to the results obtained with the wt virus (see Fig. 4A ) Likewise, there was no detectable IFN-ab release in Ad2Ts1-infected mice deficient in MyD88, an essential component of TLR9 signaling (not shown). Furthermore, the corresponding amount of empty particles (DNA-free) of Ad2Ts1 elicited no IFN-ab response in wt mice (Fig. 10C) . These data illustrate the critical role of TLR9 in the induction of IFN-ab by means of high doses of Ad2Ts1. To exclude the possibility that contaminating DNA on the surface of the virions was responsible for the TLR9dependent IFN-ab induction, we treated the mutant virions with bensonase, which destroys all kinds of free nucleic acids. Bensonase-treated Ad2Ts1 still induced an IFN-ab response in We also tested the role of endosomal escape of Ad in the in vitro induction in BMDCs of IFN-ab and IL-6. Fig. 10D shows a dose dependent production of IFN-ab by Ad2Ts1 grown at permissive temperature and Figures S12C and D the production of IFN-ab and IL-6 by wt Ad2, but no production of either of the cytokines by Ad2Ts1 grown at the restrictive temperature. Interestingly, liposomal transfection of whole Ad2 Ts1 virions (but not of empty virus particles) in BMDCs resulted in the significant production of IFN-ab that however, was critically dependent on IRF-3 (Fig. 7A) . Similarly, Ad2 Ts1 did not induce IFN-ab production in human monocyte derived DCs (Fig. S12E) . The requirement of low pH for Ad3 infection was also tested using bafilomycin A1, a drug known to inhibit the acidification of endosomes. Experiments shown in Fig. S13A , B revealed that cells treated with this drug produced significantly reduced levels of IFN-ab and IL-6 respectively, in response to Ad3. This is consistent with the notion that Ad3 and Ad7 infection of cultured cells requires low endosomal pH [70, 71] . Similar results were obtained using treatment with ammonium chloride, another acidification inhibitor known to block Ad escape from endosomes (not shown). Collectively, the data in vitro and in vivo provide evidence that the late phase of the Ad infectious entry, in which the virus escapes from the endosome, triggers an innate response characterized by the production of type I IFNs and IL-6.
Induction of type I IFNs was shown to be critical for some innate immune responses to Ads [10] .We therefore investigated whether a characteristic consequence of infection with Ad, the induction of LPS hypersensitivity [72] , might be mediated by type I IFNs. For this purpose, we infected wt and IFNabR 2/2 mice with Ads, challenged them 16 h later with LPS and measured the TNF-a response. Non-infected LPS-treated mice served as a control. Unlike the infected wt mice, the Ad3-infected IFNabR 2/2 mice did not exhibit enhanced responses to LPS (Fig. 11A) . Interestingly, LPS hypersensitivity developed also in mice injected with very small amounts of Ad. Such amounts were capable of the elicitation of IFN-b mRNA in the spleen of infected animals, but incapable of inducing detectable circulating IFN-ab ( Fig. S14A and  B) . Similar results were obtained in Ad5-GFP-infected mice (not shown). Furthermore, in IRF-7 2/2 mice that exhibit a severe impairment of the IFN-ab response to Ad a severe impairment of LPS sensitization by Ad5-GFP was also observed. (Fig. 11B) . In contrast, sensitization to LPS developed normally in Ad5-GFPinfected TLR9 2/2 mice (Fig. 11B) , which is in agreement with our finding that TLR9 is not critical for the induction of IFN-ab by Ad. Furthermore, only Ad5-GFP-infected wt, but not IFNabR 2/2 mice exhibited enhanced susceptibility to LPS shock (Fig. 11C) . Notably, also Ad vector-infected or IFN-a treated human monocyte-derived mDCs exhibited enhanced sensitivity to LPS and overproduced TNF-a upon LPS challenge (Fig. S14C) . On the whole, our results indicate that IFN-ab is an essential mediator of the LPS hypersensitivity induced by adenovirus infection.
An increased expression of the LPS receptor complex on target cells, may contribute to the enhanced reactivity to LPS [73, 74] . We therefore investigated whether macrophages of Ad infected mice overexpress the receptor components mCD14 and TLR4/ MD-2. We found that splenic macrophages from Ad infected mice overexpress mCD14 in an IFN-ab dependent manner (Fig. 11D ) but the expression of TLR4/MD-2 was only minimally affected (Fig. S14D) . Furthermore, we found increased acetylation levels of histone H4 at the TNF-a promoter in Ad-infected wt, but not in IFNabR-deficient macrophages (Fig. 11E ). This suggests that Adinduced IFN-ab increases LPS-induced TNF-a production, at least in part by epigenetic changes at the TNF-a promoter.
In the present study we investigated the IFN-ab response of mice infected with human Ads. We showed that the response is characterized by high levels of IFN-a and IFN-b, which are produced simultaneously and almost exclusively by splenic mDCs. Furthermore, the response is entirely independent of viral replication, TLR-, MyD88-, TRIF-and IRF-3-signaling, but dependent on viral escape from the endosomes, activation of the SAPK/JNK pathway and IRF-7, and subsequent IFN-ab feedback signaling. These data suggest an IFN-ab induction pathway that is different from the extracytoplasmic and cytoplasmic pathways so far described. Finally, we show that IFN-ab is a key mediator of the hypersensitivity to bacterial lipopolysaccharide, which develops, in Ad-infected mice.
As shown previously and in this study, a wide spectrum of cells including pDCs, mDCs and macrophages [10, 45, 46, 63] produce IFN-ab in response to Ad in vitro. The present finding that in vivo, in Ad-infected mice, splenic mDCs are the major source of IFN-ab is surprising, since in mice infected with different viruses (MCMV, HSV, VSV, MHV, influenza, vaccinia and Sendai viruses) [27, 57, 58, 59, 75, 76, 77, 78] pDCs activated by the TLRs constitute the major contributors to the systemic levels of type I IFNs. In the present study the expression levels of IFN-ab mRNAs in organs and cells from Ad-infected mice suggested a dominant role for splenic mDCs in the IFN-ab response. Furthermore, the IFN response to Ad was practically absent in mice depleted of CD11c high MHC II + myeloid DCs. Also, there was a striking similarity between the IFN-ab subtypes induced by Ads in the spleen of infected mice and those induced in mDC cultures in vitro. It should be emphasized that in mice infected with VSV, in which splenic pDCs are the main IFN producers, the spectrum of in vivo induced IFN-a subtypes was markedly different [76] . Finally, the finding that the response of Ad-infected mice was completely independent of TLR signaling and strongly dependent on IFN-ab feedback provide further arguments against a major role for pDCs. In a number of viral infection models, IFN-a and -b production by pDCs was mediated by TLR7/9 [13, 19, 23] and was at least partially independent of a positive IFN-ab feedback [76, 77, 79, 80] .
In variance to our data, pDCs and various types of non-pDCs [10, 42] were suggested to be responsible for the type I IFN responses to adenoviral vectors in mice. In [10] the loss of TLR9 signaling resulted in a reduction of the IFN-a response in mice infected with the recombinant species C Ad-lacZ [10] . This finding suggested a significant contribution of TLR9, and therefore of pDCs, to the Ad-induced IFN-ab response in vivo. The ratio of pDCs to mDCs (approximately 1:1) in the spleen of animals used in the above study was quite different from that we and others [50] have found (approximately 1:10). This may explain the conflicting results on the role of TLR9 in vivo, between this previous study and ours. In another study [42] the induction of IFN-ab was studied in mice with an artificially enlarged pool of DCs (due to prior pretreatment with sFLT-3L), 24 h after recombinant Ad administration. In our study naïve mice were used and 24 h after Ad infection the levels of IFN-ab were already below the detection limit. Of note also, the bone marrow stromal antigen 2 (BST2) (that is recognized by the PDCA-1 antibody used for isolation of pDCs in [42] ), was shown to be up-regulated on numerous cell types following stimulation that triggers an IFN response [81] . Thus, the use of the PDCA-1 antibody for the isolation of pDCs seems to be more reliable in the case of uninfected mice [81] .
Recently, an absolute IRF-3 dependency of the in vitro IFN-ab response of bone marrow derived macrophages to Ad has been reported [63] . In our study IRF-3 deficiency had no significant effect on the levels of IFN-ab induced in Ad-infected mice. In addition, in Ad-infected macrophages, the relatively low level of IFN-a4 mRNA and its negative regulation by the autocrine Figure 11 . Adenovirus triggered IFN-ab production is the mediator of LPS hypersensitivity. Wild-type and IFN-abR 2/2 mice (4-6 mice/ group) were infected i. p. with 1610 10 Ad3 particles or left uninfected. 16 h later the animals received 1 mg LPS i. p. or diluent only. TNF-a was determined in plasma 2 h after challenge (A). One representative experiment of three is shown. n.d.: not detectable. IRF-7 2/2 and TLR9 2/2 mice were infected i. p. with 2610 9 Ad5 GFP particles and injected 16 h later with 1 mg LPS i. p. TNF-a was determined in plasma 2 h later (B). IFN-ab signaling is required for Ad infection augmented LPS lethality. Wt and IFNabR 2/2 mice were infected with 1610 10 Ad5 GFP particles i. p. or left uninfected. 16 h later mice were injected with 3.5 mg LPS/gr. bw. The number of dead/total animals are shown at the indicated time after LPS challenge (C). Ad infection increases mCD14 expression on splenic macrophages in an IFN-ab signaling dependent manner. Wt and IFN-abR 2/2 mice were infected with 1610 10 Ad5 GFP particles i. p. or left uninfected. Expression of mCD14 was measured on the surface of F4/80 + splenic macrophages by FACS 16 h after infection (D). Ad infection increases acetylation of histone H4 in wt macrophages at the TNF promoter. Wt and IFNabR 2/2 BMMs were mock-infected or infected with 5400 Ad5 GFP particles/cell and levels of acetylation of histone H4 were measured at the TNF-a promoter with ChIP assays (E). Representative experiments of two are shown. doi:10.1371/journal.ppat.1000208.g011 feedback was different from our in vivo findings (strong induction and positive regulation by feedback). This is in agreement with the concept that in vivo macrophages make no significant contribution to the IFN-ab response to Ad. A likely explanation is that the induction of IFN-ab in macrophages requires the high numbers of virions used in vitro, and that such multiplicities were never reached in vivo. Interestingly however, in contrast to IFN-ab induction, the induction of IL-6 proceeded in the spleen of infected mice in both DC and non-DC populations. In accordance, the depletion of IFN-ab-producing mDCs in mice prior to infection lowered, but did not entirely prevent the IL-6 response. It is possible that the pathways leading to the induction of IFN-ab and IL-6 by Ad are different, at least in different cell types. Alternatively, in vivo, part of the IL-6 formed is induced indirectly via secondary mechanisms. In this context it is interesting that blockade of the p38 MAPK in mDCs in vitro had no effect on Ad-induced IFN-ab production, but partially inhibited the production of IL-6. Our data showing that IFNabR 2/2 mice exhibit strongly impaired IL-6 responses to Ad is in agreement with a previous report [10] and shows that IFN-ab is a positive regulator of IL-6 production. Moreover, our data indicate that this effect could be explained at least in part by the positive regulatory effect of IFN-ab on Ad-induced IL-6 transcription and is achieved by the alteration of the chromatin structure at the IL-6 promoter.
The experiments carried out in this study in MyD88-, TRIF-, Unc93B and various TLR-deficient mice excluded a participation of TLRs in the IFN-ab responses to Ads, including the recombinant Ad5-GFP. The only exception was the TLR9-and MyD88-dependent IFN-ab response elicited by Ad2Ts1, a mutant virus deficient in endosomal escape [5] . However, compared to all other Ads used in this study, Ad2Ts1 induced very low levels of IFN-ab, with faster kinetics and only when used in very high amounts. This is consistent with the finding that the mechanisms of type I IFN induction by Ad2Ts1 and the other adenoviruses are not the same. We suggest that the fast escape of Ads from the endosome circumvents activation of endosomal TLRs. The requirement for a longer endosomal retention time in TLR9dependent IFN-ab production has been recently demonstrated [82] . On the whole, our experiments indicate that Ad endosomal escape is required for the induction of IFN-ab in vivo and suggest a cytosolic pathway. The same requirement was ascertained in the present study for the IL-6 response.
Sensors of nucleic acids are powerful initiators of the TLRindependent cytosolic IFN-ab induction [12, 14, 15] . We excluded in this study a major involvement of the cytoplasmic RNA sensors RIG-I and MDA-5 in the induction of IFN-ab by Ads. Likewise, our study does not support a major participation of the cytosolic DNA sensor DAI/Zbp1 in this induction either. However, our study did not formally exclude the existence of all potentially redundant recognition pathways. So far reported, the pathway(s) of IFN-ab induction activated by cytosolic DNA is (are) strictly dependent on IRF-3 and minimally on IRF-7 [34, 35, 36, 37, 39] . IRF-3 was also reported to be essential for the adenoviral DNAdependent induction of IFN-ab in BMMs in vitro [63] . In agreement, in this study we show that transfection of BMDCs with naked adenoviral DNA or with whole virions of the endosomal escape deficient Ad2Ts1 results in a strictly IRF-3 dependent IFN-ab response. Evidently however, the IFN-ab response of Ad-infected BMDCs and mice is independent of IRF-3. These findings do not exclude a requirement for dsDNA recognition in the induction of IFN-ab, but suggest a different induction mechanism and show the importance of cellular compartmentalization during normal Ad entry. A possible important factor involved in cytosolic Ad sensing might be the adenoviral cysteine protease L3/23, whose activation requires the presence of Ad DNA [83] . This assumption is supported by our finding that a small fraction of the protease-lacking Ad2Ts1 still reaches the cytosol (Fig. 10F, H) , but is devoid of any IFN-abinducing activity. This enzyme, apart from its involvement in maturation of viral proteins and endosomal escape, is essential for the stepwise disassembly of Ads in the cytosol and the release of viral DNA at the nuclear pore [5] .
The absolute requirement of IRF-7 for type I IFN induction in Ad-infected mice shows that this transcription factor participates not only in the positive IFN-ab feedback, but also in the initial IFN-ab production and indeed plays a master role in the regulation of type I interferon response [29] .
A further finding of the present study is the essential role for the MAPK SAPK/JNK in the IFN-ab induction by Ads in vitro and in vivo, although the DNA-mediated cytosolic induction of IFN-ab has been reported to be independent of MAPKs activation [14, 15, 36] . A cytosolic dsDNA-signaling pathway, mediated by the RNA helicase RIG-I and MAVS, and leading to the induction of IFN-b has been recently demonstrated in human hepatoma cells [84] . This induction pathway is absent in murine systems [22, 37, 84, 85] and is therefore unlikely to participate in the IFN-ab response of Ad-infected mice.
On the whole, our findings do not support the participation of any known nucleic acid-mediated mechanisms in the elicitation of IFN-ab responses to human Ads. In this context it is interesting that, as shown here, mouse embryonal fibroblasts do not produce IFN-ab upon Ad infection, although they posses efficient cytosolic induction pathways for dsRNA or dsDNA [13, 36, 37] , the latter shown also in this study. Rather, our data support the possibility that the IFN-ab response to Ads occurs via a novel, not yet characterized cytosolic DNA or protein recognition pathway. Further studies are required to identify the viral components and the host receptors involved. We also emphasize that the known extra-and intra-cytosolic induction pathways may contribute to the IFN-ab response in a host in which Ads replicate and free viral dsRNA and dsDNA are generated.
As mentioned above, the production of IFN-ab in Ad-infected mice is strongly dependent on IFN-ab feedback signaling. Inhibition of positive IFN-ab feedback is a likely explanation for the negative regulation of the IFN-ab response, observed, in mice infected with intact Ads (expressing early genes) in this study. Likely candidates for negative regulators of the IFN-ab production are the E1A proteins. Previously, they were shown to inhibit Stat1 signal transduction [86] , which plays a role in positive feedback signaling by means of the IFN-ab receptor.
Because the adenoviral vectors used in this study correspond to some of those used in gene therapy trials, the present findings in mice may have important implications for Ad gene therapy applications. The adverse effects observed in therapeutic trials, such as systemic inflammatory response and toxicity [6, 7, 9] can be at least partly explained by the enhanced susceptibility of the Adinfected host to microbial components, such as LPS [72] and lipopeptides (unpublished results) from incoming secondary pathogens or from the patient's own flora. Our in vitro finding of a strongly enhanced TNF-a response to LPS in Ad5-GFP-infected human DCs suggests that enhanced susceptibility to LPS may develop also in patients treated with adenoviral vectors. As shown here, this hypersensitivity is mediated by viral-induced IFN-ab, which is in accordance with the role of IFN-ab as a key mediator of sensitization to LPS [87, 88] . The increased mCD14 expression on LPS target cells and epigenetic changes on promoters of relevant genes (both shown here), may at least in part explain the role of IFN-ab in the development of Ad induced LPS hypersensitivity.
Type I interferon induction was recently found in recombinant Ad-treated human cells, especially in pDCs [46, 89] and in monocyte-derived DCs in the present study. Moreover, this response was observed also in patients administered with recombinant adenoviral vectors [42, 90] . Since as shown here, IFN-a pre-treatment is capable of increasing susceptibility to LPS of human DCs in vitro, we assume that Ad-induced IFN-ab can induce LPS hypersensitivity in humans in vivo. Undesirable complications mediated by IFN-ab can occur not only during gene therapy, but also in immunocompromised patients where Ads are major pathogens [1] . Our finding that blocking SAPK/ JNK signaling inhibits the IFN-ab response to Ads, is of potential interest for prevention or treatment of the direct and indirect adverse effects of IFN-ab in Ad gene therapy.
Wt C57BL/6, C57BL/10 and 129Sv mice, as well as all knockout mice were bred under SPF conditions at the MPI. Breeding pairs of IRF-3 2/2 and IRF-7 2/2 mice were kindly provided by T. Taniguchi [54] were on a C57BL/6 background, TLR2/4-deficient mice [95] on C57BL/10 background and IFNabR-deficient mice [96] on 129Sv and for the lethality experiments on C57BL/6 background. When the strain of the mouse was not indicated C57BL/6 mice were used. Mice of both sexes, 8-12 weeks of age were used for the experiments. All of the experimental procedures were in accordance with institutional, state and federal guidelines on animal welfare.
Human Ads of species B (Ad serotype 3) and C (Ad R700, an Ad serotype 5 derivative, Ad serotype 2, Ad2Ts1 and Ad5-GFP an early gene expression defective Ad were grown, purified and stored as previously described [5, 72] . The ratio of infectious/total viral particles was determined on susceptible cells and was typically 1:20-50. If not otherwise stated, Ad2Ts1 used for the experiments was grown at non-permissive temperature 38.5uC (resulting in the absence of incorporation of the adenoviral protease L3/23 into viral capsids). Empty particles were identified by their light density in CsCl density gradients and the absence of viral DNA and were purified simultaneously with mature virions. UV inactivation of Ad was done as described previously [5, 72] using the minimal essential dose preventing viral gene expression and replication in susceptible cells. Heat inactivation was done at 56 uC for 60 min [44] . All virus preparations were LPS-free (less than 1 pg LPS/ 10 11 viral particles) as determined by the Limulus amebocyte lysate test (Pyroquant Diagnostic GMBH, Mörfelden, Germany).
MEFs, BMMs and GM-CSF induced BMDCs were generated as described [95, 97] . The purity of BMMs was higher than 98%, of BMDCs approximately 80%. BM derived pDCs were generated in the presence of Flt3L and CD11c + CD11b 2 B220 + CD62L + pDCs were MoFlo sorted (purity higher than 95%) as described [55] . MEFs were grown in a-MEM (Invitrogen). Immature, monocyte derived human DCs were obtained by incubating adherent monocytes with GM-CSF as described [98] Human mDCs were infected with the indicated amounts of Ads in growth medium containing 2% of donor serum. Mouse L-929 cells were grown in DMEM with 10% FCS. MAPK inhibitors UO126 (MEK1/2, Cell Signaling), SB203580 (p38, Sigma) and SP600125 (SAPK/JNK Calbiochem) were used at 15 mM in vitro and SP600125 at 20 mg/kg in vivo. Bafilomycin-A1 (Sigma) was used at 100 nM. The plasmids pmaxGFP (Amaxa) and RIG-IC [64, 99, 100] were purified with the Endo-Free Plasmid kit (Qiagen) and PEG purification. 
Murine IFN-ab activity was measured using an L-929 cell line (provided by B. Beutler and Z. Jiang, Scripps Research institute, La Jolla) as described [18] Human IFN-ab bioactivity was measured using the HL116 cells from G. Uzé as described [101] . The contribution of IFN-a or IFN-b to the total IFN-ab activity was determined by pre-incubating plasma for 1 h with excess amounts of neutralizing anti-IFN-b antibody (Yamasa Corporation, Japan) or control antibody. Murine TNF-a was measured by a bioassay as described [95] . IL-6 was detected with an ELISA from Pharmingen BD. Human TNF-a was detected with an ELISA from R&D Biosystems. JNK/SAPK MAPKs were detected on immunoblots with antibodies detecting all or phosphorylated isophorms of the proteins (Cell Signaling). Zbp1/DAI and b-actin were detected on immunoblots with antibodies from Santa Cruz Biotechnology.
Conventional RT-PCR, real-time RT-PCR, analysis of IFN-áâ subtypes and Chromatin Immunoprecipitation Total RNA was isolated from organs and cells with guanidinium-thiocyanate-phenol-chloroform extraction or with TRI reagent (Sigma). To exclude DNA contamination, RNA samples were treated with RNase free DNase I (Fermentas). cDNA was prepared using Expand reverse transcriptase (Roche) and oligo-dT. Conventional RT-PCRs were performed with primers as follows. [102, 103] , detecting all IFN-a mRNAs. To determine the levels of different IFN-ab subtypes the HT7900 quantitative PCR system (Applied Biosystems) was used. cDNAs were measured in duplicates or triplicates using the following genespecific assays (TaqMan Gene Expression Assays, Applied Biosystems):
IFN-alpha2 (Mm00833961_s1), IFN-alpha4 (Mm00833969_s1), IFN-alpha5 (Mm00833976_s1), IFN-alpha6 (Mm02524285_g1), IFN-alpha9 (Mm00833983_s1), IFN-alpha11 (Mm01257312_s1), IFN-alpha12 (Mm00616656_s1), IFN-al-pha13 (Mm00781548_s1), IFN-alpha14 (Mm01703465_s1),IFNbeta (Mm00439546_s1). The gene for mouse hypoxanthine guanine phosphoribosyl transferase-1 (HPRT-1, Mm00446968_m1) was used to calibrate the mRNA levels. Quantitative analysis was performed using the SDS 2.1 software (Applied Biosystems). mRNA levels were calculated by the following formula: relative expression = 2'(2(Ct(Target)-Ct(Endogenous control))*f, with f = 10 000 as an arbitrary factor.
Chromatin Immunoprecipitation (ChIP) assays were performed as described [104] . Briefly, cells were cross-linked with 1% formaldehyde and sonicated nuclear extracts were mock-immunoprecipitated or immunoprecipitated with anti-tetraacetylated histone H4 (Upstate). Recovered DNA aliquots from these samples and from input extracts were amplified with real-time PCR using the LightCycler II system (Roche) and Quantitect SYBR Green PCR Kit (Qiagen). Enrichments at specific chromatin loci are shown as the amount of immunoprecipitated DNA in the percent of total input chromatin. The following primers were used: IL -6  promoter: TGG GGA TGT CTG TAG CTC ATT and CAT  AGC GGT TTC TGG AAT TGA, TNF promoter: GGG CAG  CCC CAG AGG GAA TGA ACT C and TAT GGC AGA GGC  TCC GTG GAA AAC TCA CT, Topoisomerase 3b promoter: AGT CCG AGA ACA GCC TGG GT and AGT TGT GCT GCC CAC AGA GG, l5 promoter: TCC CCA TTG CCA GAT AGA GAC ACA and TGG GCC CAA CAG ATT AAC ACA GAG.
BMDCs were cold synchronized with saturating amounts of Ad2 and Ad2-ts1 (60 mg/ml, 0.25 ml per 4610 4 cells on a 12 mm glass coverslip) for 1 h, washed and incubated at 37uC for the indicated times. The samples were fixed with 2.5% glutaraldehyde in 0.1 M ice-cold Na-Cacodylate buffer (pH 7.2) containing 0.5 mg/ml ruthenium red for 1 h, washed with 0.1 M Na-Cacodylate buffer (pH 7.2), post-fixed with 2% OsO4 in the same buffer containing 0.5 mg/ml ruthenium red for 1 h at room temperature, and embedded in Epon as described [105] . Virus particles at the plasma membrane, endosomes and the cytosol were determined, and results expressed as means of analyzed cells (n) with standard errors of the mean. List of accession numbers/ID numbers for proteins mentioned in the text  Hemorrhagic shock and encephalopathy syndrome – the markers for an early HSES diagnosis BACKGROUND: The hemorrhagic shock and encephalopathy syndrome (HSES) is a devastating disease that affects young children. The outcomes of HSES patients are often fatal or manifesting severe neurological sequelae. We reviewed the markers for an early diagnosis of HSES. METHODS: We examined the clinical, biological and radiological findings of 8 patients (4 months to 9 years old) who met the HSES criteria. RESULTS: Although cerebral edema, disseminated intravascular coagulopathy (DIC), and multiple organ failure were seen in all 8 cases during their clinical courses, brain computed tomography (CT) scans showed normal or only slight edema in 5 patients upon admission. All 8 patients had normal platelet counts, and none were in shock. However, they all had severe metabolic acidosis, which persisted even after 3 hours (median base excess (BE), -7.6 mmol/L). And at 6 hours after admission (BE, -5.7 mmol/L) they required mechanical ventilation. Within 12 hours after admission, fluid resuscitation and vasopressor infusion for hypotension was required. Seven of the patients had elevated liver enzymes and creatine kinase (CK) upon admission. Twenty-four hours after admission, all 8 patients needed vasopressor infusion to maintain blood pressure. CONCLUSION: CT scan, platelet count, hemoglobin level and renal function upon admission are not useful for an early diagnosis of HSES. However, the elevated liver enzymes and CK upon admission, hypotension in the early stage after admission with refractory acid-base disturbance to fluid resuscitation and vasopressor infusion are useful markers for an early HSES diagnosis and helpful to indicate starting intensive neurological treatment. Since the original description of the hemorrhagic shock and encephalopathy syndrome (HSES) by Levin et al. [1] , numerous cases have been reported in the literature. Although the etiology of HSES remains unknown, this syndrome is associated with acute onset of encephalopathy, shock, watery diarrhea, severe disseminated intravascular coagulopathy (DIC), and renal and hepatic dysfunction.
As some authors have defined the HSES criteria [2] [3] [4] , patients meeting them will usually have very poor prognoses with a fatal course or severe neurological sequelae. Our experience suggests that early detection of HSES plays an important role in survival and the reduction of neurological sequelae.
We described the clinical courses of 8 patients (age range, 4 months to 9 years) who met the HSES criteria of Bacon et al. [3] , were admitted to our Intensive Care Unit between November 2001 and August 2007, and whose patient records were reviewed to detect markers for an early diagnosis of HSES.
Patients were excluded if they had an elevated plasma ammonium concentration (>130 μmol/l), historic evidence of Reye's syndrome, inadvertent heating, features of the staphylococcal toxic shock syndrome and/or the hemolytic-uremic syndrome, or if any recognized bacterial pathogens or metabolic disorders were discovered that would explain the illness.
Status epilepticus was defined as an epileptic seizure or seizures lasting more than 30 minutes or recurring within 30 minutes without recovery of consciousness. The biological investigations performed for all 8 patients included white blood cell (WBC) counts, C-reactive protein (CRP), platelet counts, hemoglobin, asparate aminotransferase (AST), and alanine aminotransferase (ALT), creatinine, base excess, creatine kinase (CK), and cell counts of cerebrospinal fluid (CSF). Serum lactate level was measured in 6 patients. DIC was defined as a decreased platelet count and an increase in fibrinogen/ fibrin degradation production. Metabolic acidosis was defined as base excess (BE) lower than -3 mmol/L. Lumbar puncture (LP) was performed upon admission for all patients except one whose LP was performed at the previous hospital and could not be performed at our center because of the CT findings of moderate cerebral edema.
Specimens of blood, urine, stool, sputum, and CSF of all 8 patients were obtained to determine any bacterial and/ or viral agents as soon as possible after admission.
Computer tomography (CT) was performed upon admission, electroencephalogram (EEG) was done within 3 hours of admission, and CT was repeated for each patient the following day.
All 8 patients needed mechanical ventilation due to coma and/or seizure. After admission, they required continuous diazepam or barbiturate infusion for seizure or brain edema with the head of the bed elevated 30°.
When hypotension was recognized, fluid resuscitation with a crystalloid or colloid and norepinephrine infusion was set up. The fluid resuscitation target was a central venous pressure (CVP) of ≥ 8 mmHg, urine output >1 ml/ kg/hr. Norepinephrine infusion was started when hypotension was refractory with fluid resuscitation. After these cultures were obtained, all the patients took broad-spectrum antibiotics until their bacterial infections were resolved.
The changes of base excess and serum lactate levels were expressed as mean ± SD. When a median was used, the range was given.
The Osaka City General Hospital ethics committee approved this retrospective analysis of patients' data and informed consent was obtained from the patients' next of kin.
All 8 patients were admitted comatose or with febrile convulsions. Five patients had a history of diarrhea and/or vomiting. The patients' ages ranged from 4 months to 9 years old (median, 1.6 years). Six patients had normal neurological development, however, 2 patients had previously been diagnosed as epileptic. Four patients were admitted in winter, between December and February. Seven patients were transferred to our center within 24 hours after the onset of coma or convulsions. Three patients survived (Table 1 ).
All 8 patients had normal platelet counts, blood pressure and a normal or slightly elevated CRP level; 7 patients had a normal hemoglobin level and renal function. All 8 patients had metabolic acidosis and abnormal serum lactate levels. Seven patients had slightly or significantly elevated liver enzymes, CK, and abnormal WBC counts. Bacterial cultures of blood and CSF were negative for all the patients, however, viral pathogens were detected by PCR in 4 patients (Tables 2, 3) .
Although abnormal cerebral edema was seen in all the patients during their clinical courses, 5 patients appeared normal or only slightly edematous as revealed on their brain CT scans upon admission. On the initial EEG, multi-focal paroxysmal discharges were seen in 4 patients, and low-amplitude patterns were seen in 4 other patients. The CSF cell counts were within a normal range in 7 patients, while the serum level of IL-6 and soluble IL-2 receptors increased with varying ranges in all the patients ( Table 4 ).
All 8 patients had hemodynamic failure within 24 hours after being admitted; therefore, fluids were infused to maintain arterial pressure with the range of fluid balance from -6 to 275 ml/kg (median, 61 ml/kg) for 24 hours from admission for hypotension. Norepinephrine was given to all patients ranging from 0.1 to 0.5 μg/kg/min (median, 0.3 μg/kg/min). Twenty-four hours after admission, 6 patients had normal renal function, and 4 patients had normal platelet counts. However, 5 patients exhibited a decrease in hemoglobin ( Table 5 ).
All the patients exhibited a severe metabolic acidosis with the BE range from -16.0 to -4.4 mmol/L (median, -10.3 mmol/L) upon admission. The acid-base disturbances were maintained with the BE range from -14.4 to -4.1 mmol/L (median, -7.6 mmol/L) at 3 hours, and from -15.2 to -3.1 mmol/L (median, -4.7 mmol/L) at 12 hours after admission with infusion of fluids and/or norepinephrine. The metabolic acidosis was refractory to intensive treatment with mechanical ventilation, infusion of fluids and/or norepinephrine at 24 hours with the BE range from -8.3 to -3.1 mmol/L (median, -4.9 mmol/L; Figure  1 ). Sodium bicarbonate for metabolic acidosis was not administered because the blood pH was kept in the normal range (7.35-7.45) with effective ventilation.
Similarly, there was a tendency to maintain the elevated serum lactate levels, which were measured in 6 patients, with the range from 2.2 to 11.5 mmol/L (median, 4.2 mmol/L) upon admission, from 2.3 to 11.8 mmol/L (median, 6.0 mmol/L) at 12 hours after admission, and from 2.1 to 10.8 mmol/L (median, 6.2 mmol/L) at 24 hours after admission.
CT scan revealed abnormalities between 24 and 72 hours after onset of coma or seizures ( Figure 2 ). When the CT revealed bilateral cortical and subcortical areas of low density, those patients had DIC, anemia and multiple organ failure. Nevertheless, the respiratory function was maintained during the clinical course of all patients.
We started intracranial pressure (ICP) monitoring (REF 110-4BT, Camino, USA) in Cases 1 and 2. In Case 1 the ICP monitoring was started from when the abnormal CT finding was discovered; however, in Case 2, it was started immediately after admission, i.e., before an abnormal CT 
In 1983, Levin et al. described a devastating disease occurring in early childhood called the hemorrhagic shock and encephalopathy syndrome [1] . HSES is not a common disease. Sofer et al. reported 20 patients diagnosed with HSES in a population of about 400,000 over an 11-year period [5] . Our center at Osaka City General Hospital is one of two tertiary pediatric centers in Osaka prefecture. The area our center services for primary referrals has a population of about 4 million residents. However, we do not in any way hypothesize that the 8 patients in our 6year study were the only cases of HSES in this large region, as compared with the 20 patients in the 11-year Sofer et al. report of a significantly smaller population [5] , as there were most likely other HSES patients who could not be transferred to our center from other hospitals due to the rapid deterioration of their conditions. Most reported cases of HSES occurred in winter [3, 5] ; as in the present study, further supporting this evidence, 50% of our patients were admitted in winter, between December and February.
The outcome of HSES was often fatal or with severe neurological sequelae [2] [3] [4] [5] [6] . In the present study, 5 patients died, and the 3 survivors had neurological sequelae. The HSES criteria have been defined in previous reports [2] [3] [4] .
The clinical presentation includes shock, coma and/or seizure, hemorrhage, diarrhea, and oliguria. Laboratory investigations reveal decreased hemoglobin and platelet counts, evidence of DIC, elevated creatinine, AST and ALT, and metabolic acidosis. However, when patients met the HSES criteria, their condition was always critical with multiple organ failure. We determined that these HSES In these cases, the first problem the physician is faced with is the difficulty of the differential diagnosis. Some reports have indicated in the differential diagnosis the diseases of the toxic shock syndrome, the hemolytic-uremic syndrome, and Reye's syndrome, among others [6] [7] [8] . In our patients, those diseases were excluded because of the lack of skin or mucosal manifestations, hemolysis, and/or blood ammonia levels. The clinical course of our patients was not indicative of any of these diseases. Heatstroke has similar clinical and pathological features to HSES; however, there was no history of over wrapping or excessive heating in any of our patients in the present study.
The most common first symptoms were seizure or coma following fever in our cases. From our experience, we have found that common febrile convulsion is often the most difficult disease in the differential diagnosis of HSES in the early stage. Concerning febrile convulsions: prolonged seizures lasting 5 to 10 minutes are relatively common, the laboratory data including CSF is near normal, CT scans reveal normal or slightly edematous conditions, and elevation of CK levels and WBC counts are often seen in cases with prolonged seizures [9] [10] [11] [12] . Even though followup CT scans could provide useful information about cerebral edema [13] [14] [15] , our results showed that the initial CT finding was not useful in making the HSES diagnosis. When an abnormality on the CT was discovered, the patient's condition was critical.
Harden et al. have reported the importance of EEG features and evolution [16] , as confirmed by the observations in the present study, the initial EEG features appeared abnormal in all patients. However, in the initial EEG features, it is usually difficult to distinguish HSES from other diseases with convulsions including febrile convulsions. Rosman has reported that the initial EEG features in patients with febrile convulsions are abnormal in as many as 88% of the patients [17] .
Dunn reported that the outcome of the status epilepticus was not associated with acidosis on admission [9] . Imuekemhe et al. reported that mean serum lactate on admission was significantly higher in patients with prolonged febrile convulsions compared to the corresponding mean value in patients with only brief convulsions [18] . Conversely, Levin et al. noticed metabolic acidosis in HSES patients upon admission [2] . Ince et al. also reported that metabolic acidosis was the common laboratory value in HSES patients upon admission [15] . Little et al. reported a marked metabolic acidosis being refractory to fluid-resuscitate in HSES patients [6] . And Idro et al.
reported that the level of base excess of < -8 mmol/l was a prodromal risk factor for death among children with acute seizures [19] .
In the present study, deterioration of the patients' conditions, especially hemodynamic failure, was dramatic up to Hours after admission 24 hours after admission. The most effective treatment for metabolic acidosis with high levels of serum lactate is the adequate and timely treatment of fluid resuscitation and vasopressor [20, 21] . All patients needed large amounts of fluids and/or norepinephrine infusion. The median rate of fluid administration needed was 61 ml/kg for 24 hours with the infusion of norepinephrine. However, neither metabolic acidosis nor abnormal serum lactate improved in this study.
Sepsis from bacterial infection was excluded by the negative bacterial cultures and the normal CRP levels. However, the hemodynamic course of our patients was very similar to severe septic shock.
The etiology of HSES is still unknown. It has been reported that cytokine storm may be associated with the progress of acute encephalopathy including HSES [22] [23] [24] as septic shock, and levels of some cytokines were useful markers for HSES [25, 26] . The serum levels of IL-6 and soluble IL-2 receptors were increased in our patients, however, the degree of the increase varied in each patient. These results suggest that the increase in cytokines may be associated with HSES. As septic shock is characterized by severe vascular leakage, this is the reason that large amounts of fluids and/or norepinephrine infusions were needed for the patients in this study.
As previously expressed, the respiratory functions of the HSES patients in the present study were maintained throughout their clinical courses. This is the most essential difference between HSES and sepsis in the state of multiple organ failure. Sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has been reported [27] , and the inflammatory response in ALI/ARDS is associated with the release of cytokines [28] .
A limitation of the present study was its small population of only 8 children. Therefore, we did not find a definitive reason the cytokine storm with HSES did not have a sig-
The changes of CT findings at the level of the basal ganglia of Case 1 Figure 2 The ICP control was difficult when monitoring was started after the abnormal CT finding was discovered in Case 1, therefore, the maximum ICP was increased to 109 mmHg. ICP monitoring started before the presence of abnormal CT findings in Case 2 in which the maximum ICP was increased to 59 mmHg; however, the CPP could be maintained above 50 mmHg. This patient was the only case with mild sequelae among all our cases. Hospital days nificant influence on the pulmonary vascular permeability, as would a prodromal marker of ALI/ARDS. It is our supposition that this difference is a key point that would detect the etiology of HSES.
Further studies are warranted to discover what kinds of cytokines would be the most useful markers. The current problem that we face in our center is that it takes considerable time and effort to get the necessary results for these cytokines.
We detected viral pathogens in 4 of 8 patients in the present study. Viral infection may be the trigger for the pathogenesis of HSES as reported in the case report by Gooskens et al. [22] . It may be associated with the most common season of HSES -winter.
Even though there were abnormalities in coagulation, hemoglobin, and renal function, they were useful to make a diagnosis of HSES, it was evident that these laboratory abnormalities were not appreciated as diagnostic markers until at least 24 hours after admission [14, 29] .
Therefore, we did not have sufficient time to make a proper diagnosis of HSES using the established criteria [2] [3] [4] . A diagnosis of HSES ought to be made within 24 hours of admission, otherwise the patients' conditions worsen and the window to provide adequate treatment closes.
The second problem is the treatment of patients with HSES. Even though prolonged metabolic acidosis and/or high levels of serum lactate refractory to large amounts of fluids and/or norepinephrine infusion are useful markers for an early diagnosis, they are not useful as markers when only respiration and circulation management is provided.
Because the brain appears to be the main target organ of HSES, in our non-surviving patients, severe diffuse brain edema with loss of differentiation between the gray and white matter was found on the CT scan during the clinical course. And similar CT abnormalities have been described in non-surviving patients in other reports [13, 15] .
The cause of brain edema following HSES remains unclear, however, Unterberg et al. reported that brain edema of traumatic brain injury was associated with various mediators including cytokines, lactate, free oxygen radicals, etc. [30] . Brain edema in HSES patients seems to occur in such a situation. Because the serum levels of cytokines and lactate had increased, an increase in vascular permeability was suggested by our patients needing large amounts of fluids. Furthermore, large amounts of fluid-resuscitate within 24 hours of admission may lead to brain edema under the state of increasing vascular permeability.
We propose that for the most efficacious management of ICP, whenever possible, ICP monitoring ought to be started before detection and observation of any decrease in the platelet count and/or any abnormal CT findings because ICP monitoring could not be performed with DIC. This is the reason that a diagnosis of HSES should be made within the early stage, i.e., within 24 hours of admission.
However, to our knowledge, there have not been any reports published proposing an effective treatment for HSES. Controlling brain edema might be the optimal therapy to help HSES patients survive. Even though there are currently only palliative therapies, e.g., mild hypothermia, infusion of fluids and osmotic diuretics, administration of anticonvulsants under mild hyperventilation, and vasoconstrictor infusion to prevent edema and to maintain the cerebral perfusion pressure (CPP). There are no reports about the efficacy of the control of ICP and CPP upon the outcome of the HSES; however, we suggest that the control of ICP and CPP is an essential part of any therapeutic treatment.
A patient characterized by coma or seizure following hyperpyrexia might be diagnosed as having common febrile convulsions. However, when such a patient also presents with elevated liver enzymes and CK upon admission, hypotension within 24 hours after admission, with refractory acid-base disturbance and an abnormally high serum lactate level, even with fluid-resuscitate and/or vasopressor infusion, these signs may be useful markers for an early HSES diagnosis and indicators to start intensive neurological treatment. HSES is not a disease that can be diagnosed easily using the current diagnostic criteria, however, HSES can be predicted in the early stage of its clinical course using these new prodromal diagnostic markers.
• When the patients met the HSES criteria, their condition was always critical with multiple organ failure.
• CT scan, DIC, EEG, or renal function upon admission did not prove useful for an early diagnosis of HSES.
• Elevated liver enzymes and CK upon admission, hemodynamic failure in the early stage after admission, and a prolonged metabolic acidosis refractory to intensive treatment were useful markers for an early diagnosis of HSES.
• Controlling brain edema might be the most important therapy to help HSES patients survive.
• HSES is a disease that should be predicted within the early stage of its clinical course. Unifying evolutionary and thermodynamic information for RNA folding of multiple alignments Computational methods for determining the secondary structure of RNA sequences from given alignments are currently either based on thermodynamic folding, compensatory base pair substitutions or both. However, there is currently no approach that combines both sources of information in a single optimization problem. Here, we present a model that formally integrates both the energy-based and evolution-based approaches to predict the folding of multiple aligned RNA sequences. We have implemented an extended version of Pfold that identifies base pairs that have high probabilities of being conserved and of being energetically favorable. The consensus structure is predicted using a maximum expected accuracy scoring scheme to smoothen the effect of incorrectly predicted base pairs. Parameter tuning revealed that the probability of base pairing has a higher impact on the RNA structure prediction than the corresponding probability of being single stranded. Furthermore, we found that structurally conserved RNA motifs are mostly supported by folding energies. Other problems (e.g. RNA-folding kinetics) may also benefit from employing the principles of the model we introduce. Our implementation, PETfold, was tested on a set of 46 well-curated Rfam families and its performance compared favorably to that of Pfold and RNAalifold. where r(σ) is the root node of τ M (σ). Since we are not using the parse tree τ M (σ) explicitely in the main text, we will write Pr(r(σ), A) as short for Pr τM (σ) (r(σ), A). 
PETfold uses a Nussinov style algorithm to calculate the consensus structure of an alignment with maximal expected overlap. The Nussinov algorithm uses dynamic programming to find the structure with the highest score. Let F (i, j) denote the maximal score of an RNA structure for the sequence s i . . . s j . Thus, we have
where s(x i ) (and s(x j )) is the score for a single-stranded position x i and s(x i , x j ) is the score for paired bases x i and x j . In PETfold the single-stranded score of position x i consists of the evolutionary reliability R sg A,T,M (i) and the thermodynamic probability 1 n u q u f −1 A (i) over all sequences s u (1 ≤ u ≤ n) in the alignment, and the base pair score of the positions x i and x j consists of the evolutionary reliability R A,T,M (i, j) and the thermodynamic probability 1 n u p u f −1 A (i,j) . The optimal structure σ can be reproduced by backtracking from F (1, L) when L is the sequence length. In PETfold, we define ex-over(σ) = F (1, L).
We present a statistical method to estimate reliability thresholds for conserved functional regions. Single stranded positions and base pair positions are collected that have a high evolutionary reliability. We write down only the base pair part. Single-stranded positions are treated analogously. For this purpose, do the following 1. Generate shuffled alignment A shuffle by shuffling the alignment columns. Then, we generate again the most likely structure under the shuffled alignment, i.e., we generate
Then, we collect all the reliability scores for base pairs that are contained in this structure, and iterate this several times:
Finally, we order them in size p 1 > p 2 > · · · > p |B| and select a significance level θ (e.g., θ = 0.01). Then the probability p ⌈θ|B|⌉ is the base pair probability p threshold such that any base pair
We applied the previously described stepwise approach on our data set consisting of 46 RNA families. We shuffled for each family 1000 times with a conservative method which mononucleotidely shuffles only columns with the same pattern of gaps and conservation. Then we averaged over the significance values of all families. Using a significance level θ = 0.01, we got a threshold for high reliable base pairs of p threshold However, the parameter tuning has indicated that the performance of reliability thresholds depend on another parameter (the weighting factor for single-stranded positions α) which has high impact in the RNA structure prediction of PETfold, and that slightly different reliability thresholds perform better for the data set.
Given two structures in bracket notation, a more stringent secondary structure evaluation can be carried out by considering all pairs of positions, and evaluate the agreement in their structural notation (i.e., dots, opening and closing brackets) in both structures. For each pair of positions (i, j), there are five possible cases. The two positions can be unpaired (4) or paired with each other (1) . Furthermore, only the left (2) (resp. right (5)) position can have an opening (resp. closing) bracket. Finally, both positions can be paired, but with different partnersi (3).
Formally, we have the following five categories (K = 5): (1) This can be evaluated by the R K correlation coefficient (K = 5) [1] . This correlation coefficient of two assignments represented by two N × K matrices of data X and Y is defined as
.
The covariance between X and Y is defined as the expected covariance between the respective k th columns X k and Y k in the matrices:
where X k = (1/N ) N n=1 X nk and Y k are the respective means of column k, and X nk are elements of X. Note that Matthews correlation coefficient (M CC) applies to the two categories (K = 2) base paired (i bp j) and not base paired (i ¬bp j) for any pair of bases (N = M (M − 1)/2 where M is length of sequence). Correction for sliding base pairing is not used.
When extending the consideration of unpaired bases, we obtain R 5 correlation coefficients of PETfold: 0.72, Pfold: 0.58, RNAalifold: 0.65. This evaluation is more strict as the two-category Matthews correlation coefficient. Nevertheless, both evaluations show almost the same differences between the three methods.
SI Table 1  A sensitive array-based assay for identifying multiple TMPRSS2:ERG fusion gene variants Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT–PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of <10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no >1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci. Chromosome rearrangements are a characteristic feature of cancer. More than 350 gene fusions, as a consequence of chromosome aberrations, have been identified (1) . While gene fusions are common in hematological malignancies, their presence in solid tumors is not as well studied due to several technical and analytic problems related to tumor heterogeneity (1) . Only very limited gene fusion events were discovered in solid tumors, mostly in sarcomas, until the recent discovery of TMPRSS2:ETS fusion genes in prostate cancer (2) . This finding has since changed the general view that gene fusions play only a minor role in the pathogenesis of epithelial tumors. Therefore, there is renewed interest in searching for fusion genes in solid tumors, due to their potential impact on basic research and clinical application as has been demonstrated in chronic myelogenous leukemia (CML) (3, 4) .
The recurrent gene fusion event in prostate cancer involves an androgen controlled gene, TMPRSS2, and members (ERG, ETV1 and ETV4) of the ETS transcription factor family (2, 5, 6) . Among these fusion genes, TMPRSS2:ERG is the most prevalent and the only member detected in the majority of reports. This fusion transcript results from $3 Mb interstitial deletion between these two loci at chromosome 21q22. It was found in approximately half (15-78%) of all prostate cancers (2, (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . As an androgen-related transcription factor controlling cell proliferation, TMPRSS2:ERG has been associated with disease pathogenesis and is a promising biomarker for prostate cancer progression, prognostication and early detection (18) (19) (20) (21) . While the presence of TMPRSS2:ETS fusion genes is highly prostate cancerspecific, its significance as a prognostic biomarker is still controversial partly because many of the clinical studies have been relatively small scale. Therefore, it is important to develop a simple and robust assay for identifying various TMPRSS2:ETS and potential fusion genes in other solid tumors. However, this could be challenging due to high heterogeneity in prostate cancer and other solid tumors, compared to leukemias and lymphomas (22) .
Several approaches that have been used previously for hematological malignancies have been applied to detect TMPRSS2:ERG exon fusion variants. These include fluorescent in situ hybridization (FISH) (2, 12, 14, 17, 23) , RT-PCR and sequencing (2, 7, 9, 13) , quantitative PCR (qPCR) (2, 8, 24) and array-based comparative genome hybridization (array CGH) (10) (11) (12) . FISH may be the most commonly used method, but it has relatively low resolution, and therefore, cannot accurately determine different fusion variants. Array CGH has a higher resolution but is costly and often fails when there is normal cell contamination.
RT-PCR and qPCR are relatively easy to perform. However, to assess multiple potential fusion variants requires multiple sets of primers and probes, and a corresponding large quantity of RNA templates. Moreover, sequencing RT-PCR products is laborious and difficult to adapt in routine clinical laboratories. Here, we describe an exon array-based detection system, combined with a RT-PCR reaction, that accurately determines multiple TMPRSS2:ERG fusion transcripts in specimens with only a minor population of tumor cells. The method adopts several features of the Virochip (25) protocol to establish a specific, sensitive and semi-quantitative assay that is very useful for analyzing highly heterogeneous solid tumors.
The cell lines described in the article were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended. Frozen unpurified prostate tissues were obtained during routine surgery, and classified pathologically by one of us. The total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The primary tumor samples were purified by Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA).
The exon and junction probes are 30-mer oligonucleotides synthesized by Integrated DNA Technologies (Coralville, IA, USA) or Illumina/Invitrogen (San Diego, CA, USA) and printed on poly-L-lysine slides at 50 mM along with Human Cot-1 DNA (Invitrogen), which is enriched for repetitive sequences, and herring sperm DNA (Promega, Madison, WI, USA), which was used as nonspecific control. The printing procedure has been described and essentially follows the manual of the DeRisi arrayer with silicon microcontact printing pins (Parallel Synthesis Technologies, Inc. Santa Clara, CA, USA) (25) (26) (27) . Arrays were postprocessed with succinic anhydride-based method for blocking before hybridization as previously described (27) . The protocols related to array printing and hybridization in this article generally can be found in the following link: http://cat.ucsf.edu/equipment/arrayer/index.html.
The RT-PCR reaction was performed with an OneStep RT-PCR kit (Qiagen) essentially following the manufacturer's protocol, except that the final reaction volume was scaled down to 20 ml. The forward (GTT TCC CAG TCA CGA TCC AGG AGG CGG AGG CGG A) and reverse primers (GTT TCC CAG TCA CGA TCG GCG TTG TAG CTG GGG GTG AG) are located at exon 6 of ERG and exon 1 of TMPRSS2 respectively, as described (2, 9) . The 5 0 -ends of both primers have the sequence of primer B (GTT TCC CAG TCA CGA TC) for the subsequent step of PCR labeling with a single primer B as described previously (25) .
The procedure is a modification of the previously reported 'Round ABC' protocol (25) . Briefly, the RT-PCR reaction was assembled at 48C in a PCR workstation and transferred to a thermocycler with the block preheated to 508C. The initial reverse transcription was performed at 508C for 30 min and followed by 958C for 15 min to activate HotStarTaq DNA polymerase as well as to inactivate the reverse transcriptases (Round A). The PCR conditions were 35 cycles at 928C for 30 s, 558C for 30 s and 688C for 1.5 min with a final extension step at 688C for 5 min (Round B). One microliter of unpurified product was subsequently used as a template for another 20 cycles of amplification to label the amplicons via a previously described 'Round C' PCR protocol (948C for 30 s, 408C for 30 s, 508C for 30 s and 728C for 1 min) with primer B and a 4:1 mixture of aminoallyl dUTP (Ambion, Austin, TX, USA) and dTTP for probe labeling (25) . The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns (Zymo Research, Orange, CA, USA), eluted in 9 ml of sodium bicarbonate (pH 9.0) and coupled with 1 ml of DMSO dissolved Cy3 NHS esters (GE Healthcare, Piscataway, NJ, USA) for 30-60 min. The Cy3-labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 ml of 10 mM Tris-HCl (pH 8.0). Then, the Cy3-labeled amplicons were diluted in water and combined with 3.6 ml of 20Â SSC, 0.5 ml of HEPES (pH 7.0) and finally 0.5 ml of 10% SDS to reach final volume of 25 ml. The mixed solution was heated for 2 min at 958C, cooled to room temperature and hybridized to the exon mapping arrays at 638C overnight essentially as previously described (25) (26) (27) . The hybridized arrays were washed and scanned with a GenePix 4000B scanner (Molecular Device, Sunnyvale, CA, USA) and analyzed by GenePix Pro 6.0 software.
Frozen prostate cancer samples were sectioned onto slides. Cell nuclei were isolated in situ with ice-cold cytoskeleton buffer (CSK: 300 mM sucrose, 100 mM NaCl, 10 mM PIPES, 3 mM MgCL 2 , 1 mM EGTA and 0.5% Triton X-100) (28) . The slides were fixed by dipping in ice-cold methanol for 3 min, followed by ice-cold acetone. After air drying the slides, they were allowed to age for at least 1 week in ethanol.
DNA-FISH was carried out according to a method for single copy loci detection (28) . The protocol was adapted with few modifications, using 50-mer oligonucleotides specific to the loci of interest and labeled with a desired hapten. Two probes were used for FISH for breakapart assay. A probe located at the promoter region of TMPRSS2 was labeled with biotin (Bio/GACTCCA GGAGCGCTCCCCAGAATCCCCTTCCTTAACCCA AACTCGAGCC). The other probe at exon 2 of ERG was labeled with 5 0 -6-carboxyfluorescein (56FAM) (56FAM/ GATCTTTGGAGACCCGAGGAAAGCCGTGTTGA CCAAAAGCAAGACAAATG). Detection of probes was achieved by using antibodies conjugated to quantum dots (Qdot) against the hapten label. Conditions were optimized to use a combination of two antibodies (1 : 200) obtained from Invitrogen-Molecular Probes TM (Qdot 655 Ã sheep anti-Bio primary antibody conjugate; Qdot 525 Ã Goat anti-FITC whole IgG primary antibody conjugate). Image acquisition was done with a Zeiss Axioplan 2e microscope (Carl Zeiss, Inc.). All pictures in the corresponding three channels were deconvolved and optical sections merged to produce 2D pictures using Axiovision 4.0 software (Carl Zeiss, Inc.) and Image J (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997-2006.)
To develop a multiplexing assay that is highly sensitive in clinical samples of high complexity, we adopted our Virochip system (25) . The key protocol, Round ABC, designed for unbiased amplification (29) , is crucial for identifying various fusion variants in this application.
Through literature review, we found that most of the TMPRSS2:ERG fusion junctions are between exons 1 or 2 of the TMPRSS2 and exons 2-5 of the ERG (2, 7, 9, 13) . Such constraints perhaps are related to whether a functional ERG protein can be made from the gene fusions (9) . Therefore, we initially used a pair of primers at exon 1 of the TMPRSS2 and exon 6 of the ERG for RT-PCR. As shown in Figure 1A , PCR products were only generated when there was a gene fusion, since the two primers are located at different genes. Subsequently, the PCR products were labeled and hybridized to an exon array for mapping the exons near the fusion junction. Printed on the array are 30-mer oligonucleotide probes derived from exons 1-3 of the TMPRSS2 and exons 1-5 of the ERG (Table 1) . Each selected sequence is represented by two complementary probes (F: forward and R: reverse complement) since sometimes PCR-labeled amplicons may bind to only one strand of the probe, based on empirical observations (25) . We observed that probes with reverse complementary (R) orientation worked consistently with our RT-PCR labeling protocol.
A prostate cancer cell line, VCaP, (30) with a TMPRSS2 and ERG fusion (2) was used for initial feasibility testing. The total RNA was subjected to RT-PCR with a pair of primers located at exon 6 of ERG and exon 1 of TMPRSS2 (2, 9) . The unpurified product was labeled and hybridized on the microarray ( Figure 1B ). Only spots corresponding to exon 1 of TMPRSS2 and exons 4-5 of ERG developed strong signals. This result indicates the fusion junction is at the exon 1 of TMPRSS2 and exon 4 of ERG, which is consistent with a previous report (2) .
To mimic a typical clinical situation, in which small population of cancer cells are present among normal host cells in a primary tumor, we spiked decreasing amounts of total RNA extracted from VCaP cells into an excess of HeLa RNA, which does not have the fusion transcripts. The detection limit is 32 pg of VCaP RNA in the presence of 100 ng of HeLa RNA (Figure 2 ). This translates into only 1-3 cancer cells in the presence of 3000 times more normal cells. The level of sensitivity is superior to previous methods for detecting fusion transcripts (24) .
To test the ability of the exon mapping array to detect and characterize TMPRSS2:ERG fusion transcripts in clinical samples, we isolated total RNA from frozen unpurified primary prostate tissues obtained during surgery. T1F  GGGCGGGGAGCGCCGCCTGGAGCGCGGCAG  T2F  ACATTCCAGATACCTATCATTACTCGATGC  T3F  GGTCACCACCAGCTATTGGACCTTACTATG  T1/2F  TGGAGCGCGGCAGGTCATATTGAACATTCC  G1F  AGGGACATGAGAGAAGAGGAGCGGCGCTCA  G2F  AGACCCGAGGAAAGCCGTGTTGACCAAAAG  G3F  GCTGGTAGATGGGCTGGCTTACTGAAGGAC  G4F  TTATCAGTTGTGAGTGAGGACCAGTCGTTG  G5F  CTCTCCTGATGAATGCAGTGTGGCCAAAGG  T1R  CTGCCGCGCTCCAGGCGGCGCTCCCCGCCC  T2R  GCATCGAGTAATGATAGGTATCTGGAATGT  T3R  CATAGTAAGGTCCAATAGCTGGTGGTGACC  T1/2R  GGAATGTTCAATATGACCTGCCGCGCTCCA  G1R  TGAGCGCCGCTCCTCTTCTCTCATGTCCCT  G2R  CTTTTGGTCAACACGGCTTTCCTCGGGTCT  G3R  GTCCTTCAGTAAGCCAGCCCATCTACCAGC  G4R CAACGACTGGTCCTCACTCACAACTGATAA G5R CCTTTGGCCACACTGCATTCATCAGGAGAG T, TMPRSS2; G, ERG. F, forward probe; R, reverse complement probe.
Many of these tumors had a substantial fraction of normal stromal cells. Total RNA (5-50 ng) from prostate cancers (n = 20) and nonmalignant hyperplastic prostate tissues (n = 10) were subjected to RT-PCR labeling and array hybridization. The results showed that 7/20 cancers but 0/10 nonmalignant samples had TMPRSS2:ERG fusion genes. To confirm the presence of the gene fusions, direct sequencing was performed for the seven samples. The sequencing data validated the exon fusion findings revealed by the array assays. Similar to other reports (7, 12, 13) , some samples clearly showed two or more bands on the agarose gel when the PCR products were subjected to electrophoresis, corresponding to two or more fusion transcripts in the same specimens. The multiple fusion transcripts in a single prostate cancer sample may reflect tumor heterogeneity or alternative splicing events. In order to map multiple fusion junctions in a single assay, we redesigned the exon array to include junction probes between exons 1 and 2 of the TMPRSS2 gene and exons 1-6 of the ERG gene ( Table 2 ). The modified probe set showed very clearly that the patient sample #15 had two fusion transcripts and also revealed the relative ratios of the two fusion transcripts through their respective signal intensities ( Figure 3 ). In this case, the two fusion transcripts are between exon 4 of the ERG fused to either exon 1 (T1G4) or exon 2 (T2G4) of the TMPRSS2. The signal intensity of T2G4 junction probe is weaker than that of the T1G4 junction probe ( Figure 3A) , consistent with the intensities of the probes within the exons. These two fusion transcripts are very likely due to alternative splicing. Figure 4 summarizes the cluster analysis (31) of the seven arrays; the results are shown in Table 3 . Multiple fusion variants were found in 4/7 positive samples. Table 3 also lists the percentages of cancer cells in the tumors, the Gleason tumor grades and the detected variants of TMPRSS2:ERG fusion transcripts. In this small sample set, there is no clear association between tumor Figure 2 . Assay sensitivity. The VCaP total RNA was serially diluted in a solution containing HeLa RNA to mimic the heterogeneous cell population in primary tumors or human body fluids. The total amount of RNA for each reaction is 100 ng. The laser power (PMT 600, 100% output) was adjusted to maximize the sensitivity of detection. Therefore, the intensity of the each expected feature (T1, G4, G5) is at the saturated level. The signal disappeared when the VCaP RNA was diluted from 1:3125 (32 pg) to 1:15625 (6.4 pg). T1G1F  CCTGGAGCGCGGCAGCCCCCGAGGGACATG  T1G2F  CCTGGAGCGCGGCAGGTTATTCCAGGATCT  T1G3F  CCTGGAGCGCGGCAGCCGTCAGGTTCTGAA  T1G4F  CCTGGAGCGCGGCAGGAAGCCTTATCAGTT  T1G5F  CCTGGAGCGCGGCAGATGCCACCCCCAAAC  T1G6F  CCTGGAGCGCGGCAGATCCTACGCTATGGA  T2G1F  ATGGCTTTGAACTCACCCCCGAGGGACATG  T2G2F  ATGGCTTTGAACTCAGTTATTCCAGGATCT  T2G3F  ATGGCTTTGAACTCACCGTCAGGTTCTGAA  T2G4F  ATGGCTTTGAACTCAGAAGCCTTATCAGTT  T2G5F  ATGGCTTTGAACTCAATGCCACCCCCAAAC  T2G6F  ATGGCTTTGAACTCAATCCTACGCTATGGA  T1G1R  CATGTCCCTCGGGGGCTGCCGCGCTCCAGG  T1G2R  AGATCCTGGAATAACCTGCCGCGCTCCAGG  T1G3R  TTCAGAACCTGACGGCTGCCGCGCTCCAGG  T1G4R  AACTGATAAGGCTTCCTGCCGCGCTCCAGG  T1G5R  GTTTGGGGGTGGCATCTGCCGCGCTCCAGG  T1G6R  TCCATAGCGTAGGATCTGCCGCGCTCCAGG  T2G1R  CATGTCCCTCGGGGGTGAGTTCAAAGCCAT  T2G2R  AGATCCTGGAATAACTGAGTTCAAAGCCAT  T2G3R  TTCAGAACCTGACGGTGAGTTCAAAGCCAT  T2G4R  AACTGATAAGGCTTCTGAGTTCAAAGCCAT  T2G5R  GTTTGGGGGTGGCATTGAGTTCAAAGCCAT  T2G6R  TCCATAGCGTAGGATTGAGTTCAAAGCCAT T, TMPRSS2; G, ERG. F, forward probe; R, reverse complement probe. grade and the presence of fusion transcripts. A relatively larger study also showed that the presence of fusion transcript was associated with tumor stage but not tumor grade (12) . Consistent with the VCaP titration study (Figure 2 ), the clinical assay can detect the fusion transcript when only 1% tumor cells is present in the prostate tissue (sample 10).
We used FISH analysis to independently confirm our array approach. Our FISH procedure (28) employed 50-mer probes that were labeled with small haptens for target hybridization in conjunction with individual hapten-specific, quantum dot conjugated antibodies for signal detection. The resolution of this method is $50 kb. We designed two probes for the FISH assays, one at the promoter region of TMPRSS2 (green in Figure 5 ) and the other at exon 2 of ERG (red in Figure 5 ). We observed heterogeneity of the FISH patterns in some primary prostate cancer samples ( Figure 5 ). It is more difficult to find interstitial deletions between TMPRSS2 and ERG in tumor samples containing low percentages of cancer cells by FISH. Therefore, we used samples that contained 480% cancer cells without detectable fusion genes to confirm the results of the arrays. The FISH experiments revealed no genomic deletions at this location for all the selected samples (#5, #7, #9, #19 and #20) that were similarly nondeleted by array hybridization.
We have established a simple assay that can concurrently profile variants of TMPRSS2:ERG fusion transcripts by combining a single RT-PCR with an exon array. The modified 'Round ABC' protocol, which was TMPRRS2e1R  TMPRRS2e1/2R  TMPRRS2e2R  TMPRRS2e3R  ERGe1R  ERGe2R  ERGe3R  ERGe4R  ERGe5R  ERGe6R  T1G1R  T1G2R  T1G3R  T1G4R  T1G5R  T1G6R  T2G1R  T2G2R  T2G3R  T2G4R  T2G5R  T2G6R   #1  #15  #17  #4  #13 #10 #18 Figure 4 . Cluster analysis of seven prostate cancer samples having fusion transcripts. The signal intensity of each feature is divided by the intensity of a nonspecific control (herring sperm DNA) to normalize the data for cluster analysis. The result is shown in Table 3 . The samples having similar fusion transcript variants were clustered together by the program. 1  30  7  T1-G4; T2-G4  2  2 0  5  3  5 0  5  4  20  6  T1-G4  5  8 0  9  6  1  6  7  9 0  8  8  2 0  4  9  8 0  8  10  1  6  T1-G2  11  2  6  12  70  7  13  20  9  T1-G4  14  1  6  15  70  8  T1-G4; T2-G4  16  20  8  17  80  8  T1-G4; T2-G4  18  50  7  T1-G2; T1-G3; T1-G4  19  80  7  20 80 7 Figure 5 . Heterogeneity of FISH patterns of interstitial deletion between TMPRSS2 and ERG in a primary prostate tumor. An unpaired green dot (TMPRSS2 probe, indicated by arrows) suggests an interstitial deletion. Nonrandom variation of FISH patterns is shown by the fact that most of the green and red signals (two different but nearby probes) are paired in each panel. This variation is expected on a heterogeneous aneuploid cancer cell population, which often makes it difficult to distinguish meaningful events from random background aberrations.
originally designed for genomic amplification (29) and has been widely adopted for chromatin immunoprecipitation (ChIP) and whole-genome DNA microarrays (ChIP-chip) (32, 33) and Virochip (25, 34) experiments, is a simple and relatively unbiased amplification procedure to semiquantitatively measure the fusion variants in a complex sample. Previously, the same simple procedure was used to obtain 83% (25 kb/30 kb) of the SARS coronavirus genome with total nucleic acids isolated from a viral culture (25) . The inclusion within the array of probes derived from individual exons and potential fusion junctions simplifies the breakpoint mapping and increases the confidence of data interpretation (Figures 3 and 4) . In contrast to some reports that used multiple primers targeted to every potential fusion junction in hematological malignancies (35) (36) (37) (38) , we used a single set of primers for target amplification (Figure 1 ). The fusion junctions were subsequently decoded by array. This design significantly reduces the problems associated with primer dimers in the multiplex PCR reaction, and creates more room for future assays to include additional fusion genes. Furthermore, most searches for fusion genes have focused on blood cancers, because the cells can be purified before analysis. The application of the previous methodologies is less useful for highly complex solid tumors that are inevitably admixed with normal cells. For example, a previously reported MLLFusionChip could not be applied to samples with cancer cells of 55-10% in 1 mg of total RNA (39) .
The current assay should facilitate a thorough compilation of the gene fusion variants in primary prostate specimens, which may be useful for stratifying the aggressiveness of prostate cancer (13) . In this regard, fusion variants of EWS with another member of the ETS family, FLI1, have been shown to be an independent predictor of disease progression in Ewing's sarcoma (40, 41) . It will be of interest to compare in transfected cells the biological activities of the different TMPRSS2:ERG variants from patients with contrasting clinical outcomes (41) .
While some studies have suggested that the presence of TMPRSS2:ERG fusions is associated with more aggressive disease or higher Gleason tumor grade, other investigators did not reach the same conclusion (12, 14, 17, 20, 23, 42) . We also did not find such an association in a small series of samples. However, all of these results are defective due to small sample size. The technology described here should make possible a larger scale investigation to find whether there is a correlation between the aggressiveness of the disease and the presence of specific fusion genes.
It is crucial to have true cancer-specific biomarkers for early cancer detection as well as for minimal residual disease monitoring, which has been extensively demonstrated in hematologic maligancies (43) . Such biomarkers could help to avoid under-or over-treatment. Thus, there is past interest (24, 44) in applying TMPRSS2:ERG fusion assays for such application since PSA and many other markers in development are not truly prostate cancer-specific (45) . A recent study reported a TMPRSS2:ERG assay with a sensitivity of detecting 1600 VCaP cells (24) . However, this level of sensitivity might not be sufficient for broad clinical application, especially with small biopsy specimens or urine samples. We were able to achieve an assay sensitivity of 532 pg of total RNA derived from VCaP cells, an equivalent to 1-3 cells (Figure 2 ). Because our assay is simple and amenable to automation, it is readily adaptable to clinical studies. While it has been challenging to adapt microarray-based technology to the clinic, some tests (e.g. AmpliChip CYP450 and MammaPrint) have been approved by FDA (46) .
The same strategy can be applied to detect other less prevalent fusion transcripts (TMPRSS2:ETV1 and TMPRSS2:ETV4) in prostate cancer (2, 5, 6) . In addition, the exon array approach can also be applied to other fusion genes, such as BCR-ABL in CML and clonal Ig/TCR rearrangements in lymphocytic malignancies. While this methodology development was motivated by the clinical need, it is generally applicable to other research requirements that are analogous to the situation for detecting fusion genes in the single cell level when a large excess of normal cells are present. For example, a developmental biologist may use a similar approach to screen mutants that have a desirable gene fusion when direct gene targeting is not feasible.
There are some shortcomings of using RNA transcripts as prostate cancer biomarkers, despite our ability to achieve very sensitive detection of TMPRSS2:ERG fusion variants. First, RNA is unstable and difficult to process in routine clinical assays. Second, commonly used drugs that inhibit androgen growth pathways, including GnRH agonists and testosterone antagonists, may diminish the production of the TMPRSS2:ERG mRNA fusion transcript, thereby producing false-negative results in patients on hormonal therapy with evolving androgenindependent tumors. Indeed, it has been reported that TMPRSS2:ERG mRNA fusion transcripts are not expressed in androgen-independent tumors in spite of the presence of interstitial deletions in between TMPRSS2 and ERG at chromosome 21q22 (10) . While FISH is useful for identifying genomic rearrangements, it has relatively lower resolution and is difficult to use in highly heterogeneous samples with small percentages of tumor cells. We have recently developed a technology, designated Primer Approximation Multiplex PCR (PAMP) for identifying breakpoints in genomic DNA without the need to purify cancer cells from normal tissues (26) . We are optimizing this assay for detecting the breakpoints between TMPRSS2 and ERG loci for primary prostate tumors to overcome any potential problems associated with RNA based biomarkers. In addition, the DNAbased assay will provide information about whether multiple fusion transcripts in a sample are derived from alternative splicing or tumor heterogeneity. The best approach may ultimately be to combine DNA and RNA based assays in a common format.
Institutes of Health (CA119335 to UCSD NanoTumor Center of Excellence for Cancer Nanotechnology, CA133634 to Y. T. Liu, NS034934 to Delivery of steric block morpholino oligomers by (R-X-R)(4) peptides: structure–activity studies Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs. Protein transduction domains as penetratin or Tat 48-60 and synthetic cell penetrating peptides (CPP) as oligoarginine have generated a large interest for their seemingly unique mechanism of membrane translocation and for their capacity to transport various biomolecules across biological membranes (1) . Both assumptions have had to be re-visited since cellular uptake does involve endocytosis (2) and transport of biomolecules does not occur as efficiently as anticipated at least at low concentrations. In a series of experiments carried out independently by several groups, CPPs mentioned above turned out inefficient in transporting uncharged splice correcting oligonucleotide (ON) analogs as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) for a large part because CPP-conjugated material remained entrapped in endocytic vesicles (3) . Accordingly, peptides or drugs (such as chloroquine) leading to endosome destabilization did significantly increase splicing correction.
We have recently described a new (R-Ahx-R) 4 -CPP (in which Arg residues are interspersed with nonnatural 6-aminohexanoic acid amino-acid spacers) which leads to efficient splicing correction at low concentration in the absence of endosomolytic agents. (R-Ahx-R) 4 is less cytotoxic and much more active to deliver splice correcting PMO and PNA in vitro than the parent oligoarginine (R n ) peptide and than the prototypic Tat 48-60 peptide (4) . Importantly, (R-Ahx-R) 4 -PMO conjugates also lead to efficient exon skipping in murine and dog Duchenne muscular dystrophy (DMD) models (5) and inhibit the replication of viruses in several murine models (6) (7) (8) (9) .
We had no clear explanation for the improved efficiency of (R-Ahx-R) 4 -PMO and (R-Ahx-R) 4 -PNA conjugates as compared to Tat or (Arg) n steric block ON constructs. Increased cellular uptake could not be the answer since, on the contrary, (R-Ahx-R) 4 -PMO conjugates were taken up less efficiently than Tat-PMO and (Arg) n -PMO conjugates in our model system (3) . Differences could originate from a different mechanism of cellular uptake with (R-Ahx-R) 4 -PMO conjugates taking profit of a more favorable route than the other CPP conjugates. Again available data did not support this hypothesis since all three conjugates were taken up by an energy-dependent pathway involving binding to cell surface proteoglycans. Along the same lines, we recently established that blocking energy-dependent processes through incubation of cells at low temperature or through ATP depletion decreased splicing correction by (R-Ahx-R) 4 -PMO conjugates and by Tat-PMO or (Arg) n -PMO ones to the same extent (10) .
Of possible relevance, (R-Ahx-R) 4 -PMO conjugates bind less strongly to heparin (taken as a model heparan sulfate) than Tat-or (Arg) n -PMO conjugates (3) . This could provide an explanation if one assumes that heparan sulfate-bound material has to be released during endocytosis in order to escape to the cell cytoplasm. While sufficient affinity is essential for cell binding and cellular uptake, a too high affinity could become detrimental at later steps, a hypothesis which we aim to investigate here.
On the other hand, the inclusion of nonnatural amino acids as aminohexanoic acid in (R-Ahx-R) 4 was expected to increase the metabolic stability of these conjugates and as a consequence to increase their biological efficiency. This assumption has to be tempered since the (R-Ahx-R) repeats are linked by arg-arg peptide bonds which are amenable to proteolysis by trypsin-like enzymes. Indeed recent studies in our group indicated that (R-Ahx-R) 4 -PMO conjugates were rapidly degraded in cells at these arg-arg bonds (11) . We have therefore investigated a (r-Ahx-R) 4 -PMO conjugate (with r standing for D-Arg) in terms of cell uptake and splicing correction activity.
The present structure-activity relationship (SAR) studies were initiated for the following additional reasons. Fluorescence microscopy evaluation of the intracellular distribution of FAM-labeled (R-Ahx-R) 4 -PMO conjugates indicated that the majority of the material was entrapped in endocytic vesicles even at concentrations leading to efficient splicing correction. It implies that the biological activity of these conjugates is due to the small (and not detectable by fluorescence microscopy) portion of material escaping from endocytic vesicles. Finally, (R-Ahx-R) 4 -PMO conjugates have shown signs of toxicity when injected to mice at >20 mg/kg dose despite their absence of cytotoxicity in cell culture experiments (12) . This presents a dosing challenge for in vivo systemic applications.
Altogether, it is clear that CPP-steric block ON conjugates need to be active at lower doses for systemic administration and clinical applications. It is hoped that a better understanding of the structural determinants required for cell binding, cellular uptake and endosome escape will be helpful for the rational design of more potent and/or less cytotoxic CPPs. The manuscript essentially aims at comparing series of (R-Ahx-R) 4 -PMO conjugates analogs differing in Arg spacer length, in hydrophobicity of the spacer and in stereochemistry of Arg. Criteria for the comparative evaluation of these conjugates include cellular uptake, splicing correction efficiency, affinity for heparin, hydrophobicity and synthetic membrane-destabilizing potential.
The antisense PMO (CCT CTT ACC TCA GTT ACA) was synthesized as described (13, 14) . CPPs were synthesized using Fmoc chemistry and purified to >95% as determined by high-pressure liquid chromatograph and MALDI-TOF mass spectrometry analysis. Conjugation, purification and analysis of CPP-PMO conjugates were described previously (3, 15) .
HeLa pLuc705 cells were cultured as exponentially growing subconfluent monolayers in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1 mM Na pyruvate and nonessential amino acids.
To analyze (R-X-R) 4 -PMO conjugates cell internalization, exponentially growing HeLa pLuc705 cells (1.75 Â 10 5 cells seeded and grown overnight in 24-well plates) were incubated in OptiMEM with FAM-labeled (R-X-R) 4 -PMO. Cells were then washed twice with PBS, detached by incubating for 5 min at 378C with 0.5 mg/ml trypsin per 0.35 mM, EDTA.4Na and washed by centrifugation (5 min, 900g) in ice-cold PBS containing 5% FBS. The resulting cell pellet was resuspended in ice-cold PBS containing 0.5% FBS and 0.05 mg/ml propidium iodide (PI) (Molecular Probes, Eugene, OR, USA). Fluorescence analysis was performed with a BD FacsCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Cells stained with PI were excluded from further analysis. A minimum of 20 000 events per sample was analyzed.
(R-X-R) 4 
Three micrograms of each (R-X-R) 4 -PMO conjugate were injected in triplicate on a 1 ml HiTrap Sepharose/heparin column (Amersham Biosciences, Freiburg, Germany) fitted on a Beckman-Gold HPLC chromatograph (Beckman Coulter, Fullerton, CA, USA). The conjugates were eluted in 30 min at a flow rate of 1 ml/min of 2.5 mM phosphate buffer (pH 7) by a linear gradient of NaCl from 70 to 970 mM. Elution of the conjugates was followed by UV absorption at 260 nm. Results are presented as eluting NaCl concentrations and expressed as the mean and standard deviation of triplicate measurements.
Total 0.1 mg of each (R-X-R) 4 -PMO conjugate were injected in triplicate on a C18 Waters Symmetry Shield 4.6 Â 250 mm column and fitted on a Beckman-Gold HPLC chromatograph. The conjugates were eluted at a flow rate of 1 ml/min of H 2 O/0.1% TFA by a linear gradient of acetonitrile from 5% to 95% in 30 min. Elution of the conjugates was followed by UV absorption at 260 nm. Results are presented as eluting acetonitrile concentrations and expressed as the mean and standard deviation of triplicate measurements.
Exponentially growing HeLa pLuc705 cells (1.75 Â 10 5 cells seeded and grown overnight in 24-well plates) were co-incubated with the (R-Ahx-R) 4 -PMO conjugates and with 20 mg/ml saponin for 30 min. The conjugates were removed and the cells were washed twice with PBS and incubation continued for 24 h in complete medium (DMEM plus 10% FBS). Cells were washed twice with ice-cold PBS, lysed with Reporter Lysis Buffer and processed as described above.
To analyze (R-X-R) 4 -PMO conjugates intracellular distribution, exponentially growing HeLa pLuc 705 cells (3.5 Â 10 4 cells seeded and grown overnight in 2 ml culture dishes) were washed with OptiMEM and incubated with 2 mM FAM-labeled (R-X-R) 4 -PMO in the absence or in the presence of 20 mg/ml saponin for 30 min in OptiMEM medium. Cells were then washed with PBS prior a co-incubation step with 10 mg/ml Transferrin-Alexa 546 (red fluorescence) and Hoechst 33342 dye (blue fluorescence) for 10 min in order to stain endosomes and nuclei, respectively. The distribution of fluorescence in live unfixed cells was analyzed on Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss, Obercochen, Germany).
Large unilamellar vesicles (LUV) were prepared as described previously (16) . In short, lipids dissolved in benzene/methanol (95:5) were freeze-dried overnight and the resulting dry lipid powder was hydrated in a buffer containing the ANTS fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (Invitrogen, Carlsbad, CA, USA) together with a DPX quencher p-xylene-bispyridinium bromide (Invitrogen, Carlsbad, CA, USA) at a final lipid concentration of 10 mM. The suspension was vigorously agitated with a Vortex, freeze-thawed 10 times and then extruded 10 times through two stacked 100 nm polycarbonate filters (Nucleopore, Whatman). Free dye and quencher were then removed by gel filtration on a PD-10 desalting column (Amersham Biosciences, Piscataway, NJ, USA). To mimic the lipid composition of late endosomes we used the following lipid mixture: dioleoylphosphatidylcholine (DOPC)/dioleoyl-phosphatidylethanolamine (DOPE)/phosphatidylinositol from soybean (PI)/bis(monooleoylglycero) phosphate (LBPA) (5:2:1:2) (23). All lipids were purchased from Avanti Polar Lipids Inc., Alabaster, AL.
Leakage of ANTS/DPX from the vesicles was measured as an increase in fluorescence intensity of ANTS upon addition of the CPP-PMO conjugates (5 mM final concentration) to 2 ml of vesicles (25 mM) (17) . Infinite dilution of the probe used to determine fluorescence of the completely unquenched probe was achieved by solubilizing the membranes with 0.1% (v/v) Triton X-100.
Most studies on basic amino-acids-rich CPPs emphasized the importance of the guanidinium side chains of arginines and of the spacing between the charged groups. Studies by Rothbard et al. (18) in particular have shown that a 6-carbon 6-aminohexanoic acid linker seemed optimal for cellular uptake as measured by the whole cell fluorescence but no data concerning efficiency in terms of cytoplasmic or nuclear delivery of a biologically functional payload was provided. We therefore designed a series of (R-X-R) 4 -PMO conjugates with X varying from 2 to 8 carbons (compounds 1-7 in Figure 1A ). The present study revealed a dependence of charge spacing with an optimum for (R-Ahx-R) 4 (in which X = 6) in terms of nuclear delivery of the PMO payload as illustrated below. Based on this first set of data, we designed a series of C6 linked-Arg peptides differing in terms of hydrophobicity (compounds 8-11 in Figure 2A ).
Since metabolic stability has often been proposed as a factor governing CPP efficiency, the D-Arg modified (R-Ahx-R) 4 , (r-Ahx-R) 4 (compound 13 in Figure 3A ), has been included.
Finally, we evaluated the splice correcting ability of (R-X-R) n -PMO conjugates with n < 3 as a possible strategy to reduce cytotoxicity.
It is now well admitted that basic CPPs interact with heparan sulfate-rich cell surface glycosaminoglycans before being internalized by endocytosis (19) . A sufficient affinity for these negatively charged proteoglycans is required for cell binding and for subsequent cellular uptake. On the other hand, too much affinity for heparan sulfate might be detrimental for the release of CPP-ON conjugates from endocytic vesicles as hypothesized in our previous publications (3, 10) .
(R-X-R) 4 -PMO conjugates with X spacers of increasing lengths (from 2 to 8 atoms) ( Figure 1A ) have thus been compared in terms of affinity for a model heparan sulfate on a Hi-trap Heparin column ( Figure 1B ). Increasing spacer length leads to decreased affinity as monitored by the NaCl concentration required for elution in keeping with published data (20) . Conjugates in this series were then compared for their ability to promote splicing correction in dose-response experiments ( Figure 1C and data not shown). Increasing the length of the spacer led to increased luciferase expression with an optimum for the C5-linked material.
Increasing the affinity for heparan sulfates thus appears being detrimental for splicing correction efficiency. Along the same lines, (Arg) 9 -PMO has an even higher affinity for heparan sulfate than (R-G-R) 4 -PMO and is less active in splicing correction [(4) and data not shown].
Compound 7 was thus expected to be more active in splicing correction than compound 5 which was not observed ( Figure 1C ). However, increasing the hydrocarbon spacer length also increases hydrophobicity which could itself be promoting unfavorable membrane interactions (see below). Increased hydrophobicity has indeed been verified by C18-column chromatography for compound 7 ( Figure 2B) . Figure 1C .
(D) Heparin affinity chromatography. Samples were treated and data were processed as described in the legend of Figure 1B . ÃÃÃ and ÃÃ Indicate statistically significant differences; NS indicates that the difference is not statistically significant.
PI uptake has been monitored in parallel as an index of cell membrane integrity. No significant PI uptake was seen at doses up to 2.5 mM for any one of these compounds except for compound 7, which leads to a concentrationdependent membrane destabilization ( Figure 4B ). This might also contribute to its lower splicing correction potential.
Since the hydrophobicity of the linker appeared to influence splicing correction efficiency, we have compared a series of PMO conjugates (compounds 8-11 in Figure 2A ) with the same spacing (a 6-carbon atom spacer as in (R-Ahx-R) 4 -PMO) but with varying sidechain hydrophobicities. Figure 1C . 5, (R-Ahx-R) 4 -PMO; 13, (r-Ahx-R) 4 -PMO. (C) Hydrophobicity chromatography. Samples were treated and data were processed as described in the legend of Figure 2B . (D) Heparin affinity chromatography. Samples were treated and data were processed as described in the legend of Figure 1B . ÃÃÃ and ÃÃ Indicate statistically significant differences; NS indicates that the difference is not statistically significant.
Some compounds in this series (11 in Figure 2A ) have hydrophobicities comparable to the parent (R-Ahx-R) 4 -PMO (compound 5) taken as a reference while others (compounds 8-10) have a significantly higher hydrophobicity, as monitored by C18-column chromatography ( Figure 2B ). These conjugates were analyzed for splicing correction efficiency at various concentrations ( Figure 2C and data not shown). Splicing correction efficiency is lower for the more hydrophobic conjugates (compounds 8-10) and remains the most active for compound 5. As expected, compounds 8-11 had similar affinities for heparin ( Figure 2D ). Therefore, differences in splicing correction in this series were largely influenced by hydrophobicity. We cannot explain why compound 11 has a lower splicing correction activity than compound 5 as their hydrophobicity and heparin affinity are similar.
Increased metabolic stability should improve biological efficiency and could in part explain the higher efficacy of (R-Ahx-R) 4 -PMO as compared to (Arg) n -PMO and Tat 48-60 -PMO, as discussed previously (3) . However, the (R-Ahx-R) 4 portion of (R-Ahx-R) 4 -PMO was found to be degraded in intact cells (11) . We therefore synthesized (r-Ahx-R) 4 -PMO (compound 13 in Figure 3A ) in which one of the two L-Arg residues in each R-Ahx-R repeat was replaced by a D-Arg (r) residue. Unexpectedly, (r-Ahx-R) 4 -PMO was significantly less efficient in splicing correction than (R-Ahx-R) 4 -PMO ( Figure 3B ). Both L-and D-Arg-containing peptides have similar hydrophobicities ( Figure 3C ). Interestingly, (r-Ahx-R) 4 -PMO has a signficantly higher affinity for heparan sulfate than the parent (R-Ahx-R) 4 -PMO ( Figure 3D ), thus pointing again to the role played by this parameter in splicing correction efficiency.
As already mentioned, (R-Ahx-R) 4 -PMO conjugates become cytotoxic in murine models at high doses. The cytotoxicity of nonviral delivery vectors for nucleic acids is generally associated to their resulting cationic charge. We therefore investigated whether reducing the number n of repeats in (R-X-R) n -PMO conjugates could be possible. A significant loss of splicing correction efficiency was found with shorter versions of these PMO conjugates as shown for (R-AbuL-R) n -PMO conjugates ( Figure 5 ).
Most (R-X-R) 4 -PMO conjugates were synthesized as fluorescent FAM conjugates to allow assessment of celular uptake by FACS analysis and by fluorescence microscopy. As seen in Figure 4 , there is no correlation between cellular uptake and splicing correction activity. Increasing the spacing between arginine residues (compounds 1-7) leads to decreased cellular uptake in parallel to heparin affinity but on the contrary leads to increased splicing correction. Remarkably, (R-Ahx-R) 4 -PMO which was the most active conjugate in this series in terms of splicing correction turned out the less efficient in terms of cellular uptake. In addition, changing the hydrophobicity of the spacer (compounds 8-11) or modifying the stereochemistry of Arg (compounds 5 and 13) had no significant impact on cellular uptake.
We next examined whether differences in splicing correction activity could be explained by differences in endosomal escape. All (R-X-R) 4 -PMO conjugates have therefore been synthesized as FAM-labeled derivatives and their intracellular distribution has been analyzed by fluorescence microscopy on live cells to avoid artefactual redistribution upon cell fixation. As shown in Figure 6A for the parent (R-Ahx-R) 4 -PMO-FAM conjugate, most of the material was distributed as punctate cytoplasmic material and none was detected in the nuclei. Splicing correction is probably due to the small amount of material which has escaped from the endocytic vesicles and remains undetectable by fluorescence microscopy analysis. Not surprisingly, a similar situation has been observed for other (R-X-R) 4 -PMO-FAM conjugates from our SAR studies and no concluding data have been provided by fluorescence microscopy comparative analysis (data not shown). Likewise, previous work from several groups including our own one had documented an increase in splicing correction upon treatment with endosomolytic agents such as chloroquine or calcium ions (3) . However, splicing correction never reached levels achieved with 2-OMe ON analogs transfected as lipoplexes and accordingly redistribution of the endosome-entrapped material could not be documented (21) . We now capitalize on a saponin treatment protocol which allows to gently permeabilize the plasma membrane. It was shown to open transient holes in the plasma membrane and to allow the passage of macromolecules while not damaging membranes from intracellular organelles (22) .
As shown in Figure 6B , the (R-Ahx-R) 4 -PMO-FAM conjugate was widely distributed within the cell with a clear accumulation in nuclei in saponin-permeabilized cells. These low molecular mass conjugates are indeed expected to diffuse freely and rapidly from the cytoplasm to the nuclei through the nuclear pores. In a different protocol, cells were loaded with Alexa-labeled Transferrin (a known marker of endosomes) and then treated with saponin. At variance with the wide distribution of the CPP-PMO-FAM conjugates, Transferrin-associated red fluorescence remained punctate in keeping with the reported minimal effects of saponin on intracellular architecture ( Figure 6C ). Along the same lines, we have verified that saponin did not lead to a significant release of (R-Ahx-R) 4 -PMO conjugate preloaded in endocytic vesicles (data not shown).
We next compared luciferase expression in doseresponse experiments in saponin-treated and untreated cells. As shown in Figure 7 for the (R-Ahx-R) 4 -PMO conjugate, splicing correction was more efficient in saponin-treated than in untreated cells. Significant luciferase expression could already be detected upon 30 min incubation in saponin-treated cells and increased to a much higher level than in untreated cells. In addition, splicing correction in saponin-treated cells reached similar levels for conjugates found much less active in nonpermeabilized cells than (R-Ahx-R) 4 -PMO ( Figure 7B) .
These data were expected if saponin permeabilization of the plasma membrane allows to bypass endocytosis and as a consequence endosome segregation. Differences in splicing correction efficiency between (R-X-R) 4 -PMO analogs were also expected to be largely abolished if caused by differences in trafficking efficiency.
Our data have confirmed that cellular uptake is not the limiting factor in the efficiency of splicing correction by (R-X-R) 4 -PMO conjugates and therefore intracellular trafficking and endosomal escape likely to be major limiting factors. To evaluate the ability of CPP-PMO conjugates to escape from endosomes we employed a liposome leakage assay. Late endosomes are characterized by a rather unusual lipid composition enriched in LBPA (23) and have a pH 5.5 lumen (24) . We therefore prepared liposomes from a lipid mixture mimicking the lipid composition of late endosomes DOPC/DOPE/PI/LBPA (5:2:1:2) and monitored the effect of low pH on the CPP-PMO induced leakage of a fluorescent dye entrapped in the lipid vesicles. We compared conjugates 1, 5, 9 and (Arg) 8 -PMO. All conjugates induced a fairly modest leakage that was strongly promoted at pH 5.5. In correlation with the data on splicing activity, (R-Ahx-R) 4 -PMO conjugate was by far the most active in this group, followed by (R-G-R) 4 endosomal escape is a major contributing factor for the efficient nuclear delivery of these CPP-PMO conjugates.
New arginine-rich CPPs have recently been proposed for the nuclear delivery of neutral ON analogs as PMO (3) or PNA (10) . They represent a significant improvement over first generation CPPs as Tat, Pen, oligoarginine or oligolysine since they allow a sequence-specific splicing correction at lower concentrations (EC 50 ranging between 0.5 and 2.0 mM) which do not lead to membrane permeabilization and, importantly, do not require endomosomolytic drugs or treatments. Importantly as well, (R-Ahx-R) 4 -PMO conjugates lead to a sustained expression of dystrophin in skeletal muscles when injected intraperitoneally (5 mg/kg) in DMD mice (25, 26) . These encouraging data should however be tempered since these may be still high doses too close to the toxic doses found in other murine models (6) . The present SAR study has therefore been initiated in order to delineate step(s) limiting the splice correcting activity of (R-Ahx-R) 4 -PMO as well as important molecular features of the (R-Ahx-R) 4 -CPP moiety.
Spacing of the guanidinium charged groups in argininerich CPPs has been extensively studied by Wender et al. (20) and found to be a key determinant of their cellular uptake. A series of (R-X-R) 4 -PMO conjugates with a X linker extending from two to eight atoms have been compared in terms of cellular uptake, splicing correction efficiency and affinity for heparin ( Figure 1B and R. Abes and H. Moulton). As expected, heparin (chosen as a model heparan sulfate) affinity decreased significantly with an increase in X linker length and with a decrease in cationic charges density ( Figure 1B ). In keeping with this observation, cellular uptake as monitored by FACS analysis decreased in parallel ( Figure 4) . However, the ranking of these (R-X-R) 4 -PMO conjugates in terms of splicing correction efficiency had a bell-shaped profile with (R-Ahx-R) 4 -PMO being significantly more efficient than (R-G-R) 4 -PMO ( Figure 1C and data not shown). These observations do strongly suggest that another step than cellular uptake is responsible for differences in splicing correction efficiency among these (R-X-R) 4 -PMO conjugates.
Whether too much affinity for heparan sulfates could be detrimental for dissociation of the heparan-bound material in endocytic vesicles and for endosomal escape is a possibilty but it is unfortunately not amenable to direct demonstration. It is worth pointing out that similar conclusions could be drawn from our previous comparison of (Arg) 9 -PMO, Tat-PMO and (R-Ahx-R) 4 -PMO conjugates. (R-Ahx-R) 4 -PMO was found more active in the splicing correction assay despite binding less efficiently to heparin and being taken up less well than the parent (Arg) 9 -PMO and than Tat-PMO (3).
The (R-Acy-R) 4 -PMO conjugate (compound 7 with a C8 linker) did not follow the ranking observed for other conjugates in this series since it had lower heparin-binding affinity but corrected splicing less efficiently than (R-Ahx-R) 4 -PMO. This might be explained by the higher hydrophobicity of its longer spacer as evidenced by an increased retention on a C18-affinity column ( Figure 2B ).
In keeping with this hypothesis, increasing the hydrophobicity of the X linker above a threshold value ( Figure 2B ) while maintaining charge spacing in a series of (R-X-R) 4 -PMO analogs ( Figure 2B ) had little impact on cellular uptake (Figure 4 ) but decreased significantly splicing correction efficiency ( Figure 2C ). Being too hydrophobic might conceivably lead to entrapment into membranes and as a consequence might be detrimental to endosomal release. The parent and most active (R-Ahx-R) 4 -PMO conjugate was rather resistant to proteolytic degradation in serum but was still cleaved by cellular proteases (11) . It was thus anticipated that the (r-Ahx-R) 4 -PMO in which some L-Arg residues have been replaced by their D-analog would become more protease-resistant and as a consequence more active in the splicing correction assay. Unexpectedly, (r-Ahx-R) 4 -PMO was significantly less active than (R-Ahx-R) 4 -PMO thus indicating that metabolic stability is not a limiting factor at least in these in vitro experiments. Whether (r-Ahx-R) 4 -PMO might be of interest for in vivo applications will have to be evaluated using transgenic murine models for splicing correction. Whether the lower biological activity of (r-Ahx-R) 4 -PMO could be due to its increased affinity for heparin is a possibility. Alternatively, earlier work from our group (11) has shown that the peptide part of (R-Ahx-R) 4 -PMO was rapidly degraded in cells thus releasing free PMOs. It is fully possible that the more stable CPP entity may decrease the rate of endosomal release of PMO. Along the same lines, linking a splice correcting PNA and a CPP (R 6 Pen in this case) by a stable linker gave rise to a lower efficiency than in the case of a reducible disulfide linker (27) .
Altogether these SAR studies have pointed to the influence of heparin affinity and hydrophobicity on the splice correcting activity of CPP-PMO conjugates. Remarkably, relatively small changes in these parameters had a rather significant impact on biological activity. Quite clearly, cellular uptake could not be an explanation since, on the contrary, we have observed in some instances an inverse correlation between cellular uptake and biological activity.
In order to determine whether differences in biological activity could be explained by differences in intracellular trafficking, we deliberately permeabilized cells by a brief treatment with saponin, using conditions known to have little impact on the internal cellular architecture (22) . Saponin treatment clearly lead to a complete re-localization of FAM-labeled conjugates ( Figure 6 and data not shown) from a dotted cytoplasmic to an homogeneous nuclear distribution in keeping with a direct membrane translocation in the presence of saponin and an endocytic process in its absence. As expected from these data, splicing correction efficiency was largely increased in saponin-treated cells for all (R-X-R) 4 -PMO (Figure 7 and data not shown) thus indicating that retention within cytoplasmic vesicles remains a major road-block even for the most active of our conjugates.
We thus tentatively conclude at this stage that (R-X-R) 4 -PMO accumulate in cytoplasmic vesicles after binding to cell surface glycosaminoglycans and endocytosis. Differences in splicing correction might thus be primarily due to the efficiency with which these conjugates escape from endocytic vesicles and reach the cytoplasm.
Since a direct evaluation of endosome leakage in intact cells could not be easily monitored, we have capitalized on synthetic lipid vesicles with a lipid composition mimicking that of the late endosomal membrane. We observed a modest but significant fluorescent dye release in the presence of (R-X-R) 4 -PMO. Importantly, the release of the probe was strongly promoted at pH 5.5 that is the characteristic pH for late endosomes. Although preliminary, these studies do indicate that more active (R-X-R) 4 -PMO conjugates in this series destabilize more efficiently these lipid vesicles than less active ones.
In conclusion, our studies support the now wellaccepted scheme of cellular internalization involving initial binding to cell surface glycosaminoglycans, endocytosis and entrapment within cytoplasmic vesicles. Most of the material unfortunately remains segregated in endocytic vesicles even for the most active of our conjugates and efforts should now be geared at improving endosomal escape. Model systems described here might turn rather useful in future SAR studies if pH-dependent liposome destabilization can be correlated with splicing correction efficiency. The most active conjugates will also have to be monitored for their biodisponibility, metabolic stability and biological activity in animal models. Immune Mechanisms Responsible for Vaccination against and Clearance of Mucosal and Lymphatic Norovirus Infection Two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. For noroviruses, the contributions of T cells to viral clearance and vaccination have not been elucidated. We report here that both CD4 and CD8 T cells are required for efficient clearance of primary murine norovirus (MNV) infection from the intestine and intestinal lymph nodes. Further, long-lasting protective immunity was generated by oral live virus vaccination. Systemic vaccination with the MNV capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human Lordsdale virus provided partial protection. Fully effective vaccination required a broad immune response including CD4 T cells, CD8 T cells, and B cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. Perforin, but not interferon gamma, was required for clearance of MNV infection by adoptively transferred T lymphocytes from vaccinated hosts. These studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent CD4 and CD8 T cell responses. More than 90% of epidemic nonbacterial gastroenteritis worldwide can be attributed to human noroviruses (HuNV) [1] [2] [3] . Infection is transmitted fecal-orally, and symptomatic infection is characterized by nausea, vomiting and/or diarrhea lasting 24-48 hours within 24 hours of exposure [4] . Despite the significant costs and morbidity of HuNV infections, no vaccine is currently available. The elderly and individuals in long-term care facilities may be more susceptible to either norovirus infection or norovirusinduced disease [5] and would be an important target population for a norovirus vaccine. The reasons for increased incidence and/ or susceptibility to HuNV disease are unknown. This is due in part to our incomplete understanding of norovirus immunity. The potential to vaccinate against these and related viruses has been demonstrated in gnotobiotic piglets, cats and rabbits [6] [7] [8] , but the immune mechanisms responsible have not been identified. The challenges for vaccine efficacy may be very different between different caliciviruses. For example, variation in MNV strains is significantly less than between HuNV strains [9] . Human volunteer studies demonstrate short-term, but not long-term, protection against homologous, but not heterologous, viral challenge [10] [11] [12] . Since HuNV belong to 3 genogroups (GI, GII and GIV) with many strains in each genogroup [4] , this lack of cross-protection is a challenge for vaccine development. Frequent exposure to noroviruses within short time periods stimulates sustained immunity and resistance to norovirus induced illness [13, 14] . Serum antibody levels in adults reflect susceptibility to infection and do not always correlate with protection [13, 14] . In children, however, serum antibody levels correlate with protection, likely reflecting short-term immunity and recent exposure [15] [16] [17] . A nonfunctional fucosyl transferase gene (FUT2) accounts for a significant proportion, though not all, of resistance to Norwalk virus infection, suggesting that other factors, yet undiscovered, may contribute to norovirus resistance [18, 19] .
In the absence of a cell culture system for HuNV, virus like particles (VLPs) that assemble when the viral capsid protein is expressed have been important for evaluating norovirus immune responses [20] [21] [22] [23] . Studies using Norwalk Virus (GI), Snow Mountain Virus (GII) and HuNoV-HS66 (GII) VLPs to evaluate immunity after infection with live virus or immunization with VLPs orally show production of T cell effector cytokines such as IL-2 and interferon c (IFN-c) and proliferation of norovirus specific T cells after in vitro restimulation with VLPs [24] [25] [26] . These studies show that T cell responses develop, but do not define their role in either clearance of primary infection or resistance to re-challenge.
Together, they suggest the potential for vaccination, but leave open important questions about the effectiveness and longevity of vaccine immune responses, mechanisms of vaccination, the viral protein targets for protective responses, and the potential for cross-protection between distantly related noroviruses.
The identification of the first murine norovirus, MNV, and its propagation in cultured cells provides a facile animal model for studies of norovirus immunity and pathogenesis [27, 28] . MNV, an enteric virus that infects tissues of the gastrointestinal tract, is spread by the fecal-oral route ( [27] and unpublished studies). The MNV genome encodes four open reading frames. ORF1 encodes a polyprotein that is cleaved into individual non-structural proteins similar to the polyprotein of HuNV [29] . ORF2 encodes the major capsid protein VP1 and ORF3 encodes a minor capsid protein.
The existence of a protein product for ORF4 has not been confirmed. In the MNV virion structure, the capsid, like that of human noroviruses, consists of 90 dimers of VP1 [30] . There are differences between the MNV virion and previously reported VLP structures. The MNV protruding domain is lifted off the shell domain by approximately 16 Angstroms and rotated approximately 40 degrees in a clockwise fashion, forming interactions at the P1 base in an infectious virion that have not been observed previously. The existence of these novel aspects of the structure are consistent with the hypothesis that MNV may undergo a capsid maturation process [30] .
Studies of MNV pathogenesis reveal an important role for interferon (IFN) and STAT-1 mediated innate immunity in resistance to infection and MNV induced lethality [27, 31] . The importance of adaptive immunity in control of MNV infection is indicated by the observation that RAG1-/-mice develop persistent MNV infection while wild type (WT) mice can clear infection with some strains of MNV [9, 27, 31] .
While MNV is an efficient enteric virus that infects many mice in research mouse colonies around the world, diarrhea has not been reported after MNV infection. Thus, MNV provides an infection only model for HuNV infection. Viral titers in tissues of infected mice have not been reported to exceed 10 6 PFU/ml, and this highest level of viral titer is obtained after infection of highly susceptible STAT1-/-mice [31] . In RAG1-/-mice and WT mice, viral titers of 10 2 to 10 4 PFU/ml are routinely observed [9, 31] . The availability of a plaque assay for MNV allows the analysis of MNV infection despite these low titers. Some MNV strains persist at a low level in WT mice, while others are cleared from intestine, spleen, liver, mesenteric lymph nodes (MLN) and feces within 7 days of infection [9, 27, 31] . Additionally, in wild type C57BL6/J mice MNV replicates maximally in the distal ileum [9] , in comparison to wild type 129S6/SvEvTac mice where replication occurs in the proximal intestine [31] . The significance of these differences is not known.
Studies of norovirus infection in human volunteers have not specifically investigated whether the infection spreads beyond the intestine to the local lymph nodes, however, it is possible that systemic invasion occurs in humans with chronic conditions or immunosuppressed hosts [32] [33] [34] [35] [36] . Additionally, viremia has been reported in infections of gnotobiotic pigs and calves [26, 37, 38] . Thus, the ability of MNV to spread to tissues other than the intestine after oral infection may not be unique, but the relationship of this aspect of MNV pathogenesis to human infection is not clear. The availability of strains that can be cleared from WT mice, such as MNV1.CW3, provides an opportunity to define the mechanisms responsible for two cardinal aspects of viral immunity: the capacity to effectively clear acute infection and the immune mechanisms responsible for effective vaccination.
B cells and MNV specific antibody are important in the clearance of primary MNV infection [39] , but the role of T cells in clearance and the potential and mechanisms of vaccination against mucosal norovirus challenge are unknown. We show here that vaccination with either live MNV or Venezuelan Equine Encephalitis replicon particles (VRPs) expressing the MNV capsid protein VP1 protect the intestine against re-challenge for at least six months. Live virus was more effective than VRP-mediated vaccination. There was partial cross protection against MNV infection after vaccination with a HuNV capsid protein. We found that both the clearance of primary infection and vaccination require the concerted efforts of CD4 T cells, CD8 T cells, B cells, and that T cells required the effector molecule perforin for maximal impact on MNV infection. The effects of specific immune cell types were tissue specific, differing between ileum and mesenteric lymph nodes. These are the first studies to demonstrate immune mechanisms responsible for norovirus clearance and vaccination.
We first determined whether we could detect short-term immunity to homologous MNV challenge and whether proteins encoded by specific MNV ORFs could elicit effective immunity. VRPs expressing ORF1, ORF2 and ORF3 of MNV1.CW3 and ORF2 of the HuNV Lordsdale (genogroup GII.4) and Chiba (genogroup GI.4) were produced for vaccination experiments. Western blots of VRP-infected cell lysates revealed proteins of appropriate sizes [29, 40] and additionally showed that hyperimmune polyclonal rabbit antisera to MNV [28] cross-reacted at low levels with VLPs from Chiba virus and Lordsdale virus ( Figure S1A ).
WT mice were vaccinated and boosted three weeks later. Two weeks after boosting, mice were challenged with MNV1.CW3 and organs titered for MNV three days later ( Figure 1A ). In these WT mice, maximal MNV replication in the intestinal tract occurs in the distal ileum [9] and viral titers could not be detected in duodenum/ jejunum (data not shown). After oral inoculation with MNV1.CW3, WT mice exhibit detectable viral titers in the distal ileum and the MLN three to five days post-infection [9, 31] . Prior infection with
Human noroviruses are the most common cause of epidemic nonbacterial gastroenteritis in the world. Despite their importance as human pathogens, little is known about how the immune system controls and clears norovirus infection, and the potential and mechanisms of vaccination remain unclear. Here, we used norovirus infection of mice to show that vaccination can provide long-lasting immunity against mucosal norovirus challenge and to identify the types of immune cells that are important in vaccination against norovirus infection. Similarly, we identified the types of immune T cells that are important for clearance of acute infection. Efficient vaccination required all three major arms of adaptive immunity: CD4 T cells, CD8 T cell, and B cells. Importantly, protective vaccination against mucosal challenge was observed after either mucosal or systemic norovirus antigen exposure. The pore-forming molecule perforin was important for T cell-mediated control of norovirus infection. Our study has important implications for understanding adaptive immunity to norovirus infection, and may provide insight into the directions to take in developing a human norovirus vaccine. either MNV1.CW1 (p = 0.0002) or MNV1.CW3 (p = 0.0009) significantly decreased MNV1.CW3 replication in the distal ileum compared to control mice infected with reovirus ( Figure 1B ). Similar decreases were observed in the MLN after vaccination with MNV1.CW1 (p = 0.0001) or MNV1.CW3 (p = 0.0003) ( Figure 1C ) compared to the reovirus controls. Similar results were observed in the spleen (data not shown). There was no statistically significant difference between vaccination with MNV1.CW1 or MNV1.CW3. This demonstrates that a protective secondary immune response develops after clearance of primary MNV infection.
ORF2 VRPs protected against MNV1.CW3 in both distal ileum (p = 0.005) and MLN (p = 0.02) compared to control VRPs expressing hemagglutinin (HA) from a mouse adapted influenza A virus [41] (HA VRP control group). Controls for VRP vaccination also included PBS. HA VRP controls were not significantly different from PBS controls across all experiments and statistical comparisons for VRP vaccination are therefore shown to HA VRP controls. ORF1 VRPs alone in the distal ileum, or in both the distal ileum and MLN when combined with ORF3 VRPs, had a small but statistically significant effect on MNV1.CW3 levels ( Figure 1B and 1C). ORF3 VRPs alone did not confer significant protection ( Figure 1B and 1C). Together these data show that vaccination with either live virus or ORF2 VRPs can confer shortterm protection against MNV challenge.
We next assessed vaccination with heterologous ORF2 proteins. Mice were vaccinated and boosted with VRPs expressing ORF2 from Chiba Virus or Lordsdale virus and challenged with MNV1.CW3. Vaccination with Lordsdale virus capsid led to statistically significant protection against MNV infection in the distal ileum, (p = 0.0007, Figure 1B ) but not the MLN ( Figure 1C ). No significant reduction in MNV titers was seen after immunization with Chiba virus capsid ( Figure 1B and 1C ). Protection after Lordsdale ORF2 VRP vaccination did not correlate with generation of cross-reactive serum IgG in these mice, measured by ELISA, despite the potential for such cross-reactivity revealed by western blot ( Figure S1B ). Fecal extracts from immunized mice yielded no measurable homotypic or heterotypic IgG or IgA (data not shown). Taken together, these data show that there is measurable functional immunologic cross protection between Lordsdale virus and MNV in the distal ileum. The lack of a correlation between serum or fecal antibody responses and protection suggested that protection may be T cell mediated.
Since older adults may be more susceptible than younger adults to norovirus infection or disease [5] , we determined whether increased age altered vaccine efficacy. Prior work has shown that mice older than 1 year of age have diminished vaccine responses to SARS virus antigens [42] . We therefore compared vaccine efficacy in adult (8 week old) and aged (14 month old) mice. Adult and aged mice were vaccinated and challenged as before. In contrast to studies using SARS virus antigens [42] , aged mice responded as well as adult mice to MNV ORF2 vaccination in both the distal ileum and MLN ( Figure 1D and 1E). Despite this protective effect, sera from vaccinated aged mice had significantly lower anti-MNV ORF2 IgG compared to adult mice ( Figure S1C ). These data indicated that protection against MNV infection occurred in the absence of robust serologic responses, again raising the possibility that T cells play a fundamentally important role in vaccination against MNV.
We next determined whether protection conferred by MNV1.CW3 or MNV ORF2 VRPs was long lived. WT mice were primed and boosted as shown in Figure 2A with MNV1.CW3 or MNV ORF2 VRPs. Mice were then challenged with MNV1.CW3 two, four, 14, or 24 weeks later and MNV titers measured three days post-challenge. Two weeks post-boost, we observed complete protection against ileal MNV1.CW3 infection after vaccination with either MNV1.CW3 (p = 0.0001) or ORF2 VRPs (p,0.0001) compared to reovirus or HA VRP controls ( Figure 2B ). At two weeks, while vaccination with either MNV1.CW3 or ORF2 VRPs limited MNV1.CW3 replication in MLN, live virus vaccination was more effective (p,0.0001) ( Figure 2C ). Live virus vaccination conferred full protection against MNV1.CW3 replication in both the distal ileum and the MLN at four, 14 and 24 weeks after vaccine boost. Vaccination with ORF2 VRPs was also protective, albeit less effective than vaccination with MNV1.CW3 ( Figure 2B and 2C). Thus both live virus and subunit vaccine induce long-term protection against MNV infection, with live virus vaccination providing more complete protection.
We next determined the mechanism(s) responsible for effective vaccination. We vaccinated mice lacking both major histocompatibility complex (MHC) Class I and b2 microglobulin (b2M) [43] (CD8 T cells deficiency [44] ), MHC Class II (CD4 T cells deficiency [45] ), or B cell deficient mice [46] ( Figure 3A) . These experiments were conducted concurrently with the experiments in Figure 2 above, as such the data from WT mice are repeated in the figure for comparison. Live MNV vaccination induced significant protection against MNV challenge in both the distal ileum and the MLN of B cell-/-, MHC Class II-/-and MHC Class I6b2M-/-mice (p,0.05 in all cases, Figure 3B and 3C). However, there was considerable variation in the efficacy of vaccination in distal ileum and MLN between different immunodeficient strains. In B cell-/-mice, after vaccination with live virus, only 2 out of 15 mice had any titer (and those two mice had less than 100 PFU of MNV) and in MHC Class I6b2M-/-mice, similar vaccination led to undetectable viral titers in the distal ileum ( Figure 3B ) but detectable titers in the MLN ( Figure 3C ). In MHC Class II-/-mice, there were detectable titers in both tissues ( Figure 3B and 3C). Results for ORF2 vaccination showed that protection required the activity of all major aspects of the adaptive immune response ( Figure 3B and 3C). Moreover, there was no protection elicited by ORF2 vaccination in either intestine or MLN tissue after vaccination of MHC Class I6b2M-/-mice with ORF2 VRPs (Figure 3B and 3C) indicating that protection by VRPs critically depends on CD8 T cells. These data demonstrated that complete protection in all tissues after vaccination with live virus required the concerted actions of B cells, MHC Class II, MHC Class I and b2M. Further, the results were consistent with tissue specific roles for B cells, CD4 T cells and CD8 T cells in the development of complete protection against MNV infection.
We next determined whether the same cell types that were required for vaccination were also required for efficient clearance of acute infection. We focused on the role of T cells in clearance since the role of B cells in clearance has already been demonstrated [39] . To determine the role of T cells in clearance of acute MNV infection we inoculated WT, MHC Class II-/-, and MHC Class I6b2M-/-mice orally with MNV1.CW3 and measured viral titers in the distal ileum and MLN three, five, seven and 21 days post-infection ( Figure 4B-4E) .
There was no significant difference in viral titer between MHC Class I6b2M-/-mice and WT mice at three and five days postinfection, indicating that MHC Class I and b2M were not required in MNV infection at early time points ( Figure 4B II-/-compared to WT mice at seven days post-infection (p = 0.04, Figure 4E ). By eight days post-infection, MLN infection was cleared. Together these data indicated that MHC Class II, and by inference CD4 T cells, were necessary for control of acute MNV infection but are not required for eventual clearance of MNV infection.
To exclude the possibility that the phenotypes we observed in MHC Class I6b2M-/-and MHC Class II-/-mice were due to abnormal immune ontogeny in knockout mice, we determined the requirement for CD4 and CD8 T cells in the clearance of primary MNV infection in WT mice depleted of CD4 and CD8 T cells. Depletion of CD4 and CD8 T cells was at least 90% effective as assessed by flow cytometry of isolated splenocytes ( Figure 5A ) and this depletion protocol is effective at depleting T cells in secondary lymphoid organs and the intestine [47, 48] . In comparison to control antibody, depletion of CD4 T cells, led to a significant increase in MNV titers in the distal ileum (p = 0.0053, Figure 5B ), but not the MLN ( Figure 5C ). In contrast, depletion of CD8 T cells led to an increase in MNV titers in both the distal ileum (p = 0.004, Figure 5B ), and the MLN (p = 0.0025, Figure 5C ).
Together, these data from primary challenges of non-immune mice lacking antigen presenting molecules or depleted of specific T cell subsets demonstrated that CD4 T cells are important for efficient MNV clearance in the distal ileum especially at days three 
We next determined whether CD4 and CD8 T cells from vaccinated mice can, alone or in combination, clear MNV infection from mucosal sites. We have previously shown that MNV infected RAG1-/-mice have high levels of viral RNA present in multiple tissues up to 90 days post-infection [27] . We therefore determined MNV viral titers in RAG1-/-mice. By 42 days postinfection, all RAG1-/-mice had consistent, high levels of MNV in both duodenum/jejunum and distal ileum ( Figure 6A and 6B) , as well as several other tissues (data not shown). These data confirmed that mice lacking adaptive immunity fail to clear MNV infection [27] .
The availability of persistently infected RAG1-/-mice allowed us to determine the role of CD4 and CD8 T cells in clearance of MNV infection using adoptive transfer of splenocytes from MNV immune WT mice into persistently infected RAG1-/-mice. Transfer of immune, but not non-immune, splenocytes signifi- To define which cells were required for MNV clearance, CD4 or CD8 T cells were depleted from splenocytes transferred into RAG1-/-recipients. Anti-T cell antibodies effectively depleted the appropriate T cell populations, as measured six days post-transfer by flow cytometry (Figure 7A ). Depletion of either CD4 or CD8 T cells individually led to a significant increase in MNV titers in duodenum/jejunum compared to control depletion ( Figure 7B , CD4 depletion p = 0.0042; CD8 depletion p = 0.0002). Depletion of both CD4 and CD8 T cells from transferred immune splenocytes caused a significant additional increase in MNV titers when compared to either CD4 depletion alone (p = 0.02) or CD8 depletion alone (p = 0.03). In the distal ileum, depletion of either CD4 T cells (p = 0.0003) or CD8 T cells (p,0.0001) led to a significant increase in MNV titers ( Figure 7C ). These data demonstrated that both immune CD4 and CD8 T cells were necessary for clearance of persistent MNV infection from the intestine.
Two major effector mechanisms for the antiviral effects of T cells are the production of IFNc and perforin mediated cytolysis [49] . We therefore adoptively transferred immune splenocytes from IFNc-/-or perforin-/-mice into persistently infected RAG1-/-mice and determined their capacity to clear intestinal MNV infection. Immune splenocytes from IFNc-/-mice were as effective as those from WT mice ( Figure 7B and 7C) . However, immune splenocytes from perforin-/-mice were less effective at clearing MNV infection from the duodenum/jejunum (p = 0.0003, Figure 7C ) or distal ileum (p = 0.0075, Figure 7B ) than cells from either WT or IFNc-/-mice, but more effective compared to transfer of non-immune cells in duodenum/jejunum (p = 0.0086) or distal ileum (p = 0.0001). Thus, while perforin was critical for efficient clearance of MNV infection from the intestine, it was not the only relevant effector mechanism.
In this paper we define the mechanisms of immunity to norovirus infection as measured by both vaccination against, and clearance of, mucosal infection. We found that it is possible to generate highly effective, and remarkably long lasting, immunity to norovirus infection by oral exposure to live virus. Further, systemic exposure to the viral capsid protein expressed in a vaccine vector resulted in effective immunity, albeit not as effective as that observed after live virus vaccination. Importantly, this shows that the MNV VP1 protein contains relevant B cell, CD4 T cell and CD8 T cell epitopes. Vaccination was effective in aged mice. Additionally, vaccination in adult mice required the concerted action of CD4 T cells, CD8 T cell, and B cells to be completely protective in the tissues surveyed. Interestingly, the activities of different components of the adaptive immune system in clearance and vaccination were tissue specific, with different cells playing roles in the intestine itself compared to the draining lymph nodes. Perforin was an important effector molecule. These data have important implications for understanding adaptive immunity to an animal norovirus, representative of a genus that causes significant disease in humans.
HuNV infection and disease is rapid, with symptoms developing within 24-48 hours of infection and lasting for a few days. Thus, we selected three days after challenge as a readout for infection in our studies, since relevant vaccine-generated immune responses would have to act very early after challenge. Lack of any of the three components of the adaptive response: B cells, CD4 T cells, or CD8 T cells significantly diminished vaccine effects generated by either live virus or VP1 capsid protein immunization, and delayed viral clearance during primary infection. This indicates that VP1 has antibody epitopes as well as MHC H-2b restricted CD4 and CD8 T cell epitopes. These data suggest that it may be necessary to engage the concerted actions of an intact immune response including both MHC Class I and MHC Class II restricted T cells and antibody responses to efficiently vaccinate against HuNV infection.
The protection against MNV infection in aged mice in the absence of robust generation of anti-MNV antibodies raised the possibility that an important component of the vaccine response is T cell dependent, a hypothesis borne out in adoptive transfer studies. Importantly, the antiviral effector perforin is important in the clearance of MNV from the intestine, suggesting that the cytotoxic T cell response is a key effector mechanism. It is possible that other cell types such as NK cells might also use perforin as a mechanism to control MNV infection. Our data do not rule out a role for IFNc in clearance of MNV infection since NK cells in recipient RAG1-/-mice can make IFNc, but do suggest that T cell derived IFNc plays at most a minor role in effector T cell function in the ileum. This argues that classical CTL assays may be a good surrogate for the development of effective vaccine-generated immune responses to HuNV.
Live virus vaccination was more effective than VRP based vaccination. The lower level of protection that we observed with ORF2 VRPs in contrast to MNV1.CW3 may be due to many factors, and this study does not provide mechanistic insights into this difference. In comparison to VLPs, VRPs may have advantages in systemic vaccination including targeting dendritic cells and intrinsic adjuvant activities [50] . These properties of VRPs may be responsible for the effectiveness of systemic single protein subunit vaccination against mucosal viral challenge in this case. However, it may be that because VRPs undergo a single round of replication at the site of inoculation they cannot generate the same breadth of immunity that is generated by live replicating virus. While VRP vaccination clearly induces some relevant effector and memory cell responses, vaccination with capsid alone may not sufficient to generate the complete antigenic repertoire required for effective immunity. Interestingly, we found some protection with the non-structural ORF1 polyprotein, suggesting that protective epitopes exist outside of the capsid protein. As the ORF1 polyprotein is expressed early after infection, it may be that these epitopes would be valuable targets for generating an efficient immune response.
Of note, vaccination with VP1 via the subcutaneous route provided significant protection despite the fact that the vaccination occurred systemically, while protection was read out at a mucosal site. This indicates that an active systemic immune response can provide protection against norovirus infection, and a mucosal vaccine may not be necessary to vaccinate against norovirus infection. Importantly, systemic vaccination was dependent on T cells, indicating that the relevant cells can traffic to the intestine after peripheral VRP-based vaccination.
These studies leave several important questions unanswered. Firstly, we used a homologous virus challenge. In nature, it is likely that hosts are repeatedly challenged with antigenically distinct noroviruses. However, the mouse norovirus strains identified so far fall into a single genogroup, GV, which likely represents a single serotype [9] . In this way murine noroviruses identified to date may present less of a challenge for the immune system than HuNV, which are distributed across 3 genogroups and appear to evolve under antibody selection [51] . In addition, we selected a strain of MNV that is cleared by WT mice. Other strains persist for prolonged periods of up to 35 days [9] . It remains to be determined whether vaccination will be effective against persistent MNV strains. It is interesting that human noroviruses can persist beyond the time frame of usual clinical symptoms [52] [53] [54] [55] . Longterm persistence might contribute to explaining the sporadic epidemics of infection in the absence of an animal reservoir. Antigenic and pathogenetic complexity will likely be a major issue for the development of norovirus vaccines. The lack of comparable variation in MNV strains limits the utility of the MNV model for assessing immunity to antigenically distinct strains. Perhaps this limitation will be overcome as additional strains of MNV are identified, sequenced, and studied. However, the fact that we observed partial cross protection between MNV and one HuNV, and the demonstration that vaccination with many different VLPs can enhance generation of cross reactive antibodies [56] provide some encouragement.
There are two ways in which murine norovirus infection may not represent the same biology as HuNV infection. The first is the lack of diarrhea in mice infected with the strains of MNV used here. It is possible that the adaptive responses that clear MNV from the intestine demonstrated here are irrelevant to the responses that may prevent human disease. In this regard, it is important to note that studies of adult mice with rotaviruses (also an infection only model), have been important to our consider-ations of rotavirus vaccines [57] . Importantly, human studies may not reveal the mechanisms of effective immunity and are based on surrogate assays of immunity, since invasive sampling of tissues may be technically difficult. Studies in piglets may be revealing since piglets develop diarrhea when infected with the HuNV strain, HuNoV-HS66 [37] . However, it is more difficult to study immune mechanisms in this system. Thus, we are left with several imperfect systems for considering what one should seek in a HuNV vaccine. Our studies in mice argue for a vaccine that induces all aspects of the adaptive immune response, and that assays for cytotoxic lymphocyte responses to HuNV infection may be an important surrogate assay for protection.
The second aspect of murine norovirus infection that is of unknown relevance to human infection is the impressive capacity of MNV to infect lymph nodes draining the intestine (this paper and [31, 58, 59] ). This may be related to the tropism of MNV for dendritic cells and macrophages [28, 59] and likely reflects spread of MNV directly from the intestine, but may also reflect seeding of the MLN from systemic sites. Considering the distal ileum alone, B cells and MHC Class I and b2M were not required for live virus vaccination, and there was significant, but incomplete, protection in MHC Class II-/-mice ( Figure 3B and 3C). Consistent with this, studies of primary clearance showed that any single arm of the adaptive response was dispensable for ultimate control of primary infection in the intestine. However, vaccination-mediated control of infection in the MLN, and clearance of primary infection from the MLN [39] , required B cells. This differential requirement for components of the immune response in different organs raises an important question about norovirus pathogenesis and lymphoid infection: are the cells infected in intestine and MLN the same? Differences in viral tropism in the two tissues might explain the differential requirement for B cells between ileum and MLN, indicating the importance of future studies on the role of immunity in norovirus cell and organ tropism.
Viruses, Viral Stocks, VRPs, Plaque Assays MNV strains MNV1.CW3 or MNV1.CW1 were used in all virus infections [9, 28, 31] . Two mutations (that result in changes in the encoded amino acids) distinguish the genomes of MNV1.CW3 and MNV1.CW1 [28] . To generate a concentrated virus stock, RAW 264.7 cells (ATCC, Manassas, VA) were infected in VP-SFM media (Gibco, Carlsbad, CA) for 2 days at a multiplicity of infection (MOI) of 0.05. Supernatants were clarified by low-speed centrifugation for 20 min at 3,000 rpm. Virus was concentrated by centrifugation at 4uC for 3 h at 27,000 rpm (90,000 g) in a SW32 rotor. Viral pellets were resuspended in PBS and titered on RAW 264.7 cells as previously described [28] . Type I Lang reovirus was kindly provided by Dr. Terrence S. Dermody (Vanderbilt University, Nashville, TN). Plaque assays were performed as previously described [28] with the following modifications. Tissues were harvested into sterile, screw-top 2-ml tubes containing 500 ml of 1-mm zirconia/silica beads (BioSpec Products, Bartlesville, OK) and stored at 280uC. To obtain viral titers in these tissues 1 ml of complete DMEM was added to each sample on ice and homogenized using a MagNA Lyser (Roche Applied Science, Hague Road, IN) prior to plaque assay. The limit of detection was 20 plaque forming units (PFU)/ml. All VRPs were produced as previously described [60] . Briefly, ORFs 1, 2 and 3 from MNV1.CW3 and ORF2 from Lordsdale virus and Chiba virus were each cloned into VRP expression vectors. Following infection of BHK cells with VRPs for 24 h, culture supernatants were harvested and cells lysed. Proteins were separated by SDS-PAGE and analyzed by western blot with polyclonal rabbit anti-MNV serum [28] . VRP titers and efficient expression of recombinant protein were determined by immunofluorescence assay using mouse antisera generated from inoculation with respective antigens. Cell lysates from MNV ORF2, Chiba virus and Lordsdale virus VRP-infected cell cultures were further purified to obtain VLPs [56] .
RAW 264.7 cells were maintained as previously described [28] . Monoclonal antibodies (MAbs) specific to CD4 (YTS191.1 [61] ), CD8 (H35 [62] ) and SFR3-DR5 (ATCC HB-151 [63] ) were produced from hybridoma cell lines in INTEGRA Celline CL1000 flasks (Integra Biosciences, Ijamsville, MD) using CD Hybridoma media (Gibco, Carlsbad, CA) as previously described [64] . All mice (or cage sentinel mice for mice deficient in antibody production) were tested by ELISA for the presence of MNV antibody prior to experiments [27] . All mice used in these studies were seronegative at the initiation of experiments.
Mice used in vaccination studies were immunized with 3610 7 PFU of MNV1.CW1 [28] , MNV1.CW3 [31] , or control Type I Lang reovirus per orally (p.o.) in 25 ml of DMEM containing 10% fetal bovine serum (Hyclone, Logan, UT) (cDMEM). VRP immunizations were with 2.5610 6 infectious units (IU) of each VRP expressing MNV1.CW3 ORF1, ORF2, or ORF3 individually or in groups of 2-3 VRPs; Chiba virus ORF2 or Lordsdale virus ORF2 in 10 ml or 50 ml volume by footpad inoculation (into the subcutaneous space) [65] on day 0 and boosted on day 21. HA VRP and PBS immunizations in 10 ml or 50 ml volume by footpad inoculation [65] on day 0 and boosted on day 21 served as controls for all VRP immunizations. Mice were challenged with 3610 7 PFU of MNV1.CW3 at specified times after boost and tissues harvested three days post-challenge. Controls for VRP vaccination included PBS or VRPs expressing hemagglutinin (HA) from a mouse adapted influenza A virus [41] . PBS served as a control for HA VRP in these experiments in the event that HA VRP had a significant effect on MNV replication. HA VRP controls were not significantly different from PBS controls in all experiments and both are presented in all figures for completeness.
RAG1-/-and all splenocyte donor mice were infected with 3610 6 PFU of MNV1.CW3 p.o. in 25 ml of cDMEM. All other mice were infected with 3610 7 PFU MNV1.CW3 p.o. In RAG1-/-mice two segments of the small intestine were harvested: a one inch section of the small intestine immediately distal to the pylorus of the stomach, (designated the duodenum/jejunum), and a one inch section of the small intestine immediately proximal to the cecum (designated the distal ileum). In all other mice the distal ileum and three mesenteric lymph nodes (MLN) were harvested. With the exception of RAG1-/-mice (inoculated at 4-6 weeks of age) and aged WT mice (inoculated at 14 months of age), all other mice were inoculated at 6-10 weeks of age.
Spleens were harvested from mice and single cell suspensions were generated [65] . Cells were counted and diluted in RPMI-1640 media (Sigma, Saint Louis, MO) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 100 U penicillin/ml, 100 mg/ml streptomycin, 10 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid), 1mM sodium pyruvate, 50 mM 2-mercaptoethanol and 2 mM L-glutamine (cRPMI). In all adoptive transfer experiments, 1610 7 cells were injected into persistently infected RAG1-/-mice by intraperitoneal (i.p.) injection in 0.5ml cRPMI.
For depletions in WT mice, 500 mg of lymphocyte-depleting antibody or an isotype-matched control antibody [SFR3-DR5, IgG2b] was administered i.p. one day prior and one day after infection. For depletions in adoptive transfer experiments, depleting antibodies were administered to RAG1-/-recipients as described above with one dose one day prior to splenocyte transfer and a second dose on the day of trnsfer. The efficacy of lymphocyte depletion in both sets of depletion experiments was monitored by flow cytometric analysis of splenocytes at the end of the experiment.
ELISA to detect binding of polyclonal anti-serum or fecal extract-derived antibody to purified MNV virions or MNV VLPs was performed as previously described [27, 56] .
All data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). Viral titer data were analyzed with the nonparametric Mann-Whitney test. All differences not specifically stated to be significant were insignificant (p.0.05).  Self-Interest versus Group-Interest in Antiviral Control Antiviral agents have been hailed to hold considerable promise for the treatment and prevention of emerging viral diseases like H5N1 avian influenza and SARS. However, antiviral drugs are not completely harmless, and the conditions under which individuals are willing to participate in a large-scale antiviral drug treatment program are as yet unknown. We provide population dynamical and game theoretical analyses of large-scale prophylactic antiviral treatment programs. Throughout we compare the antiviral control strategy that is optimal from the public health perspective with the control strategy that would evolve if individuals make their own, rational decisions. To this end we investigate the conditions under which a large-scale antiviral control program can prevent an epidemic, and we analyze at what point in an unfolding epidemic the risk of infection starts to outweigh the cost of antiviral treatment. This enables investigation of how the optimal control strategy is moulded by the efficacy of antiviral drugs, the risk of mortality by antiviral prophylaxis, and the transmissibility of the pathogen. Our analyses show that there can be a strong incentive for an individual to take less antiviral drugs than is optimal from the public health perspective. In particular, when public health asks for early and aggressive control to prevent or curb an emerging pathogen, for the individual antiviral drug treatment is attractive only when the risk of infection has become non-negligible. It is even possible that from a public health perspective a situation in which everybody takes antiviral drugs is optimal, while the process of individual choice leads to a situation where nobody is willing to take antiviral drugs. Recent outbreaks of SARS and H5N1 influenza have underlined the threat that viruses in the animal reservoir pose to the human population. Fortunately, neither SARS nor H5N1 influenza have become endemic in humans. Nevertheless, these and other events have stressed the importance of being prepared for emerging and reemerging infectious diseases.
For most infectious diseases vaccination is the preferred control measure. Indeed, vaccines generally have proven highly efficacious, providing strong and long-lasting immunity against infection, disease, and transmission. However, in case of a previously unknown infectious disease a vaccine may not be readily available. For such emerging infectious diseases the control options are limited. This is especially so for viral pathogens that cannot be treated by effective antimicrobial drugs. For these pathogens the control options are restricted to case isolation and contact tracing, promotion of changes in behavior, vaccination using vaccines with poor efficacy, and antiviral drugs. Although the efficacy of currently available antiviral drugs is also far from perfect and although antiviral drugs provide protection for a short amount of time only, an advantage of antiviral drugs over vaccines is their broad spectrum of action [1] [2] [3] [4] [5] .
For a newly arising viral infectious disease it may be possible to contain an outbreak at an early stage by means of a large-scale antiviral control program if the control program is started early and has high compliance rates, if the efficacy of antiviral drugs is sufficiently high, and if the transmissibility of the pathogen is sufficiently low [6] [7] [8] . Hence, it seems logical that all efforts should be geared towards early control of an outbreak. However, whether people will cooperate with such a containment strategy is not known. Probably, the willingness to participate in a control program depends on the (perceived) risk of infection and the consequences of the subsequent disease as compared to the (perceived) risk of taking antiviral drugs. If there are adverse effects of taking antiviral drugs it may well be that people will only be inclined to start taking antiviral drugs once the risk of infection becomes non-negligible.
In this paper we employ population dynamical and game theoretical analyses to investigate (i) under which conditions an antiviral control program can prevent an epidemic, and (ii) when people should consider taking antiviral drugs. With regard to the latter question we take two perspectives. First, we focus on the public health officer whose goal it is to minimize the total amount of damage caused by both infection and prophylactic antiviral treatment. In a second step we then compare the strategy that is optimal from the point of view of the population as a whole with the strategy that would evolve if individuals pursue their own interest.
The dilemma that an individual faces is the following. Should you take your chances and refuse antiviral prophylaxis? The price that you may have to pay upon infection may be high (death in its most extreme consequence). The potential reward is that once you have successfully recovered from infection you also reap the benefit of long-lasting immunity. The alternative is that you take antiviral drugs and so avoid the potentially high cost of infection. The drawback of this option is that you may have to take antivirals for a prolonged period of time. This has negative side-effects in the short term [9] , and may in the long run also not be harmless.
The situation is complicated by the fact that an individual's risk of infection does not only depend on whether or not the individual itself decides to take antiviral drugs but also on the decision of others. In the context of vaccination it is well known that in such a situation there can be a trend of decreasing willingness to participate in a control program, which will lead to strategies that are not optimal from the population perspective [10] [11] [12] [13] [14] . Here we ask whether similar phenomena occur in case of antiviral prophylaxis. While vaccination usually provides long-lasting and strong immunity after one or a few vaccination bouts, antiviral prophylactic therapy relies on the continuous administration of drugs. This implies that, in contrast with vaccination, individuals have more opportunities to adjust their actions to the situation in which they face themselves. Further, while the earlier studies focus on the relative perceived risk of infection as compared to vaccination, we consider infections and antiviral drugs that induce a real, albeit possibly small, risk of death. Throughout, our aim is to decipher how the optimal antiviral prophylactic control strategy is moulded by the transmissibility of the pathogen, by the risk associated with antiviral prophylaxis, and by the efficacy of antiviral drugs in reducing susceptibility, infectiousness, and mortality.
We would like to stress from the onset that we strive more for conceptual clarification than for the most precise representation of a specific system. In particular, all our analyses are based on the simplifying assumptions that individuals act rationally, have perfect information and foresight while they do not engage in projecting the epidemic, and that the details of population structure play a minor role. We are aware that these simplifying assumptions cannot be neglected in the real world, and we therefore do not believe that our model is suited to make quantitative predictions for any specific emerging infectious disease. Rather, our models serve a purpose by laying out, in an idealized context, the key factors shaping the interests of individuals and public health officers. In case of influenza vaccination others have investigated models with added layers of complexity with the goal to make quantitative predictions [13] .
Stochastic and deterministic SIRV-type epidemic models in which individuals are susceptible (S), infected and infectious (I 1 or I 2 ), recovered and immune (R), or (partially) protected against infection by antiviral prophylactic treatment (V) form the basis of the analyses. Figure 1 shows a schematic of the model. Details are given in Text S1. Throughout susceptible individuals enter the population by birth. The (natural) death rate of susceptible and recovered individuals is denoted by m, and the excess mortality while on antiviral treatment is given by c. Hence, life expectancy is m 21 in the absence of infection and antiviral control, and (m+c) 21 while on antiviral drugs. In the infected classes (I 1 and I 2 ) the excess death rates resulting from infection are given by n and n(12AVE I ), where AVE I is the antiviral efficacy for infectiousness and virulence. In the following, c and n will be referred to succinctly as the cost of antiviral prophylaxis and infection. From the susceptible class individuals move to the protected and infected classes at rates s and l. The parameter l is colloquially called the force of infection, and it depends on the prevalence of infection (Text S1). Individuals in class V are infected at a reduced rate l(12AVE S ), where AVE S denotes the antiviral efficacy for susceptibility. This implies that individuals in class V cannot be infected at all if AVE S = 1, while the antiviral drug provides no protection against infection if AVE S = 0. Finally, the rates of recovery and non-compliance are given by a and r, respectively. An overview of the model parameters and their default values is given in Table 1 . Details of the model analyses are provided in Text S1.
When will a prophylactic antiviral control program be able to prevent an epidemic? Several studies have addressed this question using simulations of metapopulation models that include household structure and other population and pathogen details [6] [7] [8] .
Here we focus on a basic model for a large well-mixed population. To evaluate whether successful invasion of the pathogen is possible we calculate the (basic) reproduction number (denoted by R 0 ), which gives the number of new infections caused by a single infected individual in a population of uninfected individuals in the early stages of an outbreak [15] . If R 0 .1 the pathogen can invade a population in which it is not yet present, while it cannot if R 0 ,1. In the case that the pathogen-induced mortality is such that it hardly affects the infectious period, the reproduction number is given by
(see Text S1 for a derivation). The first factor in equation ( Equation (1) shows that the pathogen cannot invade the population if the rate of antiviral prophylaxis exceeds a certain critical rate of antiviral prophylactic therapy s c , which is given by the solution of the equation R 0 = 1. For the default parameter values (Table 1 ) it turns out that the critical rate of antiviral prophylaxis is s c = 1.96 (yr 21 ) if antiviral drugs provide complete protection against infection (AVE S = 1). This implies that approximately two-thirds of the population needs to be protected against infection by antiviral prophylaxis in order to prevent an epidemic. This fraction increases if antiviral drugs provide partial protection against infection and subsequent transmission.
Is it possible to prevent a major epidemic with an antiviral response that is started quickly after an introduction of the pathogen? To answer this question we performed stochastic simulations in which an antiviral response is initiated after a certain number of individuals are infected (see Text S1 for details). Figure 2 shows three representative simulation runs of an epidemic in a large but finite population (10 6 individuals). If no control measures are put into place (top panel), epidemiological theory informs that a large epidemic will unfold with probability 1{R {1 0 &0:67, and the fraction of individuals that is infected once the epidemic has taken off is roughly given by the solution of the final size equation (log(12x) = 2R 0 x) [15] . For the default parameter values this means that 94% of the population will be infected, of which some 2% will die from the sequelae of infection. In a population of one million individuals this implies that more than 18,000 individuals will die during the course of an epidemic.
The situation is completely different if a large-scale antiviral prophylactic control program is initiated once a certain number of individuals is infected. The middle and bottom panels of Figure 2 show simulations in case of a perfect and imperfect antiviral drug, respectively. In both panels we assume that antiviral control is started once 100 individuals are infected, and that no individuals are exempted from antiviral drug treatment. The middle panel shows that even though the cost of antiviral prophylaxis is much smaller than the cost of infection ( Table 1 ) the number of individuals that has died by antiviral treatment at the end of the epidemic equals the number that has died from infection (8 individuals) . If the antiviral drug is imperfect (bottom panel), the duration of the epidemic is considerably longer, and many more individuals will have died from antiviral treatment than from infection (22 versus 6) . Still, the total number of deaths is orders of magnitude smaller than in the case that no antiviral control program is effective.
The simulations of Figure 2 illustrate two general phenomena. First, the number of infections and deaths is reduced dramatically by an antiviral control program that is able to successfully contain an epidemic [6] [7] [8] . Second, while the number of deaths caused by infection is proportional to the number of infected individuals (which is relatively small at an early stage of an epidemic), the number of deaths related to antiviral prophylaxis is proportional to the number of individuals that have taken antiviral drugs. The latter may be quite large, and is probably on the order of total population size. Hence, even though the individual risk of antiviral prophylaxis is small and large-scale antiviral prophylactic control appears to be the rational strategy, it may well be that the number of deaths induced by antiviral treatment exceeds the number of deaths induced by infection. Motivated by these observations we investigate in the following (i) the conditions under which a largescale antiviral control program is able to halt an epidemic, and (ii) the conditions under which rational individuals are willing to take antiviral drugs.
At what point in an unfolding epidemic does the risk of infection exceed the risk of antiviral treatment? This question is relevant because it determines the incentive for individuals to take antiviral drugs. We focus on the probability that an individual is alive after a certain time (the horizon) given that it is initially susceptible or (partially) protected against infection by antiviral control. If the probability to remain alive is larger when initially susceptible than when under antiviral treatment, the best option is not to take antiviral drugs. Otherwise the reverse is true. The formal analyses are given in Text S1. Here we summarize the main findings. 
Now let us suppose that attempts to control an outbreak at an early stage have been unsuccessful. In this case it is still of interest to determine whether and under which conditions antiviral prophylaxis should be part of a strategy aimed at pathogen eradication or containment. If antiviral treatment provides complete protection against infection (AVE S = 1) the equilibrium prevalence of infection decreases monotonically with increasing rate of antiviral control, up to the point where the pathogen cannot persist (Text S1). Furthermore, as long as the risk of antiviral prophylaxis remains small its precise value hardly affects the prevalence of infection. This is due to the fact that mortality related to antiviral treatment is negligible in comparison with natural mortality. 
i.e. if the cost of antiviral prophylaxis is small compared to the cost of infection. This implies that at equilibrium excess mortality is lowest at the point where the pathogen is just driven to extinction. If, on the other hand, inequality (2) is reversed, excess mortality increases with increasing rate of antiviral prophylaxis, so that excess mortality is lowest if no antiviral drugs are taken at all. For the default parameter values the right-hand side of inequality (2) equals 0.00025 (yr 21 ), while the cost of antiviral prophylaxis is c = 0.0001 (yr 21 ). Hence, in our default scenario excess mortality decreases with increasing rate of antiviral control up to the point where the pathogen is just unable to persist (s = 1.96 (yr 21 )).
Next we turn our attention to the different perspectives of the individual versus the public health officer. Our focus is on the antiviral treatment rate that minimizes excess mortality. Minimiz- ) and high (r = 6 (yr 21 )) rates of non-compliance, respectively. Other parameters are as in Table 1 ing this quantity with respect to the antiviral treatment rate yields the strategy that is optimal from the population perspective. Throughout this section and the next we assume that the pathogen is endemically present at the population dynamical equilibrium. As we have argued above, the optimal population strategy is such that the pathogen is just unable to persist if the risk of antiviral prophylaxis is small (i.e. if (2) is satisfied). Otherwise, the optimal population strategy is not to take antiviral drugs at all (Text S1)
In Text S1 we also show how excess mortality of a small group of individuals with antiviral treatment rate s y is calculated in a population where the majority of individuals take antiviral drugs at a rate s x . This allows one to determine the antiviral treatment strategy that will evolve at the population level by the process of individual choice. The optimal population and individual rates of antiviral treatment at the population level will be denoted by s à pop and s à ind , respectively. Figure 5 shows the results of a systematic investigation of the relation between the model parameters and the fractions of individuals taking antiviral drugs (which are determined by the antiviral control rates s à pop or s à ind ). The top panel shows the fraction of individuals taking antiviral drugs (black lines) and the associated excess mortality (grey lines) as a function of pathogen transmissibility. If transmissibility is low (b,51 (yr 21 )) the pathogen cannot persist, and there is no need to take antiviral drugs. If transmissibility is intermediate there is a positive population optimum (s à pop w0) which ensures eradication of the pathogen, while the individual optimum is still zero (s à ind~0 ). If transmissibility is high both the population and individual control rates are positive, although eradication is only achieved by the optimal population control rate.
The bottom panel of Figure 5 illustrates how the fractions of individuals taking antiviral drugs depend on the antiviral death rate. Not surprisingly, if antiviral drugs incur no cost (c = 0) then both the population and individual optimal control rates are such that the pathogen is driven to extinction. For the default parameter values this is achieved if at least two-third of the population is on antiviral drug treatment (s$1.96). If there is a cost to antiviral treatment, then the best option is to drive the pathogen to extinction if one takes the population perspective, until the risk of antiviral prophylaxis exceeds the risk of infection at the endemic equilibrium with no antiviral control (c.0.00025 (yr 21 )). Alternatively, if all individuals are allowed to flexibly adjust their own strategy, the optimal rate of antiviral prophylaxis decreases gradually with increasing antiviral death rate. In this case the optimal rate of antiviral prophylaxis is zero if c.0.00016 (yr 21 ). Notice that for intermediate cost of antiviral treatment the public health officer favours a strategy that is aimed at eradicating the disease, while the process of individual choice leads to a situation where nobody is willing to take antiviral drugs.
Unfortunately, to date there are no antiviral drugs that provide complete protection against infection and disease. For instance, an analysis of two recent trials with the antiviral drug oseltamivir shows that it provides little protection against infection with influenza, and moderate protection against subsequent shedding and disease [16] . Therefore, we will in this section study the consequences of antiviral prophylactic treatment with an imperfect antiviral drug. In the analyses below we take AVE S = 0.3 and AVE I = 0.8 as default parameter values [16] .
The relation between the antiviral efficacies for susceptibility and infectiousness, and the optimal rates of antiviral control is investigated in Figure 6 . The top panel shows the relation between the antiviral efficacy for susceptibility and the optimal fraction of individuals taking antiviral drugs, assuming that antiviral efficacy for infectiousness and virulence is fixed at AVE I = 0.8. The fact that the optimal fractions taking antiviral drugs decrease with increasing antiviral efficacy for susceptibility can be understood as follows. A decrease in the antiviral efficacy for susceptibility renders the antiviral drug less effective. However, since the cost of antiviral treatment is low while the antiviral efficacy for infectiousness is relatively high the rational strategy is to eradicate the pathogen if one takes the population perspective. With decreasing antiviral efficacy for susceptibility this is achieved by increasing the rate of antiviral control. Interestingly, the top panel indicates that if one takes the individual perspective excess mortality is highest if antiviral drugs provide complete protection against infection, since then the optimal control rate is lowest.
The bottom panel of Figure 6 shows the relation between the optimal fractions of individuals taking antiviral drugs as a function of the antiviral efficacy for infectiousness. The antiviral efficacy for susceptibility is fixed at AVE S = 0.3. The picture in this panel is more complicated than in the top panel. In particular, eradication of the pathogen is not feasible if the antiviral efficacy for infectiousness drops below a critical value (AVE I ,0.51). If the antiviral efficacy for infectiousness is just above this critical value it still is the best strategy to drive the pathogen to extinction if one If, on the other hand, the antiviral efficacy for infectiousness is low (AVE I ,0.07) it is better not to take antiviral drugs at all (s à pop~0 ) as the benefit of taking antiviral drugs do not outweigh the cost. In this region of parameter space both optimal control rates are zero. In the intermediate parameter regime (0.07,AVE I ,0.51) it is not possible to achieve eradication, but it may nevertheless be wise to take antiviral drugs. In fact, taking the population perspective, it is always better to be (partially) protected by antiviral drugs than to be fully susceptible in this region of parameter space (i.e. s à pop ??), even though eradication is not possible.
Intuitively, it may seem that one should consider antiviral treatment when faced with a highly transmissible pathogen that can kill its host. However, this line of reasoning may be inaccurate. In particular, the notion that antiviral treatment is attractive because the drugs are relatively harmless and because a potentially large number of infection-induced deaths can be prevented is not necessarily true. Our analyses show that over the course of an epidemic the death toll can be considerable if no antiviral drugs are taken, and that the number of deaths is orders of magnitudes smaller if a large-scale antiviral control program is effective ( Figure 2) . However, the analyses also show that in an epidemic that is effectively controlled by a large-scale antiviral treatment program the majority of deaths result from the use of antiviral drugs. The intuitive explanation is that although the hazard of mortality by antiviral prophylaxis is small on the short-term individual level, the total death toll may be quite high as the number of individuals that must receive antiviral drugs for an effective control effort is probably on the order of total population size. Moreover, adding to this is the fact that in the face of an imminent threat it may prove necessary to continue taking antiviral drugs for a prolonged period.
In the early stages of an unfolding epidemic the probability of infection is still small. Consequently, individuals may be tempted to put off antiviral drug treatment until the prevalence of infection and hence the probability of infection has become non-negligible. Our analyses have shown that the critical force of infection that determines at what point individuals should start taking antiviral drugs depends strongly on the adverse effects of antiviral drug use ( Figure 3 ). Hence, for purposes of successful prevention or early containment it is important that the adverse effects of antiviral drugs remain small.
Our analyses have revealed that, taking the population perspective, the best option is either to provide antiviral drugs up to the point where the pathogen is unable to invade and persist, or not to provide antiviral drugs at all (Figures 5-6) , depending on the cost of antiviral treatment and the effectiveness of antiviral treatment. If, on the other hand, one takes the individual perspective there is an incentive to take less antiviral drugs than is optimal from the population perspective, and complete prevention or eradication of the disease is rarely possible.
Interestingly, the conflict between the individual and the public health officer appears to be most pronounced when the cost of antiviral drug treatment or the effectiveness of antiviral drugs in reducing the adverse effects of infection are intermediate (Figures 5-6 ). In fact, if the cost of antiviral prophylaxis is intermediate it is possible that public health favours an aggressive containment strategy that aims at pathogen eradication, while the process of individual choice leads to a situation in which nobody is willing to take antiviral drugs. On the other hand, if the cost of antiviral treatment is high or if the effectiveness of antiviral drugs in preventing the adverse effects of infection is very low, then the conflict may become small or disappear at all (Figures 5-6) .
One aspect of our model that deserves special attention is that we have throughout assumed that the cost of both infection and antiviral treatment are in terms of an increased risk of death. This is convenient because it enables a straightforward comparison of the positive and negative consequences of infection and antiviral prophylaxis. However, although there is no question that human infections with SARS and H5N1 avian influenza bring along a risk of death, it is as yet unclear how severe the adverse effects of antiviral drug treatment may be. This is especially so for rare but possibly severe adverse effects. For instance, it is well-documented that oseltamivir (the neuraminidase inhibitor currently used to treat and prevent influenza infections) frequently leads to nausea and a number of less frequent adverse effects such as hepatitis and skin reactions [9, 17] . Recently, there have been suggestions of more serious adverse effects, including neuropsychiatric syndromes that may have contributed to a number of suicide events in Japan [18] [19] . Table 1 While we have used mortality as the currency to compare the costs and benefits of antiviral drug use, previous game theoretical studies of vaccination focused on the relative perceived risk of vaccination as compared to infection, and thereby also introduced a common currency to compare the costs and benefits of vaccination [10] [11] [12] [13] [14] 20] . Using relative perceived risk of vaccination as the basis of comparison has the advantage that it can be easily modeled. However, this approach also has some disadvantages as it assumes that the payoff loss for individuals who choose to vaccinate is a fixed quantity (the relative perceived cost of vaccination) which is unrelated to the actual number of adverse events in the population, while the payoff loss for individuals who choose not to vaccinate is proportional to the prevalence of infection, and so does not take into account the discounting of different costs and benefits (e.g., individuals who successfully recover from infection reap the long-term benefit of prolonged immunity). Ultimately, we believe that game theoretical models such as the one we have analyzed here should be refined to include the dynamics of human risk perception. In such models the perceived risks of infection and antiviral treatment are not static (as in [10] [11] [12] [13] [14] ) but dynamically adjusted, being functions of the different types of adverse events (different types of morbidity, deaths) that actually occur in the population. Of course, how such dynamical human risk perceptions can or should be modeled is not straightforward, and would necessitate adding a fair bit of sociology to our epidemic-game theoretical model.
Throughout this paper we have made the simplifying assumptions that individuals and public health officer's act rationally, have perfect information and foresight, and that the details of population structure and antiviral drug action play a minor role. These assumptions were made in order to be able to keep the analyses manageable, and to be able focus in detail on the conflict of interest. We are, of course, aware that in the real world a variety of complicating factors play a role. Therefore, our study is not intended nor suited to make quantitative predictions, but it serves to explore how the public and individual interests are shaped by pathogen transmissibility, cost of antiviral treatment, and antiviral efficacy for susceptibility and infectiousness.
It would be interesting to extend the model in a number of directions in order to be able to make specific predictions for specific viral threats. For this purpose several steps should be taken. First, depending on the precise research question some form of population structure would probably need to be taken into account. For instance, if the goal were to decide how a limited supply of antiviral drugs is best distributed across different risk groups, the model would need to include different risk groups and take into account that the stockpile of antiviral drugs or vaccines is not infinitely large [21] [22] . Alternatively, if the goal were to investigate whether local containment is possible by means of a targeted antiviral drug treatment program, it would be necessary to include spatial structure, household structure, and possible also workplace structure [6] [7] [8] . Overall, however, we believe that the present state of knowledge just barely suffices to make realistic quantitative predictions as to how effective a large-scale prophylactic antiviral drug program will be, let alone that it will be possible to make quantitative predictions when the dynamics of human choice are taken into account. This, of course, is not tantamount to saying that individual choice is unimportant.
Fortunately, none of the recent viral threats from the animal reservoir (avian influenza, SARS) has succeeded in getting a definitive foothold in the human population. As a consequence, the key epidemiological characteristics of the next emerging virus (transmissibility, infectious period, virulence) remain unknown. This is also largely true for rare but serious side-effects of antiviral drugs. This has rendered attempts to provide realistic predictions of the effectiveness of control measures such as antiviral treatment somewhat speculative [6] [7] [8] . Our model lacks much of the sophistication of the earlier models, and is not suited to make quantitative predictions. Rather, the analyses have laid out the principles guiding the decisions of rational individuals and public health officers when faced with an emerging viral threat for which antiviral drugs can be deployed as a first line of defense.
Text S1 Model structure and details of the model analyses Found at: doi:10.1371/journal.pone.0001558.s001 (0.10 MB PDF) Cross-subtype Immunity against Avian Influenza in Persons Recently Vaccinated for Influenza Avian influenza virus (H5N1) can be transmitted to humans, resulting in a severe or fatal disease. The aim of this study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for seasonal influenza A (H1N1)/(H3N2). A small frequency of CD4 T cells specific for subtype H5N1 was detected in several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T cells. We also observed that seasonal vaccination is able to raise neutralizing immunity against influenza (H5N1) in a large number of donors. No correlation between influenza-specific CD4 T cells and humoral responses was observed. N1 may possibly be a target for both cellular and humoral cross-type immunity, but additional experiments are needed to clarify this point. These findings highlight the possibility of boosting cross-type cellular and humoral immunity against highly pathogenic avian influenza A virus subtype H5N1 by seasonal influenza vaccination. I nfl uenza viruses are segmented, negative-sense RNA viruses belonging to the family Orthomyxoviridae. According to the antigenic differences in nucleoprotein and matrix proteins, 3 types of infl uenza viruses (A, B, and C) have been described. Infl uenza viruses A and B are associated with seasonal illness and death, whereas infl uenza virus C causes mild infections (1, 2) . Infl uenza A viruses are subtyped on the basis of the antigenic differences on external hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Human type A infl uenza virus subtypes have been limited to H1, H2, and H3 and to N1 and N2 (3) . Several HAs and NAs have been isolated from avian hosts; occasionally, they have been associated with human outbreaks (4, 5) .
Cytotoxic T lymphocytes play a central role in the clearance of primary infl uenza virus infection, peaking after 7-10 days; the peak in antibody titers occurs 4-7 weeks after primary infection (6) (7) (8) . Neutralizing antibodies are completely protective against secondary challenges only with closely related strains, but they are ineffective against viruses with major antigenic divergence. For this reason, current infl uenza vaccines are prepared annually on the basis of World Health Organization forecasts on the most probable infl uenza A and B virus strains thought to be circulating in the next seasonal outbreak (5, 7) . By contrast, cellular responses to cross-reactive epitopes may provide a substantial degree of protection against serologically distinct viruses (9) . The ability of infl uenza viruses to mutate and reassort their HA-NA genome segments between different animal species is a main concern because immunity generated by previous infections or vaccinations is unable to prevent infection by itself, although it may reduce virus replication and spread (8) (9) (10) .
To date, 3 infl uenza subtypes have produced pandemic disease in humans: H1N1 in 1918, H2N2 in 1957, and H3N2 in 1968 (4,11,12) . In 1997, during the avian infl uenza (H5N1) outbreak in Hong Kong Special Administrative Region, People's Republic of China, a cross-reactive cellular immune response induced by infl uenza (H9N2) was able to protect chickens from infl uenza (H5N1) (13) . Moreover, adults living in the United States who were never exposed to H5N1 subtype have shown cross-type cellular immunity to infl uenza A virus strains derived from swine and avian species (including the H5N1 subtype isolated in Hong Kong) (14) . Thus, speculation that cross-reactive T cells may decrease illness and death by reducing the replication of the new infl uenza virus, even if elicited by a different strain, is reasonable.
Avian infl uenza A viruses of the H5N1 subtype are currently causing widespread infections in bird populations. Numerous instances of transmission to humans have been recently reported in Asia and Africa, with the infection resulting in severe disease or death (>50% fatality rate). Hence, the aim of the present study was to evaluate the immune cross-reactivity between human and avian infl uenza (H5N1) strains in healthy donors recently vaccinated for seasonal infl uenza A (H1N1/H3N2). Our data indicate that infl uenza vaccination may boost cross-subtype immunity against infl uenza (H5N1), involving cellular or humoral responses or both.
Healthcare workers wishing to receive seasonal infl uenza vaccination at the Spallanzani Institute (n = 42) were enrolled. The study was approved by the local Ethical Committee; all participants gave written informed consent. Baseline characteristics of the study population are reported in the Table. Blood samples were obtained before (t0) and 30 days after vaccination (t1). The vaccine formulation was Fluarix, an inactivated and purifi ed split infl uenza vaccine (GlaxoSmithKline, Verona, Italy). The antigen composition and strains were A/California/7/2004-H3N2; A/New Caledonia/20/99-H1N1; and B/Shanghai/361/2002. Each 0.5-mL vaccine dose contains 15 μg HA of each strain in phosphate-buffered saline and excipients. Vaccine was administered intramuscularly.
Madin-Darby-canine kidney (MDCK) cells were maintained in Dulbecco modifi ed Eagle medium (DMEM) containing 10% fetal calf serum (FCS), and 2 mmol/L Lglutamine, at 37°C in a 5% CO 2 humidifi ed atmosphere. The infl uenza (H5N1) virus used was strain A/Hong Kong/156/97 (kindly provided by Paul Chan) (15) . The virus stock used as challenge antigen in the hemagglutination inhibition (HI) assay was propagated in the allantoic cavities of 10-day-old embryonated hen eggs. The allantoic fl uid was harvested 48 h postinoculation and clarifi ed by centrifugation. Virus concentration was determined by HA titration as previously described (16) , and the virus was stored at -80°C until used. The virus stock used in the microneutralization (NT) and in the cell-mediated immunity assays was propagated in MDCK cells, and the culture supernatants were collected 48 h postinoculation. The 50% tissue culture infectious dose (TCID 50 ), determined by titration in MDCK cells, was calculated by the Reed and Muench method (17) .
Infl uenza vaccine, UV-inactivated MDCK-derived infl uenza virus (H5N1), or synthetic infl uenza (H5N1) oligo-
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 1, January 2008 peptides were used as antigens for cell-mediated immunity. Infl uenza virus (H5N1) was inactivated by exposure to UV light for 10 min, and complete inactivation of UV-exposed virus was checked by infecting MDCK monolayers with undiluted preparation and by back-titrating the infectivity after 5 days postinfection. Four synthetic peptides of the infl uenza (H5N1) were purchased from Biodesign International (Kennebunk, ME, USA). The sequence of these peptides is specifi c for H5-C-terminal (15 aa), H5-middle region (14 aa), and N1-C-terminal (15 aa) and for N1middle region (16 aa), with no cross-matching with other HA and NA sequences. These peptides can bind different HLA-DRB1 alleles, as established according to the SYF-PEITHI site (www.syfpeithi.de). Specifi cally, N1-specifi c peptides can bind the following HLA-DR alleles: HLA-DRB1*0101, B1*0301, B1*0401, B1*0701, B1*1101, B1*1501. The H5-specifi c peptide from the N-terminal region binds several HLA-DR alleles (HLA-DRB1*0101, B1*0301, B1*0401, B1*0701, B1*1101, B1*1501). In contrast, the H5-specifi c peptide from the middle region did not appear to bind any HLA-DR alleles. According to the HLA-DRB1 allele frequency in the local population, these peptides can be effi ciently presented by most (up to 84%) of study participants.
Cell-mediated response was assessed by detecting intracellular interferon-gamma (IFN-γ) production by effector T cells, after antigen-specifi c stimulation in vitro to generate effector cells from memory cells (18) . Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Hypaque, Pharmacia Biotech, Uppsala, Sweden) and frozen at -150°C. Thawed PBMC in culture medium (RPMI 1640, 10% FCS, 2 mmol/L L-glutamine) were stimulated with the infl uenza vaccine preparation (1.5 μg/mL), UV-inactivated infl uenza (H5N1) (MOI 0.1), or synthetic infl uenza (H5N1) peptides (NA and HA) (1 μg peptide/mL) for 3 days and expanded for 6 additional days in the presence of recombinant interleukin-2 (IL-2) (5 IU/mL, Boehringer-Mannheim, Mannheim, Germany). On day 9, cells were restimulated with the same antigens in the presence of 1 μg/mL αCD28 and αCD49d (immunoglobulin G1 [IgG 1 ], K clones CD28.2 and 9f10, respectively; Becton Dickinson, Mountain View, CA, USA) and of Brefeldin-A (10 μg/mL, Sigma, St. Louis, MO, USA). As a negative control, a mock virus preparation, obtained with uninfected MDCK cells, or irrelevant peptides were used. To control the spontaneous cytokine production, cells incubated with only αCD28 and αCD49d were included.
In addition, the frequency of IFN-γ-producing CD4 T lymphocytes from each donor in the absence of any stimulation was used to calculate the background for each stimulation. The resulting background levels were very low in every experiment, and no differences were observed between samples obtained before (t0 0.03% ± 0.04%) and after vaccination (t1 0.01% ± 0.03%). The frequency of antigen-specifi c CD4 T cells for each study participant was calculated by subtracting the relative background levels at t0 and t1.
Cell-mediated immunity was considered positive when the net increase was >0.2%. Although retesting samples on separate occasions gave reproducible results, t0 and t1 samples for each participant were tested simultaneously to further reduce test variability.
Monoclonal antibodies coupled with phycoerythrin (PE), peridinin-chlorophyll protein (PerCP), allophycocyanin (APC), and phycoerythrin-Cy-7 (PE-Cy7) were combined for simultaneous staining. In this study the following were used: anti-CD4 PerCP (IgG1, clone SK3), anti-CD3 PE-cy7 (IgG2a, clone SK7), anti-IFN-γ APC (IgG1, clone B27), and anti-IL-2 PE (IgG1, clone 5344.111) (Becton Dickinson). Cells were stained as previously described (19) .
Multiparametric fl ow cytometry was performed by using a FACSCanto fl ow cytometer (Becton Dickinson). A total of 300,000 live events were acquired, gated on small viable lymphocytes, and analyzed with FACSDiva software (Becton Dickinson). The instrument was routinely calibrated according to the manufacturer's instructions.
The NT was performed according to a previously described procedure (20) , in agreement with indications from the World Health Organization (21) and the US Department of Health and Human Services (22) . Specifi cally, 2fold serial dilutions of heat-inactivated (30 min at 56°C) human sera were performed in 50 μL DMEM without FCS in 96-well microplates. An equal volume of infl uenza virus (H5N1) (10 3 TCID 50 /mL) was then added to each well. Uninfected-cell wells, incubated with each test serum, were included in each plate as negative controls. After 1 h incubation at 37°C, the mixtures were transferred on MDCK cell monolayers and adsorbed at 37°C for 1 h. After washing, DMEM was added, and the plates were incubated for 2 days at 37°C in 5% CO 2 . NT titer was assessed as the highest serum dilution in which no cytopathic effect was observed by light microscope inspection. All serum specimens were tested in duplicate, and t0, and t1 samples from each patient were assayed in the same plate at the same time. The results were scored by persons blinded to the study participant's identifi cation. The test results were reproducible because random replication of the assays on independent occasions gave consistent results.
The antibody titer was also established by HI test, using for challenge either the seasonal vaccine or the egg-derived infl uenza (H5N1) preparation. HI assays were performed in V-bottom 96-well plates with 0.5% chicken erythrocytes, as described (16) .
All experiments with live highly pathogenic avian infl uenza A virus (H5N1) were conducted by using Biosafety Level 3-plus (BSL3+) containment procedures (23) . All investigators were required to wear appropriate masks with HEPA fi lters.
The frequency of circulating antigen-specifi c CD4 T cells in healthy donors enrolled in the study was analyzed by fl ow cytometry, by using intracellular cytokine staining assay after the in vitro expansion of effector cells. To generate effector cells from their memory precursors, PBMC were challenged with antigen in vitro for 3 days and expanded for 6 additional days in the presence of IL-2 (18) .
Effector cells were characterized for their ability to release IFN-γ when cultured overnight in the presence of antigen. CD4 T cells were gated and analyzed for IFN-γ and IL-2 cytokine expression. A representative experiment with PBMC from a recently vaccinated healthy donor is shown in Figure 1 . Without stimuli, no cytokine production in CD4 T cells was detected (Figure 1, panel A) . However, the stimulation with the seasonal infl uenza vaccine preparation induced the production of IFN-γ by CD4 ef-fector T cells (Figure 1, panel B: 3 .2% of IFN-γ+ CD4+ T cells). Stimulation with inactivated infl uenza (H5N1) virus induced a CD4 T-cell response (Figure 1 , panel C: 1.0% of IFN-γ+ CD4+ T cells). Finally, some CD4 T cells specifi c for a pool of H5 and N1 (H5/N1) peptides were also generated in this donor (Figure 1 , panel D: 0.6% of IFN-γ+ CD4+ T cells). No IL-2 production was observed in these experimental conditions.
When the extent of CD4 T-cell-mediated immunity before and after seasonal infl uenza vaccination was compared in the healthy donors enrolled in the study, a nonhomogeneous pattern of responses was detected (online Appendix Figure; available from www.cdc.gov/EID/content/14/1/121-appG.htm. After vaccination (t1), a 2-fold variation of the frequency of antigen-specifi c T cells higher than baseline was arbitrarily considered signifi cant. According to this threshold, an increased frequency of IFNγ-producing CD4 T cells specifi c for vaccine preparation was observed after vaccination in 5 (donors 8, 11, 17, 26, 42) of 21 donors (23.8%). A slight increase of frequency of the vaccine preparation-specifi c CD4 T cells was observed in 5 donors (donors 9, 12, 33, 36, 40; 23.8%); a mild-tosignifi cant decrease was observed in the remaining donors (n = 11; 52.3%).
As shown in the online Appendix Figure, subtype (online Appendix Figure, panel B); among them, 3 were also showing an increase of the frequency of IFNγ-producing CD4 T cells specifi c for vaccine preparation (donors 11, 17, 42). Two of them, donor 11 and donor 42, had a signifi cant increase of IFN-γ-producing CD4 T cells specifi c for H5/N1 peptides (online Appendix Figure, panel C), which suggests that cross-type immunity may directly involve the HA/NA proteins. Furthermore, 3 other donors (donors 12, 16, 36) had an increased frequency of H5/N1 peptides-specifi c CD4 T cells, even if they were unable to respond to whole virus. Indeed, in some persons we also observed a signifi cant decrease at t1 in CD4 T cells specifi c for vaccine preparation (donors 2, 16, 23, 27, 30, 31, 35, 41) , specifi c for infl uenza (H5N1) (donors 4, 16, 27) , and specifi c for H5/N1 peptides (donors 2, 27, 34, 39, 41). Donors with a reduced specifi c response to vaccine preparation at t1 showed a higher frequency of specifi c CD4 T cells at t0 when compared to other donors (3.4% ± 0.88 vs. 1.29% ± 0.35, respectively, p = 0.013). Similar results were obtained when we observed the infl uenza virus (H5N1) (1.07% ± 0.47 vs. 0.14% ± 0.03, respectively, p = 0.0093) and H5/N1 peptides (1.19% ± 0.54 vs. 0.13% ± 0.07, respectively; p = 0.0018).
Because some study participants were reactive to inactivated infl uenza virus (H5N1) as well as to a peptide pool composed of 2 peptides from H5 and 2 from N1 consensus sequences, we analyzed whether this reactivity was preferentially directed against HA or NA. As shown by PBMC from a representative donor in Figure 2 , the frequency of IFN-γ-producing CD4 effector T cells was appreciable after challenge with the inactivated infl uenza virus (H5N1) (Figure 2, No specifi c CD4 T cells producing interferon-gamma (IFN-γ) were observed after challenge with H5 peptides (D). As negative control, either mock-infected culture supernatants or irrelevant peptides were used, giving results very similar to unstimulated cultures (not shown). A similar pattern was observed in 4 other study participants, supporting the hypothesis that the actual target of cross-subtype immunity could be N1.
pattern was observed in 4 other study participants, which supports the hypothesis that the target of cross-subtype immunity could actually be N1.
Human sera from the same donors were tested for HI activity against both vaccine and infl uenza (H5N1) preparations and for neutralization activity against infl uenza (H5N1) virus. Individual titers are reported in Figure 3 . A 4-fold rise in HA antibody titer is considered noteworthy, and after vaccination most donors (28/38; 73.7%) showed a noteworthy rise of HI titers against vaccine preparation, as indicated by an asterisk (Figure 3, top panel, black bars) . HI titers against infl uenza virus (H5N1) remained at undetectable levels after seasonal vaccination (data not shown), but a rise of neutralization titer >20-fold over baseline was observed in 13 (34.2%) of 38 donors ( Figure 3 , bottom panel, asterisk). All but 1 study participant also responded to seasonal vaccination by a rise in HI titers against vaccine preparation. One donor (21) showed high titers against the H5N1 subtype in NT but a low HI titer against vaccine, a unique situation in the study population. However, antibodies to both anti-infl uenza (H5N1) and infl uenza vaccine are raised by vaccination. Our fi ndings indicate that seasonal vaccination can raise neutralizing immunity against infl uenza (H5N1), which shows the existence of an antibodydependent cross-type immunity. No correlation between infl uenza-specifi c CD4 T cells and humoral responses was observed, which suggests that this type of antibody response was mainly CD4 T-cell independent.
We observed that infl uenza-specifi c CD4-effector T cells could be generated by long-term cultures in vitro and easily monitored by fl ow cytometry as IFN-γ-producing cells. When this approach was used, a small frequency of CD4 T cells specifi c for H5N1 subtype could be detected in several persons at baseline. Seasonal vaccine administration may enhance the frequency of reactive CD4 T cells, boosting the cross-subtype cellular immunity against avian infl uenza (H5N1). We also observed that seasonal vaccination raised neutralizing immunity against H5N1 subtype in a large number of donors, showing the existence of an antibody-dependent cross-type immunity. Thus, cross-reactive immunity may involve cellular and/or humoral responses, but the humoral response seems to be CD4 independent.
From the present data, N1 appears to be 1 target for cross-type cellular immunity, although we could not rule out the involvement of different (i.e., internal) antigens as possible targets of immune recognition by effector CD4 T cells. Nevertheless, in animal models, cellular immunity (mainly CLT) targeting internal proteins (i.e., NP), partly responsible for heterosubtypic protection, was not induced effi ciently by inactivated vaccines (24) . We did not use live virus, only inactivated split vaccine, whole inactivated virus, or HA and NA peptides for the infl uenza (H5N1) A/ Hong-Kong/156/97 strain. From our data, discriminating between the CD4 T-cell response against external or internal antigens in the case of vaccine preparation was not possible. For H5N1 subtype response, we can presume that the response is against the external antigens and that the results against peptides point to a specifi c response against NA.
Results obtained with the whole virus and those obtained with the H5 and N1 peptides are not in complete agreement (online Appendix Figure) . This fi nding can be explained on the basis of the substantial differences in the antigen presentation underlying the whole virus and peptides. Moreover, we observed that a high activation of specifi c cells at baseline (t0) was associated with a reduced specifi c response after vaccination (t1), which suggests that stimulation of pre-activated T cells with high dose of antigen could induce T-cell anergy (25) with consequent loss of immune response. Preliminary evidence also suggests that humoral crosstype immunity is targeting antigens differently from HA: sera from persons showing signifi cant neutralizing titers against infl uenza (H5N1) did not recognize insect cells expressing HA from the H5N1 subtype (not shown) and did not show HI activity against H5N1 subtype. N1 may possibly also be a target of humoral immunity, but additional experiments such as Western blot analysis or inhibition of NA activity (26) are needed to clarify this point.
In animals, exposure to 1 specifi c subtype of infl uenza A virus can also induce protective immunity against challenges with other subtypes. This heterosubtypic or crossprotective immunity could represent a key mechanism for facing, and limiting, new infl uenza outbreaks. In 1997, during the Hong Kong infl uenza (H5N1) outbreak, an immune response induced by an infl uenza virus (H9N2), being T cells but not antibodies, protected chickens from lethal infl uenza (H5N1) (13) . Moreover, adults living in an urban area of the United States have been described as having infl uenza-specifi c memory T cells that recognize epitopes of infl uenza A virus strains derived from swine and avian species, including the infl uenza (H5N1) strain involved in the Hong Kong outbreak in humans (14) .
Our data confi rm that persons who have never been exposed to H5N1 subtype may be able to generate a cell-mediated response against the Hong Kong infl uenza (H5N1) isolate. This cross-type response may be naturally occurring (probably as a consequence of exposure to seasonal infl uenza strains).
In mice, both CD4 T-cell-independent and -dependent antibody responses contribute to the control of infl uenza virus infection (27, 28) . Although antibodies appear to facilitate the recovery from infl uenza infection, it is generally believed that B cells cannot produce neutralizing, isotypeswitched, infl uenza-specifi c antibodies in the absence of CD4 T-cell help (29, 30) . However, other data clearly demonstrate that B cells can also produce anti-infl uenza IgA, IgM, and IgG responses independent of CD4 helper T cells (27, 31) . A non-antigen-specifi c bystander response driven by activated CD4 T cells specifi c for heterologous antigen may contribute to so-called heterosubtypic immunity (8) (9) (10) 12) . However, the ability of infl uenza virus infection to promote B-cell activation and differentiation into shortlived, isotype-switched, antibody-secreting cells may result from a combination of B-cell receptor hypercross-linking, engagement of toll-like receptors, production of cytokines, as well as triggering of innate immunity.
In our study, cellular and humoral cross-reactive immunity seemed to target antigens other than HA. Infl uenza (H5N1) cases occur mainly in young people (32) . This fi nding may be explained by hypothesizing that older people, although not previously exposed to H5N1 subtype, may have gained protective immunity by previous infections sustained by circulating infl uenza virus strains. It has also been shown that immunity to the N1 NA from the human infl uenza virus cross-reacts with the avian N1 NA virus and that this cross-reactivity protects mice against infection with the avian infl uenza virus (H5N1) (26) . All these fi ndings may be explained by hypothesizing that cross-reactive immunity is targeting the N1 NA antigen. However, whether cross-reactive antibodies to NA and CD4 T cells would be protective against illness and death, especially from infl uenza (H5N1) infection is not known. Further studies will be necessary to elucidate this point.
In conclusion, we demonstrated that vaccination against seasonal infl uenza may boost a cross-reactive immunity against an unrelated strain responsible for deadly infections in humans, i.e., the avian infl uenza (H5N1) strain A/Hong Kong/156/97. These data, together with previous experimental results from mice studies and epidemiologic reports, indicate that cross-type immunity should be considered an important component of the immune response against novel infl uenza A infections. Human Bocavirus Infections in Hospitalized Children and Adults Studies have reported human bocavirus (HBoV) in children with respiratory tract infections (RTIs), but only occasionally in adults. We searched for HBoV DNA in nasopharyngeal aspirates (NPAs) from adults with exacerbations of chronic bronchitis or pneumonia, from children hospitalized for acute RTIs, and from asymptomatic children during the winter of 2002–2003 in Canada. HBoV was detected in NPAs of 1 (0.8%) of 126 symptomatic adults, 31 (13.8%) of 225 symptomatic children, and 43 (43%) of 100 asymptomatic children undergoing elective surgery. Another virus was detected in 22 (71%) of the 31 HBoV-positive NPAs from symptomatic children. Two clades of HBoV were identified. The pathogenic role of HBoV in RTIs is uncertain because it was frequently detected in symptomatic and asymptomatic children and was commonly found with other viruses in symptomatic children. H uman bocavirus (HBoV) is a newly described human virus closely related to bovine parvovirus and canine minute virus. It is currently classifi ed in the genus Bocavirus within the family Parvoviridae. This virus was fi rst identifi ed in respiratory tract specimens from Swedish children with lower respiratory tract infections (RTIs) (1) . Nucleic acid amplifi cation has detected HBoV in respiratory samples of children with acute respiratory disease, with incidence rates ranging from 3% to 19% . However, the pathogenic role of HBoV is uncertain because other viruses have been frequently detected in HBoV-positive children with lower RTIs (range 37%-90%) (2, 3, 7, (9) (10) (11) (20) (21) (22) . The objective of this study was to describe the incidence and clinical manifestations of HBoV infections in children and adults with respiratory tract symptoms, including a control group of children without symptoms.
Respiratory samples from adults were obtained from a previous study conducted from December 2002 to April 2003 at 3 university-affi liated hospitals in the province of Quebec, Canada (24) . Two groups of patients were enrolled: those >40 years of age with chronic obstructive pulmonary disease (COPD) who came to emergency departments with exacerbation of their illness (including patients with pneumonia), and those >18 years of age without COPD who were admitted to the hospital with a diagnosis of community-acquired pneumonia. Patients were excluded from the study if they came to the hospital >7 days after onset of symptoms.
Respiratory samples from children were obtained from a case-control study, the results of which have been published (25) . Participants included children <3 years of age who were hospitalized from December 2002 to April 2003, at Laval University Hospital Center in Quebec City, Quebec, Canada. Case-patients were children admitted for an acute RTI (mostly bronchiolitis, pneumonitis, and laryngotracheobronchitis) who had a nasopharyngeal aspirate (NPA) collected as part of investigation of their illness. A specifi c questionnaire was completed at admission by a research nurse in the presence of the parents. At the end of hospitalization, charts of the children were reviewed to collect clinical and laboratory data. Eligible controls were children hospitalized during the same period for any elective surgery (ear, nose, and throat surgeries in 71% of the cases). These children had no concomitant respiratory symptoms or fever at admission. The study nurse obtained a signed consent from parents and an NPA was obtained during surgery. The original studies were reviewed and approved by the ethics committees of all participating healthcare centers.
All pediatric (from case-patients and controls) and adult (case-patients only) NPA specimens were previously analyzed by using a multiplex real-time PCR assay for infl uenza A and B viruses, human respiratory syncytial virus (hRSV), and human metapneumovirus (hMPV) (24, 25) . For symptomatic children, viral cultures and antigen detection assays were performed upon request by the treating physician. Remaining specimens were frozen at -80°C until subsequent HBoV PCR studies.
Nucleic acids were extracted from 200 μL of NPA by using the QIAamp viral RNA Mini Kit (QIAGEN, Inc., Mississauga, Ontario, Canada). A duplex HBoV PCR (TaqMan assay) was used to amplify conserved regions of NP-1 and NS-1 genes as described (14) , except that the NS-1 forward primer was replaced with primer 5′-TAG TTG TTT GGT GGG ARG A-3′. Probes were labeled with 6-carboxyfl uorescein (FAM) or tetrachloro-6-carboxyfl uorescein (TET) at the 5′ end and with a quencher at the 3′ end. Amplicons were 81 bp (NP-1) and 74 bp (NS-1), respectively. Duplex amplifi cation was conducted by using 1 μmol/L NS-1 forward primer and 0.4 μmol/L NS-1 reverse primer and the 2 NP-1 primers. Taqman probes were used at concentrations of 0.1 mmol/L for the NP-1 gene and 0.2 mmol/L for the NS-1 gene (14) . The amplifi cation master mixture consisted of 2.5 mmol/L MgCl 2 , 3.33 mg/mL bovine serum albumin, 0.2 mmol/L of each of the 4 deoxynucleotide triphosphates (Amersham Biosciences, Uppsala, Sweden), 10 mmol/L Tris-HCl, 50 mmol/L KCl, 0.625 U Promega Taq DNA polymerase (Fisher Scientifi c, Markham, Ontario, Canada) combined with TaqStart antibody (BD Biosciences Clontech, Palo Alto, CA, USA), and 3 μL DNA in a fi nal volume of 25 μL. PCR amplifi cation (180 s at 94°C and 45 cycles for 10 s at 95°C, 30 s at 58°C, and 30 s at 72°C) was performed in a Smart Cycler thermal cycler (Cepheid, Sunnyvale, CA, USA). A PCR extension step of 5 min at 72°C was performed at the end of the cycling protocol. An HBoV infection was defi ned by a positive PCR result for NP-1 and NS-1. The duplex assay had a sensitivity of 10 genome copies for NP-1 and NS-1 gene targets on the basis of quantifi cation analysis of positive control plasmids.
Half of the HBoV-positive samples were randomly selected for phylogenetic analysis, which consisted of amplifying and sequencing a 842-bp region of the VP1/VP2 genes as described (6) . The VP1/VP2 nucleotide sequences from this study, as well as prototype sequence type (ST)1 and ST2 (1), were entered into a multiple alignment generated by ClustalW software version 1.83 (www.molecularevolution.org/software/clustalw) and corrected through fi nal visual inspection with the SeqLab application (Wisconsin package version 10.3; Accelrys, San Diego, CA, USA). Phylogenetic analyses were conducted with the MEGA version 3.1 software (26) by using the distance method and the neighbor-joining algorithm with Kimura-2 parameters. Topologic accuracy of the tree was evaluated by using 1,000 bootstrap replicates.
Proportions of clinical characteristics in different groups of patients were compared by using the χ 2 test or the Fisher exact test. The Wilcoxon nonparametric test was used to compare age distribution and length of stay. Analyses were performed by using SAS software version 9.1 (SAS Institute, Inc., Cary, NC, USA).
HBoV DNA was detected in NPA samples from 1 (0.8%) of 126 symptomatic adults (71 years of age) and from 31 (13.8%) of 225 symptomatic children (mean age 17 months, median age 15 months). However, HBoV was detected more frequently (43%, p<0.001) in the 100 asymptomatic control children (mean age 22 months, median age 23 months). Another virus was detected in 22 (71%) of 31 HBoV-positive NPAs from symptomatic children. The virus most commonly co-isolated with HBoV was hRSV (16/31, 52%), followed by infl uenza A/B (3 cases), hMPV (3 cases), adenovirus (1 case), and parainfl uenza virus (1 case). Two children were infected with 2 other viruses in addition to HBoV. The median age of symptomatic children with HBoV infection (15 months) was signifi cantly greater than that of symptomatic children without HBoV infection (8 months; p<0.0001). The hospital length of stay was similar for children positive for HBoV DNA (mean 5.1 days, median 4 days) and those negative for HBoV DNA (mean 6.6 days, median 3 days) (p = 0.9).
Clinical characteristics of HBoV-positive children are summarized in the Table. There were signifi cantly fewer bronchiolitis episodes in children infected only with HBoV than in children infected only with hRSV (p<0.0001). None of the children with single HBoV infections and only 2 (6%) of all 31 HBoV-infected children were admitted to the intensive care unit. In the control group of asymptomatic children who underwent elective surgery, ear, nose, and throat surgery was more frequently performed in children with HBoV infections (36/43, 84%) than in children without HBoV infections (35/57, 61%) (p = 0.014). Ear, nose, and throat elective surgeries consisted mainly of myringotomies, adenoidectomies, and tonsillectomies.
The 1 adult with an HBoV infection was a 71-year-old man (a smoker) who came to the hospital for a COPD exacerbation and was treated with systemic corticosteroids and antimicrobial drugs. No other microbiologic agents (bacteria or viruses) could be identifi ed in his sputum or NPA. He was hospitalized for 11 days.
Sequence analysis of the HBoV VP1/VP2 genes performed on ≈50% of HBoV-positive specimens showed 2 distinct clades of viruses (Figure) . These genotypes clustered with the original strains described by Allander et al. (ST1, GenBank accession no. DQ000495, and ST2, Gen-Bank accession no. DQ000496) (1). There was no temporal link between the clades because both were equally distributed throughout the study period. No obvious relationship was found between clades and the presence or absence of symptoms.
Results from our study indicate that HBoV was rarely detected in adults with respiratory symptoms but was frequently detected in symptomatic and asymptomatic children during the 2002-2003 winter season. HBoV was detected in NPA samples from 1 (0.8%) of 126 symptomatic adults, 31 (13.8%) of 225 symptomatic children, and 43 (43%) of 100 asymptomatic children. Another virus was detected in 22 (71%) of 31 HBoV-positive samples from symptomatic children. Overall, these data do not support a pathogenic role for HBoV in acute RTIs in children.
The full spectrum of clinical diseases associated with HBoV infections and the epidemiology of this new parvovirus are not fully understood. This is particularly true for adult patients in whom few studies have been performed. Allander et al. (1) found no HBoV DNA in 112 culturenegative NPA samples from adults with respiratory symptoms. Bastien et al. (5) reported an overall rate of infection of 1.5% in respiratory samples negative for other viruses, with no differences between age groups. Maggi et al. (16) reported only 1 HBoV-positive sample from an adult with lymphoma in 62 bronchoalveolar lavages (BALs). These investigators also tested 22 nasal swabs from adults with persistent asthma symptoms and found no samples positive for HBoV. Fry et al. (10) identifi ed HBoV DNA in 1% of adults >20 years of age hospitalized with pneumonia in Thailand. Kupfer et al. (27) described a case of HBoV infection associated with severe atypical pneumonia in a patient with non-Hodgkin lymphoma who was also infected with cytomegalovirus in a BAL sample. We found 1 case of HBoV infection in an adult, which represented 0.8% of the tested population. The HBoV-positive adult did not show immunosuppression but was treated with corticosteroids for a COPD exacerbation. Overall, our results are consistent with those of previously described studies and support the fact that HBoV infection is rare in adults but may occur more frequently in those with other illnesses or immunosuppression.
Studies have reported HBoV DNA in 3%-19% of children with RTIs. Rates of detection tend to be higher in children <1 year of age (4, 10) . The incidence of HBoV infections also tends to be higher in samples from the lower respiratory tract, such as NPA or BAL (4.4%-19%) (2, 7, 9, 11, 13, 19, 21, 22) , than in nasal swabs (1%-6%) (10, 15, 16, 18) . The percentage of co-pathogens in our HBoV-positive children (71%) was comparable with those reported in the literature, with rates of co-infections ranging from 35% to 90% (2, 3, 7, (9) (10) (11) (20) (21) (22) . Moreover, coinfecting viruses detected in conjunction with HBoV in our population were similar to those described in other studies, i.e., hRSV, infl uenza A virus, and adenovirus (11, 23) .
The high frequency of HBoV detection (43%) in our asymptomatic children contrasts with the results of the few other studies that included a control group of asymptomatic children. Fry et al. (10) detected HBoV DNA in only 1% of nasal swabs from asymptomatic patients. Maggi et al. (16) did not detect HBoV DNA in nasal swabs from 51 asymptomatic children (including 30 healthy infants with a mean age of 6 months and 21 preadolescent healthy children with a mean age of 12.8 years). However, these studies analyzed nasal swabs instead of NPA or BAL samples for HBoV detection, which may result in lower rates of viral detection, as shown in symptomatic persons. Allander et al. (2) did not detect HBoV in any of 64 asymptomatic children (median age 4.1 years, range 5 months to 14 years) but used nasal swabs in asymptomatic patients and NPA samples in symptomatic patients. Furthermore, their control group was also older than our population (mean 18.6 months, median 18 months). Kesebir DNA in nasal washes from 96 asymptomatic children <2 years of age seen at a clinic compared with 22 (5.2%) of 425 various samples from symptomatic children sent to a hospital clinical laboratory. None of the previous studies used a control group consisting of children matched for age and week of admission and analyzed the same type of respiratory samples for cases and controls. Our positive results for HBoV were confi rmed by using 2 sets of PCR primers targeting different genes (NP1 and NS1) in a duplex PCR assay and by subsequent testing with a third set of primers (VP1/VP2) for sequencing. Also, sample preparation and PCR amplifi cation were performed in separate laboratory areas following the stringent quality control program of our institution. Thus, it is unlikely that our positive results were due to PCR cross-contamination. Our method was also very sensitive (detection limit = 10 genome copies), which probably enabled an increased in-fection rate compared with previous reports. We cannot exclude the possibility that prior RTIs (in the few weeks preceding sampling) occurred in our asymptomatic children hospitalized for an elective surgery or that HBoV could establish a prolonged infection in children compared with other respiratory viruses. However, the 3× higher detection rate in controls than in symptomatic children make these explanations unlikely. We did not quantify HBoV DNA load in samples from our study, which could have been different between asymptomatic and symptomatic children. Nevertheless, we detected hRSV, hMPV, and infl uenza virus RNA in <1% of the same NPA samples from those asymptomatic children compared with a rate of 43% for HBoV DNA (25) . At the very least, our results should raise concerns about the pathogenic role of HBoV in children.
We detected 2 HBoV genotypes circulating at the same time in both symptomatic and asymptomatic children during the winter of 2002-2003 in Quebec. This result is consistent with fi ndings of other groups from North America and Europe during 2002-2004 and highlights the fact that HBoV lineages do not appear to be geographically clustered (1, 6, 9, 12) . The seasonality of HBoV infection is still a matter of debate, but it seems to involve primarily the colder months of the year (9, 20, 21) . However, most studies, including ours, were performed during the typical respiratory virus season, which may have introduced a bias. Additional studies are needed to address the prevalence of HBoV outside the respiratory virus season and its role in nonrespiratory syndromes. Moreover, the possibility that this virus might be transmitted and isolated in the respiratory tract, but could cause viremia and other clinical syndromes such as gastroenteritis, should be investigated. Vicente et al. analyzed 527 stool samples from children with gastroenteritis and no respiratory symptoms and found a positivity rate of 9.1% for HBoV (with a co-infection rate of 58%) (22) .
In conclusion, our study shows that HBoV was frequently detected in both symptomatic and asymptomatic children during the winter of 2002-2003 in Quebec City. Conversely, this virus was rarely found in the adult population during the same period. Further studies are needed to establish whether this recently described parvovirus is pathogenic by using well-matched control groups and sequential samples to detect viral persistence. (1) . Numbers along branches are bootstrap values from 1,000 replicates. Scale bar shows 1 substitution for every 1,000 nucleic acid residues. Pandemic Influenza Planning in the United States from a Health Disparities Perspective We explored how different socioeconomic and racial/ethnic groups in the United States might fare in an influenza pandemic on the basis of social factors that shape exposure, vulnerability to influenza virus, and timeliness and adequacy of treatment. We discuss policies that might differentially affect social groups’ risk for illness or death. Our purpose is not to establish the precise magnitude of disparities likely to occur; rather, it is to call attention to avoidable disparities that can be expected in the absence of systematic attention to differential social risks in pandemic preparedness plans. Policy makers at the federal, state, and local levels should consider potential sources of socioeconomic and racial/ethnic disparities during a pandemic and formulate specific plans to minimize these disparities. T he threat of pandemic infl uenza has generated concern among politicians, policy makers, healthcare professionals, and the general public. For the past several centuries, major infl uenza pandemics have occurred every 10 to 30 years (1); it is widely believed that a new pandemic is "inevitable" (2) . The possibility of an imminent infl uenza pandemic has been heightened by the appearance and spread of avian infl uenza A (H5N1), which has a case-fatality ratio of >50% (3) . Although the assumption has been that avian infl uenza viruses could not directly infect humans, the transmission of infl uenza virus (H5N1) directly from chickens to humans in 1997 caused experts to reconsider that assumption (4) . Genetic changes in infl uenza virus subtype H5N1 in 2003 resulted in a new strain of the virus, which spread to multiple countries in East and Southeast Asia (5) , as well as Europe and Africa. Whether the avian infl uenza virus (H5N1) develops human pandemic potential, its spread from birds to humans and the severity of the resulting disease have heightened concerns about a possible future infl uenza pandemic.
Considerable fi nancial resources have been devoted to pandemic infl uenza preparedness planning at the federal and state levels (6, 7) ; however, resources at state and local levels may be inadequate to implement a robust preparedness plan (8, 9) . Past experience with natural disasters and current socioeconomic and racial/ethnic disparities in healthcare in the United States (10, 11) raise questions about the adequacy of plans to address the needs of disadvantaged populations. For example, in responding to Hurricane Katrina, planners apparently failed to consider that many low-income persons might lack private modes of transportation and would depend on institutional help for evacuation. Although the evacuation was successful overall (12), deaths, injuries, and illness occurred disproportionately among low-income persons in New Orleans because of economic and logistic constraints on their ability to respond to government recommendations to leave the city. Low-income and disadvantaged persons often suffer disproportionately during natural disasters and epidemics, and historical evidence demonstrates that low-income persons fared considerably worse than high-income persons during the 1918 pandemic in the United States (13) .
In this article, we describe ways in which different socioeconomic and racial/ethnic groups might fare differently in an infl uenza pandemic, on the basis of current knowledge of social factors that shape exposure and vulnerability to infl uenza virus and that infl uence the timeliness and adequacy of treatment among those who become ill. We also discuss policy decisions, made either before or during a pandemic, which might differentially affect risk for illness or death for those of low income and of specifi c racial/ethnic groups. Our purposes are to 1) call attention to potentially major and avoidable social disparities in suffering and death during an infl uenza pandemic and 2) highlight the importance of including in pandemic preparedness plans targeted strategies for minimizing or avoiding these social disparities. The following discussion is not meant to be exhaustive; rather, it is meant to provoke refl ection about how potential disparities in the effects of an infl uenza pandemic might be reduced or eliminated through appropriate planning and implementation of clinical and public health activities.
Using a conceptual framework adapted from Diderichsen et al. (14) , we systematically considered possible sources of disparities during an infl uenza pandemic by examining the following 3 levels at which underlying socioeconomic or racial/ethnic differences could lead to disparities in illness or death: 1) likelihood of being exposed to the infl uenza virus; 2) likelihood of contracting infl uenza disease, if exposed; and 3) likelihood of receiving timely and effective treatment after infl uenza disease has developed. To explore socioeconomic and racial/ethnic disparities at each level, we searched the literature for relevant fi ndings based on population-based national data (Figure, Table) .
Regardless of which strain of infl uenza virus causes the next pandemic, it will be highly transmissible between humans. Transmission of infl uenza is primarily airborne, through aerosolized respiratory tract secretions expelled during coughing and sneezing, although transmission by direct and possibly indirect contact may occur. Transmission can be expected to occur in various settings, including homes, healthcare facilities, schools, work sites, public transportation, and other settings at which people gather for social, commercial, or entertainment purposes. Higher exposure risk among particular population groups as a result of factors such as crowding and occupation could contribute to health disparities among socioeconomic and racial/ ethnic groups during an infl uenza pandemic.
Crowding, an established risk factor for many infectious diseases, can increase the likelihood of pathogen transmission. In the United States, urban poverty and Hispanic and Asian ethnicity are correlated with domestic crowding; even at higher income levels, Hispanic and Asian households are relatively more crowded than white and African-American households (15) . In addition, in the United States, low-income persons, African Americans, and nonwhite Hispanics are more likely than persons in other groups to obtain regular medical care at emergency departments and publicly funded clinics (10) , where airborne transmission of infectious agents has been documented. Because these locations typically do not segregate sick and well patients and are becoming increasingly crowded (16) , patients waiting for care in these settings are likely to have greater exposure to infl uenza viruses and other respiratory pathogens. Another source of increased exposure to infected persons is public transportation, where persons from low-income and minority households account for 63% of users (17) .
Occupational factors are also likely to lead to differential exposure risk during an infl uenza pandemic, particularly in terms of adherence to strategies that aim to limit case-patient contact with others (18) . Staying home may not be economically feasible for persons in lower wage occupations; these persons are less able to afford losing income as a result of missed work and often lack the job fl exibility that would permit them to work at home. In addition, their jobs may be necessary because they provide essential goods and services. For these reasons, parents in lower wage/lower status occupations may be more likely to keep their children in communal childcare settings-where exposure risks are relatively high-during an infl uenza pandemic, placing everyone in the family at greater risk for exposure.
Among persons who have been exposed to infl uenza virus, the likelihood of contracting disease may be modifi ed by underlying host factors and medical conditions, such as age, smoking status, nutritional status, stress levels, and cardiopulmonary disease. The infl uence that most host factors will have on the development of infl uenza during a future pandemic is uncertain; some evidence suggests that the factors affecting disease severity and death may differ from those typically observed during annual infl uenza epidemics (19) . However, given overwhelming evidence that low-income persons are generally more susceptible to infectious diseases, it is reasonable to plan on the basis of well-documented annual epidemic patterns, in which infl uenza disease development is infl uenced by factors that are differentially distributed across socioeconomic and racial/ ethnic groups. These patterns, as well as patterns of many other diseases, indicate that socially disadvantaged groups are likely to be at higher risk for infl uenza disease, particularly severe disease. The inability to predict which infl uenza virus will cause a future pandemic, together with the very limited national and global capacity to produce infl uenza vaccine in massive quantities in a short time, almost ensures that an effective vaccine will be unavailable to most or all of the population during the early stages of a pandemic and in very short supply thereafter. Even so, current plans assume that local and state public health agencies will have a primary role for distributing pandemic infl uenza vaccine. In general, however, these plans do not adequately address preventing or minimizing socioeconomic or racial/ethnic disparities in vaccine distribution and acceptance, despite evidence that such disparities have been the rule for the annual infl uenza vaccine, even among persons >65 years of age (20) . In the United States, routine use of annual infl uenza vaccine in preschool children has only recently been introduced; information focusing on school-age children is limited (21) . Nevertheless, African American/black children and children from lower income families, who are at higher risk of contracting infl uenza (22) in this country, are less likely to be up to date with other routine immunizations (23) . It is possible that, in the context of an infl uenza pandemic, vaccine-seeking and acceptance behavior and resultant coverage patterns may differ from those observed during routine vaccination efforts; however, the weight of available evidence indicates that social disparities in vac-cine coverage are likely to occur in the absence of careful planning to prevent them.
Among those who contract infl uenza, subsequent illness and death may be infl uenced by underlying factors and conditions and by the timeliness and effectiveness of various treatment modalities. Most infl uenza illnesses are self-limiting, and most infected persons during both annual infl uenza epidemics and infl uenza pandemics (including that of 1918-19) recover with only supportive care in the community. Even so, current planning efforts recognize the potential importance of reducing disease during a pandemic, through early treatment with antiviral drugs and through other forms of treatment such as respiratory support and antimicrobial agents to treat secondary bacterial pneumonia, among those with more severe disease.
In the United States, the likelihood of substantial disparities in access to timely and appropriate care under infl uenza pandemic conditions is high, given long-standing and persistent disparities in access to medical care. For example, persons with low income are ≈2× as likely as those with higher incomes to lack a usual source of healthcare (24) . Similarly, non-Hispanic black and Hispanic persons are signifi cantly less likely than non-Hispanic white persons to report having a usual primary care provider (10). Among persons who do report having a usual source of care, those who are poor or near poor and those who are non-Hispanic black or Hispanic are 2.5-4× as likely as their relatively higher income and white counterparts to rely on a hospitalbased source of primary care (24) . These same groups are also more likely to report having diffi culty obtaining timely appointments for illness or injury, which suggests problems with access to care even among those with a usual source of healthcare (10) . Language and cultural barriers to seeking and receiving medical care also may contribute to disparities. In emergency departments, for example, interpreters are frequently unavailable or underused, which has potentially adverse implications for patients' understanding of their disease or treatment and for clinical decision making Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 5, May 2008 711 Table. Factors that could contribute to health disparities among socioeconomic and racial/ethnic groups during an influenza pandemic Differences in exposure to influenza virus Crowding in households, medical facilities, public transportation Occupational factors such as inability to work from home, dependence on childcare outside of the home Differences in susceptibility to influenza disease, once exposed to the virus Host factors, including preexisting immunity, age, other underlying diseases or conditions, smoking, nutritional status, stress Vaccination status, reflecting differences in vaccine seeking and acceptance and in vaccine availability Differences in timely effective treatment, once influenza disease has developed Access to outpatient and inpatient medical care Care-seeking attitudes and behavior Financial obstacles, including lack of adequate insurance coverage Logistic obstacles, including transportation, language Quality of care Availability of antiviral treatments Appropriate inpatient treatment and quality of care (25) . In addition, the large numbers of persons who lack health insurance, as well as those who lack documentation of US citizenship, often delay seeking care because they are concerned about paying for the care or encountering legal diffi culties. Evidence from previous outbreaks suggests that antiviral drugs may be effective for treatment (26) and prevention (27) of pandemic infl uenza, and current antiviral drugs seem to be biologically effective against 1918 and 1918like viruses (28) . Because vaccine may not be available when a pandemic begins, experts have suggested that the antiviral drug oseltamivir should be stockpiled for use during a pandemic infl uenza outbreak. Recent models suggest that early use of oseltamivir may contain outbreaks if certain criteria regarding transmissibility and compliance are met (29) . However, experience with nonpandemic infl uenza indicates that oseltamivir must be given early during symptom development for it to have any substantial biological effect (30) ; modest delays may vitiate the treatment effectiveness (31) . Although plans for release and distribution of antiviral drugs are still being fi nalized, overcoming long-standing disparities in access to timely treatment by socioeconomic status, race/ethnicity, ability to speak English, and legal status will present numerous challenges to ensuring equal access to such drugs during a pandemic.
Reasons for concern about disparities in the timeliness and appropriateness of the care received by infl uenza patients who might benefi t from in-hospital care are similar. Given the predicted insuffi cient supply of hospital beds and staff during a pandemic (32), a person's access to potentially lifesaving therapies such as respiratory support and antimicrobial treatment of secondary bacterial pneumonias in an inpatient setting is likely to depend on factors that include usual source of care, citizenship status, and ability to speak English. Disparities may also occur in the quality of care received by persons who are hospitalized. Earlier US studies of persons hospitalized for pneumonia have found that blacks and "other minorities" are 71% and 79% as likely, respectively, as non-Hispanic whites to receive antimicrobial agents within 8 hours of arrival at the hospital (33) and signifi cantly less likely to have blood cultures obtained before receiving antimicrobial therapy (10) . Such disparities in quality of care would likely persist during an infl uenza pandemic.
Although reducing or eliminating socioeconomic and racial/ethnic disparities in health and healthcare has been an offi cial federal and state policy priority for 2 decades (34), such disparities remain prevalent and may inadvertently become wider when not explicitly addressed by policies designed to improve the health of the population as a whole and of disadvantaged persons in particular (35) . Given the current limitations of our public health infrastructure and the disparities in healthcare, a pandemic infl uenza outbreak in the United States is likely to disproportionately affect persons from socially disadvantaged groups. Explicit, systematic, and detailed plans are essential for overcoming the social barriers that are predicted to result in socioeconomic and racial/ethnic disparities in pandemic infl uenza illness and death. Saunders and Monet also have called for pandemic infl uenza planning that appropriately considers the needs of disadvantaged populations (36) .
The Pandemic Infl uenza Plan of the US Department of Health and Human Services (HHS) (37) does not adequately address potential social disparities in exposure, vaccination, or treatment; the possible effects of such disparities; or strategies for minimizing or eliminating them. The HHS plan (37) , the federal guidance on vaccine allocation (38) , and the recent Centers for Disease Control and Prevention (CDC) guidelines for community-level mitigation strategies (18) should be credited for calling for community engagement and inclusion of a wide variety of stakeholders in planning at the local level. Outreach to providers, community leaders, and organizations, particularly in disadvantaged communities, will be an important component of any strategy for addressing disparities during a pandemic. However, the available versions of offi cial plans do not call attention to the need for special efforts to overcome the greater barriers likely to be faced by socially disadvantaged groups.
On a US government website for pandemic infl uenza (www.pandemicfl u.gov), a question asks which groups would be especially vulnerable during an infl uenza pandemic. The answer notes that people may be vulnerable for a variety of reasons, including limited access to healthcare; limited profi ciency in English; or being disabled, homeless, economically disadvantaged, or a single parent. The response calls for faith-based and community-based organizations to develop plans "to care for dependent populations" and to "provide fi nancial aid to the poor who are unable to work and are in need of emergency income for housing, medicine, or other essential needs" (www.pandemicfl u.gov/faq/pandemicinfl uenza/pi-0001.html), which implies that attention to the needs of economically or socially vulnerable persons is not primarily a public-sector responsibility but is more a matter for private charity. The 2005 HHS plan (37) itself acknowledges that some groups may need fi nancial assistance if they are unable to work but does not indicate how that assistance would be provided or who would provide it.
Those who are still formulating plans should consider likely differences in infl uenza exposure and identify potential strategies for mitigating such disparities. Mathematical models have demonstrated that community-based interventions, such as quarantine and individual isolation, may be important for reducing infl uenza attack rates and overall incidence (29) . Most pandemic plans call for limiting public gatherings and closing schools to slow the spread of infl uenza, without adequately taking into account how implementing these strategies could differentially affect disadvantaged groups. Recent recommendations from CDC go further in recognizing the differential effect of socialdistancing measures on vulnerable communities (18) . Although CDC advocates fl exible work arrangements, income replacement, and job security to minimize the negative effects of social-distancing measures, it pays inadequate attention to those whose jobs will not accommodate these interventions. More specifi c solutions should be outlined in pandemic preparedness plans to address the economic effects of quarantine on low-income persons, who by staying home may be at risk wage loss, job termination, or both. Job security and income replacement are key components to limiting the effects of potential quarantine measures on disadvantaged persons (39) and should be extended to all persons, regardless of their type of work.
Important decisions also will need to be made concerning access to vaccination and treatment in the event of a pandemic. The federal government's Draft Guidance on Allocating and Targeting Pandemic Infl uenza Vaccine (38) provides a basic framework for allocating vaccine during the pandemic. An appendix to that document mentions (on p.17) that the principles of "fairness and equity (recognizing that all persons have equal value, and providing equal opportunity for vaccination among all persons in a priority group)" were considered when drafting the guidelines. Although the proposed schema very reasonably fi rst defi nes groups of different priority levels according to occupation and then, within the general population, according to age and pregnancy status, it does not provide explicit attention to groups who are vulnerable because of social disadvantage. Nor does it note the need for explicit attention to vulnerable social subgroups, for example, low-wage workers in prioritized occupational fi elds and low-income and minority pregnant women, infants, and toddlers. We are not questioning the rationality of defi ning major priority groups according to occupation or of using biological criteria to further prioritize within the general population. Rather, our concern is with the absence of attention to both biological and social risk factors, which must be addressed to overcome the many social barriers to equal opportunity for vaccination.
Well-documented evidence of existing healthcare disparities suggests that during a pandemic shortages of infl uenza vaccine, antiviral drugs, inpatient services, and healthcare staff will disproportionately affect persons in socially disadvantaged groups. To limit the crowds that might occur at hospitals and clinics, plans for the release of stockpiles of vaccines, medications, or both could include distribu-tion from private pharmacies or doctors' offi ces. However, because private pharmacies and private practitioners are less likely to be located in lower income neighborhoods, plans to make access to potentially lifesaving vaccines and drugs speedier and more equitable might, in fact, exacerbate disparities. Distribution plans may need to include mobile community health centers (staffed by nurses and nurse practitioners) that can travel to low-income areas, along with a variety of community medical and other service providers and nontraditional sites like soup kitchens, sheltered workshops, and transit points, which have become popular places for administering yearly infl uenza vaccine (40) . Other factors, such as the availability of transportation to a hospital, might also become more important during a pandemic. Access to a private car may be a major determinant of who is able to obtain care, presenting constraints like those that led to disparities in evacuation from New Orleans before Hurricane Katrina. To ensure that disadvantaged communities are reached and that resources are equitably allocated during an infl uenza pandemic, preparedness plans can and should involve community-based providers and organizations that are familiar with vulnerable groups.
Social group disparities in exposure, susceptibility, and access to timely and effective treatment for a variety of diseases have been well documented in the United States. Infl uenza pandemic preparedness plans that fail to explicitly provide guidelines on how to mitigate these issues could lead to decisions that may, on the surface, seem reasonable, but that are likely to exacerbate social group disparities in health outcomes. Given the existence of major disparities in health and healthcare, we cannot expect pandemic preparedness and response planning to eliminate the deep divides that exist between socioeconomic and racial/ethnic groups. These disparities can, however, be minimized through careful planning that considers and proactively addresses vulnerability at each level: exposure to disease, susceptibility to disease if exposed, and treatment of disease. Public offi cials should systematically consider the additional barriers faced by socially disadvantaged groups at each of these levels and then actively seek ways to address those barriers. Local service providers, leaders of community-based organizations and other organizations working with socially vulnerable groups, and leaders of labor unions representing low-wage service workers are likely to have valuable insights and should be included in the planning process. Plans calling for stakeholder involvement without explicitly emphasizing the need to involve representatives of socially disadvantaged groups are unlikely to be effective at minimizing social disparities during an infl uenza pandemic.
We have focused here on the United States, but similar fundamental principles-the need for systematic and concrete planning to minimize the social disparities that can be expected to occur in the face of natural disasters such as an infl uenza pandemic-apply worldwide. Countries with universal fi nancial access to healthcare and strong social safety nets will be best positioned to minimize such disparities. Countries in which large proportions of the population are impoverished or otherwise socially excluded and countries that have more limited resources and weaker public health and social welfare infrastructures will face the greatest challenges. The framework used here-considering and proactively addressing social vulnerability in exposure to pathogens, susceptibility to disease once exposed, and consequences of illness-should be applicable across national and subnational settings.  Conflict and Emerging Infectious Diseases  To the Editor: In the November 2007 issue of Emerging Infectious Diseases, Gayer et al. (1) describe how confl ict leaves populations in dire poverty, internally displaced or seeking asylum, having poor access to essential services, and consequentially vulnerable to infectious diseases. Cholera, caused by the bacterium Vibrio cholerae, is a disease that seems particularly sensitive to confl ict and deserves more consideration. Major risk factors for cholera-poverty, overcrowding, poor hygiene, contaminated food, and lack of safe drinking water (2,3)-largely resemble the consequences of war and civil fi ghting. Yet little is known about the relationship between cholera and confl ict. This lack of information may be because cholera tends to be epidemic, affecting hundreds to thousands of people across vast, war-torn regions, making it impossible for local governments, nongovernment organizations, and aid workers to control, let alone collect and analyze data.
Examination of data sources listed by Gayer et al. (1) and recent reviews (2, 3) indicate that cholera occurs 1) in countries during war and civil unrest, as exemplifi ed by the latest outbreaks among displaced populations across northern Iraq; 2) in neighboring countries, where temporary camps accommodate masses of political refugees under poor conditions, such as those in eastern Chad near Darfur, Sudan; and 3) during the postwar period when large numbers of repatriated persons return home and consequently place undue pressure on an eroded and fragile national infrastructure, as evident in Angola in recent years.
Moreover, all the countries affected by confl ict shown in the Figure by cdc.gov/EID/content/13/11/1625-G. htm) have reported cholera outbreaks (2) (3) (4) . They are also among the poorest countries in the world; the latest statistics on human development (5) indicate that compared with all developing countries, on average they have higher rates of undernourishment, refugees, child deaths, and less adequate water and sanitation facilities. Thus, more information is needed about confl ict and cholera, especially in Africa. In Response: We agree with Kelly-Hope on the propensity for cholera outbreaks to occur in confl ict-affected countries and the need to monitor and respond more effectively to such events. In 2006, cholera was reported from 33 countries in Africa, and 88% of all reported cases were from confl ict-affected countries (1) .
As highlighted in our November 2007 article on confl ict and emerging infectious diseases (2), confl ict situations present a multitude of risk factors that enhance disease emergence and transmission, over and above those in other resource-poor countries. Many such confl icts facilitate the occurrence of cholera outbreaks.
More precise research on cholera and confl ict is indeed necessary. However, despite cholera being a disease that has been around for a long time and that causes frequent outbreaks to this day, much information about this disease, beyond its relationship with confl ict, remains unknown. For example, although Vibrio cholerae persists in the environment, little is known about the exact conditions that trigger a cholera outbreak at a particular time.
Further elucidation is needed about the factors that infl uence the duration of an outbreak, disease severity, and duration of individual protective immunity after an episode of cholera.
Cholera, which is closely linked to a country's social and economic development (1, 3) , ceased to be of concern in Europe, for example, when access to potable water and sanitation improved although its cause was still unknown and antimicrobial drugs were not yet available. Today, renewed interest from the international public health community is urgently warranted, and strong initiatives are needed to help developing countries (confl ict-affected or not) fi ght against cholera and control this easily preventable disease on a global level.
*World Health Organization, Geneva, Switzerland Global Distribution of Novel Rhinovirus Genotype Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years. A cute respiratory illness (ARI) is the most frequent infectious disease of humans. Ordinary upper respiratory tract infections are usually self-limited; nevertheless, they result in major economic impact through loss of productivity and strain on healthcare systems. Lower respiratory tract infections (LRTIs) are among the leading causes of death in children <5 years of age worldwide, particularly in resource-poor regions (1) . Streptococcus pneumoniae and Haemophilus infl uenzae are important bacterial causes of ARI, although their impact is expected to decline with increasing vaccine coverage. Collectively, however, viruses dominate as causative agents in ARI. Viruses frequently implicated in ARI include infl uenza virus, respiratory syncytial virus, metapneumovirus, parainfl uenza virus, human enterovirus (HEV), and human rhinovirus (HRV).
HRVs are grouped taxonomically into Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B), 2 species within the family Picornaviridae (International Committee on Taxonomy of Viruses database [ICTVdb]; http:// phene.cpmc.columbia.edu). These nonenveloped, positivesense, single-stranded RNA viruses have been classifi ed serologically and on the basis of antiviral susceptibility profi le, nucleotide sequence relatedness, and receptor usage (2) . Phylogenetic analyses of viral protein VP4/VP2 and VP1 coding regions indicate the presence of 74 serotypes in genetic group A and 25 serotypes in genetic group B (2) .
Isolated in the 1950s from persons with upper respiratory tract symptoms (2, 3) , HRVs have become known as the common cold virus because they are implicated in ≈50% of upper respiratory tract infections (4) . Large community surveys, including the Virus Watch studies of the 1960-1970s (5), have shed light on some aspects of HRV biology and epidemiology. HRVs were also observed in LRTIs soon after their recognition (3) , and data supporting a causative association have accumulated over the past decade (6, 7) . HRVs have also been implicated in exacerbations of asthma and chronic bronchitis and are increasingly reported in LRTIs of infants, elderly persons, and immunocompromised patients (4).
The advent of broad-range molecular assays, including multiplex PCR and microarray systems, promises new insights into the epidemiology and pathogenesis of respiratory disease (8, 9) , given that a laboratory diagnosis is not routinely achieved for a substantial portion of respiratory specimens from symptomatic patients. We recently described the application of a multiplex PCR method for microbial surveillance wherein primers are attached to tags of varying mass that serve as digital signatures for their genetic targets. Tags are cleaved from primers and recorded by mass spectroscopy, enabling a sensitive, inexpensive, and highly multiplexed microbial detection. We used the multiplex MassTag PCR system (10) to investigate respiratory samples that had tested negative during routine diagnostic assessment. This previous study yielded pathogen candidates in approximately one third of cases, and in 8 cases identifi ed a novel genetic clade of picornaviruses divergent from the previously characterized clades, including HRV-A and-B (8) . To assess whether this novel clade cir- Figure) . Samples collected in Côte d'Ivoire, West Africa, were from symptomatic persons living in the vicinity of Taϊ National Park. This location was the most remote of our study; residents have limited contact with other human populations. In this location, 2 (10%) HRV-A were identifi ed in the 52 samples available for analysis (Table 1, Figure) .
In Nepal, viruses of the novel genotype were identifi ed in specimens collected during ILI surveillance or outbreaks of respiratory disease. Samples from ILI surveillance activities were collected in Kathmandu and Bharatpur. Outbreak samples were collected in the summer months from camps of >100,000 refugees from Bhutan located in Jhapa, southeast Nepal. Samples represented all age groups and were collected from December 2005 through July 2006. The novel genotype was identifi ed by independent molecular typing in both laboratories in 4 (5%) samples ( Figure) . Additional sample sets were obtained through main diagnostic laboratories in Western Australia, Denmark, and Spain, representing random respiratory specimens submitted for laboratory analysis. In 1 sample available from Western Australia, the novel genotype was identifi ed in a preterm infant with undiagnosed, wheezy LRTI. The novel genotype was also found in 5 (7%) of 70 samples from Denmark and in 6 (43%) of 14 samples with previously diagnosed HRV infection from Spain (Table 1, Figure) .
The 5% overall frequency of the novel genotype across our study samples, representing 34% of all detected picornavirus infections, and its observed global distribution, led us to analyze the accumulating sequence data for insights into their history. Rates of evolutionary change and the Time to the Most Recent Common Ancestor (TMRCA) of the novel clade were estimated by using the Bayesian Markov Chain Monte Carlo approach (BEAST package [14] ; ), applying a relaxed molecular clock with an uncorrelated lognormal distribution of rates, a GTR + I + Γ 4 model of nucleotide substitution (determined by MODELTEST [15] ), and exponential population growth. Statistical uncertainty in each parameter estimate is expressed as 95% highest probability density (HPD) values. The estimated mean rate of evolutionary change was 6.6 × 10 -4 substitutions/site/y (95% HPD = 0.3-14.6 × 10 -4 substitutions/ site/y; 38 dated samples collected over 32 mo (8,16) (S.R. Dominguez et al., unpub. data). Under this rate the mean TMRCA was estimated at 1,800 y, although with wide variance caused by the short sequence available (95% HPD = 279-5,201 y). Despite the inherent sampling error, this analysis suggests that this third clade of rhinovirus has been circulating for >250 years. The diversity observed within the novel clade and its genetic distance from other HRV/ HEV were comparable to those seen for HRV-A, -B, or the HEV species (Table 2) .
A clade of picornaviruses recently discovered in New York State is globally distributed and is found in association with community outbreaks of ARI and severe LRTIs of infants. These viruses contribute both to a substantial proportion of previously undiagnosed respiratory illness and to diagnosed, but nontyped cases of HRV infection. Similar viruses were recently characterized also in Queensland, Australia (11); California, USA (12); Hong Kong Special Administrative Region, People's Republic of China (13) ; and Germany (16) . Our fi ndings indicate the need for further investigation into this third (HRV-C) group of rhinoviruses with emphasis on epidemiology, pathogenesis, and strategies to prevent and ameliorate disease caused by HRV infection. Cost effective strategies for completing the Interactome Comprehensive protein interaction mapping projects are underway for many model species and humans. A key step in these projects is estimating the time, cost, and personnel required for obtaining an accurate and complete map. Here, we model the cost of interaction map completion across a spectrum of experimental designs. We show that current efforts may require up to 20 independent tests covering each protein pair to approach completion. We explore designs for reducing this cost substantially, including prioritization of protein pairs, probability thresholding, and interaction prediction. The best designs lower cost by four-fold overall and >100-fold in early stages of mapping. We demonstrate the best strategy in an ongoing project in Drosophila, in which we map 450 high-confidence interactions using 47 microtiter plates, versus thousands of plates expected using current designs. This study provides a framework for assessing the feasibility of interaction mapping projects and for future efforts to increase their efficiency. Mapping a complete gene or protein network evokes similar challenges and considerations as mapping a complete genome sequence. In the case of the human and model genome projects, large-scale sequencing efforts were accompanied by a series of feasibility studies27,28 which used mathematical formulations and pilot projects to explore strategies for genome assembly and the work required for each. In the case of interaction networks, similar feasibility studies are just beginning29-31. Some of the questions to be addressed are: What is the cost of completing an interactome map and what is the best strategy for minimizing that cost? Given practical cost constraints, how can the quality and coverage of interaction data be maximized? How many independent assay types are needed? How should direct pairwise tests for interaction be combined with pooled screening? What is the effect of the test sensitivity on the final cost? How should interaction predictions be incorporated, and what is their effect on the mapping cost? Which specific improvements to experimental and computational methods are likely to have the largest effect?
To approach these questions, we modeled several standard and alternative strategies for using pairwise protein interaction experiments to determine the interactome of the fruit fly Drosophila melanogaster. Our analysis shows that completing the interactome using sequential pairwise or pooled screening is probably too costly to be practical. However, this cost can be reduced substantially using a strategy that combines pooling with prioritized testing and interaction prediction. We carry out several iterations of this strategy to efficiently map 450 new high confidence interactions in Drosophila.
In contrast to a genome, the interactome has been more difficult to define. Some authors have argued32 that the "True Interactome" should be defined as all possible interactions encoded by a genome-i.e., the set of all pairwise protein interactions that occur in at least one biological condition or cell type. The assumption is that every true interaction will be detectable by some assay, and that given enough independent measurements, most of the interactome could be detected. Many assay types have been described for detecting proteinprotein interactions, a few of which have been adapted to large-scale screening1,32-34. On the other hand, some interactions may be immeasurable by any available assay, or will not arise in the conditions surveyed. Therefore, we use the term "Mappable Interactome" for the subset of true pairwise interactions that are reproducibly detectable by any given assay method or combination of methods.
To define appropriate criteria for determining when an interactome map is "complete", we distinguish between the terms saturation and coverage. Saturation measures the percentage of true interactions that have been experimentally observed at least once. Coverage we define more strictly to mean the percentage of true interactions that have been experimentally validated with high confidence such that the percentage of false interactions (i.e., the False Discovery Rate or FDR) is kept below a predetermined threshold. We treat "completion" as achieving 95% coverage of the Mappable Interactome at 5% FDR, which requires that the map include at least 95% of all true interactions with no more than 5% of the reported interactions being false.
We simulated a series of mapping strategies implementing a variety of basic and sophisticated features ( Fig. 1 ; Flowcharts of each strategy are provided in Supplementary  Fig. 1 ). All strategies were evaluated using a statistical model based on naïve Bayes to estimate saturation and coverage of the Drosophila interactome as a function of the number of interaction tests. We programmed this model with the assumption that the fly interactome contains approximately 105 interactions overall, along with estimates for the false positive rate (FPR-the probability that a non-interacting protein pair is reported as interacting) and the false negative rate (FNR-the probability that an interacting pair is reported as noninteracting). Although the magnitudes of these errors are still under debate, previous studies2,5,29,35,36 have reported Y2H error rates of FPR < 1% with FNR in the range 50-80% (note that several of these studies erroneously refer to FDR as FPR). Here, we used 0.2% FPR and 66% FNR.
Due to the high FNR of a particular assay, it becomes clear that multiple assay types will likely be needed to achieve complete coverage, and that these assays should be independent or at least only partially dependent. Although the precise correlations between different assay types have not been well studied, complementarity between assays has been widely assumed and occasionally demonstrated: For instance, protein interactions have been shown to be of substantially higher confidence if they are detected in different orientations (baitprey vs. prey-bait)2; in different Y2H screens3,8,35; by different types of Y2H system such as LexA-based vs. Gal4-based36; or by both Y2H and co-affinity purification29.
We first simulated a "Basic serial" strategy, in which all pairs of proteins are tested for interaction sequentially. Under this formulation, achieving a saturation of 95% required eight comprehensive screens, in which each protein pair was tested by eight independent assays, equivalent to ~7×108 pairwise tests assuming a total of 13,600 protein-encoding genes in fly37 (Table 1 and Fig. 2a) . Moreover, 93% of all observed interactions in this network were false positives (including 99% of interactions observed exactly once and 21% of interactions observed twice- Fig. 2b ). The false-positives predominate because, although the 0.2% FPR seems low, the number of non-interacting protein pairs is far in excess of the number of true interactions.
To ensure an overall FDR < 5%, we found that every interaction must be reported by at least three independent assays. After eight screens 55% of the interactome was covered under these conditions. The coverage goal of 95% was achieved only after 21 comprehensive pairwise screens (Fig. 2c) . This overall outcome-that the number of experiments required to reach full coverage is two to three times that required to reach saturation-was observed over a range of error parameters (Supplementary Table 1 ). Clearly, completing the interactome map under these conditions is impractical, as it would require testing 92 million protein pairs 21 separate times.
To reduce the number of tests, assays such as Y2H typically use pooled screens in which a single protein "bait" is tested for interaction against pools of protein "preys" (phase I) 38 . For pools that test positive, pairwise tests are immediately conducted between the bait and each prey in the pool (phase II-this second phase can also be conducted by sequencing3,5). The benefit of pooling is that large numbers of potential interactions can be sampled at relatively low cost. This comes at the expense of FNR, as the chance a true interaction is missed in the pool is higher than the chance it would be missed by direct pairwise tests38. Through simulation, we found that basic two-phase pooling (Pooling strategy) does indeed achieve a four-to five-fold reduction in coverage cost over pairwise testing (~4 million plates for Pooling compared to ~20 million plates for Basic-serial, Table 1 ). However, assuming the rate of interaction screening of a typical laboratory (e.g., ~2400 plate-matings per person per year), pooled screens would still require approximately 1700 person-years to achieve completion of the Drosophila protein network.
We next considered an interaction mapping strategy that, rather than treat all protein pairs equally, maintains a rank-ordered list of pairs according to their probabilities of interaction (Thresholding strategy, Table 1 ). All probabilities start at the background frequency of interaction for random protein pairs (as for Basic-serial and Pooling). Protein interactions are initially tested using pooled screening, and after each two-phase pooled experiment the probabilities increase for interactions that are observed and decrease for interactions that are tested but not observed. Unlike previous strategies, however, protein pairs with probability greater than an upper threshold (i.e., 95%) are declared to be definite "interactors" and are removed from subsequent testing (Fig. 1b) . Likewise, interactions with probability beneath a lower threshold are declared to be "non-interactors" and are also removed from further consideration. The motivation for thresholding is to more quickly exclude the overwhelming number of non-interacting protein pairs. Finally, candidate interactions are defined as those with probabilities between the upper threshold and background. When candidates are available they are always tested immediately using pairwise assays, before resorting to pooling, until their probabilities are pushed above the upper threshold or below background. The motivation for prioritizing candidate interactions is to more quickly cover the interactions likely to be positive. Overall, Thresholding resulted in more than a two-fold cost reduction compared to Pooling (Table 1 and Fig. 3a ).
Lastly, we considered whether computational prediction of interactions, based on prior knowledge and data, could hasten the time to interactome completion. A variety of prediction methods have been proposed based on evolutionary conservation39-41-i.e., transfer of interaction measurements from one species to another-or based on integrating conservation with additional features such as co-expression and co-annotation42-47. Such predictions impact the experimental design by setting the prior probabilities of interaction for each protein pair in lieu of background probabilities. In the Prediction strategy, we explored the effect of setting these prior probabilities using theoretical prediction methods simulated over a range of predetermined prediction accuracies (a range of different values for FPR, FNR, and corresponding FDR of the predictions). We found that even predictors with very high FDRs could have a major impact on the mapping cost (Table 1 and Fig. 3b) . For example, a predictor with 92.2% FDR gave a four-fold reduction in cost over Pooling, with a >50-fold reduction in cost to achieve 50% coverage and a savings of hundreds of fold in the early stages of mapping. Moreover, the 92.2% FDR means that even a predictor that makes 12 false predictions for every true one can lead to a major reduction in the cost of interactome completion. The best prediction method required approximately 385 personyears to achieve 95% coverage of the Drosophila protein network and 12 person-years to achieve 50% coverage. Thus, while obtaining full coverage of an interactome map may still be some years away, a draft scaffold providing half coverage might be feasibly achieved by a team of ~12 technicians working over a period of one year.
Given the high performance of the Prediction strategy in simulations, we explored an experimental implementation in which Drosophila protein interactions were predicted using the cross-species method of Sharan et al.39 (Fig. 4a ). According to this method, existing protein interaction networks in yeast, worm, and fly are aligned based on sequence similarity to identify conserved interaction clusters, and these alignments are used to transfer interactions that have been observed in some species but not yet in others (Fig. 4b) . A total of 1,294 interactions were predicted using this method, each of which was prioritized as a candidate with high prior probability (92.4%) based on the FDR reported by Sharan et al.39 (7.6%).
Since this prior was much greater than the background probability of other protein pairs (0.1%), we began by using the pairwise Lex-A Y2H assay48 to test all 606 predictions for which sequence-verified clones were available. Of these, 136 tested positive and 470 negative. After each 96-well plate of tests (seven plates total), the interaction probabilities were updated resulting in an increase to >99.9% for pairs testing positive and a decrease to 90.5% for pairs testing negative. Since the 136 positives now had probability greater than the upper threshold (95%), all of these could be added to the interactome map and removed from further testing.
Although the remaining 470 predictions had tested negative once, their high probability (90.5%) still prioritized them as candidate interactions. Therefore, as dictated by the Prediction strategy these pairs were tested again immediately using a second assay type.
For this second assay, Lex-A Y2H was run in a "reverse" orientation in which the two proteins cloned as bait and prey, respectively, were exchanged as prey and bait. We tested 251 of the 470 predictions for which sequence-verified clones were available in the opposite orientation. This resulted in 35 positives, elevating these interactions to probability >99.9% and adding them to the map. The pairs that tested negative in the reverse orientation were downgraded to 88.1% probability. Overall, after performing Y2H in both forward and reverse orientations, 171 new interactions were identified out of 606 protein pairs for a success rate of 28%. Although we ended our experimentation at this point, the Prediction strategy could be continued by next testing the "double negatives" (pairs testing negative in both orientations of Lex-A Y2H) using a third type of assay such as Gal4-based Y2H.
A means of predicting additional protein interactions is to probabilistically integrate many different lines of evidence into a single classifier42-47. Along these lines, we applied a machine-learning-based classifier for predicting interactions that combined many relevant features including gene expression, domain-domain interactions, conserved protein-protein interactions, genetic interactions, and shared gene annotations (Supplementary Methods). We used this approach to generate 24,798 high confidence predictions. We randomly selected 2,047 of these for testing using forward-orientation Y2H and, as above, retested the negative pairs using reverse-orientation Y2H (for which clones were available). In total, this procedure added 279 new high-confidence interactions to the map for a 13.6% success rate. Combined over both conservation-based and multiple-evidence-based predictions, 450 new protein-protein interactions were added to the Drosophila map using 47 96-well plates (Fig.  3a,b) . To establish the background rate of interaction, we also tested 2,354 randomly chosen pairs, 72 of which were positive yielding a 3% background rate (Fig. 4b) . These results show that both types of prediction are highly enriched for true interactions. Note that even if all predicted interactions were true, the expected confirmation rate would be limited by the false negative rate of the Y2H assay, equal to 1-FNR =33% in our model.
An underlying assumption of our simulations is that different assay types are conditionally independent-i.e., given that a tested protein pair is known to be positive or negative, the result of one assay is uncorrelated with that of another. To examine the extent to which this assumption holds, we compared Y2H data for protein pairs tested in both forward and reverse orientations-the two assay types used in our study. Overall, we obtained Y2H tests in both orientations for 309 conservation-based predictions (including data reported above combined with additional tests; Supplementary Data). Of these, we observed 58 positives in the forward orientation and 50 positives in the reverse orientation, for an average positive rate of 17% [(58 + 50)/(309 * 2)]. Fifteen positives were found in both orientations, representing 4.9% of the tests. Assuming all predictions are true interactions, this percentage is very close to that predicted by conditional independence, for which 3.1% of tests are expected to be positive in both orientations [17% ^ 2]. If some predictions are not true as expected, the percentages come into even better agreement-e.g., a prediction FDR of 20% predicts that 4.8% positives would arise in both orientations. A similar analysis was performed on a set of 1,572 combined-evidence predictions that were tested in both orientations, leading to similar agreement with the conditional independence assumption.
The interactions predicted by cross-species conservation were at least as accurate as we had assumed in our simulations. On the other hand, their power to prioritize interactions is dependent on the network coverage in other species, and the long-term viability of this approach will depend on obtaining greater numbers of predictions than the 1,294 that are currently available. As interactome maps progress across an ever-widening array of species, these maps might be dynamically cross-compared to continually generate sufficient numbers of candidate interactions for testing. The second set of predictions, made by integrating various lines of evidence, had a lower success rate than the predictions based solely on conservation. Their potential utility is higher, however, since the number of available predictions is nearly 20 times that of the conservation-based predictions and could be increased further by including lower confidence predictions. Even with a lower success rate, the performance of the integrated classifier was superior to the best theoretical predictor we simulated.
Predictions lead to a lower interactome mapping cost for two reasons. First, predicted protein pairs are much more likely than arbitrary pairs to be true. Second, protein pairs with high prior probabilities do not require repeated positive measurements to confirm them as true interactions. Both effects underlie the finding that 450 new predicted interactions could be added to the interaction map using just 47 microtiter plates. In contrast, the Pooling strategy would require nearly 105 plates to add this number of interactions to the map.
One might intuitively object that, rather than test predicted interactions, a better strategy would focus on the "novel" areas of the interactome that have never before been suggested by any species or data set. The problem with such an approach is that it would very quickly produce an interactome map with a very high error rate. Conversely, the rationale behind the Thresholding and Prediction strategies is that one should first clean up the map by validating predicted interactions using real experiments, and only then resort to testing random protein pairs in pools.
A second objection might be that prioritizing candidate interactions requires the corresponding Y2H baits and preys to be rearrayed in microtiter plates in different orders over the course of an interaction mapping project. While the cost of rearraying was not included in our analysis, in our lab (Finley) these costs are greatly alleviated through robotic transfer systems. Certainly, failure to rearray leads to a ~4-fold increase in cost and a ~10fold increase in the early stages of mapping (compare Pooling versus Prediction in Table 1 ).
Regardless, mapping the Interactome remains a daunting task. Our study makes it clear that achieving 95% coverage of an interactome requires many more screens than one pass through all pools or over all protein pairs. If complete coverage is to be obtained in the near future, it will be necessary to invoke better strategies for experimental design, technologies reporting fewer false negatives, or both. In terms of experimental design, we have shown that the cost of completion is reduced substantially by careful ordering of pooled screens. In terms of technology, our study underscores the importance of decreasing the FNR or of different assays that provide independent samples of a protein pair. Even if the error rates are lower than assumed here, advanced mapping strategies are still likely to be worthwhile (Suppl. Table 1 ). Here we have used two types of Y2H assay, forward and reverse orientations, to obtain multiple samples which appear largely independent. If the assays were partially dependent, multiple tests might still be worth the cost as long as they were not perfectly correlated (and the dependence could be handled quantitatively using a statistical model). In the present study, the conditional independence assumption leads to a "best-case scenario" or lower-bound on the number of interaction tests that will likely be required to achieve full coverage of an interactome. Further work will be needed to better characterize the relative dependencies among the wide range of other interaction assays that are currently available-if the current assays are highly dependent, then the required number of tests will be greater than was estimated here.
"True" reference interactomes for fly and human were generated by random sampling of interactions from the set of all possible pairs of proteins using the interaction probabilities in the String database46. Protein pairs not included in the String database were sampled using a low background probability, such that the total number of interactions in the sampled interactomes agreed with current estimates of interactome sizes30 (~100,000 fly interactions and ~260,000 human interactions). The detectability of each protein pair was independently sampled for each new assay type (representing a new type of measurement technology or new bait/prey orientation) using a 66% FNR for true interactions and 0.2% FPR for false interactions (corresponding to 82% FDR). Once an interaction was defined as "Detectable/ Undetectable", direct pairwise experiments were assumed to be 100% reproducible for a given protein pair and assay. For pooled assays, each detectable interaction in the sample space of a pool was assumed to be observed in the pool with probability equal to the pooling sensitivity (Table 1) . Pools with at least one observed interaction were declared positive. For each strategy, after every 1000 experiments the mapped interactomes were compared to the "true" interactomes and the coverage and FDR were recorded.
We used the LexA-based yeast two-hybrid mating assay48 using sequence-verified clones as previously described36 (Supplementary Methods). All new protein interactions have been submitted to the IMEx consortium (http://imex.sf.net) through IntAct49 and assigned the identifier IM-9552. The data are also available at DroID (www.droidb.org).
Detailed descriptions of the interaction probability model, the combined-evidence method for interaction prediction, the computation of thresholds, and the yeast two-hybrid test protocol appear in the Supplementary Methods.
Refer to Web version on PubMed Central for supplementary material. (a) At any given point in the project, every pair of proteins is assigned an interaction probability based on its experimental history (initially these probabilities are set to background or informed by predictions). The interaction probabilities and experimental history are used to design a 96-well plate Y2H experiment according to one of the strategies. The result of this experiment is simulated based on the detectability of the tested interactions (given the assay type) and the pooling sensitivity. The new experimental results are recorded in the history and also (b) used to update the interaction probabilities of the relevant protein pairs. The pyramid represents the ordered list of protein pairs ranked by probability. It is wider at the bottom than at the top to reflect that most pairs are negative-i.e., most pairs will have low probability and only a few pairs will percolate to the top of the list with high probability. Interactions with probability above an upper threshold are added to the mapped interactome, which is compared to the simulated "True Network" at intervals of 1,000 plates for reporting coverage and FDR. The false discovery rate (FDR) of interactions that are observed exactly once (orange), twice (purple), thrice (green), four times (yellow), and five times (cyan) as a function of the number of times they are tested with independent assays. To achieve FDR < 5% interactions should be observed at least twice when tested with < 5 independent assays, and at least three times when tested with 5-17 assays. (c) The effective coverage level at FDR < 5% is shown (red curve) by embedding the observation threshold from (b) into the curves of (a). While saturation is achieved after 8 screens, 21 screens are required for 95% coverage at FDR < 5%. * Interaction costs are given in units of total number of plates (K = Thousands, M = Millions) required for 50% or 95% coverage. When 95% coverage is achieved more than once, the greatest cost is presented. Augmented Lung Inflammation Protects against Influenza A Pneumonia BACKGROUND: Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia. METHODS AND FINDINGS: To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001). CONCLUSIONS: Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia. The annual worldwide mortality associated with pneumonia exceeds that of any other infection [1, 2, 3] . In particular, influenza pneumonia annually causes more than 40,000 deaths in the United States alone [4, 5] . Beyond the impact of seasonal influenza, episodes of pandemic influenza have accounted for as many as 50 million deaths [6] . H5N1 avian influenza has already caused more than 240 human deaths worldwide (http://www. who.int/csr/disease/avian_influenza/), and increased globalization since 1918 suggests that eventual human-to-human transmission of avian influenza may cause even greater lethality than the infamous ''Spanish flu'' [7] . Further, viral pathogens, including influenza, are considered potential agents of bioterror [8] .
The mechanisms underlying influenza-related mortality remain controversial. Progressive pneumonia following an insufficient antiviral host response is one possible cause [9] . Virally-induced excessive and/or dysregulated lung inflammation is another potential mechanism [10, 11, 12, 13] . Secondary bacterial infections have also been proposed as the primary contributors to influenzarelated mortality, due to virally-injured epithelium or virusattenuated leukocyte responses [13, 14, 15] .
We have recently reported that treatment with an aerosolized lysate of nontypeable Haemophilus influenzae (NTHi) induces profound inflammation in the lungs, yet it strongly protects mice against otherwise fatal bacterial pneumonia [16] . The induced protective phenomenon, known as Stimulated Innate Resistance (StIR), is maximal within 4 h of treatment and does not rely on recruited neutrophils or resident mast cells and alveolar macrophages. Given the profound induction of lung inflammation by this treatment, we perceived an opportunity to determine whether host inflammation contributed to or prevented mortality in influenza A pneumonia.
We demonstrate that, despite inducing inflammation greater than that observed in lethally infected animals, the aerosolized NTHi lysate results in remarkable protection against otherwise lethal influenza pneumonia, and that it can be synergistically combined with antiviral medicine for post-infectious treatment. Together, these data suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia, and indicate that innate immune resistance to viruses can be therapeutically stimulated to protect populations from pandemic influenza.
Having shown that profound inflammatory lung responses to a bacterial lysate improved survival of mice infected with noncognate bacteria [16] , we investigated the effect of stimulation of lung mucosal innate immunity on survival of influenza A pneumonia. Mice challenged by aerosol with influenza A/Hong Kong/8/68 (H3N2) (A/HK) universally succumbed to hemorrhagic pneumonia unless pretreated with aerosolized NTHi lysate (Fig. 1A) . This treatment reduced mortality .90% if delivered on the day prior to infection, .50% if delivered 3 days prior to infection, and to a lesser degree if delivered one day after infection (Fig. 1B) . Mortality occurred within 10 days of the viral inoculation, and observation for 21 days after infection showed no subsequent mortality. The protection imparted by the aerosolized lysate was paralleled by the recovery of body weight lost early in the course of infection (Fig. 1C ). Lysate-induced survival was accompanied by a significant reduction in viral load on the fourth day after challenge (Fig. 1D ).
Since prior reports have described poorer outcomes associated with virus-induced inflammation [10, 11] , we compared the inflammation induced by the NTHi lysate, inflammation induced by influenza infection, and inflammation induced by the combination of treatment and infection. Figure 2 shows that single or repeated treatment with NTHi lysate induces dramatic inflammation in the lungs, measured by cytokine levels ( Fig. 2A ) and cellular influx (Fig. 2D) .
Remarkably, while treatment with NTHi lysate acutely induces 8-fold more IL-6 and almost 40-fold more TNF than infection alone, the treated mice actually have lower levels of inflammatory cytokines in their lungs by day 3 after the infection (Fig. 2B ). By that time after treatment, the NTHi lysate-induced rise in cytokines in uninfected mice has entirely resolved (data not shown), and the inflammatory cell infiltration has fallen by more than 80% [16] . Together, this suggests that a robust early inflammatory reaction may allow for more rapid resolution of the infection-induced inflammation. Notably, the intense inflammation observed in the lungs is not seen systemically. Despite increases in IL-6 and TNF of 700-and 900-fold in the lungs, respectively, these cytokines increase only minimally and transiently in the serum (Fig. 2C ).
Since interferon signaling is essential to baseline host antiviral resistance in the lungs [17, 18] , we investigated whether treatment with NTHi lysate was capable of inducing an interferon response. In addition to the robust induction of inflammatory cytokines, we found that NTHi lysate treatment also induces significant increases in lung interferon c levels ( Fig. 2A) . However, unlike IL-6 and TNF, the fulminant influenza infections observed in untreated mice actually induce 4-fold higher levels of interferon c than the NTHi lysate treatment (Fig. 2B ). Presumably this reflects the ongoing antiviral response of the host, in contrast to the better contained infections of the treated mice.
To further characterize the lung interferon response to the NTHi lysate, we assessed gene expression using whole genome microarray analysis. As maximal resistance against S. pneumoniae is achieved by 4 h post-treatment, we compared gene expression of sham treated mouse lungs to mouse lungs 2 and 4 h posttreatment. Pathway analysis revealed interferon signaling to be among the most upregulated events following NTHi lysate treatment (p,10 211 ). Interferon signaling pathway transcripts are reported in Table 1 , showing that treatment induces expression of numerous transcripts critical for both type I (interferon-a/b) and type II (interferon-c) signaling.
Since a high level of protection against influenza virus only lasts for 3 days (Fig. 1) , prolonged protection would require repetitive dosing. To test whether tachyphylaxis to the protective effects of the aerosolized NTHi lysate occurs, we challenged mice with influenza in three different conditions: no lysate treatment, a single lysate treatment, or repetitive lysate treatment. We again observed 100% mortality in the untreated group, but found identical protection by NTHi lysate treatment whether given once one day prior to infection, or given three times (seven, four and one days prior to infection, Fig. 3A ). In both NTHi lysate treated groups, protection was associated with recovery of early weight loss by the fifth day after infection (Fig. 3B ). Observation for 21 days after the challenge showed no subsequent mortality.
An alternative to repetitive stimulation of lung innate immunity for prophylaxis during an epidemic viral infection would be to treat with the pro-inflammatory aerosol after infection in combination with an antiviral drug. We recently found that treatment with high-dose aerosolized ribavirin after exposure to influenza virus provided some survival improvement in mice [19] .
To test whether these two interventions might be effectively combined, mice were infected with influenza virus then treated with ribavirin alone, NTHi lysate alone, or a combination of the two. Ribavirin alone did not improve survival with the high level infectious challenge used in this study. However, as shown in Figure 4 , suspending ribavirin in a single dose of NTHi lysate on day 1 after infection improved survival significantly more than NTHi lysate alone (66.7% vs 20.0% survival, p = 0.013), and an additional NTHi lysate treatment on day 2 almost completely protected from mortality (93.3% survival, p,0.0001 vs. control).
No untoward effects or additional protection were noted if a third NTHi lysate dose was added on day 3. Observations through day 21 showed no subsequent mortality.
Lower respiratory tract infections are the leading cause of infectious death worldwide [1] . We have recently shown that Stimulated Innate Resistance (StIR) of the lungs induced by an aerosolized bacterial lysate can protect mice against otherwise fatal bacterial pneumonias [16] . We show here that the same treatment can prevent viral pneumonia, despite induction of lung inflam- mation of greater magnitude than that observed with lethal infection. Influenza-induced excessive and/or dysregulated lung inflammation has been recently described as a mechanism by which pandemic infections cause mortality [10, 11, 12, 13] . Consequent to these observations, considerable effort has been invested in attempts to improve outcomes of influenza-infected mice through genetic and pharmacologic suppression of inflammatory cytokine production [12, 20, 21, 22] . In contrast, our current and previous data demonstrate robust inflammation associated with the induction of protective host responses (Fig. 3a-d) . As we have previously shown that repetitive treatments result in diminishing inflammatory responses without loss of protection, it is possible that the cytokines themselves are not responsible for the protective effect. However, like our previous observations with protection against bacterial pathogens [16] , the pro-inflammatory pretreatment resulted in a significant reduction in pathogen burden, here represented by decreased viral titers (Fig. 1d) . As interferon signaling is associated with survival of viral and bacterial infections, the observation of augmented type I and II interferon signaling after NTHi lysate treatment ( Figure 3A -B, Table 1 ) supports our hypothesis that this is also an important element of anti-viral StIR, and we will explore this in the future.
One manner in which the host response to NTHi lysate differs from reported pandemic virus-induced inflammation [11] is in its restriction to the lung. While documenting massive increases in indicators of lung inflammation following treatment, there is virtually no systemic inflammation noted (Fig 3c) . Similarly, treatment with NTHi lysate induces no systemic leukocytosis (data not shown), despite the profound cellular infiltration induced in the lungs. We presume this confinement of the response to the lungs explains the lack of morbidity we have observed when mice are treated acutely even with very high doses of NTHi lysate [16] . Based on these observations, we conclude that influenza pneumonia does not kill via excessive pulmonary inflammation, but progresses through a deficit of effective inflammation.
In addition to exuberant lung inflammation, secondary bacterial infections are often considered important contributors to influenza-related mortality [13, 14, 15] . Interestingly, we here demonstrate that the host response to bacterial products actually enhances the clearance of a viral pathogen (Fig. 1D) , just as we have shown for bacteria [16] . So, while viral infections may impair antibacterial interferon-c responses [15] , we observe no evidence that the response to the bacterial lysate impedes either type I or type II interferon signaling. Rather, the response to the bacterial products seems to reinforce protective antiviral events via vigorous interferon gene expression.
Epidemic respiratory infections, as previously observed with influenza [10, 11] and SARS [23] , result in high mortality, sometimes before the pathogen is identified, and often without effective post-exposure treatment options. Seasonal influenza, though the case-mortality rate is lower, still kills ten of thousands of Americans annually [4, 5] and clinicians are faced with the problems of ineffective vaccine strategies and declining effectiveness of neuraminidase inhibitors [24, 25] . In such situations, rapidly dispersed, broad protection would be highly advantageous.
At present, the primary means to contain the spread of influenza and to prevent exposed individuals from developing disease is through the use of the trivalent hemagglutinin subunit vaccine or the live-attenuated trivalent vaccine coordinated by the United Stated Centers for Disease Control and Prevention. This strategy is limited by the fact that prevalent (potentially pandemic) strains may not be accurately predicted for inclusion in the annual vaccine, that normal adaptive immunity is required for protection against included strains, and that development of immunity takes weeks -potentially obviating the benefit in the setting of a rapidly spreading outbreak.
In this study, we showed that a single treatment with NTHi lysate was sufficient to prevent otherwise fatal influenza pneumonia (Fig. 1) . The aerosolized lysate was also administered repetitively to model the sustained protection that would be clinically desirable during an influenza epidemic. No tachyphylaxis to the influenza-protective effect was observed with successive administrations (Fig. 2) , despite the fact that the rise in lung lavage neutrophils induced by the aerosolized lysate becomes less with repeated administration [26] . These findings are consistent with the development of tolerance to the inflammatory effects of innate immune stimulation without loss of antimicrobial effector function, as recently described [27] . Alternatively, the progressive reduction in inflammation could reflect the tempering of innate immune responses by adaptive immune cells that become engaged with repetitive stimulation [28] .
In addition to the preventive benefits of NTHi lysate-induced StIR, we show that stimulation of lung innate immunity can be therapeutically combined with an antiviral medication after influenza infection to improve survival more than either treatment alone (Fig. 4) . In our current and prior work, we have reported only a modest survival benefit when the lysate is delivered 24 h after infection. However, we found that continuing induction of an antimicrobial inflammatory lung environment for up to 72 h potentiates the effects of high dose ribavirin and almost completely averts an otherwise uniformly fatal outcome. While Zheng and colleagues [22] recently reported improved influenza survival from 0% to 53.3% with the anti-viral/anti-inflammatory combination of zanamivir, celecoxib and mesalazine, we believe our current study to be the first to demonstrate such dramatic protection (improved from 0% to 93.3% survival) with an anti-viral combination known to induce inflammation. The seeming contradiction of these results may best be resolved by the hypothesis that the anti-inflammatory regimen diminished some toxicity associated with uncontrolled immune responses (i.e., reduced harmful inflammation) while our pro-inflammatory combination promoted the robust generation of defense effectors (i.e., enhanced protective inflammation).
In summary, we have shown that stimulation of lung mucosal innate immunity with a complex bacterial lysate confers striking protection against a virulent viral pathogen. Resistance to influenza is stimulated despite profound induction of lungrestricted inflammation, suggesting that excessive pulmonary inflammation is not the primary cause of influenza-induced mortality. Further, we found that the same treatment can be combined in the post-exposure setting with antiviral medication to improve survival. Taken together, we infer that therapeutic manipulation of StIR may be possible to reduce the mortality burden associated with viral pneumonias, and potentially, to protect the population against bioterror attacks.
Six to eight week old NIH Swiss-Webster mice (Charles River) were used for viral challenges. For protection studies, mice were divided into groups of 20 mice (5 for virus lung titers, 15 for survival). For bronchoalveolar lavage assays, an additional 5 mice were added for each group. Six to eight week old C57BL/6 mice were used for gene expression analysis, 6 mice per condition. Mice were handled in accordance with protocols approved by the Institutional Animal Care and Use Committees of Baylor College of Medicine and The University of Texas-M.D. Anderson Cancer Center, and euthanized if distressed.
Non-typeable Haemophilus influenzae (NTHi) was stored, grown and harvested as described [16, 26] . The cell pellet was washed and resuspended in 20 ml 0.9% sodium chloride solution. This suspension was passed three times through an EmulsiFlex C5 cell disruptor (Avestin) at greater then 10,000 psi, then diluted to 4-5 mg/ml in 0.9% sodium chloride solution by bicinchoninic assay (Pierce) and centrifuged at 15,0006g for 10 min. The supernatant was collected, the protein concentration was adjusted to 2.5 mg/ml, and the lysate was sterilized by passage through a 0.2 mm filter and frozen in 8 ml aliquots at 280uC. Treatment of mice with aerosolized lysate was performed as described [16] , delivering 8 mL of suspension during each 30 min exposure.
A clinical isolate of influenza A/Hong Kong/8/68 (H3N2) (A/ HK; Mouse Lung Pool 11-29-05) virus that had been passaged at least nine times through mice was stored as frozen stock (2.8610 7 TCID 50 /ml) in the supernatant of mouse lung homogenates [29] . Stock was diluted 1:300-1:1,000 in 0.05% gelatin in Eagle's minimal essential medium (Sigma-Aldrich) and aerosolized for 20 min to achieve LD 90 -LD 100 (target 100 TDIC 50 /mouse). Viral concentration in the nebulizer before and after aerosolization and in lung homogenates was determined by hemagglutination assay of infected MDCK cells [30] . In some experiments, 1 g of ribavirin was dissolved in 10 ml of NTHi lysate prior to aerosolization. Final ribavirin and lysate protein concentrations were 100 mg/ml and 2.5 mg/ml, respectively. Mice were challenged without pretreatment, following pretreatment on day 21, or following pretreatment on days 27, 24 and 21. On day 0, all groups of mice were exposed to the same viral aerosol. On day +4, 5 mice from each group were sacrificed and their lungs removed. Lungs were homogenized by beadbeating and the levels of virus determined. Remaining mice in each group were observed daily for up to 21 days for overt illness, morbidity and mortality. Mice were weighed on days 0 and +4, and three times weekly from day +7 until day +21.
To characterize the inflammatory host response to treatment and/or infection lung lavage and cell counts were performed as described [16] . In order to assess the response to NTHi lysate treatment, lavage samples from unchallenged mice were obtained 4 h after the final (or only) NTHi lysate treatment. In order to collect samples during acute virus-induced illness, lavage samples were also collected on day +3 following infection with influenza. Multiplex ELISA cytokine analysis was performed by Searchlight Protein Array Analysis (Pierce Biotechnology). Total leukocyte count was determined with a hemacytometer, and differential count by cytocentrifugation of 300 ml of bronchoalveolar lavage fluid at 4506g for 5 min, followed by Wright-Giemsa staining.
To better understand the gene expression response to the treatment, mice were treated with the aerosolized NTHi lysate, then euthanized after 2 h or 4 h for comparison to untreated mice. To reduce the lung leukocyte burden, the pulmonary vasculature was perfused and the airways lavaged with PBS. The lungs were mechanically homogenized, then total RNA was isolated from lung homogenates using the RNeasy system (Qiagen), and cRNA was synthesized and amplified from equal masses of total RNA using the Ilumina TotalPrep RNA amplification kit (Ambion). Amplified cRNA was hybridized and labeled on Sentrix Mouse-6 Expression BeadChips (Illumina), then scanned on a BeadStation 500 (Illumina). Primary microarray data were deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/ geo/) consistent with MIAME standards (GEO Accession GSE13740). Primary signal intensity was normalized between and within samples, and differentially expressed genes were identified based on signal change and inter-sample variation. Gene ontology analysis was performed using the NIAID Database for Annotation, Visualization and Integrated Discovery (DAVID) and the KEGG Database (GenomeNet). Differentially expressed genes were mapped to signaling pathways using Ingenuity Pathways Analysis 5.0 (Ingenuity Systems), and the pathway nodules were individually reviewed. C57BL/6 mice were used for the gene expression studies as they demonstrate similar NTHi lysate induced protection against all investigated pathogens, and because the genetically manipulated mice the authors plan to use to dissect the mechanisms of StIR are primarily on C57BL/6 backgrounds.
To characterize the interferon-related gene expression changes induced by NTHi, Table 1 presents a list of genes containing all transcripts from the Ingenuity Pathway Analysis canonical interferon signaling pathway, all detected interferon-related JAK-STAT transcripts in KEGG, and additional interferon related transcripts identified by the authors. Baseline signal intensity values of 1 were assigned to undetected control transcripts in order to avoid reporting infinite fold change values.
Summary statistics for virus in lung tissue were compared using Student's t-test. Proportions of mice surviving pathogen challenges were compared using Fisher's exact text, and log-rank comparisons of survival distribution were performed using Kaplan-Meier estimation. All data shown are representative of at least two independent experiments, and were not combined for analysis because of modest differences in virus challenge doses. Analyses were performed using SAS/STAT (SAS Institute).
For gene expression analyses, treated and untreated samples were compared to identify treatment-induced gene expression using an ANOVA-based scheme written in R (Free Software Foundation, Boston, MA), utilizing an Illumina library developed by Simon Lin, Northwestern University, Chicago, IL. A Novel Peptide Enhances Therapeutic Efficacy of Liposomal Anti-Cancer Drugs in Mice Models of Human Lung Cancer Lung cancer is the leading cause of cancer-related mortality worldwide. The lack of tumor specificity remains a major drawback for effective chemotherapies and results in dose-limiting toxicities. However, a ligand-mediated drug delivery system should be able to render chemotherapy more specific to tumor cells and less toxic to normal tissues. In this study, we isolated a novel peptide ligand from a phage-displayed peptide library that bound to non-small cell lung cancer (NSCLC) cell lines. The targeting phage bound to several NSCLC cell lines but not to normal cells. Both the targeting phage and the synthetic peptide recognized the surgical specimens of NSCLC with a positive rate of 75% (27 of 36 specimens). In severe combined immunodeficiency (SCID) mice bearing NSCLC xenografts, the targeting phage specifically bound to tumor masses. The tumor homing ability of the targeting phage was inhibited by the cognate synthetic peptide, but not by a control or a WTY-mutated peptide. When the targeting peptide was coupled to liposomes carrying doxorubicin or vinorelbine, the therapeutic index of the chemotherapeutic agents and the survival rates of mice with human lung cancer xenografts markedly increased. Furthermore, the targeting liposomes increased drug accumulation in tumor tissues by 5.7-fold compared with free drugs and enhanced cancer cell apoptosis resulting from a higher concentration of bioavailable doxorubicin. The current study suggests that this tumor-specific peptide may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC and to design targeted gene transfer vectors or it may be used one in the diagnosis of this malignancy. Lung cancer is one of the most commonly diagnosed malignancies in developed countries and is a growing problem in developing countries [1] . There are two major types of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC makes up approximately 80% of all lung cancer cases [2] and has a limited response rate to current chemotherapeutic agents, with tumor shrinkage in only 20% of patients and a two-year survival rate between 10% and 16% [3] . One major reason for this unsatisfactory outcome of chemotherapy is compromised drug delivery to the lung cancer tissues due to high interstitial fluid pressures (IFP) within the tumor [4] . Systemically administered chemotherapy cannot be adequately delivered into solid tumors because of the immature vasculature with abnormal architecture [5] and leaky, heterogeneous vessel walls [6] as well as the high IFP within tumor tissues [7, 8] . Furthermore, a lack of tumor specificity allows anti-cancer drugs to distribute indiscriminately to normal organs and tissues. Thus, cancer cells are exposed to a lower concentration of the drug than normal cells [9] , resulting in not only decreased effectiveness but also increased toxicity. Therefore, it is important to develop a strategy to enhance the amount of drugs delivered to tumor tissues in a targeted way while sparing normal tissues.
Efforts are ongoing to improve the therapeutic index of anticancer agents, either by increasing the drug concentration inside the tumor or by decreasing it in normal host tissues [10] . Compared with conventionally administered chemotherapeutic agents, lipid-or polymer-based nano-medicine drug delivery systems have the advantage of improving the pharmacological and therapeutic properties of cytotoxic drugs [11] . Most smallmolecule chemotherapeutic agents have a large volume of distribution on intravenous administration [12] and a narrow therapeutic window due to serious toxicity to normal tissues. By encapsulating drugs in nano-particles such as liposomes, scientists can significantly reduce the volume of distribution and increase the concentration of active drug within the tumor [13] . PEGylated liposomal doxorubicin (with brand names of Doxil in the US and Caelyx in Europe) [14] has been shown to significantly improve the therapeutic index of doxorubicin both in preclinical [15] [16] [17] and clinical studies [18] [19] [20] [21] . Several drug delivery systems of this kind have been approved for marketing [22, 23] .
Other than PEGylated liposomes, higher and more selective anti-cancer activity can be achieved through ligand-mediated targeting liposomes. In this novel drug delivery system, targeting moieties are coupled to the surface of liposomes to promote selective binding to tumor-specific antigens and facilitate the delivery of drug-containing liposomes to the intended cellular sites.
This system has the advantages of a higher drug-to-carrier ratio than immunoconjugates and the multivalent presentation of ligands leading to increased binding avidity [24] . Researchers have already produced liposomes conjugated with various peptide ligands that specifically target certain tumor cells or tumor vasculature [25] [26] [27] [28] [29] .
Because of the favorable selectivity and specificity, ligandconjugated liposomal anticancer drugs are a promising approach for new chemotherapy research. The use of peptides as ligands to direct liposomes to tumors represents a potentially feasible method for increasing the specificity and effectiveness of liposomecontaining drugs [26, 30] . Phage display is a technique of selecting targeting peptides, in which a peptide is expressed on the surface of a bacteriophage as a fusion-protein with one of the virion's own coat proteins [31] . Phage-displayed peptide libraries allows researchers to map protein-protein contacts such as B-cell epitopes [32] [33] [34] [35] and receptor-ligand interactions [36] . Such peptide libraries can also be used to identify organ-and cell-type-specific peptides [26, 27, [37] [38] [39] .
In this study, we used a phage-displayed peptide library to identify a novel peptide that bound specifically to NSCLC cell lines and surgical specimens from lung cancer patients. Liposomal doxorubicin and vinorelbine conjugated with this targeting peptide demonstrated enhanced accumulation of the drugs in tumor tissues and improved therapeutic index for human lung cancer xenografts in SCID mice.
We used a phage-displayed random peptide library to isolate phages that were able to bind to NSCLC CL1-5 cells. After five rounds of affinity selection (biopanning) with CL1-5, cells increased the titer of phage by 40-fold (Fig. 1A) . Enriched phages from the third to the fifth biopanning rounds were randomly selected. We then sequenced the phage clones with higher CL1-5binding activities. Using the Genetics Computer Group (GCG) software analysis, we found that these selected phages (PC3-1, PC4-1, PC4-5, PC5-2 and PC5-4) displayed the consensus motif, tryptophan (W)-threonine (T)/tyrosine (Y)-tyrosine (Y) ( Table 1) . Interestingly, the phage PC5-2 appeared in the third (PC3-1), fourth (PC4-1) and fifth (PC5-2) biopanning rounds. During the biopanning rounds, the frequency of PC5-2 increased from 20% (1/5) in the third cycle to 90% (27/30) in the fifth cycle (Table 1) . We chose to focus on the novel peptide displayed by PC5-2, TDSILRSYDWTY, for further study. In vitro phage display screening for peptides that bind to NSCLC. (A) A phage-displayed random peptide library was used to select phages that bind to the NSCLC cell line CL1-5. (B) Visualization of PC5-2 binding to CL1-5 and PC13 lung cancer cells (arrowheads) with immunohistochemical staining. The control phage did not bind to CL1-5 cells. Scale bar: 10 mm. (C) The FITC-labeled peptide SP5-2 bound to five NSCLC cell lines but not to NPC-TW01 cells as detected by immunofluorescent staining. Scale bar: 10 mm. (D) Representative photomicrographs of tumor sections from surgical specimens of human lung cancer were detected using both PC5-2 (a, arrowhead) and biotinylated SP5-2 (c, arrowhead), respectively. In comparison, the control phage or biotinylated control peptide could not bind to these surgical specimens (b and d). PC5-2 was competitively inhibited by the synthetic peptide SP5-2 (e). Mutated peptide, MP5-2, lost this competition ability (f). Scale bar: 25 mm. doi:10.1371/journal.pone.0004171.g001 Identification of phage clones specifically binding to NSCLC cells
To investigate whether PC5-2 would bind to NSCLC cells, we used immunohistochemistry to locate the phage particles in different cell types. Our results showed that PC5-2 bound specifically to NSCLC cell lines including CL1-5 and PC13 (Fig. 1B, arrowheads) . The control helper phage did not bind to CL1-5 cells. PC5-2 bound neither to other cancer cell lines, including oral cancer (SAS) and nasopharyngeal carcinoma cells (NPC-TW01), nor to normal epithelial cells (NNM) from nasal mucosa (Fig. 1B) .
The CL1-5-binding ability of PC5-2 was further confirmed by a peptide competitive inhibition experiment using immunofluorescent staining. The results showed that the binding activity of the PC5-2 phage to CL1-5 cells was inhibited by the synthetic peptide SP5-2 in a dose-dependent manner. At a concentration of 27 mg/ml, SP5-2 completely inhibited the binding activity of PC5-2 (Fig. S1 ). The control phage did not bind to CL1-5 cells, and PC5-2 did not bind to NPC-TW01 in this assay ( Fig. S1 and Text S2).
To further verify that the PC5-2 phage would bind to a target molecule expressed on the surface of CL1-5 cells, we measured PC5-2-bound cells using flow cytometry ( Fig. S2 and Text S1, S2). A control phage was used to estimate non-specific background binding (Fig. S2b) . The results showed that 42.6% of CL1-5 cells were bound by PC5-2 ( Fig. S2c) , and this binding was completely inhibited by 27 mg/ml of SP5-2 peptide (Fig. S2d ). PC5-2 did not bind to SAS or NNM ( 
To determine whether the peptide sequences displayed on PC5-2 would actually interact with NSCLC cells, we used fluorescein isothiocyanate (FITC)-labeled SP5-2 peptide (FITC-SP5-2) in place of the PC5-2 phage for a peptide-binding assay through immunofluorescent staining. FITC-SP5-2 specifically bound to all of the NSCLC cell lines we tested, including CL1-5, H460, A549, PC13 and H23, but did not bind to NNM. The same concentration of FITC-labeled control peptide (FITC-Con-P) revealed no such binding activity (Fig. 1C) . We also evaluated the magnitude and specificity of SP5-2 binding using flow cytometry. The proportions of CL1-5, H460, A549, PC13 and H23 cells bound by SP5-2 were 43.0%, 45.8%, 44.3%, 20.1% and 44.0%, respectively ( Fig. S3 and Text S2).
To determine whether this targeting ligand had an affinity for human lung cancer surgical specimens, we tested the reactivity of PC5-2 and SP5-2 with pulmonary adenocarcinoma cells using immunohistochemistry. Both PC5-2 and biotin-labeled SP5-2 (B-SP5-2) recognized the tumor cells of NSCLC surgical specimens (Fig. 1D a and c, arrowheads) , and the control phage and biotinlabeled control peptide (B-Con-P) did not (Fig. 1D b and d) . SP5-2 (TDSILRSYDWTY) competed with PC5-2 for binding to surgical specimens of pulmonary adenocarcinoma (Fig. 1De ), but the same concentration of a mutated peptide, MP5-2 (TDSILRSYDGGG) did not (Fig. 1Df) . Seventy-five percent (27/36) of the pulmonary adenocarcinoma specimens from 36 patients expressed a target molecule that was recognized by this peptide (Table S1 ). These data indicated that SP5-2 could recognize unidentified molecules expressed on NSCLC cell lines and actual cancer cells from the surgical specimens of lung cancer.
To investigate the targeting ability of the PC5-2 phage in vivo, we injected phages into the tail vein of mice bearing CL1-5-derived tumors and then recovered them after perfusion. We determined the titers of the phage in tumor masses and normal control organs (brain, heart and lungs) [26, 30] . PC5-2 showed specific homing to tumor masses with concentrations 15-fold higher than its concentration in the control organs ( Fig. 2A) . Control helper phages did not show any specific targeting to tumor tissues ( Fig. 2A) .
The tumor-homing ability of PC5-2 was further confirmed by a peptide competitive inhibition experiment, in which synthetic peptide SP5-2, injected together with PC5-2, markedly inhibited the recovery of the phage from tumor masses (Fig. 2B) . One hundred micrograms of SP5-2 inhibited 92% of PC5-2 binding to tumor masses, but the same concentration of a control peptide had no such inhibitory effect (Fig. 2B) .
From in vitro phage display screening, we identified two clones (PC5-2 and PC5-4) with a consensus motif of W-T/Y-Y (Table 1) . Like PC5-2, the tumor-homing ability of PC5-4 was also competitively inhibited by SP5-2 ( Fig. S4 and Text S2), suggesting that these two phages may bind through this motif to the same target molecule on the plasma membrane of lung cancer cells. We proposed that these three amino acid residues might play a crucial role in homing to tumor tissues. To test this hypothesis, we changed these three amino acid residues in SP5-2 (TDSILR-SYDWTY) to GGG in a mutant peptide, MP5-2 (TDSILR-SYDGGG). Although the tumor-homing ability of PC5-2 had been markedly inhibited by the peptide SP5-2, this competitive inhibition was lost in MP5-2, which contains the GGG residue instead of the WTY residues in SP5-2 (Fig. 2B) . These data indicate that the WTY residues were important for the binding ability of SP5-2 to NSCLC cells.
The tissue distribution of PC5-2 was also studied using immunostaining. We injected SCID mice bearing NSCLC xenografts with PC5-2 and then removed and fixed the tumor and control organs for localization of the phage particles. PC5-2 was found to localize in tumor tissues ( Fig. 2Cd) . At a higher magnification, the immunoreactivity of the phage was detected on the plasma membrane with some diffusion in the surrounding cytoplasm of tumor cells (Fig. 2Ce ). There was no reaction product detected on normal organs such as brain, heart and lung tissues ( Fig. 2C a-c), nor on tumor tissues treated by the control phage (Fig. 2Ci) . The specific targeting of PC5-2 to the NSCLC xenograft was inhibited by the synthetic peptide SP5-2 in the in vivo homing experiment (Fig. 2Cj ).
To determine whether the lung cancer-targeting peptide SP5-2 could be used to improve the chemotherapeutic efficacy of cancer treatment, we coupled the peptide to liposomes containing anticancer drugs. SCID mice bearing size-matched, CL1-5-derived xenografts were treated with (1) SP5-2-conjugated liposomal doxorubicin (SP5-2-LD), (2) mutant peptide-conjugated liposomal doxorubicin (MP5-2-LD), (3) non-targeted liposomal doxorubicin (LD), (4) free doxorubicin (FD), or (5) equivalent volumes of phosphate-buffered saline (PBS). All the formulations were injected intravenously (i.v.) at a total doxorubicin dosage of 8 mg/kg (1 mg/kg twice a week for a total of eight injections).
The tumors in mice that received SP5-2-LD ( Fig. 3A group a) were significantly smaller than those in the MP5-2-LD, LD, FD, and PBS groups (P,0.01) ( Fig. 3A group b-e). The tumor sizes in the LD and MP5-2-LD groups were 2.1 and 2.6 times larger than those in the SP5-2-LD group, respectively. The tumor sizes in the FD and control PBS groups were 7.0 and 8.3 times larger than that in the SP5-2-LD group, respectively (Fig. 3A) . Interestingly, the greater therapeutic efficacy of SP5-2-LD was lost when the WTY motif in the peptide had been changed to GGG in MP5-2-LD. Free doxorubicin exhibited little therapeutic efficacy at this concentration, as the tumor size in this group was only 16% smaller than that in the PBS group (Fig. 3A) .
To verify whether large xenografts would respond to SP5-2-LD treatment, mice bearing large lung cancer xenografts (500 mm 3 ) were assigned to three treatment groups. After a course of doxorubicin treatment with a total dosage of 16 mg/kg (2 mg/kg twice a week for eight injections), the tumor sizes in the control PBS and LD groups gradually increased to 3.3 and 2.1 times the tumor size in the SP5-2-LD group (P,0.01) (Fig. 3B) . These results revealed that SP5-2-LD also increased the therapeutic efficacy of doxorubicin in SCID mice bearing large lung cancer xenografts.
To further test whether SP5-2 would optimize the therapeutic index in lung cancer treatment, SP5-2-LD was used to treat a different type of lung cancer xenograft (H460-derived tumor). SCID mice bearing size-matched, H460-derived xenografts were treated with SP5-2-LD, LD, FD, or equivalent volumes of PBS, through i.v. injection at a total doxorubicin dosage of 8 mg/kg (1 mg/kg twice a week for eight injections). The final tumor size in the SP5-2-LD group was significantly smaller than those in the LD, FD and PBS groups (P,0.05). Mice in the LD, FD and PBS groups had tumors with sizes 2.0-, 8.6-and 10.0-fold larger than that in the SP5-52-LD group (Fig. 3C) . Free doxorubicin at this concentration reduced the tumor size by only 14% compared with PBS groups. Not only was tumor growth markedly suppressed in the SP5-2-LD group (Fig. 3C) , the body weight of the mice in this group increased by 10.3% (2.38 g) at the end of the treatment period. In contrast the LD-treated mice had a smaller increase in body weight of 2.4% (0.59 g) (Fig. 3D) .
To further confirm SP5-2 could increase the therapeutic index for lung cancer, we linked the SP5-2 peptide to another anticancer drug, liposomal vinorelbine (SP5-2-LV) and tested its efficacy against lung cancer xenografts. SCID mice bearing sizematched CL1-5-derived xenografts were given i.v. injections of SP5-2-LV, liposomal vinorelbine (LV), or equivalent volumes of PBS at a total vinorelbine dose of 24 mg/kg (2 mg/kg twice a week for twelve injections). The tumor-bearing mice treated with SP5-2-LV (Fig. 3E , group a) had significantly smaller tumors than the LV and PBS groups (P,0.01) (Fig. 3E, group b and c) . The tumor size in the LV group was 6.75-fold larger than the SP5-2-LV group. The average tumor size in the control PBS group was 25-fold larger than the SP5-2-LV group (Fig. 3E ). To assess side effects of the treatments, the mice were weighed twice a week. The body weight of mice increased by 5.7% (1.37 g) in the SP5-2-LV group and by 2.4% (0.58 g) in the LV group at the end of the treatment period (data not shown).
Finally, we compared the survival rates of tumor-bearing mice after treatment with SP5-2-LV, LV, or PBS over 102 days. All five animals in the PBS-treated group died (survival rate 0%). Three mice died in the LV-treated group (survival rate 40%). In the SP5-2-LV-treated group, however, the survival rate was 80%, significantly higher than the other two groups (Fig. 3F) . These experiments demonstrate that SP5-2 increased the therapeutic efficiency of liposome-encapsulated doxorubicin and vinorelbine with less toxicity.
The biodistribution and tumor localization of SP5-2-LD, MP5-2-LD, LD, and FD were estimated by measuring the intrinsic auto-fluorescence signal of doxorubicin in mice with NSCLC xenografts. Doxorubicin (M r 543.54), a small-molecule chemotherapeutic agent as its M r is ,1000, has a poor pharmacokinetic profile, and its blood concentration drops to background level within one hour after administration ( Fig. S5A and Text S2). The pharmacokinetic profile of various liposomal doxorubicin formulations, including SP5-2-LD, MP5-2-LD, and LD, were markedly greater than FD (Fig. S5A) . The area under the concentrationtime curve (AUC 0-48 hours ) of doxorubicin in tumor tissues was 10.4 mg?hr/g, 31.5 mg?hr/g, 29.0 mg?hr/g, and 59.9 mg?hr/g in the FD, LD, MP5-2-LD, and SP5-2-LD groups, respectively (Table S2 ). The mean intra-tumor doxorubicin concentration in the SP5-2-LD-treated group was 5.7-, 1.9-and 2.1-fold higher than the intra-tumor doxorubicin concentration in the FD, LD, and MP5-2-LD groups (Fig. 4A and Table S2 ).
To assess the bioavailability of the liposomal drugs, we used nuclear doxorubicin accumulation as an indicator of drug cytotoxicity [40] . The AUC 0-48 hours of bioavailable doxorubicin (i.e., bound to nuclei) for FD, LD, MP5-2-LD, and SP5-2-LD was 3.7 mg?hr/g, 6.7 mg?hr/g, 7.5 mg?hr/g, and 17.7 mg?hr/g, respectively (Table S2 ). The intra-tumor nuclear doxorubicin concentration in the SP5-2-LD group was 4.8-, 2.6-and 2.4-fold higher than the nuclear doxorubicin concentration in the FD, LD, and MP5-2-LD groups ( Fig. 4B and Table S2 ). Doxorubicin concentration inside tumor tissues and nucleus was not significantly different between the LD and MP5-2-LD groups ( Fig. 4 and Table S2 ).
To compare the drug delivery profile of the four doxorubicin formulations, we tried to detect the drug in tumor tissues using the fluorescence microscope. Images from all tumors showed that doxorubicin was visualized in the tumor nuclei one hour after SP5-2-LD was administered, but not after other formulations were injected (Fig. 5A) . Over time, areas of tumor sections with detectable doxorubicin increased. The areas with detectable doxorubicin were significantly larger in SP5-2-LD-treated tumors than that in FD, LD, and MP5-2-LD-treated tumors at each time point (Fig. 5A) . The MP5-2-LD formulation had the same distribution pattern as LD, but the regions of FD-treated tumors showed no detectable doxorubicin (Fig. 5A) .
When the tumor tissues in each treatment group (Fig. 3C) were examined by H&E staining, markedly disseminated necrotic/ apoptotic areas were present throughout the sections of SP5-2-LDtreated xenografts (Fig. 5B ). In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to identify apoptotic cells and tomato lectin was applied to detect tumor blood vessels. The tumors had larger apoptotic areas and a The greater accumulation of anti-cancer drugs in tumor tissues and more bioavailable doxorubicin in cancer cell nuclei from the ligand-conjugated liposomes further demonstrated that the SP5-2 peptide recognized the target molecule on the surface of lung cancer cells and thus increased direct drug delivery to the tumor.
The therapeutic efficacy of anticancer drugs can be achieved by enhancing the drug formulation with molecules that preferentially bind to tumor tissues [22, 26, 37, 41] . Phage display biopanning on intact cell allows for the isolation of highly specific peptides that target tumor-associated antigens. Anti-cancer regimens armed with these peptides can be used as ''cruise missiles'' that are precisely guided to the cancer cells and deliver high enough doses to kill these cells with minimal damage to normal tissues.
In this study, we identified a NSCLC-targeting peptide and demonstrated its improved therapeutic efficiency in animal models. Specifically, we found a phage clone PC5-2 that made up 90% of the selected phages binding to CL1-5 cells after five rounds of biopanning (Table 1) . Immunohistochemistry and flow cytometry assays confirmed that PC5-2 bound to NSCLC cell surface. The same binding results were observed in the cognate synthetic peptide SP5-2, which replaced PC5-2 ( Fig. 1 and Figs. S1, S2, S3). These experiments supported that this targeting peptide can specifically bind to the cell surface of NSCLC cell lines. In vivo experiments also consolidated the homing ability of the peptide. The expression of the SP5-2 peptide (TDSILR-SYDWTY) guides the phage to accumulate in NSCLC xenografts but not in normal organs (Fig. 2) . The binding activity of PC5-2 to tumor tissues was inhibited by the synthetic peptide SP5-2 (Fig. 2B) , indicating that PC5-2 interacted with NSCLC cells by its displayed peptide and not by another part of the phage particle. That the mutated synthetic peptide, MP5-2, did not inhibit the PC5-2 binding demonstrated the importance of the WTY motif in the binding activity (Fig. 2B) . Moreover, this phenomenon was observed in immunohistochemical staining of surgical specimens from human lung cancer (Fig. 1D e and f) , in animal models for ligand-targeted chemotherapy (Fig. 3A) and in tumor localization of doxorubicin delivered by SP5-2-conjugated targeting liposomes (Figs. 4 and 5A) .
Immunohistochemistry assessments on pulmonary adenocarcinoma surgical specimens demonstrated that SP5-2 has a clinical potential as imaging probes to identify NSCLC or drug delivery agents for the treatment of NSCLC. Both PC5-2 and biotinlabeled SP5-2 detected pulmonary adenocarcinoma surgical specimens in our experiments (Fig. 1D) . Seventy-five percent (27/36) of pulmonary adenocarcinoma specimens from 36 patients expressed a target molecule that was recognized by the peptide (Table S1 ). SP5-2, but not MP5-2, competed with PC5-2 for binding to pulmonary adenocarcinoma surgical specimens (Fig. 1D) , which further confirmed the specificity of this targeting ligand.
In previous studies, doxorubicin has largely been used in cancer treatment because of its broad spectrum of antitumor activity. However, the efficacy of doxorubicin in the treatment of NSCLC remains unsatisfactory with a response rate of 15% [42] . This is in part a result of suboptimal doses within the tumor due to indiscriminate drug distribution throughout the body and severe toxicity to normal tissues and organs. Liposomal encapsulation with a targeting ligand may be an effective strategy to deliver the drug directly to tumor cells. Our data revealed that this peptide markedly increased the therapeutic efficacy of liposomal chemotherapies and resulted in higher survival rates in mice with human lung cancer xenografts, and produced limited side effects on the animals (Fig. 3) . This peptide increased the therapeutic index of not only doxorubicin but also of vinorelbine, a vinca alkaloid used to treat advanced NSCLC [3] . Furthermore, we observed decreased vessel density and substantially increased cell apoptosis in tumor tissues after the targeting liposome treatment (Figs. 5B and S6). This ligand-mediated liposomal formulation is potentially superior to conventional anti-cancer therapy for NSCLC.
Our in vivo pharmacokinetic studies in mice showed that liposomal doxorubicin dramatically changed the transportation and distribution of doxorubicin in the heart, lungs, kidneys, and liver in mice (Figs. 4 and S5) . These results echo other similar investigations [43, 44] . With prolonged presence of liposomes in circulation, more doxorubicin was taken up by tumor cells than conventional doxorubicin administration (Fig. 4) ; this finding was further confirmed in fluorescence signaling of doxorubicin in tumor tissues (Fig. 5A) . This passive targeting phenomenon of non-ligand conjugated liposomes is called the ''enhanced permeability and retention effect'' [45, 46] . In our study, the doxorubicin concentration in the LD-treated group was three times that of the (Table S2) . However, the tumors in the SP5-2-LD group had even higher doxorubicin concentration that was 1.9-and 2.1fold higher than those in the LD and MP5-2-LD groups (Table  S2 ). The bioavailable drug in the nuclei of cancer cells in the SP5-2-LD was also 2.6-and 2.4-fold higher than those in the LD and MP5-2-LD groups (Table S2 ). These results indicated that this peptide directly delivered the chemotherapeutic drug to intended targets. Enhancement of drug accumulation in tumor tissues correlated with the increased therapeutic efficacy (Figs. 3, 4 and 5) . The peptide-functionalized liposomes were found to have important clinical potential in a targeted drug delivery system. In order to for them to be used clinically, however, a final targeting liposome construct will need to be selected after each component, i.e. peptide ligands, conjugation methodologies, and liposomal drugs, have been optimized.
In conclusion, using phage display peptide libraries to screen for peptides that bind to NSCLC cells, we identified several novel peptides including SP5-2 that specifically bound to the cell surface of NSCLC cells both in vitro and in vivo. Linking SP5-2 to liposomes containing doxorubicin and vinorelbine increased the therapeutic efficacy and survival rates in mice with human NSCLC xenografts because of enhanced tumor apoptosis and decreased tumor angiogenesis. Quantitation and visualization of doxorubicin levels also showed increased drug concentration in tumor tissues in this formulation, highlighting the enhancement in both the delivery and penetration of doxorubicin into the tumor. Our results indicate that the SP5-2 tumor targeting peptide may be used as imaging probes of NSCLC and targeting ligands for liposomal delivery systems to increase the efficacy of chemotherapy for NSCLC.
Lung cancer cell lines (A549, CL1-5, H23, H460, and PC13), a nasopharyngeal carcinoma cell line (NPC-TW01), an oral cancer cell line (SAS), and human normal nasal mucosal epithelial (NNM) cells were used in this study. The NNM cells were a primary culture derived from a nasal polyp [27] . The A549, H23, and H460 lines were obtained from the American Type Culture Collection. The CL1-5 line was established by Chu et al. [47] . The A549, CL1-5, H23 and H460 cells were grown in RPMI 1640 (Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, CA, USA) at 37uC in a 5% CO 2 incubator. The NPC-TW01, PC13, SAS, and NNM cells were grown in DMEM (Gibco, CA, USA) containing 10% FBS at 37uC in a 10% CO 2 incubator.
Phage display biopanning procedures CL1-5 cells were first incubated with UV-treated inactive control helper phage (insertless phage). The phage-displayed peptide library (New England BioLabs, MA, USA), which initially contained 5610 10 plaque-forming units (pfu), was then added. The bound phages were eluted with a lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) on ice. This eluted phage pool was amplified and titered in an Escherichia coli ER2738 culture (New England BioLabs, MA, USA). Recovered phages were used as input for the next round of panning as described previously [26, 27] .
Ninty-six-well ELISA plates (Falcon, CA, USA) were seeded with either cancer or control cells. Individual phage particles were added to the cell-coated plates and incubated, followed by incubation with horseradish peroxidase (HRP)-conjugated mouse anti-M13 monoclonal antibody (mAb) (Pharmacia, Uppsala, Sweden) and subsequently with the peroxidase substrate ophenylenediamine dihydrochloride (Sigma, MO, USA). The reaction was stopped and absorbance was measured at 490 nm using an ELISA reader. The selected phage clones were further analyzed using DNA sequencing. The sequencing was performed with the primer 59-CCCTCATAGTTAGCGTAACG-39 corresponding to the pIII gene sequence. The phage-displayed peptide sequences were translated and aligned using GCG program.
The synthetic targeting peptide SP5-2 (TDSILRSYDWTY), mutant peptide MP5-2 (TDSILRSYDGGG), and control peptide (RLLDTNRPLLPY) [27] were synthesized (Invitrogene, Inc., CA, USA) and purified using reverse-phase high-performance liquid chromatography to .95% purity. Conjugation of these peptides with FITC or biotin was performed through the addition of FITC or biotin to the peptide amino terminus by the same company.
Cells were plated and grown to about 80% confluence on cover slips. The cover slips were treated with 3% hydrogen peroxide plus 0.1% NaN 3 to block endogenous peroxidase activity, and then incubated with phages. After the cover slips had been washed and fixed with 3% paraformaldehyde, they were incubated with HRPlabeled mouse anti-M13 mAb and treated with peroxidase substrate. For the peptide binding assays, 30 mg/ml FITC-labeled SP5-2 or control peptide was added on each cover slip and incubated. They were counterstained with Hoechst 33258 (Molecular Probes, OR, USA) and mounted with a mounting solution (Vector, CA, USA). The cells were then examined under a Leica universal microscope. The images were merged using the SimplePCI software (C-IMAGING, PA, USA).
For localization of peptide binding on lung cancer tissues, frozen sections of NSCLC tissues were prepared and incubated with phage clones or biotin-labeled peptides. For the peptide competitive inhibition assay, phages were mixed with the synthetic targeting or mutant peptide. The slides were subjected to routine immunohistochemical staining [26, 30] . All surgical specimens were obtained from the tissue bank of National Taiwan University Hospital (NTUH) with approval from the Institutional Review Board in NTUH (IRB9461702021).
In vivo homing experiments and tissue distribution of phages SCID mice were injected subcutaneously (s.c.) in the dorsolateral flank with 1610 7 human NSCLC cells. The mice bearing size-matched lung cancer xenografts (approximately 500 mm 3 ) were injected i.v. with 10 9 pfu of the targeting or control phage. After perfusion, xenograft tumors and mouse organs were removed and homogenized. The phages bound to each tissue sample were recovered through the addition of ER2738 bacteria and titered on IPTG/X-Gal agar plates. In the peptide competitive inhibition experiments, the phages were injected along with 100 mg synthetic targeting peptide, control peptide or mutant peptide. The organs and tumor masses were fixed in Bouin's solution (Sigma, MO, USA). After fixation, the samples were embedded in paraffin blocks. The paraffin sections were deparaffinized, rehydrated, and subjected to immunostaining using the mouse M13 mAb as described above.
Peptide-conjugated liposomes containing doxorubicin or vinorelbine were prepared as described in other studies [26, 27] . Briefly, the peptide was coupled to NHS-PEG-DSPE [N-hydroxysuccinimido-carboxyl-polyethylene glycol (MW, 3400)-derived distearoylphosphatidyl ethanolamine] (NOF Corporation, Japan) in a 1:1.5 molar ratio. The reaction was completed and confirmed by quantitation of the remaining amino groups using TNBS (Trinitrobenzenesulfonate) reagent (Sigma, MO, USA). Doxorubicin and vinorelbine were encapsulated in liposomes through a remote loading method at a concentration of 1 mg of drug per 10 mmol phospholipids. Peptidyl-PEG-DSPE was transferred to pre-formed liposomes after co-incubation at a transition temperature of the lipid bilayer. There were 500 peptide molecules per liposome as described previously [48] .
SCID mice bearing NSCLC xenografts (,500 mm 3 ), were injected in the tail vein with various formulations of liposomal doxorubicin (SP5-2-LD, MP5-2-LD, and LD) and free doxorubicin at a dose of 2 mg/kg. At selected time points, three mice in each group were anaesthetized and sacrificed. Blood samples were collected through submaxillary punctures, and plasma samples were prepared. After perfusion, xenograft tumors and mouse organs were removed and homogenized. Procedures for isolating tumor cell nuclei and extracting nuclear doxorubicin were carried out according to previous reports [40, 49] . Total doxorubicin concentration was measured using a method described by Mayer et al. [49] . Total doxorubicin was quantified using spectrofluorometry at l ex 485/20 nm and l em 645/40 nm (Synergy HT Multi-Detection Microplate Reader, BioTek Instruments, Winooski, VT 05404 USA).
To correct for background fluorescence, a standard curve was obtained by spiking tissue extracts derived from mice that did not receive doxorubicin. Tissue concentrations of doxorubicin were expressed as microequivalents per milliliter of plasma or per gram of tissue. A standard doxorubicin curve was prepared in control homogenates following the same extraction procedure as above. Drug levels were estimated on the basis of doxorubicin fluorescent equivalents.
To determine the presence of the drug localized in tumor tissues, doxorubicin autofluorescence was detected using a Zeiss Axiovert 200 M inverted microscope with a 100 W HBO mercury light source equipped with a 546/12 nm excitation and a 590 nm emission filter set. Tissue sections were imaged with a FLUAR 106/0.50 NA lens and captured with a Roper Scientific CoolSnap HQ CCD camera. All images were captured in 8-bit signal depth and subsequently pseudo-colored.
Animal model for the study of ligand-targeted therapy Mice 4-6 weeks of age were injected s.c. in the dorsolateral flank with human NSCLC cells. Mice with size-matched tumors (approximately 50-100 mm 3 ) were then randomly assigned to different treatment groups and injected with SP5-2-LD or SP5-2-LV, or LD or LV through the tail vein. The dosage of SP-5-2-LD was 1 mg/kg injected twice a week for four weeks, and that of SP5-2-LV was 2 mg/kg injected twice a week for six weeks. Mice bearing large tumors (approximately 500 mm 3 ) were treated with SP5-2-LD for eight times (2 mg/kg, twice a week for four weeks). Mouse body weights and tumor sizes were measured twice a week. Tumor volumes were calculated using the equation: length6(width) 2 60.52. Animal care was carried out in accordance with guidelines of Academia Sinica, Taiwan.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining
The frozen tumor tissue sections were incubated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction mixture (Roche Diagnostics) at 37uC for an hour. The slides were counterstained with mounting medium with DAPI (Vector Laboratories). The slides were then visualized under a fluorescent microscope.
Tissues were removed from treated mice, fixed with 4% paraformaldehyde and embedded with paraffin. Blood vessels were detected by staining of Lycopersicon esculentum (tomato) lectin conjugated to biotin (Vector, CA, USA). The biotinylated lectin was visualized with streptavidin-conjugated rhodamine (Pierce, IL, USA).
We analyzed the data of phage titer, tumor volume, body weight, and doxorubicin concentration using two-sided unpaired Student's t-test. We considered a P value below 0.05 as significant for all analyses. All values are represented as mean6standard deviation.
Text S1 Supporting Information and figure legends Figure S1 Identification of PC5-2 binding to NSCLC cells. Representative microscopy images of CL1-5 cells stained with propidium iodide (a-f, red), anti-M13 monoclonal antibody (g-l, green), and merge (m-r). The binding of PC5-2 to CL1-5 cells (a, g, m) was inhibited by 3 mg/ml (b, h, n), 9 mg/ml (c, i, o), and 27 mg/ml (d, j, p) of SP5-2 in a dose-dependent manner. The control phage and PC5-2 did not bind to CL1-5 cells (e, k, q) and NPC-TW01 cells, respectively. Scale bar: 10 mm. Figure S4 Tumor homing ability of PC5-4 phage. SCID mice bearing NSCLC xenografts were injected i.v. with PC5-4, and phage was recovered after perfusion. Recovery of PC5-4 from the tumor was higher than from control organs. Targeting activity of PC5-4 to tumor tissues was inhibited by SP5-2.  IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity BACKGROUND: Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity. METHODS AND FINDINGS: Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity. CONCLUSIONS: This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease. In the recent years, emerging and re-emerging tropical infectious diseases have been shown to cause high social and economic impact. Vector-borne infectious diseases such as Dengue, West Nile have been resurging largely due to the spread of insecticide resistance, to socio-demographic changes, and to genetic mutations in the pathogens. More recently, chikungungya fever (CHIKF) has now emerged as the next important infection in South-East Asia, the Pacific region and Europe [1] [2] [3] [4] [5] , making it a major threat that requires immediate attention. Recent epidemic resurgence of CHIKF in several African and Asian countries demonstrated that infection can spread alarmingly rapidly [6] [7] [8] [9] from limited early transmission that then developed into an unprecedented and unexpected epidemic, infecting 38% of the population as occurred in Reunion island [6, 8] . The appearance of cases in Europe, the United States and other countries by travelers returning from known outbreak areas underscores the contributory factors of increased human mobility, tourism, global climate change, and increases in insecticide resistance [10] [11] [12] [13] [14] [15] . In this era of globalization, the threat of such disease epidemics should not be underestimated as such public health events could cripple public health systems and economies.
Chikungunya virus (CHIKV), which causes CHIKF, is an alphavirus of the Togaviridae family, with a 12,000-nucleotides linear, positive-sense, single-stranded RNA genome containing two large open reading frames (ORF). The first, ORF1, encodes 4 non-structural proteins (nsP1, nsP2, nsP3 and nsP4) while ORF2 encodes structural proteins that include 1 capsid protein (C), 2 major envelope surface glycoproteins (E1, E2) and 2 small proteins (E3, 6K) [8, 9] . CHIKV is transmitted by Aedes mosquitoes (mainly A. albopictus and A. aegypti).
CHIKF is an acute illness with abrupt fever, skin rash, arthralgia, and occasional involvement of the nervous system, heart and liver. Prolonged incapacitating arthralgia has sometimes been reported to persist for years [8, 9, 13] . It is of concern that the re-emerged CHIKV has caused considerable morbidity and some fatalities, whereas previously CHIKF was considered as relatively benign. Despite the fact that the clinical features of recent acute CHIKV infections from several countries have been described [16] [17] [18] , little is known about the long-term sequelae or the pathogenesis of arthropathy, and the acquisition of protective immunity remains unexplored. It has been proposed that CHIKVinduced arthritis or arthralgia is of immunopathologic origin [19, 20] . At present, there is no specific or effective treatment for CHIKF, and patient management is largely symptomatic relief and primarily anti-inflammatory drugs [8] . Given the expanding geographic range of CHIKV and its potential to rapidly cause large scale epidemics, it has become important to understand the immune and pathogenic mechanisms active during CHIKV infections in order to guide the development of targeted and effective control and treatment strategies.
Cytokines and chemokines are thought to play an important role in viral immunopathology. Although IL-2. IL-10 and IFN-c have been implicated in the pathogenesis of CHIKF [21] , a global analysis of their specific involvement with disease severity has not yet been defined. Growth factors are usually produced in response to injury. Viral infections such as CHIKV, induce cellular damage which may lead to secretion of these factors; however, limited studies have been conducted [21] . In this study, we took the opportunity to conduct a detailed study on the patients from the first outbreak of CHIKF in Singapore [22, 23] . We measured circulating levels of a wide range of cytokines, chemokines, and growth factors in 10 laboratory confirmed cases of CHIKF, and compared them with healthy individuals. We next determined which biomarker was associated with infection and/or severity. We showed for the first time that CHIKV infection induced a wide range of cytokines, chemokines, and growth factors. We subsequently found that 3 specific biomarkers, namely IL-1b, IL-6, and RANTES levels, were associated with severe CHIKF.
All 10 patients included in this study were males. Their age ranged from 22 to 65 years (median, 35 years). All except one were foreign nationals. Half of our patients were classified as severe CHIKF. We defined severe CHIKF as having a temperature of .38.5uC or pulse rate .100/min, or platelet count ,100610 9 g/ L based on studies defining severe diseases [24] [25] [26] [27] [28] . Except for the Singapore resident, none had any pre-existing medical condition. Despite being previously healthy, four non-residents developed severe illness. Fever lasted 2-10 days, and fever duration was not significantly different between those who had more severe illness and those who had not (mean, 6.6 days vs. mean, 3.8 days; P.0.05). Two patients reported persistent arthralgia lasting more than two weeks. Demographic and clinical details of the 10 CHIKF patients are summarized in Table 1 .
Among our patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%), and conjunctivitis (40%) ( Table 2 ). Gastrointestinal and constitutional symptoms were less prominent. Arthritis was observed in only one patient, who had an effusion on the right knee. None had neurologic involvement or hemorrhagic manifestation. Table 3 presents a summary of the key laboratory findings among our patients throughout the course of their illness. White cell count, hemoglobin, hematocrit, platelet count, erythrocyte sedimentation rate for most patients were within the normal range. The mean nadir platelet count (6SD) was 1996115610 9 /L. Only one patient had severe thrombocytopenia (nadir platelet count, ,100610 9 /L) during the course of his illness. Elevated C-reactive protein levels (CRP, .10.0 mg/L) were observed in 60% of patients, but the peak C-reactive protein level was not significantly different between those who classified as severely ill and those who were not (mean, 40.3 mg/L vs. mean, 9.9 mg/L; P = 0.195). The mean peak alanine and aspartate transaminases (ALT and AST) (6SD) were 58636 U/L and 50625 U/L respectively. Both ALT and AST were 2-fold greater than the upper limit of normal in one patient, who had pre-existing liver cirrhosis. None of the patients had a clinically abnormal total protein, urea or creatinine level. Among our patients, the mean nadir protein level (6SD) was 6765 g/dL, and the mean peak urea and creatinine levels (6SD) were 5.161.7 mmol/L and 101616 mmol/L respectively. Lactate dehydrogenase level was the only laboratory parameter that was significantly higher in severely ill patients, compared to those who were not (mean, 732 U/L vs. mean, 525 U/L; P = 0.047).
Profiles of 30 cytokines, chemokines and growth factors were determined by a multiplex-microbead immunoassay on acute blood samples collected upon hospitalization. The samples collected ranged from day 2 to day 19 of illness (median, day 4.5). To characterize the overall patterns, a two-way hierarchical clustering analysis was done to allow the classification of individuals according to disease severity based on the clinical features ( Figure 1 ). Evidently, this had the power to discriminate the clinical forms of CHIKF in the samples in this study from the healthy controls, with patients classified as non-severe and severe disease segregating perfectly. The levels of 8 plasma cytokines (IL-2R, IL-5, IL-6, IL-7, IL-8, IL-10, IL-15 and IFN-a) were observed to be most significantly elevated (Figure 2a ) in CHIKF patients compared to uninfected subjects (P,0.05). Among these was proinflammatory cytokine, IL-6 which was very significant. Interestingly, another proinflammatory cytokine, IL-8 was down-regulated in these patients. Anti-inflammatory cytokine, IL-10 was found to be significantly raised in most of the patients (P,0.05). The plasma concentrations of IL-2R and IL-5 were found to be increased in all patients. Levels of IFNa and IL-7 were elevated in all patients. The levels of other cytokines such as IL-2, IL-4, IL-12, IL-13, IL-17, IFN-c, and TNF-a, were only marginally increased in the CHIKF group compared with those in the uninfected group ( Figure S1 ).
Profiles of chemokines, IP-10 and MIG were shown to be significantly elevated, while Eotaxin was suppressed (Figure 2b ). There was no difference in the levels of other chemokines namely, MCP-1, MIP-1a, MIP-1b and RANTES ( Figure S1 ).
Interestingly, the levels of 4 growth factors were found to be significant in the patients, with up-regulation of HGF, FGF-basic and VEGF, with the exception of EGF which was almost totally suppressed ( Figure 2c) . It was observed that the CHIKF patients exhibited low levels of GM-CSF and G-CSF ( Figure S1 ).
Finally, in an effort to identify cytokine, chemokine, and growth factor plasmatic levels associated with severity, statistical analyses were performed after stratification of the CHIKF patients according to severity. It was observed that an increase in levels of IL-1b and IL-6, and a decrease in RANTES respectively were associated with disease severity (Figure 3a) . The levels of all other markers were not significantly different ( Figure S2 and S3).
CHIKF, an emerging arboviral infection, which induces high fever, has only been recently reported in Singapore. Up to Dec 2007, all CHIKF patients had contracted the infection overseas [22] . The first local outbreak of CHIKF occurred in Jan 2008. More than 2,500 people who lived or worked in the outbreak area were screened and a total of 13 PCR-confirmed cases were identified [22, 24] . All confirmed CHIKF cases were referred to the CDC/TTSH. Our report included 10 patients who participated in this study.
Phylogenetic analysis of the viral sequences of our patients has revealed that the circulating strains were of the Indian Ocean genotype and closely related to those from the 2006 outbreak in India [29] but without the A226V mutation, further emphasizing how remarkably rapid the disease could spread with the right environmental conditions. The attack rate in our outbreak was 0.5%, much lower than the 34% reported in Reunion Island and the 5.4% observed in Italy [13, 30] . This could be attributed to the rapid removal of human reservoirs through isolation, enhanced vector control, or the circulation of a virus strain of lower epidemic potential. Clinical features of our patients were similar to those reported in recent outbreaks [6] [7] [8] [9] [11] [12] [13] [14] [16] [17] [18] 21, 30] , indicating that although people are genetically diverse response to diseases is homogeneous across people in non-homogeneous populations. The majority of our patients was #45 years and had no premorbid condition. Unlike patients reported in the Reunion Island outbreak [6] , where the patients' underlying medical conditions could have contributed to the observed morbidities, our patients were younger and healthier. Furthermore, none of our patients was co-infected with dengue, as confirmed by RT-PCR and dengue enzyme-linked immunoabsorbent assay (ELISA)-IgM and IgG [31] . Hence, our immunologic observations can be largely attributed to acute CHIKV infection itself.
In recent years, most of the studies on CHIKF have been addressed with the clinical description of the disease [4] [5] [6] [7] [8] [9] 11, 17, 18, 21] , the molecular nature of the virus [9, 10, 19] and diagnostics methods [8] [9] [10] 24] , and the interactions of the virus with its mosquito vector, Aedes [1] [2] [3] [11] [12] [13] [14] [15] . Here, we describe for the first time the comprehensive systemic production of cytokines, chemokines, and growth factors during acute CHIKV infection which may light the path ahead in understanding the innate response to the infection. We first showed that a wide range of cytokines such as IFN-a, IL-5, IL-6, IL-7, IL-10, IL-15 were produced in response to CHIKV infection. IFN-a is a potent antiviral cytokine and has been shown to strongly inhibit CHIKV in vitro [32] . The high levels of IFN-a that we detected provide a logical explanation for how the body rapidly brings CHIKV viremia under control [8, 11] . It has been shown that the main producers of IFN-a are plasmocytoid dendritic cells [33] and monocytes [34] . The profile of circulating cytokines revealed a predominance of Type 2 cytokines. Mainly IL-5, IL-6 and IL-10 levels were increased and those of IFN-c or TNF-a were unchanged as compared to noninfected controls. This suggests that acute CHIKV infection tilts the cytokine profile to anti-inflammatory response, which would argue against the common understanding of CHIKV infection which does not really support the common description of the CHIKV infection as an inflammatory disease [8] . Alternatively, it is possible that an inflammatory response might occur earlier when the virus is actively replicating, and then gets down-regulated by a counter-anti-inflammatory response when the virus is being eliminated from circulation. High levels of anti-inflammatory IL-10 and the presence of high levels of chemokines IP-10 and MIG (ligands of CXCR3 associated with Th1-type reactions) [35] , detected here would support this hypothesis. Further studies would be needed to clarify this issue. The Type 2 cytokines detected are also important mediators of B cell growth and maturation, and thus may allow the production of high levels of persisting anti-CHIKV IgG [5] .
The detection of high levels of circulating IL-15 is of interest, since this cytokine has been shown to be a major stimulator of NK cells [36] and T cells [37] . Thus our data suggest that these lymphocytes population might be activated during acute infection and may also contribute to viral control during the acute phase of CHIKV infection. Detection of soluble IL-2R in the plasma suggests T cell activation since this molecule is secreted by activated T cells [38] . Experiments are planned to study the activation phenotype of T and NK cell subsets in acutely infected patients.
The detection of IL-7 and IL-15 is significantly interesting with regards to the immunopathology of CHIKF since CHIKV infection has been shown to induce rapidly developing and persisting arthralgia [6] . Here, 9 of the 10 patients manifested this pathology. IL-7 is known to have an important role in the development of rheumatoid arthritis [39] , while IL-15 has been associated with the development of joint inflammation [40] . It has been proposed that expansion of a particular IL15-induced NK cell subsets was responsible for this phenomenon [41] . The role of IL-15 and NK cells in the development of CHIKV arthralgia would definitively be worth investigating. We did not detect TNF-a in the plasma of the patients with acute CHIKV infection. This is surprising since this cytokine has been detected repeatedly in the blood of patients suffering from other arthritides such as rheumatoid arthritis and is known to be involved in the pathogenesis of these entities [42] . Thus, it is possible that CHIKV-induced arthralgia does not depend on TNF-a. Alternatively, TNF-a might be produced only locally. Analysis of synovial fluid or joint tissue immunohistochemistry would be necessary to provide important information on the role of TNF-a and other mediators.
Chemokines are crucial mediators of innate and adaptive immunity against various viral infections [43] . IP-10, and MIG had increased plasma levels during CHIKV infection. These two chemokines signal through the same receptor CXCR3 and thus might activate and direct migration of this T cell subset [35] . IL-8 and Eotaxin levels were lower than those of naive controls. Defining the exact contributions of these different chemokines will require further studies.
We also tested the presence of growth factors in the plasma of CHIKV-infected patients. HGF, FGF-basic and VEGF were produced at high levels and may reflect a physiological response to tissue destruction resulting for the viral infection. Interestingly, EGF levels were lower than in healthy controls. The low levels of EGF might be due to the concomitant decrease of platelets observed in infected patients since previous studies have shown that plasma levels of EGF are associated with circulating platelets [44] .
Although limited, we had access to sufficient patients to perform data analysis in relation to the severity of the disease (severe illness was defined as fever .38.5uC, or maximum pulse rate .100 beats/minute, or nadir platelet count ,100610 9 /L). Using this definition, we observed that higher disease severity was associated with increased plasma levels of IL-1b and IL-6 and a decreased level in RANTES (Figure 3b ). IL-1b and IL-6, whose levels are already high in the CHIKV infected patients, are potent endogenous pyrogens [45] [46] [47] [48] . Therefore, elevations of IL-1b and IL-6 might account for the high fever in the severe cases. The increase production of IL-1b might also mediate the development of abrupt and persistent arthralgia since this cytokine is involved in the immunopathogenesis of many arthritic pathologies such as rheumatoid arthritis [49] . On the contrary, T cell chemokine RANTES levels were significantly suppressed in severe CHIKF patients. Platelets are a major reservoir of RANTES in the peripheral circulation [50] , and severe CHIKF was characterized by thrombocytopenia. Thus, as mentioned above for EGF, thrombocytopenia can also reduce levels of circulating RANTES. Low levels of RANTES correlate with disease severity and mortality in individuals with severe malaria, who were also correspondingly thrombocytopenic [51] . Interestingly, it was observed in other studies that RANTES levels were up-regulated in dengue [48] , except for one single report from Cuba [52] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential biomarker that differentiates between these 2 clinically very similar diseases.
One limitation of this study is in the classification of disease severity as none of our patients developed neurologic or hemorrhagic complications previously reported in CHIKF patients. Nonetheless, our definition of severe illness would have included patients with sepsis, a serious form of infection commonly associated with a temperature of .38uC and heart rate of .90/ minute [24] . Furthermore, we included thrombocytopenia of ,100610 9 /L as a criteria for severe CHIKF. Marked thrombocytopenia is a common feature of sepsis [25] and has been identified as a predictor of mortality [26, 27] . The degree of thrombocytopenia is a determinant of survival and once the platelet count decreases below 100610 9 /L, mortality continues to increase, even though the risk of bleeding does not [28] . A wide spectrum of disease has been reported in CHIKF ranging from asymptomatic infections, to self-limiting febrile illness [8] , to neurologic complications, and death [17] . The ''severe illness'' cohort in our study possibly represents a more severe form of selflimiting febrile illness, an intermediate group with higher levels of viremia (data not shown) and distinctly more severe clinical features (i.e. high temperature, tachycardia, and severe thrombocytopenia). Using this clinical phenotype, we have shown in this study that immune mediators are able to distinguish very mild disease from more severe forms of CHIKF disease at the acute stage. Follow-up studies will be required to determine if long-term sequelae are indeed different between non-severe and severe clinical presentations. Elucidating the association of disease severity with two cytokines and one chemokine can be useful in order to provide early identification and monitoring of patients with severe disease. Although this study is limited by the size of the outbreak, nevertheless, based on these observations, measurement of immune mediators could be helpful for the management of future outbreaks. This study strongly suggests these biomarkers be used for measuring disease severity and be tested in outbreaks in different populations and different strains. Once confirmed, they will be useful for follow-up studies, association studies, and prognosis for public health management. More importantly, these biomarkers can potentially lead to the development of modulators to reduce disease severity and to halt disease progression. 
Ten patients who presented with acute CHIKF to the Communicable Disease Centre at Tan Tock Seng Hospital (CDC/TTSH), the national infectious disease referral centre in Singapore, during the outbreak period from January to February 2008, were included in this study. An acute case of CHIKF was defined as any case with clinical features consistent with CHIKF, and had CHIKV infection confirmed by either reverse transcriptionpolymerase chain reaction (RT-PCR) or virus isolation [53, 54] . The study was approved by the institution's domain-specific ethics review board (DSRB Reference No. B/08/026). Written consent was obtained from each patient and healthy control subject.
Plasma samples were obtained from patients during the acute phase of their illness. Data on demographic characteristics, premorbid conditions, clinical features, and routine hematological and biochemical laboratory test findings (i.e. full blood count, renal and liver function tests, C-reactive protein) were also collected. All symptomatic patients were isolated at CDC/TTSH until the febrile illness resolved and a negative CHIKV RT-PCR test was obtained. During the hospital stay, daily monitoring of body temperature, vital parameters, and blood counts were carried out. A patient was defined as having severe illness, if he had either a maximum temperature of more than 38.5uC, or a maximum pulse rate of more than 100 beats/minute, or a nadir platelet count of less than 100610 9 /L. Laboratory results were expressed as mean6SD.
In addition, plasma or serum samples from 9 healthy volunteers (who did not have a febrile illness in the preceding week and were not epidemiologically-linked to the outbreak) were also included as controls in our study.
Plasma samples collected as described above, were aliquoted and stored at 280uC until analyses were done. A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to manufacturer's instructions (Biosource Human Cytokine 30-plex Assay, Invitrogen). The following groups of cytokines were: inflammatory (GM-CSF, IL-1beta, IL-1RA, IL-6, IL-8, TNF-alpha); Th1/Th2 (IFN-gamma, IL-2, IL-2R, IL-4, IL-5, IL-10); Cytokine II (IFN-alpha, IL-7, IL-12p40/p70, IL-13, IL-15, IL-17); Chemokines (Eotaxin, IP-10, MCP-1, MIG, MIP-1alpha, MIP-1beta, RANTES); and Growth Factors (EGF, HGF, FGF-basic, G-CSF, VEGF). Briefly, 25 ul of plasma samples were diluted in 1:2 and incubated with antibody coupled beads for 2 h at RT uC. Complexes were washed twice with the use of a vacuum manifold before incubation with biotinylated detector antibody for 1 h at RT uC. Complexes were then washed twice followed by incubation with Streptavidin-phycoeryrhrin (RPE) for 30 min at RT uC. Complexes were washed thrice and incubated with wash buffer for another 3 min before detection in the Luminex 200 TM instrument. Results were acquired by the IS 2.3 software and the standard curves were plotted through a fiveparameter logistic curve setting.
To compare and analyze the expression profiles across the results, the raw cytokine values were normalized using z-score conversion based on the formula:
where x is the raw value to be converted, m is the mean of the population and s is the standard deviation (SD) of the population. The transformed value is denoted by z and exhibits positive value when the raw value is above mean and vice versa. Further, to examine the nuances and correlations masked in the full set of data, the z values are subjected to cluster analysis [55] to yield an ordered NumOfRows.x NumOfCols expression level matrix, E el . Hierarchical clustering is applied on the columns which represent the myriad of cytokine levels measurements (e.g.) based on McQuitty's or WPGMA method [56] where the distance between a pair of groups A and B is measured using the weighted arithmetic mean of all the pairwise distances between the data points in A and B. The rows which represent the suspected CHIKF patients and healthy individuals are left untouched. Seriation [57] is performed following the clustering approach to re-order the clustered data points using the minimum path length algorithm to minimize the sum of all the distances between adjacent columns.
Euclidean distance is used in both the clustering and seriation phases to measure the difference or dissimilarity, d, between data points (x 1 , y 1 ) and (x 2 , y 2 ) given by the equation:
d~ffi ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Comparisons between groups were calculated by Mann-Whitney rank sum. Further statistical analyses were done by Kruskal-Wallis test followed by Dunn's multiple comparison tests. P values of ,0.05 were considered to be statistically significant. Figure S1 Complete profile of the levels (pg/ml) of cytokines, chemokines and growth factors determined by multiplex-bead arrays from blood samples collected from CHIKV-infected patients and healthy control subjects.  Avian Influenza: Should China Be Alarmed? Avian influenza has emerged as one of the primary public health concern of the 21st century. Influenza strain H5N1 is capable of incidentally infecting humans and other mammals. Since their reemergence in 2003, highly pathogenic avian influenza A (H5N1) viruses have been transmitted from poultry to humans (by direct or indirect contact with infected birds) in several provinces of Mainland China, which has resulted in 22 cases of human infection and has created repercussions for the Chinese economy. People have been concerned whether a new pandemic will occur in the future. The eradication of pathogenic avian influenza viruses appears to be the most effective way to prevent an influenza pandemic. This paper will examine the features of H5N1, including incidence, infection, immunity, clinical management, prevention and control, and therapy in Mainland China.  Rift Valley Fever Virus NSs Protein Promotes Post-Transcriptional Downregulation of Protein Kinase PKR and Inhibits eIF2α Phosphorylation Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-β mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or α-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)–mediated eukaryotic initiation factor (eIF)2α phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2α accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2α phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2α phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts. Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen, which is distributed in sub-Saharan Africa [1] and has also caused large outbreaks in Madagascar [2] , Egypt [3, 4, 5] , Saudi Arabia [6] , and Yemen [6] . In endemic areas, RVFV naturally circulates among mosquitoes and ruminants, such as sheep, goat and cattle [7] . RVFV infection in adult ruminants causes febrile illness and a high rate of abortions, while some newborn animals less than 1-2 weeks of age develop an acute infection which results in higher mortality rates than those in adults [8] . Humans infected with RVFV usually develop an acute febrile myalgic syndrome; however, a small percentage of patients have a lethal illness that results in hepatic damage, hemorrhagic fever-like illness, encephalitis and/or retinal vasculitis [8] .
Due to the exotic origin of the virus, the potential for the aerosol transmission [9, 10, 11, 12] and serious consequences for humans and livestocks, wild-type (wt) RVFV is classified as a Risk Group 3 pathogen, that needs to be handled in a high-containment facility, e.g., a biosafety level (BSL)-4 laboratory, whereas a highly attenuated MP-12 strain of RVFV, produced after 12 serial passages of wt RVFV ZH548 in MRC-5 cells in the presence of 5fluorouracil [13] , is a Risk Group 2 pathogen. RVFV, which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome composed of S, M and L segments [14] . The S segment encodes N and NSs genes and uses an ambi-sense strategy to express the N and NSs proteins in infected cells; N mRNA encoding N protein is transcribed from the viral-sense (negative-sense) S segment, while NSs mRNA encoding NSs protein is transcribed from the antiviral-sense (positive-sense) S segment. Monocistronic M mRNA and L mRNA are transcribed from the viral-sense M and L segments, respectively. M mRNA has one large open-reading frame (ORF) which encodes the nonstructural NSm protein, a 78-kDa glycoprotein and two major viral structural glycoproteins, Gn and Gc [15, 16] . L mRNA encodes L protein, a viral RNA-dependent RNA polymerase. Both N and L proteins are required for viral RNA synthesis, while Gn and Gc function as envelope proteins [14] . NSm and NSs, both nonstructural proteins, are dispensable for viral replication in cell cultures [17, 18, 19, 20] , but are involved in viral pathogenesis [21, 22, 23] .
RVFV NSs protein is not essential for virus replication in cell cultures [19, 20] , yet plays a critical role in viral virulence [21] . A naturally occurring RVFV mutant Clone 13, which lacks approximately 70% of the NSs gene [20] , is highly attenuated in mouse, and, when reassortant viruses between wt RVFV and Clone 13 were characterized, the NSs was revealed as a major determinant of viral virulence in the mouse model [21] . The NSs localizes in the nucleus and cytoplasm in both RVFV-infected cells and NSs-expressing cells; further, the nuclear NSs, but not the cytoplasmic NSs, forms a unique filamentous structure [24] . The NSs suppresses the transcription of host mRNAs by interacting with the p44 subunit of TFIIH, an essential transcriptional factor for cellular RNA polymerase II [25] . Furthermore, the RVFV NSs binds to Sin3A-Associated Protein 30 (SAP30), which is important for maintaining the repressor complex containing histone deacetylase 3 on the interferon (IFN)-b promoter, and suppresses the IFN-b promoter activation early in infection [26] . Accordingly, the NSs protein in the nucleus of infected cells most probably exerts these host transcriptional-suppressive activities, including that of IFN production inhibition, and contributes to viral virulence [21] . In contrast, the biological function of cytoplasmic NSs is largely unknown. RVFV NSs expression promotes RVFV minigenome RNA synthesis driven by N and L protein in expression studies [27] . Because RNA synthesis of bunyaviruses occurs in the cytoplasm, NSs protein in cytoplasm may promote the minigenome RNA synthesis by unknown mechanism.
To establish that RVFV NSs exhibits a novel function, we hypothesized that, when combined, RVFV replication and NSsinduced host transcription suppression likely induces a cellular environment that is unsuitable for viral replication. Thus, to secure efficient RVFV replication, the NSs protein, in turn, alters this putative, virally unfriendly cellular environment to one that supports efficient viral replication. To test this possibility, we examined the replication of RVFV lacking the NSs gene, a mutant which was generated by employing a reverse genetics system [19] , in the presence of a host transcription inhibitor, e.g., actinomycin D (ActD) or a-amanitin; these drugs were selected because they mimicked the host transcriptional suppressive activities of the NSs. Consistent with our supposition, RVFV lacking the NSs, but not RVFV, failed to replicate efficiently in ActD-treated cells. We noted that double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2a phosphorylation suppressed the translation of RVFV lacking NSs in the presence of ActD. Further studies uncovered that RVFV NSs promoted PKR downregulation as early as 4 hours post-infection, and prevented eIF2a phosphorylation, which secured efficient viral translation. We speculate that this novel function of RVFV is important for counteracting the antiviral activities of PKR and allowing efficient virus replication and survival in infected hosts.
To explore a novel biological function of RVFV NSs protein, in addition to its host transcriptional shutoff activity, we investigated the effect of ActD, a host transcriptional inhibitor, on the replication of MP-12 lacking the NSs gene in IFN-incompetent VeroE6 cells. VeroE6 cells were mock-infected or infected with MP-12 or rMP12-rLuc, which expressed Renilla luciferase (rLuc) in place of the NSs ( Figure 1A ) at a multiplicity of infection (moi) of 3. After 1 h virus adsorption, cells were incubated in the absence or presence of 5 mg/ml of ActD. Supernatants were harvested at 16 hours post-infection (h.p.i.), and virus titers were measured by plaque assay. ActD treatment had little effect on MP-12 titers, yet it significantly reduced the titer of rMP12-rLuc ( Figure 1B) , which suggested that the RVFV NSs was important for an efficient virus replication in the presence of ActD.
To understand how the NSs protein exerted an efficient viral replication in the presence of ActD, we analyzed the status of host and viral translation. VeroE6 cells were mock-infected or independently infected with MP-12, rMP12-rLuc and rMP12-C13type, the latter of which lacks approximately 70% of the NSs ORF ( Figure 1A) , at an moi of 3. Cells were radiolabelled with [ 35 S] Methionine/Cysteine from 15 to 16 h.p.i. Cell extracts were prepared at 16 h.p.i., and the samples were applied to SDS-PAGE ( Figure 1C , top panel). In the absence of ActD, the host protein synthesis of rMP12-rLuc-infected cells ( Figure 1C , lane 5) and rMP12-C13type-infected cells ( Figure 1C , lane 7) was more efficient than that of MP-12-infected cells ( Figure 1C , lane 3). It is possible that NSs-mediated transcriptional suppression that occurred only in MP-12-infected cells, but not in cells infected with viruses lacking NSs, caused a reduction of host mRNA abundance, leading to the less efficient translation of host mRNAs in MP-12-infected cells. Efficient N protein synthesis occurred in cells infected with all three viruses in the absence of ActD, a finding which was consistent with our previous studies [19] , while for some unknown reasons the accumulation of N proteins of rMP12-rLuc and rMP12-C13type was slightly higher than that of MP-12. ActD treatment resulted in a strong inhibition of both host proteins and N protein synthesis in rMP12-rLuc-infected cells ( Figure 1C , lane 6) and rMP12-C13type-infected cells ( Figure 1C , lane 8). In contrast, ActD treatment did not inhibit N protein translation in MP-12-infected cells; rather, it moderately inhibited host protein synthesis in MP-12-infected cells ( Figure 1C, lane 4) . A similar ActD-induced, moderate host protein synthesis inhibi-
The mosquito-borne bunyavirus Rift Valley fever virus (RVFV) devastates both humans and domestic animals; it causes abortions in ruminants and complications such as hemorrhage, encephalitis, or retinal vasculitis in humans. A major RVFV virulence factor, NSs, disables host cell mRNA synthesis. Here we describe our new evidence that showed NSs working in a second way; in addition to inhibiting host cell transcription, NSs kept translation active in infected cells. It is well-established that activated protein kinase PKR phosphorylates a translation factor, eIF2a, and then this phosphorylated eIF2a suppresses translation. We found that NSs decreased PKR abundance and prevented eIF2a phosphorylation in infected cells, allowing efficient viral translation and replication. In contrast, when cells were infected with an RVFV mutant lacking NSs in the presence of transcriptional inhibitors that mimic the transcription inhibition function of NSs, the PKR reduction did not occur and phoshorylated eIF2a was accumulated, resulting in the inhibition of virus gene expression and replication. Thus, NSs functions in two ways to help RVFV replicate in mammalian hosts: its newly identified PKR downregulation function secures efficient viral translation, and its host transcription inhibition function suppresses the expression of host innate antiviral functions.
tion also occurred in mock-infected cells ( Figure 1C , lane 2). Western blot analysis of cell extracts at 16 h.p.i. clearly showed that ActD treatment strongly inhibited N protein accumulation in rMP12-rLuc-infected cells and rMP12-C13type-infected cells, but not in MP-12-infected cells ( Figure 1C , bottom panels). In summary, ActD treatment had little effect on MP-12 replication, whereas it strongly inhibited the expression of both host and viral proteins in the cells infected with MP-12 lacking the NSs gene, which resulted in poor virus production.
To further confirm that the NSs exerted an efficient viral replication in the cells that underwent ActD-induced host transcriptional suppression, 293 cells, which showed higher RNA transfection efficiencies than did VeroE6 cells (data not shown), were infected with rMP12-rLuc at an moi of 2. After virus adsorption, infected cells were mock-transfected or independently transfected with in vitro-synthesized RNA transcripts encoding chloramphenicol acetyltransferase (CAT), MP-12 NSs, or wt RVFV ZH501 NSs. Then, the cells were mock-treated or treated with ActD. Analysis of rLuc activities at 16 h.p.i. demonstrated that NSs expression had little effect on rLuc activities in the absence of ActD (Figure 2A ). In the presence of ActD, rLuc activities were clearly higher in the cells transfected with MP-12 NSs RNA transcripts or ZH501 NSs RNA transcripts than in those transfected with CAT RNA transcripts or in the mocktransfected samples (Figure 2A ). In NSs RNA-transfected cells, similar levels of rMP-12-rLuc titers were observed in both the ActD-treated and mock-treated samples, whereas the rMP12-rLuc virus titers in mock-transfected cells and CAT RNA-transfected cells were significantly lower in the presence of ActD compared to the ActD-untreated cells ( Figure 2B ). Western blot analysis showed that NSs proteins were indeed expressed in the cells transfected with RNA transcripts encoding MP-12 NSs or wt RVFV ZH501 NSs ( Figure 2C , lanes 3, 4, 7, and 8). Also NSs protein expression increased the accumulation of rMP12-rLuc N protein in the presence of ActD ( Figure 2C, lanes 7 and 8) . These data demonstrated that NSs exerted efficient rMP12-rLuc replication in the presence of ActD. We noted that ActD treatment modestly reduced the accumulation of the CAT protein ( Figure 2C, lane 6) . Probably large amounts of CAT RNA transcripts that were introduced into the cells partly overcame the translational suppressive effects that were induced by the combination of rMP-12-rLuc replication and ActD treatment.
To test the possibility that ActD treatment alone suppressed translation and RVFV NSs counteracted it, 293 cells were mocktreated or treated with 5 mg/ml of ActD. We examined the resulting polysome profiles at 16 h post-ActD treatment ( Figure  S1A ). Because ActD treatment at 5 mg/ml inhibits the transcription mediated by RNA polymerases I, II and III [28] , we expected a reduction in the abundance of cellular mRNAs, tRNAs, and ribosomal RNAs, leading to reduced abundances of polysomes. ActD treatment indeed resulted in a reduced abundance of polysomes, whereas it did not substantially alter the polysome pattern, a finding which suggested to us that ActD treatment did not abolish translational activities. To test the translational competence of the cells treated with transcriptional inhibitors, we transfected 293 cells with in vitro-synthesized RNA transcripts encoding rLuc gene. Cells were mock-treated or treated with ActD or a-amanitin, an RNA polymerase II inhibitor [29] . The rLuc activities at 16 h post-transfection were slightly increased in the cells treated with ActD or a-amanitin ( Figure S1B ), demonstrating active host translation activities in the presence of either ActD or a-amanitin. These data led to the suggestion that by combining the replication of RVFV lacking the NSs and treatment of ActD, a cellular condition that is unfavorable for translation could be induced and that NSs expression somehow altered the cellular environment from one that was translationally inactive to one translationally active.
To establish that NSs exerts an efficient viral translation in the presence of a transcription inhibitor, we tested whether coinfection of MP-12 and rMP12-rLuc increases the translation of rLuc mRNA of rMP12-rLuc in the presence of ActD. VeroE6 cells were mock-infected or co-infected with rMP12-rLuc and MP-12; rMP12-rLuc was infected at an moi. of 2, while MP-12 was infected at moi. of 0.1 or 1, as indicated in Figure 3 . The cell extracts were harvested at 16 h.p.i., and the rLuc activities ( Figure 3A ) and accumulation of viral RNAs ( Figure 3B ) were examined. As expected, ActD-treatment reduced the rLuc activities in cells infected with rMP12-rLuc alone ( Figure 3A ). Coinfection of MP-12 resulted in the reduction of both rLuc activities ( Figure 3A ) and the amounts of rLuc mRNA ( Figure 3B , lane 4) in the absence of ActD. In the presence of ActD, MP-12 co-infection also reduced the rLuc mRNA abundance ( Figure 3B , lane 9), whereas it increased rLuc activities ( Figure 3A ). The results suggested that NSs protein expressed from MP-12 S-segment promoted an efficient translation of the rLuc mRNA of rMP12-rLuc in the presence of ActD. We also noted that MP-12 coinfection did not reduce the abundance of the viral-sense rMP-12-rLuc S segment in the presence of ActD, and yet it caused the reduction of rLuc mRNA abundance ( Figure 3B , lane 9), which we believe implies that NSs expression and ActD treatment generated a cellular environment that was more favorable for rMP-12-rLuc RNA replication than for transcription.
The dsRNAs generated during viral replication activate PKR, which in turn phosphorylates eIF2a [30] . Also 59-triphosphated single-stranded RNAs activate PKR [31] . The eIF2a is a component of eIF2, which binds to GTP and Met-tRNA to deliver the Met-tRNA to the start codon in capped mRNA, forming a 43S pre-initiation complex [32] . Upon the binding of the 60S ribosomal subunits to the 43S preinitiation complex, eIF2-GDP is released from the ribosome and undergoes a GTP exchange reaction catalyzed by binding with eIF2B, and the resultant eIF2-GTP is recycled for the next round of translation initiation. The phosphorylated eIF2a binds to eIF2B with a high affinity and prevents eIF2B to be used for the subsequent eIF2-GDP-to-eIF2-GTP exchange reaction, leading to the suppression of translation initiation. Hence, the phosphorylation status of eIF2a plays a critical role in translational control [32] .
We suspected that rMP12-rLuc replication in the presence of ActD may generate dsRNAs and/or 59-triphosphated singlestranded RNAs, which activate PKR, resulting in the phosphorylation of eIF2a. When we analyzed the level of eIF2a phosphorylation in VeroE6 cells infected with rMP12-rLuc in the absence of transcription inhibitor, we found low levels of eIF2a phosphorylation from 8 to 24 h.p.i. and an efficient accumulation of N protein at 8 h.p.i. and onward ( Figure 4A -4C). In contrast, when we treated rMP12-rLuc-infected cells with ActD or aamanitin, either compound induced an efficient accumulation of phosphorylated eIF2a from 8 to 16 h.p.i., with a concomitant poor N protein accumulation ( Figure 4A -4C) and virus replication suppression ( Figure 4D ). The mechanism of reduction in the abundance of the phosphorylated eIF2a at 24 h.p.i. in the presence of ActD or a-amanitin ( Figure 4 , lanes 13 and 19) is unknown. When we treated rMP12-rLuc-infected cells with different concentrations of ActD or a-amanitin, we found that the inhibition of phosphorylated eIF2a accumulation was dependent on the concentrations used ( Figure S2 ). Also a strong correlation was seen between an increase in eIF2a phosphorylation and the decrease in both N protein accumulation and infectious virus production ( Figure S2 ). In contrast, no significant accumulation of phosphorylated eIF2a occurred in MP-12infected VeroE6 cells in the presence of ActD ( Figure 4A -4C). As expected, ActD treatment had little effect on MP-12 replication ( Figure 4D ). These data strongly suggested that the presence of highly phosphorylated eIF2a levels suppressed the translation of viral mRNAs, leading to inefficient virus production, and that the NSs protein somehow suppressed eIF2a phosphorylation, and facilitated efficient viral mRNA translation under the conditions of host transcriptional shutoff.
Because activated caspases 3, 7 and 8 can cleave PKR, releasing the biologically active C-terminus kinase domain from the Nterminus inhibitory domain, resulting in eIF2a phosphorylation [33] , we subsequently tested whether the accumulation of phosphorylated eIF2a following the combined activity of viral replication and ActD-treatment was due to induction of apoptosis [34] . VeroE6 cells, either infected with rMP12-rLuc or mockinfected, received the pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp(OMe) fluoromethylketone (Z-VADfmk), in the presence of ActD or a-amanitin ( Figure S3 ). Judging from the resulting inhibition of cleaved caspase-3 accumulation in these cells, the Z-VADfmk treatment indeed inhibited apoptosis, whereas it had little effect on the eIF2a phosphorylation status and infectious virus yield. These data suggested to us that the accumulation of phosphorylated eIF2a in cells supporting rMP12-rLuc replication in the presence of ActD or a-amanitin was caspase-independent.
Four different kinases, including PKR, PKR-like ER kinase (PERK), heme-regulated inhibitor and the general control, nondepressible-2 (GCN2), are known to phosphorylate eIF2a [35] . To determine the role of PKR in the accumulation of phosphorylated eIF2a in rMP12-rLuc-infected cells treated with ActD or aamanitin, we generated a recombinant MP-12, rMP12-PKRDE7, which expresses a dominant-negative form of PKR, PKRDE7 [36] carrying the N-terminal Flag epitope tag, in place of the NSs ( Figure 5A ). If the replication of MP-12 lacking the NSs gene in cells subjected to host transcriptional suppression activates PKR, which in turn phosphorylates eIF2a, then the virally-encoded PKRDE7 in rMP12-PKRDE7-infected cells would interfere with the PKR function, resulting in the inhibition of PKR-mediated eIF2a phosphorylation and thereby leading to efficient viral translation and virus production. VeroE6 cells were mock-infected or independently infected with rMP12-rLuc, rMP12-PKRDE7 and MP-12 at an moi of 3. After the removal of the inocula, cells were treated with ActD or a-amanitin, and cell extracts were harvested at 16 h.p.i. As expected, rMP12-rLuc replication in the presence of ActD or a-amanitin induced eIF2a phosphorylation, resulted in reduced virus replication ( Figure 5B -5D). In contrast, efficient N protein accumulation and efficient virus replication, with no significant accumulation of phosphorylated eIF2a occurred in both MP-12-infected cells and rMP12-PKRDE7infected cells in the presence of ActD-or a-amanitin ( Figure 5B -5D). This finding strongly suggested that PKR is important for eIF2a phosphorylation in cells infected with the RVFV lacking the NSs in the presence of transcription inhibitors.
To further confirm these data, viral protein accumulation and replication were analyzed in wt mouse embryonic fibroblast (MEF) cells and in Pkr 0/0 MEF cells lacking a functional PKR expression [37] . MP-12 efficiently replicated in both wt MEF and Pkr 0/0 MEF cells, and ActD treatment had little effect on N protein accumulation and virus replication ( Figure 5E , top and middle panels). rMP12-rLuc replication was not as efficient as MP-12 in wt MEF cells in the absence of ActD for an as yet unidentified reason ( Figure 5E , middle panel). In ActD-treated wt MEF cells, both rMP-12-C13type and rMP12-rLuc failed to efficiently accumulate N proteins, and rMP-12-rLuc replicated poorly, whereas rMP-12-rLuc underwent efficient N protein accumulation and viral replication in ActD-treated Pkr 0/0 MEF cells ( Figure 5E , top and middle panels). Furthermore, accumulation of phosphorylated eIF2a did not occur in Pkr 0/0 MEF cells that were infected with rMP12-rLuc in the presence of transcriptional inhibitors 
To know how the NSs suppressed the eIF2a phosphorylation activity of the PKR function, a dsRNA-binding assay was performed to test the possibility that the NSs binds to dsRNA, sequesters dsRNA from PKR, and interferes with the dsRNAmediated PKR activation. 293 cells were mock-infected or infected with rMP12-NSs-Flag carrying Flag-tagged NSs, rMP12-rLuc-Flag carrying Flag-tagged rLuc ( Figure 6A ) or rMP12-PKRDE7 ( Figure 5A ). In a separate experiment, 293 cells were transfected with in vitro-synthesized RNA transcripts encoding NSs. Lysates were prepared at 16 h.p.i. or 16 h post-transfection, and incubated with poly I:C beads (dsRNA) or poly (C) beads (ssRNA). Then the dsRNA-bound complexes were analyzed by a Western blot in which we used an anti-Flag antibody or anti-NSs antibody ( Figure 6B ). As expected, dsRNA bound to PKRDE7 [36] , whereas it poorly bound to the NSs from rMP12-NSs-Flaginfected cells and that from the NSs-expressing cells ( Figure 6B ), which suggested that NSs did not suppress PKR activation by its binding to dsRNA.
Because activated PKR undergoes a structural alteration and autophosphorylation [30] , we tested whether NSs prevented PKR autophosphorylation. 293 cells were infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, and mock-treated or immediately treated with ActD. Cell lysates were harvested at 16 h.p.i., and PKR was immunoprecipitated by anti-human PKR antibody, and the PKR bound to the protein A beads was subjected to an immunoprecipitation (IP)-kinase assay by using [c-32 P]ATP. We used 293 cells for this assay, because anti-human PKR antibody efficiently immunoprecipitated human PKR in 293 cells, but not non-human primate PKR in VeroE6 cells (data not shown). PKR is induced by IFN-a/b treatment [38] and the abundance of PKR could affect the results of the IP-kinase assay. We suspected that replication of MP-12 mutants lacking NSs may induce IFN-b production, leading to PKR induction [38] , whereas ActD treatment prevented the IFN-b production [39] . Indeed, IFN-b mRNA accumulation occurred at 8 h.p.i. in rMP-12-rLucinfected cells in the absence of ActD, but not in the presence of ActD ( Figure 6C ), a finding which suggested that ActD treatment inhibited the transcriptional induction of PKR which was induced by the type I IFN in infected 293 cells. We noted an efficient accumulation of rMP12-rLuc N mRNA at 8 h.p.i. in the presence of ActD ( Figure 6C ). Because rMP-12-rLuc replication in the presence of ActD did not induce an accumulation of phosphorylated eIF2a early in the course of the infection ( Figure 4A ), this efficient rMP12-rLuc replication probably occurred prior to 8 h.p.i. in the presence of ActD. As shown in Figure 6D , immunoprecipitated PKR from rMP12-rLuc-Flag-infected cells was phosphorylated both in the presence and absence of ActD, which led us to suggest that rMP12-rLuc-Flag replication activated PKR. In contrast, PKR phosphorylation did not occur in mockinfected cells or cells infected with rMP12-NSs-Flag or rMP12-PKRDE7 ( Figure 6D ). Most probably, a dominant-negative PKRDE7 suppressed PKR activation and prevented PKR phosphorylation [36] . To determine why we failed to detect the presence of PKR autophosporylation in rMP12-NSs-Flag-infected cells, we examined the amounts of the immunoprecipitated PKR in these samples ( Figure 6D, bottom) . Strikingly, substantial reductions in the abundance of cytoplasmic PKR occurred only in the cells supporting rMP12-NSs-Flag replication both in the presence and absence of ActD.
We subsequently tested the possibility that the NSs downregulated PKR expression or sequestered PKR into the nuclear compartment, leading to the suppression of eIF2a phosphorylation. 293 cells were mock-infected or infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, treated with ActD, and cytoplasmic and nuclear fractions of cell extracts were prepared at 16 h.p.i. Western blot analyses showed the presence of RVFV NSs both in the cytoplasmic and nuclear fractions, while rLuc and PKRDE7 signals were observed only in the cytoplasmic fraction. A substantial reduction in PKR abundance occurred in both the cytoplasmic and nuclear fractions of cells infected with rMP-12-NSs-Flag, but not in mock-infected cells and in those infected with rMP12-rLuc-Flag or rMP12-PKRDE7 ( Figure 7A ), demonstrating that NSs induced the PKR downregulation in the infected cells. The PKR downregulation also occurred in MP-12infected VeroE6 cells and in MRC-5 cells that were infected with the wt ZH501 strain of RVFV (data not shown).
To determine whether NSs expression alone induces PKR downregulation, 293 cells were mock-infected or infected with rMP12-rLuc, immediately transfected with in vitro-synthesized RNA transcripts encoding rLuc or NSs, and treated with ActD ( Figure 7B, left panel) . Cells were harvested at 16 h posttransfection. The clear reduction in the PKR abundance occurred in mock-infected cells expressing NSs, but not in those expressing rLuc ( Figure 7B, left panel) , demonstrating that NSs protein alone exerted the PKR downregulation.
We subsequently examined the requirement of the ActD treatment for the PKR downregulation. 293 cells were transfected with in vitro-synthesized RNA transcripts encoding NSs or rLuc in the absence of ActD. Analysis of cell extracts harvested at 8 h posttransfection showed the PKR downregulation in the NSsexpressing cells ( Figure 7B, right panel) , demonstrating that the NSs-mediated PKR downregulation occurred in the absence of ActD.
To further understand the mechanism of the NSs-mediated PKR downregulation, we examined whether NSs expression promoted degradation of PKR mRNA. 293 cells were mocktransfected or transfected with in vitro-synthesized RNA transcripts encoding NSs or rLuc. Then the cells were mock-treated or treated with ActD. Total RNAs were harvested at 8 h posttransfection and the expression levels of PKR mRNA were examined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis ( Figure 7C ). The relative expression levels of PKR mRNA were significantly increased in cells that were transfected with rLuc RNA transcripts or NSs RNA transcripts both in the absence and presence of ActD, which demonstrated to us that NSs expression did not promote the degradation of PKR mRNA. Efficient ActD-mediated suppression of IFN-b mRNA accumulation in rMP-12-rLuc-infected 293 cells ( Figure 6C ) led us to suggest that unexpected increases in the abundance of PKR mRNA in the RNA-transfected 293 cells in the presence of ActD were IFN-b-independent. We suspect that the RNA transcripts that were taken up by the cells induced robust PKR mRNA synthesis prior to ActD-or NSs-mediated general transcription suppression.
Since NSs expression did not decrease the abundance of PKR mRNA ( Figure 7C ), we next tested whether putative NSsmediated translational inhibition leads to a reduction in the abundance of PKR. To this end, 293 cells were mock-infected, infected with MP-12, or transfected with in vitro-synthesized RNA transcripts encoding NSs. Then cells were incubated with 100 mg/ ml of puromycin to completely shut off cellular translation or puromycin untreated, and cell extracts were harvested at 16 h.p.i. or post-transfection. As expected, puromycin treatment completely abolished the synthesis of N and NSs proteins in MP-12-infected cells ( Figure 7D, lane 4) and that of NSs in cells transfected with the RNA transcripts encoding NSs ( Figure 7D, lane 6) . We found that treatment of 293 cells with puromycin for 16 h decreased the abundance of PKR only slightly ( Figure 7D, lane 2) . Accordingly, it is highly unlikely that putative NSs-induced translational We next performed pulse-chase experiments to know whether NSs expression promoted PKR degradation. Because immunoprecipitation experiments using various anti-PKR antibodies failed to convincingly demonstrate a radiolabelled endogenous PKR signal from extracts of 293 cells (data not shown), we examined the effect of NSs expression on the stability of an expressed mutant PKR, PKRK296R, lacking kinase activity [40] and carrying a Nterminal myc tag; expression of wild-type PKR was not used due to its strong host translation suppression effects (data not shown). 293 cells were mock-transfected or transfected with pcDNA3.1-Myc-PKRK296R, a plasmid encoding PKRK296R under cytomegalovirus promoter and radiolabelled with [ 35 S] methionine/cycteine between 14 and 16 h post-DNA transfection. After pulse-radiolabelling cell extracts were prepared from some samples. In other samples, cells were transfected with in vitrosynthesized RNA transcripts encoding rLuc or NSs, and cell extracts were harvested at 8 h post-RNA transfection. Then the cell extracts were subjected to radioimmunoprecipitation analysis using anti-myc antibody, which immunoprecipitated the pulseradiolabelled, myc-tagged PKR ( Figure 7E , lane 2). After 8 h chase, the amount of the radiolabelled myc-tagged PKR was clearly reduced in the cells expressing NSs ( Figure 7E, lane 4) , but not those expressing rLuc ( Figure 7E, lane 3) . Instead, the amount of radiolabelled myc-tagged PKR was increased slightly in rLucexpressing cells, presumably due to radiolabelling of continuously synthesized myc-tagged PKR by residual [ 35 S] methionine/ cysteine.
The complete shutoff of cellular translation by puromycin for 16 h did not result in the loss of endogenous PKR ( Figure 7D ), whereas as early as 8 h post-transfection of RNA transcripts encoding NSs, the abundance of PKR decreased substantially ( Figure 7B ) without reducing the abundance of PKR mRNA ( Figure 7C ). Furthermore, NSs expression reduced the abundance of myc-tagged PKR that had been radiolabelled prior to NSs expression ( Figure 7E ). These data strongly suggested that NSs induced the downregulation of PKR at a post-transcriptional level and pointed to a possibility that the NSs promoted PKR degradation.
We next tested whether RVFV NSs downregulated PKR by promoting PKR degradation through a ubiquitin-proteasome pathway. 293 cells were infected with rMP12-rLuc or MP-12, and were immediately treated with proteasome inhibitor MG132 or lactacystin. Cells were harvested at 8 h.p.i. and analyzed in Western blotting. As expected, rMP12-rLuc replication did not induce PKR downregulation ( Figure 7F, lane 1) , while MP-12 replication induced the reduction of PKR abundance ( Figure 7F,  lanes 4) . It was evident that the treatment of MP-12-infected cells with those proteasome inhibitors suppressed NSs-induced PKR downregulation ( Figure 7F, lanes 5 and 6) , suggesting that NSs promoted PKR downregulation by the degradation through the proteasome pathway.
Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces PKR degradation [41] . Because the general PKC inhibitor, GÖ 6983, suppresses PMA-mediated PKR degradation [41] , we examined whether the NSs promoted PKR downregulation through PKC by treating MP-12-infected cells with GÖ 6983. Treatment of GÖ 6983 did not inhibit the NSs-mediated PKR downregulation ( Figure 7F, lane 8) , suggesting that PKC was not involved in NSs-induced PKR downregulation.
To know how quickly PKR downregulation occurred in MP-12infected cells, whole-cell extracts were prepared at 2, 4, 6 and 8 h.p.i. from MP-12-infected 293 cells and the amounts of NSs and PKR were determined ( Figure 7G ). In the absence of proteasome inhibitor, a substantial reduction of PKR abundance occurred as early as 4 h.p.i., where the abundance of NSs ( Figure 7G , lane 2) was not as great as that at 8 h.p.i. (Figure 7G , lane 4). In the presence of MG132, the abundance of PKR decreased only slightly, and NSs accumulation was also somewhat less efficient. A similar trend for less efficient NSs accumulation in the presence of proteasome inhibitors was also shown in the data for Figure 7F . Our observation of less efficient NSs accumulation in MG132-treated cells was consistent with the report that MG132 induces eIF2a phosphorylation through GCN2 activation and translational suppression [42] . The abundance of NSs in MG132treated cells at 8 h.p.i. and that in untreated cells at 4 h.p.i. was similar, and yet PKR abundance in the former cells was clearly higher than that in the latter cells. Taken together, these data strongly suggested that RVFV NSs promoted PKR degradation through the proteasome-dependent pathway.
The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene initially in type I IFN-incompetent VeroE6 cells [43, 44] in the presence of ActD or a-amanitin, which served as a surrogate of the host mRNA synthesis suppression function of the NSs. The NSs protein was essential for efficient virus replication in the presence of ActD or a-amanitin. We found that the replication of RVFV lacking the NSs gene in the presence of a transcription inhibitor induced an accumulation of phosphorylated eIF2a, The accumulation of phosphorylated eIF2a in the presence of transcriptional inhibitors, did not occur in VeroE6 cells that were infected with RVFV expressing a dominant-negative form of PKR (PKRDE7) and in MEF cells lacking functional PKR ( Figure 5 ). These findings suggested to us that PKR played a major role in increasing phosphorylated eIF2a; however, it is currently unclear whether other kinases, such as PERK or GCN2, have a possible role in eIF2a phosphorylation in rMP-12-rLuc-infected cells in the presence of transcriptional inhibitors. The endogenous PKRmediated eIF2a phosphorylation suppressed translation of the RVFV lacking the NSs gene, resulting in poor virus replication. Our data further suggested that the NSs promoted PKR degradation most probably through a proteasome-dependent pathway and prevented eIF2a phosphorylation, leading to efficient viral translation. (8 h) . Radiolabelled myc-tagged PKR was immunoprecipitated by anti-myc monoclonal antibody, analyzed by SDS-PAGE with 7.5% poryacrylamide gel, and visualized by autoradiography. (F) 293 cells were infected with rMP12-rLuc or MP-12 at an moi of 3, and, then, treated with 10 mM of MG132 (MG) or 50 mM of lactacystin (LA) or they were mock-treated (2) . Whole-cell lysates were collected at 8 h.p.i., and the abundance of PKR, NSs and b-actin were examined by Western blot analysis. (G) 293 cells were mock-infected (Mock) or infected with MP-12 (MP-12) at an moi of 3, and treated with MG132 (MG132) at 10 mM or they were untreated (no drug used). Whole-cell lysates were collected at 2, 4, 6, and 8 h.p.i. Anti-PKR antibody, anti-NSs antibody, and anti-b-actin antibody were used to detect PKR, NSs, and b-actin, respectively (F and G). Data are representative of two to three independent experiments (A-G). doi:10.1371/journal.ppat.1000287.g007
The past studies [25, 26, 45] and the data shown in this study illustrate that two distinct biological activities of the RVFV NSs protein worked together to secure efficient RVFV replication. Namely, nuclear NSs protein inhibits transcription of host mRNAs, including the IFN mRNAs; this activity is critical for efficient RVFV replication in IFN-competent systems [25, 45] . However, a combination of RVFV replication and NSs-mediated host mRNA transcriptional suppression possibly induces PKR activation and subsequent eIF2a phosphorylation as a combination of RVFV replication and ActD or a-amanitin treatment induced it. The NSs protein, in turn, promotes PKR downregulation as early as 4 h.p.i., and prevents eIF2a phosphorylation to secure the translation of viral mRNAs and efficient virus replication. We think that both of these two NSs functions are tightly related and protect efficient viral replication by suppressing host antiviral responses. In RVFV-infected cells, the NSs establishes general transcriptional suppression at a later stage of infection (after 8 h.p.i.) [25] , while NSs also suppresses specific IFN-b mRNA transcription at early stages of infection (about 3 h.p.i.) by maintaining repressor complex including SAP30 on IFN-b promoter [26, 45] . PKR downregulation early in infection is probably important for maintaining efficient viral translation in combination with the suppression of host IFN responses.
We suspect that the NSs-mediated PKR downregulation activity is important for RVFV replication and survival in infected mammalian hosts. RVFV-infection in rhesus monkeys showed that type I IFN is detectable around 1 day post-infection in both clinically ill surviving monkeys and lethally infected monkeys, and one dead monkey even kept high titer of IFN (120 to 480 U/ml) from 1 day post-infection [46] . In fact, the best correlation with outcome was early detection of IFN and not necessarily the height of the response. NSs suppresses IFN-b mRNA transcription early in the course of infection in cultured cells [26, 45] . Our present data suggest that the early downregulation of PKR in RVFVinfected cells might contribute to the inhibition of type I IFN induction, because PKR is known to serve as a pathogenrecognition receptor [47] . Furthermore, some of the host pathogen-recognition receptors, e.g., toll-like receptors 3 and 7, in uninfected cells located near the RVFV-infected cells, recognize virus-specific signals, e.g., viral RNAs in the virus particles or viral dsRNAs in the cell debris from infected cells, leading to type I IFN production. Because the transcriptional promoter of the PKR gene contains an IFN-stimulated response element, and IFN stimulation induces PKR mRNA transcription [38] , PKR abundance is likely increased in many uninfected cells of infected animals; RVFV needs to replicate in such cells to survive in infected hosts. The reduction in PKR abundance occurred as early as 4 h.p.i. ( Figure 7G ) and the NSs protein is produced very early in RVFV infection [48] . Immediate NSs synthesis and subsequent NSsinduced PKR downregulation in the cells, some of which have increased PKR abundance, could rapidly disarm the PKRmediated, antiviral functions and contribute to RVFV replication and survival in animal hosts.
Possible mechanisms of the accumulation of phosphorylated eIF2a in ActD-treated, rMP12-rLucinfected cells ActD treatment of uninfected cells resulted in a significant reduction of the polysome fraction, but it did not abolish the translational activities ( Figure S1 ). rMP12-rLuc replication in the cells treated with ActD or a-amanitin promoted the accumulation of the phosphorylated eIF2a, resulting in translational suppression of viral proteins (Figure 4) . The increased amounts of phosphorylated eIF2a clearly correlated with the concentration of ActD or a-amanitin in rMP12-rLuc-infected cells ( Figure S2 ). In contrast, rMP12-rLuc replication in the absence of transcriptional suppressor induced only low levels of eIF2a phosphorylation (Figure 4 ). Yet, the phosphorylation of PKR occurred in cells supporting the replication of RVFV lacking NSs both in the absence and presence of ActD ( Figure 6D ). Thus, a significant accumulation of phosphorylated eIF2a occurred only in cells in which replication of RVFV lacking NSs was combined with the treatment with transcriptional inhibitors.
Several different mechanisms are conceivable for the accumulation of phosphorylated eIF2a in rMP12-rLuc-infected in cells treated with ActD or a-amanitin (Figure 4 ). One possible mechanism relates to the eIF2a dephosphorylation step. Phosphorylation of eIF2a at Serine 51 induces a rapid synthesis of activating transcription factor (ATF)-4 mRNA, which can be translated in the presence of phosphorylated eIF2a [49] . Expressed ATF4 then induces GADD34 mRNA transcription [50] and GADD34 protein interacts with type 1 protein serine/ threonine phosphatase, PP1, and this complex dephosphorylates eIF2a to resume the cellular translation [51] . rMP12-rLuc replication in transcriptionally active cells probably induced PKR activation and eIF2a phosphorylation, the latter of which then induced GADD34 upregulation and subsequent eIF2a dephosphorylation allowing efficient viral translation. rMP12-rLuc replication in cells treated with ActD induced PKR activation, the extent of which was similar to that of infected, ActD-untreated cells ( Figure 6D ), whereas the transcription inhibitors would prevent GADD34 upregulation and subsequent eIF2a dephosphorylation, causing an accumulation of phosphorylated eIF2a, which inhibited viral translation. Other possible mechanisms relate to the failure to suppress the PKR function. Cells infected with adenovirus, human immunodeficiency virus-1, or herpes simplex virus undergo a dramatic increase in the abundance of Alu RNA, which carries a repetitive element of ,300-nt in length that is transcribed by RNA polymerase III [52, 53, 54, 55] . Alu RNA forms a stable complex with PKR, and antagonizes the PKR activation [56] . If rMP12-rLuc replication induces Alu RNA accumulation to prevent PKR activation, then the drug-induced transcriptional suppression would inhibit Alu RNA accumulation, preventing Alu RNA-mediated PKR inactivation. Another possibility is that rMP12-rLuc replication transcriptionally induces host mRNAs, and some of their gene products have PKR inhibition activities. An example of this possibility is that influenza virus replication induces P58 IPK , an inhibitor of PKR [57] . ActD or a-amanitin treatment suppresses the expression of the putative PKR inhibitor, resulting in an accumulation of phosphorylated eIF2a. Alternatively, transcriptional suppression may result in reduced amounts of ribosomal proteins, such as L18, which binds to PKR and inhibits PKR activation [58, 59] . These possibilities are not mutually exclusive, and the combination of these possibilities may contribute to the accumulation of phosphorylated eIF2a in the cells supporting replication of RVFV lacking the NSs in the presence of ActD or aamanitin.
Although many viruses suppress PKR function using various strategies [32] , poliovirus is known to promote PKR degradation in infected cells [60, 61] ; poliovirus RNA and viral protein components are required for this activity [61] , and both eIF2a and PKR are highly activated in poliovirus-infected cells before PKR downregulation occurs [60] . In contrast to poliovirus, only RVFV NSs protein was probably required for PKR downregu-lation since the downregulation of PKR occurred as early as 4 h.p.i. in RVFV-infected cells ( Figure 7G ), whereas phosphorylated eIF2a accumulation occurred around 8 h.p.i. in rMP-12-rLuc-infected cells in the presence of ActD (Figure 4 ). Furthermore, NSs downregulated PKRK296R, a non-phosphorylatable mutant PKR ( Figure 7E ). These data suggest that the phosphorylation of PKR was not essential for the NSs-mediated PKR downregulation.
A substantial reduction in the abundance of PKR occurred in both the cytoplasmic and nuclear fractions of MP-12-infected cells, but not in the mock-infected cells and in those infected with MP12 lacking NSs (Figure 7) . Expression of NSs alone resulted in a reduction in the amount of PKR ( Figure 7B ). These data established that NSs protein induced PKR downregulation. The NSs-induced PKR downregulation occurred as early as 4 h.p.i. of MP-12 ( Figure 7G ). We also demonstrated the presence of PKR mRNA in NSs-expressing cells ( Figure 7C ) and that ActD treatment had little effect on the NSs-induced PKR downregulation ( Figure 7B ). We failed to detect PKR at 16 h.p.i. of MP-12infected 293 cells, and yet we easily detected the PKR after a 16 hlong incubation of 293 cells with puromycin ( Figure 7D ). Accordingly, the putative NSs-induced translational inhibition was not a main reason for the reduction in the abundance of PKR. Furthermore, pulse-chase experiments showed that NSs expression reduced the abundance of radiolabelled myc-tagged PKR ( Figure 7E ). All of these data strongly suggested that the NSs induced PKR downregulation at post-transcriptional levels.
It was reported that treatment of a macrophage cell line with IFN-c induced PKR degradation [62] . Because NSs induced PKR degradation in ActD-treated cells and ActD treatment completely inhibited the IFN-c mRNA accumulation ( Figure 6C ), it is highly unlikely that IFN-c was involved in the NSs-induced PKR downregulation. PKC activation also potentially induces PKR downregulation [41] . Treatment of MP-12-infected cells with a PKC inhibitor, GÖ 6983, did not inhibit NSs-mediated PKR downregulation ( Figure 7D ), which implied that PKC was not involved in it. Experiments using a proteasome inhibitor suggested that the NSs promoted PKR degradation through a proteasome pathway ( Figure 7F ). MG132 can induce GCN activation and translational suppression [42] . In fact, treatment of MP-12infected cells with MG132 and lactacystin moderately reduced the accumulation of NSs ( Figure 7F and 7G) . Accordingly, a possibility still exists that the MG132-induced moderate translational inhibition affected the NSs-induced PKR degradation in MG132-treated cells. For example, if the NSs-induced PKR downregulation requires an unstable host protein, then the MG132-induced moderate translational suppression would prevent accumulation of this putative host protein, resulting in inhibition of the NSs-induced PKR downregulation. Obviously further studies are required to elucidate the mechanism of PKR downregulation mediated by RVFV NSs.
Vero E6 cells, wild-type mouse embryonic fibroblast (MEF) cells and Pkr 0/0 MEF cells [37] were maintained in Dulbecco's modified minimum essential medium (DMEM) (Invitrogen) containing 10% fetal bovine serum. BHK/T7-9 cells [63] , which stably express T7 RNA polymerase, were grown in MEM-alpha (Invitrogen) containing 10% fetal bovine serum (FBS). Penicillin (100 U/ml) and streptomycin (100 mg/ml) (Invitrogen) were added to the media. BHK/T7-9 cells were selected in medium containing 600 mg/ml hygromycin (Cellgro). An RVFV vaccine candidate MP-12 [13] and recombinant MP-12 [19] were used for the experiments, and infectivity was assessed by a plaque assay in Vero E6 cells.
Cells were treated with transcriptional inhibitors, ActD (Sigma) (5 mg/ml) or a-amanitin (Sigma) (50 mg/ml) immediately after infection or transfection. To induce the inhibition of proteasome function, cells were immediately treated with MG132 (Sigma) at 10 mM or lactacystin (Sigma) at 50 mM after infection or transfection. To suppress PKC activity, cells were treated with a general PKC inhibitor, GÖ 6983 (Calbiochem) at 100 nM immediately after infection with rMP12-rLuc or MP-12. Cells were treated with puromycin (Cellgro) at 100 mg/ml immediately after infection or transfection to inhibit cellular translation.
Standard molecular biological techniques, including a PCRbased mutagenesis method, were used for plasmid constructions. PCR fragments encoding PKRDE7 ORF with an N-terminal Flag-tag sequence were cloned between the Hpa I and Spe I sites of pProT7-S(+) plasmid [19] , designated as pProT7-S(+)-PKRDE7. PCR fragments encoding the rLuc ORF or NSs with a C-terminal Flag-tag sequence were cloned between the Hpa I and Spe I sites of the pProT7-S(+) plasmid, designated as pProT7-S(+)-rLuc-Flag or pProT7-S(+)-NSs-Flag, respectively. All of the constructs were confirmed to have the expected sequences. The PCR product of the entire human PKR ORF carrying a point mutation at K296R and the N-terminal myc tag was cloned between KpnI and XhoI of pcDNA3.1myc-His (Invitrogen), resulted in pcDNA3.1-Myc-PKR296R.
A recombinant MP-12 carrying the PKRDE7 ORF, rLuc-Flag or the NSs-Flag in the place of the NSs ORF was recovered as described previously [19] . Briefly, subconfluent monolayers of BHK/T7-9 cells were co-transfected with an S-genome RNA expression plasmid, such as pProT7-S(+)-PKRDE7, pProT7-S(+)-rLuc-Flag or pProT7-S(+)-NSs-Flag, and a mixture of pPro-T7-M(+), pPro-T7-L(+), pT7-IRES-vN, pCAGGS-vG, and pT7-IRES-vL using TransIT-LT1 (Mirus Bio Corporation). The culture medium was replaced with fresh medium 24 h later. At 5 days post-transfection, the culture supernatants were collected, clarified and then inoculated into VeroE6 cells. The supernatant of infected VeroE6 cells at 3 days post-infection was used for the experiment.
RVFV MP-12 or ZH501 NSs ORF, or CAT ORF were cloned downstream of the T7 promoter between the Kpn I and Xho I sites of the pcDNA3.1-myc-HisA (Invitrogen) plasmid. For rLuc RNA transcripts, the rLuc ORF in pRL-SV40 plasmid (Promega Corporation) was inserted downstream of the T7 promoter. Capped and polyadenylated RNA transcripts were synthesized in vitro by using mMESSAGE mMACHINE T7 Ultra (Ambion) according to the manufacturer's instructions [64] . One microgram of in vitrosynthesized RNA transcripts was transfected into 293 cells in a 12well plate with a TransIT-mRNA Transfection kit (Mirus Bio Corporation) according to the manufacturer's instructions.
VeroE6 cells in 6-well plate were infected with a series of diluted virus samples in 400 ml. After 1 h adsorption at 37uC, we removed the inocula and added 2 ml of MEM containing 0.6% noble agar (Difco Laboratories), 5% FBS and 5% tryptose phosphate broth. The cells were incubated at 37uC for 3 days. Then, 2 ml of MEM containing 0.6% agar, 100 mg of neutral red (N2889, Sigma), 5% FBS and 5% tryptose phosphate broth were added into the wells, and incubated for 16 h. The virus titers were determined in triplicate.
Cells were lysed in sample buffer and boiled for 10 min. Equal amounts of samples were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted onto polyvinylidene difluoride membranes (immobilon P; Millipore). Western blot was performed as described previously [27] . The following primary antibodies were used: anti-RVFV [27] ; anti-NSs [48] 
The IP-kinase assay was performed as described previously [65] . Briefly, 293 cells were mock-infected or infected with recombinant MP-12 at an moi of 3. Cells were dissolved in lysis buffer containing 10 mM Tris-HCl pH 7.6, 50 mM KCl, 2 mM Magnesium acetate, 10 mM 2-mercaptoethanol, 1% Triton X-100, 1 mM EDTA, phosphatase inhibitor cocktail (Sigma) and proteasome inhibitor cocktail (Roche). After centrifugation at 10,0006g for 5 min, a cytoplasmic fraction was collected. Cytoplasmic lysates were subjected to immunoprecipitation with anti-PKR antibody (Santa Cruz Biotech, K-17). Protein A beads that bound to the immunoprecipitated PKR were washed twice with a buffer-A containing 20 mM Tris-HCl, pH 7.6; 50 mM KCl; 400 mM NaCl; 5 mM 2-mercaptoethanol; 1% Triton X-100; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma); proteasome inhibitor cocktail (Roche); and 20% glycerol. The beads were further washed twice with buffer-B containing 20 mM Tris-HCl, pH 7.6; 100 mM KCl; 5 mM 2-mercaptoethanol; 1% Triton X-100; 0.1 mM EDTA; proteasome inhibitor cocktail (Roche); and 20% glycerol. Then, washed beads were resuspended in 26 kinase buffer containing 30 mM Hepes-KOH, pH 7.4; 2 mM dithiothreitol; 2 mM MgCl 2 ; proteasome inhibitor cocktail (Roche); and 10 mCi of [c-32 P] ATP (MP Biomedicals). The suspension (20 ml) was incubated at 30uC for 20 min, and then 26 SDS sample buffer was added to terminate the reaction. The samples were separated on 10% SDS-PAGE gel and visualized on an autoradiograph. A portion of the samples were also used for Western blot analysis by employing anti-PKR monoclonal antibody (BD biosciences) to show the abundance of immunoprecipitated PKR.
For the radiolabelling of host and viral proteins in infected cells, VeroE6 cells were incubated at 14.5 h post-infection for 30 min at 37uC with medium made up with MEM lacking methionine, cystine, and L-glutamine (M2289, Sigma); 1% dialyzed FBS (Invitrogen); 20 mM L-glutamine; penicillin (100 U/ml) and streptomycin (100 mg/ml). Then, Trans[ 35 S]label metabolic reagent (MP biomedicals) was directly added into the medium (100 mCi/ml). After 1 h labelling, cells were washed with PBS once and lysed in sample buffer. Equal amounts of samples were subjected to SDS-PAGE in 10% polyacrylamide gel. The gel was dried and exposed to X-ray film (KODAK BioMax XAR) overnight at 280uC. For the radiolabelling of PKR, 293 cells were mock-treated or transfected with pcDNA3.1-Myc-PKRK296R, and labelled with Trans[ 35 S]label metabolic reagent between 14 and 16 h post-DNA transfection. In some samples, cell extracts were prepared with chase using a lysis buffer. In other samples, cells were transfected with in vitro-synthesized RNA transcripts encoding rLuc or MP-12 NSs. Then, cells were washed, and incubated for 8 h in the presence of 2 mM methionine/ cysteine. At 8 h post-RNA transfection, cell extracts prepared using lysis buffer, were employed for immunoprecipitation with anti-Myc antibody (Santa Cruz: sc-40), as described in an IPkinase assay. Immunoprecipitated samples were separated on a 7.5% poryacrylamide gel and visualized on an autoradiograph. dsRNA-binding assay 293 cells were mock-infected or infected with rMP12-rLuc-Flag, rMP12-NSs-Flag or rMP12-PKRDE7 at an moi of 3. Alternatively, 293 cells were transfected with in vitro-synthesized RNA transcripts encoding MP-12 NSs. The cytoplasmic lysate was harvested at 16 h.p.i. or 16 h post-transfection, and incubated with poly C beads or poly I:C beads on ice for 45 minutes. After washing beads with buffer-A for 4 times, the beads were mixed with 26 sample buffer, and bound proteins were analyzed with anti-Flag antibody (Sigma) on a Western blot.
The luciferase assay was performed on a Renilla Luciferase Assay System (E2810, Promega Corporation) according to the manufacturer's instructions.
Total RNA was harvested by Trizol reagent (Invitrogen), and Northern blot was performed as described previously [27, 64] . Briefly, total RNA was denatured and separated on 1.2% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane (Nylon Membrane, positively charged, Roche). Northern blot analysis was performed by using strand-specific RNA probes for detecting IFN-b mRNA [64] , GAPDH mRNA [64] , rLuc mRNA [27] or RVFV antiviral-sense S-segment / N-mRNA [48] .
293 cells in 6-well plates were transfected with 2 mg of in vitrosynthesized RNA transcripts encoding rLuc or MP-12 NSs by TransIT mRNA transfection kit (Mirus). Cells were mock-treated or treated with ActD (5 mg/ml) immediately after RNA transfection. Total RNA were extracted by using an RNeasy Mini kit (Qiagen) at 16 h post-transfection. For each sample, we used 500 ng of RNA to synthesize 1 st strand cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Real-Time PCR was performed at the Real-Time PCR core facility, Sealy Center for Cancer Cell Biology, UTMB. We used an Applied Biosystems made-to-order 206 assay mix of primers and TaqMan MGB probes (FAM dye-labled) for our target gene PKR (Applied Biosystems: assay ID#: Hs01091592_m1) and predeveloped an 18S rRNA (FAM-dye labelled probe) TaqMan assay reagent (Applied Biosystems: 4352930E) for endogenous control. Separate tubes (singleplex) real-time PCR was performed with 40 ng cDNA for both target gene and endogenous control by using a Taqman Gene expression master mix (Applied Biosystems: 4370074). The cycling parameters for real-time PCR were: UNG activation at 50uC for 2 min, AmpliTaq activation at 95uC for 10 min, denaturation at 95uC for 15 sec, and annealing/extension at 60uC for 1 min (repeat 40 times) on ABI7000. Duplicate C T values were analyzed by the comparative C T (DD C T ) method, as described by the manufacturer (Applied Biosystems). The amount of target (2 2DDCT ) was obtained by normalized to endogenous reference (18S rRNA) and relative to a calibrator (one of the experimental samples). Figure S1 Effects of ActD treatment on host translation activities. (A) 293 cells cultured in 10-cm dishes were treated with 5 mg/ml of ActD (ActD) or were left untreated (Mock). Cells were harvested at 16 h post-ActD-treatment in 900 ml of Lysis buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 1% Triton X-100; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin) on ice for 5 min. The cytoplasmic lysates were collected after the removal of nucleus by centrifugation at 10,0006g for 5 min. The lysates were loaded onto a 10-50% linear sucrose gradient containing 50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 0.5 mM dithiothreitol; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin, and centrifuged at 38,000 rpm for 3 h at 4uC using a Beckman SW41 rotor. The gradients were pumped by syringe pump (Brandel) and analyzed by a density gradient fractionator (Brandel) connected to an ISCO UA-6 (ISCO Inc.) at the absorbance of 254 nm according to the manufacturer's instructions. The data were representative of two independent experiments. (B) 293 cells were transfected with in vitrosynthesized rLuc RNA transcripts and mock-treated (no drug) or immediately treated with 5 mg/ml of ActD (ActD) or 50 mg/ml of a-amanitin (Amanitin). Luciferase activities were measured at 16 h post-transfection. The data shown in the graphs (mean+/ 2standard deviation) were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s001 (4.02 MB TIF) Figure S2 Effects of different concentrations of ActD or aamanitin on eIF2a phosphorylation and N protein accumulation in rMP-12-infected cells. VeroE6 cells were infected with rMP12-rLuc at an moi of 3, and then treated with different concentrations of ActD or a-amanitin. Cell extracts and culture supernatants were harvested at 16 h.p.i. Note that ActD suppresses about 80% of RNA polymerase I activity at 0.04 mg/ml, and about 80% of RNA polymerase II and III at 4.0 mg/ml [28] , while a-amanitin suppresses nearly 100% of RNA polymerase II and about 50% of RNA polymerase III at 50 mg/ml [64] . (A) Western blot analysis of N protein, phosphorylated eIF2a, total eIF2a and a-actin in each cell extract. (B) Top panels represent the relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the untreated cells represents 100%. The middle panels and the bottom panels represent the abundance of N protein and the virus titers, respectively. The results were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s002 (4.33 MB TIF) Figure S3 Effects of pan-caspase inhibitor Z-VADfmk on PKRmediated eIF2a phosphorylation in infected cells. VeroE6 cells were mock-infected (Mock) infected with rMP12-rLuc (rMP12-rLuc) and then treated with 5 mg/ml ActD (Act) or 50 mg/ml of aamanitin (Ama) or mock-treated (M) in the presence or absence of 100 mM of Z-VADfmk. Cells and culture supernatants were harvested at 16 h.p.i. (A) Western blot analysis of eIF2a, phosphorylated eIF2a, cleaved caspase 3 using anti-Cleaved Caspase 3, Asp175, antibody (Cell Signaling Tech. #9661), and a-actin in the absence (Top) and presence (Bottom) of Z-VADfmk. The data are representative of three independent experiments. (B) The relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the mock-infected, mock-treated cells represents 100%. The average and standard deviation from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). (C) Virus titers of rMP12-rLuc from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). Found at: doi:10.1371/journal.ppat.1000287.s003 (2.29 MB TIF) Influenza Virus (H5N1) in Live Bird Markets and Food Markets, Thailand A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006–August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004–2005. Infl uenza virus (H5N1) was identifi ed in 12 of 930 samples tested. In November 2006, a total of 5 samples with infl uenza virus (H5N1) were isolated from 1 healthy chicken and 4 visceral organs obtained from 1 live bird market (chicken) and 3 different food markets (moor hen, water cock, and quail). In December 2006, a total of 5 samples with infl uenza virus (H5N1) were isolated from 5 visceral organs (quail, water cock) from 1 food market. In January 2007, a total of 2 samples with infl uenza virus (H5N1) were isolated from 2 healthy ducks obtained from 1 live bird market. In the study, 7 isolates were sequenced for whole genome analysis, and the remaining 5 To analyze the isolates, nucleotide sequences were compared with those of infl uenza subtype H5N1 viruses in Thailand, People's Republic of China, Vietnam, Indonesia, Lao, Myanmar, and Cambodia. The sequences were aligned by using the DNASTAR program (3) to elucidate and compare the genetic changes. Phylogenetic analysis was conducted by applying the PAUP program (4) with the neighbor-joining algorithm and using branch swapping and bootstrap analysis with 1,000 replicates.
In the course of the 14-month surveillance program, we isolated infl uenza virus (H5N1) from 12 samples from live birds and from bird meats obtained from the markets. Bird meats were the source of 9 virus-containing samples (5 quail, 2 moor hens, and 2 water cocks), which indicates a risk for infl uenza virus (H5N1) contamination in bird meats, especially quail. In addition, 3 highly pathogenic avian infl uenza viruses were isolated from healthy live poultry (1 chicken and 2 ducks). However, the samples that contained infl uenza virus subtype H5N1 were detected only during the 3-month winter season (November-January). A possible explanation for virus contamination in live bird and food markets may be animal movement from outbreak areas to the markets as well as an attempt to sell infected (dead or dying) birds, especially quail, as bird meat. In addition, most animals or meats in the markets came from backyard farms, where they were in unavoidably close contact with wild birds.
Phylogenetic analysis of the virus HA and NA genes indicated that all 12 subtype H5N1 isolates were part of the Vietnam and Thailand lineage (clade 1). The viruses were closely related to those investigated in Thailand (2004) (2005) as well as to other subtype H5N1 isolates in clade 1. In contrast, they differed from infl uenza subtype H5N1 viruses of the south China and Indonesia lineages (clade 2) (Figure 2) . In this study, we did not discern any Thailand isolates closely related to the south China lineage, as previously established in Lao and Cambodia (5) . Phylogenetic analysis of 6 remaining genes showed them to be also closely related to the Vietnam and Thailand isolates.
Analysis of the deduced amino acid sequences of the HA and NA proteins indicated that the viruses had characteristics of highly pathogenic avian infl uenza. The HA cleavage site consists of multiple basic amino acids RE-RRRKKR (in 1 isolate, CU-329, REKRRKKR). All infl uenza subtype viruses harbor Glu-222 and Gly-224 at the receptor binding site, indicating preferential binding to the avian receptor SA-α-2, 3-Gal. In addition, the virus sequences contain 7 glycosylation sites as previously identifi ed in most isolates from Thailand (6) . A glycosylation site adjacent to receptor binding sites may help increase virus infectivity in host cells (7) . In some isolates, polymorphisms of amino acids related to antigenic properties of the viruses at position V86A, L138Q, and K140N were observed. All 12 subtype H5N1 viruses had a 20-aa deletion in the NA protein, typical for the NA stalk region of recent subtype H5N1 isolates (2003) (2004) (2005) (2006) (2007) (8, 9) . None of the subtype H5N1 isolates had any amino acids indicating oseltamivir resistance at the crucial positions 119 (E), 275
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 11, November 2008 (H), 293 (R), and 295 (N) of the NA protein. In summary, the 12 viruses isolated from this study were similar to the viruses from other sources in Thailand, which indicates that the viruses are endemic to Thailand, are circulating in the country, and can be found in any exposed species. The route of infl uenza virus (H5N1) introduction into the markets remains to be established. We suspect that this contamination might have occurred as a consequence of animal movement from outbreak areas or from viruscontaminated cages, trucks, and equipment. Unfortunately, the original sources of animals in the markets could not be identifi ed because birds from different sources were housed in 1 or several cages. Fortunately, no human infection was found during 2007-2008 in those provinces where the viruses were isolated.
It has been known that live bird and wet markets play a major role in facilitating emergence or reemergence of infl uenza and some other respiratory diseases (10) (11) (12) . Moni-toring of live bird and food markets as an early warning system should be implemented in Asian countries where such markets are still commonplace, and routine surveillance of these markets should be conducted year-round. In addition, raw bird meats should be handled with caution, and consumption of raw bird meats should be avoided. Increased public awareness about the risks for infl uenza virus (H5N1) in association with live bird and food markets will help prevent and control subtype H5N1 infection in humans. Figure 2 . Phylogenetic analysis of the hemagglutinin (A) and neuraminidase genes (B) of infl uenza virus (H5N1) isolates. Phylogenetic trees were generated by using the PAUP computer program (4) and applying the neighbor-joining algorithm with branch swapping and bootstrap analysis with 1,000 replicates. The trees were rooted to A/goose/China/Guangdong/1/96 (H5N1). Antimicrobial and antioxidant activities of Cortex Magnoliae Officinalis and some other medicinal plants commonly used in South-East Asia BACKGROUND: Eight medicinal plants were tested for their antimicrobial and antioxidant activities. Different extraction methods were also tested for their effects on the bioactivities of the medicinal plants. METHODS: Eight plants, namely Herba Polygonis Hydropiperis (Laliaocao), Folium Murraya Koenigii (Jialiye), Rhizoma Arachis Hypogea (Huashenggen), Herba Houttuyniae (Yuxingcao), Epipremnum pinnatum (Pashulong), Rhizoma Typhonium Flagelliforme (Laoshuyu), Cortex Magnoliae Officinalis (Houpo) and Rhizoma Imperatae (Baimaogen) were investigated for their potential antimicrobial and antioxidant properties. RESULTS: Extracts of Cortex Magnoliae Officinalis had the strongest activities against M. Smegmatis, C. albicans, B. subtilis and S. aureus. Boiled extracts of Cortex Magnoliae Officinalis, Folium Murraya Koenigii, Herba Polygonis Hydropiperis and Herba Houttuyniae demonstrated greater antioxidant activities than other tested medicinal plants. CONCLUSION: Among the eight tested medicinal plants, Cortex Magnoliae Officinalis showed the highest antimicrobial and antioxidant activities. Different methods of extraction yield different spectra of bioactivities. Some medicinal plants used in traditional Chinese medicine are effective in treating various ailments caused by bacterial and oxidative stress. As new drug-resistant bacteria strains emerge, especially methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, new drugs or adjuvants have been actively searched in medici-nal plants [1] [2] [3] . New antioxidants such as plant phenolics [4] [5] [6] [7] are sought for general health maintenance, antiaging and chemoprevention.
Eight medicinal plants, namely Herba Polygonis Hydropiperis (Laliaocao), Folium Murraya Koenigii (Jialiye), Rhizoma Arachis Hypogea (Huashenggen), Herba Houttuyniae (Yux-ingcao), Epipremnum pinnatum (Pashulong), Rhizoma Typhonium Flagelliforme (Laoshuyu), Cortex Magnoliae Officinalis (Houpo) and Rhizoma Imperatae (Baimaogen) were tested for their potential antimicrobial and antioxidant properties. They have been long been used in treating of various infectious diseases, e.g. skin/wound infections, fever, cough and digestive ailments ( Table 1 , ).
The traditional method for Chinese medicine preparation is to boil the medicinal plants in water for 20 minutes to one hour. The present study aims to test the effectiveness of traditional herb preparation methods for antimicrobial and antioxidant treatments.
The rationales behind the selection of these eight plants are as follows. (1) They are commonly used in Asia. (2) They have long been used as medicinal plants. ( Reducing halitosis [11] , antioxidant [12] , antimicrobial [13] , antifungal [14] , antihyperglycemic and antihyperlipidemic properties [ [17] . Prevention of urinary infection, modulation of neutrophils and monocytes, inhibition of respiratory bacteria [18, 19] . Anti-inflammatory activity [20] . (Table 1) .
magnolol, honokiol (99.9%) and quercetin were purchased from Sigma Aldrich (USA).
Absolute ethanol (99.9%, Far East Distiller, Singapore) was diluted with water to produce 80% (v/v) solution of ethanol for extraction. De-ionized water was used for extraction (by boiling and maceration), reconstitution and dilution where appropriate. Methanol (analytical grade, Tedia, USA) was used for reconstitution and dilution in the DPPH assay.
Four strains of bacteria and one strain of yeast were used for antimicrobial tests. The test bacteria included Grampositive Staphylococcus aureus (ATCC 6538P) and Bacillus subtilis (ATCC 6633), Gram-negative Pseudomonas aeruginosa (ATCC 9027) and acid-fast Mycobacterium smegmatis (ATCC 14468). Candida albicans (ATCC 2091) was used as a representative of yeast. All microorganisms were purchased in the form of inoculation loops from Oxoid (UK). Nutrient broth with agar and Sabouraud dextrose agar (Acumedia, USA) were used for the cultivation of bacteria and yeast respectively. Mueller Hinton agar (France) was used in antimicrobial screening.
Standard antibiotic discs (diameter 6 mm) used in this study were: methicillin 5 μg, tetracycline 30 μg, carbenicillin 100 μg and streptomycin 10 μg. In our preliminary studies, these antibiotics were found to be active against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Mycobacterium smegmatis respectively. All standard antibiotic discs were purchased from Oxoid (UK). Disc containing chlorhexidine which was active against Candida albicans, were prepared by loading dry sterile filter paper discs (Whatman No. 54, diameter 5.5 mm) with chlorhexidine solution to give a total weight of approximately 100 μg of chlorhexidine per disc. The impregnated discs were dried overnight at 40°C and stored (less than five days) in a desiccator until use.
The fresh plants were kept in a refrigerator for no longer than three days prior to extraction. Cortex Magnoliae Officinalis was dried in a cool, dark room (room temperature 19°C, relative humidity 60%) and subsequently stored in a drum with silica gel desiccants until use. Before extraction, the plants were cut into 1 cm pieces with pruning scissors, except Rhizoma Imperatae and Cortex Magnoliae Officinalis which were milled into fine powder using a pulverizer mill (Christy & Norris, UK). Triplicate preparations of each sample were carried out.
Boiling, maceration and blending Two and a half grams of Folium Murraya Koenigii, Typhonium flagelliforme aerial parts and 5 g of the other plant materials, were each extracted with 200 ml of water or ethanol. Three extraction methods were employed: (1) boiling in water for 1 hour, (2) maceration for 24 hours in water or (3) 80% (v/v) ethanol at room temperature. Herba Houttuyniae was extracted using an additional extraction method that involved boiling in water for 20 minutes [36] . Additional extraction experiments were carried out on aqueous plant extracts that showed promising antimicrobial activities. Boiling time was limited to 20 minutes to minimize heat exposure. Blending-maceration was used as a non-heat extraction method with cell rupture mechanism. Blending was performed with a laboratory blender (Waring Commercial, USA) for one minute, followed by a pause and then blending for an additional minute. Maceration in de-ionized water for one hour was performed. Coarse particles were removed using Whatman No. 1 filter paper (Whatman International, UK) before evaporation.
Fresh juices of Herba Houttuyniae, Epipremnum pinnatum stem and Typhonium flagelliforme aerial parts and rhizomes were prepared in a mortar, wrapped in linen cloth and squeezed for the juices. Coarse particles were removed using Whatman No. 1 filter paper before evaporation.
The plant extracts were evaporated to dryness under reduced pressure at 40°C for ethanol extracts and 60°C for water extracts and fresh juices in a rotary evaporator (Model N1000, Eyela, Japan). The solid content of the extract was weighed. The dried extracts were stored in a freezer at -20°C.
The crude and dried extracts were characterized by their odor, appearance and texture. The weights of the dried extracts were also determined.
Preparation of extract-and standard-loaded discs Filter paper discs (Grade 54, diameter 5.5 mm, Whatman International, UK) were autoclaved at 121°C for 20 minutes and oven-dried at 40°C overnight. Plant extracts were diluted with the same extraction solvent to 50 μg/μl. Each diluted solution (2 μl, equivalent to 100 μg of the dried extract) was loaded on a sterile filter paper disc. All impregnated discs were dried in sterile glass Petri dishes placed in an oven at 40°C overnight. The discs were then allowed to condition to room temperature before use in the antimicrobial test. Solutions in methanol (5 μg/μl) were prepared for magnolol and honokiol respectively and a 1:1 solution of the two compounds (2.5 μg/μl) was made. 2 μl of the honokiol, magnolol or 1:1 solutions were loaded onto paper discs which were then left to airdry. These standard-loaded discs were freshly prepared before the antimicrobial screening experiments.
The antimicrobial activities of the extracts were determined by the Kirby-Bauer agar diffusion method according to NCCLS standards [37, 38] . Sterilized molten agar (20 ml) was dispensed to each sterile disposable Petri dish (diameter 9 cm) and allowed to solidify. Mueller Hinton agar was used for bacteria and Sabouraud dextrose agar for yeast. Microbial suspension (200 μl) containing approximately 3 × 10 6 CFU was spread evenly onto the surface of the solidified medium. The plates were allowed to dry for 15 minutes before the test discs were placed at equidistance from each other. Each plate consisted of one standard antibiotic disc and three other discs impregnated with various extracts.
After standing for 30 minutes, the Petri dishes were incubated in an inverted position at 37°C for 18 to 24 hours for bacteria and 24°C for 48 to 72 hours for yeasts. The diameters of the zone of inhibition (ZIH), defined by the clear area devoid of growth, was measured twice. The antimicrobial activities were determined by the ratio of the ZIH diameters of the extracts to that of the standard anti-biotic in the same Petri dish, whereby a higher ratio indicates a more potent extract.
Antioxidant activities of the extracts were determined with 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay [39] . The free radical, DPPH, served as the model oxidizing agent to be reduced by the antioxidant present in the extracts. The amount of dried extract subject to DPPH assay was 100 μg, the same amount used for antimicrobial screening.
The dried extract was dissolved in 1.56 ml of methanol and mixed with 40 μl of 2 mM DPPH dissolved in methanol to make up a total volume of 1.6 ml in each polyethylene microfuge tubes. The final solution was allowed to react in dim light for 15 minutes. It was then centrifuged (4000 rpm; 1165 × g, Kubota 2100 Centrifuge, Japan) for five minutes. The absorbance of the supernatant was measured at 517 nm with a UV spectrophotometer (Genesys 10 UV, ThermoSprectronic, USA). The tests were carried out in triplicates. The DPPH radical scavenging activity was calculated with the following formula:
Where A 0 is the absorbance of the control solution containing only DPPH after incubation; A 1 is the absorbance in the presence of plant extract in DPPH solution after incubation; and A s is the absorbance of sample extract solution without DPPH for baseline correction arising from unequal color of the sample solutions (optical blank for A 1 ).
Data are expressed as mean ± standard deviation (SD) of triplicates. Two-way ANOVA was used to analyze the effect of different plant materials and extraction methods on the extraction yields and DPPH radical scavenging activity while one-way ANOVA was performed to determine the effect of streptomycin, honokiol, magnolol and honokiol-magnolol combination on M. smegmatis. Both tests employed Bonferroni post hoc analysis. Student's ttest was used to compare antimicrobial activity of the extracts against the standard antibiotic. All statistical analyses were conducted with SPSS software (v.12, SPSS, USA) at a significance level of 0.05.
The extraction yields obtained from different extraction methods were analyzed with two-way ANOVA and Bonferroni post hoc analysis. Among the 11 experimental groups, Rhizoma Imperatae produced the highest yields (P = 0.001) regardless of extraction methods, followed by Cortex Magnoliae Officinalis (Figure 1 ). These two dry herbs were processed through comminution producing fine powder prior to extraction. The reduced particle size decreases the internal mass resistance for compounds to traverse through the plant matrix and increases the specific surface area for extraction. The extraction yields obtained from boiling were higher than those from other extraction methods.
Boiling Herba Houttuyniae aerial parts in water for 20 minutes or one hour produced comparable yields (P = 1.000). For Herba Polygonis Hydropiperis, Folium Murraya Koenigii and Cortex Magnoliae Officinalis, a shorter boiling time of 20 minutes was shown to be comparable to a boiling time of 60 minutes (P = 0.061, 0.053 and 0.798 respectively). While results from blending/maceration varied, this
Solid content of extracts obtained by different methods* Figure 1 Solid content of extracts obtained by different methods*. *Error bars represent standard deviation (n = 3).
method was as efficient as the boiling method in terms of solid yields (P = 0.261) of Folium murraya koenigii.
The color, texture and odor of the plant extracts were characterized (Additional file 1). The ethanolic extracts were better than corresponding aqueous extracts in retaining the natural fragrances of the plants. This may be due to the preservative ability of ethanol (i.e. reducing breakdown of organic compounds by microorganisms), its enhanced extraction capability (i.e. more fragrant components extracted) or a combination of both. Extracts obtained by boiling generally appeared darker and more turbid than those obtained by maceration. The solid content by boiling was higher than that by maceration (Figure 1 ). Boiling is more likely to damage the plant cell membrane and cell wall which act as natural filters to keep larger extraneous compounds within the cell.
Among all the extracts studied, the 100 μg of the ethanolic extract of Cortex Magnoliae Officinalis loaded on the filter paper disc demonstrated the most robust antimicrobial activities against S. aureus, B. subtilis, M.smegmatis and C. albicans, equivalent to at least 50% of the activities of the standard antibiotics. Among the test organisms, it was most active against M.smegmatis, 20% more than the standard antibiotic, streptomycin 10 μg (Student's t-test, P = 0.001) ( Table 2 ). The boiled extract of Cortex Magnoliae Officinalis had comparable antimicrobial activities to those of streptomycin 10 μg (Student's t-test, P = 0.279). These data suggest that Cortex Magnoliae Officinalis may be a potential agent to treat infections caused by M. smegmatis and Mycobacterium tuberculosis [40] . It was reported that magnolol and honokiol exhibited antibacterial activities against methicillin-resistant S. aureus and vancomycinresistant enterococci [33] , Propionibacterium sp [32] and periodontal pathogens [34] . Therefore, disk diffusion test was carried out on magnolol and honokiol individually and in combination ( Table 2 ). The one way ANOVA on the four treatment groups namely streptomycin, honokiol, magnolol and combination of magnolol and honokiol (1:1) demonstrated a significant difference between groups (P = 0.001). Bonferroni post-hoc test showed that honokiol and magnolol had comparable activities (P = 1.000) against M. smegmatis, accounting for 83.58 ± 3.06% (P = 0.015) and 82.09 ± 6.51% (P = 0.006) of those of Streptomycin 10 μg respectively. In terms of antibacterial activities, the combination of magnolol and honokiol (1:1) was comparable to the reference antibiotic (P = 1.000) but higher than either magnolol (P = 0.007) or honokiol (P = 0.017) alone. These results suggest a new discovery of synergism between magnolol and honokiol.
Ethanolic extract of Folium Murraya Koenigii and boiled extract of Herba Polygonis Hydropiperis showed 80% and 50% of the activities of streptomycin 10 μg against M. smegmatis respectively. These extracts also exhibited antimicrobial activities against S. aureus and B. subtilis. Additionally, the boiled extract of Herba Polygonis Hydropiperis was active against C. albicans. Boiling was essential for the active principles to be removed from the laksa plant, as blended and water macerated extracts showed little antimicrobial activities ( Table 3 ). The duration of the boiling process also affected the antimicrobial activities of laksa plant, whereby herbs boiled for 20 minutes were more active against S. aureus and M. smegmatis. The aerial parts of Herba Houttuyniae and rodent tuber were only active against B. subtilis and S. aureus respectively. The leaves and Rhizoma Arachis Hypogea, Rhizoma Imperatae, Rhizoma Typhonium Flagelliforme, and the leaves and stems of Epipremnum pinnatum did not show any antimicrobial activities.
An extract with a high yield, however, does not necessarily have high antimicrobial activities. For example, Rhizoma Imperatae whose yields topped all extraction methods, did not show any antimicrobial activities ( Figure 1 and Table  3 ).
The fresh juices of Herba Houttuyniae aerial parts, Epipremnum pinnatum stems and Rhizoma Typhonium Flagelliforme were tested for their folkloric use to treat wounds and various skin ailments (Table 1 ). All these fresh juices displayed some activities (less than 30% of the activity of methicillin 5 μg) against S. aureus. However, they were inactive against the rest of the test organisms. While the yields of fresh juices were lower than those of other extraction methods, antibacterial activities against S. aureus implied reduced degradation of the bioactive principles.
Among all extracts, only the fresh juices of Rhizoma Typhonium Flagelliforme and Epipremnum pinnatum leaves and stems possessed antimicrobial activities ( None of the extracts, however, inhibited Ps. aeruginosa. Both S. aureus and B. subtilis are Gram-positive, while Ps. aeruginosa is Gram-negative and has an outer lipid membrane [41] . The results suggest that the antimicrobial compounds in the extracts were unable to penetrate this lipid membrane to exert their effects inside a cell. This speculation will require further experiments to confirm.
The antioxidant activities of the dried extracts and fresh juices are presented in Figure 2 . All tested plants possessed some DPPH radical scavenging activities to a certain extent. While Cortex Magnoliae Officinalis, stems and leaves of dragon tail, laksa aerial parts, Herba Houttuyniae aerial parts and curry leaves showed high activities, rodent tuber rhizomes and aerial parts showed low activities.
The high antioxidant activities of the boiled and ethanolic extracts of the leafy materials were probably due to the extracted tannins and photosynthetic pigments. Cortex Magnoliae Officinalis is a rich source for antioxidative compounds, such as biphenols, polyphenols and tannins [42, 43] . Lo et al. found that the antioxidant effects of magnolol and honokiol isolated from Cortex Magnoliae Officinalis were 1000 times higher than those of alphatocopherol [44] . Earlier studies confirmed that several naturally occurring dietary phytochemicals, such as isothiocyanates, curcumin and Epigallocatechin-3-gallate, possessed cancer preventive properties [45, 46] .
Boiled extracts showed greater antioxidant activities than those of other extraction methods (P = 0.001). Antioxidant compounds in leafy materials are generally located in conduit structures called the apoplast and symplast [47] [48] [49] . Maceration alone is not sufficient to extract these compounds from the structures. The application of heat, in the boiling process, facilitates cell rupture and leaching,
Antioxidant activities of extracts tested by DPPH assay* Figure 2 Antioxidant activities of extracts tested by DPPH assay*. *Error bars represent standard deviations (n = 3).
thereby improving the mass transfer of these compounds from the storage organs into the boiling water. Ethanol may partially solubilize the membranes of the plant cells and storage organs, helping leach the chemicals away. However, maceration in 80% ethanol took over 24 hours and exposed the extracts to oxidative and hydrolytic degradation. This may explain the relatively low antioxidant activities of some ethanolic extracts.
The extracts of Cortex Magnoliae Officinalis, Herba Houttuyniae aerial parts and Folium Murraya Koenigii (ethanolic extract) had similar high DDPH radical scavenging activities (>85%) but markedly different antimicrobial properties ( Figure 2 and Table 3 ). The results suggest that the active components for antimicrobial and antioxidant activities do not share common biochemical pathways.
The present study discovered that (a) the ethanolic extract of Cortex Magnoliae Officinalis had 20% greater antimicrobial activities against M. smegmatis than streptomycin; (b) the boiled extract of Cortex Magnoliae Officinalis demonstrated comparable activities to streptomycin (c) the synergism of magnonol and honokiol had comparable effects to those of streptomycin; (d) the aerial parts of rodent tuber had antimicrobial activities against S. aureus. Among the tested 107 extracts, Cortex Magnoliae Officinalis had (1) potent antimicrobial activities against S. aureus, B. subtilis, M. smegmatis and C. albicans and (2) highest antioxidant activities in DPPH assay regardless extraction methods. Cortex Magnoliae Officinalis is likely a potential medicinal plant resource for developing effective antimicrobials and antioxidants. Results From a Hypothesis Generating Case-Control Study: Herpes Family Viruses and Schizophrenia Among Military Personnel Background: Herpes family viruses can cause central nervous system inflammatory changes that can present with symptoms indistinguishable from schizophrenia and therefore are of interest in schizophrenia research. Most existing studies of herpes viruses have used small populations and postdiagnosis specimens. As part of a larger research program, we conducted a hypothesis-generating case-control study of selected herpes virus antibodies among individuals discharged from the US military with schizophrenia and pre- and postdiagnosis sera. Methods: Cases (n = 180) were servicemembers hospitalized and discharged from military service with schizophrenia. Controls, 3:1 matched on several factors, were members not discharged. The military routinely collects and stores members' serum specimens. We used microplate enzyme immunoassay to measure immunoglobulin G (IgG) antibody levels to 6 herpes viruses in pre- and postdiagnosis specimens. Conditional logistic regression was used, and the measure of association was the hazard ratio (HR). Results: Overall, we found a significant association between human herpes virus type 6 and schizophrenia, with an HR of 1.17 (95% confidence interval [CI] = 1.04, 1.32). Women and blacks had significant negative associations with herpes simplex virus type 2 and cytomegalovirus; among blacks, there was a significant positive association with herpes simplex virus type 1. Among men, there was a HHV-6 temporal effect with an HR of 1.41 (95% CI = 1.02, 1.96) for sera drawn 6–12 months before diagnosis. Discussion: Findings from previous studies of herpes family viruses and schizophrenia have been inconsistent. Our study is based on a larger population than most previous studies and used serum specimens collected before onset of illness. This study adds to the body of knowledge and provides testable hypotheses for follow-on studies. Recent research has focused on infectious agents as potential players in the etiologic pathway of chronic diseases, [1] [2] [3] including psychiatric illnesses such as schizophrenia. [4] [5] [6] [7] [8] [9] [10] Due to their potential neurotropism and latency, viral organisms in particular are considered possible agents in many chronic central nervous system (CNS) disorders. 2, [11] [12] [13] Encephalitis and other conditions leading to CNS inflammatory changes often present with symptoms that are difficult to distinguish from new-onset schizophrenia. As ) are of prime interest in schizophrenia research. 14 Although laboratory-based research into herpes family viruses as possible etiologic agents for schizophrenia goes back decades, [15] [16] [17] [18] [19] [20] [21] [22] ascertaining the nature of a possible etiologic association between infection and schizophrenia is highly challenging. There have been few consistent findings between studies, which could be due to many factors, including the heterogeneity of schizophrenia itself, the use of different immunologic assays across the studies and over time as technology changes, focusing attention on a variety of different infections, and the possibility that maternal infection occurring at the right time during pregnancy may be enough to increase the risk of psychosis in offspring. 23 There is more information regarding risks associated with maternal, neonatal, or childhood infection and schizophrenia than for adult infection, with a number of studies reporting increased risk of schizophrenia among persons exposed in utero to a number of infections [24] [25] [26] or born after epidemic. 27 Little is known about potential mechanisms of action for herpes family virus infections and risk of schizophrenia.
Studies of maternal infection provide some evidence that modulation of immune response 28 and adverse effects on in utero maturation of critical brain structural and functional componenets 24 may correlate with increased risk of schizophrenia in offspring. From animal models, maternal infection can alter interleukin 6, interleukin 1b, or tumor necrosis factor a (TNF-a) in amniotic fluid or placenta and TNF-a in the fetal brain. 29 There is evidence that cytokines have an important function in the development of fetal neurons and glial cells, and abnormal levels of these (proinflammatory) cytokines may contribute to abnormal brain development, [30] [31] [32] [33] [34] at least in animals.
Findings from epidemiologic studies of herpes family viruses and schizophrenia among adults are mixed. In their 1995 review article, Yolken and Torrey 14 identified numerous published studies assessing viral (not just herpes family viruses) antibodies in serum of schizophrenia cases. Several of the studies were not interpretable because they lacked control groups or were evaluating changes in antibody titers over time. Of 21 interpretable studies, 3 reported positive significant associations with HSV-1 (or HSV unspecified), 35-37 1 study reported an association with EBV, 18 and 1 found a negative association with CMV. 38 The Military New-Onset Psychosis Project hypothesisgenerating studies afford unique opportunities to correct for some of the weaknesses identified in other studies of herpes viruses and schizophrenia. Because the US military routinely collects and stores serum samples and medical data from all active duty personnel, we are able to study large numbers of cases who have prediagnostic serum available and document the prevalence of infection prior to onset of disease. Because military members provide a serum sample at accession and about every 2 years thereafter while they remain on active duty, more than one prediagnostic sample will be available for many individuals. Sera from these routine samples are stored in the Department of Defense Serum Repository (DoDSR). 39 Informed consent is not routinely obtained when the serum is drawn; however, the DoDSR is maintained for surveillance and research purposes. Information obtained from this study may lead to more effective means of preventing, identifying, and treating new-onset schizophrenia in this population. This project was reviewed and approved by the appropriate human protection committees at the authors' institutions.
We assayed the serum of 180 individuals diagnosed with schizophrenia who had been hospitalized in a military facility with a mental health diagnosis and subsequently medically discharged or retired from the military between 1992 and 2001 and the serum of 532 controls with no evidence of any mental illness. Diagnosis date for cases was estimated as the date of first mental health hospitalization, used as a proxy for onset. Controls were selected 3:1 to cases and matched on the following variables: date of birth (6 1 year), the corresponding case's accession date 6 6 months, sex, race (black, white, other), branch of military service, and the number of serum specimens available. We attempted to obtain the same number of specimens for cases and controls: the first available specimen (usually collected during the accession medical examination), a specimen collected in the 3-to 24-month period prior to the matched case's first mental health hospitalization, and the first available after the matched case's hospitalization. Additional details on study subject selection and inclusion, serum selection and shipping, and sources and types of ancillary data have been published. 40 Sera were assayed for 6 herpes family viruses: HSV-1, HSV-2, EBV, CMV, VZV, and HHV-6.
IgG antibody to human herpes viruses were measured using microplate enzyme immunoassay. Reagents were obtained from the following sources: HSV-1 and HSV-2 from Focus Diagnostics, Cypress, CA; HHV-6 from Advanced Biotechnologies, Columbia, MD; VZV and EBV nuclear antigen from IBL Laboratories, Minneapolis, MN; and CMV from Viro-Immun Labor Diagnostika, Oberursel, Germany.
Enzyme immunoassay consists of binding serum to solid-phase antigen and subsequent reactions with enzyme-labeled antihuman IgG and enzyme substrate. The amount of color generated by the enzyme substrate reaction was measured in optical density (OD) units with a microplate colorimeter. This method of analysis was selected because it allows for high throughput measurement of antibodies using a common platform and requiring only small amounts of sample. Samples were run on plates under code in matched groups in which case and control status was not identified.
All matched case-control samples were tested on the same plates, but over the course of the study, samples were assayed on over 21 different plates per agent. To control for potential systematic error introduced by plate-toplate variation and to ensure that observed differences in OD are due to differential expression (cases or controls) and not experimental artifacts, data were normalized using the robust median normalization method which combines the within-plate and between-plates variance 41 using the following equation,
where R ijk is the raw OD of ith subject's jth blood sample, M k is the median of all the control samples in plate k, V k
is the variance of R ijk of control samples in plate k, and V b is the variance of all R ijk between plates. Therefore, S ijk is the scaled score for the ith subject's jth blood sample on plate k. To investigate the relationship between subject status (case or control) and antibody level in a matched study design, conditional logistic regression is used. Failure to account for matching in analysis may lead to biased results, usually toward the null. The conditional analysis, which has a higher (less negative) log likelihood, suggests a better ''fit'' for this data. 42 A guiding concern in regression modeling is that the relationship between the independent and dependent variables (the latter assumed to be continuous) should be as inherently ''linear'' as possible; hence, OD was analyzed as a continuous, rather than dichotomous (positive vs negative) variable. This approach also preserves the power to detect a difference between cases and controls, particularly because infection with most of these herpes viruses is ubiquitous and differences cannot be detected based on prevalence data.
For this analysis, we chose the proportional hazards (PH) model for conditional logistic regression. 42 The PH model is similar to regular conditional logistic regression but modified to allow for multiple and variable numbers of controls per case and specimens per subject. Dummy ''survival time'' to diagnosis was generated so that all samples for a given case had the same event time with corresponding controls censored at a later time. Proportional hazard regression was then used to study associations, reported as hazard ratios (HRs), between ''survival time'' and the risk factors. Because the PH assumption might not be true for all the data, but may be true among specific demographic subgroups, we performed stratified analysis on several of the matched variables.
First, we assessed the overall antibody effect for each agent separately. The analysis was stratified by the matched factors with case or control status as the outcome. Variables analyzed included antibody level, the time from serum collection to the case's diagnosis, and time in service for both cases and controls. Logistic models were developed to assess the antibody effect for modeling the agents separately as well as simultaneously.
To study the homogeneity of the agent effect across demographic levels and time, interaction terms were also evaluated. Where an interaction with demographic factor was observed, separate models were developed to explore the possibility of effect modification by those factors. For homogeneity of the agent effect across the time, we studied the interaction with time to diagnosis. Time to diagnosis was categorized as follows: greater than 2 years, 1-2 years, 0.5-1 year, less than 0.5 year before diagnosis, and after diagnosis. The interaction with time to diagnosis shows the temporal effect of antibody level and describes the consistency of risk across time periods. The interactions of the agents with demographic factors show the heterogeneity of the agent effect by those factors.
Because we used scaled values to represent measured antibody level, there is no recognized unit with which to describe increasing or decreasing levels. In this case, we chose the SD as the unit of measure. All results are reported as HRs for each increase of 1 SD of antibody level. This method also allows comparison between the effects of different antibody agents on schizophrenia. The SD of antibody level for the 6 agents ranged from 0.10 for HHV-6 to 0.59 for HSV-2.
A total of 180 cases (contributing 404 serum specimens) and 532 controls (with 1180 specimens) were included in the study population. Eight cases could only be matched to 2 controls. Table 1 shows the distribution of cases and controls by demographic factors. Overall, about 83% were males, 49% were whites, 44% were blacks, over 57% were younger than 25 years, 10% were older than 35 years, about 12% were Hispanic, and over 56% were in the army. Approximately 35% of cases had greater than 3 years of military service.
We found that antibody levels for all 6 agents were only weakly correlated (data not shown), and therefore, 
No significant effect modification was noted for sex (male vs female) or age (<25 vs !25) for any of the infectious agents (P values > .10). A significant interaction effect with race (black vs white) was found for HSV-1 (P < .01) and for CMV (P = .03), suggesting that significant differences exist in these antibody effects between black and white cases. As seen in Hazard ratio expressed per 1 SD increase of each agent antibody level.
Our hypothesis-generating study found a statistically significant positive HR between HHV-6 and schizophrenia among men and between HSV-1 and schizophrenia among blacks discharged or retired from the military with a diagnosis of schizophrenia and a history of mental health hospitalization. A negative association with HSV-2 and CMV was noted among women and blacks. Blacks dominated the results for women. These findings should be interpreted with caution, however, because they are driven by a small number of subjects (n = 80 for black cases, n = 30 for female cases, n = 22 for black females) and may be the result of type I error. Further analysis is warranted with a larger sample size. No significant associations were observed for HSV-2, EBV, or VZV among men or women. No significant association was found among whites for any agent.
Our subanalysis of HHV-6 IgG levels by time period for males around diagnosis is obviously limited by the sample size. We conducted this analysis to replicate the analytic modeling in our previous, related work on Toxoplasma gondii IgG level and risk of schizophrenia. 40 The P values of .04 in this hypothesis-generating study warrant further evaluation in the hypothesis-testing phase of our research.
More recently, studies of antibody levels in serum and cerebrospinal fluid demonstrate mixed findings. One analysis of untreated subjects with recent-onset schizophrenia found increased IgG antibody levels to CMV, decreased levels of antibodies to HHV-6 and VZV, and no differences in antibody level to HSV-1 and -2 and EBV. 9 Several other studies of cerebrospinal fluid yielded conflicting results with some reporting increased CMV antibody titers 38, [43] [44] [45] while others demonstrate no association. [46] [47] [48] Increased levels of HSV-1 antibody were demonstrated in one group of schizophrenic patients compared with normal controls, and cases with higher levels of antibody also demonstrated decreased gray matter in 2 areas of the brain. 49 Another study noted that deficit schizophrenics were more likely to have antibodies to CMV than were nondeficit patients. 50 A recent review of the literature regarding CMV and schizophrenia identified a number of studies reporting more frequent infection or higher levels of antibody in serum or cerebrospinal fluid. 51 The authors noted that studies conducted in 1992 were all null but that the serum assays utilized had been complement fixation or other less sensitive methods. They note 3 unpublished studies (M. J. Schwarz and N. Mueller, S. Bachmann; J. Schrö der; and R. H. Yolken, unpublished data) in which patients with schizophrenia were more likely to have antibodies to CMV, or had higher levels of antibodies, than did the control subjects. One of these studies (F. B. Dickerson, C. Stallings, A. Origoni, J. J. Boronow, R. H. Yolken, unpublished data) was of 415 outpatients with schizophrenia and 164 matched controls, in which the odds ratio for CMV positivity was 2.1. The authors note that patients who were seropositive were more likely to be female, black, older, and less educated. Leweke et al 9 found that CMV IgG antibody levels, but not HSV-1, HSV-2, EBV, HHV-6, or VZV, were higher among patients with schizophrenia. 9 Given the limited amount of research reported and the discordant findings among the existing articles regarding herpes viruses and schizophrenia, interpretation of our findings is challenging. Recent work has implicated HHV-6 in acute 52 and chronic 53,54 neurologic diseases. We note the negative association with HSV-2 and CMV among women and blacks and the positive association with HSV-1 among blacks. Although speculative, and limited by sample size, there is a potential for underlying genetic differences that could explain some portion of the racial differences.
There are a number of factors that could potentially account for the discrepancies observed between the various reports above and the present study. These include but are not limited to differences in diagnostic criteria for schizophrenia, all cases were adult onset, different time frames of serum collection related to illness onset, differences in laboratory assays, and multiple vs single serum specimens. In addition, our sample was drawn from the military population that differs from the general US population in several important ways. The male to female ratio in the military is much higher than in the general population, making it difficult to achieve adequate power when analyzing females separately. Comprehensive medical screening prior to entry into the military creates a healthy worker effect in the population and skews our sample toward individuals with later onset of schizophrenia. Also, our cases are a convenience sample of individuals with schizophrenia in the military. A small degree of bias introduced by any of these factors could account for the significance and direction of our findings.
This study has 2 important strengths. First, we used cases that were diagnosed and discharged from the military after a careful evaluation process. 40 A record review conducted on a sample of study subjects demonstrated a high level of concordance between the diagnoses documented in the military records and those assigned by 2 psychiatrist reviewers. 55 Also, because military applicants receive an extensive administrative and medical examination prior to accession, are directly supervised while on active duty, and have access to health care, we assumed that cases were not psychotic on accession and that the onset of psychosis would generally result in a mental health hospitalization for an expedited psychiatric evaluation. This assumption was validated by the record review. 55 Therefore, we are confident that diagnostic misclassification is not a major source of error in our findings.
In addition, the current study obtained multiple (between 1 and 3) specimens for most subjects both prior to and after onset of illness in the matched case. The second specimen, collected in the 3-to 24-month period prior to first mental health hospitalization was chosen as preonset of psychosis. The ability to analyze longitudinal specimens may be important if illness alters behaviors in a way that could impact antibody levels or if medical treatment for illness changes antibody responses.
Although this hypothesis-generating study does not resolve the issue of diverse findings between studies, we feel that our study has advantages over other studies with our high degree of diagnostic validity, adequate numbers of appropriate controls, and multiple serum specimens. It is clear that additional studies are needed to clarify the many remaining questions, particularly regarding HHV-6, CMV, and HSV-1. As part of our broad research program, we will be conducting a much larger casecontrol study with approximately 1600 cases and 2100 controls. Although herpes family viruses will not be the primary focus of this follow-on study, we intend to further explore the associations identified in this hypothesisgenerating study.  SARS-CoV: Lessons for global health  On 16 November 2002, a man developed high fever and respiratory symptoms and was hospitalized and treated for pneumonia in Foshan, China (Zhao, 2007) . After several weeks in intensive care and subsequent spread of the mysterious respiratory disease to his wife and several relatives, he recovered and was released on the 8th of January 2003. In early December, a restaurant chef in Shenzhen developed a fever with flu like symptoms and while suffering a severe disease course was hospitalized in Heyuan, infecting seven hospital staff and a patient between the 16th and 28th of December. In parallel, multiple cities around Guangzhou in Guangdong Province were reporting sporadic cases of an atypical pneumonia between the end of December 2002 and through early January 2003, oftentimes in chefs or dealers of exotic animals in wet markets and their close contacts.
While this scenario may have been repeated in earlier years given the low seroprevelance rates noted in archived serum samples (Zheng et al., 2004) , it was the super spreader events that took place in the HZS-2 Hospital in Guangzhou that catalyzed the global severe acute respiratory syndrome (SARS) pandemic. Two brothers, working in restaurants and then a third individual were all hospitalized with severe acute respiratory symptoms (SARS) in HZS2 between the 18th and 30th of January, leading a pneumonia-related hospital outbreak on 31 January 2003. While each transmitted disease to family and health care workers, the third individual infected over 30 hospital members and patients during his 48 h stay in HZS2, 21 hospital staff after transfer to a second hospital HZS3 and about 23 relatives and friends. This key super spreader event allowed the epidemic to gain critical momentum. Another major turn of events occurred in Shanxi Province after a business woman traveled to Guangdong in late February. After returning home, she subsequently became sick. At the time, knowledge regarding the developing outbreak was not common, so she transmitted the disease to eight family members and five hospital staff in Shanxi. After being transfer to a Beijing Hospital on 1 March 2003 she became the index case for the outbreak in Beijing as well (Zhao, 2007) .
Global spread of the outbreak began on the 21st of February 2003, when a physician from the HZS2 Hospital traveled to "Hotel M" in Hong Kong. Although he did not attend atypical pneumonia patients in HZS2, his health quickly deteriorated and he was admitted to a hospital in Hong Kong on the 22nd. During his short stay at Hotel M, a super spreader event transmitted the disease to 16 guests who subsequently spread the disease to Toronto, Singapore, Vietnam and then beyond. The indication of a new global emerging virus respiratory threat was first reported by Dr. Carlo Urbani, MD in Vietnam to the WHO on February 28th, resulting in a global alert notification on March 12th. Tragically, Dr. Urbani became infected while treating patients and subsequently died from SARS infection, a fate shared by many other health care professionals who were engaged in treating patients suffering from this new disease. Importantly, Dr. Urbani's sacrifice focused global resources on the new epidemic disease, resulting in the rapid implementation of public health measures, diagnostic criterion, and the scientific research community which undoubtedly saved countless of lives globally (Peiris et al., 2004) .
Within a few weeks of the HZS2 super spreader event, atypical pneumonia had spread to 25 countries across five continents, infecting some 8000 people and resulting in 774 deaths before succumbing to aggressive public health measures in early July 2003. In addition to the large outbreaks widely recognized in cities in Singapore, Taiwan, China, and Canada, smaller communities were also affected. An individual visiting a sick relative in Canada contacted the disease during the second wave of the Toronto outbreak, returning to North Carolina in late May 2003. He developed symptomatic disease 1 day after working several days on a rural college campus in central North Carolina. After being confirmed as a SARS case, the University spent ∼$40-50k screening some 42 potentially exposed individuals. Amazingly, a mystery disease whose index case was identified 6 months earlier on the other side of the globe had arrived in rural North America. It is not surprising that the panic caused by the rapid spread of an unknown highly pathogenic, fatal respiratory virus is enormous and no community is exempt. Coupled with the current globalization of the world trade, it was inevitable that the economic impact would be extensive.
Between March and May of 2003, tourist arrivals in Asia dropped between 30 and 80%, with about half of all flights being canceled depending on the country and region. As word of the mystery illness spread, the Asian Equity Market Indices declined by 15% over a 4-week period. Ultimately, the World Bank reported a ∼2% loss of East Asian GDP in the second quarter of 2003 during the SARS outbreak. The Asian Development Bank placed the global economic loss at 59 billion USD with the bulk of these losses located from mainland China (17.9 billion) and Hong Kong (12 billion) while Canada suffered losses of more than 1 billion. Most financial losses were associated with disruptions of trade, travel, investment, interruption of product supply chains and behavior changes in consumers, rather than from medical costs or the loss of human life. For comparison sake, the bovine spongiform encephalopathy epidemic in the UK, the sporadic avian influenza virus cases, and the UK foot and mouth disease epidemic caused 10-13, 15-20 and 25-30 billion in economic losses, respectively.
The SARS-CoV has revealed a clear vulnerability in the global economy. As the world economy relies increasingly on manufacturing centered in Asia and elsewhere and the transport of those goods globally, pandemic outbreaks of disease have serious global consequences including the potential collapse of the Asian, European and US equity markets, interruptions of trade and travel, manufacturing stasis and decline and reduced availability of goods, foods and medicines. Given the current interconnection of the global marketplace and the speed of transcontinental travel, significant investments in basic and applied biomedical research are essential for maintaining the health of economies and the overall public health of the global community.
Emerging infectious diseases are clearly recognized as threats to human health and a significant public health problem. New emerging pathogens lists published prior to 2002 included HIV, influenza, the filoiviruses, hantaviruses, and various arboviruses, recognizing that many emerging pathogens were zoonotic in origin. Despite the recognition that new emerging viruses are difficult if not impossible to predict, standardized lists were developed based primarily on epidemiologic trends over the past ∼50 years. Another consistent theme was the near universal agreement that coronaviruses were not emerging pathogens, but rather were only associated with relatively benign upper respiratory tract infections in humans.
Because coronaviruses have relatively high mutation and RNA recombination rates and rapidly undergo cross species transmission events in vitro and in vivo (Baric et al., 1999 (Baric et al., , 1997 Treshman et al., 1996) , we and others in the field have speculated that coronaviruses might be important emerging pathogens as early as 1996 (Baric et al., 1996) . Notably, several major coronavirus pandemics had been recorded over the past 25 years in economically important animal species. In 1978, porcine epidemic coronavirus (PEDV) emerged and has remained among the most important causes of severe gastroenteritis in swine in Europe and Asia. Sequence analysis suggested that PEDV likely evolved from human coronaviruses (Bridgen et al., 1993; Duarte and Laude, 1994) . In 1984, the less pathogenic porcine respiratory coronaviruses (PRCV) evolved from the highly virulent enteric strain, transmissible gastroenteritis virus (TGEV) (Pensaert et al., 1986) . This was accomplished by either a single nucleotide mutation or deletion in the major surface glycoprotein gene, spike (Sanchez et al., 1999; Wesley et al., 1991) . Currently, PRCV affords some level of cross protection against TGEV (van Nieuwstadt et al., 1989; Wesley et al., 1991) , although more virulent forms of PRCV have recently emerged (Saif, 1999) . A closely related arterivirus, the porcine respiratory and reproductive disease virus (PRRSV), emerged in the late 1980s in Canada and Europe, and is currently the most important swine pathogen worldwide, responsible for economic losses of $200 million/year in the US alone (Blaha, 2000; Rossow, 1998) . In 1992, a new respiratory pathogen was recognized that caused 'shipping fever' in cattle, a severe, oftentimes fatal, pneumonia. Sequence analysis suggests that "shipping fever" was caused by a respiratory bovine coronavirus (BCV-R), which likely evolved from an enteric bovine coronavirus by acquiring mutations in S and the replicase gene (Storz et al., 2000) . Concordant with this hypothesis, sequence comparisons have now indicated that host shifts, even between distant avian and mammalian species, are common events in coronavirus phylogeny (Rest and Mindell, 2003) .
Coronaviruses have emerged rapidly by mutation or recombination of existing strains altering tissue tropisms and/or species specificity in animals and despite intensive management and vaccine efforts, these "new" coronaviruses are still major problems worldwide. The emergence of a new human coronavirus from zoonotic reservoirs is not surprising given the widespread activity noted in economically important animals. In reality, epidemiologic trends for many viral pathogens are based on relatively short (<60 years) timelines, with underreporting, of disease surveillance trends in human populations. The emergence of the SARS-CoV underscores the critical need for maintaining active basic science research not only on the medically relevant human pathogens, but also on virus families that are associated with limited or benign disease outcomes in humans. The limited investment in coronavirus research primarily in Europe and the US provided key investments necessary for the rapid identification and sequencing of the SARS-CoV genome, the prompt development of rapid PCR-based detection methods, the identification of the S gene as the likely candidate target for vaccine development, potential problems in vaccines and therapeutics development, and the development of reverse genetic systems; all within a few months of emergence.
The nation hardest hit by SARS was China and the SARS-CoV epidemic underscores the importance of local research infrastructure and governmental support in controlling new emerging diseases. In the end, the successful control of the SARS-CoV is a triumph for the Chinese basic, clinical and public health research infrastructure. The Chinese research community was among the first to successfully culture the virus, sequence the genome and develop rapid RT-PCR based detection strategies (Consortium, 2004) . Despite an early belated response by governmental officials, aggressive quarantine and other public health measures demonstrated the effectiveness of classic methods of tracking cases and their contacts as an effective means of curtailing disease spread. The Chinese research community rapidly demonstrated that SARS-CoV originated from animals and that the open markets were likely sources of future disease outbreaks (Guan et al., 2003) . The closing of the animal markets has played a key role in tempering the scope and duration of SARS and will likely serve to temper the severity and scope of future outbreaks. Using epidemiology, molecular genetics and genome sequencing strategies, a detailed catalogue of the key genomic changes that contributed to disease emergence and spread have been identified and cataloged for future research (Consortium, 2004) . Other key contributions include the development of animal models, vaccines and pathogenic studies. Perhaps most importantly, a local, well-funded basic scientific community is key not only for protecting global public health, but also in protecting the health of global economies.
In many ways, we were lucky with SARS. Asymptomatic infections were rare and transmissibility was usually inefficient and typically occurred after disease onset, allowing for pubic health intervention strategies to control the outbreak (Peiris et al., 2004) . SARS highlighted the importance of rapid reporting, infection control, tracing and quarantine of contacts in controlling new emerging diseases. However, if super spreader events were more common, the story may have been dramatically different. Importantly, the outbreak has underscored critical vulnerabilities in global public health intervention strategies. Highly pathogenic respiratory viruses are clearly capable of near instantaneous global transmission, and as such rapid approaches are needed to rapidly (a) detect, (b) sequence, and (c) develop and test candidate vaccines and therapeutics to protect human populations. Safe, universal, vaccine platforms are needed that can be tailored to new pathogens as they emerge, quickly tested for safety, and then strategically used to control new disease outbreaks in human populations. Basic investment into structure, replication, genetics and pathogenesis in each virus family is justified and among the first line of defense against future emerging threats. It is no longer sufficient that only the developed world be poised and able to protect itself from new emerging diseases threats. Rapid vaccination and therapeutic platforms are needed for the developing world as well, positioned to engage local research and public health communities in controlling local disease outbreaks at the source. If control measures can be implemented rapidly, then the scope, duration and suffering in epidemics will be minimized.
The number of laboratories engaged in SARS-CoV research was enormous at the height of the epidemic and many laboratories have made significant contributions to our current understanding of SARS epidemiology, replication, cross species transmission and pathogenesis. SARS highlighted the success of the basic research community and Public Health infrastructure as the principle determinants against new emerging diseases. This special issue of Virus Research reviews only a small subset of the enormous number of high quality scientific contributions made by the global research community. Science into policy: preparing for pandemic influenza Authoratative government pandemic preparedness requires an evidence-based approach. The scientific advisory process that has informed the current UK pandemic preparedness plans is described. The final endorsed scientific papers are now publicly available. Public expectations of effective government interventions in health crises are high in developed countries. Authoritative action and provision of information to the public can help in avoiding public disquiet or panic and in mitigating the societal risks of a pandemic, complementing the direct health effects of any interventions. Conversely, disagreement over the scientific evidence base, particularly where considerable uncertainties and gaps in information exist, can open the way to debate based primarily on established beliefs and prejudices. In the face of a future event such as an influenza pandemic, the timing and precise nature of which is unknown, robust preparation will be strengthened by an agreed scientific understanding of the risks and the options for response.
The UK Government has followed an extensive process to review and confirm an agreed summary of the international evidence base. This underpins policy development on countermeasures within its pandemic influenza preparedness programme and can be of use to other countries developing pandemic preparedness plans as well.
Under the auspices of the UK Scientific Advisory Group on Pandemic Influenza, five scientific papers dealing with the main clinical countermeasures (antivirals, pre-pandemic and pandemic specific vaccines, antibiotics and facemasks) and the risk of a pandemic originating from an H5N1 virus were developed. These papers were reviewed and revised by additional national and international expert scientific reviewers and subsequently at a colloquium, convened by the Secretary of State for Health, of scientific experts. Revised papers were then submitted to the Scientific Advisory Group for final endorsement as reflecting an accurate and comprehensive summary of the state of knowledge in June 2007. The final endorsed papers have now been made publicly available as a resource to all. 1 Papers reviewing the scientific evidence base in the following areas are available at: http://www.advisorybodies.doh.gov.uk/ spi/evidence.htm (i) The use of antiviral drugs in a pandemic; (ii) pre-pandemic and pandemic specific influenza vaccines; (iii) the use of antibiotics for pandemic influenza; (iv) the use of face masks during a pandemic; and (v) the risk of a pandemic originating from H5N1.
This widely agreed scientific state of the art offers a firm foundation for complex and potentially expensive policy and procurement decisions on pandemic countermeasures. Within the UK, the papers have already informed the recently published framework 2 and policy statement. 3 They will continue to inform policy decisions across Government.
The scientific knowledge in this field is continually evolving and improving, and the UK will therefore continue to review and refine its assessment of the evidence base. Smallpox and Season: Reanalysis of Historical Data Seasonal variation in smallpox transmission is one of the most pressing ecological questions and is relevant to bioterrorism preparedness. The present study reanalyzed 7 historical datasets which recorded monthly cases or deaths. In addition to time series analyses of reported data, an estimation and spectral analysis of the effective reproduction number at calendar time t, R(t), were made. Meteorological variables were extracted from a report in India from 1890–1921 and compared with smallpox mortality as well as R(t). Annual cycles of smallpox transmission were clearly shown not only in monthly reports but also in the estimates of R(t). Even short-term epidemic data clearly exhibited an annual peak every January. Both mortality and R(t) revealed significant negative association (P < .01) and correlation (P < .01), respectively, with humidity. These findings suggest that smallpox transmission greatly varies with season and is most likely enhanced by dry weather. Smallpox is the only disease to have been eradicated worldwide [1] . Despite the success story of vaccination and other public health interventions, the number of susceptible individuals has grown to date following cessation of routine vaccination, and the threat of bioterrorist attack has led to debates on countermeasures in such an event [2] . Various mathematical studies have been conducted as part of a preparedness program, including large-scale simulation of a bioterrorist attack and the public health countermeasures against it [3] [4] [5] [6] . Theoretical studies on the spread of smallpox include not only simulations but also quantitative analysis of historical data [7] [8] [9] [10] . A statistical modeling study suggests that a small outbreak could be contained only implementing contact tracing and isolation [11] . Moreover, those who underwent vaccination in the past are believed to be still protected against severe and fatal manifestations of smallpox even today [7, 12] .
Studies on smallpox control have progressed in parallel with the development of epidemiological and statistical methods, and because of the eradication before maturation of biostatistics, many questions have remained in regards to the details of the epidemiology. Seasonal variation in smallpox transmission is one of the most pressing ecological questions playing a key role in determining the transmission dynamics, should a future outbreak occur following the deliberate release [1, 4] . For example, clarification of the seasonal preference of variola virus is crucial for identifying and forecasting the disease risk using ecological data [13] . Although seasonal occurrence of smallpox was documented early on among directly transmitted infectious diseases [14, 15] , and whereas the disease is believed to be one of the "winter diseases" in industrialized countries, even the presence of seasonality has not been investigated in detail.
The best available evidence stems from a series of studies by Sir Leonard Rogers (1868 Rogers ( -1962 [16] , who conducted epidemiologic surveys of smallpox outbreaks in India over a long period of time [17] [18] [19] . He also conducted a similar survey in England and Wales [20] . By analyzing the monthly mortality data from the late 19th to the early 20th century in these countries, Rogers argued that the smallpox epidemic in India is relatively uniform (i.e., not apparently cyclical) compared to that in England and Wales [17, 19, 21] . Further, he descriptively and implicitly suggested that there is a negative correlation between humidity and smallpox mortality, but there was little association between smallpox and rainfall [17, 18] . This effort was followed by Russell and Sundararajan [22] who supported the notion that a dry environment offers favorable conditions for smallpox transmission. These consistent findings have also been reported during the Smallpox Eradication Program (SEP), where a peak of smallpox incidence occurred from December to January in the Northern hemisphere (e.g., Indonesia and Bangladesh) and from May to June in the Southern hemisphere (e.g., Brazil) [23] [24] [25] [26] . However, the observed data during the SEP were greatly modified by intensive immunizations, and perhaps because of this, epidemics in other locations were not suggested to be seasonal [26] [27] [28] , leaving this issue yet to be clarified.
Despite the rigorous efforts before the global eradication, later progress on this issue was unfortunately subtle. Upham once revisited Rogers's dataset from India, anthropologically discussing potential reasons why the American Southwest was less infested by smallpox [29] . A time series technique was applied to historical data in Finland and England [30] [31] [32] , showing that periodicity is mainly regulated by the susceptible fraction of a population in question [33] . However, despite the analyses on the impact of vaccination and migration on periodicity, seasonal patterns of transmission have not been explicitly studied, mainly because of a lack of data precision. In a historical study examining smallpox in England from the 16th to 17th centuries, the time referred to as the "little ice age," it has been documented that longterm climatic changes did little to the smallpox transmission [34] , but this conclusion was drawn without quantitatively and explicitly analyzing the data. Instead, the quality of time series data and its impact on seasonality were discussed in relation to social backgrounds of smallpox control [35, 36] , but again no rigorous statistical analyses were made using observed data.
Accordingly, several lingering questions remain. Is smallpox transmission really seasonal? If so, is the seasonality associated with humidity? Clarification of these points will not only enhance our understanding of the pattern of smallpox transmission, but also will be crucial for identifying the seasonal preference of variola virus with some implications for bioterrorism preparedness plans. The present study is aimed at examining the presence of seasonality and clarifying the relationships between smallpox and climate. We reanalyzed various historical datasets, suggesting a new method for the analysis of time series.
Records. Seven temporal distributions of smallpox at different times and locations were extracted from historical literature. This literature review was based on references collected by tracking all the references given in the relevant publications and repeating this task until we could find no further references; the details are given elsewhere [37, 38] . Figure 1 shows the time series data by location with a monthly reporting interval. Chronologically, epidemic records for The Hague (1755-1773), Berlin (1758-1774), Zurich (1870-1887), the entire Netherlands (1870-1873), Northwest Frontier province in India (1890-1921), Shanghai (1900) (1901) (1902) (1903) (1904) (1905) (1906) (1907) (1908) (1909) (1910) (1911) (1912) (1913) , and Bombay (1902) (1903) (1904) (1905) (1906) (1907) provide monthly data of smallpox with time and were used for further analysis [17, [39] [40] [41] [42] [43] [44] [45] . The first two records contain data before the introduction of vaccination. Except for Zurich, which documents the monthly number of cases, the remaining datasets record only monthly deaths. Death data are given as the absolute number of deaths, except where indicated. With regard to the magnitude of the epidemics, the annual averages of the disaster size were 10.1 deaths (The Hague), 32.9 deaths (Berlin), 9.9 cases (Zurich), 428.6 deaths (the entire Netherlands), 5.28 deaths per 100 000 (Northwest Frontier province in India), 21.5 deaths (Shanghai), and 2.45 deaths per 100 000 (Bombay). By examining another historical record of the smallpox epidemic in Tokyo, it was found that the mean (and the standard deviation) and the median (25-75% quartile) time from onset to death were 29.1 (13.8) and 26.0 (19.0-37.0) days, respectively [46] . Thus, it is reasonable to assume that the relative frequency of death with time represents that of onset accompanied by approximately a 1 month delay. Moreover, the infection may have happened approximately half a month before the onset [9] . Meteorological variables with time were given only in Rogers's observations [17] , which contained the monthly rainfall (inch) and the absolute humidity.
First, the presence of seasonality was examined for all 7 datasets using spectral density analysis. Spectral analysis is based on the idea of a theoretical power-spectrum, which partitions the total variance of the series among sinusoidal components [47] . In other words, spectral density decomposes a time series function into a sum of sines and cosines. The density plot (i.e., correlogram) was graphically plotted to determine if a sharp peak at a period of 12 months exists, corresponding to an annual cycle (i.e., seasonality).
Second, seasonality that was evaluated using the effective reproduction number, R(t), defined as the actual average number of secondary cases per primary case at calendar time t. R(t) shows a time-dependent variation with a decline in susceptible individuals (intrinsic factors) and with the implementation of control measures (extrinsic factors). If R(t) < 1, it suggests that the epidemic is in decline (vice verca, if R(t) > 1). This approach was employed to clearly show the seasonal patterns of transmission and to partly address the issue of dependence among cases, that is, statistically, the observation of an infected individual is not independent of other infected individuals, since the disease is transmitted directly from human to human.
The following approximation was made to derive estimates of R(t). Supposing that the number of new infections at calendar time t is I(t), the transmission dynamics are described by the renewal equation using R(t) [48, 49] : 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 Year where ω(τ) is the probability density function of the generation time. The right-hand side of (1) represents secondary transmissions at calendar time t, which are determined by the number of those who were infected at time t − τ, I(t − τ), and the magnitude of secondary transmissions at time t, R(t). Since the data in the present study were recorded only monthly, the equation has to be simplified to comply with discrete points of time data. From the beginning of the history of mathematical modeling of smallpox in the late 19th century [50] , cases tended to be modeled by generation, the idea of which is applied as follows. Given the number of cases in generation i, I i , the expected number of cases in generation i + 1, E(I i+1 ) is given by
where R i is the effective reproduction number of generation i [51] . That is, the reproduction number is simply given by ratio of successive generations of cases, which was implicitly understood in history by a pioneering epidemiologist, Clare Oswald Stallybrass (1881 Stallybrass ( -1951 who applied the theory to analyze the seasonality of various infectious diseases [52, 53] .
Since the mean generation time of smallpox is approximately 15 days (i.e., half a month) [50, 54] , monthly data contains exactly two generations. Let us consider three successive generations, i, i+1, and i+2. Given the reproduction numbers R i and R i+1 , we get
Considering that the generations i and i + 1 are grouped together and reported in the same month j, the reproduction number cannot be estimated by generation i. Instead, by assuming that the reproduction numbers in the successive generations are identical, that is, R i = R i+1 (:= R j ), (3) can be rearranged as
The expected number of cases in the next generation i + 3 is given by product of I i+2 and the reproduction number in the next month j + 1, R j+1 , that is,
Given (4) and (5), the number of cases in month j + 1, C j+1 (:= I i+2 + I i+3 ) is given using C j (:= I i + I i+1 ), that is,
We assume that the expected values are sufficient to characterize Poisson distributions. This assumption indicates that the conditional distribution of the reported number of cases in month j + 1, C j+1 , givenC j is given by
Thus, for the observation of cases (or deaths with a 1 month lag) from month 0 to N, the likelihood of estimating R j is given by
By minimizing the negative logarithm of (8), the maximum likelihood estimates of the monthly-approximated reproduction numbers, R j were obtained.
Modeling. Third, to identify the characteristic factors of seasonal variation in smallpox transmissions, the relationships between meteorological variables (i.e., rainfall and humidity) and incidence (mortality) as well as the effective reproduction number were examined. To examine the influence of seasonal variables on the temporal trend of smallpox, we employed one of the generalized linear models with the construction of a Poisson regression model incorporating monthly and yearly terms [55] :
where E(C j ) is the expected number of cases (deaths) in month j, α is a constant, and β i are the regression coefficients for year or month. The relationship was investigated using both univariate and multivariate models. In the multivariate model, the year of occurrence was controlled for, but the sine and cosine of the month were not included due to colinearity with rainfall. The mortality rate ratios (MRR) for the occurrence of smallpox death were used to evaluate the impact of each meteorological variable on smallpox.
With regard to the relationship between meteorological variables and R(t), multiple linear regression analysis was employed to determine factors contributing to R(t). Because of the obvious cyclical nature of the observed data yielding an autocorrelation in the linear regression analysis (Durbin-Watson = 0.23), the monthly periodic terms (as shown in (9)) were added to the list of independent variables. The level of statistical significance was set at α = 0.05. All statistical data were analyzed using the statistical software JMP version 7.0 (SAS Institute Inc., Cary, NC, USA).
Density. The spectral densities are shown in Figure 2 which can be reasonably interpreted by comparatively examining the temporal distributions ( Figure 1 ). With regard to the data collected from The Hague and Berlin, the observations of which were made before the introduction of vaccinations, periodic epidemics (i.e., super-annual cycles) are apparent where the interepidemic period ranges from 3 to 5 years (see Figures  1(a) and 1(b) ). However, the annual cycle is not seen, and thus, the spectral densities do not show a clear seasonal pattern (Figures 2(a) and 2(b) ). On the contrary, the time series data in Zurich and Shanghai clearly revealed a peak at 12 months (Figures 2(c) and 2(f)). The entire Netherlands data covers a relatively short period of time compared to the other datasets (Figure 1(d) ) with unclear seasonal and periodic frequencies in the spectral diagram (Figure 2(d) ). Although a small peak is seen at 12 months for the data in the Northwest Frontier province in India (Figure 2(e) ), the density plot exhibits a multimodal pattern, reflecting an irregular temporal distribution (Figure 1(e) ). In the Bombay data, the annual cycle is most clearly highlighted in the temporal distribution (Figure 1(g) ), which is also reflected in the spectral density (Figure 2(g) ). Figure 3 plots estimates of the effective reproduction number as a function of calendar time. The vertical broken lines represent January in every year, while a horizontal dashed line is a reference value yielding R(t) = 1, that is, the threshold condition of an epidemic. R(t) tends to increase during the winter season for three early records (Figures 3(a), 3(b), and 3(c) ), but the annual cycles are not seen. However, the shortterm epidemic data for the entire Netherlands clearly shows that three peaks of R(t) coincide in every January with estimates above unity (Figure 3(d) ). A similar pattern is observed in Shanghai and Bombay (Figures 3(f) and 3(g) ). Figure 4 shows the spectral density plots of R(t) for the entire Netherlands and Northwest Frontier province in India. Although spectral densities of death and mortality (Figures 2(d) and 2(e)) did not exhibit a clear annual cycle, the obvious peak at 12 months is seen for both datasets in terms of R(t) (Figures 4(a) and 4(b) ). That is, seasonal patterns of smallpox transmission were reasonably shown with the use of R(t) even for the short-and long-term time series. Table 1 shows the output of univariate and multivariate models for explaining smallpox mortality in India using meteorological variables. In both models, rainfall was not significantly associated with smallpox mortality. However, significant negative association was found for humidity (adjusted MRR = 0.387 (95% confidence interval (CI): 0.311, 0.481), P < .01). Table 2 summarizes the relationship between the effective reproduction number and meteorological variables using a multiple linear regression model. On a whole, the model showed a weak predictive ability. However, humidity was again identified as an explanatory variable which significantly reduces the effective reproduction number (P < .01). No significant correlation was found between R(t) and rainfall.
The present study reanalyzed historical records of smallpox to examine the presence of seasonality and to partly clarify the characteristic factors. Although 18th century data did not show an apparent annual cycle, the remaining records reasonably showed seasonal variations either in the monthly observation or the reproduction number. In particular, even the short-term epidemic data for the entire Netherlands clearly revealed peaks of transmission every January. Although several important meteorological variables were missing (e.g., temperature and atmospheric pressure), Rogers's observation permitted investigations of a few variables as underlying factors characterizing the seasonality. Analyzing the meteorological data in India, both smallpox mortality and the reproduction number yielded significant negative association and correlation with humidity. Rainfall did not appear to be a useful predictor of seasonality.
One important message drawn from this exercise is that smallpox transmission is confirmed as seasonal and this is most likely associated with dry weather. This finding is consistent with implicit suggestions which have accumulated in the historical literature [1, 17, 19] . Whereas the data from The Hague and Berlin did not offer the relevant interpretations, their periodic peaks were also observed during the winter seasons. Assuming that these records captured mainly the large periodic outbreaks alone, it is plausible that the old data were accompanied by underreporting during less intensive years, and thus, did not precisely contain subtle seasonal fluctuations. Given that the seasonal force of infection was obvious even in the shortterm epidemic data from the entire Netherlands, not only endemic but also epidemic smallpox would greatly vary with the season and most likely would be enhanced by dry weather. Historically, virologists attempted to attribute the annual cycle to the seasonal preference of the variola virus [56] [57] [58] . To date, it is known that the variola virus could survive in an infective state under different conditions of temperature and humidity [56, 57] . However, as temperature and humidity rise above 30 • C and 55%, respectively, the virus is known to immediately lose infectivity [57] . Such a virological explanation supports the epidemiologic findings from this present study and reasonably explains the seasonal preference of the virus as a factor behind the seasonality of outbreaks. The above-mentioned point implies that we cannot ignore the seasonality even in the event of a shortterm reintroduction of variola virus due to bioterrorist attack.
A technical development in analyzing seasonal data is also notable. Since the observation of an infected individual is not independent of other infectious individuals, direct application of the generalized linear model has not been appropriate to date. One approach to resolve this issue is to employ a Bayesian method, explicitly dealing with dependence in a Poisson regression model [59] , which is, Interdisciplinary Perspectives on Infectious Diseases however, computationally complicated for nonspecialists. As an alternative, the present study suggested the use of R(t). R(t) reasonably reflects time-dependent changes in the susceptible fraction of the population in question and other various factors determining the transmission (including seasonality) [60, 61] . In particular, it should be noted that R(t) does not reflect onset or death but can represent an infection event with time, proving its potential as a marker to model seasonal and periodic transmission cycles. In addition, quantitative assessment of R(t) is theoretically important, because the amplitude of seasonal forces of infection characterizes the length of the interepidemic period [33, 62, 63] . A continued super-annual cycle mathematically requires seasonally varying forces of infection, which determines the season-specific threshold condition [64] and evolutionary dynamics of a disease [65, 66] . To the best of our knowledge, the present study is the first to suggest a quantitative method to reasonably extract the amplitude using the notation of R(t) and extending the previous efforts of Stallybrass [53] .
Most infectious diseases show characteristic seasonal variations in incidence. The present study confirms that the transmission of smallpox does vary with season. However, compared to the seasonal ecology of insects in vector-borne diseases, seasonal factors for directly transmitted diseases are far more complex, and thus, questions remain as to what exactly are the factors behind the seasonality of smallpox. At least, experimental evidence supports the role of dry weather in the dynamics of influenza [67, 68] ; a recent study found that low (dry) relative humidity in the range of 20 to 30% produced the spread of the influenza virus faster than at relative humidity in higher percentages. In fact, at a humidity of 80% or above, the study found no spread of the flu [68] . Since there are also various social factors which vary with the season, the seasonal preference of pathogens cannot be captured without sufficiently highlighting the time-varying human contact patterns, and thus, further analyses (e.g., reanalysis of small-scale outbreaks where we can adjust the contact frequency) are needed. We hope that the present study enhances the similar reanalysis of historical data, triggering an interest in investigating the relationship between the transmission of directly transmitted infectious diseases and climatic changes.
Seven historical datasets of smallpox were reanalyzed to examine the presence of seasonality and to identify the characteristic factors. Annual cycles were clearly shown not only in the monthly reports but also in the estimates of the effective reproduction number. Even for a short-term epidemic, the transmission of smallpox would most likely be enhanced by dry weather. Chinese journals: a guide for epidemiologists Chinese journals in epidemiology, preventive medicine and public health contain much that is of potential international interest. However, few non-Chinese speakers are acquainted with this literature. This article therefore provides an overview of the contemporary scene in Chinese biomedical journal publication, Chinese bibliographic databases and Chinese journals in epidemiology, preventive medicine and public health. The challenge of switching to English as the medium of publication, the development of publishing bibliometric data from Chinese databases, the prospect of an Open Access publication model in China, the issue of language bias in literature reviews and the quality of Chinese journals are discussed. Epidemiologists are encouraged to search the Chinese bibliographic databases for Chinese journal articles. The Chinese have had a long history in infectious disease control, and records of epidemics can be traced back two millennia [1] . Since the introduction of modern medicine by missionary doctors in the 19 th century [2] , modern epidemiological studies have been conducted in China, first by Western doctors, and then gradually superseded by their Chinese colleagues in the 1930s [3] . Since the 1950s, huge reductions in the incidence of infectious diseases like measles and schistosomiasis have been achieved through national vaccination programmes and environmental intervention programmes [1, 4] . The adoption of the Open Door Policy in 1978 marked the beginning of remarkable social and economic development unprecedented in China's modern history. However, rapid industrialization and urbanization are accompanied by many social problems, from the increasing rich-poor, urban-rural, coastal-interior disparity to heavy environmental pollution. Changes in disease profile with the increasing burden of non-communicable diseases as a result of an aging population with a successful one-child policy posed new challenges in the 21 st century [1] . The SARS epidemic in 2003 exposed how a lack of transparency and delayed dissemination of information on the part of the Chinese government made an epidemic of then unknown aetiology a global problem [3] .
Epidemiologists from the non-Chinese world may wonder what resources of scientific knowledge and epidemiological information China (whose health research serves a fifth of the world's population) may offer us. In 1994, the British Medical Journal published an editorial recommending to its readers the Chinese medical journals [5] . However, 13 years have gone by, and the Chinese medical and scientific literature is still largely terra incognita outside China [6] . Recent enthusiasm among Westerners in learning the Chinese language [7, 8] may rekindle their interest in this untapped resource. As Beijing prepares for the Olympics in 2008 celebrating China's arrival in the modern world, perhaps an update of the development of Chinese biomedical journals may whet the reader's appetite. This paper is intended to serve as a guide. This article will first provide a general overview to Chinese biomedical journals. Next, Chinese bibliographic databases will be described, using Wan Fang and iLib as examples. Chinese journals in epidemiology and public health will then be discussed, followed by a comprehensive examination of issues arising from switching the publication language to English, the effect on impact factors and Open Access. Lastly, the problems of language bias and quality of articles will be discussed. Three appendices are included. Appendix 1 provides additional information on bibliographic indexing of Chinese biomedical journals. Appendix 2 illustrates the historical background to the choice of language of publication using three journals as examples. Appendix 3 is a review of a survey of English language biomedical journals of China previously published in a Chinese journal.
For the purpose of this study, Chinese journals and databases discussed here are confined to that of mainland China, excluding Hong Kong, Macau and Taiwan. For a more in-depth study of the research potential of Chinese biomedical bibliographic databases, illustrated by the example of schistosomiasis research, please refer to the paper in this thematic issue by Liu et al. [9] .
Today there are more than 5000 academic periodicals published in mainland China, and around a thousand of these are related to biomedicine and health. Seventy-four journals from mainland China were indexed in 2006 Journal Citation Reports ® Science Edition (JCR) published by Thomson Scientific, of which 12 were biomedical journals and two were multi-disciplinary science journals that publish biomedical articles. Of these 14 journals, only one was published in Chinese, while the rest were in English.
Eighty-two mainland Chinese journals are indexed for MEDLINE [10, 11] , among which, 62 publish articles in Chinese, 16 in English, one in either English or German, and three in either Chinese or English. Only six of the MEDLINE-indexed mainland Chinese journals receive impact factors from JCR. All six publish articles in English (Table 1) .
Altogether, 146 mainland Chinese journals that cover subjects such as general science, biology, medicine, veterinary science, agriculture and forestry, are indexed in the PubMed journal database (some of these are indexed in MEDLINE). Of these 146 journals, 110 publish articles in Chinese, 24 in English and seven in either Chinese or English (with one in Chinese or Latin and one with missing language data). For a detailed discussion, please refer to Appendix 1.
Full texts of more than five thousand Chinese journals are now available online. There are six mainland Chinese bibliographic databases through which Chinese language biomedical journal articles can be searched and located and of which two provide English interfaces:
(a) Chinese Biomedical Literature Database (CBM) [12] , (b) Chinese Medical Current Content (CMCC) [13] , Users of traditional Chinese characters can use Yahoo! Taiwan Academia Search [20] whose mainland Chinese journal article entries are provided by iLib. In addition, Google Scholar [21, 22] , as a multi-lingual bibliographic database, also facilitates searches in the Chinese language (Table 2) As a recent paper [23] has given a detailed description and analyses of five of the Chinese bibliographic databases, the following discussion is restricted to three of them: Google scholar as related to searches in Chinese has not yet been covered by any academic paper in English and the same is true of iLib, which is not covered by [23] ; Wan Fang database, which is freely available through terminals in the British Library, will be used as an example to illustrate the wealth of biomedical journals available to us through the internet.
Google Scholar provides a convenient starting point for searching Chinese articles, of which the bibliographic data is mainly provided by VIP information, Wan Fang database and iLib (all accessed on 21 st February, 2007). For Chinese speakers, Google Scholar also provides a Chinese interface [22] .
There are two apparent advantages (especially for non-Chinese speakers) of searching for Chinese articles in Google Scholar. Firstly, Google Scholar (Chinese interface) provides 'pinyin search', i.e. using a standardised Romanised form of Chinese, known as pinyin in Chinese [24] . For example, if I type 'bing du' in the Google Scholar English interface, I will obtain journal articles with authors of the family name Bing Du. However, if I use the Chinese In additional to these functions, Google Scholar also provides links to institutional libraries and the British Library, citation records, links to related articles, and it groups different entries of the same article together. For a more structured search, the Advanced Scholar Search is needed, of which a Chinese interface is also available [25] .
As of 13 th February 2008, the Chinese links in Google Scholar provided by VIP information are linked to the PDF full text which requires subscription to VIP information. If the user is not covered by subscription, the link will be redirected to the webpage on which the title, author, abstract and keywords (all in Chinese) are displayed. The full text can then be purchased individually. Chinese links in Google Scholar provided by the Wan Fang database and iLib will directly lead to the Chinese abstract page. From there a link is provided to the full text PDF file which requires payment or subscription.
Although a previous study performed in 2005 found an English language bias in Google Scholar [26] , the search engine has evolved so quickly that a new study of its article coverage is definitely worthwhile.
Both Wan Fang database and iLib are run by Wanfang Data, an affiliate of the Chinese Ministry of Science & Technology (cf. [27] ). While Wan Fang provides access to databases of journal articles, conference proceedings, degree theses, patents, national and industrial standards and even listed companies in China, iLib is essentially a subset of Wan Fang and is restricted to journal articles only.
The Wan Fang database maintains two portals, one in Chinese [17] and one in English [18]. Cross-searches of different databases (e.g. journal articles and conference proceedings) using simplified Chinese in the domestic portal and English in the international one, are available.
The advantage of iLib over Wan Fang for journal article searches is that the interface of iLib is more user-friendly and, unlike Wan Fang, there are links to the author, the journal issue, the journal, the references cited in the paper and some related papers in the iLib database, similar to the AbstractPlus format of PubMed.
Like PubMed, the Wan Fang databases or iLib can be searched for free. However, only Chinese abstracts are available for free in HTML format. Although many Chinese journals provide English abstracts to their articles nowadays, these English abstracts are not uploaded onto the public domain by Wan Fang or iLib. To access the English abstract online, one has to download the PDF full text which requires subscription. The only exceptions are those indexed by PubMed, through which they are freely available.
A difference in the search mechanism is that in Wan Fang, one has to choose whether to search the English Online Journals category or the China Online Journals (Chinese language journals) in the first place, while in iLib, there is no separation of the journals by language. Thus, if one types 'influenza' in iLib, one will find articles published in Chinese language journals (as the English titles of the Chinese articles are actually being searched) as well as in English language journals.
Subscription or payment for full text of mainland Chinese journal articles For individual users, there are various methods of payment. However, most (if not all) of these methods apply only to users in mainland China. While VIP information accepts VISA card online payment, Wan Fang and iLib do not accept any credit cards; they accept only bank cards issued in mainland China or payment through a mainland Chinese mobile phone company, remittance via post offices or banks, or some 'pay-asyou-download cards', which provides you with a password to top-up your download credit online, using your personal Wan Fang or iLib account.
To the knowledge of the author, as of 14 th February 2007, the British Library has subscriptions to full text (PDF files) of all academic journals (both English language journals and Chinese language journals) available in the Wan Fang database (around 5700 periodicals). Readers have access to these journals through the computer terminals in the library. Below I describe in more detail what is available in the Wan Fang database.
There are 141 titles under the category of English China Online Journals. According to Wan Fang categories, eight are on agriculture, 58 on fundamental science, 24 on health and medical science, 48 on science & technology and three on social science, as of 14 August 2007 [28] . Table 3 lists 24 English language journals on health and medical science available in Wan Fang. A full list in alphabetical order is available in [29] .
Chinese language journals in the Wan Fang database Under the category of China Online Journals, there are more than 5600 titles (5638 as of March 2007). When subdivided into five categories, over a thousand titles are found to be related to health, medicine and biology (1056 as of June 2007) [30] .
Currently, it is the Chinese national standard that scientific periodicals published in mainland China in the Chinese language should contain English abstracts for every original research article and English titles for selected important articles (e.g. editorials, reviews, forums and short research articles, depending on the judgement of the editorial board) [31] . The English table of contents is available online, free of charge, through the Wan Fang database. However, the English abstracts are only available in the full text PDF file from the Wan Apart from impact factors published by Thomson Scientific in JCR, VIP Information also publishes bibliometric data of some of the journals indexed in its database [40] . A handful of journals listed in Table 4 have their VIP impact factor and immediacy index available, which can contribute towards evaluation of their quality.
Two tiers: national and provincial Guangxi Zhuang Autonomous Region in southern China now has an international board of editors [31] .
The development of the internet has prompted a drastic change in the ecology of academic publication worldwide and Chinese journals are no exception. Some observers may note that the purpose of publishing provincial journals may be to present epidemiological findings mainly of local use and serve as a local publication outlet. However, as all of these journals are now available online, the original raison d'être of provincial journals to foster the exchange of research output on a provincial level may diminish. A doctor from Sichuan can now easily download a paper published in the Shanghai Journal of Preventive Medicine, while a scientist from Guangzhou (Canton) can easily publish his/her paper in the Zhejiang Journal of Preventive Medicine. One can imagine fierce competition for good research papers among these journals in the near future and through the invisible hand of the market, some journals may prosper and attain international status while others may wither and die.
An interesting exception to the two tiers of national and provincial journals is the Journal of Preventive Medicine of Chinese People's Liberation Army, in which research articles related to public health issues in a military context, from hygiene in training camps to the temperature inside tanks, are published. Apart from those, there are also articles on civilian public health issues written by scientists in the military academy. Table 5 lists 23 journals related to tropical medicine, including journals in parasitology, HIV and tuberculosis. Table 6 lists 23 journals on non-communicable diseases, medical statistics, school health, occupational health, port/ frontier health and quarantine, evidence-based medicine and reproductive health and family planning. All but one are published in Chinese. The exception, the Journal of Reproduction and Contraception is published in English with a sister publication, Reproduction and Contraception, published in Chinese [42] . Some of these journals have been listed as Chinese core journals in 2004: three in parasitology (Table  5) , one in medical statistics, one in school health, five in Emerging Themes in Epidemiology 2008, 5:20 http://www.ete-online.com/content/5/1/20 occupational health and two in reproductive health and family planning (Table 6 ). Specialist national journals like the Chinese Journal of Parasitology and Parasitic Diseases, of the Chinese Preventive Medicine Association (CPMA) series [43] , publish papers of high academic standard in their respective specialties.
Articles of epidemiological relevance may also be found in medical university journals. It is common among mainland Chinese universities to publish university journals that contain predominantly their own research outputs. These journals number around 2000 in total, of which nearly half belong to natural sciences [44] . University journals published by medical universities or medical faculties of comprehensive universities cover the whole spectrum of medical specialities. Some are indexed in MEDLINE, like the Journal of Peking University (Health Sciences) (cf. Table 1 ). The performances of 41 of these medical university journals have been analysed recently [36] and they varied greatly. Reform proposals have been suggested [44] . [48] . On the contrary, in mainland China, with its huge population and considerable number of scientists and medical professionals, the internal market for biomedical journals is substantial enough to sustain a sizable number of Chinese language journals. Thus I suggest that the size of the prospective market (as a result of the linguistic and political divide) plays a significant role in shaping the language trend of the world's journal publication.
Through the international language of scientific communication, English language journals provide a platform for Chinese (and foreign) scientists with a broad international readership. Hopefully, some of these journals will manage to receive their impact factors from JCR. However, by switching to English and internationalising their scope (e.g. by dropping the word 'Chinese' from their titles), they face severe competition from their counterparts in North America and Europe. Nevertheless, there are a few successes so far, like Cell Research and the World Journal of Gastroenterology: WJG [49] that are now indexed in Science Citation Index Expanded and MEDLINE. As the Chinese share of the world's scientific output increases and as Chinese scientists become more fluent in English, more English language biomedical journals published in China will receive their limelight in the international arena. For further discussion, please refer to Appendix 3.
The increasing trend of using impact factors published by Thomson Scientific as an indicator in academic evaluation in universities and research institutes has received much criticism from non-English-speakers of the developing world [50] . One of the criticisms against it is the alleged language bias of the Thomson Scientific database coverage towards journals published in English and in the industrialised world [51, 52] . The 'what-if' scenarios of inclusion of non-Science Citation Index (SCI)-indexed journals upon the impact factors of SCIindexed journals have been studied and the 'hypothetical' impact factors of the non-SCI-indexed journals calculated [52, 53] . In order to better evaluate the performances of Latin American journals, SciELO publishes bibliometric indices of its own, similar to that of Thomson Scientific, using data from its database which reflect more the regional context [53] . Brazilians can now evaluate their journals using the SciELO impact factor, rather than relying solely on that published in JCR [54] . Should the Chinese do the same? At the moment VIP Information publishes bibliometric indices using data from its own database [40] (cf. Table 4 ). These data should be used in our evaluation of the quality of Chinese journals, especially in our fields of epidemiology and public health, as hardly any of these are indexed in Thomson Scientific database. Currently only a sub-set of Chinese journals receive their impact factors from VIP Information. Hopefully, in the future, more journals will receive their bibliometric data, perhaps not only from VIP Information alone, but pooling data from the other Chinese databases as well. In the long run, I envision an international collaboration between Thomson Scientific, SciELO, the Chinese databases and other bibliographic databases to provide authors and editors alike with a more accurate and comprehensive bibliometric data of journal performance by collating data across the various databases.
Open Access (OA) online publishing in China falls into two categories: non-peer-reviewed and peer-reviewed [55] . The former provides an online interface for authors to publish their papers directly online, without peerreview or other form of quality control. Examples include Qiji.cn [56] , the Chinese Preprint Service System [57] and Sciencepaper Online [58] . The latter transfers paper-based peer-reviewed journals onto the web for free access (usually in PDF format). The Alliance of open access journals (OAJs) [59] , sponsored by the Society of China University Journals in Natural Sciences, provides access to a number of OA journals, predominantly Chinese university journals. The international Directory of Open Access Journals (DOAJ) [60] also provides links to the websites of individual Chinese OA journals, including the Chinese Medical Journal [33] .
Emerging Themes in Epidemiology 2008, 5:20 http://www.ete-online.com/content/5/1/20
However, to date the proportion of mainland Chinese journals adopting the OA publishing model is small. One possible reason is that Chinese bibliographic databases, unlike their Western counterparts, provide subscribed readers with PDF full text on behalf of the journals at an affordable rate -CNY three yuans (equivalent to USD 39 cents, as of 17 August 2007 [61] ) per paper. Thus, the cost of setting up and maintaining an individual website for a journal may seem to be a potential financial disincentive. Furthermore, most OA journals, like that of BioMed Central [62] and Public Library of Science [63] , adopt an authorpay model. In the mainland Chinese context where research funding is inadequate, more often than not, authors are less willing to pay for publication in OA journals. While OA journals in the West can grant waivers to authors from low income countries because their overhead costs are met by membership fees and article processing charges paid by universities and authors from the West, for most Chinese journals this will be difficult as most of their authors come from mainland China.
However, there are reasons to believe that many mainland Chinese authors welcome the development of OA publishing [64] . Given the current limited accessibility of full text Chinese journal articles from outside China, OA journals may prove to be an option for rapid scientific communication between authors and readers from within China and without.
One may ask why bother with Chinese journal articles after all. Apart from those who do field work in China, what important epidemiological information does the Chinese literature offer us?
Avoid language bias Perhaps one important application is to avoid language bias in our literature reviews [65] . Back in 1995, Grégoire et al. [66] found that among the 36 consecutive metaanalyses that they analysed, one would produce a different conclusion had it not excluded studies based on linguistic reasons. Comparing English and German journals, Egger et al. [67] found that randomised controlled trials (RCTs) were more likely to publish in English language journals if they gave statistically significant results. This led to the worry that language bias could be introduced to reviews and meta-analyses restricted to data published in English, leading to distorted results. However, subsequent studies [68] [69] [70] found little evidence supporting this assertion. Pham et al. found that language bias led to an under-estimation of the protective effect of intervention in RCTs in complementary and alternative medicine (CAM) systematic reviews but not in that of conventional medicine [70] .
Regarding the quality of reports, trials and systematic reviews published in English and those in languages other than English (LOE), are similar [71] [72] [73] . Inclusion of studies published in LOE in systematic reviews and meta-analyses is "likely to increase precision and may reduce systematic errors" [72] , but financial budget and time constraints should also be taken into account [70] .
The quality of articles published in Chinese medical journals has led to debates in Western academia. The conclusion of a recent systematic review on the clinical effectiveness of treatment with hyperbaric oxygen for neonatal hypoxic-ischaemic encephalophathy that the "Chinese medical literature may be a rich source of evidence to inform clinical practice and other systematic reviews" [73] was disputed. In an online rapid response, Peter C. Gotzsche ("No double standards in research, please" dated 26 th August 2006) argued that Liu et al. had provided no evidence for their statement. The standard adopted by Cochrane and CONSORT by which the Chinese trials identified in [73] are judged to be of poor quality, are not "Western" as declared by Liu et al. since they are adopted internationally, including by the Chinese Cochrane Centre. Gotzsche also cited two reviews [74, 75] to argue that "Chinese trials are far more positive, on average, than trials performed in other countries". In another study, Wang and Zhang found that by 1995, the "frequency of using statistical tests in Chinese medical journals appears comparable to that in other parts of the world", but "the lack or inappropriate use of statistics remains a problem" [76] .
In spite of this scepticism, the present author agrees with Smith that Chinese medical journals are "a treasure house of medical science available for explorers" [5] provided that we evaluate the evidence published therein with no double standard. There are examples of reviews that cover Chinese journals and evaluate the evidence available, e.g. in a recent review on the effectiveness of hand-washing in preventing SARS, among the ten case-control studies identified, four were published in Chinese journals [77] .
Chinese journals are a mine of epidemiological information that is yet to be explored by the outside world.
Thanks to the development of the internet and bibliographic databases, they can now be explored with relative ease. It has been suggested that in order to be comprehensive, we should apply LILACS in our literature search to cover Spanish and Portuguese articles in our systematic Emerging Themes in Epidemiology 2008, 5:20 http://www.ete-online.com/content/5/1/20 reviews [78, 79] . Perhaps it is time to add to our list the Chinese databases and also include Chinese papers.
In 1990, Gastel and Weng [80] published a detailed overview of Chinese medical journals written for Western readers. At that time, the number of medical journals published in China was estimated to be 500, rising to 700 only four years later [5] . In 2007, around 1000 titles related to biomedicine and health, from more than five thousand academic periodicals, were published in mainland China. To see this in a bigger picture, let us take Journal Citation Reports ® (JCR) and MEDLINE as bench marks. Chinese journals (including those published by foreign publishers and thus registered the country of publication of its publisher). There were a few records whose country of publication data were missing or mistaken and were corrected for in the analysis.
According to a recent study [83] , from 1932 to 2005, there were 1093 journals (537 titles current in 2005) from greater China, including Hong Kong (n = 7, 0.6%) and Taiwan (n = 58, 5.3%) indexed in Chemical Abstracts, among which 51 (4.7%) belonged to biological sciences and 216 were health-related (yiyaoweisheng), i.e. category Q and R according to the Chinese Library Classification [84] . English language journals made up of 9.7% (n = 106) of the total. The majority of the journals indexed were established in or after 1980 (69.2%). The first journal being indexed was Chinese Medical Journal (see Appendix 2) . Up to 14 th October 2005, a total of 693610 articles had been indexed. A full list of the indexed journals with their ISSN, indexed years and number of indexed articles can be found at [85] . [94] .
Here, we observe the colonial footprint of the Japanese in the early history of Journal of the Formosan Medical Association. The end of Japanese rule brought an end to the use of Japanese among Taiwanese doctors. The decision to publish in Chinese coincided with the tide of decolonisation. The decision to split the journal into two, one in English for the publication of research output and one in Chinese for the continuing medical education at the grass-roots level, is a classic example of the dilemma between serving local needs and communicating research outputs to the world. [98] .
The above section illustrated how Chinese doctors adapt to changes in the socio-political arena by their choices of language of publication of their flagship general medicine journals. The Chinese Medical Journal, as the only English medical journal published in mainland China for many years, provided a showcase of Chinese medical achievement to the world and a channel of communication between Chinese doctors and their colleagues aboard. The Journal of the Formosan Medical Association demonstrated the process of colonisation, decolonisation and internationalisation in its switch of language from Japanese to Chinese and then to English. The Hong Kong Medical Journal is an example of how a colonial legacy has left a language heritage that fosters internationalisation in this globalising world.
The choice of language of publication by biomedical journals is often a consequence of many different sociopolitical factors. [101] . The survey covered 31 journals and gave a good summary of their basic information, bibliometrics, and details of their management, editorial board, publication and distribution. As this survey was published in Chinese with no English abstract available, and is therefore, less accessible to the average English-speaking readers, a review highlighting its major findings that are relevant to our present study will be of benefit to interested readers and is provided below.
While 19 of these 31 English language journals reported an adequate supply of submitted manuscripts, nine reported that theirs were inadequate (three did not reply to this question). Major reasons for this inadequacy were (1) dearth of scientific research output leading to dearth of manuscripts; (2) huge amount of high-quality manuscripts being drained to foreign journals; and (3) the limited capacity of writing in English on the part of some authors. The annual number of manuscripts received varied greatly (Table 7) . Eighteen journals received manuscripts from outside China, ranging from a few manuscripts per year to 30% of its total number of manuscripts received. The majority of these submissions came from other developing countries. One of the consequences of low supply of high-quality manuscripts is that the frequency of publication of journals in China is low: 19 of the 31 English language journals are quarterly or semi-annual (Table 8 ) [101] .
Another area that awaits improvement, according to Yu et al. [101] , is the unequal distribution and nonspecialisation of journals in China. Nearly half of the 31 English language journals surveyed are general medical journals, while there are no English language journals from China that are specialised in fields like epidemiology and preventive medicine. As it has been suggested [44] , general journals should merge to raise their profile while others should specialise to avoid overlap in disciplines.
Among these 31 journals, the only full-time Editor-in-Chief is that of the World Journal of Gastroenterology: WJG.
All the others work part-time for the journals. Yu et al. [101] argued that this was very disadvantageous to the development of these journals. The number of full-time editors varied across the 31 journals (Table 8 ), while 13 journals had additional part-time editors varying from one to six. Yu et al. [101] commented that in China, there are very few editors who have high academic qualifications in biomedical sciences and at the same time are proficient in the English language. More training is needed. In order to attract talent and prevent further brain-drain, Yu et al. [101] suggested that scientific editors in China should receive the same pay and benefits as scientific researchers to remove the impression that editors are second-class scientific professionals.
Twenty-four of the 31 journals received funding from the government; 20 had page-charges; six received remuneration from advertisements; six received sponsorship from the National Fund for Natural Sciences; and eight received sponsorship from other sources. Seven journals ran a deficit balance; 14 achieved breakeven and two made profits (no reply from eight journals). Given these disturbing facts, Yu et al. [101] suggested that while the Chinese government should increase its financial investment in these journals, the editorial boards should also learn how to manage the journals more efficiently.
International peer-review has been archived by thirteen of the 31 journals. Peer reviewers were drawn mainly from the West and Japan. Non-Chinese editors are found in eight journals. Apart from two Germans, they all come from English-speaking countries, and their number is limited to one per journal, with one exception which has three non-Chinese editors. All 31 journals studied are now online, of which eleven have their own websites. To different extents, they all manage their editorial process of submission, peer-review and re-submission electronically [101] .
According to Yu et al. [101] , the crux of the problem of journals in mainland China is that their model of operations remains that of planned economy, rendering them unfit to compete in today's Chinese market economy. As of 2006, eighteen of the 31 journals were distributed internationally, mainly through the agency of international publishing groups, like Elsevier, Nature, Springer and Blackwell. Through collaboration with these publishing groups, Chinese journals can benefit in terms of efficiency, economy of scales and share of the international market. Not only does this illustrate the feasibility of international collaboration, but it also provides Chinese publishers a model of development into a commercially viable publishing group of scientific periodicals. However, only seven of these 18 journals achieved an international circulation of more than 100 copies. This reflects the difficulty of breaking through into the international market.
Publishing in English is correlated to higher international visibility [6] . Using data of 2003, Yu et al. [101] showed that English language biomedical journals of China were more likely to be indexed in international databases than their Chinese language counterparts (Table 9 ). However, compared to the international English language journals, their impact was rather low (as indicated by their low impact factor in JCR). Interestingly, by moving towards an international readership, these English language journals of China fared not so well in China either (  The Complete Genome and Proteome of Laribacter hongkongensis Reveal Potential Mechanisms for Adaptations to Different Temperatures and Habitats Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats. Laribacter hongkongensis is a recently discovered, Gram-negative, facultative anaerobic, motile, seagull or S-shaped, asaccharolytic, urease-positive bacillus that belongs to the Neisseriaceae family of bproteobacteria [1] . It was first isolated from the blood and thoracic empyema of an alcoholic liver cirrhosis patient in Hong Kong [2] . In a prospective study, L. hongkongensis was shown to be associated with community acquired gastroenteritis and traveler's diarrhea [3, 4] . L. hongkongensis is likely to be globally distributed, as travel histories from patients suggested its presence in at least four continents: Asia, Europe, Africa and Central America [4] [5] [6] . L. hongkongensis has been found in up to 60% of the intestines of commonly consumed freshwater fish, such as grass carp and bighead carp [4, 7, 8] . It has also been isolated from drinking water reservoirs in Hong Kong [9] . Pulsed-field gel electrophoresis and multilocus sequence typing showed that the fish and patient isolates fell into separate clusters, suggesting that some clones could be more virulent or adapted to human [8, 10] . These data strongly suggest that this bacterium is a potential diarrheal pathogen that warrants further investigations.
Compared to other families such as Enterobacteriaceae, Vibrionaceae, Streptococcaceae, genomes of bacteria in the Neisseriaceae family have been relatively under-studied. Within this family, Neisseria meningitidis, Neisseria gonorrhoeae and Chromobacterium violaceum are the only species with completely sequenced genomes [11] [12] [13] . In view of its potential clinical importance, distinct phylogenetic position, interesting phenotypic characteristics and the availability of genetic manipulation systems [14] [15] [16] [17] , we sequenced and annotated the complete genome of a strain (HLHK9) of L. hongkongensis recovered from a 36-year old previously healthy Chinese patient with profuse diarrhea, vomiting and abdominal pain [4] . Proteomes of L. hongkongensis growing at 37uC (body temperature of human) and 20uC (average temperature of freshwater habitat in fall and winter) [9] were also compared.
The complete genome of L. hongkongensis is a single circular chromosome of 3,169,329 bp with a G+C content of 62.35% ( Figure 1 ). In terms of genome size and number of predicted coding sequences (CDSs), rRNA operons and tRNA genes (Table 1) , L. hongkongensis falls into a position intermediate between C. violaceum and the pathogenic Neisseria species. A similar intermediate status was also observed when the CDSs were classified into Cluster of Orthologous Groups (COG) functional categories, except for genes of RNA processing and modification (COG A), cell cycle control, mitosis and meiosis (COG D), replication, recombination and repair (COG L) and extracellular structures (COG W), of which all four bacteria have similar number of genes ( Figure 2 ). This is in line with the life cycles and growth requirements of the bacteria. C. violaceum is a highly versatile, facultative anaerobic, soil-and water-borne free-living bacterium and therefore requires the largest genome size and gene number. The pathogenic Neisseria species are strictly aerobic bacteria with human as the only host and therefore require the smallest genome size and gene number. L. hongkongensis is a facultative anaerobic bacterium that can survive in human, freshwater fish and 0-2% NaCl but not in marine fish or $3% NaCl and therefore requires an intermediate genome size and gene number.
The L. hongkongensis genome lacks a complete set of enzymes for glycolysis, with orthologues of glucokinase, 6-phosphofructokinase and pyruvate kinase being absent (Table S1 ). This is compatible with its asaccharolytic phenotype and is consistent with other asaccharolytic bacteria, such as Campylobacter jejuni, Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, in that glucokinase and 6-phosphofructokinase are also absent from their genomes [18, 19] . On the other hand, the L. hongkongensis genome encodes the complete sets of enzymes for gluconeogenesis, the pentose phosphate pathway and the glyoxylate cycle (Table S1) . Similar to C. jejuni, the L. hongkongensis genome encodes a number of extracellular proteases and amino acid transporters. These amino acids can be used as carbon source for the bacterium. The genome encodes enzymes for biosynthesis of the 21 genetically encoded amino acids and for biosynthesis and b-oxidation of saturated fatty acids (Tables S2 and S3 ). The L. hongkongensis genome encodes a variety of dehydrogenases (LHK_00527-00540, LHK_01219-01224, LHK_02418-02421, LHK_00801-00803, LHK_01861, LHK_02912-02913 and LHK_00934) that enable it to utilize a variety of substrates as electron donors, such as NADH, succinate, formate, proline, acyl-CoA and D-amino acids. The presence of three terminal cytochrome oxidases may allow L. hongkongensis to carry out respiration using oxygen as the electron acceptor under both aerobic conditions [type aa 3 oxidase (LHK_00169-00170, LHK_00173)] and conditions with reduced oxygen tension [type cbb 3 (LHK_00995-00996, LHK_00998) and type bd (LHK_02252-02253) oxidases]. The genome also encodes a number of reductases [fumarate reductase (LHK_02340-02342), nitrate reductase (LHK_02079-02085), dimethylsulfoxide (DMSO) reductase (LHK_02496-02498) and tetrathionate reductase (LHK_01476-01478)], which may help carry out respiration with alternative electron acceptors to oxygen (fumarate, nitrate, DMSO and tetrathionate) under anaerobic conditions. This is supported by the enhanced growth of L. hongkongensis under anaerobic conditions in the presence of nitrate (data not shown). Further studies are required to confirm if the bacterium can utilize other potential electron acceptors.
There were 441 transport-related proteins (13.6% of all CDSs) in the L. hongkongensis genome, comprising an extensive variety of transporters, which may reflect its ability to adapt to the freshwater fish and human intestines, and freshwater environments. According to the Transporter Classification Database (TCDB) (http:// www.tcdb.org/), all seven major categories of transporters are present in L. hongkongensis. Primary active transporters (class 3 transporters) were the most abundant class of transporters, accounting for 43.3% (191 CDSs) of all annotated CDSs related to transport, among which 104 belong to the ATP-binding cassette (ABC) transporter superfamily and 41 were oxidoreduction-driven transporters. Electrochemical potential-driven transporters (class 2 transporters) were the second most abundant class of transporters, accounting for 27.9% (123 CDSs) of all annotated CDSs related to transport, most of which (117 CDSs) are various kinds of porters including major facilitator superfamily (MFS) (19 CDSs), resistance-nodulation-cell division (RND) superfamily (22 CDSs), amino acid-polyamine-organocation family (8 CDSs), dicarboxylate/amino acid:cation symporter (DAACS) family (5 CDSs) and monovalent cation:proton antiporter-2 family (3 CDSs), and various heavy metal transporters which may be involved in detoxification and resistance against environmental hazards. Three different types of class 2 transporters, belonging to the DAACS, tripartite ATP-independent periplasmic transporter and
Laribacter hongkongensis is a recently discovered bacterium associated with gastroenteritis and traveler's diarrhea. Freshwater fish is the reservoir of L. hongkongensis. In order to achieve a rapid understanding on the mechanisms by which the bacterium adapts to different habitats and its potential virulence factors, we sequenced the complete genome of L. hongkongensis, compared its gene contents with other bacteria, and compared its gene expression at 37uC (human body temperature) and 20uC (freshwater habitat temperature). We found that the gene contents of L. hongkongensis enable it to adapt to its diverse habitats of human and freshwater fish intestines and freshwater environments. Genes encoding proteins responsible for survival in the intestinal environments, adhesion to intestinal cells, evasion from host immune systems, and putative virulence factors similar to those observed in other pathogens are present. We also observed, in gene expression studies, that L. hongkongensis may be using different pathways for arginine synthesis regulated at different temperatures. Phylogenetic analysis suggested that such mechanisms for temperature adaptation may also be used in bacteria found in extreme temperatures.
C 4 -dicarboxylate uptake C family, are likely involved in the transport of malate, which can be used as the sole carbon source for L. hongkongensis in minimal medium [unpublished data]. The remaining class 2 transporters were ion-gradient-driven energizers belonging to the TonB family (6 CDSs). The third most abundant class of transporters was the channels and pores (class 1), with 39 CDSs including 12 a-type channels, 26 b-barrel porins. Among the 12 a-type channels, four were mechanosensitive channels and G+C content (10-kb window with 100-b step); circles 3 to 7, red, light purple, orange, aqua and teal bars show BLAST hits to Neisseria gonorrhoeae FA 1090, Neisseria gonorrhoeae MC58, Neisseria gonorrhoeae FAM18, Neisseria gonorrhoeae Z2491 and Chromobacterium violaceum ATCC 12472, respectively; circle 8, green arcs show location of eight putative prophages; circles 9 and 12, colors reflect Cluster of Orthologous Groups of coding sequences (CDSs). Maroon, translation, ribosomal structure and biogenesis; navy, transcription; purple, DNA replication, recombination and repair; light brown, cell division and chromosome partitioning; aqua, posttranslational modification, protein turnover, chaperones; teal, cell envelope biogenesis, outer membrane; blue, cell motility and secretion; orange, inorganic ion transport and metabolism; light purple, signal transduction mechanisms; olive, energy production and conversion; lime, carbohydrate transport and metabolism; green, amino acid transport and metabolism; fuchsia, nucleotide transport and metabolism; light pink, coenzyme metabolism; red, lipid metabolism; yellow, secondary metabolites biosynthesis, transport and catabolism; gray, general function prediction only; silver, function unknown; circles 10 and 11, dark blue, dark red and dark purple indicate CDSs, tRNA and rRNA on the 2 and + strands, respectively. doi:10.1371/journal.pgen.1000416.g001 which are important for mediating resistance to mechanophysical changes. The remaining transporters belong to four other classes, namely group translocators (class 4, 9 CDSs), transport electron carriers (class 5, 16 CDSs), accessory factors involved in transport (class 8, 9 CDSs) and incompletely characterized transport system (class 9, 54 CDSs). In line with their asaccharolytic nature, the genomes of L. hongkongensis and C. jejuni do not contain genes that encode a complete phosphotransferase system. The five families of multidrug efflux transporters, including MFS (6 CDSs), RND (8 CDSs), small multidrug resistance family (2 CDSs), multidrug and toxic compound extrusion family (2 CDSs) and ABC transporter superfamily (5 CDSs), were all present in L. hongkongensis, which may reflect its ability to withstand toxic substances in different habitats [20] . 20 CDSs were related to iron metabolism, including hemin transporters, ABC transporters of the metal type and ferrous iron, iron-storage proteins and the Fur protein responsible for iron uptake regulation. In contrast to C. violaceum which produces siderophores for iron acquisition, but similar to the pathogenic Neisseria species, proteins related to siderophore formation are not found in L. hongkongensis genome. In addition to a TonB-dependent siderophore receptor (LHK_00497), a set of genes (LHK_01190, LHK_01193, LHK_01427-1428) related to the transport of hemin were present, suggesting that L. hongkongensis is able to utilize exogenous siderophores or host proteins for iron acquisition, which may be important for survival in different environments and hosts.
Except the first strain of L. hongkongensis isolated from the blood and empyema pus of a patient which represented a non-motile variant, all L. hongkongensis strains, whether from human diarrheal stool, fish intestine or environmental water, are motile with polar flagella. The ability to sense and respond to environmental signals is important for survival in changing ecological niches. A total of 47 CDSs are related to chemotaxis, of which 27 encode methyl-accepting chemotaxis proteins (MCPs) and 20 encode chemosensory transducer proteins. While most MCPs are scattered throughout the genome, the transducer proteins are mostly arranged in three gene clusters ( Figure  S1 ). At least 38 genes, in six gene clusters, are involved in the biosynthesis of flagella ( Figure S2 ).
Enteric bacteria use several quorum-sensing mechanisms, including the LuxR-I, LuxS/AI-2, and AI-3/epinephrine/norepinephrine systems, to recognize the host environment and communicate across species. Unlike the genomes of C. violaceum and the pathogenic Neisseria species which encode genes involved in LuxR-I and LuxS/AI-2 systems respectively, the L. hongkongensis genome does not encode genes of these 2 systems. Instead, the AI-3/epinephrine/norepinephrine system, which is involved in interkingdom cross-signaling and regulation of virulence gene transcription and motility, best characterized in enterohemorrhagic E. coli [21, 22] , is likely the predominant quorum-sensing mechanism used by L. hongkongensis. Several human enteric commensals or pathogens, including E. coli, Shigella, and Salmonella, produce AI-3 [23] . A two-component system, QseB/C, of which QseC is the sensor kinase and QseB the response regulator, has been found to be involved in sensing AI-3 from bacteria and epinephrine/ norepinephrine from host, and activation of the flagellar regulon transcription [21] . While the biosynthetic pathway of AI-3 has not been discovered, two sets of genes, LHK_00329/LHK_00328 and LHK_01812/LHK_01813, homologous to QseB/QseC were identified in the L. hongkongensis genome, suggesting that the bacterium may regulate its motility upon recognition of its host environment. The presence of two sets of QseB/QseC, one most similar to those of C. violaceum and the other most homologous to Azoarcus sp. strain BH72, is intriguing, as the latter is the only bacterium, with complete genome sequence available, that possesses two copies of such genes.
Before reaching the human intestine, L. hongkongensis has to pass through the highly acidic environment of the stomach. In the L. hongkongensis genome, a cluster of genes, spanning a 12-kb region, related to acid resistance, is present. Similar to Helicobacter pylori, the L. hongkongensis genome contains a complete urease gene cluster (LHK_01035-LHK_01037, LHK_01040-LHK_01044), in line with the bacterium's urease activity. Phylogenetically, all 8 genes in the urease cassette are most closely related to the corresponding homologues in Brucella species (a-proteobacteria), Yersinia species (c-proteobacteria) and Photorhabdus luminescens (c-proteobacteria), instead of those in other members of b-proteobacteria, indicating that L. hongkongensis has probably acquired the genes through horizontal gene transfer after its evolution into a distinct species ( Figure S3 ). Upstream and downstream to the urease cassette, adi (LHK_01034) and hdeA (LHK_01046) were found respectively. Their activities will raise the cytoplasmic pH and prevents proteins in the periplasmic space from aggregation during acid shock respectively [24, 25] . In addition to the acid resistance gene cluster, the L. hongkongensis genome contains two arc gene clusters [arcA (LHK_02729 and LHK_02734), arcB (LHK_02728 and LHK_02733), arcC (LHK_02727 and LHK_02732) and arcD (LHK_02730 and LHK_02731)] of the arginine deiminase pathway which converts L-arginine to carbon dioxide, ATP, and ammonia. The production of ammonia increases the pH of the local environment [26, 27] . Similar to other pathogenic bacteria of the gastrointestinal tract, the genome of L. hongkongensis encodes genes for bile resistance. These include three complete copies of acrAB (LHK_01425-01426, LHK_02129-02130 and LHK_02929-02930), encoding the best studied efflux pump for bile salts, and two pairs of genes (LHK_01373-01374 and LHK_03132-03133) that encode putative efflux pumps homologous to that encoded by emrAB in E. coli [28] . Furthermore, five genes [tolQ (LHK_00053), tolR (LHK_03174), tolA (LHK_03173), tolB (LHK_03172) and pal (LHK_03171)] that encode the Tol proteins, important in maintaining the integrity of the outer membrane and for bile resistance, are also present [29] .
In the L. hongkongensis genome, a putative adhesin (LHK_01901) for colonization of the intestinal mucosa, most closely related to the adhesins of diffusely adherent E. coli (DAEC) and enterotoxigenic E. coli (ETEC), encoded by aidA and tibA respectively, was observed ( Figure S4 ) [30, 31] . aidA and tibA encode proteins of the autotransporter family, type V protein secretion system of Gramnegative bacteria. All the three domains (an N-terminal signal sequence, a passenger domain and a translocation domain) present in proteins of this family are found in the putative adhesin in L. hongkongensis.
Moreover, a putative heptosyltransferase (LHK_01902), with 52% amino acid identity to the TibC heptosyltransferase of ETEC, responsible for addition of heptose to the passenger domain, was present upstream to the putative adhesin gene in the L. hongkongensis genome ( Figure S4 ). In addition to host cell adhesion, the passenger domains of autotransporters may also confer various virulence functions, including autoaggregation, invasion, biofilm formation and cytotoxicity. The L. hongkongensis genome encodes a putative superoxide dismutase (LHK_01716) and catalases (LHK_01264, LHK_01300 and LHK_02436), which may play a role in resistance to superoxide radicals and hydrogen peroxide generated by neutrophils.
The same set of genes that encode enzymes for synthesis of lipid A (endotoxin), the two Kdo units and the heptose units of lipopolysaccharide (LPS) are present in the genomes of L. hongkongensis, C. violaceum, N. meningitidis, N. gonorrhoeae and E. coli. Moreover, 9 genes [rfbA (LHK_02995), rfbB (LHK_02997), rfbC (LHK_02994), rfbD (LHK_02996), wbmF (LHK_02799), wbmG (LHK_02800), wbmH (LHK_02801), wbmI (LHK_02790) and wbmK (LHK_02792)] that encode putative enzymes for biosyn-thesis of the polysaccharide side chains are present in the L. hongkongensis genome. In addition to genes for synthesizing LPS, a number of CDSs that encode putative cytotoxins are present, including cytotoxins that act on the cell surface [hemolysins (LHK_00956 and LHK_03166) and RTX toxins (LHK_02735 and LHK_02918)] and those that act intracellularly [patatin-like proteins (LHK_00116, LHK_01938, and LHK_03113)] [32, 33] . Furthermore, a number of CDSs that encode putative outer membrane phospholipase A1 (LHK_00790) and collagenases (LHK_00305-00306, LHK_00451, and LHK_02651) for possible bacterial invasion are present.
To better understand how L. hongkongensis adapts to human body and freshwater habitat temperatures at the molecular level, the types and quantities of proteins expressed in L. hongkongensis HLHK9 cultured at 37uC and 20uC were compared. Since initial 2D gel electrophoresis analysis of L. hongkongensis HLHK9 proteins under a broad range of pI and molecular weight conditions revealed that the majority of the proteins reside on the weakly acidic to neutral portion, with a minority on the weak basic portion, consistent with the median pI value of 6.63 calculated for all putative proteins in the genome of L. hongkongensis HLHK9, we therefore focused on IPG strips of pH 4-7 and 7-10. Comparison of the 2D gel electrophoresis patterns from L. hongkongensis HLHK9 cells grown at 20uC and 37uC revealed 12 differentially expressed protein spots, with 7 being more highly expressed at 20uC than at 37uC and 5 being more highly expressed at 37uC than at 20uC (Table 2, Figure 3 ). The identified proteins were involved in various functions (Table 2 ). Of note, spot 8 [N-acetyl-L-glutamate kinase (NAGK)-37, encoded by argB-37] was up-regulated at 37uC, whereas spot 1 (NAGK-20, encoded by argB-20), was upregulated at 20uC (Figures 3, 4A and 4B ). These two homologous copies of argB encode two isoenzymes of NAGK [NAGK-20 (LHK_02829) and NAGK-37 (LHK_02337)], which catalyze the second step of the arginine biosynthesis pathway.
The transcription levels of argB-20 and argB-37 at 20uC and 37uC were quantified by real time RT-PCR. Results showed that the mRNA level of argB-20 at 20uC was significantly higher that at 37uC and the mRNA level of argB-37 at 37uC was significantly higher that at 20uC ( Figure 4C and 4D), suggesting that their expressions, similar to most other bacterial genes, were controlled at the transcription level. When argB-20 and argB-37 were cloned, expressed and the corresponding proteins NAGK-20 and NAGK-37 purified for enzyme assays, their highest enzymatic activities were observed at 37-45uC and 45-50uC respectively ( Figure 4E) . Moreover, NAGK-20, but not NAGK-37, was inhibited by 0.25-10 mM of arginine ( Figure 4F ).
L. hongkongensis probably regulates arginine biosynthesis at temperatures of different habitats using two pathways with two isoenzymes of NAGK. L. hongkongensis and wild type E. coli ATCC 25922, but not E. coli JW5553-1 (argB deletion mutant), grew in minimal medium without arginine, indicating that L. hongkongensis contains a functional arginine biosynthesis pathway. NAGK-20 is expressed at higher level at 20uC than 37uC, whereas NAGK-37 is expressed at higher level at 37uC than 20uC. Bacteria use either of two different pathways, linear and cyclic, for arginine biosynthesis. Similar to NAGK-20 of L. hongkongensis, NAGK of Pseudomonas aeruginosa and Thermotoga maritima, which employ the cyclic pathway, can be inhibited by arginine as the rate-limiting enzyme for negative feedback control [34] [35] [36] [37] . On the other hand, similar to NAGK-37 of L. hongkongensis, NAGK of E. coli, which employs the linear pathway, is not inhibited by arginine [35, 36] . We speculate that L. hongkongensis can use different pathways with the two NAGK isoenzymes with differential importance at different temperatures of different habitats. Phylogenetic analysis of NAGK-20 and NAGK-37 showed that they were more closely related to each other than to homologues in other bacteria ( Figure 5 ). The topology of the phylogenetic tree constructed using NAGK was similar to that constructed using 16S rRNA gene sequences (data not shown). This suggested that the evolution of argB genes in general paralleled the evolution of the corresponding bacteria, and argB gene duplication has probably occurred after the evolution of L. hongkongensis into a separate species. The requirement to adapt to different temperatures and habitats may have provided the driving force for subsequent evolution to 2 homologous proteins that serve in different environments. Notably, among all 465 bacterial species with complete genome sequences available, only Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, Roseiflexus castenholzii and Roseiflexus sp. RS-1 possessed two copies of argB, whereas Anaeromyxobacter sp. Fw109-5 and Anaeromyxobacter dehalo- genans 2CP-C possessed one copy of argB and another fused with argJ ( Figure 5 ). The clustering of argB in two separate groups in these bacteria suggests that argB gene duplication has probably occurred in their ancestor, before the divergence into separate species. The prevalence of T. thermophilus, Deinococcus species and Roseiflexus species in hot springs suggested that this novel mechanism of temperature adaptation may also be important for survival at different temperatures in other bacteria. Further experiments on differential expression of the two isoenzymes at different temperatures in these bacteria will verify our speculations.
Traditionally, complete genomes of bacteria with medical, biological, phylogenetic or industrial interests were sequenced only after profound phenotypic and genotypic characterization of the bacteria had been performed. With the advance in technology and bioinformatics tools, complete genome sequences of bacteria can be obtained with greater ease. In this study, we sequenced and analyzed the complete genome of L. hongkongensis, a newly discovered bacterium of emerging medical and phylogenetic interest, and performed differential proteomics and downstream characterization of important pathways. In addition, putative virulence factors and a putative novel mechanism of arginine biosynthesis regulation at different temperatures were discovered, further characterization of which will lead to better understanding of their contributions to the survival and virulence of L. hongkongensis, the Neisseriaceae family and other bacteria. A similar ''reverse genomics'' approach can be used for the study of other newly discovered important bacteria.
The genome sequence of L. hongkongensis HLHK9 was determined with the whole-genome shotgun method. Three shotgun libraries were generated: one small-insert (2-4 kb) library and one medium-insert (5-6 kb) library in pcDNA2.1, and a largeinsert (35-45 kb) fosmid library in pCC2FOS. DNA sequencing was performed using dye-terminator chemistries on ABI3700 sequencers. Shotgun sequences were assembled with Phrap. Fosmid end sequences were mapped onto the assembly using BACCardI [38] for validation and support of gap closing. Sequences of all large repeat elements (rRNA operons and prophages) were confirmed by primer walking of fosmid clones.
The nucleotide sequence for the complete genome sequence of L. hongkongensis HLHK9 was submitted to Genbank under accession number CP001154.
Gene prediction was performed by Glimmer [39] version 3.02, and results post-processed using TICO [40] for improving predictions of translation initiation sites. Automated annotation of the finished sequence was performed by a modified version of AutoFACT [41] , supplemented by analysis by InterProScan [42] . Manual curation of annotation results was done with support from the software tool GenDB [43] . In addition, annotation of membrane transport proteins was done by performing BLAST search of all predicted genes against the curated TCDB [44] . Ribosomal RNA genes were annotated using the online RNAmmer service [45] . Putative prophage sequences were identified using Prophage Finder [46] . Frameshift errors were predicted using ProFED [47] . CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) were searched by using PILER-CR [48] , CRISPRFinder [49] and CRT (CRISPR recognition tool) [50] .
Single colony of L. hongkongensis HLHK9 was inoculated into brain heart infusion (BHI) medium for 16 h. The bacterial cultures were diluted 1:100 in BHI medium and growth was continued at 20uC for 20 h and 37uC for 6 h, respectively, with shaking to OD 600 of 0.6. After centrifugation at 6,5006g for 15 min, cells were lysed in a sample buffer containing 7 M urea, 2 M thiourea and 4% CHAPS. The crude cell homogenate was sonicated and centrifuged at 16,0006g for 20 min. Immobilized pH gradient (IPG) strips (Bio-Rad Laboratories) (17 cm) with pH 4-7 and 7-10 were hydrated overnight in rehydration buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% IPG buffer pH 4-7 (IPG strip of pH 4-7) and pH 6-11 (IPG strip of pH 7-10) (GE Healthcare) and 60 mM DTT with 60 mg of total protein. The first dimension, isoelectric focusing (IEF), was carried out in a Protean IEF cell electrophoresis unit (Bio-Rad Laboratories) for about 100,000 volt-hours. Protein separation in the second dimension was performed in 12% SDS-PAGE utilizing the Bio-Rad Protean II xi unit (Bio-Rad Laboratories). 2D gels were stained with silver and colloidal Coomassie blue G-250 respectively for qualitative and quantitative analysis, and scanned with ImageScanner (GE Healthcare). ImageMaster 2D Platinum 6.0 (GE Healthcare) was used for image analysis. For MALDI-TOF MS analysis, protein spots were manually excised from gels and subjected to in-situ digestion with trypsin, and peptides generated were analyzed using a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems). Proteins were identified by peptide mass fingerprinting using the MS-Fit software (http://prospector.ucsf. edu) and an in-house sequence database of L. hongkongensis HLHK9 proteins generated using the information obtained from the complete genome sequence and annotation. Only spots with at least two-fold difference in their spot volume between 20uC and 37uC and those uniquely detected at either temperature were subjected to protein identification by MALDI-TOF MS analysis. Three independent experiments for each growth condition were performed.
Essentiality of Arginine for Growth of L. hongkongensis HLHK9 L. hongkongensis HLHK9 cells were grown in minimal medium M63 [51] supplemented with 20 mM L-malate as carbon source and 19 mM potassium nitrate as nitrogen source, and 1 mM each of vitamin B1 and vitamin B12. The pH of all media was adjusted to 7.0 with KOH. Essentiality of arginine for growth of L. hongkongensis HLHK9 was determined by transferring the bacterial cells to the modified M63 medium with or without 100 mM of Larginine. Escherichia coli ATCC 25922 and JW5553-1 (argB deletion mutant) [52] were used as positive and negative controls respectively. All cultures were incubated at 37uC with shaking for 5 days. Growth in each medium was determined by measuring absorbance spectrophotometrically at OD 600 . The experiment was performed in duplicate.
mRNA levels of argB-20 and argB-37 in L. hongkongensis HLHK9 cells grown in 20uC and 37uC were compared. Total RNA was extracted from culture of L. hongkongensis HLHK9 (OD 600 of 0.6) grown in conditions described in proteomic analysis by using RNeasy kit (Qiagen) in combination with RNAprotect Bacteria Reagent (Qiagen) as described by the manufacturer. Genomic DNA was removed by DNase digestion using RNase-free DNase I (Roche). The total nucleic acid concentration and purity were estimated using A 260 /A 280 values measured by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Bacteria were harvested from three independent replicate cultures. cDNA was synthesized by RT using random hexamers and SuperScript III kit (Invitrogen) as described previously [53, 54] (Table S4) . Reactions were first incubated at 50uC for 2 min, followed by 95uC for 10 min in duplicate wells. Reactions were then thermal-cycled in 40 cycles of 95uC for 15 s and 60uC for 1 min. Absolute standard curve method was used for determination of transcript level for each gene. Standard curves were made by using serial dilutions from plasmids containing the target sequences with known quantities. Housekeeping gene RNA polymerase beta subunit, rpoB, was used as an internal control. Triplicate assays using RNAs extracted in three independent experiments confirmed that transcript levels of rpoB were not significantly different (P.0.05) at 20uC compared with 37uC (data not shown). The transcript levels of argB-20 and argB-37 were then normalized to that of rpoB. Triplicate assays using RNAs extracted in three independent experiments were performed for each target gene.
The phylogenetic relationships among NAGK-20 and NAGK-37 of L. hongkongensis HLHK9 and their homologues in other bacteria with complete genomes available were analyzed. Phylogenetic tree was constructed by the neighbor-joining method using Kimura's two-parameter correction with ClustalX 1.83. Three hundred and eleven positions were included in the analysis.
Cloning and Purification of (His) 6 -Tagged Recombinant NAGK Proteins of L. hongkongensis HLHK9 Cloning and purification of (His) 6 -tagged recombinant NAGK proteins of L. hongkongensis HLHK9 was performed according to our previous publications, with modifications [53, 55] . To produce plasmids for protein purification, primers (59-GGAATTCCA-TATGCTGCTTGCAGACGCCC -39 and 59-GGAATTCCA-TATGTCAGGCTGCGCGGATCAT -39 for argB-20 and 59-GGAATTCCATATGGTTATTCAATCTGAAGT -39 and 59-GGAATTCCATATGTCAGAGCGTGGTACAGAT -39 for argB-37) were used to amplify the genes encoding NAGK-20 and NAGK-37, respectively, by PCR. The sequence coding for amino acid residues of the complete NAGK-20 and NAGK-37 was amplified and cloned, respectively, into the NdeI site of expression vector pET-28b(+) (Novagen) in frame and downstream of the series of six histidine residues. The two recombinant NAGK proteins were expressed and purified using the Ni 2+ -loaded HiTrap Chelating System according to the manufacturer's instructions (GE Healthcare).
Purified NAGK-20 and NAGK-37 were assayed for N-acetyl-Lglutamate kinase activity using Haas and Leisinger's method [56] , with modifications. The reaction mixtures contained 400 mM NH 2 OH?HCl, 400 mM Tris?HCl, 40 mM N-acetyl-L-glutamate, 20 mM MgCl 2 , 10 mM ATP and 2 mg of enzyme in a final volume of 1.0 ml at pH 7.0. After incubation at 25uC, 30uC, 37uC, 45uC, 50uC, 55uC or 60uC for 30 min, the reaction was terminated by adding 1.0 ml of a stop solution containing 5% (w/ v) FeCl 3 ?6H 2 O, 8% (w/v) trichloroacetic acid and 0.3 M HCl. The absorbance of the hydroxamate?Fe 3+ complex was measured with a spectrophotometer at A 540 [57] . Inhibition of the kinase activities of NAGK-20 and NAGK-37 were examined with and without 0.25, 0.5, 0.75, 1, 2.5, 5, 10, and 20 mM of L-arginine and incubated at 37uC for 30 min. One unit of N-acetyl-Lglutamate kinase is defined as the amount of enzyme required to catalyze the formation of 1 mmol of product per min under the assay conditions used. Each assay was performed in duplicate. Results were presented as means and standard deviations of three independent experiments. Figure S1 Physical map of the chemotaxis-related genes in L. hongkongensis. While the three gene clusters contain the transducer proteins and some of the methyl-accepting proteins (MCPs), most MCPs are scattered outside the clusters. Genes in orange are coding for chemotaxis transducer proteins; genes in green are coding for MCPs; genes in grey are coding for hypothetical proteins. The numbers refer to the coding sequences in the L. hongkongensis genome.  Malignant mesothelioma Malignant mesothelioma is a fatal asbestos-associated malignancy originating from the lining cells (mesothelium) of the pleural and peritoneal cavities, as well as the pericardium and the tunica vaginalis. The exact prevalence is unknown but it is estimated that mesotheliomas represent less than 1% of all cancers. Its incidence is increasing, with an expected peak in the next 10–20 years. Pleural malignant mesothelioma is the most common form of mesothelioma. Typical presenting features are those of chest pain and dyspnoea. Breathlessness due to a pleural effusion without chest pain is reported in about 30% of patients. A chest wall mass, weight loss, sweating, abdominal pain and ascites (due to peritoneal involvement) are less common presentations. Mesothelioma is directly attributable to occupational asbestos exposure with a history of exposure in over 90% of cases. There is also evidence that mesothelioma may result from both para-occupational exposure and non-occupational "environmental" exposure. Idiopathic or spontaneous mesothelioma can also occur in the absence of any exposure to asbestos, with a spontaneous rate in humans of around one per million. A combination of accurate exposure history, along with examination radiology and pathology are essential to make the diagnosis. Distinguishing malignant from benign pleural disease can be challenging. The most helpful CT findings suggesting malignant pleural disease are 1) a circumferential pleural rind, 2) nodular pleural thickening, 3) pleural thickening of > 1 cm and 4) mediastinal pleural involvement. Involvement of a multidisciplinary team is recommended to ensure prompt and appropriate management, using a framework of radiotherapy, chemotherapy, surgery and symptom palliation with end of life care. Compensation issues must also be considered. Life expectancy in malignant mesothelioma is poor, with a median survival of about one year following diagnosis. Malignant mesothelioma is a cancer originating from the lining cells (mesothelium) of the pleural and peritoneal cavities, as well as the pericardium and the tunica vaginalis [1] . Its distribution may be uni-or multifocal or may involve the lining cells in a continuous manner.
Before 1950, malignant mesothelioma was so rare that some pathologists even questioned its existence [2] . However, the increasing use of asbestos after the second world war led to the description of a causal relationship between asbestos exposure and the development of mesothelioma in 1960 [3] . Although its use was widely abandoned in the western world in the 1980s, the long latency period between exposure to asbestos and onset of mesothelioma, which can range from 15 to 60 years [4, 5] , meant that the mortality rates from mesothelioma have continued to rise. In the USA the annual deaths from mesothelioma peaked at 3060 in 2002 and have subsequently declined, however the incidence of mesothelioma will continue to rise in the UK until it reaches a peak in about the year 2020 [6] . According to data gathered in the US Surveillance Epidemiology and End Results programme for 1973-1992, there has been virtually a constant rate of mesothelioma in females but a consistently higher rate for males. In the 1960s a mesothelioma register was set up in the UK to systematically record the mortality rates from mesothelioma and to try to identify the incidence of tumour development without known occupational exposure. It is predicted that around 90,000 deaths will occur from mesothelioma by 2050, with 65,000 of these occurring from 2002 onwards [7] .
Asbestos is a naturally occurring fibrous silicate, and the risk of developing mesothelioma depends on the exposure to different types of the asbestos mineral fibre. The main asbestos mineral groups are serpentine fibres, which are long and curly, or amphibole fibres which are straight and rod-like. This distinction is important as the serpentine fibre shape is more easily cleared from the respiratory tract. Epidemiologic data suggests that the amphibole, crocidolite, is associated with the highest risk of mesothelioma [8] and that the serpentine fibre, chrysotile has the lowest. The diagnosis of mesothelioma is directly attributable to occupational asbestos exposure; however there is evidence that mesothelioma may result from both paraoccupational exposure (e.g.: women having laundered their husband's overalls) and non-occupational "environmental" exposure [9] . Idiopathic or spontaneous mesothelioma can also occur in the absence of any exposure to asbestos in both animals [10] and humans [8] , and a recent review suggests a spontaneous mesothelioma rate in humans of around one per million [11] .
Non-asbestos mineral fibres have also been shown to induce mesothelioma, such as erionite found in certain areas of Turkey [12] or tremolite in north-western Greece [13] , and whilst not specifically mined for commercial purposes, tremolite is often found as a contaminant of chrysotile asbestos and has been causally linked with an increased risk of mesothelioma [14] . The role of Simian virus (SV-40) in the pathogenesis of mesothelioma is more controversial; SV-40 was found to contaminate polio vaccines in the 1950s and 60s in the UK, and although it has been suggested that it is a causative factor in the development of mesothelioma [15] [16] [17] , recent studies have found no link [18] [19] [20] .
Mesothelioma is primarily a disease of adults and usually presents in the fifth to seventh decades, and 70-80% of cases occur in men. Those diagnosed between the ages of 20 to 40 years usually have a history of childhood exposure [1] . Typical presenting features are those of chest pain, dyspnoea or both [11, 21] , and in one series up to a third of patients presented with breathlessness due to a pleural effusion without chest pain [22] . When it occurs, the chest pain tends to be dull or boring in nature, but a pleuritic-type pain can occur in the presence of pleural effusions. Involvement of the mediastinal structures is well recognized but hoarseness of the voice and superior vena caval obstruction only rarely causes major symptoms. Dysphagia can also occur but this is a late finding. Unlike bronchogenic carcinomas, presentation with haemoptysis, lymphadenopathy and metastatic symptoms are unusual. A chest wall mass, weight loss and sweating are less common presentations, as is peritoneal mesothelioma although involvement may be found in up to one third of cases at autopsy. Presentation of peritoneal mesothelioma is with non-specific symptoms including loss of appetite, nausea and vomiting, diarrhoea or constipation and occasionally ascites. Small bowel obstruction is usually a late feature, and overall the prognosis of peritoneal mesothelioma is worse than pleural mesothelioma with a mean survival time of about 7 months [11] .
Physical examination is usually unremarkable except for signs of pleural effusion and pleural thickening due to tumour infiltration. Finger clubbing is more common than in benign asbestos related pleural disease, and can occur in up to 30% of cases [23] . The tumour originates mainly on the parietal pleura and spreads via the fissures, to encase the lung surfaces. Infiltration of the pericardium can result in signs of cardiac tamponade, and mesothelioma can grow along needle tracks and incisions. Blood tests can reveal an elevated erythrocyte sedimentation rate (ESR) [24] , and there have been isolated case reports of mesothelioma associated with autoimmune haemolytic anaemia [25] .
The combination of accurate history, examination, radiology and the acquisition of pathology is essential in the diagnosis of mesothelioma. A careful history of asbestos exposure is essential, and the identification of at-risk occupations are strong markers of exposure. However, the delay between exposure and presentation may naturally preclude accurate recall of occupational exposure and working conditions which may have occurred up to 60 years previously.
In those patients with a pleural effusion, sampling of the fluid for cytological examination is the first step in con-firming the diagnosis. Pleural fluid cytology is positive for malignant cells in about a third of cases [1] and if the clinical, radiological and cytological results support a diagnosis of mesothelioma then this can be accepted. However, it is uncommon for the definitive diagnosis to be made on pleural fluid cytology alone and pleural biopsy for tissue diagnosis is therefore recommended. A contrast enhanced computed tomogram (CT) scan is essential to both identify the extent of the disease, and help guide a percutaneous biopsy if the pleural fluid cytological analysis is not sufficient.
Radiological imaging is essential for the diagnosis, staging and management of mesothelioma. X-ray, CT, magnetic resonance imaging (MRI) and positron emission tomography (PET) have all been used to evaluate the disease.
Intravenous contrast-enhanced CT is the primary imaging modality for suspected pleural malignant disease. CT allows visualisation of the whole pleural surface and diaphragm and use of a 45-60 second scan delay enables the pleural surfaces to be studied whilst still allowing assessment of the mediastinal nodes [26] . A standard protocol should include the liver and adrenal glands, but in cases where there is a past history of abdominal or pelvic malignancy, the scan should also include the lower abdomen and pelvis [11] .
Distinguishing malignant from benign pleural disease can be challenging. The most helpful CT findings suggesting malignant pleural disease are 1) a circumferential pleural rind, 2) nodular pleural thickening, 3) pleural thickening of > 1 cm and 4) mediastinal pleural involvement [27] . The specificities of these findings were 100%, 94%, 94% and 88% respectively. The sensitivities were 41%, 51%, 36% and 56% respectively. The presence of bilateral pleural calcification on CT is uncommon in malignant mesothelioma [27] . A significant reduction in thoracic volume seen on CT is more common, however, occurring in up to 73% of cases according to some series [28] . Whilst these features have a high positive predictive value, absence of these signs does not reliably exclude the diagnosis of pleural malignancy.
MRI screening is not used routinely in the assessment of malignant mesothelioma, however in patients with potentially resectable disease, MRI can help to provide additional staging information over and above CT. Using gadolinium enhancement, MRI can improve the identification of tumour extension into the diaphragm or chest wall, allowing better assessment of the individual for surgical treatment. MRI also is the imaging modality of choice in those in whom intravenous iodinated contrast is contraindicated [29] .
The standardized uptake value (SUV) in PET is a semiquantitative measure of the metabolic activity of a lesion and the SUV is significantly higher in mesothelioma than in other benign pleural diseases such as pleural plaques or inflammatory pleuritis [29, 30] , and one study found PET scanning to have a 96.8% sensitivity and an 88.5% specificity at distinguishing benign from malignant pleural disease [31] . PET scanning has also increased the accuracy in diagnosing mediastinal nodal metastases [30] and therefore the combination of metabolic and anatomical information provided by PET makes it useful in the staging and preoperative evaluation of mesothelioma. PET may also help as a guide to the optimal site for CT guided pleural biopsy, and there is evidence that changes in the fluorodeoxyglucose (FDG) uptake within the tumour might indicate response to treatment suggesting its role in the assessment of response to both chemotherapy and chemo-radiotherapy [32] .
At least six different staging systems have been suggested for malignant mesothelioma, but none have been accurately shown to predict survival. Currently, a TNM staging system ( Table 1 ) similar to that used in non-small cell lung carcinoma has been proposed by the International Mesothelioma Interest Group (IMIG) [33] .
Tumour response to treatment is an important surrogate for patient benefit. The World Health Organization (WHO) criteria for tumour response were most useful for measuring bi-dimensional lesions, whereas the irregular growth pattern of mesothelioma as a rind around the chest wall makes these criteria poorly applicable [34] . More recently, the Response Evaluation Criteria in Solid Tumours (RECIST) [35] uses a uni-dimensional measurement of tumour size to assess response, but this is based on the assumption that tumours are largely spherical, so again there are limitations to the applicability of this technique in the assessment of malignant mesothelioma.
A modified RECIST criteria has now been published, however, with particular reference to malignant mesothelioma [36] . Assessment of response to treatment is now made by measuring uni-dimensional tumour thickness perpendicular to the chest wall in 2 sites at three different levels on CT.
According to the WHO classification, malignant mesothelioma can be classified as epithelioid, sarcomatoid, or biphasic based on tissue obtained by biopsy. The patho-logical diagnosis is reached with the aid of immunohistochemistry to demonstrate the presence of mesothelial, epithelial, or true sarcomatous differentiation in the malignant cells [37] . The reported diagnostic yield from CT guided biopsy varies from 60% with a single attempt up to 85% with multiple attempts [38] . Ultimately, how-ever, the highest yields are obtained with open or thoracoscopic pleural biopsy.
There is currently no individual immunohistochemical mesothelial marker that provides 100% specificity and high sensitivity for the diagnosis of malignant mesotheli- [37] and so a number of mesothelial and epithelial markers have been developed. Clearly, distinguishing a malignant pleural process from an inflammatory process is a priority and immunohistochemistry has demonstrated that mesothelioma frequently shows immunoreactivity for keratin, p53 and epithelial membrane antigen (EMA) which is unusual for benign pleural disease.
Differentiating malignant mesothelioma from other malignancies, however, can be more difficult as mesotheliomas, especially those of the epithelioid type, can mimic several other tumours. Again, immunohistochemistry can help in the differentiation of mesothelioma and markers such as mesothelin, cytokeratin 5/6, calretinin, thrombomodulin and WT-1 have been used. The most specific and sensitive markers for mesothelioma are mesothelin (in epithelioid mesothelioma), calretinin and cytokeratin 5/ 6. However, according to a recent review article, mesothelin is positive in 27% of squamous cell carcinomas, and calretinin and cytokeratin 5/6 can be raised in both squamous cell carcinoma of the lung and adenocarcinoma of both the lung and other sites [37] . Clearly there can be difficulty in making the diagnosis, and so usually a panel of two positive and two negative markers will be sufficient to confirm the presence of malignant mesothelioma.
Reliably diagnosing malignant mesothelioma early in the disease is notoriously difficult due to the variability in time to presentation from exposure in the disease.
Recently there has been much interest in the use of serum markers for the diagnosis of the disease. The ideal serum marker would be one that is able to offer: 1) early diagnosis, 2) identification of all the subtypes, 3) differentiation of malignant mesothelioma from benign pleural disease and other metastatic pleural malignancies, and 4) be able to track response to therapy and predict survival. As yet no such ideal marker exists, but studies have suggested the use of osteopontin, mesothelin or megakaryocyte potentiating factor in this role.
Osteopontin is a glycoprotein that is over-expressed in lung, breast, colorectal, gastric and ovarian carcinomas and in melanoma. Increased levels correlate with tumour progression, invasion and metastases. Although increased levels do not exclude other malignancies, recent data suggest that osteopontin has great potential use as a marker for mesothelioma [39] and has a positive predictive power for mesothelioma equivalent to Ca-125 for ovarian cancer. Studies have suggested a sensitivity and specificity for mesothelioma of 77% and 85% respectively [40] .
Mesothelin is a membrane bound glycoprotein expressed by mesothelial cells and over expressed in malignancy, especially mesothelioma [41] . Soluble mesothelin related proteins (SMRP) are serum proteins thought to be released by alternative splicing of the mesothelin protein and thereby preventing adherence to cell membranes. Serum concentrations are increased in malignant mesothelioma, and therefore this protein is a potential serum marker for the disease [42] . However, although most patients with mesothelioma have raised serum levels of SMRP, giving a sensitivity of between 80-83% and specificity between 80-100% [43] [44] [45] , it is mostly associated with the epithelioid sub-type, leading to difficulties in identifying the other sub-types of mesothelioma, especially sarcomatoid, using this marker alone [46] . Nevertheless a commercially available FDA-approved assay, MESOMARK (Fujirebio Diagnostics Inc, Malvern, PA, USA), has been recently approved for use in the monitoring of disease in epithelioid and biphasic mesothelioma, and data seems to support its usefulness [47] .
Megakaryocyte Potentiating Factor (MPF) is secreted by cells of several mesothelioma cell lines. In recent studies, MPF was elevated in 91% of 56 malignant mesothelioma patients compared with controls, and levels returned to normal after surgery in patients with peritoneal mesothelioma [46, 48] . This might make MPF useful in the monitoring of treatment response in mesothelioma.
Because there can be difficulty reaching a diagnosis of mesothelioma despite radiology, cytology and biopsy, the general management of patients with mesothelioma should be under a multidisciplinary team including respiratory physicians, oncologists, radiologists, palliative care physicians and lung cancer specialist nurses. In addition, once the diagnosis has been reached, there should be close liaison with the patient's primary care physician, and both the primary care physician and family should be warned that, at least in the UK, a post mortem examination will usually be required after the death of a patient with mesothelioma.
The general treatment strategy of mesothelioma should cover the following areas:
• Management of pleural effusions 
Management of malignant pleural effusions begins with therapeutic thoracocentesis, which assesses the response of dyspnoea to fluid removal. If symptoms do not respond to thoracocentesis, alternative causes of dyspnoea should be sought such as pulmonary thromboembolic disease, or lymphangitis carcinomatosis. Early, successful management on pleural effusions with pleurodesis is essential for the palliation of symptoms and the prevention of a trapped lung. However with repeated thoracocentesis, the pleural fluid may undergo loculation making it difficult to drain subsequently and also the risk of pleural infection increases. If a conclusive diagnosis has been made, chemical pleurodesis can be performed via a small bore intercostal chest drain (9-14F), which has equivalent success rates to larger bore chest drains with the added benefit of patient comfort [49, 50] . The ideal sclerosing agent is sterile talc, with success rates between 70 and 96% [50] [51] [52] , although care must be taken to ensure the talc particles are of the optimal calibration to prevent the rare risk of adult respiratory distress syndrome (ARDS) [53] .
If a firm diagnosis is yet to be made, and the patient is fit enough for surgery, then thoracoscopy is an extremely useful technique in the management of suspected malignant pleural effusions. This procedure allows visualisation of the pleural surface, enables histological sampling for diagnosis and allows complete drainage of the effusion followed by pleurodesis via talc poudrage.
In patients with a trapped lung, or in whom pleurodesis has failed, a pleuro-peritoneal shunt can be considered. Whilst symptoms can improve in over 90% of patients, complications (including shunt occlusion and infection) occur in 15% and therefore their use is diminishing [54] . More recently, an ambulatory pleural drainage system (Pleur x Pleural Catheter, Denver Biomedical Inc, USA) has been developed. This system enables the patient to control the pleural effusion at home by means of a long-term drainage catheter and vacuum bottles. This might help patients with trapped lungs as it both avoids the need for surgical intervention, and palliates dyspnoea [55] .
Palliative radiotherapy Radiotherapy can be used to control local tumour growth and occasionally causes regression of disease, but there is no evidence that radiotherapy alone affects survival [56] . It has, however, been shown to be helpful in the palliation of pain [57] , and approximately half the patients treated with palliative radiotherapy derive some benefit [58] . It also appears that short courses of radiotherapy (for example 20 Gy in 5 fractions) are as effective as longer courses such as 30 Gy in 10+ fractions [59] , although there is a total dose response effect [60] . Unfortunately, radiotherapy is rarely helpful in either the palliation of dyspnoea or the management of symptoms of mediastinal infiltration such as superior vena caval obstruction (SVCO), and alternative methods of treatment such as SVC stenting should be sought [11, 61] .
Whilst hemithorax irradiation may provide symptomatic benefit, no studies have shown that it prolongs survival [61] . Mesothelioma can involve large areas of the pleural cavity and use of radical radiotherapy over large fields places a range of organs such as lungs, liver, spinal cord or heart at a significant risk of dose related damage. As a result, a recent Cochrane review found no randomised clinical trial evidence to support the use of radical radiotherapy alone (or in combination with other treatment modalities) in mesothelioma [62] , and this treatment option has been shown to confer a significant mortality, with rates as high as 17% in one series [59] .
Failure to increase survival using single treatment modalities has led to a multimodality approach to treatment in mesothelioma. Combining debulking surgery with radiotherapy is the cornerstone of this approach and can both reduce systemic recurrence and influence the natural history of the disease [63, 64] . Typically, hemithorax radiotherapy is combined with extra pleural pneumonectomy (EPP -see later) and correctly staged patients with epithelioid mesothelioma can have median survival rates of 33 months [65] . Due to the large, irregular nature of malignant mesothelioma along with the close proximity of other organs, a more directed modality of irradiation which could help improve local tumour control whilst limiting exposure to surrounding organs has been developed, known as intensity modulated radiotherapy (IMRT) [66] . Despite the possibilities for improved tumour control, however, IMRT still carries a potential risk of fatal pulmonary toxicity in mesothelioma [67, 68] .
Mesothelioma may seed malignant cells in procedure scars. Whilst pain from these metastases is rare, they can become uncomfortable, and evidence has suggested that radiotherapy to intervention sites can prevent this complication [69, 70] . Whilst this practice is recommended in current guidelines [11] , evidence is emerging that it should only be offered to those patients who are symptomatic from these subcutaneous tumours as the radiotherapy itself may have side effects [71, 72] .
The role of surgery in the treatment of malignant mesothelioma is still uncertain. The three most common sur-gical procedures are surgical pleurodesis via video assisted thoracoscopic surgery (VATS), debulking surgery (also known as cytoreductive surgery or pleurectomy/decortication (P/D)) and extra pleural pneumonectomy (EPP). This comprises en-bloc resection of the parietal pleura, lung, pericardium and diaphragm and mediastinal nodes [56] . P/D allows the removal of the visceral, parietal and pericardial pleura as well as debulking the tumour and is therefore less demanding, with mortality rates < 5% [73] . P/D has limitations, however, as it does not remove the tumour completely, and the preservation of the ipsilateral lung makes postoperative radiotherapy difficult due to the risk of pulmonary side effects [74] . The impact of P/D on overall survival is contentious, with some evidence suggesting that P/D surgery via a VATS approach may provide a survival benefit in patients with advanced disease not suitable for EPP [75] , whilst other studies do not clearly distinguish the benefit of P/D over EPP [76] .
EPP is a much more demanding procedure with morbidity rates as high as 60% [77] and mortality rates of 4-9% [11, 78, 79] . However, the pneumonectomy in EPP allows the use of high dose hemithoracic radiotherapy, and this combined treatment has been shown to reduce local recurrence and prolong survival in early disease [65] . As a result, as part of the trimodality approach to treatment with surgery chemotherapy and radiotherapy, EPP has arisen as the surgical treatment of choice, albeit without clear randomised controlled trial evidence [78] . In order to clarify this issue, a large randomized trial (Mesothelioma And Radical Surgery (MARS)) is underway [80] . In this trial, EPP is sandwiched between induction chemotherapy and radical radiotherapy. The control arm offers full active tri-modality therapy, although the surgery is limited to debulking surgery. In addition patients receive the same induction chemotherapy and are given radiotherapy to any drain or port sites.
The use of chemotherapy in malignant mesothelioma aims to lengthen survival, improve quality of life and provide symptomatic relief. Currently, there is no single drug or combination therapy that could be considered as standard treatment for mesothelioma. A variety of single agent and combination regimens have been tried in clinical trials with response rates of between 0% and 45% [81] .
A review of studies by Berghmans et al [82] revealed that cisplatin was the most effective single agent for mesothelioma, with carboplatin having similar activity, and in combination with doxorubicin provided a response rate superior to other regimens studied, although there was no clear benefit in terms of survival. Single agent vinorelbine and combination treatment MVP (mitomycin C, vinblas-tine and cisplatin) have also been shown to provide good symptom relief with acceptable toxicity [83] [84] [85] .
Recently, there has been much interest in the use of the antifolate pemetrexed (Alimta; Eli Lilly). Pemetrexed exerts its effect by interrupting folate dependent metabolic synthesis of purines and pyrimidines (the building blocks of DNA and RNA), and a Cochrane review of the effectiveness of combination treatment with cisplatin/pemetrexed [81] suggests a survival benefit, albeit with increased toxicity which is reduced by the co-administration of vitamin B12 and folate supplements. Much of the evidence supporting the use of pemetrexed comes from a single study [86] in which 331 patients, fully supplemented with vitamin B12, demonstrated a median survival of 13.3 months with combination pemetrexed/cisplatin compared to 10.0 months with cisplatin alone (p = 0.05). This was associated with a significant improvement in quality of life and symptom relief compared to cisplatin alone [87] . Combination with carboplatin instead of cisplatin, however, may be an alternative regimen which provides similar efficacies and reduced side effects [88] . An alternative antifolate agent, raltitrexed, has also been shown to confer a survival benefit in combination with cisplatin, with median overall survival increasing from 8.8 months (CI 7.8-10.8) with cisplatin alone to 11.4 months (CI 10.1 to 15) with combination cisplatin-raltitrexed (p = 0.05) [89] . Although this study only demonstrated borderline significance, likely as a result of sample size, the results of these trials mean that combination cisplatin and an antifolate should be the reference regimen in mesothelioma. Recent advances now favour the use of neoadjuvant chemotherapy followed by EPP and radiation for malignant mesothelioma; Weder et al recently published their experience of a prospective trial of neoadjuvant chemotherapy consisting of cisplatin and gemcitabine followed by EPP, demonstrating a median survival of 23 months [90] . Similarly, Flores et al prospectively studied patients given induction gemcitabine and cisplatin followed by EPP.
Median survival of all patients in the study was 19 months, but those patients who completed induction chemotherapy and subsequently underwent EPP had a median survival of 33.5 months [91] .
As a result, all patients who are fit enough (ECOG performance status 0-2) should have the opportunity to discuss the merits of chemotherapy in mesothelioma with an oncologist [11] , in the knowledge that there are no published data which compares survival or symptom control in patients treated with chemotherapy or best supportive care only. The first such trial (MSO-1) [92] is complete, and the preliminary results were published in abstract form in 2007 [93] . These results suggest that the addition of chemotherapy to active symptom control with best supportive care did not infer a significant survival benefit in mesothelioma, however this trial was started before the emergence of pemetrexed.
Given that malignant mesothelioma has a poor overall prognosis, referral of patients to specialist palliative care services will be appropriate at some time in the disease course. Most patients need palliation of symptoms early on in the course of the disease and recognition of this by the patient, family and primary care physician is essential in the management of the patient with mesothelioma. The use of a central lung cancer nurse specialist provides a means by which the patient can gain access to the delivery of care needed for this disease, through the following core elements [11] :
• Communication The majority of patients who survive for more than 2 years have epithelioid histology and death from mesothelioma tends to be due to respiratory failure.
Eligibility for compensation for mesothelioma may vary from country to country. In the UK for example, a diagnosis of mesothelioma allows compensation via the Industrial Injuries Disablement Benefit, War Pensions Scheme or through a Common Law claim from the firm/firms where the asbestos exposure occurred. Patients who cannot identify occupational exposure to asbestos are not entitled for compensation, however pathological confirmation of the diagnosis is not mandatory; establishing the diagnosis and causation on the balance of probability is sufficient, although an unequivocal diagnosis obviously removes any cause for debate. Earlier this year, a new legislation was made in the UK Child Maintenance and Other Payments Act whereby dependants of persons with mesothelioma became eligible to claim for lump-sum compensation after death http://www.opsi.gov.uk/acts/ acts2008a.  Antiviral resistance during pandemic influenza: implications for stockpiling and drug use BACKGROUND: The anticipated extent of antiviral use during an influenza pandemic can have adverse consequences for the development of drug resistance and rationing of limited stockpiles. The strategic use of drugs is therefore a major public health concern in planning for effective pandemic responses. METHODS: We employed a mathematical model that includes both sensitive and resistant strains of a virus with pandemic potential, and applies antiviral drugs for treatment of clinical infections. Using estimated parameters in the published literature, the model was simulated for various sizes of stockpiles to evaluate the outcome of different antiviral strategies. RESULTS: We demonstrated that the emergence of highly transmissible resistant strains has no significant impact on the use of available stockpiles if treatment is maintained at low levels or the reproduction number of the sensitive strain is sufficiently high. However, moderate to high treatment levels can result in a more rapid depletion of stockpiles, leading to run-out, by promoting wide-spread drug resistance. We applied an antiviral strategy that delays the onset of aggressive treatment for a certain amount of time after the onset of the outbreak. Our results show that if high treatment levels are enforced too early during the outbreak, a second wave of infections can potentially occur with a substantially larger magnitude. However, a timely implementation of wide-scale treatment can prevent resistance spread in the population, and minimize the final size of the pandemic. CONCLUSION: Our results reveal that conservative treatment levels during the early stages of the outbreak, followed by a timely increase in the scale of drug-use, will offer an effective strategy to manage drug resistance in the population and avoid run-out. For a 1918-like strain, the findings suggest that pandemic plans should consider stockpiling antiviral drugs to cover at least 20% of the population. Taking into account the above assumptions, the model can be expressed as the following system of deterministic differential equations:
drug-sensitive infections
drug-resistant infections (LTF)
drug-resistant infections (HTF)
where f = β(δ A A + I U + δ T I T ), g = δ r β(δ A A r + I r,U + I r,T ), and the prime ' ′ ' denotes the derivative of the numbers in compartments with respect to the time; β is the baseline transmission rate of the sensitive strain; p is the probability of developing clinical symptoms;
δ A represents the relative transmissibility of asymptomatic infection; δ T is the relative transmissibility of treated individuals infected with the sensitive strain; δ r represents the relative transmissibility of the resistant strain (high fitness); d U and d T are disease-induced death rates of untreated and treated individuals infected with the sensitive strain, respectively; d U,r and d r,U are disease-induced death rates of individuals infected with mutants of LTF and HTF, respectively; µ A is the recovery rate of asymptomatic infection; µ U and µ T represent recovery rates of untreated and treated infected individuals, respectively; α is the rate at which treated individuals develop drug-resistance (rate of de novo resistant mutation); γ is the conversion rate of resistant strains with LTF to HTF; and q is the fraction of infected individuals which receives treatment (treatment level).
The equations for removed subpopulations are given by
where these compartments include both recovered and dead individuals following infection.
While national pandemic plans consider stockpiling antiviral drugs, it is imperative to evaluate the impact of emergence of resistance on antiviral use, particularly for the scenario of limited supply in which run-out is likely to occur. In order to demonstrate the relationship between the level of treatment and drug stockpile, we appended the following equation to the model
where M 0 is the initial size of the stockpile and M (t) represents the number of antiviral courses available at time t. We assumed that the supply is only depleted through treatment of indexed cases with a single course of oseltamivir during symptomatic infection, and no additional antiviral courses will be provided during the pandemic.
We applied a previously established technique to calculate R c [1] , and considered infectious classes in the order X = (A, I U , I T , A r , I r,U , I r,T , I T ,r ) to simplify computations. The system (2)-(4) can be written as
Taking the Fréchet derivatives F = DF and V = DV , and evaluating at the disease free equilibrium
Then, by evaluating V −1 , the control reproduction number is defined by the spectral radius of F V −1 and given by R c = max{R s c , R r 0 }, where R s c and R r 0 are expressed in (1) and (2) of the main text, respectively. 
where S 0 and S ∞ are respectively the initial and final sizes of the susceptible population. Integrating
, and qpS ′ + I ′ T , and substituting into (6), we can express the final size equation as where I U (0) is the initial number of infections at the onset of the outbreak. By integrating equation (3), we can also compute the total number of resistant cases resulted from the treatment of sensitive infections as
To investigate the effect of parameter changes on the results presented by simulations in the main text using baseline values, we performed sensitivity analyses by considering a sampling approach that allows for the simultaneous variations of several key parameters, including the basic reproduction number R 0 , the rate of de novo resistant mutations α, the rate of conversion between resistant strains γ, the relative transmissibility of the resistant strain δ r , and the probability of developing clinical disease p. Using the Latin Hypercube Sampling technique [2] , we generated samples of size n = 1000 in which each parameter is treated as a random variable and assigned a probability function. In this technique, the parameters are uniformly distributed and sampled within their respective ranges. The reproduction number was uniformly sampled from the range [1.5, 2.5] (values found in references [3, 4] ), and the rate of de novo resistant emergence was sampled from the range [0.018, 0.072] (values taken from references [5, 6] ). The corresponding range for the conversion rate of resistant strains was computed using the constraint that the fraction of treated individuals hosting resistance, which undergoes compensatory mutations and subsequently generates resistant strains with high fitness, lies between 1/5000 and 1/500 [7, 8] .
Furthermore, we considered a range of [0.5, 0.7] for the probability of developing clinical disease [9, 10] , and sampled the relative transmissibility of resistant strain with HTF from the range [0.8, 1] [8] . The same ranges of parameter values were also used in a previous study for sensitivity analyses [8] .
To evaluate the effect of parameter changes on the required stockpile and the total number of infections, we ran the simulations for different treatment levels (between 0% and 80%) in the presence/absence of resistance. These simulations, illustrated in Figure 1 , correspond to Figure 2 in the main text, and show that a substantially larger stockpile is required when treatment exceeds a certain level in a constant treatment strategy. This is due to the wide spread of highly transmissible resistance under high pressure of antiviral drugs, which in turn leads to a larger number of total infections. We further ran simulations for the optimal time t * in an adaptive treatment strategy (using a sample of size n = 100), to determine the sensitivity of the results on the parameters variation in minimizing the total number of infections. The results of this sensitivity analysis are illustrated in Figure 2 , with different sizes of stockpile (8.5%, 12%, 25%), when initial treatment levels (0%, 25%) change to 80% at time t * . Regardless of the level of stockpiles, the results show that aggressive treatment should be implemented with shorter delay after the onset of the outbreak as the basic reproduction number increases. However, the implementation of intensive treatment requires a significantly longer delay for higher initial treatment levels as the reproduction number decreases. Transmission Dynamics and Prospects for the Elimination of Canine Rabies Rabies has been eliminated from domestic dog populations in Western Europe and North America, but continues to kill many thousands of people throughout Africa and Asia every year. A quantitative understanding of transmission dynamics in domestic dog populations provides critical information to assess whether global elimination of canine rabies is possible. We report extensive observations of individual rabid animals in Tanzania and generate a uniquely detailed analysis of transmission biology, which explains important epidemiological features, including the level of variation in epidemic trajectories. We found that the basic reproductive number for rabies, R(0), is very low in our study area in rural Africa (∼1.2) and throughout its historic global range (<2). This finding provides strong support for the feasibility of controlling endemic canine rabies by vaccination, even near wildlife areas with large wild carnivore populations. However, we show that rapid turnover of domestic dog populations has been a major obstacle to successful control in developing countries, thus regular pulse vaccinations will be required to maintain population-level immunity between campaigns. Nonetheless our analyses suggest that with sustained, international commitment, global elimination of rabies from domestic dog populations, the most dangerous vector to humans, is a realistic goal. Rabies has been one of the most feared diseases throughout human history and has the highest human case-fatality proportion of any infectious disease [1, 2] . Every year over 7 million people receive post-exposure prophylaxis, and an estimated 55,000 people die from rabies [3] (more than yellow fever, dengue fever, or Japanese encephalitis [4] ). Over 99% of these deaths occur in developing countries where rabies is endemic in domestic dog populations [5] . However, the impacts of canine rabies are often overlooked, largely because human rabies deaths are now extremely rare in Western Europe and North America, where mass vaccination successfully eliminated the disease from domestic dog populations [6] . Increasing incidence of canine rabies in Africa and Asia has prompted concerns that similar strategies may not be effective in these areas [7, 8] . The critical question now is whether global elimination of domestic dog rabies is achievable. Keys to answering this question include: a quantitative understanding of the transmission dynamics of rabies in domestic dog populations, particularly the basic reproductive number, R 0 ; a quantitative understanding of domestic dog demography; and information about the practicality and effectiveness of various vaccination strategies. While recent data support the feasibility and practicality of domestic dog vaccination strategies [9] [10] [11] , there are very little quantitative data on rabies transmission dynamics [12] and the underlying demographic processes.
Transmission is the most important process underlying infectious disease dynamics [13] , but it is also the least understood. Rates of transmission are usually inferred from population patterns of disease incidence, but populationlevel analyses do not capture between-individual variation in transmission resulting from differences in behaviour, genetics, immune status, and environmental and stochastic factors, which play an important role in determining disease dynamics [14, 15] . Contact tracing has been used to directly measure case-to-case transmission, and applications of the technique to emerging infections such as SARS have generated important insights into disease transmission and control in human populations [16, 17] , but transmission processes for diseases circulating in animal populations are much harder to study.
Rabies is an acute viral encephalitis that is spread through the saliva of infected hosts [2] . Clinical manifestations vary, but the neurological phase often includes increased aggression and the tendency to bite and thereby transmit infection; rapid progression to death is inevitable [4] . These distinctive signs make transmission of rabies easier to track than that of most other diseases and provide an unusual opportunity to explore epidemiological patterns at the scale of the individual.
Here, we present data on rabies transmission in two districts of rural Tanzania, Serengeti and Ngorongoro ( Figure  1 ). We were able to monitor the spread of infection using contact-tracing methods, which were feasible due to the discrete and memorable nature of transmission events. We recorded .3,000 potential transmission events between 2002 and 2006 and reconstructed case histories of over 1,000 suspect rabid animals that illustrate heterogeneity in several aspects of transmission, including the latency, movement patterns, and biting propensity of infected individuals. Although these districts border the Serengeti ecosystem, we have argued that domestic dogs are the sole maintenance population of rabies in this community: they make up over 90% of our observations of rabid animals, and the .70 isolates that have been sequenced (from 13 host species) are all consistent with the Africa 1b canid strain [18, 19] . This is one of the most extensive datasets on individual transmission events assembled in an animal population; it has potential to shed light on critical, but often elusive, details of infectious disease transmission. We also analyze data from rabies outbreaks around the world, which provide a global and historical context for the Tanzania dataset.
Analyses of the contact-tracing data generated robust estimates of epidemiological parameters that have important implications for rabies control (Table 1 , Figures 2 and 3 , and Figure S1 ) and provide insight into how infectious disease transmission scales from individual behaviour to populationlevel dynamics. We estimated R 0 for rabies in Serengeti and Ngorongoro districts directly from infectious histories, from reconstructed epidemic trees based on the spatiotemporal proximity of cases, and from the exponential rate of increase in cases at the beginning of an epidemic. Biting behaviour of rabid dogs during the course of infectious periods was highly variable (mean bites per rabid dog ¼ 2.15, 95% confidence interval (CI) from fitting a negative binomial distribution: 1.95-2.37; variance ¼ 5.61, CI: 4.63-6.92; shape parameter k ¼ 1.33; CI: 1.23-1.42) ( Figure 3A ). The probability that an unvaccinated dog developed rabies after being bitten by an infectious animal was high (P rabiesjbite ¼ 0.49, CI: 0.45-0.52) ( Table 1) if the bitten dog was not vaccinated or killed immediately after exposure. Multiplying the average number of dogs bitten per rabid dog by the probability of developing rabies following exposure gave an R 0 estimate of 1.05 (CI: 0.96-1.14) ( Figure 3A and Table 1 ). These estimates should be regarded as lower bounds, because not all transmission events were observed (this calculation excludes rabid dogs that were killed before biting other animals or that disappeared and likely corresponded to unknown or unobserved rabid dogs in other areas; see Materials and Methods). Detailed data on the timing and location of transmission events and infections allowed us to estimate the spatial infection kernel and generation interval (distances and times between source cases and their resulting infections, respectively) ( Figure 2) and 
Canine rabies has been successfully eliminated from Western Europe and North America, but in the developing world, someone dies every ten minutes from this horrific disease, which is primarily spread by domestic dogs. A quantitative understanding of rabies transmission dynamics in domestic dog populations is crucial to determining whether global elimination can be achieved. The unique pathology of rabies allowed us to trace case-to-case transmission directly, during a rabies outbreak in northern Tanzania. From these unusual data, we generated a detailed analysis of rabies transmission biology and found evidence for surprisingly low levels of transmission. We also analysed outbreak data from around the world and found that the transmission of canine rabies has been inherently low throughout its global historic range, explaining the success of control efforts in developed countries. However, we show that when birth and death rates in domestic dog populations are high, such as in our study populations in Tanzania, it is more difficult to maintain population-level immunity in between vaccination campaigns. Nonetheless, we conclude that, although the level of vaccination coverage required is higher than would be predicted from naïve transmission models, global elimination of canine rabies can be achieved through appropriately designed, sustained domestic dog vaccination campaigns.
probabilistically reconstruct transmission networks (Videos S1 and S2). Calculating the average number of secondary cases per rabid dog during the period of exponential epidemic growth (before vaccinations were implemented) from these reconstructions gave similar R 0 estimates of 1.1 in Serengeti district and 1.3 in Ngorongoro (CIs: 1.04-1.10 and 1.26-1.42, respectively) ( Table 1 ). The more traditional approach of estimating R 0 , by fitting a curve to incidence data over the same interval of exponential epidemic growth, also produced similar estimates of 1.2 in Serengeti and 1.1 in Ngorongoro (CIs: 1.12-1.41 and 0.94-1.32, respectively) ( Table 1 and Figure 3B ). This approach is robust to underreporting (Text S1 and Figure S2 ) but should likewise be considered a lower bound, because some local control measures were instituted (such as tying or killing). We also estimated R 0 from the intrinsic growth rate of outbreaks of domestic dog rabies elsewhere in the world ( Table 2) and obtained values between 1.05 and 1.85, which are consistent with our estimates from northwest Tanzania.
For many diseases, R 0 is expected to increase with host density [12, 13, 20, 21] . Despite the domestic dog population density in Serengeti (9.38 dogs/km 2 ) being considerably higher than the dog population density in Ngorongoro (1.36 dogs/ km 2 , see Table 3 ), we were unable to detect significant differences in our estimated values of R 0 between the two districts. Nor did we find any conspicuous differences in R 0 estimated from the outbreaks listed in Table 2 , which represent a wide range of population densities. There may, in fact, be no relationship between R 0 and population density for canine rabies. On the other hand, a subtle relationship between dog density and transmission rates might be difficult to detect for a number of reasons. To investigate whether it would be possible to decipher systematic differences in R 0 across the range of values that we estimated, we simulated outbreaks using our epidemiological parameter estimates, but varied R 0 (from R 0 ¼ 1 to R 0 ¼ 2), whilst maintaining individual variance in biting behaviour (same shape parameter k, see Text S2). Although the mean estimates of R 0 from fitting to these simulated trajectories were accurate, they were surrounded by wide confidence intervals ( Figures S2 and  S5 ), suggesting that if only a small number of epidemics were sampled, any underlying relationship might not be apparent.
Several mass domestic dog vaccination campaigns were carried out in villages in the study districts during the 5-y period. We analysed the impacts of these interventions at the village level to capture the wide variation in achieved levels of vaccination coverage. We incorporated demographic processes (Table 3 gives demographic parameter estimates) and waning of vaccine-induced immunity (see Materials and Methods), because these affect the level of herd immunity within the population at any one time. There were no rabies outbreaks (defined as at least two cases not interrupted by an interval of more than one month) in villages when vaccination coverage exceeded .70%. Small outbreaks occurred in villages with lower coverage and the largest (and longest) outbreaks only occurred in villages with ,20% coverage. Observed outbreak sizes were within the range expected from the heterogeneity of biting behaviour and the coverage achieved by village-level vaccination campaigns ( Figure 4A ).
The effective reproduction number R, which describes transmission once an epidemic is underway, declined during the course of the observed epidemics ( Figure 3C ). At the level of individuals, vaccination coverage reduced the number of secondary cases per rabid dog ( Figure 4B ). More than 300 vaccinated dogs were identified by contact tracing as having been bitten by rabid animals. Only ten of these animals showed any signs indicative of rabies, although in the absence of vaccination approximately 50% (P rabiesjbite ¼ 0.49) ( Table 1) of these would have been expected to succumb to the disease. Individual actions by dog owners such as tying or killing exposed or infectious animals also had an impact. By killing rabid dogs, villagers reduced the overall average infectious period by around 16% (3.7 d for rabid animals that died from the disease versus 3.1 d for all infected animals, including those that were killed). However, there were no consistent declines through time in the number of bites by rabid dogs ( Figure S3 ). Thus we consider vaccination to have been the overwhelming factor in curtailing the outbreaks ( Figure 4A ). From our estimates of R 0 , we calculate the deterministic critical vaccination threshold for rabies elimination in rural To calculate R 0 , we excluded dogs that were killed, tied, or those that disappeared before biting any other dogs. Variability in biting behaviour means that a small number of individuals disproportionately affect transmission and can potentially spark an epidemic, but since most individuals cause few, if any, infections, R 0 is low and most introductions quickly die out ( Figure 4C ). (B) Exponential epidemic growth in Serengeti (blue, R 0 ; 1.2) and Ngorongoro (red, R 0 ; 1.1) districts. The R 0 estimates from the epidemic trajectories were relatively insensitive to the period used for fitting the exponential curve. The inset shows the distribution of R 0 estimates based on fitting to different regions of the time series. (C) The effective reproductive number, R, (averaged over three-month intervals) for Serengeti (blue) and Ngorongoro (red) districts measured from reconstructed epidemic trees that incorporate prior knowledge on who infected whom. Dots indicate the number of secondary cases resulting from each primary case (inferred from the composite tree of most likely links, with random jitter to avoid superposition on the y-axis). R 0 estimated from these reconstructions (during the period of exponential epidemic growth) was ;1. The exponential growth rates of the epidemics were estimated by fitting exponential curves to monthly time series of rabies incidence and converted to estimates of R 0 using the serial interval distribution from the contact tracing data in Tanzania (see Materials and Methods). Estimates based on weekly data are shown in parentheses. The estimated period of exponential epidemic growth, the year of the epidemic onset, and a description of the epidemic setting (where available) are listed. For populations that were partially vaccinated, we corrected our R 0 estimates by dividing by the proportion of vaccinated animals at the onset of the outbreak. Our estimates show that R 0 for canine rabies is inherently low throughout its historic global range. doi:10.1371/journal.pbio.1000053.t002 We found no effect of age on the frequency of litters for female dogs older than 3 months. Domestic dog population growth was estimated from the demography data collected in Serengeti district (r dogs ) and confidence intervals generated from bootstrapping the data. Domestic dog population densities for 2004 were estimated from 2002 national census data (with projected human population growth rates of 2.6% and 3.8% per annum in Serengeti and Ngorongoro respectively) and human:dog ratios (generated from household questionnaires). Alternate estimates of domestic dog population growth rates were extrapolated for each district (r Serengeti and r Ngorongoro ) using these data. Overall domestic dog densities are presented despite considerable variation at the village level. doi:10.1371/journal.pbio.1000053.t003 Figure 4 . The Impact of Vaccination on Transmission (A) The size of village-level outbreaks (defined as at least two cases not separated by more than one month, isolated cases are assumed to be nonpersistent introductions) in Serengeti (blue, n ¼ 138) and Ngorongoro (red, n ¼ 20) districts plotted against village-specific vaccination coverage at the outbreak onset. Coverage was extrapolated from a demographic model initialized with village-specific dog population estimates and incorporating village-specific vaccination data. Gray shading and contours correspond to the probability of observing an outbreak of a particular size or less, generated from 10,000 stochastic simulations of rabies transmission for every initial vaccination coverage (contours were calculated conditional upon .1 secondary case occurring). The inset illustrates a village-level example of the susceptible reconstruction used to calculate instantaneous vaccination coverage plotted beside rabies cases in that village.
(B) The distribution of secondary cases per infectious dog as inferred from reconstructed epidemic trees in Serengeti (blue) and Ngorongoro (red) districts, plotted against vaccination coverage in the village where the primary case occurred. Random jitter was added to prevent superposition on the y-axis.
(C) Probability of an outbreak being seeded by an introduced case under different levels of vaccination coverage. Due to heterogeneity in the transmission process outbreaks rarely occur when coverage is maintained above P crit . However if infections are frequently imported from outside the vaccinated region, at least 40% coverage would need to be maintained to reduce the probability of subsequent outbreaks (of at least ten cases) to ,0.05. doi:10.1371/journal.pbio.1000053.g004
Tanzania to be only 20% (P crit ¼ 1 -1/R 0 ), and even in areas where R 0 is higher, P crit rises to just 40% (Table 2 ). Our observations and simulations ( Figure 4 ) demonstrate that small outbreaks occur by chance even when coverage exceeds P crit and should be expected more frequently when there is individual variation in transmission (Text S2). Herd immunity declines rapidly in the interval between vaccination campaigns because of births and deaths in the domestic dog population (Figure 4 , inset). To maintain herd immunity above P crit between campaigns, therefore, requires a larger proportion of the dog population, P target , to be vaccinated (P target ¼ e (mþdþr)T P crit , where r is the rate of dog population growth, d is the death rate, 1/m is the duration of vaccineinduced immunity, and T is the interval between campaigns (see Materials and Methods)). By incorporating demographic parameters (Table 3) , we estimate that annual campaigns should therefore aim to vaccinate 60% of the dog population to avoid coverage falling below P crit .
The basic reproductive number, R 0 , is the average number of secondary infections produced by an infected individual in an otherwise fully susceptible population [20] . R 0 is the most important parameter in infectious disease epidemiology, and considerable effort has been devoted to its estimation and to understanding its implications for disease control [20, [22] [23] [24] [25] [26] , although it is important to note that some factors not incorporated in R 0 , e.g., host births as well as deaths, may also have important control implications.
Depending upon the quality and quantity of data, a number of approaches can be used to estimate R 0 . Choosing the most appropriate method and assessing its accuracy can be difficult, given the associated assumptions and shortcomings [22] . Most methods do not account for variability in the pathogenesis and behaviour of infected animals; some methods make inferences from quantities that are confounded by (often unmeasured) responses to disease incidence (e.g., epidemic size or prevalence at equilibrium); and different methods are variously biased due to measurement and process error. Although our attempts to estimate R 0 are also imperfect, they do incorporate individual variation in behaviour and pathogenesis, explicitly address several common assumptions, and have been carefully checked for biases through extensive simulations. The overall consistency in the low values of R 0 that we estimated (;1.1 , R 0 , 2) is therefore reassuring and provides optimism for the feasibility of canine rabies control by vaccination.
If R 0 increases with host density in this system, different threshold levels of vaccination coverage would be necessary to eliminate disease in different density populations [12, 20] . However, our data on individual variation in biting behaviour also illustrate that it would be difficult to detect statistical differences in the range of R 0 values that we estimated ( Figure  S2 ). Thus in practice, when only a small number of epidemics are observed, individual variation in transmission may mask any underlying variation in R 0 driven by population density. So although we cannot decipher the relationship between population density and rabies transmission, the consistency of our individual-and population-level estimates from Tanzania and from a wide range of sites around the world allow us to estimate the threshold vaccination coverage necessary to eliminate the disease.
Our estimates of R 0 predict that only relatively low levels of vaccination coverage are required to eliminate rabies (;20-45%), but there is considerable variation in empirically observed levels of coverage that have successfully controlled the disease; low levels of coverage (30-50%) have been successful in some circumstances [27] , although higher levels have also failed [28] . Our analyses suggest that these inconsistencies are, in large part, a consequence of host demography. When vaccinations are carried out in pulses, births and deaths within the host population will continuously reduce the level of herd immunity attained during campaigns (Figure 4, inset) . Turnover of domestic dogs in rural Tanzania is very high (Table 3) ; therefore, annual campaigns should aim to vaccinate 60% of the dog population to maintain vaccination coverage above P crit for the duration of the interval between campaigns. When successive campaigns have achieved this, rabies incidence has declined dramatically despite high endemic levels in adjacent areas [29] . Domestic dog population turnover therefore appears to have had a marked influence on rabies dynamics that explains the variable success of vaccination efforts. The empirically derived consensus that 70% coverage is sufficient for long-term rabies elimination [30, 31] was likely reached because it is effective as a target for annual campaigns in almost all demographic settings, including those with particularly high turnover such as those we describe from Tanzania.
There are other potential explanations and caveats. The nutritional and health status of animals might affect the development of protective immunity in response to vaccination. However, more than 97% of dogs sampled from Serengeti district developed strong antibody titres (.0.5 IU/ ml) in response to vaccination [32] , suggesting that these factors do not impair the efficacy of dog vaccination in rural Tanzania. In addition, numerous practicalities-such as occasional failures in the cold chain, improper vaccination of animals, mistaken registrations, etc.-will all reduce the level of population immunity below the estimated vaccination coverage. Furthermore, our observations and simulations confirm that small outbreaks may occur simply by chance even when coverage exceeds P crit [33] , and these are particularly likely when there is individual variation in transmission (Figure 4 ). Higher levels of coverage are therefore necessary to reduce the chance of outbreaks with greater certainty; especially where the risk from imported infections is highest ( Figure 4C ). This could be a concern if canine rabies were to be eliminated from domestic dog populations but continued to circulate in sympatric wildlife; however, canine rabies was successfully eliminated in Western Europe and North America despite the presence of wildlife hosts capable of transmission.
Thousands of people die every year from this horrific and preventable disease, because the control of canine rabies has been severely neglected in developing countries [2] . Inherent inter-annual periodicity of epidemics exacerbates the situation, with rabies only intermittently perceived as problematic [6] , as illustrated by the recent outbreak in China [34] . The problem of canine rabies has often been considered intractable in rural Africa, because of poor infrastructure, limited capacity, and the misperception that large popula-tions of wild carnivores are responsible for disease persistence. Our analyses show that global control of canine rabies is entirely feasible and that successful elimination of canine rabies in many parts of the world has likely been achieved precisely because R 0 is so low and institutional commitment to maintain high levels of vaccination coverage has been sustained [6] . Achieving vaccination coverage of 60% or more in dog populations in Africa is both logistically and economically feasible through annual vaccination campaigns [9] [10] [11] 29] . The resultant reduction in costs of human postexposure prophylaxis suggest that vaccination interventions targeted at domestic dog populations could translate into appreciable savings for the public health sector [3, 8, 29] . Furthermore, the inherently low R 0 and the tractability of rabies contact-tracing indicates that once endemic rabies is controlled, elimination could be achieved through active case detection in remnant foci of infection (much like the strategy used to eradicate smallpox [35] ); similar measures are proving effective in programmes to eliminate canine rabies in the Americas [36] . However, the most crucial step towards global elimination of canine rabies will be sustained commitment and coordinated efforts to maintain sufficient vaccination coverage in domestic dog populations.
Study areas. We collected data from two districts in northwest Tanzania: Serengeti, inhabited by multi-ethnic, agro-pastoralist communities and high-density dog populations, and Ngorongoro, a multiple-use controlled wildlife area, inhabited by low-density pastoralist communities, predominantly Maasai, and lower-density dog populations (Figure 1 ). Attributes of the dog populations in these districts are presented in Table 3 . Wildlife populations also differ in the two districts, but domestic dogs are the focus of this study because they are the only maintenance population of rabies in the area [18] .
Incidence data. Data on patients with animal-bite injuries from hospitals and dispensaries, case reports of rabid animals from livestock offices, and community-based surveillance activities were used as primary sources [18] . Visits were made to investigate incidents reported in 2002 to 2006 involving suspected rabid animals. Cases were mapped at the site of the incident (wherever possible) and villagers interviewed to evaluate the status of the biting animal, determine its case history, and identify its source of exposure and subsequent contacts (if known). The same procedure was exhaustively followed for all associated exposures/cases. Interviews were conducted with veterinary officers, local community leaders, and livestock field officers in attendance, resulting in an active reporting network. Cases were diagnosed on epidemiological and clinical criteria, adapting the ''six-step'' method through retrospective interviews with witnesses [37] . Rabies was suspected if an animal displayed clinical signs [37] and either (a) disappeared or died within 10 days, or (b) was killed, but had a history of a bite by another animal or was of unknown origin. Additional clinical criteria for wild carnivores (;10% of human exposures were caused by wild animals and ;10% of inferred transmission events involved rabid wildlife) included tameness, loss of fear of humans, diurnal activity (for nocturnal species), and unprovoked biting of objects and animals without feeding. When multiple incidents involving suspected rabid wildlife were reported on the same/consecutive days within neighbouring homesteads, we assumed a single animal was involved.
Brain samples were collected and tested for confirmation wherever possible, but despite efforts to obtain diagnostic samples, most cases reported here were suspected rather than confirmed. Inadequate sample preservation such as storage at room temperature and long intervals between sample collection and testing (during which samples underwent repeated freeze-thaw cycles) probably caused specimens to deteriorate. Composite samples of each brain necessary to achieve the highest test reliability were also rarely available. Nevertheless, a high percentage of samples from suspected cases of rabies were confirmed by laboratory diagnosis (;75%) suggesting that use of epidemiological and clinical criteria is justified and reliable [18] . Researchers are encouraged to contact the authors regarding data availability.
Vaccination data. Dog vaccination campaigns in Serengeti district in 2000 resulted in low and patchy vaccination coverage (35-40% estimated from post-vaccination household surveys). Annual campaigns conducted from 2003 onwards in a 10-km zone adjacent to the western border of Serengeti National Park achieved higher coverage levels of between 40 and 80%. In 2004, the Tanzanian government conducted vaccinations in villages in Serengeti district beyond the 10-km zone reaching 55% coverage across the remainder of the district, but in subsequent years, campaigns were less systematic and conducted in fewer villages. Vaccination in Ngorongoro was restricted to small-scale localised campaigns in the district town centre until 2004, whereupon widespread annual vaccinations were implemented with overall coverage exceeding 80% [9] . Data on the number of dogs vaccinated in each village and on each campaign date were collected from 2003 onwards.
Parameter estimation. The incubation period and duration of infectiousness were estimated for rabies in domestic dogs from records of when individual dogs were bitten, developed clinical signs, and were killed or died. Gamma distributions were fitted to these data using maximum likelihood with interval censoring to account for cases where the relevant data were only approximately known ( Figure  2 and Table 1 ). To estimate the probability distribution of the generation interval, G(t), an incubation and an infectious period were drawn from their respective distributions, a ''time-to-bite'' deviate was drawn from a uniform distribution over the interval of the infectious period, and the two intervals were summed. There was a significant correlation between the length of the infectious and incubation periods, but significance was entirely due to a single data point; we therefore treated the distributions as independent. The spatial infection kernel K(d) was estimated by fitting a gamma distribution to the distances between known source cases and animals that they contacted. Many contacts occurred within the same, or neighbouring, homesteads. In these cases, the precise distance was not always recorded, but we assumed it was less than 100 m. We therefore replaced the probability of a contact within 100 m by the probability distribution averaged over the range 0-100 m.
The basic reproductive number R 0 . (1) Direct estimates from infectious histories. Using maximum likelihood, we fitted a negative binomial distribution to data on biting behaviour of rabid dogs ( Figure 3A ). The probability of developing rabies following a bite (P rabiesjbite ) was estimated, excluding bitten animals that had previously been vaccinated, or that were either killed or vaccinated immediately after the bite, and binomial confidence intervals were calculated. R 0 was estimated as the probability P rabiesjbite multiplied by the average number of bites per rabid dog and confidence intervals were calculated using a resampling procedure. Dogs that were removed (killed or tied up) before causing secondary cases in other dogs (even if they bit people) were excluded from this calculation, as were suspect rabid dogs that either disappeared before biting other dogs or that were of unknown origin and were killed before being observed to bite other dogs ( Figure 3A ). We pooled data from both districts for this estimate because insufficient complete case-histories of rabid dogs (after excluding cases with interventions) were traced to accurately estimate R 0 for Ngorongoro (35 versus 477 in Serengeti). We also estimated R 0 directly from the distribution of secondary cases per rabid dog. Dogs that were bitten by rabid animals but did not develop rabies because of interventions (previous vaccination or being killed/vaccinated immediately after the bite) were multiplied by P rabiesjbite and added to observed secondary cases, giving an expected number of secondary cases per rabid dog in the absence of intervention and a similar estimate of R 0 (1.14, CI: 1.03-1.25) ( Figure  S1 ).
(2) Epidemic tree reconstruction. We used an algorithm for probabilistically constructing epidemic trees based on the location of cases in space and time [38] . For each suspected case (i), we chose a progenitor ( j) at random with probability p ij from all n cases preceding that case, where:
Gðt ik ÞKðd ik Þ G is the distribution of generation times, t ij is the length of time (in days) between the occurrence of case i and its potential progenitor j (G(t) ¼ 0 for t , 0), K is the spatial infection kernel, and d ij is the distance (in km) between the locations of case i and its potential progenitor j (using the average probability when distance ,100 m, see above). Because the dates that some individuals were bitten or developed rabies were only approximately known, 1,000 bootstrapped datasets were generated with the dates drawn randomly from a uniform distribution over the window of uncertainty and a consensus tree of the most probable links was determined and used to generate secondary case distributions illustrated in Figure S1 . Because transmission of rabies from livestock is recorded extremely rarely, we did not allow livestock progenitors, which considerably improved the match between known and assigned links compared to an algorithm where all species could be assigned as progenitors. All detected cases in carnivores (including domestic cat and wildlife cases) were included in the tree reconstructions using the spatial infection kernel and generation interval parameters estimated for domestic dogs. The contribution of nondomestic dog carnivores to the overall epidemic was small, and estimates of within-and between-species transmission are described elsewhere [18] . When known links between primary and secondary cases were not retained in the trees, they were correctly assigned in more than 60% of cases in both districts, indicating that probabilistic reconstruction was effective. The average number of secondary cases putatively produced from each primary case was calculated from the bootstrapped trees. R 0 was estimated as the average number of infections caused per rabid dog that was infectious during the period of exponential epidemic growth. Determining the period of exponential growth is somewhat subjective; for consistency between methods, we used the interval that gave the median R 0 value for time series regression estimates (see below). The choice of interval caused more variance in R 0 estimates for this reconstruction technique than for other methods because it averages the heterogeneous behaviour of a small number of individual animals that spark an epidemic. Thus inclusion or exclusion of particularly infectious individuals has a large effect on R 0 .
(3) Inference from the epidemic curve. A single infection will cause future cases distributed according to the probability distribution of the generation interval. Therefore the number of cases arising in any given interval is the result of those cases that occurred at times in the past whose secondary cases occur in this interval and is determined by the probability distribution of the generation interval. This intuitive description is formalized by the Euler-Lotka equation, adapted for an infection process [25] and an expression for R 0 can be obtained:
GðsÞe Àrs
We estimated the initial growth rate of the epidemic (r) by fitting an exponential curve to incidence data using a generalized linear model. We compared Akaike's Information Criterion values to determine the appropriate error structure (Poisson or negative binomial). The choice of which part of the epidemic curve the model should be fit to was subjective, therefore the model was fit to all possible sections of the epidemic curve (using a minimum of nine consecutive months) and the median, the 2.5th and the 97.5th percentile of the R 0 estimates are presented in Table 1 . Figure 3B (inset) shows that the estimate of R 0 was robust to the interval chosen for fitting the curve. We used the same method to estimate R 0 from data that we had compiled on outbreaks of canine rabies from elsewhere in the world. For these time series, we fitted exponential curves to the intervals between the first recorded case and the month (or week) with highest rabies incidence (Table 2 ) and converted the estimated growth rates to estimates of R 0 using the serial interval distribution data gathered by contact tracing in Tanzania. For partly vaccinated populations, we corrected our R 0 estimates by dividing by the fraction of dogs which were vaccinated prior to the outbreak [12] . For all the outbreaks considered, including those in Tanzania, some localized and individual control measures may have been instituted (such as tying up or killing infected animals), and therefore our R 0 estimates should be regarded as lower bounds. However simulations also revealed that for very low values of R 0 (,1.2), estimates from the epidemic trajectory can be slightly biased upwards ( Figure S2 ). This is probably because at very low levels of R 0 , most introductions do not initiate further cases and therefore a small number of individuals with higher than average biting behaviour are needed to trigger epidemics, thus biasing trajectories. The effective reproductive number R. The effective reproductive number R measures the average number of secondary cases per primary infection once an epidemic is underway. R changes through space and time depending upon the implementation of control measures, the depletion of susceptibles and the build-up of local correlations in the spatial distribution of infected and susceptible individuals. Numbers of secondary cases per rabid dog (inferred from the epidemic tree reconstructions) were calculated monthly and averaged across bootstrapped trees to give a time-varying estimate of R ( Figure 3C ). Although R declined through time in both districts, there was no apparent temporal trend in the biting behaviour of rabid dogs ( Figure S3 ), suggesting that domestic dog vaccination was the main factor responsible for reducing transmission.
Domestic dog demography. To calculate vaccination coverage and the decline in herd immunity due to population turnover and waning of vaccine-induced immunity, it was necessary to estimate the size of the domestic dog populations (N) and their rates of growth (r dogs ). We projected human population sizes in both districts using 2002 national census data [39, 40] , and we calculated human:dog ratios from household questionnaires conducted in 1994, 2003, and 2008 in Serengeti district and in 1994 and 2004 in Ngorongoro district. We then estimated dog populations from the projected human population sizes and the human:dog ratios and calculated the rate of increase of the dog population in each district (r dogs ¼ log(N t /N 0 )/t) ( Table 3 ).
An alternative estimate of the rate of domestic dog population growth was derived from demographic data collected using household questionnaires. The death rate of dogs (d) was calculated using a Cox proportional hazards model of survival from longitudinal data (n ¼ 802). When pups (dogs under 3 months of age) were excluded from the model, neither age nor sex significantly affected survival. The percapita birth rate (b) was assumed to be the product of the sex ratio (q), the average litter size (l), and frequency (/) and pup survival (s) (b ¼ ql/s). These demographic parameters were estimated from crosssectional data (309 litters) and the rate of increase was calculated (r dogs ¼ b -d). Pup survival was estimated from a subset of puppies that remained in the household, because of the unknown fate of puppies that were given away or sold. We suspect that mortality of female puppies is greater than males. However, obtaining reliable data to accurately estimate pup survival is difficult, and the result of assuming equal mortality rates is an estimate of r dogs that is more conservative with respect to vaccination coverage (i.e., results in lower population-level immunity). This estimate of r dogs (0.088 dogs/y) was similar to other estimates from the region [41, 42] and close to those calculated directly from population sizes (r Serengeti ¼ 0.090 dogs/ y, r Ngorongoro ¼ 0.102 dogs/y) ( Table 3) . A comparison of the stable age distribution (calculated from cross-sectional data assuming a roughly constant rate of population growth) was consistent with age distributions predicted from the estimated demographic parameters.
Analysis of the impacts of intervention. To evaluate whether the predicted level of vaccination coverage required to control rabies (P crit ¼ 1 -1/R 0 ) was sufficient in practice [20] , we plotted the size of village-level outbreaks (an outbreak was defined as at least two cases not interrupted by an interval of more than one month) against vaccination coverage in that village at the time of the case that initiated the outbreak.
Vaccination coverage was modeled by susceptible reconstruction using demographic parameters described above (we show the results from using the largest estimate of r dogs (0.10 dogs/y) because this gives the most conservative predictions of the impacts of vaccination, but results are very similar using the lower r dogs estimates). We assumed coverage was approximately 20% in January 2002 and that the duration of vaccine-induced immunity (1/m) was approximately 3 y (http://www.intervet.co.uk/Products_Public/Nobivac_Rabies/ 090_Product_Datasheet.asp). Numbers of vaccinated and susceptible animals within a village were adjusted according to the doses of vaccine used at village vaccination stations on each campaign date (sufficient vaccine was provided such that all animals brought to the station could be vaccinated). A time series of cases in a village and the associated susceptible reconstruction are shown in the inset of Figure 4A .
To predict the expected size of outbreaks given the observed variability in transmission, we simulated outbreaks in a starting population of 500 dogs (similar to the domestic dog population size in an average village); this choice had little effect on our results. We used our parameter estimates (Table 1) to randomly assign secondary cases and corresponding generation intervals. Each realization was seeded by a single animal and the starting population was initialized with vaccination coverage generated from a binomial distribution. For comparison with the outbreak data we conditioned each realization upon .1 secondary case ( Figure 4A ). Demographic parameters were incorporated, and 10,000 runs were completed for each starting condition. We also calculated the probability of an outbreak of a particular size or larger being seeded by one infectious case to evaluate the coverage needed to prevent outbreaks with different degrees of certainty ( Figure 4C and Figure S4 ).
If V and N denote numbers of vaccinated individuals and the total population size respectively, then vaccination coverage can be expressed as a proportion P ¼ V/N. The number of vaccinated dogs declines following a campaign as individuals die and as vaccineinduced immunity wanes (V t ¼ V 0 e -(dþm)t , where d is the death rate and 1/m is the duration of vaccine-induced immunity), whereas the total population grows at the rate of population increase (N t ¼ N 0 e rt ). To prevent sustained endemic transmission, vaccination coverage must be maintained above P crit (such that R is held below 1). From our estimates of demographic parameters and R 0 , we calculated the proportion of the population that needs to be vaccinated, P target , to prevent vaccination coverage falling below P crit during the interval, T, between campaigns: P target ¼ e (mþdþr)T P crit . This formulation for estimating the coverage needed to interrupt endemic transmission given turnover in the domestic dog population assumes that immunity from vaccination lasts an average of 1/m time units and declines exponentially. In reality, vaccine-induced immunity is likely to be closer to a fixed duration, and thus fewer dogs would be expected to lose immunity during the 1-y interval between campaigns than under the exponential model. This indicates that our estimate of P target may be slightly overestimated, although this is an important area for further investigation.
Supporting Information Figure S1 . The distributions of R 0 estimates from fitting curves to simulated epidemic trajectories generated from biting behaviour described by a negative binomial distribution (black) with mean and variance equal to observed biting behaviour or by a poisson distribution (red) with the same mean. The range of R 0 estimates from simulations spans the range of estimates from compiled outbreak data from around the world ( collection and analysis, decision to publish, or preparation of the manuscript. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences BACKGROUND: The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. RESULTS: We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. CONCLUSION: This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection. The ability to detect the presence of GMO is pivotal for consumers to be able to exercise their lifestyle choice of whether to consume, or not, food containing GMOs. Though the detection and quantification of GMO proteins using immunoassay has been reported [1] , denaturation of the protein during processing makes the method unsuitable for GMO testing and quantification of food. The durability of DNA makes it a better substrate for testing and its amplification by PCR is the method of choice, as recommended by the EC (2004/787), for detection and quantification of GMO in samples.
An alternative DNA amplification method was described by Notomi and coworkers [2] called 'loop mediated isothermal amplification' (LAMP). The LAMP assay relies on the design of a set of primers that generate stem looped (hairpin) structures during the early stage of DNA synthesis. The hairpin structures form because two of the primers used contain, at their 5' end, a reverse complement of a sequence that is present in the target further downstream of the initial binding site. Displacement primers help the formation of these hairpins at the ends of the DNA strands and once formed, these structures can be copied into a series of DNA fragments containing multiple units of the target sequence under isothermal conditions utilizing the displacement properties of Bst polymerase (see [3] ). Although LAMP was first described using a set of four primers, enhanced sensitivity was reported using an additional pair of loop primers [4] . As the reactions are performed at a single temperature, LAMP assays can be performed very quickly since there are no separate denaturation, annealing and extension steps, and as such, reactions do not require thermalcyclers. Here we assess the LAMP protocol for the detection of GMOs using primers that target event-specific sequences for transgenic MS8 and RF3 oilseed rape (Brassica napus L.) and generic GMO sequences such as the cauliflower mosaic virus 35S promoter (P-35S) and the promoter and terminator for the nopaline synthase gene (P-nos and T-nos, respectively) from Agrobacterium spp.
The LAMP technique relies on the design of an interrelated set of primers. The orientation and positions are important for self-priming through stem-looped products that drive and perpetuate the reaction.
The OSR events MS8 and RF3 arise from the insertion of two closely related transgenes from the plasmids, pTHW107 and pTHW118, respectively [5] . The former encodes the Barnase gene that give rise to male sterility, which is replaced in the latter by the Barstar gene which restores fertility: both also have the selectable marker bar which confers tolerance to the herbicide glufosinate ammonium. Though the left border of the RF3 insert has undergone rearrangement in the form of duplication and inversion [5] , the right borders of both events are relatively intact (our data [6] which agree with the two sequences in the database).
Even though the insertions of the transgenes have different breakpoints from the plasmids, they are very close so it was possible to design assays for the RF3 and MS8 events utilizing a common set of primers within the transgenes ( Figure 1 ). When used in conjunction with primers for the plant sequence at the border of each event, they are able to detect each event ( Figure 2 ). Since they have 50% of primers in common, it was important to determine whether there was any cross reaction between the assays. Specificity of the two assays was tested using plasmid DNA for each event. No cross-reaction between the two targets was observed ( Figure 2 ).
The sensitivity of the LAMP reaction was assessed in two ways: copy number detection and background in which 10 copies of the target could be detected. Copy number detection was measured by serial dilutions of known amounts of DNA containing the target sequences, either as genomic or plasmid DNA ( Figure 3 ). Reactions fail in both assays at DNA molecule number of less than 1 which is consistent with the stochastic probability of a target being present [7] .
We note that sometimes non-specific amplification can also take place, especially where the target DNA is absent (see Figure 3 ) and there is low amounts of DNA present in the reaction (cf Figures 3a and 4) . These do not form the specific banding patterns representing the different multimeric LAMP products that are characteristic for each assay and thus can be easily distinguished on a gel. Alternative banding patterns have been observed, also for low template reactions; analyses of these products show that they are formed by interactions of the primers used [8] , to form LAMP equivalents of primer-dimers. Two factors seem to be important: in the absence of target, low background DNA may aid the formation of non-specific products that go on to be amplified; and freeze-thaw repetitions may induce damage to the primers to permit the formation of 'primer intermediates' which can then be amplified. Since LAMP is capable of non-specific amplification, techniques that rely on the detection of by-products of DNA synthesis, e.g., the use of magnesium pyrophosphate precipitation [9] or the use of SyBr Green dye [10] may not be able to distinguish between real and Right border sequences of MS8 and RF3 Figure 1 Right border sequences of MS8 and RF3. Sequences of the plant (above) and transgene (below) at the right border junctions for MS8 and RF3. Highlighted sequences are the targets of the LAMP primers. The plant sequences are those shown in Table 1 : dark blue bases highlighted are the outer displacement primers, yellow and green sequences are the 5' and 3' ends of the LAMP primers respectively, and light blue sequences depict the loop primers. The sensitivity of the LAMP assay and its suitability for practical GMO detection was tested using assays for commonly-used sequence motifs, the CaMV 35S promoter (P-35S) and the Agrobacterium promoter and terminator for the nopaline synthase gene (P-nos and T-nos, respectively). These sequences are commonly used in constructs used to create approved GM events (see [11] ). RoundUp Ready™ soya construct contains both P-35S and T-nos so provided convenient template for testing the assays. We used a sample where the copy number of the GM has been well characterized and thus control the number of template in each reaction.
LAMP sensitivity was assessed by the detection of ten RoundUp Ready™ GMO targets in a background of 100 ng of genomic oilseed rape DNA (Figure 3 ). Since the C value of both species is approximately 1 pg [12] , this background DNA represents a GMO level of 0.01% for both Tnos and P-35S assays. OSR DNA was used because we did not have any soya DNA free from RoundUp Ready™. DNA extracted from our 0% CRM was shown to contain 0.002% GMO [13] . The use of 100 ng of this sample would be equivalent to adding 200 copies of the transgene sequence, considerably more than the experimental input of 10 copies. We believe the use of OSR DNA to be a valid substitute since none of the primer sequences for either assay will be present in non-transgenic soya or oilseed rape.
We have tested the upper limits of DNA that LAMP reactions can tolerate and found that up to 200 ng DNA in a 20 μl reaction, positive detection is reproducible. Above this DNA level, reactions become unreliable (data not shown).
We have found that denaturation of template was a prerequisite step prior to amplification, unlike results found by Nagamine and co-workers [14] . This can be explained by the fact that we do not use a base pair destabiliser, such as betaine, in the reaction buffer. Since we are detecting down to near single copies of templates, our results suggest no benefit to the sensitivity of the assay by their inclusion. The consistent amplification within all dilutions showed that LAMP is an 'all or nothing' reaction, with little of the tailing off effect that is often observed in PCRs with diluting templates. This makes it easy to identify positive reactions. Together with specificity and the speed at which reactions can be performed, LAMP is an excellent method for diagnostics [8, 10, 15] .
The use of CaMV 35S promoter sequence in LAMP has previously been reported as a screening method [16] .
Here we demonstrate the sensitivity and reliability of the LAMP method for GMO detection, both with generic and GMO-specific assays. The ability to perform reactions in a simple heated block or water bath without the need for thermal cycling makes testing using LAMP more accessible.
That LAMP is able to detect very small amounts of target and do that even in high amounts of background DNA makes it ideal for GMO detection. GMO testing can be performed in steps: routine screening for the presence of GMOs using generic assays such as for 35S promoters and T-nos; and if required, identification of specific events can be performed using event-specific assays. Equally, direct screening using event-specific assays is also feasible. The levels of sensitivity are orders of magnitude below the permissible threshold for GMO in food and feed (EC regulation 1830/2003), ensuring the detection of the presence of GMOs at acceptable levels and the reliable detection of any presence of unauthorised GM events, for which at present there is no legally acceptable lower limit (according to EC regulations).
Conventional oilseed rape (OSR) seed, variety 'Hearty' was a gift from Christine Lewis, NIAB. The sample was originally purchased from Monsanto UK Ltd (Cambridge, UK). DNA was extracted by grinding 2 g seed with 10 ml extraction buffer [0.5 M NaCl, 0.1 M EDTA pH 8 and 1% (w/v) SDS] in a mortar with a pestle. The sample was emulsified with 5 ml of chloroform:isoamyl alcohol (24:1) and poured into a 20 ml Falcon polypropylene tube. After centrifugation at 1000 g for 5 mins, the aqueous phase was transferred to a new tube and nucleic acids DNA from the oilseed rape MS8/RF3 was extracted from seedlings from a selfed MS8/RF3 plant [17] using DNeasy Plant DNA Extraction Kit (QIAGEN, Crawley, UK). The parent plant was genotyped to be MS8MS8/RF3rf3 using real-time PCR (data not shown). The sample was quantified using picogreen fluorescence (Molecular Probes Inc., Invitrogen). DNA containing RoundUp Ready™ soya was extracted from Soya Roundup Ready™ GMO Reference Material (Fluka Biochemika, Sigma-Aldrich, Dorset, UK) and the GMO concentration of the sample has been accurately determined by dilutions of template combined with statistical analysis [13] .
The plasmid pGreenII 0049 was a gift from Mark Smedley and Wendy Harwood of the John Innes Centre. Details of the plasmid can be found at the website: http:// www.pgreen.ac.uk/JIT/pGreen0000_fr.htm Sensitivity assessment of LAMP Detection Figure 3 Sensitivity assessment of LAMP Detection. A. Sensitivity of LAMP using genomic target. DNA from MS8/RF3 sample (16 ng.μL -1 ) was serially diluted and amplified by LAMP, in triplicate, using primers to assay for the RF3 junction. The numbers in parenthesis represent the approximate copy numbers of the target assuming that the sample represents RF3 in a hemizygous state (determined using RT-PCR data not shown) for the transgene and using 1 pg as the genome size for oilseed rape. C is the no DNA control and M represents molecular size markers. The smear (*) shows an example of non-specific amplification. B. Sensitivity of LAMP using plasmid target. Serial dilutions of the plasmid pGreenII were amplified using primers for the Pnos target. Numbers represent the calculated copy numbers of the plasmid derived from the DNA value. C is the no DNA control and M represents molecular sized markers.
Plasmids containing each event were constructed to test the specificity of the MS8 and RF3 assays separately. The junction at the right borders of the transgenes were amplified by PCR from the MS8/RF3 DNA sample using the displacement (outer) primers of the LAMP reactions, MS8-RF3 DisplR (B3c) separately with MS8 DisplF (F3) and RF3 DisplF (F3), to amplify the MS8 and RF3 junctions, respectively (see Figure 1 and Table 1 ). The fragments were cloned into pGEM-T vector (Promega, Southampton, UK) and transformed into DH5α. Clones containing the correct inserts were confirmed by sequencing.
Target sequences for P-35S, P-nos and T-nos were chosen based upon common identity between different plasmids in the EMBL database containing the promoters and terminator. The sequences of the targets and positions of the primers are provided (see Additional file 1).
Primers for each target segment have Tm's of 50-52°C (calculated using the 2 × AT, 4 × GC formula), except for the F2 and B2 regions (3' of the LAMP primers), where the Tm was 54-56°C. Primer sequences are listed in Table 1 .
For LAMP reactions, primers were purchased from Sigmagenosys (Table 1) . Reactions were performed in 20 μl containing 1 × Bst pol buffer (NEB, Ipswich, UK) with 0.4 mM each dNTP with the appropriate primers listed in Table 1 : Displacement primers were each used at a concentration of 0.2 μM; Loop primers at 0.4 μM and LAMP primers at 0.8 μM in the reactions. Enough reaction reagents, without template and enzyme, were mixed together and split into two. Template (1 μl) was added to 9 μl of the mix and the samples denatured at 95°C for 2 mins and then cooled to 4°C. Bst pol was added to the remaining reaction mix to a concentration of 3.2 U.μl -1 , mixed thoroughly, and 10 μl was added to each reaction. Reactions were incubated at 55°C for 2 hours, followed by 80°C for 10 mins to inactivate the enzyme and stored at 4°C until analysed. Aliquots of the reactions (5 μl) were run on 1.5% (w/v) agarose gels, containing ethidium bromide  Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.
DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported.
Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies.
In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.
Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] 
To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) .
To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance.
Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ).
Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera.
To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi:10.1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles).
To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown).
Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species.
We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization.
Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains.
A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] .
While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals.
A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36] . This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA).
DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the
The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA 
Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.
Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.
White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland.
Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration.
Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay.
Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate.
The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI).
The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN- 
The HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination.
Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: doi:10.1371/journal.pone.0002432.s003 (10.74 MB DOC) Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct. The development of plant virus vectors as in planta expression systems for foreign genes provides an attractive alternative biotechnological approach for peptide expression [1] [2] [3] [4] [5] . This method has been exploited in vaccine production, where small foreign peptides are expressed as a fusion with the viral coat proteins. Essentially, an insertion site has to be determined in the virus genome so that the resulting product will be displayed on the surface of the virus particle which is then propagated in plants and consequently isolated and used as antigen presenting vehicles [5, 6] . Modifications that do not interfere with the normal functions of the particular virus are a prerequisite for this peptide fusion approach. One strategy suggests that foreign gene segments could be fused to the terminus of a viral gene in a way that permits the production of both the fusion product and the native viral protein, thus avoiding interference with normal gene functions. The success of this epitope presentation strategy depends on a detailed knowledge of virus structure at the atomic level, which is only available for a limited number of viruses.
We have recently developed Cucumber green mottle mosaic virus (CGMMV) as a candidate for expressing antigenic peptides in plants [7] . CGMMV is a tobamovirus with a genome size of ∼6. 4 kb which has been well characterized both biologically [8, 9] and structurally [10, 11] . In this study, a truncated dengue virus type 2 envelope (E) protein binding region from amino acid 379 to 423 (EB4) was inserted into the end of the coat protein (CP) open reading frame (ORF) of a previously constructed CGMMV fulllength clone, pCGT7X [7] . The antigenic peptide was chosen based on a recent study that suggests its importance in enabling dengue virus to bind to specific host cell receptors (S. Abu Bakar personal communication). The present study explores the possibility of extrapolating the CGMMV antigenic epitope presentation system for developing diagnostics Figure 1 : Position of primers in constructed plasmid clones. Primers 4-8 were used to amplify the EB4 gene from pCANTAB 5E. Plasmid pCGT7X and amplified EB4 fragments were digested with HindIII and ligated together to obtain the respective plasmid clones as shown. HindIII is the insertion site of EB4 at the end of CGMMV CP. PCR amplification using primer pairs 1-9, 3-9, and 2-7 will yield amplified products of approximately 6.5 kb, 0.85 kb, and 2.2 kb, respectively. and potentially therapeutics against dengue. The study was also used to challenge the size limits of foreign gene insertion into the CGMMV vector as in the previous study the hepatitis B surface antigen (HBsAg) used was only 33 amino acids [7] .
The 45 amino acid-long EB4 protein used in this study has been previously shown to react with the dengue-specific antibody 3H5-1 (S. Abu Bakar personal communication).
Vector. The EB4 coding sequence was amplified from a pCANTAB 5E vector carrying the virus gene using 3 primer pair sets. The resulting PCR products were purified then digested overnight with HindIII restriction endonuclease, with the same treatment carried out on the full-length clone of CGMMV (pCGT7X, ∼9.0 kb) previously constructed [7] . The digested PCR products and the linearized pCGT7X were purified following 1% agarose gel electrophoresis, and then ligated to form pRT, pRG, both containing the TMV read-through sequence [12] and pCG+FSRTRE (containing the CGMMV read-through sequence) [13] , respectively ( Figure 1 ). The three primer sets used in the amplifications were as follows.
Forward RT (5 -CCAAGCTTGCCAATAGCAATTAAT-CATAGGAGTAGAGC-3 ) and Reverse E (5 -CCAAGC-TTCTCCAAAATCCCAAGCTGT-3 ) for construction of  clone pRT, Forward (RT) TGG (5 -AAGCTTGGCAATAGC-AATTAATCATAGGAGTAGAGCCG-3 ) and Reverse Q (5 -CCAAGCTTGTCCAAAATCCCAAGCTGTGT-3 ) for clone pRG, and Forward SRT (5 -CCAAGCTTCCAAATAGCA-ATTAATCATAGGAGTAGAGCCG-3 ) and Reverse E (5 -CCAAGCTTCTCCAAAATCCCAAGCTGT-3 ) for clone pCG+FSRTRE.
The templates used in the in vitro transcription reactions were synthesized through long-distance PCR (LD-PCR) in 50 μL PCR cocktails containing 1X HF Buffer of Phusion DNA Polymerase (Finnzymes, Espoo, Finland) with 1.5 mM MgCl 2 (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5 μM forward primer, CGT7dG (5 -CCGAGCTCGTAATAC-GACTCACTATAGGTTTTA-3 ), 0.5 μM reverse primer, CGMMV 3 -UTR (5 -TGGGCCCCTACCCGGGGAAAA-GGGGGGAT-3 ), 10-20 ng of DNA template, and 1 U of Phusion DNA Polymerase (Finnzymes, Espoo, Finland). The reaction was set up in 0.2 mL tubes, and the thermal cycling was conducted with initial denaturation at 98 • C for 60 seconds, followed by 30 cycles of 98 • C denaturation for 10 seconds, annealing at 63 • C for 20 seconds and elongation at 72 • C for 1 minute and 50 seconds, and finally an extension step at 72 • C for 5 minutes. The amplified product was purified through phenol-chloroform extraction followed by ethanol precipitation. The pellet was dissolved in an appropriate volume of RNase-free distilled water to 1 μg/μL and stored at −20 • C till further use. The in vitro transcription was carried out using the (Ambion, Calif, USA) High Yield Capped T7 RNA Transcription Kit according to the manufacturer protocol. Aliquots of the in vitrosynthesized transcripts were denatured and electrophoresed alongside RNA markers showing its integrity and the expected transcript size of approximately 6.5 kb. Since no DNase I treatment was done, traces of DNA template of the transcription reactions were detected. 2.5. Inoculation with RNA Transcripts. One transcription reaction was used to inoculate 2 plantlets by gently rubbing the reaction mixture over carborundum-dusted first leaf and cotyledons of 10-day-old plantlets. Mock inoculation was done by gently rubbing distilled water onto carborundumdusted first leaves. The excess inoculum was rinsed off using distilled water from the leaf surfaces 60 minutes after inoculation.
Total RNA was isolated from the new leaf of the inoculated and healthy plants using RNeasy Plant Mini Kit (QIAGEN). RT-PCR was performed using AccuPower RT/PCR PreMix (Bioneer, Daejeon, South Korea) with primers CGMMV 3 UTR (5 -TGGGCCCCTACCCGGGGAAAAGGGGGG-AT-3 ) paired with Pst I sense (5 -TAGGAAAAAACC-AGAAGATCTGCAGGAATTTTTCTC-3 ) or C5500F (5 -GTCGCTACAACTAACTCTATTATCAAAAAGGGTC-3 ). Reactions were carried out according to the manufacturer protocols. Infected plants will give a PCR-amplified product of approximately 2.2 kb (with PstI sense and CGMMV 3 UTR primers) or 0.85 kb (with C5500F and CGMMV 3 UTR primers). RT-PCR reactions were carried out for plants at 14, 21 , and 30 day-postinoculation (dpi).
Plant virus isolation procedures used in this study were modified from [8] . Infected plants showing typical symptoms were harvested, weighed, and homogenized in ice-cold 0.1 M phosphate buffer (pH 7.0 containing 1% of β-mercaptoethanol) at 1 mL/g of plant material for 10 minutes using a mechanical blender. The homogenate was filtered through 2 layers of cheesecloth and then mixed with equal volume of chloroform:butanol (1:1). The mixture was then stirred for 1-2 hours at room temperature and then the organic phase was separated from the mixture through centrifugation at 8000 g for 15 minutes. The aqueous layer was transferred to a beaker, 100 mL of NaCl (4 g/L) and PEG6000 added, and the mixture stirred on ice for 1 hour. The precipitated virus was separated from the solution through centrifugation at 10 000 g for 30 minutes at 4 • C. The resulting pellet was reconstituted in 10 mL of 0.1 M phosphate buffer pH 7.0. Any undissolved material was cleared by centrifugation at 10 000 g for 30 minutes at 12 • C. Then 0.2 M EDTA (pH 7) (50 mL/L) was added to the supernatant and the mixture subjected to centrifugation at 110 000 g for 90 minutes at 4 • C. The supernatant was discarded, and the pellet was left to air dry. The virus pellet was then reconstituted in 100 μL of distilled water and stored at 4 • C until used.
Analyses of sequences of the amplified products were carried using BioEdit Sequence Alignment Editor Software (version 6.0.5) (http://www.mbio .ncsu.edu/BioEdit/bioedit.html). The pI and charge values of the coat protein were calculated using the protein calculator developed by Chris Putnam of The Scripps Research Institute (http://www. scripps.edu/∼cdputnam/protcalc.html).
In this study, the chimeric CGMMV vectors pRT, pRG, and pCG-FSRTRE were constructed by inserting the EB4 coding sequence to the end of the CGMMV CP ORF in plasmid pCGT7X. The maps of these constructed clones are shown in Figure 1 , which indicates their respective position of the primers during amplification and cloning procedures. Maps of plasmids pCGT7X are carrying the wild-type CGMMV, and pCANTAB 5E are carrying EB4 with their respective priming sites are also as indicated in Figure 1 . The genome size of wild-type CGMMV is approximately 6.4 kb (without the plasmid backbone), and the resulting chimeric CGMMV genome would be approximately 6.5 kb in size and contains the EB4 and readthrough sequences as well as inserted HindIII restriction recognition sites and additional nucleotides enabling inframe cloning. The pRT and pRG chimeric clones were constructed with the read-through sequence of TMV (CAA-UAG-CAA-UUA). This leaky sequence meets the minimal sequence requirement for effective read-through of the stop codon [12] and had been used successfully in previous reports [14] .
The templates for in vitro transcription of these two clones were generated through LD-PCR (data not shown). The resulting amplified products (∼6.5 kb) consisted of a T7 promoter fused with the chimeric CGMMV genome carrying EB4. Transcripts produced from the two constructs were separately tested for infectivity by inoculating the host plants. After repeated attempts, both the pRT-and pRG-generated transcripts did not cause infection of the inoculated plants (Table 1 ). There was no evidence of virus genomic material in the inoculated plant tissues tested (data not shown). It is speculated that the read-through sequence of TMV may not be suitable for the CGMMV chimeric clones, hence contributing to the absence of infectious virus transcripts. To overcome this possibility, another chimeric clone (pCG+FSRTRE) was constructed using the read-through sequence AAA-UAG-CAA-UUA of the wild-type CGMMV itself ( Figure 1 ). The template for in vitro transcription, based on the pCG+FSRTRE clone, was generated through LD-PCR. The in vitro transcription products ( Figure 2) were used in inoculation studies. The new leaves of plants (Table 1 and Figure 3 ). Infection could be detected through RT-PCR when total RNA of new noninoculated leaf was used as template. Detection of the virus by RT-PCR ( Figure 4 ) suggests that the viruses had moved from the inoculated leaf to new leaves. This implies that the chimeric virus pCG+FSRTRE carrying the read-through sequence from the CGMMV genome (AAA-UAG-CAA-UUA) was infectious and that the virus particles were able to assemble. The plants, however, continued to grow without any further noticeable symptoms. Virus particles were extracted from the infected plants and an aliquot was electrophoretically separated on 15% SDS-PAGE ( Figure 5 ) resulting in two distinct suggesting that the virus population consisted of two species of coat proteins, the EB4-CP fusion (larger in size) and the wild-type CGMMV CP (smaller in size). The ratio of modified to unmodified CP was approximately 1:1.
Apart from the usage of leaky UAG amber stop codons, it has been reported that pI:charge can affect the production of viable recombinant virus [15] . The pI of the epitope is thought to be an important factor as the hybrid coat protein pI:charge value can affect epitope presentation. It was also reported that TMV was more tolerant to positively charged epitopes on its surface. Thus, it was initially speculated that the failure in expression of the foreign peptide was possibly due to the pI:charge value of recombinant CGMMV CP which was different from the wild-type CGMMV CP pI:charge value ( Table 2) . Table 2 shows the isoelectric point (pI) and charge of the wild-type CGMMV CP, the read-through recombinant CGMMV CP, and the EB4 insert. The charge of the EB4 insert is positive and potentially suitable for expression on the surface of the CGMMV CP [2] . Hence, the inserted peptide is speculated to be expressed if the pI:charge value of modified virus CP resembles the pI:charge value of unmodified virus CP. Earlier transcripts (data not shown) generated from fusion clones without a read-through sequence, where their pI values deviated significantly (>6.0) from the wildtype CP (5.08), were not able to cause infection in inoculated plants leading to the suggestion initially that pI:charge value Journal of Biomedicine and Biotechnology 410  420  430  440  450  460  470  480  490  500   510  520  530  540  550  560  570  580  590  600   610  620  630  640  650  660  670  680  690  700 Sequence not determined Deleted sequence Gaps introduced for alignment Figure 6 : Sequence analyses of RT-PCR-amplified products from putative chimeric CGMMV RNA at different days postinoculation (dpi). The sequenced alignment shows that EB4 sequence was truncated and not complete after 21 dpi. The putative chimeric CGMMV produced did not express the EB4 and its genome resembled the wild-type CGMMV. Introduced read-through sequences and extra codons are underlined. Complete EB4 sequence is aligned accordingly with the other sequences.
played an important role in virus particle assembly and infectivity. Thus, the pI value of the recombinant CP constructs was adjusted to more closely resemble the wild-type CP pI value by inserting the acidic amino acid (glutamate) to the 3 end of CP ( Table 2 ). The experiments (Table 1) , however, showed that although the pI was still higher than that of the wild type (Table 2) , the construct pCG+FSRTRE remained infectious. This implies that infectivity of the clones was not directly related to the deviation in pI value with the wild-type virus CGMMV CP.
Sequencing was carried out on RT-PCR-amplified products of viral RNA extracted from putative chimeric virus particles at 30 days postinoculation (dpi) and total plant RNA isolated from inoculated plant materials at 14 and 21 dpi to confirm the expression of EB4. Sequence analysis was done using BioEdit Sequence Alignment Editor Software (version 6.0.5) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The chimeric CP carried the complete EB4 sequence within its genome at 14 dpi ( Figure 6 ). However, the EB4 sequence appeared to be truncated at 21 dpi with an upstream portion of the EB4 sequence was not present. Only part of the downstream sequence of EB4 was detected together with the 3 untranslated region of the CGMMV. EB4 was totally undetectable at 30 dpi. Interestingly, the 5 end of introduced read-through sequence (position 490 to 501) was retained in the genome. The introduced HindIII recognition site at EB4 downstream from position 639 to 644 was found to have been deleted after 30 dpi. Even though only a single band was visible on RT-PCR screening, it was suspected that there could be products with similar sizes which could not be separated in normal agarose gel electrophoresis, therefore, the RT-PCR products from the chimeric CGMMV RNA at 30 dpi were cloned into pGEM-T Easy vector and subjected to sequence analyses. The results (Figure 7 ) confirmed the absence of the EB4 sequence, except for clone pR P3U4 (from nucleotide position of 702 to 746), where only part of the downstream sequence of the EB4 was detected together with the 3 untranslated region of the CGMMV. The 5 end of introduced read-through sequence of AAA-TAG-CAA-TTA (position 594 to 605) was retained within the genome for all sequenced clones (Figure 7) . The introduced HindIII site from position 742 to 747 was deleted for clones pR P3U1, pR P3U3, and pR P3U11 after 30 dpi.
The chimeric CGMMV sequence analyses showed one common similarity (Figures 6 and 7) , that is, that part of the read-through amber stops codon sequence, and additional nucleotides "CC-AAA-TAG" were retained for all sequenced clones. This suggests that deletion had occurred within the host plant. The sequence analysis also showed that the EB4 was not fully expressed in the putative chimeric CGMMV. : Sequence alignments of cloned RT-PCR-amplified products from putative chimeric CGMMV RNA at 30 dpi. Plasmid clones sent for sequence analysis were pR P3U1, pR P3U3, pR P3U4, and pR P3U11. The sequence alignment shows that EB4 sequence was not within the insert of plasmids, except for a partial sequence for pR P3U4 clone. Only part of the downstream sequence of EB4 was detected together with 3 untranslated region of CGMMV for pR P3U4 clone, the rest were without EB4 sequence. Introduced read-through sequences and extra codons are underlined. Complete EB4 sequence is aligned accordingly with the other sequences. The positive results from the initial RT-PCR screening of transcript-inoculated plants at 14 dpi ( Figure 4 ) suggest the presence of EB4. The EB4 was, however, not detectable at 30 dpi, and only present in some (< 50% tested) plants at 21 dpi. These findings strongly suggest that deletion had occurred within the host plants after 14 dpi. The sequence analyses in this section are summarized in Table 3 .
Plant virus vectors-based expression systems have been widely studied for their development as antigen presentation systems as well as for the production of pharmaceutically important peptides. The CGMMV has been previously shown to be suitable for expression of foreign peptide [7] . In this study, CGMMV vector was used to express a 45 amino acid EB4 gene. The integration of the EB4 gene into the end of CGMMV coat protein gene was done via a leaky UAG read-through sequence. Transcripts generated from chimeric clones of pRT and pRG carrying CAA-UAG-CAA-UUA read-through codon sequences were not infectious. This is possibly caused by the failure of self assembly [16] , and thus none of the inoculated plants was systemically infected. The assembly of CGMMV into virus particle has been shown to be essential for the viral movement through phloem [17] , hence another chimeric clone pCG+FSRTRE was constructed carrying read-through sequence (AAA-UAG-CAA-UUA) from the wild-type CGMMV genome. The clone containing this read-through signal was infectious and produced chimeric CGMMV (Table 1 and Figures 4 and 5) . It is, thus, postulated that there is a read-through signal preference between different species of tobamoviruses, in this case between TMV and CGMMV causing possibly viral particle assembly failure. It has also been shown that KGMMV, the tobamovirus, which has the closest genome similarity to CGMMV [13] also utilizes the same CGMMV read-through signal reaffirming the differences between the tobamoviruses. Additionally, unlike other plant virus vectors [15, 18] , this study reaffirms that with CGMMV pI deviation did not appear to be a factor affecting infectivity [7] .
The chimeric (carrying the EB4) and putative wild-type CGMMV were shown to coexist in the virus population of the infected plants ( Figure 5 ). Previous reports show that the efficiencies of the leaky UAG codon varied from 0.5% to 5% so that the ratio of modified to unmodified CP would be between 1:200 and 1:20 [12, 19] . However, in this study, relatively high levels of chimeric coat protein was observed ( Figure 5 ) giving a ratio of modified to unmodified CP of approximately 1:1. It has been suggested that muskmelon host plant could be producing higher levels of translation nonsense suppressor tRNA making the application of the translation read-through signal favorable in this host [7] .
Due to their relatively higher rate of mutation during replication, RNA viruses are evolving rapidly and this is the basis of their ubiquity and adaptability [20, 21] . In this study, it is shown that the EB4 gene sequence carried by the chimeric CGMMV was systemically removed during the infection process. The order of the removal of the transgene was speculated to be the 5 to 3 direction ( Figures  6 and 7) . This is further supported by the detection of two additional nucleotides together with the read-through sequence "CC-AAA-TAG" downstream from the CGMMV CP ORF. This report shows the temporal in-host truncation of the transgene from a chimeric virus in a natural host. Recent report has shown truncation occurring in transgenic plants expressing the same or similar transgenes as the chimeric virus [22] suggesting targeting by a resistance mechanism or competition with the parental virus as the mechanism involved. The exact mechanism of truncation of the transgene in our study is less clear as a previous study using the same vector and host with a different transgene did not exhibit the same instability [7] . The larger size of the EB4 peptide in comparison to the Hepatitis B epitope, however, suggests that the truncation mechanism or transgene recognition by the virus was size dependent.
In summary, we have shown that CGMMV has a readthrough codon preference and that the read-through codon for TMV was shown to be not efficient, as the chimeric CGMMV transcripts utilizing this signal were not infectious. The reported limitation of low-modified coat protein yield of this type of read-through transient expression system appears to have been overcome as relatively equal yield of chimeric and wild-type CGMMV coat protein were produced. This report also provides a rational harvesting timeline for the chimeric virus making this system exploitable for implementation in a plantation scale in the future. It can be suggested that once host plants are infected with the chimeric virus carrying the inserted foreign peptides, the optimum harvesting time would be at around 14 dpi or not more than 20 dpi in order to obtain maximum yield of the full-length transgene. Growth of the infected plants for longer periods to obtain higher yields of the chimeric virus may induce unwanted transgene deletions. This and other factors described earlier should be relevant information for the further development of CGMMV or other plant viruses as vectors for medically important peptides such as for dengue (this study) and Hepatitis B [7] viral antigens. Enhanced Hygiene Measures and Norovirus Transmission during an Outbreak Control of norovirus outbreaks relies on enhanced hygiene measures, such as handwashing, surface cleaning, using disposable paper towels, and using separate toilets for sick and well persons. However, little is known about their effectiveness in limiting further spread of norovirus infections. We analyzed norovirus outbreaks in 7 camps at an international scouting jamboree in the Netherlands during 2004. Implementation of hygiene measures coincided with an 84.8% (95% predictive interval 81.2%–86.6%) reduction in reproduction number. This reduction was unexpectedly large but still below the reduction needed to contain a norovirus outbreak. Even more stringent control measures are required to break the chain of transmission of norovirus. The estimated maximum likelihood estimates are α = 3.35 and β = 1.09, resulting in a peak generation time of 2.6, and a mean generation time of 3.6 days. Other positively skewed unimodal distributions such as the Weibull distributions did not produce a significantly better fit.
As the generation time distribution might also be a realization of a mixture of several components, we fitted the data with a mixture of 2 or 3 gamma distributed components. This did not give a significantly better fit than a 1-component model (Technical Appendix 1 Table) . 
Let t = (t 1 ,…,t n ) be the vector of observed times of symptom onset of observed cases {1, …, n}. We assume that the elements of t are ordered such that t i ≤t j for all i <j. For subsets {i k , … i k+j } ⊆ {1,…,n} with all permutations of observations within this subset are equivalent. We chose 1 possible ordering arbitrarily. We now define a transmission matrix V = (v i,j ), whose elements represent the probability that the person with time of symptom onset t i was infected by the person with time of symptom onset t j , thus
. Assuming that every case i > 2 was infected by another case in the set of observed cases, we get:
for all i > 2. For i =1, the index case, we assume that:
Furthermore, we assume that v i,j = 0 for all j < i. This assumption means that the ordering of times of infection is equivalent to the ordering of observed times of symptom onset, and more specifically, that persons cannot have infected themselves and cannot have infected persons with earlier time of symptom onset than their own. The matrix V is a lower triangular matrix and therefore does not contain cycles.
To translate the transmission matrix V to reproduction number estimates, any The expected number of secondary cases produced by case j in these possible outbreaks based on transmission matrix V is:
To translate this to an estimate of R for each day in the outbreak t; the mean R j of all cases with the same date of symptom onset is calculated, for all dates with observations:
where m represents the label of the first case with symptom onset on day t, and q the total number of cases on day t.
The likelihood that an observed time interval t i -t j represents a transmission event is determined as a product of the probability that i was infected by j and the probability that the time interval of symptom onset is t i -t j . That is,
The likelihood of any case-patient j transmitting infection to case-patient i, becomes:
Combining for all observed cases, the likelihood of a transmission matrix V becomes:
for a given value of θ, and omitting the index case (i = 1) from the multiplication. Given the parameters for the generation time distribution θ, and all dates of symptom onset t, the parameters v i,j can be estimated. To estimate v i,j , the above likelihood function was evaluated in an adaptive rejection algorithm (Metropolis Hastings sampler) obtaining sets of V matrices with relative frequencies proportional to their likelihood (2) (3) (4) .
To be reasonably certain of convergence and sufficient mixing, we have run 4 independent chains of 40,000 iterations and 3 independent chains with additional information about population structure and pathogen genotype and compared resulting estimates of reproduction numbers.
Adding additional information is possible by setting implausible transmission probabilities in the transmission matrix V to 0. This may be considered a very strong prior assumption, but we have seen (Figure 4 in main text) that the resulting reproduction numbers are not strongly influenced by this radical assumption. In a true Bayesian approach, we might have applied different weights to pairs of cases within and between camps by multiplying a matrix containing these weights with the transmission matrix V.
As described above, case-patients with a date of symptom onset on the same day are given an arbitrary order of infection within that day. Sampled transmission matrices represent all possible (noncyclic) patterns among cases, given the arbitrary order. Now any other possible pattern can be found by permutation of indexes among cases with the same date of symptom onset. Because these all have the same contribution to the likelihood such permutations do not change the likelihood: all permutations are equally likely. Such permutations also have the same reproduction numbers, only for different cases (indices). If we average over all such permutations with identical contributions, the resulting reproduction numbers do not change.
The expected time course of the reproduction number R(t) is given by the following equation:
Here, is the day of implementation of enhanced hygiene measures, G is the cumulative probability function of the generation time distribution, h t ρ is the relative reduction of the reproduction number due to implementation of hygiene measures and is the effective reproduction number without enhanced hygiene measures. u R Polyomaviruses KI and WU in Immunocompromised Patients with Respiratory Disease Polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified, mainly in respiratory specimens from children. Among 200 patients with respiratory disorders admitted to Saint Louis Hospital, Paris, France, KIPyV was detected in 8% and WUPyV in 1%. KIPyV was significantly more frequent among human stem cell transplant patients (17.8% vs. 5.1%; p = 0.01). R ecently, 2 new, distinct polyomaviruses (PyVs), KI (KIPyV) and WU (WUPyV), were identifi ed in respiratory specimens, mainly from children <5 years of age with respiratory tract infections. The fi rst retrospective studies of respiratory specimens in Sweden and Australia showed a KIPyV prevalence of 1% and 2.5%, respectively (1, 2) . Studies conducted in Australia and the United States showed a WUPyV prevalence in respiratory specimens of 3% and 0.7%, respectively (3) . Further studies conducted in Canada and South Korea have shown similar frequencies (4, 5) . In this study, we examined the prevalence of KIPyV and WUPyV in immunocompromised patients with suspected respiratory tract infections.
From January through June 2007, 265 respiratory samples were received in the laboratory of Saint Louis Hospital, Paris: 154 nasal aspirates (NA) and 111 bronchoalveolar lavage (BAL) specimens collected from 200 patients with suspected upper or lower respiratory tract infections. This hospital specializes in the management of immunocompromised patients. Respiratory samples were collected for the diagnosis of acute respiratory illness; 89% of samples were from immunocompromised patients. Their median age was 46 years (range 3.6-85.3 years). Given the observational nature of the study, French law did not require ethical approval or informed consent.
The specimens were routinely tested for infl uenza A and B viruses, respiratory syncytial virus, and parainfl uenza viruses 1, 2, and 3 by immunofl uorescence assay (Imagen; DakoCytomation, Trappes, France). Specimens positive for KIPyV or WUPyV were tested for adenoviruses; human bocavirus; human rhinoviruses; human metapneumovirus; human coronaviruses OC43, 229E, NL63, HKU1; and human PyVs BK and JC by using PCR methods (6) (7) (8) (9) (10) (11) . Total nucleic acid was extracted from 200 μL of NA, BAL, or stool specimens by using the EasyMag System (bio-Mérieux, Marcy l'Etoile, France). KIPyV was detected with an in-house real-time PCR assay targeting the VP1 gene. The primers and hydrolysis probe were designed by using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). The fi nal reaction volume was 25 μL and contained 12.5 pmol of SLKI-VP1s (5′-GGAAATACAGCTGCTCAGGAT-3′) and SLKI-VP1as (5′-CTTTGATACTTGAACCGCTTTCCTT-3′), 6.25 pmol of corresponding probe SLKI-VP1PR (5′-6FAM-C GTGACCCCACCCCTCATTACTGGTC-TAMRA-3′), 12.5 μL of TaqMan Universal Master Mix (Applied Biosystems), and 5 μL of DNA extract. The reaction was run on a 7500 Real-Time PCR System (Applied Biosystems). The specifi city of positive specimens was confi rmed by using PCR and nested PCR with primers POLVP1-39F/POLVP1-363R and POLVP1-118F/POLVP1-324R, as described (1) . The PCR products were then sequenced and compared with the previously described sequences from Sweden and Australia (GenBank accession nos. EF127906, EF127907, EF127908, EF520287, EF520288, and EF520289). WUPyV was detected by PCR as described (3) . PCR products with the expected molecular weights were sequenced by using primers AG0044 and AG0045 and compared to published sequences (GenBank accession nos. EF444550, EF444551, EF444552, EF444553, and EF444554) (3).
KIPyV was detected in 17 (6.5%) of the 265 respiratory samples and in 16 (8.0%) of the 200 patients. All cases were confi rmed by a nested PCR targeting another region of the VP1 gene. Twelve of the 17 PCR products were successfully sequenced, and all shared 100% homology with published sequences. WUPyV was detected in only 2 patients (1.0%). Genome sequencing showed 98% homology with reported WUPyV sequences.
Six KIPyV-positive patients (37.5%) had co-infections with other respiratory viruses, and 2 of them (12.5%) had a pulmonary bacterial infection (online Appendix Table, available from www.cdc.gov/EID/content/15/1/107-appT. htm). One WUPyV-infected patient who exhibited acute respiratory failure had concomitant pneumonia caused by Pseudomonas aeruginosa infection. None of the 15 patients who were positive for KIPyV or WUPyV and tested for fungi had respiratory or blood samples positive for Aspergillus spp. (Table) . Lung or sinus imaging was assessed by computed tomography scan for 12 KIPyV-positive patients. Lung parenchyma abnormalities were noted in 9 patients, and sinusitis was diagnosed for 2 patients.
Taking into consideration both the frequency of digestive symptoms in our patients and the former published detection of KIPyV in a stool sample, we looked for KIPyV infection in the available stool samples while the respiratory samples were being assessed for KIPyV (1). Strikingly 
This study shows the prevalence of KIPyV and WUPyV among immunocompromised patients with respiratory disorders. Previously, these 2 viruses had been observed mainly in young children (1) (2) (3) . Of the few adult patients with KIPyV or WUPyV infection mentioned in these studies, most were immunocompromised (3, 12) .
Considering the seemingly higher prevalence of KIP-yV in our population (8%), immunocompromised patients may be more susceptible to this PyV, as they are to JC and BK viruses (13) (14) (15) . Results from previous reports suggest a similar frequency of both KIPyV and WUPyV infections being found in respiratory specimens, ranging from 1% to 3%. In contrast, in our series, we found a likely difference between the prevalence of KIPyV (8%) and WUPyV (1%), which suggests that the replication or reactivation of the 2 viruses in the respiratory tract may differ between immunocompromised and immunocompetent patients. However, this difference requires further investigation, in particular, by using similar real-time PCR assays. Notably, a significantly higher prevalence of KIPyV infection was found among HSCT patients, which suggests that a profound T-cell defi ciency may be a factor in facilitating KIPyV replication.
As reported in other populations, our patients who yielded positive specimens for KIPyV or WUPyV had conditions ranging from a common cold to acute respiratory distress that required invasive ventilation. Respiratory coinfections, observed in other studies, had likely accounted for at least some clinical features. In the 7 of our patients in whom KIPyV was the sole pathogen detected in the respiratory tract, despite comprehensive screening for viruses, bacteria, parasites, and fungi, clinical and radiographic patterns were varied. Some of the patients had only upper respiratory tract infections, notably sinusitis, whereas others had lung parenchyma abnormalities as defi ned by computed tomographic scan imaging. However, due to the retrospective nature of the study, and therefore the lack of a control group of immunocompromised patients without respiratory symptoms, the association of KIPyV infection with the occurrence of respiratory disease cannot be stated defi nitively.
In conclusion, the seemingly higher frequency of KIP-yV shedding in immunocompromised patients (as observed with other PyVs) and the detection of KIPyV as a single pathogen in respiratory disease (e.g., as cytomegalovirus recurrence can lead to pneumonia in immunocompromised patients) together support a reactivation hypothesis. Nevertheless, a reinfection hypothesis cannot be excluded due to immunocompromised patients' increased risk of acquiring viral infection from exogenous sources Controlled prospective studies of KIPyV shedding before and during immunosuppression will help determine the pathogenic role of this virus. The clinical implication of KIPyV detection in stools and the mechanisms underlying the concomitant presence in gastrointestinal and respiratory tracts also deserve further analysis.
We thank F. Freymuth, P. Lebon, and A. Vabret for advice and technical assistance and for providing positive virus controls.
Dr Mourez is a virologist in the Laboratory of Microbiology, Saint Louis University Hospital, Paris. His research interests include the development of tests for the diagnosis of emerging respiratory viruses and the study of the circulation and molecular analysis of human respiratory viruses in pediatric and immunocompromised patients.  On the significance of Surfactant Protein-A within the human lungs Surfactant Protein-A (SP-A) is the most prominent among four proteins in the pulmonary surfactant-system. SP-A is expressed by alveolar epithelial cells type II as well as by a portion of non small cell lung carcinomas (NSCLC). The expression of SP-A is complexly regulated on the transcriptional and the chromosomal level. SP-A is a major player in the pulmonary cytokine-network and moreover has been described to act in the pulmonary host defense. By the use of cell culture or animal models the functional properties have been repeatedly shown in many aspects, often bearing surprising properties which strongly indicate the physiological importance of SP-A. To date SP-A is recognized as a molecule essential for pulmonary development, structure and function. An upcoming number of reports deals with the role of SP-A for pulmonary pathology. This article gives an overview about the state of knowledge on SP-A focused in applications for human pulmonary disorders and points out the importance for pathology-orientated research approaches using immunohistochemistry or in situ hybridization as promising methods to further elucidate the role of this molecule in adult lung diseases. The role of the surfactant system for the development of the human lung is known to be essential. Since it is synthesized by humans starting in the 28 th week of pregnancy and reaching functional levels in the 34 th week, surfactantsubstitution-therapy is a fundamental part of the treatment of premature babies suffering from Infant Respiratory Distress Syndrome (IRDS) [1] .
Pulmonary surfactant regulates dynamically the alveolar surface tension. The central role of the surfactant system for maintaining pulmonary function has been repeatedly shown by the use of cell culture or animal models [2] .
Surfactant is a complex mixture of lipids, carbohydrates and four proteins (SP-A, SP-B, SP-C, SP-D). The initial descriptions of surfactant lead back to the 1950s, but little attention was given to the surfactant proteins until the 1980s [3] . The genes coding for these proteins are located on different chromosomes. SP-B and SP-C are similarly structured hydrophobic proteins participating in the adsorption of phospholipids at the alveolar border, which leads to rapid reduction of the surface tension. The hydrophilic proteins SP-A and SP-D are members of the collectins with C-type lectin domains. SP-D together with SP-A play a role in the pulmonary defense against Gramnegative bacteria [4] .
SP-A is the major surfactant apoprotein exhibiting complex interactions and participation in processes fundamental for pulmonary structure and function with its expression restricted to alveolar epithelial cells type II. Moreover expression of SP-A was also described for a portion of NSCLC facilitating a diagnostic marker for these carcinomas [5, 6] . After characterization of the biochemical properties, a complex chromosomal organization of the genes coding for SP-A has been demonstrated [3] . The locus of the SP-A on the one hand consists of two functionally active genes and a pseudogene [7, 8] . The two active genes SP-A1 and SP-A2 on the other hand display several different alleles and splicing variants, moreover different oligomeric states have been described [3, 9] . During the development of the lung these two genes are regulated differentially, a process triggered by cAMP and glucocorticoids [10] . The SP-A1 and SP-A2 genes display a homology of 94% in their nucleotide sequences and even 96% homology in the amino acid sequences [11] . Fig. 1 , as one example, shows the transcriptional activity of the SP-A1 and SP-A2 genes determined by RT-PCR in homogenates of biopsies from NSCLC and corresponding tumor-free samples. The importance of the differential transcription of SP-A1 and SP-A2 for maintaining pulmonary function has repeatedly been demonstrated [12] . Phylogenetic analyses revealed that an ancestor proto-SP-A gene was diverged into SP-A1 and a second gene which subsequently emerged to SP-A2 and the SP-A pseudogene [3] . The high level of homology between SP-A1 and SP-A2 up to date prevents a differential analysis of the two gene products in situ. The expression of SP-A is also complexly regulated on the transcriptional level. Moreover the protein-turnover and the release of SP-A into the serum represents a further point of regulation [12] . This sophisticated regulation of the genetic activity is recognized as a further hint for the functional importance of SP-A.
In recent years the role of defects in the expression of SP-A in context with different pulmonary diseases has become an issue of scientific investigations. Initially numerous studies have been performed to elucidate the role of surfactant substitution in pediatrics [2] .
As one major function SP-A displays a protective role of the molecule in pulmonary host defense by interacting with various infectious agents such as bacteria, fungi and viruses. SP-A deficient knock-out mice -compared to wild type animals -are susceptible to infections with Pseudomonas aeruginosa [13] and the clearance of group B streptococcus is slower [14] . In accordance the defense of SP-A deficient mice against Respiratory Syncytial Virus (RSV) has been shown to be reduced and may be restored by exogenous SP-A administration [15] [16] [17] .
By mediating the attachment of Mycobacterium tuberculosis to alveolar macrophages and promoting the phagocytosis of these bacteria, SP-A is important in the pathogenesis of tuberculosis [18] [19] [20] [21] . SP-A also functionally interacts with staphylococci [22, 23] Moreover, SP-A is involved in the complex pulmonary network of cytokines as a central player, for example interacting with TNF-alpha and several interleukins [31, 12, 14] .
Therefore it is likely that defects in the expression of SP-A may be important in the course of non infectious pulmo-RT-PCR: Transcription of SP-A1 and SP-A2 in NSCLC tumors (T) and corresponding tumor-free tissues (TF) from the same cases in comparison to GAPDH nary diseases of adult patients. In the case of idiopathic pulmonary fibrosis, for example, low levels of SP-A (measured by ELISA) have been reported in broncho alveolar lavages (BAL), but elevated levels were found in the sera [32] [33] [34] . Immunohistochemical examinations of the expression of SP-A in pulmonary fibrosis demonstrated evident defects by using specimens from different diseases displaying fibrotic changes in the lungs. In good agreement with the results in BAL reduced levels of SP-A have been observed in fibrotic lungs. This reduced SP-A-expression in fibrotic lungs may be caused by two reasons: a limited number of the SP-A producing type II pneumocytes and by a clearly reduced SP-A expression of the remaining cells [35] .
Keeping in mind that surfactant substitutes are widely available due to their application in pediatrics, a growing number of therapeutic possibilities may result from these findings.
In sarcoidosis elevated levels of SP-A have been described [37] using BAL; the same applies for the sera from patients with alveolar proteinosis [38].
Since SP-A represents a central molecule in pulmonary immunoregulation as well as in host-defense it is obvious that defects in the surfactant system may have functional influence in the course of these pulmonary disorders.
Another point of research concerning SP-A is the diagnostic value of this molecule, the expression of which is restricted to the lungs. It has been reported that SP-A levels in BAL or serum from patients suffering from idiopathic pulmonary fibrosis correlate with the progression of the disease and can be used to predict survival [34, 38] . In samples from airway secretions SP-A measurements are described to be useful also for the diagnostics of pulmonary edema where elevated levels have been found compared to healthy volunteers and ARDS patients [39]. By utilizing highly sensitive RT-PCR techniques the amplification of SP-A transcripts can be used for the detection of occult metastases in non small cell lung cancer patients [5, 40] . Comparative studies of different malignomas with pulmonary localization have shown the diagnostic properties of immunohistochemically determined SP-A [6, 41, 42] . In carcinomas of occult origin localized in the lungs the diagnosis has a crucial influence on the therapy. A positive detection of SP-A represents a clear hint for a primary location in the lung [43]. Fig. 2 as an example shows the immunohistochemical detection of SP-A in a moderately differentiated adenocarcinoma of the lung using the primary monoclonal antibody PE-10 and LSAB (AEC-substrate, × 100). The positive staining in the tumor cells (reddish) in this certain case helped to manifest the diagnosis as a primary carcinoma of the lung.
However, the choice of a suitable SP-A antibody is highly important since approaches using polyclonals display cross reactions with other tumors [44] . This procedure has already become a part of pathological routine diagnosis, and -along with other markers such as the Thyroid-transcription-factor-1 (TTF-1) -the detection of SP-A (by PE-10) is a useful part of the immunohistochemical panel in pulmonary pathology.
Immunohistochemical detection of SP-A even might be utilized for forensic purposes helping to distinguish between fatal drowning and postmortem immersion [45] .
It is evident that SP-A is a molecule which already proves to be an interesting subject for medical research. However, the studies concerning the possible role of surfactantdefects in pulmonary diseases of adults have been performed mainly in different cell culture or animal models hardly analyzing adult human lung tissue. For these reasons SP-A is a promising target for histomorphological approaches using pathological specimens which exactly represent the scenarios of various diseases with all the different cell types involved which are difficult to simulate in models. With the modern tools of molecular pathology, the genetic activities of genes can be analyzed in situ, which provides evidence of the cellular activities in the context of a human native tissue. One example is shown Immunohistochemical detection of SP-A using the mono-clonal antibody PE-10 (LSAB, amonoethylcarbazole, 400×) Figure 2 Immunohistochemical detection of SP-A using the monoclonal antibody PE-10 (LSAB, amonoethylcarbazole, 400×).
in Fig. 3 : a lung section hybridized with a digoxigeninlabeled SP-A probe to analyze the transcriptional activity in situ; the reddish signals of the transcripts are visible in the cytoplasm of type II pneumocytes. When analyzing the expression of SP-A in histological sections in context with other molecules of the pulmonary cytokine network one can expect further clues for the scenarios taking place in the course of interstitial lung diseases.
Taken together, SP-A is a complexly regulated molecule with surprising properties and essential importance for pulmonary development, structure and function which is getting more and more into focus concerning various diseases of the adult lung. Thus, as an outlook, it will become an issue of pulmonary pathology which might provide promising perspectives for applications in research, diagnosis and therapy.
In situ hybridization targeting using a 663 bp digoxigenated DNA-probe complementary to SP-A mRNA Figure 3 In situ hybridization targeting using a 663 bp digoxigenated DNA-probe complementary to SP-A mRNA. Detection was achieved by Anti-digoxigenin antibody conjugated to alkaline phosphatase with NBT/BCIP as a chromogen (400×).
Publish with Bio Med Central and every scientist can read your work free of charge  Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4–myc transgene product to AtPSK4–myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transfering root explants to tissue culture. Phytosulfokines (PSKs) are a class of plant peptides discovered through the study of growth factors that mediate density-dependent growth in cell culture . isolated and identified growth factors from conditioned medium that promoted the growth at low density of asparagus mesophyll cells in tissue culture. They identified a sulfated pentapeptide [H-Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-Gln-OH, abbreviated sYIsYTQ], named PSK-a, and a sulfated tetrapeptide [H-Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-OH], named PSK-b, that were active in the asparagus cell system.
Six genes encoding PSKs (AtPSK1-6) have been identified in Arabidopsis. Each encodes a preproprotein precursor of approximately 80 residues, with the YIYTQ peptide near their C-termini . generated transgenic Arabidopsis plants (AtPSK4ox) over-expressing one of the AtPSKs, and found that root growth and callus formation were slightly enhanced in over-expression lines, but otherwise the seedlings were phenotypically indistinguishable from wild-type.
A PSK receptor was first identified in carrot (Daucus carota) as a leucine-rich repeat receptor kinase (LRR-RK) (Matsubayashi et al., 2002) . Sequence information from the carrot protein (DcPSKR1) was used to identify an ortholog in Arabidopsis, AtPSKR1 (At2g02220). described a mutant with a Ds insertion in AtPSKR1 (pskr1-1), and found that callus derived from the mutant was less sensitive to the growth-promoting effects of PSK in culture. However, they observed little difference in overall plant growth between wild-type, pskr1-1 and AtPSKR1ox, an over-expression line. The most prominent characteristic of pskr1-1 was that vegetative tissues in mature plants lost their ability to form callus. Unlike wild-type plants, leaf discs from the fully expanded leaves of pskr1-1 plants were less capable of producing callus, while unexpanded leaves retained callus-forming capacity. AtPSKR1 over-expressing plants (AtPSKR1ox) showed delayed senescence, and, as a result, leaves continued to expand, resulting in larger leaves than the wild-type.
Little is known about the proteolytic processing of the PSK propeptide precursors, other than the fact that the precursors have conserved di-basic residues 8-10 amino acids upstream from the mature peptide sequence . Di-basic residues are characteristic of substrate sites for subtilases, subtilisin-like serine proteases (Barr, 1991) ; therefore, we wished to determine whether any of the proteases encoded by the 56 subtilase genes in the Arabidopsis genome (Rautengarten et al., 2005) are responsible for cleavage of the AtPSK4 precursor. In doing so, we identified a subtilase, AtSBT1.1, that is required for cleavage of the fusion protein AtPSK4-myc. Cleavage of AtPSK4-myc is induced in root explants, suggesting that release of the peptide hormone from the precursor protein is controlled, in part, by the proteolytic processing machinery.
In studies of shoot regeneration in Arabidopsis tissue culture, we found that expression of a subtilase gene AtSBT1.1 (At1g01900) correlated with conditions for efficient shoot regeneration (Lall et al., 2004) . It was not clear what the causal connection might be between efficient shoot regeneration and expression of a gene encoding a serine protease. Most subtilases are predicted to be secreted proteins (Rautengarten et al., 2005) , so we reasoned that AtSBT1.1 could be involved in processing of an extracellular growth factor or receptor related to shoot regeneration. Growth factors that might require the action of AtSBT1.1 include peptide hormones, such as AtPSKs. AtPSKs promote callus formation in tissue culture , and are synthesized as preproproteins with signal peptides that target them to the secretory pathway and with prosequences that are processed during maturation of the peptide hormone.
To determine whether AtSBT1.1 is involved in the proteolytic processing of AtPSKs, we developed a constitutively expressed, C-terminal 4 x myc-tagged construct of the AtPSK4 precursor, 35S:ppAtPSK4-myc, and studied its processing in vivo. PreproAtPSK4 will be referred to as ppAtPSK4 and proAtPSK4 as pAtPSK4. AtPSK4 was chosen for study because it is the most abundantly expressed PSK precursor . The predicted size of pAtPSK4 with the myc tag is 12.8 kDa, but we observed a band at approximately 19 kDa on Western blots, larger than the predicted size ( Figure 1a , wt, 0 time). To demonstrate, nonetheless, that this band is pAtPSK4myc, we analyzed the partially purified myc-tagged protein and identified three peptides derived from the fusion protein by MS/MS analysis ( Figure S1 ). Thus, the larger apparent size of pAtPSK4-myc may be due to anomalous gel migration behavior or post-translational modification of part of the precursor protein. (a) Root segments from wild-type transgenic seedlings expressing the construct 35S:ppAtPSK4-myc were explanted and incubated on callus induction medium (CIM) for 1-4 days and then transferred onto shoot induction medium (SIM). Root segments from sbt1.1-1 and sbt1.1-2 mutants expressing 35S:ppAtPSK4-myc were also explanted and similarly incubated. Arrows indicate the predicted migration position for processed AtPSK4-myc. The lane marked NT is an extract from roots of non-transformed seedlings.
(b) Root segments from seedlings bearing 35S:ppAtPSK4-myc in a wild-type background were explanted and incubated on normal CIM or on B5 basal medium, without cytokinin or auxin hormones. (c) Time course following explantation for acquisition of capacity to process pAtPSK4-myc on CIM medium.
We were surprised to find that pAtPSK4-myc was not cleaved in roots of intact transgenic seedlings (Figure 1a , wt, 0 time). We suspected that failure to detect processing may have been due to seedling growth or culture conditions. We had chosen to study AtSBT1.1 in the first place due to observations that we had made about shoot regeneration from root explants (Lall et al., 2004) . Therefore, we subjected root explants from 35S:ppAtPSK4-myc seedlings to tissue culture conditions for regenerating shoots. This involves pre-incubating root segments on an auxin-rich callus induction medium (CIM), and then transferring them after 4 days to a cytokinin-rich shoot induction medium (SIM). Under these conditions, a band appeared at approximately 7 kDa in Western blots (Figure 1a , wt, lanes 1d and 4d CIM or SIM), representing the myc-tagged cleaved peptide, AtPSK4-myc. The size of the processed protein was consistent with a cut at or near the cleavage site as determined by the in vitro experiments described below. Thus, cleavage of pAtPSK4myc appears to be induced by some aspect of the culturing process.
Having identified conditions for pAtPSK4-myc cleavage, we wished to determine whether AtSBT1.1 was responsible for the proteolysis. To do so, we examined cleavage of pAtPSK4-myc in T-DNA mutants with insertions in AtSBT1.1. The gene encoding AtSBT1.1 (At1g01900) has a single intron, and sbt1.1-1 (SALK_111561) and sbt1.1-2 (SALK_108704) have T-DNA insertions in the first exon. The T-DNA lines were judged to be null mutants because AtSBT1.1 transcripts were not found in seedlings from either line ( Figure S2 ). The 35S:ppAtPSK4-myc construct was introduced into the two T-DNA mutant lines, and root explants were subjected to regeneration conditions (4 days incubation on CIM). The pAtPSK4-myc precursor was produced in these lines, but the cleavage product AtPSK4-myc peptide was not detected (Figure 1a , lanes sbt1.1-1 and sbt1.1-2). To demonstrate that the transgenic product in these lines was still cleavable, we out-crossed sbt1.1-1 bearing the 35S:ppAtPSK4-myc construct to wildtype, and demonstrated that processing was restored in root explants of the F 1 seedlings ( Figure S3 ). We concluded from these results that AtSBT1.1 is required for pAtPSK4-myc cleavage under these conditions. This finding is quite significant given that there are 56 different subtilases encoded by the Arabidopsis genome (Rautengarten et al., 2005) .
As shoot regeneration in culture is dependent on cytokinin and auxin hormones, we determined whether induction of pAtPSK4-myc cleavage required hormone treatment during CIM pre-incubation. To test this, root segments were explanted to hormone-free B5 medium as well as to CIM, and it was found that pAtPSK4-myc was cleaved on basal B5 medium at 1 and 4 days after explantation ( Figure 1b ). We concluded that the processing activity was probably induced by the wounding or handling of root tissue during explanting. We then determined how rapidly processing was induced. Cleavage of pAtPSK4-myc was first detected approximately 8 h after explanting ( Figure 1c ). Thus induction is fairly rapid, but not immediate as one might expect for a post-translational activation mechanism.
Up-regulation of AtSBT1.1 gene expression As pAtPSK4 cleavage was induced approximately 8 h following the explanting of root segments, we wished to determine whether expression of the gene encoding AtSBT1.1 was similarly up-regulated. Real-time quantitative RT-PCR was performed on extracts from root segments at various times following explanting. It was found that AtSBT1.1 was up-regulated approximately 3.5-fold starting at approximately 8 h after explanting root segments ( Figure 2a) . Thus, the up-regulation of AtSBT1.1 expression was similar to the kinetics for the acquisition of cleavage activity. The endogenous gene encoding AtPSK4 was also up-regulated after explanting, but not as much as AtSBT1.1 (approximately twofold, Figure 2a ).
We also developed promoter:GUS constructs for AtSBT1.1 and AtPSK4, and found that the gene was expressed at the cut ends of the root segments where callus formation first occurs during shoot regeneration (4 days CIM, Figure 2b ,d). Later, AtSBT1.1 and AtPSK4 continued to be expressed most intensely at sites of callus and regenerative tissue formation, both at the ends and at other wound sites along the length of the root segments (6 days SIM, Figure 2c ,e).
If pAtPSK4 is indeed a substrate for AtSBT1.1, then these proteins should occupy the same subcellular compartment. Both AtSBT1.1 and ppAtPSK4 have signal peptides, and both are predicted to be secreted proteins. To determine whether that is so, we constructed C-terminal YFP fusions and determined their location in root cells from Arabidopsis. Both AtPSK4-YFP (Figure 3a-c) and AtSBT1.1-YFP (Figure 3d-f) are associated with the periphery of the cell, coinciding with propidium iodide staining. Following plasmolysis (Von Groll et al., 2002) , most fluorescence from both YFP fusions remained associated with the extracellular matrix rather than the plasma membrane ( Figure S4 ).
To confirm that AtSBT1.1 is indeed involved in the processing of pAtPSK4-myc, we tested whether pAtPSK4 can be cleaved by AtSBT1.1 in vitro. To do so, we developed a pull-down assay using a C-terminal myc-tagged version of AtSBT1.1 (AtSBT1.1-myc) and a fluorogenic peptide substrate. We employed a similar assay in a previous study to test the activity of another Arabidopsis subtilase, AtS1P (Liu et al., 2007a) . In both cases, the tagged subtilases were synthesized in transgenic Arabidopsis because we were not able to produce active enzyme at high levels in heterologous systems.
To test AtSBT1.1-myc for activity against pAtPSK4, we generated a fluorogenic peptide called fpAtPSK4 with the sequence Abz-RRSLVLHTDY(NO2)D-OH [where Abz is a 2-aminobenzoyl fluorescent group and Y(NO2)D-OH is a 3-nitrotyrosine quencher], representing a probable subtilase recognition site in pAtPSK4. The site was chosen because di-basic amino acid residues (in this case, RR) are characteristic signatures of subtilase recognition sites (Barr, 1991) . When bead-bound protein from transgenic plants expressing AtSBT1.1-myc was incubated with fluorescence-quenched fpAtPSK4, a time-dependent increase in fluorescence was observed (Figure 4a ). Because the enzyme was produced in a homologous system, we were mindful of potential contamination by endogenous Arabidopsis subtilases in our preparations. To preclude that possibility, we thoroughly washed the beads and routinely ran controls in which the same pull-down procedure was conducted with extracts from non-transgenic seedlings and from transgenic seedlings expressing a tagged subtilase (S552A) that was mutated at the active site ( Figure 4a ). The kinetics of the reaction with the fluorogenic peptide substrate followed Michaelis-Menten kinetics, and the K m for the reaction was determined to be approximately 18 lM (Figure 4b ). The K m value lies within the range of affinity constants for fluorogenic peptide substrates with comparable subtilases, such as mammalian prohormone convertases (Basak et al., 2004 (Basak et al., , 2007 . The activity was inhibited by PMSF (Figure 4a ), an inhibitor of serine proteases. The pH optimum for the reaction was in the acidic range, centered on pH 6 ( Figure 4c) .
To determine the site at which the peptide was cleaved, the reaction products were analyzed by MALDI-TOF analysis. The first N-terminal cleavage product that appeared had a molecular mass of 862.25, indicating a preferred cutting site at Abz-RRSLVLflHTDY(NO2)D-OH (Figure 4d ). MS/MS analysis of the 862.25 peak showed a spectrum of ions consistent with Abz-RRSLVL as the initial cleavage product ( Figure S5 ). It should be noted that this cut is three amino acid residues upstream of the N-terminus of the mature peptide as has been described for PSKs in Asparagus officinalis . If the mature Arabidopsis peptide is similar, then further N-terminal proteolytic processing, as well as C-terminal processing, probably occurs to generate the active peptide.
Substrate specificity of AtSBT1.1
To determine whether AtSBT1.1 has specificity for the AtPSK4 recognition site, we conducted an alanine scan through fpAtPSK4, substituting one residue at a time for alanine (Figure 5a ). The reactions were carried out in triplicate, and the reaction rates for each of the substitutions were compared to that for the wild-type substrate. The cleavage site sequence was very sensitive to alanine substitutions. Substituting the first arginine in the di-basic residues (P6 position) resulted in a reaction rate that was less than half that of wild-type. The most sensitive positions were P2-P4, which are modestly conserved among the AtPSK precursors.
We compared the sequence of AtPSK4 to some of the most closely related AtPSKs and to PSK-related sequences in other plants. The five amino acids representing the presumed mature PSK peptide in the six Arabidopsis sequences are nearly identical (Table 1) other plant PSKs in the GenBank database, except for AtPSK6. The upstream di-basic amino acids and the leucine (P3 position) and histidine and aspartate (P1¢ and P3¢, respectively) are also conserved in Arabidopsis. To compare the activity of AtSBT1.1 with other AtPSK precursors, we developed fluorogenic peptides representing putative recognition sites for the family of AtPSKs (fpAtPSK1, 2, 3, 5 and 6, see bold residues in Table 1 ). The putative recognition sites for pAtPSK2 and 5 are the same and are represented in the fluorogenic peptides fpPSK2 and 5. The cleavage reaction was slower for fpAtPSK2 and 5 than for fpAtPSK4, and was barely detectable or not detectable at all with the other fpAtPSKs ( Figure 5b) . Thus, the activity of AtSBT1.1 is fairly specific, with most activity directed toward cleavage of pAtPSK4, followed by pAtPSK2 and 5.
The proteolytic processing of PSK4 appears to be highly regulated in the tissue culture system -in part by up-regulation of AtSBT1.1 gene expression. Among the 56 subtilases encoded in the Arabidopsis genome, AtSBT1.1 appears to be solely responsible for the release of PSK4 from its precursor in roots, because pAtPSK4-myc is not cleaved in AtSBT1.1 knock-out lines. PSKs are growth factors that stimulate callus formation in culture Sakagami, 1996, 2006) . Thus, up-regulation of AtSBT1.1 and the release of AtPSK4 from its precursor protein may be key factors in promoting the growth of cells and callus formation in tissue culture.
AtSBT1.1 functions similarly to prohormone convertases that release peptide hormones and neuropeptides from protein precursors in animal cells (Steiner, 1998) . Prohormone convertases are subtilases in the secretory pathway that cleave substrates with mono-or di-basic amino acids in the general recognition motif R/K-Xn-R/Kfl (Seidah, 1997) . In Arabidopsis, AtSBT1.1 cleaves substrates in a fairly site-specific manner, cleaving pAtPSK4 at RRSLVLflHTDY and showing preference for cleavage of proAtPSK4 over other PSK precursors. Cleavage by AtS-BT1.1 at the preferred cleavage site of the PSK4 precursor leaves three residues on the N-terminus of the five-residue peptide. This is of concern because demonstrated that even the presence of the tripeptide GGG severely reduces the activity of PSKs in an asparagus suspension cell system However, the three residues (HTD) remaining on the amino-end of AtSBT1.1processed AtPSK4 are highly conserved among the PSKs, and it is possible that the sequence is part of the active, mature peptide or serves as a recognition signal for further processing by enzymes such as tripeptyl peptidases (Book et al., 2005) . Complete proteolytic processing of PSK4 probably involves a number of other steps, including trimming of the peptide at its C-terminus .
The preferred AtSBT1.1 cleavage site within the recognition motif for proAtPSK4 is somewhat unconventional, because the di-basic residues are usually in the P1 and P2 positions, immediately upstream (on the N-terminal side) of the cleavage site (Barr, 1991) , The only caveat we have about the site is that it was identified from the cleavage product of a short fluorogenic peptide representing the putative subtilase recognition site. Other structural features of the intact Table 1 .
AtPSK precursor may be important in determining the site of cleavage, but are not found in the peptide substrates that we used to characterize the cleavage reaction. We have shown that YFP fusions of AtSBT1.1 and PSK4 accumulate in the extracellular matrix, and it is probable that cleavage occurs there simply because AtSBT1.1 has a slightly acid pH optimum and the apoplast is acidic (Bibikova et al., 1998) . PSK4 is tyrosine-sulfated , and the protein is likely to be sulfated in the trans-Golgi as are other sulfated proteins in animal cells (Baeuerle and Huttner, 1987) . Therefore, it seems reasonable that the precursor is sulfated before it is cleaved. However, the precursor does not have to be sulfated to be cleaved, because AtSBT1.1-myc can cleave the unsulfated peptide, fpAtPSK4. Sulfation, however, is important for the function of the peptide, because the sulfated peptide binds to the PSK receptor (Matsubayashi et al., 2002) .
Although there are six genes encoding PSKs in Arabidopsis, the mature peptides (YIYTQ) encoded by each gene are identical (with the exception of AtPSK6, YIYTH). Presumably, the peptides encoded by each gene should be able to bind and activate the single known receptor, AtPSKR1 . Each of the AtPSK precursors has the typical subtilase recognition site signature (di-basic amino acids) 8-10 residues upstream from the mature peptide (Barr, 1991) . However, the residues that are critical for AtSBT1.1 recognition (the four or five residues just downstream from the di-basic site) differ somewhat between AtPSK genes, and therefore AtSBT1.1 appears to be most specific for cleavage of PSK4. That suggests that other subtilases might be involved in the processing of other PSKs.
Our interest in AtSBT1.1 stems from our earlier studies of shoot regeneration in Arabidopsis (Lall et al., 2004) . We found that higher expression levels of the gene encoding AtSBT1.1 (At1g01900) correlated with the presence of the superior allele at the major QTL conditioning shoot regeneration in Arabidopsis tissue culture (Lall et al., 2004) . Higher levels of AtSBT1.1 expression may not have anything to do directly with shoot regeneration. However, higher levels of AtSBT1.1 expression might promote the proliferation of callus from which shoots are derived. Similar reasoning was used by Hanai et al. (2000) to explain the stimulatory effects of PSKs on somatic embryo formation in carrot. They concluded that PSKs might promote the proliferation of cells giving rise to somatic embryos, rather than influencing the formation of somatic embryos.
Two T-DNA insertion mutant lines for AtSBT1.1 were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). Seeds were surface-sterilized, rinsed with sterile water, and stratified at 4°C for at least 2 days in 0.1% agar. Seeds were germinated and grown vertically on agar plates containing Gamborg's B5 medium (Gamborg et al., 1968) . Root segments (5 mm) were cut and transferred to callus induction medium (CIM), which consisted of B5 medium with 5 g l )1 MES, 2.2 lM 2,4-dichlorophenoxyacetic acid, 0.2 lM kinetin and 0.8% agarose. Explants were incubated on CIM for 4 days under constant light conditions, and then transferred to shoot induction medium (SIM). SIM is prepared similarly to CIM except that it contains the hormones isopentenyladenine (5.0 lM) and 3-indoleacetic acid (0.9 lM). For AtSBT1.1-YFP and AtPSK4-YFP localization experiments, transgenic seedlings were grown on B5 plates for 7 days, and roots were used for confocal microscope examination. Screening for homozygous plants was carried out by PCR using left border (LB) T-DNA primers and the gene-specific primer pair ScrSBT1.1 (Table S1 ). The transcript level of AtSBT1.1 was evaluated by RT-PCR using primer pair sqRT-SBT1.1 listed in Table S1 .
Plasmid construction ppAtPSK4 and AtSBT1.1 were amplified from root RNA of 1-weekold Arabidopsis seedlings by RT-PCR (primer pairs pSKMPSK4 and pSKMSBT1.1, respectively, Table S1 ), and cloned into the AscI and SpeI sites of pSKM36 in-frame with a 4 x epitope myc tag (EQKLISEEDLRN). cDNA clones with error-free copies were named ppSKAtPSK4 and pSKAtSBT1.1, respectively. A mutated form of AtSBT1.1 (S552A) was generated using a QuickChange site-directed mutagenesis kit (Stratagene, http://www.stratagene.com/) with primer pairs SDM1.1 and pSKAtSBT1.1 as template. YFP C-terminal fusions were created by inserting cDNAs from above at the AscI and SpeI sites of pSKY36. The clones were named 35S:AtSBT1.1-YFP and 35S:ppAtPSK4-YFP. Promoter-GUS constructs for AtSBT1.1 and AtPSK4 were generated by amplifying 984 and 934 nucleotides using the primers pCAMSBT1.1 and pCAMPSK4, respectively. The promoters were ligated into the BamHI and PstI sites of pCAM-BIA3300.
Total protein was extracted from transgenic and wild-type plants using extraction buffer [0.1 M HEPES/KOH pH 7.0, 20 mM 2-mercaptoethanol, 0.1 mg ml )1 PMSF, 0.1% w/v Triton X-100, 1 mM EDTA, 20% w/v glycerol and protease inhibitor cocktail (Sigma-Aldrich, http://www.sigmaaldrich.com/)]. An aliquot of total protein was precipitated using trichloroacetic acid and quantified by the Bradford method (Bradford, 1976) . Reaction products were resolved by 12% SDS-PAGE and visualized by Western blotting using c-myc antibody (9E10; Santa Cruz Biotechnology, http://www.scbt.com) and an ECL kit (GE Healthcare, http://www.gehealthcare.com).
Assay for AtSBT1.1 activity in vitro AtSBT1.1-myc was affinity-purified from transgenic Arabidopsis plants as follows: 500 g of seedlings were ground in liquid nitrogen and suspended in 25 mM Tris/HCl pH 7.2, 150 mM NaCl, 0.1% Nonidet P-40 (Calbiochem, http://www.emdbiosciences.com) and 10% glycerol. Anti-c-myc agarose conjugate (200 ll; Sigma) was added to the filtered lysate and incubated for 2 h at 4°C with continuous rotation. The agarose beads with bound AtSBT1.1-myc were recovered by centrifugation at 1000 g for 3 min at 4°C. The beads were washed three times with washing buffer (25 mM Tris/HCl pH 7.2, 150 mM NaCl) and suspended in 25 mM MES/sodium acetate buffer pH 6.0. The reactions were carried out at 32°C in the same buffer supplemented with 2.5 mM CaCl 2 . Parallel purification was performed using transgenic plants transformed with a mutated form (S552A) of AtSBT1.1-myc and the empty vector to obtain material for control reactions. For fluorogenic peptide assays, 40 ll of bead-bound AtSBT1.1-myc were added to a solution containing a final concentration of 50 lM fluorogenic peptide in a buffer consisting of 25 mM MES/ sodium acetate pH 6.0 supplemented with 2.5 mM CaCl 2 . Kinetic assays were performed at 32°C and monitored as fluorescence emission at 420 nm (10 nm slit) following excitation by 320 nm (10 nm slit) in a BioTek spectrophotometer (http://www.biotek. com). The reaction was carried out in 96-well plates (Nunc, http:// www.nuncbrand.com). Control reactions were performed using the same fluorogenic peptide with bead-bound mutated AtSBT1.1 (S552A) and bead-bound myc vector only. AtSBT1.1 incubated with 1 mM PMSF, peptide and reaction buffer, and buffer alone incubated in separate wells served as additional controls. To determine the pH optimum of the reaction, a tri-component buffer system of constant ionic strength was used (Ellis and Morrison, 1982) . This buffer comprised 25 mM acetic acid, 25 mM MES, 50 mM Tris/HCl and 2.5 mM CaCl 2 .
Total RNA was isolated from ground plant tissues using an RNeasy kit (Qiagen, http://www.qiagen.com/), treated with RNase-free DNase I according to manufacturer's instructions (Qiagen), and quantified by 260/280 nm UV light absorption. A 1 lg aliquot of total RNA was reverse-transcribed using the Superscript III reverse transcription kit (Invitrogen, http://www.invitrogen.com/). Aliquots (2 ll) of cDNA were used for RT-PCR, and tenfold diluted cDNA was used for real-time quantitative RT-PCR. All primers are listed in Table S1 . For real-time quantitative RT-PCR, the efficiency of amplification of various RNAs was assessed relative to amplification of transcripts for two actin genes [actin2 (At3g18780) and actin8 (At1g49240)]. RNA samples were assayed in triplicate. Expression levels were calculated relative to actin using a comparative threshold cycle method with DDCt = DCt reference ) DCt sample , where DCt sample is the Ct value for the assay sample normalized to actin and DCt reference is the Ct value for calibration, also normalized to actin (Liu et al., 2007b) .
Root segments (5 mm) incubated on CIM were harvested and further incubated for 6 h in GUS staining solution [100 mM Tris/ NaCl buffer, pH 7, 2 mM ferricyanide, 2 mM X-gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide), 2 mM ferrocyanide, 10 mM EDTA and 0.1% Triton X-100] at 37°C in the dark. The staining solution was removed, and the tissues were dehydrated in an ethanol series from 70% v/v to absolute ethanol. Samples were visualized under the light microscope.
MALDI-TOF MS and MS/MS analyses were performed using a QSTAR XL quadrupole TOF mass spectrometer (AB/MDS Sciex, http://www.appliedbiosystems.com) equipped with an orthogonal MALDI ion source. The mass spectrometer was operated in the positive ion mode. Mass spectra for MS analysis were acquired over m/z 500-2500. After every regular MS acquisition, MS/MS acquisition was performed against the most intensive ions. The molecular ions were selected by information-dependent acquisition in the quadrupole analyzer and fragmented in the collision cell.
Subcellular localization was carried out using 35S:pSKYAtSBT1.1-YFP-and 35S:pSKYAtPSK4-YFP-expressing roots. The roots were stained with propidium iodide and examined with a laser scanning confocal microscope. Confocal microscopy was performed using a Nikon C1si confocal scanning system attached to a 90i microscope (http://www.nikoninstruments.com). The emission signals for YFP and propidium iodide were acquired using sequential scanning mode to eliminate emission signal bleed-through. The 488 line of the argon laser and 515/30 emission filters were used for acquisition of YFP images. The propidium iodide images of root cells were acquired using the 561 argon laser and 590/50 emission filter.
Cells in root segments were plasmolyzed using the conditions described by Von Groll et al. (2002) . Epifluorescent and differential interference contrast images of plasmolyzed cells were acquired using a 5 megapixel Nikon DS-Fi1 camera and Elements BR software. A 200 W mercury light source and FITC filter cube were used for image acquisition.
Additional Supporting Information may be found in the online version of this article: Figure S1 . MS/MS analysis of pAtPSK4-myc. Figure S2 . RT-PCR analysis of AtSBT1.1 transcripts in wild-type and sbt1.1-1 and sbt1.1-2 mutants. Figure S3 . pAtPSK4-myc processing is restored in F 1 hybrids resulting from out-crossing of 35S:ppAtPSK4-myc sbt1.1-1 to wildtype. Figure S4 . AtSBT1.1-YFP and AtPSK4-YFP are located in the apoplast. Figure S5 . MS/MS spectrum for the cleaved N-terminal fluorogenic peptide. Table S1 . Primer sequences used. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Protein Domain Boundary Predictions: A Structural Biology Perspective One of the important fields to apply computational tools for domain boundaries prediction is structural biology. They can be used to design protein constructs that must be expressed in a stable and functional form and must produce diffraction-quality crystals. However, prediction of protein domain boundaries on the basis of amino acid sequences is still very problematical. In present study the performance of several computational approaches are compared. It is observed that the statistical significance of most of the predictions is rather poor. Nevertheless, when the right number of domains is correctly predicted, domain boundaries are predicted within very few residues from their real location. It can be concluded that prediction methods cannot be used yet as routine tools in structural biology, though some of them are rather promising. Computational/mathematical approaches, such as structural bioinformatics [1] , structural class prediction [2, 3] , molecular docking [4] [5] [6] [7] [8] [9] , molecular packing [10, 11] , pharmacophore modelling [12] , Mote Carlo simulated annealing approach [13] , diffusion-controlled reaction simulation [14] , graph/diagram approach [15] [16] [17] [18] [19] [20] [21] , bio-macromolecular internal collective motion simulation [22] , QSAR [23] [24] [25] , protein subcellular location prediction [26] [27] [28] [29] [30] , protein structural class prediction [31, 32] , identification of membrane proteins and their types [33] , identification of enzymes and their functional classes [34] , identification of proteases and their types [35] , protein cleavage site prediction [36] [37] [38] , and signal peptide prediction [39, 40] can timely provide very useful information and insights for both basic research and drug design and hence are widely welcome by science community.
Several computational approaches aimed to the prediction of protein domain boundaries have been published during the last few years [41, 42] . Besides their intrinsic interest in genome analysis and evolution studies, they are tools that structural biologists may use to optimize the design of the constructs of the proteins, the three-dimensional (3D) structure of which must be determined [43] . While this is particularly important in structural genomics (SG), where the targets have, in general, not been deeply characterized with appropriate biochemical and biophysical tools, this can be important also for traditional hypothesis-driven structural biology projects, where a fine tuning of the construct that is inserted into the experimental pipeline -cloning, expression, purification, etc. -is often necessary in order to get suitable samples [44] .
Several information about structure prediction methods are periodically published in the framework of the CASP *Address correspondence to this author at the Department of General Chemistry, Pavia University, Viale Taramelli 12, I-27100 Pavia, Italy; Tel: +43 1 4277 52208; E-mail: oliviero.carugo@univie.ac.at initiative, the main goal of which is to promote an evaluation of computational prediction methods [45] . This is a periodical exercise, performed every two years since 1994. During CASP experiment a series of protein sequences, the 3D structure of which was determined experimentally though it was not yet published, are distributed to research groups that develop computational methods for predicting protein structural features. It is thus a blinded test, where several methods of "in silico" structural biology techniques can be compared to the reality and to each other. Nevertheless, in each CASP run, the number of targets is obviously quite limited and a prediction method that performs very well in CASP is not necessarily better than other techniques in the reality. It is necessary to make additional investigations focusing on the possibility to use these prediction methods for practical application in structural biology.
Although it is impossible to consider it a rule, it is generally easier to work with single-domain proteins than with multi-domain proteins, since the latter ones tend to be conformationally more flexible [46] . For example, the reciprocal orientation of the domains can vary and depend on the presence of other molecules. Multi-domain proteins may also be little prone to refold if, by chance, they had been overexpressed in cells lacking proper chaperones. This does not mean that multi-domain proteins cannot be studied but it implies that some care must be paid in structural biology experiments and that longer time and larger funding can be expected to be necessary to solve multi-domain proteins. It is thus extremely important to be able to predict, on the basis of its amino acid sequence, if a protein contains one or more structural domains.
CASP is divided into several sections, ranging from prediction of conformational disorder to tertiary structure prediction. Protein domain boundary predictions began to be included in the CASP initiative in 2004. The dissection of a protein into separate structural domains is in fact not trivial at all [46, 47] . It is related to the ill-definition of what a protein domain is. An amino acid segment can be in fact consid-ered to be a structural domain if i) it is a compact ensemble of atoms/residues; ii) it is an ensemble of atoms/residues that behaves as a rigid body, in the sense that it can move relative to other protein moieties without changing its shape; iii) it is a self-folding subunit; iv) it is a polypeptide segment well conserved during molecular evolution. Given the ambiguity in any quantitative definition, the real domain boundaries were defined according to the CASP7 organizers and assessors [47] . They found a reasonable consensus definition for each investigated protein, which seems to be well suitable for a structural biology analysis.
The present study is attempted to compare modern approaches for predicting protein domain boundaries and to define new prediction strategies. Here, we refer to the exercise named CASP7, organized in 2006, for which both predictions and experimental data are available on-line (http://www.predictioncenter.org/casp7/Casp7.html). In this manuscript, several tools, designed for predicting domain boundaries on the basis of the amino acid sequence, will be compared to the real domain architecture. The analysis of these data allows one to answer the following basic questions: i) Is it possible to predict, with the presently available bioinformatics tools, if a protein is made by a single domain or if it contains more than one domain? ii) What is the statistical significance of the available predictions? iii) How accurately can the domain boundaries be predicted in the cases where the presently available bioinformatics predictions work well?
Data were obtained from the CASP7 web page (http://predictioncenter.gc.ucdavis.edu/casp7/). Table  1 shows the bioinformatics tools that are freely available and that were used in CASP7. Protein domain prediction methods can be classified into three main categories [42] : i) homology prediction; ii) domain recognition; iii) new domain prediction methods. The 14 prediction methods regarded in present study include all types of approaches. The homology prediction is presented by the chop [48, 49] methods that assign the query sequence to known PDB chains. Dsp [42] uses in addition more general properties of sequence conservation throughout the protein and it can be considered as lying between domain homology and new domain predictions. Domssea [42] belongs to the domain recognition approaches. It is based on the assumption that secondary structure is a more conserved feature of proteins with similar folds than sequence. Domssea aligns the secondary structure predicted for a query protein against a database of 3D domain structures and derives the domain boundaries from the known domain with the most similar secondary structure. Robetta [50] applies BLAST/PSI-BLAST for domain homology prediction and it uses FFAS03 and 3D-Jury to find remote homologues of known domain structure. Hhpred [51] is a server for remote homology detection and for structure prediction using pairwise comparison of profile hidden Markov models (HMMs). In the foldpro [52] method the structural relevance of the query-template pairs is extracted from global profile-profile alignments in combination with predicted secondary structure, relative solvent accessibility, contact map and beta-strand pairing using support vector machines. Distill [53] provides prediction of Contact Density defined as the Principal Eigenvector (PE) of a residue contact map. This information is an important intermediate step towards ab initio prediction of protein structure and is used to identify domains. Baker generates 3D protein models using the de novo prediction algorithm Rosetta and then assigns domain boundaries using Taylor's structure-based do- maopus http://sigler.bioch.bcm.tmc.edu/CASP7-DOM/ * metadp http://meta-dp.cse.buffalo.edu [54] main identification technique. Maopus performs a template screening with PSI-BLAST and FFAS03. The SKELEFOLD approach implemented in Maopus is a de novo folding algorithm that uses vector representations of secondary structural elements; domain boundaries are defined with three sequence-based filters. In the domfold method, the output from DomSSEA, DISOPRED and HHsearch is parsed to form a consensus. Metadp [54] and NNput are meta servers that comprise a number of domain prediction methods.
Some of the bioinformatics methods provide multiple predictions. In this case, only the first, which is considered to be the more reliable, was retained for further analysis. Predicted domain boundaries were obtained from the CASP7 web page. The experimental domain boundaries were also obtained from the CASP7 web page, where they were generated by a group of expert scientists. 95 proteins are considered. Given that predictions were not deposited for each protein and for each prediction method, this results in a set of 1210 predictions [47] .
To predict, on the basis of the protein length, that a protein contains one domain or it is a multi-domain construct, a threshold value can be used. If the protein is longer than the threshold value it consists of more than one domain. On the contrary, a protein, smaller than this threshold value, would be predicted to contain only a single domain. Consequently, a true positive (tp) is defined as a multi-domain protein, which is correctly predicted to be a multi-domain protein; a multi-domain protein that is predicted to contain a single domain is defined a false negative (fn); a single-domain protein predicted to be a multi-domain protein is defined a false positive (fp); and a correctly predicted single-domain protein is defined a true negative (tn).
These four types of predictions can be used to estimate the reliability of this prediction methodology. A number of figures of merit have been used for that, like, for example, the Matthews correlation coefficient (mcc) [55] 
the values of which range from -1 to +1 (larger values indicate better predictions) and is little affected by sample heterogeneity (the number of single-domain proteins can be different from the number of multi-domain proteins).
The prediction accuracy was validated with a Jack-knife procedure. In statistical prediction, the following three crossvalidation methods are often used to examine a predictor for its effectiveness in practical applications: independent test dataset, sub-sampling test, and Jack-knife test [56] . However, as elucidated in references [26] and [27] , amongst the three cross-validation methods, the Jack-knife test is deemed the most objective that can always yield a unique result for a given benchmark dataset, and hence has been increasingly used and widely recognized by investigators to examine the accuracy of various predictors [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] .
To compare the accuracy of different methods with a random prediction we estimated numerically the probability density functions of the indices used to measure the classification validity. This approach is based on idea that the problem of domain boundary prediction using the amino acid sequence is a classification problem. Each residue is in fact predicted to belong to a certain class and it cannot belong to two different clusters at the same time. In other words, a residue can be predicted to belong to a certain domain, to another domain, or to a linker segment. The comparison between a prediction and the reality or between two predictions can thus be performed by using statistical tools that are routinely employed to compare alternative classifications [67] and that are briefly described below.
Given for example two classifications (C and K) of n residues, it is possible to count the number of cases in which residues i and j were classified in the same group in C and K (n_ss), the number of cases in which i and j were classified in the same group in C and in different groups in K (n_sd), the number of cases in which i and j were classified into two different groups in C and in the same group in K (n_ds), and the number of cases in which i and j were classified into two different groups both in C and in K (n_dd). On the basis of this description, it is possible to compute the Jaccard index (J), the Rand coefficient (R), and the Fowlkes-Mallows index (FM), which are defined as:
FM = n _ ss n _ ss + n _ sd n _ ss n _ ss + n _ ds (4) where M = n _ ss + n _ sd + n _ ds + n _ dd .
By definition, if the two classifications C and K are identical, all the indices (J, R, and FM) are equal to one. It is also important to observe that these indices can be computed independently of the fact that the classifications C and K contain the same number of clusters. This means that the values of J, R, and FM can be computed also if in one case, for example the classification C, all the residues were predicted to be in a unique domain while in the other case, for example the classification K, some residues were assigned to different domains. The only constraint to the computation of J, R, and FM is that both classifications C and K must include the same number of residues, and in the present case this is obvious.
The computation of the values of J, R, and FM is elementary. The estimation of their statistical significance is less obvious [67] . For example, it is difficult to estimate the probability that a certain value of the index J was obtained by chance. From another point of view, if J CK > J DL , where J CK monitors the similarity between the classifications C and K and J DL difference between the classifications D and L, it is clear that C and K are more similar to each other than D and L. However, it is more difficult to estimate the statistical significance of the inequality J CK > J DL . In other words, it is more difficult to estimate the probability that C and K are really more similar to each other than D and L. This depends on the fact that the probability density functions of the indices J, R, and FM are unknown and must therefore be estimated numerically on the basis of adequate simulations. Therefore, we generated a series of simulated partitions, using a Metropolis-Monte Carlo approach, by mean of the following procedure. Each partition is characterized by a series of boundaries that separate a domain and a loop and that can be located also at the N-or at the C-terminus. Given a protein containing N residues, a boundary can be any integer k with 1 k N. A series of boundaries were generated iteratively. The first (k 0 ) was randomly selected in the range (1, N); the second (k 1 ) was randomly selected in the range (1, m 0 ), where m 0 = N -k 0 ; the third (k 2 ) was randomly selected in the range (1, m 1 ) where m 1 = m 0 -k 1 ; and so on, the i th boundary (k i ) was randomly selected in the range (1,m i-1 ), where m i-1 = m i-2 -k i-1 . Two constrains were imposed during the generation of random domain boundaries within a protein. We considered that a domain must contain more than 30 residues and a loop size must be smaller than 30 residues. 10,000 random partitions into domains were generated for proteins containing 75, 100, 125, ..., 550, 575, 600 residues. It was then possible to make 49,995,000 pairwise comparisons between two partitions and the 49,995,000 values of the coefficients J, R, and FM were retained in order to determine their distributions.
As an example, Fig. (1) shows the distributions of the index R for some N values. It appears that the distribution dispersion decreases if N increases and that the maximum moves to higher R values for larger proteins. With these data, it is possible to estimate the probability pR to have R values higher than a given value Rx, simply by integrating the probability density curve from Rx to 1, and, analogously, it is possible to get the statistical significance for the other indices. 
The definition of what is a well predicted domain is obviously arbitrary and here the following conditions were used in order to select the predictions that can be considered to be satisfactory. If the domain contains N residues and it is predicted to contain M residues, and if C is the number of residues that are found in both the real and the predicted domain, a good prediction was defined as a case in which N M < 20 (6) and C min(M , N ) > 0.95 (7) For well predicted domains, we then computed the differences between the sequence position in which the domain is predicted to begin and the sequence position in which it begins in the reality (Delta_b). Note that a negative value of Delta_b indicates that the domain is predicted to begin before the real beginning along the protein sequence. Analogously, we also computed the differences between the sequence position in which the domain is predicted to end and the sequence position in which it ends in the reality (Delta_e). A positive value of Delta_e indicates that the domain is predicted to be slightly longer, at its C-terminus, than the reality.
Fig. (2) shows the distributions of the protein dimensions, measured by the number of amino acid residues, for the single-and multi-domain proteins examined in the CASP7 experiment. As expected, single-domain proteins tend to be smaller than multi-domain proteins, though some overlap between the two distributions exists. Fig. (2) . Distribution of the number of residues (nres) in the singleand muti-domain proteins examined in the CASP7 experiment.
It is thus easy to select a threshold value t and to predict that a protein contains only one domain if smaller than t and that it is multi-domain protein if larger than t. Table 2 shows the mcc values [see equation (1) ] observed at various threshold values and validated with a Jack-knife procedure for the proteins examined in the CASP7 experiment. It can be observed that the mcc values are obviously smaller for very small or large values of the threshold. On the contrary they are rather large (>0.6) for intermediate threshold values and the highest mcc (0.628) is observed with a threshold of 200 residues. This prediction approach is clearly very naive. It simply assumes that a protein domain has a little probability to be very large and, as a consequence, that larger proteins have a higher probability to contain two or more domains. A protein is predicted to contain a single domain if it contains less residues that t and it is predicted to contain more than one domain if it has a number of residues larger than t. Data are taken from the proteins examined in the CASP7 experiment.
It is interesting to compare the results of this extremely simple prediction strategy with the results obtained within the CASP7 experiment, where several prediction methods were applied to about 100 proteins. Table 3 shows the mcc values computed on the basis of the predictions deposited by the participants to the CASP7 experiment. The same classification in tp, fp, fn, and tn, which is described in the Methods section, was used. This means that if protein P contains more than a single domain and it was predicted to contain more than a single domain by using the prediction method M, this was considered a true positive (tp). On the contrary, if it was predicted to contain only one domain by the method M, the prediction was considered a false negative (fn), etc. The data of Table 3 clearly show that most of the prediction methods are less reliable than the predictions based on the very simple assumption that a small protein has a high probability to contain a single domain and that a large protein is likely to contain two or more domains. Actually, only four methods (baker, foldpro, maopus and robetta) can predict a multidomain protein better than the simple predictor (Matthews correlation coefficient larger than 0.628). What does this mean? Are these bioinformatics tools useless in structural biology? The answer is no. First, some of them seem to be rather accurate. Second, these computational techniques were not specifically trained to identify multi-domain proteins and it is thus not surprising that some of them are not suitable to discriminate mono-and multidomain proteins. However, it is reasonable to suppose that these bioinformatics tools are still immature and progress should be expected in the future. Table 4 shows the average values of the J, R, and FM indices computed by comparing predicted and real partitions [see equations (2)-(4)]. All the values tend to be large, quite close to their maximal value of 1. However, the probabilities (pJ, pR, and pFM) to observe by chance values higher than these are quite large, ranging from about 30% to about 70%. Baker, foldpro, maopus and robetta are better in predicting a partition that is closer to the real one, with J, R, and FM values that are larger and have a minor probability to be observed by chance. Not surprisingly, they are the same methods that work better to identify multi-domain proteins (see the mcc values of Table 3 ).
It must also be observed that matching between prediction and reality is slightly better for small proteins than for large proteins. For example, the probability pJ to find J values larger than those observed by comparing the reality and the predictions of the method "baker" is on average equal to 39%, it decreases to 33% for proteins shorter than 150 residues, and it increases to 43% for proteins containing more then 150 amino acids. This is actually not surprising, since it is easier to predict that a small protein contains a single domain, with, perhaps, two small N-and C-terminal segments protruding from the domain. However, it must be noted that, despite the fact that the pJ, pR, and pFM values can be used only as semi-quantitative indicators -since they are obtained from empirical statistical distributions -it is quite clear that the domain boundary predictions are still quite far from matching the reality.
We have seen in the previous chapters that the bioinformatics tools are not yet mature enough to be used as routine instruments to design structural biology experiments. However, a very positive feature of these computational methods is that when they work [see equations (6) and (7)] they work very well. The following data are shown: the percentage of domains that are correctly predicted (see text for details) PC_C, the average deviation between the real and the predicted beginning of the domain Delta_b, and the average difference between the real and the predicted end of the domain Delta_e (standard deviations of the mean in parentheses). Table 5 shows the percentage of domains that are correctly predicted [according to equations (6) and (7) ] and the discrepancy between the real and the predicted boundary in the subset of domains that are correctly predicted. It appears that only a relatively modest fraction of the domains can be considered to be well predicted, according to the criteria defined by equations (6) and (7) . The percentage of good predictions is about 30-40%, with some prediction methods behaving considerably better than the others and able to well predict about 60% of the domains. The average values of Delta_b (see Methods) are close to and lower than 0 for all the prediction methods. Also the values of Delta_e are very small, though their absolute value tends to be slightly larger than that of Delta_b. Interestingly, the Delta_e values are positive, on average, for each prediction method.
This clearly indicates that in the subset of good predictions the domain boundaries are located with very high accuracy. Actually, a deviation of 1-3 residues is probably a very minor mistake in the process of design a protein construct that has, on average, a high probability to be well folded and conformationally homogeneous. It is also interesting to observe that while the Delta_b mean values are negative, the mean Delta_e values are larger than 0, indicating that predicted domains tend to be slightly longer than real domains.
In the present manuscript we have analyzed the reliability of the predictions that were made in the CASP7 experiment and that are publicly available. It was found that most of the bioinformatics tools are able to determine if a protein is made by a single domain or if it contains more than one domain, despite a similar reliability is reached by considering only the sequence length, a much simpler strategy. Using a standard and well known statistical test, we showed that most of the predictions that can be done are not impressively better than pseudo-random predictions. It was also observed that although the reliability of the prediction methods seems to be insufficient to make them routine tools in experimental structural biology, their performance can be extremely good. When the domain is correctly identified, its boundaries are very close, within one or two residues, to the experimental ones. In conclusion, these bioinformatics applications are not yet sufficiently accurate to be used as routine tools in experimental structural biology. It is rather probable that the use of more than a single prediction method by a sort of consensus approach might improve the reliability of the predictions. Although these bioinformatics tools are still immature, progress can be expected in the future. This work was supported by the Austrian GEN-AU project BIN-II. Björn Sjöblom and Kristina Djinovic are acknowledged for helpful discussions. Financial support by Putta None is also acknowledged. One reviewer is acknowledged for a series of references that deserved citation. Scientific Abstracts   International Course on Emerging Viruses in the Amazon Region   Enhancing Time-Series Detection Algorithms for Automated Biosurveillance BioSense is a US national system that uses data from health information systems for automated disease surveillance. We studied 4 time-series algorithm modifications designed to improve sensitivity for detecting artificially added data. To test these modified algorithms, we used reports of daily syndrome visits from 308 Department of Defense (DoD) facilities and 340 hospital emergency departments (EDs). At a constant alert rate of 1%, sensitivity was improved for both datasets by using a minimum standard deviation (SD) of 1.0, a 14–28 day baseline duration for calculating mean and SD, and an adjustment for total clinic visits as a surrogate denominator. Stratifying baseline days into weekdays versus weekends to account for day-of-week effects increased sensitivity for the DoD data but not for the ED data. These enhanced methods may increase sensitivity without increasing the alert rate and may improve the ability to detect outbreaks by using automated surveillance system data. S ince the late 1990s, the threats of bioterrorist attacks, the potential for outbreaks of natural disease such as severe acute respiratory syndrome and pandemic infl uenza, and the availability of computerized data have prompted the use of automated disease surveillance systems (1) . Sources of information include clinical data, such as records of hospital emergency department visits, and nonclinical information, such as sales of over-the-counter remedies (2) .
However, human resources are limited for interpreting the large volume of available information. Thus, statistical algorithms are needed to fi lter large volumes of data, focus attention on potential public health problems, and provide an objective measure of increases in disease activity.
BioSense is a US national automated surveillance system that receives data from various sources and makes them available for public health use. The data may be viewed simultaneously by local, state, and federal public health offi cials through the Internet-based BioSense Application, which may be accessed on a jurisdiction-specifi c basis through the Centers for Disease Control and Prevention (CDC) Secure Data Network (3) . Data received include coded fi nal diagnoses and free-text chief complaints, which are assigned as appropriate to >1 of 11 syndrome groupings representing general illness categories such as respiratory and gastrointestinal illnesses (4) and to >1 of 78 subsyndromes representing more specifi c categories such as asthma or cough (5) . To identify days when disease indicator activity is higher than expected, BioSense uses a modifi ed version of the C2 algorithm, 1 of 3 algorithms (C1, C2, and C3) developed for the Early Aberration Reporting System (EARS) (6, 7) .
The C2 algorithm uses a sliding baseline of 7 consecutive recent days' counts to calculate a mean (μ) and SD (s t ). The test statistic is (x t -μ)/s t , the number of SDs by which the current value x t exceeds μ, or 0 if x t does not exceed μ. EARS uses a test statistic >3 to signal an alert (6, 7) . Owing to their simplicity, ease of implementation, and implicit correction for seasonal trends (only data from the prior 9 days are used), the EARS algorithms are widely used (8) (9) (10) . However, the algorithms do not perform optimally under all circumstances. First, because daily counts often vary by day of week, many alerts may be produced on high-count days such as Mondays and Tuesdays, and few may be produced on low-count days such as weekend days. Second, the short (7-day) baseline period may produce unstable values for the mean and SD; thus, the minimum daily count that triggers an alert may vary widely over a short period. Third, using simple count data does not account for the population at risk, which is generally unknown in these systems and which may vary, especially during crisis situations. Although C2 can be used on rates rather than counts, prior evaluations have not shown that using rates improves performance (L. Hutwagner, pers. comm.). Finally, occurrences of many disease indicators are rare, resulting in calculations for both expected values and SDs of 0; the EARS methods are not recommended in such instances. A minimum SD may be used to avoid division by zero, but if this minimum value is set to 0.2, a count of 1 will be 5 SDs above the mean and trigger a high-level alert.
This article describes and evaluates modifi cations of C2 that retain its inherent advantages, address its potential limitations, and improve its performance. We used real daily syndrome counts from 2 sources as baseline data and assessed the ability of various algorithms to detect additional counts artifi cially added to the data. Because all analyses were conducted at a constant alert rate of 1%, improvements in sensitivity were not accompanied by an increase in alerts.
Four algorithm modifi cations, designed to address shortcomings in the C2 algorithm, were tested. The fi rst modifi cation tested was stratifi cation by weekdays versus weekend days. Although many methods have been used to adjust for differing counts by day of week (11) , these methods may require customization to specifi c datasets and a long data history (up to several years). Our simple method is to stratify the baseline days used to calculate μ and s t into weekdays versus weekend days. This stratifi cation is denoted the W2 algorithm. For example, a 7-day W2 baseline for weekdays contains the most recent 7 weekdays. For unstratifi ed and stratifi ed analyses, the 2 days immediately before the index day were excluded from the baseline, a standard practice for C2, to avoid contamination with the upswing of an outbreak.
The second modifi cation tested was lengthening the baseline period. Because a 7-day period may provide insuffi cient data for an accurate and stable calculation of μ and s t , we tested baseline periods of 7, 14, and 28 days. However, because we used data from <56 days before the index day, the stratifi ed 28-day baseline will include only ≈16 days for weekend days.
The third modifi cation tested was adjustment for total daily visits. For the adjustment procedure, we used a formula in which n 0 = count of visits on the index day for the chosen syndrome (e.g., visits for the respiratory syndrome), and d 0 = the total number of facility visits on the index day, including visits that were both assigned and unassigned to any of the 11 syndromes. Σn i = total syndrome visits summed for all i baseline days. Σd i = total facility visits summed for all i baseline days. The formula for the adjusted expected value was e 0 = d 0 × Σn i /Σd i , which differed considerably from the mean of the n i if d 0 was high or low. Fewer visits for a given syndrome were thus expected on a day when the facility had fewer total visits. The estimated adjusted SD, s 0 , was taken as the mean absolute value of (n i -d i × Σn i /Σd i ) over i baseline days; that is, s 0 = Σ (abs (n i -d i × Σn i /Σd i ))/i. The test statistic adjusted for total visits was (n 0 -e 0 )/s 0 , analogous to the C2 statistic (n 0 -μ)/ s t , where μ and s t are the mean and SD of n i , the counts on baseline days. In the discussion below, we refer to this adjustment as the rate algorithm.
The fourth modifi cation tested was increased minimum value for SD. We studied minimum values of 0.2 and 1.0.
To test these modifi cations, 2 datasets were used: records of Department of Defense (DoD) facility fi nal diagnoses for September 2004-November 2007 and records of hospital emergency department (ED) chief complaints for March 2006-November 2007. The DoD data consisted primarily of data from outpatient clinics; however, ≈15% of the visits in this evaluation were from patients seen in emergency facilities and cannot currently be differentiated in the BioSense System. We studied the 11 syndrome groups designed to be indicative of infections resulting from exposure to pathogens plausibly used in a bioterrorist attack (4). The DoD data consisted of daily counts of patient visits with International Classifi cation of Diseases, 9th Revision (ICD-9)-coded diagnoses categorized into the 11 syndrome groups. The hospital ED data consisted of freetext chief complaints, which were fi rst parsed for a specifi ed set of keywords, abbreviations, and misspellings and then categorized into 10 of the syndrome groups (1 syndrome, specifi c infection, was used for diagnosis but not for chief complaint data). Some ICD-9 codes and chief complaints may be included in >2 syndromes. However, counts of different syndromes were analyzed separately, not added together, and therefore are not double-counted in the analyses. For both datasets, we analyzed counts aggregated by facility. We included facility-syndrome combinations that had mean counts >0.5 over all facility-syndrome days in the study period. Many DoD clinics are closed on holidays. Therefore, for the DoD data, 11 days (days on which federal holidays are observed and the day after Thanksgiving) were recoded as weekend days for purposes of stratifi ed algorithm calculations (5) . Because hospital EDs typically are open on these holidays, no recoding for holidays was performed for this dataset.
The mean count for each facility syndrome was calculated and categorized as follows: 0.5 to <2, 2 to <4, 4 to <6, 6 to <8, 8 to <10, 10 to <20, 20 to <40, and >40. Empirical distributions of the test statistic (e.g., number of SDs by which the observed count exceeds the expected value) were conducted separately for each dataset, algorithm, and mean count category; the 99th percentile value for each of these distributions was used as the cutoff value to defi ne an alert rate of 1%. For example, for the standard C2 algorithm in DoD data with mean count 4 to <6, a cutoff value of 3.9 was used because 1% of the facility-syndrome days had a test statistic >3.9. Because no attempt was made to fi nd and exclude real outbreaks from the data, these cutoff values defi ne an alert rate rather than a false alert rate, the latter being equivalent to 1-specifi city (12) .
At a constant alert rate of 1% for all methods, the sensitivity for detecting additional counts was calculated by performing the following steps: 1) running the algorithm to determine expected values and SDs for each facilitysyndrome-day; 2) fi nding the 99th percentile cutoff value for the test statistic for each dataset-algorithm-mean count category as explained above; 3) for each facility-syndrome day, determining whether the observed count plus additional counts is greater than or equal to the threshold value (threshold value = expected value + SD × 99th percentile cutoff value); and 4) calculating sensitivity as the percentage of days on which the additional counts would exceed the threshold value and therefore be detected. Using this method, a single computer run can calculate sensitivity for detecting single-day additional counts on all days in the dataset; if the additional counts are spread over multiple days, separate computer runs would be needed (7).
The DoD diagnosis data contained 1,939,993 facilitysyndrome days from 308 facilities in 48 states with an overall mean of 7.7 counts per facility per day; of the 11 syndromes, respiratory visits comprised the highest percentage (16% of total facility-syndrome days) and had the highest mean count (26.0 visits per facility per day) ( Table 1 ). The hospital ED data contained 768,195 facility-syndrome days from 340 facilities in 21 states and had an overall mean of 7.8 counts per facility per day; no visits for lymphadenitis and severe injury and death were included because no facilities had a mean count >0.5 per day for these syndromes.
The DoD data had a strong day-of-week effect; 16%-21% of total weekly visits occurred per day on weekdays, and only 3%-4% of visits occurred per day on weekend days and holidays ( Figure 1 ). The hospital ED data had a minimal day-of-week effect: 14%-16% of visits occurred per day on weekdays, and 14%-15% of visits occurred per day on weekend days.
The accuracy of expected value calculation was evaluated by using mean absolute residuals. For lower residuals, expected values are closer to observed values than they are for higher residuals. Similarly, the expected value calculation is more accurate for lower residuals than for higher residuals. For the DoD data, lower residuals were seen with stratifi cation (W2) and the rate algorithm: mean residual 4.2 for unstratifi ed count algorithm versus 2.2 for stratifi ed rate algorithm (Table 2) . For the hospital ED data, residuals were lower for the rate algorithm, and stratifi cation had a minimal effect. Varying the baseline duration and minimum SD had no effect on the accuracy of expected value calculation (data not shown).
The effect of modifi cations of the initial algorithm on the sensitivity for detecting additional counts was examined; each modifi cation was added consecutively (Table  3) . For the DoD data, sensitivity was 40.6% for the initial algorithm and increased to 43.9% when the rate method was used; 70.8% when the minimum SD was increased to 1.0; 79.4% when the baseline duration was increased to 28 days; and 82.0% when a stratifi ed baseline was used. Comparing the initial algoithm to the best algorithm showed a 41.4% increase in sensitivity. For the hospital ED data, sensitivity was 40.2% for the initial algorithm and increased to 64.8% for the best method (minimum SD = 1, 28-day baseline, rate method, unstratifi ed baseline); however, when the stratifi ed baseline was used, sensitivity decreased to 62.1%; the initial algorithm compared with the best algorithm showed a 24.6% increase in sensitivity. When these sensitivity calculations were stratifi ed by mean count for each facility-syndrome (data not shown), we found that the modifi cations increased sensitivity in all strata of the DoD data; for the hospital ED data, the rate method reduced sensitivity by 1.0% in the 8 to <10 count category and by 0.5% in the 10 to <20 count category, but increased sensitivity in other categories and overall. When we limited analysis to ED data with a mean count of 4 to <6 per day and explored sensitivity for detecting varying numbers of additional counts (Figure 2 ), we found, as expected, that as the number of additional counts increased, sensitivity increased. The difference between the initial and best algorithms was highest when sensitivity was ≈50% for the initial algorithm. That is, for 10 additional counts, sensitivity was 49.8% for the initial algorithm and 85.3% for the best algorithm, an improvement of 35.5%. However, if the initial C2 algorithm had either low or high sensitivity, the modifi cations had little effect.
As an example, we analyzed fever syndrome data from 1 ED. The mean count was 4.9 per day, and the 99th percentile threshold values were 3.86 SDs for the initial and 3.55 for the best algorithm. Over 632 days, the sensitivity for detecting 8 additional counts was 47.2% for the initial and 70.9% for the best algorithm (23.7% difference). Data for a 2-month period showed that the calculated SD (Figure 3 , panel A) and the threshold value (i.e., count needed to trigger an alert; Figure 3 , panel B) varied substantially for the initial algorithm but were comparatively stable for the best algorithm. During the 2-month period, 8 additional counts would be detected by initial and best algorithms on 30 days, by only the initial algorithm on 2 days, and by only the best algorithm on 19 days; neither algorithm detected the additional counts on 10 days (Figure 3 , panel C).
Our results demonstrate that simple modifi cations of the widely used C2 algorithm can substantially improve the ability to accurately recognize 1-day increases in disease syndrome activity. Depending on the dataset, mean count in the data, and the number of additional counts added, the enhanced methods may increase sensitivity by 20%-40%. These improvements were achieved without an increase in the alert rate, which was held constant at 1% for all methods. Although we chose a 1% alert rate for testing purposes, in practice, it is useful to vary the alert rate to fi t the circumstances, and the BioSense application enables the alert rate to be varied between 0.1% and 2%. Regardless of the alert rate used, the modifi ed methods have higher sensitivity. For the DoD and hospital ED datasets, sensitivity was improved by using a higher minimum SD of 1.0, a longer baseline duration of 28 days, and adjusting for total visits. Stratifying baseline days into weekdays versus weekends/ holidays increased sensitivity in the DoD data, which has a strong day-of-week effect, but modestly decreased sensitivity in the hospital ED data, which does not have such an effect. Thus, the best analytic methods depend on dataset characteristics, especially the day-of-week effect, and could be varied by manual or automated selection. These fi ndings can be used to improve both early event detection and situation awareness because accurate recognition of unusually high counts is needed for both uses.
These modifi cations were apparently effective for the following reasons. Accounting for total visits to the facility (i.e., rate method) produces a more accurate expected value and lower residuals ( Table 2 ). Although number of total visits is not the ideal denominator, in general it is better than no denominator at all. An advantage of the rate method is that calculations may be made when only partial data for a given day are available. However, adjusting for total visits may reduce sensitivity slightly in some subgroups, as we found for the hospital ED data when the mean count was 8 to <20. Stratifi cation by weekday versus weekend day improves expected value calculations when a substantial day-of-week effect exists, such as in the DoD data. When such an effect is not present, stratifi cation causes days further from the index day to be used in the baseline period, therefore producing slightly less accurate expected values. Longer baseline durations have no effect on the accuracy of expected value calculation and improve sensitivity by producing more accurate and stable SD values. Using a higher minimum SD avoids nuisance alerts that may be prompted by small fl uctuations in the daily visit count. This method also changes the distribution of test statistic values, which results in a lower 99th percentile cutoff value, which increases sensitivity for detecting moderate-to-high numbers of added counts. Using a higher minimum SD is benefi cial if disease indicators with low and high counts are analyzed; an alternate approach is to use different methods for lowversus high-count data. The issues focused on by our suggested modifi cations may alternately be addressed by various sophisticated mathematical modeling approaches. However, health departments, which are generally limited in resources and in analysis expertise, may resist use of decision-support methods that are expensive, diffi cult to implement, or not transparent to human data monitors. For example, sophisticated Serfl ing-type regression models have long been used by CDC for tracking the progress of infl uenza season (13, 14) and have been used to analyze selected data in the Bio-Sense system. However, these models have both strengths and weaknesses and have not been widely embraced for daily disease surveillance. Even if the expertise and hardware capability for applying them were made available to local health departments, many time series are unsuitable for this approach. We present simple and easily understood and implemented enhancements to C2 to extend its applicability and improve its performance. These enhancements may be applicable to other control chart-based algorithms as well.
Automated surveillance systems based on chief complaints and diagnoses have a number of uses: providing assistance in data collection; monitoring seasonal infl uenza (15) ; monitoring total ED visits during a crisis; and monitoring simple surrogates of infectious diseases, injuries, and chronic diseases during large outbreaks or disasters (16) . The utility of these systems has not been demonstrated for Additional counts Sensitivity Figure 2 . Sensitivity of detecting various numbers of additional counts, by using initial versus best algorithms for hospital emergency department chief complaint data, for selected BioSense data. Red line shows the initial algorithm (minimum SD = 0.2, 7-day baseline, count method, unstratifi ed baseline), and black line shows the best algorithm (minimum SD = 1.0, 28-day baseline, rate method, unstratifi ed baseline).
monitoring small-or intermediate-sized outbreaks or illnesses defi ned primarily by laboratory testing. Even when using these suggested modifi cations, sensitivity for detecting additional counts at the facility level remains modest. However, the utility of automated biosurveillance will be expanded with the availability of better population coverage and more specifi c data, the use of multiple data types in combination, and improved detection algorithms, such as those proposed here. The limitations of this study include using only data with a mean count >0.5 per day; analyses of sparser data might show different results. We studied only facility-level aggregation of data, selected patient types (e.g., hospital inpatients were not studied), selected data types (e.g., ED diagnoses were not studied), and broadly defi ned syndromes (the more granular subsyndromes, which are likely to yield lower counts, were not studied). Although we evaluated only a simple time-series detection method, optimizing performance of simple methods is useful before they can be meaningfully compared with more sophisticated methods, such as regression. Also, we studied effects of additional counts on single days rather than multiday outbreak effects; however, because the C2 algorithm considers data from only 1 day at a time, this is a reasonable initial approach. These results must be confi rmed by trials of multiday signal injection and performance evaluated for multiple subgroups (e.g., syndrome, day of week, season). We adopted the approach of evaluating sensitivity at a fi xed 1% alert rate defi ned empirically for each algorithm and dataset, as used by Jackson et al. (12) . Our approach is in accord with a recent review that recommended basing alert thresholds on empirical data rather than on classical statistical theory (17) . A major strength of the study is that BioSense is a national system that provided access to 2 major datasets with differing characteristics and to data from hundreds of facilities in many states. The length, geographic spread, and syndrome variation of the study datasets lend weight to the results.
The fi eld of electronic biosurveillance is in its infancy and is rapidly changing. Early work focused on attempts to detect outbreaks (early event detection) by using broadly defi ned syndromes (e.g., respiratory syndrome) based on chief complaints and diagnoses. Emphasis has recently shifted to monitoring for ongoing outbreaks (situational awareness) and for specifi c disease indicators (e.g., cough, dyspnea) called subsyndromes. The fi eld is now beginning to develop methods for case-based surveillance (i.e., automated application of a formal case defi nition using computerized data) (18) . Each data type and disease indicator may have unique characteristics that require modifi cations of standard data analysis methods. However, because the adaptation of time-series methods to recognize outbreaks will be an ongoing need, the enhanced methods identifi ed by this study are likely to have lasting usefulness. Figure 3 . Comparison of initial versus best algorithms for analysis of fever syndrome data at an example emergency department, October-November 2006. A) SD comparison. Count, fever syndrome counts; SD (initial), SD by using initial algorithm (minimum SD = 0.2, 7-day baseline, count method, unstratifi ed baseline); SD (best), SD by using best algorithm (minimum SD = 1.0, 28-day baseline, rate method, unstratifi ed baseline). B) Count threshold comparison. Count, fever syndrome counts; threshold 1, minimum count needed to trigger an alert by using initial method; threshold 2, minimum count needed to trigger an alert by using best method (for the best algorithm, which accounts for rate, 8 counts were added to total visits for calculating the threshold). C) Detection of 8 additional counts. Count, daily fever syndrome counts; count + 8, daily count plus 8 counts; both methods, 30 days with the additional counts detected by both the initial and best methods; initial only, 2 days with the additional counts detected by using initial method only; and best only, 19 days with additional counts detected by using best method only. Molecular Mechanisms of Recombination Restriction in the Envelope Gene of the Human Immunodeficiency Virus The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology. Pathogens, and viruses in particular, are subject to strong selective pressures during infection and often have characteristically high degrees of genetic variation [1] . Recombination is an important evolutionary mechanism that contributes to this genetic diversification. By creating novel combinations of pre-existing genetic polymorphisms in a single replication cycle, recombination enables greater movements through sequence space than can be achieved by individual point mutations. As a consequence, recombination provides access to evolutionary ''shortcuts''. In addition, since recombination generally involves genes that already encode functional products, the probability of producing viable progeny is higher compared to the insertion of an equivalent number of random point mutations [2] . However, the generation of recombinant forms is not an unconstrained process. Genes and genomes generally evolve through the slow accumulation of point mutations, which often requires the progressive insertion of compensatory mutations at ''linked'' sites. This coevolution permits the preservation of epistatic interactions. By simultaneously introducing several substitutions, recombination has the potential to substantially perturb such coevolved intra-genome interaction networks [2, 3] , impairing the functionality of the genes involved. Thus, the balance between the advantages of taking evolutionary shortcuts and the risk of chimeras being dysfunctional [2] determines the role played by recombination in the evolution of a given gene or organism.
Several studies have focused on the impact of recombination on the evolution of proteins, particularly in relation to directed evolution experiments [4, 5] . Two major factors have a large influence on the functionality of recombinants proteins. The first is the position of recombination breakpoint (the region where the sequence shifts from that of one parental sequence to the other) relative to the location of genetic polymorphisms within the gene. Recombinants involving a large number of non-synonymous substitutions will in fact have a low probability of being functional [2] . The second factor is the position of the breakpoints in relation to the boundaries of discrete protein folds. Breakpoints near the boundaries of these domains will in general have a smaller impact on protein folding, and hence protein function, than breakpoints occurring within them [3, 6, 7] . Recent work on Begomoviruses corroborated these findings by demonstrating that recombination events found in natural viral populations are significantly less disruptive of protein folding than randomly generated recombinants [8] .
Adaptation of pathogens, either to on-going immune pressures within individual hosts or following transmission to new hosts of the same or different species, can result in infectious outbreaks that constitute major threats for public health [9] [10] [11] [12] . The human immunodeficiency virus (HIV) is an extremely recombinogenic pathogen in which recombination has been implicated in key aspects of viral pathogenesis such as immune evasion [13] , transmissibility [14] , the evolution of antiretroviral resistance [15, 16] and cross-species transmission [9, 12] . Indeed, the remarkable genetic flexibility of HIV is underlined by its large genetic diversity. The HIV-1 population is subdivided into three groups, named M, N and O, with group M (which is responsible for the vast majority of the infections worldwide) being further subdivided into nine subtypes (named A, B, C, D, F, G, H, J and K) [17] .
Although recombination in HIV has been shown to occur at all phylogenetic levels (intra-and inter-subtype, as well as inter-group, reviewed in reference [18] ), the most widely noted impact of recombination on the genetic diversification of this virus is the frequent natural occurrence of inter-subtype recombinants in parts of the world where multiple subtypes co-circulate [19] [20] [21] [22] . When the same inter-subtype recombinant is transmitted between multiple individuals, and has therefore the potential to be of epidemiological significance, it is termed a Circulating Recombinant Form (CRF) [17] . As with the HIV-1 subtypes, CRFs form distinct clusters in phylogenetic trees and some of them contribute substantially to the pandemic. Sufficient inter-subtype recombinant sequences have been sampled to permit the detailed characterisation of variation in the locations of breakpoints both within individual genes [22, 23] , and entire genomes [24, 25, 32] . This makes HIV a particularly useful model for studying the forces that shape pathogen populations within the context of global epidemics. Here we focus on recombination within the envelope gene (env). This gene encodes two polypeptides (gp120 and gp41) that form a heterodimer at the surface of the viral particle. Trimers of these heterodimers are the functional units that are responsible for binding to the cellular receptors and co-receptors and ultimately lead to viral entry into target cells [26] . The two protein products of env are also the targets of all the neutralising antibodies identified to date [27] . By using a tissue culture system to characterise inter-subtype recombinants generated within env in the absence of selection, and assaying the functionality of recombinant genes, we produce an empirical model of HIV recombination that accurately describes recombination patterns found in viruses sampled throughout the HIV pandemic.
We used different combinations of env sequences from primary HIV-1 isolates belonging to either different group M subtypes or group O (see Materials and Methods for the list of parental isolates used) to determine the distribution of breakpoints occurring within the HIV env gene in the absence of selection. We chose combinations of isolates belonging to subtypes that are cocirculating in regions of the world from which natural intersubtype recombinant forms have emerged [28] .
In order to quantify variations in recombination rates across env we used a previously described experimental system where human T cells are transduced with HIV-1 replication-defective vectors pseudotyped with the Vesicular Stomatitis Virus (VSV) envelope [29] . As this system mimics a single cycle of viral infection in which reverse transcription products neither influence cellular survival, nor confer a specific phenotype to the transduced cells, recombinants that were produced during reverse transcription were not subjected to any selection. After cloning of the reverse transcription products in E. coli, the system enabled identification of the recombinants based on the presence of a lacZ reporter gene ( Figure 1 ). Given that known input sequences were used, such an approach enables the accurate and unambiguous localization of the breakpoint position to precise regions bounded by nucleotides that differ between the two parental sequences.
The regions of the envelope gene that were studied were chosen so as to obtain 700 to 1,500 nucleotides overlapping windows, spanning the whole of env. For each of seventeen different combinations of parental sequence pairs (Figure 2A) , a recombination rate per nucleotide and per reverse transcription run was calculated within a 50 nucleotides sliding window (with 10 nucleotides step size). These were plotted as a function of the location of the window along the gene. To evaluate whether recombination-prone regions exist within the population, data from the 17 different pairs of parental sequences were pooled and an average recombination rate was computed for the different regions, and plotted as function of the position along the env gene ( Figure 2B , top panel). Peaks and troughs were apparent all along the gene, with regions refractory to recombination being more
Recombination allows mixing portions of genomes of different origins, generating chimeric genes and genomes. With respect to the random generation of new mutations, it can lead to the simultaneous insertion of several substitutions, introducing more drastic changes in the genome. Furthermore, recombination is expected to yield a higher proportion of functional products since it combines variants that already exist in the population and that are therefore compatible with the survival of the organism. However, when recombination involves genetically distant strains, it can be constrained by the necessity to retain the functionality of the resulting products. In pathogens, which are subjected to strong selective pressures, recombination is particularly important, and several viruses, such as the human immunodeficiency virus (HIV), readily recombine. Here, we demonstrate the existence of preferential regions for recombination in the HIV-1 envelope gene when crossing sequences representative of strains observed to recombine in vivo. Furthermore, some recombinants give a decreased proportion of functional products. When considering these factors, one can retrace the history of most natural HIV recombinants. Recombination in HIV appears not so unpredictable, therefore, and the existence of recombinants that frequently generate nonfunctional products highlights previously unappreciated limits of the genetic flexibility of HIV.
common in the gp120 coding portion than in the gp41 region. The probability that breakpoints were more or less clustered across env than could be accounted for by chance (given the null hypothesis that breakpoint positions occur randomly) was determined by a permutation test ( Figure 2B , bottom panel). Six major recombination-prone or ''hot'' regions (shaded light blue areas in Figure 2B ) could be defined as env regions where breakpoint clusters were bounded by statistically significant breakpoint ''cold spot'' (p,0.05). Each of the six identified breakpoint clusters contained at least one breakpoint cluster that constituted a statistically significant recombination ''hot-spot'' (p,0.01). While these recombination-prone regions covered only slightly more than half of the whole gene (55.3%), they included 81.6% of all the breakpoints (337/413) mapped. These six hot regions are areas where recombination occurs preferentially during HIV replication, irrespective of the parental strains involved.
We next investigated the fate of these recombinants with respect to their establishment in the natural HIV-1 population. The fixation of a recombinant gene within a population is dependent on the interplay of multiple factors. Nevertheless, an obligatory component of evolution is undoubtedly the elimination by purifying selection of viruses that express dysfunctional proteins. To evaluate how profoundly this aspect of natural selection might influence the pattern of breakpoints generated by the mechanism of recombination, we determined the relative functionality of a subset of recombinant env genes.
In addition to encoding the proteins that coat the viral membrane, env also encodes a well-known functional RNA structure, the Rev responsive element (RRE). For the recombinants containing breakpoints in the RRE region the functionality of this RNA module was therefore also tested. Being involved in the regulation of the timing and the balance among the various forms of unspliced and partially or completely spliced RNAs, RRE is essential for viral replication [30] . Failure to properly regulate this process results in either a decrease or complete halt in viral production [30] . The functionality of chimaeric RREs was tested by measuring viral titres obtained upon transfection of cells with a plasmid containing the proviral sequence of the molecular clone NL4.3 of HIV-1, in which we had replaced the native NL4.3 RRE with that of the various chimaeric RREs. To uncouple the effects on RNA-folding caused by the introduced RRE sequences from those altering the amino acid sequence of expressed proteins, we used a variant of NL4.3 that does not express Env (NL4.3-Env 2 ) [31] , and a plasmid encoding the wild-type Env was co-transfected to complement the production of gp120 and gp41 proteins. In order to increase the statistical power of the analysis, additional chimaeric RREs were constructed using parental sequences other than those employed in our cell culture recombinant generation experiments (following a PCR procedure described in Materials and Methods) and tested for their functionality. As can be seen in Table 1 , the viral titres obtained with every chimaeric RRE sequence we tested were both similar to those obtained with nonrecombinant parental RRE sequences and markedly higher than that observed when the RRE was replaced with a non-viral sequence (see Materials and Methods). This result therefore clearly indicated that recombinants generated by breakpoints within the RRE generally retain the functionality of this element.
To determine the functionality of individual recombinant envelopes at the protein level, full-length recombinant envelope genes containing breakpoints of interest were constructed by successive PCR, as described in Material and Methods. Each fulllength recombinant gene was then cloned in the pcDNA3.1 expression vector, and used to transfect HEK 293T cells together with the pNL4.3-Env 2 -Luc plasmid, to generate viral particles pseudotyped with the recombinant envelope of interest. The functionality of the recombinant envelopes was then tested after transduction of HEK 293T-CD4 + -CCR5 + cells at a multiplicity of infection of 0.1, by measuring luciferase expression in these cells 48 hours after transduction. Since target cells cannot synthesize new viral envelope proteins, infection was limited to reverse transcription and, potentially, integration. The luciferase values observed therefore reflected the relative success of viral entry into the target cells. For this analysis recombinants derived from parental env sequences that yielded the strongest positive signals in this single cycle test were chosen (parental sequences A-Q461, C-CAP210, G-1033 and O-32, see Table 2 for their relative genetic distance) due to the higher reliability of the luciferase signal. The parental env sequences were used as controls. As for the functional analysis of the RRE, additional recombinants involving combinations of parental sequences -other than those involved in the experiments of recombination in cell culture, but carrying breakpoints in the same regions -were also tested. These additional recombinant env sequences were generated by PCR, as described for the reconstitution of the full-length env gene.
Luciferase values determined for each recombinant were plotted as a function of the corresponding breakpoint position ( Figure 3 ). Recombinants with breakpoints falling within the six hot regions indicated in Figure 2B were preferentially characterized. It was apparent that most of the severely defective recombinants contained breakpoints in hot regions 2 and 3 of the recombination rate distribution (Figure 3 ). Given this data, we approximated a probability of Env functionality being disrupted by breakpoints falling within each of the six high recombination-rate regions. Since the parental sequences themselves were not uniformly functional ( Figure 3 ), a situation that is probably common in nature, for each recombinant an estimate of loss of functionality was calculated by dividing the luciferase value obtained with that recombinant by the one of the least functional parental sequence involved in its generation. Recombinants displaying values between those of the two parental sequences were considered to white-grey hatched box: partially deleted and therefore non-functional U3 sequence; white boxes: HIV envelope sequences studied, env X and env Y stand for sequences of two different isolates (isolate X on the donor RNA and isolate Y on the acceptor, respectively); black box: marker lacZ' bacterial gene; grey box: partial sequence of the bacterial gene malT, inserted in the reverse orientation. The approximate location of the BamHI site (present only on the donor RNA) and of the PstI site present on both RNAs is also indicated. The path followed by reverse transcription for generating the BamHI + /LacZ' + recombinants studied in the present work is indicated schematically. doi:10.1371/journal.ppat.1000418.g001 retain functionality (and assigned a functionality value of 1). Of note, none of the recombinants yielded functionality values higher than that of the most functional parent from which it was generated. Values from recombinants containing breakpoints within the same region of the six hot regions were pooled, and a functionality loss value for each region was averaged ( Figure 3 ). The most significant loss of functionality was observed in regions 2, 3, and 6.
Natural recombination breakpoint distributions essentially mirror those of the functional recombinants generated in tissue culture
Having defined a pattern of recombination in the absence of selection and the approximate probabilities of recombination events in various parts of env yielding fully functional products, we were interested in determining whether our experimental data could explain breakpoint patterns observed in circulating recombinants. The distribution along the whole HIV genome of 691 recombination breakpoints within HIV-1 group M full genome sequences from the LANL HIV Sequence Databases (http://hiv-web.lanl. gov/) was inferred. The same approach used in Figure 2B to define the probability that at any region of the genome the breakpoints were more clustered than would be expected by chance was used, with a 200 nucleotides window. A previous analysis of HIV recombinants modelled the distribution of breakpoints and indicated a significant clustering of breakpoints in the 59 and 39 ends of the envelope gene and a lack of breakpoints between these regions [32] . Our new analysis (Figure 4 ) confirmed the propensity for breakpoints to be located at the 59 and 39 ends of the env gene and the lack of breakpoints in the majority of its internal regions in recombinants from the database.
In order to compare our experimentally determined breakpoint distribution to that found in recombinants from the HIV Sequence database, a higher-resolution view of the breakpoint distribution within the env gene was determined using the positions of 133 unambiguously unique recombination breakpoints detectable within 230 env sequences. Following the same procedure described above, but using a 50 nucleotides window to enable detection of breakpoint clusters at the same resolution as in our experimental system, we identified a series of recombination hot-and coldregions within the gene ( Figure 5A , purple graph). In a similar way to the breakpoint distribution detected in cell culture, various hot regions could be defined (light-purple boxes at the bottom of Figure 5A ), which corresponded remarkably well to recombination hot regions 1, 5 and 6 seen in cell culture (light-blue boxes). Whereas the other hot regions identified in cell culture had no corresponding counterparts in the natural breakpoint distribution, there was close correspondence between the cold-spots detected in both distributions.
Next we used the SCHEMA-based method [8] to investigate whether or not this breakpoint distribution exhibits evidence of purifying selection acting on recombinants with disrupted protein folding. This analysis indicated that breakpoints observable in natural viruses tend to occur in regions within env that were predicted to have a significantly lower impact on protein folding than randomly placed breakpoints (p,1.0610 24 for gp120 and p = 8.9610 23 for gp41, see Protocol S1). To investigate whether accounting for variations in the functionality of recombinants might reconcile the natural and experimental breakpoint distributions, we first approximated the combined effects of mechanistic recombination rate variation ( Figure 2B ) and selection for fully functional recombinants ( Figure 3 ) on the distribution of breakpoints in cell culture. Selection ''corrected'' recombination rate estimates were then used to determine the distribution of 133 expected breakpoints. The resulting distribution was used to evaluate the probability of clustering of breakpoints (green graph in Figure 5A ). Only regions 1, 4, 5 and 6 remained areas of significant clustering (light-green boxes at the bottom of Figure 5A ), a pattern very close to that found in HIV recombinants sampled from nature, with the exception of region 4 for which there was substantially less evidence of recombination within natural recombinants than was expected based on our empirical model. Indeed, when compared to the Recombinants 115A/126D, 115A/89D, 120A/89D, and 126D/120A were described previously [33] . The number (n) of individual recombinants for which the position of the breakpoint has been mapped is given on the right of each graph. A map of gp120 and gp41 domains is given as a frame of reference at the top of the figure. (B) top graph: pooled distribution of recombination breakpoints across env, obtained as described in Materials and Methods. Bottom graph: the height of the black plot at any particular position represents the probability (determined by a permutation test with 10,000 iterations) that recombination breakpoint distributions are not more clustered than would be expected by chance within a 50 nucleotides window centred on that position. Assuming that breakpoints are randomly distributed, the dark and light grey regions represent degrees of breakpoint clustering expected due to chance in 95% and 99% of the examined windows, respectively. Whereas peaks emerging above the grey regions represent possible recombination hot-spots, troughs dipping below the grey regions represent possible recombination cold-spots. The paleblue shaded areas numbered from 1 to 6 correspond to breakpoint clusters, or hot regions, as defined in the text. doi:10.1371/journal.ppat.1000418.g002
distribution found for the 133 breakpoints encountered in the natural HIV recombinants ( Figure 5B ), a remarkable overlap was observed, with the discrete statistically significant breakpoint clusters being consistently recaptured by our empirical model of env recombination. The substantial difference of recombination rates in region 4 was also clear.
Through the functional characterization of HIV envelope genes generated by recombination in the absence of selection, we retrace the early steps shaping patterns of inter-subtype env recombination found in the HIV-1 pandemic. We observe that the mechanism of recombination alone defines regions where recombination occurs at significantly higher rates than elsewhere along the gene. The existence of such regions is strongly suggestive of spatially conserved features in HIV genomes that either promote or restrict recombination between different isolates. The distribution of breakpoints within the gp120 encoding region ( Figure 2B ) is likely due to the distribution of conserved and variable regions, the latter restricting recombination because of the low degree of local sequence identity between the parental sequences [32, 33] .
Within genomic regions where sequence identity is high, a trigger for recombination could be the presence of secondary The luciferase values that were determined for the four parental strains used to generate the recombinants are represented, as a frame of reference, by lilac bars on the right. The six recombination-prone regions defined in Figure 2B are shaded in pale blue and annotated accordingly above the graph. The value of loss of functionality approximated for each region is given in bold in the top part of the graph. Three M/O inter-type recombinants were also tested (AO456, AO7810, and AO8090, breakpoint positions 6508, 7810, and 8090, respectively, of the HXB2 reference strain), resulting in a complete loss of functionality, probably due to the higher sequence divergence between the parental isolates. In order to preserve the homogeneity of the dataset to intersubtype recombinants, these recombinants are neither presented in the figure, nor were considered for the calculation of the average functionality of the recombinants, described in the main text. doi:10.1371/journal.ppat.1000418.g003 structures [34] . The highest recombination peak within the second region in Figure 2B (corresponding with the C2 portion of gp120) coincides with a recombination hot-spot that is determined by the presence of a stable RNA hairpin structure [29, 35, 36] , while the fourth hot region ( Figure 2B ) corresponds to the RRE RNA structure that is highly conserved amongst all HIV isolates [37] . It is therefore possible that RNA secondary structures also contribute to the high rates of recombination observed at some of the other recombination hot regions. Noteworthy, the functionality of the RRE was retained even when crossing genetically distant isolates as for inter-group M/O recombinants (Figure 2A) , supporting the possibility that regions of the genome harbouring functional RNA structures, which are generally more conserved within the population, provide a mechanism for crossing distantly related retroviruses and are possibly important for recombination of RNA viruses in general. With respect to selection of recombinant genes at the protein level, experiments involving lattice proteins have shown that genes encoding proteins that tolerate mutations also tend to be recombination tolerant [2] . Since the env gene displays a degree of diversity between isolates from different HIV-1 group M subtypes ( [38] and references herein) that is two to three times higher than the genome average, we anticipated that the manifest mutation tolerance of env might predispose it to high recombination tolerance. However, we show that this is not the case with certain regions within the gp120 encoding portions of env (particularly region 2 described in the present work in Figure 3 ) tending not to tolerate recombination well.
Viruses with small genomes (including all RNA viruses) tend to use overlapping genes expressed in different reading frames and to encode proteins that have multiple functions. The HIV envelope encodes for such proteins [26] , and the subtle biochemical equilibrium that regulates their functionality is very possibly limiting tolerance to recombination. The low recombination tolerance of the gp120-encoding region could only be imprecisely predicted based on computational estimates of recombination induced protein fold disruption using the SCHEMA algorithm [3] . This may have been due to either our SCHEMA analyses being based on incomplete gp41 and gp120 structures or the fact that the structures used only reflected a single conformation of these two proteins. Therefore this analysis neither takes into account the conformational changes required for Env functionality, nor the quaternary arrangement of the proteins within Env trimers. Despite these issues, the SCHEMA analysis indicated that, amongst the HIV env sequences sampled from nature, selection has been acting against recombinants with disrupted protein folding (Table S1 ). Unravelling the molecular reasons for the reduced functionality of certain recombinants could provide valuable insights into the nature of the molecular interaction networks required for proper Env function.
The specific determinants of viral fitness (or in vivo replicative capacity) are complex and poorly understood at present. The fixation of a recombinant gene within a population is likely to depend on the interplay of multiple factors. Although combining cell culture functionality data with recombination rate heterogeneity is an oversimplified view of this process, the pattern of recombination predicted by our empirical model matches remarkably well the breakpoint distributions observed in nature ( Figure 5B ). The only major deviation from this was constituted by the fourth recombination hot region observed in cell culture, which was absent from the natural breakpoint distribution ( Figures 2B and 5B ). Determining the reasons for this discrepancy will improve our understanding of the mechanisms governing the success of recombinants in nature.
Although the host immune response certainly plays a significant role in the selection of recombinant variants in vivo [13] , the similarities between the natural and experimental breakpoint distributions suggest that the forces responsible for the selection of recombinants in vivo only have limited impact on inter-subtype breakpoint patterns in env. This is most likely due to a combination of factors including mainly the complex epistatic interactions within env, the high density of fitness-determining loci within this gene, and the biochemical mechanism of recombination, which collectively constrain the fixation of genetic variability introduced by recombination. Negative fitness effects associated with recombination in env, however, should decrease with decreasing parental genetic distances [3, 6, 39] and therefore, in the context of intra-subtype recombination, the selective constraints on recombinants should be more relaxed than we have found them to be here.
Considering recombination in env in the context of the rest of the HIV genome, it is apparent that env displays the most dramatically variable natural breakpoint distribution of all HIV genes [24, 32] , and it constitutes the only gene within which there is an extended region with limited recombination (Figure 4) . Nevertheless, although less marked, breakpoint distribution patterns reminiscent of those found in env, with alternate clusters and troughs are also identifiable in several other regions of the genome such as gag and pol [32] (Figure 4) . Although little information is presently available either on differential mechanistic predispositions to recombination across these regions, or on the functionality of the resulting products, it is tempting to speculate that underlying rules such as we have defined here for env may also be operational in these other cases.
In conclusion, by experimentally reproducing the generation of HIV-1 recombinants, we demonstrate that the distinctive distribution of breakpoints found in natural viruses is strongly shaped by both the mechanism of recombination, and the relative functionality of the recombinant genes. Thus, HIV evolution might not be the relentlessly unpredictable process it sometimes seems, and exploiting this evidence to pre-empt and counter the most favoured evolutionary tactics of this virus may ultimately be an efficient means by which we can devise effective vaccines and improve drugs against the virus.
Cell culture HEK 293T, and CD4 + CCR5 + 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, penicillin, and streptomycin (from Invitrogen, CA, USA), and maintained at 37uC with 10% CO 2 . MT4 cells were maintained in RPMI 1640 medium supplemented with 10% foetal calf serum and antibiotics at 37uC with 5% CO 2 .
The parental isolates used in this study were A-115, A-120, A-899 [33] 
Single cycle recombination assays were performed using a system previously developed by our laboratory [29] . HIV-1 env fragments from group M subtypes A, C, D and G, and from group O viral DNA were amplified by PCR from infected PBMCs obtained from patients and cloned in plasmids (called genomic plasmids), which differ for the genetic marker present downstream (in the sense of reverse transcription) of the sequence in which recombination is studied (Figure 1 ). All constructs were verified by sequencing. The trans-complementation plasmids, pCMV R8.2 [40] encoding HIV-1 Gag, Pol, and accessory proteins, and pHCMV-G [41] encoding the Vesicular Stomatitis Virus envelope protein were co-transfected into 293T cells with the two genomic plasmids to produce defective retrovirus particles which were then used to transduce MT4 cells as previously described [29] . The reverse transcription products were purified from the cytoplasmic fraction of transduced cells using the method described by Hirt [42] . The purified double stranded DNA was digested with DpnI for 2 h at 37uC (in order to eliminate possible contaminating DNA of bacterial origin) prior to PCR amplification as previously described [29] . The amplified product was purified after electrophoresis on agarose gel, digested with PstI and BamHI, ligated to an appropriate plasmid vector and used to transform E. coli. Plating on IPTG/X-Gal containing agar plates allowed blue/ white screening of recombinant and parental colonies, respectively [29] . The frequency of recombination was determined by computing the number of blue colonies over the total number of colonies as described in reference [29] . Recombination breakpoints were identified by full-length sequencing of the env portion of the recombinant clones.
The recombinant and parental sequences of each pair of isolates tested were aligned using CLUSTAL X [43] . The breakpoint location of each recombinant was determined as being the central position of the interval bounded by the two closest nucleotide sites that were characteristic of each of the parental sequences). Recombination rates were calculated as follows. We define each recombination window studied with each pair of parental sequences as RwXY a-b , for a recombination window involving isolates X and Y, spanning position a to position b of env (reference sequence HXB2); a 50 nucleotides window was then considered (XY a-b Sw i , for a sliding window starting at position i of env), beginning from the 59 border of the sequence studied and the number of breakpoints (indicated as XY a-b n i ) falling within the window was counted. The resulting recombination rate per nucleotide in the sliding window XY a-b Sw i is
where XY a-b N is the total number of breakpoints characterized for the RWXY a-b pair, and 50 is the size in nucleotides of the sliding window, and F the frequency of recombination observed in the whole region studied, as defined in the previous chapter. The sliding window was then displaced with a 10 nucleotides increment (resulting in XY a-b Sw i+10 , XY a-b Sw i+20 , … ) across the recombination window, and XY a-b R i+10 , XY a-b R i+20 , … were computed. The various R values were reported in the graph as a function of the position of the midpoint of the window along the gene (i.e. the position of the 25 th nucleotide of each sliding window). For the pooled dataset reported in Figure 2B , the analysis based on the sliding window was repeated. If Swp i stands for the sliding window at position i for the pooled dataset, Rp i for the corresponding recombination rate, and q is the number of recombination window including position i, recombination rate at position i is calculated as
To statistically test for the presence of recombination hot and coldspots in the experimentally determined recombination breakpoint distributions we used a modification of a permutation test described previously [44] . Unlike in analyses of natural recombinants, the breakpoint positions approximated in our experimental procedure were not subject to biases introduced by underlying degrees of parental sequence nucleotide variability and patchiness of parental sequence sampling. Rather than explicitly accounting for these biases when placing randomised recombination breakpoints as in the permutation test described by Heath et al. [44] , our modification of the test involved the completely randomised placement of recombination breakpoints. The test essentially involved the randomised recreation of 10,000 versions of our real dataset with each version containing exactly the same number of breakpoints between the same 17 parental sequence pairs observed in the real dataset. From breakpoint distributions determined for each of these 10,000 randomised datasets we were able to work out confidence intervals for expected breakpoint density variation given the completely random occurrence of recombination. For simulating the distribution of 133 breakpoints based on the combined effects of (i) the mechanistic recombination rate and (ii) selection for functional recombinants, local recombination rate data used to generate the graph in Figure 2B were first multiplied by the respective functionality scores given in Figure 3 for each corresponding region, yielding ''functionality corrected'' rates for each region. Once the expected breakpoint distribution of 133 unique recombinants determined by this method, the number of breakpoints present in a 50 nucleotides rolling window, sliding with a 10 nucleotides increment was calculated and plotted (in Figure 5B ) as function of the position along the gene. Deviations from expected degrees of breakpoint clustering given the null hypothesis of random breakpoint locations, was tested using the same modification of the Heath et al., [44] permutation test detailed above.
Full-length sequences of recombinant env genes were reconstituted, using an overlapping PCR procedure. We separately amplified the region from the 59 end of the acceptor gene (using primer Topo59 annealing to positions 5966-5990 of the reference strain HXB2) to the breakpoint position (using a specific primer encompassing the region of the breakpoint) and from the 39 end of the donor gene (primer Donor39, HXB2 positions 8785-8819) to the breakpoint position (also in this case with a specific primer). These PCR products, overlapping by approximately 30 nucleotides around the breakpoint site, were mixed at equal ratios and used as templates to generate the full-length recombinant env gene using primer Topo59 and Donor39. All PCR reactions were run with Phusion DNA polymerase (Finnzymes, Finland) for 30 cycles. PCR products were gel purified and ligated to pCDNA3.1 Topo (Invitrogen, CA, USA). For RRE functionality assays, a portion of the envelope gene containing the RRE of pNL4.3-Env 2 -Luc (nucleotides 7646 to 8046) was replaced with the corresponding sequence of parental or recombinant envelope genes or, as a negative control, a 400 nt sequence from the Drosophila melanogaster desoxynucleoside kinase gene (DdNK). All constructs were verified by sequencing.
HIV particles were produced by co-transfection of HEK 293T cells with an expression vector for a CCR5-tropic (ADA) HIV-1 envelope [45] kind gift of Dr. M. Alizon, together with a pNL4.3-Env 2 -Luc containing either a parental or recombinant RRE sequence or DdNK. Forty-eight hours post transfection, supernatants were filtered trough a 0.45 mM filter and p24 levels were determined using the HIV-1 p24 enzyme-linked immunoabsorbent assay kit (PerkinElmer Life Sciences, MA, USA).
Reporter HIV-1 particles were produced by co-transfection of HEK 293T cells with pNL4.3-Env 2 -Luc and either an empty expression vector or an expression vector encoding either a parental or a recombinant env. For each individual recombinant variant, prior to their use for transfection, clones were verified by sequencing of the region encoding the recombinant gene as well as the vector-encoded promoter for its expression. Supernatants, containing virus stock, were harvested 48 h post transfection, and filtered trough a 0.45 mM filter. Production of viral particles was tested using an enzyme linked immunoassay for HIV-p24 antigen detection (Perkin Elmer, MA, USA) and 20 ng of p24 were used to infect 10 5 293T CD4 + -CCR5 + cells in 24 wells plates. Forty-eight hours later, cells were washed twice in PBS and lysed in 25 mM Tris phosphate, pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% triton X-100, and 7% glycerol for 10 min in a shaker at room temperature. The lysates were centrifuged and the supernatant was used to measure luciferase activity using a GloMax 96 Microplate Luminometer (Promega, WI, USA) following the instruction of the luciferase assay kit (Promega, WI, USA). For samples that yielded negative results in the luciferase assay, plasmids from at least three independent bacterial clones were tested.
The HIV-1 group M envelope sequence alignment was retrieved from the Los Alamos National Laboratory (LANL) HIV Sequence Database (http://hiv-web.lanl.gov/). The alignment was reduced to subtype reference sequences (3 strains for each where available), 53 CRF strains (2 strains for each where available) and finally 197 apparently unique recombinants. Recombination was analyzed using the RDP [46] , GENECONV [47] , BOOTSCAN [48] , MAXCHI [49] , CHIMAERA [50] , SISCAN [51] , and 3SEQ [52] methods implemented in the program RDP3BETA30 [53] . Default settings were used throughout except that: (1) only potential recombination events detected by four or more of the above methods, coupled with phylogenetic evidence of recombination were considered significant; (2) sequences were treated as linear; and (3) a window size of 30 variable nucleotide positions was used for the RDP method. Using the approach outlined in the RDP3 program manual (http:// darwin.uvigo.es/rdp/rdp.html), the approximate breakpoint positions and recombinant sequence(s) inferred for every potential recombination event, were manually checked and adjusted where necessary using the phylogenetic and recombination signal analysis features available in RDP3. Breakpoint positions were classified as unknown if they were (1) detected at the 59 and 39 ends of the alignment but could have actually fallen outside the analysed region; or (2) within 20 variable nucleotide positions or 100 total nucleotides of another detected breakpoint within the same sequence (in such cases it could not be discounted that the actual breakpoint might not have simply been lost due to a more recent recombination event). All of the remaining breakpoint positions were manually checked and adjusted when necessary using mainly the MAXCHI and 3SEQ methods (using three sequence scans and the MAXCHI matrix method) but also the LARD matrix method (generated by the LARD two breakpoint scan; [54] ), and the CHIMAERA method as tie breakers. The distribution of unambiguously detected breakpoint positions of all unique recombination events were analysed for evidence of recombination hot-and cold-spots with RDP3 as described by Heath et al. ([44] ; a window size of either 50 or 200 nucleotides and 10 000 permutations). A normalised version of the breakpoint distribution plot described in that study was used in which the local probability values of breakpoint numbers (determined by a permutation test that takes into account that local degrees of sequence diversity influence the delectability of recombination events) were plotted instead of absolute breakpoint numbers.
PDB files detailing the three dimensional structures of both gp120 (PDB ID: 2B4C, determined by X-ray diffraction, resolution of 3.3 Å , 338 amino acids, [55] ), and gp41 (PDB ID 1AIK, determined by X-ray diffraction, resolution of 2 Å , 70 amino acids, [56] ) were obtained from http://www.rcsb.org. It is important to point out that these structures are partial and that we therefore only analysed a fraction of the structural interactions involved in Env folding. We performed SCHEMA predictions of recombination induced fold disruptions using the set of natural HIV env recombinants (described above) essentially as described in Lefeuvre et al. ([8] ; See Protocol S1, Supplementary Analyses, for a description of the SCHEMA method). This involved: (1) computing protein fold disruption, or E, scores for each natural recombinant with identifiable parents; (2) based on every pair of parental sequences identified for the observed set of recombinants, simulating every possible recombinant that could have been produced with these parental sequence pairs that involved the exchange of the same number of non-synonymous polymorphisms as were exchanged during the actual recombination events; (3) calculating E scores for each of these simulated recombinants; and (4) using a permutation test to determine whether mean E scores calculated for the natural recombinants were significantly lower than mean E-scores for the same set of recombinants randomly drawn from the simulated recombinant datasets (Table S1 ). If fewer than 5% of simulated datasets had an average E score lower than that of the actual dataset (p,0.05) then this was taken to indicate that predicted fold disruptions incurred by real events were significantly less severe than if the observed distribution of breakpoints was uninfluenced by negative selection acting against recombinants with disrupted protein folding.
Protocol S1 Supplementary analyses. Schema analysis on the HIV envelope gene. Found at: doi:10.1371/journal.ppat.1000418.s001 (0.02 MB DOC)  Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission. Influenza viruses circulating in the human population are predominately type A and B, with type A being more common [1] . All influenza type A viruses originate from aquatic birds and successful introduction of these avian viruses into the human population, by either direct adaptation or reassortment with already circulating human viruses, has led to influenza pandemics of historical significance (reviewed in [2] [3] [4] 5] ). Still, documented evidence of transmission of avian influenza viruses directly from birds to humans is rare, partly because species barriers restrict avian influenza virus infection of the epithelial cells of the human respiratory tract, the primary site of influenza virus infection and spread.
Influenza A viruses possess a hemagglutinin (HA) attachment protein that binds sialic acid residues to facilitate infection of target epithelial cells. The HA of human influenza viruses preferentially binds to terminal sialic acid (SA) residues with a2,6 linkages, whereas avian influenza viruses preferentially bind to SA with a2,3 linkages [6] [7] [8] [9] . The prevalence of a2,6 SA but paucity of a2,3 SA in the human respiratory tract has been considered to restrict infection by avian influenza viruses [10] . Recent reports, however, have detected significant levels of a2,3 SA on human airway epithelium both in vitro and ex vivo, including in nasopharyngeal and tracheobronchial tissue [11] [12] [13] [14] . This SA distribution also correlated with avian influenza virus infection in vitro and ex vivo and raised the possibility that avian viruses could infect the upper airways in vivo. Therefore, although it is universally accepted that human-to-human transmission of avian influenza viruses requires adaptation of HA to switch from a2,3 to a2,6 SA usage, the cumulative data published to date indicate that SA linkages and their respective distribution in the human airways are not the sole barrier to avian influenza virus infection [15] [16] [17] . Other host factors and viral genes are likely also important determinants of infectivity.
One such host factor that may limit zoonotic transmission is the difference in host temperatures between avian and human tissues that are susceptible to influenza virus infection. Avian influenza viruses are adapted for replication in the avian enteric tract at 40-41uC . While the surface temperatures of the human respiratory tract are variable, a temperature gradient clearly exists in which the surface temperature of the proximal large airways (i.e., nasal and tracheal) average 32+/20.05uC while temperatures of the smaller, distal airways (i.e., bronchioles) are closer to that of the core body temperature, 37uC [18, 19] . While multiple transmission routes have been described for influenza viruses, the proximal airways likely represent a predominant site for human influenza virus inoculation as they provide a large exposed surface area of virus-susceptible epithelial cells [20] . These cells are directly accessible by large droplet aerosols and by way of digital inoculation of the nasopharynx and conjunctival mucosa [12, 21] . Inefficient infection by avian influenza viruses, even in the presence of a2,3-linked SA, may be due to the cooler temperature of the proximal airways compared to that of the distal airways/lung regions where H5N1 avian influenza viruses appear to replicate efficiently [22] .
Avian influenza viruses are attenuated at temperatures below 37uC and cold sensitivity of avian viral RNA replication in cell lines was linked to the presence of a glutamic acid at amino acid 627 in the avian virus polymerase subunit, PB2, instead of a lysine in the human virus PB2 [23] . Lysine substitution at residue 627 of H5N1 viruses improved virus replication in mice [24] . In addition to PB2, work utilizing human-avian reassortant viruses in MDCK cells provided initial evidence that avian glycoproteins, HA and neuraminidase (NA), may mediate temperature-dependent effects on viral growth [25] . To our knowledge, other viral genes have not been well characterized, nor the HA and NA further evaluated, in their contribution to temperature sensitivity of avian influenza viruses.
To characterize the temperature dependency of avian vs. human influenza viruses in a relevant model of the target cell types of the human airways, we utilized an in vitro model of human ciliated airway epithelium (HAE). This model closely mimics the morphological and physiological features of the human airway epithelium in vivo and has been previously used to investigate infection by diverse respiratory viruses [26] [27] [28] [29] [30] . In humans, ciliated airway epithelium is present throughout the airways, extending from the nasal cavity and large proximal airways into the distal bronchiolar airway regions. Previously, we have shown that both human and avian influenza viruses replicate well in HAE and that human and avian influenza virus cell tropism correlates with the respective distribution of the specific sialic acid linkages [13] . However, these previous studies were conducted at 37uC, reflecting conditions encountered in the distal airways [13] . Others have also utilized these airway cell systems to characterize influenza virus replication of wild-type and recombinant viruses at 35uC [14, 31, 32] . In the present study, we utilize the HAE model, in combination with influenza virus reverse genetics, to investigate the influence of temperature on human and avian influenza virus infection, replication and spread. We demonstrate that, compared to human influenza viruses, avian influenza viruses are severely restricted for infection of human airway epithelium at the temperature of the human proximal airways. Then, using different strategies to 'avianize' human influenza viruses, we show that the temperature restriction of avian viruses is closely associated with the avian HA and NA glycoproteins.
We and others have previously shown that human and avian influenza viruses infect and replicate in HAE [13, 14, 31] . Since our previous experiments were performed at 37uC, a temperature reflective of human distal airways, we have now compared human and avian influenza virus infection and growth in HAE at temperatures reflective of the proximal airways (32-33uC) and distal airways (37uC). HAE were inoculated at either 32uC or 37uC with a low multiplicity of infection (MOI; 0.01) of a representative human virus, A/Victoria/3/75 (H3N2), or an avian influenza isolate, A/Dk/Eng/62 (H4N6). Virus growth and spread throughout the epithelium at the two temperatures was measured and compared over time and infection further characterized with respect to virus-induced cytopathic effects (CPE).
At the temperature of the distal airways (37uC), the growth kinetics and mean peak titers of A/Victoria/3/75 and A/Dk/ Eng/62 reached 2.3610 8 pfu/ml and 4.7610 7 pfu/ml, respectively, by 48 hours post-inoculation (hrs pi) ( Figure 1A ). At the temperature of the proximal airways (32uC), A/Victoria/3/75 showed a modest delay in replication but still reached maximal titer of 7.8610 7 pfu/ml by 48 hrs pi. In contrast, A/Dk/Eng/62 grew very slowly, with yields at time points up to 48 hrs pi reduced by 3 to 5 logs compared to growth for this virus at 37uC or A/ Victoria/3/75 at either temperature.
In comparison to 48 hr titers, A/Victoria/3/75 titers at both temperatures and A/Dk/Eng/62 titers at 37uC were reduced at 72 hr pi and every time point thereafter, indicating reduced progeny virus production. A loss of titer was also observed for A/ Dk/Eng/62 at 32uC, but not before 120 hrs pi. To determine if loss of titer after reaching maximum levels correlated with increased CPE, we quantified adenylate kinase (AK) release by dead/dying cells into the apical compartment as a sensitive and global measure of cytotoxicity across the entire epithelial cell
Influenza type A viruses are endemic in aquatic birds but can cross the species barrier to infect the human respiratory tract. While transmission from birds to humans is rare, the introduction of novel avian influenza viruses into immunologically naïve human populations has significant pandemic potential. Avian influenza viruses are adapted for growth at 40uC, the temperature of the avian enteric tract. However, the human proximal airways, the likely site of initial inoculation by influenza viruses, are maintained at a cooler temperature (32uC), suggesting that zoonotic transmission may be limited by temperature differences between the two hosts. Using an in vitro model of human ciliated airway epithelium, we show that avian influenza viruses grow well at 37uC, a temperature reflective of distal airways, but are restricted for infection at 32uC. A panel of genetically manipulated human influenza viruses possessing avian or avian-like surface glycoproteins were also restricted at 32uC, but not 37uC, suggesting that avian virus glycoproteins are not adapted for efficient infection at the temperature of the proximal airways. Thus, avian influenza virus infection is restricted in the human proximal airways due to the cooler temperature of this region, thus limiting the likelihood of zoonotic and subsequent human-to-human transmission of these viruses. culture surface. Figure 1B indicates that substantial increases in AK levels, indicative of the onset of CPE, are first detected at 48 hrs pi for A/Victoria/3/75 at 32uC and 37uC and A/Dk/ Eng/62 at 37uC. This induction of AK coincided with peak viral titer for these viruses under these conditions (compare Figure 1A and 1B) and suggested that the loss of titer correlated with the onset of CPE. Increasing levels of AK between 48 and 96 hrs pi were directly associated with continually decreasing viral titers, further supporting this claim.
A relationship between the kinetics of virus growth in HAE and the level of CPE also suggested that CPE was a consequence of viral replication. This assertion is supported by the fact that trends in viral titers at a given time point are mirrored in AK levels detected 48 hrs later (e.g., compare viral titers at 48 hr pi ( Figure 1A ) to AK measurements taken at 96 hr pi ( Figure 1B) ). Since viral titer and AK levels could be related to the numbers of cells infected and/or the degree of virus replication within individual cells we compared titers of human and avian influenza viruses ( Figure 1A ) to the numbers of cells infected by each virus at the two temperatures over time. Immunodetection of viral antigen in inoculated HAE showed that human and avian influenza virus antigen was not detected 3 hrs pi, indicating that levels of antigen in residual viral inocula were below the limit of antibody detection (data not shown). For A/Victoria/3/75, a few isolated cells were positive for viral antigen by 6 hrs pi at 37uC, but by 24 hrs pi considerable numbers of antigen-positive cells were detected ( Figure 2A ). In agreement with our growth curves in Figure 1A , A/Victoria/3/75 infected slightly fewer cells at 32uC compared to 37uC at 24 hrs pi, but importantly, A/Victoria/3/75 spread efficiently within the epithelium at both temperatures and differences in infection at early time points became less significant over time ( Figure 2A) .
In contrast to A/Victoria/3/75, A/Dk/Eng/62 antigen was detected in only a few cells 24 hrs pi at either temperature. However, it should be noted that antigen-positive cells in en face images are viewed linearly (Figure 2A ) whereas viral titers are shown on a logarithmic scale ( Figure 1A) . Thus, an apparently small difference in titer as is seen at 24 hrs pi between A/Victoria/ 3/75 and A/Dk/Eng/62 at 37uC may correspond to a larger difference in the number of cells positive for viral antigen. While our staining also confirmed previous data that avian influenza viruses infect fewer human airway epithelial cells in comparison to human influenza virus at 37uC (Figure 2A ; [13] ), the limited extent of A/Dk/Eng/62 antigen positive cells at 37uC by 24 hr pi was still unexpected given that titers at this time were slightly greater than those for A/Victoria/3/75 at 32uC. Whether this represents a difference in yield of infectious virus per infected cell between human and avian viruses is presently not clear. Overall, A/Dk/ Eng/62 grew and spread well at 37uC, but was severely restricted for growth at 32uC and antigen positive cells were barely detectable before 48 hr pi for this virus at lower temperature.
HAE cultures infected with A/Victoria/3/75 at either 32uC or 37uC and A/Dk/Eng/62 at 37uC viewed en face exhibited loss of integrity of the epithelium although the extent of injury and time of onset varied ( Figure 2A ). Further evaluation of histological cross-sections indicated that A/Victoria/3/75 infection at 37uC, which had the highest and earliest induction of AK, resulted in the earliest evidence of morphological injury at 72 hrs pi. HAE infected with A/Victoria/3/75 at 32uC or 37uC or A/Dk/Eng/62 at 37uC all showed desquamation of the superficial layer of columnar epithelial cells with basal epithelial cells remaining attached to the matrix support by 120 hrs pi ( Figure 2B ). Similar cytopathology has been reported for A/Udorn/307/72 influenza virus infection of HAE in vitro and for clinical human influenza virus infection in vivo [29, 33] . The detection of AK in apical washes of A/Dk/Eng/62-infected HAE at 32uC suggested that this virus did eventually compromise cellular integrity at the lower temperature, but dramatic morphological effects were not seen at least for up to 120 hrs ( Figure 1B and 2B). It should be noted, however, that at 120 hrs pi, A/Dk/Eng/62-infected HAE at 32uC did display some morphological characteristics different from uninfected and infected HAE at earlier time-points. Preliminary assessment indicates that expansion of lateral spaces between the columnar epithelial cells had occurred. Although we do not know the significance of these morphological changes, we speculate these observations are the initiation of CPE that will ultimately result in similar cellular injury as seen for this virus at 37uC and human viruses at both temperatures.
In sum, for both viruses at both temperatures, detection of maximal numbers of antigen-positive cells correlated with high titers (compare Figure 1A and 2A) and increasing CPE ( Figure 1B ). By 72 and 120 hrs pi considerable loss of cells from the culture was evident and this correlated with the drop off in viral titers at these time points ( Figure 1A ). Thus, we conclude that in the context of maximal infection in which there were no additional target cells available for infection within the finite surface area of the HAE culture, ongoing replication in antigen-positive cells shown at 48 and 72 hrs pi resulted in increased cell death. This CPE led to a reduction in the number of viable, virus-producing cells and in turn, to a reduction in progeny virus. Although A/Dk/Eng/62 induced CPE when sufficient titers were generated at 37uC, one consequence of restricted replication of this avian influenza virus at 32uC was a reduction in overt CPE in HAE, even at later time points associated with considerable viral titers.
To determine whether other avian, but not human, influenza viruses display temperature dependent phenotypes, we performed multi-step growth curves with more human H3N2 isolates (A/ Eng/26/99 and A/Udorn/307/72) and A/Dk/Sing/97, an avian isolate of different subtype (H5N3). Growth of both humanderived influenza viruses tested, A/Eng/26/99 (H3N2) and A/ Udorn/307/72 (H3N2), was not significantly different between 32/33uC and 37uC ( Figure 3A and 3B). Indeed, these two additional human influenza virus strains showed even less difference in titer between temperatures than was determined for A/Victoria/3/75. Assessment of growth of avian influenza virus, A/Dk/Sing/97 (H5N3), over a 48 hr time course at 37uC showed similar growth kinetics to that of A/Eng/26/99 (H3N2), reaching titers of 7610 5 pfu/ml and 1.6610 6 pfu/ml, respectively ( Figure 3A and 3C). In contrast, at 32uC, A/Dk/Sing/97 (H5N3) failed to grow at all ( Figure 3C ). Clearly, the restriction of A/Dk/Sing/97 at 32uC compared to 37uC was an even more striking phenotype than A/ Duck/Eng/62. As the avian influenza virus strains used in this study were selected at random, with no selection for a temperature-dependent phenotype, we propose that low temperature restriction of avian influenza viruses, but not human influenza viruses, may be broadly characteristic of avian influenza viruses. The extent of restriction, however, may be variable between different virus strains.
Since the avian virus isolates used in these experiments are neither derived from samples obtained from humans nor passaged in human cells in vitro, we next investigated whether growth attenuation at low temperatures would be retained in a highly pathogenic H5N1 (A/VN/1203/04) influenza virus isolated from a fatal human case [34] . We compared infection kinetics of H5N1 (A/VN/1203/04) at 33uC and 37uC on HAE using A/Udorn/ 307/72 in parallel cultures as a human influenza virus control. As described above, A/Udorn/307/72 grew with similar kinetics at 33uC and 37uC ( Figure 3B ). A/VN/1203/04, however, exhibited slower replication kinetics at 33uC when compared to that for 37uC ( Figure 3D ). Indeed, titers were significantly decreased at 33uC vs. 37uC at 24, 48 and 72 hrs pi. In addition, only at 37uC did A/VN/1203/04 approach similar peak titers as the human A/ Udorn/307/72 virus by the end of the 72 hr time course ( Figure 3D ). Histological analyses of A/VN/1203/04-infected HAE at either temperature showed absence of obvious CPE in sharp contrast to A/Udorn/307/72 that obliterated the epithelium by 72 hrs pi ( Figure 3E ). The lack of obvious CPE after H5N1 infection contrasts reports that H5N1 induced extensive apoptosis in mammalian airway cells [35, 36] . The fact that we did not observe obvious CPE with this highly pathogenic virus warrants further investigation but is in line with the limited cell damage shown following infection with A/Dk/Eng/62 for 72 hrs ( Figure 2B ). In sum, using diverse examples of human and avian influenza viruses we have shown that avian influenza viruses, but not human influenza viruses, are restricted for infection and growth in HAE at the lower temperature of 32uC.
'Avianization' of human virus polymerase restricts growth in HAE at both 32uC and 37uC
Previously, the polymerase subunit PB2 has been shown to play an important role in host range restriction of avian influenza viruses in mammalian cells [37] [38] [39] . In influenza virus strains that circulate in humans, amino acid residue 627 in PB2 is a lysine, whereas in the majority of avian strains it is a conserved glutamic acid residue. The presence of glutamic acid at PB2 627 (avian-like) has been reported to account for the lower replication of avian influenza strains in mammalian cells and has been linked with reduced polymerase activity at lower temperature (33uC) in some cell systems [23, 24] . To assess the potential impact of this PB2 amino acid residue in restriction of avian influenza viruses at 32uC, we generated a recombinant A/Victoria/3/75 virus containing the PB2 K627E mutation and compared its growth with that of the isogenic wild-type virus in HAE at 32uC and 37uC. The K627E mutation resulted in restriction of the virus at both temperatures (Figure 4Ai ), and although titer at 32uC was 1.3 logs lower than at 37uC at 24 hrs pi, this difference was no greater than the differences in growth for wild-type virus at these temperatures (1.5 logs; Figure 4Ai ). Moreover, at the later time points analyzed, 48 and 72 hrs pi, the PB2 mutant did not show a significant difference in titer between the two temperatures. These data indicate that the K627E mutant virus was restricted for growth in HAE but that restriction was not temperature-dependent. Indeed, quantification of the numbers of infected cells identified by en face staining revealed that the K627E mutant virus infected a similar percentage of cells compared to wild-type virus at 24 hrs pi ( Figure 4Aii ) and that the mutant was capable of spread since new cells were infected by 48 hrs with similar kinetics to that of wildtype A/Victoria/3/75 at both 32uC and 37uC (Figure 4Aii ). Statistically, there was no difference between the wild-type and PB2 mutant viruses at either 32uC or 37uC at 48 hrs pi with respect to percent influenza virus-antigen positive epithelium. Together, these data suggest that the amino acid residue at PB2 627 influences viral fitness in HAE, but does not confer to a human virus the temperature-dependent phenotype of avian influenza virus infection in human ciliated airway epithelium.
Human influenza viruses with avian-like glycoproteins display restricted replication and spread at 32uC in HAE Our initial phenotype indicated that A/Dk/Eng/62 was restricted in its ability to spread from cell to cell within the epithelium at 32uC (Figure 2A ). Several events in the viral life cycle that are critical for spread, including release of progeny virions from previously infected cells and attachment and entry into new target cells, are mediated by influenza virus glycoproteins. Thus, we hypothesized that glycoprotein function could be responsible for the restricted infection of HAE by avian influenza viruses at the lower temperature of 32uC. To test whether HA and/or NA contributed to the restricted phenotype of avian influenza viruses at 32uC, we used reverse genetics to generate mutant viruses genetically altered to confer avian virus-like glycoprotein specificities on the A/Victoria/3/75 background. First, mutations in HA previously shown to switch sialic acid usage from a2,6 to a2,3 linkages (L226Q, S228G) [40] were introduced to generate the Vic-226-228HA virus. Second, we generated a reassortant virus in which the Victoria NA was replaced by that of the avian virus A/ Chick/Italy/1347/99 to generate Vic+Chick N1.
We again compared virus replication and spread of the recombinant viruses to that of wild-type A/Victoria/3/75 at the two temperatures. As stated above, replication measured for the wild-type virus was slightly compromised at lower temperature, noticeable at 24 hrs pi. Restriction at this time point was also observed during infection of HAE with Vic-226-228HA, as it had been for the PB2 mutant virus. Specifically, a 2.5 log decrease in virus growth was determined for Vic-226-228HA at 32uC compared to 37uC at the 24 hr time point (Figure 4Bi ). However, unlike the PB2 mutant virus, the difference between replication at 32uC and 37uC for Vic 226-228HA was also significant at the 48 hour time point. Moreover, this mutant virus with avian viruslike sialic acid usage spread less efficiently than wild-type at 32uC so that by 48 hrs pi the number of virus antigen-positive cells was significantly different (Figure 4Bii ). In contrast, at 37uC, Vic-226-228HA infected similar numbers of cells as the wild-type virus by 48 hrs; indeed, the mutant virus was able to spread significantly more efficiently at the higher temperature (Figure 4Bii) .
Similarly, the reassorted virus Vic+Chick N1 displayed a 2 log decrease in viral titer in HAE at 32uC compared to 37uC at 24 hrs pi. Although this difference was not appreciably greater than the difference in titer between temperatures for either wild-type virus or the PB2 mutant, Vic+Chick N1, unlike wild-type A/Victoria/3/75 and Vic 627PB2, maintained the ,2-log difference in growth at 48 hrs pi (Figure 4Ci ), suggesting this virus was more restricted at the cooler temperature. Quantification of numbers of infected cells illustrated that, like Vic-226-228HA, Vic+Chick N1 was restricted for spread at 32uC which was significant at 48 hrs, but was capable of spread similar to wild-type A/Victoria/3/75 at 37uC (Figure 4Cii ). Together these data suggest that avianizing either the HA or NA glycoprotein of an otherwise human influenza virus limits spread and subsequent infection at 32uC compared to 37uC.
We next generated a recombinant influenza virus containing both the 226-228HA and Chick N1 and tested infection and growth in HAE at 32uC and 37uC in comparison to wild-type A/Victoria/3/ 75. At 24 hrs pi, the double glycoprotein-altered virus exhibited similar restriction as observed for the other viruses. Nonetheless, an overall evaluation of the double glycoprotein-altered virus suggested that as infection proceeded, this virus was profoundly restricted at 32uC compared to 37uC (Figure 4Di ), exhibiting .2 log reduction in titer at 48 hrs. Notably, titers for the wild-type virus differed by less than 0.5 logs between temperatures at this time point. Furthermore, the double glycoprotein-altered virus was still significantly restricted at 72 hrs pi when titers at 32uC were compared to those at 37uC. The level of restriction observed for the double mutant was greater than that observed for either virus containing each of these mutations/ substitutions individually. Moreover, analysis of viral antigen positive cells at 72 hrs by en face staining of infected HAE indicated compromised spread of Victoria (226-228HA)+Chick N1 which was more severe at 32uC than 37uC (Figure 4Dii) .
Determination of CPE during these experiments revealed that the double glycoprotein-avianized virus only produced CPE at 72 hrs pi when experiments were performed at 37uC, whereas wild-type human virus produced CPE earlier and at both temperatures (data not shown). These data are consistent with the levels of CPE observed for A/Dk/Eng/62 (H4N6) and A/ Victoria/3/75 (H3N2) in our initial studies ( Figure 1B) and suggest that altering the human virus glycoproteins to avian virus-like characteristics has profound effects on infection, spread and CPE in the environment of the human ciliated airway epithelium.
One potential caveat of the recombinant viruses with avianized HA and/or NA utilized in our previous analysis was that they contained HA and NA pairs that had not co-evolved. To eliminate the possibility that the restriction we observed with these recombinant viruses was due to an imbalance between the activities of the surface glycoproteins that were not evolutionarily optimized, we next generated reassorted influenza viruses on a common genetic background, possessing human or avian glycoproteins with co-evolved pairings. This was achieved using human recombinant A/PR/8/34 (H1N1) in which the wild-type H1 and N1 glycoproteins were replaced by the H3 and N2 glycoprotein pair from A/Victoria/3/75 (generating PR8+Vic HA/NA) or the H7 and N1 glycoprotein pair from A/Chick/ Italy/1347/99 (generating PR8+Chick HA/NA, previously termed RD3) [41] . Since we and others have shown differential cell-type tropism between human and avian influenza virus in HAE [13, 14] , we next determined if avianizing the human virus HA by mutation or substitution (in the presence or absence of an avian NA) recapitulated the cell-type tropism exhibited by wholly avian influenza viruses in HAE. As shown by immunofluorescent detection in histological sections of infected HAE, PR8 containing A/Victoria/3/75 glycoproteins infected both ciliated and nonciliated cells in HAE with a tropism similar to wild-type A/ Victoria/3/75 ( Figure 5 ). In contrast, A/Victoria/3/75 with two avian-like amino acid substitutions in HA and PR8+Chick HA/ NA only infected ciliated cells, a tropism that was mirrored by wholly avian virus [13, 14] . These data clearly show that the ciliated cell tropism of avian influenza viruses is dictated by properties of the viral glycoproteins. These results correlate with the known increased sialic acid binding preference of avian HA for a2,3-linked SA, and to the presence of a2,3-linked SA on ciliated cells in HAE [8, 13, 14] .
Growth kinetics in HAE of PR8+Vic HA/NA and PR8+Chick HA/NA inoculated at equal MOI (0.01) revealed that PR8+Vic HA/NA infection and growth was efficient at both 32uC and 37uC ( Figure 6A ). PR8+Chick HA/NA grew at 37uC to identical titers as PR8+Vic HA/NA at 32uC recapitulating our data obtained for wholly human (A/Victoria/3/75) and wholly avian (A/Dk/Eng/ 62) viruses. In contrast, PR8+Chick HA/NA was severely delayed in growth at 32uC and generated titers that were .2 logs less than titers obtained for this virus at 37uC at both 24 and 48 hrs pi. Indeed, PR8+Chick HA/NA, like A/Dk/Eng/62 avian influenza virus ( Figure 1A) , was significantly restricted for growth at 32uC at 12, 24 and 48 hrs pi compared to growth at 37uC and growth of PR8+Vic HA/NA at either temperature.
As observed for wholly human and avian influenza viruses, peak titers were reached for PR8+Vic HA/NA at both temperatures and PR8+Chick HA/NA at 37uC by 48 hrs pi after which a decline in viral titer was apparent. Again, as noted in our observations with human and avian influenza viruses, the loss of viral titers with time correlated with the onset of CPE. While PR8+Chick HA/NA infection at 32uC did not result in substantial AK release until 96 hr pi, increased AK activity was detected in cultures inoculated with this virus at 37uC. AK activity measured in cultures at this temperature increased with similar kinetics and reached similar levels as AK measured in cultures inoculated with PR8+Vic HA/NA at either temperature. Furthermore, the kinetics of AK induction demonstrated that again, AK was virus and (Cii) N1 reassortant virus. Data obtained in parallel for wild-type A/Victoria/3/75 is repeated in each graph (striped bars) for comparison to the mutant (solid bars). Data shown represents the mean of the percentage of influenza virus antigen-positive epithelium across 10 different fields +/ 2SE. Differences in viral antigen positive epithelium between temperatures for each virus at 48 hrs pi is noted as significant (*p,0.05) or insignificant (NS). A one-way ANOVA model showed no significant differences between the wild-type virus and PB2 mutant at 32uC and 37uC at 48 consequential to viral replication and that, overall, CPE induced by reassortant viruses was reflective of CPE measured for human and avian influenza viruses.
En face staining of HAE at 24 hr intervals after inoculation showed PR8+Chick HA/NA spread to additional target cells at 37uC at a rate similar to that of PR8+Vic HA/NA at 32uC and correlated with the titers measured for these two viruses under those conditions ( Figure 6C and 6D ). At 32uC, however, PR8+Chick HA/NA spread was severely compromised and resembled the infection characteristics shown for A/Dk/Eng/62 (H4N6) in Figure 2A . Thus, by replacing human glycoproteins with those from an avian virus isolate, we have recapitulated the effect of temperature on infection and growth kinetics as well as the degree of cytotoxicity produced by wholly avian influenza virus interactions in human ciliated airway epithelium. The relative contributions of reduced cell-cell spread and reduced CPE by avian-like influenza viruses at temperatures of the proximal airways to in vivo infection and pathology will, however, require further investigation.
We have performed comparative studies of the infection kinetics of human and avian influenza viruses in a model of human ciliated airway epithelium at temperatures reflective of the human proximal and distal airways. Our data show that avian and avianized influenza viruses are restricted for infection and growth in HAE at 32uC but not 37uC, while human viruses infect and grow efficiently at both temperatures. Based on these data, we suggest that while the warmer temperatures of the distal airways enable comparable infection by both human and avian influenza viruses, the cooler temperatures of the human proximal airways only support efficient and robust infection of the ciliated airway epithelium by human influenza viruses. We speculate that the observed restriction for avian and 'avianized' viruses in HAE would render avian influenza viruses more susceptible to innate and adaptive immune responses that limit pathogenicity in vivo.
These results have significant impact on our understanding of why avian influenza viruses rarely undergo zoonotic transmission and why, when the rare human case does occur, that avian influenza virus infection and pathology manifest predominately in the warmer distal airways and lungs.
The inability of avian influenza viruses to replicate efficiently at cooler temperatures has been linked to the viral polymerase subunit, PB2 [23, 24] . In the present study, mutating position 627 in a human virus PB2 to an avian virus conserved residue resulted in growth restriction at both 32uC and 37uC, suggesting that this residue is important for general viral fitness in HAE, but is not responsible for the differences in infection seen at 32uC vs. 37uC. Two recent reports also found that viruses with 627E in PB2 were attenuated regardless of temperature in human bronchial epithelial cells and MDCK cells, respectively, although in other cell systems including human small airway epithelial cells, a temperature specific effect was found [24, 42] . It should be emphasized that those studies were performed in non-differentiated epithelial cells unlike our studies that use human differentiated airway epithelial cells. We and others have previously shown that differentiated airway epithelial cell models enable discrimination of attenuated phenotypes of respiratory virus infection whereas non-differentiated cells do not [26, 27, 43] . In addition, we also show using HAE, that the H5N1 strain A/VN/1203/04, which possesses a lysine at position 627 (human adaptation), is still restricted for growth at 32uC, albeit less so than avian influenza viruses that have never infected humans. The attenuation in HAE of this H5N1 isolate which possesses a ''human'' amino acid at residue 627 in PB2 suggests other residues in the polymerase subunit or other viral proteins altogether are involved in temperature sensitivity of avian influenza viruses.
In our initial experiments, spread of avian influenza viruses from cell to cell at 32uC was compromised in cultures inoculated at low MOI, suggesting a potential role for the envelope glycoproteins, HA and NA, in mediating temperature restriction. Previous work by Kaverin and colleagues also demonstrated temperature Figure 5 . Cell tropism of human, avian and avianized viruses in HAE. Representative cross-sections of inoculated HAE, fixed 24 hrs pi, were probed for viral antigen (NP; green) and a2acetylated tubulin, a marker for ciliated cells (red). Notably, the staining pattern for wild-type A/Victoria/3/ 75 was identical to that of PR8+Vic HA/NA. Arrows mark ciliated cells infected with either wild-type A/Victoria/3/75 or PR8+Vic HA/NA; arrow-head denotes non-ciliated cells infected by these viruses. These data indicate that viruses with Victoria glycoproteins were able to infect both cell types previously shown to express a2,6 SA [13] . Viral antigen was detected only in ciliated cells in cultures inoculated with Vic-226-228HA (in the Victoria background with either endogenous N2 or avian N1 or PR8+Chick HA/NA). Scale bar equals 20 mm. doi:10.1371/journal.ppat.1000424.g005 effects on growth of human-avian reassortant viruses containing avian glycoproteins [25] , although this work was performed in non-polarized MDCK cells and did not investigate additional correlates of infection such as spread and CPE. In our study, we generated recombinant influenza viruses based on the A/Victoria/ 3/75 or A/PR/8/34 genetic backbone that were engineered to contain avian-like and/or avian glycoproteins and characterized infection in HAE. Kinetic studies showed that although human influenza viruses that possessed avian or avian-like surface glycoproteins were modestly restricted compared to wild-type viruses at 37uC, these mutant viruses were able spread like wildtype viruses throughout HAE at this temperature. Wide-spread infection throughout HAE was even observed for viruses in which their endogenous HA was replaced or mutated to preferentially bind a2,3 SA, restricting tropism to ciliated cells. Efficient replication of Vic-226-228HA at 37uC in our studies corroborates previous work by Matrosovich and colleagues in which little effect of HA-specificity 'switching' on replication was noted unless a very low MOI (0.00004) was used for inoculation [44] . In contrast, Wan and Perez described more profound differences in replication in HAE at 37uC with recombinant viruses that differed only in their receptor specificity [31] . However, it should be noted that their recombinant viruses were based on an H9N2 avian strain that yielded relatively low titers, and their initial infections were performed at 35uC before incubating at 37uC [31] .
Compared to 37uC, viruses with a preference for binding to a2,3 SA, including Vic-226-228HA, were restricted for growth and spread in HAE at 32uC. Notably, the H5N1 strain examined in this study also maintains preference for a2,3 SA binding [45] ; thus, we may surmise that this characteristic of A/VN/1203/04 contributes to its attenuation observed in HAE. The contribution of a2,3 SA usage to replication of influenza viruses investigated by Hatta et al. in the upper respiratory tract of mice may have been masked in the mouse model (the 627 mutation in PB2 being more apparent) as mice express solely avian virus-like receptors (a2,3 SA) in their airways [46] . Restriction of a2,3 SA-binding viruses in HAE at 32uC was not due to a discrepancy in SA expression since HAE maintained at either 32uC or 37uC expressed similar levels of a2,6 and a2,3 SA (as detected by Sambucus nigra (SNA) and Maackia amurensis (MAA) lectin staining, respectively; data not shown).
In conjunction with the HA, the sialidase activity of NA is crucial for successful virus penetration of mucus layers for initial infection and subsequent release of progeny virions from infected cells [47, 48] . This is especially critical both in vivo and in HAE models in which the luminal epithelial cell surface is robust with glycoconjugates displaying abundant terminal sialic acid moieties that may act as false receptors for influenza viruses [49] . Using standard laboratory assays that employ small monovalent soluble substrates for cleavage by NA (MUNANA), we were not able to demonstrate any temperature-dependent loss of NA activity associated with either human or avian virus (data not shown). However, the ability of the avian virus NA to cleave biologically relevant substrates present in HAE may be compromised at 32uC vs. 37uC restricting both initial infection and subsequent spread of the virus throughout the epithelium. This is supported by our data which demonstrate restricted growth and spread of reassortant viruses containing avian virus NA, including Vic+Chick N1 and PR8+Chick HA/NA in HAE at 32uC.
In addition to their independent functions, the balance between the binding affinity of the viral HA and the sialidase activity of the NA is also critical for efficient infection. The ability of A/Victoria/ 3/75 viruses with mutations or substitutions in either the HA or NA alone to infect similar numbers of cells and replicate to comparable peak titers as for wild-type virus at 37uC implies that these viruses were not crippled by the mismatch between the specificities of their HA and NA. Replication and spread of influenza viruses that possess an avian HA paired with its ''matched'' NA was even more compromised than that of recombinant viruses with individual changes to levels seen with wholly avian viruses. Thus, viruses with co-evolved glycoprotein pairs exhibit restricted replication at low temperatures and both HA and NA genes contribute to the phenotype.
Together, these data imply that in the complex environment of the luminal surface of the human ciliated airway epithelium, the viral surface antigens have a marked effect on the extent of virus infection and that temperature plays an important role in limiting avian, but not human, influenza virus infection and spread in the cooler proximal airway regions. Given these results, we draw attention to other recently published data using the HAE model in which mutations in viruses that are growth attenuated in vivo display similar growth attenuation in HAE but not in nondifferentiated cell lines, suggesting that HAE possess discriminating properties of attenuating phenotypes of mutants of respiratory viruses [26, 27] . Admittedly, in the present study, despite restriction in both growth and spread, wild-type avian viruses and human viruses with avian or avian-like glycoproteins did eventually reach high titer at 32uC at later time points. The efficiency of infection and replication of a virus that inoculates the airway epithelium, however, is likely a critical factor in determining whether the virus is capable of establishing infection in a host that normally possesses innate and adaptive immune systems that attempt to limit virus infection and spread. At temperatures of the distal airways, avian influenza viruses displayed similar infection kinetics as human influenza viruses and would therefore, in the case of sufficient inoculum reaching these distal regions, be as likely to establish infection. Indeed, the clinical pathology findings for humans infected with H5N1 do report distal airway infection in ciliated bronchioles and lung regions [22] . Under these conditions of inoculation and infection, avian influenza viruses present in the distal airways may still be unable to spread to proximal airway regions without additional adaptation to cooler temperatures. One caveat of this prediction is that virus may be transported to proximal airway regions by innate mucus clearance mechanisms indicating that caution is required when attempting to identify proximal infection by viruses in airway secretions obtained from tracheal swabs.
In conclusion, the present study substantiates differential host temperature as a critical barrier for infection by avian influenza viruses. Since the ciliated airway epithelium of the proximal airways is a major portal for influenza virus infection and spread, accessible by multiple inoculation routes (e.g., ocular, nasopharyngeal or aerosol), the inability of avian influenza viruses to establish infection and spread in these regions would be predicted to reduce the frequency of successful zoonotic transmission. Furthermore, the ability of human influenza viruses to generate high viral titers in the human proximal airways is likely a factor in effective human-to-human transmission and the induction of airway epithelial cell cytotoxicity as shown in this study may increase particulate matter perhaps associated with virus that facilitates inoculation of new hosts. Rapid induction of cytotopathic effects by human, but not avian, influenza virus infection at the temperature of the human proximal airways may also contribute to the onset of other host defenses such as sneezing and coughing that facilitate clearance of particulate matter/virus from the airways and potentially promote transmission between human hosts.
Human airway tracheobronchial epithelial cells isolated from airway specimens from patients without underlying lung disease were provided by the National Disease Research Interchange (NDRI, Philadelphia, PA) or as excess tissue following lung transplantation under University of North Carolina at Chapel Hill (UNC) Institutional Review Board-approved protocols by the UNC Cystic Fibrosis Center Tissue Culture Core. Primary cells derived from single patient sources were expanded on plastic to generate passage 1 cells and plated at a density of 3610 5 cells per well on permeable Transwell-Col (12-mm diameter) supports (Corning, Inc.). HAE cultures were grown in custom media with provision of an air-liquid interface for 4 to 6 weeks to form differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium, as previously described [50] . Madin-Darby Canine Kidney (MDCK) cells were maintained in DMEM (Gibco-Invitrogen, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (Sigma-Aldrich, Inc.).
Influenza virus A/England/26/99 (H3N2) was isolated at the Health Protection Agency, Colindale, London, UK, during the routine surveillance program and has been minimally passaged in MDCK cells [51] . A/Dk/Singapore/97 (H5N3) and A/Dk/ England/62 (H4N6) are typical avian influenza strains that have been passaged in both embryonated chicken eggs and MDCK cells during laboratory handling. Highly pathogenic A/VN/1203/ 04 (H5N1) was biologically derived and minimally passaged in embryonated chicken eggs. A/Udorn/307/72 (H3N2) was passed in baby hamster kidney (BHK) cells and represents a clone expanded once in embryonated chicken eggs. Recombinant viruses, including wild-type A/Victoria/3/75 (H3N2) and mutants in either the A/Victoria/3/75 (H3N2) or A/PR/8/24 (H1N1) background, were generated from cloned cDNA in 293T and MDCK cell co-cultures as previously described [52, 53] . Mutant viruses were generated in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background as follows: 1) Vic 627PB2; A/Victoria/3/75 containing a lysine to glutamic acid amino acid substitution at position 627; 2) Vic-226-228HA; A/ Victoria/3/75 containing two amino acid substitutions in the HA gene (L226Q, S228G) that confer an avian-like receptor binding preference [6, 40] ; 3) Vic+Chick N1; A/Victoria/3/75 in which segment 6 containing the endogenous N2 NA gene was exchanged for the N1 NA gene from avian isolate A/Chick/Italy/1347/99; 4) Vic-226-228HA+Chick N1; A/Victoria/3/75 containing both L226Q and S228G mutations and the avian N1; 5) PR8+Vic HA/ NA; A/PR/8/34 in which the endogenous H1 and N1 were replaced with the H3 and N2 from A/Victoria/3/75 and 6) PR8+Chick HA/NA (RD3); A/PR/8/34 in which the endogenous H1 and N1 were replaced with the H7 and N1 from A/ Chick/Italy/1347/99. (RD3 was previously described as a candidate vaccine strain [41] .) The last two reassortant viruses were generated by substituting segment 4 and segment 6 from PR8 with those from either A/Victoria/3/75 (H3N2) or A/Chick/ Italy/1347/99 (H7N1). The multi-basic cleavage site in the avian H7 HA gene used in these studies was removed prior to rescue of these recombinant viruses for safety. Available accession numbers (GenBank: http://www.ncbi.nlm.nih.gov.libproxy.lib.unc.edu) are V01086 for A/Victoria/3/75 HA and CAD37074 for A/Chick/ Italy/1347/99 HA.
HAE were rinsed with PBS to transiently remove apical secretions and supplied with fresh basolateral medium prior to inoculation. Virus inoculum was diluted in PBS and applied to the apical surface of HAE for 2 hrs at either 32uC, 33uC, or 37uC, as indicated. Following incubation, viral inocula were removed and cultures incubated at 32uC, 33uC or 37uC for the duration of the experiment. Viral growth kinetics were determined by performing apical washes with 300 ml of serum-free DMEM for 30 min at either 32uC or 37uC. Washes were harvested and stored at 280uC prior to analysis. Viral titers in the apical washes were determined by standard plaque assay or tissue culture infectious dose (TCID) 50 assay on MDCK cell monolayers as previously described [13, 52, 54] .
At various points post-inoculation (pi), HAE were fixed in cold methanol-acetone (50/50) and stored at 4uC. Cultures were then permeabilized with 2.5% triton-X 100/PBS++ (containing 1 mM CaCl 2 and 1 mM MgCl 2 ) and blocked with 3% bovine serum albumin (BSA) in PBS++ before being probed with mouse antiinfluenza virus nucleoprotein (NP; Chemicon, Inc.; 1:100) and immunoreactivity detected with fluorescein isothiocyanate (FITC)conjugated anti-mouse IgG secondary antibody (Jackson Immu-noResearch Laboratories, Inc., 1:500). Fluorescent images were obtained using a Leica DMIRB inverted fluorescence microscope equipped with cooled-color charge-coupled-device digital camera (MicroPublisher; Q-Imaging, Burnaby, BC, Canada). The percentage of the epithelium positive for viral antigen as an index of percentage of infected cells was quantified over 5 images per culture by black and white pixilation of each image and computer calculation of percent black pixels after inverting the image. This technique determines percentage of black pixels in a defined area and does not account for differences in fluorescent intensity.
Viral-induced cytotoxicity was determined by measuring adenylate kinase activity in apical washes using a commercially available assay (Lonza, Inc.). Apical samples were centrifuged prior to freezing to remove any cellular contaminants present in the wash. Luminescence detected in samples from infected HAE were normalized to uninfected HAE and expressed as fold change over AK measured in uninfected (mock) HAE. Morphological assessment of cytotoxicity in HAE was performed with paraformaldehyde (PFA, 4%)-fixed histological sections (5 mm) stained with hematoxylin and eosin.
Detection of a2,3 and a2,6 linked sialic acids HAE maintained at either 32uC or 37uC for 72 hrs prior to sialic acid detection were washed, blocked with 3% BSA/PBS++ and probed with biotinylated SNA or MAA lectins to detect a2,6 and a2,3 SA, respectively (Vector Laboratories, Inc.; EY-Laboratories, Inc.; 1:100). HAE were then fixed in 4% PFA and incubated with streptavidin-alexafluor 488 (Molecular Probes, Inc.; 1:500) applied to the apical surface to detect lectin binding. Immunohistochemistry HAE fixed in methanol:acetone, were probed en face with antibody against viral NP (Chemicon, Inc.; 1:100) and FITCconjugated goat anti-mouse IgG1 and IgG2a (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:500), then embedded in paraffin. Histological sections (5 mm) were prepared and reprobed for viral antigen using standard immunofluorescence protocols. Briefly, sections were bathed in 2.5% triton-X 100/ PBS++ for 30 min, blocked in 3% BSA/PBS++ and incubated with antibodies in 1% BSA/PBS++. Primary antibodies were antiviral NP (Chemicon, Inc., as above) and anti-alpha acetylated tubulin (Zymed Laboratories, Inc.; 1:2000), a marker for ciliated cells. Secondary antibodies were FITC-goat anti-mouse IgG2a and Rhodamine red-conjugated goat-anti-mouse IgG2b (Jackson ImmunoResearch Laboratories, Inc.; 1:500). Sections were prepared with FluorSave mounting media (EMD Chemicals, Inc.) and images captured using a Leica DMIRB inverted fluorescence microscope equipped with a cooled color chargecoupled-device digital camera (MicroPublisher; Q-Imaging, Burnaby, British Columbia, Canada).
Linear mixed models were fitted to the repeated measurements of log-transformed viral titer over time that included effects for the four treatment groups (defined by virus and temperature), eight time points, and the interaction between treatment and time. We note that in a small number of cases, there were only two treatment groups (defined by temperature) and fewer than eight time points. A heterogeneous autoregressive correlation structure of order one was assumed for the repeated measurements. A joint test of the interaction terms (21 degrees of freedom) provides an assessment of the hypothesis of no differences among the four treatment groups with respect to viral titer growth (log scale). Provided this test was significant, indicating some differences among the four growth curves, pair-wise differences between the three treatment groups versus the a priori specified reference group (generally the avian strain at the lowest temperature) were carried out for each time point, and significant differences at the 0.05 level were noted. No adjustments for inflated Type I error due to multiple comparisons were made. Missing observations were assumed to be missing completely at random, based on the fact that the investigators determined a priori to remove samples at specific time points during the experiment. A multidimensional classification of public health activity in Australia BACKGROUND: At present, we have very limited ability to compare public health activity across jurisdictions and countries, or even to ascertain differences in what is considered to be a public health activity. Existing standardised health classifications do not capture important dimensions of public health, which include its functions, the methods and interventions used to achieve these, the health issues and determinants of health that public health activities address, the resources and infrastructure they use, and the settings in which they occur. A classification that describes these dimensions will promote consistency in collecting and reporting information about public health programs, expenditure, workforce and performance. This paper describes the development of an initial version of such a classification. METHODS: We used open-source Protégé software and published procedures to construct an ontology of public health, which forms the basis of the classification. We reviewed existing definitions of public health, descriptions of public health functions and classifications to develop the scope, domain, and multidimensional class structure of the ontology. These were then refined through a series of consultations with public health experts from across Australia, culminating in an initial classification framework. RESULTS: The public health classification consists of six top-level classes: public health 'Functions'; 'Health Issues'; 'Determinants of Health'; 'Settings'; 'Methods' of intervention; and 'Resources and Infrastructure'. Existing classifications (such as the international classifications of diseases, disability and functioning and external causes of injuries) can be used to further classify large parts of the classes 'Health Issues', 'Settings' and 'Resources and Infrastructure', while new subclass structures are proposed for the classes of public health 'Functions', 'Determinants of Health' and 'Interventions'. CONCLUSION: The public health classification captures the important dimensions of public health activity. It will facilitate the organisation of information so that it can be used to address questions relating to any of these dimensions, either singly or in combination. The authors encourage readers to use the classification, and to suggest improvements. Classification of public health: working definitions of functions subclasses 26 
The objective of the Public Health Classifications Project is to 'develop and endorse a higher-level classification that captures the breadth and scope of public health activity and provides a unified framework for multiple uses'. Such a unified framework will assist in improving the quality and consistency of reported information on public health activity, performance, investment and expenditure. The National Public Health Partnership funded the project in response to recommendations from the 2002 Public Health Performance Project. 1 During the early scoping stages of the Public Health Classifications Project, it became apparent that a simple, one-dimensional classification system for public health could not satisfy the needs, or reflect the diverse world-views, of its disparate potential users. To provide a single 'unified framework' for multiple public health uses, a multi-dimensional classification was needed.
In the domain of public health, a flexible and inclusive approach offers particular advantages, because there are divergent (and strongly held) views regarding what is and is not 'in scope'. By making such issues explicit, the process of developing a public health classification potentially offers a way to move towards a common language to describe public health activity in Australia, and to develop a practical tool that will improve data collection processes and the utility of public health information.
This report is the output of phase one of the Public Health Classifications Project. It introduces the concept of a multi-dimensional public health classification and describes the challenges encountered in developing it. The report presents version one of a classification of public health, outlines some potential practical applications, and proposes the next steps for phase two of the project.
A Reference Group (see acknowledgements in Appendix B) oversaw phase one of the Public Health Classifications Project and provided ongoing expert advice and comment.
The project used a formal methodology and supporting software. 2 A review of current public health definitions, concepts and relevant classifications was used to develop the scope, domain, and initial multi-dimensional structure. These were considered in a series of consultations with public health experts across Australia (see list of those consulted in Appendix B). Consultations involved both one-on-one meetings and larger group sessions. Experts identified important omissions, fine-tuned concepts, and nominated practical uses for the public health classification.
The definition of public health adopted was as follows:
Public health is the organised response by society to protect and promote health, and to prevent illness, injury and disability. The starting point for identifying public health issues, problems and priorities, and for designing and implementing interventions, is the population as a whole, or population subgroups. 3 The boundary between public health and clinical practice came up repeatedly as an issue in discussions about the scope of public health, with particular debate about whether preventive services delivered on a one-to-one basis to individuals should be considered in scope. Many agreed that immunisation was in scope because it is an activity that is 'organised' at a population level with benefits for both populations and individuals. More contentious, however, was the possible inclusion of interventions that are designed to prevent and manage chronic diseases, and that are delivered to individuals in primary care settings.
Whether or not public health is a domain solely within health or whether it includes activities in other sectors (e.g. education, transport, local government) was also debated, particularly where the public health impact of the activities in these other sectors is incidental, rather than the primary purpose of the activity.
The general approach adopted in producing the classification was to be inclusive, and to allow decisions about specific exclusions to be made at the later stages when developing individual applications and uses of the classification.
The broad structure of version one of a classification of public health consists of six top-level classes as shown in Figure 1 .
There was consensus among the public health experts consulted, that a public health classification should be multi-dimensional, and there was broad agreement on the top-level classes that should be included.
Public health functions are defined as the purpose of public health interventions, actions, activities and programs. The 'functions' class was developed from the National Public Health Partnership public health core functions 4 and includes both primary and instrumental functions (shown in Table 1 There was reasonable agreement regarding the top levels of the 'health issues' class (although its name was the subject of some debate), and the 'determinants of health' and 'settings' classes. The remaining classes are less well developed and have had limited testing through consultations.
As shown in Figure 2 , existing classifications (such as the international classifications of diseases, functioning and disability, and external causes of injuries; and various Australian standards) are available to classify the classes of 'health issues', 'settings' and 'resources'. The National Public Health Information Working Group has determined that the further development of classifications for the top-level classes of 'functions', 'determinants of health' and 'methods' are required and a priority.
Other to be classified: 
A public health classification should facilitate the organisation of information to answer key public health questions that cannot currently be answered, such as 'How much was spent last year on the prevention of obesity?' It should assist in describing what public health is, and what its characteristics are, through the development of classes that capture the functions of public health, issues of public health concern (including determinants of health), the settings in which public health operates, the population groups targeted, resources available and so on.
Potential practical applications for a public health classification include:
Explaining what public health is;
Organising information to answer key public health questions;
Promoting consistency in describing public health;
Improving data capture processes and the quality of reporting;
Contributing to higher-level classification and standards activities;
Lending structure to the design of public health information and communication systems;
Auditing the spread of activity across the public health business cycle;
Building models of good public health practice; and
Linking research, policy and practice.
Potential users of a public health classification include the various levels of government and other sectors that have an investment in public health, academics and students, researchers, evaluators, those involved in policy formulation, and anyone with an interest in public health.
The Australian Institute of Health and Welfare has indicated an interest in the longerterm development and maintenance of a public health classification.
It is recommended that phase two of the Public Health Classifications Project should:
Focus on further developing the classes of public health 'functions', 'determinants of health' and 'methods';
Develop and release a web-based version of the public health classification with facilities for eliciting structured feedback and managing contributions to the further development and refinement of the classification;
Develop a plan for ongoing development, support and governance of the public health classification;
Further specify links or relations between the public health classification and relevant existing classifications and standards (with due regard for intellectual property rights); and
Investigate inclusion of the public health classification in the Australian Family of Classifications.
A classification is an 'arrangement of concepts into classes and their subdivisions to express the semantic relations between them'. 5 The essential characteristic of a classification is aggregation according to logical rules. Standardised, shared classifications are needed if we want to compare information about entities and discern their similarities and differences.
The objective of the Public Health Classifications Project is to 'develop and endorse a higher-level classification that captures the breadth and scope of public health activity and provides a unified framework for multiple uses'. Such a unified framework will assist in improving the quality and consistency of reported information on public health activity, performance, expenditure and investment. This report is the output of phase one of the Public Health Classifications Project. It introduces the concept of a multi-dimensional public health classification and describes the challenges encountered in its development. It presents version one of a public health classification, outlines some potential practical applications, and proposes the next steps for phase two of the project.
The National Public Health Partnership defines public health as:
the organised response by society to protect and promote health, and to prevent illness, injury and disability. The starting point for identifying public health issues, problems and priorities, and for designing and implementing interventions, is the population as a whole, or population sub-groups. 7 As a sector, public health is largely funded by government. 8 In Australia the Australian Government is the major source of public health funding, while state and territory governments mostly apply the funds. 9
The public health workforce is as diverse as are its employers: there is no single or all encompassing occupation or industry group. 'general health and associated workers' who carry out aspects of public health functions on either a regular or occasional basis. 10 Some public health activities are carried out in sectors outside of health (e.g. local government, non-government organisations (NGOs), other government departments and agencies, including planning and environmental protection agencies). Some 'classic' public health functions are outsourced and funded well away from health and human services portfolios (e.g. sewage disposal, provision of safe potable water).
Public health activity is costed at the program level, 11 and effectiveness and other measures are estimated at the aggregate level as theoretical constructs (e.g. population health status, potentially avoidable mortality). It is difficult to tell when public health effort and investment is effective, even over long periods of time; the small amount of work to this end is bedevilled by the poor quality of available data, the complexity of costing public health activity, 12 and lack of agreement about what should be included. 13 Costs to society when public health fails (e.g. cryptosporidium outbreak response, effect of SARS panic) may be easier to estimate.
Available expenditure estimates suggest that there are relatively high overheads or indirect costs for public health programs and activities (e.g. design and coordination costs, costs of administering and managing complex operations). 14 Public health tends to exhibit large economies of scale and to be relatively insensitive to population size; hence unit costs may be lower in states with larger populations to absorb the fixed costs of overheads. 15
The National Public Health Partnership funded the Public Health Classifications Project in response to the 2002 Public Health Performance Project, 16 which recommended that the National Public Health Information Working Group undertake the development of a classification system for public health that could be used to further develop the categories used by the National Public Health Expenditure Project and 10 Employers include Australian, state and territory, and local governments; NGOs, Aboriginal Community Controlled Health Organisations, community services, environmental protection services, health promotion foundations, private sector organisations (e.g. pharmaceutical companies, pathology laboratories) (Riddout et al. 2002: 8) . 11 Even when program categories are artificially created, for example, state reporting against 'activity categories' in public health expenditure reporting (see AIHW 2004b). 12 Bennett 2003. 13 Abelson analysed the epidemiological and economic effects of five public health programs over decades (including programs to reduce: tobacco consumption, coronary heart disease -which some would dispute as a public health program -and road trauma), estimating costs of investment in public health interventions and benefits in terms of total return to society, and, savings to government. The 'net present value' to government of road safety programs and programs to reduce coronary heart disease was estimated as negative (expenditure greater than savings); while the benefit of immunisation for Haemophilus influenzae B disease was estimated at a 'marginal $10 million' (Abelson et al. 2003: 4) .
14 AIHW 2004b, nine public health programs in all jurisdictions. 15 Riddout et al. 2001. 16 Owen & Jorm 2002. performance monitoring by the National Public Health Partnership, and to inform a future review of the core functions for public health.
The Public Health Performance Project used the public health core functions that were endorsed by the National Public Health Partnership in 2000, to develop performance indicators for public health. 17 These core functions differ from the categories used for national public health expenditure reporting 18 , resulting in difficulties in aligning data on performance with that on expenditure. More recently, a national report of health expenditure by disease groupings excluded expenditure on public health because this was not available 'by disease' 19 -further highlighting the inadequacy of current systems for capturing information about public health activities.
The objective adopted by the National Public Health Partnership for the Public Health Classifications Project was to 'develop and endorse a higher-level classification that captures the breadth and scope of public health activity and provides a unified framework for multiple uses'.
The project objective is to develop and endorse a higher-level classification that captures the breadth and scope of public health activity and provides a unified framework for multiple uses.
During the early scoping stages of the project, it became apparent that a one-dimensional classification of public health might look very different, depending on its intended use, and user group. A simple, one-dimensional classification of public health could not satisfy all the needs, or gel with the diverse world-views, of its disparate potential users. To provide a 'unified framework for multiple uses', a multidimensional public health classification with explicit modeling of the relationships among dimensions is needed, rather than a single, mutually exclusive, hierarchy of categories. 20
This project used an ontology-building process to develop the public health classification. An ontology is an explicit formal specification of the concepts in a domain (in this case, public health), their attributes and the relations among them, which allows people to share a common understanding of the structure of information. 21
A multi-dimensional public health classification allows structure to be imposed on diverse material along different-but equally meaningful-dimensions, based on the way that public health experts and practitioners think about public health, and the ways in which they describe or classify it, or aspects of it, depending on their purpose.
In the domain of public health, this flexible and inclusive approach offers particular advantages, because there are divergent (and strongly held) views regarding what is 17 Owen & Jorm 2002 : 8. 18 NPHP 1998 , NHPC 2004 , AIHW 2004b . 19 AIHW 2004c In practice, most classifications of complex domains are multi-dimensional, either implicitly so, or explicitly constructed as such. An example in the health field is the International Classification of Diseases (WHO 1992-94) , although the relationships among the dimensions are not all set out overtly.
and what is not 'in scope'. By making such issues explicit, the process of developing a classification offers a way to move towards a common language to describe public health activity in Australia, and to develop a tool to improve data collection processes and the consistency of information about public health activity, performance, expenditure, effectiveness and returns on investment.
A Reference Group (see acknowledgements in Appendix B) oversaw phase one of the Public Health Classifications Project and provided ongoing expert advice and comment.
The project used the Ontology development 101 methodology 22 and the open source Protégé ontology-building software (from Stanford University 23 ) as the development tools. Ontology development 101 and Protégé were selected after a scan of available methods and software, because they were considered to be the most useful tools for the work, are openly available (i.e. do not require a commercial license), provide support for emerging Semantic Web standards, and have active communities of interest with strong representation from researchers and knowledge workers in health, biomedical and other related fields.
The steps followed in the public health classification building process were:
Step 1 Determine the domain and scope of the classification:
-What is the domain that the classification will cover? -For what are we going to use the classification? -For what types of questions should the information in the classification provide answers? -Who will use and maintain the classification?
Step 2 Consider reusing existing classifications.
Step 3 Enumerate important terms in the classification.
Step 4 Define the classes and class hierarchy. 24
Public health definitions and relevant classification systems, especially functional classifications, were reviewed for Step 2 and are available from the project.
The Project Reference Group workshopped the preliminary material and drafted initial responses to Steps 1 to 4. Consultations with public health content experts in a sample of jurisdictions considered the class hierarchy and its top levels, and identified important omissions. They also identified further practical uses of the classification.
Initial consultations were held in NSW from October 2004. Formal consultations were held in Brisbane, Melbourne, Canberra and Perth. Early consultations were informal, designed to seek the views of content experts on particular components (e.g. environmental health, health promotion). Later consultations were organised through Reference Group members representing various jurisdictions. Prepared material introducing the project was sent out to participants prior to each consultation. All consultations were face to face. The number of participants varied from one or two, to larger groups of up to fifteen, and the duration varied from one to three hours.
In each formal consultation, an introduction and background to the project were given with the aid of a slide presentation, then an early version of a public health classification, rendered through a Web browser, was demonstrated, concluding with 22 Noy & McGuinness 2001. 23 For more information see http://protege.stanford.edu. 24 Noy & McGuinness 2001: 5-8. the definition of public health. This was followed by a live collaborative session using the Protégé software, during which changes and additions to the class structure were made in real time. Lastly, participants were asked to identify further practical uses for a unified public health classification. An example of the agenda and other pre-consultation material that was sent to participants is in Appendix B.
The views, suggestions, and additional information captured in consultations were discussed by the Project Reference Group over a series of meetings and have informed the broad structure of the public health classification that is reported in Section 3 Results. The public health content experts who contributed are acknowledged in Appendix B.
An earlier version of this report was presented to, and discussed by, the National Public Health Information Working Group in March 2005, and this version reflects the feedback and directions given by that Group.
This section presents the results of the process of scoping the domain to be covered by a public health classification. A number of boundary issues are discussed, areas of likely agreement identified, and potential practical applications for the classification are outlined. Version one of the public health classification is presented. Issues for further consideration are highlighted in boxes.
The existing National Public Health Partnership definition of public health was adopted, as follows:
Public health is the organised response by society to protect and promote health, and to prevent illness, injury and disability. The starting point for identifying public health issues, problems and priorities, and for designing and implementing interventions, is the population as a whole, or population sub-groups. 25 Suggestions made during consultations that the Partnership definition should include references to 'evaluating' and 'measuring or achieving outcomes' were not adopted, as these were considered to be implicitly present in the definition. 26
Significant boundary issues were encountered in scoping the domain of public health, with disagreement among public health experts regarding where the boundaries are, or should be.
While most public health experts agreed, when pressed, that accounting for public health should include the activities of, and investments by, the non-health portfolios of governments (such as education and transport), local governments and nongovernment organisations (NGOs), current public health expenditure reporting is largely limited to that by State, Territory and Australian Government health portfolios. 27 One view was that the activities of other (non-health) sectors should only be counted when public health is their primary purpose (e.g. immunisation organised by local government). In practice there are major difficulties in capturing information on public health activities and expenditure by non-health sectors. 28
The boundary between public health and clinical practice came up repeatedly in discussions about the scope of public health. In many situations preventive activities in clinical practice complement broader population-based activities. At what point do 25 NPHP 1998. 26 See Appendix C for additional information on this aspect of the Project. 27 With the exception of SA which has in the past included non-health expenditures by local government, etc, in public health expenditure reporting by AIHW. 28 A more fundamental difficulty is the time and expense to collect comparable information across all sectors. they become part of 'the organised response by society to protect and promote health'? Organised interventions for promoting health and preventing illness, injury and disability include those aimed at whole populations that do not necessarily require any particular action on the part of individuals (health protection activities, e.g. the provision of clean water, clean air, sewage disposal), and those organised and delivered at the level of the population or sub-group, but requiring individuals to modify their behaviour (health promotion activities, e.g. the range of activities to reduce smoking in the community-regulations, media campaigns, organised quit lines etc). There was general agreement that health protection and health promotion are public health activities.
There was more debate in relation to preventive services delivered on a one-to-one basis to individuals. Such preventive services include screening, immunisation, and counselling and lifestyle advice to support healthy behaviour, as well as detection and management (through lifestyle changes or pharmacological means) of biological risk factors such as high blood pressure and high cholesterol. The perceived boundary between public health and clinical medicine is likely to change as new screening technologies and preventive medications become available.
Some public health practitioners argued that those individual preventive services related to communicable diseases (e.g. immunisation, contact tracing, treatment for STIs) form part of public health practice because they help to protect the health of the whole population, through herd immunity and reducing the spread of infection. A minority argued that immunisation is only a legitimate part of public health activity when it is delivered as part of a publicly organised program, such as through local government or school health services. The corollary of this point of view is that immunisations performed in general practice are not a public health activity. Alternatively, it was argued that childhood immunisation in general practice is simply the implementation strategy for an organised national approach to immunisation, one which is supported by special payments to GP's and the Australian Childhood Immunisation Register, with follow-up of those parents who do not comply.
With respect to non-communicable diseases, early detection through screening is a preventive service delivered one-to-one to individuals.
Some public health practitioners felt that this is only a public health activity when it is delivered through an organised program such as BreastScreen. Cervical screening is largely delivered through general practice, although, as with immunisation, the delivery of services in the private sector is underpinned by the National Cervical Screening Program (State and Territory recruitment programs, Pap smear registers, follow-up and reminder systems). The question is whether the taking of smears (in general practice) and the reading of smears (in laboratories), which are largely in the private sector, but essential to the implementation of the program, should be considered as public health activities.
On the other hand, opportunistic screening that is not part of an organised program, such as bone density screening for osteoporosis, was not generally considered to be a public health activity.
Even more contentious was the issue of the prevention and management of non-communicable diseases, through one-on-one counselling about lifestyle risk factors (e.g. smoking, poor nutrition, risky alcohol use and lack of physical activity), and the early detection and management of biological risk factors such as high blood pressure and high cholesterol in the prevention of heart disease and stroke. Many public health practitioners regarded these activities as clinical practice. Some suggested that a distinction can be made on the basis of whether people have symptoms or signs of disease. For example, helping people to quit smoking would be considered a public health activity when they are symptom free, but part of clinical medicine if they have any symptoms or signs of disease or a history of previously diagnosed disease. Apart from any conceptual objections to such a distinction, it would be difficult to operationalise in practice.
Further along in the disease continuum, most public health practitioners classified the effective management of chronic disease, with the goal of minimising disability and reducing complications and hospitalisations, as belonging firmly in the zone of clinical medicine. For example, the prescribing of cholesterol-lowering medication by a general practitioner, even in an otherwise healthy person, would not be considered a public health activity (although a media campaign urging people above a certain age to have their cholesterol levels checked by their GP might be regarded as public health).
These boundary issues are set out for further consideration in Box 2 in Section 3.1.6.
The Public Health Performance Project 29 envisaged that a unified classification for public health would be used to progress national public health expenditure reporting, 30 public health performance indicators, 31 and to build on the public health core functions developed by the National Public Health Partnership. 32 Potential uses and practical applications for a public health classification, identified during phase one of the current project, are summarised in Box 1.
Explain what public health is
Organise information to answer key public health questions Promote consistency in describing public health Improve data capture processes and the quality of reporting A public health classification will help to explain what public health is in a way that is recognisable and understood by the average person. It will allow description of the functions of public health, issues of public health concern, the settings in which public health activities occur, the population groups targeted by public health interventions, the resources available to public health, and so on. The process of developing a classification has the potential to unite the sector and improve understanding of the breadth of the public health effort.
A public health classification can be used to organise information to answer key questions for public health that cannot be answered currently. While agreement on the scope of public health proved contentious during consultations, formulating questions that a competent public health classification should help to answer was somewhat easier. Questions like those shown in Box 2 set a practical test for the classification.
Box 2 A public health classification should help answer questions like...
What is public health? What are the characteristics of public health?
How is public health relevant to components of the human services delivery system?
Why do public health unit costs differ across jurisdictions?
Can we describe screening in clinical settings (e.g. Pap smears taken in GP surgeries)?
What are the nature and cost of public health partnerships between health and other sectors?
Can we replicate the output of other models (e.g. current public health expenditure reporting)?
How much was spent on social marketing last year? 33
As well as organising and integrating public health information, the development of a common classification will promote consistency in describing public health, through the standardisation of definitions and terminology. This will improve data capture processes and the quality of reporting (e.g. in expenditure and performance reporting). Promoting consistency will increase the ability to compare public health information over time and across jurisdictions. 34 There is potential for a public health 33 Additional questions include other advocacy-type questions, such as: what is the relative expenditure on specific risk factors or diseases? What is the difference in expenditure on prevention of HIV/AIDS relative to other preventable diseases? Has health funding to preventive/promotive investments increased?. There are also boundary questions such as: can we describe the hospital interface with public health interventions (e.g. screening in hospitals)? Can we calculate expenditures in specific areas (e.g. product safety and protection, public health emergencies, education as a health promotive activity)? Competency questions can be used as a 'litmus test' to help determine whether the classification contains sufficient information to answer them, and whether the answers require a particular level of detail or representation of a particular area (Noy & McGuinness 2001: 5) . 34 Recent reporting of public health expenditures over several years has enabled such analyses for the first time (AIHW 2004b). classification to be used to improve jurisdictional public health financial processes (e.g. budgeting, resource allocation) and accounting systems (e.g. through developing systems that can apportion public health activities to cost centres or to aggregate Treasury outputs). 35
A public health classification will contribute to higher-level classification and standards activities through the potential membership of the Australian Family of Classifications. The development of the classification could 'fill out' the public health cells and embed public health more firmly into the 'health and related classifications matrix'. 36
A public health classification can be used to structure and design information and communications (e.g. in designing websites, structuring resources, and planning report chapters). It has practical applications in building information systems, such as a database of public health projects, using the classification to create explicit, structured information to make meaning (as well as documents) accessible and shareable. One test application proposed was for a public health equivalent of the Semantic Web Environmental Directory (SWED). 37 This could be created through web-based, universally available tools, that make it easy for public health people to describe what they do, using the classes and terms from the public health classification. Other uses are based on a broad vision of a public health classification as signposting or semantically indexing a wide range of resources (including but not limited to: thesauri, dictionaries, terminologies and definitions, scientific papers, reports and other documents, legislation, policies, information databases and indexes, case studies, stories and vignettes).
A further use for a classification identified in consultations is to audit the spread of public health activity, expenditure or investment, across the business cycle -from health problem identification and assessment to program or intervention planning and design, through to implementation and evaluation of results. This suggestion arose out of concerns that public health activity is too heavily weighted towards implementation, and that there is insufficient evaluation of interventions, and learning from and progressing beyond pilots. A related use is to examine the spread of all public health investments, for example, by Australian, state, territory, and local governments, NGOs and other investors; the links to employment and education; and public health investment by, and outcomes in, other sectors such as transport and housing.
A classification can potentially be used to help build models of good public health practice that describe the program logic for public health activities, including specification of the links between activities, expenditure and outcomes. Another suggested use for a classification is in developing a continuous improvement model to ensure that public health learns from what it does. 38
Lastly, the classification was considered to have the potential to link public health research, policy and practice, by facilitating use of a common language, and the linkage of information across these domains.
Potential users of a public health classification, who were identified along with the practical applications discussed above, are the various levels of government and other sectors that have an investment in public health. Other users include academics and students, researchers, evaluators, those involved in policy formulation, and anyone with an interest in public health.
The Australian Institute of Health and Welfare has indicated an interest in the longerterm development and maintenance of a public health classification.
During the development of the public health classification, the following principles of development were determined and agreed:
The classification system should be multi-dimensional to be able to represent the multi-dimensional nature of public health.
Different dimensions are of equal importance to public health and a range of the most important need to be considered and developed concurrently.
Existing classification systems of relevance (including Australian and international standards) should be used wherever possible in the multi-dimensional structure of the classification system.
The system should be inclusive (rather than exclusive) and deliberately broad at the top levels. Boundaries can be set (or moved) as needed for particular practical applications; they should not be used to restrict or hinder the development of a broad and inclusively scoped classification.
In addition to the definitional issues raised and discussed in Section 3.1.2 above, public health experts consulted in phase one of the project raised the important issue of whether the name of the project domain should be 'population health' or 'public health'. 39 How is the domain that public health currently works in, best described? Is 'public health' subsumed in 'population health'? Or is a 'population health approach' merely one aspect of public health practice today?
The concept of population health has its origins in the Canadian Lalonde Report in 1974, which promoted the (then radical) idea that health and well-being involve more than the health care system, and that the adoption of healthier lifestyles, and improvements in people's social and physical environments, would be the principal means of improving the health of Canadians in the future. 40 Population health, as a way of acting on the social and economic forces that structure health, builds on a tradition of public health and health promotion that goes beyond a focus on the medical, biological or lifestyle problems of individuals. 41
A population health approach can be defined as a subset of public health with a whole-of-population focus, 42 or as containing both public health and other health services. 43 Population health is not the only term that is sometimes misleadingly 39 An alternative would be to include both terms in the domain name. 40 Lalonde 1974. 41 Hayes & Dunn 1998. The population health approach is not without its critics, some of whom argue that it has been captured by the focus on the problems of individuals (e.g. overweight persons), while losing sight of the larger issues (e.g. obesogenic environments) (Raphael & Bryant 2002) . 42 Bennett 2003: 12. 43 For instance, a 'population health approach describes a comprehensive health system which ranges from public health at one end to individual health care at the other' (Buckett & Hunter 2004) . Fraser (2005) conceptualises population health as 'the health of a defined population, or a field of study that links health outcomes, determinants of health, and interventions' but notes that it is an 'ill-defined term' in the literature. The term public health has competing definitions, but is considered by many health professionals to be 'broader and more encompassing than population health' (Fraser 2005: 177) .
The decision on what to call public health is partly semantic, as the domain called 'public health' has changed over time. 'Classical' or 'traditional' public health had an external, environmental focus and produced major infrastructure projects such as sewage and safe drinking water systems, and other improvements to the human environment. Figure 3 shows changes in the conceptualisation of public health over time in two axes: populationindividual and proactive-responsive. In the figure, quadrant D describes the 'new' public health (and 'social' health, with a health equity focus) as a proactive population approach. contrasted with public health and adds to the confusion about what public health is. Figure 4 shows how such confusion can arise from the intersection of public health with other perspectives on health-such as, a population health approach, and definitions of preventive health, and primary health care.
Does it matter what the domain is called? The term 'population health' was preferred over 'public health' in several consultations during phase one of the project. A sampling of jurisdictional health departments showed that population health has overtaken public health in popularity as the name for the relevant organisational units (see Appendix E) .
At other consultations, the term public health was strongly preferred to population health as the name of the domain (although a 'population health approach' was allowed as a method used by public health). There is also a widespread view among public health experts that the general public commonly confuses or equates public health with public hospitals, or the health system funded from the public purse. Some practitioners saw the rise in the popularity of the term 'population health' as an opportunity to gain agreement on an allencompassing definition and to replace the often misunderstood term 'public health'.
As was noted in the discussion in Section 3.1.2 above, the boundary between public health and clinical medicine is contentious, and both the boundary and the components included in each are likely to change over time. There may never be complete agreement by all experts, but the act of making components and boundaries explicit can at least facilitate discussion on these difficult issues that are summarised as discussion points for further consideration in Box 2. 'Public health is the organised response by society to protect and promote health, and to prevent illness, injury and disability. The starting point for identifying public health issues, problems and priorities, and for designing and implementing interventions, is the population as a whole, or population sub-groups.' (NPHP 1998)
What is the preferred name for the domain of public health today (population health, public health, public and population health)?
How is 'organised response' defined? Is there agreement on the following examples of organised response?
a. The breast cancer screening programme supervised by BreastScreen Australia;
b. Screening for cervical cancer by GPs underpinned by registers, recall systems, and target population monitoring;
c. GPs undertaking opportunistic screening for high cholesterol, in accordance with published National Heart Foundation guidelines, in patients consulting them for an unrelated matter?
How is public health differentiated from clinical treatment services? When are treatment services -for example, treatment of sexually transmitted diseases or tuberculosis -part of public health?
Does the place of delivery of services determine that a service is or is not a public health service? For example, is an immunisation delivered in a dedicated local government or school immunisation clinic different from an immunisation delivered in a hospital emergency department?
Should the domain of public health be solely within health or should it include specific activities of other sectors (e.g. education, transport, local government) that have public health as a primary purpose? Or as a secondary purpose?
One suggested response to these questions is for a checklist approach that operationalises the agreements realised in scoping the public health domain. This could be used to determine whether an activity is public health or clinical care, for instance. The checklist components could be weighted, so that an activity that meets one 'must-have' and two out of three other criteria is defined as public health.
The checklist could test whether the activity is preventive, (e.g. primary or secondary reason for service is to prevent the need for acute care; treatment for sexually transmitted disease is to prevent transmission of disease); whether it benefits a population (this does not preclude services to individuals -the benefit could be to an individual and a population, e.g. immunisation); whether a public health response is required in addition to (any) individual treatment response required (e.g. assess area for contaminant after individual exposure, check cooling towers in response to case of Legionnaires disease, trace contacts of person diagnosed with infectious disease); whether it is an organised response, for instance, in response to a disaster, over time (e.g. immunisation register), or in scale (e.g. screening across the nation, quality assurance through pathology reference laboratories).
The most important dimensions (or top-level classes) revealed in an analysis of the National Public Health Partnership public health core functions 44 were the functions of public health, 45 and the methods that public health uses to achieve those functions.
A selection of other candidate top-level classes was made in order to focus the project. Those initially chosen for detailed examination were:
public health functions and activities or programs that funds buy (e.g. public health expenditure activities);
determinants of health, health risk and protective factors (e.g. socio-economic determinants, behavioural factors); disease, disability, and injury areas (e.g. vaccine preventable diseases) that determine intervention targets; and the public health 'toolkit'-methods, tools, and bodies of knowledge, both those specific to public health (e.g. epidemiology, health promotion techniques) and those used by but not specific to public health (e.g. management methods, policy development frameworks).
These potential classes underwent extensive development and revision and are shown in Figure 5 as they stand at the conclusion of phase one of the project (working definitions are in Table 2 ). Potential classes that were identified but not selected for detailed examination are discussed in Section 3.2.5.
Public health practitioners expressed both broad and narrow views of what a classification system for public health should include. These views reflect the range of practical applications they identified (detailed in Section 3.1.3), and their underlying requirements. For instance, for health expenditure reporting, mutually exclusive activity categories at meaningful expenditure levels are required. From a health promotion viewpoint, the ability to model the public health business cycle, and to identify gross expenditure proportions for different elements (e.g. design, implementation, evaluation) are equally important.
There was however, consensus among the public health experts consulted, that a public health classification should be multi-dimensional, and there was broad agreement on the top-level classes that should be included.
There was agreement that public health 'functions' form an important class, although there was some confusion regarding whether functions refer to the purposes of public health activities or the methods of intervention by which public health achieves its aims (see working definitions in Table 2) . 44 NPHP 1998. 45 The word 'function' is used here in the sense of 'the purpose, role or use of something'; thus, the function of public health is 'to protect and promote health, and to prevent illness, injury and disability' (NPHP 1998). There was also wide agreement that both 'health issues' and 'determinants of health' are central to public health, although there are differing views on the relative importance of individual determinants and how they should be structured at lower levels of the classification. The inclusion of a 'settings' class was also generally agreed.
The project involved extensive discussion and work regarding how to define the practice of public health, the methods and strategies used in public health interventions, and the bodies of knowledge that these draw on. There were two strong perspectives on what should be included in a classification. One perspective was restrictive and would narrow the scope of a 'methods' class to those methods that are peculiar to -or only used by -public health (e.g. population-based epidemiology, health promotion, environmental risk assessment). The other focus was on capturing all methods used by public health, including those that, while not specific to it, are employed by public health workers in the normal course of their work (e.g. administration, management, policy development).
A 'resources' class was elevated in importance when consultations reinforced the importance of the many types of infrastructure on which today's public health relies: physical infrastructure (e.g. sewers, public health laboratories), organisational infrastructure (e.g. partnerships, legislative and regulatory systems), logistical infrastructure (e.g. vaccine cold chains) -systems that are seen by some as 'joined up' resources. There were diametrically opposed views of whether infrastructure was a subclass of resources or vice versa. In the short term this has been dealt with by amalgamating the two into a 'resources and infrastructure' class.
In consultations many public health experts wanted to add a 'policy' class. There are several elements to be described. One element is the public health work of developing healthy public policy. Whether or not policy is implemented, substantial work goes into its development, and its availability can provide a head start for action on a health issue that becomes of interest. Information on existing public policy that has an impact on public health is considered by some as important to collect and integrateespecially in the absence of a national public health policy. Some public health experts were comfortable with the concept of 'policy' as a resource or as part of the public health infrastructure, however others were strongly negative -they saw that as putting policy too low in the class hierarchy. This reveals the tendency to see the toplevel classes listed in Figure 5 as a hierarchy of the factors of most importance to public health, in which case, where is policy? Where are population groups? The discussion in Section 3.2.3, which is illustrated in Figure 7 , addresses these questions.
Similarly, the addition of an 'outcomes' class was identified as important at almost every consultation, reflecting a view that outcomes (i.e. outcome indicators and their reporting 46 ) are necessary to 'close the loop' and complete the program logic for 46 For example, public health system performance measures and public health expenditure reporting. public health. This reflects a tendency to see the top-level classes listed in Figure 5 as a program logic or cycle (rather than a hierarchy of important factors) that requires information on outcomes to complete the cycle. An alternative view on the treatment of outcomes in a public health classification is that they are already captured in the classes of 'health issues' and 'determinants of health'. Section 3.2.3 also addresses these issues. Health issues Health, and well-being issues that affect health ('issues' includes: concerns, topics, problems). Health is defined (by the WHO) as 'a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity'.
Factors that influence health status and determine health differentials or health inequalities. They include, for example, natural, biological factors, such as age, sex and ethnicity; behaviour and lifestyles, such as smoking, alcohol consumption, diet and physical activity; the physical and social environment, including housing quality, the workplace and the wider urban and rural environment; and access to health care. 47
The methods used by organised public health interventions (actions, activities, programs, services) to protect and promote health and prevent illness, injury and disability, that are designed to change population exposure, behavioural or health status.
Settings in which public health activities and interventions take place, institutional and social environments, partnerships, and locations (e.g. schools, local government, hospitals, workplaces).
Resources and infrastructure, 'the means available for the operation of health systems, including human resources, facilities, equipment and supplies, financial funds and knowledge'. 48 It includes both person-time and calendar time.
Considerable development of the functions 49 or purposes of public health took place during phase one of the project. As discussed above, the National Public Health Partnership (NPHP) public health core functions 50 were analysed and distilled into the individual functions of the 'functions' class of version one of a public health 47 Based on WHO 2005 , citing Lalonde 1974 Labonté 1993 . 48 WHO 1998a Function is defined as 'the purpose, role or use of something'; thus, the function of public health is 'to protect and promote health, and to prevent illness, injury and disability' (NPHP 1998) . 50 NPHP 1998. classification presented in this report (the proposed treatment of other components of the public health core functions is shown in Table 5 ). 51 The functions as scoped at the end of phase one of the project are shown in Table 3 and their working definitions are given in Table 4 . Both primary and instrumental functions are of importance in conceptualising public health. Primary functions are ends in themselves, while instrumental functions are means to those ends, as without primary functions there would be no need to 'ensure public health capability', for instance. Instrumental functions were also described in consultations as supporting, underpinning, or crosscutting functions, as all primary functions rely on them and they do not belong exclusively to any one of the primary functions.
Although the instrumental 'build the evidence base...' function could be included in 'ensure public health capability', it is shown separately because building an evidence base and moving towards decisions informed by evidence are key features of the current context for public health.
Other functional classifications of public health were explored during the course of the project, including that portion of the OECD System of Health Accounts that is relevant to public health. 52 The OECD classification has a similar mix of classes within functions as do the public health core functions, but excludes environmental 51 The public health core functions (NPHP 1998) are shown in Table 5 . 52 'Prevention and public health services' defined (in part) as services 'mainly of a preventive nature and ... publicly provided' which include 'special public health services such as blood-bank operation, public health service laboratories, and population planning services' (OECD 2000: 44) .
health, and was structurally not helpful. 53 A functional division that followed the distinctions between primary, secondary and tertiary prevention was also explored but the classification was confusing and difficult to apply, and there are arguments that tertiary prevention in particular, has more relevance to clinical treatment services than to public health.
A comparison of the public health functions of selected other nations (see Figure 6) shows that, in the UK for instance, both primary (e.g. health promotion and disease prevention programs) and instrumental (e.g. development and maintenance of a public health workforce) functions are prominent, while the public health functions of Canada and the Americas are limited to primary functions. 54 Both the UK core functions 55 and the USA essential public health services include a specific (instrumental) partnership function for public health. In Australia, the essential importance and defining nature of inter-departmental, inter-governmental, inter-sectoral and other partnerships, in the work of public health was made clear in the expert consultations. Accordingly, version one of a public health classification proposes 'build public health partnerships' as a subclass of the 'ensure public health capability' instrumental function (see Table 3 ). 53 Dimensions used by the OECD are: population groups, service types, disease types, and settings. 54 A recent review conducted by WHO (2003), describes comparable 'essential public health functions' as 'a set of fundamental activities that address the determinants of health, protect a population's health, and treat disease... public health functions represent public goods, and... governments would need to ensure the provision of these essential functions, but would not necessarily have to implement and finance them. They prevent and manage the major contributors to the burden of disease by using effective technical, legislative, administrative, and behaviour-modifying interventions or deterrents, and thereby provide an approach for intersectoral action for health [that] stresses the importance of numerous different public health partners. Moreover, the need for flexible, competent state institutions to oversee these cost-effective initiatives suggests that the institutional capacity of states must be reinforced' (Yach 1996 cited in WHO 2003 : 1, our italics). 55 The full description of this function is 'Creating and sustaining cross-Government and intersectoral partnerships to improve health and reduce inequalities' (CMO UK 2003; see chapter 3). The UK core functions also include a specific (instrumental) research function. In Australia, although 'public health research' is one of the nine core public health activities for which public health expenditure is reported, 56 there is no corresponding function in the NPHP public health core functions. Version one of a public health classification proposes 'conduct public health research' as a subclass of the 'build the evidence base for public health' instrumental function (see Table 3 ).
A health surveillance function is common to both the UK and Canada. It is broadly specified in the UK as 'health surveillance, monitoring and analysis', while in Canada the function of 'population health assessment' is specified separately, in addition to 'health surveillance'. 57 In Australia, the first of the nine NPHP public health core functions is 'assess, analyse and communicate population health needs...' (and is proposed as 'assess health of populations' in version one of a public health classification -see Table 3 ), although expenditure on this public health activity is not currently reported in an identifiable manner.
A quality assurance function is specific for public health in both the USA and the UK. Whether such a function is pertinent to public health in Australia is a matter for discussion and has not been canvassed in consultations.
Working definitions of the public health functions proposed in Table 3 are given in Table 4 . The working definitions are based on NPHP public health core functions 58 and extensive discussion during the project. Some of the major strands that emerged in discussions, and their impact on the working definitions, are reported below.
The 'promote health and prevent disease, disability, and injury' function was initially cast as two functions, with 'promote better health' separate. In examining the mission statements and goals of health promotion and prevention units across the jurisdictions it was clear that there was no hard boundary between the promotion of health and the prevention of disease, disability and injury. In consultations it was suggested that the two functions should be married together as the distinction is increasingly blurry in practice. They have thus been joined as one function at the top level.
'Develop healthy public policy' was initially classed as a subclass of a 'promote better health' function. In consultations it was pointed out that this function, method or strategy was cross-cutting, applying to all primary functions, and should not be singled out as belonging only to one function, or as separate to all other functions. 'Policy development' was thus classed as a 'method' of intervention so that it can be applied to any or all of the public health functions in an additional dimension. 56 AIHW 2004b. 57 The post-SARS Canadian view is that: 'Among the functions of public health are health protection (e.g. food and water safety, basic sanitation), disease and injury prevention (including vaccinations and outbreak management), population health assessment; disease and risk factor surveillance; and health promotion. The public health system tends to operate in the background unless there is an unexpected outbreak of disease such as SARS or failure of health protection as occurred with water contamination... Monitor health Monitor and analyse levels of health and its determinants in populations to identify and predict trends and emerging issues ('Assess health inequalities' would be a further subclass of this).
Evaluate health risks and benefits Evaluate adverse and beneficial effects related to health and social policies and interventions, and environmental exposures.
Assess health inequalities Assess inequalities in health (level and distribution) and health gain to target interventions to improve the health of the worst-off sub-populations.
Protect from threats to health Protect from, and prevent, external threats to public health.
Prepare for threats to health Identify and prepare for potential threats to health (including communicable diseases, environmental hazards, bio-terrorism and new patterns of exposures e.g. arising from ecological change).
Respond to threats to health Respond to threats to health (including communicable diseases, environmental hazards, bio-terrorism and other disasters).
Control and mitigate risks to health Minimise or reduce the severity of risks to health (includes setting and monitoring of standards for e.g. food, air and water quality and other potential hazards, also harm minimisation measures).
Promote health and prevent disease, disability and injury Promote health and wellbeing, prevent the occurrence of disease, disability and injury; and detect disease in its early stages, through organised efforts that target populations.
Promote health and wellbeing Promote better health and well-being as it affects health (e.g. community development and community empowerment initiatives clearly differentiated from 'Prevent the occurrence of...').
Prevent the initial occurrence of disease, disability and injury (e.g. population-level campaigns to promote physical activity, tobacco control, seat belt legislation).
Detect disease, disability or injury early Detect disease, disability and risk of injury early and initiate prompt management or response (e.g. screening for cancers, newborn hearing screening).
Ensure public health capability Ensure adequate public health capacity and responsiveness by maintaining and developing the public health workforce and infrastructure, and building partnerships with other sectors of society.
Develop and maintain the public health workforce Train, maintain and develop the public health workforce. There was also a view that a 'promote better health' function should be expanded to 'enhance health and quality of life,' to incorporate the concepts of: (1) effort from non-health sectors that affects public health, and (2) quality of life and health maintenance (rather than improvement) where the presence of disease makes health improvement an inappropriate aim. Agreement on these definitional extensions was lacking in further consultations and they have not been adopted.
The working definitions in Table 4 are shown as they stand at the conclusion of phase one of the project. They should be regarded as a work-in-progress and a point to move forward from, rather than the definitive last word on the public health functions.
The relationship between the 'functions' class and other top-level classes in version one of a public health classification and the National Public Health Partnership (NPHP) public health core functions 59 is shown in Table 5 . The table illustrates how the public health classification can be used to achieve a functional equivalence to the several dimensions implicit in the NPHP public health core functions.
The multi-dimensional core functions can be classified using different top-level classes of the classification (e.g. 'health issues', 'methods'), and instances (see Figure  7 ). For example, the function or purpose of core function two (shaded in Table 5 ) is to 'Prevent and control communicable and non-communicable diseases and injuries' using the public health intervention methods of 'risk factor reduction, education, screening, immunisation and other interventions'. 
Assess health of populations
Protect from threats to health Promote health and prevent disease Risk factor reduction, education, etc classified as 'methods', and instances described as Interventions. Communicable and non-communicable diseases etc classified as 'health issues'.
Promote health and prevent disease Ensure public health capability:
Build partnerships Action with individuals, families, communities etc classified as 'methods', instances described as Interventions, and families, communities described as Population Group instances.
All. Public policy measures classified as 'methods', and instances described as Interventions. Ensure public health capability Build the evidence base for public health Plan, fund, manage and evaluate classified as 'methods', and instances described as Interventions. Programmes described as instances of Public Health Activities.
Promote health and prevent disease Ensure public health capability
Consultation, participation and empowerment classified as 'methods', and instances described as Interventions.
Protect from threats to health Promote health and prevent disease
Actions described as instances of Public Health Activities.
Promote health and prevent disease
Healthy growth and development classified as a 'health issue' (e.g. 'health and well-being').
Life stages described in Population Groups.
Protect from threats to health Promote health and prevent disease
Individual vulnerable groups described as Population Groups classified by other classes (e.g. person-level demographic descriptors in 'determinants of health'). Actions described as instances of Public Health Activities. Core function nine (shaded in Table 5 ) 'Promote, develop and support actions to improve the health status of Aboriginal and Torres Strait Islander people and other vulnerable groups' identifies important target populations, rather than describing a separate function of public health. Functions, methods, and population groups thus form three distinct dimensions (among many) of interest in a multi-dimensional classification of public health.
Few of the nine public health core functions have a one-to-one relationship with the functions of the public health classification, if the functional equivalence shown in Table 5 is accepted.
Core function four 'Promote, develop and support healthy public policy, including legislation, regulation and fiscal measures' requires special mention, as it is shown as relevant to all the functions of the public health classification. It is proposed that public policy measures are methods to address all functions rather than a function in their own right. 'Public policy development' is thus shown as a separate method in Table 6 , as is 'legislation and regulation' (which some see as enacted policy). Public health activity using these methods can have a major impact on population health. Examples include the impact on population smoking rates of legislation, regulations, and fiscal measures implemented under the policy umbrella of the Tobacco Control Strategy.
Although the multi-dimensional structure of the public health classification is quite different to the flat list structure of the public health core functions, its classes can be used in a functionally equivalent way to classify and describe the functions and other important dimensions of public health.
The public health dimensions currently scoped, and their top-level classes are shown in Table 6 . The 'functions' class has been discussed in Section 3.2.2. While there was reasonable agreement among the public health experts consulted over the top levels of the classes of 'health issues' (although its name was debated), 'determinants of health', and 'settings', the remaining classes are in the early stages of development and have not yet been subject to detailed consideration. The 'methods' class, in particular, established to describe the methods of public health intervention, is at an early stage of development. While population groups are important, it was generally agreed that they are not a toplevel class in a public health classification. As the targets of public health interventions, instances of population groups can be described by other classes in the classification, such as the person-level demographic descriptors in the 'determinants of health' class (e.g. age, sex). There was also agreement that stakeholders and partners, although important in the work of public health, did not warrant their own top-level class. As with population groups, they may also be described by other classes in the classification. This distinction is illustrated in Figure 7 , which distinguishes between classes in the classification (circles) and items to be classified (heptagons). The latter include (but are not restricted to) public health activities and programs (centre), public health interventions, public policies, outcomes (indicators that are useful for public health purposes, and those that are nationally reported), population groups, partners and stakeholders in the public health effort. Figure 7 also shows whether suitable classifications exist for use by the top-level classes, or whether they need to be developed. Existing classifications (e.g. Australian standards, international classifications of diseases, functioning and disability, external causes of injury) are available to classify major parts of the 'health issues', 'settings' and 'resources' classes. The National Public Health Information Working Group has determined that further development of classifications for the 'functions', 'determinants of health' and 'methods' classes are a required priority for the second phase of the project.
Not all public health experts will agree with the constituent parts of the classes as they stand, and some important parts are undoubtedly missing. The project anticipates feedback on these issues through making these results more widely available.
During phase one of the project, some of the practical uses that had been identified were developed in a small way in order to test the usefulness of the classification. Two examples-of public health activity from national public health expenditure reporting, and details of public policies-are detailed below. Information on some recent developments of interest in public health classification in the UK can be found at the end of Appendix B.
A selection of public health activities from public health expenditure reporting were classified using the top-level classes of the public health classification. The detail of an example public health activity is shown in Figure 8 . The symbol denotes classes and subclasses, while the symbol denotes 'instances' or individual cases, for example, an individual public health activity, partner, stakeholder, or population group.
On the left of the figure is a list of public health activities extracted from the latest public health expenditure report 60 , and to the right are the details of a selected activity, characterised by a number of 'slots' or attributes of the activity. The selected activity is Queensland's 2000-01 health promotion initiatives, on which $18.7 million was expended. The example shows the variety of health issues and determinants addressed (sun protection, healthy diet, and so on) for population groups.
Queensland's 2000-01 health promotion initiatives are classified by the public health expenditure core category of 'Selected health promotion', as used in national public health expenditure reporting, 61 while the (main) function or purpose is to 'Promote health and prevent disease, disability and injury' (using the public health functions developed in this project). Associated public health intervention methods used in the health promotion initiatives are also listed (e.g. intersectoral advocacy, community action). Partnerships and stakeholders are shown as test data.
This classification of a public health activity is much better than a one-dimensional classification at answering the questions listed in Box 2 in Section 3.1.3 as a practical test for the classification. For instance, in response to the question 'How much was spent last year on the prevention of obesity?', Figure 8 shows that public health activities for which the function is 'prevention' and the health issue is 'obesity' can 60 AIHW 2004b . 61 AIHW 2004b easily be identified, and the values in the 'expenditure' slot (attribute) for these activities can then be summed.
A selection of public health policies compiled from publicly available documents accessible on the internet were classified using the top-level classes of the public health classification. An example public health policy is detailed in Figure 9 . As previously, in Figure 9 , the symbol denotes classes and subclasses, while the symbol denotes 'instances' or individual cases, for example, a particular public health policy. Figure 9 shows detail on the Australian Government Draft National Injury Prevention Plan (NPHP 2004) and the health issues it addresses (external causes of injury, safe home environment, and so on). The plan is assigned to the function subclass 'Prevent occurrence of disease, disability, and injury'. Capture of the URL for the published policy allows rapid access to the policy through the internet. Details of the jurisdictions and/or portfolios that have endorsed the plan can be captured in additional slots.
These examples do not completely illustrate the full power of a 'third generation' multi-dimensional classification for public health, developed using a formal ontologybuilding tool such as Protégé. While nothing can replace human knowledge and intelligence in the comprehensive collection, description (classification, indexing) and use of complex information, in the future it is envisaged that semantic tagging 62 of documentation and other written resources will allow much more meaningful information to be routinely made available to humans, through machine processing of this 'computable' information. More information on this aspect of the project is presented in Appendix D.
In addition to those top-level classes discussed in detail above, other potential classes were identified in the first round of development. These included:
Geography/access to health services (e.g. urban/rural/remote geographic classification). Intervention target or focus (e.g. target population defined by age, sex, ethnicity) and intervention type. Performance measures (e.g. the national health performance framework). Precepts, principles, philosophy (e.g. equity). Service production/provision (where service is produced/provided e.g. institutional health services, non-institutional health services) and service delivery/settings (where service is delivered e.g. school, workplace, community). Sources of funds (e.g. health/non-health; levels of government). Theories and models (e.g. 'harm minimisation', 'user pays'). Time (e.g. incubation periods, time-lags, investment periods, break-even points). Workforce (e.g. public health specialists, local government workers, school nurses).
Contextual/macro-environmental/ecological factors that affect but are outside the influence of public health (e.g. factors that would be picked up in environmental scanning). Interventions as public health activities/strategies that are related but different to methods. Outcomes, including outcome indicators and reporting (e.g. national public health system performance measures), necessary to 'close the loop' and complete the program logic for public health. Policy including various views: policy development as an activity or 'method' or a cross-cutting component of all functions; policies as a class of things in existence (e.g. as in a policy register or library); policy as enhancing understanding of practice, cross-referenceable to other areas of interest. Population groups as defined in terms of attributes and characteristics from other classes (e.g. age, sex). Research/evidence allowing integration with the university sector, to link research and policy and practice, and to build the evidence base for public health. Risk factors (part of the 'determinants of health' class). Partners and stakeholders in the public health effort.
Although the project focussed on only a few selected classes, many of the other areas listed above were considered in detail. In some cases the topic area suggested has been captured in the broad structure (e.g. 'settings' have been included among the toplevel classes). In other cases, the topic area has been built into the public health classification as attributes and characteristics of classes. Some are demonstrated in the examples of practical applications in Section 3.2.4. For instance, stakeholders and population groups are shown as attributes (slots) of 'public health activities' in Example 1. 'Policy' has been represented as a register or library of existing policies in Example 2. 'Research' should be identifiable through classification using the 'methods' class (which includes the subclass 'research and evaluation methods'). 'Workforce' and 'workforce development capacity' have been included as subclasses of the 'resources and infrastructure' class, as has 'time'.
Definitional issues that were discussed during the project have been summarised as discussion points in Box 2. In Box 3 a range of other issues, raised throughout this report, are summarised for the further consideration of public health experts.
Is there agreement with the principles of development: multi-dimensional, inclusive rather than exclusive, broad rather than narrow?
Should public health classification be restricted to a domain solely within health or should it include specific activities of other sectors (e.g. education, transport, local government) that have public health as a primary or secondary purpose (e.g. immunisation organised by local government)?
Is there agreement on the top-level classes?
Are the public health functions appropriate?
Are all important functions captured (e.g. is quality assurance a public health function in Australia)?
Is the division between primary and instrumental functions clear and useful?
Are there any important subclasses that are currently missing from the first two levels of the public health classification (see Table 7 )?
What are the important characteristics of agreed top-level classes?
Phase one of the project has produced version one of a public health classification, and achieved a degree of consensus among Australian public health experts regarding its major classes, and their structure at the top levels. The classes of public health 'functions', 'determinants of health' and 'methods' of intervention have been identified as priorities for further development.
Many of the public health experts consulted during phase one of the project indicated that they were keen to continue their engagement. Most were positive about the project. They identified a range of practical applications for a public health classification that extended far beyond its uses for reporting public health activity and expenditure. The consultation process also brought to light a variety of issuesincluding areas of basic disagreement about the nature and boundaries of public health practice-that warrant more work. These are set out throughout this report in boxes.
It is proposed that the second phase of the project will further extend the availability of, and seek feedback on, the public health classification through a web-based version, and develop a proposal for its future development and support.
Because it attempts to capture the breadth of public health activity, and to serve multiple uses, the public health classification has a necessarily complex, multidimensional structure that is difficult to present adequately in paper-based forms. A web-based version, rendered in HTML, will allow interactive engagement and easier access to the structure, coverage and documentation (e.g. definitions). An early version of the classification was mounted on a test website and demonstrated in consultations with reasonable acceptance and understanding of its use as a navigation tool. A facility to collect structured feedback-rather than just adding large numbers of new classes and subclasses-and processes to compile and review this information will be needed to improve the utility of the classification for practical applications.
Developing a plan for the ongoing development and support of the classification will involve consideration of governance and maintenance arrangements, as well as the issues of access, availability and intellectual property ownership and management. Maintenance of classification systems can be difficult, time-consuming and thankless work. International classifications, like that of diseases, rely on a lengthy consensual process of experts to identify and agree upon new entries. 63 However, new capabilities made possible by the Internet and the development of the Semantic Web present opportunities to distribute the maintenance burden across many contributors, and to dramatically speed up consensual agreement. 64 These will be explored as part of scoping the requirements for ongoing development and support of the classification.
Further development of the classification will emphasise its relationships with classifications that are already in existence and widely used as standards. The public health classification, as it is currently structured, has subclasses that simply reference or point to relevant external classifications. These include (but are not limited to)
Australian standards (e.g. geographical, industry, and occupational classifications, other standards promulgated by AIHW and ABS) and the international classifications of diseases, functioning and disability, and external causes of injury 65 (see Figure 7) . In a similar vein, it is proposed to investigate the possible inclusion of the public health classification in the set of standard classifications known as the Australian Family of Classifications. 66
It is recommended that phase two of the Public Health Classifications Project should:
Focus on further developing the classes of public health 'functions', 'determinants of health' and 'methods' of intervention;
Develop and release a web-based version of the public health classification with facilities for eliciting structured feedback and managing contributions to the further development and refinement of the classification;
Develop a plan for ongoing development, support and governance of the public health classification;
Further specify links or relations between the public health classification and relevant existing classifications and standards (with due regard for intellectual property rights); and 
A number of things regarded as forming one group through the possession of similar qualities; a kind; sort. (Delbridge & Bernard 1998) Classes are the focus of most ontologies. They describe concepts in the domain. For example, the class of public health 'functions' represents all public health functions. Specific functions, for example, 'protect from threats to health', are instances of this class. A class can have subclasses that represent concepts that are more specific than the superclass. For example, we can divide the class of all public health 'functions' into 'assess...', 'protect...' and 'promote...' functions. 67 Alternatively, we can divide the class of all public health functions into primary and secondary functions. (Noy & McGuinness 2001 : 3)
An arrangement of classes in a taxonomic (subclass-superclass) hierarchy. A class hierarchy represents an 'is-a' relation, where a class X is a subclass of A if every instance of X is also an instance of A. A class hierarchy thus represents a set of classes related by inheritance. A class hierarchy is typically shown as a tree structure for single inheritance or as a lattice structure for multiple inheritance (where nodes represent classes and are connected by arcs to indicate inheritance relations).
In an ontology there is no single correct class hierarchy for any given domain. The hierarchy depends on the possible uses of the ontology, the level of the detail that is necessary for the application, personal preferences, and sometimes requirements for compatibility with other models. (Noy & McGuinness 2001 : 6-8)
A system for classifying things; in a library, a system of arranging items according to broad fields of knowledge and specific subjects within each field. To classify means to arrange or distribute in classes; to place according to class.
Example: International Classification of Diseases (ICD) (WHO 1992-94).
Computable information is information that can be readily manipulated and transformed by computers. Currently a great deal of information (on the Web and elsewhere) can be read by computers but not manipulated or understood by them. In the near future, the Semantic Web being developed by Sir Tim Berners-Lee, one of the founders of the World Wide Web, and others, will make information computable and connectable by adding semantic information, based on ontologies and classifications, to elements within text (Berners-Lee 2001).
Determinants of health are factors that influence health status and determine health differentials or health inequalities. They are many and varied and include, for example, natural, biological factors, such as age, gender and ethnicity; behaviour and lifestyles, such as smoking, alcohol consumption, diet and physical exercise; the physical and social environment, including housing quality, the workplace and the wider urban and rural 67 See Section 3.2.2 for more information on the public health functions in a public health classification. environment; and access to health care (Lalonde 1974 , Labonté 1993 . All of these are closely interlinked and differentials in their distribution often lead to health inequalities (WHO 1998a).
A part or aspect of something. For example, one dimension of public health is the settings in which public health work is carried out. A dimension is a property or construct whereby aspects of something can be distinguished (e.g. public health settings can be distinguished from public health functions and from public health methods). A dimension can also be described as a group of similar things that are from the same category of information (e.g. home and workplace settings are part of the settings dimension). Hence multi-dimensional, to have many aspects or dimensions (e.g. to provide a unified framework for multiple public health uses, a multi-dimensional classification is needed).
Disease prevention refers to measures taken to prevent the occurrence of disease, to arrest or slow its progress and to reduce its consequences. Examples of disease prevention measures include risk factor reduction, screening and early intervention.
Primary prevention of disease is directed towards preventing the initial occurrence of a disease. Secondary and tertiary prevention aim to arrest or slow the progression of existing disease and to reduce its effects through early detection of complications and appropriate treatment; or to reduce the occurrence of relapses and the establishment of chronic conditions through, for example, effective rehabilitation (WHO 1998a).
The kind of action or activity proper to a person, thing, or institution (Delbridge & Bernard 1998: 452) . The function, purpose, role or use of something; for example, the function of public health is 'to protect and promote health, and to prevent illness, injury and disability' (NPHP 1998) .
Machine readable-see computable information, Semantic Web
Information about data. Metadata can describe the fields and formats of databases and data warehouses, documents and document elements such as Web pages or research papers. Metadata management is a functional component of an information management architecture.
Example: the descriptive information provided in the 'META' tags in an HTML or XML document header that give information about the document.
A model of a particular field of knowledge -the concepts relevant to that field (e.g. the field of public health), and their attributes, as well as the relationships between the concepts. In the Protégé ontology development software, 68 an ontology is represented as a set of classes that have associated slots (attributes).
In philosophy, ontology describes a branch of metaphysics concerned with the nature and relations of being. The term has been redefined by the knowledge engineering and artificial intelligence communities to refer to a formalised description of the concepts and relationships that exist within a specific domain and all that can be represented about that domain. Ontologies can be mental models, computer models, or a combination of both. Ontologies provide a means by which characteristics of a specific representation can be assumed and behaviour predefined (Kemp & Vckovski 1998) . Multiple user views can be accommodated by providing translations between different ontologies.
An ontology defines a common vocabulary for researchers who need to share information in a domain. It includes machine-interpretable definitions of basic concepts in the domain and relations among them (Noy & McGuinness 2001) .
Ontologies are developed for the purposes of:
Sharing common understanding of the structure of information among people or software agents,
Enabling re-use of domain knowledge,
Separating domain knowledge from operational knowledge, and
Analysing domain knowledge.
Example: The (US) National Library of Medicine's Unified Medical Language System (UMLS) 'knowledge sources' and associated lexical programs for system developers. The Meta-thesaurus is organised by concept or meaning. Its purpose is to link alternative names and views of the same concept together and to identify useful relationships between different concepts.
Organised efforts focused on the health of defined populations in order to promote and maintain or restore health, to reduce the amount of disease, premature death and discomfort and disability due to disease. Programs, services and institutions here emphasize the prevention of disease and the health needs of the population as a whole. Among a broad scope of disciplines, various knowledge and skills are used, such as bio-statistics, epidemiology, planning, organisation, management, financing and evaluation of health programs, environmental health, application of social and behavioural factors in health and disease, health promotion, health education and nutrition. (IIME 2002)
Preventable conditions include many chronic, non-communicable diseases such as cardiovascular disease, type 2 diabetes, obesity, chronic lung disease; conditions amenable to early detection and treatment such as breast and cervical cancer, high blood pressure; communicable diseases such as HIV/AIDS, food borne illness, vector borne diseases, vaccine preventable diseases; intentional and unintentional injuries; many mental health problems and related conditions such as substance abuse and family dysfunction. (Straton & Sindall 2001: 1) 68 Protégé is developed by Stanford University, see http://protege.stanford.edu.
Prevention is characterised by activities that are taken to reduce the possibility that something will happen, or to minimise harm if it does occur. The prevention of illness or disability requires the identification of the factors that contribute to poor health and modifying, reducing or eliminating them, or, conversely, building and strengthening protective factors. Prevention is usually taken as a core responsibility of organised health systems-alongside the curative, restorative and palliative functions-and is a key element in achieving health improvement and the reduction of the burden of disease in society. Prevention is also an important component of many other branches of social policy (for example crime prevention, child abuse prevention), many of which also contribute, directly or indirectly, to health.
It has been customary to categorise prevention at different levels, in terms of primary, secondary and tertiary prevention. Thus the goal of primary prevention is reducing the incidence of disease by preventing its occurrence, secondary prevention aims to prevent progression of disease though early detection, usually by screening at an asymptomatic stage and early intervention, 69 and the goal of tertiary prevention includes minimisation of the impact of established disease, and prevention of complications and further disability through effective treatment and rehabilitation. While the terminology used can vary in different fields (for example a slightly different set of categories is often used in relation to mental health 70 ), the basic concepts and objectives of prevention are essentially the same.
It is often useful to think in terms of a hierarchy or spectrum of objectives for preventive activity, aimed at different points on the causal pathway, and for which there is often an important time dimension. For example, the short term aim of a preventive intervention at a certain point in time may be to change beliefs in the community about the risks of smoking; the intermediate objective may be to reduce uptake of smoking and smoking prevalence and the long term goal a reduction in rates of coronary heart disease and lung cancer. (Straton & Sindall 2001 : 1)
Prevention and public health services comprise services designed to enhance the health status of the population as distinct from the curative services, which repair health dysfunction. Typical services are vaccination campaigns and programmes. (OECD 2000: 121) Primary prevention-see also prevention, disease prevention
Primary prevention refers to the protection of health by personal and community wide effects, such as preserving good nutritional status, physical fitness, and emotional well-being, immunising against infectious diseases, and making the environment safe. There are no precise boundaries between the primary, secondary and tertiary levels of prevention. (IIME 2002) 69 A notable exception to this use of the term is found in the area of cardiovascular disease prevention and control where secondary prevention is commonly used to refer to prevention of a second heart attack. 70 In the mental health field primary prevention is further divided into approaches designated as universal, selective or indicated prevention, depending on whether they are applied to the whole population (universal) or sub-groups (selective) or those at an early stage of risk (indicated). A similar approach was used by the AIHW in development of the indicator framework for monitoring the National Health Priority Areas.
Government-funded public health activity is described as an important part of the Australian health care system, with public health activities generally representing the organised response of society to protect and promote the current and future health of the whole population or of specific subgroups of the population, which can be viewed as a form of investment in the overall health status of the nation. (AIHW 2004b: 1)
The nine public health core functions promulgated by the National Public Health Partnership (NPHP 1998) 
Public health has been defined by the World Health Organization as 'the art of applying science in the context of politics so as to reduce inequalities in health while ensuring the best health for the greatest number' (WHO 1998a cited in WHO 2003 .
Public health expenditure reporting: core public health activities
The core public health activities in public health expenditure reporting are defined as 'nine types of activities undertaken or funded by the key jurisdictional health departments that address issues related to populations, rather than individuals. Does not include treatment services.' (AIHW 2004b: 145) Government-funded public health activity is described as an important part of the Australian health care system, with public health activities generally representing the organised response of society to protect and promote the current and future health of the whole population or of specific subgroups of the population, which can be viewed as a form of investment in the overall health status of the nation. (AIHW 2004b: 1)
Public health medicine is that branch of medical practice that is primarily concerned with the health and care of populations. It is concerned with the promotion of health and the prevention of disease and illness; the assessment of a community's health needs; and the provision of services to communities in general and to specific groups within them. (AFPHM 2002a)
Research involving communities or populations, typically outside health care institutions, undertaken to identify the factors which contribute to ill-health in populations and ways of influencing these factors to prevent disease. It includes epidemiology, social and behavioural sciences, health services research on population-based health interventions, and evaluating the efficacy and effectiveness of preventive measures. (HMRSR 1998 : A6.4, Saracci 2004 Public health workforce
The public health workforce is defined as those involved in protecting, promoting and/or restoring the collective health of whole or specific populations (as distinct from activities directed to the care of sick or frail individuals). (Rotem et al. 1995 cited in Riddout et al.2002 .
Resource Description Framework (RDF) 'is a foundation for processing metadata; it provides interoperability between applications that exchange machine-interpretable information on the Web. RDF emphasizes facilities to enable automated processing of Web resources. RDF can be used in a variety of application areas; for example: in resource discovery to provide better search engine capabilities, in cataloguing for describing the content and content relationships available at a particular Web site, page, or digital library, by intelligent software agents to facilitate knowledge sharing and exchange, in content rating, in describing collections of pages that represent a single logical "document", for describing intellectual property rights of Web pages, and for expressing the privacy preferences of a user as well as the privacy policies of a Web site. RDF with digital signatures will be key to building the "Web of Trust" for electronic commerce, collaboration, and other applications' (W3C 1999).
The Semantic Web Environmental Directory describes RDF as the 'equivalent of the language for writing Web pages, HTML (HyperText Markup Language), for the Semantic Web. The Semantic Web uses RDF as the basic language for representing metadata about any kind of resource on the Web' (SWED undated).
Secondary prevention can be defined as the measures available to individuals and populations for the early detection and prompt and effective intervention to correct departures from good health. There are no precise boundaries between primary, secondary and tertiary levels of prevention. (IIME 2002)
The Semantic Web provides a common framework that allows data to be shared and reused across application, enterprise, and community boundaries. It is a collaborative effort led by W3C with participation from a large number of researchers and industrial partners. It is based on the Resource Description Framework (RDF), which integrates a variety of applications using XML for syntax and URLs for naming.
'The Semantic Web is an extension of the current web in which information is given welldefined meaning, better enabling computers and people to work in cooperation' (Berners-Lee et al. 2001) . The Semantic Web and computable information are the visions of Tim Berners-Lee, the creator of the World Wide Web (familiar to us through Google 71 and other search engines), who views this future Web as a web of data, 'like a global database', where 'information is given well-defined meaning, better enabling computers and people to work in cooperation'. Making information on the Web 'semantic' (or meaningful) means much more efficient searching 'as though it were one giant database, rather than one giant book' (Berners-Lee 1998).
The infrastructure of the Semantic Web will allow machines as well as humans to make deductions and organise information. The approach is to develop languages that express information in machine processable forms. The architectural components include semantics (meaning of elements), structure (organisation of elements), and syntax (communication). Abstract representation of data is being based on existing standards (eg RDF -Resource Description Framework) and standards yet to be defined, and is in development by the World Wide Web Consortium (W3C), in collaboration with researchers and industrial partners. 
A classification, especially in relation to its principles or laws; the department of science/s that deal with classification. A taxonomy is hierarchical, with the higher levels being larger, more inclusive and broadly defined, while the lower levels are more restrictive and specific.
Example: the classification of plant and animal life into natural, related groups in descending order: phylum, class, order, family, genus, species.
The system of terms belonging to a science, art, or subject; nomenclature.
A controlled vocabulary contains metadata about terminology to make it easier to search and maintain knowledge management systems that integrate information from multiple sources and applications.
Example: SNOMED CT ® -Systematized Nomenclature of Medicine-Clinical Terms (produced by the College of American Pathologists) is a comprehensive clinical terminology, and one of a suite of designated standards for use in US Federal Government systems for the electronic exchange of clinical health information, and is being implemented throughout the National Health Service in the UK.
Tertiary prevention consists of the measures available to reduce or eliminate long-term impairments and disabilities, minimize suffering caused by existing departures from good health, and to promote the patient's adjustment to irremediable conditions. This extends the concept of prevention into the field of rehabilitation. There are no precise boundaries between primary, secondary and tertiary levels of prevention. (IIME 2002)
A storehouse or repository, as of words or knowledge; a dictionary, encyclopedia or the like, especially a dictionary of synonyms and antonyms.
Technical thesauri are used in search-language normalisation as they specify terms to be used (preferred terms), broader and narrower terms in the hierarchy, as well as related terms (nonhierarchically related, e.g. antonyms) and non-preferred terms (synonyms for the preferred term).
Example: MeSH (Medical Subject Headings) -the (US) National Library of Medicine's controlled vocabulary, used to index articles for MEDLINE and PubMed. MeSH terminology provides a consistent way to retrieve information that uses different terminology for the same concepts.
The wicked problem concept was originally proposed by Rittel and Webber (1984) in the context of social planning. They pointed out that in solving a wicked problem, the solution of one aspect may reveal another, more complex problem. Ten rules define the form of a wicked problem, including:
1. There is no definitive formulation of a wicked problem.
2. Wicked problems have no stopping rule.
3. Solutions to wicked problems are not true-or-false, but good-or-bad.
Every wicked problem is essentially unique, and can be considered to be a symptom of another problem. (The last rule is that: The planner (designer) has no right to be wrong.) The continuing support of Richard Madden, and the additional assistance of John Goss, Tony Hynes, Daniel Aherne and Justine Boland of AIHW is gratefully acknowledged.
On behalf of the project, a big thank you to all who made time to engage with the public health classification, and for your perspectives, reactions and suggestions for improvement.
An example agenda and work-in-progress documentation used in consultations are shown in the following pages.
Consultation on Thursday 10 th December, 10-12am
Consultation Objective: to meet with content experts to model a unified public health classification that is useful and useable for multiple purposes. 
What is the domain that the classification will cover?
Public health.
Definition: Public health is the organised response by society to protect and promote health, and to prevent illness, injury and disability. The starting point for identifying public health issues, problems and priorities, and for designing and implementing interventions, is the population as a whole, or population sub-groups. (NPHP 1998) Principles: The classification should be inclusive, and deliberately broad at the top classes.
For what are we going to use the classification?
Generally, to develop a broad, generalisable public health classification that can be used to:
organise information to facilitate answering key public health questions e.g. expenditure on prevention of obesity; reflect the full scope and breadth of public health activity, in expenditure and performance indicator reporting; articulate, describe and define public health, and promote consistency in describing public health (eg through standardised instructions); build in specific content expertise in different areas of public health; relate to other high level models of health (eg through interface and reference terms); structure and design information/communications e.g. in websites or report chapters.
Specifically, a public health classification could be used to: promote standardised definitions, terminology and reporting of public health and public health functions to improve accountability across jurisdictions, eg through the development of a national Public Health Report describing public health in Australia; build systems such as a web-based database of public health projects that allows routine, bottom up, multi-dimensional reporting of public health projects; create semantic web documents that are 'marked up' for meaning (for the Semantic Web, the next generation of the world wide web) and which can be understood and manipulated by computers (e.g. computer agents can trawl semantic web documents for information to answer questions, eg what is the project expenditure, how many people work on the project, in what settings?).
For what types of questions should the classification provide answers?
Sample focus questions include:
How much was spent on prevention of obesity? Other 'advocacy-type' questions, e.g. difference in expenditure on prevention of HIV/AIDS relative to other preventable diseases, relative expenditure on specific risk factors or diseases?
Has health funding to preventive or promotive investments increased?
What is public health? How is public health relevant to components of the human services delivery system? Why do public health unit costs differ across jurisdictions?
Can we describe screening in clinical settings eg GP surgeries for pap testing?
What did we invest in social marketing last year?
Can we replicate the output of other models? (eg Public Health Expenditure Reporting, public health component of OECD Health Accounts)
The top-level public health classes listed for examination, some of which have been examined in more detail to date, are: Selected views of the classification including a practical example from public health expenditure reporting, and another from the UK, follow.
showing the main public health classes captured in the classification
(public health activities derived from Public Health Expenditure Reporting and input from the Reference Group)
Related recent developments in the UK include the development of a Public Health Information Tagging Standard -to provide website access to public health resources -and a National Public Health Language, incorporating other thesauri and vocabularies to improve web-based searching and retrieval for public health resources.
A web-based system for the classification and retrieval of public health resources was conceived by Julian Flowers of the Eastern Region Public Health Observatory (ERPHO) in the UK, as there was no system specifically suitable for this purpose. The Public Health Information Tagging Standard (PHITS) was borrowed categories from a number of extant sources, 75 and took contributions from public health specialists nationwide(see Figure 10 ).
Source: Eastern Region Public Health Observatory www.erpho.org.uk accessed November 2004. Figure 10 shows PHITS describing public health resources on the website of the ERPHO. Subjects or classes of interest can be selected from the 'Browse by subject' box on the left side of the underlying screen print. The overlying screen print shows the 'Services' class and its finer subclasses (e.g. 'Population based and preventive', 'Primary care'). The tabbed entries to the right show the types of resources available (e.g. all resources, data), and provides typical information on individual resources (e.g. 'A rapid mapping study of smoking 75 Sources included ICD10, MeSH, and SNOMED.
projects', an 'ABC of smoking cessation'), including the URL of the resource for instant access.
After its introduction on the ERPHO website, PHITS was adopted as a standard for use by all ten Public Health Observatories in England and Wales, as well as other public health organisations, such as Public Health Ireland. 76 Initially intended purely as a web site categorisation and retrieval system, PHITS has now become part of the development of a National Public Health Language for the UK.
A National Public Health Language for the UK PHITS has been integrated with the UK Health Development Authority's Public Health Information Thesaurus 77 and two other controlled vocabularies, to create the National Public Health Language (NPHL) for the UK (Figure 11) . The development of a common public health language is intended to facilitate interoperability and improve the efficiency of searching for and retrieving, public health information and resources held on websites and in databases. All organisations that were already using PHITS have agreed to move to the NPHL when version one was available (December 2004 78 ). NPHL users will have both a public health biased classification system; and a powerful, thesaurus-driven, categorisation and searching mechanism for use on web sites. 79 Figure 11 shows the entry website for online access to the NPHL (left side) and top-level classes and their definitions (right side).
UK National Public Health Language (NPHL) NPHL top-level classes initial ontology, in an iterative design process that continues through the whole of the ontology's lifecycle. 84 This iterative development style is a good fit for complex or wicked problems. Because public health is complex it is technically conceptualised as a 'wicked problem' 85 , meaning that there is no definitive formulation or solution, no 'right' or 'wrong', no absolute truth or perfect solution that holds for all cases-the best that can be achieved is a consensus of public health experts that it is good enough.
In consultations it was clear that the conceptualisation of public health is time-specific (e.g. the 'old' and the 'new' public health), includes many contested definitions and terms, as well as fuzzy borders and boundaries. There is not even agreement on what it should be called, with the terms 'population health' and 'preventive health' currently challenging 'public health'.
Two principles of development (see Section 3.1.5) address these difficulties: be inclusive; and, set rules and boundaries in applications, rather than in the development of the classification itself.
Inclusiveness is a response to the divergence of views and definitions encountered in field consultations. The project took the position that a public health classification should not exclude elements that some (but not all) consider to be an important part of public health. It should actively seek to include divergent views since its usefulness as a unified classification depends on the best coverage of the breadth of public health.
Rules and boundaries can and should be determined in practical applications rather than in the ontology. For instance, for the purposes of reporting health and public health expenditure, it may be determined that all one-to-one treatment services in clinical settings are not public health services. Another use might determine that some one-to-one clinical treatments, such as those for immunisations, sexually transmitted infections, or drug detoxification, are public health services. The decision to set a constraint or boundary for a particular application should not preclude the wider scope of a 'public health classification', which is developed as an ontology.
A single ontology can be used to develop one or more classification systems, by developing specific rules and boundaries (developed as 'constraints' in the ontology) to organise classes into a hierarchy, and to assign elements to unique classes.
Although defining and specifying classes (concepts within the domain of interest) is central to developing an ontology, the emphasis is on modeling the relationships among classes, rather than on hierarchy (broader classes contain the more specific) or mutual exclusion (an element cannot be in more than one class).
An ontology allows elements to be assigned to more than one class. This is useful, for instance, for areas (of which there are many in public health) on which there is little agreement and competing views. In a classification system, with its emphasis on mutually discrete classes, it is not so easy to do this. 84 Noy & McGuinness 2001: 4. 85 The wicked problem concept in design was described by Rittel & Webber (1984) in the context of social planning. They pointed out that in solving a wicked problem, the solution of one aspect may reveal another, more complex problem. Ten rules define the form, including that there is no definitive formulation of a wicked problem (no stopping). Solutions to wicked problems are not therefore true-or-false, but good-or-bad.
A concrete example is the categorisation of behavioural factors. Most public health experts would agree that as a determinant of health these contribute to health risk and/or protection; however some see behavioural factors as exclusively personal, while others see them as exclusively socio-economic, and some see them as both. Using an ontology, they can be classed under both categories, so that those who expect to find them under personal factors will do so, as will those who expect to find them under socioeconomic factors, as illustrated in Figure 12 . Thus all are satisfied (have found the category where they expected to), a practical result has been achieved, and an indecisive argument about where it is 'rightly' to be found has been avoided.
Sophisticated software tools are available to assist in developing ontologies. These allow multiple inheritance (as described above), definition of relationships among classes, specifications of attributes of classes, and classification of elements (instances). Aspects of public health (characteristics, attributes, etc) can be described either textually as descriptions, mathematically as values, or in terms of other classes in the class hierarchy, and can be constrained by specific rules.
Ontology development software is the backbone of the next generation of information tools. Increasingly, existing classification systems are being migrated to, or developed in, ontology building software such as Protégé 86 (used by this project). This software makes it easy to render form and content for the Web.
As the Semantic Web develops and ontologies become more widely used in Web-based applications, the development of the public health classification in an ontology can be expected to produce major productivity gains in making existing information more available and better connected. 86 More information on Protégé, and the free, open source Protégé software, are available from Stanford University at http://protege.stanford.edu/index.html.
The 'isa' relation arrows show the parent class or classes that each child class belongs to in the class hierarchy of the ontology. Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection. Influenza A virus (IAV) remains a scourge on human health [1, 2, 3] . Its antigen drifts and shifts are an ever-changing challenge for available vaccines [4, 5] . The appearance of drug resistance is the main hurdle for the development of antiviral drugs [6, 7, 8, 9] . Given the limitations of current anti-influenza A virus strategies, the need for novel strategies for prevention and treatment of IAV is evident [10] . In this regard, RNA interfering (RNAi) technology holds great promise to inhibit the replication of IAV, including H5N1 virus.
RNAi is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA) [11, 12] . In this process, the cellular complex Dicer cleaves a double-stranded RNA (dsRNA) molecule to yield doublestranded duplexes 21-25 nucleotides in length. These siRNAs then guide the RNAi induced silencing complex (RISC) to cleave target mRNAs that share sequence identity with the siRNA [13, 14, 15] . Since it was first demonstrated that adding exogenous, synthetic siRNA molecules to mammalian cells can induce RNAi, there have been rapidly expanding efforts to develop RNAi therapies that induce the degradation of target messenger RNA (mRNA) involved in genetically inherited diseases or acquired disorders [16, 17, 18, 19, 20, 21, 22] .
IAV is an enveloped, negative-stranded RNA virus. The unique property of single-stranded RNA virus itself makes RNAi an attractive approach for development of anti-avian influenza therapeutics. The single-stranded viral genome, consisting of 8 segments contained at least 10 open reading frames (ORFs), serves as template for both viral genome replication and subgenomic mRNA synthesis. It has been reported that siRNAs respectively targeting to the viral genes of polymerase 1 (PB1), polymerase 2 (PB2), polymerase A (PA), nucleocapsid protein (NP), nonstructure proteins (NS1 and NS2), matrix proteins (M1 and M2), especially those specific for NP, PA and PB1, can potently inhibit replication of influenza A viruses [16, 23, 24, 25, 26] . However, it has been reported that HIV and HCV may develop siRNAresistant mutations quickly [17, 27, 28] , and therefore abrogated the further RNAi treatment. Thus, the evaluation of long term inhibition efficiency of designed siRNAs and screening of the emergence of siRNA resistance mutants are also an important research target.
In the present study, we identified an effective siRNA targeting M2 gene (siM2), a highly conserved gene in IAV, as compared to a reported effective siRNA targeting NP gene (siNP). We further established cell lines which stably expressing the shRNAs by transducing lentiviral-shRNA vectors to Madin-Darby cannie kidney (MDCK) cells. Using these two cell lines, we evaluated long term antiviral effects of these siRNAs against IAV subtypes H1N1 and H5N1 and further screened the potential siRNA-resistant viral mutations. Our results showed that rationally designed siM2 conferred long term effective inhibition for IAV replication. It was further demonstrated that no siRNA-resistant viral mutation appeared in siM2 targeting sequence even after the virus was cultured in the shRNA expressing stable cell line for 40 passages.
Two siRNAs targeting the M2 gene were rationally designed by siRNA target designer (the sequences of siRNAs are shown in the supporting information Table S1 ) and their effect in inhibiting the virus replication was assessed in MDCK cells. Two siRNAs targeting the NP gene were included in the experiments as controls. The results showed the siRNA M-950 exhibited a good inhibition effect with dose dependent manner, while another siRNA M-126 just slightly inhibited virus replication even at a concentration of 100 nM (Fig. 1A) . Fig. 1B showed that the siRNA NP-1496 could inhibit influenza virus replication, while siRNA NP-336 had no inhibition effect, which is consistent with the previous report [25] .
The siM2 Exhibited Higher Inhibitory Effect of H1N1 Virus than siNP in Stable Cell Lines Based on the above results, the lentiviruses expressing the shRNAs M2-950 or NP-1496 were constructed and transduced into MDCK cells to establish two stable cell lines, shM2-MDCK and shNP-MDCK. MDCK cells and the MDCK cells transduced by blank lentivirus (Mock MDCK) were used as controls. The cell lines were infected with H1N1 virus at a moi of 0.005 and culture supernatants were harvested at indicated time-points to determine the virus titer by plaque assay. As shown in Fig. 2 , virus replication kinetics of Mock MDCK is similar with that of MDCK, indicating that lentivirus integration didn't influence virus replication. Virus titers in shNP-and shM2-MDCK cell cultures were 2 to 10 folds lower than the controls MDCK and Mock MDCK cultures, suggesting that virus replication had been suppressed by the expressed shRNAs in both shM2-MDCK and shNP-MDCK cells. Notably, siM2 exhibited a better inhibition effect, showing about 2-fold lower viral titer than siNP, although the expression levels of siM2 and siNP were similar (DCt siM2 = 6.68, siNP = 6.95).
The siM2 Abolished not only M2 mRNA but also siNP mRNA Accumulation in the Stable Cell Lines
We also measured the accumulation of mRNA for NP and M2 gene in infected MDCK, Mock MDCK, shM2-MDCK and shNP-MDCK cells. The mRNAs were extracted from the cells harvested at 1, 2, 4 and 24 hrs post-infection and tested by realtime RT-PCR. The mRNA expression level is normalized by copy 
To further confirm whether the suppression of NP mRNA in shM2-MDCK cells indeed affect NP protein expression, the NP protein level was tested by an indirect immunofluorescence assay. As shown in Fig. 4 , EGFP fluorescence, an indicator of shRNA expression, was detected in Mock MDCK, shNP-MDCK and shM2-MDCK but not in MDCK cells, while NP protein was detected in MDCK and Mock MDCK cells but not in shNP-MDCK and shM2-MDCK cells. The results were consistent with above viral mRNA results, indicating that siM2 indeed suppressed the NP protein expression.
siM2 Provided More Potent anti-H5N1 Viral Effect than siNP in Stable Cell Lines
We further tested whether siM2 could also inhibit the replication of a highly pathogenic H5N1 avian influenza virus. As shown in Fig. 5A , although numbers of plaques were similar in different MDCK cell lines, smaller size of plaques were only found in shM2-MDCK cells, suggesting that siM2 inhibited replication of H5N1 virus. The cell lines were also infected with different amounts of H5N1 virus and culture supernatants were collected at different time points to determine the virus titers by HA assay. The virus replication was significantly inhibited in shM2-MDCK cells at all time-points, but shNP-MDCK just offered a minor inhibition effect at early stage of the virus infection (Fig. 5B ). These results further confirmed that siM2 could provide a more potent protection than siNP against H5N1 infection.
To test if siM2 siRNA-resistant virus mutant would quickly appeared when cultured in shM2-MDCK cells, H5N1 virus was continually cultured in shM2-MDCK cells for 40 passages. Every 10 passages, the culture supernatant was collected and tested by plaque reduction assay. No obvious larger size of plaque was found. Ten plaques with relative larger size were picked to further identify potential mutation in the siRNA targeting region by sequencing. The results showed that no mutation appeared in the siM2 targeting region even after 40 passages of the cultures (Fig. 6 ).
The principal finding of this study is that rationally designed siRNA targeting influenza M2 gene (M-950) conferred effective long term inhibition against influenza A virus replication. Such high suppressive effect is not only against H1N1 influenza A virus but also against a highly pathogenic H5N1 subtype. In the previous related studies, Ge and his co-workers [25] screened siRNAs targeting to 6 conserved genes of influenza A virus and showed that NP-1496 was the best since it can confer a more than 200-folds inhibition of H1N1 virus. Li et al [29] and Tomkines et al [23] further confirmed that NP-1496 provided high anti-H5N1 effect. We therefore included NP-1496 as a positive control in this study. Our results showed that siRNA M-950 exhibited similar (Fig. 1 ) or even slight higher (Fig. 2) inhibitory effect against IAV replication as compared to that of NP-1496. A recent report by Zhou et al [30] also showed that several siRNAs targeting NP and M genes exhibited effective inhibition against influenza A virus replication in cultured MDCK cells and in animal models. However, sequences of their reported siRNAs targeting M2 gene are completely different from the siRNA M2-950. Furthermore, chemically synthesized siRNAs or plasmid based shRNAs were always delivered by transfection in previous related studies, whereas we used a lentivirus system to deliver selected shRNAs. Although the integration property of lentivirus has abrogated it to be used in human, it is helpful for our study purpose to successfully establish stable cell lines persistently expressing siRNAs.
In this study we found that siM2 not only decreased the level of M2 mRNA but also the level of NP mRNA, suggesting that siM2 has a broad inhibition manner in the process of influenza virus replication. Ge et al have reported a similar broad inhibition of siRNAs [25] . In their study, NP-1496 and PA-2087 provided a broad inhibition to H1N1 influenza virus, which not only abolished the accumulations of specific NP or PA mRNAs but also inhibited the accumulations of mRNAs for M, NS1, PB1, PB2 and PA or NP genes. A possible explanation is that some double stranded siRNAs may result in IFN responses or activate a RNA degradation pathway, e.g. Phosphorylated protein Kinase R (PKR) [9, 31, 32] . However, the mechanisms of this broad inhibition of some siRNAs are still not very clear yet. From the standpoint of viral target choice in RNAi based antiviral therapy, NP protein is required for elongation and antitermination of nascent cRNA and vRNA transcripts [33, 34] . Without newly synthesized NP, further viral transcription and replication are blocked. While, M2 plays a critical role in the assembly of infectious virus particles. Thus, the potent antiviral effect of siM2 may be attributed to its broad inhibitory effect.
Depending on the stringency of siRNA-target base pairing, siRNA treatment may cause selection of siRNA-resistant viruses, and this has been shown with HIV and HCV [17, 27, 28] , and therefore abrogated the further medication or treatments. Using lentiviral delivery system, we established stable cell lines persistently expressing shRNA, which provided a more convenient experimental approach to study long term inhibition effect of siRNAs and screen for siRNA resistant virus mutants in quasispecies in vitro. Our results showed that H5N1 virus cultured in shM2-MDCK were equally susceptible to siM2 as the original virus even after 40 passages. Moreover, sequencing of siM2 targeted region in 10 such independent plaque purified virus isolates revealed sequence identical to the parental one. The current data have shown no insertion, deletion and nucleotide substitution in the siRNA target sequence, therefore demonstrated siM2 possessed good long term inhibition effect for influenza virus replication without the problem of siRNA resistant mutants.
Taken together, all the findings about effective RNAi target, lentiviral vector delivery and the establishment of stable shRNA expressing cell lines in our study provide rational information for the development of siRNAs as prophylaxis and therapy for influenza virus infection in humans.
MDCK and Human embryonic kidney 293T cells were respectively maintained in MEM and DMEM (Invitrogene, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U penicillin G/mL and 100 ug streptomycin/mL). Influenza virus strains A/New Caledonia/ 20/1999 (H1N1) and A/Hong Kong/486/97 (H5N1) used in these experiments were prepared in MDCK cells and virus titers were determined by TCID 50 . All experiments with H5N1 virus were performed in BSL-3 laboratory.
The siRNAs targeting M or NP gene of influenza A virus were designed by siRNA target designer version 1.51 from Promega (http://www.promega.com/siRNADesigner/program/). The duplexes of designed and previously reported siRNAs were synthesized by Invitrogene (USA) (the sequences were shown in the supporting information Table S1 ). The siRNAs were reverse transfected to MDCK cells using Lipofectamine TM RNAiMAX (Invitrogene, USA) as described in company's instruction. After incubated the cells for 16,18 hrs, the cells were infected with the viruses and followed by detection of viral replication. 24 hours after infection, RNA were extracted from the cells and followed by real time RT-PCR to detect the relative quantities of replicated viral RNA.
The H1-promoter-driven shRNA cassettes were constructed by annealing two primers containing the 19-nt sense and reverse complementary targeting sequences with a 9-nucleotide loop -TTCAAGAGA-and flanking Mlu1 and Cla1 cloning sites (the sequences of shRNA were shown in the supporting information Table S1 ), and then cloned into the 39-end of the H1 promoter in the LVTHM plasmid [35, 36] . The sequences of the insertions were confirmed by DNA sequencing.
Lentiviral vectors with shRNA expression cassette were produced by calcium phosphate-mediated, three-plasmid transfection of 293T cells [37] . Briefly, 293T cells (2.5610 6 
MDCK and the stable shRNA expressing cell lines in 24-well plates were infected with viruses at moi of 0.005,0.5 (2 mg/mL trypsin was used in the infection process of H1N1). After incubation for 1 hr, the infected medium was removed and MEM without FBS was added. Cell supernatants were collected at different time points. The viral load was detected by hemagglutination (HA) and/or plaque assays as described previously [39] . Briefly, the HA assay was carried out in U-bottom 96 well plates. Serial 2-fold dilutions of virus samples were mixed with an equal volume of a 0.5% suspension of turkey erythrocytes (Lampire Biologic Laboratories, Pipersville, USA) and incubated at room temperature (RT) for 45 mins. Wells containing an adherent, homogeneous layer of erythrocytes were scored as positive. For plaque assay, serial 10-fold dilutions of virus sample were added into a monolayer of MDCK cells. After 1 hr incubation, the virus was removed and the cultures were overlaid with 1% semi solid agar-MEM. Three days after infection, plaques were visualized by staining of crystal violent.
Real-time RT-PCR was carried out as described previously [39] . Briefly, H1N1 or H5N1 virus infected MDCK, Mock MDCK, shNP-MDCK and shM2-MDCK were harvest at 1, 2, 4 and 24 hr after infection. Total RNA was extracted from the infected cell samples using RNeasy RNA isolation Kit (Qiagen, Germany) and reverse transcribed using Superscript II Reverse Transcriptase and Oligo dT primer (Invitrogene, USA), according to the manufacturer's protocol. Viral mRNA copies were measured by SYBR green M63000 Real-Time PCR System 
Indirect immunofluorescence assay was performed as described previously [40, 41] with some modification. MDCK, Mock MDCK, shNP-MDCK and shM2-MDCK cells grew on micro cover glasses (Thomas, USA) were infected with 1 moi of H1N1 virus for 6 hrs, After washed with PBS, the cells were fixed in 4% paraformaldehyde for 15 mins at RT and then permeabilized in 0.1% Triton X-100 for 3 mins at RT. After washed with PBS again, the cells were incubated with 1:50 diluted mouse anti-NP antibody (Abcam, UK) for 30 mins in dark at RT. The cells were washed three times in PBS with 1% FCS and incubated with 1:500 diluted Texas red-conjugated anti-mouse lgG (Abcam, UK) for 30 mins in the dark at RT. The cells were washed and mounted. Slides were viewed under an Olympus fluorescence microscope (Olympus, Germany).
The screening of potential siRNA resistant mutants were performed in our established stable shRNA-expressing cell lines according to previously described protocols [42] with some modification. Briefly, the shM2-MDCK cells in a T25 cm 2 flask were infected with H5N1 virus. After cultured for 2 days, the supernatants were harvested. Part of the supernatants was inoculated to shM2-MDCK for next passage of the virus culture, another part was subjected for plaque assay to determine if potential siRNA-resistant virus appeared. Every 10 passages, ten bigger size of plaques in the plaque assay were picked for sequencing to detect any mutation in the siRNA targeting region using a pair of primers: forward, 59-AAG GCA GAT GGT GCA GGC AAT-39 and reverse, 59-TAC TCC AGC TCT ATG CTG ACA-39.
Table S1
Found at: doi:10.1371/journal.pone.0005671.s001 (0.03 MB DOC) Increased expression levels of the pvcrt-o and pvmdr1 genes in a patient with severe Plasmodium vivax malaria BACKGROUND: There are increasing reports of severe clinical cases exclusively associated with Plasmodium vivax infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance. PATIENTS: Two different patients presented at the Hospital Clinic in Barcelona with P. vivax malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000. METHODS: To exclude the possibility that the patient's severe symptoms were due to Plasmodium falciparum, a nested PCR was performed. A magnetic method was used to purify P. vivax free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in P. vivax, namely the P. vivax chloroquine resistance transporter, pvcrt-o, and the P. vivax multidrug resistance transporter, pvmdr 1. RESULTS: Results demonstrated that the severe clinical symptoms were exclusively due to P. vivax. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in pvmdr1 levels and up to 21.9-fold increase in pvcrt-o levels compared to expression levels of parasites from the other patients with mild symptoms. CONCLUSION: This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of pvmdr1 and particularly pvcrt-o expression levels as molecular markers of severe disease in P. vivax. The renewed momentum for global malaria eradication has highlighted the need to further studies on Plasmodium vivax if eradication is to be achieved. Although the exact burden of disease is still a matter of debate, it is likely that it has been underestimated and that P. vivax is responsible for between 100 and 300 million clinical infections each year [1] . The emergence of worsening clinical severity and chloroquine resistance are two major factors responsible for this increasing burden.
Plasmodium vivax infections have been associated with mild symptoms such as fever, headache, fatigue, chills, and musculoskeletal pain, in particular paroxysms. Recently, however, severe complications, including renal failure, jaundice, acute respiratory distress syndrome, cerebral malaria, seizures, anaemia, hyperparasitaemia, thrombocytopenia, pulmonary edema, splenic rupture and death, have been reported in exclusive association with P. vivax [2, 3] .
Chloroquine resistance (CQR) is a major determinant of the present resurgence of malaria worldwide, including that of P. vivax [4] . Two main transporters have been studied in regard to CQR in P. vivax: the P. vivax chloroquine resistance transporter, Pvcrt-o, and the P. vivax multidrug resistance transporter, Pvmdr1 [5] [6] [7] . Interestingly, amino acid polymorphisms have not been associated with chloroquine resistance in pvcrt-o whereas pvmdr1 polymorphisms have been recently suggested to be associated with CQR in Southeast Asia [8] . This data indicates the involvement of other mechanisms in CQR in P. vivax. Likely candidates are gene amplifications and differential expression levels [9, 10] .
The European Network on Imported Infectious Disease Surveillance, TropNetEurop, is an electronic network of clinical sites that monitors imported infectious diseases in Europe http://www.tropnet.net. Since its foundation in 1999, the network has recorded 8,374 cases of malaria, of which close to 11% (930) were due to P. vivax. Worth mentioning, TropNetEurop covers approximately 10% of all malaria cases reported in Europe. Moreover, according to data from the Spanish National Center of Epidemiology and the Autonomous Government of Catalonia there have been 266 cases of P. vivax in Spain in the last six years. In addition, three cases of severe symptoms due to P. vivax in Europe have been reported elsewhere in the literature [11] [12] [13] . These figures illustrate the clinical-epidemiological importance of this parasitic disease in a supposedly malaria-free region.
Recently, CQR in P. vivax has been suggested to be associated, albeit not directly linked, with severe vivax malaria [14] . Here, the first clinical case of severe vivax malaria in Spain is presented. The data also indicates that clinical severity could be associated with increased expression levels of two parasite genes likely involved in chloroquine resistance, pvmdr1 and pvcrt-o.
Two patients presented at Hospital Clinic in Barcelona with P. vivax malaria episodes. One had severe symptoms and the other mild symptoms. The patients had travelled through the Brazilian Amazon (Manaus) for 31 and 19 days, respectively, in 2007. The patient with severe symptoms was a 30-year-old Spaniard man who had previously travelled to Kenya, in 2006. Upon his return from Brazil, he presented to Hospital Clinic in Barcelona with high fever (39°C) and a Giemsa-stained thin blood film confirmed the presence of P. vivax at a parasitaemia of 1.8%. The patient presented acute respiratory conditions, anaemia and hyperbilirubinaemia, requiring admission to the intensive care unit. The patient with mild symptoms was a 31-year-old Spaniard man who had travelled without chemoprophylaxis and who had previously visited Mexico (2006), Vietnam (2005), and India (2004). There were no records of fever episodes from these previous trips and neither of the patients had ever been diagnosed with malaria.
Two other Brazilian patients with mild symptoms presented at the "Centro de Pesquisa em Medicina Tropical, Rondônia, Brazil in 2000. Total RNA from parasites of these patients was extracted, pooled and stored in liquid nitrogen.
Five mL of infected red blood cells were obtained from each patient. One mL was used to purify genomic DNA following standard methodologies. The remaining blood was processed to isolate total RNA using the Trizol reagent (Invitrogen) according to the manufacturers' instructions. A recently described magnetic method for the isolation of matures stages of malaria parasites was used to concentrate and purify P. vivax-infected reticulocytes [15] . Giemsa-stained smears showed an absence of human leukocytes, and all the reticulocytes were infected with mature stages of the parasite (Additional file 1). Eluents were centrifuged at 800 × g for 10 minutes, supernatants discarded, and pellets used to purify total RNA. The protocol for this study was approved by the Ethical Committee of Hospital Clinic and informed consent obtained from the patients.
Nested polymerase chain reaction (PCR) was performed as previously described [16] to exclude P. falciparum infec-tions. Fragments were resolved and visualized on 2% agarose gels stained with sybr green.
Amplification reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and 45 ng of template cDNA prepared from each sample. Samples were set up in duplicate and experiments were repeated independently twice. PCR products were amplified and detected on an ABI Prism 7700 (Applied Biosystems). Cycling parameters for PCR were an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. To analyse the relative transcript levels, the threshold cycle value (Ct) of each sample was used to calculate and compare the ΔCt of each sample to that of the P. vivax housekeeping gene Sal I β-tubulin; the ΔΔCt was also calculated as in [17] to compare the transcript levels of pvcrt-o and pvmdr 1 in the patient with severe symptoms and in the patient with mild symptoms.
During the month of August, 2007, a 30-year-old Spanish tourist traveled through the Brazilian Amazon region of Manaus, where, due to gastric disturbances, he took an incomplete chemoprophylaxis consisting of proguanil and chloroquine. Upon his return to Spain 30 days later, he presented to a health center with an eight-day history of fever, chills and dry cough. He was diagnosed with lower respiratory tract infection and treated with amoxicillin-clavulanic acid 875/125 mg every 8 hours for 24 hours (3 doses) without resolution. On presenting a week later to Hospital Clinic in Barcelona, he had high fever (39°C) and jaundice; the tip of the spleen was palpable and chest auscultation was unremarkable. A thin blood smear of peripheral blood stained with Giemsa revealed P. vivax infection with a parasitaemia of 1.8%. The patient presented acute respiratory conditions requiring admission to the intensive care unit (ICU).
In the ICU, he was haemodynamically stable and blood tests revealed pancytopaenia (haemoglobin, 10 g/L; haematocrit, 29%; platelets 25 × 10 9 /L, leukocytes, 6.7 × 10 9 / L); hyperbilirubinaemia (8.3 g/dL); γ-glutamyltransferase, 146 U/L; alkaline phosphatase, 241 U/L; and prothrombin time, 76 seconds. Renal function tests were within normal limits. The C-reactive protein level was 16 mg/dL. Arterial blood gas measurement while breathing air revealed marked hypoxia (PaO2, 63 mm Hg), normocapnia (PaCO2, 32 mm Hg), low oxygen saturation (93.7%), and a blood pH of 7.49. A chest radiograph showed bilateral interstitial infiltrates and a computed tomography (CT) scan showed right midzone alveolar shadowing without parenchymal infiltrations ( Figures 1A  and 1B) .
To exclude the possibility that the patient's severe symptoms were due to P. falciparum, which is sympatric with P. vivax in Brazil, a nested PCR using P. falciparum-and P. vivax-specific primers, was performed [16] . The results demonstrated that coinfection with P. falciparum could be excluded (Figure 2A) . Moreover, sputum, blood, and nasopharyngeal swab samples obtained for culture were negative, as were serological tests for atypical respiratory pathogens, human immunodeficiency virus, histoplasma, coccidians, and paracoccidians. Together, these results demonstrated that the clinical symptoms were exclusively due to P. vivax infection.
X-ray and computed tomography (CT) scans of patient with severe Plasmodium vivax malaria Due to the severe condition of the patient, a five-day treatment was initiated, starting with an intravenous loading dose of quinine (1,200 mg) on the first day, followed by oral quinine (600 mg) every 8 hours plus oral doxycycline (100 mg) every 12 hours. The treatment was well-tolerated and parasitaemia became negative within three days. Platelet, erythrocyte and leukocyte levels were all within normal ranges in the following controls. Normal blood levels of glucose-6-phosphate dehydrogenase activity were detected and oral primaquine (30 mg/day) was commenced for two weeks to prevent a relapse. The patient recovered well and the chest CT findings were normal at two months ( Figure 1C ). There have been no recurrences during the follow-up period.
In view of the recently suggested association of severe disease in P. vivax with multidrug resistance [14] , expression levels of two genes suspected in having a pivotal role in mediating clinical resistance to chloroquine, pvcrt-o and pvmdr1, were determined. Significantly, the patient with severe symptoms had a 1.6-fold increase in pvmdr1 levels and a 21.9-fold increase in pvcrt-o levels compared to the other patient who presented to the same hospital with P. vivax infection and mild symptoms (Figure 2b ). This finding was validated by analysing parasite material obtained previously from two other patients from the Brazilian Amazon who also had mild symptoms (Additional file 2).
There are increasing reports of severe clinical cases exclusively associated with P. vivax infections, involving severe anaemia, renal failure, jaundice, cerebral malaria, seizures, respiratory failure, multi-organ failure, and death [2, 3] ; all these complications are generally believed to be exclusively associated with severe forms of falciparum malaria. Although these same clinical severity criteria are being used for P. vivax, it would be desirable to conduct prospective studies to establish a precise definition of clinical severity in P. vivax.
Expression levels of chloroquine resistance genes in severe and mild Plasmodium vivax malaria Figure 2 Expression levels of chloroquine resistance genes in severe and mild Plasmodium vivax malaria. In panel A, nested PCR to identify Plasmodium species. Amplification of gDNA from P. falciparum 3D7 strain and P. vivax from severe patient, respectively, using specific P. vivax primers (lanes 1 and 2) . Amplification of gDNA from P. falciparum 3D7 strain and P. vivax from severe patient, respectively, using specific P. falciparum primers (lanes 3 and 4) . Molecular weight ladder (lane L). A positive reaction is noted when primers for P. falciparum and P. vivax produce amplification products of 205-bp and 120-bp, respectively [16] . Molecular weight in base-pairs (bp). In panel B, relative quantification of pvcrt-o and pvmdr1 transcripts in total RNA obtained from parasites from the severe patient vs total RNA obtained from parasites from a patient from Brazil with P. vivax and non-severe symptoms also presenting to our hospital. The following oligonucleotide primers were designed for the realtime experiments using the Primer Express program (Applied Biosystems). Primers: F-pvcrt-oRT 5'-ATGTCCAAGATGT-GCGACGAT-3';R-pvcrt-oRT 5'-CTGGTCCCTGTATGCAACTGAC-3'; F-pvmdr1RT 5'-AAGGATCAAAGGCAACCCA-3'; R-pvmdr1RT5'-TCAGGTTGTTACTGCTGTTGCTATT-3'; F-pvtubulinRT 5' CCAAGAATATGATGTGTGCAAGTG 3'; R-pvtu-bulinRT 5' GGCGCAGGCGGTTAGG 3'.
The patient with severe symptoms had acute lung injury according to the definition of the American-European Consensus Conference on ARDS [18] , with acute onset, bilateral changes in chest radiography, a PaO2/FiO2 ratio of ≤ 300, and absence of clinical left ventricular failure. Importantly, these respiratory complications appeared before initiation of anti-malarial drug treatment, an observation also reported in two other severe clinical cases of lung injury due to P. vivax [19, 20] . Two main hypotheses have been proposed to explain lung damage in P. vivax malaria infections. The first suggests that there is no sequestration of P. vivax-infected reticulocytes in the deep capillaries of internal organs, postulating instead an inflammatory process due to an increase in capillary permeability associated with cytokine-induced damage in the pulmonary epithelium [21] . The second suggests cytoadherence of P. vivax-infected reticulocytes in lung capillaries, causing obstruction of blood flow and reduction of respiratory function before treatment and alveolar capillary damage and inflammation 24 to 48 hours after initiation of anti-malarial drug treatment [22] . The possibility that antibiotics given 24 hours before respiratory failure might have destroyed P. vivax-infected reticulocytes, inducing lung damage and favouring the second hypothesis, cannot formally rule out. Yet, it is clear that the lack of sequestration in P. vivax needs to be re-evaluated, as cytoadherence has been hypothesized to occur in both the spleen [23, 24] and the lungs [22] .
Chloroquine is currently the first-line treatment for P. vivax, but resistance has been rapidly increasing since it was first described in two cases of treatment failures in Papua New Guinea [4] . The clinical case reported here originated in Manaus, Brazil, where P. vivax chloroquine resistance and severe disease are now being reported [25, 26] . Interestingly, after the appearance of chloroquine resistance in P. vivax, reports of clinical severity exclusively associated with this human malaria parasite started to appear [2] . Moreover, multidrug resistance has been recently suggested to be associated, albeit not directly linked, with severe disease in P. vivax [14] . Remarkably, higher expression levels of pvmdr1 and pvcrt-o, in particular, were found in parasites from the patient with severe clinical symptoms compared to three patients with mild symptoms. The levels were increased by up to 2.9 fold in the case of pvmdr1 (when including all samples) and up to 21.9 fold in the case of pvcrt-o. Confounding effects due to sample concentrations or contamination with P. falciparum or human material were excluded by using P. vivaxspecific primers and β-tubulin as an internal control and calibrator. This data thus indicates that severe vivax disease could be associated to molecular markers.
There are increasing reports of severe disease exclusively associated with vivax malaria and this study presents the first one in Spain. The use of PCR excluded incompetent microscopy, cryptic mixed infections or sequestered P. falciparum reinforcing the need of using PCR as a new diagnostic tool to avoid default diagnosis of severe malaria as due to P. falciparum. The finding on increased expression levels of parasite genes likely involved in chloroquine resistance supports to furthering explore the potential of pvmdr1 and particularly pvcrt-o as molecular markers of severe disease in P. vivax.
Publish with Bio Med Central and every scientist can read your work free of charge  Health workers' views on quality of prevention of mother-to-child transmission and postnatal care for HIV-infected women and their children BACKGROUND: Prevention of mother-to-child transmission has been considered as not a simple intervention but a comprehensive set of interventions requiring capable health workers. Viet Nam's extensive health care system reaches the village level, but still HIV-infected mothers and children have received inadequate health care services for prevention of mother-to-child transmission. We report here the health workers' perceptions on factors that lead to their failure to give good quality prevention of mother-to-child transmission services. METHODS: Semistructured interviews with 53 health workers and unstructured observations in nine health facilities in Hanoi were conducted. Selection of respondents was based on their function, position and experience in the development or implementation of prevention of mother-to-child transmission policies/programmes. RESULTS: Factors that lead to health workers' failure to give good quality services for prevention of mother-to-child transmission include their own fear of HIV infection; lack of knowledge on HIV and counselling skills; or high workloads and lack of staff; unavailability of HIV testing at commune level; shortage of antiretroviral drugs; and lack of operational guidelines. A negative attitude during counselling and provision of care, treating in a separate area and avoidance of providing service at all were seen by health workers as the result of fear of being infected, as well as distrust towards almost all HIV-infected patients because of the prevailing association with antisocial behaviours. Additionally, the fragmentation of the health care system into specialized vertical pillars, including a vertical programme for HIV/AIDS, is a major obstacle to providing a continuum of care. CONCLUSION: Many hospital staff were not being able to provide good care or were even unwilling to provide appropriate care for HIV-positive pregnant women The study suggests that the quality of prevention of mother-to-child transmission service could be enhanced by improving communication and other skills of health workers, providing them with greater support and enhancing their motivation. Reduction of workload would also be important. Development of a practical strategy is needed to strengthen and adapt the referral system to meet the needs of patients. Prevention of mother-to-child transmission (PMTCT) has been considered a simple intervention: just giving medication to prevent viral transmission from mother to child. Now, though, PMTCT is recognized as a comprehensive set of interventions requiring capable health workers. It starts with testing pregnant women for HIV, preferably during their first antenatal visit. When giving the test result, health care workers should provide good counselling, including information about PMTCT options.
The health system should ensure that HIV-positive women receive the PMTCT services that they choose and should provide postnatal care. All along the timeline from finding out their serostatus to getting treatment for HIVrelated problems, women and their children should be followed closely. The need for comprehensive and longterm care for HIV-infected women has become a challenge for health systems, particularly where lack of coordination among different facilities is common [1, 2] .
Viet Nam's HIV epidemic is still in a concentrated phase, with the highest seroprevalence among high-risk key populations, including injecting drug users (IDU), female sex workers (FSW) and men who have sex with men (MSM). Hanoi is one of 10 provinces/cities reported to have the highest number of HIV infections per 100 000 inhabitants. After the first case of AIDS was identified, in 1992, the HIV epidemic in Hanoi increased sharply by 1994. The capital has 12 628 persons living with HIV/AIDS (PLHIV), mostly IDU from poor families. Currently, there are 3623 AIDS patients and 2081 who had died of AIDS in the city. Although HIV is predominantly concentrated among IDU and FSW, it is gradually spreading among the general population. In 2007, HIV prevalence among pregnant women attending antenatal care (ANC) clinics in the Hanoi was 0.34% [3] .
Hanoi was selected as the study site because comprehensive PMTCT care is theoretically available there. Hanoi had 45 hospitals, 290 surgeries and five international hospitals. The national hospitals in Hanoi serve as referral centres for the northern half of Viet Nam. HIV testing and counselling for pregnant women are offered at health facilities at district or higher level, but often only after the 28 th week of gestation. HIV-positive women are referred to provincial or national hospitals for ARV prophylaxis, delivery and postnatal care. Hanoi health services have sufficient supplies of prophylactic ARV to meet the demand. Antiretroviral therapy for adults is available at district level or higher. HIV-exposed infants are offered polymerase chain reaction (PCR) testing and free infant feeding formula for at least six months, while free paediatric ARV is available for children three years of age or older.
The extensive health care system in Hanoi reaches the commune level, but multisectoral and cross-programme collaboration to link the pillars of the World Health Organization's (WHO) comprehensive approach to PMTCT are weak [4] . For example, there is little collaboration between the programmes for HIV/AIDS and family planning. Our previous work suggested that a large number of HIV-infected pregnant women remain undetected by the health system [5] . In addition, a number of barriers result in failure to access PMTCT during pregnancy and delivery [6] [7] [8] [9] [10] [11] . Among the weak points identified were that HIV-infected women received inadequate information about postnatal care, but even when they had knowledge, many expressed fear of stigma and discrimination that reduced their access to care; HIV testing is not available via health services at commune level, where many pregnant women go for care and delivery; and women feared lack of confidentiality of HIV test results [4, 12] .
Our previous studies on the experiences and views of women about the provision of PMTCT in Hanoi included criticisms about the quality of services provided by health workers [4] . Other studies in Asia found that health workers were unwilling to provide appropriate care for HIVpositive pregnant women, often because of their own fear or lack of knowledge, or because of high workloads and lack of staff [13, 14] . Inadequate health care delivery may be caused by a variety of factors, but we need to identify the main issues before planning interventions to strengthen it.
We report here the health workers' perceptions on the factors that lead to their failure to give good quality PMTCT. The findings should inform the development of a more effective programme for the fourth prong of the WHO-recommended comprehensive PMTCT programme.
Sampling of study sites was based on availability of services in Hanoi and their level and function in the health care system. All hospitals in Hanoi that provide ARV, PMTCT and opportunistic infections (OI) treatment were selected, including all health facilities providing HIV testing. Additionally, all health facilities in one high-prevalence and well-resourced district were selected. In that district, interviews were carried out at all levels of the health system involved in PMTCT: district health centre, district maternity ward, district committee for family planning and mother and child health, and preventive medicine centres, including HIV testing sites. The research team also visited commune health stations in the district. Details are presented in Table 1 .
Selection of respondents was based on their function, position and experience in the development or implementation of PMTCT policies/programmes. Interviewees were first screened to check that they had appropriate positions and at least one year of experience with PMTCT, so that they could provide insightful information. They included doctors, nurses, midwives, counsellors, laboratory technicians and programme managers. Detailed information on interviewees is presented in Table 2 .
We conducted semistructured interviews with these 53 health workers about their experience in implementation of services for PMTCT, their point of view about users of their services and their perception about the challenges they faced in providing good PMTCT services in their health facility.
In addition, unstructured observations were made in nine health facilities, in waiting rooms, counselling rooms, ANC examination wards, delivery wards, postnatal wards, outpatient and inpatient clinics for ARV and OI facilities, and laboratories.
The interviewers were four trained public health and social science researchers. Institutional ethical approval was obtained from the Scientific Committee of Hanoi Medical University and written informed consent was obtained from all interviewees, who were invited to participate voluntarily. The interviews were conducted privately and anonymously. A code book was developed focusing on key findings and terminologies. The transcripts of the semistructured interviews were coded, entered and analysed by means of N-VIVO software. 
Many hospital staff explained their reasons for not being able to provide good care; the most frequently heard was high workload. Observation confirmed that the national and provincial hospitals' ANC caseload was high: the wards were always crowded. One of the main obstetrics hospitals provides ANC to between 200 and 400 pregnant women daily. None of the health facilities offering PMTCT services had recruited additional staff to provide counselling. However, the workload in ANC facilities at district and commune level was not so high, according to the respondents. "There are only very few health staff with only basic information on counselling in this hospital. Poor knowledge and skill is common problem here." Counsellor, ANC provincial hospital Especially at district or lower level, knowledge is limited regarding ARV prophylaxis and on follow-up care, such as continuing replacement feeding (RF) supplies, infant testing and services for HIV-infected mothers and exposed infants. Consequently, health workers cannot provide adequate counselling on these issues before women are discharged.
"What I can do is to provide information about her HIV test result. I know that there is a medication to prevent transmission of HIV from mother to child, but I don't know exactly. Our common practice is to refer her to provincial hospital." Midwife, ANC commune level
The staff in most health facilities reported having had limited training on PMTCT in general and on counselling in particular; that they apply knowledge and skills gained from observing colleagues conducting counselling sessions; and that refresher courses are rare. The duration of the training varied from two days to two weeks. After the training, counselling is added to their regular ANC or maternity work.
"What we do now is only to inform.
[Counsellors] lack knowledge. If they will be trained, who will train them?" Manager, ANC provincial hospital "We counsel from our experience. To me, our counselling may be not complete. We don't even know what might be our shortcomings". Nurse, ANC commune level An important point of the comprehensive PMTCT approach is that HIV-infected women should be provided with several different services -such as ARV prophylaxis, formula, counselling and HIV testing for exposed children -provided by different facilities. However, there are no inter-or intra-hospital linkages to make the PMTCT comprehensive. For example, family planning services at a national obstetric hospital are not linked to other departments, including the infectious disease department that provides ANC and delivery services for HIV-infected women. Women are seldom referred to ARV sites for clinical staging or immunological assessment. Referral to postnatal care and social support for both mothers and children is not available at the hospital exit point.
"There is no linkage with obstetric hospitals; they never directly inform us. It is very difficult to know which HIV+ patient or child has been referred to this paediatric hospital for follow-up by what hospital. We don't treat the mothers here. We only provide counselling to them. Support groups? There are some but we don't know where they are." Doctor, paediatric ARV national hospital
Many health workers stated that their task is to provide services available in their own facilities but not services provided by other departments or facilities. Lack of medications was another reason given for not being able to provide services for HIV-infected women. In many health care facilities, the ARV was not consistently available. Often even single-dose Nevirapine (NVP) was lacking, for women who were tested only at the time of labour. Even in the two PMTCT sites in the city, shortage of ARV for adults was observed to happen every few weeks. One problem with the NVP for children was that it is provided as a large bottle (200 ml) of syrup. Once it has been opened, it cannot be kept for long, but very few HIVexposed children were identified each day. That means that each bottle was not fully used, and that later, drugs were lacking when supplies ran out.
"In some periods, there is a shortage of SD-NVP for PMTCT. We could not do anything in that situation. In practice, the NVP syrup for children is very inconvenient to use. On one day, we have no more than two children to treat; we have to open one bottle for them and the rest of the syrup is unused. The syrup quickly runs out, and then we don't have medication for another child." Manager, ANC provincial hospital "We also counsel them to use condoms. If someone asks me what they should do to avoid unwanted pregnancy then I tell them. But I do not have condoms to give them for free for family planning." Counsellor, ANC district level Another issue is that there are no national guidelines on counselling and testing. Observation showed that facilities at provincial and national levels had counselling and PMTCT guidelines and protocols developed by the projects that support those facilities, but most facilities at district or lower level do not have guidelines or even access to them.
Moreover, health workers at all levels often complained about the lack of attention to the needs of health workers when they have to work in a high-risk environment.
"Among 1,000 health workers, how many want to provide care and treatment for HIV-infected patients? There is no good compensation regimen to support staff working with HIV-infected patients. There is no benefit to save the life of patients in the late period, so how could we be enthusiastic?" Doctor, paediatric ARV national hospital "We receive extra pay for providing treatment for HIVinfected people. But it is just for one health staff while all [12] staff in my department provide service. We have to share among us." Doctor, adult ARV district hospital
Fear of infection Many respondents admitted that they were afraid of HIV transmission from patients, either because they feared being injured by the patients or through an occupational accident, because they lack protective equipment. Observation at adult and pediatric ARV sites supported this finding. Health workers confront their fear of infectionTo reduce both fear and risk of infection, health workers often find ways to protect themselves, either by trying to identify which patients might be infected with HIV, by using protective equipment, or by avoid exposure to patients as much as possible.
"It is easier for us to prevent transmission if we know who among the patients is infected with HIV." Midwife, ANC provincial hospital Observation revealed good practice of precaution when health workers assisted deliveries for HIV-positive women, but not always for HIV-negative women. All health workers said they knew how to protect themselves against occupational exposure to HIV and did so very carefully if they knew who was infected with HIV.
"Staff wear protective uniforms, maintain all hygiene practices and disinfection procedure on all equipment used." Nurse, adult ARV district level However, even if health workers want to protect themselves by using protective equipment, not all health facilities can provide these means for them.
"Health workers do not have enough protective clothes. We have to use cloth coats and short gloves when assisting deliveries for HIV-infected pregnant women. So we often wear a raincoat on top of the cloth coat." Nurse, ANC provincial hospital
Although hospital managers reported that occupational exposure is rare, among the study population we found five health workers who claimed to have had an exposure to blood that they thought might have put them at risk for HIV infection, either because of needle-sticks or blood that went to their eyes. They all informed us that ARV prophylaxis for prevention of occupational exposure is free of charge at an ARV site at district hospital. But only two of them took medication because the others turned out not to need prophylaxis after closer assessment of their exposure.
Prevention of HIV transmission became a good excuse for health workers to avoid taking care of HIV-infected women, or if they had to provide care, to isolate the HIVinfected women for easier control and management.
"We offer counselling, family planning, nutrition, delivery and care after delivery at home, vaccinations for tetanus. We offer this for normal pregnancies including those with hepatitis B. Women who have high risk with HIV are referred. It is not our business." Doctor, ANC site "Not everyone understands thoroughly about stigma and dread. The more they know, the more they fear and they try to push responsibility to others." Manager, PMTCT site
Many hospital managers emphasized that although the number of HIV-infected women attending their hospitals has been increasing, the number may still account for quite a small number of women in community.
Observation at all the health facilities revealed that HIVinfected women were often placed in a separate room or area. Even at a high-level hospital, where two or three patients often had to share one bed, there was still an empty bed in the room for HIV-infected patients. The manager in an ANC facility explained:
"HIV is an infectious disease, more severe than hepatitis B. Therefore patients should be controlled carefully to avoid transmission to other patients and staff."
Not only fear of HIV infection influenced the quality of care provided to HIV-positive women. Some of the weaknesses in providing service were related to the views of health workers regarding HIV-infected people, who are often seen as drug users or sex workers, or as having a "strange appearance". The real or expected behaviour of such people also induces fear and other negative emotional responses in the health workers.
"They often have tattoos and never dress well. There are spots on their arms. Easy to recognize their bluish black lips." Doctor, ANC national hospital "They inject in our department. How can we have a good attitude toward them?" Nurse, ANC national hospital
Health workers did admit that not all HIV-infected people behaved badly towards them. However, a few bad experiences could give all staff a negative attitude about HIVinfected people in general.
"Almost all HIV-infected patients cannot be trusted. For instance, when they know they have opportunity to have care and support, they often find ways to get as much as possible. Or if they want to leave the hospital, they often lie to the nurse that the doctor already agreed. Or when they have to pay the hospital fee, they often tell the lie that they will pay tomorrow but after that they disappear." Nurse, paediatric ARV national hospital "When you have to work with them [HIV-infected patients], you will see the difficulties. It's already hard to gain trust from normal patients. Now we have to serve the very scoundrel social class and at the same time, we receive very low salary. We have to provide service because it's our responsibility but we are not happy because they [HIV-infected patients] are drug users, they are very rude. My experience shows that health workers should not be too good to HIV patients." Doctor, ARV district level Some of these attitudes are based on real experiences, but many are also based on prejudicial expectations, and women wishing to access PMTCT services will be victims of that stigma, too.
Most of the health workers agreed that the quality of care could be improved by several interventions addressing both individual and structural issues.
Reducing workload and providing better compensation for working with risks were mentioned by almost all health workers at provincial and national level as important solutions. In the interviews, hospital managers suggested that it is very difficult to hire new staff because of the limited budget allocated from the government. A better solution would be to rearrange the services in a more logical structure, for example, to replace individual pretest counselling (which is often not offered anyway) by group counselling, to use peer counsellors to provide counselling and follow-up of care (reducing the burden for health workers and improving the quality of counselling) and to improve the quality of services at a lower level to reduce the burden on the higher levels.
"If we have more equipment, we can deal with our workload with even few staff. For instance, if we are provided a video, leaflets on HIV and PMTCT, and a room with table and chairs, we can do group counselling for pregnant women." Manager, ANC national hospital "I have seen in a hospital in Thailand that peer counsellors can work in hospital to help health workers doing some administration work, providing counselling, making appointments. We may need to think about how to apply their experience." Manager, ARV district level However, many doctors and nurses are still unconvinced about the involvement of peer counsellors in the health service, because they feel that peer counsellors do not have enough capacity and the appropriate attitude to do this work, or even bring the potential for crime and corruption into the hospital.
"You can find good peer counsellors in other countries but not in Viet Nam. They have low education. They may become drug dealers or whatever, we don't know." Doctor, paediatric ARV national hospital Another possible intervention is training and updating information for health workers. Midwives and nurses said they needed to improve their basic knowledge on HIV/ AIDS and PMTCT because they received less training than doctors, while doctors preferred to have advanced PMTCT training. All of them asked to be updated routinely on PMTCT.
"Although training has often been provided to doctors but not nurses, in fact training should be conducted more often for both of them." Doctor, paediatric ARV national hospital
Regarding the system, hospital managers admitted that much needs to be done to improve the quality of service -for example, improving ARV procurement, developing detailed PMTCT guidelines taking into consideration the staff's high workload, increasing availability of high technology equipment and providing sufficient protective equipment for health workers.
"Although ARV is sufficient for all HIV-infected women in Hanoi, in the provincial hospital, there are some periods when they lack medicines. The provincial hospital should make a plan on how much medicine they need. The procurement facility should make administrative procedure short and easy for hospitals to get the medicines." Manager, ANC national hospital "We have implemented PMTCT programme for more than five years without a PMTCT guideline to instruct us. I think a PMTCT guideline should be developed with involvement of all planners and implementers." Manager, ANC provincial hospital Improvement of the referral system is seen as an important task that needs to be addressed in the near future.
Hospital managers propose to have regular meetings of the health network that provides different services for HIV-infected people.
"When there are not a lot of patients, I think it's appropriate to divide tasks among different hospitals. But we also need to link all hospitals. In practice, the linkage is loose: that makes difficulties for patient access to services. It is due to lack of information about services provided by other places, lack of active coordination to link between different services, people are too busy to think about other services besides the treatment that we can provide. Some health workers are not aware of the need to refer patients, or if they do, patients don't want to go and disclose their positive status in other hospitals, or may not go even because of lack of referral forms." Manager, ARV district hospital Some health workers also proposed that all services should be offered at one point for better coordination.
"Now we have Global Fund project providing ARV at district health centre. Why don't we provide PMTCT and paediatric treatment at the same facility? I have also heard that there are some self-help groups working at district level that can provide further support for HIV-infected people. The Ministry of Health should think about how to make one facility able to provide all services. That could help to avoid loss to follow-up, which is common among HIV-infected people." Manager, ANC national hospital
The health workers did perceive not only problems but also solutions and seemed to have some motivation to solve the problems and to provide better services for HIVpositive women seeking PMTCT services.
Studies in Viet Nam have demonstrated that both HIVinfected and non-infected women had many criticisms of ANC and delivery services, about provision of information about PMTCT and counselling, and about stigma displayed by health care workers [3, 15] . At present, the health service has not yet addressed this gap. Access to comprehensive PMTCT is still very poor, even in such a well-resourced setting as Hanoi [16] . Because the health care workers are subjected to many accusations about their performance in this context, this study was undertaken to find out their opinion regarding these gaps and weaknesses.
Health care workers usually want to do a good job and provide good care for patients. However, they are often unable to provide as good care as they would like, particularly in facilities with an overload of clients [17, 18] . Results of a survey among women who had been pregnant in Hanoi revealed that they attended and paid for ANC services at higher-level health facilities (provincial and district hospitals) rather than go to commune health stations where ANC services are free of charge [4] . High workloads were observed at provincial hospitals, while district hospitals and commune health stations appeared to have more time and space for pregnant women. Studies of ANC services in Vietnam have identified a number of weaknesses. Staff shortages and staff motivation can significantly affect the quality of service, especially for counselling, which takes a long time to do well [12, [19] [20] [21] [22] .
In the case of HIV and PMTCT, additional reasons for the unsatisfying performance included inadequate knowledge and skills due to lack of training, medicines, protective equipment and practice guidelines. Health care workers had poor knowledge about HIV and about prevention of occupational exposure to HIV, especially at district or lower levels. Even in the expected sources of expertise, the medical schools around Vietnam, 70% of medical students and 48% of lecturers recapped used needles by hand, while two thirds always cleaned their hands with antiseptic after contact with blood. Sixty percent of medical students and 37% of lecturers had been exposed directly to blood or body fluids and were worried about HIV transmission. However, 15% of the respondents recommended antibiotics for post-exposure prophylaxis, while one third proposed ARV prophylaxis [23] . These results reveal a disturbing lack of knowledge and awareness about HIV, even among the medical profession. Lack of practical needs can become an excuse for health care workers to justify their fear of HIV infection and their reluctance to provide good services for HIV-infected people [7, 24] .
As the HIV epidemic has evolved in Viet Nam, both governments and international donors have given priority to prevention and surveillance activities. The main reason is that Viet Nam has had success up to now using surveillance and containment to control infectious diseases such as polio, SARS and, more recently, avian flu. HIV/AIDS policy and practice also aims foremost at controlling the spread of the virus and has paid less attention to providing care and treatment to individuals already affected. In keeping with past experience in other epidemics, health staff perceived HIV-infected persons as sources of contamination, who should be isolated.
Health care workers are the key providers of medical care. Stigma from health care workers can reduce patients' ability to manage their infection and gain access to health care [7, 25] . Persons infected with HIV are often grouped with drug users and sex workers as marginalized, discriminated-against and criminalized elements in society, also by health workers. Stigma towards HIV-infected persons has been documented in health care settings all over the world [26, 27] . Showing a negative attitude during counselling and provision of care, treating in a separate area and avoidance of providing service at all are perceived as enacted stigma by HIV-infected patients.
On the other hand, from the health workers' point of view, these actions result from a combination of factors: high workload and personal priority-setting influenced by fear of being infected as well as distrust towards almost all HIV-infected patients because of the association with antisocial behaviors [28, 29] . When health care workers have fear and lack knowledge, they can find reasons not to focus their attention on the HIV-positive patients and give those reasons for not providing service as they think/ know they should. Moreover, health care workers are not only a source of stigma from the perspective of HIVinfected people, but can also become recipients of stigma from colleagues and family because of their exposure to HIV-infected patients.
The best and most feasible solution is to provide training and reference materials for health workers, to inform them about HIV transmission routes, universal precautions and post-exposure prophylaxis. Reduction of workload would also be important [24] . The results of this study also suggest that the quality of PMTCT service could be enhanced by improving communication and other skills of health workers and providing them with greater support and motivation.
A positive atmosphere in the health facilities should be promoted by normalizing HIV-related services, and undertaking behaviour-change communication campaigns aimed at staff of the health facilities. Feedback from service users could be used as one way to evaluate the quality of service.
In addition, health facilities should make ARV continuously available. The health workers' fear could also be reduced by ensuring that they have and use the protective clothing they need to maintain good hospital hygiene. It will be more difficult to address the issues of fear and stigma towards drug users and sex workers.
Self-help groups of both drug-using and non-drug-using women in Hanoi and other countries were able to improve the relationship and communication between health care staff and patients/clients; peer counsellors and a buddy system led to improvements in the health care provided to and accessed by the women [3, 30, 31] . Successful examples of this intervention have been documented among clubs for tuberculosis patients [32] , alcoholics, cancer patients and patients with chronic illnesses and mental problems [30] .
Continuous care and support for HIV-infected mothers after delivery was often not seen as a need to be addressed [12, 33] . In practice, the fragmentation of the health care system into specialized vertical pillars including a vertical programme for HIV/AIDS is a major obstacle to providing a continuum of care. Medical treatment, including ARV provision and medications for OI, is increasingly available but is often not accessible to PLHIV because of a weak referral system and social stigma [3, 7] . The vertical organization of the health care system and the contradictory mandates between sectors obstruct the effective collaboration and referral between different services that the women and their families need. A lack of multisectoral collaboration is a barrier to effective information exchange for patients between national staff in different facilities [34] . Providing information about topics such as abortion, clean needle exchange programmes and condoms is also politically sensitive in voluntary counselling and testing sites.
The study suggests that information on available services should be made known to health workers. Frequent meetings between different service sites should be organized, with the involvement of high-level health staff that can make decisions, to update information on services available and provide feedback on the quality of the referral system. Development of a practical strategy is needed to strengthen and adapt the referral system to meet the needs of patients. For example, comprehensive services for HIVinfected people should be provided at one service site at district level [2].
As information was collected by means of qualitative methods, the identified factors that lead to their failure to give good quality PMTCT could not be quantified and be representative for the health care worker population in Hanoi.
In conclusion, the results of this study show that health care workers also face a number of barriers in their efforts to provide good PMTCT services at different levels of the health services in Hanoi. These include a high workload, a lack of equipment and materials, lack of training and skills updating, the common fear of the type of patients who may present with HIV, and little support to improve their performance. These weak points can be addressed by a number of feasible interventions. Results of the study contribute to the picture of the PMTCT programme not only in low-prevalence settings, as in Asian countries, but also in high-prevalence settings with weak health care systems, such as African countries, and may require different interventions to improve the quality of the service. Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α(1)-Antitrypsin Variants BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted α(1)-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α(1)-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α(1)-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α(1)-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies. Lassa virus (LASV) belongs to the family Arenaviridae, which are enveloped, single-stranded RNA viruses distributed worldwide. Based on their antigenic relationships and geographic distribution, arenaviruses are divided into two major groups. The Old World group includes the prototype of this family, lymphocytic choriomeningitis virus (LCMV), and LASV, which is endemic in West African countries and causes every year thousands of human infections with hemorrhagic fever as a severe clinical manifestation [1] . The New World group includes among others Machupo, Junin, Guanarito and Sabia viruses which can cause viral hemorrhagic fever (VHF). With the exception of the New World virus Tacaribe, which was isolated from Artibeus bats, arenaviruses are rodent-borne viruses [2] .
Over the past few years great efforts have been made to find potential therapeutic and vaccination approaches in the arenavirus field (reviewed in [3, 4, 5] ). Until now there is no specific and effective treatment available to combat hemorrhagic fevers caused by arenaviruses. Administration of convalescent plasma has been reported to reduce the mortality rates of patients with Argentine hemorrhagic fever, however, 10% of immune-plasma recipients developed a late neurological syndrome of unknown origin [6] . The only existing drug used to treat Lassa fever and certain South American hemorrhagic fevers is the broad-spectrum antiviral agent ribavirin, a ribonucleoside analogue, which has shown to be partially effective if given early in the course of illness [7, 8, 9, 10] . Even though the drug is relatively inexpensive for patients in highdeveloped countries, it is still unaffordable for many of those living in West Africa and South America. Moreover, several adverse effects have been associated with ribavirin therapy in patient studies and animal models [11, 12, 13, 14, 15] . The lack of effective disease control measures as well as the discovery of new fatal arenavirus species that pose a risk of epidemic potential [16, 17] , emphasize the need for novel therapeutic interventions.
Lassa virions are pleomorphic lipid-enveloped particles that contain two single-stranded RNA segments, designated L (large) and S (small), encoding four viral proteins in a unique ambisense coding strategy. The L segment encodes the viral RNA-dependent RNA polymerase (L) and the small zinc finger matrix protein (Z) [18] ; the S segment encodes the virus nucleoprotein (NP) and the virus surface glycoprotein precursor (preGP-C) [19] . preGP-C is cleaved co-translationally into a stable signal peptide and GP-C [20] . Post-translational maturation cleavage of GP-C by the proprotein convertase site 1 protease (S1P, [21] ), also known as subtilisin kexin isozyme-1 (SKI-1, [22] ), leads then to the generation of the distal receptor-binding subunit GP-1 and the transmembrane-spanning fusion competent subunit GP-2 [23] . Together with the signal peptide these subunits form the tripartite glycoprotein spike complex on the viral surface [24, 25] .
The glycoproteins of the Old World arenaviruses LASV and LCMV were the first viral glycoproteins that were shown to be proteolytically processed by S1P [23, 26] , which normally plays important physiological regulatory roles in cholesterol metabolism, ER stress response, cartilage development and other cellular processes [21, 27, 28, 29, 30, 31] . Using systematic mutational analysis of the LCMV GP cleavage site, the consensus motif R-(R/K/ H)-L-(A/L/S/T/F) was determined, which is conserved in the glycoprotein sequences of the Old World viruses LASV, Mopeia and Mobala, as well as the New World virus Pichinde, suggesting that all arenavirus glycoproteins are cleaved by S1P [26, 32] . Indeed, more recently Rojek et al. reported that glycoproteins from the New World hemorrhagic fever viruses Junin, Machupo and Guanarito are also processed by S1P, although Guanarito possesses a protease recognition motif that differs from known arenavirus GP consensus cleavage sequences, indicating a broader substrate specificity of S1P than previously anticipated [33] .
Proteolytic activation of LASV GP-C by S1P is not necessary for transport of GP-C to the cell surface, where budding of arenaviruses occurs, but is essential for incorporation of the cleaved subunits into virions, and thus, for the formation of infectious viral particles. In the absence of GP-C cleavage, enveloped non-infectious LASV-like particles are released containing L, NP, Z protein and viral RNA but are devoid of viral glycoproteins [23] . Similar results were described for LCMV and New World hemorrhagic fever viruses [33, 34] .
In addition to its important role in the arenaviral life cycle, S1P is critical for the infectivity of Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the Bunyaviridae family, through processing of the glycoprotein Gn [35, 36] . These findings make the inhibition of S1P particularly interesting for the development of a novel antiviral therapeutic that will target pathogenic viruses known to be processed by S1P.
A successful approach to inhibit proprotein convertases involves genetically engineered antitrypsins, which are derived from a 1antitrypsin (a 1 -AT). a 1 -AT is a serine protease inhibitor (serpin) with a characteristic exposed reactive center loop (RCL), which mediates binding to the active site of its target protease. The exploration for the potential use of modified antitrypsins with an altered inhibitory spectrum has been guided by the discovery of a natural variant of a 1 -AT, known as Pittsburgh (a 1 -AT-PIT), found in a patient who had a severe bleeding disorder caused by mutation of the P1 reactive center residue of antitrypsin from methionine to arginine [37] . This substitution changed its specificity from elastase to thrombin and other coagulation proteases. Due to the introduction of a second mutation from alanine to arginine at P4 of the RCL, the engineered a 1antitrypsin variant Portland (a 1 -AT-PDX) showed high affinity for furin [38] . a 1 -AT-PDX efficiently inhibited the formation of infectious HIV, measles virus, and human cytomegalovirus progeny by blocking furin-dependent processing of glycoproteins gp160, F0 and gB, respectively [38, 39, 40, 41] . Pullikotil and coworkers used this approach for the generation of highly selective a 1 -antitrypsin variants specific for S1P by introducing various S1P recognition motifs into the RCL of a 1 -antitrypsin [42] . The adaptation of a 1 -antitrypsin towards S1P efficiently inhibited the processing of the S1P substrates SREBP-2 (sterol regulatory element binding protein), ATF6 (activating transcription factor 6) as well as CCHFV glycoprotein [42] . However, the effect of these inhibitors on CCHFV infection was not analyzed in that study. To block cleavage of the LASV glycoprotein, we generated here recombinant a 1 -antitrypsin variants mimicking the S1P recognition motifs RRIL, RRVL and RRYL that exhibited the greatest inhibitory potential based on immunoblot quantification. In addition, we used an a 1 -AT construct that contains the LASV GP cleavage motif RRLL in its RCL. Using a doxycycline regulated expression system we demonstrate that S1P-adapted a 1antitrypsin variants efficiently block proteolytic maturation of the glycoprotein precursor GP-C, whereas a furin-specific a 1 -AT had no effect on GP-C processing. Virus replication of both a replication-competent recombinant vesicular stomatitis virus expressing the LASV glycoprotein GP-C (VSVDG/LASVGP) and authentic LASV was significantly inhibited in the presence of S1P-specific a 1 -antitrypsins. The degree of inhibition of viral replication correlated with the ability of the different a 1 -antitrypsin variants to inhibit the processing of LASV GP-C.
Since glycoprotein processing by the endoprotease S1P is not only critical for virus infectivity of LASV [23] , and other arenaviruses causing hemorrhagic fever [33] , but also for members of the Bunyaviridae family [36] , further optimization based on our findings could lead to a potent and specific S1P inhibitor with the potential treatment of certain VHFs.
cDNA of the open reading frame of rat a 1 -antitrypsin (Gene Bank Accession Number NM_022519) (a kind gift from Dr. G. Thomas, Vollum Institute, Oregon Health & Science University, Portland, USA) was inserted into pSG5 and used as a template to generate S1P-specific a 1 -antitrypsin variants by recombinant polymerase chain reaction (PCR) using overlapping oligonucleotides [43] . The sequences of the oligonucleotides used are listed in Table S1 . The resulting full-length PCR products were digested
The virus family Arenaviridae includes several hemorrhagic fever causing agents such as Lassa, Guanarito, Junin, Machupo, and Sabia virus that pose a major public health concern to the human population in West African and South American countries. Current treatment options to control fatal outcome of disease are limited to the ribonucleoside analogue ribavirin, although its use has some significant limitations. The lack of effective treatment alternatives emphasizes the need for novel antiviral therapeutics to counteract these life-threatening infections. Maturation cleavage of the viral envelope glycoprotein by the host cell proprotein convertase site 1 protease (S1P) is critical for infectious virion production of several pathogenic arenaviruses. This finding makes this protease an attractive target for the development of novel antiarenaviral therapeutics. We demonstrate here that highly selective S1P-adapted a 1 -antitrypsins have the potential to efficiently inhibit glycoprotein processing, which resulted in reduced Lassa virus replication. Our findings suggest that S1P should be considered as an antiviral target and that further optimization of modified a 1 -antitrypsins could lead to potent and specific S1P inhibitors with the potential for treatment of certain viral hemorrhagic fevers.
Effect of S1P Inhibition on LASV Replication www.plosntds.org with BamHI and NheI and cloned into the tetracycline (Tet)controlled inducible mammalian expression vector pTRE2hyg (Clontech). The accuracy of all constructs was confirmed by DNA sequencing.
To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the a 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Cells were then cultured for 2 weeks under selective pressure in the presence of 500 mg/ml Hygromycin B, the selection agent for the a 1 -antitrypsin expressing plasmid, and 500 mg/ml G418, the selection agent for the rtTA (reverse Tet-controlled transactivator) cassette. The selective media were replaced every 3 days. Well-separated antibiotic-resistant cell clones were individually isolated with cloning cylinders (Sigma). Therefore, a small volume of Trypsin-EDTA (Sigma) was added and the culture dish was incubated briefly at 37uC until cells detach. Cells were then collected from inside the cylinder and transferred to individual wells of a 24-well plate for further growth in selective medium. When grown to confluence, cells were transferred to larger flasks. Protein expression was induced with 1 mg/ml doxycycline (Clontech) and analyzed by Western Blot and immunofluorescence. Stable cell lines showing similar expression levels of the various a 1 -antitrypsins were chosen for further experiments.
Vero E6 cells (green monkey kidney) were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) and CHO-K1 Tet-On cells in DMEM/F12 (Gibco), both media containing penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mmol/l) (all from Invitrogen) as well as 10% fetal bovine serum (PAN Biotech). S1P-deficient SRD-12B cells (a generous gift from Dr. J. L. Goldstein, Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, USA) were maintained as CHO cells but supplemented with 5 mg/ml of cholesterol (Sigma), 1 mM sodium mevalonate (Sigma), and 20 mM sodium oleate (Sigma) [44] .
The vesicular stomatitis virus reverse genetics system (VSV, Indiana serotype) was kindly provided by Dr. J.K. Rose (Department of Pathology, Yale University School of Medicine, New Haven, USA) and was described in detail earlier [45, 46, 47] . Recombinant VSV expressing the glycoprotein GP-C of Lassa virus (LASV, strain Josiah) designated as VSVDG/LASVGP and wild-type VSV (VSVwt) were propagated in Vero E6 cells as described previously [48] . Influenza virus A/FPV/Rostock/34 (H7N1), designated as fowl plague virus (FPV), was propagated in embryonated hen eggs and stored at 280uC until further use. Virus titration of FPV was described previously [49] . All experiments with infectious FPV were done under biological safety level 3 conditions. VSVDG/LASVGP titration was performed using a microplate format plaque assay with subsequent immunostaining as described before [50] . In brief, virus dilutions were incubated on Vero E6 cells with an overlay of 3% carboxymethylcellulose (CMC) during plaque formation. Infected cells were visualized after cell fixation with paraformaldehyde (PFA, 4%) and permeabilization with 0.3% Triton-X 100 using a specific LASV GP-C/GP-2 antibody followed by incubation with horseradish peroxidase-labeled secondary anti-rabbit antibody (DAKO). Finally, cells were stained with True Blue Peroxidase substrate (KPL).
For virus spread experiments, CHO cell lines were seeded into 96-well plates in the presence or absence of doxycycline. 24 h after induction, cells were infected with VSVDG/LASVGP or FPV and were grown without solid overlay. Cells were fixed at different time points post-infection and immunostaining was performed as described above using rabbit sera against VSV (kindly provided by Dr. G. Herrler, Institut für Virologie, Zentrum für Infektionsmedizin, Stiftung Tierä rztliche Hochschule Hannover, Germany), for the detection of VSVDG/LASVGP infected cells, and against FPV, for cells infected with FPV, respectively.
Virus titration of LASV (strain Josiah, Gene Bank Accession Number NC_004297 and NC_004296) was performed by defining the 50% tissue culture infectious dose (TCID 50 ). For this, Vero cells were grown in 96-well plates to 30 to 40% confluence. Cells were inoculated with 10-fold serial dilutions of supernatants from LASV-infected CHO cell lines grown in the presence or absence of doxycycline. The assays were evaluated at 7 to 9 days postinfection. TCID 50 values were calculated using the Spearman-Karber method [51] . All experiments involving LASV-infected samples were performed under biological safety level 4 conditions at the Philipps-University Marburg.
At 24 h post-infection, cell culture supernatants from infected cells were cleared from cell debris and pelleted in an SW-60 rotor through a 20% sucrose cushion at 52000 rpm at 4uC for 2 h. The pellet was then resuspended in PBS buffer and mixed with SDS-PAGE sample buffer. To control the intracellular expression level, cell lysates were collected simultaneously. Samples were analyzed by SDS-PAGE and Western blotting using protein-specific antibodies as indicated.
Proteins were separated by SDS-PAGE using 10% polyacrylamide gels. Immunoblotting was performed as described previously [52] . Antiserum against Lassa virus GP-C/GP-2 was also described previously [32] . Polyclonal rabbit anti-ß-tubulin antibody was purchased from Abcam (UK), and monoclonal mouse anti-Flag antibody from Sigma-Aldrich. Secondary antibodies labeled with Alexa680 or IRDye800 were from Molecular Probes Invitrogen and Biomol, respectively, and were used for visualization and quantification of detected proteins using the Odyssey Infrared Imaging System (LI-COR Biosciences).
CHO cell lines were grown on coverslips and 24 h after doxycycline-induction, cells were washed with PBS and fixed with 4% PFA in DMEM for 30 min. The fixative was removed, and free aldehydes were quenched with 100 mM glycine in PBS. Then, samples were washed with PBS and permeabilized for 10 min with PBS containing 0.1% Triton X-100. Cells were incubated in blocking solution (2% bovine serum albumin, 0.2% Tween 20, 5% glycerol, and 0.05% sodium azide in PBS) and subsequently stained with a primary mouse-anti-flag antibody (1:400) and a secondary anti-mouse antibody coupled to rhodamine (1:200, Jackson Immunoresearch). Cell nuclei were stained with DAPI (49,69-diamidino-2-phenylindole, Sigma). Microscopic analysis was performed with a Zeiss ApoTome/ Axiovert 200 M microscope using a magnification of 1:40.
Replication-competent recombinant vesicular stomatitis virus (rVSV) expressing foreign envelope glycoproteins has been demonstrated to be a suitable model system to study the role of Effect of S1P Inhibition on LASV Replication www.plosntds.org viral glycoproteins in the context of virus replication [47, 53, 54] . In the present study, we took advantage of a rVSV expressing the LASV glycoprotein GP (designated VSVDG/LASVGP) [48] . In this system biosynthesis and processing of GP was shown to be authentic compared to LASV [48] .
In an initial experiment we wanted to determine whether CHO-K1 cells are susceptible to VSVDG/LASVGP infection. The reason we chose CHO-K1 cells for our studies is the availability of a site 1 protease-deficient CHO cell line (designated SRD-12B cells), in which GP maturation is abolished and only GP-deficient non-infectious LASV particles are released [23] . Thus, this cell clone provides an ideal control for inhibition studies. Vero E6, CHO-K1, and SRD-12B cells were infected with either VSVDG/ LASVGP or wild-type VSV (VSVwt) as a control. Aliquots of cell culture supernatants were collected at different times after infection and were analyzed by plaque assay. Growth kinetics revealed that VSVDG/LASVGP grows to similar titers in CHO-K1 cells compared to Vero E6 cells which have been used in earlier studies (Fig. 1A) [48] . These data demonstrated that CHO-K1 cells support efficient VSVDG/LASVGP replication, and thus are useful tools for further investigations. As expected, VSVDG/ LASVGP lacks efficient replication in SRD-12B cells, whereas virus growth of VSVwt remained unaffected in these cells (Fig. 1A) . The reason for the low but detectable virus titers in the supernatant of VSVDG/LASVGP-infected SRD-12B cells is currently not known but has been also observed for LASV ( [23] and present study), LCMV [34] and New Word arenaviruses [33] . Glycoprotein activation by a yet unknown protease though with only very low efficiency might explain this phenomenon.
To mimic the conditions of short-term treatment, we decided to use the inducible doxycycline-dependent Tet-On expression system, which allows regulated expression of the protein of interest [55] . To determine whether treatment of cells with doxycycline interferes with viral replication, we cultivated VSVDG/LASVGPinfected CHO-K1 Tet-On cells in the presence or absence of doxycycline (1 mg/ml) for 24 h and 48 h, respectively. As shown in Fig. 1B , CHO-K1 Tet-On cells treated with doxycycline produced a virus titer comparable to cells that were cultivated in the absence of doxycycline, indicating that these conditions used in our experiments have no influence on efficient virus replication.
Generation of S1P-adapted a 1 -antitrypsin expressing cell lines Pullikotil and colleagues recently reported that various antitrypsins mimicking S1P recognition motifs are able to block processing of the S1P substrates SREBP and ATF6, although to different degrees [42] . In addition to the a 1 -AT variants shown to be most effective in that study we have chosen the LASV GP-C cleavage motif RRLL to investigate whether they also inhibit LASV GP-C cleavage. Therefore, we generated various S1Pspecific a 1 -ATs, and as a specificity control, a furin-adapted a 1 -AT, by recombinant PCR technology using the rat a 1 -AT-PIT as a template (Fig. 2A) . To facilitate their detection, we introduced a flag epitope at the C-termini of the constructs. Stable cell lines were generated and individual clones were isolated and screened for a 1 -antitrypsin expression after doxycycline induction by immunoblotting and immunofluorescence analysis. Cell lines that showed similar expression levels of a 1 -antitrypsins were chosen for further experiments (Fig. 2B and 2C ).
To test the inhibitory potential of S1P-specific a 1 -antitrypsins on proteolytic processing of LASV GP, stably transfected CHO-K1 Tet-On cells, and non-transfected wild-type CHO-K1 Tet-On cells as well as SRD-12B cells were infected with VSVDG/ LASVGP at an MOI of 0.2 in the presence or absence of doxycycline. To allow only one replication cycle, cell lysates were analyzed 10 h post-infection for detection of LASV GP cleavage by Western blot analysis using a GP-specific antiserum that recognizes both the precursor GP-C and the cleaved subunit GP-2. In CHO-K1 Tet-On cells LASV GP was efficiently cleaved, regardless of whether doxycycline was present or not. In contrast, virtually no detectable cleavage of GP was observed in SRD-12B cells that are deficient in S1P (Fig. 3A, lanes 1-4) . Without expression of the various antitrypsins efficient cleavage was detected in these stably transfected cell lines, similar to the processing of GP in wild-type CHO-K1 Tet-On cells (Fig. 3A , lanes 1, 5, 7, 9, 11, and 13) . In contrast, cells expressing the S1Padapted a 1 -antitrypsins inhibited proteolytic maturation of LASV GP (Fig. 3A, lanes 6, 8, 10, and 12) . Furthermore, our results show Effect of S1P Inhibition on LASV Replication www.plosntds.org that the presence of a furin-specific a 1 -AT did not influence LASV GP-C processing, demonstrating the specificity of the generated S1P-adapted a 1 -antitrypsins (Fig. 3A, lanes 13 and 14) . Quantification of GP-2 cleavage revealed that the a 1 -AT variant RRIL exhibited the greatest inhibitory effect on GP processing (.80% inhibition) followed by a 1 -AT RRLL (.60% inhibition), which possesses the amino acid cleavage motif of the LASV GP-C. Also a 1 -AT variants RRVL and RRYL were found to be inhibitory, although to a lesser extent (inhibition less than 50%) than the variants RRIL and RRLL (Fig. 3B) . Taken together, these data clearly demonstrate that S1P-specific a 1 -antitrypsins efficiently block the maturation cleavage of LASV GP, however, they differ in regard to their inhibitory potential.
We have shown earlier that S1P-mediated cleavage of GP-C is absolutely required for incorporation of the glycoprotein subunits into the virion envelope and thus for production of infectious LASV [23] . Therefore, we addressed the question of whether a S1P-specific a 1 -AT has the potential to prevent GP incorporation by blocking glycoprotein processing. To this end, a 1 -AT RRIL cells were infected in the presence or absence of doxycycline with either VSVDG/LASVGP or VSVwt as a control. At 24 h postinfection, viral particles released into the cell culture supernatant were purified over a 20% sucrose cushion and analyzed by means of immunoblotting. In viral particles from supernatants of noninduced a 1 -AT RRIL cells and CHO-K1 Tet-On control cells cleaved GP-2 was readily observed, whereas in the particulate material isolated from the supernatant of a 1 -AT RRIL expressing cells no glycoprotein was detected (Fig. 4A) . However, Western Blot analysis for VSV proteins revealed the release of these viral proteins into the supernatant of a 1 -AT RRIL expressing cells, which is consistent with our earlier findings that, in the absence of GP-C cleavage, enveloped non-infectious LASV-like particles containing the matrix protein Z and the ribonucleoprotein (RNP) complex, but devoid of viral glycoproteins, are still released [23] . The lower amount of VSV proteins observed in the cell lysate and supernatant of a 1 -AT RRIL expressing cells reflect lower levels of viral replication, which is due to less efficient virus spread (Fig. 4A) . In contrast to its ability to efficiently block incorporation of LASV GP into virions, the presence of a 1 -AT RRIL had no effect on the release of glycoprotein G containing wild-type VSV particles. The amount of VSV proteins detected in the supernatant from a 1 -AT RRIL expressing cells was similar to the amount of viral proteins observed in supernatants of non-induced cells and CHO-K1 cells, indicating efficient viral replication and cell-to-cell spread of VSVwt despite the presence of a 1 -AT RRIL (Fig. 4B) . Taken together, these data demonstrate that S1P-specific a 1 -antitrypsins have the potential to prevent LASV GP incorporation by inhibiting glycoprotein cleavage, which is an essential prerequisite for infectious progeny.
Virus spread is reduced in the presence of specific a 1antitrypsins Next, we wanted to know whether the observed inhibition of LASV GP processing correlates with the ability of the different a 1antitrypsin variants to inhibit virus spread. To investigate this, we established a 96-well plate assay in which infected cells are immunostained with True Blue substrate as described in Materials and Methods. Virus spread can be monitored by the appearance of characteristic comet-shaped foci, showing that the virus progeny is carried over the cell monolayer, while prevention of virus spread results in limited radial growth, due to infection of only neighbouring cells. This assay allows rapid screening of potential inhibitors [50] . Effect of S1P Inhibition on LASV Replication www.plosntds.org type cells (Fig. 5A, upper panel) . In contrast, virus spread was significantly diminished in cells expressing a 1 -AT specific for S1P (Fig. 5A, lower panel) . These data indicate that S1P-adapted a 1antitrypsins have the potential to specifically inhibit the processing of LASV GP, which in turn is required for efficient virus spread. It should be noted that the infectious foci observed in a 1 -AT RRIL expressing cells were larger compared to SRD-12B cells in which virtually no virus spread of VSVDG/LASVGP was observed, resulting in only single infected cells (Fig. 5A) . Although similar inhibition values were observed by means of immunoblot quantification (Fig. 3) , a few remaining non-detectable cleavage events may count for this limited cell-to-cell spread in a 1 -AT RRIL expressing cells. Cells expressing the furin-adapted a 1 -AT variant RVKR did not prevent virus spread. At first glance, we rather observed an enhancement of infectivity compared to noninduced cells, which might be due to an increase in the LASV cellular receptor a-dystroglycan on the cell surface [56] .
To further confirm the specificity of the a 1 -AT variants, we used fowl plague virus (FPV), which contains a hemagglutinin with a multibasic cleavage motif recognized by furin [57] . Thus, the furin-adapted a 1 -AT should prevent virus spread of FPV, while virus spread in the presence of S1P-specific a 1 -antitrypsins should remain unaffected. Fig. 5B clearly demonstrates that the most potent S1P-specific a 1 -AT variant RRIL had no effect on FPV replication, and that virus spread was found to be similar to that observed in wild type CHO-K1 Tet-On cells. In contrast, in cells expressing the furin-adapted a 1 -AT variant RVKR virus spread of FPV was drastically reduced, whereas FPV replication occurred efficiently under doxycycline-free conditions in these cells. These results demonstrate that the generated a 1 -AT variants exhibit high specificity for the corresponding proteases, which are essential for virus spread in cell culture.
To further elucidate the effect of the different a 1 -AT variants on multicycle replication, viral titers were determined. To this end, cells were infected with VSVDG/LASVGP at an MOI of 0.02 in the presence or absence of doxycycline. Cell culture supernatants were collected 24 h and 48 h post-infection and virus titers were determined by plaque assay. As shown in Table 1 , non-induced S1P-specific a 1 -AT cell lines permitted unaffected growth of Effect of S1P Inhibition on LASV Replication www.plosntds.org VSVDG/LASVGP to comparable titers, whereas virus titers were reduced in cells expressing the S1P-specific a 1 -AT variant. At 24 h post-infection virus production decreased about 100 fold in cells expressing the a 1 -AT variant RRIL compared to non-induced control cultures. The presence of a 1 -AT variant RRLL reduced the virus titer in the supernatant about 10 fold, followed by a 6.2 fold reduction of virus production in a 1 -AT variant RRVL expressing cells. The presence of the a 1 -AT variant RRYL only exhibited a very moderate inhibitory effect on viral replication (inhibition ,2 fold). Again, the presence of the furin-adapted a 1 -AT variant RVKR did not affect VSVDG/LASVGP replication compared to non-induced control cells. Our results indicate that the various S1P-adapted a 1 -antitrypsins exhibit different inhibitory potentials, due to their different recognition motifs. However, the degree of inhibition of virus replication correlated well with the inhibitory potential of the various S1P-adapted a 1 -antitrypsin variants to block LASV GP processing. Interestingly, following the inhibition of virus progeny over a time period of 48 h only the S1P-adapted a 1 -AT variants RRIL and RRLL sustained their inhibitory capacity, whereas in cells expressing a 1 -antitrypsin variants RRVL and RRYL virus production was found to recover although the initial expression levels of a 1 -antitrypsin variants were similar (Table 1 ). These data indicate that the inhibitory potential of the a 1 -AT variants RRVL and RRYL is not sufficient to efficiently suppress the formation of infectious particles by effectively blocking LASV GP-C cleavage, whereas the a 1 -AT variants RRIL and RRLL seem to be appropriate candidates for efficient inhibition of LASV propagation.
Finally, we wanted to investigate the impact of blocking S1Pmediated GP processing on virus progeny of authentic LASV. Therefore, we assessed the inhibitory potential of the most potent variant, a 1 -AT RRIL, on the multiplication of LASV (strain Josiah). For this purpose a 1 -AT RRIL cells and, as controls, CHO-K1-Tet-On and SRD-12B cells were infected with LASV at an MOI of 0.1. To induce a 1 -AT expression, a 1 -AT RRIL cells and, as a control for off-target effects, CHO-K1 Tet-On cells were cultivated in the presence of doxycycline. To determine virus titers, infectious virions released into the cell culture supernatant were analyzed by defining the 50% tissue culture infectious dose (TCID 50 ) at various times post-infection, as indicated. In noninduced a 1 -AT RRIL cells, LASV revealed a growth kinetic similar to that observed in CHO-K1 Tet-On control cultures, while expression of a 1 -AT RRIL resulted in an average 2 log10 reduction in viral titer (Fig. 6) . The difference between infectious LASV titers in the supernatant of a 1 -AT RRIL expressing cells and SRD-12B cells correlated with the limited virus spread observed in a 1 -AT RRIL expressing cells compared to single cell infections in S1P null cells (Fig. 5) . Taken together, this result highlights the inhibitory activity of modified a 1 -antitrypsins against LASV and demonstrates that inhibition of endogenous S1P is a potent strategy to reduce the production of infectious LASV progeny.
Current drug treatment of Lassa fever and certain New World hemorrhagic fevers is limited to the guanosine analogue ribavirin [7, 8, 9] . Although ribavirin therapy can reduce the mortality rates of severe clinical cases, its unavailability to most patients in West Africa and South America as well as its association with severe adverse effects including anaemia [11, 13] , teratogenicity and embryo lethality [12] , argues for the development of new alternative treatment options.
In principle, every step in the viral life cycle is a potential target for antiviral inhibitors. While current antiviral strategies in the arenavirus field mainly target virus entry [58, 59, 60, 61] or replication and assembly [62, 63, 64, 65, 66, 67] , inhibition studies of the glycoprotein activating endoprotease and its impact on viral replication are largely unexploited. Due to its central role in the arenavirus life cycle [23, 26, 33, 34] , S1P should be considered as a cellular target for antiviral drug development. In the present study we analyzed the inhibitory effect of S1P-adapted a 1 -antitrypsins on proteolytic processing of LASV GP-C and its consequences for viral replication. To our knowledge, this is the first report that Effect of S1P Inhibition on LASV Replication www.plosntds.org addresses the impact of protein-based S1P inhibition on LASV GP-C cleavage and multicycle replication. Furin-adapted a 1 -ATs have been shown to efficiently inhibit the formation of infectious progeny of other viruses (e.g. HIV, measles virus and human cytomegalovirus) [38, 39, 40, 41, 68, 69] . Using a replication-competent recombinant VSV pseudotyped with the LASV glycoprotein GP [48] , we demonstrate that proteolytic maturation of the precursor GP-C is sensitive to S1Padapted a 1 -ATs. Mutagenesis of the reactive centre loop (RCL) into the S1P recognition motif RRIL resulted in an abrogation of GP-C processing similar to that observed in S1P-deficient SRD-12B cells. The inhibitory activity of the a 1 -AT variant RRIL on LASV GP cleavage described here is in agreement with a previous study showing its inhibitory potential on the processing of the natural S1P substrates SREBP-2 and ATF6 [42] . Also an a 1 -AT variant that contains the LASV GP-C cleavage motif RRLL exhibited a high S1P inhibitory potential and was found to drastically reduce GP processing. Interestingly, this variant exhibited a 100% inhibition activity on maturation cleavage of an artificial pro-PDGF (precursor of platelet-derived growth factor) mutant that is processed by S1P due to introduction of a RRLL cleavage site, but failed to inhibit cleavage of endogenously expressed SREBP-2 [42] . These data indicate that various substrates differ in their sensitivity towards S1P inhibition.
The outcome of severe illness increased significantly with the level of viremia in Lassa fever patients [70] . Therefore, the extent of multicycle replication of LASV and thus, the load of infectious particles in its host organism have an important impact for the progress of disease. Our studies revealed that a 1 -AT variants RRIL and RRLL have a potency sufficient to sustain their inhibitory capacity during multicycle replication, which resulted in a significant reduction of virus infectivity. Inhibition of viral replication correlated with the ability of the a 1 -AT variants RRIL and RRLL to efficiently inhibit the processing of the LASV glycoprotein precursor. Although our data demonstrated that inhibition of glycoprotein cleavage by a 1 -AT RRIL reduced incorporation of the subunits GP-1 and GP-2 into virions to below detectable levels, the viral titer from a 1 -AT RRIL expressing cells was found to be greater than that obtained from S1P null cells. Based on this observation, we consider that even the most potent a 1 -AT variant RRIL failed to entirely inhibit S1P activity. However, given that S1P has important biological functions in the regulation of various cellular processes, a complete inhibition of the catalytic activity of S1P is not desirable. For a 1 -AT variants RRVL and RRYL, we observed similar inhibition values by immunoblot quantification analysis as described for CCHFV GP cleavage [42] . Though, their inhibitory activity on LASV GP-C cleavage was not sufficient to efficiently reduce virus replication of VSVDG/LASVGP. These results should be taken into consideration for experimental setups in future studies that address the impact of S1P inhibition in arenavirus replication.
The most potent a 1 -AT variant RRIL revealed a similar inhibitory potential on virus release of authentic LASV to that observed with the corresponding VSVDG/LASVGP pseudotype. Therefore, this study also demonstrates that the replicationcompetent VSV expressing the LASV glycoprotein is an excellent surrogate model for analyzing potential antivirals that target the biological function of GP and its consequence for virus replication. These studies can be performed under biosafety level 2 laboratory conditions that would otherwise require biosafety level 4 laboratory conditions [71] . Taken together, our data indicate that S1P-adapted a 1 -antitrypsins may represent a promising lead compound for the development of a new class of anti-arenavirus inhibitors.
In recent years improvements were made in the application of bioengineered serpins to combat bacterial and viral infections [39, 72] . For example, the addition of exogenous a 1 -AT-PDX, a potent and selective furin inhibitor, was found to efficiently block human cytomegalovirus infection [39] . However, in contrast to furin, which is known to recycle between the plasma membrane and the TGN via endosomal compartments, membrane-bound S1P is localized in the secretory pathway and can be sorted to endosomal compartments but not to the cell surface [73, 74, 75] . Follow-up studies with small synthetic peptides, which are derived from S1P-specific a 1 -antitrypsins described in the present work, are currently in progress and will address cellular delivery and organelle specific targeting, as well as their inhibitory potential on authentic LASV replication. In analogy to inhibition strategies of the eukaryotic subtilase furin, we previously designed and developed a cell-permeable peptidyl chloromethylketone S1P inhibitor, which contained the LASV GP-C cleavage site, designated dec-RRLL-cmk [76, 77, 78] . This irreversible inhibitor efficiently blocked the processing of LASV GP at nanomolar concentrations, however, because of cell type-dependent toxicity observed by us and others, its potential in vitro use requires further investigation [79, 80] .
Due to the essential roles of S1P in cholesterol metabolism and fatty acid synthesis, this enzyme has attracted great attention by the pharmaceutical industry. Research efforts are currently directed towards the development of S1P inhibitors that may be used in the treatment of dyslipidemia and a variety of cardiometabolic risk factors associated with diabetes and obesity [81] . Identification of specific S1P inhibitors in this therapeutic area may also be beneficial in treatment of hemorrhagic fevers caused by viruses known to be processed by S1P. Future studies Effect of S1P Inhibition on LASV Replication www.plosntds.org will have to elucidate the anti-viral efficacy of these and other novel S1P inhibitors that have been developed [82, 83] .
While most conventional antiviral drugs target proteins that are virus-encoded, cellular proteins essential for viral replication are currently considered to be alternative potential targets for antiviral therapy [84, 85, 86] . With the exception of Ebola virus, whose glycoprotein cleavage by the proprotein convertase furin is not essential for virus replication in cell culture and virulence in nonhuman primates [71, 87, 88, 89] , maturation cleavage of surface glycoproteins of several virus species by endoproteases is a key determinant for host cell tropism and pathogenicity [90] . Thus, the emergence of viral escape mutants that confer resistance due to targeted inhibition of an endogenous protease is rather unlikely. In S1P-deficient SRD-12B cells, which were persistently infected with Junin virus vaccine strain Candid 1, no virus escape variants possessing a cleavage motif other than a S1P recognition motif have evolved, indicating a low potential of arenaviruses to develop de novo a different glycoprotein maturation pathway [33] . This observation together with our findings that inhibition of S1P significantly affects LASV GP processing and virus infectivity should encourage the development of S1P inhibitors as a potential drug target to counteract infections caused by pathogenic arenaviruses.
Alternative Language Abstract S1 Translation of the abstract and author summary into French by Stephane Daffis.
Found at: doi:10.1371/journal.pntd.0000446.s001 (0.05 MB PDF)  VirHostNet: a knowledge base for the management and the analysis of proteome-wide virus–host interaction networks Infectious diseases caused by viral agents kill millions of people every year. The improvement of prevention and treatment of viral infections and their associated diseases remains one of the main public health challenges. Towards this goal, deciphering virus–host molecular interactions opens new perspectives to understand the biology of infection and for the design of new antiviral strategies. Indeed, modelling of an infection network between viral and cellular proteins will provide a conceptual and analytic framework to efficiently formulate new biological hypothesis at the proteome scale and to rationalize drug discovery. Therefore, we present the first release of VirHostNet (Virus–Host Network), a public knowledge base specialized in the management and analysis of integrated virus–virus, virus–host and host–host interaction networks coupled to their functional annotations. VirHostNet integrates an extensive and original literature-curated dataset of virus–virus and virus–host interactions (2671 non-redundant interactions) representing more than 180 distinct viral species and one of the largest human interactome (10 672 proteins and 68 252 non-redundant interactions) reconstructed from publicly available data. The VirHostNet Web interface provides appropriate tools that allow efficient query and visualization of this infected cellular network. Public access to the VirHostNet knowledge-based system is available at http://pbildb1.univ-lyon1.fr/virhostnet. Eukaryotic cells express a large panel of proteins that coordinately participate to the cellular machinery through a highly connected and regulated network of protein-protein interactions (1) . Physical architecture of model organisms and human cellular protein networks exhibits a strong robustness against random failures, and strikingly a high sensitivity to targeted attacks on highly connected and central proteins, also called 'hubs' (2, 3) . Cellular protein network is not static and its robustness may change dynamically according to various factors like tissue and cell-line origins, signals received by cellular environment or more specifically during viral infections (4) . Replication and pathogenesis of viruses depend on a complex interplay between viral and host cellular proteins both acting through a complex network of protein-protein interactions. In order to evade the cell innate immune response and/or to favour their own replication and transmission, viruses have developed strategies to hijack central functions of the cell (5) (6) (7) . Viruses also use intra-viral, i.e. virus-virus, protein-protein interactions for virion assembly or viral egress from the cell. Accumulation of functional perturbations associated with such virus-virus and virus-host protein-protein interactions may lead to severe and complex diseases, like the development of cancers (8, 9) . From a systems biology perspective, a deeper understanding of infectious diseases may rely on an exhaustive characterization of all potential interactions occurring between proteins encoded by viruses and those expressed in infected cells (10) . Thus, integration of all protein-protein interactions into an infected cellular network, or 'infectome', is a great challenge that may provide a powerful framework for virtual modelling and analysis of viral infection.
The first draft of the human cellular network, also referred to the human interactome, has been explored at the proteome-wide level by the mean of high-throughput experiments such as yeast-two hybrid screens (11, 12) or tap-tag procedure (13) . The overall quality and completeness of this human cellular network has been significantly improved thanks to systematic approaches based on text-mining and literature-curated interactions extracted from low-throughput experiments. Many generalized and specialized databases are involved in the integration of these protein-protein interactions, such as BIND (14) , MINT (15) , INTACT (16) , HPRD (17) , DIP (18) , BIOGRID (19) , REACTOME (20) , GENERIF (21) and NETWORKIN (22) . However, the low redundancy of interactions found between these databases has raised the need to unify such data resources for human and model organisms (23) . Concerning virus-virus and virus-host protein-protein interactions, few high-throughput experiments have been achieved, except some yeast-two hybrid screens completed for Herpes viruses (EBV, KSH, VZV, HSV-1) (24) (25) (26) and SARS (27) . Although some generalist databases like BIND, MINT, INTACT and HIV-GENERIF provide access to virus-virus and virus-host protein-protein interactions, no systematic approach has been reported to exhaustively mine and curate all interactions that have accumulated in scientific publications.
In this context, we have developed VirHostNet (Virus-Host Network), a public knowledge-based system specialized in the management, analysis and integration of virus-virus, virus-host and host-host interactions as well as their functional annotations in the cell. Based on an extensive scientific literature expertise, VirHostNet provides a high-confidence resource of manually curated interactions defined for a wide range of viral species. The content of this high-confidence dataset has been illustrated by the analysis of cellular functions and pathways enriched in proteins targeted by one or many viruses. An integrated cellular network has also been reconstructed from public data and combined with viral data to provide the first draft of the infected cellular network. In addition, an original Web interface has been developed, which provides multi-criteria query and visualization tools for infection network navigation. The utility of the visualisator has been exemplified by network representation of the mTOR pathway and its interplay with viruses.
A bioinformatics pipeline was developed to fully integrate virus-virus, virus-host and host-host protein-protein interactions gathered from a wide range of public databases, with those mined from scientific literature and curated by VirHostNet experts (Figure 1 ). In addition to the management of this large protein interaction resource, VirHostNet integrates contextual information concerning interacting proteins, like structural and functional annotations of proteins: Gene Ontology term (28) , KEGG pathway (29) , INTERPRO domain (30) . All these data were integrated into a knowledge-based system implemented by using PostgreSQL DataBase Management System (release 8.2.6).
The low level of redundancy observed among available databases involved in molecular interactions management has emphasized the need to integrate these heterogeneous data sources (23) . Virus-virus, virus-host and host-host protein-protein interactions and meta-data related to experimental procedures or publications were extracted from 10 databases (BIND, MINT, INTACT, HPRD, DIP, BIOGRID, REACTOME, GENERIF, HIV-GENERIF, NETWORKIN) ( Figure 1 ). Due to the heterogeneity of protein sequence identification found across these databases (i.e. gene identification number, gene name, protein accession number, protein name), NCBI and ENSEMBL protein sequence databases were chosen to unify virus and host proteins respectively (see Supplementary Table 1A) . Towards this end, the IPI database system (31) was chosen to cross-reference all the human protein sequences to ENSEMBL protein accession numbers. In addition, viral protein sequences defined at EMBL and UNIPROT were mapped on NCBI protein sequences by using BLAST Alignment software (32) . Protein cross-referencing led to the definition of nonredundant protein-protein interactions that were in many cases defined in different databases, publications or supported by distinct experimental procedures (see Supplementary Table 1B) . Thus, all information associated with non-redundant interactions, like database origin, experimental procedure description in PSI-MI 2.5 standard format (33) or PUBMED identification (PMID) number, were retrieved in VirHostNet to provide the most documented interactions. This compilation of interaction meta-data will facilitate data quality filtering based on the number of databases, methods or PMIDs used (34, 35) .
An automatic text-mining pipeline was developed and plugged into the VirHostNet system in order to prioritize scientific papers for protein-protein interaction curation. As a first step, all abstracts containing keywords related to both viruses and experimental procedures used for interactions identification (mainly yeast-two hybrid, co-imunoprecipitation, pull-down and tandem affinity purification) were extracted for an in-depth expertise. During curation, protein-protein interactions were carefully annotated according to: (i) the protein accession numbers of each of the protein interactor, the human and/or viral proteins being respectively referenced to ENSEMBL and NCBI accession numbers; (ii) the molecular interaction methods based on the PSI-MI 2.5 ontology vocabulary; and (iii) the PMIDs. Based on 1174 selected PMIDs, literature curation led to the annotation of 2186 redundant interactions in 723 papers (Supplementary Table 2 ). This effort significantly complemented data from public databases with 1297 new non-redundant protein-protein interactions. In order to provide a higher level of data accuracy, virus-virus and virus-host protein-protein interactions from public databases were also carefully inspected.
From 2294 PMIDs for which at least one protein-protein interaction was defined, database curation led to the validation of 2261 redundant interactions found in 789 papers, corresponding to 1374 confirmed non-redundant proteinprotein interactions (Supplementary Table 2 ). Strikingly, our experts confirmed 20% of BIND and GENERIF (HIV) against 90-95% for MINT and INTACT data.
One reason is that all protein-protein interactions defined by functional associations and/or genetic interactions between proteins were discarded from BIND and HIV-GENERIF.
To our knowledge, VirHostNet provides the largest and the most confident infected cellular network. This network is composed of 2671 virus-virus and virus-host non-redundant protein-protein interactions concerning 180 distinct viral species. The curated protein-protein interactions were mainly defined by low-throughput and high-throughput yeast two-hybrid screens (40%), co-immunoprecipitation (24%) and pull-down (21%) (Figure 2A ). Even if only 65% of interactions rely on a single experimental procedure, a total of 944 proteinprotein interactions (35%) were defined by at least two independent methods, in good agreement with other high-confidence databases (36) ( Figure 2B ). All these interactions were defined in 36 distinct viral families, underlying the broad taxonomical diversity provided by VirHostNet ( Figure 3A ). In addition, the distribution of interactions observed among viral Baltimore groups should allow large-scale comparative study of virus-virus ( Figure 3B ) and virus-host networks ( Figure 3C ).
In the infection network, the virus-host interactions occurred between 407 viral proteins and 1012 human proteins, suggesting the strong tendency of viruses to interact with a large number of cellular proteins. In order to characterize cellular functions targeted by the viral machinery, we performed functional enrichment analysis of host proteins interacting with viruses, by using Gene Ontology and KEGG databases and the same methodology described by Zheng and Wang (37) (Supplementary Tables 3 and 4 , respectively). The results showed that viruses interact significantly with a large panel of cellular functions (e.g. cell cycle, apoptosis, cell communication, protein transport) and with canonical signalling pathways (e.g. Jak-Stat, Toll-like Receptor, MAPK, TGF-b, mTOR). The majority of these functions and pathways have already been described to participate in either viral infectious cycle, cellular anti-viral mechanisms or viral associated diseases (38) . Interestingly, analysis of KEGG pathways revealed cellular mechanisms poorly documented in the case of viral infections. One example is focal adhesion, a pathway involved in cell contact with the extracellular matrix and in many other cellular processes including invasion, motility, proliferation and apoptosis (39) . Indeed, on 202 protein members of the focal adhesion pathway, more than 25% (59) were found significantly targeted (exact Fisher test, Benjamini-Hochberg multiple correction test P-value < 0.05) by at least one viral protein in 36 distinct viral taxons. This may suggest the central role of focal adhesion during viral infections and its potential impact on viral induced cancer development that might be associated for instance to the loss of cellular adhesion. Although cellular functions of proteins are far from being completely known and/or annotated in public databases, based on the 'guilty by association' concept the human protein-protein network may serve as a template to complete our understanding on cellular functions perturbed during viral infection. In order to include virusvirus and virus-host interactions in their cellular context, a human-human protein interaction network containing roughly 70 000 non-redundant protein-protein interactions and 10 000 proteins was built from public databases (details on interaction methods distribution are given in Supplementary Figure 1) . Thus, based on roughly 40 000 unique proteins annotated in ENSEMBL, 25% (10 000/ 40 000) are connected within the human protein network. Analysis of the infection network revealed that surprisingly 88% (881/1012) of targeted human proteins interact with at least one cellular protein. Thus, targeted proteins tend to physically interact in the cell and may probably participate in cross-linked functions and pathways. Based on protein neighbourhood or sub-networks, the human protein-protein interaction network may help to elucidate new protein regulators or modular functions associated to viral or cellular anti-viral strategies.
A user friendly and powerful Web interface based on PHP, JAVA and AJAX technologies was developed. This interface is intended to facilitate: (i) protein and contextual based queries (ii) protein-protein interaction quality filtering and display; (iii) protein-protein interaction network query (viral and host neighbours, virus-virus, virus-host, host-host sub-networks); and (iv) protein network graphical visualization. Description and examples of the database features are available in the Wiki page of the VirHostNet Web site (http://pbildb1.univ-lyon1.fr/virhost net/wiki).
Once logged-in, VirHostNet users can directly query the knowledge base by using a wide range of information concerning viral (e.g. NCBI protein name or accession number) or human proteins (e.g. ENSEMBL gene or protein accession number, NCBI gene name, REFSEQ protein accession number and UNIPROT primary and secondary accession numbers) ( Figure 4A ). AJAX technology was incorporated to control protein name and accession number availability in VirHostNet. Another important feature of the interface is batch query. It allows in-depth analysis of interaction profiles with cellular and/or viral proteins from a list of proteins defined for instance in high-throughput studies (microarray, yeasttwo hybrid). A list of genes or proteins of interest can also be assessed by the mean of contextual information, such as taxonomical information, Gene Ontology terms, KEGG pathways and INTERPRO protein domains. These properties offer a unique access to protein-protein interaction networks: (i) associated to a specific virus taxon or (ii) underlying canonical sub-cellular localization, cellular functions and pathways.
To access protein-protein interactions from a list of proteins ( Figure 4B ), users have: (i) to select all or a subset of proteins of interest; (ii) to define the kind of interactions to retrieve (virus-virus, virus-host and hosthost) and their database origin and (iii) to select the mode of navigation to perform, either protein neighbours or protein subgraph ( Figure 4C ). Neighbours are viral or cellular proteins interacting directly with a protein of interest. A subgraph (or subnetwork) is a graph made of all interactions between a set of proteins. The resulting host-host, virus-host and/or virus-virus protein-protein interactions are then given into a tabulated format in three independent tabbed panels ( Figure 4D ). For each protein-protein interaction, users have a privileged access to interaction meta-data ( Figure 4E ) and a colour code highlights interactions that have been checked by VirHostNet experts.
Beside table representation of protein-protein interactions, a more dynamic and interactive network visualization tool was specifically developed for graph representation of infection networks. This new network visualisator was fully implemented in Jung 2.0 (http://jung.sourceforge.net) as a Java Web applet. It efficiently takes into account viruses and host nodes dichotomy for both graph rendering (colour of nodes) and navigation (host and viral neighbours). The visualisator provides also sliders to dynamically filter graphs based on the number of PMIDs and experimental procedures used to identify interactions. Additional features are also provided to draw protein node size according to the number of viral or host interacting partners (i.e. their degree into the virus-virus, virushost, host-host networks) or to highlight targeted proteins. As a case study, we built a protein sub-network view of the mTOR KEGG signalling pathway that has been found significatively enriched in targeted proteins (Supplementary Table 4 ). Indeed, the modulation of PI3K-Akt-mTOR signal transduction pathway by viruses has been shown to play a crucial role in inhibition of apoptosis, cell survival, cell transformation, viral replication and viral assembly (40, 41) . To identify and compare how viruses interplay with this network, virus-host protein interactions annotated by VirHostNet were added. This mTOR-infection network is composed of 42 cellular interacting proteins (blue nodes), 10 viral proteins (coloured nodes according to viral taxonomy), 84 host-host (blue edges) and 14 virus-host (red edges) physical proteinprotein interactions ( Figure 5 ). Protein network visualization showed that cellular proteins of this pathway are highly inter-connected in contrast to the classical representation given by the KEGG pathway, underlying the extreme complexity and regulation of this pathway. Moreover, graph visualization allows identifying viral proteins targeting multiple cellular proteins (e.g. NS5A protein interacting with AKT1, PDPK1 and PIK3CB) and reciprocally cellular proteins interacting with multiple viral proteins (e.g. HIF1A interacting with LANA of Human Herpes Virus 8 type P and X protein of Hepatitis B Virus). Hence, the VirHostNet interface allows users to visualize protein interaction networks associated to any kind of GO term, KEGG pathway, list of proteins or keywords and to analyse how they interplay with viruses.
VirHostNet provides now a public access to the largest known resource of integrated virus-virus, virus-host and host-host protein interaction networks. Literature-and database-curated interactions have led to the definition of an original and high-confidence protein-protein interactions dataset. We have briefly illustrated the need of this high-confidence dataset for the characterization of cellular functions targeted by viruses. This resource may also be crucial for network-based analysis of molecular mechanisms involved during viral infections, such as cellular network properties disturbed after the connection of viral proteins. VirHostNet will also provide a backbone for automatic screening of specific protein domains or peptides motifs associated to virus-host interactions and hence may help to delineate at the proteome-wide scale footprints in both viral and host proteins sequences. VirHostNet will allow systematic prediction of virushost protein-protein interologs based on sequence homology criteria between closely related viral proteins. The knowledge-based system is also intended to integrate virus-host protein-protein interactions data derived by our team from high-throughput yeast-two hybrids experiments (Orthomyxovirus, Paramyxovirus, Flavivirus . . .). Thus, the availability of virus-virus and virus-host networks for a broad range of viruses will encourage comparative analysis and will be very helpful for the identification of molecular interactions associated to viral pathogenesis or virulence. As virus-host and virusvirus protein-protein interactions curation is one of the central features of the VirHostNet knowledge base, one of our missions is to keep these data up to date Users can select one or more proteins from the list of proteins (by default, all proteins are selected). Then, from the preferences panel, they have to choose the kind of interaction to retrieve (virus-virus, virus-host or host-host) or the database origin. Finally, they have to choose an operation ('neighbours' or 'sub-graph'). The 'neighbours' button allows users to retrieve all direct protein-protein interactions from a single protein or a list of selected proteins. The 'sub-graph' button allows users to retrieve only protein-protein interactions occurring between selected proteins (see result in Figure 4D ). (D) List of the 89 protein-protein interactions occurring between proteins of the mTOR pathway. For the interacting proteins, NCBI taxonomy name, ENSEMBL gene acc (host)-NCBI protein acc (viruses) and official gene name (host)-product name (viruses) are given. The '?' link at the end of each interaction line allows users to access interaction annotations ( Figure 4E ). In the protein-protein interactions research panel, in addition to 'neighbours' and 'sub-graph' buttons, the 'visualize' button allows from a list of interactions the graphical visualization of the resulting network (see result in Figure 5 ). (E) More detailed information concerning protein-protein interactions are available. PMID, PSI-MI method accession number and database origin of the selected interaction are given.
continuously from data published in scientific journals. The update of public databases will occur at least once or twice a year in order to keep the data as current as possible. In the next future, integration of other host species, such as mammals or insects, is envisaged. This will facilitate comparison of interaction profiles among different hosts and thus may help to elucidate the molecular basis underlying the ability of some viruses to overcome the inter-species barrier. Efforts will be made to facilitate data exchange with other generalist databases (MINT, INTACT) and to add Web2.0 capabilities to the Web interface (save, comparison and analysis of user customized networks). Altogether, VirHostNet provides an entry gate for proteome wide analysis of the virus-host system and will greatly help scientists willing to take advantage of functional genomic and systems biology to decipher viral infection, evolution and pathogenesis mechanisms and/or to rationalize anti-viral drug design.
Public access to the VirHostNet knowledge base is available at http://pbildb1.univ-lyon1.fr/virhostnet. Access can be made either anonymously (by default) or by creating a personal account (register in the account menu). On simple request, this personal account allows users to participate to the literature-curation effort. Literaturecurated and Database-curated protein-protein interactions flat files are available in a tabulated format on request. Contact V.N. (navratil@univ-lyon-1.fr) for more information. Figure 5 . VirHostNet Visualisator. Graph representation of the mTOR-infection network is drawn (right of the applet). Only interacting proteins of the mTOR pathway are represented (cellular proteins = blue nodes; host-host protein-protein interactions = blue edges). Viral interacting proteins are also added to define the infection network (viral proteins = coloured nodes according to viral taxonomy; virus-host protein-protein interactions = red edges). Cellular proteins of the network targeted by at least one viral protein (red stroke) are highlighted. The width of the edges is roughly proportional to the number of PMIDs used to describe the interaction. The width of the nodes is roughly proportional to the cellular degree, i.e. the number of cellular partners in the whole network. The taxonomical classification of viral proteins interacting with the mTOR network is given (left panel of the applet). The menu of the applet (top of the applet) allows users to define nodes and edges preferences (width, colour, labelling), interaction filtering (according to the number of methods, number of PMIDs or both), network navigation (expand viral or host neighbours) and graph layout. To obtain this figure, from the list of the 89 selected protein-protein interactions obtained in Figure 4E , users have to click on 'visualize' button. Then from the applet, in picking mode, they have to select all proteins and ask for viral proteins neighbours (in expand menu). Rfam: updates to the RNA families database Rfam is a collection of RNA sequence families, represented by multiple sequence alignments and covariance models (CMs). The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, using sensitive BLAST filters in combination with CMs. A minority of families with a very broad taxonomic range (e.g. tRNA and rRNA) provide the majority of the sequence annotations, whilst the majority of Rfam families (e.g. snoRNAs and miRNAs) have a limited taxonomic range and provide a limited number of annotations. Recent improvements to the website, methodologies and data used by Rfam are discussed. Rfam is freely available on the Web at http://rfam.sanger.ac.uk/and http://rfam.janelia.org/. Rfam is a database of sequence families of structural RNAs, including non-coding RNA genes as well as cisregulatory RNA elements. Rfam release 9.0 contains 603 families, each represented by a multiple sequence alignment of known and predicted representative members of the family, annotated with a consensus base-paired secondary structure. This so-called SEED alignment is used to build a covariance model (CM) with the Infernal software (1) . Each Rfam covariance model is searched against a nucleotide sequence database, producing a list of putative hits. Matches that score above a curated threshold are then aligned to the CM to produce a so-called FULL alignment. This process is outlined diagrammatically in Figure 1 . The Rfam database was developed as a generic approach to the annotation of structured RNA families on genomic sequences (2, 3) , but it has been widely used as a source of reliable alignments and structures for the purposes of training and benchmarking RNA sequence and secondary structure analysis software.
All Rfam models are searched against an underlying nucleotide sequence database, known as RFAMSEQ, which is derived from the EMBL nucleotide sequence database (4) . Prior to release 9.0, RFAMSEQ represented only the various species sections of EMBL. These sections contained only sequences that were considered to be of finished quality and excluded sequences from many important genomes. With release 9.0, RFAMSEQ has been expanded to include the whole genome shotgun (WGS) and environmental sequence (ENV) divisions. These changes have increased the number of sequences in RFAMSEQ by more than an order of magnitude (2 225 030 sequences in Rfam 8.0 versus 29 574 458 sequences in Rfam 9.0).
In order to make it feasible to search more than 120 gigabases of sequence with hundreds of covariance models in a reasonable time, we use sequence-based filters to prune the search space prior to applying the more accurate and more computationally expensive CMs. One of the primary limitations of the Rfam annotation pipe-line has been the use of BLAST-based sequence filters, which are likely to compromise search sensitivity. In order to address this issue at least partially, NCBI-BLAST has been replaced with a WU-BLAST search, which has been tuned for high sensitivity and low sequence similarity. A benchmark of several homology search tools has shown WU-BLAST to be the more accurate of the two methods on nucleotide data (5) . Additionally, in order to make the BLAST filters more *To whom correspondence should be addressed. Tel: +44 1223 494 983; Fax: +44 1223 494 919; Email: pg5@sanger.ac.uk ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. similar to profile HMMs, a sequence mask has been applied to each sequence in the alignment. Any nucleotide in an alignment column that has either a low frequency or is an insert relative to the majority of the rest of the sequences is 'soft masked' and not used for the BLAST word matches. These masked nucleotides do, however, still contribute to alignments that were seeded in the flanking regions. This approach has resulted in many fewer spurious hits with no detectable cost to sensitivity (data not shown), thus allowing E-value thresholds to be further relaxed. These observations together mean that the BLAST filters have been improved in terms of specificity and sensitivity.
In order to improve the species and sequence depth of individual Rfam families, more than 370 families have been expanded by an 'iteration' process, in which some sequences in the FULL alignment are chosen for promotion to the SEED alignment. The sequences selected from FULL alignments for inclusion in the SEED must pass a series of stringent quality control requirements and be manually approved by a curator. The quality control steps include: ensuring that the sequence and secondary structure are consistent with the existing SEED sequences; the sequence identity with existing SEED sequences falls within 60-95%; the sequence is not truncated with respect to the SEED alignment. The curator also ensures that the new sequences make phylogenetic sense before allowing them to be incorporated into an updated SEED. An example of the utility of iteration is the snoRNA U103 SEED from Rfam 8.1 (accession: RF00188), which contained just three sequences and spanned two eutherian mammals (human and mouse). The SEED in Rfam 9.0 after iteration contains 42 sequences and spans Eutheria, Teleost (ray-finned fishes), Iguanidae, Monotremes, Marsupials, Placentals, Aves and Chondrichthyes (cartilaginous fishes).
Phylogenetic trees have been estimated for both the SEED and FULL alignments. For the majority of the alignments we produced the trees using an accurate maximum-likelihood approach, which included models of indels (6) . However, the computational complexity of tree estimation meant that maximum-likelihood was not always possible and hence, where the number of sequences in the alignment was greater than 64, a neighbour-joining method was used instead (7). Large alignments and trees are problematic, both in terms of the computational cost of generation and the challenges of displaying them. Therefore, where the number of sequences in the alignment was greater than 1024, the highly similar sequences were filtered by sequence similarity, resulting in relatively sparse and easily presented trees that required comparatively little computing power to generate.
We are currently developing a new Rfam website, with the aim of improving the presentation of Rfam data and providing more and better tools for searching the data. The new site is now available from http://rfam.sanger.ac.uk/ and can be used to access Rfam 9.0 data. The new site lacks some features of the old site, but we aim to add all existing features and add many new ones over the coming months. Note that, at time of writing, the new website was available only at the Wellcome Trust Sanger Institute (http://rfam.sanger.ac.uk/). The two mirror sites will be updated to run the same website to coincide with the release of Rfam 9.1. The new site provides detailed overviews of Rfam families, including: a snapshot of the latest community-contributed annotation from Wikipedia (see below); tools for viewing and downloading the sequence alignments in various formats; representations of predicted secondary structure (see below); the taxonomic tree for the family; and phylogenetic trees for the SEED and FULL alignments.
Additionally, we provide several search tools in the new site. We currently support interactive searches, allowing a single RNA sequence to be searched against the whole Rfam database, and a batch search tool for searching multiple sequences against Rfam, the results of which are returned to the user via email. A new taxonomy search tool allows the user to find Rfam families that are specific to a given taxonomic level, or those found in a set of taxonomic levels that are specified by a complex, boolean query. For example, the query 'Drosophila AND Caenorhabditis NOT Mammalia' returns the two Rfam families (RF00047 and RF00533) that contain sequences from both drosophila and caenorhabditis but no sequences from any mammalian species.
New graphical representations of secondary structures have been added to the Rfam website, based on software from the Vienna RNA package (8) . We now annotate several statistics directly on secondary structure diagrams, including sequence conservation, covariation, base-pair conservation and the maximum CM scores (Figure 2 ). The sequence conservation metric uses a metric computed for each column in the alignment; this is the frequency of the most common nucleotide in each column (Figure 2A ). The covariation metric is based upon that used by RNAalifold (9) . For each base pair in the consensus structure and for each pair of sequences in the alignment, the difference in structurally consistent and inconsistent mutations is taken. Each mutation is weighted using a treeweighting scheme (10) and this value is then normalized by the number of possible mutations ( Figure 2B ). The base-pair conservation metric is the fraction of canonical base pairs (Watson-Crick and G:U) in any two columns that correspond to a base pair in the consensus structure ( Figure 2C) . The maximum covariance model score and corresponding nucleotide/base pair is computed for each node in the CM. The resulting sequence, structure and bitscores are used to produce a marked up secondary structure ( Figure 2D ).
The Rfam website now draws textual annotation of RNA families directly from the scientific community, through the online encyclopedia Wikipedia. Any updates to relevant Wikipedia articles are downloaded on a nightly basis using the MediaWiki API, verified by members of the consortium and presented on the Rfam site (11) . We consider the resulting articles to be a great improvement on the original static text because they are frequently updated, provide cross links to related articles and are generally considerably more comprehensive and informative than the original Rfam annotations that they replace.
The rate of discovery of new RNA families is accelerating rapidly, facilitated by advancements in new sequencing technologies (12, 13) and targeted computational screens (14) (15) (16) (17) . Keeping abreast of these updates whilst still ensuring the quality of alignments and secondary structures is an ongoing challenge for Rfam. We continue to evaluate new technologies and techniques as they emerge and will adopt new procedures for building and checking Rfam families as necessary.
We have been actively updating Rfam families and database crosslinks using more specialized RNA databases such as miRBase (18) , IRESite (19) , Pseudobase (20), snoRNABase (21) , the plant snoRNA database (22) , TransTerm (23) and the Yeast snoRNA database (24) . As a result of these efforts, the next release of Rfam (version 9.1) will contain more than 700 entirely new families, bringing the total number of Rfam families to over 1300.
A new version of Infernal (v1.0) is now available (http://infernal.janelia.org) and we plan to use this 0 Sequence conservation latest version to prepare the next major release of Rfam. Testing suggests that, compared with the version used for Rfam 9 (v0.72), v1.0 is faster and slightly more sensitive, whilst introducing for the first time E-values for hits returned from database searches. Although the speed increase will not be sufficient to obviate the need for BLAST filters in the Rfam production pipeline, this remains a major goal for Infernal development. Importantly, Infernal v1.0 is not compatible with the Rfam 9 CM files. Rfam/Infernal users may wish to generate new CMs from Rfam 9 SEED or FULL alignment files. We have mapped a subset of three-dimensional RNA structures found in the Protein DataBank (PDB) (25) (primarily SRP and ribosomal RNAs) to corresponding sequences in Rfam. In an initial feasibility study, we have demonstrated that RNA sequences can be retrieved from PDB files and mapped to Rfam sequences reliably. The mapping is currently performed using BLAT (26) to detect local regions of high similarity with high specificity. The positions of matches to Rfam entries are transferred to the PDB sequences, allowing us to colour threedimensional structures as in Figure 3 . We intend to rollout this mapping across all Rfam families and PDB entries using both local similarities and global matches to Rfam models. This sequence-to-structure mapping will allow us to use determined tertiary structures to calculate secondary structure as a quality control for existing families, and catalogue interactions between RNA-RNA and RNAprotein families.
A further area of active research at Rfam is how best to distribute genome annotations. We plan to make annotations available in a variety of formats including the distributed annotation service (DAS) (27) , General Feature Format (GFF) (http://song.sourceforge.net/gff3.shtml) and EMBL format, together with links to relevant genome browsers, e.g. ENSEMBL, UCSC and Genome Reviews. New Respiratory Enterovirus and Recombinant Rhinoviruses among Circulating Picornaviruses Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5′ untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses. H uman rhinoviruses (HRVs) and enteroviruses (HEVs) are leading causes of infection in humans. These 2 picornaviruses share an identical genomic organization, have similar functional RNA secondary structures, and are classifi ed within the same genus (www.ictvonline.org/virusTax-onomy.asp) because of their high sequence homology (1) . However, despite their common genomic features, these 2 groups of viruses have different phenotypic characteristics. In vivo, rhinoviruses are restricted to the respiratory tract, whereas enteroviruses infect primarily the gastrointestinal tract and can spread to other sites such as the central nervous system. However, some enteroviruses exhibit specifi c respiratory tropism and thus have properties similar to rhinoviruses (2) (3) (4) (5) . In vitro, most HRVs and HEVs differ by their optimal growth temperature, acid tolerance, receptor usage, and cell tropism. The genomic basis for these phenotypic differences between similar viruses is not yet fully understood.
HRVs and HEVs are characterized by ≈100 serotypes. Recently, molecular diagnostic tools have shown that this diversity expands beyond those predefi ned serotypes and encompasses also previously unrecognized rhinovirus and enterovirus genotypes. As an example, a new HRV lineage named HRV-C was recently identifi ed and now complements the 2 previously known A and B lineages (6-8) (N.J. Knowles, pers. comm.). The C lineage has not only a distinct phylogeny (9) (10) (11) (12) (13) (14) (15) (16) but is also characterized by specifi c cis-acting RNA structures (17) .
In this study, we screened a large number of persons with acute respiratory diseases by using assays designed to overcome the diversity of both rhinoviruses and enteroviruses circulating in humans. Whenever possible, we systematically sequenced 5′ untranslated region (UTR), capsid protein VP1, and protease precursor 3CD regions of strains. Our goals were 1) to characterize the diversity of circulating rhinoviruses and, to a lesser extent, enteroviruses, to identify putative new picornavirus variants, and 2) to assess whether recombination may drive HRV evolution, which has not been shown in natural human infections (18) .
Reverse transcription-PCR (Superscript II; Invitrogen, Carlsbad, CA, USA) was performed on RNA extracted by using the HCV Amplicor Specimen Preparation kit (Roche, Indianapolis, IN, USA), TRIzol (Invitrogen), or the QIAamp Viral RNA Mini kit (QIAGEN, Valencia, CA, USA). Real-time PCR specifi c for HRV-A, HRV-B, and HEV (19) , and a generic panenterhino real-time PCR (forward primer 5′-AGCCTGCGTGGCKGCC-3′, reverse primer 5′-GAAACACGGACACCCAAAGTAGT-3′, and probe 5-FAM-CTCCGGCCCCTGAATGYGGCTAA-TAMRA-3′), were performed in several cohort studies (Table) .
Picornavirus-positive samples were detected from patients enrolled in cohort studies in different regions of Switzerland during 1999-2008. The main characteristics of these populations, type of respiratory specimens, and screening methods are shown in the Table. The rhinovirus serotypes used for 3CD sequencing were obtained from the American Type Culture Collection (Manassas, VA, USA).
Sequencing was performed directly from the clinical specimen except for samples selected by routine isolation methods on human embryonic (HE) primary fi broblast cell lines (Table) or for HRV reference serotypes. Primers used  to amplify the 5′-UTR and the VP1 and 3CD regions are  listed in online Technical Appendix 1 Table 1A (available from www.cdc.gov/EID/content/15/5/719-Techapp1.pdf).
Full-length genome sequences of CL-1231094, a related clinical strain of enterovirus, and partial sequences of CL-Fnp5 and CL-QJ274218 were obtained as follows. RNA extracted by using the QIAamp Viral RNA Mini kit (QIAGEN) plus DNase treatment or with Trizol was reverse transcribed with random-tagged primer FR26RV-N and amplifi ed with the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) with a specifi c forward primer and FR20RV reverse primer (online Technical Appendix 1 Table 1B ) (23) . Amplifi cation products were separated by electrophoresis on agarose gels and fragments (0.6-2.5 kb) were extracted by using the QIAquick Gel Extraction kit (QIAGEN). Purifi ed products were cloned by using the TOPO TA cloning kit (Invitrogen).
Minipreps were prepared from individual colonies and clones with the largest inserts were chosen for sequencing. Sequences obtained were used to design a new forward primer (online Technical Appendix 1 Table 1 ) to advance toward the 3′ end of the genome. PCR products of 3′ genomic ends were obtained by using the BD Smart Race cDNA amplifi cation kit (Becton Dickinson, Franklin Lakes, NJ, USA) according to manufacturer's instructions. All PCR products were purifi ed by using microcon columns (Millipore, Billerica, MA, USA) and sequenced by using the ABI Prism 3130XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA). Chromatograms were imported for proofreading with the vector NTI Advance 10 program (Invitrogen). Overlapping fragments were assembled with the contigExpress module of the vector NTI Advance 10.
Alignments were constructed by using MUSCLE (24) with a maximum of 64 iterations. (For detailed analyses, see http://cegg.unige.ch/picornavirus.) Multiple FastA was converted into PHYLIP format (for tree building) with the EMBOSS program Seqret (25) . Trees were built with PhyML (26) by using the general time reversible model, BIONJ for the initial tree, and optimized tree topology and branch lengths. Trees with <50 species and larger trees used 16 and 8 rate categories, respectively. Transition/transversion ratios, proportions of invariant sites, and shape parameters of the γ distribution were estimated.
To investigate the hypothesis of recombination and map the breakpoints, we adapted the bootscanning method (27) as follows. The alignment was sliced into windows of constant size and fi xed overlap and a 100replicate maximum-likelihood (using HRV-93 as an outgroup) was computed for each window. From each tree, the distance between the candidate recombinant and all other sequences was extracted. This extraction yielded a matrix of distances for each window and for each alignment position. A threshold was defi ned as the lowest distance plus a fraction (15%) of the difference between the highest and lowest distances. The nearest neighbors of the candidate recombinant were defi ned as sequences at a distance smaller than this threshold. This distance ensured that the nearest neighbor, as well as any close relative, was always included. Possible recombination breakpoints thus corresponded to changes of nearest neighbors. Serotypes included in this analysis represented serotypes close to CL-013775 and CL-073908 on the basis of 5′-UTR and VP1 phlyogenetic trees (online Technical Appendix 2 Figure 1 , panels A, B, available from www.cdc.gov/EID/ content/15/5/719-Techapp2.pdf), as well as serotypes close to CL-135587 on the basis of VP1 and 3CD phlyogenetic trees (online Technical Appendix 2 Figure 1 , panels B, C) and whose full-length sequence was available.
Distance matrices were computed from alignments with the distmat program in EMBOSS (http://bioweb2. pasteur.fr/docs/EMBOSS/embossdata.html) by using the Tamura distance correction. This method uses transition and transversion rates and takes into account the deviation of GC content from the expected value of 50%. Gap and ambiguous positions were ignored. Final values were then converted to similarity matrices by subtracting each value from 100. 
Persons enrolled in several cohorts of children and adults with respiratory infections (Table) were screened for picornavirus by culture isolation on HE cell lines, real-time PCR specifi c for HRV-A and HRV-B (19) , or by a panenterhino real-time PCR designed to theoretically detect all rhinoviruses and enteroviruses with publicly available sequences. Of 1,592 respiratory samples tested by real-time PCR, 248 were virus positive (Table) . The 5′-UTR sequences were obtained for 77 real-time PCR or culture-positive samples and VP1 and 3CD sequences for 48 of these (Table; online Technical Appendix 1 Table 2 ). In parallel, the 3CD sequences were identifi ed for all reference serotypes. The results of this screening are summarized in online Technical Appendix 1 Table 2 , and all sequences are available from GenBank (accession nos. EU840726-EU840988).
On the basis of these results, respiratory infections caused by HRV-B might be less frequent than those caused by HRV-A, and HRV-A infections are distributed among the whole library of reference serotypes. A specifi c real-time PCR used to detect enteroviruses in respiratory specimens from some of the cohorts studied indicated that these viruses are rare in children (2.5% vs. 6.3% for HRV) and even rarer or absent in adults (0% vs. 24% for HRV) (28) .
To include all 99 HRV reference strains and new di- Table 2 ).
Characterization of HRVs newly identifi ed during 2006-2008 showed that they all belong to the same HRV-C species (9) (10) (11) (12) (13) (14) (15) (16) . Recently, Lee et al. (13) identifi ed another cluster of viruses (HRV-C′; Figure 1 , panel A) and suggested that this group was phylogenetically distinct from all other HRVs on the basis of analysis of their 5′-UTR sequences. To defi ne the phylogeny, we adapted a previously described method (23) to complete the genome sequence directly from our clinical strains (CL-Fnp5 and CL-QJ274218) that showed a similar divergent 5′-UTR (online Technical Appendix 2 Figure 1, panel A) . A condensed version (Figure 1, panel B) of the phylogenetic tree based on VP1 sequences (online Technical Appendix 2 Figure 1, panel B) indicated that CL-Fnp5 clustered with the new HRV-C clade, a fi nding further confi rmed by CL-QJ 274218 partial sequences. This fi nding supports the view that new HRVs variants described since 2006 (9-16) all belong to the same lineage.
As shown in Figure 1 , panel A, the panenterhino realtime PCR enabled detection of a new HEV strain phylogenetically distinct from all previously known HEV species and associated with respiratory diseases. Enterovirus-specifi c real-time PCRs or reference VP1 primer sets routinely used to type enteroviruses (primers 222 and 224 and nested primers AN88 and 89) (29,30) did not amplify this new genotype. We could not grow this virus on HeLa and HE cell lines. Consequently, we applied the method described above to complete the genome sequence directly from the CL-1231094 (EU840733) clinical specimen. VP1 and fulllength genome sequences showed that, albeit divergent at the 5′-UTR level, this new variant belonged to the HEV-C species (Figure 1, panels B, C) . Full-length genome phylogenetic tree ( Figure 2 ) and VP1 protein identity plots (online Technical Appendix 2 Figure 2 ) with all members of the HEV-C species indicated that this virus represents a new HEV-C genotype that shares 68%, 66%, and 63% nucleotide and 77%, 75%, and 68% amino acid sequence identity, respectively, with coxsackieviruses A19 (CV-A19), A22, and A1, the closest serotypes. This new virus was named EV-104 (www.picornastudygroup.com/types/ enterovirus_genus.htm).
Specifi c primers (Ent_P1.29/P2.13 and Ent_P3.30/ P3.32; online Technical Appendix 1 Table 1C) were then designed to amplify the VP1 and 3D regions of the 7 other samples of this cluster collected from children with acute respiratory tract infections and otitis media. VP1 nucleotide homology among these strains was 94%-98%, except for 1 distantly related sample (74%-76%), which may represent an additional genotype. Additional sequencing is ongoing to verify this assumption. At the 5′-UTR level, the strain described by Lee et al. (13) and EV-104 diverged from other members of HRV-C and HEV-C species, respectively. Thus, the 5′-UTR-based phylogeny was inconsistent with that based on VP1 sequences and suggested possible recombination events (Figure 1, panels A, B) . Because the 5′-UTR is the target of most molecular diagnostic assays, this sequence divergence needs to be taken into account in future studies.
Other studies have provided sequences of clinical strains, but genetic characterization was often limited to 1 genomic region. Our goal was to sequence 3 genomic regions for each analyzed strain to determine defi nitively whether recombination events could represent a driving force for the evolution of rhinoviruses in their natural environment. Although recombination events have been suggested for reference serotypes, they have never been shown for circulating clinical strains (18, 31, 32) . In contrast, recombination is well established as a driving force of enterovirus evolution. Thus, we completed the 5′-UTR, VP1, and 3CD sequences of 43 clinical strains by using a pool of adapted and degenerated primers (online Technical Appendix 1 Table 1A) .
Independent phylogenetic trees (online Technical Appendix 2) and similarity matrices were constructed for the 3 genomic regions. Since the last common ancestor and as depicted on the distance matrices and highlighted by boxplots of maximum-likelihood branch length distributions (online Technical Appendix 2 Figure 3 ), there are more mutations fi xed in the VP1 region than in the 3CD region, and more in the 3CD region than in 5′-UTR, which is indicative of a variable rate of evolution in these regions. Accordingly, VP1 sequences enabled genotyping of all but 3 clinical strains analyzed (online Technical Appendix 2, Figure 1 , panel B). These strains may represent rhinovirus genotypes only distantly related to predefi ned reference serotypes. In contrast, genotyping based on 3CD and 5′-UTR was less accurate, as expected. These results confi rmed that molecular typing of rhinoviruses, similarly to other picornaviruses, must use capsid sequences.
Phylogeny of the 5′-UTR, VP1, and 3CD of reference serotypes showed many incongruities caused by insufficient tree resolution or recombinant viruses as previously proposed (18, 31) . As an example, 2 VP1 clusters including HRV-85/HRV-40 and HRV-18/HRV-50/HRV-34 (online Technical Appendix 2 Figure 1 , panel B) were reorganized as HRV-85/HRV-18/HRV-40 and HRV-50/HRV-34, respectively, on 3CD (online Technical Appendix 2 Figure  1 , panel C). The differential cosegregations between these virus strains suggested recombination events. When available, full-length genome sequence bootscanning applied to all serotypes will give an estimate of the number of reference strains with mosaic genomes.
Similarly, the noncoding region, VP1, and 3CD trees showed major phylogenetic incongruities for 3 clinical isolates (online Technical Appendix 2 Figure 1 ). Two of these isolates (CL-013775 and CL-073908) were typed as HRV-67 on the basis of VP1 sequence and were closest to this serotype in 3CD, whereas the 5′-UTR cosegregated with HRV-36 (see 5′-UTR recombinant; online Technical Appendix 2 Figure 1 , panels A-C). These viruses were isolated by cell culture from 2 epidemiologically linked cases and thus represented transmission of the same virus. To confi rm the recombination, we completed the sequencing by obtaining the 5′-UTR, VP4, and VP2 sequences (EU840918 and EU840930) and compared them with HRV-36, HRV-67, and other closely related serotypes. Bootscanning analysis ( Figure 3 , panel A) enabled mapping of the recombination site within the 5′-UTR, just before the polyprotein start codon. Sequence alignment mapped recombination breakpoints more precisely between positions 524 and 553 with reference to HRV-2 (X02316). The other incongruent isolate (CL-135587) was typed as HRV-76 on the basis of VP1 sequence and was closest to this serotype in the 5′-UTR, but 3CD cosegregates with HRV-56 (3C recombinant; online Technical Appendix 2 Figure 1, panels B, C) . Similarly, we completed the fulllength sequence of this isolate (EU840726) and HRV-56 (EU840727). The same approach enabled mapping of the recombination site at the N terminus of protein 3C between positions 1511 and 1523 with reference to HRV-2 ( Figure 3, panel B) . These results demonstrate that recombination occurs among clinical rhinoviruses. In our analysis of 40 rhinovirus-positive samples collected over 9 years (3 additional samples were duplicates of 2 different viruses; online Technical Appendix 1 Table 2) for 3 genomic regions, 2 of the analyzed viruses appeared to be recombinants. The 2 documented recombinations occurred in members of the HRV-A species. The design of this study and technical issues (e.g., inability to sequence low viral loads) limited the ability to calculate a recombination rate, particularly for HRV-B and HRV-C.
Our genomic analysis of picornaviruses associated with upper or lower respiratory diseases in adults and children indicates that rhinoviruses circulating in the community are widely diverse. The large number of circulating genotypes supports the view that rhinoviruses do not circulate by waves or outbreaks of a given dominant genotype, which might explain the high frequency of reinfection during short periods. As expected, the observed variability is higher for surface capsid proteins, the targets of most immune pressure, and this region remains the only accurate one for genotyping and defi ning phylogeny. Technical constraints such as the limited amount of clinical specimens, the use of different screening methods, and the need to sequence an unknown target of extreme variability might have limited the representativeness of our sequence collection. Therefore, our study should not be considered as an exhaustive epidemiologic analysis of rhinoviruses and enteroviruses associated with respiratory diseases.
By using a systematic approach, we have identifi ed a new enterovirus genotype (EV-104) that has a divergent 5′-UTR. Undetectable by conventional methods, EV-104 could be detected by using a more generic real-time PCR assay designed to match all known available rhinovirus and enterovirus sequences. Such diagnostic tools have and will lead to constant discovery of new picornavirus genotypes (9) (10) (11) (12) (13) (14) 16, (33) (34) (35) (36) . These genotypes may represent viruses, in most instances, that have remained undetected because of insensitive cell cultures or overly restrictive molecular tools. In addition, enterovirus genotypes causing respiratory infections, such as EV-68 and CV-A21, might be underrepresented because enteroviruses are usually searched for in fecal specimens (37) .
EV-104 belongs to the HEV-C species: CV-A19, CV-A22, and CV-A1 are its closest serotypes. These HEV-C subgroup viruses are genetically distinct from all other serotypes of the species. These viruses show no evidence of recombination with other HEV-C strains and, similar to EV-104, do not grow in cell culture (29) . On the basis of our epidemiologic data, we conclude that EV-104 was found in 8 children from different regions of Switzerland who had respiratory illnesses such as acute otitis media or pneumonia. Future studies using adapted detection tools will provide more information on the range of this virus. On the basis of its genomic features and similarities with coxsackieviruses and poliovirus, EV-104 could theoreti- cally infect the central nervous system (2, 38) . Detection of new subtypes of picornaviruses indicates that viruses with new phenotypic traits could emerge, and conclusions on tropism of new strains should be substantiated by extensive experimental or clinical investigations (39) . By completing the sequence of a seemingly divergent rhinovirus (13), we assigned this virus to the new HRV-C species, thus limiting currently to 3 the number of HRV species. For the sake of simplicity, we propose to consider this virus as a member of the HRV-C clade.
Finally, we demonstrated that rhinovirus evolves by recombination in its natural host. Known to be a driving force of enterovirus evolution, rhinovirus recombination among clinical strains has never been observed. Two clinical isolates of 40 viruses analyzed resulted from recombination events and their breakpoints were identifi ed within the 5′-UTR sequence and the N terminus of protein 3C, respectively. These fi ndings are consistent with the fact that recombination breakpoints in picornaviruses are restricted to nonstructural regions of the genome or between the 5′-UTR and the capsid-encoding region (40) . Our observations provide new insight on the diversity and ability of rhinovirus to evolve in its natural host. The fact that only 2 of 40 analyzed viruses over a 9-year period were recombinants is suggestive of a lower recombination frequency in rhinoviruses than in other picornaviruses (32, 40) and might be related, but not exclusively, to the short duration of rhinovirus infection (18, 31, 32) . Recombination events occurred between HRV-A genotypes, but whether they can occur in species B and C remains unknown. Interspecies recombination is rare in picornaviruses and is mainly the result of in vitro experiments. For rhinoviruses, the different location of cre elements in each species might be an additional limiting constraint (17) .
In summary, we have highlighted the large genomic diversity of the most frequent human respiratory viral infection. Our phylogenetic analysis has characterized circulating strains relative to reference strains and has identifi ed a previously unknown enterovirus genotype. We have shown that recombination also contributes to rhinovirus evolution in its natural environment. Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR BACKGROUND: Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy. METHODS AND FINDINGS: We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log(10), and %CV <8% up to 4 log(10) dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance. CONCLUSIONS: The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings. It is estimated that 33.2 million people were infected with HIV-1 at the end of 2007; 2.5 million were children under 15 years of age, the majority of whom acquired infection through mother-tochild transmission (MTCT; [1] ). Antiretroviral therapy (ART) is effective at blocking MTCT and can markedly improve the outcome of pediatric HIV-1 infection. However, efforts to provide widespread access to ART have been hampered by the limited availability of infant diagnostic methods. [2] [3] [4] [5] [6] Methods to diagnose and monitor HIV-1 infection in resource-poor settings are usually limited to serologic assays and CD4/CD8 counts. [2, 7, 8] However, antibody based assays can reliably guide diagnosis and management only after 18 months of age following clearance of passively transferred maternal antibodies. [9] PCR based nucleic acid amplification and quantification of HIV-1 is the gold standard for early HIV-1 diagnosis in infants and for evaluating ART efficacy. [2, 5, [10] [11] [12] [13] [14] [15] [16] Several commercial nucleic acid based molecular tests are available for HIV-1 diagnosis and viral load quantification. [15] These commercial assays require relatively large volumes of blood that need to be processed for plasma, stored, and transported under special conditions. The cost, run-time, training, maintenance and infrastructure needed to perform these assays have also limited their use in low resource settings. [6, 17, 18] Practical and reliable methods to obtain, store, and transport blood samples are necessary to develop cost effective early diagnostic assays in limited-resource settings. Spotting of whole blood onto filter paper offers technical and economic advantages over conventional venipuncture methods since it simplifies sample collection and transport to reference laboratories for diagnostic testing and viral load quantification. [17, [19] [20] [21] [22] [23] [24] In the present study, we describe a LightCycler-based real time PCR assay (rtLC) to quantify viral loads using RNA extracted from filter paper containing dried blood spots. DBS prepared either under controlled conditions in a laboratory setting by research technicians or in a clinical setting by health-professionals were successfully evaluated. This assay has comparable reproducibility, diagnostic accuracy, specificity and broader linear range at a lower cost compared to probe-based commercial assays. The rtLC HIV-1 DBS assay is also capable of detecting and quantifying different clades of HIV-1 without sacrificing sensitivity.
Study Participants. In this prospective study, blood samples were drawn by trained healthcare professionals from 33 HIV-1 positive children who presented to the UMass Mother-Child HIV Program clinic for routine care between May 2005 and September 2008. Study participants ranged in age from 5 to 21 years. Four HIV-1 positive adults who were enrolled in a study of viral kinetics between 1999 and 2002 were also included in the study. Thirty patients were of U.S. origin and were infected with clade B HIV-1, while 7 were of non-U.S. origin, infected with non-B. Nineteen (51%) of the 37 HIV-1 patients were on ART and eight (22%) had undetectable RNA by Versant HIV-1 RNA 3.0 branched DNA (bDNA) assay (Siemens Healthcare Diagnostics) or Ultrasensitive Amplicor HIV-1 Monitor v1.5 assay (Roche Diagnostics) ( Table 1) . These tests were performed by trained technicians. Blood samples were also drawn from 44 HIV-1 negative individuals (27 adults, 17 children). Individual patient consent was obtained from study participants and guardians and no adverse events were associated with drawing of blood for the study. The Human Subjects Committee of the University of Massachusetts Medical School approved all studies.
In addition, DBS samples were collected between February 2008 and January 2009 by heel-stick from 19 infants born to HIV-1 positive mothers at the HEAL Africa Hospital (Goma, Democratic Republic of Congo). Informed consent for testing was obtained from the infants' guardians. These studies were exempt under the guidelines of the Human Subjects Committee of the University of Massachusetts Medical School since the samples were received in the lab as coded, de-identified DBS with no traceable patient information.
Preparation of DBS. At the UMass clinic, whole blood was drawn by venipuncture and collected in tubes with EDTA. DBS were prepared by spotting 50 ml of whole blood onto filter paper (Whatman no. 903; formerly SS 903, Schleicher & Schuell, Keene, NH). For 6 of the 23 patients, cryopreserved plasma was spiked into donor HIV-1 negative blood to prepare DBS since whole blood was not available. The spotted filter papers were allowed to dry for at least 4 hours at room temperature in a hood and placed in individual ziplock bags containing a silica desiccant (Whatman, Schleicher & Schuell, Keene, NH). DBS were stored at 220uC until further processing and testing. The plasma was recovered from the remaining blood sample by centrifugation and stored at 280uC for quantification of viral load using commercial (Roche) RNA assays.
At the HEAL Africa Hospital (Goma, Democratic Republic of Congo) site, DBS samples collected by heel stick from infants born to HIV-1 positive mothers were stored and shipped to the laboratory at ambient temperature; upon receipt in the laboratory, the DBS samples were stored at 220uC until ready to be analyzed.
To better investigate whether the rtLC DBS assay could detect and quantify across clades, in addition to the Congo DBS specimens, we prepared dried spots of whole blood or plasma from 7 non-US origin patient samples (clades A, C, D, AE, AG) spiked into HIV-1 negative donor blood.
Genotype Inclusivity Studies. Genotypic clade determination of patient viruses was done using RT-PCR followed by nested amplification of were performed to amplify env and/or gag genes from plasma HIV-1 RNA. Amplified products were cloned for bidirectional DNA cycle sequencing using an ABI model automated sequencer. Phylogenetic analyses based on env and gag nucleotide sequences were used to determine the clade specificity.
Globally Prevalent Strains. Reference viruses (13 CCR5-tropic and 1 CXCR4-tropic) were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Catalog #11412). [25] This panel represented 6 major globally prevalent HIV-1 clades (A, B, C, D, and circulating recombinant forms CRF01_AE and CRF02_AG).
RNA from these reference viral isolates was extracted using the QIAamp Viral RNA mini kit, according to manufacturer's instructions (Qiagen, Valencia, CA), eluted in 60 ml of elution buffer and then diluted 1:1000. A DBS panel was prepared by mixing 60 ml of each virus isolate with 240 ml of seronegative donor whole blood and then spotting 50 ml of this spiked donor blood onto 903 filter paper. Using the protocol described below, this dried blood spot panel was extracted and run in the rtLC DBS assay. Preparation of Standard Curve. To ensure uniformity and reproducibility in the DBS preparation and extraction process, we prepared customized DBS standards with known viral copies. An HIV-1 clade B infected patient isolate with known viral load was selected and spiked into HIV negative donor whole blood to prepare DBS in five fold serial dilutions. A series of 5 independent extractions were performed and quantified using the rtLC DBS assay in a total of 25 independent runs.
RNA Extraction. Each DBS containing 50 ml of whole blood was cut into 4 equal pieces and incubated for 5 minutes in Tris-EDTA buffer (1.0 M Tris-HCl, 0.1 M EDTA) at room temperature. HIV-1 RNA was extracted from the filter paper using Trizol reagent as lysis solution according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Glycogen (200 mg) was added to the lysis reagent as a carrier to facilitate RNA precipitation for each DBS extraction. After removing the residual filter paper, 1-bromo-2-chloropropane (BCP) was used to separate the extracted RNA from the organic phase. RNA was ethanol precipitated, eluted and further reconstituted in 40 ml of Rnase-free water containing 40 units of Rnasin Plus, an Rnase inhibitor (Promega, Madison, WI).
DNA Extraction. One spot each of DBS filter paper was processed to extract DNA using the previously described resinbased Chelex (Bio-Rad, Hercules, CA) method. [26] Briefly, the paper punched from one entire DBS was immersed in 1 mL of whole blood specimen wash solution (Amplicor HIV-1 Monitor test v1.5, Roche Molecular Systems) and rocked for 2 hours at room temperature. After a quick spin for 5 minutes at high speed, the red-tinged buffer was removed and discarded. The DBS paper was washed once, and then immersed in 250ul of Chelex resin resuspended in DNA dilution buffer (10% v/v in 10 mM Tris buffer, pH 8.3 containing 50 mM KCl). DNA kit Internal Control (3.3ul) was added to the DBS sample, mixed frequently to keep the resin in suspension, and was heated to 100uC for 1 hour, with a 10 second vortex after the first 30 minutes of heating. After a spin for 3 minutes to pellet the resin, the supernatant containing the extracted DNA was removed and stored at 280uC until HIV-reactivity of the extract was determined using Roche Amplicor HIV-1 DNA Amplicor 1.5 v kit according to a previously published protocol. [26] The cellular equivalents of DNA in each test sample were determined in a real-time Taqman PCR assay by probing for CCR5 copy number using forward primer 59-GCTGTGTT-TGCGTCTCTCCCAGGA-39 and reverse primer 59-CTC-ACAGCCCTGTGCCTCTTCTTC-39, and the corresponding fluorogenic probe 59FAM-AGCAGCGGCAGGACCAGCCC-CAAG-TAMRA 39. Known copies of plasmid carrying the CCR5 gene were used to generate the standard curve, from which the number of CCR5 copies and cellular equivalents were determined for each sample. Real-time PCR analyses on RNA extracted from infant DBS was performed as described below.
Real-time PCR. Real-time PCR amplification of HIV-1 RNA was performed in a one-step, single-tube closed system of 32 sample format using the LC-32 Roche LightCycler (Roche, Indianapolis, IN). All the samples were tested in duplicate in a 20 ml total reaction volume containing 16 ml of PCR reaction mix (Quantitect SYBR Green RT-PCR kit [Qiagen, Valencia, CA]; 0.5 mM each of forward and reverse oligonucleotide primer pairs) and 4 ml of the template. The primers were specific to a conserved region of HIV-1 LTR: 59-GRAACCCACTGCTTAASSCTCAA-39 (LTR sense; position 506 of HxB2) and 59-TGTTCGGGCGCCACTGCTAGAGA-39 (LTR antisense; position 626 of HxB2). [27] The PCR reaction was performed according to the following cycling parameters: 1) Reverse-Transcription: 50uC 20 minutes, ramp 20uC/second; 2) Activation: 95uC 15 minutes, ramp 20uC/second; 3) Amplification: 50 cycles a) 94uC 10 seconds, ramp 20uC/second, b) 52uC 20 seconds, ramp 20uC/second, and c) 72uC 20 second, ramp 2uC/ second (single data collection); 4) Melting: a) 92uC 0 second, ramp 20uC/second, b) 57uC 15 seconds, ramp 20uC/second, and c) 92uC 0 second, ramp 0.1uC/second (continuous data collection); 5) Cooling: 40uC 30 seconds, ramp 20uC/second.
HIV-1 specific amplicons were detected using SYBR Green technology according to manufacturer's instructions (Qiagen, Valencia, CA). The number of HIV-1 RNA copies in each test template was measured by its threshold cycle (C t ) and determined from the standard curve of serially diluted customized DBS standards using software for data analysis (Sequence Detector version 1.6, PE Applied Biosystems). For each experiment, a standard curve was generated from serial endpoint dilutions (586,000 to 37 copies) of the customized DBS standards. The threshold cycle values were plotted against copy numbers to construct the standard curve. Quantification of HIV-1 RNA in each test sample was back calculated and viral load was expressed as copies/ml. Using the protocol described above, DBS was extracted, run and analyzed in the rtLC DBS assay by personnel with molecular and virology expertise who were blind to the results.
Rnase-free water (4 ml) was routinely added instead of test sample to 16 ml of the master mix and used as a no-template control for every run. At the end of the assay, the specificity of each amplified product was ascertained by means of melting curve analysis. This eliminated false positive detections due to primer dimers or nonspecific amplicons. To confirm that there was no DNA contamination in the input RNA and to assess the specificity of the reverse transcribed rtLC DBS products, initial assays included PCR reactions without the addition of reverse-transcriptase enzyme. Gel electrophoresis of the amplicons was initially performed to further confirm the specificity of the products. Each sample was tested in replicates and a second DBS was independently extracted and run to verify a positive result. The test was repeated if the sample had indeterminate results, and if necessary a new spot was extracted and tested if the results of the repeat test were also indeterminate. Quality control for each experiment was assessed by the performances of the standard curve and the negative control. All DBS prepared by trained personnel were tested for this study as long as multiple filter spots were available for each patient, and the blood spots were good quality with no hemolysis or clots.
To determine whether viral load data using the Roche LightCycler system were comparable to viral load data obtained by using MyiQ TM Single-Color Real-Time I-Cycler PCR Detection System, (Bio-Rad, Hercules, CA), cDNA synthesis was carried out with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Select patient and standard DBS-RNA were used to synthesize cDNA, and then amplified and quantified using the iQ SYBR Green Supermix Kit (Bio-Rad, Hercules, CA) by using these parameters: 3 min @95C, 50 cycles of 10 sec @94C, 20 sec @52C, 20 sec @72C, and final extension for 20 sec @72C. Results were analyzed using MyiQ software, version 1.0.410 (Bio-Rad, Hercules, CA).
Sensitivity and Linearity of the Assay. Linearity of the assay was evaluated using serial dilutions of the customized standard DBS described earlier (586,000 copies to 37 copies).
Statistical Analyses. The customized DBS standards serial dilutions were tested in 25 separate runs to determine the threshold, inter-assay precision and linearity of the rtLC DBS assay. Trained statisticians performed data analyses. Probit analysis was used to determine 95% and 99% detection limits. The likelihood ratio test was used to evaluate the effect of extractions and runs; the fitness was measured by R-squared value. Spearman correlation coefficients were calculated to determine the relationship between the HIV-1 RNA levels quantified by the Roche Ultrasensitive and bDNA assays with the rtLC DBS technique. The sensitivity of the rtLC DBS assay was calculated as the number of positive results divided by the total number of samples from infected patients who had plasma viral load above the threshold level of detection of commercial assays (bDNA and Roche Ultrasensitive) expressed as a percentage. The specificity was calculated as the number of negative samples divided by the total number of known negative specimens obtained from normal healthy donors and uninfected infants expressed as a percentage. Paired Wilcoxon tests were used to determine the effect of temperature and time on RNA stability and assay performance.
The linear dynamic range of the rtLC DBS assay was initially assessed using a 5 log 10 dilution series of the customized standard DBS. The assay was shown to be linear over the entire range of 586,000 to 37 copies. A linear regression of the rtLC DBS customized standards copies on true concentrations yielded a correlation coefficient of 0.984 (P,0.001) and the fitted model is shown in Figure 1 . The slope of 1.037 closely approximates the theoretical maximum amplification efficiency of 100%; the fitted slope is slightly greater than 1 due to the several undetected cases in low range. To estimate the detection limit of the assay, we performed 22 extra runs at low concentrations (7, 17, 37, 83, 187 copies), which spread evenly in log scale and contained two concentration 37 and 187 that were already in design. We used all available data at concentrations from 7 to 937 to fit the probit model to get estimations for 95% detection limit as 135, with 95% confidence interval [82, 223]; 99% detection limit was estimated as 292, with 95% confidence interval [147, 583] ( Table 2) .
To evaluate the inter-assay precision of the assay, the data on the customized DBS standards of known concentrations (586,000 to 37 copies) were analyzed across 25 independent experiments. The results of the statistical analysis for the standard deviation (SD) and percent coefficient of variation (%CV) at each concentration level are shown in Table 3 . The coefficient of variation increased dramatically when the true concentration was approximately equal to or below the assay's detection limit.
The clinical sensitivity of the assay was determined using DBS specimens of 32 patients with known infection and viral loads above the detection threshold of the bDNA and Roche Ultrasensitive assays (Table 1 ). All 32 samples were positive using the rtLC DBS assay, yielding a clinical sensitivity of 100% for this cohort. An additional 5 DBS samples drawn from infants of indeterminate HIV-1 infection status tested positive on the rtLC DBS assay; all 5 were also positive on the commercial DNA assay. The clinical specificity of the rtLC DBS assay was determined using DBS specimens from 27 healthy HIV-1 negative adult donors and 17 HIV-1 negative infants born to HIV-1 positive women. All of these samples were negative for HIV-1 RNA, resulting in a clinical specificity of 100%. The information on the clinical specificity of rtLC DBS assay in HIV negative donors is provided in Table S1 in the supplementary section.
rtLC Assays Using Samples Collected in the Field DBS samples from infants born to HIV positive women were also prepared at a clinical site in Goma, Congo. The range of cell equivalents across these Congo DBS samples was 2,769 to 201,116 Table 2 . Calculation of the 95% and 99% detection limit of the rtLC DBS assay using probit analysis. 
The rtLC DBS assay successfully detected HIV-1 RNA in each of the DBS samples prepared from 14 viral isolates representing an international HIV-1 reference panel (clades A, B, C, D and CRF-AE and CRF-AG).
The rtLC DBS assay also successfully detected and quantified HIV-1 RNA in DBS samples prepared in our lab using whole blood samples from 23 patients (US origin) infected with clade B virus (Table 1) , and 6 patients (non-US origin) infected with nonclade B virus (Table 1) . Finally, the rtLC DBS assay successfully detected HIV-1 nucleic acids in the blood of all 5 HIV-1 positive Congo infant DBS samples that were prepared in the field and shipped at ambient temperature ( Table 4 ).
Plasma viral RNA copy numbers determined by the rtLC DBS assay were compared to results obtained using commercial assays (bDNA and Amplicor). The Spearman coefficients of rtLC DBS with the Roche and bDNA assays were 0.91 and 0.89 respectively.
In a pilot study using the i-Cylcer real-time PCR system to quantify viral load from select patient and standard DBS RNA preparations, and using Sybr Green dye for detection, we demonstrated comparable performance to that of the LightCycler system ( Figure S1 ; Supplementary section). A significant correlation between i-Cycler and LightCycler based viral loads was observed in DBS specimens (Spearman Ranks correlation, p,0.0001) suggesting that our LightCycler-based DBS assay will have universal applicability.
To evaluate the performance of this assay in limited resource settings where storage and shipment of DBS at ambient/roomtemperature (.25uC) is the norm, we investigated the effect of 37uC temperature on the stability of DBS/RNA, by comparing the detection of HIV-1 RNA in DBS samples stored at 220uC or 37uC. We also evaluated 7 days of storage at 37uC to emulate shipment of DBS from the point of preparation (clinic) to a tertiary referral center (reference-laboratory), and compared the assay results to those of identical DBS stored promptly at 220uC. No statistically significant difference in viral load was observed for 12 samples stored either at -20uC or 37uC (Wilcoxon signed rank test, p = 0.06) ( Figure 2 ). Further, to evaluate for potential loss of RNA over time in DBS samples stored at 220uC and 37uC, viral load was determined on RNA samples extracted day 1 and day 7 postpreparation of DBS. The data (available for 8 patients) for days 1 and 7 were comparable irrespective of the storage temperature (paired Wilcoxon test; data not shown).
The fitness measured by R-squared value in the simplest model, which includes only the true concentration as predictor, is 0.955, and R-squared values from models that include extraction and run effects were 0.9959 (extraction and run) and 0.9948 (extraction), respectively. The improvement of fitness by including extra extraction specific parameters and run specific parameters are marginal, and we conclude that true concentration explains most (95.5% percent) of the variation in observed concentration, and the influence of extractions and runs are limited. Measurement of Plasma HIV-1 RNA over time in Patients on ART
The rtLC DBS assay was used to monitor plasma HIV-1 RNA levels in two patients for up to one year on ART (Figures 3a and  3b) . The rtLC DBS assay showed good correlation with the Roche Ultrasensitive assay for the longitudinal follow-up of these two patients, suggesting that it may also be useful for monitoring viral load in patients on ART.
In this study, we used a nucleic acid based real-time PCR assay to detect and quantify plasma HIV-1 copy numbers on samples from 56 HIV-1 infected patients utilizing the DBS format. The assay results were comparable and correlated well with commercially available viral load assays (Siemens bDNA, Spearman r = 0.89 and Roche Ultrasensitive Amplicor, Spearman r = 0.91). In the patient cohort analyzed, the assay successfully detected all positive samples. The calculated specificity using known negative samples was 100%. The estimated 95% detection threshold was 136 copies and the dynamic range of the assay was 5 log 10 . Finally, the assay successfully detected four major subtypes and 2 CRF of HIV-1.
A little over a decade ago, we demonstrated the utility of early combination antiretroviral therapy (ARV) in infants. [28, 29] HIV infection is associated with particularly high morbidity and mortality in limited-resource settings and a recent randomized trial conducted in South Africa demonstrated reduced morbidity and mortality in infants treated with early combination ARV. [30] In a recent WHO consultants' meeting, revision of current guidelines was recommended to include routine early diagnosis and treatment of HIV positive infants under 1 year of age. [31] Nucleic acid based testing is the gold standard for early diagnosis. Commercially available assays (bDNA, Roche Amplicor Ultrasensitive, and Cobas) are relatively expensive and require significant infrastructure and technical expertise to allow transfer of technology to resource-limited settings. [2, 8] Hence access to nucleic acid testing in these settings is currently very limited. [6, 17, 18, 32, 33] Studies to detect and quantify HIV-1 have traditionally involved two-step, nested or probe based PCR [34] [35] [36] [37] ; more recently, real time PCR has been used for HIV detection and quantification. [6, 22, 24, 34, 38, 39] Multiple investigators have documented the utility of DBS sample collection for early HIV-1 detection in infants, viral load monitoring, and surveillance of seropositivity and drug resistance in laboratories and clinics that lack facilities for refrigeration or sample processing. [8, 17, 20, [22] [23] [24] 38, [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] The DBS format greatly facilitates the logistics of sample collection, processing, and shipping for limited resource settings. Whole blood saved as DBS can be transported or mailed to reference laboratories without refrigeration and has low biohazard risk. Optimal storage conditions for DBS and long term stability of DNA and RNA from DBS under different storage conditions have been extensively documented. [23, 44, 47, 51, 53] Our pilot studies with a small cohort of patient samples to assess the effect of temperature and time, albeit one week, on the stability of RNA demonstrated no difference, and were in agreement with previous larger studies. [39, [54] [55] [56] Comparable efficacy using multiple extractions and runs of our customized standards support the reliability of the DBS format and the rtLC DBS assay.
The rtLC DBS assay described herein is a one-step, walk away technique. Automation of this assay provides potential for high throughput with very small sample volumes, which makes the assay suitable for use in infants and children from whom one often has access to only small blood volumes. It is an assay system which will be cost-effective and easily adaptable to limited-resource settings, where the majority of new HIV-1 infections are seen today. The detection of PCR products by SYBR Green ensures good sensitivity. SYBR Green is relatively inexpensive compared to probe-based detection, and in general, SYBR Green detection is one cycle threshold or so more sensitive than probe-based assays.
Several previous studies have utilized SYBR Green for HIV-1 diagnosis and are reviewed by Espy et al. [57] The efficiency of this dye-based assay is also comparable with the currently available diagnostic assays on HIV. Aside from use in HIV diagnosis and quantification, SYBR Green has been widely used to detect and quantify diverse human pathogens. [58] [59] [60] [61] [62] [63] [64] [65] The data in these reports strongly support the utility and reliability of SYBR Green for detection of specific PCR amplicons above the background. The use of degenerate LTR primers in the rtLC DBS assay allows for a wide range of genotype inclusivity.
Compared to commercial assays, the rtLC DBS assay is rapid and cost-effective. The equipment used in the rtLC assay is self- contained, occupies minimal bench-space and doesn't require accessory equipments (such as a plate washer, optical density reader, and incubator; Table 5 ). Aside from the initial cost of obtaining the LightCycler instrument, a comparative analysis of assay costs and technician time reveals a 40-fold decrease in cost as well as 2.5 fold decrease in technician time (4.5 hours) associated with the rtLC DBS when compared to commercial kit based PCR assays ( Table 6 ).
The reduced equipment requirements, personnel hours, and costs compared to the commercial 'gold' standard assays make the rtLC DBS assay attractive for transfer to and use in resourcelimited settings. The success of the pilot field study on DBS from Congo emphasizes the utility and applicability of our assay although further studies with larger sample sizes are definitely warranted.
In summary, we have utilized a real-time LightCycler based PCR assay on small volumes of whole blood dried on filter paper to successfully detect and quantify viral loads across different HIV-1 clades. The use of dried blood spots provides a simple and inexpensive means for obtaining blood samples for analysis that minimizes the risk for contamination while maximizing the ability to obtain timely results. A major advantage of the rtLC DBS assay is that the amplification, real-time detection and quantification, and confirmation of amplicon-specificity by melting curve analysis are performed in a one-step, closed-tube format. In addition, viral load tests by rtLC DBS assay are substantially less expensive and logistically less intensive than commercial assays. Preliminary data suggest the adaptability of the assay into other real-time systems. Validation of these results with larger field studies would constitute a more robust evaluation and will have major implications for early diagnosis, disease management, and epidemiological-or resistance-surveillance studies in limited resource settings.
Table S1
Found at: doi:10.1371/journal.pone.0005819.s001 (0.04 MB DOC)  A Statistical Framework for the Adaptive Management of Epidemiological Interventions BACKGROUND: Epidemiological interventions aim to control the spread of infectious disease through various mechanisms, each carrying a different associated cost. METHODOLOGY: We describe a flexible statistical framework for generating optimal epidemiological interventions that are designed to minimize the total expected cost of an emerging epidemic while simultaneously propagating uncertainty regarding the underlying disease model parameters through to the decision process. The strategies produced through this framework are adaptive: vaccination schedules are iteratively adjusted to reflect the anticipated trajectory of the epidemic given the current population state and updated parameter estimates. CONCLUSIONS: Using simulation studies based on a classic influenza outbreak, we demonstrate the advantages of adaptive interventions over non-adaptive ones, in terms of cost and resource efficiency, and robustness to model misspecification. Epidemiological interventions generally remove susceptible individuals or apply some form of treatment to infected individuals in order to prevent further spread of a disease. The susceptible population may be culled, as in the case of foot-and-mouth disease [1, 2] , in which case the total population size is permanently reduced. The infected population may be quarantined, as in the case of SARS [3] , in which case total population size is unchanged but the fraction of infecteds that may be in contact with susceptibles is reduced. Most commonly, susceptibles are vaccinated (cf influenza or smallpox [4, 5] ), in which case the total number of susceptibles, but not the total population size, is reduced.
Each of these interventions incurs a quantifiable cost: culling results in additional deaths; medical treatments or quarantines result in monetary expenses; vaccination incurs both monetary expenses, and in some cases additional vaccine-induced infections. Additionally, in many situations the costs associated with each of these actions can depend upon the state of the disease within the population of interest. For example, per-dosage prices of vaccine can increase as resources become scarce as a result of an aggressive vaccination campaign. Similarly, vaccine efficacy can decrease as a result of selection for drug resistance. Such observations raise the question of how to find optimal interventions that adaptively depend on the state of the epidemic.
A key challenge to calculating optimal intervention strategies involves devising ways to characterize and explore the space of intervention policies. Most existing work on optimal intervention has required various limiting assumptions about the forms of such strategies. Ball and Lyne [6] considered optimal vaccination in terms of the allocation of vaccine doses to households of various sizes in an explicitly structured population model. Patel et al [7] considered optimal vaccination in terms of the allocation of vaccine doses to different age classes in an explicitly age-and geographically-structured population model. Tildesley et al [1] describe optimal vaccination strategies for a foot-and-mouth epidemic in which the optimized parameter is the size of the radius surrounding a point of infection within which all livestock are to be vaccinated. These methods are primarily concerned with pre-emptive interventions that can be completed before the arrival of the pathogen. Under such scenarios, there is no need to consider adaptive or sequentially updated interventions because as soon as the intervention policy is triggered, the threat of epidemic is eradicated. In real scenarios, such widespread vaccination may not be achievable. Moreover, these methods traditionally involve calculations that assume no uncertainty in key model parameters such as transmission rate, recovery rate, and mortality rate. Recently Elderd et al [8] , using Bayesian methods, demonstrated the importance of explicitly quantifying such underlying uncertainty when forecasting the expected efficacy of trace versus mass vaccination policies. Their findings demonstrate that accurate propagation of parameter uncertainty can sometimes reveal deep and troubling consequences of a proposed vaccination strategy, and they suggest that incorporation of such uncertainty could impact policy decisions.
Here we address the question of how to dynamically propagate uncertainty in order to respond to an emerging epidemic while simultaneously and continuously learning about its underlying transmission dynamics. Estimation of model parameters is facilitated by regarding the transmission dynamics as stochastic processes rather than deterministic solutions to a structural equation model. This allows us to explicitly account for uncertainty in both model parameters and disease transmission. We consider a very general class of vaccination strategies defined by a fraction of the current susceptible population to be targeted for vaccination, and a threshold number of susceptibles such that once the number of susceptibles falls below this threshold, the vaccination campaign is called off. We demonstrate the calculation and application of optimal strategies of this form when coupled with iteratively updated parameter estimates using simulations based on a well-studied influenza outbreak [9] . Our emphasis is not on the realism of the underlying SIR model (though it has been shown that even simplistic transmission models can provide good fit to actual data [10] ), but rather to describe an effective approach for combining estimation and policy calculation. Permitting greater flexibility in the form of the possible intervention renders calculation of optimal intervention strategies analytically intractable, thus requiring evaluation by Monte Carlobased methods. Once in a Monte Carlo-based framework, it becomes straightforward to couple the evaluation of intervention strategies with Bayesian procedures for performing on-line estimation of parameters of the underlying epidemic model, thereby propagating parameter uncertainty through to policy decisions.
The policies produced by this framework are optimal in that they minimize the expected cost of the epidemic and adaptive in that the optimal policy changes as a function of the state of the epidemic and the degree of uncertainty in underlying model parameters. Using extensive simulation studies we compare the distribution of costs accrued under adaptive intervention to those arising from non-adaptive policies in a variety of scenarios. Our studies show that adaptive policies perform similarly to nonadaptive policies based on perfect parameter estimates, and significantly better than nonadaptive policies based on imperfect parameter estimates. Additionally, we show that adaptive online estimation affords the method some robustness to model misspecification. These results further demonstrate the importance of accounting for such underlying uncertainties in dynamic settings and indicate the utility of adaptive policies in settings where perfect estimates and a true model do not exist. All computational methods used herein have been made freely available through the amei (Adaptive Management of Epidemiological Interventions) R package [11] .
A classic study of Murray's [9] describes the spread of influenza through the population of a British boarding school. During the course of the epidemic, which was traced to the arrival of a single infectious student, all 763 students were eventually infected. The epidemic conforms to many standard assumptions of SIR models: a population essentially closed to immigration and emigration, recovery with immunity, and nearly homogeneous mixing of susceptibles and infectives.
Viewing the transmission dynamics as a discrete time stochastic process rather than a deterministic system of coupled differential equations implies a distribution of possible outcomes for the epidemic. By conditioning on parameter values and initial conditions (S 0~7 62, I 0~1 ), Monte Carlo simulation can be used to explore the distributions of numbers of susceptible, infected, and recovered individuals, as well as total accrued cost, as functions of time. Murray provides estimates of the transmission rate (b b~0:00218) and recovery rate (v v~0:4), which we regard as the ''true'' underlying parameter values in our simulations. Additional aspects of the transmission function are discussed in the Methods section. We assume that all costs can be expressed in a common monetary cost unit. Other choices of cost functions that address the issue of nonconformable costs (e.g. lives vs dollars) are mentioned in the Discussion.
Setting the unit cost to be that of maintaining a single infected individual for one time step (cost per infected, c t~1 ), repeated forward simulation of the epidemic (Figure 1 ) indicates that the mean total cost over 40 time steps is approximately 2100 cost units ( Figure 2 ), attributable entirely to the cumulative cost of maintaining a large population of infected individuals until recovery.
We consider a relatively simple but flexible class of intervention strategies that involve vaccinating a target fraction (a) of susceptible individuals at each time step. After a round of vaccination, if the number of remaining susceptibles is less than a designated threshold (c), the vaccination campaign is discontinued. Policies defined in this way provide effective target population sizes, to which post-hoc corrections can be applied in light of knowledge of the population structure.
We assume that in a single time unit there is an upper bound on the maximum targetable fraction of susceptibles. In our simulations we set this bound to be 30%, so that several time units are required to vaccinate the majority of susceptibles. We also assume there is a period of time after the arrival of the initial infection before intervention can begin. In our examples, we assume this lag time to be 7 time units. These values are chosen purely for the purpose of demonstration, and can be assigned any value in the amei software.
The optimal variable stop time vaccination strategy can be found by searching the policy space (i.e. pairs of fractions-tovaccinate a and stopping thresholds c) for the policy that most frequently produces the lowest expected cost. The calculation of the optimal policy therefore explicitly accounts for uncertainty associated with the disease transmission and recovery processes (see Methods) under a given valuation of the model parameters. Assuming a value of 2 cost units per dose of vaccine (c v~2 ), we use Monte Carlo simulation to estimate the expected cost surface associated with variable stop time policies based on the true parameter values (Figure 3 ). The minimum expected cost is achieved under a policy of maximum (30%) vaccination and a stopping threshold of 150 individuals. Repeated simulation of the epidemic under this policy shows that in the average case (dashed line), the policy amounts to 4 time units of maximum vaccination as soon as the initial lag is over ( Figure 4 ). In situations where the number of susceptibles remaining after the lag is already below 150 individuals, no policy is implemented. The 95% central interval for the final distribution of total vaccine units dispensed is (339,581), representing variation in the total size of the epidemic at the time of the vaccination sweep, and the numbers of new infections after vaccination begins. Figure 5 shows the distribution of total costs accrued under this policy. After the end of the vaccination campaign, the uncertainty bands widen, representing variations in the costs associated with maintaining the remaining population of infected individuals until their natural recoveries. The mean total cost at time 40 is 1652 cost units, approximately a 21% reduction in total cost compared to no-intervention. 
The intervention calculated in the previous section represents a gold-standard for this particular scenario because the vaccination strategy was calculated using the same parameter values and the same SIR model formulation as the simulated disease process. In most settings it will be natural to regard the transmission model parameters as unknowns to be estimated from incoming count data describing the sizes of the susceptible, infected, and recovered subpopulations. In this section we describe the procedure for performing adaptive management of an emerging epidemic, in which we account for parameter uncertainty and its impact on vaccination strategies.
An epidemic can be effectively summarized by the disease state of the population (i.e. the current numbers of susceptible and infected individuals) and by the SIR model parameters that define the dynamics of transmission, death, and recovery. In adaptive management, the former is used to perform inference on the latter. Each time new data are collected, Markov chain Monte Carlo (MCMC) is used to sample from the current posterior distribution on model parameters. The optimal variable stop time strategy associated with each set of sampled parameter values is calculated, and the policy that most frequently minimizes the total expected cost (over all sampled parameter values) is enacted at the next time step. The fundamental difference between the adaptive policies calculated here and those calculated in the previous section is that here, the vaccination policy is a dynamic function of the current disease state and the current distribution of each parameter, whereas before, the policy was a static function of the initial disease state and the initial point estimate of each parameter.
The effectiveness of this approach can be similarly explored by repeated simulation of epidemics under adaptive management. As before, we assume an initial lag time of 7 time units before vaccination begins. Here we also introduce a cost associated with deaths (c d~4 ). Even though the ''true'' model does not include mortality, the fitted model includes a mortality parameter (m). This allows examination of the degree to which adaptive management strategies are robust to model misspecification.
Initial uncertainty regarding parameter values is expressed in the form of vague/noninformative prior distributions, as specified in the Methods. The choice of prior distributions in Bayesian models is of fundamental importance, and other possible choices are mentioned in the Discussion section. At each time step, the state of the epidemic is advanced one time step using the same ''true'' parameter values used in the previous section. Intervention strategies, however, are calculated based on the current parameter estimates. Figures 6 and 7 show the distributions of susceptible, infected, recovered, and vaccinated individuals, and total accumulated costs for repeated simulation of the epidemic under adaptive management. These dynamics can be compared to those in The tighter bound about a smaller mean is due to the ability of the adaptive strategies to methodically diminish the vaccination campaign as a function of the epidemic state. This can be seen in Figures 8 and 9 , which display the distributions of implemented vaccination strategies for each time step during the course of adaptive management. In the average case (dashed line), the maximum policy is enacted for 3 time steps, followed by a round of 20% vaccination. The uncertainty surrounding the implemented strategies indicates the degree to which the the adaptive policies are adjusted in light of data. In epidemics associated with the upper 97.5 percentile of vaccination strategies (top solid line in Figures 8 and 9 ), the adaptive policy calls for 4 rounds of maximum vaccination followed by a round of 20% vaccination, followed by a final round of 5% vaccination. In this way, the adaptive nature of the interventions enables more efficient use of vaccine resources than achieved under nonadaptive policies.
The distribution of total cost associated with the adaptive intervention simulations (Figure 7) is essentially equivalent to the distribution of costs achieved under static intervention with perfect information (Figure 5 ), indicating that even the short period of data collection prior to action produces parameter estimates that are sufficient for accurate prediction of the disease dynamics. Figure 10 shows the final posterior distributions on the four model parameters estimated from the data during one simulation of the epidemic under adaptive management. True values are indicated with a circle, mean values are indicated with an 'x', and the central 95% region of each distribution is shaded. The prior densities of each parameter for the same interval are shown in red. As mentioned above, the inference model is misspecified relative to the model being used to simulate the epidemic, in that the inference model includes a mortality parameter (m, see Methods), even though no deaths were observed in the simulated outbreaks. By coupling the policy calculations with an inference framework, the effect of such model misspecification appears to be reduced.
We can further demonstrate the utility of the adaptive approach in situations of more severe model misspecfication. To do so, we construct a simulation experiment in which the inference model upon which the adaptive management is based is as described here, but in which the underlying transmission model through which new infecteds are generated is an entirely different, non- nested transmission model with a latent infective resevoir (see the amei vignette on CRAN [11] for details). This situation more closely resembles one that may be encountered in practice, where new infections are arising from an actual disease transmission process whose dynamics are at best approximated by any mathematical characterization. Table 1 compares summaries of the posterior distribution of cumulative cost arising under adaptive management to those predicted under the optimal static policy using parameters estimated for the misspecified model based on a completely observed epidemic. It is important to recognize that the adaptive policy is at a severe disadvantage, basing its actions on parameter estimates produced simultaneously during the course of a single epidemic (and using vague prior distributions) while the static policy conditions on parameter estimates obtained from a completely observed epidemic. In spite of this, the adaptive policy achieves nearly identical costs.
We have now shown the near equivalence of the adaptive and static policies in two different scenarios. These situations indicate that the proposed methodology is efficiently and with sufficient accuracy estimating the parameters of the transmission model, such that adaptive strategies based on these on-line estimates produce equivalent outcomes to those static strategies based on full retrospective analyses. Moreover, it is simple to demonstrate that static control measures based on reasonable but imperfect parameter estimates can lead to substantially worse outcomes/ higher costs than the adaptive policies ( Table 2 ). In real situations, where actions must be based on parameter estimates made from incomplete or limited information, the practice of iterative refinement of estimates and policies is likely to result in significantly improved outcome.
We have demonstrated a novel adaptive management strategy based on a relatively simple characterization of the underlying SIR model and the epidemiological cost function. In principle, this methodological framework can readily accommodate more complicated disease dynamics such as immigration, latent infected states, missing data, and vector-communicated diseases, as well as more complicated intervention strategies that allow combined vaccination and quarantine. However, the incorporation of such features is likely to impose a heavy computational burden, and so model complexity should only be increased when additional parameters are supported (and identified) by the data and demanded by the biology. As in all Bayesian analyses, care must be taken when choosing prior distributions. In this study, our primary interests required the use of vague/noninformative prior distributions, in order to demonstrate the estimability of model parameters. In practice, informative, even pessimistic priors (i.e., overestimated infectiousness and mortality, underestimated recovery) may provide useful reference points for the adaptive policy calculations, especially in situations of acute infections for which the duration of the epidemic may be too short for incoming data to dominate the prior information. In such situations, the adaptive approach still provides the opportunity for data to inform parameter values if it becomes available, while basing interventions on current parameter estimates as determined by their prior distributions.
There is an important choice to be made in assigning costs to the various actions that comprise an intervention strategy. A monetary valuation scheme is the most straightforward, but it may be difficult to construct such a scheme that adequately represents all aspects of the decision. One alternative would be a valuation in which each cost is chosen to represent a probability of mortality. In this way, the cost to be minimized would be the expected total loss of life for the epidemic under a given intervention strategy. By assuming that the removal rate can be expressed as r~mzv, where m is the rate of disease-induced mortality and v is the rate of natural recovery from the infected state, we can set c i~1 {e r ð Þ m r , so that the cost associated with maintaining a given number of infected individuals for a unit of time is the number of infected individuals that are expected to die in a unit of time. Similarly, situations exist where it is reasonable to assign a probability of mortality to the removal of susceptibles, as in the cases of smallpox vaccination or the culling of livestock.
A related extension to this framework would involve applying a monetary constraint to a loss-of-life cost function. If we were to assume p i and p r to be, respectively, the probabilities of mortality associated with untreated infected individuals and the removal of susceptibles, and define d to be the monetary resources available for the intervention, then within this framework it is possible to find the intervention that minimizes the total loss-of-life subject to the total spending constraint d. Similarly, it would be possible to optimize with respect to some selective criterion in order to preserve vaccine efficacy rather than select unnecessarily for drug-resistant pathogens. Also note the possibility of calculating policies based on minimization of some quantile of the realized cost rather than the mean cost. This would lead to minimization of costs associated with worst case scenarios, rather than that associated with the average case scenario. These and other alternative formulations of the underlying optimization problem can be easily accommodated in the framework presented here.
The utility of adaptive interventions is especially evident in situations of an emerging pathogen with which the host population has no previous experience. In such a situation, vaccines will not be immediately available at the onset of the epidemic, and so a methodology for combining currently available actions while anticipating the future availability of vaccines would be of great use. Effective epidemiological intervention requires swift decision in consideration of the various direct and indirect costs of intervention. The methodological framework described here provides a decision theoretic basis for automating this process.
All statistical and computational methodology described here has been implemented in a freely available R package called amei (Adaptive Management of Epidemiological Interventions), which can be downloaded at http://cran.r-project.org/web/packages/ amei/index.html [11] .
We consider a standard Susceptible-Infected-Removed (SIR) model [10, 12] with no loss of immunity but with mortality. In this model, the dynamic variables at time t are the number of susceptible individuals, S(t); the number of infected individuals, I(t); the number of recovered individuals, R(t); and the number of removed/dead individuals, D(t). We assume that the population is closed to immigration such that S(t)+I(t)+R(t)+D(t) = N is constant, and any three of the dynamic variables define the fourth.
To characterize the transmission of the disease, we adopt the negative binomial form for the transmission function [13] , so that the model parameters are the transmission rate b, the overdispersion parameter k, the death rate m, and the rate of recovery to the immune class n. Under these assumptions, the SIR model is described by the following system of differential equations [12, 13] : 
The negative binomial transmission function implies that disease transmission occurs following a Poisson process in which encounters between infected and susceptible individuals are Poission distributed with the encounter rate varying according to a gamma distribution with coefficient of variation k {1=2 . Via the parameter k, the negative binomial transmission function can account for social interactions and/or network factors in disease transmission, without requiring explicit characterization of the population structure.
The SIR model formulation also leads immediately to a natural discrete time approximation for the numbers of infections (Ĩ I), recoveries (R R) and deaths (D D) arising in the unit time interval from t to t+1. Holding the total number of infected individuals I constant and integrating Equation 1 over a unit time interval gives 
Here lower case denotes the realized value of the associated capital letter random variable. In this discrete time approximation we have assumed a particular ordering of events, namely that recoveries occur first, followed by deaths from among those infected individuals who did not recover, followed by new infections. Simulation studies indicated that these assumptions, as well as other possible orderings, resulted in system dynamics that were approximately equal in expectation to deterministic solutions of the continuous time SIR model. In all forward simulations of the disease dynamic (except where noted) we assume the ''true'' underlying parameter values to be those estimated by Murray [9] , with the exception of the negative binomial overdispersion parameter k. Thus, b = 0.00218, n = 0.4, and m = 0 (no disease-related mortality). We set the overdispersion parameter to be k = 0.1, in order to produce epidemics that, without intervention, have run their course by 40 time units but such that there is variation in the size of the outbreak.
We formulate the total expected cost of the epidemic in terms of the underlying costs associated with maintaining infected individuals until recovery, suffering death, and administering vaccinations. Let c 1 a,c,s ð Þ denote the cost associated with interventions involving susceptibles when S(t) = s. Here a is the fraction of susceptibles that are moved directly into an immune/recovered class, as by vaccination, and c is the threshold below which the intervention is discontinued. Letting c v denote the cost per unit, then
We let c 2 i ð Þ denote the cost associated with interventions involving infecteds when I(t) = i. This component includes the costs associated with maintaining the non-recovered infected individuals and costs associated deaths, as in
where c t is the cost per treatment/maintenance of a non-removed infected individual, and c d is the cost per death. Assuming the initial epidemiological state is S 0 ð Þ~s 0 , I 0 ð Þ~i 0 , the expected total cost of the epidemic under intervention strategy (a,c) can be expressed recursively as
where E C t f g denotes the expected cost accumulated from time t onwards. The optimal intervention strategy (a,c) is the one that minimizes the total accumulated cost over the course of the epidemic. Two methods for calculating such strategies are as follows.
The total expected cost depends on the parameter values and the initial epidemiological state s 0 ,i 0 ð Þ. Thus, conditional on a set of parameter values, Monte Carlo simulation can be used to search over values of a and c in order to find the combination that minimizes E C 0 f g: For each combination of a and c, with a ranging from 0 to 0.7 and c from 0 to 750 in increments of 50, we conduct 100 simulations of the epidemic, using the true parameter values, in order to estimate the mean cost associated with the intervention (a,c). The strategy producing the lowest mean cost is defined to be the optimal intervention.
As above, the expected cost surface associated with a given set of parameter values (as obtained by MCMC, described below), can be explored using standard Monte Carlo methods. At each time step, MCMC is used to produce 10,000 samples from the current posterior distribution on model parameters. These samples are thinned to 100 samples, and for each of these 100 samples the optimal variable stop time vaccination strategy is calculated as described above. The adaptive strategy to be implemented at that Bench-to-bedside review: Angiopoietin signalling in critical illness – a future target? Multiple organ dysfunction syndrome (MODS) occurs in response to major insults such as sepsis, severe haemorrhage, trauma, major surgery and pancreatitis. The mortality rate is high despite intensive supportive care. The pathophysiological mechanism underlying MODS are not entirely clear, although several have been proposed. Overwhelming inflammation, immunoparesis, occult oxygen debt and other mechanisms have been investigated, and – despite many unanswered questions – therapies targeting these mechanisms have been developed. Unfortunately, only a few interventions, usually those targeting multiple mechanisms at the same time, have appeared to be beneficial. We clearly need to understand better the mechanisms that underlie MODS. The endothelium certainly plays an active role in MODS. It functions at the intersection of several systems, including inflammation, coagulation, haemodynamics, fluid and electrolyte balance, and cell migration. An important regulator of these systems is the angiopoietin/Tie2 signalling system. In this review we describe this signalling system, giving special attention to what is known about it in critically ill patients and its potential as a target for therapy. Critical illness is a life-threatening disease by definition. Patients treated for critical illness in the intensive care unit have underlying causes such as infection, trauma, major surgery, hemorrhagic shock, pancreatitis and other major insults. Despite maximal supportive care, severely ill patients treated in intensive care units are still likely to die, usually after an episode of increasing failure of multiple organs [1] .
The mechanisms that underlie multiple organ dysfunction syndrome (MODS) are not known [2] , although several have been proposed, including overwhelming infection or immune response, immune paralysis, occult oxygen debt and mitochondrial dysfunction [3] [4] [5] . Although these potential mechanisms have features in common, it is not clear whether MODS is a final common pathway or when it is engaged. The innate and adaptive immune systems, coagulation, and hormonal and neuronal signalling are undoubtedly involved and are all connected. For example, the hypoxic response is linked to innate immunity and inflammation by the transcription factor nuclear factor-κB (NF-κB) [6] . It is no coincidence that the few interventions that appear to be of benefit, although this is still under debate, have pleiotropic mechanisms of action [7] [8] [9] . Thus, it seems reasonable to study the intersections between and within cellular and molecular systems to elucidate the interactions and to develop therapeutic options.
One of the central cellular players in this system is the endothelial cell (EC). Once thought to serve as an inert vascular lining, ECs are highly heterogeneous and constitute an active disseminated organ throughout the circulatory system. ECs form the border between every organ and the bloodstream and thus with the rest of the body. The EC receives and gives signals, stores active substances of multiple systems, and regulates the passage of fluids, electrolytes, proteins and cells. The EC has a time and place dependent phenotype that is dynamically controlled, and its reactions to stimuli are specific to organ and vascular bed [10] [11] [12] [13] . The EC merits robust investigation in critical illness, as in vascular medicine [14] . genesis and organogenesis in normal physiology and in wound repair, but it is considered pathologic in tumour growth and diabetes [15] . Second, in the adult organism, ECs help to maintain homeostasis, including fluid, electrolyte and protein transport, and cell migration into and out of the vessel, and to regulate blood flow. Third, ECs react and respond to disturbances of homeostasis (for example, in inflammation, coagulation and hypoxia/reperfusion).
All three functions are involved in MODS, in which ECs are shed, blood flow regulation is hampered, vessels become leaky, cells migrate out of the vessel and into the surrounding tissue, and coagulation and inflammation pathways are activated [16] . The machinery involved -receptors, signalling pathways and effectors -is largely the same in each function, but the net effect is determined by the balance between the parts of the machinery and the context [15] .
The angiopoietin/Tie2 signalling system (Ang/Tie system) appears to be crucial in all three functions [17, 18] . The Ang/Tie system, which was discovered after vascular endothelial growth factor (VEGF) and its receptors, is mainly restricted to EC regulation and is the focus of this review. Accumulating evidence suggests that this system is nonredundant and is involved in multiple MODS-related pathways. All components of potential pathophysiological mechanisms in MODS should be viewed within their own context, because all systems are mutually dependent. Thus, examination of the Ang/Tie system might offer insight into the mechanisms underlying MODS and provide opportunities for therapeutic intervention.
The notion that the Ang/Tie system contributes to disease pathogenesis is supported by clinical studies and studies in animal models, and by the relation between symptoms of critical illness and disturbances in this system. In mice, Ang-2 over-expression in glomeruli causes proteinuria and apoptosis of glomerular ECs [19] . In a rat model of glomerulonephritis, Tie2 is over-expressed by ECs, and Ang-1 and Ang-2 are over-expressed by podocytes in a time-dependent manner during the repair phase [20] . Therefore, Ang/Tie might be involved in renal failure and repair.
Lung dysfunction is common in critical illness, and evidence of Ang/Tie involvement has been found in animal models. In a rat model of acute respiratory distress syndrome, Ang-1 reduces permeability and inflammation, whereas Tie2 deficiency increases damage [21] . In an experimental model of asthma, Ang-1 mRNA was decreased, and Ang-1 supplementation decreased alveolar leakage and NF-κB-dependent inflammation [22] . In hypoxia-induced pulmonary hypertension in rats, decreased activity of the Tie2 pathway contributed to right ventricular load, and this effect was antagonized by Ang-1 [23]. On the other hand, a causative role for Ang-1 in pulmonary hypertension has also been suggested [24] . In hyperoxic lung injury, Ang-2 is involved in lung permeability and inflammation [25] .
Ang/Tie also may contribute to critical illness in patients with pulmonary conditions. Ang-1 and Ang-2 concentrations in sputum from asthma patients correlated with airway microvascular permeability [26] . In patients with exudative pleural effusion, the Ang-2 level was increased whereas Ang-1 was unchanged [27] . Ang-2 levels are associated with pulmonary vascular leakage and the severity of acute lung injury. Plasma from patients with acute lung injury and high Ang-2 concentrations disrupts junctional architecture in vitro in human microvascular ECs [28, 29] .
Patients with cardiovascular disorders also exhibit changes in the Ang/Tie system. Circulating Ang-1 concentrations are stable in patients with atrial fibrillation, but Ang-2 concentrations are increased, along with markers of platelet activation, angiogenesis and inflammation [30] . Patients with hypertension resulting in end-organ damage have increased levels of circulating Ang-1, Ang-2, Tie2 and VEGF [31] . Congestive heart failure is associated with elevated plasma levels of Ang-2, Tie2 and VEGF, but normal levels of Ang-1 [32]. A similar pattern is seen in acute coronary syndrome [33] .
Circulating levels of components of the Ang/Tie system have been measured in patients admitted to the critical care unit. In trauma patients plasma Ang-2, but not plasma Ang-1 or VEGF, was increased early after trauma, and the level correlated with disease severity and outcome [34] . In children with sepsis and septic shock, Ang-2 levels in plasma were increased and once again correlated with disease severity, whereas Ang-1 levels were decreased [35] . The same Ang-1/ Ang-2 pattern is seen in adults with sepsis [28, 29, [36] [37] [38] [39] . The results of studies of the Ang/Tie system in humans are summarized in Table 1 . In sepsis, VEGF and its soluble receptor sFLT-1 (soluble VEGFR-1) are also increased in a disease severity-dependent manner [40] [41] [42] .The picture that emerges from these studies is that the Ang/Tie signalling system appears to play a crucial role in the symptoms of MODS. Findings in animal models and in patients suggest that Ang-1 stabilizes ECs and Ang-2 prepares them for action. The close relation with VEGF is also apparent. four (Ang-1) and two (Ang-2) subunits [48, 49] . The dissimilarity between Ang-1 and Ang-2 signalling lies in subtle differences in the receptor binding domain that lead to distinct intracellular actions of the receptor; differential cellular handling of both receptor and ligands after binding and signalling initiation may also play a role [49, 50] .
The receptors are Tie1 and Tie2 [51] . Tie2 is a 140-kDa tyrosine kinase receptor with homology to immunoglobulin and epidermal growth factor [47, 52] . Tie receptors have an amino-terminal ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain [51] .
Ligand binding to the extracellular domain of Tie2 results in receptor dimerization, autophosphorylation and docking of adaptors, and coupling to intracellular signalling pathways [47, [53] [54] [55] . Tie2 is shed from the EC and can be detected in soluble form in normal human serum and plasma; soluble Tie2 may be involved in ligand scavenging without signalling [56] . Tie2 shedding is both constitutive and induced; the latter can be controlled by VEGF via a pathway that is dependent on phosphoinositide-3 kinase (PI3K) and Akt [57] . Shed soluble Tie2 can scavenge Ang-1 and Ang-2 [56] . Tie1 does not act as a transmembrane kinase; rather, it regulates the binding of ligands to Tie2 and modulates its signalling [58] [59] [60] .
Ang-1 is produced by pericytes and smooth muscle cells ( Figure 1 ). In the glomerulus, which lacks pericytes, Ang-1 is produced by podocytes [61] . Ang-1 has a high affinity for the extracellular matrix, and so circulating levels do not reflect tissue levels, which in part is probably responsible for the constitutive phosphorylation of Tie2 in quiescent endothelium [62] [63] [64] [65] . Ang-2 is produced in ECs and stored in Weibel-Palade bodies (WPBs) [66, 67] . The release of Ang-2 from WPBs by exocytosis can be regulated independently of the release of other stored proteins [68] . Tie2 is expressed predominantly by ECs, although some subsets of macrophages and multiple other cell types express Tie2 at low levels [69, 70] . In ECs, Tie2 is most abundant in the endothelial caveolae [71] .
The Ang-1 and Ang-2 genes are located on chromosome 8. Functional polymorphisms have not been identified in the Ang-1 gene, but three have been identified in the coding region of Ang-2 [72] . In ECs under stress, Ang-2 mRNA expression is induced by VEGF, fibroblast growth factor 2 and hypoxia [44, 73] . Upregulation of Ang-2 induced by VEGF and hypoxia can be abolished by inhibiting tyrosine kinase or mitogen-activated protein kinase [73] . Ang-2 mRNA expression can be downregulated by Ang-1, Ang-2, or transforming growth factor [74] . After inhibition of PI3K by wortmannin, Ang-2 mRNA production is induced by the transcription factor FOXO1 (forkhead box O1) [75] . EC-specific Ang-2 promoter activity is regulated by Ets-1 and the Ets family member Elf-1 [76, 77] . Because Tie2 signalling is required under circumstances that usually hamper cell metabolism, its promoter contains repeats that ensure transcription under difficult circumstances, including hypoxia [78] .
Tie2 is present in phosphorylated form in quiescent and activated ECs throughout the body [62] . Signalling is initiated by autophosphorylation of Tie2 after Ang-1 binding and is conducted by several distinct pathways [54, 71, 79, 80] . Tie2 can also be activated at cell-cell contacts when Ang-1 induces Tie2/Tie2 homotypic intercellular bridges [65] . In human umbilical vein endothelial cells (HUVECs), Ang/Tie signalling resulted in 86 upregulated genes and 49 downregulated genes [81, 82] . Akt phosphorylation by PI3K with interaction of nitric oxide is the most important intracellular pathway [51, [83] [84] [85] [86] ; however, ERK1/2, p38MAPK, and SAPK/JNK can also participate in Ang/Tie downstream signalling [71, 81, 84, [87] [88] [89] [90] . Endothelial barrier control by Ang-1 requires p190RhoGAP, a GTPase regulator that can modify the cytoskeleton [80] . The transcription factors FOXO1, activator protein-1, and NF-κ B are involved in Ang/Tie-regulated gene transcription [75, [91] [92] [93] . Ang-1induced signalling is has also been implicated in cell migration induced by reactive oxygen species [94] . ABIN-2 (A20-binding inhibitor of NF-κB 2), an inhibitor of NF-κB, is involved in Ang-1-regulated inhibition of endothelial apoptosis and inflammation in HUVECs [93] . However, the downstream signalling of Tie2 varies depending on cell type and localization and whether a cell-cell or cell-matrix interaction in involved, which results in spatiotemporally different patterns of gene expression. For example, Ang-1/Tie2 signalling leads to Akt activation within the context of cell-cell interaction, but it leads to ERK activation in the context of cell-matrix interaction. The microenvironment of the receptor in the cell membrane plays a central role in this signal differentiation. Adaptor molecules such as DOK and SHP2 and the availability of substrate determine which protein is phosphorylated [95] .
After binding of Ang-1, and to a lesser extent Ang-2, Tie2 is internalized and degraded, and Ang-1 is shed in a reusable form [50] . VEGF is an important co-factor that can exert different effects on Ang-1 and Ang-2 signalling [88] . Ang-2 is antiapoptotic in the presence of VEGF but induces EC apoptosis in its absence [96] . Autophosphorylation and subsequent signalling are inhibited by heteropolymerization of Tie1 and Tie2 [59] . Although the Ang/Tie system appears to play its role mainly in paracrine and autocrine processes, its circulating components have been found in plasma. The significance of this finding in health and disease has yet to be determined.
The Ang/Tie system is an integrated, highly complex system of checks and balances ( Figure 1) [45,54]. The response of ECs to Ang-1 and Ang-2 depends on the location of the cells and the biological and biomechanical context [97, 98] . It is believed that PI3K/Akt is among the most important downstream signalling pathways and that VEGF is one of the most important modulators of effects. Below we describe in more detail how this system responds to changes in homeostatic balances under various conditions of damage and repair.
Angiogenesis, inflammation and homeostasis are highly related, and the Ang/Tie system lies at the intersection of all three processes [99, 100] . The Ang/Tie system is critically important for angiogenesis during embryogenesis, but in healthy adults its function shifts toward maintenance of homeostasis and reaction to insults. Except for follicle formation, menstruation and pregnancy, angiogenesis in adults is disease related. Neoplasia-associated neoangiogenesis and neovascularization in diabetes and rheumatoid arthritis are unfavourable events, and improper angiogenesis is the subject of research in ischaemic disorders and atherosclerosis. Finally, failure to maintain homeostasis and an inappropriate reaction to injury are detrimental features in critical illness.
A schematic model of the angiopoietin-Tie2 ligand-receptor system. Quiescent endothelial cells are attached to pericytes that constitutively produce Ang-1. As a vascular maintenance factor, Ang-1 reacts with the endothelial tyrosine kinase receptor Tie2. Ligand binding to the extracellular domain of Tie2 results in receptor dimerization, autophosphorylation, docking of adaptors and coupling to intracellular signalling pathways. Signal transduction by Tie2 activates the PI3K/Akt cell survival signalling pathway, thereby leading to vascular stabilization. Tie2 activation also inhibits the NF-κB-dependent expression of inflammatory genes, such as those encoding luminal adhesion molecules (for example, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin). Ang-2 is stored and rapidly released from WPBs in an autocrine and paracrine fashion upon stimulation by various inflammatory agents. Ang-2 acts as an antagonist of Ang-1, stops Tie2 signalling, and sensitizes endothelium to inflammatory mediators (for example, tumour necrosis factor-α) or facilitates vascular endothelial growth factor-induced angiogenesis. Ang-2-mediated disruption of protective Ang-1/Tie2 signalling causes disassembly of cell-cell junctions via the Rho kinase pathway. In inflammation, this process causes capillary leakage and facilitates transmigration of leucocytes. In angiogenesis, loss of cell-cell contacts is a prerequisite for endothelial cell migration and new vessel formation. Ang, angiopoietin; NF-κB, nuclear factor-κB; PI3K, phosphoinositide-3 kinase; WPB, Weibel-Palade body.
Angiogenesis is dependent on multiple growth factors and receptors and their signalling systems and transcriptional regulators [101] . The process is complex and encompasses the recruitment of mobile ECs and endothelial progenitor cells, the proliferation and apoptosis of these cells, and reorganization of the surroundings [102] . To form stable new blood vessels, the response must be coordinated in time and space, and the Ang/Tie system is involved from beginning to end. To prepare for angiogenesis, Ang-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism [44, 103] . Before vascular sprouting starts, focal adhesion kinase and proteinases such as plasmin and metalloproteinases are excreted [85] . Often, this stage is preceded by activation of innate immunity and inflammation [104] . Apparently, the machinery to clean up after the work has been finished is installed before the work is commenced, again illustrating the close relations among the different processes [104] .
Ang-1 maintains and, when required, restores the higher order architecture of growing blood vessels [43,44, 105, 106] . This is achieved by inhibiting apoptosis of ECs by Tie2mediated activation of PI3K/Akt signalling [107] [108] [109] . Ang-1/ Tie2 signalling is involved in angiogenesis induced by cyclic strain and hypoxia [110, 111] . Although its role is less clear, Tie1 might be involved in EC reactions to shear stress [112] . Ang-1 is a chemoattractant for ECs [83] [84] [85] , and both Ang-1 and Ang-2 have proliferative effects on those cells [98, 113] . At the end of a vascular remodelling phase, Ang-2 induces apoptosis of ECs for vessel regression in competition with the survival signal of Ang-1 [106] . This apoptotic process requires macrophages, which are recruited by Ang-2 [70, 114] .
ECs require support from surrounding cells such as pericytes, podocytes, and smooth muscle cells [63] . These cells actively control vascular behaviour by producing signalling compounds (for instance, Ang-1 and VEGF) that govern the activity and response of ECs [61] . To attract ECs, Ang-1 secreted by support cells binds to the extracellular matrix. In quiescent ECs, this binding results in Tie2 movement to the site of cell-cell interaction. In mobile ECs, Ang-1 polarizes the cell with Tie2 movement abluminal site [65] . In tumour angiogenesis and in inflammation, Ang-2 recruits Tie2positive monocytes and causes them to release cytokines and adopt a pro-angiogenic phenotype [111] .
The Ang/Tie system provides vascular wall stability by inducing EC survival and vascular integrity. However, this stability can be disrupted by Ang-2 injection, which in healthy mice causes oedema [28, 79, 115, 116] that can be blocked by systemic administration of soluble Tie2 [115] . Ang-2 can impair homeostatic capacity by disrupting cell-cell adhesion through E-cadherin discharge and EC contraction [28, 117] .
In contrast, through effects on intracellular signalling, the cytoskeleton and junction-related molecules, Ang-1 reduces leakage from inflamed venules by restricting the number and size of gaps that form at endothelial cell junctions [80, 118, 119] . Ang-1 also suppresses expression of tissue factor induced by VEGF and tumour necrosis factor (TNF)-α, as well as expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin. As a result, endothelial inflammation is suppressed [120] [121] [122] [123] .
In primary human glomerular ECs in vitro, Ang-1 stabilizes the endothelium by inhibiting angiogenesis, and VEGF increases water permeability [124] . Similar observations were made in bovine lung ECs and immortalized HUVECs, in which Ang-1 decreased permeability, adherence of polymorphonuclear leucocytes and interleukin-8 production [123] .
Reaction to injury can be seen as an attempt to maintain homeostasis under exceptional conditions. ECs can be affected by several noxious mechanisms. The Ang/Tie system is considered crucial in fine-tuning their reaction to injury and in containing that reaction. Ang-2-deficient mice cannot mount an inflammatory response to peritonitis induced chemically or with Staphylococcus aureus [125] , but they can mount a response to pneumonia, suggesting the existence of inflammatory reactions for which Ang-2 is not mandatory. Ang-2 sensitizes ECs to activation by inflammatory cytokines. In Ang-2-deficient mice, leucocytes do roll on activated endothelium but they are not firmly attached, owing to the lack of Ang-2-dependent upregulation of adhesion molecules and the dominance of Ang-1-regulated suppression of adhesion molecules [120] [121] [122] [123] 125] .
In bovine retinal pericytes, hypoxia and VEGF induce Ang-1 and Tie2 gene expression acutely without altering Ang-2 mRNA levels. The opposite occurs in bovine aortic ECs and microvascular ECs, underscoring the heterogeneity of ECs from different microvascular beds [73, 126, 127] .
Lipopolysaccharide (LPS) and pro-inflammatory cytokines can shift the Ang/Tie balance, rouse ECs from quiescence and provoke an inflammatory response. In rodents LPS injection induces expression of Ang-2 mRNA and protein and reduces the levels of Ang-1, Tie2 and Tie2 phosphorylation in lung, liver and diaphragm within 24 hours, which may promote or maintain vascular leakage. The initial increase in permeability is probably due to release of Ang-2 stored in WPBs [39, 128] . In a mouse model of LPS-induced lung injury, pulmonary oedema was found to be related to the balance between VEGF, Ang-1 and Ang-4 [129] . In a comparable model, Ang-1-producing transfected cells reduced alveolar inflammation and leakage [130] .
In choroidal ECs, TNF induces Ang-2 mRNA and protein before affecting Ang-1 and VEGF levels [131] . In HUVECs, TNF-induced upregulation of Ang-2 is mediated by the NF-κB pathway [132] , and TNF-induced Tie2 expression can be attenuated by both Ang-1 and Ang-2. Without TNF stimulation, only Ang-1 can reduce Tie2 expression [133] . Ang-2 sensitizes ECs to TNF, resulting in enhanced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin [74, 125, 134] . By inhibiting those endothelial adhesion molecules, Ang-1 decreases leucocyte adhesion [122] .
Angiopoietins can mediate the synthesis of platelet-activating factor by ECs to stimulate inflammation [90] . Moreover, both Ang-1 and Ang-2 can translocate P-selectin from WPBs to the surface of the EC [135] , and both can also increase neutrophil adhesion and chemotaxis and enhance those processes when they are induced by interleukin-8 [86, 136, 137] .
In a rat model of haemorrhagic shock, Ang-1 reduced vascular leakage, and it inhibited microvascular endothelial cell apoptosis in vitro and in vivo [107, 138] . In this model, Ang-1promoted cell survival was partly controlled through integrin adhesion [139] . It has been suggested that EC apoptosis in haemorrhagic shock contributes to endothelial hyperpermeability [140] [141] [142] . Apoptosis is one of the reactions to MODSrelated injury as demonstrated in hypoxia/reperfusion [143] .
Ang-1 and Ang-2 are involved in cell-cell and cell-matrix binding [139, [144] [145] [146] . Endothelial permeability is greatly dependent on cell-cell adhesion. The major adherens junction is largely composed of vascular-endothelial cadherin. This complex can be disrupted by VEGF, leading to increased vascular permeability [147, 148] , which can be antagonized by Ang-1 [149, 150] . ECs can also bind to the matrix through the binding of Ang-1 to integrins, which can mediate some of the effects of Ang-1 without Tie2 phosphorylation [146, 151] . At low Ang-1 concentrations, integrin and Tie2 can cooperate to stabilize ECs [151] . Ang-2 might play a role in inflammatory diseases such as vasculitis by disrupting the cell-cell junction and inducing denudation of the basal membrane [152] . Ang-1 can mediate the translocation of Tie2 to endothelial cell-cell contacts and induce Tie2-Tie2 bridges with signal pathway activation, leading to diminished paracellular permeability [65] .
In the mature vessel, Ang-1 acts as a paracrine signal to maintain a quiescent status quo, whereas Ang-2 induces or facilitates an autocrine EC response [74, 153] . In general, Ang-1 can be viewed as a stabilizing messenger, causing continuous Tie2 phosphorylation, and Ang-2 as a destabilizing messenger preparing for action [17] . Attempts to unravel the exact molecular mechanisms that control the system are complicated by microenvironment-dependent endothelial phenotypes and reactivity and by flow typedependent reactions to dynamic changes [13, 154, 155] . Hence, the EC must be viewed in the context of its surroundings -the pericyte at the abluminal site, and the blood and its constituents on the luminal site [64] . The Ang/Tie system certainly functions as one of the junctions in signal transduction and plays a key role in multiple cellular processes, many of which have been linked to MODS.
A therapy should intervene in the right place and at the right time, with the proper duration of action and without collateral damage [156, 157] . The Ang/Tie system is involved in many processes and lies at the intersection of molecular mechanisms of disease. Thus, interventions targeting this system might have benefits. As in other pleiotropic systems, however, unexpected and unwanted side effects are a serious risk. The absence of redundant systems to take over the function of Ang/Tie2 has the advantage that the effect of therapeutic intervention cannot easily be bypassed by the cell. On the other hand, because the cell has no escape, the effect may become uncontrolled and irreversible. Moreover, the exact function of the Ang/Tie system in the pathological cascade is not fully established. What we see in animal models and in patients is most probably the systemic reflection of a local process. We do not know whether this systemic reflection is just a marker of organ injury or even a mediator of distant organ involvement.
Of the three main functions of the Ang/Tie system, it is mainly angiogenesis that has been evaluated as a therapeutic target. So far, the focus of Ang/Tie modulation has been on inhibiting angiogenesis related to malignant and ophthalmological diseases and to complications of diabetes [158, 159] . In peripheral arterial occlusive disease, stimulation of angiogenesis seems a logical strategy to attenuate the consequences of ongoing tissue ischaemia. In a rat model of hind limb ischaemia, combined delivery of Ang-1 and VEGF genes stimulated collateral vessel development to the greatest extent [160, 161] . Thus far, therapy directed at VEGF has reached the clinic, but not therapy directed at Ang/Tie [162] .
Targeting homeostasis and repair/inflammation in critically ill patients is an attractive option and has already led to the development of new drugs [45, 158, 163] . From current knowledge, one can speculate about the best options for therapy aimed at the Ang/Tie system. In critical illness, Ang-1 is considered to be the 'good guy' because it can create vascular stability and thus its activity should be supported. In contrast, Ang-2 appears to be a 'bad guy' that induces vascular leakage, so its activity should be inhibited [164] .
Production of recombinant Ang-1 is technically challenging as Ang-1 is 'sticky' because of its high affinity for the extracellular matrix [165] . However, stable Ang-1 variants with improved receptor affinity have been engineered. A stable soluble Ang-1 variant has anti-permeability activity [165] . When injected intraperitoneally in mice, human recombinant Ang-1 can prevent LPS-induced lung hyperpermeability [80] . In diabetic mice, a stable Ang-1 derivative attenuated proteinuria and delayed renal failure [166] , and manipulating the Ang-1/Ang-2 ratio changed infarct size [167] . A more profound Ang-1 effect can be achieved by locally stimulating Ang-1 production. In experimental acute respiratory distress syndrome, transfected cells expressing Ang-1 reduced alveolar inflammation and leakage [130] . An adenovirus construct encoding Ang-1 protected mice from death in an LPS model, and Ang-1 gene therapy reduced acute lung injury in a rat model [21, 168, 169] . In hypertensive rats, a plasmid expressing a stable Ang-1 protein reduced blood pressure and end-organ damage [170] . If used in a disease with a limited duration, as critical illness should be, virus/plasmid-driven production of Ang-1 could easily be shut down when it is no longer needed.
Manipulating Ang-2 activity is also difficult. Ang-2 stored in WPBs is rapidly released and must be captured immediately to prevent autocrine/paracrine disruption of protective Ang-1/ Tie signalling. Soluble Tie2 or Ang-2 inhibitors should be effective [26, 171] . Neutralizing antibodies against Ang-2 might also be an option. Replenishment of Ang-2 stores could be abolished by small interfering RNA techniques or spiegelmer/aptamer approaches [25, 172, 173] .
However, no bad guy is all bad, and no good guy is all good. For example, Ang-1 has been linked to the development of pulmonary hypertension [174] . Also, under certain circumstances Ang-2 can act as a Tie2 agonist and exert effects similar to those of Ang-1 -an unexplained finding that illustrates our limited understanding of the Ang/Tie system [75] . Complete blockade of Ang-2 might also hamper innate immunity and revascularization.
Finding the right balance and timing will be the major challenge when developing therapies to target the Ang/Tie system. In the meantime, we might have already used Ang/Tie-directed therapy with the most pleiotropic of all drugs -corticosteroids.
In the airways, steroids suppressed Ang-2 and increased Ang-1 expression [26, 171, 175] . Interventions further downstream targeting specific adaptor molecules, signalling pathways, or transcription factors have yet to be explored.
In patients with malignant disease, the Ang/Tie system might serve as a tumour or response marker. In patients with multiple myeloma, normalization of the Ang-1/Ang-2 ratio reflects a response to treatment with anti-angiogenesis medication [176] . In patients with non-small-cell lung cancer, Ang-2 is increased in serum and indicates tumour progression [177] . After allogeneic stem cell transplantation in patients with high-risk myeloid malignancies, the serum Ang-2 concentration predicts disease-free survival [178] , possibly reflecting a relation between cancer-driven angiogenesis and Ang-2 serum level.
In nonmalignant disease, the levels of Ang/Tie system components correlate with disease severity [28, 29, [34] [35] [36] [37] 39 ]. However, current data are insufficient to justify the use of serum soluble Tie2/Ang levels for diagnostic and prognostic purposes. In critical illness, assessment of the Ang/Tie system in patients with different severities of disease and with involvement of different organ systems might help to define our patient population and allow us to rethink our concepts of MODS. In this way, such work may lead to enhanced diagnosis and prognostication in the future [2] .
Accumulating evidence from animal and human studies points to the involvement of the Ang/Tie system in vascular barrier dysfunction during critical illness. Many processes in injury and in repair act through this nonredundant system. Thus far, only preliminary studies in critically ill patients have been reported. Methods to manipulate this system are available but have not been tested in such patients. The response to treatment is difficult to predict because of the pleiotropic functions of the Ang/Tie system, because the balance among its components appears to be more important than the absolute levels, and because the sensitivity of the endothelium to disease-related stimuli varies, depending on the environment and the organ involved. To avoid disappointment, further experimental and translational research must be carried out, and Ang/Tie modulation must not be introduced into the clinic prematurely. Implementing the results of this research in critical care represents an opportunity to show what we have learned [2] . Ang/Tie signalling is a very promising target and must not be allowed to become lost in translation [179] .  Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites In this work, we study the consequences of sequence variations of the "2009 H1N1" (swine or Mexican flu) influenza A virus strain neuraminidase for drug treatment and vaccination. We find that it is phylogenetically more closely related to European H1N1 swine flu and H5N1 avian flu rather than to the H1N1 counterparts in the Americas. Homology-based 3D structure modeling reveals that the novel mutations are preferentially located at the protein surface and do not interfere with the active site. The latter is the binding cavity for 3 currently used neuraminidase inhibitors: oseltamivir (Tamiflu(®)), zanamivir (Relenza(®)) and peramivir; thus, the drugs should remain effective for treatment. However, the antigenic regions of the neuraminidase relevant for vaccine development, serological typing and passive antibody treatment can differ from those of previous strains and already vary among patients. This article was reviewed by Sandor Pongor and L. Aravind. The recent epidemic of the "2009 H1N1" influenza A virus (also called swine or Mexican flu) has put the world on alert since a new swine flu strain (naturally hosted by pigs) has crossed the species barrier to human and, apparently, acquired the capability for human to human transmission [1, 2] . Given earlier experiences with risks of viral pandemics such as SARS and the avian flu [3] , global control and public health surveillance mechanisms provided sequences of the new flu strain in public sequence databases within weeks of the outbreak. Here, we analyze the protein sequence of its neuraminidase with respect to sim-ilarities and differences to known strains and implications on drug treatment and vaccination.
Sequence and residue numbering in this analysis correspond to the neuraminidase [Genbank: ACP41107.1 http://www.ncbi.nlm.nih.gov/protein/227809834] representative for the new strain. Sequence analysis was carried out following an established protocol using the ANNIE resource [4, 5] . The 469 amino acid long neuraminidase (NA) protein ( Figure 1 ) is essential for release of the viral particle from the outer membrane of infected cells by cleaving sialic acid from host glycoproteins that are recognized by the viral hemagglutinin [6] . As a type II transmembrane protein, it is N-terminally attached to the membrane [7] . It consists of a tiny cytoplasmic tail at the N-terminus (residues 1 to 6) [8] followed by the transmembrane region (residues 7 to 34) that is also responsible for translocation of the protein [9] .
Next, a presumably unstructured linker region (residues 35 to 82) connects the membrane anchor to the catalytic neuraminidase domain (residues 83 to 469; Figure 1 ). Such unstructured linker regions are rich in small and polar residues and often harbour sites for posttranslational modifications [10, 11] . Probable posttranslational modification sites in the neuraminidase of the new strain are glycosylation motifs involving N88, N146 and N235, which correspond to residues that are also glycosylated in other subtype neuraminidases [12] . However, the minimal and non-specific consensus motif of glycosylation sites (Nx [ST]) is found in total 8 times in the new strain sequence with an apparent clustering (50%) in the unstructured linker region ( Figure 1 ). Interestingly, another putative novel glycosylation site N386, which is unique to the new strain, would be accessible on the surface, as seen in the structural models.
Comparing among all strains, the sequence variation is largest in the linker region, including large deleted segments. Nevertheless, this region harbours a cysteine (Figure 2 ) that can be aligned over multiple NA subtypes and is conserved in N1-N5 and N8, but not in N6, N7 and N9. Earlier reports assume that, at least in related viruses, cysteines in the non-globular region could be involved in intermolecular disulfide bridges [13] [14] [15] . Alternatively, by analogy to other influenza proteins such as hemagglutinin [16] and M2 protein [17] , it cannot yet be excluded that cysteine C49 is palmitoylated and that the anchor localizes the protein to lipid rafts [18] .
Influenza A virus protein sequences were downloaded from NCBI (as of April 29 th ). Neuraminidases were identified by BLAST (E-value < 0.001) [19] using the representative NA of the new strain as query [Genbank: ACP41107.1 http://www.ncbi.nlm.nih.gov/protein/ 227809834]. Redundancy was removed with cd-hit at a level of maximal 90% sequence identity [20] , the remain-Domain architecture (drawn with http://au.expasy.org/tools/mydomains/) Figure 1 Domain architecture (drawn with http://au.expasy.org/tools/mydomains/). Besides the labelled domains (TM ... transmembrane), grey lollipops indicate known and putative glycosylation sites and the red lollipop marks the conserved cysteine shown in Figure 2 .
Representative alignment of the sequence environment of the conserved cysteine C49 that could either serve for intermolecu-lar disulfide bridges or as palmitoylation site Figure 2 Representative alignment of the sequence environment of the conserved cysteine C49 that could either serve for intermolecular disulfide bridges or as palmitoylation site.
ing sequences were aligned with MAFFT (using L-INS-I settings [21] ) and the resulting multiple alignment was visualized and annotated in Jalview [22] . A neighbour joining tree with pairwise gap deletion, Poisson correction as distance measure and 500 bootstrap replicates (generated with MEGA [23] ) produces robust groupings consistent with previous studies [24] for the known NA subtypes (clustering of N1, N4, N5+N8 on one side and N2, N3, N6+N7+N9 on the other) and reliably places the new NA with other N1s. Interestingly, inside the N1 cluster, the new NA appeared close to the N1 of H5N1 avian flu viruses. The alignment and corresponding phylogenetic tree are available at http://mendel.bii.a-star.edu.sg/ SEQUENCES/H1N1/.
Hence, we repeated the analysis (same protocol as outlined above) for a detailed mapping of only the N1 subtype family with the difference of allowing 95% sequence identity for sequences before 2009 but keeping all new NA sequences (as of April 29 th ). A characteristic clustering emerges ( Figure 3 ) that roughly corresponds to host and geographic distributions, consistent with previous reports [25, 26] . The observed clustering is robust in respect to the method used for tree generation (same for maximum parsimony or neighbour joining trees with JTT distance and gamma-distributed variable rates). The 2009 NA is part of a cluster of avian-like swine flu H1N1 strains predominantly found in European pigs. However, previous examples of human infections from swine flu are also part of the same cluster, for example from 2005 in Thailand [27] . This indicates that, similar to the current outbreak, closely related H1N1 strains have crossed species boundaries on previous occasions as also evidenced by further reports in the literature [28] [29] [30] .
Moreover, neuroaminidases of these new H1N1 swine flu examples are more similar to H5N1 avian flu strains than other H1N1 variants found in the Americas or than that of the historic strains such as the 1918 Spanish flu [31] . This is surprising since avian flu strains typically have different hemaglutinin (HA) subtypes (e.g. H5N1). Combinations of HA and NA subtypes need to be fine-tuned to recognize the same type of sialic acid modifications to allow smooth interplay of the two proteins, which is important for the viral cycle [6] . These results support the notion that, also inside the family of N1 subtypes, a clear distinction can be made between avian-like H1N1, such as the one from the current outbreak, and other existing H1N1 strains.
The crystal structures of both the historic 1918 NA as well as the avian flu NA are available in complex with currently used drugs. We created a homology model of the new 2009 swine flu NA to map the sequence differences to the three-dimensional structure templates. Using Modeller [32] , the sequence of the new neuraminidase [Genbank Accession: ACP41107.1 http://www.ncbi.nlm.nih.gov/ protein/227809834] was modelled 50 times onto multiple templates (PDB: 2hu4 [33] , 3ckz [34] , 3b7e [35] , 3beq [35] ) and the resulting best model (as judged by DOPE score) further refined with short simulated annealing MD simulations in the presence of a bound inhibitor (zanamivir, oseltamivir or peramivir) as implemented in the Yasara Structure package [36] . The final atom-resolution models are available in PDB format at http://men del.bii.a-star.edu.sg/SEQUENCES/H1N1/.
We mapped the level of residue conservation (calculated with the evolutionary trace algorithm [37] ) from the multiple alignment of all NA subtypes to its corresponding position in the structure. The results show the strict conservation close to the neuraminidase catalytic site, which also serves as the drug binding pocket ( Figure 4A ). The remaining conserved patches (for example the sites around N104 or below N146) fit into each other and form the dimerization/tetramerization interfaces [35] . A model of the dimeric version is available in PDB format at http:/ /mendel.bii.a-star.edu.sg/SEQUENCES/H1N1/.
Next, we compared the sequences of the new strain with the related H5N1 from avian flu and H1N1 from the Spanish flu ( Figure 5 ). Among 387 residues that were structurally modelled, the "2009 H1N1" neuraminidase differs from the other two in 21 positions. The mapping to the structure ( Figure 4B ) shows that the novel sequence mutations are distributed all around the surface of the molecule leaving the hydrophobic core, but also the catalytic site, essentially untouched. Importantly, none of the new mutations appears sufficiently close to affect the drug binding pocket. For example, all 17 residues within 3 Å of the zanamivir molecule bound to the active site are fully conserved among all three strains. The closest mutation is the conservative V149I substitution at a distance of ~10 Å to zanamivir and ~7 Å to oseltavimir.
It has to be noted that indirect effects of the mutations that may alter the binding pocket also from a greater distance are difficult to assess and cannot be excluded. To this extent, we have analysed coevolution patterns in an extensive alignment of more than 6000 non-identical Influenza A neuraminidases to eventually identify connected networks of residues using the SCA algorithm [38] as implemented in [39] , but no network that would connect the surface directly to the core and catalytic site was found (see supplementary material). In fact, all positions of the observed mutations are at the surface and naturally variable, as judged by the conservation and SCA analysis, which would rather indicate that they do not have an effect on the structure of the more distant binding pocket.
Thus, we conclude that the drug binding pocket remains unchanged in the new strain and, hence, the binding Phylogenetic tree of neuraminidase protein sequences of the N1 subtype family Figure 3 Phylogenetic tree of neuraminidase protein sequences of the N1 subtype family.
behaviour of neuraminidase inhibitors such as oseltamivir (Tamiflu ® ) and zanamivir (Relenza ® ) should be unaffected. Indeed, initial clinical reports suggest that the new virus is susceptible to the two drugs [40] . Our findings support this notion and provide a molecular mechanism. Furthermore, the third currently tested neuraminidase inhibitor, peramivir, should also be effective since it also shares the same binding pocket.
Next, we review how the new mutations affect vaccine development through altering antibody interactions as well as antigenic regions. There are 3 crystal structures of related neuraminidases in complex with antibodies [41] [42] [43] . In Figure 5 , we annotate residues that are within 3 Å distance to the respective bound antibody and, hence, crucial for the interaction. Interestingly, residues in sites recognized by both NC41 and NC10 antibodies appear mutated in the new strain. This would suggest that these old antibodies (that were originally directed against N9 neuraminidase) would probably not bind to NA of the new strain. Nevertheless, using the same regions as epitope may be a viable option for novel vaccine development.
Additionally, several other known antigenic regions, partially derived from surviving patients of previous flu outbreaks (e.g. H5N1), are reported in the literature [44] [45] [46] and their location is indicated in the alignment ( Figure 5 ). Another extensive source of epitopes and antigens includ-ing neuraminidase of influenza A viruses is the immune epitope database (IEDB) [47] and the complete mapping of epitopes for the new H1N1 NA sequence is available at http://mendel.bii.a-star.edu.sg/SEQUENCES/H1N1/. While several of the new mutations are found in antigenic regions, it is also apparent that they often occur on positions that are hardly conserved among different NA subtypes. Consequently, these regions are evolutionarily more flexible and may mutate fast. This increases the risk of evading antibody responses of human hosts acquired during previous flu infection or from vaccination.
After the first wave of new patient sequences arrived, it becomes clear that there are at least two major lineages that are distinguishable by only few mutations. Most notably, N248 has mutated to Aspartate (D248) in the New York infection cluster. All intra-strain mutations available before May 8 th 2009 are indicated in Figures 4B and 5. As expected, they are predominantly found on the surface and in regions that are known to be variable from the conservation analysis. While the drug binding pocket remains unaffected by these most recent mutations, the N248D substitution changes a central part of an antibody recognition site. This has important consequences for vaccine development, forcing to either avoid this epitope or produce combined vaccines to account for the epitope in the first annotation line) is displayed as the respective mutated residue in capital letters if found in multiple patients (e.g. D for the N248D substitution) or lower-case (e.g. "i" for V241I) for single occurrences. In the second annotation row, antigenic regions are labelled as "*". Residues with < 3 Å contact to antibodies are labelled "A" for interactions derived from PDB:1ncb, "B" from both PDB:1ncb and PDB:1nmb, "C" from PDB:1nmb and "D" from PDB:2aep. variation observed in different patient groups. Although the mutation pattern will become less transparent over time it may still serve to delineate chains of transmission, retrospectively.
In summary, we provide a sequence analysis and structural modelling of the neuraminidase from the 2009 H1N1 swine flu outbreak. Besides mapping of phylogenetic relationships to other strains, we find that the sequence variation in the new strain does not seem to affect the drug binding site but may very well alter common epitopes. To allow quick analysis of future mutations that could produce drug or vaccine-resistant strains, we provide a tool for 3D visualization of the neuraminidase structure models with mapping of drug and antibody recognition sites on the supplementary webpage http://men del.bii.a-star.edu.sg/SEQUENCES/H1N1/.
NA: neuraminidase; HA: hemagglutinin.
The authors declare that they have no competing interests.
SMS did the alignments and phylogenetic trees. FLS contributed the domain architecture analysis and helped with the phylogenetic analysis. MJ did the structural models and conservation mapping. RLTC contributed the study on antigenic regions and the Jmol visualization on the webpage. SMS and FE wrote the manuscript; all authors approved the final version.
Sandor Pongor, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
The results show that this strain is phylogenetically more closely related to European H1N1 swine flu and H5N1 avian flu rather than to the H1N1 counterparts in the Americas.
Homology-modeling of the neuraminidase reveals that the novel mutations are not likely to interfere with the active site so the currently used neuraminidase inhibitors (oseltamivir, zanamivir and peramivir) will be effective against the new virus strain.
The subject is very timely and the approach is adequate. The authors may want to include analysis of more patient data that were published since the analysis was completed. More and more sequences are being published from all over the world that might be worthwhile to include into this analysis. The authors may consider establishing a periodically updated homepage, if appropriate. In summary, the analysis is careful and carried out in a commendable fashion, and the findings are highly significant.
Indeed, there have been additional "2009 H1N1" neuraminidase sequences since the submission of this manuscript for review. Between April 29 th and May 8 th , 45 new sequences became available. Overall, 3 mutations occur in multiple (S95G, V106I and N248D) while 2 mutations are restricted to single patient virus isolates (V83M, V241I), so far. We have included a section about these mutations identified in new patient sequences and mapped them to the structure. As discussed in the main text, the intra-strain variation is typically found on the surface and at positions expected to be variable as judged by the conservation analysis among all NA subtypes. Interestingly, one of the mutations among patients (N248D) is critically affecting one of the antibody binding sites.
As the virus will continue to evolve, new mutations will become available and the best way we have found to allow quick mapping and update of new sequence variation in respect to drug and antibody binding sites is to give full access to users/readers via a 3D structure visualization tool at the supplementary webpage http://mendel.bii.astar.edu.sg/SEQUENCES/H1N1/ (instructions to map new mutations are given, including an example). The key finding in the paper is that the binding cavity for neuraminidase inhibitors is unaffected as suggested by molecular modeling. This finding is of significance in the current situation of an outbreak with pandemic potential. However, one issue needs to be highlighted in this regard -mutations far away from the binding site can potentially affect the shape and or binding affinity of the binding pocket. These are not always captured by homology models, especially the issue of affinity. In principle, authors could use a conservation patterns or co-evolution measures (e.g. as in PMID: 10514373) to determine if there are interaction chains that might connect distant residues to the active site. In the least it would be useful to provide the caveat of distant changes affecting affinity in the current paper.
We totally agree with the referee that also mutations at a greater distance may affect the binding pocket under spe-cial circumstances and we have added a new paragraph to the manuscript. However, we have to admit that it remains essentially impossible to quantify the influence of non-direct interactions by theoretical means unambiguously. In our experience, co-evolution measures such as the one proposed and several others (PMID: 18056067) produce high rates of false positives and, therefore, the interpretation is difficult. We did the requested analysis with Ranganathan's SCA algorithm to eventually identify connected networks of residues using the webserver implementation at the Gerstein lab, but no network that would connect the surface directly to the core and catalytic site was found (see supplementary webpage for full details). In fact, all positions of the observed mutations are at the surface and naturally variable, as judged by the conservation and SCA analysis, which would rather indicate that they do not have an effect on the structure of the more distant binding pocket. A possible problem why the SCA could not work in this case is the high level of sequence similarity among the neuraminidases which only gives limited numbers of informative correlated mutations. This is a totally different scenario from alignments of highly divergent sequences that still have the same fold, such as the small PDZ domains analyzed by Ranganathan. In the case of a diverse family with shared fold, correlated mutations are indicative of allowed fluctuations also among structurally important residues. However, with the neuraminidases, most variation can be attributed to surface residues, which makes sense given the pressure to avoid immune responses. Additionally, sequence sampling of neuraminidases is not independent but biased by transmission chains and clusters of outbreaks. A possible but also not necessarily more precise alternative to judge indirect effects on drug binding would be to run free energy simulations with the bound drug to judge changes of affinity caused by the mutations but this is a tricky and time-consuming endeavour that would burst the scope of this current manuscript.
unspecific consensus motif: change to "non-specific" Response changed.
Phylogenetic analysis: While for sequences at this range of similarity Poisson correction may not have negative consequences it is definitely better to repeat the analysis with JTT and variable rates to see if the clustering remains the same or changes drastically.
We confirmed that the tree clustering inside the N1 subtype family is robust regarding different tree generation methods and added this also to the text. We also provide the suggested JTT distance tree with variable rates as supplementary at http://mendel.bii.a-star.edu.sg/ SEQUENCES/H1N1/. " This indicates that, similar to the current outbreak, scenarios of breaches in the species barrier between human and pigs have already arisen out of closely related H1N1 strains as also evidenced by further reports in the literature [26, 27] ." The wording of this sentence is somewhat unclear. It appears that the authors wish to state that H1N1 like strains have crossed species boundaries on other occasions, but this is not necessarily clear in the sentence.
Response changed to "This indicates that, similar to the current outbreak, closely related H1N1 strains have crossed species boundaries on previous occasions as also evidenced by further reports in the literature".
"These results support the notion that, also inside the family of N1 subtypes, a clear distinction can be made to distinguish avian-like H1N1, such as the one from the current outbreak, from other existing H1N1 strains." What would be the explanation for this? A recent recombination between an avian-like H1N1 or has it diverged from other avian like H1N1 with the recombination occurring much earlier. Could this information be superimposed in phylogenetic context on current figure 3 ?
Response This is, of course, a very interesting question and difficult to deduce from the phylogenetic tree of a single protein without molecular clock, as in our case. However, this and similar questions have already been analyzed to quite some detail for the existing strains. The current knowledge of the scenario for H1N1 is that around the late 1970s to early 1980s, a human-avian reassortant virus started to be detected in European pigs as host (PMID: 8091678). Then, this avian-like swine flu started to move from the European continent to the UK in the early 90s (PMID: 9049404). More reassortments and emergence of the N2 subtype that quickly spread is also well documented. This phylogenetic analysis plugs the new H1N1 strain into the already known clusterings among the NA subtypes and within the N1 family. Development of TaqMan(® )MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1 BACKGROUND: Anatid herpesvirus 1 (AHV-1) is an alphaherpesvirus associated with latent infection and mortality in ducks and geese and is currently affecting the world-wide waterfowl production severely. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for fast measurement of AHV-1 DNA based on TaqMan MGB technology. RESULTS: The detection limit of the assay was 1 × 10(1 )standard DNA copies, with a sensitivity of 2 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CONCLUSION: The high sensitivity, specificity, simplicity and reproducibility of the AHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method suitable for a broad spectrum of AHV-1 etiologically related application. China is currently holding the largest waterfowl population in the world and its waterfowl production industry has been characterized by an increasing expansion and rapid development during the past decades [1] . However, infectious diseases represent the biggest obstacle to successful development of this business. Anatid herpesvirus 1 (AHV-1) infection alternatively known as duck virus enteritis (DVE), or duck plague (DP) [2] , is one of the most widespread and devastating diseases of waterfowls in the family Anatidae and has severally affected the waterfowl industry since the early 1900s because relatively high mortality could be observed and a wide host range including domestic [3] and wild ducks [4, 5] , geese and swans of all species as well as other birds like coots are susceptible. Furthermore, serious carcass condemnations and decreased egg production were also observed in affected waterfowls. Like other herpesvirus-induced diseases, AHV-1 infection has latent form and the virus can be persistently shed by birds that recover from the disease [6] . This complicates the control of the disease, particularly under small-holder farming conditions prevalent in China.
The causative agent of AHV-1 is grouped in the alphaherpesviridae subfamily of the herpesvirus family [7] and the viral genome is a linear, double-stranded DNA molecule approximately 180 kb in size and its structure is similar to other alphaherpesviruses [8] . The AHV-1 genomic DNA has % G + C content of 64.3, which is the highest reported for any avian herpesvirus in the alphaherpesviridae [9] .
Since prevention and early detection are presently the most logical strategies for virus control, various diagnostic procedures including microscopic, immunological and molecular methods have been developed for AHV-1 detection, of which the polymerase chain reaction (PCR) is a powerful tool with exquisite sensitivity for detection of minute amounts of nucleic acids, even against a high background of unrelated nucleic acids. Fluorescent quantitative real-time PCR (FQ-PCR) technique has eliminated the need of sample post-amplification handling required by the conventional PCR assay and has paved the way towards fully automated detection systems now that they usually display very high sensitivity and broad dynamic capacity after optimization [10] [11] [12] . Since virus load and proliferation dynamics serve as indispensable indicators of virus-host interaction, antiviral evaluation, active/ latent infection [13] [14] [15] and guidance for therapeutic intervention, FQ-PCR is therefore of paramount importance by its exquisite virus detection and monitoring ability [16] .
The detection of AHV-1 by TaqMan real-time PCR method has only been reported by Yang [17] and with the development of technology, TaqMan Minor Groove Binding (MGB™) probes as an upgrade of the ordinary TaqMan probe has been widely used during the recent years since the following advantages: (1) The TaqMan MGB probe is characterized by the conjugation of minor groove binders which facilitates highly specific of the detection. (2) The TaqMan MGB probe contains a quencher dye that does not emit fluorescence within the detectable wavelength range and results in greater accuracy in the measurement. Therefore a TaqMan MGB-based real-time PCR method for detection and quantitation of AHV-1 is developed to serve as an alternative and improvement of the previously developed ordinary TaqMan real-time PCR method.
Following the optimization of FQ-PCR, final concentrations of primers each of 0.3 μmol/L and probe of 0.1 μmol/L were selected. The MgCl 2 concentration was balanced to 6 mM that provided optimal AHV-1 amplification. Therefore the optimized 25-μL real-time PCR reaction system for AHV-1 detection could be summarized as follows: 1 × PCR buffer, 6 mmol/L MgCl 2 , 0.2 mmol/L dNTPs, 0.3 μmol/L each primers, 0.1 μmol/L probe, 1 U Taq and 1 μL DNA template.
Following the optimization of conventional PCR, the MgCl 2 concentration was balanced to 2.5 mM and the annealing temperature of 52°C was selected. Therefore the optimized conventional PCR reaction system could be summarized as follows: 1 × PCR buffer, 2.5 mmol/L MgCl 2 , 0.2 mmol/L dNTPs, 0.5 μmol/L each primers, 1.25 U Taq and 1 μL DNA template. The optimized annealing temperature was 52°C.
The FQ-PCR amplification curves and the corresponding fluorescent quantitative real-time PCR standard curve ( Figure 1 ) were generated by employing the successively diluted known copy number of pAHV-1 for real-time PCR reaction under the optimized conditions. From the results of correlation coefficient (0.999) and PCR efficiency (86.9%) of the standard curve by the established FQ-PCR, it could be known that the standard curve and the established FQ-PCR are excellent at performance.
Different 32 AHV-1 strains kindly provided by the Avian Disease Research Center of Sichuan Agricultural University were examined with the established FQ-PCR method and these specimens all tested positive in the FQ-PCR assay, indicating that this method is sensitive and compatible with wide range of AHV-1 viruses. Ten-fold dilution series of pAHV-1 plasmid standard DNA were tested by the established real-time PCR assay to evaluate the sensitivity of the system and the detection limit was found to be 1.0 × 10 1 copies/reaction. Comparisons were made between conventional PCR and the established FQ-PCR using dilution series to calculate the end point sensitivity of each assay. The results indicate that the established FQ-PCR is around 100 times more sensitive than the conventional PCR method, detecting pAHV-1 down to dilutions of 1.0 × 10 1 , compared to dilutions of only 1.0 × 10 3 for conventional PCR.
The test using DNA from several other bacteria and viruses used as template to examine the technique's specificity showed that none of the bacteria, virus (other than AHV-1) and duck embryo fibroblast tested gave any amplification signal and the results demonstrated that the established FQ-PCR assay is of highly specific.
The intra-assay and inter-assay CV of this established FQ-PCR was in the range of 1-3% for most of the dynamic range (from 1.0 × 10 9 to 1.0 × 10 2 pAHV-1 plasmid copies/ μL), but increased to more than 6% at viral DNA loads lower than 1.0 × 10 2 pAHV-1 plasmid copies/μL and increased to more than 4% at viral DNA loads more than 1.0 × 10 9 pAHV-1 plasmid copies/μL ( Table 1 ). The results Figure 1 Establishment of the fluorescent quantitative real-time PCR standard curve. Standard curve of the AHV-1 fluorescent quantitative real-time PCR. Ten-fold dilutions of standard DNA ranging from 1.0 × 10 9 to 1.0 × 10 2 copies/μL were used, as indicated in the x-axis, whereas the corresponding cycle threshold (CT) values are presented on the y-axis. Each dot represents the result of triplicate amplification of each dilution. The correlation coefficient and the slope value of the regression curve were calculated and are indicated. demonstrated that the established fluorescent quantitative real-time PCR method was characterized by a wide dynamic range (8 logarithmic decades) of detection from 1.0 × 10 9 to 1.0 × 10 2 pAHV-1 plasmid copies/μL with high precision. However, at lower and higher dilutions quantitation was not always reproducible compared to other properly diluted samples. Therefore the dynamic range of the method was between 1.0 × 10 9 and 1.0 × 10 2 pAHV-1 plasmid copies/μL, which is relatively broad.
Viral load quantification through the established AHV-1 FQ-PCR demonstrated that the AHV-1 DNA copy number of each sample could be calculated with the CT value according to the standard curve and 100% of the samples tested were quantifiable (Table 2 ) without the need for further sample dilution or concentration.
Conventional etiological, immunohistological and serological methods [18] [19] [20] were routinely used in AHV-1 identification. However, the sensitivity is usually not high enough and the methods were time-consuming since virus propagation in cell cultures is usually required and the onset of virus-induced cytopathic effect (CPE) usually requires at least 2-3 days to develop. Titration of infectious virus in cell cultures is usually achieved by the endpoint dilution method in cell monolayer. Since titration of the virus load is labor-consuming and requires about 5 days for evaluation of virus-induced CPE, distinguishing between virus-induced CPE and non-specific cell alterations may be difficult, the established real-time PCR assay will be particularly suitable in these studies. In addition, an even more important factor is that the virus from tissues of infected birds is usually not readily adapted to cell culture system during the initial several rounds of propagations [21] .
The PCR is a rapid, sensitive and specific nucleic acid amplification technique and many conventional qualitative PCR methods for revealing merely the presence or absence of AHV-1 pathogen have been developed and well documented [22] [23] [24] . However, the conventional PCR assays are not sufficient in a variety of clinical situations. They frequently encountered problems including the risk of cross-contamination (leading to false positives) and poor quality of extracts (leading to false negatives). Moreover, the lack of fluorogenic probes in the assay results in relative lower specificity since the amplification and detection of specific PCR products are determined solely by the amplification primers. In this paper, the development of a TaqMan MGB-based real-time PCR by using fluorogenic labels and sensitive signal detection system for detection and quantitation of AHV-1 is described.
The optimized FQ-PCR detection system presented in this paper has been designed to address these issues and make it even more applicable for routine diagnostic use with several advantages over conventional PCR.
In this assay, the primers and probes have been selected on conserved DNA segments of AHV-1 genome. TaqMan Minor Groove Binding (MGB™) probes as target-specific hydrolysis oligonucleotide employed in this assay are characterized by the conjugation of minor groove binders which increases the Tm of the hybridized probe and facilitates highly specific binding to the targeted sequence [25] . Moreover this probe contains a quencher dye that does not emit fluorescence within the detectable wavelength range and results in greater accuracy in the measurement. This improvement eliminates spectral overlaps with fluorescence emitted by the reporter dye, and results in greater accuracy in the measurement of reporter-specific signals.
In view of the great sensitivity of PCR, the occurrence of false negative results is a highly underestimated problem. So an artificial construct generated by cloning of the specific target sequence into a plasmid are often used as internal controls for the amplification step. This internal positive control was incorporated into the reaction system, thus improving diagnostic conclusions, especially negative results, which is most important in the light of quarantine programs.
By carrying out direct comparisons between the established FQ-PCR method and the conventional PCR method for AHV-1 detection, the results clearly showed that overall the established FQ-PCR detection method is more sensitive and reliable when compared to conventional gel-based PCR, since it was able to detect as few as 1.0 × 10 1 DNA copies of template. Furthermore, this established AHV-1 FQ-PCR method shows more excellent characteristics such as dynamic range (from 1.0 × 10 9 to 1.0 × 10 2 pAHV-1 plasmid copies/μL, which is approximately 10 3 times broader) and sensitivity (detecting pAHV-1 plasmid down to dilutions of 1.0 × 10 1 copies/μL, which is about 2.3 times more sensitive) than other reported method [17] .
The high quality hot start Taq DNA polymerase used in this assay could minimize unspecific amplifications and increase the PCR cycling efficiency. In addition, FQ-PCR reaction and detection is all done in a closed-tube system, the need for post-amplification manipulation is removed since the detection of the PCR products occurs online during real-time PCR amplification, hence greatly reducing the risk of cross-contamination and false positive results. The optimization of the AHV-1 FQ-PCR assay was focused on the concentration of primers and probe and Mg 2+ . When all these different practical refinements are combined, the final result is a molecular diagnostic method that is not only rapid and reliable, but one that is also easy to perform and applicable to use for testing large numbers of samples since the FQ-PCR presented the benefits of increased speed due to reduced cycle time and remove of post-amplification process, offering considerable labor savings and allowing higher throughput analysis than conventional PCR assays and thus is favorable for the transition of this method from research to routine use in laboratories. This method was preliminarily mentioned in a short report [26] but related details of primers and probe sequence, specificity test, sensitivity test, reproducibility analysis, dynamic range and internal control were unavailable. By contrast, great modification and optimization have been made in this paper to improve the quality of this study.
The AHV-1 FQ-PCR assay was highly reproducible and linear over a range of eight orders of magnitude from 10 2 to 10 9 copies, allowing a precise calculation of viral DNA load in samples containing a wide range of viral DNA amounts, eliminating the need for sample dilution and minimizing sample handling. The results for intra-and inter-assay precision indicate that both intra-assay and inter-assay CVs were satisfactorily low and the assay is reproducible, even between different batches of reagents used. Probability rather than sample quality variation is the predominant cause of variability at low copy numbers [27] .
In conclusion, the FQ-PCR developed in this study is highly specific and sensitive with better parameters than conventional PCR method and is a valuable method for the detection of AHV-1. The method described in this study is especially helpful for high throughput analysis such as evaluating the efficacy of antiviral drugs and experimental vaccines for AHV-1. The research group of authors is currently using this technique to study the AHV-1 distribution characteristics in vaccinated birds and in artificially infected birds. We believe that this method could expedite related AHV-1 research in the AHV-1 viral molecular biology.
Duck embryo fibroblast (DEF) monolayer was incubated at 37°C with 5% CO 2 in tissue culture flasks with Minimal Essential Medium (MEM) that contained 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.
Anatid herpesvirus 1 (AHV-1, CHv virulent strain) was obtained from the Avian Disease Research Center of Sichuan Agricultural University (Yaan, Sichuan, China). Virus stock was added onto the surface of the cell layer which was about 90% confluency at time of infection and the maximum virus titers could usually be obtained 48 h postinfection. DNA extraction from AHV-1 infected DEF cells and tissues of AHV-1 infected ducks were performed by using TIANamp Genomic DNA extracting kit (Tiangen Corporation, Beijing, China) according to the manufacture's instructions.
The FQ-PCR assay primers and probe (named Real-F, Real-R and Real-P respectively) design was carried out using the Primer Express™ software supplied by Applied Biosystems and their sequences were listed in Table 3 . The forward and reverse primers amplified a 60 bp fragment of AHV-1 DNA polymerase gene as described (GenBank Accession No. AF064639). The fluorogenic probe was labelled at 5' with FAM (6-carboxyfluorescein) dye as reporter and labelled at 3' with TAMRA (tetra-methylcarboxyrhodamine) as quencher and 3'with MGB™ (Minor Groove Binder).
The conventional PCR amplification was carried out using primers designed using the Primer Premier™ software according to the sequence as described (GenBank Accession No. AF064639). The forward primer and reverse primer (named Con-F and Con-R respectively) sequences were listed in Table 3 and this primer pair yielded a 498 bp amplicon, in which the 60 bp FQ-PCR fragment was nested.
All probes and primers were synthesized by Genecore Corporation (Shanghai, China) and purified by corresponding HPLC system.
The real-time PCR was carried out using the ABI AmpliTaq Gold DNA polymerase system with an icycler IQ Realtime PCR Detection System (Bio-Rad Corp., Hercules, CA) according to the manufacturer's instructions. The reaction, data acquisition and analysis were performed using iCycler IQ optical system software. The Real-time PCR was performed in an 25 μL reaction mixture containing 1 × PCR buffer, 0.2 mmol/L dNTPs, 1 U Taq and 1 μL DNA template according to the manufacture's instructions. Autoclaved nanopure water was added to bring the final volume to 25 μL. The two-step PCR cycling condition was as follows: initial denaturation and hot-start Taq The conventional PCR was performed and optimized on a Mycycler™ thermo cycler system (Bio-Rad Corp., Hercules, CA, USA) with a 50 μL PCR reaction system containing 1 × PCR buffer, 0.2 mmol/L dNTPs mixture, 1.25 U rTaq (Takara Bio Inc., Shiga, Japan), 0.5 μmol/L each forward and reverse primers and 1 μL template DNA. All PCR experiments were carried out in 0.2 ml thin-walled tubes with the following cycle parameters: The mixture was subjected to initial denaturation at 95°C for 1 min, followed by 50 cycles of 95°C for 60 s, annealing for 60 s, extension at 72°C for 60 s, and one cycle of final extension at 72°C for 5 min. The amplified 498 bp product then underwent electrophoresis on 1.0% agarose gels. Electrophoresis was carried out at 100 V in a Mini-sub (Bio-Rad Corp., Hercules, CA, USA) gel electrophoresis unit and gels were viewed under a UV transilluminator. The conventional PCR reactions were optimized based on MgCl 2 concentration and annealing temperature selection criteria in a similar way as that of Real-time PCR and the selection was made by the brightness of the amplified 498 bp fragments on the agarose gel under a UV transilluminator.
An internal positive control was introduced into the FQ-PCR assay to verify the absence of DNA losses during the extraction step and of PCR inhibitors in the DNA templates. The internal positive control of pGM-T recombinant vector (designed as pB16S) consisting of Bacillus 16S rRNA gene (GenBank Accession No. AJ971894) sequence amplified with primers (IC-F and IC-R) listed in Table 3 was added into the lysis buffer at the concentration of 1.0 × 10 6 copies/μL. Real-time PCR for IC detection was carried out in a separate run, using primers and probe (named IC-F, IC-R and IC-P respectively) listed in Table 3 . The fluorogenic probe was labelled at 5' with FAM as reporter and labelled at 3' with TAMRA. The quantitative real-time PCR protocol was the same as that of AHV-1 detection. From the ratio of the calculated amount of IC to the actual amount of IC, which is shared by the specimen, the normalization could be achieved and the actual amount of AHV-1 in the specimen could be obtained. Actually this internally controlled method has been widely used in other related detection assays [28, 29] .
The 498 bp conventional PCR target amplicon band on agarose gel was cut and the DNA was recovered and purified by TIANquick DNA Purification system (Tiangen Corp., Beijing, China) according to the instruction manual of the product. The product was ligated into pGM-T vector (Tiangen Corp., Beijing, China) and transformed into E.coli DH5α competent cells. Recombinant plasmid (designated as pAHV-1) was extracted using TIANprep plasmid extraction kit (Tiangen Corp., Beijing, China). Presence of the target DNA insert was confirmed by PCR amplification and sequencing.
The standard curve of the FQ-PCR was generated by successive dilutions of the known copy number of pAHV-1.
Recombinant plasmid pAHV-1 concentration was determined by taking the absorbance at 260 nm using a Smartspec 3000 spectrophotometer (Bio-Rad Corp., Hercules, CA) and purity was confirmed using the 260/280 nm ratio. Through its molecular weight, pAHV-1 copy number was then calculated and the purified pAHV-1 plasmid DNA was then serially diluted 10-fold in TE buffer, pH 8.0, from 1.0 × 10 9 to 1.0 × 10 2 plasmid copies/ μL. These dilutions were tested in triplicate and used as quantitation standards to construct the standard curve by plotting the plasmid copy number logarithm against the measured CT values. The Bio-Rad iCycler IQ detection software created the standard curve, calculated the correlation coefficient (R 2 ) of the standard curve, standard deviations of triplicates.
Different 32 AHV-1 strains (derived from a wide spectrum of sources, subsequently confirmed through related etiological methods, and then preserved by the Avian Disease Research Center of Sichuan Agricultural University) including virulent and avirulent strains were examined with the established FQ-PCR method to test the sensitivity and compatibility of this method. In addition, the sensitivities of the conventional PCR and FQ-PCR were each determined using triplicates of different concentrations of recombinant plasmid pAHV-1. Template DNA was prepared as follows: plasmids of pAHV-1 were diluted serially in 10-fold steps from 10 10 copies/μL to 10 1 copies/μL using sterile ultra pure water. One microliter from each dilution was used as template and subjected to the conventional PCR and FQ-PCR protocol respectively. The detection limit of the conventional PCR was determined based on the highest dilution that resulted in the presence of clear and distinct amplified fragments (498 bp) on the agarose gel. The detection limit of the FQ-PCR was determined based on the highest dilution that resulted in the presence of CT value in real-time PCR detection. Within-run and between-run reproducibilities of the FQ-PCR assay were assessed by multiple measurements of pAHV-1 samples of different concentrations. The assay was conducted by assessing the agreement between the replicates in five replicates (within-run precision) and in five separate experiments (between-run precision) of the serially diluted pAHV-1 recombinant plasmid samples through performing analysis of the mean coefficient of variation (CV) values of each AHV-1 standard dilution.
Dilutions of pAHV-1 recombinant plasmid were used to determine the dynamic ranges of the FQ-PCR assay. The lower and upper limits of quantification were defined by the pAHV-1 recombinant plasmid sample concentrations possessing reasonable precision.
AHV-1 infected duck embryo fibroblast culture, allantoid fluid and other specimens including liver, brain, Bursa of Fabricius, thymus, spleen, esophagus, duodenum, ileum, kidney, lung, peripheral blood each collected from AHV-1 infected ducks were employed to assess the ability of the established FQ-PCR to detect AHV-1 in a variety of usually used samples. By this assay viral load quantification was obtained. Role of nitric oxide in management of acute respiratory distress syndrome The current mortality rate of patients suffering from acute respiratory distress syndrome (ARDS) is between 45% and 92%, with most dying within the first two weeks of the illness. In an effort to combat such an alarmingly high mortality rate, various treatment therapies such as low tidal volume ventilation strategies, corticosteroid therapy, and use of nitric oxide (NO) have been attempted in the management of patients with ARDS. Three cases which were admitted to the ICU and confirmed to have ARDS were unable to be weaned from ventilatory support, and nitric oxide therapy was initiated. It improved patients' oxygenation for short periods of time but did not affect the mortality. The patients could not be weaned from the ventilator and expired.  Avian influenza: The tip of the iceberg For some years now, we have been living with the fear of an impending pandemic of avian influenza (AI). Despite the recognition, in 1996, of the global threat posed by the highly pathogenic H5N1 influenza virus found in farmed geese in Guangdong Province, China, planning for the anticipated epidemic remains woefully inadequate; this is especially true in developing countries such as Saudi Arabia. These deficiencies became obvious in 1997, with the outbreak of AI in the live animal markets in Hong Kong that led to the transmission of infection to 18 humans with close contact with diseased birds; there were six reported deaths.[1] In 2003, with the reemergence of H5N1 (considered the most likely AI virus) in the Republic of Korea and its subsequent spread to Thailand, Vietnam, Hong Kong and China. Many countries started aggressively making preparations to meet the threat.[2] The pressure for real action from governments has increased. Most developed countries have requested increased funding for the search for a more effective vaccine, for stockpiling possibly helpful antiviral drugs, and for intensifying domestic and global surveillance.[3] Most countries, however, continue to be inadequately prepared for such an epidemic, especially with regard to animal surveillance in the farm market and surveillance among migratory birds. Even now, most countries do not have the ability to detect disease among humans in the early stages of an outbreak nor do most hospitals comply with effective infection control measures that could curtail the spread of the virus in the early stages of an epidemic. In Saudi Arabia we are rapidly implementing many of these measures.[4]  The Y271 and I274 Amino Acids in Reverse Transcriptase of Human Immunodeficiency Virus-1 Are Critical to Protein Stability Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 plays a key role in initiating viral replication and is an important target for developing anti-HIV drugs. Our previous study showed that two mutations (Y271A and I274A) in the turn RT (Gln(269)-Arg(277)) abrogated viral replication, but the replication capacity and RT activity was discordant. In this study, we further investigated why alanine substitutions at these two sites would affect viral replication. We found that both RT activity and RT protein were almost undetectable in viral particles of these two mutants, although the Pr160(gag-pol) mutants were properly expressed, transported and incorporated. Using protease inhibition assay, we demonstrated a correlation between the degradation of the RT mutants and the activity of viral protease. Our native gel analysis indicated that the mutations at 271 and 274 amino acids might cause conformational changes, leading to the formation of higher order oligomers instead of dimers, resulting in increased protein instability and susceptibility to viral protease. Thus, residues 271 and 274 are critical to RT stability and resistance to viral protease. The conservation of the two amino acid residues among different strains of HIV-1 lent further support to this conclusion. The knowledge gained here may prove useful in drug design. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1, encoded by pol gene, is a multifunctional enzyme that possesses RNA-and DNA-dependent polymerase activities as well as RNase H activity [1] . RT is indispensable for HIV-1 and it converts the single-stranded viral RNA into double-stranded DNA upon viral entry into host cells. Due to its important role in viral life cycle, RT is one attractive target for antiviral drug design [2] .
The biologically active form of HIV-1 RT is a heterodimer consisting of two subunits, p66 (66 kDa) and p51 (51 kDa). The p51 subunit is derived from p66 by proteolytic cleavage of its Cterminal domain [3] . The polymerase domain of p66 and p51, resembling a right hand configuration, consists of four subdomains, which are known as fingers, palm, thumb and connection. The fingers, palm and thumb subdomains of p66 form a nucleic acid binding cleft and the connection subdomains of the two subunits form the floor of the nucleic acid binding site [4] [5] [6] [7] .
The thumb subdomain has four a helices. Two antiparallel ahelices of them, a-H (Asn 255 to Ser 268 ) and a-I (Gln 278 to Thr 286 ), are important for holding the primer/template in position during the translocation in polymerization. The primary sequence (Val 254 to Ala 288 ) in the vicinity of these two a helices has been found to share homology with several other nucleic acid polymerases and has been termed the ''helix clamp'' [5, 8] . Extensive studies have been carried out to shed light on the relationship between the ''helix clamp'' and function of RT. The effects of alanine-scanning mutations in a-H and a-I on polymerase activity, primer/template (P/T) binding, fidelity and enzyme kinetics have been determined. While mutations in a-I do not affect P/T binding or fidelity significantly, several a-H mutants exhibit lower binding affinity, processivity and frameshift fidelity [9] [10] [11] [12] . Previous studies have demonstrated that mutations in these two helices can have significant effect on RNase H activity, minus-strand DNA transfer activity and removal of polypurine track primer [13, 14] .
Although alanine substitutions at sites 269, 270, 271 and 277 have been investigated in two studies [9, 10] , detailed studies on the functional structure of the ''turn'' (Gln 269 -Arg 277 ) between a-H and a-I were limited. In our previous study on hepatitis B virus RT, conserved residues located at the ''turn'' of helix clamp motif were found important for pregenomic RNA encapsidation during the assembly of nucleocapsids [15, 16] . Since this homologous helix clamp motif is also present in HIV-1 RT, we hypothesized that residues in the turn may play important roles in viral life cycle. Our recent study showed that alanine substitutions at 271 and 274 of HIV-1 RT drastically affected viral replication, but discordance between viral replication and RT activity was observed [17] . In this study, we confirmed our previous observations and further investigated why the two mutations abrogated viral replication. Our study demonstrated that these two mutations lead to rapid degradation of RT in viral particles, indicating that the residues of 271 and 274 are critical for maintaining the stability of HIV RT.
The parental HIV-1 proviral plasmids, pLAI.2, pNL4-3-DE-EGFP and pHEF-VSV-G, were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [18] [19] [20] . The pNL4-3-DE-EGFP based mutants (Y271A, G273A, I274A, K275A, V276A, R277A) and pLAI2 based mutants (Y271A and I274A) were constructed by sitedirected mutagenesis using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to manufacturer's instruction. The open reading frame of RT p66 subunit was amplified from wild type or mutant pNL4-3-DE-EGFP using a pair of primers and cloned into pET-28b vector (Novagen, Shanghai) to obtain expression plasmid pET-p66 with the 66 His tag at 39 terminus. The paired primers used for site-directed mutagenesis and construction of plasmids are listed in Table 1 . Cell lines 293FT, HeLa and U373-MAGI-CXCR4 CEM [21] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (Invitrogen, USA). MT2 cells [22, 23] were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics.
To prepare pseudoviruses and live viruses, 293FT cells were cotransfected with 7.5 mg wild-type or mutant pNL4-3-DE-EGFP, and 2.5 mg pHEF-VSV-G, or 10 mg wild-type or mutant pLAI.2 using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. Supernatants were collected 48 h after transfection and stored at 280uC after spinning down and filtering to remove cell debris. The cells were rinsed with ice-cold phosphate-buffered saline (PBS), scraped from each plate and lyzed in cell lysis buffer (Boehringer, Germany) and 16 protease inhibitor cocktail (Roche, USA) on ice for 30 min. Cell lysates were stored at 220uC after centrifugation at 13,0006g at 4uC for 15 min to remove cell debris.
One cycle infection assay was carried out using normalized pseudoviruses as described previously [20] . Briefly, Jurkat cells (0.5610 6 ) were infected with viral supernatants containing 250 ng p24, which was measured by Vironostika HIV-1 antigen MicroELISA kit (Biomerieux bv Boxtel, Netherlands). The virus and cells mixture was spun at 1,800 g at 30uC for 2 h. After the 2h spin infection, Jurkat cells were washed with 2 ml culture medium twice, then cultured in 24-well plates at 37uC for 48 h. The cells were collected and washed twice with PBS. After the cells were fixed with 1% paraformaldehyde in PBS for 30 min on ice, the infected cells, as determined by the expression of GFP, were measured using a FACSCalibur instrument (Becton Dickinson, USA) and analyzed with Cell Quest software (Becton Dickinson, USA) as described previously [20] .
Infection with live viruses was conducted in U373-MAGI-CXCR4 CEM and MT-2 cells. U373-MAGI-CXCR4 CEM cells were infected with normalized wild type or mutant live viruses in 24-well plate as described previously [21] . Briefly, triplicate wells (6610 4 cells/well) were infected with live viruses (60 pg p24/well) which were diluted in DMEM containing 20 mg/ml of DEAEdextran (Amersham Biosciences). After cultured at 37uC in 5% CO 2 incubator for 48 h, the cells were fixed in 1% formaldehyde-0.2% glutaraldehyde in PBS for 5 min. After washing twice with PBS, the cells were stained with 400 mg/ml of X-Gal (5-bromo-4chloro-3-indolyl-b-D-galactopyranoside), 4 mM MgCl 2 , 4 mM potassium ferrocyanide, and 4 mM potassium ferricyanide in PBS for 2 h at 37uC. The plate was washed twice with PBS and blue foci were observed under microscope. Infection with live viruses was also carried out in MT-2 cells as described previously [22, 23] . In brief, MT-2 cells (1610 4 cells/well) in 96-well plate were infected with live viruses (20 pg p24/well). After cultured for 6 days, the virus-specific cytopathic effect (CPE) was observed under microscope.
The RT activity in pseudoviruses was measured by a RT assay using colorimetric kit (Roche, USA). Briefly, viral supernatants containing 2 mg p24 were centrifuged at 4uC for 2 h at 40,0006g and the viral pellets were resuspended in 50 ml lysis buffer. Lyzed viral pellets were 10 fold serially diluted and the subsequent procedures were carried out according to the manufacturer's instructions. RT activities of mutants were calculated and compared to that of wild type pseudovirus.
Expression and subcellular localization of precursor Gal-Pol polyprotein were detected by immunofluorescence microscopy as described previously with some modifications [24] . Briefly, HeLa cells cultured on coverslip in 24-well culture plate were transfected with 600 ng pNL4-3-DE-EGFP and 200 ng pHEF-VSV-G. Two days post-transfection, the cells were fixed with 500 ml 4% paraformaldehyde (PFA) at room temperature for 15 min. After washing 3 times with PBS, 500 ml of 50 mM ammonium chloride was incubated with the cells for 10 min to neutralize residual PFA. The cells were washed 3 times with PBS and treated with 0.05% Triton X-100 for 3 min. After washing 3 times with PBS, 500 ml 10% normal rabbit serum (NRS) in PBS was added to block the slip overnight at 4uC. Primary antibody (mouse monoclonal antiintegrase, 1:100, Santa Cruz) and secondary antibody (Texas Red dye-conjugated Rabbit anti-mouse IgG, 1:100, Jackson Immu-noResearch) were incubated with samples in dark at room temperature. Following each incubation, samples were subjected to 3 washes with 1% NRS in PBS. With 3 additional PBS washes, the coverslip was mounted using fluorescence mounting medium (Dako) and observed under LSM510 Meta confocal microscope (Carl Zeiss).
Gal-Pol polyprotein and its products were tested by Western blotting as described previously with some modifications [25] . Briefly, viral proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto Hybond-P PVDF membrane (GE Healthcare, Bio-sciences). After blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated with primary antibodies (Rabbit polyclonal anti-RT, 1:3000; mouse monoclonal anti-RT, antiintergrase, anti-protease or anti-capsid, 1:500) for 1 h. Membrane was washed three times with PBS containing 0.1% Tween 20 (PBS-T) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG, 1:4000) for 1 h. After the blots were rewashed three times in 0.1% PBST, signals were visualized using ECL Western blotting substrate reagents (Amersham Biosciences, USA) and KODAK BioMax Scientific Imaging Film (Eastman Kodak).
Wild type and mutant pET-p66 expression vectors were respectively transformed into E. coli BL21(DE3) and the expression was induced with 0.3 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) when E. coli grew up to an OD600 of 0.7,1.0. E. coli expressing p66 was spun down and re-suspended in the binding buffer (50 mM NaH 2 PO4, 300 mM NaCl, 10 mM imidazole). After the bacteria were disrupted by ultrasonication and centrifuged at 15 000 g for 30 min at 4uC, the wild type and mutant p66 in supernatants were purified using Ni-NTA magnetic agarose beads (Qiagen, Germany). The purified p66 proteins were subjected to NativePage Novex Bis-Tris gel analysis and Western blotting according to the protocol recommended by Invitrogen, USA.
RT sequences of 1083 HIV-1 strains obtained from the HIV complete sequence database (http://www.hiv.lanl.gov/content/ sequence/NEWALIGN/align.html) were aligned and compared. Based on the X-ray crystal structures of HIV-1 RT (1RTH) from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), structural models of wild type and mutant RTs were analyzed using Swiss-PdbViewer software (http://spdbv.vital-it.ch/).
Statistical analysis of RT activities was performed by Student's t test using Stata statistical software. Results were considered significant at P#0.05.
The effect of six RT mutations at the helix clamp turn in HIV-1 RT on viral replication was tested with pseudoviruses. When Jurkat cells were infected with the wild type and mutant pseudoviruses (250 ng p24 virus/5610 5 cells), viral replication was almost completely inhibited in the mutants Y271A and I274A, while replication of other mutants did not show significant difference as compared to that of the wild type (Fig. 1A) . A similar result was also obtained when the cells were infected with lower amount (150 ng p24) of pseudoviruses (data not shown). To confirm the above results, replication of wild type, Y271A and I274A live viruses was further monitored in U373-MAGI-CXCR4 CEM and MT2 cells. Mutants Y271A and I274A were undetectable in U373-MAGI-CXCR4 CEM cells (Fig. 1B) and no virus-specific CPE was found in MT-2 cells (Fig. 1C) . Since reverse transcription is the first step for HIV replication after viral entry into host cells, our results suggested that the two mutations might have affected the RT activity.
RT activity and RT proteins were undetectable in pseudoviral particles of mutants Y271A and I274A
We thus interrogated the pseudovirus mutants for RT activity. The RT activity recovered from mutants Y271A and I274A were almost unnoticeable, as compared with that of wild type ( Fig. 2A) . This result was further confirmed using wild type, Y271A and I274A live viruses (data not shown). Next, we asked if the viral particles contain dysfunctional RT or the RT was not incorporated into the viral particles. By Western blotting, RT protein was basically undetectable in viral particles of these two mutants (Fig. 2B) . The results suggested that the mutations could affect the incorporation of RT into the viral particles, leading to a defect in reverse transcription which initiates viral replication.
Mutations Y271A and I274A did not affect Pr160 gag-pol expression and transportation We next investigated whether the loss of RT in viral particles of the mutants was attributed to altered expression and transportation of the precursor protein, polyprotein Pr160 gag-pol , during virus assembly. Pr160 gag-pol in lysates of cells transfected with the wild type and mutants was detected by Western blotting. As shown in Fig. 3A , similar levels of Pr160 gag-pol , Gag protein (Pr55 Gag ) and capsid protein p24 (CA p24) were found in cells transfected with the wild type or mutant constructs, indicating that the expression and stability of RT precursor protein were not affected by the mutations. Immunofluorescence staining further demonstrated a normal subcellular localization of mutant Gag-Pol polyproteins as compared to that of the wild type, suggesting that the transportation of Gag-Pol was unlikely to be affected by alanine substitutions (Fig. 3B) .
The Gag-Pol polyprotein was incorporated into mutant virions of Y271A and I274A, but the RT was degraded by viral protease
To investigate whether the precursor Gag-Pol polyprotein was indeed incorporated into pseudoviral particles of mutants Y271A and I274A, products of the Gag-Pol polyprotein, integrase (IN), protease (PR) and p24, in wild type and mutant pseudoviral particles were examined by Western blotting. The results showed that, except for RT (Fig. 2B) , all products of the Gag-Pol polyprotein, IN p32, PR p11 and CA p24, were detected in the mutants Y271A and I274A at a level similar to that of the wild type pseudoviral particles (Fig. 4A) . Thus, Pr160 gag-pol was indeed incorporated into the virions and processed properly. To study if the RTs in the viral particles of Y271A and I274A mutants were degraded by proteolysis that made them undetectable, pseudoviruses of wild type and mutants were generated in the presence or absence of indinavir, a highly specific inhibitor of HIV-1 protease. Indinavir treatment was effective, because the treatment resulted in a dose-dependent inhibition of Pr55 Gag processing into p24 for wild type and mutant viruses (Fig. 4B ). As shown in Fig. 4C , in the absence of indinavir, both RT p66 and p51 were readily detected in the wild type pseudovirus, but not in the mutant viruses. In the presence of indinavir, however, both RT p66 and p55 were markedly reduced in the wild type virus but became detectable in mutant Y271A viral particles, while RT p66 was also detectable in mutant I274A virus. We also detected the RT subunits in the viral supernatants collected at earlier time points (12, 24 and 36 hours post-transfection), but the mutant RTs were still undetectable (data not shown). These results suggested that the Y271A and I274A
RTs were degraded after incorporation of Gag-Pol polyprotein into the virions, which might be attributed to the activity of viral protease.
Since RT dimer is the stable form which remains resistant to the proteolysis, we tested whether treatment of dimerization enhancer could reduce the mutant RT proteolysis. Wild type and mutant pseudoviruses were generated in the presence or absence of Efavirenz (EFV), the most potent dimerization enhancer [26] . The results showed that the RT mutants were still undetectable in the presence of EFV (Fig. 5A) . The conformation of the wild type and mutant RT p66 was further analyzed by native gel electrophoresis followed by Western blotting (Fig. 5B) . Under native conditions, wild type p66 subunit formed homodimer. However, the formation of homodimer in mutant Y271A was markedly reduced and a higher order oligomer appeared, which might be tetramer according to its molecular weight, while mutant I274A existed as at least two higher order oligomers, which were likely tetramer and octamer based on their molecular weights. However, the exact nature of the oligomers formed by mutant p66 subunits remained to be clarified. Furthermore, the treatment with b-mercaptoethanol could partially disrupt the higher order oligomers and improve dimer formation. This result implicated that dimer formation might be necessary for the stability of RT and its resistance to proteolysis after incorporation of Gag-Pol polyprotein into the viral particles.
Amino acids at 271 and 274 are relatively conserved and big side chains may be important in maintaining RT stability and resistance to proteolysis By comparing 1083 complete sequences of HIV-1, it was found that the amino acids at 271 and 274 were relatively conserved. As shown in Table 2 , tyrosine (Y) is the predominant naturally existent amino acid at 271 residue (99.19%), while phenylalanine (F), histidine (H) and cysteine (C) occur rarely (,1%). Similarly, isoleucine (I) is the predominant naturally existent amino acid at 274, which accounts for 98.43%, while in less than 2% of all cases, valine (V) and leucine (L) are found at this site. This finding suggested that these amino acids might probably play important role in maintaining the active conformation of RT. Consistently, structural analysis suggested that amino acids at these two sites are buried in the thumb region of RT (Fig. 6A) . It was further revealed that all wild type RTs have relatively big side chains, but they are absent from the mutants 271A and 274A (Fig. 6B & 6C) . Thus, the loss of the side chains in the RT mutants plausibly leads to conformational change of RT, leading to aberrance in dimer formation and susceptibility to proteolysis by HIV-1 protease.
HIV RT plays a key role in viral replication and is an important target for development of anti-HIV drugs. However, the turn between helices H and I of HIV-1 RT thumb region (amino acid residues 269 to 277) has not been well characterized yet. To understand the structure-function relationship of this turn in details, we investigated whether alanine substitutions in this region would affect RT activity. Except for residues 269 and 270, which have been reported to have no significant influence on RT activity [9, 10] , and for residue 272, which is originally an alanine, six mutant pseudoviruses (residues 271 and 273 to 277) were constructed and viral replication was compared with that of the wild type virus. The replication of two mutants, Y271A and I271A, were found to be almost completely abolished (Fig. 1A) . This result was further confirmed in live virus system (Fig. 1B &  1C) . Since it has been reported that bacterially expressed Y271A mutant has only 1% activity of wild type enzyme [9] , we asked whether the mutants would similarly affect RT activity in the viral particles. The results showed that the RT activity was basically undetectable in viral particles of these two mutants ( Fig. 2A) . It was also found that the loss of RT activity in the viral particles might be attributed to the absence of RT in the viral particles rather than the incorporation of dysfunctional RT into the virions (Fig. 2B) . After viral entry into the cells, the first step of HIV-1 replication is reverse transcription. Our results thus suggested that replication of mutant viruses was abrogated, because the mutant RT did not exist in the virion.
Since HIV-1 RT is incorporated into the virion in the form of Pr160 gag-pol , which is transported to cell membrane where the virus is packaged, through interaction with Pr55 gag [27] [28] [29] [30] , we thus investigated whether the mutations would affect Pr160 gag-pol expression and transportation. Our results showed that Pr160 gagpol was properly expressed and transported in cells transfected with mutant constructs, Y271A and I271A (Fig. 3) , indicating that these mutations did not affect either the production of the precursor or the interaction between Pr160 gag-pol and Pr55 gag . It was further found that the Pr160 gag-pol was indeed incorporated into the virions of these two mutants, because except for RT, all other products of Pr160 gag-pol including p24, protease and integrase [31, 32] , could be detected in the viral particles of mutants Y271A and I271A (Fig. 4A) . Although it has been reported that the domain between residues 183 and 305 of RT is likely responsible for RT incorporation [33] , our study ruled out the involvement of RT was detected in wild type and mutant pseudoviruses generated in the presence and absence of EFV, the most potent dimerization enhancer, by Western blotting using mouse monoclonal anti-RT and anti-CA p24 antibodies, respectively. (B) Purified wild type and mutant RT subunit p66 were analyzed by native gel electrophoresis followed by Western blotting using mouse monoclonal anti-RT antibody in the presence or absence of b-mercaptoethanol (b-ME). Compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271A and 274A mutants formed higher order oligomers, suggestive of conformational change. doi:10.1371/journal.pone.0006108.g005 the turn (Gln 269 -Arg 277 ). Another report has suggested that two mutations in RT, L234D and W239A, led to premature cleavage of the Gag-Pol precursor and reduced levels of viral enzymes in the virions [34] . Our results also excluded that the mutations of Y271A and I271A affected the cleavage of the Gag-Pol precursor. By a viral protease inhibition assay using indinavir, we finally demonstrated that the RT mutants were degraded after incorporation of the precursor polyprotein into the virions and the degradation was associated with the activity of viral protease (Fig. 4C) . Our results indicated that the mutations in residues 271 and 274 would reduce the stability of RT inside the viral particles, rendering it susceptible to viral protease.
Post-incorporation degradation of RT has been reported previously. Wapling et al. [35] have reported that mutations of W401L and W401A in RT can inhibit RT dimerization, resulting Fig. 6A . A big side chain (blue) is found in naturally occurring residues but not in 271A. (C) Structural models of naturally existing 274I, 274V and 274L residues as well as the lethal 274A mutation of p51 subunit. The same region was highlighted above in the right panel of Fig. 6A . A big side chain (pink) is found in naturally occurring residues I, V and L, but not in 274A. doi:10.1371/journal.pone.0006108.g006 [36] . Another mutation, L289K in p66 subunit, has also been reported to be able to abrogate dimerization [37] . Another group, when they tried to generate infectious molecular clones of Simian Immunodeficiency Virus, found that glutamic acid replacement at position 287 affected the stability of RT [38] . The biologically active and stable form of HIV-1 RT is a heterodimer consisting of p66 and p51, while the immediate precursor of p66/p51 heterodimer is the p66 homodimer. Proteolytic removal of the RNase H domain in one of the p66 homodimer subunit by HIV-1 protease leads to the formation of stable heterodimer p66/p51 [39] . RT exists as an equilibrium mixture of monomers and dimers that include the p66/p51 heterodimer and the p66/p66 and p51/p51 homodimers, among which the heterodimer is the most stable and the p51/p51 homodimer is the most unstable [40] [41] [42] [43] . Plausibly, the degradation of RT as reported in previous studies may be ascribed to the inhibition of RT p66 dimerization by the mutations. The mechanism of RT degradation in the virions observed in this study, however, may be different. Our results showed that RT mutants were still undetectable in the viruses generated in the presence of a potent dimerization enhancer (Fig. 5A ). Our native gel analysis showed that the dimer form of RT mutant 271A was detected, although it was markedly reduced as compared to the wild type, while mutant 274A could not form dimer. Instead, higher order oligomers of RT were detected in both mutants (Fig. 5B ). It has been reported that higher order oligomerization may occur in HIV-1 RT [41, 44, 45] . Our results have also indicated that Y271A and I274A substitutions may change the conformation of RT, leading to oligomerization. The higher order oligomers formed in these 2 mutants, perhaps tetramer and octamer, may be associated with instability of RT mutants and their susceptibility to viral protease. Furthermore, according to the locations of residues 271 and 274 (Fig. 6A) , it is highly probable that alanine substitutions at 271 and/or 274 can affect the conformation of p51 thumb region and subsequently its interaction with RNase H domain of p66, resulting in the exposure of the 7-amino-acid p51-RNas H cleavage sequence in the p66 subunit [7, 46] and consequent proteolytic degradation by HIV-1 protease. The relative conservation of RT 271 and 274 residues and the loss of big side chains at these two sites in the mutants as shown in the structural models (Fig. 6B & 6C ) also support their important roles. We tried to demonstrate this in vitro, by treating wild type and mutant RTs with recombinant protease, but wild type RT, as well as mutant RTs, could not be digested in vitro (data not shown). This result is consistent with that reported by Abram and Praniak [36] . This could be explained by different proteolytic stability of RT in vitro and in virions.
Taken together, our study showed that alanine substitutions at residue 271 or 274 of HIV-1 RT could cause conformational changes, rendering RT unstable and susceptible to viral protease. Thus, it is demonstrated that the ''turn'' (Gln 269 -Arg 277 ) between two helices may be important in maintaining protein stability and formation of bioactive dimer. Mutations in residues 271 and 274 of RT may inactivate the virus, which may enter cells but can not replicate. These findings may prove useful in anti-HIV drug design and vaccine development. Considering the emergence of resistance to the current RT inhibitors, this turn, especially amino acid residues 271 and 274, may be an ideal new target for antiviral drug design. The agents targeting this region may be able to affect the stability or dimerization of HIV-1 RT and subsequently inhibit viral replication. Moreover, the potential drug resistant mutations in this region, especially at residues 271 and 274, may probably inactivate the virus, which prevents the appearance of drugresistant virus. CVTree update: a newly designed phylogenetic study platform using composition vectors and whole genomes The CVTree web server (http://tlife.fudan.edu.cn/cvtree) presented here is a new implementation of the whole genome-based, alignment-free composition vector (CV) method for phylogenetic analysis. It is more efficient and user-friendly than the previously published version in the 2004 web server issue of Nucleic Acids Research. The development of whole genome-based alignment-free CV method has provided an independent verification to the traditional phylogenetic analysis based on a single gene or a few genes. This new implementation attempts to meet the challenge of ever increasing amount of genome data and includes in its database more than 850 prokaryotic genomes which will be updated monthly from NCBI, and more than 80 fungal genomes collected manually from several sequencing centers. This new CVTree web server provides a faster and stable research platform. Users can upload their own sequences to find their phylogenetic position among genomes selected from the server's; inbuilt database. All sequence data used in a session may be downloaded as a compressed file. In addition to standard phylogenetic trees, users can also choose to output trees whose monophyletic branches are collapsed to various taxonomic levels. This feature is particularly useful for comparing phylogeny with taxonomy when dealing with thousands of genomes. Traditional molecular phylogeny makes use of small subunit ribosomal RNA (SSU rRNA) sequences or a few orthologous proteins. Some more recent phylogenomic studies are based on concatenation of a larger number of proteins. The ever burgeoning genome sequencing projects worldwide have prompted several whole-genome phylogenetic approaches. However, most-if not all-rely on sequence alignment at some stage and therefore depend on many parameters, such as the use of scoring matrices. As modern prokaryotic and fungal taxonomy depends more and more on the traditional phylogeny, there is an urgent need to develop alternative approaches.
CVTree provides such an alignment-free and parameterfree phylogenetic tool using composition vectors (CVs) inferred from whole genome data (1) . As a web server, it was first introduced in 2004 (2). The CV method has been effectively apaplied to phylogenetic study of viruses (3, 4) , chloroplasts (5) , prokaryotes (1, 6, 7) and fungi (submitted for publication). So far, the CV method has been cited in more than 70 papers not of our own, including some reviews (8, 9) .
Since a CV consists of 20 K (for proteins) or 4 K (for DNA sequences) components for each organism, the calculation is simple but CPU time and memory consuming. In order to catch up with the increasing amount of genomic data, we have redesigned the data processing strategy and implemented a new user-friendly web interface to improve the new CVTree server in several aspects:
(1) the inbuilt database has been enlarged and is now updated monthly from the NCBI FTP site (10). (2) Users may upload sequences of their own and carry out phylogenetic study together with genomes selected from the inbuilt database. (3) Many kinds of tree files are provided to facilitate comparison with taxonomy. Some tree files are directly uploadable to MEGA (11) or the Interactive Tree Of Life (iTOL) project (12) in order to display the results in different ways. (4) The efficiency of CVTree has been significantly enhanced to meet the requirement of treating thousands of genomes in a single run.
All these improvements make the CVTree server a useful complement to various phylogenetic projects *To whom correspondence should be addressed. Tel/Fax: +86 21 6565 2305; Email: xuzh.fdu@gmail.com, xuzh@fudan.edu.cn ß 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. such as AToL (Assembling the Tree of Life, http:// atol.sdsc.edu) or AFTOL (Assembling the Fungal Tree of Life, http://aftol.org) by providing independent verification and support to the SSU rRNA and few gene-based phylogenies (13) (14) (15) (16) (17) .
Since the algorithm used in CVTree has been described previously (1, 2, 6) , we only give a brief account here. One collects all protein products in a genome and counts the number of (overlapping) K-tuples to form a raw CV with 20 K or 4 K components, depending on whether protein or coding DNA sequences are used (both options are allowed in CVTree, but protein sequences are recommended). Furthermore, one predicts the number of K-tuples from that of K À 1-mers and K À 2-mers by using a simple Markovian assumption. The differences between the prediction and the actual counts are taken as new components of a 'renormalized' CV. One may consult (1, 2, 6) or the online user's manual (available from the CVTree home page or http://tlife. fudan.edu.cn/cvtree/help/help.pdf) for more detailed description.
The key improvement to accelerate CVTree's speed consists in avoiding repeated calculations among all jobs submitted after a major update of the database. All intermediate results of raw and renormalized CVs are kept until a major change taking place in the database. The response to a new submission may be deceptively fast if one's genome list coincides largely with that of a previous job.
In the CVTree web server, the processing is carried out in two steps ( Figure 1) .
First, the CV for each organism is calculated. CV files containing high dimensional vectors for all organisms are dumped to the hard disk. This strategy ensures that the CV is calculated only once for each organism. If the sequences of one organism have not been changed during the monthly update, the corresponding CV file will be kept.
Second, the pairwise distances between the 'renormalized' CVs are calculated to generate a dissimilarity matrix. After the dissimilarity matrix has been produced, the standard neighbor-joining method (18) generates the tree files. The program neighbor is borrowed from the PHYLIP package (19) .
CVTree reads amino acid or nucleotide sequences in FASTA format. It permits two kinds of input data: selected genomes from the inbuilt database and user's uploaded data.
The inbuilt database consists of prokaryotic genomes downloaded from the NCBI FTP site (ftp://ftp.ncbi.nlm. nih.gov/genomes/Bacteria/) and fungal genomes collected manually. Only the compressed faa, rpt and gbk files are downloaded. The ffn files are locally extracted from the gbk files by the program extractfeat in the EMBOSS package (20) . Judging by the DEFINITION line in the gbk files, files that represent plasmids, mitochondria, phages or extrachromosomal elements are not fetched. Only chromosomal sequences are used. The NCBI Taxonomy ID was extracted from the rpt file of each organism. As of 1 April 2009, there were 799 bacteria and 56 archaea genomes. More than 80 fungal genomes have been collected manually from various sequencing projects. These fungal genomes together with their origin are listed in the online user's manual. The NCBI Taxonomy ID of fungi is assigned manually. (Currently some manually collected genomes including some fungi do not contain the ffn files, therefore could not be used to perform the DNA type calculation.) We have also included a few eukaryote genomes frequently used as outgroup in previous publications to bring the total number of built-in genomes to 941. This number will grow with monthly updates.
By the way, the convention of using abbreviations for prokaryotic names has been given up, as it becomes inconvenient when organism number gets enormous. The binomina with full strain specification are used instead.
Users may upload their own genomic sequences to the CVTree web server. All sequences of one and the same organism should be included in one FASTA file. The file name (without extension) will be displayed as the organism name in the trees. For user's convenience, sequences for a number of organisms may be wrapped Figure 1 . Two-step implementation of CVTree. CVTree implements a two-step strategy to produce phylogenetic trees. In the first step, CVTree reads in each genome sequence and counts the frequency of all K-tuples. Then the CV of each organism is calculated and dumped to the hard disk (CV files). In the second step, CVTree calculates the dissimilarity matrix from the correlation between CVs. Finally, the tree files are generated by the neighbor-joining program in PHYLIP package.
into one compressed file. Many types of compressed file are accepted, such as GZIP(*.gz), BZIP2(*.bz2), ZIP(*.zip), TAR(*.tar) and RAR(*.rar). Due to disk limitation, up to 100 M disk space can be used for a user's uncompressed sequences in a project. Uploading files are restricted to 20 M at a time. These restrictions will be weakened in the future.
In the inbuilt genome page, one can use the keyword filter to pick up the species of interest. For example, for the time being entering 'Archaea' as a keyword would bring up all the 56 arachaea names, while a keyword 'Streptococcus' would show up all the 38 species/strains in this genus. A user can click on the 'Check All/Uncheck All' button to select/unselect all organisms in one click. By combined use of the keyword filter and the taxonomy selector, it is convenient to make user-specific dataset for study.
An overview of the new CVTree web server is given in Figure 2 . Once connected to CVTree's interface, a user may alternate between six pages shown in the figure, depending on how the job is being submitted and processed. We describe these pages one by one.
The CVTree home page contains a link to an online user's manual and provides several ways to get to the project page. A first-time user may choose to create a new project or load an example project for a quick start. The difference consists in that the new project will start with an empty project space and the example project will bring up a preselected list of bacteria and archaea names from the inbuilt database.
Users may recall a previous project by entering its project number. The project number also enables one to share the results with others. We suggest users always to create a new project to do their analysis, in this way the CVTree server does the garbage collection more efficiently. Any of the 'Create a new project', 'Example project' and 'Reload project' actions will redirect the user to the project page.
Parameters are set in the project page. In the CV approach, length K of the oligopeptides or oligonucleotides controls the resolution of the method and is important for getting good results. Our previous studies have shown that better results may be achieved by setting K to 5 for virus (3, 4) , 5 or 6 for prokaryotes (1,7) and 6 or 7 for fungi. Our further study on how to choose K will make the subject of a separate publication. The sequence type (DNA or protein) and email to receive the results are to be entered in the project page. For DNA sequences K may be chosen from 6 to 18 with increment 3. For protein sequences, which are recommended, K = 3-7. If an email is entered, the web page may be safely closed after the project gets running.
Using the project page, users can upload/delete their own sequences. To upload, first click the 'Browse' button (or 'Choose File' button in Google Chrome web browser) to find a sequence file locally, and then press 'Upload this file' to transfer. To delete, first select the sequences to be deleted and then press the 'Delete selected files' button. With user's files uploading or deleting the table in this page may stretch or shrink. User can select inbuilt genomes from the inbuilt genome page by clicking the 'See details' button. After that, the 'Download selected genomes' button will be activated. Clicking on this button, the user will be brought to the download page.
This page shows the organism list of all inbuilt genomes. The default view shows organism name, proteome size (or cDNA length for DNA sequences) in MB, accession number for chromosome sequences and the superkingdom label extracted from NCBI Taxonomy Browser. Full taxonomy information can be shown by putting the mouse on the organism name. Clicking on the organism name will redirect the user to the NCBI Taxonomy Browser. This table is sortable by clicking at one of the header items. This is useful, for example, when a user wants to select a few smallest or largest genomes to study. Users can see organisms from designated taxa by using the taxonomy selector. The selected taxonomy will be shown in the last column of the organism list table. By choosing the taxonomy label and typing the appropriate keywords, the user can pick up the species of interest quickly. After filtering the organism list, the user can tick the box in the table header to select or deselect all organisms in the current list. When the selection is finished, the status filter can be used to review and check the list.
When the inbuilt genomes have been selected, the 'Download selected genomes' button in the project page will be enabled. By clicking on this button, the user will be asked to wait while the selected sequence files are being prepared for downloading. Then the user will see a link appearing in download page. This link remains available as long as the project has not been destroyed or the user does not choose some other genomes to download again. 
The CVTree result page shows the run-time information and displays the final results when calculation ends.
The CVTree web server returns three kinds of result files:
(1) a dissimilarity matrix file matrix.txt: this file can be used to construct the phylogenetic trees by calling different programs of the user's choice. (2) Two Newick tree format files: NJtree.nwk for a full tree and Genus NJtree.nwk for a tree 'collapsed' to genus level. These files can be viewed in MEGA and in some other phylogenetic programs. (3) Two ASCII tree files: NJtree.txt for a full tree and Genus_NJtree.txt for a tree collapsed to genus level. These files can be displayed directly in any text editor with monospace font.
The result page appears with the five file names listed in the upper part and the NJtree.txt displayed as default in the lower window. By clicking at a file name any of the five files may be displayed.
Users get to the tree page by following the 'Show collapsed trees' link in the result page. In this page, we provide trees partially 'collapsed' to certain taxonomic level according to the NCBI taxonomy. The necessity of so doing requires some explanation. At present, the progress of prokaryotic and fungal phylogeny has made detailed comparison with taxonomy feasible. However, it is not easy to comprehend a tree with hundreds or more leaves. To simplify the job, we collapse an original genome tree to different taxonomic levels taking monophyleticity of branches as a guiding principle. For example, at the phylum level the 36 species/strains classified as Cyanobacteria do form a monophyletic branch. This branch is replaced by a single node labeled by Cyanobacteriaf36g. The reduction can only be partial, as, for example, the phylum Proteobacteria does not appear as a monophyletic group in a tree. However, three out of the five classes in this phylum do form monophyletic branches. Therefore, Alphaproteobacteriaf104g, Betaproteobacteriaf62g and Epsilonproteobacteriaf24g nodes appear when the tree is collapsed to class level. In this way, the number of leaves in a collapsed tree may be greatly reduced.
The collapsing process requires the knowledge of organism lineage. The NCBI Taxonomic Browser, though disclaimed to be a taxonomic reference, is, in fact, more dynamic and up-to-date as compared to the Taxonomic Outline of Bacteria and Archaea (TOBA) (17) or the Bergey's Manual (21) . That is why we download taxonomic information from NCBI.
Since the Genus_NJtree in the result page is generated according to the genus part of an organism's binomen, it might be different from the Genus tree given in the tree page. For example, according to NCBI taxonomy the genus Aliivibrio contains also the species Vibrio fischeri, which is classified under genus Vibrio in TOBA. Therefore, in the genus tree in the tree page we see both Aliivibriof3g and Vibriof7g, however there is only Aliivibriof1g but no Vibriof9g in the Genus_NJtree in the result page.
The neighbor-joining program or other treeing software does produce branch lengths from the dissimilarity matrix generated by the CVTree method. However, as the calibration of branch length in CVTree is a subject of current research, we recommend users pay more attention on the tree topology than branch lengths. This is especially true for the collapsed trees as the collapsing is carried out on the NJtree files directly without redefining distances.
Although the tree page appears only as a table of file names, the files can be displayed online by clicking at their names. Some tree files, listed at the lower part of the tree page, are directly uploadable to a user's iTOL personal account in order to be displayed in a different manner. In particular, the NCBI taxonomy information may be seen on the branches in the iTOL tree.
All the files in the result page and tree page are sent to the user if an email is given in the project page. More examples of output trees can be found in the online user's manual.
The new CVTree web server comes with a greater, monthly auto-updated inbuilt database, with a more user-friendly and intuitive interface and a faster data processing pipeline. A phylogenetic tree of more than 900 genomes will be calculated in several hours if the job runs from scratch. Subsequent calculations take much less time if the genome list coincides largely with a previous job. CVTree also provides a useful tool to find the phylogenetic position of the user's-specific genome data. However, there are still many eukaryote genomes not included in the new CVTree web server. These genomes will be put online when the CV method has been fully tested on these data. We will further improve the implementation of CVTree to meet the need of efficiently processing thousands of genomes. Suggestions and comments are welcome. Remission of Invasive, Cancer Stem-Like Glioblastoma Xenografts Using Lentiviral Vector-Mediated Suicide Gene Therapy BACKGROUND: Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model. METHODOLOGY/PRINCIPAL FINDINGS: We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. CONCLUSIONS/SIGNIFICANCE: In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research. Glioblastoma is the most frequent and most malignant primary brain tumor. Despite advances in neurosurgery, radiation and chemotherapy, the prognosis of patients remains poor with a median survival of 14 months [1] .
A major drawback in translational brain cancer research has been the lack of suitable animal models. Syngeneic-or xenograft tumors based on glioblastoma cell lines cultured as monolayers grow as circumscribed and highly angiogenic lesions in vivo [2] , lacking the invasive tumor cells, which represent an important feature of human glioblastoma. The invasive cells migrate away from the initial tumor mass and can cause recurrent tumors in different regions of the brain. Thus, these cells represent a major therapeutic target.
A recently established model in which glioblastoma biopsybased spheroids are serially passaged in the brains of nude rats shows highly invasive and angiogenic features [3] . Therefore, this model is well suited for the study of new therapeutic strategies. Still, reports using this or other clinically relevant models for experimental therapy are scarce. Recently, we analyzed the therapeutic potential of the HSV-1-based oncolytic Herpes vector G207 in the biopsy spheroid-based GBM model. The tumor volume in treated animals was reduced compared to control groups, but there was no significant survival advantage [4] . In contrast, the same therapy was more effective in a cell line-based animal model [5] and as a result is currently investigated in a phase I/II clinical study [6] . In the present investigation we used the invasive xenograft model to evaluate transduction and therapeutic efficacy of lentiviral pseudotyped vectors. Gammaretroviral vectors derived from the Moloney murine leukemia virus (MMLV) have been the most frequently used retroviruses for gene therapy of brain tumors [7] [8] [9] [10] . However, despite promising results in animal models, clinical trials using retroviral vector supernatants or retroviral packaging cells have failed [11] [12] [13] . One major drawback of gammaretroviral vectors is the exclusive transduction of dividing cells, since in human gliomas, the majority of tumor cells do not divide within a given treatment window. Therefore, lentiviral vectors with their ability to also transduce non-dividing cells are attractive candidates for the treatment of brain cancer. In previous studies, we have developed gammaretroviral and lentiviral vectors pseudotyped with the glycoproteins (GP) of the lymphocytic choriomeningitis virus (LCMV) [14, 15] . These vectors have a broad host range and can be concentrated by ultracentrifugation for in vivo applications. In addition, LCMV-GP is not cytotoxic, and stable recombinant packaging cell lines can be established [16, 17] . Recently, we showed that lentiviral LCMV-GP pseudotypes efficiently delivered transgenes to rat glioma cells in vivo, while resident neurons were not transduced [18] . Furthermore, we showed a significant therapeutic effect of LCMV-GP pseudotyped lentiviral vectors in the cell-line based 9L rat glioma model using the suicide gene HSV-1-tk. VSV-G lentiviral pseudotypes also showed a significant efficacy, similar to that of LCMV pseudotypes, which was mainly mediated by a bystander effect of transduced normal brain cells [19] .
In the presented work, we showed that both, VSV-G and LCMV-GP pseudotyped lentiviruses efficiently transduced human glioma cells in vitro and in vivo, whereas gammaretroviral transduction was inefficient. The gene transfer to glioma cells was efficient for both lentiviral pseudotyped vector types. However, it was more specific using LCMV-GP pseudotyped vectors, as VSV-G pseudotypes also transduced host brain cells in invasive areas. Analysis of transduced tumor cells revealed that both lentiviral vectors targeted CD133-positive as well as CD133negative cancer cells. Furthermore, transduced glioblastoma cells expressed the stem cell markers nestin and SOX2. Importantly, when evaluated for therapeutic application using HSV-1-tk as a transgene, both lentiviral vectors mediated complete tumor regression on MRI, resulting in a highly significant survival benefit (p,0.001) compared to the control groups.
The collection of human biopsy tissue was approved by the regional ethical committee. The handling of the animals and the surgical procedures were done in accordance with the Norwegian Animal Act and the local ethical committee approved the protocol.
The human embryonic kidney cell line 293T (ATCC number CRL-11268) and the TE671 cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbbeco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% glutamine. All cell lines were grown at 37uC in a humidified atmosphere of 5% CO 2 .
Tumor fragments from glioblastoma multiforme patients were obtained during surgery. Tissue specimens were taken from viable tumor areas that corresponded to regions with contrast enhancement on preoperative MRI-scans. The specimens were transferred to test tubes containing complete growth medium, and spheroids were prepared as previously described [20] . The same method was applied for tumor material passaged in nude rats. Briefly, tissue samples were minced into ,0.5 mm fragments and placed into 80 cm 2 tissue culture flasks (Nunc, Roskilde, Denmark) base-coated with 0.75% agar (Difco, Detroit, MI). The spheroids were maintained in a standard tissue culture incubator with 5% CO 2 and 100% relative humidity at 37uC. The medium was changed once a week. Spheroids with diameters between 400 and 600 mm were selected for in vitro experiments and for intracerebral implantation.
Tumors were dissociated using the Neuronal Dissociation Kit (Miltenyi, Bergisch-Gladbach, Germany) according to the manufacturer's protocol.
Cells were analyzed and sorted on a MoFlo cell sorter (Beckman Coulter, USA; former Cytomation, USA), equipped with a Coherent Enterprise 621 argon-ion laser tuned to 488 nm (used at 180 mW), and 635 nm Diode (25 mW). Two mg/ml propidium iodide -PI (Molecular Probes) were added to the samples before flow sorting to facilitate dead cell discrimination. The GFP and PI were excited at 488 nm and fluorescence was measured through 530/40 BP and 613/20 BP optical filters (all filters from Omega Optical, Brattleboro, VT, USA), respectively. Doublets were discriminated using a forward light scatter (FSC) versus pulse width. FL3 channel (in logarithmic mode) with FSC were used to display and gate out PI positive/dead cells. FSC and side light scatter (SSC) signals were detected and shown in linear mode. GFP+ cells were defined on SSC versus FL1 (in logarithmic mode) dot plot after exclusion of dead cells and debris as described above.
For analysis of CD133 expression cells were stained with allophycocyanin (APC) conjugated monoclonal CD133/1 (AC133) antibodies (Miltenyi, Bergisch-Gladbach, Germany), according to the manufacturer's general protocol for immunofluorescent staining (for 10 min in the dark at 4uC). CD133-APC was excited at 635 nm, the fluorescence was measured through 670/ 30 BP optical filter, and alive CD133+ cells were defined on SSC versus FL6 (in logarithmic mode) dot plot. Non-stained cell suspension was used as a control. GFP+ tumor cells were sorted in ''purify 1'' mode into polypropylene round-bottom Falcon tubes (Becton Dickinson Labware Europe, France) containing culture media, that were placed on ice. Aliquots from some samples at the end of the sorting were removed and reanalyzed for control of the sort purity that was greater than 98%.
Sorted cells were either cultured in neurobasal medium (Invitrogen, Carlsbad, CA) with B27 supplement (20 ml/ml; Invitrogen), Glutamax (10 ml/ml; Invitrogen), fibroblast growth factor 2 (20 ng/ml; Peprotech, Rocky Hill, NJ), epidermal growth factor (20 ng ml; Peprotech) or transferred to DMEM supplemented with 10% fetal calf serum (FCS) and 1% glutamine and grown on cover slips in 24 well plates.
Immunofluorescence staining of spheroids/adherent cells Spheroids/adherent cells were stained with human-specific mouse-anti-nestin antibodies (Millipore, Billerica, MA), goat-anti-SOX2 antibodies (R&D), mouse-anti-b2tubulinIII (Millipore) antibodies and mouse-anti-GFAP antibodies (Millipore). Primary antibodies were incubated overnight at 4uC. Alexa-Fluor647-goatanti-mouse und Alexa-Fluor647-donkey-anti-goat antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies over night at 4uC (for spheroids) or for 2h at room temperature (for adherent cells). Spheroids were examined under a fluorescence microscope (Nikon, Tokyo, Japan) and adherent cells were analyzed by confocal scanning laser microscopy (Zeiss, Jena, Germany).
The lentiviral vector plasmid pRRL.sinCMVeGFPpre was published by Naldini et al. [21] . The construction of the lentiviral vector pRRL.sinCMV-TK/eGFPpre has been described previously. The retroviral vector pMP71-eGFP-pre has been described previously [22] .
The 293T cell line was used for transient lentiviral vector production. The lentiviral vector plasmid pRRL.sinCMV-TK/ eGFPpre (5 mg) or pRRL.sinCMVeGFPpre (5 mg), the HIV gagpol-REV expression plasmid pCMV-dR8.91(12.5 mg) [21] and 2 mg of the envelope expression plasmid pHCMV-LCMV-GP [14] or pCMV-G [23] were cotransfected into 293T cells and concentrated as described previously [18] . For the production of retroviral vectors, 293T cells were transfected with 7.5 mg of pMP71-eGFP-pre, 12.5 mg of pSV-Mo-MLVgagpol, and 2 mg of the envelope expression plasmid pHCMV-LCMV-GP [14] or pCMV-G [23] . Vectors were harvested and concentrated as described previously [24] .
Vectors were titered on TE671 cells as described previously [18] .
Intracranial implantation of glioblastoma spheroids was done as described previously [25] .
Three weeks to one month after implantation, the animals were anesthetized and prepared for vector injection. The skin was withdrawn to reveal the location of the craniotomy. 2 times 10 mL of vector stocks were delivered into the centre of the tumors using a glass syringe (model 701, Hamilton, Bonaduz, Switzerland) secured in a microprocessor-controlled infusion pump (UMP 2-1, World Precision Instruments, Aston, Stevenage, UK). The injection coordinates were estimated after analyzing MRI images for each individual lesion. Vector infusion was done by convection enhanced delivery in the course of 25 min (200 nl/min for 10 min, followed by 400 nl/min for 10 min, and finally 800 nl/ min for 5 min). After infusion, the needle was left in place for 5 min to avoid vector reflux. The needle was slowly retracted and the skinfolds were closed with polyamide surgical thread. Following surgery, rats were allowed to recover in an incubator set at 35uC before returning them to the cages.
Rats bearing glioblastoma xenografts were treated by daily i.p. injections of 50 mg/kg ganciclovir (GCV, Roche, Basel, Switzerland).
Animals were euthanized and perfused with sterile saline and thereafter with 4% paraformaldehyde. Brains were removed, suspended in 30% sucrose for three days, and then snap frozen in isopentane chilled with dry ice. Coronal sections (12 mm) were prepared on a cryostat. For immunofluorescence analysis, sections were stained with human-specific anti-nestin antibodies (Millipore) for human glioblastoma cells, mouse-anti-NeuN (Millipore) antibodies for neurons, rat specific mouse-anti-nestin antibodies (Millipore) for astrocytes and progenitor cells. Primary antibodies (dilution 1:200) were incubated overnight at 4uC. Biotinylated goat-anti-mouse and goat-anti-rabbit (Vector Laboratories, Burlinghame, CA) were used as secondary antibodies (dilution 1:100) for 2 h at room temperature. Sections were incubated with Extravidin-Cy3 (Sigma, St. Louis, MO) as fluorochrome (dilution 1:200) for 1 h at room temperature. The sections were examined under a fluorescence microscope (Nikon) and analyzed by confocal scanning laser microscopy (Zeiss, Jena, Germany).
For analysis of transduction efficacy, consecutive sections (every 20.-30.) throughout the tumors were examined under a fluorescence microscope (Nikon) with an automated stage using 106magnification. The transduction volume was calculated using Nikon Lucia imaging software.
Paraffin embedded formalin-fixed tissue sections from rat brain and patient material were placed in xylene bath for 263 minutes, absolute ethanol 263 minutes, 96% ethanol 262 minutes and finally in distilled water for 30 seconds for removal of paraffin and rehydration. Epitope retrieval was performed by heating the sections at 99uC for 20 minutes in 10 mM citrate buffer at pH 6.0. The sections were incubated with a monoclonal human-specific anti-nestin antibody 1:200 in TBS/1%BSA over night at 4uC. A biotinylated goat-anti-mouse antibody (Vector Laboratories) was used as secondary antibody (dilution 1:100) for 1 h at room temperature followed by ABC-complex incubation for 30 min. Sections were developed with with 393-diaminobenzidine (DAKO Cytomation), following the manufacturer's instructions.
Using a Bruker Pharmascan 7 Tesla MR scanner (Bruker Biospin, Billerica, MA), axial T2-weighted RARE sequences were acquired (repetition time, 4,200 ms; echo time, 36 ms; slice thickness, 1 mm; field of view, 3.2 cm; matrix size, 2566256; 20 slices). During scanning, the animals were kept under anesthesia with 1.5% isofluorane (Schering-Plough, Kenilworth, NJ) mixed with 50% air and 50% O2.
Survival was analyzed by a log-rank test based on the Kaplan-Meier test using SPSS software. Differences between pairs of groups were determined by the Student's t-test. P values,0.05 were considered significant.
Human glioblastoma spheroids derived either directly from the patient (low generation) or from serial in vivo passages in the brains of nude rats (high generation), were infected with lentiviral LCMV-GP (5610 4 in 10 ml) or VSV-G pseudotyped lentiviral vectors (both 5610 4 particles in 10 ml) or with retroviral MLV-based vectors pseudotyped with LCMV-GP (1610 5 particles in 10 ml). The vectors were prepared in the same way for in vitro and in vivo experiments (see materials and methods). Both lentiviral vectors transduced patient spheroids and high generation spheroids very efficiently ( Figure 1 ). In contrast, retroviral vectors transduced only a few single cells in high generation spheroids ( Figure 1 ) and failed to transduce patient spheroids (data not shown). In conclusion, both lentiviral vectors are much more efficient in transducing human glioblastoma spheroids in vitro than retroviral vectors.
To compare the transduction efficiency of lentiviral and gammaretroviral vectors in vivo, we used a xenograft model that reflects the angiogenic and invasive features of human glioblastoma in situ. The xenograft also expresses the neural progenitor marker nestin and closely recapitulates the histology of the patient tumor ( Figure 2 ). The vectors were injected into the center of progressively growing lesions using microprocessor-controlled stereotactic infusion. The injection coordinates were estimated after analyzing MRI images for each individual lesion. The injection volume applied was 2610 ml and the vector titre 1610 7 / ml for all vectors. Transduction efficiency was evaluated 7 days after vector injection. Both lentiviral pseudotyped vectors showed very efficient transduction of the tumors ( Figure 3A,D) . When analyzed at higher magnification, both LCMV-GP and VSV-G pseudotyped lentiviral vectors showed efficient transgene delivery to nestin-positive tumor cells in solid ( Figure 3B ,E) and invasive tumor areas ( Figure 3C ,F). In contrast, the retroviral vector only transduced a few scattered tumor cells near the injection site (Figure 3 G,H) . For a quantitative comparison of transduction efficiency between the two lentiviral pseudotyped vectors, the GFP-positive areas were measured on histological slides (see material and methods). The total volume of transduced tumor tissue was 7.0563.51 mm 3 for LCMV-GP pseudotyped vectors and 4.0562.04 mm 3 for VSV-G pseudotyped vectors ( Figure 3I) . Although there was a difference in the mean, it was not statistically significant (p = 0.269) due to high interindividual differences (standard deviations).
To analyze transduction specificity, histological sections of invasive tumor areas were stained for rat specific markers NeuN (for neurons) and nestin (for astrocytes and progenitor cells). LCMV-GP pseudotyped lentiviral vectors exclusively transduced tumor cells in all invasive areas ( Figure 4A ), while normal brain cells were not transduced ( Figure 4B,C) . Also VSV-G pseudotyped vectors showed specific transduction of tumor cells in some invasive areas ( Figure 4D,E) , however, they also transduced a few host brain cells at other sites ( Figure 4G,H) .
To analyze the potential of both lentiviral vectors to infect cancer stem-like cells, transduced tumors were enzymatically dissociated and CD133 expression was measured by flow cytometry. Both vectors transduced CD133-positive and CD133-negative cells ( Figure 5A ). Although there were high interindividual differences in the fraction of total CD133-positive tumor cells in the different xenografts, both vectors showed similar efficiency in transducing CD133-positive cells, which was slightly higher than the overall fraction of CD133-positive cells (table 1) .
The GFP-positive cells from tumors transduced with LCMV-GP or VSV-G pseudotyped lentiviral vectors were sorted and cultured in neural basal medium supplemented with EGF and bFGF. Transduced tumor cells from both vectors were able to form spheroids ( Figure 5B,E) . Spheroids expressed the stem cell markers nestin and SOX2 ( Figure 5C,D,F,G) . Sorted cells were also plated under serum conditions. The cells continued to show significant expression of the stem cell markers nestin and SOX2, but also of the differentiation markers GFAP and b-tubulinIII ( Figure 5H-O) .
To evaluate the therapeutic efficacy of both lentiviral pseudotyoped vectors in the invasive xenograft model, vectors expressing the suicide gene HSV1-tk fused to eGFP were injected into established tumors when visible on MRI using the same method as described for the in vivo tropism study. The animals were treated daily with 50 mg/kg ganciclovir for 30 days starting 7 days post vector injection. Tumor growth was measured every 7-14 days by MRI. After 4 weeks of treatment, 7 out of 8 animals in both the LCMV-and the VSV-pseudotype treated groups had a complete remission on MRI ( Figure 6 ). One animal in each group had a stable disease until the end of GC treatment. All animals in the control groups developed large tumors during the treatment period of 30 days ( Figure 6 ). Both, LCMV-and VSV-pseudotype treated animals had a highly significant survival advantage (p,0.001) compared to the control groups ( Figure 7A ). There was no statistically significant difference in survival between the two treatment groups.
Upon cessation of prodrug administration, all animals developed recurrent tumors, which could be classified into three different groups ( Figure 7B-E, table 2) . The animals showed either 1) local recurrences or 2) local and/or contralateral recurrences or 3) recurrences in other distant brain areas. There was no clear difference in the recurrence pattern between the two vector types, but LCMV-pseudotyped vector-treated animals had more contralateral recurrences, whereas VSV-G pseudotyped vector-treated animals had more local recurrences ( Figure 7B -E, table 2). Histopathological and confocal microscopic analysis of the lesions revealed GFP-positive cells in all recurrent tumors demonstrating that not all transduced glioma cells were killed by ganciclovir treatment (Figure 7F-M) . In VSV-G pseudotype-treated animals, the GFP-positive surviving cells were found in invasive areas ( Figure 7F ,G), the corpus callosum region ( Figure 7H ) and also in some regions of distant recurrences. One animal also showed a focus of transduced normal brain cells at the tumor border that survived GC treatment ( Figure 7I ). In the LCMV group, most GFP-positive cells were found in the ipsilateral hemisphere, in solid and invasive tumor areas ( Figure 7J ,K,L), with only a few cells seen in the contralateral hemisphere ( Figure 7M ).
Future success of glioma gene therapy will depend on more potent vector systems that show higher transduction efficiency than the systems that are available today. In addition, the application of representative animal models that recapitulate both, the invasive and angiogenic features of patient tumors, is vital in order to minimize the huge discrepancies between the experimental results and clinical outcomes previously observed for gene therapeutic strategies for brain cancer.
To this end, we applied one of the most clinically relevant animal models for glioblastoma known. This model was established several years ago [20] and its growth pattern as well as geno-and phenotypic similarity to glioblastoma in patients has been extensively characterized [3] . A striking difference of our model compared to other cell-line based models is the highly invasive behaviour of the lesions, similar to glioblastoma in patients. Our model is based on spheroids derived from patient biopsies that are passaged serially in the brains of nude rats. First generation tumors are highly invasive and grow without signs of angiogenesis. Late generation tumors show an angiogenic phenotype, but are still invasive. Our in vitro experiments revealed that both lentiviral vectors transduced spheroids derived from both low and high generation tumors very efficiently. In contrast, retroviral vectors transduced only high generation spheroids and displayed a much lower efficiency of gene transfer than both lentiviral vectors. This difference can only be attributed to the vector backbone, as the glycoprotein which is responsible for virus entry into the cell was the same for the retroviral and one of the lentivral vectors applied (LCMV-GP). The most important feature that distinguishes lentiviral from retroviral vectors is their ability to infect non-dividing cells. It is known that glioma spheroids, especially primary biopsy spheroids, contain a significant fraction of non-dividing cells, which cannot be transduced by retroviral vectors. It has previously been shown that the cultured biopsy spheroids show a similar cell proliferation as seen in glioblastoma patients [20] .
Previous studies have demonstrated that retroviral vectors can very efficiently transduce highly proliferative monolayer cultures of glioma cell lines [14] . However, monolayer cultures change their geno-and phenotypic characteristics under long term culture and thus are not a suitable model to answer clinically relevant questions [26] .
For in vivo experiments, we selected a high generation xenograft that showed both angiogenic and invasive features and recapitulated the histology of the patient lesion. Furthermore, this xenograft showed a high level of nestin expression similar to the patient material. In translational research it is crucial to assure that the experimental tumors truly reflect the corresponding patient's tumor properties to avoid using non-relevant animal models. However, this strategy is not common practice yet, based on the simplicity of using established cell lines for in vivo experiments.
We showed a high transduction efficiency of lentiviral vectors for glioma cells in vivo, whereas retroviral vectors transduced a few scattered tumor cells near the injection track. This is in contrast to in vivo studies by others where retroviral vectors were very efficient, but as mentioned above, the models applied were non-invasive, based on cell lines cultured as monolayers [27] . Of note, the results of clinical studies using retroviral vectors showed the same low transduction efficiency as observed in our model system [12] . This finding provides further evidence that the glioma model used here has a higher predictive value for the performance of a novel therapeutic approach in the clinic than previous animal models.
The tropism for glioma cells was more specific with LCMV-GP lentiviral pseudotyped vectors, as VSV-G pseudotyped lentiviral vectors also transduced few normal brain cells in invasive areas. In previous studies using a rat glioma model, we also showed a more specific transduction of glioma cells by LCMV-GP pseudotyped vectors compared to VSV-G pseudotyped vectors [18, 19] . In fact, in these studies, the VSV-G pseudotyped vectors transduced normal brain cells at a much higher frequency than in this study. This can be explained by the mode of vector delivery, because in the previous studies, we injected the vectors both into the tumor core as well as into tumor border areas. In the present study, we injected the vectors into the tumor core only using convection enhanced delivery. We used this method because it results in a high distribution volume of drug and vector and is currently used as a delivery method in clinical studies [28] . In addition, the tumor model we apply here is highly invasive and lacks a sharply demarcated brain tumor/normal brain interface, present in the rat glioma model. Another explanation could be the species difference as we used human glioma cells in this study in contrast to rat glioma cells in the previous work. VSV-G pseudotyped vectors might have a higher tropism for human glioma cells than for rat normal host cells. However, as the receptor for VSV-G is unknown [29] , this remains a hypothesis. The targeting of cancer stem cells or cancer stem-like cells in human tumors including glioblastoma has recently evolved as a major aim in cancer therapy. These stem cells are suggested to initiate cancer and might be resistant to therapy, thus being responsible for tumor recurrence [30] [31] [32] . Yet, recent studies have initiated a controversial discussion whether cancer stem cells really exist [33, 34] . Therefore, we use the term ''cancer stem-like cells'' in our study to designate cells which have certain stem-like properties described previously. We showed that both lentiviral vectors transduced CD133-positive and CD133-negative cells. CD133-positive cells have been identified as cancer initiating cells and cancer stem cells in many different cancers including glioblastoma [30, 35] . However, more recent reports questioned these findings showing that CD133-negative popopulations can include cancer initiating cells as well [36, 37] . The efficient targeting of CD133-positive and CD133-negative glioma cells has also been described for adenoviral vectors [38, 39] .
We further demonstrated that sorted cells from tumors transduced either by lentiviral LCMV-GP or VSV-G pseudotyped lentiviral vectors had the ability to form spheroids upon culturing in neural basal medium supplemented with EGF and bFGF. Spheroids from both sorted cell populations expressed the neural stem cell markers nestin and SOX2 and showed the ability to express the differentiation markers GFAP and b-tubulinIII under serum conditions. These properties have been described for neural progenitor cells [40] as well as for cancer stem-like cells in human glioblastoma [26] . Thus, the cell populations transduced by LCMV or VSV pseudotyped lentiviral vectors showed features of cancer stem-like cells which might be an important target for therapy.
In a therapeutic approach using the suicide gene HSV-1-tk fused to eGFP, we demonstrated a highly significant therapeutic effect for both lentiviral vectors compared to control groups. Using MRI to follow tumor growth, we detected complete remission in 7 out of 8 animals for LCMV-GP and VSV-G pseudotyped vectors after 30 days of ganciclovir treatment. HSV-1-tk has been reported to be an effective therapeutic gene in previous studies [19, 41] . The limited success in clinical studies has been a result of inefficient gene delivery systems rather than lack of efficacy of the suicide mechanism [11] . However, there is still space for improvement of the prodrug delivery such as application length, treatment intervals and route of delivery. Tai et al. demonstrated that multiple cycles of prodrug application are superior over one cycle of prodrug [42] . In our study, we still detected GFP-positive tumor cells after one cycle (30 days) of ganciclovir administration in treated animals indicating that application in cycles might also improve the therapeutic effect in this setting. Further, these results demonstrate that vector-transduced tumor cells retain the ability to invade brain tissue and migrate even to distant brain regions.
In conclusion, the present study demonstrates an efficient transduction and therapy of experimental human glioblastoma by lentiviral vectors. The inefficient gene transfer by gammaretroviral vectors is in line with the results obtained in clinical trials and thus confirms the high relevance of the spheroid-based glioma animal model for translational research. A Microarray Based Approach for the Identification of Common Foodborne Viruses An oligonucleotide array (microarray) incorporating 13,000 elements representing selected strains of hepatitis A virus (HAV), human coxsackieviruses A and B (CVA and CVB), genogroups I and II of Norovirus (NV), and human rotavirus (RV) gene segments 3,4,10, and 11 was designed based on the principle of tiling. Each oligonucleotide was 29 bases long, starting at every 5th base of every sequence, resulting in an overlap of 24 bases in two consecutive oligonucleotides. The applicability of the array for virus identification was examined using PCR amplified products from multiple HAV and CV strains. PCR products labeled with biotin were hybridized to the array, and the biotin was detected using a brief reaction with Cy3-labeled streptavidin, the array subjected to laser scanning, and the hybridization data plotted as fluorescence intensity against each oligonucleotide in the array. The combined signal intensities of all probes representing a particular strain of virus were calculated and plotted against all virus strains identified on a linear representation of the array. The profile of the total signal intensity identified the strain that is most likely represented in the amplified cDNA target. The results obtained with HAV and CV indicated that the hybridization profile thus generated can be used to identify closely related viral strains. This represents a significant improvement over current methods for virus identification using PCR amplification and amplicon sequencing. Polymerase chain reaction (PCR) coupled to reverse transcription (RT) represents the most significant improvement in the area of RNA virus detection over classical cell culture based methods. In the classical culture based method, the principal mode of virus identification uses growth of the virus in permissive cells and observation of the morphological changes brought about by virus replication in the host cell [1] . Although it is possible to differentiate between cytopathic and non-cytopathic hepatitis A virus (HAV) strains due to a difference in the morphology of infected cells [2] , in practice such morphological identification is of limited value because the morphological effects are cell-line specific, and many viruses in the same genus (e.g. Enterovirus) produce rapid and similar cytopathic changes in many of the celllines normally used for virus detection. Moreover, using multiple cell-lines for virus detection is also labor intensive and time consuming, and further confirmation and identification often requires the use of additional techniques such as serotyping [1] .
Molecular methods based on viral RNA amplification by RT-PCR have evolved as rapid alternatives to cell culture for the detection and identification of viral strains [3] . For *Address correspondence to this author at the Division of Molecular Biology, Office of Applied Research and Safety Assessment (OARSA), Food and Drug Administration, 8301 Muirkirk Road, HFS-025, Laurel, MD, 20708, USA; Tel: +1-301-210-7812; E-mail: biswendu.goswami@fda.hhs.gov example, the differential identification of strains within a species is possible based on the difference in the size of the amplified PCR product (amplicon) detectable by gel electrophoresis ( [4] or single-strand conformational polymorphism (SSCP) [5] . Indeed, we have utilized agarose gel electrophoresis following RT-PCR using primer pairs straddling a 14 base insertion at the non-coding region of some HAV genomes to identify specific cytopathic strains from noncytopathic strains of HAV [4, 6] . We also reported the use of SSCP analysis following Alu 1 or Hinf 1 digestion of amplicons generated from the 3' end of the viral genome to provide differential identification of multiple HAV strains [5] . However, SSCP is a multi-step procedure involving radiolabeling of restriction fragments prior to electrophoretic separation of individual DNA strands. Consequently, this procedure works best when the restriction fragments are small enough to provide sufficient single-stranded DNA separation for effective strain identification. For genetically wellconserved viruses such as HAV, the region to be amplified for SSCP analysis has to be carefully chosen in order to represent areas of reasonable diversity [7, 8] . Due to these considerations, it has been preferable to sequence the PCR amplified DNA fragment in order to specifically identify the genotypes or strains of the viruses. While sequencing amplified PCR products is considered a precise technique for identification, PCR amplification of a mixed population of target sequences may be biased in favor of a dominant (by copy number) target such that subsequent sequence analysis may not reveal the presence of other closely related target sequences in starting populations. Putative mixed virus populations (e.g. of the same or different species) can exist in isolates obtained from environmental and infected-host samples particularly those resulting from RNA virus replication that is known to generate a sub-population of "quasi-species" [9] . Therefore, a threshold number of RNA molecules must have the same specific mutation in order to be unambiguously detectable by RT-PCR and sequencing, due to possible inhibition of amplification of a less abundant template by template competition [10] . Conversely, the dominant mutation present in a population may be preferentially amplified, and therefore, sequence analysis would represent the dominant mutant [11] . Therefore, while sequencing remains a "goldstandard" for target sequence identification, the identification of multiple viral species or tracking species mutations necessitated the development and application of a broader approach to identification prior to undertaking sequence analysis.
As an alternative to sequencing, Proudnikov et al. [12] applied a hybridization-based technique to the detection of genetic variants of poliovirus within a virus population or among viral strains. Oligonucleotide probes are synthesized and then immobilized on a solid surface. A target consisting of amplified viral complementary DNA (cDNA) then labeled and hybridized to the immobilized probes and the hybridization to the individual probes detected [12] . The presence of a change in the nucleotide sequence in the target is detected by the absence or the reduction of hybridization to the wild type probes around the change, or by the ratio of the signals generated by a mutant against a reference strain. Modifications of the above technique including the use of amplified viral complementary RNA (cRNA) were used to identify genetic variations arising during cultivation of a vaccine strain of poliovirus and the emergence of vaccine derived poliovirus in immunized patients showing signs of vaccine associated paralytic poliomyelitis [13, 14] . Application of this procedure was restricted, however, to identifying known mutations in specific virus strains.
Advances in microarray technology have allowed the identification of genetic variability over very long stretches of DNA in bacterial genomes [15] . These newly developed high density microarrays contain thousands to hundreds of thousands of oligonucleotide probes, instead of a few dozen, in a single array thereby expanding the power of identification [15] [16] [17] . In the current investigation we report the design and use of a high density oligonucleotide microarray for the identification of HAV and coxsackievirus (CV), both foodborne human pathogens. Our results indicate that the microarray hybridization technique can be applied to the identification of viruses of differing genus and species present in a sample and detect single nucleotide polymorphisms (SNP) to identify closely related viral strains belonging to the same species.
Viruses and Plasmids. Hepatitis A virus strains HM175/clone 1 and 18f, and coxsackievirus (CV) serotypes B1, A3 and A5 strains used in this study were obtained from ATCC (Manassas, VA) and further grown in FRhK4 cells [18] . The plasmid pHAV/7 contains a full length cDNA copy of wild type HAV strain HM175 cloned into the vector pGEM-1 [19] that was grown and purified as previously described [20] . HM175 clone1 and 18f are culture-adapted strains derived from continuous culture passage of the wildtype strain HAV HM175 [21] .
Oligonucleotide Arrays. All microarrays used in this study were manufactured by NimbleGen Systems Inc. (Madison, WI) using a maskless array synthesis (MAS) technology for in situ synthesis of DNA oligonucleotides directly onto glass microscopy slides [16, 17] . Oligonucleotide design was based on available complete viral genome sequences obtained from GenBank for CV (n=25), HAV (n=23), Norovirus genogroup I (n=4), Norovirus genogroup II (n=21), rotavirus (various species) segments 3 (n=11), 4 (n=19), 8 (n=11), and 11 (n=12) where n equals the number sequences obtained for each virus group. All genomic sequences within a virus group were aligned using CLUSTALX [22] , and dendrograms were generated and consensus sequences constructed based on these analyses. Examples of these dendrograms are shown for HAV and CV (Figs. 1, 2 , respectively). For the purpose of generating representative viral genomic sequences on which to base subsequent oligonucleotide designs, the HAV strains were clustered into 5 groups whose viral genome sequences were constructed as follows: i) a consensus sequence based on the seven genotype Ib (i.e. genotype I, subgenotype b) strains that clustered into group 1 which includes the HAV HM175/wt strain (M14707), ii) a sequence derived from M20273 based on the pairing of M20273 and AF314208 (genotype Ib sequences in group 2), iii) a sequence derived from the single HAV genotype II sequence (IIb) available (AY032861) and assigned as group 3, iv) two consensus sequences based on either cluster group 4 or 5 derived from fourteen genotype Ia sequences that were clustered into either of these two groups. The three consensus sequences representing cluster groups 1, 4 and 5 were obtained following a group sequence alignment and the assignment of the most frequently occurring nucleotide at positions containing nucleotide differences. The clustering of either one or two sequences within a group (as in groups 3 and 2, respectively) resulted in the selection of a single sequence representing that group. Due to the highly diverse (genetic) nature of the CV genome sequences, clustering of strains for generating a group consensus sequence was only done for serotype strains B1 and B3 (groups 1 and 2, respectively). Four additional unique strain sequences were selected as representative sequences for broadly clustered strains identified as groups 3-6. Viral genomic sequences (approximately 3000 bases) from either the 3' end of the HAV genome group sequences or the 5' end of the CV genome group sequences were submitted for design of a tiling oligonucleotide array consisting of oligonucleotides of length 29, starting at every 5 th base in every sequence, resulting in an overlap of 24 bases in two consecutive oligonucleotides [15] . Similar methods were applied to the development and tiling of oligonucleotides as probes for norovirus and rotavirus sequences on the array. The resulting array contained approximately 13,000 viral probes.
All reverse transcription (RT) reactions were completed using RNA templates obtained from linearized plasmid pHAV/7 transcribed in vitro with SP6 polymerase, total cellular RNA (1 g) isolated from virus infected cells using the RNA AqueousKit (Ambion, Austin, TX), or viral genomic RNAs (equivalent to 5 x 10 6 infectious particles) isolated directly from clarified tissue culture supernatants using the Fig. (1) . Dendrogram showing the grouping of HAV strains based on their genetic relatedness for developing viral probe sequences to be used for oligonucleotide design. HAV strains are identified by their GenBank accession number and their respective complete sequences were used to generate the dendrogram. Brackets encompass strain sequences selected to derive a group consensus sequence while arrows identify group individual (i.e. non-consensus) strain sequences for probe set development. Group sequences are designated by numbers 1-5 followed by the probe set identifier (within parentheses), and the genotype (I or II) and subgenotype (a or b) designation.
RNeasy Micro Kit (Qiagen, Valencia, CA); a mixture of oligo(dT 15 ) and random hexamers (pdN6) as primers; and AMV reverse transcriptase (Promega, Madison, WI) as previously described [4, 20] . In vitro transcribed and infected cell RNA templates represent in vitro and in vivo replication, respectively. PCR amplification with HAV or CV specific primers was carried out in 50 l reactions using 5 l of each RT reaction as template or 5ng of pHAV/7 plasmid DNA as previously described [20] . PCR products (5 l) were analyzed by agarose gel electrophoresis to confirm authenticity of product formation (data not shown).
PCR Primers. Two primers, 3399 -3423 (forward) and 7084 -7105 (reverse), were used to amplify an approximately 3.7 kb region of the HAV genome [4, 6, 20] . Tables 1  and 2 show the sequences around the primer binding sites of selected HAV strains represented on the array. Tables 3 and 4 contain the sequence alignments at the forward and reverse Dendrogram showing the grouping of CV serotype strains based on their genetic relatedness for developing viral probe sequences to be used for oligonucleotide design. CV strains are identified by their GenBank accession number and their respective complete sequences were used to generate the dendrogram. CV serotypes are given in parenthesis following the accession numbers. Brackets encompass strain sequences selected to derive a group consensus sequence while arrows identify group individual (i.e. non-consensus) strain sequences for probe set development. Group sequences are designated by the numbers 1-8 followed by the probe set identifier (within parentheses). Brackets (outer right) indicate which human enterovirus species (HEA, HEB and HEC) are represented by the CV serotype strains used to develop the dendrogram.
primer binding sites for selected CV strains. The reverse primer for CV is degenerate owing to sequence differences among strains in this region [23] . These primers amplify a 746 bp fragment from several B and A strains (data not shown).
Labeling of PCR Products and Hybridization. PCR products were purified using a spin column procedure [Qiagen or Stratagene, (La Jolla, CA)]. One g of each purified PCR product was labeled with biotin-dUTP in a primer extension reaction using random hexamers and Klenow po- forward primer GAATGATGAGAAATGGACAGAAATG a Strains are identified by their GenBank accession number. b HAV sequence alignment is presented as the positive (sense) genomic strand in 5' to 3' orientation. The primer sequence and nucleotide identity is based on the genomic sequence and nucleotide numbering of HM175 strain 18f (M59808) at nucleotide positions 3399 to 3423.
reverse primer CCGAAACTGGTTTCAGCTGAGG a HAV strains are identified by their GenBank accession number. b HAV sequence alignment is presented as the reverse complement (antisense) of the genomic strand in 5' to 3' orientation. The primer sequence and nucleotide identity is based on the genomic sequence and nucleotide numbering of HM175 strain 18f (M59808) at nucleotide positions 7105 to 7084.
lymerase (Exo -). Labeled products were purified by spin column chromatography, and concentrated by centrifugation through Microcon ® (Millipore, Billerica, MA) filters. Biotinlabeled DNA was denatured in a total volume of 20 l of hybridization solution containing 5XSSC, 0.1%SDS, 5 g poly A, and 5 g human Cot-1 DNA and 6 l used per hybridization reaction per well of a 12 well sample pod (Nim-bleGen Systems, Inc.). The microarray slide (NimbleGen) was laid on top (oligonucleotide side down) of the sample pod and held in place in a metal cassette provided by the manufacturer. Hybridization was carried out for 12h at 42 °C. The slides were washed sequentially with 2XSSC/0.1%SDS, and 0.1XSSC/0.1%SDS at 42 o C then distilled-deionized water at room temperature. The slides were then stained with a Cy3-streptavidin conjugate (Amersham Biosciences, Piscataway, NJ) as described in Jackson et al. [15] . Data Extraction and Analysis. Hybridized, Cy3-stained microarrays were scanned using an Axon GenePix ® 4200A scanner at 5 m resolution using a 532 nm laser. Fluorescence intensities of each feature (oligonucleotide probe) were extracted utilizing NimbleScan TM software (NimbleGen Systems Inc), and all subsequent data analyses were performed using MS Excel. Data were analyzed independent of comparison to a reference strain assuming that each virus strain is unique. Following normalization for background fluorescence, the fluorescent intensity of each probe (normalized probe intensity) was plotted against the genome position of each probe to generate a hybridization profile for each viral strain [15, 17] . To generate the average probe intensity for each probe set per hybridized virus strain, the sum of all normalized probe intensities for individual probes within a probe set (i.e. set of probes derived from an individual strain or group sequence) was divided by the number of probes within that set [15] .
As members of the positive-stranded RNA virus family Picornaviridae, Hepatitis A virus and coxsackievirus belong to the genera Hepatovirus and Enterovirus, respectively. At the nucleotide level there is substantial genetic diversity between these two groups with a greater within group diversity observed for coxsackieviruses than for hepatitis virus strains. Indeed, the coxsackievirus genomes are much less conserved and mutations are distributed throughout the genome [23] . Sequence analyses of small segments of different strains of HAV have led to the recognition of six genotypes (I to VI) of this virus [24] . Genotypes I, II and III have been further subdivided into two subgenotypes, a and b [24, 25] . Within each genotype the strains have greater than 85% sequence homology, whereas subgenotypes may differ from each other in up to 7.5% of nucleotide positions. Genotype I is the most prevalent HAV genotype world wide [25] . For the present investigation, complete HAV genome sequences available at the time of chip design and construction belonging to genotypes I and II were clustered into five groups (Fig. 1) . Two of these groups contain subgenotype Ia (groups 4 and 5) while sequences belonging to subgenotype Ib were clustered into two groups (1 and 2) and a single sequence of genotype II was designated group 3 ( Fig. 1) .
As members of the genus Enterovirus, the Human enterovirus A and Human enterovirus C virus species comprise most of the species assigned to CV serotype A strains with the exception of serotype A9 [23] . Coxsackievirus serotypes B1-6, coxsackievirus serotype A9 (CVA9), enteroviruses 69 and 73 and the majority of echoviruses are classified within the Human enterovirus B species that is one of the largest species group in the family Picornaviridae [26] . Fig. (2) shows the CLUSTALX analysis of the different coxsackievirus serotype strains used for designing the tiling array. For B1 and B3 strains a consensus sequence was developed based on the sequences available for individual members of these groups, whereas single sequences were used for the strains B4, B5, A16 and A21, due to extensive sequence variations between individual members of these groups [23, 26] . Since sequence diversity among the CV strains was distributed over the entire length of the genome, the first 3000 nucleotides were determined to contain sufficient sequence diversity to identify a strain without ambiguity. Identification of HAV Genotype by Microarray Hybridization. Fig. (3) shows the hybridization profile obtained with a target synthesized by PCR amplification of the plasmid pHAV/7. This plasmid contains a copy of the entire HAV sequence of wild-type HM175 strain HM175 [19, 27] that originated from an Australian outbreak, and was designated as genotype Ib by subsequent sequence analysis [24, 28] . The hybridization signals (normalized probe intensities) produced a profile indicating areas of intense hybridization at the position where the HAV sequences are clustered in the array. However, variations in the intensity of hybridization can be observed within these sequences, where the target hybridization intensities against group 1 probes (hav1Cb) differ from probes derived from groups 2 through 5 (hav2b, hav3b, hav4Cb and hav5Cb) sequences. This is more clearly observed in Fig. (4) , where the normalized probe intensities for individual probes within each group sequence present in the array were converted to average probe intensities and plotted for the target. The plot reveals that the HAV genotype 1b (HM175 wild-type) target hybridized most efficiently to probes from genotype Ib, group 1 consensus sequence (hav1cb). These results are consistent with the fact that the viral genome sequence for HAV HM 175 wt strain (14707) is a member of, and therefore most closely related to, group 1 derived probe sequences. Probes representing a closely related HAV Ib strain from group 2 (hav2b) hybridize the target about two thirds as efficiently, while probes from the more genetically distant genotype II virus (hav3b) hybridize with the least intensity. The other probe groups (hav4cb and 5cb) both representing genotype Ia consensus sequences (Fig. 1 , cluster groups 4 and 5) hybridize less efficiently than genotype Ib. Given the readily observable differences in both the normalized and average signal intensities among the genotype group sequence probes (groups 1-5) following genotype Ib target hybridization, and the fact that viruses belonging to different subgenotypes can differ by as much as 7.5% in sequence [21, 24, 28] , the data in Fig. (4) indicate that it is possible to identify HAV strains at the level of both genotype and subgenotype with this type of array.
To further explore genotype/subgenotype differentiation, different HAV strains belonging to the same subgenotype Ib sequence (Fig. 1, group 1) were hybridized to the array. As shown in Fig. (5) , HAV strains HM175 wt, clone 1 and 18f hybridize most efficiently to genotype Ib (consensus group 1) probes (hav1Cb). Lower efficiencies of hybridization are observed for all three targets against all other probe sets. These results reflect a greater target specificity for the probe set that contains target member sequences than for the other genotype Ib derived probe set (hav2b) that does not contain target member sequences (group 2 in Fig. 1) . For all target strains, the remaining probe sets yielded signal intensities equivalent to or less than intensities for probe set hav2b. Thus, in support of the interpretation of results of Fig. (4) , this array has the potential to discriminate viral targets at the level of both their genotype and subgenotype.
It is important to note that despite the variation in average probe intensities for the individual strains against probe set hav1Cb (Fig. 5) , the information as presented cannot be used to identify actual target nucleotide Fig. (4) . Hybridization profile of wild-type HAV HM175 strain target: average probe intensity. The hybridization data (normalized probe intensities) for HAV HM175 from Fig. (3) was converted to average probe intensities [15] and plotted vs each individual probe set. A given probe set represents all probes derived from their respective sequence group.
differences. For example, differences in signal height could be attributed to differing hybridization efficiencies between two different experiments. Indeed, a target derived from in vitro synthesized RNA from pHAV/7 (representing in vitro replication of the viral genome) was indistinguishable from plasmid derived target, or the virus following several rounds of replication in culture except for the peak height (data not shown). We pursued, therefore, an alternative method of analysis because the tiling array design offers the potential to distinguish between these closely related strains following hybridization by i) determining the normalized probe intensities for each target, and ii) plotting the change in signal intensity of hybridization by each target to the same probe set as the ratio (fold-change in probe intensity) vs the individual probes. As discussed by Jackson et al. [15] , this method of analysis can reveal distinct peaks with defined slopes (above background/signal noise) where changes in signal strength would occur with probes tiled further up or down stream of the nucleotide change. The presence of a mutation in the genome causes a destabilization of a number of probes around the mutation, which can be identified by the appearance of well defined peaks. Therefore, this method of analysis offers the potential to differentiate closely related strains of virus belonging to subgenotype Ib at the level of individual nucleotide differences, thereby producing data that can be used to tell them apart.
In order to complete this analysis, the two different HM175 strains designated clone 1 and the cytopathic 18f strain were again subjected to hybridization and the total normalized intensities of all probes belonging to the different HAV probe groups were plotted as in Fig. (3) . Again, we found no overall differences in the hybridization profile but rather found peaks of hybridization intensities with the strongest hybridization intensities for the group 1 (HAV1Cb) consensus sequence following calculation of average probe intensity (data not shown). The fold-change in intensity between clone 1 and 18f targets was calculated for each probe in the probe set HAV1Cb. As shown in Fig. (6) , ten well defined peaks were observed over the range of the HAV1Cb probe set and the probe number that corresponds to each peak was identified. It is important to note that due to the initial size of the graphical analysis output, it was necessary to compress the scale of the x-axis (HAV1Cb probe number) in order to fit all data points within a smaller graph. As a result, analysis of the hybridization (signal) values revealed two features not readily discernable on the graph; i) a probable single peak at probe 109 rather than what appears as two adjacent (overlapping) peaks, and ii) a possible second overlapping peak adjacent to probe 441. Since the HAV1Cb probe set (group 1) is a consensus sequence developed from the alignment of seven strains assigned to this group (Fig. 1) , there are nucleotide differences between each group member and the consensus sequence. Plotting the fold-change in intensity between clone 1 and 18f would potentially identify nucleotide sequences in a probe that are identical to clone 1 but not identical to 18f. Indeed, upon comparative analysis of clone1 and 18f amplified target sequences with HAV1Cb probes set sequence synonymous with the target sequences, one would predict a total of 11 peaks to occur by this method of analysis. We then sought to determine whether the "peak" probes contained nucleotide differences that could be mapped to nucleotide differences [e.g. single-nucleotide po- Fig. (5) . Comparison of hybridization profiles for three HAV genotype Ib strain targets. Average signal probe intensities were calculated and plotted following hybridization of targets generated as PCR products from reverse transcription of RNA derived from either in vitro transcribed pHAV/7 (black bar), HAV HM175 clone 1 infected cells (white bar) or clarified supernatant from HAV 18f infected cells (grey bar). lymorphisms (SNPs), deletions, or insertions] that exist between clone 1 and 18f (and the probe set). As shown in Table 5, we were able to conservatively detect 10 out of 11 predicted nucleotide changes in the 18f genome identifiable by this method of analysis. It is important to note that these nucleotide changes represent mutations arising in the 18f virus during its emergence as a cytopathic strain from the HM175 noncytopathic strain which were identified by direct sequencing [21] . These results demonstrate a strong correlation between results obtained by direct sequencing and array hybridization and strongly suggest that tiling arrays can be used to detect nucleotide changes instead of sequencing amplified PCR products over a much longer span of the genome in a single experiment.
Identification of CV Serotype by Microarray Hybridization. Unlike HAV strains, there is tremendous genetic diversity between CV strains, even within the same species as observed, for example, among serotype B strains although they are all members of HEV species [23, 26] . We sought, therefore, to determine whether this array hybridization technique could be used to identify a CV serotype strain target. A typical hybridization profile with a 746 bp segment amplified from CV strains is shown for CVB1 in Fig. (7,  panel A) where the data is presented as average probe intensity for all probes derived from the same group sequence, i.e. probe set. Similar to the results obtained following hybridi-zation with HAV targets, CVB1 targets hybridized very efficiently and with greatest intensity to probes (coxB1Ca) derived from a consensus sequence based on its own sequence, i.e. serotype B1 strains (Fig. 2, group 2) . As indicated by the significantly lower probe intensities, minimal hybridization was observed among the remaining 7 CV probe sets indicating a lower efficiency of hybridization to non-CVB1 sequences represented on the array. In fact, hybridization to probes representing all other (non-CV) viruses was essentially at background signal intensity. The results are consistent with the extensive sequence heterogeneity that exists between the CV serotype A and B virus strains, the members within a serotype (A or B), as well as the probe sets derived from these strains. Importantly, these results demonstrate that even with highly (genetically) diverse viruses, such as coxsackieviruses, this array design can discriminate between strains of the same (or different) virus species. We next sought to determine whether discrimination between virus strains or species was possible when the viral target contains sequences not represented by either an individual or a consensus probe set on the array. To complete this experiment, a 746 bp targets derived from coxsackievirus serotype A3 and A5 strains were hybridized to the array. CVA3 and CVA5 serotype strains are both members of HEA species, however the probes' sequence (group 7, coxA16a) for the species was derived from CVA16 (Fig. 2) . Analysis of normalized probe intensities reveal a striking reduction in the overall level of Fig. (6) . Detection of nucleotide differences between two genetically related HAV strains. Average signal probe intensities were calculated following hybridization of targets generated as PCR products from reverse transcription of RNA derived from HAV HM175 clone 1 infected cells or clarified supernatant from HAV 18f infected cells. The amplified targets (3.7 kb) derived from both clone 1 and 18f contain nucleotide sequences synonymous with the first 2.7 kb (probes 1-543) of the 3.1 kb group 1 (HAV1Cb; Fig. 1 ) consensus sequence used to develop the HAV1Cb probe set that is comprised of 608 probes. The individual points on the graph represent specific probe numbers; however, due to graphical compression of the original data, there are iterative probes not represented by individual points on the graph. Arrows identify the probe number having the peak intensity difference between clone 1 and 18f where a nucleotide(s) present in the consensus sequence is identical to nucleotide(s) in clone 1 but not identical to nucleotide(s) in 18f.
probe hybridization (normalized) intensities for CVA3 and CVA5 derived targets compared to those values obtained following hybridization with a CVB1 target (data not shown). As shown in Fig. (7, panels B and C) , this is also observed following conversion to average probe intensity. The peak average probe intensity for these hybridizations is approximately 2750 units and 900 units with CVA3 and CVA5 targets, respectively. The results indicate that in the absence of matching probe sets on the array the sequence heterogeneity between these CV targets and the existing probe sets precludes the establishment of any strong or efficient hybridization to a single probe set. It is important to note, however, that neither of these targets hybridizes with any significance to non-CV probe sets suggesting that the genetic diversity between CV targets and probe sets does not prevent or obscure virus target group (i.e. CV) identification. In addition, these hybridization profiles are not only distinct from B1 (Fig. 7) but also from one another suggesting the possibility that unique hybridization profile patterns (calculated as normalized and/or averaged probe intensity) could be used for CV serotype target identification. The results from Fig. (7) also suggest that in a single experiment it is possible to identify whether a virus belongs to group A or group B. Indeed, identification of coxsackieviruses at the level of serotype strain may be possible without single nucleotide polymorphism (SNP) analysis and limited only by the number of probe sequences/sets present on the array.
Currently, RT-PCR is the most widely used molecular method for the detection and identification of viruses in biological and environmental sources [27, 28] . Identification of genotypes of virus strains are based on the amplification of specific regions of the viral genome using gene specific primers followed by sequencing of the amplicon by standard procedures [28] . In some instances a preliminary identification is possible using the techniques of single strand conformational polymorphisms (SSCP) or restriction fragment length polymorphisms (RFLP) [5] . Multiplex PCR allows the detection of more than one species of virus in a single analyte [29] . However, these techniques have limitations on sensitivity and versatility, and require extensive prior knowledge of the sequences to be amplified. The requirement of size differences in the amplicons to be analyzed by gel electrophoresis following amplification by multiplex PCR also limits its utility.
Different strains of HAV and many enteric viruses show variable sequence diversity [23, 24, 26] . This allows easy identification of a virus at the genotype level by sequencing discrete segments of the viral genome amplified by RT-PCR. Ideally, sequencing should be done on amplicons that are known to have multiple nucleotide differences between strains. However, designing PCR primers that will capture a significant number of members of that group requires significant sequence homology, and therefore, a relatively variable region flanked by conserved regions is needed for sequence based identification. While for some virus groups such as HAV it is relatively easy to find PCR primers that can capture many members, it is much more difficult with CV genomes due to extreme sequence diversity. The length of the amplified region is another constraint for sequence based identification. Sequencing an amplicon larger that 500 bp generally will require designing multiple primers for sequence walking. Although automated sequencing techniques currently available can be used for rapid sequencing of a moderate sized amplicon, the process is still too time consuming to be used on a routine basis where a quick identification is needed. a Detection of (putative) nucleotide differences by array hybridization between clone 1 and 18f was initially based on the hybridization profile in Fig. (6) . b Probe numbers are from Fig. (6) and represent the oligonucleotide probes whose sequence contains nucleotide change(s) between clone 1 and 18f when the clone 1 target sequence is identical to the probe sequence. Nucleotide changes were indentified (grey boxed) based on comparison of the clone 1 and 18f GenBank sequences (accession numbers in Fig. (1) ) used to develop the HAV1Cb group 1 consensus probe set (Fig. 1) . The probe nucleotide range numbering is defined by the 29-mer probe and corrected to 18f nucleotide numbering from Lemon et al. [21] . c The nucleotide change and position between clone 1 and 18f as reported by Lemon et al. [21] . d Probe 120 defines a 26 nucleotide base region of 18f due to the three-base GAT deletion. e This probe identified as a potential peak overlapping with the peak at probe 441. Fig. (7) . Comparison of hybridization profiles of three CV strain targets: average probe intensity. Viral genomic RNAs isolated directly from clarified tissue culture supernatants of infected cells were used for RT followed by PCR amplification and labeling prior to array hybridization as described in Materials and Methods. The hybridization data (normalized probe intensities) were converted to average probe intensities [15] and plotted vs each individual probe set following hybridization with either CVB1 (panel A), CVA3 (panel B) or CVA5 (panel C). The underlined identifies the human enterovirus species (HEA, HEB and HEC) represented by a CV probe set.
We investigated whether hybridization of fluorescently labeled amplified DNA (target) to a microarray containing many oligonucleotide probes representing many different viral genomes can identify a virus without sequencing. Unlike sequencing, these arrays can interrogate thousands of bases of a viral genome in a single experiment [15] [16] [17] . We determined the feasibility of this approach by using labeled targets amplified from either the DNA (i.e. as recombinant plasmid) or RNA from several strains of HAV and CV. As shown in Figs. (4-6) , a single hybridization experiment using a multi-well array with different samples loaded in different wells of a 12-well sample pod can identify HAV and CVB by the unique profile generated with no ambiguity or crosshybridization to oligonucleotides representing an unrelated virus. Within the broad genus of hepatovirus of which HAV is the only species member, different genotypes which differ from each other by 5% to 8% of base positions (Fig. 1) can be identified (Figs. 4-6) . Within the same subgenotye Ib, strains such as wild type HM175 and the cell culture adapted variants including the cytopathic 18f strain differ by only 0.5% of base positions. We have shown that differentiation of these strains is possible by analyzing the ratio of the signal probe intensities generated by the isolates when hybridized to the probe sets present on this tiling array ( Fig. 6 and Table 5 ). A sequence based identification of the same 3.7 kb amplicon would require several sequencing reactions with multiple primers in order to identify nucleotide differences. In addition, mutations accumulating in the HM175 genome during its evolution into the cytopathic 18f strain can be identified by ratio analysis (Fig. 6 and Table 5 ). Thus, the present array design is suitable for identification of species (e.g. CV and HAV) and HAV subgenotypes since in the latter case the nucleotide differences are very few.
We have also demonstrated that it is possible to distinguish between CVB and CVA strains by virtue of their hybridization profiles. In addition, individual members of A and B groups show distinct and characteristic hybridization patterns. Thus, members of CVA strains such as A3 and A5 can be easily identified not only as belonging to group A CV, but also a genotype A3 or A5. More virus strains need to be examined to determine if the method is applicable to other members of this group.
The more closely the target sequence matches the probe set, the stronger the hybridization signal. Diversity between and among probe sets representing virus strains within a group such as CV increases the power of discrimination (due to heterogeneity) particularly when the target is highly similar to one of the probe sets. This enables discrimination even at least at the strain level (e.g. among strains within the same serotype group such as CV group B serotypes). This level of discrimination is lost when a target whose sequence is not represented by a probe set is not present on the array. Again, this has been shown to be problematic with highly (genetically) diverse viruses such as CV. However, despite the loss of serotype discrimination, the diverse nature of such viruses does still enable the differentiation between virus groups as shown between CVA and HAV, NV, and rotavirus.
Our results show that an oligonucleotide array incorporating thousands of probes representing genomes of multiple foodborne RNA viruses including multiple hepatitis A virus genotype strains and multiple coxsackievirus serotype (A and B) strains can be used to differentiate between virus members of either genus to identify the genotype/serotype of these viruses by array hybridization assay. Because the large number of probes can bind and detect labeled targets over a much larger area of the viral genomes, producing distinctive signal patterns for each genotype/serotype, the need for large scale sequencing is eliminated for this level of discrimination. Is the risk of multiple sclerosis related to the ‘biography’ of the immune system? Multiple sclerosis (MS) with onset in childhood offers a unique opportunity to study the infectious background of this disease but the immune reactions against infectious agents in such children have only recently been investigated. These and other epidemiological studies strongly implicate involvement of one or more infectious agents in the aetiology of MS, with Epstein-Barr virus (EBV) being the prime candidate. Rather than being the actual cause of MS, it is more probable that these agents are involved in the development of immunoregulatory pathways. These pathways, if disturbed by hygiene-related factors including an altered sequence of infections, may generate and maintain a deficit within the immunological network that facilitates, to particular early events in the development of MS, preceding the onset of MS disease by years or a decade. A framework that can serve as a guide for further epidemiological, immunologic and molecular biologic investigations is formulated. This approach may shed light on the complex natural history of MS and may lead to rational preventive and therapeutic strategies. It is possible that, in the future, MS could be prevented by vaccination against EBV in early childhood; the framework is of relevance to the design of an appropriate type of vaccine. The pathogenesis of multiple sclerosis (MS) is certainly complex and heterogeneous in nature [1] , involving an interplay between innate and environmental factors [2] [3] [4] and genetic factors, notably the polymorphism of HLA [5] . The epidemiology of this disease strongly indicates that it has emerged as a major neurological disorder in industrially developed nations over the last 150 years and is likely to be affected by hygiene-related factors [3, 6] .
According to the so-called hygiene hypothesis [7] , modern living conditions in the industrialised nations isolate infants and children from many infectious challenges that are required for the development of appropriate immunoregulatory networks. This hypothesis has been advanced to explain the rise in incidence of disease associated with immune dysregulation, including asthma, allergy, autoimmunity and at least some forms of cancer, in the industrially developed world [7] . In addition, the hypothesis suggests strategies for interventions for the prevention of such diseases, as illustrated by studies on the epidemiology of malignant melanoma which indicate that certain vaccinations, BCG, vaccinia and yellow fever, can substitute for the significant protective effect of natural B. Krone infections [8 -11] . As epidemiological investigations on MS strongly indicate that this disease is likewise affected by hygiene-related factors and by a history of infections [3, 6] , the challenge is to determine whether there is an underlying distortion of immune responses in this complex disease that could be prevented or corrected by therapeutic intervention.
Evidence from epidemiological studies of a role for any of the many common infections in the aetiology of MS is largely inconsistent [3, 6] . This could be due to the prolonged silent stage of the disease, but recent studies on MS commencing in childhood might be able to shed light on this issue [6, [12] [13] [14] . Although there is no convincing evidence that any specific active infection directly causes MS, at least 14 specified infections have demonstrable associations with this disease on the basis of serological characteristics [2, 3, 6, [12] [13] [14] [15] , with Epstein-Barr virus (EBV) emerging as the most likely single candidate for a leading aetiological role [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . This infection is unique in that the prevalence of specific antibody is consistently higher in children with MS as compared with healthy agematched controls [12] [13] [14] , whereas at the typical age of onset of MS (late 20s to early 30s) nearly all patients and control subjects have already experienced EBV infection. The two key points to emerge from these studies in relation to EBV are that all children with MS had already been infected with EBV [12] [13] [14] , and that the levels of antibody against the EBV EBNA1 antigen were very significantly higher in both adults and children with MS as compared with controls [13, 14, 16, [23] [24] [25] . In this context, infections with EBV usually occur in infancy in the developing nations where MS is rare while in the industrialised nations infection usually occurs later in life, often being delayed until adolescence or early adulthood.
At the time of diagnosis of MS in children, the earlier EBV infection had become latent and there was no serological sign of reactivation (IgM or anti-early antigen titres C80 as measured in an indirect immuno fluorescent assay) [12, 13] . Moreover, in adult patients, the MS-associated serological EBV pattern was probably established many years before the onset of clinically evident MS as prior infectious mononucleosis is a strong risk factor for MS manifesting 2-20 years (mean around 10-12 years) later [18, 25] . Taken together, these studies strongly indicate that latent EBV infection is an essential predisposing condition for the development of MS but other genetic, environmental and hygiene-related conditions appear necessary for the actual expression of the disease and the unifying condition might be a dysregulated immune response.
A distinct possibility is that other infections synergise with EBV in the aetiology of the disease and the timing of infections might be important. In this context, Chlamydia pneumoniae (CP) infection may play a special role [26] , as in a recent study on children with MS, CP-specific IgM antibody points to a high frequency of fresh, recent or reactivated infection with this pathogen at the onset of the disease [12] . A final key point is the demonstration that slight, though statistically significant, elevated levels of antibody to certain common infectious agents other than EBV and CP occur in children and adults with MS compared with age matched control subjects [12, 15, 27] . This finding does not, however, imply a direct causative relation of any infection to MS as it could reflect a more general dysregulation of immune function as a cause or consequence of the development of the disease. It is indeed likely that the elevated antibody concentrations have no significance per se for the development of MS but reflect a shift in patterns of immune reactivity away from a protection and towards enhancement of the risk of disease. Nevertheless, studies on these MS-associated infectious agents could lead to the identification of specific antigenic determinants involved in the generation and maintenance of immune dysregulation.
A hypothesis which relates MS to the 'biography' of the immune system On the basis of the available epidemiological evidence it may be postulated that MS is dependent on an infection with EBV which, owing to hygiene-related factors, occurs later than usual in life. Under such circumstances, the EBV infection might encounter patterns of immune responsiveness generated by prior infections with certain other microorganisms. In this paper we suggest a scenario in which the sequence of certain common infections results in immune dysregulation favouring the onset of MS and in the following sections this hypothesis is elaborated.
Recent studies have shown that the immune system contains a very complex network of regulatory pathways. To some extent these pathways are genetically determined, but there is growing evidence that they are critically determined by the nature and timing of infections and other immune challenges that an individual experiences earlier in life. This could be termed the 'biography' of the immune system.
The immunoregulatory pathways are based on populations of lymphocytes, termed regulatory T cells (T reg s), in which there is currently considerable interest. In the case of infectious disease, such populations may lead to rapid resolution, the establishment of latent or persistent infection or to tissue damage by autoimmune processes [28] . Accordingly, T reg s have been termed 'a dangerous necessity' [29] . This term implies that T reg s are neither 'good' or 'bad' per se but may, according to the overall pattern of responsiveness, participate in appropriate immune reactions leading to resolution of disease or in inappropriate ones resulting in immunopathology.
The temporal sequence of infections, especially initial and early ones, is crucial to the development of patterns of immune reactivity as prior contacts with other antigens may have induced cross-reactive T-helper cells competing with T reg s. As a consequence T reg s normally induced by the second pathogen may be marginalized or even eclipsed. The latter phenomenon, also known as lateral inhibition, has many parallels in biology, particularly in neurology. The locking of an immune response into an eclipsed state seems to involve an active deletion of clones of T-cells occurring as a result of reinfections or reactivations [28] . In the case of MS, infections such as those with HHV-6 [30, 31] and, possibly, with CP [12, 26] occurring before or at the time of initial or reactivated EBV infection could have such an effect.
Thus these prior contacts could have induced populations of T reg s that have a crucial role in protection against MS but also cross-react with an epitope on EBV. A subsequent infection by EBV could therefore generate a dominant population of cross-reactive T-helper-cells which could suppress or delete the T reg s.
Under normal physiological conditions, these T reg s could either suppress other populations of T-cells which would otherwise be able to induce autoimmune processes, including those involved in the pathogenesis of MS, or they could cause the expansion of a population of specific CD8 ? T-cells which would have an immune repair function. The nature of the epitope or epitopes involved in this postulated process remains unknown but, by analogy with the parallel studies on melanoma mentioned above [10, 11] , it is suggested that key host epitopes involved are coded for by certain human endogenous retroviruses (HERVs) as activation of these has been extensively documented in MS [32, 33] .
Accordingly, a challenge in MS research is to delineate patterns of MS-related immune responses [34, 35] , and the T reg s involved, that affect the risk of MS both beneficially and detrimentally and the likely targets of these responses.
The IgG-anti-EBNA1 antibody concentrations are particularly elevated in patients with MS [13, 14, 16, 18, [23] [24] [25] , and systematic studies on the T-helper cell epitopes in the EBNA1 protein revealed that CD4 ? T-cells from healthy EBV carriers matched for MS-associated HLA-DR alleles recognised several epitopes within the central region of the C-terminal domain of this protein but not other EBVencoded proteins [36, 37] . In contrast, those from MS patients recognised many more epitopes spread over the entire domain [36] . Concomitantly, the number of memory T-cells directed against this domain is increased about tenfold in MS and have been shown to be T-helper 1 cell precursors and polarised effector memory cells [36, 37] , containing a subfraction of regulatory T-cells (T reg s) [38, 39] . T reg s were suppressed in MS [40] , and high level of CD8 ? T-cell activation against EBV but not cytomegalovirus was demonstrated early in the course of MS [41] .
A search for the possible origin of competing T-helper cells was undertaken with the BLAST analytical program [42, 43] . Sequence homologies were evident over the entire expanded EBNA1 epitope with proteins from CP and HHV-6 ( Table 1 ). This possible involvement of HHV-6 and CP in T cell competition is supported by the observation that the targets of T-and B-cells which have been found to be MS-associated by systematic studies [34, 35] also have homologies in HHV-6 and CP ( Table 2) .
The central EBNA1 epitope marginalized in MS (amino acids FENIAEGLRALLARSHVER) could well have different functions in health and in MS and is therefore a major putative candidate for generation of T reg s which control the relevant immune processes. A specific or functional deficiency of T reg s in MS has only recently been recognised, and the need for a large cohort of T reg s for the resolution of experimental autoimmune encephalomyelitis has been demonstrated [44] . For the purpose of studying the potential infectious and immunological background of MS, it is relevant to search for homologies to this 'epitope No. 1' in the MS-associated pathogens [42, 43] . Interestingly, homologies to the putative epitope were found in all these pathogens (Table 3) .
It is likely that a diverse range of MS-associated infectious agents other than EBV and, possibly, CP, is involved in the generation and maintainance of the postulated immune responses associated, beneficially or detrimentally, with MS. By generating populations of T reg s or of competing T-helper cells, such infectious agents would play a role in the generation of various immunological networks based on T reg s which, in turn, would facilitate the expansion or suppression of populations of effector T cells including epitope-specific CD8 ? T-cells. The epitope recognised by these T-cells should be common to the MS-associated infectious pathogens and to one or more cellular gene products. The latter was preferentially sought in HERVs since patients with MS expressed HERV-W env at higher copy numbers as compared with controls (P = 0.00003) [32, 45] and a HERV-K18 env genotype was described as a risk factor for MS [46] .
A putative target epitope for effector T-cells in the processes suggested above ('epitope No. 2') was identified in a short peptide from the HERV-W env gene complex: MPVPSAPST. It is predicted that this peptide is presented, though only subdominantly, by diverse HLA class I molecules including Ld, A*0201, B*0702, B*5101, as determined by reference to the SYFPEIHI database for MHC ligands and peptide motifs [47] .
Only three pathogens bearing the two homologies on the same protein, which is postulated to be optimal for a co-operation of the corresponding T-cells, have been identified, namely, herpes simplex (1 and 2) virus (tegument protein), measles (nucleoprotein), and varicella (tegument). The MS-association of the serology of these pathogens (higher specific antibody concentration) [16] was confirmed in a recent study: herpes simplex-2, P \ 0.0001; measles, P \ 0.0001 and varicella, P \ 0.0001 [12] . The epidemiological evidence for the MS-association of the majority of the other pathogens in Table 3 is only weak and inconsistent. Moreover, a simultaneous occurrence of homologies to epitopes 1 and 2 was also found in a diverse range of pathogens causing respiratory and gastrointestinal infections and which have also been associated, beneficially and Specific T-cells directed against this region were found to be expanded in MS patients as compared to control individuals [36] ; the region with homologies in HHV-6 and CP proteins extends to EBNA1 amino acid position 640 * for identical amino acid; ? = conserved amino acid exchange; / = missing amino acid; arabic numbers for additional amino acids: 1 = DKK; 2 = PF; 3 = D detrimentally, with the risk of MS [48] [49] [50] . Likewise, unknown parasitic infections have recently been found to be associated with a reduced risk of MS [51] , and some parasitic organisms share the two homologies (Table 3 , footnote). In addition, preliminary studies indicate that parasite infections in MS patients lead to fewer exacerbations and this has been linked to the emergence of T reg s [52] . The relatively widespread occurrence of these two homologies explains, at least in part, why the infectious background of MS has proved so complex and difficult to define.
The MPVPSAPST-peptide is the amino-terminal part of a small hypothetical protein of 29 amino acids encoded by the complementary DNA strand of the HERV-WE1 env gene which is conserved in all homologous HERV-W sequences in the human genome. Gene transcripts of 21 of 25 open reading frames with an initiating start codon have been found in association with MS according to genetic data bank entries. Moreover, several other HERVs (-H, -K, -L) exhibit this homology. As these HERV peptides are all self-antigens, they could serve as targets, but not as inducers, of the postulated MS-protective immune response. The main HLA class I molecule A*0201 for the presentation of the peptide, with a frequency of about 30% in a European population, was shown to be associated with a significantly reduced MS risk (OR = 0.52, P = 0.0015) [53] .
Immune dysregulation in MS is likely to be an early event [18, 22, 25, 41] preceding the onset of MS disease by many years or a decade [18, 22, 25] . It should thus be emphasized that the epidemiologic observations on the possible infectious background of MS partly, if not predominantly, reflect the earliest stage in the natural history of MS. A situation similar to that postulated here has been described in malignant melanoma in which, as discussed above, cross-reactive protective immune responses are induced by homologous epitopes in BCG, vaccinia and yellow fever vaccines given at least 10 years before the onset of disease [8] [9] [10] [11] . It was suggested that melanocytes in the early stages of malignant transformation, may be eliminated or repaired by CD8 ? T-cells which recognise cells expressing a HERV peptide. This immune reaction seems to suppress the genetic activity of HERV proviruses (env genes) associated with malignant transformation [10, 54, 55] . The HERV env proteins probably impair the redox Table 2 Homologies in proteins of sero-epidemiologic MS-associated pathogens to MS associated EBV-epitopes [34, 35] 
Enhanced immune reactivity in MS patients in comparison with healthy control subjects as identified by systematic studies [34, 35] ; consensus in other proteins to the EBV sequence given by * for identical amino acid, ? for similar amino acid (conservative exchange), and / for missing amino acid; one additional homology in vaccinia virus regulation within the cells via reduced levels of glutathione peroxidase [10] . In MS there is still another environmental risk factor, namely, suboptimal levels of bio-active vitamin D [4] , which, as demonstrated in rat astrocytes, may impair via c-glutamyl transpeptidase intra-cellular glutathione levels [56] . Table 3 MS-associated pathogens and homologies in proteins thereof to candidate epitopes No. 1 (EBV EBNA1, putative T reg ) and No. 2 (HERV-W) * for identical amino acid; ? = conserved amino acid exchange; / = missing amino acid; arabic numbers for additional amino acids: 1 = TEE; 2 = AG; 3 = QKE; 4 = YCK; 5 = AT; 6 = V; 7 = V; 8 = LAT Pathogens against which higher antibody concentrations were observed as compared with control individuals. Homologies to both epitopes were also found in the following pathogens causing respiratory and gastrointestinal diseases: Bordetella parapertussis, Corynebacterium diphtheriae, cytomegalo virus, Haemophilus influenzae, human corona virus, human rota virus, Mycobacterium tuberculosis (also M. bovis, strain BCG), Salmonella enterica, Pseudomonas aeruginosa, Serratia marcescens, A and in two parasites causing gastrointestinal infections: Entamoeba histolytica, Giardia lamblia Proteins with homologies to epitope 1/epitope 2, respectively: adeno virus: DNA stabilization protein/protein V; Chlamydia pneumoniae: hypothetical protein cpB U609/hypothetical protein; Epstein-Barr virus: EBNA1/BBLF2/BBLF3; herpes simplex-1: tegument/tegument; herpes simplex-2: tegument/tegument; HHV-6: tegument/major capsid; measles virus: nucleoprotein/nucleoprotein; Mycoplasma pneumoniae: phosphate import ATP-binding protein pstB/enolase; parainfluenza-2: large protein/nucleocapsid; RSV: fusion protein precursor/glycoprotein; rubella: RNA-directed RNA polymerase/E1; vaccinia: 14K membrane protein/putative DNA-binding core; varicella virus: tegument/tegument In cell culture experiments, gangliosides of the neolacto series, such as LM1, were identified as possible relevant mediators as they suppress HERV genes [10] and induce lytic replication cycles in cells latently infected with EBV [57, 58] , thereby releasing a viral antigen which would be readily recognisable by the specific immune system. The failure of such protective mechanisms would facilitate an uncontrolled establishment of the observed extensive EBV infection of brain lymphatic tissue in MS, albeit only at a low level of virus activity [59] [60] [61] [62] . The specificity of the process for the brain may be associated with the high content of gangliosides in nervous tissue, as other gangliosides may antagonize the activity of those that possibly protect against MS.
Multiple sclerosis is certainly a complex disease entity and the pathogenic process involves more than just increased neuro-degeneration. In particular, reduced remyelination contributes to disease progression. Thus, relevant targets in addition to the HERV peptide might exist, possibly host proteins with a homology to the HERV peptide. One such candidate is neuron-specific ankyrin-2 which, owing to complementary binding sites, forms a complex with spectrin. In an animal model of remyelination of axons the latter is the target of an immune repair mechanism mediated by a monoclonal antibody [63] .
Based on epidemiological considerations, it is postulated that the relatively high risk of MS in the industrialised nations is due to hygiene-related changes in the sequence of common infections, resulting in the emergence of patterns of immune reactivity that either cause, or fail to protect against, the development of MS. Further epidemiological studies are required to determine whether the timing of EBV infection is related to the risk of MS [20] . Based on studies of altered immune responses to certain infectious agents and evidence for the expression of HERVs in MS, epitopes that have a putative role, depending on the appropriate or inappropriate activity of immunoregulatory networks, in protection against or predisposition to MS have been delineated.
Studies based on the above considerations should focus on processes that initiate MS years or a decade before manifestation of the disease. Subsequently, a better understanding of the highly complex immunopathology of MS will, hopefully, lead to the eventual development of vaccination strategies for the prevention or correction of anomalies in immune function. Such vaccines could well work by preventing or correcting hygiene-related immune dysregulation [64] . It has previously been postulated that a vaccine based on EBV should afford a high level of protection against MS [16] , but the exact nature of an efficient and safe vaccine would depend on the nature of the relationship between EBV and MS. If the framework presented in this study is in principle confirmed, the appropriate vaccine is most likely to be a living attenuated one that induces a strong T cell response to EBV epitopes but is not expressing EBV EBNA1 in latency. Evidence of Recombination and Genetic Diversity in Human Rhinoviruses in Children with Acute Respiratory Infection BACKGROUND: Human rhinoviruses (HRVs) are a highly prevalent cause of acute respiratory infection in children. They are classified into at least three species, HRV-A, HRV-B and HRV-C, which are characterized by sequencing the 5′ untranslated region (UTR) or the VP4/VP2 region of the genome. Given the increased interest for novel HRV strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of HRV strains in a 2-year study of children with acute respiratory infection visiting one district hospital in Shanghai. METHODOLOGY/FINDINGS: We cloned and sequenced a 924-nt fragment that covered part of the 5′UTR and the VP4/VP2 capsid genes. Sixty-four HRV-infected outpatients were diagnosed amongst 827 children with acute low respiratory tract infection. Two samples were co-infected with HRV-A and HRV-B or HRV-C. By comparative analysis of the VP4/VP2 sequences of the 66 HRVs, we showed a high diversity of strains in HRV-A and HRV-B species, and a prevalence of 51.5% of strains that belonged to the recently identified HRV-C species. When analyzing a fragment of the 5′ UTR, we characterized at least two subspecies of HRV-C: HRV-Cc, which clustered differently from HRV-A and HRV-B, and HRV-Ca, which resulted from previous recombination in this region with sequences related to HRV-A. The full-length sequence of one strain of each HRV-Ca and HRV-Cc subspecies was obtained for comparative analysis. We confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the 2A gene and 3′UTR of the HRV-Ca strain. Double or triple infections with HRV-C and respiratory syncytial virus and/or bocavirus were diagnosed in 33.3% of the HRV-infected patients, but no correlation with severity of clinical outcome was observed. CONCLUSION: Our study showed a high diversity of HRV strains that cause bronchitis and pneumonia in children. A predominance of HRV-C over HRV-A and HRV-B was observed, and two subspecies of HRV-C were identified, the diversity of which seemed to be related to recombination with former HRV-A strains. None of the HRV-C strains appeared to have a higher clinical impact than HRV-A or HRV-B on respiratory compromise. Human rhinoviruses (HRVs) are a highly p revalent cause of the acute respiratory infection (ARI) defined as the common cold [1, 2, 3] , which is frequently associated in children with bronchitis, bronchiolitis, wheezing, pneumonia, asthma and otitis [4, 5, 6, 7, 8, 9] . HRVs are classified in genus Enterovirus (HEVs) in family Picornaviridae [10] . HRVs are non-enveloped, single-stranded, positive-sense RNA viruses of approximately 7200 nt, composed of a 59 untranslated region (UTR), followed by a long open reading frame coding for capsid proteins VP4, VP2, VP3 and VP1, and seven non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D, and terminated by a short 39UTR and poly A tract.
More than 100 serotypes of HRV are known, which have been classified into two species, HRV-A and HRV-B, according to comparative alignment of nucleotide fragments of VP1 [11, 12] , VP4/VP2 [13] and 59UTR [14, 15] , and more recently, on complete genome nucleotide sequences [16] . Moreover, some genomic sequences have been found not to cluster with HRV-A and HRV-B species, which suggests the existence of other species (HRV-C and HRV-D) [16] . A new species of HRV-C was recently identified worldwide by comparative analysis of VP4 or VP4/VP2 genes [7, 17, 18, 19, 20, 21] and 59UTR [14, 15] .
However, discrepancies have appeared in the classification of some of the new HRV-A or HRV-C strains, depending on the size and location of the nucleotide sequence in the viral genome and on the phylogenetic methods used for direct analysis of HRV sequences [3, 7, 14, 15, 17, 18, 20, 21, 22, 23] .
These recent data underline the lack of knowledge about the biodiversity of HRV strains and their worldwide distribution [14, 17] . Moreover, little is known about the characteristics and diversity of HRVs circulating in a given area in a short period of time. In the present study, we looked for HRVs in a 2-year collection of nasopharyngeal swabs (NPSs) of children with ARI visiting a district hospital in Shanghai, and compared sequences in two regions previously defined for genetic classification of HRV serotypes [13, 15] . Our study showed a high diversity of HRV species and genotypes, and a prevalence of the novel HRV-C species in NPSs of children with bronchitis and pneumonia. This biodiversity appeared to result partly from recombination events in the 59UTR, between HRV-C strains and those close or similar to HRV-A species, which led to the suggested classification of HRV-C into at least two subspecies.
Eight hundred and twenty-seven samples were collected from a group of children consulting the Shanghai Nanxiang Hospital during a 2-year period, and tested for 17 respiratory viruses using a multiplex RT-PCR (mRT-PCR). Sixty-four samples (7.7%) were positive for HRV, according to the length of the amplified fragment in the VP4/VP2 region visualized on agarose gel (data not shown). A larger fragment of 924 nt, including part of the 59UTR (starting at nt 163) and the VP4/VP2 genes (ending at nt 1086), was amplified and cloned into plasmid vectors for genetic analysis (Table 1) . Only one sample, N1, could not be amplified and was amplified in two steps in the 59UTR (nt 163-552) and in the VP4/VP2 region (nt 528-1086), respectively. Analyses of different clones for each sample allowed the identification of multiple infections: sample N16 was shown to contain one HRV-A and one HRV-B (N16A and N16B, respectively), while N58 contained one HRV-A and one HRV-C (N58A and N58C, respectively) (see below). In order to further characterize the 66 HRVs identified in the 64 samples, the nucleotide sequences located in the 59UTR (285 nt) and the VP4/VP2 genes (420 nt) were chosen to allow comparative alignment with sequences of reference serotypes and field strains available in GenBank.
We first compared and classified Shanghai strains according to their VP4/VP2 sequences. The pairwise nucleotide divergence in the VP4/VP2 region ranged from 0 to 72.2%. Twenty-seven HRVs (40.9%) showed .81% nucleotide identity with the closest HRV-A clusters, and five HRVs (7.6%) showed .88.8% nucleotide identity with HRV-B clusters ( Table 2 ). The remaining 34 strains (51.5%) diverged from HRV-A and HRV-B species by .47.3% in their VP4/VP2 nucleotide sequence ( Table 2) . These strains showed from 68.3 to 100% nucleotide identity with each other and were related to the recently described HRV-C strains, NAT001 and NAT045, isolated in California, USA [18] , C024, C025 and C026 in Hong Kong [20] , and QPM in Australia [7, 24] ( Fig. 1 ). Strains N34, N35 and N68 were closely related to the recently identified strains C025 and NTA001 with .95.9% nucleotide identity, whereas the 31 remaining HRV-C strains showed only 74.4-86.4% nucleotide identity with six other recent strains (QPM, NAT001, NAT045, C024, C025 and C026; Table 2 ), which were classified tentatively as HRV-C species [7, 18, 20, 24] . Classification of the strains into three different species was also demonstrated by construction of a phylogenetic tree using aligned VP4/VP2 sequences (Fig. 1) .
To characterize and classify further the Chinese HRV strains, 59UTR sequences were considered (Table 3 , Fig. 2 ). They were compared to the 59 UTR of all 101 reference HRVs, to those of 26 new strains identified in children with respiratory illness in Wisconsin (indicated as W) [15] , and to those of other recently identified HRV-C strains [14] . Pairwise nucleotide divergence between the three HRV species was 0.7-64.3%, and a limit of ,9% divergence between genotype pairs was chosen for similar genotype assignment in one species [15] . New genotypes were identified when they had 9-30% pairwise nucleotide divergence from the nearest serotype in the same species (Table 3) . Fifty-five HRVs shared .94.4% nucleotide identity with strains already identified, and 11 HRVs showed 9.5-20.9% nucleotide divergence with the nearest known HRVs. They may represent newly discovered genotypes. These strains clustered with HRV-A (N6) or HRV-C species (N4, N8, N21, N62, N63, N67, N24, N25, N28 and N32) ( Table 3) .
Most surprisingly, 20 of the 34 strains classified as HRV-C by comparative analysis of VP4/P2 sequences ( Table 2) were related more closely to HRV-A strains when their 59UTRs were analyzed, and showed incongruent clustering in phylogenetic trees (Figs. 1 and 2). The nucleotide sequences in the 59UTR of these strains were related closely to those of formerly identified QPM, NAT001, NAT045, C024, C025 and C026 HRV-C strains, and to some of the W strains recently identified as HRV-A [15] (Fig. 2) . However, our 20 strains clustered together with these six strains in two major branches in the phylogenetic tree constituted a subspecies of HRV-C called HRV-Ca (Table 3 , Figs. 2 and 3) . The fourteen other HRV-C strains formed another unique branch of HRV-C subspecies, called HRV-Cc, which clustered differently from other species of HRV-A and HRV-B, and from subspecies HRV-Ca in the phylogenetic tree based on 59UTR sequences (Table 3 ; Figs. 1 and 2). These strains were related closely to some W strains of HRV that were classified as HRV-C [15] (Fig. 2 , Table 3 )
In order to characterize more precisely the differences observed in the 59UTR between HRV-Ca and HRV-Cc subspecies, and to localize possible recombination sites in the 59UTR of the genome of HRV-Ca subspecies, bootscanning and similarity plot analyses were conducted in the gene fragment of 868 nt that included the 59UTR and adjacent capsid genes. HRV-Ca nucleotide sequences were scanned against sequences of N10, R16 and R52, which are considered as representative strains of HRV Cc subspecies and HRV-A and HRV-B species, respectively. Stretches of nucleotide sequences that were closer to HRV-A (R16) than to HRV-Cc (N10), flanked by sequences related to HRV-C could be detected in the 59UTR of HRV-Ca strains, as exemplified with HRV-Ca N25 strain ( Fig. 3a and b) . These HRV-A-related nucleotide stretches were thus flanked by putative recombination sites. These sites were located differently among the HRV-Ca strains, delimiting HRV-A-related stretches that ranged from 150 to 400 nt in length (Fig. 3c ). While variable among the strains, the identified recombination sites were all located inside the 59 UTR and none of them was identified in the downstream VP4/VP2 coding sequence. HRV-A-related nucleotide sequences and putative recombination sites were also found in the 59UTR of the previously described HRV-C strains C024, C025, C026, NAT001, NAT045 and QPM (Fig. 3) . The results corroborated the clustering observed in the phylogenetic tree based on 59UTR sequences (Fig. 2) , since strains gathered in the same HRV-Ca subcluster (for example N24, N25, N28 and N32, or N4, N7, N8, N21, N36 and N46) displayed the same recombination pattern. These subclusters revealed different recombinant lineages, each of which originated from independent recombination events.
In order to further characterize the genome of the HRV-Cc subspecies, for which no full-length sequence was yet available, we sequenced the remaining genes that covered the whole coding sequence and 39UTR of N10 strain, which was chosen as the representative of this subspecies (Tables 2 and 3 ). The full-length N10 genome sequence was compared to those of the HRV-Ca subspecies strains C024, C025 and C026, and to that of N4 strain, which was sequenced as the representative of the HRV-Ca subspecies. The genome sequences were also compared to those of the HRV-A strains N13 and R44, and to the HRV-B strains R14 and R52 (Table 4 ). The full-length nucleotide sequence of N10 strain contained 7111 nt, excluding the poly(A) tract, which was shorter than sequences from HRV-A and HRV-B strains, but similar to those of HRV-Ca strain N4 and other related strains (C024, C025 and C026). The 2144 aa lengths of the polyprotein and of each of the individual proteins of N10 were slightly different from those of HRV-A and HRV-B species, but similar to those of other HRV-C strains. The most divergent amino acid length between HRV was observed for the VP1 protein that was shorter in HRV-C species ( Table 4 ). The unique putative cleavage (M/S) site between VP4 and VP2 protein identified previously for QPM, C024, C025 and C026 strains [20] was also observed for N10 and N4 strains. It was different from those of the HRV-A strains N13 and R44 (Q/S), and from those of the HRV-B strains R14 and R52 (N/S) (data not shown).
Alignment of the VP1 amino acid sequence of HRV-Cc strain N10 with those of other HRV-A and HRV-B species and HRV-Ca subspecies, designated in Table 4 , showed structural features typical of HRV-C species [16, 20, 25] (data not shown). In particular, footprints including deletions in the BC, DE and HI loops and conserved amino acids potentially involved in Inter-Cellular Adhesion Molecule 1 (ICAM-1) receptor binding [7, 11, 20, 24] were conserved within the HRV-C species (data not shown). Bootscanning and similarity plot analysis conducted on the genomic sequences of N4 (HRV-Ca), N10 (HRV-Cc), R16 (HRV-A) and R52 (HRV-B) confirmed that N4 featured a 59UTR sequence that was related to the R16 sequence (stretch I), followed by a capsidic sequence related to the N10 sequence. N4 nonstructural sequence (2A to 39UTR) was related more closely to N10 than to R16 and R52 sequences. However, in stretch II (nt 3300-3500 according to N4 numbering), N4 strain (HRV-Ca) was closer to R16 (HRV-A) than to R52 (HRV-B) or N10 (HRV-Cc), which resulted in high bootstrap values between N4 and R16 2A sequences (Fig. 4) . This may have been the result of a recombination event that occurred in the 39 part of the 2A-encoding sequence of the parental strain N4, and which involved an HRV-A strain. Nevertheless, the HRV-A parental strain or ancestral strain could not be identified since the closest HRV-A 2A nucleotide sequence available was ,80% identical to that of N4 in this region.
In contrast from nt 6,550 to the 39 end (stretch III in Fig. 4) , the N10 strain genome was found to be closely related to that of N4, with nucleotide identity .98%. This result is corroborated in Figure 5 , which shows a phylogenetic analysis of the 39UTR sequences of N10 and N4 compared to those of HRV-Ca subspecies and HRV-A and HRV-B species. This suggests that N4 and N10 strains share a common recent ancestor through recombination. 1 and 2 ) and on local recombination in 59UTR (see Fig. 3 ). b Strains closely related with more than 93% identity. c These strains clustered differently when based on VP4/VP2 sequences (see Table 2 and Figs. 1 and 2) . doi:10.1371/journal.pone.0006355.t003
Clinical outcome from HRV strains isolated from pediatric outpatients Among the pediatric patients, 46 were males and 18 females, and their age ranged from 5 months to 14 years. The majority of HRV infections were diagnosed between 2 and 6 years of age (84.6%). Bronchitis (73.4%) and pneumonia (26.6%) were highly prevalent in children with comparable incidence in HRV-A and HRV-C infections (Table 5) . Moreover, the ratio of pneumonia over bronchitis (36.2%) was comparable to that in the whole cohort of 827 children (40.7%). Only one child among the 64 HRV-positive patients had asthma and was co-infected with HRV-C, influenza A virus (IAV) and respiratory syncytial virus (RSV) ( Table 5) , whereas 100 of the 827 patient were diagnosed with asthma. HRVs were isolated throughout the 2 years, with a predominance of HRV-C viruses in the cold season (Table 5) . Interestingly, different HRV genotypes were detected within the same period (for example, N1 and N4, N9 and N11/N12, N44/N48 and N51, and N55/N56 and N62/N67), with a larger diversity and distribution of individual or paired HRV-A genotypes compared to HRV-C strains, which clustered in closely-related genotypes (Fig. 2, Tables 2 and 3) . Conversely, N4 and N21 strains of samples collected at 10 months interval showed 99.8% identity (Fig. 2) .
Single HRV infection was diagnosed in 42 children and coinfections were identified in 22 patients (Table 5) , with 17 double and five triple infections. The viruses most often identified in HRV co-infection were RSV (six cases) and human bocavirus (HBoV; four cases), and two patients were co-infected with HRV, HBoV and RSV (Table 5 ). There was no difference between HRV-Ca or HRV-Cc subspecies and any of the clinical or epidemiological data (data not shown).
In this report, we looked for HRVs in a 2-year collection of NPSs from children with ARI visiting a district hospital in Shanghai, and found a high diversity of HRV strains that belonged to different species and genotypes. We characterized by RT-PCR and sequenced 66 HRVs, among them 27 HRV-A, five HRV-B, and 34 HRV-C strains. When sequencing the VP4/VP2 region of the HRV genome, several recent studies have identified new strains of viruses from children and adults with ARI, asthma, or otitis, which are clustered differently from HRV-A and HRV-B, and have been classified into a novel HRV-C species [7, 8, 17, 18, 19, 20, 21, 25, 26, 27, 28] . Other groups have also identified novel HRV-C strains by sequencing the VP1 gene [29] or the 59UTR [14, 15] . The different sizes and locations of the regions amplified in the HRV genomes renders difficult comparative genetic analysis. Recently, Palmenberg et al. (2009) have finalized the full-length genome sequences of all HRV-A and HRV-B reference strains, and identified structural features of these two species and the novel HRV-C species [16] . In our study, we identified 34 HRVs (51.5%) that clustered differently from HRV-A and HRV-B in a phylogenetic tree that was established on the basis of VP4/VP2 sequences, which were related to recent strains classified in the novel HRV-C species (Fig. 1, Table 2 ). Fourteen HRV-C strains (41.2%) segregated from the other 20 strains (58.8%) that were closely related to HRV-A in their 59UTR sequence (Fig. 2) . This led us to propose a classification of two HRV-C subspecies, HRV-Cc and HRV-Ca. In previous studies [15] . These strains clustered with our field strains within the HRV-Cc subspecies. Moreover, 17 strains that clustered with HRV-A, and had 12-35% pairwise nucleotide divergence from the nearest reference serotype [15] , clustered within the two major branches of HRV-A and HRV-Ca strains (Fig. 3) . Therefore, we cannot ensure that some of the 17 strains were HRV-A or HRV-Ca strains. Kiang et al. (2008) have identified five novel HRVs out of 24 clinical samples (20.8%), which segregated from HRV-A and HRV-B, and were classified as HRV-C, and three additional strains (12.5%) that also clustered with QPM, C024, C025, C026, NAT001 and NAT045 [14] (Fig. 2) . However, the field HRV strains of these previous studies were sequenced using a 59UTR that did not match fully our sequence and that of Lee et al. (2007) [15] , and could not be included in the present study for comparative analysis. Interestingly, the five strains identified in California in 2007 [14] and N42 and N45 from our study were closely related to strain W37 isolated in Wisconsin in the late 1990s [15] , and to NAT001 isolated in the winter of 2004 in California [18] , which confirms that similar genotypes of HRV-Ca are widespread [17] .
The strains of HRV-C species identified in the present study were characterized by analyzing the 59UTR, VP4, and part of VP2 (Fig. 3) . This approach showed the advantages of covering only 59NCR, VP4/VP2, VP1 or 3D genome fragments. Analyzing sequences that covered the 59UTR and the downstream VP4/ VP2 capsid region allowed identification of co-infections when several clones were sequenced, and helped to locate the recombination sites in strains of the HRV-Ca subspecies. Thus, this region of the genome may be useful for building a database of the novel strains that are circulating worldwide.
The genome of HEVs is subject to frequent recombination [30, 31, 32, 33, 34, 35, 36] , with interspecies exchanges observed in the 59UTR [37] . Palmenberg et al. (2009) have observed intraspecies recombination in three HRV-A, with structural characteristics and phylogenetic evidence that suggests a novel clade D classification [16] . Tapparel et al. (2009) observed phylogenetic incongruities in 59 UTR, VP1 and 3CD sequences of two clinical isolates of HRV-A related to recombination [38] . We observed incongruent clustering of N12, N44 and N48 strains of HRV-A species in the phylogenetic trees based on the 59UTR or the VP4/VP2 regions of their genomes (Figs. 1 and 2, Table 3 ), which suggests intraspecies recombination in the 59UTR.
We observed one co-infection with HRV-A and HRV-B (N16A and N16B), one with HRV-A and HRV-C (N58A and N58C), and three co-infections of HRV-A and HRV-B with HEVs that may favor recombination events. Previous comparison of genome sequences between 34 HRVs showed only limited recombination events and a pattern of genetic diversity lower than that observed with other picornaviruses [25] . The presence in HRV-C subspecies of sequences that share 90.5-98.6% identity with HRV-A strains (Table 3) suggests that recombination events occurred between HRV-C and HRV-A. Bootscanning of the 59UTR of HRV-C strains also showed different sites and lengths of recombination (Fig. 3c) , which suggested that there were several independent events that led to several groups of HRV-Ca genotypes, which formed clusters in the phylogenetic tree (Fig. 2) .
Comparative analysis of the full-length nucleotide sequences of two field strains of different HRV-C subspecies (N4 and N10) with those of other HRV species suggested that multiple interspecies recombination events occurred in the 59UTR and in the NS2A protein gene, and that recombination also occurred in the 39UTR between N4 and a strain close to N10. These findings are in agreement with those observed for other HEVs, for which recombination events in the capsid-encoding sequence are very rare, probably because of structural constraints that restrict the functioning of chimeric capsids [31] . This result appeals for the full-length genome sequencing of the major representatives of the HRV-C species, in order to establish a clear understanding of the evolution and classification of the novel virus into subspecies. Comparison of the coding sequences of N10 HRV-Cc with other strains of HRV-Ca subspecies [20] , including our field strain N4, showed high similarities in the lengths of the 11 proteins, their cleavage sites, and the structural features of VP1. These characteristics and the absence of growth in cell culture, noted in our laboratory and by others (data not shown), support the classification of the novel strains into a unique HRV-C subspecies.
Our clinical specimens all originated from NPSs from pediatric outpatients. The remarkable outcome of the study is the large diversity of genotypes that has circulated in a relatively small group of people in a district of Shanghai during a 2-year observation. Although some clusters of similar genotypes in a limited period of time were observed, co-circulation of different genotypes and HRV species and subspecies, and co-infections with two HRV species were observed. The prevalence of the novel HRV-C in our specimens (4.1%) differed from previous studies that associated the prevalence of the novel variant with severe disease outcomes, which ranged from influenza-like illness or infection of the low respiratory tract [17, 28] to asthma exacerbation, bronchiolitis, and febrile wheeze [7, 8, 15, 18, 20, 21, 29, 39] . All our patients showed bronchitis or pneumonia, with no etiological correlation with any of the species or subspecies of HRV. Only one patient co-infected with HRV-C, IAV and RSV was diagnosed with asthma among the HRV-positive patients (1.6%), whereas 100 of the 827 children had asthma (12%). The difference observed with previous studies, 44.6% [8] and 12% [29] asthma in HRV-positive patients, may be related to the criteria for enrolment. Moreover, none of the patients in our study were hospitalized, which makes comparison with hospitalized children difficult [20, 24] . Another criterion to consider in the trend to correlate clinical symptoms with HRV infection is the presence of co-infecting pathogens. In our study, four strains of HBoV and six strains of RSV (17.6%) were identified in association with HRV-C (11.7%). HBoV and RSV are common viruses diagnosed in ARI, which are often associated with HRV [40, 41] , and HBoV was identified in .50% of children co-infected with HRV [20] . Nevertheless, the incidence of HBoV in ARI and in severe outcomes remains elusive [42] . More studies need to be carried out on large numbers of samples from severe and mild diseases, to identify any obvious role of HRV sequence diversity and association with other pathogens in disease severity. Since a large diversity of recombination in HRVs has become obvious, we must be aware of the occurrence of novel HRVs that may become highly virulent.
This study was approved by the ethical committee of Shanghai Nanxiang Hospital and written informed consent was obtained from the parents of the children.
Clinical specimens (n = 827) from NPSs were collected from children under 14 years old, who experienced a lower respiratory tract infection, and who were consulting the pediatric department of Shanghai Nanxiang Hospital during the period October 2006 to October 2008.
Total RNA was extracted from NPS specimens using QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany), and stored at 280uC. RNA was amplified using the Qiagen One Step RT-PCR Kit. A five-tube mRT-PCR was used for virus identification as previously described [43, 44] . Tube 1 targeted IAV, influenza B virus, RSV, and human metapneumovirus; tube 2, parainfluenza viruses 1 to 4; tube 3, HRV and influenza C virus; tube 4, human coronaviruses (HCoVs) 229E-HCoV, OC43-HCoV, NL63-HCoV and HKU1-HCoV; and tube 5, adenovirus and HBoV. Amplified products were analyzed in 0.5 mg/ml ethidium bromide/2% agarose gel.
Samples that showed positive results for HRV were amplified again using specific primers P1-1F and VP4/2R, located in the 59UTR and VP2 gene, respectively (Table 1) . One strain of HRV-C (N1) could not be amplified using the P1 and VP2 extreme primers and was amplified using primers in 59UTR and VP4/ VP2, respectively (Table 1 ). In brief, 2.5 ml of extracted RNA was mixed with 56 buffer and 0.4 mM dNTPs, 0.2 mM of each of the primers, and 1 ml of enzyme mix, and diethylpyrocarbonatetreated ultrapure water was added to a final volume of 25 ml. Amplification programs included reverse transcription at 50uC for 30 min, inactivation at 95uC for 15 min, followed by 40 cycles at 94uC for 30 s, 50uC for 30 s, 72uC for 70 s, and final extension at 72uC for 10 min. The amplified DNA products were detected by ethidium bromide-agarose gel electrophoresis. The lengths of P1-VP2, VP4-VP2 and P1-P3 amplicons were 924, 559 and 390 nt, respectively. DNA products were extracted from agarose gels by using QIAquick Gel Extraction Kit (Qiagen), and were ligated into pMD20-T vector (Takara Biotechnology, Dalian, China), and at least two recombinant plasmids were sequenced in Biosune Sequence Company and Life Biotechnology in Shanghai, China. Sequences of different clones of N16 and N58 showed identities for either HRV-A or HRV-B strains. More plasmids were sequenced for these strains to confirm that the two patients were originally coinfected with two different HRV species.
Sequences of three complete genomes of HRV were obtained for strains N4 (reference R3061207002 collected on December 7, 2006) , N10 (R3070614001 collected on June 14, 2007) and N13 (R3070719007 collected on July 19 2007). Primers used for the amplification of viral genomes were designed after multiple alignments of sequences from the genomes of different HRVs available in GenBank (Table 1) . Overlapping amplified DNA products were obtained after PCR of cDNA that was obtained using oligodT and a Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Mannheim, Germany), following the manufacturer's protocols. Briefly, 10.4 ml viral RNA was mixed with 1 ml oligodT and heated at 65uC for 10 min, and then kept on ice for 2 min. After addition of 4 ml 56buffer, 0.5 ml Protector RNase Inhibitor, 2 ml dNTPs, 1 ml DTT, and 1 ml RT enzyme, the reaction was incubated at 50uC for 1 h, inactivated at 85uC for 5 min, and stored at 220uC. Amplification of a 3D region of N4, N10 and N13 HRV strains was carried out by nested-PCR using Takara EXTaq (Takara Biotechnology) and specific primers (Table 1) , for 35 cycles of 30 s at 94uC, 30 s at 55uC, and 70 s at 72uC. To amplify VP1 (upstream of 2A) sequences of N4 and N13 strains, nested PCR was carried out using Takara LATaq with GC buffer I(Takara Biotechnology), specific primers (Table 1) , and incubation for 35 cycles of 30 s at 94uC, 30 s at 60uC, and 4 min at 68uC. The fragment VP2-3D of N10 HRV strain was obtained by seminested PCR and specific primers VP2 F, and 3D inner and outer reverse primers (Table 1) , using Takara LATaq with GC buffer II, for 35 cycles of 30 s at 94uC, 30 s at 60uC, and 6 min at 68uC. The terminal part of the whole genome was obtained by rapid amplification of cDNA ends using 59/39 rapid amplification of the cDNA kit, following the manufacturer's protocol (Roche). To perform 59 terminal RACE, 4 ml of 56cDNA buffer, 2 ml dNTPs, 1.25 ml specific primer 1 (10 mM), 9.2 ml RNA, 1 ml control primer neo1/rev (12.5 mM), 1 ml control RNA, 1 ml RT enzyme, and 0.6 ml RNase inhibitor (Roche) were mixed and incubated for 55uC for 60 min, followed by inactivation at 85uC for 5 min, and stored on ice. The product was purified using the Qiagen PCR Purification Kit and eluted with 30 ml deionized distilled water. A polyA tail was added to the cDNA, by mixing 9.5 ml DNA with 1.25 ml 106 reaction buffer, 1.25 ml (2 mM) dATP, and after incubation at 95uC for 3 min, the reaction was chilled on ice for 2 min. After addition of 0.5 ml terminal transferase, the reaction was incubated at 37uC for 30 min, inactivated at 70uC for 10 min, and kept on ice. Nested PCR was performed by using the Expend High Fidelity PCR kit (Roche). A mixture of 2.5 ml poly-dA-tailed cDNA, 0.5 ml oligodT-anchor primer 37.5 mM, 0.62 ml SP2 primers (10 mM) (Table 1) , 0.5 ml control neo2/rev primer (12.5 mM), 0.5 ml dNTP, 0.35 ml enzyme, 2.5 ml 106 buffer, and 18 ml ddH 2 O was incubated for 40 cycles of 30 s at 94uC, 30 s at 60uC, and 30 s at 72uC. To perform 39 terminal RACE, the method was similar to normal two-step RT-PCR using 3D inner and outer F primers (Table 1) .
Sequence alignment, phylogenetic analyses and recombination analysis DNA sequences used for P1-P2 gene analysis were based on HRV-16 nt 178-462 and those used for VP4/VP2 gene analysis were based on HRV-16 nt 626-1045. Multiple sequences were aligned using Clustal X [45] . The multiple-sequence alignment was subjected to phylogenetic analyses using programs in the PHYLIP package (v3.6). Bootstrap analysis was performed using SEQBOOT, with a replicate number of 1000. Then, DNADIST and NEIGHBOR were used to obtain distance matrices with the F84 parameter, and a transition/transversion ratio of 4. Consensus trees were computed by CONSENSE, and then re-rooted with RETREE. The final tree was visualized and edited with MEGA version 4 [46] .
Recombination analysis was carried out by using Recombination Detection Program v.3.22. Manual bootscanning was performed by using the Juke-Cantor algorithm and the neighbor-joining method [47] , with a window size of 200 nt, a step size of 20 nt and 100 replicates. Pairwise identities between sequences were determined with SimPlot software method [48] ,with a window size of 200 nt and a step size of 20 nt. Pairwise homology matrices were obtained by using CLC Combined Workbench 3.0 software (CLC bio, Aarhus, Denmark).
The original P1-VP2 sequences described in this study were deposited in GenBank under accession nos. GQ223119 to GQ223136. The VP4-VP2 sequences were deposited under the nos GQ223137 to GQ223181, and the P1-P2 sequences under the nos GQ223182 to GQ223226. The full length genomes sequences of N4, N10 and N13 strains were deposited under the nos. GQ223227, GQ223228, GQ223229, respectively. Poster Exhibition  Background: Biliary atresia (BA) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. The incidence rate of BA is higher in Asian countries, occurring in approximately 1 of 8,000 (Asian Countries ) to 1 of 18,000 (European countries) live births. Early identification and prompt intervention is very important. To im prove the early diagnosis, we used proteomic technology to screen serum biomarker for BA. Methods: Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were employed to screen serum biomarkers specific to BA sera from idiopathic neonatal hepatitis. After pretreatment including albumin and immunoglobulin (IgG ) depletion, sera were subjected to 2-DE and there after image analysis. The differentially expressed protein spots were identified by MALDI-TOF-MS. Result: From optimized 2-DE gel images, thirty-four spots were differentially expressed and identified by MALDI-TOF-MS to be eight proteins. Overall, kininogen 1 variant was under expressed and alpha-1-B-glycoprotein, leucine-rich alpha-2-glycoprotein 1, 'SP40,40', A1BG protein, vitamin D-binding protein/group specific component , apolipoproteinA-, AQGV 3103 were over expressed in BA group com pared to idiopathic neonatal hepatitis. Conclusion: 2-DE based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of BA, though further validation is needed.
S. Somani 1 , A. Somani 2 , A. Jain 3 , V. Dixit 3 1 Suvidha, 2 Navjeevan Hospital, 3 IMS, BHU, Varanasi, India
Background: A variety of autoreactive antibodies are detected in patients with chronic liver disease. This prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. Methods: Study population included 53 patients (75% males), who met defined criteria for chronic liver disease. Detailed clinical, laboratory and sonographic evaluation was done. Sera were tested for ASMA, anti-LKM type1, AMA, APA, ANA, by standard methods. P<0.05 was considered significant. Results: Among various etiologies for chronic liver disease, Hepatitis B was most common (28%), followed by alcohol (19%), autoimmune hepatitis in 15%, Hepatitis C (6%) and miscellaneous (2%). 30% of patients were labeled as cryptogenic after detailed investigations. ANA (>1/80) was positive in 100% of definite AIH, 33% of HCV related CLD but at titer of >1/40, 66.6% of HCV related CLD & 60% of probable AIH were found positive. ASMA (>1/40) was positive in 6% of HBV related CLD, 10% of alcohol related CLD, 33% of definite AIH, 40% of probable AIH, 33% of HCV related CLD but ASMA in titer of >1/80 was positive only in 33% oh definite AIH. APA was detected in 12.5% of cryptogenic CLD, 13.3% of HBV related CLD & 20% of alcohol & probable AIH related CLD each. AMA was detected in 1% of cryptogenic, HBV, AIH (definite) & HCV related CLD each, and 2% of alcohol related CLD & 100% of PBC. Conclusions: Apart from AIH there is high prevalence of ANA & SMA in HCV related CLD while other antibodies has low prevalence in non-AIH related CLDs. This study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of CLD especially of mixed etiology.
Conclusions: OA infusion did not lower ammonia levels or improve survival.
Results: The mortality (50.0%) of patients in lamivudine group with MELD score from 30 to 40 was lower than that (86.1%) of control group ( 2 =23.319, P=0.000). Univariate analysis showed that mortality was significantly related to age (P=0.005), MELD score (P=0.009), treatment method (P=0.000), pretreatment HBV DNA load (P=0.000), the decline of HBV DNA load during therapy (P=0.006) and encephalopathy (P=0.007). In multivariate analysis, in patients with MELD scores 30-40, treatment method (P=0.004), pretreatment HBV DNA load (P=0.009), decline of HBV DNA load during therapy (P=0.014) and encephalopathy (P=0.019) were independent predictors of mortality; for MELD scores above 40, only MELD score (P=0.015) was independent predictive. Conclusions: Lamivudine treatment significantly decreases the 3 month's mortality of patients with MELD score 30-40, and a low viral load pre-treatment and quick decline of HBV DNA load are good predictors for the survival of lamivudine treatment. Background/Aims: Early identification of patients with Fulminant hepatic failure (FHF) who need a liver transplantation is very important. To construct a prediction model for early diagnosis and prognosis of FHF, we studied dynamics of metabolic profiles using a D-galactosamine/lipopolysaccharide (GalN/LPS)-treated mouse model. Methods: BALB/c mice were used to construct FHF model and sacrificed for blood collection at 4, 5, and 6 hour after treatment, respectively. Levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (PLS-DA). Results: Distinct clustering differences were observed 5 and 6 h after treatment between survival and dead groups. At 5 h, plasma levels of some metabolites differed significantly between survival, dead and control groups. Ketogenesis and the TCA cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. PLS-DA indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. The Y-predicted scatter plot in PLS model assigned samples to survival or dead groups using an apriori cutoff of 0.10 with 100% sensitivity and specificity. Similar results were observed in 11 FHF patients with different outcomes.
PE012 Association between Polymorphisms in the Interleukin-10 Gene Promoter and Hepatitis B-related Acute Liver Failure Conclusions: The PLS model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of FHF.
IL-10 promoter are associated with the susceptibility to hepatitis B-related ALF in the Chinese population. IL-10 A-592C may be a regulatory polymorphism that affects gene regulation.
Hepatocyte Cell Death in ACLF: Mechanism and Significance -An Immunohistochemical Study. P. Sakhuja 1 , A. Rastogi 2 , S. S Hissar 1 , A. Singh 1 , A. Kumar 2 , R. Gondal 1 , S.K. Sarin 1 1 GB Pant Hospital, 2 Institute of Liver and Biliary Sciences, New Delhi, India Background: Acute on chronic liver failure (ACLF) is defined as acute hepatic insult complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. Caspases play an essential role in apoptosis. COX-2 is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. We studied the immunohistochemical expression of COX-2 and caspase-1 in liver tissue to assess their role in pathophysiology and in predicting outcome of ACLF. Method: A retrospective analysis of 50 liver biopsies with clinical diagnosis of ACLF was undertaken. Patients were divided into two groups A and D based on clinical outcome (Alive/Died respectively). Immunohistochemical analysis for COX-2 and caspase was performed on 39 and 36 cases respectively and scored from 0-8 as per intensity and distribution. Score 6-8 indicated high intensity with focal to diffuse distribution, and was considered significant. Results: Etiology of acute liver failure was viral or alcoholic. Increased expression of Caspase was observed in 10/21 cases in group D and none of the cases in group A(n=15) (p=0.001). Increased expression of Cox-2 was observed in 4/21 cases in group D and none of the cases in group A (n=18) (p=0.052). Conclusion: Increased immunoreactivity of caspase in liver biopsies of patients of ACLF may indicate worse prognosis and its important role in the pathophysiology of ACLF. Immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future.
Conclusion: hHGF expression vector (pCMV-hHGF) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hHGF in vivo.
J.H. Kim 1 , K.W. Kim 1 1 Asan Medical Center, Seoul, Korea Background: Splenic artery embolization (SAE) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (LT). Thus, the purpose of this study was to estimate SAE effect on the basis of changes in caliber of related vessels and splenic volume on pre-SAE and serial post-SAE CT scans in LT recipients. Methods: Between 2003 and 2007, among 73 LT recipients who underwent SAE and serial follow-up CT, 43 with no compounding factor that may obscure SAE effect were included in this study. They underwent CT before and after (1week, 1month, and 1year) SAE. A radiologist retrospectively measured diameters of CA, CHA, SA, SV and splenic volume on serial CT scans. Their diameters and splenic volume on each CT were compared with those on the prior and pre-SAE CT. The difference was compared using repeated-measures ANOVA tests. Results: CAs decreased between 1week and 1month after SAE (P<.05), but were stable before 1week and after 1 month. CHAs increased within 1week (P<.05) but decreased between 1week and 1month (P<.05) and remained stable after 1month. Compared with pre-SAE CT, CHAs were larger for 1month after SAE. SAs continuously decreased for 1year (P<.05). SVs decreased for 1 month (P<.05) and remained stable after 1 month. Compared with pre-SAE CT, SAs and SVs were smaller from 1week after SAE and on. Splenic volume continuously decreased for 1year except a period between 1week and 1month. Conclusion: The increase of hepatic arterial flow persists for 1month after SAE, but returns to baseline thereafter. The decrease of portal flow may lasts for at least 1year after SAE.
Poster Exhibition -Cholangioca and Other Liver Neoplasm Poster Session, Hall 5B Background: Now, RFA has becoming an important practice of HCC therapy. In this study, we evaluated whether RFA therapy for metastatic liver tumor has a beneficial effect on patients' survival. Methods: Forty six patients were treated by RFA for metastatic liver tumor from July 2001 through February 2008 in our hospital, of the 46, 33 patients were metastasis either form colon or stomach cancer. These 33 patients were analyzed in this investigation. Cumulative survival rate from initial RFA therapy was calculated by Kaplan-Meier method. Predictive factors for survival were identified using Cox proportional hazard regression model. Results: The mean age of the 33 patients were 64. 6 (range, 40-79) . The mean size of the tumor is 28mm (range,8-70mm) and the numbers of tumor foci are 2.7 nodules range,1 18. The survival rates of patients treated by RFA were 49.5% at 3 years and 31.8% at 5 years in colon cancer, 15.6% at 3 years and 15.6% at 5 years in gastric cancer. In this series of 33 patients, primary cancer: colon (P=0.002 odds ratio 0.132 95%CI 0.037-0.473), younger patients ( 64) (P=0.041 odds ratio 0.312 95%CI 0.102-0.955) and multiagent chemotherapy (P=0.012 odds ratio 0.223 95% CI 0.069-0.723) were significantly correlated with better survival. Conclusion: The survival of patients treated by RFA for metastatic colon cancers had better survival than those of gastric cancers. In addition, good indication of RFA is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies.
Utility of Contrast Enhanced Ultrasonography with Sonazoid in Radiofrequency Ablation (RFA) for Liver metastasis E. Goto 1 , S. Shiina 1 , R. Tateishi 1 , R. Masuzaki 1 , K. Enooku 1 , T. Sato 1 , J. Imamura 1 , T. Goto 1 , Y. Sugioka 2 , H. Ikeda 1,2 , H. Yoshida 1 , M. Omata 1 1 Department of Gastroenterology, University of Tokyo, 2 Department of Clinical Laboratory, Tokyo, Japan
Background & Aims: Contrast enhanced ultrasonography (CEUS) with Sonazoid is effective for liver metastasis because enhance defect in Kupffer imaging is well delineated. The aim of this study is to investigate the detection ability of CEUS and the utility of Sonazoid in RFA for metastasis liver tumors. Material & Methods: From January 2007 to December 2007, a total of 346 liver metastatic nodules in 87 patients (62 colon cancer, 13 breast cancer, 3 gastric cancer, 3 islet cell tumor, and 6 others) admitted to receive RFA were studied. The detection ability of liver metastasis was compared between CEUS and conventional US using enhanced CT as reference standard. The mean numbers of treatment session of RFA were compared between patient treated with CEUS assistance and historical controls matched for size and number of tumors. Results: The detection rate was 78.6% with conventional US and 96.4% with CEUS (P=0.0004). 83 nodules in 25 patients were not detected by conventional US and detected after injection of Sonazoid. In addition, 12 nodules in 2 patients were detected not by CT but only by CEUS. The mean number of session was 1.5±0.5 as compared to 2.0±1.0 in the historical controls (P<0.001). Conclusions: CEUS with Sonazoid is useful for detection of liver metastasis. Sonazoid is an excellent supportive agent in RFA of liver metastasis.
Background/aims: Carcinogenesis of intrahepatic cholangiocarcinoma (ICC)-associated liver fluke infection accumulated genetic and epigenetic alterations. Cholangiocarcinoma cell line (KKU-M213) is adenosquamous carcinoma which rare variants and not commonly found in ICC. However, interactions of liver fluke-associated ICC proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. Objectives: To analyze the whole genome-wide genetic alterations in KKU-M213 using microarray comparative genomic hybridization. Methods: DNA of KKU-M213 and matched-sex reference were differentially labeled with fluorescence dries (Cy3 and Cy5) and mixed together with cot-1 DNA. The mixture was hybridized on array with spotting 2,464 human bacterial artificial chromosomal (BAC) clones in triplicate and mapped these directly onto human genome sequence. The genetic alterations were classified the DNA copy-number variations according to the intensities of log2 ratio (Cy3/Cy5) as DNA copy-number loss/gain and deletion/amplification. Results: the whole genomic alterations in KKU-M213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. Chromosomal amplifications were detected on 4q13.1, 4q21.1, 4q21.2, 5p tel, and 5p15.3, whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3. Conclusions: The whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. This recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated ICC carcinogenesis.
artificial chromosomal (BAC) clones in triplicate and mapped these directly onto human genome sequence. The genetic alterations were classified the DNA copy-number variations according to the intensities of log2 ratio (Cy3/Cy5) as DNA copy-number loss/gain and deletion/amplification. Results: the whole genomic alterations in KKU-M213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. Chromosomal amplifications were detected on 4q13. 1, 4q21.1, 4q21.2, 5p tel, and 5p15.3 , whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3 . Conclusions: The whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. This recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated ICC carcinogenesis. Acknowledgements: This work was supported by Faculty of Medicine, KKU, Thailand (Grant No. I51117 Background/Aims: We studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (ICC). Thirty patients with unresectable ICC or intrahepatic recurrence of ICC after surgery were studied. Comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in 5 patients and 5-fluorouracil (5-FU) in 7 patients. Eighteen patients were treated without port system. Results: Disease was stable in 5 patients with adriacin and lecithin-added lipiodol emulsion and in 3 patients with 5-FU. Disease was progressed in 4 patients with 5-FU. The mean survival period was 20.8 months in patients with adriacin and lecithin-added lipiodol emulsion, 9.3 months in patients with 5-FU, and 10.5 months in patients without port system (p=0.02, p=0.04). Conclusion: Arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of ICC after surgery.
PE038
S. Kaur 1 , T. Kaur 1 1 Department of Biophysics, Panjab University, Chandigarh. India Background: A number of dietary factors have been involved in the pathogenesis of cholelithiasis. Cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.Aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. Methods: Balb/c mice was divided into two groups control in this group animals were fed normal chow diet, High fat diet group in this group (2% cholesterol,0.5% sodium cholate, 5% butter fat and 15% coconut oil) mixed with chow diet was fed to the mice for 4 weeks. Cell apoptosis and proliferation was assayed in gallbladder epithelial cells. Histological analysis of gallbladder sections were done with hematoxylin and eosin staning. Results: Mice fed high fat diet had apoptotic as well as necrotic epithelial cells. Rate of proliferation was enhanced after 24 and 48 hrs in mice fed high fat diet group as compared to the control group. The histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet.
Conclusion: Results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. It has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. Background: Obstructive jaundice can be caused by malignancy. The treatment can be drainage by biliary stenting. In advanced malignant jaundice, the stent placement is often difficult. Objective: To evaluate the success rate of malignant obstructive jaundice evaluation of ERCP and success rate of stent placement. Methods: Retrospective study based on data of ERCP from October 2004 until July 2008. Results: We evaluated 139 patients who has done ERCP examination, 131 (94,2 %) patients have clinical diagnosis of obstructive jaundice. There were 73 (55,7%) male and 58 (44,3%) female, age range 20 -84 (median age was 51). There were no malignancy in 66 (50,4 %) patients; malignancy in 48 (36,6%) patients and 17 (13%) patients need further evaluation.. From 114 patients, 57 (50 %) patients attempted to have stent placement, 50 (43,9 %) patients do not and 7 (6,1 %) patients have no data. We done descriptive study on 57 patients attempted to have stent placement, 32 (56,1 %) patients succeed in stent placement whereas 25 (43,9 %) failed. Malignancy was showed to be a factor of stent failure (malignancy: 23 fail and 10 success (30,3 %) vs non malignancy: 2 fail and 22 success (91,7%)). Conclusion: ERCP can identify the cause of obstructive jaundice in 87 % patients. The success rate of stent placement was 56,1 %. The success rate of biliary stenting in malignant obstructive jaundice was 30,3 % whereas in non-malignant cases was 91,7 %. Papillary carcinoma was the most frequent cause of malignant obstructive jaundice. Background: In Hydatid disease of the liver cystobiliary fisula (CBF) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. Aim: To study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. Patients and Methods: Fourteen patients with complicated cysts were divided into 2 groups; group A: nine patients presented with cholangitis, and group B: five patients had history of jaundice. In all patients, the diagnosis of CBF was confirmed by ERC (Endoscopic Retrograde Cholangiography). Preoperative endoscopic sphincterotomy (ES) was done in group A with retrieval of hydatid daughter cysts. Seven patients (subgroup A1) were subsequently submitted to surgery entailing endocystectomy in 5 and hepatic resection in two. The remaining 2 patients in group A (subgroup A2), were managed by endoscopic therapy only. Patients of group B (n=5), were not submitted to preoperative ES and were subsequently managed by hepatic resection in one patient and endocystectomy in four. Results: There was no mortality in the studied group. Postoperative bile leak occurred in four cases in group B. In contrast, none of the patients who were submitted to preoperative ES (subgroup A1) had bile leak. All patients received albendazole treatment. Conclusion: ERC is important in confirming the diagnosis of CBF. Also, therapeutic ERC has a place in the treatment algorithm of CBF as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery.
V. Singh 1 , G. Singh 1 , G.R. Verma 1 , V. Gupta 1 , S. Ghosh 1 , R. Gupta 1 , R. Kapoor 1 , N. Sharma 1 , A. Bhalla 1 , S.K. Mahi 1 1 
Background: Endoscopic palliation in malignant hilar biliary obstruction requires ERCP. However, contrast injection leads to cholangitis. Recently, contrast-free metal stenting with or without MRCP has shown encouraging results. However, MRCP and metal stents are costly. There have been no reports on the use of air cholangiography in these patients. Methods: We prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. Results: Ten patients with unresectable malignant hilar biliary obstruction were studied. Air cholangiography detected type II obstruction in 8 and type I in 2 patients which is similar to MRCP. All patients underwent unilateral plastic stenting. A successful endoscopic drainange was achieved in 100% patients. Cholanngitis occurred in none and there was no 30-day mortality. No major complications were observed. Conclusion: Air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. However, it requires a larger study. Introduction: A description of IgG4-related sclerosing cholangitis (IgG4-SC) without pancreatic lesion has recently been reported. In addition to imaging, diagnosis relies on findings of elevated serum IgG4 and immunodetection of invading IgG4-positive cells. Here we report a case of IgG4-SC with only slight common bile duct abnormalities and normal pancreatic findings. Case Study: A 65-year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. His blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an IgG level of 2820 mg/dl (IgG4 374 mg/dl). Although ERCP did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. Echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. Due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. Based on the combination of eosinophilia, elevated serum IgG4 levels, image findings and a negative result for helminth, IgG4-SC was suspected. Liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. The IgG4-positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. Summary: This case displayed two unusual features that are not generally observed with IgG4-SC: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. Introduction: Various systemic diseases have been reported to be associated with IgG4. Although steroids are effective in the treatment of IgG4-related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. We encountered a case of autoimmune pancreatitis with sclerosing cholangitis (AIP-SC), in whom CA19-9 was elevated with episodes of exacerbation and an elevated serum IgG4 concentration. IgG4 staining was also useful for the diagnosis. Case Study: An 81-year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of CA19-9 (304) seven years previously. Her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. Four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of CA19-9 (419) elevation and liver dysfunction. Cholangiocarcinoma was suspected based on ERCP, but was not confirmed by histologic findings of bile duct biopsy. Elevated IgG4 and other test results established the diagnosis of AIP-SC, so steroid therapy was initiated, after which symptoms and laboratory findings improved. This recurrence of CA19-9 elevation (634) was diagnosed as a relapse of AIP-SC based on an increased IgG4 level and histologic findings. Summary: Some papers have reported that IgG4-positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. This might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue.
S. Kaur 1 , T. Kaur 1 1 Department of Biophysics, Panjab University, Chandigarh, India Background: Cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. Aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. Methods: The study was carried out on gallstones, bile and serum of patients. Gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. Blood of the patients was divided into two groups with gallstones and without gallstones patients. Trace elements and various biochemical estimations were carried out. Clinical history of the gallstones patients was recorded from the hospital records. Results: Trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. Iron was the main element in mixed gallstones. In pigmented gallstones magnesium and zinc were the major trace elements. Liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. Clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. Conclusion: Results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. The concentration of products derived from the free radicals reactions increases with degree of inflammation. Such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. Background and Aims: Diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (AIDS).Therefore there is a need to do a research about the risk factors of gallbladder diseases in HIV/AIDS patients. So it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. The aim of this study was to find the prevalence and varieties of gallbladder diseases in HIV/AIDS patients. Methods: A cross sectional study was performed in patients with HIV/AIDS who visited Ciptomangunkusumo Hospital, Jakarta. The risk factors (route of transmision,CD4,ARV,hepatitis) and clinical presentations were studied.Ultrasonography examinations were performed to detect gallbladder annormalities. Results: 68 patients with HIV/AIDS match the study criteria. There were gallbladder abnormalities in 22 (32.4%) subjects, which 19 (27.9%) had acalculous cholecystitis and 3 (4.4%) had cholecystitis with cholelithiasis. On bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of ARV to gallbladder abnormalities (p = 0.000; 0.000; 0.004; 0.012). However, there was no association between age, sex, transmision route of HIV, hepatitis and CD4 to gallbladder abnormalities. Conclusion: HIV/AIDS patients are susceptible to opportunistic gallbladder infection. Acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of HIV/AIDS patients in this study.
Poster Exhibition -HBV Poster Session, Hall 5B
Long-Term Stopping Therapy T.B. Trung 1 , P.H. Phiet 1 1 University Medical Center, Hochiminh City, Vietnam Background: Among the approved nucleos(t)ide analogues therapies for chronic hepatitis B, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. Objectives: the aim of the study was to determine the YMDD variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. Methods: 16 blood samples from lamivudine resistant patients were collected after long-term stopping therapy. The YMDD variants are detected using technique PCR Restriction Fragment Length Polymorphism (PCR-RFLP) at HCMC University Medical Center Results: After stopping lamivudine treatment 25 months (6-72 months) YMDD mutants were detected in 14 (87,5%) of 16 patients. Among them 13 (92.9%) had the most important M204V/I mutant, 1(7 1%) had accompanying L180M mutant. It means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. Many of patients have the features characterized for the patients in immune tolerance phase (young age, HbeAg positive, normal ALT). The treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. Conclusion: The most important M204V/I mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. The options of retreatment for this patients when necessary are limited due to cross-resistance. The management of chronic hepatitis B should be followed strickly the recommendations of specialized association to avoid this problem. Background/Aim: Whether liver stiffness measurement (LSM) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis B (CHB) is uncertain. Methods: We prospectively recruited consecutive patients with severe acute exacerbation of CHB (alanine aminotransferase or ALT >10x upper limit of normal). The relationship of ALT levels and LSM were serially assessed and liver biopsy was performed after ALT normalization. Results: Eleven patients (10 male, median age 43 years) were followed up for 25 weeks; 9 patients received anti-viral therapy. Overall, LSM was positively correlated with ALT levels (r=0.67, P<0.001). At initial presentation, the median serum ALT and LSM was 1136 (581-2210) IU/l and 26.3 (11. 1-33. 3) kPa. A progressive reduction in LSM was observed during subsequent visits in parallel with the reduction of ALT levels. Even after the normalization of ALT at week 12, LSM of 9 patients continued to drop at week 25. At the last visit, the median ALT was 27 (11-52) IU/l and LSM was 7.7 (4.7-10.8) kPa. Among the 5 patients who had liver biopsy performed at week 25, 4 patients had F2 fibrosis (LSM 5.7-8.1 kPa) and 1 patient had F3 fibrosis (LSM 8.6 kPa). Conclusions: LSM using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis B. LSM should be assessed after normalization of ALT levels in order to accurately assess the degree of fibrosis.
H.C. Lai 1 , S.W. Lai 1 , K.F. Liao 1 , C.S. Liu 1 , T. Lin 1 , C.C. Lin 1 1 China Medical University Hospital, Taichung, Taiwan Background: In 2007, chronic liver disease was the seventh leading cause of death in Taiwan. Hepatitis B and hepatitis C are two major causes of chronic liver disease in Taiwan. The purpose was to investigate the seroepidemiology of hepatitis B surface antigen (HBsAg) and hepatitis C virus (HCV) antibody in Taiwan. Method: This was a hospital-based cross-sectional study. We analyzed viral hepatitis data from 2695 subjects who received health checkups at one medical center in Taichung from 2003 to 2004. All subjects were divided into three age groups, including 20-39, 40-64 and 65. This study emphasized the prevalence of HBsAg and HCV antibody by gender and age. The statistical analysis was performed by t test and 2 . Result: There were 1526 men (56.6%) and 1169 women (43.4%). The mean age was 49.2 (standard deviation 12.2, range 20-84). The overall prevalence of HBsAg was 14.7%, with statistically significant difference(SSD) between gender (17.4% for men vs 11.2% for women, p <0.001). The prevalence of HBsAg was decreased with age in men, with SSD (p <0.001), and also decreased in women, without SSD (p =0.08). The overall prevalence of HCV antibody was 5.2%, without SSD between gender (4.8% for men vs 5.7% for women, p =0.272). The prevalence of HCV antibody was increased with age both in men and in women, with SSD (p <0.001). Conclusion: We hope this study can provide the epidemiological data for further studies of hepatitis B and hepatitis C virus infection in Taiwan.
S.M. Wu 1 , X. Zhou 2 1 Wuhan Medical Treatment Center, 2 Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, China E-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. Chronic hepatitis B virus infection is regarded as a chronic inflammatory process. To examine the possible involvement of E-selectin in the etiology of chronic HBV infection, we analyzed two polymorphisms of E-selectin and determined the plasma souble E-selectin levels in patients with chronic HBV infection and controls. The frequency of C allele of the A561C polymorphism was significantly increased in patients with LC campared with controls. No significant positive association was observed between the G98T polymorphism and chronic HBV infection. But in patients with LC, divided according to the Child-Pugh classification, the frequency of T allele was of significant difference between Child'class A and class B plus C. Plasma Levels of soluble E-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. In the liver cirrhosis group, levels of sE-selectin were significantly decreased from Child' class A to class C. In each group, patients with C allele of the A561C polymorphism showed higher soluble E-selectin levels than those with A allele. This is the first report describing the association between E-selectin polymorphisms and HBV-related hepatic fibrosis. Our data showed the A561C polymorphism of E-selectin gene is associated with disease progression in patients with HBV infection and controls the expression of plasma soluble levels, the G98T polymorphism may be related to fibrotic severity in patients with liver cirhosis. Background: Chronic hepatitis B (CHB) patients with high serum HBV-DNA and normal serum alanine aminotransferase (ALT) levels might be considered for treatment if histopathological findings show fibrosis stage 2 or more. However, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. Objective: This study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis B patients with high serum HBV-DNA and normal serum ALT levels. Patients and method: This was an open-label study in CHB patients with high level serum HBV-DNA levels between January 2007 and October 2008. Patients were included if they showed normal serum alanine aminotransferase (ALT) level at two measurements within a 3-month interval and had fibrosis stage > 2 on liver biopsy specimens. Patients were treated with entecavir 0.5 mg/day or telbivudine 600 mg/day. The primary endpoint was the reduction or undetectable of serum HBV-DNA at 24 week and 48 week of treatment, while the secondary endpoint was hepatitis B e antigen (HBeAg) seroconversion. Results: During a 2-year period, 37 CHB patients with high level serum HBV-DNA with normal ALT two times with 3 months interval underwent a liver biopsy. Twenty-eight (75.7%) of 37 pts showed fibrosis stage 2 on histological findings (Metavir score). Twelve of these 28 patients received nucleoside analogues, 7 (58.3%) of them were men. Patients' median age was 42 (range: 24-52) years. There were 5 patients with stage-2, 6 patients with stage-3 and 1 patient with stage-4 fibrosis. Eleven (91.7%) patients had genotype B virus. At baseline, the mean serum ALT level was 32 + 11.8 U/L and mean HBV-DNA level was 2.48 x 10 6 IU/mL, ranging from 1.23 x 10 3 to 2.4 x 10 7 IU/mL. Six patients received entecavir and the other six received telbivudine therapy. Undetectable HBV-DNA was achieved by 9 (75.0%) patients at week-24 and 2 (16.7%) patients at week-48 of treatment. One patient who had the highest HBV-DNA level had viral load reduction to 1.6 x 10 4 IU/mL at week-48 of treatment. Two out of 5 patients with positive HBeAg achieved HBeAg seroconversion at week-48 of treatment. Conclusion: This preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis B patients with high serum HBV-DNA and normal serum aminotransferases levels.
J. Chen 1 , X.J.. Wu 1 , Y. Wang 1 , G.Q. Wang 1 1 Department of Infectious Diseases, Peking University First Hospital, Beijing, China Background: The dysfunction of T cells may represent a mechanism of hepatitis B virus (HBV) persistence. Programmed death-1 (PD-1) and its ligands, PD-L1/PD-L2, are new members of CD28/B7 family, as co-stimulatory molecules expressing on T cells and Antigen Present Cells (APCs). Their engaging can downregulate the T cells function, including proliferation, cytokines secretion and cytotoxicity. In periphery blood, PD-1 was upreguated on virus specific-T cells, leading to the impairment of T cells. Blocking the PD-1/PD-L can improve the function of T cells. Methods and Patients: 21 patients with chronic hepatitis B (CHB) were treated by pegylated IFN -2b (PegIntron from Schering-Plough, once a week, 0.5 or 1 g/kg/weight). The periphery blood were taken at 0 weeks, 4 weeks, 8 weeks, and 12 weeks. Periphery blood mononuclear cells (PBMC) were isolated from fresh heparinized blood by Ficoll-Hypaque (density: 1.077g/L) density gradient centrifugation. Then the cells were incubated with APC-conjugated anti-PD-1 antibodies. The PD-1 expression on lymphocytes was detected by flow cytometry (FCM). Results: The PD-1 expression on lymphocytes at 0 weeks was 14.47±5.8%, at 4 weeks was 9.68±3.75%, at 8 weeks was 6.95±2.39%, at 12 weeks was 6.08±1.31% (p<0.05). Conclusion: Treatment with IFN -2b can downregulate the PD-1 expression on lymphocytes and may partially restore the function of T cells. To investigate the effects of nucleoside analogs therapy in hepatitis B related acute-on-chronic liver failure, we treated 55 HBV related acute-on-chronic liver failure patients with entecavir. as control, the remaining 74 were not treated with nucleoside analogues. Results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (P>0.05). Although entecavir greatly reduced HBV replication during different therapy times (P<0.001), the MELD score and liver function (ALT, albumin, bilirubin, prothrombin time) had no significant changes (P>0.05). Further more, we analyzed the MELD score and liver function in different HBV-DNA level patients .No significantly difference was observed (P>0.05). There is no significant correlation between HBV-DNA level and MELD score in different therapy times (P>0.05).The HBV-DNA level between patients with over 3 months and less than 3 months survival patients showed no significant difference either (P>0.05). However, MELD score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (P<0.05). These results suggest HBV-DNA loading may not be a direct factor to increased liver injury and suppression of HBV replication may not reduce the severity of liver failure in HBV related acute-on-chronic hepatitis.
S. Firdoos 1 , U. Adeeb 1 , A. Mehmood 1 , M. Gill 1 1 Islamabad Specialists Clinic, Islamabd, Pakistan Background: Before the availability of ETV, it was common to use ADV for treatment of chronic hepatitis B patients. Primary nonresponse and suboptimal response is a common problem with ADV treatment.
METHODS: We wanted to study the outcomes of Entacavir therapy in this subset of patients. Study was conducted between April 2007 to April 2008. We enrolled 30 CHB patients who had Non response to 12-24 weeks of 10 mg ADV therapy. Non response and suboptimal response was defined as non dimunition of at least one log of HBVDNA from baseline after 12 weeks of therapy and Persistence of 3 log10 after 24 weeks of therapy respectively.They were switched to 1mg entacavir before breakfast daily for at least 12 months.They had serial ALT CBC and HBVDNA measured every 12 weeks. Results: Out of 30 patients 20 male and 10 were female. Only 8 patients were HBeAg(+).Mean HBVDNA level prior to ADV exposure was 6.5 log copies/ml.Mean duration of exposure to ADV was 26 weeks.5 patients lost to F/U.We did intention to treat analysis. 15 out 30 (50%) patient has, undetectable level of HBVDNA after 12 weeks of therapy labelled as group 1.6 out of 30 (20%) had HBVDNA level reduced by a mean of 3 log 10 copies/ml labelled as group 2.on week 24 treatment analysis all 15 patients from group 1 was HBVDNA undetectable, 2 additional patients from group 2 had undetectable HBVDNA. Conclusion: Entacavir therapy results in rapid suppression of HBVDNA levels in majority of patients with primary nonresponse or partial non response to ADV therapy. Background: Except for serum ALT level, baseline factors predictive of therapeutic response to lamivudine in patients with HBeAg-positive chronic hepatitis B remain largely unknown. We thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. Methods: A total of 116 treatment-naïve HBeAg carriers who had pre-therapy serum ALT level> 5xULN and received lamivudine for 18 months reimbursed by the National Health Insurance were prospectively enrolled. HBeAg seroclearance and combined HBeAg seroclearance, ALT normalization as well as undetectable HBV DNA at the end of therapy were defined as primary and secondary endpoint, respectively. The pre-therapy viral factors including viral load, genotype, precore stop codon (PC)/ basal core promoter (BCP) status, and pre-S deletion were determined to correlate with therapeutic endpoints. Results: The frequency of patients with detectable PC mutation (G1896A), BCP mutation (A1762T/G1764A), and pre-S deletion at baseline was 22.4%, 21.6%, and 12.1%, respectively. After completing 18-month lamivudine therapy, overall HBeAg seroclearance rate was 56.0%. Patients with HBeAg seroclearance had a higher prevalence of baseline PC mutation than those without (30.8% vs, 11.8%, P= .015). By multivariate analysis, the odds ratio of patients with PC mutation to develop HBeAg seroclearance was 3.33 (P= .024). In addition, the presence of PC mutation also correlated with the combined response. Conclusions: For HBeAg-positive chronic hepatitis B patients with serum ALT> 5xULN, PC mutation could predict a higher HBeAg seroclearance rate at the end of 18-month lamivudine therapy.
The Efficacy of Adefovir Dipivoxil against All Patterns of Lamivudine Resistant Hepatitis B D.J. Kim 1 , Y.D. Park 1 , Y.G. Kwon 2 , H.G Seo 1 1 Daegu Fatima Hospital, 2 Kunngpook National University Hospital, Daegu, Korea Background: Our aim was to evaluate the efficacy of adefovir dipivoxil (ADV) and determine patient-dependent or laboratoroy variables that are predictive of HBeAg loss and IVR for hepatitis B patients resistant to lamiduvine. Also we evaluated the activity of ADV against all patterns of lamivudine-resistant HBV. Method: 179 HBV-infected patients with lamivudine resitance received ADV for 6 months. Quantitative HBV DNA, HBeAg/anti HBeAg, ALT was checked every 3-6months. The HBV polymerase of 161 patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. Result: There is no significant difference in all patterens of HBV mutation about HBV DNA reduction at 24W, 48W, 72W. There is no significant difference in all patterens of HBV mutation about ALT normalization at 24W, 48W, 72W. Conclusion: Adefovir dipivoxil demonstrated similar potent anti-HBV efficacy regardless of the different patterns of lamivudine-resistant HBV mutations.
G. Novelli 1 , M. Rossi 1 , V. Morabito 1 , F. Pugliese 1 , P. Berloco 1 1 La Sapienza University, Rome, Italy Background: Hepatitis B (HBV)-related end-stage liver disease is one of the most common indication for liver transplantation (LT). A number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. We valued the possibility to improve the Model End-stage Liver Disease (MELD) of patients awaiting liver transplantation using a albumin dialysis: Molecular Adsorbent Recirculating System (MARS). Methods: We treated 34 patients (19 male and 15 female) with a mean age 49.5. Inclusion criteria: serum bilirubine > 15mg/dl, MELD 25, INR > 2.1, Encephalopathy Grade II. All patients were treated with MARS mean 9±2.5 hr cycles and mean 9 treatments (range 3-15). All patients received standard medical treatment in addition to MARS dialysis. The patient survival was valued at six months. Results: We obtained a significant change of cytokines levels as Interlukine 6 (p< 0.02) and Tumor Necrosis Factor alfa (p<0.01) in association with an improvement of kidney, hepatic and hemodynamic parameters. At the end of MARS treatments we observed a significant reduction of MELD score (p<0.003). The results of MELD show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p<0.005). Twenty patients lived and 14 dead for clinical complications. Conclusion: The improved MELD score with MARS gave patients on LT waiting list more time of survival, thus allowing them more opportunity for liver transplantation.
Entecavir for Treatment of Lamivudine-refractory Patients Chronic Hepatitis B H.T. DAT 1 , P.T.T. THUY 1 1 MEDIC Medical Centre, Ho Chi Minh, Vietnam Lamivudine treatment is associated with frequent development of resistant hepatitis B virus. This incidence especially is higher in longer time of treatment and loss of treatment benefit. Entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with Lamivudine resistance. In this study, we evaluate the efficacy, the safety of Entercavir in treatment of Lamivudine-refractory patients chronic hepatitis B. Sixty chronic hepatitis B patients with evidence of Lamivudine resistance were randomly divided into two groups in proportion of 3:1. Group I (n=45) used Entecavir 1mg/day, group II (n=15) used Lamivudine 100mg/day. Treatment time was 48 weeks. Histology, ALT, HBVDNA were evaluated in the end of the treatment. Age, sex, ALT, HBVDNA, genotype, HBeAg were analyzed to evaluate their influences to the treatment. The results have showed HBVDNA<2000 copies/mL in Entecavir group 37.78% vs. 0% Lamivudine group (p<0.01). HBVDNA negative in Entecavir group was 17.77% and incidence of seroconversion of HBeAg was 8.82%. ALT was normal in Entecavir group 77.77% vs. 26.66% in Lamivudine group (p<0.001).Histologic improvement in Entecavir group was 37.77% vs.6.66% in Lamivudine group (p<0.05). Patients with HBeAg negative, genotype B, low viral load were shown better results. Entecavir was shown to be efficacious in treatment for chronic hepatitis B patients experienced with Lamivudine resistance. Entercavir is safe, with almost no side effects. Factors such as HBeAg negative, genotype B, low viral load seems to be better in response to treatment. Recurrence or mutation of Entecavir resistance should be studied further in future.
J.M. Kim 1 , S.K. Hwang 1 , B.H. Choe 1 1 Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea Backgrounds: By analyzing the characteristics of children with chronic hepatitis B who have lost HBsAg by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis B in children. Methods: A total of 75 HBeAg positive children (< 18 y-o) were recruited who have visited Kyungpook National University Hospital from Mar. 30, 1999 to May 8, 2008 . They were treated with lamivudine for at least 6 months. HBeAg seroconversion occurred during lamivudine treatment in 49 out of 75 children. They were divided into HBsAg clearance and non-clearance group. Parameters influencing treatment results were analyzed according to HBsAg loss. Result: Thirteen out of the 49 (26.5%) patients with HBeAg seroconversion were classified as HBsAg clearance group, while 36 (73.5%) as non-clearance group after lamivudine treatment. Twenty five of 49 patients with HBeAg seroconversion were under 6 years old, in 10 (10/25, 40%) of whom HBsAg loss occurred as well. Twenty four of 49 patients were over 6 years old, in 3 (3/24, 12.5%) HBsAg loss occurred, that showed significantly difference (p-value= 0.029, OR: 4.667, CI: 1.094-19.902) compared to younger group. Age was significantly lower in HBsAg clearance group (5.1±4.3 years) than non-clearance group (8.2±5.0 years) (p=0.043), but no difference was observed in other parameters. Anti-HBs appeared in 12 patients. Conclusion: In the treatment of HBeAg positive chronic hepatitis B with lamivudine, age was significantly lower in HBsAg clearance group than non-clearance group. Background: Dysfunction of T cells may represent a mechanism of hepatitis B virus (HBV) persistence. Programmed death-1 (PD-1) and its ligands, PD-L1/PD-L2, are members of CD28/B7 family, was reported to transfer inhibitory signal, leading to the dysfunction of T cell.
Background: Hepatitis B viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. Mutations of HBsAg allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels.
Methods: Immunohistochemical analysis of tissue samples from 56 patients with chronic hepatitis B (CHB), 12 acute hepatitis B (AHB) patients and 10 health controls was performed. Results: PD-1 was positively expressed on lymphocytes infiltrating the portal area.PD-L1 expression was the same as PD-1,also expressed in interlobular.PD-L2 expressed on kupffer cells and dendritic cells.PD-1-,PD-L1-,and PD-L2-positive cells express index of CHB patients were much more than that of health controls and AHB patients(p 0.05).Between groups in CHB,the expression rate increase with the disease progression (p 0.05).
Methods: 58 chronic hepatitis B patients with both positive for HBsAg and HBsAb were studied.Serological markers of HBV were detected by ELISA and microparticle enzyme immunoassay. HBV DNA levels were determined by fluorescent quantitative PCR, S gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer AU400. Correlation test was conducted to evaluate their dependablity. Conclusion: Overexpression of PD-1 and PD-L within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in HBV infection.
Results: The level of HBsAg and HBsAb was 254.4±68.3 S/N and 39.4±38.1 mIU, respectively. HBV DNA was detectable in 46 patients. Fifty-one mutations of S gene were detected in 38 patients, and the relating amino acid substitution was at the sites of 36, 39, 47, 63, 77, 89, 90, 115, 126, 129, 139 and 154. Eight (15.7%) out of 51 mutations were located at the "a" determinant region in 14 patients, while no mutation was found at the sites of 124, 137 and 147. However, the mutation did not affect HBV replication. HBV DNA was positive correlated with HBeAg. Conclusions: Change in HBsAg antigenicity due to S gene resulted in concurrent HBsAg and HBsAb. The existence of HBsAb did not affect HBV replication. The damage of liver failure in those patients was slight. Background: HBV infection is common in Bangladesh. We often encounter young patients incidentally detected with HBeAg negative chronic hepatitis B (CHB) in our clinical practice. However the characteristics of these patients is yet to be studied in this country. The aim of this study was to study the characteristics of young Bangladeshis incidentally detected with HBeAg negative CHB. Methods: We did percutaneous liver biopsies of 36 CHB patients aged between 8 to 20 years. They were all HBeAg negative with persistently normal or raised serum ALT values. We did pre-core mutation (PCM) study in 4 patients who were randomly selected. Results: 56% patients had significant necro-inflammation (HAI-NI >3), while significant fibrosis (HAI-F >2) was seen in 17.6%. Serum ALT (cut off 42 U/L) was raised in 38.2%, while high HBV DNA load (>10 5 copies/ml) was observed only in 26.5%. PCM was negative in all 4. Conclusion: Although CHB patients between 10-20 years of age are supposed to be in immune clearance phase, which is characterized by low HBV DNA and HBeAg positivity, the study shows that HBeAg negative CHB is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. This challenges our current concept about immune clearance state of HBV infection, although much larger study is needed to draw any specific conclusion. Background: HBV infection is common in Bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis B is yet to be studied in this country. Methods: We did percutaneous liver biopsies of 88 CHB patients aged between 8 to 20 years. Results: Significant necro-inflammation (HAI-NI >3) was seen in 79.6% patients with HBeAg positive and 56% patients with HBeAg negative CHB, while significant fibrosis (HAI-F >2) was seen in 20.3% and 17.6% patients in these two groups respectively. Serum ALT (cut off 42 U/L) was raised in 37% HBeAg positive and 38.2% HBeAg negative patients, while in these two groups 87% and 26.5% patients respectively had high HBV DNA load (>10 5 copies/ml). Conclusion: HBeAg negative CHB is an entity that can also be seen in young population. A significant percentage of both HBeAg positive and negative patients may have considerable hepatic involvement.
Profile of HBeAg +ve Chronic HBV Infection in Bangladesh M. Mahtab 1 , S. Rahman 1 , F. Akbar 2 , F. Karim 1 , A. Shrestha 1 , M. Khan 1 , M. Kamal 1 1 Bangabandhu Sheikh Mujib Medical University, 2 Toshiba General Hospital, Dhaka, Bangladesh Background: Inactive HBV carriers constitute the major reservoir of HBV. Present management guidelines provide inadequate treatment modalities. They are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. This is due to lack of information about their extent of liver damage. Aim of this study was to assess extent of liver damage in HBeAg +ve patients, unaware of their infection. Methods: In this retrospective study, records of 206 HBeAg +ve CHB patients from our pool of 561 CHB patients were reviewed. They were tested for HBsAg, HBeAg, HBV DNA, anti-HCV and serum ALT. All underwent per-cutaneous liver biopsy. Results: 78.2% (161/206) patients were males and 21.8% (45/206) females. They were between 8-45 years of age. ALT was raised >2times UNL in 17% (35/206). 92. 2% (190/206) patients had high HBV DNA (>10 5 copies/ml), while low HBV DNA (<10 5 copies/ml) was seen in 7. 8% (16/206) . In high HBV DNA group, significant necro-inflemmation (HAI-NI >7) was seen in 48. 9% (93/190) and significant fibrosis (HAI-NI >3) in 24. 7% (47/190) . Figures were 37.5% (6/16) and 31.3% (5/16) respectively in low viral load group. None tested positive for HCV infection.
Conclusion: Study indicates that machinery should be developed to characterize undetected HBV carriers in developing countries by conducting multi-center clinical studies. We have shown that considerable number of patients, unaware of their HBV infection, suffer from progressive liver damage. The overall strategy of management of chronic HBV infection should also be revisited.
High Viral Load Does Not Necessarily Represent Significant Liver Damage in Patients with Chronic HBV Infection in Bangladesh M. Mahtab 1 , S. Rahman 1 , F. Akbar 2 , F. Karim 1 , A. Shrestha 1 , M. Khan 1 , M. Kamal 1 1 Bangabandhu Sheikh Mujib Medical University, 2 Toshiba General Hospital, Dhaka, Bangladesh Background: In general, it is assumed that patients with chronic hepatitis B virus (HBV) infection with high viral load exhibit increased liver damages. Treatment guidelines also emphasize on reducing viral load. These observations were mainly accumulated from developed countries. >80% chronic HBV carriers live in the developing nations, but little is known about relationship between HBV viral load and extent of liver damage in these countries. In this study, we addressed this issue. Methods: In this retrospective study we reviewed records of 306 CHB patients from our pool of 561 patients. All had high HBV DNA (>10 5 copies/ml). 62. 1% (190/306) were HBeAg +ve and 37.9% (116/306) HBeAg -ve. They were alsotested for anti-HCV and serum ALT. All underwent per-cutaneous liver biopsy. Results: 51.1% (97/190 ) HBeAg +ve patients with high HBV DNA had non-significant hepatic necro-inflammation (HAI-NI <7); this figure was 53.4% (62/116) in HBeAg -ve patients. Non-significant hepatic fibrosis (HAI-F <3) was observed in 75. 2% (143/190) and 69.8% (81/116) in HBeAg +ve and -ve patients respectively. None tested positive for HCV. Conclusion: Correlation doew not exist between viral load and liver damage in CHB in Bangladesh. Many with both HBeAg +ve and -ve CHB with high HBV DNA do not have significant hepatic necro-inflammation and fibrosis. Further study may be needed to find out influence of other factors on liver damages in CHB in Bangladesh. Most of these patients have not been characterized and treatment modalities have not been defined for them. Background/Aims: Expression of intrahepatic hepatitis B core antigen (HBcAg) is related to the immunopathogenesis of hepatitis B virus (HBV) infection. The role of HBV genotype and basal core promoter (BCP) mutation in expression of HBcAg was investigated. Methods: Seventy HBeAg-positive chronic hepatitis patients (genotype B in 52 and C in 18; BCP T1762/A1764 mutation in 16) were enrolled. Clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic HBcAg. The effects of HBV genotype and BCP T1762/A1764 mutation on the expression of HBcAg were further evaluated by in vitro assays. Results: Cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic HBcAg were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. Fifty-eight (80.6%) of these patients expressed a high level of HBcAg. In multivariate analysis, cytoplasmic localization of HBcAg correlated only with low serum viral load (P=0.045) and BCP mutation (P=0.04). High expression level of HBcAg also correlated with high serum viral load (P=0.015) and BCP wild-type sequence (P=0.037). In vitro assays supported that HBV BCP mutant had lower subcellular expression of HBcAg compared with BCP wild-type strain. Conclusions: HBV BCP mutation and viral load but not genotype contributes to the expression of intrahepatic HBcAg.
Hepatitis B virus (HBV) genotypes show distinct geographical distributions and virological and clinical differences. In some of genotypes, specific substitutions and mutations have been described in association with hepatitis B e (HBe) protein expression and viral replication. In this study, genetic characteristics of HBV genotype E (HBV/E) were investigated using clinical samples obtained from 12 Hepatitis B e antigen (HBeAg)-positive, and 11 anti-HBe-positive asymptomatic carriers (ASCs) in West-Africa. Full-genome analysis of isolated HBV strains revealed strong association between precore (PC) mutation and HBeAg to anti-HBe seroconversion.
Furthermore, using 53 partial genome sequences, correlation among HBeAg/anti-HBe status, viral load and key mutations were analyzed. The data showed that PC mutation is associated with HBeAg seroconversion and enhanced viral replication efficiency. Comparison between HBV/E and HBV/D strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (CURS/BCP) regions. It has been known from the previous phylogenetic studies, that HBV/D and HBV/E cluster together in trees reconstructed on X and preCore/Core ORFs. In addition, this study, demonstrates that in spite of the high sequence similarity of CURS/BCP region, the seroconversion-related mutation patterns are different between HBV/E and HBV/D in ASC. Further studies are needed to clarify the clinical significance of the regulatory sequence similarity between HBV/E and HBV/D.
Necro-Inflammation and Fibrosis P. Siddappa 1 , P. Kar 1 , B. Das 2 , R. Gondal 1 , M. Asim 1 1 Maulana Azad Medical College, 2 ICPO, New Delhi, India Background: Chronic hepatitis B(CHB) is an important cause of morbidity and mortality. Methods: Pilot study involving 30 patients of CHB, were equally randomized to receive either Adefovir or Lamivudine for 6 months. Quantification of serum and hepatic HBV DNA levels by Real time PCR and liver biopsy done at start and end of 6 months. Results: After 6 months there was significant and comparable reduction in Serum and hepatic HBV DNA viral load and liver biopsy showed significant reductions in HAI scores in both the groups. Serum ALT which was elevated to 2 or more times normalized in both the groups. In the Adefovir group 2 patients became HBeAg negative and 2 patients who were HbeAg negative at the start of therapy remained so. In the Lamivudine group one patient became HBeAc negative and 2 patients who were negative at the start of therapy remained so. In the Adefovir group 4 patients became HBV DNA (qualitative test) and in the Lamivudine group 2 patients became HBV DNA negative. There was strong correlation between serum and hepatic HBV DNA levels both before and after the completion of therapy. Conclusion: Both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after 6 months without complete clearance of virus. There was not enough evidence to show therapeutic advantage of one drug over the other. The serum and hepatic HBV DNA levels correlate well with eachother before and after treatment. Aim: Assessing efficacy and safety of treatment of chronic hepatitis B in children with pegylated IFN. Materials and Methods: 13 children (9 boys and 4 girls) aged 11-17 years with CHB treated with PEG-IFN alfa-2a, 100 g/m 2 /week during 48 weeks, 5 HBeAg-positive and 8 HBeAg-negative children, 4 previously treated with recombinant interferon. No child had liver disease greater than grade 2, stage 2. Serum HBV DNA was quantified at baseline, TW 4, ("RVR") TW 24, TW 48 (ETR) and W 72 (SVR) with RT PCR method (Roche TaqMan). ALT activity, haematology and adverse events were monitored. Results: After 4 weeks treatment median HBV DNA level decreased from 7.8x10 3 IU/mL at baseline to 1.43x10 2 IU/mL (p<0.01). "RVR" -undetectable HBV DNA at TW4 was observed in 6/13 children and associated with lower pretreatment ALT levels <25 IU and pretreatment viral load <750 IU/mL. All children with "RVR" were HBeAg-negative pretreatment. At TW 24 and TW 48 seven children including all with "RVR" had undetectable HBV DNA. 5 children achieved SVR (undetectable serum HBV DNA in W 72), among them 3 with "RVR". In 2/6 children with "RVR" HBsAg disappearance was observed since TW 48. Leukopenia was reported in 7 children, thrombocytopenia in 3. No adverse events were observed following dose modifications. Conclusions: 1. PEG-IFNalfa-2a is a good therapeutic option for children with CHB, in particular with HBeAg-negative CHB 2. Low pretreatment viral load and "RVR" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (PSP) and HBsAg among appliances and practitioners worked in those places. Methods: 63 beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, 682 workers were investigated in questionnaire. The HBsAg in appliances of PSP and employee was detected by RIA. Results: The rate of HBsAg among appliances of PSP was 2.13%. The rate of HBsAg in large-, medium-and small-sized appliances was 0.63%, 2.67% and 3.70%. The rate of HBsAg has different( 2=6.68 P 0.05). The rate of HBsAg among appliances of beauty parlors, barbering shops and footbath inns was 2.97%, 0.61% and 3.42%. Different appliances had different rate of HBsAg, such as the rate of acne needle and the forceps was 5.13% and 4.17%. The positive of HBsAg amongworkers in PSP was 7.13%. The rate of HBsAg among workers in large-, medium-and small-sized PSP was 7.34%, 8.33% and 2.94%. The rate of HBsAg among workers in beauty parlors, barbering shops, footbath inns and bathing centers was 9.01%, 6.37%, 4.35% and 7.29%. The HBsAg rate among workers was different in different works, the rate was higher in tattoo workers (13.33%), pedicures workers (12.68%), Massagists (8.03%). Conclusions: It is important to enhance the sanitizing management in PSP and improve workers KAP) of HepB. And we should promote health education to enhance the knowledge of Hepatitis B control and build up supervision consciousness. Background: Integration of hepatitis B virus (HBV) DNA into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of HBV-related carcinogenesis. However, the molecular mechanism of integration remains unclear. Here we describe a potential mechanism of HBV integration and identify that Ku70 and Ku80, the gatekeepers of non-homologous end-joining (NHEJ) repair pathway, can serve as targets for anti-hepatitis virus integration. Methods: Using I-Sce endonuclease-based system, we induced a DNA double-strand break (DSB) in human hepatoma cell line HuH-7. The cells were then incubated with serum from patients with chronic HBV infection. PCR amplification and direct sequencing were used to detect the inserted sequence in the site of DSB. Finally, we employed TaqMan-based real-time PCR assay to quantify the integrated HBV DNA and evaluate the effects of shRNA on HBV integration. Result: When HuH-7 were exposed to viral serum and incubated for several days, HBV DNA was detected in integrated form at the exact site of DNA damage. Furthermore, small interference RNA (siRNA) targeted against gatekeeper genes for NHEJ can down-regulate NHEJ repair and even the frequency of HBV integration. Conclusion: Thus, this project provided us with the first direct evidence that DNA double-strand breaks are potential targets for HBV integration. The study has also shown that shRNAs targeted against gatekeeper genes for NHEJ can regulate the frequency of HBV integration. Objective: To screen proteins of human pancreas cDNA library interacting with HBsAg protein. Methods: The library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain Y187. The reconstructed plasmid pGBKT7-HBsAg was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp. The transformed AH109 mated with Y187 containing the library plasmid. The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X--gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5 . The plasmids in DH5 were extracted, sequenced and analyzed by bioinformatic methods. Results: Sixteen proteins interacting with HBsAg were founded. Conclusions: These results show that HBsAg protein may be related with metabolism of glucose and lipid.
Comparison of the Sensitivity and Specificity of the Elecsys ® HBsAg II Assay with other Available Assays in China for Detection of HBsAg J.D. Jia 1 , L. Wei 2 , X.X. Zhang 3 , Y.L. Mao 4 , L.L. Wang 5 , Z.L. Gao 6 , J.L. Hou 7 , J. Zhang 8 , W. Melchior 9 , W. Van der Helm 9, 10 1 Beijing Friendship Hospital, Beijing, China, 2 Beijing People Hospital, Beijing, China, 3 Ruijin Hospital, Shanghai, China, 4 Beijing 302 Hospital, Beijing, China, 5 West China Hospital, Chengdu, China, Guangzhou, China, 7 Guangzhou Nanfang Hospital, Guangzhou, China, 8 Shanghai Public Health Clinical Centre, China, 9 Roche Diagnostics Ltd, Rotkreuz, Switzerland, 10 Conclusions: In this patient population the prevalence of HBsAg positive and anti-HCV were much higher than reported in community studies. Genotypes 1 and 6 accounted for most of HCV. These very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention.
Hepatitis B is one of the major diseases of mankind that kills about one million persons each year in the world. Accoring to primary study about 3% of Iranian population is chronic HBV carriers. Among Iranian cirrhotics, 70-84% has evidence of exposure to HBV and 51-56% is carriers. Because increase demand of blood transfusion, high blood dependent patients and long term window period of HBV infection, any controlling HBV infection program in blood donors can enhance the blood safety and public health.
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In this descriptive study included all the blood donors that referred to Dezful blood transfusion center during 2005-2008. All the blood donors screened for HBs Ag by using Enzyme immuno assay and repeatedly reactive (R.R) samples confirmed by HBC-Ab or confirmatory (neutralization) tests. The data analyzed by using SPSS 11.5.
We found that in the first year 1.052 % were repeatedly reactive and 1.045 % confirmed. The results for other years as the followed: 0.782 %(R.R) and 0.770 % confirmed and in the last 0.725 % (R.R) and 0.700 % confirmed.
The repeated blood donors increase in this period (42.11 %, 50.15 % and 51.68 % respectively).
Aim: We aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. Methods: Decision analysis model was used to study the incremental cost-effectiveness ratios (ICER), i.e. the additional cost (in USD) required to achieve undetectable HBV DNA or HBeAg seroconversion for a patient at 2 years in America and Hong Kong. Entecavir was used as a continuous monotherapy. Lamivudine and telbivudine would be shifted to entecavir if HBV DNA was detectable at month 6 and continued otherwise with drug resistance treated by add-on adefovir. Weighted event rates based on previous reports were estimated for analysis.
According our study, although the prevalence was higher than other region in our province, the HBV prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . Good following up those strategies to enhance the blood safety recommended.
Results: Telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable HBV DNA from lamivudine at 2 years. Entecavir was least effective and most costly for HBeAg seroconversion.
Conclusions: Telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in Hong Kong. H. Tang 1 , G.L. Zhang 1 , Y.X. Li 1 , R.Q. Tian 1 , M. Liu 1 , X. Li 1 1 Tianjin Life Science Research Center, Tianjin Medical University, Tianjin 300070, China MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 18 to 25 nucleotides that play critical roles in a wide spectrum of biological processes. We investigated whether the miRNAs-silencing machinery influences HBV replication or antigen expression. On the basis of ELISA and MTT, the effect of 328 miRNAs on the HBsAg expression and cell proliferation was examined. Three microRNAs efficiently inhibited HBsAg expression without significant effect on the proliferation of HepG2 2.2.15 cells compared to LacZ control. Subsequently, bioinformatics analysis were used to predict targets for the three miRNAs, and the prediction results were conformed by cDNA microarray analysis. The target region in HBV genome and the 3'UTR region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative RT-PCR and western blot. The results demontrated that miRNA may play an important role in replication and gene expression of HBV.
Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made, the pathogenesis of HBV infection is still elusive. There's an urgent need to elucidate the mechanisms of HBV-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-HBV treatment. Herein, we applied a two-dimensional gel electrophoresis and MALDI-TOF/MS based comparative proteomics approach to globally analyze the host response to HBV by using an inducible HBV-producing cell line HepAD38. Of the 23 differentially expressed proteins identified, glucose regulated protein 78 (GRP78) was one of the most striking proteins elevated by HBV replication, which was confirmed by real-time PCR and western blotting. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in HBV-transfected HepG2 cells. Reversely, GRP78 overexpression led to HBV suppression. The expression levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were determined by enzyme linked immunosorbent assay (ELISA). Immunofluoresce further revealed a positive correlation between the expression levels of GRP78 and HBsAg in both HBV-transfected HepG2 cells and HBV-infected human liver tissues. Altogether, these data demonstrate for the first time that GRP78 is an endogenous anti-viral factor in HBV-transfected HepG2 cells and may serve as a potential prognostic indicator of viral status in anti-HBV therapies. Background/Aims: To evaluate the predictors of response to long-term treatment of adefovir dipivoxil (ADV) in patients with emerging lamivudine (LAM)-resistant hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. Methods: One-hundred-thirty-four LAM-resistant HBeAg-positive CHB patients were treated with ADV for a median of 28.0 months (range, 18 -54 months), following LAM therapy for a median of 25.5 months (range, 5 -66 months). 65 patients (48.5%) were switched from LAM to ADV monotherapy, 43 (32.1%) were switched to ADV with 1 month of LAM overlap therapy, and 26 (19.4%) were switched to ADV with 3 months of LAM overlap therapy. The influence of baseline parameters on treatment response to ADV in patients with LAM-resistant HBeAg positive CHB was analyzed. Result: During the follow-up period, 28 (20.9%) of 134 patients achieved complete response, defined as normalization of ALT level, negative HBV DNA by a Digene hybrid capture assay and achievement of HBeAg loss. Sixteen (11.9%) patients achieved HBeAg seroconversion. Twenty-eight (20.9%) patients developed ADV-related mutations during ADV treatment. In multivariate analysis, virological response at 3 months (OR=9.70, 95% CI: 32.63-35.78, p=0.001), defined as serum HBV DNA levels less than 5 log10 copies/mL or a reduction in serum HBV DNA levels greater than 2 log10 copies/mL after 3 months of ADV therapy, independently predicted complete response. Conclusions: Virological response at 3 months was the strongest predictor of ADV response in LAM-resistant HBeAg-positive CHB patients. Background/Aims: To explore the effects of HBV DNA level HBV genotype/subgenotype on the pathogenesis of severe liver diseases in Chongqing. Methods HBV DNA level was analyzed in patients with severe liver diseases in retrospect,and HBV genotype/subgenotype HBV DNA level and HBeAg were determined in patients with hepatocellular carcinoma (HCC,n=78 ), Liver cirrhosis(LC, n=58),chronic hepatitis B(CHB, n=20) and acute on chronic liver failure(ACLF, n=42). Results HBV level from high to low with CHB were LC, ACLF and HCC in turn(P 0.05). HBV genotype was mainly genotype B.The rate of genotype B and C were 51. 3% and 47.4 respectively in HCC patients, 60.3% and 39.7 in LC patients, 55% and 40 in CHB patients, 55% and 40 in ACLF patients. The percentage of genotype B/C in ACLF patients was higher in compared with other groups. But the distribution of HBV genotype among groups was not statistically different(P 0.05).Subgenotypes of genotype B were almost Ba but one. Subgenotypes of genotype C were mainly Ce in Chongqing area, and there was no statistical difference among the 4 groups (P 0.05). Conclusion: HBV DNA level seems not to be a determining factor at end point of severe liver disease. Both genotype B and C of HBV can lead to severe liver diseases, and there are more mixed infections by different genotypes in ACLF.
The Efficacy of Switching to Entecavir (ETV) Monotherapy in Japanese Lamivudine (LVD)-Experienced Patients. Background: This study aims to determine the efficacy of switching to 0.5mg ETV daily in chronic hepatitis B (CHB) patients previously treated with LVD. Method: Retrospective analysis of CHB patients (n=134) previously on 100mg LVD daily and switched to 0.5mg ETV daily. Results: LVD-experienced patients were divided into three groups based on HBV viral load at time of switching to ETV (<2.6 log10 copies/mL; 2.6-5.0 log10 copies/mL and >5.0 log10 copies/mL). Detection of LVD-resistant virus at the time of switching was higher in the group with HBV DNA 2.6 log10 copies/mL (76% in both 2.6-5.0 and >5.0 log10 copies/mL groups versus 23% in <2.6log10 copies/mL group) and was higher in patients treated with LVD for 3 years (52% versus 24% for patients on <1 year of LVD). A year after switching to 0.5mg ETV daily, HBV DNA undetectable rates were 100% (42/42), 94% (17/18) and 43% (6/14) for <2.6, 2.6-5.0 and >5.0 log10 copies/mL groups, respectively. ALT normalization occurred in more than 90% patients at the end of the first year of switching to ETV for all three patient groups. Only one patient in the 2.6-5.0 log10 copies/mL group, who had LVD-resistant mutants at the time of switching, developed ETV resistance during follow-up. Conclusion: Switching from LVD to ETV maintains or improves viral suppression and ALT normalization, especially in patients with viral load <5.0 log10 copies/mL.
Background/aims: We investigated the association between on-treatment HBsAg decline and sustained response in patients treated with PEGASYS±lamivudine. Methods: HBsAg levels were measured retrospectively pre-treatment and at weeks 12, 24, 48 and 72 using the Abbott Architect HBsAg assay in sera from 542 patients (83% Asian) treated with PEGASYS alone (180 g qw; n=271) or combined with lamivudine (100mg qd; n=271) alone for 48 weeks as part of a large multinational trial. Response was measured 6 months post treatment. Results: More patients treated with combination therapy had >1 log decline in HBsAg from baseline to week 48 ( Figure) . HBsAg level <1500 IU/mL at week 12 was associated with higher rates of response to PEGASYS±lamivudine 6 months post treatment ( Figure) . Data comparing HBsAg and HBV DNA as on-treatment predictors of response will be presented. Conclusion: On-treatment HBsAg monitoring may be useful for predicting response in patients treated with PEGASYS.
Y. Wakui 1 , J. Inoue 1 , Y. Ueno 1 , T. Shimosegawa 1 1 Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan Background/aim: Chronic hepatitis B patients are clinically treated with nucleot(s)ide analogues and IFN-. Nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and IFN-has disadvantages of limited effectiveness and side effects. Therefore, novel antiviral drugs are still needed. In this study, the suppressive effect to the replication of HBV was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (PPAR) and , respectively. Methods: The cytotoxicity of bezafibrate and rosiglitazone to HepG2 cells was examined with MTS assay, and the concentration of 50% cytotoxicity (CC50) was calculated. HepG2 cells were transiently transfected with the plasmid containing 1.3-fold HBV genome of a genotype B strain. After 24 hours of transfection, rosiglitazone and bezafibrate was added to the cells. Using the medium at day 3 after the addition of drugs, HBV DNA was quantified with real-time PCR. Results: The CC50 of bezafibrate and rosiglitazone in HepG2 cells were 250 M and 150 M, respectively. The amount of HBV-DNA in the medium was decreased when the density of bezafibrate was over 200 M, but the density demonstrated considerable cytotoxicity. In contrast, rosiglitazone of 5 M, which showed no cytotoxicit, decreased the amount of HBV DNA. The 50% effective concentration (EC50) was calculated to be 9.8 M.
Conclusions: In this study, it was suggested that the replication of HBV was inhibited by rosiglitazone of the density without cytotoxicity. The mechanism is uncertain and being investigated now.
Q. Zheng 1 1 Center for Liver Diseases, The First Affiliated Hospital, Fujian Medical University, Fuzhou, P. R. China Background: The objective of this study was to evaluate the early virologic response for prediction of achievement of HBeAg seroconversion and hepatitis B virus (HBV) DNA negativity after two years of lamivudine treatment in chronic hepatitis B (CHB) patients. Methods: In this retrospective study, adult patients with chronic hepatitis B (255 HBeAg-positive and 122 HBeAg-negative) were treated with lamivudine (100 mg/day), and followed-up up to 72 months. Response and resistance to the treatment were assessed during the treatments with lamivudine. Results: It was found that gender, age, baseline levels of ALT and HBV DNA, serum HBV DNA at week 24 (P =0.019 , OR = 0.442) were closely related to the achievement of HBeAg seroconversion, undetectable HBV DNA level and emergence of drug resistance after 2 years of lamivudine treatment. HBeAg positive patients with baseline serum HBV DNA in 10 6 -10 8 copies/ml and serum HBV DNA 10 3 copies/ml at week 24 showed high response rate of ALT normalization rate (91.7%), undetectable HBV DNA rate (84.5%), HBeAg seroconversion rate (55.2%), as well as low drug resistance rate (25.4%) after 2 years of treatment. Similarly, HBeAg negative patients with serum HBV DNA 10 3 copies/ml at week 24 could achieve high 2-year response rate of ALT normalization rate (72.41%), undetectable HBV DNA rate (82.3%), and low drug resistance rate (15.5%). Conclusion: Serum HBV DNA 10 3 copies/ml at 24-week provide the best prediction of 2-year lamivudine treatment response. Background/aims: Unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis B (CHB), with increasing rates of HBsAg clearance observed in patients who respond during post-treatment follow-up. HBsAg clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of HCC and increased survival. Methods: In a randomised multinational study, patients (HBeAg-negative) received 180µg PEGASYS+placebo (n=177); 180µg PEGASYS+100mg lamivudine (n=179); or 100mg lamivudine (n=181) for 48 weeks, and were assessed 6 months post-treatment. From this initial study, 230 of those who had received PEGASYS±lamivudine and 85 patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. HBsAg clearance at yearly post-treatment follow-up visits up to 5 years post-treatment was analysed. Results: HBsAg clearance in patients treated with PEGASYS±lamivudine increased post-treatment (3% at 1 year to 6%, 8%, 11% and 12.2% at years 2, 3, 4 and 5). At year 5, 28 PEGASYS-treated patients (12.2%) had cleared HBsAg compared with 3 (3.5%) of lamivudine-treated patients (P=0.022). 16/28 PEGASYS-treated patients had anti-HBsAg (HBsAg seroconversion). Detailed analysis of the 5-year follow-up data will be presented. Conclusion: The ability of a finite course of PEGASYS to induce sustained response with increasing HBsAg clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in HBeAg-negative patients with CHB. Background/aims: Recent studies suggest that quantification of HBsAg levels early during treatment can be used to predict post-treatment response to PEGASYS. Elecsys ® HBsAg II (Roche) is a sensitive assay for the detection of HBsAg. This assay can be used for the quantification of HBsAg levels using a simple dilution algorithm. We compared results obtained using the Elecsys ® HBsAg II method with those of a commonly used quantification assay. Methods: HBsAg levels obtained using the Elecsys ® HBsAg II assay were compared with those obtained using the Abbott Architect ® HBsAg assay for a total of 40 samples from patients infected with HBV genotypes A (n=8), C (n=1), D (n=29) and F (n=2). Samples were diluted 1:100 in diluent provided by the manufacturer. Samples with HBsAg levels >250 IU/mL were retested at a final dilution of 1:1000. Samples with HBsAg levels <0.05 IU/mL were retested undiluted. Results: Overall, HBsAg levels measured with the two assays correlated well (R 2 =0.9718) over a wide range (3-3x10 5 IU/mL). Discrepancies in HBsAg levels >±20% were reported for a minority of the samples (n=15), mainly distributed evenly above and below the ideal line (n=11). In the four low titre (range 3-9x10 3 IU/mL) samples with greatest discrepancy Elecsys ® underestimated values (in two cases by >50%). Conclusion: The Elecsys ® HBsAg II assay provides a simple and reliable means for determining HBsAg levels. This simple assay format could be used to provide useful information during on-treatment monitoring of HBsAg levels in patients with chronic hepatitis B undergoing therapy.
Conclusions: HBV/A has been increasing in CHB patients in Japan as the consequence of AHB, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of HBV/A to induce chronic hepatitis. The spread of HBV/A infection in Japan should be prevented by universal vaccination programs. Introduction: Chronic viral hepatitis is common in end-stage renal disease (ESRD), from endemic hepatitis B (CHB) and nosocomial hepatitis C (CHC). Reduced outcomes post-renal transplant were reported thus CHB and CHC cirrhosis became contraindications to listing. However, these predated effective anti-viral therapies. We reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. Methods: Prospective database of ESRD patients with viral hepatitis referred for renal transplantation was reviewed. Results: 110 patients were assessed. 23 patients underwent kidney transplantation. Two were cirrhotic and had liver/kidney transplantation; both died within 6 months. 21 were non-cirrhotics, of whom 20 are alive. 14/20 have functioning allografts; predictors were normal ALT and low viral load. Of the 87 non-transplanted, 31 had cirrhosis; 17/31 received anti-virals. Mortality was 35% -7 liver-related (3 hepatoma,1 bacterial peritonitis,3 sepsis -5 inactive cirrhosis); 4 non-liver related (2 cerebral,1 haemorrhage,1 renal -3 inactive cirrhosis). 12/20 surviving cirrhotics received anti-virals. In non-transplanted non-cirrhotics, mortality was 18%; 80% of survivors had inactive disease. 23 CHB patients received Lamivudine; 11 Adefovir (Lamivudine resistance). 11 CHC patients received IFN-based therapy. Conclusion: Excellent outcomes are achieved in ESRD patients with CHB/CHC post-renal transplant, in absence of cirrhosis. Normal ALT/non-detectable viral load can predict graft function. However, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. The role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed.
The Truncated HBc149 Interferes with Replication of Hepatitis B Virus J.C. Han 1,2 , X.B. Pan 1,2 , L. Wei 1,2 , K. Deng 1,2 1 Institute of Hepatology, 2 Peking University People's Hospital, China Hepatitis B virus (HBV) capsids play an important role in production of progeny virus and other elements of the virus life cycle. Misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. HBV capsids can be assembled in vivo and vitro from the dimeric HBV core protein (HBcAg). The interaction of single and dimeric HBcAg with some truncated HBcAg is verified in vitro. The truncated HBcAg consists of the first 149 amino acids and lacks the C-terminal, 34-residue RNA-binding domain. Method: we transiently transfected hepG2.2.15 with pcDNA3.1HBc149 by Fugene6.after 48 and 96h, HBVDNA, HBeAg and HBsAg in culture supernatant were detected and cell subjected to Southern blot and Immunofluorescence analysis. Result: The level of HBsAg and HBeAg had gentle change, we found that HBVDNA decreased at 96h after transfection( 10 3 10 4 copies/ml P<0.01) ,but replication intermediates obviously decreased from 48h. Some positive signal of HBcAg located around the nuclear and conglomerated in cytoplasm compared to the control. Conclusion: The truncated HBc149 can inhibit replication of hepatitis B virus. Misdirection of capsid assembly and formation of aberrant particles could be an important cause.
Y.P. Li 1 , R.C. Li 1 1 
Objective: To assess the long-term efficacy of recombinant yeast derived hepatitis B vaccine in infant s born to HBsAg and HBeAg carrier mother. Methods: A total of 273 neonates born to HbsAg, HBeAg both positive mothers were vaccinated with 5 ,5 ,5 g doses of recombinant yeast derived hepatitis B vaccine by 0 ,1 , and 6 months schedule. They were all followed for 129 years after the primary vaccination. Results: Twelve infant s (6.63 %) become HBsAg positively conversed in 9 year after primary vaccination ,and the positive rate of HBsAg in 1-9 year was 0.72 %-6.8 % ,17.18 % of child in no/ lowly respond become HBsAg positively. At the ninth year, the positive rates of anti-HBs were 60 % above. Anti-HBs positive rates and immunity level were higher at 3-5 year old by repetition immunity than others. Conclusion: The recombinant yeast derived hepatitis B vaccine have good immunogenecity and long-term protective efficacy to HBV interruption of perinatal transmission , a booster dose seems necessary in aged 3-4 years to the mother with HBsAg and HBeAg.It is high risk tobecome HBsAg positively in the baby of norespones to hepatitis B vaccine.
CHB Patient Group-initiated Programme to Improve Awareness, Adherence and Treatment Outcomes in Asia Pacific N. Leung 1 1 Founding Chairperson of ASIAHEP Background: Worldwide, over 400 million people live with chronic hepatitis B (CHB); 300 million in Asia Pacific. Regional survey data from 1,500 patients in 10 countries showed a lack of knowledge and understanding of CHB, its severity and impact on quality of life. This initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. Methods: The patient groups met in Hong Kong in May 2008 and identified common needs to: (1) improve educational resources; (2) raise awareness; (3) increase diagnostic yield; and (4) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. Results: A patient engagement programme was developed for people with newly diagnosed or known CHB. The programme comprises: -Detailed information about CHB -A health-tracking tool for self-monitoring of blood tests and treatment progress -Detailed information for carers/family -A patient-physician communications video (including role-play) -Mobile phone text messages providing advice and compliance/appointment reminders Conclusion: This programme was developed to address the needs of patients and clinicians. Improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. The programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. Acknowledgement: The CHB patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from GlaxoSmithKline.
Serum HBV RNA Level Reflects the Potency of Nucleos(t)ide Analogue Y.W. Huang 1,2 , K. Chayama 3,4 , M. Tsuge 3,4 , S. Takahashi 3,4 , T. Hatakeyama 3,4 , M.Y. Lai 2,5 , H.L. You 1 , J.T. Hu 1 , C.J. Liu 2,5 , P.J. Chen 2,5 , D.S. Chen 2,5 , S.S. Yang 1 , J.H. Kao 5, 6 1 Liver Unit, Cathay General Hospital Medical Center, 2 Background and aims: Serum HBV RNA is detectable in patients treated with lamivudine (LMV) or entecavir (ETV) (Hatakeyama, 2007 and Huang, 2008) . The aim of this study was to determine the clinical significance of serum HBV RNA levels in patients treated with nucleos(t)ide analogues of different potency. Methods: Serum HBV RNA was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (ADV), 5 with LMV, and 6 with ETV). Serum HBV RNA was quantified by reverse transcription of HBV nucleic acid extract with subsequent real-time PCR. Results: HBV RNA was detectable in 11 patients as follows: 2 of 9 in ADV (22%), 3 of 5 in LMV (60%), and 6 of 6 in ETV (100%) (p = 0.002). Mean log serum HBVDNA levels at baseline were 10.1 ± 0.6 for ADV, 6.6 ± 2.0 for LMV, and 9.5 ± 0.9 for ETV, which were comparable between less potent ADV and most potent ETV (p = 0.14). During antiviral therapy, peak log HBV RNA level of patients with ETV was significantly higher than that of those with ADV or LMV (3.2 ± 0.9 vs. Background: In the phase III clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. The objective of this study is to compare the anti-HBV activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis B (CHB) patients in a randomised way. Methods: The patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. Eligible patients were treatment-naïve HBeAg(+) CHB patients with HBV DNA levels 500,000 copies/mL. The primary endpoint is the proportion of patients with HBeAg loss. Preliminary results are presented here. Results: Thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. At week 24, 26% of patients had HBeAg loss. At week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had HBeAg loss. At week 24, 63% of patients had negative HBV DNA by Amplicor PCR (<300 copies/mL). At week 36, all of patients in both groups had negative HBV DNA by PCR and 73% in clevudine alone and 80% in combination group had normal ALT. Conclusion: Clevudine demonstrated good serologic response as well as significant viral suppression and ALT normalization. With this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. Background/Aims: To determine the reasonable number of clones for HBV quasispecies analysis. Methods: 16 chronic hepatitis B patients were enrolled with HBVDNA levels range from 4~10 log copies/ml. HBVDNA was extracted. HBV reverse transcriptase (RT) gene encompassing the overlapping surface S gene was amplified by polymerase chain reaction, then cloned and sequenced. Ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. The characteristics of HBV quasispecies including Shannon entropy and genetic distance were calculated. Results: The Shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in RT gene or in S gene (p<0.01). While the Shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in RT gene nor in S gene (p>0.05). The number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). The Shannon entropy and genetic distance in three different clones group had no correlation with HBV DNA levels (p>0.05). Conclusion: Although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. The information contained in 20 clones per sample could well represent the quasispecies characteristics. The clone number was not necessary modulated according to different HBV DNA levels. Background: Recent studies reported that basal core promoter mutation (A1762T and G1764A) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and HCC. But the effect of the double mutations on the activity of enhancer II/basal core promoter is still uncertain. Objectives: To evaluate the influence of nt1762 A/T and nt1764 G/A mutations on HBV enhancer II/basal core promoter activity. Methods: The PCR fragments of HBV enhancer II/basal core promoter (nt1601 to nt1815) from the serum-derived genotype B HBV DNAs of one HBV carrier aged 66 and one HBV related hepatocellular carcinoma patient aged 26 were introduced into the pGL3-Basic-Vector from Promega via restriction sites of Xho I and Hind III. The nt1762 A to T and T to A, the nt1764 G to A and A to G mutations were carried out by GeneTailor Site-Directed Mutagenesis System from Invitrogen. The promoter activity was evaluated by comparing firefly luciferase measurement with Renilla luciferase as the internal control using the Dual-Luciferase Reporter Assay System from Promega. Results: The luciferase reporter assay results indicated that the 1762 T to A combined with 1764 A to G mutations increase (P<0.001) while the 1762 A to T combined with 1764 G to A mutations decrease (P<0.05) the HBV enhancer II/basal core promoter activity significantly. Conclusions: Associated with increased risk of hepatocellular carcinoma, A1762T and G1764A double mutations of hepatitis B virus reduce the enhancer II/basal core promoter activity. Background/Aims: A substantial proportion of chronic hepatitis B (CHB) patients with mildly elevated alanine aminotransferase (ALT) have significant fibrosis. We evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. Methods: One hundred five CHB patients with ALT less than two times the upper limit of normal underwent liver biopsy. Multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. Results: There were 27 patients in the low normal ALT group, 21 in the high normal ALT group, 37 in the low elevated ALT group, and 20 in the high elevated ALT group. Fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). The age, platelet count and grade of inflammation were factors associated with significant fibrosis. Progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. The stage of fibrosis, ALT group and antiviral therapy were significant predictive factors for progressive liver disease. Conclusion: Liver biopsies should be recommended in patients over 35 years with mildly elevated ALT levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. Background: Four nucleos(t)ide analogues (NAs) are currently approved for the treatment of HBV infection in China. However, long-term benefits are limited by the emergence of drug-resistant viruses. Methods: Patients accepted the examination based on physician's instruction. HBV reverse transcriptase gene was amplified from serum via nested PCR and sequenced directly. Results: Well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. In patients receiving NA monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (LAM), 35/333 receiving adefovir (ADV), 0/57 for entecavir (ETV), and 9/17 for telbivudine (L-dT). The mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 NAs. M204I (32%), M204V+L180M V173L (32%), and M204I+L180M (21%) were identified as major mutant patterns of LAM monotherapy. N236T A181 substitution was the dominant ADV-resistant mutation. T184 substitution was the dominant ETV-resistant mutation always accompanied with LAM-resistant mutation. L-dT-resistant mutation was M204I L180M exclusively. ADV-resistant mutation was frequently seen in LAM-resistant patients receiving ADV sequential therapy rather than those receiving ADV add-on therapy. Controversial LAM/ADV-resistant mutations including A181T, V214A, Q215S and I233V were detected in some patients singly or with the well-recognized drug-resistant mutations. Interestingly, the drug-resistant mutations were also observed in a few of patients naïve to NAs. Conclusions: The exploration of HBV drug-resistant mutation profile in large clinical samples furthers our understanding of HBV drug-resistant status in China with implications for administrating anti-HBV therapy more reasonably.
Toll-like Receptor (TLR) 2, TLR4 and CD4+CD25+CD127low/-regulatory T Cells Correlate with Hepatitis B Virus Infection Y. Zhang 1 , J.Q. Lian 1 , C.X. Huang 1 , X. Wei 1 , J.P. Wang 1 , P.Z. Wang 1 , X.F. Bai 1 1 Center of Infectious Diseases, Tangdu Hospital, The 4th Military Medical University, Xi'an, China Background: TLRs play a crucial role in sensing and initiating innate antiviral response and Tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. In this study, we determined TLR2 and 4 expression and Treg frequency, as well as their function in the effect of HBV infection. Methods: TLR2 and TLR4 expression on monocytes and circulating CD4 + CD25 + CD127 low/-Tregs were determined by flow cytometry in 16 AHB, 42 CHB, 22 AsC and 20 NC. Spearman correlation was performed to investigate associated variables on Treg or TLRs. PBMCs were stimulated with HBeAg or HBcAg and the TLRs profile was examined. Result: TLR2 expressions were up-regulated in CHB and AsC, while TLR4 were increasingly expressed in AHB and AsC. Treg frequency in CHB was significantly higher than that in NC. In CHB, the increased TLR2 negatively correlated with HBV DNA loads and Treg frequency negatively correlated with TLR4 expressions. TLR2 was up-regulated after HBeAg stimulation in both NC and CHB. Conclusion: Increased Tregs may be associated with CHB and there might be possible interactions between HBeAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance. (6.7±1.35) . HBV-DNA was quantitatively determined by polymerase chain reaction (PCR) technique, and HBV genotype was determined by PCR microwave gene chip technique. Antiviral efficacy was assessed using measuring the following scales: the ALT normalization rate, HBV-DNA negative conversion rate and the HBeAg/anti-HBe seroconversion rate. Results: Among 55 serum specimen, HBV genotype distribution was 38 genotype C, 14 genotype B, and 3 genotype non-B or C respectively. In genotype B, ALT normalization rate was 78.57%(11 cases), HBV-DNA negative conversion rate was 35.71%(5 cases) and the HBeAg/anti-HBe seroconversion rate was 14.28%(2 cases). In genotype C, ALT normalization rate was 76.31% (29 cases), HBV-DNA negative conversion rate was 47.37%(18 cases) and the HBeAg/anti-HBe seroconversion rate was 18.42%(7 cases). The efficacy of adefovir dipiroxil showed no significant differences between genotype B and C in the treatment of chronic hepatitis B P>0.05 . Conclusion: Adefovir dipiroxil is an effective antiviral drug. HBV genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis B.
The Effect of Anti-HBV Drugs on Albumin and Bilirubin Levels, and Platelet Count H. Yoshida 1 , H. Taniguchi 1 , R. Nagano 1 , K. Sakitani 1 , E. Seki 1 , T. Serizawa 1 , Y. Ito 1 , H. Mizuno 1 , Y. Mitsuno 1 , R. Nakata 1 , M. Omata 2 1 Japanese Red Cross Medical Center, 2 University of Tokyo, Japan Background/Aim: We assessed the efficacy of anti-HBV drugs on the liver function. Methods: Patients with HBV-related disease followed at our center between 2005 and 2007 were enrolled. Lamivudine (100mg), lamivudine (100mg) +adefovir (10mg), or entecavir (0.5mg) was administered to the patients with detectable HBV DNA and elevated ALT. Liver function (ALT, ALB, and T.Bil) and platelet count were observed. ALT, ALB, T.Bil, and platelet count of treated group at pretreatment, year 1, and year 2 were compared with untreated group. Results: Eighty six patients with positive HBsAg were enrolled between Jan 2005 and Dec 2007. Seven patients (2 acute infection, 1 overlap infection with HCV, 7 lost of follow up) were excluded. In total 76 patients were followed up for a median follow up of 26 (range 2-39) months. Of 76 patients, 32 received anti-viral treatment. Twenty one patients were treated with lamivudine, 4 with lamivudine+adefovir, and 7 with entecavir. The mean of levels of pre-treatment-year1-year2 were ALT:129-37-43 (U/L), ALB:4.0-4.4-4.4 (g/dl), T.Bil:1.0-0.8-0.7 (mg/dl), and Plt:14.3-15.4-16.0 (x10 4 mcl) respectively. Markers of untreated group (n=44) (at baseline-year1-year2) were ALT:33-40-35 (U/L), , T. Bil:0.8-0.8-0.9 (mg/dl), and Plt:18.7-18.0-18.5 (x10 4 mcl) respectively. Although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year2. Conclusion: Treatment with anti-HBV drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. Background: Currently, HBeAg-negative chronic hepatitis B(CHB) is increasing. But there are still controversial on the treatment of HBeAg-negative CHB with ALT 2×ULN. We have investigated the clinical efficacy of nucleotide analogues(NAs) in the treatment of HBeAg-negative CHB with ALT 2×ULN. Methods: The data of patients who were treated by NAs for more than 2 years and with ALT 1 2×ULN (n=87) , ALT 1×ULN(n=43) and ALT 2×ULN(n=88) were collected, and 12W and 24W virologic response, 48W and 96W complete response, virologic breakthrough and clinical resistance were analyzed. Results: Compared with the base line, HBV DNA level in all three groups were significantly decreased (P 0.01), and there was no significant difference between ALT 1 2×ULN group and ALT 2×ULN group. The viral load was significant decreased in ALT 1×ULN group at 12W, 24W and 36W (P<0.05). Virologic response at 12W and 24W complete response at 48W and 96W was 71.3%, 82.8% , 82.8% and 86.2% respectively in ALT 1 2×ULN group and was 51.2%, 60.5%, 65.1% and 74.4% respectively in ALT 1×ULN group. There was no significant difference between ALT 1 2×ULN group and ALT 2×ULN group. Virologic response at 12W and 24W and complete response at 48W were significant decreased (P<0.05) in ALT 1×ULN group. There was no significant difference among the three groups in virologic breakthrough and clinical resistance. Conclusion: HBV replication can be satisfactory inhibited by NAs in HBeAg-negative CHB patients with ALT 2×ULN, which suggests that in these patients the indication of ALT is different from HBeAg-positive patients.
Quantitative HBeAg Assay as a Predictive Factor of HBeAg Seroconversion Induced by PEG-IFN -2a Therapy to HBeAg-positive Chronic Hepatitis B Y.Y. Zhu 1 , J. Dong 1 , Y.T. Chen 1 , J. Chen 1 , J.J. Jiang 1 1 Liver Diseases Research Center, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China RP, 350005 Background: To find predictive factor for HBeAg seroconversion in the treatment of HBeAg-positive chronic hepatitis B (CHB) by PEG-IFN -2a. Methods: HBeAg-positive CHB patients were given PEG-IFN -2a treatment for 48 weeks. Clinical data were collected every 3 months. Receiver Operator Characteristic (ROC) curve was employed to calculate positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity.
Results: Sixty-five patients completed PEG-IFN -2a therapy. Among them, 17 (26.15%) were found HBeAg seroconversion and 19 (29.23%) were found HBeAg loss at cessation of therapy. None of age, gender, ALT level and HBV DNA load at baseline had relationship with HBeAg seroconversion. HBeAg level of baseline was correlated to HBeAg seroconversion, with P value as 0.003 (Table 1) . According to ROC curve, supposed AUC as 0.705 and P value as 0.042, the PPV, NPV, sensitivity and specificity of HBeAg level as 61 at 12 week were 0.361, 0.862, 0.765 and 0.521, respectively. Supposed AUC as 0.828 and P value as 0.001, the PPV, NPV, sensitivity and specificity of HBeAg level as 15 at 24 week were 0.452, 0.912, 0.824 and 0.646, respectively. The HBeAg level (s/co) and decreased degree (percentage) at 12 week and 24 week were significant related to HBeAg seroconversion (Table 2) . Conclusion HBeAg level at baseline and at 12th and 24th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for HBeAg seroconversion. Background: It is well documented that perinatal transmission is the major cause of chronic HBV infection in China. The aim of this study was to evaluate the efficacy of interruption of HBV intrauterine infection with hepatitis B immunoglobulin (HBIG) in pregnant women with HBeAg positive. Methods:: A prospective randomized controlled trial was adopted. Each subject in the trial group (28 cases) was given 200 IU HBIG intramuscularly every 4 weeks from 28-week of gestation, while each subject in the control group (24 cases) received placebo in the same way. The cord blood of newborns were collected for detecting HBsAg, HBeAg and HBV-DNA. Results: For newborns, HBeAg positive rate in trial group was 21.4%(6/28).HBeAg positive rate in control group was 79.2%(19/24). There was significant difference in HBeAg positive rate of newborns between the two groups( P < 0.01, RR = 0.27). HBV-DNA positive rate in trial group was 25%(7/28). HBV-DNA positive rate in control group was 83.3%(20/24). There was significant difference in HBV-DNA positive rate of newborns between the two groups( P < 0.01, RR = 0.30). HBV-DNA load of 7 cases of newborns in trial group was lower than that of their mothers(T = 28,P = 0.02). There was no significant difference in HBV-DNA load between women and their newborns after delivery in control group (T = 81.5,P > 0.1). Conclusion: It is effective and safe to prevent HBV intrauterine infection with HBIG from the 28(th) wk in pregnant women with HBeAg positive. ), especially, the cirrhosis and HCC cases obviously more in both HBeAg and anti-HBe patients are negative than HBeAg-negative but anti-HBe positive patients (.36.7% vs 26.7%; 7.9%vs5.5%, p ) .The prevale nce of pre-core G1896A mutate have no significant difference regardless of HBV serum marker status or the state of illness. Conclusion: Recent 5 years the HBeAg-nagative chronic hepatitis B patients are gradually increasing in Yunnan province. While the HBeAg disappear but no anti-HBe serum transfer, and the virus still active replication -It may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. The clinical significant of pre-core G1896A mutate remain unknow.
Efficacy of Interferon for Chronic Hepatitis B Patients with Normal or Paranormal ALT Z. Liu 1 , J.Z. Guo 1 , Y.J. Lin 1 , Y.J. Zhang 1 , Z.W. Lang 1 1 Beijing Ditan Hospital, Beijing, China Background: We reported Interferon treatment for 4 cases with normal or paranormal ALT but in which liver histologic exam showed G2-4 and/or S3-4. Methods: 4 patients were male with an average age of 28 years.Mean ALT was 46.7 IU/L and HBV DNA level was 3~5 log10copies/ml. Two patients were HBeAg positive; one patient was both negative for HBeAg and anti-HBe and one patient was anti-HBe positive. Liver biopsy showed G2~G4 and S3~S4 respectively. 3 patients were treated with IFN-alpha, Liver biopsy was repeated after 1 year.Only one patient had received combination therapy with IFN and adefovir after 10 months treated IFN monotherapy and liver biopsy was taken after 1.5 years. Results: All patients got normal ALT after 1 year treatment. HBV DNA was undetectable in 3 patients. 2 patients with initial positive HBeAg cleared. But 3 patients still were anti-HBeAg negative.Liver biopsy showed change fromG4 S3-4 to G2 S2-3 in 1 patient; fromG2 S3 to G3 S4 in 1 patient and no change in the other 2 patients. Conclusions: Though ALT and HBV DNA improved after 1 year treatment, histological improvement is not satisfying. 1 patient's improvement in liver histology may be due to seroconversion before treatment and adding Adefovir after 10 months of interferon therapy. After 8 months of combination therapy we did liver biopsy again. The other 3 patients were HBeAg negative, but HBeAb were also negative, liver biopsy was taken 1 year later without combination of nucleoside analogs.
Evaluation of Long Term Efficacy of Hepatitis B Vaccination R.C. Li 1 , J. Gong 1 , J.Y. Yang 1 1 
Objective: To evaluate the long term effectiveness of preventive HBV infection and to monitor the incidence of hepatitis B in children to see possible impact on the program of Long An that was launched in 1986. Methods: (1) Set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis B. (2) To serologically evaluate the effect of the program, a stratified random sampling of 1000 subjects in 1987 birth cohorts was recruited for long term follow up at the age 1-20 years. (3) Cross-sectional seroepidemiolgical survey was carried out in the county in 1985 before the program and 20 years later. HBsAg , anti-HBs and anti-HBc were tested by RIA. Results: The average coverage of hepatitis B vaccine was 89. 1 %. At 20 years after vaccination, the seropositivity for HBsAg in population of 1-20 years has decreased from 16. 0 % to 2. 3 %, the annual effectiveness was 89. 7 %. HBV accumulated infection rate was 3. 5 %, protective rate was 96. 2 %. The incidence of acute hepatitis B was 1. 60 per 100,000 in population aged 1 -20 years , it decreased by 91.3 % as compared with the incidence of 18.38 per 100,000 in same age group in 1985 -1987. Conclusion: Mass hepatitis B vaccination program in Long An County has proved to be effective in control of HBV chronic infection and incidence of acute hepatitis B. Background and Aims: Although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis B virus (HBV), especially during HBeAg seroconversion. Methods: Nucleotide sequences of HBV precore/core genes from 5 time points were analyzed in four cohorts of chronic hepatitis B, interferon-induced seroconverters (IS, N=9), interferon non-responders (IN, N=9), spontaneous seroconverters (SS, N=9) and non-seroconverters (SN, N=9), followed during 60 months on average. Only patients with genotype C were used. Viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. Results: Analysis of 1800 nucleotide sequences showed that the nucleotide genetic distance of seroconverters (IS and SS; 9x10 -3 substitutions/site and 8.5x10 -3 subsititutions/site, respectively) was similar to that of non-seroconverters (IN and SN; both 7x10 -3 substitutions/site) before seroconversion. Compared to that of nonseroconverters (IN and SN; substitutions/site and 7.5x10 -3 substitutions/site, respectively) the viral diversity of seroconverters (IS and SS; 13x10 -3 substitutions/site and 12x10 -3 substitutions/site, respectively) was significantly higher after seroconversion (p<0.05) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p<0.05) while it almost didn't change in non-seroconverters irrespective seroconversion. Phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. Conclusions: The distinctly higher viral diversity after seroconversion in HBeAg seroconverters could be related to increased HBV-specific T-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity.
Adefovir Dipivoxil 10mg (ADV) Resistance at 5 yrs in Chinese HBeAg+ve Chronic Hepatitis B (CHB) J.L. Hou 1 , Y.Z. Wang 2 , X.Q. Zhou 3 , J.Q. Niu 4 , Y.M. Wang 5 , H. Wang 6 , Y.M. Mao 7 , K.F. Barker 8 1 NanFang Hospital, Guangzhou, PRC, 2 JiNan Infectious Disease Hospital, JiNan, PRC, 3 Ruijin Hospital, Shanghai, PRC, 4 1st Hospital of Jilin University, Changchun, PRC, 5 Xinan Hospital, Chongqing, PRC, 6 People's Hospital, Beijing, PRC, 7 Renji Hospital, Shanghai, PRC, 8 GlaxoSmithKine R&D, London, UK Background: Long term ADV provides clinical and histological improvement in CHB, but may lead to emergence of treatment associated resistant mutations. We report on ADV resistance data from Chinese HBeAg positive subjects treated for 5 years. Methods: 480 HBeAg positive CHB subjects were randomized in an initial 52 weeks controlled ADV study (with a 12 weeks placebo period in half of patients) and then offered open label ADV treatment for a further 208 weeks. A total of 474, 456, 443, 421 and 390 subjects completed the 1 st , 2 nd , 3 rd ,4 th and 5 th yr, respectively. At the end of each year samples were analysed from those subjects with protocol-defined HBV DNA breakthrough for the rtN236T or rtA181V ADV mutations associated with resistance. Sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. Results: At the end of the 1 st yr, none of the 45 subjects with HBV DNA breakthrough had either mutation. Sera were available for analysis from 137, 199, 228 and 187 subjects with viral breakthrough at the end of the 2 nd , 3 rd , 4 th and 5 th yr, respectively, with new mutations identified in 6, 20, 24 and 20 subjects at the same timepoints. Of the cumulative 70 subjects at the 5 th yr analysis 31 had rtN236T, 29 had rtA181V, and 10 had both mutations. Conclusion: Treatment with ADV in Chinese HBeAg positive CHB subjects for up to 5yrs resulted in a cumulative rate of 14.6% (70/480) ADV resistance-associated mutations with HBV DNA breakthrough. Background and aims: To evaluate the predictive significance of rapid virologic response (RVR) for achieving an end-of-treatment virologic response (ER) or HBeAg seroconvertion and the predicting indicator of nonresponse (NR). Methods: 205 patients with chronic hepatitis B were treated with ADV and prospectively observed to 48 weeks. We assessed the values of virus load reduction at weeks 4, 8, and 12 weeks to predict the ER and HBeAg seroconversion. The association between less reduction of viral load at 12 and 24 weeks and nonresponse was also analyzed.
Results: Of 146 ETV-treated patients enrolled in ETV-901, 98 met criteria for inclusion into 5 year ETV treatment analyses. The proportion of patients achieving efficacy endpoints through 5 years of ETV therapy is presented the table.
Results: After 48 weeks of therapy, serum HBV DNA levels decreased with a median 4.07±2.47 log10 copies/mL. Twenty-three(11.2%) of patients had ER. Twenty-six (12.7%) patients achieved HBeAg seroconversion. HBV DNA <4 log 10 copies/mL at week 4 predict both ER and HBeAg seroconversion. HBV DNA> 4log10 copies/mL at 4 weeks but decline to <4log10 copies/mL at 12 weeks or 24 weeks both can predict ER and HBeAg seroconversion. Less than 2 log 10 HBV DNA reductions at 24 weeks might predict NR.
Conclusions: The majority of patients experienced durable serum HBV DNA suppression (94%) and ALT normalization (80%) after 5 years ETV therapy.
Conclusions: The virologic response within 4 weeks could be useful for prediction of ER and HBeAg seroconversion of adefovir therapy. Failing to EVR might not predict NR. Objectives: To determine the accuracy of HBcIgM in diagnosing AHB and the correlation between HBcIgM and liver inflammation (ALT), bilirubin & biosynthetic functions (albumin,PT). Result: A total of 233 patients were included: in 40 patients, ADV was added on LAM (add-on therapy), and in 193 patients, LAM was switched to ADV (switch therapy). During 16.5 months of follow-up, 25 patients developed ADV resistance (rtA181V and/or rtN236T) and all had undergone switch therapy. The cumulative probability of ADV resistance at the 24th month was 17.5%. Although add-on therapy induced no ADV resistance, it failed to show significant superiority over switch therapy (P=0.220). In multivariable analysis, female (odds ratio [OR], 2.75; 95% confidence interval [CI], 1.22-6.17; P=0.014), liver cirrhosis (OR, 2.39; 95% CI, 1.07-5.33; P=0.033), and age >45 yr (OR, 3.31; 95% CI, 1.14-9.66; P=0.028) were independent risk factors of ADV resistance.
Methods: A retrospective cross-sectional study involving patients with HBcIgM positivity between June 2006-December 2007, satisfying the definition for AHB and CHBF,and fulfilling the exclusion criteria was performed. HBcIgM test were done by using Microparticle Enzyme Immunoassay (MEIA) and results were expressed as an Index value.HBcIgM positivity was defined as Index value of >1.00 Results: 74 patients were positive for HBcIgM and 60 fulfilled the criteria(33 AHB, 27 CHBF).HBcIgM was significantly higher in AHB compared with CHBF(median 3.46 vs 1.70;p <000.5).The HBcIgM arbitary Index value of 2.76 was highly sensitive(97%) and specific(85%) in diagnosing AHB with high accuracy(AUROC 0.964;95% CI:0.925-1.003;p<0.0005).Among patients in both groups, there was a weak, but significant negative correlation between HBcIgM and PT above control(r = -0.309,p =0.016).However, among patients with CHBF,the negative correlation between HBcIgM and PT above control was moderately strong(r = -0.557,p =0.003).There was also a weak, but significant positive correlation between HBcIgM and albumin in with CHBF(r = +0.434,p =0.024).
Conclusion: ADV add-on therapy developed no ADV resistance during the observation period. Therefore, add-on therapy is recommended to LAM-resistant CHB patients with genotype C who have any risk factors for development of ADV resistance: female, liver cirrhosis, and age >45 yr.
HBcIgM-hepatitis B core IgM antibody AHB-acute hepatitis B,CHBF-chronic hepatitis B flare ALT-alanine transaminase,PT-prothrombin time PE117 Detection of Emerging Drug Resistance Mutations Associated with Major Approved HBV Antivirals Using a Novel Line Probe Assay (LiPA). J. Doutreloigne 1 , F. Shapiro 1 , R. Maertens 1 , E. Van Assche 1 , E. Sablon 1 1 Hepatitis Diagnostics Unit, Innogenetics NV, Belgium Background: In study ETV-022, ETV demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (LVD). This study (ETV-022/-901) presents efficacy and safety results for patients who received 5 years continuous ETV treatment.
Background/Aims: An increasing number of antiviral drugs are being used to treat chronic hepatitis B virus (HBV)-infected patients. However, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. Effective treatment strategies must therefore take possible drug resistance (DR) into account with respect to monitoring and selection of alternative drugs. We evaluated the use of an updated INNO-LiPA HBV DR v2+v3 reverse hybridization assay versus sequence analysis to detect resistance mutations.
Methods: The study evaluates ETV-treated nucleoside-naïve HBeAg (+) patients who completed ETV-022 and enrolled into ETV-901 with a treatment gap 35 days. The proportion of patients with HBV DNA <300copies/mL, ALT normalization, HBeAg loss or HBeAg seroconversion was evaluated at Week 240.
Background: ETV resulted in improved liver histology compared to LVD at 1 year. Histologic data for patients on ETV for a median of 6 years is evaluated.
Methods: 273 clinical samples (from untreated HBV patients or treated with different antivirals; HBV genotypes A-H) were tested for mutations with the LiPA assay and sequencing. For LiPA, samples were extracted with the QIAamp® DNA blood mini kit (Qiagen), and then tested on the LiPA strips. Sequencing-derived reference data were subjected to phylogenetic analysis (Kodon version 3.10 Applied Maths, Neighbour joining, with Kimura-2 parameter). Sequential samples from 9 patients were evaluated as well.
Methods: ETV-treated patients completing ETV-022 or ETV-027 received ETV (1.0mg daily) in ETV-901. Primary endpoints included 2-point decrease in Knodell necroinflammatory score, no worsening of Knodell fibrosis score and improvement in Ishak fibrosis score (IFS) ( 1-point decrease) vs. baseline. Secondary endpoints included proportions with HBV DNA<300 copies/mL, ALT normalization, and IFS normalization in patients with advanced fibrosis/cirrhosis. Results: Quasi-perfect concordance (> 99.6%) was obtained between the two assays for the samples tested. No indeterminate results were observed. For one sample, LiPA provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. For sequential samples, LiPA was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough.
Results: ETV treatment led to significant histological improvements and improved IFS in 96% (55/57) and 88% (50/57) of patients respectively. Of 10 patients with baseline fibrosis/cirrhosis (IFS 4), all demonstrated 1-point improvement in IFS (median change of -3). Conclusions: LiPA accurately detects the complex quasispecies nature of HBV and can help unravel the dynamics of emerging HBV resistance during treatment with different antiviral drugs. Like its predecessor, it is useful for the monitoring and early detection of drug resistance.
Conclusions: Long-term ETV therapy in nucleoside-naïve CHB patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. Precore (PC, G1896A) and basal core promoter (BCP, A1762T and G1764A) mutations of HBV are important for predicting the risk of hepatocellular carcinoma (HCC). We developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (RFMP) to detect A1896 and T1762/A1764 mutations, and applied it to analyze their clinical significance in type B liver diseases (N=336), including 35 HCCs, 118 liver cirrhosis (LC), 157 chronic hepatitis B (CHB), and 25 HBsAg-positive with low level viremia (inactive HBsAg carrer, IHC). We devided patients into 4 major groups according to the presence of wild (W) or mutant (M) genes in BCP/PC regions; W/W, W/M, M/W and M/M gene types. Each proportion was 9.5%, 3.6%, 41.4% and 23.5%, respectively. Mixed infection (X) was also found as minority; 4 W/X, 28 M/X, 15 X/W, 4 X/M and 13 X/X. Disease distributions (HCC, LC, CHB and IHC) in each group were as follows; [W/W (n=32)] 3.1%-6.3%-90.6%-0; [W/M (n=12)] 0-16.7%-58.3%-25%; [M/W (n=139)] 9.4%-42.5%-46%-2.2%; [M/M (n=79)] 11.4%-45.5%-20.1%-13.9%. These results suggest that, in Korea where only genotype C has been identified, BCP dual mutation is predominant (>73.2%), while BCP wild alone is only 13.1%. Especially, A1896 mutation alone without BCP mutation (W/M type) is uncommon, while BCP mutation alone without A1896 mutation (M/W type) is most common. It might be suggested that prognosis of wild type in BCP and PC region (W/W type) is much better than that of M/W or M/M types. Background/Aims: Entecavir is a potent inhibitor of HBV DNA polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis B (CHB) patients. The aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through 1 year in CHB patients. Methods: From 2007 May to 2008 October, we reviewed 82 patients (mean age 46±12 years, male:female=57:25) who were diagnosed as CHB patients (HBeAg (+) 64). Forty-seven patients (57.3%) had been treated with 0.5 mg of entecavir and 35 (42.7%) with 1 mg of entecavir, respectively. Mean follow-up period was 25±17 weeks. HBV DNA was quantified by bDNA assay with a lower limit of detection of 141,500 copies/mL.
Results: Median HBV DNA levels before therapy was 7.64 log10 copies/mL and the median decreases from baseline in HBV DNA were -4.19, -4.31, -5.09, -5.31, and -5.59 log10 copies/mL at 4 (n=39, p<0.001), 12 (n=62, p<0.001), 24 (n=42, p<0.001), 36 (n=22, p<0.001), and 52 (n=15, p =0.053) weeks of follow-up, respectively. At baseline, overall median ALT was 106 IU/L and the proportions of patients with normal ALT were 13%, 48%, 56%, 71%, 67%, and 69% at baseline (n=82), 4 (n=42), 12 (n=63), 24 (n=44), 36 (n=24), and 52 weeks (n=16) after entecavir therapy, respectively. Thirteen cases (15.9%) of HBeAg seroconversion were noted. Background: Hepatitis B virus (HBV) infection is a major risk factor for the progression of liver diseases. Because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with HBV infection. The aim of the present study was to determine the risk factors for the occurrence of HCC. Methods: A total of 620 patients who tested positive for hepatitis B surface antigen and were referred to Chiba University Hospital between February 1985 and March 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of HBeAg, ALT, HBV-DNA level, and PLT. Result: HCC was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). Multivariate analysis revealed that age [compared with young patients: odds ratio (OR) = 1.07, 95% confidence interval (CI) = 1.03-1.11] and PLT level (compared with patients with low PLT level: OR = 0.99, 95% CI = 0.98-0.99) were the predictive factors for HCC occurrence. In patients with age more than 35 years, the HBV-DNA level (compared with <5.0 log copies/ml: OR = 3.29, 95% CI = 1.40-11.5) and PLT level (OR = 0.99, 95% CI = 0.98-0.99) were the predictive factors for HCC occurrence. Conclusion: Advanced age and low PLT level were the risk factors for HCC occurrence in patients with HBV infection irrespective of the PLT level at baseline. In patients with age more than 35 years, viral load was also a risk factor for HCC. Results: Before treated by LPS, the total MAPK p38 level of PBMCs have no significant difference among the healthy control group, different stage groups with HBV infection, however, after treated with LPS, The phosphorylated MAPK p38 (pTpY180/182) in healthy control group are significant elevate than HBV infected groups(120.0±52 vs 80.2±84.8, p<0.05). In the two groups which HBsAg, HBV DNA are positive, alanine aminotransferase elevate than normal and HBsAg positive, but HBV DNA lower under the detect limited level, after treated by LPS the pTpY180/182 although lower than healthy control yet, but significant elevated than themselves before treated by LPS(86.6±38.6 vs117.8±21.3, 63.3±24.7 vs 93.1±21.8; p<0.05).Otherwise, in the group of both HBsAg and HBV DNA are positive, but ALT is normal, before and after treated by LPS, the level of pTpY180/182 have no significant difference. Conclusion: MAPK p38 is a important signal transduction pathway which involving in inflammation and immune response, especially, MAPK p38 activated up-regulate the IFN-gamma mRNA. According to the result shown, we propose a hypothesis, HBV infection and virus active replication inhibit the MAPK p38 activated, consequent on host immunotolerance and HBV persistence, thus, MAPK p38 may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. Van der Helm 10 1 Siriraj Hospital, Bangkok, Thailand, 2 Ramathibodi Hospital, Bangkok, Thailand, 3 Phyathai Hospital, Bangkok, Thailand, 4 Singapore General Hospital, Singapore, 5 National University Hospital, Singapore, 6 Changi General Hospital, Singapore, 7 Cheil General Hospital, Korea, 8 Hwasun Jeonnam University Hospital, Korea, 9 St Mary Hospital, Korea, 10 Roche Diagnostics Ltd, Rotkreuz, Switzerland Background/aims: Hepatitis B virus (HBV) surface antigen (HBsAg) is one of the most important markers for diagnosis of acute and HBV infection. High sensitivity of HBsAg assays can reduce the diagnostic window during course of disease. In addition, the presence of HBV mutants may be affected by the performance of the HBsAg kit. Therefore, the technical performance of the Elecsys ® HBsAg II assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. Methods: Nine HBsAg screening centers in Thailand, Korea and Singapore compared the sensitivity of Elecsys ® HBsAg II assay with that of their routine testing procedure -Abbott Architect ® (7 centers), Abbott AxSym ® (1 center) and Bayer Advia ® Centaur HBsAg assays (1 center) using preselected seroconversion panels (n=5), recombinant HBV mutant panels (n=13) and routine clinical practice samples (n=1,863). Results: The sensitivity of Elecsys ® in seroconversion samples was equivalent to the Architect ® assay, but more sensitive than the AxSym ® and Advia ® Centaur assays (26 vs 23 and 24 vs 18 positive bleeds, respectively). There was concordance between the Elecsys ® and Architect assay results with respect to potentially cross-reactive samples (99.74%). The Elecsys ® and Architect ® assays detected all recombinant mutant samples, whilst AxSym ® and Advia ® Centaur failed to detect three and nine samples, respectively. Conclusion: Elecsys ® HBsAg II assay was not only highly sensitive and specific when compared with established HBsAg screening assays, but also reliably detected HBsAg mutants. Therefore, this attractive assay is suitable for HBV diagnosis and assessing safety of blood products. Background: Recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis B (CHB) patients treated with switch-to therapy than those treated with add-on therapy. We compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant CHB. Methods: A prospective cohort study was performed in 49 patients with lamivudine-adefovir combination therapy and 77 patients with adefovir monotherapy for lamivudine-resistant CHB over 18 months. Result: Biochemical response was achieved in 41 patients (83.7%) treated with combination therapy and in 77 patients (92.8%) treated with monotherapy (p=0.101). Virologic response was observed in 19 patients (38.8%) in combination therapy and in 26 patients (31.3%) in monotherapy (p=0.383) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy (8.9±4.8 months vs. 13.9±5.0 month, p=0.001). Cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p=0.033). HBeAg loss was found in 6 patients (16.2%) in combination therapy and 17 patients (20.5%) in monotherapy (p=0.485). Biochemical breakthrough was found in 17 patients (20.5%) with monotherapy significantly more frequent than 3 patients (6.1%) with combination therapy (p=0.026). Genotypic resistance to adefovir was developed in 1 patient (2.0%) in combination therapy and 9 patients (10.8%) in monotherapy Conclusion: To achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant CHB patients, adefovir in combination with lamivudine is preferable. Background/Aims: Adefovir dipivoxil (ADV) effectively inhibits both wild-type and lamivudine (LAM)-resistat chonic hepatitis B virus (CHB) replication. The aims of this study were to determine the factorts associated with antiviral effect of ADV in LAM-resistant CHB. Methods: One hundred-eighteen LAM-resistant CHB patient (74.6% HBeAg-positive) were treated with ADV plus LAM (n=96) or ADV monotherapy (n=22) for a mean of 33.0 months. Restriction-fragment mass polymorphism analysis was used for detection YMDD and ADV mutants. Results: Fifty-eight patients (49.2%) achieved complete response(CR) defined as HBV-DNA levels <2000 copies/ml and ALT normalization. Twenty-eight patients (23.7%) achieved initial vilologic response(IVR) defined as HBV-DNA levels <10 4 copies/ml within the first 6 month of treatment. 47 (53.4%) of 88 HBeAg-positive patients exhibited HBeAg loss and 31% seroconverted to anti-HBe Ab. Five (4.23%) patients developed ADV-related mutations. Factors associated with IVR were pretreatment level of ALT (P=0.00), AST (P=0.00), pretreatment HBV DNA level (P=0.03), HBeAg negativity (P=0.01) and HBeAb positivity (P=0.00). Factors associated with CR were IVR (P=0.00), HBeAb positivity (P=0.01), pretreatment level of ALT (P=0.01), AST (P=0.02) and y-Glutamyl Transferase (P=0.04). Age, sex, presence of liver cirrhosis, pretreatment HBV DNA level and the type of YMDD mutants were not related to an CR during ADV treatment. Conclusions: ADV therapy achieved CR in more than 49% of LAM-resistant CHB. Factors associated with CR were IVR, HBeAb positive status, high base line ALT, AST, GGT levels.
Q.J. Sheng 1 , Y. Ding 1 , X.G. Dou 1 1 Department of Infectious Disease, Shengjing Hospital Affiliated to China Medical University, Shenyang, 110004, China Objectives: To study a kinetics of hepatitis B virus during 12-week and 24-week of treatment with enticavir (ENT); To compare the detecting results of HBVDNA levels from different detection reagents. Methods: Thirty-seven cases of chronic HBV infections were selected randomly, treated with daily dose of ENT 0.5-1.0mg (0.5mg for nucleoside-naïve patients, 1.0mg for lamivuding-refractory patients). Evaluation indexes: Serum HBVDNA, HBV serological markers, and liver function tests. HBVDNA levels were measured by PCR assay, using both domestic reagents and Roche Cobas Amplicor,. The lower limits of measure level of HBVDNA were 1000 copies/ml and 300 copies/ml, respectively. Results: Mean baseline of HBVDNA was 5.24log10copies/ml for detection using domestic reagents and 5.70log10copies/ml for that using Roche Cobas Amplicor, (P>0.05). The ratios of cases with undetectable (<1000 copies/ml) HBVDNA at week-12 and week-24 were 46.2% and 72.9%, respectively. The ratios of cases with undetectable (<300 copies/ml) HBVDNA at week-24 were 54.5%. Among the cases whose HBVDNA were lower than 1000 copies/ml(using domestic reagents), the ratio of HBVDNA lower than 300 copies/ml(using Roche Cobas Amplicor) was 94.7%. Conclusions: ENT can suppress HBV DNA rapidly no matter the patients with ALT elevation or not. There is a concordance on HBVDNA levels detection between domestic reagents and Roche Cobas Amplicor. Background The study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccDNA and tDNA and sera HBV DNA, HBsAg to probe reliable marks for evaluation of therapy endpoint. Methods The quantity of hepatocellular cccDNA and tDNA and sera HBVDNA were assayed by FQ-PCR, and sera HBsAg by ELISA in 4 CHB patients over 2 years nucleoside analogue therapy satisfied the China criteria of therapy endpoint (therapy group)and 12 CHB patients without antiviral therapy and sera HBVDNA<10 3 copies/ml(control group). Results: The quantity of hepatocellular cccDNA, tDNA and sera HBVDNA, HBsAg in therapy group were lower than that of control group,but low level hepatocellular cccDNA in therapy group could be detected. Conclusion: Long term nucleoside analogue therapy may consume hepatocellular cccDNA with decreasing of hepatocellular tDNA and sera HBVDNA, HBsAg; Although the patients have satisfied the China criteria of therapy endpoint, low level of hepatocellular HBVcccDNA were detected, cessation of therapy may cause relapse.
Peptides that Lead Nuclear Entry of Nucleocapsid of Hepatitis B Virus in HepG2.2.15 Cells X.B. Pan 1,2 , L. Wei 1,2 , J.C. Han 1,2 , K. Deng 1,2 1 Peking University Hepatology Institute, 2 Peking University People's Hospital Background: The nuclear entry of nucleocapsid is a key step for the HBV life cycle and the formation of covalently closed circled DNA (cccDNA). It has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (NLS). Whereas HBcAg was primarily distributed in cytoplasm and no marked cccDNA was detected in HepG2.2.15 cells. Methods: We designed peptides containing a cell-penetrating sequence (RRRRRRR) and a nucleocapsid binding sequence (GSLLGRMKGA) with/without a classic nuclear localization sequence (PKKKRKV) and these sequences were linked by a soft linker Acp. HepG2.2.15 cells were treated with the peptides at levels of 0 M, 25 M and 50 M for 4 days. Results: Compared with that of control cells, the results showed HBV DNA levels in culture medium decreased at least 2 log10 both in 50 M of peptide RRRRRRRAcpGSLLGRMKGA treatment group and RRRRRRRAcpGSLLGRMKGAAcpPKKKRKV treatment group; whereas HBsAg and HBeAg increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. The signal strength of cytoplasmic HBcAg increased at about 2.5-fold in both groups. In RRRRRRRAcpGSLLGRMKGAAcpPKKKRKV treatment group, nuclear HBcAg increased about 3.2-fold and obvious cccDNA signal was detected by southern blot. Conclusion: Our results implied that the NLS of core protein likely does not expose to surface of nucleocapsid in HepG2.2.15 cells, the artificial peptide containning NLS binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccDNA. Our study presents a tool for study on cccDNA formation and nuclear entry of nucleocapsid.
B. Tang 1 , J. Xia 1 , Y.M. Wang 2 , H.F. Wang 1 1 Liver Failure Treatment and Research Center, 302rd Military Hospital, 2 The Dept.
Aims: To establish a reliable real-time fluorescence quantitative (RFQ) PCR method to quantify HBV cccDNA, basing on LightCycler system and Taqman probe. Methods: HBV genotypes A-G were aligned to obtain a conserved sequence, crossing rcDNA gap, which was used to design cccDNA primers and Taqman-MGB probe. Also another pair of primers for quantify HBV total DNA (tDNA) was designed, utilizing the same probe. To increasing specificity, we added plasmid-safe ATP-dependent dnase (PSAD) digestion step just before cccDNA PCR amplification. A standard curve from 9 standard plasmid samples, from 2.0×10 9 to 2.0×10 1 copies, was created to examine our system. 50 HBV cccDNA samples with known-amount were quantified by creating standard curve from 5 standard samples. Results: The standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. The slope (regression coefficient) of standard curve was -3.141, mean square error was 0.0990 and regression coefficient was -1.0. All of these key indexes measured up. In 50 tests, we got right results if the starting templates of cccDNA copies were between 10 2~1 0 9 copies. The range was superior to commercial HBV kits. By quantifying 16 samples containing different amounts of cccDNA and rcDNA, digested or undigested, PSAD digestion would eliminate 10 7 rcDNA molecules, leaving 10 2 cccDNA molecules untouched. The test specificity was maintained up to 1:100,000 ratio of cccDNA:rcDNA. Conclusions: The RFQ-PCR based on LightCycler system for HBV cccDNA quantification is reliable, sensitive, with high specificity and low cost. Background: To study the changes of Toll-like receptor (TLR)3 on dendritic cells derived from peripheral blood mononuclear cells(MoDC) and its role in the pathogenesis of chronic hepatitis B(CHB) and chronic severe hepatitis B(CSHB). Methods: The expressions of TLR3 on 10000 MoDC were stimulated by poly I:C, and then were determined by flow cytometry in 20 healthy controls, 28 patients with CHB and 30 patients with CSHB.The level of interferon IFN-was determined by ELISA. The differences of expression of TLR3 on MoDC and serum IFN-among the three groups of study subjects were determined by student-t test.The correlation between TLR3 and IFN-were determined by linear correalation test. Results: The values of mean fluorescence intensity(MFI) of TLR3 on MoDC of the healthy controls, patients with CHB and CSHB were 1593.00±349.65,1369.56±287.08,and 1203.96±192.40.The serum IFN-(pg/L) of respective groups was 172.66±37.96,107.98±31.15 and 72.06±29.58. There was a gradual decrease of these values from the group of healthy controls to the group of patients with CHB and CSHB .Significant positive correlations between TLR3 and serum IFN-were found. Conclusion: TLR3 may have a role in the pathogenesis of CHB and CSHB.
Background/Aim: To evaluate the efficacy and safety of entecavir treatment in patients with HBeAg-positive chronic hepatitis B who had not previously received a nucleoside analogue. Methods: Fifty-five patients received 48-week entecavir 0.5mg/d therapy. Serum HBV DNA load was measured with quantitative real-time-PCR. Alanine aminotransferase (ALT) activity, HBeAg, anti-HBe-antibodies, HBV DNA level in serum were evaluated at baseline, week 2, 12, 24 and 48 during therapy. Evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. Results: HBV DNA levels declined sharply by around 3 log10 copies/mL during the first two weeks, with a highly significant reduction (p<0.0001) at week 2 and thereafter, as compared to those at baseline; 31%, 51% and 78% of the patients had undetectable serum HBV DNA levels at week 12, 24 and 48 respectively. Highly significantly decreasing serum ALT (p<0.0001) occurred during the first 2 weeks of the study. At week 48, ALT levels were normalized in 84% of the patients. HBeAg seroconversion (HBeAg negative, HBeAb positive) was achieved in 7.3% and 14.5% of patients by 24 and 48week. At the end of 24 th and 48 th weeks, complete response (ALT normalization and HBV DNA and HBeAg loss) was observed in 11% and 15%, respectively. There was no evidence of drug resistance or adverse effect in CHB patients treated for up to 48 weeks. Conclusion: Entecavir treatment through 48 weeks was well tolerated and resulted in continued benefit for patients with HBeAg-positive chronic hepatitis B. Aim: To assess the associations of single nucleotide polymorphisms of the MxA gene promoter and sustained treatment response of chronic hepatitis B or C patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. Methods: To clarify the impact of MxA gene promoter polymorphisms on sustained treatment response of chronic hepatitis B or C patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of MxA gene promoter polymorphisms at nt -88 G/G, -88 G/T, -88 T/T and nt -123 C/C, -123 C/A , -123 A/A alleles in individuals with interferon treatment. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. Results: The sustained treatment response rate was higher in patients with the nt -88 G/T and nt -123 C/Aalleles in the MxA promoter SNP . The Meta-analyses yielded summary estimatesodds ratio (OR) were 2.07 [95%CI (1.58, 2.7), P <0.00001] and 1.9 [95%CI (1.32, 2.73), P =0.0006] of the nt -88 G/T and nt -123 C/A alleles, respectively. Conclusion: MxA gene promoter polymorphisms at nt -88 G/T and nt -123 C/A may be useful as a marker to predict the sustained treatment response of chronic hepatitis B or C patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. Background: To determine whether HBV with the same characteristics causes dissimilar mutations in different hosts. Methods: Full-length HBV genome was amplified and linked with pMD T18 vector. Positive clones were selected by double-restriction endonuclease digestion (EcoRI and HindIII) and PCR. Twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (S)]. BioEditor, Clustal X and MEGA software were used to perform phylogenetic and mutational analysis. Potential immune epitopes were determined by the Stabilized Matrix Method (SMM), SMM-Align Method and Emini Surface Accessibility Prediction. Result: All of the 27 sequences were genotype C, the inner-divergence for the mother and son was 0%-0.8%.13 specific nucleotides differed from the other pubished genotype C isolates were co-exist in the mother and her son. AA 1-11 deletion in preS1 was the dominant mutation in the mother (14/18). The 1762T/1764A double mutation existed in all clones of the mother, 3 of them were also coupled with G1896A mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential HLA class I epitopes and one B cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the P gene. Conclusion: The son was infected HBV from his mother, and discrepant mutation occurred in the mother and her son during infection. Background/aims: Nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis B. This randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after 104-week therapy in patients with compensated chronic hepatitis B in Taiwan. Methods: We analyzed 114 Taiwanese patients from GLOBE trial receiving telbivudine 600mg (n=58) or lamivudine 100mg (n=56) once daily for 104 weeks. The primary efficacy endpoint was therapeutic response with serum HBV DNA <5 log10 copies/ml and either hepatitis B e antigen (HBeAg) loss or alanine aminotransferase (ALT) normalization. Results: The therapeutic response at week 104 was 74.8% in telbivudine group versus 50% in lamivudine group (p=0.005). More patients with telbivudine achieved nondetectable serum HBV DNA (<300 copies/ml) (p=0.024) and ALT normalization (p=0.021) at the end of treatment. The cumulative resistant rate was significantly lower in those with telbivudine treatment (p=0.0032). The rate of HBeAg seroconversion was comparable in both groups (p=0.407). Although a lower percentage of patients in lamivudine group (83.9%) reported adverse events than those in telbivudine group (89.7%), the difference was not significant. Conclusions: Telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis B in Taiwan. Background: Tumor necrosis factor-(TNF-) plays a pivotal role in the viral clearance and host immune response to HBV, and the capacity for TNF-production in individuals is influenced by a major genetic component. The studies of TNF--308 gene promoter polymorphism in chronic HBV infection have reported apparently conflicting results. Objective: To derive a more precise estimation of the relationship between the polymorphism of TNF--308 gene promoter and chronic HBV infection. Method: Meta-analysis was done of 22 case-control studies in relation to TNF--308 gene promoter, involving a total of 4338 chronic HBV infection cases and 3013 controls. The pooled odds ratios (ORs) for the risk associated with the genotypes of GA, AA, and GA+AA (A-allele carriers) compared with the GG genotype were calculated.
Results: Overall meta-analysis indicated that -308A heterozygotes (GA) had 22% decreased risk of developing CHB with a borderline significance (OR = 0.78; 95% CI: 0.60-1.02; P = 0.065). For the -308A allele homozygotes (AA) and carriers (GA+AA), the pooled ORs both indicated a significantly decreased risk of CHB (OR = 0.39; 95% CI: 0.21-0.73; P = 0.003; and OR = 0.74; 95% CI: 0.57-0.96; P = 0.026, respectively) ( Table 1 ). In the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (GA and AA) in Mongoloid populations in all genetic models. However, no significant associations were found in Caucasoid. Conclusion: The meta-analysis suggests that the TNF--308A allele is a low-penetrant protective factor for chronic HBV infection, especially in Mongoloid. Aim: To define the potential role of PD-1/PD-Lpathway in different HBV infection status; we examined the expression of PD-1on CD8+ T cells in PBMC of patients with CHB and AEHB infection. Methods: The PD-1 level on CD8+ T lymphocytes and the number of HBV specific CD8+ T lymphocytes in patients and healthy controls were analyzed by flow cytometry. PCR was used to measure the serum HBVDNA levels. Results: The level of PD-1 expression on CD8+ T cells in CHB patients was higher than that in AEHB patients and healthy individuals. Compared to AEHB patients, lower frequency of HBV-specific CD8+ T cells was detected in CHB patients. There was an inverse correlation between the strength of HBV-specific CD8+ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8+ T cells in CHB and AEHB subjects. However, PD-1 expression was not associated with ALT levels. Conclusion: Our results confirm previous reports that HBV specific CD8+ T-cell response in the peripheral blood is more intense in patients with AEHB than in CHB with persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8+ T cell response. observed in 10.9% of patients with a mean age of 51.8±12.9. The ethnic composition was 53.1% Chinese; 27.3% Malay; 14% indigenous Sabahans; 2.9% indigenous Sarawakians; 1.8% Indians and 0.8% others. Chinese patients were on average, older (mean 45.6±14.5 years), Indians patients had higher mean alanine transaminase and indigenous Sarawakian patients had the highest rate of cirrhosis (P<0.0001). During the study period, 20.7% of patients were on treatment and they were significantly older than those who were not on treatment (mean age 44.6 ± 14.5 vs 40.7 ± 14.3). Lamivudine was the first agent used in 86.9% of cases. Conclusions: In Malaysia, CHB remains a public health issue and significantly afflicts males in the productive age groups and of Chinese ethnicity. The observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. Background/Aims: Liver stiffness measurement (LSM) has been validated for predicting fibrosis stage in patients with chronic hepatitis C. However, studies on LSM for chronic hepatitis B (CHB) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. This study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with CHB Methods: Thirty three patients who had taken liver biopsy and LSM at Korea University Ansan Hospital between March 2008 and October 2008 were enrolled. Necroinflammatory activity and fibrosis stage were assessed by Metavir system. Activity, fibrosis, and the sum of both score were included for the correlation analysis with LSM Results: Among 33 patients, 29 (87.9%) were male, and median values were as follows: age, 42 (20~54); AST, 49 IU/L (19~627); ALT, 62 IU/L (8~778); Total bilirubin, 1.03 mg/dL (0.4~2.39); LSM, 10.1 kPa (4.2~43.5). Fibrosis stages were F1 in 4 (12.1%), F2 in 9 (27.3%), F3 in 12 (36.4%), and F4 in 8 (24.2%) patients. Spearman correlation coefficient with LSM were 0.457 (p=0.008) for activity, 0.694 (p<0.001) for fibrosis stage, and 0.724 (p<0.001) for the sum of activity and fibrosis. In linear regression analysis, only the sum of activity and fibrosis remained to be significant. Conclusions: Not only fibrosis but also activity was an important factor for determining LSM for CHB. It would be more appropriate to consider both activity and fibrosis for interpretation of LSM in patients with CHB Background: HBeAg seroconversion is a key goal of CHB therapy. HBeAg kinetics may predict HBeAg seroconversion during treatment. We aim to develop a robust HBeAg quantitative method as the value of HBeAg quantitation is undefined and data is limited. Methods: We evaluated two commercially available qualitative HBeAg assays (Abbott Architect, Siemens Centaur) for their linear range and validated them against Paul-Ehrlich Institute (PEI) standards. HBeAg levels were determined from samples of untreated and telbivudine-treated CHB patients. Results: As a pre-requisite for quantitative use, the linear range for the Architect (0.5-43 PEIU/mL) and Centaur (0.05>5 PEIU/mL) assays were defined. Architect was selected for further investigation. HBeAg levels of 44 untreated patients (mean HBV-DNA 9.8 log10 copies/mL, mean ALT 166.7 IU/mL) varied from 0.4 to 1073.5 PEIU/mL (median 161.7 PEIU/mL). In 23 patients (mean HBV-DNA 9.9 log10 copies/mL, ALT 166 IU/mL) treated with telbivudine for 12 weeks, baseline HBeAg levels varied from 1.6 to 664 PEIU/mL (median 150.7 PEIU/mL). After 12 weeks of telbivudine treatment, median HBeAg level was 4.4 PEIU/mL, with 76% decline from baseline (median decline 95.1 PEIU/mL, range 1.4-657.6 PEIU/mL). Individual HBeAg decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline HBVDNA. Conclusion: HBeAg quantitation is feasible and robust with Architect HBeAg assay. HBeAg decline occurred in all telbivudine-treated patients, and was not correlated to HBVDNA. Whether the magnitude of HBeAg decline is predictive of future HBeAg seroconversion merits further investigation.
Experience from the Combined GLOBE (NV-02B-007/CLDT600A2302) and 015 (NV-02B-015) Study Clinical Safety Database. C. Avila 1 , R. Laeufle 2 , W.B. Bao 3 1 Novartis Pharma AG, Fabrikstrasse 6, Basel, Switzerland, 2 Novartis Pharma AG, Basel, Switzerland, 3 Novartis Pharma, East Hanover, US Background: Creatine phosphokinase (CK) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). Normal CK levels are affected by muscle mass and elevated levels are described during the natural course of CHB. 22% of patients in the GLOBE study had pretreatment Grade 1-4 CK elevations. Methods: We reviewed data from this combined study clinical safety database, and describe the experience of CK elevation and its relationship to adverse event reports of muscle related symptoms. Results: The frequency of new onset of Grade 3-4 CK elevations in telbivudine-treated patients (combined database ITT population) was 1.8% (15/847), 6.3% (53/838), 3.2% (27/826) and 5.1% (40/786) from weeks 0-24, 24-52, 52-76 and 76-104 respectively. The frequency of Grade 3-4 CK elevations for all patients from week 0-104 was 12.6% (107/847). The majority of Grade 3-4 CK elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within 1 or 2 visits and were not associated with more frequent muscle-related adverse events. Cumulative data from this combined database showed no relationship of the degree of increased CK to acute or persistent muscle disease. Conclusion: CK elevations are associated with HBV disease and were also common during the GLOBE and 015 trials and were not predictive of the development of muscle related symptoms. Onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. Backgrounds: Leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (Ob-RL). Epidemiologic studies showed that obesity is one of the factors associated with HBV related hepatocellular carcinoma. Methods: Huh7 cells were transiently transfected with 1.3 copies of HBV-replicon plasmid. After 48h, cells were harvested and total RNA of the cells were extracted and reverse-transcribed into cDNA. Long form and short form leptin receptor (Ob-RL, Ob-RS) mRNA transcription levels were assayed by Real-time PCR respectively. And mRNA transcription levels and protein expression of Ob-RL and Ob-RS in HepG2.2.15 cells were also detected. Results: After transfected by 1.3 copies HBV-replicon plasmid, the mRNA transcription level of Ob-RL was inhibited significantly (**P<0.01), but the mRNA transcription level of Ob-RS did not change, and the Ob-RL protein expression was reduced. In HepG2.2.15 cells, the mRNA transcription level of Ob-RL was also significantly lower than the mRNA transcription level of Ob-RL in HepG2 cells, while the mRNA transcription of Ob-RS in HepG2.2.15 and HepG2 cells didn't show significant difference. Besides, the protein expression level of Ob-RL in HepG2.2.15 was also lower than it in HepG2 cells. Conclusion: HBV replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in HBV infections.
The Results of Entevavir Treatment in Patients with Chronic Hepatitis B S. Kose 1 , G. Akkoclu 1 , M. Turkeri 1 , A. Gozaydin 1 1 The Ministery of Health Tepecik Training and Research Hospital, Izmir Purpose: We evaluated the short and long term effectiveness of entecavir. Patients and Methods: Those patients had received diagnosis of chronic hepatitis B. Their pretreatment transaminases, HBsAg, anti-HBs, HBeAg, anti-HBe, HBV-DNA were checked and a liver biopsy and a resistance test for Lamivudine (LAM) and Adefovir (ADV) were performed. A total of 44 patients who were taking entecavir for at least 24 weeks were included in the study. Findings: The biochemical and virologic response were observed in 88.8 % at 3 and 6 months and in 75 % at 12 months. In 9 HBeAg positive patients who had received therapy previously, the biochemical response was observed in 88.8 % at 3 and 6 months and in 100 % at 12 months. The virologic response in 77.7 % at 3, in 88.8 % at 6, and 100 % at 12 months. Posttreatment HBeAg seroconversion did not develop. In 9 HBeAg negative patients the biochemical and the virologic responses were observed in 88.8 % at 3 months and 100 % 6 and 12 months, respectively. In 17 HBeAg negative patients had received therapy previously, the biochemical response was observed in 76.4 % at 3, in 100 % at 6 and 12 months. The virologic response in 94.1 % at 3, in 100 % at 6 and at 12 months. Conclusion: In our study, a higher therapy-response rate was achieved, especially in HBeAg negative patients. In HBeAg positive patients biochemical and virologic response rates were high. Background/Summary: Patients with liver disease are known to have a higher prevalence of glucose intolerance. Preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. Individuals with type II diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. There is now emerging epidemiological data to suggest that Hepatitis C virus (HCV) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. While these investigations suggest an epidemiological association between HCV infection and diabetes, large controlled studies are required to observe association between HBV infection and diabetes. The present study was designed to study the relative proportion of Diabetes mellitus in patients suffering from hepatitis B virus (HBV) infection. Background: In EASL 2008, we reported significant liver disease among HBeAg negative patients with serum HBV-DNA level <4log10copies/ml (i.e. 2.0x3log10IU/mL) and alanine aminotransferase (ALT) <55IU/L. This reflects fluctuating nature of these levels. The aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of CHB patients. Methods: Clinical records of HBeAg negative treatment naïve CHB patients with at least one serum HBV-DNA <4log10copies/ml were reviewed. Results: There were 194 (62.5% male, median age 48.0 years) CHB patients with negative HBeAg and HBV-DNA <4log10 copies/ml (Roche COBAS Amplicor PCR assay, LOD<300copies/ml). 32 had serial HBV-DNA measurements within 2 years; 7 of them (21.9%) had increase serum HBV-DNA level by >1log10 copies/ml; 2 patients had associated serum ALT elevation from normal (normal range <55 IU/L), 4 had persistent normal ALT, and one had persistently raised ALT. 5 (15.6%) had serum HBV-DNA level decrease by >1log10 copies/ml. Another 20 patients had HBV-DNA levels fluctuating within 1log10 copies/ml. 159 HBeAg negative patients with single HBV-DNA measurement showing <4log10 copies/ml had serial serum ALT measurements within 3 years. 35 (22.0%) patients had intermittent / persistently raised ALT; while 124 (78.0%) patients had persistently normal ALT. Conclusions: HBeAg negative CHB is common among Chinese. Serial serum HBV-DNA and ALT measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. Background: To determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. Methods: A model was built in order to obtain the age-specific infection frequency Q(t) for neonates(n), infants(i), children(c), and adults(a). The infected group can either become HBsAg(+) or anti-HBs(+)* based on F(t). Q(t) can be found from P(t) = [Q(t) x (1 -F(t)) x CRs + Q(t) x F(t) x (1 -CRas)], where P(t) represents the proportion of the late anti-HBs(+)** group and CRs/CRas denote natural conversion of HBsAg/anti-HBs. To test the model, cross-sectional serologic marker data in Korea were used. Because F(t), CRs, and CRas were known(F(n)=0.1, F(i)=0.5, F(c)=0.8, F(a)=0.95, CRs=0.015, CRas=0.02), in order to determine Q(t), only P(t) values were needed, which were evaluated from logistic modeling using the glm() function of S-PLUS. Results: The infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were 15.4%, 33.3%, 16.6%, and 34.7%, respectively. Each group's likelihood of infection compared to adults was then: neonates 170.2 times more likely, infants 33.5 times, and children 0.9 times, making a strong case for neonatal and infantile vaccination for the studied region. Conclusions: The HBV infection model can be used for determining the most cost-effective strategy for HB vaccination in nations where longitudinal data are not available. And where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness.
The Effect of Telbivudine on Peripheral Blood Regulatory T cells and Its Significance in Patients with Chronic Hepatitis B X.C. Pan 1 , F. Yang 1 , M. Chen 1 1 
Objective: To investigate the effect of Telbivudine on peripheral blood regulatory T cells and its significance in patients with chronic hepatitis B. Methods: 36 patients with HbeAg positive chronic hepatitis B were recruited and receiving telbivudine treatment for 9 months. Before and during 3 6 9 months of treatment , Flow cytometry was used to detect the proportion of peripheral blood Tregs; real-time PCR was used to detect the levels of HBV DNA in surum, markers of hepatitis B virus infection were detected by ELISA assay and levels of alanine aminotransferase in serum were measured. Results: The proportion of peripheral blood Tregs in patients with CHB was significantly higher than that in healthy controls and decreased over 6 or 9 months of treatment to a level comparable to that of healthy controls. After 3 months of treatment, The rate of ALT normalization in patients which the proportion of peripheral blood Tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood Tregs was reduced (P<0.01). 3, 6 or 9 months of telbivudine treatment resulted in negative HbeAg in 4(11%) patients, 7(19%) patients or 9(25%) patients respectively. Within 9 months of treatment, 7 (19%) patients seroconverted from HBeAg to anti-HBe , in which the proportion of peripheral blood Tregs had decreased to a level comparable to that of healthy controls over 3 or 6 months of treatment. Conclusion: During antiviral treatment with subsequent reduction of the viral load or ALT levels, the proportion of Tregs decreased to a level similar to that of normal healthy controls. In addition, seroconversion from HBeAg to anti-Hbe was prone to be established in patients which the proportion of Tregs decreased quickly at the early phase of antiviral treatment with Telbivudine. Background: In China a part of patients with ALT <1.5×ULN and HBV DNA >10 5 copies/ml will advance into hepatic cirrhosis even hepatoma. So these patients should not only be monitored but also be treated. This study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha 2a (peg-IFN -2a) and entecavir in treating naive patients with ALT <1.5×ULN and HBV DNA>10 5 copies/ml. Methods: Nine patients with HBsAg positive over 6 months and ALT<1.5×ULN HBV DNA >10 5 copies/ml were taken as research subjects. Before treatment,liver biopsy was used to assess histological damage. Patients were treated with peg-IFN -2a 180 g /week for 48 weeks, and in the first 12 weeks entecavir 0.5 mg/day was applied, then it was stopped. Results: 1 Liver biopsy showed that 7 patients had mild inflammation. 2 After 12 weeks' treatment , HBV DNA level in all patients decreased to less than 10 4 copies/ml, and after 24 weeks' treatment(12 weeks after entecavir was stopped)HBV DNA in all patients was less than 10 3 copies/ml. 3 Normal ALT was seen in all patients after 12 weeks' treatment and 24 weeks' treatment. 4 None of the patients had peripheral neuropathy with combining treatment. Conclusions: 1. Bulk of patients with ALT <1.5×ULN and HBV DNA>10 5 copies/ml had mild inflammation and need treatment. 2 Combing treatment of peg-IFN and entecavir was safe and effective to this group. 3 It proved that it was safe for patients to stop treatment with entecavir after short time use. Background & Aims: Quantification of serum HBV DNA levels is important to monitor viral replication in chronic hepatitis B (CHB) patients. Both Abbott RealTime HBV and Roche COBAS Amplicor HBV Monitor are updated fully automatic commercial assays for HBV DNA quantification. The aim of this study is to compare the performance of these two assays on the HBV DNA quantification in CHB patients. Methods: Serial serum samples from 30 CHB patients were collected at the baseline and at days 4, 7, 10 and 14 and weeks 3, 4, 8 and 12 after the commencement of therapy. Genotype was determined by sequence alignment. Abbott and Roche assays were employed for HBV DNA extraction and quantification according to the instructions of manufactories. Results: HBV DNA quantification results of Abbott assay was significantly correlated with those of Roche assay (r=0. 972, P<0.0001). For genotype C, the difference in HBV DNA levels [median (range): 1.22 (-0.16-2.36) log units] measured by these two assays was significantly higher than that for genotype B [0.66 (-0.52-1.63) log units, P<0.0001]. Moreover, the difference in serum HBV DNA levels after 12 weeks antiviral treatment [1.18 (-0.21-1.76) log units] measured by these two assays was significantly higher than that in baseline serum HBV DNA levels [0.68 (-0.12-1.24) log units, P<0.0001]. Conclusion: The quantification results of Abbott RealTime HBV showed a good correlation with those of Roche COBAS Amplicor HBV. But the performances of these two assays have significant difference in the quantifications of serum HBV DNA levels in genotype C patients and in patients after 12 weeks antiviral therapy. Background: Guidelines suggest Hepatitis B Virus (HBV) vaccination to all Hepatitis C Virus (HCV) infected patients and healthcare workers. We attempted to find out HBV vaccination status in our HCV infected population, and healthcare workers.
Methods: Prospective survey of 100 consecutive HCV infected patients and also doctors and paramedical staff in our hospital. Results: Major sources of viral infection in study patients (58 males; average age 40 years -range 18 to 65 yrs) were reused syringes (38 pts). Twenty had a household member infected with HCV. Twenty were co-infected with HBV. Eighty five of HCV infected patients were not vaccinated.against HBV. Twenty five of them (29%) had financial reasons and 45 patietns (52%) had lack of awareness. Out of 30 doctors, 12 and 15 did not know about their HBV and HCV status respectively, but none was known to have either of these infections. Four (13%) were not vaccinated against HBV. Out of 29 paramedical staff, 1 was HCV positive, 11 each were unaware of their HBV and HCV status, and remaining were negative for these markers. Thirteen of them (44%) were not vaccinated against HBV. Conclusion: Thirty eight percent HCV infected patients were infected by reuse of syringes. Eighty five percent were not vaccinated against HBV, out of which 52% had no awareness about it, whereas 29% could not financially afford it. A significant number of paramedical staff and some doctors were also not vaccinated Background: Early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis B. Methods: A multi-center clinical study. Patients were injected interferon alpha 2b 5 Million IU subcutaneously every other day for 24 weeks and 24-week follow-up was followed. Results: 53 patients (44 male) were enrolled, 27.5 ±9.1 years old. 24 hours after administration, hepatitis B virus (HBV) load decreased significantly (6.96±1.03log copies/ml, P<0.05) from baseline (8.00±0.93 log copies/ml). HBV load was 4.94±1.47 and 5.25±1.58 log copies/ml at week 24 and 48, respectively. At the two points upwards, complete response rate was 4.2%(2/48) and 7.9%(3/38), partial response rate was 35.4%(17/48) and 34.2%(13/38), respectively. At week 4 and 12, HBV DNA levels of complete responder and partial responder were lower than those of non-responder (P<0.05) at week 24. At baseline, on hour 12, day 2, 3, week 2, 4 and 12, HBV DNA levels of complete responder were lower than those of non-responder at week 48 (P<0.05). Multiple linear regression showed that baseline HBV DNA was the independent variables to predict the response at week 24 and 48. Conclusions: Interferon alpha 2b was effective in treating patients with HBeAg positive chronic hepatitis B. It could decrease the HBV DNA level rapidly. Early HBV DNA levels were predictive to response at the end of treatment and follow-up. Baseline HBV DNA level was the independent predictor of the response at the end of treatment and follow-up. Aim: To investigate features of PD-1 expression on peripheral Tcells and PD-L1 expression in liver in chronic hepatitis B (CHB) patients in immune clearance phase. Methods: PD-1 expression on total peripheral T cells were evaluated by using flow cytometry. Immunostaining was performed according to the EnVision ChemMate methods. The degree of PD-L1 expression was scored and assessed according to the percentage and staining intensity of positive cells. Results: Compared to health control, the percentage of total peripheral T cells expressed PD-1 was elevated in CHB with repeatedly increasing ALT level. No specific association between the percentage of PD-1 positive and the mean fluorescence intensity MFI of PD-1 expression on total T cells with serum viral load were found. But ALT level was correlated with the MFI of PD-1 expression on total CD8+T cells significantly. PD-L1 is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. Conclusion: The MFI of PD-1 on CD8+T cells plays important role in regulating the immune-host interaction in CHB in immune clearance phase. And PD-1 expression on T cells is correlated with high immune inflammatory refection. Aim: To study the quantity, characteristic of HBV-specific T-cell and the extent of liver damage in chronic hepatitis B (CHB) patients with different HBeAg status. Methods: 103 CHB patients were enrolled and divided into two groups according to the HBeAg status, and the liver damage index were analyzed. The frequency and Foxp3 expression of CD4 + CD25 + regulatory T cells (Treg) were measured, as well as the frequency and phenotypic molecules expression of HBV-pentamer+ T-cell. HBV specific T-cell responses including cellular proliferation and IFN-production, with or without anti-PD-L1 and/or anti-CTLA-4 blocking, were also observed. Results: The demographic characters, serum ALT, AST levels, the frequency and Foxp3 expression of CD4 + CD25 + Treg were similar, while the serum HBV DNA levels were higher in HBeAg+ patients (P <0.05). The liver necroinflammation was comparatively more severe in HBeAg-patients (P =0.056), but the median percentage of liver cirrhosis was much higher in HBeAg+ patients (P <0.05). The difference of HBV-specific T-cell frequency was not significant between two groups, while the expression levels of PD-1 and CTLA-4 on HBV-specific CD8 T cells were significantly higher in HBeAg+ patients (P both <0.05). Combined using of anti-PD-L1 and anti-CTLA-4 mAb significantly increased the cellular proliferation in either HBeAg+ or HBeAg-patients, but only markedly enhanced the IFNproduction in HBeAg+ patients. Conclusion: HBeAg persistency could probably induce higher expression of PD-1 and CTLA-4 on the HBV-specific T cells and result in T-cell impairment, high HBV DNA load and high percentage of liver cirrhosis in HBeAg+ CHB patients.
Hepatocyte Apoptosis in Patients with Chronic Hepatitis B Y. Liu 1 , K. Wang 1 1 
Background: To investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (iNOS) in hepatic tissue in the patients with chronic hepatitis B CHB . Methods: we observed 37 cases with CHB and 10 normal controls. Transferase-mediated-UTP-biotin nick-end labling ( TUNEL) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (iNOS) in biopsy samples .The serum level of ALT HBV-DNA grading of necroinflammatory activity and staging of fibrosis were also assessed. Results: Hepatocytes in all CHB liver tissues were positively stained by TUNEL in various degree. In contrast, control tissues did not show DNA fragmentation. A significant correlation was seen between apoptosis index (AI) and necroinflammatory grading ((r=0.404, P=0.015) and serum iNOS level r=0.465, P=0.004 . It did not correlate with fibrosis stage and serum alanine aminotransferase level. Conclusion: The oxidative stress.in patients with CHB may reflected the apoptosis of hepatocyte. Apoptosis involves in liver injury of CHB,but with no significant correlation to serum level of ALT. Objectives: To investigate the genotype-dependent development of lamivudine resistance in hepatitis B virus (HBV). Methods: 215 patients with chronic hepatitis B who had been treated with lamviudine for more than 1year, and become lamviudine resistance were analysed for the HBV genotypes and cumulative rate of RT region mutant with standard DNA sequencing technology. Results: Among the 215 patients, 60 patients were infected with HBV genotype B (HBV/B)(27.9%), and 155 with genotype C (HBV/C)(72.1%). In the HBV/B patients, 16/60(26.7%) were of subtype Ba, and 44/60(73.3%) were of of subtype Bj. The cumulative type and YMDD mutation rates in patients with genotype C were showed as L180M+M204V (67/155,43.2%) > L180M+M204I (52/155,33.5%) > M204I (36/155, 23.3%), while in patients with genotype B as L180M+M204V 36/60(60.0%) > M204I(24/60, 40.0%), none of L180M+M204I. Conclusions: Our results indicated that in patients with lamivudine resistance, HBV genotype C (HBV/C) were higher than genotype B (HBV/B). In both genotypes the combined mutations (180+204 sites) were found more than the single 204 site, showed some significance for monitoring lamivudine resistance. 
Background & Aims: IL-35, a novel identified inhibitory cytokine specifically produced by regulatory T cells (Tregs), is an Ebi3-IL-12 heterodimer encoded by Epstein-Barr-virus-induced gene 3 (Ebi3) and interleukin-12 alpha (Il12 ). The aim of the study is to determine the expression levels of IL-35 in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients in different phases. Methods: A total of 36 treatment naïve CHB patients, including 17 in immune-tolerant phase [group 1, ALT: 21 (12-51) U/L, serum HBV DNA: 2.48 x 10 9 (6.30 x 10 6 -1.20 x 10 10 ) copies/ml] and 19 in immune-clearance phase [group 2, ALT: 221 (71-1530) U/L, serum HBV DNA: 5.10 x 10 8 (1.62 x 10 6 -1.77 x 10 10 ) copies/ml] were enrolled in the study. The relative mRNA expression levels of Ebi3, Il12 and FOXP3 were determined by semi-quantitative PCR. Results: The significant correlations were observed between the expression of Ebi3 and Il12 (r=0.661, P<0.001), Ebi3 and FOXP3 (r=0.388, P<0.05), Il12 and FOXP3 (r=0.431, P<0.01). The relative expression levels of Ebi3 and Il12 in PBMCs were significantly higher in group 2 when compared with those in group 1 (1.46 ± 0.23 vs 0.80 ± 0.10 and 1.26 ± 0.19 vs 0.65 ± 0.09, P<0.05, respectively). Furthermore, the relative expression levels of Ebi3 and Il12 in group 2 were significantly correlated with ALT levels (r=0.473, r=0.474, p< 0.05, respectively), but not with serum HBV DNA levels.
Conclusions: The expression levels of IL-35 in PBMCs were significantly higher in CHB patients in immune-clearance phase than that in immune-tolerant phase. Increased IL-35 expression levels were associated with liver injury. Background: There are a number of oral antivirals approved for chronic hepatitis B. Lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from 20% at one year to 50% at three years. Therefore, an alternative or add-on treatment is necessary. Adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis B. We did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis B infection. Methods: A comprehensive literature search was performed using the following databases: Medline, Cochrane, and Embase. A total of 3 randomized controlled trials were retrieved and analyzed. Outcomes measured were virologic response, biochemical response and resistance rates. Results: Meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (OR 1.04, 95% CI 0.50-2.15, P=0.92). Likewise, in terms of biochemical response, both regimens were equally effective (OR 1.06, 95% CI 0.47-2.40, P=0.90). One study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (P= 0.0182) after the first year of therapy. Conclusion: In patients with lamivudine-resistant chronic hepatitis B infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response.
R. Safadi 1 , Q. Xie 2 , Y.G. Chen 3 , Y.K. Yin 4 , L. Wei 5 , S.G. Hwang 6 South Korea, 7 Bnai Zion Medical Center, Haifa, Israel, 8 Beijing Friendship Hospital, Beijing, China, 9 Novartis Pharma AG, Basel , Switzerland Background: LdT produces greater viral suppression than LAM. We investigated whether patients receiving LAM can benefit from switching to LdT. Methods: HBeAg positive and negative persistently viraemic patients (median HBV DNA 5.0 (LdT), 5.3 (LAM) log 10 copies/mL) and LAM treated for 3-12 months, were randomized to either switch to LdT or continue LAM. We report the benefit of LdT switch assessed by primary treatment failure (TF, <1 log HBV DNA decline) and viral breakthrough (VB, >1 log above nadir). Results: 17% (21/122) of the LdT switch and 15% (18/124) continuing LAM patients had pre-existing M204 mutations at screening. TF was 5% (LdT) versus 21% (LAM, p<0.05). In patients with >24 weeks prior LAM treatment, TF was 10% (LdT) versus 41% (LAM). 83% LdT TF (5/6) was associated with resistance at screening versus 56% LAM TF. In LdT switch with <24 weeks prior LAM, no LdT TF occurred versus 12% LAM. In HBeAg positive, TF occurred in 6% (LdT) versus 29% (LAM). Among HBeAg positive with >24 weeks prior LAM treatemtn, VB was 19% (LdT) versus 44% (LAM, p<0.05). Differences were not significant for HBeAg positive with >24 weeks LAM or for HBeAg negative regardless of duration of prior LAM treatment. Conclusions: Early switch to LdT is associated with better virological outcomes in these patients. Persistent viraemia for >6 months on LAM treatment is associated with a high risk of TF and VB. For these patients, genotypic analysis is recommended prior to screening. Objective: The aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis B with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . Methods: eAg positive chronic hepatitis B naïve patients with alanine aminotransferase (ALT) more than 2 ULN were assigned to receive 100 mg of lamivudine, 10mg of adeforvir, or 0.5mg of entecavir once daily, respectively. Patients in the interferon group were administrated with 5,000,000 IU of 2a interferon on every other day, and the therapeutic duration lasted for another three months after eAg-Ab seroconversion appeared. HBV DNA and eAg-Ab in the serum were tested during the off-treatment period of 12 months. Results: Thirty four patients in lamivudine group of 148 cases got eAg-Ab seroconversion after treatment with 20±5 months of average duration, and the viral rebound ratios in the off -treatment 6 an 12 months follow up period were 20.6 7/34 and 40.1 15/34 , respectively. In adeforvir group were 19 4/21 and 33. 7/21 . In enticavir group were 20 3/15 and 46.7 7/15 . In interferon group was 16.2 6/37 in the off-treatment 6 months follow up period. Conclusions: We conclude that eAg-Ab seroconversion in the treatment of eAg positive chronic hepatitis B patients is the goal but not an endpoint of therapy physicians should aim at. To gain everlasting effect, longer duration of treatment may be needed. Background: Universal hepatitis B(HB) vaccination of HBsAg negative people (especially infants) is widely recommended and practised. Objective: To assess whether there is robust evidence of protective efficacy to back such practice. Methods: This Cochrane review included randomised trials identified from six databases through detailed electronic searches. Trials comparing HB vaccine versus placebo/another vaccine, in HBsAg negative persons were included without any restrictions. The primary outcome was HB infection (developing HBsAg or anti-HBc). Robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(ITT) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario Results: Twelve trials were eligible among 2964 citations; all were methodologically poor (high risk of bias). Data from four trials could be included in meta-analysis. Efficacy of vaccination varied with the type of data analysis. Available-case analysis suggested efficacy in reducing risk of developing HBsAg (RR=0.17;95%CI=0.09-0.31;n=1341) and anti-HBc (RR=0.42;95%CI=0.31-0.57;n=1235). ITT analysis results varied depending on the model chosen (Table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not.
The available evidence on efficacy of HB vaccination in HBsAg negative people is not robust; there are serious limitations in quality and quantity. Background: Open-label rollover study (ETV-060) assessed histologic improvement in CHB patients on at least 3 years ETV therapy. Methods: 100% nucleoside-naïve patients and 98% lamivudine (LVD)-refractory patients from ETV-053 and ETV-052 studies, respectively, entered ETV-060 study and received ETV at 0.5/1mg for greater than 96 weeks. Improvement in Knodell necroinflammatory (NI) score and Knodell fibrosis score at Weeks 48 and 148 were studied. Results: At Week 148, 95% of nucleoside-na ve patients and 56% of LVD-refractory patients achieved HBV DNA <400copies/mL. Furthermore, 95% of nucleoside-na ve patients and 93% of LVD-refractory patients had normalized ALT levels. Mean platelet counts in both naïve and LVD-refractory patients were improved at Weeks 48 and 148 compared with baseline.
Conclusions: Naïve and LVD-refractory CHB patients showed significant improvement in liver histology after 3 year ETV therapy, and improved DNA and serum ALT levels. Results: 84.7 percent of patients were younger than 30 years old, 15.3 percent were older than 30 in this study. 48.0% patients' mothers were HBsAg positive. High levels of serum HBV DNA were founded in all patients, >10 7 copies/ml were 78.6%. Only 5 cases (5.1%) whose liver inflammation grade were G0, the rest patients were mild inflammation, in which G1 were 64 cases (65.3%), G2 were 29 (29.6%); there were 56 patients (57.1%) had no signifecant liver fibrosis, the rest 42 cases (42.9%) had different fibrosis, among those S1 were 23 cases (23.5% , S2 were 14 14.3% , S3 were 5 5.1% , none of patients had cirrhosis. The fibrosis stages of higher ALT level were markedly severer than lower ALT in patients with normal ALT P < 0.01 . Conclusions: Most of patients with chronic hepatitis B virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. Although ALT testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher ALT level, so liver pathology should be recommended to judge illness correctly. Background/aims: Hepatitis B virus infection (HBV) is a global health problem. In Bangladesh, 5-7% of people are HBsAg positive. This study was carried out to evaluate the efficacy and safety of peginterferon alfa-2a in chronic hepatitis B patients. Methods: A total of 60 patients with chronic hepatitis B, 32 (53.3%) were HBeAg positive (group A) while 28(46.7%) were HBeAg negative (group B) were included in this study after meeting the following criteria: age 18 to 60 years, HBsAg positive for more than 6 months, serum HBV-DNA was >5 log(10) copies/mL and ALT more than two times the upper normal limit. They were given peginterferon alfa-2a (180 microgram once weekly) for 24 weeks and followed for an additional 24 weeks. RESULTS: After 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or HBVDNA levels below 20,000 copies per milliliter was significantly higher in HBeAg positive patients (59 percent and 43 percent, respectively) than among HBeAg negative patients (45 percent and 33 percent). Loss of hepatitis B surface antigen occurred in 3 patients in group A, as compared with 1 patients in the group B (p<0.01). Adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. CONCLUSIONS: Patients with HBeAg positive chronic hepatitis B had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a. Background: The effect of hepatitis B vaccination on individuals with isolated anti-HBc in endemic areas is not clear. We investigated the prevalence of individuals positive for anti-HBc only and their antibody response after hepatitis B vaccination in a single healthcare center. Methods: The study included 1,812 healthcare workers. After screening for HBsAg and anti-HBs, the individuals negative for both HBsAg and anti-HBs were examined for anti-HBc and were vaccinated with a recombinant hepatitis B vaccine at 0, 1, and 6 months. The serum anti-HBs level was measured after the vaccination. Results: Of the subjects, 334 (220 females) were negative for both HBsAg and anti-HBs. Forty (2.2%) subjects had isolated anti-HBc, including more males (60.0% vs. 30.6%) and older people (45.7±8.2 vs. 37.3±8.5 years), compared with individuals negative for all of the viral markers. The anti-HBs seroconversion rate and anamnestic response in the individuals with isolated anti-HBc after the first vaccine injection were 60% and 27.5%, respectively. In the 294 persons who were negative for all hepatitis B viral markers, the seroconversion rate after the first vaccination was 52.6%. The anti-HBs seroconversion rate did not differ between the isolated anti-HBc positive individuals and those negative for all hepatitis B markers (89.5% vs. 96.6%) after the full course of vaccination. Conclusions: Serum HBsAg and anti-HBs tests are sufficient for screening before hepatitis B vaccination, especially in healthcare workers. 
Objective: To understand the quantity and distribution of CD83 + mature dendritic cells in patients with hepatitis B virus in immune tolerant phase. Methods: There were 30 immune tolerant phase patients with hepatitis B virus infection (fibrosis stages were S0), 10 immune clerance phase patients, 10 non-active status patients and 5 healthy controls involved in our research. The quantity and distribution of CD83 + mature dendritic cells in liver were determined by immunohistochemical staining. Result: The liver inflammation grades were between G1-G2 in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between G2-G4. There were a small amount of CD83 + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. The quantity and distribution of CD83 + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p 0.05 .The number of CD83 + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p 0.05 , the CD83 + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. Conclusion: CD83 + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis B virus clearance. Lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. Background: Local hospitals provide obstetric services including antenatal care to women normally living in the Mainland China, whose prevalence of hepatitis B carrier is unknown. Objectives: Compare prevalence of HBV carrier of pregnant women from the Mainland China with local counterparts and discuss the implications of results. Materials and Methods: Antenatal serological results were retrieved from corporate laboratory information system databases. Pregnant women from the Mainland China were identified by a specific set of temporary-allocated identity number during January 2007 -October 2008. Results: 7491 pregnant local residents and 1397 pregnant women from the Mainland China underwent antenatal serological tests for hepatitis B surface antigen. Positive hepatitis B surface antigen results were more frequent in pregnant women from the Mainland China (12.7%) than in local pregnant women (8.17%) (p<0.001). Discussion: Because infected pregnant women can transmit the hepatitis B virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis B immune globulin to the infant at birth and immunization later on. However, early repatriation to the Mainland China, which is common, will make completion of immunization program difficult. These babies will be at a higher risk to be infected by HBV, particularly when breast-fed by HBV carriers. Their return to Hong Kong later will dilute the effects of local immunization program. The volume of work derived from the provision of obstetric services to women from the Mainland Chinese is larger with regard to medication, counseling and immunization for babies born to HBV carriers.
Immunosuppressive or Anticancer Therapy K. Hirano 1 , T. Kodani 1 , S. Sato 1 , Y. Narita 1 , T. Kikuchi 1 , T. Genda 1 , K. Iijima 1 , K. Ogawa 1 , T. Ichida 1 1 
Background/Aim: We compared the prevention of HBV reactivation in (HBsAg)-positive patients with HBsAg-negative patients who were positive for antibody to (anti-HBc) and/or (anti-HBs) undergoing immunosuppressive, anticancer or molecular target therapy. Methods: From Sep 2004 to Nov 2008 HBsAg-positive patients and 22 anti-HBc and/or anti-HBs-positive patients were enrolled in this study. We compared with 2 groups about background disease, age, blood examination, and nucleoside analogues. Results: In HBsAg-positive patients mean age were 56.6±10.2 years old, median AST levels were 30 (18-706) IU/L, and median ALT levels were 36 (13-544) IU/L for 8 (47%) haematological disease and 9 (53%) collagenosis disease. In anti-HBc and/or anti-HBs-positive patients mean age were 71.3±9.6 years old, median AST levels were 20 (9-106) IU/L, median ALT levels were 16.5 (5-247) IU/L for 17 (77%) haematological disease and 5 (23%) collagenosis disease. Serum HBV-DNA levels >5.0 log copies/ml were 8 (47%), 2.6~5.0 were 7 (41%), <2.6 were 2 (12%) in HBsAg-positive patients, and serum HBV-DNA levels <2.6 were all cases in anti-HBc and/or anti-HBs-positive patients. 14 (82%) of HBsAg-positive patints received nucleoside analogues (7 LAM and 7 ETV), and 12 (55%) of anti-HBc and/or anti-HBs-positive patients received nucleoside analogues (8 LAM and 4 ETV). Mean duration of treatment for 14.2 months in HBsAg-positive patients, and for 4.1 months in anti-HBc and/or anti-HBs-positive patients, the resistance virus occurred to 3 (75%) of 4 HBsAg-positive patients treated with LAM for collagenosis disease more than two years. Conclusion: When HB carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent HBV reactivation with ETV. Background: Adefovir dipivoxil is used for the initial treatment of chronic hepatitis B or rescue treatment of lamivudine-resistant chronic hepatitis B, and exhibits excellent antiviral activity. However, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis B patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. But the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. The aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis B virus in lamivudine-resistant patients not treated with adefovir dipivoxil. Methods: The existence of adefovir resistance-related mutations was examined in 240 lamivudine-resistant chronic hepatitis B patients with breakthrough hepatitis and 100 antiviral-naïve chronic hepatitis B patients. Both polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and directly sequencing of PCR product were used to detect resistant viruses. Results: rtA181T mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis B patients. There was no rtN236T detected in the two groups. Conclusion: Our results suggest that the rtA181 mutant virus were present in a few lamivudine-resistant chronic hepatitis B patients before they have been treated with adefovir dipivoxil, but the rtN236T mutant was not detected in any of the two groups. The rate of adefovir resistance-related mutations in polymerase gene of hepatitis B virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. Objective: In this study, we tried to detect and identify the special protein of HBV related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. To find new opinion on the developing of chronic liver disease. Methods: The sera of health adult, HBV related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The arrays of every group were analysised by clustering analysis and to establish disease predictive model. Then the sample was eluted with different pH Tris, trypsinization on-chip, mass determination and peptide database comparison. Results:
According CM10 chip we find 18 protein with obviously deviation (P<0.05) among HBV related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.
Clustering analysis for the data from SELDI-TOF-MS confirmed 42 differentially expressed proteins. Then we developed disease predictive mathematic models ( Decision Tree Model, DT model ) with average validity up to 73.9 .
The 5805 Da protein peak was identified to be Chondroitin sulfate synthase 2 (CHSS2), which is a potential molecule involved in the pathologic process and a potential serum marker for the HBV related hepatic diseases as well. Conclusions: Our results suggest that SELDI-TOF-MS is a usefull technique for differential expressed proteins screening and analysis in HBV related chronic liver disease. CHSS2 may be useful during the developing of HBV related chronic liver disease. Backgound: Clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis B patients. However, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. This study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic HBV infection. Methods: 46 patients with chronic HBV infection who visited Korea University Ansan Hospital and Guro Hospital between May 2007 and May 2008 were included. 24 patients had chronic hepatitis B (group A) and 22 had liver cirrhosis (group B). Early virologic response was defined as HBV DNA less than 200 IU/mL at week 12. Early biochemical response was defined to be normalization of ALT (<45 IU/L) at week 12. Result: Pretreatment HBV DNA levels were higher in group A compared with group B (8.06 log IU/mL vs 7.09 log IU/mL, p=0.06). Pretreatment ALT levels were not significantly different between the two groups (166 IU/L vs 139 IU/L, p=0.725). The rate of early virologic response was significantly higher in group B compared with groups A (72.7% vs 33%, p=0.01). The rate of early biochemical response were not significantly different in both groups (75% vs 72.7%, p=0.725). Conclusion: Clevudine is considered to be safe and effective in cirrhotic patients with chronic HBV infection as well as chronic hepatitis B patients. Long term safety and efficacy need to be evaluated in the future. Objective: The aim of this study was to evaluate the role of nucleos(t)ide analogues against HBV reactivation in immunosuppression. Methods: Non-active HBsAg carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. The outpatients or in-patients from April 2007 to July 2008 were enrolled. The nucleos(t)ide analogues were used in cancer patients 1-2 weeks before chemotherapy, and the duration lasted 6-12 months according to patients' compliance after completion of chemotherapy. Patients with other diseases used nucleos(t)ide analogues in 1-3 months before using glucocorticoids or other immunosuppressive agents, and continued to use for 6-12 months after accomplishing the course of immunosuppressant treatment. The characheristics and clinical manifestations about HBV reactivation were investigated. Results: Of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. The amount of HBV DNA was detected in the first, third, sixth and 12th month after the use of nucleos(t)ide analogues. After chemotherapy or immunosuppressant treatment, only 9.4% (3 / 32) of them suffered from HBV reactivation, which presented with HBV DNA positive and abnormal liver function. Conclusion: Non-active HBsAg carriers would appear potential incidence of HBV reactivation during use of chemotherapy or immunosuppressant. Nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis B in immunosuppression and to improve clinical prognosis. Background: HBV therapies are evolving toward combination antivirals. This study evaluated the combination of clevudine (CLV), a potent nucleoside analog, with tenofovir dipivoxil (TDF). Methods: A phase I, single-arm, multi-dose study in 18 healthy adult volunteers to evaluate pharmacokinetic and safety interactions between CLV and TDF. Subjects received 21 days of CLV 30mg followed by 7 days of CLV 30mg +TDF 300mg. PK profiles were obtained on Days 1, 21 and 28. CLV AUC and Cmax were compared on Days 21 and 28. Day 28 tenofovir PK was compared to historical data. Safety assessments were conducted throughout. Results: 18 subjects were enrolled (13M/5F);17 completed the study. The mean (range) age was 37y (22-49) and body mass index (kg/m2) was 27.6 (22.9-31.9). 22 AEs were reported by 6 subjects, with 10 AEs reported during CLV-only dosing and 12 AEs reported during CLV+TDF dosing. AEs included nausea (2) and pharyngolaryngeal pain (2). The majority of the AEs were mild. There were no clinically significant changes in ECGs or laboratory parameters. Comparisons of CLV AUC and Cmax on Day 21 and 28 revealed no significant impact of TDF upon the plasma CLV exposure (D28/D21 AUC ratio=0.98, D28/D21Cmax ratio=0.89). There is no significant effect of CLV on tenofovir when comparing AUC and Cmax of TDF to historical values. Conclusion: Safety and pharmacokinetic results demonstrate that CLV and TDF may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic HBV infection.
S. Kuznecovs 1,2 , I. Kuznecovs 1 , K. Jegina 2 , G. Kuznecova 1 1 
Background: Dolichyl (Dol), the main lipid intermediator of Dolichyl Phosphate Cycle (DPC) has been reported to be elevated in urine of patients with multidrug resistance in cancer. Drug resistance poses a major threat to nucleoside analogue-based therapies for chronic HBV infection. Methods: With focus on a risk predictor for susceptibility to the development of HBV drug resistance the present study was carried out to estimate urinary levels of Dol in chronic HBV infection. The samples obtained every week before and during the course of treatment from 42 patients with HBV. The occurrence of exacerbations of chronic HBV were registered for 2 years. Dol in urine was assayed by HPLC method. Results: The normal amounts of Dol in healthy persons urine (n=1500) are 6,0 + 10,0 mkg/mmol creatine. During the period of observation 36 (86%) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis B virus to antiviral drugs. From this group of HBV patients 35 (98%) have had elevated urinal Dol excreation (45,8±5,2 g/ml vs . 8,2±1,9 g/ml, p<0.0001) in more than 3 months of observation. Conclusion: There is a reason to suggest that elevated urinal Dol detected in patients with exacerbations during HBV treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. The interest drawn to the employment of Dol as a predictor for exacerbation of chronic HBV is explained by the role of DPC in P-glycoprotein regulation in human hepatocytes. Background: A significant proportion CHB patients treated with ADV have a suboptimal response, increasing the risk of disease progression and development of resistance. We report clinical results from patients who either failed or relapsed following ADV therapy and were subsequently switched to ETV. Methods: Study ETV-079 was a randomized, open-label study comparing antiviral efficacy of ETV (0.5mg/day) vs ADV (10mg/day) in nucleoside-naïve HBeAg-positive patients. After up to 96 weeks of treatment in ETV-079, 24 patients treated with ADV (13 suboptimal responders) rolled over into study ETV-901 (1.0mg/day). HBV DNA viral suppression and safety was evaluated during 48 weeks of ETV treatment. Results: At entry to ETV-901, the median HBV DNA was 5.72log10 copies/mL. Median exposure to ETV (1.0mg) in ETV-901 was 46 weeks and 18 patients currently remain on study therapy. At Week 24, the mean reduction in HBV DNA was 4.5log10 copies/mL and 8/16 (50%) reached HBV DNA levels <300 copies/mL. Nine patients have achieved Week 48 and all have achieved HBV DNA <10 4 copies/mL and 8/9(89%) had HBV DNA levels <300 copies/mL. No patients experienced virologic breakthrough on ETV. The safety profile of ETV in ADV-treated patients remained consistent with the previously reported experience. Conclusions: The majority of patients who were suboptimal responders or virologic rebounders following ADV treatment in study ETV-079, experienced rapid reductions in HBV DNA levels when switched to ETV. HBV DNA levels continued to decline to undetectable levels with 48 weeks of ETV treatment.
Y. Li 1,2 , T. Han 1 1 Tianjin Third Central Hospital, 2 
Aim:To quantify hepatitis B virus (HBV) total DNA and covalently closed circular DNA (cccDNA) in liver biopsies and sera which from Chronic hepatitis B(CHB) liver cirrhosis of hepatitis B(LC) and hepatitis B relevance hepatocellular carcinoma(HCC) patients, and analysis HBV replication under the circumstances of different diseases. Methods:Total HBV DNA and cccDNA in serum and liver biopsy samples were measured in 21 CHB 23 LC and 25HCC patients by the real-time PCR assay. Results: The levels of total HBV DNA in serum,intrahepatic total HBV DNA, intrahepatic cccDNA, as well as the proportion of intrahepatic cccDNA in total HBV DNA decreased progressively in CHB,LC and HCC ,moreover CHB had significantly higher levels of total HBV DNA in serum and liver biopsy samples than LC (log [total serum HBV DNA] P = 0.024;log [total intrahepatic HBV DNA] P = 0.034); CHB and LC had significantly higher levels of intrahepatic cccDNA and the proportion of intrahepatic cccDNA in total HBV DNA than HCC(P <0.01); cccDNA couldn't be detect in all patients'serum. In CHB ,the levels of serum's total HBV DNA,intrahepatic total HBV DNA and cccDNA in HBeAg-positive group had significantly higher than the HBeAg-negative group(P <0.01) ,but in LC only intrahepatic total HBV DNA had statistical difference between HBeAg-positive and negative group (P=0.026) , no statistical difference between HBeAg-positive and negative group in HCC. Conclusions: The replication activity of hepatitis B virus in CHB,LC were higher than HCC, HBV reproduction reduced significantly in HCC. Duplication of HBV in LC was lower than CHB but had no statistical difference. The levels of HBV reproduce in HBeAg-positive group was higher than HBeAg-negative group of all three desease. 
Background: Clevudine is a pyrimidine analogue with potent and sustained antiviral activity against HBV in the 24 week therapy. The present study assessed the efficacy and viral resistance of 48 week clevudine therapy in patients with chronic hepatitis B. Method: A total of 42 patients (26 HBeAg positive and 16 HBeAg negative) who were received clevudine 30 mg once daily for 48 weeks were included in this analysis. Serum HBV DNA was quantified by real time PCR assay. Result: At week 48, median reductions of serum HBV DNA from baseline were 4.98 log10 IU/mL (5.00 log10 IU/mL for HBeAg positive and 4.95 log10 IU/mL for HBeAg negative) and 76.2% of patients showed undetectable serum HBV DNA (<60 IU/mL) (65.4% for HBeAg positive and 93.8% for HBeAg negative). The normalization of ALT levels (<35 IU/L) was achieved in 81.0% (88.5% for HBeAg positive and 68.8% for HBeAg negative). 15.4% of HBeAg positive patients showed HBeAg loss or seroconversion. HBV DNA negativity at week 48 was associated with HBeAg negativity (P =0.037), HBV DNA <2,000 IU/mL at weeks 4 and 24 (P =0.019 and 0.001, respectively). Two HBeAg positive patients showed viral breakthrough with M204I mutation during 48 week. Conclusion: Clevudine therapy in patients with chronic hepatitis B showed potent virologic responses at week 48, especially in those with HBeAg negativity and complete early virologic response (HBV DNA <2,000 IU/mL at weeks 4 and 24). But clevudine resistance can occur in HBeAg positive patients.
Background: Serum alanine aminotransferase (ALT) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. Current standards for "normal" ALT level were defined by using populations that included persons with subclinical liver disease. There is no study regarding normal level of ALT and its modulating factors in healthy Thai people. Objective: To definitions of Normal ranges for serum ALT level in Thai people. Design: Prospective observational study Setting: Phramongkutklao hospital and Army Institute of Pathology Pramongkutklao Medical center(A.I.P.), Bangkok, Thailand Participants: 200 persons who were first-time blood donors from August through December 2007 were negative for anti-hepatitis C virus(HCV), negative HBsAg(HBV), and had no contraindications to donation. Measurements: Univariate and multivariate analyses examined associations between clinical and laboratory factors and ALT levels. Normal ranges for ALT were computed from the population at lowest risk for liver disease. Results: Serum ALT activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. Normal ranges for serum ALT level in Thai people upper limits for men 26 U/L and for women 21.67 U/L. Conclusion: In men serum ALT is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. The normal range of ALT should be defined for male and female separately. Background: The open-label rollover study ETV-060 was conducted after ETV phase II clinical study ETV-047 for nucleoside-na ve adult CHB patients in Japan. In this analysis, we report ETV long-term efficacy and safety in patients who were switched from 24-week LVD treatment to ETV therapy. Methods: Ninety-seven percent (33/34) of LVD-treated patients from ETV-047 were rolled over into ETV-060 treated with 0.5mg of ETV. Thirty patients completed 96 weeks of ETV therapy and were evaluated for HBV DNA level, ALT normalization, HBeAg seroconversion, resistance and safety. Results: Comparing to baseline before switching to ETV, after 96 week of ETV treatment, the proportion of patients achieving undetectable HBV DNA (<400 copies/ml) increased from 21% to 90%. Increases were also observed for ALT normalization (81% to 90%) and HBeAg seroconversion (10% to 19%). Three patients had detectable HBV DNA at Week 96 after ETV treatment and samples from two were tested for resistance. Neither demonstrated substitutions associated with ETV or LVD resistance. Five patients had Grade 3-4 laboratory abnormalities, including increased AST/ALT and increased lipase levels. Conclusions: Switching patients from LVD therapy to ETV resulted in increased proportions of patients achieving HBV DNA suppression, ALT normalization and HBeAg seroconversion, with no evidence of ETV resistance. ETV was well tolerated during treatment. Backgrounds/aims: Hepatitis B virus (HBV) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. Prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. The aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. Methods: Cancer patients with positive HBsAg who underwent cytotoxic chemotherapy in a tertiary medical center from January 2005 to June 2008 were included. Prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within 6 months after the completion of chemotherapy. All patients were followed up even after withdrawal of lamivudine. Results: 115 patients were enrolled. Twenty-nine patients (23.7%) had hematologic malignancies and eighty-six (76.3%) had solid tumors. Median follow-up duration was 15.9 months and twenty-six patients (22.6%) experienced the prophylaxis failure: viral breakthrough (11 patients, 9.6%), withdrawal flare (15 patients, 13.0%). YMDD mutation developed in four patients. Withdrawal flare occurred at a median 2.5 months after discontinuation of lamivudine. Using log-rank test and Cox multi-variate analysis, our results showed that the type of underlying malignancies (HR 2.46, 95% CI, 1.11-5.43; P=0.026) and baseline HBV DNA titer (HR 3.91, 95% CI, 1.63-9.39; P=0.002) were significant independent risk factors for antiviral prophylaxis failure. Conclusion: Cancer patients with high viral load of HBV and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. Back Ground: The usefulness of hepatitis B virus (HBV) DNA and HBV core-related antigen (HBcrAg) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis B. Method: A total of 32 events of HBV reactivation due to withdrawal of lamivudine (LAM) or emergence of mutants resistant to LAM or adefovir dipivoxil (ADV) virus were analyzed in 25 patients with chronic hepatitis B (20 men, median age 56 years [range: 30-66]). They were followed monthly for serum ALT, HBV DNA and HBcrAg before, during and after the treatment. Result: High ALT flare (ALT > 100 IU/ml) after viral breakthrough or withdrawal was related with baseline HBeAg positivity (p=0.016), HBcrAg level at HBV DNA elevation (p=0.038) and duration from HBcrAg elevation to salvage therapy (p=0.044). In multivariate analysis, HBcrAg > 4.0 log U/ml (OR 22.9, 95%C.I. 1.7-304.1, p=0.018) and Salvage therapy after 8 weeks from HBcrAg elevation (OR 10.6, 95%C.I. 1.6-71.4, p=0 .015) were selected as related factor with high ALT flare. After appearance of resistant-virus or withdrawal of LAM or ADV therapy, HBV DNA re-elevated without increase of HBcrAg, then HBcrAg elevated with HBV DNA. Re-elevations of ALT occurred in 27 of the 32 (84%) events. In 23 of the 27 (85%) events, ALT re-elevated within 8 weeks from the start of HBcrAg increase. Conclusion: HBcrAg was useful for timing the re-elevation of ALT after HBV DNA re-elevation induced by drug-resistant virus or withdrawal of LAM or ADV therapy. Background and Objectives: The aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (ADV), and modeling the viral dynamics. Methods: The studied object was a Chinese CHB patient with lamivudine mutation. Used the LVD + placebo for 12 weeks' therapy. Then switched ADV for another 95 weeks. After that stopping the treatment and following up for 24 weeks. Based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and CTL cells, respectively. Results: Selected the model parameters, the simulation data of HBV DNAs of our model are good in agreement with the clinical ones. Observe that after 24 weeks' treatment cessation, the benefit (HBV DNA < 250 copies/mL) for suppressing HBV replication can still be kept. Numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs 9 years to replace all infected cells by normal ones. Conclusion: For LVD mutation patients, LVD+ placebo to AVD therapy scheme may help patients to suppressing HBV replication. Further researches are promising. Acknowledgments: This work is jointly supported by the NNSF of China Background: G-A-1896 pre-Core mutants (P-C-mt) cause HBeAg-negative chronic hepatitis (CHB) in genotype D infected Mediterranean adults. We studied their emergence during chronic HBV infection in children. Methods: Eighty consecutive HBsAg carriers (50/30 males/females, age 11y, range 0.2-17y) with vertical (66%) or intra-familial (16%) transmissions were followed-up for 12.5y (range 1-25 y). HBV genotype and HBeAg status were determined at the admission, HBeAg/anti-HBe every 2 years thereafter. During the follow-up, HBV-DNA was measured in 185 sera (1-7 sera/patient) (Cobas-Amplicor, Roche); P-C populations were characterized by direct sequencing (DS), by oligo-hybridization (OHA) and allele-specific-PCR (AS-PCR) with 30%, 10% and 0.1% sensitivities, respectively. Results: Seven children were genotype A and 73 D; 70 (87.5%) were HBeAg-positive. Fifty-five (78.6%) underwent HBeAg/anti-HBe seroconversion (median age 13y, range 1.3-27y). Baseline HBV-DNA (cp/ml) was lower in seroconverters (7.9+1.9 vs 9,4+0,6, ANOVA p=0.012). DS/OHA P-C-mt were 4.2% at the admission and 45.8% after follow-up; AS-PCR P-C-mt 33.3% and 50% respectively. After seroconversion 47 (85.5%) became inactive carriers, 6 (14.5%) lost HBsAg (5 genotype D/P-C-wt); 8 P-C-mt had CHB. HBV-DNA (cp/ml) was lower in 31 P-C-wt than in 14 P-C-mt inactive carriers (3.42+1.05 vs 4.58+0.91; ANOVA p=0.007). Conclusions: In genotype-D infected children P-C-mt is selected progressively after HBeAg/anti-HBe seroconversion to become predominant in HBeAg-negative CHB. Early and efficacious immune control of HBV replication avoids P-C-mt selection and leads to HBsAg loss. , a, , d, k, u, m, n , k1 ,k2 and k3. Here k1, k2, and k3 are the rate of CTL production and dead, killing virus, respectively. Results: The patients with HBV DNA levels less than 1000 copies/ml were reported in 17.5% (7/40). A patient whose HBV DNA levels were higher than 40000 copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. The simulation data of our model are in agreement with the patient's HBV DNA data. Our simulation also shows that it needs to spend about 18 years for clearing all infected cells. Conclusion: The simulation result implies that some Chinese patients may need long term's therapy to clear all infected cells. Patients' CTLs assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' HBV DNAs are higher than undetectable levels. Background/Aim: Recent reports have shown that programmed death 1(PD-1) expression is associated with T cell exhaustion and persistent viral infection. We studied longitudinally 28 chronic hepatitis B(CHB) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(PEG-IFN-) in 12-16 weeks to determine the relationship between PD-1 expression levels on T cells and early reduction of viral load induced by treatment. Methods: Our investigations were focused on three points: baseline (time point 1, T1), treatment weeks 4-6(time point 2, T2) and treatment weeks 12-16(time point 3, T3). PD-1 expression on total CD4 and CD8 T cells in CHB patients during antiviral therapy was detected by flow cytometry. Serum Hepatitis B virus (HBV)-DNA load was measured by real time polymerase chain reaction. Results: Between T1 and T2, PD-1 expression on total CD8 (P<0.01) and CD4 T cells (P<0.01) dropped concurrently with treatment-induced HBV-DNA decline(P<0.01). Between T2 and T3, however, only the HBV-DNA levels reduced significantly (P<0.01). Conclusion: Early suppression of HBV replication induced by antiviral treatment results in a significant decrease in PD-1 expression on total CD8 and CD4 T cells in CHB patients.
C. Zhao 1,2 , W. Zhang 2,3 , X.C. Tian 1 , C.Y. Fang 2,3 , H.J. Lu 2,3 , P.Y. Yang 2,3 , Y.M. Wen 1,2 Background/Aims: Hepatitis B virus (HBV) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. The interactions between hepatitis B surface antigen (HBsAg) and host cells still remain largely unknown and need to be explored in detail. Methods: Differential protein expression profiles of HepG2-S-G2( Stably expressing HBsAg cell line) and HepG2-Neo-F4 ( control cell line) were compared using two dimensional gel based differential proteomic approach. Cell proliferation assay and survival assay were used for further studies on the candidate protein.
Results: Compared with the control down regulation of 44 proteins and up regulation of 38 proteins were found in HepG2-S-G2 cell. All these regulated proteins were identified by MS/MS and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. Among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein 78kD (GRP78/Bip), heterogeneous nuclear ribonucleoprotein (hnRNP), Far upstream element-binding protein (FUSEbp), Rho GDP Dissociation Inhibitor (GDI), cystatin B and some scaffold proteins. GRP78, an important chaperone protein involved in multiple functions in host cells, was consistently decreased in HepG2-S-G2 and in Huh7 cell transiently transfected with HBsAg expression plasmid. Decreased CRP78 inducing by HBsAg or blockage of RNAi consistently led to the less resistance to staurosporine-induced cell death. Conclusions: These results revealed a possible pathogenesis induced by HBsAg via GRP78. Background/Aim: To evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory HBeAg-positive chronic hepatitis B. Methods: Eighty-five HBeAg-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented YMDD mutations and persistent viremia were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. The primary efficacy measure was virological response. The secondary efficacy measure was serological response (HBeAg loss rate and HBeAg seroconversion rate) and ALT normalization rate. Results: After 24 weeks of therapy, mean reduction of HBV-DNA level, the percentage of patients with HBV-DNA lower than 5 log10 copies/ml and the percentage of patients with HBV-DNA level decrease of more than 2 log10 copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group (2.58, 2.21 log copies/ml vs. 1.02 log copies/ml, 92.3%, 88.5% vs. 33.6%, 76.9%, 75.8% vs. 28.8%; P < 0.001, respectively). At the end of 52 weeks, mean reduction from baseline in serum HBV-DNA level at was 0.08, 4.25, and 4.12 log10 copies/mL in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. ALT normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients (62%, 55% vs. 8%, P < 0.001, respectively). A similar pattern was observed in HBeAg loss among three groups. Conclusions: Adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory HBeAg-positive chronic hepatitis B. Aim: To find the prevalence of HBV virologic flare as defined by HBV DNA viral load of > 2,000 IU/mL in inactive chronic hepatitis B (HBV DNA less than 2,000 IU/mL), HBeAg-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. Methods: We retrospectively analyzed medical records of the patients who have attended Hepatitis Clinic, Siriraj Hospital from January 1, 2002 to February 28, 2008 . The patients were eligible if they were naïve to any treatment and HBV DNA less than 2000 IU/mL at entry. Co-infection with HIV and/or hepatitis C virus were excluded. HBV DNA measurement determine by Roche Amplicor ® (detection limit of 60 IU/mL). HBV virologic flare was defined as HBV DNA more than 2000 IU/mL during follow up period. Result: There were 84 patients with mean follow up time was 598 days with annual prevalence of HBV virologic flare of 12.8, 4.8, and 4.2% for the first, second and third year of follow up, respectively. Initial HBV DNA level was the only predictor that can predict reactivation. No patients with HBV DNA at entry below detection limit developed flare and the patients with HBV DNA above 740 IU/mL had 22 times higher chance to develop flare during follow up. Conclusion: HBV DNA flare is not uncommon in inactive chronic hepatitis B patients. Most of the virologic flares occur in the first year. The most important predictor or virologic flare is higher HBV DNA at beginning. Background: Multi-drug resistant HBV developed with multiple antiviral agents. There existed difficulty in dealing with Multi-drug resistant HBV. Methods: Retrospective analysis of 23 consecutive patients who exhibited chronic hepatitis B associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. Multiple drug-resistant mutations were detected in those patients by DNA direct sequencing. Result: Before multiple drug-resistant HBV emerged, 20 patients accept sequential antiviral therapy, 3 patients accept NA monotherapies. There were 16 cases of rtA181T/V rtM204V/I mutation, 2 cases of M204V/I +N236T mutation, 4 cases of A181T/V+M204V/I +N236T mutation, 1 case of L180M+A181T/V mutation. 14 cases received rescue therapy of Interfronand HBV DNA level of 8 cases decreased; Other 9 cases received combination treatment and HBV DNA level of 4 cases decreased. Conclusion: The main reason of multiple drug-resistant mutations was sequential antiviral therapy. Another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. De novo combination of antiviral agents should be recommended. Combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV. Interfron may be one choice for HBV of multiple drug-resistant mutations. Background: To furnish basis for an accurate evaluation of HBeAg negative chronic hepatitis B (e CHB), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e CHB. Methods: A study is performed in 120 Chinese e CHB patients (106 men and 14 women; mean age SD, 34.3 9.4 years). The relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by Spearman's rank correlation test or Kruskal-Wallis test by applying STATA 7.0 software. Result: Negative correlation is shown between the grade and leucocyte count Methods: 80 patients with HBeAg-positive compensated CHB with HBV DNA >6 log10 copies/ml, serum ALT 2 x ULN were divided two groups:one treated with Telbivudine and the other treated with Entecavir. Results: Baseline characteristics were well balanced between treatment groups. At 12wk of the treatment the HBV DNA undetectable rates of HBeAg-poitive patients in the Telbivudine group and the Entecavir were respectively 50 52.5 (p 0.05), the rates of HBeAg negative were 10 0 respectively, the rates of HBeAg seroconversion were 20 5 respectively; At 24wk of the treatment the HBV DNA undetectable rates of HBeAg-poitive patients in the Telbivudine group and the Entecavir were respectively 80 70 (p 0.05), the total rates of HBeAg negative were 20 15 respectively, the total rates of HBeAg seroconversion were 27.5 17.5 respectively(p 0.05).No adverse reactions were found in both groups Conclusion: There was no significant difference in HBV DNA undetectable rates between two nucleotide analogs in short-term (24 weeks).The Telbivudine group has better effect in HBeAg seroconversion rate than the Entecavir group in early stage,but no statistical significance.
J.W. Song 1,2 , Z. Xin 1 , J.X. Tang 1 , L. Yao 1 , B. Wu 1 1 Sun Yat-sen University, 2 Zhuhai Sinochips Biosci. Co.,Ltd., China
Background: To develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis B virus Pre-C/BCP mutation assay. Methods: A thin film optical biosensor were applied for amplification the microarray signal in situ. And HBV sites 1762, 1764,1768,1770 and 1896 were selected as the targets and the microarray were be fabricated. The 5 mutated plasmids contained 1762, 1764,1768,1770 and 1896 sites and 30 HBV sera were be tested in our study and all the plasmids and sera PCR products were be assayed by really time PCR and sequencing. Results:
The biosensor based microarray signal can be easily record by digital camera or even by the naked eyes And the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; 2. The sensitivity of the detection limitation of sera HBV load is 2X10E3 copies/ml with 95% reproducibility. The concordance index of 50 times negative and mutated plasmids were 98%(Kappa=0.932). 3. 30 sera samples of HBV>10E4 load and 20 sera of HBV negative tested by PCR fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was 0.7619. Conclusion: Our biosensor-based microarray for Pre-C/BCP mutation assay were a both sensitive and accurate method. And its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. Objective: To investigate the frequencies of CD4 + CD25 + regulatory T cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis B, we assayed the differences among HBsAg-positive and healthy subjects by flow cytometry. The results might offer some experimental evidence to explain the high rates of HBV persistent infection in vertical transmission of HBV from HBV-infected mothers. Methods: 12 newborns born from HBsAg positive mothers were recruited , 10 healthy subjects being used as a control group. The cord blood and peripheral blood of mothers were collected respectively .Frequencies of CD4 + CD25 + regulatory T cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis B were analyzed by flow cytometric analysis. Result: The number of CD4 + CD25 + regulatory T cells/PBMCs in the cord blood of newborns born from HBsAg positive mothers 4.49 ±1.18 significantly exceeded that in normal controls 2.26 ±0.97 ,P 0.001 ;And newborns born from HBsAg positive mothers presented a much higher fraction of cord blood CD4 + CD25 + /CD4 + 9.62 ±2.34 than those in normal controls 7.93 ±1.43 ,P P=0.0025 P 0.05 . Conclusions: The results indicate that the proportion of CD4 + CD25 + regulatory T cells in HBsAg positive mother cord blood was higher than those of healthy cord blood. Objective: To study the clinical features of chronic severe hepatitis B with negative hepatitis B e-antigen (HBeAg) and positive hepatitis B e-antigen (HBeAg) Methods: A total of 353 in-patients with chronic severe hepatitis B were recruited into the study and divided into two groups according to the HBeAg status. The serological chemistry data, hepatitis B virus (HBV) DNA quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. Results: Of the 353 in-patients, 236(66.8%) patients were HBeAg-negative. 117(33.2%) patients were HBeAg-positive. The ratio of HBeAg-positive patients was significantly higher than that of HBeAg-positive patients (p<0.05).The average age of HBeAg-negative patients was older than that of HBeAg-positive patients (P=0.048). The serum HBV DNA level of HBeAg-negative patients was significantly lower than that of HBeAg-positive patients (5.49±2.02) vs(6.64±1.41) log copies/ml (p<0.01).The ratio of patients who had a serum HBV DNA level less than 5log copies/ml in HBeAg-negative patients was significantly higher than that in HBeAg-positive patients (41.8% vs 11.9% ,p=0.000). There was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. Conclusions: The study suggested that serological chemistry data, morbility of complications and prognosis of the disease of HBeAg-negative patients mimics that of HBeAg-positive patients. The HBeAg-negative patients had a higher level of age, while a lower level of serum HBV DNA. To reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis B patients with negative hepatitis B e-antigen. Background: The emergence of LAM-resistant virus greatly limits the efficacy of therapy and induces the liver injury. The aims of this study were to assess the related factors of LAM-resistant mutation in HBeAg positive CHB patients. Methods: Thirty-five patients carrying LAM-resistant with HBeAg positive were enrolled in this study. All of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. Age, viral load, levels of ALT, types of mutation and HBV genotype was monitored. Result: The median year of mutation found was 23months. 85.71% were genotype C and 14.29% were genotype B. The mutation of L80I, L80V, G173L, L180M, M204V and M204I were detected. The emergence rates were 34.3%(12/35), 25.7%(9/35), 17.1%(6/35), 60%(21/35), 57.1%(20/35), 54.3%(19/35) respectively. The rate of patients with two or three mutation were much more than one or four mutation. 62.9% patients were found to have significant histological findings, even 5 had established cirrhosis. Two had no histological finding. One had rtL80I and rtM204I. The other had rtL80V, rtL180M and rtM204V. The number of resistant mutation has no significant finding with histological finding, basic ALT level and basic viral load. Conclusions: The emergence rate of L180M, M204V and M204I were higher than that of L80I, L80V, G173L in HBeAg positive CHB patients with LAM-resistance. Most of them have two or three LAM-resistant mutation regardless of histological finding severity, level of basic ALT and viral load. We must select the efficacious method to treat the patients with LAM-resistant. Objective: To investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis B. Methods: Forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.Each group consisted of 22 patients, 22 patients in foscarnet sodium group were treated with foscarnet sodium twice daily 3.0g given by intravenous infusions ,in addition to general therapy for 28 days.The other 22 cases were treated without any form of antiviral therapy as control.All patients were followed up for 6 months.The HBV markers, quantification of HBV-DNA, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . Results: Clinical symptoms were improved in Two groups patients, meanwhile alanine aminotransferase (ALT) and total serum bilirubin (TBiL) decreased. Compare ALT and TBiL at the end of trentment, there were no significant differences between the two groups (p>0.05). In foscarnet sodium treatment group, the level of serum HBV-DNA descreased from (6.993±0.898) log copies/ml to( 4.033±1.286) log copies/ml (p<0.05), the rate of HBV-DNA descrease of more than two log was 81.1% 18/22 . In the control group, the level of serum HBV-DNA descreased from( 7.068±0.938) log copies/ml to (5.188±1.926 )log copies/ml, the rate of HBV-DNA descrease of more than two log was 45.4% 10/22 .A comparison of serum HBV-DNA showed significant differences between the two groups(p<0.05) Conclusion: foscarnet sodium administered can inhibit HBV replication in treating severe chronic hepatitis B.It can rapid lower the level of serum HBV-DNA obviously.But the relapse rate was 47.0% in foscarnet sodium treated at the end of follow up period Objective: Evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis Bpatients failed with lamivudine therapy in the Chongqing area. Methods: Thirty-two eligible patients were enrolled who had documented LVD failure.In the double-blind phase,patients were randomized(4:1)to ETV1.0mg/d (n=28)and placebo (n=4) for 12 weeks.In the open-lable phase ,patients received ETV 1.0mg/d for 240 weeks.HBV-DNA level,liver function tests,HBV serology and safety assessments were conducted. Results: The mean reduction in HBV DNA levels at week 12 was 4 05 logl0 copies ml in ETV group compared to 0.08logl0 copies ml in placebo group(P< 0 05). The mean of HBV DNA levels after 240 weeks of ETV treatment decreased to 2.58logl0 copies m1 The proportion of HBV DNA<3log10copy/ml raised from 0 at baseline to 6.25% at week 8,to 15.6% at week 24,to 50% at week 96,and raised to 57.14% at week 240.There were two patients with HBsAg seroconversion and four patients with HBeAg seroconversion at the end of study. The mean of ALT became normal at week 12 and remained normal throughout week 240.There was one patient who had a severe adverse event during the trail. Conclusion: The findings from this study demonstrated the antiviral activity and safety of ETV in adults with CHB who have failed LVD PE198 showing delayed response on T cells as increased on day 7.The mRNA expression of IL-5 and IL-2 showed no response to HBV vaccine but highly regulated in TT after day 7(p=0.00,0.001).MyD88andTRAF 6(p=0.042)upregulated in HBV vaccine group followed by of IFN-(0.012)no change of IFN found in TT Conclusions: i) HBV vaccine stimulates innate response by day 3 which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows MYD88 pathway and releases IFN-.ii) Whereas T cells Marjory involved in TT showed delayed immune response.iii) Identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. Objective: To Compare TH1/TH2 cytokines' dynamic change and its clinical significance in hepatitis B e antigen-positive patients treated with Telbivudine. Methods: Twelve hepatitis B e antigen-positive patients treated with telbivudine.The blood sample was collected at baseline, week 4, week 8, week 12, week 24 and week 48 and stored at -70C; serum IL-2, IL-4, IL-6, IL-10, TNF-and IFN-were tested at each time point by Cytometric Bead Array (CBA), Compare TH1/TH2 cytokines' dynamic change at different time point in each group and Compare TH1/TH2 cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . Results:The level of Th1 type cytokines in complete response group are Obviously higher than the group of partial response non-response and breakthrough,but the level of Th2 type cytokines are lower than the group of partial response, non-response and breakthrough. Conclusions: Th1/Th2 cytokines is essential for the regulation of the immune function of the body. After treated with Telbivudine, the level of Th1-type cytokines in the complete response group increased significantly, while the level of Th2 cytokines declined trend.
A. Soamni 1 , S. Somani 2 , A. Jain 3 , V. Dixit 3 1 Navjeevan Hospital, 2 Suvidha, 3 
Background: Chronic infection with hepatitis B virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.The characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. Methods: 61 incidentally detected asymptomatic hepatitis B surface antigen positive (IDAHS) subjects having HBsAg positivity for >6 month presenting to our liver clinic were enrolled after appropriate consent. Detailed clinical, laboratory and sonographic evaluation was done. They were divided into two groups according to presence or absence of e antigen. Group A -HBeAg + (n=48) Group B -HBeAg -(n=13) Results: Most of our patients (49%) were young adults (21-30 years) with male to female ratio of 3.6:1. Approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. Most of our patients were asymptomatic, and Fatigue was most common symptom found in 16%. All demographic and biochemical parameters other than AST & ALT were comparable in both groups. Among HBeAg negative 48 (79%) subjects, HBV DNA level >10 5 copies/ml was found in 31%. Subjects with positive HBeAg as compared to non-replicative infection (antiHBe positive and HBV DNA negative) had more frequent elevation of transaminase levels (62% versus 31%, p<0.05). AntiHBe antibody was positive in all HBeAg negative subjects. Mean age of seroconverted (antiHBe positive) individuals was a decade older than HBeAg positive. Conclusion: From our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. HBsAg testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. Background and Objectives: This research was carried out to determine the prevalence of HBcAb among the HBsAg negative first-time blood donors who had referred to Khorramabad and Borujerd centers for blood donation. Materials and Methods: This study was established on a descriptive cross-sectional basis in which HBsAg test (ELISA) was primarily performed on all of the donors having referred to Khorramabad and Borujerd blood centers; then, out of all those referred 1000 subjects, who were first-time and HBsAg negative, were selected for furthur investigation. The information concerning age, gender, job, blood transfusion, and HBV vaccine injection was included in the questionnaire of the study. HBcAb (total & IgM) and HBsAb tests were performed on the selected donors. Data were collected and finally the prevalence rate of HBcAb was determined. Results: The results of the study showed that out of 1000 HBsAg-negative first-time blood donors, only 47 were HBcAb+, from which 27 were HBcAb (total)+, and 3 were HBcAb (IgM)+. 18 were both HBsAb+ and HBcAb+, and 53 were seropositive only for HBsAb. Conclusions: It was demonstrated that the first-time blood donors who are seronegative for HBsAg marker will be easily identified through HBcAb test if they are in the so-called core window period of the virus. Meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted HBV infection. So, detecting this marker will remarkably reduce the chance of latent cases of HBV infection and help promote blood safety. 
Background: Tumor necrosis factor-(TNF-) plays a pivotal role in the viral clearance and host immune response to HBV, and the capacity for TNF-production in individuals is influenced by a major genetic component. The studies of TNF--308 gene promoter polymorphism in chronic HBV infection have reported apparently conflicting results. Objective: To derive a more precise estimation of the relationship between the polymorphism of TNF--308 gene promoter and chronic HBV infection. Method: Meta-analysis was done of 22 case-control studies in relation to TNF--308 gene promoter, involving a total of 4338 chronic HBV infection cases and 3013 controls. The pooled odds ratios (ORs) for the risk associated with the genotypes of GA, AA, and GA+AA (A-allele carriers) compared with the GG genotype were calculated. Results: Overall meta-analysis indicated that -308A heterozygotes (GA) had 22% decreased risk of developing CHB with a borderline significance (OR = 0.78; 95% CI: 0.60-1.02; P = 0.065). For the -308A allele homozygotes (AA) and carriers (GA+AA), the pooled ORs both indicated a significantly decreased risk of CHB (OR = 0.39; 95% CI: 0.21-0.73; P = 0.003; and OR = 0.74; 95% CI: 0.57-0.96; P = 0.026, respectively) ( Table 1 ). In the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (GA and AA) in Mongoloid populations in all genetic models. However, no significant associations were found in Caucasoid. Conclusion: The meta-analysis suggests that the TNF--308A allele is a low-penetrant protective factor for chronic HBV infection, especially in Mongoloid. Method: 20 HBV transgenic mice were randomly divided into physiologic saline group and Matrine Injection group. Another10 normal mice at the same species and age with HBV transgenic mice were regarded as the normal group. The mice in Matrine Injection group were administrated at dosage of 82.2 mgkg -1 d -1 by intraperitoneal for 30 days. The mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. The contents of HBV DNA in serum and liver were quantitated by PCR. And the spleens were separated for cultivating dendritic cells. The surface molecules of dendritic cells were tested by flow cytometry. IFN-mRNA and TNF-mRNA in liver were tested by RT-PCR. Result: There was no significant difference of the serum HBV-DNA level between physiologic saline and Matrine Injection groups. The content of serum HBV-DNA after treatment showed a significant decrease in two groups. The content of serum HBV-DNA in Matrine Injection dropped significantly as compared with that in the physiologic saline group. But there was no significant difference in the content of HBV-DNA in liver between physiologic saline and Matrine Injection groups. The expression level of MHC-II on dendritic and hepatic IFN-r mRNA and TNF-a mRNA showed a significant decrease in HBV transgenic mice than normal mice. In comparison with physiologic saline group the expression level of them in Matrine Injection group showed a significant increase. Conclusion: Matrine Injection was effective on depressing HBV-DNA in HBV transgenic mice. Its antiviral action may be achieved through regulating MHC-II on DCs surface and promoting the production of antiviral factor such as IFN-and TNF-. Purpose: To stimulate non-specific immune response capacity as the main content of the study to explore the HBV-DNA and non-specific immune responses in the relationship between the low response capability, Methods: 190 cases of asymptomatic carriers, double-blind, randomized into Mycobacterium FU 36, lamivudine and traditional Chinese medicine for the treatment group, Mycobacterium FU36 with traditional Chinese and lamivudine with traditional Chinese medicine were in the control group, a total of 12 weeks of treatment, follow-up six months after the termination of treatment.
Results: different treatment of HBV -DNA effect of the existence of significant differences; P> 0.01, the performance of different types of asymptomatic carriers negative rate of HBV-DNA there is a significant difference; P> 0.01, as well as the performance of the different types of asymptomatic carriers continued application A treatment plan presented HBV-DNA rebound rate there is a significant difference; P> 0.01, Conclusion: HBV-DNA and non-specific immune responses in response to the lower capacity, anti-HBV therapy is not associated with non-specific immune response capacity or improve Is the anti-HBV drugs alone can not solve the asymptomatic carriers in anti-HBV therapy where the cause of the problem, solve the asymptomatic carriers in the anti-HBV treatment although the need for anti-HBV drugs with non-specific immune activation synchronous drugs on the basis of the Joint application , but the simultaneous combination of two drugs rather than as a result of HBeAg and HBV-DNA can HBeAg-positive asymptomatic carriers receive HBV-DNA negative effect of the results. Background: Adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (LAM)-resistant hepatitis B virus (HBV) replication and resistance to this drug is infrequent compared with LAM. In this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in LAM-resistant chronic hepatitis B (CHB) patients. Methods: The subjects were 87 CHB patients with LAM-resistance who had received adefovir for more than 12 months (range 12-39 months). The initial viral response (IVR) was defined as HBV DNA <4.0 log copies/mL. The adefovir resistant mutant was assayed at baseline and every 6 months during adefovir administration. Results: IVR was observed in 38% of patients. The cumulative emergence rates of adefovir resistance were 2.6% at 6 months, 10.4% at 1 year, 14.6% at 2 years and 21% at 3 years. In univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline HBV DNA > 6 log copies/mL (P=0.003) and IVR (P<0.0001). The presence of precore mutation and type of YMDD mutants were not related. In multivariate analysis, only IVR was an independent factors affecting the emergence of adefovir resistant virus (P<0.0001). Conclusion: IVR is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in LAM-resistant CHB patients. For IVR-negative patients, the change of therapeutic options such as add-on LAM or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. Background: Elevated HBV DNA is strongly associated with the risk of disease progression. This study investigated the early viral suppression effects of ETV and LVD in nucleoside-naïve Chinese patients with active HBeAg (+) CHB.
Methods: This open-label study was conducted in 5 major hospitals in China. At study entry all patients had HBV DNA levels 10 7 copies/mL, elevated ALT (1.3-10xULN) and compensated liver function. Patients received either 0.5mg ETV or 100mg LVD daily. HBV DNA measurements were taken at baseline and at Weeks 2, 4, 12 and 24 during treatment, using Roche Cobas Amplicor assay (LLOD 300 copies/mL). Results: A total of 97 patients were enrolled; 42/50 ETV patients and 40/47 LVD patients completed 24 weeks of treatment. At baseline, mean HBV DNA levels were 8.45 0.81 in ETV group and 7.67 1.76 log 10 copies/mL in LVD group (P<0.05). The mean change in HBV DNA from baseline (log10 copies/mL) was -2. 96±0 NGO's/Funding-agencies representative at APASL2009-conference need to address-this-issue. We NGO-representatives from developing-nations need exposure to research treatments used by European/American experts. Do we all failed in addressing socio-economic issues of cancer-sufferers? We need to address these socio-economic issues of affected population in resource-poor-nations. Background: A garlic derivative S-allylcysteine (SAC) has anti-cancer effect in human prostate and colon cancers. We aimed to investigate the effect of SAC and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. Methods: The orthotopic liver tumor model using a metastatic liver cancer cell line MHCC97L labeled with luciferase gene was applied. SAC was given at day 7 after tumor implantation at 1mg/g/day, or 1mg/g/day combined with low dose Cisplatin for 5 weeks. Tumor growth and metastasis were monitored by Xenogen in vivo imaging system. Hepatic stellate cell (HSC) activation and tumor-associated macrophage (TAM) in the tumor tissue were detected by -SMA and ED1/ED2 staining. Tumor micro-vessel density (MVD) and apoptosis were also analyzed. In vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. Results: Tumor growth was inhibited by SAC combined with Cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. The observation of Xenogen IVIS was confirmed by histopathological examination. The HSC activation by -SMA staining in the liver tumors was suppressed by SAC and Cisplatin treatment accompanied with less TAM infiltration. Consistent with in vivo study, in vitro functional study also demonstrated that SAC not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of Cisplatin. Conclusion: SAC treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. Background: Anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (HCC). Although a sorafenib has survival benefits in patients at advances stages of HCC, there seem to be several serious concerns to employ this agent for chemoprevention against HCC. Branched-chain amino acid (BCAA) reportedly inhibits the incidence of HCC in patients with insulin resistance (IR). However, the possible mechanism is still obscure. The aim of the current study was to examine the effect of BCAA on hepatocarcinogenesis under the condition of IR, especially in conjunction with angiogenesis. Methods: The effect of BCAA on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic Otsuka Long-Evans Tokushima Fatty rats was examined. We also performed an in-vitro study to elucidate the possible mechanisms involved. Result: Treatment with BCAA markedly inhibited the glutathione-S-transferase placental form (GST-P)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. The hepatic expression of the vascular endothelial growth factor (VEGF), a potent angiogenic factor, was also attenuated. BCAA treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of VEGF. These results indicate that BCAA exerted a chemopreventive effect under the condition of IR via suppression of VEGF-mediated angiogenesis. Conclusion: Since BCAA is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against IR-based HCC in the future. 
Background: Krüppel-like factor 8 (KLF8) is a member of transcription factors. Whether and how KLF8 signaling pathways contribute to Hepatocellular carcinoma (HCC) development and progression is unknown. This study investigated role of KLF8 in Hepatocellular carcinoma cell line HCCLM3 proliferation, invasiveness and Epithelial to Mesenchymal Transition (EMT). Methods: The expression of KLF8 in different liver cell lines was detected by Quantitative real-time PCR and Immunocytochemistry. We used small interfering RNA (siRNA) to down-regulate KLF8 expression in HCCLM3. The change of proliferation and invasive ability of KLF8 down-regulated HCCLM3 was investigated by MTT Reduction Assay and Trans-well Invasive Assay respectively. The change of proliferation, invasiveness and EMT related gene in KLF8 down-regulated HCCLM3 was evaluated by Quantitative real-time PCR. Result: KLF8 protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. HCCLM3 has the highest KLF8 level. Decreased KLF8 expression can notably inhibit the proliferation (p<0.001, n=6), mobility and invasiveness of HCCLM3 (p<0.001, n=8). We found that the mRNA level of N-cadherin, Fibronectin and Vimentin is much higher than that of E-cadherin in HCCLM3. The expression of Cyclin D1, Focal Adhesion Kinase (FAK) and fibroblast markers including N-cadherin and Fibronectin was obviously suppressed in KLF8 down-regulated HCCLM3. Conclusion: KLF8 plays an important role in the process of HCCLM3 proliferation, invasiveness and EMT. Background: Insulin resistance (IR) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. The aim of this study was to elucidate the possible mechanisms of IR on the liver fibrosis development and hepatocarcinogenesis using obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Methods: To induce liver fibrosis, 1.0ml/kg of pig serum was injected twice a week for 4 weeks in the OLETF and LETO rats. In the hepatocarcinogenesis model, glutathione-S-transferase placental form (GST-P)-positive pre-neoplastic lesions were induced by a single injection of 200mg/kg of diethyl nitrosamine (DEN). We also performed in-vitro studies to examine the mechanistic insights. Results: The liver fibrosis development and GST-P-positive pre-neoplastic lesions were both markedly accelerated in OLETF. In the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (HSC) also increased in OLETF along with augmentation of the hepatic collagen content and transforming growth factor-b1. In the DEN model, the neovascularization was up-regulated in OLETF almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. Our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated HSC and augmented the neovascularization. Conclusion: These results indicated that the IR status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated HSC proliferation and hepatic neovascularization, respectively, in the rat.
N. Wakui 1 , T. Ikehara 1 , R. Takayama 1 , M. Takahashi 1 , K. Shiozawa 1 , H. Nagai 1 , M. Watanabe 1 , K. Ishii 1 , K. Iida 1 , Y. Igarashi 1 , Y. Sumino 1 1 
Case: A 68 years old man diagnosed with chronic hepatitis C regularly visited our hospital. In April of 2005, ultrasonography revealed a tumor 20mm in diameter in S2 of the liver and another tumor 15mm in diameter in S6/7 of the liver. The patient was hospitalized for further examination. Computer tomography (CT) revealed that the tumor localized in S6/7 presented a pattern of hypervascular hepatocellular carcinoma (HCC). For the tumor localized in S2, the following were revealed. 1) Contrast-enhanced ultrasound findings: A tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. 2) CT: No tumor was detected. 3) SPIO-MRI (T2 weighted imaging): Iso-low intensity images were obtained. Although these imaging findings indicated FNH, the patient was HCV positive. In order to disprove the possibility of HCC, a biopsy was performed on the tumor at S2 in the liver. The resulting diagnosis was well-differentiated HCC. Discussion: Until now, a characteristic finding of FNH has been spoke-like vasculariation, which is considered diagnostically quite important. However, some recent cases of HCC have been reported to present FNH-like vascularization. From now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of HCC should be considered and a close examination may be needed. Chronic infection with HCV is problem .Clinical management of chronic HCV depend on extent liver fibrosis .Liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. Serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. Diagnostic accuracy panel of Non-invasive serum biomarkers for hepatic fibrosis (Fibrosure , APRI score, Forn's score) versus liver biopsy. 20 HCV patients subjected for: APRI, Forn's , Fibrosure scores PCR quantitative HCV-RNALiver functions .Lipid Profile CBC . Ultrasound guided liver biopsy. FORNS score; AUROC (0.917) with 95% CI(0.791-1.042) for(fof1) vs. (f2f3f4) while(0.688)with 95% CI(0.464-0.91)for(fof1f2) vs.(f3f4). Cutoff(>6.9)sensitivity for significant fibrosis(f2f3f4)and extensive fibrosis (f 3f4)were (100%) and with low specificity ,with accuracy(40%) and (20%)respectively.-APRI score; AUROC(0.792)with 95% CI( 0.568 -1.015)comparing(f0f1) vs.(f2f3f4)while was(0.875)with 95% CI(0.703 -1.047)for( f0f1f2)vs.(f3f4).Cutoff(<0.5) had low sensitivity and specificity(100%)with accuracy(60%)for significant fibrosis and(80%)for extensive fibrosis.-Fibrosure(fibro-acti test); showed best AUROC(1.00)in different fibrotic stages with 95 % CI (1.00-1.00).Cutoff(>0.59) sensitivity(50%)for significant fibrosis and(100%)for extensive fibrosis while specificity(100%)in all fibrotic stages. The PPV (100%)for significant and extensive fibrosis .NPV and accuracy(75%, 80%)respectively for significant fibroses,while it was (100%) for extensive fibrosis respectively.Significant correlation between liver biopsy and Fibro-test(P0.002)and Acti-test(P0.000).Significant correlation between liver biopsy hepatitis activity score and APRI (P 0.047)and FORNS score (P0.000). Conclusion: FORNS score wasn't considered since does not discriminate between significant and extensive fibrosis. Low sensitivity of APRI prohibtes detection of minmal fibrosis and allow undetermined results. Fibrosure classified all cases of chronic HCV sufficient to wave liver biopsy PE215 Introduction: HCC is the 6 th common cancer. Global increase of hepatitis B and C infection, the incidence of HCC steadily increasing. Egypt seroprevalence of HCV in Nile delta 20 -35%. AFP had limited sensitivity 60% and specificity 90% for small HCC. GPC-3 oncofetal protein over expressed in HCC. Evaluating validity of Glypican-3 as early detector of HCC.:10 healthy controls and 40 HCV positive patients:10 patients chronic hepatitis C virus infection.10 patients compensated cirrhosis [child-Pugh class A and B].10 patients decompensated cirrhosis [child-Pugh class C]. 10 patients HCC. liver functions: ALT, AST, Bilirubin(T), Albumin, GT.Tumor markers: AFP and GPC-3.Viral markers: HCV antibodies, HBs Ag and HBc Ab. The median value of GPC-3 in HCC, DC, CC significantly higher than chronic hepatitis and control groups. No significant correlation between AFP and GPC-3. AUROC of AFP 0.85 & AUROC of GPC-3 0.84. The diagnostic Sensitivity of AFP (20 ng/ml) 70% with PPV 53.8%. The specificity85% with NPV 91.9%. While the diagnostic Sensitivity of GPC-3 (2 ng/ml) 100% with PPV 27%. The specificity 42.2% with NPV 100%. Combined serial approach of AFP and GPC-3 improved specificity to 87.5%. Conclusion:GPC-3 although a serological test for early detection of HCC, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. The diagnostic signature approach for simultaneous determination of AFP and GPC-3 improve prediction accuracy of HCC patients in those showing seronegativity to AFP. Results: Patients with HCV infection (n=388) were significantly older (mean age, 69 years) than patients with dual virus (n=75, 65 years) and HBV infection (n=752; 60 years) (p< 0.0001). The male-to-female ratio for HBV, dual virus and HCV group was 5.2, 3.4 and 1.3, respectively (p< 0.0001). Patients in the HBV group more often had higher total tumor volume (mean, 409 cm 3 ) than the dual virus group (244 cm 3 ) and HCV (168 cm 3 ) group (p< 0.0001). No significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. Patients in the HCV group had a significantly poor survival in comparison to the HBV group only in the subset of patients with small tumor volume (< 50 cm 3 ) in the Cox proportional hazards model (relative risk: 1.44, p=0.041). Conclusions: Dual HBV and HCV virus infection does not accelerate the speed of HCC formation in patients with chronic hepatitis B, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of HBV and HCV infection. Background: Patients presenting with HCC is not infrequent in our clinical practice. The aetiology vary ranging from HBV, HCV, NASH and alcohol. The aim of this study was to see the aetiology of HCC in Bangladeshi patients.
Methods: In this retrospective study, records of 976 patients who attended our OPD between July 2004 to August 2008 were reviewed. Patients having hepatic SOL and/or heterogeneous echotexture of liver on USG and/or CT scan were included. Diagnosis of HCC was confirmed at USG guided fine needle aspiration cytology with or without elevated serum AFP (>500 ng/ml). Results: Of the 976 patients, 75% (732/976) had HBV infection. HCV infection was diagnosed in 17% (165/976). NASH was responsible for 5% (49/976) cases, alcohol in 1% (10/976), while in the rest 2% (20/976) cases no specific aetiology could be established. Conclusion: The study shows that HBV is the commonest cause for HCC in Bangladesh followed by HCV. Background: The aim of this study was to determine whether the hepatitis B virus (HBV) DNA viral load and antiviral therapy is associated with hepatocellular carcinoma (HCC) recurrence. Methods: This retrospective study involved 93 patients who underwent hepatic resection or radiofrequency ablation for initial HCC curative treatment. The patients were divided into four groups. Fifteen patients with low serum HBV DNA levels ( 4 log10 copies/ml) at the time of initial HCC treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before HCC appeared (pre antiviral therapy group; pre-TG). Thirty-four had low serum HBV DNA levels without antiviral therapy (low virus group; LVG). Fourteen had high serum HBV DNA levels and received antiviral therapy after HCC appeared (post antiviral therapy group; post-TG). Thirty patients had high serum HBV DNA levels without antiviral therapy (high virus group; HVG). Results: The cumulative HCC recurrence rates at 3 years in the HVG, LVG, pre-TG, and post-TG groups were 68.9%, 42.2%, 44.3%, and 57.1%, respectively. There were significant differences in the HCC recurrence rates between the HVG and LVG groups (P = 0.016), and between the HVG and pre-TG groups (P = 0.013). The recurrence rate was lower, though not significantly, in the post-TG group than in the HVG group (P = 0.18). Conclusions: Not only HBV DNA viral load but also antiviral therapy is associated with HCC recurrence. Antiviral therapy before HCC appears is important for patients with high serum HBV DNA levels to prevent HCC recurrence. Background/aims: Few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (HCC) patients, especially those treated by repeated transcatheter arterial chemoembolization (TAE). To determine risk factors for death and determine prognosis in patients treated with repeated-TAE, we evaluated clinical data. Methodology: We retrospectively analyzed clinical parameters of 224 unresectable HCC patients treated with repeated-TAE from January 1997 to December 2007. TAE was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. Factors affecting survival were evaluated using multivariate analysis after univariate analysis. Next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with JIS and CLIP score methods. Results: Multivariate analysis revealed that bilobular HCC, alpha-fetoprotein ( 400 ng/ml), tumor invasion of the portal vein, tumor size ( 10 cm), and albumin (<2.8 g/dl) were related to poor prognosis, Using those 5 factors, we developed a new prognostic scoring system. The 50% survival period was 29.2 months for all subjects, while it was 54.6, 29.2, 15.0, 5.6, and 1.0 months for those with scores of 0, 1, 2, 3, and 4 or over, respectively (P<0.0001), using our new system. CLIP score was not useful to predict prognosis, while JIS score was better. However, subjects with JIS scores of 1 and 2 were difficult to differentiate. Conclusion: Our scoring system was easy to perform and the results showed that repeated-TAE was effective for unresectable HCC with a score of 2 or less.
Local Ablative Therapies and Intrahepatic Pressure C. Kawamoto 1 , A. Yamauchi 1 , K. Kaneko 1 , N. Miyagi 1 , K. Kani 1 , T. Aoyama 1 , K. Yakabi 1 1 Saitama Medical Center, Saitama Medical University, Japan Background: Some of the unexpected recurrence observed after radiofrequency ablation (RFA) might be caused by increased intratumoral pressure. The present study examined the relationship between local ablative therapies and intrahepatic pressure. Methods: A. Basic study: Under general anesthesia, laparotomy was performed on 19 pigs. A LeVeen needle and a percutaneous ethanol injection (PEI) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. Ablation was performed as follows: 1. RFA. 1) Single-step method: After fully deploying the electrode, the power was initially applied at 30 W, then increased in increments of 10 W/min until power roll-off. 2) Multi-step method: The array was deployed in 8 steps. At each step, the power was fixed at 30 W until power roll-off. 2. PEI. Injection of ethanol (2 ml). B. Clinical study: We examined the multi-step RFA and PEI for HCC. Under local anesthesia, intratumoral pressure was monitored. 1. RFA. 39 patients with a mean tumor size of 15.3±4.9 mm were studied. 2. PEI. In 10 patients with a mean tumor size of 21.4±8.8 mm, 5 to 10 ml of ethanol was injected per session. Results : A. Basic study: The intrahepatic pressures were: single-step method, 154.5±30.9 mmHg; multi-step method, 24.1±18.2 mmHg; and PEI, 12.0±8.5 mmHg. B. Clinical study: Intratumoral pressure was 39.5±27.9 mmHg for RFA and 12.2±9.0 mmHg for PEI. Conclusion: These results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. Background: A late evening snack (LES) is recommended for liver cirrhosis. However, no clinical study has evaluated the nutrition status and the effect of LES in cirrhotic patients with hepatocellular carcinoma (HCC). We investigated the effect of LES undergoing hepatic arterial infusion chemotherapy (HAIC) in patients with HCC. Method: Nineteen patients with HCC were enrolled. Ten patients were LES group, and nine were control group. In the LES group, the patients received LES supplementation with a branched-chain amino acid (BCAA)-enriched nutrient mixture. In the control group, the patients received ordinary food. There were no significant differences in relation to age, gender, etiology, Child-Pugh scores, tumor stage, clinical responses to HAIC between two groups. Blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. Results: The non-protein respiratory quotient (npRQ) and molar ratio of branched-chain amino acid to tyrosine (BTR) were significantly improved in the LES group but not in the control group. There were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. Background & Aims: Hepatocellular carcinomas (HCCs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular HCCs is not fully elucidated. Cytokeratin (CK) expression profiles may also be useful prognostic indicators, and specifically CK19 may reflect metastastic potency in HCCs. This study was to assess the prognostic implication of tumor vascularity and its relation to CK19 expression in HCC patients. Methods: A total of 150 patients who underwent surgical resection for HCC were enrolled. Tumor vascularity was evaluated according to arterial enhancement pattern on CT scans and CK19 expression was evaluated using tissue microarray methods. Clinicopathologic data were analyzed using Kaplan-Meier and Cox proportional hazard model. Results: During follow-up period, 91 (60.7%) patients experienced tumor recurrence. Forty-five patients (30%) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for CK19 expression (p=0.001) and shorter disease-free survival (p=0.023) than patients with hypervascular HCCs. In addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular HCCs (p=0.001). Hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. Following treatment of recurred HCCs, hypovascular tumors showed poor response to transarterial chemoembolization (TACE), which resulted in shorter overall survival than hypervascular tumors (P=0.057). Conclusions: These results demonstrate that tumor hypovascularity in HCCs is associated with positive CK19 expression, early tumor recurrence, poor TACE response and poor survival. Therefore, tumor vascularity may also be a prognostic indicator in HCC patients. Background: Hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. Myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(HCC). In this study, we determine gene expression changes in two different models of HSCs activation and investigate whether induction-activated HSCs(iHSCs) gene expression changes are different from culture-activated HSCs(aHSCs). Methods: HSCs were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell lines C5F. Twenty-seven thousands and one hundred gene expression between quiescent HSCs(qHSCs), aHSCs and iHSCs was analyzed by microarray and confirmed by real-time RT-PCR and Western blot. Results: Sixteen hundreds and seventy-one probe sets were differentially expressed in aHSCs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. Seven hundreds and eleven probe sets were differentially expressed in iHSCs. Induction-activated HSCs showed specific gene expression patterns including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSCs activation. Induction-activated HSCs might play a important role in invasion and metastasis of HCC. Conclusions: Induction-activated HSCs gene expression patterns are different from aHSCs. Culture-activated HSCs does not properly regulate gene expression in HSCs, suggesting that iHSCs may be considered the model for the study of HSCs biology in HCC. 
Background: Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis is important for tumor growth. Ephrin receptors are related with vascular system development and the polymorphism of EphB1 in the carcinogenesis of digestive tract has been reported. Our aim was to examine the polymorphsims of EphB 1 with the occurrence of Hepatocelluar carcinoma in Korean population. Methods: Genomic DNA was extracted from 182 patients with hepatocellular carcinoma (HCC), 266 healthy subjects. EphB 1 polymorphism was determined by polymerase-chain reaction-based assays, and the association with HCC was investigated. Results: With regard to EphB 1 polymorphism, A/A genotype at rs11926992, T/T genotype at rs7644369, A/A genotype at rs6776570, T/T genotype at rs3821502 and G/G genotype at rs6766459 were significantly associated with HCC but these were not associated with clinical characteristics of HCC. Conclusions: Five out of seven polymorphisms on Ephb1 gene were statistically associated with HCC, in the Korean population. Therefore, more studies of Ephb1 gene polymorphisms including various risk factors should be performed to use as genetic markers of HCC occurrence. Background: We aimed to compare the results of hepatectomy for HCC in patients older than 70 years old with those for younger patients. Methods: Clinicopathological data and outcomes for 155 elderly patients and 333 younger patients with HCC who underwent hepatectomy between 1992 and 2007 were retrospectively compared. Results: Although postoperative delirium was more common in the elderly group, there were no significant differences between the 2 groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. The overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. Multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. Conclusions: Elderly patients with preoperative alcohol abuse should be followed closely, even after R0 surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. Background: Little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. Methods: Among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group A), while 46 patients were only transfused with packed red cells (group B), 43 patients were only transfused with fresh frozen plasma (group C), and 141 patients had no transfusion (group D). Results: Group C had significantly fewer postoperative complications and a shorter hospital stay than group A. Preoperative coagulation was significantly worse in group C. Survival was significantly better in groups C and D than in group A. Conclusions: Perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. Background: This study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. Methods: Perioperative risk factors for liver failure after hepatectomy were analyzed in 191 patients with hepatocellular carcinoma. Results: Liver failure occurred postoperatively in 16 patients, 3 of whom died. The hyaluronate/GSA-Rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. All 3 patients who died had a hyaluronic acid/GSA-Rmax ratio 500 mg min/dl. Conclusions: To reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. The hyaluronate/GSA-Rmax ratio can predict liver failure after hepatectomy, and a ratio greater than 500 mg min/dl is a relative contraindication to liver resection. The patient was a 73-year old Japanese man with chronic hepatitis C(CH-C) who achieved a sustained virological response(SVR) to interferon(IFN) therapy. As a result the liver functions were normalized and the histological findings of the liver also improved. However, 13 years after SVR, mild liver dysfunction was noticed along with a marked increase of tumor markers. Several modalities revealed huge liver tumors about 13 cm in greatest diameter in the left lobe invading the bile ducts and another tumor about 3 cm diameter in segment V. We performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (HCC). Only mild fibrosis development could be observed in the adjacent non-cancerous lesions. We successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. Recent studies revealed that the risk of developing HCC still exists even after SVR. Since most of HCC that develop in patients with SVR are usually detected within 5 years, several investigators speculate that HCC is already present but too small to be detected at the time of completion of IFN therapy. This speculation is not the case in our patient, since SVR was achieved 13 years ago and no HCV-RNA could be detected when HCC appeared. Therefore, another possible mechanism should be considered. An annual follow-up with strict surveillance program for HCC should be performed for more than 10 years after the completion of IFN therapy. Background/Aims: In order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (HCC) we analyze the expression of 8-Hydroxydeoxyguanosine (8-OHdG) in the liver tissue of the HCC patients with and without hepatitis viral marker. Methods: Patients undergoing hepatic resection for the first HCC from 1995 to 2004 were enrolled into the study. Only the cases that took no alcohol or small amount of alcohol were enrolled. 24 cases were negative for hepatitis B surface antigen (HBsAg) and antibody to hepatitis C virus (HCVAb) (NBNC group). 24 were positive for HBsAg and negative for HCVAb (B group). 21 were positive for HCVAb and negative for HBsAg and antibody to hepatitis B core antigen (C group). Staining with hematoxylin and eosin (H&E) and Berlin-Blue, and immunohistochemical staining for 8-OHdG were performed using the non cancerous liver regions. The degree of 8-OHdG immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per 1000 hepatocytes. Results: The labeling index of 8-OHdG for NBNC group is 19.3(±55.3), significantly lower (p=0.014) than that for B group 49.7(±89.5), and also lower (p=0.016) than that for viral group (B group and C group)(35.4±71.8).
The labeling index of 8-OHdG had no correlation with Grading, Staging, fatty and iron deposit among all cases. Conclusions: There is possibility that oxidative stress might not associate with the carcinogenesis of HCC in some cases without hepatitis viral infection. Background: No effective chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. Recently, it has reported that vitamin K2 (VK) and angiotensin-converting enzyme inhibitor (ACE-I) exert anti-angiogenic activity. The aim of the current study was to elucidate the combination effect of the clinically used VK and ACE-I on cumulative recurrence after curative treatment, especially in consideration of neovascularization. Methods: VK (menatetrenone; 45 mg/day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36 to 48 months after the curative therapy for HCC. The cumulative recurrence and several indices were analyzed. Results: A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment of VK or ACE-I for 36 months. Conclusions: The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization. Aim: The aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (HCC) patients surviving more than 5 years after hepatectomy. Materials & Methods: Retrospective study was carried out on 823 HCC patients who underwent curative hepatectomy between 1973 and 2002. Clinicopathologic factors in 5-year survivors and patients who died within 5 years were compared. The prognostic factors affecting survival were examined among the 5-year survivors. Results: There were 290 patients who survived for more than 5 years after initial hepatectomy, and 83 of those patients survived for more than 5 years after HCC recurrence. The overall 3-, 5-, 10-and 20-year survival rates were 57.4%, 40.9%, 17.2%, and 8.5% respectively. In multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than 1000 ng/mL, and absence of microscopic vascular invasion were favorable independent factors associated with 5-year survival. Negative hepatitis C virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. Multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<.05). Conclusions: The results suggest that patients without underlying cirrhosis who have a solitary HCC that does not demonstrate vascular invasion or high AFP levels might survive for longer than 5 years after the initial hepatectomy. Close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. The history of the use of carbon ion radiotherapy (CIRT) for treating hepatocellular carcinoma (HCC) goes back to 1995, when clinical trials were initiated at the National Institute of Radiological Sciences. We have already reported that CIRT used for the treatment of HCC is safe and effective, and that it causes only minor liver damage. In a phase II clinical trial, the local control and cumulative overall survival rates were 94 % and 35 % at 5 years, respectively. However, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for CIRT because of the high risk of radiation injury of the digestive organs. In order to extend the indication of CIRT, we have challenged the CIRT for such patients under the use of spacers. A case was a 67-year-old female with 8cm tumor in Segment 4. In radiological findings, the tumor revealed typical enhancement pattern for HCC, and was near the EC junction. She had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. She could undergo the 42.8 GyE/2-fraction CIRT after the placement of GORE-TEX Soft Tissue Patch under the laparoscopic procedure. Up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. This method seems to have a promising efficacy for extension of the indication of CIRT to the patients with tumors adjacent to the gastrointestinal tract. Background: Previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. On the basis of these finding, we firstly analyzed the effect of Bortezomib(proteasome inhibitor) on human HCC cell line. We also reported that HHM/DIP1/GCIP1 was early marker for human hepatocarcinogenesis. HHM was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. We analyzed change of HHM signal by Bortemib. Method and Result: We used HCC cell line (HuH7, HLF, HepG2) . The inhibitory effect of Bortezomib was evaluated using MTT assay. 20nM Bortezomib significantly inhibited proliferation of HCC cell line. The inhibitory effect by 20nM Bortezomib was similar with 10 M Cisplatin. On the other hand, Bortezomib has no inhibit effect in isolated hepatocyte from Rat. In this condition, we analyzed the expression of Cyclin D1, Phospho-Rb and HHM in HCC cell line by Western Blot analysis. Expression of Cyclin D1, Phospho-Rb decreased, but HHM was increased with time. Next we analyzed cell cycle by FACS. Bortezomib induced HCC cell line into cell cycle arrest in G2/M. The transcriptional activity of HHM was also activated by Bortezomib administration using pTimer-promoter-HHM plasmid. Conclusion: Bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. The induction of HHM by Bortezomib might be related with cell cycle arrest. Bortezomib will be a useful drug for HCC.
Neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study M. Kitade 1 , H. Yoshiji 1 , R. Noguchi 1 , K. Kaji 1 , T. Namisaki 1 , Y. Aihara 1 , H.
Background/aim: Non-alcoholic steatohepatitis (NASH) may progress to liver cirrhosis, and finally hepatocellular carcinoma. Recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. We therefore examined the role of angiogenesis in the hepatocarcinogenesis of NASH in both experimental and human study. Methods: As an experimental NASH model, Zucker (Z) rats, which naturally develop leptin receptor mutations, and their lean littermate (L) rats were fed a choline-deficient, amino acid-defined (CDAA) diet. In human study, 11 patients with NASH-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and 13 with simple fatty liver (FL) were enrolled and underwent clinico-pathological examinations. Immunohistochemical analysis of 4-hydroxy-2-noneal (4-HNE) and CD34 were employed for detection of reacrive oxidative stress (ROS) and angiogenesis in the liver tissues, respectively. Results: In experimental NASH model, both groups showed marked steatohepatitis by feeding CDAA diet. In sharp contrast, the development of glutathione-S-transferase placental form (GST-P)-positive pre-neoplastic lesions and HCC could be observed only in the L-rats. The hepatic neovascularization was also significantly increased only in the L-rats. In human study, Both NASH and FL exerted a marked elevation of ROS. In sharp contrast, significant development of hepatic neovascularization was observed only in NASH, whereas almost no neovascularization could be observed in FL. Conclusion: In conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in NASH. Background: Paternally expressed gene 10 (PEG10), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(HCC). The aim of this study was to generate transgene mice expressing PEG10 in the liver under the control of mouse albumin (ALB) promoter and study the integration, transcription, expression of PEG10 gene in the transgenic mice Methods: The linearized 1716bp transgene fragments, which contained ALB promoter and structural gene of PEG10, were microinjected into fertilized eggs of mice. Then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. All the newborn mice were screened and identified by PCR detecting genomic DNA in tail tissue. As the transgene was driven by the ALB promoter, we examined its expression in the liver of transgenic mice by RT-PCR and western blotting. Results: The transgene fragment was microinjected into the male pronucleus of 3741 fertilized oocytes. The injected eggs were implanted into oviducts of 94 pseudo-pregnant foster mothers, of which 22 mice became pregnant and give birth to 108 offspring. 65 of them died from unknown reason. Among the 43 offspring, 3 were identified to carry PEG10 cDNA as demonstrated by PCR, and PEG10 transgene could be expressed successfully in the liver of the established transgenic mice. The ratio of transgene integration were 6.97% (3/43) by PCR. Conclusions: The PEG10 transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in HCC. Background/Aims: Brivanib alaninate is the L-alanine ester prodrug of BMS-540215, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. It is being developed in treating hepatocellular carcinoma (HCC), a disease highly prevalent in Asia-pacific region. This analysis investigated whether BMS-540215 exposure was different between Asian and non-Asian subjects. Methods: A population pharmacokinetic (PPK) model was developed with data collected in 125 subjects (78 non-Asian, 47 Asian) with advanced and metastatic solid tumors (including HCC) from 2 clinical studies. Potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. Model-based simulation was performed to examine BMS-540125 exposure in Asian and non-Asian patients following brivanib doses of 800 mg QD (Phase III dose). Results: The PPK of BMS-540215 was characterized by a 2-compartment model with first-order absorption and elimination. Clearance was found to slightly increase with body weight (p<0.01). However, effects of age, gender and race on clearance were not statistically significant. The median of apparent clearance in Asian was 9.5% lower than that of non-Asians, which was adequately explained by 16% lower body weight in Asians. There was substantial overlap in steady-state BMS-540215 AUC of Asian and non-Asian patients, simulated based on their observed body weight distributions in these patient groups. Conclusions: BMS-540215 PK can be adequately described by a linear 2-compartment model; exposures in Asian and non-Asian subjects are similar following brivanib doses of 800 mg QD. Background/aims: Hepatic resection is the standard treatment for hepatocellular carcinoma. In some patients with multiple HCC, one-block resection can not be feasible due to either the tumor location or the reserved liver function. In this study, we attempted to analyze the outcome of multiple-site resection or combined resection and RFA in patients with multiple HCC. The prognostic factors for postoperative survival were also investigated.
Methods: Among 507 patients who received resection from January 1996 to August 2006, 58 patients had a radiologically detected multiple HCC. Patients with multiple HCC were divided into: group A, patients treated with one-block resection (n=40) and group B, patients with multiple-site resection or combined resection and RFA (n=18). Results: In group B, 6 received multiple-site resection and 12 underwent combined resection and RFA. The clinicopathological variables and postoperative complication rate were not significantly different between the two groups. The 5-year disease-free survival rates for group A and B were 24.1% and 18.3%, respectively (p=0.386). The overall survival rates were also not significantly different (36.9% vs. 39.4%, p=0.528). The multivariate analysis revealed that radiological tumor number 3, Edmondsons-Steiner grade (III-IV) and indocyanine green retention rate at 15 minutes> 10% were adverse prognostic factors for overall survival. Conclusions: Active treatments including multiple-site resection and combined resection and RFA showed similar treatment outcomes compared with one-block resection in patients with multiple HCC. The prognosis after treatment was associated with tumor number, tumor grade and ICG R15. Background: Nasopharyngeal carcinoma (NPC) is endemic to southern China. Mortalities are mostly associated with secondary metastases. Novel treatments for NPC metastases are thus urgently needed. We aim to test the efficacy of a physiologically stable gold compound, gold (III) meso-tetraarylporphyrin 1a (gold-1a), in treating intrahepatic NPC metastasis in athymic mice. Methods: Twenty million of C666-1 human NPC cells were injected into the livers of athymic mice to induce primary tumors. Gold-1a was administrated by intraperitoneal injection. Survival times, tumor volumes and degrees of metastasis of the animals were evaluated. Intratumoral microvessel density was determined by immunohistochemical staining for CD34. Tube formation by MS1 mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. Gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. Cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. Result: Gold-1a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. The compound induced tumor tissue necrosis and reduced tumor microvessel formation. In in vitro studies, gold-1a inhibited tube formation and proliferation of MS1 cells, and downregulated the expression of stanniocalcin 1 (Stc1), which plays roles in angiogenesis. Furthermore, our preliminary data showed that overexpression of Stc1 in MS1 cells rescued cells from gold-1a-induced death. Conclusion: Gold-1a is a novel anticancer agent that prolongs survival of the NPC metastases-bearing mice. It inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of Stc1. 
Tbx3 is a transcriptional repressor that is important for embryonic development. Overexpression of Tbx3 was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. Tbx3 promote carcinogenesis by bypass cellular senescence via suppression of p14 ARF . Our resent studies revealed that two key motifs composed of 7 + 3 residues are essential for its transcriptional repression. Based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. We found that TAT-tagged peptides (TAPs) effectively transduced hepatoma HepG2 and BEL7404 cells at almost 100% efficiency and inhibited cell growth in a dose dependent manner. Further studies revealed that the TAP treated cells underwent up-regulate apoptosis via suppression of p14 ARF both at mRNA and protein levels, demonstrating the potential of novel TAPs for anti-HCC treatment in the future.
Safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma K. Kakisaka 1 , H. Kuroda 1 , K. Kasai 1 , Y. Takikawa 1 , K. Suzuki 1 1 Iwate Medical University Background and Purpose: A tendency of the aging in patients with hepatocellular carcinoma (HCC) is predominantly seen in Japan. In fact, the mean age of patients with HCC in our institute in 1990 was 60.1 years old, while that in 1999 was 67.8 years old. It is not still remained whether the percutaneous radiofrequency ablation (RFA) therapy in elder patients with HCC is safety and equal in therapeutic usefulness compared to the non-elder patients with HCC. Subjects and Methods: Two hundred six cirrhotic patients with HCC (376 tumor nodules) received RFA therapy curative intent since August, 2006 were enrolled. We divided all patients into two groups: over 65 years (elder group: n=135) and under 65 years (non-elder group: n=71), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. Results: The characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. Although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under RFA respectively, total occurrence rate of complications did not differ between two groups. Conclusion: RFA therapy is safety and effective even in elder patients with HCC, although their care is necessary to prevent any complications which are often occurred during the RFA therapy. Background and Purpose: The aim of this study is to evaluate whether administration of the branched-chain amino acid (BCAA) enriched nutrient (namely, aminoleban EN, Ostuka Pharmaceutical Company, Japan) might improve protein-energy malnutrition (PEM) status and quality of life (QOL) in cirrhotic patients with HCC receiving RFA therapy. Subjects and Methods: Thirty-five cirrhotic patients with HCC who had received RFA therapy from October 2005 to October 2006 in our institute were randomized into two groups: diet with supplementation of aminoleban EN (EN group: 20 patients, 420 kcal/day) and diet only (control group; 15 patients). The total intakes of calories (30-35 kcal/kg) and protein (1.0-1.5 g/kg) were equal between tow groups. The primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related QOL by ShortForm-8 questionnaire (SF-8). Results: Total intakes of calories and protein were similar during the one year after RFA. No significant differences in event-free survival rate were seen between two groups. However, decreased serum albumin levels and one (general health perception) of domains in SF-8 were significantly improved in EN group compared to the control group. Conclusion: Supplementation of BCAA-enriched nutrient may improve the impaired liver function and QOL after RFA therapy. Large scale prospective study should conduct to confirm these results near the future. 
Backgrounds and aims To investigate the effects of selective Cox-1 and Cox-2 inhibitor on proliferation and apoptosis of HCC cell. Methods Hep3B and SNU 387 cells were treated with NS-398 and SC-560. MTT assay, caspase 3/7 activity assay and TUNEL assay were performed. Cox protein and mRNA expression were measured by Western blot and real time RT-PCR. Results In Hep3B cell line, Cox-1, Cox-2 (50, 100, 200 uM) and combination (25+25, 50+50, 100+100 uM) treatment after 24hr showed a significant dose dependent inhibitory effect on cell growth (p<0.05). Cox-1, Cox-2 (100 uM) and combination (50+50, 100+100 uM) treatment after 24hr significantly increased caspase 3/7 activity (p<0.05) and induced apoptosis (p <0.05). However, the combination treatment could not showed a additive effect to Cox-1 or Cox-2 inhibitor (p>0.05). In SNU 387 cell line, Cox-1 inhibitor and combination treatment showed a inhibitory effect on cell growth (p <0.05) similar to Hep3B cell line but any of treatment could not induce apoptosis significantly (p >0.05). In Cox protein and mRNA expression, SNU 387 cell line showed significant Cox-1 predominency (p=0.037) but Hep3B cell line showed Cox-2 predominency (p=0.032). Conclusions In HCC cells, no additive effect of the combination treatment of Cox-1 and Cox-2 inhibitors could be anticipated. The apoptosis inducing effect of Cox inhibitor could be different between HCC cell lines. More studies for the mechanism of different response to Cox inhibitor between cell lines is needed. Background: The aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (UFT) (phase I part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (HCC) at the recommended dose (phase II part). Methods: Patients eligible for study had histologically confirmed, chemo-naïve advanced HCC, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. The therapy consisted of mitoxantrone dosages (6, 8 and 10 mg/m 2 /day) intravenously on day1 and oral administration of UFT 300 mg/m 2 on day 1 through day 21. The treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. Results: A total of 25 patients were entered into the study. All had a good ECOG performance status score of 0-1. In phase I part, dose limiting toxicities occurred in all three patients (two patients: grade 4 neutropenia, one patient: grade 3 creatinine elevation) given mitoxantrone at dosage of 10 mg/m 2 /day, and the recommended mitoxantrone dosage was 8 mg/m 2 /day. Among 19 patients administered at the recommended dosage, one patient (5.3%) achieved a partial response, 8 patients (42.1%) had stable disease and 10 patients (52.6%) had progressive disease. One-year survival proportion, median survival and median progression free survival were 26.3%, 8.4 months and 2.5 months, respectively. The most common toxicities were grade 3-4 leucopenia (15.4%) and neutropenia(10.3%). Conclusion: Mitoxantrone 8 mg/m 2 with UFT 300 mg/m 2 /day is recommended dose. This regimen is generally well tolerated, but appears to have little activity for advanced HCC. These findings do not support its use in practice, and further trials with this regimen in patients with advanced HCC are not recommended. The study assessed the benefits of 3-D reconstruction of spiral CT scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. We retrospectively analyzed 33 cases of children with such tumors treated in past 5 years.The patients were examined by 3-D reconstruction using 64 slice spiral CT. In 28 cases, the volume of tissue removed exceeded 1/3 the entire volume of the liver. In 5 cases, the excised tissue represented less than 1/3 of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. Pathological diagnoses included hepatoblastoma (n = 20), hepatocellular carcinoma (n = 4), mesenchymal hamartoma (n = 4), teratoma (n = 1) and adenoma (n = 4). All children had curative resections with tumor-free microscopic margins. 3-D CT imaging can provide high quality images and accurate location of the tumors. It could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. With its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. Background: To investigate the association between C-509T polymorphism of transforming growth factor (TGF)-1 gene and HBV-related hepatocellular carcinoma (HCC). Methods: Patients with HBV infection (196 cases were HBV carriers, 379 cases were HCC) and 299 healthy volunteers were enrolled. The polymorphism of TGF-1 gene C-509T was identified by polymerase chain reaction-restriction fragment length polymorphism method. The concentrations of plasma TGF-1 were measured by enzyme linked immunosorbent assay (ELISA). TGF-1 mRNA expression was quantified by real-time PCR. A recombinant construct containing -509C>T variant as promoter and CAT as reported gene was transfected into HepG2 cells. The reporter gene CAT was detected with ELISA. Results: The CT genotype at position -509 of TGF-1 gene prevailed in all three groups, the frequency of genotype CC and allele C at -509 in HCC were significantly higher than those of the HBV carriers and controls. The plasma TGF-1 concentration among the three genotypes did not show any significant difference in three groups. However, both the TGF-1concentration and liver mRNA levels were statistically higher in patients with CC genotype than in those with TT genotype in the HCC group. Reporter gene CAT was elevated when HepG2 were transfected with -509C-CAT recombinant construct compared to that with -509T-CAT one (p<0.05) Conclusion: The presence of C allele at position -509 may play an important role in the development of HBV-related HCC through influencing TGF-1 expression both at mRNA level and protein level. Background: To assess diagnostic value of N-glycan markers in identifying hepatocelluar carcinoma (HCC) from liver fibrosis after HBV infection. Methods: A total of 273 cases of HBV related liver fibrosis (n=128) and HCC (n=145) patients as well as matched healthy controls (n=120) were recruited. Routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. N-glycome of serum protein was profiled by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based ABI3130 sequencer. Results: The abuncance of a single agalacto biantennary glycan (NG1A2F, peak 4) was increased in liver fibrosis and decreased in HCC, while that of a branching triantennary glycan (NA3Fb, peak 9) was decreased in fibrosis and increased in HCC. The efficacy of the log ratio of above two N-glycan abundance [log (p9/4)] was similar to AFP in differentiation HCC from fibrotic patients. With logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining AFP with N-glycan analysis(Cscore B) were increased 7-10% compared to AFP. Log(p9/4) was even more powerful in monitoring the progresison of HCC with the specificity improved 16% and accuracy improved 8% compared to that of AFP. Besides, log(peak 9/4) was correlated well with other tumor markers and TNM stages. Conclusions: The log ration of the abundance of a branching triantennary glycan (NA3Fb, peak 9) to a single agalacto biantennary glycan (NG1A2F, peak 4) and the model combining AFP with N-glycome markers are promising in HCC diagnosis and progression monitoring. The low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (RFA) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. However, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. Objective: We aimed to compare the necrotic zone produced using different electrode withdrawal techniques. Methods: Eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled RFA electrode (Cool-tip radiofrequency system, Valleylab) 1-2 mm/second using energy-dependent (20 vs.40 vs.60 vs.80 watts) and temperature-dependent (80 vs. 90 0 C) techniques. Horizontal 
Mathematical modeling suggests an impractical number of radiofrequency ablation (RFA) zones needed in order to ablate a medium-large hepatic tumor. However, overlapping RFA zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. Objectives: To compare the necrotic diameter in single (Group1), dual overlapping (Group2) and dual non-overlapping (Group3) ablation. Methods: Single (n=5) and dual (overlapping n=18; non-overlapping n=6) ablation zones were created in ex vivo porcine livers using Cool-tip RFA electrodes. Necrotic diameter was measured at the midpoint (MaxD) of the single and the two distinct RFA zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (MaxD-O), corresponding to the point of overlap in Group2. The RFA electrode was withdrawn 2.5 and 4.1 cm before re-ablating for Group2 and Group3, respectively. Results: Despite no difference in end-RFA temperature between 3 groups (Group1=75.6+7.4Cvs.Group2=73.9+7.3Cvs.Group3=74.8+4.8C; p=0.873), MaxD was significantly greater (p=0.011) in Group2 (4.1+0.7cm) as compared to Group1 (3.3+0.6cm) and Group3 (3.3+0.4cm), with no difference between Group1 and Group3(p=1.00). Further proof of synergism between two overlapping ablations is that the MaxD-O in Group2(4.3+0.6cm) was larger than MaxD of Group1 (p=0.042) and Group3 (p=0.028), and was similar to MaxD of Group2 (p=1.00). Conclusions: Overlapping two RFA zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. This may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. Background: Hepatocellular carcinoma (HCC) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis B and C viruses. The present study was undertaken to evaluate the association of glutathione-S-transferase (GST) T1 and M1 null genotypes and microsomal epoxide hydrolas e(mEPHX) polymorphisms with hepatitis virus related HCC risk in Indian population. Subjects and Methods: Three groups of subjects were considered viz. control (n=169), chronic viral hepatitis (n=174) and HCC (n=63). PCR-RFLP was used for this polymorphic study. Genotype distributions between categories were compared using the 2 test; odds ratios (ORs) and 95% CI were calculated to express the relative risk. Results: Presence of GSTM1 null genotype significantly (p<0.05) decreased the risk for HCC development among chronic viral hepatitis subjects. However, GSTT1 null genotype was associated with an increased risk for HCC by 2.23 and 1.42 times among control and hepatitis subjects respectively. In case of mEPHX, Tyr113His and His113His genotypes significantly (p< 0.05) reduced the risk of HCC development in both viral hepatitis and control subjects. In case of mEPHX exon 4 genotypes, Arg139Arg imposed an approximate 2 fold risk for HCC development in the two groups. Combination of heterozygous mutant genotypes at mEPHX exons 3 & 4 also imposed around 2 fold risk (non-significant) for HCC. Conclusions: Polymorphic forms of GST and mEPHX share an association with viral related HCC risk in Indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related HCC. 
Background : the association between type 2 diabetes mellitus (DM2) and hepatocarcinoma (HCC) has been identified in the last ten years. Methods: to clarify the temporal relationship between DM2 and HCC and the possible effects of antidiabetic therapy on HCC risk, we recruited 465 patients with HCC compared with 490 control subjects without liver diseases and 618 cirrhotic patients. Results: prevalence of DM2 was 31.2% in HCC, 23.3% in cirrhotic and 12.7% in control group. In univariate and multivariate analysis, the odds ratio (OR) for HCC in diabetic patients were respectively 3.12 (CI 2.2-4.4; P <0.001) and 2.2 (CI 1.2-4.4; P= 0.01). OR in univariate analysis were higher in male than in female patients. In 84.9% of the patients DM2 pre-exists the diagnosis of HCC from a mean time of 141.5 months. Moreover, the insulin treatment was more frequent in diabetic HCC patients than controls and we report an OR for HCC of 2.99 (CI 1.34-6.65; P= 0.007) in patients treated with insulin or sulfonylureas, and an OR of 0.33 (CI 0.1-0.7; P= 0.006) in patients treated with metformin.
Conclusion: our study confirms that male patients with type 2 diabetes mellitus have a significantly increased risk of HCC independently of other cofactor such as HBV, HCV and alcoholic abuse. DM2 is a pre-existing disease in most HCC patients and suggests that insulin and sulphonylurea treatments in DM2 are associated with an increased risk of HCC development, while metformin may have a protective effect. Background & Aims: Over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to HCC. The purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by LOH or CGH, and search the new tumor suppression genes for HCC. Methods: Primary HCC and corresponding non-tumor liver tissues were obtained from surgery. Serologically, 13 cases were with hepatitis B virus infection and 12 cases were with hepatitis C virus infection. Four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.Affymetrix GeneChip, U133A, was used to compare the loss and gained gene expression in liver needle biopsy samples (n=54). Results: After adjusting by chromosome arm length, 16p, 17p, 19p, 19q and 22q showed higher gene loss-expression ratio (>= 10 loss / cM) in the comparison between normal samples and tumor samples; 1q, 2p and 4q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. More than 50 genes showed different loss expression level in this study. For example, CD10 was loss expression in all non-tumor samples comparing to four normal samples. Ficolin 3 and ficolin 2 were loss expression in HCC samples with HBV infection and with HCV infection, respectively. Conclusion: Our results revealed the potential tumor suppression genes and the genomic region they harbored. Further study is needed to validate the observation. Background/Aims: Hepatocellular carcinoma is common malignancy in human, accounting for 1 million deaths in the world annually. Caspase 8, as an initiator caspase, is involved in the induction of apoptosis. Survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of HCC with unfavorable prognosis. Methods: To explore the possibility that the epigenetic alteration of caspase 8 and survivin genes is implicated in the development and progression of HCC, promoter methylation of two genes was analyzed in 73 cases of primary HCC by methylation specific PCR. The relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. Results: The methylation of caspase 8 (34.2%, 25/73) and survivin (32.9%, 24/73) demonstrated a negative correlation with immunohistochemical expression of capsase 8 (47.9%, 35/73) and survivin (43.8%, 32/73) (p=0.042 and p=0.001 respectively). Methylation of caspase 8 and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p=0.026 and p=0.032). Survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p=0.001). By survivial analysis, the negative caspase 8 expression and positive survivin expression showed worse prognosis in HCC, that was statistically insignificant (p>0.05).
Conclusion: In conclusion, caspase 8 and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in HCC. injection was recently reported to be effective against HCC with PVI, though the therapy is not always applicable for the patients with arterial abnormality. Therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. Methods: The combined therapy was conducted in 7 HCC patients with PVI. Transcatheter arterial embolization with 1mg/kg cisplatin powder (IA call) was performed against intralobar lesions, followed by external radiation targeted for PVI (60Gy in 2 Gy fractions). The following variables were evaluated with the survival rate: gender, age, viral etiology, Child's class, performance status, and location of PVI. Results: One (16%) patient showed complete response and another two (33%) partial response. Two (33%) showed no change, and one (16%) showed progress of disease. The survival rates at six months among overall patients were 85.7%. Adverse events were limited to nausea and appetite loss. One of the patients with partial response underwent curative resection, and is still alive without any recurrence for 530 days. Conclusions: The combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. This therapy is suggested to be a useful alternative therapy for the patients with extensive PVI. Recently, the injection port has been used for hepatic arterial infusion chemotherapy (HAI) in Japan. HAI is usually used for the treatment of multifocal bilobar tumors of the liver or HCCs combined with portal vein tumor thrombosis (PVTT), not amenable to TACE. This study examined the efficacy and toxicity of repeated hepatic HAI using lipiodol suspension mixed with cisplatin powder. Methods: From April 2005 to September 2008, 20 patients with inoperable advanced HCC were enrolled in this study. All received cisplatin powder (50mg) and Lipiodol (2ml) suspension, with an intervening 4 weeks interval. The drugs were delivered from an injection port. 13 patients had HCC with PVTT, and 7 had HCC without PVTT. 11 patients with liver function of Child grade A, 7 of grade B, and 2 of grade C were enrolled. Result: The mean number of HAI given during the follow-up period was 4.1 times. We found complete response in 1 case, partial responses in 6, no change in 6, and progressive disease in 7. The overall response rate was 35.0%. The 1-year survival rate was 36.7% and the 2-year survival rate was 12.1%. Although 3 patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. Conclusion: Chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced HCC. Background: Recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. The gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. However, there are still few reports of mRNA expression of Vascular Endothelial Growth Factor (VEGF) receptors in HCC. Methods: The relative mRNA level of VEGF and its receptors (KDR and flt-1) was analyzed using quantitative RT-PCR in 51 patients with HCC. Matched samples of HCC (T) and non-tumor liver tissue (NT) were obtained by fine needle (21gauge) biopsy. Results: Gene expression level of VEGF and flt-1 was significantly higher in HCC than NT (VEGF; p<0.001, flt-1; p<0.001). According to the clinicopathological findings, gene expression level of VEGF and KDR in HCC was significantly high in hypervascular HCC compared to hypovascular HCC (VEGF; p=0.002, KDR; p=0.042). Additionally, flt-1 tended to be expressed higher in hypervascular HCC than hypovascular HCC (p=0.09). Moreover, gene expression level of VEGF, KDR and flt-1 tended to be higher in advanced-stage HCC than early-stage HCC. Conclusion: Not only VEGF but KDR and flt-1 were highly expressed in hypervascular and advanced HCC. Aims: Fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". Fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. Therefore, this study was designed to examine the role of fgl2 in tumor development. Methods: Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (HCC) model on nude mice (established from high metastasis HCC cell line MHCC97LM6) were obtained. Results: Hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8 + T lymphocytes and vascular endothelial cells. Hfgl2 mRNA was localized in cells that expressed hfgl2 protein. Fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. Conclusion: The hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . The therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least 2 cycles, when responses to treatment were evaluated by Eastern Cooperative Oncology Group criteria. Results: Of 146 patients treated (male, 79%; median age, 64 years), 64% had Child-Pugh A, and 31% had B. 90% had either metastasis or vascular invasion. 71% had metastasis and 49% had vascular invasion. On the basis of independent assessment, three (2.1%) patients achieved a complete response, thirteen (8.9%) had a partial response, and 42 (28.8%) had stable disease. There was no grade 3/4 drug related toxicities. Median overall survival was 6.9 months. Conclusion: Combination therapy of IFN +5-FU has modest efficacy in HCC. Background: AMT is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). Preclinical and phase I data in healthy volunteers showed a favourable safety profile for AMT. This pilot study should examine efficacy and safety of AMT in the patients with advanced hepatocellular carcinoma (HCC). Methods: Thirteen patients with advanced HCC (TNM stage III-IV), who did not respond to existing therapy, were treated with i.m. AMT at 0.066 ml/kg up to a maximum volume of 5 ml twice daily for 1-10 months. Primary study objectives: Clinical Benefit Response (CBR). Secondary objectives: safety of AMT, tumor response according to WHO-RECIST criteria, quality of life (QOL) and iimmunomodulatory effects. The effects were evaluated by cytokine production of PBMCs before and after the treatment. Results: There were no significant safety issues. Four and 3 patients showed positive and stable responses for CBR, respectively. Tumor response was 1 PR, 6 SD and 6 PD. Even in the patients with PD, 2 and 1 patients showed positive and stable responses for CBR. QOL data showed clear improvement.
Immune monitoring demonstrated effects of AMT on the functional immune parameters in about half of patients. In the patients with PR, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. Conclusion: These results suggest that a promising rate of patients with advanced HCC respond clinically to the AMT treatment without significant safety issue and AMT has some immuno modulatory capacities. Background/Aims: Dysplastic nodules are important due to premalignant potential. The aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. Methods: A total of 5 patients (mean age: 64±9 years old, male 4) with dysplastic nodules which suspected as malignant nodule (mean size 1.38±0.22 cm) was enrolled from 48 cases of liver cirrhosis undergone ultrasonography-guided biopsy from December 2006 to January 2008. The etiologies of liver cirrhosis were as follows; alcohol (1 patient), hepatitis B virus (2), and hepatitis C virus (2). Results: Hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. The nucleus was round or oval shaped and the nucleus membrane was irregular. The nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. Glycogen granules and lipofuscin pigments were abundant. Sinusoid formation was poorly developed and collagen fiber bundles were increased. The hepatocytes of rosette formation and bile ductules cell made of canal of Hering, which was dilated and microvilli was decreased. The number of canal of Hering was 7, which was composed of 1.7±0.8 with hepatocyte and 2.1±0.7 with bile ductule cell, respectively. There was no oval cell in the canal of Hering, which was relatively well developed. Schwann cells were clustered together in nerve plexus. Therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. Conclusions: This study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma.
DCP is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< 3cm in diameter R. Kuromatsu 1 , A. Takata 1 , N. Fukushima 1 , S. Sumie 1 , M. Nakano 1 1 Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine Background and aims: The aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (RFA) and hepatic resection (Hr) for single hepatocellular carcinoma (HCC) < 3cm in diameter. Methods: Between 1999 and 2007, 219 patients with single nodule < 3cm in diameter and Child-Pugh grade A were treated by Hr and RFA, and recurrence rate and survival rate using Kaplan-Meier method, and important risk factors for recurrence using Cox's proportional-hazards regression model were analyzed. Factors used for multivariate analyses were age, gender, viral marker, tumor diameter, AFP, AFP-L3, DCP, and platelet count. Results: Mean age was 66 years old, M/F ratio was 136/83, Hr/RFA was 59/160, and mean observation period was 1194 days. Five-year survival rates, and 2-year local recurrence-free + intrahepatic metastasis-free rates were not significant between Hr group and RFA group (82/72%, 92/89%). In RFA group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was DCP (P=0.0034). Tumor diameter was not significant for recurrence. In Hr group, there was no risk factor for recurrence. In pathological analyses of Hr group, DCP had a tendency to associate with microvascular invasion (P=0.065).
Conclusions: RFA was effective for HCC < 3cm in diameter and DCP < 40 mAU/ml. Hepatic resection should be selected for single HCC with DCP > 40 mAU/ml even though HCC <3cm in diameter. Background: Radiofrequency ablation (RFA) is now a common treatment for small hepatocellular carcinoma (HCC). However, critical complications after RFA such as rapid intrahepatic dissemination have been reported. In this study, we investigated the method how to estimate the malignant potential of small HCC by dynamic CT before RFA. Methods and results: Firstly, 61 HCCs less than 3cm in diameter were analyzed. Those tissues were classified into 4 groups as followed, small nodular type with indistinct margin (type E), simple nodular type (type 1), simple nodular type with extranodular growth (type 2), confluent multinodular type (type 3). In the type 2 and 3 groups, portal invasion over vp1 were observed more frequently than those in the type 1 and E groups. At the next step, these 61 HCCs were classified into above-mentioned 4 types by two radiologists according to the shape of early stain or defect of delay phase of dynamic CT before operation. The accorded rate was 87% between those classifications. Next, 45 patients, which had solitary HCC less than 3cm in diameter and treated with RFA, were classified into those 4 types by dynamic CT before RFA. The recurrence rate and prognosis of those patients were examined. In the type 2 and 3 groups, the recurrence rate was higher and significant worse prognosis was showed than those in type 1 and E group . Conclusion: It was suggested that HCC with type 2 and 3 might process higher malignant potential and RFA should be carefully performed on those types of HCC. Background/Aims: Hypoxia-inducible factor-1 (HIF-1 ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. IL-8 became known to replace HIF-1 functions in the other cancer cell lines. The aim of this study was to evaluate whether IL-8 may induce angiogenic factors without HIF-1 by inflammation signal of hypoxic condition. Methods: HIF-1 knockdown cell lines of HCC (Huh7 and HepG2) were constructed by RNA interference tools, and cultured under normoxia (21%O2, 24 hours) and hypoxia (1%O2, 24 hours) conditions. Following transfection, the amounts of HIF-1 , IL-8, angiogenic factors and matrix metalloproteinase (MMP) were examined using RT-PCR and western blotting, respectively. Results: The expression of HIF-1 , angiogenic factors, MMP, IL-8 was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and MMP was partially blocked in HIF-1 knockdown HCC cell lines. NF-B inhibitor suppressed angiogenic effects by blocking IL-8 activity. Conclusion: These data suggest that IL-8 induced tumor angiogenic factors in HIF-1 knockdown HCC cell lines. Background There were some reports that liver caner related to the levele of sera HBVDNA Our research focused on the relationship between the quantity of hepatocellular HBV cccDNA , tDNA and liver cancer. Methods The samples included the liver tissue of 24 CHB patients (CHB group) and the para-liver cancer tissue of 26 primary liver cancer patients (PHC group) The quantity of hepatocellular HBVcccDNA, tDNA were assayed by FQ-PCR in both groups. Result: The quantity of hepatocellular HBV cccDNA in CHB group was 11.56±16.14 copys/cell higher than PHC group(3.96±9.27 copys/cell), P=0.011; The quantity of hepatocellular HBV tDNA in CHB group was 57.38±91.64 copys/cell higher than that of PHC group(35.07±93.03 copys/cell),P=0.13.; Conclusion: The quantity of hepatocellular HBVcccDNA, tDNA can not be used as predictors of liver cancer for hepatitis B patients. Hepatic cancer predominantly occurs in Males. This is almost a commonsense to most of us. But the detailed mechanisms underlying such phenomenon are still not well-known. The average age of liver cancer patients are about 30-40 years old. So most female patients have udergone pregnancy at least one time. Pregnancy is a very important event before or during the development of liver cancer in females. In this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. So it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. In this study, 100 female SD rats are randomly divided into two groups: pregance group and controll group. Rats in both groups are injected IV Diethylnitrosomine(a chemical carcinogen). In pregnance group, rats are raised together with male rats in 3:1 ratio(3 female, 1 male) to make every rats undergo pregnancy. While in controll group, rats are coupled with spermaduct-ligated male rats. The size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. The survial rate is also significantly higer than that in controll group. We conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. Acknowledgement: This project was sponsed by the National Natural Science Foundation of China. The number of the grant is 30600727:
The use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation N. Yamashiki 1,2 , Y. Sugawara 1,3 , S. Tamura 3 , R. Tateishi 2 , H. Yoshida 2 , J. Kaneko 3 , Y. Matsui 3 , N. Kokudo 1,3 , M. Omata 2 1 Organ Transplantation Service, 2 Department of Gastroenterology, University of Tokyo, 3 Department of Surgery, University of Tokyo Background: The recurrent hepatocellular carcinoma (rHCC) after liver transplantation (LT) can occur in 10 to 20 % of transplant recipients despite with a careful patient selection. For the surveillance of rHCC, frequent measurement of alpha-fetoprotein (AFP) and annual CT scan is commonly used. However, the usefulness of AFP is not clear. We report the update of our experience using our surveillance protocol. Methods: Between 1996 and March 2008, 330 adult living donor LT were performed at the University of Tokyo. Among them, 100 recipients with HCC in their explanted liver were subjected to analysis. We used monthly measurement of AFP and des-gamma carboxy prothrombin (DCP) with annual dynamic CT scan. Results: 83 met Milan criteria pre-operatively and 12 did not. 5 were incidental HCC. rHCC was experienced in 9 patients at 10 (2-24) months after LT. Recurrence sites were graft (1), lung (2), bone (3), and multiple organs (3). rHCC was first suspected from elevation of tumor markers in 8; AFP in 4, DCP in 2, and both AFP and DCP in 2. rHCC was confirmed with CT scan (7) or MRI (2) in 4 (0-6) months after the first sign of rHCC. When the cutoff level of AFP>20ng/ml was used, the sensitivity and specificity for rHCC were 66% and 100%. Six cases were treated surgically of which two achieving prolonged survival. Conclusions: Although the confirmation of the rHCC sites required multiple imaging studies, AFP measurement was useful as for the first sign of rHCC.
Purpose: To evaluate the therapeutic effect of heated (60 C) lipiodol via hepatic artery administration in VX2 rabbit liver cancer model. Materials and Methods: Thirty male New Zealand white rabbits were randomly divided into 3 groups with 10 rabbits for each group. VX2 carcinoma cells were surgically implanted into the left liver lobe. The tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching 2 to 3 cm. Under the anaesthesia, transcatheter hepatic arterial embolization was performed and Doxorubicin-lipiodol (37ºC) (1 mL), lipiodol (60ºC) (1 mL) and control (physiological saline (37ºC) (1 mL)) were injected into hepatic artery of the 3 different groups. One week later, the volume of tumor was measured by ultrasonograph again. The serum of all rabbits was collected before injection and at 4 and 7 days after injection and the level of aspartate aminotransferase (AST) was checked. The survival period of 3 groups of rabbits after treatment was also recorded. During the last course of their disease, the rabbits were given some analgetics to relieve suffering. Results: The tumors' growth rate in lipiodol (60ºC) Background/Aims: Hepatocellular carcinoma (HCC) is one of the male-dominant cancers, and hepatitis C virus (HCV) is one of the causes of HCC. It was reported that androgen receptor (AR) is expressed in HCC and its surrounding tissues. Androgen signaling and AR may be involved in hepatocarcinogenesis. In this study, we investigated whether HCV interacts with androgen signaling in human hepatocytes. Methods: HCV protein expression vectors were co-transfected with AR-expression vectors and AR-responsive element-driven reporter vector into immortalized human hepatocytes (IHHs) and human hepatoma cell lines. Kinase inhibitors were used to examine the activation of the Akt, MAPK, and JAK/STAT3 pathways. Real-time PCR and Western blotting were performed. Cell culture grown HCV (HCVcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of 5 -androgen-1 -ol-3-one. Results: HCV enhances AR-responsive gene expression in the presence of androgen. HCV core protein has the strongest effects and induced AR activation associated with JAK/STAT signaling. HCVcc enhances VEGF mRNA expression and angiogenesis. Conclusions: HCV core protein is an enhancer in androgen signaling and can be expected to play an important role in HCV-related hepatocarcinogenesis. 
Background: To evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the Chinese traditional Jianpiliqi (JPLQ) formula in the treatment of advanced hepatocellular carcinoma (HCC). Methods: Patients with advanced HCC, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide (6 mg / m 2 ) administration from days 1-14, and an oral administration of JPLQ formula twice daily from days 1-28. Each cycle was composed of 28 days and treatment could expand up to 4 cycles before evidences of intolerable toxicity or disease progression. Result: One patient had partial response, one had minor response, 15 showed stable disease and 15 (46.9%) had disease progression. Total disease control rate was 53.1%, median survival time was 5.8 months (2-23.9 ms), and time to progression was 4.2 months (1-16.9 months). The incidences of grade 1-3 abdominal distention and nausea/vomiting were 65.6% and 37.5%, respectively. Increases in GGT occurred in 4 patients (2 grade 2, 1 grade 3, and 1 grade 4) and increases in serum creatinine in 2 patients (1 grade 3 and 1 grade 4), respectively. Conclusion: Compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with JPLQ showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced HCC; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. 
Aim: To investigate the effect of osteopontin (OPN) expressions down-regulated by RNA interference (RNAi) on the invasion and metastasis of human hepatocelluar carcinoma (HCC). Methods: HCC cell line (HCC-LM3) was transfected with the chemically synthesized small interfering RNA (siRNA) in study arm and with non-specific siRNA in control arm. Real-time PCR and Western blotting were used to quantify the mRNA and OPN protein levels. The malignant phenotypes including cellular growth rates, colony formation and Matrigel invasion activities of the HCC cell line were analyzed. Results: In study arm OPN mRNA expressions decreased 75% and OPN protein decreased 80% compared to those of blank arm. The number of formed colonies and migrating numbers of the cells in vitro decreased significantly (64.6% and 59.6% respectively) in study arm compared to these of blank controls (p<0.05). The parameters in the control arm did not differ from those of the blank arm (p>0.05). Conclusion: The specific siRNA was able to reduce OPN expressions at both the mRNA and protein levels and significantly diminished the invasiveness of HCC cells. Methods: The expressions of MIF and VEGF in HCC and adjacent tissues were detected from 4 patients. Specific siRNA targeting MIF gene was synthesized, and transfected into the HCC cell lines (PLC and HepG2) in study group and non-specific siRNA was used in controls. The mRNA and protein expressions of MIF and VEGF were examined by PCR and western blot. Results: MIF and VEGF mRNAs were overexpressed in the HCC tissues compared with adjacent tissues (RQ=8.91±1.85 and 3.00±0.86, P 0.01). The mRNA and protein expressions of MIF and VEGF of HCC cell lines significantly decreased in study group compared with controls (P 0.01). VEGF mRNA levels decreased 75.6%±2.6%; 79.8%±1.2% in PLC, and 73.6%±4.6%; 80.7%±2.2% in HepG2 cells when disposed with siRNA 50nM and 100nM. VEGF protein levels also significantly reduced in study group P 0.01 . Conclusions MIF and VEGF mRNAs were overexpressed in the HCC tissues in vivo, and MIF siRNA was able to knock down the expressions of MIF and VEGF in HCC cell lines in vitro.
Y.Y. Li, Y.C. Zhang, Y.J. Zhou, Y.M. Wei
Aim: To identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (HCC) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. Methods: HCC cells and normal liver cells from resection samples of 4 patients were laser capture microdissected. Micro-RNA was isolated from them for linear amplification then cRNA was tested with whole genome microarray. Differentially expressed genes were screened. Results: The quality control of this technique was satisfactory with RNA Integrity Number>7, A260/A280 ratio for cRNA measurement=2.0~2.1 and good pictures for microarray. Compared with normal liver tissues, HCC had 1361 differentially expressed genes, with 607 being up-regulated and 754 being down-regulated genes respectively. Among the top ten ranked up-and down-regulated genes (total 20), 5 genes were known as HCC differentially expressed genes, 11, known as other tumors expressed genes previously.
Four unknown tumor related genes (DEPDC1B, ASPM, FCN2 and BBOX1) were detected in this study. Conclusion: The combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of HCC. Background/Objective: Young patients present with large HCC on initial presentation are not uncommon. Our aim is to study the computed tomography(CT) imaging of HCC and the clinical features of this special group of patients. Methods: 521 HCC patients had CT imaging of liver peformed in a three year period, 385 patients had CT imaging peformed at the time of initial HCC diagnosis in our centre and were selected. They were divided into three age groups: Young patients with age </= 40 year-old(Group 1), Age 41 to 60(Group 2), Age >60(Group 3) to study imaging and clinical factors. Univariate and multivariate analysis by Cox regression model done to look for prognostic predictive factors. Results:
Infiltrative tumour in CT scans, symptomatic presentation, Child's and TM staging are prognostic factors in HCC. Conclusion: Young HCC patients have larger infiltrative tumour in initial CT scans and more being symptomatic. Age is not an independent prognostic factor.
Aim: To investigate the expression change of NK cells receptor NKG2D from human peripheral blood in patients with primary carcinoma of liver and study the relationship between NKG2D expression and cytotoxicity of NK cells. Background/Aims: Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3) is a specific protein produced by hepatocellular carcinoma(HCC), which is more valuable than AFP in the diagnosis of HCC. Aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (SELEX). Our aims were to select the aptamers against AFP-L3 from a self-designed ssDNA library for potential application in diagnosis of HCC. Methods: A random ssDNA library and its corresponding primers were designed and synthesized. Aptamers against AFP-L3 were selected by SELEX. Individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and characterized. Results: A ssDNA library of 78 nucleotides with 40 random nucleotides in middle were designed and used for the selection. The binding rate of library against AFP-L3 was increased from 5.1% to 48.2% after 13 round selection. Seven aptamers (S1 to S7) were isolated, and their sequences in random region and secondary structures were different from each other. All aptamers could bind AFP-L3 in a different extent, and the dissociation constants of S4 and S7 are 650nmol/L and 132nmol/L. Conclusions: Aptamers for AFP-L3 are successfully screened out and could bind AFP-L3 specifically. Methods: Flow cytometry was used to determine the number of NK cells and the expression of NK cells receptor NKG2D from human peripheral blood in patients with 20 case primary carcinoma of liver 23 case hepatitis B cirrhosis 20 case hepatitis B and 20 healthy cases and enzyme mark instrument was used to detect cytotoxicity of NK cells in all cases. Results: killing rate of NK cell for K562 cell,NKG2D expression level of NK cells, and the number of NK cells in the patients with primary carcinoma of liver decreased significantly P<0.05 compared with those in the healthy subjects and hepatitis B group ,And decreased a little compared with those in the hepatitis B cirrhosis (P>0.05).The activity of NK cells showed a obvious positive-correlation with the number of NK cell and expression level of NK cell receptor NKG2D. Conclusion: The cytotoxicity of NK and the NKG2D expression of NK cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .The activity of NK cells is closely related to the NKG2D expression level of NK cells. enhancing the NKG2D expression level of NK cell may provide a new idea for Adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein AFP in serum and tissues for primary hepatic cancer(PHC). Methods: Sixty-six PHC and 32 cirrhotic patients were enrolled. In PHC patients,male /female was 48:18, age was 55.4 ± 9.91.of them, 16 patients were defined as stage a-a. In cirrhotic patients, male /female was24:8, age was 53.3 ± 5.81. SerumGPC3 was detected using ELISA. Serum AFP was detected using electrochemiluminescence. The hepatic expressions of GPC3 and AFP were measured using immunohistochemistry in 21 PHC and 6 cirrhotic patients. Results: The cutoff value of AFP diagnosis for PHC was 400 g / L or more, AFP positive in PHC patients was 28.8% (19/66); The cutoff value of GPC3 diagnosis for PHC was 300ng / L or more, GPC3 positive was in 47.0 % (31/66), P = 0.031. In a-a stage PHC patients,the positive of GPC3 and AFP was 62.5% (10/16), 0(0/16), respectively,P = 0.000. In serum AFP negative or positive patients, the positive of GPC3 was 46.7% (14/30) , 47.2% (17/36), respectively,P = 0.964. The relationship between GPC3 with age, sex, Child-pugh grade, HBV infection, tumor size and metastasis were not observed.The positive expression of GPC3 and AFP in hepatocellular carcinoma tissue was 85.7% (18/21), 4.8% (1 / 21), respectively, P = 0.000. Neither GPC3 ,nor AFP in the paracarcinomatous and cirrhotic tissue, was expressed. Conclusions: Diagnosis of Glypican-3 protein for primary hepatic cancer is superior to AFP.GPC3 can be regared as a early marker to diagnosis PHC. 
Objective: To investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. Methods: HCCLM3-RFP cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum. The fluorescent areas of HCCLM3-RFP were photographed daily and repeated in 4 consecutive days after curcumin treatment for obtaining cell growth curves. The cell morphologic changes were also observed. Cell invasion experiment was performed with Boyden chamber array. The RFP-expressing human HCC xenograft model in nude mice was established to study the anti-tumor effects of curcumin. The CTC was detected by FACS. The expression of cyclinD1 and MMP-2 was detected by SYBR Green Real-time PCR. Results: After incubation with 20 M, 10 M and 5 M curcumin respectively for 24, 48 and 72 hours, the growth of HCCLM3-RFP was significantly inhibited and some morphologic changes were observed. The mean tumor size in nude mice treated with curcumin since day 21were significantly less than those of the control group(p 0.01). The mean metastasis area of lung and the number of CTC in curcumin group on day 35 were remarkably less than in the control group(p 0.01). The mRNA levels of cyclin D1 p 0.01 and MMP-2 p 0.05 in curcumin group on day 35 were significantly lower than in the control group.
Conclusion: Curcumin can inhibit the proliferation and invasion of HCCLM3 cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin D1 and MMP-2 in mRNA levels.
Phosphorylated ERK is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study Z. Zhang 1 , Y.H. Wang 1 1 
Background: Sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (HCC) and thus sets the new standard for the first-line treatment of advanced HCC. However, it remains unresolved to predict the drug sensitivity in treating HCC with sorafenib. Pretreatment pERK level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that pERK may be a potential biomarker for treatment of HCC with sorafenib. Methods: The effects of sorafenib and 5-Fluorouracil on cell proliferation were evaluated by cell viability assay in four types of HCC cell line (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6), with different metastatic potential and basal pERK expression. Levels of pERK expression were determined by immunocytochemical analysis and quantification, along with Western Blot analysis. Correlation analysis was carried out between the IC50 values of drugs and Mean Optical Density values of pERK. Results: The basal pERK levels increased stepwise in cell lines in accordance with their metastatic potential. Sorafenib inhibited ERK phosphorylation at a concentration between 5 and 20 M dose-dependently, while no changes were observed after 5-FU treatment. Correlation analysis between the IC50 values and MOD values of pERK revealed that the effects of sorafenib were significantly correlated with basal pERK levels (Spearman r=-0.8671, P=0.0003). On the other hand, the resistance to 5-FU were significantly associated with basal pERK expression in these HCC cell lines (Spearman r=0.7846, P=0.0009). Conclusions: In this vitro study, pERK was confirmed to be a useful biomarker predictive of sensitivity in treating HCC with sorafenib. The RAF/MEK/ERK pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in HCC. 
Background: To investigate the dynamic expression of IGF-II and IGFBP-3 and its alteration of bcl-2 in HCC. Methods: HCC models were induced with 2-FAA on male SD rats. Morphological changes of livers were observed and the dynamic changes of liver or serum IGF-II, IGFBP-3, and bcl-2 were quantitatively analyzed by ELISA. The expression and distribution of liver IGF-II were observed by immunohistochemistry. Result: Hepatocytes from granule-like degeneration to a typical hyperplasia to HCC and the progressing increasing of the levels of hepatic IGF-II after rats induced by 2-FAA. The levels of IGF-II in hepatoma and sera were significantly higher than any of other groups. The positive relationship of IGF-II was found between liver and sera (P<0.01). The IGFBP-3 levels in hepatoma were significantly lower than that in other groups (P<0.01) and the progressing increasing of the levels of hepatic bcl-2 expression during the course. The levels of bcl-2 in hepatoma tissues were significantly higher than those in normal and degeneration ones. The immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl-2 in rat hepatoma. Conclusion: Hepatic IGF-II, IGFBP-3 and bcl-2 may participate in hepatocyte canceration and accelerate the occurrence and development of HCC. The expression of IGF-II and IGFBP-3 could be useful molecular markers for early diagnosis and prognosis of HCC. Background: This study was done to assess the etiological role of hepatitis B virus (HBV), hepatitis C virus (HCV) and aflatoxin B1(AFB1) in development of hepatocellular carcinoma (HCC) in Bangladesh. It was also investigated whether alpha-feto protein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) has any diagnostic advantage over each other Methods: Fifty five histologically proven HCC patients were tested for serological markers of hepatitis B and hepatitis C, and AFB1-DNA adduct. During the diagnosis, they were also investigated for liver function tests, AFP PIVKA-II. Results: Out of fifty five HCC patients, 41(74.5%) were found positive for serological markers of HBV, 21(38%) for HCV and 15(27%) for both. Eight cases (14.5%) were negative for the markers of HBV and HCV. However, none had AFB1-DNA adduct above normal range. Both PIVKA-II and AFP is strong marker for HCC with satisfactory level of sensitivity and specificity; but PIVKA-II is more sensitive (92.7%) and AFP is more specific (96%). Conclusions: HBV and HCV is the major etiological agent responsible for the development of HCC in Bangladesh. Background: To investigate the influences on the malignant transformation of hepatocytes through the intervention of NF-B activation pathway. Method: HCC models were induced with 2-FAA on SD rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. Morphological changes were observed by HE staining. NF-B expressions were detected by IHC. The relationship between NF-B expression and pathological characteristics in HCC and non-HCC were analyzed. Results: Rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated HCC. Thalidomine can repress the morphologic change of liver cells. There were only punctiform denaturations at the early and middle stage; Nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. The IHC results demonstrated that NF-B level was significantly higher than those in normal ones, and the NF-B level of livers in HCC was higher than those in thalidomide group. An increasing tendency of NF-B was found from normal to HCC. NF-B in HCC were significantly higher than those in NC. The NF-B levels with thalidomide intervence raised first and decreased later. NF-B expressions in HCC were higher than that in their non-cancerous tissues. No positive relationship presented between NF-B expression and histological differentiation grade or the number of tumor, and size of tumor. Conclusion: Decrease NF-B expression can inhibit HCC development and NF-B is expected to be a new molecular target of HCC therapy. Method: The cellular distributions of VEGF expression in HCC tissues were investigated by immunohistochemistry. The levels of total RNA and VEGF were quantitatively detected in HCC, their paracancerous, and distal cancerous tissues, respectively. Simultaneitily, serum VEGF were analyzed in patients with chronic liver diseases for clinical values. Results: The positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of HCCs. The incidence of VEGF was 63.9% in HCC tissues, 78.3% in non-encapsulated HCCs, and 90.9% in HCCs with extrahepatic metastasis, respectively. No significant difference was found between hepatic VEGF and HCC diameter or differentiation degree. The specific concentration (pg/mg liver) of VEGF expression was significantly higher (P<0.01) in HCC than their paracancerous or distal cancerous tissues, respectively. The circulating VEGF was abnormally elevated in HCC. If the cut off values was more than 280 pg/mL, the incidence of serum VEGF was 88.4% in HCC, 14.3% in chronic hepatitis, and 10% in liver cirrhosis, respectively. The combined VEGF and AFP can increase positive rate up to 94.2% for HCC. Conclusion: The VEGF overexpression is a useful marker for vascular invasion and metastasis of liver tumors. Background: Hepatocellular carcinoma (HCC) represents a major health problem world wide. It accounts for 90% of all primary liver cancers and is the fifth most common malignancy (1). Objectives: Evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of Viscum (Fraxini 2) on tumour recurrence. Methods: 60 patients with HCC were enrolled in the study. Group 1 include 30 patients and were treated with radiofrequency thermal ablation (15 patients of them received viscum 2 by subcutaneous route for 2 years). Group 11 included 30 patients with HCC and were treated by TACE (15 patients of them received viscum 2 subcutaneously for 2 years). Results: Group 1 patients showed total ablation in 70% with persistant inactivity during 2 years follow up. Group 11 did not show significant difference from Group 1 as regards relapse rate nor the performance status. Complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with TACE. Viscum 2 did not significantly arrest tumour recurrence. Conclusion: Non surgical patients with HCC can achieve curative treatment with radiofrequency with minmal side effects. TACE is a palliative treatment option for large HCC.
A new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of 5-7 cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. In this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. 40 patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between 7 and 10 cm in its maximum diameter. All had a pretreatment helical (triphasic) CT study for accurate delineation of the whole extent and vascularity of the mass. Two needles were sufficient to cover the whole extent of the mass in 23 patients (57%) while in the remaining 17 patients (43%) three needles were necessary. The procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. No major complications. Follow up spiral (triphasic) CT was done 2 weeks after ablation revealed percentage of tumour necrosis of 90% or more in 30 patients (75%), 70-90% in 6 patients (15%) while in the remaining four patients (10%) the percentage was 50-70% necrosis. In conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. Results: The median value of GPC-3 in HCC, DC, CC was significantly higher than chronic hepatitis and control groups. No significant correlation found between AFP and GPC-3. AUROC of AFP was 0.85 & AUROC of GPC-3 was 0.84. The diagnostic Sensitivity of AFP (20 ng/ml) was 70% with PPV 53.8%. The specificity was 85% with NPV 91.9%. While the diagnostic Sensitivity of GPC-3 (2 ng/ml) was 100% with PPV 27%. The specificity was 42.2% with NPV 100%. Combined serial approach of AFP and GPC-3 improved the specificity to 87.5%. Conclusion:GPC-3 although it is a serological test for early detection of HCC, it showed limited specificity, where It is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. The diagnostic signature approach for simultaneous determination of AFP and GPC-3 may improve the prediction accuracy of HCC patients in those showing seronegativity to AFP.
Outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: Retrospective analysis in an Asian regional hospital W.M. Yip 1 , K.F. Li 1 , K.K. Li 1 , M.L. Szeto 1 1 
Background: Hepatocellular carcinoma (HCC) is a common cancer worldwide causing substantial mortality. Although surgical resection is a form of curative treatment in HCC, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. Transarterial chemoembolization (TACE) is a treatment option for inoperable HCC and it was proven by randomized control trials that TACE can prolong survival in selected patients. The aim of this study is to evaluate the survival and the prognostic factors in patients with advanced HCC treated by TACE. Methods: Seventy four patients with inoperable HCC diagnosed from January 1998 to December 2003 were analyzed retrospectively in this study. Only patients with unresectable HCC or who refused operation were included. Patients with advanced cirrhosis, extrahepatic metastasis or previously treated HCC were excluded. Multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. Results: The median survival of the study patients was 213.5 days. The cumulative survival rates at 1 year, 2 year and 3 year were 28.4%, 12.2% and 6.8% respectively. By multivariate analysis, superselective cannulation performed in TACE (hazard ratio: 0.47, 95% CI: 0.23-0.95, p=0.034), embolization with gelfoam (hazard ratio: 0.30, 95% CI: 0.11-0.80, p=0.017), treatment interval more than 45days (hazard ratio: 0.33, 95% CI: 0.15-0.72, p=0.006), Child-Pugh grade B (hazard ratio: 5.62, 95% CI: 2.11-14.97, p=0.001), and pre-treatment serum FP level (hazard ratio: 2.93, 95% CI: 1.50-5.73, p=0.002) were independent predictors of survival. Conclusions: Survival of patients with inoperable HCC is still grave despite treatment. This study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. Result: Age <65, total bilirubin (TB) <2.0mg/dl, albumin (Alb) 3.5g/dl, prothrombin time (PT) 70%, platelet counts (Plt) 10.0 10 4 /mm 3 , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of CLIP 0-1 HCC patients. Of CLIP 2-6 HCC patients, TB <2.0mg/dl, Alb 3.5g/dl, des-gamma-carboxy prothrombin (DCP) <100mAU/ml, absence of vascular invasion, and type of treatment were correlated with survival. The following factors were related to survival by multivariate analysis: CLIP 0-1 HCC patients; age, Alb, single nodule, and absence of vascular invasion, CLIP 2-6 HCC patients; age, TB, Alb, alpha-fetoprotein (AFP) <100ng/ml, DCP, absence of vascular invasion, and type of treatment. Conclusion: Age, albumin, vascular invasion were significant predictors of survival both CLIP 0-1 and CLIP 2-6 HCC patients. CLIP 0-1 HCC patients: single nodule; CLIP 2-6 HCC patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival.
The role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. S. Sumie 1 , R. Kuromatsu 1 , K. Okuda 1 , E. Ando 1 , A. Takata 1 , N. Fukushima 1 , M. Sata 1 1 
Background; The presence of microvascular invasion (MVI) as the risk factor in hepatocellular carcinoma (HCC) is controversial. The aim of this study was to determine the outcomes and predictive factors after hepatic resection for HCC with MVI. Methods; One hundred and ten patients who underwent curative resection for HCC were included in this retrospective study. The risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of MVI were also evaluated. Result; Multivariate analysis showed that cirrhosis and MVI were identified as independent risk factors for recurrence-free survival. The 2-year recurrence-free survival rates for patients with and without MVI were 44.6% and 76.7%, respectively. Multivariate analysis showed that the number of tumors, presence of MVI, and IM were identified as independent predictors of disease-specific survival. The 5-year disease-specific survival rates for patients with and without MVI were 59.3% and 92.0%, respectively. By univariate analysis, MVI was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (IM). Gross classification proved to be the only independent predictive factor for MVI by multiple logistic regression analysis. The gross classification could be evaluated by preoperative imaging diagnosis. Conclusion; MVI is strongly associated with recurrence and survival in HCC patients after curative resection. Furthermore, gross classification of HCC can be helpful in predicting the presence of MVI. Background: HCC is a common cause of cancer morbidity and mortality. PXD101 is a novel, low molecular weight, histone deacetylase inhibitor. This phase I study aims to determine dose limiting toxicity (DLT) and maximum tolerated dose (MTD). Methods: Patient eligibilities include unresectable disease, ECOG 2, adequate organ functions. PXD101 was given intravenously on day 1-5 every 3 weeks; dose levels were: 600 (level 1), 900 (level 2), 1200 (level 3) and 1400 mg/m 2 /day (level 4). DLTs are defined as grade 4 hematological toxicity or grade 3/4 non-haematological toxicity during cycle 1 (according to NCI CTC v3), or treatment delay >2 weeks. The MTD is defined as the dose below which > 2 of 3 or > 2 of 6 patients experiencing DLT. Results: 18 patients were entered; level 1 (3), level 2 (3), level 3 (6) and level 4 (6). Grade 3/4/5 toxicities in cycle 1 included: raised ALT 1/0/0, diarrhea 1/0/0, abdominal distension 1/0/0, anaemia 1/0/0. A total of 56 cycles were administered; overall grade 3/4/5 toxicities: raised ALT 1/0/0, bilirubinaemia 1/0/0; cardiac ischaemia 1/0/0; diarrhoea 1/0/0, abdominal distension 2/0/0, anaemia 2/0/0; variceal haemorrhage 1/0/0; hypercalcaemia 1/0/0; hyperkalaemia 0/1/0; hyponatraemia 1/1/0; infection 2/0/0; liver dysfunction 0/2/0; muscle weakness 1/0/0; abdominal pain 1/0/0; prolonged QTc 1/0/0; syncope 1/0/0; seizure 1/0/0. There were 3 SD and 15 PD. Conclusion: At the maximum dose of 1400 mg/m 2 /day, MTD has not been reached. PXD101 is very well tolerated. Sponsor: The Division of Cancer Treatment and Diagnosis, National Cancer Institute, USA.
Tumor thrombus (PVTT) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (HCC) patients and its molecular mechanism is still unclear. In this study, we first established a HCC cell line named CSQT-01 from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . This cell line was composed of polygonal shaped cells and its peaks of the chromosome number was 69 and 77. Study on stem cell biology in this cell line suggests that CD133 cells represent about one fourth of the tumor cell population and CD133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. With this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found 20 proteins expressed differentially between primary tumor and the PVTT. from these proteins, AnnexinV, Prx , CycB were selected for further analysis to find potential biomarkers of PVTT in hepatocarcinogenesis. For clinical study, we recommended a new tumor thrombus type system ( type I IV) according to anatomic features of portal vein and tumor thrombus of HCC developing modes, then evaluate this type system to predict prognosis of HCC patients. The retrospective data of 406 HCC patients with PVTT underwent resection shows that the 1y, 2y, 3y overall survival rates were 44.7 , 26.3 and 19.7 for type I, 29.9 , 20.6 and 12.4 for type II, 25.0 , 12.5 and 3.6 for type III, 27.3 , 0 and 0 for type IV, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of HCC patients with PVTT.
Polyprenol could decrease the risk of hepatocarcinogenesis in HBV G. Kuznecova 1,2 , S. Kuznecovs 1,2 , I. Kuznecovs 1,2
Background: Over-expression of P-glycoprotein (Pgp) is associated with liver cancer development from HBV . Glycoprotein synthesis in malignant tissues is limited by Dolichyl Phosphate (DolP). The aim of the present study was to investigate the effect of polyprenol (PP) which provides a DolP substitute in regulation of N-glycosylation on Pgp over-expression in the development of liver cancer in HBV infection. Methods Human hepatocytes, infected with HBV and human hepatocarcinoma HEP 3B cell line were used. Pgp was assessed by an immunohistochemical technique. DolP fractions were analysed by HPLC methods. Results It is confirmed that plasmatic membrans of hepatocytes cells contain 7,9 -9,4% of Pgp (the total protein amount) as a resistance marker. HBV infected cells differ from normal hepatocytes in Pgp content by 4-5 times and HEP 3B cells differ by 10-12 times The study showed 5-fold DolP decrease in HBV infected cells and 10-fold DolP decrease in HEP3B cells. The investigations demonstrate that the situation can be changed by treatment with DolP and PP. The DolP concentration in HBV infected hepatocytes was returned to the normal level. It is established that DolP in the concentration 10 -6 M aid 6-8-fold reducing Pgp in membranes of HBV infected cells. Background: Metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. Snail was recently highlighted as a critical transcriptional factor for tumor metastasis. Method & Result: Real time RT/PCR and Western blot analysis demonstrated that Snail mRNA and protein, respectively, were induced by 12-Otetradecanoylphorbol-13-acetate (TPA) in hepatoma cell HepG2. Blockade of gene expression of Snail by antisense oligodeoxynucleotide and/or siRNA technique can prevent not only the TPA-triggered EMT/cell migration and growth inhibition of HepG2 but also TPA-induced down-regulation of E-cadherin and up-regulation of p15INK4b. Moreover, the TPA-triggered promoter activation of p15INK4b was also prevented. On the other hand, two of the HepG2 clone overexpressing Snail, namely S7 and S15, had a scattered fibroblastic morphology and acquired higher motility than parental HepG2. Also, the proportion of G0/G1 phase of S7 and S15 was higher than that of parental HepG2, consistent with the longer doubling time of both cells. Semiquantitative RT/PCR analysis demonstrated a greatly elevated gene expression of Snail accompanied with decreased E-cadherin and increased p15INK4b in both Snail-overexpressing cells. On the transcriptional level, p15INK4b promoter activity was 2.6-fold higher in S7 as compared with parental HepG2. Furthermore, electrophoretic mobility of DNA fragments encompassing proximal p15INK4b promoter can be retarded by incubation of nuclear extract of S7. Conclusion: Our results demonstrated that Snail play diverse trans-regulatory roles in HepG2. Notably, we suggested that Snail may upregulate p15INK4b gene expression by directly activating its promoter.
A. Schmitt-Graeff 1 , R. Fischer 1 , M. Grosse-Perdekamp 1 , O. Skalli 2 1 Universityhospital Freiburg , 2 Louisiana State University Health Sciences Center, Shreveport Background/Aims: Synemin is an intermediate filaments (IF) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and F-actin. We have previously shown that synemin is expressed in resident hepatic stellate cells (HSC) and myofibroblasts (MF) in hepatic inflammation and fibrosis. In the present study we evaluated systematically the expression of synemin in a large cohort of western European hepatocellular carcinoma (HCC). Methods: Single and double immunolabelin for alpha-smooth muscle actin (SMA), vimentin, CD31, CD34, CD68, CEA, CD10, cellular retinol-binding protein1 (CRBP-1) and synemin were performed on 75 paraffin-embedded HCCs and 10 controls. Results: Synemin-positive HSCs/ MFs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. Tumour cell plates of the trabecular and pseudoglandular types of HCC were covered by scattered synemin-positive cells outlining sinusoidal structures. A subpopulation of these cells showed features of pericytes while others resembled endothelium. This pattern correlated with the degree of differentiation and was not observed in poorly differentiated HCCs which generally contained rare intratumoral MFs. Conclusion: The presence of synemin-positive HSCs/MFs in the vicinity of HCCs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. Since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population.
A.S. Butt 1 , A. Ahmed 1 , S. Hamid 1 , W. Jafri 1 , H. Ali Shah 1 1 
Aim: To estimate the prevalence of viral marker negative HCC and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive HCC. Methods: Medical records of patients diagnosed to have HCC visiting Aga Khan University Hospital, Karachi during January 1998 to December 2007 were reviewed. Patients were divided in to NBNC-HCC(those who have negative HBsAg and Anti-HCV antibody)and viral HCC(those who have positive HbsAg and Anti-HCV antibody)group. Results: Out of 433 patients 68(15.7%) had NBNC-HCC. Over all mean age was 57.48± 10.9 years and 69.5% were males. The proportion of HCC detected under surveillance was significantly smaller in NBNC-HCC group(p0.02). There was no difference in distribution of age, gender, BMI, child score, bilirubin, serum albumin, prothrombin time and Alfa Feto protein in both groups. However, patients with viral-HCC were found to be more thrombocytopenic(52.67±86.7vs.226.15±153.9,p<0.001) and had hepatopulmonary syndrome. On liver biopsy greater proportion of moderate to poorly differentiated HCC was observed in NBNC group(19.2%vs.7.9%,p<0.001). HCC measuring 5cm in diameter(60.3%vs.41.9%, p 0.01), non -solitary HCC(p0.038) and portal thromboses(p0.01) were strongly demonstrated in NBNC-HCC group. Involvement of right hepatic lobe and extra hepatic tumor spread was greater in NBNC-HCC group but that difference was not statistically significant. Out of 178 patients who underwent for liver transplantation(0.5%),TACE(36.7%),Resection(1%),ethanol ablation(2%) and chemotherapy(2%), poorer responses were observed in NBNC-HCC group (p 0.04). Conclusion: HCC secondary to NBNC-cirrhosis is not uncommon. Patients with NBNC-HCC tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. 
Background: Hepatocellular carcinoma is a common malignancy in Asia and is related to the high prevalence of chronic viral hepatitis. We examined the clinical features, treatments and survival rates in Asian Americans with HCC. Methods: Retrospective cohort study of 278 HCC patients who presented to the UCLA Liver Cancer Center in Los Angeles, California, USA from September 2000 to December 2007. Results: Two hundred and seventeen of 278 (78%) HCC patients were male, 58% and 30% had HBV and HCV infection respectively, and 73% had cirrhosis. HCC patients detected by surveillance had smaller tumor sizes, more within the Milan and UCSF criteria, lower HCC Tokyo system scores and had improved 1,3,5 year overall patient and disease free survival rates compared to HCC patients who presented with symptoms (p<0.01 to p<0.0001). By multivariate analysis, independent predictors of patient survival were tumor volumes greater than 5cm (HR1.48, p=0.04), AFP per unit log10 increase (HR 1.12, p=0.02), HCC Tokyo score per unit increase (HR 1.3, p<0.0001), liver transplantation (HR 0.14, p<0.0001), hepatic resection (HR 0.18, p<0.0001), RFA (HR 0.17, p<0.0001), TACE (HR 0.44, p=0.0006), and Hepatitis B infection (HR 0.71, p=0.03). Factors associated with disease free survival were age per year increase (HR 1.01, p=0.03), MELD per unit increase (HR 0.97, p=0.0049), liver transplantation (HR 0.23, p<0.0001), and hepatic resection (HR 0.39, p<0.0001). Conclusion: HBV and HCV infection accounts for the majority of HCC in Asian Americans. HCC detected by surveillance resulted in treatments which improved overall patient and disease free survival.
Treatment of small ( 2cm) HCC tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. Indeed, surveillance program for the early detection of small HCC tumour is imperative to facilitate curative treatment, and hence better survival. Discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small HCC tumour marker herein identified using proteomics. Experimental design: A total of 76 liver tissues were evaluated by 2-DE analysis. Differentially expressed proteins were unequivocally identified by MALDI-TOF/TOF and validation of the best candidate from protein to gene levels. Indirect ELISA assay was developed to detect soluble vimentin from 152 serum samples. Results: Vimentin was significantly over-expressed in small HCC tumours compared to non-malignant controls and maintained expression in >2cm tumours using 2-DE analysis. Blind verification displayed over-expression of vimentin in both transcripts and proteins levels. Soluble vimentin was significantly detected at high level in small HCC as well as in overt HCC tumours. Receiver operating characteristic analysis showed vimentin exhibited 40.91% sensitivity and 87.50% specificity in detecting small HCC at a cutoff of 245ng/ml. Combined diagnostic performance of soluble vimentin and serum AFP increases the detection sensitivity and specificity to 50.53% and 98.15%, respectively. Conclusion: In this context, over-expression of vimentin is associated with the favourable 2cm sub-class of HCC thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients.
Purpose: Our recent comparative oncogenomic analysis in mouse model has identified YAP (Yes associated protein) as a novel oncogene in HCC. However, its clinical significance is unknown. In this study, we aimed to investigate the clinical values of YAP as an independent prognostic marker in HCC. Experimental Design: A total of 177 HCC cases with retrospective clinicopathologic and follow-up data were recruited in this study. Both tumor and adjacent non-tumor tissues were examined for immunoreactivity of YAP expression by immunohistochemistry. Clinicopathologic features and YAP expression were investigated with Pearson 2 test. HCC-specific disease free survival and overall survival with YAP expression were analyzed by Kaplan-Meier curves and Log-rank test. Cox regression was used to test the independence and magnitude of the effects. Results: YAP was found over-expressed in HCC (62.1%) with nuclear expression pattern. Positive YAP immunoreactivity was significantly correlated with worse tumor differentiation grade (P=0.021) and high serum alpha-fetoprotein (AFP) level >400 ng/ml (P<0.001). Kaplan-Meier plot and Cox Regression showed that YAP was an independent predictor for HCC-specific disease free survival (hazard ratio, 1.653; 95% CI, 1.084-2.522; P=0.02) and overall survival (hazard ratio, 2.226; 95% CI, 1.312-3.778; P=0.003). Conclusions: YAP expression in HCC is correlated with tumor differentiation and serum AFP level. It served as an independent prognostic marker for HCC. Background: Integrative analysis of global protein and mRNA expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. Methods: We used a 2D-PAGE approach to profile and compared the global protein expression profiles of hepatitis B virus-related HCC tissues, adjacent non-tumor liver tissues, normal liver tissues and HCC cell lines. Subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. We compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from 6 research centers in different countries. Results: We identified 66 proteins dysregulated in HCC. Hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, HCC tissue, then to HCC cell lines. According to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. Ontology analysis of the genes with consistent dysregulations at both mRNA and protein levels identified specific pathways down-regulated during the progression of HCC. The inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. Results: In pre-S/surface regions, HCC patients had higher frequencies of pre-S deletions, amino acid substitutions at codon 4, 7, and 81 in pre-S1 genes, at the start codon in pre-S2 genes, and at codon 68 in surface genes. But they had a lower frequency of amino acid substitution at codon 2 in pre-S2 genes than those without HCC. In BCP/precore regions, HCC patients had higher frequencies of C or G1753, A1762/T1764, T1846, and A1899 than those without HCC. Multivariate analysis showed that pre-S deletions, I68T in surface gene, T1762/A1764, and A1899 were independent factors for HCC. The HBV with a complex mutation pattern (pre-S deletion, T1762/A1764, and A1899) rather than a single mutation was associated with HCC. Patients with combined mutations of T1762/A1764 and pre-S deletion, T1762/A1764 and A1899, pre-S deletions and A1899, and T1762/A1764, pre-S deletions and A1899 had a 7.81, 7.7, 7.0, and 16.88 fold increased risk of HCC, respectively, compared to patients with wild-type at both or three genomic regions. Conclusions: Pre-S deletions, I68T in surface gene, T1762/A1764, and A1899 were independent factors for HCC. Combination of these viral mutations appeared increasing HCC risks.
High peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection H.X. Xu 1 , X.D. Zhu 1 , P.Y. Zhuang 1 , W.Zhang 1 , H.Chuan Sun 1 1 
Background/Aims: Angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (HCC). Placental growth factor (PlGF), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of HCC. Methods: Expression of PlGF was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from 105 patients underwent hepatectomy for histologically proved HCC. Prognostic values of PlGF and clinicopathological factors were evaluated. Result: PlGF staining was mainly on the cytoplasm of tumor cells or hepatocytes. The mean integrated optical densities of peritumoral and intratumoral density of PlGF were 0.018±0.018 and 0.006±0.007 respectively. Peritumoral PlGF density was significantly higher than that in tumor (p<0.001), and this result was also validated in another cohort of 37 patients by quantitative real-time reverse transcription-PCR (p=0.007). Intratumoral density of PlGF was not correlated with common clinicopathological factors (eg, TNM stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (OS) (p=0.425) and time to recurrence (TTR) (p=0.419). However, peritumoral density of PlGF, which was correlated with tumor size (p=0.003) and intrahepatic metastasis (p=0.004), was a prognostic factor for both OS (p=0.015) and TTR (p=0.018). In multivariate analysis, peritumoral expression of PlGF was also an independent prognostic factor for OS (p=0.015, RR: 2.500 95% CI: 1.191-5.253 ) and TTR (p=0.045, RR: 2.013 95% CI: 1.014-3.996). Conclusion: Peritumoral expression of PlGF in HCC patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. Purpose: To further research RFA in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (HCC). Methods: 25 cases were treated with transhepatic artery chemoembolization (TACE) + radiofrequency ablation (RFA) + introportal vein chemotherapy (PVC) + percutaneous ethanol injection (PEI) (four combined group) and this method was compared with 23 cases that were performed TACE + PEI (two combined group) . The serum level of AFP was measured respectively after 2 and 6 months, CT scan and color Doppler ultrasound were measured after treatment for six months.
Results: The serum level of AFP declined in two groups after 2 months. For treatment after six months, AFP in four combined groups was rose lower than two combined group (X 2 =5.06, p<0.05). CT and Doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (X 2 =8.29, p<0.01) and blood supply X 2 =6.81, p<0.01), relapse and mortality are also less. Conclusions: RFA in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced HCC.
Poster Exhibition -HCV Poster Session, Hall 5B
On the average, hepatitis C virus infects 1.2% of the population worldwide. In Egypt, the prevalence rates reach 20% in some areas. Ability of the virus to persist in about 75-85% of infected individuals is related to the virus higher mutation rate. Six major HCV genotypes have been identified. Genotype 4 seems to be confined to the Middle East and Central Africa. Extra hepatic syndromes have been reported in up to 1/3 of HCV patients. We aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis C. The study group included 60 selected patients with chronic hepatitis C having various haematological problems. We studied hepatitis C virus genotypes using RT-PCR. We found among 60 patients , 57 genotype 4 (95%) and 3 patients genotype 1a (5%).28 patients (46.66%) were diagnosed as chronic hepatitis C with associated thrombocytopenia, 16 patients (26.66%) were diagnosed Mixed Essential Cryoglobulinemia(MEC), 13 patients (21.66%) were diagnosed Non-Hodgkin's lymphoma, and 3 patients (5%) were Aplastic anemia. Positive serum cryoglobulins level was found in 20 patients (33.3%).No significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (P>0. 05). We did not find correlation between HCV genotype and specific extrahepatic haemological disorder in HCV infected patients. Several environmental, genetic and immunological factors may contribute in disease progression. Results: In the targeted area 111 shops of barbers were successfully interviewed and total 715 questionnaires were filled by both groups. The mean age were found in both groups of Barbers (n=186) and Clients (n=529), 28.47 years. The both groups showed that there are no any drugs which can protect us from diseases. Both of the groups were not vaccinated for hepatitis B diseases. Regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the Hakeems. Those who knew Hepatitis as liver disease, were 73 (39.2%), out of 186 barbers only 68 ( 36.6%) were knowing about Hepatitis-B&C, When we enquired about routes of HBV& HCV transmission only (37%) replied correct routes of transmission in both groups. About HBV vaccination (49.7%) were aware, only (14.8%) were vaccinated against HBV. 60% barbers claimed for disinfection of instruments before shaving (88.9%) claimed for use of new blades. In the sero-surveillance the HBV found was very low and HCV became epidemic (5.7% -14.4 %) respectively. Conclusion: The both groups need awareness for transmission. The use of new blade for the clients reduces the burden of HBV and HCV. The study highlights the roles of male sex, older age, and genotype 1b in the progression from CHC infection to HCC. Patients with higher HCV viral load potentially tend to develop HCC; however, HCC occurrence could be prevented using antiviral treatment. These two points need to be clarified further by a larger study population with longer follow-up period. An approach have recently been described that retroviral vectors encoding T cell receptor (TCR) genes are used to redirect the specificity of normal peripheral blood lymphocyte (PBL)-derived T cells to recognize the tumor antigens. The therapy in which T cells have been genetically modified with TCR genes to recognize HCV would represent a novel approach for the treatment of HCV infections and HCV-related malignancies. We have previously shown that HCV+ liver transplant patients that have received HLA disparate liver allografts have HCV reactive T cells of host origin in their peripheral blood that are restricted by the donor HLA molecules. Initial studies indicate that the TCRs expressed by HCV reactive T cell clones from these patients have relatively high affinity for their ligands. We have cloned and expressed two TCRs which mediate recognition of the 1406-1415 and 1073-1081 epitopes from the HCV NS3 protein. The results indicate that these TCR transduced T cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. Most importantly, the TCR transduced T cells could also recognize HCV+ hepatocellular carcinoma cells. These data suggest this high affinity HCV-specific TCR might have potential new immunotherapeutic implications. Background: Factors associated with SVR in patients without an RVR remains unclear. Methods: 200 HCV-1 (100 for 24 and 48 weeks, separately) and 150 HCV-2 (100 for 24 weeks, 50 for 16 weeks) patients were randomized to peginterferon-alpha-2a and ribavirin for analysis. Results: Multivariate analysis showed that treatment duration and a complete EVR were the strongest independent factors associated with an SVR. A higher SVR rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cEVR (table 1). The best levels of viral loads in predicting cEVR at week 4 were < 10 4 IU/mL (table 2) . Conclusion: It was crucial to achieve a cEVR with adequate treatment duration in patients who failed to achieve an RVR. 
Our aim was to evaluate the impact of some biochemical, histological and viral factors on both EVR and SVR in patients with genotype 1 chronic hepatitis C (CHC) treated with peginterferon plus ribavirin. Patients and methods: We evaluated retrospectively 188 naïve patients with CHC treated with peginterferon plus ribavirin at standard weight-based doses for 48 weeks. Biopsies were assessed for inflammatory activity and fibrosis. Steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: >5%, >5-33%, 34-66%, >66%. Biopsies were also assessed for stainable iron using the Brissot scoring system. All patients were evaluated for metabolic syndrome (MS) using the NCEP-ATP III criteria. Results: EVR was achieved in 115/188 pts (61.17%) while SVR occurred in 82/115 (71.3%). After adjusting for sex and age, independent factors that negatively interfered with both EVR and SVR were: fibrosis score, steatosis, iron score, HOMA-IR index and viral load. After excluding the patients with MS criteria (n=32), EVR was observed in 102/156 (65.4%) and SVR in 82/102 (80.4%). Factors that independently influenced both EVR and SVR were: fibrosis score, steatosis, iron score and viral load. Conclusion: Fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both EVR and SVR in genotype 1 CHC patients, regardless the presence of MS. If MS is present, high HOMA-IR index can also additionally impair viral response.
Issue/Argument: Asia has rising cases of hepatitisB/C. Alcohol/Food-habits cause high prevalence in rural/tribal areas. Lack of monitoring/follow-up complicates management. vaccines emerge as hope. Clinical-trials of vaccines debated-issue. Design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. We focus on controversies identified in international/regional/local CME/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. Critical task is protect interests of vaccine-subjects in face of substantial-risks. Determine if hepatitis-Vaccine-volunteers will have access to treatment during trial. Access to Vaccine-trial-outcome. Interaction with seniors 19 th APASL-Congress from developed-countries will give voice to such burning-issues. Methodology: Researchers/pharmaceuticals/Govt-policy planners need to develop forum to solve these problems. NGO's can play pivotal role. Obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, Risky vaccine-development. Conclusion: counselling/right to withdraw from trial be made basic guideline. Apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. Researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. Need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community.
Recommendations: Researchers/NGOs should come together at 19 th APASL-congress platform to form workgroup to settle these issues. We shall raise our this burning issue & present Hepatitis-Prevention-Advocacy plan of our NGO graphically to APASL-2009 participants. Results: 53% patients expressed that alternative-medicines-Rx most important factor to cope with hepatitis. higher scores of QOL (ANOVA p < 0.001) correlated with alternative-medicines-Rx. Our NGO-initiative suggests that over 70% patients will need well trained specialist for home-based-care unit. Conclusions: Life-span/QOL of Hepatitis-sufferers depends on appropriate-palliative-care. NGO-personals should be trained in Palliative-care-services. Our data is being used for palliative care advocacy. Field of Spiritual/psycho-social/community support is fertile ground for further investigations. Such use of complimentary Indian medicinal plant extracts needs further evaluation in a large group in multicentre trial.
Treatment with Adacolumn in patients with hepatitis C related who have undergone kidney transplantation: Preliminary study G. Novelli 1 1 La Sapienza University Introduction: Patients who have undergone kidney transplant and suffer from hepatic C related (HCV) cannot be treated with standard therapy (PEG-IFN combined with ribavirine) due to acute rejection risk. Furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. Monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. Our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:Adacolumn®(OTZUKA). Methods: The Adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. These carriers absorb granulocytes and monocytes/macrophanges through FCR receptors. Six patients were treated in our department. All patients were affected by virale genotype 1b. Patients underwent five 1hour treatments for five consecutive days according to protocol. Results: During treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. At 3 rd month follow up we observed a significant decrease in plasma HCV-RNA in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered CD4+ and CD8+ where positive was observed at 3 rd month. In another patient, even though immunomodulation improved, there was no reduction in viremia. Conclusions: Considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. Background: Patients who have undergone kidney transplant and suffer from hepatic C related (HCV) cannot be treated with standard therapy (PEG-IFN combined with ribavirine) due to acute rejection risk. Furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. Monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. Our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:Adacolumn®(OTZUKA). Methods: The Adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. These carriers absorb granulocytes and monocytes/macrophanges through FCR receptors. Six patients were treated in our department. All patients were affected by virale genotype 1b. Patients underwent five 1hour treatments for five consecutive days according to protocol. Results: During treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. At 3 rd month follow up we observed a significant decrease in plasma HCV-RNA in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered CD4+ and CD8+ where positive was observed at 3 rd month. In another patient, even though immunomodulation improved, there was no reduction in viremia. Conclusions: The treatment was found to be safe without hemodynamic or infective complications. Considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase.
M. Sharaf-Eldin 1 , H. El Batae 1 , N. Abd EL-Ghaffar 2 , W. Rasheed 2 1 Tanta Faculty of Medicine , Egypt., 2 National Research Centre, Cairo, Egypt Aim: We aimed to characterize serum cytokine levels of interleukin-1 Beta (IL-1 ) and interleukin -6 (IL-6) in HCV infected patients & in patients with hepatocellular carcinoma (HCC) in comparison to control group and their possible use as markers of disease progression. Patients and Methods: Sixty Patients were divided into three groups: Group I: twenty HCV infected patients without cirrhotic changes. Group II: twenty HCV infected patients with liver cirrhosis (LC). Group III: twenty HCV infected patients with HCC and 20 healthy subjects as control group. All patients and control group were subjected to biochemical and serological tests, anti HCV, HCV (RT-PCR) and cytokines measurements of serum IL-1 & serum IL-6 levels. Results: Showed a high statistically significant elevated serum IL-6 and IL-1 levels in patients with chronic HCV infection in comparison to control group. Highly statistically elevated levels of IL-6 and IL-1 in liver cirrhosis and higher levels were found in HCC group in comparison to control group. The levels of Il-6 and IL-1 increased significantly in HCV infected patients as the disease progress. Conclusion: Serum IL-1 , and IL-6 levels are elevated in patients with hepatitis C-related liver diseases, especially in LC and HCC patients. Their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum IL-1 and IL-6 as markers for Liver disease progression in HCV-infected patients instead of invasive techniques.
Atsushi Tanaka 1 , Naoko Hanawa 1 , Mitsuhiko Aiso 1 , Yoriyuki Takamori 1 , Hajime Takikawa 1 1 Teikyo University School of Medicine Background and Aim: Pegylated interferon (PEG-IFN) therapy is not indicated for many cases with HCV-related cirrhosis due to various adverse effects. However, patients with HCV cirrhosis are at high risk for development of hepatocellular carcinoma (HCC). Thus we have introduced low-dose PEG-IFN treatment for patients for compensated HCV cirrhosis. Patients and Methods: Selection criteria for low dose PEG-IFN is 1) compensated HCV-related cirrhosis, and 2) either the elderly (>65) or presence of thrombocytopenia (<8.0x10 6 / l). We have treated patients who met these criteria with low-dose PEG-IFN, consisting of either PEG-2a 90 g/1 -2w or PEG-2b 0.5 g/kg/w+ribavirin 200mg/d. [Results] Twenty patients with compensated HCV cirrhosis (all patients genotype 1b) have been treated with low-dose PEG-IFN (PEG-2a:9, PEG -2b+rib:11 . The age, platelet counts (x10 6 / l), and ALT (IU/l) of 20 patients at baseline were 64.1±8.3, 7.8±4.6, and 90.4±68.6 respectively. All patients were well tolerated. Low-dose PEG-IFN has been continued 59.6±43.8 weeks on average. Although viral response was not detected, biological response (BR), defined as maintenance of ALT within normal range, was obtained in 12 patients (12/20=60%). Of note, neither development of HCC nor decompensated cirrhosis was observed in these 12 BR cases. By contrast, HCC and decompensation developed in 5 and 1 patients respectively among 8 patients who failed to achieve BR. Conclusion: Low-dose PEG-IFN treatment was safe and well tolerable, and could potentially prevent HCC or decompensation in patients with liver cirrhosis when BR was obtained. Aims: To study the efficacy of peginterferon and ribavirin in treating chronic hepatitis C (CHC) with genotype 6a in Hong Kong Chinese. Methods: To assess sustained virological response (SVR) (serum HCVRNA< 500IU/mL) at 6-months follow-up. Results: Nine patients with genotype 6a CHC (included from Jan2003 to Dec2007) received peginterferon and ribavirin. Mean age: 50 (range 28-64). Mean ALT before treatment: 96 IU/L (range 29-173 IU/L). Seven patients had liver biopsy performed, only one showed stage 3-4 fibrosis and others showed active hepatitis without advanced fibrosis. Mean serum HCV-RNA: 9.17x10 5 IU/mL(range 9.0x10 3 -3.3x10 6 IU/mL). Six patients had received peginterferon alfa-2b (1.5mcg/kg/week), other 3 received peginterferon alfa-2a (135mcg/week). Ribavirin dosage ranged from 600mg-1000mg/day depending on body weight and baseline haemoglobin. Treatment durations were 48-52 weeks in 7 patients, 24-26 weeks in 2 patients as one showed rapid virologic response at week 4 and the other was intolerant to side effect of peginterferon. Eight patients had early virologic response at week 12 and one had >2log drop of HCVRNA. Eight patients had end-of-treatment response. Eight patients (88.9%) achieved SVR at end of follow-up. Two patients who received only 24-26 weeks of combination therapy also achieved SVR. The one who failed to achieve SVR was at older age of 60 and had advanced fibrosis. Conclusions: The efficacy of pegylated interferon and ribavirin in treating Chinese patients with chronic hepatitis C genotype 6a can achieve high sustained virologic response rate of 88.9%.
T. Bharati 1,2 , P. Kar 2 , A. Mohammad 2 , K. Mariappan 1 , J. Annamalai 1 , R.
Introduction: HCV is a recognized cause of HCC. Information on HCV genotypes in HCC are scanty in India. Methods: A total of 154 HCC cases from Delhi, 96 HCC cases from Madurai and 246 cases of chronic hepatitis without HCC were controls in the study. RT-PCR for HCV RNA and genotyping were carried out in all the cases Results: In group-I, HCV RNA was positive in 26.58% HCC cases in which genotype 3 was found in 57.1% Genotype 1 was observed in 26.2% HCC cases. Whereas 16.6% cases remained nontypable. In group-II, HCV RNA was positive in 23.95% HCC cases, with genotype 3 in 30.4% cases, Genotype 1 in 52.2% cases and Genotype 4 in 4.34% cases. However, 13.04% cases remained nontypable. Out of the 246 control cases, 187 were CH and 59 were cirrhosis. In CH group, HCV RNA was positive in 22.45% cases in which, genotype 3 was detected in 71.4% cases whereas Genotype 1 was observed in 21.4% cases . However 7.1% cases remained nontypable. In Cirrhosis group, HCV RNA was positive in 37.2%cases. Genotype 3 was found in 59.1% cases. While genotype 1 was present in 31.8% cases and 9.1% cases remained nontypable. Conclusion: Genotype 3 in Delhi and Genotype 1 in Madurai were predominant. in HCC cases. Our study demonstrates that no particular HCV genotypes were associated with HCC and genotype did not appear to influence the development of HCV-associated HCC. 
Background/Aims: The standard treatment for chronic hepatitis C infected with HCV genotype-1 is a combination of pegylated interferon alfa and ribavirin for a 48weeks. It is unclear if 24weeks treatment is possible for patients showing a rapid virologic response (RVR) without compromising the sustained virologic response (SVR) in Korea. Method: Between June 2005 and July 2008, among patients chronically infected with the HCV genotype-1 (HCV-1) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), 20 consecutive paients who had low pretreatment viral load ( 1.5x10 6 copies/mL) and RVR were treated for 24weeks and then followed up for 24weeks. The HCV RNA was quantitatively assessed pretreatment, at 12weeks of treatment and was qualitatively assessed at 4weeks of treatment, the end of treatment (24weeks), 24weeks after end of treatment. RVR was defined as undetectable HCV RNA at the 4weeks. Results: Baseline characteristics of patients was as followed; age (30-65years:mean 45years), BMI (21-27kg/m²:mean 23.5kg/m²), HCV RNA titer (0.3-1.4×10 6 copies/mL:mean 0.5×10 6 copies/mL), ALT (5-751IU/L:median 77IU/L). Among the 20 patients, all patients (100%) had sustained virologic response (SVR). Conclusions: HCV-1 infected patients with a low baseline HCV RNA concentration ( 1.5×10 6 copies/mL) who had HCV RNA negative at week 4 of treatment may be treatment for 24weeks without compromising sustained virlolgic response. However, an additional trial will be needed to optimize the treatment duration. 
Background/Aims: Acute hepatitis C (AHC) has a high chronicity rate of up to 50~84% if it is not treated. Although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of AHC in Korea yet. The aim of our study was to investigate the clinical course and treatment outcome of AHC in single center of Korea. Methods: We performed a retrospective analysis of 35 patients who were diagnosed with AHC during the period from May 1996 to December 2007. The diagnosis of AHC was based on seroconversion to anti-HCV antibody or the clinical and biochemical diagnostic criteria satisfactory to AHC and on the presence of HCV RNA in first serum sample. The spontaneous resolution was defined as loss of HCV RNA in serum for 6-month in untreated group, and in treatment group, the sustained virological response (SVR) was defined as a index of treatment success. Results : Thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. In treatment group, nine of thirteen (69%) acquired SVR, and two of six (33%) showed spontaneous resolution in untreated group. Ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. Conclusion : Compared with untreated group, there was higher SVR rate in treatment group (33% vs. 69%). So early interferon treatment in acute hepatitis C should be considered. Background: This study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegIFN) therapy in chronic hepatitis C patients. Methods: Sixty chronic hepatitis C patients treated with pegIFN -2a or -2b in combination with ribavirin from 1st July 2004 to 30th July 2008 were included in this study. Treatment responses were evaluated and thyroid functions were assessed every 4 weeks. Results: Seventeen patients (28.3%) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (BMI). The proportion of patients with a high viral load (a serum HCV RNA titer >750,000 IU/mL) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group(88.2% vs. 48.8%, p=0.005). Among patients with HCV genotype 1, the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegIFN-based therapy(SVR, p=0.024; relapse, p=0.017). The female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, OR 9.44, p=0.009; high HCV RNA titer, OR 8.40, p=0.030) . Conclusion: The risk of thyroid dysfunction during pegIFN therapy for chronic hepatitis C was found to be higher for women and for those with a low BMI and a high viral load.
Background: In peginterferon alpha2b (PEG-IFN 2b) and ribavirin (RBV) combination therapy for 48weeks for patients with chronic hepatitis C, it is still difficult to predict which patients will achieve sustained viral response (SVR) at the completion of this therapy. Aim: To predict SVR and non-SVR (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (HA) levels. Methods: Eighteen patients were enrolled and their serum HA levels were measured before therapy, and after the 1st, 2nd, 3rd, and last trimesters during therapy. Results: Eleven patients achieved SVR and 7 became relapsers. All patients showed higher HA levels in the 1st trimester than the pretreatment levels. In the SVR group, 3 of 11 (27.2%) patients in the 2nd, 5 of 10 (50.0%) in the 3rd, and 9 of 11 (81.8%) in the last trimester showed lower HA levels than the pretreatment levels. By contrast, in the relapser group, none in the 2nd, 1 of 6 (16.7%) in the 3rd, and 1 of 7 (14.3%) (p<0.05) in the last trimester showed lower HA levels than the pretreatment levels. This study revealed that as the 48-week therapy went on, HA levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving SVR. However, HA levels of relapsers tended to continuously be above the pretreatment levels. Conclusion: Determination of changes of serum HA levels during PEG-IFN 2b and RBV therapy predicts SVR and non-SVR at the completion of this therapy. Results: The most frequent lymphomas were with high malignancy (40%), intermediate (32.5%) and low degree (27.5%). Cryopathy was negative (87.5%). The presence of viral markers was performed soon as possible after the NHL diagnosis, at the same time (52.5%) or during the first year of evolution (32.5%). The prevalence of the HCV infection was 15%, comparable to the one in the control patients group (22.68%), admitted in a gastroenterological clinic. On the other hand, this prevalence is significantly increased compared to the one in the general Romanian population (4.9%). The patients with NHL and HCV infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of HCV infection. The prevalence of HBV infection in the tested patients was 2.5%, being lower than that of HCV infection (2.5% vs. 15%, p = 0.113) but comparable to the one in the general population (2.5% vs. 6.3%, p = 0.525). Conclusions: The prevalence of HCV infection in the patients having NHL was 15%, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between HCV infection and NHL.
Iron hepatic overload and hepatitis C D. Damian 1 , M. Grigorescu 1 , M.D. Grigorescu 1 , T. Zaharie 1 1 Third Medical Clinic, Cluj Napoca, Romania Aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. Method: 212 patients with chronic hepatitis C were included, to whom we tested the blood iron level. In order to evaluate the hepatic iron accumulation we performed the Perls staining, using a qualitative analysis and a semiquantitative scoring system (Deugnier). Results: From a total of 212 patients, 16.5% presented increased blood iron level (p = 0.000). The evaluation of the liver iron loading was performed in 54 patients, some having normal blood iron level (n = 34) and others (n= 20) increased (p = 0.007). The stainable iron was observed in 27 patients. The iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. The total iron score Deugnier was low. The increased blood iron correlated with the ALT and GGT levels, the necroinflamatory activity and fibrosis. No correlationships between stainable iron and increased blood iron. The presence of liver iron accumulation only correlated with the fibrosis degree. Conclusions: Of the 212 patients to whom we tested the blood iron, 16.5% had increased levels. The Perls staining was positive in 50% of the patients. The iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis.
Response Patients whose HCV RNA Became Negative at 12 -16 Weeks T. Ide 1 , T. Arinaga 1 , K. Ogata 1 , I. Miyajima 1 , K. Kuhara 1 , R. Kuwahara 1 , M.
Background/aim: Chronic hepatitis patients whose HCV RNA became negative at 4 weeks of PEG-Interferon/ribavirin treatment achieved excellent SVR(sustained viral response) rate of almost 90%. However, in patients whose RNA became negative after 20 weeks, the SVR rate is very low. Since many patients became RNA negative at 12-16 weeks, it is important to clarify the characteristics of the patients. Material and Method: Among 234 patients, 41 became RNA negative at 12-16 weeks and the therapy completed (total 48-60 weeks). The characteristics were analyzed by using sex, age, weight, BMI, ALT, GTP, hemoglobin, platelet counts, Ccr, hyaluronic acid, the mutation of HCV core region (aa70, 91) and interferon sensitivity determining region, adiponectine, HOME-IR, RNA dynamics, dose and the treatment period. Result: The SVR rate was 75.6%(31/41). Because all 10 patients of non SVR were female, we compared these 10 patients and 15 female SVR. The platelets counts were low in non SVR (non SVR 15.0 ± 2.8 (x10 4 /mm 3 ) vs SVR 20.2 ± 4.8 (P<0.05)). The mean dose of ribavirin was lower (447 ± 126 mg/day) in non SVR (P<0.05) than in SVR (625 ± 127). Conclusion: As for the characteristics of the patients whose HCV RNA became negative at 12-16 weeks but became non SVR, female, low platelet count and low dose of ribavirin were important factors. In the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use 400mg and 600mg alternately) PE329 Background: Chronic hepatitis C virus (HCV) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in China. The combination treatment of peginterferon alpha (PEG-IFN alpha) plus ribavirin (RBV) is recommended as a standard care for HCV infections, which can improves hepatic markers and eradicates the virus in about 50% of patients. However, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. Several viral, hepatic, and patient-related factors influence response to therapy. Methods: In our clinical practice, a total of 77 interferon-naïve patients (61% male; median age 47 years) with chronic hepatitis C include 11 cirrhotic patients (no genotyping) received PEG-IFN alpha-2a 180 mcg/week plus RBV 900-1200mg/day for 48 weeks and follow up 24 weeks. Results: show that the patients have more RVR and EVR rate (54% and 90.9% respectively). While the SVR (undetectable HCV-RNA 24 weeks after treatment completion) rate is only 51.5% In conclusion: comparing with the data of clinical trail, the RVR, EVR and EOTR were higher, while SVR was the same in Chinese patient with chronic hepatitis C patients received the combination therapy of PEG-IFN plus RBV. The reason of high relapse was still unknow. Although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved EVR.
S. Nakamoto 1 , F. Imazeki 1 , K. Background/Aims: Recently amino acid (aa) substitutions in hepatitis C virus (HCV) core region (double wild (DW); arginine at aa 70, leucine at aa 91) were reported to be associated with sustained virological response (SVR) in a combination therapy of peginterferon and ribavirin. We evaluated the viral factors influencing treatment response. Methods: Nucleotide sequences of core region were determined directly in 104 patients with genotype 1 and high viral load ( 100 KIU/ml) treated with peginterferon-alpha 2b and ribavirin for 48 weeks. Rapid virologcal response (RVR) was defined as more than 2 log decrease of HCV-RNA during the first four weeks of therapy and early virological response (EVR) as that during the first 12 weeks. SVR was defined as negative HCV-RNA 6 months after the end of treatment and non-virological response (NVR) as less than 2 log decrease of HCV-RNA during the treatment. Results: DW at aa 70 and 91 was shown in 17/44 (35%) patients with RVR and in 5/44 (11%) with non-RVR (p=0.003), in 25/72 (35%) with EVR and in 1/28 (3.6%) with non-EVR (p=0.001), in 16/37 (43%) with SVR and in 6/44 (14%) with non-SVR (p=0.003), and in 1/24 (4%) with NVR and in 25/79 (32%) with non-NVR (p= 0.005). In multiple logistic regression analysis, DW was significantly associated with RVR, EVR, SVR and NVR. Conclusions: DW at aa 70 and 91 in HCV core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. Medicine and Hepatology, Henry Dunant Hospital, Athens, Greece, 3 Biometrics, IST GmbH, Mannheim, Germany, 4 
Background: Among patients with chronic HCV treated with pegylated interferon and ribavirin, the highest sustained virologic response (SVR) rates are achieved in patients with a rapid virological response (RVR). Here we investigate how the time taken to become HCV RNA undetectable influences the probability of relapse during untreated follow-up. Methods: Data from 569 patients treated for 48 weeks with peginterferon alfa-2a (40KD) 180µg/week plus ribavirin 1000/1200mg/day were included in the intent-to-treat analysis. Response was classified as RVR, complete early virological response (cEVR) slow responder and non-EVR. Results: There was a correlation between the time required to become HCV RNA undetectable and the relapse rate after stopping treatment. Patients with an RVR had the lowest relapse rate (4%); this increased among patients with slower responses. Conclusion: There was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. Background: Rapid virologic response (RVR; HCV RNA <50IU/mL) at week 4 of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an SVR. Patients with detectable HCV RNA at week 4 have a lower probability of achieving an SVR than those with an RVR; further subdivision of these patients may be useful in predicting outcomes. Methods: We conducted a retrospective analysis including 569 genotype 1 patients treated for 48 weeks with peginterferon alfa-2a (40KD) 180 g/week and ribavirin 1000/1200 mg/day. Patients were categorized as RVR and non-RVR. Those without an RVR were further subdivided into detectable but unquantifiable, 3, 2, 1 or <1 log10 drop in HCV RNA. The proportion of patients with undetectable HCV-RNA at week 12 and achieving an SVR was calculated within each category. Results: RVR and non-RVR patients had an 88% and 43% rate of SVR respectively. Among non-RVR patients, rates of SVR depended on the categorical response at week 4: detectable but unquantifiable HCV RNA, 77%; 3 log10 drop in HCV RNA, 61%; 2 log10 drop, 43%; 1 log10 drop, 27%; and <1 log10drop, 13%. Independent of week 4 response, undetectable HCV RNA at week 12 was also highly predictive of SVR. Conclusions: Patients achieving an RVR have high rates of SVR. Among patients who do not achieve an RVR a more precise prediction of SVR can be achieved by considering the extent of viral load reduction at week 4 and week 12.
Retrospective Japanese Validation Study of FibroTest and ActiTest in patients with chronic hepatitis C. N. Nagata 1 , T. Mine 1 1 
Background: Fibrotest (FT) and Actitest (AT) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis C virus. The aim of this study was to perform a validation study the discordances between FT and AT(FT/AT) and liver biopsy in patients with chronic hepatitis C in Japan. Methods: 117 serum samples of chronic hepatitis C patients sended at -80°C at the Biochemistry Department of Pitié Salpetrière Hospital were analysed between july and august 2007. FT/AT components were assessed on thawed sera for 110 patients. 96 from 110 patients had liver biopsy at the moment of serum analysis. Liver biopsy fibrosis and activity scores were assessed by a pathologist in Japan according to METAVIR scoring system. For each individual test-FT/AT the following statistical analysis were performed Result: FT observed AUROC for the diagnosis of advanced fibrosis was 0.81 and after adjustment according to the prevalence of different stages of fibrosis the AUROC was 0.89. This difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -F0 and F4-and high prevalence of adjacent intermediate stages -F1 and F2). The observed AUROC of FT for the diagnosis of precirrhosis and cirrhosis was 0.86 and the observed AUROC of AT for the diagnosis of moderate to severe activity was 0.74 . Conclusion: These results are similar to those observed in all independent validations worldwide. Background: Accurate monitoring of HCV-RNA level throughout anti-HCV therapy is key factor for predicting sustained virological response (SVR). Real-time detection polymerase chain reaction (RTD-PCR) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical PCR. Aim: To compare RTD-PCR based assays; Cobas Ampliprep/Cobas TaqMan (CAP/CTM) and recently developed Abbott RealTime HCV for HCV RNA quantification and measurements differences by 2 assays in different genotypes. Methods: In total, 253 serum samples were used including, 135, 39, 24, 15, and 40 with genotypes 1b, 2a, 2b, 3a and 4 respectively were tested quantatively for HCV-RNA by CAP/CTM and Abott RealTime. Results: Good correlation between two assays as overall (r=0.96) with correlation coefficient (R) in genotypes 1b, 2a, 2b, 3a ranged between 0.99 to 0.98 and least in genotype 4 (r=0.78). Mean differences between CAP/CTM and Abott RealTime was significnat in genotypes 1b and 4. Significantly HCV-RNA genotype 4 underestimation by CAP/CTM (4.3+0.9 log IU/ml) than Abbott RealTime (4.8+0.9 log IU/ml; P=0.01). In genotype 1b, significantly higher HCV-RNA measurement by CAP/CTM (5.7+1.7log IU/ml) than Abbott RealTime (5.0+1.4log IU/ml, P=0.001). Two HCV genotype 4 samples showed measurement differences (CAP/CTM minus Abbott RealTime) of -3.75 and -1.68 log IU/ml. Studying genotype 4 sequences within 5 UTR , target for CAP/CTM RT-PCR amplification, revealed nucleotide polymorphisms at positions A107, A165, T203, A205, and A243. Conclusion: Different measurement efficiency by commonly used CAP/CTM in different genotypes compared to Abbott RealTime. 6 New York, New York, USA, 7 Vertex Pharmaceuticals, 8 Cambridge, MA, USA, 9 Duke Clinical Research Institute , 10 Duke University, Durham, NC, USA Background: PROVE1 is a placebo-controlled study of 250 subjects with genotype 1 chronic hepatitis C randomized to 48 weeks of peginterferon-alfa-2a 180 ug/week (P) plus ribavirin 1000-1200 mg/d (R) (PR48, n=75), or 3 regimens of 750 mg q8h telaprevir (TVR) with PR: TVR/PR for 12wks followed by PR for 0wks (T12/PR12, n=17), 12wks (T12/PR24, n=79) or 36wks (T12/PR48, n=79). The impact of African American race (AA) and bridging fibrosis on sustained virologic response (SVR) was examined. Methods: Subjects with cirrhosis were excluded from study. Fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within 2 years. ITT analysis was performed. Results: Overall, SVR was achieved by 41% of subjects in the PR48 group, 35% in T12/PR12 group, 61% in T12/PR24 group, and 68% in T12/PR48 group. Subgroup analyses indicated SVR was improved with TVR/PR (TVR/PR arms pooled) vs PR48 alone in AA subjects (44% (8/18) vs 11% (1/9)), and in subjects with bridging fibrosis (69% (22/32) vs. 26% (5/19)). Adverse events leading to discontinuations were more frequent in the TVR/PR groups (21% vs. 10%). Rashes, gastrointestinal events and anemia were more common in the T/PR arms, and rashes were more frequently severe (7% vs 1%). Conclusions: TVR-based treatment for 24 or 48 weeks was associated with an increase in SVR rates compared to PR48. Subgroups with impaired response to standard Peg-IFN/RBV therapy appeared to benefit from the addition of telaprevir. Adverse events leading to discontinuation were more frequent in TVR-based regimens. Background and aims: Induction of Type I IFNs is a core issue in antiviral responses and must be tightly controlled. The protein kinase TBK1 is critically involved in virus-triggered type I IFN signaling. In previous studies, an alternatively spliced isoform of TBK1, termed TBK1s, was identified to be induced in both human and mouse cells. Bound to RIG-1, it is able to disrupt the interaction between RIG-I and VISA. This study was designed to observe the expression of TBK1s in HCV-infected patients. Methods: Total RNA was extracted from samples of peripheral blood mononuclear cells obtained from 11 HCV patients, 9 HCV patients treated with IFN-/ribavirin and 9 healthy controls, and subjected to real -time PCR using the primer-probe sets for human Tbk1s, Tbk1 and IFN-genes. Results: The TBK1s expression was significantly elevated in HCV-infected patients, while treatment of HCV-infected patients with IFN-/ribavirin resulted in down-regulation of TBK1s to the normal level. Conclusions: The study strongly supports the idea that expression of TBK1s is correlated with HCV infection, and indicates that TBK1s may play an important role in the regulation of HCV infection.This work was supported by NSFC(30571643, 30672380, 30700702 Background: Infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. The interrelation between the syndrome of iron overload (SIO) and SVR achievement is studied during chronic HCV infection treatment. Methods: 68 patients with chronic HCV infection (genotyping: 1-34; 1+3-5; 3-22; 2-6; 4-2) are investigated. SIO criteria: iron increase-more than 37 mkMol/l, ferritin -more than 200 mkMol/l, percent of transferriny saturation with iron (%Tf) -more than 50%. Results: SIO revealed in 10 patients (14.7%): 5 patients -1 genotype (2 assotiative with a diabetes) and 5 patients-genotype 3 (3 -in combination with liver steatosis and obesity). Venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. RVR: SIO -5 patients, normal metabolism -48; EVR: 6 and 54, SVR: 4 and 54 relatively. 5 nonresponding patients (SIO) had steatosis and diabetes, hereditary hemochromatosis (C282Y/H63D) is verified in 1 case. Increase of ferritini values and %Tf during therapy and positive HLA-A3 and HLA-B7 is registered in nonresponding patients. Conclusions: SIO in HLA-A3, HLA-B7 and C282Y/H63D positive patients is independent predictor of nonresponse during peginterferon alpha-2a (40 kd)" ribavirin treatment.
A.P. Srivastava 1 , G. Dogra 2 , S. Sachdeva 1 , N. Nigam 1 , A. Chakravarty 2 1 Dr. RML Hospital, New Delhi (India)-110001, 2 Maulana Azad Medical College & Associated Hospitals, New Delhi, India Background: Hepatitis C virus (HCV) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.
Genotyping and assessment of viral load in HCV patients are vital for designing therapeutic strategies. We aimed to determine the pattern of HCV genotypes and its association with viral load and biochemical profile. Methods: 71 HCV RNA positive patients were included in the present study attending the medical-OPD and wards of Dr RML Hospital, a tertiary care hospital in New Delhi during 2006-2008. HCV genotyping was carried out by restriction fragment length polymorphism (Buoro et al 1999) followed by the type specific primers from the core region (Ohno et al 1997) . Viral load estimation was carried out by Taqman real time PCR system using previously described method (Martell et al 2000) . Result: 67.6% of cases were having genotype 3 (3a, 3b, 3f & 3i) followed by genotyping 1 (1a & 1b) in 26.8% and genotype 2 in 5.6%. There was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. Genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. Parentral mode of transmission was accounted for the 68% of all the infected cases. Conclusion: HCV genotypes 3 and 1 accounted for 94% of our cases. The genotype 1 is associated with higher degree of disease severity as assessed by viral load. Also two unusual subtypes 3i and 3f were identified from this geographical region.
A.P. Srivastava 1 , G. Dogra 2 , S Sachdeva 1 , N. Nigam 1 
Background: The development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. Regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. Polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mRNA, thus regulating cytokine production. We aimed to determine the polymorphism of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) genes in chronic hepatitis C patients. Methods: 40 HCV RNA positive patients were included in the present study conducted during 2006-2008. 25 healthy controls were also included. Genomic DNA was extracted by using Q1A amp DNA blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of Vidigal et al 2002.
Result: Genotyping of -308-promoter variant of TNF-alpha was performed by PCR. Polymorphism in the TNF-alpha (G/G, G/A and A/A allele) was different between HCV patients and healthy controls. IL-10 variants (C/T, C/C) were more frequent among HCV patients as compared to healthy controls. Conclusion: Genetic polymorphism analysis on IL-10 promoter have indicated that distribution pattern of IL-10 polymorphism was significantly different between controls and HCV patients. Furthermore, polymorphism in promoter region of TNF-alpha (-308) was found, though the difference was not significant. Since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. Introduction: The assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. Although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. Fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. Our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis C genotype 4. Methods: Subjects were 50 patients with chronic hepatitis C genotype 4. All biopsies were scored using METAVIR system by two independent pathologists. Fibro-test was done with (Biopredictive, Houilles, France) for the assessment of liver fibrosis. Sensitivity, specificity, PPV and NPV were measured for distinguishing between different degrees of severity of fibrosis. Results: patients (45 male and 4 female) age ranged 21-56 years, liver biopsy showed 4% (F0), 40% (F1), 20 % (F2), 28% (F3), 8% (F4). The efficacy of Fibrotest is 63.26%, sensitivity 83.3%, specificity 53%, positive predictive value 50% and negative predictive value 85%. Conclusion: Fibrofast has a low performance in assessment in fibrosis in chronic hepatitis C genotype 4. Introduction: Liver biopsy is the reference method for assessing liver fibrosis. However, it is invasive, costly and has some limitations. European Liver Fibrosis (ELF) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. Our aim is to test the performance of ELF markers in an independent cohort of patients with chronic hepatitis C genotype 4. Methods: Subjects were 199 patients with chronic hepatitis C genotype 4. All biopsies were staged for fibrosis using METAVIR system by two independent pathologist. ELF markers were done by (Diagnostic & Operations, England) and fibrosis scores were derived using the published ELF algorithm. The area under the curve (AUC) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (PPV) and negative (NPV) predictive values for distinguishing between different stages. Results: patients (179 male and 20 female), age was ranged 25-51 years, liver biopsy showed 2% (F0), 27% (F1), 34% (F2), 26% (F3) and 11% (F4). ELF markers had no correlation with fibrosis score where r = -0.003, P = 0.963, AUCs: 0.469, Specificity 87.9 %, Sensitivity only 9.3 %, PPV: only 6.03 %, NPV: 61.5 % and efficacy 58.2%. Conclusion: The performance of ELF marker is low and can not be used for assessment of fibrosis in chronic hepatitis C genotype 4. Background: The PROVE2 trial is a randomized, placebo-controlled study that assessed the safety and efficacy of 750mg q8h telaprevir (TVR) combined with 180 g/week PEG -IFN alfa-2a (P) ± 1000-1200mg/day ribavirin (R) in chronic HCV genotype 1-infected treatment-naïve patients without cirrhosis. Methods: Overall, 323 patients received TVR + PR for 12 weeks (T12/PR12; n=82), TVR + PR for 12 weeks then PR for 12 weeks (T12/PR24; n=81), TVR + P for 12 weeks (T12/P12; n=78), or to PR for 48 weeks (PR48; n=82). Primary endpoint: sustained virologic response (SVR, undetectable HCV-RNA 24 weeks post-treatment). Results: Baseline characteristics were well balanced across groups. Numerically higher SVR rates were observed in patients receiving T12/PR24 (69%; p=0.004 for difference vs. PR48) than T12/PR12 (60%), T12/P12 (36%) or PR48 (46%). Relapse rates were lower in the T12/PR24 group (14%) than the T12/PR12 (30%), T12/P12 (48%) and PR48 (22%) groups. The relapse rate in patients receiving T12/PR24 with 4-week and 12-week undetectable HCV-RNA was 7% (3/45). The AEs occurring more frequently with the T/PR regimen were pruritus, rash, asthenia, nausea and anemia. In the T/PR arms, 12 patients discontinued due to rash, 2 discontinued due to pruritus, and 2 patients due to anemia. Conclusion: These results showed that a telaprevir-based regimen led to significantly higher SVR rates than PR, and indicate that this regimen could shorten the overall treatment duration from 48 weeks to 24 weeks for most patients infected with HCV genotype 1.
A.C. Cardoso 1 , C. Stern 1 , R. Moucari 1 , N. Giuily 1 , P. Bedossa 1 , P. Marcellin 1 1 Hopital Beaujon Background/Aim: This study evaluated the effect of the response (SVR) to therapy on fibrosis stage, as assessed by LS, in patients with advanced fibrosis (F3) or cirrhosis (F4). Methods: HCV patients with F3 or F4 who received interferon-based treatment were studded. LS was assessed after treatment (median delay of 36 months, 2-206) in patients with or without SVR. Correlations between LS and clinical and treatment characteristics were analyzed. Results: 114 patients were included: male gender (72%), mean age (54±9 years), diabetes (26%), mean BMI (26±6 kg/m2), genotype 1 (59%). 33% had SVR. LS was performed 0-3, 3-6, >6 years following treatment. By linear regression, the median of the LS was independently associated with SVR (p=0.005) and diabetes (P=0.008). SVR patients had lower LS (8.4 kPa; range 3.3-45) than non SVR patients (15.7 kPa; range 5.3-75) (p<0.001). Among the SVR patients the median LS was lower when the delay between LS and the end of treatment was longer (10.9,8.8,6. 3) (p=0.02). On the opposite, among the non-SVR patients the median LS was not significantly different (p=0.57). The median of liver stiffness was higher in patients with diabetes (p=0.006). BMI and dyslipidemia did not influence the median of the LS. Conclusion: In patients with advanced fibrosis or cirrhosis, LS was lower in patients with SVR and decreased with time while it was higher and did not decrease in non-SVR patients. LS could be important for assessment of fibrosis stage during the post-treatment follow-up. To study peripheral blood and intrahepatic Natural Killer (NK) cells in patients with chronic hepatitis C in relation to disease activity and severity of hepatic fibrosis. Patients & Methods: Fifteen untreated patients with histologically-proven chronic hepatitis C, and 12 matched healthy subjects. The NK cells and Natural Killer T (NKT) cells were identified in fresh whole blood samples using two-color flow cytometric assay as CD3 -CD56 + and CD3 + CD56 + positive cells. Immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against CD56 for detection of NK cells and rabbit polyclonal antibody against smooth muscle actin (SMA) for identification of activated hepatic stellate cells (HSCs). Results: Patients with chronic hepatitis C showed significant decreases in the percentages of NK cells and NKT cells in peripheral blood. A negative correlation was found between serum HCV RNA levels and the percentages of peripheral blood NK cells and the intensity of intrahepatic NK cells. The percentages of circulating NK cells and NKT cells and the intensity of intrahepatic NK cells were inversely correlated with the METAVIR fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated HSCs. Conclusion: Patients with chronic hepatitis C had significant deficiency in circulating NK and NKT cells as well as in intrahepatic NK cells. This may provide a possible mechanism for the suppression of innate immunity against HCV. Background: HCV infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. The virus is classified in six genotypes and more 50 subtypes, which are related distinct with antiviral therapies reply. In Brazilian Amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype 1 (74%) followed by genotypes 3 (25%) and 2 (1%). However, epidemiological research in populations of risk to the infection still is scarce. Aim: To determine HCV genotypic frequency in 116 blood donors, 55 patients with blood transfusions multiples, 57 patients in hemodialysis and 52 drugs users in the State of Pará, Brazilian Amazon. Methods: Using Real Time PCR and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. Results: In blood donors, HCV distribution was constituted by genotypes 1 (93.1%) and 3 (6.9%). In multitransfunded patients occurs maximum prevalence of genotype 1 (100%), probably reflect of genotype 1 specific transmission of blood donors population. On the other hand, in hemodialysis patients had been detected genotypes 1 (85.7%), 2 (3.6%) and 3 (10.7%), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . In drug users occurs the biggest frequency of genotype 3 (38.1%) with prevalence of genotype 1 (61.9%), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype 3. Conclusions: The genotype 1 possesses the biggest frequency in different population. Moreover, through HCV genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. Virol. 2007 ). We established real-time polymerase chain reaction (PCR) assays for the easy detection of these HCV mutations. Methods: Plasmids p-core-W, including wild type HCV core coding region (70R and 91L), and p-core-M, including mutant type HCV core (70Q/H and 91M), were constructed by cloning and PCR-based mutagenesis for control vector of wild type core and that of mutant core, respectively. Using serially diluted forms of these vectors, SyBr Green-based real-time PCR detections with mutation-specific primers were performed. Results: Analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least 10 copies, 10 copies, 1000 copies, and 10 copies of 70-wild, 70-mutant, 91-wild, and 91-mutant, respectively. Each primer could clearly distinguish the difference between p-core-W and p-core-M at the same copy numbers. Concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-W versus p-core-M could be distinguished. On the other hand, for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. Conclusions: This method could represent a useful alternative for the detection of genotype 1b HCV core amino acid substitutions 70 and 91 and be reliably applied for rapid screening.
Efficacy and tolerability of HCV treatment in Asian patients according to age and genotype at a tertiary centre in Western Australia N. Saroj 1 , N. Kontorinis 1 , T. Lorenzo 1 , M. Marion 1 , S.L. Chen 1 , W. Cheng 1,2 1 Royal Perth Hospital, 2 Centre for International Health, Curtin Introduction: Race and ethnicity can influence efficacy and tolerability to treatment in HCV. The higher response rate in Asians is thought to be associated with better adherence and tolerability. Objectives: (1) To evaluate the adherence according to age and genotype (2) To assess the effect of age on treatment efficacy (3) Background: Hepatitis C virus (HCV) infection is a major health problem. There is huge regional variation in its prevalence and genotypic distribution. Voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. Reliable statistics are not available for the entire country, particularly for the rural areas. It is important to know local situation and rationalize use of limited resources. Methods: Retrospective study of the records of patients attending the Free Liver Clinic (FLC) of our hospital located in a rural area of Pakistan, and those screened for HCV infection prior to voluntary blood donation. Results: Patients at FLC (324 out of 1638 [20%; males 65%] were found to have higher chances of being reactive for HCV antibodies as compared to voluntary blood donors (121/804 [14%]; p = 0.004; OR 1.39 -95% CI = 1.11 -1.75). Out of a total of 1022 HCV reactive patients, 904 (88%) were found to be positive on HCV RNA testing. Out of a total of 166 typeable genotypes, 125 (75%; 95% CI = 68.7 -81.9, estimated odds = 3.05) were infected with a single genotype, and only 7 patients (4%) were infected with genotype 1, either alone (n=4) or in combination with 3a. Conclusions: One out of every 5 people tested in our FLC is seropositive for HCV, and 14% of "healthy" voluntary blood donors have the same results. Genotype 1 is very rare in our region.
S.A. Batool 1 , S.Z. Abbas 1 1 Department of Gastroenterology, Muhammad Hospital, Mirpurkhas, Pakistan Background: Hepatitis C viraus (HCV) infection is common in our region. Data is not available on success rates of conventional Interferon (INF) based products here. We attempted to find out the dominant genotype, and to determine the success rate of conventional INF-based treatment in eradicating HCV. Methods: Retrospective case series study of HCV infected patients' records treated with 14 different brands of INF. Results: 320/1858 (17%) of all patients tested were positive for HCV antibodies. HCV-RNA was tested by PCR for 1022 patients, of which 904 (88%) turned out to be positive. Genotype type 3 was the dominant genotype -found in 118/168 (70%) patients. 101 men and 57 women were treated with various brands of INF with the same manufacturer's brand of Ribavirin. The overall ETR achieved was 100/142 (70%) -58/85 (68%) men and 42/57 (74%) women. 41/62 (66%) of genotype 3 achieved ETR. There was no significant difference in average ages for those who achieved good ETR and those who did not (39 years each). The ETR achieved by different brands ranged from 48% to 91%. SVR was achieved by 17/30 patients. Conclusions: 17% of all people tested positive for HCV antibodies, of which about 88% had evidence of active HCV infection. ETR achieved by different brands averaged 70%. This was 74% in female sex, although age did not appear to be a factor in determining a favourable ETR. Patients & Methods: A total of 439 consecutive diabetic patients of either sex were evaluated for HCV and HBV infection by using Enzyme Linked Immunosorbant Assay (ELIZA-3) along with serum ALT levels. On the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. Different variables were: Age, sex, BMI, area of residence (rural or urban), type and duration of DM, smoking, literacy and ALT. Results: Males 50.3% and females 49.7%. Age ranged from 18 to 95. Majority were married (98.4%), from rural area (70.4%), had type-2 DM (99.8%), normal weight (39.2%), normal ALT(60.1%) and non-smokers (78.6%). Seroprevalence for HCV, HBV and both were 25.1%, 1.8% and 1.6%. Two groups were made, sero +ve and sero -ve. Raised ALT (59.2%) was significant (P<0.05) factor while all others variables were insignificant (P>0.05). Conclusion: HBV and HCV infections are more prevalent in DM with increased ALT levels. While HCV infection is more common than HBV in patients with DM. Hepatitis C virus (HCV) envelope proteins (E1 and E2) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. The expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. In order to obtain glycosylated, correctly-folded HCV envelop proteins in large quantities, we optimized the DNA sequences of HCV envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (HEK) 293 cells. Both proteins were detected intracellularly, with a small portion secreted into supernatant. In order to enhance secretion, truncated forms of envelop proteins including E2 TM, E2384-661, E2484-661 were also expressed. Both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. In addition, we also expressed the codon-optimized HCV receptors CD81 and Claudin-1 in 293 cells. By comparing the expression level of codon-optimized sequences and the sequences that were obtained from cDNA library by PCR, we found that codon-optimization enhance protein expression significantly in 293 cells. These results not only lay solid foundation for further research concerning the mechanism of HCV entry, including the optimal pH and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. Background: Prolactin (PRL) is an immunoregulatory hormone secreted from lymphocytes, however, PRL induction in relation to Hepatitis C virus (HCV) infection has not been elucidated. Methods: Serum PRL levels were measured in both 232 subjects of our HCV cohort study and 31 male patients of the hospital, who were chronically infected with HCV. Furthermore, serum PRL levels were compared in 27 male patients before and after interferon therapy. We measured expression of PRL mRNA level in PBMCs in 12 male patients, and also investigated PRL mRNA of PBMCs collected from 5 healthy men that stimulated by HCV produced by Huh7.5 cells in vitro. Result: Serum PRL levels were significantly higher in the HCV-infected subjects than in the controls (p< 0.01). They were significantly higher in HCV-infected male subjects than in the controls (p< 0.001). Serum PRL levels were significantly higher in male patients than in the controls (p<0.01). Serum PRL levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p<0.05). The levels of PRL mRNA in PBMCs derived from HCV-infected patients were significantly higher in 12 male patients than in the controls (p<0.001). Conclusion: The high levels of PRL expression are associated with HCV infection in carriers. Background and Objectives: Hepatitis C virus (HCV) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.Current clinic standard therapy is interferon alpha (IFN-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. Until recently, development of a HCV cell culture system (HCVcc) provides a suitable tissue culture system to study the complete HCV life cycle. In this study, we tested the effect of IFN omega (IFN-)-a member of type interferon on HCV compared with IFNbased on HCV 1b replicon and HCVcc. Methods: We compared IFN-and IFN-2a effects on HCV RNA replication and protein expression, as measured by ribozyme protection assay and western blot. We also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) treated with different interferon type and concentration with western blot analysis. Results: HCV RNA and protein level were inversely related with IFNconcentration and compared with IFN-2a, at the same concentration, the HCV RNA and protein levels treated with IFN-were lower than that treated with IFN-2a p 0.05 .Also based on the HCV RNA analysis, EC50 of IFN-was 10 folds lower than IFN-2a. IFNs increased intracellular p-STAT1 level at a dose dependent manner and compared the same concentration of IFN-and IFN-2a, p-STAT1 protein level was higher in IFN-treated group p 0.05 . Conclusions: These results demonstrate distinct antiviral effect of IFNcompared with IFN-2a and this difference maybe partly caused by the stronger stimulation of IFN receptor . Outstanding antiviral activity of IFNmay be useful for developing new HCV treatment strategies. Background & Aim: Hepatitis B virus (HBV) infection with undetectable levels of hepatitis B surface antigen (HBsAg) is called an occult infection, which although has been described among subjects with chronic hepatitis C liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the Indian subcontinent. Materials and Methods: We investigated HBV-DNA PCR in serum samples of 260 HBsAg negative subjects with chronic HCV-related liver disease, and 70 apparently healthy volunteers negative for HBsAg and anti-HCV as control. Results: Serum samples found positive by at least two independent PCR assays were considered HBV DNA positive. HBV-DNA was detected among 19 HCV-related chronic liver disease (CLD) patients (7.3%), which was higher (p = 0.2) as compared with the control volunteers (4.3%). It was more frequent (37.5%) in 24 anti-HBs negative/anti-HBc positive patients than in 180 anti-HBs/anti-HBc positive (5 %, p < 0.05). HCV RNA by qualitative PCR was significantly (p <0.001) higher in occult HBV compare to non-occult. HCV genotype 1b was predominantly associated with occult HBV (73%), especially among subjects with hepatocellular carcinoma (HCC) (p<0.05) as compared to non-occult HBV cases. Though not significant, frequency of occult HBV infection was higher than healthy controls and HCV 1b genotype was significantly associated in patients with HCC. Conclusion: This study suggests that in all HBV-endemic areas, the possibility of occult HBV in patients with HCV should be considered and HBV-DNA should be performed.
J. Zhao 1,2 , W.D. Cai 2 , L. Chen 2 , Y.X. Gan 2 , M.L. He 1 , X.R. Wang 1 1 
Background: Besides HIV and syphilis, hepatitis C virus (HCV) is also rapidly spread among men who have sex with men (MSM). This study was designed to identify the prevalence of these 3 sexual transmitted diseases in MSMs in Shenzhen, China. Methods: A cross sectional study was conducted by using time location sampling method from April to July, 2008. 831 MSM participants (including 454 Male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in 37 venue-date-time randomly selected from 48 active venues. Results: Results were analyzed using SPSS. 736 blood samples were collected for HIV, syphilis and HCV test. Participated MSMs were between the age of 18 to 51 years (25.5±5.9) with a majority of 20-29 years (73.7%). Most of them finished junior high school education (74.9%). 84.2% had high level of knowledge on modes of transmission and prevention. Likewise, 56.0% MSMs have ever sold sex to men, 38.3% of them were self identified as gay, 35.1% as bisexual. 78.3% MSMs had multiple male sexual partners and 50.3% MSMs always used condom. 48.6% of them had sex with women in the past 6 month, and the condom use rate decline to 35.1% during both male and female sex. HIV positive rate is of 6.7% and syphilis for 18.3%, HCV is only found in 3 cases (0.4%). Conclusions: A greater number of the participants have both male and female sex partners. This survey shows that HCV infection rate is still low among MSMs in Shenzhen, although the HIV and syphilis rate is high and continuing increased in the past few years.
The Change of Insulin Sensitivity in Hepatitis C Patients with Normal Insulin Sensitivity S.G. Park 1 , Y.K. Cho 1 , J.W. Lee 1 , J.W. Yun 1 , H.J. Kim 1 , W.K. Jeon 1 , B.I.
Background: Hepatitis C virus (HCV) infection is associated with a high prevalence of diabetes mellitus (DM). Insulin resistance (IR) is known to play a crucial role in the development of DM in Chronic hepatitis C (CHC) patients. We prospectively investigated the change of insulin sensitivity in CHC patients during 5-year period, and analyzed factors significantly associated with IR. Methods: Subjects consisted of 62 non-cirrhotic CHC patients with normal alanine aminotransferase (ALT) and normal insulin sensitivity (CHC group), and healthy control group of 172 subjects matched by age, sex, body mass index and life styles. We compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of IR at the end of follow up period in both groups. The change of insulin sensitivity and metabolic parameters and development of IR was analyzed, and factors associated with development of IR were evaluated. Results: IR developed in 22.5% of 62 CHC patients and 5.2% of 172 normal individuals (P<0.001). HCV infection per se and genotype 1 were independent risk factors of IR. Initial fasting glucose 90-100 mg/dL, fasting insulin 10 uIU/mL, HOMA-IR 2.3-2.7 were significantly associated with development of IR in CHC group. Conclusions: HCV infection is independent risk factor of IR. Even if CHC patients with normal insulin sensitivity, careful monitoring for IR is necessary.
Prevalence of Viral Hepatitis C in Latvia I. Tolmane 1,2 , B. Rozentale 1,2 , J. Keiss 1 , F. Arsa 1 1 SA Infectology Center of Latvia, 2 Riga Stradin's University Background and aim: Viral hepatitis C (VHC) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. To date chronic hepatitis C affects over 170 million individuals worldwide. Chronic VHC is a leading cause of cirrhosis and hepatocellular carcinoma. The aim of this study was to investigate how many residents of Latvia, that are over 18 years of age have been exposed to VHC (anti-HCV prevalence) and how many are infected at the moment (HCV-RNA prevalence). Until now such research has not been performed in Latvia. Methods: From the register of general practitioners there were randomly selected 26 GP's from different regions of Latvia, 60 persons over 18 years of age were selected out of each GP register and tested for anti-HCV with screening test (ELISA). In case of positive result antibodies were confirmed with Western-Blot reaction and person was tested for HCV-RNA (PCR). Results: In total 1591 person was invited by general practitioners for the test and 1442 persons responded (response rate 90.6%). Confirming test (Western-Blot) was positive in 34 participants and out of which HCV RNA test was positive in 25 patients. Conclusions: There are 2.4% of people exposed to hepatitis C virus in Latvia and 1.73% are infected with hepatitis C virus, respectively, 1734 infected persons per 100 thousand individuals.
Genetic variation in the IKK/NF-B pathway and the live fibrosis progression in chronic hepatitis C R. Sho 1 , K. Ishii 1 , R. Ishii 2 , H. Watanabe 2 , K. Sugahara 2 , Y. Nishise 2 , K. Okumoto 2 , T. Saito 2 , S. Kawata 2 , A. Fukao 1 1 Department of Public Health, 2 Department of Gastroenterology, Yamagata University Faculty of Medicine Background/Aims: I B kinase/NF-B (IKK/NF-B) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. We carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding NF B inhibitor kinase alpha and beta (IKBKA and IKBKB; the major components of IKK/NF-B pathway) to the progression of live fibrosis in chronic hepatitis C. Methods: Based upon the common single nucleotide polymorphisms (SNPs; minor allele frequency(MAF) 0.05) and linkage disequilibrium (LD) information derived from the HapMap, we selected 5 and 3 tag SNPs from IKBKA, and IKBKB, respectively, for genotyping. By using melting curve analysis, SNPs were genotyped in 217 chronic hepatitis C patients, including 80 patients with hepatocellular carcinoma. Association between common genetic variations in IKBKA/IKBKA and platelet count (Plt) was tested by both genotype-and haplotype-based approaches. Results: We succeeded in genotyping a total of 8 tag SNPs that efficiently capture common variation across the 32 kb-block of IKBKA and the 25 kb-block of IKBKB. For each of genes tested, 5 haplotypes were found in population studied. All SNPs were in Hardy-Weinberg equilibrium, but no significant association was observed between any single tag SNP or haplotype and decreased Plt in patients analyzed. Conclusions: Our data suggest that it is unlikely that polymorphisms within the IKBKA and IKBKB genes are involved in the progression of live fibrosis in chronic hepatitis C. Further studies on genetic variations in other NF-B-related genes in chronic hepatitis C are needed.
Background: Hepatitis C virus infection is a major burden after liver transplantation. The effective treatment for patients who underwent liver transplantation has not been well established. Management of these patients is the most challenging task. Cyclophilins are essential host factors for HCV replication. We report here the efficacy of divided administration of IFN plus cyclosporine A in the treatment of chronic hepatitis C patients who failed Peg-IFN or IFN combined ribavirin. Patients and method: We prospectively included 59 patients (median age, 63) with genotype 1b and, failures to combination IFN plus ribavirin or combination pegylated IFN plus ribavirin. The present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. The induction therapy comprised intravenous 1 MU IFN every 4 hours for the first 3 days, 1.5 MU IFN every 6 hours for the next 4 days and 2 MU IFN every 8 hours for the following 3 weeks, totaling 168 MU of IFN . The intensified therapy was induction therapy shortened to 2 weeks. The maintenance therapy comprised of pegylated IFN 2b and ribavirin. CsA was given 4 times daily during the induction and the intensified therapies. Ribavirin was given twice daily during the maintenance therapy. Results: The end treatment response and sustained virological response rate of the present study were 73 % (43/59) and 59% (35/59), respectively. The relapse rate was 19 %(8/43). Non-responders was 16 % (3/19). All adverse effects were completely reversible. The treatment protocol was well tolerable. Conclusion: We concluded that our protocol should be effective in failures to the previous combination therapies. Host factor targeting treatment will become a promising treatment option.
Cyclophilin Targeting Treatment is a Promising New Anti-HCV Treatment K. Inoue 1 , T. Watanabe 1 , S. Yoshiba 1 1 
Background: Hepatitis C virus (HCV) is the most common cause of chronic liver disease. However, the efficacy of currently available treatments is limited.
We recently reported the effects of combined interferon-/cyclosporin A treatment. Cyclophilins are associated with HCV replication and bind cyclosporin A. Which cyclophilins are closely associated with HCV replication remains controversial. In this study, several cyclophilins were found to be essential host factors for HCV replication and HCV replication was rescued by overexpression of cyclophilin A in the presence of cyclosporin A. Methods: We evaluated the effect of cyclosporin A and its analogues on the replication of HCV in vitro using several types of HCV replicon. The gene expression of representative cyclophilins and Pin-1 was knocked down using small interfering RNA2 (siRNA) to identify cyclophilins associated with HCV replication. The specificity of the effect of siRNA was confirmed by western blot analysis. The effect of overexpression of cyclophilins on HCV replication in the presence of cycloporin A was also studied. Results: Cyclosporin A and its analogues suppressed HCV replication in a dose dependent manner. Cyclophilin F, cyclophilin LC1 and cyclophilin LC2 as host factors which are closely associated with HCV replication, in addition to the previously reported cyclophilin A. Knockdown of chclophilin B showed little effect on HCV RNA replication. Cyclophiln-dependent HCV replication varied among the three HCV replicon cell-lines used.
Overexpression of cyclophilin A rescued HCV replication in the presence of cyclosporin A. Conclusions: These findings suggest several cyclophilins are essential host factors for HCV RNA replication. Thus potent cyclophilin inhibitors have the potential to be anti-HCV drugs. Background/Aims: Hepatitis C Virus (HCV) genotypes 1-6 have a worldwide distribution. Types 1a and 1b are predominant in Northern Europe and North America, and in Southern and Eastern Europe and Japan, respectively. Type 3 is endemic in south Asia and is variably distributed in different countries. Genotype 4 in Egypt, genotype 5 in Central and South America and genotype 6 is common in China, Japan and South East Asia. In Pakistan 3a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after 24 weeks of therapy. The aim of this study is to find out HCV Genotypes in newly diagnosed chronic hepatitis C patients. Methods: This observational study was conducted in chronic hepatitis C patients. All patients had raised ALT levels for last 06 months, had positive polymerase chain reaction (PCR) for HCV RNA by real time method and liver biopsy was done in all patients under National program for prevention and control of hepatitis during year 2006 -2007. Genotyping was done on Roche Genotyping Kit. Data was analyzed by SPSS 13.0 Results: Out of 164 patients, 85.9% (n=141) were genotype 3a. 6.1% (n=10) were genotype 3b. 3.0% (n=5) were genotype 1a. n= 01 had genotype 1b. 4.2% (n= 7) had mixed genotype (3a,3b/1a,1b,3a,3b). Conclusion: Majority (85.9%) of chronic hepatitis C patients were genotype 3a which is associated with favorable outcome after 24 weeks of interferon and ribavirin therapy and only 3.0% had genotype 1a in this cohort.
S.T. Zhou 1 , Y. Zhao 1 , F.J. Zhang 1
Background/aims: As human immunodeficiency virus (HIV) infected children who are receiving antiretroviral therapy (ART) are living longer in China, comorbidities of hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection should be carefully considered when making management decisions. However, the coinfection rate of either HBV or HCV is unknown in HIV-infected children in China. We evaluated the seroprevalence of HBV and HCV in the China national pediatric ART cohort of HIV-infected patients.
Methods: Patients were selected from HIV infected children medically eligible for ART who were enrolled into the China national pediatric ART cohort since 2004. Interviews, medical assessment, serology for HBsAg, anti-HCV antibody, transaminase levels, and HIV serostatus and CD4 counts at baseline of patients were obtained. Results: 42 of 763 HIV-infected children were HBsAg seropositive (5.50%; 95%CI: 3.88%-7.12%), and 69 of 662 children were anti-HCV antibody seropositive (10.42%; 95%CI: 8.09%-12.75%). Only age was associated with HBV coinfection. Multivariate analysis revealed that children infected with HIV through contaminated blood or transfusion of blood products were 6.35 times more likely to be anti-HCV antibody positive than those infected with HIV through other routes. And children from central China provinces, Henan, Anhui, Shanxi, and Hubei were 2.5 times more likely to be HCV seropositive.
Conclusion: The high seroprevalence of HBV and HCV coinfection in HIV-infected children attending China national pediatric ART cohort calls for routine screening for hepatitis viral coinfection and modification of the management of HIV-infected children in China. Background: BMS-790052 is a first-in class and highly selective hepatitis C virus (HCV) NS5A inhibitor with picomolar in vitro potency against genotypes 1a and 1b. In a SAD study with healthy subjects, BMS-790052 was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. Methods: The objectives of this randomized, double blind, placebo-controlled, SAD study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of BMS-790052 in patients with genotype 1 chronic hepatitis C (CHC). Treatment naïve or experienced patients were randomized to receive 1, 10, or 100 mg of BMS-790052 or placebo. Results: All BMS-790052 single doses were well tolerated and had a safety profile similar to that of placebo. Following oral administration, BMS-790052 was readily absorbed with dose proportional exposures over the studied dose range. The mean terminal half-life of BMS-790052 was approximately 12 hours. Mean decline in HCV RNA 24 hours after a single 1, 10 and 100 mg dose of BMS-790052 was 1.8 log10 (range 0.18 to 3.0 log10), 3.2 log10 (range 2.9 to 4.0 log10) and 3.3 log10 (range 2.7 to 3.6 log10), respectively. The 100 mg dose resulted in a mean decline of 3.6 log10 (range 3.0 to 4.1 log10) 48 hours after dosing, which was maintained at 144 hours. Conclusions: Single doses of up to 100 mg of BMS-790052 were safe and well tolerated in patients chronically infected with HCV genotype 1. BMS-790052 produced a robust decline in HCV RNA and has a pharmacokinetic profile that potentially supports once-daily dosing. Background: The global infection rate of HCV is approximately 3%, and nearly 3.2% in China. Only 42%-46% of patients with genotype 1b can achieve sustained virological response (SVR) after antivirus therapy, nearly half of them experienced treatment failure. The study aimed to determine HCV-1b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. Methods: 19 patients with genotype 1b accepted antiviral treatment of IFN plus Ribavirin for 48 weeks, and long-term follow-up after therapy. 2 patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). Sera were reserved at baseline, 12W, 48W and 4-year after therapy. HCV-RNA was extracted. HCV full-length ORF was amplified by RT-nested-PCR and sequencing. Result: 9 of the 19 patients achieved SVR (47.4%). From sequence alignments of relapser at baseline and 48W, we find that p7, NS5A and NS4A have higher mutation rate both in nucleotide and amino acid level (7.41% and 6.35%, 4.08% and 5.63%, 4.32% and 5.56%, respectively). But there is no significant difference in the alignments of 48W and 4-year after therapy, the mutation rate is lower. Mutation rates of the non-responder among baseline, 12W, 48W and 4-year after therapy are very low. Conclusion: Antivirus effect is correlated with specific HCV sequences in chronic hepatitis C, mutations in HCV non-structure protein p7, NS5A and NS4A have important impacts on treatment outcome in IFN-based therapy. Background: The results of antiviral therapy for hepatitis C (HCV) have improved recently with the use of peg-interferon (peg-IFN)/ribavirin therapy. However, age of patients are concerned because of side effects and safety.
As we known, a few studies have targeted therapy in elder with chronic HCV. Aim: We reviewed the results of interferon based antiviral therapy in the elderly with chronic HCV at our institution. Methods: Patients were defined as elderly if they were 65 years and elder who received therapy for HCV. The prescribed treatment duration, end of treatment response were mention. The data recorded included laboratory tests, adverse events (AE), dose modification, and withdrawal rate of therapy. Results: 304 of chronic HCV patients treated with peg-IFN/ribavirin between Nov 2004 and Feb 2008. 50 patients were older than 65 years old. The mean age of the elder patients was 70.3 ± 4.8 years old. 21 were male and 29 were female. Histological studies showed 18 with cirrhosis. Almost all patients had experienced AE/side effects. The most common abnormalities were anemia and neutropenia. Therapy was discontinued in 22% (11/50). The rate of dose modification was 46% (18/39) patients who received 24 weeks therapy. Transaminases were normalized in 66% (33/50) after 24 weeks treatment and sustained in 66% (29/44) one year later. Conclusion: The elder patients are more at risk of developing AE while on treatment. Most patients should be discontinued or decreased dosage of medication. However, the elder patients with chronic HCV can be treated successfully. Background: In the general population the incidence of interstitial lung disease is estimated to be 0.03% and has also been reported with the use of interferons. The higher reporting rate of IP in Japan has created interest and warrants further investigation. Methods: Using both data from 12 randomized clinical trials (ex-Japan) and the Roche world-wide safety database (ADVENT), the frequency of IP was estimated in patients treated with peginterferon alfa-2a ± ribavirin. IP was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. Results: One case of IP was reported among the 6180 patients included in the clinical trials (0.02%). In the ADVENT database considering the estimated 926,000 patients with cumulative exposure to peginterferon alfa-2a (42,600 in Japan and 883,400 US/ROW) the 228 reported cases of IP represent a rate of 0.02% with a proportional reporting ratio (PRR) of 1.7 (p<0.0001). Of these cases, 140 were reported in Japan (PRR 2.9; p<0.0001), 34 in the USA (PRR 0.7; p=0.06) and 54 ROW (PRR 1.2; p=0.11) representing reporting rates of 0.3% in Japan and 0.01% in the USA and ROW. Japanese patients with reported IP were older (66 versus 50-55 years) and were more likely to have been treated with peginterferon alfa-2a monotherapy (81% versus 21-39%). Furthermore, the yearly incidence rate has remained unchanged. Conclusions: The apparently higher rate of IP reported in Japan may result from differences in patient demography, diagnostic criteria and treatment patterns. The overall incidence of IP remains low. Background: Hepatitis C virus (HCV) infection carries a significant risk for development of insulin resistance (IR) and/or diabetes (DM). Recently, retinol-binding protein 4 (RBP4) has been reported as a protein contributing to IR. This study aimed to assess the different expression of serum RBP4 between chronic HCV infection (CHC) patients and non-CHC controls. Methods: Serum RBP4 was measured in 105 treatment-naïve CHC patients and its correlation with the homeostasis model assessment of insulin resistance index (HOMA-IR), liver histology, virology and metabolic factors was investigated. Patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. Another 100 sex-and age-matched non-CHC adults served as the controls. Results: The mean RBP4 level of controls tended to be higher than that of CHC patients (32.46 ± 20.92 vs 25.48 ± 13.13 g/mL, P=0.07). The mean RBP4 level of 34 IGT control-group subjects was 43.7 ± 24.0 g/mL, which was significantly higher than that of 34 NGT (24.4 ± 13.0 g/mL, P<0.001) and 32 DM controls (29.0 ± 19.5 g/mL, P<0.01). In contrast, the mean RBP4 level (22.2 ± 10.3 g/mL) of 32 DM/CHC patients was not significantly different from that of NGT/CHC (24.9 ± 10.5 g/mL, n=28) and IGT /CHC (28.1 ± 15.8 g/mL, n=45) patients. Amongst CHC patients, there was a significant decreasing linear trend of RBP4 dependent of both histological grading and staging progression, whilst a significant increment of HOMA-IR was found. Conclusion: Serum RBP4 is dysregulated in CHC patients. Introduction: Sustained Viral Response (SVR) in Hepatitis C treatment with Interferon Alfa and Ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. Pregabalin, an orally effective GABAsergic drug is not metabolized via Cytochrome P450 and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.This study evaluates the addition of Pregablin to standard agents in achieving SVR by reducing side events. Methods: Thirty patients with chronic hepatitis C {mean age -46 years, male: female -2:1,Genotype(G)1(n=29), G6 (n=1), Fibrotic score F2-3 (n=24) and F4 (n=6), mean BMI > 29 Kg/m 2 , initial viral load > 800,000 IU/ml} were randomized to Pregablin100mg (n=15) or Duloxetine 20mg (n=15) both orally daily with Interferon Alfa 2a 180mcg sq once a week and Ribavirin 1200mg daily for 48 weeks. Myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. All were tested for rapid viral response, early viral response and end treatment viral response and SVR.
Results: At the end of 48 weeks, in the Pregablin arm, 14(97.3%) completed the therapy without interruption, one stopped due to excessive somnolence. Duloxetine arm -10(66.6%) completed with interruptions, 4(22.2%) withdrew from the trial due to side events, one left the country. 9(62.2%) achieved SVR in pregablin arm and 5(33.3%) with Duloxetine. Conclusions: Pregablin may be considered with IFN and RBV for better adherence and compliance in achieving SVR in treatment of Chronic Hepatitis C. Larger randomized studies are needed to confirm the findings. In this study we extended this treatment approach to on treatment nonresponders (defined as having detectable HCV-RNA after at least 24 weeks of SOC). Methods: So far, 5 pts. HCV-RNA pos. after 24 weeks of SOC (3 male, 2 female, genotype 1:4; genotype 3a:1, 3 with cirrhosis) participated in this protocol; 4 were treatment naïve pts, 1 relapser to two previous therapies (24 and 48 weeks). 20 mg/kg/d SIL was given for 14 days, SOC was continued. HCV-RNA was quantified by TaqMan (Roche Diagnostics, USA) at monthly intervals on standard treatment and weekly after starting SIL. Results: All patients received at least 24 weeks of SOC, at week 24 3 had a log drop < 3, two patients had detectable but unquantifiable HCV-RNA (< 15 IU/mL). After 14 days of SIL all 5 had undetectable HCV-RNA, in one HCV-RNA increased to 100 IU/ml and recived after a second course of SIL. .All 5 patients are still on SOC and are HCV-RNA negative. Conclusion: SIL iv. Is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy.
Poster Exhibition -Imaging Modalities Poster Session, Hall 5B Background: Levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. Our institution performs Sonazoid-enhanced ultrasonography using a Toshiba APLIO80 that is set to a PS low images, as generally recommended. The resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. These staining images were then compared to Advanced Dynamic Flow (ADF) images of a hyperechogenic nodule recorded using Levovist-enhanced ultrasonography. Methods: The subjects were five nodules who had undergone Sonazoid-enhanced ultrasonography. Two patients had experienced a recurrence of HCC after TACE, while three patients had a hyperechogenic nodule of HCC that had never been treated. One patient with HCC after TACE was imaged at a PS low. The second patient with HCC after TACE and the three patients with HCC showing a high echoic nodule, were imaged using ADF. Results: In the patients with HCC after TACE, the remaining tumor was difficult to observe in both the vascular phase and the Kupffer phase taken at a PS low. In the other patients, however, images taken using ADF clearly showed the residual tumor. Also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of HCC, the HCC was more easily detectable in the ADF images than in those taken at a PS low. Conclusion: Hyperechogenic perfused nodules are easier to identify in images taken using ADF than in images taken using PS low.
Y. Komorizono 1 , T. Shibatou 1 , K. Sako 1 1 Nanpuh Hospital
Background: This study aimed to evaluate the usefulness of Sonazoid enhanced radiofrequency ablation under real-time virtual sonography (RVS) guidance in a series of patients with hepatocellular carcinoma (HCC). Method: Twenty-five patients with a solitary HCC tumor measuring < = 2.5 cm in greatest dimension were enrolled in this study. Eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. All patients were easy to scan by multiple detector CT (MDCT), but not by conventional ultrasound ( Conclusions: The combination of the RVS system with Sonazoid-enhanced US appears to have a high potential for use on patients that are difficult-to-scan by US examinations for percutaneous radiofrequency ablation. Background & Aims: Contrast enhanced ultrasonography (CEUS) with Sonazoid can be expected to be useful not only for detection of tumor but also for US guided ablation therapy because Kupffer imaging lasts for long time. The aim of this study is to investigate the usefulness of Sonazoid in RFA for HCC. Material & Methods: A total of 716 HCC nodules in 316 patients admitted to receive RFA were studied. The detection ability of HCC was compared between CEUS and conventional US using dynamic CT as reference standard. The effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of RFA in patient treated with CEUS assistance and that in historical controls matched for tumor and background conditions. Results: The detection rate was 83.5% in conventional US and 93.2% in CEUS (P=0.04). Sixty-nine nodules in 52 patients were not detected by conventional US and detected after injection of Sonazoid. The mean increase in detected tumor number with contrast enhanced US were well correlated with serum albumin level (P=0.016). CEUS was not superior to conventional US in patients with low albumin level. The mean number of session was 1.33±0.45 as compared to 1.49±0.76 in the historical controls (P=0.0019). Conclusions: CEUS with Sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. The decrease in the mean number of sessions compared to historical controls suggested that Sonazoid is an excellent supportive agent in RFA treatment of HCC. 
Direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. In the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (CFD) software in patients with primary liver cancers. Methods: Patients with fibrotic or non-fibrotic livers were analyzed. According to preoperative MD-CT, mesh models of portal branches were constructed. CFD software (Fluent 6.2, Fluent Inc.) was employed for flow simulation. On the Fluent 6.2, changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. The simulation was also performed 14 days after the operation using DICOM data obtained at that time. Results: Relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. The sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day 14 in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day 14. These results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. Discussion: Computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. Aim: To determine if ROI analysis can characterize washout in Hepatocellular Carcinoma (HCC) better than visual analysis. Methods: Surgically proven HCCs from a single institution were studied. The patients' gender, age, date of scan, date of surgery were recorded. 94 patients with pre-operative triphasic (n=67) and quadriphasic CT scans (n=27) were included. A representative section containing the lesion was selected for each case. The HU change between the precontrast and arterial (HUabsolute hypervascularity) and the HU change between the peak attenuation and late portovenous phases (HUabsolute washout) were recorded. Cases were deemed positive if the HU change was more than the standard deviation (11 HU). This was compared against visual analysis to determine if our method would increase sensitivity of CT for HCC. Results: The mean patient age was 63.7 years (range 19 to 84 years); there were 77 males and 17 females. The mean duration between surgery and the scan was 39.5 days (range 1 to 348 days). Peak enhancement was seen in the early portal venous phase in 76.6% cases. The mean HUabsolute washout was 23.8 HU (range -5 -54). ROI analysis detected 86/94 cases (91.5%). This was 12.8% more than visual assessment, which detected 74/94 cases. This was statistically significant (p=0.014). Conclusion: Visual assessment of lesion density is subjective. Quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. Background: Abdominal ultrasonogram(USG) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(HCC). But it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. We investigated the characteristics of HCCs that was missed in the USG but was detected in the CT. Methods: Total 122 patients who were diagnosed with HCC between December, 2003 and February, 2008 , were enrolled and analysed retrospectively. All patients were performed with a USG prior to a spiral CT. The period between USG and spiral CT was limited within 1 month. We investigated age, gender, cause(HBV, HCV, alcohol), the size of HCC(the length of long diameter), stage(modified UICC), Child-Pugh Grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of HCC, regenerative nodules(RNs), and the tumor location at segement 8 as the possible related factors.
Results: The mean period between USG and spiral CT was 3.04±5.70 days. The diagnostic accuracy rate to HCC was 84.4%(103/122). There was no interobserver variation. In analysis of associated factors, there was no statistical significance in age, gender, cause(HBV, HCV, alcohol), stage(modified UICC), Child-Pugh Grade, cirrhosis, portal vein thrombosis, diffuse type of HCC, regenerative nodules(RNs) (P > 0.05). There was statistical correlation in the tumor size less than 2 cm, the solitary tumor and location at segement 8. (P > 0.05). Conclusion: Tumor size less than 2 cm, solitary lesion and location at segment 8 are significant factors to miss HCCs in USG diagnosis.
S. Somani 1 , A. Somani 2 , A. Jain 3 , V. Dixit 3 1 Suvidha, 2 Navjeevan hospital, 3 
Background: Histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. Earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. Our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. Methods: It was a retrospective single center study done between June 2003 and May 2007. Trucut Liver biopsy needle was used in all patients. Demographic, clinical and histological characteristics between the two groups were evaluated. Insufficient biopsy was defined as a sample with less than 6 portal spaces. We reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. Results: Out of 256 liver biopsies done in this period after excluding 16 patients we included 240 patients, 144 in Group A(60%, blind approach) and 96 in Group B (40%, sonographic guided approach). Mean age was 38±12.4 years and male: female ratio was 1.6:1. Biopsy was sufficient in 76% in Group A and 94% in Group B (p < 0.05). Minor complications occurred in 58% in Group A and 49% in Group B which was not significant. Major complications occurred in 2.8 % in Group A and 1.2% in Group B which was statistically significant. Mortality was 1.2% in Group A and 0.4% in Group B which was statistically significant. Conclusion: Our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality.
Poster Exhibition -Liver Fibrosis Poster Session, Hall 5B Background/Aims: HMG-CoA reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. Therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (Ato) or rosuvastatin (Ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. Methods: Liver cirrhosis was induced by thioacetamide (TAA, 200 mg/kg, I.P.) twice a week, for 12 weeks. Acute damage was induced by two consecutive TAA injections (200 mg/kg in a 24 h interval). Rats were treated concurrently with TAA only or TAA and either Ato or Ros daily by nasogastric gavage. Another group was treated with TAA+pentoxifyline (PTX), an agent with known antifibrotic effect through a different mechanism and served as positive control. Results: Presented in the Conclusions: The lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by TAA. Purpose: The development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. The present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. Strain Rate Imaging is a new method based on Tissue Doppler Imaging (TDI). The usefulness of Strain Rate Imaging in assessing the degree of hepatic fibrosis was evaluated. This time, it mede comparative study with Fibroscan in 11 cases. Methods: Strain Rate Imaging was performed using a diagnostic ultrasound system (Aplio TM , Toshiba Medical Systems Corporation, Tochigi, Japan) in a total of 47 subjects:25 in the chronic hepatitis group, 12 in the cirrhosis group, and 10 in the normal control group. TDI-Q, the Tissue Doppler analysis software installed in the Aplio system, was used for analysis. Measurement was performed five times from the epigastrium, with the ROI size set to 10 mm and the derivative pitch to 3 mm.
Results: (i): Both scores were largely reproducible among the different laboratories. However, compared to the histological findings, the error ratio was 77% for all results calculated by Fibrotest and Actitest. (ii): Calculated scores varied among F2 (9%), F3 (31%), F3-F4 (6%), and F4 (54%) (Fibrotest), as well as A1/A2 (48%), A2 (9%), A2-A3 (5%), and A3 (38%) (Actitest).
Results: The mean strain value was 0.156 in the chronic hepatitis group, 0.055 in the cirrhosis group, and 0.26 in the normal control group.The correlation was not thought to be Fibroscan. Conclusion: The results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. It is expected that the accuracy of the Strain Rate Imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination.
Conclusion: Despite reproducibility of Fibro-and Actitest results among the six laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. Furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. Thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. Background: Current knowledge attributes connective tissue growth factor (CTGF/CCN2) a crucial role in enhancing TGF-actions during hepatic fibrogenesis. Recently, we demonstrated that caffeine leads to an upregulation of PPAR in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of 15-deoxy-12,14 -prostaglandin J2 (15-d-PGJ2) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se, its physiological ligand 15-d-PGJ2 is required for exerting its inhibitory effect on CTGF synthesis. Aim and Methods: This study compares serum concentrations of 15-d-PGJ2 in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non-liver disease sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (n= 43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPAR inducing (i.e. CTGF inhibitory) drugs such as caffeine.
Results: Presented data show that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2 ± 5.9 µg/L) display impressingly higher serum concentrations of 15-d-PGJ2 than healthy probands (mean 2.3 ± 1.0) and Caucasian patients with non-liver disease (mean 2.7 ± 1.4 µg/L). Similar results are found in Chinese patients with fully developed HCC (mean 1.3 ± 0.7 µg/L) compared to Chinese healthy controls (mean 0.4 ± 0.2 µg/L). We identified the predictors of tumor recurrence using Cox-regression model.
Introduction: Non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. Aims/Methods: (i) Haptoglobin, ALT, GGT, alpha 2-macroglobulin, apolipoprotein A1 and bilirubin in sera of 4 patients with histological proven fibrosis (F1-F4, A1-A3) were determined in 6 different quality-controlled laboratories. Interlaboratory variations of the calculated Fibrotest Score for staging and Actitest Score for grading (both BioPredictive TM ), and their error ratios compared to biopsy results were calculated. (ii) The variability of obtained Fibrotest/Actitest Scores depending on 64 differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the German Association of Laboratory Medicine was determined and the frequency distribution of the results calculated.
Results: A total of 51 patients (mean age, 54.3±9.8 years; male, 78.4%) were included. Median follow-up duration was 14.2 months (range, 5.6-37.3) and 20 patients (39.2%) experienced local tumor recurrence during the observational period. Multivariable analyses showed that low P2/MS level (relative risk, 0.98; 95% confidence interval [CI], 0.96-0.99; P=0.035) and serum alpha-fetoprotein level >100 ng/mL (relative risk, 5.41; 95% CI, 1.59-18.18; P=0.007) were independent risk factors for tumor recurrence. Patients with P2/MS level <45.0 revealed 3.79-fold (95% CI, 1.05-13.76; P=0.042) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with P2/MS level >45.0. However, tumor size, Child-Pugh score, and hepatitis B virus DNA level failed to significantly affect the time-to-recurrence. Conclusion: Our study suggests that lower P2/MS value, which means more severe liver fibrosis, is an independent predictor for HCC recurrence after RFA. Background/Aims: Despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. The aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. Methods: The prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the Institute of Gastroenterology and Hepatology, CNUH between March 2008 and September 2008. Osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy X-ray absorptiometry (DEXA) at lumbar spine and femoral head. The variables taken into consideration were: sex, body mass index (BMI), presence of cholestasis, severity and duration of liver disease. Results: Total 45 patients (male 32 and female 13, respectively) were estimated for association of liver disease and osteoporosis. Of these, 39 patients were estimated for bone density of lumbar spine and neck of femur by dual X-ray absorptiometry (DEXA). Morning blood samples were taken for hormonal and biochemical analysis from all patients. Among 39 patients, 25 patients (64%) were found to have osteopenia or osteoporosis. There was no statistically significant correlation between age, BMI, severity and duration of liver disease, PTH, Vitamin D, ALP and IGF-1. Conclusion: There is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. Although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. Background: To build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis B. Methods: Liver biopsy and routine laboratory tests were performed in 391 patients with chronic hepatitis B. Using Multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by ROC curve. Results: After Spearman analysis, factors such as age, platelet count(PLT), international rate(INR), total bilirubin(TBIL), albumin(ALB), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), total bile acid(TBA) and cholinesterase(CHE) were found to be correlated with liver fibrosis P 0.01 . Three models (s 2, s 3, s=4, respectively) were built by PLT, INR, ALB, GGT and CHE, which were independent predictors after multiple Logistic regression analysis.Finally, Fibrosis Score (FS) was calculated to predict different liver fibrosis stages. ROC curve analysis revealed that the AUC of FS was 0.784 in model1 (s 2), 0.768 in model2 (s 3) and 0.806 in model3(s=4) fig1 .The cut-off FS in model1 was at 7.09 with 67.4% sensitive, 79.3% specificity and the accuracy was 71.1%. The cut-off FS in model2 was at 5.67 with 75.0% sensitive, 67.7% specificity and the accuracy was 72.9%. The cut-off FS in model3 was at 3.65 with 71.4% sensitive, 78.5% specificity and the accuracy was 73.7%. Conclusions: The predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis B. Background: To investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. Methods: Normal male New Zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (CCl4) group and a complex group. Pathologic changes in ascending aortas and livers were observed. The levels of serum alanine aminotransferase ALT , lipid, C-reactive protein (CRP) were also determined. Results: Significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. Compared with the control group, serum ALT and lipid levels in CCl4 group were increased significantly (P<0.05), but no difference of arterial intima-media thickness (IMT) and I/M ratio between these two groups. The levels of serum ALT, lipid, CRP and IMT in two high fat diet groups were significantly increased compared with the control group (P<0.05). The level of serum ALT in the complex group was significant higer than that in the high fat diet group, but the I/M ratio was just opposite (all P<0.05), and there was no difference of IMT between the two groups. Conclusions Rabbits treated with CCl4 can elevate serum lipid levels, but can not induce atherosclerosis. Though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition.
Higher Values of Liver Stiffness in Males with Mild Chronic Hepatitis C C. Stern 1 , A.C. Cardoso 1 , R. Moucari 1 , A.D. Pumpo 1 , N. Giuily 1 , P. Bedossa 1 , P. Marcellin 1 1 Hopital Beaujon Background/aim: Liver stiffness (LS) measured by FibroScan (EchoSens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. We evaluated the impact of factors on LS results in mild chronic hepatitis C (CHC). Methods: CHC patients with METAVIR Fibrosis stage 1 at liver biopsy and a reliable LS exam were eligible. All patients had no prior antiviral treatment.
The LS values were compared to clinical and biochemical data. Results: 93 patients were included with the following characteristics: mean age 50 11, male gender (46%), mean BMI 23 2.7, median LS 5.8 kPa (3.2-21.8), diabetes (7%), genotype 1 (61%), METAVIR activity A1 (86%), A2 (10%), steatosis at biopsy 30% (92%), mean glucose 4.9 1, abnormal ALT (78%), abnormal GGT (47%), HOMA (2 1.2). The LS values were associated wtih male gender (median 6.1 in males vs 5.2 in females) (p=0.04), BMI (p=0.03), ALT (p=0.006), GGT (p=0.02) and glucose levels (p=0.04). No association was found between LS and activity stage (p=0.34) or steatosis (p=0.14). In the linear regression, the only factor independently associated with higher LS was gender (p=0.038). In men, higher LS was related to levels of ALT (p=0.005), but not to necro-inflammation grade (p=0.4). In women, LS was not associated with ALT levels, but with BMI (p=0.045) and GGT levels (p=0.035). Conclusion: In patients with mild CHC, liver stiffness values are higher in males. These results suggest that different cut-off for fibrosis stage 1 should be proposed according to gender.
Aims: To investigate the effects of Shuanghu Qinggan Granule(SQG) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats.
Methods: 60 SD rats were divided into 6 groups, normal control groupAmodel groupB, SQG largeC1, middleC2small dose groupsC3 and silymarin positive contrast groupD. The rats of BC1C2C3D were injected with carbon tetrachloride for 8 weeks. The rats of C1C2C3 were then administered with SQG for 8 weeks. The rats of D were then administered with silymarin for 8 weeks. Results The liver structure of rats of B was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. The hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of C1C2C3D reduced significantly. There was no fiber hyperplasia in liver tissues of rats of C1C2C3D. Blood serum HA CP P levels in rats of B were significantly higher than those in AC1C2C3D. Conclusion: SQG has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of SQG was, the more effective the results would be. Conclusions: None of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in HBeAg positive patients. Two markers out of 7 sophisticated biomarkers provide additional diagnostic information in HBeAg negative patients. Before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised.
Objective: To evaluate the efficiency and safety of "TINMAX" HB-3 Herbal Compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis B. Methods: A double-blind randomized method was employed. 60 patients of hepatofibrosis or cirrhosis post hepatitis B were separated into study group ("TINMAX" HB-3 group) and control group (natural vitamin group) by randomized method. The course was 52 weeks. Patients visited once every 12 weeks and the last visit at 12 weeks after the cessation of treatment. Part of patients had liver biopsy before and after treatment. Before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type III procollagen and collagen IV) were tested, and ultrasound evaluation was performed. Results: 60 patients enrolled in the evaluation. 58 patients completed the evaluation according to the protocol. 20 patients had liver biopsy twice, 10 from the study group and 10 from the other one. At the end of therapy, the total effective rate of hepatofibrosis in histopathology is 74.13% in the study group, much higher than that of 21.95% in the control group (P<0.05). The total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was 70.10%, much higher than that of 30.24% in the control group (P<0.05). The total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was 76.08%, much higher than that of 19.41% in the control group (P<0.05). The drugs of adverse event had not happened in both groups. Conclusion: "TINMAX" HB-3 herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis B.
W.H. Sha 1 , Xiaohui Zeng 1 , Yuyuan Li 1 1 GI Department, First municipal hospital of Guangzhou, Guangzhou Aim: To investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. Methods: Competitive radioimmunoassay was used to determine the serum level of collagen type ( C), laminin (LN) and hyaluronic acid(HA) in 193 patients with different severity degree of chronic liver diseases, and in 30 healthy subjects. Results: The serum levels of C, LN, and HA in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. Of which the highest of C, LN, and HA were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of HA is highlight. The combination detection of serum C, LN, and HA is more valuable than single index. Conclusion: Joint detection of serum C , LN, and HA is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. 
Aims: To investigate the mechanism of Fuzheng Huayu decoction (FZHY) on hepatic stellate cells (HSCs) activation relating to TGF-1 signal transduction pathway. Methods: HSCs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. At day 4 after isolation, cells were stimulated with 100pM TGF-1 for 24h, then incubated with 10% FZHY pharmacological serum or 10 M T R -I inhibitor (SB-431542) for 24h. Protein expression of -SMA, Smad3 was assayed by immunofluorescent stain; Total protein expression of -SMA, T R -I, Smad2/3 and nuclear expression of Smad3 was analyzed by Western blotting. Results: FZHY pharmacological serum significantly decreased expression of -SMA, T R -I, and inhibited Smad3 nuclear expression and translocation in TGF-1 stimulated HSCs. Conclusions: Fuzheng Huayu decoction can prevent HSCs activation through TGF-1 signaling transduction pathway in HSCs, which may be the important molecular pharmacological mechanism of Fuzheng Huayu decoction action against liver fibrosis. Background: Fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. Aim of this study is 1) to study the involvement of endoplasmic reticulum stress (ER stress) in the occurrence of seatohepatitis and 2) to obtain the evidence of resolution of fibrosis by changing the diet. Methods: Non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (MCDD) for 10 weeks. Methionine-choline-control diet (MCCD) instead of MCDD was given for the last 2 weeks in an experimental group. Fibrosis and inflammation was determined by several tissue stainings. Gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time PCR. Expression of caspase-12, caspase-7, and glucose-regulated protein 78 was evaluated to clarify the presence of ER stress Aim against liver fibrosis relating to hypoxia and angiogenesis regulation. Methods: The rats were divided into normal, model, SA-B and Perin control group. Rats in SA-B and Perindopril group were administrated with SA-B and Perindopril respectively. Liver fibrosis was induced by ip dimethylnitrosamine (DMN) for 4w. Fibrosis degree was observed by Sirius red staining. Col-I protein expression was analyzed by Western blot; Col-I , VCAM-1, ICAM-1, HIF-1 and vWF expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography.
Result: Compared to normal group, Col-I, HIF-1 , ICAM-1, Results: 1) Changing the diet from MCDD to MCCD triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. 2) Immunohistochemistry, immunoblotting, and RT-PCR analysis all indicated the occurrence of ER stress in steatohepatitis while it recovered immediately after changing the diet from MCCD to MCDD.
vWF protein expression and gelatinase activity in liver tissue were increased obviously in model group, while SA-B and perindopril treatment significantly decreased these protein expressions and gelatinase activity.
Conclusions: This simple experiment clearly shows that the changing diet from steatohepatitis-causing MCDD to MCCD triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. ER stress is involved in the process. Background: Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. Under injury conditions, hepatic stellate cells (HSCs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens I, III, and IV. Gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in Asia. Gypenosides are the major saponins derived from G.
pentaphyllum. In previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by CCl4, and anti-proliferative effect in rat isolated HSCs. Methods: In cultured HSCs model, we detected type1 procollagen protein and mRNA by Western blot and RT-PCR. Result: we found that G. pentaphyllum inhibited type1 procollagen protein expression in 66% at 48 hours. Furthermore, G. pentaphyllum also inhibited type1 procollagen 1 and 2 mRNA expression in 39% and 11% respectively. In addition to transcriptional inhibition, we found that G. pentaphyllum also enhanced the degradation rate of type1 procollagen protein. Base on the effect of enhancing protein degradation, we used some protease inhibitors like CA-074 Me, z-FA-fmk, AEBSF, TPCK and TLCK to identify the potential target of G. pentaphyllum. On the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of G. pentaphyllum. Conclusion: G. pentaphyllum reduced type1 procollagen protein by inhibiting transcription and enhancing protein degradation. Aim: Excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. The present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. Methods: Wistar strain albino rats were selected for this study. The rats were divided into 4 groups: (i) control, (ii) ginger treated (200mg/kg b.w. orally, 30 days) (iii) diabetic (50 mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. The lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. Results: Ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. Diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. Further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. Conclusion: We summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. This results also reveal that ginger possess hepatoprotective properties in diabetic cases.
Anti-Fibrotic Action M. Naime 1 , S. Ali 1 1 Hamdard University
Rhizomes of Valeriana jatamansi (family, Valerianaceae) have long been used in Indian subcontinent by the traditional healers for the treatment of various diseases. This study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. Crude extract (50% ethanolic) at a dose level of 800 mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. Administration of the extract for 9 weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. Morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. DPPH assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. Further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. The study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ 3 H]-thymidine incorporation by hepatic DNA in extract treated rats. Results suggest that V. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis.
with Chronic Hepatitis C C. Wongjitrat 1 , S. Chainuvati 1 , A. Manuyakorn 1 , S. Aroonparkmongkol 2 , T. Tanwandee 1 1 Mahidol University, 2 
Background: Leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. Leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. In human chronically infected by HCV, the role of leptin-associated fibrosis of the liver is still unclear. There is no data in Thai patients chronically infected by HCV regarding leptin level and its correlation with hepatic histology and fibrosis.The purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in Thai patients chronically infected by HCV. Methods: Sixty-six patients (31 men, 35 women) with chronic HCV infection and liver biopsy was done within 3 months were enrolled. Fasting blood samples were obtained and serum leptin levels were measured by ELISA. BMI, blood sugar, liver function test, lipid profile, HCV RNA viral load and HCV genotype were also measured and related to histological findings. Results: Mean serum leptin levels were significantly higher in women than in male. There was a significantly correlation between serum leptin and BMI (r = 0.469, P < 0.001). Leptin levels were not associated with hepatic fibrosis (r = 0.166, P = 0.183) and necroinflammation (r = 0.203, P = 0.102). Steatosis was significantly associated with severe necroinflammation (r = 0.261, P = 0.034), but not fibrosis (r = 0.22, P = 0.076). Conclusions: These findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in Thai patients chronically infected with HCV. Background and aim: Liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. Even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. The aim of the present study is to assess insulin resistance, cytokines and CRP levels in patients with liver cirrhosis and control subjects. In additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, Child-Pugh classification, spleen size, TNF-?, IL-1?, IL-2RES, IL-6, IL-8, IL-10, CRP and Hs-CRP. Material and method: A total of 79 patients with cirrhosis of different etilogy (49 male, 30 female) were included into the study. As controls, 50 (23 male and 27 female) subjects were taken. The two groups were compared with each other in terms of glucose, insulin, C-peptid, HOMA-IR, TNF-?, IL-1?, IL-2RES, IL-6, IL-8, IL-10, CRP and Hs-CRP levels. In the second part of our study, the liver cirrhosis group was divided into two subgroups: Patients with HOMA-IR value >2.7 as insulin resistance positive, and those with HOMA-IR value >2.7 as insulin resistance negative. These two groups, i.e. , HOMA-IR positive and HOMA-IR negative, were compared in terms of age, sex, etiology, Child-Pugh classification, spleen size, TNF-?, IL-1?, IL-2RES, IL-6, IL-8, IL-10, CRP and Hs-CRP levels. Results: In liver cirrhosis group, glucose, insulin, C-peptid, HOMA-IR, TNF-?, IL-2RES, IL-6, CRP and Hs-CRP levels were determined to be significantly higher than controls. Between patients with HOMA-IR positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, Child-Pugh classification, spleen size, TNF-?, IL-1?, IL-2RES, IL-6, IL-10, CRP and Hs-CRP levels, but IL-8 level was seen to be significantly low in patient HOMA-IR positive. Conclusion: In patients with liver cirrhosis, the levels of glucose, insulin, C-peptid, HOMA-IR, TNF-?, IL-2RES, IL-6, CRP and Hs-CRP increase with respect to normal population. Determination of increased HOMA-IR level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. In the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, Child-Pugh classification, spleen size, TNF-?, IL-1?, IL-2RES, IL-6, IL-10, CRP and Hs-CRP levels. The determination of statistically lower level of IL-8 in patients with HOMA-IR positive with respect to those with HOMA-IR negative does not indicate similarity with the studies carried out earlier. ) in patients (48.08%) than in controls(14%)In group I HLA-B5 significantly increased in patients (60%)as compared to controls (14%) . in group II HLA -B5 significantly higher in patients (45.46%)than controls (14%) also HLA-AW19 significantly higher (40.91%) in patients than controls (12.67%).in group III HLA-AW19 significantly increased in patients (46.67%) compared to controls.No significant association between HLA antigens and cases with HBV or HCV infection. Conclusion: The significantly high association of HLA-AW19 and HLA-B5 in patients with hepatic Schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal Schisto . Antigens predispose to liver affection.Individuals possessing HLA-AW19 appear to be more prone to severeform of liver disease 
Background: ATP8B1 mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the Mainland China before. The aim of this study was to elucidate the role of ATP8B1 mutation in Mainland Chinese patients with progressive intrahepatic cholostasis and low GGT. Methods: 24 children who presented with progressive intrahepatic cholostasis and low GGT were admitted in a tertiary pediatric hospital in eastern China. ABCB11 gene was analyzed firstly to exclude BSEP deficiency. Afterwards, all the encoding exons and their flanking areas of ATP8B1 gene were sequenced in the remaining 19 patients in whom only one or no mutations of ABCB11 were found. Results: 10 mutations of ATP8B1 gene were found in 9 patients. I694N had been reported in Taiwanese patients with PFIC1, and the others were novel. P209T and IVS6+5T G were linkage and found in 4 of 9 patients, including 2 homozygote and 2 heterozygote. Liver biopsy had been performed in 6 patients with ATP8B1 mutations and 5 with ABCB11 PE408 mutations. Variety portal fibrosis was showed in 2 patients with ATP8B1 mutations and 4 patients with ABCB11 mutations. Giant cell transformation was detected in one patient with ATP8B1 mutations and 4 patients with ABCB11 mutations. 1 Laboratory of Exercise Biochemistry, Taipei Physical Education College, Jhongcheng Rd., No. 101, Sec.2, Taipei City-11153, Taiwan, ROC Background/aim: Generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. This study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. 
Methods: Zucker rats lean (200±10gms) and obese (450±15gms) were divided into control and acute hypoxia groups. The acute hypoxia treatment was performed in a hypoxic chamber at 14% oxygen consumption. Objectives: To compare the diagnostic value of morning urine copper to zinc (Copper/Zinc) ratio and 24 hour urinary copper excretion in Wilson's disease (WD) children.
Results: In the results, acute hypoxia caused a significant (p<0.05) decrease in major antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione (GSH) content in lean groups when compare to their controls. The decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. However, this decrease was not significant in case of CAT, GSH-Px activities and GSH content. The MDA levels (lipid peroxidation marker) were higher in obese rats compare to lean rats.
Methods: Morning urine and 24 hour urine were collected from 96 patients over three years age who were hospitalized in a tertiary pediatric liver service. Each patient was re-evaluated according to WD Scoring system, and was assigned to one of the three groups: WD, suspecting WD, and non-WD. 24, 6, and 64 cases were assigned to WD, suspecting WD, and non-WD respectively. Urine copper and zinc concentration was determined simultaneously by using Inductively Coupled Plasma Mass Spectrometry.
Conclusions: The higher hepatic MDA values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. From this study it is concluded that decrease of antioxidant enzymes (except GR) with acute hypoxia treatment were more in lean group compared to with that of obese group.
Results: The morning urine copper/zinc ratio and 24hr urinary copper excretion correlated well (r=0.758, P < 0.001). The median of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, 24h copper excretion, and 24h zinc urinary excretion were 0.370, 0.394, 87.1 and 398.7 in WD group, and 0.051, 0.061, 24.2 and 358.9 in the non-WD group respectively. The differences of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, and 24h copper excretion were significant (Z-value -6.502, -6.020 and -6.208 respectively, all P values < 0.000 CHD1L is a recently discovered oncogene localized at 1q21, one of the most frequently amplified chromosomal regions in HCC. Herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of CHD1L is associated with its interaction with Nur77, a critical member of a p53-independent apoptotic pathway. As the first cellular protein identified to bind Nur77, CHD1L inhibits the nucleus-to-mitochondria translocation of Nur77, and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( Figure 1 ). Further study found that C-terminal Macro domain of CHD1L is responsible for the interaction with Nur77, and a CHD1L mutant lacking residues 600-897 failed to interact with Nur77 and prevent Nur77-mediated apoptosis. We also find that CHD1L confers cellular chemoresistance to drugs that induce apoptosis via the Nur77-mediated pathway, which may lead to the identification of new therapeutic targets for HCC treatment.
Background/aim: Accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. The current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. Methods: Sprague-Dawley rats of young (3 months) and old (12 months) were divided into four groups; young control (n=5), young exercise (n=5), old control (n=5) and old exercise (n=5). 90 minutes of swimming exercise was given to the exercise group for a period of two weeks. Results: The estimated antioxidant enzyme activities including, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were decreased with age and significantly (p<0.05) increased with exercise training. However, elevated protein carbonyls and MDA levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. The significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (SDH) activity was observed with response to swim training in old rats. Conclusions: This data implied that swim exercise training could revert the oxidative damages in liver. This was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. To sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats.
Results: PEG10 was expressed in L02/PEG10 (Fig.1) . PEG10 accelerated the growth of L02. After treatment with 400mM H2O2 for 24 h, the inhibitory rate of L02/PEG10 cells was 32.5%; the chromosomal condensation and ladder-like DNA fragmentation were not observed (Fig.2) .
Methods: HepG2 or HepG2.2.15 were co-cultured with Jurkat cells, with blocking test by adding anti-PD-1 antibody. The PD-1 expression was detected by flow cytometry (FCM); Cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (ELISA); Cytotoxic test of T cells were measured by methyl thiazolyl tetrazolium (MTT).
Conclusions: Over-expression of PEG10 can significantly promot L02 proliferation and ameliorate apoptosis-inducing effects of H2O2 on L02. Results: The PD-1 expression on Jurkat cells was induced by Hepatoma cells, the expression rate were 16.17±6.5% (by HepG2) and 17.43±6.8% (by HepG2 2.2.15), respectively. The cytokines IL-2 level (202.9±53.0 pg/ml), INF-level (88.6±4.6 pg/ml) and IL-10 level (63.7±13.4 pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (IL-2, 102.9±53 pg/ml, INF-, 39.3±4.2 pg/ml and IL-10, 34.6±13.7 pg/ml, respectively. p < 0.05). The cytotoxic test (OD value) was markedly higher in blocking group (0.29±0.06) than that of control group (19±0.09 p<0.05) . Conclusion: The PD-1 expression on lymphocytes can be induced by Hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking PD-1/PD-L1 interaction. Background: Hedgehog (Hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). Our aim to observe the expression change of Hh pathway on rat hepatic regeneration . Materials and methods: Adult male Sprague-Dawley rats underwent approximately 70% partial hepatectomy (PH) or sham operation (SO). Liver specimens were collected at 2, 6, 12, 24, 36, 48, 72 , and 168h after PH or SO. Hedgehog expression was determined in mRNA level by RT-QPCR as well as in protein levels via immunohistochemical staining and western-blotting. Results: SO treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after PH. We found Sonic hedgehog(Shh )and Glioblastoma (GLi1-3) mRNA expression in the regenerating liver arrive at its peak at as early as 24 h and returned to its physiologyical level 168 h later. It is similar to the change of proteins (Shh and Gli1) .As seen from immunohistochemistry experiments; SHH protein was expressed uniquely in regenerating hepatocytes. Similarly, PH induced over expression for Shh protein occurred from 12 h with a peak level at 36 h after surgery. But Gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. Conclusion: Hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as PH. Background: To investigate whether PEG10, an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (H2O2) induced cellular apoptosis. Methods: PEG10 gene was stable transfected into L02. Cellular gene expression was determined by RT-PCR, Western blot and immunocytochemistry. Cell proliferation was analyzed by MTT. After treatment with different concentrations (50-400 mM) of H2O2, cell proliferation inhibition rate was measured by MTT. Morphological changes of apoptotic cells were determined by Hoechst33342 staining, DNA fragmentation was observed by agarose gel electrophoresis.
Hua Tang 1 , Xiao-Yan Tang 1 , Min Liu 1 , Xin Li 1 1 Tianjin Life Science Research Center, Tianjin Medical University, Tianjin 300070, China We determined how AFP modulates the proliferation of hepatoma cells. A recombinant adenovirus expressing siRNA against AFP (Adv-AFPsiRNA) was created and found that it reduced expression of AFP specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. Local treatment using Adv-AFPsiRNA caused significant repression of the growth of hepatoma derived HepG2 cells in xenograft in nude mice. Knockdown of AFP resulted in an obvious delay in the G1/S transition of cell cycle, but did not affect apoptosis in HepG2 cells, as analyzed by flow cytometry and TUNEL assay. Also, differential expressions of some genes related to the cell cycle, including SKP2, Cyclin D1, Csk and EBAG9 were identified by microarray and RT-PCR in HepG2 cells and HepG2 cells with knocked down AFP. These results suggest that endogenous AFP is a critical determinant of the growth of hepatoma cells. Hematopoietic stem cell (CD 34+) therapy can improve liver function in patients with cirrhosis. These cells can be mobilized into peripheral blood using Granulocyte colony stimulating factor (GCSF). This study was undertaken to assess feasibility and safety and of GCSF induced CD 34+ cell mobilization and its impact on liver function in patients with cirrhosis. Patients with liver cirrhosis (Cryptogenic or alcoholic with 6m abstinence) with CPT > 7 and < 12, and splenic diameter < 17 cm were included. GCSF injection was given for 5 days (5mcg/kg/dose). Baseline & day-6 CD 34+ counts in peripheral blood were done by flow cytometry. Follow up was weekly for 4 weeks and then monthly. CPT was compared at baseline and 6 months. 9 Patients (median age 51 y, range 33-64 y, 8 males; etiology: 6 alcohol, 3 cryptogenic; median CPT 10, range 8-12) were included. CD 34 + cell counts at baseline and day 6 were 2(0-3) and 15 (13-41) respectively (median, range). Side effects were fever in 8, allergic reaction in 1 and increase in splenic size in 1 (excluded). In follow up, 3 patients died (1, 3 & 4m after therapy, 1 after OLT), 1 lost to follow-up. 4 patients showed improvement in 10 -7) at 6-month follow-up. GCSF treatment is safe and yields adequate CD 34+ cells in peripheral blood. In short term it results in improvement in liver function in patients with cirrhosis PE415 Molecular cloning and Transcriptional analysis of KCTD9 gene promoter B. Pi 1 , J.S. Wang 1 , M.F. Han 1 , Y.Y. Zhou 1 , X.J. Liu 1 , X.P. Luo 2 , Q. Ning 1 1 Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2 Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Aim: Our previous work has shown that high expression of KCTD9, a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis B(SCHB). To further understand the gene transcription and regulation, KCTD9 promoter was cloned and gene transcription was studied. Methods: A full length of isolated promoter and series of 5' truncated promoter of KCTD9 gene was subcloned into the luciferase report vector pGL2-basic to form the promoter-report constructs. The KCTD9 promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing HBV X,C and S protein respectively or stimulated with cytokines (IL-2, IFN and TNF ) in 293 T cells to investigate KCTD9 gene regulation upon both viral factors and host cytokines. Result: A 759bp KCTD9 segment upstream of ATG translation start site was evidenced to contain potential regulative domains. An important regulation site located between -268bp and -81bp upstream of ATG translation start site. Based upon the luciferase activity assay, IL-2 was able to upregulate the transcription of KCTD9 whereas there was no effect from neither HBV viral proteins nor IFN and TNF . Conclusion: Here we first successfully cloned the full length promoter of KCTD9. IL-2 significantly enhanced the transcription of KCTD9, a gene which has been shown to be involved in T cell activation and disease severity of SCHB from our group. This work was supported by NSFC(30571643, 30672380, 30700702) 1 1 Istanbul University, Cerrahpasa Medical School, 2 Marmara University, 3 Okmeydani Teaching Hospital, 4 
Background: The treatment in chronic hepatitis C virus (HCV) is not highly effective, and cost, duration, and side effects are challenging. Predicting favorable factors of response to treatment would make it possible to give it only responsive patients. Recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. Hepatocyte damage in HCV is mediated by cytotoxic T-cells. Apoptosis primarily developed by the interaction between Fas antigen on hepatocyte and Fas ligand on T-cell corresponds to a main mechanism for hepatocyte damage. Methods: In this study, we aimed to detect any relationship between apoptotic markers (Fas, Fas ligand, Fas-associated death domain, caspases 3,8, and 9, insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. Additionally, any relationship between these parameters and the other ones predicting the response to therapy including ALT level, viral load, genotype, and gender were studied. Results: The study includes the patients in 4 centers managing chronic HCV infection. All parameters were studied in 180 patients. Study results revealed that histological activity index is correlated with CD95 staining density, caspase 8 intensiveness, and portal and parenchymal Fas ligand scores. Fibrosis is also seen to be correlated with the same parameters. Apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. Conclusion: Apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment.
S. Gao 1 , D. Xi 1 , J.W. Guo 1 , C.L. Zhu 1 , X.P. Luo 2 , Q. Ning 1 1 Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2 Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science Objective: This study was designed to explore the opportunity of microRNA interference technique in the inhibitory application of human fgl2, human Fas and TNFR1 expression. Methods: The eukaryotic expression plasmids of human fgl2, Fas and TNFRI genes were constructed and have successfully expressed hfgl2, hFas and hTNFRI protein. miRNA expression plasmids of hfgl2, hFas and hTNFRI complimentary to the sequence responsible for hfgl2, hFas and hTNFRI respectively were also constructed, meanwhile an irrelevant miRNA plasmid was used as control. By respective cotransfection of p-hfgl2miRNA and pcDNA3.1-hfgl2, p-hFasmiRNA and pcDNA3.0-hFas, p-hTNFRI miRNA and pcDNA3.0-hTNFRI expression construct into 293T cells, the inhibition of hfgl2, hFas and hTNFRI expression were analyzed by quatitative real time PCR and western blot. Results: The experiments showed the significantly inhibitory effect of p-hfgl2miRNA on hfgl2, p-hFasmiRNA on hFas and p-hTNFRI miRNA on hTNFRI expression at 48h post-transfection both at RNA level and at protein level, as well in 293T cell lines the inhibitory efficiency reached as high as 89.3% for hfgl2, 87.5% for hFas and 80% for hTNFRI, respectively. Conclusions: The study demonstrated the constructs of p-hfgl2miRNA, p-hFasmiRNA and p-hTNFRI miRNA successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl2, Fas and TNFRI contribute to. This work was supported by NSFC30571643, 30672380, 30700702; 2005CB522901, 2007CB512900 PE418 Influence of the ID2 on the Anti-Tumor Activity of Histone Deacetylase Inhibitor in Hepatocellular Carcinoma Cells R. Tsunedomi 1,2 , S. Harada 1 , N. Iizuka 1 , M. Oka 1 1 Dept. of Digestive Surgery and Surgical Oncol., Yamaguchi Univ. , 2 Research Fellow of the Japan Society for the Promotion of Science for Young Scientists Background: Our recent study revealed that levels of the Inhibitor of DNA binding/differentiation 2 (ID2) were associated with the progression of HCV-related hepatocellular carcinoma (HCC) and can affect susceptibility of HCC cells to histone deacetylase (HDAC) inhibitors. We here aimed to investigate how and whether ID2 expression affected on the anti-tumor activity of sodium butyrate (NaB), one of HDAC inhibitors. Methods: Two HCC cell lines, HLE and HuH-7, were used for gene targeting experiments. The ID2 over-expressing and knockdown cells were subjected to MTS assay to evaluate the susceptibility to NaB. Time-course of the expressional change of Bcl-2 and Bcl-xL genes after NaB administration was measured by real-time RT-PCR. Result: Upregulation and downregulation of ID2 levels in HCC cells resulted in decreased and increased susceptibility to NaB, respectively. We observed that after NaB administration, the ID2 expression was induced gently, the Bcl-2 expression was greatly increased immediately, and the Bcl-xL expression was decreased to less than half once and then recovered. These increase and recovery of the expression of anti-apoptotic genes were inhibited in the ID2 knockdown cells. In the ID2 overexpressing cells, the Bcl-2 expression was more upregulated than mock-transfected cells.
Conclusion: In HCC cells, ID2 influences the susceptibility to the HDAC inhibitor by regulating the expression of anti-apoptotic genes caused by the HDAC inhibitor stimulus. We suggest that ID2 could serve as a fascinating marker predictive of response to HDAC inhibitors.
The role of Zinc Finger Protein 146 in Liver Cancer M.M.Y. Waye 1,2 , T.L. Yeung 1,2 , J.L. Zhu 1,2 1 The Chinese University of Hong Kong, 2 Croucher Laboratory for Human Genomics Background/aims: The aim of this research project is the characterization of a Krüppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using hepatocellular carcinoma as a disease model. Zinc finger protein 146 (ZNF146), also named as Only Zinc Fingers protein (OZF), is a 33kDa nuclear zinc finger protein consisting solely of ten C2H2 zinc finger motifs of the Krüppel type. Like most of Krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. The ZNF146 gene is amplified in 15-25% of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. It is thus proposed that overexpression of the ZNF146 may contribute to the development or progression of hepatocellular carcinoma. Methods: We used flow cytometry, microarray, green fluorescent recombinant protein, RT-PCR site-directed mutagenesis, and transfection to study the effect of expression of ZNF146. Results: Our results shown that ZNF146 was over-expressed in two human HCC cell lines HepG2 and Hep3B. Expression profiles of ZNF146 over-expressing shown that genes related to the p53 tumor suppressor activity or DNA damage, repair response and control were deregulated upon overexpression of ZNF146. Conclusions: ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage. Background: In the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of DNAzyme 10-23 in vitro cleavage on the target RNA molecules as well as for rapid, accurate, high-throughout evaluation of varied DNAzymes on their counterpart RNA molecules. Methods: DNAzyme named Dz-HCV-9 specific to hepatitis C virus (HCV) ORF AUG were designed and synthesized. Dz-HCV-Mis-9 with mismatched substrate-recognition domains, Dz-HCV-Mut-9 with mutant catalytic domains, antisense oligonucleotide ASON and nonsense oligonucleotide NSON were synthesized respectively as controls. A chimeric oligonucleotide of 29nt containing both RNA and DNA bases was designed and synthesized as the substrate: 5' FAM-GT AGACCGUGCACCAUGAGCACGAAUCCT-BHQ 3', corresponding to the330-354 nt (underline) of HCV genome(gi: 329873) The reporter FAM/BHQ was incorporated at the 5' and 3' end, respectively. Under simulated physiological conditions (37 ), kinetic characterization of RNA-cleaving DNAzyme was analyzed in a real-time way. Factors that influencing DNAzyme cleavage were analyzed. Results: Dz-HCV-9 specific to HCV ORF AUG could cleave target RNA at A•U site, a continuous change of fluorescence intensity was monitored. While the control oligonucleotides couldn't cleave RNA, there were no change of fluorescence intensity. Factors that influencing DNAzyme cleavage concluded different substrate-recognition domain, Mg 2+ concentration and pH. Conclusion: A real-time monitoring system for kinetic characterization of RNA-cleaving DNAzyme was successfully established in the first time.
The Study on the Apoptosis of Hepatoma Cells Synergeticly Induced By Plasmid-Mediated Anti-Angiogenesis and Immunopotentiation Therapy P.Y. Li 1 , Q. Zhang 1 , Y. Chang 1 , J.S. Lin 1 , D.A. Tian 1 1 
Background: Angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. We want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. Methods: Mouse endostatin eukaryotic plasmid (pSecES) and mouse IL-12 (interleukin 12) eukaryotic plasmid (pmIL-12) were extracted and purified from E. coli. H22 hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pSecES, pmIL-12, pSecES+pmIL-12 or pcDNA3.1 naked plasmid DNA respectively into implantation sites repeatedly. Tumor formation and its weight was evaluated. Tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by CD31 staining, HE staining and TUNEL assay respectively. Results: Inoculated mice received pSecES, pmIL-12 injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcDNA3.1 injection. There were much more tumor apoptosis cells in pSecES+pmIL-12 group (19.9±5.5 per 400× microscope field P<0.05) than any other single plasmid injection group (400× microscope field: pSecES 11.3±4.1, pmIL-12 14.6±3.2, pcDNA3.1 1.4±1.3). Conclusion: Tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. (20, 40, 80, 160, 200 ng/mL) in a serum-free medium for 24 h. Cell proliferation was measured by Brdu incorporation analysis, untreated WB-F344 cells were taken as controls. After treatment with Wnt3a (160ng/mL) for 24 h, subcellular localization and protein expression of -catenin in WB-F344 cells treated and untreated with Wnt3a were examined by immunofluorescence staining and Western-blot analysis. CyclinD1 mRNA expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (RT-PCR). mRNA levels of some phenotypic markers (AFP, CK-19, ALB) and two hepatic nuclear factors were measured by RT-PCR. Expressions of CK-19 and AFP protein were detected by Western-blot analysis. Results: Wnt3a promoted proliferation of WB-F344 cells. Stimulation of WB-F344 cells with recombinant Wnt3a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical Wnt target gene cyclinD1. After 3 d of Wnt3a treatment in the absence of serum, WB-F344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as AFP, CK-19 following activation of the canonical Wnt signaling pathway. Conclusion: The canonical Wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. The expression level of Bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. Results: HBx/51 showed the most sensitive towards Dox treatment, and truncated Bid (tBid) was also only detected in this cell line. The level of Bax was also increased in HBx/51 cells. Conclusions: The carboxy-terminal of HBx may enhance the processing of Bid into tBid, which may contribute to increased sensitivity of the cell towards the Dox treatment.
cell homeostasis were performed with concentrations of oxysterol (3x10 -5 -10 -4 M) faraway from the physiological and/or pathological one (0.2 and 2x10 -7 M). In our study, we asked the effects of oxysterols (7K and 5'6S) on hepatoma cell lines homeostasis. To this purpose we used concentrations similar to those described in physiological or pathological conditions. Sub-physiological (10 -9 M) to pathological (10 -7 M) oxysterol (7K and 5'6S) concentrations were used to stimulate HepG2 cells. A surprising pro-proliferative effect of 5'6S at sub-physiological (10 -9 M) concentration was observed. This behaviour was confirmed by the synergic increase of ERK1/2 levels. FACS analysis revealed an early progression of cells in S phase at the lowest concentration of 5'6S, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in G2/M phase. Apoptosis was absent at all concentration used, except for the highest one (10 -5 M). At this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. Effectively, both 7K and 5'6S, at all concentration used (except for 10 -9 M), induced cell senescence (revealed by SA-ß-gal staining and SIRT1 and p21 over-expression). In conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. The main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of 5'6 secosterol at low concentration. Mortalin, a member of HSP70 family protein, has been shown to play an important role in hepatocellular carcinoma (HCC). It has been reported that mortalin is binding to the C-terminal of p53, which acts as a safety guard and is a commonly mutated gene in HCC. In this study mortalin was silenced by specific shRNA in PLC/PRF/5, a HCC cell line constitutively expressing p53ser249, and normal liver cells MIHA, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p53 dependent way in PLC cells. Tunel staining positive cells were only found in the PLC cells mortalin knockdown group, and apoptosis associated protein, such as p53, Bax, Bcl-xl, cleaved-caspase 3, have been screened by western blot after transfection. Quantitative-PCR data also showed that p53 mRNA level are upregulated about 2 folds in mortalin knockdown group compared with the control groups in liver tumor cells. Two p53 inhibitors, PFT-and PFT-, which can reverse this apoptosis was applied to demonstrate p53 dependent way. In summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p53 in PLC cells, but had no effect on normal cells. The clinical application of this study suggested that motalin specific shRNA might be a potential anti-cancer drug for HCC. Background: NAFLD can proceed to NASH and are at risk of cirrhosis and HCC. Aim was to study profile of Bangladeshi NAFLD patients. Methods: 52 patients with NAFLD were included. Of them 59.6% were males and 40.4% females. Patients were between 12-60 years of age. They presented with dull right upper abdominal ache and/or incidental detection of raised ALT/AST and/or fatty liver on ultrasonography. All tested negative for hepatitis B and C. None had history of alcohol. All underwent per-cutaneous liver biopsy for histopathology. They were also tested for DM, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis C. Their BMI and BP were recorded. Results: 88.5% had NASH. 63.0% of them were males and rest 37.0% females. 11.5% had NAFL. Of them 50% each were males and females.
Majority had NASH. 47.8% were obese and 41.3% had dyslipidaemia. 28.3% had hypertension, 28.3% insulin resistance and 13% were diabetics. 6.5% had hypothyroidism. None had hepatitis C. ALT was raised in 72% and AST in 40%. Although all patients with NASH did not have elevated ALT, it was raised in majority, contrary to AST, which was normal in most. Conclusion: Majority NASH patients in Bangladesh are obese. Other leading causes of NASH include dyslipidaemia, hypertension and insulin resistance. Some NASH also had diabetes and hypothyroidism. This study also reveals that elevated ALT in patients with NAFLD is suggestive of fibrosis, although normal serum ALT does not exclude NASH. The study further suggests that ALT is superior to AST in predicting NASH. Background: Non-alcoholic fatty liver disease is prevalent in obese patients. Liver biopsy remains the best diagnostic tool for confirmation. We tried to find out the correlations of laparoscopic parameters with histology and laboratory data. Besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. Methods: In the period of one year and five months,126 morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. Results: Laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in Table 1 . Besides, important parameters with relationships to laboratory data were summarized in Table 2 Department of Internal Medicine, Seoul National University Hospital Gangnam Healthcare Center, Seoul, South Korea, 2 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea Background/aims: Hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (NAFLD). Recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. Among them, BARD score and Guha's simple panel are easy to use in clinical practice. In this study, we evaluated the efficacy of BARD score and Guha's simple panel as a noninvasive fibrosis marker in Korean NAFLD patients. Methods: Data from 79 patients with biopsy-proven NAFLD in Seoul National University Hospital from 2000 to 2007 were used. BARD score and Guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. Results: Stage 0 fibrosis were found in 67 patients, stage 1 in 4, stage 2 in 2, stage 3 in 1 and stage 4 in 5. The relationship between fibrosis stage and BARD score ( = 0.43, p < 0.001) was statistically significant. All patients with advanced fibrosis (stage 3-4) had BARD score greater than 2. Mean values from original Guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. However, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < 0.001). Conclusions: Our data suggest that Bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the Guha's simple panel may be needed to use as a fibrosis marker in Asian NAFLD patients.
S.K. Mohan 1 , S. Subramaniam 2 , S. Subramaniam 3 1 Assistant Professor, Department of Biochemistry, Saveetha Medical College & Hospital, Saveetha University, T.N, INDIA., 2 Consultant, Department of Biochemistry, Apollo Hospitals, Chennai, T.N, INDIA. , 3 Department of Biochemistry, Apollo First Med Hospitals, Chennai, T.N, INDIA. Background: Non-Alcoholic Fatty Liver Disease (NAFLD) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. Non-Alcoholic Steato Hepatitis (NASH) is a severe form of NAFLD and progresses to the end stage of liver disease. It is becoming the leading cause for referral to liver clinics in most areas. The prevalence of NAFLD in Indian population is estimated around 7 -13%. The NAFLD has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. Aim: To evaluate the combination of Inter Cellular Adhesion Molecule -1 (ICAM -1), Adiponectin and Type-IV collagen, a new biomarker profile for NASH in patients with NAFLD. Methods: 76 patients with NAFLD and age & sex matched 68 normal healthy individuals as controls were selected for this study. Levels of Serum ICAM -1, Adiponectin, Type-IV collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. Results: Serum ICAM -1 & Type -IV collagen levels were significantly increased in patients with NASH among the NAFLD patients compared to controls. The Serum Adiponectin levels were significantly reduced in patients with NASH among the NAFLD patients compared to controls. Compared to liver function test parameters and lipid profile levels, NASH profile has got positive negative predictive value among the NAFLD patients. Conclusion: In patients with NAFLD, NASH profile Test -a simple, noninvasive and reliable to predict the presence or absence of NASH. Background/Aim: Oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( TGF-) and tumor necrosis factor-( TNF-) in patients with NAFLD and compare it with patients of chronic viral hepatitis (CVH) and healthy controls (HC). Methods: Lipid peroxidation was studied by estimating plasma malondialdehyde (MDA) levels as per the methodology described by Buege and Aust and TGF-& TNF-levels were measured by ELISA kits (Ray Biotech, USA, & Diaclone, UK) in the stored sera in 10 biopsy proven patients with NAFLD (M: 4, F: 6, Mean age: 41.7 ± 11.0 yrs), 15 patients with CVH ( M:14, F:1, Mean age: 33.7 ± 11.4 yrs) and 5 HC (M:5, Mean age: 25.2 ± 2.7 yrs). Results: There was no difference in mean plasma MDA levels amongst patients with NAFLD (17.28 ± 3.6 mol/L), CVH (15.29 ± 2.3 mol/L) and HC (16.79 ± 1.2 mol/L). Serum TGF-levels between NAFLD (0.56 ± 0.41 ng/mL) and CVH (0.52 ± 0.25 ng/mL) patients and HC (0.58 ± 0.57 ng/mL) were also comparable. Though patients with CVH (17.2 ± 27.0 pg/mL) and NAFLD (7.5 ± 6.3 pg/mL) had higher levels of TNF-than HC (5.5 ± 1.1 pg/mL), the difference was not significant statistically. Conclusion: Lipid peroxidation, TGF-and TNF-need to be studied in a larger number of patients with NAFLD. Background/Aim: Burnt out nonalcoholic fatty liver disease (NAFLD) may be responsible for cirrhosis and hepatocellular carcinoma (HCC) in the absence of other causes. Aim of this study was to evaluate the surrogate markers of NAFLD in patients with cryptogenic cirrhosis (CC) and cryptogenic HCC (CHCC). Methods: Sixty five patients with CC and 31 patients with CHCC were analyzed for the presence of abnormal body mass index (BMI) and type 2 diabetes mellitus (DM Aim: To investigate the relation of phosphatidylethanolamine N-methyltransferase PEMT gene G175A single nucleotide polymorphism (SNP) with the susceptibility to nonalcoholic fatty liver disease NAFLD . Methods The genotypes and allele frequencies of PEMT exon 8 SNP G175A were analyzed by using PCR-RFLP in 51 NAFLD patients and 50 controls.
Results: The G to A variation of the PEMT gene G175A SNP was significantly higher in NAFLD group compared with controls. The frequencies of GG GA and AA genotypes were 58.8 39.2 and 2.0 in NAFLD and 78.0 22.0 and 0.0% in controls (P=0.038 . The A allele of the PEMT gene was significantly more frequent in NAFLD group (21.6%) than that (1.0%) in controls P=0.042 .There were significant differences in serum levels of cholesterol, triglyceride, HDL-C and LDL-C between GG and GA/AA genotypes P <0.05 .
N. Assy 1,2 , G. Lipez 3 , S. Korem 3 , M. Grozovski 3 1 Sieff Hospital, Safed, Israel, 2 2Technion Institute, Faculty of Medicine, Haifa, Israel, 3 Ort Braude College, Karmiel, Israel Background: Previous studies reported increase in serum protein C and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (NAFLD).
Conclusion People with PEMT gene G175A SNP were more susceptible to develop NAFLD Aim: 1) Determine whether there is a relationship between NAFLD, Protein C and Paraoxonase levels in quiescent and in regenerating rats fatty liver 2) determine the effect of ISA on hepatic "protein C" and paraoxonase mRNA. PE439 Methods: Forty-eight SD rats were treated with fructose enriched diet (FED), or FED with Metformin (2 mg/Kg/d), FED with Rosiglitazone (3 mg/Kg/d), or the combination of both drugs for 5 wks. 30% PHX was performed at WK 5. Protein C, paraoxonase mRNA expressions, lipids, MDA were measured before and 24 hours after PHX. Results: Hepatic "protein C" mRNA was higher in rats with fatty liver than control rats (+105%, p<0.01) whereas hepatic paraoxonase mRNA was lower in rats with fatty liver than control rats (-28%, p<0.005). Hepatic protein C and paraoxonase mRNA increased in rats with fatty liver in regeneration (+116%, p< 0.01, and +15%, p<0.01 respectively). The combination of metformin and rosiglitazone decreased hepatic protein C expression at 24 hours after PHX by -170% (p<0.001) and increase paraoxonase mRNA by +50% (p<0.01). Serum paraoxonase correlates with serum protein C (r=-0.2), MDA (r=0.4), Background: Non-alcoholic steatohepatitis (NASH) is a type of non-alcoholic fatty liver disease (NAFLD), and may progress to hepatic fibrosis and cirrhosis. The pathogenesis of NASH remains unclear. The aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. Methods: Male SD rats weighing 70-80g were obtained. Twenty animals were randomly divided into two groups. In the model group, five animals were fed with high lipid forage that includes 3% cholesterol and 20% lard for 6 weeks, five were fed for 12 weeks, while the control group ate normal foods. The animals were sacrificed after 6 weeks. The animals were sacrificed after 6 weeks and 12 weeks. Liver and blood serum were collected while the serum levels of ALT, AST, TG and TC were measured. The pathology of liver was observed by HE staining. Western blot was used to investigate the expression of arginase in control and model group.
TG (r=-0.23). Conclusion: Hepatic "protein C" mRNA levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (ISA) whereas hepatic paraoxonase mRNA levels are low at baseline, up regulated during liver regeneration and increase after treatment with ISA.
Results: Vacuolization were observed extensively in hepatic cells in the model group after 6 weeks and 12 weeks of high-fat diet. It is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. Western blot showed that the level of arginase II increase in the liver of model group rats as compared to the control group. Furthermore, the level of arginase was higher in liver samples obtained from model rats that were 12 weeks on a fat diet as compared to rats that were only 6 weeks on the same diets. Conclusion: The level of arginase II was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase II is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis.
Methods: C57BL6/J mice were fed with MCD diet to induce hepatic fibrosis and rosiglitazone was given in treated group. Effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of MMP-2/9, TIMP-1/2 mRNA and protein detected by RT-PCR and Western blot respectively.
The Ethanolic Extract of Fructus Schisandrae Chinensis Decreased Hepatic Triglyceride Level in Mice Fed with a High Fat/Cholesterol Diet Results At week 8, fibrosing NASH models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated MMP-2/9 mRNA and protein, up-regulated TIMP-1/2 mRNA and protein. Rosiglitazone significantly reduced MCD-induced fibrosis by induced MMP-2/9 expression and reduced TIMP-1/2 expression by activating PPAR .
S.Y. Pan 1 , Z.L. Yu 2 1 Beijing University of Chinese Medicine, 2 Hong Kong Baptist University Effects of the ethanolic extract of Fructus Schisandrae Chinensis (EtFSC) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for 8 or 15 days. Single dose of EtFSC (1 or 5 g/kg/day, i.g.) increased the serum triglyceride (TG) level (40 and 142%, respectively), but decreased hepatic total cholesterol (TC) level (15 and 16%, respectively) in normal mice. The hypertriglyceridemia produced by EtFSC was suppressed by the co-administration of fenofibrate. The induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic TC levels (up to 165%) and hepatic TG levels (up to 528%) in mice. EtFSC treatment (1 or 5 g/kg/day for 7 days, i.g.) significantly decreased the mouse hepatic TG level (by 35%) and slightly increased the hepatic index (by 8%). Whereas fenofibrate treatment (0.1 g/kg/day for 7 days, i.g.) significantly lowered the hepatic TG level (by 61%), it significantly elevated the hepatic index (by 77%) in hypercholesterolemic mice. The results indicate that EtFSC treatment can invariably decrease hepatic TG in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. Aim: To investigate the influence of multiple gene polymorphisms in the susceptibility of NAFLD. Methods: The data of single nucleotide polymorphisms (SNPs) in 201 NAFLD patients who had at least one of the genetic variations at the sites of TNF--238, adiponectin -45 and leptin-2548 were analyzed. The genotypes were determined by using PCR-RFLP. Our previous studies showed that the variations of these sites increased the susceptibility of NAFLD. Results: The prevalence of NAFLD in adiponectin variation alone group (n=45) was 35.6%; in TNF-alone group (n=33) 42.4%; in leptin alone group (n=54) 35.2% (p>0.05). In comparison with the above groups with single SNP, the prevalence of the groups with two gene variations of TNF-plus adiponectin (57.9%, n=19) increased significantly (p<0.05). However the prevalence of other two groups i.e. adiponectin plus leptin (34.6%, n=26) and TNF-plus leptin (40.0%, n=15) did not differed significantly from those of groups with single SNP (p>0.05). The prevalence in the group with three gene variations (55.6%) differed significantly from all (p<0.05) except that of TNF-plus adiponectin group (p>0.05). The metabolic features of the NAFLD patients in the 7 groups mentioned above were not different significantly (p>0.05). Conclusion: NAFLD is a polygenic disease. Multiple gene polymorphisms may, but not always, increase the susceptibility of NAFLD.
Chronic Hepatitis B Patients with Nonalcoholic Fatty Liver Disease R.D. Zheng 1 , C.R. Xu 1 , J. Chen 1 , B.F. Chen 1 1 Southeast Hospital Background: To investigate clinical pathological characteristic in HBeAg negative Chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD). Methods: We measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST) in HBeAg negative chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD). And we detected Hepatitis B Virus marker, HBV-DNA, counted body mass index, insulin resistance index and observed pathological characteristic. All these patients with diagnosis were confirmed by clinical and pathological evidence. Result : The body mass index, homeostatic model assessment (HOMA) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in HBeAg negative chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD) than HBeAg negative chronic hepatitis B patients. But the alanine aminotransferase (ALT), aspartate aminotransferase (AST), HBV DNA levels were significantly lower in HBeAg negative CHB patients with NAFLD than in HBeAg negative chronic hepatitis B patients. Histologic features in HBeAg negative chronic hepatitis B(CHB) patients with nonalcoholic fatty liver disease (NAFLD) are in zone 3 predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. Conclusion: The HBeAg negative chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. To carry out liver biopsy selectively for the patients with HBeAg negative chronic hepatitis B having metabolic factors, which is helpful for early diagnosis in HBeAg negative chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD). Aims: To investigate the preventive effect of Cordyceps sinensis and its possible mechanism on apoptosis of NAFLD. Methods: Rats were randomly divided into basic diet group (B group), pathologic group (NASH group) and cordyceps sinensis group(CS group).The latter two groups were administered with high-fat diet to establish NAFLD animal models. CS group were treated with CS at the 9th week after high fat diet. Rats were sacrificed at the end of the 18th week. Biochemical examination were used to detect superoxide dismutase (SOD) of liver tissue. Hepatocyte apoptosis was assessed in each group using the TUNEL assay and immunohistochemistry for activated Bax Bcl-2 Caspase-3 and NF-kB P65. Results: (1) Compated with the B group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of NFSH group. SOD lever was significantly decreased (P<0.01) and TUNEL-positive cells were significantly increased (P<0.01). Immuunohistochemistry test demonstrated active Bax Caspase-3was increased (P<0.01) while no apparent change was observed in Bcl-2. (2) In CS group, only diffusive steatosis but not inflammation or fibrosis was found. SOD lever was increased than that of NASH group (P<0.05). TUNEL-positive cells and active Bax Caspase-3 were significantly decreased (P<0.05 P<0.01) that those of NASH group. Bcl-2 and NF-kB P65 were increased (P<0.01) than those of NASH group. Conclusions: Hepatocyte apoptosis is a prominent feature of NAFLD. Cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of SOD, decreasing express of Bax and increasing express of Bcl-2 and NF-kB P65. Background: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease. Insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of NASH. We aimed to evaluate the efficacy of thiazolidinediones in the treatment of NASH. Methods: We have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in NASH, through MEDLINE, EMBASE, AMI, Cochrane Central Register of Controlled trials. Data were abstracted from each study and disagreements were resolved by consensus. Dichotomous outcomes were reported as relative risk with 95% confidence interval based on fixed-effects model. Results: We included three trials, two evaluating pioglitazone and another rosiglitazone. A total of 171 patients were involved in the analysis. Thiazolidinediones was noted to improve liver function tests. It was effective in the reduction of steatosis among patients with NASH (RR 0.67, 95% CI 0.52-0.87). It was found to be beneficial in improving ballooning necrosis (RR 0.79, 95% CI 0.66-0.95). It was also found to improve lobular inflammation (RR 0.72, Background: It is well known that the weight reduction is effective for ALT normalization in patients with non-alcoholic fatty liver disease (NAFLD). The necessary condition for ALT normalization is still unclear. To clarify the necessary and sufficient condition for ALT normalization, we investigated the effects of body fat decrease in NAFLD patients by body composition analyzer. Methods: Forty-six NAFLD patients (23 male, 23 female, mean age 49.8±12.9 years old) with abnormal ALT levels were evaluated. The volume of skeletal muscle, body fat and BMR were examined by using the body composition analyzer (In Body 720; Biospace Co. Ltd., Tokyo Japan). All patients were received an individualized diet consultation by dietician every 4 weeks for 6 months. Daily energy was BMR (basal metabolic rate) x1.2 kcal and protein was 1.0-1.2g per ideal body weight. Result: Twenty-eight of 46 patients (60.9%) were achieved normal ALT level. In ALT normalized group, the body weight and fat loss were 3.6±2.3 kg, 3.0±1.5kg (2.8±1.7 %body fat) respectively. On the other hand, in cases with ALT remained abnormal level, the body weight and fat loss were 0.5±1.5kg, 0.4±1.6kg (0.5±1.6 %body fat). Conclusion: Our results demonstrate that the fat loss of 3 kilograms or more was necessary to normalize ALT level in NAFLD patients.
A. Somani 1 , S. Somani 2 , A. Jain 3 , V. Dixit 3 1 Navjeevan hospital, 2 Suvidha, 3 
Background : NAFLD is often clustered within families and the causes include both genetic and environmental factors. Family studies done thus far have been limited by small sample size. To examine the familial patterns , we performed a prospective study to see (a) Whether NAFLD is more common in first degree relatives (b) Genetically determined risk factors associated for clustering. Methods: First degree relatives of histologically confirmed NAFLD patients and spouses (controls) were included after excluding other causes of fatty liver. Those having raised transaminases >3 months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. They were divided into three groups. Group I Patients Group II First degree relatives Group III Spouses Results: NAFLD was more prevalent among first degree relatives then spouses (37% and 17%, p<0.01). Anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. HOMA-R was similar in Group I and II (p=0.073), but was significantly different in Group I and III (p= 0.0001) and Group II and III (p=0.0001) respectively. Metabolic syndrome was present in >70% of patients and were comparable in three groups except for fasting glucose > 110, which was present in 76%, 77% and 57% of patients in group I, II and III respectively. Majority (>50%) of our patients among groups I, II and III were having only steatosis while NASH was present in 20%, 13% and 14% of patients.
A. Somani 1 , V. Dixit 2 , A. Jain 2 , S. Somani 3 1 Navjeevan hospital, 2 IMS, BHU, VARANASI, 3 Suvidha Background: Normal levels of alanine aminotransaminase (ALT) have been demonstrated in NAFLD patients. ALT levels are also modulated by age, gender, BMI, fasting glucose, and serum triglyceride levels. We performed a prospective study of patients with histologically confirmed NAFLD and having ALT < 1.5 times and compared them with those having raised ALT to determine (a) clinico-pathologic features of NAFLD patients with normal ALT (b) to observe any differences between them. Methods: Patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. Participants were divided into two groups (a) Those having ALT > 1.5 times normal (n=97) (b) Those having normal ALT (n=47) Results: Mean age was comparable with slight male predominance. There were significant differences in anthropometric measurements like BMI (p=0.0001) and WHR (0.90±3.57 and 0.88±3.23, p=0.0071). Mean BP, lipid profile, fasting glucose, insulin, and HOMA R were comparable. There were significant differences in both mean AST (50.2±5 and 37.4±3.21, p=0.0001) and ALT (104±7.29 and 69.9±11.5, p=0.0001) levels. Metabolic syndrome was present in >75% of patients and Individual components were comparable except for increased waist circumference which was significantly more in those with raised ALT (78.35% and 44.68%, p<0.001). Majority of our patients were having only steatosis, while NASH was present in (27.82% and 8.5%, p<0.05) of patients. Conclusion: NAFLD can exist in patients with normal ALT values. Although more work is needed to determine who should be screened for NAFLD and how such individuals should be evaluated, this study is a step toward the identification and characterization of NAFLD patients with normal ALT. We can suggest that patients having metabolic syndrome or insulin resistance, despite having normal ALT, should be screened for NAFLD. Also ALT values should be adjusted for variables like BMI to appropriately screen NAFLD patients. Background: Scientific evidence has demonstrated that Traditional Chinese Medical (TCM) approaches and products can be beneficial for managing Non-alcoholic Fatty Disease (NAFLD), but few rigorous criteria of patterns of TCM therapy are available to guide practitioners in deciding the CAM interventions. Objectives: To evaluate criteria of patterns of TCM therapy for the management of NAFLD identified by biomedicine. Methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. Descriptive analysis, factor analysis, and cluster analysis were involved. Results: (1) Background/Aim: Serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. The aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (NAFLD). Methods: A cross-sectional study was performed among the employees of Zhenhai Refining & Chemical Company Ltd., Ningbo, China. Results: The study included 8925 subjects (6008 men) with a mean age of 43 years. The prevalence rate of NAFLD and hyperuricemia was 11.78% and 14.71%, respectively. NAFLD patients had significantly higher level of serum uric acid than controls (370.3 ± 86.6 vs. 321.1 ± 82.6 mol/L; P < 0.001). The prevalence rate of NAFLD was significantly higher in the subjects with hyperuricemia than those without hyperuricemia (24.75% vs. 9.54%; P < 0.001), and the prevalence rate increased along with serum uric acid levels (P value for trend < 0.001). Multiple regression analysis showed that hyperuricemia was associated with increased risk for NAFLD (odds ratio [OR]: 1.291, 95% confidence interval [CI]: 1.067 -1.564; P < 0.001). Conclusion: Serum uric acid level is significantly associated with NAFLD, and increased serum uric acid level is an independent risk factor for NAFLD. Background: Development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. However, the cellular and molecular events in the early development of alcoholic liver diseases (ALD) and the contributory effects of a high fat diet are not fully understood. Methods: This study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. Results: Ethanol-fed rats exhibited high blood ethanol levels (0.85+0.31%) and significant increases in serum ALT (67.7+ 13.4 unit/L), AST (136.3+ 60.2 unit/L), and ALP (400+108.9 unit/L) when compared with control rats (p<0.01, respectively). Histopathological examination found unevenly raised Knodell scores (5.33+1.15 in the ethanol-fed livers vs. 0.33+0.58 in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. However, typical steatosis lesions were absent. qPCR demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (ADH1, P<0.05; cytochrome P450 2E1, CYP2E1, P<0.05; GSTA2, P<0.01; PPAR , P<0.05), and genes coding for pro-inflammatory cytokines (IL-1 , p<0.01 vs. control livers; TNF-p<0.05; TGF -, p<0.05; RANTES P<0.05), ECM components and proteinases (collagen-1, P<0.01; SMA, p<0.01; MMP -9, P<0.05 and TIMP-1, P<0.05). Conclusion: Chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ALD. The aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by Molecular Adsorbent Recirculating System (MARS) in 15 ACLF patients versus 15 patients treated with Medical Standard Therapy (SMT) that presented alcoholic liver disease etiology and similar Model End-stage Liver Disease (MELD). Methods: MARS Group: Fifteen (10 male and 5 female) patients were treated with MARS® (Gambro). Five patients were excluded by study.The number of MARS applications was about 9, the length of applications was about 10h. SMT Group: Fifteen patients (10 male and 5 female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. Three patients were excluded by the study. The patients were valued during 30 days from inclusion with a survival follow up a three months. Results: MARS Group: we observed a significant changes in levels of IL-6(p<0.01), IL-1(p<0.002), IL-8(p<0.04) and TNF-alfa (p<0.03) in association with improvement of hepatic growth factor (p<0.001). The patient's survival at three months was 50%. SMT Group: we observed only a significant changes in IL-1(p<0.01) and TNF-alfa (p<0.2). The patient's survival at three months was 33%. Conclusion: The MARS liver support device has corrective effects on disturbed pathophysiology of ACLF and may be used to enhance spontaneous recovery or as bridge to transplant.
A Study of Protective Effect of Centella Asiatica in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Liver Injury N. Haleagrahara 1 , S. Chakravarthi 1 , P. Kumar 1 1 International Medical University, Malaysia Background: Centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster.
Results: PPC significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. Phosphorylation of apoptosis signal-regulating kinase 1, p38 mitogen-activated protein kinase, and protein kinase C, as well as activation of nuclear factor-kappa B, were markedly suppressed by PPC. These effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ROS)-generating enzymes, including cytochrome P450 2E1, acyl-CoA oxidase, and NADPH oxidases, in addition to restoration of ethanol-induced increases in Toll-like receptor 4 and CD14. PPC also decreased the pro-apoptotic proteins Bax and truncated Bid, thus inactivating mitochondrial permeability transition. Furthermore, PPC suppressed overexpression of transforming growth factor-1 and hepatic stellate cell activation, which retarded hepatic fibrogenesis. Conclusion: PPC exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ALD as a result of inhibition of alcohol-induced ROS production. Background: Dysctamnus dasycarpus has used for the promotion of health in South Korea. But, there were rare a report concerning the hepatotoxicity. We report cilinical features of liver injury by Dysctamnus dasycarpus. Method: Eighteen patients diagnosed as acute toxic hepatitis by Dysctamnus dasycarpus in Chungnam national university hospital between January 2005 and Arpil 2008 was enrolled. Toxic hepatitis was diagnosed by RUCAM score ( 4). The medical records were reviewed, retrospectively. Result: Eleven patients (61%) were female and the mean age was 53.5. Most common symptom was jaundice. Initial laboratory findings were as follows(mean value): WBC 6126/uL, Hemoglobin 13.4 g/dL, Platelet 212×10 3 /L,ALT 1375IU/L, total bilirubin 11.3 mg/dL, alkaline phosphatase 159 U/L, GGT 244U/L, prothrombin time(INR) 1.1. The mean hospitalization was 21.5days. Peak laboratory findings were as follows: ALT 1382IU/L, total bilirubin 15mg/dL. Recovery time of each biochemical finding was as follows: ALT 37days, total bilirubin 39.4 days. Recovery rates of ALT and total bilirubin were 27.8% and 38.9%, 89.9% and 89.9% at 4 weeks, 8 weeks, respectively. The main biochemical pattern of hepatotoxicity was hepatocellar (72.2%) type. Prednisolone was prescribed in six patients. Progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. Other one patient had prolonged jaundice (117 days). But, all patients had recovered without sequelae. Conclusion: In South Korea, Liver injury by Dysctamnus dasycarpus was more frequent in women. The main pattern of hepatotoxicity was hepatocelluar type. Most patients had prolonged icteric phase and hospitalization. Patients were recovered by supportive management after drug cessation or prednisolone therapy. In Korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. However, clinical study of the herbal medicine is not conducted in a structured manner. We report three cases of toxic hepatitis caused by the intake of Dictamnus Albus. The first patient, a 44 year old woman was admitted due to nausea after ingestion of liquor containing Dictamnus albus for 2 months. Total bilirubin was 2.55 mg/dL AST/ALT 774/1,424 IU/L on admission. Liver biopsy observed hepatocyte necrosis and cholestasis. The elevated bilirubin and transaminase returned to normal 2 weeks later after cessation of Dictamnus albus. The second patient, a 64 year old man was admitted due to jaundice after ingestion of boiling Dictamnus albus for 3 months. Total bilirubin was 11.37 mg/dL AST/ALT 2,010/2,522 IU/L on admission. Liver biopsy observed pericellular fibrosis and necrosis. The bilirubin decreased slowly compared to the transaminase and normalized 3 months later after cessation of Dictamnus albus. The third patient, a 48 year old man was admitted due to jaundice after ingestion of liquor containing Dictamnus ablus for 1 month. Total bilirubin was 12.8 mg/dL AST/ALT 1,097/1,869 IU/L The hepatocyte necrosis was observed by liver biopsy. The elevated bilirubin and transaminase levels normalized 1 month later after cessation of Dictamnus albus. All patients had negative viral markers and non-specific ultrasonographic findings. The above mentioned three cases demonstrate that liver may have been damaged by Dictamnus albus, which indicated clinical characteristics. Background/Aims: CMILI poses a diagnostic challenge as no tests are available to confirm the causality. The aims of this study were 1) to evaluate clinical features and patterns of CMILI and 2) to assess the likelihood of causality among patients with liver impairment and exposure to Chinese Medicine (CM) by a multidisciplinary approach. Method: Between 5/2005 and 8/2007, patients who had liver derangement and CM or proprietary CM exposure within six months managed in the United Christian Hospital were studied. Clinical features and the CM were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and CM experts. Literature search of relevant herbs in Chinese and western journals were performed. CM samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. The likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. Results: There were forty-six cases of suspected CMILI, nineteen cases with alternative causes of liver diseases were excluded. Twenty-seven cases of CMILI proceeded to detailed analysis. Median age of patients was 51 (21-76) with female predominance. The median duration of CM exposure to presentation was 20 (1-150) days. Majority of them (82%) had hepatocellular liver injury pattern. One case of adulteration with NSAID and erroneous substitution of herb was identified respectively Causality were classified as unlikely, possible, probable and highly probable in 3, 12, 8 and 3 patients respectively. Conclusion: A multidisciplinary approach allows systemic evaluation of suspected CMILI.
Mouse Model I. Nassar 1 , T. Pasupati 1 , I. Segarra 1 , J.P. Judson 1 1 International Medical University, Kuala Lumpur, Malaysia Background: Imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome P450 pathway. Acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via P450 pathway. This study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. Methods: Four groups of male ICR mice (30-35g) were used. The mice were administered either saline solution orally, imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg and IP acetaminophen 700 mg/kg (study group). The mice (n=4 per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of 15, 30 minutes, 1, 2, 4, 9 and 12 hours and liver samples obtained by dissection. H&E stained liver sections (3µm thick) were histopathologically analysed. Results: The liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately.
The damage increased gradually with time, peaked at 2 hours and then resolved completely by 6 hours. Liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after 12 hours duration. Conclusion: Co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. This may be due to the fact that both drugs are metabolised by the cytochrome P450 pathway in the liver. Background: A higher risk of Antituberculosis drug (ATT) induced hepatotoxicity has been reported in Indian subcontinent compared to the western counterparts. Slow acetylator genotype of N-acetyltransferase2 (NAT2) and cI genotype of Cytochrome P4502E1 (CYP2E1) gene are two known risk factors associated with this disease. CYP2E1 gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. Therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. Objectives: To analyze the allelic distribution of NAT2 and CYP2E1 gene in patients of pulmonary tuberculosis who developed ATT induced hepatitis Materials and Methods: The study included cases of pulmonary tuberculosis (190) and ATT induced hepatitis (95). Polymorphism of NAT2 and CYP2E1 gene was studied by PCR-RFLP method in both these groups. Results: Occurrence of ATT hepatotoxicity was 17.89%. There was a higher prevalence of slow acetylator genotype particularly NAT2*5/*7 and NAT2*6/*7 in patients with hepatotoxicity compared to patients without hepatotoxicity (72.09% vs 44.2%, P value < 0.05). No association of CYP2E1 RsaI polymorphism could be considered with ATT hepatotoxicity. However, DraI C/D genotype of CYP2E1 appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (OR 5.06, P value < 0.05). Conclusion: The study demonstrates that patients with slow acetylator genotype particularly NAT2*5/*7 and NAT2*6/*7 and heterozygous mutant C/D genotype of CYP2E1 gene are predisposed to develop antituberculosis drug induced hepatotoxicity. Regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. Background: Drug-induced liver injury is the most common adverse drug reaction. We often use two kinds of diagnostic scales to evaluate suspected patients. However, we still can't diagnose accurately without the direct drugs history and the pathological evidence. Methods: Twenty-seven drug-induced liver injury cases with liver biopsies from 2001 to 2008 were reviewed retrospectively by Maria and Japanese scale. Result: There were 11.1% of cases with increasing eosinophils. Herbs (29.63%) were the most common suspected drug and unknown drugs intake history (14.81%) were described in these cases. The high possibility and possibility were 25.93%, 29.63% by Maria scale and 85.19%, 3.7% by Japanese scale, respectively (P=0.015). Conclusions: Japanese scale seems more sensitive than Maria scale in these cases. However, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. Early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. It is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. Background: Previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. The study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. Materials and Methods: The 25 days old male Sprague-Dawley rats were subcutaneous injected daily with methamphetamine (10 mg/kg body weight) for 15, 30, 60 and 120 days. Control group received equal volumes of vehicle. The liver tissues were extracted to measure the activities of SOD, catalase, glutathion reductase (GR), and glutathione peroxidase (GPx), and the level of glutathione. Western blot were used to measure the expression of Rho and phosphor-ezrin-radixin-moesin (p-ERM). Results: Compared with vehicle group, treated with methamphetamine for 15 and 30 days, the activities of liver SOD, GPx, and catalase were significantly decreased. In 60 and 120 days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. The levels of glutathione production also had the same trend. The activities of GPx and catalase on vehicle group gradually reduced following the days of treatment. However, administration of methamphetamine resulted to a lower activity of catalase through the treated days. There was no difference on the activity of GR between vehicle and methamphetamine group. The expression of Rho and p-ERM were also increased by methamphetamine treated for 30 days. Conclusion: These results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. Finally, the situation of mechanism needs taking in advantage discussion. Background: To observe intervening effects of preventive and theraptical treatment of Radix Sophorae Tonkinensis's polysaccharides(RSTP) on alpha-naphthylisotheganate(ANIT)-induced cholestasis in mice. Methods: Kunming mice intoxicated with ANIT 50mg/Kg orally and treated with RSTP 50mg/Kg for 7 days before ANIT exposure and for 2 days after ANIT exposure respectively, the general condition,mortality rate and serum ALT activity are obeverated. Result: It was found that by preventive treatment the general condition and mortality rate were improved, serum ALT activity reduced.By therapeutic treatment,the general condition deteriorated,mortality rate and serum ALT activity increased. Conclusion: The preventive treatment of RSTP reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. Background: The product's instruction pointed out that in some patients polyphenolic acids' salt from Salvia Miltiorrhiza(PPAS-SM) may lead to a temporary increase in serum ALT activity.So we observe effects of PPAS-SM on alpha-naphthylisotheganate(ANIT)-induced cholestasis in mice. Methods: 24 hours after intoxicated with ANIT 50mg/Kg orally, Kunming mice were treated with PPAS-SM 50, 25,10mg/Kg/days for 2 days orally, then serum ALT activity was measured. Result: All doses of PPAS-SM led to rise of serum ALT activity in mice, most obvious in group of high dose.But the general situation and mortality rate did not increase significantly. Conclusion: PPAS-SM lead to rise of serum ALT activity in mice with damaged liver.The auther suggests as a double-edged sword,the antioxidant PPAS-SM may have a prooxidative effect in some condition too. *This project was supported by grants from Shanghai Municipal Education Commission under High School High-Tech Characteristic Development Programme (NO SMEC Finance (2005) 81) PE467
A. Somani 1 , A. Jain 2 , V. Dixit 2 , S. Somani 3 1 Navjeevan hospital, 2 IMS, BHU, VARANASI, 3 Suvidha Introduction: Hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (TNF-). Several studies have shown that serum levels of (TNF-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. It has been shown that plasma levels of TNF-correlate with the severity of hepatic encephalopathy (HE) in fulminant hepatic failure. However, still there is very few published data regarding the relationship between serum levels of TNF-and the presence or severity of HE in patients with chronic liver failure. Methods: The aim of this study is to determine the relationship between serum levels of TNF-and clinical grades of HE in patients with chronic liver failure. This prospective study included 149 consecutive male patients with alcoholic cirrhosis in various clinical grades of HE (according to West Haven criterion). Detailed clinical, biochemical and sonographic examination was done in all patients. Circulating levels of TNF-was measured using solid-phase ELISA. Results: The mean±SEM values of serum TNF-at presentation in patients with MHE (n=37), grade 1 (n=17), grade 2 (n=41), grade 3 (n=44), and grade 4 (n=10) were 6.2±0.4, 9.5±0.6, 15±0.7, 26.3±1.7, and 46±5 .9 pg/ml, respectively. Significant Positive correlation was found between serum levels of TNF-and severity of HE (Correlation Coefficient = 0.7). Conclusion: From the present study we can suggest that there is significant relationship between TNF-and HE in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. Background: Acute hepatitis A (AHA) is one of the most common infectious diseases and usually a self-limiting disease. Although extrahepatic manifestations are not common, a few cases associated with acute renal failure (ARF) have been reported.
Methods: We reviewed clinical features of AHA patients complicated with ARF (group A) and compared with non-complicated AHA patients (group B). Medical records of 191 patients with AHA were reviewed between January 2003 and December 2007. We experienced 11 patients (5.8%) with ARF associated AHA. Result: There were no differences between group A and group B in sex ratio and age. The peak value of ALT (median: 6133 IU/L vs 1685 IU/L, p<0.001), Alkaline phosphatase (median: 235 IU/L vs 201 IU/L, p=0.03), prothrombin time (INR, median 1.72 vs 1.09, p<0.001) was significantly higher in group A than B. Nine patients (81.8%) recovered completely with hemodialysis (6 patients, 66.7%) and only conservative management (3 patients, 33.3%), while 1 patient underwent liver transplantation and 1 patient died due to fulminant hepatic failure. There were 4 patients who underwent kidney biopsy. Two patients were diagnosed as acute tubular necrosis and 2 patient as acute interstitial nephritis and IgA nephropathy. Conclusion: AHA patients with ARF had higher ALT and more prolonged prothrombin time. The prognoses were poorer than those without ARF. However, ARF patients with nonfulminant AHA had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. Background/Aims: To investigate the HEV infection among different animals and people with special profession, and to analyse the genotype of HEV isolated in this study. Methods: Serum and fecal samples were collected from various animals and people with special profession in the south suburbs of Beijing. HEV antigen and anti-HEV antibody were detected by DAS-ELISA. HEV RNA was extracted from fecal samples and amplified by RT-nPCR. The nucleotide sequence homology and phylogenetics of HEV strains isolated from swine were analysed. Results: The anti-HEV antibody positive rate of adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. The HEV antigen positive rates of adult swine, cow, sheep and younger swine were 2.65% (6/226), 4.35% (8/184), 1.45% (3/207) and 9.75% (16/164), respectively. The HEV antigen and anti-HEV antibody positive rate of professional group was 0.40% (1/247) and 42.51% (105/247) respectively. The HEV RNA positive rate of fecal samples from younger swine was 22.89 %(19/83). 16 of 19 samples were HEV RNA positive by PCR with primers of HEV ORF1 and ORF2. The sequence analysis of the 16 samples showed that there were 2 groups designated as BJ-1 (11/16) and BJ-2 (5/16). The nucleotide homology of BJ-1 and BJ-2 was 99%. Phylogenetic analysis of HEV ORF2 indicated that both of them belonged to genotype 4d. Conclusion: Phylogenetic analysis of HEV ORF2 indicated that HEV isolated in the south suburbs of Beijing belonged to genotype 4d. 6 Bracops Hospital Brussels , 7 St. Jan Hospital, Bruges , 8 CHU Brugmann, 9 CHU Sart Tilman, Liège , 10 Gent University Hospital , 11 ZNA Middelheim, Antwerp Hepatitis Delta virus is a subviral satellite requiring hepatitis B virus to propagate, usually leading to severe, chronic liver disease. As data on epidemiology and management practice of HDV infection in Belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in 2008. Results of 32 patients are reported. Background/ Aims: Hepatitis A is an acute infectious disease that is transmitted by fecal-oral root. Because the incidence of hepatitis A has been increased in Gwangju and Chonnam province of Korea recently, hepatitis A patients in Chonnam National University Hospital employees had been increased. So we investigated the seroprevalence of IgG anti-HAV in hospital empolyees less than 40 years old. Methods: We analysed seroprevalence of anti-HAV IgG from 1,002 hospital employees (men: 190, women: 812) . Serum ALT and bilirubin at admission were 2,979 1,711 IU/L and 3.9 2.4 mg/dL, respectively. These levels were elevated up to 3,397 1,789 IU/L and 6.8 3.7 mg/dL, respectively. ANA was positive in 81 patients (64.3%). Age, duration from peak-ALT day, duration from peak-bilirubin day, ALT level, and peak-bilirubin level were not different between ANA(-) patients and ANA(+) patients. In the while, sex, duration from symptom-onset day, and bilirubin level, and peak-ALT level were significantly different. In 51 (62%) of 81 patients with positive ANA, ANA was followed after 1 month and ANA became negative in 38 patients (74.5%). Among 13 patients with positive ANA after 1 month, titer decreased from the baseline in 7 patients, showed no interval change in 4, and increased in 2. Conclusions: Positive ANA result is not rare in patients with acute hepatitis A. It is considered that ANA transiently appear during the course of acute hepatitis A and then, disappear with the improvement of acute hepatitis. (89), 2007(107). The clinical data such as sex, admission period, AST, ALT, total bilirubin, prothrombin time, CRP, ALT normalization time did not show difference. Just WBC and GTP were higher on 2008 group. The older age patients were more on 2008 group. The patients admitted mainly on April, May, June, July (84%) on 2008 while admitted even on past years. Conclusion: Acute hepatitis A ptients is increasing. It is occurring in older age people and mainly on specific period. The more concern to prevention should be needed. Background: We analyzed the 5' non-translated region (5'NTR), non-structural proteins 2B and 2C of hepatitis A virus (HAV) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in HAV are responsible for the range of clinical severities. Methods: Our accumulated HAV strains of 5'NTR (nt 200 and 500), entire 2B and 2C from 25 Japanese patients with sporadic hepatitis A, consisting of 7 patients with fulminant hepatitis (FH), 5 with severe acute hepatitis (AHs), and 13 with self-limited acute hepatitis (AH), in whom the sequences of all 3 regions were available, were subjected to phylogenetic analysis. Results: FH patients had fewer nucleotide substitutions in 5'NTR, had a tendency to have more amino acid (aa) substitutions in 2B, and had fewer aa substitutions in 2C, than AH patients. Four FH and 2 AHs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis A and genomic variations in 5'NTR, 2B and 2C of HAV. Conclusions: Our study suggests that genetic variations in some parts of HAV might cooperatively influence replication of the virus, and thereby affect virulence. Viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis A. Aims: The incidence of acute viral hepatitis A in adults is increasing very much in South Korea, 2008. The aim of this study was to the clinical features and course in Daejoen and its surrounding area. Methods: Forty seven patients admitted as acute viral hepatitis A in Chungnam national university hospital between January 2008 and June 2008 were enrolled. The medical records were reviewed, retrospectively. Results: The mean age was 30.5. Common occupations were company employee and studuents. Most common symptom was jaundice. Presumptive infection sources were raw fish or shellfish and raw meat. Initial laboratory findings were as follows(mean value): WBC 6126/uL, Hemoglobin 13.4g/dL, Platelet 212×10 3 /L, AST 2268IU/L, ALT 2755IU/L, total bilirubin 5.2mg/dL, alkaline phosphatase 207U/L, GGT 379U/L, prothrombin time(INR) 1.2. Hospitalization was 21.5days. Peak laboratory findings were as follows: ALT 3181IU/L, total bilirubin 8.8mg/dL. Leukopenia (<4000/uL) and thrombocjtopenia (<10×10 5 /L) were ocurred in sixteen and six patients, respectively. Recovery time of each biochemical finding was as follows: ALT 20.2 days, total bilirubin 26 days. Recovery rates of ALTand total bilirubin were 74.4% and 74%, 88.5% and 92% at 4 weeks, 8 weeks after diagnosis, respectively. Prolonged jaundice (115days) was detected in one patient. All patients were recovered by supportive management. Conclusions: In South Korea, acute viral hepatitis A was more prevalent in young adults, recenlty. Presumptive infectious sources were raw fish or 1 Guangxi Center for Disease Prevention and Control shellfsh and raw meat. If it can not change the food style that many Korean enjoy raw seafood, vaccination for adults must be considered to prevent it.
Objective: To assess the safety and immunogenicity of a new inactivated hepatitis A vaccine (Vero Cell). PE479
Methods: 1507 subjects were selected in Gongcheng city of Guangxi Zhuang Autonomous Region, and the clinical trail was carried out according to the random, double-blind and parallel principle from January to August, 2005. After vaccination by 0, 6 schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (GMT) were tested by the competitive inhibition ELISA. Results: After immunization, the systemic and local reaction rates of adults were 8.80% and2.67%, which was no significantly statistical difference compared with control group, 12.41%and 4.41%; while the rates of children were 10.60and 2.28%%, and no significant statistical difference compared with control group, 10.71%and2.86%. One month after first dose of vaccination, the seroconversion rates of children and adults were 88.2% and 93.8%, and one month after second dose of vaccination, the rates were all 100%, the GMTs of children and adults were 16447mIU/m1 and 8555mIU/ml, which was significant statistical difference in children compared with control group, 1946 mIU/ml and 5881 mIU/ml, respectively.
Methods: IgG anti-HAV was measured in a total of 3905 subjects under the age of 50, who visited Hanyang University Seoul and Guri Hospitals between January 2001 and May 2008.
Results: Fig. 1 shows the relatively low positive rates of the antibody in ages of 11 to 30 and the lowest rates of 9.9% and 9.2% in the age group of 16 to 20, following the ages of 21 to 25 with rates of 12 Background: Some viruses encode proteins that affect their cap-independent internal ribosomal entry site (IRES)-mediated translation and their replication. It was recently reported that hepatitis A virus (HAV) proteases interact with intracellular dsRNA-induced retinoic acid-inducible gene (RIG-I)-mediated signaling, but it remained unknown whether HAV proteins have any effects on HAV IRES-independent translation. In this study, we investigated the effects of 7 HAV non-structural proteins on their IRES-mediated translation using a reporter assay. PE480
Methods: The bicistronic reporter constructs, termed pSV40-HM175-IRES, pSV40-A1-IRES, pSV40-A2-IRES, pSV40-F1-IRES, and pSV40-F2-IRES, contain the SV40 promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by HAV IRES, and are derived from strain HM175, acute convalescent hepatitis clones A1, A2, fulminant hepatitis clones F1, F2, respectively. Human hepatoma cell lines were co-transfected with pSV40-HAV-IRES and each HAV protein-expression vector. Luciferase activity was determined 48 h after transfection. were from other countries within Asia, Africa, Middle East, and Eastern Europe. Patients of a wide age range were affected by hepatitis delta (mean age 46.0, median 47.0, range 19-79). 18 (40%) of 45 were co-infected with HCV. Hepatitis B virus (HBV) DNA was detectable in 43 (78%) patients and negative in 8 (15%) patients. All hepatitis delta patients were extracted from a prior study conducted by this collaboration. There were 1,191 chronic HBV carriers. 18 (1.51%) were HBV/ HCV/HDV infected. 32 (58%) of 55 patients carried a diagnosis of cirrhosis compared to 262 (22%) of 1191 chronic HBV patients. 13 (72%) HCV co-infected patients had evidence of cirrhosis while 4 (22%) patients did not. Conclusion: Individuals with HBV/HDV co-infection have higher rates of cirrhosis. Individuals with HBV/HCV/HDV infection have rates of cirrhosis significantly higher than individuals with either chronic HBV infection or HBV/HDV co-infection. Testing for HDV should be performed in all patients, especially those with advanced liver disease or high risk behavior. Clinical characteristics were compared between the patients with significant endoscopic findings (group A) and without such findings (group B). Peak AST and ALT level were higher in group A (p<0.01). There were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group A and group B Conclusion: Significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. Severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. In patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. Background: In Japan, hepatitis E virus (HEV) testing is not allowed as routine one. To study the role of HEV testing, we checked 22 sera of the patients diagnosed as etiology-obscure acute liver injury. Methods: We have seen 54 cases of acute liver injury from January 2004 through December 2007 in our hospital and 25 cases of them were etiology-obscure. In 25 cases, 22 were retrospectively tested for HEV-IgM, HEV-IgA and HEV-RNA (RT-PCR) by direct sequence method on stored sera taken at the time of presentation. Result: Two of 22 cases (9.1 ) were positive for both HEV-IgM and HEV-IgA and one case was positive for HEV-RNA. In 54 cases of acute liver injury, the cause of virus was 31 cases (57.4%) and unknown was 8 cases (14.8%). HEV was occupied in 3.7% in all cases and 6.5% in the cases caused by virus. One of the two cases had been misdiagnosed as "drug induced hepatitis". HEV of genotype 3 was detected in one case and its nucleotide sequences of HEV showed quite a high degree of similarity to the reported one at closed city in the same year. Conclusion: HEV is not rare in Japan and the HEV testing can reverse the diagnosis of acute liver injury. HEV testing sould be used as routine one for acute liver injury.
Association of Progesterone Receptor Gene with Hepatitis E Disease Severity in Pregnancy P.D. Bose 1 , B. Das 2 , A. Kumar 1 , P. Kar 1 1 Maulana Azad Medical College, 2 
Background/Aims: Incidence of Fulminant Hepatic failure (FHF) in Hepatitis E is high in pregnancy particularly during 3 rd trimester when there is an altered status of hormone and immunity. Progesterone receptor (PR) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. Till now, no data is available whether PR can play any role in Hepatitis E disease severity during pregnancy. PROGINS, a haplotype of PR consisting of 320-bp insertion in intron G together with point mutations in exons 4 and 5 is associated with increased stability and higher transcriptional activity. The aim of the study is to analyze PR mutation (PROGINS) and m RNA expression in hepatitis E virus infected pregnant women with AVH and FHF. Methods: A total of 68 AVH and 32 FHF cases were studied. Blood and placental tissue were collected from the Medicine and Gynecology wards of LNJP hospital, New Delhi. Cases were screened for acute viral markers by commercially available ELISA kit. Extraction of DNA from blood and RNA from placental tissue was done by Qiagen kit. Mutation in PR was detected by PCR-RFLP. Semiquantitative rt-pcr for PR expression was performed in placental tissue using beta-actin as internal control. Results: PR mutation (PROGINS) was significantly more in FHF compared to AVH (28.1% vs 10.3%, P value<0.05). Protein expression was found higher in PROGINS carriers. Conclusion: Progesterone receptor mutation (PROGINS) may have a role in the Hepatitis E disease severity in pregnant women. Results: The hepatitis E was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis B. In the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. The incidence of complication in the superinfected group was higher than the simple infection. The transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis.
Methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and RT-PCR detection. Result: Positive rate of HEV Immunohistochemistry ranged from 90% to 100% (Fig. 1) . Hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (Fig. 2) , and two genotype 4 HEV which closely related to many strain isolated from patients with sporadic acute hepatitis been detected.
Conclusion: The patients infected with hepatitis E of young men were frequently. Jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men.
Conclusion: Additional public-health concerns might be placed on pork safety and the risk of HEV infection via the consumption of undercooked pork products.
Poster Session, Hall 5B 
Aim: Esophageal varices (EV) recurs frequently after endoscopic variceal ligation (EVL) or endoscopic injection sclerotherapy (EIS). We retrospectively investigated risk factors for early recurrence of EV after endoscopic treatment. Methods: We treated 110 patients with EV, who had no past history of EV, at Ehime Prefectural Central Hospital from October 2005 to June 2008. Of those, 78 (71%) were observed for at least 2 months after treatment and enrolled. We divided them into rupture cases at initial endoscopic treatment [(bleeding group; n=25 (32%)], and cases with preventive EVL or EIS performed [preventive group; n=53 (68%)]. All received periodic upper endoscopy examinations to confirm recurrence or no recurrence of EV. Results: Recurrence of EV occurred in 18 of all subjects and the average period after treatment was 6.5±3.0 months. The recurrence rate was significantly higher in the bleeding group (11/25) as compared to the preventive group (7/53) (P=0.0026). There was a significant relationship between recurrence of EV and hepatic reserve function (Child-Pugh A+B, C; 11/64, 7/14 respectively; P=0.0083). In logistic multi-variant analysis, EV rupture at initial treatment and Child-Pugh C were risk factors for recurrence. In contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (EVL or EIS), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. Conclusion: In cases with EV rupture at initial treatment or Child-Pugh C, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations.
Endoscopic Cyanoacrylate Injection: Less Oil for Less Ectopic Embolism C.Z. Li 1 , L.F. Cheng 1 , Z.Q. Wang 1 , F.C. Cai 1 , Q.Y. Huang 1 , E.Q. Linghu 1 1 General Hospital of Chinese PLA Background and Aim: Endoscopic injection sclerotherapy with N-butyl-2cyanoacrylate (NBCA, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. The present study aimed to determine whether less amount of iodized oil preload in NBCA injection helps in decreasing ectopic embolism. Methods: From January 1997 to April 2006, 2 different methods of endoscopic NBCA injection, "Sandwich method" and "modified Sandwich method" (in which iodized oil preload was minimized), were applied on 635 GV cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. Results: Altogether 5 cases of ectopic embolism occurred in the whole group (0.8%), including 3 cases of splenic infarction, 1 case of transient paralysis and 1 case of minor infarction of the lung. The modified Sandwich method showed some superiority over original method in decreasing ectopic embolism (0/276 vs. 5/359, p=0.049). Less cough during procedure was also found with the modified method (3/276 vs. 18/359, p=0.006). Conclusions: Less amount of iodized oil preload in endoscopic NBCA injection is beneficial to decrease ectopic embolism. Background: Portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. Hepatocellular carcinoma (HCC) and low serum sodium (SNa) are manifestations of end-stage liver disease (ESLD) and are associated with poor survival in decompensated cirrhosis patients. Therefore, we aimed to determine the relationship between hepatic venous pressure gradient (HVPG) and the development of HCC or low SNa in decompensated alcoholic cirrhosis patients. Methods: Child-Pugh scores, MELD scores, and HVPG at baseline, and the development of low SNa (SNa <130 mEq/L) or HCC during follow-up were analyzed prospectively in 170 patients with decompensated alcoholic liver cirrhosis. The predictive values of different risk factors for the progression to the ESLD were investigated by multivariate analysis and the Kaplan-Meier method Results: Twenty-four patients developed HCC during the follow-up period. In the multivariate analysis, only baseline HVPG>15mmHg was an independent predictive factor for the development of HCC (relative risk (RR)=1.128, P<0.05). Those with HVPG >15 mmHg showed a significantly shorter time for the development of HCC on Kaplan-Meier analysis. Twenty patients developed low SNa during follow-up. Initial HVPG was also an independent predictive value for the development of low SNa in the multivariate analysis (RR=1.169, P<0.05). Those with HVPG>15 mmHg also showed significantly shorter times for the development of low SNa on Kaplan-Meier analysis. Conclusions: In decompensated alcoholic cirrhosis, HVPG may be a useful predictive factor for the development of HCC and low SNa, both of which are characteristic of ESLD and poor prognosis.
The Effectiveness of the Treatment of Octreotide on Chylous Ascites after Liver Cirrhosis D.X. Zhou 1,2 , H.P. Hu 1,2
Background: Octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. Thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. Methods: Eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between January 2003 and May 2008. Octreotide was given to the six patients, while the remaining two were treated as a control. All patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. All the necessary care was individually given to the patients during the therapy Results: All patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. The volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from 2000 ml to 50 ml with a clear appearance and negative qualitative analysis of chyle For those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. Conclusion: Octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. It appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin <38g/L were the best predictors of large varices. A model using these predictors in a validation cohort study is planned.
Background-Aim: Cirrhosis is associated with raised acute phase proteins (APP), irrespective of infection. It is, however, unclear whether their values differ significantly or whether a particular APP might be more indicative of infection, and these questions were addressed in our study. Methods: We measured serum CRP, fibrinogen, ferritin, haptoglobin, 2-microglobulin, C3, C4, and C1 inhibitor in 88 consecutive, cirrhotic patients, on admission. All patients were investigated according to a standard protocol for infection. Child-Pugh scores (CPS) were calculated. Results of APP were expressed as means SEM and compared with the Mann-Whitney test. Results: 19 (21,6%) patients, median age 60 years, (CPS: A=0; B=7; C=12), were diagnosed with infection (spontaneous bacterial peritonitis=7; pneumonia=5; septic shock=4; extensive cellulitis=1; Listeria Monocytogenes meningitis=1; viral infection=1), while 69 (78%) patients, median age 59 years, (CPS: A=25; B=28; C=16), showed no infection. Although most APP values were raised, there was no statistically significant difference between patients with or without infection, or among different CPS groups, except for CRP, which was significantly more raised in patients with infection (p<0.01). This difference remained even after CPS A cases in the non-infection group were excluded from analysis. Interpretation: A significantly raised CRP in cirrhosis would seem to be independent of CPS staging and should prompt a thorough work up to exclude infection. By contrast, the discriminating power of all other APP in the face of possible infection is negligible. The predictive value of CRP towards infection is under investigation prospectively. Although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. Here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (EISL). Patients and Methods: In 2000-2008, we performed endoscopic treatment in 215 patients with portal hypertensive varices. Among those, four cases of hemorrhagic rectal varices were treated with the combined EVL and sclerosing technique. The etiology of portal hypertension included oen idiopathic portal hypertension and three HCV cirrhosis. All patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. Results: Hemostasis was obtained easily by the EVL initially. Furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (EVIS) using 5% ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. Conclusions: It is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. The EISL technique is useful to control the initial and recurrent bleeding from rectal varices.
T. Hirano 1 , T. Okada 1 , J. Yamanaka 1 , Y. Iimuro 1 , N. Kuroda 1 , K. Oh 1 , Y. Yoshida 1 , J. Fujimoto 1 1 
Aim: Interferon (IFN) therapy is a powerful treatment for HCV-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (HCC). However, thrombocytopenia is a common side effect of IFN treatment, often leads to discontinuance without insufficient therapeutic effect. In this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis C cirrhosis and portal hypertension. Patients and Methods: Out of 41 patients who underwent LS in our department during Aug 2003 and December 2007, 13 patients associated with portal hypertension. Among these patients, three patients had HCC, and they were simultaneously underwent partial hepatectomy after splenectomy. Platelet count, operative time, blood loss, complications and length of stay were calculated. Results: Thirteen patients underwent laparoscopic splenectomy; their mean age was 59 years (range 20 to 68 years). Six patients were Child's class A and seven patients were class B. Mean operative time was 178 minutes (range 97 to 255 minutes). Blood loss was little, and none required transfusion with packed red cells. A hand-assisted laparoscopic technique was used in four cases (30.8%). Average length of stay was 12.7 days. There have been no major complications during follow-up. Platelet counts improved from a preoperative mean of 62000/ul (44000 to 108000) to 273000/ul (126000 to 469000) postoperatively. Six patients are ongoing IFN treatment without remarkable thrombocytopenia. Conclusion: Laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. It may allows these patients by reversing thrombocytopenia. Background: Hepatic encephalopathy (HE) is a significant cause of mortality in advanced cirrhosis patients. L-acyl-carnitine has been suggested as an alternative treatment for patients with HE patients. To assess the clinical efficacy of acetyl-L-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. Methods: We performed a randomized placebo-controlled, cross-over study. We administered acetyl-L-carnitine to group 1 during 3 months first then placebo during later 3 months, and administering acetyl-L-carnitine to group 2 alternatively. Results: Between January 2008 and February 2008, thirty two selected cirrhotic patients were enrolled in this study. Following randomization, the patients were divided into two groups (group 1=14, group 2=18). During administering acetyl-L-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p=0.005, vs. p=0.001 respectively). However, during administering placebo period, serum ammonia level changes were not significant. In group 1, the first recurrence cases of hepatic encephalopathy were more than group 2(group 1=5, group 2=2), and the first recurrences were occurred during first 3 months in all groups. Conclusion: Our study demonstrates that acetyl-L-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. Sodium (Na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. Aim: To assess the relationship between the MELD score and urinary excretion of Na+ in non-azotemic cirrhotic patients. Methods: Fifty four cirrhotic patients with ascites and normal serum creatinine (<1.5 mg/ml) were admitted and placed on a low sodium diet (4g/day), while all diuretics were withdrawn for 3 days. The electrolytes (Na + , K + , Na + / K + ) were measured in a random urine and both the volume and Na + concentration of urine collected for 8 h after administration of furosemide 80 mg i.v. were determined. Results: Table. Conclusions: The MELD score was significantly correlated with the degree of impairment of urinary Na+ excretion. The ratio of Na + /K + in a random urine specimen and furosemide-induced Na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. Background: Portal hypertensive gastropathy (PHG) is common finding in patients with liver cirrhosis and portal hypertension. Despite portal hypertension remains the crucial trigger for the development of PHG, the relationship between portal hypertension and PHG has not been widely investigated. Methods: Fifty-three cirrhotic patients (48 males, mean age 51 years) who were performed hepatic vein catheterization between November 2006 and August 2008 were prospectively included in this study. The degree of PHG was assessed according to the Third Baveno International Consensus Workshop, and classified three degrees as no, mild and severe. The hepatic venous pressure gradient (HVPG=WHVP-FHVP) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. Result: HVPG values did not differ between the patients without PHG (13.28±4.72 mmHg) and those with PHG (14.91±3.84, p=0.187), nor between those with mild (15.31±3.69 mmHg) or severe PHG (14.27±4.12mmHg, p=0.323). The degree of PHG and HVPG did not differ regarding the etiology of the cirrhosis(p=1.0, p=0.085) nor regarding the Child Pugh classification(p=0.085, p=0.738). No correlations were found between the degree of PHG and Child Pugh score, age, with or without ascites, albumin, bilirubin, creatinine, MELD score and the degree of gastroesophageal varices. Conclusions: Our data show that the presence and the severity of PHG does not correlate with the degree of HVPG, and that correlate with esophageal varices in patients with liver cirrhosis. Introduction: Phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. There are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. Case description: A 61-year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. A colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. Abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. A follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. Superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. A right hemicolectomy was performed because of the colonic obstruction. Gross findings on operation showed thickening of the cecum and ascending colon. Microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. Here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in Korea. Spontaneous bacterial peritonitis (SBP) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. Although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. Furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than 50% of patients with suggestive clinical manifestations of SBP. Therefore, diagnosis of SBP is based on the polymorphonuclear leucocytes (PMN) cell count in the ascitic fluid. The Hybrizep kit (Fuso Pharmaceutical Industries, Osaka, Japan) detects the DNA of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. Here we present a case of the patient for whom the Hybrizep kit was used to detect the causal pathogen of SBP. A 76-year-old man had been admitted for the treatment of ascites and esophageal varices. One week after the admission, he complained abdominal pain and fever. Because the PMN cell count in ascites fulfilled the criteria of SBP (1141/mm 3 ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. On the following day of the onset, in situ hybridization showed the positive signals by the EK probe, which detected the genomeic DNA of E.coli species. However, the ascitic fluid culture was negative. This case suggested that the Hybrizep kit was useful for the rapid diagnosis of SBP with high sensitivity. 
Background: It has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. This study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. Patients and Methods: From May 2007 to May 2008, 144 cirrhotic patients were performed the doppler ultrasonography. 91 of these patients were accessed the severity of varices endoscopically. The three dimensional volume of spleen was measured from a length, width and thickness on sonography. Results: The splenic volume (415.2ml vs 505.6ml, p=0.048) and blood flow of main portal vein (12.6cm/s vs 15.1cm/s, p=0.012) were statistically significant different in alcoholic (38/144) and non-alcoholic (105/144) cirrhosis groups. The splenic volume (600.1ml vs 384.4ml, p=0.001), damping index (0.52 vs 0.38, p=0.024), and blood flow of main portal vein(12.5cm/s vs 15.7cm/s, p=0.006) were statistically significant different in esophageal variceal groups (55/91) and non-esophageal variceal groups(36/91). The only splenic volume (669.4 ml vs 461.7 ml, p=0.004) were statistically significant different in gastric variceal groups (24/90) and non-gastric variceal groups (66/90). The hemodynamic parameters venous ammonia and CFF at baseline and after one month of treatment with lactulose. MHE diagnosed by abnormal psychometry and/or P300ERP.Response defined by normalization of abnormal test parameters. Results: MHE diagnosed in 35(58%) patients. Of 35 patients 26 (74%) had both abnormal psychometry and P300ERP whereas30 (86%) alone had abnormal psychometry, 31 (89%) had abnormal P300ERP.CFF was <39Hz in 28(80%) patients. MHE recovered in 55% with treatment and CFF >39Hz was seen in 22(69%) of 32 patients. CFF sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy before and after treatment is shown in table. Conclusions: Critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with MHE. Background/Aims: Endoscopic injection of N-butyl-2-cyanoacrylate (Histoacryl) is an effective treatment of varix bleeding. But nontarget embolizations and septicemia are unwanted complications. We evaluate the risk factors for complications. Methods: Thirty-three patients with esophageal or gastric varix bleeding received endoscopic Histoacryl therapies (54 procedures). Baseline varix size, CTP score were checked. Serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. Average volume of Histoacryl per each session was 1.6 mL, and dilution volume ratio of Histoacryl/lipiodol was 1/1 or 1/2. Results: Average of CTP score was 8.0 ± 1.7. Three cases of septicemia were correlated with CTP score rather than session frequency or injection volume. Two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio (1/2) and lipiodol volume rather than Histoacryl volume or CTP score. Conclusion: CTP score, lipiodol volume and dilution ratio of Histoacryl/lipiodol were significant risk factors for complications.
Detection of Circulating Toll-Like Receptor 2 and 4 and CD4+CD25+ Regulatory T Cells in Patients with HBV-Related Liver Cirrhosis X.Q. Wang 1 , Y. Zhang 1 , X.F. Bai 1 , J.Q. Lian 1 1 
Background : To detect circulating CD4 + CD25 + regulatory T cells and toll-like receptor(TLR)2 and TLR4 expression on the peripheral blood mononuclear cells (PBMCs) of patients with HBV-related liver cirrhosis (LC), and to explore the correlation between them. Methods: PBMCs isolated from 14 LC patients , 21 chronic hepatitis B (CHB) patients and 16 normal controls(NC) were stained with fluorescent labeling anti-TLR2-PE, anti--TLR4-APC, anti--CD14-FITC monoclonal antibodies and anti-CD4-PerCP anti-CD25-FITC anti-CD127-PE. Samples were collected and detected of three-color immunofluo rescence by flow cytometry. Results: The expression of TLR2 and TLR4 were significantly up-regulated in patients with LC than those in the controls.The expression of TLR2 was significantly increased in patients with LC than those in patients with CHB, but there were no differences of TLR4 expression between LC and CHB.Treg/CD4 + T cells were significantly increased in patients with CHB than those in patients with NC and LC, but there were no differences between LC and NC. There were no correlation between the expression of TLR2,TLR4 and Treg in patients with LC . The expression of TLR2 and TLR4 on PBMCs in patients with LC were positive correlation.The expression TLR4 and HBV DNA level were negative correlation in patients with LC. Conclusion: The expression of TLR2 and TLR4 were up-regulated on PBMCs in patients with LC. It seems to be expression of TLR2 and TLR4 invovlved in the pathogenesis of LC.
Evaluation of 13C-Phenylalanine Breath Test for the Measurement of Hepatocyte Function in Patients with Chronic Liver Disease Z.J. Bao 1 , D.K. Qiu 2,3 , X. Ma 2,3 , G.S. Zhang 1 , Y.Q. Huang 1 , Z.P. Fan 2,3 , S.M. Yin 1 1 Huadong Hospital, Fudan University, 2 Renji Hospital, Shanghai Jiao Tong University School of Medicine, 3 
Background: The objective is to investigate whether the 13C-phenylalanine breath test(PBT) would be useful for the evaluation of hepatic function in patients with chronic hepatitis B, liver cirrhosis and minimal hepatic encephalopathy (MHE). Methods: L- [1-13C] phenylalanine was administered orally in a dose of 100 mg to 80 patients with liver cirrhosis, 20 with chronic hepatitis B and 20 healthy subjects. The PBT was measured at 8 different time points (0, 10, 20, 30, 45, 60, 90, 120 min) to obtain the values of Delta over baseline, percentage 13CO2 exhalation rate and cumulative excretion (Cum). The relationships of the cumulative excretion with the 13C-%dose/h and blood biochemical parameters were investigated. Results: The 13C dose h -1 at 20 and 30 min combined with the cumulative excretion at 60 and 120 min showed correlations with the chronic liver diseases, especially Child-Pugh score and MHE or not. And the data showed correlations with serum albumin hemoglobin platelet and Child-Pugh score. Prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at 60 and 120 min values decreased by degrees in healthy controls, Child-Pugh A, B, and C patients (P<0.01). Similar results of PBT were in the patients with and without MHE, while only prothrombin time prolonged and total bilirubin increased (P<0.05). Conclusions: The PBT can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and MHE. The %13C dose h-1 at 20 min, %13C dose h-1 at 30 min and cumulative excretion at 60 min may be the key value for determination at a single time-point.
Branched Chain Amino Acids in Improving Survival and Decreasing Risk of Liver Failure among Cirrhotic Patients: A Meta-Analysis H. Flores 1 , E.L. Ang 1 , N. IV Estanislao 1 1 Philippine General Hospital Background: The state of a patient's nutritional status greatly affects disease outcome. Among cirrhotic patients, approximately 60-90% are in a state of protein-energy malnutrition. Hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. Several studies have shown than branched chain amino acids (BCAA) may be of benefit for this purpose. It is the aim of this study to evaluate the effectiveness of diet plus BCAA compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. Methods: Pubmed, Cochrane, and EMBASE search was done for articles which compared the clinical effects of BCAA supplementation versus diet alone among patients with liver cirrhosis. The following free-text terms and MESH words were used -"Branched chain amino acids", "amino acids, branched chain", "BCAA", "liver cirrhosis", "cirrhosis", "Randomized Controlled Trials'" and "Meta-analysis". After critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (RevMan 4.2). Results: 3 RCTs were included for analysis with a total study population of 836. Combination of the studies showed a significant decrease in the risk of liver failure (OR 0.45, 95% CI 0.25-0.82, p=0.009) and a trend towards benefit in improving survival (OR 0.57, 95% CI 0.27-1.17, p=0.12). Conclusions: The overall trend appears to show benefit in the use of Branched Chain Amino Acids for patients with cirrhosis with respect to liver failure and survival.
Background: The proxisome prolifrator-activated receptor gamma (PPAR ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. On the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. The present study was designed to investigate the effect of specific PPAR ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. Methods: Cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. Both cirrhotic and sham rats were kept for 28 days after the operation. Different groups of sham and cirrhotic animals received saline, or 5, 10 or 15 mg/kg pioglitazone, daily during last 5 days of the fourth week after the surgery. Another 2 groups of BDL or 2 groups of sham rats received L-NAME, a non selective inhibitor of nitric oxide synthase, alone or along with 5 mg/kg pioglitazone for 5 days. On day 28, rats were killed 1 hour after ethanol administration and the area of gastric lesions was measured. Results: The ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (P < 0.001). Pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. Concurrent treatment of L-NAME and pioglitazone decreased the ulcer index in BDL rats more than the groups that received L-NAME or pioglitazone alone. Conclusion: We conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase.
Inhibition of Phosphodiesterase 5 -a Novel Therapeutic Strategy for Portal Hypertension L. Halverscheid 1 , P. Deibert 2 , B. Pannen 1 , R. Schmidt 2 , M. Roessle 2 , W. Kreisel 2 1 University Hospital Duesseldorf, Germany, 2 University Hospital Freiburg, Germany Introduction: The NO-cyclic GMP system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. Clinical data have shown that inhibitors of phosphodiesterase 5 (PDE5) lower portal pressure in cirrhotics. Methods: We monitored in rats the effects of the PDE5 inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to 60 minutes after the drug. The drugs were administered intravenously into the tail vein at 1 (group A), 10 (group B), and 100 g/kg body weight (group C). 0.9% NaCl was the control. N = 7 for each group. Results: The most prominent changes were observed in the vardenafil B group: Mean arterial and portal venous pressure decreased (-9%, -8%), as well as portal venous, hepatic arterial, and systemic vascular resistance (-31%, -30%, -12%). Portal venous and sinusoidal flow increased (+31%, +13%). In the vardenafil C and sildenafil B and C groups there was an increase of portal venous flow by 20-30%, an increase of sinusoidal flow by 12-30%, and a decrease of portal venous resistance by about 25%. There was a trend for reduction of portal venous pressure. Conclusions: Vardenafil and sildenafil influence portal hemodynamics in the rat. Portal venous flow increases by 20-30%, portal venous resistance decreases by >25%. Dependent on the dose, portal venous pressure decreases significantly. These data yield further evidence that PDE5 inhibitors may be a novel therapeutic option for portal hypertension.
Groups of Liver Cirrhosis E. Havrilyuk 1 1 Lviv National Medical University Introduction: Rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. But it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. Aims & Methods: 3713 autopsy cases performed in Lviv regional hospital in 2004-2007 were analyzed. Screening of slides with liver tissue allow to select 580 cases (15,6%) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. Results: According to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease (49,8%), viral hepatitis (7,8%), nonalcoholic steatohepatitis (14,8%), secondary biliary cirrhosis (2,9%), cardiac sclerosis (0,9%), combined lesions (11,9%) and cryptogenic cirrhosis (11,9%). Analysis shows that in 287 cases (49,5%) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in 105 cases (18,3%) -from posthemorrhagic anemia caused by rupture of esophageal varicose. In the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions (60%) and biliary cirrhosis (6,7%) are more frequent. Conclusion: Analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. Probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation.
Patients with Viral Cirrhosis K. Mumtaz 1 , S. Ahmed 1 , H. Ali Shah 1 , S. Hamid 1 , W. Jafri 1 Background and Aims: Increased nitric oxide (NO) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (PHT). We investigated the relative roles of constitutive NOS (eNOS) and inducible NOS (iNOS) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (EIPHT) Methods: EIPHT was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for 6 months. The concentration of NO, expression of NOS (eNOS and iNOS) mRNA and protein was measured in EIPHT and sham operated control animals. Results: Rabbits with EIPHT compared with controls had raised portal pressure (in mmHg-14.34±1.78 vs 6.30±0.60;p<0.05;1mo; 14.91±0.56 vs 7.04±0.42; p<0.05, 3mo; 19.8±3.10, vs 10.2±4.80;p<0.05), arterial hypotension (in mmHg-65.40±3.2 vs 80.04±1.40, p<0.05, 1mo; 62.90±5.40 vs 76.05±2.60,p<0.05, 3mo; 65.85±2.50 vs 79.1±5.10, p<0.05,6mo), splenomegaly (in g-0.92±0.14 vs 0.60±0.13,1mo; 0.90±0.16 vs 0.62±0.04, 3mo; 0.97±0.12 vs 0.67±0.07, 6mo), normal liver functions and preserved hepatic architecture at 1,3 and 6 mo. Serum levels of NO2 as well as the NO3 were significantly elevated in EIPHT rabbits as compared to the controls. The expression of eNOS, at the level of mRNA, was significantly increased in EIPHT rabbits consistent with increased levels of expression of iNOS as compared to the controls. The eNOS but not iNOS protein expression was elevated in EIPHT than control rabbits. Conclusion: Vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by eNOS and partly by iNOS gene up-regulation.
Liver Cirrhosis Contributed to the Immunocompromised Status by Shedding the Membranous TNFRII T.N. Lin 1 , C.H. Chao 1 , I.S. Sheen 1 , Y.P. Ho 1 , W.T. Chen 1 , C.J. Lin 1 , C.T. Yeh 1 , C.Y. Lin 1 1 Liver Research Unit, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan Background & Aim: Patients with decompensated liver cirrhosis (DLC) were regarded as immunocompromised, reflected by high incidence of bacterial infection. Paradoxically, the proinflammatory cytokine like TNFincreased significantly in patients with DLC even in the face of this immunocompromised status. On the other hand, regulatory T cell (Treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of TNF-effect through soluble TNFRII. Here, we studied the role of Treg cells and TNFRII in patients with decompensated liver cirrhosis. Patients and Methods: 33 healthy volunteers and 78 cirrhotic patients were enrolled. The percentage of Treg cells were enumerated by flow and serum levels of IL-10, TGF-and TNF-by ELISA. Results: The percentage of Treg cells increased significantly in patients with DLC associated with increased serum levels of IL-10 and TGF-. In addition, these Treg cells were mainly memory type reflected as high CD45RO. Furthermore, the TNFRII expression increased significantly on these Treg cells of DLC. Interestingly, these membranous TNFRII on Treg cells could be shed-off. Lastly, we found the serum soluble TNFRII concentration increased significantly in patients with DLC when compared with normal volunteers. Conclusion: Our results demonstrated memory Treg cells with high TNFRII expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of TNF-by shedding membranous TNFRII and contributed to the immunocompromized status of DLC. Background: Portal pressure measured as hepatic venous pressure gradient (HVPG) correlates with severity of portal hypertension and the development of complications. HVPG measurement is invasive. Recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. This study was conducted with the aim to study the correlation between portal pressure as measured by HVPG and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. Methods: Between August and September 2008, consecutive patients with portal hypertension were included and were subjected to HVPG measurement and fibroscan (Echosens, France). Results: Of the 18 patients with portal hypertension, both HVPG and liver stiffness were measurable in 12[9 (75%) males; mean age 37.5(10.6) years]. The etiological distribution was HBV related cirrhosis in 3 patients, HCV cirrhosis in 3, cryptogenic cirrhosis in 2, alcoholic cirrhosis in 2, HBV and alcoholic cirrhosis in 1and primary extra-hepatic portal vein obstruction in1. The mean HVPG and liver stiffness of this group were 13.9 (5.1) mm hg and 26.6 (16.5) kPa respectively. There was a strong positive correlation between HVPG and liver stiffness [r = 0.708; p = 0.01]. Conclusions: Non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. Liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension.
Endpoints were rate of rebleeding and mortality till day 30 after inclusion and to see for any adverse events. Results: The bleeding was stopped in all 15 patients (100%). Rebleeding till day 30 was observed in 4 (26%) patients (2 each in group A and B). Total 2 patients (13%) died (1 each in both groups) due to rebleeding. Transfusion needs were higher in group A (4.2±2.8 versus 2.3±2.1, p<.05). Serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. Conclusion: Prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. However transfusion requirements were significantly decreased in patients receiving prolonged treatment. Serious adverse effects leading to treatment discontinuation are rare.
Poster Exhibition -Miscellaneous Poster Session, Hall 5B Background: It is important to know the prevalence of ATP7B gene mutations of different geographical areas to justify the local screening strategies for Wilson disease (WD). Materials: Eleven unrelated Lithuanian families, including 13 WD patients were tested. Genomic DNA was extracted from whole venous blood using a salt precipitation method. Firstly, semi-nested PCR technique was used to detect the c.3207C>A (p.H1069Q) mutation. Patients not homozygous for c.3207C>A (p.H1069Q) mutation were further analyzed. The 21 exons of the WD gene were amplified in a thermal cycler. Direct sequencing of the amplified PCR products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. Results: Total of 13 WD patients (mean age 26.4 years; range 17-40; male/female, 3/10) presented with hepatic disorders and 16 their first degree relatives were studied. Some of WD patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia 3; Fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). Twelve of 13 (92.3%) WD patients have had c.3207C>A (p.H1069Q) mutation, 6 of them in both chromosomes, 5 were presented as compound heterozygotes with additional c.3472 -82delGGTTTAACCAT, c.3402delC or c.3122G>A (p.R1041Q) mutation. For one patient with liver cirrhosis and psychiatric disorder no mutations were found. Out of 16 first degree WD relatives 11 (68.7%) were heterozygous for c.3207C>A (p.H1069Q) mutation. Conclusion: c.3207C>A (p.H1069Q) missense mutation is characteristic for Lithuanian WD patients. Even 92.3% of WD patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. Background: Diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. We investigated the relationship between glutamic-pyruvic transaminase (GPT) and high-density lipoprotein cholesterol (HDL-C) in diabetic patients. Methods: With informed consents, we recruited outpatients with diabetes at a hospital in rural area in Taiwan in [2004] [2005] [2006] [2007] . Anthropometric measures, blood tests and urine screening were examined in diabetic patients. Results: Overall, there were 1241 diabetic patients aged 19-91 years enrolled in this study and 660 (53.2%) of them had low HDL-C. Diabetic patients with the highest quintile of GPT had higher average of body mass index (p<0.0001), diastolic blood pressure (p = 0.003), but lower average of HDL-C (p<0.0001) compared with diabetic patients with the lowest quintile of GPT. The prevalence of obesity (45.7% vs. 24.8%, p<0.0001) and low HDL-C (64.6% vs. 48.3%, p<0.0001) were higher in diabetic patients with highest quintile of GPT than in diabetic patients with lowest quintile of GPT. In the multivariate logistic regression, diabetic patients with highest quintile of GPT had higher odds ratio (OR) of low HDL-C compared with diabetic patients with lowest quintile of GPT (OR = 1.88, 95% confidence interval [CI] = 1.21-2.92). The corresponding OR of low HDL-C in patients aged 70 years and older was 3.30 (95% CI = 1.15-9.47). Conclusion: High GPT is one of factors associated with low HDL-C in diabetic patients.
The Effect of Desferrioxamine as supplement to Cefotaxime in the Treatment of Spontaneous Bacterial Peritonitis NA. Seda 1 , M. El-hamamsy 2 , R. El-wakil 3 , M. Al Azizi 4 1 Ain Shams Specialized Hospital, 2 Faculty of Pharmacy,Ain Shams University, 3 Faculty of medicine,Ain Shams University, 4 Faculty of Pharmacy ,Ain Shams University Background: Oxidative damage lead to cell damage, organ dysfunction and death in sepsis. Desferrioxamine (DFX), an antioxidant iron Chelators. The aim was to assess the efficacy of Desferrioxamine supplemented to Cefotaxime in the treatment of Spontaneous Bacterial Peritonitis (SBP) in cirrhotic patients. Methods: thirty patients divided into two groups: Group I (n=15) with SBP and receiving Cefotaxime (1g IV every12 hours) alone and Group II (n=15) with SBP receiving Cefotaxime (1g IV every 12 hours) with desferrioxamine (500mg IM twice daily).All patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. Results: The concomitant administration of Desferrioxamine with Cefotaxime significantly at (p<0.001)and(p<0.01) improved the therapeutic outcome and the cure rate after 5 days of treatment as compared to patients using cefotaxime only. Conclusions: Desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. Oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. Bacteremia due to this organism is an extraordinary finding. We describe here a case of Oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. A 51-year-old male man was admitted to hospital with 9-month duration of debility, poor appetite and abdominal distension. Decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. Laboratory results showed positive serum anti-HCV and high serum HCV RNA level. The patient received a therapeutic regimen of pegylated interferon alpha 2a plus ribavirin after being admitted to hospital. During hospital stay, a fever of 2-day duration with shivering occurred to the patient. Three blood cultures were drawn, which all grew Oligella ureolytica in pure culture. The organism was identified by the Viteck 2 Compact (Biomerieux, France). Additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at 42.7C. Tests for nitrite reduction and motility resulted negative. The organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. A 14-day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. Within 2 days, the patient became afebrile. Background: Endoscopic ultrasound (EUS) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. An unremarkable EUS exam and negative hepatology workup often leads to a liver biopsy. EUS may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. Aim: To assess the feasibility and efficacy of EUS guided core biopsy of the liver in a porcine model. Methods: Female pigs were used and live procedures were performed under general anesthesia. A linear echoendoscope was used and the liver identified endosonographically. Transgastric core biopsies of the liver were obtained with a 19 gauge Quick-Core ultrasound biopsy needle (Wilson-Cook) and sent for histopathologic evaluation. Live animals were euthanized at the end of the procedure and necropsy performed. Results: Core biopsies of the liver biopsy were obtained in 4 animals (1 cadaver and 3 live anesthetized). A total of thirteen needle passes were made (mean 3.25; range 2 -4 needle passes per animal) and a visible core of tissue obtained. The maximum length of liver tissue obtained was 10 mm and considered adequate for assessment as more than one such specimen could be obtained. Microscopic evaluation confirmed liver tissue. No complications were noted. Necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. Conclusion: EUS-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing EUS examination for abnormal liver tests. Background: As the common indexes, ALT, AST and PLT play an important role in disease diagnosis, treatment and prognosis. Many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis B and C patients whose ALT level was persistently normal. A large sample investigation showed that the serum level of ALT in healthy persons is lower than the normal reference value. This study re-evaluated the normal serum level of ALT, AST and PLT. Methods: 3815 people were enrolled in the study between Sep. and Oct. 2007. The platelet count and serum ALT and AST levels were measured. Frequencies, One-Sample Kolmogorov-Smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. Result: In the five groups, there is significant difference in ALT and AST levels between male and female. In group 1, the ALT and AST levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. In the three groups the PLT level is significantly different between male and female, and the serum level in male is higher than female. There is significant difference between different age groups Conclusion: The serum levels of ALT, AST and PLT are all significantly different between male and femal. There is significant difference between different genders and age groups for PLT. The serum level of PLT is higher than the reference value. Background/aims: Myeloproliferative disorders (MPD) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for 50% cases of hepatic venous thrombosis (HVT) and 35% of portal venous thrombosis (PVT) in western series. Latent form of MPD lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. A point mutation at Val617Phe of Janus kinase 2 tyrosine kinase gene (JAK2 V617F mutation) occurs in high proportion of the patients with MPD. This non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various MPD. This test may be useful in diagnosing latent form of MPD in splanchnic venous thrombosis Methods: 232 patients with confirmed pyogenic liver abscesses admitted from 1995 to 2007 in our institution were included. There were 145 men and 87 women ranging in age from 19 to 91 years. The medical records were reviewed for clinical, laboratory and radiographic characteristics. Results: Among 232 patients, 81 (34.9%) experienced at least one complication. There were 67 pulmonary (pleural effusion, pneumonia, empyema) complications, 16 septic shock, 12 acute renal failure, 2 abscess rupture, 2 pseudomembranous colitis, and 2 pericardial effusion. The predictive factors for its complications were: systemic inflammatory response syndrome (SIRS, 2 factors), thrombocytopenia ( 80,000/mL), hypoalbuminemia ( 3.0g/dL), elevated AST or ALT (>200 IU/L), hyperbilirubinemia ( > 2.0 mg/dL), K. pneumonia, air within abscess cavity (p<0.05). Conclusions: The incidence of complications in the pyogenic liver abscess was 34.9%. The various predictive factors of complication should be monitored carefully. Further large scaled study should be warranted. Background/Aims: Hepatic iron deposition is a common feature in chronic hepatitis C (CH-C), however, whether it could enhance the progression of fibrosis or not is controversial. The aim of this study was to evaluate the status and significance of hepatic iron deposition in the Korean patients with chronic hepatitis C. Methods: Untreated, 78 CH-C patients who underwent liver biopsy were included. The hepatic iron was assessed by Scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. Clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. Results: Hepatic iron staining was positive in 26(33%). Among 26 patients with hepatic iron deposition, serum levels of ferritin (p=0.005) and -fetoprotein (p=0.002), and body mass index(BMI) (p=0.039) were significantly elevated. There was no significant association between the degree of hepatic iron deposition and fibrosis stage (p=0.321), although elevated levels of serum hyaluronic acid (p=0.049), -Glutamyl transpeptidase (p=0.028), and prothrombin time (p=0.012) were associated with advanced fibrosis. Conclusions: Hepatic iron deposition in Asian-Pacific CH-C patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. Therefore, phlebotomy might not commonly applicable to this area. Further studies on the pathogenic role of iron in CH-C in Asian-Pacific countries are warranted. A late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. Limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative Human hepatic stem cells(hHpSCs) transplantation. hHpSCs are located in the ductal plates in fetal and Canals of Hering in adult livers [Schmelzer et al.(2006) ]. Hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [SchmelzerE etal(2006) ].hHpSCs express CD326(EpCAM)marker. Scjelzer etal demonstrated that during embryogenesis 90% of the EpCAM positive cells had hepatoblast phenotype. In animal study, on transplantation of freshly isolated hHpSCs in SCID mice results in mature liver tissue expressing human-specific proteins. Recently, we(Aleem etal 2008)have shown clinical improvement in study in patients with Crigler-Najjar syndrome, Biliary atresia using hHpSCs infusion. In the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with MELD score >30. hHpSCs were sorted using MACS with CD326 antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. All the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. Ascites was decreased and changed Encephalopathy grade into normal level was observed. MELD score system falling to normal level from >30 to <22 after infusion. 1 The Aga Khan University patients. The APRI of 1.5 in combination with a cut-off HA of 300 ng/ml can best detect patients with moderate to severe fibrosis (stages 2-4). It has a PPV of 93.7%. Also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of 98.9%). But the APRI of 1.5 in combination with different HA as cut-off points is not possible to detect patients with no or mild fibrosis.
Objectives: Terlipressin is used in esophageal variceal bleed (EVB) along with Endoscopic Band Ligation (EBL) for 3days (UC). Due to its high cost, it was stopped <3days (SC) who could not afford & were stable after achieving hemostasis with EBL. We retrospectively assessed the efficacy of SC Vs UC of Terlipressin for control of EVB and length of stay. Conclusion: The APRI of 1.5 in combination with HA 300 ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages 2-4) is an easy and accurate method.
Methods: Patients with EVB who had achieved hemostatsis with EBL from Jan 2004-Dec 2005 were included. All were managed on standard protocol on hospital variceal bleeding pathway. The course of Terlipressin as SC or UC was based on patient's inability to afford the cost of hospitalization and Terlipressin. The Efficacy of Terlipressin in the control of EVB was defined based on Baveno III criteria. Results: Total of 117 patients were admitted during the study period. Out of them, 66 received UC & 51 SC of Terlipressin. The base line characteristics were comparable except younger age in SC. There were 2 re-bleed (3%) in UC and 1 (2%) in SC Terlipressin group. The length of stay was shorter in SC group. (2.47±0.57 vs 6.15±2.92 days). Conclusions: SC seems as effective as UC Terlipressin in the control of EVB after initial control of hemostatsis with EBL and may reduces the length of hospital stay. RCTs are needed to assess this as all stable patients may not need to continue Terlipressin for 72 hours. Background/Aims: To analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (CEVB), and establish a simple model for timely predicting death rsik of the patients.
Outcome of patients with gastro-oesophageal bleeding in a tertiary center J. Wat, W.H. Li, M.T. Cheung
Methods: The medical documents of CEVB patients were reviewed retrospectively and the data were collected. Univariate and multivariate Logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. The multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated.
Objectives: To determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. Method: This is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. The study period was from January 2000-October 2008. Data were collected from our hospital computer system and records.
Results: In univariate regression, the significant positive variables for death outcome were DBIL, AKP, K, WBC and PLT, and the significant negative variables were TP, AP, A/G, Na, Cland Ca 2+ . The variables entered the multivariate regression are ALT, TBIL, DBIL, GP, A/G, Cr, Na + , Cl -, Ca 2+ , WBC, Hb, PLT. The sensitivity, specificity and accuracy of the regression model for predicting death of CEVB patients were 97.1%, 95.1% and 95.8%. Results: A total of 172 patients were included in this study, with 122 male and 55 female. Their mean age was 61.9. The initial failure rate in endoscopic haemostasis was 15.1%. The 5-day and 6-week mortality rates were 11.8% and 21.9 %, respectively. Poor Child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable HCC, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of 6-week mortality (p < 0.05). Mean duration from index bleed to first rebleed was 15.1 months. Poor Child's grading and presence of inoperable HCC were associated with both early or multiple rebleed (p <0.05). Overall, 37.2% of our patients developed rebleed before their variceal eradication. 5-year survival in patients with Child's A, B and C were 65%, 22%, and 10%, respectively (Log rank test p 0.000).
Conclusions: The liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for CEVB patient death risk, especially DBIL, A/G and Ca 2+ . The established model shows a excellent predictive performance.
An Imbalance in Plasma Amino Acids of Advaced Cirrhotic Patients Impairs the Maturation Of Dendritic Cells Via Mtor/S6K Signaling Pathway E. Kakazu 1 , Y. Ueno 1 , Y. Kondo 1 , K. Fukushima 1 , M. Shina 1 , J. Inoue 1 , K. Tamai 1 , M. Ninomiya 1 , T. Shimosegawa 1 1 Division of Gastroenterology, Tohoku University Hospital Conclusion: Although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor Child's grading and inoperable HCC. This will result in significant bleeding-related death and poor overall survival. Further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis.
Background: We have demonstrated that extracellular branched-chain amino acids (BCAAs), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (J Immunol. 179: 2007) . However, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (DCs). Methods: We used human PBMCs and CD1c+DCs in this study. We made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n=100) was defined as the healthy control medium (HCM); whereas that from advanced cirrhotic patients (n=50) was defined as the advanced cirrhotic  Reading chest radiographs in the critically ill (Part II): Radiography of lung pathologies common in the ICU patient This is part II of two series review of reading chest radiographs in the critically ill. Conventional chest radiography remains the cornerstone of day to day management of the critically ill occasionally supplemented by computed tomography or ultrasound for specific indications. In this second review we discuss radiographic findings of cardiopulmonary disorders common in the intensive care patient and suggest guidelines for interpretation based not only on imaging but also on the pathophysiology and clinical grounds.  Cytomegalovirus infection in critically ill patients: a systematic review INTRODUCTION: The precise role of cytomegalovirus (CMV) infection in contributing to outcomes in critically ill immunocompetent patients has not been fully defined. METHODS: Studies in which critically ill immunocompetent adults were monitored for CMV infection in the intensive care unit (ICU) were reviewed. RESULTS: CMV infection occurs in 0 to 36% of critically ill patients, mostly between 4 and 12 days after ICU admission. Potential risk factors for CMV infection include sepsis, requirement of mechanical ventilation, and transfusions. Prolonged mechanical ventilation (21 to 39 days vs. 13 to 24 days) and duration of ICU stay (33 to 69 days vs. 22 to 48 days) correlated significantly with a higher risk of CMV infection. Mortality rates in patients with CMV infection were higher in some but not all studies. Whether CMV produces febrile syndrome or end-organ disease directly in these patients is not known. CONCLUSIONS: CMV infection frequently occurs in critically ill immunocompetent patients and may be associated with poor outcomes. Further studies are warranted to identify subsets of patients who are likely to develop CMV infection and to determine the impact of antiviral agents on clinically meaningful outcomes in these patients. Cytomegalovirus (CMV) is a major  herpes virus and a significant human pathogen. Infection is common with seroprevalence rates increasing steadily from 65% among 40 to 49 year olds to 91% in those aged 80 years or over [1] . After primary infection, CMV, like other  herpes viruses, establishes lifelong latency. In immunocompetent individuals, asymptomatic viral shedding may be detectable in saliva or urine; however, cell-mediated host immune responses prevent the development of overt CMV disease.
In contrast, CMV infection has been shown to lead to significant disease in immunocompromised hosts such as those with HIV infection or transplant recipients. End-stage HIVinfected patients with a CD4 lymphocyte count of less than 50 cells/mm 3 are at the highest risk of developing CMV retinitis [2] . In transplant recipients, CMV disease occurs in 11 to 72% of patients especially in the first three months after transplant while the patients are receiving maximum immunosuppression [3] . In addition to febrile syndrome and end-organ disease directly as a result of viral replication, immunomodulatory characteristics of CMV may contribute to opportunistic infections, allograft rejection, and higher mortality in transplant recipients. Clinical trials have shown that preventive approaches utilizing antiviral agents have lead to a reduction in the rates of CMV infection and disease, and indirect sequelae associated with CMV [3] [4] [5] . Currently, prophylaxis or periodic monitoring and antiviral therapy targeted towards patients with viral replication are routinely employed at many transplant centers. APACHE : Acute Physiology and Chronic Health Evaluation; ARDS: acute respiratory distress syndrome; BAL: bronchoalveolar lavage; CI: confidence interval; CMV: cytomegalovirus; HHV: human herpesvirus; HSV: herpes simplex virus; ICU: intensive care unit; IL: interleukin; MeSH: medical subject headings; NF-B: nuclear factor-B; OR: odds ratio; PCR: polymerase chain reaction; SAPS: simplified acute physiology score; SOFA: sepsis-related organ failure assessment, TNF: tumor necrosis factor; VAP: ventilator-associated pneumonia.
It has increasingly come to be recognized that critically ill patients who are traditionally considered immunocompetent may also be at risk for CMV infection. For example, septic insult as a result of bacterial or fungal infections has the potential to promote the release of immunomodulatory cytokines and lead to reactivation of CMV [6, 7] . Reactivation from the latency rather than primary infection is believed to be the cause of CMV infection because none of the critically ill CMV seronegative patients developed CMV infection as opposed to 13 to 56% of seropositive patients [8, 9] . Several observational studies have shown an association between CMV infection in critically ill patients and poor clinical outcomes [8, 10, 11] . However, available data are limited by relatively small sample sizes, diversity in patient populations studied, difference in methodological assays employed for CMV, and variability in reported outcomes that preclude generalizability of the results of the individual reports. The objectives of this review are to summarize the frequency and predictors of CMV infection, and outcomes in critically ill immunocompetent patients with CMV infection. Additionally, we discuss the pathophysiologic basis of CMV reactivation and the implications of these data for optimizing outcomes in critically ill patients.
English-language reports of published studies on CMV infection in critically ill immunocompetent patients were identified through November 2008 by cross-referencing the following medical subject headings (MeSH) keywords and text words: cytomegalovirus, immunocompetence, critical illness, intensive care units, intensive care, reactivation, sepsis, and shock. Databases searched included PubMed, EMBASE, Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials. Bibliographies of original articles were manually reviewed for additional articles. Non-Englishlanguage reports were also identified in PubMed using the same keywords in order to supplement our search.
We included studies in which: critically ill immunocompetent adults were monitored either retrospectively or prospectively for the development of CMV infection in the ICU and; the rate of CMV infection was explicitly reported. CMV infection was defined as evidence of positive viral cultures, antigenemia, and/or DNAemia by PCR from blood or a clinical specimen. Patients were considered to be immunocompetent if they were not solid organ or hematopoietic stem cell transplant recipients, not infected with HIV, did not have primary immunodeficiencies, and were not recipients of immunosuppressive agents such as calcineurin-inhibitors, anti-TNF- drugs, antilymphocyte antibodies, or chemotherapeutic agents for treating cancer. We excluded studies in which an increase in CMV serologic titers in the absence of viremia was the sole evidence for CMV infection.
Two of the authors independently searched articles and extracted the following data for analyses: study design, inclusion criteria, type and frequency of CMV assays, rate of CMV infection, rate of CMV IgG positivity, the time elapsed from ICU admission to CMV infection, risk factors for CMV infection, and outcomes (i.e. mortality, duration of ICU stay). Any discrepancies were resolved by review and discussion. Authors of published studies were contacted if reported data required further clarification. Additional mortality data was provided in one study [12] .
The initial database search identified 524 English-language and 77 non-English-language studies. After review of the title and abstract and manual search for bibliographies of the potentially relevant articles, 26 studies were selected for fulltext review . Ten studies were excluded after full-text review because: CMV infection was diagnosed based on an increase in titers [19] [20] [21] [22] [23] [24] ; the study was not explicitly conducted in the ICU [25] ; or the rate of CMV infection was not reported [26, 27] . Data from one institution with overlapping study cohorts in two articles were analyzed only once to avoid duplication in the results [16, 28] . We found three studies in which CMV disease was sought as etiology of acute respiratory distress syndrome (ARDS) or ventilator-associated pneumonia (VAP) [29] [30] [31] . Considering that CMV disease (organspecific symptoms or signs plus the detection of CMV in organ biopsy samples by histopathology) is a distinct entity with worse outcomes than CMV infection, the data from these studies were summarized separately.
Thus, we identified a total of 13 studies that have described CMV infection in immunocompetent critically ill patients with sample sizes ranging from 23 to 237 (Table 1) [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . These included nine prospective observational studies [7, 8, [10] [11] [12] [13] [14] [15] [16] , three retrospective studies [9, 17, 18] , and one study where insufficient details were available to determine the type of the study [6] . CMV IgG was positive in all the subjects in four studies [10, 11, 14, 16] , not measured in four studies [12, 15, 17, 18] , and positive in 28 to 94% of the subjects in five studies [6] [7] [8] [9] 13] . Other inclusion criteria were sepsis in four studies [6, 7, 16, 17] , mediastinitis in one study [13] , simplified acute physiology score (SAPS) II of 41 or higher in one study [10] , prolonged ICU stay in three studies [8, 9, 16] , fever for more than 72 hours in one study [18] , and shock or organ failure in two studies [12, 16] . There were five studies in which corticosteroid use was documented [8, 12, [16] [17] [18] . In one study, eight of 48 patients were recipients of long-term corticosteroid therapy or had a malignancy [12] . In a case-control study, 22 of 40 of the cases and 13 of 40 of the controls were recipients of short-term corticosteroids ( 3 months) [18] . The details of corticosteroid use were unavailable in three studies [8, 16, 17] . 
Based on nine prospective studies that assessed CMV infection in all study subjects, the rate of CMV infection ranged from 0 to 36% with the median rate of 25% [7, 8, [10] [11] [12] [13] [14] [15] [16] . The specimens used to assess CMV infection included blood, urine, and respiratory secretions. All the studies used blood to assess CMV infection. Blood was used solely in four studies [7, 11, 12, 15] , whereas urine and respiratory specimen in addition to blood samples were used in two [13, 16] and four studies [8, 10, 14, 16] , respectively. The rate of CMV infection was reported separately based on the type of specimens in these studies (Table 1 ). Considering only studies that assessed for viremia, the CMV infection rate ranged from 0 to 33% with the median rate of 20%. Thereafter, we reported the rate of CMV infection diagnosed based on the presence of viremia.
Next, studies were categorized based on the frequency of virologic monitoring because it can influence the rate of CMV infection. In five studies in which CMV infection was monitored at least weekly, the rate of CMV infection ranged from 6 to 33% with the median of 32% [7, 8, 10, 11, 16] . In contrast, CMV infection rate was 0.8 to 2.1% in two studies in which assessment for CMV infection was performed only once, 1.8 to 4 days after ICU admission [12, 15] .
There are three methods of diagnosing CMV infection: viral cultures, antigenemia, and PCR assays [32] . Culture-based assays (conventional and shell-vial cultures) are considered obsolete because of their low sensitivity and time-consuming nature. The antigenemia assay is based on direct detection of the CMV protein pp65 using monoclonal antibodies. It is sensitive and quantitative, although it requires sufficient leukocytes in peripheral blood and is more labor-intensive than the PCR assays. Finally, the PCR assays have been considered gold standard given their high sensitivity and rapid turnover time, although these are not fully standardized [33] . In our review, the assays used to assess CMV infection were viral cultures in five studies [8, 10, 13, 14, 16] , antigenemia in three studies [7, 12, 16] , and PCR in six studies [7, [10] [11] [12] 14, 15] . CMV infection rate was 0 to 20% (median 4%), 0 to 32% [ 
(median 18%), and 0 to 33% (median 17%) by viral cultures, antigenemia, and PCR, respectively in these indices. PCR assay and antigenemia were performed in all patients in two studies [7, 12] . Twelve of 82 patients developed CMV infection detected by the PCR assay compared with six of the 82 patients by antigenemia. No cases of CMV infection were diagnosed solely by antigenemia. Lastly, we focused on three studies in which the PCR assay was used at least weekly because this method is currently widely utilized to monitor CMV infection; the rate of CMV infection was 32 to 33% in these studies [7, 10, 11] .
Based on five studies where CMV infection was assessed at least weekly, the mean (or median) time to onset of CMV infection ranged from 4 to 28 days [7, 8, 10, 11, 16] . Among studies where PCR was used, the mean (or median) time ranged from 4 to 12 days [7, 10, 11] . A PCR assay seems to help diagnose CMV infection earlier than the antigenemia assay. In a study that used both methods for the diagnosis of CMV infection, the median time between onset of sepsis and CMV infection determined by PCR and antigenemia were four days (range 1 to 23 days) and 11 days (range 1 to 23 days), respectively [7] .
Candidate variables assessed as predictors for CMV infection varied for different studies and primarily included demographic and clinical characteristics, and severity of illness markers. Age did not appear to be a risk factor for CMV infection [7, 8, 10, 11, 13, 16, 18] and the association between CMV infection and gender was inconsistent [7, 8, 10, 11, 13, 16, 18] . Other risk factors identified included mechanical ventilation at admission (odds ratio [OR] 8.5, 95% confidence interval [CI] 1.1 to 66.5 for high-grade CMV viremia, i.e. CMV PCR > 1000 copies/ml) [11] , bacterial pneumonia [8] , and sepsis (OR 4.62, P = 0.02) [10] . Corticosteroid use was a risk factor in one study [8] . In a retrospective case-control study where variables used in a multivariate logistic regression model were not clearly documented, neither corticosteroid use nor sepsis was a risk factor for CMV infection [18] . Transfusion within 24 hours of admission was associated with high-grade viremia, i.e. CMV viral load greater than 1000 copies/ml (OR 6.7, 95% CI 1.1 to 42.7) [11] ; however, no association between CMV infection and transfusion during hospitalization was documented in two other studies [18] . The mean number of packed red blood cell transfusion was larger in patients with CMV infection than in those without it (22.3 units vs. 11.2 units, P = 0.002); however, this difference was not statistically significant after controlling for other risk factors [8] . Malignancy was not associated with CMV infection [9, 10, 18] . None of the disease severity scores including Acute Physiology and Chronic Health Evaluation (APACHE) II score [7, 8, 11, 13] , sepsisrelated organ failure assessment (SOFA) score [16] , or SAPS II [10, 18] correlated with a risk of CMV infection.
Organ dysfunction Organ dysfunction was reported in three studies [8, 13, 18] . One study showed a higher incidence of hepatic dysfunction (international normalized ratio > 1.5 or total bilirubin > 2.5 mg/ dL) in CMV infection group (70% vs. 36%, P < 0.047) [8] . In two studies, renal failure was observed more frequently in those with CMV infection (55 to 58% vs. 32 to 33%, P < 0.05 in each study) [13, 18] .
Based on six studies where the duration of ICU stay was assessed, the mean (or median) duration of ICU stay ranged from 33 to 69 days in patients with CMV infection as compared with 22 to 48 days among those without (P < 0.05 in each study) [8] [9] [10] 13, 16, 18] .
The mean (or median) duration of mechanical ventilation ranged from 21 to 39 days in patients with CMV infection compared with 13 to 24 days in those without CMV infection in four studies (P < 0.05 in each study) [8, 9, 16, 18] .
Nosocomial infection was more frequently observed in patients with CMV infection as compared with those without CMV infection in one study (75% vs. 50%, P = 0.04) [18] .
A higher level of CMV viremia was associated with death or continued ICU hospitalization at 30 days in one study [11] . An OR of combined outcome of death or continued ICU hospitalization at 30 days was 1.7 (95% CI 1.2 to 2.4) for each logarithmic increase in maximum CMV viral load measured [11] .
Mortality rate in critically ill patients with CMV infection was 29 to 100% as compared with 11 to 74% in those without CMV infection. Except for two retrospective studies [9, 18] , no other studies showed a significant difference in the mortality rates between those with and without CMV infection. CMV viremia at any level was associated with death or continued ICU hospitalization at 30 days (OR 5.7, 95% CI 2.1 to 15.6) [11] .
Human herpesvirus (HHV) -6 and -7 have been associated with a greater risk of developing CMV disease in transplant recipients [34] [35] [36] [37] . Furthermore, HHV-6 and HHV-7 have been shown to be significant contributors to morbidity and poor outcomes, particularly when concurrent infection with CMV exists [35, [38] [39] [40] . Thus, it is of interest to investigate whether the association between CMV and other herpes viruses exists in critically ill patients.
HHV-6 infection has been frequently observed in critically ill patients [12, 15] . In one study, HHV-6 infection occurred in 53.5% of all patients (54/101) requiring hospitalization in the ICU as compared with none of the healthy volunteers [15] . In another study, HHV-6 infection occurred in 54% of all ICU patients with at least two organ failures as opposed to 15% of those with less than two organ failures [12] . HHV-6 viremia was assessed by the PCR assay at a mean of day 4 and day 1.8, respectively [12, 15] . Potential association between CMV and HHV-6 infection could not be assessed because only one patient developed CMV infection in each study. Both studies failed to show an association between HHV-6 infection and mortality. HHV-7 infection was observed more commonly in healthy volunteers (18/50, 36%) as compared with ICU patients (14/101, 14%; P = 0.002) in one study [15] .
Three studies evaluated herpes simplex virus (HSV) infection in conjunction with CMV infection. In one study, HSV was isolated in the bronchial aspirate of 8 of 25 patients (32%) consisting of six of eight patients with CMV reactivation and 2 of 17 patients without CMV infection (P = 0.004) [16] . In other studies, none or only one patient developed co-infection of CMV and HSV, therefore, the association between CMV and HSV could not be evaluated [8, 17] .
CMV disease and acute respiratory distress syndrome or ventilator-associated pneumonia CMV pneumonia was diagnosed by open-lung biopsy while investigating the etiology of pulmonary disease in 29 to 50% of critically ill patients with ARDS or VAP (Table 2 ) [29] [30] [31] . In a study in which open-lung biopsy led to the diagnosis of CMV pneumonia in 30% of critically ill patients with ARDS, lung tissue culture was positive only in 10% of these patients [29] . The sensitivity/specificity of bronchoalveolar lavage (BAL), blood, and urine culture for the diagnosis of histologically proven ventilator-associated CMV pneumonia was 53%/92%, 20%/83%, and 13%/62%, respectively [31] . Clinical outcome data were largely lacking; however, there was no difference in duration of ICU stay in one study [31] .
Our review demonstrates that depending on the methodological assay used and the patient populations studied, CMV infection occurs in 0 to 36% of the critically ill otherwise immunocompetent hosts in the ICU. Among the most frequently studied inciting event for CMV infection in these patients is sepsis [6, 7, 13, 16, 17] . The risk of CMV infection was five-fold higher in patients with sepsis even when controlled for age and the initial severity of illness [10] . In a murine model of CMV infection, cecal ligation and puncture resembling post-surgical intraabdominal sepsis led to reactivation of latent CMV in the lungs and ultimately pulmonary fibrosis [41, 42] . The propensity of sepsis to promote CMV infection may result from its pleiotropic effects on the host immune system. Pro-inflammatory cytokine production such as TNF- and IL-1 in the early phase of sepsis has the potential to activate NF-B and other transcription factors that are key in the reactivation of CMV from latency [43, 44] . The later phase of sepsis, characterized by the generation of immunosuppressive cytokines such as IL-10 and IL-4 is often referred to as compensatory anti-inflammatory response syndrome [45, 46] . Once latent virus is reactivated, these cytokines may further enhance CMV replication. Indeed, in lung transplant recipients, elevated levels of IL-10 in the BAL and/or plasma were associated with delayed CMV clearance [47] . A sustained high level of IL-10 in patients with sepsis has been associated with poor outcomes, presumably due to excessive anti-inflammatory effects [48] .
Transfusion within 24 hours of admission was identified as a risk factor for high-grade CMV viremia in critically ill patients [11] . This association may be explained by potential transmission of CMV by blood products, but more likely by the immunomodulatory effect of transfusion per se. Previous studies have shown that allogeneic blood transfusion resulted in a reduction in T-helper cells, induction of suppressor T cells, and suppression of natural killer cell activity [49] . The transfusionrelated immunosuppression has been associated with clinically important sequelae such as improvement of renal allograft survival, increased risk of tumor recurrence, and postoperative infections [50] [51] [52] . The risk of CMV transmission by leukocyte depleted blood products is at least as low as by CMV seronegative blood products [53, 54] , supporting the hypothesis that transfusion-related immunomodulatory effect plays a major role in CMV infection in critically ill patients if transfusion were truly a risk factor. However, these data were not available for most studies. For example, only one in three studies that evaluated transfusion as potential risk factors for CMV infection reported use of leukocyte depleted blood products explicitly [18] .
A body of literature based largely on serologic assays for the diagnosis of CMV suggests that severe burn injuries are a major risk factor for CMV infection [55, 56] . At least a four-fold rise in serologic titers suggestive of CMV reactivation has been documented in 45 to 56% of the burn patients [19, 21, 22] . Recently, in a study where patients with severe burn injuries comprised a subset of critically ill patients, CMV viremia using PCR was observed in 55% (11/20) of the burn patients [11] . Burn injuries are associated with profound changes in cell-mediated immunity and a predominant Thelper 1 cell response that may facilitate CMV infection [57] [58] [59] . Susceptibility to sepsis due to the loss of skin integrity in these patients may also contribute to the risk of CMV infection.
Attempts to utilize the severity of 'critical illness' to predict CMV infection have not shown a correlation between scoring systems such as APACHE II or SAPS II and the risk of CMV [7, 8, 10, 11, 13, 16, 18] . Severity of illness scores have typically (page number not for citation purposes)
been assessed in the first 24 hours after ICU admission whereas CMV infection does not usually occur until late in the ICU stay. Additionally, these scores are based on age, physiologic parameters, basic laboratory values, and chronic medical conditions and may not be necessarily representative of host immunologic deficits that lead to CMV infection.
CMV infection rate was 0.8 to 2.1% in two studies where the PCR assay was performed only once at a mean of 1.8 and 4 days following the onset of illness requiring ICU admission [12, 15] . The median time to first detectable CMV viremia was 12 days (range 3 to 57 days) in a study where the PCR assay was performed thrice weekly [11] . Thus, it appears that CMV infection is a rare event very early in the course of critically ill patients and that most infections develop between 4 and 12 days after the onset of illness requiring ICU stay, which could lead to a hypothesis that CMV infection may coincide with the development of compensatory anti-inflammatory response syndrome, and not with the initial surge of pro-inflammatory cytokines.
A key question is whether CMV infection adversely affects outcomes in critically ill patients. Virtually all studies have documented that CMV infection was related to prolonged mechanical ventilation and duration of ICU stay in patients with CMV infection. CMV infection has also been associated with organ system failure and at least two studies have documented significantly higher mortality rates in patients with CMV infection compared with those without it [9, 18] . Thus, although these data do not prove a causal association as CMV infection may have been more likely to develop or diagnosed in sicker patients, existing evidence suggests that CMV infection is associated with poor outcomes even in immunocompetent critically ill patients. We believe that a causal association between these can only be assessed by carefully conducted clinical trials designed to show whether suppression of CMV has a mitigating effect on the severity of illness.
Another major unresolved issue is whether CMV infection is associated with overt disease or clinical manifestations directly attributable to this virus in critically ill patients. CMV infection in immunocompetent patients generally presents with mononucleosis-like symptoms including fever and malaise with liver enzyme abnormalities [60, 61] , which are typically benign. However, 31 to 42% of the hospitalized patients with CMV infection may have organ involvement [60, 62] and rarely life-threatening CMV infection has also been reported [63, 64] . In critically ill patients, 10% (2/20) of those with CMV infection eventually developed severe CMV disease (pneumonitis, neurologic disease) in one study [10] . CMV pneumonia has also been diagnosed in 29 to 50% of patients with ARDS or VAP [29] [30] [31] ; however, this does not necessarily mean that CMV is the cause of ARDS or VAP. Critical illness due to serious pulmonary disease may predispose these patients to CMV infection in the lungs. In a cohort study in the ICU, 17% of critically ill patients who experienced fever for three or more days had CMV infection [18] . Current guidelines for the evaluation of new fever in critically ill adult patients list transfusion-asso- ciated CMV mononucleosis as a cause of fever [65] . However, it remains to be determined whether and how often CMV produces febrile syndrome and whether coexistent infection with HHV-6 is a contributor to this entity as shown in the transplant setting [34, [36] [37] [38] [39] [40] .
Experimental studies have shown that ganciclovir prevented murine CMV reactivation and the development of pulmonary fibrosis in immunocompetent mice with sepsis [42] . Two retrospective studies where small subsets of ICU patients received antiviral agents for CMV infection have yielded inconclusive results and data on the utility and efficacy of antiviral therapy for CMV in critically ill immunocompetent patients are largely lacking [17, 18] . Employment of potent antiviral therapy in all critically ill patients may be impractical, logistically infeasible, and potentially harmful given a large number of ICU patients and potential adverse effects of ganciclovir such as bone marrow suppression or teratogenicity. A more prudent approach may be to identify subgroups of patients at high risk for developing CMV infection and targeting antiviral prophylaxis towards these patients. These subgroups may include patients with sepsis, persistent fever, or those receiving transfusion. An alternative approach is to employ antiviral therapy only in those with CMV viremia. Regardless, carefully conducted clinical trials are warranted to discern the impact of antiviral agents on clinically meaningful outcomes before employing antiviral therapy or even considering routine monitoring of CMV in critically ill patients.
Several limitations of our study deserve to be acknowledged. We found considerable heterogeneity in the methodology used to assess CMV infection and in patient characteristics. As noted in the Results, the frequency and type of CMV monitoring influenced the rate of CMV infection. Although all the studies in this review were conducted in the ICU, the overall mortality of the studied patients ranged from 5 to 71% [7, 15] , suggesting that study populations were significantly diverse. Furthermore, these studies were published over a period spanning nearly two decades in different regions with diverse clinical practices. Considering the heterogeneity of available data, quantitative analyses such as meta-analysis can be misleading [66, 67] and our results are therefore presented in a descriptive fashion only. Second, while we excluded the recipients of iatrogenic immunosuppressive agents that enhance the risk of CMV reactivation, critically ill patients in whom corticosteroids were employed were included. Controversy abounds whether corticosteroids alone without other immunosuppressive agents lead to reactivation of CMV from latency or merely promote the replication of activated virus [68] [69] [70] .
Corticosteroids were employed in a subset of patients in 5 of 12 studies in this review. Given that corticosteroid use is a common practice in the ICU [71] , these studies reflect clinical scenarios encountered by care providers and therefore their inclusion in this review was deemed appropriate.
In summary, accumulating data suggest that CMV infection is a frequent occurrence in critically ill patients. Considering a large number of patients requiring ICU level of care, the scope of impact of CMV infection in these patients may be equally or potentially wider than in other immunocompromised hosts traditionally recognized to be at risk for CMV infection. For example in the USA, an estimated 383,000 cases with sepsis require ICU admission as opposed to 28,360 solid organ transplant cases yearly [72, 73] . Mortality rate in patients with sepsis ranges from 20 to 50% and approaches 70% in those with multiple organ failure [74] . Furthermore, the incidence of sepsis and the number of sepsis-related deaths appear to be increasing [73, 74] . Precise identification of the role of CMV as a contributor to outcomes in these patients may therefore have far reaching implications. Subsets of critically ill patients who are at risk for developing CMV infection and for poor outcomes remains to be determined. These studies have significant implications for future investigations to determine the potential benefits and for guiding the study design to evaluate the impact of antiviral agents on clinically meaningful outcomes in critically ill patients.
The authors declare that they have no competing interests.
RO participated in the study design, literature search, data acquisition, interpretation of the data, and the drafting of the manuscript. NS participated in the study design, literature search, data acquisition, interpretation of the data, and the revision and editing of the manuscript. Both authors read and approved the final manuscript.
• CMV infection occurs in 0 to 36% (median 25%) of critically ill patients between 4 and 12 days after ICU admission, especially those with sepsis, requiring mechanical ventilation, and receiving transfusion.
• CMV infection is associated with poor outcomes; however, it is not known whether the causal association exists, that is, CMV is truly a pathogen or CMV infection is just an indicator of immunosuppression.
• It remains to be determined whether CMV produces febrile syndrome or end-organ disease directly in critically ill patients.
• Further studies are warranted to identify subsets of patients who are at high risk of developing CMV infection and to determine the role of antiviral agents on clinically important outcomes in critically ill patients. Heliox reduces respiratory system resistance in respiratory syncytial virus induced respiratory failure INTRODUCTION: Respiratory syncytial virus (RSV) lower respiratory tract disease is characterised by narrowing of the airways resulting in increased airway resistance, air-trapping and respiratory acidosis. These problems might be overcome using helium-oxygen gas mixture. However, the effect of mechanical ventilation with heliox in these patients is unclear. The objective of this prospective cross-over study was to determine the effects of mechanical ventilation with heliox 60/40 versus conventional gas on respiratory system resistance, air-trapping and CO2 removal. METHODS: Mechanically ventilated, sedated and paralyzed infants with proven RSV were enrolled within 24 hours after paediatric intensive care unit (PICU)admission. At T = 0, respiratory system mechanics including respiratory system compliance and resistance, and peak expiratory flow rate were measured with the AVEA ventilator. The measurements were repeated at each interval (after 30 minutes of ventilation with heliox, after 30 minutes of ventilation with nitrox and again after 30 minutes of ventilation with heliox). Indices of gas exchange (ventilation and oxygenation index) were calculated at each interval. Air-trapping (defined by relative change in end-expiratory lung volume) was determined by electrical impedance tomography (EIT) at each interval. RESULTS: Thirteen infants were enrolled. In nine, EIT measurements were performed. Mechanical ventilation with heliox significantly decreased respiratory system resistance. This was not accompanied by an improved CO2 elimination, decreased peak expiratory flow rate or decreased end-expiratory lung volume. Importantly, oxygenation remained unaltered throughout the experimental protocol. CONCLUSIONS: Respiratory system resistance is significantly decreased by mechanical ventilation with heliox (ISCRTN98152468). Respiratory syncytial virus (RSV) is the most important causative agent of lower respiratory tract disease (LRTD) in infancy [1] . Approximately 100,000 infants are annually admitted with RSV-induced bronchiolitis in the USA, and the number of hospitalizations is increasing [2] . Because of this, RSV-associated disease imposes a major burden on health care resources [3] .
There is no effective therapy against RSV available, prevention can only be achieved through passive immunisation using monoclonal antibodies [4] . RSV LRTD is pathophysiologically characterized by sloughed necrotic epithelium, excessive mucus secretion, bronchial mucosal oedema and peribronchial inflammation that contributes to airway obstruction resulting in increased airway resistance with subsequent air-ANOVA: analysis of variance; ARDS: acute respiratory distress syndrome; CO 2 : carbon dioxide; Cstat: static compliance; EELV: end-expiratory lung volume; EIT: electrical impedance tomography; ELISA: enzyme-linked immunosorbent assay; ET-CO 2 : end-tidal carbon dioxide; FiO 2 : fraction of inspired oxygen; LRTD: lower respiratory tract disease; MAP: mean airway pressure; MV: mechanical ventilation; OI: oxygenation index; PaCO 2 : partial pressure of arterial carbon dioxide; PaO 2 : partial pressure of arterial oxygen; PEEP: positive end-expiratory pressure; PEFR: peak expiratory flow rate; PICU: paediatric intensive care unit; PIP: positive inspiratory pressure; Ptrach: intratracheal pressure; relative Δ EELV : relative change in end-expiratory lung volume; Rlung: lung resistance; Rrs: respiratory system resistance; RSV: respiratory syncytial virus; SPO 2 : oxygen saturation; V D : dead space; VI: ventilation index; Vte: expiratory tidal volume.
trapping and respiratory acidosis [5, 6] . Although the majority of infections run a mild disease course, mechanical ventilation (MV) for up to 10 days is necessitated in approximately 2% to 16% of previously healthy hospitalised infants due to severe lower respiratory tract infection including bronchiolitis or pneumonia [1, 7, 8] .
Helium is an inert gas with a density that is one-seventh that of air. In addition, carbon dioxide (CO 2 ) diffuses more easily through helium than through air [9] . With helium, a more laminar flow is preserved in narrowed airways, resulting in lower resistance to gas flow allowing for increased bulk flow [10] . Based on these properties, MV with heliox could be considered in mechanically ventilated infants with RSV LRTD. Its use in these patients has been studied once but with inconclusive results [11] .
We hypothesized that the use of heliox in mechanically ventilated infants with RSV LRTD would result in decreased respiratory system resistance (R rs ). In addition, MV with heliox would result in less air-trapping defined by the relative change in end-expiratory lung volume (EELV), and improved CO 2 clearance. The objective of our study was to test this hypothesis in a prospective, double cross-over intervention trial comparing heliox 60/40 with conventional gas (nitrox) using lung function testing and electrical impedance tomography (EIT) measurements.
The study protocol (ISCRTN98152468) was approved by the hospital's Institutional Review Board and written informed consent was obtained from patients before enrollment.
Eligible for inclusion were infants younger than 12 months of age with a virologically confirmed clinical diagnosis of RSV LRTD (either a positive direct immunofluorescent assay or ELISA) who were admitted to the nine-bed paediatric intensive care unit (PICU) facility of the VU university medical center for MV during the RSV seasons (autumn and winter) between 2005 and 2007. Infants were excluded if no informed consent was obtained, fraction of inspired oxygen (FiO 2 ) was more than 0.4, corticosteroids were used prior to admission, they were on high-frequency oscillatory ventilation or a haemodynamically significant congenital heart defect (i.e. significant left-to-right shunting with or without pulmonary hypertension) was present.
Patients were in supine position, intubated with an uncuffed endotracheal tube size 3.5 or 4.0 mm, and put on a timecycled, pressure-limited ventilation mode (Pressure Control, AVEA ventilator, Cardinal Health, Yorba Linda, CA, USA). Aims of ventilation were transcutaneously measured oxygen saturation (SpO 2 ) 88 to 92%, and partial pressure of arterial carbon dioxide (PaCO 2 ) 45 to 65 mmHg (if pH >7.25). Inspir-atory times were fixed at 0.5 seconds, positive end-expiratory pressure (PEEP) was set 1 to 2 cmH 2 O below total PEEP (i.e. extrinsic PEEP + intrinsic PEEP). The flow-time curve was observed thoroughly throughout the study period in each patient to examine if expiration was complete in order to prevent dynamic hyperinflation. Patients were sedated with midazolam and morphine, paralysis was achieved using intravenous rocuronium. Endotracheal suctioning was performed 30 minutes prior to the start of, but not during, the experimental protocol. Bronchodilators (either nebulized or intravenous) or ketamine were not used before or during the study period.
Arterial blood samples were drawn from an arterial line to determine PaCO 2 and partial pressure of arterial oxygen (PaO 2 ). End-tidal carbon dioxide (ET-CO 2 ) concentration, and expiratory tidal volume (V Te ) were measured at the airway opening. ET-CO 2 was measured using a side-stream Microstream (Philips Medical Systems, Best, The Netherlands) and V Te was measured with a proximal flow sensor connected to the AVEA ventilator (Cardinal Health, Yorba Linda, CA, USA). The ventilator is designed to detect which gas is used and adjusts its pneumotachograph automatically in order to measure the correct V Te .
A chest radiograph was obtained and evaluated by one pediatric radiologist in each patient prior to the start of the experimental protocol to evaluate the presence of hyperinflation (defined by a depression of the diaphragm below the sixth anterior rib) or an infiltrate (described as opacities with irregular markings without loss of volume) [12] .
The experimental protocol started within 24 hours of PICU admission and lasted for 90 minutes. At four intervals (T = 0 (baseline), T = 30, T = 60, and T = 90 minutes) data were collected and respiratory variables measured. At T = 0 and T = 60, patients were ventilated with nitrox. At T = 30 and T = 90, patients were ventilated with heliox (helium 60%, oxygen 40%). Ventilator settings were kept constant throughout the experimental protocol.
Positive inspiratory pressure (PIP), intratracheal pressure (Ptrach), mean airway pressure (MAP), PEEP, SpO 2 , ET-CO 2 , respiratory rate and V Te were measured. Ptrach was measured with a pressure transducer placed at the distal end of the endotracheal tube. Blood samples were drawn for the determination of the PaO 2 , PaCO 2 and pH. Static compliance (C stat ), R rs and peak expiratory flow rate (PEFR) were measured using the AVEA ventilator (Cardinal Health, Yorba Linda, CA, USA) according to the manufacturer's manual. In summary, R rs was defined by the ratio of the airway pressure differential to the inspiratory flow 12 ms prior to the end of inspiration. Lung resistance (R lung ) was defined by the ratio of the tracheal pressure differential to the inspiratory flow 12 ms prior to the end of inspiration.
At each interval, EIT measurements were made using the Göttingen Goe-MF II EIT system (Cardinal Health, Yorba Linda, CA, USA). Sixteen electrodes (Blue Sensor BR-50-K, Ambu, Denmark) were applied circumferentially around the infant's chest at the mammary line. A 30 second reference measurement at 13 Hz scan rate was recorded. All further measurements were referenced to this measurement. All other measurements were made at a scan rate of 44 Hz for 180 seconds. A 5 mA peak-to-peak, 50 kHz electrical current was injected at each adjacent electrode pair, and the resultant potential differences were measured at the remaining adjacent electrode pairs. Subsequently, all adjacent electrode pairs were used for current injection, thus completing one data cycle. The impedance map was built using the back-projection image reconstruction algorithm [13] . It calculates the relative impedance ΔZ, defined by (Z inst -Z ref )/Z ref (where Z inst is the instantaneous local impedance and Z ref the reference impedance, determined from each cycle of current injections and voltage measurements in each pixel).
Both the respiratory and cardiac components of the EIT signal were identified in the frequency spectra generated from all EIT measurements (Fourier transformation). The EIT data was lowpass filtered with a cut-off frequency of 2 Hz to eliminate small impedance changes synchronous with the heart beat [14] .
The calculations performed on the sums of values from all pixels of the 32 × 32 pixel matrix EIT image were described as 'global'. In addition, sums of values from the left and right lung regions were described separately, and the entire EIT image was divided into 64 regions-of-interest (32 left and 32 right lung) from anterior to posterior as previously described by Frerichs and colleagues [15] . Ventilation-induced tidal volume (ΔZ VT ) was quantified by measuring the relative ΔZ from the highest point at end inspiration to the lowest point at end expiration, and an average ΔZ was calculated from multiple breaths. Changes in ΔZ VT were calibrated to volume using the known V T . The relative change in end-expiratory lung volume (relative ΔZ EELV ) was determined by measuring the median impedance from the lowest point at expiration during the sampling time (Z EELV ) [16] . The relative ΔZ EELV was normalized to volume (relative Δ EELV in ml) by multiplying the median impedance with the ratio V T /ΔZ VT .
The oxygenation index (OI) was calculated as follows: (FiO 2 × 100 × MAP in cmH 2 O)/PaO 2 in mmHg. The ventilation index (VI) was calculated as follows: (PaCO 2 in mmHg × respiratory rate × (PIP -PEEP in cmH 2 O))/1000. VI is used as determinant for CO 2 elimination because the respiratory rate, PIP, and PEEP were kept constant throughout the study period [17] . Dead space (V D ) was calculated according to the Bohr-Enghoff equation: V D = V Te × (1 -(P ET-CO2 /PaCO 2 )) [18] .
As no data on relative Δ EELV in mechanically ventilated infants with RSV LRTD were available, we performed a power analysis after inclusion of all patients using the paired t-test.
The data were analyzed with one-way repeated measures analysis-of-variance (ANOVA) with Tukey post-hoc testing between T = 0 versus T = 30, T = 30 versus T = 60, and T = 60 versus T = 90. P < 0.05 was accepted as being statistically significant. Data are expressed as mean ± standard deviation unless stated otherwise. Statistical analysis was performed using SPSS version 15.0 (Chicago, IL, USA).
Thirteen patients were included in 11 EIT studies; good-quality EIT signals were obtained from nine patients. Descriptive data, ventilator settings and baseline data of respiratory system mechanics and gas exchange are summarized in Table 1 . Although three patients were born prematurely (one at 32 weeks and two at 36 weeks' gestation), none of the patients had chronic lung disease. Hyperinflation was present in 10 patients, four of these patients also had infiltrates. Ten patients had hypercapnia (PaCO 2 >45 mmHg) and seven infants had PaO 2 /FiO 2 less than 200 at baseline (T = 0). Tidal volume remained constant throughout the experiment (Figure 1 ). Leakage around the uncuffed endotracheal tube was less than 5% in all patients.
Mechanical ventilation with heliox had an overall significant effect on R rs (P < 0.001; Figure 2 ). R rs decreased from 69.1 ± 6.9 cmH 2 O/L/sec at T = 0 to 50.2 ± 6.0 cmH 2 O/L/sec (P = 0.020) after 30 minutes of ventilation with heliox. After reintroduction of nitrox, R rs increased significantly to 70.7 ± 7.2 cmH 2 O/L/sec (P = 0.016) but decreased again to 42.9 ± 3.3 cmH 2 O/L/sec (P = 0.001) when heliox was reintroduced.
Course of tidal volume Course of tidal volume.
(page number not for citation purposes) R lung was not significantly influenced by MV with heliox ( Figure  3 ).
PEFR was not significantly improved by MV with heliox compared with nitrox (P = 0.520; Figure 4 ). C stat was 1.9 ± 0.4 L/ cmH 2 O at T = 0 and not significantly different throughout the study (P = 0.214; Figure 5 ).
The mean relative Δ EELV ± standard deviation at T = 0 was 76.6 ± 15.1 ml. With an estimated reduction of 25% with heliox, nine patients were needed to recruit in order to detect a statistically significant difference with α 0.05 and β 0.90. The degree of airtrapping as defined by the relative Δ EELV in ml was overall not significantly reduced by heliox (P = 0.493; Figure  6 ). This was due to differences in response to MV with heliox. Five patients showed a reduction in relative Δ EELV when heliox was introduced, and when conventional gas was reintroduced relative Δ EELV increased in only three patients (Table 2 ). There were also patients who had an increase of relative Δ EELV with heliox that was either reversed or increased when conventional gas was reintroduced.
To investigate if a time-dependent effect of heliox could be found, the change in relative Δ EELV was correlated with the change in R rs for T = 30 to T = 0 (R 2 0.068, P = NS), T = 60 to T = 30 (R 2 0.110, P = NS) and T = 90 to T = 60 (R 2 0.498, P = 0.01).
Fractional ventilation (i.e. the distribution between left and right lung), as well as the center of ventilation of the left and right lung, also remained constant throughout the study period (Table 3) . Table 4 summarizes the effect of mechanical ventilation with heliox on indices of gas exchange and V D /V T . Elimination of CO 2 defined by the VI (P = 0.661), as well as a reduction in V D /V T (P = 0.929) was not positively influenced by MV with heliox. Importantly, oxygenation as defined by the OI (P = C stat = static compliance; EIT = electrical impedance tomography; N/A = not available; PaCO 2 = partial pressure of arterial carbon dioxide; PaO 2 = partial pressure of arterial oxygen; PEEP = positive end-expiratory pressure; PEFR = peak expiratory flow rate; PIP = positive inspiratory pressure; Pt = patient; R rs = respiratory system resistance.
0.477) and alveolo-arterial oxygen gradient (Aa-DO 2 ) remained unaltered throughout the study period.
The major finding of our study is that MV of infants with RSV LRTD with heliox 60/40 resulted in a significant reduction of the respiratory system resistance.
Increased R rs resulting from airway narrowing due to sludging, excessive mucus secretion, edema, and possible bronchoconstriction has been described in mechanically ventilated infants with RSV LRTD [19] [20] [21] [22] [23] . Measures to alleviate increased R rs such as nebulisation of bronchodilators or nitric oxide have yielded inconclusive results [20, 22, 24, 25] . However, these studies are methodologically different compared with ours. For instance, we excluded patients with chronic lung disease or congenital heart disease.
The decrease in R rs led not to an improved CO 2 clearance as defined by the VI or a reduction in PEFR. Some explanations for this may be proposed. First, it is uncertain how much of the observed reduction in R rs could be partitioned to the ventilator circuit or the endotracheal tube because no endotracheal suctioning was performed during the study. Increased mucus production during RSV LRTD is common, and may further obstruct the airways [26] . As the AVEA ventilator is able to calculate the R lung , we also studied if MV with heliox resulted in a reduction in R lung , but were unable to demonstrate this. This could mean that MV with heliox does not affect the resistance of the small airways of the infants; it cannot be ruled out, how-
Effect of mechanical ventilation with heliox on respiratory system resist-ance Effect of mechanical ventilation with heliox on respiratory system resistance. Data are expressed as mean ± standard deviation. * P < 0.05 T = 30 vs T = 0; ** P < 0.05 T = 60 vs T = 30; *** P < 0.05 T = 90 vs T = 60.
Effect of mechanical ventilation with heliox on lung resistance Effect of mechanical ventilation with heliox on lung resistance. Data are expressed as mean ± standard deviation.
Effect of mechanical ventilation with heliox on peak expiratory flow rate Effect of mechanical ventilation with heliox on peak expiratory flow rate. Data are expressed as mean ± standard deviation.
Effect of mechanical ventilation with heliox on static compliance Effect of mechanical ventilation with heliox on static compliance. Data are expressed as mean ± standard deviation. ever, that the resolution of the AVEA's signal of R lung (1 decimal) might not be sufficient enough to detect true differences in R lung in small children with little tidal volume. Second, the measured R rs in our patients is lower than previously reported in mechanically ventilated infants with RSV LRTD designated to have an obstructive disease phenotype [20, 22, 27] . This could indicate that our patients had mild-to-moderate airway obstruction, although hyperinflation suggesting airway obstruction on chest radiograph was present in all but one patient. Unfortunately, there is no gold standard for the radiological definition of hyperinflation especially in mechanically ventilated infants. Furthermore, the degree of air-trapping might vary between patients, indicating that severe RSV LRTD necessitating MV is a heterogeneous disease in which patients express to a varying degree both restrictive and obstructive disease characteristics explaining why some patients had a PaO 2 /FiO 2 ratio of less than 200 or a C stat less than 0.3 ml/cmH 2 O/kg in our study. This assumption opposes the previously proposed dichotomization of RSV LRTD by Hammer and colleagues, who have observed that mechanically ventilated infants with RSV LRTD showed either a disease pattern compatible with acute respiratory distress syndrome (ARDS) or a disease pattern characterized by increased airway resistance [27] . Although our study was not designed to investigate differences in clinical phenotype, we would dare to challenge this dichotomy in clinical phenotype for several reasons. Hammer and colleagues included prematurely born infants with chronic lung disease and infants with congenital heart disease [27] . C rs is significantly lower in these patients compared with healthy infants [28] [29] [30] . In addition, the term 'bronchiolitis' to describe RSV LRTD is strictly speaking a histopathologic diagnosis and hampered by universal differences in its clinical interpretation [31] . Controversy exists about whether differences in parameters for gas exchange correlate with clinical phenotype [32, 33] .
The lack of improved CO 2 clearance in our study is compatible with the observations by Gross and colleagues [11] . They were unable to demonstrate a beneficial effect on PaCO 2 of various heliox mixtures (ranging from 50%/50% to 70%/30%) compared with T = 0 (PaCO 2 45 ± 10 mmHg) in 10 mechanically ventilated infants with moderate severe RSV LRTD. It should be mentioned, however, that our study population was probably more ill than theirs based on a higher T = 0 PaCO 2 and lower PaO 2 /FiO 2 ratio. Previously, we did observe a beneficial effect of heliox in a small infant with obstructive airway Effect of mechanical ventilation with heliox on relative change in end-expiratory lung volume Effect of mechanical ventilation with heliox on relative change in endexpiratory lung volume. Data are expressed as mean ± standard deviation. Negative values indicate a decrease in relative change in end-expiratory lung volume (relative Δ EELV ). EIT = electrical impedance tomography.
disease [34] . This disparity in results cannot easily be explained except for the fact that this particular patient had severe respiratory acidosis.
EIT is a non-invasive bedside technique to assess global and regional lung volumes that has primarily been used in acute lung injury or ARDS [35] . Hinz and colleagues have shown that compared with the validated nitrogen-washout method it is an appropriate tool to study EELV in critically ill patients [16] . To our knowledge, the use of EIT in the determination of the dynamic process of air-trapping in patients with small airway disease has not been used before, although its use in this disease condition can be rationalised. In our study, MV with heliox did not result in a universal reduction of air-trapping as defined by the relative Δ EELV . However, there were some patients who seemed to benefit from MV with heliox as they did show a reduction in relative Δ EELV that was reversed by MV with conventional gas. Several explanations for the non-universal reduction in relative Δ EELV may be proposed. First, not all alveoli have the same degree of hyperinflation due to the difference in time constants throughout the lung, indicating that hyperinflation is a regional phenomenon rather than a global problem [36] . This would implicate that the technique of EIT may be insufficient to detect regional differences in viralinduced small airway disease due to heterogeneity of the disease, a problem that can be overcome by increasing the reso-lution of the EIT signal. In favor of EIT, however, is the study by Adler and colleagues showing that with EIT dynamic hyperinflation could be adequately monitored [37] . Second, during the study no endotracheal suctioning was performed. Increased mucus production could obstruct the airways, resulting in the collapse of alveoli that is reflected by a decrease in EELV. As tidal volume remained constant throughout the experiment, we think that not performing endotracheal suctioning did not influence our results ( Figure 5 ). Third, if there is a difference in expression of clinical phenotype of RSV LRTD a universal response in relative Δ EELV would not be expected. Some patients responded with a decrease in relative Δ EELV whereas others did not in our study. Also, redistribution of ventilation within each lung or between the left and right lung was not significantly influenced by MV with heliox. This is in line with a heterogeneous clinical phenotype of RSV LRTD.
There are some limitations to our study that should be mentioned. First, the small sample size of our study. This sample size does not allow discrimination between responders and non-responders nor a categorization of clinical phenotype based on chest radiographs, but this should be the subject of further research. Second, patients were paralyzed throughout the study, thus prohibiting spontaneous breathing and mucus clearance by the patient itself. We choose to do so to eliminate any confounding effect of spontaneous breathing on the Data are expressed as mean ± standard deviations. Aa-DO2 = alveolo-arterial oxygen gradient; OI = oxygenation index; VI = ventilation index; V D /V T = dead-space/tidal volume ratio.
(page number not for citation purposes) degree of dynamic hyperinflation in order to truly assess the effect of MV with heliox. However, our findings require re-evaluation in spontaneously breathing mechanically ventilated infants. Supportive therapy maintaining spontaneous breathing could very well be a key element while awaiting therapeutic modalities for mechanically ventilated infants with RSV LRTD [38] . Third, the measurements of our study were not blinded because connection of the heliox and the measurements were conducted by one investigator (MK). However, this might have introduced measurement bias. Fourth, ventilation with heliox may have influenced the tidal volume measurements of the AVEA ventilator. The AVEA is equipped with the Bicore CP100™ pulmonary mechanics monitor that has been validated previously [36, 39] . Finally, the AVEA performs in a similar way with respect to tidal volume measurement when heliox is used [40, 41] .
MV with heliox significantly reduced R rs in mechanically ventilated infants with RSV LRTD with a heterogenous effect on the degree of hyperinflation and CO 2 elimination. These findings warrant further study in order to identify a subgroup of mechanically ventilated infants with RSV LRTD who might benefit from MV with heliox.
• MV with heliox decreases respiratory system resistance in RSV LRTD.
• MV with heliox does not reduce air-trapping in RSV LRTD.
• MV with heliox does not improve gas exchange in RSV LRTD.
• RSV LRTD may actually be a heterogeneous disease. Regulation of the apoptosis-inducing kinase DRAK2 by cyclooxygenase-2 in colorectal cancer BACKGROUND: Cyclooxygenase-2 (COX-2) is over-expressed in colorectal cancer (CRC), rendering tumour cells resistant to apoptosis. Selective COX-2 inhibition is effective in CRC prevention, although having adverse cardiovascular effects, thus focus has shifted to downstream pathways. METHODS: Microarray experiments identified genes regulated by COX-2 in HCA7 CRC cells. In vitro and in vivo regulation of DRAK2 (DAP kinase-related apoptosis-inducing kinase 2 or STK17β, an apoptosis-inducing kinase) by COX-2 was validated by qRT-PCR. RESULTS: Inhibition of COX-2 induced apoptosis and enhanced DRAK2 expression in HCA7 cells (4.4-fold increase at 4 h by qRT-PCR, P=0.001), an effect prevented by co-administration of PGE(2). DRAK2 levels were suppressed in a panel of human colorectal tumours (n=10) compared to normal mucosa, and showed inverse correlation with COX-2 expression (R=−0.68, R(2)=0.46, P=0.03). Administration of the selective COX-2 inhibitor rofecoxib to patients with CRC (n=5) induced DRAK2 expression in tumours (2.5-fold increase, P=0.01). In vitro silencing of DRAK2 by RNAi enhanced CRC cell survival following COX-2 inhibitor treatment. CONCLUSION: DRAK2 is a serine–threonine kinase implicated in the regulation of apoptosis and is negatively regulated by COX-2 in vitro and in vivo, suggesting a novel mechanism for the effect of COX-2 on cancer cell survival. Colorectal cancer (CRC) remains a leading cause of cancer death, with worldwide one million new cases each year and as many as half a million cancer deaths annually (Boyle and Leon, 2002) . Cyclooxygenase-2 (COX-2) expression is increased in the majority of colorectal tumours (Eberhart et al, 1994) and this induction is associated with advanced tumour stage and correlates with poor clinical outcomes (Sheehan et al, 1999) . Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, show anti-neoplastic effects in vitro (Richter et al, 2001; Sheng et al, 1997) and human studies have demonstrated their use to be associated with a reduced incidence of colorectal neoplasia Sandler et al, 2003) . Although more recent studies have confirmed the chemopreventive activity of COX-2selective NSAIDs (Arber et al, 2006; Baron et al, 2006; Bertagnolli et al, 2006) , it is also clear that their long-term use is associated with an unacceptable increase in the risk of cardiovascular events (Bertagnolli et al, 2006; Bresalier et al, 2005) .
The anti-neoplastic properties of these agents result from the inhibition of prostaglandin generation, particularly that of prostaglandin E 2 (PGE 2 ), the most abundant in vivo product of COX-2 activity in CRC cells (Pugh and Thomas, 1994; Rigas et al, 1993) . Although it appears that PGE 2 modulates various processes that are fundamental to tumour cell survival, such as altered proliferation and susceptibility to apoptosis (Sheng et al, 1997 (Sheng et al, , 1998 (Sheng et al, , 2001 Tang et al, 2002) , the precise molecular mechanisms remain unclear. A strong rationale exists therefore to generate a more complete understanding of the downstream targets of COX-2 activity (Doherty and Murray, 2009 ). This may lead to the development of more refined therapies, with side-effect profiles that allow their generalised use. Previous studies examining gene regulation by COX-2 in CRC cells have focused on long time points and have used relatively high doses of NSAIDs (Zhang and DuBois, 2001) . With this in mind, we set out to explore early changes in gene expression in CRC cells resulting from low-dose treatment with a selective COX-2 inhibitor, to improve our understanding of the early signalling events downstream of prostaglandin production. One candidate gene that we have identified, DRAK2 (DAP kinase-related apoptosis-inducing kinase 2 or STK17b), is one of a family of serine threonine kinases that share the ability to induce apoptosis (Sanjo et al, 1998) . The aim of this study was to explore the relationship between COX-2 and DRAK2 as a potential downstream regulator of cell survival in CRC. 
All cells were grown in culture at 371C in a humidified 5% CO 2 incubator. HCA7 cells were kindly donated by Susan Kirkland (ICRF, London, UK). HCA7 cells were cultured in DMEM with 10% FBS, supplemented with 1 mM sodium pyruvate and 100 mg ml À1 kanamycin, to approximately 90% confluence before treatment. HT29 cells were purchased from the ATCC (Rockville, MD, USA) and maintained in McCoy's 5A medium containing 1.5 mM L-glutamine, 10% FBS, penicillin 100 U ml À1 and streptomycin 100 mg ml À1 . SC236, a selective COX-2 inhibitor, was a gift from Dr Peter Isakson (Searle, Skokie, IL, USA). PGE 2 was purchased from Cayman (St Louis, MO, USA). Staurosporine was purchased from Calbiochem (San Diego, CA, USA). A validated siRNA against a target sequence in exon 3 of the DRAK2 (STK17b) gene was purchased from Ambion Inc (Austin, TX, USA). siRNA to scrambled DRAK2 sequence target (scrambled/negative control) was in vitro transcribed from oligonucleotide template using Silencer siRNA construction kit (Ambion Inc) . pEGFP-N1 vector was purchased from BD Clontech (San Jose, CA, USA).
Total RNA was isolated from cells and tissue following homogenisation in RNA lysis buffer (Qiagen GmbH, Hilden, Germany) supplemented with 1% b-mercaptoethanol. Extraction was performed using RNeasy Midi kits (Qiagen GmbH). RNA quality was determined by agarose gel analysis and RNA concentration was determined by spectrophotometry (GeneQuant pro; Amersham Biotech, Bucks, UK).
Total RNA was isolated as outlined above from HCA7 cells treated for 4, 6 and 8 h with SC236 (5 mM) or vehicle. cDNA was synthesised using the Custom SuperScript ds-cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Samples from various time points were pooled and underwent in vitro transcription using the ENZO IVT kit (Affymetrix, Santa Clara, CA, USA) to form biotin-labelled cRNA. This was fragmented and three independent biological samples for both control and treated conditions were then hybridised to Affymetrix U95Av2 GeneChips according to Affymetrix protocols. A detailed description of microarray analysis is included as Supplementary methods.
Total RNA (1 mg) was reverse transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. DRAK2, COX-2 and vascular endothelial growth factor (VEGF) were quantified by RT -PCR using SYBR Green Universal Master Mix (Roche Diagnostics Corp., Indianapolis, IN, USA) . Reactions were carried out in a 96-well format in the ABI 7700 Sequence Detector (PerkinElmer/Applied Biosystems, Warrington, Cheshire, UK). Results were then normalised to 18S rRNA amplified from the same cDNA mix and expressed as fold induction compared with the controls. cDNAs were amplified using the following primer pairs: DRAK2, AAAATAGGGCATGCGTGTGAA and TATTATACC AATATTCCACATATCTGTTGCT; COX-2, TTGTACCCGGACAGG ATTCTATG and TGTTTGGAGTGGGTTTCAGAAATA; VEGF, CA TGCAGATTATGCGGATCAA and TTTGTTGTGCTGTAGGAAGCT CAT. Bax and Bcl-xl quantification was performed using TaqMan pre-developed assay reagents according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA).
HCA7 cells were grown on glass chamber slides and treated with SC236 (5 mM) or vehicle control for 24 h. Slides were air-dried, fixed (methanol) and permeabilised (cold acetone). Slides were then blocked with 1% BSA in PBS (2 h) and incubated with a 1 : 50 dilution of anti-DRAK2 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 41C. After serial washes, the slides were incubated with a 1 : 250 dilution of fluorescently labelled donkey anti-goat Alexa Fluor 488 (Molecular Probes, Leiden, the Netherlands) and subsequently propidium iodide (Molecular Probes) diluted at 1 mg ml À1 in PBS as a nuclear counterstain for 5 min. Slides were mounted with fluorescent mounting media (Dako, Carpinteria, CA, USA) and imaged with an LSM510 Zeiss Axioplan-2 upright confocal microscope (Carl Zeiss, Göttingen, Germany).
HCA7 cells were seeded at a density of 0.5 Â 10 6 cells per well in six-well plates and were treated with SC236 (5 mM) or vehicle control for 24 h. Cells were detached using Versene for 5 min at 371C and floating and adherent cells were then pelleted by centrifugation and washed in a solution of 2% BSA suspended in PBS. We performed FITC -Annexin V/propidium iodide labelling was performed using a TACS Annexin V -FITC Apoptosis detection kit (R&D Systems, Abingdon, UK). Briefly, cells were re-suspended in a 100 ml working stock of binding buffer, propidium iodide and Annexin V -FITC conjugate and incubated in the dark for 15 min. Cells were re-suspended in binding buffer before analysis in a FACScalibur flow cytometer (Becton Dickinson, Oxford, UK) with measurement of fluorescence emission at 530 nm (FL1 channel) and at 4575 nm (FL3 channel). Four quadrant analyses using CellQuest (Becton Dickinson, Oxford, UK) software allowed quantification of cell populations according to labelling characteristics. The results were verified by staining/morphology by confocal microscopy.
HT-29 cells were seeded at a density of 0.5 Â 10 6 cells per well in six-well plates and allowed to adhere overnight, then cotransfected with 3 mg of DRAK2 siRNA and 1 mg of pEGFP-N1 (as a marker to select transfected cells) DNA per well using Fugene 6.0 (Roche Diagnostics Corp.) with a 2 mg:1 ml ratio of RNA to transfection reagent. Transfections were performed in OptiMEM, 1 ml per well (final concentration of siRNA, approximately 200 nM). A negative control siRNA to a scrambled target in the DRAK2 gene (without sequence homology to any other known transcript as verified by BLAST analysis) was used in the mock transfection controls. Populations of both mock-and positively transfected cells were subsequently treated with either SC236 (5 mM), staurosporine (1 mM) or vehicle control for 24 h. Adherent and floating cells were then pelleted, washed and then re-suspended in 250 ml of PBS with 7-aminoactinomycin D (7-ADD; Molecular Probes) at a final concentration of 20 mg ml À1 and incubated on ice for 20 min in the dark. Samples were analysed by flow cytometry using an established method for assessment of cell viability in transfected cells selected according to GFP fluorescence (Vezina et al, 2001 ).
The protocol was approved by the ethics (medical research) committee of Beaumont Hospital, Dublin and all patients provided written, informed consent. Colon cancer tissue and matched normal colonic mucosa were obtained from patients at the time of surgery and were immediately placed in RNAlater solution (Qiagen GmbH). Total RNA was extracted from the tissue samples as above.
Additional patients with newly diagnosed distal CRCs were recruited under a separate protocol, approved by the Irish Medicines Board and Beaumont Hospital Ethics Committee, for treatment for 5 -7 days with the selective COX-2 inhibitor rofecoxib at a dose of 25 mg daily. Tumour was sampled endoscopically at day 0 and again on completion of therapy and was placed in RNAlater. Compliance with drug therapy was monitored by measurement of whole-blood monocyte COX-2 activity as previously described (Panara et al, 1995) .
Analysis of tumour/normal differences were performed using twotailed Student's t-test. Differences in gene expression across time points were analysed by ANOVA with Bonferroni multiple comparisons test. Linear correlation was assessed using Pearson's test. t-Test, ANOVA and correlation statistics were performed using In-Stat, version 3.0 (GraphPad Software, La Jolla, CA, USA).
We examined the regulation of apoptosis by COX-2 in a human CRC cell line. HCA7 cells have previously been reported to express high levels of COX-2 with abundant PGE 2 generation (Sheng et al, 1997) . Apoptosis was quantified by fluorescence labelling with FITC-conjugated antibody to Annexin V and propidium iodide assessed by flow cytometry (Figure 1 ). Following a 24 h treatment with the selective COX-2 inhibitor SC236 (5 mM), a significant decrease (P ¼ 0.01) in HCA7 cells viability was observed ( Figure 1B ), mirrored by a doubling of the proportion of the cell population gated to the early apoptosis phase (Annexin V positive, propidium iodide negative) ( Figure 1B ). The observed increase in apoptosis at low micromolar doses of SC236 was prevented by coadministration of exogenous PGE 2 (1 mM), whereas more marked increases in apoptosis seen with higher doses of SC236 were not rescued. A similar effect was noted with regard to effects on cell proliferation (see Supplementary Figure S1 ). The ability of PGE 2 to promote cell survival confirmed that at low micromolar doses the effects of SC236 are largely dependant on its ability to inhibit COX-2.
High-density oligonucleotide arrays identify SC236mediated changes in gene expression in HCA7 cells
Having confirmed that COX-2 inhibition causes an apoptotic phenotype in HCA7, we used high-density oligonucleotide microarrays (HDONAs) to examine early global changes in gene expression associated with the abolition of prostaglandin production. Total RNA from cells treated with SC236 (5 mM) or vehicle control for 4, 6 or 8 h was pooled and used to probe Affymetrix HGU95Av2 GeneChips that feature probe sets for over 12 000 different human transcripts. Three independent biological replicates were assayed for each condition (i.e. vehicle control or COX-2 inhibitor).
Robust Multichip Average (RMA)-based analysis was performed to compare expression measures. Magnitude of changes in expression of individual genes was small in most cases, reflecting the early time points used. Correspondence analysis (CoA) of RMA-based measures, however, highlighted significant global differences in gene expression between control and treated samples ( Figure 2A ). This technique, similar to principal components analysis, represents expression (hybridisation) as points projected in three-dimensional space and demonstrates a consistent alteration in the transcriptome of cells treated with COX-2 inhibitor. Expression of DRAK2 (STK17b) consistently showed strong induction with COX-2 inhibition. This novel serine -threonine kinase is a member of the death-associated protein (DAP) kinase family and like other family members has been implicated in the control of apoptosis (Sanjo et al, 1998) . However, a potential function in the regulation of cell death and viability in cancer has not been explored to date. A comparison of our expression data set with another recently published HDONA experiment (Levitt et al, 2004 ) (evaluating selective COX-2 inhibition in a breast cell line) revealed a cluster of genes regulated by COX-2 in both systems (see Supplementary data; Table S1) and differential expression of DRAK2 once again featured as a strong signal. It was thus chosen for detailed validation and further analysis.
Differential DRAK2 expression was confirmed by quantitative RT-PCR (qRT-PCR) on template from HCA7 cells. Induction of DRAK2 expression was observed as early as 4 h after treatment with SC236 and was inhibited by co-administration of PGE 2 ( Figure 2B) . A 4.4-fold induction of DRAK2 expression (relative to control) was observed with SC236 (5 mM) after 4 h (P ¼ 0.001), a response that was attenuated on co-incubation with prostaglandin (SC236 vs SC236 þ PGE 2 , P ¼ 0.01), suggesting that the changes in DRAK2 expression were dependant on variations in prostaglandin generation. Indirect immunofluorescent staining for DRAK2 in HCA7 cells showed a corresponding increase in levels of DRAK2 protein with COX-2 inhibitor treatment, with development of intense nuclear staining for DRAK2 at 24 h post-treatment with SC236 ( Figure 2C ).
We examined the expression of both DRAK2 and COX-2 in a bank of colorectal tumour samples using total RNA extracted from tumour and normal mucosa sampled from the freshly resected tumours of 10 patients with CRC (not taking aspirin or NSAIDs). The levels of COX-2 transcript (normalised to 18S rRNA) were elevated in the majority of tumour samples relative to normal mucosa ( Figure 3B , mean 2.4-fold increase, P ¼ 0.006). DRAK2 expression showed an opposite pattern, with relative suppression of DRAK2 expression in tumour compared to normal from the same patient ( Figure 3A , mean decrease approximately 50%, P ¼ 0.003). A negative correlation between the ratio of DRAK2 and COX-2 expression (in tumour relative to normal) was noted ( Figure 3C , R ¼ À0.68, R 2 ¼ 0.46, P ¼ 0.03) reflecting an inverse relationship between the tumour/normal differences for the two genes across the patients sampled.
Our observations suggested a suppression of DRAK2 expression, either directly or indirectly, by the actions of COX-2-derived prostaglandins in tumour samples. To test this hypothesis further, five patients with newly diagnosed CRC (not taking aspirin or NSAIDS) were recruited to a pilot study. Endoscopic biopsies of tumour tissue were obtained before and after a short course (5 -7 days) of treatment with the COX-2-selective inhibitor rofecoxib at a dose of 25 mg daily, a dose that has been demonstrated to selectively inhibit COX-2 in vivo (Ehrich et al, 1999) . The study was conducted before the withdrawal of rofecoxib by the manufacturer. qRT-PCR demonstrated an induction in DRAK2 expression ( Figure 4A ; mean 2.5-fold increase, P ¼ 0.01) in tumour from each of the patients treated with rofecoxib, reflective of the changes seen with COX-2 inhibition in vitro. The expression of a panel of additional genes was also evaluated for comparative purposes ( Figure 4B ). Although no significant change in several genes that modulate apoptosis (Bax, Bcl-xl) was observed, a significant decrease in VEGF expression was noted, with a mean reduction of close to 40% (P ¼ 0.01). This pro-angiogenic factor has previously been linked to COX-2 activity and reduced VEGF expression has been observed in animal models following treatment with rofecoxib (Yao et al, 2003) . This finding provided additional validation of the suppression of COX-2 activity in the tumours of these patients. 
Finally we evaluated the phenotypic activity of DRAK2 in cell systems. We suspected DRAK2 expression was already significantly suppressed in HCA7 cells, given high levels of COX-2 expression whereas HT-29 cells showed lower levels of COX-2 with higher levels of DRAK2 expression. Following transient transfection of HT-29 cells with a commercially available sequence validated siRNA to DRAK2, significant repression of DRAK2 transcription was seen ( Figure 5A ). To counteract the effects of low transfection efficiency and maximise detection of a phenotype associated with DRAK2 silencing, a GFP expression vector was cotransfected to 'gate'-transfected or 'silenced' cells in subsequent assays to quantify cell survival using an established method (Vezina et al, 2001) . The percentage of viable cells (without 7-ADD uptake) and non-viable cells (with 7-ADD uptake) was calculated in mock-transfected cells and in DRAK2 siRNA-transfected cells selected (gated) by GFP fluorescence. Loss of cell viability was observed in mock-transfected HT-29 cells following treatment with both SC236 and staurosporine ( Figure 5B ). In contrast, the siRNA-transfected (GFP-gated) cells showed no loss of cell viability following treatment with the COX-2 inhibitor SC236. They did however still show a significant loss of viability with staurosporine, suggesting a specific effect on the pathway involved in the action of SC236.
Traditional NSAIDs have anti-neoplastic properties, but their prolonged use is limited by their association with gastrointestinal side effects, particularly the risk of gastrointestinal haemorrhage. The new-generation coxibs have also shown promise for chemoprevention of CRC but are now considered by many to carry an unacceptable risk of thrombotic events. Our aim was to identify novel 'effector' pathways operating downstream of COX-2 in tumours, so that this knowledge might allow future selection of more refined therapeutic targets.
There are a number of ways in which COX-2 may promote cancer progression, either through an effect on cancer cells, the associated immune response or angiogenesis in response to the tumour. There is evidence that COX-2 expression protects cells from apoptosis and conversely that treatment of CRC cells with selective COX-2 inhibitors causes cell-cycle arrest, growth inhibition and induction of apoptosis (Richter et al, 2001; Sheng et al, 1997 Sheng et al, , 1998 . Evidence from both animal models (Hansen-Petrik et al, 2002) and human studies (Sinicrope et al, 2004) suggests that modulation of cell survival by altered susceptibility to apoptosis is equally important in vivo.
However, the precise mechanisms by which COX-2 exerts these effects in tumour cells are unclear. Enhanced COX-2 expression in cells alters susceptibility to tumour necrosis factor-related apoptosis-inducing ligand by reduction in membrane death receptors (Tang et al, 2002) and may also alter the threshold for intrinsic pathway activation (Tang et al, 2002) . Over-expression of COX-2 leads to the generation of prostanoids, particularly PGE 2 , and signalling through the various EP receptors, several of which have been implicated in carcinogenesis (Watanabe et al, 1999; Sonoshita et al, 2001; Mutoh et al, 2002) . PGE 2 stimulates TCF-b-catenin-mediated gene transcription (Castellone et al, 2005; Shao et al, 2005; Eisinger et al, 2007) , possibly by activation of EP2 and EP4 receptors (Fujino et al, 2002) . Some of the important effects of PGE 2 are probably also related to its ability to transactivate the epidermal growth factor receptor through a number of distinct mechanisms (Pai et al, 2002; Shao et al, 2003; Al-Salihi et al, 2007) . It seems likely therefore that prostaglandins generated by CRC cells do not exert their activity by a single mechanism but have a range of downstream signalling targets.
We made use of HDONAs to identify 'early' target genes downstream of COX-2. Earlier studies in other cell systems have demonstrated the ability of COX-2 to regulate gene expression, such as the expression of MDR1 (Patel et al, 2002) . Specific studies of differential gene expression in colon cancer cells (using suppressive subtractive hybridisation and differential screening) have previously demonstrated altered expression of genes involved in cell proliferation and viability (Zhang and DuBois, 2001) , however high NSAID doses and long time points make a direct comparison with our expression studies difficult. We chose to look at early time points following treatment with the highly selective COX-2 inhibitor SC236 to identify crucial early responses. COX-2 independent effects of SC236 have been described (He et al, 2006) . However, we chose to use SC236 at a dose which was associated with only modest changes in rates of apoptosis but where we observed rescue by co-incubation with PGE 2 , in the belief that study of a more subtle but specific phenotypic change would yield more significant findings.
We identified DRAK2 as showing consistent changes in expression. This predominantly nuclear serine -threonine kinase has similarity to the DAP kinases and is involved (at least in certain cell types) in initiation of apoptosis (Sanjo et al, 1998) . Unsupervised hierarchical clustering of our expression data (Supplementary Figure S2 ) selected a cluster of genes showing a similar pattern of upregulation with COX-2 inhibition. This cluster of genes have either known nuclear localisation within the cell or have a bipartite nuclear localisation signal, which suggests their ability to traffic to the nucleus. Many of these are known to be involved in transcriptional regulation or in the induction or potentiation of apoptosis. Prior expression analyses have previously identified nuclear proteins as significantly over-represented in genes induced by aspirin treatment in colon cancer cells (Hardwick et al, 2004 ) and a mechanism regulating the nuclear trafficking of DRAK2 has recently been suggested (Kuwahara et al, 2008) .
We have confirmed that DRAK2 expression in colon cancer cells in vitro is regulated by COX-2 ( Figure 2 ) and also demonstrated an in vivo relationship between COX-2 and DRAK2 expression in human CRC (Figure 3 ). Both COX-2 and DRAK2 have been implicated in the modulation of T-lymphocyte function, with opposing phenotypic consequences. Activated T cells in patients with SLE markedly upregulate and sustain COX-2 expression and resist inactivation and cell death (Xu et al, 2004) . Indeed, COX-2 expression has been shown to mediate lethal T-cell activation (Brewer et al, 2003) . By contrast, over-expression of DRAK2 in activated T cells enhances apoptosis in the presence of IL-2 (Mao et al, 2006) , and studies in mice with targeted disruption of DRAK2 have demonstrated that this protein is an important negative regulator of T-cell activation, raising the threshold for activation through the T-cell receptor (McGargill et al, 2004; Friedrich et al, 2005) and acting as an important regulator of T cells response and survival (Schaumburg et al, 2007; McGargill et al, 2008) .
Although it is clear that DRAK2-mediated cell death is cell-type and context dependent, there is a growing body of evidence to indicate that its function is directly related to the regulation of cell survival. In addition to in vivo evidence from DRAK2 transgenic mice (Mao et al, 2006) , induction of DRAK2 in vitro in a variety of contexts and cell types augments apoptosis (Sanjo et al, 1998; Kuwahara et al, 2006; Mao et al, 2006) . We also show that silencing DRAK2 expression in HT29 cells promotes cell survival and abrogates the effects of a COX-2 inhibitor in vitro. These findings confirm published observations on the effect of DRAK2 silencing by RNAi on susceptibility of rat colon cancer cells to UV-induced apoptosis (Kuwahara et al, 2006) . We also recently observed that DRAK2 may be important in the ability of COX-2 to protect cardiomyocytes from doxorubicin-induced apoptosis (Neilan et al, 2006) , suggesting that the modulation of DRAK2 expression may not be confined to cancer but may also be important in the positive effects of COX-2 on cell viability and tissue healing in other systems. For the moment, the precise mechanisms by which DRAK2 impacts on cell survival are unclear. Recent observations suggest that the ribosomal kinase p70S6, involved in cell-cycle regulation, is a substrate for DRAK2 (Mao et al, 2009) , highlighting a possible involvement in regulation of cell-cycle dynamics. In an analysis of publicly available expression data (Whitfield et al, 2002) , we have noted that DRAK2 forms part of a cluster of genes showing similar cyclical changes in expression with cell-cycle periodicity in HeLa cells (Supplementary Figure S3 ; Supplementary Table 2 ). There is an interesting degree of overlap (summarised concisely in Supplementary Figure 4) between this panel and COX-2-regulated genes identified by our gene expression analysis and a number of other gene discovery studies that examined the effects of COX inhibitors (Iizaka et al, 2002; Levitt et al, 2004) .
How does the identification of the regulation of DRAK2 by COX-2 further our understanding of the role of COX-2 in cancer? One of the most consistent observations about COX-2 in cancer is its contribution in vivo to the risk of systemic metastases (Tomozawa et al, 2000; Yao et al, 2004) . This fact likely explains the impaired survival observed in individuals whose tumours express COX-2 (Sheehan et al, 1999) . Previous observations about the effects of DAP kinase (the best-characterised member of the kinase family to which DRAK2 belongs) in cancer cells may provide a unifying explanation, linking the ability of this DAP kinase to regulate apoptosis to a suppression of metastatic potential (Inbal et al, 1997) . The most plausible explanation is that the enhanced cell survival conferred by DRAK2 silencing (by COX-2) provide a resistance to various forms of cell death and possibly crucially to anoikis (matrix detachment-induced cell death) (Hofmann et al, 2007) , the form of cell death that may be the most important in deciding the viability of circulating tumour cells and their ability to form distant metastases. Rooting human parechovirus evolution in time BACKGROUND: The Picornaviridae family contains a number of important pathogenic viruses, among which the recently reclassified human parechoviruses (HPeVs). These viruses are widespread and can be grouped in several types. Understanding the evolutionary history of HPeV could answer questions such as how long the circulating lineages last shared a common ancestor and how the evolution of this viral species is shaped by its population dynamics. Using both strict and relaxed clock Bayesian phylogenetics we investigated 1) the substitutions rates of the structural P1 and capsid VP1 regions and 2) evolutionary timescale of currently circulating HPeV lineages. RESULTS: Our estimates reveal that human parechoviruses exhibit high substitution rates for both structural P1 and capsid VP1 regions, respectively 2.21 × 10(-3 )(0.48 – 4.21 × 10(-3)) and 2.79 × 10(-3 )(2.05 – 3.66 × 10(-3)) substitutions per site per year. These are within the range estimated for other picornaviruses. By employing a constant population size coalescent prior, the date of the most recent common ancestor was estimated to be at around 1600 (1427–1733). In addition, by looking at the frequency of synonymous and non-synonymous substitutions within the VP1 gene we show that purifying selection constitutes the dominating evolutionary force leading to strong amino acid conservation. CONCLUSION: In conclusion, our estimates provide a timescale for the evolution of HPeVs and suggest that genetic diversity of current circulating HPeV types has arisen about 400 years ago. Parechoviruses belong to the Picornaviridae family which includes other pathogenic viruses such as foot-and-mouth disease virus (FMDV), hepatitis A virus, enteroviruses and rhinoviruses [1, 2] . The Parechovirus genus includes two species: human parechoviruses (HPeV) and the zoonotic Ljungan virus. HPeV are non-enveloped pathogens with a single-stranded genomic RNA of positive polarity with around 7.400 nucleotides organized into a single long open reading frame in between a 5'UTR and 3'UTR. The open reading frame can be divided into three main regions: P1 (encoding capsid proteins VP0, VP3, VP1), P2 (nonstructural proteins) and P3 (nonstructural proteins, including the viral RNA polymerase) [2] [3] [4] . In HPeV, out of the three capsid proteins that constitute the monomeric units of the viral icosahedric-shaped capsid [3] , VP1 protein plays a crucial role in cell entry via interaction of an Arg-Gly-Asp (RGD) triplet with integrins on the cell surface [5] . However, some HPeVs (among which the type 3 strains) lack the RGD motif in VP1, and their mode of cell recognition and entry is less clear [6] . Typing of HPeV is based on the VP1 sequence providing a reliable locus to type all the identified HPeV strains as described for enteroviruses by Oberste et al [7] . As a result, the majority of HPeV available nucleotide data concerns the VP1 gene.
In general, HPeV is transmitted by the oral-fecal route causing in most cases relatively mild respiratory and gastrointestinal symptoms [2, 8] , though conditions such as bronchiolitis [9] and severe neonatal infections [10, 11] have also been reported. HPeV1 and HPeV2 were first isolated in 1956 and classified by serotyping as enteroviruses, respectively echovirus types 22 and 23 [3, 12] . HPeV3 was first described in 2004 [6] and is associated with more severe conditions related to CNS symptoms [10, 11, 13] . Subsequently, improvements in HPeV-specific screening tools allowed a successful identification of HPeV4 and HPeV6 throughout North America, Japan and Europe [14] [15] [16] [17] [18] . Moreover, an HPeV variant originally classified as HPeV2-Connecticut was reclassified as HPeV5 [14] . Currently, sequences have become available for two novel types that were recently isolated in Pakistan and Brazil [19, 20] and in the Netherlands one more novel type was identified (HPeV14, [21] ). Unfortunately sequences of the HPeV types 9 to 13 are not available for analysis yet. Of all types, HPeV1 and HPeV3 are the most prevalent strains [11, 22] .
Understanding the mechanisms underlining pathogenicity and persistence of pathogens in human populations is an important aspect of disease epidemiology and control. Fixation of mutations into nucleotide substitutions, a key principle behind phylogenetic signatures, is shaped by major evolutionary forces such as selection (molecular adaptation deriving from an increasing fitness of a corresponding phenotypic trait) and genetic drift (stochastic gene sampling process at reproduction) [23, 24] . A useful tool to detect and measure selection in viral gene sequences is the ratio between synonymous (dS) and nonsynonymous (dN) substitutions. Whereas a ratio above 1.0 is an indicator of positive selection operating at the amino acid sequence level [25] , significantly lower values are generally referred to as purifying selection and refer to preservation of the phenotypic trait.
RNA viruses yield the highest mutation rates of all groups of pathogens which is approximately six orders of magnitude higher than in most DNA organisms [23, 26] . In the context of viral population genetics, substitution or evolutionary rates can be defined as the number of fixed mutational changes that accumulate in the population per nucleotide site per unit of time [27] . This rate is driven by the short-generation times of viruses and their error-prone RNA polymerase proteins lacking proofreading activity. Combined with their small genomes, these characteristics make RNA virus ideal models for evolutionary research [23, 28, 29] . In addition, recombination events may also play a role in RNA virus evolution [23] . While lacking a fossil record, evolutionary histories of RNA viruses can be calibrated because they represent 'measurably evolving populations', in which genetic diversity accumulates over a timescale of human observation [30] . Their evolutionary history and population dynamics can be reconstructed by means of genealogy-based coalescent approaches using nucleotide sequences sampled over an epidemiological time frame in order to estimate timed viral ancestry as well as the rates of genetic change [27, 29] . The most advanced methods operating on time-stamped sequence data use Bayesian Metropolis-Hastings Markov Chain Monte-Carlo (MCMC) algorithms that accommodate for the uncertainty of phylogenies rooted in time. Here, we estimated the substitution rates for the P1 and VP1 regions of HPeV with such a Bayesian approach, which provides a statistical framework for evolutionary analysis [31] .
The identification of several novel types within the last few years may be conceived as a relatively recent introduction of HPeV into the human population, but this is not necessarily the case. By reconstructing the evolutionary history of HPeV we shed light on this issue. We investigated when current HPeV diversity emerged by determining the time of divergence from the most recent common ancestor (TMRCA).
Dataset 1 comprised 29 nucleotide sequences from the P1 structural region (2291 nt) from different HPeV isolates (12 sequences of HPeV1, 1 sequence of HPeV2, 4 sequences of HPeV3, 5 sequences of HPeV4, 2 sequences of HPeV5, 3 sequences of HPeV6, 1 sequence for HPeV7 and 1 sequence for HPeV8). Dataset 2 comprised 199 nucleotide sequences of the VP1 capsid region (647 nt) (117 sequences of HPeV1, 2 sequences of HPeV2, 40 sequences of HPeV3, 18 sequence of HPeV4, 9 sequence of HPeV5, 10 sequences of HPeV6, 1 sequence of HPeV7, 1 sequence of HPeV8 and 1 sequence of HPeV14). To date, sequences of HPeV9-13 have not been made available [32] . The accession numbers of the sequences from both data sets are available in Additional file 1. Sampling date (year) for dataset 1 (1956-2007) and for dataset 2 (1975-2007) was either collected directly from Genbank record or following direct contact with the relevant authors. Multiple alignments of the P1 and VP1 regions of HPeV were conducted in ClustalW [33] and sequences were edited manually with Se-Al v2.0 [34] .
Overall evolutionary rates for P1 and VP1 regions were measured as the number of nucleotide substitution per site per year (s/s/y). Relevant parameters were summarized as the median of posterior distributions by Bayesian coalescent Markov chain Monte Carlo algorithm implement in the Bayesian Evolutionary Sampling Trees (BEAST) software package version 1.4.8 [31] . To identify the optimal substitution model we performed a maximum likelihood analysis using the Modelgenerator package [35] . The model that best fit both sequence datasets was General Time Reversible (GTR) model with a discretised γ-distribution (GTR+Γ), allowing for nucleotide rates to vary among sites within the protein coding sequence alignments. Codon partitions (1+2)+3 were applied to both alignments, keeping first and second positions (mostly to non-synonymous changes) in one partition and the third position (related to increase redundancy and prone to synonymous changes) in a separate partition [36] . Relative rate parameters were estimated in separate for each partition, in order to accommodate rate variation at the third codon position.
We employed both strict and relaxed lognormal molecular clocks, the latter allowing rate variation among branches [37] . The coefficient of variation (σ r ) was used as a quantification of the rate variation among branches (σ r > 0.2 was considered as significant rate variation among branches) ( Table 1) . A constant size demographic model was used as coalescent prior. Each alignment of both data sets was analyzed using Markov Chain Monte-Carlo (MCMC) computations run over a sufficient time to achieve convergence of the chains, which was analyzed by inspection of the MCMC samples using TRACER 1.4 [38] . The 95% highest posterior density (HPD) interval is the shortest credible interval that contains 95% of the samples values. Statistical uncertainties of the substitution rates and the TMRCA were summarized as the lower 95%, median, and upper 95% values of the HPD. Out of the tested models (GTR + Γ, both with strict and relaxed lognormal molecular clocks), the clock model that performed better was the lognormal molecular clock, which yielded the highest marginal likelihood. Clock models were also compared in terms of Bayes Factors (BF, Table  2 ). The relaxed model clock following a lognormal distribution was also supported by the highest log 10 BF as suggested [39] .
The fact that a relaxed lognormal molecular clock fits best to our data was consistent with an estimated coefficient of variation of 0.29 and 0.41 (respectively, for dataset comprising P1 and VP1 regions) that reflected significant rate heterogeneity, thus rejecting a strict molecular clock. The resulting trees for each run were summarized using Tree-Annotator and the maximum clade credibility tree was visualized with FigTree v1.1.2 [34] . BEAST xml files are available as additional files 2, 3, 4 and 5.
Overall selective pressures acting on VP1 antigenic region were estimated by using the CODEML program in the PAML package [40] . We used site models M7 (with a discrete distribution of 10 categories and accounting for sites not allowed to be positively selected) and M8 (estimates dN/dS for an extra class (p11) of sites, accounting for positively selected sites with dN/dS>1). Models were compared by means of likelihood ratio test and statistical support was taken from the Bayes-Empirical-Bayes output (BEB, see additional file 6: Log-likelihood and parameter estimates for PAML analysis) [40] . To detect adaptative molecular evolution, we used the complete dataset 2.
We first identified the best-fitting substitution model for the HPeV sequences using the Modelgenerator package (GTR + Γ) [35] , and tested whether the evolution of the P1 and VP1 genetic regions was better described by a strict or relaxed lognormal molecular clock. A relaxed lognormal molecular clock provided a better fit to both datasets according to Bayes Factor (BF) analyses (P1: log 10 BF = 7.03 and VP1: log 10 BF = 27.8, Table 2 ). This is in accordance with significant rate variation among the branches of the inferred phylogeny as measured by a non-zero coefficient of variation (σ r ) obtained with the relaxed molecular clock analysis (P1: σ r = 0.29; VP1: σ r = 0.41) (see Methods for details). Using the available P1 and VP1 dated sequences of HPeV, our analysis inferred a similar rate of nucleotide substitution for both regions (P1 median: 2.21 × 10 -3 s/s/y, 95% HPD [0.48 × 10 -3 , 4.21 × 10 -3 ]; VP1 Table 2 ). The higher rate indicated for the VP1 region is possibly related to its antigenic properties, perhaps reflecting a difference in the level of gene expression or mirroring the involvement of the VP1 capsid protein in the viral entry mediated by cellular integrins.
Despite our study focused on the available sequences of HPeV, more accurate estimates could probably be obtained with broader and more homogenous sampling timescale, preferably for all types. Yet, this may be a daunting task because it is difficult to obtain older samples and some of the HPeV types e.g. HPeV2, HPeV4, HPeV5 and HPeV6 appear to be relatively rare (see e.g [17, 18] ). Moreover, a common pitfall on estimating evolutionary rates is its underestimation due to mutational saturation of synonymous sites [41] [42] [43] [44] . By using a gamma distributed substitution model, we assured that rate variation among sites was allowed. Therefore, the effect of possible saturation of synonymous sites was alleviated by permitting a proportion of these sites to change at a higher rate [43] . In addition, we used partitioning in codon positions that allows different codon positions to have different substitution rates (and different amount of rate heterogeneity) (see Methods for details) [31, 45] thus further accommodating rate variation among synonymous and non-synonymous positions.
The high rates of evolutionary change obtained in this study are in accordance with the evolutionary rates of other RNA viruses [32, 33] . Consistently, HPeV replication mechanism relies on an RNA-dependent RNA polymerase that lacks proofreading capacity. This increases the number of mutations incorporated in viral genomes over time and settles the ground for a relatively rapid genetic diversification [46] . The evolutionary rates of a few members of the Picornaviridae family have been studied. Despite the fact that most of the studies used different evolutionary frameworks, the rate of evolutionary change estimated in this study for the capsid region of HPeV VP1 is 1) faster than the rate of Hepatitis A virus [47] , 2) resem-bles the rate estimated for the antigenic region of Echovirus 71 [41, 48, 49] and finally 3) it is nearly one order of magnitude lower than the rates of poliovirus (2.09 × 10 -2 s/s/y) [48] or FMDV (2.7 × 10 -2 s/s/y) [50] .
RNA viruses are the most suitable object of study for rates of change and divergence times. This is due in large part to the rapid rate at which they evolve allowing genetic diversity to accumulate within a timescale approximately the same as mutations are fixed in viral populations [29] . Yet, a deeper understanding of the replication machinery of HPeV (e.g. generation times, fidelity of RNA polymerase) may deliver insights on the molecular basis of these high rates of evolutionary change [44] .
According to our analysis based on 199 HPeV VP1 available sequences, these viral species diverged from their most recent common ancestor (MRCA) at the year 1600 (95% HPD [1427-1733]) ( Figure 1 , Table 2 ). Moreover, and focusing on the two most recently isolated types (HPeV7 [19] , HPeV8 [20] . Taken together, we suggest that the genetic diversity of the currently circulating HPeV types has arisen around 400 years ago (Figure 1 ).
The wider 95% Bayesian credible intervals obtained for the estimates using dataset 1 composed by the total of 29 available P1 sequences to date (Table 2) probably reflect a less heterochronous sequence data. Yet, an identical timescale was obtained when performing the MCMC approach with the dataset comprising the P1 region (1603, 95% HPD [940-1883]) ( Table 2 ). Despite holding new pieces to solve the puzzle of HPeV origins, the evolutionary rates and the timescales for the most recent common ancestor and type lineage-splitting events, may be better framed once a larger number of sequences are available [51] . However, the overlapping of the 95% Bayesian credible intervals obtained in our analysis for both genomic regions indicates that our estimates on the TMCRA of the HPeV lineages are robust ( Table 2) .
One facet of fast evolving RNA viruses that induce acute infections (as the case of HPeV) is that they are likely candidates for jumps between species boundaries [29] . While the latter appears to be clearly established for e.g. SARS-CoV or influenza H5N1, a zoonotic link remains to be elucidated for HPeV. Because Ljungan virus shares a close phylogenetic proximity with HPeV virus, it is likely that both species have had a common ancestor [52] . Moreover, the reservoir host for Ljungan virus is Myodes glareolus, a widely distributed rodent commonly named as bank vole [4] . Despite the connection of Ljungan virus infection and human disease still remains to be clarified, bank voles are recognized as the reservoirs of other infectious agents, e.g. Puumala Hantavirus [53] and have been linked to a significant number of outbreaks over Europe [54] [55] [56] .
Bayesian time-scaled phylogeny of HPeV based on VP1 sequence analysis Figure 1 Bayesian time-scaled phylogeny of HPeV based on VP1 sequence analysis. Maximum clade credibility tree obtained with BEAST with a constant size coalescent prior showing lineage splitting events (nodes A-F) since the most recent common ancestor to the presently circulating HPeV types. The divergence times correspond to the mean posterior estimate of their ages (in years). For the TMRCA, the correspondent 95% Bayesian credible intervals are shown (median 1600). Time axis is shown in years and ranges from the TMRCA to the present year. Deeper and some subtype nodes with posterior probability of higher than 0.8 are pointed out. Each colour corresponds to a specific HPeV, as indicated in the box on the right. The dashed grey circle depicts the extent of genetic diversity of the sampled HPeV strains. HPeV-1-"Harris-like" strains (*) clustered separately from the contemporary HPeV-1.
In search for the driving force that shapes the evolution of the HPeVs, we looked at the ratio of non-synonymous-tosynonymous substitutions (the dN/dS ratio) [24] . For most codons in the VP1 region the ratio is < 0.1 (Figure 2 ). We noticed a few sites that tend to escape from purifying selection displaying dN/dS values > 0.3 (position Q61, A119, G203), or even > 1.0 (position N202 of our alignment, see additional file 7), however with statistically poor support (see additional file 6, Log-likelihood and parameter estimates for PAML analysis).
Also other studies have found an overall low dN/dS ratio for the HPeVs [1, 57] . Our analysis confirms on a codon level that throughout the structural region strong purifying selection is dominant, leading to the conservation at the level of the amino acid sequence. Future analysis may shed lights not only in a unified framework of evolution for this viral species but also help preventing major burdens associated with HPeV pathogenicity.
The HPeV are highly prevalent human RNA viruses and thus far no studies have addressed the evolutionary history of these pathogens. The Bayesian analysis presented here first indicates that the structural P1 and the capsid VP1 region of this viral species evolve at a high rate of evolutionary change (~10 -3 substitutions per site per year). Additional genomic and epidemiological data will help to reveal the relation between such rates and the widespread of this viral species. We also show that the currently circu-lating HPeV types have shared a common ancestor around four centuries ago. Since then, HPeV evolved into different lineages that have spread widely. Overall, a strong tendency for phenotypic conservation could be observed, suggesting that genetic drift plays an important role in the generation of the diversity within the regions under investigation. In summary, by delivering insights into the evolutionary mechanisms of HPeV, this study provides the foundations for a unified understanding of HPeV evolution. HIV/AIDS prevention in China: A challenge for the new millennium China’s first HIV infection was officially reported in 1985 and by the end of 1996, there may have been up to 200,000 people affected nationwide. In 2001, this figure probably exceeded 600,000. By 2003, the predicted number of HIV cases had reached 1.5 million. At least 80,000 individuals now have fullblown AIDS. China may soon have the largest HIV-infected population in the world, possibly 6 million cases by 2005. With infection rates rising at about 30% per year, it is feared this figure might exceed 10 million by 2010. Although the Chinese government was initially slow to accept the problem, in the late 1990s definite changes began occurring. In 2003 Premier Wen Jiabao publicly shook the hand of an AIDS patient and his government promised to introduce a range of free HIV-related services. Large preventive education campaigns are now underway. Unfortunately, there will still be many obstacles in controlling the epidemic and preventing further spread of this disease. Without doubt, China faces a serious predicament in the new millennium, and one which will pose numerous challenges for preventive medicine. China's first AIDS case was officially reported in 1985 in Yunnan Province, adjacent to the border with Burma (1). By the first half of 1995, a total of 1774 patients had been identified (2) . In 1996 it was estimated that there were probably between 150,000 and 200,000 people affected nationwide (3) . According to the Chinese Ministry of Health however, there were only 8303 cases officially recorded by October 1997 (3) . By September 2001, this official number had risen to 28,133, although estimates suggest that it most likely exceeded 600,000 (4) . Until 2002, the Chinese government maintained that there were only about 30,000 HIV cases nationwide, although their official count was later revised 25-fold upwards to 840,000. By 2003 the United Nations had estimated that the number of cases was closer to 1.5 million (5) . In the first half of 2001 alone, China's HIV incidence rate increased by 67% (6) , and at least 80,000 people now have full-blown AIDS (7) . The Chinese Academy of Preventive Medicine finally acknowledged in 2001 that they may soon have the largest HIV infected population in the world, possibly 6 million cases by 2005 (8) . With infection rates now rising at about 30% per year, the United Nations estimates that China may have more than 10 million HIV positive patients by 2010 (9) . Given this alarming situation, we considered it necessary to conduct an in-depth literature review on the topic. We conducted a detailed literature search using standard internetbased medical search engines. We also retrieved some older documents from library archives and identified other material from the reference lists of published papers. An up-to-date reference list of HIV/AIDS in contemporary China appears at the end of our manuscript.
The three phases
Most epidemiologists agree that HIV spread through China in three distinct phases (10) . The first phase (the entry phase) occurred between 1985 and 1988, involving a small number of imported cases, and was officially viewed as a 'foreign' problem (4) . In 1987, China's Health Minister suggested the disease might be curtailed because sexual promiscuity and homosexuality were limited. HIV infected foreigners were subsequently banned from living in China, and it was also noted that it was illegal for Chinese citizens to have sex with foreigners (10) . The second phase (the spreading phase) occurred between 1989 and 1993, with the detection of HIV cases among minority groups from Western province communities that bordered Myanmar, Laos and Vietnam (4) . The third phase (the expansion phase) seems to have begun around 1994 (4), although several aspects of the 'expansion phase' and the 'spreading phase' were probably overlapping. In 1995, an article appeared in the scientific literature describing a mother and her two daughters who had become HIV infected after donating plasma (11) . This was followed by a Chinese newspaper story in 2000 titled 'Strange disease in a Henan village shocks top officials,' and one which later appeared on the US Embassy web site (12) .
Blood selling in Henan Province probably created China's first significant HIV reservoir. Traditional Chinese taboos against blood donation made blood-buying a profitable venture, with entrepreneurs visiting many rural areas and collecting blood from poor villagers and townsfolk (8) . The first case of HIV acquired through plasma donation was reported in 1995 (11) . In the 1990s it was estimated that 70% to 90% of Chinese blood reserves were obtained from people who sold their blood commercially (2) . Approximately 1 million poor farmers trying to supplement their incomes regularly donated blood in Henan province during this period (6) . Unsanitary conditions and limited or non-existent infection control procedures contributed to the early spread of HIV. However, it was the practice of donating plasma only that enabled multiple donations from the same individual, who was presumably encouraged by the notion of multiple payments. In this manner, blood was centrifuged and the plasma removed, before pooling red blood cells from multiple donors and then reinjecting the mixture back into the original donor (10). Plasma selling not only disseminated HIV infection more widely, but it also ensured high viral titres in recipients of contaminated blood. It is estimated that up to 250,000 blood donors became HIV positive from blood donation during the 1990s (13) . In Donghu village for example, over 600 villagers sold their plasma to blood companies, among whom 231 became HIV positive (13) . In Wenlou village Henan, over 60% of the population are now HIV positive (6) . The overall HIV prevalence among former blood donors now appears to range from 1% to 40%, depending on the individual village (13) .
Although the Chinese government initially responded to the Henan outbreak by banning journalists and international aid workers (13) , it eventually addressed the issue of unsafe blood donation and officially banned the practice of pooled donations. Their countermeasures were reasonably successful, with few blood-donation-related HIV cases appearing after 1996 (8) . Nonetheless, the spreading phase probably never ended as such; rather it was superseded by the expansion phase as HIV positive individuals began unknowingly infecting others. This situation was facilitated by a number of interrelated and complex social factors that China faced as it rapidly industrialised and social freedoms increased. HIV now has a firm foothold in Chinese society, with HIV-1 subtypes now including A, B, Thai B, C, D, E, F and G, as well as BC and BB recombinants (4).
Intravenous drug users represent one of the most important social groups for HIV transmission in contemporary China, with approximately 60% to 70% of reported HIV infections occurring within this demographic (4) . Most appear to be men aged between 20 and 29 years of age (8) . Interestingly, some investigations have revealed that the HIV prevalence rate may be higher among female drug users than males (1) . Either way, their overall number has certainly increased rapidly in recent years. For example, there were officially 170,000 drug users nationwide in 1993, a figure which increased to 400,000 in 1994 (2) . By 2001, the official population of drug users was put at 900,000 (4). The prevalence of HIV among drug users, although variable from study to study, is known to be uniformly high. In Yunnan province, bordering Burma, almost half of all Heroin users are infected. In Xinjiang province, some studies have shown a HIV prevalence rate of 85% among drug users (8) . It has been demonstrated that after 1 year of injecting drug use, the prevalence of HIV infection reaches 68% (1) . HIV infection also occurs in conjunction with Hepatitis C Virus (HCV) and other blood-borne diseases. A recent study of drug users in Sichuan province showed the prevalence of HCV and HIV to be 71% and 11% respectively (14) . HIV has now spread to all 30 provinces throughout China (6) . At present, Yunnan has the highest number of HIV infected persons, representing approximately one-quarter of all reported cases. In Dehong for example, more than 1% of all pregnant women now suffer from HIV (15) . Although Yunnan is one of China's poorest provinces, increased foreign trade in recent years has paradoxically improved the economy while simultaneously facilitating the spread of HIV (16) .
HIV in China is also being spread via sexual contact and heterosexual transmission now accounts for about 7% of all cases (4). There are a few reasons for this trend. Firstly, an increasing proportion of the country's youth are no longer waiting until marriage before they have sex (6) . This large demographic of 15 to 24 year old persons is estimated at approximately 210 million (6) . A history of multiple sex partners is also increasing among young Chinese, especially in the more urban areas. Traditional Chinese conservatism has made it difficult to talk openly about sexual matters, particularly in the older generation. Lack of public health knowledge regarding HIV/AIDS also represents a problem, with many contemporary Chinese knowing very little about sexually transmitted diseases and their prevention (5) . A recent survey showed that less than 3% of people were aware that condoms minimized the risk of HIV infection and only 21% knew they could be infected through sexual intercourse. Furthermore, almost 20% of the respondents had never even heard of HIV or AIDS (5) .
Prostitutes represent another important social group for HIV transmission, and similar to injecting drug users, their numbers have been steadily increasing in recent years. According to the Ministry of Public Security, 137,000 prostitutes and their clients were arrested in 1990, 240,000 in 1992 and 400,000 in 1993 (2) . This number had risen to 700,000 by the late 1990s (1). The overall number of prostitutes in modern China is now believed to exceed 3 million. Among them, high levels of HIV and other sexually transmitted disease have been revealed. Research in Yunnan and Guangxi demonstrated the HIV prevalence among sex workers to be about 5% and 11%, respectively (6) . Concurrent gynaecological infections may also facilitate sexual transmission of HIV, particularly in rural communities (6) . In this regard, prostitute arrest records have revealed that the majority of commercial sex workers have at least one active, sexually-transmitted disease (17) . Prophylactic usage is not widely practiced among Chinese sex workers. Estimates of condom usage among prostitutes ranges from 1% to 30% (1). Condom usage usually depends on the customer. Some studies have shown that most prostitutes have never had formal sex education (17) . Many of them are also intravenous drug users, while sexually transmitted diseases are commonly treated with over-the-counter medicines. As prostitution is officially illegal in China, many infected sex workers are understandably reluctant to seek help or make themselves available for HIV testing and counselling.
Their customer demographic is also concerning. A recent study of prostitution users showed that their main clients were middle-class men under the age of 35. Men with the highest incomes were considerably more likely to have bought sex than those less well-off (6) . In this manner, commercial sex workers may transmit HIV to their married clients, who will then transfer it to their families. Another factor compounding prostitution usage is the traditional social preference for male children, which became particularly acute following China's one-child policy (10) . There is now an increasing demographic trend for males to outnumber females, with the rate currently believed to be about 120 males for every 100 females (18) . This situation has resulted in less available women for sexually active males, which in turn, encourages or even necessitates prostitution usage. The problem may not be temporary either. From current figures it is anticipated that, over the next decade, around 10 to 15 million young Chinese men will have zero future prospects for marriage. This large demographic of unmarried men may be destined to find no sexual outlet during their lives other than commercial prostitution (10).
Homosexual transmission is another probable HIV vector in China, with the number of gay men currently estimated at between 5% and 7% of the total male population (18) . Men who have sex with men probably represent a group of between 2 and 8 million people (19) . A survey of Chinese college students in the 1990s suggested that up to 8% may have homosexual tendencies (2) . Interestingly, around 80% of gay Chinese men are currently married (6) . Approximately 2% of married Chinese in rural areas engage in homosexual activities, a higher rate than for urban areas (4). Although homosexuality was removed from the official list of mental disorders in 2001, it remains illegal and highly stigmatized throughout Chinese society (1) . The problem may worsen in future years if China's homosexual remains highly marginalised. In the year 2000 for example, gay men comprised one-third of all AIDS patients in two dedicated AIDS care hospitals in Beijing (6) .
Furthermore, safer sex appears not to be widely practiced among China's homosexual community. For example, a recent study showed that condom usage, either with heterosexual or homosexual partners, occurred less than 10% of the time (6) . Another investigation revealed that homosexual Chinese men may often engage in bisexual behaviour and/or paid sex with commercial sex workers, thereby expanding their range of potential contacts (20) . This latter point is particularly important, as HIV prevalence is probably increasing among homosexual communities within China. A study of homosexuals recruited in Beijing for example, demonstrated that the HIV prevalence was about 3% and was independently associated with being older than 39 years and having more than 20 male sexual partners (19) .
Another of China's major HIV carriers appears to be the increasing number of itinerant workers who are for the most part, young, poorly educated labourers, in a sexually active period of their life (10) . During the Maoist era, a strict system of household registration kept most people confined to the immediate area in which they lived. With China's increasing economic prosperity and personal freedoms, such limitations were lifted and people were freely able to travel around. Rapid modernisation and industrial development also contributed to this demographic (4). The situation was compounded in poorer rural areas, where many young people left their hometown in search of a more prosperous life in urban centres (18) . Additionally, there were around 40 million workers who became unemployed when previously state-owned enterprises failed due to free-market economic reform. Itinerant workers now number at least 120 million (6) . Most of them are between 15 and 45 years of age, and by the nature of their demographic, represent a floating population that is difficult to educate, monitor and treat. They typically travel between their rural hometowns and cities seeking itinerant work, thereby providing an ideal vector for the extensive dissemination of HIV infection to remote areas (10) .
Although prostitution is illegal in China as previously noted, commercial sex workers are readily available to itinerant workers and other travelling subpopulations such as longdistance truck drivers (21) . Again as previously noted, condom usage is not widely practiced among prostitutes, HIV knowledge and preventive measures are not well known; and like their nomadic clients, commercial sex workers themselves are also a very mobile subgroup. China's itinerant workforce does not represent the only mobile vectors however. Newfound economic prosperity and individual freedoms have made international travel possible for many, with almost 100 million people visiting China in 2002 and around 17 million Chinese travelling abroad (21) . These individuals may also provide additional vectors for HIV dissemination and importation.
Although China's first HIV patient was reported almost 20 years ago, official government reaction was initially unrealistic and suboptimal. This situation was also compounded by a general community belief that HIV was simply a 'foreigners' disease that must be kept out (3) . Most early programs targeting HIV seem to reflect such notions. Even early predictions of HIV prevalence were hopefully optimistic, suggesting that it might only affect between 50,000 and 250,000 by the year 2000 (2) . As previously noted however, the real figure was probably double or triple these early government estimates. In many ways, Chinese officials were reluctant to acknowledge that their country was beginning to suffer from a disease, which was at the time, often associated with sexual promiscuity or moral degeneration (8) . The HIV issue appears to have been essentially ignored until the summer of 2002 (7), by which time the national HIV reservoir had probably reached a critical figure of 1 million. The problem was not solely due to central government inaction however, with local government officials in Henan province (one of the worst hit areas) often detaining aid workers, journalists and other health workers in futile attempts to cover up their own regional epidemics (7) .
Although the gallant efforts of various individuals undoubtedly occurred behind closed doors for many years during China's growing HIV epidemic, official and visible government response was essentially delayed until the late 1990s. In January 1997, the Chinese government announced that it would take positive steps to keep HIV infections below 1.5 million by 2010 (3). In November 2001, an unprecedented international meeting on HIV/AIDS was held in Beijing, combining the talent of over 2700 health care experts (22) . Two important AIDS symposiums were also held in Beijing in November 2003, where guest speaker Bill Clinton warned that HIV might hinder China's continued economic progress (9) . However, it was the unprecedented and highly symbolic handshake between Premier Wen Jiabao and an AIDS patient in late 2003 that reverberated strongly across the country (10). On World AIDS Day, December 1, virtually every major media outlet published the handshake photo acknowledging this formerly taboo subject. Premier Wen even told the AIDS patients: 'You must have confidence. All of society cares about you' (5) . In a country steeped in tradition and heavily influenced by symbolism, the message was clear. It was also the first time a high ranking official from the ruling party finally confronted the type of discrimination that HIV positive Chinese are burdened with on a daily basis (10) .
On World AIDS Day 2003, China's health minister finally acknowledged that their government was probably not doing enough to fight the emerging HIV epidemic. He offered to expand four important services: free HIV testing, free antiretro-viral drugs, free care for HIV-infected mothers and free education for AIDS orphans (5) . Most importantly the testing and treatment would be free for those who could not afford to pay for it themselves (23) . Aggressive public health education and prevention campaigns are now underway in China, although their effectiveness may be limited and are yet to be adequately demonstrated (24) . Nevertheless, in Yunnan Province, the government recently issued Regulation 121 which focuses on aggressive HIV education. Hotels with prostitutes must now offer condoms (15) . A new HIV vaccine is also being developed at the Chinese Academy of Medical Sciences, and one which uses distinctly Chinese primates as the research instrument (25) .
China's health care system
Although positive steps have now been taken by the Chinese government in combating HIV/AIDS, numerous challenges remain. One issue is the structural inadequacy of China's current health care system that was graphically revealed during the SARS outbreak (26) . Tuberculosis offers another pertinent example of how these shortcomings may compound the situation. China now has twice the level of multi-drug resistant tuberculosis of other countries (7) . Providing affordable and accessible health care to the general public has also become increasingly difficult following the introduction of free-market reforms in 1978. In this manner, the rural health cooperatives that once provided health insurance and affordable health care have now been effectively abolished (7) . Furthermore, the 'middle tier' community hospitals which formerly dispensed a great proportion of total health care have also disappeared (27) . Such facilities will be essential to the success of any future HIV treatment and prevention programs. Social inequalities are also problematic, as increasing numbers of Chinese hospitals now charge fees for services, thereby excluding those who cannot afford to pay. Drug users, prostitutes and itinerant workers represent important HIV-affected sub-populations that have limited capacity to pay for medical care and drug treatments.
Although antiretroviral medications (such as indinavir, efavirenz and nevirapine) are now available from Chinese hospitals, most HIV positive individuals still rely on traditional Chinese medicine (1). Many patients also alternate between traditional Chinese medicine and standardised pharmaceutical regimes, which in turn, creates problems with drug compliance and resistance. In one Beijing study for example, 20% to 30% of the subjects had become resistant to nevirapine after 9 months (24) . The costs of these drugs are also prohibitive for many Chinese HIV patients, although the price has fallen considerably in recent years. In 2001 for example, the average annual cost was US$10,000 (22) . By 2004, this had fallen to US$200 or US$300 (7) . Unfortunately, the drop in price was not uniform among all drug types and from all drug companies. For example, the main drug of choice for treating HIV in Africa and Thailand still remains prohibitively expensive in China (24) . Although China has recently offered to provide free HIV drug treatment for those who cannot afford it themselves, it remains to be seen whether the country can keep such a bold promise. Nevertheless, it is estimated that these costs may be minimised to about US$30 million per year, a figure which is probably affordable in the current economic climate (7) .
The accurate identification of HIV positive individuals represents another barrier to effective public health interventions in China's HIV/AIDS epidemic. For example, although an estimated 80,000 Chinese HIV patients now have symptomatic AIDS, less than 5% have been identified (24) . General knowledge regarding the biological mechanisms and public health implications of HIV also remains problematic. There appears to be a false sense of security among ordinary Chinese that HIV is a foreigners' disease which only affects marginal communities (26) . A previous community survey demonstrated that only 3% of Chinese people were aware that HIV was transmitted through an exchange of bodily fluids. However, in the same study, 54% of participants thought that sharing chopsticks could spread the disease (8) . Lack of preventive knowledge is another serious issue hampered by limited HIV-related knowledge. Aside for hampering the ultimate success of public health intervention programs, this type of biological ignorance also stigmatizes people already carrying the virus, forcing them even deeper underground and away from potential sources of help.
HIV/AIDS clearly represents one of the greatest challenges for preventive medicine in modern China. There are at least 1 million HIV positive individuals at present, and this figure may reach 10 million by the year 2010. However, the situation is not hopeless. In particular, the SARS epidemic provided an important learning experience and a key to the future battle against HIV/AIDS in China. Firstly, it revealed some important deficiencies in the medical and political infrastructure. Secondly, and perhaps crucially, it captured the attention of the international community and China's top leadership (28) . This may indicate tentative acceptance of the comprehensive response needed to combat the nation's greatest public health challenge. In some ways, Thailand can be seen as a role-model for HIV prevention within developing countries. Thailand has been particularly successful in reducing new HIV infections among prostitutes and military recruits with their 100% condom program (6) , and this approach may be appropriate for China. Either way, there will be many obstacles in controlling the epidemic, not the least of which will be to change public perceptions of HIV and raise awareness of preventive measures. Whether the Chinese government can muster the resources needed to keep its bold promises is also unknown. What is certain is that China now faces a serious epidemic in the new millennium, and one which will pose numerous challenges for public health and preventive medicine. Emerging zoonotic diseases: An opportunity to apply the concepts of nidality and one-medicine The use of animals as sentinels of human disease revolves around the concept of nidality. That is, an agent of disease occupies a particular ecologic niche and alterations in that niche will change the function of that agent relative to traditional host-agent-environment relationships. Nidality is a derivation of the root word nidus. Nidus is defined as a nest or breeding place, particularly a place where microbes such as bacteria, fungi, viruses, as well as other organisms and larger parasites, are located and multiply. Application of the concept of nidality and development of prevention strategies has most frequently been associated with military campaigns and interruption of tick-borne infections. Modern usage of the phrase “one-medicine” was popularized in the United States and Europe by Calvin Schwabe and the concept is attributed to Rudolph Virchow. It is applied today to the study of zoonotic disease and interventions in rural agricultural communities that share close living arrangements between people and their families, their pastoral work environment, and the animals for which they care. Integration of the two concepts of one-medicine and nidality provides an opportunity to apply a systems approach (i.e. general systems theory) to dealing with emerging zoonotic diseases in today’s global agricultural and industrial settings. The use of animals as sentinels of human disease revolves around the concept of nidality. That is, an agent of disease occupies a particular ecologic niche and alterations in that niche will change the function of that agent relative to traditional host-agent-environment relationships (1) . Nidality is a derivation of the root word nidus. Nidus is defined as a nest or breeding place, particularly a place where microbes such as bacteria, fungi, viruses, as well as other organisms and larger parasites, are located and multiply. Application of the concept of nidality and development of prevention strategies has most frequently been associated with military campaigns and the interruption of tick-borne infections (2) .
Modern usage of the phrase "one-medicine" was popularized in the United States and Europe by Calvin Schwabe and the concept is attributed to Rudolph Virchow (3). It is applied today to the study of zoonotic disease and interventions in rural agricultural communities that share close living arrangements between people and their families, their pastoral work environment, and the animals for which they care. A recent movement in the United States, the "Medicine and Public Health" initiative (http://www.sph.uth.tmc.edu/mph/) recognized that human clinical medicine and public health communities had developed parallel but separate enterprises and there was a need to reassess this division. Medicine (focusing primarily on the health of individuals) and Public Health (with a population perspective) needed to find innovative and joint solutions to improve the health of the people of the United States. Similar schisms are evident between veterinary medicine and the agricultural industry in the United States and initiatives are underway to bridge these gaps. Integration of the two concepts of one-medicine and nidality provides an opportunity to apply a systems approach to dealing with emerging zoonotic diseases in today's global agricultural and industrial settings.
In the late 1990s, our School of Public Health embarked on a series of Bioterrorism Awareness Seminars for health care students and primary care providers, i.e. family practitioners, pediatricians, general internists, nurse clinicians, physician assistants, and others. I struggled with the development of a communications approach that would capture the interest of the audience, especially prior to the events of September 11, 2001. The presentation of information needed to raise a clinician's awareness for the early diagnosis of a bioterrorism incident was considered by many to be esoteric, an unlikely scenario, and not relevant to the daily practice of medicine in the United States.
As a public health veterinarian, I knew that many of the biological organisms identified as potential agents of bioterrorism occur naturally in North America, specifically Texas which is situated on the southern border of the United States of America. Texas has enzootic foci of anthrax, tularemia, and plague. Mosquito-borne encephalitides such as Saint Louis Encephalitis, Eastern Equine Encephalitis, Western Equine Encephalitis, and most recently West Nile Virus occur in our geographic region. We have had cases of hemorrhagic fever (Ebola alice) in primate colonies in Texas. Dengue is endemic in countries immediately to our south. Vibrio species infections are identified in association with our seafood and shellfish industry in the Gulf of Mexico. It is not unusual for sporadic human cases of infection with these organisms to be diagnosed by clinicians in Texas. The teaching challenge was to accomplish our goal of "heightened awareness" for agents of bioterrorism and also make the seminars relevant to the daily practice of medicine (4).
There is an overall lack of knowledge regarding the natural history and ecology of these zoonotic organisms (5) . There is an abysmal lack of understanding among the general public and media of the natural occurrence of many biological organisms in the agricultural and animal industry setting as well as in free-ranging animal populations. The natural history, normal ecology, and epidemiology of potential agents of bioterrorism provide scenarios for explaining risk to the human population at large. Understanding the natural history of these organisms and their role in agriculture and free-ranging animal populations provides a context for health care providers to make appropriate risk assessment decisions.
When first confronted with the emergent threat of bioterrorism I used a mental paradigm based on the principles of nidality. Instead of considering the biological niche of the organism in nature, however, I chose to define the agent's nidality from an epidemiological perspective. Every organism that causes disease in human populations has an expected epidemiologic presentation. The epidemiologic pattern provides clues as to whether the clinical presentation is "expected" or "un-expected" in that physician's range of practice experience. A corollary to the expected/un-expected clinical paradigm is the potential route of exposure to the organism in a particular geographic and/or occupational setting (6) . Routes of exposure can be categorized as "natural" or "un-natural" and may in fact influence the constellation of signs and symptoms observed by the clinician. I now employ these concepts in teaching and instructional activities to prepare health care providers in biodefense preparation and response.
The paradigm that I employ is as follows: ‫ع‬ Expected and natural clinical presentation and epidemiologic circumstances ‫ع‬ Unexpected but natural clinical presentation and epidemiologic circumstances ‫ع‬ Unexpected and unnatural clinical presentation and epidemiologic circumstances Let me illustrate by providing examples of the three clinical presentations of anthrax in humans: cutaneous; gastrointestinal; and inhalational.
In South Texas it would be usual and expected to diagnose a sporadic case of human anthrax. The presentation would normally be a single cutaneous eschar on a visible surface such as the hand or arm. The epidemiologic history would most likely involve contact with a known diseased animal. The latest example was an adult male who cut himself while removing the hide (skinning) from a buffalo (American Bison) that had died at a game ranch. The geographic area had recently experienced a severe die-off of white tailed deer from anthrax. This clinical presentation and epidemiologic history would be usual and normal in South Texas.
It is rare to diagnose gastrointestinal anthrax in humans in Texas. Animal protein is abundant. Cattle raised for human consumption are inspected. The hunting culture is one of harvesting healthy and active game animals, not culling old and/or impaired specimens. When anthrax epizootics occur in free-ranging populations, they are in remote areas with minimal human interaction.
However, one could envision a scenario of ranch workers and/or unwitting hunters harvesting anthrax infected cattle or deer for the meat. Diagnosis of a cluster of gastrointestinal anthrax cases in a family or several acquaintances with a history of hunting their own food would be unexpected, but given the epidemiologic evidence, natural.
It is difficult to develop a scenario for the state of Texas where any occurrence of primary respiratory infection with the anthrax organism would be expected. However, Laredo, Texas is the largest inland port in North America. Ten thousand tractor trailer trucks cross the Mexico-United States border every day. If a trailer contained a cargo of anthrax infected animal hides, it is feasible that the truck driver and or illegal passengers riding in the trailer could be exposed through the respiratory route. This fictional example would certainly represent unexpected and unnatural circumstances.
All three of the preceding scenarios for cutaneous, gastrointestinal, and primary respiratory infection with the anthrax organism share several epidemiologic characteristics. Bacillus anthracis occupies a natural ecologic niche in the southwestern United States and parts of Mexico. The number of individuals involved in each scenario was small. The clinical presentations followed classical expectations. The epidemiologic picture matched the clinical presentation. Thus, the information presented to my clinical audience was relevant to their practice of medicine today in the state of Texas and hopefully raised their level of knowledge regarding the natural history of one of the potential agents of bioterrorism. I conclude with "clues" to understanding how the epidemiologic picture may differ if the anthrax exposures were man-made and intentional rather than a part of what is expected in our part of the world: the epidemiology would be wrong; there would be large numbers of human cases; there may be concomitant deaths of other animal species; and, there may be unusual strains of the organism.
The concepts of nidality and one-medicine provide the opportunity to explore the natural history of agents of disease; the inter-relatedness of human activity, animal industry, and free-ranging animal populations. Application of the principles espoused by the Medicine and Public Health initiative provides a framework for human medical clinicians, public health practitioners, and the veterinary medicine community to interact and address the threat of bioterrorism in a reasoned and scientific manner based on effective risk assessment. Disease ecology and the global emergence of zoonotic pathogens The incidence and frequency of epidemic transmission of zoonotic diseases, both known and newly recognized, has increased dramatically in the past 30 years. It is thought that this dramatic disease emergence is primarily the result of the social, demographic, and environmental transformation that has occurred globally since World War II. However, the causal linkages have not been elucidated. Investigating emerging zoonotic pathogens as an ecological phenomenon can provide significant insights as to why some of these pathogens have jumped species and caused major epidemics in humans. A review of concepts and theory from biological ecology and of causal factors in disease emergence previously described suggests a general model of global zoonotic disease emergence. The model links demographic and societal factors to land use and land cover change whose associated ecological factors help explain disease emergence. The scale and magnitude of these changes are more significant than those associated with climate change, the effects of which are largely not yet understood. Unfortunately, the complex character and non-linear behavior of the human-natural systems in which host-pathogen systems are embedded makes specific incidences of disease emergence or epidemics inherently difficult to predict. Employing a complex systems analytical approach, however, may show how a few key ecological variables and system properties, including the adaptive capacity of institutions, explains the emergence of infectious diseases and how an integrated, multi-level approach to zoonotic disease control can reduce risk. The growing problem of globally emerging infectious diseases (EIDs) has prompted a substantial effort by the biomedical research establishment to identify the causes and recommend action. As reported in the most recent of a series of volumes (1), a main finding is that the current episode of global infectious disease emergence is the result of a convergence of factors involving complex interactions among numerous variables. This includes genetic, biological, social, economic, political, ecological, and physical environmental factors, and calls for an interdisciplinary research agenda. It is also concluded that "human development and large scale social phenomena are closely associated to infectious disease threats at a global level," which points to the need for research focused on "social and ecological factors affecting infectious disease emergence" (1) .
The phenomenon of globally emerging infectious diseases requires understanding biological systems in the broadest sense and dealing with their extraordinary complexity. This includes processes operating at the level of transmission and evolution of a pathogen within and among host species and humans. It extends to and includes processes involving ecosystems and regional environmental change occurring on a global scale (2) . In fact the scale and magnitude of anthropogenic activity has reached a point of virtual co-dominance with natural processes of energy and material flows globally (3) .
Understanding these kinds of processes traditionally has been the domain of classical ecology, or natural history, plus systems ecology (4, 5) . Adding to this are recent ecological perspectives and models applied at the molecular, cellular level, and organismal levels (6) , and others addressing the complexity, multiple variables, cross-scale influences, and dynamic behavior at the level of natural ecosystems (7) , and social-ecological systems (8) .
Along with the research at the organismal level and below, that aimed at the level of social-ecological (coupled humannatural systems) is critical to the development of the comprehensive scientific framework necessary for understanding zoonotic infectious disease emergence in particular. Not only does this new area address the dynamic behavior of complex, large scale systems, but also bridges theory from the traditionally separate biological and social science disciplines, thus contributing to the interdisciplinary research agenda also called for in the above reports. The purpose of this paper is to consider how regional and global zoonotic disease emergence trends might be explained on the basis of current thought in biological ecology including the very recent developments new to the field of infectious disease ecology. Here we draw on ecological science as broadly defined as a basis for identifying causal mechanisms of zoonotic disease emergence, the ultimate goal being to enhance disease prevention and control programs.
Several authors have categorized causal factors of infectious disease emergence, including explicitly citing 'ecological' ones involving land use change (9) (10) (11) (12) or 'land use drivers' (13) , human movement (10, 12) , encroachment and wildlife translocation (10, 11) , rapid transport (9, 10) and climate change (11, 12) . Most recently, the Institute of Medicine (1) described these along with others (13 categories of factors in all) and a model stating the major categories of factors have historically converged to bring about the current global emerging infectious disease crisis. Ecological factors are described as one of four major categories of factors that have converged with social, political, and economic factors; genetic and biological factors; and physical environmental factors. We take a different approach to understanding the interaction of the above factors and their causal relationships by focusing on disease emergence as an ecological-evolutionary phenomenon influenced by human factors. Our interest is in how human factors interact with natural processes and, in particular, how mechanisms operating at levels meaningful to understanding pathogen transmission and evolution can result in regional and ultimately global phenomena (i.e., regional endemism, epidemics, or global pandemics).
As a first step we distinguish between the two broad categories of human factors, 'demographic and societal' and 'disease intervention and policy' suggested previously by Gubler (10) ( Table 1) . This categorization differentiates between factors associated with specific kinds of environments or ecosystems and those involving biological and policy factors not so associated. However, both can be described as part of a single ecological framework involving interaction of systems of essentially natural versus human design, respectively. Our focus is on the first category from the standpoint of how disease emergence is explained by ecological concepts and principles. This includes some relatively new models and theory not previously used to explain the current trend in increasing emerging infectious diseases. We present a general model of zoonotic disease emergence on this basis. We also discuss recent explanations based on complexity theory for how human behavior and ecosystems interact to contribute to disease emergence. Classical ecology, or natural history, has been the basis and mainstay of infectious disease research since its origins with 'Koch's Postulates' and subsequent development of microbiology and zoonotic disease epidemiology during the 19th and 20th Centuries (14, 15) . Throughout much of its early history, zoonotic disease research involved this descriptive, empiricallybased ecology: identifying the life cycle, transmission, incidental and natural hosts of pathogens, along with demographic, life history, dispersal, and habitat attributes of reservoirs and vectors. A substantial theoretical dimension has developed, beginning with Ross's mathematical analysis of malaria transmission (16) and extending to Anderson and May's (17) recent synthesis Infectious Diseases of Humans.
Although essential to designing effective prevention and control programs, empirical field based, disease ecology has been neglected in recent years (18) . Fortunately, theoretical disease ecology, stimulated largely by the work of Anderson, May and others has flourished and led to a significant syntheses involving application of ecological-evolutionary biology to the study of infectious diseases (19, 20) . Parallel to this, systems ecology has begun to extend its domain by applying complexity theory to emerging infections with at least initial suggestions of its implications (6) . This development in particular, along with observations from several decades of applications of systems ecology to natural resources and economic development (8, (21) (22) (23) , have resulted in important insights of significant potential in understanding zoonotic disease emergence as a cross-scale process. This area uses complex systems theory applied to coupled, human-natural systems to explain how processes such as local phenomena can result in a cascade of effects ultimately reaching global proportions. The finding suggests this crossscale behavior is controlled by relatively few variables, and is mitigated by social and ecological resilience. The loss of this resilience in ecological systems is observed to lead inevitably to unpredictable events or the 'surprise' characteristic of complex systems generally. This combination of socialecological systems and resilience theory helps explain the unpredictability of disease emergence events. It represents another potentially useful area of application to understanding emerging infectious diseases along with those areas generally considered within the domain of ecology mentioned above: population ecology and genetics, community ecology, and systems ecology.
Population ecology, genetics and disease emergence Of particular relevance to disease emergence is the explanation provided by theoretical population biology, already mentioned, of how host (including human) population size determines whether or not a pathogen can persist in a population. The accumulated findings demonstrate thresholds exist, depending on the type of pathogen and host population, below which a pathogen cannot be sustained. Considered in light of the exponentially increasing size of human and domestic host and vector populations in the world, the breaching of thresholds of pathogen persistence can explain much of the increase in emerging infectious diseases.
This can be explained as follows. Although zoonotic disease emergence is not entirely a tropical phenomenon, it is mostly associated with tropical developing regions undergoing the most rapid population growth and ecological changes. Prior to the post-WWII economic era, most regional ecosystems in the tropics consisted of relatively scattered human settlements, and only a few large cities (>500,000) (24) . These were separated by large expanses of cropland and pastureland and relatively undisturbed forest. Since then, in what has been the most rapid period of large scale ecological transformation in human history, the pattern has essentially reversed (25) . The once scattered settlements and few large cities have coalesced into expansive megacities and surrounding periurban settlements with only remnant patches of undisturbed forest remaining in a sea of cropland, scrub, and ecologically degraded lands. Dacca, Bangladesh, which grew from a population size of 200,000 to 13 million from 1970 to 2003, is one of many examples.
Thus the existence of population density-dependent thresholds for disease emergence is particularly relevant (26, 27) . This explains the abrupt transitions of urban diseases between non-persistence to endemic and endemic to epidemic behavior as population densities of susceptible humans, hosts, and vectors reach critical densities. The classic illustration is that of measles which, given its particular transmission rate, requires human settlements with population sizes in excess (>250,000) of what historically existed in most pre-industrial states and geographically isolated populations even today (28). Thus, for example, many infectious diseases endemic on continents have not become established on islands despite their occasional introduction and the occurrence of local outbreaks. The same mathematical ecology that explains why measles and virtually all diseases have threshold densities, explains the much lower thresholds existing for vector-borne diseases such as arboviruses (29) . Particularly noteworthy is the theoretical demonstration that the pathogen 'reproductive rate' increases with the square of vector population density. This indicates threshold densities can fast be breached as domestic and peridomestic hosts and vectors expand (or re-expand) their geographic ranges (once they are introduced or re-introduced) and increase their densities. This helps explain the explosive re-emergence of dengue and dengue hemorrhagic fever in the American Tropics as vector populations responded to relaxed controls and new breeding habitats associated with urbanization (30) . This phenomenon can be likened to the gradual build-up of 'dead and down' wood across a forested landscape with a history of fire suppression. The build-up of fuel, like that of host or vector populations, becomes an 'accident waiting to happen' when a single ignition event in one locality, similar to a single infection event, spreads to the entire region.
Another consequence of the dramatically increased densities of humans, host reservoirs, and vectors is the increased number of pathogen genomes. The resulting increased levels of genetic variability can accelerate microbial adaptation, including evolution of pathogenesis, and antimicrobial resistance. Genetic variability increases with population size and density through a variety mechanisms including mutation. The probability of producing more virulent variants not only increases with host population size but also with crowding and co-mingling of different host species (31) . In general, parasite (pathogen)-host relations naturally constitute a co-adaptive/ evolutionary 'dance' along the pathogenicity threshold, which is likely to be crossed with greater frequency due to unnatural anthropogenic disturbances (32) independent of increasing population sizes and pathogen genetic diversity.
The study of ecological communities and the 'community ecology' theory it has yielded includes a number of principles and mechanisms that describe how human disturbances as well as natural environmental variation can contribute to any of the above population level factors (33) . There are a number of implications to zoonotic disease emergence, although most have not yet been described in terms of disease emergence or in the medical, public health, or zoonotic disease literature. Of critical significance from this area of ecology is the general principle of community assembly. Research has demonstrated that communities of arranged predicably in terms of 'assembly rules' (34) . This order, in terms of the spatial distribution, composition and the abundance of each species in an ecological community, is affected by interspecific interactions (predation, competition, and parasitism). Density independent factors (e.g., weather, natural catastrophes) play an important, but a more ephemeral role in most ecosystems. The process of community assembly (and disassembly) has been particularly well demonstrated through studies of insular ecosystems, the so-called 'species area relationship,' and the phenomenon of faunal collapse (35) (36) (37) . A principal outcome and the ecological significance of this body of research first described in terms of the process of 'habitat fragmentation', has been identified as the principal mechanism by which human land and resource use contributes to species extinction (38) . Although initially debated, a significant amount of evidence has since accumulated resulting in its general acceptance and applicability, particularly to tropical forest ecosystems (36, 39, 40) .
The critical significance of habitat fragmentation and related human disturbances to disease emergence stems from it contribution to the disassembly of orderly natural communities. For example, human activities such deforestation, use of pesticides, and various forms of pollution often result in the loss of predators. In fact, carnivorous mammals typically are the first species to disappear following forest fragmentation. Their local extinction represents the loss of 'top down' natural control in ecological communities. This can in turn result is an increase in abundance, even 'hyper-abundance' (41) , of species such as rodents and biting insects.
Community disassembly and the resulting loss of natural population control mechanisms for such species generally is associated with the conversion of natural landscapes to urban and agriculture landscapes. Broad spectrum pesticide use and habitat simplification, along with habitat fragmentation, are important contributing factors. The reduction in species diversity can contribute to the phenomenon of 'ecological release' in remaining species, whose predators, competitor and parasites are reduced in numbers or eliminated. Some of these may be already serving as zoonotic reservoirs or vectors. If so, ecological release may result in their proliferation. This helps explain why many emerging zoonotic diseases occur in areas where settlement and agricultural expansion recently have encroached into tropical forests. A similar phenomenon, associated with the regrowth of forests in developed regions, can lead to zoonotic disease emergence. The pattern of reforestation in Eastern North America during the past half-century resulted in increased habitat favored by forest edge adapted species such as whitetail deer and white-footed mice. White-tailed deer, the principle host of the adult tick Ixodes scapulari, reinvaded the area and with few predators and competitors the population exploded. This pattern of ecological change arguably has been a major factor in the emergence of Lyme disease in this region.
An extension of the concept of 'ecological release' has been suggested to explain the invasive species phenomenon in which super-successful introduced (alien) species have escaped from their natural complement of parasites (42) , as well as predators and competitors. This may explain in large part our most successful invasive domestic species, Rattus and Aedes species, which are hosts and vectors of some of our current and historically most problematic urban zoonotic diseases.
In sum, zoonotic infectious disease emergence can be explained in part as a consequence of the disruption of natural ecological communities, and the breakdown of naturally existing predator-prey, competitive, and host-parasite relationships that tend regulate and stabilize species' abundance. This can occur through the use of non-selective pesticides through changes in land use and land cover that affect the distribution and abundance of species. It should be noted that the disassembly of natural ecological communities that results from habitat fragmentation is a protracted process compared to exponential decay. Depending on the extent of habitat loss and other factors such as the sizes, shapes and spatial relationships of remaining fragments, the process may require decades, centuries or longer as communities 'relax' toward a new equilibrium (36, 43, 44) . It follows that the frequency of disease emergence can be expected to follow a similar path: highest rates at first, followed by gradual decline as an ecosystem 'stabilizes'.
The application of systems thinking to ecological communities centers on the ecosystem concept and has been the basis of significant research activity in ecological science since the 1960's (4) . Much of the focus has been on describing and understanding energy and nutrient fluxes across different kinds of ecosystems, and more recently emphasizing the human dimensions of global environmental change (45) . Although global scale ecosystem change has been considered in reference to human and infectious diseases (13, 46) , no systematic framework has yet been presented in this regard. The evidence presented has been anecdotal.
However, the development and application of systems theory independently of and largely outside the realm of mainstream systems ecology (47) shows significant promise in providing a basis for more systematic interpretation of the role of ecosystem change in disease emergence. Recent thinking draws in particular on the application of the theory of 'complex adaptive systems' to ecological systems in which ecosystems are represented as scaled, self-organizing, far from equilibrium, evolutionary, and non-linear (6) . Organization, diversity, and resilience are important 'emerging' properties of complex systems-often equated with a 'healthy' state. 'Surprise', or qualitative disagreements of system behavior with a priori expectations, is another property (48) . Their association with loss of system resilience and increased vulnerability applies both to increasing inflexible social systems 'managing' and attempting to 'control' natural variables (i.e., vectors, pathogens) and that of the ecosystems whose component variables (e.g., vector and pathogen populations) are targeted for management or control.
Alteration of natural disturbance regimes (via control of natural variation via flood control projects for example) reduces resilience, while secondary disturbance events (wildfires, storms, floods, and earthquakes) precipitate events caused by crossscale influences (e.g., a thunderstorm igniting a fire locally that spreads regionally as a result of fuel build-up from years of artificial fire suppression). Regional environmental change such as that associated with population growth development often does not accommodate the need to maintain resilience (21) . Such ecosystems have been described as 'over-connected' or 'brittle' and, as stated above, 'accidents waiting to happen' (8, 22) . Floods, often associated with waterborne disease outbreaks, occur more frequently and with greater severity as a result of lost resilience in natural systems due to attempts to 'control' (in contrast to manage) natural variation. Conversion of upland forests to plantations or cropping systems for more 'efficient' natural resource production is another, as is agriculture generally. Both result in a reduction of heterogeneity in ecosystems that tend to 'buffer' against disease outbreaks, which can spread more readily across the more uniform landscape including immunologically vulnerable domesticated species. Gunderson et al. (8, 23) describe this as general pattern of institutional behavior. It begins with the targeting of a natural variable for control, followed by increasing institutional efficiency and inflexibility in the control methods used. As the target variable and ecosystem changes and initial signs of failure are ignored, the ultimate result is a crisis. This 'pathology of regional resource and ecosystem management' and is apparently applicable to infectious disease management as well.
The above body of ecological theory and observations involving specific emerging infectious disease cases suggests a causal schema that links ecological phenomena on the scale of pathogen transmission and evolution to regional and global transformations. This is represented by Figure 1 , focusing on the role of demographic and societal factors in disease emergence (shown in Table 1 ). This schema is constructed using the generally adopted view of human-environment interaction in which the impact of human population and technology is taken as the driving force, or ultimate cause. Clark et al. (25) elaborate on this in their seminal treatment on global and regional change. Here, the combination of population, technological capacity, and sociocultural organization act as the system drivers of regional environmental change. These and 'mitigating' forces are in turn influenced by 'human behavior', referring to patterns of actions and the rationales giving rise to them. Broadly speaking, these forces and their affects on ecosystems represent the 'ecological factors' in social-ecological systems, while human behavior represents the 'social factors' for the purposes of discussion.
This schema represents zoonotic disease emergence from the perspective of the ecosystems within a regional environment, the large scale processes involved, and the associated ecological effects and processes involved in disease emergence. Thus, referring to Table 1 , the factors under 'Demographic and Societal Changes' can be identified in reference to particular ecosystems and processes (i.e., urban and urbanization, agriculture and agricultural intensification, forest and forest alteration), and associated factors operating on regional and global scales. These are unprecedented population growth, unplanned and uncontrolled urbanization, non-biodegradable packaging of consumer goods, and modern transportation, all contributing to conditions that promote pathogen transmission and persistence.
For zoonotic diseases, which by definition involve the jumping of a pathogen from its natural host to humans, and in some instances extension of its host cycle to include humans, conditions can be described for this simple schema as follows. The likelihood or frequency of transmission events change when the natural host or pathogen changes, humans change, or the ecosystem supporting both changes. Thus, fundamentally the processes influencing transmission of zoonotic pathogens can be described as a consequence of one or a combination of three possible kinds of change: expansion of the habitat or geographic range of a host, of a pathogen or both; expansion of human's habitat or geographic range; or change in the habitat or ecosystem occupied by both humans and the natural host. Evolutionary adaptation of pathogens is omitted from the figure. However, it can be assumed any factor contributing to increased likelihood of transmission, as well as increased population size of hosts and pathogens will contribute to the potential that new, increasingly pathogenic, infectious, antimicrobial resistant variants will emerge. Anthropogenic climate change, while not incorporated in the diagram, can be considered to potentially contribute to disease emergence through its contribution to habitat alteration.
Our review of ecological theory and the resulting complex model described here demonstrates the limitation of classical disease ecology and natural history. For example, without incorporation of population genetic theory in ecology as 'evolutionary ecology', along with the concepts of pathogen spillover and cross-scale ecosystem dynamics, classical disease ecology cannot explain recent emergence events like those involving HIV/AIDS, SARS, avian influenza, E. coli 0:157, dengue, malaria, West Nile virus, and Nipah virus, among others. The resurgence of vector populations, having acquired pesticide resistance and lost predators and competitors as natural controls after a regimen of inappropriate pesticide use, similarly is explained by modern ecology and evolutionary biology. This has direct application to control and intervention policies and practices. For example, it points to the need to adopt control strategies that integrate landscape and habitat management with careful rotation of targeted chemical and biological agents (30) . Developing such strategies requires detailed field studies, built on traditional natural history and classical disease ecology, but supplemented with advanced molecular techniques, as well as ecological research that takes into account the community and systems level dimensions of a particular pathogen-host complex.
The continual expansion of human populations since prehistoric times, and particularly since the advent of settled agriculture with its associated domesticated animals exposing humans to their pathogens as well as those spilling over from wild animal populations, has incrementally added to the pathogen load through successive invasions by different organisms over time (49, 50) . Well established principles of population ecology, applied via mathematical epidemiology as Anderson and May and others so aptly have done, readily explain why, ceteris paribus, infectious disease incidence should generally be increasing with human population size, as it has in the world's poorest and most populous regions. Yet in spite of what are in general commonly understood principles, and warning signs that went unheeded in the 1970's and 1980's (10, 30), biomedical and public health institutions were unprepared for the recent surge in emerging infectious diseases (1, 9, 10, 51) . Not until the 1990's, prompted in part by the HIV/AIDS pandemic and the failure of 'quick-fix' solutions based on drugs, vaccines, and pesticides (18) , did the biomedical science and public health communities begin to launch a significant response.
In light of the complexity of the factors involved, this lack of preparedness should perhaps not be surprising. As explained by the infectious disease ecology described here, zoonotic disease emergence involves biological processes operating on the scale of molecules and cells to that of coupled, regional scale human-natural systems. The latter involve political economic factors and policies driving regional environmental change, spreading geographically across the globe. It is this process of globalization-its ecological underpinnings and consequences-to which the current EID global trend of zoonotic disease emergance can be largely attributed.
Lack of awareness of the ecological implications of Fig. 1 Diagram depicting infectious disease ecology causal schema. The aggregate variable at the top of the diagram, representing population and consumption, along with mitigating socio-cultural attributes, is the driver or 'forcing function' responsible for land use and land cover change characteristic of a particular region. The result is varying degrees (and types) of urbanization, agricultural intensification (including food production and distribution), and alteration of natural habitat. These changes at the level of landscapes and habitat produce conditions influencing the ecological and evolutionary dynamics of vector and host species and vector/host-pathogen dynamics. In turn, these conditions facilitate the geographic expansion, novel appearance, or increased epidemic activity of a disease.
regional environmental transformations, and of their synergy with the ecological and evolutionary consequences of inadequate or inappropriate policies or methods of vector control and disease prevention, have unwittingly promoted disease emergence. The 'ecological' changes taking place as a result of public health agencies' actions (or inaction) involve pathogen biology and are small scale in time and space: selection for vector pesticide and antimicrobial resistance. However, the cumulative effect of micro-scale processes involving pathogen adaptation and host range expansion (or re-expanding) can ultimately produce regional and even global consequences. However, policy action and inaction outside the domain of biomedical or public health agencies has produced ecological changes at a historically unprecedented rate and scale. Urbanization, agricultural and food production intensification, alteration of natural habitat, along with concomitant loss of ecosystem functions, have transformed entire regional ecosystems during the past 50 years. The role of urbanization cannot be underestimated. The direct land use changes associated with urbanization effectively concentrate human, animal reservoir and vector populations at unprecedented densities. But urbanization also is strongly tied to socio-economic and cultural factors, along with human migration dynamics, affecting agricultural and food production intensification, as well as rural and natural ecosystems. In the case of food production and distribution, dramatically increased contact with and transport of wildlife (e.g., bush meat trade in Africa), and increased ruralurban transport and concentration of wild species for the exotic food market (e.g., civet cats in Guangdong, China), is another contributor to increasing disease emergence. It can be assumed these impacts on wildlife populations also contribute to changes in the composition of ecological communities on a regional scale (52) , and often result in a hyper-abundance of small mammal species low in the food chain, which are likely to serve as human disease reservoirs. Similar effects of ecological disruption appear to generally apply to invertebrate commu- Whether a disease 'jumps' to the regional population and the global human population scale is determined by quite different processes. These depend on demographic and transport patterns related to processes, like urbanization, influencing pathogen transmission and behavior operating on the regional and global scales. Physical environmental processes such as climate variability (on the right side of the figure), which are episodic by nature, include short term and small scale variations in the form of seasonal storms (e.g., monsoons), for example, as well as larger time and space scale variations. These include, for example, decadal and regional scale changes in weather patterns such as El Niño. These climate changes and weather events can precipitate floods and droughts. These act as cross-scale mediators that directly affect disease reservoir and vector populations, or pathogens (e.g, dispersal via flood waters). They cause the disease to jump from a smaller demographic unit to a larger one (e.g., from a single village to a district). Human Factors such as land use and land cover changes (Table 1 and Fig. 1 ) produce ecosystem changes contributing or magnifying the cross-scale processes. For example, urbanization and deforestation increase the magnitude of floods and droughts resulting from natural or anthropogenic climate variation. nities (53) , the kinds of organisms most commonly serving as human disease vectors. The estimated acceleration of natural species extinction rates by orders of magnitude over nonanthropogenic extinction rates (54) gives an indication of the scale and magnitude of change in natural communities, particularly in the tropics. In these regional ecosystems, the original extent of tropical forest has declined to a fraction of its original area with concomitant affects on biodiversity (38) .
Large scale agricultural expansion (a common proximate cause of tropical deforestation) has decelerated and all but ended in many regions. This suggests that further contributions from what historically has been a main factor in natural habitat alteration, ecosystem disturbance and biodiversity loss, will decline. Yet agricultural intensification has replaced expansion with technologies that further impact native biodiversity (55) . The reduction in plant species richness that accompanies agricultural intensification leads to changes in the community composition of the pest complex-herbivorous insects, their natural enemies (predators and parasites), and the microbial community attacking crops (56) .
The schema described here demonstrates how regional environmental change, involving ecological dynamics such as demographic or landscape transformations on the scale of decades, interact with change on the scale of host-parasite/ pathogen dynamics. How these cross-scale mechanisms produce regional or global scale disease emergence patterns is beyond the realm of conventional epidemiology, or analytical approaches generally. This may explain why such changes either are not apparent or their implications in terms of disease emergence are a low priority to biomedical and public health agencies. Such cross-scale processes are however characteristic of ecosystems, whose dynamics involve interaction of variables and operate on vastly different time and space scales, involving natural processes that are discontinuously distributed as shown in Figure 2 .
Change in such systems is difficult to understand employing conventional analytic approaches alone, but require new thinking and analytical methods drawn from complex systems theory. The discontinuous character of the processes or variables makes their interaction intuitively improbable except for the mediating effect of 'cross-scale influences' as illustrated in Figure 2 . Examples include the eruption of Hantavirus in the American Southwest, thought be associated with weather events precipitated by El Nino Southern Oscillation. Storms events are similarly episodic, and represent a discontinuous form of variation transporting pathogens via flood waters in domestic and natural environments. On the other hand, massive environmental events, like the recent Southeast Asian tsunami, are potentially capable of producing epidemics across an entire region, and may episodically extinguish some zoonotic diseases by temporarily destroying reservoir and vector populations and habitat. The roles of resilience and vulnerability in determining the severity of such events, both in terms of social systems and ecological systems is another critical aspect of social-ecological systems behavior (8) .
The recent worldwide upsurge of zoonotic infectious diseases, involving the resurgence of a growing number of diseases previously believed under control or the emergence of newly recognized diseases, has been attributed to a list of global factors characterized in terms ranging from microbial adaptation and land use to changing ecosystems, breakdown of public health, and poverty (1, 57) . The categorization provided by Gubler (10) , elaborated here based on an expanded view of infectious disease ecology, provides the basis for a schema describing the causal relations involving factors previously identified, along with hypothesized mechanisms.
The complicated nature of this problem, which obviously entails numerous interacting variables operating on different time and space scales pose a significant challenge to biomedical science and epidemiological research as well as public health intervention. However, the current trend of increasing global emerging infectious diseases is linked with another issue of 'global governance', sustainable development, with which disease control and prevent strategies must be integrated. Here too, the problems of politically stability, population growth, unplanned urbanization and economic development, income disparity, and environmental degradation, are all integral to the solution.
This interdisciplinary imperative challenges what historically has been an increasing disciplinarily focus in infectious disease research. Greater investment in research has succeeded in revealing more detail about diseases within specific disciplines. Yet this arguably has been at the cost of greater disintegration rather than integration among disciplines. The division is widest between the genetic and biological aspects of disease and health on the one hand and the social, economic, political, and physical environmental factors on the other. The disciplinary distinctions within infectious disease research of course belie the true transdisciplinary nature of this and most problems in the global heath and environment arena (59). The above disciplinary divisions largely reflect the gap between disciplines focusing on systems at or below and above the level of the organism or species. The former involve disciplines addressing the genetics, pathogenesis, or immune response within particular organisms (humans, vectors of pathogens), for example, or disease as a statistical phenomena at the species population level (the mainstay of epidemiology). The latter deal with social or ecological phenomena, essentially representing higher levels of biological organization, within which the organism-level processes operate but that also involve many other variables or processes operating on a wide range of biological scales (from genes to the biosphere) characterized by cross-scale influences, and interactions at multiple societal and ecological levels.
It follows that for intervention to be globally effective, in addition to rebuilding public health infrastructure based on the comprehensive view of infectious disease ecology presented here, at least three elements are required:
1. a multilevel ecosystem approach, involving cross-scale integration. 2. incorporated ecological theory and data for the specific disease system, 3. local scale intervention using a participatory approach that matches pathogen management with sustainable development across ecosystem and institutional scales. Globalization and emerging governance modalities This paper explores the possibilities for global governance effectively dealing with the international transmission of disease. First, zoonotic regulation and control pose a special case for public health agencies, and this paper proposes a propositional model for an effective public health stance. Second, globalization dynamics are briefly reviewed in terms of an emerging consensus on the need for global governance in public health. Third, a brief examination of global governance modalities suggests that a strong global governance case has distinct limitations (despite its possible justification); an exploration of contemporary directions in global governance follows. Finally, the paper examines the phenomenon of contemporary zoonotic control within the conditions of an effective regulatory regime. The global governance of zoonoses presents a special case for global public health. The structural framework and dynamics of public health define the parameters of how emerging mechanisms of global governance are likely to influence the control of zoonoses. I begin this paper with a review of some of the endemic tensions that exist within efforts to extend public health into a global framework. I then discuss how the dynamics of contemporary globalization influence the transformation of both social and governmental behavior. I review the notion of "regimes" of governance that embrace trans-national and supra national entities. Finally, I inquire about the conditions for an effective regime for zoonoses control via global governance.
Efforts to control infectious disease lie within the writ of state authority and are bound by the framework of public health. Wherever society has legitimized private capital as to some degree separate from that of the state, a structural tension exists between the state's interest in defining health on behalf of its subject population, and the resulting regulation of private inter-ests. When the state does not permit the existence of private capital within a framework of civil society (and the structure of rights implied), national state structures pursue public health as part of their collective national responsibility.
This structural tension with private interests in the context of global governance informs zoonoses because (a) the processes of contemporary globalization are changing accepted notions of the role of the state (of which more later), and (b) scholars of public health have increasingly come to recognize that the definition of health itself (which the state must act to protect and promote) is flexible, constantly changing, and subject to the transformations occurring in modern, globalized consumer societies (1) . Indeed, a review of competing definitions of public health by the Institute of Medicine (IOM) in the United States in 1988 led the IOM to define public health as "fulfilling society's interest in assuring conditions in which people can be healthy" (2) . This broad definition, were it to be taken seriously and public policy aligned with it, would create a politics in which state power potentially could invade every private activity that challenges health. Pursued with vigor the political power of the state could develop into a national "health police".
The proposition that the state should regulate directly the "upstream" causes of health/disease is advocated by 'the social production of disease' school 1 and is opposed by those concerned that pursuing public health with such means will lead to excessive state regulation of private interests. Ticklish regulatory issues are also raised by the so-called new public health in which individuals are held to be primarily responsible for their own health. In the latter regulatory regime, responsibility shifts from the state (e.g. to regulate noxious industries) to the individual (e.g. to take responsibility in avoiding the products of noxious industry.) The regulation of smoking falls very much within this paradigm. Current controversy over eating behaviors, resulting obesity, and the impact on the "public's" health are of the same character, as are efforts to hold mothers criminally responsible for ingesting "dangerous" substances during pregnancy (ranging from methamphetamines to alcohol and tobacco). As political scientists look at these issues, health as a value always stands in dynamic tension to other rights and values (e.g. wealth, liberty, rectitude, etc.) What societies negotiate at any given moment is the relative authority to be given one value weighed against another. The ultimate policy questions become how much authority will health values and those who espouse and affect them have in society, and at the opportunity cost of what else (3)?
Two oft-conflicting notions of authority contend within the contemporary public health paradigm. Optimizing the public's health in a world of expanding threats requires increased amounts of state intervention. However, in part because of the prospect of this increasing state intervention, and to side-step the challenge to the power of private interests associated with the creation of dangers to the public's health, some would shift the ground for responsibility for health to individuals, who would be required to take on new responsibilities to calibrate their social behaviors (where to go, where not to go, what to eat, what not to eat) to promote their own health.
Public health always involves some construction of "the public" and its presumptive interests as defined by state authority. Public authority over the monitoring and control of historic zoonoses has involved a full range of extensions authority of state authority to those areas from which such disease arises. And, while some of these extensions are well established, others reappear in response to changing social demands and technologies. As new diseases emerge, each simultaneously is "fitted" into the prevailing model of regulation, even as they challenge that application. National and transnational regulation of beef herds under threat of a BSE infection is a case in point. Equally important, however, has been the level of state fiscal support for public health. Even as the dynamics of globalization increase health threats to the public, the prevailing ideology of neo-liberalism through which state policy is filtered leads to a diminution of state authority and diminished resources for public health disease control (5).
The goal of global governance of zoonoses lies in creating an effective regime of regulation. Below I will examine the notion of global regulatory regimes. Here, I seek to determine where in our understandings of public health as a regime of national regulation we find the necessary properties to be effective. The core argument is that a global governance regime of zoonoses regulation would need to be modeled on these characteristics.
This seemingly self-evident proposition conceals a greater subtlety, for the current regime of globalization has relied on the state and its capacity to effect purposeful public health policy. Indeed, political scientists will categorize states as weak or strong in part by their ability to create and maintain effective policy processes. Weak states can create, but not necessarily implement public policy (6) . Much of the world continues to live in "weak" states in which governments (central as well as regional and local) have difficulties ranging from grave to impossible to carry out policy intentions, particularly those regarding public health 2 . In these circumstances, maintaining good public health becomes impossible.
Proposition Two: Effective public health is dependent on sufficient social investment Overall, public health is losing comparative budget parity with the rising costs of curative medicine in the developed nations. In the United States, with overall medical costs once gain breaking out into double digit annual increases, the relative share of national budgets devoted to public health declines. Especially in times of economic downturn, and the absence of compelling crisis conditions, social investment in public health is uncertain in most national budgets.
Proposition Three: Sufficient social investment is dependent on prevailing political and economic ideology Neo-liberal economic and political ideology call for reduced taxes, a dismantling of welfare state structures, increased individual responsibility for social consumption, deregulation of the private sector, and reduced governmental spending. Those who see global public health structures as incapable of meeting the public health challenges spawned by globalization, find the major culprit in neo-liberal policies weakening the public sector (7) . Adoption of necessary regulatory regimes may be viewed as a kind of social investment. The unwillingness of the U.S. and China to participate in Kyoto for the reason that to do so would threaten continued levels of economic development is a testament to the power of neo-liberalism. The endorsement of the protocols by Russia this week (in addition to the relative advantage this gives Russia within the accord group) is a measure of its relative irrelevance to the current politics and economics of Russia.
Proposition Four: Inequality is detrimental to good public health A growing consensus holds that contemporary globalization is increasing inequality. A long-range macro analysis of inequality and health status holds that as inequality declines, overall levels of health improve, and the reverse (8).
Proposition Five: Regime corruption is detrimental to good public health
Regime corruption manifests itself when particularized interests subvert public policy for their own benefit. Persistent regime corruption diverts public policy from its intended purposes. It follows that public health's successes or levels of regime corruption significantly determine failures.
Proposition Six: Good public health is directly linked to positive social, economic, and political development Uneven or ineffective development results in poverty and weak regime states. The 'social determinants of health' school holds, importantly, that good developmental policies contribute more overall to the health of the public than medically oriented individual intervention, no matter how sophisticated and successful the latter. Good development policies lead to improved population health; uneven and unsuccessful development leads to inequality, poverty, and deficient provision of clean water, effective sanitation, adequate shelter and diet, as well as the political problems that follow from these conditions 3 .
Proposition Seven: To achieve policy success, public health needs to be able to value its own successes
The overall goal of public health is to reduce or eliminate the incidence of specific diseases. When public health practices result in lessened disease threats, the relative value of public health in the policy process wanes. In an odd way, public health is successful when things don't happen, when people do not become ill; it is about negative instances, which are notoriously difficult to "count". Tying this observation to proposition two above, public health's budgetary fates rise during times of crisis and suffer during times of normality (9) .
Proposition Eight: Public health suffers from the politics of focused expertise and technology
In a related manner, public health funding tends to lose out when public policy is oriented toward producing focused expertise and technology. Public sector investment in "health" has reached very high levels: the National Institutes of Health in the United States received $27,066,782,000 in FY2003. These massive levels of investment have produced spectacular successes in knowledge creation, the invention of non-invasive and minimally invasive surveillance of the body, and a vast array of medical interventions. At the system level, however, the multiplication and diffusion of highly technologized interventions results in rising expectations for medical care, and increased overall medical care costs, which crowd public health spending in national and sub-national budgets.
By contrast much important public health work is low tech. In a corollary to Gresham's law, high tech drives out low tech in budget contests (just as specialized medicine trumps primary care, and cutting edge proprietary pharmaceuticals trump generics). Some exceptions to this proposition may exist with emerging tools for micro surveillance devices.
Proposition Nine: Achieving public health is a moving target: notions of acceptable levels of health change over time; new diseases are constantly developing
Health is a relative value. Achieving it is an uncertain objective. Potentially the demand for health-especially as defined by medical interventions-may be infinite in a social climate in which individuals seek and receive new interventions to extend life or improve some aspect of bodily well being (10) . These observations clash with the languages and perspectives of the policy process in which notions of attacking problems, defeating social ills, or achieving victory in another war on something are commonplace. These rhetorical tropes serve well-recognized strategic and tactical means within the policy processes for mobilizing support, achieving agenda positions, and gaining budgetary allocations. When applied to public health, however, they create unrealistic notions of what can and cannot be accomplished within the frame of health by those practices we term public health. The result is that rhetorically, we are always in some ways losing the public health battle (11, 12) .
My hypothesis is that any regime of zoonotic control will be subject as well to all of these propositions. Whatever governance regime is developed for zoonotic control at whatever level (local, regional, national, global), the effectiveness of such controls will depend on the degree to which the values and institutional practices suggested by these propositions is found to apply.
The contemporary era of globalization dates from the mid-1960's. A set of similar global transformations took place during the period 1870-1914. In both cases heightened economic integration resulted from increases in international trade, finance, and investment. While this earlier globalization took place within the framework of the national state, contemporary globalization has expanded beyond the nation state through transnational economic actors, multinational corporations. The result has been to create a new political space within which the immense transactions of the global economy take place. While new economic institutions have developed in contemporary globalization, corresponding political governance has not: economic space has expanded beyond political space (13, 14) .
The dynamics of contemporary globalization compress time and space, creating a more immediately available world of goods, services, communications and interactions of all kinds (including military) (15) . Massive amounts of capital are aggregated at the global level, circulating in currency flows that dwarf anything previously known. These capital concentrations provide immense power to private capital to transform societies, often at the expense of a relative reduction in the power and authority of national governments, who in comparative terms lose the capacity to control their own policy agendas (14) . Economic interdependence brings unparalleled efficiencies in goods and information exchanges, but at the price of a hyper-sensitivity to negative economic effects within the system, as the 1997 Asian currency crisis revealed. Sudden disease outbreaks such as SARS can also have large and immediate negative economic effects that rapidly ripple throughout the system. The actual collapse of national economies from global financial instability such as that of Indonesia in 1997-98 can drastically reduce national income, with society-wide negative health consequences 4 .
Contemporary globalization, operating through its primary vehicle the multinational corporation, shifted the manufacturing centers of the world into the former developing world. Essential to the success of this relocation has been the creation of constantly innovating transportation and communication systems that move goods with steadily increasing rapidity and lowering costs, and a global information system based on constantly innovating communications technology and computer networks (16) . As is well known, these advances enhance the spread of disease throughout the world and vastly complicate efforts to establish effective controls.
We are just beginning to glimpse how changes in the ownership and extension of global media are coupling with other elements of our information societies and their networking capacities. Already it is obvious that basic consumption patterns related to health are changing, as societies become more consumer oriented. Changes in diet, work and living patterns are associated with goods through which we fashion our identities and make choices. Castells, like Harvey, sees the rate and nature of change the central element of how globalization impacts society, a process that he calls the creation of network societies. This contemporary process of change has the apparent property of being high recursive; elements of change in one dimension affect another through chains of reciprocal causation, working themselves back to the initial causal elements and producing new, and often unexpected, effects in the process. Social theorists make strong analogies to how ecological systems function recursively in making these operations. When these new and complex recursive processes occur within the social stew of hyper-urbanization, the outcomes are highly unpredictable (17) .
Globalization is also marked by new means of wealth creation and distribution, inducing labor forces to move toward aggregations of capital most of which are urban. This process has touched off the largest migration in human history (18) . The 21st century has become the urban century as for the first time in human history more people live in cities than in rural areas. One component of this migration is cross-border, legal and illegal migrants seeking work. This pattern, however, is dwarfed by within-country migrations which have brought hundreds of millions of people into cities, now perceived as the critical nodes of global production, and therefore the locus of jobs. In many of these cities, especially the "newly large" cities of the developing world, or the mega-cities of Asia, living and working conditions resemble those of Dickensian England. At the current edge of the 21st century's world, industrialism coupled with urbanization has reproduced the social conditions of the 19th century. The unchecked growth of mega cities as survival harbors make them the new reservoirs for the lethal combination of poverty, crowding, insufficient sanitation, impure water and disease.
The non-urban world has also been fundamentally transformed, as logging, mineral extraction, and oil exploration and production have brought virtually every hectare of global space under the economic gaze of capital exploitation. In search of ever greater food supplies, fisheries throughout the world have been driven to collapse and forests pushed back for agricultural development, especially when monoculture drives out smaller scale diversified agriculture. The agricultural equivalent to global financial interdependence lies in the possibility of irreversible environmental shifts such as drought in China, which under current financial arrangements could cause market forces to eliminate from global demand those who cannot afford grain. Structural inequalities coupled with civil unrest (another kind of inequality) already produce widespread hunger, malnourishment and famine. An environmental disaster in a large population, most obviously China or India, could have devastating effects within the world system. This, of course, is the great fear environmentalists have for dependence on mono-cropping, species loss, and water management 5 .
In sum: contemporary globalization has dramatically increased global wealth, through innovation and joining new capital to massive labor forces; it has also produced a distribution system that promotes inequality on a scale previously unseen. Stunning innovations in productive capacity, communication, and transportation have imposed new technologies throughout the world. World populations have increased in number and concentration as the world has rapidly urbanized; ecological imbalances have been intensified and with them the conditions for the spawning and transmission of new diseases increased. Contemporary patterns of global industrialization create profound ecological challenges from resource depletion.
One further note on globalization as a market phenomenon. Contrary to traditional theories of economics, the market is not self-governing. Classical and neo-classical economics can assure us that markets will create efficient price levels and send appropriate signals to organize supply and demand, but markets also routinely produce externalities, sometimes of massive, negative proportions, and are inseparable from the business cycle. As global wealth increases, the business cycle produces spectacular wealth during periods of boom and spectacular poverty and despair during periods of bust: the greater the extent and interdependence of the world system, the greater the attenuation of these extremes.
In the industrial progression of national economies, the early periods of raw industrial growth were followed by the imposition of regulatory regimes designed to mitigate the human costs of industrial development. Markets may be efficient for exchanging goods, but they are not effective in representing the controls on excess that modern industrial human populations historically have come to demand from the economic schemes within their societies. The replication of these economic excesses at the global level is producing similar movements toward global governance (regulation). In a world still governed by the sovereignty of nation states, however, achieving the required and effective regulation is a daunting challenge.
Part Four: Governance regimes: Strong and weak programs of global governance
The compelling questions for global governance are what should be governed and how? The fundamental environmental/ bio-health questions for global governance include: whether to seek remedies eliminating the causes of negative health and environmental outcomes; or to pursue limited programs that seek to mitigate effects at the margin.
A similar tension exists within the discourse of global governance: should global governance seek to regulate the processes of globalization themselves, or should efforts concentrate on regulating effects? While the two can be seen as opposite sides of the same coin, these can fairly be termed the strong and weak programs of global governance.
The strong program envisions institutions that have a direct capacity for regulating (with sanctions) individual actors, whether they are nations acting on behalf of private interests (or their own state corporations), or private interests, and individual actors themselves. The weak case envisions governance mechanisms that operate largely through existing institutions (including states) and require their compliance to effect action. These positions define the antipodes of a governance continuum. The current politics of global governance distributes advocacy for various structures or mechanisms along this continuum.
Obviously, the strong governance program most directly addresses the operation of globalization actors at the transnational level; conversely, this position acknowledges the degree to which state sovereignty has already been compromised by cross border globalization (14) . A propensity for the strong program in large part depends on one's view of the global condition. If it is seen as a "ticking time bomb" e.g. unrelenting global warming with catastrophic consequences such as the possible melting of the Ross ice shelf and rising sea levels, one views the world in crisis and moves toward a discourse that promotes more radical changes necessary to sustain society under threat. The scale of the threat, it would be argued, justifies actions that impinge drastically on the traditional institutions of the state and its sovereignty. A paradigmatic example may be the Montreal treaty on CFC regulation.
The biological version of this scenario might be a conviction that current global practices previous mirror conditions in human history that led to the emergence of new diseases or their epidemic spread: a warming climate, the rapid and large scale movement of populations, novel mechanisms of transportation that permit rapid communication of peoples, and destruction of existing barriers between native forests, agricultural land, urban concentrations, and compromised water and sanitation systems (19) . Convinced of this scenario, one might seek to impel regulatory settlements intended to reduce population flows into cities, reduce the destruction of forests by agricultural incur-sion, curtail global warming, etc., actions that could only take place by directly interdicting the economic forces creating them.
But such measures are unlikely. Policy processes do not work that way. Crises discourse may serve to modify the content of policy talk, but for large scale regulatory efforts to take place, the catastrophe in whose name such actions are taken must already have occurred. People are only convinced of the severity of truly profound crises until their very occurrence has validated that severity. The purpose of crisis dynamics and its role in the strong governance program is to legitimize lesser efforts. It is in this terrain that global governance is emerging.
Within weak programs for global governance two basic types prevail; one seeks to reform existing institutions of global governance, the other to fashion novel regulatory regimes.
Nayyar and Court indicate the assumptional base of the reform model:
The endeavor should be to make the market-driven process of globalization conducive to a more egalitarian and broadbased development pattern. The object of such a design should be to provide more countries with opportunities to improve their development prospects and more people within these countries to improve their living conditions. It would have to be supported by a new institutional set-up. This would mean providing global public goods, such as world peace and a sustainable environment, as well as regulating global public bads, such as international crime whether trade in drugs, arms, people, or [human] organs. It will be necessary to reform existing institutions and to introduce new rules or create new institutions. Some of these would require a system to correct for the failures of unregulated or liberalized international markets, while other initiatives will be needed to build up missing markets (14) .
From this proposal spring reforms of existing governance institutions including the United Nations 6 , the International Monetary Fund, the World Bank, and the World Trade Organization. Nayyar and Court are sensitive to the needs for differential application of regulatory rules for countries at different levels of development, for the need to make global governance more inclusive (and not just a rich nation's club), to provide a voice for those currently excluded, and to specify the conditions under which countries can opt out or exit from multi-lateral rules. Their reforms include the desirability for articulating the obligations of transnational corporations as well as their rights, including some version of an international regime of antitrust 7 . Like similar global governance proposals, acknowledgement is made of existing institutions that already constitute core arrangements of governance on which this larger program could be built, e.g. UNCTC, UNCTAD, the Organization for Economic Cooperation and Development (OECD), the International Finance Corporation (IFC), and the International Centre for Settlement of Investment Disputes (ICSID).
The regime approach to global governance seeks to identify the existence of these institutions and practices in relation to specific problems they would regulate. Oran Young defines regimes as "sets of rules, decision-making procedures, and programs that define social practices, assign roles to the participants in these practices, and govern their interactions" (21) .
Regimes differ in terms of their functional scope, geographical domain, and membership. Regimes are empirical, and may be unstable; they form in response to some situations and not others. Regimes have proliferated within environmental governance largely in response to discrete issues. Examples would include the Great Lakes water quality regime, the Antarctic Treaty System, and the European transboundary air pollution regime.
Regimes may be nested in larger institutional structures, e.g. that for high seas fishing, which is subject to the more encompassing law of the sea. Often regimes are "lightly administered", generating compliance with minimal organizational resources ("governance without government"). While regimes often do not seek to provide comprehensive systems of public order for large geographic regions, they may occasion participation by states as well as inter-governmental actors. The result, Young suggests, is that regimes tend to form horizontally rather than vertically; they represent a "complex pattern of decentralized order." (21) Since its inception in 1946 and consistent with its predecessor organizational versions, the WHO has ascribed to the weak regulatory program, utilizing the terms employed in this section. Its constitutional function is the direction and coordination of international health work, including setting international norms and standards for health, and technical cooperation among members. Evidence of its weak program nature can be seen in those instances in which it has abjured political involvement (especially concerning Eastern Bloc membership issues in the 1960's) (22) . An issue beyond the immediate scope of this paper is the examination of current efforts within WHO to ascertain those that are part of regime formation and implementation, a la Young.
In an effort to apply these diverse observations about public health, global governance and globalization, I turn to Frederick Murphy's 1998 presentation of emerging zoonoses in the special issue of the journal by the same name. I choose Murphy as a text because of his effort to capture in this brief review not only the range of factors implicated in emerging zoonoses, and the specifics of the most prevalent instances of that date, but also because he focuses on specific proposals to address what he sees as the need for an effective response. I seek to put this conclusion in the context developed in this essay.
"…an emerging zoonosis is 'a zoonosis that is newly recognized or newly evolved, or that has occurred previously but shows an increase in incidence or expansion in geographical, host or vector range'. Emerging zoonotic diseases have potentially serious human health and economic impacts and their current upward trends are likely to continue." Examples include: avian influenza, Bovine Spongiform Encephalitis (BSE) and the Nipah virus (23) .
Writing before the emergence of SARS, but in the context of BSE in the UK, HIV/AIDS, Sin Nombre and West Nile Virus, Murphy's specific concern is the adoption of "unique strategies" that will build more on fundamental research than "traditional" approaches. Included will be the rebuilding of "a cadre of career-committed professionals with a holistic appreciation of several medical and biologic sciences" (5) .
He sees a persistent and to some extent alarming increase in emerging disease episodes (nearly all of which involve zoonotic or species-jumping infectious agents) including at the microbial/virologic determinant level mutation, natural selection, and evolutionary progression.
Among individual host determinants he identifies acquired immunity and physiologic factors. Host population determinants include host behavioral characteristics and numbers as well as societal, transport, commercial, and iatrogenic factors. Environmental determinants include ecological and climatologic influences.
The remainder of his review focuses on ecologic factors, especially those exemplified by arbovirus diseases. He seeks to identify lessons learned from Venezuelan equine encephalitis epidemics, the equine morbillivirus outbreak in Australia, from Ebola hemorrhagic fever, rabies epidemics, from the hantavirus pulmonary syndrome epidemic and from bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans.
Murphy's policy argument is to extend the discovery-tocontrol continuum to the full range of zoonotic diseases. The discovery-to-control structure draws on elements from fundamental scientific research, to the creation of an effective practitioner community, to creating and sharing data bases drawn from increased national and sub-national surveillance, to the full range of actions required in the final control stages of zoonotic diseases. And at each step, he is sensitive to their costs (without necessarily employing the various languages of public policy that apply). He recognizes the extensive nature of the final phases of the project.
More expensive and specialized expertise and resources come into play in the final phases of the discovery-to-control continuum: public health systems, including rapid casereporting systems, surveillance systems, vital records and disease registers, staffing and staff support, logistic support, legislation and regulation, and expanded administration; special clinical systems, including isolation of cases, quarantine, and patient care; and public infrastructure systems, including sanitation and sewerage, safe food and water supplies, and reservoir host and vector control.
My interest in Murphy's review and analysis stems precisely from its initial assumption that the beginning point for eventual control begins with discovery. It certainly seems a reasonable place to begin with. However, most of the above argument about globalization suggests, paradoxically, otherwise. That argument says, in effect, that if we continue along the road that globalization is taking us, we may be "producing" diseases in ways that overwhelm the capacity of systems to deal with them if they are actuated at the point of discovery.
Is this a silly way to reason? I don't think so. Defining zoonoses in terms of a set of problems within the discovery-tocontrol continuum is analogous to the weak program for global governance. It accepts the premise that important public health situations constitute problems to be solved by intense, expert, scientific driven problem solving techniques.
But these issues may more rightly be dilemmas than problems. A dilemma differs from a problem in that the situation it "contains" will not produce a "solution" at the level of analysis at which it stated. Simply: within a dilemma one cannot continue to do what one is doing-the thing that produces the "problem"-and gain an effective solution. In the above analysis, the world cannot continue to embrace and support the activities of contemporary globalization and meet the public health problems it will create. To resolve the dilemmas that constitute global public health requires changing (perhaps in radical ways) the ways that globalization works, so that it will produce different results. Problems say: do more; dilemmas say: do something different. This is what proposals for global governance purport to do. The all-important question is, even were they to succeed, through the reforms proposed, would these be sufficient to create transformations in globalization behavior yielding different outcomes? One should never say never. I am tempted to argue, nevertheless, that we cannot solve the problem of creating an effective regime of zoonotic control because such a regime, as posited, would not change the conditions of the global economy that are producing the very diseases one wants to control.
This does not mean however, that one might not embrace projects or obtain results that are important steps in this direction. For example, I have made much of the tension between the current world of infectious disease in which there is a large, and perhaps ever increasing, need for more research, more science, more data bases, more trained personnel and international cooperation on policy implementation, all of which cost money (and involve additional contribution of resources on the part of the state), and the trend under current globalized regimes of neo-liberalism to actually weaken the state, to shrink public spending and to weaken its regulatory hold. Following this logic suggests to me two conclusions.
One, we will fall behind in the kind of program Murphy advocates because the political and economic forces that promote global neo-liberalism are stronger than those that promote public health (including zoonoses control). In the contest of values and public policy discussed in section one of this paper, particularized interest will triumph over more generalized interests. Two, some disease will present itself with such threat and virulence that its consequences to existing society cannot be ignored. In the face of this manifest crisis (perhaps a breakout of the current Thai-based avian flu virus), public health intervention will go to the top of the policy list. Not only public monies will be made available, but also private sector funds. William Gates or Warren Buffet, or George Soros or some other combination of the world's largest holders of private wealth may seek to intervene to transform the direction of current public policy. And that might turn the tide on the existing threat.
Or not. Within the last century only the flu pandemic of 1918-19 has constituted as great and widespread a threat to human population as the HIV/AIDS pandemic. Yet threats of this magnitude have failed to mobilize an effective interventionist regime. While it is useful to speculate on some of the reasons that such a regime has not emerged (e.g. the disease emerged outside the developed world (non "us"), and then proliferated in stigmatized populations, wide spread denial of the size and extent of the disease by many governments, etc.) the combination of urgency of impact (disease incidence continues to rise) and failures in the establishment of an effective governance regime (typified by the failure of the U.S. government to contribute its full share to the global fund) shows that the HIV/AIDS pandemic has new lessons for us about how crisis dynamics do and do not work to effect appropriate responses.
Before proceeding to a conclusion, two final words. On risk. Like health, risk is a socially constructed category that changes within the complex norms and values of a given society or culture. What is an acceptable level of risk at one period of time, may not be in another; what is an acceptable level of risk for one group (e.g. others), may not be for another (e.g. ourselves) 8 . Notions of risk are negotiated within the political process; notions of crises, their relative severity and the amounts of resources to be devoted to them emerge from the complex processes of bargaining within the political process. When the site of the political process is global, rather than national, and when the mechanisms for decision making lack the force of national sovereignty, the complex calibrations of acceptable risk compound exponentially.
On technology. We are wise enough to realize that technology can bring astonishing immediate benefits, but also through unexpected recursive feedback loops, create yet new and unanticipated problems, perhaps as severe as those they were designed to fix. Nevertheless, at the very least microtechnology and nano-technology hold enormous promise for assisting with surveillance tasks to control disease spread. Biobased sensors that can be made cheaply and sensitive down to the individual molecule level promise new ways of scanning large numbers of people (e.g. airport arrivals) cheaply and with minimal intrusiveness. Nano-tech sensors operating within the bodies of suspected infectious persons are also being studied. To see the individual as the broadcasting site for disease surveillance strikes many as a further draconian incursion into the oppression of individual liberty…but to return to our opening theme, this tension between private interest (including the self) and the public's health is endemic.
This is not a pessimistic paper, although it may read like one. It makes a simple argument. Globalization is about the deployment of capital throughout the world through marketprivileging mechanisms. Globalization has a powerful up side in the vast wealth that has been created through its mechanisms, and the benefits that it brings to millions of people every day. (The World Bank for example estimates that 400 million people in China have been lifted from poverty by the mechanisms of the current capitalist based society.) But globalization also has its dark side, some of which is eluded to above, and which can be easily amplified in reference to the millions throughout the world who work in conditions that are unregulated, dangerous and underpaid 9 ; to the emergence of a world-wide sex industry tied to human trafficking that acts as a particularized HIV/AIDS reservoir; to the wanton disregard of private enterprises in many parts of the world to environmental destruction; to corruption and theft in the higher levels of some of the largest corporations in the world, global trafficking in arms and drugs, etc. 10 My argument is in the end simple: the dynamics of globalization have become the major "factor" in the social production of disease, including those elements that are intimately associated with the emergence of zoonoses. Directly addressing how these globalization dynamics impact human populations to produce disease must be a program for global governance 11 . It must look beyond a discovery-to-control paradigm for emergent diseases, to a research-to-prevention-to-discovery-to-control paradigm. Within these mechanisms of governance lay the difficult tasks of regulating capital in its excesses. This-to repeat my earlier point-was the basic issue in the development of national economies, and it is of necessity the basic issue in the creation of global governance. To succeed, these issues must enter the discourses of global governance in a direct way (and not be viewed as some exotic side issue). I conclude by repeating a portion of the previous quote from Nayyar and Court. Global governance means: "This would mean providing global public goods, such as world peace and a sustainable environment, as well as regulating global public bads, such as international crime whether trade in drugs, arms, people, or [human] organs. It will be necessary to reform existing institutions and to introduce new rules or create new institutions. Some of these would require a system to correct for the failures of unregulated or liberalized international markets…" 9 An especially egregious instance exists in China, in which hundreds of thousands, perhaps millions of peasants moving into cities have over the past decade been employed in myriad construction projects, including many proceeding under the approval of government authority. A current suit is being conducted on behalf of these workers who are owed $42 BILLION in unpaid wages. This is a country in which annual individual income is still a pittance. It is no secret who benefits and who loses from such a situation (18) . 10 The next great crisis in globalization has already begun to emerge. As capacity spread through out the developing world for labor-cheap manufacturing, countries such as Bangladesh became early players in the inexpensive manufacture of world goods. The rapid integration of China and India with their enormous labor forces into world markets is threatening to overwhelm other countries in this role, creating capital movement and resulting unemployment and deepening poverty. One adds to this the opening wedge of service industry outsourcing from the developed word, from low level work, e.g. call centers, to the highest levels of professional training. Radiologists in the United States (average income north of $300,000) may be replaced by those in India (average income of around $25,000); similar issues exist in engineering, computer programming and medical technology. Singapore, acutely aware of the implications for this threat is investing heavily to upgrade the country's professional labor force to better gain niches in scientific and technological research. As the deputy minister for development told me some months ago, "we can make all the capacity we need to stay competitive; we rent brains. It is cheaper and more efficient." (The opportunity in question arose from the U.S. government's decision in 2002 not to pursue stem cell research.) In this view of research, only a portion of it needs to be site specific. 11 This argument accords with the general thrust of Burci and Vignes in their recent review of WHO, in which they argue for a strong step beyond what they see as the legal timidity of the organization in the direction of a stronger "normative stance" (22) . Generation and Characterization of Novel Human IRAS Monoclonal Antibodies Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions. Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine [1] . Based on their physiologic and pharmacological properties, imidazoline receptors are classified into three main types: I1R, I2R, and I3R [2] [3] [4] . I1R and I2R have been implicated in hypertension and psychiatric disorder regulation, respectively, while I3R may be involved in insulin secretion [5] [6] [7] [8] [9] . Compared with mitochondrial I2R, which resides within the monoamine oxidase protein [10] , the clonidine-preferring imidazoline binding sites (known as I1R) are localized to plasma membrane fractions [11, 12] and specifically to synaptic plasma membranes [13] .
A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus [14] . hIRAS is a larger protein of 1504 amino acids consisting of an NH 2 -terminal phox (PX) domain, 5 putative leucine-rich repeats, a predicted coiledcoil domain, and a long COOH-terminal region. Several evidence supported the identity of native I1R and IRAS protein in tissue distributions, ligand binding properties, some cellular functions and downstream signal pathways [14] [15] [16] [17] [18] . The murine form of IRAS, Nischarin, truncated at the N-terminal 244 amino acids including the PX domain compared with the hIRAS, was a soluble cytosolic protein involved in cytoskeletal organization [19] . It has been shown that decreasing the expression of rat IRAS or Nischarin in PC12 rat pheochromocytoma cells could attenuate the activation of extracellular signal-regulated kinase (ERK) or reduce the radioligand binding to I1R, which further supported that hIRAS or Nischarin might serve as I1R itself, or at least a functional subunit of I1R [20] . Recently, Molderings et al. have reported that the "I1-imidazoline receptors" mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belonged to the S1P-receptor family, in particular, representing a mixture of sphingosine-1-phosphate (S1P)1-and S1P3-receptors and/or heterodimers of both. It was then proposed that an increased expression of IRAS or Nischarin may improve the receptor-trafficking from cytosolic S1P-receptors to the cell membrane and thereby increase the number of binding sites in the plasma membrane for radioligand binding [21] . In our previous study, we also reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells [22] . Despite intensive efforts, the molecular base of the I1R had not yet been elucidated.
To elucidate the functional and structure properties of I1R, several different epitope-specific antisera against IRAS have been raised in rabbits [23] . Because of IRAS splice variants or nonspecificity of these antisera, more sizes of IRAS (≈33, ≈85, ≈170 KDa) have been visualized in various tissue and cells, which limited their uses on western blot. IRAS was reported to target to the endosomes by a combined action of a PX domain and a coiled-coil region. The PX domain, consisting of 10-130 amino acids, was first identified from the sequence analysis of two SH3 domain-containing cytosolic components of NADPH oxidase, p47phox and p40phox [24] . Therefore, in the present study, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) . This development has great utility for immunoblotting, indirect immunofluorescent staining, immunoprecipitation, and flow cytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.
Protein. E. coli BL21(DE3) (F-ompT gal dcm hsdSB (rB-mB-) λ (DE3) (Novagen) cells were transformed with recombinant plasmid, pET43.1-IRAS(10-120aa). Transformants were selected from ampicillin-containing Luria Bertani (LB) lates. Selected colonies were cultured in ampicillincontaining LB media. Isopropyl-β-D-thio galactopyranoside (IPTG) was added to a final concentration of 1 mM. The incubation was continued for 3 hours at 30 • C. Cells were harvested, mixed with lysis buffer (phosphate buffered saline [pH 7.3], 1.0 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, 0.5 mg/mL lysozyme (Roche)), and sonicated. The high-speed supernatant which contained the pET43.1-IRAS(10-120aa) soluble protein fraction was loaded onto 10 mL Ni 2+ -NTA agarose columns. The fusion protein was eluted with lysis buffer with 400 mM imidazole. Eluted proteins were electrophoresed on SDS-PAGE gels. Expression yields were analyzed with the Quantity One quantitative software (Bio-Rad) based on relative band intensities of comassie blue stains. Purified proteins were detected with western blot using anti-His mAbs (Santa Cruz).
Immunosorbent Analysis (ELISA). The purified NusA-IRAS (10-120aa) , diluted to 10 μg/mL in 50 mM carbonate salt buffer (pH 9.6), was coated on plates at 100 μL aliquot per well, 4 • C overnight. The wells were washed three times with PBS-Tween buffer (0.05% Tween 20 in PBS). The coated wells were blocked with 200 μL of 3% BSA for 1 hour at 37 • C and then incubated with 150 μL monoclonal antibodies against NusA-IRAS (10-120aa) fusion protein with different dilutions (from 1 : 1000 to 1 : 25600). After incubation for 1 hour at 37 • C, the wells were incubated with 150 μL horseradish peroxidase-conjugated goat antimouse IgG (dilution 1 : 5000, Santa Cruz) for 1 hour at 37 • C after thorough washing. Peroxidase activity was detected using o-phenylenediamine and H 2 O 2 as enzyme substrates. Color development was stopped with 2 M of H 2 SO 4 and the absorbance was measured at 490 nm using Microplate Reader (Bio-Rad).
The purified NusA-IRAS (10-120aa) protein (50 μg in a volume of 80 μL) was mixed with an equal volume of Freund's complete adjuvant. The antigen-adjuvant mixture was injected into 6 female BALB/c mice and was followed by three booster injections at 2-week interval in incomplete Freund's adjuvant. The mouse with the highest antibody titer tested by ELISA was boosted intraperitoneally with 100 μg NusA-IRAS (10-120aa) protein without adjuvant 3 days before the cell fusion. Feeder layer cells were prepared 1 day prior to fusion. Splenocytes from mice with the highest ELISA antibody titers were fused with murine myeloma cells SP2/0 following standard procedures [25] . Culture supernatants were collected after fusion and initially screened by ELISA with purified NusA-IRAS (10-120aa) fusion proteins as antigens. Positive hybridoma clones were selected with the limiting dilution method, and stable hybridoma clones were obtained after 3 cloning cycles. Isotypes of antibodies were identified with a mouse subisotype panel (Bio-Rad). The pristine-primed BALB/c mice were injected intraperitoneally with 1 × 10 6 hybridoma cells per mouse in order to acquire abundant mAbs. The ascitic fluid was collected and mAbs were purified with a protein A/G affinity column (Amersham Pharmacia Biotech).
Transfected cells were harvested 48 hours after transfection. Total cell lysate preparation and western blot analysis were performed according to previously described procedures [26] . In brief, cell lysates were prepared, electrophoresed on SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% [v/v] Tween 20) and incubated with IRAS or GFP mAbs (Cell Signaling Technology Inc). Blots were incubated with horseradish peroxidase (HRP) conjugated goat antimouse antibodies (Santa Cruz) after primary antibody incubation, and blots were developed with enhanced electrochemiluminescence (ECL, Cell Signaling Technology Inc).
The cellular localization of the IRAS protein was identified according to previously described procedures [27] . Cells on glass cover slips were rinsed with PBS, fixed with 3% paraformaldehyde for 30 minutes, and permeabilized with 0.2% [v/v] Triton X-100/PBS. Permeabilized cells were incubated with IRAS mAbs and TRITC-conjugated affinipure goat antimouse by PCR amplification of the human IRAS cDNA (AF082516) using the following oligomers: sense, 5 -CGGGATCCA-TGGAGCGGGAAGCCGAGCCG-3 , and antisense, 5 -CGGAATTCATAGAAGTGAAAATGCAAGAAGTG-3 . The full-length human IRAS was obtained by PCR amplification of the entire coding region, and the resulting 4512-bp PCR product was ligated into the pEGPC1 and PCMV-myc vectors in a similar fashion using the following oligomers, respectively: sense, 5 -CGCGAATTCTATGGCGACCGC-GCGCACCTTCG-3 , and antisense, 5 -CGGGATCCTAGC-CGGTGAGCTCGACAGGC-3 , sense, 5 -CGCGAATTC-GGATGGCGACCGCGCGCACCTTC-3 , and antisense, 5 -CCGCTCGAGCTAGCCGGTGAGCTCGACAGGC-3 . All plasmid sequences were confirmed by sequencing analysis.
IRAS is a large protein comprising of 1504 amino acids. Its NH 2 -terminal phox domain is important for membrane association and cellular localization. The N-terminal of IRAS (10-120aa) covering the PX domain was cloned into the pET-43.1a(+) (Figure 1(a) ) [29] . The supernatant fusion protein was purified by His-tag affinity purification and was subsequently used to generate the monoclonal antibody. The dissolved protein yielded one major band of 78 kDa expected molecular weight (Figure 1(b) ) with high purity and integrity. The NusA protein used as a control generated the 66 kDa expected molecular weight. The recombinant protein was also confirmed with western blot using anti-His mAbs (right column, Figure 1(b) ). BALB/c mice (n = 3) were immunized with the NusA-IRAS (10-120aa) fusion protein, and blood was collected from the mice after multiple injections. Antibody titers were tested by ELISA on plates coated with the NusA-IRAS (10-120aa) protein (data not shown). The 2 mice with the highest titers were sacrificed and spleens from both mice were fused to myeloma cells following standard procedures. Individual hybridomas was grown and 125 hybridomas were further characterized. Supernatant from the growing hybridoma clones was screened with ELISA. Screening was performed on plates coated with NusA-IRAS (10-120aa) , NusA protein, and GST-IRAS (10-120aa) fusion protein to determine antibody specificity. A total of 5 hybridomas (DA041, DD015, BE073, BA022, and AH021) reacted selectively with the NusA-IRAS (10-120aa) protein in all 3 assays and were further evaluated. Isotype analysis revealed that all mAbs were of the IgG1 subtype.
The immunoreactivities of the 5 representative mAbs with NusA-IRAS (10-120aa) were shown in Figure 2 , all of which specifically recognized a 78 kDa protein band which corresponded to the purified NusA-IRAS (10-120aa) protein, but not to the 66 kDa NusA protein. Anti-His mAbs reacted with both proteins in the same experiment (Figure 2(a) ) as controls. We evaluated the specificity of mAbs in mammalian cells by inserting IRAS cDNA into expression vectors to allow the production of GFP fusion proteins under the control of a CMV promoter. The pEGFPC1 and pEGFPC1-IRAS plasmids were separately transfected into HEK293 cells. Western blot analysis with the anti-GFP antibody demonstrated that chimeric proteins were expressed and migrated separately at expected molecular masses of approximately 19 or 27 KDa (Figure 2(b) ). However, the expected 190 kDa band whose molecular weight corresponded to the full-length IRAS protein was only detected with the mAbs DA041, DD015, and BE073 in GFP-IRAS expressed cells. The remaining BA022 and AH021 mAbs were negative (Figure 2(b) ). The same samples were also incubated with preimmune serum with no reactivity (data not shown). Results revealed that all 5 selected mAbs specifically recognized bacterially expressed NusA-IRAS (10-120aa) proteins, but only 3 mAbs recognized recombinant IRAS in mammalian cells.
IRAS mAbs were used to detect the cellular localization of IRAS protein based on specificity characterized by western blot analysis. The pEGFPC1-IRAS plasmid was transfected into HEK293 cells. IRAS protein localization was tested by fluorescence confocal microscopy after 48 hours transfection. GFP-IRAS was primarily located in the cytoplasm in a punctate pattern (Figure 3(a) ) with concentration onto discrete loci and spot fluorescence (panel A, E, I, Journal of Biomedicine and Biotechnology M, and Q), as confirmed in previous studies [30] . The mAbs DA041, DD015, and BE073 were used as probes to detect a predominantly punctate cytosolic distribution as expected (panel B, F, G). However, the mAb BA022 homogenously detected the fluorescence distribution over the cytoplasm (panel N) and the mAb AH021 scored negatively (panel R). Merged images revealed that the GFP-IRAS fluorescence significantly coincided with the immunostaining with mAbs DA041, DD015, and BE073 (panel D, H, and L), but was not reflected by the mAbs BA022 and AH021 (panel P and T). Further, the punctate fluorescence patterns appeared to be unique to the mAbs DA041, DD015, and BE073. In addition, we also detected the endogenous IRAS protein with monoclonal antibody DA041 in PC12 cells, showing a strong signal associated with the membrane and faint signal in the cytoplasm (data not shown). These results suggest that the 3 mAbs specifically detect IRAS proteins by immunofluorescence, similar to immunoblotting results. Flow cytometry is a rapid, quantitative, multiparameter cellular analysis based on the measurement of visible and fluorescent light emission. We chose the representative mAb DA041 for further characterization based on the strong signals detected by immunoblotting and immunofluorescence. The IRAS cDNA was cloned into the mammalian expression vector PCMV-myc and was expressed in HEK293 cells. The mAb DA041 and the c-myc mAb (used as a positive control) resulted in significant increases in fluorescence intensity when compared to cells incubated with preimmune serum (Figure 3(b) ). The binding efficiencies were 30.77% by the mAb DA041 and 48.26% by the c-myc mAb. No reactivities were detectable in preimmune sera (0.01%). These results indicate mAb DA041 is effective for detecting IRAS protein by flow cytometry.
We next characterized IRAS mAb ability to recognize IRAS proteins in their native states by immunoprecipitation. Cell extracts were immunoprecipitated with the mAb DA041 and analyzed with mAb DA041 immunoblotting after 48-hour transfection with PCMV-myc-IRAS. IRAS was selectively immunoprecipitated from cell lysates that expressed the myc-IRAS protein (approximately 175 kDa), and those which corresponded to the predicted size of the full-length IRAS ( Figure 4) . Immunoprecipitation of lysates with mouse normal IgG did not result in detection of protein species. Lysates were immunoprecipitated with the c-myc mAb to confirm mAb specificity, and similarly sized bands were detected. No corresponding band was visualized when the negative control c-myc mAb was used to immunoprecipitate cell lysate of the transfectant expressing the empty PCMV-myc vector. Similar results were obtained with the mAbs DD015 and BE073 under the same conditions, and the remaining mAbs BA022 and AH021 scored negatively. Therefore, the 3 isolated antibodies specifically recognized native full-length IRAS protein products.
Our results suggested that the mAbs DA041, DD015, and BE073 were reactive for both immunofluorescence and immunoblotting. Therefore, these mAbs likely recognized linear epitopes in the IRAS protein. In addition, the 3 mAbs were capable of recognition of native full-length IRAS proteins by immunoprecipitation. However, the mAbs BA022 and AH021 specifically recognized bacterially expressed IRAS immunogens and did not detect recombinant IRAS in mammalian cells. This could be related to the different IRAS folding patterns, since misfolded IRAS proteins could result in the exposure of unique immunogenic epitopes different from native proteins. The mAbs BA022 and AH021 could be used as backup reagents to safeguard against antibodyspecific artifacts. In addition, analyzing the overall homology of the amino acid sequence of the PX domain, Sorting Nexins 13 (SNX13) is the most relative protein to IRAS [31] . We found monoclonal antibody DA041 against hIRAS could not react with SNX13 by immunofluorescence assay (data not shown). It was proposed that these antibodies developed here were specific to the PX domain of IRAS.
In summary, we generated specific mAbs directed against the human IRAS N-terminal. The mAb DA041 exhibited the best performance in a variety of assays including immunoblotting, immunoprecipitation, indirect immunofluorescence stainning, and flow cytometry. Specific mAbs may provide an ideal reagent for further investigation of the function of IRAS proteins. Molecular and physiologic basis of quinoline drug resistance in Plasmodium falciparum malaria 30 years before the discovery of the pfcrt gene, altered cellular drug accumulation in drug-resistant malarial parasites had been well documented. Heme released from catabolized hemoglobin was thought to be a key target for quinoline drugs, and additional modifications to quinoline drug structure in order to improve activity against chloroquine-resistant malaria were performed in a few laboratories. However, parasite cell culture methods were still in their infancy, assays for drug susceptibility were not well standardized, and the power of malarial genetics was decades away. The last 10 years have witnessed explosive progress in elucidation of the biochemistry of chloroquine resistance. This review briefly summarizes that progress, and discusses where additional work is needed. Human malarias are caused by infection with one of five malarial (Plasmodium) parasites (Plasmodium ovale, P. malariae, P. knowlesi, P. vivax and P. falciparum), which occurs during a female Anopheles mosquito blood meal (P. knowlesi is zoonotic, the others pass human to human). P. falciparum infections account for 95% of malaria mortality worldwide, so historically most malaria research has focused on this species. However, there is also a critical need for basic research on the others, particularly P. vivax. In P. falciparum malaria, sporozoites injected during the blood meal migrate through the skin via multiple routes, localize to the liver within hours, invade hepatocytes and are then released into the blood after approximately 2 weeks as large merosomes containing 5-10,000 new haploid merozoites. These then rapidly invade red blood cells (RBCs) [1] and proceed through an amazing differentiation and feeding cycle that is 45-50 h long. An initial 'ring' differentiates into a single trophozoite, which then differentiates to 8-36 new mero zoites. These are then released from lysed infected RBC (iRBC) and re-invade RBCs once again. The resulting increased parasitemia generates the well known symptoms of fever, anemia, and so on, which without treatment can ultimately progress to severe or cerebral malaria, coma and death. Sporadically, iRBCs commit to gametocyte differentiation via poorly understood pathways. Haploid gametocytes ingested by another mosquito feeding on an infected human, differentiate to male and female and combine in the mosquito to produce diploid stages that ultimately result in the injection of new sporozoites via mosquito saliva during another blood meal.
Most clinical manifestations of malaria are associated with the iRBC cycle. Not coincidentally then, nearly all known antimalarial drugs act against the iRBC. Understanding unique parasite biochemistry within the iRBC is essential since it is intricately linked to the molecular mechanisms of drug action and drug resistance for virtually all known antimalarial drugs. The past 10 years has shown that as these mechanisms are increasingly defined in molecular detail, this guides the rapid development of additional inexpensive antimalarials active against chloroquine (CQ) resistant (CQR) P. falciparum. Promising advances with partially protective vaccines notwithstanding, new antimalarial drugs will be direly needed for at least the next two decades, and probably longer. The above is just a glimpse at a very complex host-parasite biology that varies for different species and that protects multiple parasite stages from effective immune-system intervention in multiple ways. Childhood versus adult versus pregnancy-associated malarias differ, and common co-infections involving malarial parasites and other microbes pose additional therapeutic challenges. As a consequence, multiple vaccines will most likely be needed to eradicate malaria. Thus, it is imperative that equal emphasis be placed on inexpensive drug development and other approaches for controlling malaria.
This review briefly summarizes progress related to inexpensive antimalarial drug development on three fronts: Molecular and physiologic basis of quinoline drug resistance in Plasmodium falciparum malaria It is a comprehensive picture of all three that teaches us how to circumvent quinoline antimalarial drug resistance. To date, three of the best five antimalarials of all time have been quinolines, and recent work suggests that, with a comprehensive understanding, additional inexpensive, effective quinolines can be developed quickly.
Rapid RBC growth and subsequent multiplex asexual division presents severe metabolic challenges. The parasite has adapted to these challenges by acquiring the ability to rapidly digest millimolar levels of hemoglobin (Hb) acquired from the RBC cytosol. Hb digestion via elegant internalization and proteolysis within a unique lysosomal-like organelle termed the digestive vacuole (DV) is essential for parasite survival. The toxic by product, heme, released upon Hb proteolysis, cannot be degraded by the parasite because it lacks the heme oxygenase pathway. Instead, the parasite is one of only a handful of organisms on the planet that detoxifies copious amounts of heme by crystallization to hemozoin (Hz), as described later. Thus, this pathway is a specific, biochemically unique drug target. Key molecular details of this pathway have been elucidated in recent years. In parallel, drug-resistance researchers are defining how drug-pathway interactions are 'hijacked' by the drug-resistant parasite via subtle alterations in DV bio chemistry and physiology. In a desperate time, some optimism is on the horizon.
The most successful antimalarial drug to date is the 7-chloro-4-amino-quinoline, chloroquine, which targets the Hb digestion pathway. The mechanism is not completely understood [1-6] but a critical facet is the inhibition of ferriprotoporphyrin IX (FPIX) heme crystallization to Hz [5-10]. As early as 1964 [11] the likelihood of important CQ-FPIX interactions was realized, and the idea gained momentum via the work of Fitch [12] . The Hz crystal unit is a curious 'head to tail' FPIX dimer, wherein oxygens, coordinating adjacent irons in the two tetrapyrrole rings, are donated by FPIX propionic acid side chains [13] . Formation of this dimer within the DV competes with the formation of the better studied µ -oxo dimer. In simple aqueous solutions the µ-oxo dimer is favored over the head to tail dimer. Overall, equilibria between multiple chemical forms of FPIX within the DV is poorly understood. However, it is known that both FPIX dimerization and Hz crystallization are highly pH dependent, and that Hz formation is accelerated by lipids and is obligatory for parasite survival.
A number of drugs, including CQ, potently inhibit the formation of Hz in vitro. Provocatively, in vitro IC 50 for Hz crystallization sometimes (but not always) correlates with antimalarial IC 50 in vivo [14] . However, this does not mean that every quinoline drug acts on the same chemical form of FPIX or at the same pH optimum. The kinetics of Hz inhibition for different drugs against various CQ sensitive (CQS) or CQR strains of malaria are just beginning to be defined in vivo, because crystals form in an irregular shape and are exceedingly difficult to image quantitatively within the live parasite [15] .
Different molecular models for how CQ inhibits Hz formation have been proposed, including the lysosomotrophic model wherein changes in DV pH caused by CQ diffusion titrate either Hb digestion, FPIX derivatization, Hz crystallization, or some combination. Alternatively, CQ might inhibit a heme/enzyme complex that forms as an intermediate. However, most evidence suggests that CQ and other anti malarials bind directly to one or more chemical forms (monomers, dimers) of FPIX [5, 6, 14, [16] [17] [18] [19] [20] . These data suggest that quinolines directly inhibit Hz formation by sequestering one or more precursors of the crystal unit, or perhaps by interacting with growing faces of the Hz crystal [17] . It then follows that the CQ resistance mechanism involves the disruption of one or more of these CQ-FPIX interactions. Importantly, since FPIX is made by the host (not the parasite), the target cannot be mutated, and since its release is an inevitable consequence of essential Hb digestion its availability cannot be reduced. Thus two of the most common routes to drug resistance are eliminated (e.g., mutation or decreased expression of target). To become CQR the parasite must therefore either compartmentalize the drug in a different way, alter signal transduction related to death or modify the target in some biochemically unique way. Indeed, Bray et al. extrapolated CQ-binding data and determined that intracellular target affinity differs for CQS versus CQR parasites [9] , so some biochemical transformation of FPIX for CQR versus CQS parasites seems probable. Another study demonstrated this directly in vivo [17] . Recently, we have been the first to determine the atomic-level structures of noncovalent CQ, quinine (QN), future science group amodiaquine (AQ) and quinidine (QD)-µ-oxo dimer FPIX complexes [18, 19] and these structures reveal a number of routes for altering FPIX-drug interactions. Remarkably, we have also discovered that when CQ and FPIX are added together under conditions that mimic that of the DV, a covalent (dative Fe-quinolinal N) FPIX-CQ complex is formed [20] . These discoveries enhance our understanding of how drugs inhibit Hz crystallization, and define important molecular features of CQR malaria. That is, noting the presence of at least two different CQ-FPIX complexes, two different FPIX dimers and the different pH dependencies for complex and dimer formation, we learn that there are several quite simple routes that the parasite can take to titrate multiple drugheme target interactions. Indeed, slightly altered DV pH, volume, ionic or lipid composition for CQR versus CQS parasites [21, 22] can easily alter the ratio of monomeric versus dimeric FPIX, the ratio of µ -oxo versus head to tail dimer, or ratios of multiple drug-heme complexes. Since even closely related quinoline antimalarials have different preferences and affinities for various chemical forms of FPIX, these data suggest that heme target-affinity differences for CQR parasites seen in vivo are easily explained via alterations in physiologic parameters (DV pH, volume, ionic and lipid composition and so on) known to exert enormous influence on Hz and drug-heme chemistry [23] .
FPIX monomers and dimers have different affinities for lipids. Lipids and/or monoacyl glycerols found within the DV provide convenient catalysts or scaffolds for the formation of Hz [24, 25] , and different quinoline drugs have different affinities for both monomer and dimer in aqueous solution. Thus, understanding heme monomer-dimer equilibria is essential [14] . De Dios and colleagues have recently explored pH and drug perturbations of the monomer-µ-oxo dimer equilibrium in some detail, and have also measured how this is influenced by drugs and micelles of different charge that, to a first approximation, model various possible lipid phases [26, 27] . In brief, remarkably, CQ is now known to favor binding to FPIX dimers whereas QN clearly favors the FPIX monomer [14] . In free solution, CQ actively promotes the formation of the µ-oxo dimer whereas QN stabilizes the monomer, both in a highly pH-dependent fashion [26] . Addition of a model lipid phase reveals pH-dependent lipid head group dependencies for monomer-dimer equilibria in the presence of quinoline drugs [27] . These are due to different aqueous versus lipid solubilities of monomeric versus dimeric FPIX, as well as different solubilities for these species complexed with quinoline drugs. This partitioning behavior is remarkably drug specific, even for related quinolines such as CQ compared with QN. Thus, even subtle changes in lipids or pH has profound effects on the efficiency of Hz production and has vastly different influences on the ability of even closely related drugs (e.g., CQ vs QN) to inhibit Hz formation. This makes sense since a nondrug-associated form of FPIX successfully partitioned into (or on) a lipid phase is likely to be essential for Hz production at the rate that has recently been directly measured in vivo [15] , and because drugs, pH and lipid composition individually and synergistically perturb FPIX-lipid partitioning. Evidenced by reciprocal patterns of CQ resistance versus QN resistance in some strains (see later), these data further highlight that CQ and QN are really quite different drugs, and that the design of inexpensive second generation quinolines active against CQR malaria should independently target CQ and QN pharmacophore scaffolds.
While this progress in elucidating molecular details of quinoline pharmacology is critical, importantly, a recent report suggests that drug-heme interactions are likely not the only way in which the drug exerts a toxic effect -it is only one layer of a more complex collection of effects [28] . In this paper, cytotoxicity of CQ against different stages of iRBC parasites was quantified for both CQS and CQR parasites. Surprisingly, CQ was found to be nearly as toxic to rings and schizonts as to trophozoites. Although recent work by Elliot and colleagues shows that ring parasites do indeed begin degrading some small amounts of Hb, as proposed earlier, schizonts do not metabolize Hb nor produce Hz as trophozoites do [29, 30] . The simple conclusion then is that different forms of free FPIX are not likely to be the only targets of quinoline drugs. Even more surprisingly, this study also demonstrated that P. falciparum CQ-resistance transporter (PfCRT) mutations confer approximately similar levels of CQ resistance to all three iRBC stages [28] . Since a fully formed DV membrane does not exist for ring stage parasites, this suggests that the PfCRT protein (see later section on PfCRT) is likely to have additional locations within the parasite, and more than one physiological function. The next few years of research will yield a more complex, but more complete molecular picture of quinoline antimalarial pharmacology. Although some new pharmacophores are in development, the three major classes of antimalarial drugs are the quinolines (e.g., CQ, mefloquine [MQ] and QN), the antifolates (pyrimethamine and cycloguanil), which both poison nucleotide biosynthesis by limiting essential folate cofactors, and the reactive endoperoxides (artemisin and derivatives) whose action is not well understood. Resistance to the first two classes is profound and widespread, and resistance to the third may be beginning to appear [31] . CQR parasites were first noticed 10-20 years after the introduction of the compound and CQ resistance was well documented in 1959 in Asia. Attempts to eliminate malaria (via insecticides and prophylactic use of CQ) possibly further promoted CQ resistance.
CQ resistance is both spreading and continuing to evolve via additional selective pressure. Continued evolution of CQ resistance, artemisin resistance, MQ resistance, QN resistance and so on, continuously produces new genotypes and pharmacologic phenotypes. When resistance to multiple drugs is seen, the strain is often called multidrug resistant (MDR). Molecularly speaking there are many MDR genotypes. Thus, a sometimes overlooked aspect of antimalarial drug discovery is that we are only in the midst of an ongoing phenomenon; what is relevant for one subspecies of CQR P. falciparum is not necessarily relevant for another. It is crucial to understand 'subtypes' of CQ resistance and to develop inexpensive therapy that is effective against multiple subtypes.
Several important subtypes can now be described pharmacologically, genetically and (to some extent) in terms of their physiology and biochemistry. As elucidated in several other reviews there are many parallels between drug resistance in tumor cells versus P. falciparum [6, 8, 23] . A key role for ATP-binding cassette (ABC) proteins was emphasized early on, however, this cannot explain all phenomena [8, 23, 32, 33] . Many reports underscore an important role for ion transport in drug resistance phenomena [34] [35] [36] [37] and it is now generally well accepted that dysregulation of ion transport is likely to be central to multidrug resistance in tumor cells, malarial parasites and some bacteria. This suggests that altered drug accumulation in MDR cells may be due, at least in part, to consequential alterations in pH gradients, volume and/or other biophysical parameters that control diffusion or accumulation of drugs; that signal transduction regulated by ion transport is a key player in resistance (this is certainly now clearly recognized for apoptotic signal transduction vs tumor multidrug resistance); and that the chemistry of drug-target interactions is manipulated via ion transport such that drug activity is then altered (e.g., the special case of highly pH-dependent FPIX-quinoline interactions described earlier).
Biochemical and physiological features shared among MDR tumor cells, malarial parasites and certain Gram-negative bacteria include altered intracellular accumulation of the drugs to which cells or micro-organisms are resistant, and that certain 'chemo-modulators' (e.g., the calcium channel blocker, verapamil [VPL]) can partially reverse the drug resistance. Although it is still not precisely understood how VPL functions as a chemoreversal agent, it is reasonable to expect that concepts from the study of MDR tumor cells will apply to MDR malarial parasites, and vice versa. Such was the hope when pfmdr genes were first cloned [38, 39] and shown to be homologous to the hsmdr1 gene, which is overexpressed in MDR tumor cells. It was initially thought that an ABCprotein drug pump for CQ must therefore exist in drug-resistant P. falciparum, similar to the drug pump proposed for tumor cells (human multidrug resistance protein 1 [HsMDR1], also known as Pgp) believed by many investigators to directly translocate vinblastine and many other anti-tumor drugs.
However, subsequent work showed that some drug-resistant P. falciparum strains do not encode mutated or overexpressed pfmdr [40, 41] , and that other genetic events must therefore be important. In parallel once again to tumor drug resistance, at the same time an increasing importance of the overexpression of other genes (e.g., ion transporter and mutant pro-and antiapoptotic genes) was recognized for MDR tumor cells [42, 43] . Similar to P. falciparum multidrug resistance (PfMDR) protein versus malarial multidrug resistance (see later), the precise role of HsMDR1 protein in tumor multidrug resistance has been questioned for some time as pathology data have not correlated HsMDR1 overexpression with clinically relevant tumor multidrug resistance as strongly as was initially suspected [36, 43, 44] .
It is now clear that there are multiple molecular layers to multidrug resistance pathways and that these continue to evolve. Elucidating the molecular function of proteins that appear to be membrane transporters and that are mutated in CQR malarial parasites (which are often also MDR) remains a critical area of study and is briefly summarized in the last section of this review. future science group Initial genetic studies: cg2 versus Na + /H + exchange; the wrong gene but the correct physiology A genetic definition of CQ resistance began with the cloning of pfcg2 [45] , a technical tour de force essentially started years earlier via the successful creation of CQS × CQR cross progeny [46] . Correlation between specific pfcg2 sequence and CQS or CQR status was found for all but one strain of P. falciparum examined. The one exception, strain Sudan 106/1, turned out to have a special utility in characterizing the true CQ resistance determinant (see later section on PfCRT). Prior to that work, Lanzer et al. concluded that mutated PfCG2 protein was a dysregulated Na + /H + exchanger (NHE) that also pumped CQ out of the cell [47, 48] . Wellems et al. questioned this because subcellular localization revealed PfCG2 in vesicle-like structures near the parasitophorous vacuolar space and the DV [49, 50] , not within the plasma membrane as envisioned by Lanzer et al [47] . Follow-up studies by Bray and Ward did not measure any Na + dependency for CQ resistance, arguing against a strong role for NHE in influencing CQ transport or any other feature of CQ resistance [51] . Most recently, quantitative trait loci (QTL) analysis, availability of the P. falciparum genome and novel single-cell imaging in an extensive series of drug-resistant progeny suggested that altered NHE and cytosolic pH (pH cyt ) are indeed related to resistance, but that the relevant ion exchanger is not PfCG2 (see later section on P. falciparum Na + /H + exchanger [PfNHE]), and that resistance to QN is more influenced by these changes than CQ resistance [52] .
Wellems and colleagues subsequently proved, via direct transfection experiments, that mutant PfCG2 did not confer CQ resistance [53] . Simultaneously, the Roepe group measured alkaline pH cyt for some but not all CQR parasites, and early NHE data by that group [54] led to conclusions similar to those of Bray and Ward [51] . Thus, attention then focused on another gene found within the same 36 kbp fragment that harbored pfcg2, namely, pfcrt [55] . Results described in this and additional papers show that PfCRT protein is the ultimate determinant of CQ resistance in P. falciparum malaria, that it resides within the DV membrane, and is not directly involved in pH cyt regulation or plasma membrane NHE (see later section on PfCRT) [56, 57] .
Along with the now recognized dominant role of PfCRT, P. falciparum MDR protein 1 (PfMDR1) is now thought to only modulate cross-resistance patterns in CQR parasites (see later). Meaning, this transporter modifies the rank order of resistance to other drugs such as MQ [58, 59] . Other proteins, including PfNHE1, which is a true NHE, are now suspected to complement drug resistance even further [52, 60] . This degree of genetic complexity involving at least three membrane transporter genes is daunting, but is to be expected. Early explanations for MDR phenomena in either tumor cells, bacteria or parasites were (in hindsight) a bit too optimistically reductionist.
More genetics; the elusive role of PfMDR1
As mentioned, early studies of CQ resistance in P. falciparum showed resistance was associated with decreased drug accumulation that was reversed by the ion-channel blocker VPL [33, 61] . Similar phenomena had been seen in MDR tumor cells. Thus, Wirth and colleagues screened P. falciparum for hsmdr1 homologs and identified pfmdr1 and pfmdr2 [38] . While both encode proteins expressed in drug-sensitive P. falciparum, a MQR strain showed elevated pfmdr1 [54] . MQ is chemically similar to CQ, so at the time resistance pathways were expected to overlap. In support of this, another group found pfmdr1 to be upregulated in some CQR P. falciparum [39] . However, subsequent experiments showed that pfmdr1 overexpression did not correlate with CQ resistance [62] . This was not surprising since Wellems et al. had earlier shown that CQ resistance did not segregate with the pfmdr1 chromosome 5 locus in progeny from a CQS × CQR genetic cross [46] . Proposals arguing against a dominant role for HsMDR1 protein in tumor resistance were also being made during this period, as ion transporters were found to be alternatively expressed in some MDR tumor cells well before overexpression of HsMDR1 [42] .
On the other hand, polymorphisms in pfmdr1 were also associated with CQ resistance early on [63] . While CQS isolates had identical PfMDR1 sequences, there were five changes in CQR isolates. In strains K1 and ITG2, N86Y was the only change. CQR strain 7G8 had four changes; Y184F, S1034C, N1042D and D1246Y [63] . The Y184F mutation was postulated as not likely to be involved in CQ resistance since it was also found in CQS strains. Thus, the pfmdr1 overexpression hypothesis was revised to suggest that CQR strains expressed mutant PfMDR1 but did not necessarily overexpress wild-type [63] . (Which pfmdr alleles are considered 'mutant' versus 'wild-type' is open to interpretation, wild-type is used here to refer to the allele found in the CQS strain HB3.) Interestingly, CQR K1 versus 7G8 polymorphisms appear to be geographically biased [64] [65] [66] .
future science group Subsequently, when MQR P. falciparum were selected to higher levels of MQ resistance, pfmdr1 was found to be amplified [67] . Halofantrine resistance and QN resistance increased with increasing pfmdr1 whereas AQ resistance did not [67] . However, when CQR strain K1 was selected against halofantrine it did not result in MQ resistance or amplification of pfmdr1 [68] . In a more recent study, which used allelic exchange of pfmdr to probe these questions, incorporation of pfmdr1 7G8 polymorphisms into a CQS strain not previously exposed to a drug had no effect on CQ resistance, but incorporating wild-type pfmdr1 into a CQR strain expressing mutant pfmdr1 did decrease the level of resistance by half [58] . CQS strains expressing mutant pfmdr1 alleles showed some mild QN resistance and altered sensitivity to MQ [58] . Variations on this theme have also been described by Fidock and colleagues [59] . Collectively, these data suggest that PfMDR1 effects are subtle and strain-specific. This brings us to our current understanding: it seems unlikely that mutations in pfmdr1 confer CQ resistance in and of themselves [69] , but they (or overexpression of certain isoforms) provide an important modulatory effect [70] .
As previously mentioned, early on, Wellems et al. showed that pfmdr1 was unlikely to directly cause CQ resistance since the relevant region of chromosome 5 did not segregate with the CQ resistance phenotype in genetic cross progeny [46] . A follow-up paper suggested that CQ resistance segregated with the pfcg2 gene on chromosome 7 [45] , but this paper also showed that one CQS strain (Sudan 106/1) carried CQ resistance-associated pfcg2 yet was nonetheless CQS. The 36 kbp chromosome 7 locus harboring pfcg2 that segregated with CQ resistance was thus re-examined and a previously unrecognized gene, now known as pfcrt was found [55] . Mutations in pfcrt are the central determinant of P. falciparum CQ resistance. The 13 exons span 3.1 kbp and encode a 424 amino acid, 48.6 kDa protein. Mutant pfcrt alleles found in CQR parasites contain a number of point mutations that confer amino-acid substitutions, with the pattern depending on the region of the globe from which the CQR parasite originates. In general CQR parasites from southeast Asia and Africa carry seven to eight point mutations, whereas South American CQR strains carry five [55] . Novel patterns continue to be discovered [65] and now at least 12 distinct CQ resistance-associated PfCRT isoforms have been described.
The pattern of mutations provides identification of the probable geographic origin of a CQR isolate. The number of mutations apparently required for CQ resistance explains two riddles, namely, why CQ resistance took so long to appear on a large scale and why it had been impossible to create CQR strains from CQS strains in the laboratory via drug selection. However, if one begins with Sudan 106/1, a strain that harbors all but one of the mutations required to complete a CQR-pfcrt allele, CQR strains can be rapidly created in the laboratory by CQ selection [57] , and the final mutation required to complete the CQR-pfcrt allele [55] is found in these selected strains. In performing this experiment, Cooper et al. also found PfCRT substitutions that are not known to exist in the wild but that confer unusual and scientifically informative drug-resistance profiles [57] .
Similar to PfMDR1, PfCRT is localized to the DV membrane and is a polytopic, integral membrane protein that is likely to perform some type of transport function [21, 22, 55, [71] [72] [73] [74] [75] [76] . Similar to the case for HsMDR1 most initial hypotheses for its function suggest either ion or drug transport or both, since, again, CQR parasites accumulate less antimalarial drug in a given time relative to CQS and quinolinal antimalarial drugs (like anti-tumor drugs), which are hydrophobic weak bases. In fact, CQ and related drugs are dibasic and the DV is now known to be quite acidic. So, fold concentration of CQ within the DV (where the heme-drug target is found) is dependent upon the square of the net pH gradient and would be as much as 10 5 -10 6 -fold by the predictions of weak base partitioning theory, depending on relative permeabilities of neutral, +1 and +2 CQ species [6]. Thus, even very subtle changes in DV pH would have very significant consequences. Progress on these and related questions is summarized later.
As mentioned previously, one additional genetic event that tailors CQ resistance caused by PfCRT, may be the mutation and/or over expression of PfMDR1, but this cannot fully explain all MDR phenotypes. Another contributing event, recently identified by QTL analysis [60] , involves one or more genes encoded by a segment of chromosome 13. This fragment is hypothesized to contain genes encoding for proteins with unclear homology to known proteins, however, it also encodes homologs to a V -type ATPase subunit and to the NHE protein family. By combining future science group QTL analysis with improved pH cyt measurements, we have recently shown that Pf NHE dysregulation is likely to be linked to one route to increased QN resistance [52] . In addition, additive QTLs on chromosomes 5 and 7 were found as expected (the identified fragments contain pfcrt and pfmdr1) [60] . Pairwise effects were also detected between chromosome 13 and a chromosome 9 locus. The NHE homolog encoded within chromosome 13 was named PfNHE1. This protein is the second largest eukaryotic NHE yet identified (surpassed only by a NHE for the related apicomplexan Toxoplasma gondii), and has several unusual features. Bioinformatic ana lysis and cross reactivity with anti-TgNHE antibody [Chen D, Pleeter P, Roepe PD, Unpublished Data] show that PfNHE is localized to the plasma membrane. Polymorphisms that encode variable DNNND repeats in the predicted PfNHE protein sequence have been found in progeny of a CQS × CQR cross, as well as a range of field isolates and additional laboratory strains showing variable QN resistance [60] .
Prior to the availability of the P. falciparum genome, Ginsburg et al. quite logically suggested a plasma-membrane NHE must exist to decrease cytosolic acid caused by anaerobic glycolysis [77] . As mentioned earlier, Lanzer et al. suggested that pfcg2 found close to pfcrt on chromosome 7 encoded this NHE [48] , but this was disputed, since PfCG2 is actually peri-vacuolar [50] , not plasma membrane localized, and because the putative PfCG2-NHE homology was based on sequence ana lysis that did not account for very high AT content in malarial genes [49] . Regardless, the field has come full circle and the initial conclusions by Lanzer et al. were indeed partly correct (the physiology was correct, but not the genetic explanation). In our hands single-cell photometry (SCP) analysis of pH cyt for intra-erythrocytic parasites under continuous physiologic perfusion indeed show elevated pH cyt for some CQR parasites, but not all [54, 55, 57] . A corollary we suggested is that the relative size of the net cytosolic-DV pH gradient might be a more important parameter for CQ resistance versus QN resistance, rather than steady-state pH cyt or digestive vacuolar pH values alone [54] . Overall, a range of pH phenomena and genetic changes consistent with changes in pH regulation are associated with QN resistance and CQ resistance. As described [52] , we recently optimized localization of the pH probe BCECF exclusively to the parasite cytosol to avoid complexities in interpretation from earlier studies, and showed that elevated pH cyt is well correlated with QN resistance and increased apparent PfNHE activity [60] . We now believe there are at least two physiological signatures for QN resistance that segregate with the two genetic descriptions, and that one is alkaline pH cyt [52] . Technical details of PfNHE measurements have recently been questioned [78] , but these issues have hopefully been clarified [79] .
Genetic definition of CQ resistance and related phenomena is a major breakthrough, but to develop drugs and other therapies the altered biochemistry and physiology linked to that genetics must also be elucidated. As described in the first part of this review a chief drug target is FPIX in the DV, so studies of DV biochemistry are particularly important.
A central characteristic of DV biochemistry and physiology is the high pH gradient (acid inside) that the organelle has. Regulation of DV pH is not fully understood, but it includes a V -type H + ATPase that hydrolyzes cytosolic ATP to pump H + into the DV [80] . Interestingly, although conflicting data have been published [81] it is now generally accepted that changes in DV pH and volume are linked to CQ resistance caused by PfCRT [21, 22, 74, 76] . These are further predicted to affect drug, lipid, metabolite and osmolyte traffic in and out of the DV. However, since the DV is a subcellular organelle for an intracellular parasite, precise quantification of these perturbations is quite challenging. It requires the development of novel, overlapping approaches, as described later.
Our group attempted the first DV pH measurements for living intra-erythrocytic parasites under physiologic perfusion using the pH probe acridine orange (AO) and novel SCP [71, 82] . Our initial hypothesis was that, owing to the dibasic nature of CQ, even subtle increases in DV pH would significantly lower DV concentrations of CQ and thus cause CQ resistance. Therefore, relative to CQS, CQR parasites might show slightly elevated DV pH or different DV pH behavior upon addition of CQ [83] , or perhaps both. We and others have actually found that mutant PfCRT in CQR parasites causes more acidic (lower) DV pH. The initial AO data in support of this conclusion generated controversy (as most new technologies tend to do), but a number of different, complementary methods from several labs were subsequently developed and strongly supported the initial AO conclusions [21, 22, 74, 76, 79] .
Nonetheless, lower DV pH for CQR parasites appeared paradoxical, because simplistically it is predicted to concentrate more drug within the DV by weak base effects. It is difficult to future science group see how concentrating more drug at the site of action works to confer drug resistance, but as pointed out [6, 82] in hindsight the physiology is obvious when examined alongside the chemistry of the principle CQ target, FPIX released from Hb. As mentioned, iRBC parasites detoxify FPIX by crystallization to Hz. In aqueous solution, FPIX dimerizes to a µ-oxo dimer. FPIX has two propionic acid side chains, thus µ-oxo FPIX is a tetraprotic amphipathic weak acid with four equivalent titratable groups, and a pKa near DV pH [6, 82] . As pH is dropped even subtly (0.1-0.3 units), because the titration curve of FPIX dimer with four equivalent pKa is exceedingly steep (shown in [82] ), even low levels of soluble dimer convert to insoluble (aggregated) dimer over a very narrow pH range (<0.5 units [82] ). CQ and other drugs bind well to soluble FPIX but not to aggregates. In addition, acid aggregation of FPIX accelerates conversion to Hz (to which drugs also do not bind well). For these reasons, lowering DV pH by as little as 0.1 units is actually predicted to be a potent pathway to CQ resistance [6] . Ingeniously, CQR parasites titrate one drug target to lower levels, and also bias pHdependent FPIX chemistry. That is, even though at DV pH FPIX monomer is likely to be more abundant than µ-oxo dimer [26] , FPIX equilibria are pulled away from monomer by acid aggregation phenomena. As explained in the first section of this review, it is now known that this simple picture is a bit more complex, and also involves quinoline drug-specific effects (e.g., CQ vs QN) on monomer-dimer equilibria and drug-FPIX aqueous versus lipid partitioning [26, 27] . The overall point is that ion dependent CQ resistance DV biochemistry drives a number of chemical conversions that will act to disrupt quinoline drug-FPIX interactions in very potent ways. However, importantly, lower DV pH caused by CQ resistance-associated mutant PfCRT that correlates with a VPL reversible CQR phenotype can only be one aspect of the CQ resistance mechanism and cannot explain all quinoline drug resistance. For example, evidence suggests lower DV pH may not be directly related to QN resistance, and might even be related to MQ hypersensitivity in some cases [56, 57] . As described earlier, added effects of proteins encoded by other identified loci (PfMDR, PfNHE) may hold clues to this complex spectrum of multidrug resistance phenomena. In addition, better definition of the molecular origins and repercussions of this altered pH is required. How does it occur? Is PfCRT a H + -coupled metabolite transporter that when mutated to a CQR form becomes partially uncoupled? Does PfCRT interact with the DV H + ATPase? Does PfCRT transport a counterion (i.e., Cl -) to shunt membrane potential in the presence of a high change in pH, such that CQ resistance mutations alter anion flux to indirectly cause a greater pH change? Do PfCRT CQ resistance mutations change substrate specificities for a facilitative diffusion transporter? These questions all have very different predicted consequences.
Some cell-based experiments have attempted to address these questions. Lanzer and colleagues successfully expressed PfCRT in oocytes and measured pH, membrane potential and certain ion conductances across the oocyte membrane [83] . They noted that oocytes expressing PfCRT exhibited an altered transmembrane pH gradient and membrane potential due to H + leak and somewhat nonspecific cation conductance. They proposed that PfCRT activates endogenous oocyte ion transporters in some way. Multiple molecular models that explain these observations are possible, but overall the data further highlight a role for PfCRT in ion and/or osmolyte traffic.
Another study followed ion dependencies for DV pH and volume regulation by imaging these parameters for live parasites under perfusion with a medium of altered salt composition [22] . Importantly, CQR parasites showed increased DV volume relative to CQS parasites, suggesting that pH and volume regulation are linked for the organelle, as is found for other acidic vesicles or lysosomes. However, fast transient changes in Clgradients across the DV membrane did not lead to rapid changes in the DV transmembrane pH gradient, indicating no direct coupling of Cland H + transport. On the other hand, fast transient changes in DV Clgradients were found to strongly influence DV volume. These effects were strongly CQ-and VPL-dependent and differed dramatically for CQS versus CQR parasites. The overall conclusion was that PfCRT mediates transport of an important DV osmolyte (presumably peptides, di-peptides or amino acids released from Hb digestion) and that this transport is altered for CQR parasites.
Taken together, the bulk of the evidence suggests that altered DV pH for CQR parasites is an indirect consequence of altered osmolyte traffic promoted by PfCRT mutation. This might be an explanation for why parasites treated in different ways (resulting in various levels of relevant osmolytes produced by a finely tuned metabolism) could perhaps show different DV pH in some studies [81, 84] . If altered osmolyte traffic that then indirectly perturbs normal pH regulation is the explanation for altered DV physiology future science group linked to PfCRT mutations, it begs the obvious question: what is the normal physiological function of PfCRT? An obvious attractive possibility suggested early on is that PfCRT might transport products of Hb digestion (peptides, dipeptides and/or free amino acids) [73, 22] , since these are among the most important DV osmolytes, since products of Hb digestion are unique osmolytes and PfCRT is a unique transporter with a unique sequence, and because when CQ, QN and other drugs are superimposed upon amino acid structures (e.g., CQ vs the cationic amino acid lysine), many interesting similarities can be noted. For example, the superposition (overlay) of a dipeptide N-terminal lysine (a common Hb amino acid) and CQ shows similar charge and a similar carbon chain flanked by nitrogen atoms. As another example, an OH group is bound to one chiral center of QN that is two s bonds away from the positively charged quinuclidine nitrogen, and a near identical spatial arrangement of stereochemically sensitive atoms is also found for one isomer of serine. Interestingly, QN and QD differ in their stereochemistry at this center yet show different PfCRT-isoform dependent pharma cology [57] . Perhaps not coincidentally then, if PfCRT did transport peptides or amino acids, substrate recognition would also be stereo-selective since only l-amino acids are found in Hb.
Ultimately, genetics and cell physiology can only take us so far, and cannot fully resolve speculation regarding substrates of PfCRT or define the thermodynamics and kinetics of any proteinmediated transport. Elucidating the remaining questions will require detailed molecular studies with PfCRT, PfMDR1, PfNHE and perhaps other proteins yet to be described. However, the P. falciparum genome is anomalously AT rich, and some genes (including pfcrt) can have significant stretches of AT content that are 80% or more. These do not translate well in convenient heterologous systems such as bacteria or yeast. In fact, in the case of pfcrt and pfmdr1 they do not translate at all. That is unfortunate, since techniques and strategies for defining transporter function have been elegantly laid out in bacteria and yeast model systems for the past four decades. Proteoliposomes, kabackosomes, Goffeau membranes, and Menendez phase separated yeast vesicle preparations would all prove incredibly useful for defining the molecular mechanism of CQ resistance, if only pfcrt cDNA could be expressed in either bacteria or yeast.
Hanbang Zhang working the Roepe laboratory succeeded in a brute force resolution to this dilemma. Noting the success of earlier gene design that allowed MSP-1 expression in Escherichia coli [85] , Zhang back-translated the PfCRT amino-acid sequence using preferred yeast codons, then designed a set of overlapping 40-mer oligonucleotides that encoded both strands of the theoretically optimized sequence, and constructed an entirely synthetic gene via nested PCR methods. This enabled the expression of high levels of PfCRT protein in either Saccharomyces cerevisiae or Pichia pastoris, in either a constituitive or inducible fashion, respectively [72] . Analysis of ion transport for plasma membrane inside-out vesicles produced from these strains provided further evidence that PfCRT protein plays a role in ion transport [72] , and purified membranes along with equilibrium binding studies, using 3H-CQ, provided the first direct evidence that PfCRT protein indeed binds CQ [73] . The latter is a central prediction for nearly all molecular hypotheses for PfCRT. Although not often cited, the same paper also provided the first direct molecular evidence that PfCRT likely transports CQ, via flow dialysis experiments using inside-out yeast plasma membrane vesicle with or without PfCRT protein [73] . The simplest model consistent with these results is that CQ transport by PfCRT is facilitative downhill diffusion at a rather low turnover. Different thermodynamic models for CQ transport (e.g., PfCRT as a CQ exchanger or active transporter) have also subsequently been proposed based on different approaches that measure 3H-CQ flux for live parasites [86, 87] . Distinction between these models will require additional direct transport experiments at much higher kinetic resolution.
At the time, pfcrt was (to our knowledge) the largest synthetic gene ever constructed by overlapping PCR, nonetheless, success enticed others to attempt the reconstruction of a yeastoptimized pfmdr1 gene, which is over three times the size of pfcrt. After numerous challenges were overcome, this too was achieved and unusual ATPase activity of PfMDR1 was rather quickly quantified via plate-based phosphate release assays [88] . Relative to other ABC transporters involved in drug-resistance phenomena, PfMDR1 has unusually high K m and V max for ATP hydrolysis. Also, interestingly, antimalarial drugs only mildly stimulated PfMDR1 ATPase activity, consistent with the notion that PfMDR1 exerts only mild affects on antimalarial drug resistance. With methods for in vitro future science group analysis of PfMDR1 in hand, a follow-up study quickly defined which amino acid substitutions in the curious 7G8 PfMDR1 isoform confer its unusual and quite specific ATPase activity [89] . The S1034C mutation in 7G8 PfMDR1 is now known to abolish antimalarial drug-stimulated PfMDR1 ATPase activity. Although this molecular-level work is only just beginning, it has already significantly refined proposals for the molecular mechanisms of CQ resistance and antimalarial multidrug resistance.
Direct demonstration of CQ binding to PfCRT was a very important result, but equilibrium binding studies with radio-labeled CQ obviously do not allow for definition of the drug binding site. To answer this question, the Roepe and Wolf laboratories designed and synthesized various CQ photoaffinity probes. One particularly novel probe, haboring both per-fluoro-azido and biotin tags via flexible linkers attached to the aliphatic terminus of CQ, proved to be enormously useful. It has recently led to the identification of the CQ binding site in HB3 isoform PfCRT, and has also quickly quantified relative affinities of other drugs (QN, VPL and so on) versus various PfCRT isoforms [90] . An obvious extension of this work would be the definition of CQ binding-site differences (if any) for other PfCRT isoforms, and perhaps similar studies with PfMDR1.
The chemistry developed for construction of these photoaffinity probes leads to a rather straightforward design and synthesis of fluorescent CQ derivatives. Some dansyl-CQ derivatives actually turn out to be more active against CQR strains of P. falciparum than CQ, and are useful for imaging subcellular localization of CQ [91] . A more convenient probe that can be followed with more routinely available lasers and fluorescent microscopes, NBD-CQ, has also recently been developed [92] . Combined with vesicles and proteoliposomes as described for drug binding studies [73, 90] this probe is currently allowing us to define putative CQ transport by PfCRT in very detailed molecular and thermodynamic terms.
In principle, genetics, physiology and biochemistry related to altered FPIX chemistry and membrane transport now defines CQ resistance. It is my suspicion that permutations of the above will ultimately be shown to define resistance to all other quinolines (QN, AQ, MQ and so on), to acridines, xanthones and reactive endoperoxides, but probably not to antifolates, for which resistance involves other distinct pathways [93] . That is, at least three genes (pfcrt, pfmdr1 and pfnhe) have been identified that, in various allelic combinations, confer what we now understand to be a spectrum of antimalarial multidrug resistance phenomena. The molecular function of at least two of these is becoming clearer via a combination of new chemical biology, artificial gene construction, and tried and trusted membrane biochemistry techniques. The physiologic and pharmacologic consequences of their function is at least qualitatively defined, and although there is still much more to do at the molecular level, the field has come a very long way in the 9 years since the identification of PfCRT. We can be hopeful that, based on this information, new quinolines effective against CQR malaria will prove increasingly easier to design and synthesize. In fact, the author suggests that this hope is even now being realized [91, 94, 95] . This work complements continued uses and combination therapies involving other quinoline derivatives such as pyronaridine (an aza-acridine) [96] , piperaquine [97] and isoquine [98] as well as other heme-binding pharmacophores such as the very exciting xanthones pioneered by Riscoe and colleagues [99] . History repeatedly teaches us that even with the emergence of CQ resistance, inexpensive quinolines and related compounds can still be effective.
In summary, the PfCRT protein binds CQ as is predicted from several models for its function. Azido-biotin-CQ now clearly defines that binding site and distinguishes drug binding preferences for various PfCRT isoforms [90] . CQ resistance-associated mutant PfCRT shows altered CQ binding, promotes altered diffusion of CQ and perturbs osmolyte equilibrium within the DV, which then alters DV pH and volume regulation.
The latter influences FPIX chemistry and multiple FPIX-drug interactions in critical ways. Taken together, a more complex, but clearer molecular model for the mechanism of trophozoite CQ resistance is now possible [Paguio M, Cabrera M, Roepe PD, Submitted]. However, importantly, we should not neglect that trophozoite/DV-specific models for CQ resistance are clearly only one part of the story. Other effects of CQ and other layers of CQ resistance need to be elucidated [28] . Other aspects of CQ pharmacology must be present at the ring and schizont stages of P. falciparum development. Additional physiological functions for PfCRT at the ring and schizont stages must also exist and when altered by mutation also cause CQ resistance for these stages. A key question future science group is how are these physiologic functions related to putative ion or osmolyte transport? For ring stages, based on the recent work of Elliot et al. it seems possible, at least in theory, that controlling Hz chemistry is again, at least, part of the explanation [29] . But for schizonts, the answer must be different. Perhaps mutant PfMDR1 and PfNHE further modulate drug resistance in the presence of mutant PfCRT for these stages as well. Their molecular level function can now also be studied using 'yeast-optimized' recombinant proteins and novel drug probes as described for PfCRT, so additional rapid progress is on the horizon.
It is true that great strides have been made in understanding antimalarial drug resistance at a molecular level, and it is also true that this information is now turning out to be of great value in developing new, inexpensive, secondline antimalarials. 'Molecular medicine', well developed for western cancer and cardiology clinics, has (in theory) finally come to the malaria clinic -genes that dictate how the disease might respond to therapy have now been identified, and additional molecular tools developed with that knowledge are leading to new clinical successes. The function of proteins encoded by these genes is becoming understood. High-throughput screening methods such as the SybrGreen assay have led to fast, inexpensive ways to screen for novel antimalarials active against CQR malaria, at least at the pre-clinical stage. Multiple plasmodial genomes have become available, giving us unique insight into comparative genomics and many new drug targets to explore. New anti malarial drug leads are indeed rapidly appearing.
Yet, the expense and often difficult logistics of Phase II and III drug trials still prevent full implementation of these advances. The entire world yearns for vaccine development, but optimistically that is still many years in the future. In the meantime, inexpensive drugs are still desperately needed. In 5-10 years, we hope that additional emphasis will have been placed by the entire global community on the rapid development and deployment of inexpensive anti malarial drugs. More funding and effort directed towards a multi-tiered, balanced approach to malaria treatment and prevention is very much needed. For over 60 years, inexpensive anti malarial drugs have saved the lives of many millions of malaria victims. In theory, there is no scientific roadblock that cannot be overcome such that this continues to be the case well into the future.
It has proven quite difficult to either increase or decrease expression of Pf NHE protein to further test how Pf NHE may be linked to QN resistance, but a new paper reports decreased PfNHE levels for three parasite strains (GC03, 1 BB5, 3 BA6) by stable transfection with a truncated 3´-untranslated region [100] . The transfectants from these experiments do not show statistically significant changes in resting (steady state) pH cyt as would be expected based on previous work [52] , but they do show small increases in QN sensitivity consistent with Pf NHE protein being involved in QN resistance as proposed [60, 52] . Unfortunately, in [100] only strains with low levels of QN resistance were amenable to transfection, whereas decreased Pf NHE expression for strains with Executive summary n Over the past 9 years, a clear picture of the genetic alterations that accompany evolution of chloroquine (CQ) resistance in Plasmodium falciparum malaria has become available. P. falciparum CQ-resistance transporter (PfCRT) mutations must be present and these confer a tenfold decrease in CQ-mediated growth inhibition and toxicity. Mutations and/or elevated expression of P. falciparum multidrug-resistant protein 1 (PfMDR1), perhaps along with mutations in P. falciparum Na + /H + exchanger, further tailor quinoline drug resistance, and perhaps resistance to other classes of compounds that also target the parasite digestive vacuole.
n To understand the physiological repercussions of these genetic alterations, additional cell culture and computerized microscopy methods have been developed. These are now rapidly providing new information on a number of key cell biological questions, including organellar biogenesis, CQS versus CQR parasite fitness, and metabolism.
n To uncover the molecular details behind how mutant proteins link to resistance phenomena function, heterologous expression, purification and reconstitution of at least two of them (PfCRT and PfMDR1) has recently been accomplished. Combined with new 'chemical biology' approaches, the field is now poised to fully describe the molecular mechanism(s) of CQ resistance.
n Taken together, these advances and others are accelerating development of new second-line antimalarial drugs active against CQR malaria.
future science group high levels of QN resistance would have been particularly informative. Also, the authors of [100] were unable to consistently clamp parasite pH cyt to acid values in the absence of Na + , formally leaving open the question of whether decreased levels of Pf NHE do indeed (as obviously expected) lead to a lower rate of Na + /H + exchange. More work is needed with these and other transfectants, and better methods for analyzing Na + /H + exchange for the intracellular parasite would clearly be helpful. Regardless, it is essential to recognize that effects on H + transport and steady state pH due to lowering Pf NHE expression [100] are certainly not expected to be analogous to the effects predicted from analysis of multiple pairwise loci influences on PfNHE activity [52] . Clearly, levels of QN resistance span a wide range, and Pf NHE effects on QN resistance are complex, require multiple loci [52] and, as reported earlier, do not necessarily segregate exclusively with elevated pH cyt [52] . Overall, as emphasized throughout this review, we now realize that multiple genetic alterations confer the molecular and physiologic basis of quinoline resistance patterns, and so it is to be expected that resistance phenotypes will involve multiple overlapping pathways. Interaction Between Humans and Poultry, Rural Cambodia Because avian influenza H5N1 infection risks are associated with exposure to infected poultry, we conducted a knowledge, attitudes, and practices survey of poultry-handling behavior among villagers in rural Cambodia. Despite widespread knowledge of avian influenza and personal protection measures, most rural Cambodians still have a high level of at-risk poultry handling. Because avian influenza H5N1 infection risks are associated with exposure to infected poultry, we conducted a knowledge, attitudes, and practices survey of poultryhandling behavior among villagers in rural Cambodia. Despite widespread knowledge of avian influenza and personal protection measures, most rural Cambodians still have a high level of at-risk poultry handling.
T he circulation of the highly pathogenic H5N1 avian influenza (AI) strain throughout Asia since late 2003 (1) , and more recently in Europe and Africa, has resulted in considerable concern for the potential of a new pandemic. In Cambodia, outbreaks of HPAI A/H5N1 infection were first reported in poultry in early 2004 (2) . Since 2005, 6 human cases have occurred (100% fatal); the 2 most recent cases occurred in early 2006 (3, 4) .
Most Cambodians live in rural areas and raise animals for consumption (2) , typically keeping poultry, swine, or cattle close to the home. Because H5N1 infection has been associated with exposure to infected poultry (5-10) and little is understood of the perceptions of rural farmers regarding AI (11), we conducted a knowledge, attitude, and practices survey of poultry handling in rural Cambodia to estimate the extent of interactions between humans and poultry, to understand practices in poultry handling among villagers, and to develop interventions designed to increase reports of poultry deaths and safe poultry handling.
We conducted a 2-stage household based cluster survey (12) with a goal of 500 participants: 20 persons >15 years of age in each of 25 villages from Prey Veng and Kampong Cham Provinces. The sampling frame of eligible villages within these provinces were those located in H5N1 high-risk communes, as defined by the Food and Agriculture Organization of the United Nations training program for village animal health workers. The villages were selected with probability proportional to size. For the second stage, we randomly selected the first household within each village. Subsequently, households were selected by proximity until 20 eligible participants were enrolled in each cluster.
Verbal consent was obtained from all participants. All were interviewed by using a structured questionnaire designed to collect information on demographics, basic hygiene practices, quantity of poultry owned, poultry death reporting, practices when deaths occurred, knowledge and attitude of sick and dead poultry, and knowledge of AI.
Twenty-three villages were included in Kampong Cham (11) and Prey Veng (12) Provinces ( Figure 1 ). Four hundred sixty respondents from 269 households completed the questionnaire. Most were women (60%), farmers (88%), and persons who had completed less than primary schooling (57%). The median number of household members was 5 (range 1-16), and 77% of all households included children <15 years of age.
Many households owned chickens (97%) and ducks (39%) (Figure 2 ), although the size of most poultry flocks was small (Table) . Almost all poultry were free ranging (100% of chicken flocks; 96% of duck flocks), and mixing of the poultry with pigs and other domestic animals was common. Respondents reported that they use poultry feces for manure (77%), touch sick/dead poultry with bare hands (75%), eat poultry that died from illness (45%), eat wild birds (33%), let children touch sick/dead poultry with bare hands (20%), and gather dead wild birds for consumption (8%).
During the previous 6 months, of the 260 households that owned poultry, 162 (62%) experienced poultry deaths; however, only 18 (7%) reported these deaths to local authorities. Half of the respondents (n = 231) believed that it was important to report any poultry deaths because the death may be due to AI (61%) or because the poultry owners may receive management advice from the village veterinarians (39%). Among these 231 respondents, many did not report poultry deaths because they did not know how (41%), were in the habit of not reporting poultry deaths (31%), believed they would have a problem selling poultry if they reported deaths (18%), did not know the risks of AI (7%), or feared poultry culling (5%). Among those respondents who did not believe reporting deaths was important, the reasons provided included the following: "the number of poultry deaths were too few" (62%), "poultry are not as important as cattle" (18%), "no help would be provided from veterinary staff or authorities" (13%), or "because mortality was similar to previous years" (7%). Of respondents that experienced poultry deaths, 62% buried or burned dead poultry, 53% prepared them for food, 22% threw away the dead poultry, 3% used them to feed other animals, and 2% prepared them for sale or gave them to their neighbors.
Participants had learned about AI from television (81%) and radio (78%). Thirty-one percent of respondents were able to describe AI symptoms in humans, and 72% believed that AI is a fatal disease among poultry that can be transmitted to humans. Most respondents believed it is unsafe to touch sick or dead poultry with bare hands (67%), eat wild birds (70%), let children touch sick or dead birds with bare hands (83%), and eat meat or eggs that are not fully cooked (86%). Sixty-one percent of respondents mentioned at least 1 of the recommended behavioral practices that protect against AI infection.
General media reports about AI through radio and television broadcasts appear to have been effective at reaching rural people. However, despite high awareness and widespread knowledge about AI and personal protection measures, most rural Cambodians still often practice atrisk poultry handling. Anecdotally, we also reported that family members of H5N1-infected patients, who knew about AI risks, still prepared dead or sick poultry for household consumption during massive die-offs, because they observed that neighbors with the same behavior did not become sick (Institute Pasteur in Cambodia, unpub. data). These findings provide evidence that high awareness does not necessary lead to behavior change. Behavior change involves comprehensive and multidisciplinary intervention, which combines risk perception communication and feasible and practical recommendations, including economic considerations. We speculate that it is hardly feasible to sustain good poultry-handling practices if access to personal protective equipment is cost prohibitive, particularly when disease occurrence poultry die-offs are common. Further studies are needed to determine appropriate behavior change strategies in Cambodia.
We did find that many of the villagers were willing to report poultry deaths but did not know how. However, this finding should be interpreted in light of some limitations. We observed difficulties and frustrations among farmers whose flocks underwent culling after identification of H5N1 viruses in their flocks because compensation has not yet been approved by the government of Cambodia. In contrast, Thailand and Vietnam have introduced compensation along with the introduction of poultry vaccination in Vietnam and the reduction of backyard poultry ownership in Thailand in an effort to protect the commercial poultry industry. Thus, it is difficult to envision effective control strategies in Cambodia based exclusively on culling. Coincidentally, Vietnam has reported far fewer H5N1 outbreaks in poultry and humans since the introduction of the vaccination program, while Cambodia detected 4 outbreak sites in domestic poultry and 2 unrelated human cases in 2006. The real effect of a no-compensation policy on willingness to report poultry deaths needs to be assessed. Not surprisingly, direct contact with poultry and poultry products was common among household members. Transmission of H5N1 from poultry to humans, even in circumstances in which human-poultry interactions are regular and intense has been limited; however, as the virus continue to circulate and evolve among poultry, bird-tohuman transmission may increase. In this context, improvement in risky practices can only be achieved through relentless behavior change efforts. Because lack of knowledge does not appear to be a factor, intervention programs must include feasible options for resource-poor settings that have limited materials for personal protection (water, soap, rubber gloves, masks) and must offer farmers alternative methods to safely work with poultry on a daily basis. Bird Migration Routes and Risk for Pathogen Dispersion into Western Mediterranean Wetlands Wild birds share with humans the capacity for moving fast over large distances. During migratory movements, birds carry pathogens that can be transmitted between species at breeding, wintering, and stopover places where numerous birds of various species are concentrated. We consider the area of the Camargue (southern France) as an example to highlight how ad hoc information already available on birds’ movements, abundance, and diversity can help assess the introduction and transmission risk for birdborne diseases in the western Mediterranean wetlands. Avian influenza and West Nile viruses are used as examples because birds are central to the epidemiology of these viruses. B irds are the only terrestrial vertebrates that share with humans the peculiarity of traveling in a few hours across national and intercontinental borders. The record for distance covered in a single year belongs to the arctic tern (Sterna paradisaea), which travels ≈50,000 km between Antarctica and northern Scandinavia. As a whole, billions of birds travel between continents twice a year in only a few weeks (1) . During these yearly migrations, birds have the potential of dispersing microorganisms that can be dangerous for public as well as animal health (2, 3) . For instance, birds are believed to be responsible for the wide geographic distribution of various pathogens, including viruses (e.g., West Nile, Sindbis, influenza A, Newcastle), bacteria (e.g., borrelia, mycobacteria, salmonellae), and protozoa (e.g., cryptosporidia). Insight into the ecology of bird populations is necessary to understand the epidemiology of bird-associated diseases. Furthermore, data about avian movements might be used to improve dis-ease surveillance schemes or to adapt preventive measures. However, solid bridges between ecology and human medicine are still lacking.
We explored the bird sector, in an attempt to provide general ideas on bird abundance, migration, geographic origin, and interspecies mingling. We focused on the Camargue area, an alluvial lowland covering some 140,000 ha in the Rhône Delta. As other Mediterranean wetlands (Figure 1) , the Camargue is a major rallying point for Palearctic birds that are migrating between the great continental masses of Eurasia and Africa. This area is the current focus of intense sampling to study 2 pathogens closely associated with wild birds: avian influenza (AI) virus and West Nile virus (WNV). These 2 viruses have very different transmission cycles and ecology: AI viruses have a waterborne transmission, and ducks are their main natural reservoirs (4) (5) (6) (7) (8) ; WNV has a vectorborne transmission, and passerines are believed to play a major role in the amplification cycle (9) (10) (11) . However, both viruses are known to be carried by reservoir birds during migration and have been associated with emerging disease transmission risk for humans and domestic animals (2, 5, 7, 11, 12) . For both of them, the avifauna abundance, diversity, and departure origin may be of key importance in terms of disease transmission. We use these 2 viruses as examples in our discussion of the risk for dispersion of bird-carried pathogens into Mediterranean wetlands. We address the following questions: 1) What are the main geographic origins of birds observed in western Mediterranean wetlands? 2) How abundant and diverse in species are they during the year cycle? 3) When are interspecies contacts between birds from different origins most likely to occur? To address these issues, we used crude empiric indexes, which are known to have biases yet prove valuable within the scope of our objectives. Readers interested in modern ecologic methods used to study wildlife diseases in natural populations may refer to general publications on host-parasite systems (13) (14) (15) .
Migration research is constantly changing, and new methods are always emerging. Historically, information about the movements of individual birds was first acquired through ringing studies. Bird ringing (also known as bird banding) consists of catching birds and attaching a small individually numbered metal or plastic ring to their legs or wings. Ring-recovery data are obtained when ringed birds are resighted, recaptured, hunted, or found dead. In Europe, large-scale ringing projects have been conducted, mostly between the 1950s and 1980s, and they represent a wealth of information that has not yet been fully exploited.
Data recovered from birds ringed from 1950 to 1975 at the Station Biologique de la Tour du Valat in the Camargue were collected from annual reports. Seven species of waterbirds were chosen to illustrate various migratory patterns. We selected 4 species of the Anatidae family, known to have different geographic origins, including 3 dabbling ducks, i.e., ducks that search for their food primarily in surface water (mallard, Anas platyrhynchos, n recovered = 434; green-winged teal, A. crecca, n = 3,903; garganey, A. querquedula, n = 181) and 1 diving duck, i.e., a species that mostly searches for its food under water (tufted duck, Aythya fuligula, n = 313). We also took the example of the common coot (Fulica atra, n = 99), a diving bird of the Rallidae family that frequently shares ponds with ducks. The common snipe (Gallinago gallinago, n = 54) is an example among waders, i.e., shorebirds that feed in muddy swamps and coastlines. Finally, the purple heron (Ardea purpurea, n = 39) is an ardeid species that lives in reed beds and marshes. All these species are large or hunted, which explains the high number of rings recovered. We only considered data recovered from birds ringed in the Camargue area and later reported outside France.
Since the 1950s, a large amount of data have been collected at the Station Biologique de la Tour du Valat thanks to bird counts, netting records, and field ornithologists' observations (see supplemental, online Technical Appendix Table l, indicating the methods used for each bird genus; available from www.cdc.gov/EID/content/ 13/3/365-appT1.htm). This information was used to create a database with a row for each of the 289 avian species regularly observed in the Camargue (16) . Strictly pelagic birds were not taken into account as they do not have any contact with terrestrial vertebrate species. Quantitative data were completed on the number of birds (abundance) and number of bird species (diversity) observed monthly in the Camargue. Three categories of migrating birds were considered, depending on the area from which they come: incoming birds from sub-Saharan Africa in spring and those arriving in autumn either from continental Europe or from Scandinavian and the Siberian tundra and taiga. Analyses were performed for all species and separately for species of the Anatidae family (ducks, swans, geese) and waders (shorebirds of the families Scolopacidae and Charadriidae), which are essentially associated with wetlands or coastlines.
Regular bird counts provide information on bird populations for the studied area and therefore give an idea of potential contacts between species that share similar biotopes. Since September 1964, the Camargue duck and coot populations have been estimated every winter (17) . The count was made monthly by the same observer from a plane flying at an altitude of 200 feet. One hundred brackish lakes and marshes used by waterbirds as resting places were counted. The arrival of the plane made dabbling ducks fly off, which is necessary for detecting them and identifying their species. To count diving ducks, it was necessary to turn the group of birds around by using the plane. Results of the winter 2004-05 counts were used as examples.
Ringing recoveries provide a valuable insight into the origins and dispersion areas of bird species. Figure 2 illustrates that western Mediterranean wetlands provide habitat for birds from a wide geographic range: all European countries but also other areas in the Mediterranean Basin, central and northern Asia, and sub-Saharan Africa. Ringed common coots and common snipes were mostly reported from continental Europe and Mediterranean areas, whereas mallards and common teals were also found in more northern places, including the former Soviet Union and Scandinavia. The pattern was slightly different for tufted ducks, for which >40% of recoveries were located in areas of taiga and tundra. Garganeys were recaptured in very distant places far north (Siberia, Finland), far east (Kazakhstan, Altai), and far south (Senegal, Mali) of the Camargue. In contrast to the previously described species, purple heron rings were recovered only from areas located south, including 4 countries in the Guinea Gulf in Africa (Benin, Côte d'Ivoire, Ghana, and Sierra Leone). As a whole, we discerned 3 broad areas from which Mediterranean waterbirds come and potentially disperse pathogens: continental Europe, northern Siberia and Scandinavia, and sub-Saharan Africa.
Monthly abundance (number of individual birds) and diversity (number of species) in the Camargue are presented respectively in Figures 3 and 4 for birds originating from the 3 major areas of provenance described above. These figures show how many birds are in the Camargue, just as monthly photographs of bird populations do. A corresponding table indicates monthly abundance of each species (online Technical Appendix Table 2 , available from www.cdc.gov/EID/content/13/3/365-appT2.htm.).
As many as 111 bird species might disperse pathogens from sub-Saharan Africa into the Camargue. Broadly speaking, birds coming from sub-Saharan Africa become rapidly and simultaneously abundant and diverse in spring, are still numerous in summer, and decrease in winter. The pattern is different if one considers solely ducks, as only 3 duck species fly south to tropical Africa, namely, the northern pintail (Anas acuta), the garganey, and the northern shoveler (Anas clypeata). Conversely, numbers and species diversity are high for waders, which are mainly passage visitors, especially in spring and late summer.
A total of 53 species might introduce pathogens from northern areas into the Camargue. Abundance is highest in April and October-November with a higher peak in autumn, notably because of juvenile birds. Species diversity is high during winter and low from May to July. The opposite pattern was observed for sub-Saharan species. This pattern is even clearer for birds of the Anatidae family: They are abundant from October to January and in very small numbers from March to September. In contrast to ducks, waders are mainly transient visitors, and only a few individual birds spend the winter in the Camargue. Their number is greatest in spring and autumn. Up to 135 species could be involved in pathogen dispersion from continental Europe. Their abundance is highest from February to April and later from September to November. Species diversity remains high year-round with peaks in spring and autumn due to migrating passage visitors. The pattern observed for Anatidae species is the same as the 1 we described for Arctic species: birds are abundant in autumn and winter and in very small numbers in spring and early summer. However, the number of duck species remains stable year-round. Indeed, in species such as the mallard or the red-crested pochard (Netta rufina), some birds are sedentary whereas others are migratory. Waders show a constant level of species diversity because migration staggers over several months, but numbers are highly variable throughout the year.
The results of the winter 2004-05 waterfowl counts are presented in Figure 5 for the species mentioned in Methods. Other species are also present, such as the northern pintail or the common shelduck (Tadorna tadorna).
Garganeys are present in small numbers in September and February-March, but from an airplane they cannot be distinguished from common teals. These counts show that numerous species, with various migratory patterns, congregate on the same wetlands during the long winter period and therefore easily transmit waterborne pathogens such as AI virus. Most wintering birds are still present in March, when the first African migratory birds have already returned to breed in the Camargue or make a stop for refueling before flying further north. For instance, as many as 11,550 black-tailed godwits (Limosa limosa) were counted in the Camargue in March 2005.
Moreover, the movement and abundance of birds vary greatly from 1 year to another because of movements influenced by weather conditions. For example, the duck population in the Camargue was estimated at ≈60,000 ducks in March 2005 compared with only ≈40,000 the previous year, when climatic conditions in Europe were warmer. 
Maps of ring-recovery data and graphs of monthly variations in bird abundance and diversity show that western Mediterranean wetlands such as the Camargue are a hub for birds from all origins (Central Asia, Siberia, northern and Eastern Europe, western Africa, and the Mediterranean basin) and that numerous birds of various species are seasonally aggregated in similar habitats. Under the hypothesis that risk for dispersion of pathogens into the Camargue is correlated with the number of birds and bird species encountered in a given area, these indices are helpful to determine periods at higher risk for introduction and emergence of birdborne diseases. We recall that these empiric estimates are skewed, which is briefly discussed with the perspectives below.
The risk for introduction of African pathogens in Mediterranean wetlands would be highest from March to July, which corresponds with spring migration and breeding for birds. Conversely, in autumn, birds return to Africa and are more likely to introduce pathogens originating from the north than from the south (Table) . Of the 111 species that come every year to the Camargue from different countries of sub-Saharan Africa, most are insectivorous passerines that spend winter in Africa and breed in Europe; among aquatic birds, waders are the most numerous.
Up to now, no evidence exists that birds migrating from sub-Saharan regions play a major part in the epidemiology of AI viruses. However, under the assumption that this area became an important epicenter for AI viruses, ducks would likely have the highest probability of introducing AI viruses in Mediterranean wetlands, even if they are less numerous than waders. Indeed, recent studies in Europe showed that overall AI virus prevalence in waders is really low compared with that in dabbling ducks (7) . WNV, which is transmitted by arthropod vectors, could potentially be introduced by any species of bird that comes from disease-endemic areas in Africa, is exposed to mosquito or tick bites (18) , and sustains high viremia levels. Insectivorous passerines are the most numerous and thus may be particularly suspected. WNV dispersion by birds migrating from sub-Saharan Africa might explain why an outbreak occurred in 2000 in the Camargue, even though the virus had not been observed there since the 1960s (19) .
Pathogens may be introduced into Mediterranean wetlands by birds coming from northern areas of Scandinavia and Siberia. The risk would be higher from September to December when Arctic bird abundance reaches its peak (Table) . In spring, the northern birds observed in the Camargue have recently spent a long time in southern lands so that their associated probability of introducing pathogens originating from Scandinavia or Siberia is rather low. Waterbirds and granivorous passerines, which do not need to fly further south to find food supplies throughout the cold season, could introduce pathogenic microorganisms that could be transmitted later between wintering birds when densities are high. Waders, which migrate from Siberia and stop in the Mediterranean wetlands in autumn before crossing the Mediterranean Sea, could contaminate other bird species before pursuing their flight. As a whole, 53 species seen in the Camargue come from Arctic areas, which is half the number of species that come from sub-Saharan Africa or continental Europe. As a result, the probability of pathogens being introduced from Arctic areas should be lower than from birds of these 2 other areas. Another scenario can nevertheless be considered: if birds coming from northern areas disseminate a pathogen all along their migration route, then this pathogen would also infect continental European species and the probability of its being introduced into the Mediterranean wetlands would depend on the arrival of both Arctic and continental birds. AI viruses are likely to be introduced in autumn by ducks that breed in northern Europe and Siberia, especially since numbers are high because of the presence of juveniles. Furthermore, surveillance studies of wild ducks showed that the prevalence of AI viruses is primarily high in juveniles (5, 7, 20) . Conversely, WNV activity has never been reported in Scandinavia and Siberia, probably because the transmission cycle cannot be maintained in these northern biotopes.
Autumn and winter are the 2 seasons during which the transmission of bird pathogens originating from continental Europe would be most likely (Table) . Indeed, in spring, the introduction of pathogens from continental Europe is less probable because birds have been absent from this area for 5 or 6 months. As previously seen, up to 135 species have the potential to introduce pathogenic agents in the Camargue. Granivorous passerines, birds of prey, and waterfowl are among the species that come in large numbers to take advantage of the Mediterranean wetlands' temperate climate during winter. Aquatic birds, which need unfrozen ponds to feed, show variations in their movements, depending on climatic conditions. For instance, if a cold spell occurs in eastern or northern Europe, the number of green-winged teals in the Camargue increases (17) . These weather-associated movements might at certain times prove essential in pathogen dispersion within European and Mediterranean wetlands.
Surveys of wild waterbirds in Europe have shown that AI viruses are frequently found (21) (22) (23) (24) , which means that waterbirds arriving from continental Europe might often be carriers of AI viruses. Similarly, because WNV activity was recently reported in Romania (25) and the Czech Republic (26) , wild birds migrating in autumn from these countries to the Mediterranean basin could introduce WNV, either because of a high viremia level or because they carry infected ectoparasites. If the virus managed to overwinter in a reservoir host or a vector, it could then be responsible for an outbreak the next summer, when mosquito vectors are abundant (27) .
Several factors affect the risk for bird-to-bird transmission: bird abundance or density, bird diversity, species receptivity and sensitivity to pathogens, interspecies interactions, and environmental conditions (14) . For watertransmitted pathogens such as AI viruses, risk for transmission may be associated with the number of ducks congregated in the same pond, particularly in autumn and winter ( Figure 5 ). This crowding of wintering species, in addition to the permanent presence of a transient population of birds using wetlands to stop off during migration, could allow AI viruses to circulate and be maintained because of rapid dissemination on shared water. For vector-transmitted pathogens such as WNV, transmission possibilities depend both on the bird reservoir density and on the dispersion abilities and activity periods of the arthropod vectors.
The risk for interspecies transmission of disease is particularly problematic when wild and domestic species are involved. Ducks are aquatic birds that are most likely to come in contact with free-range poultry, especially because the presence of congeners can induce migrating wild ducks to make a stopover. Captive-bred mallards, used for hunting purposes and voluntarily put in the wild to attract other ducks, are particularly likely to share pathogens with their migratory congeners and facilitate the transmission of diseases to other domestic species. The risk is different for domestic chickens or turkeys, which are more likely to have contact with granivorous birds. Conversely, waders are rarely in direct contact with human-raised species.
Bird-carried pathogens are above all susceptible to being spread worldwide because of human activities such as legal or illegal trade of wild and domestic birds or bird products (28) . The mechanism for the introduction of WNV into America in 1999 is not known with certainty, but a plausible scenario is the importation of an infected bird (29, 30) . Similarly, the highly pathogenic AI strain H5N1 was isolated in Belgium from crested hawk-eagles (Spizaetus nipalensis) smuggled by air travel (31) . In Asia, transmission of H5N1 influenza virus has mainly been the result of human activity such as live-poultry markets and the international trade of birds, bird products, or contaminated equipment (32) (33) (34) (35) .
The ornithologic data we have presented are merely crude estimates. Ring-recovery data, for instance, are subject to strong biases related to where and when the ringing was conducted but also to high variability in the probability of reporting marked animals among areas (36) . Similarly, our estimates of bird abundance and diversity are basic indices associated with the number of birds heard, seen, or caught in the Camargue (see online Technical Appendix Table 1 ). These estimates do not take into account 2 important sources of error: detection error, related to the fact that the probability of detecting a bird is <1, and survey error, associated with spatial and temporal variability (37) . Since our motivations were merely to show that information already available on birds may lead to better understanding of animal and human health issues associated with birdborne pathogens, these biases do not invalidate our objectives.
The results obtained were helpful to identify key groups of species likely to introduce pathogens from a given area at a given time of year. We voluntarily chose to focus on birds and leave pathogens aside, but studies of diseases in natural bird populations are obviously critically needed. Ecology, the science of interactions between living organisms and their physical environment, has been extended to include microorganisms. Understanding the relationships between organisms (such as hosts, pathogens, predators, competitors) and their environment is the aim of disease ecology. As studying the dynamics of systems with many hosts and pathogenic agents is complex, efforts should primarily focus on a few specific bird-pathogen models.
Mathematical modeling may help to predict specific bird-pathogen interactions and to identify key parameters that need to be better estimated through additional research. Long-term records enable establishment of databases, which would illustrate bird-pathogen relationships in natural conditions. These data would focus on hosts, their migration, population age, behavior, and so forth. Host-pathogen interactions should be described by using data such as antibody prevalence in different age classes, frequency of virus isolation, and characterization of the strains involved. Complementary laboratory and field experiments within a controlled environment might also provide relevant information. All these investigations should gradually make it possible to gather valuable baseline data to test specific hypotheses and gain new insights in bird-pathogen relationships in Mediterranean wetlands. Prolonged extracorporeal membrane oxygenation therapy for severe acute respiratory distress syndrome in a child affected by rituximab-resistant autoimmune hemolytic anemia: a case report INTRODUCTION: Autoimmune hemolytic anemia in children younger than 2 years of age is usually characterized by a severe course, with a mortality rate of approximately 10%. The prolonged immunosuppression following specific treatment may be associated with a high risk of developing severe infections. Recently, the use of monoclonal antibodies (rituximab) has allowed sustained remissions to be obtained in the majority of pediatric patients with refractory autoimmune hemolytic anemia. CASE PRESENTATION: We describe the case of an 8-month-old Caucasian girl affected by a severe form of autoimmune hemolytic anemia, which required continuous steroid treatment for 16 months. Thereafter, she received 4 weekly doses of rituximab (375 mg/m(2)/dose) associated with steroid therapy, which was then tapered over the subsequent 2 weeks. One month after the last dose of rrituximab, she presented with recurrence of severe hemolysis and received two more doses of rrituximab. The patient remained in clinical remission for 7 months, before presenting with a further relapse. An alternative heavy immunosuppressive therapy was administered combining cyclophosphamide 10 mg/kg/day for 10 days with methylprednisolone 40 mg/kg/day for 5 days, which was then tapered down over 3 weeks. While still on steroid therapy, the patient developed an interstitial pneumonia with Acute Respiratory Distress Syndrome, which required immediate admission to the intensive care unit where extracorporeal membrane oxygenation therapy was administered continuously for 37 days. At 16-month follow-up, the patient is alive and in good clinical condition, with no organ dysfunction, free from any immunosuppressive treatment and with a normal Hb level. CONCLUSIONS: This case shows that aggressive combined immunosuppressive therapy may lead to a sustained complete remission in children with refractory autoimmune hemolytic anemia. However, the severe life-threatening complication presented by our patient indicates that strict clinical monitoring must be vigilantly performed, that antimicrobial prophylaxis should always be considered and that experienced medical and nursing staff must be available, to deliver highly specialized supportive salvage therapies, if necessary, during intensive care monitoring. Autoimmune hemolytic anemia (AIHA) in children is usually characterized by a severe course with a mortality rate of approximately 10% [1] . The required prolonged immunosuppressive therapy often leads to steroid dependence [2] . The administration of non-steroidal immunosuppressive drugs such as cyclosporine A, cyclophosphamide and azathioprine, has been used in the past [1] [2] [3] [4] . Nowadays, the use of monoclonal antibodies such as rituximab, has given promising results for pediatric refractory AIHA [5] [6] [7] , with sustained remissions in the majority of patients. Nevertheless, potentially life-threatening infections are known to occur with rituximab [7] . In the event of rituximab failure, there is no general consensus or guidelines available indicating precisely how to manage resistant forms of AIHA. Heavy immunosuppression consisting of the combined use of cyclophosphamide and high-dose steroids may be considered [8, 9] .
We report the case of an 8-month-old Caucasian girl referred to us for observation due to intense pallor, jaundice, lethargy and fever. Serological evaluations revealed severe anemia (Hb = 2.8g/dL) with a strongly positive direct antiglobulin test and high-titer warm IgG autoantibody. AIHA was diagnosed and steroid therapy with intravenous methylprednisolone at 2mg/kg/day was administered for 5 days (Figure 1 ). An adequate Hb increase was obtained and the child was discharged after 10 days with oral prednisone at 2mg/kg/day. During the subsequent months, several attempts were made to taper off the prednisone, but the patient had developed steroid dependence. Considering this dependence on high steroid doses, a therapeutic course with four doses of rituximab was performed (375mg/m 2 /dose) at weekly intervals ( Figure 1 ). Before rituximab infusion, serum immunoglobulin levels were normal and subpopulation lymphocyte counts were within the normal range. The treatment with rituximab was well tolerated and the patient received intravenous substitutive therapy with commercially available immunoglobulin preparations (400mg/kg, every 3 weeks for 6 months). One month after the end of the first course of rituximab, while still receiving low-dose steroids, the patient presented with a clinical relapse of AIHA, so prednisone was increased to 2mg/kg/day and two further rituximab infusions were performed ( Figure 1 ). After these infusions, B lymphocytes became undetectable and the count returned to normal values 8 months after treatment. The patient remained in clinical remission and free from immunosuppressive drugs for 7 months, before presenting with a further relapse. A more intensive treatment was performed ( Figure 1 ) with cyclophosphamide 10mg/kg/ day for 10 days and methylprednisolone 40mg/kg/day for 5 days, which was tapered over 20 days. Hb level increased and the patient was discharged 10 days later in good clinical condition, without any antifungal or antiviral prophylaxis.
Two weeks later, the child was referred to the Emergency Room for respiratory failure, persistent fever and abdominal pain. Laboratory examination showed an Hb level of 12.8g/dL, total leukocyte count (WBC) of 710/µL, absolute neutrophil count (ANC) of 90/µL, a platelet count (PLT) of 339,000/µL, and low levels of immunoglobulin (IgG = 360mg/dL, IgA = 10mg/dL, IgM = 33mg/ dL). Chest X-ray and CT scan revealed an interstitial pneumonia ( Figure 2 ). Therapy with amikacin, ceftazidime, G-CSF and voriconazole was started. Within a few hours, her clinical condition deteriorated and the patient developed Acute Respiratory Distress Syndrome (ARDS), which required immediate admission to the intensive care unit (ICU). Acceptable gas exchange was initially maintained by non-invasive continuous positive airway pressure ( Figure 3 ). Serologic tests showed a level of Aspergillus galactomannan antigen of 0.8. All tested virus and microbial antigens were negative. On day 4, concomitantly with an elevation of WBC from 400 to 10,400/µL (ANC = 4900/µL), respiratory conditions precipitated and endotracheal intubation and mechanical ventilation were started ( Figure 3) . A protective ventilatory strategy with tidal volume of 6mL/kg and positive end expiratory pressure (PEEP) of 10cmH 2 O was instituted.
In the following days, gas exchange deteriorated and PEEP levels rose to 17cmH 2 O. Recruitment manoeuvres, prone positioning, and high doses of inhaled nitric oxide (NOi) were necessary to maintain viable gas exchange. Endotracheal instillation of porcine surfactant and a trial with High Frequency Oscillation were ineffective. On day 10, owing to the refractory hypoxia, worsening hypercapnia, and chest X-ray evidence of a pneumomediastinum, the patient was placed on venous-venous extracorporeal membrane oxygenation (ECMO) (Figure 3) . A double lumen 15 french catheter (Maquet, Jostra Medizintechnik AG, Hirrlingen, Germany) was inserted into the right internal jugular vein. The ECMO circuit consisted of a polymethylpentene membrane oxygenator, permanent life support and a centrifugal Rotaflow pump (Maquet, Jostra Medizintechnik AG, Hirrlingen, Germany). ECMO was started with a blood flow of 0.8 to 0.9 L/minute and gas flow of 1 L/minute of 100% oxygen. Following the institution of ECMO, respiratory rate decreased from 45 to 10 breaths/minute, and it was possible to stop NOi.
After commencing caspofungin with voriconazole, WBC, ANC and C reactive protein (CRP) slowly decreased, while pulmonary function slightly improved. On day 19, a multidrug-resistant Pseudomonas aeruginosa was isolated from bronchoaspirate ( Figure 3 ). In spite of antibiotic reinforcement with levofloxacin, Pseudomonas aeruginosa antibiogram showed increased resistance to all antibiotics and to colimicine which was started on day 26. On day 28, a sudden increase in resistance on the return part of the circuit caused a massive thrombosis in the oxygenator. The entire circuit and the cannula were immediately changed and ECMO restarted within 2 hours. Following the development of pulmonary embolism, the gas exchange rapidly worsened and NOi had to be restarted. An echocardiographic assessment showed right ventricular dilatation, with paradoxical septal wall motion and pulmonary hypertension (systolic pressure 90mmHg). Prostacyclin and sildenafil improved the right heart function and effectively attenuated pulmonary hypertension. In the following days, WBC, ANC and CRP slowly decreased, while pulmonary function improved. Thirty days from ICU admission, ECMO was stopped, the patient rapidly restored her spontaneous ventilatory functions and she was extubated 10 days later. She was finally discharged from the ICU on day 44. At 16-month follow-up, the patient is alive and free from immunosuppressive drugs. At the time of last follow-up, Hb level was 13.5g/dL, reticulocyte count was 11 × 10 9 /L, with total bilirubin, lactic dehydrogenase and haptoglobin all within the normal ranges.
AIHA in children younger than 2 years is in some cases characterized by a resistance to corticosteroids or dependence on high steroid doses and subsequent development of severe side effects [2] . Splenectomy or immunomodulating agents have frequently been used, but there is no consistent demonstration of their efficacy in controlling hemolysis [3] . Immunosuppressive drugs such as azathioprine, cyclosporine A, or cyclophosphamide, alone or in combination reduce steroid dependence and sometimes control hemolysis [1] [2] [3] [4] . Clinical experience with monoclonal antibodies appears encouraging. In particular, rituximab is increasingly being used off-label, for difficult-to-treat auto-immune diseases and presents the advantage of inducing a selective B-cell depletion, sparing cellular immunity mediated by T cells and natural killer cells. Even though prospective controlled studies are not currently available, the efficacy of rituximab has been shown in pediatric studies. Quartier et al. [5] treated five pediatric refractory AIHA patients, who achieved a complete remission within 15 to 22 months after rituximab therapy. These results were confirmed by Zecca et al. [6] in a group of 15 children treated with rituximab. Four other children were treated by Motto et al. [7] , with the achievement of complete remission. Nevertheless, the prolonged impairment of antibody production leads to an increased risk of viral and bacterial infections. For this reason, monthly intravenous immunoglobulin infusions are recommended for a minimum of 6 months following completion of therapy and prophylaxis for P. jirovecii pneumonia is also suggested [7] .
The patient described in our report received four rituximab infusions in an off-label setting, followed by two additional doses over 6 months. Clinical remission was achieved for 7 months after which it was possible to interrupt steroid treatment. The pattern of immune reconstitution after rituximab therapy revealed persistently low immunoglobulin levels, partially corrected by the substitutive therapy. Immunoglobulin levels reached their normal range in 18 months and the lymphocyte subpopulations returned to normal range in 16 months. It is, nevertheless, difficult to quantify the real role of rituximab in this heavy immunosuppression, since a combined therapy with high doses of methylprednisolone and cyclophosphamide was subsequently started. Even though our patient did not present any early side effects related to the rituximab infusions, a prolonged follow-up should be carried out to monitor and prevent long-term side effects of rituximab, which are still unknown.
When our patient relapsed, an alternative treatment was required, since therapies with steroids, rituximab and intravenous immunoglobulins proved to be ineffective. The role of splenectomy in refractory AIHA is still controversial [1] [2] [3] [4] . Although effective vaccinations are available, this surgical treatment should be avoided in children younger than 6 years of age, due to the risk of developing severe bacterial infections. According to local policy, drug-based immunosuppressive therapy is to be preferred. We therefore decided to adopt a combined therapy approach, with high doses of methylprednisolone (40mg/kg/day for 5 consecutive days), which was then tapered down over 20 days, and cyclophosphamide (10mg/kg/day for 10 consecutive days). This approach appeared to be feasible and encouraging, since we had previously successfully treated two pediatric cases of refractory AIHA with an identical approach [8, 9] . The administration of methylprednisolone and cyclophosphamide increased the already significant immunodepression which had resulted from prior therapies and further contributed to the severity of the infectious complication presented by our patient that required ECMO therapy. While in the ICU, the patient underwent various ventilatory treatments, some of which are not considered conventional. Modern ventilatory strategy in ARDS aims to provide viable gas exchange with high oxygen concentration and PEEP, while minimizing the injurious effects of mechanical ventilation by using low tidal volume ventilation (6mL/kg) [10] . Although other techniques such as the prone position, NOi and recruitment manoeuvres are effective in improving gas exchange, they did not prove effective in terms of survival [10] . Nevertheless, before ECMO, the only means of providing minimal acceptable oxygenation was to use both NOi and the prone position. Despite using low tidal volumes, a respiratory rate of up to 45 breaths/minute was necessary to obtain acceptable CO 2 levels, and the occurrence of pneumomediastinum demonstrated that we were unable to provide an effective protective ventilatory strategy. Thus, ECMO was the only real means of providing such a strategy, while allowing adequate gas exchange.
Refractory AIHA in pediatric patients is a challenging disease that forces us to weigh up the risks and benefits of heavy and prolonged immunosuppressive therapies that can reduce or even eradicate the hemolysis, despite the risk of infectious complications. For this reason, we feel that prolonged viral and fungal prophylaxis therapy should always be considered, during and after the immunosuppressive therapy. Furthermore, strict clinical monitoring should be carried out, even when no evident symptoms are present. In our patient, we did not administer any prophylaxis and clinical monitoring was probably delayed for too long after discharge. Resolution of the infectious complication was possible thanks to an advanced intensive care assistance, which consisted of ECMO and the management of its related complications.
This case study shows that rituximab-resistant AIHA in young children represents a significant challenge, requiring aggressive immunosuppressive therapy, which may potentially cause severe life-threatening complications. Nowadays, it is not clear which is the best immunosuppressive agent to be administered in the event of rituximab failure. We found that the combination of methylprednisolone and cyclophosphamide could be a valid alternative, based on previous experience. Nevertheless, a universal therapeutic flow-chart is still lacking and should be defined, which considers new therapeutic strategies such as alemtuzumab [11] or hematopoietic stem cell transplantation [12] . What is clear, however, in the case of heavy immunosuppressive therapy, is the importance of strict patient monitoring during and after immunosuppressive therapy and an antimicrobial prophylaxis, particularly for fungal agents and P. jirovecii.
AIHA, autoimmune hemolytic anemia; ANC, absolute neutrophil count; CT, computed tomography; WBC, leukocyte count; PLT, platelet count; ARDS, acute respiratory distress syndrome; ICU, intensive care unit; PEEP, positive end expiratory pressure; NOi, inhaled nitric oxide; ECMO, extracorporeal membrane oxygenation; CRP, C reactive protein. Regulated Ire1-dependent decay of messenger RNAs in mammalian cells Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box–binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1. The ER is responsible for folding and processing proteins entering the secretory pathway and uses a variety of mechanisms to adjust its capacity in response to changes in the folding burden. This collection of mechanisms, termed the unfolded protein response (UPR; Ron and Walter, 2007) , is activated by stress caused by environmental stimuli and diseases such as viral infection (Tardif et al., 2004; Bechill et al., 2008) and multiple myeloma (Carrasco et al., 2007) . In metazoans, components of the UPR are essential for developmental processes requiring high levels of secretion such as the differentiation of plasma cells (Gass et al., 2002; Iwakoshi et al., 2003) and pancreatic  cells (Harding et al., 2001; Zhang et al., 2002) . The UPR restores ER function both by increasing its capacity and decreasing the load of new proteins through transcriptional induction of secretory pathway components and general translational attenuation. One of the key players in the UPR is Ire1 (inositolrequiring enzyme 1), a conserved transmembrane protein with a luminal domain that senses protein misfolding in the ER.
The resulting oligomerization of Ire1 leads to activation of its cytoplasmic kinase and endoribonuclease domain. The main function of the kinase appears to be activation of the nuclease, which requires binding of ATP or ADP in the active site of the kinase (Papa et al., 2003) . The nuclease in turn cleaves two specific sites in the mRNA encoding XBP-1 (X-box-binding protein 1), a conserved UPR transcription factor, which leads to XBP-1 activation and translation through removal of a regulatory intron.
We have recently shown that Ire1 in Drosophila melanogaster cells independently mediates the cleavage and degradation of mRNAs encoding proteins that traverse the secretory pathway (Hollien and Weissman, 2006) . This new branch of the UPR, which we call regulated Ire1-dependent decay (RIDD), has the potential to selectively relieve the burden on the ER while clearing the translation and translocation machinery for the subsequent influx of new proteins induced by the UPR. Studies on the regulation of specific messages suggest that the RIDD pathway also operates in mammalian cells (Tirasophon et al., 2000; Iwawaki et al., 2001; Oikawa et al., 2007; Iqbal M aintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositolrequiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1. ments of RIDD. By expressing wild-type and mutant variants of Ire1-, we find that the nuclease activity of Ire1 is required for both splicing and RIDD. However, these two outputs can be differentially triggered, revealing an unexpected complexity in Ire1 activation.
To determine whether the RIDD pathway functions in mammals as well as in Drosophila, we used a mouse embryonic fibroblast (MEF) cell line established by Lee et al. (2002) in which the Ire1- gene has been disrupted. We stably introduced human Ire1- (hIre1) into these cells using flippasemediated, site-directed recombination , which allowed us to insert the wild-type and hIre1 variants discussed in the next sections into the same sites within the genome. In response to various forms of chemically induced ER stress, the reconstituted cells (referred to here as hIre1 R ) but not the Ire1 knockout (Ire1 / ) cells induced the splicing of XBP-1 et al., 2008; Lipson et al., 2008) . Mammals express two isoforms of Ire1: Ire1- is expressed ubiquitously, and Ire1- is expressed in intestinal epithelial cells. Overexpression of Ire1- leads to cleavage of its own message in COS-1 cells (Tirasophon et al., 2000) and reduced levels of the message encoding CD59 (complement defense 59) in HeLa cells (Oikawa et al., 2007) . Ire1- also appears to mediate the degradation of insulin transcripts in pancreatic  cells under chronic high glucose conditions, perhaps promoting cell survival during extreme chronic stress (Lipson et al., 2008) . Ire1- appears to have alternative targets as well, mediating cleavage of the 28-S ribosomal RNA (Iwawaki et al., 2001) and the mRNA encoding microsomal triglyceride transfer protein, an ER chaperone important for the assembly of lipid transport vesicles (Iqbal et al., 2008) . Together, these examples of Ire1 function in mRNA decay may explain observations that Ire1 in metazoans has a broader range of physiological outputs than XBP-1 splicing (Zhang et al., 2005) .
In this study, we take advantage of mouse fibroblasts lacking Ire1 activity, both to confirm that RIDD is conserved in mammalian cells and to investigate the functional require- and Fig. S1 ). Although 120 mRNAs fell into this category in the array data, the magnitudes of the changes in expression were generally small (many were twofold or less) compared with those seen in Drosophila cells, where many RNAs were downregulated by 5-10-fold. Despite the relatively small changes for individual messages, the effect of down-regulating mRNAs at the ER surface may profoundly impact the folding burden of the ER. For example, the redistribution of certain nascent proteins from the ER to the cytosol during ER stress significantly impacts cell survival, although the effect on translocation is similar in magnitude to RIDD (Kang et al., 2006) .
To limit false positives, we applied a series of strict criteria for identifying the most likely RIDD targets (see Materials and methods); the results are shown in Table I . We confirmed the regulation of several of these targets by reverse transcription followed by quantitative real-time PCR (qPCR; Fig. 1 and Fig. S1 ). To determine whether the down-regulation was a general response to ER stress or was restricted to DTT, we also treated cells with tunicamycin (Tm), which inhibits N-linked glycosylation or thapsigargin, which disrupts calcium homeostasis. All candidate RIDD (see Fig. 3 B). Both cell lines strongly induced immunoglobinbinding protein (BiP), an abundant ER chaperone, in response to ER stress ( Fig. 1) , which is in agreement with previous observations that BiP induction is Ire1 independent in mouse fibroblasts (Lee et al., 2002) . As expected, induction of ERdj4, a transcriptional target of the Ire1/XBP-1 branch of the UPR , was stronger in the hIre1 R cells compared with Ire1 / cells, especially in response to DTT, a reducing agent which disrupts disulfide bond formation in the ER (Fig. 1 A) .
To investigate the RIDD pathway, we took an unbiased, microarray-based approach. We induced ER stress in the Ire1 / and hIre1 R cells with DTT, purified total RNA from treated and untreated cells, amplified and labeled these samples, and hybridized them to whole-genome MEEBOChip arrays. As expected, treatment of our hIre1 R cells with DTT led to the induction of classical UPR targets to levels comparable with that seen by others in wild-type cells ( Fig. S1 ; Lee et al., 2003) .
Consistent with the RIDD pathway functioning in these cells, we observed that several RNAs were down-regulated in response to ER stress in the hIre1 R but not the Ire1 / cells ( Fig. 1 genome encode membrane or ECM proteins, as do 6 of the 7 confirmed targets that displayed increased decay rates. The seventh confirmed target, Blos1, encodes a protein that does not appear to contain a membrane-spanning domain itself but is part of a complex detected in both cytosolic and peripheral membrane fractions (Starcevic and Dell'Angelica, 2004) . We consistently observed partial splicing of XBP-1 in our hIre1 R cells in the absence of ER stress, which is likely caused by overexpression of hIre1 in these cells. To confirm that the mRNA decay we observed was not an artificial product of additional stress or Ire1 activity caused by overexpression, we measured the mRNA decay rates of two confirmed RIDD targets in NIH-3T3 cells (Fig. 2) . As in our reconstituted cells, NIH-3T3 cells displayed increased decay rates and lower abundance for RIDD targets in the presence of DTT. Collectively, our data indicate that RIDD is a general, conserved pathway associated with folding stress in the ER.
Because the parent Ire1 / cell line lacks Ire1 activity, we could probe specific functions of Ire1 by inserting variants of hIre1 targets tested were down-regulated in response to these three forms of ER stress in an Ire1-dependent manner (Fig. 1 , A-C).
Several observations indicate that the RIDD pathway, if not the specific targets, is conserved in mammalian cells and Drosophila. First, the down-regulation of mouse target mRNAs was independent of XBP-1 (Fig. 1 D) . Using RNAi to deplete Ire1 from cells lacking a functional XBP-1 , we found that Ire1-dependent down-regulation of several target mRNAs occurred in the absence of XBP-1. As a control, in cells lacking XBP-1, ERdj4 was induced only to the level seen in Ire1 / cells, and this induction was insensitive to Ire1 depletion ( Fig. 1 D) . Second, the down-regulation of many mouse targets was achieved through an increase in their decay rates. We inhibited transcription with actinomycin D and monitored mRNA levels over time in the absence and presence of ER stress ( Fig. 2 and Fig. S2 ). For 7 of the 11 targets tested, the decreases in mRNA abundance were dependent not on transcription but on increased rates of mRNA degradation. Third, as in Drosophila cells, the set of targets in mouse cells is highly enriched for mRNAs encoding membrane proteins. 17 of the 22 targets in Table I that were assigned localization annotations in the mouse little effect on splicing or RIDD targets. However, cells treated with both 1NM-PP1 and ER stress degraded RIDD targets to similar levels as those expressing the wild-type Ire1 (Fig. 4 , D and E). As a further control, we confirmed the XBP-1 independence of RIDD in these cells. XBP-1 is transcriptionally induced in our cells in a largely Ire1-independent manner and therefore does not occur to a significant extent in cells treated with 1NM-PP1 alone. Therefore, we depleted XBP-1 from cells expressing Ire1-I642G using RNAi, and although this blocked containing point mutations into these cells in an isogenic manner. Using this approach, we expressed an hIre1 variant containing a single point mutation (D847A) that has been previously shown to compromise the nuclease activity of hIre1 while leaving the kinase activity intact (Tirasophon et al., 2000) . For these experiments, we used C-terminally Flag-tagged versions of both the wild-type and mutant Ire1 and confirmed by Western blotting that the hIre-D847A variant was expressed at similar levels to the wild type (Fig. 3 A) . Consistent with a lack of nuclease activity, cells expressing hIre1-D847A did not support XBP-1 splicing in the absence or presence of ER stress (Fig. 3 B) and induced ERdj4 only to the level seen in Ire1 / cells (Fig. 3, C and D) . All cell lines induced BiP by similar amounts, indicating that they are experiencing a similar level of ER stress. However, unlike the Ire1 R cells, cells expressing hIre1-D847A displayed no change in the abundance of RIDD targets upon induction of ER stress (Fig. 3, C and D) . These results indicate that the nuclease activity of Ire1 is required for degradation of RIDD targets, which is consistent with a mechanism in which Ire1 directly cleaves these RNAs in response to ER stress.
Recently, it has been shown that for yeast and human Ire1, the cytoplasmic kinase activity can be bypassed using a small molecule that binds to the enlarged ATP pocket of an engineered Ire1 variant (Papa et al., 2003; Lin et al., 2007) . This variant, I642G in hIre1, has very little activity in the absence of the ATP analogue 1NM-PP1 (4-amino-1-tert-butyl-3-[1'-naphthyl methyl]pyrazolo[3,4-d] pyrimidine). Binding allows activation of the nuclease while simultaneously inhibiting the kinase activity of Ire1-I642G. In HEK293 cells (Lin et al., 2007) and MEF cells (Han et al., 2008) , hIre1-I642G splices XBP-1 and induces the UPR upon binding 1NM-PP1 alone, even in the absence of ER stress. To test whether activation of Ire1 is sufficient for degradation of RIDD substrates, we introduced hIre1-I642G into Ire1 / cells and tested its activity in the absence and presence of 1NM-PP1 and ER stress (Fig. 4) . Treatment of mouse cells expressing hIre1-I642G with 1NM-PP1 was sufficient to induce splicing of XBP-1 (Fig. 4 A) and transcriptional induction of the Ire1/XBP-1 target ERdj4 (Fig. 4 B) . This Ire1 activity was not caused by a general induction of ER stress, as cells lacking Ire1 or expressing the wild-type hIre1 were insensitive to 1NM-PP1 (Fig. 4) , although the drug did suppress the Ire1-independent induction of ERdj4 by DTT in all strains through an unknown mechanism (Fig. 4 B) . The 1NM-PP1induced activation of hIre1-I642G was not caused by increased levels of the protein, as assayed by Western blotting (Fig. 4 C) . Thus, our data are consistent with a 1NM-PP1-induced conformational change in the Ire1 variant itself, as proposed previously (Papa et al., 2003) .
In contrast to XBP-1 splicing and ERdj4 induction, no degradation of RIDD substrates was observed with 1NM-PP1 alone, indicating that XBP-1 splicing and RIDD are separable functions of Ire1 and have distinct requirements for activation (Fig. 4, D and E) . Treatment of cells expressing Ire1-I642G with DTT or Tm alone (in the absence of 1NM-PP1) had very BiP, an ER chaperone which binds Ire1 and is titrated off by misfolded proteins during ER stress. A second possibility is that other factors activated by ER stress are necessary for RIDD. Although new transcription of such a factor is not required (Fig. 2) , there may be proteins recruited or activated by ER stress that are important for mRNA decay but not for splicing. Finally, the complement of mRNAs available for targeting to this decay pathway may change upon subjecting cells to ER stress. Certain signal sequences are sensitive to the ER folding environment and influence how associated ribosomes partition between the ER and cytoplasm (Kang et al., 2006) ; thus, a substantial change in the pool of mRNAs associated with the ER membrane under stress conditions could affect the RIDD pathway.
The fact that the two outputs of Ire1's nuclease activity, RIDD and XBP-1 splicing, can be differentially activated reveals an unanticipated complexity in the UPR. Growing evidence suggests that various sensors associated with the UPR are activated under different conditions, allowing for specific responses to different forms of ER stress in different cell types (Gass et al., 2002; DuRose et al., 2006) . This customization of responses may extend to activation within Ire1 itself. Activation of XBP-1 leads to a protective remodeling and expansion of the secretory pathway, whereas RIDD reduces the load of incoming proteins. Under certain physiological situations, e.g., during plasma cell development, activating the XBP-1-dependent remodeling pathway without inducing RIDD may be more beneficial; thus, cells may induce an active state of Ire1 that is similar to the 1NM-PP1-mediated activation described in this study. In contrast, conditions such as viral infection may call for a more destructive response that limits the load of incoming proteins, which is analogous to the effects of reduced translation mediated by the PERK (PKR-like ER kinase) branch of the UPR (Harding et al., 2000) . Intriguingly, both hepatitis C virus and human cytomegalovirus induce Ire1 but appear to block downstream effects of XBP-1 (Tardif et al., 2004; Isler et al., 2005) . However, hepatitis C virus protein production is increased in the absence of Ire1 (Tardif et al., 2004) , suggesting that an alternate activity of Ire1 such as RIDD may be attenuating viral protein synthesis. It remains to be determined what role RIDD may play in viral infection and other physiological stress conditions, but the ability of this pathway to function in mammalian cells and the potential to decouple it from XBP-1 splicing could allow for a more specific and effective response to changing conditions within the ER.
Establishment of Ire1-expressing cell lines and RNAi Ire1- / MEFs were obtained from R. Kaufman (University of Michigan Medical Center, Ann Arbor, MI), and XBP1 / MEFs were obtained from L. Glimcher (Harvard School of Public Health, Boston, MA). All cells were maintained at 37°C and 5% CO 2 in DME supplemented with 10% fetal calf serum, Gln, and antibiotics. We generated Ire1- / MEFs containing induction of ERdj4 by DTT, RIDD remained intact when cells were treated with both 1NM-PP1 and DTT (Fig. 4 F) .
To determine whether the lack of RIDD in our 1NM-PP1induced cells is merely a threshold effect, we treated cells with higher concentrations of 1NM-PP1, such that no further XBP-1 splicing or ERdj4 activation could be achieved with 1NM-PP1 alone (Fig. 4, G and H) . The levels of XBP-1 splicing in cells expressing hIre1-I642G treated with 30 µM 1NM-PP1 were as high or higher than those seen in wild-type cells treated with low concentrations of DTT (Fig. 4 G) or with Tm ( Fig. 4 A) , where RIDD was clearly functioning. Note that although XBP-1 splicing appears insensitive to low concentrations of DTT in Fig. 4 G, we observed partial splicing shortly after the addition of DTT followed by rapid recovery before changes in gene expression could be reliably measured. For cells expressing wildtype hIre1, ERdj4 and RIDD appeared to be activated in parallel by low DTT concentrations, suggesting that RIDD is not limited to extreme ER stress (Fig. 4 H) . In contrast to splicing and activation of ERdj4, no change in mRNA abundance for RIDD targets was detected in cells expressing hIre1-I642G and treated with 1NM-PP1 alone, even at high concentrations, although the cells were still capable of inducing RIDD when treated with 1NM-PP1 and DTT together (Fig. 4 H) . In summary, although XBP-1 splicing and downstream transcriptional induction can be artificially induced with 1NM-PP1, RIDD cannot.
Our results indicate that in mammalian cells, RIDD requires an active Ire1 nuclease domain but does not, per se, depend on Ire1's kinase activity. ER stress-dependent degradation of RNAs by hIre1-I642G was seen at concentrations of 1NM-PP1 1.5-fold higher than those that inhibited the kinase activity of the protein in vitro (Papa et al., 2003) and sixfold higher than those that inhibited kinase activity in HEK293 cells (Lin et al., 2007) . These data, together with the observation that RIDD does not require new transcription, are consistent with a direct mechanism of mRNA target cleavage by Ire1, although we cannot rule out an alternative role for Ire1's nuclease activity and/or the involvement of other nucleases.
However, our data also indicate that activation of Ire1's nuclease is not sufficient for RIDD. This observation represents a mechanistic divergence between RIDD and Ire's well-established role in XBP-1 splicing, which is induced by 1NM-PP1 activation of hIre1-I642G. There are several potential explanations for this. It may be that Ire1 assumes a distinct conformation or oligomerization state when activated by 1NM-PP1 versus ER stress and that although the former is sufficient for splicing XBP-1, the latter is required for RIDD. For example, it was recently found that yeast Ire1 forms higher order oligomers that lead to higher levels of RNase activity (Aragon et al., 2009; Korennykh et al., 2009) . Such oligomers may form only in the presence of misfolded proteins or, conversely, in the absence of binding to relative mRNA abundance for ERdj4 and Blos1 (H) in various concentrations of 1NM-PP1 and/or DTT. Measurements are as described for A and B. (A-H) Cells were treated with 2 mM DTT, 3 µg/ml Tm, and/or 7 µM 1NM-PP1 for 5 h unless otherwise indicated. The means and SD for three to five independent experiments are shown.
proteins on NuPage Bis-Tris 4-12% acrylamide gels (Invitrogen), transferred them to nitrocellulose membranes, and probed blots using primary polyclonal anti-Flag antibodies (Sigma-Aldrich) at a 1:2,000 dilution followed by secondary peroxidase-conjugated anti-rabbit antibodies (Jackson Immuno-Research Laboratories) at a 1:5,000 dilution. As a loading control, we also probed blots with polyclonal anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; ProSci, Inc.). We visualized the immunoblots using a chemiluminescent assay (ECL; GE Healthcare).
Online supplemental material Fig. S1 shows the array data for hIre1 / and hIre1 R cell lines, a comparison with published data for UPR targets, and confirmation of RIDD target regulation measured by qPCR. Fig. S2 shows decay rate measurements for 11 RIDD candidates in the presence and absence of DTT. ferritin-like protein recombination target (FRT) sites for site-specific transgene expression by transfection with pFRTlacZeo (Invitrogen), and stable clonal integrants were selected with Zeocin . We then transfected the Ire1- / FRT cells with the pOG44 ferritin-like protein recombinase vector (Invitrogen) and FRT vectors containing hIre1 under the control of the human ER-1 promoter (Lin et al., 2007) . Quik-Change mutagenesis (Agilent Technologies) was used to make the point mutations in the hIre1 coding sequence. Multiple independent isogenic clones were analyzed for transgene mRNA and protein expression with identical findings. The ATP analogue 1NM-PP1 was a gift from C. Zhang and K. Shokat (University of California, San Francisco, San Francisco, CA; Bishop et al., 2000) .
To deplete cells of Ire1 or XBP-1 by RNAi, we transfected 3 × 10 4 cells with 150 ng total of a mixture of four different siRNAs (QIAGEN). After 48 h, we treated cells with DTT and/or 1NM-PP1, and purified RNA as described in the next section. We typically attained 90% knockdown as measured by qPCR.
We passaged and plated Ire1 / and hIre1 R cells into 150-cm 2 flasks for collection of microarray samples. After a 3-d growth (at 70% confluency), we either left cells untreated or induced ER stress by adding 2 mM DTT for 6 h. We then harvested the cells and purified total RNA using TRIZOL (Invitrogen). To generate labeled samples for array hybridization, we amplified 0.5 µg RNA in a single round using the amino allyl Message-Amp II aRNA kit (Applied Biosystems). As a reference for the array hybridizations, we also amplified and labeled a pool of RNA samples from NIH-3T3, Ire1 / , and hIre1 R cells (both untreated and DTT treated). In parallel, we synthesized unlabeled cDNA from 2 µg of total RNA for qPCR measurements to confirm the array data. We repeated the entire experiment for a total of three times.
We hybridized 5 µg each of a Cy5-labeled experimental sample and the Cy3-labeled reference pool to whole-genome mouse arrays at 65°C for 48 h. The arrays were produced in-house using the MEEBOChip platform. We extracted and processed image data using GenePix 6 (MDS Analytical Technologies), normalized each array to achieve a mean Cy5/ Cy3 ratio of 1.0, and removed spots containing low signal or poor signal uniformity. To simplify the analysis, we disregarded spots for which one or more samples did not pass these quality control measures and for which the SD of the three measurements was >30% of the mean. For the remaining spots (representing 25% of the total spots on the array), we calculated the log 2 ratio of DTT treated/untreated for each of the three replicates in the two cells lines. We then performed hierarchical clustering of the averaged data (Fig. S1 ).
To select robust RIDD candidates, we applied the following criteria to the 122 spots in clusters displaying Ire1-dependent down-regulation. First, we required the candidate RNA to be down-regulated by 1.5-fold (log 2 [DTT/untreated signal] ≤ 0.58) in at least two of the three replicates and in the mean of the three replicates. Second, to select those RNAs whose down-regulation was truly Ire1 dependent, we required that the targets be down-regulated 1.5-fold more in the hIre1 R cells compared with the Ire1 / cells and display no more than a 25% decrease in signal in response to ER stress in the Ire1 / cells. Lastly, to rule out artifacts caused by higher expression of the candidate RNAs in the hIre1 R cells in the absence of ER stress, we also required that the mean signal intensity in the presence of ER stress was lower (by 15%) in the hIre1 R cells compared with the Ire1 / cells. 26 RIDD candidates fit these criteria; these are listed in Table I. qPCR and XBP-1-splicing assays We purified RNA samples for all experiments using TRIZOL and synthesized cDNA from total RNA samples using Superscript II (Invitrogen). We then performed qPCR measurements using the primers shown in Table S1 . We measured each sample in triplicate using the Opticon (Bio-Rad Laboratories) or Realplex (Eppendorf) qPCR machines and normalized them using the signal from Rpl19, which did not change significantly relative to total RNA input concentration in any of the treatments used. We used mock cDNA samples containing no reverse transcription to ensure that the qPCR signals arose from cDNA and not from contaminating genomic DNA or other sources. To quantify the amount of XBP-1 splicing in each experiment, we amplified cDNA using primers surrounding the splice site (Table S1 ) and ran products on 2% agarose gels.
We washed cells in PBS and lysed them in radioimmunoprecipitation assay buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% Nadeoxycholate, and 0.1% SDS) with added protease inhibitors. We resolved Empirical Relationship between Intra-Purine and Intra-Pyrimidine Differences in Conserved Gene Sequences DNA sequences seen in the normal character-based representation appear to have a formidable mixing of the four nucleotides without any apparent order. Nucleotide frequencies and distributions in the sequences have been studied extensively, since the simple rule given by Chargaff almost a century ago that equates the total number of purines to the pyrimidines in a duplex DNA sequence. While it is difficult to trace any relationship between the bases from studies in the character representation of a DNA sequence, graphical representations may provide a clue. These novel representations of DNA sequences have been useful in providing an overview of base distribution and composition of the sequences and providing insights into many hidden structures. We report here our observation based on a graphical representation that the intra-purine and intra-pyrimidine differences in sequences of conserved genes generally follow a quadratic distribution relationship and show that this may have arisen from mutations in the sequences over evolutionary time scales. From this hitherto undescribed relationship for the gene sequences considered in this report we hypothesize that such relationships may be characteristic of these sequences and therefore could become a barrier to large scale sequence alterations that override such characteristics, perhaps through some monitoring process inbuilt in the DNA sequences. Such relationship also raises the possibility of intron sequences playing an important role in maintaining the characteristics and could be indicative of possible intron-late phenomena. The apparent lack of pattern of composition and distribution of bases in DNA sequences have been one of the enduring problems of molecular biology. Chargaff's rule provided a clear relationship between the numbers of guanines and cytosines, and between adenines and thymines in duplex DNA sequences, understood well by the Watson-Crick model. However, no clear relationship has been found as yet between the occurrences of these four bases in an individual strand of a gene sequence, although much work have been done on understanding nucleotide frequencies and base distributions in DNA sequences. To cite a few examples, Sueoka [1] proposed a unitary theory based on genetic and evolutionary considerations which attempted to account for the main characteristics of the distribution of DNA base composition in nature. The author considered the GC content of DNA sequences of organisms and mutations of GC base pairs to AT base pairs and vice versa over many generations from which a distribution function of the GC content of DNA molecules at equilibrium was obtained based on assumptions of rather uniform mutation and selection pressures affecting base pair conversions.
Goldman and Yang [2] proposed a codon-based model for the evolution of protein-coding DNA sequences using a Markov process to describe substitutions between codons. They used codon level information to model synonymous and asynonymous nucleotide substitution applicable to homologous sequences with no insertion/deletion gaps or with gaps removed. A 61661 matrix of codon substitution rates (excluding the three stop codons) is used, assuming that mutations occur at the three codon positions independently and only single-nucleotide substitutions are permitted to occur at any instant. Using several constraints and refinements on the nucleotide substitution rates, the transition/ transversion bias and amino acid differences, they show that their codon based model gives better phylogenies than simple nucleotide substitution model. However, the possible patterns arising from this model is very large and computationally very slow requiring Monte Carlo simulations, maximum likelihood methods and other approximations to arrive at quantitative results. While the model is useful for pairwise distance measures and for phylogenies, a relationship defining base composition in a DNA sequence is not clearly realised.
In a recent paper, Qi Ding et al [3] formulated an approach to determine a linear regression model for DNA sequences. By regarding a DNA primary sequence as a random process in time and defining the nucleotides' random distribution functions in three ways based on chemical structures they proposed two methods to measure their similarities. Relating the random distribution functions by a linear regression equation enabled them to construct six new models to analyse the DNA sequences and quantify their similarities and dissimilarities. The optimal model can be chosen based on the amount of information contained or lost in the process.
Several other studies have also focussed on nucleotide asymmetries in DNA sequences to gain an insight into correlations, if any, in base composition and distribution. Arnold et al [4] used tetranucleotide frequencies in a third order Markov chain to predict the frequencies of longer oligonucleotides in the yeast genome and observed that the oligonucleotide frequencies depended strongly on base composition. Freeman [5] considered several prokaryotic DNA genomic sequences and found, from base composition asymmetries like purine excess over pyrimidines and coding strand excess, that the global minima of the purine excesses correlated with the origin of replication, and the maxima with the likely terminus for prokaryotic genomes and that such a prominent correlation between the purine excess and replication direction probably leads to excess pyrimidine accumulation in the sense strand and accordingly should increase the less mutationally vulnerable purine content of the coding strand. Prusak and Grzybowski [6] observed that there is a strong non-random distribution of nucleotides in the cytochrome b sequence in several species with the highest differences at the third codon position which is also the codon position of the strongest compositional bias; some species like quail, frog, python and elk appeared to prefer C over A in the light DNA strand whereas species belonging to the artiodactyls contained fewer pyrimidines compared to other species investigated.
Such studies serve to also highlight the complexity of base arrangements in a DNA sequence and the difficulties in finding any inherent pattern or signal sequences in such arrangements, especially in a character based representation of the hundreds and thousands of nucleotides comprising the sequences. However, a graphical representation can be expected to provide visual clues to any inherent pattern or regularity as distinct from a purely random distribution of the bases which could be expected to generate a corresponding random distribution in the representative plot. Indeed, a purine-pyrimidine sequence map proposed by Peng et al [7] provided a visual rendition of the growth of purine and pyrimidine numbers in a DNA sequence, which was interpreted by the authors as indicative of an inherent fractal nature in the purine-pyrimidine structure of the DNA sequences. The chaos generator representation of Jeffreys [8] with its double-scoop depletion pattern reflected largely the abundance, or sparseness, of various dinucleotides and higher combinations, and also showed characteristic variations for different classes of organisms.
Graphical representations of DNA sequences assigning the four individual nucleotides to designated axes on a Cartesian grid provide a more direct visual indication of the progression of nucleotides in a sequence, akin, in some respects, to the Wilson cloud chamber for particle tracks. The first graphical representation of a DNA sequence was proposed by Hamori and Ruskin [9] based on a 3D Cartesian axis system that generated a visual map of the sequence of bases in a selected DNA sequence. A 2D representation was proposed by Gates [10] and rediscovered independently by Nandy [11] and Leong & Morgenthaler [12] , with different axes identifications for the four bases. These were followed subsequently by many different approaches to render a visual representation of the DNA sequences [13, 14] , and their applications have been found to be very useful in elucidating different characteristics of DNA sequences that are not easily accessible in other ways. For example, Gates [10] referred to large scale structures seen in the plots of some sequences; such structures were also reported by Nandy and Nandy [15] , and recently by Larionov et al [16] who reported, inter alia, the presence of gigantic palindromes in mouse and human chromosomal sequences. Liao et al [17] have shown that 2D graphical methods that convert a DNA sequence to a series of co-ordinates and therefrom construct distance matrices can be used for computation of molecular phylogeny without need for multiple alignments; Wang et al [18] constructed a 3D representation in which a DNA sequence could be denoted mathematically and a similarity matrix constructed for multiple sequences to derive a phylogenetic tree by virtue of the fuzzy theory. Lo et al [19] have shown using a 3D trajectory method that global views of DNA sequences can be obtained such that different types of DNA sequences can be easily distinguished and any local differences and similarities between two DNA sequences can also be easily observed.
Furthermore, numerical characterisation techniques based on graphical representations have enabled quantitative estimations of sequence similarities and dissimilarities [see review 14] . Basically there have been two approaches for numerical characterization, both of which use the graphical representation to map a DNA sequence into a set of numbers. One approach using geometrical mapping proposed by Raychaudhury and Nandy [20] have been found to be useful for several calculations based on the 2D graphical representation [14] , and extended recently to an abstract 20D modelling for protein sequences [21] , where individual sequences are indexed by numerical descriptors. The other approach is to use matrix methods by forming ratios of graph theoretic and Euclidean distances between nodes of the graphical plots, first formulated for DNA sequences in Randic et al [22] . Since invariants associated with matrix formulation are well-known, individual sequences can be indexed by one or more such invariants of various orders; it is expected that these would be sufficiently characteristic of the underlying sequences to enable unique characterization. This technique has been the most widely used method of choice for the researchers in this field who have defined different types of matrices to construct various invariants to describe the DNA sequences. However, the difficulties associated with computing various parameters for very large matrices that are natural for large sequences have restricted the numerical characterizations to leading eigenvalues and the like [14] .
In principle, however, many of the indices used to characterize numerically DNA representations are graph invariants that describe the distribution of nodes and/or node-node connections in these graphs. In the parlance of graph theory, many authors have referred to some of these indices as Topological Indices (TIs) and applications have been made not only to DNA sequences but also to proteins, viral surfaces, RNA secondary structures and small molecules [23, 24] . Consequently, the method is of more general application taking into consideration that the type of graph representations referred to above have been extended from DNA/ RNA to the study of other types of relevant biological sequences. In particular, González-Díaz et al. extended these representations to the study of protein sequences [25] and Mass Spectra outcomes of proteins and/or protein serum profiles in parasites [26] , toxicoproteomics and diagnosis of cancer patients [27, 28] . In any case, the various numerical parameters of DNA/RNA graph representations (TIs or otherwise) may be used not only to study sequence-sequence similarity but also to fit Quantitative Structure-Activity Relationship (QSAR) models. These QSAR connect structural information with the biological function of a molecular entity under study and may be used to predict unknown entries. Structure here refers not only to drug structure but also to DNA sequence, RNA sequence or secondary structure, and protein sequences or 3D structure [see review 28] .
Thus, the utility of graphical methods in revealing different types of hidden structures and similarities/dissimilarities in and between DNA and other biological sequences can be considered to be well demonstrated.
In this light, a perusal of the representative patterns of conserved gene sequences appears to indicate a possible relationship between the numbers of the various nucleotides in conserved gene sequences. Here we use the 2D graphical representation method to show that plots of the conserved gene sequences trace out apparently curved paths that are also visually similar across species for the same gene. The nature of these curves is seen to generally imply a so far undescribed quadratic relationship between intra-purine and intra-pyrimidine differences, whereas the null hypothesis would have indicated random directionless walks. With such empirical relationship between the two basic nucleotide differences, we propose a probable mutation path to explaining the relationships. We then hypothesize that the parameters of such relationships could be a property of the underlying gene sequences and speculate that extensive alterations of such genes by accretion or deletion of DNA fragments would be s only if the modified sequences subscribe to the same basic parameterised relation.
Here we use the random walk system envisaged in the Nandy plot [11] based on a 2D Cartesian grid where the four bases are assigned to the four cardinal directions: guanine (g) to the positive x-direction, thymine (t) to the negative y-direction, adenine (a) to the negative x-direction and cytosine (c) to the positive y-direction. The method to plot a DNA sequence is to start at the origin and take a step in the positive x-direction for a guanine base, in the negative x-direction for an adenine, positive y-direction for a cytosine and the negative y-direction for a thymine, and proceed likewise for each succeeding base in the sequence, starting each step from the end of the last one taken. This way a succession of bases in the original DNA sequences is represented by a succession of points in the 2D plot, the overall trace being a representation of the distribution of bases in the DNA sequence (see e.g., Fig. 1 human beta globin). The axes essentially represent the excess of guanine over adenine along the x-axis and the excess of cytosine over thymine along the y-axis; thus the plots are basically of instantaneous values of intra-pyrimidine, intra-purine differences as we proceed along the sequence. The end point of such a curve will be given by (N G -N A , N C -N T ), where by N A , N C , N G, N T we mean the total number of adenines, cytosines, guanines and thymines in the sequence being plotted. GC-rich sequences therefore plot mostly in the first quadrant, AT-rich sequences in the third quadrant on this axes system.
Once the co-ordinates are available, we use an Excel spreadsheet to plot the graph and apply the Add Trendline feature of the Excel software to fit the best polynomials for our analysis, with axes transformation where required to conform to the software's curve fitting engine. Fig. 1 shows a plot of the human beta globin gene complete cds generated using the above algorithm. We note that a DNA sequence that consists of a succession of short segments each having a complete mix of a,g,c,t with equal contributions of each of the bases within each of the segments would be expected to generate a dense cluster of points around the origin; a random distribution of the a,g,c,t along the sequence could be expected to generate a random walk. The human beta globin gene sequence complete cds inclusive of all introns and exons (Fig. 1) shows a distinct pattern where the bases appear to follow one another with some regularity, with the total extent of the representative plot arising from the non-equal composition of the bases in the sequence; other beta globin sequences produce similar plots implying that the human beta globin gene cds is not an arbitrary random sequence. Fig. 2 shows the close similarity of the shapes of the plots of three sequences of histone H4 genes of wheat, maize and chicken, demonstrating that these sequences are not random but have a close kinship in base distribution. A randomisation of the bases in the human beta globin gene [29] produces, on the other hand, a simple linear plot (Fig. 3) in the third quadrant of the axes system as can be intuitively expected for an unorganised mixture of the four bases along the sequence.
To further demonstrate that the base distribution in these gene sequences are non-random, and generally true for conserved gene sequences, we have generated graphical representations of several conserved genes, a selection of which are shown in Fig. 4 . Plots of several alpha globin genes are included here to show that there is shape similarity between the same genes from different species indicating that entire gene sequences inclusive of introns and exons have close similarities. This can also be seen in the plots of several histones, tubulins and heat shock proteins, of which some representative samples are given in Figs. 4 and 5.
With our observation that the sequences of the conserved genes, both intronless and with-introns, have close similarities, we can start to enquire whether these sequences have any discernible patterns. We notice that the general nature of the DNA walks on the 2D representation as per the Nandy prescription [11] shown in the above figures is directional and curvilinear. A simple 2 nd degree polynomial produces reasonable fits. A selection of the plots with the trendlines is shown in Fig. 6 and 7 corresponding to the sequences shown in the previous two figures. A list of the details of the fits and statistics are given in Table 1 .
While this nature of the base distributions is found across many different sequences of eukaryotic genes, it is also very much evident in the case of viral sequences like the H5N1 neuraminidase which are known to mutate very rapidly; other plots from a sample of over 600 sequences of the H5N1 neuraminidase show similar quadratic forms. Interestingly, plots of the wheat, maize and chicken histone H4 genes, which are also intronless genes, can also be fitted by polynomials of degree 2, similar to the case of the viral genes. Chicken beta globin gene with intron sequences that are quite different compared to the mammalian genes plot in the first quadrant, but it also can be fit by a quadratic polynomial. This is however not true of all gene sequences. The mouse beta globin gene sequence representation in the 2D framework gives a very poor fit for the quadratic function (R 2 = 0.06) but much better statistics with a cubic polynomial (R 2 = 0.53) ( Table 1) ; the human delta globin gene also shows very good statistics when fitted with a cubic polynomial (R 2 = 0.98). Some sequences where significantly large segments are at variance with the overall pattern too cannot be put into such slots. The rat myosin heavy chain gene sequence where the intron sequences have been hypothesized to have grown through accretion of large fragments [30] , for example, presents a highly compact form on the 2D plot [15] and cannot be fit by the simple polynomials we have used so far. However, sufficiently large numbers of sequences are seen to follow the apparent quadratic relationship with good statistics that it is of interest to try to understand the underlying pattern.
The equations that fit the curvilinear patterns of the base distributions with reasonable statistics are of the form
and
where a,b,c,d are parameters,
and n A , n C , n G , n T are the instantaneous values of the numbers of a, c, g, t present up to the particular position (x,y) on the sequence, starting the count from the beginning, i.e. the 59-end, of the sequence. These are our empirical equations connecting the intrapurine (x) and intra-pyrimidine (y) numbers obtained from the observations of the patterns on the 2D graphical plots. While the majority of the plots shown are well-represented by such polynomials of the second degree, the fits could be improved in some instances by fitting higher degree polynomials as mentioned earlier; e.g., in the case of the human beta-globin gene a polynomial of the third degree yields better statistics (R 2 = 0.94) than the second degree (R 2 = 0.81), for the human delta globin gene the statistics for the quadratic and cubic fits are R 2 = 0.87 and R 2 = 0.98, respectively. We, however, consider the second degree form for now for conformity without excessive loss of statistical significance.
The origin of such a relationship as in equations 1 and 2 could be traced to mutational changes in a sequence, where we restrict our analysis for the moment to transitional types of mutations since this is the dominant mode. Consider a mutation of a cytosine to thymine in one strand of a DNA in a GC-rich sequence. The opposite strand, calling it strand 2 for convenience, will initially have a bulge for the original paired guanine, and the event leads to following possibilities [31] : (a) the DNA repair mechanism reverse mutates the thymine to cytosine in strand 1, thus negating the effect of the original mutation; (b) the guanine in strand 2 is replaced by an adenine; and (c) a third possibility in case the damage repair coincides with replication, that the DNA is elongated by pairing the mutated thymine in strand 1 with a new adenine in strand 2 and addition of a cytosine in strand 1 to match the guanine in strand 2 left over after the original mutation, i.e. creation of a T-A pair and addition of a new C-G pair. Such an event would be quite rare, especially in coding regions since it will alter the reading frames unless the total change leads to addition of three base pairs; several intronless gene sequences indeed show very small contribution from the quadratic term compared to the interrupted genes, e.g. petunia hsp70G ( Table 1 ). The example of the mutation event of cytosine to thymine in strand 1 can be considered to change the intra-pyrimidine difference, n C-T , in strand 1 and trigger a change in the intrapurine difference, n G-A , in strand 2. The two changes can be related by
where the first term on the right relates to the probability of the G to A change in strand 2 and the second term to the probability of the third type of response, i.e. DNA elongation by addition of a C in strand 1; the (1) and (2) in equation 4 are strand identifiers and we have defined
We expect q,p since the probability of DNA elongation will be significantly lower than effecting only a replacement of the 
Using recursion and keeping up to first order terms in q/p, equation (7) reduces to
where by O3 we mean terms of higher orders (dropping the conventional form O(3) so as not to confuse with the strand number indicators). For many mutations over a long sequence, this takes the form
where a,b are redefined constants with b,a. This equation relates changes in the intra-pyrimidine numbers in strand 1 to intrapurine numbers in strand 2 where the n C-T and n G-A are as defined in equations 5 and 6 above. Now, Chargaff's rule states that
and since from Watson-Crick rule we know n C 1 ð Þ~n G 2 ð Þ and n C 2 ð Þ~n G 1 ð Þ, and similarly for A,T, then from the above we have
as a consequence of which,
Excluding some pathological cases or very short segments where one or the other type of nucleotide is conspicuous by its absence, e.g., as in poly-adenylation segments where only A's dominate and the others are absent, in a real gene sequence we can expect the second term within each of the square brackets above to be%1, implying that
which transforms Eq. (9) to 
or, dropping the strand number indicator since all quantities now refer to the same strand,
From similar considerations for the AT-rich sequences considering the mutations of adenine to guanine for example, we can obtain the equation
where n C-T and n G-A are as defined in Eqs. 5 and 6. Equations 16 and 17 are similar to equations 1 and 2 and conform to the general shapes of the sequence plots. This shows that the path traced out by the nucleotide sequence of a gene follows some pattern that can be ascribed to the accumulated effects of spot mutations over evolutionary time scales. The actual plots of the gene sequences in the 2D grid representation show reasonable conformity with the predicted curves with variations that could be ascribed in part to the higher order terms and other factors described below. It is to be noted that equations 16, 17 have been derived independent of any graphical representations or reference frames, and they are functions of the instantaneous values of the base counts only. The 2D Nandy representation here used happens to be a natural and convenient reference frame to plot the outcome of these equations. Equations 16-17 have been developed on the basis of transition type mutations only. Transverse mutations are much less frequent than the transition mutations and will affect these equations to a smaller extent. Consider a mutation of a cytosine to an adenine. This will result again in a bulge in the guanine in the opposite strand and the repair mechanism will either re-establish the cytosine or replace the guanine with a thymine, or replace the guanine in strand 2 with a thymine and add a CG base pair to the DNA thus elongating it. This will reduce the n C-T (1) by 1 unit and also reduce the n G-A (2) by 1 unit as well as have, as before, a small probability of adding an extra guanine in strand 2 by virtue of the additional base pair elongating the DNA. Thus we will have an equation of the type of Eq.4 again except that the coefficients in this instance will be much lower in value than for these parameters in the transition type mutation case. Again, we end up with equations of the form 16 to 17, this time for transverse type mutations, but with much smaller parameter values, so those equations can be considered to be quite general.
It is also possible that as a result of mutations more than one base pair are added during the repair and replication phases. Such actions can be expected to lead to further higher order terms being added to equations 16, 17 . That the third order terms seem to be required for good fits to some of the gene sequences has already been noted earlier. Such modifications to the intra-purine, intrapyrimidine relationship can result in deviations from the smooth distribution that first order approximation equations like Eq.16, 17 would imply. This could be the underlying reason for the deviations between the actual representative plots of the various sequences and their fitted curves observed in several graphs.
There is a further assumption that underlies the applicability of this analysis. We assume that in the case of the conserved gene sequences inclusive of introns and the coding segments, extensive restructuring through recombinations have not taken place. Indeed the first order formalism developed here implicitly assumes that all changes to a DNA sequence that have resulted in the form that we observe now have taken place through mutational changes only, and recombinations and transpositions have not had any major impact on the sequences.
A couple of points of interest arising from these equations may be mentioned here. First, homologous sequences of conserved genes of the intronless or with-introns varieties that have the same general shape on the 2D plots therefore have similar describing equations, and the coefficients of the variables have similar order of magnitude values. This would seem to indicate that these gene sequences have inherent characteristics that are expressed by the values of the parameters of the describing equations, whereby major deviations in base distributions that necessitate large departures from the characteristic values could be inimical to the functioning of the gene and thus would either be rejected, or would render the gene ineffective. A case could be made from the human alpha globin 1 pseudogene: although it shares a reasonable degree of homology with the functional alpha 1 globin gene and has the 3 exon-2 intron architecture of the globin family, its 2D plot shows a wide variation in base distribution from the alpha globin gene (Fig. 8) , and the coefficients of fitted polynomial also are quite different from the other mammalian alpha globin family fits (Table 1) . It may also be mentioned that transposons are found in many instances to insert segments into genes which are then excised out in successive replication cycles. If DNA sequences have inherent characteristics, which are encapsulated by the polynomial expression, and the inserted segments lead to incompatibility with such characteristics, then such excisions can be understood.
We note that reversion of mutated genes to ancestral forms is not totally unknown. A case in point is the recent study of reversion of mutant hothead gene in Arabidopsis thaliana to genes that existed in plants of two or more generations ago [32] . The authors have hypothesized that a template-directed restoration of ancestral DNA passed on in an RNA cache could underlie the mechanism of such reversion; the existence of such a mechanism that lies outside the DNA genome could lead to the high-frequency modification of DNA sequences in a template-directed manner, perhaps by the postulated RNA cache that could allow it to persist for several generations. While Lolle et al's RNA cache [32] would need to carry an exact duplicate of the ancestral sequence, in the case of gene sequences of the types considered here that may or may not contain intron segments and could be quite large, we could postulate existence of some as yet unknown mechanism for monitoring conformity with the overall intra-purine intra-pyrimidine base distribution pattern, as perhaps for long range correlations [7, 33] . In this connection it is also interesting to consider the possibility of the existence of some error correcting code in DNA sequences as speculated upon by Liebovitch et al [34] . They considered the DNA sequence as a digital code of four symbols and speculated that since the integrity of modern information encoding is secured by having error correcting codes built in, DNA sequences might also have such codes to allow repair enzymes to protect the fidelity of nonreplicating DNA and increase the accuracy of replication. In such a case if a linear block error correcting code is present in DNA then some bases would be a linear function of the other bases in each set of bases. Although the authors were unable to find any such simple code in the lac operon and cytochrome c gene they investigated, the suggestion remains an intriguing possibility nevertheless. Given that we are considering highly conserved genes whose functions are important to the survival of the organism, mechanisms such as these would provide a survival advantage and could be used under conditions that compromised the continued functioning of the organism or the requirements of the monitoring process.
Second, the basic patterns, and therefore the describing equations, have the same form irrespective of the intron content of the genes considered here. This could be indicative of the monitoring process hypothesized above also functioning irrespective of whether the sequences are intronless or intron-rich. Since mutations and other evolutionary changes have led to modifications in the coding sequences within the requirements of maintaining protein functionality, the introns may have a role to play such as maintaining the structure of base arrangements so that the restrictions implied by the equations 16, 17 can continue to apply. We could perhaps consider for support for this contention the globin genes where the beta globins separated from the alphas quite late, but the introns of the beta globin cluster are generally longer than the alpha globins while the coding sequences, though with differences, remain at almost the same length. If sustained, such a hypothesis could lend support to the intron late theory.
We note in passing that for the majority of the gene sequences of the vertebrates, the 2D plots display an overall shape that is concave going in the clockwise direction; correspondingly, the coefficient of the second degree variable is negative. This is seen even in the case of the chicken beta globin gene where the large intron component makes it GC-rich, the human beta globin gene which is AT-rich, as also the alpha globin genes of the horse, rhesus monkey and human which are all GC-rich. Plots of some viral sequences such as the H5N1 neuraminidase, on the other hand, have concavity in the opposite direction and the sign of the coefficient of the second degree variable is positive. Interestingly this is also seen in the case of the wheat histone H4 gene, which is claimed by some authors to have viral features [35] ; their origins from before the eukaryote split could also be a factor. Whether this is just chance coincidence or whether it is symptomatic of some deeper characteristic arising from base composition and distribution differences in prokaryotic and eukaryotic sequences remains an intriguing question.
In summary, our observations have shown that the 2D plots of intra-purine versus intra-pyrimidine differences in conserved gene sequences exhibit an apparent pattern in base distribution of the sequences that mimic the behaviour essentially of a polynomial of degree 2, and in some cases of degree 3. This is found over a wide cross section of sequences, from e.g., the members of the globin family, the histones, tubulins and heat shock proteins. Viral sequences such as the H5N1 neuraminidase, although known to mutate rapidly, also exhibit similar structure. We have seen that this may arise from the non-symmetrical mutation repair mechanism where e.g., a cytosine mutating to thymine in a GCrich sequence could lead to negating the mutation, to replacing the original paired guanine with adenine, or elongating the DNA by addition of a CG pair along with coupling the thymine with an adenine. Equivalent considerations apply to AT-rich sequences as well.
Since these observations appear true for intron-rich sequences also, the intron sequences may play a regulatory role in preserving sequence integrity as indicated by the intra-purine intra-pyrimidine relationships permitting greater flexibility in changes in coding sequences.
Not unexpectedly, we have seen that homologous genes have characteristic equations where the coefficients of the describing polynomials are quite close. Assuming that the DNA fidelity processes fit to this scheme of preferential arrangement of bases in conserved segments, our observations raise the possibility that DNA fragments, introduced into such segments by processes such as transpositions, that do not conform to the overall fit may be preferentially excised by the replication machinery to retain the integrity of the host sequence. If our observations here in gene sequences are extendible further to genomic sequences then it would imply that not all genetic modifications would be sustainable. Willingness of Hong Kong healthcare workers to accept pre-pandemic influenza vaccination at different WHO alert levels: two questionnaire surveys Objective To assess the acceptability of pre-pandemic influenza vaccination among healthcare workers in public hospitals in Hong Kong and the effect of escalation in the World Health Organization’s alert level for an influenza pandemic. Design Repeated cross sectional studies using self administered, anonymous questionnaires Setting Surveys at 31 hospital departments of internal medicine, paediatrics, and emergency medicine under the Hong Kong Hospital Authority from January to March 2009 and in May 2009 Participants 2255 healthcare workers completed the questionnaires in the two studies. They were doctors, nurses, or allied health professionals working in the public hospital system. Main outcome measures Stated willingness to accept pre-pandemic influenza vaccination (influenza A subtypes H5N1 or H1N1) and its associating factors. Results The overall willingness to accept pre-pandemic H5N1 vaccine was only 28.4% in the first survey, conducted at WHO influenza pandemic alert phase 3. No significant changes in the level of willingness to accept pre-pandemic H5N1 vaccine were observed despite the escalation to alert phase 5. The willingness to accept pre-pandemic H1N1 vaccine was 47.9% among healthcare workers when the WHO alert level was at phase 5. The most common reasons for an intention to accept were “wish to be protected” and “following health authority’s advice.” The major barriers identified were fear of side effects and doubts about efficacy. More than half of the respondents thought nurses should be the first priority group to receive the vaccines. The strongest positive associating factors were history of seasonal influenza vaccination and perceived risk of contracting the infection. Conclusions The willingness to accept pre-pandemic influenza vaccination was low, and no significant effect was observed with the change in WHO alert level. Further studies are required to elucidate the root cause of the low intention to accept pre-pandemic vaccination. In 2005 the World Health Organization recommended its member states to revise or construct a preparedness plan for pandemic influenza. The WHO also set up a system of influenza pandemic alert levels. Phases 1-3 include capacity development and response planning, while phases 4-6 signify the need for response and mitigation efforts. 1 By August 2008, 47 countries had prepared such a plan. 2 The recent spread of infection with a novel influenza A virus (H1N1 subtype) of swine origin ("swine flu") has prompted governments to review and carry out their pandemic responses, including vaccination strategies.
Modelling studies have shown that vaccination is an effective measure to reduce infection, hospitalisation, mortality and morbidity. 3 Since the supply of vaccines will be limited at the beginning of the influenza pandemic, prioritisation in the administration of the vaccines has been one of the major components in pandemic preparedness. Governments in different countries have issued consultation documents outlining their proposed vaccination policies. 4 5 In nearly all countries with a preparedness plan, healthcare workers are listed as the priority group for mass vaccination. 6 7 The American College of Physicians position statement supports measures to increase the supply of influenza vaccine and antiviral drugs in the strategic national stockpile. 8 Pre-pandemic H5N1 vaccine is now ready and stockpiled by many countries. The first batch of H1N1 vaccine is expected to be available by the end of 2009 or early 2010. 9 More than 30 governments have placed their orders. 9 For example, Hong Kong has decided to buy five million doses for its population of seven million, and the UK Department of Health has ordered 130 million doses of vaccine for its population of 61 million. However, healthcare workers' acceptance of the H5N1 and H1N1 vaccines is unknown. A survey conducted in May, when the pandemic level was already at phase 5, revealed that the general public in Hong Kong did not perceive swine flu (H1N1 influenza) as a threatening disease and did not think an outbreak to be highly likely. 10 The uptake of pre-pandemic vaccination among healthcare workers is a concern as the uptake of seasonal influenza vaccine is often low. In most studies, fewer than 60% of healthcare workers were vaccinated against seasonal influenza in various clinical settings. The most common barriers were fear of side effects, uncertainty about the vaccine's efficacy, and misconceptions about the vaccination and the infection. [11] [12] [13] [14] [15] One study in the UK of 520 participants found that 57.7% of healthcare workers expressed willingness to accept vaccination with the stockpiled H5N1 vaccine. Willingness was associated with perceived risk and benefits, and previous seasonal vaccination. 16 The aim of our study was to investigate the stated acceptability of pre-pandemic vaccination (H5N1 or H1N1 vaccine) and its associated factors among public hospital based healthcare workers in the departments of accident and emergency medicine, internal medicine, and paediatrics in Hong Kong. We also investigated the effect of escalation of the WHO pandemic influenza alert level on the acceptability of pre-pandemic vaccination.
The first survey was conducted from January 2009 to March 2009. The WHO influenza pandemic alert level assigned to H5N1 during that period was phase 3. Phase 3 signifies an animal or human-animal influenza reassortant virus that has caused sporadic cases or small clusters of disease in people but has not resulted in human to human transmission sufficient to sustain community level outbreaks. Limited human to human transmission may occur under some circumstances, such as when there is close contact between an infected person and an unprotected carer. However, limited transmission under such restricted circumstances does not indicate that the virus has gained the level of transmissibility among humans necessary to cause a pandemic.
We recruited healthcare workers only in public hospitals in this study because 94% of secondary and tertiary healthcare services in Hong Kong are provided by these hospitals. Pandemic influenza patients will be primarily treated in these hospitals-as occurred in the outbreak of SARS (severe acute respiratory syndrome), when public healthcare workers were at the highest risk for contracting infection. All department heads (42 departments with 8508 doctors and nurses) of emergency medicine, internal medicine, and paediatrics units under the Hong Kong Hospital Authority were contacted through emails, invitation letters, or telephone calls to obtain approval to send the questionnaires to their staff. These departments were chosen as their staff are at the highest risk of exposure to patients with influenza. We also sent invitation letters to the physiotherapy and occupational therapy departments under the Hospital Authority.
Self administered, anonymous questionnaires were used in both surveys. The questionnaire consisted of five sections with 17 questions: (1) demographics, patient contact, and history of seasonal influenza vaccination in 2008-9; (2) willingness to accept pre-pandemic vaccination with H5N1 vaccine; (3) willingness to accept pandemic vaccination with H1N1 vaccine (the second survey only); (4) perception of risk and seriousness of the pandemic influenza; and (5) suggestions on deployment of duties during pandemic flu, opinion on mandatory vaccination, and the possible ways of disposal for nearly expired vaccines. The participants were requested to send the completed questionnaires via internal mail.
The second survey was conducted in May 2009 when the WHO pandemic influenza alert level assigned to H1N1 influenza (swine flu) was phase 5. Phase 5 signifies human to human spread of the virus into at least two countries within one WHO region. Although most countries are not affected at this stage, the declaration of phase 5 is a strong signal that a pandemic is imminent and that the time to finalise the organisation, communication, and implementation of the planned mitigation measures is short. 1 During this phase 5 period, we repeated our questionnaires in the three specialties in one hospital. All questionnaires were collected within two weeks, before the announcement of phase 6 by the WHO.
Descriptive statistics were performed. Using cross tabulations, we analysed univariate associations between intention to accept vaccine and the following variables: sex, age (≤30 v >30 years), specialty, job title, years working in health services, work site, weekly number of contacts with patients, whether the respondents had seasonal flu vaccination in 2008-9, how likely they thought they were to get flu if there was a pandemic, and how seriously they thought a pandemic would affect their lives. We tested the statistical significance of the associations using Pearson's χ 2 test, Fisher's exact test, or trend tests where appropriate. Trend tests were used to test associations between one binary and one ordinal variable, and for two ordinal variables if a trend was apparent in the data. Multiple logistic regression was used to evaluate independent predictors of intention to accept vaccine. Demographic variables and variables on vaccination history and attitudes were tried in the models. A flexible modelling approach was adopted, and variables were retained in the model if they had P<0.1.
We compared the results in the second survey with the results from the same hospital in the first survey to assess the effect of escalation in pandemic alert level on the willingness to accept vaccination. Differences in characteristics between the two surveys were evaluated using Fisher's exact test.
Of the 4006 questionnaires distributed for the first survey, 1866 were completed and returned, giving a response rate of 46.6%. Of the 42 targeted units, 31 (73.8%) participated (including emergency medicine, internal medicine, and paediatrics units), representing 20% of all doctors and nurses working in these units in Of the 810 questionnaires distributed in the second survey, 389 were completed and returned. The response rate was 48.0%. Details of the second survey are shown in figure 2. All three invited departments had also participated in the first survey. The age and sex distribution of respondents were similar to those of the respondents in the same hospital in the first survey. Demographics of all respondents in both surveys are shown in table 1.
The overall intention to accept pre-pandemic vaccination (H5N1 vaccine) was only 28.4% for the first survey, which was conducted at WHO influenza pandemic alert phase 3. The level of intention to accept increased to 34.8% in the second survey, when the WHO alert phase was level 5. Responses from three departments in the hospital where both surveys were conducted are shown in table 2. No significant changes in the level of intention to accept pre-pandemic vaccination (H5N1 vaccine) was observed, despite the escalation to phase 5 because of the wide spread of H1N1 virus (swine flu).
The proportion of healthcare workers intending to accept pre-pandemic vaccination (H1N1 vaccine) was 47.9% when the WHO alert level was at phase 5. The respondents who were willing to accept H5N1 vaccines were more likely to accept H1N1 vaccines as well (91%); in contrast only 23.6% of those who declined H5N1 vaccination expressed an intention to accept H1N1 vaccination (P<0.0001).
The most common reasons for intending to accept vaccination were "wish to be protected" and "following Health Authority's advice" (fig 3) . The most common reason for refusal was "worry about side effects," and other reasons included "query on the efficacy of the vaccine," "not yet the right time to be vaccinated," and "simply did not want the vaccine" (fig 4) . More than half of the respondents thought that nurses should be the first priority group to receive the vaccines, followed by doctors and allied health professions, and then similar ratings for non-clinical and administrative staff. About half of the respondents (52.2% in the first survey and 56.0% in the second) wanted their family members to receive the vaccines as well. All the above responses remained constant in the different WHO alert phase levels.
Univariate associations between intention to accept H5N1 vaccination and other variables at WHO alert phase 3 are shown in table 3. Male sex, working in a specialty other than internal medicine, being a doctor, having fewer years of work in the health services, having received seasonal influenza vaccination in 2008-9, and perceptions that they were likely to contract the influenza and that a pandemic would seriously affect their lives were all significantly associated with greater intention to accept vaccination. In multiple logistic regression models for intention to accept vaccination (table 4), all of these variables remained significant except for specialty, which became marginally significant. At WHO alert phase 5, only having received seasonal influenza vaccination in 2008-9 and younger age were found as significant associated factors for intention to accept H5N1 vaccination in multiple logistic regression (table 4) .
For H1N1 vaccination, the factors showing a significant association with intention to accept at WHO alert phase 5 after adjustment by multiple logistic regression included younger age; having received seasonal influenza vaccination in 2008-9, and the perception that they were more likely to contract the pandemic influenza. The results are shown in table 5.
The surveys conducted at WHO pandemic alert phases 3 and 5 found a consistently low level of willingness to accept pre-pandemic influenza vaccination among hospital based healthcare workers, especially in those working in the allied health professions. This is particularly surprising in a city where the SARS outbreak had such a huge impact. The intention to accept vaccination against H1N1 influenza (swine flu) in our study was less than 50% even at WHO alert phase 5. On 21 May 2009, the WHO stated that 41 countries had officially reported 11034 cases of swine flu, including 85 deaths. 17 This was the time when our survey was conducted. It was surprising that neither this information nor the escalated WHO alert phase affected the intention to accept pre-pandemic vaccination. Vaccination is one of the potentially effective measures that can reduce mortality and morbidity from pandemic influenza. On 13 July 2009, the WHO also recommended that all countries should immunise their healthcare workers as a first priority to protect the essential health infrastructure. 18 However, the effectiveness of this measure depends heavily on the uptake rate in those groups assigned high priority. Therefore, the low potential acceptance of this vaccine warrants our attention, with a view to improving acceptance.
The factors with the strongest association with intention to accept pre-pandemic or pandemic vaccine were history of seasonal influenza vaccination and perceived risk of contracting the infection. The strong association between acceptance of seasonal and pre-pandemic vaccination also suggests similar barriers exist for both vaccines. Efforts to improve the uptake of seasonal influenza vaccination by healthcare workers should therefore be a part of the pandemic preparedness plan, as disseminating correct information may be more difficult at time of crisis, and the health belief model could be applied to improve the acceptance of pre-pandemic vaccine as in seasonal influenza vaccination. 19 The major barriers to vaccination we identified were fear of side effects and questions about its efficacy. This suggests that public and hospital health agencies need to provide more information to the staff, especially to those with higher levels of anxiety and doubt.
In our study younger staff and staff whose working experience was less than 5 years were more willing to accept vaccination. This again implies that the experience of the SARS outbreak did not enhance the willingness to accept the vaccination.
Comparison with another study The willingness of Hong Kong healthcare workers to be vaccinated against H5N1 virus is very low when compared with the study done in a UK NHS trust, where more than half of the staff were willing to accept pre-pandemic vaccination when surveyed at a similar WHO alert level (phase 3). 16 The uptake rates for seasonal influenza vaccine were higher among our participants than in the UK study (32.9% for Hong Kong and 15.6% for UK healthcare workers), whereas the proportion willing to be vaccinated against H5N1 influenza in the UK study (57.7%) was double that in our survey (28.4%). Whether this higher willingness to accept was only temporary as a result of the well publicised H5N1 outbreak at a poultry farm in the UK when the survey was started remains to be explored.
The uptake rate for seasonal influenza vaccine in Hong Kong varies among the target groups. A previous study on patients attending a general outpatient clinic, of whom half had chronic illnesses, found a vaccination rate of 27%, but without correlation with sex, occupation, or household income. 20 The uptake rate among elderly people aged >65 years living in the community was also low (26.9%-36.4%). 21 In contrast, more than 90% of elderly people living in institutions received influenza vaccine, which was delivered on site. 22 The overall vaccination rate for elderly people in Hong Kong could be within the range reported from surveys in the UK, Italy, France, Germany, and Spain conducted from 2003 to 2007 (41.3%-78.1%). 23 As for healthcare workers, both Hong Kong and European countries face a low uptake rate. The 32.9% vaccination recorded in the current study is close to the ranges reported for the UK (15.9%-25.2%), Italy (13.3%-23.2%), Spain (20.5%-28.9%), Germany (22.0%-27.5%), and France (15.8%-22.2%). 23 The Department of Health in Hong Kong provides a comprehensive immunisation programme from birth to 12 years old. There seems to be no major generic barrier to vaccination in Hong Kong, as the uptake for childhood immunisation programmes is high (84.7%-99.6%), 24 and a recent survey indicates a high level of willingness (88%) to accept human papillomavirus vaccine, 25 similar to that recorded in the UK (89%). 26 While cultural differences could affect the acceptance of vaccines in general, we believe there are common barriers to influenza vaccination that exist across geographical regions and racial groups. 27 The findings of this study can therefore serve as a reference for other countries that are planning to offer the H1N1 vaccine to their healthcare workers.
Strengths and weaknesses of this study To our knowledge, this is the largest study conducted to assess the willingness of healthcare workers to accept pre-pandemic influenza vaccination, and it provides important information on barriers to vaccination. Campaigns to promote vaccination should consider addressing the knowledge gap of staff and the specific target groups for intervention. Our study also captured the effects of change in WHO alert level on people's perception and willingness to accept vaccination.
The main limitation of this study is the response rate of below 50%. The low response rate may have resulted in a biased sample. Another caveat is the lack of details for the non-responders. Nevertheless, the characteristics of the participants matched the overall staff profile, and the participating specialty departments represented over 70% of our target population. An additional limitation is that the study only documented what people said they would do and thus may not reflect the actual vaccine uptake rates. A follow-up study will be needed to capture the true uptake rates and factors associated with vaccine uptake when it is available. Further qualitative studies such as focus group discussion or semi-structured interviews could help to consolidate and supplement the findings.
We believe this information can assist governments to design their pandemic vaccination plan for healthcare workers, taking into account their opinions on these contentious issues. A successful vaccination strategy does not just protect the health of healthcare workers but also can limit the transmission between the health sector and the community, a lesson from the SARS outbreak. Qualitative studies are being conducted by our group to explore barriers faced by healthcare workers in the uptake of vaccination. With the reported low level of willingness to accept pre-pandemic vaccination in this study, future work on intervention to increase vaccination uptake is warranted.
We thank Dr Ian Stepheson for his invaluable advice and experience in constructing the questionnaires and generous support from the chiefs of service in the participating departments. We also thank Ms Kate TC Ng for organising the questionnaire administration. Contributors: JSY Chor and PKS Chan designed the study, interpreted the findings, and wrote the manuscript; KLK Ngai collated the survey data; WB Goggins and SYS Wong performed statistical analyses; MCS Wong, N Lee, TF Leung, TH Rainer administered the survey; S Griffiths supervised the study. All authors, external and internal, had full access to all of the data (including statistical reports and tables) in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis. Funding: This study did not receive any external funding. Competing interests: None declared. Ethical approval: The study was approved by the Survey and Behavioural Research Ethics Committee of the Chinese University of Hong Kong. Data sharing: no additional data available.
The WHO recommended that healthcare workers in all countries should get top priority for vaccination against the influenza A H1N1 virus of swine origin
The acceptance of seasonal influenza vaccination in healthcare workers worldwide is low. One study conducted at WHO alert phase 3 showed 57.7% of UK healthcare workers would accept the pre-pandemic H5N1 vaccine
The willingness to accept vaccination against both influenza A subtypes H5N1 and H1N1 among hospital based healthcare workers in Hong Kong was low Neither the change in WHO pandemic alert levels nor the experience of the SARS outbreak affected the potential acceptance of the vaccines Barriers identified include fear of side effects and doubts about the efficacy of the vaccines The strongest associations with the intention to accept vaccination were a history of seasonal influenza vaccination and perceived likelihood of being infected Evolutionarily Conserved Herpesviral Protein Interaction Networks Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species. Herpesviruses are subdivided into three taxonomic subfamilies (a, b and c) based on both genomic composition and biology according to a well-known phylogeny [1, 2, 3] (Figure 1A ). While all herpesviruses are structurally similar, the different subfamilies are highly divergent in genome size, content and organization. The genome size ranges from 120 kbp for varicella-zoster virus (VZV), which belongs to the a-herpesviruses, to 240 kbp for human cytomegalovirus (hCMV), a member of the b-herpesviruses [4, 5] . Gene-coding potential is reflected in the size of the genomes with VZV containing 70 open reading frames (ORFs) and hCMV containing ,170 ORFs. The overlap between the protein sets of the five viruses clearly supports the known phylogeny, but there are also some proteins shared among viruses not consistent with the phylogeny (Figure 2A ). Although the three subfamilies are thought to have diverged from a common ancestor around 400 million years ago (McGeoch 2006) , they still contain a set of 41 core orthologs present in all herpesviruses [6, 7] . Herpesviral core proteins are generally involved in fundamental aspects of viral morphogenesis (e.g. DNA replication, DNA packaging, structure and egress), and are consequently often essential for replication in cell culture [8, 9, 10] .
Several genome-wide yeast-two-hybrid (Y2H) studies of proteinprotein interactions in eukaryotes have been published over the last years, including Saccharomyces cerevisiae [11] , Caenorhabditis elegans [12] , Drosophila melanogaster [13] , Plasmodium falciparum [14] and Homo sapiens [15, 16] . The first complete genome-wide interaction study, however, was published for the E.coli phage T7 [17] . With their relatively small genomes and few genes, viruses seem the ideal candidates for studying protein-protein interactions on a genome-wide level and to address the generally low coverage of Y2H measurements in a systematic way. It is therefore surprising that not more genome-wide studies of intraviral interactions have been performed to date. With the exception of bacteriophage T7 [17] and Vaccinia virus [18] , most of the studies of viral interactions have been performed on small RNA viruses [19, 20, 21, 22] . Recently more studies have been focusing on larger DNA viruses. In addition to our previous studies of VZV and Kaposi's sarcoma-associated herpesvirus (KSHV) [23] , a study by Calderwood and colleagues identified 43 interactions between viral proteins in Epstein-Barr virus (EBV) [24] . Two Y2H studies on herpes simplex virus 1 (HSV-1) and KSHV have also focused on interactions between structural components of these viruses [25, 26] . To add to our understanding of intraviral interactions in herpesviruses we present in this article the first interactomes for herpes simplex virus I and murine cytomegalovirus (mCMV), in addition to a second and independent interactome for Epstein-Barr virus. Based on these data we are able to compare five related interactomes, obtained using a standardized experimental setup for all five species. From the comparison and extensive experimental testing by CoIP we conclude that i) genome-wide interaction studies are sufficiently sensitive for between-species comparisons to identify the basic sunflower structure of the interaction networks and their common core, ii) interactions are to a large degree conserved between orthologs in herpesviruses, iii) comparing interactomes from several species can improve the low coverage of individual Y2H measurements and iv) biologically relevant interactions which may not be apparent from the interactome of a single species, often become obvious when multiple interactomes are aligned and compared.
To study intraviral protein-protein interactions of herpesviruses we recombinatorially cloned the individual open reading frames of HSV-1, mCMV and EBV into the yeast-two-hybrid (Y2H) vectors pGBKT7-DEST and pGADT7-DEST and tested all pairwise intraviral protein interactions using an array-based Y2H strategy [27] . To address the issue of false negative interactions, viral proteins containing transmembrane domains were cloned both as full-length and as intracellular and/or extracellular domains. From the mCMV Y2H analysis we observed that 33% of the tested preys, and 40% of the baits, gave positive interactions. Similar results were observed with HSV-1 and EBV with ,1/3 of the clones yielding positive interactions (Table S1). In total, the Y2H  analysis revealed 111 interactions for HSV-1, 406 for mCMV and  218 for EBV (Figures 1B, S1 and S2, Tables S2 and S13) . Combined with our previously published interactomes for VZV (173 interactions) and KSHV (123 interactions), we obtained altogether 1,031 intraviral interactions in five herpesviral species (Tables S2 and S13 ). To evaluate the coverage of our five interactomes we performed an extensive literature search which identified 257 previously published interactions for these herpesviruses (including human cytomegalovirus (hCMV) homologues). Of these 257 interactions we were able to detect 24 (9.3%) in at least one virus ( Figures S3 and S4 and Tables S3 and S14). When comparing our EBV interactome with the recently published EBV network by Calderwood et al., 6 out of 43 (13.9%) interactions could be confirmed [24] . Such low confirmation rates are common to Y2H studies, even for studies within the same species, which in general suffer from low coverage [28, 29, 30] . For instance, in a previous study of human interactions only 2.3-8.4% of known interactions were identified [15] . On the other hand, this implies that ,3% of the interactions found in the present study have been published so far in the literature or identified in previous genomewide screens in the case of EBV. In the case of HSV-1 our study added 102 new interactions to the network of already known interactions (coloured grey in Figure 1B ). As is typical for such interaction networks, no apparent structure can easily be recognized.
A comparison of the five herpesviral networks revealed that the degree distribution differed from cellular networks, local clustering was not as high as expected in small-world networks of this size (Figures 1C and S5 and Table S4 ), and attack tolerance and robustness were increased compared to cellular networks ( Figure 1D and S6), probably reflecting that the viral interactome in itself only represents a minor part of the complete interactome of the infected cell. In a previous study we observed that the topology of the KSHV and VZV networks approached that of cellular networks as the viral interactomes were connected into a human interactome [23] . The observations presented here confirm our previous findings, and indicate that herpesviral PPI networks share an evolutionarily conserved topology.
Apart from general topological features, herpesviral interactomes were also compared on the level of individual interactions. For this purpose, we used the orthology assignments based on sequence similarity and gene order (Table S5 ) [31] . Species within the same subfamily are generally characterized by higher sequence similarity between orthologous proteins. They also share more orthologous proteins with each other than species from different subfamilies ( Figure 2A ). In previous inter-species comparisons [32] , very few interactions were found to be shared between different species (yeast, worm, fly). Unlike the previous comparative studies, the five different interactomes analysed in this study were obtained using exactly the same experimental protocols. Nevertheless, we still observed little overlap between the networks of the five herpesviruses. Of 488 (409 non-redundant, i.e. conserved interactions are only counted once) interactions between proteins conserved in more than one species, 140 (61 non-redundant) (28.7% or 14.9% non-redundant, respectively) interactions were conserved between at least two species. For any two herpesvirus species, we compared the number of interactions between proteins conserved in both species against the number of interactions found in both species ( Figure 2B ). Although the pair
Herpesvirus proteins interact with each other in a complex manner throughout the infectious cycle. This is probably best exemplified in the process where a large number of viral proteins come together to form new viral particles which are subsequently released from the infected cell. A more detailed understanding of how viral proteins interact with each other might assist the development of drugs which may inhibit these interactions and consequently block viral replication. Here we present three genomewide studies of protein-protein interactions in the herpesviruses herpes simplex virus I, murine cytomegalovirus and Epstein-Barr virus. Altogether we identified 735 interactions in the three viruses, most of which have not previously been reported. By combining these studies with our previously published studies for Kaposi's sarcomaassociated herpesvirus and varicella-zoster virus we were able to perform a comparative analysis of interactions in five related viral species. We observed that a high proportion of interactions were conserved between the different species, despite a low degree of sequence conservation. This implies that by comparing interaction data, we were able to increase the coverage of our viral networks and thus obtain a better and more complete picture of interactions between herpesviral proteins. [6, 7] . (B) Intraviral protein-protein interaction network for HSV-1. The proteins are coloured according to their conservation in the herpesvirus phylogeny: the blue nodes are core proteins conserved in all five viruses, two nodes (pink) are conserved in a and c herpesviruses, several red ones in a herpesviruses and the grey ones are specific to HSV-1. Edges indicate observed interactions in HSV-1, and red edges indicate previously reported interactions. The protein interaction network was generated using the Cytoscape software (www.cytoscape.org) [58] .. (C) node degree distribution on a linear or logarithmic (inset) scale. The herpesviral networks can be approximated by power law distributions (Table S3 ). (D) Simulations of deliberate attack on HSV-1 in comparison to two human networks by removing their most highly connected nodes in decreasing order. After each node is removed, the new network characteristic path length (average distance between any two nodes) of the remaining network is plotted as a multiple or fraction of the original parameters. The herpesviral networks consistently exhibited a higher attack tolerance, as the increase in path length is considerably smaller. doi:10.1371/journal.ppat.1000570.g001 wise overlaps observed were small, they were nevertheless significantly higher than observed with randomized orthology assignments ( Figures 2C and S7 ). Randomized orthology assignments for each pair of herpesviruses were obtained by first selecting the sub-network of conserved proteins between the two species, and then randomizing the orthology assignments for these sub-networks.
A similar analysis was performed for all five networks taken together. First, networks were divided into interactions conserved within a subfamily or between different subfamilies, and the number of interactions conserved in 2, 3 or 4 species in each category was evaluated. We furthermore compared the number of interactions conserved in 2, 3, 4 and 5 species against the results for randomized orthology assignments and found in each case a significant enrichment ( Figure 2D ). This shows that despite the low coverage of the Y2H system significant conservation can still be observed.
Herpesviruses share a set of 41 core orthologous proteins which are conserved throughout the three subfamilies (Table S5 ) [31] . These core orthologs comprise approximately half of the genome of HSV-1, VZV, EBV and KSHV but less than 25% of mCMV. They can be further subdivided into a group of 31 orthologs with relatively high sequence similarity (approximately 30-60% sequence similarity), and a group of 10 orthologs with little similarity (approximately 16-30% similarity) (Table S6 ). Based on this orthology assignment, we generated an overlay of all protein interactions between the core orthologs detected in any of the five herpesviruses (Core network, Figure 3A ). Of a total of 283 (218 In each rectangle, the value above the lines indicates the observed number of homologous interactions detected in both herpesviruses (in green). The value below the line (in black) gives the total number of interactions detected in the first species (indicated in columns) between proteins which have orthologs in the second species (indicated in rows). On the diagonal, the total number of interactions is shown for each virus. (C) Distribution of the number of conserved interactions between HSV-1 and mCMV for 1000 random orthology assignments (blue line) in comparison to the true number of conserved interactions (red vertical line). For each pairwise comparison, subnetworks were selected between proteins conserved in both viruses and then the orthology assignments between the proteins were randomized. Accordingly, the size and degree distribution of the subnetworks does not change. (D) Comparison of the number of interactions conserved in 2, 3, 4 and 5 species for 1000 random orthology assignments (yellow boxes) to the true number of interactions conserved in that many viruses (red line). Random orthology assignments were created in a similar way as for Figure 2C . non-redundant) core protein interactions detected, 113 (48 nonredundant, 39.9%) were found in more than one species (Table  S7) . For the core network, we did not observe a correlation between sequence similarity and the number of conserved interactions detected ( Figures 3B and 3C ). For example, the interaction between the two tegument proteins UL11 and UL16 in HSV-1 was also detected in mCMV and EBV, although sequence similarity of UL11 and its orthologs across subfamilies is quite low (28%). This interaction was interestingly also observed for HSV-1 in a recent report by Vittone and colleagues [33] . In addition, interactions were not preferentially conserved between closely related species ( Figures 3D and S8) . Accordingly, overlaps between the interaction sets in the core network were not correlated to the true phylogeny of herpesviruses ( Figure 3E ). Indeed, the highest overlap was observed between HSV-1 (a-subfamily) and mCMV (b) which belong to lineages separated early in herpesvirus evolution [7] . However, since our phylogenetic trees are based on relative overlaps between the different species, we cannot exclude that a more complete set of core interactions might have allowed for better separation of the subfamilies. In contrast, when also including subfamily-and species specific interactions (i.e. the complete interaction network of the five herpesviruses with the characteristic sunflower structure, see Figure 4A ), the analysis yielded a phylogeny that was consistent with the known evolutionary relationships ( Figure 4B ). This indicates that the presence of conserved subfamily specific interactions provides sufficient conserved and non-conserved interactions to accurately separate the subfamilies from each other.
In the overlay of all five herpesviral networks ( Figure 4A , sunflower structure), the core network is indicated as a central node common to all herpesviruses. Subfamily-and species-specific networks are attached (as leafs) to this core. Only few connections exist between the subfamily-specific networks due to few shared proteins outside of the core. Our data provides evidence that the viral core network is extremely dense while the non-core network appears relatively sparse. However, since non-core interactions were tested in at most two species, and not in five as the core interactions, the non-core network may be equally dense. Indeed, no consistent difference was observed between the number of intraviral core and non-core interactions when considering each network separately (Table S8) .
To further evaluate whether interactions between orthologous proteins are conserved we used co-immunoprecipitation to test 92 interactions predicted from 55 interactions detected in KSHV for the corresponding orthologs in HSV-1, mCMV and EBV. 11/19 (58%) of the predicted interactions could be confirmed by CoIP in HSV-1, 12/18 (67%) in mCMV and 36/55 (65%) in EBV, in comparison to 29/55 (53%) in KSHV itself ( Figures 5A and S9A and Table S9 ). The percentage of core-derived orthologs that were confirmed by CoIP significantly correlated with the number of species in which the interactions were detected in Y2H screens, suggesting that the accuracy increases with the number of positive assays ( Figure S9B , Table S10 ). As negative controls, ten interactions which were not detected in any of the Y2H screens were tested in four viruses (39 interactions in total, Table S10 ). Although the confirmation rate of these negative controls seems relatively high (6/39 (15%)), it is still significantly smaller than for the predicted interactions and correlates well with the confirmation rates of interactions observed in 2, 3 and 4 species ( Figure  S9B ). Due to the low coverage of the Y2H system many (particular weak) interactions were most likely missed, and the positively tested controls may be examples of such interactions. It also suggests that, although our core interactome is very dense, it has not yet reached full coverage. Since the confirmation rate by CoIP for the Y2H interactions in KSHV is not higher than for the predicted interactions in HSV-1, mCMV and EBV, we conclude that a high percentage of interactions between core orthologs are conserved despite low sequence similarity of some of the orthologs across subfamilies.
To further assess the level of completeness of our core network, we evaluated the average number of new interactions added to the core network with each new Y2H screen ( Figure 5B ). If core interactions indeed are conserved, as indicated by our predicted interactions, we would expect the coverage to increase with each new herpesviral interactome. Although the number of newly discovered interactions steadily decreased with each new screen, saturation does not seem to be reached yet. Thus, although coverage for the core network could be increased, a significant fraction of interactions might still be missing.
Finally, to determine if conserved intra-viral interactions allow viral proteins to interact across different species, we tested four interactions which were detected in at least two herpesviruses in the original screens by Y2H and LUMIER (luminescence-based mammalian interactome mapping) pull-down assays ( Figure S10A and B) [34] . While Y2H in general yielded few cross-species interactions, we detected a larger number of interactions by LUMIER ( Figure S10B ). The cross-species interactions between the HSV-1 UL11 and UL16 tegument and between the HSV-1 UL19 and UL35 capsid orthologs were mainly observed within a specific subfamily, in accordance with previous observations by Schnee et al. [35] . For the two other interactions, involving orthologs with both a high and low degree of sequence similarity based on Table S6 , we saw a more promiscuous interaction pattern. HSV1 UL14 for example was able to interact with HSV-1 UL33 and its orthologs in all five species, suggesting that sequence similarity might be a poor predictor of interspecies interactions in herpesviruses. Additionally, we tested 4 core and 4 noncore VZV baits against prey libraries of all five viruses. As expected, the intraspecies analysis (VZV baits against VZV preys) yielded the highest fraction of positive interactions (2.8%), compared to 0.5% positive interactions in the cross-species screens. Of the positive crossspecies interactions we observed 4 core-core, 15 core-noncore and 2 noncore-noncore interactions (Table S11 ). When the number of positive interactions was correlated to the number of interactions tested for each class, we observed a significant enrichment of positive interactions for the core-core and core-noncore classes compared to the noncore-noncore class ( Figure S11 ).
Most core proteins are essential, and a majority can be found in herpesvirus virions composed of an icosahedral capsid of 162 capsomers, an amorphous tegument layer and a lipid bilayer membrane with embedded glycoproteins. Using the high-coverage core network, a map of conserved protein interactions in herpesviral particles was generated ( Figure 6A and S12). One outstanding example for a highly connected protein in this virion map is the mCMV M51 ortholog (HSV-1 UL33, VZV Orf25, EBV BFRF4 and KSHV Orf67.5), which interacted with 14 tegument proteins. Since (i) 11 of the 14 interactions (79%) of this protein were found in more than 1 species, (ii) most Y2H interactions were confirmed even under high concentrations of the competitive HIS3 inhibitor 3amino-1,2,4-triazole which can be used to suppress non-specific Y2H interactions ( Figure S13A and S13B), and (iii) a majority of interactions were confirmed by CoIP (Table S12) , we considered them as high-confidence interactions. Furthermore, 4 of the 5 interactions conserved in 4 herpesviral species are M51 interactions. One example is the interaction of mCMV M51 orthologs (HSV-1_UL33/VZV_25/EBV_BFRF4/KSHV_67.5) with M53 orthologs (HSV-1_UL31/VZV_27/EBV_BFLF2/KSHV_69), which also interact in 4 species with M50 orthologs (HSV-1_UL34/ VZV_24/EBV_BFRF1/KSHV_67). M50 and M53 and their orthologs are involved in the nuclear egress of viral capsids and are well-characterised in mCMV, HSV-1, EBV and pseudorabiesvirus [36, 37, 38, 39] . Both M50 in mCMV and its ortholog UL34 in HSV-1 recruit protein kinase C to the nuclear membrane, which subsequently phosphorylates lamins to dissolve the nuclear lamina allowing the capsids to reach the inner nuclear envelope [38, 40] . In mCMV, we confirmed 17/22 (.75%) (Table S12) of M51 interactions by CoIP, and showed that M51 is targeted to the nuclear membrane by M50 and co-localizes with both M50 and M53 ( Figure 6B ). Our results suggest that M51 and its orthologs are part of a larger protein complex and may be involved in nuclear egress. Since most of its interaction partners are present in the virion tegument we hypothesize that it plays a role in tegument formation, and represents a possible link between DNA packaging, nuclear egress and tegumentation.
Here we present an extensive study of intraviral protein-protein interactions for the three herpesviruses HSV-1, mCMV and EBV, using Y2H as the main experimental method. By combining the results with our previous studies of interactions in VZV and KSHV we were able to compare the interactomes of five related herpesviral species. Although there was little overlap between the five viral networks according to the Y2H maps, we were able to show that interactions between core orthologous proteins are to a large degree conserved between species of different subfamilies. By generating a separate network of interactions between core proteins of five herpesviruses, we were also able to overcome the coverage problem of Y2H and to identify interactions of interest from the common network which were not apparent in each single network.
While the overlaps between the different interactomes were generally quite low, there was still a significant enrichment of conserved interactions between orthologous proteins for any pair of the five species ( Figures 2C and S7A to I). The same holds true for the conservation between all five viruses. Interactions observed in two, three or four species were all enriched significantly as compared to background expectations ( Figure 2D ). This argues that, although troubled with false negative and false positive interactions, Y2H as a technique is still sufficiently sensitive and specific to obtain data for a comparative analysis of related interactomes. Similar observations were also made for the recently published Campylobacter jejuni interaction network, where highly significant overlaps were found with both the Escherichia coli and Helicobacter pylori interactomes [41] . It is very likely that true overlaps between the herpesvirus interactomes are higher, but that due to false negative interactions we only observe modest overlaps. There are numerous reasons why interactions may be missed in the Y2H system, including improper folding of fusion proteins and post-translational modifications. In an attempt to address some of these issues we cloned all the viral proteins containing transmembrane domains as both full length and extra/intracellular fragments, which has been reported to increase sensitivity [42] .
Our observations indicate that intraviral interactions between core proteins are conserved, and as a result we are not able to separate the Y2H interactomes into their phylogenetic subfamilies solely based on their core interactions. However, when interactions involving subfamily-specific proteins present in at least two of the virus species were included, we were able to generate a correct phylogenetic tree. This implies that interactions involving subfamily-specific proteins are at least partly conserved. Indeed, several of the interactions predicted from KSHV and confirmed in EBV by CoIP involved subfamily specific proteins. From published literature there are several examples of core interactions being conserved between species of different herpesviral subfamilies, e.g. the interactions between HSV-1 UL31 and UL34 [36, 37, 38, 39] , HSV-1 UL15 and UL28 [43, 44] and the HSV-1 UL54 self-interaction [45, 46] . Indeed, much of what is currently known about herpesvirus biology is derived from studies of Herpes Simplex Virus and extrapolated to other species. Our study indicates that it is effectively possible to transfer intraviral interactions between orthologous proteins from one species to another. Thus, by generating an overlay network from several genome-wide Y2H screens in related species, the large number of false negative interactions within each individual analysis can be overcome and a more complete picture of the core interaction network obtained.
In general, interactions are transferred between different species based on the sequence similarity between the corresponding proteins. In addition, one might expect interactions among orthologous proteins with high sequence similarity to have a higher likelihood of being conserved. Yu and colleagues found that interactions could be confidently transferred from one species to another if the joint sequence identity of the interacting orthologs was .80% [47] . However, since none of the herpesviral core proteins shares such a high degree of sequence similarity across subfamilies, these criteria cannot be applied to herpesviruses. Furthermore, no correlation was observed between sequence similarity and the number of species in which an interaction was observed in the Y2H experiments. Thus, our results show that sequence similarity alone seems to be insufficient for predicting herpesviral interactions from one species onto another. Our analysis of cross-species interactions indicates an enrichment of interactions involving core proteins (either core-core or core-noncore). The detailed cross-species analysis of the interaction between the major capsid protein (MCP) and the smallest capsid protein (SCP) (HSV-1 UL19-UL35, Figure S10B ) only yielded 1 intraspecies interaction by Y2H, however 4 by LUMIER, indicating that this interaction is conserved despite being observed in only one species by Y2H. While capsid proteins and interactions are thought to be highly conserved, most of them were indeed only observed in one species in our genome-wide Y2H screens. However, three of the four observed capsid interactions (HSV-1 UL19-UL35, UL18-UL38, UL18-18 and UL35-UL35) have been published previously (Tables S3 and S14) , and, in addition, the LUMIER analysis resulted in an increased number of cross-species interactions. The cross-species interactions between the two tegument proteins HSV-1 UL11 and UL16 (and their orthologs), as well as between the two capsid proteins HSV-1 UL19 and UL35 (and their orthologs), were mainly observed between species within the same herpesviral subfamily. A similar observation has recently been reported by Schnee and colleagues [35] , and may indicate that some binding sites are more conserved within herpesvirus subfamilies. The other two interactions could be detected in a larger number of cross-species interactions by both Y2H and LUMIER. HSV-1 UL14, for example, was observed to interact with all orthologs of HSV-1 UL33 by LUMIER. Previous reports suggested that HSV-2 UL14 shares certain similarities with cellular chaperones which may account for its promiscuous binding pattern [48] .
In the core network derived from the overlap of all five herpesviruses, mCMV M51, and its orthologs HSV-1 UL33, VZV ORF 25, EBV BFRF4 and KSHV ORF 67.5, show up as intraviral hubs with a number of conserved interactions. For instance, the interaction between M51 and M53 was observed in all species apart from HSV-1. Interestingly, when retesting UL33 interactions under more stringent conditions (Figure S12) , the corresponding interaction between UL33 and the M53 ortholog in HSV-1, UL31, is clearly one of the positive interactions on both 2.5 and 5 mM 3AT. These interactions were not included in the HSV interactome to prevent an overrepresentation of interactions tested more than once. While not much is known about M51, M53 has been extensively documented to be involved in nuclear egress through its binding to M50 [35, 38, 49, 50] . The interaction between M53 and M50 was confirmed in this study in four viruses. In addition, from our study of interactions in VZV we observed an association between the ortholog of M51 (ORF25) with the M50 ortholog (ORF24) [23] , and retesting of HSV-1 UL33 interactions also revealed its binding to HSV-1 UL34 lacking the transmembrane region ( Figure S12 ). Finally, immunofluorescense studies indicated that M51 co-localizes with both M53 and M50 when using fluorescent fusion proteins. These results suggest a possible role for M51 in nuclear egress through its interactions with M53 and/or M50. As interactions between orthologs of M51 and M53 were observed in members of all three subfamilies, it is likely that this represents a conserved function of M51. Previous studies have indicated that the M51 ortholog in HSV-1 (UL33) is involved in packaging of DNA [51] , and that it interacts with at least one of the subunits of the terminase complex (UL28) [52] . In the data presented here, UL33 was observed to interact with UL15 and UL28 in three different species. These results suggest that UL33 represents an association between packaging and egress. Studies done in HSV-1 have indicated that UL33 is associated with the external surface of capsids [53] , which would make such a dual role reasonable. While it is not known exactly how UL33 associates with the capsid, the interaction observed between M51 and the smallest capsid protein (m48.2) in mCMV and EBV suggests a possible manner of association.
In summary, this study suggests that a distinctive network topology is still present in all vertebrate herpesvirus species although herpesviruses co-evolved with their hosts for millions of years. Moreover, it provides evidence (i) that interactions and hence functions of proteins may be more conserved than their sequence and (ii) that a common core of protein interactions is conserved in all herpesviruses. We hope that the data presented will inspire future herpesvirus research and facilitate the selection of potential targets for antiviral therapy [54] .
The nucleotide sequences for all ORFs were obtained from the ncbi (http://www.ncbi.nlm.nih.gov/), and cloned into the Y2H vectors pGBKT7-DEST and pGADT7-DEST by recombinatorial cloning [55] (Protocol S1). All clones were sequence verified.
Yeast strains AH109 and Y187 were transformed using 1 mg of prey (pGADT7-DEST) or bait (pGBKT7-DEST) plasmid DNA, respectively, and grown on SD medium lacking either leucine (-leu) or tryptophane (-trp). Prey-and bait-expressing yeast were arrayed in a 384-pin format using a Biomek 2000 workstation (Beckman-Coulter) (4 replicas for each interaction tested), and mated in an all-against-all matrix approach [27] . Diploid colonies were grown for 2 days at 30uC on SD -leu-trp plates, and subsequently transferred to selective SD -leu-trp-his plates. Interactions were considered positive if at least 3 out of 4 colonies grew (Protocol S1).
pGBKT7-DEST and pGADT7-DEST were co-transfected into HEK-293 cells by means of calcium phosphate, and superinfected with recombinant vaccinia virus (vTF-7) expressing T7 RNA polymerase (NIH AIDS repository) at a MOI of 10. After 24 h cells were lysed, and precipitation of proteins was done using 1 mg of either anti-myc (Santa Cruz) or anti-HA (Roche) antibodies in addition to protein G Sepharose beads. Precipitates were separated by SDS-PAGE, and western blots initially reacted with the anti-myc and anti-HA antibodies, and secondary, peroxidaseconjugated anti-mouse IgG or anti-rat IgG antibodies (Jackson). The CoIP was scored positive if a co-precipitate was detected in at least one direction (Protocol S1).
HeLa cells were grown on a cover slip until ,50% confluence, and subsequently transfected with 1 mg of DNA for each of the fluorescent vectors analyzed, either alone or in combinations, by means of Effectene (Qiagen). Cells were incubated for 24 h, and fixed by incubating with 4% paraformaldehyde for 30 min at RT. Coverslips with fixed cells were mounted in Vectashield Mounting Medium (Vector Labs), and imaged on an OLYMPUS BX61 microscope.
Literature interactions were identified by combining automatic text mining and manual curation. A set of ,87000 MEDLINE abstracts on herpesviruses was screened using ProMiner [56] for occurrences of proteins of any of the five viruses considered. Subsequently, 565 abstracts were selected containing a reference to interactions and at least two different proteins of the same virus. Physical interactions were then extracted manually from the corresponding articles.
From the five individual networks an overlay network was created by merging orthologous proteins and interactions between orthologous proteins. Orthology relationships were assigned based on Davison [31] . The overlay network was then used to predict interactions between core proteins and to analyze network characteristics (Protocol S1).
For all core orthologous proteins the average pairwise global sequence similarity across all five viruses was calculated. Global similarity was used to avoid a distortion of the results by short but high local similarities between orthologous proteins. For an interacting pair of core proteins, the similarity was calculated as the geometric mean of the average similarities for the corresponding proteins.
The distance metric used to construct the phylogenetic tree ( Figure 3E ) for the complete and core network, respectively was based on the relative interaction overlaps. Accordingly,
where S ij is the number of shared interactions between species i and j and C i and C j are the total number of interactions for species i and j. In this case, only interactions between proteins conserved in at least two species or all species for the core network were considered for each species-specific network. The phylogenetic tree was generated using the neighbour-joining algorithm of the PHYLIP package [57] with 10,000 bootstrap samples.
Protocol S1
Found at: doi:10.1371/journal.ppat.1000570.s001 (23 KB PDF) Figure S1 Protein network in mCMV. The protein interaction network was generated using Cytoscape software (www.cytoscape. org) [58] . Interactions previously reported in the literature are indicated with red edges. The colours of the nodes indicate in which herpesviral species a specific protein is conserved. [16, 17] . After each node is removed, the new network characteristic path length (average distance between any two nodes) of the remaining network is plotted as a multiple or fraction of the original parameters. The herpesviral networks consistently exhibited a higher attack tolerance, as the increase in path length is considerably smaller. Figure S13 Protein interaction partners of HSV-1 UL33 tested by Y2H. HSV-1 UL33 cloned in pGADT7 (prey) was tested against a variety of interaction partners cloned in pGBKT7 (bait). As negative controls, each bait was tested against the empty pGADT7 prey vector (left plates) while the UL33 prey was tested against the empty pGBKT7 bait vector (right plates). (A) Evaluation of mated yeast clones on double and triple selective plates with empty pGADT7 vector used as a control. (B) Evaluation of mated yeast clones on increasing amounts of 3-AT (0, 2.5, 5, 10 mM) with empty pGADT7 vector as a control. Self-activation of UL15, UL16 and UL21 at 0 mM 3-AT was suppressed at 5 mM 3-AT, while the interactions with UL33 were still found to be positive. Found at: doi:10.1371/journal.ppat.1000570.s014 (0.08 MB PDF)
Table S1 Summary of prey and bait hit-rates for HSV-1, mCMV and EBV. Overview of the total number of preys and baits included in the Y2H screens, including the number of preys and baits which yielded interactions. The total number of preys and baits exceed the total number of proteins tested due to many of the proteins being cloned as both fragments and full-length proteins. Table S8 Average degree values for core and non-core proteins in all viruses. Average degree of core vs non-core proteins for all five interactomes. P-values were calculated with a Wilcoxon rank test between the degree values of core and non-core proteins. Found at: doi:10.1371/journal.ppat.1000570.s022 (0.06 MB PDF) Table S9 Ortholog protein interactions (predicted from KSHV) tested by Y2H and CoIP. List of orthologous interactions predicted from the KSHV interactome [23] which were tested by co-immunoprecipitation in HSV-1, mCMV and EBV. Results from the Y2H analysis of the predicted interactions are also indicated. Found at: doi:10.1371/journal.ppat.1000570.s023 (0.00 MB PDF) Table S10 Negatively predicted orthologous protein interactions (predicted from core interaction network) tested by Y2H and CoIP. Ten interactions were predicted to be negative, based on the fact that they were not observed in any of the five viral interactomes, and analysed by co-immunoprecipitation. Found at: doi:10.1371/journal.ppat.1000570.s024 (0.02 MB PDF)
Table S11 Analysis of interspecies interactions. VZV core and noncore baits were analysed for Y2H interactions against prey libraries of VZV, HSV-1, mCMV, EBV and KSHV. The species of the interacting prey is included, in addition to whether it was a core or a noncore protein. Table S12 M51 interactions tested by CoIP. Interactions observed with mCMV M51, or with other orthologs of M51, with a subset of interaction partners from the Y2H analysis. Tegument proteins, and other virion components, were determined based on whether they were reported to be present in the CMV virion [59, 60] .  Insertion/Deletion Polymorphism of Angiotensin Converting Enzyme Gene in Kawasaki Disease Polymorphism of angiotensin converting enzyme (ACE) gene is reported to be associated with ischemic heart disease, hypertrophic cardiomyopathy, and idiopathic dilated cardiomyopathy. In this study, we investigated the relationship between Kawasaki disease and insertion/deletion polymorphism of ACE gene. Fifty five Kawasaki disease patients and 43 healthy children were enrolled. ACE genotype was evaluated from each of the subjects' DNA fragments through polymerase chain reaction (PCR). Frequencies of ACE genotypes (DD, ID, II) were 12.7%, 60.0%, 27.3% in Kawasaki group, and 41.9%, 30.2%, 27.9% in control group respectively, indicating low rate of DD and high rate of ID genotype among Kawasaki patients (p<0.01). Comparing allelic (I, D) frequencies, I allele was more prevalent in Kawasaki group than in control group (57.3% vs. 43.0%, p<0.05). In Kawasaki group, both genotype and allelic frequencies were not statistically different between those with coronary dilatations and those without. ACE gene I/D polymorphism is thought to be associated with Kawasaki disease but not with the development of coronary dilatations. Kawasaki disease is an acute systemic vasculitis and its diagnosis is made on clinical features. Main complication of the disease is coronary artery lesion that may result in myocardial infarction or sudden death. Coronary artery aneurysms or ectasia develop in approximately 15-25% of untreated children (1) . Since the intravenous immune globulin (IVIG) and aspirin therapy have been introduced, its mortality rate has decreased to 0.1%, but cardiac sequelae continues to occur in about 13% of Kawasaki disease patients (2) .
The etiology of Kawasaki disease is largely unknown despite the various suggested hypotheses. Based on epidemiologic and clinical manifestations, it is thought that Kawasaki disease is caused by some infectious agents (3) (4) (5) (6) (7) (8) (9) (10) . Hypercytokinemia and hyperchemokinemia have also been observed and are thought to cause vascular injuries by inflammatory reaction and immunologic activation (11) . Genetic factors are also thought to have influences on the development and progress of Kawasaki disease (12, 13) . Up to present days, Kawasaki disease is thought to be an infectious disease manifested by immunologic reaction in genetically susceptable person.
Angiotensin converting enzyme (ACE) breaks down the potent vasodilator, bradykinin to its inactivate metabolite and catalyzes angiotensin I to angiotensin II. Angiotensin II promotes hyperplasia and hypertrophy of vascular smooth muscle cells, induces the production of proinflammatory cytokines and causes endothelial dysfunction by free radical generation. By playing an important role in cardiovascular regulatory system, ACE gene has been proposed to be associated with various cardiovascular diseases, such as ischemic heart disease, hypertrophic cardiomyopathy, idiopathic dilated cardiomyopathy, and vascular hypertrophy (14) . Among the different ACE genetic loci, the insertion/deletion polymorphism coded within intron 16 has been studied in many literature to find the association with such diseases.
However, there has been few studies regarding the association between the polymorphism of ACE gene and Kawasaki disease. The present study investigates whether the I/D polymorphism of ACE gene (DD, ID, II) is associated with the prevalence and severity of Kawasaki disease among Korean pediatric populations.
Fifty five Kawasaki patients (mean age 28.2±25.2 months) diagnosed at Ewha Womans University Mokdong Hospital from January 2001 to June 2003, and 43 healthy children (mean age 28.5±17.2 months) were enrolled. Kawasaki disease was diagnosed by its clinical features, that is fever lasting for at least 5 days, accompanied by 4 of the 5 classical signs: 1) bilateral bulbar conjunctival injection; 2) pharynx, injected and/or dry fissured lips, strawberry tongue; 3) changes of the peripheral extremities in the acute phase or periungal desquamation in the subacute phase; 4) nonvesicular rash; 5) cervical adenopathy, ≥1.5 cm. The diagnosis was made if he/she had typical manifestations even before less than 5 febrile days of illness. All patients were treated with immunoglobulin (2 g/kg) on the day of diagnosis. High dose (50 mg/kg/day) aspirin was given from the day of diagnosis and its dosage was changed to 5 mg/kg/day after 2 nonfebrile consecutive days.
Two dimensional echocardiography was done to evaluate cardiac complications. Coronary arterial lesion was defined as following: 1) inner diameter that is >3 mm in children <5 yrs old and >4 mm in children ≥5 yrs old; 2) internal diameter of a segment ≥1.5 times that of an adjacent segment; or 3) lumen with irregular surface. Among these patients, 17 showed coronary dilatations and 38 did not.
Informed consent was obtained from his/her parents prior to the participation in the study. The study was approved by the hospital's ethics commitee.
Each of the subjects' DNA was extracted from whole blood at the time of diagnosis using QIAamp DNA Blood Mini Kit (Gene Company LTD., Chai Wan, Hong Kong). DNA fragments were amplified through polymerase chain reaction (PCR), which was carried out in a total volume of 10 L containing 50 ng of genomic DNA, 200 mM dNTPs, 0.3 mM/mL of each primers (5′ -CTGGAGACCACTCCCATCCTTTCT); (5′ -GATGTGGCCATCACATTCGTCAGAT) in PCR buffer with 0.5 units Taq DNA polymerase (Takara, Shiga, Japan). After the initial denaturation step (10 min at 95℃), 35 cycles were repeated for 30 sec at 94℃, 30 sec at 52℃, 90 sec at 72℃, and 5 min at 72℃. DNA fragments were then separated by electrophoresis on 2.5% agarose gel.
Student's t-test was used to compare the demographic characteristics between the two groups. Chi square test and Fisher exact test were performed to compare the genotype and allelic frequencies between the two groups. Its frequencies were compared by odds ratio. p value less than 0.05 was considered significant.
The mean age of Kawasaki group (28 boys and 27 girls) was 28.2±25.2 months, and that of the control group (30 boys and 13 girls) was 28.5±17.2 months. Among 55 Kawasaki disease patients, coronary dilatation was observed in 17 patients (8 boys and 9 girls, Table 1) Frequencies of ACE genotypes (DD, ID, II) were 12.7%, 60.0%, 27.3% in Kawasaki group, and 41.9%, 30.2%, 27.9% in control group respectively, indicating low rate of DD genotype (p<0.01, odds ratio=0.2) and high rate of ID genotype (p<0.01, odds ratio=3.3) among Kawasaki patients (Table 2) .
Comparing allelic (I, D) frequencies, I allele was more prevalent in Kawasaki group than in control group (57.3% vs. 43.0 %, p<0.05, odds ratio=1.78, Table 3 ).
In Kawasaki group, both genotype (DD, ID, II) and allelic frequencies were not statistically different between those with coronary dilatations and those without ( 
The ACE gene is localized on chromosome 17q23 and is characterized by a major insertion/deletion polymorphism consisting of the presence or absence of a 287-base pair Alu repeat sequence within intron 16 (15) . Angiotensin converting enzyme is an ectoenzyme found on the external surface of the endothelial and epithelial cell membranes. It enhances the synthesis of angiotensin-II, that promotes proliferation, migration, and hypertrophy of vascular smooth muscle cells. Angiotensin II also induces the production of proinflammatory cytokines and matrix metalloproteinases (16, 17) . Moreover, the increased free radical generation by angiotensin-II contributes to endothelial dysfunction (18, 19) .
In humans, the ACE activity is partly under genetic control. It is suggested that about half of the interindividual difference in ACE levels may be accounted for its polymorphism (19, 20) . It is reported that mean ACE levels were lowest for II homozygotes, highest for DD homozygotes, and intermediate for ID heterozygotes (18, 21) . Danser et al. explained the higher ACE levels observed in subjects with D allele than those with the II genotype by the sequence harbored in the insert of ACE gene. This sequence was said to be very similar to a silencer element (22) . But it is not clear whether increased levels of ACE actually affects the levels of angiotenisn II because the renin-angiotensin system is regulated by feedback mechanism (22) .
Angiotensin converting enzyme is mainly produced by vascular endothelial cells (23) . In Kawasaki disease, the associated endothelial cell damage subsequently lowers the ACE level. It is reported in some literature (24, 25) that serum ACE levels are significantly attenuated during the acute phase, and recovered during the convalescent phase of Kawasaki disease.
Slowik et al. (26) reported that the II genotype of ACE gene contributes to vascular dilatation at the site of aneurysm by 1) increased bradykinin activity, 2) another polymorphism responsible for vascular dilatation that is in linkage disequilibrium with ACE I/D polymorphism, 3) degeneration of endothelial cells, or 4) lack of vascular remodeling.
There is pathological difference between adult coronary artery disease (CAD) and that caused by Kawasaki disease. CAD caused by Kawasaki disease is characterized by vascular intimal thickening, whereas the adult CAD is characteri-zed by atherosclerotic lesions initiated by atheroma and plaque formation. Thus, the pathophysiologic mechanism of myocardial ischemic development is also different; in adult CAD, plaque rupture and thrombus formation plays the important role, whereas in Kawasaki disease, coronary arterial narrowing by intimal hyperplasia is responsible (24) .
In this study, the ID genotype was more prevalent (59.3 vs. 30.2%, p<0.01), and the DD genotype (12.9 vs. 41.9%, p< 0.01) was less prevalent in Kawasaki group than in control group. And I allele was more prevalent in Kawasaki group than in control group. However, both genotype (DD, ID, II) and allelic frequencies were not statistically different between Kawasaki disease patients with coronary dilatations and those without. Therefore, ACE gene I/D polymorphism was thought to be associated with the prevalence of Kawasaki disease but not with the development of coronary lesions. These results are similar to the report by Wu et al. (13) that the DD genotype was present in lower frequency among Kawasaki patients and ACE polymorphism was not associated with coronary aneurysmal formation. These results are different from the study by Takeuchi et al. (27) that the II genotype of ACE gene is more prevalent in Kawasaki disease and those with coronary aneurysm. These inconsistent results may be accounted for different ethnic traits of the individual population.
In conclusion, the ID genotype is present in a significantly higher frequency and DD in lower frequency among Kawasaki disease patients than in control subjects. In addition, there are no significant association between ACE I/D polymorphism and the coronary artery aneurysm formation in Kawasaki disease patients. Because the study groups are relatively small size in number, it is difficult to generalize these results. In this study, the coronary arterial dilatation was observed in 17 out of 55 Kawasaki patients. This is higher than that in previously reported literature. It can be accounted for either 1) longer duration from the onset of illness to the infusion of immunoglobulin, 2) other factors that may affect the development of coronary arterial lesions in Kawasaki disease, such as different ethinicity or 3) selection bias caused by limited number of enrolled institution and small number of patients. IVIG is recommended to be given within the first 10 days of illness and, if possible, within 7 days of illness. Since the duration of febrile days before the diagnosis and the IVIG infusion were 4.6±1.6 days (range, 2-8 days) in our study groups, the first explanation seems less likely.
Further study is required to clarity the association. Table 5 . Deletion/insertion allelic prevalence of angiotensin converting enzyme gene of Kawasaki patients with and without coronary dilatation Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors. Viruses need to interact with host macromolecules to hijack the cellular machinery and replicate. These interactions are essential for viruses to target endocytic pathways and penetrate host cells, to recruit cellular transcription and/or translation machinery, and to achieve intracellular migration and viral particles assembly. But viruses also encode virulence factors that induce a substantial alteration of host cell functions and genetic programs to increase virus replication and spreading. For example, specific viral factors stimulate survival pathways to prevent apoptosis of infected cells or inhibit cell signaling involved in immune response. Among these pathways, IFN-a/b signaling represents a prime target for viruses because of its critical role in the induction of both innate and adaptive antiviral immune responses [1] . IFN-a/b transduce signals through direct binding to a cell surface receptor composed of two transmembrane subunits, IFNAR1 and IFNAR2c [2] . This interaction activates IFNAR1/IFNAR2c associated kinases Tyk2 and Jak1 that subsequently phosphorylate STAT2 and STAT1 transcription factors. Activated STAT1 and STAT2, altogether with IRF9, form the Interferon-Stimulated Gene Factor 3 that binds IFN-stimulated response element (ISRE) promoter sequences to induce a large antiviral gene cluster. As a consequence, most viruses that are pathogenic in vertebrates have evolved virulence factors both to block IFN-a/b expression and signal transduction downstream of IFN-a/b receptor.
Human parainfluenza virus type 1 (hPIV1) and human parainfluenza virus type 3 (hPIV3) are important human pathogens that belong to Respirovirus genus (Paramyxoviridae family; [3] ). These viruses are responsible for upper respiratory tract infections and colds, but often spread to the lower respiratory tract causing bronchitis, bronchiolitis and pneumonia in young children and immuno-compromised patients. hPIV3 infection is also suspected to exacerbate chronic airway inflammatory diseases like asthma [4] . Sendai virus and bovine parainfluenza virus type 3 (bPIV3) are animal counterparts of hPIV1 and hPIV3 that infect mouse and cattle, respectively. Respirovirus genome is a single-strand, negativesense RNA molecule that encodes six structural proteins (Mononegavirales order). While hemagglutinin-neuraminidase (HN) and fusion (F) are membrane glycoproteins associated with the envelop of hPIV3 particles, the nucleoprotein (N), the phosphoprotein (P) and the viral polymerase (L) form the ribonucleocapsid complex. The matrix protein (M) is at the interface between glycoprotein tails and ribonucleocapsids.
The P gene of Respirovirus encodes for P but also for a panel of accessory proteins by site-specific editing of P mRNA and usage of overlapping open reading frames (ORFs). In all Respirovirus except hPIV1, the co-transcriptional insertion of one G residue at an editing motif midway of P mRNA leads to the expression of a chimeric protein called V. The V proteins of bPIV3 and Sendai virus bind MDA5 and suppress double-stranded RNA-stimulated IFN-b production, thereby contributing to the virus evasion of host immune response [5] . Surprisingly in hPIV3, multiple stop codons localized downstream of the editing site prevent the normal expression of a full-length V protein. As a result, P mRNA molecules edited by the addition of one G residue encode for the 242 amino acid (AA)-long N-terminal residues of P followed by only six additional AA (see Materials and Methods and [6] ). But P mRNA molecules edited by the addition of five G residues encode for D, a protein exhibiting a large and specific C-terminal domain of unknown function ( Figure 1A ). Besides co-transcriptional edition, an overlapping ORF embedded in the first half of the P mRNA allows the expression of a single C protein (hPIV3 and bPIV3) or a nested set of four proteins called C9, C, Y1 and Y2 (Sendai virus and hPIV1). The C proteins of Sendai virus and hPIV1 have a high degree of sequence homology and have been studied in details. They are involved in the regulation of viral RNA synthesis [7, 8] , the inhibition of innate immune response [9] and potentially contribute to the budding of viral particles [10] [11] [12] . In particular, the C protein of Sendai virus both inhibits IFN-b production [13, 14] and blocks interferon signaling downstream of IFN-a/b and IFN-c receptors [15] [16] [17] . The C proteins of hPIV3 and bPIV3 only share ,35% of sequence homology with the C proteins of Sendai virus and hPIV1, but they have also been shown to target interferon expression and signaling [5, 18] . Although expression of the C protein of hPIV3 (hPIV3-C) is essential to virulence in vitro and in vivo [19] and explains hPIV3 ability to block IFN-a/b signaling [20] , host proteins that bind hPIV3-C remain unknown. (A) Organization of the gene P of hPIV3 that encodes for three proteins: P, D and C. Whereas conventional transcription and translation lead to the expression of the phosphoprotein P, co-transcriptional insertion of five G residues at the editing site by the virus RNA polymerase leads to the expression of a chimeric protein called D. Insertion of one G residue can also occur during transcription but two stop codons immediately downstream of the editing site prevent the expression of the protein V that is specific of Paramyxoviridae. The protein C is encoded by an overlapping opened reading frame (ORF) embedded in P mRNA. (B and C) HEK-293T cells were transfected with expression vectors encoding GST alone or fused to the C proteins of measles virus (MV-C), hPIV3 (hPIV3-C), or Nipah virus (Nipah-C), and tested for the interaction with endogenous STAT1 (B) or GRB2 (C). Total cell lysates were prepared 48 h post transfection (cell lysate; middle and lower panels), and copurifications of endogenous cellular proteins were assayed by pulldown using glutathione-sepharose beads (GST pull-down; upper panel). GST-tagged C proteins were detected by immunoblotting using anti-GST antibody, while endogenous STAT1 and GRB2 were detected with specific monoclonal antibodies. doi:10.1371/journal.ppat.1000587.g001
Respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immunocompromised individuals. Their RNA genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. Among them, the C protein plays a critical role by blocking cellular response to type I interferons, the main antiviral cytokines secreted during virus infections. To provide molecular basis to this activity, we found that the C protein of human parainfluenza virus type 3 (hPIV3-C), the most frequent human Respirovirus, interacts with STAT1, a key component of type I interferon receptor complex. But hPIV3-C was also found to interact with GRB2, an adaptor molecule involved in cellular response to Epidermal Growth Factor (EGF), and to enhance cell response to this cytokine. This pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. Thus, our findings show that hPIV3-C not only inhibits the antiviral response but also stimulates cellular response to EGF, which benefits virus replication and induces an excessive inflammation of airways during infection.
In an attempt to answer this question, we performed a yeast two-hybrid screen and we report here the identification of STAT1 and GRB2 as direct interactors of hPIV3-C. Although binding to STAT1 accounts for hPIV3-C ability to block IFN-a/b signaling, the interaction with GRB2 was unexpected. This adaptor protein bridges growth factor receptor tyrosine kinases (RTKs), like Epidermal Growth Factor (EGF) receptor, to the mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Upon engagement by their ligands, RTKs autophosphorylate on tyrosine residues to recruit adaptor proteins containing phosphotyrosine binding (PTB) or Src homology 2 (SH2) domains like GRB2 [21] . Once associated to RTKs by its SH2 domain, GRB2 recruits the guanine nucleotidereleasing factor son-of-sevenless (SOS) to activate Ras. Downstream events include MAPK/ERK kinase (MEK1/2) activation, which in turn phosphorylates ERK1/2. Finally, phosphorylated ERK1/2 directly or indirectly activates numerous cellular targets including transcription factors (e.g. Elk1, SAP1, SAP2, c-Fos, CREB, SRF) but also cellular factors that control mRNA translation like eukaryotic initiation factor 4E (eIF4E) or small ribosomal subunit S6 protein [22, 23] . Growth factor binding to RTKs regulates a multiplicity of cellular processes including proliferation, differentiation and survival. In the respiratory tract, this signaling cascade has been shown to trigger inflammation and mucus secretion by epithelial cells [24] [25] [26] [27] , suggesting a critical role in innate immunity [28] . However, excessive activation of this pathway could benefit to virus replication by inhibiting IFN-a/b signaling [29] and promoting infected cell survival [25] . Altogether, these data provided a rational to investigate the functional impact of hPIV3-C expression on IFN-a/b vs EGF receptor and MAPK/ERK signaling pathways.
The C protein of hPIV3 interacts directly with STAT1 and GRB2
To identify cellular targets of hPIV3-C, this viral protein was used as bait in the yeast two-hybrid system to screen a human cDNA library. The screen was performed at saturation with a 10-fold coverage of the library (50.10 +6 diploids), and positive yeast colonies growing on selective medium were analyzed by PCR and sequencing to identify binding partners of hPIV3-C. STAT1 and GRB2 were the main interactors of hPIV3-C identified in the screen with 5 and 150 yeast colonies corresponding to these cellular proteins, respectively. In both cases, cDNA clones retrieved from the screen corresponded to full-length STAT1 and GRB2 in frame with the Gal4-AD transactivation domain. To validate these interactions in human cells, GST-tagged hPIV3-C was expressed in HEK-293T cells and purified with glutathion-sepharose beads. As shown in Figure 1B and 1C, endogenous STAT1 and GRB2 co-purified with hPIV3-C. Highly divergent C proteins from measles virus (MV-C) and Nipah virus (Nipah-C) failed to do so, thereby demonstrating the specificity of identified interactions. Binding to STAT1 provides molecular basis to the inhibition of IFN-a/b signaling by hPIV3-C [18] , and parallels the interaction previously identified between Sendai virus C protein and mouse STAT1 [15] . Altogether, this suggests that STAT1 is a specific cellular interactor of Respirovirus C proteins. In contrast, binding to GRB2 is unexpected and suggests a new function for hPIV3-C that we decided to investigate.
hPIV3-C has opposite effects on IFN-a/b and EGF signaling pathways
The adaptor protein GRB2 plays a critical role in coupling signal from growth factor receptors to MAPK/ERK signaling pathway. To address the question of hPIV3-C interference with this pathway, we used a trans-reporter gene assay that measures Elk1 activation by ERK1/2. In this system, Elk1 transcription factor is fused to the DNA binding domain of Gal4 (Gal4-DB) and binds the promoter sequence of a luciferase reporter gene. Upon stimulation with a growth factor like EGF, Elk1 is activated as assessed by a significant increase in luciferase expression. Surprisingly, we observed a 6-fold enhancement in this cellular response to EGF when 36FLAG-tagged hPIV3-C was expressed in HEK-293T cells (Figure 2A ). Same results were obtained when using hPIV3-C without a tag (14-fold enhancement) or tagged with the red fluorescent protein Cherry (7-fold enhancement). In contrast to hPIV3-C, neither MV-C nor Nipah-C enhanced Elk1 activity upon EGF stimulation (Figure 2A ) whereas expression levels of hPIV3-C, MV-C and Nipah-C were similar in this system ( Figure 2F , left panel). Elk1 activity was also enhanced by hPIV3-C expression in Vero and Hela cells as well as BEAS-2B and A549, two epithelial cell lines that originate from the respiratory tract, which is the tissue targeted by hPIV3 in vivo ( Table 1 ). The effect of hPIV3-C in these different cell lines was highly significant (see p-values in Table 1 ) although relatively modest when compared to HEK-293T cells. This is probably because our reporter system requires the co-transfection of four plasmids and Vero, Hela, BEAS-2B and A549 cells are more difficult to transfect than HEK-293T. In parallel experiments, cellular response to IFN-a/b was monitored using a cis-reporter gene, of which expression is controlled by five ISREs. As previously reported [18] , we found that hPIV3-C efficiently blocked IFN-a/b signaling ( Figure 2B ) as opposed to what we observed for the EGF pathway. Again, MV-C or Nipah-C was unable to do so. Altogether, these results show that hPIV3-C enhances the cellular response to EGF in addition to its ability to block IFN-a/b signaling.
We also determined if similar effects on the EGF pathway were observed in infected cells. HEK-293T cells were infected with hPIV3 (MOI = 3) and then transfected with Elk1 activity reporter plasmids. Infection of HEK-293T cells was confirmed by anti-hPIV3 hemagglutinin-neuraminidase (hPIV3-HN) immunostaining and flow cytometry analysis ( Figure 2E ). Like hPIV3-C alone, hPIV3 infection enhanced Elk1 activity upon EGF stimulation ( Figure 2C ). Interestingly, hPIV3 infection induced a significant level of Elk1 activity in the absence of EGF stimulation. This suggests that in addition to hPIV3-C interaction with GRB2, other mechanisms modulate MAPK/ERK pathway during hPIV3 infection.
Finally, to demonstrate that enhancement of Elk1 activation by hPIV3-C is completely dependent on ERK1/2 activation, HEK-293T cells were pre-treated with MEK1/2 inhibitor U0126 before stimulation with EGF. This molecule targets MEK1/2 and totally abrogates downstream phosphorylation and activation of ERK1/2 [30] . As shown in Figure 2D , Elk1 activation was blocked by U0126, whereas hPIV3-C expression was maintained ( Figure 2F , right panel). This demonstrates that hPIV3-C is acting through ERK1/2 stimulation. Altogether, these results support a model where hPIV3-C interaction with GRB2 enhances cellular response to growth factors as assessed by an increased activation of MAPK/ ERK pathway.
Phosphorylation of ERK1/2, eIF4E and small ribosomal subunit S6 protein are stimulated by hPIV3-C expression or hPIV3 infection To further document hPIV3-C impact on MAPK/ERK signaling pathway, we compared the kinetic of ERK1/2 phosphorylation in HEK-293T cells expressing hPIV3-C or not. Cells were transfected hPIV3-C Impact on IFN-a/b and EGF Pathways In addition to these three plasmids, cells were co-transfected with an expression vector encoding 36FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-36FLAG. 12 h after transfection, cells were starved and 6 h later EGF was added at a final concentration of 100 ng/ml. After 24 h, relative luciferase activity was determined. (B) HEK-293T cells were transfected with pISRE-Luc, a plasmid containing a luciferase gene of which expression is controlled by five ISREs, and pRL-CMV. In addition to these two plasmids, cells were co-transfected with an expression plasmid encoding 36FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-36FLAG. 24 h after transfection, 1000 IU/ml of recombinant IFN-b were added. After 24 h, relative luciferase activity was determined. (C) HEK-293T cells were infected with hPIV3 (MOI = 3) and then transfected with pFA2-Elk1, pGal4-UAS-Luc, pRL-CMV vectors. 12 h later, cells were starved during 6 h and stimulated with EGF at a final concentration of 100 ng/ml. After 24 h, relative luciferase activity was determined. (D) Same experiment as (A) but 20 mM of MEK1/2 specific inhibitor U0126 was added as indicated. (A-D) All experiments were achieved in triplicate, and data represent means6SD. (E) HEK-293T cells were infected as in (C), and hPIV3-HN expression determined by immunostaining and flow cytometry analysis. (F) HEK-293T cells were transfected to express 36FLAG-tagged MV-C, hPIV3-C or Nipah-C as described in (A) and (B), and relative expression levels were determined 36 h later by western blot analysis (left panel). In a parallel experiment, HEK-293T cells were transfected to express hPIV3-C and were cultured with or without EGF in the presence or absence of U0126 as described in (D). hPIV3-C expression level was determined by western blot analysis (right panel). doi:10.1371/journal.ppat.1000587.g002 with 36FLAG-tagged hPIV3-C or a control plasmid and 24 h post transfection, they were starved before stimulation with EGF. ERK1/ 2 phosphorylation was determined at 10, 30 and 120 min after stimulation. As illustrated by one representative experiment in Figure 3A , EGF stimulation induced ERK1/2 phosphorylation in control cells but signal was markedly and reproducibly increased by hPIV3-C expression at maximum phosphorylation time point (1.4 to 2.8 fold increase; p = 0.005; n = 4). We then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mRNA translation, the translation initiation factor eIF4E and the ribosomal protein S6 ( Figure 3B ). Before EGF stimulation, low levels of phosphorylated eIF4E and S6 were detectable in mock-treated cells ( Figure 3B and 3D). hPIV3-C expression had virtually no effects on this background. Thus, eIF4E and S6 phosphorylation levels were determined at different time-points after EGF stimulation. Because ERK1/2 activation precedes eIF4E and S6 phosphorylation, maximal phosphorylation occurs at later time points and was determined at 30 min, 2 h, 6 h and 24 h after stimulation. As observed for ERK1/2, phosphorylation levels of eIF4E and S6 were enhanced by hPIV3-C expression when stimulating the cells with EGF.
To validate these observations in infected cells, HEK-293T cells were infected with hPIV3 (MOI = 3) and 24 h later, cells were starved for 12 h before stimulation with EGF. Like hPIV3-C expression alone, hPIV3 infection enhanced ERK1/2 phosphorylation at the peak of induction, i.e. 10 min after adding EGF to the cells ( Figure 3C ). Interestingly, hPIV3 infection of A549 cells also enhanced ERK1/2 phosphorylation but the induction profile was different. Indeed, ERK1/2 phosphorylation was not significantly increased at the peak of induction, but the signal was boosted by hPIV3 infection at late time points ( Figure S1 ). The same profile was observed when eIF4E and S6 phosphorylation levels were analyzed in infected HEK-293T cells. hPIV3 infection sustained the phosphorylation of these two translation factors at late time points, but showed no increase at the peak of stimulation, i.e. 30 min after adding EGF to the cells ( Figure 3D ). This could relate to the fact that hPIV3 infection also induces low levels of eIF4E and S6 phosphorylation in the absence of EGF stimulation ( Figure 3D ). This is reminiscent to what was observed for Elk1 ( Figure 2C ), and suggests that hPIV3 infection induces a basal activation of MAPK/ERK pathway leading to the constitutive phosphorylation of downstream targets.
Altogether, these data demonstrate that hPIV3 infection or hPIV3-C expression alone both enhance MAPK/ERK pathway activation in EGF-stimulated cells. Several RNA viruses require an activated MAPK/ERK pathway to produce viral components and replicate properly (for review see [31] ). To test if the same was true for hPIV3, cells were treated for 2 h with MAPK/ERK pathway inhibitor U0126 and infected with hPIV3 (MOI = 1). Two days after infection, cell surface expression of hPIV3-HN was detected by immunostaining and flow cytometry. U0126 completely blocked the expression of hPIV3-HN in hPIV3-infected cells (Figure 4 ), whereas the same inhibitor had no effect when cells were infected with MV ( Figure S2 ). Altogether, this demonstrates that MAPK/ERK signaling is essential for the expression of hPIV3 proteins and suggests that hPIV3 manipulates this pathway to increase replication efficiency.
The C-terminal region of hPIV3-C binds STAT1 and GRB2
To better understand how hPIV3-C targets both the IFN-a/b and EGF signaling pathways, we characterized the functional domains of hPIV3-C that bind STAT1 and GRB2. To do so, we generated by PCR a full matrix of hPIV3-C overlapping fragments and tested their ability to interact with either STAT1 or GRB2 in the yeast two-hybrid system ( Figure 5 and 6 ). Both forward and reverse primers were designed every 75 nucleotides along hPIV3-C sequence and fused to appropriate tails to allow gap-repair recombination with linearized Gal4-DB yeast two-hybrid vector ( Figure 5A ). All possible combinations of forward and reverse primers were used to amplify hPIV3-C fragments. Finally, corresponding PCR products were transformed in a yeast strain expressing Gal4-AD fused to either STAT1 or GRB2, and growth on selective medium was used to detect potential interactions. A 124 (AA)-long peptide encompassing position 76 to 199 located in the C-terminal half of hPIV3-C was sufficient to bind STAT1 ( Figure 5B ) or GRB2 ( Figure 6A ). In an iterative process, we then generated a second, a third and a fourth set of hPIV3-C fragments corresponding to one-by-one AA deletions ( Figure 5C -E and Figure 6B -D), allowing to further reduce the STAT1 and GRB2 binding motifs to minimal peptides. A 106 AA peptide encompassing residues 90 to 195 of hPIV3-C was sufficient to observe the interaction with STAT1 ( Figure 5E ). The binding region to GRB2 was virtually the same, encompassing AA 97 to 195 ( Figure 6D ).
The C-terminal region of hPIV3-C required to bind STAT1 and GRB2 in the yeast two-hybrid system is highly conserved among Respiroviruses ( Figure 7A ) and suspected to fold into a structured coiled-coil domain [18] . Furthermore, virtually the same C-terminal region of Sendai virus C protein (AA 85-204) was previously reported to mediate the interaction with mouse STAT1 [32] . To further validate our observations performed in the yeast two-hybrid system, we retested by co-affinity purification the ability of hPIV3-C fragment encompassing AA 90-195 (hPIV3-C 90-195 ) to interact with STAT1 and GRB2 in HEK-293T cells. GST-tagged hPIV3-C 90-195 was expressed together with 36-FLAG-tagged STAT1 or GRB2, and purified with glutathionsepharose beads. Full-length hPIV3-C and the N-terminal region encompassing AA 1-89 (hPIV3-C 1-89 ) were used as positive and negative controls, respectively. As shown in Figure 7B and 7C, hPIV3-C 90-195 interacted with STAT1 and GRB2, whereas hPIV3-C 1-89 did not. Although hPIV3-C 90-195 interacted with GRB2 as efficiently as full-length hPIV3-C ( Figure 7C ), interaction with STAT1 was weaker suggesting that more residues contribute to the stabilization of this interaction ( Figure 7B ). Altogether, these results confirm that AA 90-195 of hPIV-3 include both STAT1 and GRB2 binding sites. As described in Figure 2 , HEK-293T, Hela, Vero, A549 or BEAS-2B cells were transfected with pFA2-Elk1, pGal4-UAS-Luc, pRL-CMV to measure the activation level of MAPK/ERK signaling pathway. Cells were co-transfected with plasmids encoding 36FLAG-tagged MV-C, hPIV3-C or Nipah-C or the corresponding empty vector pCI-neo-36FLAG. 12 h after transfection, cells were starved and stimulated 6 h later with 100 ng/ml of EGF. After 24 h, expression of luciferase was quantified. Results were normalized so that reporter activity in cells transfected with a control vector equals 1. Experiments were performed in triplicates and data represent means6SD. doi:10.1371/journal.ppat.1000587.t001
hPIV3-C Impact on IFN-a/b and EGF Pathways Although STAT1 and GRB2 essentially bind to the same region of hPIV3-C as demonstrated above, it remained unclear whether these interactions are mutually exclusive. To answer this question, a competition experiment was designed where GST-tagged hPIV3-C was co-expressed with STAT1 in the presence or absence of GRB2 ( Figure 7D ). In this setting, GRB2 expression prevents STAT1 copurification together with GST-tagged hPIV3-C. This validates our finding that STAT1 and GRB2 interact with the same region of hPIV3-C, and demonstrates that STAT1 and GRB2 compete for hPIV3-C binding. Interestingly, GRB2 interaction with hPIV3-C was not affected by STAT1 expression (Figure 7D and data not shown), suggesting that GRB2 has a higher affinity for hPIV3-C than STAT1.
Both the N-and C-terminal domains of hPIV3-C are required for its activity
We finally tested if hPIV3-C 90-195 was able, like full-length hPIV3-C, to block IFN-a/b signaling and enhance cellular response to EGF stimulation. First, cells were transfected with 36FLAG-tagged hPIV3-C, hPIV3-C 90-195 or hPIV3-C 1-89 together with the IFN-a/b reporter plasmid, and stimulated 24 h later with recombinant IFN-b. Reporter gene expression was determined 24 h post transfection and found to be inhibited exclusively by full-length hPIV3-C ( Figure 8A ). Although this may reflect the weakness of hPIV3-C interaction with STAT1 ( Figure 7B ), this also indicates that both the N-terminal and C-terminal regions of hPIV3-C are required to block IFN-a/b signaling, even if only the C-terminal region is required for the binding to STAT1. The same constructs were tested using the Elk1 activity reporter plasmids ( Figure 8B ). Again, only full-length hPIV3-C was able to enhance Elk1 activation upon EGF stimulation whereas full-length hPIV3-C and hPIV3-C 90-195 were expressed at similar levels in transfected cells ( Figure 8B , upper right panel).
Because GRB2 binding to hPIV3-C and hPIV3-C 90-195 were essentially equivalent in co-affinity purification experiments, we hypothesized that the N-terminal region of hPIV3-C was required for its proper subcellular localization. Thus, hPIV3-C, hPIV3-C 90-195 and hPIV3-C 1-89 were expressed in fusion downstream of the red fluorescent protein Cherry. As shown in Figure 8C , Cherry alone or fused to hPIV3-C 90-195 localized both in the nucleus and the cytoplasm of transfected cells. In contrast, full-length hPIV3-C essentially accumulated at the cellular membrane whereas hPIV3-C 1-89 was in the nucleus. Although we have no explanation for this unexpected localization of hPIV3-C 1-89 , these observations show that only full-length hPIV3-C is able to target the cellular membrane where both IFN-a/b and EGF signaling are triggered.
Paramyxoviridae have evolved various mechanisms to block IFNa/b response, in particular signaling downstream IFNAR1/ IFNAR2c receptor [20, 33] . Although members of Pneumovirinae subfamily have specific genes to encode inhibitors of IFN-a/b signaling pathway, those expressed by other Paramyxoviridae (i.e. Paramyxovirinae subfamily) are encoded by overlapping reading frames embedded within the gene P. Rubulaviruses express V proteins that target STAT1 and/or STAT2 for ubiquitination and degradation, while Morbilliviruses and Henipaviruses V proteins essentially impair STAT1/2 phosphorylation, activation and nuclear translocation. In addition, Morbilliviruses and Henipaviruses also encode for C proteins of which role in the inhibition of IFNa/b response has been a matter of debates [34] [35] [36] [37] . Recent reports showed that Morbillivirus C proteins only have a minor role in the inhibition of IFN-a/b signaling [37] , but are essential to block IFN-a/b induction [38] . Whether Henipavirus C proteins can directly block IFN-a/b or promote viral replication through alternative mechanisms is unclear [35] . In contrast, it has been clearly established that Respirovirus C proteins are potent inhibitors of IFN-a/b signaling [5, 18, [39] [40] [41] . In this report, we show that hPIV3-C, but not MV-C or Nipah-C, directly interacts with STAT1 and efficiently inhibits IFN-a/b signaling. In addition, we identified a minimal STAT1 binding domain that encompasses AA 90-195 of hPIV3-C, a region suspected to fold into a coiled coil. Interestingly, this conserved domain is localized within the STAT1 binding region shared by all four isoforms of Sendai virus C protein [32] . Together, these results confirm the capacity of hPIV3-C to block IFN-a/b signaling pathway [18] , provide molecular basis to this inhibition and clarify the fact that Respirovirus C proteins are functionally distinct from Morbillivirus and Henipavirus C proteins.
In addition to STAT1, we show that hPIV3-C interacts directly with GRB2 and enhances MAPK/ERK signaling downstream of EGF receptor (EGFR). Our data give the first example of a Paramyxoviridae protein that contributes to the stimulation of EGFR and MAPK/ERK pathway and provides molecular basis to this activity. This pathway has been known for decades as a prime target of DNA tumor viruses and oncogenic retroviruses, and its activation represents an essential step toward carcinogenesis [3, 42] . But recent data demonstrate that non-oncogenic RNA viruses also activate this signaling cascade to support viral replication and spreading [31] . Whether it is activated upon EGFR engagement or other means, MAPK/ERK pathway regulates a multiplicity of cellular processes including proliferation, differentiation, development, cell survival and inflammation. As a consequence, how the activation of MAPK/ERK pathway promotes viral replication is a complex question. Interestingly, two non-oncogenic RNA viruses associated with acute respiratory tract infections have been recently reported to modulate the EGFR pathway. Both human respiratory syncytial virus (hRSV), a member of Paramyxoviridae like hPIV3, and a rhinovirus that belongs to Picornaviridae family activate EGFR and MAPK/ERK pathway [25, 27] . Infection of epithelial cells by these viruses stimulates the processing and activation of EGFR ligands by membrane matrix metalloproteinase and subsequent engagement of EGFR through autocrine/paracrine mechanisms. Experiments performed on rhinovirus show that viral replication and TLR3 engagement by viral RNA are both required to activate the EGFR and MAPK/ERK pathway [27] . In this report, we show that hPIV3 infection also activates MAPK/ERK pathway in the absence of external stimuli, a phenomenon that possibly relies on the engagement of pathogen recognition receptors. Although hPIV3-C alone is unable to activate this pathway, our data suggest that expression of this virulence factor enhances MAPK/ERK activation above normal level in infected cells, thereby contributing to viral replication and pathogenesis.
Induction of MAPK/ERK pathway by RNA viruses has numerous consequences on cell biology. First, it results in increased expression of inflammatory factors, in particular cytokines and chemokines that recruit cellular effectors of immunity [27, [43] [44] [45] [46] [47] . MAPK/ERK pathway was also reported to block the antiviral response induced by IFN-a/b, making its activation beneficial to virus replication [29] . Another consequence of MAPK/ERK pathway activation is the induction of mucin production by infected epithelial cells [24, 27] . Although mucin expression is a critical innate defense system, excessive production of mucus results in the obstruction of airways and delays the elimination of pathogens. Finally, it has been demonstrated in vitro that upon hRSV infection, activation of EGFR and MAPK/ERK pathway sustains viral replication by retarding the death of infected cells [25] . Altogether, these data suggest that although a moderate activation of MAPK/ERK pathway contributes to the innate response against viruses, an excessive activation leads to deleterious inflammation, inhibition of IFN-a/b response, airway obstruction and infected cell survival Figure 6 . Identification of a minimal hPIV3-C region interacting with GRB2. Fragments of hPIV3-C were generated and tested for their ability to interact with GRB2 following the procedure described in Figure 5A . (A) The first iteration identified a GRB2 binding region of 124 AA. After two (B), three (C) and four additional rounds (D), this domain was finally reduced to a minimal GRB2 binding motif of 99 AA. This binding domain, encompassing position 97 to 195, is contained in the STAT1 binding region of hPIV3-C previously identified in Figure 5 . doi:10.1371/journal.ppat.1000587.g006 hPIV3-C Impact on IFN-a/b and EGF Pathways [28] . Therefore, it is tempting to speculate that hPIV3-C interaction with GRB2 and EGFR pathway participates in such deregulation of airway epithelium homeostasis to promote hPIV3 replication and spreading. A consequence of such perturbations could be an aggravation of chronic inflammatory airway diseases like asthma or chronic obstructive pulmonary disease as already suggested by epidemiological links with Paramyxoviridae infections and in vivo models [48, 49] .
Besides its effects on immune response, activation of MAPK/ ERK pathway has direct consequences on viral replication as assessed by in vitro experiments. It is now well documented that MAPK/ERK pathway inhibition with U0126 or PD098059 deeply impairs the replication of numerous RNA viruses including hRSV ( [50] ; and for review see [31] ). Similarly, we show in this report that MAPK/ERK pathway inhibition prevents hPIV3 protein expression in infected cells as assessed by hPIV3-HN detection. In influenza virus infected cells, membrane accumulation of influenza virus hemagglutinin (HA) induces lipid-rafts clustering that leads to MAPK/ERK pathway activation and nuclear export of viral ribonucleoprotein complexes to achieve A and B) hPIV3-C 90-195 and hPIV3-C 1-89 were tested for their ability to modulate IFN-a/b or EGF signaling. (A) As described in Figure 2 , HEK-293T cells were transfected with pISRE-Luc and pRL-CMV to determine the activation level of IFN-a/b signaling pathway. Cells were co-transfected with expression plasmids encoding 36FLAG-tagged full-length hPIV3-C (C FL ) or fragments. 24 h after transfection, 1000 IU/ml of recombinant IFN-b were added. After 24 h, relative luciferase activity was determined. Experiments were performed in triplicates, and data represent means6SD. (B) As described in Figure 2 , cells were transfected with pFA2-Elk1, pGal4-UAS-Luc and pRL-CMV to determine the activation level of MAPK/ERK signaling pathway. Cells were co-transfected with expression plasmids encoding full-length hPIV3-C (C FL ) or fragments. 12 h later, cells were starved during 6 h and stimulated with EGF at a final concentration of 100 ng/ml. After 24 h, relative luciferase activity was determined. Experiments were performed in triplicate, and data represent means6SD. As a control, relative expression levels of C FL , C 90-195 and C 1-89 were determined by western blot analysis (upper right panel). (C) Full-length hPIV3-C, hPIV3-C 90-195 or hPIV3-C 1-89 was expressed in fusion downstream of the red fluorescent protein Cherry to determine subcellular localization in HEK-293T cells. 24 h after transfection, cells were fixed with PFA, permeabilized and labeled with DAPI to stain nuclei. Single confocal sections show Cherrytagged protein fluorescence in red and DAPI staining in blue. doi:10.1371/journal.ppat.1000587.g008 viral particles assembly [51] [52] [53] . Because hPIV3 replication cycle is only cytoplasmic, mechanisms involved are necessarily distinct. A possible link between MAPK/ERK pathway and hPIV3 protein expression lies in the fact that among downstream targets of this pathway are essential factors of cellular translational machinery.
We show that hPIV3-C expression enhances the phosphorylation of S6 and eIF4E. The small ribosomal subunit protein S6 is the major phosphoprotein of eukaryotic ribosomes with five phosphorylation sites (Ser235, Ser236, Ser240, Ser244, and Ser247). Two families of serine/threonine kinases phosphorylate S6 in vitro: S6K1/2 and p90 ribosomal S6 kinase (RSK). Recently it has been shown that MAPK/ERK signaling pathway activates RSK family members that contribute to S6 phosphorylation on Ser235/236 thereby stimulating cap-dependent translation [23] . In addition, eIF4E that interacts with the cap structure and brings translation initiation factors together with the small ribosomal subunit via the scaffold protein eIF4G, undergoes regulated phosphorylation on Ser209 upon MAPK/ERK pathway activation. This phosphorylation event is dependent on eIF4Gassociated MAPK signal-integrating kinases, Mnk1 and Mnk2 [22] . eIF4E is believed to be the least abundant of all initiation factors and therefore considered as a perfect target to regulate protein synthesis. Even though there is no direct link between eIF4E phosphorylation and the enhanced translation observed, the fraction of phosphorylated eIF4E dramatically increases following treatment of the cells with growth factors, hormones and mitogens. Therefore, eIF4E phosphorylation has been associated with increased translation rates. hPIV3 mRNAs are capped and polyadenylated like their host counterparts. Thus, S6 and eIF4E phosphorylation together with a high level of viral gene transcription may contribute to a rapid switch toward viral protein synthesis within infected cells.
Specific biochemical investigations are still required to decipher how hPIV3-C can both inhibit IFN-a/b signaling and enhance EGFR and MAPK/ERK pathway. When searching the literature for viral proteins that target GRB2, we found specific reports on NS5A from hepatitis C virus and ORF3 from hepatitis E virus [54, 55] . Although NS5A inhibits MAPK/ERK activation induced by exogenous EGF, ORF3 was described as an activator of MAPK/ERK pathway like hPIV3-C. Both NS5A and ORF3 exhibit a proline-rich motif (PXXP) to bind the Src homology 3 (SH3) domains of GRB2, but there is no such motif in hPIV3-C suggesting that other mechanisms mediate STAT1 and GRB2 binding. Interestingly, these two cellular proteins exhibit SH2 domains. Such domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein. Although there is no evidence that hPIV3-C becomes phosphorylated, we have tested hPIV3-C interaction with mutant STAT1 and GRB2 exhibiting SH2 domains disabled for the interaction with phosphotyrosine residues. These mutants were not affected for the interaction with hPIV3-C (data not shown). This suggests that hPIV3-C either binds distinct regions of STAT1 and GRB2, or interacts with a region of the SH2 domain that does not involve the phosphotyrosine binding site. Finally, our results also show that full-length hPIV3-C is required to modulate IFNa/b and MAPK/ERK pathways since AA 90-195 that bind STAT1 and GRB2 are unable to do so when expressed alone. Full-length hPIV3-C was also required to observe a localization at the cell membrane, suggesting a link with its activity. Interestingly, the N-terminal 23 residues of Sendai virus C protein act as a membrane targeting signal [56] . But the N-terminal residues of hPIV3-C (AA 1-89) were unable to do so, and sequence analysis did not show any conservation with the C protein of Sendai virus. Thus, hPIV3-C tertiary structure is apparently required to target this protein at the cell membrane. This specific localization could both sequester STAT1 to prevent the stimulation of IFN-target genes and contribute to the aggregation of GRB2-SOS complexes to enhance MAPK/ERK signaling [57] . Altogether, this suggests that hPIV3-C interaction with STAT1 and GRB2 represents a potential target for the development of antiviral molecules against hPIV3 and possibly other members of Respirovirus genus.
Plasmid DNA constructs P-encoding sequence from hPIV3 wild-type strain (DF042505) was amplified by RT-PCR (Titan One tube; Roche Applied Science) from total RNA purified from infected cells (RNeasy kit; Qiagen). Amplification was performed using the following hPIV3-P specific primers flanked with Gateway cloning sites: 59-gggga-caactttgtacaaaaaagttggcatgGAAAGCGATGCTAAAAACTATC-AAA and 59-ggggacaactttgtacaagaaagttggttaTTGGCAATTATT-GACATCTTCATTGAAC. PCR products were cloned using TOPO TA Cloning kit (Invitrogen) into TOPO vector. A total of 21 clones were analyzed to establish the sequence of hPIV3-P (GenBank ID: EU719627). Interestingly, 8 clones were not edited, 11 clones were edited by the addition of one G residue, and 2 clones were edited by the addition of 5 G residues. One of the plasmids containing the unedited sequence of hPIV3-P was selected and subsequently used as a template to clone hPIV3-C.
DNA sequences encoding full-length hPIV3-C or fragments corresponding to AA 1-89 or 90-195 were amplified by PCR from p(hPIV3-P)-TOPO and cloned by in vitro recombination into pDONR207 (Gateway system; Invitrogen) as previously described [58] . Similarly, MV-C was amplified from p(+)MV323 that contains the full genome of measles virus wild-type strain Ichinose (kindly provided by Dr. K. Takeuchi, [59] ). Nipah-C was amplified from NiV-P plasmid (kindly provided by Dr. TF. Wild; [60] ). GRB2 coding sequence was amplified from the human spleen cDNA library used to perform the yeast two-hybrid screen (Invitrogen). The pDONR207 plasmid containing STAT1 was previously described [58] . Viral or cellular coding sequences were subsequently transferred by in vitro recombination from pDONR207 into different Gateway-compatible destination vectors (see below) following manufacturer's recommendation (LR cloning reaction, Invitrogen). To perform yeast two-hybrid experiments, coding sequences were recombined into pPC86 (Invitrogen) to be expressed in fusion downstream of the activation domain of Gal4 (Gal4-AD) or into pDEST32 to be expressed in fusion downstream of the DNA binding domain of Gal4 (Gal4-DB). In mammalian cells, GST-tag and 36FLAG-tag fusions were achieved using pDEST27 (Invitrogen) or pCI-neo-36FLAG vector, respectively [61] . We also used pCI-neo (Promega) and pmCherry-C1 (Clontech) to express proteins without a tag or in fusion downstream of Cherry, respectively. These two plasmids were made Gateway-compatible using the Gateway vector conversion system (Invitrogen).
HEK-293T, Hela and Vero cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-Invitrogen) containing 10% fetal bovine serum, penicillin, and streptomycin at 37uC and 5% CO 2 . A549 and BEAS-2B cells were maintained in F-12K medium (Gibco-Invitrogen) containing 10% fetal bovine serum, penicillin, and streptomycin at 37uC and 5% CO 2 . hPIV3 (strain C243) was amplified and titrated on Vero cells following recommendations of ATCC (American Type Culture Collection). Recombinant MV-EGFP virus used in Figure S2 has been hPIV3-C Impact on IFN-a/b and EGF Pathways previously described [62] . Infections were performed for 2 h at 37uC in Optimem (Gibco-Invitrogen). Later on, cells were washed and incubated in fresh culture medium for 24 or 48 h. To detect viral replication, cells were recovered and incubated in PBSparaformaldehyde 3.2% for 20 min. After extensive washing with PBS, cells were permeabilized with PBS-Triton 0.05% for 15 min, and then incubated with a monoclonal antibody specific to hPIV3-HN (M02122321, Abcam). Cells were washed and incubated in the presence of an anti-mouse Cy3-conjugated antibody (Jackson Immunoresearch). After extensive washing, cellular immunostaining was analyzed using a FACSCalibur flow cytometer (BD). When specified, cells were pre-treated with MEK1/2 specific inhibitor U0126 (20 mM final; Promega) for 2 h before, during and after infection to study the impact on hPIV3 infection.
To perform co-affinity purification experiments, cloned ORFs were transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST fusion, and to pCI-neo-36FLAG vector [61] for 36FLAG-fusion. Cell transfections were performed using Lipofectamine 2000 (Invitrogen). Unless specified otherwise, 5610 5 HEK-293T cells were dispensed in each well of a 6-well plate, and transfected 24 h later with 600 ng of each plasmid DNA per well. Two days post transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). Cell lysates were incubated on ice for 20 min, and then clarified by centrifugation at 14,0006g for 10 min. For pull-down analysis, 400 mg of protein extracts were incubated for 1 h at 4uC with 25 ml of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).
Purified complexes and protein extracts were resolved by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% NuPAGE Bis-Tris gels with MOPS running buffer (Invitrogen), and transferred to a nitrocellulose membrane. Proteins were detected using standard immunoblotting techniques. 36FLAGand GST-tagged proteins were detected with a mouse monoclonal HRP-conjugated anti-36FLAG antibody (M2; Sigma-Aldrich) and a rabbit polyclonal anti-GST antibody (Sigma-Aldrich), respectively. Specific antibodies were used to detect endogenous STAT1 (clone-1; BD Biosciences), GRB2 (clone-81; BD Biosciences), phospho-ERK1/2 (clone-12D4; Upstate), ERK1/2 (CT; Upstate), phospho-eIF4E (Ser209; Cell Signaling), eIF4E (Cell Signaling), phospho-S6 235-236 (Ser235/236; Cell Signaling), S6 (clone-54D2; Cell Signaling) and hPIV3-HN (M02122321; Abcam). Secondary anti-mouse and anti-rabbit HRP-conjugated antibodies were from GE-Healthcare. Densitometric analysis of the gels was performed using a specific module of Photoshop CS3 Extended (Adobe Systems Inc.).
Yeast two-hybrid screening and gap-repair procedure Our yeast two-hybrid protocols have been described in details elsewhere [58] . Briefly, pDEST32 plasmid encoding Gal4-DB fused to hPIV3-C was transformed in AH109 yeast strain (Clontech), and used to screen by mating a human spleen cDNA library cloned in the Gal4-AD pPC86 vector (Invitrogen) and previously established in Y187 yeast strain (Clontech). Yeast cells were plated on a selective medium lacking histidine and supplemented with 10 mM 3-amino-triazole (3-AT; Sigma-Aldrich) to select for interaction-dependent transactivation of HIS3 reporter gene. AD-cDNAs from [His+] colonies were amplified by PCR and sequenced to identify the host proteins interacting with hPIV3-C.
The gap-repair procedure was used to map the minimal portion of hPIV3-C interacting with STAT1 and GRB2. As previously described [63] , both forward and reverse PCR primers were designed along the sequence of hPIV3-C and fused to specific tails allowing yeast-based recombination in Gal4-DB two-hybrid vector. Matrix combinations of forward and reverse primers were used to amplify fragments of hPIV3-C by PCR. AH109 yeast cells expressing AD-fused STAT1 or GRB2 were co-transformed with 5 mL of each PCR product in the presence 50 ng of linearized pDEST32 vector to achieve recombinatorial cloning by gaprepair. Fragments of hPIV3-C fused to Gal4-DB were then tested for interaction with AD-STAT1 or AD-GRB2 by plating yeast cells on selective medium lacking histidine and supplemented with 10 mM of 3-AT.
Luciferase reporter gene assay HEK-293T, Hela or Vero cells were plated in 24-well plates (2610 5 cells per well). One day later, cells were transfected with either pISRE-Luc (0.3 mg/well; Stratagene) or pFA2-Elk1 (0.3 mg/well; Stratagene) and pGal4-UAS-Luc plasmids (0.3 mg/ well; provided by Dr. Y. Jacob) together with pRL-CMV reference plasmid (0.03 mg/well; Promega). Cells were simultaneously cotransfected with 0.3 mg/well of pCI-neo-36FLAG, pCI-neo or pmCherryC1 expression vectors encoding viral proteins as specified. 24 h after transfection, cells were stimulated with IFNb (Biosource) at 1000 IU/ml or starved for 6 h then stimulated with EGF (Upstate) at 100 ng/ml. 24 h post transfection, cells were lysed, and both firefly and Renilla luciferase activities in the lysate were determined using the Dual-luciferase Reporter Assay System (Promega). Reporter activity was calculated as the ratio of firefly luciferase activity to reference Renilla luciferase activity, and normalized so that positive control activity equals 100. When indicated, cells were treated with U0126 (Promega) at 20 mM final concentration upon EGF stimulation. Subcellular localization of Cherry-tagged hPIV3-C FL , C 1-89 and C 24-well plates containing coverslips were seeded with HEK-293T cells (2610 5 cells per well). One day later, cells were transfected with pmCherryC1 expression vector alone or encoding hPIV3-C FL , hPIV3-C 1-89 or hPIV3-C 90-195 . 36 h after transfection, cells were incubated with PBS-PFA 4% for 20 min at RT, then treated with PBS-Triton 0.05% for 15 min at RT to permeabilize the cells. Finally, cells were incubated for 10 min at RT in a PBS-PFA 4% solution containing DAPI (49-6-Diamidino-2-phenylindole) at 10 mg/ml. Preparations were mounted using Fluoromount-G (Southernbiotech), and imaging performed using a SP5 confocal miscroscope (Leica).
Figure S1 ERK1/2 phosphorylation is enhanced by hPIV3 infection in A549 cells. A549 cells were infected with hPIV3 (MOI = 3) and after 24 h, cells were starved for 12 h before stimulation with 100 ng/ml of EGF. Phosphorylation of ERK1/2 was determined by western blot analysis at 10 min, 30 min and 2 h post stimulation. hPIV3 infection was confirmed by anti-hPIV3 hemagglutinin-neuraminidase (hPIV3-HN) immunoblotting. Found at: doi:10.1371/journal.ppat.1000587.s001 (7.34 MB TIF) hPIV3-C Impact on IFN-a/b and EGF Pathways Figure S2 MEK1/2 inhibitor U0126 has no effect on MV protein synthesis. HEK-293T cells were left untreated (A) or treated with 20 mM of U0126 for 2 h (B). Then, cells were mocktreated or infected with a recombinant MV strain expressing EGFP (MOI = 1) and cultured with or without U0126 (A and B, respectively). 48 h after infection, EGFP expression was quantified by flow cytometry analysis. Found at: doi:10.1371/journal.ppat.1000587.s002 (7.34 MB TIF)  A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. To sense infection, the innate immune system preferentially responds to conserved molecular signatures of microbes that are absent from normal host cells. There is currently great interest in determining what molecular features of pathogens are detected, and how these features are sensed by the innate immune system. A better understanding of the fundamental principles of how immune responses are stimulated is critical for the design of more effective vaccines, adjuvants, and immune therapeutics.
One important class of ligands sensed by the innate immune system is nucleic acids. Several distinct nucleic acid sensors have been described and have been found to be distributed to distinct subcellular locations and exhibit distinct specificities for different nucleic acid ligands. A common theme is that nucleic acid sensors tend to induce expression of type I IFN genes and, thus, appear to be particularly important for initiating immune responses to viruses.
including Listeria monocytogenes, Mycobacterium tuberculosis, Legionella pneumophila, Francisella tularensis, group B Streptococcus, and Brucella abortus, it appears that induction of type I IFN is via a novel cytosolic pathway and is independent of TLRs. It has been suggested that these pathogens trigger the cytosolic DNA-sensing pathway, but neither the host sensors nor the bacterial ligands that trigger cytosolic induction of type I IFNs by these pathogens have been identified. Intriguingly, evidence from L. monocytogenes suggests that bacterial multidrug efflux pumps are required for induction of type I IFN , and raises the possibility that a small molecule substrate of these pumps, rather than DNA, is the bacterial trigger of type I IFN gene expression.
Nucleotide second messengers are critical transmitters of signaling in all living things. cAMP is used by bacteria and eukaryotes alike, whereas certain nucleotide second messengers are unique to bacteria. For example, guanosine tetraphosphate (ppGpp) is a key regulator of the stringent response in bacteria (Srivatsan and Wang, 2008) . Another nucleotide second messenger, cyclic-di-GMP (c-di-GMP), is a relatively recently appreciated cyclic ribonucleotide (Ross et al., 1987) synthesized by bacteria from two GTP precursors that are hydrolyzed and ultimately circularized via 5-to-3 monophosphate linkages. The bacterial diguanylate cyclases that synthesize c-di-GMP all contain a characteristic GGDEF domain, whereas the phosphodiesterases that specifically degrade c-di-GMP to pGpG contain an EAL domain. The GGDEF domain, and presumably c-di-GMP, appears to be limited to Bacteria (Galperin et al., 2001) and is not found in Archaea or Eukarya. c-di-GMP appears to play complex roles as a second messenger in most bacterial species, and regulates diverse processes, such as motility, biofilm formation, and virulence gene expression (Tamayo et al., 2007) . The molecular mechanisms by which c-di-GMP regulates these biological processes in bacteria are only beginning to be understood. It appears that c-di-GMP is capable of specific binding to regulatory proteins containing the PilZ protein domain (Cotter and Stibitz, 2007) . The PelD protein of P. aeruginosa is an additional non-PilZ-containing c-di-GMP sensor protein (Lee et al., 2007) .
Several recent reports have suggested that c-di-GMP can stimulate a variety of signaling pathways in mammalian cells in vivo. One early report demonstrated that 50 µM of exogenous c-di-GMP inhibited growth of human colon cancer cells (Karaolis et al., 2005a) without toxicity against normal kidney cells. Two additional reports have indicated that c-di-GMP can function as an adjuvant. One group demonstrated a highly significant (>200-fold; P < 0.001) induction of anti-ClfA IgG2a titers when mice were vaccinated with ClfA and c-di-GMP as compared with vaccination with ClfA alone (Karaolis et al., 2007a) . Another group found a similar induction of anti--Gal titers when c-di-GMP was used as an adjuvant (Ebensen et al., 2007) . Both groups also found increased T cell responses in mice injected with c-di-GMP. Karoalis et al. (2007a) also showed that c-di-GMP has immunostimulatory effects in vivo and in vitro on innate cell populations, Toll-like receptor (TLR) 3, 7, 8, and 9 are transmembrane nucleic acid receptors that reside in intracellular compartments, where they detect endocytosed or autophagocytosed nucleic acids Medzhitov, 2007) . TLR7, 8, and 9 all require the signaling adaptor MyD88 to initiate downstream signaling, whereas TLR3 requires the signaling adaptor TRIF. Thus, MyD88 / Trif / double-knockout cells are deficient in signaling through all known TLRs that sense nucleic acids (Hoebe et al., 2003; Yamamoto et al., 2003) .
Interestingly, recent work has established that MyD88 / Trif / cells are capable of responding to nucleic acid ligands via cytosolic immunosurveillance pathways. Cytosolic RNA appears to be sensed by two RNA helicase-containing proteins, RIG-I and Mda5 (Yoneyama et al., 2004) . Interestingly, RIG-I and Mda5 do not perform redundant functions and appear to respond to distinct classes of viruses (Gitlin et al., 2006; Hornung et al., 2006; Kato et al., 2006; Pichlmair et al., 2006; . Signaling by RIG-I and Mda5 requires a common adaptor molecule called MAVS (also known as IPS-1, Cardif, or VISA; Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005; Sun et al., 2006) . MAVS recruits the TBK1 kinase that phosphorylates and activates the IRF3 transcription factor. Other transcription factors such as ATF2/c-Jun and NK-B form a coordinated DNA-bound complex with IRF3, and are together required for robust activation of the IFN- promoter (Maniatis et al., 1998; Panne, 2008) .
Many cell types also appear to respond to the cytosolic presence of DNA Stetson and Medzhitov, 2006a) . The cytosolic response to DNA does not require MAVS, but does require TBK1 and IRF3 in most cell types Stetson and Medzhitov, 2006a; Sun et al., 2006) . Recently, a putative cytosolic sensor of DNA was identified, and was named DNA-dependent activator of IFN regulatory factors (DAI; previously known as DLM-1 or ZBP1; Takaoka et al., 2007) . Knockdown experiments indicated that DAI was involved in sensing cytosolic DNA in the L929 fibroblast-like and RAW macrophage-like cell lines (Takaoka et al., 2007; Wang et al., 2008) . DAI was expressed in other cell types, such as mouse embryonic fibroblasts (MEFs), but was dispensable for the response to cytosolic DNA in these cell types, suggesting that additional uncharacterized nucleic acid sensor proteins also exist . Indeed, cells from DAI knockouts appear to respond normally to cytosolic DNA (Ishii et al., 2008) , possibly because of redundancy with other cytosolic DNA sensors.
Although type I IFNs are primarily considered to be antiviral cytokines (Stetson and Medzhitov, 2006b) , there is growing appreciation for their complex role in bacterial infections as well. Indeed, it is clear that type I IFNs are induced by many, if not all, bacterial pathogens, and can contribute to diverse outcomes in vivo (Coers et al., 2000; O'Riordan et al., 2002; Opitz et al., 2006; Stetson and Medzhitov, 2006a; Henry et al., 2007; Roux et al., 2007; Stanley et al., 2007; Charrel-Dennis et al., 2008) . For several bacterial pathogens, (a) the c-di-GMP compound was chemically synthesized and >98% pure by HPLC (not depicted), (b) phosphodiesterase treatment of c-di-GMP resulted in a product that is no longer stimulatory ( Fig. 1 E) , and (c) macrophages deficient in all TLR signaling and unable to respond to LPS still responded to c-di-GMP (see below). Thus, these observations suggest that c-di-GMP is sensed in the cytosol, resulting in a potent induction of type I IFN in bone marrow macrophages.
We performed microarray experiments to compare the global transcriptional response induced by cytosolic c-di-GMP with that induced by cytosolic DNA (Fig. 1 F and Table S1 ). Whole transcriptome spotted MEEBO microarrays (Verdugo and Medrano, 2006) were used in these experiments. The results indicated that the transcriptional profile of cells stimulated by cytosolic c-di-GMP was very similar to the transcriptional profile of cells stimulated with cytosolic DNA. Induction of type I IFNs and known type I IFN-inducible genes dominated the transcriptional response, but other genes (e.g., Il6, Cd86, and Cxcl9) were also strongly induced ( Fig. 1 F and Table S1 ). Although the transcriptional profiles of cells stimulated with c-di-GMP and DNA were highly similar, a small number of genes were reproducibly preferentially induced by c-di-GMP as compared with DNA (e.g., HtrA serine peptidase 4 and claudin 23; Table S1 ). Collectively, these results suggested that similar but not identical downstream signaling pathways are triggered by DNA and c-di-GMP.
It was previously reported that HEK293 cells stably transfected with various TLRs failed to respond to c-di-GMP (Karaolis et al., 2007a) , but the reason for this failure was unclear. For example, HEK293 cells might lack an essential accessory protein. To rule out a role for TLRs in sensing of c-di-GMP, we tested responses in Myd88 / Trif / double-knockout macrophages, which are deficient in all TLR signaling (Yamamoto et al., 2003) . Both wild-type and Myd88 / Trif / macrophages showed a robust induction of IFN- after c-di-GMP stimulation (Fig. 2 A) . As expected, the Myd88 / Trif / macrophages did not respond to LPS (Fig. 2 A) . These results indicated that TLR signaling is not required for responsiveness to c-di-GMP, and are consistent with c-di-GMP signaling via a cytosolic surveillance pathway.
Robust transcription of the IFN- gene requires the coordinate activation of several transcription factors, including IRF3 and NF-B (Panne, 2008) . We therefore expected that the c-di-GMP-induced signaling pathway would also require these factors to induce type I IFN. IRF3 activation requires phosphorylation by the TBK1 kinase, which leads to IRF3 dimerization and nuclear translocation (Fitzgerald et al., 2003; Sharma et al., 2003; Hemmi et al., 2004; McWhirter et al., 2004; Perry et al., 2004) . To address whether TBK1 is required for activation of type I IFN by c-di-GMP, we tested including monocytes, macrophages, and granulocytes. c-di-GMP induced dendritic cell maturation and led to the production of various cytokines and chemokines, including TNF, IL-1, IP-10, Rantes, and CXCR4 (Karaolis et al., 2007a) . However, c-di-GMP did not stimulate induction of type I IFNs by plasmacytoid dendritic cells. Impressively, preinjection of 2.5 mg/kg c-di-GMP protected against subsequent lethal challenge with Klebsiella pneumoniae or Staphlyococcus aureus (Karaolis et al., 2005b; Karaolis et al., 2007b) . Despite the impressive in vivo biological effects of c-di-GMP, the mechanism by which c-di-GMP stimulates host immunity remains unknown.
In this study, we provide evidence that mammalian cells survey their cytosol for the presence of c-di-GMP. We find that c-di-GMP triggers a transcriptional response virtually indistinguishable from the response triggered by cytosolic DNA. Like the response to DNA, the response to c-di-GMP requires the TBK1 kinase and IRF3 transcription factor. However, the pathway for sensing c-di-GMP can be distinguished from that for sensing DNA in certain cell types and, moreover, appears to be distinct from all other known cytosolic sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.
We hypothesized that c-di-GMP might be sensed by a cytosolic sensor leading to the induction of type I IFNs. To test this hypothesis, we compared the ability of overlayed versus transfected c-di-GMP to induce type I IFN in bone marrow macrophages. At the concentrations used (up to 25 µg/ml), overlay of c-di-GMP did not elicit a significant response ( Fig. 1 A) , although IFN- production could be detected at higher concentrations (50 or 100 µg/ml; not depicted). In contrast, delivery of c-di-GMP to the cytosol by transfection elicited robust activation of IFN- in a dose-dependent manner ( Fig. 1 A) . A significant response was detected with a concentration of c-di-GMP as low as 2.5 µg/ml (equivalent to 3.6 µM). These results suggested that c-di-GMP signals via a cytosolic immunosurveillance pathway.
We tested whether compounds related to c-di-GMP were capable of stimulating IFN- expression when delivered to the cytosol. We measured induction of endogenous type I IFN protein by bioassay ( Fig. 1 B) , and induction of IFN- and IFN-5 mRNA levels by quantitative RT-PCR ( Fig. 1 , C and D). No detectable IFN- production was observed upon transfection with GTP, cGMP, ppGpp (a different bacterial nucleotide second messenger), or pGpG (hydrolyzed c-di-GMP), whereas the cytosolic response to 3.3 µg/ml of transfected c-di-GMP was of a similar magnitude to that of 3.3 µg/ml of transfected DNA (pdA:dT) and RNA (pI:C), which are known cytosolic inducers of type I IFN. We do not believe that the cytosolic induction of type I IFNs in response to c-di-GMP is caused by a contaminant (such as LPS) because a role in IFN--mediated antiviral immunity (Tenoever et al., 2007) , and found that Ikbke / macrophages responded normally to c-di-GMP (Fig. 2 C) .
To examine the role of IRF3 in c-di-GMP induction of IFN-, we tested the ability of c-di-GMP to induce signaling in macrophages lacking Irf3. We observed that Irf3 / macrophages failed to induce IFN- in response to c-di-GMP ( Fig. 2 D) . We also tested Irf7 / macrophages and observed a partial requirement for IRF7 by the c-di-GMP pathway (Fig. 2 D) . Loss of Irf7 is likely compensated for by Irf3 in macrophages. The partial requirement for Irf7 may reflect a c-di-GMP responses in Tbk1 / macrophages (Fig. 2 B) . We observed very little IFN- induction by c-di-GMP in cells lacking Tbk1, suggesting that TBK1 is required for c-di-GMP signaling. We also observed that Tbk1 is required for the induction of IFN- by pdA:dT ( Fig. 2 B) , in agreement with a recently published report (Miyahira et al., 2009) . Although the data indicate a key role for Tbk1 in the response to c-di-GMP, it is formally possible, albeit unlikely, that Tbk1 protein rather than Tbk1 kinase activity is required for the response. We also tested the requirement for the TBK1related kinase Ikbke (also called IKK or IKK-i), which plays (Jiang et al., 2005) using recombinant IFN- as a standard. *, P < 0.01 as compared with transfected c-di-GMP (Student's t test). (B) Bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and type I IFN was assessed after 6 h by bioassay as in A. *, P < 0.001 as compared with unstimulated cells (Student's t test). (C) Bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and transcription of the IFN- gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.001 as compared with unstimulated cells (Student's t test). (D) As in C, but transcription of the IFN-5 gene was assessed. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.01 as compared with unstimulated cells (Student's t test). (E) c-di-GMP, treated or untreated with snake venom phosphodiesterase, was transfected at 3.3 µg/ml into B6 bone marrow macrophages and analyzed after 6 h for IFN- production by bioassay, as in A. *, P < 0.02 as compared with c-di-GMP treatment alone (Student's t test). Results in A-E are representative of at least three independent experiments. Data are means ± SD (n = 3). (F) Bone marrow macrophages were transfected with poly dA:dT (DNA) or with c-di-GMP. Total RNA was isolated after 6 h of stimulation. Probes were amplified and hybridized to whole-transcriptome spotted MEEBO microarrays. Each dot represents a single gene, and its position is determined by its induction in response to DNA (x axis) or c-di-GMP (y axis). Most genes lie on the diagonal, indicative of similar induction ratios by both treatments. Selected highly induced genes are labeled. Two independent microarray experiments gave similar results.
transcription factors, as well as phosphorylation of the p38, JNK, and ERK MAP kinases, in response to c-di-GMP. Activation of IRF3 involves phosphorylation, dimerization, and translocation into the nucleus. We tested whether treatment with c-di-GMP resulted in significant nuclear accumulation of IRF3. As expected, we found that c-di-GMP resulted in significant translocation of IRF3 to the nucleus (Fig. 3 A) .
We also tested whether transfected c-di-GMP is capable of activating NF-B. This was particularly important to establish in light of a previous report that exogenous (untransfected) c-di-GMP failed to activate NF-B (Karaolis et al., 2007a) . There have also been inconsistent reports in the literature as to whether cytosolic DNA induces NF-B Stetson and Medzhitov, 2006a; Sun et al., 2006) . We found that c-di-GMP treatment led to the activation of NF-B, as assessed by using an electromobility gel shift assay, with similar kinetics to that of transfected pI:C and pdA:dT ( Fig. 3 B) . The probe-bound complex was confirmed to contain NF-B p65 by supershifting with an anti-p65 antibody ( Fig. 3 B) . We confirmed these results by using a RAW role for Irf 7 signaling as part of a positive feedback loop involving signaling through the type I IFN receptor (Ifnar). Indeed, Ifnar / macrophages exhibited a diminished response to c-di-GMP (Fig. 2 E) . Irf5 and Irf1 did not seem to be required for responsiveness to c-di-GMP (Fig. 2 F) . In addition, macrophages deficient in Rip2, Nalp3, or doubly deficient in Nod1/2 responded normally to c-di-GMP (unpublished data).
We validated our results by examining production of a second secreted molecule known to be a target of the Tbk1-Irf3 signaling axis, the chemokine Rantes (Ccl5). Rantes production was assessed by ELISA (Fig. S1 ). As expected, we found that Rantes was induced by c-di-GMP in a manner dependent on Tbk1, Irf3, and Ifnar, but largely or entirely independent of MyD88/Trif, Irf1, Irf5, Irf7, or Mavs (Fig. S1 ).
Our genetic data suggested that the cytosolic responses to DNA, RNA, and c-di-GMP share a common downstream signaling pathway. If this were true, we would expect to be able to detect biochemical activation of the NF-B and IRF3
Myd88 / Trif / mice were transfected with 3.3 µg/ml c-di-GMP or pdA:dT (DNA), or overlayed with 100 ng/ml LPS as indicated. After 6 h of stimulation, induction of IFN- mRNA was analyzed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) As in A, except Tnfr1 / or Tnfr1 / Tbk1 / macrophages were analyzed. Tbk1 deficiency is embryonic lethal, but viability can be rescued on the TNF receptor 1 (Tnfr1)-deficient background. (C) As in A except Ikbke (IKK/IKK-i)-deficient mice were analyzed. (D) as in A, except Irf3 / and Irf7 / macrophages were analyzed. (E) as in A, except macrophages deficient in the type I IFN receptor (Ifnar / ) were analyzed. (F) as in A, except Irf5 / and Irf1 / macrophages were analyzed. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.05; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test) . discrepancy between our results and those of Karaolis et al. (2007a) , it is more likely that we were able to detect MAP kinase activation because transfection of c-di-GMP triggers more robust responses than overlayed c-di-GMP, and because we examined later time points.
Cell type-specific responses to cytosolic DNA and c-di-GMP Collectively, these results suggest that there are strong similarities in the signaling pathways triggered by the cytosolic presence of c-di-GMP and other nucleic acids, such as DNA and RNA. However, c-di-GMP is not structurally similar to these nucleic acid ligands. The DNA molecules capable of provoking cytosolic responses are double stranded and >25 deoxyribonucleotides in length Stetson and Medzhitov, 2006a) . In contrast, c-di-GMP is composed of two guanosine ribonucleotides (not deoxyribonucleotides) in an unusual cyclic conformation. c-di-GMP also lacks features consistent with recognition by RIG-I or Mda5, such as 5triphosphate, polyuridine, or double strandedness . Therefore, we considered whether distinct sensors might be responsible for triggering responses to DNA/RNA and c-di-GMP, and if so, whether the responses could be distinguished in certain cell types. macrophage cell line stably transfected with an NF-B luciferase reporter. We observed activation of the NF-B luciferase reporter by transfected c-di-GMP (Fig. 3 C) . Both the gel shift and reporter assays showed that the activation of NF-B by transfected c-di-GMP was not as strong as observed with transfected pdA:dT and pI:C. The subtle difference in NF-B induction between cytosolic DNA and c-di-GMP may suggest that there are distinct signaling components upstream of NF-B in the two pathways. Nonetheless, we concluded that transfected c-di-GMP is capable of activating NF-B. The relatively low levels and late time course of NF-B induction by c-di-GMP (as compared with induction by LPS), as well as the fact that we transfected c-di-GMP into cells, may explain why NF-B induction was not previously observed in response to c-di-GMP (Karaolis et al., 2007a) .
We also tested whether MAP kinase pathways are activated by c-di-GMP. A previous report found that overlay of c-di-GMP induced ERK but not p38 in human macrophages (Karaolis et al., 2007a) . In contrast, we found that cytosolic c-di-GMP stimulated p38, ERK1/2, and JNK phosphorylation in mouse bone marrow macrophages transfected with c-di-GMP (Fig. 3 D) . Although a difference between human and mouse macrophages may explain the Data are means ± SD (n = 3). *, P < 0.05 compared with unstimulated cells (Student's t test) . tion, and is downstream of two cytosolic viral RNA sensors, RIG-I and Mda5 (Sun et al., 2006) . To test if c-di-GMP signals through the cytosolic RNA-sensing pathway, we tested c-di-GMP responses in Mavs / and Mda5 / macrophages. Loss of Mavs or Mda5 did not affect the induction of IFN- by c-di-GMP (Fig. 5, A and B; and Fig. S1 C) , suggesting that c-di-GMP does not signal through the cytosolic RNA signaling apparatus (Gitlin et al., 2006; Kato et al., 2008; . DAI (encoded by the Zpb1 gene) is a recently identified molecule that has been proposed to function as a sensor of cytosolic DNA (Takaoka et al., 2007; Wang et al., 2008) . We found that bone marrow-derived macrophages from Zbp1deficient mice (Ishii et al., 2008) still responded to c-di-GMP (Fig. 5 C) . However, as previously reported (Ishii et al., 2008) , Zbp1-deficient macrophages also responded normally to DNA (Fig. 5 C) . These results imply that macrophages express at least one cytosolic DNA sensor that is distinct from In addition to bone marrow macrophages, we observed induction of IFN- by cytosolic c-di-GMP in a variety of other cell types, including peritoneal macrophages, conventional dendritic cells, L929 cells, and RAW 264.7 macrophages (Fig. 4, A-D) . These cells also responded to transfected DNA and RNA. Interestingly, however, we were unable to detect significant induction of type I IFNs by c-di-GMP in MEFs or in 293T cells, even though the cytosolic pathways for detecting DNA and RNA are intact in these cells (Fig. 4 , E and F). These results suggest that at least one component of the host signaling pathway responding to c-di-GMP is distinct from that used for responses to cytosolic RNA or DNA, and is differentially expressed in different cell types.
Known DNA and RNA sensing pathways are not required for responses to c-di-GMP MAVS (also known as IPS-1) is an essential adapter protein in the cytosolic RNA pathway leading to type I IFN induc- Data are means ± SD (n = 3). n.d., not detectable. *, P < 0.01; and **, P < 0.001 as compared with unstimulated cells (Student's t test) . and measured their responsiveness to YAC-1 target cells ex vivo. Because NK cells respond to in vivo injection of poly I:C, a prototypical IFN inducer, we expected that c-di-GMP might also activate NK cells. Indeed, NK cells obtained from B6 mice injected with c-di-GMP responded to YAC-1 cells, as measured by intracellular cytokine staining for IFN- (Fig. 6 B) . However, NK cells obtained from Irf3/7 / mice stimulated with c-di-GMP were nonresponsive to YAC-1 target cells. As a negative control, NK cells stimulated ex vivo with PBS did not produce IFN-. These observations indicate that c-di-GMP can stimulate cytokine and NK cell responses in vivo, and these responses require the IRF3/7 transcription factors.
To obtain a preliminary indication as to whether adaptive immune responses could be stimulated by c-di-GMP, we immunized mice with human serum albumin (HSA) and tested whether coinjected c-di-GMP could function as an adjuvant, as previously reported (Ebensen et al., 2007; DAI, and at least one of these uncharacterized DNA sensors, or another independent sensor, may respond to c-di-GMP.
To validate our findings in vivo, we injected mice intraperitoneally with c-di-GMP (200 nmol per mouse) and measured the production of type I IFNs in the blood 18 h later. B6 mice injected with c-di-GMP produced an IFN response to c-di-GMP, and this response was abolished in Irf3/7 double-deficient mice (Fig. 6 A) . Interestingly, single-deficient Irf3 / mice responded to c-di-GMP in vivo (unpublished data), implying a role for IRF7 in responses to c-di-GMP in vivo. To determine whether cytokine induction by c-di-GMP affected cellular responses in vivo, we collected splenic NK cells from mice injected with c-di-GMP Figure 5 . c-di-GMP does not stimulate known cytosolic pathways for sensing nucleic acids. (A) Mavs / and littermate wild-type macrophages were transfected with 3.3 µg/ml c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA), and transcription of the IFN- gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) Mda5 / and wild-type control macrophages were stimulated and analyzed as in A. (C) Zbp1 / (DAI knockout) and wild-type control macrophages were stimulated and analyzed as in A. SeV, Sendai virus. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.01; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test) .
(A and B) B6 and Irf3/7 / mice were injected intraperitoneally with 200 nmol c-di-GMP, and (A) serum IFN was measured by bioassay 18h later, or (B) 36 h after injection, splenic NK responsiveness to YAC-1 target cells (or PBS control) was measured ex vivo by intracellular staining for IFN-. (C) B6 and Irf3/7 / mice were injected intraperitoneally with HSA ± 200 nmol c-di-GMP, and 2 wk later serum IgG1 specific for HSA was determined by ELISA. The experiments in A and B were repeated twice, and the experiment in C was performed once. Horizontal bars represent means. **, P < 0.01; and *, P < 0.05 (Student's t test) . respond well to c-di-GMP. Given the marked chemical dissimilarities among DNA, RNA, and c-di-GMP, it is perhaps not surprising that these nucleic acids appear to be recognized by different sensors. Indeed, we favor the idea that there are multiple cytosolic sensors for nucleic acids leading to induction of type I IFNs. The specificities of these various sensors will require further dissection. For example, although DAI knockout cells still responded to c-di-GMP, it remains formally possible that DAI is just one of several redundant sensors for c-di-GMP. These possibilities can be addressed once additional cytosolic nucleic acid sensors are identified.
Our results suggest that cytosolic sensing of c-di-GMP is relatively specific. Other related small nucleic acid compounds such as GTP, cGMP, ppGpp, and pGpG did not trigger transcriptional induction of type I IFNs (Fig. 1) . In addition, c-di-GMP that was hydrolyzed by snake venom phosphodiesterase did not induce type I IFN (Fig. 1) . However, our results cannot eliminate the possibility that a metabolite of c-di-GMP, rather than c-di-GMP itself, is the true molecular moiety that is sensed in the cytosol. Elimination of this possibility awaits identification of the c-di-GMP sensor and biophysical or crystallographic characterization of its binding to c-di-GMP. Our results also cannot eliminate the possibility that c-di-GMP triggers type I IFN expression indirectly, for example, by stimulating the synthesis of a host ligand that functions as the "true" proximal trigger of type I IFN gene expression. It is also possible that c-di-GMP acts "pharmacologically" by disrupting host physiology in a way that results in type I IFN expression. Moreover, because the signaling triggered by c-di-GMP and cytosolic DNA are very similar, it is possible that the c-di-GMP pathway is a branch off of the DNA-sensing pathway rather than a fully independent pathway. However, even if these alternative models are correct, our data indicate that the pathway downstream of c-di-GMP contains at least some novel components and is at least partially independent of known cytosolic or TLR signaling pathways.
Several bacterial pathogens, including L. monocytogenes, L. pneumophila, F. tularensis, M. tuberculosis, and group B Streptococcus, have been reported to induce type I IFNs (Coers et al., 2000; O'Riordan et al., 2002; Opitz et al., 2006; Stetson and Medzhitov, 2006a; Henry et al., 2007; Roux et al., 2007; Stanley et al., 2007; Charrel-Dennis et al., 2008) . The mechanism by which these pathogens induce type I IFN resembles that of c-di-GMP: in all cases, type I IFN induction is independent of MyD88/Trif and TLRs, requires TBK1 and IRF3, and (with one exception; unpublished data; Opitz et al., 2006) is independent of the cytosolic RNA-sensing pathway. It is widely assumed that bacterial DNA, perhaps released after bacterial cell lysis, is the relevant ligand that triggers type I IFNs in response to pathogens. There are no data that eliminate or confirm this possibility, as sensors required for IFN induction in response to cytosolic bacteria have not yet been reported. However, it is noteworthy that in all cases, induction of type I IFNs by cytosolic bacteria requires expression of an auxiliary secretion system, namely the ESX-1 system of M. tuberculosis, the Francisella pathogenicity island-encoded 2007a). Serum was collected from immunized mice 2 wk after a single intraperitoneal injection. Mice immunized with HSA alone did not mount a significant antibody response to HSA. In contrast, mice injected with HSA plus c-di-GMP produced variable but significant titers of HSA-specific IgG1 antibodies, confirming that c-di-GMP can function as an adjuvant in vivo. Importantly, our preliminary data indicated that the adjuvant effect of c-di-GMP depended on IRF3/7, as antibody responses in Irf3/7 / mice immunized with HSA and c-di-GMP were significantly (P < 0.05) reduced as compared with immunized B6 mice (Fig. 6 C) . Further studies will be required to establish the mechanism by which c-di-GMP functions as an adjuvant, but our results are consistent with a recent report indicating that the IRF3/7 kinase, TBK1, is required for adaptive immune responses in a DNA-vaccine model (Ishii et al., 2008) .
It has been well established that innate immune responses are initiated in response to certain microbial ligands that are evolutionarily conserved and that can be distinguished from selfligands. Nucleic acids appear to be a favored target of immune recognition, and are sensed by a variety of endosomal and cytosolic sensor proteins. The cyclic dinucleotide c-di-GMP is a bacterial second messenger that exhibits several characteristics that are desirable in an immunostimulatory ligand: it is produced by numerous species of bacteria, it is a critical regulator of bacterial physiology, and it is not similar to host molecules. Indeed, several previous reports have demonstrated that c-di-GMP can provoke potent immune responses when injected in vivo into mice (Karaolis et al., 2005a; Karaolis et al., 2005b; Karaolis et al., 2007a; Karaolis et al., 2007b) . However, the mechanism by which c-di-GMP stimulates immune responses remained unclear. Our data provide evidence that c-di-GMP is sensed by a novel cytosolic immunosurveillance pathway. Responsiveness to c-di-GMP is independent of TLRs that monitor the extracellular/endosomal compartments and is strongly potentiated by transfection of c-di-GMP into the host cell cytosol. Moreover, c-di-GMP provokes a transcriptional response highly reminiscent of that triggered by the cytosolic presence of DNA or RNA, and involves activation of TBK-1, MAP kinases, and the IRF-3 and NF-B transcription factors. Thus, we propose that c-di-GMP is sensed in the cytosol.
How many different cytosolic sensors exist for nucleic acids? Our data suggest that sensing of c-di-GMP occurs via a novel cytosolic immunosurveillance pathway. Known nucleic acid sensors include Mda5 and RIG-I, which sense viral RNA and signal via MAVS, as well as DAI, a putative sensor of DNA that signals independently of MAVS. Based on the finding that most cell types, including MEFs, can still respond to DNA in the absence of DAI (Ishii et al., 2008) , it has been proposed that an additional DNA sensor must exist and that MEFs express both of these sensors . Our results now suggest that there is at least one additional nucleic acid sensor in the cytosol, because we find that MEFs do not B. Beutler (The Scripps Research Institute, La Jolla, CA), and RAW-B cells were obtained from G. Barton. Animal protocols were approved by the University of California, Berkeley Animal Care and Use Committee.
Reagents. c-di-GMP was synthesized as previously described (Kawai et al., 2003) , poly I:C was obtained from GE Biosciences, pdA:dT (poly(dA-dT): poly(dA-dT)) was purchased from Sigma-Aldrich, ppGpp (guanosine-3,5bisdiphosphate) was obtained from TriLink Biotechnologies, pGpG (diguanosine) was purchased from IBA GmbH, GTP and cGMP were obtained from Sigma-Aldrich, purified LPS was purchased from InvivoGen, and Sendai virus was obtained from Charles River Laboratories. Theiler's virus was the gift of M. Brahic (Stanford University, Stanford, CA).
Cell culture. L929, RAW 264.7, and MEF cell lines were cultured in DMEM containing 10% FBS, glutamine, and penicillin-streptomycin. For bone marrow-derived macrophages, bone marrow cells from femurs and tibias were cultured for 7 d in RPMI 1640 media containing 10% FBS, glutamine, penicillin-streptomycin, and10% CSF from 3T3 cells, with feeding on the fourth day of growth. For conventional dendritic cells (GM-CSFdendritic cells), bone marrow cells were cultured for 5 d with RPMI 1640 containing 10% FBS, glutamine, penicillin-streptomycin, -mercaptoethanol, and GM-CSF, with fresh media added on the second and fourth days of growth. Peritoneal macrophages were elicited by injection of 2 ml 4% thioglycollate (Fluid Thioglycollate Medium; BD), and were obtained 4 d later by lavage of the peritoneal cavity with RPMI 1640.
Cell stimulations (transfections). Cells were transfected using Lipofectamine 2000 (LF2000; Invitrogen) according to the manufacturer's protocol. All nucleic acid stimulants were mixed with LF2000 at a ratio of 1 µl LF2000/1 µg nucleic acid, incubated at room temperature for 20-30 min, and added to cells at a final concentration of 3.3 µg/ml (96-well plates) or 4 µg/ml (6-well plates). For pI:C, 2 mg/ml of the stock solution was heated at 50°C for 10 min and cooled to room temperature before mixing with LF2000. Transfection experiments were done for 6 h, unless otherwise stated in the figures. For phosphodiesterase treatment of c-di-GMP, 0.5 µg/µl c-di-GMP was incubated for 2 h at room temperature in Optimem buffer (Invitrogen), with 15 mM MgCl 2 and 1 U Phosphodiesterase I (GE Healthcare).
Quantitative PCR. Stimulated cells were overlayed with RNAlater (Applied Biosystems) and stored. RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's protocol, and was treated with RQ1 RNase-free DNase (Promega). 0.5 µg RNA was reverse transcribed with Superscript III (Invitrogen). Platinum Taq DNA polymerase (Invitrogen) and EvaGreen dye (Biotium) were used for quantitative PCR assays and analyzed with a real-time PCR system (StepOnePlus; Applied Biosystems). All gene expression values were normalized to Rps17 (mouse) or SP9 (human) levels for each sample. The following primer sequences were used: mouse Ifnb, (forward) 5-ATAAGCAGCTCCAGCTCCAA-3 and (reverse) 5-CTGTCTGCTGGTGGAGTTCA-3; mouse Ifn5, (forward) 5-TGACCTCAAAGCCTGTGTGATG-3 and (reverse) 5-AAG-TATTTCCTCACAGCCAGCAG-3; mouse Rps17, (forward) 5-CGCC-ATTATCCCCAGCAAG-3 and (reverse) 5-TGTCGGGATCCACC-T CAATG-3; human IFN, (forward) 5-AAACTCATGAGCAGTCT-GCA-3 and (reverse) 5-AGGAGATCTTCAGTTTCGGAGG-3; and human SP9, (forward) 5-ATCCGCCAGCGCCATA-3 and (reverse) 5-TCAATGTGCTTCTGGGAATCC-3.
Type I IFN bioassay and luciferase reporter assay. Cell-culture supernatants from stimulated cells were overlayed on top of ISRE-L929 IFN reporter cells (Jiang et al., 2005) and incubated for 4-6 h (96-well plate). The reporter cells were lysed in Passive Lysis Buffer (Promega) for 5 min at room temperature, mixed with firefly luciferin substrate (Biosynth), and measured on a luminometer (LmaxII 384 ; MDS Analytical Technologies). Levels of type I IFN were calculated from a standard curve using recombinant mouse IFN- (R&D Systems). secretion system of F. tularensis, the Dot/Icm system of L. pneumophila, or the MdrM multidrug efflux pump of L. monocytogenes. It is tempting to speculate that a small, bacterially derived molecule such as c-di-GMP could be transported or leak through these secretion systems. It has not yet been possible to test this idea directly because all of these bacterial species encode numerous c-di-GMP synthases, and a strain lacking all c-di-GMP synthesis has not been reported. In any case, interpretation of these experiments would be complicated by the fact that c-di-GMP plays important regulatory roles in bacterial physiology and pathogenesis. Nevertheless, our results with synthetic purified c-di-GMP suggest that in addition to DNA, c-di-GMP is a candidate for a conserved molecule unique to bacteria that is responsible for triggering transcription of type I IFN genes. In light of a recent report that bacteria appear to be able to synthesize c-di-AMP (Witte et al., 2008) , it is interesting to consider whether additional nucleic acids produced specifically by bacteria might also trigger host immunosurveillance pathways. Non-nucleic acid small molecules, such as the drug DMXAA, also appear to be able to stimulate the TBK1-IRF3 axis (Roberts et al., 2007) . Indeed, there may be multiple redundant pathways for cytosolic sensing of bacteria. Collectively, the available evidence suggests that host cells may sense a wider array of bacterial ligands than was previously appreciated.
Our results may have important implications for the design of new adjuvants and vaccines. DNA vaccines have attracted considerable enthusiasm as an approach for protecting against a variety of infectious diseases (Wang et al., 2001; Yang et al., 2004) , but there are safety concerns about the insertional mutagenic potential of DNA vaccines and/or their potential to trigger pathogenic anti-DNA autoimmune antibody responses (Schalk et al., 2006) . The potency of DNA vaccines appears to derive from their ability to stimulate the TBK1 and innate cytosolic DNA-sensing pathways . Thus, our demonstration that a synthetic nonself non-DNA molecule such as c-di-GMP can stimulate an in vitro and in vivo innate and adaptive immune response (Fig. 6 ) similar to that induced by DNA, without similar autoimmune or mutagenic risks, suggests that c-di-GMP might have valuable application as a small-molecule adjuvant. Understanding the molecular basis of c-di-GMP signaling in mammalian cells will be a crucial step toward achieving this aim.
Mice and cell lines. Bone marrow macrophages were derived from various mouse strains, including Mavs / (Sun et al., 2006) , Mda5 / (Gitlin et al., 2006) , and Zbp1 / (Ishii et al., 2008) . Wild-type C57BL/6 and Tnfr1 / mice were from the Jackson Laboratory. Myd88 / /Trif / mice were obtained from G. Barton (University of California, Berkeley, Berkeley, CA). Irf1 / , Irf3 / , Irf5 / , and Irf 7 / mice were obtained from T. Taniguchi (University of Tokyo, Tokyo, Japan). Tbk1 / /Tnfr1 / mice were generated by crossing Tbk1 / mice (provided by W.-C. Yeh, University of Toronto, Toronto, Canada) with Tnfr1 / mice. Rip2 / mice were obtained from R. Medzhitov (Yale University, New Haven, CT). Nod1 / /Nod2 / mice were obtained from D. Portnoy (University of California, Berkeley, Berkeley, CA). Nalp3 / mice were obtained from V. Dixit (Genentech, South San Francisco, CA). ISRE-L929 IFN reporter cells were obtained from Gene Expression Profiling in Cells with Enhanced γ-Secretase Activity BACKGROUND: Processing by γ-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains (ICDs) that, following nuclear translocation, modulate the transcription of different genes regulating a diverse array of cellular and biological processes. Because the list of γ-secretase substrates is growing quickly and this enzyme is a cancer and Alzheimer's disease therapeutic target, the mapping of γ-secretase activity susceptible gene transcription is important for sharpening our view of specific affected genes, molecular functions and biological pathways. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes and molecular functions transcriptionally affected by γ-secretase activity, the cellular transcriptomes of Chinese hamster ovary (CHO) cells with enhanced and inhibited γ-secretase activity were analyzed and compared by cDNA microarray. The functional clustering by FatiGO of the 1,981 identified genes revealed over- and under-represented groups with multiple activities and functions. Single genes with the most pronounced transcriptional susceptibility to γ-secretase activity were evaluated by real-time PCR. Among the 21 validated genes, the strikingly decreased transcription of PTPRG and AMN1 and increased transcription of UPP1 potentially support data on cell cycle disturbances relevant to cancer, stem cell and neurodegenerative diseases' research. The mapping of interactions of proteins encoded by the validated genes exclusively relied on evidence-based data and revealed broad effects on Wnt pathway members, including WNT3A and DVL3. Intriguingly, the transcription of TERA, a gene of unknown function, is affected by γ-secretase activity and was significantly altered in the analyzed human Alzheimer's disease brain cortices. CONCLUSIONS/SIGNIFICANCE: Investigating the effects of γ-secretase activity on gene transcription has revealed several affected clusters of molecular functions and, more specifically, 21 genes that hold significant potential for a better understanding of the biology of γ-secretase and its roles in cancer and Alzheimer's disease pathology. Introduction c-Secretase is an unconventional aspartyl protease (composed of PS1, NCT, Aph-1 and Pen2) with an intramembranous catalytic site that is typical of the class of intramembrane-cleaving proteases (I-CliPs) (for review, see [1, 2] ). Via the processing of its substrates and freeing of their intracellular domains (ICDs), c-secretase regulates a multitude of signaling pathways and biological processes by influencing gene transcription. This is exemplified by the processing of the Notch receptor and the Notch signaling pathway (for a review, see [3] ). After specific ectodomain shedding via tumor necrosis factor a converting enzyme (TACE) (Fig. 1, step 1) , Notch is further cleaved intramembraneously by c-secretase (Fig. 1, step 2) . The intracellular domain of Notch (NICD) is freed to enter the nucleus, where it interacts with the transcription factor CSL (Fig. 1,  step 3 ). With help from the coactivator Mastermind, CSL is converted from a transcriptional repressor to a transcriptional activator. CSL as an activator leads to the expression of Notch target genes (Fig. 1, step 4) , like the Hes or Hey family. Hes1, a transcriptional repressor, inhibits the transcription of NC3C1 (Fig. 1,  step 5 ). Enhanced c-secretase activity, through its cleavage of Notch, leads to increased transcription of specific genes (Fig. 1, step 4) that repress the expression of other genes (Fig. 1, step 6) to influence a multitude of biological processes. For example, the processing of Notch by c-secretase is crucial for hepatoblast differentiation [4] , epidermis and hair follicle differentiation [5] , alveolar differentiation in mammary glands [6] , maintenance of skin appendages [7] , intestinal stem cell specification [8] , induction of satellite cells after injury and maintenance [9] and neural specification of embryonic stem cells [10] .
The directions in which c-secretase activity can up-and downregulate gene transcription following its cleavage of a variety of substrates is further exemplified by the processing of Amyloid-b (Ab) precursor protein (APP), one of the better-known c-secretase substrates. The successive processing of APP by BACE1 and csecretase indeed leads to the production of Ab peptides (a causative agent in the pathogenesis of Alzheimer's disease (AD)), and APP-intracellular domains (AICDs) which, following associ-ation with the adaptor protein Fe65 and nuclear translocation, are able to suppress the expression of the major Apolipoprotein e (ApoE)/lipoprotein receptor LRP1 by binding directly to its promoter [11] . Thus, APP processing is also involved in the regulation of brain ApoE and cholesterol metabolism through LRP1 [11] . As ApoE4 is the major known genetic risk factor for late onset Alzheimer's disease (LOAD) and since AICD production depends on c-secretase, the latter is implicated in the sporadic form as well. In contrast to LOAD, which correlates directly with age, early onset familial Alzheimer's disease (FAD) is genetic and is mainly caused by mutations in presenilin1 or presenilin2 (PSEN1 or PSEN2), leading to loss of physiological or gain of toxic functions. Murine specific loss of Psen1 in the forebrain has been shown to affect certain aspects of memory [12, 13] . However, it remains difficult to correlate the loss of four murine PSEN alleles with the mild single PSEN allele mutations in FAD [14, 15] . c-Secretase is thus directly or indirectly implicated in the pathogenesis of both FAD and LOAD, making this protease an attractive therapeutic target for the prevention and/or treatment of AD. c-Secretase inhibitors/modulators have indeed reached clinical phase III trials [16] .
With an increasing number of reports about new c-secretase substrates and the transcriptional effects of their ICDs being potentially implicated in the pathogenesis of AD or several types of cancer, we see a need for a basic overview of genes and molecular functions that are transcriptionally affected by c-secretase activity.
In an effort to identify specific alterations of gene transcription as a result of c-secretase activity, the transcriptomes of two CHO cell lines (biological triplicates were used in each case) with To identify genes whose transcription is affected by c-secretase activity, two starkly contrasting conditions were analyzed by cDNA microarray: genetically engineered enhanced c-secretase (left panel) and pharmacologically inhibited c-secretase (right panel) in CHO cell lines. For a schematic depiction of the strategy, the Notch-1 receptor signaling pathway is used as an example. After processing by the Furin protease and when activated by binding to its ligands Notch-1 is cleaved at the S2 position by the TACE protease, generating a substrate for c-secretase (1, 7) . Under enhanced (left panel) or inhibited (right panel) c-secretase activity, the cleavage of the substrate controls the release of the Notch intracellular domain (NICD) (2, 8) . With enhanced c-secretase, increased numbers of NICDs enter the nucleus and interact with CSL (3), leading to the transcription of target genes like Hes1 and Hey (4). The Hes1 transcription repressor inhibits transcription of target genes like NC3C1 (5), with the final consequence being reduced production of NC3C1 mRNA (6) . Thus, enhancing c-secretase leads simultaneously to gene-dependent increase (in the case of Hes/Hey) or decrease (in the case of NC3C1) of mRNA copy numbers. With inhibited c-secretase, reduced numbers of NICDs (9) lead to the transcription of less Hes1/Hey (10), to reduced inhibition of target genes like NC3C1 (11) and consequently to increased production of NC3C1 mRNA (12) . Inhibiting c-secretase thus leads to gene-dependent decrease (in the case of Hes/Hey genes) or increase (in the case of NC3C1) of mRNA copy numbers. Following mouse cDNA microarray analysis of both transcriptomes, top scoring candidates were evaluated and validated by real time PCR and further analyzed for changes of transcript levels between healthy and AD human brain cortices. doi:10.1371/journal.pone.0006952.g001 enhanced and inhibited c-secretase activity were analyzed and compared (strategy depicted in Fig. 1 -exemplified by Notch processing). The S-1 cell line overexpresses the four components of c-secretase (NCT, Aph1a, PS1 and Pen2) and was characterized by a marked increase in the level of PS1 heterodimers and an associated 8-fold increase in c-secretase activity compared to untransfected controls [17] . The other cell line consisted of the original parental wild type CHO cells incubated with DAPT, a well-known c-secretase inhibitor. We strategically chose those two conditions, overexpression of c-secretase and inhibition of its activity, to amplify the activity-dependent effects on gene transcription levels (i.e., amplification of the signal from the cDNA microarray). To reduce potential effects due to changes in the protein levels of the c-secretase subunits as opposed to changes in its activity that we are interested in, we used chemical inhibition (DAPT) of c-secretase activity instead of gene silencing, which ultimately leads to changes in protein levels [18] . Biological functions have indeed been reported mainly for the c-secretase subunit PS1, independently to the c-secretase activity. However, because treatment of CHO cells with DAPT has been recently reported to exacerbate the secretion of exosomes [19] , we cannot exclude at this stage that some detected genes may be exosomerelated in response to the DAPT treatment. The gene transcription levels of the two cell lines were analyzed using a mouse cDNA microarray ( Fig. 1 and Material and Methods) [20] because of the absence of a readily available DNA microarray based upon hamster gene sequences or cDNA clones. This lack has been noticed, and cross-species reactivity of a mouse microarray hybridized with CHO-derived samples has been investigated recently by De Leon Gatti et al. [21] . This group generated an EST-based CHO microarray and compared it with results from a mouse microarray and vice versa. They state that cross-species hybridization yielded 89.6% overlap in their arrays, noncontradicting results and led only to a decrease in sensitivity resulting in detection of fewer differentially expressed genes. Accordingly, we probably have not detected all differentially expressed genes, but we have detected a significant amount, including clusters of functional relevance. For example, Neprilysin, an Ab-degrading enzyme of functional relevance to AD that has been previously shown to be transcriptionally downregulated in PSEN1/PSEN2 double knock out fibroblasts and to exhibit reduced activity under chemical (DAPT) inhibition of c-secretase in mouse neurons [22] , was not detected in the current study. Collectively, this supports the use of CHO cells with a mouse microarray. The microarray data set discussed in this publication has been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE16379.
The mouse microarray consistently detected the four human csecretase subunits overexpressed in the S-1 cell line (Table 1) . By applying a cut-off based on the false discovery rate (FDR, i.e., the probability to wrongly accept a difference between the two conditions) with a p value of 0.005, we found 2658 EST clones (1981 genes) to be differentially expressed, with 1241 EST clones of increased and 1417 EST clones of decreased transcription upon enhanced c-secretase activity (Supplemental Material, Dataset S1 and Dataset S2).
Mapping clusters of genes of GO functions transcriptionally susceptible to c-secretase activity levels resulted in a GO hierarchydependent tree that will provide further orientation for c-secretase research. Functional clustering of 2658 differentially expressed sequences (1981 genes, Supplemental Material, Dataset S3) was performed using the FatiGo tool [23] . Comparing the representation of functional groups of genes throughout the entire mouse genome with their representation within the group of differentially transcribed genes allowed us to see whether clusters of genes of a specific functional group were enriched in the differentially expressed set. Clusters of over-and underrepresented genes were detected (Fig. 2) . The gene functions ''transcription regulator activity'', ''kinase regulator activity'', ''catalytic activity'' and ''binding '' were found to be overrepresented among the 2658 sequences (1981 genes) that were differentially transcribed. The cluster of ''molecular transducer activity'', through its subclusters in the GO hierarchy: ''receptor activity'' GO0004872, ''transmembrane receptor activity'' GO 0004888 and ''neurotransmitter receptor activity'' GO0030594, as well as the cluster of ''transporter activity'', via its subcluster of ''ion transporter activity'' GO0015075, were underrepresented (Fig. 2 , blue boxes). This is significant since neurotransmitter activity and transmembrane receptors are well within the focus of current AD research [24] .
Supporting our hypothesis that c-secretase has a role in multiple transcriptional regulatory activities, the GO cluster of ''transcription regulator activity'' is overrepresented through both its subclusters ''transcriptional activator activity'' GO0016563 and ''transcriptional repressor activity'' GO 0016564 (Fig. 2 , red boxes. Single member genes of each cluster are annotated in Supplemental Material Dataset S3). A well-described gene within the activator cluster is b-catenin (CTNNB1, FC = 3, p = 0.001), whereas an example of a gene in the cluster of ''transcriptional repressor activity'' is HES1. Hes1 (FC = 5.4, p = 7.69E-04) is a transcription factor that has previously been reported as a downstream target of the Notch signaling pathway [25] (Fig. 1) .
Like the examples above, 56 other transcription-related genes were found to be differentially transcribed with enhanced csecretase activity (Supplemental Material, Dataset S4). Consistent with these findings, several known substrates of the enzyme were detected on the microarray as well ( Table 2 ). This suggests a possible feedback mechanism by which the augmented processing of these substrates by c-secretase might lead to their altered transcription. The overrepresentation of genes in the clusters of enzymatic activity, such as ''kinase regulator activity'' GO0019207 and ''catalytic activity'', through four distinct GO subclusters (''isomerase activity'' GO 0016853, ''ligase activity'' GO0016874, ''hydrolase activity'' GO0016787 and ''transferase activity'' GO 00167740- Fig. 2 , red boxes), is broad in terms of the type of enzymatic activity and further shows the diversification of the downstream effects of enhanced c-secretase activity. The most complex cluster of molecular function that is overrepresented among the differentially transcribed genes identified in our microarray analysis is the GO function termed ''Binding''. This cluster is overrepresented through six subclusters and several subclusters of these ( Fig. 2 , lower part). Consistent with transcription regulation, the binding subclusters of ''nucleic acid binding'' GO0003676 and ''nucleotide binding'' GO0000166 are overrepresented. The cluster of ''ion binding'' GO0043167 is overrepresented as well as the cluster of ''protein binding'' GO0005515. A consistently overrepresented subcluster of the latter is ''cytoskeletal protein binding'' GO0008092 ( Fig. 2 ). Cytoskeletal proteins have long been known to play a role in AD and Tauopathies. They are targets of the cell polarity Wnt pathway, and their dynamics have recently been shown to be affected by AICD [26] .
''Receptor binding'' GO0005102 also includes the Notch ligand and known c-secretase substrate Jagged 2 [27, 28] , as well as the asecretase ADAM 10 [29] , four members of the Wnt family (Wnt6, 7a, 9b and 10a) and, the aforementioned b-catenin. Indeed, the translocation of b-catenin is mediated by ADAM 10, which is of the same functional cluster [30] .
By clustering transcriptionally affected genes, we demonstrate that neurotransmitter, transcription regulator and enzymatic activities, transmembrane receptor and cytoskeletal proteins functional groups are affected by c-secretase activity in their mRNA copy numbers.
For specific analysis of single genes, the fifty most prominently transcriptionally altered genes were evaluated by real time PCR. Mouse code based primers worked reproducibly and specifically for 35 genes. Among them, 21 genes were found to be differentially transcribed with enhanced c-secretase activity ( Fig. 3 upper panel, annotations lower panel). The highest increase in transcription Categories within the Molecular Function GO hierarchy that were over-and under-represented among the genes that were differentially transcribed in cells with enhanced c-secretase activity. Red boxes display GO terms that were overrepresented; blue boxes indicate GO terms that were under-represented. Black boxes represent main molecular functional clusters and arrows point toward according subclusters. The clustering of 1981 differentially transcribed genes was performed with DAVID and the FatiGo tool [23] . doi:10.1371/journal.pone.0006952.g002 levels was detected for UPP1, a gene encoding an enzyme (Uridine phosphorylase, UPase) directly implicated in the processing of uridine. UPP1 was confirmed by real time PCR to have a 39.2-fold increase in transcription levels ( Fig. 3 upper panel). Uridine is a strong sleep-promoter and is crucial for RNA, DNA and membrane biosynthesis [31] . Because of the latter, a lack of uridine (caused by increased UPase) would thus first damage cells with a large membrane to cytoplasm ratio, one of the most extreme ratios being found in neurons due to their axon and dendrite structure [32] . Interestingly oral administration of uridine has improved AD phenotypes [33, 34] . The protein Upp1 also interacts with Vimentin [35] , the distribution of which is characteristically altered in FAD fibroblasts [36] .
The Notch-dependent transcriptional repressor Hes1 was also confirmed by real time PCR with a 7-fold increase in mRNA levels under enhanced c-secretase activity (Fig. 3) .
Importantly, we found several key players of the three Wnt pathways to be transcriptionally altered in response to enhanced csecretase. We confirmed one of these, Wnt3a, to be increased by 2.8-fold in S-1 cells (Fig. 3 ). Aoyama et al. have reported that Wnt3a can influence Notch protein levels and increase Notch1 activation [37] , which increases the effect of enhanced c-secretase even further through substrate enhancement. Thus, enhanced csecretase activity may lead to increased WNT3A transcription, which in turn can increase the protein levels of the NICD-carrying c-secretase substrate. This proposed enhancement of the canonical Wnt pathway is further supported by the recent observation that b-catenin (the central protein that also ties PS1 to the pathway) modulates the level and transcriptional activity of Notch1/NICD through their direct interaction [38] . Several proteins, including Frizzled and Disheveled (Dvl), relay the Wnt signal along the canonical pathway between Wnt and b-catenin. We found that they both show increased gene transcription in our microarray analysis. DVL3 was confirmed by qPCR to increase in mRNA copy numbers by 3-fold (Fig. 3) . Taken together with the microarray data showing differential transcription of PROC, DKK, LRP5/6, GBP, AXIN, b-catenin, C-JUN and CYC D (all part of the canonical Wnt pathway-for further details see 'Discussion'), our data suggest a strong transcriptional effect on this pathway by c-secretase activity and resulting alterations in gene expression. CYC D has also been reported to be downregulated by Protein tyrosine phosphatase receptor type G (PTPRG) [39] . We confirmed by real time PCR that PTPRG transcript level is reduced by 515-fold (Fig. 3) . AMN1 (levels down 978-fold) has also been connected with cell cycle regulation in yeast, but its role in mammals is not well known. TERA, a gene of unknown function (levels down 24-fold), has also been associated with Wnt antagonism (see Fig. 3 and results of human cortex analysis). b-actin served as housekeeping gene.
Protein interaction data suggest Wnt pathways as a major target of c-secretase susceptible gene transcription
In order to see whether c-secretase affects the transcription of genes encoding interacting proteins, an interaction map of encoded proteins was generated with the string 8.0 data bank exclusively relying on evidence-based data. Clusters of protein interactions suggest the Wnt signaling pathways as a major focus of c-secretase-affected candidates (Fig. 4 , highlighted in grey). Indeed, we found several members of the canonical Wnt pathway, but also some interactors of the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway, to have c-secretase activity susceptible gene transcription (Fig. 4) . Some of these genes have been confirmed by real time PCR as well as DIGE experiments (Egger et al., unpublished). The largest decrease in gene transcription occurred for the gene encoding the protein Ptprg. This single-pass type I membrane protein dephosphorylates protein tyrosine phosphate and was recently suggested as a candidate tumor suppressor gene in nasopharyngeal carcinoma [39] . The same group reported functional evidence for a critical interaction of Ptprg with the extracellular matrix, which induces cell arrest, changes in cell cycle status and downregulation of cyclin D1 [39] . The latter is strongly affected by the canonical Wnt pathway. Ptprzeta and beta, structurally similar to Ptprg, interact with Psd95 [40] , which directly interacts with Wnt3a [41] . We could confirm that WNT3A transcripts show an increase of 2.8-fold (Fig. 3) . Further, Wnt3a has also been reported to interact directly with LRP1 ( Fig. 4, lower right) , a stimulator of the Wnt5a signaling pathway [42] and a known c-secretase substrate tying c-secretase to a major AD risk factor, ApoE [43] . Porcn, another protein that interacts with Wnt3a [44] , shows a three-fold increase in transcript level by the microarray experiment. Porcn also interacts with Wnt 6 (4-fold increase in microarray) as reported by the same group and is the first player of the canonical Wnt pathway as displayed by the Kegg database (mmu04310, http://www. genome.jp/dbget-bin/show_pathway?mmu04310). Wnt3a interacts with Frizzled 1 [45] , which showed a 5-fold increase in mRNA levels by our microarray. Following the canonical Wnt pathway, the first intracellular protein of the Wnt signaling cascade is ''Disheveled''. As confirmed by real time PCR, DVL3 mRNA is increased by 3-fold with enhanced c-secretase activity. Next, with the help of Gbp (microarray reports a 4-fold increase of Gbp2), Gsk-3b is inhibited, which in turn inhibits b-catenin. As made apparent by the graphical overview of interacting proteins encoded by genes we found to be transcriptionally susceptible to csecretase activity, b-catenin plays a central role, linking different proteins involved in different Wnt pathways (Fig. 4) . Furthermore, b-catenin has been found to function as a major node connecting PS1 and several proteins that are encoded by genes that we found to be differentially transcribed (Fig. 4) . b-catenin transcription was shown by the microarray to be 3-fold decreased. It interacts directly with cdh15 (which showed a 2.4-fold increase in transcript levels as confirmed by real time PCR, Fig. 3) , with Cdh1 (a known c-secretase substrate [46] ), with PS1 (the c-secretase catalytic subunit) and other proteins encoded by candidate genes reported by the microarray. In the context of the canonical Wnt pathway, b-catenin affects c-myc (CMYBP 3-fold increased on the microarray), c-jun (3-fold decreased on the microarray) and cyclin D (4-fold decreased on the microarray) -the latter, as mentioned, is also downregulated by Ptprg. Dvl3 however also interacts with other proteins encoded by candidate genes, among which Nkd1 is of special interest since it links the canonical with the planar cell polarity pathway where it has a different effect on Dvl. The Planar cell polarity pathway through several players, among them Rac (3fold decreased on the microarray) affects gene transcription, as we hypothesize for c-secretase activity changes. Through a chain of different mediators, the planar cell polarity Wnt pathway affects the cytoskeleton. Our microarray has reported some of these mediators to be differentially transcribed as c-secretase activity is enhanced; RhoA transcript levels for example are 3-fold decreased. Rock, which is known to directly interact with the csecretase substrate CD44 (CD44/Rho Family GTPase/ROCK2) [47, 48] , is transcriptionally affected too. Our top candidate UPP1 has only one interaction partner that was also reported to be differentially expressed by the microarray, the cytoskeleton protein vimentin (Vim) [35] . Vimentin itself is not new to AD research, as altered Vim distribution patterns were observed in FAD fibroblasts [36] . Also, UPP1 transcription is regulated by the transcription factor Oct3/4, as is the transcription of another candidate, called SPP1 [49] . Spp1 is a direct interaction partner of the aforementioned c-secretase substrate CD44 and strongly affects Ca 2+ levels [50] . It directly interacts with several proteins encoded by candidate genes, including PKCA, which itself directly interacts with Aplp2, a well-known c-secretase substrate, and Csnk2b, which directly interacts with b-catenin, thus closing the circle.
Csnk2b also directly interacts with Shmt1, which has enhanced transcription of 3.5-fold (Fig. 3) , and has been further confirmed in DIGE experiments (Egger et al., unpublished) . The third Wnt pathway mentioned is the Wnt/Ca 2+ pathway which includes, among others, Plc (Plcb1 5-fold increase on the microarray), CaMKII (4-fold decrease on the microarray) and Calpain (3-fold decrease on the microarray). Our mapping of genes differentially transcribed with c-secretase activity shows that they encode proteins that directly interact with each other, with many of them being members of Wnt pathways.
Our modeling of extreme levels of c-secretase activity in CHO cells has revealed c-secretase-dependent differences in transcript levels of specific genes. One of the major known risk factors for developing Alzheimer's disease is carrying the ApoE4 allele.
Recently it was shown that ApoE through LRP1 regulation is connected with c-secretase [43] , which supports the hypothesis of a potential role of c-secretase in sporadic AD. c-Secretase is also directly implicated in the inheritable familial early onset forms of AD (FAD), as most cases are caused by mutations in PSEN1, the gene encoding for PS1, the catalytic center of this enzyme.
To investigate whether changes in gene transcription that coincide with alterations of c-secretase activity levels also differ between sporadic Alzheimer's and healthy human brain tissue, we evaluated our top scoring c-secretase affected genes in human AD and healthy cortices. Based on b-actin as housekeeping gene, we found one c-secretase affected gene, TERA, to be significantly differentially transcribed in the AD brain relative to the normal brain. Real-time PCR results showed an average two-fold increased TERA transcript levels (P2 = 0.04) in human AD cortices compared to healthy controls (Fig. 5) .
Altogether, the Wnt antagonism gene TERA represents a new candidate for differential expression with c-secretase activity as well as in AD brain cortex tissue. Whether it is implicated in the pathogenesis of AD requires further investigation.
Since the discovery of the roles for NICDs and AICDs in gene transcription, the notion of c-secretase as a major player in pathologically altered gene transcription patterns has been steadily gaining ground with new substrates and their transcriptionally active ICDs being identified regularly. To investigate the impact of c-secretase activity on gene transcription, we compared two starkly contrasting situations: genetically engineered enhanced human csecretase activity and pharmacologically inhibited c-secretase activity in CHO cell lines. By investigating the effects of enhanced c-secretase activity on gene transcription using cDNA microarray analysis, we could show that the canonical, the planar cell polarity (PCP) and the Ca 2+ /Wnt pathways are transcriptionally affected through more than a dozen of Wnt signaling players (summarized in Fig. 6 ). From Proc and Wnt outside the membrane, through Frizzled and Dvl, to b-catenin and down to cell cycle regulating genes, the canonical Wnt pathway is the most affected of Wnt pathways. Several genes of the PCP Wnt pathway as well as Ca 2+ / Wnt pathways were found to be differentially expressed too (Fig. 6) . One of the cell cycle regulating genes is CYC-D, which itself is Figure 5 . Selected relative gene transcript levels in AD cortices. Real time PCR validated genes differentially transcribed in cells with enhanced c-secretase activity were selected and their gene transcript levels analyzed in ten to twelve AD and healthy human cortical brain tissue samples. Only the transcript levels of TERA, a gene of unknown function, is significantly altered with a two-fold increase in AD cortices. Note that TERA transcript levels were significantly reduced in cells with enhanced c-secretase activity (Fig. 3) . Relative quantification of gene transcription in CHO cells as well as in brain tissue used b-actin as housekeeping gene. Healthy control levels are displayed on the left part of each diagram, AD transcript levels on the right. Dashed lines indicate mean values for healthy controls (green) and AD cases (red). Double-headed arrows indicate tendencies of differences between groups. P2 values obtained from t-test are indicated in black boxes of the upper part of each diagram. doi:10.1371/journal.pone.0006952.g005 regulated by one of the most c-secretase-dependently altered genes reported by us, PTPRG.
Functional clustering of the microarray data revealed the overrepresentation of the ''receptor binding'' cluster, which includes four different Wnt signaling molecules and b-catenin. b-Catenin also finds itself in the center of interactions of proteins encoded by strongly differentially expressed genes. Components downstream of the canonical Wnt pathway, like c-myc, c-jun and cycD, influence the cell cycle, the latter as mentioned is downregulated by protein tyrosine phosphatase receptor type c (Ptprg). Interestingly, we found PTPRG transcription to be strongly decreased in cells with enhanced c-secretase. Barnea et al. [51] identified a subfamily of PTPRs, defined by the carbonic anhydrase-like domain in the extracellular region of PTPRG, and described its expression during hippocampal formation, and in septal and midline thalamic nuclei in the cortex of newborn rats (in contrast to the expression pattern in adult rats, which is reduced to the hippocampal formation). Several groups have shown a connection between alterations in receptor tyrosine phosphatases' expression levels and c-secretase [52, 53] . However, we report here for the first time, to our knowledge, the transcriptional connection between the receptor tyrosine phosphatase type gamma and c-secretase.
TERA, a gene that we found to be decreased in transcription (down by 23.5-fold), has been connected to brain development and Wnt antagonism as well. TERA is decreased to minimal transcript levels with enhanced c-secretase activity (Fig. 3) . This gene, encoding a phosphoprotein of unknown function, is upregulated in squamous cell carcinoma (SCC), adenocarcinoma (AC), and colon, ovary, rectum and stomach tumors [54] (suggesting associations with Notch?). It has also been reported that TERA gene expression is increased in day 13 embryonic (E13) and decreased in E17 cortex and maintains low, but consistent expression levels in the subventricular zone (SVZ) [55] . The expression pattern in earlier rather than later stages of brain development and in the location of neuronal stem cell niches, like the SVZ, suggest possible roles for Tera in regenerative processes and raise questions about its function if the gene is being shut down in degenerative disorders like AD [55] . Tera expression has further been found to be maintained in neural progenitors and downregulated during non-neural differentiation, and was shown to have appreciable expression in embryonic stem cells in a screen Figure 6 . Involvement of c-secretase-dependently transcribed genes in Wnt pathways. Several key players of the canonical Wnt pathway (green panel) were reported by our microarray to increase (red quadrangles) or decrease (blue quadrangles) in transcript levels under conditions of enhanced c-secretase activity compared to inhibited activity. b-Catenin is a central node connecting Wnt-Frizzled-Dishevelled to a downstream effect influencing the cell cycle (see also Fig. 4 and interactions of encoded proteins). For better understanding, selected genes that were not detected by the microarray are displayed as well (dashed lines black quadrangles). CycD was reported to be regulated by PTPRG, one of the top scoring candidates for c-secretase affected gene transcription. Nkd, which we found to be increased in transcript levels, connects the canonical Wnt pathway with the planar cell polarity pathway (blue panel). CD44, a well-known c-secretase substrate, interacts with SPP1. SPP1 and UPP1, two strong candidates are both under the control of the same transcription factor Oct3/4, as has been suggested for TERA [92] . UPP1 directly interacts with Vimentin (see also Fig. 4) , a known player in AD and a cytoskeletal protein. Crucial genes of the Wnt/Ca 2+ pathway (grey panel) were also found to be differentially expressed in our array. All together, c-secretase activity influences the transcript levels of genes of the canonical, the planar cell polarity and Ca 2+ Wnt pathways. doi:10.1371/journal.pone.0006952.g006 for functional genes in ES cells that implicated Wnt antagonism in neural differentiation [56] .
TERA and the anti-mitotic exit network antagonist 1 (AMN1) map to chromosome 12p11, which is interesting when considering the fact that chromosome 12 has been discussed to contain an unknown LOAD locus for over a decade, and in a recent study including 492 LOAD cases [57] [58] [59] . In our study, AMN1 transcription is decreased by 978-fold with enhanced c-secretase activity. The function of AMN1 is not known. However, several expression pattern based studies suggest it functions as a cilia gene in sensory neurons [60] . Another typical cilia gene is intraflagellar transport protein 81 (IFT81) which, among a dozen of known cilia genes, was also shown by the microarray to be differentially expressed with altered c-secretase activity (see also Fig. 3 ). More and more evidence has been emerging over the last years that primary cilia, in parallel to their well-established functions in sight, smell and mechanosensation, are key participants in intercellular signaling [61] . The importance of monocilia for the regeneration of olfactory neurons has only been better understood recently [62] . Subventricular zone (SVZ) astrocytes, providing glia as well as neurons for the mammalian olfactory bulb, have primary cilia [63] . They give rise to type C cells, which in turn generate neuroblasts [64] that migrate in the adult brain from the SVZ to the olfactory bulb along the cerebrospinal fluid (CSF) flow. The CSF flow depends on the beating of the ependymal cilia [65] . Cilia genes are not only relevant to the maintenance of adult regeneration in the brain since they uphold the constant flow of the CSF, but also because they are directly implicated in cell cycle control. Polycystins, for example, control the cell cycle through three major pathways with one depending directly on b-catenin [66] . A study of inversin has further shown that flow shear stress as sensed through cilia may regulate the Wnt signaling pathway through b-catenin [67, 68] . Given that fluid flow is crucial for the transport of neuroblasts in the SVZ, one could expect that bcatenin and the Wnt signaling pathway that connects our candidates are also functionally relevant to the cilia genes found in this study. We found both genes of unknown function TERA and AMN1 to be decreased in transcription with enhanced csecretase activity. TERA and AMN1 can be connected to neural stem cells through several types of cancer, neural differentiation (in the case of TERA) and through the role of monocilia for neurogenesis (in the case of AMN1). All in all, we have demonstrated that AMN1 and TERA are genes of basically unknown function that are worthy of further investigation to understand their roles in neurogenesis, cancer and c-secretase biology.
We further report here that UPP1 transcript levels are increased with enhanced c-secretase activity (by 39.2-fold). UPP1 encodes for uridine phosphorylase (UPase), an enzyme that catalyzes the reversible phosphorylytic cleavage of uridine and deoxyuridine to uracil and ribose-or deoxyribose-1-phosphate [69] . UPP1 expression has been extensively connected to cancer, stem cells and inflammation such as multiple sclerosis [70] [71] [72] [73] [74] [75] [76] [77] . UPase is induced by vitamin D3 and a mixture of inflammatory cytokines, Interferon gamma, TNF-alpha and IL-1, with the latter two being upregulators of Ptprg [78] . Increased UPP1 transcript levels, associated with enhanced UPase activity cleaving uridine, would potentially have inhibitory effects on several pathways downstream of uridine, like RNA/DNA and membrane synthesis, as well as protein glycosylation, which would in turn trigger long-term neurodegeneration. Particularly, decreased membrane synthesis, in the case of synaptic membranes, would also reduce synaptic activity and plasticity. In support of that, TNF-a and IL-1, inducers of UPP1, alter lipid metabolism and stimulate production of eicosanoids, ceramide and reactive oxygen species that potentiate CNS injuries and certain neurological disorders [33] . Interestingly, this hypothesis offers an explanation for the multitude of beneficial effects of orally administered DHA and uridine on memory, neuronal health, regeneration and membrane synthesis in traumatic and chronic neuropathological conditions [33, 34] .
The presented work demonstrates that c-secretase is capable of influencing single gene transcription. However strong the impact will prove to be on the protein level of each single gene, we have further observed transcriptional effects spanning several genes throughout clearly defined pathways. This puts forth the possibility of much stronger effects on the target functions of these pathways than the small impact on the individual genes transcriptional or translational levels might indicate. In support of this hypothesis, we have observed that the proteins encoded by those genes interact with each other and are part of the Wnt pathways. Evaluation of the impact of these pathway-specific accumulative effects needs further investigation. This should include physiological and pathological in vivo experiments on both the transcript as well as protein levels. For c-secretase to serve as a therapeutic target, it is indeed crucial to sharpen our view of its role and influence over gene transcription and biological functions.
The S-1 cell line overexpressing Flag-Pen2, Aph1-a2-HA, PS1 and NCT-GST [17, 79] was derived from the Chinese Hamster Ovary (CHO) c-30 cell line [80] 
CHO parental cell line triplicates were exposed for 24 hrs to the c-secretase inhibitor DAPT (10 mM) in DMSO (0.05%), and S-1 cells were treated for the same time with DMSO (0.05%). Cells were next washed twice with PBS and total RNA was extracted, amplified, reversely-transcribed, labeled and hybridized to a 17 k mouse cDNA microarray chip produced by the DNA array facility of Lausanne (DAFL, see below).
Total RNA extraction: was performed using the RNeasy Mini Kit (Qiagen, Basel, Switzerland), in the absence of DNAse treatment. RNA quality was assessed using the RNA 6000 Nanochip assay (Agilent Technologies, Meno Park, USA) and RNA concentration was determined using the ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, USA). Three independent experiments were performed.
RNA amplification: a single round of amplification was performed with 3 mg of total RNA using the MessageAmp RNA Amplification Kit (Ambion, Austin, USA) and following the protocol provided with the kit. Next, 5 mg of amplified RNA was mixed with 9 mg random primers (Cat. No. 4819001; Invitrogen, Carlsbad, USA) in 19 ml of water, heated for 5 minutes at 70uC and then immediately transferred to ice.
Reversed transcription and labeling: was performed for 2 hrs at 42uC in a final reaction volume of 40 ml containing 1X SuperScript II buffer (Invitrogen), 40 units RNasin (Promega, Madison, USA), 10 mM DTT, 0.5 mM dATP, dGTP, dTTP, 0.2 mM dCTP, 0.1 mM of either Cy3-dCTP or Cy5-dCTP (GE Healthcare, Uppsala, Sweden) and 400 units of SuperScript II reverse transcriptase (Invitrogen). The RNA strand was hydrolyzed by adding 2 ml 500 mM EDTA and 4.5 ml 1 M NaOH and heating at 65uC for 15 minutes; the solution was then neutralized by adding 2.5 ml 1 M Tris (pH 6.8) and 4.5 ml 1 M HCl. The labeled cDNA was purified using the Qiagen MiniElute PCR Purification Kit (Cologne, Germany), eluting in 50 ml of EB buffer according to the manufacturer's instructions. The Cy3 and Cy5 labeled targets were combined and mixed with 400 ml of TE, 20 mg Cot 1 DNA (Invitrogen), 10 mg polyadenylic acid (Sigma, St. Louis, USA) and 10 mg yeast tRNA (Sigma). This mixture was concentrated to a final volume of 19.4 ml using a Microcon YM-30 filter (Millipore, Billerica, USA) according to the manufacturer's instructions. 20X SSC and 10% SDS were added to final concentrations of 3X and 0.4%, respectively, in a final volume of 24 ml. This mixture was heated for 2 minutes at 98uC, pipetted immediately onto the cDNA microarray and, after covering with a glass cover slip (Erie Scientific, Portsmouth, USA), placed in a humidified chamber (Telechem, Sunnyvale, USA) and allowed to hybridize at 64uC for 20 hrs. Slides were then washed at room temperature twice for 5 minutes in 2X SSC, 0.1% SDS, twice for 1 minute in 0.2X SSC, once for 1 minute in 0.1X SSC and once for 5 minutes in 0.1X SSC, 0.1% Triton X-100. After drying, slides were scanned on a microarray scanner (Agilent Technologies) and the resulting TIFF images were analyzed using the GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, USA). The mouse cDNA microarrays used in this study consisted of approximately 17,000 PCR products generated from cDNA clones and control DNAs spotted onto Nexterion AL slides (Schott, Mainz, Germany). A complete description of the slides and their content can be obtained from the Lausanne DNA Array Facility (http://www.unil.ch/dafl). The microarray data set discussed in this publication has been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE16379. Note that Hamster genomic sequence information is not yet sufficiently available to the research community. Consequently no commercial hamsterspecific microarrays were available at the time of the experiment. However, the strategy to use a microarray from a closely related species is not new and has proven successful before [81] .
The analysis was performed with open source R software packages (http://www.r-project.org/ and http://www.BioConductor.org/). Gene expression was quantified with the array package using print tip group lowess normalization without background subtraction. The resulting measures of expression for each array are the log2 ratios (M values) and the average log2 intensities (A value) of Cy3 and Cy5 signals. Statistics of differential expression between the different groups of samples were calculated with a linear model fitted by the limma package.
Total RNA was isolated with the RNeasy mini kit following the manufacturer's protocol for adherent cells in the case of CHO cell cultures. For the isolation of total RNA from brain tissue, the TRIzol reagent was used as described in the human samples section. RNA was dissolved in water, which was followed by ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, USA) quantification and pico chip quality control analysis (6000 Nanochip assay Agilent Technologies, Meno Park, USA).
Total RNA was reverse transcribed with our standard laboratory protocol. 1 mg of total RNA was dissolved in 4 ml of RNase-free water (Ultrapure DNase/RNase free water, Invitrogen Carlsbad, USA)) and premixed with 0.5 mg of oligo dT primer (synthesized by Eurogentec Seraing, Belgium) dissolved in 1 ml RNase-free water. The RNA/oligo dT premix was heated to 70uC for 5 minutes in a standard PCR machine (TProfessional Basic Gradient, Whatman Biometra Goettingen, Germany). The machine was paused to add 4 ml of 5X Buffer (ImProm-II M28A, Promega Madison USA), 4 ml of MgCl 2 (25 mM) (Promega Madison USA), 1 ml dNTP Mix (10 mM U151B, Promega Madison USA), 1 ml RNase inhibitor (RNasin Plus N261A 40 u/ul Promega Madison USA), 1 ml of ImProm-II Reverse Transcriptase (Promega Madison, USA) and 4 ml RNasefree water. The PCR machine program was continued after pausing at 25uC for completion of reaction mixes with 60 min at 42uC and 15 min at 70uC. cDNA was kept at 4uC on wet ice for short-term or at 280uC for long-term storage.
Reverse transcription products were used without purification for real time PCR at equivalent of 0.5 ng/ml RNA in 384 well plates. Samples were used as biological triplicates and each one was additionally pipetted as a triplicate. Reaction volumes were 10 ml consisting of 5.02 ml SYBR Green (Power SYBR Green Master Mix #4367660 Applied Biosystems, Cheshire UK), 1.49 ml RT-PCR product at 0.5 ng/ml input RNA equivalent (0.75 ng/rxn) and 3.49 ml of 3 mM Forward and Reverse primer mix. 384 well plates were prepared with a liquid handling robot (Freedom EVOware Tecan Trading AG, Switzerland) and read for relative quantification with Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Cheshire UK). Primers (synthesized by Eurogentec Seraing, Belgium) for CHO cDNA were based on mouse code, which was aligned with rat and human code, preference was given to aligning sequences (Table 3) . Sequence specificity was determined via nBlast. b-actin was used as housekeeping gene [82] [83] [84] [85] [86] [87] [88] [89] for CHO as well as human cortex templates with the forward sequence: CCTTCAACACCCCAGCCATGTACG and the reverse sequence: CCTTCAACACCCCAGCCATGTACG.
Results were analyzed by the DDCt method [90] and significance was calculated via students t-test. b-actin was used a normalizer to determine DCts. DDCts were calculated against the mean of DAPT treated WT-CHO DCts or the mean of healthy human brain cortex DCts. Results were expressed as relative quantification by 2ˆ-(DDCt) [90] .
Human brain tissue was kindly provided by the Joseph and Kathleen Bryan Alzheimer's Disease Research Center, Duke University Medical Center. The Autopsy and Brain Donation procedures have been approved by the Duke University Institutional Review Board (IRB) and cortical brain tissue was obtained as described by [91] . 12 AD post-mortem confirmed cortical samples as well as 12 healthy cortical samples were obtained in dry ice. Cortical samples were of both genders, different ages, ApoE stati and Brack stages.
Isolation of total RNA: ,50 ug of total cortex tissue were scraped off on dry ice three times for biological triplicates of each sample. TRIzol reagent (Invitrogen Carlsbad, USA) was used according to manufacturer's protocol for total RNA isolation.
RNA was dissolved in water, which was followed by ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, USA) quantification and pico chip quality control analysis (6000 Nanochip assay Agilent Technologies, Meno Park, USA).
Dataset S1 EST clones with increased transcription under enhanced c-scretase activity compared to inhibited c-secretase activity. By applying a cut-off with a p value of 0.005 based on the false discovery rate (FDR, i.e. the probability to wrongly accept a difference between the two conditions), we found 2658 EST clones to be differentially expressed, with 1241 EST clones of increased with enhanced c-secretase activity compared to inhibited csecretase activity. FC = Fold change; adj,P,Val = adjusted P-value Found at: doi:10.1371/journal.pone.0006952.s001 (0.20 MB XLS)
Dataset S2 EST clones with decreased transcription under enhanced c-secretase compared to inhibited c-secretase activity. By applying a cut-off with a p value of 0.005 based on the false discovery rate (FDR, i.e. the probability to wrongly accept a difference between the two conditions), we found 2658 EST clones to be differentially expressed, with 1417 EST clones of decreased transcription with enhanced c-secretase activity compared to inhibited c-secretase activity. FC = Fold change; adj,P,Val = adjusted P-value Dataset S4 EST clones of transcriptional relevance differentially transcribed under enhanced c-secretase compared to inhibited csecretase activity. By applying a cut-off with a p value of 0.005 based on the false discovery rate (FDR, i.e. the probability to wrongly accept a difference between the two conditions), we found 2658 EST clones to be differentially expressed with enhanced c-secretase activity compared to inhibited c-secretase activity. Among them 56 imply transcriptional relevance. FC = Fold change; adj,P,Val = adjusted P-value. Found at: doi:10.1371/journal.pone.0006952.s004 (0.03 MB XLS)  Bayesian Phylogeography Finds Its Roots As a key factor in endemic and epidemic dynamics, the geographical distribution of viruses has been frequently interpreted in the light of their genetic histories. Unfortunately, inference of historical dispersal or migration patterns of viruses has mainly been restricted to model-free heuristic approaches that provide little insight into the temporal setting of the spatial dynamics. The introduction of probabilistic models of evolution, however, offers unique opportunities to engage in this statistical endeavor. Here we introduce a Bayesian framework for inference, visualization and hypothesis testing of phylogeographic history. By implementing character mapping in a Bayesian software that samples time-scaled phylogenies, we enable the reconstruction of timed viral dispersal patterns while accommodating phylogenetic uncertainty. Standard Markov model inference is extended with a stochastic search variable selection procedure that identifies the parsimonious descriptions of the diffusion process. In addition, we propose priors that can incorporate geographical sampling distributions or characterize alternative hypotheses about the spatial dynamics. To visualize the spatial and temporal information, we summarize inferences using virtual globe software. We describe how Bayesian phylogeography compares with previous parsimony analysis in the investigation of the influenza A H5N1 origin and H5N1 epidemiological linkage among sampling localities. Analysis of rabies in West African dog populations reveals how virus diffusion may enable endemic maintenance through continuous epidemic cycles. From these analyses, we conclude that our phylogeographic framework will make an important asset in molecular epidemiology that can be easily generalized to infer biogeogeography from genetic data for many organisms. Phylogenetic inference from molecular sequences is becoming an increasingly popular tool to trace the patterns of pathogen dispersal. The time-scale of epidemic spread usually provides ample time for rapidly evolving viruses to accumulate informative mutations in their genomes [1] . As a consequence, spatial diffusion-among other processes-can leave a measurable footprint in sampled gene sequences from these viruses [1] . Reconstructing both the evolutionary history and spatial process from these sequences provides fundamental understanding of the evolutionary dynamics underlying epidemics, e.g. [2, 3] . It is also hoped that these insights can be translated to effective intervention and prevention strategies [4] and elucidating the key factors in viral transmission and gene flow over larger distances is central in formulating such strategies, e.g. [5] .
Phylogeographic analyses are a common approach in molecular ecology, connecting historical processes in evolution with spatial distributions that traditionally scale over millions of years [6] . Many popular phylogeographic approaches [7, 8] can be remiss in ignoring the interaction between evolutionary processes and spatial-temporal domains. One first reconstructs a phylogeny omitting spatial information and then conditions the phylogeo-graphic inferences on this reconstruction [1, 9] , exploiting nonparametric tests to evaluate the significance of this conditional structure, e.g. [7, 10, 11] . To draw conclusions about the epidemic origin or epidemiological linkage between locations, however, we require a reconstruction of the dispersal patterns and process throughout the evolutionary history. Considering locations as discrete states, this boils down to the well-known problem of ancestral state inference [7] . Parsimony is a popular heuristic approach to map characters onto a single phylogenetic tree [12] . Unfortunately, parsimony reconstructions ignore important sources of model uncertainty, including both uncertainty in the dispersal process as well as in the unknown phylogeny [13] . In addition, minimizing the number of state exchanges over a phylogeny is misleading when rates of evolution are rapid and when the state exchange probabilities are unequal [14] .
Probabilistic methods draw on an explicit model of state evolution, permitting the ability to glimpse the complete state history over the entire phylogeny and conveniently draw statistical inferences [15] [16] [17] . These analyses typically employ continuoustime Markov chain models for discrete state evolution analogous to common nucleotide, codon or amino acid substitution models [18] . In contrast to parsimony, maximum likelihood-based reconstructions incorporate branch length differences in calculat-ing the conditional probability of each ancestral state given the observed states at the phylogeny tips [14] . Bayesian reconstruction methods enable further generalization of this conditional probability analysis by removing the necessity to fix the Markov model parameters to obtain ancestral states and the necessity to specify a fixed tree topology with known branch lengths. Bayesian inference integrates conclusions over all possible parameter values but to achieve this, however, requires prior probability distributions for all aspects of the model. While probabilistic methods have been previously presented in a bio-or phylogeographic context, in particular Bayesian methods that integrate over phylogenetic uncertainty and Markov model parameter uncertainty [19] , viral phylogeography studies have rarely made use of these developments. This may be a consequence of low awareness of existing software implementations for arbitrary continuous-time Markov chain models [20, 21] or a lack of appreciation for the uncertainty intrinsic in these reconstructions and the ease with which one can formally access epidemiological linkage through probabilistic approaches. A recent phylogeographic study of influenza A H5N1 introduces a heuristic non-parametric test to evaluate whether parsimonyinferred migration events between two particular locations occur at significantly high frequency [22] . Null distributions for these frequencies arise from randomizing tip localities after false discovery rate correction to control for simultaneous testing issues. Although this procedure addresses concerns about statistical inference on sparse frequency matrices, the multiple comparison correction still results in a conservative estimate of significant migration events. Fully probabilistic approaches may further ease statistical inference, yet similar tests remain lacking for likelihoodbased phylogeographic models.
Advances in evolutionary inference methodology have frequently demonstrated how novel approaches can be appended to a sequence of analyses, in many cases starting from alignment to parameter estimation conditional on tree reconstructions. For example, demographic inference has involved genealogy reconstruction, estimating a time scale for the evolutionary history, and coalescent theory to quantify the demographic impact on this tree shape [23] . It is well acknowledged that such sequential procedures ignore important sources of uncertainty because they generally purge error associated with each intermediate estimate.
With the advent of novel computational techniques like Markov chain Monte Carlo (MCMC) sampling, it has become feasible to integrate many of the models involved and simultaneously estimate parameters of interest. Demographic inference is a wellknown example of genealogy-based population genetics that benefited from these advances [24, 25] . Bayesian MCMC methods also enable ancestral state reconstruction while simultaneously accounting for both phylogenetic and mapping uncertainty. Although this adds much needed credibility to ancestral reconstruction [13] , phylogeographic analysis would benefit even more from fully integrating spatial, temporal and demographic inference.
Here, we implement ancestral reconstruction of discrete states in a Bayesian statistical framework for evolutionary hypothesis testing that is geared towards rooted, time-measured phylogenies. This allows character mapping in natural time scales, calibrated under a strict or relaxed molecular clock, in combination with several models of population size change. We use this full probabilistic approach to study viral phylogeography and extend the Bayesian implementation to a mixture model in which exchange rates in the Markov model are allowed to be zero with some probability. This Bayesian stochastic search variable selection (BSSVS) enables us to construct a Bayes factor test that identifies the most parsimonious description of the phylogeographic diffusion process. We also demonstrate how the geographical distribution of the sampling locations can be incorporated as prior specifications. Through feature-rich visual summaries of the space-time process, we demonstrate how this approach can offer insights into the spatial epidemic history of Avian influenza A-H5N1 and rabies viruses in Africa.
The highly pathogenic avian influenza A-H5N1 viruses have been present for over a decade in Southern China and spread in multiple waves to different types of poultry in countries across Asia, Africa and Europe [26] . As a result, highly pathogenic A-H5N1 is now a panzootic disease and represents a continuous threat for human spill-over. Strong surveillance has been in place since these viruses caused extensive outbreaks, but the source and early dissemination pathways have remained uncertain. Because parsimony analysis has attempted to shed light on the latter [22] , A-H5N1 provides an ideal example for comparison with Bayesian phylogeographic inference. Rabies is endemic in Asia and Africa, where the primary reservoir and vector for rabies virus (RABV) is the domestic dog. Phylogenetic analysis has revealed several genotypes of lyssaviruses (family Rhabdoviridae); genotype 1 has been found responsible for classical rabies, a fatal disease in terrestrial mammals throughout the world [27, 28] . Here, we explore the phylogeographic history of RABV in domestic dogs in West Central Africa, using recently obtained sequence data, and evaluate the role of viral dispersal in maintaining RABV epidemic cycles.
We examine the evolution and spatial dispersion of two viral pathogens, Avian influenza A-H5N1 and rabies, to demonstrate the strengths and limitations of our discretized stochastic model for phylogeography.
To reconstruct the spatial dispersion patterns of Avian influenza A-H5N1, we analyze the hemagglutinin (HA) and neuraminidase (NA) gene datasets previously compiled by [22] . Both datasets contain whole gene sequences from 192 A-H5N1 strains sampled from 20 localities across Eurasia. [22] explore these genes individually, as well as concatenated together, through a strictly parsimony-based ancestral reconstruction method. Our Bayesian approach builds upon stochastic models and naturally affords
Spreading in time and space, rapidly evolving viruses can accumulate a considerable amount of genetic variation. As a consequence, viral genomes become valuable resources to reconstruct the spatial and temporal processes that are shaping epidemic or endemic dynamics. In molecular epidemiology, spatial inference is often limited to the interpretation of evolutionary histories with respect to the sampling locations of the pathogens. To test hypotheses about the spatial diffusion patterns of viruses, analytical techniques are required that enable us to reconstruct how viruses migrated in the past. Here, we develop a model to infer diffusion processes among discrete locations in timed evolutionary histories in a statistically efficient fashion. Applications to Avian Influenza A H5N1 and Rabies virus in Central and West African dogs demonstrate several advantages of simultaneously inferring spatial and temporal processes from gene sequences. quantification of uncertainty in both the ancestral state reconstructions and the underlying phylogeographic process. Further, as we are able to infer plausible root positions unlike the original analysis, we are not required to include outgroup sequences. To model sequence evolution, we employ the [29] (HKY85) CTMC model of nucleotide substitution; we include discrete gamma-distributed rate variation [30] and assume an unknown, constant population-size coalescent process prior over the unknown phylogeny [31] . Exploratory analyses using the less restrictive Bayesian skyline plot model indicate that the demographic prior has little influence on the phylogeographic inference (data not shown). Figure 1 summarizes the Bayesian maximum clade credibility (MCC) trees for the A-H5N1 HA and NA segments. An MCC tree is a point-estimate characterizing the posterior distribution of trees and represents the tree topology yielding the highest product of individual clade probabilities in their posterior sample [2] ; branch lengths in these MCC trees are posterior median estimates.
We further annotated the tree nodes with their most probable In combination with other topological differences between the trees, this difference strongly suggests past reassortment events between both segments, with the progenitor virus of the basal Hong Kong clade and a chicken strain from Hebei having acquired an NA segment from different lineages. Such events are not surprising given frequent reports of A-H5N1 reassortment in China, e.g. [26] , and the particular reassortment event for the basal Hong Kong clade has very recently been confirmed [32] .
Despite different time scales for HA and NA, most probable location states agree on Guangdong as the predominant location of these sequences throughout the majority of their evolutionary history. As an indication of the A-H5N1 epidemic origin, we consider the inferred location at the root of the trees (Figure 1 ). In the HA tree, Guangdong and Hong Kong share a vast majority of the posterior mass, neighboring locations in which surveillance efforts report early Avian influenza cases [33, 34] . In the NA tree, although Hong Kong and Guangdong still obtain marginally higher support than other locations, all posterior root state probabilities are much closer to their prior probability. The substantially deeper NA root explains this difference as the depth greatly increases uncertainty on the root state. Table 1 quantifies differences in ancestral state reconstruction uncertainty between the HA and NA trees using the Kullback-Leibler (KL) divergence measure (see Methods). The NA tree results in considerably lower KL divergence than the HA tree, signifying a much smaller deviation of the posterior distribution of the root location from the prior. However, lack of phylogeographic structure in the data does not contribute to this difference because the NA trees return a lower association index (AI). This measure of spatial admixture is based on a sum across all nodes in the tree of the complement of the frequency of the most abundant location among all descendent taxa weighted by the depth of the node in the tree [35] , and thus bears some relationship with an entropy value for descendent taxa locations. The AI rescales this sum by its expectation for randomized location assignments and results in low values for relatively strong phylogeny-locality correlation whereas AI values close to one reflect complete spatial admixture. If the basal Hong Kong clade and a chicken strain from Hebei have indeed acquired a different NA through reassortment, the root state might be difficult to interpret for NA and is not necessarily the same as that for HA. Therefore, we also list uncertainty measures for the marginal posterior distribution of the most recent common ancestor (MRCA) of the Gs/GD lineage, named after the A/ goose/Guangdong/1/96 strain very close to this node (indicated in Figure 1 ). KL divergence is again lower for this node in the NA phylogeny, but the difference is not as pronounced as for the root node. Table 1 also explores the effects of distance-informed priors and BSSVS on location reconstruction. In general, the distance- Figure 1 . Maximum clade credibility (MCC) phylogenies for hemagglutinin (HA) and neuraminidase (NA) genes of Avian influenza A-H5N1. We color branches according to the most probable location state of their descendent nodes. We use the same color coding as [22] . To the upper left of both phylogenies are their root location state posterior probability distributions. A white arrow indicates the A/goose/Guangdong/1/96 sequence; a filled white circle identifies the most recent common ancestor of the Gs/GD lineage named after this strain. doi:10.1371/journal.pcbi.1000520.g001 informed priors furnish little advantage while inferring the root locations for both the HA and NA trees. If anything, KL divergences are slightly smaller for models involving distanceinformed priors than those with flat priors. For these data, this finding is unsurprising as physical distances can be poor proxies for inverse-diffusion rates when dispersal results from a heterogenous mix of migratory birds, transport of poultry and poultry products, and trade of wild birds [36] . Finally, we also investigated the uncertainty that is accommodated by averaging over plausible trees by analyzing the HA data using a fixed tree topology and branch lengths ( Table 1 ). The state reconstructions for the Gs/GD node in the fixed tree topology appear to ignore some uncertainty in comparison to integrating trees, which is not that evident for the root node. Although state reconstruction uncertainty is expected to be correlated among nodes, we also compared the KL divergence summed over all internal nodes, indicating much higher KL divergences using a fixed tree topology, e.g. for HA, m jk~C : 292 vs 523 for integrating trees and a fixed tree respectively.
Under BSSVS, we assume a truncated Poisson prior that assigns 50% prior probability on the minimal rate configuration, comprising 19 non-zero rates connecting the 20 locations. This model strongly favors reduced parameterizations. A sensitivity analysis with respect to larger Poisson prior means reinforces that the data prefer a minimal number of rates, as increasing the mean leads to lower overall marginal likelihoods ( Table 2 ). BSSVS has a strong impact on root location reconstruction ( Figure 2 ). Many localities that are weakly supported as the root location without BSSVS obtain negligible posterior probability under BSSVS. Consequentially, BSSVS leads to larger KL divergences for both the HA and NA root nodes ( Table 1 ), suggesting that these reduced models more efficiently exploit the information content of the data. Interestingly, the posterior support for Guangxi increases under BSSVS at the expense of Guangdong in the HA phylogeny ( Figure 2 ). This may be an artifact of the reversible CTMC assumption we enforce. Specifically, at the tips of the phylogeny, several pathways of migration into Guangxi are highly likely. Assuming reversibility dictates that migration out of this location occurs as well; placing these emigrations deeper in the phylogeny is most consistent with the data. Because many locations already receive very low posterior probabilities at the GsGD node, the increase in posterior probability for a few locations now seems to outweigh the marginal reductions in posterior probabilities for most other locations and results in lower KL divergences at this node.
By specifying a prior on the number of non-zero rates, we are able to construct Bayes factor (BF) tests for significance of individual rates ( Figure 3 ). To visualize the epidemiological linkage that this test establishes, we employ Google Earth to display all rates with a non-zero expectancy that results in a BF larger than three. The majority of well-supported rates (16 out of 25 for both genes) are concordant between HA and NA. Some variation in support for different migration pathways between HA and NA was also noted in the original parsimony analysis [22] . Importantly, Guangdong presents as an end-point in three wellsupported epidemiological links in HA as well in NA. For HA, four migration links previously identified using the parsimony sFDR test (Guangdong to Fujian, Bangkok to Vietnam, Uthai Thani to Phitsanulok, and Qinghai to Novosibirsk) are also present in our well-supported symmetric rates. We can, however, not confirm epidemiological linkage directly between Guangdong and Indonesia. Despite having more supported rates by this Bayes factor test as compared to the parsimony sFDR test, it remains difficult to We report the Kullback-Leibler divergence between the posterior and prior location distributions of the root and the GsGD most recent common ancestor (MRCA), as well as a phylogeographic association index. We analyze genes independently, assuming equal phylogeographic models (Shared) and by fixing the HA phylogeny through phylogeographic models with prior rates proportional to a constant (C) or distance-informed (DI) and using Bayesian stochastic search variable selection (BSSVS). doi:10.1371/journal.pcbi.1000520.t001 univocally identify the pathways seeding remote localities as Japan and Indonesia, and to connect the eastern diffusion network with the Chinese/Russian inlands. Distance-informed priors do not have strong influence on the Bayes factor test for significant rates. The presence of reassortment amongst the gene segments obfuscates phylogenetic inference for concatenated HA/NA sequence data. In this respect, it is interesting to note that previous parsimony reconstructions on a phylogeny for the concatenated HA and NA segments result in fewer significant diffusion rates compared to the separate analyses; [22] found 2 for the concatenated alignment vs. 5 and 10 for HA and NA separately. The Bayesian framework enables a flexible combination of the data without having to specify a single phylogeny for both segments. To this end, we share the instantaneous rate matrix L between both segment phylogenies and sample all parameters in a single MCMC analysis. Without BSSVS, sharing the rate matrix results in slightly higher KL divergences for both the root node and the Gs/GD node in the HA and NA phylogenies (Table 1 ). Figure 4 illustrates the well-supported rates based on the Bayes factor test of the shared rate matrix with a distance-informed prior. The shared data bring to light two possible pathways seeding the remote localities of Japan and Indonesia; these pathways suggest Guangxi and Hunan as possible source for Indonesia, and Hunan and Hebei as possible source for Japan.
A major advantage of the current phylogeography implementation is the ability to infer the migration process in natural time scales. The panels in Figure 5 summarize the temporal dynamics of A-H5N1 spatial diffusion inferred using the shared rate matrix (KML files, Dataset S1 and S2 for HA and NA respectively, which enable visualizing the spread over time in Google Earth are available as supporting information). The lines connecting different locations represent branches in the MCC tree on which state exchanges occur and circle areas reflect the number of branches maintaining a particular state at that time point. By May 1997, Avian influenza lineages have accumulated in Guangdong, where the virus was originally isolated from a farmed goose [33] , and to a large extent in Hong Kong Southeast Asia resulting in severe A-H5N1 outbreaks in 2003. Finally, A-H5N1 virus also spreads to the west in a second major transmission wave. Since this occurs after a major outbreak in migratory waterfowl at Qinghai Lake in Northern China, migratory birds could play a prominent role in this dissemination pathway [37] .
We investigate the ''Africa 2'' lineage of rabies transmitted by African dogs. This lineage forms one of the most divergent African rabies virus clades [28, 38] . The data set we analyze here comprises 101 complete nucleoprotein (N) gene sequences sampled across 12
African countries including Chad, Niger, Cameroon, the Central African Republic, Benin, Sierra Leone, Mali, Mauritania, Guinea, Ivory Coast and Burkina Faso [39] . Figure 6A illustrates the location-annotated MCC phylogeny and demographic history for the African dog rabies lineages. We make this initial inference without either BSSVS or a distanceinformed prior. To allow for variation in the underlying coalescent process giving rise to the phylogeny, we assume a piece-wise constant multiple change-point model on the effective populationsize with 20 coalecent-interval groups [25] . As generally observed for rabies viruses [28] , there exists strong signal of phylogenetic clustering according to sampling location. This observation is also These two locations are geographically distant from each other, but they both host viruses from the most basal lineage in the phylogeny ( Figure 6A ). Root inference is somewhat different using BSSVS and a distance-informed prior on the rates ( Figure 6B ). In this case, a more central location, Niger, obtains the highest posterior probability (0.144) but the KL divergence for the root state reconstruction is only marginally greater than zero (0.0645). The exploitation of BSSVS contributes to this effect; as for Avian influenza A-H5N1, distance-informed priors, alone, on the rates have little impact (data not shown).
Although geographic origins remain elusive, we are able to identify locations that are epidemiologically linked using the BF test under BSSVS (Figure 7) . Panel A in the figure highlights the rates yielding a BFw3. The resulting migration graph is markedly parsimonious with a distinctive East-West axis running along the Southern border of the Saharan desert. To glean how this graph reflects the migratory process acting along the rabies phylogeny, panel B projects each of the branches of the MCC phylogeny onto the geographic map. In this projection, we translate each branch into a geographic link that connects the branch's most probable starting and ending location states. The height of a link represents the relative length of the time elapsed on the link's corresponding branch, while the color gradient reflects the relative age of the migration. Many recent (magenta) migration events that occur in a relatively short time contribute to the well-supported East-West axis.
Although the best supported rates mainly form an East-West axis, many transitions along this axis occur in the last three decades; this suggests that the axis is not representative of a relatively slow unidirectional migration wave. Figure 8 reports the migration pathways over the last thirty years. These migration events accumulating over time, contingent on the estimated time of the branches on which they occur, demonstrate that RABV diffusion in West Africa is characterized by different simultaneous migration events in various directions rather than a unidirectional pattern, and that most of these migrations are short-range, occurring between neighboring countries.
The Bayesian phylogeographic inference framework we present here incorporates the spatial and temporal dynamics of gene flow. In this study, we focus on pathogen diffusion because viral sequence sampling on a time-scale commensurate with the rate of substitution permits the inference of spatial patterns in real-time units. In addition, elucidating the phylodynamics of viral epidemics has important implications for public health management. We selected the Avian influenza A-H5N1 example to allow a convenient comparison of Bayesian ancestral state inference with the previous parsimony analysis; on the other hand, statistical analysis of the rabies migration in Africa up to this point has been largely unexplored. Both zoonoses represent a clear threat to human health. The frequent transmission of A-H5N1 from poultry or wild birds to humans suggest that the virus could emerge as or contribute genetically to the next human flu pandemic. Although the lack of a human-to-human transmission mechanism means that rabies will not emerge as a purely human disease, rabies infection causes a fatal neurological disease and at least 55,000 people die from this disease every year, mainly in the developing world [40] . A Bayesian statistical approach presents many advantages over parsimony inference of ancestral states. First, MCMC offers a computational technique to integrate over an unknown phylogeny and unknown migration process as the former is not directly observable in nature and the latter is poorly understood. Accommodating this lack of knowledge protects against potentially severe bias, but can reduce the power to make inferences; phylogeographic analyses are no exception to this. One can regard this uncertainty itself as a 'mixed blessing' because whilst it can hamper drawing definitive conclusions [13] , it protect us from making overstated conclusions. For example, parsimony analysis of the influenza data establishes an epidemiological link between Guangdong and Indonesia [22] . Bayesian inference does not confirm this conclusion and phylogenetic analysis of more recently obtained sequence data now identifies the progenitors of Indonesian strains in the Chinese province of Hunan [26] , a site which our shared analysis also identified as a possible source. Further, unlike parsimony, likelihood-based probabilistic methods consider branch lengths in ancestral reconstructions. The impact of the tree depth on root state reconstructions for the A-H5N1 genome segments clearly illustrates the importance of branch lengths. Moreover, probabilistic methods allow for estimating the relative posterior probability of each location state at any position along the phylogeny; this ability is indispensable in a hypothesis testing framework. As introduced by [19] in a different setting, we also demonstrate how phylogeographic parameters can be estimated from different genomic segments without assuming the same evolutionary history. H5N1 reassortment, however, will have not have fully unlinked HA and NA evolutionary histories and partially shared ancestry may lead to overstated credibility in some aspects of the phylogeographic inference.
Bayesian inference also proffers particular benefits within the class of likelihood-based methods, for example, by allowing for straightforward approaches to control model complexity. BSSVS naturally provides a BF test to identify significant non-zero migration rates. Further prior specification easily incorporates geographical detail of the sequence data. Although distanceinformed priors appear to have little impact on the phylogeographic analyses presented here, both BSSVS and informed priors furnish new opportunities for hypothesis testing when comparing competing prior scenarios of the diffusion process. Examples include ''gravity models'' [41] in which infinitesimal rates become functions of the host population-sizes at the end-point locations and a priori structurally-fixed graphs [19] . Finally, it has been recognized that an MCMC-based Bayesian framework is wellsuited to bring together information of different kinds [42] . The BEAST software, which has a strong focus on calibrated phylogenies and genealogies, elegantly illustrates this by offering a large number of complementary evolutionary models including substitution models, demographic and relaxed clock models that can be combined into a full probabilistic model [43] . By adding spatial reconstruction to this arsenal of evolutionary models, the full probabilistic inference now brings us much closer to biogeographical history reconstruction from genetic data.
Our primary motivation for exploiting BSSVS to select among all possible migration graphs is to elucidate the limited number of epidemiological links that appropriately explain the viral diffusion process. This parsimonious set both informs major modes of migration and reduces the high statistical variance that burdens estimation of all pairwise transition rates. Following this argument, less uncertainty on node state reconstructions would be expected when focusing on a parsimonious parameterization of the instantaneous rate matrix. The A-H5N1 analysis indeed indicates lower uncertainty of root state reconstructions. However, for some other internal nodes, we note the opposite behavior. We attribute this to the reversibility assumption in the rate matrix. Selection of reversible rates by BSSVS imposes more balanced transitions in the phylogeny among locations that could have unidirectional links in reality. Therefore, work is in progress to develop non-reversible models that may better fit a spatially expanding epidemic like A-H5N1 or recurring epidemic influenza emergence through sourcesink dynamics [2] . Considerable technical hurdles remain to incorporate BSSVS procedures under such models. Because BSSVS places non-negligible probability on structural zeros in the rate matrix, we can not guarantee that all resulting rate matrices are diagonalizable, challenging stable computation. Bearing in mind the reversibility assumption, we pass no judgement on the origin of the A-H5N1 epidemic based on the frequency by which a location is present in well-supported rates, as was previously done in the parsimony analysis [22] . Instead, we focus on node location state reconstructions throughout the phylogeny and their posterior probabilities. Figure 1 suggests that, although Hong Kong and Guangdong both receive posterior support as root location states, the dominant location throughout the phylogeny and hence the more likely hub of diffusion is Guangdong. An inherent assumption of the discrete model of location change is that ancestral viruses necessarily reside at only the sampled locations of the extant viruses. In this respect, it is important to realize that the CTMC process should describe the underlying spatial dynamics more accurately as the sampling density increases. E.g., for A-H5N1 [26] , provide more recently obtained sequence data across a larger set of geographic locations; the data could inform further pathways seeding remote localities that remain elusive in our present analysis. In addition to tackling the reversibility assumption, it may also prove necessary to relax constant diffusion rates through time to realistically model phylogeographic processes in many situations. Covarion-like models [44] and allowing different diffusion matrices across different time intervals in the phylogeny may help achieve these aims.
Our rabies phylogeographic analysis confirms a longstanding presence of this viral lineage in West Africa [28] . The virus appears to have a constant population size for about 150 years during which, extrapolating from the more recent spatial dynamics, diffusion occurs continuously with no particular directionality ( Figure 6A ). These continuous dynamics explain why we can not achieve precise root state location inference based only on samples from the last 20 years. In the light of the constant population dynamics, however, the location of the MRCA may be epidemiologically irrelevant as the location probably does not necessarily represent the ultimate source of the rabies endemic. We note that our analysis does not include all currently available strains originating from Chad, which may add to the weak East-to-West dispersal signal revealed by a recent parsimony analysis [39] . Our analysis confirms the model proposed for dog RABV in general; that is, of a series of spatially distinct clusters that experience relatively little contact among them [28, 39] . By providing a time scale for the seeding of these spatial clusters, we again demonstrate a clear advantage of the Bayesian inference over parsimony analysis. The ability to draw migrations over time also promotes a more precise dissection of local and temporal RABV movement on smaller geographical scales. After migration, the virus appears to establish local populations maintaining the viral lineage for at least a limited amount of time. These dynamics are reminiscent of a metapopulation model with continuous turnover of locally established viral populations. A long-standing rabies presence in West Africa is not surprising; already recognized since the late 60s, the territory plays a major role in the rabiescanid ecological balance [45] . It remains a remarkable feat that an acute and mainly fatal disease achieves prolonged endemicity. Because disease-induced mortality can rapidly deplete the number of susceptibles in a population, one expects epidemic cycles with oscillatory dynamics to occur. Rabies cycles and traveling waves have been well documented in wildlife across Europe and North America, e.g. [46] , and more recently [47] , demonstrate such cycles in African dogs. Because their periodicity is notably shorter than expected from epidemiological models, the authors argue that intervention responses also impact the epidemic cycles [47] . Importantly, there is also a remarkable phase synchrony in rabies outbreaks across southern and eastern Africa, most pronounced for distances up to 1,000 km [47] . For oscillating systems in particular, it is well known that dispersal can generate population synchrony [48] . Because previous studies illustrate that even limited amounts of relatively local dispersal can generate synchrony in cyclical dynamics over large spatial scales [48] , and that the resulting synchrony tends to decline as distance increases and varies through time [49] , [47] argue that dispersal could enforce synchrony in dog rabies epidemics across different countries. Our analysis clearly reveals rabies dispersal as a continuous dynamic process that could indeed be essential in maintaining epidemic cycles. As argued by [39] , however, the rate of dispersal is probably not sufficiently high to explain the short epidemic cycles as suggested by [47] . Nevertheless, we underscore that sustained and coordinated responses across political boundaries are necessary to control domestic dog rabies in Africa.
Many questions in evolutionary biology require a biogeographical perspective on the population under investigation. We hope to have demonstrated that Bayesian phylogeographic framework can contribute significantly to evolutionary hypothesis testing, and, although we have focused on viral phylodynamics, this approach is generally applicable in molecular evolution. Employing geographically-informed priors delivers a first step in incorporating GIS information. Future developments like irreversible CTMC processes may offer even more biological realism.
For many spatial scales and problems, geography can naturally be partitioned into a finite number of discrete sites fS k g for k~1, . . . , K. Examples of these situations include individual cities, islands or countries. Starting from the observed data, at the tips of the phylogeny F we record discretized locations X~(X 1 , . . . , X N ), where X i [fS k g pin-points the sampling site of taxon i. Unobserved in the spatial process are the locations of the most common recent ancestor X root drawing from root distribution p root , the times at which the descendent taxa move and amongst which discrete sites these moves occur, a process which ultimately gives rise to X. Conditioning on X root and the unobserved locations realized at each internal node (X Nz1 , . . . , X 2N{2 ) [13, 16] , suggest modeling the instantaneous locations X (t) for taxa along each branch in F as independent continuous-time Markov chains (CTMCs). CTMCs are the same processes one commonly exploits to model sequence character evolution [15, 50] . Although many readers are familiar with CTMCs, we here highlight several chain properties to which we turn later when discussing CTMC modeling limitations. CTMCs are the simplest stochastic processes that emit discrete outcomes as a continuous function of time. The processes are memoryless, in that the probability of transitioning to a new location only depends on the current location and not the past history. A K|K infinitesimal rate matrix L~fl jk g completely characterizes the CTMC process. Rate matrix L contains non-negative off-diagonal entries and all rows sum to 0, yielding a stochastic matrix upon exponentiation. Solving the Chapman-Kolmogorov equation that specifies the behavior of the chain yields the finite-time transition probabilities p jk (t)~Pr X (t)~S k j X (0)~S j À Á . In matrix form,
Determining the finite-time transition probabilities involves matrix exponentiation, generally accomplished through an eigen-decomposition of L. Here, we restrict ourselves to infinitesimal rate matrices that yield only real eigen-values and eigen-vectors. Any matrix similar to a symmetric matrix ensures a real eigen-decomposition; consequentially, we formulate
where m is an overall rate scalar, S~fs jk g is a K|K symmetric matrix and P~diag(p 1 , . . . , p K ). Infinitesimal rate matrices of this form generate reversible Markov chains, such that p j l jk~pk l kj and p j p jk (t)~p k p kj (t), ð3Þ placing many restrictions on the underlying geographic process. We discuss these limitations and modeling extensions that allow for irreversible chains in the Discussion. In its most general timereversibile (GTR) form, L contains (Kz2)(K{1)=2 free parameters, with P donating K{1 together with mS's K(K{1)=2 off-diagonal entries. Following standard practice, we normalize entries in S such that m measures the expected (with respect to P) number of transitions per unit time t. One illuminating perspective from which to view a CTMC is that of a random walk on a graph G. From this perspective, the possible realizations of the chain fS k g correspond to the vertex set of G. Between the vertices lie edges that record the infinitesimal transition rates. For example, between S j and S k sits l jk . As a continuous-time random walk, a particle, starting at vertex S j at time 0, first waits an Exponential amount of time with rate {l jj and then randomly decides to which neighboring vertix S k to move with probability {l jk =l jj . Now on S k , the process repeats. Neighboring vertices are those for which a single edge connects them. For character evolution, ''complete'' graphs find almost exclusive use, such that edges exist between all pairs of vertices. At a minimum, however, the graph must remain ''connected'', such that it remains possible to walk between any two vertices on G.
Bayesian stochastic search variable selection. When GTR models find use modeling nucleotide substitution, most of the K(K{1)~12 possible transitions have non-neglible probability of occurring and are observed over the evolutionary history. Such is unlikely to be the case for geographical locations; given that there may be many sites and each taxon only has one location (the equivalent of just one single alignment site), we expect most transitions to rarely, if ever, occur. Consequentially, we suspect a priori that many infinitesimal rates are zero. From a statistical perspective, so many degrees of freedom fit to the limited data lead to extremely high variance estimates. These poor estimates arise not only for L, but, more critically, for inference of the unobserved ancestral locations and X root . We circumvent this sparse data problem by invoking BSSVS to select a parsimonious parameterization of L. BSSVS enables us to simultaneously determine which infinitesimal rates are zero depending on the evidence in the data and efficiently infer the ancestral locations. As a beneficial by-product of BSSVS, directly quantifying the evidence about which rates are non-zero furnishes both the most likely migration patterns and the ability to test between competing migratory hypotheses.
BSSVS is traditionally applied to model selection problems in a linear regression framework, in which statisticians start with a large number of potential predictors X 1 , . . . , X P and ask which among these associate linearly with an N-dimensional outcome variable Y. For example, the full model becomes Y~½X 1 , . . . , X P bze, where b is a P-dimensional vector of regression coefficients and e is an N-dimensional vector of normally distributed errors with mean 0. When b p for p~1, . . . , P differs significantly from 0, X p helps predict Y, otherwise X p contributes little additional information and warrants removal from the model via forcing b p~0 . Given potentially high correlation between the predictors, deterministic model search strategies tend not to find the optimal set of predictors unless one explores all possible subsets. This exploration is generally computationally impractical as there exist 2 P such subsets and completely fails for PwN.
Recent work in BSSVS [51, 52] efficiently performs the exploration in two steps. In the first step, the approach augments the model state-space with a set of P binary indicator variables d~(d 1 , . . . , d P ) and imposes a prior p b ð Þ on the regression coefficients that has expectation 0 and variance proportional to a P|P diagonal matrix with its entries equal to d. If d p~0 , then the prior variance on b p shrinks to 0 and enforces b p~0 in the posterior. In the second step, MCMC explores the joint space of (d, b) simultaneously.
To map BSSVS into the phylogeography setting, we consider selection among the 2
random graphs in which each of the K(K{1)=2 edges either exists or does not exist in G. Let d jk be the binary indicator that an edge exists connecting S j and S k . An equivalent parameterization specifies that l jk~0 when d jk~0 and l jk w0 otherwise. So, rate matrix L plays an analogous role to the regression coefficients in BSSVS. An important difference is that l jk [ (0, ?) while b k [ ({?, ?), mandating alternative prior formulations.
Prior specification. To specify a prior distribution over d~fd jk g, we assume that each indicator acts a priori as an independent Bernoulli random variable (RV) with small success probability x. The sum of independent Bernoulli RVs yields a Binomial distribution over their sum W~X jvk d jk . In the limit that x%K(K{1)=2, this prior conveniently collapses to
where g~x|K(K{1)=2 is the prior expected number of edges in graph G. We entertain two prior choices for P. Diagonal vector (p 1 , . . . , p K ) is the stationary distribution for the CTMC when all edges are included in the graph G. For this complete graph, as the length of the random walk t??, one expects that X (t)~S k with probability p k . One natural choice says that there exists no spatial preference over the long-run and fixes p k~1 =K for all k. However, sites may expound spatial preference over the long-run; for example, such preference can relate to known or unknown quantities such as population-size or geographic size of the site. In these situations, we estimate P simultaneously with the rest of the model by imposing the flat prior (p 1 , . . . , p K )*Dirichlet(1, . . . , 1). Also non-informatively for small values, we take m*Exponential (1) .
To complete the CTMC specification, we assume that all unnormalized rates in S are a priori independent and Gammadistributed with prior expectation m jk and variance d jk |v jk , following in the vein of Bayesian SSVS. However, little previous work on prior formulation helps inform our choices of m jk and v jk . This represents a critically important area of research. A common, yet arbitrary choice in the Bayesian phylogenetic literature assumes that rates draw from Exponential distributions, forcing m jk~vjk . We follow this practice in light of there being no obvious way to elicit information on the variance of these rates. Finally, we explore two choices for setting the means. The first assumes no preference over rates, setting all m jk~C , where C is an arbitrary constant; as, after normalization, only ratios of infinitesimal rates participate in the data likelihood, the actual value of C has no influence on the likelihood. The second is informed by the geographical distance between sites.
prior. Considerable additional information exists about the sites fS k g and remains unused. Most notably, the geographic distances d jk between (the centroids) of sites is readily measurable. A priori we may believe that more distantly separated sites have the smallest infinitesimal migration rates, yielding
Other information is also surely helpful and application-specific. One example involving human hosts quantifies the availability of motorized transportation, such as air flights, between sites. We explore the utility BSSVS and distance-informed priors in our phylogeographic models. Bayes factor test of significant diffusion rates. The Bayes factor (BF) for a particular rate k contributing to the migration graph is the posterior odds that rate k is non-zero divided by the equivalent prior odds,
where p k is the posterior probability that rate k is non-zero, in this case the posterior expectation of indicator d k . Since we employ a truncated Poisson prior with mean g~log 2, that assigns 50% prior probability on the minimal rate configuration (K{1), the prior probability q k reduces to
We consider rates yielding a BFw3 as well supported diffusion rates constituting the migration graph.
A strength of the Bayesian approach we exploit in this paper is the ability to integrate together into a joint model of spatial locations X and aligned molecular sequence data Y~(Y 1 , . . . , Y N ) collected from the N taxa. The joint model affords a natural way to incorporate uncertainty about the unobserved phylogeny F and the character substitution process giving rise to Y. We take a standard statistical phylogenetic approach and assume that a separate CTMC characterized by w generates Y. While we discuss specific choices about this process in the Results sections, we do assume that the sequence and location CTMCs are independent given F, enabling us to write the joint model posterior distribution as Likelihoods Pr X jF, L ð Þ and Pr Yj F, w ð Þ follow directly from Felsenstein's pruning algorithm [15] , efficiently integrating over all possible locations and sequences at the root and internal nodes in F.
We approximate the joint posterior (8) and its marginalizations using MCMC implemented in the software package BEAST [43] . We employ standard transition kernels over the parameter spaces of F and w. To sample realizations of L, we consider random-walk operators on the continuous portions and a specialized ''bit-flip'' operator on the Bernoulli rate indicators d jk . [53] discuss this transition kernel further. Finally, in many situations, inference on the posterior distribution of the root and internal node states is of paramount interest. We implement a pre-order, tree-traversal algorithm in BEAST that allows researchers to generate realizations of the root and internal node states following [20] and produce posterior summaries. Importantly, this procedure is not limited to phylogeographic models, making general ancestral state reconstruction available in BEAST for the first time.
Summarizing posterior location uncertainty. An important statistical question asks to what extent the data inform our inference when fitting different phylogeographic models. A model of low statistical power makes poor use of the information in the data, while a successful model exploits this information to generate posterior distributions that are maximally different from prior beliefs. One primary outcome of a Bayesian phylogeographic study is the marginal posterior distribution of the root location Pr X root jX, Y ð Þ . We calculate the Kullback-Leibler (KL) divergence [54] from the root location prior P to summarize this information gain,
where 0| log 0~0. When the posterior and prior distributions are equal, d KL~0 .
In the examples in this paper, we fix p k~1 =K and d KL achieves its maximum log K when the posterior places all estimable mass on a single location. From this perspective, log K{d KL plays the role of a measure of dispersion [55] or uncertainty. As a simple numerical summary, we also use d KL to explore the utility of BSSVS and distance-informed priors on drawing inference from phylogeographic models. Larger values signify that the model extracts more information from the data. To calculate KL divergence, we employ a uniform discrete distribution as reference distribution. Association index. Following existing phylogeographic approaches, we finally score the degree of spatial admixture using a modified association index (AI) [35] . For a given phylogeny F and tip locations X, we obtain the association value d AI by summing over each internal node n,
where c n counts the number of sampled locations descendent to n and f n is the frequency of the highest frequency location amongst these descendents. Similar to [10] , we report the posterior distributions Pr d AI jX, Y ð Þ and the AI compares these distributions to those obtained by random permutation of the tip locations X. Deviation from these permuted distributions reflected in low AI values suggests phylogeographic structure whereas AI values close to 1 suggest spatial admixture.
Visualizing phylogeographic diffusion. To summarize the posterior distribution of ancestral location states, we annotate nodes in the MCC tree with the modal location state for each node using TreeAnnotator, and visualize this tree using FigTree (available at http://tree.bio.ed.ac.uk/software). To provide a spatial projection, we convert the tree into a keyhole markup language (KML) file suitable for viewing with Google Earth (http://earth.google.com). We introduce the temporal information on the marked-up tree using the TimeSpan KML-function to animate viral dispersal over the time. Example KML files for the Avian Influenza A HA and NA genes are included as supplementary files and software to convert annotated trees to KML is available from the authors on request.
Dataset S1 KML file for H5N1 diffusion over time as inferred from HA Found at: doi: 10  The Model Repository of the Models of Infectious Disease Agent Study The model repository (MREP) is a relational database management system (RDBMS) developed under the auspices of models of infectious disease agent study (MIDAS). The purpose of the MREP is to organize and catalog the models, results, and suggestions for using the MIDAS and to store them in a way to allow users to run models from an access-controlled disease MREP. The MREP contains source and object code of disease models developed by infectious disease modelers and tested in a production environment. Different versions of models used to describe various aspects of the same disease are housed in the repository. Models are linked to their developers and different versions of the codes are tied to Subversion, a version control tool. An additional element of the MREP will be to house, manage, and control access to a disease model results warehouse, which consists of output generated by the models contained in the MREP. The result tables and files are linked to the version of the model and the input parameters that collectively generated the results. The result tables are warehoused in a relational database that permits them to be easily identified, categorized, and downloaded. T HIS PAPER describes the model repository (MREP) of models of infectious disease agent study (MIDAS), which is a tool developed to organize and catalog the models, results, and suggestions for using results of the MIDAS and store them in a relational database for future use and reference. The database is a repository of epidemiological-based infectious disease models and their derivatives (e.g., inputs, parameters, and outputs). These products will be stored to allow easy retrieval via a query method. This section gives a brief explanation of the MIDAS and the rationale for the MREP, and identifies other related tools in the literature. Section II presents the architecture and design of the MREP. The MREP's key design elements include the ability to link all related model components together and to include a version control tool as part of the relational data base management system (RDBMS) data model. Section III describes the content of the MREP, including the information about the MIDAS models and the studies they comprise. Section IV summarizes key features of the MREP and discusses future enhancements.
The MIDAS is a research partnership between the National Institutes of Health (NIH) and the scientific community to develop computational models for policymakers, public health workers, and researchers to help them make better-informed decisions about emerging infectious diseases-both man made and naturally occurring. The MIDAS researchers are working to develop models that can assist the public health community understand how best to respond during outbreaks and epidemics. The MIDAS consists of seven research groups and one centralized informatics group.
1) MIDAS Objectives: The MIDAS Research Groups develop epidemiological models that represent host-pathogen relationships, disease epidemiology and forecasting systems, and pandemic response strategies. They also focus on informationdriven research rather than hypothesis-driven investigations. The MIDAS model developers use real or simulated data available through the MIDAS Web site. As a collaborative network of scientists, the MIDAS investigates using computational and mathematical models that will prepare the nation to respond to outbreaks of infectious diseases by providing policymakers and public health officials with reliable and timely information that can be used to prepare for infectious disease outbreaks.
2) Epidemiological Models: Epidemic models represent powerful tools for gaining insight into how the dynamics of an epidemic are affected by interventions. In order to understand and control the spread of pathogens, it is essential to establish some of the key parameters associated with disease transmission. Fundamental to the dynamics of an epidemic are two quantities: the basic reproduction number (R0) and the generation time (Tg) of the pathogen [1] . The R0 is the average number of secondary cases produced by each primary case at the start of an epidemic in a previously unaffected population. The Tg is the average time between infection of index cases and the secondary cases they produce. The R0 is a measure of the transmissibility of the strain in the population, and largely determines the proportion of the population that will be infected in a pandemic. The ratio R0/Tg is a measure of an epidemic's rate of growth.
Many epidemiological models are based on a compartmental, Sampling Importance Resampling (SIR) framework; the host population is partitioned into those that are susceptible, infected, or immune to a particular pathogen. These models assume that the rate at which new infections are acquired is proportional to the number of encounters between susceptible and infected individuals, and leads to an effective reproductive ratio that depends on a threshold density of susceptibles. Thus, the models depend not only on parameters intrinsic to the disease such as latent and infectious periods, but also on contacts between infectious and susceptible hosts. Historically, structuring the population at risk into compartments permits subpopulations of varying risks to be represented. Compartmental models of this kind implicitly assume that the host population is well mixed, such that the probability of infection is equal for all.
However, social network structures are clearly not always well mixed, and the complexities of host interactions may have profound implications for the interpretation of epidemiological models and clinical data. To overcome the simplifying assumption of classical transmission models, a new type of model is gaining recognition. Agent-based models (ABMs) represent an important new approach for describing interacting heterogeneous agents. The heterogeneous aspect of agents enables more sophisticated and complex environments to be described by ABMs approaches. Also, it is typical to introduce a geospatial dimension into the framework so that both time and geographical patterns are represented. Thus, every ABM run identifies infected persons where they live and work in general, and their movements within groups (referred to as social networks) that influence disease spread. ABMs have been used to describe phenomena from social systems to immune systems, both of which are distributed collections of interacting entities (agents) that function without a leader. Simple agents interact locally according to simple rules of behavior, responding in appropriate ways to environmental cues and not necessarily striving to achieve an overall goal. An ABM consists of a set of agents that encapsulate the behaviors of the individuals that make up the system, and model execution consists of emulating these behaviors [2] .
3) Models of Influenza Transmission: The major focus of the MIDAS research partnership has been pandemic influenza. In response to the MIDAS mission (see http://grants.nih.gov/ grants/guide/notice-files/NOT-GM-06-106.html), a number of large ABMs describing influenza transmission were developed. The main purpose of the models was to examine possible intervention strategies that would protect the general public from morbidity and mortality should an influenza pandemic strike. One of the initial objectives in all disease transmission studies is to determine the basic R0 value. The goal of intervention is to reduce R0 below the self-sustaining threshold of R0 = 1. The R0 of a future newly emergent influenza strain is unknown, but estimates for previous pandemics are available. For example, an estimate of 1.89 was obtained for the pandemic of 1968 in Hong Kong [3] , and the pandemic of 1957 in Great Britain (GB) was estimated to be between 1.5-1.7 [4] . Also, the reproductive number of the first wave of the 1918 pandemic in the United States was estimated as 2-3 [5] . There is historical evidence that these three influenza pandemics were explosive. The results reported by the MIDAS also suggest that the pandemics can be controlled to some degree. By comparison, childhood diseases such as rubella, pertussis, and measles have R0 values in most populations in the range 7-15 [6] , and consequently, are much less controllable.
To estimate the effect of various interventions on the spread of pandemic influenza, the effect of specific interventions on transmission rates needs to be quantified. Generally, estimates of the proportion of infections that occur in the various social contexts such as households, schools, workplaces, and communities have been reported. Then, estimates of relative effect of intervention measures on transmission in each context will need to be established. It would also be useful to know how implementation of a particular measure might disrupt contact patterns in other social contexts. For example, we would like to know the extent to which household and community contacts are increased when schools are closed.
There is scant information on the proportion of transmission that occurs in different social contexts. The best data available only allow the proportion of transmission in households to be quantified [7] , [8] . Therefore, while models can give some insight into the likely benefit of single or combined interventions, that insight is somewhat limited by this lack of data. To some extent, the results depend on the assumptions made by the modelers about transmission in the different contexts. The degree of uncertainty does depend on the specific control measures. Modelers can arguably better project the possible effects of antiviral and vaccine use, case isolation, and household quarantine than the effects of school closure, mask use, banning of mass-gatherings, or nonspecific social distancing measures.
The most recent MIDAS study examined the effectiveness of a set of proposed targeted, layered containment strategies that combined a number of transmission interventions currently available to public health planners in the United States [9] . These include nonpharmaceutical social distancing measures and antiviral treatment and prophylaxis. All three MIDAS models examined the same set of interventions, although each of the three implemented the interventions using different approaches. The three sets of models also examined the sensitivity of the effectiveness of the intervention combinations to thresholds for triggering the interventions, levels of case ascertainment and compliance, and the transmissibility of the circulating pandemic strain. The intervention scenarios examined reflect those being considered now by the United States Homeland Security Council and Department of Health and Human Services (DHHS).
An important goal of MIDAS was creating a repository for storing and managing the computerized models, model results, model parameters, and the specifications used to develop the models. The MREP was developed to house the code being developed by the MIDAS research groups into a professional, organized, and controlled environment. The guiding premise is that responding to an emergency requires a process that can be activated in a controlled, orchestrated manner; this premise also allows legacy code to be identified and past results reproduced.
The MREP needs to fit into and support the emergency response process. To support ease of access, the MREP is implemented using relational database management technology and a Webbased interface.
In addition, there are other compelling reasons for developing the MREP, including promoting quality assurance and enhancing productivity during day-to-day activities. Losing track of the exact versions of the model that generated specific model results is easy. By imposing a structure that links together all model components, houses these connected components in a centralized database, and annotates those components, we can preserve and reuse essential linkages.
In summary, the MREP provides the following capabilities to the MIDAS: first, a process for responding to an emergency event. Characteristics of MIDAS models across all modeling groups can be quickly identified and linked to relevant documentation. Second, a quality assurance mechanism for registering models into the MREP. For a model to be part of the MREP, it must: 1) have a name; 2) be linked to a readily available description; 3) be linked to a contact; 4) identify the date and time of creation; and 5) specify the terms of distribution. The registration system is consistent with the process identified by Le Novère et al. [10] . Third, a tracking mechanism that catalogs, locates, and identifies the different versions of the models involved in different experiments that comprise MIDAS studies. Fourth, the MREP has productivity features that allow modelers to efficiently locate previous model versions and reuse the code in new models. Fifth, the MREP maintains inventories of work developed by the research groups in a locatable form.
1) MREP Scope: A repository is a collection of resources that can be accessed to retrieve information. Repositories often consist of several databases tied together by a common search engine. The MREP consists of a collection of infectious disease models and pertinent information about those models including: model specifications, inputs (static), parameters, results, source code, object code, scripts for compiling the object code, scripts for executing the model, user manuals, and other model documentation including published manuscripts.
Each element in the MREP is part of a relational database. This permits them to be linked to each other so that the inputs to a specific model and the results generated by running that model using those inputs are connected. Therefore, one major feature of the MREP is tracking and connecting all of the components of a model, which allows researchers to revisit previously generated results. A second important feature is that the linked components can be unified as part of the information retrieval process. This enables a query engine to present information to a user without having to browse irrelevant information.
In summary, the MREP is important to the MIDAS scientific research environment because it links specific model runs with the explicit model code and input data with the corresponding output data. It also provides a link to the specifications that guided model development. If researchers wish to identify how specific interventions were implemented and rerun a particular model or analyze it in any way after the run is completed, the MREP provides all the information necessary.
The MIDAS MREP appears to be unique among computerized epidemiological model repositories. However, a number of quantitative biology-based model repositories exist and provide a useful point of comparison.
We performed a literature search and identified other model repositories similar in some degree to the MREP. Specifically, we sought applications that maintained a database where models (model code, data inputs, and outputs) were shared by users in a controlled environment (i.e., an environment that connects model components). We identified a number of existing relevant model repositories and we contrast five of those that catalog and share information about a specific set of models and model runs. A number of online archives and/or data repositories from a number of nonmodeling applications were also identified.
The repositories described later offer information about a specific class of models to their user communities, and, in this context, they are similar in their capabilities to the MREP. None of them, however, catalogs infectious disease models and none of them attempts to maintain a version-controlled environment that offers code to the users from a stable, documented environment provided by a version-managed system. Nevertheless, all of the examples offer their users annotated models, and all are concerned about providing reliable programs with usable documentation.
1) Biomodels Database: The BioModels.net project describes itself as an international effort to: 1) define standards for model curation; 2) define vocabularies for annotating models with connections to biological data resources; and 3) provide a free, centralized, publicly accessible database of annotated computational models in Systems Biology Markup Language (SBML) and other structured formats [11] (for detail, see http:// www.ebi.ac.uk/biomodels/).
The database component of BioModels.net is especially designed for working with annotated computational models: each model is carefully reviewed and augmented by human annotators on the BioModels.net team to add metadata linking the model elements to other biological databases and resources. The BioModels database at the European Bioinformatics Institute (EBI) system is a true database, featuring browsing, crossreferencing, searching, and facilities for visualization, exporting models in different formats, and remote application programming interface (API) access.
The BioModels Database is a data resource that allows biologists to store, search, and retrieve published mathematical models of biological content. Models present in the BioModels Database are peer reviewed, annotated, and linked to relevant data resources, such as publications, databases of compounds and pathways, controlled vocabularies, and similar items. All models use SBML as their standard form of representation.
The premise of this tool is that researchers must be able to exchange and share their results. The development and broad acceptance of common model representation formats such as SBML is a crucial step in that direction, allowing researchers to exchange and build upon each other's work with greater ease and accuracy.
To make assembling useful collections of quantitative models of biological phenomena easier, establishing standards for the vocabularies used in model annotations as well as criteria for minimum quality levels of those models is crucial. The BioModels.net project aims to bring together a community of interested researchers to address these issues.
2) CellML-A Biological Model-Based Repository: This application is similar to the BioModels Database application and uses a markup language called CellML developed specifically for describing biological processes contained in CellML [12] . The repository is a Web site that stores and exchanges computer-based mathematical models. This site allows scientists to share models described by the CellML markup language. It also enables them to reuse components from one model in another, thus accelerating model building (for detail, see http://www.cellml.org/examples/repository/index.html).
The CellML language is an open standard based on the Extensible Markup Language (XML) and is designed to describe biological models. The CellML also includes information about model structure (how the parts of a model are related to one another), model mathematics (equations that describe the underlying biological processes), and metadata (additional information about the model that allows scientists to search for specific models or model components in a database or other repository).
3
The premise behind this MREP is taken from the AnyBody Web site [13] . The site describes how the cost of musculo-skeletal injuries is rapidly increasing, while fundamental understanding of the mechanical functions of the body is increasing at a dramatic rate. However, it notes that there are still many unknown questions and problems researchers are addressing. For example, the AnyBody Web site indicates that the number of ergonomic-based injuries caused by excessive use of the computer mouse is exploding, yet the actual cause of many of these injuries remains a mystery. Policymakers and others, therefore, find it difficult to issue guidelines to reduce the problems. Also, research into human locomotion has historically been relegated to experimental studies (for detail, see http://anybody.auc. dk/).
The stated purpose of the AnyBody project is to develop mechanical models of different elements of the human body, and then, perform detailed studies of the behavior of these models.
Typical model examples include the analysis and optimization of tools and workplace layout, and designing sports equipment and hand tools for maximum efficiency. A unique software system called the AnyBody Modeling System was developed to conduct necessary research into causes and treatments of musculo-skeletal injuries.
There are four major aims of the AnyBody project. The first is to develop methods for analyzing movement strategies and tendon, muscle, and joint forces in humans performing specific manual tasks. The second is to investigate what numerical simulation can teach us about the function of the human body. The third is to use the analysis for ergonomic optimization of tools, workplaces, and man/machine interfaces. The fourth is to provide an MREP to enable interested researchers to share the models. Overall, AnyBody identifies useful information and models that can be shared by researchers interested in studying musculo-skeletal injuries.
This repository contains software for manipulating and learning probabilistic-logical models [14] . The aim is to construct an MREP that will allow dissemination of software for probabilistic-logical models and facilitate comparisons among competing approaches (for detail, see http://www.informatik.uni-freiburg.de/∼kersting/plmr/).
The site notes that probability models are important methods for representing uncertainty, and mentions that various probabilistic frameworks include Bayesian networks, hidden Markov models, and stochastic context-free languages, along with other popular tools for describing appropriate scenarios exhibiting uncertainty. It notes that these types of models have been applied to problems in diagnosis, forecasting, automated vision, sensor fusion, manufacturing control, speech recognition, and computational biology. However, traditional approaches have a major drawback-they have a rigid structure, and therefore, lack of versatility in representing complex models.
To overcome these limitations, the site states that various researchers have recently proposed logical extensions of classical probabilistic models incorporating the notions of objects and object interconnections.
This repository consists of a database of software, documentation, and links to developers. The models are not specifically designed to be general purpose; rather, they are instructional in nature and represent a basis for discussion. The potential scope for the models in this repository is huge, with many different methods for defining and describing probabilistic-logical models.
The cancer intervention and surveillance modeling network (CISNET) is a consortium of investigators sponsored by the National Cancer Institute "whose focus is using modeling to improve our understanding of the impact of cancer control interventions (e.g., prevention, screening treatment, etc.) on population trends in incidence and mortality" [15] . They use models to project future trends and help determine optimal cancer control strategies (for detail, see http://cisnet.cancer.gov/about/). The CISNET also describes using a comparative modeling approach in which each modeler focuses on an individual area. However, whenever possible, they develop a common "base" question that allows comparison across all models using a set of common population inputs. Then, they develop a common set of intermediate and final outputs.
To aid in this process of model description and comparison, the CISNET has developed the Model Profiler, an Internet-based application. Each CISNET team has a private model profile Web site on which it maintains model profile information and controls what parts of the profile are shared with other teams. By using a core documentation format that is the same for each group, the published profile information can be compared among models. The system allows modelers to describe their models, and allows interested readers to read about, compare, and contrast simulation models.
The sites described earlier we believed were fairly representative of the repositories available to researchers. Our literature search indicated that other sites are available. SigPath is described by Campagne et al. [16] as an information management system that stores both quantitative information on cellular components and their interactions, and the basic reactions governing those interactions; EcoCyc, which is described as a comprehensive database resource for Escherichia coli by Keseler et al. [17] ; JWS Online, described by Olivier and Snoep as a repository of kinetic models describing biological systems that can be interactively run and interrogated over the Internet [18] ; and the Database of Quantitative Cellular Signaling, which Sivakumaran et al. describe as a repository of models of signaling pathways [19] .
A number of model repositories are identified in the literature. Many of these models support computational biology applications, and certainly, by some measures are more sophisticated than the MREP we describe here. One manifestation of sophistication is using a markup language based on XML concepts that imposes a standard method for describing and representing models common to a repository. We assert that ABMs are conceptually less mature than computational biology models, and broader in their scope (i.e., agents can represent genes, proteins, and cis-regulatory elements at one end of the spectrum, and represent people, states, and countries at the other end). We chose not to grapple with the notion of trying to develop a common taxonomy to describe agent-based epidemiology models. Rather, we focused on building a repository that captures ABMs in whatever form they were developed and creates a common set of documentation and annotations, so that the model can be understood (at some level) and reused should the need arise.
The MREP is designed to house, manage, and allow users to run infectious disease models from an access-controlled disease MREP. The MREP contains source code of disease models that have been developed by external developers and tested in a production environment. Different versions of models used to describe various aspects of the same disease are housed in the repository. During registration, models are linked to their developers, to a paper or PubMed reference that describes the model, to the name and contact information of model creators, to the date and time of creation, and to the terms of distribution. In addition, a code, available from the versioning software described later, is used to distinguish between different model versions. The annotations captured during the model registration process also identify a model's purpose, specifications, and relevant features.
The MREP also houses, manages, and controls access to a disease model results warehouse, which consists of output generated by the models contained in the MREP database. The results, tables, and files will be linked to the version of the model and the input parameters that collectively generated them. They will also be stored in a relational database to permit them to be easily identified, categorized, and downloaded.
The MREP includes a version control system (also referred to in the literature as a configuration control system) as one of its core elements. The system manages the source code, documents, graphics, and related files. Version-control software provides a database that is used to keep track of the revisions made to a program by all the programmers and developers involved in it.
The MREP uses Subversion as its version control system [20] . Subversion is a free, open-source application that is licensed under the Creative Commons Attribution License (see http://svnbook.red-bean. com/en/1.0/). The MREP also includes a Graphical User Interface (GUI) application that allows the user community to use the MIDAS models more easily. The GUI interfaces with Subversion, the database management system, and the computer environment. The GUI allows the user access to the model source code, provides an interface to input data sets, permits results to be viewed directly or downloaded to a user workstation, and provides a mechanism to submit the models for rerunning.
The MREP is comprised of the following major components: 1) a source code repository and version control system; 2) a model documentation tree; 3) a data warehouse (input and output data sets); and 4) an application interface (API) consisting of a database, browser, and graphics user interface components that allow the model user to develop input data sets, run the codes, and browse output results.
A standard system development approach was used creating the initial version of the MREP using approximately two fulltime equivalents (FTEs) over a 13-month period.
B. Architecture 1) Overview: There were two features that were considered extremely important by the developers to include in the MREP design. These features include: 1) a version control system that provides configuration control over model source code and 2) complete flexibility regarding output formats. The first of these features was a response to an absence of model standards (such as SBML) in the storage of source code. The most common model development languages represented in the MREP are anticipated to be C, C++, Java, and Matlab. To maintain control in a language-free environment, using a version control element was deemed essential.
The second feature was the ability to support a common output format. All of the models generate results in different formats, including Concurrent Versions System (CVS), text, and Portable Document Format (PDF), among others. The MREP offers these results to users in the received format. Users can view results directly or by downloading a file and using a viewer that they supply. Fig. 1 represents a high-level logical data model for the MREP. Five primary components comprise the architecture, including the MIDAS Compute Server; a File System; a relational database management system (RDBMS); a Version Control System; and an External Systems Gateway. A description of each is provided later.
2) MIDAS Compute Server: The MIDAS cluster is the central computational resource for the MIDAS research groups and is referenced in Fig. 1 as the cluster. The primary interaction that an analyst using the system will have with the cluster is through interacting with the job queuing system. Additionally, code maintainers/developers will have full access to all of the Linux system's features as needed to make changes to the code.
The MIDAS cluster is managed by Cluster Resource's MOAB (see http://www.clusterresources.com/pages/products/ moab-cluster-suite.php) [21] , an advanced cluster scheduler capable of optimizing scheduling and node allocations. MOAB allows site administrators to control job scheduling, priority, and where jobs are run.
3) File System: The file system is part of the MIDAS cluster resource and is an integral part of the cluster system. A second system element is primarily used to archive study results in the MREP warehouse. At present, the archival file system is configured with 2 terabytes (TB) of disk storage that will eventually be expanded to 14 TB. Users interact with the file system to associate simulation run I/O data locations with metadata that will be used to identify and tag achievable simulation results. The configuration control system also interacts with this system. 4) Relational Database System: The database management system platform will be housed by the MIDAS Web portal, which serves as the interface for electronic information exchange for the MIDAS network. The MIDAS portal is accessible to the public, but only registered users can access and provide information to the private section of the portal.
The MIDAS portal runs on RTI's Oracle Application Server 10 g (v. 9.0.4.1.0) server farm and uses Oracle technology [22] to manage the information within the repository (see www.oracle.com/appserver/index.html).
The database system tags metadata that reference specific simulation run results with the input and output data sets and source code associated with those runs. The database system will allow users to perform keyword searches to identify repository elements assigned to those metadata. a) Data model-hierarchal design: The database design will maintain tables of metadata that describe the following entities:
Projects-a collection of studies with a common set of objectives; Studies-a collection of runs that were produced by one or more models; Models-the core code that is designed to describe computer environments that generate the runs; Model versions-a specific instance of a model to handle the production of runs having a specific set of attributes; and Runs-a set of information referred to as results that describe a single realization of a simulated epidemic.
Each realization is associated with a unique set of parameter values or alternatively is associated with a repeated set of parameter values. In the last situation, the results are referred to as a run replicate. Each model run produced by a specific model version is housed in the results data warehouse and consists of information that is part of the experiment. Fig. 2 defines the interconnections between the entities. A study is either linked to a published manuscript that defines project aims and study results or to a document that describes an MIDAS study that is part of a larger MIDAS project effort to examine specific hypotheses about disease spread and containment. For example, the DHHS project examined the problem of whether influenza could be stopped at its source (i.e., South East [SE] Asia), and if so, what methods of containment are important. Two studies from this project are part of the MREP: the Emory SE Asia Study and the Imperial SE Asia Study.
In a second example, the combined project focus is to address the problem of "what can be done to mitigate pandemic influenza if it gets established in the U.S." The specific objectives of the combined study are to assess the feasibility and effectiveness of different types of interventions strategies. The types of interventions specifically exclude prepandemic vaccines and limit the available quantity of a partially efficacious vaccine, but utilize as much antiviral treatment as required. Two paradigms are the focus of this study: the entire United States and a large city (Chicago). Six studies (and corresponding models) examine this problem. Two of these models simulate transmission in the continental United States, three others represent transmission in the city of Chicago, and the sixth presents the results of a historical review of the 1918 Spanish influenza epidemic. The last study helped justify the design of the intervention strategies.
Each model is composed of one or more model versions. Usually, a modeler attempts to describe the complete set of simulations through parameter manipulation. However, in many instances, it is likely that new and novel interventions will not be anticipated by the code developer. In these instances, it is common for the developer to create a different version of the model to handle a subset of the simulated interventions. One of the explicit objectives of the MREP is to track the different model versions and to link them to the results they produce.
The run is the lowest unit of analysis of the MREP. Each run constitutes a single replicate of a single set of parameters or a summary run over all replicates having a common set of parameters.
b) Query engine: The overall plan is to populate the MREP with models that are initially in accordance with MIDAS goals, which are currently limited to infectious disease models. Eventually, we anticipate including models from a broader perspective; namely, models that are relevant to any pathogen and appropriate transmission environment. This will potentially necessitate using a query tool to readily identify models of interest. For example, the MIDAS ABMs use synthetic population data that describe a specific geographical region. The model results identify individuals that influence disease spread within that region. The query tool enables users to identify a set of model results that pertain to a region of interest. Then, by using the geospatial identifiers that are tagged to individual model results, users can drill down into subregions and neighborhoods that are affected by epidemics. Please note that it is possible to use the search keys to identify the studies that focus on a particular type of intervention strategy, and then, by examining Model Version details, determine the implementation details to decide a target computer to rerun the model. This could be done to replicate earlier results or to begin the process of modifying parameters to assess new interventions.
5) Version Control: The version control system maintains the various model version source codes and executables. Each model version in the MREP is maintained in Subversion, the free, open-source version control system that manages files and directories over time. A tree of files is placed into a central database. This database is similar to an ordinary file server, except that it remembers every change made to files and directories. This allows the user to recover older versions of data, results, and/or code and to examine their change history.
Subversion is a distributed application; therefore, it can access its database across networks. This allows people to use Subversion on different computers and fosters collaboration by allowing various people to modify and manage the same set of data from their respective locations. Furthermore, progress can occur more quickly because there is no single conduit through which all modifications must occur. Because the work is versioned, we prevent the possibility of losing that conduit if an incorrect change is made to the data, in that the change can be easily undone.
Subversion is a full version control system. The MREP only uses a small subset of Subversion's functionality. The MREP user interface allows registered users to check out model executables that the user may then run on the RTI cluster. Another MREP interface allows the user to browse the source code tree for any model of interest. The MREP Subversion server is hosted on a virtual Linux host using VMWare Server software. 6) External Applications: An important component of Fig. 1 , referred to as External Systems, is a general set of tools that are available outside of the MREP. These tools are used to visualize, process, and analyze results from the MREP. The data from the MREP are served up via a HyperText Markup Language (HTML) portal. These data can be downloaded to the user's workstation or can be visualized directly from the MREP. For example, many of the data files in the MREP are stored as PDF, text, or.xls files, and can be viewed directly from the repository using Adobe Acrobat Reader, a text editor, or Microsoft Excel, respectively. Other files can be downloaded and imported into external systems available to specific users.
The results/outputs of production runs will also be housed in the MREP. The model results will only include outputs from registered models. For a model to be registered, it must be placed under version control.
When output from the model is used in a paper submitted for publication or otherwise presented publicly, or when the model's code is declared stable (by the developer), the model is a candidate for inclusion in the repository. If it has not been developed under version control, it will be moved to Subversion, tested, and moved to the MREP data warehouse.
The model code will be annotated with the following set of metadata: name of model, link to model description, contact information, date of model creation, distribution terms, model specifications, model implementation of those specifications, disease, region of analysis, types of intervention strategies, computer resource requirements, user's manual (link), validation measures (link to supporting manuscript), and compile and/or run scripts. The information about model specification and how those specifications were implemented is particularly important for explaining model differences. For example, if an implementation strategy calls for a reduction of 50% in model contacts, it is plausible to implement the strategy by halving the number of people contacted or alternatively maintaining the same number of persons in the contact network while halving the number of contacts with each person. The model results for each of the implementations could vary significantly.
2) Model Results: Model results are also captured in a model results warehouse and linked to the model that generated them. Each result unit is tagged to a second set of metadata that includes model name, version number, developer, intervention, parameter file, fixed input file, and script used to generate results.
3) Model Inputs: Model inputs are also placed in the repository and linked to the model that uses the inputs and the corresponding results that are generated. Each set of inputs is tagged to a set of metadata defined at the model version level: model name, version number, developer, intervention, and parameter location (including name, type, and range of values).
Either a query tool is used to locate repository results or a complete list of models is displayed, and the user selects from the list. These results are then available for download and display. The process proceeds according to the following five steps.
In step 1, query keys are specified: the study and/or model and/or results are selected that meet the user-specified search criteria. For example, disease, geographic description, objective of study, model name and version number, and model developer.
In step 2, the query tool identifies the model in the repository with the specified attributes and displays the information. At this point, the user can either access the annotations or drill down to lower-level (run-level) linked results (results are displayed and/or downloaded if desired), or identify input data files and scripts that run the model.
In step 3, the model identified by the earlier steps can be checked out of Subversion. Please note that all version updates are performed by the MREP administrator and that model development occurs under version control. When a model is modified, debugged, and tested, it can be entered back into Subversion, but only as new model version. The modified model is then entered into the repository.
In step 4, the model is loaded into the MREP. This step annotates the model with both descriptive text and model metadata as part of the check-in process. An HTML file is created that connects the model, results, and input data; it also creates directories for source code, object code, inputs, results, and scripts.
In step 5, model results are loaded into the model result warehouse and metadata specified at the run level, including model name, version number, developer, result category, replicate, link to parameter list, and location of results.
Currently, four projects, 12 studies, five models, six model versions, and 538 runs are loaded into the MREP.
The vaccine project is a single study project that uses a single influenza-based model of a medium-sized city in the United States. It was developed by the Emory Group headed by Ira Longini. The study consists of six runs generated from a single epidemiological model of disease spread. The main hypothesis behind the vaccine distribution study is to assess whether targeted antiviral prophylaxis (TAP), taken prophylactically, is effective in containing influenza. The authors conclude that TAP is nearly as effective as vaccinating 80% of the population, and further, that vaccinating 80% of children less than 19 years of age is almost as effective as vaccinating 80% of the entire study population.
The Influenza Containment-SE Asia was developed by the MIDAS network and was completed in September 2005. It consists of two studies and two models. Both studies simulated disease transmission in regions that included some part of Thailand. The Imperial SE Asia model was developed by Ferguson et al. [23] and the Emory SE Asia model was developed by Longini et al. [24] . The overall hypothesis of the project was to examine if it is possible to contain Avian Influenza in the place of origin before it becomes a pandemic. The Emory SE Asia study represents a region of rural Thailand and consists of one model and 18 runs. Sixteen of the runs were produced by a single version of the model. However, the developers used a special version (the second) of the model that simulates the impact of geographically targeted antiviral prophylaxis (GTAP) to produce two other runs. All runs are loaded in the MREP. The Imperial model represented all of Thailand plus a perimeter region around its border that extended into its four border countries. Only the baseline (no intervention) run, produced by the Imperial SE Asia model, is loaded into the MREP at this time.
The Influenza Containment-United States and Great Britain project examined whether pandemic flu could be mitigated in the United States, assuming containment in SE Asia failed. This project was developed in collaboration with the DHHS and consisted of three studies: the Imperial Assessment study, the Epicast assessment, and the EpiSims assessment. The Imperial Assessment study consisted of two models: one describing disease transmission in the United States and the second describing transmission in GB. The GB model served as a counterpoint for the United States model, suggesting some interesting influences of geography and national patterns of behavior on disease spread.
The Epicast DHHS experiment described disease in the United States and examined influences on the spread of disease on a population derived from the U.S. 2000 Census data. The principal investigator of the Imperial assessment study is Ferguson et al. [4] . The MIDAS principal investigator of the Epicast assessment study is Ira Longini [25] .
The EpiSims assessment study simulated disease behavior in a mid-size city in the United States and used the same type of containment strategies that were used by the other two U.S. experiments. The EpiSims DHHS experiment consisted of a single model and generated 516 runs, involving a complete factorial design of nine binary variables and a partial design that examined the influence of three more variables. A complete set of 516 runs is loaded in the MREP for this study. The baseline GB run is also loaded into the MREP. Four national U.S. Epicast runs are currently loaded in the MREP.
The Combined project is also a U.S.-based study. It represents a refinement of the Influenza Containment-U.S. and GB project. Specifically, it examines a more complete set of social distancing interventions with the goal of determining practical strategies for implementation at the state and local level. This is an ongoing study that consists of six sets of experiments: two U.S. experiments, three Chicago-based experiments, and a historic study that examined certain characteristics of the 1918 influenza pandemic. Each study is associated with a single model with a single version per model. The objective of the combined study is to simulate a set of scenarios at the city and the national level. The scenarios are designed to address specific concerns by various federal agencies. The entire study amounts to about 150 distinct scenarios, each with multiple (thousands of) runs. This is an ongoing study that will be loaded into the MREP. Currently, only the principle results are loaded into the MREP; the code that generated the results has not yet been loaded.
A set of economic runs has also been generated as part of this study. The economic models involve assessments of cost-effective interventions with respect to containing disease spread.
The MREP represents a one-of-a-kind resource for housing and cataloging infectious disease models. The strength of the MREP's design is its data model hierarchy that accurately portrays the stages of a study and its derivatives, at least from the MIDAS perspective. This data model begins with a high-level study as represented by an overall set of study objectives and design specifications and culminates at a low level with a set of runs (results) that contribute to assessing those objectives. In between the studies and the runs linked to those studies are the experiments that represent the different and independent points of view characterized by different research groups and the models those groups used to generate their results. The final data model design element is based on the assumption that containment strategies are not always accommodated within a single model; a change in the model code is sometimes the favored approach for representing simulated behavior differences, that is, the containment strategy responses.
A second important element of the MREP is its use of a recognized code versioning application to house different model versions. Using Subversion fosters a highly controlled environment that promotes quality assurance/quality control (QA/QC) activities through a rigorous association among different model versions-their inputs and the results the input data set and model version generate.
A final feature of the MREP is the absence of standards associated with including models results. The MREP permits Statistical Analysis Software (SAS), text, Excel, images, and virtually any output format for which a reader exists. Results can be left in the source environment or downloaded onto a user's workstation.
In the future, we plan to add an epidemiology-based ontology and develop a separate query tool. This is in part a substitute for a markup language. This tool will identify all models within the MREP (using the ontology information) that reference specific model parameters and connect those parameters to the values assigned by the study developers. This will provide a convenient mechanism for identifying and comparing assumptions across models within the MREP. Escape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization. The current AIDS pandemic is the result of genetically diverse viral subtypes and circulating recombinant forms (CRFs) of HIV-1 group M, of which subtypes A, C, and D account for a majority of infections worldwide [1, 2] . A key source of this genetic diversity is the viral env gene, which encodes the envelope (Env) glycoproteins, gp120 and gp41 (reviewed in [3] ). On the virion, monomers of non-covalently associated gp120 and gp41 subunits trimerize to form 'spikes', and together these facilitate entry into a target cell. Env has a complex conformation and undergoes substantial rearrangements in both subunits upon gp120 binding to CD4 and coreceptor [4, 5, 6] . Env also contains the principal targets for neutralizing antibodies (Nab) [7, 8] , and epitopes are targeted in both Env subunits [9] . However, many potential neutralization targets are transiently or not exposed on the trimeric form of virion-associated Env, including the V3 domain, CD4-induced epitopes, and the CD4 binding site [10, 11, 12] . Despite these limitations, most HIV-1 infected patients develop robust Nab responses against their autologous virus, particularly those infected with subtype C [13, 14, 15, 16, 17, 18, 19] .
To confer potent and broad neutralization, it is expected that an epitope will need to possess at least four properties: (i) exposure on the virion-associated native Env trimer, (ii) conservation across diverse HIV-1 variants, (iii) immunogenicity, and (iv) lack of autoreactivity. To date, there are no epitopes that meet these criteria. However, our knowledge of the epitopes that are recognized by Nab during natural infection with diverse HIV-1 is somewhat limited. It is not known which or how many epitopes are targeted by the initial autologous Nab response, what proportion of these epitopes is strain-specific or shared, how antigenicity differs between patients or viral subtypes, or what the predominant escape mechanisms are. Furthermore, there is mounting evidence that at least some of these parameters differ between HIV-1 subtypes (reviewed in [20] ). We, and others, have characterized the autologous Nab response as it first develops, and have observed high titers of autologous Nab activity against the infecting strain in most (but not all) patients [13, 14, 15, 16, 17, 18, 19] . In addition we have reported higher Nab titers in subtype C infected patients compared to subtype B infected patients that were evaluated in parallel, prompting our group and others to propose that there are differences in antigenicity between subtype B and C Envs [15, 20, 21, 22] . Recent studies honed in on regions that could be involved in early subtype C autologous Nab responses, and these included the V1V2 hyper-variable domain and the C3 to V4 subregion of gp120 [23] . However, these regions could not account for all of the Nab activity present in patient plasma, suggesting the involvement of additional determinants. Furthermore, we have demonstrated that both V1V2-dependent and -independent pathways are utilized for escape from Nab during chronic subtype C infection [24] . In addition, we have shown a strong association between mutations in the a2 helix region of C3 and neutralization resistance, although these mutations did not directly alter neutralization sensitivity when transferred between sensitive and resistant Envs from linked transmission partners [25] .
Temporal studies of HIV-1 have demonstrated that HIV-1 undergoes recurrent cycles of escape from autologous Nab [17, 19, 26, 27] , and escape also occurred during infection with a chimeric SIV-HIV-1 (SHIV) virus in response to vaccine-induced Nab [28] . Yet, our knowledge of the specific molecular events that lead to escape remains incomplete. Shifting carbohydrate moieties in and around the outer surface of gp120, as well as changes in the hyper-variable domains, have been proposed as general mechanisms used by HIV-1 and SHIV to alter neutralization epitopes, although most of these studies are based on subtype B Envs. Nab escape in SHIV-infected macaques has been shown to involve glycan changes in the V1, V2, and V3 domains, perhaps by shielding conserved epitopes such as the CD4 binding site [29, 30, 31] . Consistent with this finding, a recent study identified V1V2 as the major determinant of strain-specific autologous Nab in macaques infected with two different strains of SHIV [32] , and this domain was also shown to be the principal determinant of inherent Nab resistance for HIV-1 strain JRFL [10] . Subtype B HIV-1 can also escape from autologous Nab by shifting N-linked carbohydrates on the outer domain of gp120 with little involvement of V1V2 [17] . Others have demonstrated that subtype B viral escape could also occur in the absence of frank changes in glycosylation, with no clear mutational pattern emerging [33] . In experimental SIVmac infection, the emergence of N-and O-linked carbohydrates in the V1 and V4 hypervariable domains has been shown to confer escape from autologous neutralization [34, 35, 36] . Furthermore, the presence of specific glycans in V1 reduced the immunogenicity of SIVmac in the context of an experimental infection [37] . Taken together, these studies hint at the complexity of HIV-1 neutralization and escape, but also suggest that common themes may exist.
Thus, unlike cytotoxic T lymphocyte (CTL) epitopes and their escape mutations, which are frequently predicted by the association of viral sequence polymorphism and HLA alleles, Nab epitopes and escape pathways in Env can be inherently difficult to identify based on sequence alone. We have therefore undertaken a molecular approach to define these in subtype C HIV-1 Env during early infection. Using a pseudovirus-based assay that facilitates evaluation of individual, patient-derived Envs, we analyzed the neutralizing ability of longitudinal plasma samples against contemporaneously-derived, autologous Envs from two subtype C seroconvertors who generated potent Nab against the infecting Env. Sequential neutralization escape variants emerged in both patients, and we used Env domain exchange and sitedirected mutagenesis approaches to map the pathways involved in Nab escape at multiple time points throughout the first two years of infection. Nab escape in these two patients was driven by V1V2dependent and -independent pathways, and these pathways exhibited different levels of stability over time. Yet, spatial representation of the sequence changes in gp120 indicated that immune pressure was directed at the same Env regions in both subjects. The derivation of autologous monoclonal antibodies (Mabs) from one patient demonstrated how a single potential glycan change in V1V2 afforded simultaneous resistance against multiple antibodies. These studies therefore provide a detailed look at Nab escape in subjects recently infected with the most predominant subtype worldwide, and demonstrate that the flexibility of Env facilitates the use of multiple mechanisms.
We previously demonstrated that 9 out of 11 subtype C infected subjects from the ZEHRP cohort developed robust autologous Nab responses against the infecting Envs, with 50% inhibitory (IC50) titers often exceeding 1:3,000 within the first few months of infection [15] . For two of the subjects who developed potent autologous Nab and were identified as viral p24 antigen positive (Table 1) , we sampled the emerging quasispecies by single genome PCR amplification, cloning, and sequencing of biologically functional env genes from longitudinal plasma and PBMC DNA samples. For both subjects, the 0-month Envs were cloned at the first seropositive time point estimated to be within 48 days of infection, and longitudinal timing was calculated in months from
A significant obstacle to developing an HIV vaccine is the potential for the virus to escape from the immune response induced by immunization. We previously showed that subjects in a Zambian cohort developed potent neutralizing antibody responses shortly after becoming infected by subtype C HIV-1, and here we have extended those findings to demonstrate that cycles of viral escape occurred in two of these subjects despite a potent immune response. We investigated the determinants of immune escape, and found that a single common mutational pathway was not sufficient to facilitate viral escape. Instead, we demonstrate that multiple strategies, including potential changes in glycosylation pattern, direct alteration of an epitope sequence, and cooperative envelope interactions, were used independently or together to evade neutralization. We also recovered individual monoclonal antibodies from one of the subjects and found that a single mutation can confer escape from different neutralizing antibody specificities. The studies demonstrate the remarkable flexibility of subtype C HIV-1, and suggest that the envelope glycoproteins are uniquely equipped to adjust to the specific properties of the immune response in each newly infected host. this point forward [38] . Samples from five subsequent time points over the first two years of infection were evaluated. A subset of Envs was chosen to represent the diversity of the circulating quasispecies at each time point (see arrows in Fig. S1A and B) , and was evaluated for sensitivity to neutralization by each contemporaneous (simultaneously collected) plasma sample using the JC53-BL (Tzm-bl) pseudovirus assay [15, 24, 25] . The IC50 Nab titer for each plasma-Env combination was calculated from each virus infectivity curve using a growth function. Fig. 1 shows that the median IC50 titer of the 0-month Envs (designated according to the first seropositive time point; see Table 1 ) was higher than the contemporaneous Envs at each time point, indicating repeating cycles of neutralization resistance. This difference in median IC50 titer was statistically significant at all time points for 185F. However, for 205F, the median IC50 titer differed significantly at only two time points, probably due to the wide range of Nab sensitivities observed for the contemporaneous Envs of this subject (Fig. 1B) . Nevertheless, Nab-resistant variants were present at each time point and these were neutralized by subsequent plasma samples, indicating continued induction of a de novo Nab response (data not shown).
To gauge whether breadth developed within the window of evaluation, cross-neutralizing activity of a single plasma sample from subject 185F (23-months) and subject 205F (20-months) was measured against heterologous 0-month subtype C Envs from six other subjects in the same cohort (Fig. 2) . Consistent with previous studies of early subtype C infection [15, 16] , Nab in 185F and 205F was mostly strain-specific. However, each of these plasma samples neutralized 2 of the 6 heterologous Envs with an IC50 of greater than 1:100, and in two cases approaching 1:1000. Interestingly, 205F plasma potently neutralized the 185F Env (red dot on right point plot), but the reciprocal was not observed (green dot on left point plot). Thus, Nab in these plasma samples was directed against predominantly strain-specific targets, but were also capable of recognizing some common epitopes by approximately two years after infection.
For 185F, the median Nab IC50 titer for the 0-month Envs was significantly greater than the contemporaneous Envs at every time point using a Mann-whitney test (Fig. 1A) . Using the criteria of at least a 100-fold decrease in sensitivity to neutralization compared to the median of the 0-month Envs, a resistant Env (highlighted in green) from each time point was selected for in-depth investigations into Nab escape. At 28-months, three different Nab resistant Envs were selected because the phylogenetic tree indicated that multiple lineages of resistance were circulating at this time point (Fig. S1A ). For 205F, the difference between 0-month and contemporaneous Envs reached significance at only two of the five time points, although there were Envs at each time point for which Nab activity was undetectable at the highest dilution of plasma tested (1:20, Fig. 1B ). These Envs, which were often 1000fold less sensitive to Nab than the median of the 0-month Envs, were selected for detailed studies of Nab escape. Two genetically diverse Envs from distinct lineages were also selected to represent the 20-month time point in 205F (Fig. S1B ).
We next investigated the adaptations that were responsible for escape from contemporaneous Nab in 185F and 205F. The 0month Envs were potently neutralized by plasma from all subsequent time points ( Fig. 1A and B) and were used to provide a neutralization sensitive background, which remained more than 95% conserved at the amino acid level with subsequent variants and could be used to investigate the molecular determinants of escape for each Nab resistant variant. To do this, two approaches were used: (i) where sequence changes were limited in the Nab resistant Env, site-directed mutagenesis was used to introduce potential escape mutations into the 0-month Env and (ii) where multiple sequence changes were present in the Nab resistant Env, larger Env subregions (i.e. V1 to V5, V3 to V5, V1V2, etc.) were transferred from the Nab resistant Env into the 0-month Env. The neutralization sensitivity of the chimeric and parental Envs was then evaluated using plasma contemporaneous with the Nab resistant Env.
For 185F, the 5-month Nab resistant EnvPB1.1 was chosen to determine which sequence adaptations were responsible for early Nab escape. Fig. 3A demonstrates that the chimeras containing either the region spanning the V1 loop through the end of the V5 domain (V1V5) or the V3 loop through the V5 domain (V3V5) from the 5-month Env displayed a level of resistance similar to the parental Env. However, the Nab sensitivity of the chimera containing the entire V1V2 domain (V1V2) was unchanged compared to the 0-month Env, despite a K192Q change in V2 (Fig. 3C) . We therefore surmised that Nab resistance was heavily dependent upon the V3V5 subregion, in which there were 3 residues that differed between 0-month EnvPB3.1 and 5-month EnvPB1.1. These changes were an E335A in the first position of the a2 helix, and two changes in the V5 region: I459T, which may also impact CD4 binding, and S463N ( Fig. 3C ; based on HXB2 numbering). None of these changes altered any of the predicted Nlinked glycosylation sites. To assess its individual contribution to Nab resistance, each amino acid change was introduced into the 0month EnvPB3.1. The V5 mutations I459T and S463N each independently produced a decrease in neutralization sensitivity, while these mutations combined recapitulated the Nab resistance level of the V3V5 chimera ( Fig. 3B ). In contrast, the E335A change in the a2 helix did not decrease neutralization sensitivity when introduced by itself into the 0-month Env (Fig. 3B ).
Together, these findings indicate that the combined V5 mutations facilitated neutralization resistance at 5 months, while the changes in the a2 helix and the V2 loop did not contribute to a detectable level, at least within the context of the 0-month Env. A different scenario was observed for early escape in 205F. For the 2-month escape variant EnvPB2.3, the V1V5 region contained determinants for Nab resistance (Fig 3D) , and this entire region differed from 0-month EnvPL6.3 by only 3 amino acids (Fig. 3E ). Two changes were located within V1 (an N134S substitution that introduced a potential N-linked glycosylation site near the Nterminal V1V2 stem and a Y140P substitution; numbered according to Fig. S2 ). In contrast to 185F, introduction of the two changes in V1 decreased Nab sensitivity by more than 10-fold in the context of the 0-month Env (Fig. 3D) . A third change, A453T, was located within the b23 region and has the potential to impact CD4 binding (Fig. 3E ). This residue by itself reduced Nab sensitivity by about 3-fold ( Fig. 3D ). Thus, for 205F, complete Nab resistance at 2-months could be achieved by sequence changes in V1, including addition of a potential N-linked glycan, and a change in a region involved in CD4 contact [39] .
Although the mutagenesis studies strongly suggested that the changes in V5 at 5-months were Nab escape mutations, these specific residues were not maintained in the subsequent Nab resistant Envs (Fig. S3) . However, the sequence of the V5 domain continued to evolve over time, suggesting ongoing selective pressure from Nab. At the last time point analyzed, 28-months, genetically distinct lineages of Nab escape variants were circulating (Fig. S1A ). The chimera-mapping approach revealed that these different Env variants had acquired resistance through at least two distinct mutational pathways (Fig. S4 , see bottom 3 panels). More detailed mapping revealed that for 28-month EnvPL5.1, the V5 domain continued to contribute to Nab resistance (Fig. 4A) , retaining the major escape pathway operative at 5-months. By contrast, 28-month EnvPL3.1 achieved a similar level of resistance through an escape pathway that required the gp41 ectodomain in addition to the cognate V1V5 domain (Fig. 4B ). For this Env, the V1V5 region from the 28-month Env independently conferred only partial escape onto the 0-month Env, while insertion of the 28-month ectodomain alone had no effect on Nab sensitivity. Further dissection revealed somewhat surprisingly that the V1V2 domain was a major contributor to Nab resistance in the context of the gp41 ectodomain, while the V3V5 region in this context did not appear to contribute to escape. Thus, a cooperative interaction between the gp41 ectodomain and V1V2 appeared to be conferring Nab resistance in this Env.
Nab escape in 205F is primarily determined by V1V2 over time
Having found that changes in the V1V2 and b23 regions drove early escape from Nab in 205F, we investigated whether this pattern was maintained over time. Env chimeras were created for 205F using Nab resistant Envs from five time points over a 26month follow-up period (Fig. 1B) and evaluated using the same approach as for 185F. V1V2 was the major determinant of Nab resistance at all time points analyzed (see Fig. S5 ). By contrast, the V3V5 region alone had little effect on resistance, although in combination with V1V2 it clearly contributed to escape.
In an effort to more precisely define Nab targets and escape pathways in 205F, B cell hybridomas were generated from viably frozen PBMC samples collected at 49 months after infection, which were the earliest available sample of this type. A 0-month Env (clone PB1.1) was used to screen for neutralizing activity in the hybridoma supernatants, and two hybridomas produced monoclonal antibodies (Mabs; 6.4C and 13.6A) that neutralized this and other 0-month Envs ( Fig. 5A and B, respectively) . Surprisingly, the 0-month Envs were not equally sensitive to neutralization by the two Mabs, despite being very homogeneous in sequence and potently neutralized by patient plasma (Fig. 1B) . For Mab 6.4C, 0-month EnvPL6.3 was moderately more sensitive to neutralization than the other two 0-month Envs (Fig. 5A ). In contrast, neutralizing activity for 13.6A was not detectable against EnvPL6.3, but the other two 0-month Envs were neutralized at levels similar to those observed with Mab 6.4C (Fig. 5B ). The 2month EnvPB2.3 was neutralized by both Mabs (Fig. 5A and B). Neutralizing activity against Envs cloned at 8-months or beyond, however, was undetectable for both Mabs ( Fig. 5A and B), providing strong evidence that these Mabs could be representative of those elicited during early infection and that the later Env variants had developed resistance mutations that protected against both specificities. An identical pattern was observed for the purified 6.4C and 13.6A Mabs, with a mean IC50 against the sensitive 0-months Envs of 39 and 156 ng/ml, respectively (data not shown). Envs from 8-months and beyond were not neutralized at 10 mg/ml of either purified Mab (data not shown).
Resistance against Mabs 13.6A and 6.4C involves loss and gain of predicted glycan addition sites in V1V2
Neutralization of the 205F chimera panel by each Mab localized differences in sensitivity to the V1V2 domain (data not shown). Examination of the V1V2 sequences of 0-to 26-month 205F Nab resistant Envs revealed that each one differed in length, pattern of predicted glycosylation sites, and sequence (Fig. S2) . However, all of the Nab resistant Envs from 8-months and beyond had acquired a mutation that created a potential glycosylation site in V2 at position 197 (highlighted in yellow in Fig. S2 ). Furthermore, 0-month EnvPL6.3 was the only 0-month Env that was resistant to 13.6A, and it lacked a potential N-gly site in V1 relative to the other Envs (highlighted in yellow in Fig. S2 ). Thus, Figure 2 . Moderate neutralization breadth of plasma from 185F and 205F against heterologous 0-month Envs. A single plasma sample from 185F (23-months) and 205F (20-months) was evaluated for neutralizing activity against heterologous Envs acquired during acute/early subtype C infection of six subjects. The Nab IC50 titer for each plasma-Env combination is shown on the vertical axis on a log10 scale. Each data point represents a single plasma-Env combination. Arrows indicate the Nab IC50 titer for the autologous plasma-Env combination; all other data points represent heterologous Envs. The plasma sample is indicated below each point plot. doi:10.1371/journal.ppat.1000594.g002 A and B) , and 205F 2-month PB2.3 (D) and chimeras or mutants from each of these Envs in the corresponding 0-month Env was evaluated using contemporaneous plasma. Pseudoviruses were created by expressing each Env with an HIV-1 env-deficient backbone, and their infectivity for JC53-BL13 (Tzm-bl) cells was evaluated in the absence or presence of serially-diluted patient plasma with luciferase as a quantitative measure. Percent virus infectivity relative to no test plasma is plotted on the vertical axis; the reciprocal of the plasma dilution is plotted along the horizontal axis on a log10 scale. Each curve represents one Env against serial plasma dilutions, and error bars represent the standard deviation of at least two independent experiments using duplicate wells. The legends list the parental Nab resistant Env followed by the chimeric and mutant Envs created in the 0-month Env background. Amino acid alignments are shown to indicate the sequence differences between the different Envs for 185F (C) and 205F (E). The color of the text corresponds to the curves on the graph. Only regions that contained differences are shown. doi:10.1371/journal.ppat.1000594.g003 . 185F Nab resistant variants at 28-months utilize distinct escape pathways. Neutralization of 28-month EnvPL5.1 (A) and 28month EnvPL3.1 (B) and the chimeric Env pseudoviruses generated from each of these Envs in the 0-month Env background was evaluated using the 28-month plasma sample in JC53-BL13 cells with luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. The panels below each graph indicate the region that was transferred from the 28-month Nab resistant Env (PL5.1 in A and PL3.1 in B; gray boxes) into 0-month EnvPL3.1 (white boxes). (C) Amino acid alignment of the V5 region for the 0-month and 28-month Envs. doi:10.1371/journal.ppat.1000594.g004
we hypothesized that a different array of potential glycan addition sites determined the pattern of sensitivity to the two Mabs. Fig. 6A shows the naturally occurring patterns of these predicted glycan sites that were detected in the early 205F Envs with the positions of the sites of interest indicated in red (V1) and blue (V2).
To define the effects of these potential glycan addition sites in V1 and V2 on sensitivity to the two Mabs, both sites were introduced into 0-month EnvPL6.3, which carried neither (Fig. 6B) . For Mab 13.6A, introduction of the V1 predicted glycan site into 0-month EnvPL6.3 resulted in a dramatic increase in neutralization sensitivity (Fig. 6C ). In contrast, for 6.4C, introduction of the V1 predicted glycan produced a moderate decrease in sensitivity (Fig. 6D) . Introduction of the V2 predicted glycan site into EnvPL6.3, with or without the V1 predicted glycan site, resulted in strong protection against both Mabs (Fig. 6C and D). Thus, predicted glycosylation at this site in V2 potentially tracked with protection against both Mabs (for a summary of longitudinal Envs and glycan sites see Table 2 ). These results also demonstrate that while both Mabs target a V1V2-dependent epitope, they recognize distinct structures.
The detection of a 0-month Env that was resistant to one of the Mabs was unexpected given the early timing and high sensitivity of these Envs to patient plasma. Therefore, the frequency of this predicted glycan site in V1 during acute/early infection was investigated using 21 uncloned single genome amplified V1V4 sequences from a p24-positive, antibody-negative sample (1-Mar-03) and 31 from the 0-month sample (27-Mar-03), which was antibody positive [38] (see Table 1 ). These sequences were combined with the five cloned 0-month Envs from this study, and a highlighter plot was created using the HIV Database (Fig. S6) . Forty-four out of 57 sequences (77%) were identical throughout V1V4, and all of these contained the predicted glycan addition site in V1. Two variants (including EnvPL6.3), both from the antibody positive time point, carried an identical G to A mutation that abrogated the predicted V1 glycan addition site. Taken together, these observations provided strong evidence that the founder virus, like the 0-month Envs PB1.1 and PL4.1, carried the predicted glycan site in V1, and that the mutation in 0-month EnvPL6.3 arose shortly after transmission, but circulated only transiently.
In a previous study, we demonstrated that subtype C infected seroconvertors mount robust Nab responses against their autologous viruses during the early stages of infection [15] . Here we have extended those findings to demonstrate that cycles of viral escape occur despite potent Nab and that these cycles involve multiple mechanisms and regions of Env. In subject 185F, early Nab escape required amino acid substitutions in V5 that were independent of glycosylation. It is possible that these changes directly altered an epitope in V5, as this region may be accessible to Nab on the Env trimer. However, attempts to remove Nab activity with a V5 peptide were unsuccessful, and Env chimeras in which unrelated Envs were engineered to carry the 185F 0-month V5 sequence lacked biological activity (data not shown). Thus neither of these approaches allowed definitive identification of a V5 epitope, and the latter suggested that the V5 domain itself, or the proximal region of gp120, likely evolved in concert with adjacent regions of the protein. Another possibility is that the early changes in V5 created conformational changes that protected a distinct target. The N-terminal region of V5 has been shown to contain contact sites for both CD4 and Mab b12 [39] , and the escape mutations could therefore have influenced exposure of epitopes such as the CD4 binding site. These two alternatives, epitope mutation or masking, are not mutually exclusive, and it is conceivable that V5 changes could protect from more than one antibody specificity. This is clearly the case for 205F, where a single amino acid change in V2 creating a potential glycan addition site resulted in resistance against two distinct Mabs.
At later time points in subject 185F, the flexibility of the Env structure provided alternative mutational pathways to resist neutralization. Escape pathways in 185F oscillated between changes localized to the gp120 outer domain (V3V5), and conformational masking strategies that required interaction between spatially separated Env subregions ( Fig. 7A and B) . V5 or V3V5 was the major determinant at 5-, 14-, and 17-months, and also in one of the 28-month Nab resistant Envs (see Fig. S4 for neutralization curves). However, in Envs from two other time points (11-and 23-months), V3V5 did not independently confer resistance, but appeared to require contributions from V1V2. In Nab resistant Envs from three time points (20-, 26-, and 28months), the gp120 V1V5 region and the gp41 ectodomain were both required for resistance. Further mapping for one of these Envs demonstrated that the V1V2 domain contributed in large part to this phenotype, but the V3V5 domain in this context did not. Interestingly, some of the V1V2 domains in subject 185F contained changes in the predicted glycosylation pattern relative to the 0-month Env, while others had sequence changes that would not alter the original glycosylation pattern (Fig. 7A : 5-PB1.1, 11-PL5.1, 23-PL5.1, and 28-PL5.1).
A novel finding is that Nab resistant Envs at 28-months utilized distinct Env sub-regions to block the same Nab pool. This provided a striking example of convergent, intra-patient evolution during early infection. One pathway was heavily dependent on the V5 domain, while the other exhibited V1V2 and gp41 codependence. The V5 domains of these two Envs contained the same predicted glycosylation shift (Fig. 7A : green and white spheres in 28-PL3.1 and 28-PL5.1) but differed in primary amino acid sequence (Fig. 4C) . This raises the possibility that the V5dependent Env contained mutations that directly confer epitope escape, while the V1V2-dependent Env retained the target but escaped through indirect mechanisms. In addition, both V1V2 and the regions flanking V5 are proximal to the CD4 binding site and could therefore alter its exposure, as has been proposed for changes in V2 and V5 in the context of a SHIV infection [30] . Thus, in examining a single subject in great detail, we have uncovered remarkable flexibility in the pathways of viral escape during early infection. These results further highlight how the plasticity of the Env hyper-variable domains coupled with complex conformational interactions could provide numerous options for escape.
In contrast to subject 185F, Nab escape in 205F was driven predominantly by changes in the V1V2 domain ( Fig. 8A and B) . A preference for potential glycan shifts in V1V2 became evident from the spatial representation of gp120, where in the 14-month Env, four predicted glycosylation site changes are observed in V1 alone (Fig. 8A : green and white spheres in Env 14-PB5.4). The importance of V1V2 for escape was further illustrated by the demonstration that two predicted glycan sites, one in V1 and one in V2, influenced neutralization by two Mabs derived from this same subject. Thus, this study is the first to identify specific mutations that confer autologous Nab resistance at the single antibody level. This made possible several observations that were not apparent from polyclonal plasma. First, a single substitution can confer resistance against multiple antibody specificities within an individual. While we did not formally show that glycosylation at the substituted site was responsible for resistance against Mabs 6.4C and 13.6A, there is strong evidence from other studies to support that this is the case. Second, different pathways of escape also operate at the single antibody level. Resistance against 13.6A could be achieved either by addition of the predicted glycan site in V2 or by loss of the predicted glycan site in V1. Interestingly, only the modification of V2 was retained in subsequent escape variants, suggesting that it could have been more advantageous in terms of escape and or maintenance of replication fitness. Third, mutations that confer escape from multiple monoclonal antibody specificities do not necessarily confer escape from the entire polyclonal Nab milieu in plasma. The V2 modification in the 8-month Env conferred complete resistance against 6.4C and 13.6A at 10 mg/ ml, but only partial escape from patient plasma ( Fig. S5 and data not shown). This finding suggests that escape determinants mapped against plasma will only reflect the dominant Nab specificities that are present at relatively high concentration (able to inhibit virus infectivity at greater than a 1:100 dilution in our assay), but other lower titer Nab specificities could also drive escape mutations. The relative contributions of different antibody specificities in plasma will undoubtedly vary among subjects, and potentially even within a subject over time, resulting in the need for customized escape pathways that are driven by each dominant Nab response. Thus, to derive a complete picture of autologous Nab and escape, it will be necessary to recover and characterize individual Mabs with different specificities, as was done here and recently by others to dissect the B cell response in subjects with neutralization breadth [9] . These studies demonstrate how the HIV-1 subtype C Env is uniquely equipped to respond to the current immune response of each individual host by adjusting its pathways of escape.
The V2-based mutation that conferred resistance against 6.4C and 13.6A appeared during the first eight months of infection; however, 13.6A and 6.4C were recovered from memory B cells circulating 41 months later. As such, it was not possible to determine whether related B cells were circulating during early infection in 205F. The V1-based change that conferred resistance against 13.6A was present at 0-months (at which time the subject was seropositive), but only transiently. If this mutation occurred in response to immune pressure from 13.6A, then this antibody must have been present within ,48 days from the calculated time of infection. Indeed we have observed very low level neutralizing activity (IC50 = ,1:40) in the 0-month plasma of 205F against 0-month Envs [15] and (data not shown), consistent with this concept. While others have observed that the very early antibody response (within the first ,40 days after infection) lacks neutralizing activity and is directed predominantly against gp41 [40] , our findings raise the possibility that viral neutralization and escape could occur earlier than previously thought. Individual Mabs, derived from B cells early in infection The blue gp120 backbones were generated by homology modeling of the 0-month Env sequence onto the CD4-liganded HIV-1 YU-2 gp120 structure [54] , with modeled V1V2 and V3 loops as described previously [25] . Red indicates amino acid changes relative to the 0-month Env sequence. Spheres indicate changes in potential N-linked glycosylation sites (green = loss, white = gain and tested against founder virus Envs, may be required to detect this initial Nab activity. It will therefore be important to determine whether Nab activity and viral escape is present in other subtype C infected subjects at very early time points, as well as whether early escape mutations are associated with decreased viral fitness. The blue gp120 backbones were generated by homology modeling of the 0-month Env sequence onto the CD4-liganded HIV-1 YU-2 gp120 structure [54] , with modeled V1V2 and V3 loops as described previously [25] . Red indicates amino acid changes relative to the 0-month Env sequence. Spheres indicate changes in a potential N-linked glycosylation sites (green = loss, white = gain Spatial properties of escape pathways Importantly in this study, not all sequence changes were linked directly with Nab escape. For example, one of the first sequence changes that was present in the 185F 5-month Env was a substitution in the first position of the a2 helix (E335A); however, this change did not alter Nab sensitivity to contemporaneous plasma when introduced into the 0-month Env. Reversal of this mutation (A335E) in the 5month escape variant also did not increase its sensitivity to autologous Nab (Murphy et al., in preparation) . The 205F Nab escape variants also exhibited variation in the a2 helix beginning at 14-months, but again this region did not appear to contribute independently to Nab escape. These findings support that a2 was not targeted directly by Nab in these instances, despite ongoing sequence evolution. The a2 helix has been linked to autologous and heterologous Nab sensitivity of subtype C Envs from Zambia, South Africa, and India by our group and others [23, 25, 41] . However, its exact role(s) in Nab sensitivity or escape remains undetermined [23, 25] . We have speculated that the a2 helix plays an ancillary role in Nab escape, and perhaps participates in maintenance of the tertiary structure of the gp120 outer domain or the quaternary structure of the trimer [25, 42] . The findings presented here support our earlier findings, but do not rule out the possibility that the a2 helix is targeted by Nab in some instances, or by low titer Nab specificities. Studies are ongoing in our laboratory to more precisely define the role of changes in the a2 helix in the context of autologous Nab. In addition to the a2 helix, sequence variation was observed in V1V2, V5, and other regions of the outer domain in both 185F and 205F Envs ( Fig. 9A and B, respectively) . However, in 185F, the V3V5 region had the strongest effect on Nab resistance, while in 205F V1V2 was the major determinant. A companion study of four subtype C infected seroconvertors and our own previous study of subtype C chronically infected subjects reported consistent findings, in that V1V2 was commonly involved in Nab escape, but to varying degrees [24] and (Moore et al., in press). Importantly, the combined biological results and spatial analysis of these two subjects demonstrate how the perpetual flexibility of the V1V2 and V5 domains provides a formidable defense against Nab. This could be due in part to their ability to simultaneously mask multiple epitopes through limited changes, but may also involve direct escape.
Although these studies were conducted on a small number of subjects, it is still beneficial to work toward developing a mechanistic model that can explain the underlying complexity of escape pathways between and within subjects. The results presented here provide the basis for such an endeavor. First, the escape pathways observed here appear to define Nab resistance through a combination of direct and indirect mechanisms. Direct epitope changes may be sufficient in the setting of limited antibody specificities, such as during the early phase of infection, while indirect 'masking' or cooperative mechanisms may be required later when multiple antibody specificities are circulating. However, it will be important to expand and confirm these studies by characterizing Nab escape in additional subjects. The frequency, timing, and underlying basis for convergent escape pathways within a subject will require further investigation, as will the significance of regions of subtype C Env that appear to be under positive selection but do not contribute directly to escape from plasma Nab. Lastly, it will be important to determine if different escape pathways share any common conformational basis or point to specific regions of the Env that should be incorporated into or excluded from vaccine immunogens.
Informed consent and human subjects protocols were approved by the Emory University Institutional Review Board, and the Figure 9 . Sequence variation occurs in similar regions of Env in 185F and 205F. A 3-dimensional representation of the sequence variation in gp120 over time in 185F and 205F is shown in panels (A and B) , respectively. The gp120 backbones were generated by homology modeling of the 0-month Env sequences of 185F and 205F onto the CD4-liganded HIV-1 YU-2 gp120 structure [54] , with modeled V1V2 and V3 loops as described previously [25] . Blue to green to red indicates degree of sequence conservation (high to low) within the alignment. doi:10.1371/journal.ppat.1000594.g009
University of Zambia School of Medicine Research Ethics Committee. Written Informed consent was obtained from human subjects.
The Zambia Emory HIV Research Project (ZEHRP) was established in Lusaka in 1994 to provide voluntary HIV-1 testing and counseling, long-term monitoring, and health care to cohabiting heterosexual couples. Details of the cohort have been described elsewhere [43] . Briefly, HIV-discordant couples enrolled in studies of transmission are monitored for seroconversion of the negative partner at three-month intervals, at which time the participants also receive preventative counseling and condoms. Banked plasma samples from seronegative partners are tested for p24 antigen by ELISA to identify individuals with acute infection [38] . The two subtype C infected seroconvertors studied here were participants in this cohort and were identified as p24 antigen positive and seropositive by rapid test and western blot, as described in [38, 44] . Plasma viral loads were determined using the Roche Amplicor HIV-1 assay. None of the subjects received antiretroviral therapy during the evaluation period.
Conditions for single genome PCR amplification of full-length gp160 (plus Rev, Vpu, and partial Nef coding sequences) from the genomic DNA of uncultured peripheral blood mononuclear cells and cDNA from plasma have been described previously [38, 44] . The viral env amplicons were directionally T/A cloned into the CMV-driven expression plasmid pcDNA3.1-V5HisTOPO-TA and screened for biological function as pseudoviruses following co-transfection with an Env-deficient subtype B proviral plasmid (SG3Denv) into 293T cells [45] . Seventy-two hours later, supernatant was collected and used to infect JC53-BL13 (Tzmbl) cells. At 48 hours post-infection, b-gal staining was performed and each well was scored positive or negative for blue foci.
DNA sequencing of env genes was carried out by Lone Star Labs, Inc. (Houston, TX) utilizing the ABI Prism H Automated DNA sequencer 377XL and Big Dye TM Terminator Ready Reaction Cycle Sequencing Kit. Nucleotide sequences were edited and assembled using Sequencher v4.7, translated using Se-Al v2.0all, and nucleotide or amino acid alignments were created using Clustal W v1.83. Neighbor joining phylogenetic trees were generated by Clustal W v1.83 using gap-stripped nucleotide sequences of the complete env gene, and reliability of branching orders was assessed by bootstrap analysis using 1,000 replicates. Trees were visualized using NJ Plot. Aligned sequences were imported into the Highlighter tool to analyze viral diversity (http://www.hiv.lanl.gov/content/ sequence/HIGHLIGHT/highlighter.html). Sequences have been deposited into Genbank under the accession numbers GQ485312-GQ485447.
Patient plasma samples were evaluated for neutralizing antibody activity against virions pseudotyped with autologous patientderived viral Envs using a single round reporter assay described previously [17, 45] . Briefly, JC53BL-13 (Tzm-bl) cells were plated and cultured overnight. Two thousand infectious units of each pseudovirus was combined with five-fold dilutions of heatinactivated patient plasma and incubated for 1 hour at 37uC. Normal heat-inactivated human plasma was added as necessary to maintain a constant overall concentration. The virus-Ab mixture was then added to JC53BL-13 cells, and after two days, the cells were lysed, and the luciferase activity of each well was measured using a luminometer. Background luminescence was determined in uninfected wells and subtracted from all experimental wells. Percent infectivity was calculated by dividing the number of luciferase units at each plasma dilution by the value in the well containing no test plasma. The dilution of patient plasma that inhibited 50% of virus infectivity (IC50 titer) was determined using the Microsoft Excel 2004 for Mac Growth Function. Each experiment was performed independently at least twice with duplicate wells.
Chimeric Envs were constructed using a domain exchange strategy that has been described previously [24, 25] . The primer sequences and their HXB2 locations are shown below. A PCR screen was performed to identify transformants in which the fragments ligated together in the correct orientation using forward primer EnvA and the reverse primer that was used to amplify the exchanged domain. Colonies that were positive by PCR screen were inoculated into LB-Ampicillin broth for overnight cultures, and the plasmid was prepared using the QIAprep Spin Miniprep Kit. Env chimeras were then screened for biological function as described above. For chimeras that produced functional Env pseudotypes, the plasmids were re-transfected into 293T cells on a larger scale to produce a working pseudotype virus stock. Transfection supernatants were collected at 72 hours post-transfection, clarified by low speed centrifugation, aliquoted into 0.5 ml or less portions, and stored at 280uC. The titer of each pseudovirus stock was determined by infecting JC53-BL13 cells with 5-fold serial dilutions of virus as described previously. All Env chimeras were confirmed by nucleotide sequencing of the entire env gene. The 0-month Env backbones (minus the target domains) with pcDNA3.1 vector sequences were PCR amplified using primers that anneal to conserved regions adjacent to the target domain primers. These primers amplify away from the target domains. A 59 phosphate group (Phos) was added to these primer sets during synthesis to facilitate ligation to the target domain amplicons. The primer sequences and their HXB2 locations were as follows:
For subject 185F, V1V5 domain backbone, forward primer 59-Phos-atgagggacaattggagaagtg-39 (HXB2 nt 7647 to 7668) and reverse primer 59-Phos-gctttaagctttgatcccataaac-39 (HXB2 nt 6576 to 6553); V1V2 domain backbone, forward primer 59-Phoscaagcctgtccaaaggtctct-39 (HXB2 nt 6831 to 6851) and reverse primer 59-Phos-gctttaagctttgatcccataaac-39 (HXB2 nt 6576 to 6553); V3V5 domain backbone, forward primer 59-Phos-atgagggacaattggagaagtg-39 (HXB2 nt 7647 to 7668) and reverse primer 59-Phos-ccttacacacacaatttctac-39 (HXB2 nt 7118 to 7098); ectodomain backbone, forward primer 59-Phos-aagatatttataatgatagta-39 (HXB2 nt 8271 to 8291) and reverse primer 59-Phostttttctctctccaccactctcc-39 (HXB2 nt 7754 to 7732); V5 domain backbone, forward primer 59-Phos-atgagggacaattggagaagtg-39 (HXB2 nt 7647 to 7668) and reverse primer 59-Phos-catgttatgtttcctgcaatg-39 (HXB2 nt 4527 to 4507).
The PCR amplification conditions for the 0-month Env backbones were 1 cycle of 95uC for 3 min; 35 cycles of 95uC for 1 min, 50uC to 60uC for 30 s (the optimal annealing temperature was determined for each primer set), 72uC for 10 min; 1 cycle of 72uC for 15 min; and storage at 4uC. The amplification conditions for the target domains were the same, except the extension time at 72uC was reduced to 30 s. The 25 ml PCR mixtures contained 50 ng of each primer, 10 ng of the plasmid template, 2.5 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphate, and 16 reaction buffer. PfuUltra II DNA polymerase (Stratagene) was used to generate the blunt-ended PCR amplicons, which were digested with DpnI to remove contaminating template DNA and gel purified from an agarose gel using the QIAquick Gel Extraction Kit (QIAGEN) prior to ligation. Each target domain DNA fragment was then ligated to the purified 0-month env backbone to produce a chimera using T4 DNA ligase (5 U/ml; Roche) at 4uC overnight. The ligation reaction mixture (usually one-third of the volume) was transformed into maximum-efficiency XL2-Blue Ultracompetent cells (1610 9 CFU/mg DNA; stratagene) so that the DNA volume did not exceed 5% of the cell volume. The entire transformation was plated onto LB-ampicillin agar plates, generally resulting in 10 to 50 colonies per ligation reaction.
To investigate whether individual amino acid sequence differences contributed to the neutralization resistant phenotype, PCR-based site-directed mutagenesis was used to introduce an amino acid change as described previously [24] . Briefly, a set of primers was used that each had either the wildtype or mutated sequence. The primer sequences and their HXB2 locations are shown below, where the substituted nucleotides are underlined. All mutants were confirmed by sequencing the entire env gene.
The PCR amplification conditions were 1 cycle of 95uC for 1 min; 18 cycles of 95uC for 50 s, 60uC for 50 s, 68uC for 8 min; and 1 cycle of 68uC for 7 min. The 25-ml PCR mixtures contained 63 ng of each primer, 5 ng of the plasmid template, 0.2 mM deoxynucleoside triphosphate, and 16reaction buffer. PfuUltra HF DNA polymerase (Stratagene) was used, amplicons were digested with DpnI to remove contaminating template DNA, and 2 ml was transformed into maximum-efficiency XL10-Gold ultracompetent cells (5610 9 CFU/mg DNA; Stratagene). Half of the transformation was plated onto LB-ampicillin agar plates, generally resulting in 10 to 50 colonies per reaction. All mutants were confirmed by sequencing the entire env gene.
185F 
Human B cell hybridomas were generated from viable frozen PBMC samples from subject 205F based on a protocol of EBV immortalization of peripheral blood B cells as described previously [46, 47, 48] . This protocol includes immortalization of pre-selected (CD22+, IgM2, IgD2, IgA2) memory B cell populations that are cultured in medium supplemented with immunostimulatory CpG sequences and irradiated allogeneic PBMC, as described in [49, 50, 51] . EBV-transformed B cell cultures were screened for Mabs that neutralized the 205F 0-month Env and that bound envelope glycoproteins by ELISA [52, 53] . The method used to screen for neutralizing Mabs is a modification of the Tzm-bl (JC53-BL13) luciferase reporter cell assay originally developed by Wei et al. [15, 17, 24, 45] . EBV-transformed B cell hybridomas with neutralizing activity were cloned in the presence of CpG and irradiated PBMC. Culture supernatant was collected from the two hybridomas, 6.4C and 13.6A, and used in the neutralization studies to map targets and escape. Figure S1 Phylogenetic tree of longitudinal 185F and 205F Envs. A. A neighbor-joining tree was generated using Clustal W v. Figure S2 Amino acid sequence alignment for 205F Envs. Three 0-month Nab sensitive Envs and six Nab resistant Envs from subsequent time points were selected for study from 32 Envs. Env clones are indicated by the time point (in months), source (PB = PBMC DNA or PL = plasma), and clone number. Sequences are shown in reference to the 0-month EnvPB1.1, with amino acid differences indicated by the letter, and deleted residues indicated by a dot. Domains that were transferred into the 0-month Env to create chimeras are as follows: V1V5 (blue, gray and green; HXB2 nt 6557 to 7634), V1V2 (blue; HXB2 nt 6557 to 6876), V3V5 (green; HXB2 nt 7110 to 7634). Major Env domains are indicated above the region, and the a2 helix is underlined. Two potential N-linked glycan addition sites of interest in V1 and V2 (NXS or NXT where X is any residue but proline) are highlighted yellow. Found at: doi:10.1371/journal.ppat.1000594.s002 (0.37 MB PDF) Figure S3 Amino acid sequence alignment for 185F Envs. Two 0-month Nab sensitive Envs and ten Nab resistant Envs from subsequent time points were selected for study from 58 Envs. Env clones are indicated by time point (in months), the source (PB = PBMC DNA or PL = plasma), and clone number. Sequences are shown in reference to the 0-month Env PL3.1, with amino acid differences indicated by the letter, and deleted residues indicated by a dot. Domains that were transferred into the 0month Env to create chimeras are as follows: V1V5 (blue, gray, and green; HXB2 nt 6577 to 7646), V1V2 (blue; HXB2 nt 6577 to 6810), V3V5 (green; HXB2 nt 7119 to 7646), gp41 ectodomain (yellow; HXB2 nt 7755 to 8270). Found at: doi:10.1371/journal.ppat.1000594.s003 (0.50 MB PDF) Figure S4 The major determinants of Nab resistance in 185F Envs change over time. Neutralization of 185F parental and chimeric Env pseudoviruses was evaluated using longitudinal plasma samples that are indicated in each panel. Each plasma sample was contemporaneous with the Nab resistant Env, and neutralization sensitivity was evaluated in JC53-BL cells using luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 reciprocal plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. All chimeras were created in the 0-month EnvPL3.1 background (red lines). In the legend, the parental Env clones are indicated, followed by each chimera that contains the indicated region from the Nab resistant Env in the 0-month Env. 'ecto' stands for the gp41 ectodomain. Found at: doi:10.1371/journal.ppat.1000594.s004 (0.22 MB PDF) Figure S5 The major determinants of Nab resistance in 205F Envs are in V1V2. Neutralization of 205F parental and chimeric Env pseudoviruses was evaluated using longitudinal plasma samples that are indicated in each panel. Each plasma sample was contemporaneous with the Nab resistant Env, and neutralization sensitivity was evaluated in JC53-BL cells using luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 reciprocal plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. All chimeras were created in the 0-month EnvPL6.3 background (red lines). In the legend, the parental Env clones are indicated, followed by each chimera that contains the indicated region from the Nab resistant Env in the 0-month Env. Found at: doi:10.1371/journal.ppat.1000594.s005 (0.14 MB PDF) Figure S6 Highlighter plot showing nucleotide mismatches in the gp120 V1V4 region for 205F Envs. Single genome amplified, uncloned V1V4 sequences derived from the 1-Mar-03 sample (n = 21) or the 27-Mar-03 sample (n = 31) from 205F and V1V4 sequences from single genome amplified cloned Envs from the 27-Mar-03 sample (n = 5) were subjected to Highlighter analysis (www.hiv.lanl.gov). Ticks represent mismatched bases compared to the master sequence listed at the top. The three 0-month Envs that were analyzed for neutralization by 6.4C and 13.6A in Fig. 11 are boxed in red. PB = uncultured PBMC DNA; PL = plasma. The G.A mutation (diamonds) located near the beginning of the sequence represents the loss of a potential N-gly site in V1 that tracks with resistance against 13.6A. Found at: doi:10.1371/journal.ppat.1000594.s006 (0.11 MB PDF) Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE BACKGROUND: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family. PRINCIPAL FINDINGS: We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements. CONCLUSIONS: The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. Sialic acids are Nor Osubstituted terminal monosaccharides with a nine-carbon backbone highly expressed on eukaryotic cell surfaces [1] . Sialylation of glycoproteins and glycolipids modulates a wide range of biological and pathological events including early development [2] , tumorigenesis [3] , viral and bacterial infection, and immunity [4, 5] . In vertebrate systems, N-acetylneuraminic acid (Neu5Ac) is the metabolic precursor of all known naturally occurring sialic acids [6] . Neu5Ac is synthesized in the cytosol from UDP-N-acetylglucosamine (UDP-GlcNAc) by four consecutive reactions; and UDP-GlcNAc is a derivative of fructose-6phosphate and the end-product of the hexosamine biosynthesis pathway (Figure 1 ).
The first two steps of the biosynthesis of Neu5Ac from UDP-GlcNAc are catalyzed by the bi-functional enzyme UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE). GNE contains an N-terminal epimerase domain and a C-terminal kinase domain [7] . The epimerase domain converts UDP-GlcNAc to Nacetylmannosamine (ManNAc), which is then phosphorylated at the 6 position by the kinase domain. GNE is feedback-inhibited by the activated form of Neu5Ac, i.e., cytidine-monophosphate Nacetylneuraminic acid (CMP-Neu5Ac). The kinase domain belongs to the ROK (Repressor, ORF, Kinase) family. The ROK family consists of a set of bacterial proteins that include repressors for sugar catabolic operons, and sugar kinases [8] . Gne is the only known gene in the entire human genome that encodes a ROK domain-containing protein.
Three protein isoforms have been described for human GNE, where isoform 1 is ubiquitously expressed and is believed to be responsible for the basic supply of sialic acids. Isoforms 2 and 3 are generated by alternative splicing and show tissue specific expression patterns. Isoforms 2 and 3 have reduced epimerase activities but almost intact kinase activities and may fine-tune the production of sialic acids [9] . Wild type GNE forms homohexamer in solution [10] , and allosteric regulation of the epimerase and kinase activities of GNE is important for the normal function of the protein [10, 11] . Mutations in the epimerase domain lead to the rare congenital metabolism disorder sialurea, which results in the production of high levels of Neu5Ac due to loss of the allosteric feedback control of the UDP-GlcNAc 2-epimerase activity by CMP-Neu5Ac [12] . Late onset autosomal recessive inclusion body myopathy, which is also known as hereditary inclusion body myopathy (hereinafter referred to as HIBM), and allelic Nonaka myopathy are neuromuscular disorders that are caused by a number of different mutations within the gne gene. The mutations are located at either the epimerase domain or the kinase domain [13] and lead to hypoactivity of the enzyme [11] . Mutagenesis and enzymatic activity analysis revealed that the activities of the epimerase domain and the kinase domain are interrelated such that a single mutation in one domain could affect the activities of both domains [11] . Here, we solved the structure of the dimeric GNE kinase domain in the ligand-free state. The structure reveals the dimerization interface of the kinase domain and also suggests a possible hexameric assembly of the protein. Furthermore, the structure provides insights into the relationship between GNE mutations and GNE-related metabolism disorders.
Overview of the GNE kinase domain monomer
The overall structure adopts a typical bi-lobal kinase architecture. Both the N-lobe and the C-lobe have the a/b fold. Each lobe consists of a central b-sheet flanked by a-helices on both sides of the sheet. The last helix C-terminal to the C-lobe is part of the Nlobe and perpendicular to the interfacial helix of the C-lobe. Residues 475-498 of the N-lobe are invisible in the electron density map (Figure 2 ).
The GNE kinase domain contains a type I zinc-binding motif GHx 9-11 CxCGx 2 G(C/H)xE, which forms an HC3 type zincfinger with residues H569, C579, C581, C586. The zincbinding motif is a characteristic feature for all ROK family members [14] . The kinase domain also contains a DxGxT type ATP-binding motif [15, 16] . The side chains of this ATPbinding motif residues point toward the cleft between the Nlobe and the C-lobe. Comparison with the actin/hexokinase/ hsp70 ATPase domains suggests that the disordered residues 475-498 form part of the binding pocket for the adenosine moiety of ATP [17] and are located near the DxGxT ATPbinding motif. Taken together, these findings suggest that the ATP binding pocket of the GNE kinase domain is located in the cleft between the two lobes.
Previous deletion mutations study has suggested that the GNE kinase domain is responsible for dimerization, while a segment of residues between the epimerase and kinase domains, residues 360-382, is a potential site for trimerization [18] . Our gel filtration data ( Figure 3) show that the kinase domain exists predominantly as a dimer in the solution, with small amounts of monomer and a higher order oligomer. The apparent molecular weight of the oligomer fits a hexamer of the kinase domain (see also below). However, the possibility of a tetramer [19] cannot be completely ruled out due to the low resolution of the gel-filtration column at this molecular size The GNE kinase domain was crystallized in space group P3 1 21 with three molecules in the asymmetric unit. Protein interface and assembly analysis using the PISA server [20] suggests that two of the three molecules dimerize through the C-lobe with an average buried surface area of 1587 Å 2 per molecule ( Figure 4 ) whereas the third molecule dimerizes with a two-fold symmetry related molecule through the same C-lobe ( Figure 5a ). The solvation free energy gain upon formation of the interface, D i G, is 224.2 kcal?mol 21 , indicating that the dimer interface is very stable and may not simply be a crystal packing artifact.
A crystallographic hexamer can be produced when a two-fold rotational symmetry operation is applied to the three molecules (one and a half dimers) in the asymmetric unit ( Figure 5 ). In this hexamer, the N-lobes of three kinase molecules are pointing to the same side of the ''hexamerization plane'', while the N-lobes of the other three molecules are pointing to the opposite side of the plane (Figure 5b ). This assembly mode of the kinase domain allows locating the epimerase domain further away from the hexamerization plane and is consistent with the proposition that the interdomain segment (residues 360-382) is the site of trimerization [18] .
Structural homology search of the GNE kinase domain using the FATCAT (Flexible structure AlignmenT by Chaining Aligned fragment pairs allowing Twists) server [21] revealed the top four non-redundant hits to be PDB codes 2aa4, 1xc3, 1z05 and 1z6r. All these structures contain the signature zinc-binding motif of the ROK family. The structure of E. coli putative ManNAc kinase (PDB 2aa4) was the top hit with twist-adjusted r.m.s. deviation (opt-rmsd value) of 1.94 Å . The structure of a putative sugar kinase from Bacillus subtilis was the second best hit (PDB 1xc3). The other two homologous structures were transcription repressors that belong to the ROK family (PDB: 1z05 and 1z6r). Vibrio cholerae transcriptional regulator (PDB: 1z05) is a homolog of the E. coli Mlc protein (PDB: 1z6r). The latter is a transcriptional repressor that controls the expression of malT, the central transcription activator of the E. coli maltose system [22] . The structure of the GNE kinase domain aligns well with the E. coli Mlc structure: the N-lobe of GNE kinase domain aligns to the E-domain of Mlc and the C-lobe aligns to the Odomain of Mlc. It is interesting to note that the Mlc O-domain is responsible for the oligomerization of Mlc protein [22] in a way similar to the dimerization of GNE kinase through the C-lobe. However, these four structures do not contain sugar ligands that would help inform on a substrate binding mode for GNE.
To evaluate the putative sugar binding site, the sequence of the GNE kinase domain was aligned with that of E. coli glucokinase complexed with glucose (PDB: 1sz2) [23] , which is the closest homologous structure containing a bound substrate currently available in the PDB data bank. The five residues involved in sugar binding are conserved in GNE (N516, D517, E566, H569, E588, GNE numbering). These five residues are arranged to accommodate the sugar substrate ( Figure 6 ). Two residues, H569 and E588, are located in the ROK family zinc-binding signature motif and H569 directly coordinates the zinc ion. This finding suggests that zinc may play a catalytic role in sugar substrate binding, as well as a structural role.
Since the identification of the relationship between gne mutations and HIBM [13] , more than 60 mutations have been found to be associated with HIBM [24] . Among these mutations, 25 missense mutations at 23 unique sites are located in the kinase domain of the GNE protein. These 23 mutation sites can be classified into 4 different groups based on their solvent accessibility, and their locations ( Table 1 ).
The first group of residues I557, G559, V572, and G576 is located at the dimerization interface of the C-lobe and mutation of these residues may interfere with dimerization of the kinase domain. It is noteworthy that kinase domain dimerization does not affect the solvent accessibility of G576 (Table 1 ), indicating that G576 is not directly involved in dimerization. The amino acid side chain of a mutant at this position would point into a hydrophobic niche that also accommodates the side chain of L574 from another monomer. The G576E mutation would exert both charge and space hindrances on the side chain of L574 and thus disrupt the dimerization (Figure 7) , consistent with the previous observation that the G576E mutant of the full length GNE remains as a trimer [11] .
The location of this group of residues is also close to the residues involved in sugar substrate binding (Figure 6b ). Residues V572 and G576 are located on the zinc-binding signature motif of the ROK family (Figure 6b, 8b) , which could play both a functional and a structural role. Mutations of these residues could thus also affect the sugar substrate binding affinity of the kinase domain indirectly.
The second group of residues includes those located at the interfacial helices between the N-lobe and the C-lobe, i.e. N519, A524, F528, G708, and M712. Since the interlobar cleft is the site of ATP and carbohydrate binding as well as where phosphoryl transfer occurs, mutation of these residues could change the interlobar movement during catalysis and thus affect the kinase activity of the protein. For example, the first identified HIBM-related mutation, M712T [13] , would likely abolish the hydrophobic interaction of the side chain of M712 with that of L523 from the C-lobe helix (Figure 8a ). In the previous study [11] , the M712T mutation has been shown to cause a 30% reduction in the kinase activity without affecting the epimerase activity of full length GNE. On the contrary, mutations of other residues in this second group reduce not only the kinase activity but also the epimerase activity of the full length protein ( [11] , Table 1 ). This suggests that the kinase domain is allosterically coupled to the epimerase domain. The structure of the full length GNE is needed to fully understand the coupled effects of the kinase and epimerase domains.
The third group currently includes residue P511. P511 has the highest relative solvent accessibility (.40%) among the 23 mutation sites and is located on a loop region of the structure. The underlying mechanism for the association of P511H and P511L mutations with HIBM is elusive without further data, but mutation of a proline to any other residue type will inevitably change the flexibility of the loop region around this residue and could thus change the allostery of full length GNE in the higher-order oligomeric state.
The fourth group of residues includes all the rest of mutation sites in Table 1 . All residues have hydrophobic side chains and low solvent accessibilities, and are located within secondary structural elements. Mutations of these residues may disrupt the secondary structural elements at given mutation sites, and could interfere with the hydrophobic interactions of the secondary structure elements that stabilize the protein quaternary structure.
We show here the 3D structure of the N-acetylmannosamine kinase domain of GNE, the only ROK family kinase encoded in the human genome. The kinase domain dimerizes through an Data of missense mutations were extracted from reference [11] and [24] . b UDP-GlcNAc epimerase and ManNAc kinase activities are percentage values relative to the corresponding activities of the full length wild type GNE. Data extracted from reference [11] . c Oligomeric state of the full length mutant GNE. Data extracted from reference [11] . d Relative solvent accessibility of the residue calculated using the DSSP program [30] and normalized according to values in reference [31] . A value of 1 means full exposure of the residue while a value of 0 means the residue is fully buried. e The type of the secondary structure element the residue is located at was assigned using the DSSP program [30] . doi:10.1371/journal.pone.0007165.t001
interface at the C-lobe. This is consistent with mutagenesis data from other groups on the full length GNE protein [11, 18] . The crystallographic hexamer, which consists of a trimer of kinase dimers, could serve as a prototype of a proposed full length GNE hexamer. Structure comparison of the GNE kinase domain with previously studied proteins revealed potential substrate binding sites at the interlobar cleft and also the structural and functional importance of the signature zinc-binding motif of the ROK family. Four groups of missense mutations associated with hereditary inclusion body myopathy are classified and their effects on the enzymatic activity can mostly be explained by the structure model.
The cDNA template encoding the kinase domain of GNE was codon optimized for overexpression in E. coli and synthesized commercially (Codon Devices, Inc.) The DNA fragment encoding GNE residues 406-720 was PCR amplified and subcloned into the pET28-MHL vector (gi:134105571) using an In-Fusion dry-down PCR cloning kit (ClonTech). Protein was overexpressed in E. coli BL21(DE3) CodonPlus-RIL cells (Stratagene) grown in terrific broth medium. The culture was grown at 37uC in a LEX bubbling system (Harbinger Biotech. & Engineering Corp.) until OD 600 reached 3.0. The temperature of the culture was then lowered to 15uC and the cells were induced with 0.5 mM isopropyl 1-thio-b-D-galactopyranoside and allowed to grow further overnight. Cells were harvested by centrifugation and flash frozen in liquid nitrogen and stored at 280uC until purification. Frozen cells were thawed and resuspended in 10 mM HEPES buffer (pH 7.5) containing 500 mM sodium chloride, 5% glycerol, 2 mM b-mercaptoethanol, and supplemented with 5 mM imidazole, and mechanically lysed using a microfluidizer (Microfluidics, model M-110EH) at 1,000 bar pressure. The lysate was clarified by centrifugation. GNE protein was bound with nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) at a ratio of 2.5 mL 50% Ni-NTA flurry per litre of cell culture. The bound protein was washed twice with the same HEPES buffer containing 30 mM or 75 mM imidazole, and finally eluted with the HEPES buffer supplemented with 300 mM imidazole. The elutant containing the GNE protein was further purified by Supderdex-75 size exclusion chromatography (GE Healthcare). The eluted fractions were pooled, concentrated to a final concentration of 40 mg per mL, and stored in a buffer containing 10 mM HEPES, pH 7.5, 500 mM sodium chloride, 5% glycerol and 5 mM dithiothreitol. The purity of the protein was better than 95% judging from SDS-PAGE gel.
Selenomethionine (SeMet) labelling of the protein was carried out using prepacked M9 SeMet growth media kit (Medicilon) following manufacturer's instructions.
The ligand-free form crystals were grown at room temperature in sitting drops. A final concentration of 5 mM ADP, 1:100 chymotrypsin (w/w) were added into the protein stock solution and 0.5 mL protein solution was mixed immediately with 0.5 mL well solution containing 15% polyethylene glycol (PEG) 4000, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 5.6 and set up for vapour diffusion crystallization. The SeMet crystal used for structure determination was grown in 14.55% PEG4000, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 6.0, with 1:100 chymotrypsin (w/w) and 5 mM ADP in a sitting drop setup. Crystals grew to a mountable size within 24 hours. Paratone oil was used to cryo-protect the crystals.
Diffraction data of a selenomethionyl derivative of the GNE kinase domain were collected at beamline 19ID of the Advanced Photon Source (APS) at a wavelength of 0.9792 Å . Initial phases were obtained by single wavelength anomalous diffraction with SOLVE and density modification with RESOLVE [25] . For model building, the phases were combined with data collected at APS beamline 23ID-B at a wavelength of 0.9793 Å (see Table 2 ). The refined model of the target resulted from iterative application of density modification with DM and RESOLVE, interactive model building with COOT [26] , coordinate and B-factor refinement with REFMAC [27] and PHENIX [28] , and geometry validation with MOLPROBITY [29] . Diffraction data and refinement statistics are summarized in Table 2 . The current model was deposited at the Protein Data Bank with PDB ID 3EO3.
Datapack S1 Standalone iSee datapack -contains the enhanced version of this article for use offline. This file can be opened using free software available for download at http://www.molsoft.com/ icm_browser.html. Found at: doi:10.1371/journal.pone.0007165.s001 (ICB) Text S1 Instructions for installation and use of the required web plugin (to access the online enhanced version of this article). Found at: doi:10.1371/journal.pone.0007165.s002 (PDF)  Increased Host Species Diversity and Decreased Prevalence of Sin Nombre Virus Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence. D uring the past 60 years, the number of emerging pathogens affecting humans has substantially increased (1) . Of these emerging infectious diseases, 62% are zoonotic (2) , meaning they are naturally hosted by, and persist in, wildlife but also affect human populations. The ecological factors associated with zoonotic disease emergence are likely complex and are poorly understood. Most often, because of limited time, resources, and the exigencies of the situation, outbreak investigations of emerging diseases seek only to discover the pathogen responsible for the disease in humans. But ecological studies are of critical importance to long-term containment of zoonotic disease emergence; they are the only way to ascertain the wildlife source of the disease, the dynamics of the host-pathogen relationship, and the ecological factors associated with an outbreak. Knowledge of all these factors is needed to proactively protect the public from zoonotic diseases; without this knowledge, new diseases will continue to emerge. The worldwide distribution of these largely zoonotic diseases suggests a globally distributed mechanism for their emergence.
Anthropogenic factors-including pollution, land-use conversions, and climate change-likely contribute to disease emergence by several mechanisms (3), one of which has been hypothesized to be decreased species diversity. The number of species currently being lost, as well as the rate of species loss, is unprecedented (4); these losses generally have negative effects on ecosystem functioning (5, 6) . It likely is not coincidental that areas where many zoonoses are emerging among humans are the same areas where loss of species is accelerating, e.g., Central Africa (Ebola, monkeypox, Marburg virus), West Africa (Lassa virus, HIV-2), Southeast Asia (Nipah virus, severe acute respiratory syndrome, avian influenza), and South America (dozens of strains of hantaviruses and arenaviruses).
Lyme disease, a vector-borne zoonosis, is affected by loss of species by a process known as the dilution effect (7), whereby increasing species diversity decreases disease prevalence by diluting the availability of competent hosts with increased numbers of noncompetent hosts. Little research on the dilution effect has been carried out beyond its effect on Lyme disease (8) , yet the global implications of the phenomenon-if the effects are applicable to other types of diseases and transmission dynamics-could have substantial and enduring effects on human health and conservation.
Hantaviruses provide a model system in which to test the dilution effect in directly transmitted zoonoses. Since their initial discovery in the Western Hemisphere in 1982, several dozen hantavirus strains have been found, each hosted by a unique rodent species (9); novel hantaviruses have recently been discovered in shrews (10, 11) . Natural hosts are asymptomatic and chronically infected; intraspecies spread is hypothesized to be through bites (12) . Humans become infected with hantavirus by inhaling aerosolized excreta from infected rodents (13) . Occasionally hantavirus pulmonary syndrome (14) develops; this syndrome has a mortality rate of almost 40% and no prophylaxis, treatment, or cure (15) . Most of the 506 confirmed cases in the United States have been caused by Sin Nombre virus (SNV). Studies have found that low diversity ecosystems dominated by the rodent hosts for 3 distinct hantaviruses had high infection prevalence in the host (16, 17) , suggesting a role for species diversity. Although the mechanism of disease dilution would differ in directly transmitted zoonoses (e.g., hantaviruses), as opposed to vector-borne diseases, a dilution effect could occur if 1) individuals of the host species remain as species diversity decreases, 2) the disease is spread within the host species through direct encounters (such as biting), and 3) presence of other species causes encounters among the host species to decrease.
Other ecological factors could affect the number of intraspecific deer mouse (Peromyscus maniculatus) encounters, including increased density of deer mice and vegetative factors that lead to variation in population numbers (e.g., available cover and forage) ( Table 1 ). Some studies have found high SNV prevalence in host populations when deer mice densities were high (18, 19) . However, although the concept of density-dependent transmission is not unique to hantaviruses, its applicability to the deer mouse-SNV system has been elusive. SNV prevalence also has been shown to vary with habitat characteristics and quality (15, 18, 19) , although interpretation of this variation has been difficult because SNV prevalence varies as much within as among habitat types (20) .
In this study we examined small mammal populations in 5 forested sites over a 3-year period, October 2002 through September 2005. We monitored mammal species diversity, deer mouse densities, and SNV infection prevalence in the mammals to test the hypotheses that 1) areas of higher mammal species diversity would exhibit lower prevalence of SNV infection in host populations, 2) areas of higher host density would contain higher infection prevalence of SNV in the host populations, and 3) vegetative factors could be related to prevalence of SNV infection among deer mice.
We sampled small mammals at 5 (21) .
To sample as many different mammal species as possible, we set up a trapping web 200 m in diameter (22) at each site and used 4 trap types: Sherman (H.B. Sherman Traps, Tallahassee, FL, USA), handmade wire mesh, Tomahawk (Tomahawk Live Trap Co., Tomahawk, WI, USA), and pitfall. Each station included an aluminum folding Sherman live trap and a custom-built mesh live trap (23) of similar dimensions (7.6 cm × 8.9 cm × 22.9 cm). Two sizes of Tomahawk live traps were used to trap larger animals; a 61 cm × 17.8 cm × 17.8 cm trap was placed at each 50-m trap station, and a 91.4 cm × 25.4 cm × 30.5 cm trap was placed at each 100-m trap station. Pitfall traps were made by using a 19-L bucket (30-cm All captured animals were treated as if they were infected with SNV, and standard precautionary methods were implemented (25) . After point of capture was recorded, animals were transferred from traps to sealable plastic bags or, if too large, left in the trap and brought to the center of the web, where they were weighed and measured and examined for age, sex, reproductive status, scarring, or other notable characteristics. Retroorbital blood samples were collected by using heparinized microcapillary tubes and either placed in cryovials and frozen in liquid nitrogen or placed in serum separator tubes and refrigerated for no more than 1 week before testing. Infection prevalence was determined by ELISA (26) . Infected deer mice were counted 1 time (time of first capture).
During the first 2 years of the study, to obtain tissue samples for a companion study, deer mice were euthanized in a chloroform chamber (25) . The resulting specimens were tagged and stored at the Museum of Vertebrate Biology at Portland State University. All other animals captured were marked with ear tags and released at the point of capture. During the last year of the study, deer mice were also tagged and released. To determine whether removal affected subsequent capture rates within the same trapping period, the differences between the number of captures on the first and last day of the trapping period were calculated and averaged, then compared between removal and replacement sampling with the Welch 2-sample t-test. Because no significant differences were found between the first 2 years and the last year of the study (t = 0.50, p = 0.63, df = 8), data from all 3 years were analyzed together. This research was conducted under the auspices of federal, state, and city permits, and it complied with the American Society of Mammalogists' guidelines for animal care and use (27) .
Deer mouse density was calculated by using the Distance program (28) . Mammal species diversity was measured by using the Simpson diversity index (D S ) (29) , which takes into account both richness (number of species) and evenness (number of individuals within each species) and ranges from 0 (least diversity) to 1 (maximal diversity). D S further represents the probability of interspecies encounters (30) . Pairwise comparisons of D S values among parks was conducted by using the Student t test; differences of D S were divided by the square root of their variances (30) . To minimize the possibility of type 2 errors resulting from multiple comparisons, a statistically conservative Bonferroni correction was made (α = 0.05/10 comparisons, or 0.005) (31) . Deer mouse densities were compared pairwise by using the Welch 2-sample t-test. Logistic regression with binomial errors was initially used to assess the association between infection prevalence and deer mouse density and species diversity. However, the resulting models showed such extensive overdispersion that we considered logistic regression to be an unsuitable statistical method for these data (32) . Accordingly, we used nonlinear regression analysis.
An analysis of similarity returns a statistic (R) based on a Bray-Curtis dissimilarity measure, which considers the difference of the mean ranks between and within groups. Most values fall between 0 and 1; 1 is the most dissimilar. Significance is assessed by comparing the observed value of R to the permutation distribution of R (33). Again, because of multiple comparisons, a Bonferroni correction was made such that α = 0.005 (31) . We then used stepwise (backward) logistic regression with binomial errors to assess the association between infection prevalence and vegetative characteristics.
Although only 5 sites were examined, the intensity of the sampling yielded a total of 5,057 individuals from 21 species, resulting in a thorough species inventory over a gradient of diversity in small mammal ecological communities. Deer mice averaged 62% of all captures (Table 2) and were the dominant species at all sites. Mammal species diversity differed significantly among sites (p<0.001; Table  2 ), except sites 3 and 4 (p = 0.1). Densities varied spatially and temporally; all parks exhibited the highest densities during year 2 (Table 3) . Interannual variances of densities were large due to seasonal differences in capture rates, such that no statistical differences in densities were found either within or among parks. Infection prevalence also varied, although it remained consistently low at 4 of the 5 sites.
During year 1, infection prevalence was significantly higher at site 1 than at sites 2 and 3 (p<0.001) but not different than at sites 4 and 5 (p = 0.20 and 0.32, respectively). Site 1 was the only site where infection prevalence significantly increased between years 1 and 2 (p = 0.005); thus, infection prevalence at this site was significantly higher than at any of the other parks during year 2 (p<0.001). High infection prevalence was maintained at site 1 during year 3. Although the rate for site 2 increased significantly between years 2 and 3 (p = 0.035), prevalence remained significantly higher at site 1 than at any other park during year 3 (p<0.01).
Using nonlinear regression, we found a significant negative relationship between infection prevalence and mammal species diversity. Infection prevalence increased as diversity decreased, up to an inflection point where the rate of infection increased exponentially (Figure) . No regression model was able to account for the association between infection prevalence and density of deer mice, either alone or with species diversity in the model.
A pairwise analysis of similarity was used to compare sites floristically; all parks differed significantly from each other (p<0.001). Stepwise backward logistic regression with binomial errors found no association between infection prevalence and any vegetative factors alone or in combination with other vegetative factors.
Population densities fluctuated synchronously at all sites, yet infection prevalence increased significantly at only 1 site, which suggests that factors other than density alone are involved in disease transmission. If, as hypothesized, transmission were through aggressive encounters (12) , SNV would spread most efficiently in an ecosystem composed solely of deer mice, where every encounter would be a potential disease-transmitting encounter. As more spe-cies, and more individuals within those species, are added to the community, the number of potential disease-transmitting encounters decreases because species other than deer mice are nonhost (not competent, or nonamplifying) species. This type of decreased intraspecies interaction has been termed "encounter reduction" (34) and would occur if increasing species diversity increases the number of competitors in an ecosystem, thereby increasing the amount of time a host species has to spend securing limited resources (food, nest sites), in turn decreasing the time spent on intraspecies encounters.
An increase in species diversity, in combination with an increase in the densities of individuals within those species, as we observed in this study, should also mean an increase in the number of predators of the rodent host species. It is reasonable to hypothesize that predators keep rodent numbers under control, in turn limiting pathogen spread both among rodents and into human populations, although it has been difficult to empirically support this hypothesis (35) . Our results suggest that predators control infection prevalence not by controlling the density of host species but instead by an unrelated mechanism, possibly encounter reduction. When predators are present in the ecosystem, host species should spend more time in the nest, in hiding, or within the familiarity of their territory, all to avoid predation and all likely to decrease intraspecies encounters. This hypothesis is supported by the fact that capture ratebut not density-was highest at site 1 during year 2 relative to all other parks (p<0.01), which means that deer mice were moving about and encountering traps more often. We hypothesize that when predation and competition are decreased or absent, for this small mammal community at a Simpson diversity index ≈0.43, a zoonotic release of predatory and competitive controls appears to have occurred, in which SNV infection prevalence increased drastically. This hypothesis would account for the lack of differences in infection prevalence rates at sites 2-5; although the Simpson index for these sites varied significantly; the threshold for zoonotic release had not been breached at any of those sites. Above the threshold level, sites would maintain a low level of infection, or perhaps locally lose infection altogether. In this study, SNV infection prevalence was either so low during some seasons at some sites as to be virtually undetectable by traditional trapping techniques or ephemerally absent. In particular, SNV was undetected or absent most often at the most diverse site (no SNV was detected in 8 of 12 seasons at site 5, in 6-7 seasons at sites 2-4, and in 1 season at site 1). Our system differs from the Lyme disease system, which depends on a vector that is not host specific (black-legged tick) to transmit the disease. Here, in contrast, presence of nonhost species in the small mammal community will not directly affect the transmission of SNV; instead, the behavior of members of the natural host species will be affected, decreasing SNV transmission rates among competent hosts through encounter reduction. Increased diversity in both the Lyme disease and SNV systems appears to lead to decreased disease prevalence, although the mechanisms differ. Another difference between the 2 systems is the threshold relationship between species diversity and SNV prevalence, which suggests that the shape of the dilution curve may be mechanism dependent and is the reason we proposed the term "zoonotic release." Given that many hantaviruses are hosted by generalist rodent species (i.e., those able to exploit a broad variety of ecological resources) that dominate ecosystems as species diversity decreases (e. Host density should likewise be considered a factor in this phenomenon because density increased before infection prevalence increased. However, the result of the logistic regression between density and infection prevalence, although significant, was marked by considerable overdispersion, suggesting that this was the wrong model, and its significance was greatly overestimated (32) . Additionally, at all parks deer mouse density increased but infection prevalence did not, clearly indicating that density is not the sole driver of infection prevalence in this system. A logistic regression with both density and mammal species diversity in the model showed similar overdispersion. Our results suggest that dependence on both density and frequency play a role in SNV transmission, which may be one of the reasons it has been so hard to determine their respective roles in the transmission of hantaviruses (36) . More extensive studies should therefore be undertaken wherein species diversity, density, and frequency of encounters are carefully measured to determine their respective roles in disease transmission.
The finding that infection prevalence of a directly transmitted zoonosis may be inversely related to species diversity has implications for human health. The toll in illness and death from emerging zoonotic diseases is high, and outbreak investigations are costly (37) . These investigations often fail to identify the source of a pathogen, let alone answer the question of why an outbreak occurred at a given time and place. If the host species or vector is found, eradication usually is neither possible nor desirable, particularly when the species are as ubiquitous as deer mice. Prophylaxis is difficult when transmission is airborne, as in hantaviruses, for which potentially everyone in a region is at risk. Ecosystem-level control may be the best way to protect the public from the increasing threat of many zoonotic diseases. Wildlife also are at risk for infection with novel pathogens, and the factors underlying wildlife disease emergence are similar to those in humans (38) ; a dilution effect may therefore help protect wildlife as well. For example, a study of West Nile virus suggested that increased bird species richness depressed the prevalence of the virus in ecosystems (39) . Thus, wildlife could be protected in 2 ways: first, from dilution of diseases that are potentially harmful to them and second, from maintenance of healthy ecosystems.
Extension of a dilution effect to directly transmitted diseases has implications for conservation as well. Although protecting species diversity is a cause that would seem universal in its appeal, conservationists often are perceived as being overly biocentric and having little concern 1016 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15 for human welfare. In addition, many benefits derived from maintaining diverse ecosystems are difficult for the layperson to decode and seem far removed from daily life such that despite scientific research, unparalleled loss of species caused by anthropogenic factors continues at an unabated rate. Conservation likely will not succeed without the support of the general public, who in turn influence the environmental policies our society embraces. To gain support of the general public, tangible human benefits from conservation should outweigh the immediate-usually economicgains of nonconservation land use (40) . Linking human health to biodiversity could be just the benefit for gaining the public's support of conserving biodiverse ecosystems. Protection from disease is a tangible objective; it is easily understood and translated and it has direct benefits for all. As a consequence, extension of a dilution effect to directly transmitted diseases could have broad conservation implications by raising the public's concern about conservation in a manner that has yet to be emphasized. Chinese-like Strain of Porcine Epidemic Diarrhea Virus, Thailand Since late 2007, several outbreaks of porcine epidemic diarrhea virus (PEDV) infection have emerged in Thailand. Phylogenetic analysis places all Thai PEDV isolates during the outbreaks in the same clade as the Chinese strain JS-2004-2. This new genotype PEDV is prevailing and currently causing sporadic outbreaks in Thailand. , is an enveloped, single-stranded RNA virus belonging to the family Coronaviridae. The PEDV genome contains genes for the following proteins: pol1 (P1), spike (S) (180-220 kDa), envelope (E), membrane (M) (27-32 kDa), and nucleocapsid (N) (55-58 kDa) (2) . The M protein is a structural membrane glycoprotein, which plays an important role in the assembly process; the S surface glycoprotein harbors the specific host cell receptor binding sites (3) .
During late 2007, the PED outbreak appeared first in Nakornpathom province before spreading throughout the country. Pig losses from the recent PED outbreaks were extensive. Obvious clinical signs were severe diarrhea (Figure 1 , panel A) and dehydration with milk curd vomitus in suckling piglets. Most of the affected farms reported the disease first in farrowing barns and subsequently lost 100% of newborn piglets. Pigs of all ages were affected and exhibited degrees of diarrhea and inappetite, which varied by their ages. Boars and sows had mild diarrhea and anorexia for a few days and recovered within a week. In piglets that died, the small intestinal wall was congested and intestinal contents were watery with undigested milk curd ( Figure 1, panel B). Segmental enteritis was indicated by segmental disappearance of intestinal lacteal caused by malabsorption in affected intestinal parts ( Figure 1 , panels C and D). Atrophic enteritis, characterized by blunting of the intestinal villi and sloughing of intestinal epithelium, occurred in all affected piglets (Figure 2 , panel A). Immunohistochemical tests, performed by using monoclonal anti-PEDV S protein (JBT Biotechnology Laboratory, Seoul, South Korea), demonstrated dark brown staining in intestinal epithelial cells (Figure 2, panel B) . Massive feedback of piglet feces and minced piglet guts to gestating sows was recommended by local veterinary practitioners to prime the sow's immune response and pass protective immunity to the piglets. At affected farms, the outbreak lasted <3 weeks.
Samples from 8 provinces (24 farms) in Thailand from December 2007 through March 2008 were submitted to the veterinary diagnostic laboratories of Kasetsart University and Chulalongkorn University. A total of 33 porcine samples were confirmed as positive for PEDV by reverse transcription-PCR (RT-PCR) (4) before virus isolation (Table) . Published primers (5) were used for generating the PEDV 651-bp partial S gene. Primers were designed to amplify the PEDV M gene and yielded the amplified product of 715 bp on the basis of CV777 and Br1/87. Products were purified by using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) and were sequenced by 1st BASE Pte Ltd (Singapore).
Nucleotide and deduced amino acid sequences of the 33 PEDV isolates were aligned, edited, and analyzed with ClustalX version 1.83, Bioedit version 7.0.5.2, and MegAlign software (DNAStar Inc., Madison, WI, USA), respectively. Phylogenetic trees were generated by using partial S and full-length M genes, including the deduced amino acid sequences with selected reference PEDV strains, by applying the Jotun Hein method in the MegAlign software. To assess the relative support for each clade, bootstrap values were calculated from 1,000 replicate analyses.
The M gene sequence analysis of 31 PEDV isolates obtained in Thailand indicated that the nucleotide sequence of the entire M gene was highly conserved. All recent PEDV isolates in Thailand had 99.3%-100% nucleotide homology. The lowest sequence identity (96.5%) was with the Chinese strain, EF185992/LZC, and the highest sequence identity (99.2%-99.7%) was with the Chinese strain, JS-2004-2, and concurrent isolates from the National Institute of Animal Health, Thailand, M_NIAH 07-08 (data not shown).
All 33 PEDV isolates had 97.0%-98.8% DNA sequence identities of the S gene with each other. Our findings demonstrated that the recent PEDV isolates in Thailand were genetically diverse in their S genes either within Our results indicated that the recent Thai PEDV isolates clustered in the same group were highly homologous with the Chinese strains, JS-2004-2 and LJB/03. They were responsible for the recent PED outbreak in Thailand and able to produce pathologic effects similar to the Chinese isolates (6) . Notably, 08NP04, isolated 4 months after the first outbreak, had the highest identity to JS-2004-2. Also, 08NP04 and 07NP01 (the first isolates in 2007) originated in the same geographic area. The Chinese-like strain of the virus might have gained entry into Thailand via unknown routes as early as December 2007. In addition, rendering trucks traveling from farm to farm might have encouraged widespread transmission of the disease. Structural differences in the partial S gene could help elucidate pathogen- esis and antigenic structures of the recent PEDV isolates because S glycoproteins are responsible for inducing the virus neutralizing antibodies and known to be highly conserved in PEDV strains (7) . Continuing investigation of PEDV isolates will contribute to the prevention and control of this virus.
The phylogenetic relationship of the Thai PEDV strain indicated that the recent Thai PEDV isolates differed genetically from previous Thai isolates. Despite precautions, sporadic outbreaks continue to occur. In addition, disease transmission frequently occurs due to the purchase of new stock with improper gilt acclimatization and biosecurity. Immunity induced through vaccination, currently unavailable in Thailand, does not provide lifelong protection from this virus. However, vaccination is recommended to encourage specific immunity to PEDV in all stock when an acute outbreak occurs. Undoubtedly, effective biosecurity is a key management tool for PED prevention and control.  Optimism/pessimism and health-related quality of life during pregnancy across three continents: a matched cohort study in China, Ghana, and the United States BACKGROUND: Little is known about how optimism/pessimism and health-related quality of life compare across cultures. METHODS: Three samples of pregnant women in their final trimester were recruited from China, Ghana, and the United States (U.S.). Participants completed a survey that included the Life Orientation Test - Revised (LOT-R, an optimism/pessimism measure), the Short Form 12 (SF-12, a quality of life measure), and questions addressing health and demographic factors. A three-country set was created for analysis by matching women on age, gestational age at enrollment, and number of previous pregnancies. Anovas with post-hoc pairwise comparisons were used to compare results across the cohorts. Multivariate regression analysis was used to create a model to identify those variables most strongly associated with optimism/pessimism. RESULTS: LOT-R scores varied significantly across cultures in these samples, with Ghanaian pregnant women being the most optimistic and least pessimistic and Chinese pregnant women being the least optimistic overall and the least pessimistic in subscale analysis. Four key variables predicted approximately 20% of the variance in overall optimism scores: country of origin (p = .006), working for money (p = .05); level of education (p = .002), and ever being treated for emotional issues with medication (p < .001). Quality of life scores also varied by country in these samples, with the most pronounced difference occurring in the vitality measure. U.S. pregnant women reported far lower vitality scores than both Chinese and Ghanaian pregnant women in our sample. CONCLUSION: This research raises important questions regarding what it is about country of origin that so strongly influences optimism/pessimism among pregnant women. Further research is warranted exploring underlying conceptualization of optimism/pessimism and health related quality of life across countries. The psychosocial constructs of optimism and pessimism have been under study for several decades. Optimism is associated with more active coping strategies, lower levels of psychological distress [1] [2] [3] [4] [5] , health-enhancing behavior [6] , higher immune functioning [7] , better health outcomes [8] and even lower mortality. [9] [10] [11] On the other hand, pessimism has been shown to have prophylactic effects in certain circumstances. In particular, pessimism can insulate people from the psychological consequences of failure, including anxiety, depression, and diminished self-esteem. [12] Thus, the impact of optimism and pessimism is potentially enormous yet still very unclear.
In this context, even less is known about differences across cultures. While several studies have shown levels of optimism/pessimism to vary across cultures, [1, [13] [14] [15] [16] [17] [18] [19] findings have been inconsistent in terms of which cultural groups are more or less optimistic. No research to date has compared Asian, African, and Western cultures in the same study.
Recent research among pregnant women has yielded interesting, albeit similarly inconsistent, findings. Lobel et al. [8] found that women who were the least optimistic had babies of the lowest birthweight, even when controlling for gestational age. Moyer et al. [20] found that optimism/pessimism among Ghanaian pregnant women was inversely associated with knowledge of HIV and previous HIV testing. In other words, those who were not tested prior to their pregnancy and had the least knowledge of HIV were the most optimistic. The authors also found that, when compared to a similarly aged sample of nonpregnant women in the United States (U.S.), the Ghanaian women were significantly more optimistic. [20] The construct of health-related quality of life is one variable that has been linked to optimism/pessimism in past research. [21] In one study, researchers found that, even when health status was controlled, pessimists had significantly worse health-related quality of life (HRQOL) scores than optimists or so-called "realists." [21] In that study, pessimists were those who expected disproportionately negative outcomes associated with their Hepatitis C diagnosis, optimists were those who expected few negative outcomes associated with their Hepatitis C diagnosis, and realists were those who had a fairly accurate perception of the impact Hepatitis C was going to have on their lives. Optimists' HRQOL scores in this study mirrored the scores of the general U.S. population, even though the population being studied (chronic hepatitis C patients) has been shown to have significantly lower QOL than the general population.
The results of the research to date suggest that further examination of the cross-cultural issues in optimism/pes-simism and health-related quality of life among pregnant women is warranted. This research was undertaken to explore the differences among a three-sample matched cohort of pregnant women at the same stage in their pregnancies in Ghana, China, and the U.S. The specific aims of this research were to 1) identify if and to what extent optimism and pessimism vary across similar populations of pregnant women in three different countries; 2) determine if and to what extent self-assessed quality of life scores vary among similar populations of pregnant women in three different countries, and 3) determine if optimism and/or pessimism is predictive of or associated with current self-perceived health status and/or selfassessed Health-Related Quality of Life (HRQOL) and how that might vary by culture.
China Data were collected from women presenting for prenatal care at the obstetric outpatient clinic at the Peking University First Hospital between May and July 2006. As one of the largest and most well-known academic medical centers in Beijing, Peking University First Hospital draws both public and private patients from in and around Bejing. Clinics average 600 pregnant women per week and 3000-3500 deliveries per year.
Data were collected from women presenting for prenatal care at the Obstetrics and Gynecology Clinic at the Noguchi Research Institute/Medical School at the University of Ghana in Accra, Ghana, between May and July 2005. This facility is housed in a public hospital that is also the largest government hospital in Ghana. Patients from all over Ghana travel to this clinic to receive their care. Clinics average 500-600 pregnant women per week and 10,000 -12,000 deliveries per year. 
At all three sites, pregnant women in their last trimester of pregnancy who were 18 years old or older were asked to participate in this research. Women facing an imminent health crisis or those in active labor were excluded. At all three sites, research assistants talked patients through an informed consent form. Translators were used when nec-essary. Surveys were administered verbally to all participants in Ghana as part of a larger study. [20] In China and in the U.S., surveys were designed to be self-administered, but women were given the option to have the survey administered verbally. (None chose this option.)
The instruments used for the survey in each study location varied slightly, but for the purposes of this analysis, each study site used three key instruments -a demographic questionnaire, the Life Orientation Test (LOT-R), and the Short form 12, or SF-12. The instruments were pilot tested separately in each location, and minor modifications were made to ensure comprehension and comparability across sites. In China and Ghana, the instruments were translated into the dominant language of the region and backtranslated into English by native bi-lingual speakers. The original and back-translated versions were compared for consistency, and any inconsistencies were resolved by discussion and consensus among the research team.
A Demographic and Health Questionnaire was used to measure patient characteristics that may be associated with optimism, pessimism, HRQOL, or pregnancy outcomes. These include age, number of pregnancies, other medical conditions, previous treatment for mental health problems such as depressed mood or anxiety, previous use of anti-depressants, and self-perceived health status. Women were also asked to rate their perception of the difficulty of their pregnancy on a scale of 1 to 4, with 1 being "extremely easy" and 4 being "extremely difficult."
The Life Orientation Test -Revised (LOT-R) [22] is a revision of the original Life Orientation Test [23] . It assesses optimism/pessimism using a series of questions that inquire about an individual's attitudes in daily life. This instrument has been widely validated [24] and used in both Ghana [25] and China. [14] Its 10 items generate an overall score, as well as two possible subscales: affirmation of optimism and affirmation of pessimism. The participant answers each item based on a 5-point scale, with response options ranging from strongly disagree to strongly agree. A higher score relates to a greater level of optimism.
The Short Form 12, or SF-12, [26] is a 12-item quality of life instrument derived from the Short Form 36, or SF-36, an instrument used and validated around the world to determine self-assessed health-related quality of life (HRQOL). The SF-36 and shortened SF-12 generate not only summary scales of mental and physical functioning (MCS and PCS), but also a profile of patients' HRQOL across eight domains: physical functioning (PF), role physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role emotional (RE), and mental health (MH). According to SF-36 developers, 12 of the original SF-36 items accounted for at least 90% of the variance in PCS-36 and MCS-36 in both general and patient populations, and those same 12 items reproduced the profile of the eight SF-36 health concepts sufficiently for studies in which the length of the instrument may be prohibitive.
All research protocols and survey instruments were reviewed and approved by the institutional review boards at the U.S. and foreign institutions participating.
Pregnant women presenting for prenatal care were approached and asked to participate in a research study. If they expressed an interest, women were asked how far along they were in their pregnancies. Those in their final trimester were taken through a consent form. A research assistant (and translator, when necessary) answered any questions the participant might have and made sure women had a copy of the consent form to keep.
Data were gathered using paper and pencil forms (China, U.S.) and verbal interviews (Ghana). No identifying information was collected from Ghanaian participants, given that collecting post-delivery follow-up data was anticipated to be extraordinarily difficult and thus was not attempted. Hospital registration numbers were collected from Chinese and U.S. patients to allow for post-delivery follow-up for a separate research protocol than that described here. Hospital registration numbers were removed from the original survey and replaced with a unique ID number once the registration number was recorded in a separate location for follow-up purposes.
Responses from the hard copies of the self-administered surveys (China and the U.S.) and the interviewer-administered surveys (Ghana) were entered into an Excel spreadsheet and cleaned.
Cleaned data from each site (China, N = 251; Ghana, N = 101; U.S., N = 311) was combined into one large dataset. A data subset was created by matching women on three key variables: maternal age, number of weeks pregnant at the time of enrollment, and number of previous pregnancies. Women were matched within 3 years of age (average age difference across matched sets = 0.85 years); within 1 pregnancy (average difference in number of pregnancies across matched sets = 0.48); and within 5 weeks of gestational age (average difference in gestational age across matched sets = 2.66 weeks). This matching schema was undertaken to attempt to create a sample that was as similar as possible on key variables that could influence optimism/pessimism. Previous research has suggested that age can be related to optimism/pessimism [27] , as well as number of previous deliveries [20] . We also postulated that women at different stages of pregnancy might feel differently in terms of optimism/pessimism, and thus wanted to reduce the impact of stage of pregnancy on our results. Given the one child policy in China, "number of previous pregnancies" was used as a key variable rather than number of previous deliveries. This resulted in a matched sample of 168 women, 56 in each country, that were predominantly nulliparous. We refer to these threeway matches as "matched sets" throughout the manuscript.
Frequencies and descriptive statistics were calculated. Means across the three groups were compared using ANOVA with post-hoc pairwise comparisons. In addition, multivariate linear regression analysis was used to create a model to identify the factors most strongly contributing to the overall LOT-R scores. Variables that were significantly associated with the LOT-R in univariate analysis were entered into the multivariate model. The model was reworked using only those variables that maintained significance until the best-fit model was identified. Interactions between key variables were examined as well. A pvalue of .05 was taken to be statistically significant. Table 1 illustrates the demographic characteristics of women in the sample. Note that two of the three variables upon which women were matched (age and number of previous pregnancies) were not significantly different (see Table 2 ). In fact, the average difference in age across the matched sets was 0.857 years, with a maximum difference of 3 years. The average difference in number of previous pregnancies was .482, with the highest difference being 2.0, reflected in only 4 of the 56 sets. Women from China were enrolled significantly later in their pregnancies than women in the U.S. (p = .04, mean difference of 1.52 weeks), yet the average difference in gestational age across the matched sets was 2.66 weeks.
All of the women in the Ghanaian sample were married, as were a majority from the China sample (98.2%), and in the U.S. sample, a smaller yet significant percentage of the women (76.8%) were married. Educational variables were assessed slightly differently in Ghana than in China and the U.S., making it difficult to compare across countries. That said, it is clear that the U.S. sample included women with higher levels of education than both China and Ghana (p < .001). Ghanaian women in our sample had Table 2 illustrates the health-related variables assessed across the three samples. Note that while the number of pregnancies was not significantly different across the three groups, the number of previous deliveries was significantly different at p < .001.
Women from our sample in China reported having significantly easier pregnancies than women from our Ghanaian and U.S. samples (p < .001), and far more women in the U.S. sample report ever seeing a healthcare professional for help with emotional issues (p < .001). Table 3 ). Both the LOT-R optimism subscale and the LOT-R pessimism subscale indicate significant differences across country samples as well. (See Figure 1 .) Figure 2 illustrates differences in self-assessed healthrelated quality of life across the samples. Physical Functioning (PF) was significantly different across the country samples (ANOVA, p = .049), but the difference was primarily between Ghanaian and U.S. samples (post-hoc pairwise comparisons, p = .038). General Health (GH) was significantly different as well (ANOVA, p = .001), and here the difference was primarily between Chinese and Ghanaian samples (p = .001). Vitality (VT) showed significant differences across country samples as well (ANOVA, p < .001), with pronounced differences between Chinese and U.S. samples (p < .001) and Ghanaian and U.S. samples *Ns may total more than 56. Two separate questions were asked: "Have you ever been treated... yes/no" and "Are you currently being treated...yes/no " Thus the number "currently being treated" is a subset of the number who have "ever been treated." ANOVAs were conducted using the "ever treated" and "never treated" groups for comparison.
(p < .001), but not Chinese and Ghanaian samples. Finally, Role Emotional (RE) scores were also significantly different across country samples (ANOVA, p < .001), but the difference was most pronounced between Ghanaian and U.S. samples (p < .001).
Among all the women across the three samples, univariate analysis indicated that overall LOT-R optimism/pessimism scores were significantly associated with country of origin (p = .001; Ghanaian women sampled had the highest scores, U.S. women sampled had scores in the middle, Chinese women sampled had the lowest scores), educational attainment (p = .046; optimism increased with education level), whether women worked for money (p = .006; working women were more optimistic than nonworking women), and number of previous deliveries (p = .004; those with the fewest and the most deliveries were the most optimistic -those with 2 previous deliveries were the least optimistic).
Women who had never seen nor were currently seeing a health care provider for emotional issues were more optimistic than those who had ever seen or were currently seeing a health care provider for emotional issues. (p = .002, p = .001 respectively) Treatment for mental health issues with medication was another variable that was associated with LOT-R scores: those ever treated and those currently being treated with medication were significantly less optimistic than those who had never been treated. (p < .001, p = .005 respectively)
Women's self-reported experience of the ease or difficulty of the current pregnancy was also associated with LOT-R scores (p = .007). Those who reported having the easiest pregnancies had the highest levels of optimism.
LOT-R scores were positively correlated with SF-12 mental health summary scores (p = .001), vitality subscale scores (p = .041), and mental health subscale scores (p < .001).
The LOT-R optimism subscale was significantly associated with the same scales (MCS, p = .018; VT, p < .001; MH, p = .001), as well as the general health subscale (GH, p = .002). The LOT-R pessimism subscale was associated with the mental health summary score (p = .013), the role physical subscale (p = .032), the role emotional subscale (p = .034), and the mental health subscale (p = .038).
Note that LOT-R scores were not associated with the selfreported presence of any ongoing health issues.
Multivariate linear regression analysis indicated four key variables that predicted approximately 20% of the variance (adjusted R square = .199) in overall LOT-R scores: country of origin (p = .015), working for money (p = .025), level of education (p = .001), and ever being treated for emotional issues with medication (p = .002). No significant interactions were identified.
These data suggest that in a three-country cohort of pregnant women matched on age, number of weeks pregnant, and number of previous pregnancies, significant differences existed with regard to both optimism/pessimism and health-related quality of life. According to the LOT-R, Ghanaian pregnant women in our sample were the most optimistic and Chinese women in our sample were the least optimistic of the matched sets. Interestingly, Ghanaian women sampled also reported higher levels of pessimism than Chinese and U.S. women sampled. This is consistent with research that suggests optimism and pessimism may not be mutually exclusive constructs. [28] Physical functioning, general health, vitality, and role emotional subscales of the SF-12 also indicate significant differences by country per our samples -U.S. women sampled had the lowest physical functioning and vitality scores, Ghanaian women sampled had the highest physical functioning and general health scores, and Chinese women sampled had the lowest general health scores and highest vitality scores.
LOT-R Optimism/Pessimism Scores by Country (*ANOVA: p < 0.001) Figure 1 LOT-R Optimism/Pessimism Scores by Country (*ANOVA: p < 0.001).
LOT-R = overall optimism; higher score = greater level of optimism LOT-OPT = optimism subscale; higher score = greater level of optimism LOT-PESS = pessimism subscale; higher score = greater level of pessimism Correlates of optimism/pessimism included country of origin, educational attainment, working for money, number of previous deliveries, and having ever seen a healthcare provider for emotional issues. Self-reported ease or difficulty of the pregnancy was also associated with LOT-R scores.
Perhaps most striking in these findings is that we were able to create a multivariate linear regression model that predicted approximately 20% of the variance in overall LOT-R scores. The key variables were country of origin, working for money, level of education, and ever being treated for emotional issues with medication. What is particularly fascinating about this model is that country of origin somehow remains a strong factor in the modeleven with variables that might be seen as proxies for country of origin (education, working for money, ever being treated for emotional issues). It raises a question about the potential differences that underlie being Ghanaian, Chinese, or American that yield differential LOT-R scores.
It also raises questions about what accounts for the remaining 80% of the variance. Further research with more comprehensive instrumentation is necessary to begin to answer these questions.
There are several limitations to this research. First, the study design does not allow for the definitive determination of a causal relationship -we can only report observed associations. Second, in each setting, the women recruited represented a convenience sample from one hospital in one city, minimizing the ability to generalize based upon our findings. Third, we created our matched sets based on three variables we believed were most important to control for at the outset: maternal age, gestational age, and number of previous pregnancies. Given the one child policy in China, we did not match on number of previous deliveries -as that would have eliminated all but the nulliparous women in Ghana and the U.S. and reduced our sample size to an untenably low level. However, upon analysis it was discovered that the number of previous deliveries was significantly different across countries. In China, women were more likely to have had a previous pregnancy that did not result in a delivery. Whether these were induced or spontaneous terminations is not known, although previous research indicates that more than half of women in China have had at least one abortion. [29] It is possible that Chinese women who have had the same number of pregnancies as their African and North American counterparts but have not had as many deliveries are in some way inherently different. Our univariate analysis did indicate that number of deliveries was associated with overall LOT-R scores (p = .004), but this significance was not sustainable in the multivariate logistic regression model.
Note as well that the samples differ significantly on a few key variables. First is in terms of educational attainment. In Ghana and China, approximately a third or respondents were high school graduates or less, compared to only seven percent in the U.S. Our data suggest that educational attainment is associated with optimism/pessimism and health-related quality of life. What is less clear is through what mechanism. Further research is needed to disentangle these associations. The second key difference in the samples is related to their reports of current and previous treatment for emotional issues. While nearly 40% of U.S. women in our sample reported ever seeking care for emotional issues, only 5% of Chinese women in our sample said the same. While this may reflect true differences in need for mental health care, more likely it reflects social norms and access issues surrounding treatment for mental health issues. Nonetheless, such differences are indicative of the likely many other variables that would differ across cultures that were not measured in this study and that might have an effect on optimism/pessimism or selfassessed health-related quality of life. Finally, the samples were likely to be very different in terms of standard of living. Women in Ghana were recruited from a public hospital, suggesting lower income participants, whereas women in China and the U.S. were either 'public' or 'private,' depending upon the type of insurance being used. We attempted to assess income to sort out these differences in a meaningful way, but there were numerous challenges. The first is the distinction between family and individual income. In China, family income may or may not include extended family. There may be four -or even six -adults in one household, awaiting the arrival of a single child. In Africa, family income was challenging for some respondents to answer, whether due to lack of knowledge or the challenge of translating non-currency income into survey response options. In addition, assuming for a moment that our survey instrument accurately collected and recorded a representative gradient of incomes in each culture, how does one compare being 'low income' in the U.S. versus being 'low income' in sub-Saharan Africa? The combination of missing data and uncertain interpretation of our income-related items made meaningful comparisons challenging at best, misleading at worst, thus income data were removed from analysis.
Another limitation to this research is that we were unable to suitably control for or verify "difficult" pregnancies. We did not have independent corroboration of women's reported symptoms or reported ratings of the ease or dif-ficulty of their pregnancies. Our sole measure of the ease or difficulty of women's pregnancies was their self-report. That said, the LOT-R measures what is referred to as dispositional (trait) optimism/pessimism -or something that is seen as relatively stable over time. Other measures of situational (state) optimism may have been more responsive to changes over time and may have been influenced by easy or difficult pregnancies. However, for the purposes of this research, we believe that a measure of dispositional optimism/pessimism is the most appropriate and that it should not have been differentially influenced by women's pregnancies. This is corroborated by the finding that despite reporting the easiest pregnancies of the three groups of women, Chinese women in our sample reported the lowest levels of optimism.
Another limitation to this research is the uncertainty surrounding the comparability of the constructs of optimism/pessimism across cultures. In a review of Chinese folk wisdom of behavioral health, researchers cite that for Chinese subjects, being optimistic means to be able to accept one's current life conditions positively rather than to expect good things to occur in one's life. [30] This is a more present-focused interpretation of optimism, rather than the future-focused interpretation most commonly used in the West. In the West, optimism is defined as the expectation that good things will happen to you in the future. Given these potentially contradictory definitions, future research needs to identify and clarify definitions of optimism and pessimism as commonly understood in different areas of the world.
Similarly, it is unclear how Ghanaians would articulate the concept of optimism. Research in Africa has shown that optimism has been inversely correlated to income and a number of other indicators of higher standards of living and positively correlated with a number of variables associated with deep poverty. [31] This finding -if realraises several critical questions. Are those who are optimistic in the midst of poverty the ones that survive? Is optimism a result of the perception that if one is extremely poor it can't get much worse, so it must be getting better? Are those with higher incomes and better standards of living so afraid of losing it all that they steel themselves against that potential by assuming the worst? Clearly more research is needed to begin to tease out some of these issues.
One final limitation is the inconsistency of data collection methodology across sites. Data were colleted verbally in Ghana, whereas data were collected via self-reported surveys in China and the U.S. We do not believe this difference significantly impacted the outcome variables in question, however it is possible there may be differences attributable to the method of data collection. 
In conclusion, these results raise some critical questions worthy of further exploration. Rigorous studies of crosscultural differences are not only methodologically, but also logistically, very challenging. That said, research needs to address the question of whether the underlying constructs of optimism/pessimism and health related quality of life are conceptualized similarly across cultures. In addition, further research is warranted that explores the potential link between psychosocial constructs such as these and measurable health outcomes. Only then can researchers and practitioners explore the possibility of interventions to influence what have typically been seen as stable constructs. Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecularbased diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.
Validated diagnostic tests that confirm the presence of rabies virus or a lyssavirus variant have been the foundation of rabies control strategies in many countries. Historically, histopathological techniques such as the Sellers Stain technique [1] were used to determine the presence of Negri bodies as rabies virus-specific antigen, however due to poor sensitivity and specificity this technique is no longer recommended by the World Health organization (WHO). The Fluorescent Antibody test (FAT) [2] relies on the ability of a detector molecule (usually fluorescein isothiocyanate) coupled with a rabies specific antibody forming a conjugate to bind to and allow the visualisation of rabies antigen using fluorescent microscopy techniques. Microscopic analysis of samples is the only direct method that allows for the identification of rabies virus-specific antigen in a short time and at a reduced cost, irrespective of geographical origin and status of the host. It has to be regarded as the first step in diagnostic procedures for all laboratories. Autolysed samples can, however, reduce the sensitivity and specificity of the FAT. The Rabies Tissue Culture Infection Test (RTCIT) [3] and the Mouse Inoculation Test (MIT) [4] are based on the propagation and isolation of the virus. These diagnostic tests are used to detect virus particles either directly in tissue samples (FAT) or indirectly in animals and in tissue culture (MIT and RTCIT, respectively). The rationale for the use of virus isolation (RTCIT/MIT) from a sample where there is a suspicion of infection with rabies virus is always recommended, especially when Koch's postulates are likely to be met. Such amplification of the viral pathogen facilitates additional molecular analysis to be undertaken, including sequencing of the viral isolate and subsequent phylogenetic analysis. Conventional diagnostic tests for rabies (FAT, RTCIT, MIT) are not labour intensive and rely upon low throughput. The FAT can be completed in less than two hours. In contrast, both the RTCIT and MIT require longer turnaround times (4-days and 28-days, respectively).
The fluorescent antibody virus neutralisation (FAVN) test [5] and the Rapid Fluorescent Focus Inhibition Test (RFFIT) [6] utilise a similar principle, to measure the level of virus neutralising antibody in vaccinated individuals. 'Indirect' serological methods, including the FAVN and RFFIT measure the host response to infection/vaccination only and do not detect the presence of infectious virus/antigen directly. However, host antibody detection (FAVN/RFFIT) is an indirect tool to measure the presence of rabies virus in a non-immunised individual by evaluating the host response to infection. The test may lack sensitivity and specificity, and the interpretation of the test results may be difficult as the host response to infection varies substantially between individuals. As such, the negative predictive value of serological tests for rabies diagnosis is considered poor. Therefore, serological assays are not suitable as diagnostic tools for routine rabies testing.
These internationally approved methods have provided accurate and timely information of animal and human rabies cases thereby supporting surveillance for rabies and providing a greater understanding of the epidemiology of this disease (Box 1). For numerous laboratories in rabies-endemic regions in the developing world, cost and simplicity are critical factors in the delivery of disease diagnosis and cannot be neglected, even when the principal consideration is for rapid diagnosis. Therefore, cost and simplicity need to be considered if new technologies are to be adopted in the regions of the world where they are most needed.
Molecular tools based on the detection and manipulation of the genetic information of the virus are becoming more widely accepted and accessible for the diagnosis of rabies. The advent of molecular biology is changing the face of diagnostic virology generally enabling high throughput and short turnaround-time analysis of samples. In the 21 st century, it is expected that diagnostic virology techniques for high throughput rabies virus detection will progress rapidly towards the use of molecular diagnostics replacing more conventional testing techniques such as virus isolation and histopathology. It is also possible that immunological tests, measuring 'surrogate' markers such as cytokines and electrolytes, will augment the standard diagnostic approach; nevertheless they will continue to remain oddities outside the realms of the routine diagnostic laboratory and be confined to a few reference laboratories. Semi-automated or automated instruments and robotics-based techniques are useful when large numbers of the same test are undertaken and these tests will continue to increase in popularity and use, especially in central reference laboratories rather than in each local or regional facility. New technological advances will undoubtedly be faster, more accurate and may, in time, offer a cost-effective alternative to traditional rabies diagnostic tests. These paradigm shifts including modern advances in technology will lead to the effective control of rabies in animals and wildlife [7] (Box 2). This review provides information on some of the latest developments and diagnostic techniques for determining the presence of rabies virus or nucleic acid in diagnostic samples.
The principal focus of this review is to highlight the new developments in virology related to techniques for the diagnosis and surveillance of rabies. Literature reviews were identified through Web of Science, PubMed and Scopus using various search phrases. This review also drew on information provided to international organisations, mainly WHO and OIE, funded by the UK Department for Environment, Food and Rural Affairs (Defra) in an advisory context on diagnostic and surveillance strategies for rabies. This review however, does not reflect the views of Defra, WHO or OIE. This review provides information on some of the latest developments and diagnostic techniques for determining the presence of rabies virus in diagnostic samples. Our aim is to provide a viewpoint on the current thinking in diagnostic virology for rabies, reflecting the 'neglected' nature of this tropical disease and the contrasting needs of diagnostic laboratories in developed and developing countries.
Development of Rapid Immunohistochemical Test (dRIT) in the evaluation of suspect rabies tissue samples A direct Rapid Immunohistochemical Test (dRIT) for the postmortem detection of rabies virus antigen in brain smears has been developed [8] . Using a cocktail of highly concentrated and purified biotinylated monoclonal antibodies, rabies antigen can be detected by direct staining of fresh brain impressions within 1 hour. This test employs anti-rabies monoclonal antibodies specific for the nucleoprotein, a viral protein produced in abundance during productive infection. The FAT is based on antibodies specific for the same protein but, being conjugated to fluorescein isothiocyanate, requires a fluorescent microscope to visualise any specific antibody bound to viral protein within the test sample [2] . In contrast, the new Box 1. Key Learning Points dRIT antibody cocktail is biotinylated such that following a short incubation with a streptavidin-peroxidase complex, antibody-antigen binding complexes can be visualised through the addition of the substrate, 3-amino-9-ethylcarbazol. Performed on brain tissues, the dRIT has proven as sensitive as the FAT for fresh specimens [9, 10] . Brain impressions stained using the dRIT technique can be read within one hour and the antibody cocktail used has been shown to detect classical rabies virus strains (genotype 1) that have been assessed [11] . Currently, the FAT is routinely used to detect virus antigen in badly decomposed sample material. For the purpose of testing samples in the developing world where suitable cold storage for samples is often unavailable, this factor is important in the development of new tests. This obstacle has been overcome through evaluating sample preservation in phosphate buffered 50% glycerol at a range of temperatures for different time periods prior to testing for virus antigen. Glycerol saline solutions have been previously recognised as suitable storage media for tissue samples in the absence of cold storage [12] (Box 2). Using the dRIT in field studies in Tanzania, viral antigen could be detected in samples after considerable time periods post collection regardless of the regimen of glycerol preservative used [11] . Applications of the dRIT to analyse field samples in other rabies endemic regions have also proven highly successful. Field trials in Chad sought to study the dRIT in direct comparison to the FAT to attempt to confirm previous studies as to the incidence of rabies within a district known to be endemic. In this study, results between the two tests were 100% in agreement [9] and the only issue regarding use of the dRIT over the FAT was the need for the dRIT kit to be stored refrigerated prior to use. The dRIT will enable developing countries to perform routine rabies surveillance at greatly reduced cost and without the need for prohibitively expensive microscopic equipment along with the expertise and financial input needed to maintain them. The cost effectiveness of the dRIT will allow knowledge and technology transfer to areas of the developing world that currently are unable to diagnose rabies cases.
Another method for the detection of rabies virus antigen from postmortem samples is a recently developed rapid immunodiag- www.plosntds.org nostic test (RIDT) based on the principles of immunochromatography [13] . The immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of various analytes including viruses [14] . Briefly, suspect material is subjected to a test device similar to a lateral flow device. Conjugated detector antibodies attached to two different zones on a membrane indicate the presence of viral antigen. Preliminary validation studies with a limited number of samples showed that the RIDT might have great potential as a useful method for rabies diagnosis without the need for laboratory equipment [13] . However, thorough validation including various circulating variants of RABV and other lyssaviruses is still needed before this test could be relied upon and be used as an alternative for the gold standard FAT.
Reverse-transcriptase polymerase chain reaction (RT-PCR)
Various conventional RT-PCR protocols for the diagnostic amplification of lyssavirus genome fragments have been published (Tables 1-3 ). Since primers were selected from conserved regions of the genome, most assays amplify parts of the nucleoprotein (N-) gene as earlier proposed [15] . In generic approaches intended to detect all lyssaviruses either hemi-nested or fully nested amplifications are used and have applications for both antemortem (saliva, CSF, brain) and postmortem samples (principally brain tissue) ( Table 2 ). Some of these diagnostic procedures are also applied for further virus characterization, including sequencing reactions [16] or restriction fragment length polymorphism (RFLP) [17] . Also, strain-specific RT-PCRs have been developed to distinguish various rabies virus (RABV) strains in a particular region [18] .
Classical RT-PCR assays proved to be a sensitive and specific tool for routine diagnostic purposes [19, 15] , particularly in decomposed samples [20, 21, 22] or archival specimens [23, 24] .
The sole detection of amplified RT-PCR products by gel-based systems however, especially when using hemi-nested RT-PCRs generates the risk of cross-contamination, does not allow an exact quantification of genome copies and does not include tests for specificity [25] . Hybridisation methods [26] and PCR-ELISA methods were established to overcome these difficulties [27] , although these techniques have not become universally accepted. Additionally, many laboratories now use partial sequencing to confirm the detection of a lyssavirus and obtain data that can be used in a phylogenetic analysis of viruses circulating in a specific region. The importance of sequencing the PCR products was highlighted in an experimental study [28] . This study demonstrated that although the nested RT-PCR was shown to be the most sensitive of the diagnostic techniques employed, host genomic amplicons of the same size as the target amplicons were observed on the agarose gels, which were subsequently confirmed as false positives following direct sequencing [28] .
With the introduction of fluorogenic probes, detection of sequence specific templates can be achieved in real-time. Specificity is ensured by an inherent hybridization reaction, and cross-contamination is avoided due to the closed tube nature of the test [29, 30] . Consequently, for RABV and other lyssaviruses, various PCR assays using TaqMan technology have been described (Tables 4-5) . A generic real-time TaqMan-PCR for the detection and differentiation of lyssavirus genotypes 1, 5, and 6 has also been developed [31] . This assay utilises a pan-lyssavirus primer set, which has been shown to amplify a large panel of representative lyssaviruses, with probes specifically designed to discriminate between classical rabies virus and the European Bat Lyssaviruses type-1 and -2 (EBLV-1 and EBLV-2). PCR assays using TaqMan technology have applications for antemortem and postmortem samples. The pan-lyssavirus primer www.plosntds.org set can also be used in conjunction with a specific dye such as SYBR Green to allow for rapid detection of the amplicons. Validation of probe based assays relies on the availability of representative viruses or nucleic acid. However, for some lyssavirus genotypes only a limited number of viruses or sequences are available for primer/probe design, and they may not represent the genetic diversity of all current variants that are circulating. Single mutations for the North American RABV strains [32] in the region of the primers or the probe can alter the sensitivity of the PCR. Thus the genetic diversity among lyssaviruses may hamper the use of a single assay for all lyssaviruses. As such scanning surveillance may benefit more from the use of a pan-lyssavirus primer SYBR green assay rather than a strain or specific based assay.
The use of a rapid automated NASBA technique was successfully applied to the ante-mortem saliva and cerebrospinal fluid (CSF) of four rabies patients in Thailand and shown to have a ten-fold increase in sensitivity compared to RT-PCR [33] . The assay detected rabies viral RNA as early as two days after onset of symptoms. The NASBA technique involves the use of three enzymes (reverse transcriptase, RNase H and T7 RNA polymerase) to synthesise multiple copies of target RNA under isothermal conditions. Briefly, a large number of RNA copies are generated using a pair of specific primers, one of which contains the T7 RNA polymerase binding site, and the other which has an electrochemiluminescence detection region attached to the 59 end. The Table 3 . Conventional, gel-based PCR-assays for the detection of lyssavirus species other than RABV. www.plosntds.org amplified RNA is detected using an automated reader, enabling rapid throughput testing. It is relatively easy to use and the whole process from extraction to detection can take as little as four hours. This technology has already been applied for point of care testing of bacterial pathogens [34] and viral pathogens [35, 36] . The NASBA technique has also been adapted to investigate rabies virus replication in situ, whereby the relatively lower isothermal temperatures of NASBA compared to in-situ RT-PCR ensure that cell integrity is maintained [37] .
LAMP offers an alternative DNA amplification method to the polymerase chain reaction for applications to the ante-mortem saliva and CSF testing. The originators of the technique suggest that it amplifies with high specificity, efficiency and without the need for thermal cycling [38] . Amplification is achieved through the specific binding of two inner and two outer primers to the target sequence. The inner primers initiate strand synthesis whilst the outer primers displace the inner primers, allowing them to self-anneal to the nascent strand. This creates hairpin structures that trigger further strand synthesis that in turn lead to concatenation of the target sequence [38] . Polymerisation and strand displacement are achieved using a single enzyme, Bst 1 DNA polymerase. The technique is rapid, generating large quantities of target sequence within minutes. For the amplification of RNA viruses, a reverse transcription step is undertaken prior to the LAMP reaction. Primer sets have been successfully developed to detect a range of pathogenic viruses including West Nile virus [39] , Japanese Encephalitis virus [40] , Foot and Mouth Disease virus [41] and Chikungunya virus [42] . To assess the applicability of LAMP to the detection of rabies virus we designed a primer set using PrimerExplorer V4 software (Eiken Chemical Company Ltd., Japan) that can detect the Challenge Virus Standard (CVS) fixed strain of rabies virus (Table 6 ). In addition to the standard set of four primers, two further loop-binding primers have been added to increase the rate of strand displacement and synthesis [43] . The reverse transcription and LAMP reactions were undertaken simultaneously (RT-LAMP) in a single tube at 65uC using a www.plosntds.org thermostable reverse transcriptase, hence avoiding the step process inherent in an RT-PCR. Target amplification was monitored by the incorporation of the double stranded DNA binding fluorophore picogreen (Figure 1a) or by separation on a 1% agarose gel (Figure 1b) . At a constant temperature of 65uC, CVS RNA could be detected within 30 minutes. Development of RT-LAMP assays for use in diagnosis and surveillance is challenged by the considerable sequence variation observed within the rabies virus genome [44] that can frustrate specific primer design. Preliminary attempts at this suggest that multiple combinations of primers (up to 12 different primers) can lead to sensitive, rapid amplification of RABV genomes from a wide range of geographical locations. The use of isothermal amplification has the benefit of reducing the technological requirements of thermal cycling used in RT-PCR. This in turn offers the opportunity, when linked with lateral flow devices, to develop surveillance protocols where testing can take place in the field or in less sophisticated laboratories.
Microarray linked to sequence independent PCR amplification offers the ability to rapidly identify pathogenic viruses from postmortem samples [45, 46, 47] . We have undertaken a study that has demonstrated the ability of a microarray to detect each of the seven lyssavirus genotypes (VLA Weybridge, unpublished data). The microarray is composed of oligonucleotide probes 70 nucleotides in length and includes probe sets for each of the seven classified genotypes and sets for the unclassified lyssaviruses. The www.plosntds.org diagnostic capability of the array was illustrated showing the ability of the array to detect RABV in a human case of rabies as the amplified RNA bound specifically to the classical rabies virus (genotype 1) probe set ( Figure 2 ).
Development of a novel ultrasensitive and stable potentiometric immunosensor A stable potentiometric immunosensor for the detection of various analytes of interest from complex real world samples such as blood, serum and milk has been described [48] . The biosensor detects enzyme labelled immunocomplexes formed at the surface of polypyrrole coated, screen-printed gold or silver electrodes. Detection is through a secondary reaction that produces charged products with a shift in potential, being measured by local changes in redox state, pH and/or ionic strength. The magnitude of the change in potential is directly related to the level of target in the matrix such that the assays are quantitative and the numerical output is rapidly transmissible. The bioassays produce very rapid results (5-15 minutes), are highly reproducible (%CV,5%) and are ultra-sensitive (,50 fM). Thus this sort of biosensor offers the rapidity of production of signal of a lateral flow device with the sensitivity of third generation ELISAs. Immunoassays can be developed in a sandwich or competitive format for small (e.g. Digoxin; MW 780 Da) or large (e.g. hepatitis B surface antigen; .300 kDa) molecules [49] . In addition to immunoassays, it is also possible to detect specific nucleic acids and whole cells using the technology. Due to the method of production of the electrode strips, the base assays are inexpensive (,US$1 per strip) as is the detection hardware (US$2,000). In addition, most immunoassays can be readily adapted to this format with minimal additional optimisation. Potentiometric immunosensors for detecting the rabies virus nucleoprotein are in progress and offer the ability to rapidly screen complex non-clarified matrices, possibly at pen-side, in a cost-effective manner.
Serological assays are not suitable as diagnostic tools for routine rabies testing as the host response to infection varies substantially between individuals. However, serology is still useful, particularly to monitor the development of the immune response. We would suggest that detection of rabies antibodies in serum and CSF, early after presentation and in the absence of a history of vaccination may be a positive indicator for a therapeutic intervention. Viral pseudotypes, the core of one virus coated with envelope protein derived from a second virus, offers a safe alternative to the use of pathogenic viruses in neutralisation assays. Using pseudotypes expressing genotype 1 CVS-11 glycoprotein, high titre stocks (1.3-3.2610 5 infectious units/ml) were produced that proved 100% specific and highly sensitive compared with neutralisation titres achieved using the FAVN [50] . A high correlation was also observed (r = 0.89). Using pseudotypes expressing EBLV-1 (genotype 5) and EBLV-2 (genotype 6) G-proteins, neutralising antibody titres broadly correlated with the degree of G-protein diversity. A vaccine study in Tanzania compared the two assays with pseudotypes showing 100% specificity and 94.4% sensitivity to the FAVN with a high correlation of antibody titres (r = 0.92). Incorporation of Lagos bat virus (genotype 2), Mokola virus (genotype 3) and Duvenhage virus (genotype 4) G-proteins, as well as lacZ as a reporter gene, makes the pseudotype assay an attractive option for serosurveillance in Africa and other resource limited countries. In addition, as the pseudotype assay uses substantially less input serum (10 ml) compared to FAVN and RFFIT, multiple tests can be undertaken on samples where collection volumes are limited or valuable e.g. bat sera.
Due to the neurotropic nature of rabies virus, infection results in enormous viral replication in the CNS in the final stage of the disease that leads to massive antigen and viral genome concentrations. This makes detection of viral antigen in brain tissue by tests such as the FAT or the dRIT [7] very robust and relatively simple to perform, and these have become rapid gold standard tests. As for detection of viral genome, approaches are now available which process multiple specimens from nucleic acid extraction through to genetic typing, with significantly reduced risks of contamination. In addition, the use of TaqMan RT-PCR or similar technologies on robotics platforms, allow for rapid largescale rabies detection, typing and quantification in real time [32, 31, 51] . The development of PCR-based methods (Box 2) provided an alternative method for post mortem rabies diagnosis [26] , and the possibility of ante mortem diagnosis of human rabies [52] . RT-PCR methods invariably involve multiple transfers of nucleic acids between different tubes. Coupled with the high sensitivity of PCR methodologies, any small amount of contam- Figure 1 . Amplification of rabies virus RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP). For each reaction 1 mg RNA, prepared using TriZol (Invitrogen) from normal mouse brain (1A, circles; 1B 2) or infected mouse brain (1A, triangles; 1B, +) was added to a reaction mixture containing all six primers at concentrations indicated in Table 1 ination will undoubtedly produce false-positive results. Attempts have been made to adapt RT-PCR to reduce manipulations thereby reducing contamination risks. The visualisation of PCR products by gel electrophoresis exposes facilities and operators to large quantities of amplified material and thus many adaptations have been directed at replacing this step. New and improved rapid diagnostic tools for rabies using Taqman technology have been developed that avoid cross-contamination due to the closed tube nature of the test [29, 30] . A further benefit of RT-PCR has been to enable practical molecular characterization of rabies viruses [26] that has added significantly to the understanding of virus evolution and epidemiology. This approach has superseded the use of monoclonal antibodies for typing and characterising new strains of rabies virus. This has provided the evidence to support the delineation of lyssaviruses into genotypes [53] (Box 2) and was used for the classification of another four putative members of the genus [54, 55] . Also, this technique was a prerequisite for the understanding of the molecular biology of lyssaviruses [56] and underpinned future developments in rabies diagnosis and prevention. It is likely that in the future microarray techniques in combination with sophisticated bioinformatics and arrays with a hierarchical set of probes will provide an alternative to rapid virus discovery and characterisation.
The development of 'real-time' RT-PCR techniques allows the quantification of this RNA in 'real-time,' giving a relatively quick and reliable method for the measuring levels of viral RNA. PCR based techniques are not currently recommended by the WHO for routine post-mortem diagnosis of rabies. However, in laboratories with strict quality control procedures in place and demonstrable experience and expertise, these molecular techniques have been successfully applied for confirmatory diagnosis and epidemiological surveys. For these reasons, it is likely that international bodies will accept their use in the future for routine rabies diagnosis. Reverse transcription PCR has been reported to confirm rabies diagnosis intra-vitam in suspect human cases, when conventional diagnostic methods have failed and post-mortem material is not available (Box 1) [57] . Rabies virus RNA can be detected in a range of biological fluids and samples (e.g. saliva, CSF, tears, skin biopsy sample and urine). Owing to the intermittent shedding of virus, serial samples of fluids such as saliva and urine should be tested but negative results should not be used to exclude a diagnosis of rabies. All positive PCR results should be sequenced to confirm the origin of the virus and rule out possible contamination. In terms of the RNA concentrations in the brain, the sensitivity especially of nested or real-time PCRs may be beyond the threshold needed for routine post-mortem testing. Also, contamination of negative samples could lead to an unjustifiable administration of a high number of costly post exposure prophylaxis and would produce false data for the rabies surveillance. However, with the introduction of accreditation for laboratories, quality control measures are being implemented in a growing number of laboratories worldwide. Such quality controls for diagnostic rabies PCRs should encompass several measures, including the inclusion of appropriate positive, negative, and inhibition controls in assay runs. The consistency and the interassay reproducibility should also be ensured over time by monitoring performance. Only if laboratories meet the required standard [58] , can PCR fulfil its full potential. The use of PCR should not be restricted only as a confirmatory diagnostic test for decomposed samples but also as a powerful tool for virus typing Figure 2 . Microarray identification of rabies virus RNA prepared from a brain sample from a confirmed case of human rabies. Total RNA was extracted using TriZol (Invitrogen) and treated with DNase I prior to conversion to double stranded DNA [45] . Non-specific amplification was achieved using a DNA polymerase (Klen Taq, Sigma) and the products were labelled through binding of Alexa Fluor 555 reactive dye (Invitrogen) to amplicons. Labelled target DNA was denatured at 95uC and chilled on ice before dilution in hybridization buffer and addition to the microarray slide. Hybridization occurred at 50uC overnight. Slides were washed and the target-probe binding was captured using GenePix Pro 6.1 software (Molecular Devices). Statistical analysis of the data was conducted using DetectiV software [64] . doi:10.1371/journal.pntd.0000530.g002
and molecular epidemiology studies. The lack of standardization is a major obstacle to the general use of PCR for rabies diagnosis, especially in developing countries.
It is evident that the RT-PCR dominates genetic detection of rabies virus and it seems probable that this technique will dominate rabies diagnosis in the 21 st century. However, we should not discount alternatives that have the benefit of isothermal amplification that will enable implementation in laboratories where access to thermal cyclers is an obstacle. A NASBA technique was successfully applied to the saliva and CSF of four living patients with rabies and detected rabies viral RNA 2-days after the onset of symptoms. This technique has also been adapted to investigate rabies virus replication in-situ. LAMP also falls into this category and can be adapted for use with lateral flow devices thus making its application very simple.
Existing assays for rabies virus antibody prevalence studies either require high containment facilities or do not distinguish between neutralising and non-neutralising antibodies [59] [60] . Recently however, a neutralisation assay using retroviral pseudotypes was described [50] , not bound by the restrictions listed above and also allowing a choice of endpoint reporter proteins (bgalactosidase, green fluorescent protein or luciferase) [61] . A further benefit of this technique is its adaption to using small volumes of sera thus making them useful for surveillance.
Currently, high-throughput rabies virus molecular detection methods augment standard diagnostic tests or are in the process of development and refinement for use alone. As we progress through the 21 st century, it is possible that these techniques will replace conventional tests (Box 1). Obstacles to adoption include cost, complexity and local acceptance of their use. It is also possible that immunological tests by measuring 'indirect' markers such as cytokines and electrolytes will increase in use. These tests however, will probably remain in the realm of large reference laboratories where resources allow the development of novel assays. As far as semi-automated or automated instruments and robotics-based techniques are concerned, they are useful when large numbers of the same test are undertaken such as surveillance and companion animal testing and these tests will continue to increase in popularity and use, especially in central reference facilities. There is a clear need to simplify molecular diagnostic techniques so these tests can be applied universally in developing and developed countries. It is likely that new developments will focus on generating low volume and yet affordable diagnostic tests for rabies. More use will be made of point-of-care (POC) diagnostic testing using portable extraction techniques linked to PCR machines with the use of lyophilised reagents to overcome coldchain dependencies in tropical countries. In the 21 st century, these technologies will have a demonstrable impact on people living in developing countries, especially where rabies is still considered a 'neglected' disease. By contrast in the developed world, these new technological advances will undoubtedly be faster, more accurate and cost-effective leading to a 'Theragnostics Approach' that combines therapeutics with diagnostics for the human treatment of rabies. Interest in treating human rabies aggressively is gaining momentum, largely due to the reported success in treating a 15year-old girl, in whom clinical rabies developed one month after she was bitten by a bat, using a combination of therapeutic coma with antiviral drugs whilst allowing for the host immune system to confer immunity -The 'Milwaukee Protocol' (Box 2) [62, 63] . Bioinformatics, genomics, proteomics, and functional genomics are the molecular biology tools that are essential for the progress of molecular 'theragnostics', where both early diagnosis and monitoring of serology are critical factors for the successful treatment of a rabies patient. In addition, theragnostics could eliminate the unnecessary treatment of patients for whom rabies immunotherapy is not appropriate i.e. immunosuppressed patients, resulting in substantial drug cost savings for the healthcare system. Ducks: The “Trojan Horses” of H5N1 influenza Abstract Wild ducks are the main reservoir of influenza A viruses that can be transmitted to domestic poultry and mammals, including humans. Of the 16 hemagglutinin (HA) subtypes of influenza A viruses, only the H5 and H7 subtypes cause highly pathogenic (HP) influenza in the natural hosts. Several duck species are naturally resistant to HP Asian H5N1 influenza viruses. These duck species can shed and spread virus from both the respiratory and intestinal tracts while showing few or no disease signs. While the HP Asian H5N1 viruses are 100% lethal for chickens and other gallinaceous poultry, the absence of disease signs in some duck species has led to the concept that ducks are the “Trojan horses” of H5N1 in their surreptitious spread of virus. An important unresolved issue is whether the HP H5N1 viruses are maintained in the wild duck population of the world. Here, we review the ecology and pathobiology of ducks infected with influenza A viruses and ducks’ role in the maintenance and spread of HP H5N1 viruses. We also identify the key questions about the role of ducks that must be resolved in order to understand the emergence and control of pandemic influenza. It is generally accepted that wild duck species can spread HP H5N1 viruses, but there is insufficient evidence to show that ducks maintain these viruses and transfer them from one generation to the next. Avian influenza is caused by type A viruses of the family Orthomyxoviridae. The influenza A viruses infect primarily free-living aquatic birds, and they are classified by their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. All 16 HA and 9 NA subtypes have been isolated from aquatic birds; wild ducks are the main reservoir. The viruses cause asymptomatic or low pathogenic infection in these natural hosts. 1 However, certain strains of influenza A virus have crossed the host range barrier and infected other species, including humans. These viruses are the source of the influenza pandemics that emerge at irregular intervals. 1, 2 The H5 and H7 subtypes are of particular concern because they can become highly pathogenic (HP), causing systemic illness and death in both avian and mammalian species, including humans. 2 The H5N1 virus that emerged in Asia in 1996 is unique among the HP avian influenza (HPAI) viruses in that it has continued to circulate in avian species for more than a decade and has spread to more than 60 countries in Eurasia (http://www.who.int/csr/ disease/avian_influenza/en/). While the H5N1 HPAI viruses are 100% lethal to chickens and gallinaceous poultry, they often cause asymptomatic infection in some species of domestic and wild ducks. These ''silent spreaders'' of H5N1 HPAI viruses are therefore referred to as ''Trojan horses''. [3] [4] [5] Clearly, ducks play a complex and vital role in the biology and the overall natural history of influenza, including H5N1 HPAI viruses.
Ducks are members of the subfamily Anatinae, which contains most species of anserine birds. This subfamily is nearly cosmopolitan in distribution, and its members occupy almost all aquatic habitats. The ecology of these birds, summarized in Figure 1 , facilitates the maintenance and spread of avian influenza viruses. Although human influenza A isolates and the currently circulating H5N1 HPAI viruses typically infect the upper respiratory tract, the primary site of infection in ducks is the intestine. 6 Avian influenza viruses enter the environment when the host defecates or drools, and they then infect susceptible hosts as they feed and drink. Avian influenza virus replication has been observed in the respiratory tract, 6 but the contribution of this site to maintenance of infection in the population is unresolved. Specifically, fecal shedding of H4N7, H7N3, and H11N9 virions from experimentally infected mallard ducks persists longer and at higher titers than tracheal shedding. 6 When a large number of birds roost on a small pond (for example, in the staging ⁄ marshalling areas), as many as 10 10 EID 50 •g )1 •d )1 infectious virions are estimated to enter the environment in the fecal matter of each infected duck. 6 Further, avian influenza viruses are stable in water 1,7 and have been isolated from the surface of ponds containing a large number of waterfowl. 8, 9 Although aerosol transmission cannot be dismissed, the larger number of positive cloacal than tracheal swabs, the high fecal virus titer, and the stability of the virions in water suggest that lowpathogenic avian influenza (LPAI) viruses persist in duck populations through fecal-oral transmission. 1 This mechanism could partially explain the higher prevalence of infection in surface-feeding (dabbling) ducks than in diving ducks that typically feed in deeper water. 10 Surveillance data suggest year-round transmission of avian influenza viruses within duck populations. The prevalence of infection exhibits an annual cyclical pattern in both North American 1,11 and Eurasian 12 duck populations (Figure 1 ), peaking before and during the fall migration as a result of the influx of immunologically naïve juveniles. 1, 9, 10, 13 Experi-mentally infected white Pekin ducks have shed virus for more than 3 weeks after inoculation. 3, 14 Coupled with limited morbidity and serum antibody response, 3 infected birds are likely to shed virus during the first few weeks of the fall migration, dispersing it along their numerous migration corridors. However, the prevalence of infection is much lower along the migration routes and at the wintering grounds than at the marshalling areas. 9, 12, 15, 16 This disparity may reflect the development of immunity to circulating virus subtypes within the duck population or a decline in transmission because of population dispersal. 13 In general, prevalence of infection is higher at the wintering grounds and spring nesting sites in duck populations from Europe than in North American populations (Figure 1 ). The most likely explanation for this difference is random variation, since surveillance studies from multiple areas in North America and in Europe often obtain slightly different prevalence values in the duck populations. Many factors can affect prevalence including, but not limited to, the size of the duck population, sampling location, and time of collection. Thus, the few multi-year studies that exist likely exemplify the variation that one would observe if additional sampling sites were included in the studies, and not the differences in geography between Europe and North America. 16 Prevalence is at its lowest Figure 1 . Overview of the annual movement and behavior of migratory ducks and their role in interspecies transmission. During spring and fall migration, the ducks rest and feed for a few days to weeks at numerous stopover sites (wetlands, lakes, or ponds) along the migration route. The length of stay and the aquatic habitat allows the transmission of influenza viruses to and from the domestic duck populations. Domestic ducks that become infected are likely to maintain the virus locally and increase the probability of its spread to other species. In the diagram, solid arrows indicate confirmed routes of transmission of LPAI and ⁄ or HPAI viruses between species. The dashed line represents a probable but unconfirmed route of transmission. The graphs indicate the average prevalence of low-pathogenic avian influenza in North American and European duck populations during 3 stages of the annual migration. 10, 16 during the spring migration but increases again after the breeding season, when the ducks have moved to the molting and staging areas. 11, 13, 16 It is not clear how the duck population acquires avian influenza viruses during the spring of every year. Infectious virions may persist through the winter in the frozen waters of the breeding areas and reinfect the ducks when they return in the spring. 1, 6 Alternatively, the duck populations may carry the viruses throughout the entire migration. Year-round prevalence in the ducks supports the latter, although persistence in the frozen habitats could play a role in the perpetuation of the viruses. 10 Some virus subtypes are isolated more frequently than others. 10, 11 Three HA subtypes, H3, H4, and H6, are common in both North American and European ducks, 11, 12 and the most prevalent subtype combinations in both areas are H4N6 and H6N2. 16 Explanations vary for why certain HA and NA subtypes (and combinations) are common or rare in wild birds. The general hypothesis is that these subtypes likely have the highest fitness, with replication rates balanced by a level of virulence that sufficiently increases transmission probability to the level where infection in the next cohort of birds is almost guaranteed. It is speculated that this could be largely influenced by the functional balance between HA binding affinity and NA enzymatic activity. 17 Although the H6 gene is of Eurasian origin and is widely distributed in North American ducks, genomic analysis of viruses suggests limited intercontinental exchange between Eurasia and the Americas. 18 Therefore, novel viral genotypes must arise via mutation and reassortment of the genomes in circulation within a specific geographic area. The marshalling areas provide such an opportunity by attracting populations of ducks from various breeding areas and migration corridors, with each population harboring a potentially different combination of subtypes. 16 Coinfection of ducks with two or more virus subtypes is common, 19 as is reassortment, 14 and emergent strains that are most virulent to gallinaceous poultry can have low pathogenicity in the duck hosts. 20 However, the role of ducks in the maintenance and spread of influenza viruses, and especially in the emergence of novel genotypes, appears to depend on their migratory behavior. Specifically, ducks that migrate annually are likely to spread influenza viruses along the migration routes, primarily by exposing the resident and domestic duck populations at the numerous stopover sites. 10, 16, 21 In contrast, domestic and resident ducks maintain the viruses in close proximity to other species and have been implicated in the spread of both LPAI and HPAI viruses to domestic poultry and terrestrial birds. 20, 22, 23 
Low-pathogenic avian influenza LPAI viruses can pass through the upper digestive tract of ducks and replicate in the lower intestinal tract without causing clinical manifestations of disease. 3, 6 Further evidence that the intestinal tract is the target organ of LPAI viruses in ducks includes the replication of virus in the lower intestinal tract, but not in the lungs, after direct inoculation into the crop and rectum 6 and high fecal virus titers after intravenous inoculation. 3 The specific site of LPAI virus replication is believed to be the crypts of Lieberkühn in the large intestine. 3 The other potential target organ for LPAI viruses in ducks is the respiratory tract. Lungs of mallard ducks intranasally inoculated with LPAI viruses showed mild pneumonia, and lymphocyte and macrophage infiltration within 2 days. Immunostaining for viral nucleoprotein revealed intermittent staining of respiratory epithelial cells but no viral replication in the lung tissue. 24 This evidence indicates that the respiratory tract and not the lung tissue itself is the primary target of infection.
The species diversity of ducks may also play an important role in the pathogenicity of influenza viruses. Mallard duck embryos inoculated with LPAI virus have significantly lower mortality rates than inoculated Muscovy duck embryos; however, in regards to replication, the LPAI viruses behave in a different way. Viral antigens were found in the internal organs (nasal sinuses, pharynx, trachea, bronchi, lung, and air sacs) of the mallard duck embryos, but not in those of the Muscovy duck embryos. The reason for this mortality ⁄ virus replication paradox in mallard ducks is unclear but is in keeping with the evidence that mallard ducks are considered to be the main reservoirs of LPAI viruses in nature. 25 Although several studies have examined the serum antibody response in both naturally and experimentally infected ducks, knowledge of the avian immune response to influenza viruses is very limited. 26 White Pekin ducks inoculated with an H7N2 LPAI virus developed negligible serum hemagglutination inhibition (HI) antibody titers despite fecal shedding of virus until day 7 post-inoculation. Animals reinoculated 46 days later with the same virus strain had a marked antibody response, but virus could not be isolated from any of the organs. These results and the lack of a secondary immune response after inoculation with formalin-inactivated virus suggested that the rapid immune response in re-infected birds may restrict influenza infection to short time scales. 3 It is noteworthy that prior infection does not protect ducks against subsequent infection with other virus subtypes. For example, ducks infected with an H4N6 subtype are protected from reinfection with the same virus, but they shed virions for 8 days after challenge with an H11N3 isolate. 27 These data have applications in the field, where isolation of influenza virus from migratory waterfowl is infrequent during the winter, potentially indicating the existence of a significant level of immunity in wintering ducks acquired from previous influenza infections. Further illustration of this is seen from a study of wild waterfowl in Italy over six winter seasons, in which 17 of the 20 viruses isolated were of the H1N1 subtype, suggesting that the wintering ducks had some degree of immunity to the other subtypes of circulating influenza strains. 28 Highly pathogenic avian influenza Several experimental studies have investigated the pathogenicity of H5N1 HPAI viruses (isolated since 2002) in ducks. Cherry Valley Pekin ducks inoculated with a 2003 HPAI H5N1 strain isolated from duck meat at a quarantine inspection station in China developed neurologic signs, including blindness and head shaking, although none died. High virus titers were detected in the respiratory organs (lung and trachea), brain, liver, kidneys, and colon, and microscopic lesions were observed in the brain (viral encephalitis), heart (myocarditis with degeneration and necrosis of myocytes), and bursa (mild lymphoid follicular hyperplasia). 29 Viral neurotropism and pancreatotropism have been observed in multiple other studies of recent HPAI virus isolates. Ducks lethally challenged with these H5N1 HPAI viruses showed severe neurologic signs, including torticollis, incoordination, tremors, and seizures. 30, 31 Immunohistochemistry positivity was recorded in the cerebrum and brain stem, and in situ hybridization detected virus in the neurons and glial cells of the cerebral gray matter, further confirming the strong neurotropism of post-2002 isolates. 30, 31 Although the route of entry of virus into the central nervous system has not been determined, at least two different hypotheses have been proposed, including ascending transmission of virus via vagal, olfactory, and trigeminal nerve fibers, and penetration of the blood-brain barrier. 32, 33 Another recurring characteristic of recent H5N1 HPAI viruses in ducks is that virus titers are frequently higher in oropharyngeal swabs than in cloacal swabs. 30, 34 Pharyngeal excretion of H5N1 HPAI viruses has been suggested to originate from the lung and ⁄ or air sac, as only these tissues have shown immunohistochemical evidence of virus replication. Preferential pharyngeal excretion suggests that pharyngeal swabs, as well as the customary cloacal swabs, should be taken when conducting surveillance of avian influenza viruses in wild ducks. 34 Otherwise, the prevalence of H5N1 HPAI may be underestimated. Additional studies of the role and rates of respiratory transmission of H5N1 HPAI viruses in ducks are needed, especially as they relate to cloacal excretion.
In 1996, the parental virus (A ⁄ Goose ⁄ Guangdong ⁄ 1 ⁄ 96; A ⁄ Gs ⁄ GD ⁄ 1 ⁄ 96) of currently circulating H5N1 HPAI viruses emerged in southern China. Genetic evidence revealed that this virus originated from H5 LPAI viruses carried from northern Japan by wild ducks or other migratory birds. 15, 35 A reassortant H5N1 HPAI virus subsequently emerged in poultry at farms and live animal markets in Hong Kong in 1997. Genetic analyses showed that the H5 HA gene of the reassortant virus was derived from an A ⁄ Gs ⁄ GD ⁄ 1 ⁄ 96-like virus, while the remaining gene segments were derived from low-pathogenic viruses: the N1 NA gene came from A ⁄ Teal ⁄ Hong Kong ⁄ W312 ⁄ 97 (H6N1) virus, and the internal genes from A ⁄ Quail ⁄ Hong Kong ⁄ G1 ⁄ 97 (H9N2) virus. 36 The reassortant virus caused the first lethal infection in humans (6 deaths among 18 known cases) by direct bird-to-human transmission. 37 Between 1999 and 2002, H5N1 HPAI viruses with the H5 HA gene of A ⁄ Gs ⁄ GD ⁄ 1 ⁄ 96-like viruses but with a diversity of genotypes in the other genes, re-emerged multiple times in Hong Kong. 37 The first indication of the spread of H5N1 HPAI viruses from domestic to wild species of aquatic birds occurred in Kowloon and Penfold Park in Hong Kong, 38 where 19 different duck species were infected. Some species, including the Red-Crested Pochard (Netta rufina), were highly susceptible (19 ⁄ 20 died), whereas others, including the Bahama Pintail (Anas bahamensis), were less susceptible (4 ⁄ 21 died).
The next major event in nature was the massive die-off of waterfowl at Qinghai Lake in China. [39] [40] [41] Four different genotypes of H5N1 HPAI viruses co-circulated in the waterfowl there; one of these became dominant and spread westward to India, Europe, and Africa. Notable features of the dominant Qinghai Lake H5N1 HPAI isolates were the acquisition of a lys627 mutation in the PB2 gene and the absence of pathogenicity in mallard ducks. 42 The lys627 mutation has been found to be associated with pathogenicity in mammalian species, 43, 44 suggesting that it may have been generated while the virus was replicating in a mammal. The virus was likely transmitted from domestic ducks to wild ducks en route to Qinghai Lake.
The role of duck species in the westward spread of the Qinghai-H5N1 virus remains controversial. Circumstantial evidence from global wildlife surveillance supports the hypothesis that migratory birds, including wild ducks, have contributed to the current Eurasian endemic of H5N1 HPAI viruses. 45 Surveillance studies in Thailand in 2004 showed that most domestic grazing ducks infected with H5N1 HPAI viruses were asymptomatic 4 and that the initial spread of H5N1 HPAI viruses to chickens and humans corresponded to the movement of grazing ducks. 4, 46 In fact, infected domestic ducks grazing on man-made wetlands (e.g., harvested rice fields and irrigation canals) may maintain the infection and spread it to wild birds that feed at the same sites. If these wild birds are migratory and experience limited morbidity, they in turn can disperse HPAI viruses widely (Figure 2) , as suggested by the high genetic similarity of HPAI isolates from Africa, Europe, and the Middle East to the Qinghai-H5N1 virus. 37 
The evolution of H5N1 HPAI viruses by reassortment with LPAI viruses in the aquatic bird reservoir played an important part in the genesis of the multiple genotypes, clades, and subclades of Asian H5N1 HPAI viruses and is ongoing. 37 However, it is the ever-increasing poultry industry that provides the reassortment interface between wild and domestic avian species. The number of domestic ducks, chickens, and other poultry continues to increase, but biosecurity and separation of species is not always taken into account. Ducks raised in a closed high-biosecurity system in Thailand were protected from infection while H5N1 HPAI viruses were circulating among backyard ducks, open house ducks, and grazing ducks. 4 Therefore, biosecurity can prevent the spread of influenza viruses from wild to domestic ducks.
Live poultry markets (wet markets) have been identified as a risk factor in the genesis of novel influenza viruses 49 and were identified as the source of the human outbreak of HP H5N1 viruses in Hong Kong. The ban on ducks, geese, and later, quail, together with improved biosecurity (clean days), have markedly reduced the influenza virus diversity in the Hong Kong wet markets. 37 Live poultry markets are being phased out in Hong Kong, and in the interim no live poultry can be carried over from 1 day to the next. Taiwan plans to close all live poultry markets by 2009, and Shanghai authorities are reducing the number of wet markets. Overall, however, the role of live poultry markets in the emergence and control of pandemic influenza has been largely ignored. Universal closure of live markets would make biological sense but is difficult in regions where refrigeration is not widely available.
Vaccination has been accepted as an option for the control of HPAI by the Food and Agriculture Organization of the United Nations and the World Organization for Animal Health. Emphasis is placed on the use of vaccine to facilitate eradication. The continued use of poultry influenza vaccines without an eradication plan has immediate benefits but also long-term consequences. The difficulty with continued vaccine usage is that it promotes genetic variation and allows shedding of virus in the absence of disease signs, thus creating the potential for epicenters of virus spread. Further, while both inactivated oil emulsion whole-virus H5 vaccines and recombinant NDV vaccines containing H5 HAs are highly efficacious in chickens, the recombinant NDV vaccines are less efficacious in ducks. The experience in Vietnam illustrates these points. In 2005, after 61 human cases of HP H5N1 virus infection and 19 deaths, universal poultry vaccination and reduction of duck populations were implemented, with dramatic results. In 2006, there were no human cases of H5N1 influenza. However, in 2007-2008, there were 14 human cases of HP H5N1 virus infection and 10 deaths (http://www.who.int/ csr/disease/avian_influenza/country/cases_table_2009_03_11/ en/index.html). The difficulty of controlling H5N1 HPAI viruses in ducks by vaccination and the enormous task of vaccinating sufficient poultry to maintain ''herd immunity'' remain daunting obstacles.
Influenza in humans is considered a non-eradicable disease due to periodic introduction of viruses from their natural reservoir, wild migratory birds -mainly ducks. The culling of wild birds is not an option. The sole current option is biosecurity and eradication of HP influenza from domestic poultry. The longer-term goal will be to understand the genetics of natural resistance in ducks and to introduce these traits into domestic animals. 
There is consensus that the migratory waterfowl of the world (predominantly wild ducks) serve as the natural reservoirs of all influenza A viruses, which cause asymptomatic infection in these birds. Influenza viruses have probably co-evolved with ducks over millennia, establishing an equilibrium between hosts and parasites so that neither suffers a significant loss of biological fitness; the evidence being minimal signs of disease in the hosts and the annual isolation of common subtypes. The unanswered question is whether these migratory bird species are the reservoirs of the currently circulating H5N1 HPAI viruses. Until the emergence of the Asian H5N1 HPAI viruses, the available data indicated that each new outbreak of HP H5 or H7 virus died out or was stamped out and that subsequent HP strains emerged from the low-pathogenic H5 and H7 virus reservoir.
All species of birds tested to date support replication of some HP H5N1 strains and, providing they are not killed rapidly, could contribute to the spread of H5N1 HPAI viruses. The present review has concentrated on ducks, some species of which are susceptible to H5N1 HPAI virus-caused disease and death while others (e.g., mallards) are quite resistant. 34, 50 Therefore, ducks that are infected but are naturally resistant to disease could have contributed to the spread of H5N1 HPAI viruses westward from Qinghai Lake in 2005 to Europe, Africa, India, and the Middle East. An unanswered question is whether the H5N1 HPAI viruses are being carried back to the duck breeding areas and are infecting the next generation. Extensive surveillance in the migratory pathways in Europe and Asia has provided no evidence of H5N1 HPAI viruses in the new generation of birds after the breeding season.
While all duck species tested to date are susceptible to lethal infection with H5N1 HPAI viruses, some species, including the mallard and Pintail ducks, are less susceptible and many survive. It is in these relatively resistant species that the H5N1 HPAI viruses could be maintained. However, surveillance studies to date in these species have detected no H5N1 HPAI viruses in breeding or juvenile birds. Experimental studies show that some mallard ducks continue to shed virus for up to 17 days, allowing the development of humoral immunity and subsequent selection of antigenic variants within the same bird. 23 If this occurs, it could be argued that a limited number of ducks would be sufficient to maintain the virus in nature. Continued surveillance is needed to determine whether H5N1 HPAI viruses are maintained in nature by a small number of naturally resistant ducks that are long-term virus shedders.
While naturally resistant ducks can be argued to have been involved in the spread of H5N1 HPAI viruses from Qinghai Lake to the rest of Eurasia, it is difficult to explain why H5N1 HPAI viruses have not spread to susceptible species in the Philippines, Australia, and the Americas, which are on the direct flyways of migratory waterfowl. More than 6AE6 million birds migrate from Eastern Asia to Alaska yearly (alaska.usgs.gov ⁄ science ⁄ biology ⁄ avian_influenza ⁄ migrants_tables.html). Despite intense surveillance in Alaska, no H5N1 HPAI viruses have been detected to date, and influenza viruses of Eurasian lineage are introduced into the Americas only rarely. 18 The major spreaders of influenza in domestic poultry are humans. As described by , from the molecular epidemiology data, transmission of H5N1 influenza in domestic poultry is perpetuated largely through movement of poultry and poultry products rather than continued reintroduction of viruses from migrating birds. 42 The alternative reservoir, the domestic duck population, has a higher likelihood of perpetuating H5N1 HPAI viruses. Prospective surveillance continued to isolate H5N1 HPAI viruses from apparently healthy ducks, geese, and chickens in Southeast Asian poultry markets during 2004-2006. Naturally resistant ducks might not be expected to show disease signs, but the absence of morbidity in highly susceptible geese and chickens is surprising. The widespread use of vaccine in chickens may explain this observation, but vaccine has been used less in geese and ducks. An alternative possibility is that the susceptible poultry had cross immunity as the result of exposure to co-circulating influenza viruses. Experimental studies have demonstrated that chickens previously infected with H9N2 virus and then inoculated with H5N1 HPAI virus become infected and shed virus but do not show disease signs. 51 The continuing co-circulation of multiple subtypes of LPAI viruses in domestic poultry could explain why a small percentage of susceptible domestic species can appear healthy while shedding transmissible levels of H5N1 HPAI virus. To provide answers to these unresolved questions about the role of domestic species, it will be necessary to establish long-term prospective surveillance in domestic poultry in the hypothetical ''epicenter zones,'' including China, Indonesia, Vietnam, Egypt, and Nigeria. It is noteworthy that in these regions, control of H5N1 HPAI virus is attempted by the continuing use of vaccines.
An area that has been surprisingly neglected is the genetics of ducks, the ultimate reservoir species of all influenza A subtypes. The wild duck reservoir contributes some or all of the genes of future pandemic strains in humans and future panzootic strains in domestic poultry. Immune mechanisms in ducks are currently understudied, and the molecular basis of resistance of some duck species to lethal infection is unresolved. Sequencing of the genome of the mallard duck is warranted, as it could provide insight into the factors that contribute to markedly reduced influenza virus pathogenicity.
Because wild ducks are the main reservoir of all influenza A viruses and the ultimate source of future pandemics, members of the scientific community who are interested in understanding the emergence and control of pandemic influenza should direct their attention to the following questions:
• Do ducks (wild or domestic) serve as the reservoirs of the Asian H5N1 HPAI viruses? • What genomic characteristics of ducks are associated with natural resistance in some species? • Is antigenic diversity driven naturally in ducks or is it the consequence of vaccine usage? • What dose of vaccine antigen is required to prevent transmissible levels of excretion of H5N1 HPAI viruses by ducks of different species (and geese and swans)? • Is eradication of Asian H5N1 HPAI viruses achievable?
• Can the use of transgenic animals containing the natural resistance gene(s) of mallard ducks prevent pathogenic influenza virus infection? Non-Hemagglutinating Flaviviruses: Molecular Mechanisms for the Emergence of New Strains via Adaptation to European Ticks Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE. Tick-borne encephalitis virus (TBEV) causes up to 14,000 human cases of tick-borne encephalitis (TBE) across Eurasia annually [1, 2] . TBE outbreaks are now registered in about 30 European countries with a recorded morbidity increase of about 400% during the past 30 years [3] . TBEV is a member of the tick-borne flavivirus (TBFV) group that, together with mosquito-borne and no-known vector virus groups comprise the genus Flavivirus within the family Flaviviridae. Human pathogens within the genus Flavivirus include Japanese encephalitis virus, Dengue virus and Yellow fever virus that cause annual epidemics of fever, encephalitis and hemorrhagic fever in the tropics and some sub-tropical regions [4, 5] .
In its natural habitat, TBEV is maintained by transmission between infected and non-infected ticks when they co-feed on small forest animals [6] [7] [8] . Humans are incidental hosts for ticks and may become infected by a feeding infected tick. The clinical manifestations caused by TBEV range from inapparent infections and fevers, with complete recovery of patients, to debilitating or fatal encephalitis. The proportion of fatal human infections varies widely in different regions and in different years. The factors that determine disease severity are poorly defined but correlations between viral subtype and disease severity have been described. TBEV strains are currently divided into 3 closely related subtypes, i.e. western-European (WE), Siberian (SIB) and Far Eastern (FE) [9] . FE TBEV is recognised as the most virulent pathogen with a 20-40% case fatality rate. The SIB subtype is considered less virulent (7-8% case fatality rate) but chronic disease occurs more frequently (1-3%) . Western European strains are the least virulent with case fatality rates lower than 2%. However, a range of clinical manifestations, from asymptomatic to encephalitic is observed for all TBEV subtypes [1, 2] and the underlying basis for this has not yet been adequately explained.
Conventionally, each TBEV subtype has been associated with distinct geographic ranges within the Old World region of the northern hemisphere, hence the groupings Far East, Siberia and Western Europe [9] . However during recent decades the epidemiology of the TBFV appears to have been changing, with SIB TBEV becoming the dominant subtype apparently gradually replacing the WE or FE subtypes that previously appeared to monopolise many regions [10] [11] [12] [13] [14] [15] [16] . Moreover, the SIB subtype is being isolated more frequently from patients who develop the most severe forms of encephalitis, with the virus invading the entire brain in contrast with the more focal virus localization observed previously. Over a period of time, the most severe cases of TBE have been more frequently associated with the SIB strains than with the FE strains [17] , indicating that this is not an artifact of increased surveillance. Whilst these reports are disturbing they have not as yet been addressed at the molecular virological level.
TBEV virions are spherical particles with an ,11 kb-RNA genome embedded in a capsid that is surrounded by a lipid envelope mainly containing a virus envelope (E) glycoprotein. This E protein plays a key role in many stages of the virus life cycle; it mediates virus binding to receptors on the cell surface (adsorption) which triggers receptor-mediated endocytosis. Exposure of the endocytosed virus to the acid pH converts the native E protein dimers into fusogenic trimers [18, 19] ; the latter promote fusion of virion and endosomal membranes thus releasing viral RNA into the cytoplasm. The E protein also plays the major role in inducing the host immune response and mediates hemagglutination (HA), i.e. the ability of virions to agglutinate avian erythrocytes; for decades HA has been used in routine diagnosis [20] .
Whilst most strains of TBEV show HA activity, during the past 10 years atypical HA-deficient strains have been isolated with increasing frequency in Europe from both ticks and infected patients. More than 40 HA-deficient strains are now recognized and they all exhibit reduced pathogenicity for mice when compared with HA-competent strains. They are also called ''antigenically deficient'' (AD) strains, in contrast to the more common antigenically competent (AC) strains; the term ''AD'' is derived from the strict correlation between loss of HA and immunoprecipitating activities [21] . The AD-viruses were also deficient in complement-fixation and neutralization tests when analysed using either hyperimmune antisera or sera obtained from patients recovered from TBE [21] . Here, using molecular methods of analysis, we show that HA-deficiency is linked with the adaptation of SIB TBEV strains to western European Ixodes ricinus ticks reflecting altered, E protein-mediated, receptor/fusion functions. We also illustrate how this might result in continued westward dispersal and emergence of new highly pathogenic virus variants (see Fig. S1 ).
Yar-strains of TBEV i.e. Yar71, Yar 114, Yar 46-2, and Yar 48 were isolated in the European part of Russia (Yaroslavl' region) between 1999-2001 and details for their isolation are listed in Table 1 . All 4 Yar viruses had equivalent infectivities when compared with the Vasilchenko (Vs) strain of TBEV (see Methods) used as the positive control virus (Table 1 ) and produced comparable concentrations of E protein when analysed by Western blot (data not shown). However, they were completely negative in HA tests over a range of pH 5.75-7, regardless of whether they were prepared in newborn mice or in PS cells. The control Vs virus and pGGVs virus, recovered from the infectious clone (see Methods), produced high positive HA titres (1:640-1:1280) at pH 6.2 and this was therefore the pH of choice for all subsequent tests (Table 1) . Thus, the 4 Yar isolates can be defined as HA-or AD-deficient in common with the other 40 strains that have been isolated in Europe and were also identified as HA-and AD-deficient [21] .
Yar-viruses belong to the ''Baltic'' group of the Siberian TBEV subtype Phylogenetic analyses based on a 1110-bp fragment of the E gene (positions 1114-2224 in Vs virus L40361) showed that the Yar-viruses belong to the SIB subtype of TBEV [9, [22] [23] [24] [25] [26] , which includes the control Vs virus (see Methods). Fig. 1 illustrates the overall branching pattern in agreement with previously published results [24, 25] . Three Siberian sub-clusters I, II and III were clearly identifiable and supported by high bootstrap values. Yarviruses were grouped with strains of sub-cluster III designated ''Baltic'' [27] , isolated only in Europe, in contrast to strains of clusters I and II that were found across Europe and Asia. Separate phylogenetic analyses based on the C, prM, E and NS1 genes produced trees that were congruent with the one presented (data not shown).
To identify amino acid(s) responsible for the loss of HA-activity, we sequenced Yar viruses and aligned them with 290 available TBEV E sequences ( Fig. 2A) . One amino acid 175N in the E protein was common to all HA-deficient Yar-viruses, in contrast with the highly conserved 175T. Therefore, the substitution T for N at amino acid position 175 was introduced into a TBEV infectious clone (IC) designated pGGVs [28, 29] to generate mutant virus IC-T175N that produced HA titres similar to parent Vs virus and also control pGGVs virus rescued from the infectious clone (Table 1 ). Since no other common amino acids were identified that distinguished Yar-viruses from the other strains, we hypothesised that individual non-shared amino acid substitutions might be responsible for the HA-deficient phenotype. The alignment in Fig. 2A revealed 3 non-conserved mutations, each of which was unique to one of the Yar strains, i.e. D67G (Yar 46-2), E122G (Yar 71 and Yar 114) and D277A (Yar 48). When compared with the parent pGGVs virus, each of these mutations increased net charge and hydrophobicity of the E protein and was surface orientated, mapping on the most protruding loops of the E protein in its native dimeric conformation (Fig. 2B) .
The loss of HA activity in TBEV has only previously been reported in relation to selective adaptation of TBEV to ticks. One substitution, E87K was generated during the propagation of a WE Figure 1 . Phylogenetic analysis of Yar strains. MEGA version 4 [43] was used to align E genes (between nucleotide positions 1114-2223 of Vs virus genome) of SIB TBEV strains (accession numbers are specified). Tree topology was reconstructed by Neighbor-Joining. The Tamura-Nei model was used for estimation of evolutionary distances [44] . Bootstraps were based on 1000 replications; values below 90% are hidden. The scale bar shows the number of nucleotide substitutions per site. Geographic origins of SIB strains and clusters I, II and III are shown on the right hand side of the tree. Yar strains are highlighted using triangles. KFDV was used as the outgroup. doi:10.1371/journal.pone.0007295.g001 strain to I. ricinus ticks [30] and two substitutions, E122G and T426I respectively, followed a few SIB TBEV passages in H. marginatum ticks [31] . In support of our observations, the E protein surface in these independently reported HA deficient viruses, is predicted to be of either positive or neutral charge, as we have described for the substitutions, D67G, E122G and D277A respectively.
Whilst the molecular details of interactions between virions and erythrocytes remain unknown it has been suggested that HA activity might be mediated by the trimeric E protein in its postfusion conformation rather than by native E dimers [32] . Five of these trimers form a fusion pore that enables fusion between the viral and cellular endosomal membrane thus releasing the viral RNA into the cellular cytoplasm. Therefore we mapped the chimaeric Yar-simulated mutants onto the crystal structure of the trimeric (post-fusion) conformation of the E protein [18] . This demonstrated that the 3 Yar-virus mutations are located along the most protruding parts of the lateral surface of the trimer (Fig. 2C ). Therefore they are likely to be able to make direct contact with the erythrocyte surface and/or with each other.
Thus, we hypothesised that each of these three amino acid substitutions could individually abolish HA deficiency in TBEV. To test this hypothesis experimentally we used a TBEV infectious clone to engineer three mutant viruses IC-D67G, IC-E122G and IC-D277A (See Methods) that simulate the wild-type viruses Yar 46-2, Yar 71/Yar 114 and Yar 48 ( Table 1 ). The introduction of any one of the three mutations into the parent HA positive pGGVs virus rendered it HA negative (Fig. 3 ).
All three engineered mutants demonstrated delayed growth in PS cells, most visible during the first 24 hours (Fig. 4) . Nevertheless, they all subsequently reached titres similar to pGGVs HA-positive virus by day 3 (Table 1) . Although, mutant IC-D67G exhibited better growth characteristics than IC-E122G and IC-D277A it was reproducibly slightly lower than the control pGGVs virus.
All 3 natural HA-negative isolates (Yar71, Yar 46-2, and Yar 48) displayed small-plaque phenotype (1 mm, Fig. 4B ), in . Abbreviated comparative alignment of TBEV E protein sequences (complete version is available on request). TBEV strains are specified by subtype (FE-, SIB-or WE-subtypes) and GenBank accession numbers. HA-disabling mutations are encircled. The ''tick-specific'' amino acids that differentiate I. persulcatus-transmitted viruses (shadowed) from the I. ricinus-transmitted viruses are highlighted in yellow; those that increase hydrophobicity are boxed and surface-faced amino acids are marked with asterisks (*). The fusion peptide is underlined. (B) Mapping of HA-disabling residues onto native dimeric conformation of E protein crystal structure [45] (1SVB.pdb); the monomer is shown as it lies on the virion membrane. The ''persulcatus'' and ''ricinus'' residues are highlighted in orange; fusion peptide is in green and HAdeficient amino acids (arrows) are red. The position of Yar substitution D277A (red coloured) coincides with the position of ''ricinus/persulcatus'' substitution (orange colour is masked). (C) Residues 67, 122 and 277 (purple spheres) are mapped onto the E protein in trimeric post-fusion conformation (1URZ.pdb) [18] . The fusion peptide is highlighted in green and can be seen protruding out of the virion membrane towards the endosomal membrane. Different subunits of the trimer are coloured in red, blue and yellow. doi:10.1371/journal.pone.0007295.g002 comparison with control Vs strain which produced much larger plaques (3 mm) but only two corresponding mutations, E122G and D277A caused plaque size reduction in the engineered viruses (0.7 to 1.5 mm as shown in Fig. 4B ). Clearly, as yet unidentified additional mutations contributed to the small plaque phenotype of Yar 46-2 (D67G). Similarly Yar 48 virus formed smaller plaques (1 mm) than the corresponding IC-D277A mutant (1.5 mm) demonstrating that HA activity and plaque phenotype are not always determined by the same amino acid.
Vs virus is different from many other laboratory-maintained TBEV strains; in cell culture it develops cytopathic effect (cpe) relatively slowly [22] . All tested mutants showed no increase or decrease of cpe in comparison with the control infectious clone pGGVs, even at high multiplicity of infection (10 PFU/cell).
After ip inoculation, the control virus pGGVs produced a relatively high morbidity rate (65%) in correspondence with previous results [29] . In contrast, all three engineered virus mutants exhibited lower neuroinvasiveness as determined by morbidity rates (Fig. 4C ). Mice observed for 21 days following ip inoculation with IC-D67G produced antibodies against TBEV, indicating that they had been infected (data not shown).
HA-deficiency correlates with increased TBEV reproduction in feeding ticks and tick-to-tick transmission efficiency Studies on the reproduction and tick-to-tick transmission efficiency of IC-D67G, IC-E122G and IC-D277A were carried out using a novel tick/mouse laboratory model initially developed by Labuda et al [7] . In the first set of experiments, 30 ticks were infected by injection in the leg with each engineered virus as described in Methods and TBEV titres in salivary glands were measured in each of 6 ticks at each time point, i.e. on 2 nd , 7 th , 14 th and 21 st day following infection. The reproduction characteristics of IC-E122G and IC-D277A in fasting ticks were similar to those of control pGGVs virus whereas the titres of IC-D67G were significantly lower (Fig. 5A) .
On the 21 st day post-infection, fasting infected ticks were allowed to feed on mice and virus titres were measured in each of 6 individual ticks. This analysis was employed for each engineered virus, 3 days after feeding. Fig. 4A shows that infectivity of IC-E122G had increased approximately 1000-fold and IC-D277A and IC-D67G had increased approximately 300-fold, whereas for control pGGVs virus the increase of virus titre was about 10-fold. Since 6 ticks were used for each tested virus for each time-point, the differences in titres between different virus mutants were statistically significant, based on Student t-tests (p,0.05).
In the second set of experiments, the efficiency of virus transmission from infected adult female ticks to uninfected nymphs during co-feeding on mice (tick-to-tick transmission; see Methods) was evaluated by estimating the proportion of infected nymphs. In addition, the titres of virus in each recipient nymph were estimated in a plaque assay. The results show in each case, that HA deficiency directly correlated with increased TBEV titres in nymphs, following feeding and also tick-to-tick transmission efficiency (Fig. 5B) .
We analysed 290 TBEV E protein sequences for the presence of amino acid substitutions similar to those that resulted in the loss of HA in TBEV, i.e. acidic (positively charged) residues that were replaced by hydrophobic and/or neutral amino acids (i.e. glycine) and localized on the virion envelope protein surface ( Table 2 ). In total, 5.8% (including Yar-viruses) of the strains exhibited similar mutations. Strains with potentially increased charge and/or surface hydrophobicity were identified in all three TBEV subtypes, i.e. FE, SIB and WE; they were isolated from different geographical regions and a variety of hosts including ticks, rodents and humans.
Notably, the substitution D67G was detected in 7 viruses isolated only from mammalian hosts and human patients. No other correlation between isolation source, geography or virus subtype specificity was observed. A significant number of TBEV E proteins have been sequenced partially (227 viruses of 290 available from GenBank), therefore it could not be excluded that TBEV strains with increased surface charge/hydrophobicity are quite common in nature. Indeed among 63 completely sequenced E proteins those with increased charge/hydrophobicity on the virion surface comprise ,20% (including Yar viruses).
We also compared E proteins of TBEV strains isolated from I. ricinus (WE-strains) with those isolated from I. persulcatus (FE-and SIB-strains), to identify group amino acids that might be involved in TBEV adaptation to these two different tick species (Fig. 2A ). Ten amino acid differences were revealed that have previously been localised in hypervariable clusters of the envelope protein [33] and five showed overall increased E protein hydrophobicity in the I. ricinus-transmitted WE-strains in contrast with FE-and SIBstrains ( Fig. 2A) . Four of these ten substitutions were localised on the virion surface, with two (S47A and S88G) increasing the surface hydrophobicity of ''ricinus'' strains and two (D178E and D277E) being conserved (Fig. 2B) . Remarkably, the position of the ''tick''-specific (i.e. I. ricinus or I. persulcatus) D277E amino acid substitution overlapped with the D277A substitution of Yar 48 ( Figs. 2A and 2B) . Two substitutions that also increased the hydrophobicity of strains adapted to I. ricinus were localised either inside the E protein (T115A) or on the membrane-oriented side (S267A), i.e. under the virion surface. Four substitutions (T427A, T431S, V433I and L437V) were located in the transmembrane domain of the E protein, with one (T427A) more hydrophobic for ''ricinus'' strains. Louping ill virus (LIV), a TBEV-related virus which is transmitted by I. ricinus in the UK, also showed the same more hydrophobic pattern and localisation of ''tick-specific'' amino acids as the I. ricinus-transmitted WE TBEV strains ( Fig. 2A) . Discussion TBE is currently reported in more than 30 countries of Eurasia and causes significant outbreaks of encephalitis in 16 countries, including 13 EU Member States (Austria, the Czech Republic, Estonia, Finland, Germany, Greece, Hungary, Latvia, Lithuania, Poland, Slovak Republic, Slovenia, Sweden) and three non-EU Member States (Norway, Russia and Switzerland) [3] . The clinical manifestations of TBE in endemic regions vary widely, from inapparent and febrile infections, with recovery of patients, to debilitating or fatal encephalitis, even in some vaccinated individuals [1, 2] and no adequate explanations have as yet been produced.
Here, we have investigated the molecular mechanisms and epidemiological implications of the emergence of unusual TBEV strains originally identified as HA-or AD-deficient [21] . In contrast with most TBEV strains, these novel viruses fail to agglutinate avian erythrocytes, show reduced antigenic characteristics and replicate relatively poorly in mammalian cells. We demonstrated that these strains display increased hydrophobicity and positive charge on their virion surface. By engineering genetically modified viruses we proved that these distinct surface characteristics, including HA-deficiency, are caused by any one of three single amino acid substitutions D67G, E122G or D277A in the E protein. These mutations significantly increased virus reproduction in feeding ticks and increased the efficiency of tickto-tick virus transmission when infected and uninfected ticks co-fed on the same animal. Thus mutations leading to HA-and ADdeficiency are directly associated with selection for enhanced virus transmission between ticks, a process that facilitates virus survival in the natural habitat [7] .
Although I. ricinus ticks can be routinely maintained in laboratories, SIB TBEV is normally associated with transmission by I. persulcatus, which is not as readily available for laboratory experiments. Nevertheless, WE TBEV strains show ,100% transmission efficiency in their natural tick vector I. ricinus (manuscript in preparation). Thus, logically, Yar viruses (i.e. SIB TBEV strains) should be transmitted efficiently in their natural vector I. persulcatus. Geographically, I. ricinus and I. persulcatus overlap in Europe and numerous reports describe the isolation of Siberian strains from I. ricinus which is now recognised as a second vector for SIB TBEVs [11, 34] . We therefore propose that the driving force behind the westward dispersal of these HA-deficient strains is their adaptation to newly-encountered European I. ricinus ticks. This hypothesis is also supported by our analysis of E protein comparative alignments between FE, SIB and WE subtypes that revealed more hydrophobic ''ricinus'' amino acid patterns, compared with ''persulcatus'', in correspondence with tick preference of WE or FE/SIB strains respectively. Louping ill virus, which is transmitted by I. ricinus in the UK, shares this ''more hydrophobic'' pattern with the WE TBEVs.
For many viruses, HA-activity is recognised as a reflection of receptor [35] [36] [37] or low-pH dependent fusion activity [38, 39] . It was suggested that HA activity of flaviviruses is mediated by fusion activity of the E protein [32] because it is optimal at pH 6.2 which promotes conversion of native E protein dimers into fusion-active trimers [18, 19] . This implies that the Yar mutations identified herein destabilise trimer-trimer contacts or contact between trimers and erythrocyte membranes, thus preventing HA activity. Alternatively, these mutations may impact on both E protein functions, ie virus adsorption to the cell surface and pH-dependent fusion of virions with endosomal membranes. Therefore, depending on charge and hydrophobicity, tick cell receptors may restrict ''easy'' entry of WE-strains into I. persulcatus or SIB-strains into I. ricinus. Clearly these barriers to infection are not absolute since Vs virus (SIB TBEV) has a limited capacity to replicate in I. ricinus (Fig. 5 ). Phylogenetic analysis also supports our hypothesis; it was proposed that WE-strains diverged from ancestral FE-and SIB lineages [40] , implying that the emergent WE subtype adapted to I. ricinus from I. persulcatus by evolving a more hydrophobic E protein (Fig. 2) . The coincidence of increased hydrophobicity of the virion surface for I. ricinus-adapted WEstrains and SIB Yar viruses presumably reflects a similar molecular requirement for different viruses to adapt to the same host. Alternatively, the emergence of the atypical Yar viruses may result from adaptation of TBEV to both tick species. Indeed SIB TBEV strains have been isolated from both I. ricinus and I. persulcatus on numerous occasions [11, 34] and regular switching between them cannot be excluded. These data might explain the apparently increasing dissemination of SIB TBEV in Europe; in a few decades this virus could reach more western territories, possibly even the UK where I. ricinus is the vector for the Louping ill virus that is closely related to TBEV.
The mutant IC-D67G was distinct from other HA-deficient TBEV strains since there was no obvious correlation between loss of HA activity and significantly reduced growth in mammalian cells. The Yar 71 virus, with the corresponding D67G mutation was isolated from a fatally infected individual (Table 1 ) and ominously, similar substitutions have also been detected in other TBEV isolates from hospitalised patients with encephalitis (Table 2 ). It is possible that due to altered surface charge and hydrophobicity, strains with D67G might be more able to penetrate the human blood-brain barrier (neuroinvasiveness) or more rapidly spread between human neurones, with no correlation to reduced mouse neuroinvasiveness. This would explain the recently discovered association of the most severe form of human encephalitis with the SIB strains [17] .
The molecular basis of antigenic deficiency of Yar viruses has not been elucidated but it might be related to the increased incidence of the most severe forms of TBE having been associated with SIB subtype [17] . Clearly more studies are required before we can understand, at the molecular level, the implications of the phenomenon of HA-and AD-deficiency in terms of the development of TBE from fever to encephalitis as an interplay between virus neuroinvasiveness and ability to evade the immune response.
Thus, the emergence of HA-deficient TBEV mediated by adaptation to different tick species might represent a mechanism for the westward dissemination of SIB TBEV, increased TBE incidence in Europe, and might also be the reason for encephalitis in humans (see Fig. S1 ). To confirm and develop these ideas, future research should focus on large-scale genomics and transmission studies of TBEV isolates recovered from patients and ticks.
Porcine embryo kidney cells (PS) were used to produce TBEV stocks, to recover mutant viruses, for plaque assay and studies of cytopathogenicity. Yar-strains of TBEV i.e. Yar71, Yar 114, Yar 46-2, and Yar 48 were isolated in the European part of Russia (Yaroslavl' region) between 1999-2001 (Table 1 ) and stored as 10% mouse brain suspensions. SIB TBEV strain Vasilchenko (Vs) and its infectious clone (pGGVs) used as control viruses have been described previously [22, 28] . I. ricinus ticks were bred in the Institute of Zoology, Slovak Academy of Science, Bratislava [7] .
The RNA of each Yar virus was extracted from 200 ml of 10% infected mouse brain suspension or infected PS cell supernatant using Total RNA Isolation System (PROMEGA). The RT-PCR was used to amplify the 59-C-prM-E gene region of Yar viruses as described [28, 41] . PCR products were directly sequenced using a Taq BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences of the C-prM-E region of Yar-viruses were deposited in GenBank with accession numbers EU444077, EU444078, EU444079 and EU444080 (Table 1 ).
Nucleotide and deduced amino acid sequences were aligned using program package BioEdit [42] . Phylogenetic analyses were conducted using MEGA4 [43] . Tree topology was reconstructed by the Neighbor-Joining method and the Tamura-Nei model was used for estimation of genetic distances [44] . The reliability of the tree was evaluated by bootstrapping based on 1000 replications.
The unique amino acid substitutions in the E protein of each Yar virus were mapped onto the crystal structure of the native dimeric [45] or low-pH induced trimeric [18] Cytopathic effect (cpe), plaque assay and virus growth cycles in PS cells
The routine protocols for cpe, plaque assay and growth curve experiments were described in detail previously [25, 28, 29, 41] . Briefly, for cpe and growth curve experiments PS cells in 24-well plates were infected with viruses at a multiplicity of infection (moi) of 1 PFU/cell, in four replicates. To estimate cpe, the inoculum was replaced with RPMI medium after virus adsorption for 1 h at 37C. Infected cell monolayers were initially examined by microscopy and then stained with 0.1%naphthalene black at 24, 48, 72, 96 or 120 h post-infection for further examination of the extent of cpe. For plaque assay, original virus stocks were 10-fold serially diluted and after 1-h of virus adsorption at 37uC infected monolayers were overlaid with 1% SeaPlaque Agarose (Cambrex, USA). After incubation at 37uC for 5 days monolayers were fixed with 10% formol saline and stained with 0.1% naphthalene black. For virus growth curve experiments, after virus adsorption for 1-h at 37uC, monolayers were washed 5 times with serum-free RPMI medium and overlaid with 1 ml of medium containing 2% FCS. The supernatant medium from infected cells was collected at 2, 10, 12, 16, 20 and 24 hours pi and frozen at 270uC. The titres of infectious virus were determined by plaque assay.
Hemagglutination assay PS cells were infected at a moi of 0.1 PFU/cell in 500 ml culture flasks and infectious supernatant medium was collected at day 5pi. The TBEV virions with estimated initial virus titers of 228610 6 PFU/ml were concentrated 100 times using 7% polyethylene glycol (PEG) in the presence of 2.4% NaCl overnight at 4uC and precipitated by centrifugation at 8000 rpm for 3 h. The resulting pellet was resuspended in 500 ml of PBS. For routine hemagglutination test [20, 46] , 50 ml of the concentrated virus sample (10 8 PFU/ml) were placed in the first well of 96-well plates and diluted two-fold in borate buffer (pH 9.0). Then 100 ml of a suspension of 0.5% newborn chick erythrocytes was added to each well. Results are recorded as the reciprocal of the maximum virus dilution that produced agglutination.
The ligation in vitro of two overlapping plasmids, pGGVs H and pGGVs 660-1982del produces full-length infectious clone of TBEV strain Vs [28, 29] . The megaprimer-mediated domain swapping mutagenesis technique [47] was utilized to introduce point mutations in pGGVs 660-1982H (Fig. 6 ). For the Hi-Fi PCR, a pair of primers was used to amplify regions of about 200 nucleotides (megaprimer); one primer contained the appropriate point mutation within the E gene. Subsequently, the megaprimer was used to amplify the pGGVs 660-1982 H template in 15 cycles of circular PCR at 95uC for 30 sec, at 60uC for 30 sec and at 72uC for 5 min. Each cycle of the PCR produced nicked dsDNA molecules, with nascent (circular) DNA strand originating from template bacterial dsDNA and the other one (nicked) from newly amplified PCR product. Following 15 cycles, the accumulation of amplified linear complementary ssDNA strands resulted in the formation of annealed circular (twice nicked) molecules. To facilitate screening, the dammethylated bacterial (template) DNA was digested with 40 U of DpnI (New England Biolabs) at 37uC for 1 h. Following this, PCR products were electroporated into AbleK bacterial cells (Stratagene) and selected clones were completely sequenced.
To recover engineered viruses, the mutated plasmids, constructed on the basis of pGGVs 660-1982 H, were ligated with pGGVs 660-1982del to restore the full-length cDNA of TBEV as described previously [29] . Each mutant full-length clone was subsequently linearised by Sma I and used for SP6 transcription to produce full-length RNA. The SP6-transcribed RNA was transfected into PS cells using Lipofectin reagent (Invitrogen) according to the manufacturer's protocols. Infectious supernatant medium was collected on day 5 pi. The presence of virus in infected cells was confirmed by immunofluorescence microscopy using monoclonal antibodies specific for flavivirus E proteins [46] and by RT-PCR. The entire E-gene was sequenced to ensure no additional substitutions appeared during the genetic manipulation or initial virus replication in PS cells.
Unfed adult females of I. ricinus ticks were inoculated with virus under a stereo zoom microscope (Wild M 400, Wild Heerbrugg AG, Switzerland) into the coaxial plate of the second pair of legs using a digital microinjector TM system (MINJ-D-CE; Tritech Research, Inc.; USA). Clean nitrogen served as a gas source to produce an injection pressure of 20 psi ( = app. 1.38bar). The injection interval was set to 1.0 sec. Hollow glass needles with a microscopically fine tip were prepared using a P-30 Micropipette puller (Sutter Instrument Company, USA).
To investigate virus reproduction in fasting ticks, groups of 45 female ticks were infected with 500 PFU/tick of one TBEV strain. Infected ticks were incubated at room temperature (2464uC) and 85-90% RH in a desiccator for 21 days. At 2, 7, 14 and 21 days pi salivary glands of 6 ticks were dissected, individually homogenized and the concentration of infectious virus was estimated by plaque titration.
Virus tick-to-tick transmission experiments were carried out essentially as described previously [6] [7] [8] . Two of 45 infected adult female ticks were allowed to feed simultaneously on Balb/C mice for 3 days with 15 uninfected I. ricinus nymphs that were attached in close proximity (1-1.5 cm) to the feeding donor females. Surviving nymphs and salivary glands of donor females were used to determine the titres of infectious virus using plaque assays. The co-feeding transmission rate was estimated as the proportion of nymphs (%) that became infected.
Experimental animal procedures were performed in accordance with the guidelines for care and maintenance of animals (Act of the Government of the Slovak Republic 2003 regulating the use of experimental animals). All animal experiments were approved by the State Veterinary and Food Administration of the Slovak Republic (permission numbers 12284/03-220 and 2362/06-221). The ethical permission to carry out the work with mice was obtained from the Ethical Review Committee of the Institute of Virology, Slovak Academy of Sciences''.
Ten adult ICR mice were inoculated intraperitoneally (ip) with 2000 PFU/mouse. Mice were observed for 21 days and morbidity rate was estimated as the proportion of animals that showed clinical symptoms that included hind-leg paralysis. Sick and healthy mice were tested for the presence of anti-TBEV antibodies by HA-inhibition test [20] .
Statistical analysis was performed on the data obtained from virus replication studies in PS cells and ticks, neutralization and neuroinvasiveness test using EXCEL and SigmaPlot 11 software (Systat Software Inc., USA). Standard errors of mean (SEM) were estimated for each dataset. Between-groups comparisons were performed using unpaired, two-tailed Student's t-test. Values of p,0.05 were considered as significant. 
Conceived and designed the experiments: MAK VVP ML BK EAG TSG. Diagnosis and management of drug-associated interstitial lung disease Symptoms of drug-associated interstitial lung disease (ILD) are nonspecific and can be difficult to distinguish from a number of illnesses that commonly occur in patients with non-small-cell lung cancer (NSCLC) on therapy. Identification of drug involvement and differentiation from other illnesses is problematic, although radiological manifestations and clinical tests enable many of the alternative causes of symptoms in advanced NSCLC to be excluded. In lung cancer patients, high-resolution computed tomography (HRCT) is more sensitive than a chest radiograph in evaluating the severity and progression of parenchymal lung disease. Indeed, the use of HRCT imaging has led to the recognition of many distinct patterns of lung involvement and, along with clinical signs and symptoms, helps to predict both outcome and response to treatment. This manuscript outlines the radiology of drug-associated ILD and its differential diagnosis in NSCLC. An algorithm that uses clinical tests to exclude alternative diagnoses is also described. The diagnosis of drug-associated interstitial lung disease (ILD) involves three elements: clinical suspicion, differentiation from other parenchymal lung diseases using computed tomography (CT) and other clinical tests for alternative disease, and a compatible histological pattern. This review article will discuss the radiological evaluation of a patient with non-small-cell lung cancer (NSCLC) suspected of suffering from drug-associated ILD, the diagnosis and management of drug-associated ILD, and the development of a diagnostic algorithm designed to distinguish gefitinib ('Iressa')-associated ILD from other forms of parenchymal disease.
The onset of drug-associated ILD during therapy for advanced NSCLC usually occurs within a few weeks of the start of treatment (Thomas et al, 2000; Gupta et al, 2002; Kudrik et al, 2002; Read et al, 2002) . Indeed, retrospective analysis of the first 152 patients in Japan to experience gefitinib-associated ILD showed that 475% of cases occurred within 3 months, with the majority of these occurring within 4 weeks.
The symptoms of drug-associated ILD, as with all forms of the condition, include rapidly developing breathlessness and a dry and unproductive cough, together with fever (Thomas et al, 2000; Gupta et al, 2002; Kudrik et al, 2002; Read et al, 2002) . Such symptoms are nonspecific and can occur with a large number of common illnesses often associated with NSCLC, or they may be due to cancer progression or lung cancer therapy. These patients are also prone to pneumonia and many have radiation-related injury as a result of prior treatment. Cardiovascular causes of the symptoms, such as fluid overload, congestive heart failure and pulmonary embolus, are not uncommon. Differentiation of drug-associated ILD from these illnesses is difficult and the diagnosis is usually made by exclusion. Highresolution CT (HRCT) is recommended on first suspicion of ILD to provide an assessment of the parenchymal nature of the cause of symptoms.
The radiological manifestations of drug-associated ILD, although heterogeneous and nonspecific, enable many of the alternative causes of symptoms in advanced NSCLC to be excluded. There is no specific radiological pattern of parenchymal change connected with drug-associated ILD. Furthermore, in the early stages of disease, patients with symptoms secondary to drug reaction may have a normal chest radiograph.
High-resolution CT allows a more precise assessment of the presence, pattern and distribution of parenchymal and airway abnormalities than a chest radiograph. It has the advantage over lung biopsy of providing an overall view of the extent and pattern of parenchymal involvement rather than being limited to a small region, which may not be representative of the overall pattern of disease. However, there is limited information on the correlation between the findings on HRCT and histological patterns in drug-associated lung disease. Data based on a small number of cases suggest that the different histological patterns of drug reaction are not reflected by distinctive HRCT findings (Cleverley et al, 2002) . *Correspondence: Professor NL Müller; E-mail: nmuller@vanhosp.bc.ca Despite these limitations, it seems reasonable to approach the radiological manifestations of drug-associated lung disease by the use of the underlying histological pattern (Ellis et al, 2000; Myers et al, 2003) . Using such an approach, the most common manifestations can be classified into diffuse alveolar damage, hypersensitivity pneumonitis, organising pneumonia, nonspecific interstitial pneumonia (NSIP) and eosinophilic pneumonia (other less common drug reactions are not discussed here). Any one histological pattern can be caused by a number of different drugs. Furthermore, similar histology is found in other conditions that are not associated with drug use, such as idiopathic interstitial pneumonias, viral, bacterial and fungal pneumonia, pulmonary haemorrhage or leukaemia and collagen vascular disease. Occasionally, HRCT may demonstrate findings that are highly specific for the diagnosis, including increased attenuation in amiodarone lung and areas of fat attenuation in lipoid pneumonia.
Diffuse alveolar damage is characterised histologically by the presence of alveolar airspace and interstitial oedema, hyaline membrane formation and proliferation of type II pneumocytes (Rossi et al, 2000; Cleverley et al, 2002) . In relation to drug-associated pulmonary disease, it occurs most commonly with cytotoxic agents such as bleomycin and, less commonly, with aspirin and narcotics (Rossi et al, 2000; Cleverley et al, 2002) . The corresponding radiological features are also found in adult respiratory distress syndrome (ARDS). The chest radiograph shows bilateral patchy or homogeneous airspace consolidation involving mainly the middle and lower lung zones (Rossi et al, 2000; Cleverley et al, 2002) . High-resolution CT demonstrates extensive bilateral ground-glass opacities and dependent areas of airspace consolidation (Rossi et al, 2000; Erasmus et al, 2002) ( Figure 1A ).
A number of drugs may result in hypersensitivity pneumonitis, including methotrexate, cyclophosphamide and antidepressants such as fluoxetine and amitriptyline (Ellis et al, 2000) . The radiological and HRCT findings are identical to those seen in hypersensitivity pneumonitis secondary to the inhalation of organic dust and consist of bilateral ground-glass opacities and/ or small poorly defined centrilobular nodular opacities (Ellis et al, 2000; Cleverley et al, 2002) . The majority of patients also demonstrate lobular areas of air trapping, although this is less Figure 1 High-resolution CT images demonstrating radiology of drug-associated ILD. (A) A 77-year-old man with diffuse alveolar damage secondary to amidarone; note the extensive bilateral ground-glass opacities, airspace consolidation and bilateral pleural effusions. (B) A 36-year-old woman with hypersensitivity pneumonitis secondary to sertraline; note the extensive bilateral ground-glass opacities and lobular areas of air trapping (arrows). (C) A 69-year-old man with BOOP-like reaction to amiodarone; note the mild reticulation and bilateral areas of consolidation and ground-glass opacities in a predominantly peribronchial distribution. (D) A 47-year-old man with NSIP reaction to bleomycin; note the extensive bilateral ground-glass opacities with mild superimposed reticulation. (E) A 47-year-old man with eosinophilic pneumonia reaction to dilantin; note the patchy bilateral areas of consolidation involving the peripheral regions of the upper lobes. apparent than in extrinsic allergic alveolitis to airborne agents (Ellis et al, 2000) ( Figure 1B ).
Organising pneumonia, also known as bronchiolitis obliterans organising pneumonia (BOOP)-like reaction, has been reported most frequently in association with methotrexate, cyclophosphamide, gold, nitrofurantoin, amiodarone, bleomycin and busulphan (Cleverley et al, 2002; Erasmus et al, 2002) . The chest radiograph shows patchy bilateral areas of consolidation, masses or nodules, which may be symmetric or asymmetric (Müller et al, 1990; Ellis et al, 2000) . In a few patients, the disease manifests with a lone mass. On HRCT, areas of consolidation often have a predominantly peripheral or peribronchial distribution (Müller et al, 1990; Ellis et al, 2000) ( Figure 1C ).
Nonspecific interstitial pneumonia is one of the most common forms of drug-associated pneumonitis. Nonspecific interstitial pneumonia is characterised histologically by homogeneous alveolar wall thickening by fibrous tissue and mononuclear inflammatory cells. The reaction is seen in association with a variety of drugs, the most common being methotrexate, amiodarone and carmustine (Erasmus et al, 2002) . The corresponding radiographical and HRCT findings usually consist of patchy or diffuse ground-glass opacities (Rossi et al, 2000; Erasmus et al, 2002) ( Figure 1D ). On disease progression, there may be evidence of fibrosis with development of a reticular pattern and traction bronchiectasis. In some patients, the fibrosis is patchy in distribution and predominantly peribronchovascular, a pattern most commonly seen in patients receiving nitrofurantoin. Late chemotherapy lung may predominate in the upper and lateral parts of the lung.
Eosinophilic pneumonia is characterised histologically by the accumulation of eosinophils in the alveolar airspaces and infiltration of the adjacent interstitial space by eosinophils and variable numbers of lymphocytes and plasma cells. Peripheral blood eosinophilia is present in p40% of patients. Eosinophilic pneumonia secondary to drug reaction is seen most commonly in association with methotrexate, sulphasalazine, para-aminosalicylic acid, nitrofurantoin and nonsteroidal anti-inflammatory drugs. Chest radiography and HRCT show bilateral airspace consolidation, which tends to involve mainly the peripheral lung regions and the upper lobes (Rossi et al, 2000; Cleverley et al, 2002 ) ( Figure 1E ).
Alternative diagnoses to drug-associated ILD in NSCLC include progression of the cancer, infection, radiation-related lung injury, fluid overload, congestive heart failure and pulmonary embolus. Additionally, some lung cancer patients may develop BOOP or other steroid-responsive inflammatory disorders that cannot be clearly related to drug therapy. Other possible causes of dyspnoea that do not give infiltrates include comorbid diseases such as chronic obstructive pulmonary disease (COPD) and aspiration of food and saliva (particularly in patients with vocal cord paralysis or brain metastases).
In the USA, pulmonary embolism is particularly common in patients with lung cancer, with as many as 20% of patients estimated to develop a deep vein thrombosis or pulmonary embolism during the course of their disease (Lieberman et al, 1961; Sack et al, 1977; Lee and Levine, 2003) .
Most episodes of pneumonia in patients with lung cancer are due to bacteria. This is particularly the case when risk factors of neutropenia, endobronchial lesions, underlying COPD and aspiration are present. Although opportunistic infections are not common, fungal infections should be considered if the patient has received a high dose of corticosteroids. Viral infections with herpes simplex, cytomegalovirus or respiratory syncytial virus may also rarely result in pneumonia in patients who have received highdose corticosteroids or very intensive chemotherapy.
The development of lung fibrosis following radiation therapy is well documented and is usually confined to the radiation port (Abid et al, 2001; Aviram et al, 2001) . With the use of threedimensional radiation portals, however, resulting infiltrates from radiation may not result in the traditional straight-edged infiltrate and may be more difficult to distinguish from other entities.
Patients with lung cancer who present with respiratory failure should undergo systematic investigation. Pulmonary function tests, such as measurement of forced expiratory volume in 1 s, carbon monoxide diffusing capacity and measurement of arterial oxygen saturation with pulse oximetry, are commonly used. These tests ascertain the type of defect, for example, obstructive or restrictive ventilatory defects, which helps establish the cause, such as an exacerbation of any underlying obstructive airways disease versus interstitial disease. They also indicate the severity of the disease that helps determine the need for further assessment and treatment.
A standard chest CT scan is commonly performed to exclude a diagnosis of disease progression or pulmonary embolism. However, as discussed previously, obtaining high-resolution cuts is very helpful (HRCT) if drug-associated ILD is suspected.
Bronchoscopy is useful to evaluate some NSCLC patients with dyspnoea to assess extension of the cancer and to exclude an opportunistic infection using bronchioalveolar lavage. However, the value of a bronchoscopy in establishing the diagnosis of drugassociated ILD is less clear, since bronchioalveolar lavage is not specific for drug-associated disease, and biopsies obtained by transbronchial biopsy are small and often do not yield enough tissue to make this distinction. Open lung biopsy is rarely performed in NSCLC patients with respiratory distress since most patients have advanced disease with a limited prognosis. Furthermore, procedures requiring surgery are not usually believed to arrive at a definitive diagnosis of drug-associated ILD.
Patients with mild symptoms or pulmonary function abnormalities (such as a decrease in diffusing capacity of o20% from baseline or no change in oxygen desaturation during exercise), or with transitory or slight radiographical infiltrates are monitored using pulmonary function tests, symptoms and usually CT scans. Diagnostic evaluation and treatment for drug-related lung disease is considered in those patients who experience dyspnoea at rest or on mild exertion, have a X20% decrease in carbon monoxide diffusing capacity, or experience oxygen desaturation at rest or during exercise. Patients whose radiographical infiltrates are extensive or progressive are also considered for therapy.
Retrospective analysis of the adverse-event reports from patients diagnosed with ILD following gefitinib treatment is also difficult as there is often limited or heterogeneous clinical information, no pathology result and no access to the results of radiological investigations.
A diagnostic algorithm has therefore been developed to assess the accuracy of the reports of drug-associated ILD among Japanese patients receiving gefitinib. This approach to differential diagnosis used an algorithm developed to aid early diagnosis of drugassociated ILD in clinical practice.
The algorithm used both radiological and clinical evidence to exclude alternative diagnoses, such as infection, tumour progression, heart failure and pulmonary embolism, to arrive at a presumptive diagnosis of drug-associated ILD (Figure 2 ; Table 1) . Patients were categorised based on the strength of evidence supporting the diagnosis of drug-associated ILD (category 1, good supporting evidence for ILD; category 2, limited supporting evidence for ILD; category 3, no supporting evidence for ILD).
In the first assessment, adverse-event reports from Japanese patients receiving gefitinib for NSCLC were evaluated using the algorithm, with available clinical information and radiographical reports but without access to chest radiographs or HRCT films. These findings were then compared with those of a second assessment, conducted by an independent panel of expert radiologists and physicians. The panel assessed the radiological and clinical findings for the same patient population using a set of standardised criteria (Cleverley et al, 2002) .
The initial 152 reported patients with NSCLC in Japan who had experienced adverse events involving the lungs while receiving treatment with gefitinib were included in the first assessment of the algorithm. Of these, 135 were included in the second assessment because radiological examinations, including 47 with CT imaging, were available. Approximately 20% of these patients (23 out of 135) were considered by the expert panel not to have drug-associated ILD, highlighting the difficulty in diagnosing drug-associated ILD in patients with NSCLC.
These results were then compared with those obtained using the algorithm. A large proportion (17 out of 23) of patients not considered by the panel to have drug-associated ILD had been categorised as having 'good' or 'limited' supporting evidence for drug-associated ILD. The initial algorithm based on the terminology used in radiology reports, without considering differential diagnosis criteria, was not adequate. Infection, heart failure and tumour progression can be differentially diagnosed and excluded using additional radiological clinical data.
Therefore, the algorithm is now used to enable the clinician to make a diagnosis having first undertaken all the necessary steps in the clinical examination and investigation. This approach is applied to the prospective nested case -control study to investigate the relative risk and risk factors for ILD in NSCLC patients in Japan treated with and without gefitinib. An independent case review board will review each reported case of ILD using the information gathered from the algorithm.
General principles in the management of acute respiratory distress in patients with lung cancer are influenced by the multiple causes of respiratory failure that are associated with cancer, lung cancer therapy and the presence of comorbid disease. In addition, the diagnosis may remain uncertain, even when invasive procedures are performed, and empirical therapy for the likely causes is frequently given. Finally, respiratory failure in patients with cancer results in high mortality (Groeger et al, 1999) requiring aggressive assessment and therapy.
There are no firm guidelines for the treatment of drug-associated ILD and therapy tends to be on an empirical basis. Withdrawal of the drug suspected of causing the ILD is the first step in treatment. For patients in respiratory failure, high-dose methylprednisolone (250 mg four times a day i.v.) for several days is commonly used. If the patient responds, then the dose is reduced (0.5 -1 mg kg À1 day À1 orally) for several weeks before being gradually tapered. For patients in respiratory distress, methylprednisolone (1 mg kg À1 day À1 or 60 mg day À1 ) is commonly used, again with gradual dose reduction. Low-dose methylprednisolone (10 -20 mg) is prescribed for patients with mild radiographical or pulmonary function abnormalities, particularly if oral corticosteroids are contraindicated (White and Stover, 1984; Baughman et al, 1994; American Thoracic Society, 2002) .
Immunosuppressive agents such as azathioprine have been used as steroid-sparing agents in the treatment of drug-associated ILD, particularly in chronic cases of bleomycin-associated ILD (Maher and Daly, 1993) . These agents are useful for patients in whom corticosteroids cannot be tapered or who cannot tolerate corticosteroids. It is also advisable to avoid combining agents associated with ILD, such as bleomycin and possibly mitomycin, with other agents that cause lung damage, such as oxygen and radiation, as this may result in worsening of the lung damage. If radiotherapy is indicated in a patient who has experienced mild bleomycin toxicity, then concomitant low-dose corticosteroid may minimise any further lung damage. Following resolution of the drug-associated ILD, some patients are susceptible to exacerbations on subsequent insults (e.g. during a respiratory infection) and may require further treatment.
In cancer patients, drug-associated ILD is most commonly observed during mitomycin, paclitaxel, docetaxel or gemcitabine therapy. Table 2 outlines the types of injury reported with these agents and their response to therapy (Buzdar et al, 1980; Chang et al, 1986; Goldberg and Vannice, 1995; Rivera et al, 1995; Ramanathan and Belani, 1996; Bookman et al, 1997; Merad et al, 1997; Piccart et al, 1997; Semb et al, 1998; Vander Els and Miller, 1998; Dunsford et al, 1999; Thomas et al, 2000; Fogarty et al, 2001; Read et al, 2002; Barlesi et al, 2004) . Acute pneumonitis, both interstitial and noncardiac pulmonary oedema pattern, has been observed with mitomycin. Response to high-dose methylprednisolone has been reported within 24 -48 h of therapy; however, approximately 60% of patients developed ongoing and persistent lung disease (Rivera et al, 1995) . Chronic pneumonitis, similar to bleomycin-type pneumonitis, has also been reported during mitomycin therapy, which responded to prednisone therapy (Buzdar et al, 1980; Chang et al, 1986) . As with bleomycin, it has been suggested that oxygen therapy be avoided during mitomycin therapy, although the evidence for this is not as established as that for bleomycin (Klein and Wilds, 1983) .
Infusion hypersensitivity is very common during paclitaxel therapy. Pretreatment with an antihistamine, a corticosteroid and an H2 blocker largely prevents or ameliorates this reaction (Bookman et al, 1997) . Mild interstitial pneumonitis with transitory infiltrates has also been reported; observation or lowdose corticosteroids resulted in a good response, even when this occurred in conjunction with radiotherapy (Goldberg and Vannice, 1995; Ramanathan and Belani, 1996) . Fluid retention syndrome during docetaxel therapy is very common and is associated with dyspnoea. This reaction can be prevented by dexamethasone (Piccart et al, 1997; Semb et al, 1998) . Interstitial pneumonitis and ARDS-like pattern have also been reported with docetaxel, particularly when it is used in combination with radiotherapy or gemcitabine. Both reactions respond well to corticosteroid therapy; however, when docetaxel is used in combination with radiotherapy or gemcitabine, the response to corticosteroid therapy can be variable to poor, with some deaths reported (Merad et al, 1997; Dunsford et al, 1999; Read et al, 2002) .
Self-limiting dyspnoea is common during gemcitabine therapy. Additionally, many patients experience a mild capillary leak throughout the lung, similar to that seen in ARDS. However, these patients are asymptomatic and have normal pulmonary function; therefore, gemcitabine therapy can be continued without the need for corticosteroids. Gemcitabine-associated ILD has also been reported and the response to corticosteroid therapy is variable. As with docetaxel, more deaths due to ILD have been reported when gemcitabine is used in combination with other therapies (radiotherapy or chemotherapy) (Vander Els and Miller, 1998; Dunsford et al, 1999; Thomas et al, 2000; Fogarty et al, 2001; Barlesi et al, 2004) .
In the management of patients with NSCLC, it is often necessary to treat patients with an agent that has been previously associated with ILD. In this scenario, it is important to consider the patient's previous response to that therapy, the severity of the lung damage and the respiratory distress, the presence of fibrosis and the previous response to corticosteroid therapy. For some patients, the benefits may outweigh the risks and therapy may be re-instituted with concomitant low-dose prednisone (10 -20 mg day À1 ); however, for some patients and/or agents the potential for causing further lung damage may be too great.
In summary, lung cancer patients receiving systemic therapy frequently develop dyspnoea and infiltrates. It is difficult to make a specific diagnosis in most cases because of the difficulty of performing invasive procedures in this patient population. Radiological assessment, and HRCT in particular, can play a key role in establishing a diagnosis of drug-associated ILD; however, in the vast majority of cases, the radiological manifestations of drugassociated pulmonary injury are nonspecific, making an accurate diagnosis difficult. Corticosteroids are indicated for suspected drug-associated ILD; however, the outcome is variable unless the patient develops respiratory failure, in which case the mortality is high.
As a result, clinicians are reluctant to withhold corticosteroid therapy if there is any indication of drug toxicity, further complicating the diagnosis. However, once a patient responds to corticosteroid therapy, the decision to re-institute a drug suspected of causing ILD is made on an individual basis. In this article we described an algorithm developed to assess the incidence of drugassociated ILD in Japanese patients receiving gefitinib for NSCLC. Such an algorithm, once validated, may be a useful tool in the differential diagnosis of ILD in patients with cancer and help clarify some of the apparent discrepancies in the incidence and reporting of ILD. Buzdar et al (1980) , Chang et al (1986) , Goldberg and Vannice (1995) , Rivera et al (1995) , Ramanathan and Belani (1996) , Bookman et al (1997) , Merad et al (1997) , Piccart et al (1997) , Semb et al (1998), Vander Els and Miller (1998) , Dunsford et al (1999) , Thomas et al (2000) , Fogarty et al (2001) and Read et al (2002) . a Even when paclitaxel is used in conjunction with radiotherapy. b When docetaxel is used in combination with radiotherapy or gemcitabine, the response is variable to poor. Primary biliary cirrhosis Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis and cirrhosis of the liver and may need liver transplantation in the late stage of disease. PBC primarily affects women (female preponderance 9–10:1) with a prevalence of up to 1 in 1,000 women over 40 years of age. Common symptoms of the disease are fatigue and pruritus, but most patients are asymptomatic at first presentation. The diagnosis is based on sustained elevation of serum markers of cholestasis, i.e., alkaline phosphatase and gamma-glutamyl transferase, and the presence of serum antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex. Histologically, PBC is characterized by florid bile duct lesions with damage to biliary epithelial cells, an often dense portal inflammatory infiltrate and progressive loss of small intrahepatic bile ducts. Although the insight into pathogenetic aspects of PBC has grown enormously during the recent decade and numerous genetic, environmental, and infectious factors have been disclosed which may contribute to the development of PBC, the precise pathogenesis remains enigmatic. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at adequate doses of 13–15 mg/kg/day, up to two out of three patients with PBC may have a normal life expectancy without additional therapeutic measures. The mode of action of UDCA is still under discussion, but stimulation of impaired hepatocellular and cholangiocellular secretion, detoxification of bile, and antiapoptotic effects may represent key mechanisms. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarizes current knowledge on the clinical, diagnostic, pathogenetic, and therapeutic aspects of PBC. Primary biliary cirrhosis (PBC) [1] is an immune-mediated chronic progressive inflammatory liver disease that leads to the destruction of small interlobular bile ducts, progressive cholestasis, and, eventually, fibrosis and cirrhosis of the liver without medical treatment commonly necessitating liver transplantation. Addison and Gull [2] have first described a disease with a PBC-like picture in 1851, but the term "primary biliary cirrhosis" was coined in 1949 when a cohort of 18 patients with characteristic features of PBC was published [3] . PBC, predominantly affecting middle-aged women, is characterized by biochemical markers of cholestasis, serum antimitochondrial autoantibodies (AMA), and lymphocytic infiltration of the portal tracts of the liver [4] . Histologically, the hallmark of the disease is damage to biliary epithelial cells (BEC) and loss of small intrahepatic bile ducts accompanied by significant portal tract infiltration with CD4 and CD8 T cells, B cells, macrophages, eosinophils, and natural killer cells [5, 6] .
Being one of the first conditions in which specific autoantibodies were recognized, PBC is regarded as a "model autoimmune disease." Both environmental factors and inherited genetic predisposition appear to contribute to its pathogenesis [7] . Although there has been tremendous progress in unraveling potential pathophysiologic factors in PBC over the past years [7] , the actual impact of each of the identified genetic and environmental associations is still controversial. It is the aim of this article to review the current knowledge of major pathologic features in PBC and to try to combine these findings to an overall picture of the pathogenesis of PBC.
The most frequent symptoms in PBC are fatigue and pruritus, occurring in up to 85% and 70% of patients, respectively [8, 9] . Median survival in untreated individuals has been reported to be 7.5 to 16 years [1, 10] , but has largely improved since the introduction of ursodeoxycholic acid (UDCA) therapy and liver transplantation. Patients that are treated with UDCA at an early stage of the disease and respond well to therapy may reach normal life expectancy [11] [12] [13] [14] . However, the beneficial mechanisms of UDCA treatment are still incompletely understood, and about one third of patients fail to adequately respond to UDCA monotherapy. In the second part of this review, we will therefore summarize the rationale behind UDCA therapy and give an overview on future therapeutic options currently under study.
PBC occurs in individuals of all ethnic origins and accounts for up to 2.0% of deaths from cirrhosis [15] . It primarily affects women with a peak incidence in the fifth decade of life, and it is uncommon in persons under 25 years of age. Incidence and prevalence vary strikingly in different geographic regions (as does the quality of epidemiological studies related to PBC), ranging from 0.7 to 49 and 6.7 to 402 per million, respectively [16] [17] [18] [19] [20] [21] [22] [23] . The highest incidence and prevalence rates are reported from the UK [16, 21] , Scandinavia [17] , Canada [18] , and the USA [19, 22] , all in the northern hemisphere, whereas the lowest was found in Australia [20] . There is no clear evidence to support or exclude the concept of "a polar-equatorial gradient," as it has been reported for other autoimmune conditions [24] .
Increased awareness of the condition and the increasing availability of diagnostic tools, in particular serological testing, have led to a more frequent and earlier diagnosis of PBC [25] . More than half of patients diagnosed today with PBC are asymptomatic at presentation [26, 27] . They generally attract attention by findings of elevated serum alkaline phosphatase (AP) and/or total serum cholesterol, especially in asymptomatic patients often during routine checkup. A diagnosis of PBC is made "with confidence" when biochemical markers of cholestasis, particularly alkaline phosphatase, are elevated persistently for more than 6 months in the presence of serum AMA and in the absence of an alternative explanation [28, 29] . In PBC patients, AMA is generally present in high titer. Low-titer AMA may not be specific and may disappear on retesting [30] .
Compatible histological findings confirm the diagnosis and allow staging before therapeutic intervention, but in many cases, histological workup is not necessary to diagnose PBC [29] .
Serum AP and γGT are commonly elevated and define, together with AMA, the diagnosis of PBC. Mildly elevated serum aminotransferases (ALT, AST) are usually observed in PBC but are not diagnostic. Increased serum levels of (conjugated) bilirubin as well as alterations in prothrombin time and serum albumin are late phenomena in PBC like in other cirrhotic states and unusual at diagnosis. However, serum bilirubin is a strong and independent predictor of survival [31] with a high impact on all established models for prognosis.
Serum cholesterol is commonly elevated in patients with PBC, alike other cholestatic conditions. The increased cholesterol level in PBC is largely caused by the presence of LpX [32] . LpX is an abnormal lipid particle which is characteristic for cholestatic liver disease and that is directly derived from biliary lipids that regurgitate into the blood [33] . Upon lipoprotein fractionation, LpX is usually found in the very low-density lipoprotein fraction but is very different from other lipoproteins. In contrast to normal lipoproteins, which have a core filled with neutral lipid, LpX consists of liposomes (with an aqueous lumen) of phospholipids and free cholesterol. LpX is not taken up in atherosclerotic plaques and may reduce the atherogenicity of low-density lipoprotein (LDL) cholesterol by preventing LDL oxidation [34] . Accordingly, the increased serum cholesterol in PBC patients is not associated with increased risk for cardiovascular disease. In contrast, long-lasting PBC is associated with the occurrence of xanthomata and xanthelasma. Hypercholesterolemia in PBC patients is responsive to statin treatment. Long-term treatment with UDCA also reduces serum cholesterol [35] .
Though not diagnostic, thyroid-stimulating hormone levels should be assessed in any patient who is believed to have PBC due to the high association of PBC with thyroid dysfunction, mainly caused by Hashimoto thyroiditis.
AMA autoantibodies are pathognomonic for PBC and lead to the diagnosis with a high specificity and sensitivity. AMA-positive individuals, even if no signs of cholestasis and/or liver inflammation are present, are very likely to develop PBC. Mitchison et al. [36] , in a small study, evaluated liver pathology of 29 asymptomatic AMApositive (levels>1:40) individuals lacking AP elevation. At inclusion, all but two had abnormal liver histology, and in 12, findings were diagnostic for PBC. A 10-year followup of these subjects revealed that 24 of the 29 remained AMA-positive; all 24 developed biochemical evidence of cholestasis and 22 became symptomatic [37] , confirming a high positive predictive value of positive AMA testing for the development of PBC.
Sensitivity of AMA, however, although high, is limited. Investigators have reported patients who clinically, biochemically, and histologically have all the features of PBC despite consistently negative AMA testing both by immunofluorescence and with the most specific immunoblotting and immunoenzymatic techniques [38] [39] [40] [41] [42] . Overall, AMA seems to be negative in 5% of patients who otherwise have all the features typical for PBC [43] and an identical autoreactive CD4 T cell response to the critical autoantigen, PDC-E2 [44] . This small group of AMA-negative patients, however, may erroneously include patients with PBC-like symptoms induced by causes other than autoimmunity, such as patients with mutations in the ABCB4 (MDR3) gene [45] . These patients secrete reduced amounts of phospholipids into the bile, which is harmful to hepatocytes and cholangiocytes.
The pattern of serum immunoglobulin fractions in PBC is characterized by an elevation of serum IgM [46] , possibly due to an abnormal chronic B cell activation by Toll-like receptor-dependent signaling [47] . However, specificity of this finding is limited and IgM levels are not commonly used as a diagnostic criterium.
Nonspecific antinuclear antibodies (ANA) and/or smooth muscle antibodies are found in serum of one third of patients with otherwise clear-cut PBC [48] , but are of limited diagnostic value. In contrast, specific ANA directed against nuclear body or envelope proteins such as anti-Sp100, presenting as multiple (6) (7) (8) (9) (10) (11) (12) nuclear dots at indirect immunofluorescence staining and anti-gp210, presenting as perinuclear rims have shown a specificity of >95% for PBC, although their sensitivity is low. These specific ANA can be used as diagnostic markers for PBC in the absence of AMA titers [29] .
Ultrasound examination of the liver and biliary tree is obligatory in all cholestatic patients in order to differentiate intrahepatic from extrahepatic cholestasis. When the biliary system appears normal and serum AMA are present, no further radiologic workup is necessary. Abdominal lymphadenopathy, particularly in the hilar region of the liver, is seen in 80% of patients with PBC [49] .
Transient elastography (TE) has been introduced as a new, simple and noninvasive imaging technique for determining the degree of fibrosis in patients with chronic liver diseases, mainly chronic hepatitis C [50] . Corpechot et al. [51] compared liver stiffness as determined by TE (Fibroscan R ) to histological findings obtained by liver biopsy in 101 patients with PBC (n = 73) or primary sclerosing cholangitis (PSC; n = 28) and showed a highly significant correlation of liver stiffness with both degree of fibrosis and histological stage. Further studies in independent cohorts of PBC patients are warranted before TE can be regarded as an established alternative to liver biopsy in the staging of chronic cholestatic liver disease. Still, TE appears attractive as a screening tool in future therapeutic trials, as it may help overcome the limited staging accuracy of liver biopsy due to heterogeneous distribution of inflammation and fibrosis in PBC.
A liver biopsy is not anymore regarded as mandatory for the diagnosis of PBC in patients with elevated serum markers of cholestasis and positive serum AMA [28, 29] , but may be helpful in excluding other potential causes of cholestatic disease and in assessing disease activity and stage. A liver biopsy may also be helpful in the presence of disproportionally elevated serum transaminases and/or serum IgG levels to identify additional or alternative processes. Histological staging of PBC (stage 1 to stage 4) is determined by the degree of (peri)portal inflammation, bile duct damage and proliferation, and the presence of fibrosis/cirrhosis according to Ludwig et al. [52] and Scheuer [53] . Stage 1 disease is characterized by portal inflammation with granulomatous destruction of the bile ducts, although granulomas are often not seen. Stage 2 is characterized by periportal hepatitis and bile duct proliferation. Presence of fibrous septa or bridging necrosis is defined as stage 3 and cirrhosis as stage 4 [52] . Findings of fibrotic or cirrhotic changes (stage 3 or 4) are accompanied by a worse prognosis [54] . Florid duct lesions as defined by focal duct obliteration and granuloma formation are regarded as typical for PBC. The liver is not uniformly involved, and features of all four stages of PBC can be found in one biopsy specimen. The most advanced histological features are used for histological staging.
At diagnosis, the majority of patients are asymptomatic and present e.g. for workup of elevated serum levels of AP or cholesterol [55, 56] . In symptomatic patients, fatigue and pruritus are the most common complaints and have been reported in 21% and 19% of patients at presentation, respectively [27, 57] . Unexplained discomfort in the right upper quadrant of the abdomen has been reported in approximately 10% of patients [58] . In the majority of asymptomatic and untreated patients, overt symptoms develop within 2 to 4 years, although one third may remain symptom-free for many years [27, 56] .
During the course of the disease, up to 80% of PBC patients complain of chronic fatigue impairing quality of life and interfering with daily life activities [8, 59] . No correlation with the severity of the liver disease could be demonstrated [59] , but there is an association with autonomic dysfunction (in particular orthostatic hypotension) [60] , sleep disturbance and excessive daytime somnolence [60] , and, although weak, depression [61] , which might necessitate treatment for themselves. The exact pathophysiological mechanisms leading to chronic fatigue in PBC and other cholestatic diseases are not unraveled so far. Standard therapy of PBC with UDCA and even liver transplantation may fail to improve this often disabling symptom.
PBC is more frequently associated with pruritus than other chronic cholestatic liver diseases. During the course of the disease, pruritus occurs in 20% to 70% of patients and can often be the most distressing symptom [62] . It develops independently of the degree of cholestasis and the stage of disease. Its pathogenesis remains poorly understood and the potential pruritogens in cholestasis are undefined. The therapeutic efficacy of anion exchange resins like cholestyramine, the pregnane X receptor agonist, rifampicin, plasmapheresis, and albumin dialysis as well as nasobiliary drainage led to the conclusion that in cholestasis putative pruritogens accumulate in the circulation, are secreted into bile, and undergo an enterohepatic cycle. Itch could then be induced locally in the skin or in neuronal structures. Bile salt metabolites, progesterone metabolites, histamine, and endogenous opioids, among others, have all been proposed as causative agents. However, evidence for a key role of any of these suggested pruritogens in cholestasis is weak [63] .
Variceal hemorrhage or other signs of portal hypertension secondary to fibrosis or cirrhosis are uncommon at first presentation, but have occasionally been described [64] . However, portal hypertension as determined by measurement of the portohepatic pressure gradient (PHG) is common in PBC, and a stable or -under treatmentimproved PHG is a predictor of survival [65, 66] .
Patients with PBC have been reported to be at increased risk of osteoporosis in some studies [67] , but reports in the literature remain contradictory. Patients with advanced PBC may be at particular risk for osteoporosis and may present with osteoporosis and yet be otherwise asymptomatic from their liver disease. The development of osteoporosis in PBC patients has been attributed to both, decreased osteoblast activity and increased osteoclast activity [68] . Metabolism of vitamin D is normal in PBC, but malabsorption of both calcium and vitamin D may occur predominantly in latestage disease. Pancreatic insufficiency and celiac disease, which are associated with PBC [69] [70] [71] , may further aggravate malabsorption.
When secretion of bile and bile salts is insufficient, i.e., bile salts drop below the critical micellar level in the duodenum, malabsorption of both fat and fat-soluble vitamins may ensue. Serum levels of vitamin A and E have been shown to be low in a minority of patients with primary biliary cirrhosis prior to development of jaundice [72] . Osteomalacia is barely seen in PBC, as a liver transplant is performed in most patients before the development of this complication of prolonged deep jaundice.
Though often asymptomatic, recurrent urinary tract infections have been reported in up to 19% of women with PBC [73] , and a potential pathophysiologic relevance of Escherichia coli strains has been suggested.
Reports on the rate of breast carcinoma in women with PBC differ and reported either an increase in risk [74, 75] or equal risk [18, 76] compared to a healthy population.
Hepatocellular carcinoma in late-stage PBC has been reported to occur at rates similar to other kinds of cirrhosis [77] [78] [79] , but seems to be more frequent in men with PBC:
One study reported an average HCC prevalence of 5.9% in advanced PBC (4.1% in women but 20% in men) [77] .
A number of mostly immune-mediated diseases are commonly observed in patients with PBC. Thyroid dysfunction is frequently associated with PBC, often predating its diagnosis [80] . Sicca syndrome is seen in up to 70% of patients [81] . Incomplete or complete CREST syndrome (calcinosis cutis, Raynaud syndrome, esophageal motility disorder, sclerodactyly, teleangiectasia) is not uncommon [82] . Celiac disease has been reported in up to 6% of patients [72] and is by far more commonly associated with PBC than inflammatory bowel diseases [83] .
A florid bile duct lesion with damage to BEC and subsequent destruction of small bile ducts is the histopathologic hallmark of PBC. The exact pathogenetic mechanisms responsible for BEC damage in PBC remain unknown. Increasing experimental evidence, however, suggests that the florid bile duct lesion in PBC is initiated by environmental trigger(s) acting on a genetically susceptible individual [84] .
Genetic factors have an impact on PBC pathogenesis that is stronger than that in nearly any other autoimmune disease [85] [86] [87] . Accordingly, a concordance rate of about 60% was seen in monozygotic twins (as opposed to nearly 0% for dizygotic twins) [88, 89] . A significantly increased incidence of PBC is seen in relatives of PBC patients [89, 90] . The relative risk of a first-degree relative of a PBC patient is 50-to 100-fold higher than for the general population [91] , yielding a prevalence rate up to 5-6% [19, 90, 92] . Interestingly, among affected monozygotic twins, though the age of disease onset is similar, progression and disease severity vary, emphasizing the role of epigenetic and probably environmental factors [89] .
It has been difficult to identify distinct susceptibility genes in PBC so far. Genetic associations in PBC were shown with major histocompatibility complex encoded genes. PBC is apparently associated with the DRB1*08 family of alleles, although marked variation is observed between different ethnic groups. Association with the DRB1*0801-containing haplotype is seen in populations of European origin, whereas in populations of Asian origin, an association is seen with the DRB1*0803 allele. A protective association has been described with DRB1*11 and DRB1*13, but once again, significant population differences are observed [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] . Both associations of PBC with the DRB1*08 allele as well as the protective association of DRB1*11 and DRB1*13 were recently confirmed in the largest series ever reported including 664 unrelated patients from Italy [103] . The odds ratio for developing PBC was 3.3 for DRB1*08-positive subjects, whereas it was reduced to 0.3 for subjects positive for DRB1*11, to 0.7 for DRB1*13, and to 0.1 for carriers of both DRB1*11 and DRB1*13. This study again highlighted the relevance of geographic variation, with marked differences in allele association between northern and southern Italy.
Associations have also been reported with polymorphisms of genes involved in innate or adaptive immunity. Allelic variations of tumor necrosis factor α (TNFα) and of cytotoxic T lymphocyte antigen 4 (CTLA-4), a key regulator of the adaptive immune system, have been repeatedly associated with susceptibility to different autoimmune diseases (such as type I diabetes mellitus and systemic lupus erythematosus) and were also associated with PBC [104] [105] [106] [107] [108] [109] . Although an association of CTLA-4 variants and susceptibility to PBC could not be demonstrated in all studies [110, 111] , Poupon et al. [112] most recently confirmed a potential role of TNFα and CTLA-4 variants in the pathogenesis of PBC. In 258 PBC patients and two independent control groups of 286 and 269 healthy volunteers, the authors investigated distribution of newly identified htSNPs of 15 selected candidate genes: two related to immunity encoding for CTLA-4 and TNFα, ten genes related to bile formation encoding hepatobiliary transporters, and three related to adaptive response to cholestasis encoding nuclear receptors. In a case-control analysis, only haplotype-tagging single nucleotide polymorphisms (htSNPs) in CTLA-4 and TNFα showed differences in distribution between PBC and controls, confirming their potential role in the pathogenesis of PBC. In contrast, htSNPs of the ten transporter genes as well as the three nuclear receptor genes under study were equally distributed, confirming previous studies of htSNPs in key transporters without major impact [1, 113, 114] . A strong association of the allelic variant TNFα rs 1799724 (C/T) with disease progression was shown. Most interestingly, a strong association with disease progression was also shown for AE2 rs 2303932 (T/A), a gene encoding for the apical anion exchanger 2 (AE2) in cholangiocytes and hepatocytes. In both cases, presence of the variant was associated with delayed disease progression. In a multi-variate Cox regression, the AE2 variant rs2303932 (T/A) was an independent prognostic factor for disease progression in PBC under UDCA treatment, in addition to serum bilirubin, alkaline phosphatase, and serum albumin levels which are established surrogate markers of prognosis in PBC [112] . Most recently, a landmark genetic association study was published [332] . Associations with the risk of disease were unravelled for 13 loci across the HLA class II region, two SNPs at the interleukin 12 alpha (IL12A) locus, one SNP at the interleukin 12 receptor beta 2 locus, and one previously described SNP at the CTLA4 locus. Associations with more than 10 further loci were described. Various associations with other loci have been described in individual populations, mostly of limited size. However, the vast majority have not been confirmed in independent cohorts, and to date, none of the genetic associations described in PBC have been proven sufficiently [84, 88, 108] .
Future genetic linkage studies in affected families as well as association studies in large cohorts of unrelated patients may disclose genetic variants conferring susceptibility or influencing progression and severity of disease. Such linkage studies are awaited for PBC [115] .
It remains speculative whether the female preponderance (gender ratio up to 10:1) reflects an X-chromosomelinked locus of susceptibility. Alternatively, a protective role of Y-linked genes could be assumed, or just a gender-specific exposure to environmental triggers like cosmetics [116] or nail polishers [117] , as discussed below. However, speculation on a pathomechanistic role for X chromosomal genes was supported by the observation of an increased frequency of X chromosome monosomy in PBC as well as in other autoimmune diseases. This increase in X chromosome monosomy might lead to haploinsufficiency for specific X-linked genes and thereby increase disease predisposition [118] . Case reports of PBC in patients with Turner syndrome (45, X0) also supported this hypothesis [119] .
Estrogen signaling has also been proposed to play a role in the homeostatic proliferative response of cholangiocytes in PBC. Accordingly, studies on polymorphisms in estrogen receptor genes revealed associations with the disease, at least in some populations [120] . At the tissue level, cholangiocytes from PBC patients in the earliest disease stages (but not cholangiocytes from normal controls) express estrogen receptors [121] . Agents able to modulate estrogen-receptor-mediated responses (such as tamoxifen) have therefore been proposed as novel, BEC homeostasis targeting therapies, and case reports support this hypothesis [122, 123] , but as yet, this potentially interesting therapeutic approach has not undergone formal assessment in clinical trials.
Most recently, altered expression of hepatic microRNA (miRNA) has been described in liver tissue of PBC patients [124] . Certain miRNA negatively regulate protein coding gene expression and may play a critical role in various biological processes. However, a causal link between altered miRNA expression and the development of PBC still remains unproven.
Despite strong evidence for a genetic background in PBC, epidemiological studies have early suggested a role for environmental factors in triggering and/or exacerbating PBC [20, 125, 126] . A significant role for environmental factors was supported by the identification of geographic disease "hot spots," as first reported in the northeast of England, using formal cluster analysis. The original UK analysis reported an increased frequency of PBC in former industrial and/or coal mining areas [127] . Another recent study from New York examined the prevalence of PBC and PSC near superfund areas and reported significant clusters of PBC surrounding toxic sites [128] . In synopsis, these observations gave rise to the hypothesis of a chemical environmental factor, potentially associated with contaminated land, which could either trigger disease or cause disease through a direct toxic effect [84] . This hypothesis would also provide one possible explanation for the tissue tropism of PBC if the toxin or toxins are excreted into bile (and thereby concentrated in the biliary tree) [84] . The observation that hormone replacement therapy and frequent use of nail polish are linked to the risk of developing PBC further supports the potential impact of environmental factors in the pathogenesis of PBC [117] . Smoking also seems to be a risk factor for PBC and has been demonstrated to accelerate progression [117, 129] . Associations of exposure to chemical environmental compounds and xenobiotics (including drugs, pesticides, or other organic molecules) with various human autoimmune diseases have been described as summarized in [86] .
Xenobiotics may contribute to the pathogenesis of PBC by triggering autoimmune reactions. Different mechanisms for the induction of autoimmunity by xenobiotics have been proposed [86, 130] . A potential direct toxic effect of xenobiotics my cause cell death by apoptosis or oncosis, inducing the generation of immunogenic autoepitopes. In addition, chemical modification of native cellular proteins by removal and/or exchange of a hapten has been shown to change processing in antigen-presenting cells and may lead to the presentation of cryptic, potentially immunogenic peptides. Furthermore, xenobiotics may have the potential to modify host proteins to form neoantigens. Neoantigenspecific T cells and B cells, once primed, may cross-react with the formerly inert native autoantigens. In accordance with this hypothesis, Amano et al. [131] studied a number of xenobiotics with a structure similar to lipoic acid, a residue on the E2 epitope of the pyruvate dehydrogenase complex (PDC-E2), the main autoreactive antigen identified in PBC so far in PBC. Replacement of lipoic acid by certain xenobiotics enhanced the reactivity of PBC sera against the PDC-E2 epitope. Particularly, one of the xenobiotics, 2-nonynoic acid, induced reactivity of PBC sera stronger than that of the native lipoic acid residue. Interestingly, the methyl ester of 2-nonynoic acid has a viol-/peach-like scent and is used as an ingredient in perfumes. It is ranked 2,324th out of 12,945 chemical compounds in terms of occupational exposure with an 80% female preponderance due to its use in cosmetics.
Among the environmental factors that have been suggested as potential causative agents in PBC, particularly different bacteria have been discussed. In early histologic lesions in PBC, non-caseating granulomas are observed, as seen in other granulomatous liver diseases including sarcoidosis [1] , drug reactions, and, most interestingly, infections. Furthermore, non-caseating granulomas are unique to PBC when compared to other autoimmune pathologies. This has led to suspicion of a microbial basis for PBC [132] . In support of this hypothesis, certain bacteria were found to contain PDC components fully cross-reactive with the mammalian form. It was proposed that exposure to these homologues could trigger crossreactive immunity. In favor of a bacterial etiology, recent data suggest that Toll-like receptor ligands induce an augmented inflammatory response in PBC. In combination, presence of cross-reactive antigens in a proinflammatory environment would theoretically be able to break tolerance [133, 134] .
With this theoretical background in mind, early studies associating various bacteria with PBC re-attract interest: E. coli has been reported to be present in excess in the feces of patients with PBC. In addition, the incidence of urinary tract infections often induced by E. coli is high in PBC patients [73, 135] , and history of urinary tract infections increases the risk of PBC [117] . Another microorganism that has been proposed as a candidate for the induction of PBC is Novosphingobium aromaticivorans [136] . Titers of antibodies against lipoylated bacterial proteins of this ubiquitous organism, which metabolizes organic compounds including estrogens, were 1,000-fold higher compared to those against E. coli in patients with PBC, but no antibodies were observed in a large cohort of healthy subjects. Lactobacilli and Chlamydia, which show some structural homology with the autoantigen (although reactivity against them is considerably less than that against either E. coli or N. aromaticivorans), have also been implicated as putative pathogens, as have Helicobacter pylori and Mycobacterium gordonae [137] [138] [139] [140] . Recently, a case of PBC following lactobacillus vaccination for recurrent vaginitis was reported. The vaccine contained Lactobacillus salivarius, which exerts a high homology to the beta-galactosidase of Lactobacillus delbrueckii (LACDE BGAL [266] [267] [268] [269] [270] [271] [272] [273] [274] [275] [276] [277] [278] [279] [280] , and cross-reactivity of patients' autoantibodies against the human PDC-E2 212-226 epitope and LACDE BGAL [266] [267] [268] [269] [270] [271] [272] [273] [274] [275] [276] [277] [278] [279] [280] was found. Affinity to the Lactobacillus epitope was higher than to the native mammalian, suggesting that antimicrobial reactivity may have preceded that to the self-mimic [141] . However, the AMA status of this patient before repetitive lactobacillus vaccination could not be assessed and causal relation of lactobacillus exposure and development of PBC remains speculative also in this study.
Despite these intriguing associations, no compelling data have been provided to show that one individual infectious agent can reproducibly be detected in patients with PBC. Although attractive, the model of bacterial infections as cause of PBC is thus supported by little direct evidence. Further objective data are warranted, obtained either from prospectively followed cohorts or through case-control epidemiological approaches, confirming a role for bacteria in triggering PBC [84] .
An alternative infectious agent has recently been proposed as trigger of PBC when a human retrovirus was identified both in liver tissue and hilar lymph nodes from PBC patients. EM analysis of liver tissue obtained from PBC patients revealed retrovirus compatible particles in BECs. In periportal lymph nodes, mouse mammary tumor virus (MMTV) was detected and correlated with aberrant distribution of PDC-E2 in perisinusoidal cells. Homogenates of these periportal lymph nodes also had the capacity to infect BEC cultures inducing marked phenotypic change, and this effect could be abolished by irradiation of the culture media, suggestive of an infectious agent [142, 143] . Retroviral infection hypothetically could cause BEC damage either through a direct viral cytopathic effect, through cross-reactivity between viral protein and self-PDC, a "molecular mimicry" model, or virus-induced apoptosis [84] . Retroviral infection would also provide explanations for some key phenomena in PBC. PBC can recur rapidly after transplantation with all of the clinical manifestations including the detection of AMA in serum [144] , the aberrant expression of the AMA-reactive protein on BEC [145] , and histologic evidence of disease in up to 45% of patients [146] . In this respect, the observation of an association of more potent immunosuppressive therapy following transplant and earlier and more aggressive recurrence of PBC [147] is also of interest. MMTV replication is regulated in part by a progesterone-responsive glucocorticoid regulatory element in the promoter region, offering an alternative explanation for the female preponderance seen with PBC [148] .
These findings attracted attention in the field and were acknowledged by other investigators who pointed out the need for clinical trials with antiretroviral therapies [149] . Subsequently, in a small non-randomized pilot study, therapy with Combivir (lamivudine + zidovudine) improved inflammatory scores, normalized AP, and reduced bile duct injury in patients with PBC [150] . These findings await confirmation in a randomized, controlled trial.
Unfortunately, major findings of the outlined in vitro studies could not be reproduced by independent groups, and others raised concerns that these findings might mainly reflect contamination or technical artifacts [151] . In an independent study, a large number of sera of PBC patients and healthy controls did not show reactivity against MMTV encoded protein, and no detectable immunohistochemical or molecular evidence for MMTV was found in liver specimens or peripheral blood lymphocytes [152] . It was also speculated that beneficial effects of antiretroviral therapy could be partly explained by anti-apoptotic properties of nucleoside analogs [151, 153] . Furthermore, mechanisms by which human betaretrovirus would enter human cholangiocytes are also not identified.
A more recent study, however, strengthened the case for involvement of retroviruses in (immune-mediated) liver disease: Sera of 179 patients with diverse chronic liver diseases and 31 controls were tested for reverse transcriptase activity and presence of human betaretrovirus by polymerase chain reaction. Reverse transcriptase activity was detected in 73% of autoimmune hepatitis patients, 42% of PBC subjects, 35% of patients with viral hepatitis, 22% of liver patients without viral or autoimmune pathogenesis (non-alcoholic fatty liver disease and alcoholic liver disease), and 7% of control subjects. In polymerase chain reactions, 24% of PBC samples were positive for human betaretrovirus compared to 13% in autoimmune hepatitis, 5% in other liver diseases, and 3% in non-liver disease control subjects [154] . If these data can be confirmed, a retroviral compound in the pathogenesis of immunemediated and viral liver disease seems attractive, though not specific for PBC. Thus, despite some intriguing findings, the pathogenetic relevance of retroviruses in the development of PBC remains enigmatic.
Appendectomy, other abdominal surgeries, and tonsillectomy were significantly more frequently reported in patients with PBC in an epidemiological study in North America [155] . However, an earlier population-based case control study conducted in England did not show such associations [156] . More recently, another case-control study provided evidence that there was at least no association between PBC and the occurrence of appendectomy and pointed out the selection bias present in the previous study done in North America [157] . The linkage to appendectomy was theoretically attractive, since a PBC-specific immune response to the highly conserved caseinolytic protease P of Yersinia enterocolitica in 40% of patients with PBC was reported [158] . It is noteworthy, that infection with Y. enterocolitica is one of the major causes of acute terminal ileitis mimicking acute appendicitis [159] .
Autoimmunity is a phenomenon of dysregulated immune response against self-antigens. If persistent, this can result in inflammatory tissue damage. The immune response to antigens is tightly controlled by various pathways whose deregulation may lead to autoimmune responses. Genetic predisposition and environmental factors affect the susceptibility to such deregulation [86] .
Tolerance against self-antigens is achieved in the lymphopoietic differentiation in early life, when highaffinity self-reactive lymphocytes are deleted in the primary lymphoid organs, thymus, and bone marrow. Second, in the periphery, there is activity of a subset of T lymphocytes, T regulatory cells (Tregs), which are dedicated to regulatory function expressing the CD4 and CD25 surface markers and the transcription factor forkhead box P3 (FOXP3). Additional backups along the maturation process of lymphocytes are described, limiting the induction and expression of autoimmunity. These regulatory mechanisms include apoptosis pathways, cytokines and their receptors, chemokine signaling, T cell-T cell interactions, and intracellular signal transduction. Accordingly, loss of self-tolerance could involve multiple faults, most of which are of genetic origin.
It is worth mentioning that autoimmunity, defined by the presence of autoantibodies and autoreactive lymphocytes does occur naturally. It appears that such naturally occurring autoantibodies and autoreactive lymphocytes are modulators for the suppression of early infections, clearance of apoptotic bodies, immune surveillance against cancer cells, among others, as reviewed in [160] . In this not yet completely unraveled system regulating immune response, the mechanisms responsible for the development of autoimmunity and autoimmune diseases remain enigmatic.
Imbalance of T cell regulation can be sufficient on its own to initiate or propagate autoimmunity in various chronic inflammatory diseases such as inflammatory bowel disease (IBD) or rheumatoid arthritis. In line with this concept, recent transgenic animal models highlight the role of T cell regulation in the development of autoimmune diseases. For instance, IL-2 receptor −/− mice were shown to develop severe anemia and IBD [161] , possibly due to the decreased numbers of Tregs facilitating autoimmune reactivity in the presence of proliferating (and probably, activated) T-cells. Absence of the IL-2 receptor in these animals leads to proliferation of T cells and decreased numbers of Tregs.
Another hypothesis is the concept of molecular mimicry based on the similarity of pathogen and host antigenderived epitopes recognized by the immune system [162, 163] , which render bacteria and viruses candidates for the induction of autoimmune disease. This mechanism has first been suggested to be responsible for the development of rheumatic fever, and though this could never be confirmed, there is evidence suggesting associations of infectious triggers for several systemic autoimmune diseases including multiple sclerosis [164, 165] , systemic lupus erythematosus (SLE) [166] , and rheumatoid arthritis [167] . Autoantigens are unable to elicit a primary immune response themselves. However, T cells stimulated by a pathogenic cross-reactive epitope can recognize such targets. As a prerequisite, the pathogen-derived cross-reactive epitope has to be sufficiently different from the host-derived epitope. The role of infectious agents in development of autoimmunity has recently been reviewed elsewhere [168, 169] .
Xenobiotics represent another environmental factor foreign to human organisms, and may induce immune reactions or have the potential to modify host proteins and render them more immunogenic. Examples include drugs, pesticides, or other organic molecules. A number of xenobiotics have been associated with several human autoimmune diseases. Chemicals which were linked to autoimmunity include mercury in glomerulonephritis [170] , hydrazines in SLE [171, 172] , iodine in autoimmune thyroditis [173] , and halothane in drug-induced hepatitis [174, 175] . Halothane-induced liver disease occurs when susceptible individuals develop immune response against trifluoroacetylated (TFA) self-proteins upon halothane exposure. Noteworthy is that the lipoylated E2 domain of human PDC is also recognized by anti-TFA [176] .
As a further trigger of autoimmunity, it has been speculated that increased cell turnover or, more specifically, increased cell apoptosis may lead to exposure of otherwise rarely exposed antigens and induction of immune response to self. Enhanced apoptosis has been implicated in several autoimmune diseases, including Hashimoto's thyreoiditis [177] . This hypothesis would also provide an appealing explanation for the tissue specificity of most autoimmune reactions despite the often ubiquitous expression of the targeted autoantigen.
However, apoptosis is genuinely designed to actually prevent inflammatory reactions to cell death, and ingestion of apoptotic cells by macrophages induces the expression of anti-inflammatory cytokines such as TGF-β and IL-10, both promoting the expression of Tregs, suppressing an autoimmune response during apoptosis [160] . While apoptosis per se is a non-inflammatory process, it can lead to abnormal antigen presentation, especially of previously sequestered antigens. Evidence for a role in the development of autoimmune disease, however, is limited and particularly in organ-specific autoimmune diseases [178] . Whether apoptosis-related mechanisms lead to PBC is unclear [179] , but cholangiocytes in PBC may undergo increased cell turnover, e.g., due to metabolic stress, resulting in an inadequate immune response [179] . Strikingly, BECs in patients with PBC seem to be under significantly increased apoptotic stress compared to healthy controls or patients with other causes of inflammatory reactions in the liver, such as chronic viral hepatitis or PSC [180] [181] [182] . However, it is yet to be elucidated whether this effect is really a cause of autoimmunity in PBC or rather the consequence of increased inflammation.
PBC is associated with other autoimmune diseases, both within individuals and among families, reflecting the "clustering" characteristic for autoimmunity [183] . PBC was one of the first conditions in which the presence of autoantibodies in the serum was identified and in which the antigen specificity of this autoreactive response was characterized [5] . It is therefore often referred to as a "model autoimmune disease." The predominant autoreactive antibodies in PBC are AMAs, which, with a high sensitivity and specificity, are virtually diagnostic for PBC when detected in serum. The so far identified targets of AMA are all members of the family of 2-oxo-acid dehydrogenase complexes (2-OADC). This includes the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), the 2-oxo-glutaric acid dehydrogenase complex (OGDC-E2), and the dihydrolipoamide dehydrogenase binding protein (E3BP) [184] , all localized within the inner mitochondrial matrix, catalyzing oxidative decarboxylation of keto acid substrates. The targeted E2 subunits all have a common N-terminal domain containing single or multiple attachment sites for a lipoic acid cofactor to lysine. Previous studies have demonstrated that the dominant epitopes recognized by AMA are all located within these lipoyl domains of the target antigens [185] [186] [187] [188] . The autoreactive CD4 and CD8 T cells infiltrating the liver in PBC recognize the same PDC-E2 domain (peptides 159-167 and 163-176) [189, 190] , and the same accounts for the dominant autoreactive B cells [191] . CD8 T cells isolated from livers of patients with PBC have been found to exert cytotoxicity against PDC-E2 pulsed autologous cells (peptides 159-167) [192] , supporting the hypothesis of a T cell response contributing to bile duct injury in PBC.
It remains a mystery how PDC-E2 and other epitopes localized to the inner membrane of mitochondria become targets of autoimmune injury in PBC. One working hypothesis, largely enforced by the Gershwin group, is that modifications of 2-OADC by xenobiotics may alter these self-proteins to cause a breakdown of tolerance facilitating an autoimmune response.
This group identified a 12-amino acid residue peptide of the inner domain of PDC-E2 containing the lipoic acid cofactor carrying 173 lysine to elicit the strongest reactivity of purified sera from PBC patients when compared to a number of other potential epitopes. They subsequently could show that reaction of the same PBC sera was significantly increased by lipoylation of this epitope [193] .
Subsequently, the identified PDC-E2 residue was modified by replacing lipoic acid with a series of similar but distinct synthetic structures. Following this modification, PDC-E2-specific autoantibodies from patients with PBC reacted with higher affinity to the modified epitopes than to the native PDC-E2 peptide. The structure inducing maximal affinity was demonstrated to be derived from 2-nonynoic acid, a compound widely used in cosmetics [116] .
Xenobiotics, in studies of the same group, were also shown to induce PBC-like features in different animal models. Immunization of rabbits with 6-bromohexanoate (6BH), coupled to bovine serum albumin (BSA), led to break of tolerance to PDC-E2 as judged by the detection of AMA [194] . In guinea pigs, immunization with the compound 6BH-BSA led to the development of histological lesions typical of autoimmune cholangitis with the concurrent appearance of AMA, albeit with a long latency of 18 months [195] . Most recently, these efforts resulted in the development of an inducible animal model of PBC in C57BL/6 mice. In these animals, 2-octynoic acid coupled to BSA after a short follow-up of 8-12 weeks induced manifest autoimmune cholangitis, typical AMA, increased liver lymphoid cell numbers, an increase in CD8 liverinfiltrating cells, and elevated levels of serum tumor necrosis factor α and interferon γ. Remarkably, unlike many other animal models of PBC, the reported immunogenic response was liver-specific, and no inflammation was found in organs other than the liver [196] . However, the model still has disadvantages, e.g., lacking the development of fibrosis.
An alternative but complementary concept for loss of self-tolerance in PBC is the idea of underlying immune deficits. This concept is based on clinical and experimental evidence. PBC exhibits clustering with various autoimmune disorders, both within individuals and family. Moreover, in PBC there are reduced levels of Tregs suppressing immune reactions against self [197] . In two genetically manipulated mouse strains, spontaneous occurrence of a PBC-like lymphoid cholangitis together with positivity for anti-PDC-E2 within several weeks of life was reported when either transgenic expression of a dominant negative TGF-β II receptor (dnTGF-β RII) or transgenic disruption of the IL-2 receptor alpha that is highly expressed on Tregs was performed [198, 199] . Interestingly, B cells had a suppressive effect on the inflammatory response in the dnTGF-β RII model of PBC [200] . A role for IL-2 signaling defects in development of PBC was supported by a report of a PBC-like liver disease in a child with inborn deficiency of IL-2 receptor alpha [201] . An additional mouse model of NOD.c3c4 was described to develop autoimmune biliary disease and was found to test AMA-positive [202] . In all models outlined here, the biliary epithelium is infiltrated with CD4 and CD8 T cells, whereas granulomas and eosinophilic infiltration are seen only in NOD.c3c4 mice.
Conversely, IL-17 has recently been shown to be involved in various autoimmune disorders. In PBC liver tissues, density of IL-17(+) CD4 lymphocytic infiltration (Th17) was higher than in healthy liver. Although enhanced density of Th17 cells is not specific to PBC, it is in line with the observation of the decreased number of Tregs and may illustrate the counterplay of Tregs and Th17 cells in PBC [203] .
There has been impressive progress in unraveling pathogenetic factors of PBC over the past decade. In favor of a genetic background, an outstanding concordance rate among monozygotic twins has been identified, as well as various genetic associations. Clustering of distinct HLA alleles and, among others, allelic variations of TNFα, CTLA-4, and anion exchanger 2 (AE2), respectively, have been described. In addition, there is compelling evidence for an environmental factor required for the development of PBC. Xenobiotics capable of modulating mammalian proteins to form neoantigens have been shown to induce pathologies resembling PBC in animal models. Furthermore, various infectious agents have been associated with PBC in humans, although direct experimental evidence for their role in the pathogenesis is limited. Lastly, disruption of AE2 [204] and genes involved in the regulation of immune response, such as the TGF-β II or IL-2 receptor or the AE2, gives rise to histologic and serologic changes mimicking PBC in different animal models and may be contributing to susceptibility for PBC in human.
However, a common causative pathway to PBC, if it exists, could not yet be identified, and it is still controversial which of the outlined factors predispose most to PBC. It may well be speculated that the search for "the cause" of PBC might lead into the dark, and the identified pathogenetic factors may contribute to a different extent in each patient. Why should the cause of PBC not be variable as is the clinical picture? The latter is highly variable: We define PBC today as the presence of cholestasis and AMAs, but only 90-95% of patients are AMA-positive and some AMA-positives hardly develop PBC. Most patients respond to UDCA treatment, but one third does not and disease progression is highly variable, to mention only the most obvious variabilities. The clinical picture that, by convention, we call PBC may well emerge from an individual composition of the described and other pathological factors.
Jones [84] suggested a model of PBC development distinguishing "upstream" from "downstream" events. "Upstream" in this model refers to the causes of BEC loss, ductopenia and cholestasis, which are unique to PBC (and probably unique to each individual patient) and include genetic and toxic factors, infectious agents, and immune-mediated events. "Downstream" of these initiating mechanisms, nonspecific pathologic events occur resulting in bile duct damage, hepatocyte injury, inflammation, and fibrosis independently of the primary -individual variablecause. "Downstream" events such as hydrophobic bile salt retention aggravate the underlying injury, further promoting hepatic and cholangiocellular damage [205] .
This pathogenetic model provides an explanation for the limited efficacy of immunosuppressive drugs in PBC. Agents such as prednisolone, in the treatment of PBC of limited efficacy, may mainly affect upstream mechanisms. In clinical trials, these agents have, however, largely been evaluated in relatively advanced, symptomatic patients. In these individuals, downstream processes may have become predominant.
The balance of evidence in PBC remains strongly in favor of an autoimmune process in which the autoreactive attack is directed at epitopes within self-PDC-E2. The following factors can, in principle, contribute to this breakdown of self-tolerance and have been suggested for PBC:
1. There is strong and compelling evidence to support molecular mechanisms of cross-reactivity between the PDC-E2 lipoic acid co-factor and environmental xenobiotics inducing auto-immunogenity of modified self-PDC-E2. Animal modeling data would argue that a cross-reactive B cell response induced to xenobioticmodified self-PDC can, through a process of epitope spreading driven by antigen-specific cross-reactive B cells, translate into the breakdown of T cell tolerance responsible for the effector T cell mechanisms thought to be directly responsible for BEC loss. The clinical implication of this observation again is that immunomodulatory approaches to therapy should play a role particularly in the earliest stages of PBC. 2. Alternatively or in addition, molecular mimicry mechanisms with cross-reactivity between self-PDC and bacterial or potentially viral structures may support or induce breakdown of tolerance.
3. Most data on genetic associations with disease point to loci or genes involved in immune function. Altered regulation of self-tolerance can possibly support or induce immune reaction against self-PDC-E2, expressed normally or aberrantly by BECs. 4. Primary events of cholangiocellular apoptosis or cell damage have been suggested that could lead to aberrant presentation of self-antigens or create an inflammatory environment potentially triggering immune dysregulation. Suggested mechanisms include metabolic stress, possibly triggered by dysfunction of transporters involved in cell maintenance like AE2. This mechanism was suggested [179] based on the finding that an AE2 variant is a strong and independent prognostic factor for disease progression in PBC under UDCA therapy [112] . Furthermore, secretion of directly toxic environmental compounds into the bile or viral infection would trigger cholangiocellular damage.
BECs, either as an associated process to outlined factors or as part of the homeostatic mechanism designed to retain BEC function, may lead to altered self-recognition.
Each of these mechanisms may occur simultaneously or sequentially and, to a variable extent, result in the breakdown of tolerance and immune-mediated liver pathology. Once an autoimmune reaction is initiated, various vicious cycles are conceivable. Inflammatory reaction secondary to loss of tolerance will lead to further cholangiocellular damage and apoptosis, increasing presentation of (altered) self-PDC and increase autoreactivity. Once bile duct loss and cholestasis are established, retention of bile salts and other toxic compounds perpetuates damage to BECs and subsequently to the entire liver. Maintenance of these vicious cycles may be supported by an immune system that is genetically dysregulated and insufficiently capable of suppressing autoimmune reactions.
The outstanding paradox in PBC pathogenesis remains the tissue tropism of the immune attack on the small intrahepatic bile ducts, although the mitochondrial targets are ubiquitously expressed proteins. An increased vulnerability of the primarily affected BECs therefore is a prerequisite in the pathogenesis of PBC. Staining of BECs from PBC livers with monoclonal antibodies against the mitochondrial PDC-E2 autoantigen showed a specific reaction at the apical surface which was not found in controls [206, 207] . Other non-PBC-related mitochondrial proteins show the expected cytoplasmic pattern [208] . It was subsequently demonstrated that the apical staining is due to a complex between (auto-)antimitochondrial IgA and PDC-E2, giving rise to speculations that IgA might be a player in the immune-mediated destruction of BECs [209, 210] . Apoptosis of BECs has been proposed as a cause of aberrant neoantigen presentation, responsible for activation or attraction of autoreactive T lymphocytes or antibodies. Unlike other cell types [211] for which autoantibody recognition of PDC-E2 is abrogated during apoptosis, probably by glutathiolation of the lysine-lipoyl moiety of PDC-E2, the antigenicity of PDC-E2 persists in the apoptotic BECs in which glutathiolation does not occur [211, 212] .
The course of apoptotic markers in patients with PBC peaks in the middle stages of the disease (stages II-III) rather than in earlier stages. This could solely support a role of apoptosis as a trigger for loss of self-tolerance, with probably only a little number of cells being affected rather than being the main initiating event in PBC. In a putative positive feedback loop, apoptosis, autoreactive T cell response, cholestatic bile salt retention, and failed replicative homeostasis may result in the destruction of BECs and bile duct structures.
UDCA has been administered in Chinese traditional medicine as a remedy for liver diseases and other disorders in the form of black bear's bile since the time of the T'ang dynasty (618-907AD) and was reestablished in the late 1950s in Japan as a choleretic agent with gallstonedissolving and anticholestatic properties. In the Western literature, the beneficial effects of UDCA on serum liver tests for patients with hepatic disorders were first reported in the 1980s [213, 214] , and UDCA has since been established for the treatment of PBC. Today, it is the only FDA-approved drug and standard therapy for PBC. UDCA was shown to improve serum biochemical markers such as bilirubin, AP, γGT, cholesterol, and IgM levels [215] [216] [217] [218] [219] [220] . UDCA may slow down histologic progression to liver cirrhosis [219, 221] , improve quality of life, survival free of transplant, and overall survival [11] [12] [13] [14] 222] . It is safe and side effects are few [223] . However, the mechanisms of action of UDCA in chronic cholestasis remain enigmatic [224] . About a third of patients is not sufficiently controlled with UDCA monotherapy [12, 13] , which drives the search for additional therapeutic approaches (Fig. 1) .
Cholestasis leads to retention of bile salts and other potentially toxic constituents of bile not only systemically but particularly in hepatocytes. This promotes hepatocellular damage and, in a chronic state, can induce the development of fibrosis and cirrhosis of the liver. As recently summarized by Paumgartner and Pusl [225] , the general biochemical and physiologic principles in treating cholestatic liver disease can be broken down to "(1) reduction of hepatocellular uptake of bile salts and other organic anions;
(2) stimulation of the metabolism of hydrophobic bile salts and other toxic compounds to more hydrophilic and less toxic metabolites; (3) stimulation of orthograde secretion into bile and (4) stimulation of retrograde secretion of bile salts and other potentially toxic cholephils into the systemic circulation for excretion by the kidney; (5) protection of injured cholangiocytes against toxic effects of bile salts; (6) inhibition of apoptosis caused by elevated levels of bile salts; and (7) inhibition of fibrosis" [225] .
Treatment with UDCA addresses most of these postulated principles as recent clinical and experimental data suggest [224, 226, 227] . Still, no relevant effect of UDCA on (1) bile salt uptake and (2) bile salt metabolism [228, 229] has been demonstrated in man.
Hepatic secretion When patients with PBC are treated with UDCA, serum levels of bilirubin [216] and endogenous bile salts [230] decrease. This effect of UDCA seems to be mediated by posttranscriptional rather than transcriptional mechanisms, as mRNA levels of key transporters like the conjugate export pump, ABCC2/MRP2, and the bile salt export pump, ABCB11/BSEP, are not affected by UDCA in man. Enhanced hepatic protein levels of BSEP, but not MRP2, have been observed under UDCA treatment in patients with gallstones [228] and may contribute to improved elimination of bile salts [231] . In an animal model of cholestasis, UDCA increases the density of Bsep and Mrp2 in the canalicular membrane of the rat by stimulating transporter targeting and insertion into the membrane [232] by a cooperative protein kinase C α/protein kinase A-dependent mechanism [224, [233] [234] [235] . An integrin-dependent dual signaling pathway involving mitogen-activated protein kinases (MAPK) Erk 1/2 and p38 MAPK has been shown to mediate UDCA-induced canalicular BSEP insertion in normal [236, 237] , but not cholestatic [238] hepatocytes. Whether UDCA-induced kinase-mediated phosphorylation of carriers contributes to enhanced canalicular transporter density and activity needs to be studied in more detail [224, 235, 239] .
Dilution of bile and the flushing of bile ducts were postulated as important functions of cholangiocellular bile formation [225] . In PBC, expression of the hepatic AE2 and biliary bicarbonate secretion by AE2 are impaired [240, 241] . UDCA stimulates both AE2 expression [242] and bicarbonate secretion [241] .
Retrograde secretion of bile salts and other potentially toxic cholephiles As an adaptive mechanism during cholestasis, basolateral conjugate transporters such as ABCC3/ MRP3 are upregulated and cholephiles such as bilirubin glucuronides, which are not adequately secreted into bile, can leave the liver cell via the basolateral route. In contrast to the postulated increase in retrograde secretion [228] , UDCA diminishes basolateral efflux due to the effective stimulation of orthograde secretion of potentially toxic compounds into the bile.
Protection of cholangiocytes Bile, with its high concentration of hydrophobic bile salts, exhibits extracellular cytotoxicity in vitro, but does not cause cholangiocyte injury under physiological conditions. In PBC, inflammatory bile duct injury may be aggravated by hydrophobic bile salts. Two mechanisms have been discussed by which UDCA protects the biliary epithelium against toxic effects of bile, i.e., relative reduction of hydrophobic bile salts and enrichment of phospholipids in bile. When administered at recommended doses of 13 to 15 mg/kg/day, UDCA content may rise up to 50% of total bile salts [243] and to an even higher percentage when the dose of UDCA is increased [244, 245] . In consequence, bile composition is shifted towards less toxic and less hydrophobic bile salts.
UDCA administration in patients with gallstones increases expression of MDR3, a phospholipid flippase, in the liver [228] . This might explain the stimulation of biliary phospholipid secretion by UDCA, as described in patients with PSC [246] . Secreted phospholipids form mixed micelles with bile salts, thereby mitigating their toxic effects on cholangiocytes.
Inhibition of apoptosis Hydrophobic bile salts induce hepatocellular apoptosis [247] [248] [249] by death receptordependent [250, 251] and -independent [252, 253] mechanisms, an effect that may become relevant when bile salts accumulate in the liver in cholestatic states. UDCA exerts anti-apoptotic effects in experimental in vivo models [247, 249, 252] as well as in vitro in primary human hepatocytes [254] . This anti-apoptotic effect may contribute to the alleviation of liver injury during UDCA treatment.
Inhibition of fibrosis Release of chemokines and cytokines by injured cholangiocytes and infiltrating inflammatory cells [255] as well as hepatic stellate cell proliferation induced by bile salts may play a role in fibrogenesis [256] . UDCA has been reported to delay development of severe fibrosis and cirrhosis in PBC [221] . This may be related to the aforementioned anticholestatic and anti-apoptotic effects rather than direct antifibrotic effects of UDCA. The bile salt derivatives 6-ethyl CDCA (6-ECDCA) and nor-UDCA inhibit fibrosis in the bile-duct-ligated mouse. While 6-ECDCA seems to mediate this antifibrotic effect via farnesoid X receptor (FXR) and small heterodimer partner (SHP) [257] , the mechanism of action of nor-UDCA remains yet unclear [258] .
Immunomodulatory properties of UDCA have been controversially discussed in the past [259] [260] [261] [262] . The glucocorticoid receptor in rat hepatocytes is activated by UDCA in a ligand-independent way, whereas suppression of IFN-γinduced MHC class II expression was found to be glucocorticoid-receptor-dependent [263] . It remains to be defined whether glucocorticoid receptor activation is unique to UDCA, and thus therapeutically relevant, or might be shared by endogenous bile salts.
Comprehensive reviews on molecular actions of UDCA have been published in the recent past [205, 224, [264] [265] [266] .
UDCA is currently considered the mainstay of therapy for PBC [28, 29] . Randomized, double-blinded, placebocontrolled trials have consistently shown that UDCA, administered today in standardized doses of 13 to 15 mg/kg/day, improves serum biochemical markers including bilirubin [215] [216] [217] [218] 220] , an important prognostic marker in PBC [267] . A number of studies demonstrated an improvement of histological features by UDCA. In a combined analysis of four clinical trials including a total of 367 patients [268] and in an independent in 103 patients [221] , UDCA therapy was associated with delayed progression and a marked decrease in progression rate from early-stage disease to late histologic stages. Despite these effects on disease progression and severity, the assessment of the effect of UDCA treatment on long-term survival has been more difficult. A combined analysis of three randomized controlled trials including 548 patients in total treated with UDCA for up to 4 years revealed improved survival free of liver transplantation in patients with moderate or severe disease [222] . Another combined analysis of five studies, all with a follow-up of at least 4 years, quantified the reduction in the risk of death or liver transplantation in patients treated with UDCA to 32% [269] . A long-term follow-up study in a cohort of 225 patients, in part overlapping with the aforementioned analyses, reported 10-year survival without liver transplantation to be significantly higher in UDCA-treated patients compared with survival predicted by the Mayo model [270] . Recent long-term trials from France, Spain, and The Netherlands have shown comparable results [11] [12] [13] 271] . The survival rate of patients in early stages of the disease who biochemically responded to therapy was similar to that in the control populations. "Response" here is defined as a decrease in AP to <40% of pretreatment levels or normalization at 1 year (Barcelona criteria) [12] or serum bilirubin <1 mg/dl, AP≤3 × N and AST≤2 × N after 1 year of UDCA treatment (Paris criteria) [13, 14] . An additional nonrandomized controlled study from Greece confirmed that in most patients with PBC treated with UDCA (particularly those who are in early stage of disease), the 10-year survival is comparable to that in the general population [272] . Despite these intriguing findings, the beneficial effects of UDCA on survival have repeatedly been questioned: A large randomized Swedish trial failed to confirm an effect of UDCA on disease progression and survival at a dose of approximately 8 mg/kg/day [273] . It was concluded from this and other studies that doses of UDCA lower than 10 mg/kg/day are of little benefit. In the follow-up of patients in a US [274] and a Canadian trial [217] , UDCA had no significant influence on incidence of endpoints liver transplantation or death. Also, a meta-analysis of 11 randomized trials could not confirm a significant effect of UDCA on survival and incidence of liver transplantation [275] . In this meta-analysis, however, six studies with only 2 years of follow-up were included, as were two studies administering UDCA at low doses of 10 mg/kg/day or less. Other meta-analyses suffered from similar shortcomings [276, 277] and may therefore have missed a beneficial effect of UDCA. Accordingly, metaanalyses which only included long-term trials with followup of at least 2 years and those using an effective dose of UDCA of more than 10 mg/kg/day verified that treatment with UDCA significantly improves quality of life and transplant-free survival and delays histologic progression in early-stage patients [278, 279] . Current guidelines therefore recommend to treat PBC with UDCA using doses of 13 to 15 mg/kg/day and to start treatment early [28, 29, 280] .
About two thirds of patients treated according to these recommendations respond adequately as defined by the Barcelona or the Paris criteria and may have a normal life expectancy. The most recent study from The Netherlands comprising 375 patients with a mean follow-up of 9.7 years again stressed the importance of early treatment showing a clear survival benefit for patients treated in early stages of disease with normal serum bilirubin and albumin levels at the start of therapy [14] .
For the remaining one third of patients who fail to achieve biochemical response according to Paris and Barcelona criteria or who are at an advanced histological stage at start of medical treatment, therapeutic options are limited to date and novel approaches are needed.
Corticosteroids and other immunosuppressive agents have been evaluated for therapeutic use in PBC. In a 3-year, placebo-controlled trial including 36 patients with PBC, prednisolone significantly improved serum AP levels, IgGs, and AMA and diminished deterioration of liver histology, whereas bone loss was aggravated [281] . Combination of UDCA and prednisolone in comparison to UDCA alone resulted in a significant improvement of histological features [282] . It is yet unclear whether increased expression of AE2 isoforms contributes to the beneficial effect of combined treatment with UDCA and corticosteroids in PBC. Experimentally, enhanced expression of alternative AE2 isoforms and enhanced transport capacity for bicarbonate in human cholangiocytes and in a hepatocyte cell line were demonstrated [283] . AE2 expression and biliary bicarbonate secretion is usually diminished in PBC [241] .
Serious side effects of long-term glucocorticoid treatment may outweigh the potential benefit. In this respect, the introduction of budesonide, a nonhalogenated corticosteroid with an extensive first-pass metabolism has been a promising innovation. A 2-year controlled double-blinded trial included 39 patients with early-stage PBC and compared treatment with UDCA plus budesonide against UDCA plus placebo. Combination therapy improved biochemical and histological features and reported only few corticosteroid-related side effects [284] . These promising results, however, could not be confirmed in a subsequent study from the Mayo Clinic. Here, adding budesonide for 1 year in 22 patients with suboptimal response to UDCA alone had only marginal effects on serum bilirubin and AP levels. In contrast, the Mayo risk score increased and there was a significant worsening of osteoporosis [285] . This open trial, however, included latestage patients, which may in part explain the disappointing results. As found in a short-term pharmacokinetic study, administration of budesonide to cirrhotic PBC patients leads to high plasma levels of budesonide associated with serious adverse effects and should therefore be avoided [286] . A Finish study [287] included only patients with stages I to III (n=77) in a 3-year randomized trial and used a lower dose of budesonide. The effects of budesonide plus UDCA were compared to UDCA alone. A significant improvement of histological features was observed in the combination group on top of the beneficial biochemical effects of UDCA alone. Long-term controlled trials are required to define whether combination of UDCA with budesonide provides a significant benefit in patients with early-stage PBC inadequately responding to UDCA monotherapy.
Other immunosuppressive agents including azathioprine, cyclosporine, mycophenolate mofetil, or methotrexate and drugs with antifibrotic properties including penicillamine, colchicine, and silymarin have not been shown to markedly improve the natural course of the disease or were associated with significant toxicity during long-term treatment [1, [288] [289] [290] [291] [292] [293] [294] [295] [296] .
Novel concepts for medical therapy of PBC, alone or in combination with UDCA, have recently been considered particularly for use in patients with incomplete response to UDCA. Among others, antiretroviral, immunomodulatory, and antioxidant approaches were evaluated.
A human betaretrovirus has been controversially debated as a potential pathogen in PBC as outlined above. An antiretroviral strategy has therefore been tested in PBC: Lamivudine in combination with zidovudine (Combivir) normalized AP and reduced bile duct injury in a 1-year pilot trial including 11 patients [150] . This finding still awaits confirmation by a randomized, placebo-controlled study.
The peroxisome proliferator-activated receptor α (PPARα) agonist bezafibrate was reported to improve serum liver tests in PBC [297] and should undergo more extensive evaluation in patients with PBC with an incomplete response to UDCA.
Future anticholestatic strategies in PBC are targeted towards stimulation of transcription of key hepatocellular and cholangiocellular transporters in order to improve the secretory capacity of the cholestatic liver. First results of pilot studies using the FXR agonist 6-ECDCA are eagerly awaited.
Pruritus UDCA is an accepted treatment of cholestatic pruritus in ICP (intrahepatic cholestasis of pregnancy). However, its effect on pruritus is variable in PBC [217] . At present, no convincing data for an antipruritic effect of UDCA in PBC are available [298] .
Both peripherally acting pruritogens and central nervous dysfunction have been implicated in the pathogenesis of cholestatic pruritus [9] . According to these two concepts, most therapeutic interventions currently under study for cholestatic pruritus are either directed towards elimination of so far undefined pruritogens or modulation of central neurotransmission. The pharmaceutic options currently recommended include [29] : (1) anion exchange resins, such as cholestyramine and colestipol, which bind anions and amphipathic molecules and reduce their intestinal absorption and systemic accumulation. Despite extensive clinical experience, suggesting a beneficial effect in up to 90% of patients, no large clinical trials have evaluated the efficacy of exchange resins [298] . In patients lacking adequate improvement, treatment with (2) rifampicin may be helpful [298] [299] [300] [301] . Rifampicin is a semisynthetic antibiotic and as a potent pregnane X receptor (PXR) agonist leads to the induction of hepatic microsomal enzymes. Thereby, it may promote metabolism of potential pruritogens. As a thirdline option, (3) opioid antagonists (naloxone, nalmefene, naltrexone) have been found to reduce itch severity in patients with PBC [298, [302] [303] [304] . (4) The selective serotonin reuptake inhibitor sertraline [305, 306] has been reported to improve cholestatic pruritus in small clinical trials and has most recently been considered as a fourth-line treatment option [29, 280] . Other experimental approaches include 5-hydroxytryptamine receptor type 3 antagonists, cannabinoids, subhypnotic doses of propofol, plasmapheresis, albumin dialysis, and nasobiliary drainage in desperate cases, although adequate trials are lacking [29, 63] . Liver transplantation should be considered in serious cases in which all other strategies have failed, even if liver function is still conserved [29] .
Specific medical therapies of fatigue associated with chronic cholestasis have not yet been defined. UDCA treatment seems to have no or limited beneficial effects on fatigue in PBC [217, 307] . Oral supplementation with antioxidants showed promising results in a pilot study, but had no beneficial effect in a randomized, placebocontrolled, crossover trial [308] . Independent of the underlying disease, altered serotoninergic neurotransmission has been implicated in the development of fatigue [309] . The 5-HT3 serotonin receptor antagonist ondansetron, however, did not significantly reduce fatigue compared with placebo in a randomized, placebo-controlled trial [310] . The selective serotonin reuptake inhibitors fluvoxamine and fluoxetine also failed to exert beneficial effects on fatigue in this patient group [311, 312] .
In a series of 21 PBC patients with excessive daytime sleepiness [313] , the centrally acting agent modafinil was investigated in an open-label study [314] . Significant improvement was seen in Epworth Sleepiness Scale scores and fatigue severity as assessed by PBC-40 fatigue domain score, but the drug had to be stopped due to side effects in a considerable part of the patients before the end of the study.
In contrast to pruritus, fatigue may not improve significantly following liver transplantation.
Independent of the etiology, bone mineral loss is a recognized complication of cholestatic liver diseases. In PBC, it moderately increases absolute and relative fracture risk compared to the general population [315, 316] . Treatment of the underlying liver disease with UDCA does not prevent bone loss in PBC [317, 318] . Calcium and vitamin D supplementation is usually recommended in osteoporosis, although their role in preventing osteoporosis and fracture in chronic liver disease is unclear [319, 320] . Alternative interventions to prevent and treat osteoporosis in liver disease have only been tested in a few studies.
Parenteral calcitonin, tested in one controlled trial for 6 months, was ineffective in halting bone loss in patients with PBC [321] , whereas hormone replacement therapy was demonstrated to improve vertebral bone density in PBC [319, 322, 323] . In large clinical trials, however, hormone replacement therapy was associated with serious side effects, including the risks of breast cancer, coronary artery disease, and thromboembolism, limiting its use for the prevention of fractures in osteoporosis [324] [325] [326] .
Use of bisphosphonates is controversial. In a placebocontrolled trial, cyclical etidronate did not significantly improve bone density [327] . Also, a 4-year treatment with clodronate plus calcium/vitamin D did not improve osteopenia in women with PBC in a prospective study [328] . In two controlled trials however, alendronate significantly improved spine and femoral bone mineral density compared with placebo and had greater antiresorptive activity than etidronate [329, 330] .
According to current guidelines [29] , supplementation with calcium (1,000-1,200 mg/day) and vitamin D (400-800 IU/day), though not evidence-based, should be considered in all patients with cholestatic liver disease. Alendronate or other bisphosphonates are recommended at a T score < −2.5 (DEXA) or following pathological fracture.
Liver transplantation is the treatment of choice in patients with late-stage PBC. Indications are decompensated cirrhosis with treatment-resistant ascites, recurrent spontaneous bacterial peritonitis, encephalopathy, recurrent variceal bleeding, or hepatocellular carcinoma. In highly selected patients (see above), treatment-resistant pruritus in the absence of decompensated cirrhosis may be an indication for transplantation, and so is severe osteoporosis [28, 331] . Survival rates of 80% to 90% at 5 years have been reported. The disease recurs in up to 30% at 10 years after transplantation, but then usually displays a mild course under immunosuppressive treatment [331] .
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design 16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences. During the last decades, different nucleic-acid-based detection techniques have been developed in order to employ identification based on single-nucleotide variations in both genotyping and detection experiments on a multiplicity of targets. These techniques allowed distinguishing alleles and correctly assessing the genotype at the singlebase level.
In particular, 16S rRNA gene sequences have been used to resolve bacterial phylogeny and taxonomy issues in different contexts. The DNA sequence coding for the small ribosomal subunit has been by far the most common genetic marker employed by the scientific community, because of: (i) its presence in almost all bacteria, often existing as a multi-gene family, or operons; (ii) the function of the 16S rRNA gene has not changed over time, suggesting that random sequence changes are an accurate measure of time (evolution); and (iii) the 16S rRNA gene (more than 1500 bp) is large enough for informatics purposes (1) with large stretches of conserved regions and few different loci.
DNA microarrays represent one of the most popular platforms in molecular technologies, allowing a highthroughput format for the parallel detection of 16S rRNA genes from environmental samples (2) . DNA chips have been developed as a preferred device for the identification of different microorganisms based on 16S gene sequences. The multiplicity of species which can be arrayed on a single-DNA chip allows a high multiplexing capability, with the possibility of identifying many different targets at one time (3) . Single-base variations by microarray analysis can be detected by differential hybridization techniques using allele-specific oligonucleotide probes (4), or by enzyme-mediated detection methods (5) . One of the most critical points of the molecular recognition procedures is the design of the specific probes needed to perform the entire analysis. In genotyping experiments, this is accomplished on the basis of the already-known information about each single-base variation. In detection experiments, on the other hand, in order to explore whether a certain target sequence is present in a DNA sample or not, the main problem is searching for a priori not yet identified specific positions that can discriminate exactly between one target and another.
In hybridization-based techniques, mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Although this has been the most frequently applied technique, it is characterized by many hindrances which make hybridization-based strategy function poorly in high-complexity biological samples. Therefore, for analytical and diagnostic purposes, hybridization is generally combined with some other selection or enrichment procedures. Enzyme-mediated ligation methods, on the other hand, rely on interrogation of a mutation by a couple of oligonucleotides annealing immediately adjacent to each other on a target DNA, with one of the probes having its 3 0 -end complementary to the point mutation. In this case, the search is for a single base that characterizes a species against all the others in a group of interest. The presence of a point mutation is assessed by the ligation of the two adjacent oligonucleotides, which occurs only when both are correctly base-paired (6) . The Ligation Detection Reaction (LDR) (7) , for instance, represents a reliable technique for identifying one or more sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of targetnucleotide sequences. This enzymatic in vitro reaction is based on the design of two oligonucleotide probes for each target sequence: a probe specific for the variation (called 'Discriminating Probe', or DS), which is 5 0 -fluorescently labeled, and a 5 0 -phosphorylated 'Common Probe' (or CP), starting one base 3 0 -downstream of the DS. The previously polymerase chain reaction (PCR)-amplified sample, the oligonucleotide probe pairs and a thermostable DNA ligase are blended to form a mixture: the two probes hybridize consecutively along the template and the DNA ligase joins their ends only in the case of a perfect match. This reaction is cycled to increase product yield. The PCR-LDR approach, usually, is associated to the hybridization onto a Universal Array (UA), where a set of artificial sequences, called Zip-codes are arranged (7) . This entire approach was proven to be rapid, flexible and easily adaptable from one target to another, useful, for example, in environmental monitoring (8, 9) , forensics (10) and the food industry (11, 12) .
Here we present Oligonucleotide Retrieving for Molecular Applications (ORMA), a series of integrated scripts in Matlab, which performs an accurate search of all the positions able to specifically discriminate one species among homologous ones, based on the 16S rRNA gene sequence. ORMA also performs an accurate selection of high-quality oligonucleotide probes to be used in molecular applications. Automated and computerbased methods can be very useful for performing accurately and rapidly all the requested operations, through the many steps between the original, complete, set of sequences and the final list of application-oriented probes.
The problem of designing specific oligonucleotide probes for the identification of target species has already been addressed by a certain number of software (13) (14) (15) (16) . At present, there is no preferential reference strategy for designing microarrays for species identification based on 16S rRNA sequences: many authors rely on academic software (17, 18) , others develop their own scripts (19, 20) . Among the currently available academic software, ARB (21) and PRIMROSE (22) are very diffused, both being tools implemented specifically on 16S rRNA, structured for interacting with and retrieving sequences from specific databases and operating a probe design on the basis of the phylogenesis of the species under analysis. Also, some commercial software, like Oligo 7 (Molecular Biology Insights, Cascade, CO, USA) (23) or AlleleID (Premier Biosoft, Palo Alto, CA, USA) (24) have been applied for probe design in a pathogen characterization experiment (25) . In this article ORMA was used for determining sets of LDR probe pairs in microbiological-related contexts (water safety and food safety applications, respectively). The approach was evaluated and validated using the probe pairs derived from ORMA-determined discriminating positions on a set of cyanobacteria 16S rRNA sequences belonging to 18 different species; the results were compared to those of a previously published study (8) . Secondly, a set of LDR probe pairs for the discrimination of 13 mastitis-or intoxication-related pathogens species in bovine milk was designed and experimentally evaluated. The tool, although here applied on 16S rRNA, can be used on any set of highly correlated sequences.
ORMA scripts were developed under Matlab 6.1 (Mathworks, Natick, MA, USA) environment (Release 12.1). No additional toolboxes are required. All statistical analyses and representations were made by the same software. Probe designs and simulations were run onto a hp Workstation xw4100, with a Dual-core 3.2 GHz. Intel Processor and 2.5 GB RAM. ORMA functions and m-code are available for free upon request.
Overall structure. ORMA overall structure is tree-like, with a main function that, sequentially, recalls all the side scripts needed to perform each requested operation. The software also comprises a series of scripts for retrieving oligonucleotide sequences, quality-check them and design probes for different applications, such as Ligase Detection Reaction (LDR) or Minisequencing/Primer Extension probes. The overall procedure is accomplished in four main steps (Figure 1, Supplementary Figure 1 ): (i) sequence importing and processing; (ii) discriminating positions finding; (iii) designing of the candidate probes, starting from the positions found and (iv) ranking (i.e. assignment of a quality score to each) and exporting of the candidates (in tabular format).
(i) Sequence import and processing. The search for discriminating positions on 16S rRNA starts from the import of a set of already-aligned sequences (which can be optionally used for the creation of consensus sequences, grouping them in homogeneous clusters, before being used for the discriminating position search algorithm).
Standard multiple-alignment formats (Clustal-like, Multi Sequence Files, or aligned FASTA format) can be used. A careful check of multiple alignment scores should be made, in order to avoid designs on sequence datasets of distantly related species, which can occur in base misalignments. The scripts also include a procedure for consensus determination from a set of user-defined sequences, according to four different rules: (a) majority rule, in which the consensus base is the most frequently present in the aligned sequences and no degenerated bases are used. In case of equal occurrences, 'N's are used in the consensus; (b) threshold rule 'simple', which assigns a specific base to the corresponding position in the consensus only if its frequency is above a given threshold. Different thresholds can be set for gaps and bases. Degenerated bases are not used and are substituted by 'N's in the consensus; (c) threshold rule 'complex', which comprises also degenerated bases. The algorithm is the same as point (b) option, but requires a threshold for substituting positions with multiple bases above the threshold with the corresponding IUPAC code degenerated base and (d) ARB-like algorithm, with separate thresholds for gaps and bases. All the bases above the given threshold are used to compute eventual degenerated bases.
For each of these four options, consensus score accuracy is calculated, as the percentage of original sequences that carried the same base as the consensus in each position. (ii-iii) Design of candidate probes. We have implemented a Single Base Seeker (SBS) algorithm, for the determination of positions able to discriminate one sequence among a set of homologous ones. The discriminating position finding procedure can be summarized as follows in four basic steps: (a) Choice of a user-defined subset of sequences of the dataset (indicated as the 'positive set'). The remaining sequences are used as a group of the discriminating positions must be different from; these are addressed, in the present article, as the 'negative set'. 'Positive' and 'Negative' sets differ for the fact that every consensus in the 'positive set' group will be subjected to probe design, whereas those of the 'negative set' will not; (b) For each sequence, determination of a list of the positions of nondegenerated bases; (c) For each position on point b, calculation of a score as the sum of all the sequences carrying the same base as the considered sequence, in the same position. If the only sequence carrying the base is the tested one, the position is set as discriminating and (d) Re-calculation of the score on point c, substituting to each (eventual) degenerated base its two or three alternatives (an 'N' automatically flags the position as nondiscriminating).
ORMA, then, retrieves the sequences flanking each of the putative discriminating positions. Actual oligonucleotide design is dependent on the molecular application chosen. The maximum length and the thermodynamic model for calculation of the parameters of the probes can be specified by the user. For the LDR experiments here described, two oligonucleotide probes are designed, one upstream (Discriminating Probe, DS, comprising the discriminating position) and one downstream (Common Probe, CP) of each position.
(iv) Discriminating position related filters and scores. The putative discriminating positions and related candidate probes are subjected to a series of constraints and quality filters. The software keeps track of all the designed candidates, assigning a quality score, depending on how many filters they pass. The current options of the script on the discriminant base are: (a) limiting the range of positions, in order to exclude candidates insisting on positions too close to the 5 0 -or 3 0 -end of the sequences, where, usually, the majority of errors in the alignment or characterization of the sequences occur and (b) testing the presence of other species with probes insisting on the same position, thus excluding eventual interactions between a single CP and multiple DS, with subsequent non-specificity. The candidate probes can also be filtered and ranked according to their thermodynamic properties (length, melting temperature, number of degenerated bases, low complexity regions), evidencing the candidates having a certain length, a melting temperature comprised in a user-specified range, having no more than the inputted number of degenerated bases (which can be a real issue for the oligonucleotide specificity), having short homopolymeric regions and not comprising short tandem repeats. Then, ORMA calculates some specific statistics for the qualitative evaluation of the candidates designed on consensus sequences, compared to the original dataset (i.e. the subset of sequences from which every consensus is built): (a) the intra-group score, as the number of initial sequences having the same discriminating base as the consensus and (b) the inter-group score, as the number of sequences other than those used for that consensus having the same discriminating base as the candidate one. This latter score is calculated only when the consensus were created inside ORMA, starting from a single-global alignment. These scores allow the choice of probes that best discriminate between the target and the non-target sequences (i.e. having the highest intra-group and the lowest inter-group score). The software output can be exported as a comma-separated spreadsheet reporting: (a) the list of all the discriminating bases, grouped per species, with absolute (referring to the global alignment) and relative (referring to the specific consensus) positions of the discriminating base, and the base distributions of all the other consensus sequences in the same position; (b) the thermodynamic parameters of the candidate probe pairs, including the T m , the length of DS and CP probes and the number of degenerated bases in each and (c) the qualitative filtering and the specificity-related scores, including the sequence score, as the average of the consensus scores along all the bases constituting the DS and CP, with penalties for degenerated bases.
Cyanobacteria dataset experiment. The complete cyanobacteria 16S rRNA data set comprised a total of 352 sequences, which were organized by phylogenetical similarity and grouped in a total of 18 clusters, as described in (8) . Multiple alignments of all the sequences was performed by ClustalW (26) and the resulting file was imported into ORMA, where 18 consensus, one per cluster, were built. Consensus sequences were determined following the 'ARB-like' algorithm (as described in 'Materials and Methods' section and in Supplementary Methods), setting 50% as the threshold for gaps and 40% as the threshold for other bases. Melting temperature calculations followed the 'salt-adjusted' method, with 50 mM Na + and 0% formamide. Candidate probe pairs were filtered on the basis of their length (minimum 25 nt, maximum 60 nt per probe), melting temperature (63-688C) and number of degenerated bases (maximum 4), on both DS and CP. The best probe pairs for all the species were selected, according to their best intra-and inter-group scores. We required that no less than 80% of the sequences constituting each of the 18 clusters carried the same base as the consensus in the candidate discriminating position (intra-group score). When only one candidate was designed or the intra-group score of the best candidate was below 80%, we still picked that candidate for further evaluations. On the other hand, the inter-group score was set to be below a 2% threshold, with the same exceptions as above. The 'Unicyano' probe, which allowed the identification of any of the species in the study, was the one proposed by Castiglioni et al., with minor refinements for adjusting its melting temperature. At first, the LDR mix made by all probe pairs (250 fmol/ml each probe) was tested on specific synthetic templates (perfectly complementary to each probe pair) to assess the feasibility of the LDR procedure with the ORMAdesigned probe pairs. Then, a total of 14 DNA samples, corresponding to 13 cyanobacteria species (kindly provided by MIDI_CHIP project partners, http://www.cip .ulg.ac.be/midichip/) (Table 1) , were tested in duplicate, independent, LDR experiments, with both ORMA and Castiglioni et al. probe pairs.
Milk-pathogen dataset experiment. Milk pathogensrelated 16S sequences were retrieved from RDP-Ribosomal Database Project II (release 9.51, http:// rdp.cme.msu.edu/) (27) for a total of 738 sequences and divided into 13 subgroups, according to their phylogenetic classification. Only sequences of length >1200 bp and flagged as of 'good' quality were retrieved. Each subgroup was aligned independently in ClustalW, since the overall number of 16S sequences was >500 (above the maximum limit of the alignment tool) and imported into ORMA. The consensus sequence for each group was calculated with the same parameters specified for the cyanobacteria data set. Then, a new multiple-alignment step was performed before proceeding to actual probe design. One probe pair for each of the main six subspecies of the Streptococcus group (Streptococcus agalactiae, S. bovis, S. equi, S. canis, S. dysgalactiae S. uberis) was designed; moreover, the Staphylococcus aureus probe pair was designed independently from all the remaining coagulase negative Staphylococci (grouped in the 'Staphylococcus, no aureus' probe), because of its relationship with outbreaks of mastitis in dairy ruminants (28) and with major health issues, like food-related intoxications (29) . In order to have the best homogeneity among the species within each group, the design was actually performed in three rounds: (a) Salmonella spp. was aligned against Escherichia coli and related spp. consensus sequence only; (b) S. canis was aligned against Streptococcus group sequences only; (c) All the remaining positions were selected considering the alignment of all other subspecies. One probe pair per species was designed, except for Campylobacter spp. for which two probe pairs were evaluated in terms of reproducibility and specificity. The thermodynamic parameters were the same described for the cyanobacteria data set, except for the melting temperature, which was required to be in the range 67-698C. The inter-group score of the candidates was required to be above a threshold of 80%, as in the cyanobacteria dataset. Probe pair specificity was checked by both RDP II database and BLAST (Basic Local Alignment Search Tool, http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi) (30) analysis, carefully examining the 3 0 -region of the discriminating probe, in order to exclude any interaction between probe pairs targeting different species. LDR probe pairs were mixed at a final concentration of 1 pmol/ml and tested on 13 DNAs from ATCC reference strains (LGC Promochem, Middlesex, UK) and bacterial collections (Supplementary Table 1 ). Genomic DNA was extracted following the protocol described by (31) , PCR amplified and analyzed in duplicate, by separated LDR reactions.
PCR and LDR/Universal Array approach. Complete experimental procedures concerning the amplification of 16S rRNA sequences (including primers and thermal cycling), LDR mixes, Universal Arrays preparation and hybridization are reported in Supplementary Data. Data analysis. All arrays were scanned with ScanArray 5000 scanner (Perkin Elmer Life Sciences, Boston, MA, USA), at 10 mm resolution, with different acquisition parameters on both laser power and photo-multiplier gain, in order to avoid saturation. Intensities of fluorescence (IF) were quantitated by ScanArray Express 3.0 software, using the 'Adaptive circle' option, letting diameters vary from 60 to 300 mm. No normalization procedures on the IFs were performed.
To assess whether a probe pair was significantly above the background (i.e. was 'present' or not), we performed a one-sided t-test (a = 0.01). At the same time, also the type II error was calculated and 1-b used as the estimate of the power of the statistical test. The null distribution was set as the population of 'Blank' spots (e.g. with no oligonucleotide spotted, n = 6) IFs. Two times the standard deviation of pixel intensities of the same spots was added to obtain a conservative estimate. For each Zip-code, we considered the population of the IFs of all the replicates (n = 4) and tested it for being significantly above the nulldistribution (H 0 : m test = m null ; H 1 : m test > m null ).
Signal-to-noise ratios, SNR p and SNR np were calculated, for each 'present' and 'non-present' probe pairs, respectively, indicating the ratio between the mean IF of each probe pair and the mean 'Blank' IF, divided on the probe-type.
Searching, designing and selecting oligonucleotide probes for molecular applications experiments on sets of highly similar sequences, such as the 16S rRNA, is a non-trivial procedure, which involves many complex and timeconsuming steps. In this article, this procedure was accomplished by the use of ORMA, an integrated architecture of Matlab scripts. The 16S rRNA, a gene sequence of more than 1500 bp, is the preferred genomic target for analyses in the microbiological field (17) (18) (19) (20) . It should be noted that 16S region is commonly used in taxonomical classifications involving in silico alignment and procedures for its two basic properties: (i) 16S presents highly conserved regions which can be used to correctly align all the sequences in the database; (ii) on the other side, 16S presents highly polymorphic regions that can be used in clusterization, phylogenetic tree construction and molecular discrimination of microbiological families even very close one to each other (32) . Use of an automated method for discriminating positions determination, probe retrieval and filtering has obvious and evident advantages over the manual design, often used in previously published papers (8, (33) (34) (35) . These advantages become more significant with increasing dimension of the databases and of the sequences length. ORMA can perform all these operations with user-specified parameters in an automated way and calculates a series of Where sequencing has been performed, the result of the classification is also reported. Sample ID refers to the numbers used in Figure 2 .
a Clonal DNA from environmental sample.
b Specific indicates that only the probe corresponding to the species was present; non-specific means that no probe was present (except for the universal cyanobacteria probe); aspecific means that the species-specific probe was present, but also other probes showed an IF significantly above background signal. The number of replicates is reported within brackets.
c According to RDP II database, release 9.60.
qualitative parameters which help in the choice of candidate probes that best discriminate between the sequences of the positive and those of the negative set. The general idea of these scores is to distinguish the sequences/groups which are of interest in a given experiment from those who aren't and that can potentially have a cross-contamination with the positive set, because they could be amplified by PCR, contributing to the molecular complexity of the sample. In this article, performances of ORMA were evaluated by considering the experimental evidences coming from the design of LDR probe pairs on two different 16S rRNA datasets. First, a new set of cyano-specific probe pairs was designed and compared to the original one (8), generated on the same database of sequences. Then, the tool was used to setup LDR probe pairs for the identification of pathogenic species present in bovine milk.
Species-specific probe pairs were designed in a single round, starting from the whole dataset of 352 ClustalWaligned cyanobacteria 16S rRNA sequences, imported, converted and grouped into 18 group-specific consensus sequences by ORMA. Calculated consensus sequences were highly similar, (ClustalW score = 87.31 AE 2.13, n = 18), had a high consensus score (average score 89.20 AE 4.16, n = 352) and a very low content of degenerated bases (average < 2%, max = 6%). ORMA identified a total of 192 candidate probe pairs for the 18 species, with an overall duration of the whole procedure of less than 5 min (Table 2 ). More tests on speed performances of the SBS algorithm on simulated data available as Supplementary Data and Supplementary Figure 2 . One probe pair per species was chosen, according to its ranking after ORMA filtering steps. The probe pair for Anabaena + Aphanizomenon group was flagged as inadequate by ORMA, having six degenerated bases in the CP, which could negatively influence its thermodynamical properties. However, this probe pair insisted on the only discriminating position found for that cluster. The mix containing all probe pairs was tested on the corresponding synthetic templates and, as expected, all except Anabaena+Aphanizomenon gave positive results. Duplicate LDR experiments on 18 probe pairs (17 species-specific + 1 universal) were carried out on 14 16S rRNA PCR products. We performed side-by-side tests of the same DNA samples by the two probe pairs datasets, ORMA and the one described in Castiglioni et al., comparing their performances and specificity. Probe pairs used in Castiglioni et al. identified correctly (P < 0.005, average beta power of the test: 0.85) 6 out of 14 analyzed DNAs (in both duplicate LDR), whereas other two completely failed. Six other DNAs somehow showed a degree of aspecificity (i.e. the correct probe pair was present, but non-specific probe pairs were also called present) (Table 1, Figure 2 ). Cyanobacteria universal probe pair was called as statistically over the background in all the experiments. Evaluations on ratio of signal intensities suggested that hybridizations went well and were not responsible for the aspecificity. In fact, excluding non-specific signals, SNR np had an average value of 1.18 AE 0.61 and SNR p varied between 10 and 680, with an average of about 149 (data not shown). The Anabaena + Aphanizomenon probe pair of Castiglioni et al. study resulted specific on both synthetic and environmental samples (data not shown). This probe pair, however, was designed with its DS insisting on a position which did not discriminate univocally the Anabaena + Aphanizomenon consensus from the consensuses of the other species. Thus, it would never be identified by ORMA as discriminating (because of the way the algorithm is built). Instead, the presence of some internal mismatches (especially the one on the second base before the 3 0 -end of the DS) is probably the reason for this finding. In fact, the mismatch gives instability to the 3 0 -end of the DS when annealing on the 16S rRNA sequences of species other than those of Anabaena + Aphanizomenon cluster, impeding the ligase to join the two adjacent end of the DS and CP oligonucleotides.
ORMA designed probe pairs have been capable of correctly identifying (P < 0.005, average beta power of the test: 0.85) 12 out of 14 analyzed cyanobacteria samples, on both replicates. Also in this experimental set, the cyanobacteria universal probe pair was called as statistically over the background in all the experiments (as expected, since this probe pair and the ones used in Castiglioni et al. coincided). Performances of the LDR procedure, in terms of signal-to-noise ratios were comparable to those obtained with the Castiglioni et al. probe set, having a SNR np of 1.1 AE 0.26 and a SNR p ranging from 7 to 387 (average $131) (data not shown), indicating a certain variability. In this case, we had no signs of aspecificity in the experiments (Figure 2 ), even in those cases which were critical with Castiglioni et al. probe pairs. In fact, probes were chosen in order to maximize the intra-group similarity (i.e. having the maximum number of sequences in the positive set carrying the discriminating base) and minimize the possibility of an inter-group cross-talk (i.e. having a minimum number of sequences in the negative set carrying the discriminating base) (Figure 3 ). The average of intra-group scores of the candidates was 95.1% AE 10.1% (n = 17), varying in the range 60-100%. The minimum value was that of the cluster of Gleotheceae, in which we had only five sequences, whereas 13 out of the 17 clusters were characterized by a score of 100%. Intergroup scores, on the other hand, were always very low, with an average of 0.4% AE 1% (n = 17). Thus, where ORMA probe pairs failed, we had a false negative call (with the cyanobacteria universal probe pair called as 'present'), but not a false positive. Experiments on the two Nostoc DNAs gave no results on the species-specific probe pair; anyway the presence of a cyanobacterial DNA was correctly assessed by the Universal probe pair. Sequencing of the two products revealed that one of them has been correctly classified by microbiological methods, whereas the other DNA was very uncertain and classified as 'cylindrospermum' (58% confidence) by RDP 'Classifier' tool, release 9.60. [On 22 May 2008, RDP II database for cyanobacteria (release 9.61) underwent a major change in hierarchical classification of the species. The taxonomies here presented refer to older versions, which at present, can be found within genus GpI of On the x-axis, the IDs of the tested samples (see Table 1 for full description) are reported. On the y-axis, the probe pair name is reported. The line 'Other' represents the mean of all the remaining Zip-codes in the universal arrays that were not associated to any actual probe. Experiments on Nostoc samples were repeated twice on different DNAs because of the failure of the first test. Halotolerans probe pair in one replicate of sample 8 (classified as Nostoc) has a P-value of 0.02, above the threshold of 0.01 chosen for significance.
family Family I, philum cyanobacteria.] Both sequences found very little similarity with our probe pairs, with internal mismatches and a different base in the discriminating position. Anyway, the probe pair itself was successfully tested on the synthetic template. The failure of ORMAdesigned Anabaena + Aphanizomenon probe pair suggests the possibility of making a re-design in the near future, increasing the number of sequences of the database and improving the information content of the dataset. Another strategy could be designing probe pairs on subclusters of Anabaena + Aphanizomenon, building new consensuses from more homogeneous groups; in this way, the presence of such two species would be assessed by multiple probe pairs and not only by one.
16S rRNA sequences of pathogens contaminating bovine milk or related to bovine mastitis were used to design LDR oligonucleotide probes by ORMA, providing a further confirmation of its reliability and specificity. In this study, three round of design were actually performed, in order to have the best homogeneity between the species used in each round. A single round would have caused the loss of discriminating positions due to misalignment of some species (e.g. Salmonella) which are somehow different from all others. ORMA found a total of 392 candidate positions (34, 4 and 354 in the design for Salmonella, S. canis and all remaining species, respectively), which were selected according to the quality ranking scores assigned by ORMA. In this experiment, ORMA calculated only the intra-group score, but not the inter-group score, because of the fact that the sequences for each group were imported separately and the software was unable to recall the position corresponding to discriminating ones in all the sequences constituting each of the consensuses. The candidate probes were all characterized by an optimal specificity of the discriminating base, as suggested by the intra-group scores which were above 90% in 11 out of 14 cases. The scores were, in any case, above the fixed threshold of 80%, having an average of 94.0% AE 6.9% (n = 14). Also in this case, the lowest score (i.e. 80%) was that of the cluster (i.e. S. equi) constituted by the lowest number of sequences (n = 5). The final evaluation on the candidate probe pairs was made by RDP and BLAST checks, because of the multiplicity of species, whose 16S rRNA gene was amplified by means of universal primers, potentially present in milk-derived matrixes and the lack of a complete internal negative set in ORMA. The probes were slightly longer than the ones on cyanobacteria dataset, with an average length of about 40 nt, with very homogeneous melting temperatures (mean T m = 67.6 AE 0.4, n = 28) and a very low number of degenerated bases (only the DS probe for S. equi had 1 degenerate base) ( Table 3 ). The consensus scores for both the DS and CP confirmed the overall quality of the probes (average score of 96.5 AE 4.2, n = 28, with 60% of the probes having a score >99%).
The procedure showed optimal specificity, with excellent signal-to-noise ratios, as shown in detail in the article of Cremonesi and co-workers (36) . Results were in complete concordance with sample identification made by ATCC; only probes associated to the supposed species were present (P-values always <0.005), whereas all remaining probes were well below any acceptable P-value for the t-test (Figure 4) . In this dataset, SNR p varied from 4.31 to 238.3, with an average of 34.28; at the same time, SNR np varied between 0.12 and 0.83, with an average of 0.48 AE 0.18. The two probes on Campylobacter species (insisting on two different positions) performed nearly the same in terms of specificity, both giving P-values far below the acceptance threshold of 0.01, whereas performances in terms of signal intensity varied, with one probe having average IFs about 2-fold higher than the other, in both replicates, suggesting a somehow different sensitivity in the two competing probes.
Thus, ORMA helped in developing a reliable PCR-LDR-UA assay, which allowed the identification of pathogenic species in milk, based only on 16S rRNA gene, whereas other assays (37) needed multiple genes. The molecular procedure permitted the discrimination between the most frequently isolated or emerging The exact probes sequence from ORMA is reported. For synthesis purpose, any degenerated base was substituted with inosine (I). The description of the reported columns is the same as those in Table 2 .
pathogens in mastitis (e.g. S. aureus, S. agalactiae, S. uberis), or potentially dangerous for human health (e.g. E. coli and related species, Salmonella, S. aureus and Bacillus spp). Streptococcus spp. was identified at the species level, even in the cases, like the one of S. uberis and S. parauberis, where the molecular identification on the basis of the 16S rRNA gene required PCRs with species-specific primers. Moreover, the ORMA-based LDR technique represents a significant improvement of the existing detection methods for Mycoplasma spp. strains (38) , known to be contagious causes of intramammary infection in herds, overcoming the long and laborious standard-detection methods based on microbiological procedures (36) . These results confirmed the ability of this tool to determine discriminating positions in complex datasets.
The ability to identify 'fingerprint' positions within a set of homologous sequences, like those of 16S rRNA gene, is the main feature of ORMA. To achieve optimal results, the starting set of sequences should be carefully selected, because, if sequences are characterized by many lowsimilarity regions, the determination of terminally discriminating position could be biased by badly aligned subsequences. In that case, a different algorithm (actually not included, but under development) for the determination of detection probes by means of the hybridization strategy, can be more appropriated. On the other side, using sequences nearly identical one to each other can cause the opposite behavior, where no discriminating positions can be determined. A careful grouping of the sequences in clusters (as we did for both of our examples, building 18 consensus out of 352 sequences in cyanobacteria dataset and 13 consensus out of 752 sequences in milk pathogens dataset) is strongly suggested. In this latter application three rounds of design were applied, in order to compensate the non-perfect homogeneity of some species. Experimental results demonstrated the correctness of this approach and the specificity of the probe pairs obtained with this design strategy. Experimental data on the 16S rRNA cyanobacteria and milk-pathogens dataset demonstrated that ORMA specifically addressed discriminating positions within a set of highly similar sequences. Nonetheless, our tool identified a total of 192 and 392 candidate positions, respectively. The intra-and intergroup scores were demonstrated to be very helpful in determining the best probes for discrimination and avoiding cross-talk between species. ORMA is a bioinformatic tool for the search and determination of single-discriminating positions among a set of highly homologous sequences and represents a The scale varies between non-significance (>0.05) to high-significance (<0.005). The line 'Other' represents the mean of all the remaining Zip-codes in the universal arrays that were not associated to any actual probe. Complete association between samples numbers and names is given in Supplementary Table 2. significant improvement from other contexts where enzyme-based techniques have been employed on already known single-nucleotide polymorphisms (SNPs) (39) or on entire subsequences (11) . This unique feature makes ORMA completely different from all the other available software for probe design in detection experiments. During the past years, academic software for species detection have been developed. ProDesign (13) is a tool based on a 'spaced seed algorithm' for the determination of probes capable of discriminating multiple pathogenic species, at different hierarchical levels. Similarly, YODA (14) performs design tasks on complete genomes against non-target species. TOFI-beta (15) implements a suffixtree-based algorithm for isolating suitable candidate probes from a target genome and filters the list according to thermodynamical and specificity requirements. These three software are implemented for the design of probes for hybridization-based detection assays. PathogenMIPer (16) , instead, is based on a different strategy (i.e. molecular inversion assays), which starts from the selection of unique sequences on a reduced dataset and then does a global comparison to all those potentially matching.
All these software perform smart designs where the probes have to be selected on the whole genomic DNA; this is the typical pipeline in contexts where no preselection of the target sequences has been made, which is not the case of ORMA. In fact, in both the presented datasets, the molecular complexity of the genomic material has been reduced by PCR on the 16S rRNA. The probe pairs design, then, was performed only on the basis of a specific subset of the whole 16S dataset, limited for the specific environment in which the target species have to be detected: cyanobacteria DNA was selected and amplified by cyano-specific PCR primers, while milk pathogens 16S rRNA sequences, although amplified by universal primers, were compared only to context-specific species. The double check in RDP and BLAST, performed after the complete probe pair design by ORMA, confirmed that our choice to work with such a reduced dataset was indeed correct, because the detected species accounted for the majority of the biological diversity present in the target matrix (i.e. milk). Moreover, many of the aforementioned software perform the specificity checks by extensive BLAST searches, which is a reasonable choice for designing specific probes starting from the whole genomic DNA; in case of datasets with limited complexity (or in which the complexity has been reduced by means of molecular procedures), this approach results too computationally intensive and unnecessary for the scope. ARB (21) and PRIMROSE (22) are tools widely used for the classification and the phylogenesis of bacterial species, structured for interacting with databases specific for the same molecular target (i.e. 16S rRNA) and operate a probe design on the basis of the phylogenesis of the species under analysis. None of these two software, however, is built specifically for the determination of discriminating positions within a set of very similar sequences and they provide probe design functionality only for hybridization assays or PCR primers. When used for probe design in detection application, the strategies are based on internal mismatches or on unique stretches of nucleotides (40) .
In this case, the discrimination power resides more in the decreased melting temperature of mismatched duplexes, rather than on a perfectly matched base pair between the probe and the target. Although our tool was applied on the design of probes for a specific technique (LDR) and on a specific target gene (16S rRNA), the software is not limited to this combination. LDR technique approach implied the retrieval of a pair of sequences, one of which (the DS probe) insisted on the discriminating position, whereas the other (the CP probe) is designed to anneal one base 3 0 -downstream of the discriminating position; the design of probes for minisequencing application would have implied only the determination of one probe with its 3 0 -end one base before the variation. At the same time, the design of a reporting probe for a TaqMan Real-time PCR assay would have implied the determination of one oligo with the single-base variation in the middle of the sequence. Due to its modular structure and to the straightforwardness of other applications from the already implemented one, probe sets retrieval and filtering methods could be easily added, starting from the discriminating positions found by the SBS algorithm. A further extension to hybridization probes/standard PCR primer design will be evaluated, changing the strategy for determining the positions to be tested. Provided that the initial database of sequences is accurate, updated and complete as much as possible, ORMA can retrieve discriminating positions and design specific probes on every set of sequences. Its implementation, in fact, is not based on an internal database of sequences (which are, instead, retrieved and loaded from external resources) and can be extended to any gene. In any case, the database should be critically built by only context-specific sequences. Standard procedures, like PCR with specific primers, can help in isolating only the subsets of sequences which constitute the actual database from those completely unrelated to the biological context under investigation, avoiding any interference with actual probes, as exemplified by the cyanobacteria dataset experiment. Sequences of off-target or distantlyrelated species could negatively act in the process of multiple-alignment, leading to poorly aligned datasets and biased designs. Since the databases, cyanobacterias in particular, are constantly and frequently upgraded, ORMA capability of determining discriminating positions can be refined, depending on the completeness of the initial datasets (both positive and negative set). Moreover, the continuous changing of classification and the addition of new sequences make an exhaustive and definitive design of the best cyanobacteria probes absolutely not trivial.
ORMA represents a good alternative solution to the troublesome problem of searching specific positions within a large set of homologous 16S rRNA sequences and provides tools for performing a series of probe-related operations, such as sequence retrieval, filtering and scoring, allowing the user to have a set of candidates on which the actual and definitive selection can be done. The calculation on intra-and inter-group scores allows the selection of highly specific probes for molecular applications, covering the highest number of species of the positive set and having the lowest interaction with the negative set.
In silico checks versus public databases (e.g. RDP or BLAST) are necessary only in case of lack of a reference among the sequences imported in ORMA or when the species eventually present in the biological context under study are too many for being comprised into a reasonably small negative set (e.g. all the microorganisms potentially present in bovine milk). Appropriate experimental designs, comprising context-specific PCRs for reducing the molecular complexity of the target can also be helpful. A complete set of major thermodynamic parameters are reported in the output, helping the researcher to carefully select the best probes. Our data assessed and demonstrated the performances of ORMA in designing probes for molecular applications on 16S rRNA gene and their feasibility for experimental use, with improved specificity and sensitivity. Initial psychological responses to Influenza A, H1N1 ("Swine flu") BACKGROUND: The outbreak of the pandemic flu, Influenza A H1N1 (Swine Flu) in early 2009, provided a major challenge to health services around the world. Previous pandemics have led to stockpiling of goods, the victimisation of particular population groups, and the cancellation of travel and the boycotting of particular foods (e.g. pork). We examined initial behavioural and attitudinal responses towards Influenza A, H1N1 ("Swine flu") in the six days following the WHO pandemic alert level 5, and regional differences in these responses. METHODS: 328 respondents completed a cross-sectional Internet or paper-based questionnaire study in Malaysia (N = 180) or Europe (N = 148). Measures assessed changes in transport usage, purchase of preparatory goods for a pandemic, perceived risk groups, indicators of anxiety, assessed estimated mortality rates for seasonal flu, effectiveness of seasonal flu vaccination, and changes in pork consumption RESULTS: 26% of the respondents were 'very concerned' about being a flu victim (42% Malaysians, 5% Europeans, p < .001). 36% reported reduced public transport use (48% Malaysia, 22% Europe, p < .001), 39% flight cancellations (56% Malaysia, 17% Europe, p < .001). 8% had purchased preparatory materials (e.g. face masks: 8% Malaysia, 7% Europe), 41% Malaysia (15% Europe) intended to do so (p < .001). 63% of Europeans, 19% of Malaysians had discussed the pandemic with friends (p < .001). Groups seen as at 'high risk' of infection included the immune compromised (mentioned by 87% respondents), pig farmers (70%), elderly (57%), prostitutes/highly sexually active (53%), and the homeless (53%). In data collected only in Europe, 64% greatly underestimated the mortality rates of seasonal flu, 26% believed seasonal flu vaccination gave protection against swine flu. 7% had reduced/stopped eating pork. 3% had purchased anti-viral drugs for use at home, while 32% intended to do so if the pandemic worsened. CONCLUSION: Initial responses to Influenza A show large regional differences in anxiety, with Malaysians more anxious and more likely to reduce travel and to buy masks and food. Discussions with family and friends may reinforce existing anxiety levels. Particular groups (homosexuals, prostitutes, the homeless) are perceived as at greater risk, potentially leading to increased prejudice during a pandemic. Europeans underestimated mortality of seasonal flu, and require more information about the protection given by seasonal flu inoculation. Medical interest in Influenza A, H1N1 has been considerable [1] . However, despite dramatic warnings in the media, little is known about behavioural responses to pandemic threats. 'Common sense', lay beliefs about those most likely to be at risk, and the appropriate behaviours to adopt to avoid infection, are often not taken into account by medical practitioners. Such beliefs have been shown to influence adherence and self-care behaviours [2] . During the SARS and Ebola outbreaks, association of the viruses with Chinese or African 'others' permitted Europeans to feel relatively safe from infection [3] , and contributed to the victimisation of some Chinese in Toronto [4] . Stockpiling by the worried well can rapidly lead to shortages; cancellations of travel and closure of businesses can rapidly have profound economic consequences [5, 6] . Faced with concerns about their mortality, individuals may turn to others for reassurance, but these social networks, by sharing their uncertainties, may sometimes contribute to greater stress [7] .
The international threat posed by H1N1 calls for a necessary pooling of international data, both by medical teams and by social scientists. Particular concern has been expressed about the pandemic spreading to Asia, and the potential for mixing with other variants, such as avian influenza [8] . Our team in the UK, Portugal and Malaysia sought to explore initial responses to the pandemic influenza threat. Responses to a new pandemic can be very time specific, with public reactions liable to change almost daily with media coverage [9] . Particularly important may be the gathering of data during the escalating responses that accompany a WHO pandemic alert phase 4 and 5 [10] . The WHO raised their flu alert to level 5 on 29 th April [11] , a mass media information campaign began in the UK on 5 th May 2009. We collected data from 30th April 2009 (at which time there had been 9 deaths and 212 confirmed cases worldwide) until 6th May 2009 (31 confirmed deaths, 1569 cases). By 6 th May there had been 27 confirmed cases in Europe, but none in Asia.
Our study sought to gather a snapshot of the attitudinal and behavioural responses during the early stages of a pandemic, knowledge about the differences between seasonal and pandemic flu, and those groups seen as most 'at risk' from infection. Understanding such attitudes and levels of knowledge may have important public health implications for information campaigns aimed at encouraging appropriate precautions against infection, while comprehending risk perceptions can help identify those groups most likely to be at risk of stereotyping and prejudice during a pandemic.
Following ethical approval by the relevant University ethics boards in London and Malaysia, data was collected from a total of 328 respondents (mean age 31.2, SD 13.37, 62% female). A paper version of the questionnaire was distributed in Malaysia, with students recruiting 180 respondents from their own classes, and community members from residential areas and local offices in Kuala Lumpur (age range 18-70, mean age 29.0 (SD 13.36), 59% female)). Response rate was generally good, with 180 out of 200 approached to participate (90%) completing the questionnaire. In Europe, data was collected between 30 th April and 6 th May 2009 from 158 respondents (age range 18-69, mean age 33.9, SD 12.8, 68% female) via an online questionnaire in English or Portuguese, linked to the website http://www.swinefluques tionnaire.com. The website link was pasted onto a variety of general, non-health networking websites (e.g. 'I love London'). Respondents were primarily from the UK and Portugal but also included 30 respondents living outside these countries and resident in Finland (19 respondents), Poland (6 respondents), Malta (3 respondents) and France (2 respondents). Ten non-European based residents were then removed from the online survey before analysis.
Respondents were asked to complete a questionnaire 'about perceptions of swine flu' which comprised a number of closed and open-ended format questions. Beliefs about comparative risk were analysed for a number of group members associated with risk in previous pandemics. We included these 'high risk' groups on the basis of previous research on representations and reactions to HIV/AIDS, the Ebola virus and SARS [3, 9, [12] [13] [14] . 'Outgroup' members, who may be marginalized by society (prostitutes, homosexuals) have been linked to higher risk in previous epidemics [12, 13] , while the association of poverty with disease spread in previous infections [3] led us to include homeless people. Concerns about the risk of respiratory diseases from proximity to animals [3] meant we included pig farmers and farmers in general in our list of risk groups. We also included the elderly and immune compromised, two groups at higher risk from seasonal influenzas. Respondents completed closed-format 3point scales, indicating the extent to which they believed these groups were more at risk than me, the same risk as me or less at risk than me.
Anxiety indicators were assessed through two closed-format questions assessing personal worries about catching the virus (measured on a 4-point scale, from very concerned to not at all concerned), as well as questions assessing friends and families' estimates of the risk (4 point scale, from very high to very low). We used closed format questions for two questions assessing the purchase, or intention to purchase, specific items, such as face masks ("Have you already bought (or are you intending to buy) anything in preparation for a swine flu epidemic (e.g. face masks, food, tissues, cleaning materials)?" (yes/no responses). We accompanied this with an open-ended question for those who indicated they had/intended to purchase an item ("what have you bought?") with responses categorised for frequency in Malaysia and Europe, with the most common categories reported below. We also assessed whether respondents expected to modify their public transport use as a result of the threat (3 point scale: increase transport use, decrease transport use, or remain the same, collapsed into the categories 'less' and 'much more or the same' for analysis), and whether respondents intended to cancel or delay travel plans (yes / no). An open-ended question asked 'how can you protect yourself from infection?' with the most frequent responses coded into categories by our research team in Malaysia and Europe.
In Europe only, we asked additional closed-ended questions about stopping or reducing eating pork as a result of the pandemic (yes/no), and the precautionary desire for anti-viral drugs at home ('would you like to have your own anti-viral drugs at home just in case'? 'Have you tried to obtain any anti-viral medicines to keep just in case?' (both yes/no answers)). We asked about the mortality rate in ordinary, seasonal flu (five point scale, under 50,000, 50-000-99,000, 100,000-249,000, 250,000-500,000 and over 500,000), as well as whether the symptoms of swine flu differed from seasonal flu, and whether seasonal flu vaccination provided immunity against swine flu (both yes/no answers). Throughout, statistical analysis for structured questions was through chi-square analyses and Pearson product moment correlation.
As shown in table 1 approximately a third of respondents reported they would use public transport less (116/320) or had contemplated cancelling or delaying flights (124/ 312), with this response more pronounced in Malaysia (respective x 2 (1) = 21.91, 49.20, both p < .001). Few had already bought products (25/328), but 41% (74/180) of our Malaysians were preparing to do so (x 2 (1) = p < .001 for differences between Malaysia and Europe). Most likely to be purchased were masks (mentioned 46 times in Malaysia, 14 in Europe) and food (14 times Malaysia, 5 times in Europe). Asked in the free response question how to avoid infection, 37% (121/328) of our respondents cited washing hands and good hygiene, 28% (92/328) wearing a mask, 13% avoiding infected others and 12% (39/328) shunning crowded places.
Five groups were seen as particularly at risk by more than half of our respondents: those with weakened immunity, pig farmers, the elderly, the homeless and prostitutes/ highly sexually active. Malaysians were more likely to see pig farmers, general farmers, homosexuals and prostitutes as at greater risk (respective x 2 (2) = 68.03, 11.44, 31.82, and 12.10, p < .001 for each), Europeans were more likely to see the elderly and those with weakened immunity at risk (respective x 2 (2) = 8.27, 3.49, p < .05). Whilst around half (165/325) of our respondents reported they were at least 'somewhat concerned' about being a victim of the pandemic, this anxiety was stronger in Malaysia, where 71% (127/178) indicated they were at least 'somewhat concerned' (x 2 (3) = 91.67, p < .001). Nearly three quarters of our overall respondents (241/325) felt they had at least some control over whether they were infected. Europeans were more likely to have discussed their fears with their friends (90/142) (x 2 (1) = 66.56, p < .001). Across the sample, personal perceptions of risk about the pandemic were related to those of families and friends (respective rs .57, .58, p < .001). Those most anxious about personally being a victim of the outbreak were the most likely to reduce their use of public transport (r (320) = .48, p < .001) and cancel/delay air travel (r (322) = .37, p < .001).
In our additional (Europe only) data, respondents underrated the dangers of ordinary season flu, with 64% (95/ 148) claiming that this killed under 100,000 worldwide (actual numbers are between 250,000 and 500,000) [15] . 26% (38/148) of our European respondents wrongly believed that a vaccination for seasonal flu gave immunity against swine flu. The same percentage believed swine flu symptoms differ from those of seasonal flu. While only 3% (4/148) had already obtained anti-viral drugs for use against swine flu, 32% (47/148) claimed they would like to have these at home in case of infection. Few (7%, or 10/ 148) claimed they had stopped or reduced their eating of pork as a result of the pandemic.
At present, it is unclear as to whether the outbreak of Influenza A, H1N1 will prove to be a "false alarm", or whether the virus will mutate and spread in a new, more dangerous form, perhaps this Autumn [16] . Our data collection in the early stages of the pandemic/pandemic of Spring 2009 suggests that respondents felt they had some control over potential infection. Respondents identified 'washing hands', avoidance of infected people, avoidance of crowded areas and mask wearing as strategies for avoiding infection, reflecting generally approved public health measures [17] . Malaysians were particularly anxious about a pandemic, despite the lack of cases of this influenza in Malaysia during our research period, probably reflecting the recent avian influenza alert in this country [8] . As with previous health alerts, personal anxieties can feed behavioural changes [18] , with many Malaysians contemplating significant changes in their use of transport, and anticipating the purchasing of goods, particularly masks, in preparation. European respondents were particularly likely to have discussed the pandemic with their friends, while a quarter of respondents overall had discussed the pandemic thereat with their family. Our correlational data suggests that such conversations may reinforce existing levels of anxiety. Practitioners need to be aware that rumours spread fast during times of pandemic threat, with significant risks of emotional as well as physical 'contagion' between populations [19] . Any increase in anxiety can lead to rapid behavioural changes that can soon lead to shortages, and enhance the desire for medication available at home.
An unrealistically optimistic belief that others are at greater risk than ourselves can reduce our willingness to enact healthy behaviours [20] . During pandemics, particular 'out-groups' may be vulnerable to discrimination [21] . Although respondents correctly identified groups such as the immunocompromised as at greater risk [22] , half our respondents saw the sexually active as at greater risk, almost a third of Malaysians suggested homosexuals were at more risk of infection. This may reflect a popular belief in Malaysia that homosexuals are likely to be already immunocompromised through infection with HIV/AIDS. The homeless were also perceived as at greater risk in both Malaysia and Europe. Political and health authorities need to be wary of increased stereotyping and prejudice towards particular societal groups during an influenza pandemic. Our Europe-only data suggested that individuals underestimate the threat of regular seasonal flu, while a quarter of our respondents incorrectly believed seasonal flu and swine flu symptoms were different, and that seasonal flu vaccination could help immunise against swine flu. Despite major media and governmental campaigns across Europe, there is obviously still a need for greater information with respect to symptom logy and immunisation against infection.
Our study was a rapid, cross-sectional analysis in response to a particular outbreak, and as such suffers from a number of limitations. Although this research was unique in tracing initial behavioural responses to this pandemic, our respondents were not true random samples in either continent, and we assessed only a small range of potential behaviours. Our Malaysian sample was drawn from one large city -Kuala Lumpur -and may therefore not be representative of other, more rural populations in that country. Similarly, our European data was drawn primarily from the UK and therefore cannot be seen as representative of the whole continent. Self-report biases in questionnaire completion may mean that our respondents were unwilling to provide openly prejudicial responses, whilst our study in Europe was further limited by including only those who had access to the Internet. To fully model likely behavioural changes, and their consequences for public health services, larger, more representative longitudinal studies are now needed to track public anxieties and health behaviours in the continuing battle against pandemic influenzas.
Numerous studies have identified behavioural interventions valuable in prevention of epidemic/pandemic influenza, but we know little about individuals' own perceptions of risk at the beginning of a pandemic, which groups in society they believe most at risk of infection and how they have changed and intend to change their behaviours as a pandemic develops. Our findings suggest culture and individual anxiety are important predictors of behavioural responses to pandemic influenza, with higher levels of anxiety about swine flu in Malaysia compared to Europe, and with greater levels of behavioural change in Malaysia. Particular 'out-groups' (e.g. prostitutes, homosexuals) were judged to be at relatively high risk of infection, with Malaysian respondents particularly likely to emphasise the infection risk in these groups. Such judgements of risk may have important implications for the equitable treatment of socially marginalised group, particularly as the pandemic continues to accelerate worldwide. Note Asterisks indicate significant regional differences (Europe vs. Asia) using Pearson chi-square statistic * p < .05; ** p < .01. We reran these analyses using logistic regressions controlling for age and sex, with similar results to the chi-square analyses. Further details of these findings are available from the first author. Percentages are rounded so may not all always add to 100. Ns range from 312-328 due to some missing data from Malaysia.  On managing complex adaptive systems motivated by biosystems application to infections Many attempts to control Complex adaptive systems (CAS) have failed. Here we try to learn from biosystems to derive some principles for CAS management. An application to managing infections is given. Complex adaptive systems are special cases of complex systems. They are complex in that they are diverse and made up of multiple interconnected elements and adaptive in that they have the capacity to change and learn from experience.
i.1) CAS consists of adaptive (capable of learning) entities. Some properties of such systems cannot be understood just by studying the constituents. They are called emergent properties [3, 4] .
i.2) Examples of CAS include the brain where the emergent property is cognition which is a property of the brain not of individual cells. We propose that IS is a CAS with tolerance as an emergent property since tolerance is a property of IS as a whole not individual B or T or macrophage etc.... cells. In fact almost every biological, social and financial system is a CAS.
i.3) Emergent properties imply that reductions approach is not suitable for CAS. Hence these systems have to be studied as a whole in addition to the reductionest approach. It is crucial to see that both approaches complement each other and are not contradictory [5] .
i.4) CAS have some generic properties e.g. they are open (connected to other systems). Interactions in CAS are nonlinear hence long range predictions in CAS are highly unlikely. Typically they have a network (e.g. evolving network) structure. Optimization in such systems is multiobjective. Control of most CAS is difficult, it should be targeted and integrated i.e. using diverse approaches not just one. Most CAS show delay and memory behavior. Spatial effects should not be neglected in many CAS.
Most, but not all, CAS are decentralized.
i.5) CAS should be studied as a whole in addition to studying its constituents. Hence in vivo experiments are crucial to understand them. i.6) CAS are difficult to control for the following reasons: They are nonlinear. They are open. Delay is a generic property of CAS. Some failure cases in controlling CAS are the Measles-Mumps Rubella (MMR) vaccination and the change of fish type in Lake Victoria [2] . Therefore in the next sections we try to learn from some CAS systems to derive some principles for "managing" CAS.
By management we mean taking decisions with as least bad side effects as possible. And if bad side effects occurred then decisions are taken to remove or contain them as best as possible.
Some of the basic concepts for optimization in CAS are:
ii.1) Multi-objective optimization [6] especially metaheuristics i.e. methods which contain random element and which give a good approximate solution to the optimization problem within a reasonable time.
ii.2) Game theory specially evolutionary game theory [7] . In some cases it is important to include the evolution of strategies or players c.f. the immune system deals with almost continuously evolving antigen e.g. bacteria, viruses or macroparasites.
ii.3) Fractals and networks which play a crucial role in nonlocal spread of effects e.g. infections.
ii.4) Collective animal behavior. Some of its principles include [8] : Non-selfish key individuals, positive and negative feed back, diversity and threshold responses.
From the above results we conclude the following:
iii.1) CAS are extremely difficult to control hence managing CAS may be a more feasible goal than controlling them.
iii.2) Diverse and integrated approaches to the problem are crucial. In may cases single approaches to the problem may lead to counter-productive results. Also this increases the robustness of the management.
iii.
3) The existence of non-selfish key individuals is necessary to solve many problems.
iii.4) The goals should be multi-objective and only guide lines (metaheuristic) approaches should be adopted. One cannot attempt exact determination or control in nonlinear and open systems. Hence no detailed plans should be laid out, instead guide lines may be more realistic.
iii.5) Small world network structure should be followed hence a kind of hierarchical network with short cuts can significantly improve the efficiency of the system [9] .
iii.6) Feedback hence adaptive management is expected to be more efficient than decisions taken once and for all. Also considering the system as game theory with evolving strategies or opponents may help.
iii.7) Emergent problems "messes" happen and will happen. Now the above conclusions are applied to an overview of infection management. We propose to call such problems "emergent problems" [10] . An emergent problem is characterized by:
1) It has several reasons not just one.
2) It cannot be solved locally (e.g. by one country) hence "collective efforts" are needed.
3) It needs a long time to be solved hence evolution in solution strategies has to be taken into consideration.
Collective efforts needed to solve such emergent problems require large scale cooperation. Humans are known not to be good in cooperation. In fact a core problem in most of the above problems is the tragedy of commons [11] which simply states that in the case of finite resources e.g. drugs (which is always the case) a conflict exists between individual's interest and the group's (common) interest e.g. rich and poorer countries. However it is known that for such cooperation to exist a feeling of imminent danger has to exist [12].
Therefore early preparations to deal with such problems are not expected to be as efficient as should be.
It is clear that the absence of non-selfish key individuals is a key reason for the inability to solve this problem.
However some clusters of cooperators do exist. This should be accompanied by retaliation against non-cooperators to lower their benefits from defection (c.f. prisoner's dilemma game [7] ). Some benefits from our discussion are: i) Diverse approaches to the problem is highly desirable e.g. using multi-drugs to reduce the danger of drug resistance (c.f. the sole dependence on Tami flu for avian influenza).
ii) The network (long range) effects are crucial in many infections e.g. foot and mouth disease, SARS, Avian flu and H1N1 flu.
iii) We should take into account the evolution of antigens (c.f. the new strains of Tuberculosis). Prevalence of human herpesvirus 6 antibodies and DNA in allogeneic stem cell transplant patients: two-year single centre experience INTRODUCTION: Human herpesvirus 6 (HHV-6) has been recognized as a potentially significant pathogen in hemopoietic stem cell transplant (HSCT) recipients. Different clinical manifestations have been described, including fever, skin rash, bone marrow suppression, and encephalitis. MATERIALS AND METHODS: A retrospective review of a group of 26 adult recipients of allogeneic HSCTs was conducted. Serum samples taken before transplant were examined for the presence of specific anti-HHV-6 IgM and IgG antibodies. After transplantation, quantitative real-time PCR was used to determine viral load in plasma samples from days 0–80 post-transplant. RESULTS: HHV-6 DNA was detected in plasma samples in 8 (30%) of the 26 recipients between days 18 and 40 after transplantation. All of them developed fever of unknown origin and over 50% had graft-versus-host disease features. Three individuals from this group died during detectable HHV-6 viremia. Another two recipients showed a single positive PCR result at a later time. Infection with HHV-6 was thus confirmed in 10 (38.5%) of the 26 graft recipients. CONCLUSIONS: There is a high frequency of detectable HHV-6 viral load in stem cell transplant recipients in Poland. Further investigation to monitor HHV-6 reactivation in graft recipients will be important to improve outcome for these patients. Human herpesvirus type 6 (HHV-6) belongs to the subfamily β-herpesvirinae and is closely related to HHV-7 and HHV-5 (formerly known as cytomegalovirus -CMV) [5] . Most people have had a primary HHV-6 infection in early childhood [3] and the virus is widespread in the human population, as shown by the presence of specific antibodies in 90% of healthy adults [17] . As a result of the primary infection, HHV-6 is assumed to establish a latent infection and viral DNA can be detected in salivary glands [7] , monocytes [10] , and early bone marrow progenitor cells [13] .
HHV-6 is reported to be a causative agent of exanthema subitum [20] and has been associated with a broad spectrum of diseases, including febrile convulsions [11] , encephalopathy [9, 18] , hepatitis [6] , and lymphoproliferative disorders [15] . An active HHV-6 infection can cause fatal disease in an immunocompromised host, including patients after stem cell transplantations (SCTs), but fatal outcome in immunocompetent persons due to HHV-6 infection is extremely rare [14, 16] .
HHV-6 presence in plasma or serum was documented in 33-48% of hemopoietic stem cell transplant (HSCT) recipients using molecular techniques [22] . The peak viral load occurs early after transplantation and usually within the first four weeks after transplantation [4, 21] . Allogeneic SCT, advanced hematological disease, young age, gender mismatch between the donor and recipient, and treatment with corticosteroids are commonly reported risk factors associated with HHV-6 infection after transplantation [4, 21] .
In a recent study we summarized retrospective results of the determination of HHV infection status in allogeneic stem cell transplant recipients. As infections with HHV-6 are rarely accompanied by clinical symptoms, our first aim was to compare HHV-6 infection status, measured as viral DNA presence in the blood in the post-transplantation period and patients' anti-HHV-6 serological status by detection of IgG and IgM antibodies. This allowed us to determine both the presence of active infection and whether a recent infection was a result of primary contact with the virus or the reactivation of latent virus, which should be valuable information in terms of comparing HHV-6 infection status and clinical status of an infected patient.
Another virus belonging to the β-herpesvirinae subfamily, HHV-5 (CMV), is one from the most important viral pathogens causing clinical infections in patients receiving immunosuppressive treatment. As there is information in published data about a connection between HHV-5 and HHV-6 reactivation in graft recipients, our aim was to determine the appearance of simultaneous reactivation of both β-herpesviruses. We believe that this is probably the first report from Poland involving this kind of examination in HSCT recipients.
This retrospective study involved patients who received an allogeneic HSCT and were hospitalized at the Hematology, Oncology, and Internal Medicine Clinics, Medical University of Warsaw. In the period under consideration (December 2004 to October 2006) there were 38 patients receiving allogeneic stem cell transplants. The criteria for patient selection for the study included the appearance of at least one syndrome from those listed below within a period of 100 days after HSCT: pneumonia, appearance or intensification of graft-versus-host disease (GvHD), skin rash, or pyrexia of unknown origin. Introduction of these criteria resulted in a group to 26 adult receivers of HSCTs ( Table 1) . Monitoring of clinical status of the patients and viral load in blood samples comprised a period of 180 days after HSCT.
IgG and IgM antibodies against HHV-6 were measured from the panel of 26 serum specimens, collected once before HSCT, from the patients with different hematological disorders using a commercial enzyme immunoassay (PanBio). The results obtained with both HHV-6-specific IgG and IgM tests were expressed in PanBio units (PBU). The PBU is calculated as the ratio of the sample absorbance to the cut-off absorbance and multiplied by 10 (absorbance of sample/mean absorbance of cut-off ×10).
The collection of plasma samples from all patients for HHV-5 PCR investigations, according EBMT guidelines, began at a median of 3 days after transplantation (range: 1-7 days) and lasted until a median of 105 days (range: 30-180 days). Routine collection of plasma samples was performed once a week until the 100th day after allogeneic HSCT, thereafter once every two weeks, up to 180th day after HSCT. The median number of blood samples per patient was 12 (range: 3-21). A total of 294 samples from the 26 patients were collected. For the purpose of recent study, viral DNA was extracted from 200 µl of each plasma sample using a QIAmp ® DNA Blood Mini Kit (Qiagen) in accordance with the manufacturer's instructions and retrospectively used for detection of HHV-6 DNA.
For the detection of HHV-6, a real-time PCR assay with fluorescent probes complementary to the sequence lying within the amplified product was used. The tests were run on a LightCycler 2.0 instrument (Roche) with the commercial quantitative MutaREAL ® HHV-6 kit (ALPCO). All samples were examined according to the manufacturer's instructions. Every tested sample was amplified with an internal control (positive control of the DNA extraction and amplification process). Each amplification reaction also included, besides the tested samples, positive HHV-6-specific controls and a negative control of the DNA extraction and amplification process.
HHV-5 DNA was detected using the commercial real-time test CMV Quant Kit ® (Roche) developed for the LightCycler 2.0 instrument. The test uses internal SCORPIONS™ fluorescent probes for the PCR amplification product. Analogously to HHV-6 detection, for every sample an internal control was added and the amplification was performed in the presence of amplification-specific controls (positive, negative, and extraction process control).
Specific IgM antibodies were present in the serum of 2 (8%) patients, while the others were negative (92%). Twenty of the 26 persons (77%) had IgG antibodies against HHV-6 before HSCT and the other 6 (23%) were negative. Of the group of 16 patients who had no detectable HHV-6 during the entire post-transplantation period, 10 (62.5% of the HHV-6-negative patients) had a positive result only for the anti-HHV-6 IgG test, but no anti-HHV-6 IgM, in serum sample taken directly before transplantation, 4 (31.3%) were both IgM and IgG negative, and the remaining patient had anti-HHV-6 antibodies of both classes ( Table 1) .
The serological status of the 10 patients who had HHV-6 DNA detected in one or more blood samples was very similar: 7 patients (70%) had only IgG, 2 patients (20%) had no IgG or IgM antibodies, and one patient had HHV-6-specific IgG and IgM.
Regarding the amplification results of DNA isolated from plasma using HHV-6-specific PCR, expressed as the presence of exponential increases in fluorescence, products were detected in 29 samples (10%) taken from 10 patients (38%). Two of them (8%) had HHV-6 DNA in only a single positive sample and another 8 (30%) had positive results in two or more subsequent tests. In the patients with two or more plasma samples containing HHV-6 DNA, viremia was observed between days 18 and 40 after transplantation (Table 1 ). In the two patients who had a single HHV-6-positive blood sample, viral DNA was detected at a later time. The quantitative results obtained by the MutaREAL ® HHV-6 test in all positive cases were at low levels, between 700-1600 copies/ml. HHV-5 DNA was detected in the plasma samples collected from four HHV-6-negative patients. In all four cases, HHV-5 DNAemia was observed in the typical period of 40-65 days after transplantation and, moreover, a second HHV-5 viremia course occurred in two patients starting at days 103 and 123 after transplanta- Abbreviations: ALL -acute lymphoblastic leukemia, AML -acute myeloid leukemia, CML -chronic myeloid leukemia, MDSmyelodysplastic syndrome, rPBSCT -related peripheral blood stem cell transplantation, uPBSCT -unrelated peripheral blood stem cell transplantation, uCBSCT -unrelated cord blood stem cell transplantation, rBMT -related bone marrow transplantation, uBMT -unrelated bone marrow transplantation, -: negative, +: positive in one plasma/serum sample, ++: positive in two or more plasma samples. Abbreviations: ARDS -acute respiratory distress syndrome, CNS -central nervous system, GvHD -graft-versus-host disease, N/A -not applicable, -: negative, +: positive in one plasma/serum sample, ++: positive in two or more plasma samples.
tion. None of the HHV-6-positive patients had HHV-5 DNA in their plasma samples during the 180-day period.
Overall mortality in the entire group of HSCT recipients during the first 180 days after transplantation was 34.6% (9 of the 26 patients) and the most frequent direct cause of death was acute respiratory distress syndrome (6 patients), in some patients accompanied by sepsis (2 patients), pneumonia (1 patient), or central nervous system (CNS) infection (1 patients). Other death causes included pneumonia (1 patient), bleeding within the CNS during infectious meningitis (1 patient), and shock during subsequent HSCT (1 patient). Seven of the 9 patients died during the first 34 days after HSCT (Table 2 ). In the HHV-6-positive patients, mortality was 50% (5 out of 10 patients). Four of those patients died during the HHV-6 viremia period, which occurred within the first 34 days after HSCT. Table 2 summarizes the clinical features and the times and causes of death of the HSCT recipients.
Herpesviruses persist throughout life after primary infection. Viral proliferation occurs either spontaneously or under conditions of immunosuppression. Reactivation can lead to illnesses that typically differ in their clinical presentation from the disease associated with the primary infection. After SCT, reactivating members of the β-herpesvirinae subfamily (among them HHV-6) frequently cause serious, sometimes life--threatening disease [8, 12] . HHV-6 infection or reactivation in these individuals has been associated with a delay or suppression of marrow engraftment [12] , pneumonia [2] , skin rash, and fever [16] . Although it is difficult to prove an etiologic association of the virus with these disease events, their propinquity with HHV-6 activity in the absence of other possible causes suggests that at the very least a subset of these events is due to HHV-6 activity.
In the present study we found that 38% (10 individuals) of the graft recipients developed HHV-6 infection ( Table 1) . Eight patients (30%) had detectable viral DNA levels in two or more samples in subsequent tests during the six weeks following SCT and another two (8%) had a single sample positive at a later time. It is likely that the HHV-6 infection that occurred after transplantation was due to activation of the virus in the bodies of the recipients, since most of the recipients were immune to HHV-6 and no virus was found before SCT. However, we do not know whether HHV-6 strains detected in the present study were derived from the donor or were transfered from other sources to the seronegative recipients. The virus probably remains latent in the body after primary infection, as do other human herpesviruses. If the HHV-6 was derived from the donor, it must have been latently infecting the donor's hemopoietic stem cells and was activated to replicate after transfer to the recipient. In other cases, the virus was probably reactivated from the recipient's own body by factors such as a profound immune dysfunction or an allogeneic reaction after transplantation.
We did not find any correlation between viremia and the applied conditioning regimen or anti-GvHD prophylaxis. All of the HHV-6-positive graft recipients had fever of unknown etiology during the six weeks after SCT and half of them (4 persons) had acute GvHD features. Sixty-three percent of the described patients had pneumonia and 38% skin rash. No Epstein-Barr virus or CMV DNA was found in all the plasma samples during HHV-6 onset ( Table 1) .
The exact association between HHV-6 reactivation and mortality found in this study is not clear. Three of the patients (38%) with detectable HHV-6 DNA levels died during viremia shortly after transplantation, all due to coexisting pneumonia of unconfirmed etiology. Acyclovir in typical doses was used as an antiviral prophylaxis during this period, but without any visible clinical success. Studies in vitro have shown that HHV-6 DNA replication is inhibited by ganciclovir, foscarnet, and cidofovir, but not by acyclovir [1] .
There is a high frequency of detectable HHV-6 viral load in SCT recipients and it may lead to an increased risk of fatal symptomatic disease [19] . The availability of quantitative real-time PCR means that results are available in a clinically helpful time-frame, which should assist with implementing timely therapeutic intervention and assessing response to treatment. Further investigation to monitor HHV-6 reactivation on a larger group of SCT recipients will be important in improving outcome for these patients. The infection of primary avian tracheal epithelial cells with infectious bronchitis virus Here we introduce a culture system for the isolation, passaging and amplification of avian tracheal epithelial (ATE) cells. The ATE medium, which contains chicken embryo extract and fetal bovine serum, supports the growth of ciliated cells, goblet cells and basal cells from chicken tracheas on fibronectin- or matrigel-coated dishes. Non-epithelial cells make up less than 10% of the total population. We further show that ATE cells support the replication and spread of infectious bronchitis virus (IBV). Interestingly, immunocytostaining revealed that basal cells are resistant to IBV infection. We also demonstrate that glycosaminoglycan had no effect on infection of the cells by IBV. Taken together, these findings suggest that primary ATE cells provide a novel cell culture system for the amplification of IBV and the in vitro characterization of viral cytopathogenesis. Embryonated egg inoculation is commonly used to amplify avian RNA viruses. Successful viral replication mainly depends upon viral adaption to and replication in the cells of the embryonic and extraembryonic tissues. Growth retardation and malformation of the embryo are the main indicators of viral reproduction in this system and signal the proper period for harvesting the amplified virions from the amniotic or chorioallantoic fluids.
The low efficiency of viral amplification may prevent etiology determinations, intensive surveillance of epidemiology and the tracing of potential natural domestic hosts for viral pathogens during an outbreak. For instance, avian metapneumovirus (MPV), which primarily attacks the respiratory tract, replicates inefficiently in embryonic eggs. Diagnosis of MPV infection is mainly dependent on the detection of viral RNA or viral antigens, but not viral isolation [5, 6] . In addition, to investigate the epidemiology of avian influenza viruses (AIV) requires efficient and sensitive viral detection and amplification system for comprehensive collection of the viruses from waterfowl and migratory birds [17, 31] . However, the wildbird isolated AIV are usually mildly or nonpathogenic to chicken embryos [20, 26] and the efficiency of viral isolation is often unsatisfactory in embryonated eggs [11, 19] .
It has been shown that serial passage using chicken embryos enforces the selection of adapted viruses and inevitably alters the genetic codes of the primary viral isolates, especially for genes involved in cell adsorption and viral replication [8, 9] . The environment of the viral replication may also selectively modify the original cell tropism and pathogenesis of the isolated virus [15, 25] . It has been shown that a serially embryo-passaged of infectious bronchitis viruses (IBV) M41 strain exhibited significantly attenuated pathogenesis within the oviduct compared to the parental strain [7] . In addition, egg-mediated epitope alterations in amplified isolates might attenuate the protective efficiency of these viruses when used to vaccinate against the challenge of a wild-type virulent strain [16, 24] . For instance, after egg adaption, human influenza A virus, originally showing a2-6 sialic acid tropism only, exhibits increased hemagglutinin (HA) binding affinity to a2-3 sialic acid-containing gangliosides due to amino acid substitutions in the vicinity of the receptor binding site of the HA protein [8, 15] . For primary viral isolates, providing natural host cells for replication might eliminate the selection pressure of an exotic growth environment and avert the alteration of cell tropism and consequent genomic mutations.
Recently, a study reported a primary culture system for tracheal epithelial cells from an embryonic day (E) 17 chick embryo, in which dissociated culture cells exhibited ciliary movement and were positive for the expression of pan-cytokeratin [33] . In addition, the global profile of mRNA expression in the cultured cells showed that cytokeratin 14 (K14), a basal cell marker, was highly expressed. However, the protein expression of cell-type specific markers and the existence of mucin-secreting goblet cell are not well-characterized. In this study, we developed a novel culture system to isolate, amplify and passage chicken tracheal epithelial cells in an efficient manner. The avian tracheal epithelial (ATE) cell types were identified using immunocytostaining, and the ratio of cell types is statistically illustrated. We further demonstrate that primary ATE cells support IBV replication. The susceptible cell types and the effect of glycosaminoglycan (GAG) on IBV attachment to ATE cells was also investigated.
Two IBV, 2575/98 and 2296/95, were isolated in Taiwan and maintained using serial passage through specific pathogen-free (SPF) eggs [29] . IBV were inoculated into the embryonic eggs at the E9 stage and harvested at the E11 stage from chorioallantoic fluid. The original viral titers of 2575/98 and 2296/ 95 are 10 7.4 EID 50 /0.1 mL and that of 2296 reached 10 9.6 /0.1 mL, respectively [14] . The applied viral titer of both IBV was 1 · 10 5 EID 50 /mL.
Plaque-purified Taiwan-isolated Japanese encephalitis virus (JEV) strain RP-9 [28] was used as a positive control for the study of GAG effect on the virus-cell attachment. The adsorption of JEV was performed with serum-free RPMI medium at 37°C for 1 h. Virus-infected cells were incubated in RPMI containing 2% fetal bovine serum (FBS). Virus titers were determined by plaque assay in BHK-21 (baby hamster kidney) cells, which were maintained in RPMI 1640 with 5% FBS, 2 mM L-glutamine, 100 mg/mL of streptomycin, and 100 IU/mL of penicillin. The medium and culture reagents are obtained from Invitrogen (Carlsbad, CA, USA).
Tracheas were obtained from one-day-old SPF chicks and rinsed in DMEM medium (Invitrogen) under sterile conditions. After the removal of surrounding adipose and muscular tissues, tracheas were digested with dispase I solution (2.5 U/mL dispase I) (Roche, Nutley, NJ, USA) for 2 h at 37°C to disrupt the basal membrane. Forceps were used to force the outflow of detached cell sheets of tracheal epithelium from the tracheal lumen. The epithelial cells were harvested and further digested with collagenase I (1 mg/mL, Roche) for 5 min at 37°C. The cells were gently pipetted, and the tissues were homogenized into small cell clumps. FBS was added to the digesting solution to stop the reaction. The cells were centrifuged at 1000 rpm for 5 min to remove residual digesting enzymes. The cell pellets were resuspended in ATE medium, containing 10% FBS (Invitrogen), 10% chicken embryo extract (CEE) (US Biological, Swampscott, MA, USA; or self-made [27] ), 1% glutamine (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 1% nonessential amino acids (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) in DMEM-F12 medium (Invitrogen). Cells were seeded on 2% matrigel-or 20 lg/mL fibronectin-coated 24-well or 96-well plates (Corning Inc., Corning, NY, USA). When the cells reached confluence, the cultured cells were passaged by digestion with both 0.01% (w/vol) protease (type XIV, Sigma-Aldrich) and 2.5 U/mL dispase I for 5 min at 37°C.
GAG compounds, including the heparin (H), heparan sulfate (HS), dextran sulfate (DS) and chondroitin sulfate (CS), were obtained from Sigma-Aldrich. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay kit was obtained from Sigma-Aldrich. FITC-conjugated BSI-B4 isolectin (Sigma-Aldrich) was used as a marker for basal cells from the tracheal epithelium [12] . Commercial culture media for human and rat airway epithelial cells were obtained from CELLnTEC advanced cell system (Postfach, Bern, Switzerland) and CELL Applications Inc. (San Diego, CA, USA), respectively.
Cells or cryosections of infected trachea were fixed in 4% cold paraformaldehyde and permeabilized with 0.3% Triton-X 100. Immunocytochemistry was performed with the following primary antibodies: E-cadherin (1:500, BD Biosciences, Franklin Lakes, NJ, USA), b-tubulin IV and pan b-tubulin (1:100, Sigma), ZO-1 (1:100, Zymed-Invitrogen), mucin 5AC (1:50, Abcam, Cambridge, MA, USA), cytokeratin 14 (1:100, Convance, Princeton, NJ, USA), smooth muscle actin (SMA, 1:50, DakoCytomation, Glostrup, Denmark) and vimentin (1:500, Convance). Immune-serum of IBV-infected chicken, screened by ELISA, was used for the detection of IBV antigens in infected cells (1:200) [30] . Fixed cells were washed twice with 0.1% Tween-20 in phosphate buffer saline (PBS). Appropriate fluorescence-tagged secondary antibodies (all from Jackson ImmunoResearch, West Grove, PA, USA) were used for visualization. Blue 4 0 ,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counter-staining. Images of immunostaining were captured using a fluorescent microscope (Nikon ECLIPSE 80I) or confocal microscope (LSM510 Meta, Zeiss).
Total RNA was extracted from TW2575/98infected ATE cells using TRIzol TM C&T (Protech, Taiwan) according to the manufacturer's protocol. Positive-sense and negative-sense viral RNA (0.5 lg) were reverse transcribed into cDNA using SuperScript TM III reverse transcriptase (RT) (Invitrogen) with oligo dT and sense primer 5 0 -ACT GAA AAT GAT AGT GTT ATG-3 0 , respectively. PCR was performed with a proofreading DNA polymerase (KOD-Plus, Toyobo, Osaka, Japan). The PCR cycling conditions consisted of 28 cycles at 94°C for 30 s, 62°C for 30 s, and 68°C for 1 min on a PCR machine (ASTEC 818). The primer sequences for detection of the nucleocapsid gene of IBV were 5 0 -AAT GCA TCT TGG TTT CAA GC-3 0 and 5 0 -TCC TCA TCT GAG GTC AAT GC-3 0 ; for detection of GAPDH, the sequences were 5 0 -GTG AAG GTC GGT GTG AAC G3 0 and 5 0 -GGT GAA GAC ACC AGT AGA CAC TC-3 0 .
The effect of GAG on JEV and IBV infections was measured in BHK-21 cells and ATE cells, respectively. The cells were seeded at a density of 5 · 10 3 cells/well in 96-well plates. The GAG were serially diluted and applied at 0, 7.5, 15, and 30 lg/mL for BHK-21 cells and at 0, 0.5, 1.0, 2.0 mg/mL for ATE cells. The BHK-21 and ATE cells were infected with 500 plaque-forming units of JEV and 10 3 EID 50 of IBV at 37°C for 1 h. Cells were washed three times with serum-free medium, and then normal growth medium was added. The number of infected cells per 96-well, revealed by IBV immunocytostaining, was manually counted at 8 h post-infection (h.p.i.) from five individual fields. No obvious cell damage was observed after GAG treatment or viral infection at 8 h.p.i. [18, 28] .
For the isolation of tracheal epithelial cells, the intact cell sheet of the tracheal epithelium was first isolated from dispase-treated tracheas (Fig. 1A) . The epithelial membrane sheet was further mechanically disrupted into small pieces by pipetting. Floating unattached ATE cells rapidly underwent cell death or growth arrest after a one-day culture. The optimal cell matrix for ATE cells was evaluated; both 2% matrigel or 20 lg/mL fibronectin efficiently promoted cell attachment, but gelatin, collagen I, collagen IV and laminin were less effective (data not shown). The primary culture system for mammalian tracheal epithelial cells is well-established. To culture the ATE cells, specialized commercial media for the tracheal epithelia of humans and rats were first tested (described in the Materials and methods section). None of them supported cell growth or long-term viability of ATE cells. Adjustment of the concentration of retinoic acid, a differentiating factor for tracheal epithelial cells [10] , did not affect attachment or cell growth of ATE cells. Increasing FBS from 10% to 20% in the culture medium modestly enhanced the viability of the cells (data not shown). We speculated that unidentified chicken nutrients essential for the promotion of ATE cell growth were absent in the culture medium. CEE, an essential supplement for the culture of mouse neural crest stem cells [27] , was added to DMEM-F12 medium with 10% FBS. We found that CEE significantly improved cell growth and reduced cell death of the primary ATE cells (Figs. 1B and 1C) . The cell number of initial harvest from one trachea on day 1 is about 2 · 10 5 cells. With this optimized culture medium, isolated cells displayed increased MTT activity, and the celldoubling time was 46 h during the first 6 days of culture (Fig. 1D) . The ATE cells could sustain proliferative activity for 3-5 passages. These results show that the established culture system provides sufficient nutrients and proper 
Tracheal epithelial cells are connected to each other by E-cadherin molecules [23] . Among the isolated ATE cells, 88.3 ± 13.2% expressed E-cadherin and a gap-junction molecule, ZO-1 [23] . E-cadherin-negative cells, including SMA-positive muscle cells and vimentin-positive fibroblast cells, accounted for 5.0 ± 1.4% and 3.5 ± 1.2% of the cultured cells, respectively, indicating the high purity of this primary culture system. In tracheal epithelial cells, b-tubulin IV/b-tubulin, mucin 5AC and cytokeratin 14 (K14) are specific markers of ciliated cells, goblet cells and basal cells, respectively [12, 13, 23] . Basal cells also show high affinity for GSI-B4, a plant lectin that specifically binds the glycoconjugates of basal cells of tracheal epithelia [12, 13] . Figure 2 shows that these three major cell types -ciliated cells, goblet cells and basal cells -were all present in the ATE population, and accounted for 22.8 ± 9.1%, 44.8 ± 9.7% and 41.6 ± 8.4% of the ATE cells, respectively.
To determine whether ATE cells support the replication of IBV, two egg-adapted Taiwan IBV strains, 2575/98 and 2296/95, were used in this study. These two IBV viruses cannot infect primary chicken embryonic fibroblast cells or immortalized DF1 chicken fibroblast cells. The ATE cells (5 · 10 4 ) were infected with 50 lL 2575/98 (EID 50 = 10 5 /mL) for 1 h at 37°C. At 24 h.p.i., IBV proteins were detected in 2 500-3 000 cells by staining with chicken anti-serum against IBV (Fig. 3B) . We found that the IBV only infected N-cadherin + tracheal epithelial cells, revealing that neither smooth muscle cells nor fibroblast cells are the major target cells for IBV infection (Figs.  3D and 3E) . The infection rate gradually increased to 55.3 ± 12.8% of the total cells at 72 h.p.i. (Fig. 3C) , as estimated by immunocytostaining for IBV antigens in all adherent cells. Similar results were observed in 2296/95-infected cells (data not shown). In addition, both positive-and negative-sense viral RNA present were detected in the cytosol, and positive-sense viral RNA could be detected in the supernatant of ATE cells at 24 h.p.i. (Fig. 3F) , demonstrating that mature IBV virion could be produced and released from the cells. These results indicate that cultured ATE cells support viral infection, replication and spreading of IBV.
It has been reported that IBV can infect both ciliated cells and goblet cells in the tracheal epithelium [1, 22] . However, whether basal cells are also susceptible to IBV infection remains obscure. We infected isolated intact tracheas with 50 lL 2575/98 (EID 50 = 10 5 /mL) for 48 h and found that IBV protein expression only colocalized with b-tubulin IV-or mucin 5AC-positive cells (Figs. 4A and 4B) . No viral protein was detected in the K14-positive basal cells of the tracheal epithelium (Figs. 4C and 4D). To further examine whether basal cells are resistant to IBV infection in vitro, 5 · 10 4 ATE cells were also infected with 50 lL of IBV 2575/98 strain (10 5 EID 50 /mL) for 48 h. In vitro results consistently showed that viral proteins of IBV were mainly detected in ciliated cells and goblet cells, but not in basal cells (Figs. 4E-4H). Similar cell tropism results were obtained when IBV 2296/95 or a higher dosage of viral loading was used (data not shown). These in vivo and in vitro experiments clearly delineate the cell tropism of IBV in the avian respiratory tract.
Virus binding to host cell surface receptors is critical for determining tissue tropism and viral pathogenesis. It has been shown that H or HS may serve as a coreceptor in fibroblast cells for infection by the IBV Beaudette strain, but not the M41 strain [18] . In this study, highly sulfated GAG, such as H and DS, and common GAG, such as HS and CS, were tested to determine whether they possess different neutralizing effects on binding of Taiwan-isolated IBV to ATE cells. JEV was used as a positive control for the evaluation of the GAG effect [28] . Figure 5A shows that both H and DS at 7.5 lg/mL effectively blocked JEV infection in BHK-21 cells, as previously reported [28] . Complete inhibition of JEV infection was achieved when the concentrations of H and DS were elevated to 30 lg/mL. Although low concentrations of HS and CS did not affect JEV infection, a modest neutralizing effect was observed when the concentrations of HS and CS were elevated to 30 lg/mL. In contrast to the JEV results, even when all of the tested GAG were applied at 2.0 mg/mL, no significant inhibitory effects were observed on the infection of primary ATE cells by TW2575/98 (Fig. 5B ). In addition, the GAG did not alter the cell tropism of IBV on cilia cells or goblet cells (data not shown). These results strongly suggest that the binding affinity of GAG for IBV is too low to interfere with viral entry into tracheal epithelial cells. In this study, we showed that primary ATE cells exhibit the same cell composition as tracheal epithelia and can be passaged and amplified in a convenient and efficient way. These cells support IBV viral replication and viral release, providing an ideal system to amplify respiratory viruses and characterize their pathogenesis.
In studies of tracheal infection, ciliated cells and goblet cells have been shown to be the major target cells of IBV [1, 22] . Our results with primary ATE cells also showed that IBV viral protein can be detected in both ciliated cells and goblet cells. This cell tropism may account for the pathogenesis of IBV, such as the ciliostasis in observed IBV-infected TOC and the reduction of sialic acid secretion [3, 22] . In addition, we are the first to demonstrate that IBV does not appear to infect K14-positive basal cells. In an uncomplicated IBV-infected chick, clinical signs such as gasping, coughing, tracheal rales and nasal discharge persist for only 5-to-7 days and disappear within 2 weeks [3, 22] . In the recovery stage, unaffected basal cells may be responsible for epithelial hyperplasia after desquamation of the ciliated and goblet cells and reconstruction of the epithelium of the injured respiratory tract to reestablish normal physiological functioning [12, 13, 22] . The insensitivity of basal cells to IBV infection also suggests that coinfection with other viral or bacterial pathogens is required to disrupt basal membrane integrity and cause hemorrhage in IBV-infected tracheas. At present, quantification of the virulence of IBV is still a problem [3, 22] . Measuring ciliostasis in TOC is not a sensitive approach for determining the severity of respiratory tract injury caused by IBV infection [21] . In addition, different species of chicken may also vary in the vulnerability of their respiratory tissue to IBV infection [2, 22] . In our ATE system, IBV shows high affinity for ciliated cells and goblet cells. Cell tropism, viral replication and viral spread can be determined and quantified by immunocytostaining, suggesting that ATE cells could serve as a quantitative platform to determine the pathogenesis of IBV. It has been shown that sialic acid and HS may help IBV bind to its receptor on the cell membrane of target cells [18, 32] . Receptor binding triggers endocytosis and delivers the viral genome into the cytosol through pHdependent membrane fusion [4] . In this study, we demonstrate that none of the tested GAG interfered with IBV binding to ATE cells. Further investigation is required to test whether this ineffectiveness is due to a lack of the XBBXBX H consensus sequence in the S protein sequences of TW IBV.
In this study, we established a primary ATE cell culture system for the maintenance and amplification of epithelial cells of the chicken respiratory tract. Using these culture conditions, immortalized cell lines could potentially be established by overexpression of oncogenes, telomerase or anti-apoptotic genes. Both primary cells and respiratory cell lines will provide a new window to reveal viral life cycles, viral persistence, virus-cell interaction and the pathogenesis of avian respiratory viruses. Using Dynamic Stochastic Modelling to Estimate Population Risk Factors in Infectious Disease: The Example of FIV in 15 Cat Populations BACKGROUND: In natural cat populations, Feline Immunodeficiency Virus (FIV) is transmitted through bites between individuals. Factors such as the density of cats within the population or the sex-ratio can have potentially strong effects on the frequency of fight between individuals and hence appear as important population risk factors for FIV. METHODOLOGY/PRINCIPAL FINDINGS: To study such population risk factors, we present data on FIV prevalence in 15 cat populations in northeastern France. We investigate five key social factors of cat populations; the density of cats, the sex-ratio, the number of males and the mean age of males and females within the population. We overcome the problem of dependence in the infective status data using sexually-structured dynamic stochastic models. Only the age of males and females had an effect (p = 0.043 and p = 0.02, respectively) on the male-to-female transmission rate. Due to multiple tests, it is even likely that these effects are, in reality, not significant. Finally we show that, in our study area, the data can be explained by a very simple model that does not invoke any risk factor. CONCLUSION: Our conclusion is that, in host-parasite systems in general, fluctuations due to stochasticity in the transmission process are naturally very large and may alone explain a larger part of the variability in observed disease prevalence between populations than previously expected. Finally, we determined confidence intervals for the simple model parameters that can be used to further aid in management of the disease. Feline Immunodeficiency Virus (FIV) infects numerous feline species worldwide [1] . This Lentivirus from Retroviridae family is closely related to Human Immunodeficiency Virus (HIV) and Simian Immunodeficiency Virus (SIV) [2] . This is a virus of major importance because it is lethal to the domestic cat (Felis silvestris catus) and can affect several other cat species, most of which are threatened or endangered e.g., the European wildcat F. s. silvestris in Europe [3] [4] [5] . There is thus a need to better understand the risk factors affecting the spread and patterns of persistence of FIV in natural populations of domestic cats.
In natural domestic cat populations, FIV is mainly transmitted through bites arising from aggressive or sexual contacts [3, [6] [7] [8] [9] [10] . As a consequence, the spread of FIV in domestic cat populations is highly influenced by the mating system; a higher FIV prevalence is observed in aggressive and polygynous cat populations that involve more fights and bites than in much less aggressive and promiscuous urban ones [8, 9] , where FIV can be absent [11] .
Basically, factors affecting cats'aggressiveness can be divided into two categories. At the individual level, some cats are more aggressive than others. Typically, this is the case for dominant males [8, 9] or orange cats [12] . In the field, they are generally more often infected than subordinates, females or other colour morphs [8, 12, 13] . At the population level, the overall aggressiveness of cats largely depends on the population social structure. A male-biased sex-ratio may make the entire population more aggressive, making virus transmission more efficient and, thus, lead to higher disease prevalence.
Until now, to our knowledge, all studies of FIV risk factors have focused on individual risk factors. Factors that may increase the overall virus transmission rate are at least as important for controlling the disease spread but, paradoxically, have been largely overlooked until now. Here, we investigate how some characteristics of cat populations, such as cat density or sex-ratio, e.g., as indicators for aggressiveness in contacts within the population, may act as population risk factors that increase or decrease the virus prevalence within populations.
Understanding the factors that may increase the FIV transmission rate within populations requires the sampling of a set of neighboring cat populations (which, until now, has rarely been done), and then examination of how FIV prevalence correlates with the suspected risk factors. For that purpose, we sampled 15 cat populations in North-Eastern France and measured, within each population, FIV prevalence in males and females. We found significant variability in disease prevalence between populations, especially in males. We also measured five social indicators in order to measure how they correlated with FIV prevalence.
Commonly, risk factors are analyzed with logistic regression models. However, these models are built on the assumption that individuals become infected independently of each other; a hypothesis that contradicts the fundamental communicable nature of infectious diseases [1, 14, 15] . Moreover, as described below, assumptions of independence lead to underestimate the variability in disease prevalence between populations that would be observed in the absence of risk factors.
Our method is inspired by previous works based on the comparison of stochastic dynamic models of the disease spread within host populations to the data [14, [16] [17] [18] [19] [20] . The idea is that each combination of population risk factors leads to a different model. Our objective is to determine the model (i.e. the combination of risk factors) that best fits the data. Beyond the simple analysis of the risk factors associated with FIV, this work aims to understand why the variability observed in our disease prevalence data is so large -data on disease prevalence in males exhibited significant extra-Binomial variations. Can we isolate population risk factors that would explain particularly high disease prevalence in some populations? Does the spatial aggregation of populations with high virus prevalence help to explain the variability in disease prevalence? Or, in contrast, is the large variability observed in disease prevalence a natural consequence of the transmissible nature of the virus?
The work presented here supports this last hypothesis: random fluctuations in the transmission process lead to much greater variation in disease prevalence than with a simple Binomial distribution, underlying classical risk factor analyses. So the simplest model describes well the data and explains the large variability observed in disease prevalence between cat populations without invoking any risk factor. Finally, we determine confidence intervals for the model parameters. The model is very simple, explains the data well, and hence constitutes an interesting tool for further understanding and control of the spread of FIV in these cat populations. The approach developed here can easily extend to many host-parasite interactions.
The field work has been made by qualified people according to current French legislation. Accreditation has been granted to the UMR-CNRS 5558 (accreditation number 692660703) for the program.
Fifteen spatially separated rural cat populations were sampled during 2007 in North-Eastern France near the city of Nancy (Fig. 1, black rectangles) . The distance separating neighboring cat Figure 1 . The study area. We identified three metapopulations (grey areas). Studied cat populations are represented with black rectangles and solid arrows represent the suspected interactions between the studied populations. Some unstudied populations may interact with the studied ones (dashed arrows) and are represented by white rectangles. doi:10.1371/journal.pone.0007377.g001 populations varied from 1.2 to 4 km. The study zone covers a territory of approximately 250 km 2 . In order to delimit the study area, we considered the geographical characteristics that might limit movements between the studied cat populations and those outside of the studied area. The spatial organisation of geographical barriers suggests that the populations may be organized into three distinct metapopulations, with rare contacts between cats of different metapopulations (Fig. 1, grey areas) . At a finer scale, behavioral observations reveal that males can disperse between populations along roads. By adopting a basic assumption that populations are considered connected when they are not too distant from each other (i.e. less than 2 km) and are connected by roads, we propose a connection network between the different populations (see Fig. 1 , solid arrows). Unfortunately, it was not possible to establish a fully isolated perimeter (at least in relation to spread of FIV), so some populations of the study area are in fact connected to unstudied populations ( Fig. 1 , white rectangles -the connections to the study populations are represented by dashed arrows). In particular, Saulxure-Les-Vannes is connected to several study populations. However, it was not considered for sampling because of potential bias due to a cat culling programme there. The village of Sepvigny, which is connected to Champougny, could not be sampled for technical reasons.
Most of the cats were captured using baited traps; others being caught directly in their owner's houses. Upon capture, cats were anaesthetized with an intramuscular injection of ketamin chlorhydrate (Immalgène 1000 15 mg/kg, Rhône-Mérieux) and acepromazin (Vétranquil 5.5% 0.5 mg/kg, Sanofi). They were marked permanently using an electronic passive integrative transponder (pig-tag) to allow all individuals to be identified in case of recapture. For each cat, we have recorded, among other data, information on sex, age and serological status in relation to FIV. Blood samples were taken from the jugular vein and the cats were then released. The ELISA method (SNAP Combo +, Idexx) was used to detect the presence of FIV-specific antibodies, which generally identifies virus carriers [6] . All positive sera for FIV were confirmed by Western blot analysis [21] . FIV was scored as present or absent for each sampled cat.
2.1 General approach. The approach we use here is very similar to the classical approach based on multifactorial logistic regression, which consists of: -Step 1: Choice of a model H 0 against which the data is compared. In the case of the classical logistic regression approach, it is assumed that all individuals have a same probability p to be infected, independently of the other individuals' status. As a result, under model H 0 the distribution of the number of infected cases in the population follows a binomial distribution of parameter p and N, where N is the number of individuals of the population. -Step 2: Some of the model parameters are expected to depend on risk factors.
Choosing p as a function of risk factors means that each individual has its own probability of being infected (depending on its characteristics in terms of risk factors). It is classically assumed that the logit of p is a linear function of the different risk factors: logit(p) = a 0 +ga i X i , where X i denotes the i-th risk factor value for the individual. -Step 3: Model selection process. Different models are defined by setting some coefficients (a i ) to 0. Hence, the probability of being infected only depends on the risk factors which associated coefficients are non-zero. The different models are compared (usually using an Akaïke Information Criterium, AIC) to determine which model best describes the data.
Note that there are two equivalent ways of presenting the classical approach. Firstly, the expected proportion of infected captured individuals is taken as a function of risk factors plus a random term based on a centered binomial distribution. Secondly, the probability that each captured individual is infected is taken as a function of risk factors, with random fluctuations in expected proportions naturally arising from these probabilities. Here, we present the second format because it allows us to easily illustrate how our approach is, in fact, a natural extension of the classical one.
The main difference between our approach and the classical one comes from the model used to describe the data. It is quite obvious that for transmissible diseases the probability of one individual being infected is not independent of the infection status of the other individuals [1, 14, 15] . Here we consider the probability of individuals becoming infected as the result of a dynamic process of between-host virus transmission (described in the next section). These types of models are widely recognized as common tools for representing infectious disease data.
We also make some minor changes to steps 2 and 3. In step 2, the logit function is chosen in the classical approach mainly because the model parameter p is bounded by 0 and 1. Since, as described below, our model parameters are not bounded by 1, we have no reason to consider their logit value. Lastly, in step 3 for model comparison we use likelihood ratio tests (LRT) rather than AIC. LRTs are chosen to test one particular assumption, which is here whether the simplest model, i.e. where no model parameter depends on risk factors, is sufficient to describe the data.
2.2. The dynamic epidemiological model -Model H 0 . The aim of this framework is to study population risk factors, i.e. factors that affect the rate at which the virus is transmitted within the population. Individual risk factors, i.e. factors that make some individuals more prone to infection than others in the same population, are not studied here.
Our mathematical model extends the classical Susceptible-Infected (SI) model (Fig. 2) . We assume that all individuals of each population are equivalent, apart from their sex, the effect of sex on FIV transmission being too significant to be ignored. Indeed, of the 250 males captured in the study, 58 were seropositive (23.2%) compared to 22 of 249 (8.8%) females, which is highly significant (x 2 = 13.80, 1 df, p,10 24 ). Moreover, males and females play different roles in the transmission of FIV [8, 12] . Since females rarely bite, they can be considered as non-transmitting of the virus. The sexual structure of the model is simply represented by splitting classes S and I into two sub-classes, one for each sex.
The age of individuals is not considered in our model, even though it may affect their behavior and, thus, their risk of becoming infected [3, 8, 13] . Moreover, due to long FIV infection duration, an accumulation of infected cases develops in older age cohorts. To represent the effects of age in a simplified way, we assume that the mean age of cats in the population may act as a risk factor for FIV transmission. This is justified since, here, we mainly focus on the global prevalence of FIV within populations without reference to the age-distribution of infections.
We assume a proportionate mixing law for the incidence function of FIV between males, which is more appropriate in social species [22, 23] . Transmission between males of the same population occurs at a rate b M /M, where M is the total number of males in the population, and susceptible females are infected by infected males from their population at a rate b F /M. The constants b M and b F are proportional to the rate at which males are involved in fights and to the rate at which females mate, respectively. We assume constant numbers of males (M) and females (F) within each population, whereby dead cats are instantaneously replaced by newborn cats. Since vertical transmission is very unlikely in the field [7, [24] [25] [26] , all newborns are classified as susceptible to infection. Infected cats die at a rate a. Susceptible cats also die, but since they are instantaneously replaced by susceptible (and thus equivalent) newborn cats, their death is not explicitly modeled.
For the sake of simplicity, we assume that populations are not explicitly connected, such that the numbers of infected cats in the different populations are independent random variables. To avoid the definitive extinction of the virus from the populations we assume regular infections from an external source, e.g. another population. Males and females are infected from external sources at a rate e M /M and e F /F, respectively. The rates e M and e F are termed the external transmission rates.
The model is based on a continuous-time Markov process. Since we consider independent populations and constant numbers of males and females, the following set of (M+1)(F+1) ordinary differential equations describes the model (see [27] for an example of demonstration of differential equations representing continuoustime Markovian processes).
For 0#m#M and 0#f#F we have:
where p m, f (t) is the probability of having exactly m infected males and f infected females in the population at a time t (I M = m and
In this model, the spread of FIV in males is independent of the number of infected females. As a result, the probability of finding exactly m infected males in the population (given by p m~P F f~0 p m, f ) is independent of the female transmission rates (b F and e F ) and hence of the proportion of infected females in the population. The distribution of the number of infected males given by the model can also be compared with male infection prevalence data, independently of female infection prevalence. We define this model as the ''male transmission model''. It is equivalent to a classical SI model [28] .
Models H 1 . As discussed earlier, our purpose here is to measure the influence of some factors on the rate at which the virus spreads within or between populations. Two types of risk factors are tested here. The first ones concern the impact of demographic parameters (such as the number of cats within the population) on the virus transmission rate between cats of the same population. The second ones are not really risk factors. Behavioral observations suggest networks of connectivity between the different populations. The objective is to estimate whether introducing this information on the probability of disease reintroductions within populations produces significant predictive improvements, compared to models where external reintroduction rates are simply constants.
Firstly, we try to improve the goodness-of-fit of the observed data by assuming that both within-population transmission rates b M and b F depend on the demographic characteristics of the cat population:
where SR obs , N obs , M obs , AF obs and AM obs are the observed values for the sex-ratio, the population size, the number of males in the population (M obs = SR obs N obs ) and the mean age of captured males and females, respectively; considering these characteristics is intuitive since all of them may affect the social structure of the population and, hence, the transmission rates of
are the (linear) parameters that quantify the effects of these five demographic characteristics on the transmission rates b M and b F . Note that, a priori, the coefficients can have negative values and hence predict negative transmission rates. We fix a minimum value (10 24 ) below which b M and b F cannot fall since negative transmission rates are not allowed in the model. For the sake of simplicity, we assume that the external transmission rates e M and e F are not affected by the risk factors presented above.
We define as H 0 the model where b M~b 0 M and b F~b 0 F , the four model parameters (b 0 M , b 0 F , e M and e F ) being positive. As a general definition, models involving other parameters are called H(l), where l denotes the set of free (non-zero) parameters in the model that are notb 0 M , b 0 F , e M and e F . Then we investigate the possibility that, all other parameters being equal, the external transmission rates (e M and e F ) may differ between cat populations due to their spatial organization. Indeed, behavioral observations suggest a network of contacts between the different populations (see Fig. 1 , solid arrows), which can be simplified by dividing the study area into three distinct metapopulations (see Fig. 1 , grey areas). Since we do not model spatial structure explicitly, we assume that connectivity between populations affects external transmission rate. We define the resulting ''neighboring'' models and ''metapopulation'' models as follows.
A potential neighboring network has been suggested by behavioral observations (see Fig. 1 ). Intuitively, when there is a high FIV prevalence in males in neighboring populations, the external transmission rate of FIV should be higher. For this reason, we propose that the external transmission rate of FIV within a population could be considered as an affine function of the number of infected males in the neighboring populations (I neigh obs ):
We refer to this model as the ''neighboring model'' H neigh (l), where l denotes the set of free parameters in the model (in addition to b 0 M , b 0 F , e 0 M and e 0 F that are always freely variable). The metapopulation model considers that viral exchange is more intense between populations from the same metapopulation than between populations from different metapopulations. A simple way to test this hypothesis is to assume that populations belonging to the same metapopulation have the same external transmission rate, and that this external transmission rate differs between populations from different metapopulations. We define the ''male metapopulation model'' H M meta l ð Þ, where e i M represents the value of e M in metapopulation i, and l denotes the set of free parameters in the model (in addition to to b 0 M , b 0 F , e 1 M , e 2 M and e 3 M that are always freely variable). Note that in this model the only parameter that differs between cat populations is e M , which varies among metapopulations (e 1 M , e 2 M , e 3 M depending on the metapopulation). Finally, we also define the ''female metapopulation model'' H F meta l ð Þ, which is strictly equivalent to H M meta l ð Þ, except that it pertains to female external transmission rates.
2.4. Comparing models to data. Models cannot be directly compared with data because they predict distributions for the total number of infected and susceptible males and females in the population, whereas data are just samples of the real total numbers, i.e. the probability of capture is strictly below 1. To simplify, we assume that the total number of males and females in the populations are proportional to their observed values, i.e. M = M capt (1+p NC ) and F = F capt (1+p NC ), where p NC is a constant (1/(1+p NC ) is the proportion of captured cats) and M and F are the real numbers of males and females within the population, respectively. Based on the ratio between the number of cats captured through baited traps and the number of cats observed through intense monitoring in each population, we estimate that p NC is equal to 0.3 in average.
We assume that FIV is present in this area for a long period of time, corresponding to the stationary state of the distribution. So data are compared to this state. Note that the fact that the distribution is stationary does not mean that the population is at the equilibrium (i.e. endemic state), but only that epidemic, endemic and extinction events may succeed, and this being considered a population has a time-independent probability of being in each of its possible states.
Stationary distributions of the model, i.e. probabilities of finding exactly m infected males (for all 0ƒmƒM) and f infected females (for all 0ƒf ƒF ) in the population, generate a distribution of possible outcomes d 0 for the total number of cats. To incorporate the fact that data are missing for non-captured individuals, we add a hypergeometric sampling element to the distribution d 0 (in other words data are the result of a random sampling of the entire population). This leads to the distribution d to which data can be compared [29] :
where H x,y,z is the hyper-geometric law of integer parameters x, y and z, which is defined when max(y,z) #x for all integers t satisfying t #min(x,y) and z2t #x2y by:
The distribution d is then equal to the distribution d 0 after sampling a proportion 1/(1+p NC ) of the population. In other words, d 0 is the asymptotic distribution of the number of infected males and females, after sampling a proportion 1/(1+p NC ) of the population.
2.5. Model selection. Each of the models presented above can be summarized by the set of parameters that may vary freelyother parameters being fixed. Let us consider a model H. For each value h of the free parameters in the freely variable parameter space H (h is a vector of the values of all the free parameters), we can calculate for each population k the probability of generating the number of infected males and females actually observed. We call it L k (h|D k ), where D k represents the data restricted to population k; D k is defined by the number of infected males and females in population k.
Since we assumed that populations are independent, we can easily calculate the likelihood of the data D with the model H:
Now, if we consider two models H 1 and H 2 , the two models are compared using the maximum likelihood ratio statistics defined by:
We use the classical approximation that, under regularity conditions, the likelihood ratio follows a x-square distribution with r degrees of freedom, where r is the difference in the number of free parameters between models H 2 and H 1 .
2.6. Determining confidence intervals for the parameters. Another important objective of mathematical modeling is to calibrate the selected model, i.e. the model selected from the previously described process (see Section 2.4 above) by determining confidence intervals for its parameters. We consider a model H with a given set of freely varying parameters that defines a vector (h); i.e. each component of h is a parameter of the model. Within each population, the model predicts a distribution for the number of infected cats (male or female). Each possible model outcome (defined as a vector of 30 integers representing the number of infected males and females within each of the 15 populations) has a probability of occurrence. What we want to determine is the values h of the free parameters for which the observed data is a plausible outcome of the model. We accept that the data is a plausible outcome of the model when its likelihood is within the range of likelihood values of typical model outcomes, as described below.
For each vector h, we determine the threshold L 0.05 (h) such that 95% of the model outcomes have a likelihood value larger than L 0.05 (h). We now look at the likelihood of the observed data under the model parameters, defined above as L(h|D). Again we explore the parameter space. The confidence region H C can be defined as H C = {hMH / L(h|D).L 0.05 (h)}. Thus, for H C the observed data is a likely model outcome. Since the model often has several free parameters, then the 95% confidence interval is, in fact, a region of the multi-dimensional parameter space of the free parameters (H). For that reason we use the term ''confidence region'' rather than ''confidence interval''.
Finally, note that in the models the only parameter value we fix a priori is the mortality rate of FIV infected individuals (a). Since the model is analyzed at equilibrium, changing the mortality rate of infected individuals only results in a change in time scale. To remain consistent with cat-FIV interaction characteristics, we fix a = 0.0208 month 21 , so that infected cats have a 4-year life expectancy [8] . The model time unit is the month.
2.7. Computational procedure. Computationnal procedures are performed with Matlab. Stationary distributions of FIV prevalence in males and females are obtained by resolving the linear system corresponding to dp m,f /dt = 0. Maximum of the likelihood function are computed using a conjugate gradient method.
The cat number, sex-ratio, mean age of the males and females and percentage of FIV positive males and females captured in each population are given in Table 1 .
A total of 499 cats were sampled and tested for FIV in the 15 populations. There was large variability in the number of cats sampled due to large differences in population sizes, ranging from 13 cats in Clerey-la-Côte to 71 in Sauvigny. The overall sex-ratio is close to 50% but with differences between populations, although it does not differ statistically from a 50:50 binomial distribution (x 2 = 17.21, 15 df, p = 0.31). However, in Ruppes the sex-ratio is rather high (0.79) and this value significantly differs from 0.5 when applying a Bonferroni correction for multiple tests (p,0.05).
For each captured cat, we estimated its age following Pascal and Castanet [30] , and then the mean age of males and females in each population. For the entire study area the mean age is 3.08 years for males and 3.55 years for females; ranging from 1.54 years in Champougny to 5.25 years in Jubainville for males and from 2.32 years in Ruppes to 5.60 years in Barisey-la-Côte for females. It is also interesting to note that there is a strong correlation between the mean age of males and females in the studied populations (r = 0.85).
Finally, as previously documented, the global prevalence of FIV differs greatly between sexes (23% in males compared to 9% in females), with an average FIV prevalence in the entire study area of approximately 16%. There is significant variability in FIV prevalence between populations, especially in males, where data show significant extra-Binomial dispersion (Fisher's exact test with Table 1 . Total number of sampled cats, adult sex-ratio, number of FIV seropositive individuals (FIV+) and mean age of captured males and females in each population. 
Here we perform a rapid analysis of the mathematical model, this type of model having been studied in more detail elsewhere [28, 31] . For the sake of simplicity, we focus on the real distribution of FIV prevalence in males (the results are thus independent of b F , F and e F ); we assume p NC = 0, i.e. all individuals of the population have been sampled.
First, we look at the distribution of FIV prevalence in males for arbitrarily fixed values of the parameters: b M = 0.025, M = 50 and e M = 0.01 (Fig. 3a, solid line) . For clarity, we plot the distribution of FIV prevalence as a continuous line, although the distribution is discrete. The probability of finding no infected cats in the population is high (17%). The mean prevalence of FIV is 12.66% and in 95% of the model outcomes the FIV prevalence ranges between 0 and 32%. This predicted distribution of FIV prevalence in males differs from a binomial one (a distribution frequently used in risk factor analysis, [8, 9] ) having the same mean (Fig. 3a , dashed line). For a binomial distribution, the probability of finding no infected individuals in the population is much smaller (0.1%) and the confidence interval for FIV prevalence is [0.01; 0.20].
In Fig. 3b we analyze the effect of the external transmission rate on the mean and standard deviation of FIV prevalence in males. We focus on the distribution conditioned to non-extinction and, in parallel, we plot the probability of FIV extinction from the population (dashed line, right axis). Unsurprisingly, the probability of FIV extinction decreases with increasing external transmission rate (e M ). More interestingly, below a given threshold (here e M = 10 23 ) the distribution of FIV prevalence is not affected by e M , meaning that infrequent infections of FIV from external sources have almost no effect on FIV transmission within already infected populations. Under these circumstances, external infections only affect the frequency of extinction of the virus. Above the threshold, the mean prevalence of FIV increases with e M . Thus, external infections are an important component of FIV transmission, even within already infected populations.
This result may have important implications. For example, in our data only two of the 15 populations have no infected males. This indicates that the external transmission rates of FIV within our populations must be large enough such that there are infected males in at least 13 out of the 15 populations. Under such external transmission rates, is the spread of FIV within already infected populations affected by external infections or is external infection only important for the long-term persistence of the virus? This question will be addressed later when we provide estimates for the parameters.
3. Analysis of the observed data using the dynamic model 3.1. Effect of demographic risk factors. Now we consider the full model, including both males and females, and compare how integrating the different risk factors increases the goodness-offit to our observations using likelihood ratio tests. We performed the data analysis with each of the following values for the proportion of non-captured cats (p NC ): 0.15, 0.30 and 0.45. Since the results obtained from these three values are very similar, we only show those obtained for p NC = 0.30. It is important to note that from now on, likelihoods are calculated with the distributions of FIV prevalence in males and females, without removing the cases of extinction (i.e. we use the distribution d). The probability of observing zero infected individuals is an important characteristic of the models, and removing extinction cases would lead to lose very important information, especially relating to the external virus transmission rate.
To start with, we look at the impact of the population characteristics (the sex-ratio in captured cats SR obs ; the estimated population size, i.e. the number of captured cats N obs ; the number of captured males in the cat population M obs = SR obs N obs ; and the mean age of captured males and females, AM obs and AF obs , respectively). Results are summarized in Table 2 . The only significant effect we found is associated with the effect of mean age of males (x 2 = 4.09, 1 df, p = 0.043) and females (x 2 = 5.335, 1 df, p = 0.02) on the male-tofemale transmission rate (b AM F and b AF F , respectively). Interestingly, there is a negative correlation between the mean ages of males and females and FIV prevalence (b AF
). This means that the effect of age on FIV prevalence observed here is not due to the accumulation of FIV cases with age. The p values are rather large (p = 0.02 and p = 0.043), especially considering the large number of tests performed. Unfortunately, we cannot apply a simple Bonferroni correction for multiple tests because of dependence among the different tests performed. Due to the strong correlation between the mean age of males and females, it is not surprising that both variables have the same significant effect on the male-to-female transmission rate. It seems more likely that only one of the two variables has a real biological effect, the effect of the other one being due to correlation. Due to the strong correlation between the two variables, we cannot rule out a role for mean age of males on the male-to-female transmission rate. H neigh e neigh F , respectively). Under these circumstances, we find no significant improvement in the models compared to H 0 (see Table 2 ). In summary, we found two potential risk factors for FIV: the mean ages of males and females that influence the FIV prevalence in females. These two factors are certainly linked because a large correlation exists between the two variables. Yet, considering the number of tests we performed and the relatively high p values we obtained, we cannot exclude the possibility that the simplest model We remove female prevalence data from the analysis because there is no female to male transmission of the virus. We determine the confidence region of the transmission rates b M and e M, of the ''male transmission model'' parameter space for three different values of the proportion of non-captured cats: p NC = 0.30 (Fig. 4a,  b) , p NC = 0.15 and p NC = 0.45 (Fig. 4b) . In Fig. 4b we superimpose these three confidence regions; only showing their boundaries. We conclude that p NC has a slight impact on the edge of the confidence region. If we project the region onto the b M 0 axis we obtain a 95% confidence interval for the male-to-male transmission rate b M ([1. Finally we look at the impact of the external transmission rate e M on FIV spread in already infected populations. We estimate the average size of a population as the mean number of observed males per population multiplied by 1+p NC , which is equal to 21 for p NC = 0.30. We divide the mean number of infected hosts calculated with the model for a population of average size conditioned to FIV non-extinction by the value obtained with the same parameters, but with an external transmission rate a hundred times lower. We denote R as this value minus one
, where I is the mean number of infected individuals in the population. R is a proxy of the impact of external infections on FIV transmission in already infected populations. If external infections have a small effect compared to the withinpopulation transmissions, then R will be close to 0 (see Fig. 3c ). In contrast, if external infections have an important effect compared to the within-population transmissions, then R will be quite larger than 0.
For p NC = 0.30 we calculate R in a square region of the male transmission rate (b M and e M ) parameter space (Fig. 4) and we superimpose on the same graph the edge of the confidence region. We observe that in the upper left corner of the parameter space (Fig. 4c) R is around 0.02, which means that at low external transmission rates, external infections only increase by 2% the prevalence of FIV and, so, have a very limited impact on the spread of FIV in already infected populations. In contrast, in the lower right corner of the confidence region R is around 2.5, which means that frequent external infections greatly increase FIV prevalence even in already infected populations.
prevalence in females. Now we investigate for which parameters values in the model including both males and females data are a plausible outcome of the model. We focus on the simplest model. First, it is interesting to know if, and for which set of parameters, the simplest model can fit the data. Second, since the effect of the mean age of populations is not highly significant, we do not believe it makes biological sense to take this factor into account here.
Here, the parameter space is four-dimensional, so we cannot plot the confidence region. Since we are interested in determining the parameters directly influencing FIV prevalence in females, we simply plot the projection of the confidence region in the female transmission rate (b F , e F ) parameter space (Fig. 5) . 5 shows that there is an important dependency between b F and e F . Increasing the value of e F increases the mean prevalence in females and so the parameter b F must be decreased in order to explain the observed data. As a first approximation, the confidence region can be characterized by the relationship 0:015ƒb F z3:46e F ƒ0:11.
Interestingly, the confidence region crosses the X and Y axis (see Fig. 5 ). This means that even if one of the two rates (b F or e F ) equals zero, then the model can still explain the data. In other words, the data may be explained by considering only infection of females by males of the same population, without external infections or, conversely, by only considering infections by males from other populations without within-population male-to-female infections. Overall, we cannot determine which source of infection for females (internal or external) is the most important in our study area.
In the previous section we have seen that data are a plausible outcome of the simple model for a large region of the parameters. In the present section we show how the simple male-transmission model (where the transmission rate is independent of risk factors) (Fig. 6a) . For comparison we show the same graph using a binomial model (assuming independence between individuals regarding FIV, Fig. 6b) .
As seen previously, the dynamic model predicts a very large variability of FIV prevalence in males within population (see Fig. 6a ), which is larger than with the binomial model (see Fig. 6b ). As a result observed FIV prevalence in males is always in the 95% confident region for the dynamic model (see Fig. 6a ), but not for the binomial model (see Fig. 6b ).
In Fig. 6c we show the variance predicted by the different models (with maximum likelihood estimations of their parameters) and we compare it with that estimated from the data (usinĝ
14, where p i is the prevalence of FIV in males in population i and p p is the mean FIV prevalence). Again results show that the binomial model predicts a smaller variance than what is observed in the field (FIV prevalence data in males show around 73% more variance than what is expected by the binomial model), whereas the dynamic model shows an overestimated variance compared to what is estimated from data (but only around 42% larger).
To investigate whether the variance estimated from data is consistent with predictions of the dynamic model, we study the distribution of FIV prevalence in males expected by the male transmission model with maximum likelihood estimation of the parameter. In each population we simulate a male FIV prevalence according to the distribution d and then we estimate the variance in FIV prevalence in males between the 15 populations. We run 10,000 replicates and obtain a theoretical distribution of the estimated variance of FIV in males (ŝ s 2 , Fig. 6d ). We find that the observed value ofŝ s 2 (black bar) is in fact a plausible outcome of with the dynamic model.
The spread of a transmissible disease in a host population is a dynamic process where the probability of individuals becoming infected depends on the number of infected individuals in their neighborhood. Nowadays, dynamic models of epidemics are widely accepted as efficient tools to help understand the spread and management of infectious diseases (see e.g. [32] [33] [34] [35] ). So it is not surprising that stochastic versions of these models have emerged during the past decade as the best way to analyze infectious diseases data (see e.g. [1, [14] [15] [16] [17] [18] [19] 20, 36, 37] ). Methods based on the comparison of stochastic epidemic models to data hence constitute natural tools to estimate how different factors may affect the spread and impact of infectious diseases.
Our dataset exhibits large variability in FIV prevalence in both males and females among populations. However, a rapid study of the dynamic model shows that, in such a model, great variability in FIV prevalence may be expected. The rate at which susceptible individuals become infected depends on the proportion of infected individuals in a population. If, by chance, the proportion of infected individuals becomes large then the number of new infections will increase, maintaining high infection prevalence for the next generation. By contrast, a low proportion of infected individuals decreases the number of infections in subsequent generations.
To investigate the possibility that the cats density, the sex-ratio, the number of males or the mean age of cats within the population may act as risk factors influencing the disease transmission rate, we performed a statistical analysis of the data using the sexuallystructured SI model. Which population characteristics correlate with large FIV prevalence and so explain, in part, the variability in FIV prevalence? We found no such factors, except for mean ages. Interestingly, these ages have a negative effect on FIV prevalence in females, despite the accumulation of cases that occurs with age. One possible explanation is that the presence of older territorial males in some populations ensures greater social stability, which decreases the rate of at-risk (mating) contacts. Reversely, a negative correlation between FIV prevalence and age of cats could be due to the additional mortality induced by the virus. However, considering the weak impact of the infection on the lifeexpectancy of individuals, this explanation seems rather implausible to us.
In fact, these effects are not highly significant (p = 0.02 for the mean age of females and p = 0.043 for the mean age of males). Determining whether or not age affects the probability of becoming infected by FIV would require i) correction for the multiple tests performed and ii) correction for the effect of the accumulation of cases with age. Since this is beyond the scope of the work presented here, we cannot make definite conclusions on the effect of age.
The cat populations observed in this study are of small size, and certainly are not large enough to retain the virus over long periods of time. Since we detected infected cats in 14 out of the 15 populations (and infected males in 13 of them), we can assume regular viral exchange between populations. Previous theoretical studies have shown the importance of the spatial dispersal of the FIV virus between populations [38] . Due to the topographic isolation of our study area, it seems reasonable to assert that viral exchange between the studied populations is primarily responsible for the reintroduction of the virus into populations where it has become extinct. We proposed two different virus dispersal networks between the populations, but neither significantly improved the goodness-of-fit to the observed data. Although our observations are most likely insufficient to capture the exact dispersal network between populations, the networks we analyzed should be quite realistic, because they are consistent with the natural barriers in the study area.
Lastly, it is important to note that a spatial correlation in FIV prevalence between connected populations can be observed only if external infections have a substantial impact on FIV prevalence within the population. An analysis of the confidence region of the male transmission parameters shows that the impact of external infections on FIV prevalence within populations is very limited for the smallest values of the external transmission rate (see Fig. 4c ). In this case, the connectivity between populations cannot be revealed by a corresponding correlation in FIV prevalence. In contrast, for the highest values of external transmission rate in the confidence region, we can expect a correlation in FIV prevalence between connected populations. To sum up, the fact that no spatial correlation in FIV prevalence is observed may simply be due to the fact that external infections are relatively rare and thus play almost no role for disease prevalence in already infected populations.
Logistic regression models are still widely used for the analysis of risk factors associated with infectious diseases, even though their over-simplified independence hypothesis is largely recognised as a limitation to their use [1, 14, 15] . The main difference between the two approaches, based on binomial and dynamic models, comes from the variability expected by their respective H 0 models, as illustrated in Fig. 3a . Binomial models predict much narrower distributions than dynamic models. The consequence is illustrated in Fig. 4 , where we can see that the simple SI model accounts for the observed variability in FIV prevalence in males for a wide range of parameters. In contrast, the binomial test on the distribution of the infected cats among the 15 populations rejects the global binomial distribution hypothesis (p<0.006). To explain the data with a logistic regression model that assumes binomial distributions, additional risk factors need to be invoked. With dynamic models, risk factors are not required to explain the variability in the male disease prevalence observed here. The implication of that is that bringing evidence for population risk factors in infectious disease requires large sample sizes. In our present case n = 499 is not large enough and further sampling is required to bring evidence of population risk factors for FIV transmission.
The model developed here is quite simple. In particular, it does not account for a potential difference in individuals' infectivity between the acute and chronic phase of the infection. Such levels of complexity could be added to the method. This would make the model more realistic but also more complex, which was not our purpose here. The most important conclusion of the paper, i.e. that dynamic models predict much more variability than models where individuals are independent and hence are sufficient to explain highly variable prevalence data, would remain true for more complex model. Another model assumption is that we neglected the contacts with populations outside the study area (white rectangles in Fig. 1 ). Since we did not find an important effect of the number of infected neighbors on the disease prevalence in populations, we are confident that adding the neglected populations would not deeply affect our results.
The approach developed here is general and can easily extend to a wide variety of cat populations, but also to other host-parasite systems. It facilitates selection of the best model to describe data, which can be calibrated by determining confidence regions for the model parameters. The model can be used, for example, to test virtual management plans and to look at the expected results in the entire confidence region. This should assist in predicting the success one might expect with different management strategies. In the case of FIV, this study could help to rationalize the use of potential future vaccines or castration campaigns to limit the spread of the virus between males.
In the case of FIV, the approach gives us a 95% confidence interval for the model parameters, in particular for the basic reproductive number R 0 ([0.626, 1.942]), with a maximum likelihood estimate of 1.285. This value appears rather low, meaning that virus transmission is rather rare at the level of the population. This is not surprising, since experimental results indicate that most of the virus present in the saliva is not infectious [39] , suggesting a weak efficiency in disease transmission [7] . Given the high frequency of fights between males in such populations and the low rate at which males acquire the infection (around once every four years in a hypothetical scenario where all males are infected), our results are consistent with the concept of a low probability of virus transmission from bites [9] . Multiplex primer prediction software for divergent targets We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus. Researchers employ numerous approaches for viral detection and discovery, including metagenomic sequencing (1) (2) (3) , microarrays (4) (5) (6) (7) (8) (9) or multiplex PCR followed by other methods of characterization such as mass spectrometry (10) (11) (12) (13) (14) , suspension arrays (15, 16) or amplicon sequencing (17) . Multiplex PCR followed by analysis of amplified products fills a niche for viral identification when singleplex PCR has failed or there are a few dozen likely candidates but the expense of metagenomic sequencing or high-density microarrays is unwarranted (18) . However, multiplex primer design for many highly divergent targets is challenging since no universally conserved primers may exist, and finding sets of primers likely to function well in multiplex (e.g. isothermal T m 's, no primer dimers) adds to the complexity of finding conserved primer candidates. Primer design software that requires a multiple sequence alignment (MSA) as input can be problematic for diverse target sets, as MSAs can be difficult to construct, exhausting memory or available time before an alignment is completed. Even if an alignment does complete for divergent target sets such as all members of a family of RNA viruses or gene homologues across species, alignments may show little nucleotide sequence conservation, and multiple primers are required to amplify all targets. Considering the challenges of primer design for targets showing sequence variation, it is not surprising that many of the PCR-based assays in the literature are predicted to fail to detect desired targets when compared against available sequence data, and this problem is worst at higher taxonomic levels like family (19) .
Most currently available multiplex primer prediction tools require an MSA (20) (21) (22) (23) (24) . None of those tools build multiplex sets in which no primers in the set are predicted to form primer-dimers, although some avoid homodimers. We attempted to run a number of alternative software tools for multiplex or degenerate primer prediction for several species level target sets ranging in size from 41 to $6000 sequences: Primaclade (24) , FastPCR (www .biocenter.helsinki.fi/bi/programs/fastpcr.htm), GeneUp (25) , PDA-MS/UniQ software (26) , Greene SCPrimer (20) and HYDEN (22) . Only Greene SCPrimer and HYDEN could handle the two smallest, species-level target sets (Norwalk virus and FMDV), and none of the tools completed for the larger target sets that we attempted to run. Therefore, we designed the MPP software to avoid the requirement of an MSA and to scale better for large and diverse target sets. MPP builds a multiplex-compatible set of primers capable of amplifying all target sequences, attempting to minimize the number primers in the set.
We set out to determine a set of highly conserved 'universal' primers for viruses, akin to the highly conserved 16S rRNA universal primers for bacteria. Throughout, we use the term 'detected' to mean that there should be at least one PCR product. This is a loose definition of 'detected' adopted for convenience in the following discussions, and we recognize that a PCR product may prove insufficient for viral characterization. We predicted a set of 'universal viral primers' for all available complete genomes of all viruses, and found that the number of universal viral primers would be impractical to implement, even if short, highly conserved priming sequences were used. Then we predicted family-level primer sets for every viral family, as well as for several highly diverse species of RNA viruses, for primers of a traditional length as well as nontraditional, shorter, more highly conserved primers, which are more likely to amplify novel, unsequenced viruses and which could be an alternative to degenerate primers. While the software uses a greedy algorithm that may settle at a local minimum which is above the true minimal set, it generated primer sets for each viral family with fewer than half the number of primers that would be expected without optimization. Although family-level primer sets for some families are too large to be practical, 64% of the families had primer sets of no more than 20 primer pairs, quite feasible for multiplex PCR.
Finally, we empirically multiplexed a set of 16 short (10-mer) primers designed for the Poxviridae family. This demonstrated specific amplification of the expected viral fragment from vaccinia Lister strain. This preliminary demonstration suggests that specific amplification with family-level multiplexed sets of short primers might be feasible for viral detection and discovery, particularly as a means for selective enrichment of viral target for downstream amplicon characterization (e.g. sequencing or probe hybridization).
Here, we outline a greedy algorithm used to calculate conserved sets of multiplexed primers to amplify fragments from each member of a target set of sequences, and provide a more detailed description in the Supplementary Methods. First, we enumerate all candidate oligos fitting user-specified requirements for length, T m , and lack of hairpin formation. We rank pairs of these by the number of targets in which that pair occurs within a distance range d 1 bases to d 2 bases of one another, where these might be specified so as to bound a reasonable range for a downstream characterization method such electrophoretic discrimination of bands, probe hybridization or sequencing. The most frequent pair is selected as primers. The process is repeated for the remaining targets that would not have an amplicon from the first pair, with the added consideration that new primers selected be predicted not to form dimers with other primers already selected, as well as can be predicted based on nearest neighbor thermodynamic predictions (27) , although free energy calculations cannot predict with certainty that primer dimers will be excluded in practice. There is an option to bin primers into reaction subsets, if desired. With binning, primers are added to a bin until that bin contains b primers, at which point a new bin is begun, following the same process. Binning primers in smaller groups avoids exclusion of the most highly conserved oligos because of primer dimer free energy constraints. The universal set of primers is the set of selected primers to amplify all genomes in the target set. The primers within a bin should be multiplexed into a single PCR reaction, but each bin should be run separately. This is a simple binning strategy, and alternative strategies could be employed such as starting a new bin with any primer pair that dimerizes with other previously selected primers regardless of the number of primers in the bin, but this could have the possible disadvantage of bin explosion to numerous singleplex or small multiplex reactions. The graph-based algorithm of MuPlex (28, 29) is another binning strategy that could be incorporated. Output on the number of primers rejected due to the various filters (T m , hairpin free energy, etc.) is printed to standard output after each round of primer pairs are selected and also cumulatively, so a user can monitor if/which parameters might be too stringent. If an alternative set of primers is desired that does not overlap with the set selected, one can replace with N's the subsequences matching the selected primers and their reverse complements in the target sequences and rerun the software with the modified input sequences.
The script find_amplicons.pl predicts all the amplicons that should be generated by a list of (multiplexed) primers mixed with a set of sequences. This PCR-simulating script lists amplicon sequences, their length and position, and the forward+reverse primer combination to yield each product, as well as a summary file of the fragment length distributions enabling a quick assessment of how well each target sequence can be discriminated based purely on amplicon length patterns. We used this script to check whether some viral primer sets are predicted to generate amplicons from the human genome.
For all T m and free energy predictions, we used the following Unafold settings: [Na+] = 0.2 M, [Mg+] = 0.0015, annealing temperature = 30 C, and strand concentration of each strand = 10 -7 M, making the total strand concentration of both strands = 2 Â 10 -7 .
The MPP algorithm described here focuses on finding conserved primers, and does not require that the primers be family-or species-specific. For the runs predicting family-specific primers, we designed viral family primers that were unique relative to nontarget viral families by replacing any substring of 17 nt or longer that occurs in any non-target family with a substring of 'N''s, using the suffix array software vmatch (http://www.vmatch.de/). MPP eliminates oligos with N's from consideration as primers. This approach is simple, although it risks being overly strict by eliminating some potentially successful candidate primers, since it disallows those cases where a single nonunique primer pairs with a unique primer, a pair of nonunique primers are too far apart to actually generate an amplicon in a nontarget sequence, or candidate oligos partially overlap a nonunique stretch of N's.
In general, searching for primers from sequence that is specific to one set of targets and excluding candidate substrings present in nontarget sequence is a useful strategy to design signatures for pathogen detection. This can be done by replacing nonunique or nonspecific substrings with N's in the sequences input to MPP using software such as vmatch.
For runs predicting universal viral primers including all viruses at once in the target set, all viral complete genomes and segments downloaded from publicly available sequence databases (Genbank, Baylor, TIGR) as of 25 April 2007 were used. Draft sequences with multiple contigs were merged into a single sequence entry, with contigs separated by 1000 N's, a stretch sufficiently long (>d 2 ) so that primer pairs would not be designed to fall on different contigs, although there were very few draft sequences in contigs where this was necessary. Because of the large numbers of sequences in two families, only the MP segment sequences from Orthomyxoviridae and the L segment from Bunyaviridae were included, as these are the more conserved segments, reducing the number of targets by 23 017 sequences. The total number of target sequences for these all virus runs were 11 477. We predicted a multiplex set of universal 10-mers without binning the primers into separate reactions, but it was necessary to use a very low x dimer and x homodimer of À11 kcal/mol to be possible to predict multiplexed primers to amplify every target. For the next calculations, we subdivided the primers into sub-reactions with 20 primers per reaction bin, to avoid excluding primers that were predicted to form primer dimers, and raised x dimer and x homodimer to -7 kcal/mol. Primer sets of length 5-18 were predicted ( Figure 1 ). To assess the effect of removing T m constraints, we generated universal primer sets with length but no T m requirements, and compared the primer counts to those with T m constraints ( Figure 2 ). Primer sets are available by contacting the authors.
We evaluated how the growth of sequence availability could affect the size of the universal set of viral primers, as well as our ability to detect unknown/unsequenced viruses. We predicted a universal viral primer set for all viral genomes and segments available as of 1 January 2004, totaling 9965 sequences, for primers of length 7-15 nt ( Figure 2 ). It was not necessary to exclude any Orthomyxoviridae or Bunyaviridae segments, because these segments were not so deeply sequenced at that time. We then determined how much of the 2007 sequence data would have been detectable using the 2004 primer sets (Figure 3 ).
To predict how contamination with human DNA might affect the ability to detect specific amplification of viruses, the average number of amplicons for the human genome was also predicted based on these primer sets with 20 primers per bin ( Figure 4 ). The effect of reducing the multiplex size to 10 primers per reaction on human genome amplification was calculated by dividing priming sets in half, with 10 primers per reaction.
Subdividing viral targets into families, we used an updated set of sequences, downloaded 5 February 2009. For familylevel primers, we computed primer sets using three alternative parameter settings (Table 1) : (i) 17-21-mer primers with T m = 55-60 C, primers not checked for uniqueness; (ii) same length and T m specifications as in (i) but eliminating from consideration as primers any oligos of at least 17 nt that occur in nontarget viral families, as described earlier; and (iii) 10-15-mer primers with T m = 40-45 C. These primers are available as Supplementary Data.
We also generated primer sets for several species with high sequence diversity: HIV-1, FMDV, Norwalk virus and influenza A segments HA and NA (summary in Tables 2 and 3, primer sequences available as Supplementary Data). MPP parameters were the same as in Table S1 , with T m = 35-50 C for 10-mer primers and T m = 55-70 C for 17-18-mer primers, and sequences were downloaded in 2007 or 2008 (influenza only). To illustrate the challenges of designing primers from an alignment, we aligned these organisms using MUSCLE (30) when possible. For the HIV-1 and influenza A segments HA and NA, MUSCLE ran out of memory before completing. An alternative alignment tool, ClustalW (31), had completed only a small fraction of the alignment after running for days. Therefore, for these large data sets, a random selection of $35 sequences for each target was aligned with MUSCLE, and this alignment was used to build a profile Hidden Markov Model (HMM) (hmmbuild) using HMMer (http://hmmer.wustl.edu/). (32) The full sequence set was then aligned to the HMM using hmmalign. For Norwalk virus and FMDV, we designed multiplex-degenerate primer sets using GreeneSCPrimer (20) and HYDEN (22) , but the other three targets sets were too large for those programs. None of the other primer prediction programs we tried (as indicated in the 'Results' section) would scale to handle even these two smallest target sets.
For an empirical demonstration of our algorithm, we tested a multiplex set of 16 short, 10-nt primers designed for the Poxviridae viral family against commercially purified vaccinia virus extracts. The primer sequences are provided in Table 4 along with the predicted single amplicon for vaccinia Lister strain. We chose the Poxviridae family multiplex for these first empirical demonstrations because extracted viral nucleic acid was readily available for experiments. All primers were purchased from Integrated DNA Technologies (Coralville, IA, USA) and resuspended to 100 mM stock solutions in TE buffer (pH 8.0, Teknova, Hollister, CA, USA). Working solutions containing equimolar concentrations of each of the 16 primers were used in all experiments. Purified, quantitated vaccinia Lister strain DNA was purchased at a concentration of 1.3 Â 10 4 copies/ml in nuclease-free water from Advanced Biotechnologies, Inc. (Columbia, MD, USA). All PCR experiments were prepared using the Superscript III RT-PCR kit from Invitrogen (Carlsbad, CA, USA). We selected the RT-PCR kit in order to establish a protocol that could later be readily applied to additional multiplex viral family reactions with viral DNA and/or RNA. Each 25 ml reaction contained 1 Â SSIII buffer, 1 U of SSIII RT/ Taq enzyme, 4.8 mM MgSO 4 , 0.1 mM each primer, and a viral template mass of 2.7 pg ($10 4 copies). Tests were performed in triplicate and corresponding negative controls were run under identical conditions except that viral template was replaced with nuclease-free water (Ambion, Austin, TX, USA). All reactions were thermocycled on the Bio-Rad DNA Engine (Hercules, CA, USA) as follows: one cycle of 2 min at 94 C; 40 cycles of 15 s at 94 C, 30 s at 43.9 C, 1 min at 68 C; one cycle of 5 min at 68 C. In line with our goal to create a protocol applicable to both DNA and/or RNA templates, we verified that inclusion of an RT step (one cycle of 30 min at 45 C) does not alter the outcome of the subsequent PCR when working with DNA template (data not shown). As such, for all Poxviridae 16-plex results reported here, an RT step was not used. Another goal was to establish reaction conditions that can be applied to any multiplex viral primer set without the need for re-optimization, assuming the primer set is designed with the same general parameter contraints (T m s, etc.) used to compute the 16-plex presented here. As such, we first optimized master mix conditions, where it was determined that a 4.8 mM MgSO 4 concentration resulted in most optimal amplification. Similarly, we determined the optimal annealing temperature based on results from annealing temperature gradient experiments. A more detailed discussion on the optimization of reaction conditions for amplification with our short primer viral multiplexes is the subject of a separate paper in preparation (Hiddessen and co-workers).
This multiplex was compared against the human genome, predicting 233 amplicons between 50 and 1000 bp from the Poxviridae 16-plex. For empirical tests against background human nucleic acids, we followed the same reaction conditions as above. In these tests, vaccinia DNA was held at a constant concentration of 2.7 pg (1.3 Â 10 4 copies), and serial dilutions of human genomic DNA (Novagen, Madison, WI, USA), were added to the vaccinia PCR reaction mix, starting at 2.7 pg and titrating down over 4 orders of magnitude to 2.7 Â 10 -4 pg. These experiments were performed using mass ratios of vaccinia:human DNA at 1 : 1, 10 : 1, 100 : 1, 1000 : 1 and 10000 : 1. These correspond to copy number ratios of vaccinia:human genomes of 1. All PCR experiments were analyzed on 3% agarose TBE gels containing ethidum bromide that were purchased from Bio-Rad (Hercules, CA, USA). Blue juice TM 10Â loading dye was purchased from Invitrogen (Carlsbad, CA, USA) and diluted to 2Â before use. A 50-bp DNA ladder was purchased from Novagen (Madison, WI, USA). For analysis, 15 ml from each separate 25 ml PCR reaction were combined with 2 ml of of loading dye and 15 ml of the loading-dye/product mixture was loaded per well and electrophoresed for 1 h 40 min at 85 V.
For confirmation of amplicon sequence, 9 ml of amplified PCR product was mixed with 4 ml of ExoSap-it (USB, Cleveland, OH, USA) and incubated at 37 C for 15 min followed by a denaturation step at 80 C for 15 min. Sanger sequencing was then performed with the BigDye V3.1 Terminator Kit (Applied Biosystems, Inc., Foster City, CA, USA). Single reactions contained 4 ml of Ready Reaction Mix, 0.2-mM primer, 2 ml 5Â Sequencing Buffer, 11.5 ml of nuclease free water (Applied Biosystems, Inc.) and 2 ml of post-Exosap-it PCR product. The sequencing reaction used the following thermocycling profile: 1 cycle of 94 C for 1 min; 25 cycles of 94 C for 15 s, 38 C for 30 s and 60 C for 4 min. Two microliters of sequencing reaction product was combined with 18 ml of Hi-Di Tm Formamide (Applied Biosystems, Inc.) and run on the ABI 3130 (Applied Biosystems, Inc.). The results were analyzed using Sequencing Analysis v5.2 (Applied Biosystems, Inc.).
Predicting a set of universal viral primers that are all mixed in a single large reaction, we predict that 1008 primers of length 10 nt would be required to ensure amplification of at least one fragment of length 80-620 bp from every viral genome. These are predicted to generate a mean and maximum number of amplicons per viral genome of 13.2 and 948, respectively. Binning the primers into smaller sets of 20 primers/bin doubled the number of primers required, as Figure 1 shows the fraction of viral genomes amplified versus the number of primers, for primers ranging from 6 to 18 nt in length. About 2000 binned 10-mers are required to amplify 100% of sequenced viruses, generating a mean of 1 and maximum of 2.9 amplicons per genome, on average. Using traditional-length 18-mer primers nearly doubles the number to $3700 primers. The incomplete curve for 7-mers was from inappropriate settings for this oligo length, since some genomes do not contain pairs of 7-mers with the required T m , as given in the Supplementary Tables S1 and S2, within the desired amplicon length range. The concave curves showing diminishing returns reflect biased sequence availability rather than any particularly highly conserved primers: the primers that amplify the largest number of sequences are all from influenza, as far more sequences are available for this species than for any other.
Increasing availability of sequence data on universal primer sets
The increase in sequence data requires $700 more 10-mer primers to amplify all sequenced viruses in 2007 compared to 2004 (Figure 2 ). While the increase in the number of sequences used between the two dates was only $15%, the number of primers required increased by 48%, illustrating the substantial increase in diversity represented by the additional sequence data. Figure 2 also shows that removing all T m constraints allows fewer primers to be used since no conserved primers are eliminated due to T m , as some AT rich subsequences tend to be fairly conserved. Figure 3 shows that a universal primer set predicted using the 2004 sequence data would amplify only 35% of the 2007 sequences using primers of at least 10 nt in length. Shorter primers increase this fraction to over 60%, due to the higher likelihood of occurrence and conservation of shorter oligos, but even so, a multiplex of 7-mers is not guaranteed to amplify a fragment from every virus. The minor differences in the number of genomes detected between 12, 13, 14 and 15-mers can be attributed to the facts that a greedy but not necessarily optimal algorithm is used to select one solution from among many, that the primers in a particular set depend on the T m ranges we used as well as length differences, and the unpredictable nature of novel viral sequences accumulated between 2004 and 2007, rather than any real difference in the ability to detect genomes among those primer sets.
If all human DNA cannot be removed from the sample, simulations indicate that on average, multiplexes of 10-mer primers are expected to produce hundreds of amplicons from the human genome, which would appear as a smear on a gel ( Figure 4 ). With primers of length 11-14, there is a large variation among bins. While most short sequences do occur in the human genome (33) , an amplicon requires that two occur in proximity, and primers !11 bases make this a sporadic event for bins with only 10 primers. Nonetheless, imperfect sample purification to eliminate eukaryotic nucleic acids could be problematic for universal viral priming using primers shorter than 15 bases, particularly for multiplexes of 10 or more primers.
Family identification by sequencing products from universal 10-mer primer set versus randomly amplified fragments
If universal viral primers amplified fragments from a newly emerged virus, would the product sequences show similarity to others in the same family? We predicted (2).
The number of family-level primers for each family, and the number of genomes available for generating those primer sets, is given in Table 1 , for three alternative settings: short primers of 10-15 nt with T m 40-45 C, standard length primers of 17-21 nt and T m 55-60 C, and the same but requiring that each primer subsequence of at least 17 nt be unique to the target viral family relative to other viral families. At least one amplicon of 200-800 bp was required from every genome. Hypothetically, the worst-case scenario to amplify a target set of N sequences would require 2N primers. MPP requires on average only 37% or 45% of this number, for primers of length 10-15 nt or 17-21 nt without the requirement for family specific primers, respectively (averaged across families, omitting those families for which the computations were not run to completion). The most diverse families, in particular Bunyaviridae, Geminiviridae, Polydnaviridae, Reoviridae, Retroviridae, Siphoviridae and Orthomyxoviridae require so many primers that actually applying family-level amplification is probably infeasible. For these, more restricted target sets may be necessary, such as limiting to a single segment for the segmented families or to subclades, and possibly the incorporation of primers with degenerate or inosine bases. Some families with many genomes can be amplified with relatively few primers, such as Coronaviridae, Hepadnaviridae, Poxviridae, Togaviridae, Microviridae and Polyomaviridae. Using primers of length 10-15-mers or 17-21-mers, 66% or 63%, respectively, of the viral families have primer sets of 40 or fewer primers (20 primer pairs), which is feasible for typical multiplexes.
The sizes of the family-level primer sets show a trend for an increase with the number of available sequences in the family ( Figure 5 , P = 0.14). There is no clear relationship between the number of primers in the set and whether the genomes are single or double stranded RNA or DNA. All family primer sequences are available as Supplementary Data.
Primer design with the MPP software indicates that relatively few primers are required to amplify all sequenced genomes of HIV-1, FMDV and Norwalk virus (Table 2) , and these can be calculated in minutes. Influenza A HA and NA segments demand large numbers of 10-mer or 17-18-mer primers and hours to calculate, so one could break these into subgroups, possibly by serotype, as shown for several HA serotypes in Table 3 . The percentage of genomes amplified versus the number of primers used, for primers of either 10-mers or 17-18-mers, is shown in Figure 6 . This plot shows that a large fraction of targets are amplified with only 2 primers, and the addition of subsequent primers shows diminishing returns in amplifying fewer, more divergent targets not detected by the initial, more conserved, primer pair, although the true diminishing returns depend on the extent to which available sequence data is an unbiased representation of diversity. The more traditional method of attempting to find primers from a MSA would be problematic, probably requiring manually designed primer multiplexes or highly degenerate primers. GAAGAAGCG starting at 9071. These regions are too far apart to be used as primers for most polymerases used in diagnostic PCR protocols, where amplicons must typically be less than 300 bases long for efficient amplification. A recently published study (35) selected primers from the 5 0 LTR U5 end to the Gag-Pol start (5 0 -TAGC AGTGGCGCCCGA-3 0 and 5 0 -TCTCTCTCCTTCTAGC CTCCGC-3 0 ), but a comparison against available genomic data indicates that 487 of the 1175 genomes (41%) do not contain a sequence match for this primer pair, so may fail to be amplified. For influenza A segment HA, the size of the longest conserved region from the 95% consensus is only 5 bases, and for segment NA, only 6 bases, insufficient for even a single primer. For FMDV and Norwalk virus, the longest 100% conserved regions are 9 and 6 bases, respectively. The MPP software makes it straightforward for a nonexpert to predict a multiplex-compatible set of primers to amplify all targets, even for enormous and heterogeneous target sets that cannot be aligned.
For comparison, we considered other software options for designing primers for these heterogeneous viruses. FastPCR (www.biocenter.helsinki.fi/bi/programs/fastpcr .htm) and GeneUp (25) were the only programs that did not require an MSA as input. The FastPCR algorithm for group-specific PCR (i.e. universal amplification) designs PCR primer pairs individually for each target sequence without regard for primer conservation among targets, and then compares each primer pair to the other targets. This is a brute force strategy that is only suitable for small target sets and short target sequences (appropriate for gene lengths, but not for viral genome-length sequences). We ran the FastPCR software on our internal servers, but it did not complete 'group-specific PCR' for the smallest data set, Norwalk virus, after running for 18 h, and for 'multiplex PCR' gave the error message 'No compatible combination of pair primers for multiplex PCR found'.
GeneUp simulates PCR with pairwise combinations of candidate primers which pass length, T m , GC%, and palindrome filters against all target sequences, and uses a greedy algorithm to build a primer set to amplify all of the targets. Since testing all possible pairwise oligonucleotide combinations against each target explodes in time and memory for large target sets, a cap on the maximum number of candidate primers to be tested must be imposed, presumably using the most common oligos, although this is not explicit in the paper. Then a Grey shaded entries indicate where calculations were not run to completion. In other cases (not shaded) where fewer than 100% of targets were predicted to be amplified, the algorithm failed to find primer pairs that met all the required specifications for the remaining targets, that is, primers in the right length, T m , and amplicon length range, with hairpin and dimer avoidance with other primers already selected to be in the set, could not be found. None of the calculations that completed required more than the 16 GB of RAM that was available. PCR simulation (performing a text search for the primer in the target sequence) of each pair versus each target is performed. Unfortunately, the most common oligos may not occur in the correct orientation or distance to serve as primer pairs, so that multiple iterations of the entire process must be performed before a set covering all targets is obtained. MPP, in contrast, uses an efficient ranking algorithm to favor pairs of primer candidates that will produce amplicons of the right size in the most targets. Because of the MPP hashing algorithm and data structures, those targets that are amplified is easily determined without simulating PCR. A copy of the GeneUp software for testing could not be obtained from the authors. The PDA-MS/UniQ approach (26) uses a hash index of 4-mers and scoring heuristic to identify common regions in the target sequences with the most shared tetramers. Simulating combinations of candidate primers selected from these common regions is then performed using a genetic algorithm. Their genetic algorithm is a more scalable approach than those above, and the quality of the solution may be improved with more compute power, although a potential disadvantage of genetic algorithms is that they can be slow, particularly if there are very many possible combinations. For computational tractability, they limited primer size to 12 nt, and avoidance of hairpins and primer dimers was not modeled. This software was not available for download or on a public web server. The other software all required MSA as input. While these tools could not be tested on larger (e.g. family level) primer prediction problems where alignments would not be possible or appropriate, we nevertheless attempted to run these tools on the alignable species-level target sets. The Primaclade (24) webserver timed out for the smallest alignments we tested, Norwalk and FMDV. CODEHOP (23) requires protein alignment as input so is not appropriate for whole-genome (nucleotide) alignments.
GreeneSCPrimer (20) did generate a number of degenerate primer candidates from the MSAs for Norwalk and FMDV, requiring the user to manually select a combination of forward and reverse groups from a set of options. We ran GreeneSCPrimer using length, T m , etc. settings mirroring or more lenient than those we used for Table 3 (T m = 55-65 C, GC% = 20-80%, length 17-25 bp, 100% coverage, product size 80-620 bp, allowed T m difference 10 C, others left as defaults). HYDEN (22) also generated degenerate candidates from a MSA, although it does not check T m and the length is limited to a single value rather than a range. For our tests using HYDEN, we used a length of 18 rather than 17 because of the lack of T m control, and allowed 0 mismatches. The GreeneSCPrimer option requiring the fewest total primers for the Norwalk set required 18 primers, four of which had either 2-fold or 4-fold degeneracy so the actual number of priming sequences would be 26, compared to a total of 20 non-degenerate primers predicted by MPP (Supplementary Data). HYDEN generated four degenerate primers covering only 34 of 41 sequences, each with 3-or 4-fold degeneracy for Norwalk, which translates to 15 priming sequences in the reaction. One would need to find primers to amplify the remaining seven sequences. Small degenerate priming sets (e.g. four primers in this case) are less expensive to purchase, but because of dilution effects from the many sequence combinations actually present (15 priming sequences in the PCR), sensitivity may be reduced compared to nondegenerate priming. However, using a smaller set of degenerate signatures such as those from HYDEN may be preferable, and is a capability that could improve MPP in a future version.
For FMDV, MPP predicted six nondegenerate multiplex compatible primers to amplify all targets. GreeneSCPrimer generated a number of candidates, and manual inspection identified that the best of those primer combinations would require 6 primers, one of which had 2-fold and another had 3-fold degeneracy, totaling 9 actual priming sequences in a reaction (Supplementary Data). This compares with two primers each with 4-fold degeneracy using HYDEN (eight priming sequences in a reaction), to amplify 98% (183 of 187) targets. Again, the small number of signatures predicted by HYDEN is desirable for some applications, although the degeneracy is high and these must be supplemented to pick up the few outlying sequences
The aim of the MuPlex software (28, 29) is to partition primer pairs into multiplex-compatible bins for SNP genotyping, and it does not employ any algorithm during primer selection to minimize the number of primers to amplify all targets, yielding a one-to-one correspondence between number of primer pairs versus number of targets. However, the MuPlex graph-based algorithm to partition primers into bins is more sophisticated than the simple binning scheme of MPP. In future work, MPP could be used to identify a universal set of primers and the backend of MuPlex used to optimize how they are binned into separate reactions.
In summary, no software except MPP was capable of predicting primer sets for the larger target sets we examined or generating a multiplexed primer set without a MSA for any of the target sets we examined, even the smallest ones containing only a single species. Only HYDEN and GreeneSCPrimer, both requiring MSA inputs, completed for the two smallest target sets, selecting smaller sets of degenerate primers than the nondegenerate MPP primers. If target sets are sufficiently conserved so that a reasonable MSA can be built, and degenerate primers are acceptable, these tools may be preferable over MPP. However, for larger and more diverse target sets, only MPP completed.
Using the experimental conditions described in the 'Methods' section, we tested the Poxviridae 16-plex (Table 4 ) against vaccinia virus, Lister strain DNA. As assessed by agarose gel analysis, we achieved specific amplification of the predicted 617-bp amplicon for the target vaccinia genome (lane 3, Figure 7 ). The band does not appear in the no template control shown in lane 4 ( Figure 7 ).
To confirm that the amplicon observed in gel analysis was a specifically amplified product from vaccinia virus, Lister strain, we sequenced the product according to the procedures described above. An analysis of the high-quality sequence read data taken from the electropherogram yielded a 95% identity (maximum) to vaccinia virus, Lister strain (AY678276.1) with a query The forward and reverse primers (FP, RP) in bold are predicted to amplify vaccinia Lister with the indicated product length (617-bp amplicon size).
coverage of 99% and an e-score of 0 (no data shown).
These values indicate that the sequenced product was vaccinia DNA and not amplification from an exogenous nucleic acid source, e.g. host cell. Notably, our multiplex reaction did not produce a smear (lane 3, Figure 7 ) that would be indicative of nonspecific priming of either the target or exogenous host cellular nucleic acids. While the exact manufacturer's extraction protocol is proprietary, it is generally known that a standard sucrose gradient ultracentrifugation step is used to enrich for the viral capsids prior to viral nucleic acid extraction. However, to the best of our knowledge, no nuclease digestions are performed prior to viral capsid lysis. Furthermore, while the viral 'extract' contains a mixture of both viral DNA and cellular nucleic acids, the exact proportions of host cell and viral nucleic acids cannot be determined. Thus, our results indicate that a multiplex of 16 primers of 10 nt each amplifies only the specific predicted band from vaccinia.
In some applications, such as clinical or biodetection applications, the exogenous nucleic acids from other eukaryotic sources, notably human sources, may be present in varying and unknown concentrations. To test the effects of background human genomic DNA on the performance (amplification specificity) of the Poxviridae 16-plex primer set, we conducted PCR reactions across a series of mass ratios of vaccinia Lister DNA:human genomic DNA at 1 : 1, 10 : 1, 100 : 1, 1000 : 1 and 10000 : 1. These correspond to approximate copy number ratios of vaccinia : human genomes of 1.3 Â 10 4 : 0.82, 1.3 Â 10 4 : 0.082, 1.3 Â 10 4 : 0.0082, 1.3 Â 10 4 : 0.00082 and 1.3 Â 10 4 : 0.000082, using genome sizes of 1.9 Â 10 5 bp and 3 Â 10 9 bp for vaccinia and human DNA, respectively. For context, there are 6.58 pg or two copies of the genome in one human cell. Our algorithm predicted 233 amplicons between 50 and 1000 bp from the human genome. In experiments, this would appear as a 'smear' on the agarose gel, which indeed was observed for the mass ratios of 1 : 1 and 10 : 1 (Figure 7 , lanes 6 and 7, respectively). However, even at a ratio of 10 : 1 vaccinia:human DNA (lane 7), the 617-bp vaccinia amplicon is clearly visible on the gel, despite the numerous nonspecific amplicons. At ratios of 100 : 1, 1000 : 1 and 10000 : 1 (lanes 8, 9 and 10, respectively, Figure 7) , the smear is drastically reduced to nonexistent (at the resolution of the agarose gel), and the vaccinia amplicon is readily visible. For comparison, we tested the 16-plex primers against the same mass of human DNA in the absence of vaccinia DNA at 2.7 pg (the 1 : 1 ratio mass), 0.027 pg (100 : 1 mass) and 2.7 Â 10 À4 pg (10000 : 1 mass) (lanes 12, 13, and 14, respectively, Figure 7) . The data show a similar smear at 2.7 pg as was observed when vaccinia DNA was present (lane 6). However, the low-intensity 617-bp amplicon from vaccinia is visible in lane 6 (with vaccinia) while a similar amplicon is not present in lane 12 (without vaccina). These results provide evidence that amplification with these family level primer sets will depend on viral titers in the actual sample, and that there are cases where amplification will either not be possible or require additional sample purification steps. However, as discussed further in the next section, specific amplification with a short-primer multiplex could be used for selective enrichment of viral targets by generating amplicons with known sequence, which may be feasible for viral detection if combined with a probebased amplicon detection method such as TaqMan Õ or Luminex bead based suspension arrays (http://www .luminexcorp.com/). We compared recently published conserved Orthopoxvirus primers (36) to the 148 Poxviridae genomes, and computational predictions suggest that 67 of the available genomes might not be amplified by the two primers they designed for the Orthopox genus. These included a number of monkeypox, ectromelia, several vaccinia and a couple of variola strains. However, in permissive hybridization conditions it is possible that primers would anneal despite mismatches to target, allowing more of the targets to be amplified. Four conserved Orthopoxvirus primers from an earlier publication (37) , before many of the Poxviridae genomes were available, do not match 53 genomes, including a number of monkeypox, ectromelia, camelpox and one variola minor genome. The primers in (37) included inosine bases, which we replaced with each possible A, T, G and C base in all possible combinations for our primer-target comparison. Thus, published Orthopoxvirus primers may fail to amplify many desired targets.
Primer design for amplification and detection of divergent target sequences can be challenging, and this problem will only grow as sequencing technologies improve. Some methods are limited in scalability, particularly those requiring a MSA as input. Developing a PCR multiplex is often a tedious mix-and-match process from among primers originally designed to work in singleplex. We describe the MPP algorithm based on hashing of conserved k-mer subsequences that requires no MSA and where multiplex-compatible primer sets are built de novo to avoid primer dimer and hairpin formation to the extent that can be predicted based on free energy calculations.
The algorithm seeks to minimize the number of primers to amplify all targets, although because the algorithm is heuristic and not an exhaustive search of all combinations of primers, the smallest primer sets may not always be selected. This is an NP complete problem (38) , so an exhaustive search for the global optimum is only practical for very small target sets. It is also beyond the scope of the current software to predict priming with mismatches between target and primer, which may occur under nonstringent hybridization conditions or for mismatches at the 5 0 end of a primer. While allowing for such priming would reduce the number of primers needed to amplify all targets, it would substantially slow the algorithm and increase memory requirements. Nor do we claim to have taken all steps to optimize (e.g. through parallelization, see Supplementary Methods) for speed or memory, but instead present this software as a simple embodiment of one alternative to MSA for multiplex primer design.
The software handles a large number of input sequences, although for very diverse targets the predicted primer multiplex may be too large to be empirically feasible. For many target sets, such as those including all the genomes of a species, predicting a universal primer set requires only minutes up to a few hours, although for inputs with thousands of sequences a run may take days. We used MPP to design a universal primer set for a target set of all virus genomes, and showed that even if short primers are used, thousands of priming sequences would be required to amplify all sequenced viruses. We then applied MPP to design multiplex family level primers for every viral family, as well as for some diverse species target sets too large for other available primer prediction software.
In addition to finding primers for viral detection, another application of MPP could be to design multiplex primers for homologs in a gene family. The user can specify an appropriate product length range to ensure amplification of an adequate span across the gene. MPP could also be used to design multiplex primer sets for unrelated target sequences, for example, multiple bacterial and viral species or gene families, in a single reaction. Since no sequence alignment is required, there is no need for any sequence conservation among targets. Recent work showed that a large-scale multiplex of 800 primer pairs specifically designed to detect a diverse set of genes from nine pathogens improved sensitivity on a microarray by up to 1000-fold (39) . The MPP software could be used to design multiplex primer sets for similar work.
We demonstrated the application of the algorithm experimentally using purified vaccinia DNA, amplifying the expected band with a Poxviridae short primer multiplex PCR, and confirmed the band's expected sequence. We provided an example of the effect of background nucleic acids by spiking human DNA into the PCR reaction over a series of mass ratios. While a band for vaccinia was visualized by slab gel electrophoresis for all mass ratios, background DNA smears were also clear at the higher ratios. At a mass ratio of 1 : 1 vaccinia:human DNA, there are 2.7 pg or 0.82 copies of the human genome present in the reaction. To place this into context, there are $6.58 pg or two copies of genomic DNA in one human cell. Thus, the results from the 1 : 1 mass ratio reaction represents the impact that $41% of the total DNA content from one human cell could have on the results, when analyzed by slab gel electrophoresis. In a clinical sample, there could be a much greater number of human cells and DNA. Amplification with these family level primer sets will depend on viral titers in the actual sample, and these experiments show that there are cases where amplification will either not be possible or require additional sample purification steps. While short primer PCR multiplexes may enable amplification of diverse target sets, they will not match the specificity of longer primers. Combination of these short primer multiplexes with digitized, microfluidic-based picoliter reactions (40) and/or next-generation microfluidic-based sample purification technologies that have the ability to isolate target from contaminating nucleic acids may help to overcome this limitation. However, even in the absence of such technologies, a highly conserved short primer multiplex could enrich amplification products for members of a particular viral family compared to randomly amplified or unamplified sample. Then it would need to be followed by product sequencing or array hybridization, such as TaqMan Õ or Luminex bead-based suspension arrays (http://www.luminexcorp.com/), to provide more specific information about the organism(s) present, since electrophoretic banding patterns may contain unexpected bands, particularly if the sample contains a significant amount of nonviral nucleic acids.
Previous studies have shown the utility of short primer singleplex PCR using 9-mers or 10-mers followed by gel electrophoresis for genetic fingerprinting of eukaryotes and bacteria (41, 42) . For viruses, with much smaller and more diverse genomes, the large numbers of 9-mer or 10-mer primers required to generate at least one band from every virus as predicted by our analyses implies that primer size would need to be as short as 5-mers to rely on a gel banding pattern using only one priming sequence for fingerprinting viruses (unpublished data). However, the analyses here predict that imperfect sample purification to eliminate eukaryotic nucleic acids could be problematic for universal viral priming using primers shorter than 15 bases, particularly for multiplexes of 10 or more primers. Nanda et al. (43, 44) were able to achieve sufficient viral isolation from cell culture samples to allow viral identification using viral PCR with priming sequences as short as pentamers, so the problem of contaminating host nucleic acids for specific, short primer PCR of viruses is not insurmountable. They found specific pentamer PCR to be several logs more sensitive than nonspecific amplification, provided that they purified encapsidated viral nucleic acids prior to PCR. Another method that has been used for virus discovery is VIDISCA (Virus discovery cDNA-AFLP) using restriction enzyme digestion, adaptor ligation, and PCR by priming with the adaptor sequence (45, 46) . This method, like the pentamer priming used by (43, 44) , requires prior separation of encapsidated viral nucleic acids, as it generates fragments from any DNA present, viral, host or otherwise. Multiplex PCR with primers 10-15 nt in length may be yet another alternative strategy lying between these nonspecific methods and PCR with standard primers of at least 18 nt, as we have shown that it can add some measure of specificity for a viral family.
In summary, we applied the MPP software to generate multiplex-compatible primer sets for every viral family and several divergent viral species and experimentally demonstrated application of one multiplex set to show nonrandom amplification with a set of short primers.
Primer sets are available as supplemental material or by request from the authors. The MPP software is freely available for academic and nonprofit use at http://mpp .llnl.gov. Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short-hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify a shRNA that potently activates the IFN pathway in human cells in a sequence- and 5′-triphosphate-dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation. A specific double-stranded RNA (dsRNA) structure, $21-22 bp dsRNA with 3 0 overhangs, plays a critical role in initiating both microRNA (miRNA)-and small interfering RNA (siRNA)-mediated gene silencing, as it is the structure recognized by the RNA interference (RNAi) machinery, the RNA-induced silencing complex (RISC) (1) (2) (3) . Except for preformed siRNA duplexes of $21 bp, the RISC-loaded small RNAs are generated by a ribonuclease (RNase) III-like enzyme that is found in virtually all eukaryotic organisms. This enzyme, aptly named Dicer for its ability to cleave a variety of larger (>30 bp) dsRNA molecules into the $21 bp dsRNA with a characteristic 3 0 overhang of 2 nt, is a multidomain RNA-binding protein and itself a component of RISC.
The primary sequence of the RNAs is not important in RISC formation, and RNAi can suppress virtually any target as long as rules of sequence complementarities between the small RNA and the target RNA are satisfied.
dsRNAs are also a type of pathogen-associated molecular pattern (PAMP) that are detected by cellular innate immunity sensors named Pattern Recognition Receptors (PRRs) (4) . The interaction between a PAMP and a PRR triggers activation of the interferon (IFN) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of RNAi. IFN induction is especially problematic in antiviral studies employing RNAi, where the antiviral effect of IFN must be distinguished from that of RNAi.
Typical IFN-inducing structure patterns include dsRNA of certain length, single-stranded RNA (ssRNA) containing 5 0 -triphosphates (5 0 -ppp), the dsRNA analogue polyinosinic-polycytidylic acid (poly I:C), and certain dsDNA molecules. These RNA patterns are generally believed to possess 'non-self' properties to allow the cell to recognize foreign (often viral) RNAs specifically. Various forms of siRNA duplexes have been reported to trigger IFN induction both in vitro and in vivo (5) (6) (7) (8) (9) , probably through the cell surface-and/or endosomeexpressed Toll-like receptors (TLRs), including TLR3 and TLR7 (6, 8, 9) . Short-hairpin RNAs (shRNAs) expressed from a DNA plasmid have also been shown to activate IFN (10) . The double-stranded form of these RNAs is below the size limit of the stem-loop RNAs that can be detected by the RNA-activated protein kinase (PKR) (11) and is probably detected by other cytoplasmic PRRs. Two cytoplasmic RNA helicases, retinoic-acid-inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5), signal to the IFN-b promoter when activated by specific RNA structures (12) (13) (14) . Although both PRRs signal through the mitochondrial antiviral signaling protein MAVS/Cardif/VISA/IPS-1 (15) (16) (17) (18) , studies of ligand specificity suggest that RIG-I and MDA5 are parallel sensors with overlapping substrates. For example, although both PRRs are activated by poly I:C in cell culture systems (12, (19) (20) (21) (22) (23) , MDA5 appears to be more important in mediating the poly I:C response in vivo (13, 14) . In addition, RIG-I can bind and respond to ssRNAs bearing 5 0 -ppp, whereas MDA5 is not activated by 5 0 -ppp-containing RNA (24, 25) . Finally, several cytosolic sensors for dsDNA has been recently reported (26) (27) (28) (29) (30) (31) . Nevertheless, current data on what constitutes effective substrates for either PRR are incomplete and sometimes controversial. Here we report for the first time that shRNAs delivered by lentiviral transduction triggered IFN activation and that RIG-I and MAVS, but not MDA5 or TLR3, mediated the IFN activation triggered by intracellularly expressed shRNA, which could activate both IFN-a and IFN-b promoters. IFN activation depended on sequence, a 5 0 -ppp and correct processing of the RNA hairpin by Dicer; it was independent of promoter choice, presence of blunt ends, route of delivery and RNAi potency.
GS5 and LH86 cells have been described earlier (32, 33) . Huh-7 and 293FT cells were maintained in DMEM supplemented with 10% FBS. We used the following antibodies: anti-CyPA (Biomol, Plymouth Meeting, PA, USA); anti-CyPB (AfEnity BioReagents, Rockford, IL, USA); anti-Ku80, anti-Flag and anti-actin (Sigma-Aldrich, St Louis, MO, USA); anti-IFN stimulate gene (ISG)15 (Rockland Immunochemicals, Gilbertsville, PA, USA); anti-NS5A (Virogen, Watertown, MA, USA) and anti-NS3 (in-house). GSB1 and H801 cells have been described earlier (34) . Poly I : C was purchased from Sigma-Aldrich, and synthetic hairpin RNA was purchased from Integrated DNA Technologies (Coralville, IA, USA). Synthetic siRNA was purchased from Ambion (Austin, TX, USA).
Protein contents of cell lysate were quantified with the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA), and an equal amount of total protein was loaded in each lane. Samples for IRF-3 dimerization assay were run on a polyacrylamide gel under non-denaturing conditions (35) . Other samples were denatured and separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Proteins were then transferred onto a nitrocellulose membrane and stained with the appropriate antibodies with the SNAP i.d. TM system (Millipore, Worcester, MA, USA) according to the manufacturer's instructions.
For luciferase assays, cells were seeded to a confluency of 50%, and for all other assays, cells were seeded to a confluency of 30%. The next day, transfections of DNA plasmids and synthetic RNAs were performed with Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
Plasmids pGL3-IFNA1, pGL3-IFNB, pRL-TK, pCMV-Flag-IRF-3 and pCR3.1-IRF-7A have been described earlier (36) . shRNAs were expressed from a human immunodeEciency virus (HIV)-based lentiviral vector (32, 37) , and sh-PCAF was constructed on the basis of a previously reported sequence (38) . Plasmid sh-B971/H1 was constructed by cloning of the DNA fragment encoding the sh-B971 RNA into pSilencer 3.0-H1 (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The RIG-I and TLR3 constructs have been described (39, 40) . The RIG-I C construct encodes Flag-tagged, C-terminal 707 aa of human RIG-I cloned into a bicistronic expression vector modified from pBICEP-CMV-1 (Sigma-Aldrich, St Louis, MO, USA), in which the CMV promoter was replaced with the elongation-factor-1 promoter. The MDA5, MDA5-C constructs were kindly provided by Fujita (12) . HCV genotype 2a NS3-4A protease was expressed from the pCMV-3Tag-1a plasmid (Stratagene, La Jolla, CA, USA).
293FT cells were seeded in 24-well plates and were transfected 16 h later with 400 ng of a shRNA expression vector, 40 ng of pGL3-IFNA1 or pGL3-IFNB, 20 ng of pRL-TK and 50 ng of pCR3.1-IRF-7A. Cells were collected 48 h after transfection. Luciferase assays were performed with the Dual-Glo Õ Luciferase Assay system reagents (Promega, Madison, WI) and luminescence quantified with a Modulus Microplate reader (Turner BioSystems, Sunnyvale, CA, USA). Ratios of firefly luciferase (from the pGL3 vectors) to Renilla luciferase (from the pRL-TK vector) were calculated, and that of the sh-B971 sample was normalized to 100%.
Sequences of shRNA are shown in Table 1 . Lentiviral vector production and transduction were performed as described earlier (37) . Viral vectors were pelleted by ultracentrifugation at 50 000g at 4 C for 3 h and resuspended in a volume of PBS that was 1% of the original medium volume. The titers of the concentrated vectors were then measured with a p24 ELISA kit (ZeptoMetrix, Buffalo, NY, USA).
Real-time reverse transcription PCR (RT-PCR) was performed as described earlier (32) . The primers used were OAS1 forward, 5 0 -AGG TGG TAA AGG GTG GCT CC-3 0 and OAS1 reverse 5 0 -ACA ACC AGG TCA GCG TCA GAT-3 0 ; RIG-I forward 5 0 -GAG GCA GAG GAA GAG CAA GAG G-3 0 and RIG-I reverse 5 0 -CGC CTT CAG ACA TGG GAC GAA G-3 0 ; GAPDH forward 5 0 -TCA CTG CCA CCC AGA AGA CTG-3 0 and GAPDH reverse 5 0 -GGA TGA CCT TGC CCA CAG C-3 0 . The primers for HCV detection were 5 0 -CGC TCA ATG CCT GGA GAT TTG-3 0 and 5 0 -GCA CTC GCA AGC ACC CTA TC-3 0 .
For flow cytometry, GS5 cells were fixed 48 h after treatment in a solution of 2% paraformaldehyde and analyzed with a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Mean GFP intensity was plotted, and that of the sh-NTC sample was normalized to 100%.
Total RNA from transiently transfected 293FT cells was extracted with RNA STAT-60 (Tel-Test, Friendswood, TX, USA) and separated on a 7.5% urea polyacrylamide gel. The transfer of RNA onto nitrocellulose membrane and hybridization were performed according to standard molecular biology protocols. The probe for detecting the expression of sh-B971 and its variants was a synthetic DNA oligomer corresponding to the bottom strand of sh-B971. Radioactive labeling of the probe was performed with an end-labeling protocol with T7 polynucleotide kinase (Ambion, Austin, TX, USA). The exposure and detection of the radioactive signal was performed with a Typhoon Imager (GE Healthcare, Piscataway, NJ, USA) with Quantity One software (Bio-Rad, Hercules, CA, USA).
A short-hairpin RNA directed at CyPB induces IFN production in human embryonic kidney cells
To investigate the potential role of the cyclophilins (CyPs) in HCV replication (41), we delivered several shRNAs directed at mRNAs of three CyPs into HCV replicon cells by means of a lentiviral vector, using a murine U6 promoter to drive the expression of the shRNA ( Figure 1A ) (37) . We observed a discrepancy between two anti-CyPB shRNAs (B971 and B710) in their relative efficiency in knocking down CyPB expression and in suppressing HCV. Lentiviral vector sh-B971 was less efficient in knocking down CyPB expression but potently inhibited HCV NS5A expression in a human hepatoma cell line containing replicating HCV RNA ( Figure 1B , left). Viral inhibition was independent of CyPB knockdown, as control medium from transfected 293FT cells that did not contain any lentiviral vector particles, generated by omission of the packaging plasmids during transfection, also inhibited HCV replication ( Figure 1B , right) without affecting CyPB expression. The fast kinetics of viral inhibition (complete inhibition with 48 h, data not shown) was also more consistent with IFN than with RNAi-based inhibition. The presence of IFN in the lentiviral vector preparation of sh-B971 was confirmed by strong induction of 2 0 -5 0 -oligoadenylate synthetase 1 (OAS1), a classic IFN-induced gene, in both naı¨ve Huh-7 and the HCV replicon cell line (GS5) treated with the medium ( Figure 1C ). In addition, HCV replication in an IFN-resistant HCV replicon cell line (H801), in contrast to that in a wildtype replicon cell line (GSB1) (34), was not inhibited by the sh-B971 medium ( Figure 1D ), suggesting the lack of additional viral inhibiting agents in the sh-B971 medium. Expression of sh-B971 in 293FT cells also induced dimerization of IRF-3, confirming the activation of the IFN production pathway in these transfected cells ( Figure 1E ). Finally, sh-B971 was able to activate both IFN-a and IFN-b promoters, although the activation of the IFN-a promoter required coexpression of IRF-7, which is normally expressed at very low levels in 293-based cells ( Figure 1F ). These results demonstrate that sh-B971 is a potent activator of IRF-3 and IRF-7, master regulators of IFN expression in human cells.
We next investigated the role of the different viral/ exogenous RNA sensors, RIG-I, MDA5 and TLR3, in sh-B971-triggered IFN production. Mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (DN) mutants of RIG-I and MDA5, were transfected into 293FT cells with shRNAs and an IFN-b promoter reporter construct. The signaling to IFN-b promoter and the expression of the PRR proteins were then examined 48 h after transfection. In the absence of sensor proteins, the sh-B971 increased activation of the IFN-b promoter by 2.6-fold ( Figure 2A ). Coexpression of MDA5 or TLR3 did not increase or decrease sh-B971's ability to activate IFN-b promoter relatively to the negative control shRNA (sh-NTC), but in the presence of RIG-I coexpression, the induction of IFN-b promoter by sh-B971 was increased to $30-fold. Moreover, ectopic expression of a DN mutant of RIG-I (RIG-I C), but not that of MDA5 (MDA5-C), completely abrogated IFN promoter activation by sh-B971. With the exception of TLR3, which required prolonged exposure of the western blot to be detected, the cytoplasmic sensors and their mutants were expressed at comparable levels ( Figure 2B ). Moreover, activation of IRF-3 ( Figure 1E ) and IFN promoters ( Figure 1F ) in 293FT cells, which do not contain a functional TLR3 signaling pathway (42) , indicates that TLR3 plays a negligible role, if any, in IFN induction by sh-B971. The combination of sh-B971 and RIG-I produced the highest level of IFN-b promoter activity, which were confirmed by western blotting showing that endogenous ISG15 induction was only detectable in cells cotransfected with sh-B971 and wild-type RIG-I ( Figure 2B ). To confirm further that biologically active IFN was released from these cells, we applied the culture medium of the transfected 293FT cells to an HCV replicon cell line (GS5) in which NS5A-GFP expression is used for monitoring viral RNA replication (43) . HCV replication in this cell line is extremely sensitive to IFN, and the effect of the cytokine can be readily measured as the change in the mean GFP intensity of the treated cells. As shown in Figure 2C , culture medium from sh-B971 efficiently suppressed HCV replication, resulting in a decrease in Figure 1 . A small-hairpin RNA directed at CyPB induces IFN production in human embryonic kidney cells. (A) Sequence of sh-B971, which was expressed from a self-inactivating human immunodeficiency virus (HIV) vector with a murine U6 promoter (59) . (B) Inhibition of HCV expression by culture media of sh-B971-transfected 293FT cells. GS5 cells were treated with culture supernatant taken from 293FT cells transfected with various shRNA plasmids with (left) or without (right) the packaging plasmids overnight. Cells were then cultured in fresh media for an additional 6 days before being lysed for western blotting. (C) OAS1 induction by culture supernatant from 293FT cells transfected with sh-B971. Huh 7 and GS5 cells were treated with culture supernatant from 293FT cells transfected with either sh-Luc or sh-B971 for 24 h before RNA extraction and real-time RT-PCR analysis. OAS1 RNA level was normalized to that of GAPDH RNA. (D) Transfected culture media failed to suppress HCV replication in an IFN-resistant cell line. HCV replicon cells were cultured as described earlier (34) and then treated with the indicated culture medium from transfected 293FT cells. HCV RNA was analyzed with real-time RT-PCR. (E) IRF-3 dimerization in response to sh-B971 expression. Flag-IRF-3 was cotransfected with a shRNA into 293FT cells. Cells were lysed 24 h after transfection, and total cell lysate was separated on a polyacrylamide gel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (F) IFN-a and IFN-b promoter activation by sh-B971 expression. Sh-NTC, sh-C454 (an shRNA directed at CyPC), or sh-B971 was cotransfected along with luciferase reporter plasmids with or without IRF-7. The ratios of firefly luciferase readings to Renilla luciferase readings were plotted. the NS5A-GFP intensity within 48 h of treatment. Cotransfecting wild-type RIG-I produced a medium with stronger inhibition, whereas the RIG-C drastically suppressed the antiviral effect of the medium. Finally, real-time RT-PCR analysis revealed that sh-B971, but not the negative control shRNA, strongly activated expression of endogenous RIG-I, a well-characterized ISG whose induction requires paracrine/autocrine action of IFN (44, 45) . As expected, poly I : C activated RIG-I expression in the same assay ( Figure 2D ). These results, taken together, show that RIG-I is the cellular sensor that mediates the IFN induction by sh-B971.
The majority of the shRNAs that we use in the lab do not activate RIG-I expression and IFN signaling despite having essentially the same structure as sh-B971, so we wanted to determine whether the sequence of sh-B971 is distinctive enough to trigger the production of IFN. We first tested a synthetic siRNA duplex with the same target sequence as sh-B971. This siRNA (si-B971-syn) should resemble the final Dicer product of sh-B971 except for the 5 0 -ends. The synthetic siRNA contains 5 0 -OH groups, whereas the Dicer products probably Figure 3A ) while failing to activate IFN production, as measured by the GFP-HCV assay ( Figure 3B ). To determine whether the sequence of the intact hairpin RNA before Dicer cleavage is sufficient to trigger IFN, we tested a synthetic shRNA (sh-B971-syn) that had exactly the same sequence as the predicted intracellular sh-B971 transcript generated by the U6 promoter. Again, the 5 0 -end of the synthetic sh-B971 had a 5 0 -OH group instead of any phosphate. Sh-B971-syn behaved similarly to si-B971-syn in that it knocked down CyPB expression without activating IFN response (Figure 3) . These results suggest that the 5 0 -end status of sh-B971 is important for IFN activation, consistent with the previously finding that a 5 0 -triphosphate is required for RIG-I activation (24, 25) .
To determine the contribution of the individual residues of the sh-B971 sequence, we introduced a series of point mutations into the shRNA and tested them for IFN induction. We changed the first nucleotide from A to G, C, or T while maintaining base-pairing between nucleotides +1 and +47. These mutant shRNAs lacked the ability to activate IFN production (Table 1) . Changing the +1 nucleotide to G while leaving the +47 nucleotide intact also abolished IFN activation by the shRNA (A1/G), as did the reciprocal mutation U47/C. The importance of the first nucleotide was further confirmed by the inability of sh-B971+1 to activate IFN. The target of sh-B971+1 was shifted 1 nt downstream on the CyPB mRNA, producing an shRNA starting with a G at the +1 position. The presence of an A at the +1 position was not, however, sufficient to render a shRNA competent for IFN activation, as replacing the first nucleotide of the sh-NTC with an A did not generate an IFN-inducing shRNA (NTC-A and NTC+1). These results indicate that a protruding/unpaired A at the end of the hairpin or the RNA duplex, a potential result of 'breathing' at the end of the dsRNA, is not sufficient to trigger IFN induction as previously suggested (38) .
Two point mutations located farther into the stem structure of the shRNA (9G9 and B18A1) also reduced its ability to induce IFN even though the base-pairing was perfectly maintained in these mutants. Finally, replacing the 9-nt hairpin loop with a 7-nt loop that had been previously shown to abolish shRNA-mediated RNAi (loop A mutant) (46) eliminated sh-B971's ability to induce IFN, suggesting the importance of RNA processing in the induction. To determine whether the inability of the mutant shRNAs to induce IFN was due to lower expression levels, we performed northern blotting analysis of the shRNA expression on the wild-type and two mutants. The mutants A1/G and Loop A were chosen because their final siRNA products have exactly the same sequence as that of the wild-type sh-B971 and can thus be detected with the same efficiency by the same probe. Although sh-A/G and sh-Loop A were clearly unable to activate IFN-b promoter ( Figure 4A ), they were both expressed at levels comparable to those of the wild-type sh-B971 product ( Figure 4B) . Interestingly, the final siRNA product of sh-Loop A was slightly smaller than those of sh-B971 and sh-A1/G, suggesting that cleavage did occur and perhaps occurred one or 2 nt into the stem to compensate for the shorter loop.
Blunt-ended siRNA has been previously reported to be stronger inducers of IFN than the siRNAs with overhangs (47) . Indeed, a previously reported IFN-inducing shRNA, sh-PCAF (p300/CREB-binding protein-associated factor), contains a blunt end (38) and was more potent in activating IFN than sh-B971 ( Figure 5A ), which is predicted to form an overhang of 2-3 Ts at each end of the final siRNA. We therefore constructed a version of the sh-B971 that would be blunt at the end that is not processed by Dicer by adding two extra As to the 5 0 -end of the shRNA. This modification (Blunt sh-B971) did not increase the ability of sh-B971 to activate IFN-b promoter ( Figure 5A ). We confirmed, in two independent experiments, that IFN induction by sh-PCAF was also mediated by RIG-I. First, cotransfection of DN RIG-I resulted a 50-to 100-fold inhibition of IFN induction by sh-PCAF ( Figure 5B ), whereas wild-type RIG-I increased IFN induction by several fold in the same assay. Second, when HCV NS3-4A protease, which cleaves MAVS, thereby blocking the RIG-I pathway, was coexpressed with either sh-B971 or sh-PCAF, IFN induction by these shRNAs were severely compromised ( Figure 5C ), further substantiating a role of the RIG-I and MAVS pathway in mediating IFN induction by both the blunt-ended sh-PCAF and the sh-B971 with overhang. The proper expression of NS3-4A protease was confirmed by western blotting ( Figure 5D ).
To assess the contribution of the promoter choice in IFN activation by intracellular expressed shRNA, we expressed sh-B971 from another commonly used pol III promoter, the human H1 promoter. Both the original, mU6-driven sh-B971 and the H1-driven sh-B971 activated IFN-b promoter ( Figure 6A ) and resulted in secretion of IFN into the transfected cell-culture media, which in turn suppressed HCV replication ( Figure 6B ). Proper expression of the siRNA ( Figure 6C ) and the subsequent knockdown of CyPB expression ( Figure 6D ) all appeared normal for sh-B971 expressed from the H1 promoter plasmid, which has a backbone different from that of our lentiviral vector carrying the mU6 promoter. These data suggest that IFN induction by sh-B971 is not restricted to a particular promoter or expression construct. Further supporting this conclusion was the observation that the expression cassette by itself, removed and isolated from the lentiviral plasmid by restriction digestion, could also activate IFN production in transfected 293FT cells (data not shown).
To this point, all the IFN induction experiments were done with transient transfection of DNA vectors and it was possible that certain features of the double-stranded plasmid DNA are responsible for IFN induction. We first tried to address this point by transfecting just the shRNAexpressing cassette, generated either by PCR or restriction enzyme digestion, into 293FT cells and confirming that these fragments of $200 bp were sufficient to trigger IFN induction (Supplementary Figure S1) . To definitively rule out any contribution by dsDNA, we used a lentiviral transduction system which has been suggested to express shRNAs that can escape detection by PRRs and IFN activation (48) . We produced lentiviral particles containing shRNAs from 293FT cells using standard . Sh-B971 expressed from an H1 promoter triggers IFN activation. Sh-B971 expressed from an H1 promoter was capable of (A) activating IFN-b promoter and (B) triggering IFN production to inhibit HCV replication in GS5 cells. (C) Intracellular levels of U6-and H1-driven sh-B971 products. RNA extraction and northern blotting were performed as described in Figure 4B . (D) Knockdown of CyPB expression by sh-B971 expressed from an H1 promoter. methods, centrifuged them to separate the vectors from the IFN-containing media, and then used them to infect naı¨ve 293FT cells ( Figure 7A ). Both sh-B971 and sh-PCAF vectors induced IFN production when delivered as concentrated lentiviral particles, measured both by HCV suppression ( Figure 7B ) and by OAS induction ( Figure 7C ) in Huh-7 cells. To rule out the possibility that residual IFN in the concentrated viral particles was responsible for these results, we added 100 U/ml IFN to the negative control vector sample before the concentration step. This preparation, designated sh-NTC*, was not able to trigger IFN production in naı¨ve 293FT cells, suggesting that the concentration step effectively removed the soluble IFN from the viral particle pellet. Proper knockdown of the siRNA target of sh-B971 was confirmed by this route of shRNA delivery ( Figure  7D ). To prove definitively that IFN induction by the shRNAs was mediated by the lentiviral infection route, we tested the effect of an inhibitor of HIV reverse transcriptase, Nevirapine, on IFN induction by sh-B971 and sh-PCAF. As shown in Figure 7E , inclusion of Nevirapine at the time of transduction effectively blocked the ability of both shRNAs to induce IFN in the transduced cells, suggesting the importance of the reverse transcription step in the expression of the shRNAs delivered by the lentiviruses. To determine whether lentiviral vector-delivered shRNA can trigger IFN induction in cells other than 293FT cells, we transduced a human hepatoma cell line, LH86, which has been reported to produce IFN upon viral infection (33) , and examined IFN induction in these cells. Culture medium from LH86 cells transduced with sh-PCAF contained biologically active IFN, which suppressed HCV replication in GS5 cells ( Figure 7F ), indicating that the ability of shRNAs delivered by lentivirus to induce IFN response was not limited to 293FT cells.
It has been reported that certain chemically synthesized and phage polymerase in vitro transcribed siRNAs can non-specifically induce IFN responses and produce offtarget effect via various PRRs, including TLRs. However, the induction of IFN response by shRNAs and its underlying mechanisms have not been as well studied. The actual number of shRNAs that are capable of triggering IFN response will certainly be larger than the few that have been reported in the literature, yet very little is known about the unique characteristics of the select shRNAs and the pathway that they use to activate IFN production. The present study identifies RIG-I, but not MDA5 or TLR3, as the mediator for activation of IFN responses by two shRNAs that are distinct in sequence and structure but both capable of IFN induction in human cells. This was demonstrated by induction of IRF-3 dimerization, activation of IFN promoters, induction of endogenous ISGs (ISG15, OAS and RIG-I), and secretion of IFN, all of which depended on RIG-I and its downstream adaptor, MAVS. In addition, we show that delivery of these shRNAs via lentiviral transduction does not reduce their IFN-inducing capacity, indicating that the ability of lentiviral vector transduction to avoid IFN induction by shRNAs, as reported previously (48), may not be universally applicable to all the shRNAs. Specific recognition of dsRNAs or ssRNAs bearing 5 0 -triphosphates by RIG-I is presumably determined mostly by structural features other than the nucleotide sequence of the RNA. Yet IFN activation by sh-B971 exhibited a stringent dependence on specific nucleotides at multiple positions of the shRNA. An AA dinucleotide at the beginning of the U6 transcript has previously been suggested to result in aberrant transcription, and preserving a C/G sequence at positions À1/+1 suggested to avert IFN induction (38) . We indeed observed a strict requirement for an adenylate at the +1 position of sh-B971 for RIG-I recognition and IFN activation, but we observed no difference in expression levels or the apparent sizes of the sh-B971 RNAs bearing either an A or a G at the +1 position. Furthermore, mutations introduced elsewhere in the shRNA also abolished or diminished sh-B971's ability to activate IFN, suggesting additional sequence requirement for efficient RIG-I recognition and IFN triggering. Despite these results, because we were not successfully in cloning and sequencing the vectorexpressed siRNA, we cannot exclude the possibility that the adenylate at the +1 position interferes with transcription and that the resultant abnormal transcript contributes to IFN induction.
Interestingly, the loop A mutant, which contains a predicted loop of 7 nt, generated a siRNA duplex inside the cells that is slightly smaller than that of the shRNAs with a wild-type hairpin loop, suggesting the processing by Dicer into the stem, perhaps fulfilling the requirement of a length of 9 nt for the hairpin loop (46) . This mutant form of sh-B971 was not, however, able to trigger IFN activation.
Despite the abilities of both sh-B971 and sh-PCAF to activate the RIG-I pathway, the two shRNAs are unrelated in sequence. Two short stretches of siRNA sequences, GUCCUUCCAA and UGUGU, that have been previously defined as IFN-or cytokine-activating motifs (8, 9) are not found in either sh-B971 or sh-PCAF. Any common sequence motifs of IFN-activating shRNAs, if any, remain to be defined. The two shRNAs also differ in that one is predicted to contain one blunt end and the other two ends with overhangs. These results suggest that, although blunt ends may increase siRNA's ability to be recognized by RIG-I (47), they are not required for IFN activation by an endogenously expressed shRNA. The best-characterized RNA structure motif recognized by RIG-I is the 5 0 -ppp, which is absent from virtually all the cellular RNAs as a result of either 5 0 -capping or internal cleavage before their appearance in the cytoplasm. A synthetic shRNA that has the same sequence as sh-B971 but lacks the 5 0 -ppp failed to induce IFN, suggesting the 5 0 -end status of the intracellularly expressed sh-B971 contributes to IFN activation. Whether or not the 5 0 -end of an shRNA is capped has not been investigated. Murine U6 RNA does not contain the trimethylguanosine cap that is present on mRNAs and other U small nuclear RNAs; instead it contains a g-monomethyl phosphate cap at its 5 0 -end (49) . Capping of heterologous transcripts produced from the mU6 promoter, however, requires a stem loop at the 5 0 -end of the transcript and an AUAUAC sequence immediately after (50) . Most shRNAs, including sh-B971 and sh-PCAF, would not meet these requirements and thus should contain unmodified 5 0 -ppp. Similarly, no evidence of a cap structure for H1 transcripts could be found in the literature. We attempted to express sh-B971 using a miRNA expression cassette and the pol II promoter (51) . The primary transcript generated with this construct would be capped at 5 0 -end by a trimethylguanosine cap and the final siRNA duplex would bear a monophosphate at the 5 0 -ends of both strands because of Drosha and Dicer cleavage. This version of the sh-B971 vector was much weaker in its ability to trigger IFN activation. Unfortunately the intracellular expression of the RNA duplex was also much weaker and barely detectable by northern blotting. In addition, no knockdown of the target CyPB mRNA was seen with this miRNA-based sh-B971 (data not shown). As a result, whether sh-B971, if expressed at higher level from this construct, could effectively activate IFN remains unclear.
So far as we know, ours is the first report of IFN activation in the target cells by shRNAs delivered by lentiviral transduction. A previous report of IFN induction by lentiviral vector-expressed shRNA only examined the IFN generated in the vector-producing cells, which then up-regulated IFN-stimulated genes in the transduced cells (10) . The distinction is important as lentiviral vectors used in a gene-therapy setting will likely be purified and free of any IFN that has been generated during the vector preparation step, but IFN activation in the target cells would pose a more serious concern. Our data suggest the importance of screening shRNAs for IFN induction in the transduced cells in vitro before largescale studies. An HIV reverse transcriptase inhibitor efficiently blocked IFN production by both sh-B971 and sh-PCAF when delivered by transduction, indicating the virion-encapsulated RNA was not able to trigger IFN activation. In this respect, it is interesting to note that positive-stranded RNA viruses, which produce dsRNA intermediates in the cytoplasm during replication (52) (53) (54) (55) , often replicate in membrane enclosed vesicles (56) , This sequestration of viral dsRNA in membranous structures may shield the RNA from the cytoplasmic PRRs and contribute to a successful infection.
IFN-induction and RNAi by shRNAs appear to be independent functions of the same RNA (57). Our results also showed that IFN-induction by sh-B971 is independent of its ability to suppress target mRNA expression through RNAi. On the other hand, it might be possible to screen for duel functional siRNAs that confer therapeutic benefits by both RNAi and immunostimulation (58) . For example, siRNAs that target either viral genomes or cellular cofactors of the viruses can be screened for their ability to trigger IFN activation in hopes of find 'super siRNAs' with increased efficacy against IFN-sensitive viruses. The early diversification of influenza A/H1N1pdm Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification. Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 – July 9, 2009, including relatively large samples from the US states of Wisconsin and New York. Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures. Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns.  Immunoglobulin Superfamily Virus Receptors and the Evolution of Adaptive Immunity  Obligate intracellular pathogens depend on cell-surface molecules to attach and enter into host cells. Pathogen receptors may be highly specialized proteins, such as complement receptors or neurotransmitter receptors, or more ubiquitous components of cell membranes, such as integrins or sialic acid-containing oligosaccharides. The immunoglobulin superfamily (IgSF) of molecules contains several members that are expressed at the cell surface, bind diverse ligands, and contribute to a variety of cellular activities, including adhesion and immune responses. Many viruses have usurped the adhesive properties of IgSF proteins to mediate attachment (Table 1) . Strategies used by viruses to engage IgSF receptors provide clues to general mechanisms by which IgSF proteins bind different types of ligands, including antigens.
Members of the IgSF have diverged in sequence and function. However, all contain domains with the characteristic immunoglobulin fold, which is defined by two opposing antiparallel b-sheets connected in a unique manner [1, 2] . The core of the immunoglobulin fold is formed by four b-strands (B, C, E, and F) augmented with three to five additional b-strands (A, C9, C0, D, and G) to yield several distinct subtypes [1, 2] . Most common are the Vset and C-set immunoglobulin domains, which are named according to their occurrence in the variable and constant regions of immunoglobulins, respectively. A third type, the I-set, is an intermediate structure between the V-and C-sets found frequently in cell-surface receptors. Immunoglobulin domains rarely occur in isolation but typically form concatenated chains, often with a V-set or I-set domain at the N-terminus.
Biochemical and structural analyses of interactions between viruses and their cognate IgSF receptors reveal several striking similarities. First, in cases in which structural information about virus-receptor complexes is available, the viral attachment proteins exclusively bind to the most membrane-distal, N-terminal domain (D1) of the IgSF receptors [3] [4] [5] [6] [7] [8] [9] [10] . While structural information about com-plex formation is lacking for the IgSF receptors carcinoembryonic antigen-related cell adhesion molecule, nectin-1, nectin-2, and signaling lymphocyte-activation molecule (SLAM), biochemical studies also implicate their respective D1 domains in virus binding [11] [12] [13] [14] . Second, viruscontacting residues lie towards the upper ''tip'' of the IgSF D1 domain. Third, the viral receptor-binding region engages the CC9FG b-sheet of the IgSF receptor D1 domain. Fourth and finally, almost all of the receptor domains interacting with viruses belong to the V-type IgSF fold. The single exception, the D1 domain of ICAM-1, belongs to the I-set type, which is structurally similar to the V-set domain.
Although the database of viral proteins in complex with IgSF receptors is still quite small, interactions of viruses with their receptors parallel the recognition mode of immunoglobulins, which also recognize their cognate antigens via residues at the tip of their N-terminal, V-set domains. The case of the receptor-binding head domain of reovirus attachment protein s1 in complex with the D1 domain of its receptor, junctional adhesion molecule-A (JAM-A) [9] , serves to illustrate this point ( Figure 1A ). The JAM-A homodimer strikingly resembles the dimer formed by the V-set domains of the light and heavy chains of immunoglobulins. In both structures, the two V-set domains face each other with similar orientations. Moreover, residues in the receptor required for virus attachment reside in bstrands and intervening loops that juxta-pose the complementarity determining regions (CDRs) of antibody molecules. Thus, residues known to interact with ligands map to corresponding regions near the tip and one side of the V-set domains. These similarities extend beyond reovirus receptor JAM-A. Other IgSF virus receptors, such as the coxsackievirus and adenovirus receptor (CAR) [5] and HIV receptor CD4 [4] , also recognize their viral ligands via residues that partially overlap with the CDR region of immunoglobulins ( Figure 1B -F). CAR forms a homodimer via its D1 domain that is very similar to the JAM-A homodimer [15] . CD4 also forms homodimers, albeit via its D4 domain [16] .
The immunoglobulin fold predates the evolution of vertebrates. Genomes of invertebrate organisms encode numerous molecules that belong to two families with homologs in vertebrates: the JAM/cortical thymocyte marker of Xenopus (CTX) family and the nectin family [17] . Vertebrate counterparts of these genes are found in discrete blocks, and many are now diversified to encode molecules that function in adaptive immunity, including CD3 and SLAM [17] . Invertebrates do not encode recombination-activating genes (RAGs) and generally display only limited antigen-specific immunity. Therefore, the core structural element of adaptive immunity, the immunoglobulin fold, evolved prior to a mechanism to generate a highly diversified antigen-specific repertoire.
Similarities in mechanisms of ligand engagement by IgSF pathogen receptors and immunoglobulins, coupled with the evolution of the immunoglobulin fold prior to the existence of the vertebrate adaptive immune system, suggest the possibility that primitive members of the JAM/CTX and nectin families evolved to become soluble adaptive immune mediators in modern vertebrates. One attractive hypothesis is that soluble forms of pathogen receptors served as precursors to molecules of the adaptive immune system. Soluble receptors would neutralize viral [18] . Expression of a soluble pathogen receptor followed by duplication within the primitive genome and acquisition of mutations that permitted recognition of additional pathogens could confer a strong selective advantage. Upon introduction of RAGs into the vertebrate genome, such a gene family would have been primed to express molecules akin to present-day immunoglobulins. Alternatively, membrane-anchored forms of IgSF molecules that arose in primitive invertebrates may have been maintained in the genome due to their cell-adhesion functions, followed by the serendipitous introduction of mechanisms for the secretion and generation of diversity. In this scenario, pathogens may have contributed to the evolution of the modern adaptive immune system at much later evolutionary times. Is there evidence that favors either of these potential evolutionary mechanisms? In addition to similarities in their ligandbinding strategies, many of the closest structural homologs of JAM-A are immunoglobulins, which raises the possibility that immunoglobulins are more closely related to JAM-A than to other IgSF molecules. A search for structural homologs of the JAM-A D1 domain using the Dali algorithm [19] provides support for this hypothesis. The closest structural homologs of the JAM-A D1 domain are immunoglobulin domains, with the highest Dali Z-score of 14.6 for an IgAk variable domain (PDB code 2FBJ) ( Table 2) . Other IgSF proteins with similarity to JAM-A D1 have significantly lower Z-scores. The Z-scores correlate well with root mean square deviations for superpositions of JAM-A D1 with immunoglobulins, which also are lower (i.e., more similar) than the corresponding values for superpositions of JAM-A D1 with other IgSF proteins. This homology search can be extended to CAR, neural cell adhesion molecule, and nectin-like molecule 1, which result in Z-scores that are generally higher for the superposition of their D1 domains with immunoglobulins than with other cell adhesion molecules. In urochordates (Ciona) and cephalochordates (Branchiostoma), evolutionarily close relatives of the vertebrates, there are homologs of JAM/CTX and nectin IgSF molecules with features of membrane receptors. Ciona encodes only a single JAM/CTX-like molecule and two nectin-like molecules [20] . In humans, these molecules are all part of a single linkage group involved in immune function [17, 20] . Taken together, these results suggest that relatively few JAM/CTX and nectin family IgSF molecules were maintained in invertebrates, and the expansion and duplication resulting in the evolution of immunoglobulins may have occurred after the introduction of these molecules into the vertebrate genome.
There also is evidence of expansion of IgSF molecules in invertebrates. For example, like many immunoglobulins, chitin-binding protein (CBP) of Branchiostoma is a close structural homolog of JAM-A (Table 2 ). Variable region-containing (V) CBPs contain a V-type immunoglobulin domain with extensive sequence diversity in the N-terminal region [21, 22] . This diversity is thought to result from high haplotype variation, including variable copy number, polymorphisms, and potential for alternative splicing [23] . Another of the closest structural homologs of JAM-A is Down syndrome cell adhesion molecule (Dscam), an IgSF member of the more evolutionarily distant invertebrate Drosophila (Table 2) . Dscam is an immune mediator found in clusters of variable exons flanked by constant exons [24, 25] . Thousands of different Dscam molecules can be generated via alternative splicing, a mechanism that is highly conserved across insect orders [26] . Secreted isoforms of Dscam circulating in insect hemolymph contribute to phagocytic uptake of bacteria. While the structural similarities between JAM-A and VCBP or Dscam may not indicate a direct evolutionary relationship, it is clear that diversification and secretion of soluble forms of IgSF molecules can occur in invertebrates and raise the possibility that pathogens have had selective influence on the diversification and secretion of these molecules. Thus, IgSF proteins that served as precursors to soluble adaptive immune effectors may have diversified both prior to and following their introduction into the vertebrate genome. A more thorough examination of IgSF members in invertebrates may clarify mechanisms that led to the evolution of modern adaptive immune mediators and the role of JAM/CTX family molecules in this evolutionary process.
The evolution of JAM family members prior to the biochemical means to efficiently and extensively diversify antigen receptor genes, along with the structural similarities in the binding surfaces of virus receptors and immunoglobulins, provides strong support for the contention that viruses and perhaps other pathogens that engage IgSF receptors contributed to the selection of humoral mediators of adaptive immunity. These observations provide a new framework for understanding how pathogen-host interplay during a prolonged period of evolutionary struggle may have led to the development of antigen-specific immune responses in vertebrates.
We thank Jim Chappell and the PLoS Pathogens reviewers for insightful suggestions and critique of the manuscript.  Peptide-Mediated Cellular Delivery of Oligonucleotide-Based Therapeutics In Vitro: Quantitative Evaluation of Overall Efficacy Employing Easy to Handle Reporter Systems Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments. Oligonucleotide-based strategies which can be used to modulate a vast variety of cellular functions represent a promising alternative to conventional therapies (for a review see: [1, 2] ). Among the different oligonucleotides with therapeutic potential are aptamers, transcription factor-binding decoy oligonucleotides, ribozymes, triplex-forming oligonucleotides (TFO), immunostimulatory CpG motifs, antisense oligonucleotides, small interfering RNAs (siRNAs) and antagomirs. Nowadays, these potential macromolecular drugs are generally either relatively easily derived by rational design (e.g. antisense or siRNA) or straightforward selection processes (e.g. aptamers). One of their main advantages over protein-or peptide-based approaches comprises the high specificity for their target while being non-immunogenic. However, despite these advances, a major impediment to the development of nucleic acid-based strategies for treatment and prevention of diseases is the relatively inefficient means to effectively deliver these macromolecules into the desired target cells. Although viral vectors have been widely used to transfer genetic material into cells [3, 4] , they bear an inherent risk for the patient to encounter severe immunological responses or even develop cancer [5] [6] [7] [8] . As a result of these problems much attention has been paid in recent years to the development of non-viral delivery systems. This conception *Address correspondence to this author at the Institut für Molekulare Medizin, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; Tel: +49-451-500-2745; Fax: +49-451-500-2729; E-mail: restle@imm.uni-luebeck.de includes an assortment of fairly unrelated approaches yielding various degrees of enhanced cellular uptake of nucleic acids. Currently, liposomes and cationic polymers are used as a standard tool to transfect cells in vitro. However, these procedures are characterized by a significant lack of efficiency accompanied by a high level of toxicity rendering them mostly inadequate for in vivo applications. In this context cell-penetrating peptides (see below) represent an interesting alternative as they generally are less toxic than liposomes or cationic polymers. Moreover, they are commonly better suited to transfer cargo into different cell types like non-adherent cells and primary cells, which are hard to transfect using commercially available standard protocols. The most advanced approaches in the field, which are not subject of the present article, are complex carrier systems combining vantages of assorted strategies to generate nanoparticles with better defined properties aimed towards enhanced uptake as well as intracellular trafficking in combination with cell-specific functionalities. For example, there are attempts to combine peptides with cationic liposomes [9] [10] [11] [12] [13] [14] [15] [16] or polyethyleneimine (PEI) [17] . Other strategies are aimed towards the synthesis of high or low molecular weight branched polymers and/or peptides [18] [19] [20] [21] [22] or dendrimers [23, 24] . Even more complex systems are particularly promising with respect to in vivo delivery [25] [26] [27] [28] [29] .
In this review we will report about particular aspects of non-viral oligonucleotide delivery in vitro, pinpointing the current limitations, and provide quantitative means for determining where the bottlenecks of such strategies at present are. The focus of this article is recent progress in the field of peptide-mediated cellular delivery of siRNA and steric block oligonucleotides in cell tissue culture as a starting point for further developments illustrated by own experimental data. Our intention is not to provide the reader with easy solutions on how to solve the existing problems encountered with such approaches but give some hints where to start optimizing a particular approach.
The idea of using peptides as carriers goes back some twenty years when it was discovered that the HIV-1 transactivating protein Tat is taken up by mammalian cells [30, 31] . A few years later, the Antennapedia homeodomain of Drosophila melanogaster was shown to act similarly [32] . Later on, it could be shown that peptides derived from Tat and Antennapedia as well as other proteins are capable of transporting macromolecular cargo molecules into cells [33] [34] [35] . Based on such promising results, a rapidly expanding field focusing on the so-called cell-penetrating peptides (CPPs), also referred to as protein transduction domains (PTD), began to develop. Since the first reports about Tat, a large number of naturally occurring as well as engineered CPPs have been discovered [36] [37] [38] [39] [40] [41] [42] . Table 1 gives an overview of selected "classical" CPPs. Generally, CPPs are short polycationic sequences of less than 30 amino acids that are able to translocate different cargoes (e.g. nucleic acids, peptides and even entire proteins) into cells. The only common characteristic of these peptides appears to be that they are amphipathic and net positively charged at physiological pH. Frequently the cargo is covalently attached to the CPP which can be achieved by expression as a fusion construct or by chemical coupling (for a review see: [43] ). In particular cases, cargo and carrier bind each other non-covalently through mainly ionic interactions [40, 44, 45] .
Despite the widespread interest in peptide carriers, the mechanisms underlying the cellular translocation of CPPs are poorly understood. Early work relied upon fluorescence imaging or flow cytometry analysis of chemically fixed cells to examine intracellular localization of fluorescently labeled peptides in the absence or presence of cargo. According to these experiments peptides appeared to be internalized very rapidly within minutes even at 4 °C. From such observations it was concluded that CPPs penetrate cell membranes by an energy-independent mechanism [46] [47] [48] [49] [50] . Although it had been reported quite early on that certain fixation procedures may cause artefacts leading to an overestimation of the cellular uptake rates [51] [52] [53] the dimension of this problem was not commonly recognized until a side by side comparison of fixed and living cells was published [54] . Based on these findings, many groups re-examined their data. However, despite considerable technical improvements, there are still puzzling controversial results concerning the exact mechanism of CPP uptake. Though in most cases endocytosis has been suggested to be the main route of internalization (Fig.  (1A) ), substantial difficulties are encountered identifying the exact pathway ( [42, 55] and references therein). Prior to endocytosis CPPs interact electrostatically with the extracellular matrix of the cell surface mostly through binding to negatively charged glycosaminoglycans, i.e. heparan sulfate proteoglycans [56] [57] [58] [59] . Recent studies indicate that the uptake mechanism of CPPs can be influenced by the attachment of cargos. For example, Richard et al. [54, 60] reported a colocalization of Tat 48-59 with markers of clathrin-mediated endocytosis, whereas Fittipaldi et al. [61] found a caveolae/lipid raft-dependent process for a Tat-GFP fusion protein and Wadia et al. [62] described a macropinocytotic uptake pathway for a fusion construct of Tat peptide with Cre recombinase. In summary, the precise mechanism of internalization remains elusive and strongly depends on the properties of both CPP and cargo as well as on the transfection conditions and the cell lines used [63] [64] [65] [66] [67] [68] .
As opposed to the majority of CPP applications reported, which rely on covalent linkage of carrier and cargo, limiting their general use considerably as a new construct has to be generated as well as tested for any given nucleic acid cargo, we will focus in this article on a peptide termed MPG which forms highly stable non-covalent complexes with nucleic acids (Fig. (1) ). The peptide is a derivative of the original MPG peptide described by Morris and coworkers [47] and differs by five amino acids in the hydrophobic part. These changes result in an alteration of the overall structure of the peptide towards a higher tendency of adopting a helical conformation [69] . Accordingly, the two peptides behave penetratin (Antp 43-58 ) RQIKIWFQNRRMKWKK [203] transportan GWTLNSAGYLLGKINLKALAALAKKIL [204] TP10 AGYLLGKINLKALAALAKKIL [205] Oligoarginine (R8) RRRRRRRR [50] MAP KLALKLALKALKAALKLA [163] MPG GALFLGFLGAAGSTMGAWSQPKKKRKV [47] MPG GALFLAFLAAALSLMGLWSQPKKKRKV [69] differently with respect to their interaction with artificial lipids as well as Xenopus oocytes [70, 71] and most probably, their exact mechanism of uptake is not the same. Besides ionic interactions responsible for the initial peptide/nucleic acid complex formation, hydrophobic peptide/peptide interactions drive the maturation of large nanoparticles in a sandwich-like assembly reaction (Fig. (1B) and Fig. (2) ).
In recent years, RNA interference (RNAi) has gained a lot of interest as a tool for functional genomics studies and probably equally important as a promising therapeutic approach for the treatment of various diseases [72, 73] . RNAi is a highly evolutionally conserved and specific process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homologous mRNA sequences [74, 75] . Mechanistically the process can be divided into two steps. An initiator step where dsRNA is cleaved by dicer, a member of the RNase III family, into 21-25 nt long small interfering RNA (siRNA) fragments [76] . In a consecutive step, these fragments are transferred to RISC (RNAinduced silencing complex) where one of the strands, the so called guide strand, serves as a molecular template to recognize homologous mRNA that is cleaved by Argonaute [77, 78] , a protein component of RISC. Once the guide strand is bound to RISC this complex can undergo many rounds of mRNA binding and cleavage ( [79] , Fig. (3A) ). To circumvent application of long double stranded RNAs, which inevitably trigger an interferon response, it is sufficient to extracellularly supply 21 nt long dsRNAs [80, 81] . Alternatively, siRNAs can be expressed endogenously using DNA vectors which code for short hairpin (sh) RNAs [82] [83] [84] . These shRNAs are than cleaved by dicer to siRNAs. Short hairpin RNA constructs have advantages over siRNA because the effects of these constructs can lead to a more stable and long-term result. On the other hand, besides the fact that shRNAs might interfere with the microRNA pathway [85, 86] , this strategy requires a gene therapy approach in the long run [87] . For this reason we will not cover shRNAs.
As described above, siRNAs represent a valuable tool to inhibit the expression of a target gene in a sequence-specific manner. In the following section, selected examples of CPPmediated siRNA delivery will be presented which are summarized in Table 2 . Only a few studies describe the covalent attachment of nucleic acid cargo and peptide carrier (confer Table 2 ). In one approach, simple mixing of siRNA targeted against GFP or CDK9 and Tat peptide did not generate any measurable RNAi effect whereas cross-linked siRNA-Tat 47-57 led to a significant down-regulation of the target proteins. However, high concentrations of siRNA (about 300 nM) had to be used [88] . Both LF-and Tat 47-57 -mediated transfections resulted in a perinuclear localization of siRNA. In contrast, fluorescently labeled Tat 47-57 without cargo was mainly found in the nucleolus, suggesting that interactions with RISC influence subcellular localization. In another approach, significant uptake of siRNAs targeted against luciferase or GFP could be observed after disulfide coupling the 5'-end of the sense strand to penetratin or transportan [89] . Compared to LF2000, slightly higher levels of transfection were achieved. Interestingly, after LF2000-mediated transfection, basal luciferase activity returned to normal levels one day earlier than after CPP-mediated transfection although the same concentration of siRNA was applied. A remarkably strong RNAi effect in hard to transfect primary neuronal cells was reported by Davidson et al. [90] . Here, siRNAs directed against several endogenous proteins were coupled to penetratin via a disulfide bond. The observed down regulation of the target proteins after peptide-mediated siRNA delivery was found to be far more effective compared to LF2000. This was in part attributed to the toxicity of the lipids. As one of the first groups to report on Tat 48-60 -or penetratin-mediated siRNA delivery in vivo, Moschos et al. showed, that intratracheal administration of the conjugates did not lead to any intensification of the knockdown of the target gene p38 mitogen-activated protein kinase in mouse lungs in comparison to unmodified non-formulated siRNA [91] . Strikingly, it was found that the peptides alone triggered a detectable decrease in target gene expression and that the penetratin-conjugate induced elevated levels of the immune markers IFN-, TNF-, and IL-12p40 in lung tissue.
Besides technical difficulties arising from the syntheses of conjugates consisting of short cationic or hydrophobic peptides and highly negatively charged siRNAs, Dowdy and his group [92] present a rather critical point of view referring to previous studies with CPP-siRNA-conjugates. They claim that the successful delivery described therein is solely the result of excess free peptide, which leads to additional complexation, and thereby cellular import of the siRNA. This is in accordance with Turner et al. [93] , who were the first to observe that careful purification of CPP-antisense-conjugates abrogates their biological effect. Among other things, this might be the reason why most of the studies reporting on successful peptide-mediated delivery of siRNAs use a noncovalent complexation approach (confer Table 2 ).
In 2003, Simeoni et al. [94] were the first who noncovalently complexed siRNA with the peptide MPG. At a 1:10 ratio of negative nucleic acid to positive peptide charges a decrease in luciferase activity of about 80 % was detectable in HeLa or Cos-7 cells. This effect was further enhanced to about 90 % down-regulation by a mutation in the NLS sequence of the carrier peptide (MPG NLS ), presumably due to an increased delivery to the cytoplasm, where RISC is localized. Recently, Veldhoen et al. [55] used a derivative of the MPG peptide for the delivery of siRNA, which will be described in the chapter "MPG -mediated delivery of siRNA and steric block oligonucleotides". Leng et al. [21] presented promising results with a prospect for cell-specific siRNA delivery. Different versions of a branched histidine/ lysine-polymer (H3K8b) yielded up to 80 % knockdown of the target gene in several cell types. Structure-function studies revealed an important role of the composition of the histidine-rich domain as well as its position within the peptide and the branches for siRNA delivery, whereas size and surface charge did not have any effect. Furthermore, the toxicity was much lower than for the commercial cationic lipids Oligofectamine and LF2000. Finally, the attachment of the tripeptide RGD, an integrin-ligand, slightly enhanced siRNA delivery and turned this carrier into a cell-specific system. A  Fig. (1) . Simplistic scheme of peptide-based nucleic acid delivery systems (A). Interaction of CPP and cargo is either achieved by covalent attachment or by non-covalent complexation through mainly ionic interactions. In case of non-covalent complex formation, a further assembly of cargo/carrier complexes occurs, leading to the formation of large nanoparticles (confer Fig. (2) ). In case of covalently joined molecules a similar scenario is less likely, yet cannot be excluded. Prior to the translocation process the particles attach to the cell surface by ionic interactions of positively charged CPP residues with negatively charged membrane components. Subsequently, complexes are taken up via an endocytotic pathway. Although less likely, direct penetration cannot be excluded and may occur simultaneously. Once inside the cell, the cargo has to escape from vesicular compartments, otherwise it eventually gets degraded in the lysosome. Red: negative charges, blue: positive charges, green: hydrophobic domains. Three-dimensional model of MPG /siRNA interactions (B). The model was generated by iterative rigid body docking cycles of siRNA (PDB 1R9F) and peptide using the program Hex 4.2 [201] . The PDB file of MPG was generated with the program ICM (Molsoft LLC) taking into consideration different secondary structure predictions and energy minimization protocols. Out of many docking solutions particular ones were picked for illustration purposes using the program Chimera [202] . The phosphate backbone of the siRNA is shown in red, the nucleobases in light gray. Aliphatic, aromatic and hydrophobic residues of the peptide are shown in green, positive charged residues in blue and the remaining amino acids in gray. It is assumed that formation of larger particles is driven by hydrophobic peptide/peptide interactions generating free positive charges where other siRNA molecules can interact. This eventually drives complex formation in a sandwich or mesh like assembly reaction. In principle such a scenario holds true for any given nucleic acid cargo. similar concept has very recently been used by Kumar et al. [95] for a specific delivery approach into the brain. A peptide derived from rabies virus glycoprotein (RVG) interacts specifically with the nicotinic acetylcholine receptor (AchR) on neuronal cells to enable viral entry. The authors could show that the biotinylated form of the 29-amino-acid peptide (YTIWMPENPRPGTPCDIFTNSRGKRASNG) was taken up by neuronal cells. In order to transport nucleic acids with this vehicle, R 9 was conjugated to RVG peptide. Systemic treatment of mice with siRNA in a non-covalent complex with this modified peptide promoted a highly specific cellular import of siRNA only into cells expressing AchR. Even more important, an antiviral siRNA treatment resulted in successful protection of mice against encephalitis caused by Japanese encephalitis virus (JEV). This is the first study to report on a non-toxic method to deliver siRNA across the blood brain barrier which could help to circumvent dangerous and ineffective injections into the brain. To date it presents one of the most promising tissue-specific delivery approaches which might be expandable to other in vivo applications.
Along these lines, most studies today are performed with the aim of CPP-mediated siRNA delivery in vivo. Although many of them are already showing promising results, e.g. concerning tumor-targeting and ocular delivery [96] [97] [98] , this is beyond the scope of this review and will be discussed elsewhere in this issue [99, 100] .
With the aim to increase the endosomal escape of siR-NAs after peptide-mediated delivery, Lundberg et al. [101] rationally modified penetratin to form a CPP (termed EB1) with improved endosomolytic properties. They achieved a pH-dependent conformational change of the peptide to a higher degree of helicity by the replacement of two basic amino acids with histidines and the N-terminal addition of six amino acids. In this study, several CPPs were compared in a non-covalent approach by measuring the overall cellular stearyl-R8 n-c EGFP, MAP2B primary rat hippocampal neurons [206] R8-MEND (siRNA/stearyl-R8 core) n-c luciferase HeLa [207] uptake via fluorescence and biological effect of siRNA targeted to luciferase mRNA. Penetratin-as well as TP10mediated transfection did not lead to any silencing of luciferase gene expression, despite high amounts of intracellular siRNA [101] and in contrast to previous reports using siRNA-penetratin-conjugates [90] or TP10/DNA-complexes [102] . EB1-mediated delivery of 100 nM siRNA led to approximately 50 % reduction of luciferase activity. This silencing effect was slightly better than for bPrPp and in the same range as for MPG NLS , but still not as pronounced as for LF2000-mediated transfection of 100 nM siRNA. As it was described earlier, that addition of a pH-sensitive peptide derived from hemagglutinin (HA2) can promote endosomal escape [62] , the authors linked HA2 to penetratin [101] . It turned out that although HA2-penetratin improved the silencing effect when coincubated with penetratin, EB1 was more potent than this combination of peptides. Together with confocal microscopy studies the authors concluded that the lack of biological effect after penetratin-mediated siRNA delivery is due to a lack of endosomal escape and that EB1 has a superior endosomolytic activity in comparison to HA2penetratin.
Endoh et al. [103, 104] very recently presented an innovative strategy, called CLIP-RNAi (i.e. CPP-linked RBPmediated RNA internalization and photo-induced RNAi) combining delivery of a specific RNA sequence with enhanced photoinduced release of RNA from endosomes. This goal was accomplished by fusing the U1A RNA-binding domain (RBD) to the Tat peptide and extending the siRNA with a short stretch of nucleotides specifically recognized by this RBD. These complexes were efficiently internalized but exhibited a punctuate cytoplasmic localization pattern, indicative of endosomal entrapment. However, photostimulation of a fluorophore attached to the peptide led to a redistribution of complex into the cytosol followed by efficient RNAi-mediated gene silencing.
Human pre-mRNAs contain on average eight expressed sequences (exons) with an average length of 150 nt and up to 60 intervening sequences (introns) which can vary in length between 35 and 10,000 nt, therefore comprising up to 90 % of each transcriptional unit. In the nucleus, ribonucleoprotein complexes called spliceosomes recognize exon-intron boundaries and catalyze the precise removal of introns and subsequent joining of exons in a process called RNA splicing [105] [106] [107] . Additionally, each primary transcript can yield different mature RNAs through alternative splicing, thereby expanding the information content and versatility of the transcriptome, e.g. through the production of protein isoforms. A recent study of 10,000 human genes revealed, that at least 70 % of all multi-exon genes are alternatively spliced [108] . There are several different types of alternative splicing, amongst others affecting transcription start sites, splice sites, polyadenylation sites or even whole introns and exons. Disruptions of these intricate splicing patterns are tightly coupled with human pathophysiology, either as a determinant or a direct cause of disease or as a modifier of disease susceptibility and severity [109] . Among these diseases are -thalassemia, cystic fibrosis, muscular dystrophies, Frasier syndrome, certain kinds of dementia and cancer. A more de-tailed description of the underlying mechanisms is beyond the focus of this article and can be found in a number of reviews [110] [111] [112] [113] . López-Bigas et al. [114] proposed that 60 % of mutations that cause disease lead to splicing defects rather than changes in the amino acid sequence. Two common forms of mutations are depicted in Fig. (3B) . On the one hand, a mutation in the splice donor can favor recognition of a cryptic splice donor and result in a mutant mRNA containing additional intronic sequences (part I). On the other hand, a mutation in the splice acceptor can lead to skipping of a whole exon and result in a shortened mRNA (part II). Both scenarios have been used in the context of antisense oligonucleotide-mediated approaches targeting alternative splicing. The use of antisense oligonucleotides interacting with mRNA to affect protein production goes back some 20 years [115, 116] . Since then, three principle mechanisms have been exploited for this purpose (for a review see: [117, 118] ): (I) the oligonucleotide/RNA duplex forms a substrate for endogenous RNase H, leading to mRNA cleavage; (II) the oligonucleotide/RNA duplex prevents the productive assembly of the ribosomal complex or arrests a ribosomal complex already engaged in translation, in both cases affecting protein biosynthesis; (III) the oligonucleotide/RNA duplex alters pre-mRNA splicing in the nucleus. The following section will focus on the last approach with the aim to treat splicing disorders and give examples of possible applications for CPPs in this context.
Different forms of human -thalassemia are caused by mutations within in the -globin intron 2, which activate cryptic splice sites and thus lead to the formation of nonfunctional transcripts (Fig. (3B part I) ). Those aberrantly used sites can be blocked by antisense steric block oligonucleotides, which leads to the synthesis of functional protein [119, 120] . Kole and his group adopted this principle for the development of a splice correction assay [121] . In this model system, a firefly luciferase construct leads to the synthesis of inactive enzyme because the reporter gene pre-mRNA is interrupted by the human -globin intron 2 containing an aberrant splice site. Upon binding of a steric block oligonucleotide, correct splicing is restored which in turn yields a functional luciferase protein. To achieve this, the oligonucleotide has to be delivered to the nucleus. Furthermore, only oligonucleotides that don't activate RNase H are applicable [2] , e.g. phosphorodiamidate morpholino oligomers (PMO, [122] ), locked nucleic acids (LNA, [123] ), peptide nucleic acids (PNA, [124] ) or 2'-O-methyl-modified oligonucleotides (OMe). In addition to their inability to activate RNase H, most of these modifications confer higher affinity to the target RNA and increased resistance against enzymatic degradation than unmodified versions. Compared to RNAibased model systems, this assay is less susceptible to side effects like cytotoxicity or off-target effects because the reporter gene activity is turned up rather than turned down. The splice correction assay has been successfully applied for the analysis of several carrier systems [24, 93, [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] . In the following section, selected examples will be presented, which are summarized in Table 3 . In contrast to many noncovalent CPP-mediated siRNA delivery approaches, efficient splice correction was only achieved with conjugates of peptide and steric block oligonucleotide. Astriab-Fisher et al. [128] described delivery of OMe RNA phosphorothioate oligonucleotides linked via a disulfide bridge to Tat peptide and penetratin. A few hours after transfection, the CPP-oligonucleotide conjugates were detected both in cytoplasmic vesicles and in the nucleus and caused a dose-dependent increase in luciferase activity. These findings are in contrast to results of Turner et al. [93] , who could not find a biological effect for several CPP-oligonucleotide conjugates in a HeLa cell assay for Tat-mediated transactivation of the HIV-1 long terminal repeat. The authors observed vesicular uptake but no nuclear import for their highly pure conjugates. Interestingly, the rate of uptake could be enhanced by addition of free CPP to the conjugates, though still no biological activity was detected. Based on these findings, Turner et al. [93] concluded that these free CPPs form complexes with CPP-cargo conjugates, which play a significant role in the uptake process. This is in accordance with observations by Meade et al. [92] for the uptake of CPP-siRNA conjugates described above. Moulton et al. [136] achieved correction of missplicing at low micromolar concentrations of a R 9 F 2 -PMO conjugate but not with complexes of peptide and PMO. The steric block activity of the R 9 F 2 -PMO conjugates could be further increased with longer spacers whereas variations in the conjugation chemistry did not result in any differences. Furthermore, transfection rates were higher than for conjugates with Tat peptide, penetratin or a Tat peptide analogue. Using the HIV-1 transactivation assay mentioned above, Turner et al. [137] could show that most CPP-oligonucleotide conjugates attained biologic activity only through co-administration of the endosomolytic substance chloroquine. Fluorescence microscopy analyses revealed that this treatment released fluorescently labeled conjugates from endosomal compartments into the nucleus. Besides the addition of chloroquine, different endosome disrupting strategies have been evaluated using the splice correction assay, for example co-treatment with endosome-disruptive peptides [129] or photochemical internalization [138] (see chapter "Strategies to enhance endosomal escape"). However, the most promising results have been achieved with two newly developed derivatives of classical CPPs (reviewed in [130] ). The modification of oligoarginines with non-natural, uncharged amino acids [139] led, amongst others, to the peptide (R-Ahx-R) 4 , in which Ahx represents a six-atom aminohexanoic acid spacer. Abes et al. demonstrated that in contrast to Tat or oligoargine, PMO-conjugates of this peptide led to dose-dependent splice correction at low micromolar concentrations in the absence of endosomolytic agents. The underlying mechanism for this superior activity is not clear yet, as the uptake of (R-Ahx-R) 4 constructs was less efficient than the uptake of Tat or oligoarginine constructs and also involved endocytotic routes [126] . The second peptide is a derivative of penetratin, to which six arginine residues were added at the N-terminus (R 6 Pen). R 6 Pen-PNA conjugates were shown to promote efficient splice correction at low concentrations and in the absence of endosomolytic agents [127] . Again, uptake of R 6 Pen-conjugates seemed to involve endocytosis and there was hardly any difference in splice correcting activity regardless of the nature of the linker used for conjugation, e.g. a stable thioether versus a reducible disulfide linker [130] .
Part II of Fig. (3B) illustrates a phenomenon that represents a strategy for the treatment of Duchenne muscular dystrophy (DMD). DMD is a severe progressive neuromuscular disorder caused by several different mutations in the dystrophin gene that abolish the production of functional protein [140] . Depending on the location of the mutation, the corresponding exon is skipped by covering the responsible splice sites with steric block oligonucleotides. This allows the transcription of internally deleted, but largely functional, dystro-phin proteins and converts a severe DMD into a milder Becker muscular dystrophy phenotype. A more detailed description of this approach and its application in a number of animal models can be found in several excellent recent reviews [141, 145] . Successful systemic delivery of splice switching oligonucleotides with or without chemical modifications (PMO, LNA, OMe) has been accomplished via injection of naked nucleic acids [146, 147] , with the help of viral vectors [148] , through re-implantation of ex vivo manipulated stem cells [149] or in combination with CPPs [150] [151] [152] [153] [154] . For the latter purpose, several studies were carried out with novel derivatives of arginine-rich peptides containing different numbers of non-amino acids, e.g. aminohexanoic acid and/or -alanine. These CPP-PMO conjugates showed higher serum stability, less endosomal trapping and led to efficient exon skipping in myoblasts and mice at lower dosages than the splice switching PMO alone [152, 153] . Yin et al. [154] used a PNA-modified splice-switching oligonucleotide conjugated to Tat, a muscle specific peptide (MSP) or different functional domains of the adenovirus capsid protein VP1 (AAV6, AAV8) and examined exon skipping efficiency in vitro and in vivo. Surprisingly, both after transfection and intramuscular injection, the activity of these PNA-peptide conjugates was not significantly better than that achieved by naked neutral PNA, presumably due to endosomal trapping.
Intracellular trafficking represents one of the major limitations of current non-viral nucleic acid delivery approaches [155] . In other words a large percentage of intracellular cargo molecules are entrapped in vesicular compartments and thus will not trigger the desired effect. Moreover, degradation or retrograde transport might further reduce the number of active molecules. So in order to determine the overall efficacy of a given delivery approach it is essential to know the numbers of intact cargo molecules inside the cell along with the minimal numbers of molecules required to cause a particular effect. Based on such information one can easily calculate the percentage of bioactive molecules. In the following chapter we will briefly describe selected examples of variable suitability for a quantitative determination of nucleic acids in a cellular context.
In principle, either the peptide or the cargo can be labeled by a reporter group, e.g. a radioisotope [156] or a fluorophore [157] . Fluorescent peptides or cargos have been quantitatively evaluated by FACS [54] or fluorescence correlation microscopy (FCS) [158] or FRET [159] . In all cases, it is crucial to distinguish between internalized and membraneassociated signals. For this purpose, a simple wash step with just buffer is not sufficient to completely remove membranebound peptide/cargo-complexes [54, 55] . Extracellularly bound complexes can be efficiently removed for example by enzymatic digestion with trypsin [160] , acid wash [161] or heparin treatment [55, 162] . Alternatively, discrimination between intra-and extracellular material is possible through chemical modification of extracellular components [163] or fluorescence quenching [164] .
Having established that only intracellular signals are taken into account, it is still challenging to distinguish between intact and degraded forms of peptide or cargo. In two studies, a fluorescence-based quantification method was combined with either HPLC analysis [163] or "cell activity by capillary electrophoresis" [165] to verify the integrity of cargo and carrier.
Recently, a technique to measure cellular uptake of CPPs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was reported by the group of Burlina [166] [167] [168] . This quantification is based on the addition of an internal standard, i.e. a peptide with a stable isotope label. The method has been used to determine the amount and stability of intact internalized peptides, e.g. Penetratin, R9 and several novel CPPs, and can also be used for the quantification of peptidic cargoes, e.g. an inhibitor of protein kinase C [166] .
Varga et al. [169] [170] [171] developed an "integrative systems" approach, combining quantitative experiments and computational modeling studies of vector uptake and trafficking kinetics, with the aim to take multiple potentially rate-limiting cellular and molecular processes into account. By applying their mathematical model to plasmid delivery with either Lipofectamine, several PEI-based vector formulations or an adenoviral vector, they could successfully predict experimentally observed effects and identify endosomal escape as the most important rate-limiting intracellular barrier for non-viral vectors. Recently, Zhou et al. [172] applied a similar strategy for the characterization of a novel lipopolymer (WLSP). This carrier shows an increased rate of endosomal escape compared to conventional PEI-based carriers.
With the aim to quantify rhodamine-labeled plasmid DNA in cellular compartments while avoiding problems arising from subcellular fractionation, like recovery and leakage, Akita et al. [173] developed a novel quantitative strategy, the confocal image-assisted three-dimensionally integrated quantification (CIDIQ) method. To distinguish endosomes/lysosomes and the nucleus from the cytosol, they were stained with LysoSensor DND-189 and Hoechst 33258, respectively, and sequential Z-series images were captured by CLSM. By applying this quantification method, the authors could show that due to a rapid endosomal escape, Lipofectamine Plus delivered more plasmid into the nucleus than R8 or stearylated R8. Hama et al. [174] used the same method in combination with TaqMan PCR to evaluate the uptake and intracellular distribution of plasmid DNA after delivery with viral as well as non-viral vectors. Due to superior cell surface binding, the efficiency of cellular uptake was significantly higher for Lipofectamine Plus than for adenovirus whereas intracellular trafficking, i.e. endosomal escape and nuclear transfer, were essentially the same. However, to achieve comparable transgene expression, 8000-fold higher intranuclear plasmid numbers were required in case of Lipofectamine Plus. This finding suggests a difference in nuclear transcription efficiency after non-viral delivery.
In another approach, Jiang et al. [175] extended the 3' end of the sense strand of a siRNA with a nuclease-resistant DNA hairpin to obtain a so-called "crook" siRNA. This modification had no effect on RNAi-mediated reporter gene inhibition and served as a primer for a filling-in reaction followed by PCR. Parameters were chosen so that the initial rate of template amplification correlates with the initial con-centration of the "crook" siRNA. Under these conditions, quantification of attomolar siRNA levels per cell was possible after liposomal transfections with Oligofectamine.
A highly sensitive method was developed by Overhoff et al. [176] for the detection of siRNA after phosphorothioatestimulated uptake [177] and adapted for the quantification of siRNA or steric block oligonucleotides after non-covalent peptide-mediated delivery ( [55] and Laufer et al., manuscript in preparation, see chapter "MPG -mediated delivery of siRNA and steric block oligonucleotides"). This so-called liquid hybridization assay is based on the extraction of total cellular RNA and the subsequent hybridization in solution of a radioactively labeled probe which is complementary to the oligonucleotide to be detected. Finally, following PAGE analysis, absolute amounts of internalized oligonucleotide can be quantified with high accuracy down to ~10 molecules per cell using internal standards [55] . In addition to this outstanding sensitivity, no amplification step is needed and only intact oligonucleotides are taken into account.
Considering the multitude of available CPPs, nucleic acid cargos and cellular as well as animal model systems, a comparison of different delivery strategies seems nearly impossible. In this context, we have for the first time undertaken a detailed side by side comparison of two different model systems using the peptide MPG as delivery agent. In the following paragraph we present own experimental data to exemplarily illustrate particular aspects regarding current limitations of peptide-based delivery systems. In contrast to many other CPP procedures, which rely on covalent linkage of carrier and cargo, the peptide MPG forms highly stable non-covalent complexes with nucleic acids, displaying binding constants in the low nanomolar range ( [55] , A. Trampe, unpublished data). The high flexibility of this non-covalent approach can be exploited to easily transport a wide variety of nucleic acid cargos without having to synthesize a new construct for each oligonucleotide. We have used MPG for the delivery of siRNAs in the context of an RNAi-based reporter system and for the delivery of steric block oligonucleotides in the context of the splice correction assay described above [121] . After MPG -mediated transfection of a luciferase-targeted siRNA, we observed strong inhibition of reporter gene readout with an IC 50 in the subnanomolar range [55] . After MPG -mediated transfection of a luciferasetargeted steric block oligonucleotide (further on also referred to as ON-705), we observed a moderate up-regulation of reporter gene readout, representative of low splice correcting activity (Laufer et al., manuscript in preparation). One possible explanation for the different degree of reporter gene regulation could be the different intracellular target sites, i.e. the cytoplasm for siRNAs and the nucleus for splice correction oligonucleotides. To attain more information about the subcellular localization of MPG -oligonucleotide complexes, we performed confocal laser scanning as well as conventional fluorescence microscopy studies with fluorescently labeled siRNAs or steric block oligonucleotides. In both cases, a punctuate non-homogenous distribution of the nucleic acids inside the cells was observed. This pattern is indicative of an accumulation of nucleic acids in endocytotic vesicles, which was verified by coincubation with LysoSensor (Fig. (4A) ). A quantitative computational analysis of fluorescence microscopy data yielded an average of approximately 50 % colocalization between endosomes and siRNA (Fig. (4B) ). In contrast to earlier assumptions that CPPs directly traverse the lipid bilayer, it has commonly become accepted that for most peptide-cargo combinations endocytosis plays a major role in cellular uptake. As described above and in the chapter "Strategies to enhance endosomal escape" below, administration of endosome disruptive substances like chloroquine can greatly increase endosomal release of trapped nucleic acids. Chloroquine is a weak base, non-charged at neutral pH but charged at pH 5.5 [178] . It is able to pass easily through membranes in its uncharged form, but becomes protonated and accumulates within acidic vesicles in its positively charged, membraneimpermeable form. Although its exact mode of action has not yet been resolved, it is generally accepted that chloroquine works via prevention of endosome acidification which in turn increases the residence time of cargo within the endosomes eventually resulting in a higher probability of transfer to the cytoplasm. Fluorescence microscopy analyses in the presence of 100 M chloroquine yielded two quite contrary outcomes. While the localization of siRNA did not change after addition of chloroquine (Fig. (4C) ), for the steric block oligonucleotide the picture changed completely (Fig. (4D) ). In the latter case, a diffuse fluorescence all over the cytoplasm with an accumulation of ON-705 in the nucleus could be observed. Nonetheless, considerable amounts of nucleic acid molecules were still visible as a punctual pattern, which indicates that the chloroquine treatment liberates only a certain fraction. On the whole, these qualitative observations are in full agreement with the observed biologic effects in the absence or presence of chloroquine (Fig. (5A) ). For MPG -mediated transfection of siRNA, even under conditions where the amount of bio-available siRNA was severely limited, only a minor increase in RNAi (ca. 30 %) was measurable. For MPG -mediated transfection of steric block oligonucleotide, on the other hand, a dramatic increase of reporter gene up-regulation by a factor of 50 -100 was observed. However, in both cases, the overall uptake did not change upon incubation with chloroquine ( Fig. (5B) ), which proves that the endosomolytic substance does not interfere with uptake but leads to a re-distribution of internalized nucleic acids. The underlying mechanism for the different effects triggered by chloroquine in case of peptide/siRNA and peptide/steric block oligonucleotide complexes remains unclear. Though, this is a good example that the cargo can substantially affect the properties and thereby intracellular trafficking of a particular carrier system. Ultimately, to assess the overall efficacy of this carrier system, we were interested to elucidate which percentage of molecules taken up after MPG -mediated delivery is biologically active. To derive such information, the exact intracellular amount of intact oligonucleotide, the corresponding reporter signal and the minimal number of molecules necessary to trigger a specific degree of reporter gene modulation have to be known. For the quantification of internalized cargo, we adapted a highly sensitive method first described by Overhoff et al. [176] , enabling us to detect intracellular oligonucleotide amounts down to 10 copies per cell [55] . The method is based on the liquid hybridization of a radioactively labeled probe with the corresponding oligonucleotide in cellular lysates. In this context it should be noted that a stringent heparin wash following the transfection procedure is crucial to avoid an overestimation of intracellular nucleic acid molecules due to complexes attached to the outside of the cell membrane [54, 55] . In order to correlate the numbers derived from the quantification experiments with the minimal number of molecules essential to trigger the observed effect, an independent assay had to be established. The gold standard in this case is microinjection as this technique enables one to deliver definite amounts of nucleic acids with a high degree of bioavailability into the cytoplasm or the nucleus of a mammalian cell along with a low toxicity profile and great accuracy. Considering that after cytoplasmic microinjection only 12 siRNA molecules are sufficient for halfmaximal inhibition of reporter gene expression, one can estimate that of the 10,000 molecules measured after MPGmediated transfection, only ca. 0.1 % are biologically active ( Table 4) . For the splice correction assay, the numbers are different in terms of absolute numbers but the outcome remains the same. Compared to the 300,000 molecules sufficient for maximal splice correction after nuclear microinjection, of the 70,000,000 molecules required following peptide-mediated delivery only ca. 0.5 % are biologically active ( Table 5 ). In both cases >> 99 % of internalized oligonucleotides are most likely retained in endosomes and subsequently degraded in lysosomes after peptide-mediated delivery. Frankly, this is a sobering result and puts in numbers how much room for improvement there actually is. Though it certainly is not legitimate to generalize these findings, there are countless reports in the literature suggesting similar limitations for the majority of non-viral strategies.
According to actual conceptions, siRNA enters a multiple-turnover pathway with one siRNA molecule capable of RISC-mediated cleavage of 50 or more mRNA molecules [79] . Even though the fate of steric block oligonucleotides is not really clear, it can be assumed that per splicing event one molecule is used up and translated into a functional mRNA molecule (e.g. single-turnover pathway). As a result the number of molecules needed to trigger an apparent effect is much higher in the splice correction assay compared to the RNAi-based reporter system (confer Table 4 and Table 5 ). On the other hand, being a single-turnover pathway, the splice correction assay should be much more sensitive to even minor changes in intracellular steric block oligonucleotide concentrations whereas the catalytic nature of the multiple-turnover RNAi mechanism might mask such small variations. This is in accordance with the data described above.
Taken together, based on the results presented as well as unpublished data (Laufer et al., manuscript in preparation) , the splice correction assay appears to be the superior tool for a quantitative assessment of nucleic acid delivery strategies.
As outlined above, endosomal release is one of the major rate-limiting steps for cellular delivery of macromolecules via cationic lipids, polyplexes and especially CPPs. In the following chapter we will present some examples of how to increase endosomal release. Transfections were performed with 2.1 M MPG and 1 nM siRNA or 10 g/ml LF2000 and 0.02 nM siRNA, i.e. in the range of the IC50 value [55] . Quantification was performed after 24 h according to the liquid hybridization protocol [55] . Molecules per cell were calculated based on the cell number seeded for transfection. For microinjection experiments, molecules per cell were calculated on the basis of the injection volume. Endosome-disrupting substances, like chloroquine, calcium or sucrose, were used to significantly enhance the activity of antisense PNA oligonucleotides conjugated to Tat, oligoarginines or oligolysines [125, 179, 180] . This effect did not result from increased uptake, but rather improved bioavailability in the cytoplasm or nucleus after endosomal escape. Takeuchi et al. [181] showed that by incubation of the target cells with pyrenebutyrate, delivery of arginine-rich peptides could be shifted from endocytic uptake to direct membrane translocation, yielding a rapid distribution of the peptide throughout the cytoplasm, even at 4 °C. Pyrenebutyrate acts as a counteranion and, by interacting with the positively charged peptide, increases the overall hydrophobicity, thereby facilitating a direct translocation through the lipid bilayer, as earlier shown with artificial membranes [182] . This method, which works only in the absence of a medium or serum, was successfully applied for administration of a fluorescent protein and an apoptosis-inducing peptide into dividing as well as non-dividing cells [181] . However, in general the strategies described above are not feasible for in vivo applications, due to high cytotoxicity or other undesirable secondary effects.
The imidazole group of histidine (His) can absorb protons in the acidic environment of the endosome, leading to osmotic swelling, membrane disruption and eventually nucleic acid escape. Accordingly, Lo et al. [183] modified Tat, which can bind and condense DNA through ionic interactions but has no acidic residues that can promote endosomal release, with different numbers of His residues. Highest reporter gene expression could be achieved after plasmid delivery with a Tat peptide covalently fused to 10 His residues (Tat-10H). Insertion of two additional cysteine residues into Tat-10H further enhanced stability of peptide/DNA complexes and transgene expression through formation of interpeptide disulfide bonds. Youngblood et al. [184] evaluated the influence of the stability of arginine-rich peptide PMO conjugates on cellular uptake and antisense activity. They could show that the stability is affected by the amino acid composition and the type of linkage to the cargo. Moreover, they found that degraded fragments could not escape anymore from endosomal or lysosomal compartments.
Another concept makes use of photosensitive substances, which induce the release of macromolecules from vesicles by light exposure. This so-called photochemical internalization (PCI) has, in the past, been used for intracellular delivery of a large variety of macromolecules (reviewed in [185] ) and, more recently, for the endosomal release of nucleic acids after delivery mediated by liposomes [186, 187] , polyplexes [188] or CPPs [138, 189] . Shiraishi et al. [138] investigated the biological activity of PNAs conjugated either to Tat, R7 or KLA-peptide in combination with a PCI treatment. Depending on the peptide, nuclear as well as cytosolic antisense effects could be enhanced by up to two orders of magnitude. Similar results were presented by Folini et al. [189] for a PNA targeting human telomerase reverse transcriptase conjugated to Tat. In both studies, lower nucleic acid doses were sufficient, thereby reducing the probability of off-target effects. In light of encouraging data from ongoing anticancer clinical trials employing photodynamic therapy [190, 191] , an in vivo application of PCI seems feasible and will be discussed in more detail by Oliveira et al. [192] in this issue. Furthermore, target specificity could be increased by local illumination of cells or tissue. Viral fusion proteins drive the fusion process between the viral membrane and the endosomal host cell membrane in a pH-dependent manner, which is required to translocate the viral genome into the cytoplasm after receptor-mediated endocytosis. Fusogenic peptides, usually hydrophobic, rich in glycine residues and found at the amino terminus of these proteins, were shown to have membrane perturbing and lipid mixing activities [193] . Many well studied representatives of this group are derived from the fusion sequence of influenza virus HA or HIV-1 gp41 [194] and have been used to improve the transfection efficiency of non-viral delivery systems [195] .
Addition of the influenza-derived dimeric peptide diINF-7 to LF2000/siRNA complexes had no effect on the particle size of ca. 120 nm, but significantly improved gene silencing activity of siRNAs targeting the epidermal growth factor receptor or the K-ras oncogene [196] . Similar results were obtained for plasmid delivery through addition of a fusogenic peptide derived from herpes simplex virus glycoprotein H to Lipofectamine/DNA complexes [197] . To the same end, Futaki et al. [198] used the peptide GALA, which was specially designed to mimic the function of viral fusion sequences, together with various commercially available cationic liposomes. Although they could not detect significant differences in cellular localization, plasmid transfection efficiency was increased and liposomal dosage could be reduced.
PEI covalently modified with the HIV-1 gp41-derived peptide HGP led to a 38-fold increase of gene expression after plasmid DNA delivery and also enhanced siRNAmediated knockdown of GAPDH by approximately 2-fold [199] . 30 % of cells incubated with PEI-HGP polyplexes showed not only the punctuate plasmid DNA staining observed with PEI polyplexes alone, but also a diffuse fluorescence throughout the cell, indicative of endosomal release of vectors. This would explain the observed increase in transfection efficiency, since the overall uptake was unaffected.
Wadia et al. [62] were the first to use the influenza hemagglutinin-derived fusogenic peptide HA2 in combination with a CPP. Delivery of a Tat-HA2 conjugate with increasing concentrations of a Tat-Cre conjugate enhanced reporter protein activity, presumably through an increased release from macropinosomes. The same fusogenic peptide, linked to a polyarginine-p53 conjugate, promoted release of p53 from macropinosomes and subsequent translocation to the nucleus, accompanied by an enhanced anti-cancer effect [200] .
The development of delivery systems for therapeutic oligonucleotides is a fast growing field. Owing to the enormous potential of short nucleic acids as alternative drugs such a growth is not unexpected. Besides viral vectors there is a highly diverse and constantly increasing number of non-viral systems evolving. However, despite considerable progress achieved in recent years, even the most advanced systems either lack the efficiencies required for downstream drug development or do show a substantial degree of toxicity or both. Of the many factors which limit their use, cellular uptake of the cargo/carrier complexes and subsequent intracellular trafficking to reach the target site are the most important. In addition to such essential considerations there are various additional parameters to be taken into account like serum stability, pharmacokinetic features and tissue barriers as well as target cell specificity. Now the question arises where to start optimizing a given delivery system. Currently, there is a clear trend towards in vivo testing. In principle such a development is a step in the right direction since many of the experimental data derived from artificial cell tissue culture systems with established cell lines are not applicable to the in vivo situation. On the other hand, it is questionable to what extent such animal experiments will eventually pay off as long as important fundamental problems remain largely unsolved. As outlined above, our quantitative studies along with microscopic analyses of siRNAs and steric block oligonucleotides using either a peptide or a commercially availably cationic lipid as carrier clearly show that less than 0.1% -5% of molecules taken up are involved in a biological response, i.e. RNAimediated down regulation or splice correction-mediated up regulation of reporter gene activity. Evidently, the vast majority of internalized cargo never reaches the target. This implies that uptake per se is not the limiting factor here. Although there are no such detailed quantitative numbers available in the literature for other systems, there are countless reports showing that to various degrees this applies to almost any non-viral delivery approach currently available. Taken together, if one would succeed to optimize intracellular trafficking this holds the potential to boost overall efficacy by up to 3 orders of magnitude. Moreover, it is reasonable to assume that such fundamental cellular restrictions can be adequately investigated in tissue culture without the use of animal models. So it might be worthwhile to reconsider the concept of maybe premature in vivo testing by moving backwards one step and first optimizing the systems with regard to intracellular limitations before dealing with the next level of complexity. Accordingly, in vitro model systems like the ones described above for siRNAs or steric block oligonucleotides are valuable tools to study particular aspects of nucleic acid delivery. However, currently available data are based on studies using a variety of different cell lines and techniques, which renders a direct comparison of different delivery approaches impossible. In this context it would be highly desirable to introduce standardized protocols for in vitro testing (e.g. the splice correction system developed by Kole and coworkers [121] ) together with methods for detailed quantitative analyses (e.g. the liquid hybridization protocol [55, 176] ). This would facilitate a direct quantitative comparison of exceedingly diverse approaches on at least the cellular level.
One reason for the problem with intracellular trafficking of oligonucleotides arises from the mode carrier/cargo complexes are taken up by cells. Today it is well established that the majority of these complexes are taken up via endosomal pathways and therefore end up in vesicular compartments from which they have to escape in order to reach their target. Although there are many attempts reported to trigger endosomal escape by various strategies, they either proved to be toxic or did not achieve sustained success. Additionally, there might be a further reason for the encountered difficulties to overcome these intracellular barriers. It is not unreasonable to speculate that during evolution cells might have evolved mechanisms to avoid large amounts of foreign nucleic acids freely floating in the cytoplasm and thus safely contain them in vesicular compartments where they are eventually degraded. Alternatively they might be exported by retrograde transport. Co-evoluting viruses evidently have developed strategies to circumvent such defense mechanisms. So it might be somewhat naive to expect that a rather simple man-made carrier system is capable to efficiently overcome such an intrinsic barrier. Current developments towards more complex and elaborate carrier systems take into account such considerations. In any case it appears there is no simple solution for this problem.
In conclusion, despite significant progress in the field of nucleic acid delivery in vitro as well as in vivo, there still is a long way to go before this will become a standard procedure in the clinic. Certain problems like endosomal escape are known for more than twenty years and still far from being resolved. In order to develop new strategies, more information about intracellular processes involved in nucleic acid trafficking is needed. Moreover, it would be desirable if the field would move from a qualitative description towards a quantitative evaluation preferentially using standardized model systems. This would allow for comparison of different approaches with one another. While animal studies are inevitable in the long run, there still is a lot of room for improvement on the cellular level. So it might be worthwhile to fathom how far we can push the different systems on this level.  Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSA(PAM)), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSA(PAM). Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSA(PAM), indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization. People living in areas of intense transmission of Plasmodium falciparum parasites acquire protective immunity to malaria during childhood, and the bulk of mortality and severe morbidity from P. falciparum malaria is therefore concentrated among young children. Protective immunity acquired in response to P. falciparum exposure appears to be mediated mainly by IgG antibodies specific for variant surface antigens (VSA) that mediate sequestration of infected erythrocytes (IEs) in various tissues (reviewed by Hviid, 2005) . Despite pre-existing protective immunity, women become highly susceptible to P. falciparum infection when they become pregnant, and pregnancyassociated malaria (PAM) is a major cause of mother/ offspring morbidity (Guyatt and Snow, 2001; 2004) . However, in areas of stable P. falciparum transmission, susceptibility to PAM rapidly declines with increasing parity, consistent with acquisition of PAM-specific protective immunity (reviewed by Hviid, 2004) . PAM is caused by P. falciparum-IEs selectively accumulating in the placental intervillous space through VSA PAM-mediated adhesion to chondroitin sulphate A (CSA). VSAPAM differ in several ways from VSA expressed on IEs obtained from males and non-pregnant females. Thus, only VSAPAM mediate binding to CSA in vitro (Fried and Duffy, 1996) and only VSAPAM-expressing IEs are consistently not recognized by IgG in the plasma of P. falciparum-exposed women who have never been pregnant or by IgG in plasma from similarly exposed men (Beeson et al., 1999; Ricke et al., 2000) . These observations, and the fact that plasma levels of VSAPAM-specific IgG increase with increasing parity (Fried et al., 1998; Ricke et al., 2000) , are consistent with evidence that these antibodies are the mediators of protective immunity to PAM (Duffy and Fried, 2003; Staalsoe et al., 2004) .
The molecular identity of VSAPAM remains controversial, although current evidence points to VAR2CSA, an interclonally conserved member of the PfEMP1 molecules encoded by the multigene var family. Thus, transcription of the gene encoding VAR2CSA is increased among CSAadhering and placental isolates, VAR2CSA is exposed on the surface of CSA-adhering IEs (Salanti et al., 2003; 2004; Tuikue Ndam et al., 2005) , and plasma levels of VAR2CSA-specific IgG increase with increasing parity and correlate with protective immunity to PAM (Salanti et al., 2004) . However, the importance of VAR2CSAspecific antibodies relative to antibodies specific for other putative VSA PAM in acquired protective immunity to PAM remains to be established. The clonal analysis of memory B cells represents a powerful tool to dissect the immune response to complex pathogens such as P. falciparum (Lanzavecchia et al., 2006) . In this study, we used an improved Epstein-Barr virus (EBV) immortalization method (Traggiai et al., 2004) to analyse memory B cells from multiparous PAM-exposed women. Frequency analysis and isolation of specific monoclonal antibodies identified polymorphic, linear and conformationdependent epitopes in VAR2CSA as dominant targets of the human memory B-cell response to PAM.
We first used flow cytometry to screen plasma from 27 PAM-exposed and recently pregnant multigravidae for IgG antibodies capable of staining P. falciparum-IEs expressing VSAPAM Ricke et al., 2000) . We selected three donors (one parity 2 and two parity 3 women) with high VSAPAM-specific plasma antibody levels and used frozen peripheral blood mononuclear cells (PBMC) obtained 1 month post-partum. Memory B cells were immortalized with EBV in the presence of CpG oligonucleotides and allogeneic, irradiated PBMC as described (Traggiai et al., 2004) . A total of 5760 replicate cultures of 100 immortalized B cells per well were set up, and after 3 weeks the culture supernatants were screened for their capacity to stain erythrocytes infected with each of three P. falciparum lines. Two of the lines (FCR3-CSA and NF54-VAR2CSA) had been previously selected in vitro to express VSA PAM, characterized by reactivity with IgG from multiparous women and lack of reactivity with IgG from P. falciparum-exposed men ( Fig. 1) (Fried et al., 1998; Beeson et al., 1999; Ricke et al., 2000) . The third line (3D7-SM) was selected to express non-PAM-type VSA equally recognized by IgG from P. falciparum-exposed men and women ( Fig. 1) Jensen et al., 2004) . Supernatants from 105 of the polyclonal B-cell lines stained one or both of the VSAPAM-expressing lines. The frequency of VSAPAM-reactive polyclonal supernatants varied from 6/1920 [0.3% (95% confidence interval: 0.1-0.7%)] to 33/1344 [2.5% (1.8-3.4%)] in the three donors. These results suggest that the frequency of VSAPAM-specific B cells can be high (at least up to 1 in 4000 memory B cells) in recently pregnant multigravidae. The higher memory B-cell frequencies in the present study compared with earlier reports for PfEMP1 (Dorfman et al., 2005) and total P. falciparum antigens (Fievet et al., 1993; Migot et al., 1995) probably reflect the efficient method of B-cell immortalization employed here.
Cloning of EBV-immortalized IgG + B cells from 28 of the VSAPAM-specific lines by limiting dilution resulted in eight clones producing VSAPAM-specific IgG1. Lines were selected for cloning on the basis of their IgG synthesis Flow cytometry analysis of human VSA-specific plasma IgG reactivity with the surface of P. falciparum-IEs. Labelling of FCR3-CSA, NF54-VAR2CSA and 3D7-SM by IgG in individual plasma samples from P. falciparum-exposed pregnant women ( ), from sympatric men (᭢) and from non-exposed adult control donors (᭹) are shown. and growth characteristics. Six of the clones (PAM1.4, PAM2.8, PAM3.10, PAM5.2, PAM6.1, PAM7.5) produced antibodies recognizing antigens on the surface of erythrocytes infected by both the VSAPAM-expressing lines used to screen for antibody specificity (Table 1) . Antibodies from the two remaining clones (PAM4.7 and PAM8.1) only recognized FCR3-CSA. In contrast, none of the monoclonal antibodies recognized the 3D7-SM control line not expressing VSAPAM ( Fig. 2A-C) . Testing of monoclonal antibody reactivity with erythrocytes infected by a panel of additional parasite lines provided further evidence that all were indeed specific for PAM-type VSA expressed on the surface of CSA-adhering IEs (Table 1) . However, the monoclonal antibodies did not all recognize all VSAPAM-expressing lines, probably because the epitopes they recognize are polymorphic. IgG antibodies produced by a control B-cell clone (D7) did not recognize any of the tested parasite lines. Monoclonal antibody recognition patterns for individual parasite lines were tested in parallel, and repeated assessments of recognition patterns yielded consistent results.
The flow cytometry evidence of antibody reactivity with antigens on the surface of IEs expressing VSAPAM and the absence of reactivity with non-PAM-type VSA (Table 1) was confirmed by immunofluorescence microscopy of live IEs ( Fig. 2D and E). Denaturing Western blots of the VSAPAM-expressing sublines yielded single, distinct bands (of similar size for each antibody) when probed with PAM3.10, PAM5.2, PAM6.1 and PAM7.5 monoclonal antibodies ( Fig. 2F , and data not shown). Proteins were not detected when blots were probed with the monoclonal antibodies PAM1.4, PAM2.8 or PAM4.7 (data not shown) despite their reactivity with the surface of intact VSAPAMexpressing IEs (Table 1) , pointing to reactivity with conformation-dependent epitopes. No bands were observed when the monoclonal antibodies were used to probe Western blots of the non-PAM-type VSAexpressing parental lines (Fig. 2F , and data not shown).
PAM8.1 was not tested by Western blotting with IEs expressing VSAPAM, but was tested with VAR2CSAspecific constructs (see below).
The high molecular weight of the proteins detected by Western blotting (Fig. 2F ) suggested that the monoclonal antibodies were specific for members of the so far bestcharacterized family of VSA, PfEMP1 (Leech et al., 1984) . This family includes VAR2CSA (predicted molecular weight: 355 kDa), which is the only PfEMP1 described so far that has the characteristics expected of VSAPAM (Salanti et al., 2003; 2004) . We therefore used a panel of recombinant proteins spanning the entire extracellular b. 3D7 (Walliker et al., 1987) was originally cloned from, and appears genetically identical to, NF54 ( Delemarre and Van der Kaay, 1979) . c. Line used in screening of B-cell supernatants for production of VSAPAM-specific IgG. n.d., not determined.
VAR2CSA-specific IgG in pregnancy-associated malaria 337 part of VAR2CSA from 3D7 ( Fig. 3A ) and FCR3 (data not shown) to examine the antigen specificity of the monoclonal VSAPAM-specific IgG antibodies further. Antibodies PAM2.8, PAM3.10, PAM5.2, PAM6.1 and PAM7.5 tested positive in 3D7-VAR2CSA domain-specific ELISA ( Fig. 3A and Table 2 ), while antibodies PAM2.8, PAM3.10, PAM4.7, PAM5.2 and PAM8.1 tested positive in the FCR3-VAR2CSA ELISA (Table 2) . Control ELISA employing scrambled constructs and constructs from other PfEMP1 not implicated in the pathogenesis of PAM were consistently completely negative (data not shown). VAR2CSA constructs produced in Escherichia coli cells that should promote disulphide bond formation in secreted proteins (Barfod et al., 2006) were also consistently negative in ELISA (data not shown). Each of the VAR2CSA-reactive monoclonal antibodies had absolute specificity for either DBL3-X (PAM2.8, PAM6.1 and PAM8.1; originating from two donors) or DBL5-e 
VAR2CSA-specific IgG in pregnancy-associated malaria 339 (PAM3.10, PAM4.7, PAM5.2 and PAM7.5; also originating from two donors) ( Fig. 3A and Table 2 ). This pattern of reactivity was confirmed when the monoclonal antibodies were used to detect surface-expressed 3D7-VAR2CSA domains on transfected Jurkat cells in a flow cytometry assay (Fig. 3A) . Competition ELISA to examine the epitopes recognized by the DBL3-X and DBL5-e-reactive monoclonal antibodies showed that the DBL3-X-reactive antibodies PAM2.8 and PAM6.1 targeted antigenically distinct epitopes (Fig. 3B ), while two (PAM3.10 and PAM7.5) of the DBL5-e-reactive antibodies appeared to target neighbouring or overlapping epitopes (Fig. 3C) .
Human VAR2CSA DBL3-X-specific monoclonal IgG antibodies recognize epitopes that vary between parasite isolates
The pattern of monoclonal antibody recognition of IEs varied between parasite isolates. This is consistent with the finding that the var2csa sequence is composed of conserved stretches separated by stretches with substantial interclonal diversity (Duffy et al., 2006a; Trimnell et al., 2006) , and the prediction that B-cell epitopes in VAR2CSA DBL3-X locate mainly to polymorphic, surface-exposed parts of VAR2CSA . We therefore cloned and sequenced 43 VAR2CSA DBL3-X domains from placental parasite isolates. A subset of 29 of these domains selected to represent the overall VAR2CSA DBL3-X diversity was expressed as Baculovirus recombinant proteins and used in ELISA to test the specificity of the three VAR2CSA DBL3-X-specific monoclonals. The PAM2.8 antibody reacted with 25, PAM6.1 with eight and PAM8.1 with 20 of the domain variants (Fig. 4A) . A multiple sequence alignment of all the proteins indicated that the main difference between the PAM8.1-negative and -positive proteins was a C-terminal 16-amino-acid stretch that maps to a polymorphic region of 3D7-VAR2CSA DBL3-X, which is predicted to be a surfaceexposed loop (Fig. 4B) . Residues in this region either were deleted in the PAM8.1-negative proteins or had a different amino acid composition compared with the PAM8.1-positive variants (Fig. 4A) . To substantiate this possibility we constructed a chimeric protein where the 16-amino-acid stretch from a PAM8.1-positive domain variant (FCR3) was transferred to the corresponding site in a PAM8.1-negative variant lacking this sequence (3D7) (Fig. 4A, bottom) . The recombinant proteins corresponding to the unmodified FCR3 sequence and the chimeric construct both tested positive in Western blots probed with PAM8.1, in contrast to the recombinant protein representing the authentic 3D7 sequence (Fig. 4C) , thus confirming the predicted position of the PAM8.1 epitope. It was not possible to predict the exact targets of PAM2.8 and PAM6.1 by multiple alignments of the primary sequences.
Human monoclonal antibody PAM1.4 effectively selects for expression of VSA PAM and increased transcription of VAR2CSA
PAM1.4 stained VSAPAM-expressing IEs, but did not yield any bands in Western blots, and did not react with any of the VAR2CSA constructs when tested in ELISA or by flow cytometry (Tables 1 and 2 ). These observations are compatible with recognition by this antibody of a conformational epitope in VAR2CSA, but also with recognition of an unidentified non-VAR2CSA PAM-specific IE surface antigen. To address this question, we tested the ability of PAM1.4 to enrich VSAPAM-expressing IEs in two parasite lines (EJ24 and EJ27) initially expressing non-PAM-type VSA and only marginally recognized by PAM1.4 ( Fig. 5A and B, and data not shown). Although both isolates were originally obtained from the peripheral blood of pregnant women, and thus expected to express VSAPAM, isolates expressing non-PAM VSA -such as EJ24 and EJ27 -are occasionally found (Ofori et al., 2003, and our unpublished data) . Remarkably, a single round of PAM1.4 antibody selection of EJ27 ( Fig. 5C and D) and EJ24 (data not shown) resulted in rapid emergence of IEs uniformly recognized by PAM1.4 and expressing VSAPAM ( Fig. 5C and D). Quantitative real-time polymerase chain reaction (PCR) analysis of the isolates showed increases in var2csa transcription in response to the selection for PAM1.4 reactivity (EJ24: twofold and EJ27: 30-fold). In addition, EJ24 acquired reactivity with the VAR2CSAspecific antibodies PAM2.8, PAM3.10, PAM6.1 and PAM7.5 following selection for PAM1.4 reactivity ( Table 1) . EJ27 did not acquire additional reactivity following PAM1.4 selection, probably because of interclonal C. Western blots of recombinant 3D7-and FCR3-specific VAR2CSA DBL3-X constructs, and of the above-mentioned chimeric construct, probed with loading control antibody V5 (left) and PAM8.1 (right). MW, molecular weight. differences in the VAR2CSA epitopes recognized by the other monoclonal antibodies. Taken together, these findings are consistent with VAR2CSA being the antigenic target of PAM1.4.
We have shown that it is possible to interrogate the memory B-cell repertoire of malaria-immune donors to estimate frequencies of P. falciparum-specific B cells, and to isolate specific monoclonal antibodies with specificity for the VSA repeatedly implicated as the main targets of acquired protective immunity to malaria. We have used this approach to demonstrate that PAM can result in acquisition of high frequencies of B cells producing IgG with specificity for VSA PAM, and in particular VAR2CSA, strengthening previous evidence that these antigen specificities are critically important in acquired protective immunity to PAM. We furthermore show that VSAPAM-specific memory B cells acquired in response to PAM primarily target polymorphic, conformationdependent epitopes that are reproduced by Baculovirusproduced recombinant antigen constructs. Our data thus underscore the importance of VAR2CSA in acquired B. Pre-selection non-PAM VSA-type recognition pattern of EJ27 by IgG in plasma from P. falciparum-exposed men and women and in plasma from non-exposed adults. C. Reactivity of PAM1.4 antibody (heavy line) and negative control antibody (thin line) with the surface of erythrocytes infected by the EJ27 after a single round of selection for reactivity with PAM1.4. D. Post-selection VSAPAM-type recognition pattern of EJ27 by IgG in plasma from P. falciparum-exposed men and women and in plasma from non-exposed adults.
immunity to PAM. However, the findings reported here and elsewhere also suggest that var2csa diversity (Duffy et al., 2006a; Trimnell et al., 2006) is driven by protective immunity to PAM, a situation that may complicate development of VAR2CSAbased vaccines against PAM (Beeson et al., 2006) . IE adhesion to CSA, which is thought to be a critical element in the pathogenesis of PAM (Fried and Duffy, 1996) , is mediated by VAR2CSA as documented by recent knockout studies (Viebig et al., 2005; Duffy et al., 2006b) , and several CSA-adhesive domains have been identified in the antigen (Gamain et al., 2005) . Recent studies in mice suggest that vaccination can elicit broadly reactive antibodies that can block VAR2CSAdependent IE adhesion to CSA (Gamain et al., 2004; Bir et al., 2006) , but whether such antibodies are ever produced in humans in response to PAM, how clinically relevant they are, and whether they can be induced by vaccination in humans remain unanswered questions.
Understandably, present research is highly focused on the identification of functionally constrained epitopes in these domains that are critical for IE adhesion to CSA and of intergenomically conserved epitopes that may serve as targets of antibodies interfering with it. Human monoclonal antibodies appear to be a powerful tool in this research.
All P. falciparum parasites used in this study were grown in 0 + erythrocytes (Cranmer et al., 1997) . 3D7, FCR3 and NF54 are long-term in vitro cultured lines. All expressed non-PAM-type VSA, meaning that intact IEs were recognized to a similar extent by IgG in the plasma of P. falciparum-exposed men and sympatric, multigravid women in a flow cytometry assay of VSA expression (Fig. 1 , 3D7-SM) . The 3D7 subline 3D7-SM was derived by human plasma antibody selection of 3D7 for expression of non-PAM-type PfEMP1 associated with severe malaria in children as described Jensen et al., 2004) . The VSAPAM-expressing subline 3D7-BeWo was selected by repeated panning of IEs on the choriocarcinoma line BeWo as described elsewhere (Haase et al., 2006) . Parasites were considered as expressing VSAPAM if the level of labelling of intact IEs by IgG in a panel of plasma samples from P. falciparum-exposed multigravid women was significantly higher than the level in plasma from sympatric men (Fig. 1, FCR3 -CSA and NF54-VAR2CSA). The characteristics of the plasma IgG recognition pattern of VSAPAM and non-PAM-type VSA have been documented in detail elsewhere (Ricke et al., 2000; Staalsoe et al., 2001) . Sublines of FCR3 and NF54 (FCR3-CSA and NF54-CSA respectively) were selected for expression of VSAPAM by repeated panning of IEs on CSA in vitro (Fried and Duffy, 1996; Ricke et al., 2000) . NF54-CSA was further selected for IE reactivity with rabbit antiserum specific for VAR2CSA DBL5-e, resulting in subline NF54-VAR2CSA (Salanti et al., 2004) . Additional sublines of FCR3 (FCR3-A745 and FCR3-CD36) expressing non-PAM VSA were selected by repeated panning on CSA-negative CHO cells (CHO-A745) and recombinant CD36, respectively, essentially as described for BeWo and CSA selection. Isolates EJ24 and EJ27 were obtained from the peripheral blood of pregnant, P. falciparum-exposed women and adapted to in vitro culture . Both isolates were selected for expression of VSA reacting with the human VSAPAMspecific monoclonal antibody PAM1.4 (see below), essentially as described , but using Protein A-coated magnetic microbeads, as VSAPAM-expressing IEs are prone to non-specific labelling by second-step antisera (Creasey et al., 2003; Rasti et al., 2006) .
Peripheral blood mononuclear cells (PBMC) from P. falciparum-exposed, recently pregnant multiparous women were isolated and cryopreserved as described (Hviid et al., 1993) . At the day of use, PBMC were thawed and IgG + memory B cells were isolated using CD22 microbeads (Miltenyi) followed by cell sorting as described (Traggiai et al., 2004) . Cells were immortalized at 100 cells per well in multiple 96-well plates using EBV in the presence of CpG ODN2006 (Microsynth, Switzerland) (Hartmann and Krieg, 2000) and irradiated PBMC as described (Traggiai et al., 2004) .
Polyclonal B-cell culture supernatants were screened by flow cytometry for IgG reactivity with the surface of intact, unfixed erythrocytes infected by FCR3-CSA, NF54-VAR2CSA and 3D7-SM. VSAPAM-reactive B-cell lines, selected on the basis of their rate of IgG synthesis and growth rates, were cloned by limiting dilution as described (Traggiai et al., 2004) and the selectivity of the human monoclonal antibodies produced by the clones for IEs expressing VSAPAM was confirmed as above. The reactivity of the antibodies with the surface of wet-mounted antibody-labelled IEs was further verified by immunofluorescence microscopy, using an LSM5 scanning microscope (Carl Zeiss MicroImaging) (Salanti et al., 2004) . The IgG subclass of all the human monoclonal antibodies was determined by ELISA and verified by flow cytometry using isotype-specific antibodies (Megnekou et al., 2005) .
Parasite cultures were enriched for erythrocytes infected by late trophozoite/schizont-stage parasites by exposure to a strong magnetic field (Paul et al., 1981; Staalsoe et al., 1999) . Protein extracts of purified IEs were prepared with 2% SDS in PBS containing complete protease inhibitor (Roche, VAR2CSA-specific IgG in pregnancy-associated malaria 343
Basel, Switzerland). The extracts were boiled in denaturing loading buffer and separated in pre-cast tris-acetate 5-8% SDS gradient gels (Invitrogen, Tåstrup, Denmark) with trisacetate running buffer (Invitrogen), employing pre-stained broad-range molecular weight markers (Bio-Rad, Herlev, Denmark) . The separated proteins were transferred to PVDF membranes by wet blotting in transfer buffer containing 20% isopropanol, 20 mM tris-acetate and 0.1% SDS, followed by blocking with 5% skimmed-milk powder in TBS-T buffer. Membranes were incubated with the human monoclonal antibodies or a monoclonal mouse anti-exon 2 antibody, followed by incubation with a secondary anti-human or anti-mouse AP-conjugated antibody (Sigma, MO, USA) and developed using a 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium solution (Sigma). Baculovirus-produced proteins and a pre-stained ProSieve protein marker (Cambrex) were run on pre-cast 4-12% SDS gradient gels (Invitrogen, Tåstrup, Denmark) with NuPage MOPS SDS running buffer (Invitrogen). Proteins were transferred to a nitrocellulose membrane by wet blotting using a buffer of 20% methanol, 25 mM Tris and 192 mM glycine. Following blocking in 5% skimmed-milk powder in TBS-T buffer, membranes were incubated for 1 h with either a 1:5000 dilution of horseradish peroxidase-conjugated loading control antibody anti-V5 (R960-25, Invitrogen) or a 1:1000 dilution of PAM8.1. The PAM8.1-probed membrane was further incubated with a 1:1000 dilution of a secondary anti-human IgG antibody (P0214, Dako Cytomation). Membranes were developed using 3-amino-9-ethyl-carbazole tablets dissolved in acetone, 50 mM sodium acetate and 30% H2O2.
Regions of re-codonized 3D7-var2csa (PFL0030c) and FCR3-var2csa covering the entire exon 1 were subcloned into the pBAD-TOPO vector, transferred with the V5 and HIS tag to the pAcGP67-A transfer vector (BD Biosciences), produced as recombinant proteins in Baculovirus-infected insect cells, and purified as described (Salanti et al., 2004) . We have previously shown that Baculovirus-produced VAR2CSA constructs are conformationally intact, as they induce production of rabbit antisera reactive with native VAR2CSA on the surface of IEs (Barfod et al., 2006) . The following regions (indicated by encoded amino acids numbers) were produced: -1967, N: 1380-1579, O: 1559-1992, Q: 1776-2131, R: 1965-2666, S: 1981-2336, U: 1981-2524, V: 2147-2666, X: 2210-2666 and Y: 2317-2666 . Different variants of DBL3-X were cloned and produced as described . For the cloning of the chimeric construct composed of 5′ 3D7-VAR2CSA DBL3-X and 3′ FCR3-VAR2CSA DBL3-X, we used the primers 5′:
cggaattcGATACAAATGGTGCCTGT and 3′: CATTTCTTT TCATTCTTACCATTATTAGTGCA to generate the 3D7specific, and 5′: AAGAATGAAAAGAAATGTATTAATTC and 3′: atttgcggccgcATATACTGCTATAATCTCC to generate the FCR3-specific part of the chimera. These primers amplify a slightly smaller PCR product than the original primers used for making the FCR3 and 3D7 DBL3-X constructs, and this is reflected in the smaller molecular size of the chimeric construct. The two PCR products were gel-purified and used in a second PCR using the two outer primers to generate a PCR product consisting of 5′ 3D7 and 3′ FCR3, with an EcoRI site and a NotI site. The PCR product was cloned into a modified pAcGP67-A vector (BD Biosciences) and expressed in insect cells as described (Salanti et al., 2004) .
VSAPAM-reactive monoclonal IgG-containing supernatants were tested in ELISA (Dodoo et al., 2000) for reactivity with the recombinant VAR2CSA proteins produced in Baculovirusinfected insect cells. In addition, the epitope specificities of monoclonal antibodies targeting DBL3-X (PAM2.8 and PAM6.1) and DBL5-e (PAM3.10, PAM5.2 and PAM7.5) were analysed by competition ELISA. PAM3.10 and PAM6.1 were purified on ÄktaXpress (GE Healthcare, Brøndby, Denmark) using a HiTrap Protein G HP 1 ml column with subsequent desalting on a HiPrep 26/10 desalting column (GE Healthcare). Purified IgG was biotinylated using EZ-link maleimide-PEO solid phase as described by the manufacturer (Pierce, Bonn, Germany). Microtitre plates (Nunc, Roskilde, Denmark) were coated with recombinant DBL3-X (8.3 mg ml -1 ) or DBL5-e (10.4 mg ml -1 ) in PBS (1 h, 37°C). After blocking of the plates, biotinylated PAM3.1-specific IgG (0.72 mg ml -1 ) or biotinylated PAM6.1-specific IgG (2.5 mg ml -1 ) and increasing concentrations of the competitor monoclonal culture supernatants were added to triplicate wells. PAM1.4 (unknown VSAPAM-specificity) and PAM2.8 (DBL3-X-specific) were added as negative controls to DBL3-X-coated and DBL5-e-coated plates respectively. Bound biotinylated IgG was detected by incubation (1 h, room temperature) of wells with horseradish peroxidase-conjugated streptavidin (1 mg ml -1 , 100 ml well -1 ; Pierce, Bonn, Germany).
The human T-cell line Jurkat (Gillis and Watson, 1980) was cultured in RPMI 1640, supplemented with 25 mM HEPES and L-glutamine (Gibco, Tåstrup, Denmark), 10% FCS, 100 IU ml -1 penicillin and 100 mg ml -1 streptomycin. Two million cells were seeded into each well of a six-well plate and transfected with 3-4 mg of plasmid DNA (see above) and 4 ml of DMRIE-C transfection reagent (Invitrogen) according to the manufacturer's instructions. Within 48 h of transfection, the cells were washed and re-suspended at 1 ¥ 10 6 ml -1 in PBS supplemented with 2% FCS. Cells (1 ¥ 10 5 ) were incubated with human monoclonal antibodies or with a haemagglutinin mouse antibody for 30 min followed by two washes and labelling by secondary FITC-conjugated anti-human IgG or anti-mouse IgG antibody. Flow cytometry analysis was essentially as above.
Recombinant VAR2CSA DBL3-X constructs from 29 genotypically distinct P. falciparum isolates were produced in Baculovirus-infected insect cells and tested in ELISA essentially as described above. The three-dimensional structure of the 3D7-VAR2CSA DBL3-X sequence (PFL0030c, amino acids 1217-1559) was modelled in silico as described elsewhere . Briefly, the crystal structure of EBA-175 F1 (PDB code 1ZRO chain A) (Tolia et al., 2005) , which has 28% sequence identity to the 3D7-VAR2CSA DBL3-X domain, was used as a template. The model was evaluated with respect to locations of conserved cysteine bridges and buried hydrophobic residues in the structures of DBL domains from EBA-175 F1 and F2 (Tolia et al., 2005) and Pk-a DBL (Singh et al., 2005) .
Quantitative real-time PCR was performed on cDNA from unselected and PAM1.4 antibody-selected isolates EJ24 and EJ27 using a Rotorgene thermal cycler system (Corbett Research, Cambridge, UK) and a primer set specific for a highly conserved part of the var2csa DBL-4e domain, targeting all var2csa genes without bias (Salanti et al., 2003; Tuikue Ndam et al., 2005) . Selection-induced changes in var2csa transcription were quantified as described (Salanti et al., 2003) . The OptAIDS project: towards global halting of HIV/AIDS  We face a unique, transitory opportunity in the history of the HIV/AIDS epidemic, because we have collectively pooled money faster than the epidemic has grown [1] . Can we then seize the moment and halt this epidemic now? Most scenarios for the future of HIV/AIDS project modest reductions spread out over decades [2] . The very timescale of such projections, beyond the persistence time of all models, makes them unreliable [3] . Can we do better, quicker?
The OptAIDS project was conceived as a means to address this issue. Its implementation thus far has been twofold: a workshop held in July 2008 and this supplement on the eradication of AIDS. The aims of the project are to address two questions:
1. Can we optimally spend our way out of the HIV/AIDS epidemic?
2. Can we work together to build a World Halting AIDS Model (WHAM) that would permit us to estimate the quickest way to halt HIV/AIDS, monitor our success, and adjust our strategy as we go?
The OptAIDS project grew out of a frustration with existing attempts to tackle the disease. AIDS exceptionalism means that HIV/AIDS is handled differently from other public-health epidemics, which has likely been detrimental [4, 5] . Consequently, much of the funding of HIV/AIDS efforts has been for qualitative observations of the expanding epidemic rather than quantitatively effective intervention.
Although fund accumulation has recently outpaced the epidemic, we argue that plans to spend donor money are too long range in the face of a growing epidemic [6] . Longrange scenarios have no reality to them, so that only shortterm solutions -those that fall within the persistence time of their models -have any possibility of being realistic [3] . Furthermore, disease is a global problem that is only tackled locally [7] ; epidemics cross borders, whereas we fund mostly local or national "solutions".
The OptAIDS project was an outgrowth of the Stop Afghan AIDS project [8] . This project was led by mathematical modellers planning to continuously adapt their models to new data and predicting what data should be collected. The Stop Afghan AIDS project showed how it should be possible to intervene quantitatively in an epidemic. The usefulness of modelling in complex systems is not new. Mathematical models of the economy tell us whether a decrease in income tax will result in an increase in investment or an increase in imported consumer goods. Mathematical models of the atmosphere tell us what the effects of carbon dioxide emissions or of nuclear wars may be. Mathematical modelling is used routinely in such things as aircraft design and the design of traffic systems [9] .
So too, epidemics are quantitative creatures with predictable thresholds. Models that can be adapted to new results and to changes in control policy have been identified as an integral part of disease-control programs [10] . Modelling-led interventions were instrumental in halting the 2001 Foot and Mouth outbreak in the UK [11] . A mathematical model of the dynamics of measles in New Zealand developed in 1996 successfully predicted an epidemic in 1997 and was instrumental in the decision to carry out an intensive immunisation campaign in that year. While the epidemic began some months earlier than anticipated, it was rapidly brought under control, and its impact on the population was much reduced [12] .
The West African Onchocerciasis (river blindness) Control Program successfully used modeling to supplement intervention programs [13] . By using clearly delineated endpoints, these models helped convince donors and the scientific community that the aims of the program were achievable [14] . As a result, mathematical models have retained a role in subsequent policy discussions [15] . Insights from mathematical models during the SARS epidemic helped determine how serious the epidemic might become, as well as the impact of proposed control measures. These models provided important guidance to public-health authorities at a critical time when little other information was available. Insights from the models showed that, if unchecked, the virus could cause a significant epidemic, but that basic epidemiological control measures -patient isolation, contact tracing, etc -could have a substantial impact on the extent of the epidemic. Subsequently, these control measures played a major role in limiting the spread of the 2003 epidemic [16] .
Weather prediction models provide a workable analogy. Such models consist of continually updatable inputs, that must adapt to an enormous array of incoming data [17] . Short-term predictions, especially those associated with discrete, extreme weather events such as floods and hurricanes, have proven useful in supporting emergency management strategies, unlike events such as earthquakes or acid rain, which have longer lead times [18] . Complex mediating models which themselves have explanatory power and which embody techniques of modeling can be refined and passed down to successor models [9] . The virtue of mathematics in such a context is that it forces clarity and precision upon the conjecture, thus enabling meaningful comparison between the consequences of basic assumptions and the empirical facts [19] .
Existing scenarios for HIV control have typically been spread out over two or more decades [20] , which means that the reliability of their predictions is low. The basic concept of OptAIDS is to spend more money up front, effectively, based on the best models and their parameters we can formulate, with the goal being a rapid halt to the epidemic with the fewest additional cases. This means that models can be shorter term and therefore more relia-ble, because we stay within the models' persistence time. OptAIDS emphasises continuous monitoring to check the accuracy and adjust the parameters of the global model. Mathematically, this is an optimal halting problem.
The OptAIDS workshop was the first of its kind: a scientific meeting held simultaneously in both a real world location and also Second Life ® http://secondlife.com, a virtual landscape that allows real-time communication.
The broad topic was the eradication of AIDS using optimal spending models, but this encompasses an enormous number of issues surrounding the AIDS epidemic. Topics covered included the impact of circumcision, the effect of traditional medicine, prevention strategies for countries with nascent epidemics and the difficulties of developing an HIV vaccine.
MITACS http://www.mitacs.ca gave us a Can$10,000 workshop grant for this meeting, which was held on July 29, 2008. Given that this amount would only cover a few airfares, we decided to allow people to participate via Second Life ® . Second Life ® allows the creation of avatars [21] , so that users can participate in the world. Within the virtual world, you can talk to other people's avatars, upload PowerPoint slides and manipulate objects within the environment.
Twenty-two people convened over the day in Toronto (Figure 1) , including ten presenters. The traffic count during the day indicated 500 avatar arrivals in Second Life ® , four of whom were presenters. The workshop was advertised to pertinent groups in Second Life ® and open to the avatar public. The Second Life ® building includes a location where the original slide presentations can be viewed ( Figure 2 ). Two screens were used in Toronto, showing the slides and the audience (represented by avatars), while Second Life ® participants could lecture by having their avatar stand near a virtual screen in Second Life ® . Everyone could hear everyone else. A second virtual screen showed a live camera view of the audience in Toronto (Figure 3) . The all-day meeting had only a few short interruptions for technical reasons. A summary and follow-up discussion was presented at the annual meeting of the Society for Mathematical Biology shortly afterwards.
The four speakers presenting via Second Life ® were located in Poland, Seattle, Denmark and Los Angeles. The technology allowed for interactive discussion, so speakers in Toronto faced questions from Second Life ® participants all around the world, while Second Life ® speakers had their Powerpoint presentations shown on a screen in Toronto (operated by their avatars in Second Life ® and simultaneously by the organisers in Toronto), and faced questions from Toronto and other Second Life ® participants. The size of the turnout in Second Life ® demonstrated the effectiveness of virtual conferencing; many more people were able to attend the conference than would have been feasible otherwise.
The event was covered by the National Post, which reported on the innovative use of Second Life ® in an academic setting. All the presentations remain in Second Life ® http://slurl.com/secondlife/Silver Bog/21/27/22 as posters that can be clicked on by anyone interested. Speakers can be asked to show up personally as avatars to go over the slides.
The aim of this supplement is to discuss AIDS as a global phenomenon and address issues surrounding its eradica-tion. Due to the scale of the epidemic, a great number of sub-issues arise. In thinking of AIDS as a global pandemic, we need to tackle the disease from as many directions as possible. Some of the articles involve mathematical models, others involve a thorough examination of the state of resources, or an understanding of the effect of the disease on society.
This supplement comprises fifteen articles (including this introduction), divided into six themes:
1. History 2. Resources 3. Demographics 4. In-host models 5. Computation 6. Spending our way out of the epidemic Theme 1 comprises an introduction and overview of mathematical modeling [22] , as well as a history of AIDS in Africa and its effects on human development [23] . Theme 2 is concerned with the various resources that comprise our intervention arsenal: the allocation of resources [24] , cost-effectiveness of prevention [25] , antiretroviral pricing [26] , the effects of migration upon availability of health professionals [27] , and the relation- ship between mathematical models and resource allocation [28] .
Theme 3 looks at the effects of demographic changes in China on HIV [29] and the spread of HIV among men who have sex with men [30] . Theme 4 examines in-host modeling -a crucial element in tackling the disease, often overlooked by epidemiologists -by proposing new methods for evaluating the efficacy of antiretroviral treatment [31] and examining antioxidant supplementation as HIV therapy, with a focus on injecting drug users [32] .
Theme 5 looks at using virtual epidemics to understand real ones [33] and develops an epidemic simulator of an agent-based, data-driven disease model [34] . Finally, Theme 6 examines the question at the core of the OptAIDS project: spending our way out of the AIDS epidemic [6] .
The collection of articles in this supplement run the gamut of topics related to HIV/AIDS. They examine the disease from a global perspective, in an attempt to untangle many of the problems associated with the epidemic. However, we view this as a starting point: the next step is for policymakers and the donor community to embrace the idea of global eradication. Only by working together can we combat this disease.
AIDS is the fourth worst infectious disease of all time, resulting in more deaths per day over the past 25 years than occurred on 9/11/2001 [35] . Over 33 million adults are now infected with HIV, many in the developing world, where resources are scarce and infrastructure is struggling under the weight of this burgeoning epidemic. The HIV/ AIDS epidemic is often spoken of in terms of "reducing the spread" [36] , or achieving "sustainable financing" [37] However, this special issue demonstrates that, despite the immensity of the epidemic, eradication is not only possible, it is feasible. The time has come to stop thinking locally and to start acting globally. Early Assessment of Anxiety and Behavioral Response to Novel Swine-Origin Influenza A(H1N1) BACKGROUND: Since late April, 2009, a novel influenza virus A (H1N1), generally referred to as the “swine flu,” has spread around the globe and infected hundreds of thousands of people. During the first few days after the initial outbreak in Mexico, extensive media coverage together with a high degree of uncertainty about the transmissibility and mortality rate associated with the virus caused widespread concern in the population. The spread of an infectious disease can be strongly influenced by behavioral changes (e.g., social distancing) during the early phase of an epidemic, but data on risk perception and behavioral response to a novel virus is usually collected with a substantial delay or after an epidemic has run its course. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the Influenza A(H1N1) outbreak during the first few days of widespread media coverage (April 28 - May 5, 2009). We find that after an initially high level of concern, levels of anxiety waned along with the perception of the virus as an immediate threat. Overall, our data provide evidence that emotional status mediates behavioral response. Intriguingly, principal component analysis revealed strong clustering of anxiety about swine flu, bird flu and terrorism. All three of these threats receive a great deal of media attention and their fundamental uncertainty is likely to generate an inordinate amount of fear vis-a-vis their actual threat. CONCLUSIONS/SIGNIFICANCE: Our results suggest that respondents' behavior varies in predictable ways. Of particular interest, we find that affective variables, such as self-reported anxiety over the epidemic, mediate the likelihood that respondents will engage in protective behavior. Understanding how protective behavior such as social distancing varies and the specific factors that mediate it may help with the design of epidemic control strategies. An ongoing outbreak of novel influenza A(H1N1), colloquially referred to as ''swine flu,'' has caused over 200,000 confirmed cases (as of 28 August 2009 [1] ). Because of under-reporting, the actual number of people infected is substantially larger, particularly considering that many countries have ceased testing for A(H1N1) [1] . As human-to-human transmission became widespread in at least one region of the world, WHO rapidly declared the outbreak an imminent pandemic [2] and with widespread human infection, WHO declared a phase 6 pandemic on 11 June 2009, where it remains at the time of submission [3] . The virus appears to have a higher reproduction number and somewhat higher case fatality ratio than recent seasonal influenza viruses [4, 5] , and has certainly caused great concern in the population, fueled by both extensive media coverage and an initially high level of uncertainty about mortality rates and transmissibility of the virus.
Mathematical and computational models are useful for predicting the fate of an epidemic, and while such models have become increasingly complex and realistic in recent times, a key ingredient is often ignored: human behavioral responses to the threat and/or presence of a disease [6] . How people assess risk of infection and how such risk assessment drives behavioral change is of great interest as individual social distancing can greatly affect the spread of an epidemic [7, 8, 9] . While the effect of behavioral change in response to perceived health threats on the spread of infectious diseases has been investigated theoretically for some time, particularly in the context of sexually transmitted diseases [8] , recent work has started addressing the problem in a broader context that is also applicable to the spread of influenza [6, 7] . This work has a strong, though as yet under-explored relationship to work on risk perception and health threats [10, 11, 12] .
Data on risk perception and behavioral response in the general population have rarely been collected right from the outset of an epidemic. Instead, they are usually gathered with a substantial delay in the case of influenza A(H1N1) [13] , after the epidemic has run its course, as in the case of SARS [9] , or before sustained human to human transmission is established, as in the case of highly pathogenic avian influenza A(H5N1) [14] . However, the feasibility of halting or mitigating the spread of an infectious disease is highest during the very early phases of an outbreak, and thus data on behavioral response during this time would provide valuable information for public health policy and research. Here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the outbreak during the first few days of widespread media coverage (April 29 -May 5, 2009) of the emergence of novel swine-origin Influenza A(H1N1).
The research presented here was approved by the Stanford University Non-Medical Human Subjects Institutional Review Board on 28 April 2009 (Protocol #16782).
We fielded in internet-based survey starting on 29 April 2009 using Opinio survey software [15] . The URL for the survey is (https://opinio.stanford.edu/opinio/s?s = 1403). The sampling universe for this study was adults 18 and older with access to a networked computer. The initial seed for the sample was generated using social networking software, and a request sent to a standing subject pool comprised of Stanford alumni and social science students at a nearby community college maintained by the Institute for Research in the Social Sciences at Stanford University. The survey was picked up by a variety of internet media sources including several science general media blogs. Directly following publicity in these blogs, we received the most responses. Table 1 summarizes the sample.
The survey was designed to get a rapid assessment of respondents' affective state, sources of information on the emerging pandemic, and the behaviors undertaken for protection while minimizing respondent burden. As such, it included only 17 questions.
Questions probing subjective assessment of risk perception, level of anxiety, and ability to avoid flu infection were asked on a 9 point ordinal scale with anchors at the extrema (''very high'', ''very low'') and the center (''intermediate''). Subjective emotional status (i.e., respondents' affective state regarding the epidemic) was anchored by the terms ''very calm'' through ''intermediate'' to ''very anxious.'' Comparative questions of subjective risk percep-tion for eight health threats were asked using a five-point ordinal scale with anchors at all points: ''very low,'' ''low,'' ''intermediate,'' ''high,'' and ''very high.'' Questions regarding media (both respondents' frequency of getting information from a particular source and their judgment of each source's accuracy) were asked on a five point ordinal scale with anchors at all points (''very often/ accurate'' to ''never/almost certainly inaccurate''). Respondents' knowledge of swine flu was assessed with a series of six True/False questions. Respondents gave free-text responses to questions about their current age, the number of people currently living in their household (including themselves) and their zip code if they currently live in the United States. Respondents who reported not currently living in the United States were asked to report their current country of residence in a free-text field. A screen-shot of the full survey instrument is included in the Supplementary Material.
For our analysis of participants' response to the threat of swine flu, we use a variable we call ''survey day.'' The survey went online in the evening of 28 April, Pacific time, so we combined responses from 28 and 29 April into a single day. This combined day of 28-29 April represents survey day 1.
Subjects were asked to state the number of contacts in the past 24 hours. Contacts were defined by close physical contact as operationalized by a face to face conversation of more than two words in the presence of another individual or physical exposure involving skin contact such as a handshake, hug, or contact during sporting activities. Respondents were provided five ordered categories: less than 5, 5-10, 11-20, 21-50, 51-100, more than 100. Handcock and Jones [16] discuss the phenomenon of heaping and related problems for statistical inference in answering epidemiological questions regarding contact number. Structuring responses within broad ordinal categories avoids many of the pitfalls of contact-heaping encountered in epidemiological investigations.
To measure the response in epidemiologically relevant behavior to information on the potential pandemic, we asked a series of questions about protective actions taken by the respondents. In the survey, we asked: ''Given the current status of the epidemic, which of the following precautionary actions will you take?'' Avoid people who cough/sneeze Avoid large gatherings of people Wash hands more often Avoid people who are in contact with infected people Avoid public transportation Avoid school/work Avoid travel to infected areas Use disinfectant Wear a mask Not all of these behaviors are necessarily effective or recommended protective measures (e.g., wearing a mask), but our aim was to gauge people's attempt at self protection so even non-efficacious behavioral change is interesting in that it indicates willingness to act on the part of the respondent.
We constructed an index of protective behavior by summing the answers to the questions regarding actions taken to avoid influenza infection. The index ranged from 0-9, with larger values indicating more protective measures taken. Using a binomial GLM with canonical logit-link, we modeled the protection index as a function of covariates. Our primary interest was the possible mediating effect of affective variables on action taken to protect against swine flu infection. To evaluate the hypothesis that respondents' affective state (subjective anxiety, fatalism about infection) predicts protective measures, we include in the model demographic (age, gender), epidemiological (household size, number of contacts, survey day), and media (source of information on the outbreak) conditioning variables. For the media variables, we constructed dummies with a value of 1 corresponding to answers of ''very often'' or ''often'' and a value of 0 for all other responses to the question of ''How often do you use the following sources to get information about swine flu?'' With such a large number of conditioning variables, in addition to the affective variables of greatest interest, there is a distinct danger of overfitting the GLM. To address this problem, we used likelihoodbased model selection [17] to search the model space set up by our conditioning variables [18] .
Of the nine protective behaviors, increased hand-washing is both the simplest and probably most effective at curbing transmission. As such, it is strongly advocated in infection control educational material [19] . In addition to our tests for predictors of the protection index, we therefore also tested the effect of measured covariates on the odds of increased hand washing using a binomial GLM again with canonical logit-link.
A concern regarding the relationship of people's self-reported anxiety and their protective behavior is that some people might generally be more anxious regarding health. We probed general anxiety by asking about respondents' anxiety with regard to a number of infectious, chronic, and violent threats to health. We asked a series of questions probing respondents' perceived subjective risk on a 5-point scale for a variety of health threats, including other infectious diseases (A(H5N1) ''bird flu'', seasonal flu, HIV/AIDS), chronic diseases (heart disease, diabetes, cancer), and violence (unintentional accidents, terrorism). We calculated the correlation matrix for answers to these threat questions and used Principal Components Analysis (PCA) to explore potential structure in the responses to different categories of threat [20] .
We begin by presenting descriptive results of the survey and follow with our primary analytical questions from the survey, namely, testing the hypothesis that respondents' affective state mediates their protective action.
We gathered 6,249 responses from 28 April to 5 May 2009. Table 1 presents descriptive statistics of the sample. Figure 1 presents the distributions of respondents' contacts within the 24 hours prior to taking the survey. Figure 2 presents the means of the subjective threats. Swine flu had a mean second only to injury, and the highest among the infectious sources of threat. The mean of perceived threat from swine flu fell above the Bonferroni-corrected 95% confidence interval for all other threats but unintentional injury. Figure 3 presents the frequency distribution of perceived personal risk. There is a notable bimodality to this plot. This apparent bimodality is not simply attributable to sampling error since the difference between the responses = 4 vs. those = 5 vs. those = 6 is in excess of 300. Further analysis using finite mixture models [21] provides strong statistical support for the reality of the bimodal pattern (results not shown). While the majority of respondents felt that their personal risk was low, there is a second mode rating their risk as intermediate ( = 5) . This same bimodal pattern can be seen in the frequency distribution of personal empowerment (i.e., ability to avoid infection) shown in figure 4 . While most respondents indicate that they are confident they can avoid infection, a substantial second mode appears at the intermediate value. Figure 5 shows the frequency distribution of protective behaviors. We can see that nearly 80% of respondents report washing hands more frequently, while very few avoid work or school or wear protective masks. Figure 6 shows the means for respondents' information sources. Not surprisingly, the most common source of information reported was the Internet. Again, mean values are plotted with their 95% Bonferroni-corrected confidence intervals. With the exception of social-networking tools (e.g., Facebook, Twitter), all other media sources are statistically indistinguishable from each other, with the social-networking tools being used significantly less.
The results of the model for the protection index show a number of robust trends (table 2). In particular, we find that age increases and male gender decreases the protection index.
Receiving a large amount of information from the internet, television, and health officials all increase the protection index while receiving large amounts of information from print media, friends, or social networking media has no effect. The number of household members has no discernible effect, though the number of contacts outside the home does. For the ordered factor ''contacts,'' the first category (,5 contacts in the past 24 hours) is the reference category. Interestingly, relative to respondents with the fewest number of contacts, all other contact categories have reduced protection indices, indicating that people with fewer contacts take more protective actions. Not surprisingly, residence in Mexico has a large positive effect, while residence in Canada or Europe decreased the index. The day that the survey was taken (29 April = 1) had a negative effect on the index, indicating that respondents took less protective action as the epidemic proceeded. Respondents' reported subjective anxiety has a substantial impact on the index with high anxiety increasing protection, supporting our hypothesis that affective state mediates protective behavior.
Increased hand-washing showed similar trends to the model for the protection index (table 3) . Male gender decreases while age and survey day increase the odds of increasing hand-washing.
Receiving a large amount of information from the internet, radio, television, and health officials increase, while living in Europe or Australia/New Zealand decrease the odds. As with the overall protection index, perception of risk and subjective anxiety significantly increase the odds of increased hand-washing modestly. 
A key epidemiological question is how people's affective status and protective behaviors undertaken change as the epidemic proceeds. To develop a measure for this, we cross-tabulated individual values of the protection index and affective status by survey day. Pearson's chi-square test for independence of both tables was strongly significant (affective: x 2 = 135.6, df = 48, p,0.001; protection: x 2 = 113.1, df = 54, p,0.001), indicating substantial departure of cells from the expected values. To visualize the pattern of departure from the expected values, we calculated an expected tables taken as the cross-product of the marginals of the observed table normalized by the grand sum. We combined rows of these tables to simplify the presentation, plotting the difference between observed and expected tables for a high, medium and low emotional status/protection index respectively. For example, a value of 251 on the calm affective status on day one means that there were 51 fewer responses in the calm categories than would be expected by the overall marginal distribution of responses across all days.
In figures 7 and 8, we plot the change in respondent's protective behavior and emotional status over the first week of the survey. The lines represent the differences between observed and expected frequencies of responses for the 9-point scale simplified to three levels each. We see that by day three of the survey (May 1st), the relative number of people reporting a calm emotional state was very high, while the number of people reporting high values of the protective index declined dramatically. We interpret these results to indicate that people's response to a potential pandemic is quite sensitive to media reports.
In general, individuals' survey responses to perceived risk for the eight health threats were only moderately correlated, with pairwise correlations typically well under 0.5. PCA did not reveal that a substantially reduced number of dimensions explained these correlated data -six principal components were required to explain 85% of the variance. Nonetheless, some intriguing PC loadings revealed themselves. In particular, the second PC, which explained 15.6% of the variance in the data, showed strong positive correlations with swine flu (r = 0.516), bird flu (r = 0.530), and terrorism (r = 0.467). All three of these threats receive a great deal of media attention and their fundamental uncertainty are likely to generate an inordinate amount of fear vis-a-vis their actual threat [22] .
Our results indicate that respondents' behavior varies systematically with covariates from demographic, epidemiological, media, and affective domains. People's anxiety about swine flu and the preventative actions they took to avoid infection declined as the perceived gravity of the novel outbreak waned. Overall, subjective risk perception was low and people's belief in their ability to avoid infection was high. Both of these distributions nonetheless showed a marked bimodality, with a large proportion of respondents indicating a higher subjective risk and more protective actions taken than the majority (Figures 3-4) .
The results of our statistical modeling suggest that respondents' deployment of protective behavior is mediated by their subjective anxiety level, controlling for demographic, epidemiological, and geographic variables. There is good and bad news in this result. The literature on risk perception and public health shows that there is generally a very weak correlation between people's anxiety over a particular risk and the probability of death or disability arising from that risk [11, 12] . Overall, it is unclear whether anxiety over perceived risk will lead to efficacious protective behaviors [10] . This said, by far the most common protective behavior reported in our survey was increased hand-washing, which has been shown to be effective at removing Influenza A(H1N1) virus from subjects' hands [23] and is promoted by CDC and other health organizations as an effective infection control intervention [19] .
One curious result from the model for the protection index is that people with the fewest contacts have marginally higher protection indices. There are two potential explanations for this finding: (1) individuals with small social support networks (and consequently, few contacts outside the home) are more anxious, making it more likely that they will take greater protective actions or (2) people concerned about infection and taking relatively many protective actions also reduce the number of contacts they have and therefore had a small number of contacts in the past 24 hours. The first explanation is consistent with work in social epidemiology on the role of social networks in mediating infection risk [24, 25] Because of the nature of the sample, we are unable to evaluate the direction of causality that leads to this result. Nonetheless, it remains an intriguing hypothesis.
Many questions about this Novel Swine-Origin influenza A(H1N1) virus remain. Of particular concern is the possibility that the virus could mutate during the flu season in the southern hemisphere and selection could drive it to become more virulent as it returns to the northern hemisphere in Autumn. Worryingly, such a pattern of multiple waves with an increased proportion of the total influenza-associated mortality burden has been reported for all three past influenza pandemics [26, 27] . Finding a means to simultaneously communicate to the public the structural uncertainty inherent in projecting pandemics and the seriousness of a pandemic after the media frenzy about its emergence has died down remains a major challenge to public health.
Pharmaceutical interventions such as vaccines and antiviral drugs may form a strong line of defense, but the efficacy of such measures remains unclear and depends on the particular biology of a given pathogen. This is exacerbated by people's reluctance to be vaccinated [28] . With more than 300 infectious diseases emerging within less than a century [29] , the threat of pandemic influenza is only the latest out of many public health threats posed by infectious diseases in a globalized world. Unlike pharmaceutical interventions, non-pharmaceutical interventions like social distancing may be effective in halting or at least mitigating an epidemic independent of the specific biology of a pathogen, and thus provide a reliable set of strategies to combat infectious diseases that warrant further attention [30] . Our results that people do not rely on social networking tools to the extent that they rely on other media may have implications for CDC strategies for spreading public health information via social media [19] . In particular, public health messages spread via social media will need to backed up by information spread via more traditional channels, which respondents list as being common sources of trusted information on the outbreak. Our study is subject to a number of weaknesses. The advantage of our internet-based sampling strategy is the ability to quickly deploy a survey and thereby track responses in near real-time. The clear disadvantage of this strategy is a sacrifice of population representativeness. Despite its general availability on the internet, our sample shows a pronounced bias for highlyeducated respondents living in the Western United States. These biases clearly limit the generality of our results, but the large number of respondents filing out the survey as information on the potential pandemic changed nonetheless provides a uniquely valuable data source. Within one week (the cutoff point for the current analysis), we had a sample of 6,249 responses. In contrast, the telephone-based study of Rubin et al. [13] employed a random-digit-dial sampling design, allowing a more representative sample of the general UK population, but their sample was only 997 respondents and the survey was undertaken after media attention had abated, beginning 8 May 2009. Nonetheless, the results reported in this paper are largely congruent with our own results and we see the studies as strongly complementary.
Our respondents began filling out the survey on the day that WHO upgraded the pandemic threat category of the H1N1 outbreak from 4 to 5 and spans the week in which the number of WHO-confirmed cases increased tenfold. While our sampling design is subject to many of the usual criticisms of internet-based surveys and is not necessarily representative of the general population, the unparalleled immediacy, longitudinal nature, and the large number of respondents it contains make our data set unique and scientifically important for the study of the spread of information and distribution of risk perception and behavioral change during the most uncertain time (i.e. the initial phase) of an epidemic of a virus novel to the human population.  Accurate noise projection for reduced stochastic epidemic models We consider a stochastic susceptible-exposed-infected-recovered (SEIR) epidemiological model. Through the use of a normal form coordinate transform, we are able to analytically derive the stochastic center manifold along with the associated, reduced set of stochastic evolution equations. The transformation correctly projects both the dynamics and the noise onto the center manifold. Therefore, the solution of this reduced stochastic dynamical system yields excellent agreement, both in amplitude and phase, with the solution of the original stochastic system for a temporal scale that is orders of magnitude longer than the typical relaxation time. This new method allows for improved time series prediction of the number of infectious cases when modeling the spread of disease in a population. Numerical solutions of the fluctuations of the SEIR model are considered in the infinite population limit using a Langevin equation approach, as well as in a finite population simulated as a Markov process. The interaction between deterministic and stochastic effects in population dynamics has played, and continues to play, an important role in the modeling of infectious diseases. The mechanistic modeling side of population dynamics is well known and established. 1, 2 These models typically are assumed to be useful for infinitely large, homogeneous populations, and arise from the mean field analysis of probabilistic models. On the other hand, when one considers finite populations, random interactions give rise to internal noise effects, which may introduce new dynamics. Stochastic effects are quite prominent in finite populations, which can range from ecological dynamics 3 to childhood epidemics in cities. 4, 5 For homogeneous populations with seasonal forcing, noise also comes into play in the prediction of large outbreaks. [6] [7] [8] Specifically, external random perturbations change the probabilistic prediction of epidemic outbreaks as well as its control. 9 When geometric structure is applied to the population, the interactions are modeled as a network. 10, 11 Many types of static networks which support epidemics have been considered. Some examples include small world networks, 12 hierarchical networks, 13 and transportation networks of patch models. 14 In addition, the fluctuation of epidemics on adaptive networks, where the wiring between nodes changes in response to the node information, has been examined. 15 In adaptive network models, even the mean field can be high dimensional, since nodes and links evolve in time and must be approximated as an additional set of ordinary differential equations.
Another aspect of epidemic models, which is often of interest, involves the inclusion of a time delay. The delay term makes the analysis significantly more complicated. However, it is possible to approximate the delay by creating a cascade consisting of a large number of compartments. 16 For example, one could simulate the delay associated with a disease exposure time with several hundred "exposed" compartments.
These model examples are just a few of the types of very high-dimensional models that are currently of interest. As a result of the high dimensionality, there is much computation involved, and the analysis is quite difficult. In particular, real-time computation is not currently possible. However, there are usually many time scales that are well separated ͑due typically to a large range in order of magnitude of the parameters͒ when considering such high-dimensional problems. In the presence of well-separated time scales, a model reduction method is needed to examine the dynamics on a lower-dimensional space. It is known that deterministic model reduction methods may not work well in the stochastic realm, which includes epidemic models. 17 The purpose of this article is to examine a method of nonlinear, stochastic projection so that the deterministic and stochastic dynamics interact correctly on the lower-dimensional manifold and predict correctly the dynamics when compared with the full system. Because the noise affects the timing of outbreaks, it is essential to produce a low-dimensional system that captures the correct timing of the outbreaks as well as the amplitude and phase of any recurrent behavior.
We will demonstrate that our stochastic model reduction method properly captures the initial disease outbreak and continues to accurately predict the outbreaks for time scales, which are orders of magnitude longer than the typical relaxation time. Furthermore, in practice, real disease data include only the number of infectious individuals. Our method allows us to predict the number of unobserved exposed individuals based on the observed number of infectious individuals.
For stochastic model reduction, there exist several potential methods for general problems. For a system with certain spectral requirements, the existence of a stochastic center manifold was proven in Ref. 18 . Nonrigorous stochastic normal form analysis ͑which leads to the stochastic center mani-fold͒ was performed in Refs. [19] [20] [21] [22] . Rigorous theoretical analysis of normal form coordinate transformations for stochastic center manifold reduction was developed in Refs. 23 and 24. Later, another method of stochastic normal form reduction was developed, 25 in which any anticipatory convolutions ͑integrals into the future of the noise processes͒ that appeared in the slow modes were removed. Since this latter analysis makes the construction of the stochastic normal form coordinate transform more transparent, we use this method to derive the reduced stochastic center manifold equation. Figure 1 shows a schematic demonstrating our approach to the problem. We consider a high-dimensional system along with its corresponding reduced low-dimensional system. In this article, two types of low-dimensional system are discussed: a reduced system based on deterministic center manifold analysis and a reduced system based on a stochastic normal form coordinate transform. Regardless of the type of low-dimensional system being considered, a common noise is injected into both the high-dimensional and lowdimensional models, and an analysis of the solutions found using the high and low-dimensional systems is performed.
In this article, as a first study of a high-dimensional system, we consider the susceptible-exposed-infected-recovered ͑SEIR͒ epidemiological model with stochastic forcing. As previously mentioned, we could easily consider a SEIR-type model where the exposed class was modeled using hundreds of compartments. Since the analysis is similar, we consider the simpler standard SEIR model to demonstrate the power of our method. Section II provides a complete description of this model. Section III describes how to transform the deterministic SEIR system to a new system that satisfies the spectral requirements needed to apply the center manifold theory. After the theory is used to find the evolution equations that describe the dynamics on the center manifold, we show in Sec. IV how the reduced model that is found using the deterministic result incorrectly projects the noise onto the center manifold. Section V demonstrates the use of a stochastic normal form coordinate transform to correctly project the noise onto the stochastic center manifold. A discussion and the conclusions are contained, respectively, in Secs. VI and VII.
We begin by describing the stochastic version of the SEIR model found in Ref. 26 . We assume that a given population may be divided into the following four classes that evolve in time:
͑1͒ Susceptible class s͑t͒ consists of those individuals who may contract the disease. ͑2͒ Exposed class e͑t͒ consists of those individuals who have been infected by the disease but are not yet infectious. ͑3͒ Infectious class i͑t͒ consists of those individuals who are capable of transmitting the disease to susceptible individuals. ͑4͒ Recovered class r͑t͒ consists of those individuals who are immune to the disease.
Furthermore, we assume that the total population size, denoted as N, is constant and can be normalized to S͑t͒ + E͑t͒ + I͑t͒ + R͑t͒ = 1, where S͑t͒ = s͑t͒ / N, E͑t͒ = e͑t͒ / N, I͑t͒ = i͑t͒ / N, and R͑t͒ = r͑t͒ / N. Therefore, the population class variables S, E, I, and R represent fractions of the total population. The governing equations for the stochastic SEIR model are Schematic demonstrating the injection of a common noise into both the high-dimensional system and its associated low-dimensional system.
where i is the standard deviation of the noise intensity D i = i 2 / 2. Each of the noise terms i describes a stochastic, Gaussian white force that is characterized by the correlation functions
Additionally, represents a constant birth and death rate, ␤ is the contact rate, ␣ is the rate of infection, so that 1 / ␣ is the mean latency period, and ␥ is the rate of recovery, so that 1 / ␥ is the mean infectious period. Although the contact rate ␤ could be given by a time-dependent function ͑e.g., due to seasonal fluctuations͒, for simplicity, we assume ␤ to be constant. Throughout this article, we use the following parameter values: = 0.02͑yr͒ −1 , ␤ = 1575.0͑yr͒ −1 , ␣ =1/ 0.0279͑yr͒ −1 , and ␥ =1/ 0.01͑yr͒ −1 . Disease parameters correspond to typical measles values. 26, 27 Note that any other biologically meaningful parameters may be used as long as the basic reproductive rate R 0 = ␣␤ / ͓͑␣ + ͒͑␥ + ͔͒ Ͼ 1. The interpretation of R 0 is the number of secondary cases produced by a single infectious individual in a population of susceptibles in one infectious period.
As a first approximation of stochastic effects, we have considered additive noise. This type of noise may result from migration into and away from the population being considered. 28 Since it is difficult to estimate fluctuating migration rates, 29 it is appropriate to treat migration as an arbitrary external noise source. Also, fluctuations in the birth rate manifest itself as additive noise. Furthermore, as we are not interested in extinction events in this article, it is not necessary to use multiplicative noise. In general, for the problem considered here, it is possible that a rare event in the tail of the noise distribution may cause one or more of the S, E, and I components of the solution to become negative. In this article, we will always assume that the noise is sufficiently small so that a solution remains positive for a long enough time to gather sufficient statistics. Even though it is difficult to accurately estimate the appropriate noise level from real data, our choices of noise intensity lie within the huge confidence intervals computed in Ref. 29 . The case for multiplicative noise will be considered in a separate paper.
Although S + E + I + R = 1 in the deterministic system, one should note that the dynamics of the stochastic SEIR system will not necessarily have all of the components sum to unity. However, since the noise has zero mean, the total population will remain close to unity on average. Therefore, we assume that the dynamics is sufficiently described by Eqs. ͑1a͒-͑1c͒. It should be noted that even if E͑t͒ + I͑t͒ = 0 for some t, the noise allows for the reemergence of the epidemic.
One way to reduce the dimension of a system of equations is through the use of deterministic center manifold theory. In general, a nonlinear vector field can be transformed so that the linear part ͑Jacobian͒ of the vector field has a block diagonal form where the first matrix block has eigenvalues with positive real part, the second matrix block has eigenvalues with negative real part, and the third matrix block has eigenvalues with zero real part. 30, 31 These blocks are associated, respectively, with the unstable eigenspace, the stable eigenspace, and the center eigenspace. If we suppose that there are no eigenvalues with positive real part, then orbits will rapidly decay to the center eigenspace.
In order to make use of the center manifold theory, we must transform Eqs. ͑1a͒-͑1c͒ to a new system of equations that has the necessary spectral structure. The theory will allow us to find an invariant center manifold passing through the fixed point to which we can restrict the transformed system. Details regarding the transformation can be found in Sec. III A, and the computation of the center manifold can be found in Sec. III B.
Our analysis begins by considering the governing equations for the stochastic SEIR model given by Eqs. ͑1a͒-͑1c͒. We neglect the i i ͑t͒ terms in Eqs. ͑1a͒-͑1c͒ so that we are considering the deterministic SEIR system. This deterministic system has two fixed points. The first fixed point is
and corresponds to a disease free or extinct equilibrium state. The second fixed point corresponds to an endemic state and is given by
To ease the analysis, we define a new set of variables, S, Ē , and Ī, as S͑t͒ = S͑t͒ − S 0 , Ē ͑t͒ = E͑t͒ − E 0 , and Ī͑t͒ = I͑t͒ − I 0 . These new variables are substituted into Eqs. ͑1a͒-͑1c͒.
Then, treating as a small parameter, we rescale time by letting t = . We may then introduce the following rescaled parameters: ␣ = ␣ 0 / and ␥ = ␥ 0 / , where ␣ 0 and ␥ 0 are O͑1͒. The inclusion of the parameter as a new state variable means that the terms in our rescaled system which contain are now nonlinear terms. Furthermore, the system is augmented with the auxiliary equation d / d = 0. The addition of this auxiliary equation contributes an extra simple zero eigenvalue to the system and adds one new center direction that has trivial dynamics. The shifted and rescaled augmented system of equations is
where the endemic fixed point is now located at the origin. The Jacobian of Eqs. ͑5a͒-͑5d͒ is computed to zeroth order in and is evaluated at the origin. Ignoring the components, the Jacobian has only two linearly independent eigenvectors. Therefore, the Jacobian is not diagonalizable. However, it is possible to transform Eqs. ͑5a͒-͑5c͒ to a block diagonal form with the eigenvalue structure that is needed to use the center manifold theory. We use a transformation matrix P consisting of the two linearly independent eigenvectors of the Jacobian along with a third vector chosen to be linearly independent. There are many choices for this third vector; our choice is predicated on keeping the vector as simple as possible. This transformation matrix is given as
Using the fact that ͑S , Ē , Ī͒ T = P · ͑U , V , W͒ T , then the transformation matrix leads to the following definition of new variables: U, V, and W,
The application of the transformation matrix to Eqs. ͑5a͒-͑5c͒ leads to the transformed evolution equations given by
The Jacobian of Eqs. ͑8a͒-͑8d͒ to zeroth order in and evaluated at the origin is
which shows that Eqs. ͑8a͒-͑8d͒ may be rewritten in the form
where x = ͑U͒, y = ͑V , W͒, A is a constant matrix with eigenvalues that have negative real parts, B is a constant matrix with eigenvalues that have zero real parts, and f and g are nonlinear functions in x, y, and . In particular,
Therefore, the system will rapidly collapse onto a lowerdimensional manifold given by center manifold theory. 32 Furthermore, we know that the center manifold is given by
where h is an unknown function. Substitution of Eq. ͑14͒ into Eq. ͑8a͒ leads to the following center manifold condition:
In general, it is not possible to solve the center manifold condition for the unknown function h͑V , W , ͒. Therefore, we perform the following Taylor series expansion of h͑V , W , ͒ in V, W, and :
where h 0 , h 2 , h 3 , h ,¯are unknown coefficients that are found by substituting the Taylor series expansion into the center manifold condition and equating terms of the same order. By carrying out this procedure using a second-order Taylor series expansion of h, the center manifold equation is
where ⑀ = ͉͑V , W , ͉͒ so that ⑀ provides a count of the number of V, W, and factors in any one term. Substitution of Eq. ͑17͒ into Eqs. ͑8b͒ and ͑8c͒ leads to the following reduced system of evolution equations, which describe the dynamics on the center manifold
We now return to the stochastic SEIR system given by Eqs. ͑1a͒-͑1c͒. The shift in the fixed point to the origin will not have any effect on the noise terms, so that the stochastic version of the shifted equations is
As Eqs. ͑19a͒-͑19c͒ are transformed using Eqs. ͑7a͒-͑7c͒, the ␣ = ␣ 0 / scaling, the ␥ = ␥ 0 / scaling, and the t = time scaling, the noise also is scaled so that the stochastic transformed equations are given by
where 043110-5 Noise projection for epidemic models Chaos 19, 043110 ͑2009͒
The stochastic terms 4 , 5 , and 6 in Eqs. ͑20a͒-͑20c͒ are still additive Gaussian noise processes. However, Eqs. ͑21a͒-͑21c͒ show how the transformation has acted on the original stochastic terms 1 , 2 , and 3 to create new noise processes, which have a variance different from that of the original noise processes. Also note that we have suppressed the argument of 4 , 5 , and 6 in Eqs. ͑20a͒-͑20c͒. The time scaling means that these noise terms should be evaluated at .
The system of equations given by Eqs. ͑20a͒ and ͑21c͒ is an exact transformation of the system of equations given by Eqs. ͑1a͒-͑1c͒. We numerically integrate the original stochastic system of the SEIR model ͓Eqs. ͑1a͒-͑1c͔͒ along with the transformed stochastic system ͓Eqs. ͑20a͒-͑20c͔͒ using a stochastic fourth-order Runge-Kutta scheme with a constant time step size. The original system is solved for S, E, and I, while the transformed system is solved for U, V, and W. In the latter case, once the values of U, V, and W are known, we compute the values of S, Ē , and Ī using the transformation given by Eqs. ͑7a͒-͑7c͒. We shift S, Ē , and Ī, respectively, by S 0 , E 0 , and I 0 to find the values of S, E, and I. Figure 2 compares the time series of the fraction of the population that is infected with a disease I, computed using the original stochastic system of equations of the SEIR model with the time series of I computed using the transformed stochastic system of equations of the SEIR model. Although the two time series shown in Fig. 2 generally agree very well, there is some discrepancy. This discrepancy is due to the fact that the noise processes 4 4 , 5 5 , and
It is tempting to consider the reduced stochastic model found by substitution of Eq. ͑17͒ into Eqs. ͑20b͒ and ͑20c͒, so that one has the following stochastic evolution equations ͑that hopefully describe the dynamics on the stochastic center manifold͒:
͑22b͒
One should note that Eqs. ͑22a͒ and ͑22b͒ also can be found by naïvely adding the stochastic terms to the reduced system of evolution equations for the deterministic problem ͓Eqs. ͑18a͒ and ͑18b͔͒. This type of stochastic center manifold reduction has been done for the case of discrete noise. 27 Additionally, in many other fields ͑e.g., oceanography, solid mechanics, fluid mechanics͒, researchers have performed stochastic model reduction using a Karhunen-Loève expansion ͑principal component analysis, proper orthogonal decomposition͒. 33, 34 However, this linear projection does not properly capture the nonlinear effects. Furthermore, one must subjectively choose the number of modes needed for the expansion. Therefore, even though the solution to the reduced model found using this technique may have the correct statistics, individual solution realizations will not agree with the original, complete solution.
We will show that Eqs. ͑22a͒ and ͑22b͒ do not contain the correct projection of the noise onto the center manifold. Therefore, when solving the reduced system, one does not obtain the correct solution. Such errors in stochastic epidemic modeling impact the prediction of disease outbreak when modeling the spread of a disease in a population.
Using the same numerical scheme previously described, we numerically integrate the complete stochastic system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ along with the reduced system of equations that is based on the deterministic center manifold with a replacement of the noise terms ͓Eqs. ͑22a͒ and ͑22b͔͒. The complete system is solved for U, V, and W, while the reduced system is solved for V and W. In the latter case, U is computed using the center manifold equation given by Eq. ͑17͒. Once the values of U, V, and W are known, we compute the values of S, Ē , and Ī using the transformation given by Eqs. ͑7a͒-͑7c͒. We shift S, Ē , and Ī, respectively, by S 0 , E 0 , and I 0 to find the values of S, E, and I. Figures 3͑a͒ and 3͑b͒ compare the time series of the fraction of the population that is infected with a disease I, computed using the complete stochastic system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ with the time series of I computed using the reduced system of equations of the SEIR model that is based on the deterministic center manifold with a replacement of the noise terms ͓Eqs. ͑22a͒ and ͑22b͔͒. Figure 3͑a͒ shows the initial transients, while Fig. 3͑b͒ shows the time series after the transients have decayed. One can see that the solution computed using the reduced system quickly becomes out of phase with the solution of the complete system. Use of this reduced system would lead to an incorrect prediction of the initial disease outbreak. Additionally, the predicted amplitude of the initial outbreak would be incorrect. The poor agreement, both in phase and amplitude, between the two solutions con-tinues for long periods of time, as seen in Fig. 3͑b͒ . We also have computed the cross correlation of the two time series shown in Figs. 3͑a͒ and 3͑b͒ to be approximately 0.34. Since the cross correlation measures the similarity between the two time series, this low value quantitatively suggests a poor agreement between the two solutions.
Using the same systems of transformed equations, we compute 140 years worth of time series for 500 realizations. Ignoring the first 40 years of transient solution, the data are used to create a histogram representing the probability density p SI of the S and I values. Figure 4͑a͒ shows the histogram associated with the complete stochastic system of transformed equations, while Fig. 4͑b͒ shows the histogram associated with the reduced system of equations with a replacement of the noise terms. The color-bar values in Figs. 4͑a͒ and 4͑b͒ have been normalized by 10 −3 .
One can see by comparing Fig. 4͑a͒ with Fig. 4͑b͒ that the two probability distributions qualitatively look the same. It is also possible to compare the two distributions using a quantitative measure. The Kullback-Leibler divergence or relative entropy measures the difference between the two probability distributions as
where P i,j refers to the ͑i , j͒th component of the probability density found using the complete stochastic system of transformed equations ͓Fig. 4͑a͔͒ and Q i,j refers to the ͑i , j͒th component of the probability density found using the reduced system of equations ͓Fig. 4͑b͔͒. In our numerical computation of the relative entropy, we have added 10 −10 to each P ij and Q ij . This eliminates the possibility of having a Q ij =0 in the denominator of Eq. ͑23͒ and does not have much of an effect on the relative entropy. If the two histograms were identical, then the relative entropy given by Eq. ͑23͒ would be d KL = 0. The two histograms shown in Figs. 4͑a͒ and 4͑b͒ have a relative entropy of d KL = 0.0391, which means that the two histograms, while not identical, are quantitatively very similar. However, one cannot rely entirely on the histograms alone to say that the solutions of the complete system and the reduced system agree. As we have seen in Figs. 3͑a͒ and 3͑b͒, the two solutions have differing amplitudes and are out of phase with one another. It is important to note that these features are not picked up by the histograms of Fig. 4 .
To project the noise correctly onto the center manifold, we will derive a normal form coordinate transform for the complete stochastic system of transformed equations of the SEIR model given by Eqs. ͑20a͒-͑20c͒. The particular method we use to construct the normal form coordinate transform not only reduces the dimension of the dynamics, but also separates all of the fast processes from all of the slow processes. 25 This technique has been modified and applied to the large fluctuations of multiscale problems. 17 Many publications [19] [20] [21] [22] discuss the simplification of a stochastic dynamical system using a stochastic normal form transformation. In some of this work, 19, 22 anticipative noise processes appeared in the normal form transformations, but these integrals of the noise process into the future were not dealt with rigorously.
Later, the rigorous theoretical analysis needed to support normal form coordinate transforms was developed in Refs. 23 and 24. The technical problem of the anticipative noise integrals also was dealt with rigorously in this work. Even later, another stochastic normal form transformation was developed. 25 This new method allows for the "͓removal of͔ anticipation… from the slow modes with the result that no anticipation is required after the fast transients decay" ͑Ref. 25, p. 13͒. The removal of anticipation leads to a simplification of the normal form. Nonetheless, this simpler normal form retains its accuracy with the original stochastic system. 25 We shall use the method of Ref. 25 to simplify our stochastic dynamical system to one that emulates the long-term dynamics of the original system. The method involves five principles, which we recapitulate here for completeness:
͑1͒ Avoid unbounded secular terms in both the transformation and the evolution equations to ensure a uniform asymptotic approximation. ͑2͒ Decouple all of the slow processes from the fast processes to ensure a valid long-term model. ͑3͒ Insist that the stochastic slow manifold is precisely the transformed fast processes coordinate being equal to zero. ͑4͒ To simplify matters, eliminate as many as possible of the terms in the evolution equations. ͑5͒ Try to remove all fast processes from the slow processes by avoiding as much as possible the fast time memory integrals in the evolution equations.
In practice, the original stochastic system of equations ͑which satisfies the necessary spectral requirements͒ in ͑U , V , W͒ T coordinates is transformed to a new ͑Y , X 1 , X 2 ͒ T coordinate system using a near-identity stochastic coordinate transform given as
where the specific form of ͑Y , X 1 , X 2 , ͒, ͑Y , X 1 , X 2 , ͒, and ͑Y , X 1 , X 2 , ͒ is chosen to simplify the original system according to the five principles listed previously, and is found using an iterative procedure. To outline the procedure, we provide details for a simple example in Appendix A. Several iterations lead to coordinate transforms for U, V, and W along with evolution equations describing the Y-dynamics, X 1 -dynamics, and X 2 -dynamics in the new coordinate system. The Y-dynamics have exponential decay to the Y = 0 slow manifold. Substitution of Y = 0 leads to the coordinate transforms 
All of the stochastic terms in Eqs. ͑25a͒-͑25c͒ consist of integrals of the noise process into the past ͑convolutions͒, as given by Eqs. ͑26͒ and ͑27͒. These memory integrals are fast-time processes. Since we are interested in the long term slow processes and since the expectation of G equals e −Ꭽ ‫ء‬ E͓͔, where E͓͔ = 0, we neglect the memory integrals and the higher-order multiplicative terms found in Eqs. ͑25a͒-͑25c͒ so that
Note that Eq. ͑28a͒ is the deterministic center manifold equation, and at first order, matches the center manifold equation that was found previously ͓Eq. ͑17͔͒. Substitution of Y = 0 and neglecting all multiplicative noise terms and memory integrals using the argument from above ͑so that we consider only first-order noise terms͒ leads to the following reduced system of evolution equations on the center manifold:
The specific form of F and G in Eqs. ͑29a͒ and ͑29b͒ is complicated and is therefore presented in Appendix B. We numerically integrate the complete stochastic system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ along with the reduced system of equations that is found using the stochastic normal form coordinate trans form ͓Eqs. ͑29a͒, ͑29b͒, ͑B1a͒, and ͑B1b͔͒. The complete system is solved for U, V, and W, while the reduced system is solved for X 1 = V and X 2 = W. In the latter case, U is computed using the center manifold equation given by Eq. ͑28a͒. As before, once the values of U, V, and W are known, we compute the values of S, Ē , and Ī using the transformation given by Eqs. ͑7a͒-͑7c͒. We shift S, Ē , and Ī, respectively, by S 0 , E 0 , and I 0 to find the values of S, E, and I. 5. ͑Color online͒ Time series of the fraction of the population that is infected with a disease I, computed using the complete stochastic system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ ͑red solid line͒, and computed using the reduced system of equations of the SEIR model that is found using the stochastic normal form coordinate transform ͓Eqs. ͑29a͒, ͑29b͒, ͑B1a͒, and ͑B1b͔͒ ͑blue dashed line͒. The standard deviation of the noise intensity used in the simulation is i = 0.0005, i =4,5,6. The time series is shown for ͑a͒ t =0 to t = 40 and for ͑b͒ t =40 to t = 100. computed using the complete stochastic system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ with the time series of I computed using the reduced system of equations of the SEIR model that is found using the stochastic normal form coordinate transform ͓Eqs. ͑29a͒, ͑29b͒, ͑B1a͒, and ͑B1b͒ ͔. Figure 5͑a͒ shows the initial transients, while Fig. 5͑b͒ shows the time series after the transients have decayed. One can see that there is an excellent agreement between the two solutions. The initial outbreak is successfully captured by the reduced system. Furthermore, Fig. 5͑b͒ shows that the reduced system accurately predicts recurrent outbreaks for a time scale that is orders of magnitude longer than the relaxation time. This is not surprising since the solution decays exponentially throughout the transient and then remains close to the lower-dimensional center manifold. Since we are not looking at periodic orbits, there are no secular terms in the asymptotic expansion, and the result is valid for all time. Additionally, any noise drift on the center manifold results in bounded solutions due to sufficient dissipation transverse to the manifold. The cross correlation of the two time series shown in Fig. 5 is approximately 0.98, which quantitatively suggests there is an excellent agreement between the two solutions.
Using the same systems of transformed equations, we compute 140 years worth of time series for 500 realizations. As before, we ignore the first 40 years worth of transient solution, and the data are used to create a histogram representing the probability density p SI of the S and I values. Figure 6͑a͒ shows the histogram associated with the complete stochastic system of transformed equations, while Fig.  6͑b͒ shows the histogram associated with the reduced system of equations found using the normal form coordinate transform. The color-bar values in Figs. 6͑a͒ and 6͑b͒ have been normalized by 10 −3 .
As we saw with Figs. 4͑a͒ and 4͑b͒, the probability distribution shown in Fig. 6͑a͒ looks qualitatively the same as the probability distribution shown in Fig. 6͑b͒ . Using the Kullback-Leibler divergence given by Eq. ͑23͒, we have found that the two histograms shown in Figs. 6͑a͒ and 6͑b͒ have a relative entropy of d KL = 0.0953. Since this value is close to zero, the two histograms are quantitatively very similar.
In addition to computing the cross correlation between the solution of the original system and the solutions of the two reduced systems for i = 0.0005, we have computed the cross correlation for the case of zero noise as well as for noise intensities ranging from = 5.0ϫ 10 −10 to = 5.0ϫ 10 −5 . These cross correlations were computed using time series from t = 800 to t = 1000. For the deterministic case ͑zero noise͒, the cross correlation between the time series which were computed using the original system and the reduced system based on the deterministic center manifold is 1.0, since the agreement is perfect. The cross correlation between the original system and the reduced system found using the stochastic normal form is also 1.0. Figure 7 shows the cross correlation between the original system and the two reduced systems for various values of .
One can see in Fig. 7 that the solutions found using the reduced system based on the deterministic center manifold compare poorly with the original system at very low noise values. Furthermore, as the noise increases, the agreement between the two solutions gets worse. On the other hand, Fig. 7 shows that the solutions computed using the reduced system found using the normal form coordinate transform compare very well with the solutions to the original system across a wide range of small noise intensities.
We have demonstrated that the normal form coordinate transform method reduces the Langevin system so that both the noise and dynamics are accurately projected onto the lower-dimensional manifold. It is natural to consider ͑a͒ the replacement of the stochastic term by a deterministic period drive of small amplitude and ͑b͒ the extension to finite populations. These cases are discussed, respectively, in Secs. VI A and VI B. . 6 . ͑Color online͒ Histogram of probability density p SI of the S and I values found using ͑a͒ the complete stochastic system of transformed equations for the SEIR model with mortality ͓Eqs. ͑20a͒-͑20c͔͒ and ͑b͒ the reduced system of equations of the SEIR model with mortality that is found using the stochastic normal form coordinate transform ͓Eqs. ͑29a͒, ͑29b͒, ͑B1a͒, and ͑B1b͔͒. The histograms are created using 100 years worth of time series ͑starting with year 40͒ for 500 realizations, and the color-bar values have been normalized by 10 −3 . Cross correlation between time series found using the original stochastic system of transformed equations and the reduced system of equations based on the deterministic center manifold ͑"circle" markers͒ and cross correlation between time series found using the original stochastic system of transformed equations and the reduced system of equations based on the stochastic normal form coordinate transform ͑"square" markers͒. The cross correlation is computed using time series from t = 800 to t = 1000.
A single time series realization of the noise might be thought of as a deterministic function of small amplitude driving the system. One could rederive the normal form for such a deterministic function. However, since our derived normal form holds specifically for the case of white noise, we show that a simple replacement of the stochastic realization with a deterministic realization does not work. As an example, one could consider the following sinusoidal functions: 1 1 = cos͑10t͒/8000, ͑30a͒ 2 2 = sin͑4t͒/8000, ͑30b͒
where 4 4 , 5 5 , and 6 6 are given by Eqs. ͑21a͒-͑21c͒. Using Eqs. ͑30a͒-͑30c͒ or some other similar deterministic drive, the solution computed using the reduced system based on the deterministic center manifold analysis will agree perfectly with the solution computed using the complete system of equations. On the other hand, since the reduced system based on the normal form analysis was derived specifically for white noise, the transient solution found using this reduced system will not agree with the solution found using the complete system. It is possible to find a normal form coordinate transform for periodic forcing, but the normal form will be different than the one derived in this article for white noise. Figures 8͑a͒ and 8͑b͒ compare the time series of the fraction of the population that is infected with a disease I, computed using the complete system of transformed equations of the SEIR model ͓Eqs. ͑20a͒-͑20c͔͒ with the time series of I computed using the reduced system of equations of the SEIR model that is found using the stochastic normal form coordinate transform ͓Eqs. ͑29a͒, ͑29b͒, ͑B1a͒, and ͑B1b͔͒, but where the stochastic terms of both systems have been replaced by the deterministic terms given by Eqs. ͑30a͒-͑30c͒. Figure 8͑a͒ shows the initial transients, while Fig. 8͑b͒ shows a piece of the time series after the transients have decayed. One can see in Figs. 8͑a͒ and 8͑b͒ that although the two solutions eventually become relatively synchronized with one another, there is a poor agreement, both in phase and amplitude, throughout the transient.
The solutions to the original system and both reduced systems are continuous solutions based on an infinite population assumption and are found using Langevin equations having Gaussian noise. It is interesting to examine the effects of general noise by using a Markov simulation to compare solutions of the original and reduced systems.
The complete system in the original variables ͑see p. 2͒ will evolve in time t in the following way: transition rate ͑s − 1,e + 1,i͒ ␤si/N ͑s,e − 1,i + 1͒ ␣e ͑s,e,i − 1͒ ␥i ͑s + 1,e,i͒ N ͑s − 1,e,i͒ s ͑s,e − 1,i͒ e ͑s,e,i − 1͒ i.
Using a total population size of N =10ϫ 10 6 , we have performed a Markov simulation of the system. After completing the Markov simulation, we divided s, e, and i by N to find S, E, and I. Figure 9͑a͒ shows a time series, after the transients have decayed, of the fraction of the population that is infected with a disease I. The results reflect both the mean and the frequency of the deterministic system. Performing the simulation for 500 realizations allows us to create a histogram representing the probability density p SI of the S and I values. This histogram is shown in Fig. 9͑b͒ , and one can see that the probability density reflects the amplitude, which var- ies with the population size of S and I. The color-bar values in Fig. 9͑b͒ have been normalized by 10 −4
The complete system in the transformed variables has the stable endemic equilibrium at the origin. To bound the dynamics to the first octant, we use the fact that s Ն 0, e Ն 0, and i Ն 0 to derive the appropriate inequalities for the transformed discrete variables u, v, and w. These inequalities can be found in Appendix C as Eq. ͑C1͒. These inequalities enable us to define new discrete variables Y 1 , Y 2 , and Y 3 given by Eqs. ͑C2a͒-͑C2c͒ in Appendix C.
In the Y i variables, we define evolution relationships similar to those found in Eq. ͑31͒. The complete transformed system will evolve in time according to the transition and rates given by Eq. ͑C3͒ in Appendix C.
After performing a Markov simulation of Eq. ͑C3͒ with a population size of N =10ϫ 10 6 , we can compare the dynamics of the transformed system with the dynamics of the original system by transforming the Y i variables in the time series back to the original s, e, and i variables. Dividing by N yields S, E, and I. Figure 10͑a͒ shows a time series, after the transients have decayed, of the fraction of the population that is infected with a disease I. The mean and the frequency agree with those found from the Markov simulation of the original system. We have performed the simulation for 500 realizations, and a histogram representing the probability density p SI is shown in Fig. 10͑b͒ . The color-bar values in Fig. 10͑b͒ have been normalized by 10 −4 . One can see in Fig.  10͑a͒ that the relative fluctuations of the I component have nearly doubled. While the fluctuation size was 0.152 for the original system, it is 0.310 for the transformed system. Additionally, the two histograms shown in Figs. 9͑b͒ and 10͑b͒ have a relative entropy of d KL = 0.9519, which means they are not in agreement. Because the simulation of the stochastic dynamics in the complete system of transformed variables does not qualitatively ͑or quantitatively͒ resemble the original stochastic system, we cannot expect that the reduced system will agree with either the original or the transformed systems. Therefore, much care should be exercised when extending the model reduction results ͑which show outstanding agreement͒ derived for a specific type of noise in the limit of infinite population to finite populations with a more general type of noise.
We have considered the dynamics of a SEIR epidemiological model with stochastic forcing in the form of additive Gaussian noise. We have presented two methods of model reduction, whereby the goal is to project both the noise and the dynamics onto the stochastic center manifold. The first method uses the deterministic center manifold found by neglecting the stochastic terms in the governing equations, while the second method uses a stochastic normal form coordinate transform.
Since the original system of governing equations does not have the necessary spectral structure to employ either deterministic or stochastic center manifold theory, the system of equations has been transformed using an appropriate linear transformation coupled with appropriate parameter scaling. At this stage, the first method of model reduction can be performed by computing the deterministic center manifold equation. Substitution of this equation into the complete stochastic system of transformed equations leads to a reduced system of stochastic evolution equations.
The solutions of the complete stochastic system of transformed equations as well as the reduced system of equations were computed numerically. We have shown that the individual time series does not agree because the noise has not been correctly projected onto the stochastic center manifold. However, by comparing histograms of the probability density p SI of the S and I values, we saw that there was a very good agreement. This is caused by the fact that although the two solutions are out of phase with one another, their range of amplitude values is similar. The phase difference is not represented in the two histograms. This is a real drawback when trying to predict the timing of outbreaks and leads to potential problems when considering epidemic control, such as the enhancement of disease extinction through random vaccine control. 35 To accurately project the noise onto the manifold, we derived a stochastic normal form coordinate transform for the complete stochastic system of transformed equations. The numerical solution to this reduced system was compared with the solution to the original system, and we showed that there was an excellent agreement both qualitatively and quantitatively. As with the first method, the histograms of the probability density p SI of the S and I values agree very well.
It should be noted that the use of these two reduction methods is not constrained to problems in epidemiology, but rather may be used for many types of physical problems. For some generic systems, such as the singularly perturbed, damped Duffing oscillator, either reduction method can be used since the terms in the normal form coordinate transform which lead to the average stochastic center manifold being different from the deterministic center manifold occur at very high order. 17 In other words, the average stochastic center manifold and deterministic center manifold are virtually identical. For the SEIR model considered in this article, there are terms at low order in the normal form transform, which cause a significant difference between the average stochastic center manifold and the deterministic manifold. Therefore, as we have demonstrated, when working with the SEIR model, one must use the normal form coordinate transform method to correctly project the noise onto the center manifold.
In summary, we have presented a new method of stochastic model reduction that allows for impressive improvement in time series prediction. The reduced model captures both the amplitude and phase accurately for a temporal scale that is many orders of magnitude longer than the typical relaxation time. Since sufficient statistics of disease data are limited due to short time series collection, the results presented here provide a potential method to properly model real, stochastic disease data in the time domain. Such longterm accuracy of the reduced model will allow for the application of effective control of a disease where phase differences between outbreak times and vaccine controls are important. Additionally, since our method is general, it may be applied to very high-dimensional epidemic models, such as those involving adaptive networks. From a dynamical systems viewpoint, the reduction method has the potential to accurately capture new, emergent dynamics as we increase the size of the random fluctuations. This could be a means to identify new noise-induced phenomena in generic stochastic systems. Comparison of distance measures in spatial analytical modeling for health service planning BACKGROUND: Several methodological approaches have been used to estimate distance in health service research. In this study, focusing on cardiac catheterization services, Euclidean, Manhattan, and the less widely known Minkowski distance metrics are used to estimate distances from patient residence to hospital. Distance metrics typically produce less accurate estimates than actual measurements, but each metric provides a single model of travel over a given network. Therefore, distance metrics, unlike actual measurements, can be directly used in spatial analytical modeling. Euclidean distance is most often used, but unlikely the most appropriate metric. Minkowski distance is a more promising method. Distances estimated with each metric are contrasted with road distance and travel time measurements, and an optimized Minkowski distance is implemented in spatial analytical modeling. METHODS: Road distance and travel time are calculated from the postal code of residence of each patient undergoing cardiac catheterization to the pertinent hospital. The Minkowski metric is optimized, to approximate travel time and road distance, respectively. Distance estimates and distance measurements are then compared using descriptive statistics and visual mapping methods. The optimized Minkowski metric is implemented, via the spatial weight matrix, in a spatial regression model identifying socio-economic factors significantly associated with cardiac catheterization. RESULTS: The Minkowski coefficient that best approximates road distance is 1.54; 1.31 best approximates travel time. The latter is also a good predictor of road distance, thus providing the best single model of travel from patient's residence to hospital. The Euclidean metric and the optimal Minkowski metric are alternatively implemented in the regression model, and the results compared. The Minkowski method produces more reliable results than the traditional Euclidean metric. CONCLUSION: Road distance and travel time measurements are the most accurate estimates, but cannot be directly implemented in spatial analytical modeling. Euclidean distance tends to underestimate road distance and travel time; Manhattan distance tends to overestimate both. The optimized Minkowski distance partially overcomes their shortcomings; it provides a single model of travel over the network. The method is flexible, suitable for analytical modeling, and more accurate than the traditional metrics; its use ultimately increases the reliability of spatial analytical models. Health service research is concerned with the investigation of how social, financial, organizational, technological, and behavioral factors affect access to health care, the quality and cost of health care, and ultimately health and well-being [1] . Distance plays a vital role in studies assessing spatial disease patterns as well as access to hospital services. In a highly complex health care environment, even micro-geographic differences in the availability of tertiary services can affect access to care [2, 3] . The study of distances from patient homes to the nearest hospital is an example where distance is often studied as a crude but objective indicator of geographic accessibility to hospital services [4] . In such situations, the measurement of actual travel distance (or travel time) on a road network is clearly the most appropriate method [5] . Health service research, however, encompasses a much broader investigation area, where spatial analytical models are employed to assist in the provision of effective accessibility to health care services. Distance is often used indirectly in these types of analysis as one of the parameters defining the model's thrust and its results.
In a rapidly changing physical and social environment, transportation means and travel modes change quickly as do epidemic transmission modes, overturning traditional ways of conceptualizing and measuring distance [6] . A commonly-used distance metric is the Euclidean distance, a straight line distance measurement between two points, 'as the crow flies' [7, 8] . This method is simple and intuitive, but very few are the applications where it can yield accurate distance estimates. An alternative, well-known distance metric is the Manhattan, or taxi-cab distance: as its name suggests, it is most appropriate for grid-like road networks, typical of many North American cities, characterized by a rectangular city block pattern. The Manhattan metric measures distance between points along a rectangular path with right angle turns [9, 10] . Most commonly, travel along road networks involves a mixture of Euclidean, Manhattan, and curvilinear trajectories. There is no firm consensus on methods for selecting a distance metric [11] , nor is there much published information on the extent to which Euclidean, Manhattan, and road distances relate to one another in applied distance analysis [12, 13] .
Travel along a complex, or mixed network can be usefully modeled by a class of distance metrics, known as Minkowski distance [14] , which is a general distance metric, of which the Euclidean and Manhattan metrics are special cases. This array of metrics provides flexibility and generality, in that, within a single class of metrics, a range of parameters can be selected; therefore, a single yet flexible method for measuring distance can be defined for the optimal estimation of distance on a variety of empirical road networks. One further important aspect is the funda-mental role of time in accessing health care services: if distance is a crude estimate of accessibility, travel time is a more relevant estimate. Travel time computation is no longer a prohibitively time consuming and computationally intensive task, thanks to powerful GIS software, hardware, and rich road network datasets [14] [15] [16] [17] . However, actual travel time on a road network is highly variable due to local (spatial and temporal) conditions which are hardly predictable and controllable. Because of this characteristic, travel time computations lack general validity, requiring adjustments to account for specific temporal conditions, e.g., weekend vs. weekdays, rush vs. non-rush hours, season, and weather, as well as local spatial conditions, e.g., local traffic congestion, lane closures, or proximity to amenities or popular destinations. All these reasons hamper the implementation of travel time computations in spatial analytical models, since even local analytical models require the definition of a single rule for distance measurement. A crude solution to this problem is the use of average travel time in spatial models; a more realistic solution can be obtained through the use of Minkowski distance: an optimal value of Minkowski distance can be selected to model travel time on a complex road network.
Spatial data tend to exhibit characteristics that negatively impact the statistical properties of quantitative models, decreasing their reliability: spatial analytical models are designed to mitigate these negative effects. The most crucial properties of spatial data are spatial dependence (near things tend to be more similar than distant things) and non-stationarity (inconstant variability of phenomena across space) [18] . Two broad categories of spatial analytical models include spatially autoregressive (SAR) methods, which deal with spatial dependence [19] , and geographically weighted (GWR) methods, which deal with spatial non-stationarity [20] .
In empirical situations, spatial dependencies and non-stationarities take up specific forms, which are a function of many factors, including the nature of the phenomena under investigation and the representation of space underpinning the model. For this reason, a simplistic application of spatial analysis, one that does not carefully model the salient aspects of phenomena, often fails to fulfill the model's primary objective, which is to enhance the model reliability. The transition from a simplistic to a customized implementation of spatial analysis requires the calibration of each parameter defining the analysis: one of the most crucial parameters, affecting the analytical results, is the distance measurement method.
Cardiac catheterization is a procedure that is performed to determine presence or absence of coronary artery blockages. The procedure involves the percutaneous insertion of a catheter into the arterial system, after which it is guided into the aorta where the coronary arteries are positioned. Contrast dye is then injected into the coronary arteries so that blockages can be located and identified. In some instances, cardiac catheterization can lead to immediate use of percutaneous coronary intervention with balloon angioplasty and the insertion of coronary stents that open up partially or completely blocked arteries to restore blood flow. In some instances, this procedure is performed in stable patients where distance and travel times are a minor concern. In other instances, however, the procedure is done urgently, and for such situations, consideration of distances and travel times become a central consideration in the planning of health services.
In the context of an applied study of distance between patient residence and a tertiary cardiac catheterization facility in a large city, this paper analyzes the effectiveness of a selection of distance metrics in providing a useful model of travel distance and travel time along an urban road network. The comparison of different metrics leads to the identification of a metric that is conceptually sound and computationally effective. The metric thus identified is experimentally used in a spatial autoregressive model analyzing the spatial distribution of cardiac catheterization cases in the city.
The study area encompasses the City of Calgary, one of the largest Canadian cities, with approximately 1 million residents [21], distributed over a large geographic area (roughly 750 Km 2 ), characterized by diversity of population, housing type, residential density, and accessibility to heath services.
Cardiac catheterization is an invasive procedure for patients experiencing cardiovascular symptoms and defines coronary anatomy, left ventricular and valvular function; it provides important prognostic information for individuals affected by cardiovascular conditions [22] . During the study period the procedure was only performed at the Foothills Medical Centre, located in the northwest of the city.
Three types of data are used in this study: cardiac catheterization patient database, postal code locations, and the Calgary road network. Cardiac catheterization patient data were obtained from the Alberta Provincial Project for Outcome Assessment in Coronary Heart Disease (APPROACH), an ongoing data collection initiative, begun in 1995, producing information on all patients undergoing catheterization in Alberta [22] . The data are released at the postal code spatial aggregation level. Data were extracted for Calgary residents only and catheterizations performed over the year 2002, resulting in a total of 2, 445 catheterization cases, distributed over 2, 138 postal codes.
A postal code conversion file (PCCF) [23] was obtained from Statistics Canada. Only postal codes that have at least one catheterization case are retained for the analysis. It shall be observed that postal code locations refer to the primary residence of catheterization patients, not to the place where symptoms were felt or where emergency care was first administered.
The Calgary road network data were obtained from the University of Calgary data holdings, based on street information collected and compiled in 2005 by DMTI Spatial [24] . This road network was used to calculate shortest road distances from patient residence location to hospital for cardiac catheterization services.
Straight line (Euclidean) distance and Manhattan distance are often used in health service research [25] . Each of these distance metrics may appropriately estimate distance in some parts of a study area, but their application at the city level tends to yield large errors in areas that depart from the dominant pattern, and may lead to highly inaccurate distance estimations. One of the reasons for using Euclidean and/or Manhattan distance is the relative ease of their implementation; in contrast, it is more problematic to design algorithms implementing actual road network distance in spatial analytical models.
In order to reduce the error associated with the Euclidean and Manhattan metrics while maintaining the computational simplicity of a single, intuitive mathematical formula, the general Minkowski metric is examined, to devise a single method that best approximates the average pattern of an empirical road network. Optimizing values of the Minkowski formula are calculated for road distance as well as travel time; the results are compared with more traditional distance measures in the context of assessing geographic accessibility to cardiac facilities. The Minkowski distance has the potential to provide a more accurate estimate of road network distance and travel time than the Euclidean and Manhattan metrics.
A set of 2, 138 distances between each patient's postal code of residence and the Foothills hospital are calculated according to each of the distance measurement methods considered. The geographic locations of each postal code from the PCCF and the hospital are recorded in latitude and longitude; therefore, in order to implement distance computations, the road network is projected using an equidistant projection system, which is chosen in order to preserve distance and produce consistent distance measurements [26] . Latitude and longitude coordinates are then converted into Eastings and Northings, i.e., x and y values, expressed in kilometers. Alternative methods could have been used, for example the great circle distance formula [27] , which, however, provides rougher estimations. The ArcGIS 9.3 [28] Geometry calculator was used to calculate the x and y coordinates based on the projected dataset and the resulting x and y values were used in the distance formulas defined below.
Euclidean [7, 8] , Manhattan [9, 10] , and Minkowski [14] distance can be calculated by the formula:
where, for this application: d is the distance between a patient's residence and the hospital;
x i , y i are the geographic coordinates of the centroid of each postal code of residence;
x j , y j are the geographic coordinates of the Foothills hospital.
The generic p parameter in Equation 1 can be replaced by the value 2 to yield the well known Euclidean distance; the value 1 would yield the Manhattan distance, and all the intermediate values in the in the [1 <p < 2] interval yield an array of Minkowski distances ( Figure 1 ).
The computation of road network distances (shortest distance between two locations along a road network) is implemented directly in GIS software [28] using a shortest path algorithm. It is recognized a priori that this method depicts the actual travel trajectory and is likely to produce the most accurate distance estimates for patient travel routes to a hospital. Likewise, travel time calculated over the road network using an appropriate algorithm is likely to yield the most accurate travel time estimates. The basic need for road distance calculation is a road network with information on all the segments constituting the network, as well as on all existing constraints such as prohibited left or right turns, one way streets, etc.
Travel time calculations, likewise, are implemented in GIS software [28] . Additional information required for travel time calculation include estimated speed and length of road segments, along with an algorithm capable of taking all these factors into consideration [15] . The Network Analyst extension within ArcGIS [28] enables the modeling of spatial networks and provides the tools for road distance calculations [28, 29] and travel time calculations [15, 16] from multiple postal code locations to a hospital. Figure 1 illustrates road network distance, along with the distance metrics considered in this study.
A simple procedure can be implemented to select, within the [1 ≤ p ≤ 2] interval, the value of the parameter p in the distance formula that best approximates distance along a given road network. In light of the applied focus of this analysis, an empirical solution to this problem is sought for the set of 2, 138 patient-to-hospital distances. As a first step for determining the best Minkowski distance, Equation 1 is transformed into Equation 2:
Two new quantities are defined as X = (x i -x j ) and Y = (y iy j ), and replaced in Equation 2, which is also further modified by means of a logarithmic transformation. Equation 2 is solved for p values in the [1 ≤ p ≤ 2] interval, sampled at regular intervals. The set of distances thus calculated are considered approximations of the road distance. A simple regression model is defined, where the dependent variable is the road distance, and the independent variable is, in turn, the distance obtained by each p value. Goodness-offit, residuals, and other regression diagnostics are then compared over the entire interval [30, 31] . This simple method helps assess and rank the various coefficients, identifying the one that produces the highest R 2 value, which is considered the best Minkowski coefficient.
The procedure is then modified to determine the optimal Minkowski coefficient for travel time: conceptually, this experiment is less straightforward, because travel time is a measure of time, whereas Minkowski remains a measurement of distance in space. Once travel time is computed, in order to make the transition between time and space, in terms that are valid both conceptually and computationally, the concept of speed, a simple ratio between space and time, is introduced. Average speed over the city is calculated, yielding the following values: the average distance traveled in one hour is 58.71 km; conversely, the average time required to travel 1 kilometer is 1.02 minutes. For the sake of simplicity, in order to make the argument more intuitive, these values were rounded to an average speed of 60 km/h or 1 minute to travel 1 kilometer. It shall be observed that these values are obtained under optimal conditions, i.e., without considering delays due to rush hour, traffic congestion, traffic lights, stop signs, road closures, weather, etc.
Travel time calculations are then used as the dependent variable, while the independent variables remain unchanged. To this end, actual travel times are converted
to distances, using the average travel time, and replaced in the procedure for the calculation of the optimal Minkowski p value to approximate travel time. Once the two optimal Minkowski coefficients are identified, standard descriptive statistics are used to analyze and compare the four distance metrics and the two empirical distance measurements.
The results of the regression models estimated to identify the optimal Minkowski coefficients are summarized in Figure 2 . Figure 2a summarizes the regressions for the road distance coefficient optimization, and Figure 2b for the optimization of the travel time coefficient. Figure 2 shows different values of a goodness-of-fit indicator (R 2 ) obtained from a regular sample of p values throughout the [ 
Visual illustration of road distance and distance metrics the methods used to measure distance, it displays the second largest standard deviation and range. Travel time can be immediately compared with all the distance metrics, as 1 minute corresponds to 1 kilometer: in comparison with road network distance, travel time displays a lower standard deviation and a shorter range, with a minimum travel time of 1.51 minutes, and a maximum of 26.76 minutes, under optimal conditions. Conversely, its mean displays the highest value, 12.11 minutes. Shorter range and higher mean are likely due to the opposite effects of speed limits on road segments of different length: in general, longer travel paths include large segments that occur on major roads, where speed limits are higher, whereas shorter trips tend to occur on minor roads, which are associated with lower speed limits. As a consequence, a model that takes speed into consideration produces a shorter travel range. Consistently, the standard deviation is lower. The higher mean value suggests that overall speed limits tend to slow down travel; that is, segments with low speed limits have a large impact on the overall travel pattern throughout the road network.
Euclidean distance tends to underestimate road distance, as shown by the mean and range; the standard deviation is lower than for road distance, with values that are fairly close to those for travel time. This is probably due to the smoothing effect of a uniform distance model, which produces lower values than actual road distance for curvilinear segments and the most complex paths.
Manhattan distance tends to overestimate road distance and produces values consistently larger than those of Euclidean distance. Its mean value is very close to the road distance mean, but it also presents the largest standard deviation and the largest range. This may suggest that the Calgary road network contains several parts that follow the Manhattan pattern (hence a similar mean value), but the presence of different patterns in the same network increases its error (large standard deviation).
In comparing Euclidean and Manhattan distances, Manhattan distance produces a close approximation of the mean, and only slightly overestimates the standard deviation, whereas Euclidean largely underestimates both values. For the range, both metrics produce approximately the same error, though with opposite sign. This suggests that, overall, Manhattan is a better model than Euclidean for the Calgary road network.
The value of p = 1.54 best approximates road distance in the Minkowski formula. Indeed minimum and maximum values are close approximations, whereas mean and standard deviation underestimate road distance. This result can be considered satisfactory, as it indicates that the error is minimized for individual measurements, but overall the method displays the aforementioned smoothing effect, whereby mean measurements tend to be slightly smaller than actual ones, with overall lower variations around the mean. It shall be observed that this distance metric is approximately half-way between Euclidean and Manhattan; however, all the descriptive statistics present values that are closer to the Euclidean than the Manhattan results.
The value of p = 1.31 best approximates travel time in the Minkowski formula. Interestingly, the descriptive statistics suggest that this metric is the best approximation of road distance; indeed a close approximation. As noted,
Determination of optimal p values for Minkowski distance Figure 2 Determination of optimal p values for Minkowski distance. travel time presents features that differ from distance, i.e., larger mean and lower range: this combination is hard to achieve by the class of metrics considered, because p values closer to 1 (Manhattan) produce larger means, whereas p values closer to 2 (Euclidean) produce lower ranges. In light of these considerations, a relatively low p value, such as p = 1.31 most closely approximates this pattern, rendering a model that is less extreme than Manhattan, as shown by a lower standard deviation. Overall the metric p = 1.31 provides a model of travel that is intermediate between measured road distance and travel time measured under optimal conditions. This is supported by almost all the descriptive statistics; the greatest shortcoming of this metric is its poor approximation of the mean. This value produces the best of all the metrics examined.
Finally, it shall be noted that the travel time pattern would be very different if measurements were to consider less favorable conditions. The most common impediments to fast travel in Calgary include traffic congestion, e.g., rush hour, and severe winter weather conditions. Traffic congestion tends to affect major roads more heavily, where feasible speed can easily be reduced by 20-25% of the speed limit, whereas its effect is generally lesser on minor roads. Severe winter weather conditions are likely to have a comparable effect on major roads, but they will also have comparable or worse effects on minor roads. However, a speed reduction from 80 to 60 km/h on long road segments significantly impacts travel time, lowering the maximum and range values, and increasing the mean value. Conversely, an equivalent or greater speed reduction from 50 to 40 or 35 km/h on shorter road segments is likely to have only a minor impact on the overall travel time. Seeking to approximate such travel pattern via a Minkowski p value is therefore unlikely to produce better results. Table 2 presents a selection of summary statistics of the differences between each empirical measurement and the distance metrics considered. These are only global results, overshadowing the performance of each distance model throughout the city. These results suggest that, globally, the differences produced by each model are modest.
For road distance, the smallest differences are achieved by the Manhattan and Minkowski (p = 1.31) metrics, with Manhattan producing the overall best result. The same metrics produce the best results for travel time, with Minkowski (p = 1.31) producing the lowest standard deviation. Median and mean differences are lower than 2 kilometers and just over 2 minutes, respectively. However, standard deviations tend to be quite high, relative to the mean. Figure 3 and Figure 4 illustrate graphically these differences, providing greater spatial detail. Differences between road distance and each distance metric are presented in Figure 3 , and those between travel time and each metric in Figure 4 . The figures display spatially the magnitude of the error associated with each distance metric in each part of the city.
With respect to road distance (Figure 3 ), Euclidean distance produces only negative errors, but in some peripheral areas of the city these errors are large in absolute value. Manhattan distance produces mostly positive errors, and overall it produces better results, as the areas characterized by the highest absolute errors are reduced to a triangle west of the hospital and the far southeast corner of the city. The Minkowski metric with p = 1.54 improves over the Manhattan metric results, by resolving the area of large residuals in the southeast corner; conversely, the area of high residuals west of the hospital is moderately larger. The best results are produced by the Minkowski metric with p = 1.31, as for the entire eastern part of the city errors are contained in the interval 0-2.5 km, and greater errors remain only in some peripheral northwest areas.
The area west of the hospital consistently emerges as an outlier, despite its close distance to the hospital. Careful observation of the topography of the area reveals that the hospital is located on the east side of a hill, with a river running to the west of the hill. Therefore, no immediate access is possible from the west side of the river to the hospital, and patients are left with no option but to travel a considerable distance either northbound or southbound With respect to travel time ( Figure 4 ) the improvement obtained by the two Minkowski metrics is more evident throughout the city. Areas of large residuals remain in the far southeast and southwest corners, and, in general each metric performs worse in the west than in the east part of the city. The latter observation is counterintuitive, since the hospital is located in the northwest; however, that part of the city, closer to the foothills, is characterized by a more complex topography. Moreover, in a city like Calgary, urban design and age of communities play an important role, as, over the decades, rectangular patterns have alternated with such patterns as crescent and cul-de-sac, and other typologies of urban connectivity. Likewise, it shall be observed that the most peripheral areas are very recent developments, and likely the planned road connections had not been completed during the study period.
Within the scope of a larger project, the association between cardiovascular disease and socio-economic variables was recently analyzed [32] . Specifically, the relationship between cardiac catheterization and socio-economic variables was analyzed by means of a multivariate spatially autoregressive model. While distance does not explicitly enter in these models, it is one of the key parameters defining the spatial weight matrix (19) , which represents the neighborhood definition, hence the model's ability to cope with spatial dependencies, and ultimately the reliability of the model estimates. Figure 5 provides an example of how different distance metrics affect the neighborhood configuration defined by a spatial weight matrix. Locations in the figure represent census tract centroids, as these relatively larger spatial units are used in these regression models. The lines connecting these locations indicate whether or not 2 close locations are considered neighbors and included in the estimation of spatial dependence. Other parameters contribute to the neighborhood definition: generally the most influential param-eter is the number of nearest neighbors, complemented by a distance decay function and a weight [33] . Figure 5a shows the connectivity defined by the Euclidean metric, while Figure 5b corresponds to the Minkowski metric optimizing travel time (p = 1.31). A close comparison of the two plots (aided by the superimposed circles) reveals how the modification of the distance metric substantially alters the neighborhood configuration.
Each neighborhood configuration, such as the ones presented in Figure 5 , forms the basis for the definition of a spatial weight matrix, which is one of the crucial elements that define a spatial regression model, differentiating it from a standard (non-spatial) model. Through the spatial weight matrix, the neighborhood configuration ultimately affects the variance of the model estimates, hence their reliability. As an example, Table 3 shows the variation in the parameter estimates and the regression diagnostics determined by the two alternative neighborhood configurations depicted in Figure 5 . The regression model analyzes the socio-economic variables significantly associated with cardiac catheterization: the main predictors are family status, income, and educational attainments; the spatial distribution of these variables is used to identify areas of social and economic concern. This model thus provides an effective analytical tool to support policy decisions, providing guidance for the initiation of targeted, localized preventative health measures [32] .
A succinct summary of the parameters associated with each independent variable is presented in Table 3 , along with a small selection of regression diagnostics. While the coefficients (beta) linking each independent to the dependent variable remain substantially unchanged, the Minkowski distance leads to increased values of their associated t test: the t values increase thanks to a reduction of the variance associated with the estimates. All the independent variables benefit, in varying degrees, from the modified distance model. Likewise, the regression diagnostics indicate that the distance model does not appreciably affect the overall goodness of fit (represented by the Differences between road distance and distance metrics Figure 3 Differences between road distance and distance metrics. pseudo-R 2 ), but it does noticeably impact the autoregressive coefficient and, more importantly, the spatial dependence in the regression residuals.
These results confirm the importance of an accurate distance model to enhance the reliability of spatial analysis for health service research.
This study compares four different distance metrics, i.e., Euclidean, Manhattan, and Minkowski distance (the latter for two different coefficients), and contrasts them with road distance and travel time, respectively, in the context of applied health services research. The Euclidean metric is the most common and intuitive measure of distance; Manhattan is another common distance measure, but very rarely does either of them provide a close approximation of an empirical road network, such as an urban network.
Road distance, conversely, provides an accurate measurement, but it is prone to local features, and does not provide a single model of travel throughout the urban network. Travel time is, arguably, the most relevant estimate of distance, but its calculation introduces further specificities, as temporal anomalies are added to the local features, further reducing its generality. For these reasons, Minkowski distance is a promising solution: it provides a general model of travel throughout an empirical network; it possesses a large range of parameters, which enhance its flexibility; it can be easily calculated; and it can provide a less crude approximation of travel along a road network.
Euclidean distance is widely used in distance analyses in the literature [25] but it tends to underestimate road distance and travel time. Manhattan distance, on the contrary, tends to overestimate road distance and travel time.
The use of either of these two metrics in any spatial anal-Neighborhood configurations determined by different distance metrics ysis may result in inaccurate results [10] . Ideally, travel time provides the most accurate estimate of a patient's travel from their place of residence to a given health facility [15] . While accessibility studies may profitably employ this method, its use in spatial analytical models is inhibited by the particularity of its calculation, which tends to be heavily affected by local features, both in space and in time.
A simple, empirical optimization procedure led to the identification of the coefficients, in the Minkowski formula, that best approximate road distance and travel time, respectively. Summary statistics and cartographic representations consistently indicate the value p = 1.31 as the best coefficient, i.e., the one that leads to the most accurate approximation of both road distance and travel time.
The model of distance based on this coefficient was experimentally introduced in spatial analytical routines, to define neighborhood connectivity and determine the spatial weight matrix for multivariate spatial regression analysis.
The enhanced reliability of the spatial analytical model based on the optimal distance model far outweighs the cost of the computational procedure that leads to the coefficient selection. The advantage of the procedure discussed in this paper can be best appreciated by considering that empirical measurements of distance and travel time cannot be implemented in spatial analytical modeling, exactly because of their empirical nature and the great impact they receive from local features and conditions. For this reason, the optimized Minkowski coefficient represents a valuable compromise between an approach that is often simplistic (i.e., Euclidean and Manhattan metrics) and the ideal, but impractical sophistication of empirical measurements (i.e., road distance and travel time, respectively).
This study has some limitations. Most importantly, it is limited to one class of distance metrics, i.e., Minkowski, whereas other metrics could be considered: Mahalanobis distance is just one example [34] . There are locational inaccuracies in the patient data [35] as well as in the road network; additional errors are likely to derive from the algorithm used for the distance calculations. Travel time calculations are based on assumptions, referred to as optimal conditions that tend to represent an ideal, but unlikely situation. The hypothesis of optimal conditions should be lifted, and more realistic conditions should be entered in the model, e.g., rush hour, or severe winter weather, and should be considered not just individually, but also jointly. An array of optimal Minkowski coefficients should consequently be calculated for the varying conditions, leading to the final identification of a stochastic optimum. The entire procedure is also based on the strong assumption of a single transportation mode: it can be argued that optimal conditions approximate ambulance travel, but the catheterization registry used in this analysis was not limited to patients who were transported directly from their residence to the tertiary catheterization facility. The APPROACH registry also includes patients initially admitted to hospitals or emergency facilities without catheterization facilities who were subsequently transferred to a tertiary center for this procedure under less urgent circumstances. Accuracy in travel route selection and in travel time estimates are most relevant to patients with more emergent cardiac conditions requiring rapid transportation from their residence to the catheterization facility. This limitation can be addressed by a twofold model, optimizing for ambulance and private vehicle travel. Still, road network travel is a reasonable assumption for urban environments, but is unlikely to be the sole mode of transportation to emergency care from rural and remote locations. The proposed approach was tested, as an example, on a spatial autoregressive model; however, virtually all spatial analytical techniques involve some distance measurement. The impact of alternative distance measurements was examined in this implementation through an analysis of the spatial weight matrix, but distance is likely to impact other analytical techniques in further, different ways [19, 20] .
Given its advantages and limitations, Minkowski distance appears to be most usefully implemented in spatial analytical modeling; however, other useful applications can be envisaged, particularly in geographic areas characterized by paucity or unreliability of spatial data, or by high dynamism. Examples include urban or regional road networks of countries with poor spatial digital records, or characterized by high population mobility or varying transportations routes, for example due to seasonal variations. In all such cases, a model of travel in the area, obtained by the method discussed in this paper, can provide rough, but reasonable distance estimates, potentially useful for facility planning, or as initial input for more sophisticated analyses.
The proposed method provides a single model of travel on an urban road network, via the identification of an optimal coefficient within a class of distance metrics, known as Minkowski metrics. The coefficient can be optimized to approximate different distances, e.g., road distance or travel time, under varying conditions. The resulting distance model can be usefully input in spatial analytical models, providing a method for the estimation of less simplistic spatial analytical models, by means of a more accurate representation of distance. Such models yield more reliable estimates, hence more effective tools for health service planning and management. Evaluation of Five Decontamination Methods for Filtering Facepiece Respirators Concerns have been raised regarding the availability of National Institute for Occupational Safety and Health (NIOSH)-certified N95 filtering facepiece respirators (FFRs) during an influenza pandemic. One possible strategy to mitigate a respirator shortage is to reuse FFRs following a biological decontamination process to render infectious material on the FFR inactive. However, little data exist on the effects of decontamination methods on respirator integrity and performance. This study evaluated five decontamination methods [ultraviolet germicidal irradiation (UVGI), ethylene oxide, vaporized hydrogen peroxide (VHP), microwave oven irradiation, and bleach] using nine models of NIOSH-certified respirators (three models each of N95 FFRs, surgical N95 respirators, and P100 FFRs) to determine which methods should be considered for future research studies. Following treatment by each decontamination method, the FFRs were evaluated for changes in physical appearance, odor, and laboratory performance (filter aerosol penetration and filter airflow resistance). Additional experiments (dry heat laboratory oven exposures, off-gassing, and FFR hydrophobicity) were subsequently conducted to better understand material properties and possible health risks to the respirator user following decontamination. However, this study did not assess the efficiency of the decontamination methods to inactivate viable microorganisms. Microwave oven irradiation melted samples from two FFR models. The remainder of the FFR samples that had been decontaminated had expected levels of filter aerosol penetration and filter airflow resistance. The scent of bleach remained noticeable following overnight drying and low levels of chlorine gas were found to off-gas from bleach-decontaminated FFRs when rehydrated with deionized water. UVGI, ethylene oxide (EtO), and VHP were found to be the most promising decontamination methods; however, concerns remain about the throughput capabilities for EtO and VHP. Further research is needed before any specific decontamination methods can be recommended. During an influenza pandemic, a shortage of filtering facepiece respirators (FFRs) may occur if manufacturing production is unable to meet the demand or if FFR stockpiles become depleted. According to a 2006 report from the National Academies' Institute of Medicine, over 90 million N95 FFRs will be needed to protect workers in the healthcare sector during a 42-day influenza pandemic outbreak (Bailar et al., 2006) . Guidance provided by the Centers for Disease Control and Prevention (CDC) states that once an FFR is worn in the presence of an infected patient, it should be considered potentially contaminated and not be reused by the same person or a coworker (CDC, 2007) . A contaminated FFR could potentially serve as a fomite and lead to self-inoculation or spread of the organism to patients and other healthcare workers. Guidance from the Occupational Safety and Health Administration (OSHA) considers *Author to whom correspondence should be addressed. Tel: +412-386-4001; fax: +412-386-6864; e-mail: rshaffer@cdc.gov FFRs to be one-time-use devices when used in the presence of infected patients and advises employers and employees to only reuse FFRs during a pandemic if FFRs are in short supply and the device has not been obviously soiled or damaged (e.g. creased or torn), and it retains its ability to function properly (OSHA, 2007) .
One possible strategy to reduce the impact of a respirator shortage would be to apply a biological decontamination process (e.g. such as those used in hospital settings for infection control) to inactivate the influenza virus that may be on the FFR. If the treatment did not deteriorate the FFR or leave potentially toxic residues on the FFR, then it could be available for subsequent reuse by the original user. Until recently, no data were published on the effects of decontamination on FFR performance. Viscusi et al. (2007) measured the laboratory filtration performance of one N95 model and one P100 model FFR that were exposed to 20 different biological decontamination treatments. They found that filtration performance after onetime decontamination treatments using bleach, ethylene oxide (EtO), microwave oven irradiation, ultraviolet germicidal irradiation (UVGI), and hydrogen peroxide (vaporized and liquid forms) was observed to have filter aerosol penetration values that remained less than the National Institute for Occupational Safety and Health (NIOSH) certification criteria. It was also found that decontamination using an autoclave, 160°C dry heat, 70% isopropyl alcohol, and soap and water (20-min soak) caused significant degradation to filtration efficiency.
Expanding on that research, the goal of this study was to further evaluate five of the decontamination methods examined in the previous study using a more diverse set of nine models of NIOSH-certified FFRs to determine which decontamination methods should be considered for future research studies. The biological decontamination methods used in this study include: (i) UVGI, (ii) EtO, (iii) vaporized hydrogen peroxide (VHP), (iv) microwave oven irradiation, and (v) 0.6% aqueous solution of sodium hypochlorite (hereafter referred to as 'bleach'). Following treatment by each decontamination method, FFRs were evaluated for changes in physical appearance/odor (observational analysis) and laboratory performance (filter aerosol penetration and filter airflow resistance). Additional experiments were then conducted to examine the material properties of the FFRs in an attempt to rationalize some of the findings in the laboratory performance evaluation and observational analysis. The advantages and disadvantages of the various decontamination methods (including throughput capacity and possible health risk to the user) were also assessed.
Nine respirator models were used in this study, of which six models [three N95 FFR models (N95-A, N95-B, and N95-C) and three surgical N95 respirator models (SN95-D, SN95-E, and SN95-F)] constitute a random sampling from those N95 FFR models present in the US Strategic National Stockpile (SNS). Healthcare workers often use surgical N95 respirators, which are NIOSH-approved N95 FFRs that also have been cleared by the US Food and Drug Administration (FDA) for marketing as medical devices. Surgical N95 respirators are designed to be fluid resistant to splash and spatter of blood and other infectious materials and thus may respond differently to the decontamination processes than N95 FFRs. Three models of P100 FFRs (P100-G, P100-H, and P100-I) were randomly selected from models commercially available at the time of the study and included because they were considered likely to be more resistant to filtration efficiency degradation and thus offer a more rigorous basis of comparison. All respirators were purchased and verified to be from the same respective manufacturing lot at the beginning of the study to minimize any lot-to-lot variation as well as to ensure consistency during FFR filtration performance testing. FFRs used in this study consisted of electrostatically charged polypropylene filters (electret filter media).
The experimental conditions and parameters for the five decontamination methods and the 'asreceived' (control) method are summarized in Table 1 . All laboratory experiments were conducted under standard laboratory conditions (21 -2°C and relative humidity of 50 -10%) on triplicate sets of FFRs.
Observational analysis. All post-decontamination and control FFR samples were inspected and scrutinized carefully for any visible sign of degradation or changes that could be noted in texture or 'feel' of the respirator (softness, pliability, coarseness, roughness, etc.). All samples were sniffed for any discernible odor or smell.
Filter aerosol penetration. A Model 8130 Automated Filter Tester (AFT) (TSI, Inc., St Paul, MN, USA) was used to measure initial filter aerosol penetration for all post-decontamination and control FFR samples. All tests were conducted at room temperature with a continuous airflow of 85 -4 l min À1 in accordance with NIOSH certification test procedures (NIOSH, 2007) for challenging N-series filters, with two exceptions: all filters were tested for filter aerosol penetration without any relative humidity pretreatment or NaCl aerosol loading. Collecting the data in this manner allows consistency with previous work (Viscusi et al., 2007) . Filter aerosol penetration levels were determined using a Plexiglas test box as previously used and described by Viscusi et al. (2007) or an appropriately sized test fixture supplied by the respective FFR manufacturer, as was the case for models N95-C, SN95-D, and P100-H.
Filter airflow resistance. For all control and postdecontamination FFR samples, a TSI Model 8130 AFT was also used to measure initial filter airflow resistance in millimeters of water column height pressure (mmH 2 O). It must be clarified that the NIOSH certification test for inhalation airflow resistance for FFRs is not performed using the TSI 8130 AFT but is executed in accordance with NIOSH Standard Test Procedure RCT-APR-STP-0007, which specifies the use of a different calibrated apparatus incorporating a vacuum source and manometer (NIOSH, 2005) . For this evaluation, it was convenient to report the filter airflow resistance obtained from the TSI Model 8130 AFT because filter aerosol penetration and filter airflow resistance results are generated simultaneously and the intent is to determine changes in filter airflow resistance. This methodology was used previously by the National Personal Protective Technology Laboratory (NPPTL) .
The primary experimental design called for 162 FFRs (nine different FFR models  six test conditions  three samples per test condition) to be tested by observational analysis, for filter airflow resistance and for filter aerosol penetration. The 162 FFRs in the design included 135 post-decontamination FFRs and 27 control FFRs (no decontamination).
For statistical analysis, the six test conditions (see Table 1 ) comprised one control group and five decontamination treatments. A one-way analysis of variance (ANOVA) test was performed for each of the nine FFR models for filter aerosol penetration and filter airflow resistance (for 18 total tests). Thus, each model was treated independently due to its inherent uniqueness (difference in number of filter layers, hydrophobicity, materials of construction, etc.). Results were considered statistically significant if the P-value was ,0.05. Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, part of Microsoft Office Professional Edition 2003) . No statistical analysis of the subjective observational analysis data was done.
Additional secondary experiments were subsequently conducted on the FFRs to understand better their material properties. This information can be . FFRs and a chemical indicator placed in an individual standard poly/paper pouch. EtO exposure for 1 h followed by 4 h of aeration. FFRs were shipped to and from a commercial facility specializing in low-temperature sterilization methods and were tested within 72 h of receipt.
VHP STERRADÒ 100S H 2 O 2 Gas Plasma Sterilizer (Advanced Sterilization Products, Irvine, CA, USA), single 55-min standard cycle. FFRs and a chemical indicator placed in an individual Mylar/Tyvekä self-seal pouch. FFRs were shipped to and from a commercial facility specializing in low-temperature sterilization methods and were tested within 72 h of receipt.
Commercially available 2450 MHz, Sharp Model R-305KS (Sharp Electronics, Mahwah, NJ, USA) microwave oven with revolving glass carousel, 1100 W (manufacturer rated); 750 W ft À3 experimentally measured; 2-min total exposure (1 min each side of FFR). A paper towel was placed on the revolving glass plate for insulation to protect the FFRs from melting onto the glass plate. Using a power setting of 10 (maximum power), FFRs were placed faceseal-side down, initially, to reduce the risk of faceseal component materials melting onto the paper towel due to elevated temperatures reached by the glass plate when microwaved for 2 min. Ambient cooling of the glass plate was maintained between trials.
Thirty minutes submersion in 0.6% (one part bleach to nine parts of deionized water) aqueous solution of sodium hypochlorite (original concentration 5 6% available as Cl 2 ). Manufacturing specification: 6.00 -0.06% (w/w) available chlorine; Cat no. 7495.7-1, CAS no. 7732-18-5 (Ricca Chemical Company, Pequannock, NJ, USA). After treatment, FFRs were hung on a laboratory pegboard and allowed to air-dry overnight with assistance from a freestanding fan.
Evaluation of decontamination methods for FFRs 817 used to further optimize the decontamination methods and/or explain some of the findings from the observational analyses or laboratory performance evaluation experiments. Dry oven experiments. To investigate the effects on filter aerosol penetration at various dry heat temperatures and to determine if these effects were similar to those of FFRs that underwent microwave oven irradiation, new FFRs were placed in a Fisher Scientific Isotemp 500 Series laboratory oven (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at temperatures ranging from 80 to 120°C. Filter aerosol penetration was measured after samples cooled to ambient temperature.
Hydrophobicity testing. A qualitative assessment of water affinity for each FFR filter media layer was performed to determine the hydrophobic/ hydrophilic nature of the various layers for the nine different FFR models. For this experiment, it was hypothesized that the number of layers and the nature of the outer layer (surface of the FFR most distant from the wearer) and the inner layer (surface of the FFR closest to the breathing zone of the wearer) would provide insight into any model-specific effects associated with liquid chemical-based decontamination methods. A circular swatch ($5 cm in diameter) was cut from additional, new as-received samples of each FFR model. Following layer separations, a 100 ll aliquot of deionized water was pipetted onto the surface of each side of each layer (front and back). Two FFR models incorporated layers of plastic webbing, presumably to support shape; these layers were not tested because they are not filtering layers. A layer was noted as hydrophilic when it absorbed the water droplet. A layer was noted as hydrophobic when the water droplet beaded on the layer's surface.
Chlorine off-gassing experiments. To quantify observations of discernable odor from FFRs following bleach decontamination, a series of off-gassing experiments was conducted using a Model 4340 Chlorine Gas Analyzer (Interscan Corp., Chatsworth, CA, USA). Chlorine off-gassing was measured from FFRs after bleach treatment as described in Table 1 . A subset of four FFR models was chosen for testing based on the various combinations of water repellency discerned from the hydrophobicity experiments described previously: N95-A (outer hydrophobic layer/inner hydrophilic layer), N95-B (outer and inner hydrophilic layers), SN95-E (outer and inner hydrophobic layers), and SN95-F (outer hydrophobic layer/inner hydrophilic layer). Bleach off-gassing tests were conducted after a bleach decontamination treatment by immediately placing the FFR face up inside a plastic bag which was open to room air on one side. This setup was designed to minimize air fluctuation within the bag. The detector's sample tube inlet was positioned under the inside of the FFR and all tests were conducted at a flow rate of 0.5 l min À1 . FFRs were tested under four conditions: (i) immediately after a 30-min submersion in bleach, (ii) dried overnight after a 30-min submersion in bleach, (iii) a 30-min submersion in bleach, immediately rinsed (under a flowing stream of deionized water for $1 min) and then dried overnight, and (iv) a 30-min submersion in bleach, then dried overnight followed by rinsing with deionized water.
Changes to the FFR materials of construction caused by each decontamination treatment are summarized in Table 2 . Respirator component materials melted on all six FFRs from two models (SN95-E and P100-I) during microwave oven irradiation. EtO and UVGI were the only methods that did not cause any observable physical changes to the FFRs.
For each 'FFR model/decontamination treatment' combination, the average initial filter aerosol penetrations are summarized in Table 3 . Not all the 135 All three physical samples of two different models (SN95-E and P100-I) melted partially. SN95-E filtration material melted in areas adjacent to the metallic nosebands. P100-I melted in various locations of the inner foam faceseal comfort lining. Both models were considered unwearable following treatment and subsequently were not evaluated for filter aerosol penetration or filter airflow resistance. These results indicate that for all tested FFR samples that did not melt, FFR filtration performance was not adversely affected by the decontamination process. Most of the ANOVA tests for initial filter aerosol penetration were nonsignificant (P . 0.05), (Table 4 ). In terms of average initial filter aerosol penetration, only P100-I yielded a significant difference by treatment (P 5 0.0438), which appeared to be primarily driven by the increased filter aerosol penetration levels for the UVGI treatment (0.012 versus 0.008% for the control). Although statistically significant, this difference in levels of filter aerosol penetration is practically insignificant because the penetration levels still are far less than expected levels for this class of FFRs (,0.03%).
For each 'FFR model/decontamination treatment' combination, the average initial filter airflow resistances are summarized in Table 3 . The six FFRs in which melting occurred could not be tested for filter airflow resistance. For the remaining 129 postdecontamination samples tested, average initial filter airflow resistance measurements were 17.0 mm H 2 O. Previous studies using the same test method on 21 models of NIOSH-approved N95 FFRs observed filter airflow resistance levels between 7 and 30 mmH 2 O . For filter airflow resistance, three of the nine ANOVA tests, including N95-B (P 5 0.0035), SN95-D (P 5 0.0170), and SN95-F (P 5 0.0014), showed significantly different means (see Table 4 ). Although statistically significant, the levels of differences in filter airflow resistance between treatments are not practically meaningful as small changes in filter airflow resistance are unlikely to be noticed by the user (Vojtko et al., 2008) . 
The degree to which temperature affects initial filter aerosol penetration and component melting was observed to be model specific (Figs 1 and 2) . The average initial penetration (n 5 3) for each N95 model is shown in Fig. 1 . Only three tested N95 FFR samples had filter aerosol penetrations .5% (therefore failed to maintain their expected filtration efficiency level of !95%). These three failing samples were one SN95-D (5.37% at 110°C) and two N95-C (5.18 and 5.37%, both at 120°C). Five of the SN95-D samples could not be analyzed following treatments of 100°C (one sample), 110°C (two samples), and 120°C (two samples) because their inner moisture barrier melted into the filtration media rendering those samples unsuitable for testing. For the three P100 FFR models, average initial filter aerosol pene-tration values for P100-G and P100-H exceeded 0.03% beginning at 100°C for P100-G and beginning at 90°C for P100-H (Fig. 2) . P100-I averaged an initial filter aerosol penetration value ,0.03% for all evaluated temperature increments with the exception of one 110°C temperature experiment. This unexpectedly high average result was due to a single test (%P 5 0.096).
All nine FFR models demonstrated differences in their number of media layers and the hydrophobicity of their filter media (Table 5 ). Common to all three models of surgical N95 respirator was the fact that their outer layer was hydrophobic. This is not surprising since surgical N95 respirators cleared by the US FDA undergo fluid resistance testing and are used as barriers against disease transmission by airborne respiratory fluids, including blood, and other small infectious droplets (Bailar et al., 2006) . The N95 FFRs and P100 FFRs varied by having either hydrophobic or hydrophilic outer and inner layers. All middle layers, with the exception of those that were plastic webbing, were hydrophobic on both sides.
Initial concentrations of chlorine gas (2-12 p.p.m.) were measured on FFRs wet with bleach immediately following submersion for 30 min (Fig. 3) . FFRs that were treated using bleach and allowed to air-dry overnight (as described in Table 1 ) had initial concentrations of $0.05 p.p.m. followed by no detectable off-gassing (0 p.p.m.) after the initial data point. FFRs which were submerged in bleach, immediately rinsed (entirely under a stream of deionized water for $1 min) and then allowed to air-dry overnight had concentrations similar to FFRs which were not rinsed, indicating that the water rinse had no effect. When FFRs were rehydrated by rinsing with deionized water following overnight air-drying, low-level chlorine off-gassing concentrations were measured at $0.1 p.p.m. (Fig. 4) . 
The goal of this study was to evaluate five decontamination methods using nine FFR models from three FFR types (three N95 models, three surgical N95 respirator models, and three P100 models) to determine which methods should be considered for future research studies. The five decontamination methods were selected based on previous research from the NPPTL laboratory (Viscusi et al., 2007) . Criteria for assessing methods of decontaminating disposable N95 FFRs have been suggested by the National Academies (Bailar et al., 2006) ; the decontamination method must remove the viral threat, be harmless to the user, and not compromise the integrity of the various elements of the respirator. This manuscript utilizes and expands upon the second and third criteria. For purposes of discussion, a suc-cessful FFR decontamination method is considered to be a physical or chemical treatment which does not degrade laboratory performance (filter aerosol penetration and filter airflow resistance) beyond expected performance levels, is able to be performed on enough FFRs in a short period of time to be practical in the event of a pandemic-induced shortage, and should not pose any additional health risk to the user. In this study, assessment of potential health risks (e.g. possible dermal contact with residuals and/or inhalation of off-gassing residuals) was done using the observational analysis data, off-gassing test results, and general knowledge of the physical/ chemical characteristics of the decontamination method. Chemical off-gassing is of particular concern because of the close proximity of the FFR to the wearer's face and breathing zone. A limited assessment of the throughput capability was also done Evaluation of decontamination methods for FFRs 823 using general knowledge of the various decontamination methods. Additional studies on dry heat laboratory oven exposure and FFR media layer hydrophobicity were conducted to collect data on various aspects of FFR resilience and construction in order to further optimize decontamination strategies and assess the practicality for FFR decontamination during a shortage. In the following sections, the results of laboratory performance testing and observational analysis, additional testing, and assessment of throughput and health concerns will be discussed for each of the five decontamination methods evaluated in order to provide recommendations on which decontamination methods should be considered in future research studies.
Bleach is available as an aqueous solutions containing 5-15% sodium hypochlorite (active ingredient) which is a highly active oxidizing agent known to be effective against a broad spectrum of bacteria and viruses (Rutala and Weber, 1997; McDonnell and Russell, 1999) . Bleach decontamination did not affect the FFRs' filter aerosol penetration and filter airflow resistance. The metallic nosebands of all models that had them were slightly tarnished following decontamination and the inner nose cushion on the SN95-E FFRs was discolored. Throughput capability of a bleach method similar to the one used in this study is likely to be high; the main limiting factors are the size of the vessel containing the bleach and FFRs, adequate space to dry the FFRs, and sufficient time for air-drying.
All FFR models had a scent of bleach following overnight air-drying. Residual bleach remaining on FFRs is of concern given its known health effects. Hypochlorite powder, solutions, and vapor can be irritating and corrosive to the eyes, skin, and respiratory tract. For example, Nixon et al. (1975) reported that a 5.25% sodium hypochlorite solution caused severe irritation to human skin over a 4-h exposure. Other studies also reported skin irritation for long-term exposure down to a 1% solution (Eun et al., 1984; Habetes et al., 1986; Hostynek et al., 1990) . Low concentrations of bleach have been shown to trigger respiratory events in asthmatics and sensitized individuals (Medina-Ramon, 2005; Mirabelli et al., 2007) . The chlorine off-gassing measurements showed that overnight air-drying significantly reduced off-gassing; however, when the FFR was rehydrated with deionized water, an increase in offgassing was measured. This observation may be significant when viewed in light of the moisture in the exhaled breath of an individual; it gives rise to the possibility of an individual being exposed to low levels of chlorine (,0.2 p.p.m.) from a bleachdecontaminated FFR. Comparing Table 5 and data shown in Fig. 4 , a relationship between hydrophobic-ity of outer and inner respirator surface layers to offgassing concentration could not be established.
Considering the potential health risks, the bleach method evaluated in this study is not recommended for further study without modification. Possible modifications worth further investigation would include reduced initial bleach concentration, chemical methods for neutralizing residuals, additional rinse steps, and more aggressive air-drying procedures.
EtO is used in a wide range of work settings as a sterilant or fumigant, including healthcare, diagnosis, and treatment facilities; medical products manufacturing; and libraries and museums (NIOSH, 1981) . EtO decontamination did not affect the filter aerosol penetration, filter airflow resistance, or physical appearance of the FFRs in this study. The EtO process used in this study has a 5-h total processing cycle (1-h EtO exposure followed by 4 h of aeration) and has a 4.8 ft 3 (0.14 m 3 ) chamber volume (3M, 2007) . The 5-h total processing time may be a limiting factor in the timely processing of a large volume of FFRs. Residual EtO remaining on FFRs following EtO vapor-phase decontamination is not believed to be a concern because the sterilization process includes a final aeration cycle of 4 h to remove residual EtO gas.
VHP has been shown to be sporicidal at temperatures ranging from 4 to 80°C, with sterilant concentrations ranging from 0.5 to ,10 mg l À1 (Joslyn, 1991) . VHP decontamination for a single warm cycle did not significantly affect FFR filter aerosol penetration or filter airflow resistance. The only visible physical effect on the FFRs was a slight tarnishing of the metallic nosebands. The VHP process used in this study has a short cycle time (55 min) and a usable processing volume of 3.5 ft 3 (0.1 m 3 ) (Advanced Sterilization Products, 2007) . Although the 55-min cycle time is short compared to the lengthy EtO total process time, the throughput capability of VHP processing is limited by the fact that cellulose-based products (e.g. cotton, which may be present in some head straps or some FFR layers) absorb hydrogen peroxide and can cause the STERRADÒ cycle to abort due to low hydrogen peroxide vapor concentration. Significant levels of residual hydrogen peroxide vapors off-gassing from FFR materials following the STERRADÒ process are unlikely and not of concern because the vapors decompose readily into water vapor and oxygen, both of which are environmentally benign (Advanced Sterilization Products, 2007) .
Biological decontamination of FFRs using a domestic microwave oven is an attractive idea since it has the advantages of convenience and short treatment times. The decontamination method used here treats the microwave oven as a source of dry heat, similar to other studies. Elhafi et al. (2004) demonstrated that four avian viruses (infectious bronchitis virus, avian pneumovirus, Newcastle disease virus, and avian influenza virus) were inactivated on dried cotton swab samples using a domestic microwave oven for as little as 20 s. Rosaspina et al. (1994) demonstrated destruction of Mycobacterium bovis dried onto scalpel blades after 4 min of microwave exposure.
Of the nine FFR models that underwent microwave oven irradiation, filter aerosol penetration and filter airflow resistance were not affected for seven models. Material components melted on the two remaining models. Correlation could not be established for filter aerosol penetration results between dry oven-treated and microwave oven-irradiated samples. In microwave oven irradiation tests, all three SN95-D samples had penetration values ,5% and did not melt; however, some SN95-D samples partially melted at 100, 110, and 120°C during dry oven treatment (Fig. 1) . All SN95-E samples and all P100-I samples partially melted in the microwave oven, but no melting was observed for these two models, even at 120°C following dry oven treatment (Table 3 , Figs 1 and 2) .
The throughput capability of a method similar to the one in this study was limited by microwaving one FFR at a time; however, the 2-min treatment time per FFR was relatively short. Although it is likely that processing more than one FFR at a time is feasible (limited only by the internal volume of the oven), maximizing throughput was beyond the scope of this investigation. No known health risks to the user were identified. The data presented here suggest that the dry microwave oven irradiation method requires improvement before it could be recommended for decontamination and subsequent reuse. Possible modifications worth further investigation would include microwave irradiation of wet FFRs, shorter exposure times, and lower power settings.
UVGI has been demonstrated to be effective for the disinfection of drinking water and wastewater (Sykes, 1965; Angehrn, 1984; Lazarova et al., 1999; Craik et al., 2001; Lazarova and Savoye, 2004; Wu et al., 2005) and for hospital air disinfection as a method for controlling airborne infectious disease (Macher et al., 1992; Nardell, 1993; CDC, 1994; Gorsuch et al., 1998; Miller and Macher, 2000) . This study found that UVGI treatment did not affect the filter aerosol penetration, filter airflow resistance, or physical appearance of the FFRs. Throughput capability of a method similar to the one in this study is benefited by a relatively short ir-radiation time (30 min); however, it is limited by the available working surface area of a biosafety cabinet equipped with a UV-C source or other area being irradiated by a UVGI source. No known health risks to the user were identified.
These findings are exploratory and the data presented in this study are applicable only to the FFRs and decontamination methods tested; other FFRs may be more easily degraded while others may be less affected and slight modifications to the decontamination methods could result in different findings. Future studies are still needed to evaluate whether the decontamination processes evaluated in this study will inactivate infectious microorganisms (or appropriate surrogates), if FFR decontamination influences respirator fit, and the effect of multiple decontamination treatments on FFR performance. Future studies should also investigate the depths that infectious organisms (or appropriate surrogates) penetrate into each FFR layer, assess the relative cost of various decontamination strategies, and determine how effective various decontamination methods are at reducing the number of viable virus in all layers of the FFRs. Recent work in the NPPTL laboratory toward developing a system for studying the virucidal capability of decontamination methods for FFRs appears promising (Fisher et al., 2009) .
The effects of the various decontamination methods on the laboratory performance (filter aerosol penetration and filter airflow resistance) and physical appearance of FFRs were found to be model specific. The respirators tested have differences in their design, materials of construction, and hydrophobicity of their layers (including the filter media layers). Microwave oven irradiation melted all six samples from two FFR models. The remainder of the FFR samples that were evaluated exhibited average initial filter airflow resistances 17.0 mmH 2 O and average initial sodium chloride filter aerosol penetration values 1.86% for N95 FFRs and 0.012% for P100 FFRs. Although there were statistically significant differences found between control respirators and those that have undergone decontamination for both filter aerosol penetration and filter airflow resistance, the practical significance is minimal as the range of numerical differences is quite small. The scent of bleach remained noticeable on all FFR models following overnight drying and low levels of chlorine were found to off-gas from bleach-decontaminated FFRs when rehydrated with deionized water, thus giving rise to the possibility of low-level exposure to a subsequent wearer.
In light of these results, the microwave oven irradiation and bleach decontamination methods investigated in this study were determined to be the least desirable among the five methods tested for consideration in future studies. UVGI, EtO, and VHP were found to be the most promising decontamination methods; however, concerns remain about the throughput capabilities for EtO and VHP. Further research is needed before any specific decontamination methods can be recommended.  Antiviral Activity of Some Plants Used in Nepalese Traditional Medicine Methanolic extracts of 41 plant species belonging to 27 families used in the traditional medicine in Nepal have been investigated for in vitro antiviral activity against Herpes simplex virus type 1 (HSV-1) and influenza virus A by dye uptake assay in the systems HSV-1/Vero cells and influenza virus A/MDCK cells. The extracts of Astilbe rivularis, Bergenia ciliata, Cassiope fastigiata and Thymus linearis showed potent anti-herpes viral activity. The extracts of Allium oreoprasum, Androsace strigilosa, Asparagus filicinus, Astilbe rivularis, Bergenia ciliata and Verbascum thapsus exhibited strong anti-influenza viral activity. Only the extracts of A. rivularis and B. ciliata demonstrated remarkable activity against both viruses. Plants have long been used as a source of medicine from ancient time to today all over the world. In developing countries the availability of modern medicines is limited. So traditional medicine is still the mainstay of health care and most drugs come from plants. Although many plants have long been recognized and widely used in Nepalese traditional medicine, some are relatively unexplored and not arrived to mainstream medicine (1) . Therefore, the search on new drugs must be continued and natural products from plants, microorganisms, fungi and animals can be the source of innovative and powerful therapeutic agents for newer, safer and affordable medicines (2, 3) . On the other hand the screening of plants as a possible source of antiviral drugs has led to the discovery of potent inhibitors of in vitro viral growth (4) (5) (6) (7) (8) (9) (10) (11) .
Therefore, the present investigation was carried out to assess the antiviral effects of some native plants used by the local people belonging to Gurungs and Thakalis of Manang and Mustang districts that lie in the Annapurna Conservation Area Project (ACAP). Permission for the field study as well as the collection of voucher specimens was received from the headquarters of ACAP in Pokhara. The plants were selected on the basis of ethnopharmacological records, so the prospect of finding new bioactive compounds is always promising. The name  of the plants, respective families, the parts used for the  extract preparation and traditional uses of the plants are  listed in Table 1 .
The dried and powdered plant material (each 10 g) was extracted successively with n-hexane, dichloromethane and methanol in a soxhlet extractor for each 8 h. Evaporation of the solvent followed by drying in vacuum gave the respective crude dry extract. Only methanol extract was used for the antiviral assay, n-hexane and dichloromethane extracts were not included because of their insolubility in medium and high toxicity to the cells. Each 2 mg of the extract was dissolved in 10 ml dimethylsulfoxide (DMSO) before adding tissue culture medium supplemented with 2% fetal calf serum (FCS, GIBCO Life science technologies, Paisley, UK) and stocked at a concentration of 2 mg ml À1 .
Madine-darby canine kidney (MDCK) and African green monkey kidney (Vero) cells (cell bank of the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany) were maintained in Eagle's minimal essential medium (MEM) supplemented with 5% FCS (GIBCO, Paisley, UK). The exponentially growing cells were harvested and seeded at a cell density of 60 000/well in a 96 well microtiter plate (8 mm diameter, Falcon Plastic, NJ) and incubated for 24 h at 37 C with 5% carbondioxide in a 90% humidified chamber so as to form confluent monolayers.
Human influenza virus A/WSN/33 (H1N1) London was obtained from the strain collection of the Institute of Medical Microbiology, University Greifswald, Germany, and propagated in embryonated hen eggs for 72 h. The infected allantoic fluids were harvested, the hemagglutination (HA) titer and virus infectivity were determined on MDCK cells and the virus stock was stored at À70 C.
Herpes simplex virus type 1 (HSV-1, strain KOS) was obtained from the strain collection of the Consiliar and Reference Center for Alpha Herpes Virus Infection, Institute of Virology and Antiviral Therapy, University Jena, Germany and propagated in Vero cells. The virus infected cells were frozen and thawed and the virus suspension was titrated on Vero cells and stored at À70 C (7).
The cellular toxicity of extracts on Vero and on MDCK cells was assessed by dye uptake method using neutral red (12) in 96-well tissue culture plates (8 mm diameter, Falcon Plastic, NJ). Only living cells are able to manage the active uptake of neutral red. Confluent monolayers of cells were treated with 100 ml 2-fold serial dilutions of extracts prepared at concentrations of 200, 100, 50 and 25 mg ml À1 in four replicates and incubated at 37 C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatant was removed and 200 ml neutral red solution (0.005%) in optimum was added. The microtiter plate was further incubated for 3 h at 37 C. After removal of the supernatant, the dye incorporated by the viable cells was extracted with 100 ml ethanol/water/glacial acetic acid solution (50 : 50 : 1) by shaking for 15 min. The absorbance was measured on an ELISA reader using Ascent software at 540 nm. The cytotoxic concentration that caused the reduction of viable cells by 50% [CC 50 ] was calculated from dose-response curve.
Antiviral activity was determined by dye uptake assay using neutral red as described by Mothana et al. (7) . Non-cytotoxic extracts were tested in concentrations of 100, 50, 25, 12.5 and 6.25 mg ml À1 . The antiviral tests of cytotoxic extracts started with the half of the individual CC 50 . The extracts were diluted 1 : 2 by medium. Confluent monolayers of Vero and MDCK cells were treated with 100 ml of extracts in four replicates for 30 min. After that Vero cells were infected with 30 TCID 50 of HSV-1 and MDCK cells with 30 TCID 50 of influenza virus A and incubated for 72 h at 37 C. TCID 50 (tissue culture infectious dose) is the virus dose that leads to the infection of 50% of the cells. The virus suspension and dilution medium without samples were added, respectively, to the cell cultures to serve as the virus control and cell control. The supernatant was replaced by 200 ml neutral red solution (0.005%) and the cells were incubated for 3 h at 37 C. After removal of the supernatant, the dye incorporated by viable cells was eluted with 100 ml ethanol/water/glacial acetic acid solution (50 : 50 : 1) by shaking for 15 min. The absorbance was measured at 540 nm and the percentage protection was calculated by the following formula (13):
where, (OD T ) V , (OD C ) V and (OD C ) M correspond to absorbances in virus infected cells with test compounds, virus infected cells without test compounds and the mock infected control (assay without viruses), respectively.
Amantadine HCl and acyclovir were used as reference compounds in concentrations of 0.1, 1, 10 and 100 mg ml À1 .
In this study, 43 methanolic extracts from 41 different plant species belonging to 27 families (Table 1) were screened for their antiviral activity against herpes simplex virus and influenza virus A by dye uptake assay. By methanolic extraction, a broad spectrum of compounds with different polarity can be obtained. As prerequisite for antiviral tests, the cytotoxicity of the extracts against virus-host cells was investigated. The results are summarized in Table 2 .
The extracts of Androsace strigilosa, Anemone rivularis, Delphinium brunonianum, Euphorbia longifolia and Thalictrum cultratum exhibited strong cytotoxicity in Vero cells with CC 50 (the concentration that causes the reduction of viable cells by 50%) ranging from 12.5 to 25 mg ml À1 . A moderate cytotoxicity was observed for the extracts of Asparagus filicinus, Bergenia ciliata, Primula involucrata and Saussurea auriculata with CC 50 ranging from 30 to 50 mg ml À1 . Other eight extracts showed very mild toxicity while rest of the extracts were non-toxic at 100 mg ml À1 .
Similarly, in MDCK cells extracts of Artemisia caruifolia, D. brunonianum and E. longifolia showed strong toxicity with CC 50 ranging from 19 to 25 mg ml À1 . A moderate toxicity was exhibited by the extracts of A. strigilosa, A. rivularis, Asparagus filicinus, Dicranostigma lactucoides, Hyoscyamus niger, Thymus linearis and Zanthoxylum armatum with CC 50 ranging from 30 to 50 mg ml À1 . Other three extracts demonstrated very low toxicity while rest of the extracts were non-toxic at 100 mg ml À1 .
Antiviral activity against HSV-1 was shown by 11 extracts at non-cytotoxic concentrations. The IC 50 values (the concentration that protects 50% of the cells against destruction by viruses) ranged from <6.25 to 82 mg ml À1 . The highest activity against HSV-1 with IC 50 values <6.25 mg ml À1 was observed for the extracts of A. rivularis, B ciliata, Cassiope fastigiata and T. linearis. Moderate activity was shown by Cotoneaster integrifolius (IC 50 18 mg ml À1 ) and Clinopodium umbrosum (IC 50 19 mg ml À1 ). Weak activity (IC 50 50-82 mg ml À1 ) was found in the extracts of Bistorta affinis, Juniperus squamata, Oxytropis williamsii, Rhododendron anthopogon and Rubus foliolosus.
Antiviral activity against influenza virus A was shown by 20 extracts at non-cytotoxic concentrations. The IC 50 values ranged from <6.25 to 97 mg ml À1 . The highest activity was shown by the extracts of A. filicinus, A. rivularis and Verbascum thapsus with IC 50 < 6.25 mg ml À1 . In addition, the extracts of Allium oreoprasum, A. strigilosa and B. ciliata also exhibited high activity (IC 50 values from 8 to 10 mg ml À1 ). Moderate activity (IC 50 values from 17 to 50 mg ml À1 ) was demonstrated by 11 extracts. Weak activity (IC 50 values from 78 to 97 mg ml À1 ) was shown by three extracts ( Table 2 ). The extracts of A. rivularis and B. ciliata were found to be highly active against both viruses.
The results of this work justify the potential of some of the investigated plants for the production of bioactive compounds. The phytochemical knowledge about these plants is so far very limited. The active principles present in A. rivularis are still unknown. Phytochemical investigation of A. rivularis revealed the presence of flavonoids, terpenoids and bergenin (14, 15) .
Bergnia ciliata is known to contain phenolic compounds (16) . Polyphenols, especially high polymeric procyanidines possess strong anti-influenza viral activity (17) , which is in agreement with our previous study (18) . In our previous study (19) , methanol-water extract of Bergenia ligulata, which is taxonomically closely related to B. ciliata, inhibited the growth of influenza virus A in cell culture with IC 50 of 10 mg ml À1 . The extract also inhibited the viral protein and nucleic acid synthesis (18) . In the present study, the methanol extract of B. ciliata inhibited the influenza virus A and HSV-1 indicating that the genus Bergenia could be the source of potent antiviral drugs. Again potent activity of A. rivularis against both viruses indicated the high prospect of finding antiviral drugs in Saxifragaceae family.
No antiviral compounds have previously been isolated from A. filicinus. The plant is known to contain steroidal saponins (20, 21) , furostanol glycosides (22) and furostanosides (23, 24) . The phytochemicals possibly responsible for the high activity of C. fastigiata against HSV are not described. Some Cassiope species are reported to contain flavonoid glycosides (25) . Similarly, the compounds responsible for the high anti-influenza viral activity of A. oreoprasum and A. strigilosa are not reported elsewhere.
Likewise, no antiviral constituents have been isolated from C. integrifolius, C. umbrosum and T. linearis. Other members of the genus Cotoneaster, have been found to possess phenolic glycosides (Cotoneaster orbicularis, 26), flavonols and isoflavones (Cotoneaster simonsii, 27). From the other member of the genus Clinopodium, C. chinensis var. parviflorum, oleanane triterpene saponins have been isolated (28) .
Whereas for the extract of V. thapsus, antiherpes activity has been reported (29); our study revealed only the strong anti-influenza viral activity. However, no antiviral compounds have previously been isolated. The plant is known to contain phenylethanoid and lignan glycosides (30) . On the other hand, the phytochemicals responsible for anti-influenza viral activity could be different from anti-herpes activity and also the amount of active constituents present in the plants depends on the geographical distribution, season of collection and climatic and ecological condition at the collection site. Looking at the chemical structures of the already identified compounds, most of these substances should be extracted by methanol. The foregoing extraction by more lipophilic solvents (n-hexane and dichlormethane) alleviates the methanolic extraction and the planned fractionation.
Comparing the use of plants in traditional medicine and their antiviral activity, a direct correlation could be established for some plants, e.g. A. oreoprasum, A. strigilosa (anti-influenza activity) and T. linearis (antiherpes activity). For other plants, e.g. C. fastigiata, which exhibited potent anti-herpes activity, this cannot be recognized till now.
The extracts that exhibited only medium and low activity, could also be the source of potential antiviral drugs because the bioactive compounds may be present in too low concentrations to show effective antiviral activity at non-toxic concentration. Further fractionation and separation of extract(s) may reveal potent antiviral activity (31) .
Our results indicate that several plants used in Nepalese traditional medicine could be the lead to potential antiviral drugs, which possibly provide molecules with drug-like properties and with incredible structural diversity. Besides, the results are useful for rationalizing the use of medicinal plants in primary health care in Nepal. The phytochemical characterization of the extracts, the identification of the responsible bioactive compounds and the elucidation of the mode of action and quality standards are necessary. Predicting intention to treat HIV-infected patients among Tanzanian and Sudanese medical and dental students using the theory of planned behaviour - a cross sectional study BACKGROUND: The HIV epidemic poses significant challenges to the low income countries in sub Saharan Africa (SSA), affecting the attrition rate among health care workers, their level of motivation, and absenteeism from work. Little is known about how to deal with deterioration of human resources in the health care systems. This study aimed to predict the intention to provide surgical treatment to HIV infected patients among medical- and dental students in Tanzania and Sudan using an extended version of the Theory of Planned Behaviour (TPB). METHODS: Four hundred and seventy five medical- and dental students at the University of Dar es Salaam (mean age, 25 yr) and 642 dental students attending 6 public and private dental faculties in Khartoum (mean age 21.7 yr) completed self-administered TPB questionnaires in 2005 and 2007, respectively. RESULTS: Both Tanzanian and Sudanese students demonstrated strong intentions to provide care for people with HIV and AIDS. Stepwise linear regression revealed that the TPB accounted for 51% (43% in Tanzania and Sudan) of the variance in intention across study sites. After having controlled for country and past behaviour, the TPB in terms of attitudes, subjective norms and perceived behavioural control accounted for 34% and moral norms for an additional 2,3% of the explainable variance in intention. Across both study sites, attitudes were the strongest predictor of intention followed in descending order by subjective norms, moral norms and perceived behavioural control. CONCLUSION: The TPB is applicable to students' care delivery intentions in the context of HIV and AIDS across the two SSA countries investigated. It is suggested that attitudes, subjective norms, moral norms and perceived behavioural control are key factors in students' willingness to treat AIDS and HIV infected patients and should be targets of interventions aimed at improving the quality of health care delivery in this context. is currently recognized by the World Health Organization to suffer an intermediate HIV and AIDS prevalence of 1.6% [1] . The HIV epidemic poses significant development challenges to the low income countries in SSA [2] . It affects the attrition rate among health care workers, their level of motivation, professional practices and absenteeism from work [2] . To date, little is known about how to deal with the deterioration of human resources in the health care systems.
Due to the method of transmission of HIV virus through direct contact with blood, the risk for cross-infection comes into particular focus in medical and dental practices [3] . Over 90% of the HIV infections occurring among health care workers annually stem from developing countries where occupational safety is a neglected issue [4] . Although the risk of transmission in health care settings has been recognized to be low, fear of illness, contagion and death has influenced health workers' attitudes and thus the quality of care provided towards patients with HIV [5] [6] [7] . Increased personal risk, lack of necessary skills, knowledge gaps, difficulties in dealing with staff worries and concern about loosing other patients are the most frequent complaints [8] [9] [10] [11] [12] . A recent publication focusing on dental students in Khartoum, Sudan, revealed that half of the participants reported a need for further education across HIV and AIDS related issues, suggesting they are not adequately prepared for treating HIV infected patients [8] . Unacceptable knowledge and practice as well as gaps in the availability and access to policies and protocols on the part of health care workers have been observed in several sub-Sahara African countries [11, [13] [14] [15] [16] [17] . According to the World Health Organization (WHO), dentists have a professional and ethical responsibility to treat patients with HIV and AIDS [18] [19] [20] . The importance of training dental and medical staff to provide health care to HIV infected patients at the same level as non-infected people have been widely recognized [17, 19] . As future health care workers, the attitudes of dental and medical students towards delivering high quality care for HIV infected patients are of particular concern. Effective promotion of quality health care delivery in the context of HIV and AIDS requires a thorough understanding of the psychosocial determinants of students' intention to provide care to HIV infected patients. Ajzen's theory of planned behaviour (TPB) is a valuable model for identifying the determinants of health behaviours, including quality health care provision for patients with HIV and AIDS [21] .
The TPB [21] constitutes a promising framework for understanding and predicting social behaviours ( Figure  1 ). The TPB includes perceived behavioural control on a level with attitude and subjective norm as predictors of behavioural intention. This theory implies that the three predictors influence subsequent behaviour indirectly through behavioural intention. The TPB posits that behavioural intention is a function of attitude, reflecting a favourable or unfavourable evaluation of the particular behaviour and subjective norm, referring to the perceived social pressure to perform the behaviour. Perceived behavioural control reflects the ease or difficulty associated with performance. Attitudes, subjective norms and perceived behavioural control are underpinned by behavioural, normative and control beliefs, respectively. A number of studies have suggested that past behaviour has a residual effect on behavioural intention after the TPB has been taken into account [22, 23] . Thus, Ajzen [21] suggested that the TPB is open to the inclusion of additional variables if it can be shown that they capture a significant proportion of the outcome variance.
The TPB has been applied successfully to a range of domains, including HIV related behaviours, particularly condom use [24] [25] [26] [27] [28] . With respect to occupational behaviour, the TPB has predicted health workers' use of gloves, their intention to provide home-care for HIV infected patients, their adherence to universal precautions for venipuncture and their intention to provide professional labour support [29] [30] [31] [32] [33] . The TPB has been used previously in sub-Saharan African settings to predict HIV protective behaviours, however mostly by small scale studies [27] . It has been advocated that the applicability of socio-cognitive models to the African context should be systematically addressed considering the need for theory-based research in the planning of effective HIV and AIDS related educational programs [34, 35] .
This study extends analyses of external variables within the TPB in the context of health care delivery by adding past behaviour and moral norms from Triandis' Theory of Interpersonal Behaviour [36] . Personal normative belief or moral norms represents a measure of the personal feel-
The theory of planned behaviour (Ajzen 1991) Figure 1 The theory of planned behaviour (Ajzen 1991).
Subjective norm Attitude Intention Behaviour ings of moral obligations or responsibility to perform or refuse to perform a given behaviour. Studies of moral norms in the context of the TPB were reviewed by Conner and Armitage [24] who estimated that across investigations moral norms predicted an additional 4% of the variance in intention after controlling for the TPB. Moreover, studies on the Theory of Interpersonal Behaviour have consistently shown that moral considerations are significant predictors of behavioural intentions in the presence of the TPB [37] . Focusing on medical and dental students from Tanzania and Sudan, this study aims to predict the intention to provide surgical treatment to patients with HIV and AIDS as part of future professional work, using the TPB and moral norms. The hypotheses of the present study were:
-attitudes, subjective norms and perceived behavioural control will each contribute positively and statistically significantly to the prediction of intention to provide surgery treatment to HIV and AIDS infected patients -moral norms will add significantly to the prediction of behavioural intention over and above the TPB
A cross-sectional survey was carried out from June to September 2005 at Muhimbili University College of Health and Allied Sciences (MUHAS) at the University of Dar-es-Salaam. The target population consisted of students attending the faculties of dentistry and medicine. A total of 1,021 (862 medical and 159 dental) students were enrolled at the college in 2005. Six hundred students (100 students in each study year) attending the 1 st to the 5 th study year were invited to participate. A total of 476 students agreed to participate and 454 students (response rate 75.6%), mean age 25.6 (sd 2.6), 22.5% females completed supervised self-administered structured questionnaires at the faculty in class-room settings.
A cross-sectional study was carried out from April to May 2007 among a census of dental students attending dental faculties in Khartoum, the capital city of Sudan. A list of all the dental faculties was obtained from the Ministry of Higher Education and lists of all registered students in their 3 rd , 4 th and 5 th years were obtained from all faculties through the Dean's office. The faculties included in this study were both publicly and privately funded. Moreover, they represented all available dental faculties in Sudan, making potentiating admission from all over the country. The total number of dental students registered by the time of the survey was 782 out of which 642 students (response rate 82%), mean age 21.7, (sd 1.9), 82% females completed self-administered, anonymous questionnaires in supervised (by teaching assistants) class-room settings. The main reason for non-participation was absenteeism on the day of the data collection. More information regarding the sampling, recruitment and data collection is available in a previous publication [8] .
Written informed consent was obtained from all participants in both countries. A formal ethics waiver was received from the research committee at the University of Science and Technology in Sudan, the Research Committee at MUHAS, Tanzania and from the Regional research committee of Norway (REK VEST).
Identical survey instruments were used at both sites. Before administration in the field, the questionnaire was reviewed by experienced local researchers, dental academics and health administrators. The survey instrument was adapted from instruments previously employed in SSA [38] . In Tanzania, the survey instrument was constructed and completed in English. In Sudan, the survey instrument was constructed in English, translated into Arabic (the Sudanese national language) and then back translated into English by independent language experts. The survey instrument covered socio-demographic factors and each component of the TPB developed according to the guidelines proposed by Ajzen and Fishbein [39] .
Intention to provide surgical treatment to patients infected with HIV/AIDS as part of future professional work was measured by three items. E.g. "How likely is it that you will provide--------". The respondents indicated their subjective probability along a five point response scale from (1) very unlikely to (5) very likely. A sum score was constructed from the three items.
Attitudes-were measured by 5 items, three positively worded and two negatively worded. Responses were recorded on a five-point scale ranging from (1) strongly disagree to (5) strongly agree. A sum score was constructed after positively worded items were reversibly scored.
Subjective norms were measured by 4 items in relation to all my patients, community leaders, my family and my teacher at the college. E.g. "My teacher at my college wants me to provide surgical treatment to HIV and AIDS infected patients as part of future professional work". Responses were indicated on five-point scales ranging from (1) strongly disagree to (5) strongly agree. A sum score was constructed from the 4 items.
Perceived behavioural control was assessed using one item, "How easy or difficult is it for you to provide surgical treatment to patients infected with HIV and AIDS as part of future professional work? Responses were rated on a scale ranging from (1) very difficult to (5) very easy.
Moral norms were assessed using 3 items "I I feel guilty if I do not provide ----------". "I get a bad conscience if I do not provide surgical treatment and "It is morally wrong for me not to provide surgical treatment ". Responses were indicated on a scale ranging from (1) strongly disagree to (5) strongly agree. A sum score was constructed from the 3 items.
Past behaviour was assessed using a dummy variable in terms of "Have you ever participated in the clinical treatment of HIV and/AIDS infected patients". Answers were provides as (1) yes and (0) no.
Confirmatory factor analysis, CFA, with AMOS 16 was employed to test the hypothesized measurement model with respect to intention, attitudes, subjective norms and moral norms, specifying the relationship between the observed variables (indicators) and the underlying latent variables (concepts). Thus CFA was used to test whether the Tanzanian and Sudanese data were consistent with an a priori hypothesized 4-factor model. The parameters of the model were estimated with maximum likelihood (ML) estimation. Missing data were assumed to be missing at random and was handled using the direct approach in AMOS 16 [40] .
The adequacy of overall model fit was estimated using chisquare test statistics and the following supplemental fit indices, root-mean-squared error of approximation (RMSEA), the comparative fit index (CFI) and Akaike's information criteria, AIC. In line with the conventional recommendations of Hu and Bentler [41] , a good model fit was indicated by a RMSEA less or equal to .06, a CFI greater or equal to .90 and with models having lower AIC being more plausible. The statistical significance of parameter estimates are the critical ratio (CR) representing the parameter estimate divided by its standard error. Based on a level of 0.05, the test statistics need to be <± 1.96 before the null hypothesis can be rejected. All other analyses were performed using SPSS 15.0 (SPSS, Inc, Chicago, Illinois, USA). Internal consistency reliability of-and bivariate associations among the theoretical constructs were assessed using Cronbach's alpha and Pearson's correlation coefficients, respectively. Unadjusted and adjusted marginal means and 95% confidence intervals for the components of the TPB and the construct of moral norm were estimated using General Linear Models, GLM (ANOVA). Linear multiple regression analysis was applied to predict intention from the TPB and external variables. The effect of the independent variables were expressed in terms of standardized regression coefficients (betas) and tested for statistical significance by means of F-test. The fit of the model was reported in terms of the squared multiple correlation coefficient (R 2 ).
Of the 475 Tanzanian students, 17% were in the younger age group (below or equal to 22 yr), 77.5% (368) were males, and 25.4% (121) were in their 5 th study year. The corresponding figures pertaining to the Sudanese participants were 69.6% (438), 28% (177) and 36% (231), respectively. Table 1 depicts the percentage distribution of participants according to socio-demographics and country of residence. All socio-demographic characteristics varied substantially and statistically significantly across the two cultures considered. . All factor loadings (standardized regression weights) were in the expected direction and were statistically significant at CR>1.96, with inter-factor correlations ranging from 0.46-0.78 (Tanzania) and from 0.50-0.68 (Sudan). Thus, all inter-factor correlations were below the threshold of 0.80 which is set as cut off to indicate poor discriminative validity [41] . Table 2 depicts unadjusted and adjusted marginal means and 95% confidence intervals for the components of the TPB and the construct of moral norm by country of residence. Country differences were estimated after controlling for potential confounding effect from sociodemographic variables (age, gender, parental education, study year) using General Linear Models, GLM (ANOVA). In both countries, students had on average positive attitudes, strong moral norms and strong intentions regarding care delivery to patients with HIV and AIDS. Both groups of students had on average moderately strong subjective norms and perceived less control regarding this behaviour. Tanzanian students had on average more positive attitudes and stronger intentions, perceived control, moral norms and subjective norms compared to their Sudanese counterparts. Internal consistency reliability in terms of Cronbach's alpha ranged from 0.83 (moral norms/subjective norms) to 0.44 (attitudes) in Tanzania and from 0.81 (attitudes/intention) to 0.45 (subjective norms) in Sudan.
In Tanzanian and Sudanese students, the TPB components, moral norms, and past behaviour were statistically significantly associated with intention. In Tanzanian students, Pearson's correlation coefficients ranged from r = .52 between attitudes and intention to r = .14 between past behaviour and intention. In Sudanese students, Pearson's correlation ranged from r = .54 between intention and attitudes to r = .03 between intention and past behaviour. Table 3 depicts the results from hierarchical linear regression analysis assessing the fit of the extended TPB model among Tanzanian and Sudanese students. Country and past behaviour were entered in the first step explaining 15.7% (R 2 change = 0.157, p < 0.001) of the variance in intention. Adding attitudes, subjective norms and perceived behavioural control in step 2 increased the explained variance by 33.4% (R 2 change = 0.334 p < 0.001). Moral norm added in step 3 raised the explained variance by 2.3% (R 2 change = 0.023 p < 0.001). A total of 6 variables accounted for 51.4% of the variance in intention (43.7% and 43.9% in Tanzania and Sudan, respectively). In the final equation, attitude was by far the strongest predictor of intention (beta = 0.35), followed in descending order by subjective norms (beta= 0.22), moral norm (beta= 0.17), perceived behavioural control (beta = 0.16) and past experience (beta = 0.06). The effect of country (beta = 0.35, p < 0.001) in step 1 was reduced to (beta = 0.04, p = 0.099) in the final third step, whereas the strength of the effect from past behaviour was maintained from step 1 (beta= 0.08, p < 0.001) to step 3 (beta= 0.06, p < 0.001). Statistically significant two-way interactions occurred, in terms of country × attitudes (R 2 change 0.007, F change = 13.7, p < 0.001) and country × subjective norms (R 2 change 0.009, F change = 17.0, p < 0.001). Stratified analyses suggested that the relationship between attitude and intention and between subjective norms and intention were statistically significantly stronger in Sudanese-than in Tanzanian students.
This study supports the applicability of an extended version of the TPB to students' intended care delivery for patients with HIV and AIDS in two culturally different sub-Saharan African countries. According to the CFA, the extended TPB questionnaire reflected four concepts across the study sites in terms of attitudes, subjective norms, moral norms and intention, thus lending support to it's within construct validity, formally. This appears to imply that the four constructs underlying the TPB questionnaire are discrete measures that can be reliably assessed in Tanzanian and Sudanese students and that those measures can be reported as four summary scores.
A total of six variables explained 51% of the variance in health care delivery intentions across study sites. After having controlled for country and past behaviour, attitudes, subjective norms and perceived behavioural control accounted for an additional 34% of the explainable variance in intention. This finding is consistent with those of previous studies, whereby the TPB has explained 68% of nurses' intention to adhere to universal precautions, 48% of health workers' intention to provide home care for HIV infected individuals, 35% of nurses' intention to care for SARS patients and 70% of nurses intended labour support [29] [30] [31] [32] [33] . In line with a growing body of research supporting the role of perceived moral obligations as an independent predictor of intention, moral norms contributed 2.3% to the explained variance in students' intentions after controlling for the TPB variables. This factor which has moral connotations and represents personal feelings of responsibility has been considered to be important in the adoption of several health related behaviours [36, 37] . Conner and Armitage [25] found that moral norms contributed an additional 4% of the variance in intention after controlling for the TPB across various behaviours. The present results suggest that the TPB and its extended version is useful in the SSA context in terms of identifying correlates of health care delivery that can be targets in interventions aimed at improving health care delivery to HIV infected patients.
A major shortcoming of the TPB model has been its inability to account for the influence of past behaviour [42, 43] . Evidence from meta-analytical reviews suggests that the addition of past behaviour to the TPB explains on average 7% of the variance in intention [25] . In this study, past experience significantly predicted intention after controlling for the TPB and moral norms and left the cognitive variables of the model almost unaffected. This suggests that the TPB provides a fairly accurate description of the intention formation process considering HIV and AIDS related care delivery among Tanzanian and Sudanese students. Students decide upon care delivery for HIV infected patients mainly as a consequence of situation specific expectations of the behaviour itself and to a lesser extent because they have engaged in similar care delivery previously.
Tanzanian and Sudanese students had on average strong intentions to provide surgical treatment to HIV infected patients as part of their future professional work. In contrast, in a study of Taiwanese nurse students' care intentions, almost all stated that they did not intend to care for HIV infected patients [44] . In this study, intended health care delivery was primarily driven by attitudes followed in descending order of importance by social norms, moral norms and perceived behavioural control in both countries. This finding is congruent with findings in other studies of treatment delivery and compliance with precautions in health care workers [29] [30] [31] [32] . This finding is also similar to that reported by Sauls [33] , who identified attitudes as more influential in determining health care delivery intentions than subjective norms. In contrast, Vermette and Godin [30] found perceived behavioural control to be the strongest influencing factor of nurses' intention to provide home care for HIV infected people. Students who did not express confidence in their ability to circumvent difficulties associated with care delivery, who evaluated care delivery negatively and who felt less normative pressure from colleagues at the faculty and less moral obligations to act were less likely to have strong intentions. These findings add insight to faculty administrators and educators to further develop strategies to increase students' intention to care for patients with HIV and AIDS. Providing sufficient and adequate protective equipments, routinely practicing infection control measures and protocols and providing up to date continuing education and training exercises should improve students' ability to overcome perceived obstacles related to care delivery. Even more important is the enhancement and reinforcement of students' positive attitudes through verbal expression of approval, persuasive messages based on strong arguments, substantial rewards and psychological support by faculty staff to encourage and acknowledge their efforts. Students' decision to provide surgical treatment to HIV infected patients was also influenced by their expectations that faculty colleagues would approve their provision of such treatment. In planning intervention programs, one approach which could be particularly important among students having less care delivery experience is to train peer leaders to communicate the importance of quality care for HIV infected patients. Interventions might also benefit from making students' focus on moral obligations by increasing their awareness of others' needs and their perception that providing quality care could help relieve such needs.
Some limitations of this study should be considered when drawing inferences based upon its results. In Tanzania, dental and medical students were recruited from one university, thus the representativeness of the findings to other undergraduate-and post-graduate students is unknown. Self-selection might also be a potential limitation since the students who chose to participate might differ from those who did not implying that only students with interest in health care delivery for HIV infected patients responded to the survey invitation. Students might have over reported intention to provide surgical treatment to HIV infected patients because of social desirability bias. However, a general effect of low reliability is weak associations between variables. Thus, the magnitude of the correlations presented, and the findings harmonizing with the TPB indicate acceptable reliability as well as validity of the results. Past experience as assessed in this study was limited to the extent that it provided no information about the level and frequency of HIV and AIDS related care delivery. Whereas the present findings support the notion that the TPB model is applicable in the sub-Saharan African context, it says nothing about the validity of the model per se, only that the TPB is just as useful in sub Saharan Africa as in other industrialized country contexts.
The TPB is applicable to students' intention to provide health care to patients with HIV and AIDS across two sub-Saharan African countries. It is suggested that attitudes, subjective norms, moral norms and perceived behavioural control are key factors in students' decision to treat HIV infected patients and should be targeted in interventions aimed at improving health care quality in the context of HIV and AIDS.
ful to Dr. Elizabeth Lyimo who was responsible for collecting data in Tanzania. Pattern Recognition Receptor–Dependent Mechanisms of Acute Lung Injury Acute lung injury (ALI) that clinically manifests as acute respiratory distress syndrome is caused by an uncontrolled systemic inflammatory response resulting from clinical events including sepsis, major surgery and trauma. Innate immunity activation plays a central role in the development of ALI. Innate immunity is activated through families of related pattern recognition receptors (PRRs), which recognize conserved microbial motifs or pathogen-associated molecular patterns (PAMPs). Toll-like receptors were the first major family of PRRs discovered in mammals. Recently, NACHT–leucine-rich repeat (LRR) receptors and retinoic acid–inducible gene–like receptors have been added to the list. It is now understood that in addition to recognizing infectious stimuli, both Toll-like receptors and NACHT-LRR receptors can also respond to endogenous molecules released in response to stress, trauma and cell damage. These molecules have been termed damage-associated molecular patterns (DAMPs). It has been clinically observed for a long time that infectious and noninfectious insults initiate inflammation, so confirmation of overlapping receptor-signal pathways of activation between PAMPs and DAMPs is no surprise. This review provides an overview of the PRR-dependent mechanisms of ALI and clinical implication. Modification of PRR pathways is likely to be a logical therapeutic target for ALI/acute respiratory distress syndrome. (TLRs 1-9 and TLRs 11-13) in mice have been defined (14) . TLRs 3, 7, 8 and 9 are expressed intracellularly, whereas TLRs 1, 2, 4, 5, 6 and 10 are expressed on the cell surface. TLRs are expressed on a range of immune cells including macrophages, dendritic cells, B cells and certain types of T cells, as well as on certain nonimmune cells, such as endothelial cells, smooth muscle cells and epithelial cells that lie at potential sites of entry, including the skin and the respiratory, intestinal and genitourinary tracts. The expression of TLRs is modulated by activation, matu-ration or differentiation of the different cell types (15, 16) .
TLR proteins are a family of type I transmembrane receptors characterized by an NH2-terminal extracellular leucine-rich repeat (LRR) domain, which mediate the recognition of their respective PAMPs, and a COOH-terminal intracellular tail containing a conserved region called the Toll/interleukin 1 (IL-1) receptor (TIR) homology domain. The TIR domain is the defining motif of the TLR/IL-1 superfamily, and it is likely to be one of the earliest signaling domains to have evolved (17) . TLRs can recognize a diverse range of PAMPs, generate inflammatory signals to coordinate innate immune responses and modulate adaptive immune responses. The list of TLR ligands is growing. However, the ligand for TLR10 and mouse TLR8 remains unknown at present. Activation of TLRs initiates two major pathways: the MyD88-dependent pathway, which is used by all TLRs except TLR3, resulting in the activation of nuclear factor (NF)-κB and activator protein-1 (AP-1); and the TRIF-dependent pathway, which is initiated by TLR3 and TLR4, resulting in the activation of type I interferons (IFNs) (13, 18, 19) . Expression of numerous proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-6, IL-12 and IFNs, is one of the major outcomes of the activation of the pathways (15) . TLR signaling is summarized and shown in Figure 2 .
RLRs as DExD/H-containing RNA helicases are expressed in the cytoplasm in a variety of cells, including immune and nonimmune cells. Unlike membranebond TLR3, TLR7 and TLR9, which are localized on the endosome and recognize viral double stranded RNA, singlestranded RNA and DNA, respectively, RLRs are cytoplasmic proteins that recognize viral RNA produced as a consequence of viral replication (20, 21) . RLRs consist of three family members: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) (21, 22) . Role of PRRs in mediating inflammation and organ injury. Infection causes PAMP release, but also causes tissue and cell damage and subsequent DAMP release. Similarly, injury caused by trauma or various other factors not only leads to DAMP release but also renders the patient more susceptible to infection and therefore PAMP release. In turn, the PAMPs and DAMPs act through PRRs, which include TLRs, NLRs and RLRs, to activate the innate immune system, yet they can also contribute to persistent and deleterious systemic inflammation and organ injury, including ALI. Structurally, RIG-I and MDA5 contain a DExD/H box RNA helicase domain and two caspase-recruiting domain (CARD)like domains required for eliciting downstream signaling pathways (23, 24) . The C-terminal region of RIG-I contains a repressor domain (RD), which inhibits downstream signaling. The MDA5 C-terminal region is similar to the RD of RIG-I; however, its function is not clear.
LGP2 contains a DExD/H helicase domain and an RD, but lacks the CARD-like region.
LGP2 was suggested to play an inhibitory role in virus-induced response, because the LGP2 RD binds the RIG-I RD and suppresses signaling as a consequence of interfering with the self-association of RIG-I (20, 25, 26) .
RIG-I is essential for the recognition of a series of RNA viruses, which include Sendai virus, Newcastle disease virus, influenza virus, vesicular stomatitis virus and Japanese encephalitis virus (27) . MDA5 is required for the recognition of other RNA viruses, including picornaviruses such as encephalomyocarditis virus, Mengo virus and Theiler virus (13, 27) . Thus, RIG-I and MDA5 have specificities in their detection of RNA viruses, presumably through recognition of distinct structures of viral RNA (24, 28) .
Recent studies revealed a pathway of RLR regulation of NF-κB. RIG-I/MDA5 CARD domains, through a CARD-containing adaptor protein, IFNβ promoter stimulator 1, also known as mitochondrial antiviral signaling protein, and CARD adaptor inducing IFNβ, ultimately activate IRF3 and NF-κB (29, 30) . The role of RLRs in the mechanism of ALI has not been elucidated.
The NLR family is a group of recently identified cytoplasmic PRRs that contain more than 23 members in humans (15) . The major role of NLRs is to recognize cytoplasmic microbial PAMPs and/or endogenous danger signals and initiate immunological responses, although the physiological function of most NLRs is poorly understood at present (31) . Members of the NLR family are categorized into at least five subfamilies according to their N-terminal structure, including NODs (nucleotidebinding oligomerization domain-1), NALPs (NACHT-, LRR-and pyrindomain-containing proteins), IPAF (ICE-protease activating factor), NAIPs (neuronal apoptosis inhibitor factors) and class II transactivator (CIITA) (32) .
The NLR family shares a domain organization consisting of a C-terminal LRR domain, a central nucleotide-binding NACHT domain, and an N-terminal protein-protein interaction domain composed of a CARD, pyrin domain (PYD) or baculovirus inhibitor of apoptosis repeat (BIR) domain (33) . NODs and IPAF contain CARD effector domains, whereas NALPs have PYD domains, and NAIPs possess BIR domains.
The functions of NOD1, NOD2, IPAF and NALP3 are more studied. NOD1 and NOD2 are the first NLRs that are reported (TLR2 in association with TLR1 or TLR6), TLR4, TLR5 and TLR11 are localized on the cell surface for ligand  recognition. TLR3, TLR7 and TLR9 are localized in the endosome for ligand recognition in the  lumen of endosome. All TLRs, except TLR3, recruit MyD88, and TLR1, TLR2, TLR4 and TLR6 recruit the additional adaptor TIRAP, which links the TIR domain with MyD88. TLR3 and TLR4 recruit TRIF. TLR4 requires the additional linker adaptor TRAM, which links the TIR domain of TLR4  with TRIF. Stimulation of the cells with TLR1, TLR2, TLR5, TLR6 and TLR11 ligands initiates the  MyD88-dependent pathway whereas TLR3 ligands initiate the TRIF-dependent pathway. TLR4 activates both MyD88-dependent and TRIF-dependent pathways. In the MyD88dependent pathway, MyD88 recruits the IRAK family of proteins and TRAF6. In turn, TRAF6 activates TAK1. The activated TAK1 activates the IKK complex, which activates NF-κB subunits. The activated TAK1 also activates the MAPK pathway. In the TRIF-dependent pathway, TRIF interacts with RIP1 and TRAF6. Activated TRAF6 and RIP1 activate NF-κB and MAPKs. TRIF also interacts with TRAF3 and activates TBK1/IKKi, which activate IRF3 and IRF7. Cells stimulated with TLR7 and TLR9 ligands activate NF-κB and MAPKs via the MyD88-dependent pathway. To induce type I IFNs, MyD88 associates with the IRAK family of proteins. IRAK1 and IKKα activate IRF7. IRAK1 also interacts with TRAF3 and activates IRF7. The activated NF-κB subunits and IRFs are translocated to the nucleus. NF-κB and MAPKs initiate the transcription of inflammatory cytokine genes whereas IRFs initiate the transcription of type I interferons. (The figure is adapted from [13] and used with permission from Elsevier.)
to have a direct function as PRRs in the recognition of peptidoglycan (PGN)derived peptides. NOD1 senses γ-D-glutamyl-meso-diaminopimelic acid (that is,-DAP) that is derived from Gramnegative bacteria (34) , whereas NOD2 senses muramyl dipeptide, which is from both Gram-positive and Gram-negative bacteria (35, 36) . When NODs bind with PGN-derived peptides, they rapidly form oligomers, which lead to the recruitment of the receptor-interacting protein 2 (RIP2) kinase through CARD-CARD interactions (37) . This complex of NOD1-RIP2 or NOD2-RIP2 then recruits the inhibitor of NK-κB kinase complex (IKK), leading to the activation of NF-κB. Activation NOD1 and NOD2 can also initiate an MAPK pathway that leads to the activation of p38 and ERK. In addition, NOD1 signaling can activate JNK as well (38) . NALP proteins are characterized by the presence of an N-terminal pyrin effector domain (39) . Several NLRs, namely NALP1, NALP2 and NALP3, have an important role in activation of proinflammatory caspases through formation of inflammasome (37, 40) . The inflammasome is a multiprotein complex of more than 700 kDa that is responsible for the activation of caspases 1 and 5, leading to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18 (41, 42) . Martinon et al. have presented different caspase activation platforms in which different components constitute the various inflammasomes (43) . Two types of NALP inflammasome are better studied: the NALP1 inflammasome that is composed of NALP1, the adaptor protein ASC, caspase-1 and caspase-5 (41) , and the NALP2/NALP3 inflammasome that contains NALP2 or NALP3 CARDI-NAL, ASC and caspase-1 (44) .
TLRs 1-10 are expressed in lung tissue (45) , and individual TLRs are differentially regulated in specific lung cell populations in response to microbial stimula-tion. TLR2, TLR4, TLR5 and TLR9 are the most likely to be involved in recognition of bacteria in the lungs (46) (47) (48) . A study by Bernard et al. has shown that ALI/ ARDS induced by lipopolysaccharide (LPS) is a major cause of mortality among humans (49) . The LPS membrane receptor complex is composed of several accessory molecules, which include phosphatidylinositol-anchored CD14, TLR4, MD2 and MD1 (50) . The LPSbinding protein (LBP) enhances the binding of LPS to its receptor (51) . Absence of CD14, MD2 or LBP abrogates most LPS responses (51, 52) . Expression of functional TLR4 has been found in many cell types in the lung (45) , and LPS-induced lethal shock and ALI have been shown to be TLR4 dependent (53) (54) (55) . Thus, TLR4 plays a critical role in the mechanism of infection-related ALI.
A recent study has shown that respiratory infections in the human lung initiated by TLR2 agonist lipoteichoic acid (LTA, a component of Gram-positive bacteria) and TLR4 agonist LPS (a component of Gram-negative bacteria) exhibit different inflammatory responses (56) . The study was performed on healthy subjects with LPS or LTA instillation into the contralateral lung. Alveolar macrophages (AMφ) isolated from bronchoalveolar lavage fluid were analyzed by multiplex ligation-dependent probe amplification. The results show that whereas both LPS and LTA elicited neutrophil recruitment, only LPS instillation was associated with activation of neutrophils (PMN) (CD11b surface expression and degranulation) and consistent rises of chemokine/cytokine levels. Moreover, LPS but not LTA activated AM, as reflected by enhanced expression of proinflammatory mediators and increased spontaneous cytokine release upon incubation ex vivo. Remarkably, only LTA induced C5a release. These data suggest that stimulation of TLR2 or TLR4 results in differential pulmonary inflammation, which may be of relevance for understanding the differences during Gram-positive and Gram-negative respiratory tract infection (56) .
Pulmonary endothelium is a major component of the alveolar-capillary unit, and is susceptible to injury from noxious agents that are either inhaled or delivered to the lung through the pulmonary circulation (57) . In ALI, pulmonary endothelium plays a major role by (a) altering metabolic activity to affect pulmonary and systemic homeostasis, (b) mediating polymorphonuclear PMN adhesion to promote PMN infiltration, (c) changing PMN barrier permeability to cause pulmonary edema and (d) secreting cytokines and chemokines to induce lung inflammation (58) .
A recent study by Andonegui and colleagues has shown that endothelial cells (ECs) are the key sentinel cells for detecting infection by Gram-negative bacteria and recruiting PMN to peripheral tissues (53, 59) . Indeed, previous studies have shown that direct activation of circulating PMN with LPS is not sufficient to induce their sequestration within the lung (53) . Interactions of PMN with ECs seems important for the process of PMN sequestration into the lung (53, 60) . LPS stimulates the CD14 and TLR4 complex, which in turn activates NF-κB (61) and increases the expression of adhesion molecule E-selection, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (62, 63) . The TLR4 signaling also leads to production and release of various bioactive molecules, including IL-1β, IL-6, TNF-α, chemokines and nitric oxide (64) , all of which are actively involved in the development of ALI.
More importantly, TLR4 signaling can also upregulate other TLRs, such as TLR2, and thus amplify the inflammatory responses. Although TLR2 is predominantly expressed in the first-line host defense cells (monocytes, macrophages, dendritic cells and PMN) (65, 66) , its expression is low in ECs and epithelial cells (67) . Studies from our laboratory showed that LPS through TLR4-and MyD88-dependent signaling induced TLR2 upregulation in ECs (68) . We have demonstrated that TLR4 signaling, through activating NF-κB, upregulates TLR2 expression in ECs, and this process is enhanced by oxidant signaling generated by PMN NAD(P)H oxidase. The functional relevance of NAD(P)H oxidase in mediating TLR4-induced TLR2 expression in ECs is evident by markedly elevated and stable ICAM-1 expression as well as augmented PMN migration in response to sequential challenge with LPS and peptidoglycan (68) . Thus, TLR2 activation, signaled by TLR4 and as regulated by PMN NAD(P)H oxidase, is an important mechanism responsible for amplifying PMN transmigration to sites of infection ( Figure 3 ).
The TLR4-TLR2 interaction suggests a highly coordinated, oxidant-mediated upregulation of TLR2 in response to LPS. When one considers the interactions of the innate immune system as microbes are first encountered, the value of such temporal organization is significant. For example, Gram-negative bacteria persist in tissues and, if they are not immediately killed through the activation of PMN, complement and other antimicrobial factors, they may spill out systemically and result in septic shock. Survival in the face of such infections depends on the innate immune system, which must be able to monitor and respond to pathogens over a prolonged period of time. Given the need for a prolonged response to bacterial infection, it has always seemed somewhat surprising that response to LPS is temporally finite. This endotoxin tolerance means that within hours after exposure to LPS, innate immune cells are incapable of responding again to a rechallenge (69) . But it is now clear that as LPS sensitivity wanes, the immune system has at its disposal the capability of marshaling responses via oxidative metabolites and their ability to upregulate other TLRs (70) . The subsequent means of responding to bacteria depend on the ability of the innate immune system to destroy microbes and enhance the release of alternative immune stimuli. The TLRs that are utilized are the ones that bind the constituents of degrading bacteria, such as lipopeptides, PGN, heat shock proteins and CpG DNA. It seems plausible that activated PMN may even alter the phenomenon of LPS tolerance, at least in a localized context, by setting into action a positive feedback loop at sites to which PMN are chemoattracted (71) . This would enhance inflammatory responses locally and help fight infection. However, in a setting of posttrauma SIRS or ALI, the primed PMN activation serves as an amplifier to cause enhanced PMN infiltration and organ injury.
Extensive PMN influx into the lungs is one of the characteristics of ALI. Studies have shown that excessive induction of proinflammatory cytokines in PMN and delayed PMN apoptosis are associated with higher mortality and more severe organ dysfunction in sepsis patients (72, 73) . Human PMN express all TLR mRNA except TLR3. TLR2 are more abundant than TLR4 on PMN (74, 75) . PMN recruitment to the lung after LPS inhalation is primarily dependent on TLR4-NF-κB signaling (76, 77) . The PI3K/Akt pathway was also reported to be involved in TLR4-induced expression of IL-1β, TNF-α and chemokine macro phage inflammatory protein (MIP)-2 (78) .
We have reported that TLR4, through regulating G-protein-coupled receptor kinases (GRKs), promotes PMN migration. We demonstrated that MIP-2 induces GRK2 and GRK5 expression in PMNs through phosphoinositide-3-kinase (PI3K)-γ signaling, and LPS-activated signaling through the TLR4 pathway transcriptionally downregulates the expression of GRK2 and GRK5 in response to MIP-2. The reduced expression of GRKs lowers chemokine receptor desensitization and markedly augments the PMN migratory response. These data indicate that TLR4 modulation of PMN surface chemokine receptor expression after the downregulation of GRK2 and GRK5 expression is a critical determinant of PMN migration (79) (Figure 4) .
A recent study explored a novel role of mTOR complex 1 (mTORC1) in TLR2and TLR4-induced PMN activation (80) . Administration of rapamycin, an inhibitor of mTORC1, decreased the severity of lung injury after intratracheal LPS or PAM (a TLR2 ligand) administration, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-α and IL-6 in bronchoalveolar lavage fluid. These results indicate that mTORC1 activation is essential in TLR2-and TLR4-induced PMN activation, as well as in the development and severity of ALI.
The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. However, a recent study showed that Cblb also has an unexpected function in acute lung inflammation (81) . Cblb attenuates the sequestration of PMN in the lungs after administration of LPS. In a model of polymicrobial sepsis in which acute lung inflammation depends on TLR4, the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of WT Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for NF-κB in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of PMN is impaired in the absence of Cblb. These data reveal that Cblb regulates the TLR4mediated acute inflammatory response that is induced by sepsis (81) .
PMN apoptosis is a crucial injurylimiting mechanism of inflammatory resolution. Circulating PMN undergo constitutive apoptosis that results in the shutdown of secretary capacity and allows PMN recognition and removal by macrophages (82, 83) . Several inflammatory agents, such as LPS, TNF, IL-8, IL-6, IL-1 and granulocyte colony-stimulating factor (G-CSF), can delay apoptotic response, providing PMN with a longer life span, which in turn allows the PMN to accumulate at local tissue sites of inflammation/infection (84, 85) . Protein 53 (p53) is a transcription factor that is important in multicellular organisms, where it regulates the cell cycle and promotes apoptosis. Modulation of p53 by nutlin-3α diminished the response of PMN and macrophages to stimulation through TLR2 or TLR4 as well as attenuated LPS-induced ALI. NF-κB has been reported as a modulator of apoptosis in inflammatory cells (86) . p53 can negatively regulate NF-κB activity by decreasing binding of NF-κB to the promoters of genes for proinflammatory cytokines. In p53 -/mice, the inflammatory process and severity of ALI in response to LPS are enhanced (87) .
AMφ account for approximately 95% of airspace leukocytes (88) . Tissue damage induced by LPS is mediated mainly by inflammatory products released from AMφ (89, 90) , thus activated AMφ play a critical role in the development of ALI (91) . LPS inhalation induces AMφ to produce and release inflammatory mediators TNF-α, IL-1β and MIP-2 in a TLR4dependent manner (92) , which further result in the recruitment of PMN into the lower respiratory tract and activate other cell types, including epithelia and endothelia (93) .
TLR4 is constitutively expressed in AMφ, and TLR2 can be induced in response to LPS or proinflammatory cytokines. The inducible TLR2 expression might be important in responding to other bacterial components from Grampositive bacteria (48) .
The role of CD44 in the regulation of LPS-TLR signaling in macrophages has recently been reported (94) . CD44 is a transmembrane adhesion molecule and hemopoietic CD44 has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. Following intratracheal LPS treatment, CD44 -/mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid and a marked increase in NF-κB DNA-binding activity in lung tissue in vivo and in macrophages in vitro. Furthermore, CD44 -/mice were more susceptible to LPS-induced shock. The study further found that the induction of the negative regulators of TLR signaling IL-1R-associated kinase-M, Toll-interacting protein and A20 by intratracheal LPS in vivo and in macrophages in vitro was significantly reduced in CD44 -/mice. Collectively, these data suggest that CD44 plays a role in preventing exaggerated inflammatory responses to LPS by promoting the expression of negative regulators of TLR-4 signaling (94).
Impairment of the alveolar epithelial barrier is important in the development of ALI. Under physiologic conditions the epithelial barrier is less permeable than the endothelial barrier; thus, destruction of epithelial barrier integrity prompts a progressive influx of protein-rich fluid into the alveoli (95) . On the other hand, the loss of epithelial integrity represents an impairment of physiologic transepithelial fluid transport and further inhibits the reabsorption of alveolar edema (96) . TLRs are critical for airway epithe-lial cell recognition of inhaled pathogens and for innate immune signaling. In cultured human lung epithelial cells, mRNA of all TLRs has been detected (97, 98) . TLR2, TLR3, TLR5 and TLR6 have the highest expression, and the ligands for these TLRs increased IL-8 and vascular endothelial growth factor (VEGF) production in normal human bronchial epithelial cells (99) . TLR2 is a heterodimer with TLR1 and TLR6, and each of these is present on the airway epithelial surface. TLR3, which recognizes dsRNA or poly(I:C), is located in endosomes in unstimulated human bronchial epithelial cells (100) . TLR5 is also present on the airway epithelial surface where it can interact with epidermal growth factor receptor (EGFR). Studies have shown that TLR ligands stimulated IL-8 and VEGF production via EGFR and the downstream signaling that might include MAP kinases and NF-κB (101, 102) . Interestingly, heat shock proteins (Hsp), such as Hsp72 and Hsp90, appear to be intimately involved in the recognition of LPS. Extracellular Hsp72 released from virally infected airway epithelial cells induces IL-8 express in human bronchial epithelial cells, resulting in the recruitment and activation of PMN via TLR4 (103, 104) . Type II alveolar epithelial cells can also be activated by LPS mediated through TLR4 signaling and in turn promote pulmonary inflammatory processes (105, 106) .
A study by Togbe et al. of whether TLR gene dosage contributes to infection has demonstrated that overexpression of TLR4 augmented an LPS-induced bronchoconstrictive effect, as well as TNF-α and CXC chemokine ligand 1 (keratinocyte-derived chemokine) production (55) . The study further showed that PMN recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were dependent on TLR4 gene dose. Therefore, the TLR4 expression level determines the extent of acute pulmonary response to inhaled LPS, and TLR4 may thus be a valuable target for immunointervention in acute lung inflammation as a result of infection.
In addition to the TLRs, NLRs are critically involved in the sensing of bacterial pathogens. NOD1 senses diaminopimelic acid-containing peptidoglycan present in Gram-negative bacteria, whereas NOD2 senses the muramyl dipeptide present in most organisms. Because of the apparent lack of direct effects on cell signaling induced by activators of NLRs, it is suggested that their role in pathogen sensing is one of cooperation with the TLRs (107) . However, studies suggested that the actions of NOD1 vary between cell types and, unlike those seen with LPS, the in vivo effects may be independent of leukocyte activation. For instance, Cartwright and colleagues have shown that although selective activation of NOD1 in macrophages has no apparent effect, in vascular cells NOD1 activation results in the profound induction of NOSII and shock in vivo (108) .
NOD2 is thought to be important in the maintenance of a healthy gut barrier because individuals who carry a defective NOD2 have an increased risk of Crohn disease and other intestinal disorders (109) .
Although the importance of TLR family in sensing pathogens is well recognized, it is also plausible that they may function in noninfectious diseases because TLR expression is also regulated in conditions other than infection. Indeed, growing evidence has shown that PRRs play a central role in the mechanisms of noninfectious ALI.
Several TLRs not only have the ability to recognize more than one ligand, but often recognize ligands with completely different chemical structures. Such TLR activation does not occur under normal circumstances but only when there is a change in the environment that either leads to the release of endogenous ligands from a cellular compartment, or leads to the modification of endogenous mediator that gives them the ability to activate TLRs (110, 111) . Because of the association of many endogenous ligands with tissue injury, the nomenclature of DAMPs has been suggested. The best characterized DAMPs include those products released from cells in response to stress or undergoing abnormal death, including Hsp60, Hsp70, the extra domain A of fibronectin, oligosaccharides of hyaluronic acid and high-mobility group box 1 (HMGB1). Most of these ligands act as agonists of TLR2 or TLR4, or both receptors (112) .
Resuscitated hemorrhagic shock (HS) often promotes the development of lung injury by priming the immune system for an exaggerated inflammatory response to a second, often trivial, stimulus, the so-called "two hit hypothesis" (113) .
We used a simplified animal model of the two-hit paradigm to address the mechanisms of HS-primed PMN migration and lung inflammation (114) . In this model, animals are subjected to a nonsevere resuscitated HS (hypotension at 40 mmHg for 1 hour, followed by a small dose of intratracheal LPS. Although neither shock nor LPS alone induces injury, the combination caused lung PMN accumulation and increased 125 I-albumin transpulmonary flux. Findings from this model have suggested that the mechanisms underlying the priming of PMN and inflammation involve a complicated receptor cross-talk process and interaction between PMN and AMφ, which is described below.
We demonstrated that LPS-TLR4 signaling upregulates TLR2 expression in AMφ, and HS-activated PMN play a critical role in the mechanism of TLR2 upregulation (115) . This cross-talk between TLR4 and TLR2 in AMφ results in the amplification of expression of cytokines and chemokines in response to the bacterial products LPS and PGN, and subsequently leads to enhanced PMN sequestration in the lung. These findings reveal a novel mechanism underlying HSprimed lung injury, namely that HS-activated PMN that were initially sequestered into the alveoli can instruct AMφ to upregulate TLR2, thereby sensitizing AMφ to TLR2 ligands and promoting enhanced lung inflammation.
How does HS-activated PMN enhance TLR4 upregulation of TLR2 in AMφ? We found that reactive oxygen species (ROS) derived from PMN NAD(P)H oxidase play an important role in amplifying the TLR2 upregulation (116) . Studies have also shown that lack of endogenous NAD(P)H oxidase in the AMφ caused a decrease in TLR2 expression in response to LPS stimulation; however, the decrease was restored when the AMφ was coincubated with PMN isolated from wild-type mice subjected to HS. These results indicate that although the endogenous NAD(P)H oxidase in AMφ is also involved in the signaling, the exogenous oxidants from PMN NAD(P)H oxidase are essential for inducing amplified TLR2 expression in AMφ in response to LPS.
The TLR2 gene promoter contains multiple binding sites for transcriptional factors, which include NF-κB, CCAAT/enhancer binding protein, cAMP response element-binding protein and STAT (signal transducer and activator of transcription) (117) . Of these, NF-κB has been reported to regulate TLR2 expression in response to cytokines and mycobacterial infection (117, 118) . It has been demonstrated that LPS-TLR4-induced TLR2 upregulation in AMφ is largely mediated through the NF-κB signaling pathway, because the NF-κB inhibitor IKK-NBD significantly decreased LPS-induced TLR2 expression in AMφ (115) . Although oxidants are involved in the NF-κB signal transduction pathway (119) (120) (121) , their molecular targets have not yet been defined. The contribution of redox regulation and location of potential redox-sensitive sites within the NF-κB activation pathway are the subjects of controversy (119) .
Recently we reported that HMGB1/TLR4 signaling mediates the HS-induced increase in TLR2 surface expression and decrease in TLR4 surface expression in the lung as well as in mouse lung vascular ECs (MLVEC) (122) . These alterations in TLR4 and TLR2 expression result in HMGB1-mediated activation of NAD(P)H oxidase and expression of ICAM-1 in MLVEC that is TLR4 dependent in the early phase and switches to being TLR2 dependent in the late phase following HS. More importantly, the HS-induced surface expression of TLR2 contributes to an enhanced activation of MLVEC and augmented pulmonary PMN infiltration in response to the TLR2 agonist PGN. Thus, the study demonstrates a novel mechanism underlying HS-augmented lung inflammation, namely that induction of increased TLR2 surface expression in lung endothelial cells, which is induced by HS/R and mediated by HMGB1 activation of TLR4 signaling, is an important mechanism responsible for EC-mediated inflammation and organ injury following HS (122) .
We have shown that HS-induced PMN NAD(P)H oxidase activation is mediated by HMGB1-TLR4 signaling. HMGB1 was originally defined as a nuclear protein that functions to stabilize nucleosome formation, and it also acts as a transcription factor that regulates the expression of several genes (71) . HMGB1 can be secreted by innate immune cells in response to microbial products or other inflammatory stimuli (123, 124) . HMGB1 is also released by injured cells and is known as one of the main prototypes of the emerging DAMPs (125) (126) (127) . HMGB1 was initially identified as an inflammatory cytokine that is a late mediator of lethality in sepsis (123, 124) . However, recent studies suggest that HMGB1 acts as an early mediator of inflammation, contributing to the development of ALI after hemorrhage (128) , and hepatic injury after liver ischemia-reperfusion (129) .
We found in our study that HS/R activates the TLR4-MyD88-IRAK4 signaling pathway through HMGB1, and further activates p38 MAPK and Akt pathways to initiate PMN NAD(P)H oxidase activation. PMN NAD(P)H oxidase-derived oxidants, in turn, mediate TLR4-TLR2 cross-talk in AMφ and sensitize AMφ response to TLR2 ligands, which act in a positive feedback manner to amplify pulmonary PMN infiltration and inflammation (130) .
We have also addressed a fundamental question regarding how HS globally regulates PMN infiltration in the lungs. We have shown that HS, through alarmin HMGB1, induced IL-23 secretion from macrophages in an autocrine and TLR4 signaling-dependent manner. In turn, IL-23, through an IL-17-G-CSF-mediated mechanism, induced PMN egress from bone marrow. Therefore a sustained and HS-primed migration of PMN was maintained. We have also shown that β-adrenergic-receptor activation by catecholamine of macrophages mediated the HS-induced release of HMGB1. These data indicate that HS, a global ischemia/ reperfusion stimulus, regulates PMN mobilization through a series of interacting pathways that include neuroendocrine and both innate and acquired immune systems (131) .
Hyaluronan (HA) is a massive sugar polymer in the extracellular matrix. Under physiologic conditions, HA exists as a high-molecular-weight polymer (>106 D) and undergoes dynamic regulation resulting in accumulation of lower molecular weight species (10-500 kD) after tissue injury. HA fragments can trigger innate immune responses in a manner that overlaps with both Grampositive and Gram-negative organism recognition pathways (132) . It has been demonstrated that fragmented HA accumulates during tissue injury (133) (134) (135) . CD44 is required to clear HA during tissue injury, and impaired clearance of HA results in unremitting inflammation. Additionally, fragmented HA stimulates the expression of inflammatory genes by inflammatory cells at the injury site (136) . Recently, Jiang et al. demonstrated that HA fragments require both TLR2 and TLR4 to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. In a noninfectious lung injury model, mice deficient in both TLR2 and TLR4 showed an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis and decreased survival (132) . Lung epithelial cell overexpression of high molecular mass HA protected mice against ALI and apoptosis, in part through TLR-dependent basal activation of NF-κB. The exaggerated injury in TLR2-and TLR4-deficient mice appears to be due to impaired HA-TLR interactions on epithelial cells. These studies demonstrate that host-matrix component HA and TLR interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from ALI (136) .
Mechanical ventilation (MV) provides life-saving support for many patients with respiratory failure (137) . However, mechanical stresses produced by MV can induce lung injury, termed ventilator-induced lung injury (138) . Evidence from animal experimental studies has demonstrated that MV per se can induce inflammatory responses (139) (140) (141) . A recent report has demonstrated that TLR4, but not TLR2, played a role in development of the inflammatory response after shorttime MV (142) . MV not only causes ventilator-induced lung injury in healthy animals, but also exacerbates damage in the injured lung (143) . TLR4 blockade reduces pulmonary inflammation caused by the combination of LPS and MV (144) . In the mechanism of hyperoxia-induced ALI, TLR3 expression and activation seems impotent. Exposure of human epithelial cells to hyperoxia in the absence of an exogenous viral pathogen significantly increased TLR3 expression. In vivo studies showed that both the absence of TLR3 via gene deletion in mice and the presence of an anti-TLR3 antibody in wild-type mice conferred significant protection in a hyperoxia-mediated lung injury model (145) .
The inflammasome is a multiprotein complex that mediates the activation of caspase-1, which promotes secretion of the proinflammatory cytokines IL-1β and IL-18, as well as pyroptosis, a form of cell death induced by bacterial pathogens. Members of the NLR family, including NLRP1, NLRP3 and NLRC4, and the adaptor ASC are critical components of the inflammasome that link microbial and endogenous danger signals to caspase-1 activation.
The role of NALP3 in mediating noninfectious ALI has been revealed by two recent studies. Gasse and colleagues reported that uric acid locally produced in the lung upon bleomycin (BLM)-induced DNA damage and degradation triggers NALP3 inflammasome activation, and in turn causes lung injury (146) . Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1β production, lung inflammation, repair and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88 and IL-1R1 pathways and TLR2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor (146) . Babelova et al., however, reported the role of biglycan, a ubiquitous LRR proteoglycan of the extracellular matrix, in mediating ALI through interacting with TLR2 and TLR4 on macrophages (147) . The study showed that in macrophages soluble biglycan induces the NLRP3/ASC inflammasome and subsequent activation of caspase-1 and release of mature IL-1β without need for additional costimulatory factors. This is caused by the interaction of biglycan with TLR2/4 and purinergic P2 × 4/P2 × 7 receptors, which induces receptor cooperativity. Furthermore, ROS formation is involved in biglycan-mediated activation of the inflammasome. By signaling through TLR2/4 biglycan stimulates the expression of NLRP3 and pro-IL-1β mRNA. These results provide evidence for direct activation of the NLRP3 inflammasome by biglycan and suggest a fundamental paradigm of how tissue stress and injury are monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response (147) .
Susceptibility and response to infectious disease is, in part, heritable. Potential associations between clinical outcome from sepsis and many inflammatory cytokine gene polymorphisms, innate immunity pathway gene polymorphisms and coagulation cascade polymorphisms have been observed.
We may yet be able to tease out the complex influence of genetic variation on susceptibility and response to infectious disease (148) .
In 2000, Arbour and colleagues reported that two polymorphisms of the TLR4 gene were present in a higher proportion of individuals who are hyporesponsive to inhaled LPS (149) . This finding led to a number of studies investigating the potential impact of these TLR4 polymorphisms on the course of infectious diseases and the development of septic shock and TLR4 polymorphisms (150) (151) (152) . These polymorphisms do not seem to confer sus-ceptibility to all Gram-negative infections, because other groups (153, 154) have shown no correlation in other infectious diseases such as meningococcal disease, and again one should be mindful that very significant hypofunctioning TLR4 alleles are likely to have a very strong negative selection pressure across the generations (155) . TLR-2 polymorphisms have also been linked to susceptibility to staphylococcal infection (156) and lepromatous leprosy (157, 158) . Although some of these studies are still relatively small in scale, they reinforce the important role of TLRs in pathogen recognition and immune response in humans. CD14 polymorphisms, key accessory molecules for TLR signaling, have been associated with increased prevalence of positive bacterial culture findings and sepsis attributed to Gramnegative infections in a critically ill population (159) , as well as the susceptibility to chronic Chlamydia pneumoniae infection in patients with coronary artery disease (160) .
The discovery of the importance of PRRs in the pathogenesis of SIRS and organ injury, including ALI, has led to a therapeutic strategy targeting PRRs. TLRs are the most extensively studied family of PRRs, and thus recently developed new drugs mainly target TLRs and are either agonists of TLRs to enhance immune responses against infectious agents or antagonists designed to reduce inflammation due to infection or autoimmune responses (161) . The approaches to modulating TLR activity have focused on the following aspects: (a) ligands or analogues such as Eritoran (E5564) from Eisai (Woodcliff Lake, NJ, USA), a synthetic analogue of bacterial lipid A that inhibits LPS from activating cells through the TLR4/CD14/MD2 complex (162) (163) (164) ; (b) monoclonal antibodies, soluble receptors and other accessory proteins, such as a natural soluble form of TLR2, found in mouse plasma and breast milk, which acts to block TLR2 ligand stimulation (165) , and a member of the TLR/IL-1 receptor family (TIR8 or SIGIRR) that inhibits NF-κB signaling and may be an endogenous inhibitor of the TLR system (166); (c) signal transduction blockers; many of the key molecules in the signaling pathways for each TLR have been identified and are considered potential drug targets (167) (168) (169) . The structural bases of TIR domain interactions between TLRs and adapters such as MyD88, Mal, TRAM and TRIF have been modeled, and small peptidic sequences based on the TIR domain BB loop or peptidomimetics of this region have been made that can block the interactions (110, 169, 170) . (d) siRNA and antisense; studies with knockout mice suggest that deficiency in individual TLRs has limited consequences for animals under normal conditions, but exhibits impact under conditions of specific infectious challenge (110, 171, 172) . However, siRNA sequences themselves may be ligands for intracellular TLRs (173) ; thus attention to the design of appropriate sequences is necessary.
Drugs targeting TLRs have not yet been clinically applied to the treatment of ALI, but because of the critical role of PRRs in the development of ALI, targeting of PRRs has opened up a productive area for the therapy of ALI.
Current pharmacotherapy has not been highly successful in increasing patient survival in cases of ALI/ARDS. Since PRRs were recognized, their significance in the mechanisms of ALI has been quickly identified. The combined activation of these different receptors may result in complementary, synergistic or antagonistic effects that modulate the process of ALI. Therefore, modification of PRR pathways is likely to be a logical therapeutic target for ALI/ARDS. However, a complete understanding of the role of PRRs in the mechanism of ALI requires further "decoding" of these multiple receptor interactions.
This work was supported by the National Institutes of Health Grant R01-HL-079669, National Institutes of Health Center Grant P50-GM-53789, and a VA Merit Award.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper. Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection. Feline leukemia virus (FeLV) is a retrovirus of great veterinary importance that was discovered more than 40 years ago [20] and infects domestic cats and some related small felids worldwide [18, 22, 32] . An infection with FeLV may cause disorders of hematopoietic cells, a state of immunodeficiency and fatal neoplasia. Common symptoms are fever, anaemia, anorexia and weight loss. The prevalence of FeLV infection has been decreasing in the past few years. In Switzerland, FeLV prevalence was found to be 3% in healthy and of up to 13% in ill cats [29] . Decreasing prevalence is the consequence of identification and segregation of infected cats, and of extensive vaccination programs. However, cases of recurrence keep occurring and emphasise the importance of an accurate diagnosis [24] . FeLV is mainly transmitted directly from cat to cat. Transmission occurs through contact with saliva via licking, mutual grooming and sharing of food or water dishes or trough bites [5, 6] . Infectious FeLV can also be transmitted via faeces and milk [8, 34] . FeLV RNA was also detected in urine, but, its infection potential has not been demonstrated so far [1] . Pathogenesis is well understood if infectious pressure is high. Infection starts in the oropharynx where the virus first replicates in tonsils and local lymph nodes [37] , from which the virus spreads to the bone marrow, thymus, spleen and intestine through infected lymphocytes. Bone marrow infection is an important hallmark of FeLV pathogenesis as the virus replicates extensively in bone marrow and infects blood precursor cells first, and many organs and tissues thereafter, including salivary gland, tonsils, pharyngeal, urinary bladder, gastric, intestinal, colonic, pancreatic and endometrial epithelia, lymph nodes throughout the body, bone marrow, and spleen [36, 37] . FeLV infection presents a variety of outcomes [11, 16-18, 25, 27, 35, 37, 38] , which are influenced by both host and virus factors. Known host resistance factors include age and immune system status. Known virologic factors are virus strain, subtype and viral infection dose [18] . The outcome of infection is still a rather controversial issue. In the past, infection outcome was classified as viremic, non-viremic, and transiently viremic based on results of virus isolation, immunofluorescence assays and/or antigen detection [27] . Viremic cats are characterized by continuous expression of p27 viral antigen. FeLV infection is not contained due to a lack of FeLV specific immunity [3, 4, 18] . Viremic cats continuously shed virus, thereby posing a risk of infection to susceptible cats, and usually succumb to FeLV-associated diseases (anaemia, immunosuppression, and neoplasia). In transiently viremic cats, viremia is overcome after a few weeks post-infection (p.i.). However, transiently viremic cats remain provirus positive [15] . In addition, some nonviremic cats were shown to have localized infection characterized by virus replication in certain tissues, such as mammary, salivary and urinary epithelium [8, 12, 34] . This additional form of FeLV infection was termed atypical or sequestered infection. New sensitive molecular assays have been described recently for the use in detection and quantification of FeLV provirus DNA and viral RNA [14, 39, 42] , resulting in a more sensitive measure for FeLV exposure. The spectrum of host response categories was refined accordingly. Especially in p27 negative cats, the outcome should be reevaluated based on the presence or absence of FeLV proviral DNA in blood or bone marrow. The spectrum of host response categories was thus reclassified into: abortive (no virus detected after exposure), regressive (p27-negative, provirus positive after or without transient antigenemia) and progressive (persistently p27 positive, virus isolation, provirus positive, FeLV RNA positive) [16] . Whether cats that show no signs of infection (abortive) are truly immune or just resistant, is still open to debate. In this context, a very interesting finding was that FeLV infection can be transmitted by contact with faeces [8] with no apparent viremia and infection involving the bone marrow, and constant negativity for proviral DNA in blood -which would be classified as abortive infection. However, in the study of Gomes-Keller et al. [8] FeLV provirus could be detected in several organs and the cats showed seroconversion, indicating that infection indeed had occurred. It was concluded that under low infectious pressure a different pathogenesis may take place in which bone marrow is not involved. Since FeLV transmission at very low infection levels appears to be the most natural way of infection, many cats may show this yet uncharacterized outcome. Cats that test negative for FeLV by conventional diagnostic methods could still have been infected and constitute a relevant portion of the cat population affected by the virus. The prevalence of FeLV infection may therefore have been underestimated, a factor that can have important consequences for FeLV control management, e.g. in Iberian lynxes, which seem to be particularly susceptible for FeLV infection and for which a correct estimation of FeLV spreading potential is of great importance [32] . It was thus the aim of this study to further characterize the course of infection after exposure to low infectious pressure and to test the hypothesis that low FeLV doses applied to young cats may lead to seroconversion even when the infection does not progress through bone marrow. The virus may not be completely eliminated and a limited viral replication in tissues may lead to a development of a weak immune response. Three groups of 7 cats were exposed to different, low doses of FeLV. Blood p27 antigen, proviral DNA and viral RNA were measured in blood at regular intervals until week 20 p.i. and in popliteal and mesenteric lymph nodes, bone marrow, spleen, kidney, urinary bladder, lungs, thymus, myocardium, parotid gland, and pancreas after euthanasia. Immune response against FeLV was assessed by the detection of antibodies by ELISA to FeLV whole virus and to FeLV p45 (the recombinant env-gene product), by immunofluorescence assay to feline oncornavirus-associated cell membrane antigen (FOCMA), and by Western blot analysis to the separated FeLV components.
Twenty-six, 9 weeks-old Specific Pathogen-Free (SPF) male kittens were obtained from Liberty Research, Inc. (Waverly, NY, USA). Animals were kept under barrier conditions and under optimal ethological and hygienic conditions in one group of 5 cats (uninfected control group) and three groups of 7 cats each (group 1K, 10K, and 100K, based on viral infectious dose). Prior to the beginning of the experiment the cats were tested by PCR, RT-PCR and serology and shown to be negative for FeLV, FIV, Herpes-, Corona-, Calici-, Parvovirus and feline hemotropic mycoplasmas.
At the age of 16 weeks, each kitten of groups 1K, 10K, and 100K were infected once oronasally with FeLV-A/Glasgow-1 [19] by introducing 0.2 mL of virus suspension into each nostril and 0.6 mL into the mouth. The virus suspension for group 1K contained a total of 1 000 focus-forming units (FFU), the one for group 10K 10 000 FFU and group 100K 100 0000 FFU. The viral stock origin and infectivity was the same as described in earlier works [15, 41] .
Blood samples were collected under sedation (0.01 mg/kg midazolam (Dormicum Ò , Roche Pharma AG, Reinach, Switzerland) and 10 mg/kg ketamine (Narketan Ò , Vétoquinol AG, Belp, Switzerland)) prior to challenge at week À7, À5, À3, and then weekly, starting from week 0 until week 6 p.i., later in biweekly intervals until week 20 p.i. Blood samples were obtained by jugular venipuncture using 5 mL syringes and blood was immediately transferred into EDTAtubes. 400 lL of EDTA blood was submitted for haematology analysis, 200 lL of EDTA anticoagulated whole blood were aliquoted for DNA extraction. Plasma was obtained by centrifuging approximately 2 mL of EDTA blood at 1 700· g for 10 min. Blood and plasma samples were immediately frozen at À80°C until they were processed.
For determination of FeLV proviral loads, total nucleic acids were extracted from a blood volume containing 10 6 white blood cells using the MagNa Pure LC Total Nucleid Acid Isolation Kit (Roche Diagnostics AG, Rotkreuz, Switzerland). The extracted total nucleic acids were analyzed by real-time TaqMan PCR as described in [39] using the 2· TaqMan Ò Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a ABI 7500 sequence detection system (Applied Biosystems) and under the following cycling conditions: an initial denaturation of 20 s at 95°C was followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. For each run, a glyceraldehyde-3-phophate dehydrogenase (GAPDH) pseudogene of which one copy is present in the genomic DNA of feline cells [33] was also quantified as described [39] using the 2· TaqMan Ò Fast Universal PCR Master Mix and the same PCR run conditions as for FeLV provirus. FeLV proviral DNA amounts were normalized to feline GAPDH by dividing FeLV copy numbers by fGAPDH copy numbers to calculate FeLV copies per cell. Viral RNA in plasma samples was extracted from 200 lL of plasma (either from 5-sample pools or from single samples) using the MagNa Pure LC Total Nucleic Isolation Kit and quantified by real-time TaqMan reverse transcriptase (RT)-PCR as described [39] using a ABI 7500 sequence detection system.
The presence of plasma FeLV p27 antigen was determined using a sandwich ELISA as previously described [26] . FL-74 feline lymphoblastoid cell line permanently expressing FeLV), which was considered 100%. Samples reaching > 5% of the positive control signal were considered positive [14] .
The plasma samples were also analysed for the presence of antibodies to FeLV whole virus, to FeLV p45 (the non-glycosylated form of gp70 surface unit of the envelope glycoprotein), and to FOCMA. Anti-FeLV p45 and anti-FeLV whole virus antibodies were measured by ELISA as described [21, 25] , using 100 ng of p45/well and 100 ng of gradient purified FL-74 FeLV, respectively. Plasma was used at a dilution of 1:200 and antibody levels assessed by comparison with predefined control antisera [25] . Antibody to FOCMA was measured at week 0 and week 20 p.i., by indirect cell membrane immunofluorescence as described [2] . FL-74 cell culture medium was tested for the absence of FCV, FHV, FPV, FCoV, FIV, hemotropic mycoplasma and presence of FeLV by RT-PCR/PCR as described [9, 13, 23, 31, 39, 44, 45] . The culture was consistently free of the unwanted contaminants. The cat sera were titrated at 4-fold dilutions from 1:4 to 1:256. Samples showing a minimal titre of 1:4 were considered to be FOCMA positive. In addition, samples from week À3 and week 20 p.i. were examined for the presence of antibodies to FeLV gp70, p27 and p15(E) [26, 30] by Western blot analysis as described [28] .
Cats of group 10K and group 100K were euthanized at week 20, and tissue samples from popliteal and mesenteric lymph nodes, bone marrow, spleen, kidney, urinary bladder, lungs, thymus, myocardium, parotid gland, and pancreas were collected within 30 min post-mortem. Samples were snap-frozen in liquid nitrogen. Ten mg of tissue were used for the extraction of DNA using MagNA Pure LC DNA Isolation Kit II (Tissue kit, Roche Diagnostics). FeLV provirus was quantified by real-time PCR as described [39, 40] . To guarantee a comparable sensitivity, samples yielding less than 15 000 fGAPDH copies per reaction were re-extracted until a suitable concentration was reached. In addition, samples were collected under sterile conditions from mesenteric lymph node, urinary bladder, lungs, thymus, and myocardium for virus isolation. Tissue samples were co-cultured with FEA cells and supernatants of the co-cultures collected at days 4, 8, 12, 16, 20, 24 , and 28 post-inoculation were tested for p27 antigen by ELISA as described [7] . Bone marrow samples were cultured in medium and supernatants collected at days 18 and 21 were used for total nucleic acid isolation (MagNA Pure LC Total Nucleic Acid Isolation Kit, Roche Diagnostics) and samples were tested for the presence of FeLV RNA. Additionally, supernatants were tested by p27 ELISA [26] .
Statistical analyses were carried out using R software version 2.9.0. (The R Foundation for Statistical Computing, Vienna, Austria). Longitudinal effects (time) on antibody titres of the different groups were compared to each other by multivariate analysis of variance (MANOVA). A p value < 0.05 was considered to be statistically significant.
With one exception, cats did not become provirus positive at any time point tested. The one cat (JCR2) that was found to be positive belonged to the group challenged with the highest of the three doses of FeLV (100 000 FFU). Proviral DNA was detected from week 2 p.i. on with a peak at week 3 (3.33 copies/cell) (Fig. 1) . Similarly, viral RNA was consistently detected only in samples of the provirus-positive cat (JCR2) until week 12 and additionally at week 18 once more. The highest viral RNA load measured was at week 2 p.i. (Ct-value of 22.95, Fig. 1 ). Viral RNA was not detectable in pooled plasma samples (5 samples each pool) of any of the provirus-negative cats at week 0, 10, and 20 p.i. In addition, samples from week 2, 3, and 4 were tested individually, as the viral RNA peak usually occurs in this time period; the results were negative, too.
p27 antigen was detected in plasma of one (JCR2) of the 26 cats tested. This cat became transiently positive from week 3 p.i. to week 4 p.i. (Fig. 1) . p27 was not detectable at any time point in all other cats.
By measuring antibodies against FeLV p45 and whole virus by ELISA, seroconversion was observed in one cat. In the p45 ELISA, this cat (JCR2) had a positive response throughout weeks 8 to 20 with a peak of 26% of the positive control at week 14, and throughout weeks 4 to 20 with a peak of 54% of the positive control at week 20 in the FeLV whole virus ELISA (data not shown). Analysis of the remaining animals showed no statistically relevant difference between the groups in p45 as well as whole virus ELISA (P MANOVA 0.53 and 0.47, respectively Figs. 2 and 3). Prior to challenge, all samples were negative for the presence of antibodies directed against FOCMA. At week 20 p.i., antibodies to FOCMA were detected in 4 cats (KCT1, JCU3, JCO2, JCP1, 57%) of group 10K and 4 (KDA1, JCR2, KCQ1, JCS1, 57%) cats of group 100K. Of these 8 cats, the PCR-positive cat (JCR2) reached the highest titre (1:64), the other 7 cats developed antibody titres which ranged from 1:4 to 1:16. No negative control group and group 1K animal had a detectable titre (Tab. I). In Western blot analysis, sera from 2 cats of group 10K (JCU3, JCS2) showed a weak reactivity to the upper band of the p15(E) protein at a serum dilution of 1:40 as well as at 1:100. However, the same cats showed some reactivity to the upper band of p15(E) already at week 0 p.i. (Fig. 4) . In the PCR-positive cat (JCR2) the presence of antibodies against FeLV was confirmed at a dilution of 1:100. All other cats from group 100K, 10K and 1K had either a completely negative Western blot pattern or showed cross-reactive bands already present at week À3 (cats KDA1 and JCS2, Fig. 4 ).
At week 20 post-challenge, cats of groups 10K and 100K were euthanized and different tissue samples were collected and analysed for the presence of FeLV DNA sequences. Popliteal and mesenteric lymph nodes, bone marrow, spleen, kidney, urinary bladder, lungs, thymus, myocardium, parotid gland, and pancreas were tested. FeLV DNA sequences were found in organs of the cat (JCR2) positive for FeLV provirus in blood. The copy numbers were in average very low and ranged from 5.6 to 704.25 copies/reaction (3.4 · 10 À5 to 0.0024 copies/cell, data not shown). The popliteal lymph node showed the highest FeLV proviral load (0.0024 copies/cell). No FeLV DNA sequences were detected in tissue samples of all other cats. Virus isolation from urinary bladder, myocardium, lungs, mesenteric lymph node, thymus, and bone marrow was negative for all cats. 
The aim of this study was to investigate to what degree cats can become infected by low dose oral FeLV infection without detectable involvement of the bone marrow. Even though FeLV prevalence has reportedly decreased during the past years, FeLV is still among the most important infectious diseases of cats. Hence, a deep knowledge of all aspects of FeLV pathogenesis and biology is essential. Recently, a novel course of infection was postulated that Week 0
Week 20
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Week deviates from the classical course involving a phase in which bone marrow is infected. In this alternative course, cats exposed to low loads of virus via faecal route seroconverted without involvement of bone marrow and without showing any other sign of infection [8] . [16] . In this study, we demonstrated that a single oronasal exposure to FeLV as low as 10 000 FFU is sufficient to elicit an antibody response and to lead to seroconversion in at least 18% (lower 95% confidence interval of the distribution) of the cats, demonstrating that the postulated alternative course of infection may occur in a considerable portion of the animals. We propose to designate this outcome of infection as abortive infection with seroconversion. No hallmark of infection was observed in the cats that received the smallest of the three doses (1 000 FFU), for which individual local innate immunity might have been enough to contain viral replication and to lead to abortive infection without a detectable adaptive immune response. However, under field conditions, multiple exposures in the range of 1 000 FFU or lower may occur giving rise to seroconversion or even provirus positivity.
Considering that the assay for proviral DNA is able to detect one copy per reaction, we believe that the consistent PCR-negativity in blood of the cats that seroconverted is a strong indicator for the absence of bone marrow infection, a confirmation that an alternative course of infection may indeed exist, where the bone marrow is not involved [1] . FeLV provirus may still be integrated into the genome of the cells of some tissues but, in most cases, at levels beyond detection. In the study presented here, FeLV DNA sequences were not detectable in the organs tested. However, the presence of FeLV provirus in tissues other than the ones we analysed can not be excluded and should be investigated further. In localized infection where FeLV replication is confined to certain tissues, presence of plasma viral RNA loads is regarded a sensitive parameter for infection. RNA loads reflect ongoing viral replication somewhere in the cat's body even in sequestered places. However, detection of RNA is generally less frequent than that of proviral DNA [39] . Seroconversion was observed by use of the FL-74 cell membrane immunofluorescence test. This assay originally had been designated FO-CMA test and was thought to detect antibodies at a non-virion tumour-associated antigen [2] . Later, it became clear that the FOCMA phenomenon can be explained not by a tumour specific antigen but by viral proteins including FeLV p15(E), gag-polyproteins and gp70 of FeLV subtype C present on the FL-74 cell membrane [43] .
The FL-74 cell membrane immunofluorescence test seems to be the most sensitive method to detect seroconversion compared to ELISA and Western blotting. This may be due in part to the less stringent test conditions, which allow antibodies with lower affinity to bind to their epitopes, and in part to the fact that in the FOCMA assay the binding targets are presented in a native conformation, thus allowing a better binding of conformation-specific antibodies. Induction of cytotoxic T-cells or virus-neutralizing antibodies by oral low dose infection, or Th1 cytokine expression that might have been induced by the experimental infection were not measured. Instead, antibody response was used as marker of infection. It is unclear to what degree the alternative pathway may be relevant to the spread of FeLV infection. We speculate that in most cases cats will overcome the infection for good. In very few cases -when the infected cats undergo immunosuppression due e.g. to concomitant FIV infection -full-blown FeLV infection with bone marrow involvement and viral shedding may develop. It will be important to determine to what extent this may occur.
In conclusion, the present study provides additional evidence for the existence of an alternative FeLV infection course that leads to development of antibodies without involvement of the bone marrow. The results may be important for the surveillance of the FeLV status of a cattery or a cat population. Most likely a cattery cannot be considered free of FeLV as long as cats with FeLV-reactive antibodies are present. The potential for overt infection cannot be neglected. A large and accurate collection of peptidase cleavages in the MEROPS database Peptidases are enzymes that hydrolyse peptide bonds in proteins and peptides. Peptidases are important in pathological conditions such as Alzheimer's disease, tumour and parasite invasion, and for processing viral polyproteins. The MEROPS database is an Internet resource containing information on peptidases, their substrates and inhibitors. The database now includes details of cleavage positions in substrates, both physiological and non-physiological, natural and synthetic. There are 39 118 cleavages in the collection; including 34 606 from a total of 10 513 different proteins and 2677 cleavages in synthetic substrates. The number of cleavages designated as ‘physiological’ is 13 307. The data are derived from 6095 publications. At least one substrate cleavage is known for 45% of the 2415 different peptidases recognized in the MEROPS database. The website now has three new displays: two showing peptidase specificity as a logo and a frequency matrix, the third showing a dynamically generated alignment between each protein substrate and its most closely related homologues. Many of the proteins described in the literature as peptidase substrates have been studied only in vitro. On the assumption that a physiologically relevant cleavage site would be conserved between species, the conservation of every site in terms of peptidase preference has been examined and a number have been identified that are not conserved. There are a number of cogent reasons why a site might not be conserved. Each poorly conserved site has been examined and a reason postulated. Some sites are identified that are very poorly conserved where cleavage is more likely to be fortuitous than of physiological relevance. This data-set is freely available via the Internet and is a useful training set for algorithms to predict substrates for peptidases and cleavage positions within those substrates. The data may also be useful for the design of inhibitors and for engineering novel specificities into peptidases. Database URL: http://merops.sanger.ac.uk Peptidases (proteases or proteinases) are enzymes that hydrolyse the peptide bonds between amino acids in a protein or peptide chain. Hydrolysis of such bonds is required for removal of targeting signals (signal and transit peptides (1), ubiquitin (2), SUMO (3) and NEDD8 (4) peptides), the release of a mature protein from its precursor (5) , the switching off of a biological signal by degradation of the signal protein (6) , and for widespread catabolism of proteins for recycling of the amino acids. When proteolysis occurs unchecked, then diseases can result, such as Alzheimer's (7) , osteoarthritis (8) , emphysema (9) , tumour invasion (10) and acute pancreatitis (11) . Pathogens use peptidases to enter the host and to degrade host proteins for food (12) .
Peptides and proteins have been widely used to characterize the specificity of peptidases, but frequently the substrates chosen have been physiologically irrelevant. One of the most popular substrates has been the oxidized insulin B-chain, because this is a peptide without tertiary structure, and cleavage depends solely on the preference of the peptidase (13) . (The terms 'specificity', 'selectivity' and 'preference' are used interchangeably here.) However, peptidase preference is exactly that: a preference only. Researchers often find that after prolonged exposure to a peptidase other bonds are degraded, albeit slowly, once none of the preferred bonds remain. If the peptidase preparation is not pure, then there is the danger that some of the observed cleavages are due to contaminating peptidases.
The bond in a substrate where hydrolysis occurs is known as the 'scissile bond'. In the Schechter and Berger nomenclature (14) , residues on the left-hand side of the scissile bond (towards the N-terminus) are numbered P1, P2, P3, etc. and residues on the right-hand side (towards the C-terminus) are numbered P1 0 , P2 0 , P3 0 , etc. with cleavage occurring between P1 and P1 0 . The substrate-binding pocket in the peptidase that accommodates the P1 residue is known as the S1 pocket, and that accommodating the P1 0 residue is the S1 0 pocket.
Predicting where a peptidase will cleave in a native protein is difficult. Where cleavage does occur in a protein is due to a combination of the preference of the peptidase and the availability of bonds in the substrate. Although the preference of the peptidase can be quite simple, for example trypsin (MEROPS identifier S01.151) cleaves lysyl and arginyl bonds (15) and caspase-3 (C14.003) cleaves only aspartyl bonds (16) , very often peptidase preference is cryptic. It is relatively easy to predict trypsin cleavages in a denatured protein, but few lysyl and arginyl bonds will be cleaved in a native protein. This has proved useful for researchers wishing to separate structural domains in a multidomain protein using limited proteolysis (17) . It is not possible to predict where in a peptide cathepsin B (C01.060) will cleave, for example, despite its known preferences for a hydrophobic residue in the S2 pocket and arginine in S1 (18) .
Even though for some peptidases the specificity has been clearly defined, in all probability only a few bonds will be susceptible to cleavage in a mature protein. A protein will have few bonds flexible enough to thread into a peptidase active site if the protein is in a native state, because of the stabilizing interactions within and between secondary structure elements within the substrate. It is widely assumed that the susceptible bonds will be within surface loops and interdomain connectors. However, once a bond is cleaved and the tertiary structure perturbed, further bonds may become susceptible.
Most studies of the action of a peptidase on a supposed physiological substrate are performed in vitro. It may be, however, that in vivo peptidase and substrate do not meet, either because of a physical boundary, such as being in different intracellular (or extracellular) compartments, because inhibitors inactivate the peptidase, the cleavage sites are inaccessible because the substrate is bound to another protein, or the environment is unsuitable and the peptidase is not active.
Despite the importance of protein cleavage, there has been no centralized repository for cleavage data collection and no attempt to curate these cleavages by mapping them to residue positions in protein primary sequence databases. Given that nearly all proteins are eventually degraded, and that any one protein can be degraded by several different peptidases often by cleavages at multiple peptide bonds, the potential total number of cleavages will always exceed the number of known proteins. Up until recently each cleavage had to be characterized biochemically, which meant N-terminal sequencing of the products, a timeconsuming and labour-intensive task. Now that proteomic analyses are possible, where cell lysates or similar samples are subjected to cleavage by a peptidase, peptides isolated, composition determined by mass spectroscopy, and possible source protein(s) determined from the composition (19) , the amount of data is set to rise exponentially. This makes it vitally important that the information be accurately stored and curated. Such a collection made readily available would provide a comprehensive training set for algorithms and software for the prediction of physiological substrates and cleavage positions.
The classification of peptidases into clans and families was first published in 1993 (20) , and this was converted into an Internet resource, the MEROPS database (21), in 1996. The database was extended to include nomenclature and bibliographies, and has been developed over the years to be a one-stop shop for researchers with an interest in proteolysis. The collection of known cleavages in substrates which was started in 1998 (22) has now been added to the MEROPS database. For each peptidase there is a page listing known substrates, and, where enough substrates are known, the peptidase summary has displays to show peptidase specificity. For each protein substrate, the sequence is displayed showing where cleavage occurs and which peptidase performs that cleavage. In addition to the MEROPS collection, there is also a collection of physiologically relevant protein cleavages assembled by the CutDB database (23) and more specialist collections of substrates for individual peptidases or peptidase families, such as CASBAH for caspases (24) .
The primary source of protein cleavage information is the published literature. Search profiles have been developed for use at PubMed (25) and Scopus (http://info.scopus.com/). These are updated regularly and currently include $500 names that are known to be used for peptidases. These retrieve a set of $250 potentially interesting abstracts each week. There is much redundancy, in that a single article may be retrieved by several search terms. Once a nonredundant list of articles has been obtained, the abstracts are reviewed to select the subset that is to be included in MEROPS. In a typical week, 50-60 references come through this filter. Keywords, including the MEROPS identifiers for the relevant peptidases, are manually attached to each and these determine which pages in MEROPS the reference will appear on. If from the abstract it is clear that the paper contains substrate-cleavage data these are entered immediately into the MEROPS collection. Periodically, the bibliography in MEROPS for a peptidase is reviewed to find substrate cleavages with a preference for peptidases without any substrates in the MEROPS collection.
If the substrate is a protein, it is mapped to a UniProt protein sequence database entry (26) initially by name and species. Each cleavage in the protein is mapped to a specific residue in the UniProt entry. Frequently the residue number reported in the paper refers to a position in the mature protein, and to map this to the UniProt sequence the length of any signal peptide and/or propeptide has to be added. The UniProt accession, the P1 residue number, the CRC64 checksum for the sequence and the MEROPS identifier for the peptidase are stored. In addition other information may be retained, including whether the cleavage is deemed by the authors of the source paper to be physiological or not, whether the substrate was in native conformation, the pH of the reaction, and the method used to identify the cleavage. The four residues either side of the scissile bond are also stored so that the cleavage position can be recalculated should the UniProt protein sequence change, and to provide the data for what amino acids are acceptable in the binding pockets S4-S4 0 for each peptidase. A bespoke program (in Perl) was written to add each cleavage in a protein substrate to ensure consistency; the program connects to the locally installed version of UniProt so that each cleavage position can be confirmed as the data are entered. Some data were acquired from proteomics studies. Again a bespoke program was written to parse the data from the Excel spreadsheets available as Supplementary Data to the published papers. Some cleavages were acquired from the CutDB database, but these have been manually checked against the original reference and the UniProt sequence. Once again a bespoke program was written to collect the data, translate the provided substrate Protein Identifier to a Uniprot accession, check that a cleavage event was not already present in the MEROPS collection (and add the CutDB accession number if it were), and add new cleavage events to the MEROPS collection, reporting any inconsistency between the P4-P4 0 residues and the sequence in the UniProt entry.
The data collected are non-redundant. If more than one paper reports the cleavage at the same position in the same protein by the same peptidase, then only data from the paper published first is retained. If several peptidases cleave the same protein in the same position, each is considered a different cleavage event and each is entered. There is no attempt to map cleavages to isoforms of a protein, unless different isoforms were used by the original researchers. Synthetic substrates that differ only in leader (for example benzyloxycarbonyl, succinyl or tosyl) or reporter groups (for example aminomethylcoumarin, naphthylamide or nitroanilide) are considered different cleavages even if all are performed by the same peptidase.
The MEROPS website has two displays to show peptidase specificity. Both use the data from natural and synthetic substrates, but show only naturally occurring amino acids. The first display is a logo which uses the WebLogo software (27) . Residues P4-P4 0 from all the substrates for a peptidase are treated as an alignment. The observed frequency for each amino acid in each position is calculated as a bit score, the maximum possible being 4.32 bits. An amino acid is shown in the logo (in single-letter code) if the bit score exceeds 0.1. The logo is also shown as a text string, where if a single amino acid predominates at one position (i.e. the bit score exceeds 0.4) the letter is shown in uppercase, and if more than one amino acid predominates in any position a letter is shown in uppercase when the bit score exceeds 0.7 and in lower case if the bit score is between 0.1 and 0.7.
The second display is a frequency matrix, which is an 8 Â 20 matrix with residues P4-P4 0 along the x-axis and all amino acids along the y-axis. The amino acids are ordered so that those with similar properties are adjacent. The order is Gly, Pro, Ala, Val, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg and His. Preference is calculated in terms of the percentage of substrates with each amino acid in each position, and a different shade of green is used for each tenth percentile interval. The number of times a residue occurs at each position is shown.
The UniProt accession for each substrate with a known cleavage site was used to search the UniRef50 database (clusters of sequences that have at least 50% sequence identity to the longest sequence) (28) . If a UniRef50 entry was found, then all the UniProt accessions included in that entry were extracted and the sequences retrieved from the UniProt database in FastA format. Short fragments were excluded and the remaining sequences were then aligned with MUSCLE (29), using the default parameters and performing two iterations over the complete alignment to minimize gaps. Because each UniRef50 entry contains sequences sharing 50% or more sequence identity, the program is very quick, and the resulting alignment approximates to an alignment of orthologues. However, some UniRef50 entries will also contain closely related paralogues.
Sequence alignments were generated and highlighted to show not just conserved residues but also peptidase preference. For each cleavage the residues P4-P4 0 were highlighted to indicate whether the residue in each sequence had been observed in any substrate at that position for the peptidase in question. Residues identical to the sequence where the cleavage is known are shown with a pink background. Replacements observed in other substrates are shown with an orange background. Where no substrate for this peptidase is known with this amino acid in this position the residue is shown with a black background. The term 'atypical' is used for an amino acid that has not been observed in a particular binding pocket in any known substrate for a peptidase.
The MEROPS cleavage collection
The MEROPS cleavage collection contains 39 118 cleavages (as of 7 August 2009). The number of cleavages that can be mapped to entries in the UniProt database is 34 606, the remaining 4512 consisting mainly of synthetic substrates. The number of different entries in the UniProt database to which cleavages are mapped is 10513. The number of cleavages that are designated as 'physiological' is 13 307; whereas 20 187 cleavages are designated 'nonphysiological' and 2677 cleavages are designated 'synthetic'. The remaining 2947 are cleavages in peptides that can not be mapped to UniProt because: they are too short; they are significantly modified, such as the non-alpha peptide bond between ubiquitin and its target protein; they are derived from phage displays; they are theoretical cleavages or because it is unclear whether the cleavage is physiological or not. The data are derived from 6095 publications. The number of cleavages in common between the MEROPS and CutDB collections is 5876, of which 3424 were originally found in the literature by the CutDB researchers. The number of cleavages from the CutDB database that failed to make the MEROPS collection, excluding 892 isoforms and 35 duplicates, was 560 (9.5%), mostly due to being mapped to the wrong residue or sequence. The CutDB curators have been informed of these discrepancies. Because the CutDB database includes only cleavages thought to be of physiological relevance and those that can be included in their proteolytic pathways, it has fewer cleavages than in the MEROPS collection. It does not include cleavages in synthetic substrates and those peptides used solely to map peptidase specificities, or general purpose processing enzymes such as signal peptidases and methionyl aminopeptidases.
There are 2415 different peptidases recognized in the MEROPS database (excluding hypothetical peptidases from model organisms). Substrate cleavages have been collected for 1086 peptidases (45%); for the remainder any cleavage positions in substrates are either unknown or have not yet been found in the literature. Only 312 peptidases have had ten or more cleavages collected, and it is only these for which there is enough data for further analysis. The total number of cleavages for these 312 
The residues from P4-P4 0 were collected for each substrate cleavage. Figure 1 shows the number of peptidases showing some selectivity for one or two residues in each binding pocket from S4 to S4 0 . Clearly, many peptidases have extended substrate-binding sites with preferences beyond S1, with 52 showing a preference in the S4 pocket. There are a few peptidases that have a preference at S5 (30), but a preference so far from the scissile bond is rare. It is conceivable that mitochondrial intermediate peptidase (M03.006), which removes a transit octapeptide from the N-terminus of proteins synthesized in the cytoplasm but destined for the mitochondrial matrix, may have a preference as far away from the cleavage site as S8 (31) . Preference on the prime side of the scissile bond is thought to rarely extend beyond the S1 0 pocket, but Figure 1 shows that 32 different peptidases have a preference in S4 0 .
Exopeptidases cleave near protein termini, and because the binding pockets do not exist are unable to accept any amino acids in some positions. Dipeptidases can only accept residues in the S1 and S1 0 pockets; aminopeptidases are unable to accept any residue in S4-S2, carboxypeptidases in S2 0 -S4 0 , dipeptidyl-peptidases in S4 and S3, tripeptidylpeptidases in S4 and peptidyl-dipeptidases in S3 0 and S4 0 . Some omega peptidases (peptidases which do not cleave normal peptide bonds but release substituted amino acids such as pyroglutamate or cleave isopeptide bonds, such as many deubiquitinating enzymes) may also be unable to accept any residue in certain positions, or it is not possible to interpret cleavages in terms of P4-P4 0 , for example for isopeptidases. There are 36 peptidases with 10 or more cleavages that cannot accept any residue in S4, 35 for S3, 26 for S2, 15 for S2 0 , 22 for S3 0 and 25 for S4 0 . Table 1 shows the number of peptidases showing some selectivity in each binding pocket from S4 to S4 0 for amino acid properties (where 'acidic' is Asp or Glu; 'basic' is Arg, His or Lys; 'aliphatic' is Ile, Leu or Val; 'aromatic' is Phe, Trp or Tyr; and 'small' is Ala, Cys, Gly or Ser). Properties are taken from Livingstone and Barton (32) . Only the categories with the fewest amino acids, and those that do not overlap (with the exception of His, which can also be considered aromatic) have been used. If categories such as 'hydrophobic' and 'polar' are used then nearly every binding pocket is highlighted because each category contains more than half of the amino acids. Most preference is directed towards the S1 (201 different peptidases) and S1 0 (160 different peptidases) pockets. The commonest preferences are for a basic amino acid in the P1 position, small amino acids in P1 and P1 0 , and an aliphatic amino acid in P1 0 . No aromatic amino acids were observed in P4 0 in any of the substrates of these 312 peptidases. For each amino acid category preference was most pronounced in the S1 pocket with the exception of aliphatic amino acids, where most peptidases have a preference in the S1 0 pocket. Preference for acidic amino acids is very rare except in the S1 pocket, and similarly aromatic amino acids are rarely preferred except in S1 and S1 0 .
The preference for individual amino acids is shown in Table 2 . It is clear from the table that cysteine is an S3 S2 S1 S1' S2' S3' A count is made whenever an amino acid occurs in one binding pocket in 40% or more of the substrates. There are 15 peptidases that have a preference for two amino acids in a binding pocket: walleye dermal sarcoma virus retropepsin (A02.063, Asn or Gln in S2), sapovirus 3C-like peptidase (C24.003, Glu or Gln in S1), SARS coronavirus picornain 3C-like peptidase (C30.005, Gly or Gln in S1), peptidyl-peptidase Acer (M02.002, Gly or Pro in S1), vimelysin (M04.010, Phe or Leu in S1), carboxypeptidase M (M14.006, Arg or Lys in S1 0 ), carboxypeptidase U (M14.009, Arg or Lys in S1 0 ), dactylysin (M9G.026, Leu or Phe in S1 0 ), chymase (S01.140, Phe or Tyr in S1), tryptase alpha (S01.143, Lys or Arg in S1), trypsin 1 (S01.151, Lys or Arg in S1), plasmin (S01.233, Lys or Arg in S1), flavivirin (S07.001, Lys or Arg in S2), dipeptidyl aminopeptidase A (S09.005, Ala or Pro in S1) and kumamolisin (S53, 004, Glu or Gly in S3). Many peptidases show a preference in more than one binding pocket. There are 13 peptidases with a preference for all eight binding pockets, another 13 with a preference in seven, five peptidases in six, three in five, eight in four, 24 in three, 47 in two and 89 in only one. The number of peptidases showing a preference for an amino acid in a binding site is shown. Only those 312 peptidases with 10 or more known substrate cleavages are included. An amino acid must occur at that position in 40% or more of substrates. Therefore, it is possible for two amino acids to be preferred in any one binding pocket, as is the case for trypsin 1 where there is a preference for either Lys (59% of substrates) or Arg (41%) in S1.
There are 202 peptidases that show a preference, of which 13 show a preference at all eight sites, 13 for seven sites, five for six sites, three for five sites, eight for four sites, 23 for three sites, 49 for two sites and 88 for one site. unwelcome amino acid near a cleavage site. Only the peroxisomal transit peptide peptidase shows a preference for cysteine binding to the S2 and S1 pockets. Tryptophan is also rare around cleavage sites, with only tryptophanyl aminopeptidase (M9A.008, a preference in the S1 pocket) and mast cell peptidase 4 (Rattus) (S01.005, in the S2 pocket) showing a preference; however, this may have more to do with the fact that tryptophan is the rarest of the amino acids. Asparagine is also very rare in the proximity of a cleavage site, one of the few examples being the specialist peptidase legumain (C13.006) which only cleaves asparaginyl bonds (33) . Histidine is also a rare preference, with only three peptidases showing any preference for it, namely chymosin (A01.006; S4), carnosine dipeptidase I (M20.006; S1 0 ) and Xaa-methyl-His dipeptidase (M20.013; S1 0 ). Methionine is also not preferred by most peptidases, exceptions being methionyl aminopeptidases (M24.001, M24.002), where the preference is as expected for methionine binding in the S1 pocket, some members of the peptidase Clp family (S14) and the unsequenced Met-Xaa dipeptidase (M9B.004). The gpr peptidase (A25.001) shows a preference for Met binding to S4. The commonest preference is for arginine binding to the S1 pocket, which occurs in over fifty peptidases. However, arginine is relatively rare outside the P1 position. There are peptidases that show a preference for Gly, Pro and Val for every binding pocket in the range S4-S4 0 . Peptidases showing unique preferences are listed in Table 3 .
Despite there being a large number of substrates collected, the specificity of some peptidases can not be explained in terms of S4-S4 0 preferences. These peptidases 
Specificity logos and frequency matrices present the user with a visual representation of peptidase specificity. An example specificity logo is shown in Figure 2 . From the logo and the cleavage pattern string it is clear that caspase-3 has an absolute requirement for Asp in the S1 pocket (position 4, only one cleavage after Glu is known) and a preference for Asp in S4. There are minor preferences for Glu in S3 and Gly or Ser in S1 0 .
While the logo indicates which amino acids are acceptable in each position, it does not indicate which amino acids are unobserved. These are shown in the frequency matrix, and an example is also shown in Figure 2 for caspase-3. In this example Asp occurs in the P1 position in all 413 substrates, Asp occurs in P4 in almost half the substrates, while Glu occurs in P3 in 27% of substrates. Note that in this frequency matrix every amino acid occurs in positions P4-P2 and P1 0 -P4 0 , but tryptophan is observed only once in P4, P2, P1 0 and P4 0 . This gives an indication of the minimum number of substrate cleavages that has to be collected for a peptidase before definite conclusions about specificity in all binding pockets can be drawn.
A substrate alignment is shown in Figure 3 . The density of residues highlighted in black is high, implying that this cleavage position is very poorly conserved and thus may not be physiologically relevant. The logo is shown at the top with the frequency matrix below. The cleavage pattern is a textual representation of the logo, where the scissile bond is shown as a red cross, and the binding pockets separated by forward slashes. The preferred residue is shown in uppercase if the preference is strong. The number of cleavages on which these data are based is given in parentheses. For the logo, the binding pockets S4-S4 0 are shown along the x-axis, where 1 is S4, 2 is S3, etc. The bit score is shown on the y-axis. The height of the letter is proportional to the bit score. The letters are coloured to indicate amino acid properties: blue for basic, red for acidic, black for hydrophobic and green for any other. In the frequency matrix below the logo, each cell shows the number of substrates with an amino acid occupying one of the positions P4-P4 0 . Cells in the matrix are highlighted in shades of green where the greater the preference, i.e. the more often an amino acid occurs at that position, the brighter the shade. Cells are highlighted in black if the amino acid is unknown at that position for any substrate.
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Protein sequence alignments were constructed for every substrate where the cleavage had been assumed in the literature to be of physiological significance. The total number of alignments generated was 3141. A selection of cleavage sites which were not conserved in all homologues included in the same UniRef50 database entry are listed in Table 4 . Only those cleavages by peptidases with at least 20 known substrates are included.
There are a number of possible causes for a cleavage site not to be conserved which are listed below.
(1) The UniRef50 entry might include paralogous sequences which although at least 50% identical to the sequence with the known cleavage, might be processed or degraded differently and there is no evolutionary pressure to maintain the known cleavage site. Where a cleavage site was not conserved, a paralogue was identified in an alignment as a second protein from the same species that was clearly not a splice variant. (2) UniRef50 entries contain many translated genes from genome sequencing projects; gene finding in eukaryote genomes is notoriously difficult and it is possible that erroneous gene building has resulted, for example, in the loss of the exon encoding the cleavage site or the inclusion of part of an intron in its place. (3) It is also probable that for some peptidases there are not enough substrates known to be sure that any amino acid is really excluded from a particular binding site. The number of substrates known for each peptidase is included in Table 4 , because the greater the number of substrates the more likely that an amino acid is really atypical and not just unobserved. (4) The alignment is incorrect. This is unlikely given the close relationship between the sequences, which are all 50% or more identical; however there are situations where an insert or deletion occurs within the range P4-P4 0 . (5) Some endogenous cleavages (for example removal of signal and transit peptides) may be the result of more than one cleavage, because aminopeptidases nibble away the N-terminus (1), and may thus be incorrectly mapped to the specificity of the leader peptidase. (6) It is theoretically possible that if the substrate and peptidase are from the same organism both will have evolved to accommodate a change in the cleavage position. (7) A single residue mismatch may also be due to a single-base sequencing error. Potential errors of this kind can be identified using a codon dictionary, provided the atypical residue could be the result of a single base change, and that it is the only residue not conserved, regardless of the number of sequences in the alignment. (8) Some cleavages regarded as 'physiological' are actually fortuitous. If a cleavage site is extremely poorly conserved it is unlikely to be physiologically relevant. 
---P----(4)
Cytochrome C Where it is possible to suggest a cause why a cleavage site is not conserved this is indicated in Table 4 by the letters a-h. Included in category d, where insertions and or deletions occur in the homologous cleavage sites, is 50S ribosomal protein L7Ae (UniProt accession P12743). There are N-terminal extensions to most homologues so that the known methionyl aminopeptidase 2-cleavage site is not aligned. Five of these sequences may be derived from erroneous gene builds (point b). The UniRef50 database entry for 60S ribosomal protein L10 (P27635) includes a wide range of species (the cleavage is known in the human protein) and the peptidase performing the cleavage (granzyme B) is not present in Paracoccidiodes brasiliensis, where the substrate cleavage is also not conserved. The replacements that are reported as atypical in hemoglobin subunit alpha (P69905) by Schistosoma cathepsin D (A01.009) (34) are the rarest naturally occurring amino acids, tryptophan and cysteine, and despite there being 109 known cleavages for this peptidase, this may still not be enough to properly exclude these rare amino acids. On the other hand, this is the cleavage of a host protein by a parasite peptidase and the specificity may have adapted to limit the availability of hosts.
None of the cleavages listed in Table 4 has been assigned to cause f above, namely where changes in the substrate cleavage site may be mirrored by changes in peptidase specificity. Without modelling the substrate binding sites, if that were possible, detecting this situation is difficult. However, the autolytic processing of cathepsin E (P43159) may be such an example (35) .
In some cases, a poorly conserved cleavage site may represent a pathological condition in the species where the cleavage was first identified. For example, despite there being few cleavages for cathepsin H, the reported cleavages in the BID protein (36) are in particularly poorly conserved regions (see Figure 3 ). Cleavage of the BID protein leads to the induction of apoptosis. That the cleavage sites are not well conserved amongst mammalian orthologues is not surprising given that the cytoplasmic substrate and the lysosomal peptidase should not meet under normal circumstances. The mouse protein in which the cleavage was identified may therefore be unusually susceptible to cleavage should the lysosomal membrane be ruptured.
The specificity logos and frequency matrices for all peptidases with 10 or more known substrate cleavages are already available in the MEROPS database. Alignments are also available for all protein substrates that have a corresponding UniRef50 entry, showing conservation of both physiological and non-physiological cleavages. The next release of the database will include tables showing comparative peptidase specificity in terms of preference for both amino acid and amino acid type. 
The MEROPS database includes over 39 000 cleavages in substrates (synthetic and naturally occurring) which have been collected from the literature. These are classified as physiological or non-physiological, depending on whether the substrate is naturally occurring and if it is in native conformation. At least one substrate is known for 45% of the different peptidases identified in the MEROPS database. Displays in the database give insights into peptidase specificity and to the conservation of cleavage sites amongst orthologous proteins. The data provide a substantial training set for algorithms to predict peptidase substrates and cleavage positions in those substrates. The data may also be useful for the design of inhibitors and engineering novel specificities into peptidases. By examining the conservation of cleavage sites in protein substrates in terms of peptidase substrate binding sites, it is clear that there are a number of cleavages where atypical replacements occur. Many of these can be explained by gene build or sequencing errors, inserts or deletions in the region around the cleavage site, or the alignments contain one or more paralogues in which cleavage may be absent or different. In a few cases it is possible that more than one peptidase is involved in processing, or there may not be enough known substrates for some peptidases to be sure that an atypical replacement is really unacceptable. A number of substrate cleavages that may be fortuitous and not of any physiological relevance have been identified.
This cleavage set is freely available and can be downloaded from the MEROPS FTP site (ftp://ftp.sanger.ac.uk/ pub/MEROPS/current_release/database_files/ Substrate_search.txt). Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins BACKGROUND: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. METHODOLOGY/PRINCIPAL FINDINGS: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. CONCLUSIONS/SIGNIFICANCE: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus. Dengue is one of the most important vector-borne viral diseases in humans. However, the interaction between dengue virus (DV) and host cells is only partly understood. Therefore, there is an urgent need to develop new tools to gain insight into the viral journey through host cells.
As a member of the Flavivirus genus in the Flaviridae family, DV is a small, positive strand RNA enveloped virus. There are four serotypes of dengue virus (DV1-4). Their genome encodes a polyprotein precursor of at least seven non-structural proteins and three structural proteins which are the capsid protein (C), the membrane protein (M) and the envelope glycoprotein (E) [1] . The polyprotein is processed co-and post-translationally by cellular signalase in the lumen of the rough endoplasmic reticulum (ER) and by a viral protease in the cytosol [1, 2, 3] . The nascent C protein contains a C-terminal hydrophobic domain that acts as a signal sequence for translocation of the immature form of M, the prM, into the lumen of the rough ER. Two adjacent prM C-terminal transmembrane domains are responsible for prM membrane anchoring and E translocation into the ER [2] . prM and E associate into heterodimers at ER membranes [4, 5] where they assemble with the viral RNA/C complex to form progeny virions [1] . During the egress of virions through the secretory pathway, prM protein is cleaved by the trans-Golgi resident furin protease to form the M envelope protein and the soluble pr segment, which is released into the extracellular medium upon particle secretion [6] . prM cleavage marks maturation of flavivirus virions [7, 8] . Cleavage of prM is intimately correlated to change of conformation of envelope protein complexes. Although it was thought that prM cleavage is a prerequisite for E dimerization, recent studies show that change of conformation most probably occurs at low pH in the TGN and allows cleavage of prM by furin [6, 9, 10] .
prM and E proteins from flaviviruses, such as yellow fever virus [11] , Japanese encephalitis virus (JEV) [12, 13] , West Nile virus (WNV) [14] and tick-bone encephalitis virus (TBEV) [15, 16] , are able to assemble into subviral particles in the absence of any other viral component. Subviral particles and infectious virions are coproduced in infected cells, assemble in an immature form, and subsequently undergo the same maturation process and display similar fusion activity as infectious viruses [17, 18] . Therefore, subviral particles could be a precious tool for research on cell biology of DVs. Although there have been attempts by several groups to obtain DV RSPs, either their production was inefficient or the sequence of DVs structural proteins had to be substantially modified [19] before they could efficiently generate RSPs. For example, the furin cleavage site on prM had to be mutated to establish the DV2 RSPs producing CHO cell line because it was found that the DV2 RSPs cause cell-cell fusion [20, 21] . Others replaced a portion of the carboxy-terminal region of DV1 and DV2 E genes with the corresponding sequence of JEV to observe significant RSP secretion [22, 23] . In either case, these modifications may interfere with the interactions with cytosolic proteins and, possibly, with the maturation and folding of the structural proteins.
Here we describe the efficient production of native RSPs of the four DV serotypes by using a codon optimization strategy. Codons of the DV prME gene were replaced with those preferentially used in mammalian cells. Optimization was first applied to the DV1 prME gene. We found that this experimental approach could efficiently increase the intracellular expression level of the E glycoprotein in human cells and therefore enhance the production of DV1 RSPs. We have established a DV1 RSPs producing stable cell line (HeLa-prME). Our data by immunofluorescence and electron microscopy show that most of E protein co-localizes with markers of the ER, where RSPs accumulate. Biochemical analysis of secreted RSPs demonstrates that they contain E homodimers and M, indicating that RSPs have trafficked through the secretory pathway and that maturation process has occurred. Using the HeLa-prME cell line, we have developed a semi-quantitative assay to screen a 122 genes cellular membrane trafficking siRNA library and identified genes that may be involved in the secretion of DV. From our screen, 23 genes show a significant reduction in DV1 RSPs secretion whereas for 22 genes RSPs levels were increased in cell supernatants. These data provide a proof of concept that RSPs of DVs and the producing cell line are safe tools that can be used in the study of viral egress. Finally, the optimization strategy was applied to DV2, DV3 and DV4 prME, and RSPs for all four DV serotypes were efficiently produced. These native RSPs are, therefore, a safe mimicry of virions that can be used to study viral and cellular requirements for virus assembly and egress.
To generate DV RSPs, we first transfected HeLa cells with native DV1 prME gene derived from the DV1 FGA/NA d1d [24] strain, which contained the signal sequence of vesicular stomatitis virus G (VSV-G) envelope glycoprotein inserted in frame upstream of the prME cDNA. 48 hours post-transfection, expression of dengue E protein was monitored by flow cytometry on permeabilized transfected HeLa cells with the 4E11 monoclo-nal antibody against dengue E protein. E protein was detected in transfected cells at low expression level ( Figure 1A) . Analysis of the prME gene sequence revealed that 15% of nucleotide triplets were rarely used in mammalian cells so that its codon usage was not adapted for efficient expression in this cell system. Thus we designed and synthesized an optimized DV1 prME (prMEopt) gene in which the rarely used codons were replaced with those preferred by mammalian cells, without changing the amino acid sequence ( Figure S1 ). More than 70% codons were modified for prME optimization. In addition, we also removed the negative cisacting sites (such as splice sites, poly(A) signals, etc) which might have negatively influenced expression. The optimized gene resulted in high and stable expression levels in transfected mammalian cells ( Figure 1B ). Both the mean fluorescence intensity (MFI) and percentage of positive cells increased by almost 3 fold. E protein was hardly detected without cell permeabilization, indicating that its localization was intracellular and was not expressed at cell surface (data not shown).
A HeLa cell line that stably expresses DV1 prM and E envelope proteins was then established by transducing HeLa cells with a retroviral vector pCHMWS-prMEopt-IRES-Hygromycin. Hygromycin-selected cells were designated HeLa-prME. Several single colonies of HeLa-prME were used in this study and they showed similar results. The HeLa-prME cell line has already been propagated for more than thirty passages in the presence of hygromycin and has kept a stable prME expression level. Culture of HeLa-prME cells in the absence of hygromycin resulted in a significant reduction of prME expression (data not shown).
The intracellular distribution of DV1 E glycoprotein was analyzed in fixed HeLa-prME cells by co-labeling with Erp72, ERGIC-53 and Golgin-97, which are proteins of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the Golgi apparatus, respectively. As expected, the DV1 E glycoprotein mainly distributed in the ER in HeLa-prME cells, as shown by immunofluorescence, where it colocalized with Erp72 ( Figure 2A -D), whereas it showed no colocalization with ERGIC-53 ( Figure 2E -H) and Golgin 97 ( Figure 2I -L). Interestingly, E and Erp72 were also enriched in a perinuclear compartment, which is not usually labeled by anti-Erp72 antibody and other ER markers (data not shown), suggesting that it has been induced by prM and E viral protein expression. In addition, no staining was observed at the plasma membrane, confirming the previous flow cytometry result. Altogether, our data demonstrate that the DV1 E glycoprotein is efficiently expressed and enriched in the endoplasmic reticulum in the HeLa-prME stable cell line.
The endoplasmic reticulum is the assembly site for flaviviruses. In order to verify whether this ER staining reflects the presence of E-containing assembled particles, we analyzed the HeLa-prME stable cell line by transmission electron microscopy (TEM) on thin sections of fixed, epon-embedded cells ( Figure 3A ). We found that HeLa-prME, but not control HeLa cells (data not shown), displayed dilated ER membranes with aligned round particles and elongated parallel tubules. Particles were ,20 nm in diameter and homogenous in size and shape. The aligned particles that we have observed in HeLa-prME cells are similar to the stacked viral particles which have been described in cisternae of the rough ER in infected insect and mammalian cells [25, 26, 27, 28] . To confirm that these structures contain viral proteins, we labeled thawed cryosections with the 4G2 antibody that recognizes the E protein ( Figure 3B -D). We found antibody binding for the E protein Figure 2 . Subcellular localization of the DV1 E protein in HeLa-prME cells. HeLa-prME cells were fixed, permeabilized, and stained for E protein (green) and for cellular marker antigens (red). DAPI staining was used to label cell nuclei (blue). Erp72, ERGIC-53 and Golgin-97 are proteins of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and Golgi apparatus, respectively. The scale bar represents 10 mm. doi:10.1371/journal.pone.0008325.g002 present in the lumen of cisternae, where it is localized to electron lucent, round particles and tubular structures. In addition, E protein is also found on amorphous, electron dense material present in these cisternae ( Figure 3B ). To confirm the nature of these cisternae by other than morphological criteria we performed a double labeling of the 4G2 antibody with the ER resident protein calreticulin. We found antibody binding for calreticulin present on the 4G2 positive cisternae ( Figure 3C ), unequivocally identifying them as ER. In addition, we found a lower but significant amount of label for the E protein present in the Golgi stack and in vesicles in the Golgi area ( Figure 3D ).
Thus, our observations indicate that expression of prM and E DV1 envelope proteins induces the formation of RSPs in an ERderived compartment.
We then investigated if the RSPs observed by EM in HeLa-prME cells could undergo maturation and be secreted like DV.
Cell lysate (CL) and clarified supernatant (SN) concentrated by ultra-centrifugation were analyzed by Western blotting followed by incubation with either the anti-E monoclonal antibody 4E11 ( Figure 4A -B) or an anti-DV1 serum from a human patient ( Figure 4C -D). In CL samples, a 50 kDa monomeric form of the E glycoprotein was predominantly observed ( Figure 4A ,C), whereas high levels of 100 kDa E glycoprotein homodimers were readily detected in SN samples ( Figure 4A -D). This suggests that whereas in HeLa-prME cells the majority of viral proteins are localized in the early pre-Golgi secretory pathway in an immature state, secreted RSPs have passed through maturation stages in the Golgi apparatus thus resulting in homodimerization of E proteins. To further confirm the presence of E dimers in SN and exclude the possibility that dimerization resulted from treatment for electrophoresis, the SN were incubated in the presence or absence of a cross linker (3, 39-dithiobis [sulfosuccinimidylpropionate]; DTSSP) and then subjected to Western blotting using the anti-E 4E11 antibody. Under these conditions, we observed that E dimer/ monomer ratio was lower in non-treated RSPs compared to the Figure 3 . Electron microscopy analysis of the HeLa-prME cell line. HeLa-prME (DV1) cells were fixed and either prepared for epon embedding (A), or for immuno labeling on thawed cryosections (B-D). A), Round particles are found aligned in the lumen of the ER (arrows) together with tubular structures (arrowheads). B) Labeling for the E protein is present in the lumen of cisternae. It is localized to electron lucent, round particles (arrows) and tubular structures (arrowheads). In addition E protein is also found on amorphous, electron dense material present in these cisternae (asterix). C) Double labeling of E protein (10 nm gold, black arrows) and the ER marker calreticulin (15 nm gold, white arrows). Calreticulin is present on the limiting membrane of the cisternae, which contain round particles positive for E protein labeling. D) Label for the E protein is also found within the Golgi stack (G) and on vesicles in the Golgi area. N = nucleus, all scale bars represent 200 nm. doi:10.1371/journal.pone.0008325.g003 DTSSP treated (data not shown). These data indicate that, in SN, a significant proportion of E protein is present as a homodimer and detection of E monomers most likely results from partial dissociation under the denaturing conditions of electrophoresis. E homodimers were no longer detected when samples were heated in presence of dithiothreitol ( Figure 4B ). In addition, the prM protein could be readily detected with the anti-DV1 serum in CL but not SN samples ( Figure 4C ). By contrast, when the HeLa-prME cells were treated with NH 4 Cl, which inhibits acidification of the trans-Golgi compartment and, hence, the activity of furin protease and prM cleavage, prM was also found in SN ( Figure 4D -E). To our surprise E dimers were also detected in these conditions. Interestingly, the E monomers from SN exhibited slower electrophoretic mobility than those of CL samples, which suggests that E glycoproteins have acquired complex N-glycans in the Golgi apparatus. This was confirmed by N-Glycosidase F (PNGase F) and endoglycosidase H (EndoH) digestion ( Figure 4 F-G), which cleaves nearly all types of N-glycan chains or only high mannose and some hybrid N-glycan chains from glycoproteins, respectively. Whereas E protein in CL was sensitive to both treatments, only PNGase F digestion increased the electrophoretic mobility of E protein in SN, demonstrating that RSPs had acquired EndoH-resistant, complex N-glycans in the Golgi apparatus before secretion. Moreover, we found that secretion of DV RSPs was altered by incubation of cells at 15uC and 20uC, which block cargo transport from ERGIC to cis-Golgi and exit from the trans-Golgi, respectively, and by brefeldin A treatment, which inhibits exit from ER and induces fusion of Golgi membranes with ER (data not shown). Altogether, homodimerization of E, acquisition of complex sugars and efficient cleavage of prM indicate that prM and E viral proteins have correctly assembled in the ER into RSPs which have trafficked through the secretory pathway before secretion into the cell medium.
To study the dynamic of RSP production, we performed a pulse chase experiment on overnight starved HeLa-prME cells that were metabolically labeled with S 35 -methionine for 1 hour and subsequently chased for 24 hours in normal medium. Supernatant and cell lysate were collected at the specified time points after chase; RSPs were immuno-precipitated using the 4E11 anti-E monoclonal antibody and then analyzed by autoradiography. Detection of prM or M in this assay results from co-immunoprecipitation and, therefore, indicates efficient interaction with E protein. Secretion of E and M proteins was detected from the 8 hours time point on, with a significant increase at 24 hours postchase ( Figure 5A ). Consistently, high levels of E and M proteins were found in cell lysates at all time points with a substantial Figure 5B ). Two major protein products were found in cell lysates, with an apparent molecular weight corresponding to E and prM proteins. Bands of higher molecular weight were also present and could be oligomeric forms of E and prM or M. Interestingly, the pattern of immunoprecipitated proteins from cell supernatant was slightly different. Higher proportions of E dimers were found and a band corresponding to the molecular weight of M but not prM was detected at the 24 hours time point, indicating that most secreted RSPs are mature. Our data demonstrate that newly synthesized proteins need 8 hours to be translocated through the secretory pathway and released into the supernatant as mature RSPs.
To further characterize secreted DV1 RSPs, we performed sucrose gradient fractionation on RSPs concentrated from supernatant of HeLa-prME cells. Supernatants were ultracentrifuged and RSPs-containing pellets either resuspended in PBS or in 0.5% Triton-X100 containing PBS to solubilize E from lipid membranes, as described [29] . As expected, Triton-X100solubilized E protein and RSP-associated E protein appeared in distinct fractions of a discontinuous 20 to 60% sucrose gradient ( Figure 6 ). The amount of E glycoprotein in each fraction was quantified. E glycoprotein in non-treated sample sedimented in fractions containing 20% to 30% sucrose ( Figure 6 , black bars) whereas in Triton-X100 treated samples, E protein was solubilized and detected in fractions at the top of the gradient (Figure 6 , white bars). These data further confirm that the RSPs formed in HeLa-prME are secreted into culture medium from which they can be easily purified by ultracentrifugation. Moreover, we have obtained preliminary evidence that DV1 RSPs and DV1 viral particles isolated from HeLa-prME and mammalian infected cells, respectively, sediment at similar densities on 10-60% continuous gradient of sucrose (Dr Philippe Despres, Institut Pasteur, personal communication).
Our results show that the DV1 RSPs produced by HeLa-prME cell line mimic maturation and secretion of DV1, thus providing a useful tool to study the interaction between DV and host cells during viral egress. To identify host factors that could either enhance or reduce production of DV RSPs, we first developed a quantitative assay to relatively quantify levels of secreted particles in supernatant of HeLa-prME cells. The chemiluminescence dotblot (CLDB) assay is based on the concentration of RSPs from cell supernatant on PVDF membranes, followed by detection of E with a specific horseradish peroxidase (HRP)-conjugated antibody and quantification of substrate-induced luminescence using a luminometer. Our data using purified E protein of known concentrations showed that, when ranging between 400 pg to 40 ng, E protein on PVDF membrane displayed a very good linear correlation with the luminescence density in CLDB assay ( Figure 7A ). The CLDB was first used to estimate the RSPs yield from HeLa-prME cell line, and we found that the concentration of E protein in supernatant of HeLa-prME cell line was around 500-1000 ng/ml under the culture condition used for siRNA transfection (data not shown). We then screened a siRNA library that consisted of 122 genes which target cellular membrane trafficking using the HeLa-prME cell line. Non-targeting siRNA (NT) and siRNA targeting DV1 prME were added as controls. Library and control siRNAs were transfected in triplicates on 96-well plates. Levels of RSPs secreted by siRNA transfected HeLa-prME were measured by CLDB assay from 40 microliters of supernatant from each well. Levels of E protein in cell supernatant were expressed in relative luminescence units and ratios to that of NT controls were shown in Figure 7B . T test was used to assess the statistical significance of differences between each sample and NT. Differences were considered statistically significant when P,0.05. We observed that targeting of 23 genes resulted in significant reduction of DV1 RSPs amounts in supernatants whereas targeting of 22 other genes induced a significant increase. For instance, our results showed that downregulation of ADP-ribosylation factor 1 (ARF1), which regulates secretory membrane transport [30] , resulted in 3 fold decrease of DV1 RSPs in cell supernatant, suggesting its involvement in the secretion of DV. Some genes whose down-regulation enhanced levels of DV RSPs in the supernatant are involved in endocytosis, such as the three dynamins which show a 2 to 4 fold increase in dengue RSPs secretion. These proteins are involved in the budding process or in the transport of vesicles [31] . Such an enhancement might have been due, at least in part, to a blockade in the re-internalization of secreted RSPs by HeLa-prME cells.
Although further experiments are required to confirm the screening data, our study validates the use of DV RSPs-producing HeLa-prME cell line in combination with the CLDB-based quantification strategy as a promising system to facilitate the identification of cellular factors involved in DV secretion.
The codon usage of DV2, DV3 and DV4 prME genes differed substantially from that of mammalian cells and, therefore, was optimized as described for DV1 by replacing more than 70% of the codons ( Figure S1 ). RSPs of the four serotypes were first produced in 293T cells by transient expression of prME genes. Particles were purified and detected by Western blotting using either the anti-E 4E11 monoclonal antibody or a mixture of sera from four patients who were infected by all dengue serotype ( Figure 8A-B) . With the anti-E 4E11 antibody, monomeric and dimeric forms of the DV1 E protein could be readily detected whereas weaker signals were observed for the other three serotypes, because of limited affinity of the antibody ( Figure 8A ) [32] . E proteins of all DVs were detected using the mixture of sera ( Figure 8B ) with a signal of similar intensity, which suggested that RSPs of four serotypes could be generated to comparable levels. Monomeric E proteins of four serotypes displayed slightly different electrophoresis mobility, which could be due to a differential level of N-glycosylation. E glycoproteins of DV1 and DV3 have two N-glycosylation sites at Asn 67 and Asn 153, whereas those of DV2 and DV4 have only one at Asn 67 [33] . Dimeric E protein was observed in DV1, DV3 and DV4 but not in DV2. Besides E, the prM protein was also detected in RSPs produced by transient transfected 293T cells. Recently, we have established HeLa-prME and 293T-prME cell lines of DV1, DV2 and DV3 using the same procedure described for DV1 HeLa-prME. We have compared the maturation of RSPs produced by both cell types using SDS-PAGE and silver staining of the gel ( Figure 8C ). Supernatants from parental HeLa and 293T cells were used as controls in the experiment. We found that, whereas only a small fraction of prM was cleaved in the RSPs produced by 293T-prME cell lines, cleavage of prM was much more effective in RSPs from the HeLa-prME cell lines. This result indicates that efficacy of maturation is cell type dependent. RSPs of the four serotypes were further analyzed by sucrose gradient. As for DV1, RSPs of DV2, DV3 and DV4 were concentrated in fractions containing 20% to 30% sucrose ( Figure 8D) . Altogether, our results demonstrated that the strategy of codon optimization was successful in leading to the production of RSPs of all serotypes with comparable efficacy and similar sedimentation properties.
In this study, we have used a codon-optimization strategy to establish and characterize stable cell lines that produce RSPs for the four dengue serotypes. Previous studies had reported the production of DV RSPs in which the glycoproteins were mutated either to avoid host cell fusion or to delete the ER retention signal. For example, DV1 RSPs were not secreted effectively and DV2 RSPs could not form unless the transmembrane domain of the E glycoprotein, which contains an ER retention motif was replaced with that of JEV [22, 23, 34] . Our ability to obtain the efficient production of RSPs of all serotypes without any change in the amino acid sequence is likely the result of codon optimization for expression in mammalian cells, which has been proven to be an Figure 6 . Sucrose gradient analysis of DV1 RSPs. The supernatant from HeLa-prME cells was concentrated and resuspended in PBS or 0.5% Triton-X 100 containing PBS. RSPs were then centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm (Beckman SW-41Ti rotor) for 2.5 hours at 4uC. Fractions of 0.5 ml were collected and E content was measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis. doi:10.1371/journal.pone.0008325.g006 Figure 7 . Application of the DV RSP-producing HeLa-prME cell line and CLDB to screen a small library of siRNA which targets 122 genes involved in membrane trafficking. A) The correlation between luminescence density and amount of E protein on PVDF membrane in CLDB assay. B) The screen results of the siRNA library which targets 122 genes involved in membrane trafficking. Non-targeting siRNA (NT) and siRNA targeting DV1 prME were used as controls. Level of E protein in cell supernatant of each siRNA was expressed as its ratio to that of NT controls. Two-sided Student's t test was used to assess the statistical significance of differences between each sample and NT. Differences were considered statistically significant when P,0.05. Genes inducing either a significant decrease or increase in RSPs production are shown in gray and black columns, respectively. doi:10.1371/journal.pone.0008325.g007 , respectively (B) . C) Production of RSPs by 293T-prME and HeLa-prME stable cell lines. DV1-DV3 RSPs in supernatants were analyzed by SDS-PAGE and silver staining of polyacrylamide gels. Bands corresponding to the approximate molecular weight of E monomers and dimers, as well as prM and M are indicated by arrows. Supernatants from parental 293T and HeLa cells were used as controls. D) Analysis of RSPs of 4 serotypes by sucrose gradient. RSPs were centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm for 2.5 hours in 4uC. Fractions of 0.5 ml were collected and measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis. doi:10.1371/journal.pone.0008325.g008 effective method to increase the expression level of glycoproteins from various viruses [35, 36] . Thus, gene optimization significantly enhanced expression of prME glycoproteins in transfected cells, and this over-expression increased both RSP production and secretion. However, it is also possible that using other viral strains may result in different efficacy of RSPs production, regardless of codon optimization. To our knowledge, this is the first report of RSPs using the native prM and E envelope proteins for the four serotypes of DVs in mammalian cells.
We have characterized the maturation of DV1 RSPs produced by the HeLa-prME cell line. Sucrose gradient and sedimentation analysis demonstrated that the DV1 RSPs are concentrated in fractions containing 20-30% sucrose and are sensitive to detergent treatment. Although prM/E heterodimers were not found in our experiments, possibly because prM/E interaction is weak, we observed processing of prM into M protein in HeLa-prME cells. This cleavage was sensitive to NH 4 Cl treatment, which inhibits acidification of the trans-Golgi compartment and, hence, the activity of furin protease. Newly synthesized E proteins first form heterodimers with prM proteins in the ER of host cells and then rearrange as homodimers during the process of secretion [37, 38] . The rearrangement from heterodimer to homodimer was first thought to require cleavage of prM by furin in the trans-Golgi to form M and soluble pr proteins [39] . A recent study, however, has shown that such rearrangement is mainly caused by the progressive acidification of the milieu along the secretory pathway, which facilitates prM cleavage by furin [6, 10] . If rearrangement is a pre-requisite for prM cleavage to occur, it could explain, at least in part, why E homodimers were still observed in our study following treatment with the acidotropic reagent NH 4 Cl. The precise mechanism underlying their presence in RSPs under these experimental conditions, however, requires further investigation. Finally, we found by pulse-chase experiments that 8 hours are necessary for production and secretion of RSPs. Altogether, these results show that mature DV1 RSPs are efficiently produced by the stable HeLa-prME cell line.
We have studied the subcellular localization of DV1 E and RSPs in the HeLa-prME cell line. By immuno-staining of fixed cells and fluorescence microscopy, we have shown that most of the E protein is localized in the ER compartment, where dengue glycoproteins are synthesized [1] . The fact that we did not detect any significant signal of E in ERGIC and Golgi apparatus could be explained by a very low amount of protein in these organelles. In these conditions, saturating signals in the ER would mask its detection in other compartments along the secretory pathway. The fluorescent microscopy results were in accordance with our electron microscopy data. Analysis of cell sections by transmission electron microscopy has revealed that E proteins in the ER are associated with both round particles and tubular structures. The tubules could either be intermediate forms of RSPs assembly or result from accumulation of prM and E proteins in the ER. Budding of virions from cellular membranes depends on assembly of viral structural proteins that generate pushing and/or pulling forces simultaneously to induce curvature of membranes, which is necessary for particle formation and membrane fission. It is possible that assembly of overexpressed prM and E proteins in the ER allows formation of long tubules but that the fission event is a limiting factor. The secretion pathway was also investigated by temperature block or pharmacological experiments. Incubation at either 15uC, to stop traffic between the intermediate compartment and the cis-Golgi, or at 20uC, to block exit from the trans-Golgi network, or BFA treatment, to block the exit from ER, significantly reduced secretion of RSPs. These results demonstrate that RSPs traffic through ER, ERGIC and Golgi compartments before being secreted out of the cell.
The efficient production of DV1 RSPs by HeLa-prME cell line and the fact that RSPs could mimic the maturation and secretion processes allowed us setting up an assay to study the interaction between DV and host cells during egress, a step which has received little attention in comparison to DV viral entry and replication [40, 41, 42, 43] . We have validated our assay by using a siRNA library preferentially targeting genes involved in cellular transport. As expected, a number of genes involved in the secretory pathway resulted in reduced release of RSPs in the cell supernatant, whereas other factors were associated with an opposite effect. Clearly, the identification of cellular factors involved in the egress process will be helpful to understand the maturation of DV and its pathogenicity. Moreover, our system will allow comparing the effect of cellular factors using RSPs assembled from the four dengue serotypes to test whether there are strain-specific interactions with host proteins.
HeLa and 293T cells maintained in our lab [44] were cultured in DMEM containing 10% fetal bovine serum. Purified anti-E 4E11 and 4G2 monoclonal antibodies were provided by Dr. A. Amara and P. Despres (Institut Pasteur, France), respectively. Purified anti-DV1 mouse IgG and sera from four patients infected by the four dengue serotypes (1) (2) (3) (4) , respectively, were kindly provided by Dr. Philippe Buchy (Institut Pasteur, Cambodia). The native (non-codon optimized) DV1 (strain FGA/NA d1d) prME gene containing construct was provided by Dr A. Amara. The prME sequences of DV1 (strain FGA/NA d1d), DV2 (strain FGA/02), DV3 (strain PAH881/88) and DV4 (strain 63632) were codon-optimized and synthesized by the Geneart Company (Regensburg, Germany) and subcloned into pcDNA or retroviral vector pCHMWS-IRES-Hygromycin (kindly provided by Dr. Rik Gijsbers, from Molecular Medicine at Katholieke Universiteit Leuven) using BamHI and XhoI restriction sites. A nucleotide sequence encoding for the signal peptide of VSV-G (MKCLLY-LAFLFIGVNC) was included upstream of each prME gene. Sequences of codon-optimized prME genes are provided as Supporting Information ( Figure S1 ).
To produce the retroviral vector for delivery of the prME-opt gene into HeLa cells, the pCHMWS-prME-opt-IRES-Hygromycin, pcDNA-VSV-G and p8.71 (modified HIV provirus coding for gag and polymerase) plasmids were co-transfected into 293T cells. The cell supernatant containing infectious VSV-G-pseudotyped retroviral particles was harvested 48 hours post-transfection and used to infect HeLa cells. Two days after infection, cells were selected in culture medium containing 500 mg/ml of hygromycin for two weeks. Selected cells (HeLa-prME) were tested by flow cytometry for E expression and were maintained in DMEM +10% FBS +500 mg/ml of hygromycin.
HeLa cells were transfected with pcDNA-prME, pcDNA-prME-opt or a pcDNA empty vector using calcium phosphate precipitate method. Two days after transfection, cells were detached by incubation in 10 mM EDTA at 37uC for 10 min, fixed in 2% paraformaldehyde, and then permeabilized in 0.1% Triton X-100. After washing, the cells were incubated with a mouse anti-E antibody (4E11 
For fluorescence microscopy on fixed cells, HeLa-prME cells were grown on glass coverslips. Cells were fixed, permeabilized, and incubated with anti-DV human sera (1:200) and anti-Erp72 Rabbit polyclonal Ab (1:200, Stressgen Bioreagents, Ann Arbor, MI, USA), anti-ERGIC-53 mAb (1:1000, Alexis Biochemicals, Farmingdale, NY, USA), or anti-Golgin-97 mAb (1:50, Invitrogen, Carlsbad, CA, USA), followed by the incubation with corresponding secondary antibody conjugated with FITC or TRITC. Nuclei were stained with DAPI and coverslips were mounted on glass slides for analysis. Fixed cells were visualized under AxioObserver Z1 inverted motorized fluorescent microscope using the ApoTome module and piloted through the Axiovision 4.6 software and images were acquired through the MRm AxioCam high resolution CCD camera (Carl Zeiss, Germany).
HeLa-prME cell line was fixed at 28 hours post-trypsination and processed for EM and immuno-EM.
For conventional TEM, cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1.5 hr at room temperature and post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) for 1 hour at room temperature. Then cells were embedded in 2% agarose and cell blocks were post-fixed with 2% uranyl acetate in 30% ethanol, dehydrated in graded series of ethanol and embedded in epoxy resin. Ultrathin sections were cut with a Leica Ultramicrotome UCT (Leica Microsystems; Vienna, Austria) and collected on 400-mesh formvar coated copper grids. Sections were stained 45 minutes with 4% aqueous uranyl acetate and 5 minutes with lead citrate.
For immuno-EM, cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4), and embedded in 12% gelatin. Blocks were infiltrated with 2.3 M sucrose for cryoprotection, mounted on specimen holders and frozen in liquid nitrogen. Cryosections were cut with a Leica EM UC6/FC6 Microtome (Leica Microsystems, Vienna, Austria). Thawed cryosections were labeled with anti-E 4G2 antibody, rabbit polyclonal antibody against ER resident protein calreticulin (Abcam, Cambridge, MA, USA) and protein-A gold (10 nm and 15 nm) obtained from Utrecht University (Utrecht, The Netherlands) and used as described before [45] . Double labeling was performed sequentially, using for each antibody a different size of protein A gold. Unspecific binding of the second protein A to the first antibody was blocked by incubation with 1% glutaraldehyde as described before [46] . The grids were viewed with Philip CM10 electron microscope at 80 kV and images were taken with KeenView camera (Soft Imaging System, Lakewood, CO, USA) using iTEM 5.0 software (Soft Imaging System GmbH).
Supernatants of HeLa-prME or its parent HeLa cells were harvested and cleared by centrifugation at 3,000 rpm for 15 minutes and 10,000 rpm for 30 minutes. Clarified supernatants were then concentrated by ultracentrifugation at 28,000 rpm for 2.5 hours. Pellets were then resuspended in 100 ml of Phosphate buffered saline (PBS). For the production of immature RSPs, the HeLa-prME cells were cultured in medium containing 20 mM of ammonium chloride (NH 4 Cl).
To generate RSPs of dengue 2, 3 and 4, pcDNA constructs containing the optimized prME genes (10 mg each) were transfected into 293T cells separately. The DV1 pcDNA-prME construct was transiently transfected as control. Supernatants of transfected 293T cells were harvested, clarified and concentrated as mentioned above.
After ultracentrifugation, RSPs were resuspended in 100 ml PBS to which 33 ml of 4X NuPAGE LDS (lithium dodecyl sulfate) sample buffer (Invitrogen) was added. RSPs were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4,12% NuPAGE Novex Bis-Tris Gels (Invitrogen). For analysis of cell lysates by SDS-PAGE, HeLa-prME cells were detached by incubation in 10 mM EDTA at 37uC for 10 min and resuspended in 500 ml of PBS. Cell suspensions were then mixed with 167 ml 4X of sample buffer and then sonicated before electrophoresis of samples as described above. For immunodetection, proteins were blotted from gels onto polyvinylidene difluoride (PVDF) membranes. The membrane was blocked overnight in 5% milk in PBST solution and then incubated with anti-E antibody (4E11, 1:1000) for 1 hour. After washes, the membrane was incubated for 1 hour at room temperature with a horseradish peroxidase-labeled goat anti mouse IgG polyclonal antibody. The membrane was finally visualized using ECL Western blot detection reagents (Invitrogen) and Amersham Hyperfilm ECL (GE Healthcare, Waukesha, WI, USA). For silver staining, the gel was fixed for 30 minutes and incubated with sodium thiosulfate for 30 minutes at room temperature. After three washes, the gel was incubated with Silver Nitrate for 40 minutes and developed for 15 minutes in sodium carbonate solution (25 g/L). EDTA solution (40 mM) was used to stop the development.
For the endoglycosidase treatment, RSPs or HeLa-prME cell lysates were treated with 500 U of EndoH or PNGase F at 37uC for 3 hours according to the manufacturer's instructions (New England Biolabs, Beverly, MA, USA), and subsequently analyzed by Western blot.
A chemiluminescence dot-blot (CLDB) method was developed to quantify RSPs. Briefly, 40 ml of supernatant of either HeLa-prME cell line or purified E protein solution at known concentration were blotted onto PVDF membrane through a Dot Blot 96 System (Biometra, Goettingen, Germany). The membrane was blocked overnight in 5% milk in PBST solution, incubated with anti-E antibody (4E11, 1:10,000) for 1 hour and then for 1 additional hour with a peroxidase-labeled goat anti mouse IgG polyclonal antibody (1:10,000; ZYMED). ECL Western blot detection reagents (Invitrogen), diluted five times, were mixed and added to the membrane and the luminescence intensity was measured using the Microbeta luminometer (PerkinElmer, Waltham, MA, USA).
For sucrose gradient analysis, concentrated RSPs were ultracentrifuged in a 20 to 60% discontinuous sucrose gradient at 28,000 rpm (Beckman SW-41Ti rotor) for 2.5 hours in 4uC. All sucrose solutions were prepared with HEPES buffer (20 mM). Fractions of 0.5 ml were collected and levels of E were measured using the CLDB assay. Alternatively, RSPs were treated with 0.5% Triton X-100 for 1 hour before sucrose gradient fractionation, for RSPs denaturation.
The human membrane trafficking siRNA library targeting 122 genes and the corresponding transfection reagents were purchased from Dharmacon (#G-005500; Dharmacon Research Inc, Lafayette, CO, USA). For the screen, the HeLa-prME cells were seeded in eight 96-well plates with 10,000 cells per well. 24 hours later, 10 pmol of each siRNA was added to each well together with the transfection reagent. siRNA targeting DV1 prME (target sequence: AGATC-CAGCTGACCGATT) and non-targeting siRNA (NT) (Dharmacon Research Inc) were used as controls. Each plate contained in triplicates 15-16 siRNAs from the library, as well as positive (DV1 prME) and negative (NT) siRNA controls. Culture medium was changed two days post-transfection, the supernatant from each well was harvested after an additional 48 hour incubation and then cleared by centrifugation at 4000 rpm for 15 min. 40 ml of supernatant from each well were used to measure by CLDB the levels of RSPs secreted by siRNA transfected HeLa-prME. Two-sided Student's t test was used to assess the statistical significance of differences between each sample and the non-targeting control. Differences were considered statistically significant when P,0.05. Levels of E protein in cell supernatant were expressed in relative luminescence units and ratios of experimental conditions to controls, set as unity, were calculated. Figure S1 The four optimized DV prME sequences. Each optimized prME gene has a BamH I restriction enzyme site, a kozak sequence GCCACC, a signal sequences from VSV-G, and a Xho I restriction enzyme site. Found at: doi:10.1371/journal.pone.0008325.s001 (0.03 MB DOC) Breaking the Waves: Modelling the Potential Impact of Public Health Measures to Defer the Epidemic Peak of Novel Influenza A/H1N1 BACKGROUND: On June 11, 2009, the World Health Organization declared phase 6 of the novel influenza A/H1N1 pandemic. Although by the end of September 2009, the novel virus had been reported from all continents, the impact in most countries of the northern hemisphere has been limited. The return of the virus in a second wave would encounter populations that are still nonimmune and not vaccinated yet. We modelled the effect of control strategies to reduce the spread with the goal to defer the epidemic wave in a country where it is detected in a very early stage. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a deterministic SEIR model using the age distribution and size of the population of Germany based on the observed number of imported cases and the early findings for the epidemiologic characteristics described by Fraser (Science, 2009). We propose a two-step control strategy with an initial effort to trace, quarantine, and selectively give prophylactic treatment to contacts of the first 100 to 500 cases. In the second step, the same measures are focused on the households of the next 5,000 to 10,000 cases. As a result, the peak of the epidemic could be delayed up to 7.6 weeks if up to 30% of cases are detected. However, the cumulative attack rates would not change. Necessary doses of antivirals would be less than the number of treatment courses for 0.1% of the population. In a sensitivity analysis, both case detection rate and the variation of R0 have major effects on the resulting delay. CONCLUSIONS/SIGNIFICANCE: Control strategies that reduce the spread of the disease during the early phase of a pandemic wave may lead to a substantial delay of the epidemic. Since prophylactic treatment is only offered to the contacts of the first 10,000 cases, the amount of antivirals needed is still very limited. On June 11, 2009 , the World Health Organization (WHO) declared Pandemic Phase 6 based on sustained community transmission in more than one WHO region [1] . By the end of September 2009, in most European countries the impact has been limited, which is likely due to the dampening effect of the summer months. What the future holds is difficult to predict as past pandemics have been rather variable because of the complex interactions of immunity, pathogenicity, season and other factors [2] . Many countries have established a stockpile of antivirals for the treatment of the (severely) sick. We examine how a control strategy in the very beginning could defer the peak of the epidemic by using only a very small part of the stored antivirals for prophylactic treatment.
There are a number of possible public health measures that may be used to stop or slow down the spread. Border control has been extensively discussed in this context to delay the international spread of influenza. However, in order to achieve a significant delay, more than 99% of air travel would have to be stopped [3] . As has been shown for SARS, entry screening methods are unlikely to detect more than 10% of imported infections and the positive predictive value of temperature screening is low especially at the beginning of a pandemic [4, 5] . It is therefore inevitable that importation occurs. For the management of imported cases, other measures may be used and include contact tracing, isolation and quarantine, as well as post exposure prophylaxis. It is unclear; however, which public health strategy could be effective in preventing the spill-over from imported cases and slow-down the transmission within the general population. Even less clear is how long these measures should be maintained, particularly once that domestic transmission has started.
Mathematical models can be used to aid in decision making and have been increasingly applied to analyse the potential impact of containment strategies, pharmaceutical interventions and public health measures on the course of a novel influenza pandemic [6] [7] [8] [9] . Before the emergence of the novel influenza A/H1N1, virus modelling studies have suffered from the high number and uncertainty of necessary assumptions [10] . Even during the first months of the pandemic with the novel virus A/H1N1 only limited knowledge has emerged about the characteristics of the new virus [11, 12] . Therefore, we do not stress the particular timing and severity of a certain baseline scenario, but rather concentrate on the effect of the intervention in the model that should allow an estimation of the possible effects of these interventions.
To construct possible baseline scenarios we used some of the estimates of properties of the novel virus that have been published based on data from the initial outbreak in Mexico by Fraser [11] . Similar estimates for R0 and a little higher estimates for the generation time were found in the USA [13] . We constructed a deterministic model with the following goals:
(1) To model a possible evolution of an epidemic in Germany including assumptions about importation and domestic spread using the present knowledge about the virus; (2) to quantify the potential impact of public health measures, such as case detection, case isolation, quarantine of contacts, and the use of antiviral medication for therapy and post exposure prophylaxis, with a given effectiveness, on the initial evolution of the epidemic; (3) to identify possible conditions, which -if known -favour the adaptation of measures or the termination of the control strategies.
Until June 5th, the rate of imported cases has been stable in Germany. The first identified cases in Germany were confirmed on April 29 th , and until June 5 th a total number of 49 confirmed cases had been reported ( Figure S1 (A)). Of these 41 (84%) had a known travel history to Mexico, the United Kingdom or the USA; of the remaining 8 cases 7 had been contacts to one of the imported cases and for one case the source case was unknown but might have been related to contacts with travellers (airport worker). The average number of imported cases during this time period (38 days) corresponds to 1.1 cases per day, however, 28 of the 41 imported cases were reported during the 10 days prior to June 5 th (corresponding to 2.8 imported cases per day). We considered two different scenarios of case importation ( Figure S1 (B)): firstly, a constant number of imported cases per day (five or ten cases per day), and secondly, an exponentially growing number of imported cases limited to 120 imported cases per day. The exponential growth rate was determined by assuming an ''import-R0'' of 1.1 and the same generation time as for the transmission inside Germany.
An example for the prevalence of symptomatic cases resulting from our SEIR model is shown in Figure S2 for three different values of R0 (1.34, 1.58, and 2.04). For an R0 of 1.58 the point estimate of the timing of the peak after introduction of the first case would be 10 to 11 weeks (depending on the number of imported cases, compare Table 1 ), the point estimate for the peak prevalence of the population infected would be 4.3%, for the total attack rate of the population infected 44.8% and for the population diseased 38.5% (Table 1) . Depending on the three R0 the cumulative proportions of children that develop symptoms are 48%, 67%, and 79%, and the cumulative proportion of symptomatic adults 17%, 34%, and 54%, respectively. As a result of the higher susceptibility of children in the model the peak proportion of infected children is reached 18, 12 or 8 weeks (126, 82, or 56 days) after the first infected case, roughly 1-1.5 weeks earlier (i.e. 11, 8 or 6 days) than in adults (data not shown). Figure S3 shows how the peak is delayed for an assumed R0 of 1.58 and 5 imported cases per day when the first 500 cases are managed with a combination of intensive case-based measures (CCM1), followed by 10,000 with management mainly restricted to members of the household (CCM2; for details see Methods section). The effect of the number of household focused interventions (CCM2) on the delay of the peak is dependant on the basic reproduction number R0 and the sensitivity of the surveillance system ( Figure S4 ). When R0 is at least 1.58 and not more than 30% of cases can be detected, saturation occurs relatively early. Even if 50% of the cases can be detected or R0 is as small as 1.34 the peak of the epidemic can not be deferred any more after management of approximately 10,000 cases with CCM2. In general can be said: the higher R0 the earlier management with CCM2 becomes ineffective. This can only be balanced to a certain degree by a higher sensitivity of the surveillance system.
The following considerations are done on the basis of 5 imported cases per day and R0 equal to 1.58.
Effect of sensitivity of the surveillance system. The delay of the peak increases with the proportion of detected cases. When 10% of cases are detected and these are followed-up with CCM1 for the initial 500 cases (but no CCM2) the delay is 6 days, but can be raised to 20 days (gain of 2 weeks) when case detection is improved to 30%. The combined approach of 500 cases targeted with CCM1 and additional 10,000 cases with CCM2 the gain based on the improved case detection results in an increase for up to 6 weeks (11 to 50 days; Figure S3 , Table 2 ).
Separate analysis of the effect of CCM1 and CCM2. In the example above 55% (6 of 11 days; 10% case detection rate) or 40% (20 of 50 days; 30% case detection rate) of the delay, respectively, is already achieved through CCM1 alone ( Table 2) . Table 1 . Characteristics of the baseline scenario without preventive interventions under the assumption that each day 5 cases were imported to Germany. This holds similarly in the scenario of the exponentially growing number imported cases -of course under the assumption that already in the beginning the surveillance system detects a percentage of the imported cases. Effect of R0. All of the above calculations are very sensitive to the value of R0. If R0 is as low as 1.34 it is much easier to delay the peak. If it is high, such as 2.0 or more, and the number of imported cases is at least 5 per day, then the effect of CCM1 and CCM2 can delay the peak at most by 8 weeks (57 days; Table 2 ), but only when case detection rate is high (50%).
The personnel and personnel time that is needed to implement such measures depends on the number of local health departments involved, their capacity and many other factors. The maximum number of antivirals, however, can be estimated. Assuming that each case has 15 contacts that merit attention for CCM1 and would get antiviral post exposure prophylaxis, this would result in a maximum of 500615 ( = 7,500) treatment courses (as the number of doses for treatment equals the number of doses for prophylaxis). Further, if the household members of 10,000 cases are given antiviral prophylaxis this would amount to another 10,00063 ( = 30,000) treatment courses, in total 37,500 treatment courses.
We present here a model how public health measures can contribute to the delay of an epidemic wave with the novel influenza virus A/H1N1 when the epidemic is detected in a very early stage. Delaying the pandemic spread is an important achievement because it gains time for other measures and preparations, such as early assessment of the virus' characteristics, activation of surge capacities or vaccine production and the development of a vaccination strategy [10] .
The transmission parameters of our model were derived from the initial analysis of the epidemic in Mexico by Fraser [11] and a constant or slowly increasing influx of imported cases as observed for one month after the identification of the first case in Germany. This is also in agreement with European data showing a constant proportion of travel-related cases over time (the travel related cases within Europe between April 16 and June 2 also seem to remain constant over time [14] ).
It has been shown before that so called targeted layered containment strategies, a combination of antiviral prophylaxis and non-pharmaceutical interventions, can be effective in reducing the transmission of pandemic influenza [8] . We extended this approach by analyzing the effect of a more intensive phase including contact tracing, identification and management of contacts outside of the household (CCM1), followed by household centred measures (CCM2).
The assumed effectiveness for CCM1 indicates that a corresponding strategy would be very effective in reducing the spread of the epidemic, if R0 is moderate or low and if the number of imported cases does not increase rapidly. In contrast, the household centred measures (CCM2) continue to gain time, even when larger amounts of cases are imported per day. In the model, the effect of CCM1 and CCM2 rapidly decreased for higher Table 2 . Delay (in days) of the peak of the epidemic wave for adults in Germany as a function of the number of imported cases (column A), the number of CCM1 and CCM2 treatments, R0 and the case detection rate (10%, 30% or 50%). values of R0, indicating that this intervention should be stopped when the delay becomes only marginal. To our knowledge, the question of conditions that would lead to stopping interventions (or the lowered effects of specific measures) has so far been studied only in the context of community mitigation strategies and interventions, such as school closures [15] .
Modelling studies analyzing preventive measures often assume that the intervention will be available for a large part or the whole population [6, 8, 16] . E.g. Carrat et al. analysed the effect of combined interventions targeting up to 70% of households in their ''small-world-like'' model [17] . In contrast, our model focuses on the initial stage and the first few thousand cases in Germany, and on the delay that can be achieved. We have restricted the analysis of maintaining the less intensive CCM2 strategy for 10,000 courses. The maximum amount of antivirals used in this approach corresponds to treatment courses for less than 0.1% of the German population and 5% of the amount available for seasonal influenza in the winter season. After the end of isolation, the treated contacts were assumed to remain susceptible to the infection. However, these conservative assumptions result only in a shift (depending on R0 significant) of the epidemic curve and virtually no reduction of the overall attack rate.
As we have shown, a number of factors are important when effects of public health interventions are considered. First, the rate of seeding has significant impact on the delay that can be achieved by the interventions. Other published models start their simulation with one infection, a random number each day or an increasing number at a reduced R0. In contrast, our analysis is based on the observed number of cases. Second, the sensitivity of the surveillance system to detect cases is important. In reality, detection of cases without travel history to countries with community transmission of novel influenza A/H1N1, i.e. domestic cases, may be difficult as the clinical picture of the disease has proven to be often non-specific. Therefore, sensitivity to detect cases may be relatively low. Given these limitations we have provided a number of different scenarios (case detection rates of 10, 30 and 50%) addressing how the described interventions may impact the epidemic.
As a note of caution it must be mentioned that these calculations should of course not be mistaken as a prediction. It was for example not possible to validate the assumptions about the effectiveness of the interventions using real data.
Other limitations are: (1) While before the start of the novel A/ H1N1 epidemic it was difficult to make realistic assumptions, it is now easier to do so now, since first estimates can be drawn for a number of parameters of the novel influenza virus. Nevertheless, many pieces of information are uncertain and may change due to more information coming to light, or due to real changes of the virus and its epidemiology. (2) The effect of season is not taken into account, we expect that the virus is more easily transmitted in the fall or winter time [18, 19] . (3) The proportion of asymptomatic cases and their contribution to transmission is still unknown. (4) Lacking realistic alternative information we distinguished only two age groups, children and adults. (5) The evolution of the number of cases imported is unknown and will probably change over time. (6) The age dependence of susceptibility in Germany is unknown and is likely to differ from the one in Mexico. (7) The sensitivity of surveillance is unknown, and therefore the true proportion of cases detected is also unknown. If persons were infected in Germany from a source without travel history, then they may have been more easily missed, especially since initial surveillance efforts usually focus on diseased persons with travel history. (8) The rigor of public health measures is likely to vary among different local health departments. However, it is plausible that the measures taken contributed to the delay of the initial spread of the infection, because, until mid June at latest, virtually no tertiary cases had been detected by close surveillance of contacts and the surrounding of cases.
In the model the change from CCM1 to CCM2 was suggested on a population level. In reality, of course, there might not be a real threshold and the strategy might change depending on the individual resources of local health authorities. Of course with the change of the epidemiologic picture more rigorous measures of social distancing, such as school closures may be implemented. When leaving the intensive phase of contact tracing and case management (CCM1), we believe that CCM2, or a strategy with similar effect, might be well suited to follow after because it focuses on the household. This is a much more amenable unit and is based on the knowledge that being a member of a household with a confirmed case is the highest single risk factor for influenza infection [6] .
In conclusion, despite the many possible pitfalls and caveats of our study we believe that we have demonstrated the possible impact of a sequential strategy on the spread of the novel influenza virus A/ H1N1 in a country where imported cases start the epidemic.
Cased-based information was used to assign reported confirmed cases in Germany and status of either imported or domestic. Cases with travel history of more than 7 days before onset of symptoms (two times the maximal incubation period) were considered domestic.
(a) Assumptions. We assumed that at the outset of the epidemic the entire population is fully susceptible to infection with the influenza A/H1N1 virus. Infectiousness was assumed to be equal in symptomatic and asymptomatic persons. This is based on the rationale that a lower degree of infectiousness is coupled with unrestricted mobility resulting in a higher number of potentially infectious contacts. In comparison, a higher degree of infectiousness in symptomatic patients is compensated by the reduction of the number of contacts, because patients are isolated and bedbound.
Assuming that the epidemiologic and virologic characteristics are similar to the epidemic in Mexico allowed us to use the values as described by Fraser [11] . They found the ''most likely'' basic reproductive number was 1.58, range 1.34 to 2.04, and estimated a generation time of 1.91 days (95% confidence interval 1.3-2.71). They distinguished children (,15 years of age) and adults (. = 15 years of age) and found that children were 2.06 (95% confidence interval 1.60-3.31) as susceptible as adults. The assortativity of mixing between children and adults was estimated as 0.5 (95% confidence interval 0.00-0.72) -an assortativity of 0 corresponds to a completely random mixing, whereas 1 corresponds to fully assortative groups. Finally, they found that 86% (95% confidence interval 69%-100%) of the infected persons become symptomatic. We considered three different scenarios of R0, namely R0 equal to 1.34, 1.58 or 2.04 and used the point estimates for all other parameters. The model does not incorporate assumptions about the severity of disease or how severity might alter infectiousness.
Lastly we needed to make assumptions about the effectiveness of the public health measures and the sensitivity of the surveillance system. Assumptions are made for the effectiveness of two approaches that combined several case-based public health measures (combination of case-based measures; CCM):
(1) Intensive measures, including isolation and therapy of cases, contact tracing, quarantine and post-exposure prophylaxis of selected contacts in-and outside of the household (CCM1). Because CCM1 consumes many resources it is assumed that CCM1 will only be sustained in the first phase. We model two scenarios with the first 100 or up to 500 cases cared for by CCM1 countrywide. (2) Less-intensive measures, focusing on the household, including isolation and therapy of cases, quarantine and post-exposure prophylaxis of household contacts (CCM2). Thus, we assume that following the initial 100-500 confirmed index cases there will be no contact tracing any more, i.e. no more post exposure prophylaxis for non-household contacts.
CCM1 and CCM2 are set to be 75% and 50% effective in reducing secondary cases, respectively. We modelled four scenarios: in the first and second, CCM1 is maintained for the first 100 cases followed by 5,000 or 10,000 cases cared for with a CCM2 strategy, in the third and forth, CCM1 is maintained for 500 cases followed by 5,000 and 10,000 cases with a CCM2 strategy, respectively.
To include the effect of surveillance we made assumptions about the number of imported cases that are recognized. For this purpose we varied the proportion of recognized imported cases from 0%, 10% and 30% to 50%. The assumed sensitivity of the surveillance system reflects the probability (10%, 30% or 50%) to detect domestic cases. A higher probability to detect imported cases would have led effectively to a reduced total number of imported cases per day in the model.
(b) Construction. We used -similar to Fraser [11] in their description of the outbreak in La Gloria -a generalised agestratified deterministic SEIR model to describe the spread of the disease [20, 21] .
We used the following assumptions about the age distribution and size of the population of Germany: 71,000,000 adult population (. = 15 years of age), 11,000,000 children (,15 years of age).
The complete model is described in the Appendix S1. Figure S1 Confirmed imported (red) and domestic (blue) cases in Germany by date of onset of symptoms (A Figure S3 Delay of epidemic curve. The ''most likely'' Ro of 1.58 from the study of Fraser et al. (Science, 2007) was used and case detection rates of symptomatic cases were set to 10% and 30%, respectively. Ro is assumed to be 1.58, and each day five cases were imported. The household and non-household contacts of the first 500 detected cases were treated with a combination of case-based measures that include contact tracing, quarantine, and post-exposure prophylaxis (CCM1); and the household contacts of the next 10,000 cases were managed with strategy CCM2, which includes only preventive measures in the household of the cases.  European Hedgehogs as Hosts for Borrelia spp., Germany  . Members of the B. burgdorferi s. l. group are the most common vectorborne pathogens of humans in central Europe (3) . The role of hedgehogs as hosts for these pathogens is, therefore, of considerable epidemiologic interest. Hedgehogs are a common synanthropic species that live in urban, suburban, and rural environments (4) and are known to carry not only the hedgehog tick, Ixodes hexagonus, but also the most common European tick, I. ricinus (2, 5) . Both of these ticks are known vectors of B. burgdorferi s. l. and tickborne encephalitis virus; I. ricinus is the most important vector of both throughout Europe (1, 5) . To date, however, only limited information has been available on the role of the hedgehog as a host or reservoir for B. burgdorferi s. l. in Germany.
We report the presence of 3 species of the B. burgdorferi s. l. group in European hedgehogs from Germany. To our knowledge, this is the fi rst report of these species in hedgehogs in this country and the fi rst report of B. spielmanii (A14S) (6) from this host.
The investigated hedgehogs came from 2 sources: 9 from the ≈40 in an experimental plot in the city of Karlsruhe, state of Baden-Wuerttemberg, and the remainder from wild hedgehogs that had been brought to hedgehog care centers from various areas of Germany. All hedgehogs had died naturally, and tissue samples were taken from 43 animals (kidneys from 43, heart from 22, bladder from 33).
The bodies had been frozen at -17°C before the samples were taken.
DNA isolation was done by using the Maxwell 16 Instrument and System (Promega, Madison, WI, USA). Tissue samples were 3×3×3 mm. To detect B. burgdorferi s. l., we used 2 PCR protocols. The fi rst was a nested PCR done according to the method of Rijpkema et al. (7) . The target for the PCR was the spacer region between 5S and 23S rRNA genes of B. burgdorferi s. l. The nested primers generated a product of 226 bp. The amplifi ed products were analyzed by agarose gel electrophoresis. The second protocol, a LightCycler-PCR hybridization assay (Roche Diagnostics, Mannheim, Germany) (8), simultaneously detects and genotypes the 3 genomic groups of B. burgdorferi s. l. This assay was specifi c for B. burgdorferi senso stricto, B. garinii, and B. afzelii (8) but also amplifi ed B. spielmanii and B. valaisiana. The target for the PCR was the OspA gene.
The PCR products of both systems were sequenced. For DNA sequencing reaction, the fl uorescence-labeled didesoxynucleotide technology (Applied Biosystems, Darmstadt, Germany) was used. The sequenced fragments were separated, and the data were collected with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained sequences were then analyzed and compared by using BLAST (www. ncbi.nlm.nih.gov/BLAST).
For 6 hedgehogs, Borrelia spp. could be clearly defi ned by using both gene sequences. Two additional animals had positive results, but sequencing was not possible because of either too little DNA or a mixed infection. B. spielmanii DNA was detected in the kidneys of 2 hedgehogs: 1 from Karlsruhe and 1 from 30 km west of this city in the German federal state of Rhineland-Palatinate. When sequences were compared by using BLAST, 4 BLAST sequences (AM055823, AM055822, DQ133518, AY 995900) showed 100% similarity with B. spielmanii. B. garinii was detected in the heart of 2 animals (from Berlin and Karlsruhe); B. afzelii in 3 animals (in the kidney of 2 from Hamburg and Karlsruhe and in the bladder of 1 from Rhineland-Palatinate). A single animal (from Karlsruhe) had B. afzelii in the kidney and bladder and B. garinii in the heart. Preliminary results have also shown that ticks collected from hedgehogs from the Karlsruhe site were infected with B. afzelii (an I. hexagonus nymph and an I. ricinus female) and with B. spielmanii (an I. ricinus female, a nymph, and a larva) (Skuballa et al., unpub. data).
These results show, that hedgehogs harbor at least 3 of the 5 recognized Borrelia genospecies found in Germany, all of which are known (B. afzelii, B. garinii) or are strongly suspected (B. spielmanii) of being pathogens for humans (9, 10) . To our knowledge, ours is the fi rst report of B. spielmanii from hedgehogs, a Borrelia sp. that is usually associated with rodents, especially with garden and hazel dormice (10) . That Borrelia spp. infections commonly occur in European hedgehogs is likely. However, questions remain about the role of these pathogens in regulating the populations of European hedgehogs and about the status of these common synanthropic mammals as a reservoir host of B. burgdorferi s. l. in periurban and rural environments. Expression of the VP2 Protein of Murine Norovirus by a Translation Termination-Reinitiation Strategy BACKGROUND: Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAA UG. PRINCIPAL FINDINGS: Here, we confirm that MNV VP2 expression is regulated by termination-reinitiation and define the mRNA sequence requirements. Efficient reintiation is dependent upon 43 nt of RNA immediately upstream of the UAA UG site. Chemical and enzymatic probing revealed that the RNA in this region is not highly structured and includes an essential stretch of bases complementary to 18S rRNA helix 26 (Motif 1). The relative position of Motif 1 with respect to the UAA UG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine. CONCLUSIONS: The termination-reinitiation signal of MNV most closely resembles that of influenza BM2. Similar to other viruses that use this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the VP1 stop codon. Our data also indicate that accurate recognition of the VP2 ORF AUG is not a pre-requisite for efficient reinitiation of translation in this system. For most eukaryotic mRNAs, translation initiation is a 59-enddependent process beginning with recognition of the cap structure by the cap-binding complex eIF4F [1] and (usually) recognition of the AUG codon of the first open reading frame (ORF) on the mRNA by the scanning ribosome complex [2] . This 59-end dependence is a problem faced by many RNA viruses with polycistronic genomes and elaborate strategies have been developed to facilitate access of ribosomes to downstream open reading frames (ORFs). Amongst these, a number of unconventional translation strategies have been described [3] . These include leaky scanning of 40S subunits past the start codon of the first ORF [4] , the possession of intercistronic internal ribosome entry signal [5] , programmed ribosomal frameshifting during elongation [6] and stop codon suppression at the termination step [7] [8] . Another strategy that has evolved to allow expression of a downstream ORF is termination-reinitiation (also referred to here as stop-start). In this process, ribosomes translate the upstream ORF but following termination, a proportion of 40S subunits remain tethered to the mRNA and go on to reinitiate at the start codon of the downstream ORF. This termination-dependent reinitiation strategy allows the coupled expression of products from adjacent ORFs and thus the production of a defined ratio of gene products.
Termination-reinitiation in virus systems [9] was first described in the synthesis of the BM2 protein of the orthomyxovirus influenza B virus [10] and subsequently in expression of VP2 of feline calicivirus (FCV) of the genus Vesivirus [11] [12] [13] and VP10 of the calicivirus rabbit haemorrhagic disease virus (RHDV) of the genus Lagovirus [14] . A related phenomenon is also seen in expression of the M2-2 protein [15] [16] of the paramyxovirus respiratory syncytial virus (RSV) and the M2-2 protein [17] of pneumovirus of mice (PVM). In FCV, the stop codon (UGA) of the major capsid stop-start protein VP1 overlaps the start codon of the minor capsid protein VP2 (AUGA) (the stop-start ''window''). Efficient termination-reinitiation depends upon several factors, including the close proximity of the stop and start codons, the transit of ribosomes along the VP1 mRNA up to the stop codon and a stretch of some 70-80 nucleotides (nt) of mRNA upstream of the stop-start window whose primary sequence, rather than the encoded protein, is key. This region of the mRNA, termed the termination upstream ribosomal binding site (TURBS), is needed for the retention of post-termination 40S subunits [11] . A short sequence of the TURBS (termed Motif 1) that is complementary to part of helix 26 of 18S rRNA likely acts to tether the 40S ribosomal subunit to the mRNA post-termination, allowing time for the ribosome to acquire the factors necessary to initiate on the downstream ORF [12, 14, 18] . The TURBS may also act by recruitment of eukaryotic initiation factor 3 (eIF3) or eIF3/40S complexes [13] . Recent studies of termination-reinitiation in the expression of the orthomyxovirus influenza BM2 protein have revealed a requirement for a shorter stretch of mRNA (45 nt) upstream of the stop-start window, but nevertheless, the RNA contains a similar TURBS Motif 1 [19] . From RNA secondary structure probing, it has been proposed that this stretch may be displayed on the apical loop of a stem-loop structure that may form following transit of the ribosome through the region and termination at the upstream ORF stop codon [9, 19] .
In this paper, we describe an analysis of termination-reinitiation in the expression of the VP2 protein of murine norovirus (MNV), a calicivirus of the genus Norovirus. The VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG, consistent with a terminationreinitiation strategy, and a stretch of bases (59 UAUGGGAA 39) complementary to 18S rRNA helix 26 is present upstream. Using a luciferase-based reporter plasmid, we show that VP2 is expressed by termination-reinitiation and provide evidence consistent with a functional interaction between the coding region of the VP1 mRNA and 18S rRNA. The formation of mRNA secondary structure within the TURBS is also investigated. Overall, our data suggest that the mechanism of VP2 expression is broadly similar to that of the other caliciviruses and influenza B. However, in contrast to what was observed with the FCV signal [13] and seen here with influenza BM2, termination-reinitiation at the MNV signal shows resistance to the initation inhibitor edeine. Thus the mechanism by which the AUG of the downstream ORF is recognised may differ.
To investigate termination-reinitiation in the synthesis of the MNV VP2 protein, a 255 bp fragment of viral cDNA was cloned between the SalI and BamHI sites of the dual-luciferase reporter vector p2luc [20] . The cloned fragment, which contained 203 bp of sequence information upstream of the UAAUG stop-start window, and 52 bp downstream was suspected, on the basis of work with other viruses (see Introduction), to contain all of the required sequences for termination-reinitiation. The cDNA fragment was cloned in such a way that the Renilla and Firefly luciferase ORFs were in frame with the stop and start codons respectively of the termination-reinitiation motif to give an ORF configuration 59 rlucVP1-VP2fluc 39 ( Figure 1 ). This vector, named p2luc-MNVwt, contains a T7 RNA polymerase promoter allowing synthetic mRNAs to be generated to investigate the stopstart process in in vitro translation reactions.
The translation of in vitro synthesised wild-type (wt) mRNA from p2luc-MNVwt was carried out in FlexiH rabbit reticulocyte lysate (FlexiHRRL) supplemented with 140 mM KCl (see Materials and Methods) and gave products of the expected sizes (upstream rlucVP1 ORF, ,42 kDa, downstream VP2fluc ORF, ,64 kDa, Figure 1b ). The molar ratio of VP2fluc to rlucVP1 (taking into account the methionine content of the two proteins) was typically in the region of 1:10. Thus, initiation on the downstream ORF occurred at a frequency of about 10% of that of the upstream ORF. That this was indeed the product of the second ORF was further confirmed by comparing the migration of RRL translation products from mRNAs derived from p2luc-MNVwt that had been linearised at different points within the second ORF (data not shown). Termination-reinitiation is distinct from IRES-mediated expression of downstream ORFs as translation through the upstream ORF is an absolute requirement [11, 14, 16] . In order to establish whether this is also the case for MNV expression, a premature in-frame stop-codon was inserted close to the end of the rluc ORF but upstream of VP1 sequence information (219 bp upstream of the authentic rlucVP1 termination codon). If the expression of VP2fluc is a result of termination-reinitiation, translating ribosomes would be unable to reach the AUG start Figure 1 . Minimal sequence requirements for MNV termination-reinitiation. A) Schematic of the p2luc-MNV reporter mRNA. The termination-reinitiation region (203 nt upstream and 52 nt downstream of the UAAUG motif) was cloned into the SalI and BamHI sites of the p2luc reporter plasmid. HpaI run-off transcripts for in vitro translation were generated using T7 RNA polymerase. The location of the T3 promoter present in the structure mapping construct p2luc-MNV-T3 is indicated. B) Deletion analysis of MNV termination-reinitiation. A series of p2luc-MNV variants were prepared with stepwise, in-frame deletions from the 59 end of the inserted viral sequence. The wild-type (wt), premature stop (ps) and deletion mutant plasmids were linearised with HpaI and run-off transcripts translated in FlexiH RRL at a final RNA concentration of 50 mg/ml in the presence of [ 35 S]-methionine and 140 mM added KCl. The products were resolved by 12% SDS-PAGE and visualised by autoradiography. The number of nucleotides of viral sequence remaining up to the AUG start codon of the MNV ORF is shown below the gel. The product of the full-length or truncated versions of the rlucVP1 ORF (predicted size of MNVwt is 42 kDa) is marked rluc, and the VP2fluc product (predicted size, 62 kDa) is marked fluc. The MNV ps rluc product is the shortest (predicted size, 33 kDa). RRF denotes the relative reinitiation frequency in comparison to MNVwt (set at 100). The figure in brackets represents the ratio of the intensity of the fluc and rluc products (adjusted for methionine content and expressed as a percentage) for the MNVwt mRNA. doi:10.1371/journal.pone.0008390.g001 codon of VP2fluc in the mutant mRNA and the ORF could not be translated. As is clear in Figure 1b , the introduction of a premature stop codon into the rluc/M1 ORF abolished expression of the VP2/fluc product, but had no effect on synthesis of the upstream ORF (rlucVP1ps, ,33 kDa). These data are thus consistent with a termination-reinitiation strategy for the expression of the VP2 protein and confirm a requirement for translation through the upstream ORF.
Expression of MNV VP2 Is Dependent on ,40-43 nt Upstream of the UAAUG Motif Previous work has suggested that viral termination-reinitiation events show little dependence on sequence information downstream of the ''stop-start'' window but require 45-250 nt of upstream primary sequence [11,14,16.19] . In order to determine the minimal sequence requirements for termination-reinitiation in VP2 expresssion, deletions of increasing size were made from the 59 end of the inserted viral information (Figure 1b) . The stop-start product was synthesised efficiently with up to 43 nt of VP1 information present upstream of the UAAUG motif, and to a lesser extent with 40 nt. However, deletion to 37 nt or less abolished expression of the termination-reinitiation product ( Figure 1b) . These data indicate that only 40 nucleotides of VP1 primary sequence immediately upstream of the stop-start window are required for termination-reinitiation in vitro, although 43 nt are required for full activity.
Termination-Reinitiation of MNV VP2 Synthesis Is Dependent upon an mRNA Sequence with Complementarity to 18S rRNA In FCV, RHDV and influenza B, it has been shown that termination-reinitiation requires a closely conserved primary sequence element (referred to as Motif 1) that is complementary to a region of helix 26 of 18S rRNA [11] [12] 14] ). The position of Motif 1 varies somewhat, with the 59 base 73 nt (RHDV), 63 nt (FCV) or 34 nt (influenza B) upstream of the stop codon of the first ORF. Mutational analysis has revealed that this sequence is essential for the stop-start process [12] [13] [14] [18] [19] . Within the ,43 nt minimal region of the MNV VP1 RNA required for VP2 expression, a stretch of bases with a similar level of complementarity to 18S rRNA is also found (Figure 2a , complementary bases are shown in italics). To investigate whether this region plays a role in termination-reinitiation in VP2 expression, two point mutations were made to disrupt potential mRNA:rRNA pairs ( Figure 2a ). In the first, the A at -31 was mutated to a G (p2luc-MNV GU), creating a presumably slightly weaker putative U-G base pair between the rRNA and mRNA. In the second, the G at -32 was changed to a C (p2luc-MNV CC), which would act to disrupt the interaction between 18S rRNA and mRNA. As can be seen in Figure 2b , the latter mutation greatly reduced expression of the VP2fluc product, supporting the idea that an interaction between the 18S rRNA and the mRNA just upstream of the termination-reinitiation site is required. In the mutant where pairing was predicted to be maintained (p2luc-MNV GU) termination-reinitiation was clearly detectable, although the efficiency was reduced somewhat compared to that of wild-type mRNA.
The experiments described above confirm the existence of Motif 1 and its role in reintiation in MNV. It was therefore of interest to determine the context of this 18S rRNA complementary region within the global RNA secondary structure of the minimal functional sequence, and to compare the structure with that determined for the influenza BM2 signal [19] . To achieve this, a bacteriophage T3 promoter was inserted upstream of the viral sequence of the p2luc-MNV.61 plasmid ( Figure 1b ). The plasmid was linearised with BamHI, T3 run-off transcripts synthesised and the RNA end-labelled with [ 33 P]-cATP. The labelled transcripts were subjected to limited chemical and enzymatic probing prior to analysis on denaturing polyacrylamide gels. The chemical probes used were imidazole and lead acetate, specific for cleavage of single stranded regions. Enzymatic probes were RNases T1, U2 and CL3, which preferentially cleave single-stranded G, A and C residues respectively, and RNase CV1, which cuts in helical regions in double-stranded or stacked conformations. A representative stucture mapping gel is shown in Figure 3 and in Figure 4 , the data are mapped onto mfold predictions of the secondary structure of the ''stop-start'' region.
Structure probing analysis of the MNV signal revealed that, like the BM2 signal, the mRNA in the region essential for terminationreinitiation is not highly structured. This was especially evident from the chemical probes, with most residues sensitive to imidazole and lead cleavage. The enzymatic probes were also active against the majority of bases in the region and consistent with this, CV1 probing identified very few double-stranded or stacked bases. We also noticed a few CL3 cuts at residues other than C, although the reason for this is uncertain. Minimal free energy mfold predictions, performed using the online server of Zuker (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) indi- cated that the most stable RNA fold was the bulged stem-loop shown in Figure 4 . However, the correspondence between this mfold and the mapping data was not absolute. Whilst in general, the single-stranded probes displayed more activity against regions of the model predicted to be single-stranded than they did against predicted helices, there were anomalies. For example, residues G51-52 were sensitive to RNase T1, yet were predicted to be in a double-stranded region (stem 2). Generally, the predicted duplexes showed more reactivity to single-stranded probes than one would expect for stable double-stranded stretches. Therefore, it seems likely that the RNA in this region is metastable, potentially adopting a number of co-existing structures. In our model, the sequence complementary to 18S rRNA is sequestered between two putative stems (stems 2 and 3; Figure 4 ) at a location similar to that found with BM2 [19] . Given that the termination-reinitiation process requires the ribosome to translate through the VP1 ORF, secondary structure in the RNA upstream of the ''stop-start'' window would be unwound and perhaps remodelled as the ribosome transits to the termination codon. Toeprinting of ribosomes paused at initiation codons has shown that the 59 edge Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH-) and the position of known RNase U2 and T1 cuts, determined empirically. Products were analysed on a 10% acrylamide/7M urea gel containing formamide. Data was also collected from 6% and 15% gels (gels not shown). Enzymatic structure probing was with RNases T1, U2, CL3 and CV1. Uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). The number of units of enzyme added to each reaction is indicated. Chemical structure probing was with imidazole (I, hours) or lead acetate (Pb; mM concentration in reaction). The water lane (W) represents RNA which was dissolved in water, incubated for four hours and processed in parallel to the imidazole-treated sample. The sequence of the probed RNA and the inferred secondary structure is shown in Figure 4 . of the ribosome is some 12 to 13 nt from the first base of the AUG [21] . This would place the 59 edge of the terminating ribosome (with the UAA codon in the A-site) close to residue C78 on our mRNA. Thus a terminating ribosome would prevent formation of the secondary structure, conceivably releasing the 18S rRNA complementary region for interaction with the ribosome (see Discussion). An alternative structure can be predicted under such circumstances, shown in the inset box in Figure 4 . In this structure, the 59 arm of the original stem 2 is predicted to pair with alternative bases to generate a new stem (stem 29) with Motif 1 forming part of the apical loop. This alternative fold is attractive for a number of reasons. By displaying Motif 1 on an apical loop, this could promote 18S rRNA binding and ribosome tethering [19] . Until ribosomes transit through this region, Motif 1 would remain within a larger structure with potentially reduced access to the ribosome which could, at least in part, account for the observation that the signal does not appear to function as an IRES. The deletion analysis of Figure 1 is also consistent with a role for this alternative structure as the functional ''end-point'' maps to the start of the 59 arm of stem 29. Furthermore, most, if not all, viral TURBS have the potential for base-pairing between regions flanking Motif 1 [18] . Nevertheless, it should be noted that the structure mapping data are not fully consistent with this alternative structure, for example, there is considerable sensitivity to RNase T1 cleavage within the 59 arm of stem 29. This is considered further in the Discussion section.
Efficient termination-reinitiation seems to require the close proximity of the stop and start codons [12, 14, 19] . To investigate whether this is also the case for MNV, the authentic stop codon of rlucVP1 (in the context of the fully functional MNV49; see Figure 1b ) was mutated from UAA to CAA such that the first ORF was extended by 13 amino acids (MNV49.1 Figure 5 ). The separation of stop and start codons by such a distance in BM2 is known to reduce reinitiation about 10-fold [19] , but with the MNV signal, only a three-fold reduction in flucVP2 synthesis was observed ( Figure 6 ). A possible explanation for this lies in the fact that the ''new'' stop codon is itself embedded within a second potential stop-start sequence (UGAUG) which could facilitate some reinitiation, but perhaps at a lower frequency, as it would not necessarily be spaced appropriately with respect to Motif 1. Another possibility is that 40S subunits terminating at the downstream stop-start sequence can reinitiate, at a reduced frequency, at the correct (upstream) AUG, despite the increased spacing, with the 40S subunit remaining tethered to the mRNA and ''snapping-back'' to the normal position of reinitiation. In an attempt to distinguish between these possibilities, additional constructs were prepared in which point mutations were introduced into pMNV.49 such that the termination and start codons in the two stop-start regions were changed separately and in combination (MNV49.2 to MNV49.8; see Figures 5 and 6) . From this analysis, it is evident that modification of the authentic termination-reinitiation motif reduces reinitiation, irrespective of whether the stop or start codon is eliminated. Alteration of the AUG codon had the most effect, with reinitiation reduced to 8-38% of the wild-type level. When the natural termination site was changed such that termination now took place 13 or 15 amino acids downstream, the frequency of termination-reinitiation was also reduced, to 25-49% of the wild-type level. In MNV49.8, where termination of the upstream ORF occurred 30 amino acids downstream of the natural site, very little reinitiation was seen (8% of the wild-type level), indicating that the ribosome is unable to locate the authentic AUG from such a distal termination site. In the translation of this mRNA, an additional product was seen (asterisked in Figure 6 ) whose size is consistent with a fusion of the encoded ORFs (this is considered in the Discussion section). Reinitiation events that take place at either the authentic or the downstream stop-start motifs would produce polypeptides that differ in size by only 13 amino acids, thus we would not expect to be able to distinguish them by SDS-PAGE, and this is clear in Figure 6 , where the reinitiation products show very similar electrophoretic mobilities. Thus we cannot say with confidence whether a particular AUG (or both) is used. However, the substantial reintiation activity displayed by MNV49.7, an mRNA in which both AUGs were changed, indicates that non-AUG codons can act as reinitiation codons, although probably at reduced efficiency. This is consistent with other work demonstrating that reinitiation can occur at non-AUG codons within the context of a termination-reinitiation signal [12, 14, 19] . Whilst in principle, reinitiation of translation of the MNV 49.7 VP2fluc ORF, following termination, could occur at the next available AUG, this is located 54 amino acids from the natural stop-start signal and initiation here would produce a substantially shorter product that would have been detectable by SDS-PAGE. Thus in this mRNA, a significant proportion of ribosomes (25% of the wild-type level) that terminate 13 amino acids downstream of the authentic stop-start site can reinitiate in an AUG-independent manner within the stop-start window.
The precise mechanism of termination-reinitiation is not known, but the sensitivity of FCV VP2 protein expression to the translation initiation inhibitor edeine suggests that the reinitiation process bears at least some similarity to standard initiation at AUG codons [13] . To ascertain whether edeine sensitivity is a general feature of termination-reinitiation, we analysed the effect of the peptide on the activity of the MNV and BM2 signals (with FCV as a control) using translation time-courses (Figure 7 ). Reactions were programmed with the relevant mRNA and at various times an aliquot was removed, edeine added (to 5 mM) and the aliquot reincubated such that the total time of translation was 60 minutes. To determine the time of first appearance of the termination and reinitiation products, identical reactions were also performed in which the elongation inhibitor cycloheximide replaced edeine. In the edeine experiments, it was evident that for FCV and BM2, only a trace of ''stop-start'' product was synthesised at the early time points. In these experiments, the vast majority of ribosomes did not reach the stop-start window until at least 7.5 minutes had passed (as shown in the cycloheximide time course experiments [data not shown; see legend to Figure 7 ]), thus the trace of VP2fluc seen likely corresponds to the product of infrequent internal initiation at the VP2fluc AUG or is derived from those few ribosomes that had reached the stop-start window prior to edeine addition. At later time points, however, the termination-reinitiation product steadily accumulated, with the ratio of the upstream and downstream ORFs stabilising after 30 minutes (at a reinitiation frequency of ,4%). Thus for the FCV and BM2 signals, when edeine is present prior to arrival of ribosomes at the stop-start signal, it greatly inhibits termination-reinitiation, but has little effect on translation post-reinitiation. Unexpectedly, the MNV signal responded differently, with the termination-reinitiation product being more evident at early times post-edeine addition (in comparison to FCV and BM2). At these early time points, few ribosomes would have reached the stop-start window prior to edeine addition, thus the MNV signal shows increased resistance to the effects of edeine. Examination of the kinetics of synthesis of the two ORFs (Figure 7d ) reveals that in all cases, the frequency of termination-reinitiation at early time points was higher than that seen at the steady state. This is indicative of a titration effect; early in the time course, when fewer ribosomes have loaded onto the mRNA (due to the earlier addition of edeine), the greater frequency of reinitiation may reflect the increased relative abundance of a necessary factor. The molecular basis of the resistance to edeine seen with the MNV signal is difficult to explain. It may be that recognition of the stop-start motif is indeed blocked by edeine but somehow, a proportion of initiation complexes still recognise the AUG present in the second pentanucleotide motif (UGAUG; see above) on the mRNA.
In this paper we show that expression in vitro of the murine norovirus VP2 protein occurs by coupled translation terminationreinitiation. The process requires the close proximity of stop and start codons, a defined region of mRNA upstream of the stop-start window that includes a functional TURBS Motif 1 and translation by the ribosome through this region up to the site of terminationreinitiation. Secondary structure mapping indicates that the RNA in this region is weakly structured, with Motif 1 loosely embedded in the 59 arm of a putative stem-loop structure. The MNV signal thus exhibits many of the features and functional characteristics of the stop-start signals of FCV, RHDV and influenza B. The Figure 1 ] which acts as the ''wild-type'' reference construct [WT] in these experiments), in which the stop and start codons of the termination-reinitiation signal were altered. The figure shows the primary sequence and three-frame translation of the relevant region of the mRNA encoded by each construct. The natural stop-start motif is shown in pink and emboldened text, the downstream fortuitous stop-start motif in pink. Mutations within the mRNA sequence are highlighted by uppercase, red emboldened characters. The upstream rlucVP1 ORF is highlighted in grey, as is the downstream VP2fluc ORF where this is known.
Likely key methionines (start codons) or their replacement amino acid are highlighted in green. doi:10.1371/journal.pone.0008390.g005 molecular mechanism of termination-reinitiation remains to be fully elucidated, however. Central to the discussion is the TURBS and in this context the purpose of the identified Motifs, the role (if any) of RNA secondary structure, and the functional requirement for translation through the TURBS.
Regarding Motif 1, it is clear that in all studies so far, mRNA mutations that would destabilise an interaction with 18S rRNA reduce or abolish reinitiation and changes not predicted to affect pairing having a lesser effect or none at all. Recently, the reciprocal experiment was performed, where mutations were introduced into the relevant region of (yeast) 18S rRNA. Their effect on termination-reinitiation was found to be highly consistent with a role for mRNA-18S rRNA pairing [18] . These experiments confirm a role in tethering through rRNA, although do not rule out the contribution of other factors, for example, binding of eIF3 [13] . A comparative alignment of the MNV signal with other known or suspected termination-reinitiation signals (Figure 8 ) reveals that Motif 1 is always present and that the stop and start codons of the termination-reinitiation site are in close proximity to each other. What does vary is the spacing between the two elements, from only 26 nt in the case of BM2 to 29 nt in MNV, 53 nt in FCV, 61 nt in RHDV and 62 nt (the longest) in the Lagovirus European brown hare syndrome virus. It is not clear whether the ''additional'' sequences present in viruses with longer TURBS have a role in termination-reinitiation. Deletion analysis of the FCV and RHDV TURBS has revealed some dispensible sequences -there may be some flexibility in the spacing of Motif 1 that allows other biological information to be accommodated into the TURBS without affecting function in stop-start. However, there is little sequence conservation between the signals of viruses of different genera, arguing against the presence of other primary sequence motifs. Another stretch of bases of functional consequence has been identified in FCV and RHDV, namely TURBS Motif 2, which is located closer to the stop-start window than Motif 1 and is speculated to help position ribosomes correctly at the reinitiation codon [12, 14] . Recent work has shown that the functional requirement for Motif 2 is in its participation in a basepaired region that forms between this motif and a stretch of bases immediately upstream of Motif 1 [18] ; see Figure 8 . This basepairing has previously been noted from structure predictions of the signal of FCV [13] and direct RNA secondary structure probing of BM2 stem 2 [19] and the MNV stem 2 (see Figure 4) . Based on the observations of Luttermann and Meyers [18] , the formation of this stem is likely to be important to termination-reinitiation in the BM2 and MNV systems. Indeed, it is noticeable that in the deletion analysis of the MNV signal, and that of BM2 [19] , those deletions that would affect formation of stem 2 showed reduced activity in termination-reinitiation (Figure 1b, Figure 4) .
Despite this progress, the occurence and role of RNA secondary structure within viral TURBS is poorly understood. Direct structure probing and mfold analysis indicates that the RNA upstream of the stop-start window is metastable and whilst the secondary structures proposed for FCV [13] , BM2 [19] and MNV (this study) are superficially similar, the largely single-stranded nature of the TURBS weakens these models and their comparison. The insertion of a premature termination codon upstream of the TURBS blocks reinitiation, ruling out the possibility that VP2 expression occurs by ribosome recruitment to a conventional, structured, viral IRES or by shunting from the untranslated region of the upstream ORF. The requirement for translation through the TURBS may simply reflect the need to deliver ribosomes to the stop-start window, but it could also indicate a requirement to remodel the TURBS, conceivably by alteration of RNA secondary structure or displacement of a bound factor. Based on chemical and enzymatic RNA structure probing of the BM2 signal and folding predictions (mfold), it has been suggested that transit of the ribosome to the stop-start window leads to melting of one stemloop structure and the formation of an alternative structure that has Motif 1 displayed on its apical loop [19] . The position of MNV Motif 1 relative to the stop-start window is very similar to that of BM2 suggesting that the same remodelling could operate (Figures 4  and 8) . However, whilst transit and termination of the ribosome would destabilise the identified secondary structure (Figure 4) , liberating TURBS motif 1 in close proximity to helix 26, it is not clear whether this motif would subsequently be displayed as part of an alternative secondary structure. Whilst mfold analysis of the MNV region present locally upstream of the terminating ribosome does suggest an alternative secondary structure, further work will be needed to confirm this possibility.
Studies on the FCV signal have revealed that the reinitiation process occurs in the standard fashion by the criterion of sensitivity to edeine, but it is distinct in being completely independent of eIF4G or the eIF4F complex [13] . Analysis of the MNV signal here provides further evidence that the process deviates from the standard mechanism. First, like BM2 [19] , there appears to be efficient use of non-AUG codons to reinitiate translation, indicating a relaxed requirement for the full complement of initiation factors, which would include eIF1 and eIF1A, thought to play important roles in locating and correct recognition of the AUG start codon [22] [23] . Secondly, in contrast to what has been observed with FCV and BM2, the MNV signal is more resistant to treatment with edeine. Edeine does not inhibit binding of the eIF2/GTP/Met-tRNAi ternary complex to the 40S ribosomal subunit, nor Met-tRNAi/40S complex scanning, but there is a complete failure of AUG codon recognition, so that scanning continues past all AUG codons, and, probably as a secondary consequence, there is no ribosomal subunit joining [24] [25] . The relative insensitivity of the MNV signal to edeine suggests that recognition of the AUG start codon during reinitiation may not require a scanning ternary complex. It is not clear why the FCV and BM2 signals respond differently to edeine, especially as the organisation of the BM2 signal (with regard to the position of Motif 1 and the primary sequence of the stop-start window) is so similar to that of MNV. Another observation that hints at nonstandard reinitiation mechanisms relates to the the translation pattern seen with the MNV49.8 transcript. In this mRNA, the two termination-reinitiation windows (the natural UAAUG and the fortuitous downstream UGAUG) were mutated to eliminate the stop codon in each case. In translations of this mRNA, where termination occurs 30 amino acids downstream of the authentic site, very little termination-reinitiation product was seen, but an additional product was synthesised whose size is consistent with that of a fusion of the two reporter ORFs (asterisked in Figure 6 ). The origin of this protein is uncertain. It could have arisen through a ribosomal frameshift event, although no obvious conventional frameshift signals are present in the region of overlap between the two ORFs [26] [27] . It could also represent the outcome of a failed attempt to terminate and subsequent resumption of translation by ribosomes on the downstream ORF. Further work will be required to elucidate the nature and origin of this product and how it relates to the mechanism of termination-reinitiation.
Plasmids used to assay termination-reinitiation were based on the p2luc reporter vector [20] . Sequences encompassing the stopstart signal of MNV (203 bp of sequence information upstream of the VP1 stop codon and 52 bp downstream) and FCV (97 bp upstream of the VP1 stop codon and 14 bp downstream) were generated by PCR (using Pfu polymerase [Roche]) from, respectively, plasmids pT7:MNV (kind gift of Dr Ian Goodfellow, Imperial College, London) and pSG-2/3* [13] , a kind gift of Dr Tuiya Pöyry, University of Cambridge. The PCR products and p2luc were digested with SalI and BamHI and ligated together. Sequences were confirmed by dideoxy sequencing (using the facility at the Department of Biochemistry, University of Cambridge). The influenza B termination-reinitiation assay plasmid (p2luc-BM2wt; 250 bp upstream of M1 stop-codon, 18 bp downstream) was described previously [19] .
Site-directed mutagenesis was performed using the Quikchange II site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. For large deletions (greater than 48 bp) a modification of the manufacturer's protocol was used with the primers containing ,30 bp of complementary sequence either side of the site of deletion, as described previously [28] . Mutagenesis to introduce insertions longer than 6 bp was performed in two steps [29] , by first subjecting mutagenesis reactions (containing either the sense or antisense primer) to three cycles of PCR, then mixing the reactions and performing a further 18 cycles according to manufacturer's instructions.
Reporter plasmids were linearised with HpaI and capped run-off transcripts generated using T7 RNA polymerase as described [30] . Messenger RNAs were recovered by a single extraction with phenol/chloroform (1:1 v/v) followed by ethanol precipitation. Remaining unincorporated nucleotides were removed by gel filtration through a NucAway spin column (Ambion). The eluate was concentrated by ethanol precipitation, the mRNA resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry.
Unless otherwise stated, mRNAs were translated in FlexiH rabbit reticulocyte lysate (FlexiHRRL, Promega) programmed with 50 mg/ml template mRNA. Typical reactions were of 10 ml and composed of 60% (v/v) FlexiHRRL, 20 mM amino acids (lacking methionine), 500 mM MgOAc, 2 mM DTT, 5U RNAse inhibitor (RNAguard, GE Healthcare Life Sciences), 130 mM-160 mM KCl (optimised for each batch of FlexiHRRL) and 0.2 MBq [ 35 S]methionine. Reactions were incubated for 1 h at 30uC and stopped by the addition of an equal volume of 10 mM EDTA, 100 mg/ml RNase A followed by incubation at room temperature for 20 minutes. Samples were prepared for SDS-PAGE by the addition of 10 volumes of 2X Laemmli's sample buffer [31] , boiled for 3 minutes and resolved on 12% SDS-PAGE gels. The relative abundance of products on the gels was determined by direct measurement of [ 35 S]methionine incorporation using a Packard Instant Imager 2024.
A plasmid encoding the putative termination-reinitiation signal of MNV (p2luc-MNVwt) was modified by site-directed mutagenesis to include a T3 RNA polymerase promoter 30 bp upstream of the minimal required viral sequence generating plasmid p2luc-MNV-T3. RNA for structure mapping was prepared by in vitro transcription of BamHI-digested p2luc-MNV-T3 using T3 RNA polymerase. Transcription reactions were performed on a 200 ml scale essentially as described [30] . Structure mapping was performed using a 59 endlabelling procedure as described previously [30, 32] . All probing reactions were performed in a final volume of 50 ml and contained ,40,000 c.p.m. 59 33 P-end-labelled transcript, 10 mg Escherichia coli rRNA and the relevant enzymatic or chemical probe. Further details are provided in the legend to Figure 3 . Interaction of the HIV-1 frameshift signal with the ribosome Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome. The ribosome translocates mRNA and bound tRNA molecules accurately in order to maintain the reading frame. This process results in movement of the ribosome along the mRNA by three nucleotides toward the mRNA's 3 0 -end. Translocation of mRNA and tRNAs is a property of the ribosome itself (1, 2) , however binding of elongation factor G (EF-G) and subsequent hydrolysis of GTP strongly catalyzes it (3) . Although the ribosome acts as its own helicase, stable folded structures within the coding regions of mRNA affect the rate of translocation, and more seriously, may trigger a change of reading frame (4, 5) . Such frameshifting mRNA elements play a crucial role in the translational control of viral proteins via -1 programmed ribosomal frameshifting (-1 PRF) where the reading frame has shifted by one base toward the mRNA 5 0 -end. The -1 PRF requires both the mRNA slippery sequence at the ribosome coding sites as well as a downstream structural element that resists unfolding, representing a physical barrier to the mRNA translocation machinery. While in many cases the downstream barrier constitutes a hairpin (H)-type pseudoknots (6, 7) , on a rare occasion it can also be a simple stem-loop structure (8) (9) (10) (11) . It is of interest to note that although these pseudoknots or stem-loops also trigger ribosomal pausing at the slippery site, consistent with notion of them acting as physical barriers, the extent of pausing shows no correlation with frameshift efficiency (12) .
The crystal structure of the ribosome in complex with mRNA has revealed that the mRNA is in a singlestranded conformation in the narrow downstream tunnel (13, 14) . The ribosome therefore has to unwind mRNA secondary structure through its mRNA helicase activity (15, 16) . A mechanistic basis for mRNA helicase activity has been proposed involving ribosomal proteins S3, S4, S5 at the mRNA entrance and rotational movement of the head of the 30 S subunit (13, 15) . The 9 Å and the torsional restraint models (17) propose that -1 PRF is dependent on the mechanical tension induced when a pseudoknot resist unfolding by a moving translating ribosome. Possible effects of such tension were directly observed in the cryo-electron microscopic (Cryo-EM) images of eukaryotic ribosomes stalled in the process of -1 frameshifting in complex with eEF2, tRNA and a frameshifting mRNA pseudoknot (18) . The opposing actions of translocation catalyzed by eEF2 and resistance to unfolding by the mRNA strand generate strain that deforms the P-site tRNA which may weaken the codonanticodon interaction and promote the shift by one nucleotide into the 5 0 direction.
The notion that mechanical stability of mRNA structural element is crucial for -1 PRF, has triggered mechanical unfolding experiments of individual mRNA pseudoknot, mutants as well as some of the constituent hairpins using optical tweezers hairpins (19) (20) (21) . Some of these experiments suggest that frameshift efficiencies correlate with unfolding forces rather than the free-energy difference between the folded and unfolded state (21) . This indicates that -1 PRF is kinetically controlled, as has been proposed previously (22) .
In HIV-1, translational frameshifting leads to synthesis of the Gag-Pol fusion protein which gives rise to the viral protease, reverse transcriptase (RT) and integrase. This HIV-1 RNA frameshifting signal is a potential target for antiviral therapy (23) (24) (25) (26) . The exact structure of this RNA frameshifting signal has been the subject of debate. Jacks and collaborators initially proposed that it is a stem-loop structure downstream of the slippery sequence that is essential for efficient frameshifting (4) . Alternative structures have subsequently been proposed-reviewed by Brierley and Dos Ramos (27)in which the stimulatory RNA folds as a pseudoknot (28, 29) ; a pseudoknot with an RNA triple helix motif (30) and two-stem helix containing a three-purine bulge (8) . Recently however, two independent nuclear magnetic resonance (NMR) studies have shown that, in the absence of the ribosome, the fold is a long hairpin ( Figure 1 ) with an internal three-nucleotide bulge (9, 10) . A more recent structure-function analysis of the ribosomal -1 FS signal of two human HIV-1 isolates (31) favors the two-stem helix model of Dulude et al. (8) . The internal loop of the long stem-loop introduces a distinct bend between the lower and upper helical regions, a structural feature which, remarkably is also often seen with frameshifting pseudoknots. It has been proposed that the lower stem and the bend serve to initiate contacts between the upper stem-loop and the ribosome. Subsequently the lower stem melts allowing the slippery sequence to bind at the decoding site (10, 32) . Based on the identification of position +11 as the limit of accessibility of an RNA double helix approaching the ribosome (15), we previously proposed that the upper segment of the lower stem and the bulged region could be structured and/or contact the ribosomal surface (9) . NMR studies also pointed out that the upper stem, rich in conserved G-C Watson-Crick base pairs, is highly stable whereas the bulge region and the lower stem are much less so, and may readily unfold/melt. We therefore decided to unfold individual HIV-1 hairpins using optical tweezers, which complement existing methods, such as thermal denaturing, chemical probing or NMR spectroscopy, that address local features within the context of the entire global RNA structure. In principle, optical tweezers aid in applying forces locally to a folded RNA molecule in a way that may be more similar to in vivo conditions than, for example, thermal or chemical denaturing. In the case of a hairpin, the mechanical force will act locally on the 5 0and 3 0 -ends of the RNA unzipping the stem base-pair by base-pair toward the loop. A similar situation may be found in the ribosome where the translocation movement will generate a force pulling the 5 0 -end of the mRNA inside the ribosome.
Questions, however, remain about the structure of the HIV-1 frameshift signal when ribosomes are present and bound at or downstream of the slippery sequence. Here, using Escherichia coli ribosomes, we address this question and probe possible interactions of the HIV-1 frameshift signal with the ribosome by using toeprinting and chemical probing assays. The ability of this eukaryotic mRNA frameshifting signals to promote -1 PRF in the prokaryotic translational machinery has been previously demonstrated (33, 34) .
The translocation of mRNA was assayed by toeprinting as described (35, 36) . mRNA (1 mM) was annealed to primer (2 mM) in 50 mM K-Hepes (pH 7.0) and 100 mM KCl by heating to 90 C for 1 min and placing at room temperature until the temperature reached 45 C. To form the complexes, tight-couple ribosomes (2-5 mM) (37) from E. coli MRE600 were added to 0.6 mM of mRNA in 60 mM NH 4 Cl, 10 mM Tris-Acetate (pH 7.4) and 20 mM MgCl 2 and incubated at 37 C for 10 min. A first tRNA (4 mM) was added to fill the P site by incubation at 37 C for 10 min and aliquot (0.6 pmol mRNA) was removed to ice for later extension. A second tRNA (4 mM) was added to fill the A site by incubation at 37 C for 10 min and aliquot (0.6 pmol mRNA) was removed to ice for later extension. EF-G was added in buffer (50 mM Tris-HCl (pH 7.6), 20 mM MgCl 2 , 100 mM NH 4 Cl and 1 mM DTT, 1.5 mM GTP) such that the final concentrations of GTP and EF-G were 300 and 1 mM, respectively. Reactions were incubated at 37 C for 10 min, and aliquots (0.6 pmol mRNA) were removed from each reaction lacking (-G) or containing (+G) EF-G. Each of the aliquots was then extended in parallel (38) with (5 0 -CTTTATCTTCAGAAGAAAAACC-3 0 ) primer, and the product were resolved by 8% denaturing PAGE.
Stepwise translocation experiments were done as previously described (15) . Tight-couple 70 S ribosomes (1 mM final concentration) from E. coli MRE600 (39) were incubated with mSP-HIV-1 mRNA (1 mM) in 30 ml binding buffer (10 mM Tris-HCl (pH 7.4), 60 mM NH 4 Cl, 10 mM Mg(OAc) 2 , 6 mM b-ME) for 10 min at 37 C, followed by addition of tRNA Phe (1 mM) and a further 10 min incubation to fill the P site. Aliquots (5 ml) of this reaction were then added to separate tubes containing either GTP (600 mM) (F), GTP + tRNA Leu (1 mM) (L), GTP + tRNA Leu , EF-G (1 mM) (L'), GTP + tRNA Leu , EF-G + tRNA Gly (1 mM) (G), GTP + tRNA Leu , EF-G + tRNA Gly + tRNA Lys (1 mM) (K), GTP + tRNA Leu + tRNA Gly + tRNA Lys (K'), in binding buffer (final volume, 10 ml). These tubes were incubated for 10 min at 37 C and then placed on ice for the primer extension in toeprinting analysis (4 ml).
To footprint, binding of mRNAs was performed by incubating 70 S ribosome (in the range of 50-750 pmol according to the mRNA tested) with mRNA (10-50 pmol) in 50 ml reaction buffer A (20 mM MgCl 2 , 150 mM NH 4 Cl, 80 mM potassium cacodylate, pH 7.2) at 37 C for 10 min. A first tRNA (320 pmol) was added to fill the P site by incubation at 37 C for 10 min. A second tRNA (320 pmol) was added to fill the A site by incubation at 37 C for 10 min. mRNA-tRNA-70 S complexes were then purified by ultrafiltration (MICROCON YM-100 100 000 Da, Fisher scientific LABOSI). The ternary complex was diluted in 250 ml of buffer A and distributed into 50 ml aliquots (2 pmol mRNA). Chemical probing (38) was performed by addition of 2, 4 or 8 ml dimethyl sulfate (DMS; 1:10 dilution in 95% ethanol), 2, 4 or 8 ml kethoxal (KE; 19 mg/ml in H 2 O) on 50 ml aliquot, followed by incubation at 37 C for 10 min. All modification reactions were stopped by addition of 150 ml 95% ethanol and 5 ml 3 M sodium acetate followed by rapid mixing. KE-modified samples were adjusted to 25 mM potassium borate (pH 7.0). The pellets were resuspended in 200 ml of 0.3 M sodium acetate, 2.5 mM EDTA and 0.5% SDS (with addition of 25 mM potassium borate for KE samples), extracted three times with phenol, twice with chloroform and resuspended in 10 ml H 2 O (for DMS samples) or in 10 ml 25 mM potassium borate (for KE samples). Primer extension reactions were performed as described (38) .
Purified tRNA Lys , tRNA fMet and tRNA Phe were purchased from Sigma, tRNA Gly and tRNA Leu were gracefully donated by Henry Grosjean.
Messenger RNAs were prepared by in vitro transcription. Plasmid pGENE32 is pUC118 containing a region of phage T4 gene 32 (40) from nucleotide position -54 to +84 (where +1 is the translational start) downstream of an engineered T7 promoter sequence. The introduction of slippery sequence in pGENE32 was performed by sitedirected mutagenesis kit (Stratagene). Transcripts with stem-loop were obtained by in vitro transcription of synthetic genes flanked upstream by T7 RNA polymerase promoter region and downstream by a BamHI restriction site. The synthetic genes were constructed by shotgun ligation of 10 DNA fragments (24-30-mers) covering both strands and ligated in the KpnI and BamHI sites of pGENE32. All transcripts have been purified by denaturing PAGE.
His-tagged EF-G was purified from pET24b-fusA in E. coli BL21(DE3) as described (41) .
RNA was synthesized from a template obtained by polymerase chain reaction (PCR) from bases 3821 to 628 of the pBR322 DNA plasmid, where the frameshifting RNA signal from HIV-1 was cloned into the EcoRI and HindIII restriction sites and a T7 promoter was appended to the template in the course of the PCR reaction (42) . The DNA components of the handles were prepared by PCR from pBR322. Handle A (pBR322 bases 3821 to 3) was biotinylated, and one of the primers used to amplify handle B (pBR322 bases 30 to 628) was purchased with a 5 0 digoxigenin group.
RNA and DNA handles were resuspended in 10 mM sodium phosphate buffer (pH 6.4), and incubated at a ratio of $1:1:1 at 90 C for 1 min and transferred to room temperature to cool down gradually, and subsequently diluted to a final concentration (of RNA) of $1 mM.
Five microliters of anti-digoxigenin-coated polystyrene beads (0.3 nM; diam. 0.49 mm) were mixed with 1 ml of the DNA-RNA hybrid ($1 mM) in binding buffer (10 mM Tris buffer [pH 7.0], 250 mM NaCl, 10 mM MgCl 2 , 0.4% w/v BSA), and incubated at 4 C for overnight on a rotator.
Sample cells were preassembled prior to use. Two thin strip spacers (thickness $200 mm) were positioned $5 mm apart on the center of a pre-cleaned microscope slide, and epoxy applied at the outer edges of the spacers. A streptavidin functionalized 24 Â 40 mm, no. 1.5 coverglass (Xenopore) was then put on the top of the slide. Before introducing the bead and RNA mixture, the sample cell was surface-coated with acetylated BSA by incubation with binding buffer for 30 min at room temperature and washed with 1 ml of the binding buffer, to prevent any sticking of beads to the surface. The bead and RNA mixture was then introduced into the sample cell, and incubated for 30 min at room temperature, and finally washed with 1 ml of binding buffer to remove any unbound beads and RNA.
Molecules were stretched in 10 mM Tris buffer (pH 7.0), 250 mM NaCl, 10 mM MgCl 2 , or alternatively in 10 mM Tris-acetate (pH 7.4), 60 mM NH 4 Cl, 6 mM b-mercapto, 20 mM MgCl 2 . Unfolding/refolding parameters were statistically indistinguishable for both conditions. The spring constant of the optical tweezers was 0.1-0.2 pN nm -1 .
The extension of the unfolded single stranded HIV-1 hairpin, x SS was computed as x SS (F) = Áx + L HP for convenience, with F = (F 1 +F 2 )/2 the unfolding force and Áx the increase in extension ( Figure S2 ). The increase in contour length (expressed in number of nucleotides) was subsequently computed using the worm-like chain model for polymer elasticity assuming a stretching modulus of 1000 pN, a persistence length of 1 nm (43,44) and inter-phosphate distance of 0.59 nm. Alternatively, one may choose to do the computation taking into account F 1 and F 2 explicitly, where it is found that
Þ þ L HP . Then however, one needs to fit the section of the force versus extension curve up to the unfolding event with a worm-like chain model in order to compute the extension of the handles, x H F 2 ð Þ at F 2 . When performing this more laborious analysis on a subset of our data, we only find a 1-2 nucleotide difference in contour length compared to the analysis that utilizes the applied approximation
Standard free-energies at zero force were computed for each trajectory according to
Fdx using the worm-like chain model with persistence length of 1 nm and stretching modulus of 1000 pN (43) .
We used a toeprinting assay to monitor the position and/ or structure of the HIV-1 hairpin during the movement of the ribosome along mRNA. This primer-extension inhibition assay has been shown to be a powerful tool for mapping the position of mRNA within 30 S and 70 S ribosomal complexes that contain tRNA (45) . When RT encounters the ribosome (a so-called hard-stop), it terminates cDNA synthesis thereby generating a highly specific toeprint. Alternatively, RT may also stall at mRNA structural elements further downstream from the ribosome that prove too hard for RT to unwind, resulting in what is generally called an extended toeprint.
We first demonstrated that under simple experimental conditions (where tRNA was non-enzymatically delivered at the ribosomal A site), toeprinting allows localization of the ribosome on the mRNA containing the wild-type HIV-1 slippery sequence (mSP-HIV-1) but not the downstream hairpin. The sequences and secondary structures of the mRNA constructs used are derived from T4 gene 32 mRNA in which we introduced the HIV-1 frameshifting signals as shown in Figure 1 . In the case of the mSP-HIV-1 construct, two distinct toeprints are observed at +15 and +16 when tRNA Phe is bound at the P site ( Figure S1A , lane F), in correspondence with the two possible reading frames created by the slippery sequence. A third toeprint at +17 appears when tRNA Leu subsequently binds at the A site ( Figure S1A , lane F 0 ). This is thought to be due to a conformational change in the ribosomal complex following A-site binding so that a single position of the tRNA in the A site results in a doublet of bands (at positions +16 and +17) (46, 47) . It is important to note that the signal at +15 did decrease, indicating that binding of the tRNA Leu at the A site assisted in the positioning of the mRNA with tRNA Phe preferentially bound to the u +1 u +2 u +3 codon adjacent to the leucine codon u +4 u +5 a +6 . After EF-G catalyzed translocation of tRNA Leu to the P site ( Figure S1A , lane L), the toeprints at +16, +17 were moved to +20 and +21, corresponding to a 4-nt translocation event. We cannot conclude if a toeprint at +19 exists since this band is also present in the control lane (without ribosome). However, if it exists, this toeprint is very weak. When tRNA Gly is subsequently added to the EF-G containing reaction mixture ( Figure S1A , lane G), translocation proceeds further giving the expected toeprints at +22 and +23 for tRNA Gly bound to P site and paired with the g +7 g +8 g +9 codon. A weak toeprint at +24 may indicate a low population of tRNA Gly bound to P site and paired with the g +8 g +9 a +10 glycine codon in the +1 frame. The addition of tRNA Lys (Figure S1A, lane K) triggered translocation leading to a toeprint at +25, which corresponds to a post-translocation complex with tRNA Lys in the P site bound to the a +10 a +11 g +12 lysine codon and leaving an empty A site.
We then applied this assay to a gene 32 message (mSP-SL-HIV-1), which contains the mRNA frameshifting signal (Figure 2A) . In order to avoid any unnecessary ambiguity in interpreting the toeprints, we substituted the phenylalanine codon (u +1 u +2 u +3 ) for a methionine codon (a +1 u +2 g +3 ) (mSP-SL-HIV-1; Figure 1 ) to avoid complications due to the presence of the slippery site. This allows tRNA binding in only a single unique reading frame.
When tRNA fMet was bound at the P site, a doublet of toeprints at positions +U16/+G17 was detected ( Figures  S2 and 2A) . We note the existence of a stop at position +17 in the control lane (in the absence of ribosomes). However, the signal in presence of ribosome is substantially stronger and therefore is interpreted as a ribosomal toeprinting signal. tRNA Leu4 was subsequently bound to the A site ( Figures S2, lane 3, and 2A , lane M 0 ) which resulted in the disappearance of the +16 toeprint. The +16 and +17 toeprints are hard stops, independent of the reverse transcription activity indicating that the mRNA secondary structure unfolds during cDNA synthesis as previously seen for a stem-loop (48) and pseudoknots (49) . However, in addition to these hard stops downstream extended toeprints can also exist indicating that mRNA interactions with the ribosome 5'-(N 42 ) aAGGAaauaaa aug uuu aaa cgu aaa ucu (N 68 )-3' can extend beyond the usual 15 nucleotides buried in the ribosome-mRNA track (48, 50) . Unlike hard stops, extended toeprints are generally dependent upon change of temperature, RT concentration, or the source of RT. Therefore, we varied the temperature and also tested different RTs to look for extended toeprints in the case of mSP-SL-HIV-1. Two additional reverse transcription stops were identified within the 3 0 region of the stemloop at positions +47 (very weak) and +43 (Figures 2A  and 3A ). These extended toeprints are weak in comparison to the hard stop at +17 but are absent in the control lane ( Figure 2A , lane C, in the absence of the ribosome). The +43 and +47 toeprints indicate that a fraction of the RT molecules halted when the enzyme approached or encountered bulge region in the HIV-1 hairpin. In order to test the contribution of the bulge region to the extended toeprint, we changed the GGA-bulge by a CCC-bulge (mSP-SL-CCC-HIV-1, Figure 1 ). The intensities of both toeprint signals (at +43 and +47) decreased ( Figure 2B ) and were reproducibly of lower level than the RT stops found in the control lane -70 S. Interestingly, the substitution of the three purines in the bulge by pyrimidines also decreases frameshifting efficiency (8, 32) .
U-G C-G C-G U-A U-A C-G C-G G-C G-C U-A C-G U-A A-U G-U A-U A-U G-C G-U A A G 12 G C A A46
We subsequently monitored the position of mSP-SL-HIV-1 within the ribosome allowing a single or multiple rounds of EF-G catalyzed translocation. The signal at positions +43 and +47 disappeared to give a new toeprint at +39 (Figure 2 , lane L, and 3B), indicating that the lower stem readily gave way upon just a single translocation step. An identical result was observed in the mSP-SL-HIV-1 RNA which has a wild-type slippery sequence (first codon is u +1 u +2 u +3 ) indicating that codon substitution to a methionine codon did not affect the observed toeprints except for the +47 signal that is ambiguous ( Figure  S1B ). Furthermore upon a single translocation, the +16/+17 toeprint signals disappeared without, however, any occurrence of a new toeprint at position +19 (lane L). The disappearance of the mSP-SL-HIV-1 +16/+17 toeprints upon addition of EF-G is intriguing. We also observed the same phenomenon with the construct containing the wild-type slippery sequence ( Figure S1B ), and interestingly also with an mRNA containing a pseudoknot (BWYV) bound to ribosome (51) . This phenomenon is characteristic to mRNAs containing downstream structural elements, however its cause still need to be elucidated. Ribosomes are known to change conformation upon EF-G binding (52) (53) (54) (55) . One may speculate that this either blocks RT access directly or stabilizes the mRNA structure.
Any subsequent addition of tRNA Gly (lane G) and tRNA Lys (lane K) in presence of EF-G did not further affect the position of the extended toeprint, which remained at +39, indicating that further movement of the mRNA through the ribosome was impaired.
We next tested an mRNA with an additional codon between the AUG and the start of the lower stem (mSP-Tyr-SL-HIV-1 RNA) extending the spacer, which should at least allow for one round of translocation to be visualized before the ribosome stalls. The toeprints at the new positions A+16/U+17 were unambiguously identified ( Figure S3 ), which places the boundary region contacting the ribosome upstream from the bulge region. These bases are in the upper stem for the mSP-SL-HIV-1 RNA. In Figure 2C , where experimental conditions were tuned as to specifically detect the extended toeprints, the toeprint signals in the region +17 prove somewhat weaker than in Figure S3 , but are always present. As expected, with the longer spacer, a band at +19 appeared upon addition of EF-G ( Figure 2C, lane L) . Interestingly, the extended toeprints did not change and appeared at the exact same positions at A + 46 and U + 50 (corresponding to the A + 43 and U + 47 in mSP-SL-HIV-1 RNA) ( Figure 2C ). Subsequent addition of EF-G and tRNA Tyr and tRNA Gly produced the same changes in the toeprint pattern as for the mSP-SL-HIV-1 RNA (a shift of 4-nt from +46 to +42). We note that in this case the +19 toeprint remains the strongest toeprint in the upper region of the gel (by comparison with the +17 toeprint) ( Figure 2C , lanes Y and G) indicating that translocation, as before, seems to be impaired with the downstream upper stem.
Toeprinting assays fail to provide information on more detailed structural changes that the mRNA might undergo upon binding to the ribosome. Therefore, a footprint assay was performed to obtain structural information upon the interaction of mSP-SL-HIV-1 mRNA with 70 S ribosome-tRNA complexes. In these complexes the ribosome is positioned with the slippery sequence at the decoding site. Results of the chemical probing with KE, 1-cyclohexyl-2-morpholino-carbo-diimide-bmetho-ptoluene sulfonate (CMCT) and DMS are shown in Figure 4 . For free mSP-SL-HIV-1 mRNA the chemical modification patterns and levels of reactivities are identical to those previously published (9) . Nucleotides in the apical ACAA teraloop as well as A +43 and A +44 from the bulge are found to be reactive to DMS (Figure 4) , while nucleotides G +41 and G +42 were reactive to KE. In the lower stem most of the guanine, adenine and uracil bases are accessible to the chemical probes indicating poor stability (9) .
Subsequently, mSP-SL-HIV-1 mRNA was probed in complex with the ribosome and tRNA. For each chemical probe tested, bands that were present in a control lane of unmodified mRNA incubated with ribosome were not taken into account. In the complex, the characteristic strong protections at the guanine nucleotides in the Shine-Dalgarno sequence (G -11 , G -10 ) are clearly seen. Base A +1 , of the methionine codon is protected from chemical modifications demonstrating, as previously described (56), Watson-Crick pairing with the anticodon of tRNA fMet . Interestingly, nucleotide A +6 experienced an increase in reactivity similar to what has previously been seen for a mRNA containing a hairpin (selenocysteine incorporation sequence SECIS) in complex with 30 S subunit and tRNA fMet (57) . In the spacer and hairpin regions, most of the changes in chemical reactivities were detected in the lower stem that is supposed to be close to the ribosomal surface. Bases G +7 , G +8 and G +12 were protected from modification by KE (Figure 4) . The reactivity of G +9 toward KE slightly increased in a way similar as A +10 toward DMS.
Since toeprinting experiments suggested reduced structural stability of the lower stem, we investigated the mechanical stability of the HIV-1 hairpins. Individual hairpins, sandwiched between two differentially endlabeled hybrid DNA-RNA handles were unfolded using optical tweezers (42) . Molecules were tethered between a streptavidin-coated glass cover slip and anti-digoxygenin coated polystyrene beads (diameter 1 mm), and stretched by moving the piezo-actuated microscope stage while holding the bead with optical tweezers. Force versus extension curves were computed taking into account this experimental geometry (58) . Stage velocities were 67 nm/s, so that the system remained at or close to thermodynamic equilibrium as confirmed by the overlap of the stretching and relaxation force versus extension curves ( Figure 5B ). In addition, repeated unfolding and folding can be observed within single force versus extension curves ( Figure 5B) , a further indication that the loading rate is sufficiently low as to assure thermodynamic equilibrium. The increase in contour length (expressed in number of nucleotides) upon unfolding was subsequently computed using the worm-like chain model for polymer elasticity assuming a stretching modulus of 1000 pN, a persistence length of 1 nm (43,59) and a inter-phosphate distance of 0.59 nm (see 'Materials and Methods' section). Results are summarized in Figure 5C , yielding a mean increase of 25.3 AE 3.4 nm (mean AE SD) nucleotides, consistent with the contour length of the upper stem of the HIV-1 hairpin. The mean unfolding force is 12.8 AE 1.0 pN (mean AE SD), whereas the standard free-energy change at zero force was found as ÁG = 10 AE 2 kcal/mol (mean AE SD). Refolding statistics are summarized in the Figures S4 and S5 and yield a decrease of contour length of 26.0 AE 4.7 nm (mean AE SD) at an average refolding force of 13.1 AE 1.1 pN (mean AE SD), and ÁG = 11 AE 3 kcal/mol (mean AE SD), essentially unchanged from the unfolding statistics, as one would expect when at thermal equilibrium.
Extended toeprints (albeit weak ones) at +43 and +47 in the pre-translocation state indicate that a fraction of RTs was incapable of unwinding part of the lower stem and bulge region when encountering the HIV-1 hairpin bound at the ribosome. Since such toeprints do not occur with free hairpins, contacts with the ribosome (34) may be responsible for these signals. Extending the spacer sequence by an extra codon yields an identical toeprint; not surprising as it is hard to imagine that a longer spacer would stand in the way of forming ribosomal contacts. This will be discussed further below. Furthermore, we showed that these toeprints are dependent on the structure of the bulged region of the frameshifting signal. The subsequent replacement of the GGA-bulge with a CCC-bulge, known to reduce -1 PRF efficiency (8, 32) , practically erases the +43 and +47 toeprints, indicating such a mutation affects either contacts with the ribosomes or stability of the hairpin itself. We tend to favor the former possibility as mechanical unfolding of free hairpins indicates the lower stem is fairly weak to begin with. Certainly it remains possible that contact with the ribosome can stabilize part of the lower stem. If such stabilization was to occur, it does not prevent further translocation as a single cycle of EF-G catalyzed translocation moves the toeprint to +39 providing direct evidence for melting of the lower stem. Interestingly, the extended toeprint at +39 remains even under conditions where further translocation is allowed. This suggests that the upper stem is capable of inhibiting translocation of a sizeable fraction of ribosomes. Our mechanical unfolding study supports the notion of a weak lower stem since no transition in the force versus extension curves indicative of unfolding of solely the lower stem have been observed. Although on occasion we suspect such a transition (by visual inspection of the force versus extension data, typically at forces $6 pN and lower), the increase in extension in those cases should be $4-5 nm (by theory), too small to reliably distinguish from thermal fluctuations at those force levels. We note that were these transitions to occur in the steeper part of the force versus extension curve (F > 6 pN), we should be able to detect a shift of the curve toward the right consistent with unfolding of the lower stem. However, the increases in contour lengths ( Figure 5C ) observed are consistent with unfolding of solely the upper stem. Not on any occasions have we observed increases in contour lengths consistent with simultaneous unfolding of the lower and upper stems. Since unfolding is hierarchical, the lower stem has to unfold before the upper stem does. We conclude that the lower stem therefore has much reduced mechanical stability compared to the upper stem. Quantitative assessment of the stability of the lower stem is currently not possible in the existing experimental geometry, and requires higher resolution experimental designs (60) , if at all possible. The question naturally arises how these findings accord with the observed +43 and +47 toeprint and the dependence of frameshifting upon the lower stem and bulge regions. It seems unlikely that tensions as low as a mere 6 pN and below, can trigger a frameshift (61) . Therefore, it is appealing to consider that unfolding of the hairpin in the presence of ribosomes will differ significantly from that when ribosomes are absent. When adhering to a tension-dependent mechanism of -1 frameshifting this suggest that contacts with the ribosome's exterior surface may indeed stabilize the part of the lower stem of the hairpin. Single-molecule mechanical unfolding experiments in the presence of ribosomes are considered, but beyond the scope of this article. Our chemical probing experiments provide further insight into the structure of the HIV-1 frameshift signal when bound to the ribosome. The chemical reactivity pattern indicates that almost the entire structure of the hairpin is maintained. Nucleotides from the ACAA tetraloop and the GGA bulge region remained reactive to chemical probes whereas nucleotides in the upper stem were unreactive. As expected, most of the changes in chemical reactivity of the bases are concentrated in the lower stem. The changes observed in the spacer at positions +7, +8 are consistent with this segment of the RNA being engaged in the 30 S mRNA tunnel (13) . Further downstream in the spacer sequence, nucleotides G +9 , A +10 and G +12 that are expected to be located in the vicinity or at the ribosomal helicase center indeed experienced a change in reactivity. In this segment of the RNA, the DMS reactivity of nucleotide A +11 remained mostly unaffected by the presence of the ribosome. Unfortunately, our chemical probing experiments could not provide useful information on the 3 0 strand of the lower stem. In summary, the data demonstrate that the 5 0 region of the lower stem experiences structural changes when contacted by the ribosome.
A number of other observations, although of no direct consequence for the interpretation of the extended toeprints, are of interest. First is the observation of the occurrence of an apparent 4-nt translocation step during transfer of tRNA Leu from A site to P site. The disappearance and appearance of respectively the +43 and +39 toeprints indicate that the addition of EF-G triggered a movement of the mSP-SL-HIV-1 RNA inside the ribosome of four nucleotides. However, we note that this 4-nt shift is detected on the 3 0 strand of the mRNA hairpin that is located outside the ribosome and therefore may not necessarily present a 1:1 reflection of what happened at the coding site. On the other hand, single translocation cycles with only the slippery sequence (mSP-HIV-1) also show a 4-nt step. In this experiment we analyzed the toeprint signals that result from A site, P site tRNA binding and subsequent EF-G catalyzed translocation. The mSP-HIV-1 toeprint at +15 corresponds to tRNA Phe that recognizes the overlapping codon, one nucleotide upstream (u -1 u +1 u +2 ). Binding of tRNA Phe to the u -1 u +1 u +2 codon results in an optimal spacing of seven bases between the SD sequence and the start codon (62) . The toeprint at +16 corresponds to tRNA Phe paired to codon 1 (u +1 u +2 u +3 ). Interestingly, overlapping codons u +2 u +3 u +4 and u +3 u +4 u +5 are not pairing with tRNA Phe because no toeprints were observed at +17 and +18. This is most likely due to unfavorable spacing between the P-site codon and the Shine-Dalgarno sequence (47, 63) . After P-site filling and the first round of translocation, the toeprint at +19 is the expected signal for a post-translocation complex with tRNA Leu in the P site bound at the u +4 u +5 a +6 codon. However, the major toeprint signal is at position +20 suggesting that in the post-translocation complex the P site-bound tRNA Leu is interacting with the u +5 a +6 g +7 codon. This corresponds to a 4-nt translocation event. Subsequent translocation cycles seem to force the toeprints back into register. It remains unclear what gives cause to this behavior. We note that a 4-nt translocation has been observed for punctuated mRNA with an extra, unpaired nucleotide between codons (64). Interestingly, Leger and collaborators proposed that slippage and repairing of Psite-bound tRNA Phe in the -1 frame would leave an unpaired nucleotide between the Phe and Leu codons (32) . But this is unlikely at equilibrium in our assay because A-site binding of tRNA Leu mostly repositioned tRNA Phe into the canonical frame removing any unpaired nucleotide between Phe and Leu codons.
The P-site tRNA binding triggered a major RT stop at position +17 while only a weak signal at position +16 (the 'classical toeprint'). Previous toeprinting assays under identical conditions but with different phage T4 mRNAs showed a variety of toeprints signals confined to the +14 to +17 range (45) . Thus toeprinting assays are very sensitive to the type of mRNA tested and we note that the toeprint signals with the mSP-HIV-1 and mSP-SL-HIV-1 mRNAs respectively at +16 and +17 fall into this range. Could the observed difference between the two mRNAs be attributed to the hairpin? With unstructured mRNA, we expect a pulling force on the spacer mRNA out of the ribosome purely based on entropic arguments. Such a force presumably exposes base +16 giving rise to the classical toeprint. In the case of the HIV-1 hairpin, physical interactions of the ribosome with the hairpin may provide some sort of strain relieve allowing +16 to slightly relax back into the ribosome. On the other hand, how could such a mechanism be consistent with +16/+17 toeprint obtained when an additional codon was inserted in the spacer, between the AUG codon and the hairpin? If the hairpin provides strain relieve would one not expect the mRNA to recede further back into ribosome, not only protect +16 but also perhaps +17. However, lacking any quantitative information about tension in the spacer, such extra movement may be too small to also protect +17. These are intriguing possibilities and raise questions that require the design of new experiments, well beyond the scope of this work, for answering.
Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to the UUA codon of the HIV-1 slippery sequence (65) . The presence of the GCrich upper stem strongly enhances frameshifting efficiencies. This level of mRNA slippage is enhanced further by the bulge and lower stem. We show here that when the slippery sequence is bound at the decoding site, the ribosome directly influences the spacer sequence in the lower stem that very likely enters the mRNA track. In addition, extended toeprints localized in the upper segment of the lower stem support a physical interaction between this region and the ribosome. These results are in agreement with the importance of the lower stem and bulge regions for the -1 frameshift (8, 32, 66) . It is conceivable that this interaction enhances -1 frameshifting at the particularly slippery UUUUUUA sequence (67) . Outdoor environments and human pathogens in air Are pathogens in outdoor air a health issue at present or will they become a problem in the future? A working group called AirPath - Outdoor Environments and Human Pathogens in Air was set up in 2007 at University College London, UK with the aim of opening new discussion and creating a research network to investigate the science and impacts of outdoor pathogens. Our objective in this paper is to review and discuss the following areas: What is the source of human pathogens in outdoor air? What current, developing and future techniques do we need? Can we identify at-risk groups in relation to their activities and environments? How do we prepare for the anticipated challenges of environmental change and new and emerging diseases? And how can we control for and prevent pathogens in outdoor environments? We think that this work can benefit the wider research community and policy makers by providing a concise overview of various research aspects and considerations which may be important to their work. Low moisture and nutrient levels, combined with high levels of ultraviolet (UV) radiation mean that the atmosphere is inhospitable to microbial life [1] . The huge volume of air outdoors compared to air indoors also helps to dilute the concentration of microbes and reduce the level of exposure. Nevertheless, we need to ask: are pathogens in outdoor air a health issue at present or will they become problematic in the future? The multidisciplinary working group, AirPath was organised to review and discuss the problem of pathogens. Four two-day meetings over a period of 18 months from July 2007 have generated contributions from more than 30 participants, over 20 oral presentations, and various round-table discussions, which were recorded on DVD. In order to summarise the wealth of knowledge contributed by the multidisciplinary group, the panel (the authors of the present paper) has arranged the discussion into five themes: 1. What is the source of human pathogens in outdoor air? 2. What current, developing and future techniques do we need? 3.
Can we identify at-risk groups in relation to their activities and environments? 4. How do we prepare for the anticipated challenges of environmental change and new and emerging diseases? and 5. How can we control for and prevent pathogens in outdoor environments? Participants were selected on the basis of their involvement, for example, as president or executive committee members of related professional and academic bodies such as the British Aerobiology Federation, the Aerosol Society and the International Association of Aerobiology; their work in related UK organisations, such as the Health Protection Agency (HPA), hospitals and Defence Science and Technology Laboratory (DSTL); or their research in relevant fields such as epidemiology, meteorology, geoinformatics, and natural resource management. Researchers outside the UK were invited to give an international dimension and network of collaboration to the UK participants. Rather than focus on a narrow topic, the aim of the AirPath working group is to explore the complex and multidisciplinary facets of research connected with the outdoor environment and human pathogens in air, analyse their potential implications, and investigate applications of this research for the well being of society.
At the first two meetings, participants were given a topic and asked to prepare a one-page literature review to support their presentations; each topic was assigned to two participants from different disciplines in order to ensure cross-disciplinarity of the discussion. These presentations now form the core of our review, and all the discussions from all the meetings were recorded. We have conducted an extended literature search and review to ensure that the content is representative, comprehensive and connected to the five main areas arranged below.
Participants discussed four main areas that are known to contribute pathogens to the outdoor air and have proved to be linked with human health -1. Natural environments 2. Engineering environments 3. Agriculture and 4. Waste treatment ( Figure 1 ). As shown in Figure 1 , air pathogens from environmental sources are diverse in terms of type of source and aerosolisation factors, and it is moreover possible that some pathogens are under-reported because a number of them cause similar respiratory symptoms, e.g. coughing and sneezing [2, 3] . Because there are numerous types of pathogens released to the outdoors and given the publishing constraints, the list of pathogens from different sources can be found in the cited references in Figure  1 . In addition, although many environmental pathogens are restricted to limited geographic areas, it may be naïve to assume that pathogens previously restricted to specific locations will not shift with impending global climate change; or we may find that our current knowledge is based on inadequate data and we have yet to discover the actual distribution of a number of these pathogens [2, 4] . The pathogens cited in the references may not yet be relevant to the UK, but could pose a future threat due, for example, to climate change. Moreover, various pathogens can potentially be released from waste treatment facilities; how will the mounting levels of composted green waste, food waste and other traditional landfill materials, as well as increasing bio-solid applications to land, impact on the pathogens in air?
What types and how many pathogens are we exposed to? Is our environment changing and impacting on our health? To answer these questions, we require various techniques to support our research, such as sampling, detection and identification, monitoring, transport models, and laboratory experimentation (Table 1) . Kuske reviewed the current and emerging technologies for the study of bacteria in outdoor air in 2006 [5] . We searched the literature from 2006 onwards to extend Kuske's review and paid close attention to the study of viruses. Some studies show that climatological factors can influence viral disease transmission, e.g. respiratory syncytial virus [6] . The environmental sampling of human viruses is therefore an area that we regard as important in order to prepare for new and emerging diseases in our time. Table 1 highlights the diverse skills and knowledge outside the traditional microbiology and aerobiology fields, which can be adopted to understand pathogens in the outdoor air. Kuske's review [5] contributes largely with regard to sampling and detection and identification in Table 1 . Our review has added, for example, the spatial and temporal monitoring issues, epidemiology and computational fluid dynamic models, and the application of laboratory experimental facilities. Moreover, a variety of examples were given on the study of viruses in the air.
Environmental sources of pathogens in outdoor air Figure 1 Environmental sources of pathogens in outdoor air.
Engineering environments Waste treatment Agriculture Urban environments [12, 16] Ventilation & air conditioning systems [8] Water/Soil/ Biosphere [16] Desert storms [14] Landfills [15] Composting [10, 15] Flooded buildings [13] Sewage treatment plants [7] Disposal of biosolids [17] Crop and livestock production [9, 15] Land application of biosolids [17] Can we identify at-risk groups in relation to their activities and environments? It is important to better understand the risk factors associated with the outdoor air transmission of infections, but the best approach is not always straightforward. Epidemiology is the study of the link between exposure, outcome and confounders, but the measurement of exposure to outdoor pathogens in the air is difficult, as it is problematic to conduct controlled laboratory experiments, for example, to expose people to pathogens within the laboratory. In addition, the technology to obtain accurate organism counts may not yet be available. Moreover, it will be a challenge to evaluate the possible environmental influences such as climatic conditions and proximity to a source, when we assess exposure levels, especially retrospectively [6] [7] [8] . Furthermore, it is not always clear how the outcomes should be measured [9, 10] . Many respiratory infections do not have a definitive causal organism [3] . Infections may be asymptomatic and most infections have numerous subtypes [3, 8] . Age, gender, social class, health, exposure to pollution, and a variety of other factors are the potential confounders that must be addressed in future epidemiology studies.
From the AirPath point of view, to carry out an exposure assessment -that is, to estimate and measure the amount of infectious pathogens entering our bodies through inhalation -is already a complex and multidisciplinary science without the added component of using exposure data and disease outcomes to predict risk factors. We think that Air-Path has contributed towards forming a technical network, as shown in the output in Table 1 , as well as a medical network to further clarify a comprehensive and systematic exposure assessment. 
Sampling Sampling of bioaerosols (bacteria and fungi) is widely reviewed [5] . Bioaerosols can be collected on various media depending on the type of microbial detection, and can be collected according to their size to estimate their deposition on the respiratory system. All of these sampling techniques have pros and cons regarding the issues of size separation, sampling volume and time, biological recovery, and particle removal efficiency as well as the choice of subsequent analytical and detection methods. The sampling and quantification of viruses is less widely studied. One recent study has developed methods for airborne influenza and avian influenza virus, which is currently one of the biggest concerns of public health [11] .
Detection and identification of pathogens has changed since the development of different molecular methods and innovative approaches other than culture methods [5] . The existing detection methods can be divided into two levels: generic and specific. Generic detection gives information about whether the particles are biological materials, microbes or living cells, e.g. bioluminescent measurement of ATP using continuous flow luminometer and mass-spectrometry. Specific methods such as micro-arrary and immuno-assays can tell us what kind of microbes are detected and identified. Other new techniques have been proposed for bio-detection, for instance, by characterising the size and shape of bioaerosols, pollens and fungal spores under microscope [18] and analysing fluorescence spectrum of bacteria [5] .
It is widely recognised that background biological and chemical materials and their continuous environmental fluctuation will significantly influence monitoring. Air movement, sunlight/UV radiation, humidity, rainfall, and inversions are some of the environmental factors that need to be considered during monitoring. Another consideration is where and when to sample with regard to spatial and temporal relevance [19] . For example, the release of pathogens can cause a significant downwind hazard which requires a wide area and long period of sampling [7] .
Transport/Transmission models Epidemiology studies can link disease cases together and develop a disease transport and transmission model [8] .
However, it will not always explain the mechanism. Moreover, it requires a significant number of cases in order to develop a model. The use of computational fluid dynamic (CFD) models and tracer gas simulation has demonstrated that the Severe Acute Respiratory Syndrome (SARS) virus can travel and disperse outdoors through air, and became a source of pathogens to other indoor environments [12] . A similar technique has been used in the modelling of aerosols and chemical pollutants in streets, waste treatment facilities, and other pathogen sources outdoors [7] .
Because pathogens travel in air, it is inevitable that the biological activity will be influenced by the environment. Data can be collected from field studies to determine the impact of environments on the fate and behaviour of pathogens. However, since the pathogens and environment vary and fluctuate frequently, it is not easy to build this scientific link using field data alone. Some studies have investigated the viability and environmental limits of airborne viruses and bacteria using a rotating drum and controlled climate environmental chambers [20] .
Studies from both field and laboratory settings indicate that environments and environmental factors can significantly impact on the fate and behaviour of bioaerosols and health risk. It is generally recognised that the environment is constantly changing, either physically, climatically, socially, or a combination of all three. Not only is the environment ever changing, but the types of diseases are also changing. The presence of new and emerging diseases is one of the most urgent threats to humanity across the globe [3, 11, 12] . Climate change is a high priority issue that everyone is facing; and it may be that environmental pathogens will respond to climate change as well. Climate change not only affects the pathogens in air, but also their source, source strength and aerosolisation mechanisms (e.g. through extreme weather conditions) [13, 14] ; these are fairly unexplored at present.
New and emerging diseases are a major health concern because we do not know much about them. We do not yet know how best to prevent and control them and, most importantly, we often do not know how to treat them, but it is likely that genetics and the state of the immune system of the global population plays a key role in disease prevalence [2] . With advances in medical treatment, greater numbers of disease-susceptible groups, such as immuno-compromised individuals are expected to survive longer in the overall population. This trend may change our understanding of pathogens because many unexpected microbes and infection pathways can appear within this transforming population.
Once pathogens are in the air, one of the few things we can do to minimise the health risk is to source control (reduce/eliminate the source and its strength and the aerosolisation factors, as well as potential dispersal interception near the source) [15] . Modelling the movement of bioaerosols in order to advise people to prepare or even evacuate is another option for reducing the health risk [10] . Although the physical properties and transport of aerosols can be predicted in most environments, it is not always known how biological properties change in the aerosol dynamics pathway and during outdoor transport (e.g. aggregation, scavenging and deposition). Source control is not always possible, for instance, if the sources of pathogens are unknown. The longer it takes to identify the source, the higher the risk it poses to people.
Because of the differences in the type of sources, the nature of the pathogens and their geographical prevalence, various research and policy approaches have been taken by different regulatory bodies. Using Legionellosis as an example, guidelines and regulations are available from various government agencies, cooling tower manufacturers and industrial trade organisations to control and prevent the growth and dispersal of these particular pathogens. Although guidelines and regulations are widely known and implemented, outbreaks of Legionnaire's disease are not uncommon. Some studies report that the pathogens can travel up to 12 km from the suspected source and still cause infections [8] , thus indicating a more complicated relationship between source and disease transmission than was previously understood.
There is a significant knowledge gap in understanding and assessing the risk of outdoor pathogens in air. In order to complete the transmission pathway and set up regulation, control and prevention measures, we need to better understand and identify pathogens at their source, identify the aerosolisation mechanisms and dispersal plume, have adequate qualitative and quantitative techniques to detect the pathogens in different media, and understand the deposition process and required dose for infection. As in Skyes et al.'s analysis, a risk assessment framework was used to assess the potential health risks from bioaerosols from composting [10] . Current knowledge of pathogen risk assessment in air is far from complete; without knowing the infectious dose and actual risk to society, there is little motivation on the part of governments or manufacturers to research methods of control.
In our complex, changing world, some environmental health problems are not straightforward to identify until a very serious health impact occurs. When health problems do emerge, we may only have a very short period of time and opportunity in which to react, identify and restore the damaged natural environment. As a result, we strongly assert the necessity to understand the environment and its relationship with pathogens in air before health hazards associated with the relevant pathogens can appear. In the present paper we have briefly reviewed and presented our views on various issues. Our next step is to encourage and support focused multidisciplinary research in order to fill the missing knowledge gaps and translate research into practice and policy. Combination strategies for pandemic influenza response - a systematic review of mathematical modeling studies BACKGROUND: Individual strategies in pandemic preparedness plans may not reduce the impact of an influenza pandemic. METHODS: We searched modeling publications through PubMed and associated references from 1990 to 30 September 2009. Inclusion criteria were modeling papers quantifying the effectiveness of combination strategies, both pharmaceutical and non-pharmaceutical. RESULTS: Nineteen modeling papers on combination strategies were selected. Four studies examined combination strategies on a global scale, 14 on single countries, and one on a small community. Stochastic individual-based modeling was used in nine studies, stochastic meta-population modeling in five, and deterministic compartmental modeling in another five. As part of combination strategies, vaccination was explored in eight studies, antiviral prophylaxis and/or treatment in 16, area or household quarantine in eight, case isolation in six, social distancing measures in 10 and air travel restriction in six studies. Two studies suggested a high probability of successful influenza epicenter containment with combination strategies under favorable conditions. During a pandemic, combination strategies delayed spread, reduced overall number of cases, and delayed and reduced peak attack rate more than individual strategies. Combination strategies remained effective at high reproductive numbers compared with single strategy. Global cooperative strategies, including redistribution of antiviral drugs, were effective in reducing the global impact and attack rates of pandemic influenza. CONCLUSION: Combination strategies increase the effectiveness of individual strategies. They include pharmaceutical (antiviral agents, antibiotics and vaccines) and non-pharmaceutical interventions (case isolation, quarantine, personal hygiene measures, social distancing and travel restriction). Local epidemiological and modeling studies are needed to validate efficacy and feasibility. Many countries have developed pandemic preparedness plans in response to the threat from pandemic influenza [1] , to attempt containment of the virus or to reduce the pandemic's impact. The influenza A (H1N1-2009) pandemic has underscored the importance of such plans, with the World Health Organization (WHO) calling for the activation of pandemic plans worldwide [2] . Although the WHO has made public guidelines for developing pandemic plans [3] , the comprehensiveness and standards of pandemic plans differ widely across different countries and continents [4] [5] [6] . To ensure the success of these plans, it is necessary to adopt a combination of different strategies.
Although there are existing historical data on the possible success of strategies used in previous pandemics such as personal hygiene, school and workplace closures, and social distancing, these are often anecdotal and difficult to interpret [7, 8] . Mathematical models provide a platform for the assessment of multiple interventions in an environment where individual parameters can be altered. The recent increase in mathematical modeling studies on pandemic interventions suggests the effectiveness of these strategies and provides guidance for policy makers. Although the 2009 pandemic has spread rapidly, these combination strategies can be applied in populations yet to be severely affected, for the second wave, or for the next pandemic [9, 10] . This systematic review aims to determine the individual components that constitute combination strategies, and the quantitative impact of these combination strategies in reducing pandemic spread and morbidity.
This study explored available mathematical modeling publications on the effectiveness of combination strategies for an influenza pandemic. To obtain papers on the effectiveness of combination strategies, data for this review were identified by the authors through searches of the PubMed search engine for English language articles and articles translated into the English language. The authors used the following search terms to focus on modeling studies, and those which had a focus on pandemic preparedness and strategies -influenza and pandemic and (preparedness or strateg* or model*); influenza and modeling or modelling. The search included all published articles listed on PubMed from 1990 to 30 September 2009there were few articles on influenza pandemic planning or modeling before this period.
Abstracts were reviewed where available to determine if a study met the inclusion criteria and the full manuscript was obtained for further scrutiny. Snowball searches by hand were performed on the reference lists of articles meeting the inclusion criteria to find additional studies.
The inclusion criteria were primary mathematical modeling papers that compared and reported the quantitative effectiveness of combination strategies (two or more strategies used together) versus individual strategies for human pandemic influenza. Mathematical modeling papers were those which used quantitative predictive methods to determine the likely impact of strategies, and had descriptions of these methods which could be reproduced or verified. All influenza preparedness strategies were considered, including pharmaceutical and nonpharmaceutical public health strategies. These articles would allow clear comparison on the advantages of combination strategies over and above the impact of individual strategies. An explanation of some of the key strategies are found in the appendix.
Mathematical modeling articles that described the effectiveness of multiple singular strategies but did not analyze the quantitative effect of combination strategies were excluded. Articles that referred to general pandemic preparedness without quantitative evidence, or provided only qualitative discussion were also excluded. Reviews without primary data, articles in abstracts without full publication, and unpublished studies were excluded as their methodology and results could not be verified.
Mathematical models are based on input variables which are assumptions made based on available evidence in specific scenarios. One important assumption is the reproductive number (Ro), which is the average number of secondary infections generated by a single case in a completely susceptible population. No attempt was made in this review to homogenize data across studies for comparison; on the contrary, the heterogeneity of data provides public health professionals with evidence of the effectiveness of strategies across a wide range of assumptions and scenarios. We have instead listed the different types of models used, and the scenarios, interventions, and countries where they were applied.
The search yielded a total of 1,920 papers including overlaps. Of these, 162 used mathematical modeling techniques and on closer review, 144 were excluded based on the exclusion criteria listed in Methods. The remaining 18 studies were included for analysis, together with one additional study identified from the snowball searches ( Figure  1 ). The selected modeling papers that show the effectiveness of combination strategies in increasing the impact of individual strategies are listed in Additional files 1 and 2 [11] . The following sections highlight key findings on the effectiveness of combination strategies in these modeling studies on pandemic influenza.
Zoonotic influenza such as H5N1 influenza is endemic in several countries, and there is interest in containing a highly virulent pandemic at the earliest sign of localized efficient human-to-human transmission. Two key modeling studies suggested a high probability of success for rapid containment of an influenza epicenter with combination strategies under favorable conditions [9, 10] . These studies formed the basis for the epicenter containment strategies recommended by the WHO. Longini showed that antiviral prophylaxis alone could contain a pandemic influenza virus with reproductive number (Ro) less than 1.7; while 70% household quarantine alone was effective up to Ro of 1.7. A combination of quarantine and antiviral prophylaxis was effective up to Ro of 2.1; while a combination of pre-pandemic vaccination, household quarantine and antiviral prophylaxis was effective for Ro of 2.4 [9] . Ferguson found that antiviral prophylaxis for contacts only would have a 90% chance of containing a virus with a Ro less than 1.25, while antiviral prophylaxis for contacts and all individuals in a 10 km zone would have a 90% chance with Ro less than 1.7 [10] . Combined anti-viral prophylaxis and either school and workplace closures or area quarantine provided a similar chance of containment with Ro of 1.7 to 1.8, while a combination of all three strategies would contain a virus with Ro of 1.9 and allow for greater initial surveillance errors [10] .
Combination strategies can be used to reduce the global spread of the influenza virus [12, 13] . Redistribution of limited antiviral drugs can help contain pandemics or reduce the global attack rate (AR) [12] . If global antiviral stockpiles are limited, non-cooperative strategies where countries keep their antiviral stockpiles for their own use can only contain a pandemic influenza virus with Ro less than 1.5; in contrast, if redistribution of 25% of stockpiles from countries that have them to countries that do not, a pandemic with Ro up to 1.9 may be contained, and overall AR reduced by 25% at higher Ro [12] .
Another example of combination strategy is reduction of pandemic spread through air travel. Suspension of 99.9% of air travel can only delay individual national epidemics by up to four months, while a combination of local strategies reducing influenza transmission by 40% can delay pandemic spread by up to 10 months [13] . A combination of vaccination and travel restrictions may delay epidemic growth, allowing vaccination of susceptible individuals [14] . With a pandemic starting in July in Asia, the number of United States (US) metropolitan cases was 102.4 million -0.1% daily vaccination alone reduced this to 73.0 million, and vaccination together with travel restriction reduced this to 56.9 million [14] .
Combination strategies may have substantial impact in reducing the global spread of resistant viruses. For example, if the probability of emergence of anti-viral drug resistance was 1%, antiviral monotherapy was associated with overall AR of 67% and resistant AR (RAR) of 38% [15] . In contrast, early combination chemotherapy was associated with reduced AR of 58% and RAR of 2%, while sequential multi-drug chemotherapy was associated with AR of 57% and RAR of 3%.
During the pandemic, several studies found that combination strategies delayed the spread of the virus, reduced the overall number of cases, and delayed and reduced the peak AR much more than individual strategies which may be ineffective if used alone [16] [17] [18] [19] .
A study using individual-based modelling in the United Kingdom and United States examined the effects of antiviral treatment and prophylaxis, vaccination, case isolation, household quarantine, school and workplace closure and travel restrictions in pandemics with Ro of 1.7 to 2.0. It found that external or internal travel restrictions alone would delay spread by two to three weeks only if more than 99% effective [16] . Reactive school and work-Flow diagram for selection of combination strategy modeling studies Figure 1 Flow diagram for selection of combination strategy modeling studies.
place closures alone did not impact on overall AR, but reduced peak AR by about 40%; antiviral treatment and prophylaxis within the household reduced overall AR by 35% and peak AR by 45%.; while household quarantine alone reduced overall AR by 10% and peak AR by 20%. Combination antiviral treatment and prophylaxis, and household quarantine reduced overall AR by 40% and peak AR by 60%. Combination school and workplace closure, antiviral treatment and prophylaxis, and household quarantine reduced overall AR by more than 60% and peak AR by more than 80%. Combination antiviral treatment and prophylaxis, school closure and 20% pre-pandemic vaccination reduced overall AR by more than 60% and peak AR by more than 75%. Combination antiviral treatment and prophylaxis, household quarantine, school and workplace closure, and effective border control reduced overall AR by more than 70% and peak AR by more than 90% [16] .
Similarly, another individual-based stochastic simulation model in Chicago evaluating the effects of antiviral treatment and prophylaxis, quarantine, isolation, school closure, community and workplace social distancing showed that social distancing alone may reduce overall AR by 60% for pandemic Ro of 1.9 but combination antiviral treatment and prophylaxis, quarantine, social distancing, and school closure could reduce overall AR by more than 90% for similar pandemic Ro of 1.9 [17] .
Another study in France examined the effects of antiviral treatment and household prophylaxis, vaccination, household quarantine, school and workplace closure at the individual and community level [20] . Treatment only with anti-viral drugs did not affect AR substantially. Antiviral prophylaxis of 90% of household contacts reduced AR by 50%. Vaccination of 70% of the population within one day reduced AR by 80%. A combination of antiviral treatment and prophylaxis, and household quarantine reduced AR by 90% [20] .
An Australian individual-based stochastic simulation model assessed the effects of non-pharmacological pandemic mitigation measures of case isolation, school closure, workplace non-attendance and community contact reduction [21] . For a pandemic with Ro of two, school closures alone reduced AR by 20%, case isolation by 40%, workplace non-attendance by 15%, and social distancing by 25%. In contrast, combination of all these measures reduced AR by more than 95% [21] . A deterministic compartment model using InfluSim based on a small community of 100,000 population assessing the effects of antiviral treatment, case isolation and social distancing showed that case isolation and social distancing could reduce overall AR by 25%, and antiviral treatment alone by 20%, compared with a reduction of 40% with a combination of case isolation, social distancing and antiviral treatment [18] . The triple combination strategy could delay the peak by one month compared with 10 days for the first two strategies [18] .
Another study using a deterministic model with a stochastic simulation component based on Italy examined the effects of household antiviral prophylaxis, pre-pandemic vaccination, and social distancing via closure of all schools, public offices and public meeting places [22] . In a pandemic with an attack rate of 35%, vaccination alone reduced AR by up to 10% even at vaccine efficacy levels of 70%; antiviral prophylaxis alone for even the entire pandemic duration reduced AR by up to 6% only; and social distancing alone reduced AR by less than 1%. However, a combination of all three measures reduced AR by up to 30% [22] .
The relative success of interventions depends on the transmissibility of the pandemic, which is commonly reflected in the Ro. In an influenza pandemic with higher Ro, the effectiveness of interventions is reduced and individual interventions are commonly ineffective. However, across most scenarios, combination strategies maintain some effectiveness as shown clearly in the studies on containment by Longini [9] and Ferguson [10] .
A stochastic agent-based discrete-time simulation model in the United States examining the effect of antiviral prophylaxis, vaccination, school closure and travel restriction found that for a pandemic influenza virus with Ro of 2.4, unlimited antiviral prophylaxis and best vaccination program may reduce cases by 64% and 34% respectively, while school closure within seven days of pandemic onset may reduce cases by 14%, social distancing within seven days by 6%, and travel restrictions exceeding 90% was ineffective [19] . However, a combination strategy of all of these measures may reduce cases by 99.8% [19] . The effectiveness of any strategy in delaying the pandemic or reducing the AR is highly dependent on the Ro. For example, for a pandemic with Ro of 1.6, individual strategies of prophylaxis, vaccination, or school closures had very high effectiveness [19] . However, once the Ro increased beyond 2.0 (which is similar to the Ro for the 1918 pandemic), individual strategies were much less effective, whereas combination strategies still maintained effectiveness across a range of Ro.
An individual-based model in Italy assessing the effects of household, school and workplace antiviral prophylaxis, vaccination, international air travel restriction, social distancing via school closure and closure of some public offices showed that without any interventions, importation of pandemic influenza would occur 37 to 77 days after the first case elsewhere in the world. Air travel restric-tion would delay introduction by one week to one month. For a pandemic with Ro of 1.7, travel restriction and social distancing did not affect overall AR, household prophylaxis reduced AR by 50%, and vaccination reduced AR by 0 to 40%. A combination of antiviral prophylaxis, social distancing, vaccination, and travel restriction reduced AR by more than 90% [23] . For a pandemic with Ro of 2.0, travel restriction in fact increased overall AR by 1% and peak AR by 20%. Household prophylaxis reduced AR by 35%, while vaccination reduced AR by 0 to 30%. A combination of antiviral prophylaxis, social distancing, vaccination, and travel restriction reduced AR by 80%.
An individual-based stochastic model in Hong Kong looking at the effects of antiviral prophylaxis, case isolation and household quarantine reported that in a pandemic with Ro of 1.8 and AR of 74%, household quarantine could reduce AR to 49%; household quarantine and isolation to 43%; household quarantine with anti-viral prophylaxis to 44%; household quarantine, isolation and antiviral prophylaxis to 40% which was recommended. Although adding contact tracing and quarantine of all contacts to the latter combination strategy reduced AR to 34%, the number of people under quarantine would be excessive. Therefore, contact tracing was not recommended [24] .
Another study examining the effects of antiviral treatment and prophylaxis, home quarantine and social distancing based on a community of a million population with the assumption that pandemic influenza was introduced by an undetected airline passenger, found that if a pandemic Ro was 3.0, individual interventions would result in increased transmission while combination measures may break community transmission [25] . This was similarly shown by Ciofi and colleagues for a pandemic with Ro of 2.0 [23] . A deterministic compartmental model evaluating the effects of antiviral treatment and prophylaxis, vaccination, case isolation and air traffic reduction globally demonstrated that individual strategies such as case isolation and air travel restrictions may result in higher peak AR even though overall AR could be reduced [26] .
A study in Taiwan evaluated the effects of enhanced ventilation, use of respiratory mask and vaccination on pandemic influenza transmission in a school [27] . Vaccination alone of 80% of children was effective in preventing the spread of the virus but this was only if a suitable vaccine was available, which is often not the situation. A combination of masks and ventilation, or a combination of vaccination and masks achieved similar effectiveness [27] .
Many modeling studies were performed as a result of H5N1 influenza threat and an impending pandemic, but all have used parameters based on historical pandemics and existing studies on the influenza transmission. In addition, these studies provided sensitivity analyses across a wide range of influenza parameters. As such, they are directly relevant to the 2009 influenza pandemic which has an Ro of between 1.2 to 1.6 [28] , similar to the 1957 and 1968 influenza pandemic [16] , and for future pandemics. At the same time, the 2009 influenza pandemic provides the opportunity to study unknown variables to validate and refine these models.
All of these modeling studies in various settings, and using different models and assumptions, consistently show that combination strategies are more effective compared to individual strategies. Given the lack of good experimental, observation or controlled studies on these strategies, and the difficulties of performing trials during a pandemic, it is difficult for policy makers to know the effectiveness of their policies. These modeling studies provide policy makers with a suggestion of the effectiveness of different combination strategies. At the same time, new models will have to be developed using local data to provide realistic outcomes for local settings. The diverse methodology available from these studies provides sufficient information for countries to build and validate their results locally.
Although the use of individual-based and other stochastic models provide better data resolution, deterministic models mentioned in this review show similar outcomes [18, 22, 23, 27] . These deterministic or simple stochastic compartmental models are much easier to build and may provide rapid results for policy making. This is especially true in countries where the vast amounts of data required for individual-based and complex stochastic models may not be available compared with high-income countries where most sophisticated models were built.
The use of combination strategies necessitates the availability of resources and feasibility for each individual component. For example, stockpiling of pharmaceutical agents is an integral part of preparedness plans and currently widely adopted in well-resourced countries. The increase in anti-viral drug resistance underscores the importance of combination drug use and provides policy makers with recommendations for their stockpiles [15] . Combination stockpiles of sufficient amounts of different antiviral drugs such as oseltamivir, zanamivir and adamantanes will allow for early combination chemotherapy or sequential multidrug therapy which was modeled to be effective against antiviral resistance when a small secondary stockpile was used to augment a primary stockpile [15] . The United States Federal stockpile is composed of 80% oseltamivir and 20% zanamivir, and several million doses of rimantadine from previous stockpiles [29] . The United Kingdom has purchased additional antiviral drugs to ensure it has a total stockpile for 50% of its population, comprising 68% oseltamivir and 32% zanamivir [30] . Bacterial pneumonia results in substantial morbidity and mortality among pandemic influenza cases [31, 32] . Antibiotics should therefore be considered for stockpiling [31] . Stockpiles should take into account common locally circulating bacteria, and recommended amounts range from 10 to 25% of the population [33] . In contrast to antiviral drugs that are not widely used, antibiotics can be part of a rolling stockpile which ensures sufficient stockpiles without expiry issues. Vaccination against bacterial infections should likewise be considered.
From the effectiveness of combination strategies in reducing global spread of influenza or resistant viruses [12] [13] [14] [15] , resource-rich countries should consider redistributing their resources for the greater global benefit and their own benefit if they have yet to be affected by the pandemic. Controlling local outbreaks through combination strategies can reduce global spread, and countries affected early during the pandemic should be provided with assistance [13] .
Vaccines are part of many combination strategies and modeling has shown that introduction of a vaccine four months after the pandemic virus has arrived has limited effectiveness, while stockpiling prototype pandemic vaccines could reduce overall AR [16] . Therefore countries were stockpiling H5N1 vaccines as candidate pandemic vaccines [34, 35] . However, if the pandemic influenza virus is totally different from the vaccine virus, the vaccines would be of negligible effectiveness. Investments are needed to develop new vaccines with greater cross-protection against conserved viral regions; vaccine libraries to quickly produce candidate vaccines; better adjuvants and antigen-sparing strategies to increase production capacity; and modes of administration for improved immunogenicity and cross-protection [36, 37] .
Although some individual strategies may seem very effective, they may not be feasible and models assist policy makers in avoiding potentially disastrous decisions. Social distancing has been widely used in epidemics [7] but their impact remains unclear and highly dependent on disease severity, transmission, and risk groups affected. Local interventions such as school closures may be effective if done early, decisively, and for prolonged periods [20, [38] [39] [40] . A United Kingdom model based on a 1957like pandemic showed more than 20% case reduction if the Ro were low (<2) and schools were closed early, but less than 10% case reduction in pandemics with high Ro [38] . A French study showed that prolonged closure and limiting contact among children outside school may reduce cases by 17% and peak AR by 45% [39] . However, school closures and limiting social contact may be socioeconomically difficult to achieve. Another study found that total closure of schools and workplaces reduced AR by 95%. However, the socio-economic impact would be unimaginable [20] . Similarly, most modeling studies found that travel restrictions alone did not impact overall AR [13, 16, 19, 23] . Reducing air travel has been modeled to be effective in delaying pandemic spread if nearly 100% reduction can be achieved [13, 16] , and will be difficult if not impossible to achieve [41] . If used alone, local epidemic severity may increase because restriction-induced travel delays can push local outbreaks into high epidemic season [14] .
Although combination strategies are more effective than individual measures, not all combination strategies may be feasible. Active surveillance, isolation of cases, and quarantine of close contacts are important interventions during epicenter containment. These interventions may reduce the Ro of the disease to below one and contain the outbreak. However, it is often difficult to ensure total compliance with these measures and if used alone, will result in missed cases due to surveillance failures, isolation facility exposures, and quarantine failures as shown in the SARS experience [42] . A Hong Kong modeling study found that although contact tracing and quarantine of all contacts was effective, it was not feasible because the number of people under quarantine would be excessive [24] . Therefore combination strategies enable policy makers to leverage on the effectiveness of some measures and reduce potential negative impact of others.
For combination strategies to work, they have to be tailored for each scenario at organizational, community, national, and international levels. To facilitate integration of interventions into effective combination strategies, more evidence is needed through targeted research, for example, the effectiveness of non-pharmaceutical interventions (e.g. personnel cohorting, school closures or reduction in air travel). In the absence of definitive studies, mathematical modeling studies provide an effective means of assessing the effectiveness of these strategies.
A limitation of this study is the restriction of our searches to the PubMed database. While we have made attempts to include additional articles from snowball searches, there is the potential for other published or unpublished studies to be missed from other databases and private sources. Other intrinsic limitations of modeling studies exist, and include the fact that they are based on theoretical epidemiology and not fully based on clinical or epidemiological evidence. For example, widespread use of pandemic vaccines raises safety concerns, and widespread use of antiviral drugs raises concern for antiviral resistance. Viral transmission during treatment with anti-viral drugs is also not well understood. It is therefore important to perform clinical and epidemiological studies during pandemic or seasonal influenza to understand the effectiveness and impact of these interventions. Models are also highly dependent on the assumptions and input variables, and are specific for a local context. However, if these limitations are understood by decision makers, modeling provides a reflection of the possible outcomes, helps to delineate possible strategies for inclusion, and avoids costly errors.
Modeling studies show that combination strategies increase the effectiveness of individual strategies, guard against individual failures, and may reduce socio-economic impact. In the initial phases of an influenza pandemic, combination strategies provide the opportunity to contain the novel virus or delay its spread, allowing unaffected areas within a country and other countries to activate preventive strategies. During a pandemic, combination strategies allow for different strategies to have synergistic effect in reducing the impact of pandemic influenza, and the socio-economic impact of individual interventions. Finally, combination strategies protect against failure of individual interventions and should be considered in preparedness plans.  Let the sun shine in: effects of ultraviolet radiation on invasive pneumococcal disease risk in Philadelphia, Pennsylvania BACKGROUND: Streptococcus pneumoniae is a common cause of community acquired pneumonia and bacteremia. Excess wintertime mortality related to pneumonia has been noted for over a century, but the seasonality of invasive pneumococcal disease (IPD) has been described relatively recently and is poorly understood. Improved understanding of environmental influence on disease seasonality has taken on new urgency due to global climate change. METHODS: We evaluated 602 cases of IPD reported in Philadelphia County, Pennsylvania, from 2002 to 2007. Poisson regression models incorporating seasonal smoothers were used to identify associations between weekly weather patterns and case counts. Associations between acute (day-to-day) environmental fluctuations and IPD occurrence were evaluated using a case-crossover approach. Effect modification across age and sex strata was explored, and meta-regression models were created using stratum-specific estimates for effect. RESULTS: IPD incidence was greatest in the wintertime, and spectral decomposition revealed a peak at 51.0 weeks, consistent with annual periodicity. After adjustment for seasonality, yearly increases in reporting, and temperature, weekly incidence was found to be associated with clear-sky UV index (IRR per unit increase in index: 0.70 [95% CI 0.54-0.91]). The effect of UV index was highest among young strata and decreased with age. At shorter time scales, only an association with increases in ambient sulphur oxides was linked to disease risk (OR for highest tertile of exposure 0.75, 95% CI 0.60 to 0.93). CONCLUSION: We confirmed the wintertime predominance of IPD in a major urban center. The major predictor of IPD in Philadelphia is extended periods of low UV radiation, which may explain observed wintertime seasonality. The mechanism of action of diminished light exposure on disease occurrence may be due to direct effects on pathogen survival or host immune function via altered 1,25-(OH)(2)-vitamin-D metabolism. These findings may suggest less diminution in future IPD risk with climate change than would be expected if wintertime seasonality was driven by temperature. Many infectious diseases of public health importance exhibit predictable periodicity, with major increases in incidence during a specific season of the year [1] . Empirical evidence of such "seasonality" has been noted by physicians for centuries, and has been prominent enough to become a part of our vernacular (e.g., "cold and flu season") [1, 2] . Despite this wealth of experiential evidence, the mechanisms underlying seasonality are poorly understood, especially diseases characterized by person-to-person transmission [1] .
Invasive bacterial disease due to Streptococcus pneumoniae and other respiratory pathogens exhibits striking seasonality in its occurrence [3] [4] [5] [6] [7] . Pneumococcal infections are a common cause of severe, community-acquired illnesses, including community-acquired pneumonia requiring hospitalization, bacteremia, and meningitis [8] . While the introduction of antibiotics dramatically reduced the case fatality rate (CFR) for pneumococcal disease, the current CFR for bacteremic pneumococcal disease is still estimated at 5-10% in the United States, and may be twice as high among the elderly and in cases of meningitis [9, 10] . The emergence of antimicrobial resistance to beta-lactam agents, macrolides, and other antibiotic classes is an important clinical concern [11] . Although the introduction of conjugate pneumococcal vaccines has been associated with a reduction in disease incidence [12] , the recent increase in invasive infection by non-vaccine serotypes [13] , which may be highly resistant to commonly-used antimicrobials [14] , suggests that this microorganism will persist in challenging both the medical and public health communities.
The incidence of IPD peaks in the winter months, with annual periodicity [4, 5] , but the forces that drive this characteristic seasonality are unknown. Wintertime seasonality of communicable respiratory diseases are often assumed to be driven by seasonal changes in environmental conditions (e.g., diminished ultraviolet radiation (UV) exposure, decreased temperature) but such associations may be confounded by other seasonally varying factors, including population behaviours (e.g., clustering indoors), co-occurrence of other infections (e.g., influenza) [4] , and frequency of laboratory testing [15] .
A more thorough understanding of the effect of environmental factors on seasonal IPD incidence could offer significant insight into pathogenesis, improve disease forecasting, and help determine the likely direction of pneumococcal disease incidence in the face of global climate change [16] . Our objective was to investigate how environmental factors influence IPD occurrence in Philadelphia County. We used both traditional analytic methods (i.e., Poisson regression with seasonal smoothers) and a novel case-crossover method to examine the effects of acute weather fluctuations on IPD occurrence. Both methods reduce confounding by environmental, behavioural, and infectious influences that might otherwise distort the observed magnitude of environmental effects on disease risk.
Philadelphia County encompasses an area of 369 km 2 in south-eastern Pennsylvania, and is coterminous with the City of Philadelphia (population 1,517,550 in the year 2000 [17] ). The population receives public-health services from the Philadelphia Department of Public Health (PDPH). IPD has been a notifiable condition in the Commonwealth of Pennsylvania since 2002; Pennsylvania uses the uniform case definition endorsed by the National Notifiable Diseases Surveillance System [18] . A case is considered "confirmed" when a consistent clinical syndrome occurs in association with the isolation of S. pneumoniae from a normally sterile site (e.g. blood, cerebrospinal or pleural fluid). Data on IPD case occurrence in Philadelphia was obtained from PDPH records, and included date of onset, age, sex, race and ethnicity of the patient, isolation site, and fatal outcome (if known).
Meteorological data including temperature, relative humidity, wind speed, atmospheric pressure, and precipitation for the period from 2002 to 2006 was obtained from the weather station at Philadelphia International Airport, located eight kilometres southwest of Philadelphia's city center [19] . Information pertaining to air quality in Philadelphia County during the years of interestincluding concentrations of lead, ozone, sulphur oxides and particulate matter-was obtained from the Environmental Protection Agency [20] . Because daily readings were taken at various locations throughout the region, the arithmetic means of the air quality values were used as exposure variables. UV index forecast estimates for Philadelphia during the same period were retrieved from the National Weather Service Climate Prediction Center [21] . Clear-sky UV indices represent an integral of measured UV radiation levels weighted by the ability of the different UV wavelengths to cause skin erythema. The issued UV index is a similar measure, which accounts for the effect of clouds on radiation transmission; because of inconsistencies in cloud measurement during the study period, we used the clear-sky UV index as our exposure variable.
Rates of invasive pneumococcal disease were calculated using demographic data for Philadelphia County from the year 2000 US Census, as well as 2006 population estimates from the Bureau of the Census, with linear interpolation and extrapolation used to generate estimates for population by age and sex in other years [17] . We evaluated the seasonality of disease occurrence through construction of periodograms and autocorrelograms [15, 22] for weekly case counts. As yearly periodicity was observed, we estimated seasonal and year-on-year trends in IPD occurrence using Poisson regression models that incorporated sine and cosine oscillators, with 52 week (annual) frequencies (i.e., incorporated fast Fourier transforms) [7, 22] .
Using these parameters, the expression for the expected number of case counts for a given week, E (Y) is given by:
where cases is an autoregressive model term reflecting the cumulative case count in the month prior to case occurrence, (i.e., cases = ).
The phase-shift of the composite waveform generated by combining sine and cosine components of the above equation can be approximated as the arctangent of β 2 /β 3 , and can be used to estimate the timing of peak disease incidence [22] . We also included model terms that controlled for longer term trends in incidence of invasive pneumococcal disease, which may have reflected the initiation of surveillance, the introduction of public funding for conjugate pneumococcal vaccination [12] , changes in medical diagnostic practices, or other long-term changes in real or apparent pneumococcal epidemiology. As yearon-year trends in disease occurrence reflected a non-linear increase in disease risk, we used models that incorporated both linear and quadratic yearly terms.
The quadratic model term was statistically significant, but is difficult to interpret, thus we present our final Poisson model with separate linear yearly terms for the period from 2002 to 2003, and the period from 2004 to 2007 [23] . We evaluated the impact of environmental exposures on weekly IPD incidence by incorporating exposure variables into Poisson models both individually, and using a backwards elimination algorithm (with variables retained for P < 0.2 [24] ).
To explore the possibility of effect modification by subject characteristics, we evaluated stratum-specific estimates of effect for age categories and genders. Heterogeneity of effects across strata was assessed using meta-analytic techniques, including both graphical inspection and calculation of meta-analytic Q-statistics [25] . We further explored the sources of between-stratum heterogeneity through construction of meta-regression models that estimate the contribution of group-level covariates to between-stratum variation in effects [25] .
We used a case-crossover approach to evaluate acute (i.e., daily) associations between environmental exposures and IPD occurrence. This approach provides a means for evaluating the association between brief, transient exposures and rare outcomes. The design is characterized by "self matching", in that cases serve as their own controls. In the context of environmental epidemiology, a "case" is a day on which a case occurred, while a "control" is an appropriately selected day on which a case did not occur [6] . We used a time-stratified 2:1 matched case-crossover design in which hazard periods were defined as the reported date of IPD onset from Philadelphia County public health.
Beginning on January 1, 2002 the person-time at risk was divided into three-week time strata. Control periods were chosen by matching the hazard period by day of the week within the stratum, and could both precede, both follow, or straddle the hazard period [26, 27] . Random directionality of control selection was used in order to avoid biases that can occur with unidirectional or uniform bidirectional control selection [26] . The 1-3 day incubation period of S. pneumoniae was used to estimate the lag days between acute environmental occurrence and case onset, or plausible effect period [28] . We also evaluated effects during the period immediately preceding incubation (i.e., 4-6 day lags) to evaluate the possibility that environmental conditions might affect risk via enhanced transmission of S. pneumoniae. We evaluated the effects of both raw environmental exposures, and quantile ranks within time strata through construction of conditional logistic regression models [24] . Analyses were performed using SAS version 8.01 (SAS Institute, Cary, NC) and Stata version 9.1 (Stata Corporation, College Station, TX). Both spectral decomposition and construction of autocorrelograms identified annual periodicity of infection, with peak incidence in mid-February (phase = 6 weeks) (Figures 1 and 2) . Strong statistical evidence for seasonal oscillation was obtained from Poisson regression models (P for seasonal oscillation < 0.001). A significant annual increase in incidence was seen throughout the study period, though this was more marked prior to 2004 (IRR per year 1.34, 95% CI 1.08 to 1.66) than subsequently (IRR 1.22, per year 95% 1.16 to 1.34) (Figure 3 ). We found no clear trends in the incidence of IPD or case-fatality in individual age groups, and no significant heteroge-
neity was detected between age groups with respect to year-on-year trends in incidence or case-fatality (see Additional File 1 and Additional File 2).
In univariable models the risk of IPD increased with several seasonally oscillating environmental exposures, including temperature, humidity, pressure, air pollution, and UV radiation, as shown in Table 2 . Risk of IPD increased with average weekly barometric pressure, sulphur and nitrous oxides, and decreased with average weekly temperature, relative humidity, and UV index. However, after controlling for seasonal oscillation and longer term temporal trends, only cooling-degree days (i.e., average number of degrees above 18°C), maximum temperature, and clear-sky UV index were independently associated with case occurrencein a final multivariable model ( (Figure 4 ).
Evaluating associations between environmental and meteorological exposures and IPD risk using a case-crossover approach, we identified an inverse association between ambient levels of sulphur oxides and disease risk during the likely incubation period (Table 3) . No other significant associations between environmental exposures and risk were identified either during the incubation period, or in the period immediately prior to incubation. In particular, occurrence of IPD was not affected by daily changes in clear-sky UV index, temperature or coolingdegree days, in contrast to associations on longer time scales described above.
Notwithstanding the existence of vaccination and effective antibiotic therapy, invasive pneumococcal disease remains an important source of population morbidity and mortality. The seasonality of IPD is well recognized, but poorly understood. Epidemiological mechanisms invoked to explain this pattern have included co-occurrence of other infectious diseases [4] , wintertime social gatherings [5] , and seasonal oscillation in immune function [29] . However, concurrent seasonal changes in a variety of environmental, behavioural, and epidemiogical exposures make identification of causal associations particularly challenging [30] . We attempted to address this challenge by using analytic approaches that should control for seasonal confounders, known and unknown, at different time scales. At a weekly time scale we found increases in UV radiation to be most strongly associated with decreased numbers of invasive pneumococcal disease cases, though average temperatures also appeared to influence disease risk. At short time scales, fluctuations in ambient air quality, as manifested by differences in concentrations of sulphur oxides, were associated with changes in risk. The direction of this association was at variance with existing models relating air pollution to pneumonia occurrence [3, 31, 32] .
The casual (as opposed to causal) association between low UV radiation in the winter and surges in respiratory disease has been noted previously [33] , and has been proposed as an important driver of influenza seasonality, but has not to our knowledge been evaluated in a way that accounts for coincident seasonal changes in other seasonally oscillatory factors. The degree to which such seasonal oscillation can result in "just so" stories that lead to misattribution of causation to non-specific seasonal exposures is highlighted in the univariable analyses we conducted without including seasonal oscillators. In these models, a variety of environmental conditions, including weather variables and air quality indices, were strongly associated with IPD risk. However, after controlling for non-specific seasonality, only UV radiation (and, more weakly, temperature) were associated with disease risk; indeed, the apparent protective effect of UV radiation was actually strengthened after controlling for seasonal oscillation.
The interpretation of such a model is that increases in UV radiation reduce IPD risk, even after accounting for the fact that IPD risk is maximal during low-UV periods of the year.
An important consideration is whether changes in UV radiation, operating at a weekly time scale, constitute a biologically plausible mechanism that explains seasonal oscillation in pneumococcal disease risk. Indeed, there are several mechanisms that may have substantial biological plausibility. Modulation of risk may occur through direct effects of UV radiation on host immune function: Dowell Figure 1 Periodogram Constructed from Spectral Decomposition of Weekly Pneumococcal Case Counts. Spectral density is represented on the y-axis, and can be conceptualized as a measure of goodness-of-fit for oscillatory regression models at different frequencies. The large peak at a frequency of 51 weeks suggests that invasive pneumococcal disease is a process that oscillates with annual periodicity (and is, in other words, compatible with wintertime seasonality). The two peaks at lower frequencies are lower harmonics illustrating bi-and tri-annual behaviour.
Spectral Density reviewed a variety of immunological changes associated with diminished UV radiation exposure in experimental settings, and noted that in granulocyte and monocyte function were reduced during periods of short light exposure [33] . UV radiation also influences the production of 1,25-(OH) 2 -vitamin D, which has important immunomodulatory functions [34] . Namely, enhanced maturation of macrophages, macrophage secretion of bactericidal substances such as lysozomal enzyme phosphatase and hydrogen peroxide [33, 35] , and secretion of antimicrobial peptides (including cathelicidins and defensins) by both immune cells, and respiratory tract epithelium [36] . Vitamin D deficiency is associated with a marked increase in the risk of pneumonia [37] , and most human vitamin D is acquired via sun exposure. Thus, extended periods of low UV light could result in an increased susceptibility to S. pneumoniae infection resulting from a lack of vitamin D production, though the week-to-week fluctuations in risk described in this paper may be too rapid to represent a vitamin D effect.
An alternative mechanism of action of UV radiation in reducing IPD risk could be direct effects of radiation on pathogen survival. Many bacterial respiratory pathogens, including pneumococcus, are transmitted in respiratory secretions over short distances (i.e., via "large droplet transmission"), and thus encounter the physical environment directly during transmission events. The bactericidal effects of UV-B radiation have been well known for decades; such radiation inactivates bacteria by causing harmful genetic mutations through creation of pyrimidine dimers [38] . A model of UV effect via diminished transmissibility, rather than decreased host susceptibility, is supported by our finding that UV effect is strongest in the youngest individuals in the population (i.e., toddlers), where disease risk is likely to be driven by mobility, contact with peers, prolonged carriage and carelessness with respiratory secretions. We found very little protective effect against IPD in oldest individuals, whose risk may be more strongly linked to immune senescence than to high rates of contact with infectious contemporaries [39, 40] .
An unexpected finding in our case-crossover analysis was the identification of increased levels of ambient sulphur oxides with diminished risk of IPD. This may represent a chance association, due to the established association between sulphur dioxide and adverse respiratory outcomes [32] , as well as the previously described correlation between ambient sulphur dioxide and pneumonia risk [3] . Nonetheless, this association may warrant further exploration; for example, ambient air pollutants might have adverse effects on respiratory tract pathogens as well as hosts.
Other findings in this study are also worthy of comment; in Philadelphia, the wintertime peak in pneumococcal incidence occurred about six weeks later than previously been described in U.S. adults [5] . In addition, a gradual increase in cases was observed during the 5-year study period. We suspect that this increase is less likely to represent a true surge in disease rates, which have actually been falling in the U.S. with the introduction of 7-valent conjugate pneumococcal vaccine [41] ; rather, we suspect that this increase, which is more attenuated after 2003, represents the gradual increase in reporting that commonly fol-lows implementation of new infectious disease reporting requirements [42] .
Like any observational study, ours has several limitations. First among these is our heavy reliance on public health surveillance data which is known to suffer from underreporting of notifiable infectious diseases [43] . Thus our data set may be incomplete, consisting of only a subset of the cases of IPD in Philadelphia during the study period. However, selection bias will only be introduced if the notification of infectious diseases to public health was correlated with meteorological patterns (e.g., cases which occur during days with higher UV index have a greater likelihood of being diagnosed and reported), which seems unlikely [44] . Second, we obtained weather data from a single site at Philadelphia International Airport, and air pollution and UV data were averaged over several locations throughout the county, which may not represent the true exposure status of individual cases. This is 8 0 a n d o v e r probable non-differential misclassification, and will bias the results towards the null hypothesis. The effects seen here are most likely conservative, weakening the strength of observed associations and suggesting our estimates tend towards the lower bound [44] .
In summary, we described the occurrence of IPD in a major U.S. urban center and found that incidence was associated with marked wintertime seasonality that may be partly explained by diminished exposure to UV-B radiation in winter months. Further study is needed, but this result is consistent with observed patterns of respiratory infectious disease, and would be consistent with several biologically plausible models of effect. As the nature of future changes in UV radiation related to climate change are more difficult to predict than general changes in temperatures or precipitation patterns, the implications of these findings for future pneumococcal disease epidemiology are unclear [45] . Nonetheless, we believe this obser-vation goes some way towards explaining the notable seasonality of IPD, and could conceivably lead to novel disease control strategies through improved understanding of this common and virulent infectious disease.  Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection BACKGROUND: Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection. METHODOLOGY/PRINCIPAL FINDINGS: However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5(−/−)). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5(−/−) mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5(−/−) BMDMs. CONCLUSIONS/SIGNIFICANCE: We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs. Listeria monocytogenes is a Gram-positive, facultative intracellular, food-borne pathogen that causes severe disease in pregnant and immunocompromised hosts [1] . L. monocytogenes is also an important model organism that has been used for decades to study bacterial pathogenesis, immunology and cell biology [2] [3] [4] [5] .
The intracellular life cycle of L. monocytogenes has been described in detail [1] . L. monocytogenes can enter either phagocytic or nonphagocytic cells, where it is initially contained in a membranebound vacuole that matures through the endocytic pathway. Following acidification, the bacterium escapes from a vacuole into the host cell cytosol by secreting a cholesterol-dependent poreforming cytolysin, listeriolysin O (LLO) [6] [7] [8] . The precise mechanism by which LLO induces vacuolar destruction and bacterial escape into the cytosol is not completely understood.
Subsequent to infection, approximately 10% of internalized bacteria are in the cytosol as early as 10 minutes post infection while approximately 80% of the bacteria are in the cytosol after 90 minutes [9] . Once in the cytosol, L. monocytogenes express the bacterial protein ActA to facilitate bacterial motility and ultimately cell-to-cell spread [10] [11] [12] . Intercellular spread begins between 3 and 5 hours post infection [13] [14] [15] . Following cell-to-cell spread, bacteria are contained within double-membrane vacuoles in a newly infected cell. The bacteria utilize two bacterial phospholipases (PI-PLC and PC-PLC) as well as LLO to escape from spreading vacuoles [1] .
Recently it has been suggested that host cells may utilize autophagy as a defense against intracellular pathogens [16] [17] [18] . Autophagy is a mechanism by which cytoplasmic components, including long-lived proteins and damaged organelles (peroxisomes, ER, and mitochondria) are enveloped within specialized double-membrane-bound vesicles that deliver their cargo to the lysosome for degradation [19] [20] [21] . It has been hypothesized that basal levels of autophagy occur continuously inside of cells [20] . An increase in autophagic activity can be stimulated by amino acid starvation, hormone signaling, cytokines, TLR stimulation, immunity related GTPases and microbial infection [16] [17] [18] 20] . However, the mechanism by which substrates are targeted for autophagic degradation is unknown [20] .
L. monocytogenes has been shown to interact with the host autophagic machinery. Rich et al. (2003) reported that approximately 92% of chloramphenicol treated L. monocytogenes were surrounded by double-membrane vacuoles in J774 macrophage-like cells 21 hours post infection. The number of these chloramphenicoltreated bacteria captured by autophagic-like membranes decreased in the presence of autophagy inhibitors [22] . Subsequently, Py et al. (2007) showed that L. monocytogenes induces autophagy, as measured by microtubule associated light chain 3 (LC3) lipidation and colocalization of LC3 with intracellular bacteria. Further, Py et al., (2007) provided evidence that L. monocytogenes replicate more efficiently in autophagy-deficient (atg5 2/2 ) mouse embryonic fibroblasts (MEFs) compared to wild-type MEFs, suggesting that an Atg5-dependent process directly inhibits the replication of wildtype L. monocytogenes in mammalian cells. Using similar assays to test for autophagic induction, Birmingham et al. (2007) demonstrated that RAW 264.7 macrophages transfected with LC3-GFP, exhibited colocalization of transfected LC3-GFP with L. monocytogenes. However, the authors of this study did not observe that wild-type L. monocytogenes grew better in atg5 2/2 MEFs than in wild-type MEFs. Thus the role of Atg5 in the control of wild-type L. monocytogenes replication in cultured transformed MEF cells remains controversial. Py et al. (2007) and Birmingham et al. (2007) reported that L. monocytogenes lacking LLO did not induce autophagy, as measured by LC3I lipidation and colocalization. Further, both ActA and the bacterial phospholipases (PlcA and PlcB) were reported to play a role in escaping autophagy, as measured by LC3I colocalization and bacterial growth in the presence or absence of Atg5 [23, 24] . This suggested that autophagy may play a role in the control of wild-type L. monocytogenes replication that is effectively inhibited by the action of bacterial phospholipases and/or actin polymerization. However, this hypothesis has not been directly tested in primary cells. Yano et al. (2008) showed that autophagy in Drosophila hemocytes was induced in response to L. monocytogenes infection and was dependent upon detection of peptidoglycan by the cytosolic receptor PGRP-LE as well as on bacterial expression of LLO. Lastly, Zhao et al. (2008) revealed that mice lacking Atg5 in macrophages and neutrophils had a slight increase in susceptibility to L. monocytogenes as measured by bacterial levels in spleen and liver three days after infection. However, this work did not address whether the role of Atg5 in vivo was due to autophagy or an autophagy-independent role of Atg5. Furthermore, Zhao et al. (2008) did not address whether Atg5 controls L. monocytogenes replication in primary cells.
We re-evaluated the hypothesis that primary macrophages use autophagy as a defense against invading L. monocytogenes. Here we present a detailed cellular study that strongly suggests L. monocytogenes induces bacterially targeted autophagy early in infection as a result of phagosomal membrane damage caused by cytolysin expression. However, in contrast to earlier findings in transformed MEFs we report that either autophagy has no role in control of L. monocytogenes replication or that an as yet undiscovered bacterial virulence factor effectively circumvents autophagy.
To examine the amount of cellular autophagy induced by L. monocytogenes, we monitored the conversion of LC3-I to LC3-II in wild-type bone marrow derived macrophages (BMDMs) isolated from C57/B6 mice, infected with L. monocytogenes [25, 26] . Activation of autophagy triggers LC3-I lipidation resulting in the conversion of LC3-I to LC3-II, which is measured by a mobility shift on SDS-PAGE [27] . Within 40 minutes of infection with wild-type L. monocytogenes, the amount of LC3-II increased compared to uninfected cells (Fig. 1A) . Dhly (LLO-minus) L. monocytogenes and heat-killed bacteria stimulated almost no LC3-I to LC3-II conversion, as compared to wild-type L. monocytogenes over a 60 minute time course of infection ( Fig. 1B and data not shown) . Therefore, we concluded that while infection alone induced very little autophagy, wild-type bacteria secreting LLO induced increased amounts of autophagy. Further, we found that during a 60-minute infection, the absence of the bacterial proteins ActA and either of the phospholipases did not significantly affect the amount of autophagy activation as measured by LC3-I lipidation (Fig. 1B) .
In order to examine the degree of autophagy induction in individually infected cells, we monitored L. monocytogenes colocalization with LC3 in infected BMDMs isolated from transgenic mice expressing LC3-GFP [28] . Over the course of three independent experiments, we found that within 60 minutes after infection with wild-type L. monocytogenes, approximately 15% of the bacteria colocalized with LC3-GFP, whereas Dhly (LLO-minus) bacteria showed no detectable colocalization with LC3-GFP ( Fig. 2A,B) . Given that infection is asynchronous, these observations represented bacteria at various stages of early infection: entry into cells, within intact phagocytic vacuoles or in the process of escape from the phagosomes. As LC3-GFP did not co-localize with Dhly L. monocytogenes, LC3-GFP was either targeting phagosomes damaged by LLO pores or bacteria in the cytosol.
The data presented above support the observation that LLO is required for LC3-GFP colocalization with L. monocytogenes. To determine if LLO was the only L. monocytogenes determinant of pathogenesis necessary to induce autophagy, we examined the interaction of LC3-GFP in BMDMs with the non-pathogenic Gram-positive bacterium Bacillus subtilis engineered to express and secrete LLO or a related cholesterol dependent cytolysin, perfringolysin O (PFO). These modified bacteria have previously been shown to escape into the host cell cytosol, although the efficiency of escape is less than that observed for wild-type L. monocytogenes [29, 30] .
B. subtilis expressing LLO induced an increased amount of LC3-I lipidation at 60 minutes post infection as compared to wild-type B. subtilis (Fig. 3A) . Further, within 40 minutes after infection, B. subtilis expressing LLO or PFO co-localized with LC3-GFP equal to or greater than twice as often as wild-type L. monocytogenes (Fig. 3B ,C). As with Dhly (LLO-minus) or heat killed L. monocytogenes, wild-type B. subtilis did not co-localize with LC3-GFP ( Fig. 3B ,C). Live video microscopic imaging of cells infected with B. subtilis expressing LLO clearly showed LC3-GFPcontaining vesicles, approximately 1 mm in diameter, surrounding the bacteria (Fig. 3D ).
These data are consistent with the hypothesis that phagocytic vacuolar membranes damaged by LLO or PFO induce phagosome-targeted autophagy. However, it remained possible that the autophagic machinery was not recruited to the damaged primary vacuole, but rather to the bacterial surface upon exposure to the host cell cytosol.
To directly test whether membrane damage induced by LLO was sufficient for autophagy activation during the early stages of infection, we incubated macrophages with liposomes containing either active or heat-killed LLO (HK-LLO) in the presence of Texas Red dextran as a fluid-phase marker [31, 32] . As previously reported, LLO-containing liposomes were phagocytosed, their contents released and the dextran delivered to the cytosol. Of the .300 liposomes counted at each time point over three independent experiments, we found that liposomes encapsulating wild-type LLO had a peak of LC3 colocalization (8%) at 25 minutes post-infection (Fig. 4A,B) . The liposomes encapsulating HK-LLO showed less than 2% colocalization with LC3 at the same time point (Fig. 4B ). Because only ,10% of phagosomes harboring LLO-loaded liposomes are competent to rupture (as measured by dye release; KD Lee, personal communication), 8% was not statistically significantly different from this 10% expectation, while 2% was statistically significantly different from both the expected (10%) and observed (8%) values (p..99 by z-test, using a binomial distribution). These data are consistent with a model in which LLO-mediated membrane damage is sufficient to induce LC3 colocalization in the absence of bacteria.
Does Autophagy Promote or Prevent L. monocytogenes Escape from a Phagocytic Vacuole?
We hypothesized that a damaged phagosomal membrane was targeted by autophagy and that this interaction could ultimately affect the ability of the bacteria to survive inside of cells and/or escape from a phagosome. The capacity of L. monocytogenes to escape from phagocytic vacuoles was examined in both fibroblasts and macrophages that lacked functional autophagy due to the targeted disruption of the essential autophagy gene atg5 [33, 34] . L. monocytogenes escaped at an increased rate from primary vacuoles in the presence of autophagy in MEFs (Fig. 5A ). In contrast, the presence of autophagy had no measurable effect on escape rates in bone marrow-derived macrophages (Fig. 5B) . Further, contrary to a previous report, bacterial mutants lacking the secreted phospholipases that contribute to vacuolar escape had similar levels of cytosolic entry in both wild-type and atg5 2/2 MEFs as well as BMDMs (Fig. 5A, B) [24] . Finally, autophagy did not significantly affect the growth of wild-type L. monocytogenes in either MEFs or BMDMs (Fig. 5C, D) . Therefore, while LLO stimulated autophagy, there was no observable consequence in bacterial escape or growth in BMDMs. However, the decreased escape observed in atg5 2/2 MEFs suggests that under some conditions, autophagy may enhance, rather than prevent, bacterial escape.
There is mounting evidence that autophagy contributes to the innate immune response to microbial pathogens [16] [17] [18] . Pathogens that grow and or reside in the cytosol, such as: Shigella flexneri, Francisella tularensis, and Group A Streptococci lyse the host phagosomal membrane, escape into the cytosol and subsequently induce autophagy, as measured by LC3 colocalization, lipidation and electron microscopy [35] [36] [37] . Bacteria that reside in pathogen-modified vacuoles, such as Legionella pneumophila, Salmonella Typhimurium, and Mycobacterium tuberculosis, have also been reported to stimulate autophagy, also as measured by the above mentioned techniques [18, [38] [39] [40] [41] [42] . In both groups of bacteria that induce autophagy, it remains unclear if cytosolic translocation of bacteria and/or bacterial products, and/or phagosomal membrane damage, is the signal that induces autophagy. It is formally possible that all three induce autophagy under some conditions. In Drosophila, autophagy is induced by cytoplasmic peptidoglycan sensors, and the signaling pathway involved is independent of the known pathways that induce the expression of antimicrobial peptides [43] . Thus it is possible that L. monocytogenes induction of autophagy is multi-step process, with LLO falling upstream of an as-yet-unidentified receptor. However, the data presented here, with LLO-containing liposomes indicate that, while these other putative steps may play a role during bacterial infection, the presence of damaged membranes alone is sufficient to induce LC3 colocalization. The role of autophagy, if any, during L. monocytogenes infection is only just beginning to be appreciated. Previous studies have provided evidence that L. monocytogenes triggers autophagy, and that autophagy limits bacterial replication [23, 24, 43] . Further, bacterial mutants lacking the pore-forming cytolysin LLO fail to trigger autophagy [23, 24] . Zhao et al. (2008) recently reported that infection of mice lacking Atg5 in macrophages and neutrophils exhibited a modest increase in L. monocytogenes susceptibility.
In this study we confirmed that wild-type L. monocytogenes, but not Dhly mutants, triggered autophagy. However, our data from infection of atg5 2/2 BMDMs provided no evidence that autophagy had a measurable effect on bacterial escape from phagosomes or on intracellular growth. In contrast, autophagy slightly increased phagosomal escape and intracellular growth in transformed MEFs, which potentially highlights differences in autophagy utilization and/orfunction between various cell types. Additionally, our data provided no evidence that either ActA or bacterial phospholipases affect autophagy as measured by LC3I lipidation and autophagy-restrictive intracellular growth (some data not shown). Finally, by using heterologous expression of LLO and purified LLO encapsulated in liposomes, we showed that LLO activity was not only necessary but sufficient to trigger autophagy. These results are consistent with a model wherein phagosomal membrane damage induces autophagy.
One major difference between our studies and others that studied the interaction between autophagy and L. monocytogenes is that we used primary cultures of BMDMs lacking Atg5, while others used transfected RAW macrophage-like cells and atg5 2/2 MEFs, respectively [23, 24, 34] . Additionally, we examined phagosomal escape much earlier during infection. The majority of wildtype L. monocytogenes escape the vacuole within the first 30 minutes following entry, however Py et al. (2007) assayed escape at four hours post-infection [44] . Since L. monocytogenes begin to spread from cell to cell as early as three hours post-infection and phospholipase mutants have a known defect in escape from a secondary vacuole, it is possible that the defect in growth observed by Py et al. (2007) was due to a defect in escape from vacuoles of secondarily infected cells [13, 45, 46] Autophagy plays a role in cellular homeostasis. For example, smooth ER, excess peroxisomes, damaged mitochondria and rough ER have been shown to be selectively targeted and removed from cells by the autophagic machinery [47] [48] [49] [50] . We propose that some or all of these organelles are targeted by autophagy due to membrane damage. For example, chemically damaged mitochondria are selectively targeted and removed by autophagy [49] . Selective removal is thought to be due to the disruption of the inner membrane by pores which occurs upon depolarization, a signal which may be similar to pore formation due to cytolysin expression [49] . Our hypothesis is further supported by two reports showing that two pore forming toxins secreted by extracellular pathogens, Vibrio cholerae cytolysin (VCC) and the vacuolating cytotoxin from Helicobacter pylori (VacA), also induce autophagy in the absence of infection [51] [52] [53] . Autophagic bodies were identified inside of cells exposed to VCC [52] . Similarly, we noted non-specific induction of autophagy (diffuse puncta formation), when LC3-GFP BMDMs were exposed to increasing concentrations of extracellular LLO (data not shown). Further autophagic induction in response to VacA was blocked when the channel forming activity of the toxin was inhibited [53] . Therefore, autophagy may be a general response to membrane damage, and as a result may have evolved as a mechanism to recognize and target intracellular pathogens.
There are numerous examples of pathogens evading and exploiting host cell biology to promote their pathogenesis. It is likely that the autophagic response to intracellular pathogens is no exception. We suspect that L. monocytogenes has acquired the means to avoid or circumvent autophagy by yet undiscovered mechanisms. This is an area of current interest in the lab. We hope to gain a better understanding of how bacterial pathogens have evolved to recognize and evade host cellular processes.
The wild-type Listeria monocytogenes strain used was 10403S [54] . The Dhly (DP-L2161; [55] ), DactA (DP-L3078; [56] ) and DplcAB (DP-L1936; [46] ) mutants contained in-frame deletions of the respective genes in the 10403s background.
The wild-type Bacillus subtilis strain used was DP-B1066 [30] . It contains a vector-only insertion with no hemolysin. DP-B980 and DP-B1512 contain single copies of hly and pfo from L. monocytogenes and Clostridium perfringens, respectively, under the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter p spac from plasmid pAG58-ble-1 [29] . B. subtilis were grown on brain-heart infusion (BHI) agar without antibiotics at room temperature. Transformed mouse embryonic fibroblasts (MEFs) were a kind gift from N. Mizushima [36] . The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS) and passaged a maximum of 5 times when the cells reached approximately 80% confluency. MEFs with a passage of greater than 5 were not used for experimentation.
Wild-type (C57/B6), microtubule light chain associated-green fluorescent fusion protein (LC3-GFP) and atg5 2/2 bone marrowderived macrophages (BMDMs) were isolated from six-to eightweek-old female mice and cultured as previously described [25, 26, 28] . The atg5 2/2 mice were generated by mating Cre recombinase lysozyme M mice (Jackson labs, Strain # 004781) with atg5 flox/flox mice [34, 57] . one hour, washed three times with PBS containing calcium and magnesium, and gentamicin was added at a concentration of 50 mg/mL one hour post-infection. MEFs were washed and gentamicin (50 mg/mL) was added at 60 minutes post-infection.
For infections with B. subtilis: B. subtilis (from colonies scraped from a plate) were added to 5 mL of BHI broth containing 1 mM IPTG. Cultures were grown at 37uC with shaking to an optical density at 600 nm OD 600 of 0.8 to 1. 
After infection, coverslips containing infected BMDMs were washed once with PBS and fixed in 4% paraformaldehyde for 30 minutes at 37uC. Coverslips containing cells were incubated with blocking buffer (PBS, 0.1% Triton X-100, 2% BSA) for 30 minutes at 37uC. The primary antibodies used were: anti-GFP (combination of two monoclonal murine antibodies, Roche, 1:200 dilution) and rabbit polyclonal anti-L. monocytogenes (Difco, 1:2,000 dilution). Secondary antibodies used were Alexa-Fluor conjugated goat antimouse IgG1 and goat anti-rabbit IgG (Invitrogen, 1:1000) respectively. Both primary and secondary antibodies were diluted in block buffer and incubated for one hour at room temperature followed by three washes with TBST buffer (25 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% Triton X-100). Coverslips were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories).
L. monocytogenes-infected cells were stained as described, followed by incubation with a secondary Alexa 488-labeled anti-rabbit polyclonal antibody (Molecular Probes, 1:2,000 dilution), and F-actin was stained with tetramethylrhodamine B isothiocyanatephalloidin (Sigma, 1:1,000 dilution). Both primary and secondary antibodies as well as phalloidin were incubated at room temperature for 60 minutes followed by three washes with TBST buffer. Coverslips were mounted as described above. Bacterial escape was quantified by the number of actin associated (red, phalloidin-positive) fluorescent bacteria over the total number of intracellular green fluorescent bacteria at 30, 60, 90 and 120 minutes post-infection. The percentage of escape was determined by dividing the number of phalloidin-positive (escaped) bacteria by the number of total intracellular bacteria [58] . Numbers were obtained from the average of a minimum of three experiments.
An MOI of ,5:1 was used to infect BMDMs (1610 7 L. monocytogenes and 2610 6 BMDMs). Cell samples were rinsed once with PBS containing calcium and magnesium and lysed with 2X Laemmli sample buffer (125 mM Tris HCl pH 6.8, 4% sodium dodecyl sulfate, 30% glycerol, 1% b-mercaptoethanol, 0.005% bromophenol blue). The Laemmli buffer was added directly to the infected cells and scraped off the tissue culture treated Petri dishes using a cell scraper. The samples were collected into Eppendorf tubes, boiled for 5 minutes at 95uC and loaded on 12% pre-cast NuPage gels (Invitrogen, Carlsbad, CA). Gels were run in 1 X 3morpholinopropane-1-sulfonic acid (MOPS) buffer (Invitrogen). Gels were transferred to polyvinylidene fluoride (PVDF) membrane at 15V for 1 hour on a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). The membrane was blocked for 1 hour at room temperature with Odyssey blocking buffer (LI-COR, Nebraska, NE, catalogue no. 929-97301) diluted 1:1 with PBS. The membrane was incubated overnight at 4uC with mouse monoclonal anti-LC3 antibody (courtesy of R. Kopito and B. Riley, Stanford University) at 1:500 in blocking buffer containing 0.02% Sodium Azide and 0.1% Tween-20. The membrane was washed three times for 10 minutes with TBST. The membrane was incubated with the LI-COR secondary antibody covalently attached to an infrared-emitting fluorophore IRdye-680 and IRdye-800 (LI-COR Biosciences, Lincoln, NE (goat anti-mouse IgG, 0.5 mg)) for 1 hour at room temperature. The membrane was washed three times for 10 minutes with TBST followed by one wash with 1X PBS. Membranes were scanned at 700 nm or 800 nm using the Odyssey Infrared Imaging System (LI-COR Biosciences), and quantitated using the accompanying software package (Odyssey V 3.0). Anti-gamma-tubulin, mouse monoclonal (clone GTU-88, Sigma catalogue no. T6557) was used as a loading control. Westerns were repeated three independent times.
Liposomes (at a concentration of 9 nM) containing either LLO or heat killed LLO (LLO was inactivated by incubation at 70uC for 10 minutes) were prepared as previously described [31] . Liposomes were incubated with BMDMs on coverslips at a 1:10,000 dilution (ratio of liposome-containing media to cell media) and rinsed with 37uC fresh cell media after three minutes of incubation with the BMDMs. The coverslips containing cells were fixed as described above. At least 300 liposomes were counted per time point and the experiment was repeated three independent times.
An MOI of ,5:1 L. monocytogenes was used to infect BMDMs (1610 7 bacteria and 2610 6 BMDMs). Growth curves were done as previously described [26] .
Imaging for L. monocytogenes-and B. subtilis-infected LC3-GFP BMDMs:
Cells were imaged with an Olympus IX71 epifluorescence microscope using the 100X objective for L. monocytogenes and 60X objective for B. subtilis and 30 frames per time point were randomly selected. Images were collected and color-combined using Metamorph software (Universal Imaging) and subsequently scored for colocalization of L. monocytogenes and B. subtilis with discrete LC3-GFP containing vacuoles. Images are representative of observed results, repeated in three independent experiments. Images were compiled in Adobe Photoshop software (Adobe, San Jose, CA).
Samples were viewed at 6006 magnification with a Nikon TE300 inverted microscope. Microscopy was performed on an Olympus IX-70 or IX-80 microscope equipped with a cooled CCD camera. For live imaging, samples were loaded onto a stage heated to 37uC by a fan and antibody staining was not used. Media was changed every ,60 minutes during long time sequences. Images were collected and color-combined using Metamorph software (Universal Imaging). Large collections of photomicrographs were used for counting cytosolic contents; each graph represents .100 bacteria or liposomes, and three trials for each time point. Figures were compiled in Adobe Photoshop software. Bars are SEM (standard error of mean), where n = 3. Definition of Mafa-A and -B haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis) The major histocompatibility complex (MHC) class I B gene/allelic repertoire was investigated in a pedigreed population of cynomolgus macaques of mixed Indonesian/Malaysian origin. The Mafa-B alleles detected in this cohort are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations. This suggests the fast evolution of Mafa-B alleles due to adaptation to new environments. In contrast to humans, the B locus in Old World monkeys displays extensive copy number variation. The Mafa-B and previously defined -A gene combinations segregate in families and thus allowed the definition of extended haplotypes. In many cases it was possible to assign a particular Mafa-I allele to one of these Mafa-A/B haplotypes as well. The presence of a large number of stable haplotypes in this cohort of animals, which was pedigreed for up to eight generations, looks promising for developing discriminative MHC typing tools that are less cumbersome. Furthermore, the discovery of 53 unreported Mafa-B sequences expands the lexicon of alleles significantly, and may help in understanding the complex organisation of the macaque B region. The cynomolgus macaque (Macaca fascicularis), also known as the crab-eating or long-tailed macaque, is widely used as an animal model in biomedical studies. Currently this species is applied as often as the commonly used rhesus macaque (Macaca mulatta). Cynomolgus monkeys are used as models for infectious diseases, such as AIDS, SARS and tuberculosis, as well as for transplantation research (McAuliffe et al. 2004; Wiseman et al. 2007; Aoyama et al. 2009; Mee et al. 2009; Reed et al. 2009 ). Owing to use of macaques in immune-related research, thorough investigations of their major histocompatibility complexes (MHC) are required. The MHC represents a multigene family in which the proteins play a key role in the generation of adaptive immune responses in vertebrate species. The class I and II genes of the MHC display abundant polymorphism that has a profound impact on features such as disease susceptibility, organ transplantation, and reproduction success.
The MHC systems in humans (HLA) and in other primate species have been studied extensively (Bontrop 2006) . The orthologues of the classical HLA-A and -B genes, which are involved in the presentation of intracellularly processed peptides to cytotoxic T cells, are present in the rhesus and cynomolgus macaque (Boyson et al. 1996; Krebs et al. 2005) . However, in these animals the genes have undergone several rounds of duplication and display copy number variation (Anzai et al. 2003; Daza-Vamenta et al. 2004; Otting et al. 2005) . Whereas in humans only one copy of the HLA-A and -B genes is present, in macaques seven A-like genes are distinguished. On each haplotype, one polymorphic gene is observed, named Mamu-A1 or Mafa-A1, in combination with one or two oligomorphic genes designated Mamuor Mafa-A2 up to -A7, respectively (Otting et al. 2007; Pendley et al. 2008; Campbell et al. 2009; Kita et al. 2009 ). The same organisation is also applicable to the pigtailed macaque (Macaca nemestrina) (Lafont et al. 2007; Wu et al. 2008) .
The situation for the HLA-B orthologues in macaque species is even more complicated. In one rhesus macaque, the MHC region was completely sequenced, yielding one complete haplotype of 5.3 megabase-pairs. On this haplotype, 19 distinct Mamu-B genes were present, of which 14 genes have the potential to code for bonafide proteins (Anzai et al. 2003; Daza-Vamenta et al. 2004; Bonhomme et al. 2008; Doxiadis et al. 2009 ). For the MHC of cynomolgus macaque a BAC-based contig map was constructed (Watanabe et al. 2007) . Although the degree of gene multiplication is less than in the rhesus macaque, this contig map still contains 12 distinct Mafa-B like loci. Sequencing studies at the cDNA level, however, have shown that only two or three genes per haplotype are transcribed at considerable levels (majors) in rhesus-and in cynomolgus macaques (Krebs et al. 2005; Otting et al. 2005 Otting et al. , 2008 Pendley et al. 2008) . At least one other B-like gene, characterised by low levels of polymorphism and transcription (minors), is present on all haplotypes. It has been designated Mamu-I, Mafa-I, and Mane-I in the respective species of macaques (Urvater et al. 2000; Robinson et al. 2003) . On the completely sequenced MHC-region of the rhesus macaque, this locus is designated as the Mamu-B3 gene (Daza-Vamenta et al. 2004; Doxiadis et al. 2009 ).
The sequencing of macaques from different geographic areas has shown that each population has its own characteristic set of Mamu/Mafa-A and -B alleles, and only a few alleles are shared between cohorts/populations (Krebs et al. 2005; Otting et al. 2008; Campbell et al. 2009 ). This is in contrast to the data that were observed for the MHC class II sequences obtained from these species (Otting et al. 2002; Doxiadis et al. 2006; O'Connor et al. 2007; de Groot et al. 2008) . Moreover, the interspecies sharing of MHC class I alleles in rhesus and cynomolgus macaques is in the same order of magnitude as the intraspecies sharing (Otting et al. 2007) .
We have access to cynomolgus macaques that have been pedigreed for eight generations, and the origin of the animals was determined based on mtDNA analyses. In an earlier study, we showed that the Mafa-A alleles are mostly unique for this population. Hence, a unique set of Mafa-B alleles is expected to be present in the same animals. The question is whether these B-alleles segregate in a stable linkage to the already described Mafa-A sequences in these animals. Should this be the case, MHC typing on this cohort may be then performed using less cumbersome techniques: for instance, those based on microsatellite or SNP analyses.
Furthermore, expanding the lexicon of Mafa-B alleles may provide more insight into the organisation of the macaque B-region, and may help in the definition of different lineages and loci, resulting in a more appropriate nomenclature.
The cynomolgus macaques used in this study had originally been kept at the University of Utrecht, where the animals were housed in social groups for up to eight generations. Recently, however, the colony of 135 animals was transferred to the new facilities at the Biomedical Primate Research Centre (BPRC), for the purpose of behavioural studies. The BPRC had access to blood samples drawn during health-checks, and lymphoblastoid cell-lines were established. The origin of the animals was determined by mitochondrial DNA (12S rRNA) analyses (Doxiadis et al. 2003; , and the founder animals appear to have originated either in the Indonesian islands or in continental Malaysia.
cDNA, cloning, and sequencing For all animals used in this study RNA was isolated from lymphoblastoid B-cells (Rneasy kit, Qiagen) and subjected to One-Step RT-PCR, as recommended by the supplier (Qiagen or Promega). The primers 5′MBS: AATTCATGGCGCCCCGAACCCTCCTCCTGC and 3′ MBS: CTAGACCACACAAGACAGTTGTCTCAG were used that anneal specifically to Mhc-B transcripts in macaques (Boyson et al. 1996) . Furthermore, for a subset of the animals the generic class I primers 5′ GGACTCA G A A T C T C C C C A G A C G C C G A G a n d 3 ′ TCTCAGTCCCTCACAAGGCAGCTGTC were used. The final elongation step was extended to 30 min to generate a 3′dA overhang. The RT-PCR products were cloned using the InsT/Aclone kit (Fermentas) or the PCR cloning kit (Qiagen). After transformation, 32 to 48 colonies were picked for plasmid isolation. Sequencing reactions were performed using the BigDye terminator cycle sequencing kit, and samples were run on an automated capillary sequencing system (Applied Biosystems Genetic Analyzer 3100).
Sequences were analysed using Sequence Navigator Software version 1.0.1 (Applied Biosystems) and MacVector™ version 10.6.0 (Oxford Molecular Group), followed by manual adjustments. After the alignments of all Mamuand Mafa-B exon 1-4 sequences using the MacVector software, version 10.6.0, phylogenetic analysis was performed with the phylogeny.fr pipeline (Dereeper et al. 2008 ) using maximum likelihood (ML) of the software PhyML 3.0 with the substitution model HKY85 with 4 categories, the gamma shape parameter of 0.407, a transition/transversion ratio of 2.130, and a SH-like approximate Likelihood-Ratio Test (aLRT) for statistical test of branch support. For tree rendering the pipeline uses the program TreeDyn 198, and the output tree is rooted using the mid-point rooting method.
The Mafa-B alleles have been named according to published nomenclature proposals (Klein et al. 1990; Ellis et al. 2006) ; however, the number of Mafa-B lineages has exceeded 100, and for the lineage numbers three digits have been introduced. In this document, the designations that were published previously are extended with a zero, and contain five ciphers. Two alleles that are highly similar receive the same lineage number, but any difference is indicated by the allele number (fourth and fifth cipher). If two alleles have synonymous base-pair differences, they receive an identical allele number, and the difference is indicated by a sixth and seventh digit. For example, Mafa-B*0950101 has synonymous differences in comparison to Mafa-B*0950102 and non-synonymous ones as compared to Mafa-B*09502. The novel alleles were submitted to the EMBL-EBI database (accession numbers FM212793-FM212843, FM246485-FM246500, FN423784, FN546179, and FN546180) and to the Non-human primate section of the IMGT/MHC Immuno Polymorphism Database (Robinson et al. 2003) .
In the cohort of 115 cynomolgus macaques, three to eight different Mafa-B sequences per animal were detected, with varying levels of transcription. The sequences, of which at least three identical clones were present, were reported as alleles. In most cases, these alleles were also confirmed in different animals. In total, 69 Mafa-B alleles were present, of which 16 were previously described by other research groups (Uda et al. 2005; Lafont et al. 2007; Pendley et al. 2008; Wu et al. 2008; Campbell et al. 2009; Kita et al. 2009 ). The other 53 sequences have been submitted to EMBL-EBI and to the MHC-NHP database (Robinson et al. 2003) , and have been catalogued. A list of the Mafa-B alleles is provided, including the accession numbers, and the reference animals (Table 1) .
In most animals, sequences that are alleles of the Mafa-I gene, the equivalent of the oligomorphic Mamu-I locus, were detected (Urvater et al. 2000) . The Mamu-I/Mafa-I locus has the characteristics of a nonclassical, with low levels of polymorphism and transcription. Only 12 Mafa-I sequences met the criterion of three identical clones, and eight of them have been submitted as novel alleles (Table 1) . Nevertheless, the Mafa-I gene appears to be present on all haplotypes.
The Mhc-A region-derived class I alleles of the animals in this cohort were sequenced in an earlier study (Otting et al. 2007 ). In those analyses, primers were used that are specific for Mhc-A alleles in macaques. However, in some animals the Mafa-A1 locus was not amplified by this primer set. In the present study, RT-PCR was performed with macaque Mhc-B-specific primers, and with the generic class I primers for those animals previously lacking a Mafa-A1 sequence. The use of these generic primers resulted in the detection of six new alleles for the Mafa-A1 locus, and one for both the Mafa-A2 and the -A5 genes (Table 1) , and as such extends the earlier reported data.
Since pedigree data were available, it was possible to determine the combinations of Mafa-B alleles on one chromosome (haplotype). Most haplotypes contain two major alleles, in combination with one or two alleles with lower levels of transcription, or minors, as based on the number of picked clones within a PCR sample. Unfortunately, it can not be excluded that some alleles are incorrectly considered as minors due to primer inconsistencies. Haplotypes with only one or three majors were also observed. Moreover, it was possible to extend these Mafa-B combinations with specific -A region configurations/haplotypes that were described in an earlier study (Otting et al. 2007 ). Combinations of Mafa-A and -B alleles that are segregating, and are observed in at least two related animals are listed (Table 2) . Although more than one Mafa-A gene is present on the cynomolgus chromosome, only alleles of the highly polymorphic Mafa-A1 locus are provided for sake of convenience. Six additional Mafa-A/B haplotypes were seen in only one animal, whereas nine sequence combinations were ambiguous, and are not listed in the table. Further analyses on these animals, and on their offspring are needed to find out if these are recombinations.
The number of at least 24 distinct haplotypes is high, though they appear to be stable entities in this population of macaques; recombination was seldom observed. Only one case of crossing over between the Mhc-A and -B region is seen; Mafa-A1*03101 is present in conjunction with two different Mafa-B combinations (7 and 8 in Table 2 ). For eleven of the 24 Mafa-A/-B haplotypes it was possible to add an associated Mafa-I allele. Preliminary studies with DRB-microsatellites de Groot et al. 2008 ) indicate that the Mafa-A/B haplotypes are also linked to DRB-STR patterns. Further investigation should reveal whether in the future these animals and their offspring can be typed for the class I alleles by means of this extremely fast and accurate typing technique. In our cohort of animals, 16 Mafa-B alleles were detected that were already described in studies on other cynomolgus populations. To determine whether these alleles were arranged in haplotypes that are shared between populations a comparison was made. Three of these combinations were observed. Pendley and coworkers have already described the sharing of B*01201/B*05001/2 and B*04501/B*05101 allele-combinations in Indonesian and Mauritian cohorts (Pendley et al. 2008) , which illustrates that the Mauritian animals originate in the archipelago. Probably the animals were introduced to the island by merchant ships in the Dutch Golden Age (Sussman and Tattersall 1986) . Both haplotypes are present in our animals, and are listed, respectively, as 13 and 15 in Table 2 . The first one was extended to A1*06001/ B*0110102/B*01201/B*05001. In Pendley's cohort this combination of Mafa-B alleles is seen in an animal that also transcribes Mafa-A1*06003. This allele differs by two basepairs from Mafa-A1*06001. With genotyping based on reference-strand conformational analysis (RSCA), Krebs and co-workers found the combination B*0430101/ B*0440101/B*0460101 in a cohort of Mauritian animals (Krebs et al. 2005) . In our animals, however, we observed this set without B*0430101 (Table 2 , haplotype 14). It is possible that this allele is a minor, and therefore was probably missed in our cloning procedures. In the RSCA study the B*0430101 peak is also low in comparison to the peaks of the other two alleles. The three shared haplotypes were present in cohorts that, like most of our animals at the BPRC, originate in the Indonesian Islands. Sharing of haplotypes with the recently described Filipino cynomolgus macaques was not observed (Campbell et al. 2009 ).
The restricted sharing of alleles in different populations of cynomolgus macaques, and moreover the recombination of similar alleles into other haplotypes in these populations, suggests that the diversity within the Mafa-B region has been the result of recombination and reshuffling of B-like loci during evolution. The cohort under study has been pedigreed for up to 8 generations, however, the haplotypes listed are observed in maximal five generations of related animals. The finding that within this cohort only one crossing between Mafa-A and Mafa-B is observed may be due to this relatively small number of generations. The fact that each cohort of animals has its own set of alleles and haplotypes necessitates investigation of the MHC for each cohort under study. Only within a breeding colony are the haplotypes more or less stable and predictable, and once the haplotypes are inventoried, robust MHC typing based on microsatellite analyses may be performed. Recombinations can be traced by using microsatellites spanning the whole MHC region. Fig. 1 . The phylogenetic tree shows that cynomolgus and rhesus sequences are fully intertwined, and each clade contains alleles of both species. In total, 17 sets of alleles were observed that are identical for all exons (Table 3) , and this number is in the same order of magnitude as the shared alleles among different populations of cynomolgus macaques. For instance, 25 Mafa-B alleles were described in the cohort Filipino cynomolgus macaques, of which three alleles were shared with animals of Indonesian origin (Campbell et al. 2009 ). Next to these shared alleles are several that differ by only one or two basepairs. Identity at the predicted amino-acid level for these alleles was not investigated. The sharing of haplotypes between cynomolgus and rhesus macaques was also investigated. Only one combination of three Mafa-B sequences was present in the rhesus macaque, and interestingly this was the B*0110102/ B*01201/B*05001 combination mentioned above. Moreover, this Mamu-B*03601/2/B*04501/2/B*03701 haplotype is one of four combinations that is shared by Indian and Chinese rhesus macaques, apart from a few basepair differences. The cynomolgus version differs by three basepairs in Mafa-B*01201 from the Indian rhesus macaque haplotype. The presence of the haplotype in different cohorts of rhesus monkeys and in the Indonesian/Mauritian cynomolgus macaques suggests that its ancestor was already present before the separation of both species. The stability of the haplotype during macaque evolution may have been caused by a significant advantage in the combat of intercellular pathogens. It is also possible that the sharing of the haplotype results from hybridisation. Molecular studies have revealed that introgression from rhesus macaques to cynomolgus monkeys has occured into the Indo-Chinese peninsula ). Fig. 1 Phylogenetic analyses of Mafa-B alleles detected in this study and Mamu-B alleles in known haplotypes of Indian and Chinese rhesus monkeys. The analyses are based on the exon 2, 3 and 4 sequences. Alleles that seem identical in this three may have basepair differences in other exons. The two species of macaques are indicated by different colors. Mafa/Mamu-B alleles of the one shared haplotype are depicted in green. The extension of allele-names with mi means that these are minors; alleles with relatively low transcription Similarities in the genetics of the macaque MHC class I regions are at this stage unfortunately not reflected in the nomenclature for Mhc-B alleles. The recent renaming of Mhc-A alleles has led to a nomenclature in which the distinct A loci and lineage numbers are compatible for all macaque species. For the Mhc-B region in macaques, however, it is not yet possible to assign the transcribed alleles to distinct B loci on the chromosome (e.g. Mamu-B1, -B2, -B3 etc.). However, it would be useful to adjust the lineage numbers (first three ciphers after the asterisk) so that the same numbers refer to similar sequences in different macaque species. Should more information become available in the near future on the number and order of B genes/loci on the macaque haplotypes, the allele designations may then easily be extended by a cipher following the B, without affecting the lineage numbers. In Vitro Viability and Cytotoxicity Testing and Same-Well Multi-Parametric Combinations for High Throughput Screening In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats. A significant advance in the field of early medicine was Paracelsus' recognition that all compounds have the capacity to be poisonous depending upon dosage [1] . This observation makes toxicity testing a necessary and critical industry practice to identify and define safety thresholds for all new potential chemotherapeutics. The advent of in vitro cytotoxicity testing has greatly streamlined this process and is now considered to be a nearly compulsory activity starting at target validation and continuing through medicinal modification.
Unlike animal-based toxicology testing, there are clearer definitions and greater agreement for what constitutes cytotoxicity in vitro. Classically speaking, a compound or treatment is considered to be cytotoxic if it prevents cellular attachment, causes dramatic morphological changes, adversely affects replication rate, or leads to a reduction in overall viability [2] . It should be noted that the manifestation of these effects is greatly dependent on length of compound exposure and mechanism of cytotoxicity [3] .
A host of new assays have been described and utilized which measure biomarkers of cellular stress or specific signaling events more proximal to initial cytotoxic insult (i.e. glutathione, caspases) [4] . These methods offer early indication of potential cytotoxicity, but are typically relegated to secondary screening because they are more difficult to employ as endpoint assays due to the transient nature of the biomarker and kinetic differences associated with cell death progression [5, 6] . Therefore assay chemistries predicated upon the detection of changes in membrane integrity remain the gold standard for in vitro cytotoxicity testing.
Many methods exist for the assessment of membrane integrity, including several classic dye inclusion, exclusion and lysosomal accumulation techniques [7, 8] . Although well *Address correspondence to this author at the Research Department, Promega Corporation, 2800 Woods Hollow Road, Madison, WI, USA; E-mail: andrew.niles@promega.com validated, these methods are poorly suited for HTS implementation due to low sensitivity, multiple processing steps or miniaturization problems associated with higher plate densities [9, 10] . Recent advances in reagent formulations accommodate these miniaturized formats and are fully compatible with automated dispensing systems and integrated detection instruments. These reagents deliver the linearity, sensitivity, and robustness (Z' Values) necessary for properly interrogating large chemical libraries for cytotoxic risk. The most acceptable assay format is known as "add-mixmeasure", whereby reagent chemistry is delivered directly to the test well.
Viability assays are designed to measure activities attributable to cellular maintenance and survival. These activities are typically metabolic biomarkers such as ATP and mitochondrial reductase potential, but can include homeostatic "housekeeping" enzyme activities. The underlying assay premise is that these activities are directly proportional to viable cell number after a treatment period. During relatively short incubation periods (8 hr or less), a reduction or complete cessation of these biomarker activities is strongly indicative of overt cytotoxicity by catastrophic membrane damage (i.e. primary necrosis) [11] . A reduction in biomarker activity compared to control in longer incubations indicates either a reduction in normal cellular division rate (cell-cycle arrest) or cell death by programmed elimination mechanisms such as apoptosis [12, 13] . Because viability assays measure the relative number of cells remaining after treatment, they offer significant utility even during long compound-contact periods (72 hr).
The reductive capacity inherent within viable cells can be conveniently measured using the redox indicator, resazurin [14, 15] . Resazurin is soluble in physiologically buffered formulations and can be added directly to growing cultures in a one-step, homogeneous addition. Unlike the tetrazolium chemistries (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-carboxanilide-2H-tetrazolium (XTT), (4-[3-4-iodophenyl]-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate) WST-1, 5-(3-carboxymethoxyphenyl)-2-(4, 5-dimethyl-thiazoly)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) which measure reductive capacity by colorimetric means, the product of resazurin reduction (resorufin) can also be measured using fluorescence with a fluorometer equipped with 560nm excitation and 590nm emission filter set [16] [17] [18] .
Resorufin product formation as a result of reagent contact with viable cells is accumulative and proceeds as a function of time. Although somewhat dependent upon cell type, metabolic rate and number per well, 2-4 hr of incubation with the reagent is typically sufficient to generate signal windows for adequate statistical discrimination in HTS. ( Table 1 ) Fluorescent resazurin reductase assay chemistries have been adapted to 1536 well formats, but are typically most useful at lesser plate densities (384 and 96) due to practical limitations associated with prolonged incubation periods [19, 20] .
Resazurin-based chemistries continue to offer significant value for HTS because the reagents are robust and stable, well-validated, and easy to use. The single greatest attribute however, is that resazurin reagents generate amongst the most cost-effective data on a per well basis [21] . The major disadvantages of employing the chemistry are that the reagent is subject to interferences from compounds with inherent reductive capacity (such as ascorbic acid, glutathione, coenzyme A, dithiothreitol etc.) or those with intrinsic fluorescence characteristics [22] [23] [24] . Lastly, extended reagent incubations (2hr or longer) may produce toxic affects or augment affects produced from the experimental compound [25] .
Eukaryotic cells contain a diverse abundance of proteolytic activities which act in a concerted effort to maintain homeostasis. Recently, a constitutive and conserved aminopeptidase-like, proteolytic biomarker profile was identified in mammalian cells that can be harnessed for assessing viability in cell culture [26] . The assay utilizes the cellpermeant protease substrate glycyl-phenylalanyl-aminofluorocoumerin (GF-AFC) that is delivered to wells in a neutral buffer. The substrate, which lacks an amino-terminal blocking moiety (i.e. Boc, Ac, or Cbz), can be processed by aminopeptidase(s) within the cytoplasm. The AFC product released after proteolysis of the substrate is proportional to viable cell number. Viability can be measured using a fluorometer equipped with a 380-400nm excitation source and a 505nm emission filter.
The selective detection of viable cells by this method is possible because the proteolytic activity towards the GF-AFC substrate is dependent upon the continued maintenance of membrane integrity. This live-cell, proteolytic activity decays within seconds after a cytotoxic event, so non-viable cells do not contribute appreciably to fluorescence generation. A 30 minute incubation of reagent and cells at 37ºC is typically sufficient for generating a workable signal window, but the incubation can be extended without detriment if work-flow scheduling does not permit shorter periods.
A major advantage of this assay is that it can be conveniently multiplexed with other compatible assay chemistries to deliver more per-well information ( Fig. 1 ) [27] . This feature is particularly attractive for normalizing spectrallydistinct fluorescent or luminescent data sets such as genetic report assays [28] . Multiplex applications for viability and cytotoxicity will be handled in greater detail in a later section of this review. Lastly, although red-shifted from much of the problematic coumarin spectrum, the assay is susceptible to fluorescence interferences from test compounds [29] . Additional interferences should be expected from protease inhibitors or from color quenching chemical entities. 
ATP quantification is a widely accepted method for assessing viability because healthy cells contain closely regulated levels of the biomarker [30, 31] . Non-viable cells not only lose the ability to synthesize ATP, but also contain en- dogenous ATPases that rapidly deplete the existing ATP. Luciferase reaction chemistry is used to quantify ATP, the limiting reactant, by generation of a luminescent signal that is proportional to viable cell number [32, 33] .
All commercial ATP quantification reagents contain luciferase enzyme and luciferin substrate supplied with a lytic buffer. Cell lysis is necessary to liberate cellular ATP and is typically facilitated by detergents in the buffer. The most useful formulations for HTS applications also contain thermostable recombinant luciferase and ATPase inhibitors which allow for "glow-type" luminescence that produces a sustained signal with a half-life on the order of 5 hr [34, 35] . Prolonged maintenance of the luminescent signal allows for batch processing of plates and obviates any inter-plate kinetic variability.
The principle advantages of an ATP assay include that it is currently the most rapid and sensitive HTS method available for assessing viability. For instance, as few as 10 cells can be detected in limiting dilution series of eukaryotic cells within 10 minutes (Fig. 2) . The assays also benefit from relatively high signal to background ratios, which enable routine miniaturization into 1536 well formats [36, 37] . Furthermore, luminescence signals are not encumbered by intrinsically fluorescent test compounds [38] . Despite an abundance of advantages for HTS implementation, ATP assays also carry liabilities. Because the ATPdependent luciferase reaction rate is susceptible to temperature flux, equilibration to constant room temperature is required to minimize thermal gradients created by plate well position (edge-effects). Also, like other metabolic assay chemistries used to measure viability, ATP is (rarely) subject to positive or negative modulation by test reagents independent of loss of viability leading to misleading data [39, 40] . Lastly, all commercial formulations of ATP-detection chemistries are susceptible to luciferase inhibition to various degrees by small molecule compounds. Recent evidence suggests that specific formulations may greatly minimize the extent of this inhibition [41] .
Cytotoxicity assay chemistries are formulated to detect loss of membrane integrity associated with cell death. The current most useful cytotoxicity biomarkers are constitutive, conserved and relatively stable, high-abundance enzymes "released" into the extra-cellular environment (culture medium) following loss of membrane integrity. Unlike viability assays whereby a decline in cell response is strongly inferred to be caused by cell-cycle arrest or death, detection of cytotoxic biomarker activities outside the cellular compartment after treatment is proof-positive for a compound's cytotoxic effect. Unfortunately, activity-based surrogates of cell death have a finite enzymatic half-life, which can limit their utility during long compound/cell contact incubations (48-72 hr) [3, 27] . Enzymatic decay as a function of time in culture medium can lead to underestimation of the absolute level of cytotoxicity [27] . The quantitative HTS (qHTS) approach eliminates a majority of this error because the kinetics of cytotoxicity is intrinsically linked to dose [42] .
LDH activity has a long history as a preferred marker for cell death with in vitro cell models [43, 44] . In HTS applications, the activity of LDH is indirectly measured by an enzymatically-coupled reagent chemistry comprised of lactate, NAD + , diaphorase and an appropriate redox dye such as a tetrazolium compound or resazurin. Although the result of LDH activity can be measured by a change in absorbance, fluorescence detection is favored because of greater sensitivity and the potential to miniaturize to high-density plates.
The implementation of LDH assays for cytotoxicity testing in HTS has been historically limited because most commercial assay protocols require a cell-free sample transferred into a separate assay plate. Reagents that are compatible with viable cells are available to detect release of LDH, thus enabling a single addition homogeneous assay format [45] . Now LDH release can be routinely measured in HTS formats after a short incubation (10-20 minutes) using a fluorometer equipped with a 560nm excitation source and a 590nm collection filter (Fig. 3 ) [46] . LDH assay detection is a robust and cost-effective means of assessing cytotoxicity, but is susceptible to background 
Well Number signal from serum sources of LDH found in supplemented growth media [47, 48] . Exogenous sources of LDH greatly elevate background values and negatively impact the overall signal window. This experimental detriment can be largely mitigated by careful pre-screening of serum sources for cell culture prior to LDH assay. LDH assays are also subject to assay artifacts by compounds that inhibit the LDH activity and by standard fluorescence and color quenching interferences [49] [50] [51] .
A specific proteolytic profile associated with cell death has been recently identified and can be measured in a manner similar to LDH assays [26] . This method employs sequence-specific (Ala-Ala-Phe), fluorogenic or luminogenic peptide substrates delivered directly into assay wells via a buffered vehicle. The proteolytic activities present in viable cells are not detected by this method because normally compartmentalized proteases are not in contact with the impermeant substrates. However, protease(s) released into the culture medium as a result of cell death cleave the substrates releasing a detectible reporting molecule. The fluorescent format of the assay utilizes the highly-efficient rhodamine 110 (R110) fluorophore which is measured in as short as 30 minutes by a fluorometer with 485nm excitation and 520-30nm emission filters [52, 53] . The luminescent format employs an engineered luciferase formulation to detect the luciferin product of cleavage thereby generating a glow-type signal which peaks at 15-20 minutes, stably persists for 3-4 hr, and is measured by standard luminometry.
Dead cell protease detection is advantageous in HTS and secondary screening applications because of improved sensitivity, linearity with respect to different degrees of cytotoxicity, reduction in background and artifactual hits, and choice in detection format (Fig. 4) . These attributes allow the technology to be applied in 1536 well formats as easily as in lower density plates [54] . The dead cell protease detection method is subject to enzymatic activity decay due to the kinetics of cytotoxicity, biomarker inhibition, and standard interferences associated with the detection molecules. Fig. (4) . Protease Assay Sensitivity and Linearity. CytoTox-Glo™ (Promega Corporation) was added to serial dilutions of viable and non-viable Jurkat cells and luminescence measured after 30 minutes.
The single parameter assays described above offer convenient and predictive methods to view the cytotoxic land-scape of chemical libraries. When certain combinations of the assays are added as same-well multiplexes however, they provide not only complementary but additive benefit [55] . Although the concept of multiplexed screening is well appreciated for image-based technologies, it is emerging as a more viable option for plate-based, homogeneous assays because implementation is simple, accessible, and cost effective [56] .
Reagent compatibility is a major consideration in samewell multiplexing. There is an absolute requirement that individual signal readouts are distinct and can be parsed for meaningful data set analysis. Also, reagent formulations must not compromise functional aspects of the chemistries. For instance, lytic assays for ATP measurement or caspase activation must be conducted in a sequence-specific order after first measuring experimental cytotoxicity.
Several informative combinations of viability and cytotoxicity chemistries are currently employed in HTS formats to mitigate false determinations. These multiplexes address systematic error caused either by disparate cell numbers delivered to assay wells or random compound interferences with reporter molecules [55] . Except in cases of cell-cycle arrest prior to loss of membrane integrity, or long term exposure models, measures of viability and cytotoxicity are inversely proportional (Fig. 5) . Therefore, non-conforming data sets can easily "flag" problem compounds for further analysis by orthogonal methods [57] .
Several classic examples of fluorescent viability and cytotoxicity assays are known [58] . These methods rely on an impermeant DNA intercalating dye which labels cells with damaged membranes and a permeable dye that is sequestered inside viable cells after esterase cleavage of a labeling group. Variations of these traditional chemistries are rarely employed where total fluorescence from assay wells is measured, because of poor sensitivity from simple stoichiometric dye binding events in the non-viable cell population and requisite washes required for removal of unbound dye and serum-derived esterases. Stable, non-viable cell esterase activity is particularly problematic for assay utility as it greatly impacts assay background values and constricts signal windows.
A new, more amenable method for HTS environments combines the protease substrates for viability and cytotoxicity allowing for simultaneous measurement of viable and non-viable cell populations (Fig. 6) . This method delivers broad linear responses (0-50,000 cells/well) and can measure as little as a 2% change in viability in both the AFC and R110 channels [59] . Furthermore, the reagent can be concentrated and delivered into assay wells in a substantially reduced volume to accommodate a third spectrally distinct reagent.
Triplexes created by uniting the fluorescent viability and cytotoxicity multiplex with luminescent chemistries are advantageous for several reasons. First, it is possible to conduct immediate confirmation testing of apparent cytotoxic compounds in the same well by leveraging the statistical power of orthogonal detection of viability (viable cell aminopeptidase, dead cell protease and ATP biomarkers). The economy of this consolidated testing is noteworthy in terms of compound usage, culture set up, and operator man-hours. Second, the mechanism of cell death can often be elucidated when combined with caspase detection reagents, allowing for partial characterization of a compound's potential. It should be noted however, that caspase activity can be transient and immeasurable in long-term experiments due to the kinetics of apoptosis and caspase biomarker decay. Therefore, caspase-negative data sets should be rescreened at multiple compound exposure periods. This type of characterization of pathway dependent cytotoxicity is useful for continued annotation of a library, because compounds causing necrotic profiles at micromolar concentrations are rarely therapeutically useful and may offer unacceptable cytotoxic risk. And third, viability and cytotoxicity can be used to normalize luminescent data from other assays (Fig. 7) . Single parameter genetic reporter responses are susceptible to experimental variation due to differential transfection efficiencies, cell number delivered to the well (clumping) or to cytotoxicity caused by the test compound [55] . Viability and cytotox-icity data can therefore help reconcile aberrant data points or explain decreases in genetic reporter signals due to cell death.
Mixed detection combinations of the protease biomarkers are also available that offer benefit in screening applications. In this method, viability is measured first by the addition of the GF-AFC reagent followed by the addition of the luminogenic dead cell reagent chemistry. The use of disparate detection platform methodologies is known to greatly reduce the statistical potential for assay interferences associated with reporter molecules [60] . Therefore this combination of chemistries is favored if a library is uncharacterized with regard to the relative abundance of luminogenic inhibitors or which may cause fluorescence interference. Unlike the fluorescence/fluorescence multiplex, fewer convenient triplex options exist. For example, the chemistry can be configured with a fluorogenic caspase activation chemistry (R110 fluorophore) to deliver data relating to possible induction of apoptosis (Fig. 7A) [61, 62] . Again, negative caspase induction results should be verified by rescreening to verify necrotic response profiles.
Fully-luminescent formats are highly desirable for HTS environments because of the aforementioned advantages over fluorescent methods. Another remarkable feature of luminogenic assays is the persistent and stable glow-type signal produced after reagent addition (Fig. 8) . This attribute can be harnessed to produce both viability and cytotoxicity values from the same well. In this method, the non-lytic, luminogenic cytotoxicity assay chemistry is first delivered to the assay well and cytotoxicity measured. Next, a lytic agent is added to assay wells to artificially release the remaining dead cell protease from viable cells. The net signal derived after complete lysis reflects the contribution of all the cells in the assay well. Because the luminescent signal is additive and proportional, the viable cell population can be indirectly determined by subtracting the cytotoxic values from the total values. This method is particularly useful for determining cytotoxicity in long incubation protocols where dead cell biomarker activity may not fully reflect actual cytotoxicity. One caveat of this method is that both viability and cytotoxicity measurements utilize the same biomarker and are subject to the same interferences.
In vitro cytotoxicity testing technologies for HTS continue their refinement on many fronts. High Content Screening (HCS) and label-free methods have gained some degree of popular following recently, but require expensive instrumentation, complicated analysis software packages and offer relatively low throughput. With burgeoning costs associated with drug discovery efforts, straight-forward, validated, homogeneous assay chemistries remain the choice as a simple and cost-effective means of assessing cytotoxic burden. A. 
Depending upon the goals of the screen, either viability or true cytotoxicity assay chemistries can be employed. Cytotoxicity assays based on membrane integrity changes are positive-readout assays most typically indicated for shorterterm exposure models (48 hr or less). These assays may not always accurately estimate the absolute degree of early or late stage cytotoxicity due to the kinetics of biomarker emergence or degradation. Viability assays measure the level of biomarker activity inversely correlated with cytotoxicity and therefore may be used at any endpoint during a compound/cell incubation period.
Each biomarker of viability and cytotoxicity has advantages and disadvantages. The choice of which surrogate of health or death to use is greatly influenced by the available detection format and extent of assay time required to reach a result ( Table 2) . Fluorescent formats require short to lengthy incubations with the sample whereas luminescent formats tend to deliver maximal signal windows in a rapid but prolonged fashion. Luminescent formats offer additional utility over fluorescent methods with regard to detection sensitivity, but are also typically more costly to use on a per well basis.
All assay chemistries are subject to experimental error or interferences which can lead to false interpretation of data sets. Multiplexed combinations of compatible assays offer a convenient and cost-effective manner to address variation (by response normalization), flag non-conforming orthogonal data points and increase per well content. Although greatly informative, special attention should be paid to experimental design and the interpretation of multiplexed data sets due to the transient or kinetic nature of mechanistic biomarkers. Fig. (8) . Bioluminescent Protease Assay Signal. Stability Cyto-Tox-Glo™ Reagent was added to blends of experimentally lysed and untreated HL-60 cells. Luminescence was measured using a BMG Polarstar™ in kinetic mode for 3 hr.  Relapsing macrophage activating syndrome in a 15-year-old girl with Still's disease: a case report INTRODUCTION: Macrophage activating syndrome is a severe, potentially life-threatening condition that may accompany Still's disease. It is characterized by fever, hepatosplenomegaly, lymphadenopathy, severe cytopenia, serious liver dysfunction, coagulopathy and neurologic involvement. The principal treatment for patients with this syndrome includes etoposide 150 mg/2 M twice a week for two weeks, dexamethasone 10 mg/2 M for two weeks and cyclosporine 3 mg/kg to 5 mg/kg for a longer period. Cases of relapse of macrophage activating syndrome are relatively rare. CASE PRESENTATION: We describe the case of a 15-year-old Iraqi girl with Still's disease who developed macrophage activating syndrome with acute respiratory distress syndrome that required resuscitation and mechanical ventilation. Following intensive treatment, including high dose steroids and cyclosporine, the patient improved significantly. Two weeks after cyclosporine was discontinued, however, she was readmitted with an acute relapse of macrophage activating syndrome manifested by spiking fever, arthralgias, maculopapular rash and leukocytosis. This time the patient recovered following the reintroduction of treatment with cyclosporine and the addition of mycophenolate mofetil (Cellcept). CONCLUSION: We believe that cyclosporine is a cornerstone for the treatment of Still's disease. We recommend continuing this medication for several weeks following the patient's clinical recovery in order to prevent macrophage activating syndrome relapses. Macrophage activating syndrome (MAS) is a severe, potentially life-threatening condition that is characterized by fever, hepatosplenomegaly, lymphadenopathy, severe cytopenia, severe liver dysfunction, coagulopathy and neurological involvement [1] . The principal treatment for patients with this syndrome includes etoposide 150 mg/2 M twice a week for two weeks, dexamethasone 10 mg/2 M for two weeks and cyclosporine 3 mg/kg to 5 mg/kg for a longer period of time. Relapsing cases of MAS are rela-tively rare. However, there are several case reports that describe a relapse happening after a short treatment or after the dose of cyclosporine was decreased rapidly [1] .
Still's disease (SD) is a rheumatic disease of unknown etiology characterized by prolonged fever, arthralgias and/or arthritis and maculopapular rash. One of its grave complications is MAS, which can occur in up to 15% of reported cases [2] .
We describe a rare case of a young patient with SD who presented with an acute relapsing pattern of MAS. Following intensive treatment, she recovered and has remained free of symptoms for the past 20 months. A 15- year-old Iraqi girl was admitted with a fever of 39°C which lasted for more than two weeks. A week prior to admission, she had complained of a sore throat, fever and maculopapular rash on her limbs, abdomen, palms and back. The patient was treated with doxycycline 150 mg twice a day, but due to her prolonged fever she was hospitalized. On admission, her physical examination was unremarkable. However, her blood count revealed leukocytosis of 23,000 (4000 to 10,000/mm 3 ), with 90% neutrophils, hemoglobin of 12.6 Gr% (12 to 16 Gr% with MCV 75 fl (77 to 91 fl)), erythrocyte sedimentation rate (ESR) at 92 mm/1 hour, C-reactive protein (CRP) at 16 (0-1 mg%) and serum ferritin level at 2267 ng/ml (10 to 120 ng/ml).
The patient's electrolytes, kidney and liver functions and coagulation profile were all normal. Her antinuclear antibodies, rheumatoid factor, c-ANCA, p-ANCA, anticardiolipin antibodies, lupus anticoagulant, antiparietal cell antibodies, anti-smooth muscle antibodies, anti-histone antibodies and anti-mitochondrial antibodies all showed negative results. Serological tests for CMV, EBV, HSV, HAV, HBV, HCV, HIV, adenovirus, parainfluenza, Coxsackie, influenza, Toxoplasma and Brucella were all negative. Serology for parvovirus was positive for IgG antibodies as well as for Coxiella burnetii antibodies (Qfever). A total body computed tomography (CT) scan was performed but did not show any pathology except for a 16-cm enlarged spleen. Bone marrow (BM) biopsy showed no evidence of lymphoma or any other type of malignancy.
Based on the patient's clinical and laboratory studies, a diagnosis of Still's disease was made. Three days after admission to the hospital, her liver enzymes became elevated. Two days later, the patient complained of shortness of breath with saturation of 85% at room air. Her chest Xrays were compatible with acute respiratory distress syndrome (ARDS). Her serum ferritin level rose to 10,648 ng/ ml; her CRP rose to 27.7; and her ESR was at 102 mm/1 hour. A complete blood count showed anemia with hemoglobin values of 7.7 Gr%, a platelet count of 75,000 Gr% and white blood cell count of 18,000 (neutrophils 93%).
The patient's liver function tests showed the following results: ALT 101 U/l (6 to 53 U/l), AST 369 U/L (2 to 60 U/l), alkaline phosphatase 329 U/l (40 to 200 U/l), GGTP 1055 U/L (10 to 80 U/l), total bilirubin 39 micromol/l (0 to 17 micromol/l), and LDH 10,800 U/l (300 to 620 U/l). Her blood gases showed PO 2 76 mmHg (85 to 90 mmHg), PCO 2 31.8 mm/Hg (30 to 44 mmHg), HCO 3 17.1 mmol/l (18 to 24 mmol/l) and pH 7.34 (7.38 to 7.42). Her coagulation profile disclosed a new alteration in the INR (international normalized rate) with values of 1.57 (1 to 1.4) and hypofibrinogenemia of 90 mg% (normal values at 140 to 400 mg%). Polymerase chain reaction (PCR) tests for CMV, EBV and HSV were performed on the patient and the results were all negative. A second BM biopsy, which was performed in order to rule out malignancy (possibly indicated by spiking fever, arthralgias, sore throat, leukocytosis and maculopapular rash), revealed impressive hemophagocytosis. The serum level of beta 2 microglobilin was higher than 12,000 mg/ml (normal value <2000), serum IL-2 receptor (IL-2R) was 1000 IU/ml (normal value <500 IU/ml) and serum tumor necrosis factor (TNF) was lower than 20 pg/ml. The patient was transferred to the intensive care unit and was intubated and ventilated with high positive end expiratory pressure (PEEP) levels.
A diagnosis of SD associated MAS was made and the patient was treated with high-dose intravenous methylprednisolone 1 g daily for three days, cyclosporine 5 mg/ kg daily and supportive noradrenalin due to her low blood pressure (50/30 mmHg). With this treatment her fever gradually diminished after two days and laboratory values showed a slow improvement: a decline in leukocytosis to normal range 9.5 (4-0 10E9/l) with 75% neutrophils, a decline in serum ferritin levels to 900 ng/ml (10 to 120 ng/ml), ESR at 30 mm/1 hour and CRP at 2.9. Her serum fibrinogen and the coagulation profile became normal. Her platelet count normalized after nine days of treatment. Her repeated serum level of beta 2 microglobilin declined to 2600 mg/ml (normal value <2000). Her serum IL-2R was 560 IU/ml (normal value <500 IU/ml) and her serum TNF was lower than 20 pg/ml.
Due to the patient's clinical and laboratory improvement, the dose of cyclosporine was reduced to 3 mg/kg and that of solumedrol was reduced to 100 mg three times daily. After 10 days, the patient was successfully extubated and discharged with oral treatment. Two weeks later the patient was readmitted due to spiking fever, arthralgias and maculopapular rashes on her palms. A physical examination revealed jaundiced eyes and her laboratory tests showed serious liver dysfunction, with ALT 791 U/l, AST 611 U/l, alkaline phosphatase 404 U/l, GGTP 2105 U/l, LDH 1683 U/l, and total serum bilirubin at 177 micromol/l. An abdominal ultrasonography revealed that the patient had a normal size liver with a 16.5 cm enlarged spleen and normal pancreas and common bile duct. Her kidneys also appeared normal. Repeat blood tests showed leukopenia of 3200, which further declined to 1100, hemoglobin 11 gr%, platelets 217,000, ESR 92, CRP 4 and ferritin 2680 ng/ml. Her lipid profile disclosed hypercholesterolemia of 15.1 mmol/l (normal range 3 to 5 mmol/ l), HDL 1.66 mmol/l (normal value > 0.91 mmol/l), LDL 11.11 mmol/l (normal range 0 to 3.4 mmol/l) and hypertriglyceridemia 5.0 mmol/l (normal range 0 to 2.3 mmol/ l).
PCR tests for CMV, EBV and HSV were also repeated and gave negative results. The biomarkers test was also repeated and showed her serum level of beta 2 microglobulin to be higher than 9000 mg/ml (normal value <2000). Her serum IL-2R was 850 IU/ml (normal value <500 IU/ ml) and serum TNF was lower than 20 pg/ml. A liver biopsy was then performed and showed a mild chronic inflammatory infiltration with no evidence of bile tract enlargement, foamy macrophage (hypercholesterolemia hypertriglyceridemia state), mild sinusoidal fibrosis and macrophage with intracellular iron pigmentation.
The official interpretation of the patient's condition was that of non-specific changes. An additional BM biopsy with aspiration was performed, again indicating severe hemophagocytosis. The patient was treated again with cyclosporine 5 mg/kg, solumedrol 1 g for three days and mycophenolate mofetil 500 mg three times daily. With this treatment the clinical conditions and the laboratory tests of the patient improved and she was discharged with the treatment of oral prednisone, cyclosporine and mycophenolate mofetil.
For the last 20 months the patient has remained completely asymptomatic while being treated with prednisone 5 mg per day (after a very slow tempering) and cyclosporine 75 mg per 2 days. Eight months after she recommenced this treatment, the prescription for mycophenolate mofetil was stopped.
Still's disease (SD) was described for the first time in children by George Still in 1896. It is characterized by a fever of approximately 39°C which continues for more than seven days, arthralgias or arthritis of two weeks duration or longer, and macular and/or maculopapular rash which is non-pruritic and salmon pink in color. Sore throat, lymph node swelling, hepatomegaly and/or splenomegaly with abnormal liver function studies, may also be present [3] . Typically, laboratory studies show an elevated ESR, which is accompanied by leukocytosis with the predominance of granulocytes. Normocytic normochromic anemia with hemoglobin values less than 10 g/dl and reactive thrombocytosis are seen in the majority of patients. Altered liver function studies are relatively common. However, liver biopsy findings are usually non-specific [4] .
The serum ferritin level is high in patients with SD, with values of 2,500 to 10,000 or even higher in 70% of patients reported. These high levels most probably reflect an acute phase response, since hepatocytes responding to inflammatory cytokines increase ferritin synthesis [5] . Since ferritin levels are not that high in other rheumatologic diseases, it was suggested as a candidate for a serologic marker for the diagnosis and monitoring of the response to treatment of SD. However, other studies suggest that the portion of glycosylated ferritin (less than 20% of the total value of ferritin) is a more specific finding for the diagnosis of SD, in contrast to other rheumatologic diseases where the value is much higher [6] . Interleukin-6, TNF alpha, interferon gamma and interleukin 18 may be elevated during the active phase of the disease, but serology for antinuclear antibody and rheumatoid factor are negative [7] .
The etiology of the disease is unknown and a variety of infectious triggers have been suggested including rubella, echovirus 7, mumps, cytomegalovirus, Epstein-Barr virus, adenovirus, parainfluenza, parvovirus, Coxsackie, influenza, herpes, and hepatitis B and C [8] . Possible bacterial etiology including Yersinia enterocolitica and mycoplasma pneumonia has also been raised [9] . Genetic factors such as HLA-B17, B18, B35 and DR-2 have been suggested as a predisposition for SD [10] .
The diagnosis of SD is made using major and minor criteria. However, it requires the exclusion of infectious mononucleosis or parvovirus B19 infection, malignancy (particularly lymphoma), or other rheumatologic diseases such as polyarthritis nodosa and systemic lupus erythematosus [2] .
Macrophage activating syndrome (MAS) is a severe, potentially life-threatening condition which may complicate chronic rheumatic diseases, especially systemic onset juvenile chronic arthritis. MAS is characterized by fever, hepatosplenomegaly, lymphadenopathy, severe cytopenia, serious liver dysfunction, coagulopathy and neurologic involvement. Early diagnosis of this condition is important because of the disease's aggressive clinical course.
Clinical and biological features of MAS closely resemble reactive hemophagocytic lymphohistocytosis (HLH) and the disease is in fact considered today as a subclass of HLH (secondary), which is induced by heterogeneous disorders including infections, malignant tumors and medications such as gold therapy, Aspirin and other non steroidal antiinflammatory drugs [11] . The exact underlying mechanisms of MAS have not yet been clarified.
The diagnosis of MAS in a patient known to have a rheumatic disease must be suspected when the patient shows signs of systemic derangement, fever, hepatosplenomegaly, bleeding tendency, leukopenia, thrombocytopenia, increase in liver enzyme values and coagulations disturbance [12] . Hypofibrinogenemia is one of the most important clues for the diagnosis of MAS since patients usually have high fibrinogen levels due to their underlying inflammatory disease. An increased fibrinolysis is most probably due to uncontrolled activation of the macrophages and the production of plasminogen. A decrease in factor II and factor VII + X values is also observed in some reported cases [13] . A bone marrow smear may show macrophage hemophagocytosis. Another biological indicator of MAS is an increased level of serum triglyceride that can be related to the patient's extensive production of cytokines, such as tumor necrosis factor alpha, which reduce the lipoprotein lipase activity. Interleukin-1 and interferon gamma are also overproduced in a patient with MAS [1] .
In 1994, the Histocyte Society conducted the first international treatment study for patients with HLH and they recommended treatment with etoposide 150 mg/2 M twice a week for two weeks, dexamethasone 10 mg/2 M also for two weeks, cyclosporine 3 mg/kg to 5 mg/kg for a long period and metotrexate in cases where the patient's nervous system is already affected. In resistant cases, several modes of therapy were described including high dose steroids, cyclosporine, antihuman thymocyte globulin (ATG), intravenous immune globulin (IVIG), plasma exchange and allogeneic bone marrow transplantation [14] . Recently, IL-1 beta receptor antagonist (Anakinra) was used with success in severe cases of HLH [15] .
This case is remarkable for its special pattern of an acute relapsing MAS. The exact mechanism underlying the relapsing pattern of the disease is not well understood. However, our patient fully recovered following long-term treatment with steroids, mycophenolate mofetil and cyclosporine. We cannot evaluate the exact contribution of each medication to the patient's positive response. Nevertheless, we believe that the prolonged use of cyclosporine treatment played a major role in her recovery.
We recommend continuing treatment with cyclosporine for several weeks following the patient's clinical and laboratory recovery in order to prevent a MAS relapse. Treatment with IL-1 beta receptor antagonists should also be considered in future cases. Multiple lung abscesses due to acinetobacter infection: a case report Acinetobacter species are well-known causes of nosocomial infections. Recent increasing evidence emphasize on the role of these pathogens in community-acquired infections. We report a case of a 16-yr-old female with fever, sore throat, productive cough, malaise and the presence of lung consolidation with multiple abscesses on radiographic examination. The patient had no significant medical history. After a detailed diagnostic work-up the diagnosis of community acquired Acinetobacter pneumonia with multiple lung abscesses was made. The Acinetobacter stain was susceptible to a variety of antimicrobial agents and the patient's condition improved rapidly. A new computed tomography chest scan, three months later, confirmed full recovery. The presence of lung abscesses due to Acinetobacter infection is an extremely uncommon manifestation of the disease. This case underlines the emergent role which these, often multi-drug resistant, bacteria may play in the future, perhaps in community infections as well. The presence of lung abscesses due to Acinetobacter infection is an extremely uncommon manifestation of the disease. This case underlines the emergent role which these, often multi-drug resistant, bacteria may play in the future, perhaps in community infections as well.
A 16-yr-old female presented to the emergency department with a two-day history of fever up to 39°C, sore throat, productive cough and malaise. Initially a diagnosis of acute tonsillitis was made and the patient was treated for a seven-day period with penicillin. Unfortunately the patient remained febrile up to 38°C, with persistent productive cough and malaise. Ten days after her first visit she was admitted to the pulmonary department for further evaluation. The patient until then had no significant medical history.
On admission she had low-grade fever (37,4°C). The rest of the clinical findings were unremarkable. Blood tests demonstrated normal white cell count (9500/μl), anemia (hematocrit 32,6% and hemoglobin 10,7 gr/dl), mild thrombocytosis (512000/μl), increased erythrocyte sedimentation rate (ESR = 109 mm/hr) and C-reactive protein (CRP = 49,6 mg/dl). Serum biochemistry tests were within normal limits. Arterial blood gases were normal as well. The chest radiograph disclosed a right upper lobe consolidation with the presence of at least three abscesses within the lobe. A spiral computed tomography of the chest demonstrated the consolidation within the right upper lobe with multiple lung abscesses (Figure 1 ). No other evidence of parechymal damage, lymph nodes enlargement or pleural fluid was visible. Heart and great vessels visualized without any pathology.
Cardiological examination including heart ultrasonographic analysis and dental examination did not demonstrate any pathological evidence. Skin tests for M. tuberculosis, atypical mycobacteria and Quanti-FERON TB Gold test were negative. Serum component analysis, rheumatoid factor, antinuclear antibodies, antineutrophilic cytoplasmic antibodies, immunonoglobin levels, serologic tests for hepatitis A, B, C, D, human immunodeficiency virus and common viruses did not reveal any abnormality. Finally sputum cultures in three different samples revealed the presence of Acinetobacter baumannii and the diagnosis of community-acquired pneumonia with multiple abscesses due to Acinetobacter baumannii infection were made based on this evidence. The stains were susceptible to multiple antibiotics and treatment with ciprofloxacin and amoxicillin with clavoulanic acid was initiated for a three month period of treatment. A significant clinical improvement in the patient's condition was noted only a few days later which was reflected in her laboratory investigation as well. Three months later a new computed tomography scan of the chest was obtained revealing an almost completely healed right upper lobe ( Figure 2 ). The patient remains asymptomatic until now.
Acinetobacter spp are aerobic gram-negative coccobacillus with preference for warm, ambient environments, which have received a great interest during the last two decades due to its ability to accumulate, with multiple mechanisms, multi-drug resistance stains. As a result these bacteria have become, a sometimes lethal, etiologic factor mainly in intensive care unit (ICU) infections, ventilator associated pneumonia (VAP), and in communityacquired infections and represent an emergent public health problem [1, 2] .
Typically this pathogen has been described as a cause of infection in tropical or subtropical climates, more often during summer, in ICU patients or in long term residents at health care facilities. Ventilator-depended patients are at greater risk but other factors such as recent surgery, central vascular lines, tracheotomy, enteral feeding and treatment with new generation antibiotics, like thirdgeneration cephalosporins, fluoroquinolones or carbapenems, may contribute as well. The majority of affected patients have underlying comorbidities mainly chronic obstructive pulmonary disease, renal disease, diabetes mellitus, alcoholism and heavy smoking [1, 3, 4] .
Nosocomial outbreaks have also been described due to health care professionals with colonized hands and poor personal hygiene. These people could act as opportunist carriers of an epidemic stain. Contaminated ventilators or respiratory care equipment as well as intrahospital transmission may also contribute to the beginning of an outbreak [1, 4] .
The computed tomography scan of this 16-y-old female revealed the presence of multiple abscesses at the right upper lobe Figure 1 The computed tomography scan of this 16-y-old female revealed the presence of multiple abscesses at the right upper lobe.
After a three-month period of treatment a new computed tomography scan demonstrated a full recovery Figure 2 After a three-month period of treatment a new computed tomography scan demonstrated a full recovery.
Recently Turkey's earthquakes in Marmara area, as well as others physical disasters like the South Asia tsunami, demonstrated that vulnerable populations are also at risk to Acinetobacter infections due to contamination and cross-infection inside hospital settings [1]. Moreover as a result of the ongoing war conflicts in Afghanistan and Iraq some cases of multi-drug resistant Acinetobacter outbreaks have also been reported bringing reminiscent of similar reports during the Korean and Vietnam wars. It seems that in cases like these multiple factors can contribute including contamination of wounds in the battlefield, local food, environmental spread and cross-infection in the local military or abroad hospitals [5] .
Community-acquired infections due to these pathogens are still rarely reported, mainly in warm and humid climates, and pneumonia. Fulminant course with an acute onset of dyspnea, cough, and fever, is the most common manifestation. Most patients suffer from various comorbidities. Leung et al in a three-year retroprospective study involving 19 patients with community-acquired pneumonia (CAP) due to Acinetobacter baumannii and 74 patients with health care associated pneumonia (HAP) concluded that CAP appears to be a unique clinical entity with a high incidence of bacteremia, ARDS, disseminated intravascular coagulation (DIC), and death, when compared to HAP [6] . In another study involving thirteen patients Chen et al concluded that Acinetobacter baumannii can be considered as an etiologic factor in communityacquired lobar pneumonia in patients with fulminant course in the warmer and humid periods of the year and also in younger alcoholic patients. The authors also suggested that a good sputum smear originated from the lower respiratory tract if it contains >25 leukocytes per high power (100×) field on microscopic examination early on to establish the diagnosis and that a combination of a third-generation cephalosporin and an aminoglycoside is adequate as empirical treatment [7] . Falagas et al in a recent review highlighted six case series with community-acquired Acinetobacter infections involving 80 patients. Of these 51 had pneumonia and 56% (45 patients) died. Moreover in the same review 26 case reports involving 43 patients had been identified. Pneumonia was present in 38 of them and from the others two had meningitis, one had native valve endocarditis, one had soft skin infection and one had an ocular infection. Chronic obstructive pulmonary disease, renal disease, diabetes mellitus, excess consumption of alcohol and heavy smoking were the most common risk factors. According to the same review on 12 retrospective or prospective studies the range of isolation of Acinetobacter from patients suffering from community-acquired pneumonia was 1, 3% to 25, 9% [8] .
A variety of antibiotics can be used if the infection has been caused by a susceptible stain including broad-spectrum cephalosporins, β-lactam-β-lactamase inhibitor combinations, carbapenes, and fluoroquinolones alone or in combination with aminoglycosides. Multi-drug resistant infections, including carbapenes, aminoglycosides and fluoroquinolones resistance, are challenging, however the combination of intravenous or inhaled colistin with tigercycline, imipenem, rifampicin or azithromycin seems reasonably enough to achieve satisfactory efficiency, at least in vitro, due to synergic or addictive effects which these combinations offer. On the other hand it's unclear if these antibiotic combinations provide adequate therapeutically results in vivo [1, 2, 9, 10] .
To our knowledge only three other cases of lung abscesses and three more of pneumatoceles due to Acinetobacter infection that have been described until now but none has been presented with multiple abscesses on admission. Myrianthefs et al reported a case of a 77-year old woman with multi-drug resistant Acinetobacter baumannii lung abscesses who underwent splenectomy after multiple traumas and remained for a long period in an ICU. Fortunately this patient recovered [11] . Hunt et al reported three cases of ICU pneumonia with the formation of pneumatoceles due to Acinetobacter infection. Unfortunately none of them survived [12] . Yen et al in a 23 cases retrospective review of pediatric lung abscesses reported only one case of secondary abscess due to Acinetobacter baumannii infection. Although only two deaths occurred in this group of patients it's unclear if one of them was the Acinetobacter baumannii infected patient [13] . Finally Yang et al reported a case of a 35-year-old male with right lobe necrotizing community-acquired pneumonia and lung abscess formation one week after his admission, who achieved a slow but fully recovery [14] .
In conclusion we presented a case of right lobe community acquired pneumonia with the formation of multiple lung abscesses due to a susceptible Acinetobacter baumannii stain. This is a rare manifestation of a community acquired disease.
ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; ICU: intensive care unit; VAP: ventilator associated pneumonia; CAP: community acquired pneumonia; HAP: health care associated pneumonia; ARDS: adult respiratory distress syndrome; DIC: disseminated intravascular coagulation.
Written informed consent was obtained from the patient's legal guardian for publication of this case report and A novel anti-mycobacterial function of mitogen-activated protein kinase phosphatase-1 BACKGROUND: Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α (TNF-α), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Tuberculosis (TB) remains a major cause of morbidity and mortality in the world, especially in the developing countries [1] . The disease is caused by Mycobacterium tuberculosis (MTB) and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization (WHO) estimated that there are 9.2 million new TB cases around the world in 2006 [1] .
In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity [2] . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) have been implicated as important cellular signaling molecules activated by mycobacteria [3] . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv [4] . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes (PBMo) leading to cytokine expressions via the interaction with PKR [5] . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated.
Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases (DUSPs). Some DUSPs are also known as MAPK phosphatases (MKPs) [6] [7] [8] . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout (KO) macrophages in response to lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment [9] . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity [10] . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF) [10] , thus demonstrating the role of MKP-1 in signal transduction.
Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities [10, 11] . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus (S. aureus), a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus [12] . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus [12] . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.
In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down [13] . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts [13] . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E (IgE) receptor (FcεRI) and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 [13] .
These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs [13] . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK [13] . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.
Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation.
It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts [6] . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction (QPCR) method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys (TLR2 agonist), poly IC (TLR3 agonist), or LPS (TLR4 agonist) for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment ( Figure  1A ). These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys ( Figure 1A ). In contrast, poly IC did not induce MKP-1 ( Figure 1A ). The results indicate that different inducers showed differential up-regulation of MKP-1 expression.
LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro [9, 12] . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression (Figure 1B) .
Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG (MOI = 1 CFU/cell), there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 ( Figure  2A ). The effects lasted for at least 6 hours ( Figure 2A ).
To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours ( Figure 2B ). The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.
It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages [14] . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 (inhibitor for MAP kinase kinase [MEK] or ERK1/2), SB203580 (inhibitor for p38 MAPK), SP600125 (inhibitor for JNK), and CAPE (inhibitor for NF-κB) for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced ( Figure 3 ). In addition, using higher dose of SB203580, we showed that the inhibition is increased further (data not shown). On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG ( Figure 3 ). These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. 
Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.
In Figure 4A , BCG stimulated MKP-1 expression (lanes 1 and 2). In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased (lanes 2 and 4). The results showed that the siRNA does abrogate the levels of MKP-1 mRNA.
To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA ( Figure 4B , lanes 1-3). However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA ( Figure 4B , lanes 4-6). Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein.
As stated in the literature [9] , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression.
To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported ( [5] and data not shown). Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA ( Figure 4D ). With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways [7] .
The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 [7] . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes (data not shown) and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA ( Figure 5 ). Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation ( Figure 5 ). The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA.
This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response (Figure 2) . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK ( Figure 3 ). Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 ( Figures 4D and 5) . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression.
Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK [14] . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation [15] . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors (for p38 MAPK and ERK1/ 2) was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression.
On the basis of the literature results on LPS effects ( Figure  6 ), the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α [9] . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS (Figure 4C ).
There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 [13] . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST [6] . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1.
Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members [13] . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts [13] . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here.
It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs [13] . It was shown that there are interactions between JNK and ERK1/2 pathways [16] .
Here, we showed that the sustained activation of JNK blocks ERK activation ( Figure 6 ). In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 [13] .
In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings (Left). In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated (Middle), since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 (Right) may be explained by events similar to the DUSP2 effects. In this case (Right), there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA (data not shown). Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 ( Figure 6 ) may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction ( Figure 6 ).
In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment ( Figure 6 ). Taken together, the role of MAPK crosstalks need further exploration. (3) TNF-α, 30 cycles (TM = 56°C), upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ (Invitrogen, USA) was run along with the PCR products.
To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH (as internal control) were assayed by the gene-specific Assays-on-Demand reagent kits (Applied Biosystems, USA). All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold (C T ) method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample (ΔC T ). The C T value of the treated cells was compared with that of the untreated or mock-treated cells (ΔΔCT). The relative gene expression of the targeted genes (fold induction) was calculated as 2 -ΔΔCT .
Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit (Pierce, IL, USA).
Western blot was done as described [20] . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes (Schleicher & Schuell), and followed by probing with specific antibod-ies for Actin, MKP-1 (Santa Cruz Biotech., USA), phospho-p38 MAPK, phospho-ERK1/2 (Cell Signaling, USA). After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) as per the manufacturer's instructions.
Transfection of siRNA into human monocytes was done as described [21] . MKP-1 siRNA included (i) MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; (ii) MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and (iii) MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi (ratio = 1:1:1, 200 nM, Invitrogen, USA). Stealth™ RNAi Negative Control Duplex (200 nM) was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent (Polyplus Transfection, USA) according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above.
Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05. Localization of Low-Density Detergent-Resistant Membrane Proteins in Intact and Acrosome-Reacted Mouse Sperm Mammalian sperm become fertile after completing capacitation, a process associated with cholesterol loss and changes in the biophysical properties of the sperm membranes that prepares the sperm to undergo the acrosome reaction. Different laboratories have hypothesized that cholesterol efflux can influence the extent and/or movement of lipid raft microdomains. In a previous study, our laboratory investigated the identity of sperm proteins putatively associated with rafts. After extraction with Triton X-100 and ultracentrifugation in sucrose gradients, proteins distributing to the light buoyant-density fractions were cored from polyacrylamide gels and microsequenced. In this study, a subset of these proteins (TEX101, basigin, hexokinase 1, facilitated glucose transporter 3, IZUMO, and SPAM1) and other molecules known to be enriched in membrane rafts (caveolin 2, flotillin 1, flotillin 2, and the ganglioside GM3) were selected to investigate their localization in the sperm and their behavior during capacitation and the acrosome reaction. These molecules localize to multiple sperm domains, including the acrosomal cap (IZUMO, caveolin 2, and flotillin 2), equatorial segment (GM3), cytoplasmic droplet (TEX101), midpiece (basigin, facilitated glucose transporter 3, and flotillin 2), and principal piece (facilitated glucose transporter 3). Some of these markers modified their immunofluorescence pattern after sperm incubation under capacitating conditions, and these changes correlated with the occurrence of the acrosome reaction. While GM3 and caveolin 2 were not detected after the acrosome reaction, flotillin 2 was found in the equatorial segment of acrosome-reacted sperm, and IZUMO distributed along the sperm head, reaching the post- and para-acrosomal areas. Taking into consideration the requirement of the acrosome reaction for sperm to become fusogenic, these results suggest that membrane raft dynamics may have a role in sperm-egg membrane interaction. Mammalian sperm are highly specialized cells that undergo a complex series of differentiating processes to become fertile. These processes start in the male reproductive tract with epididymal maturation and continue in the female tract, where sperm undergo capacitation and become fully able to fertilize an oocyte [1, 2] . Molecular events implicated in the initiation of capacitation include lipid rearrangements in the sperm plasma membrane that are coupled to changes in ion fluxes and to several signaling cascades. Among the changes in lipids, capacitation has long been thought to involve loss of cholesterol, leading to a different organization of the sperm plasma membrane.
The release of cholesterol appears to be necessary for the signaling events that accompany capacitation [3] [4] [5] , including the preparation of the sperm to undergo an agonist-induced acrosome reaction. However, our understanding of how sterol efflux couples to the regulation of signal transduction pathways intrinsic to capacitation remains rudimentary. Years after the description of the plasma membrane fluid mosaic model [6] , specialized platforms clustering signaling molecules were identified [7] . These structures are highly enriched in cholesterol and glycosphingolipids; this lipid composition provides their characteristic biochemical properties (insolubility in nonionic detergents such as Triton X-100 at 48C and light buoyant density after centrifugation in a sucrose gradient). Although several limitations should be taken into account [8] , sucrose light buoyant detergent-resistant membranes (DRMs) have been widely used as a first approach to identify membrane rafts [9] [10] [11] [12] [13] [14] [15] . Current concepts attribute important signaling properties to the existence of membrane rafts acting to bring protein assemblies together [16] [17] [18] . In the case of mammalian sperm, it has been hypothesized that cholesterol release during capacitation modifies the function/location of proteins in sperm raft microdomains [19] [20] [21] [22] [23] [24] . In an effort to elucidate the mechanisms by which the release of cholesterol couples to signaling events in the sperm, we previously conducted a proteomic analysis of the sperm light buoyant fractions and identified a number of proteins, including hexokinase 1 (HK1), testis serine proteases 1 and 2, TEX101, hyaluronidase (SPAM1), facilitated glucose transporter 3 (SLC2A3), lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin (BSG), and CRISP1 [20] .
Sperm are highly polarized cells, and their membranes can be categorized into different compartments. In intact sperm, the plasma membrane on the head surrounds the anterior acrosome, the equatorial segment, and the postacrosomal region. In the tail, the membrane covers the anterior part of the flagellum (midpiece) containing the mitochondria and the posterior portion of the tail (principal piece) containing the fibrous sheath surrounding the outer dense fibers [25] . These sperm compartments should be taken into account when the role of DRM proteins is analyzed; therefore, in this study we used antibodies against a subset of these proteins to investigate their localization in mouse sperm and their behavior during capacitation. Using filipin staining, the cholesterol-rich regions have been mapped to the plasma membrane overlaying the acrosome [5, 26, 27] . However, our findings indicate that proteins present in the light buoyant fractions, as well as other molecules known to distribute dynamically in membrane rafts, localized in multiple sperm compartments, including the anterior head, equatorial segment, cytoplasmic droplet, midpiece, and principal piece. When the behavior of these molecules during capacitation was studied, no changes were found in the tail-resident proteins. On the other hand, while some of the molecules in the head were lost, others change their immunofluorescence pattern; these changes were observed only in sperm that had undergone acrosomal exocytosis. IZUMO, present exclusively in the acrosomal region in intact sperm, spread to the post-and para-acrosomal regions of the sperm head in acrosome-reacted cells, while flotillin 2 (FLOT2), originally in the dorsal acrosome and midpiece, appeared in the equatorial segment. Considering that sperm acquire fusogenic capacity after the acrosome reaction, these changes in DRM-associated proteins could indicate their involvement in sperm-egg interaction.
All chemicals were reagent grade and were purchased from Sigma or Fisher unless otherwise stated. Molecular weight standards and all reagents used for SDS-PAGE were from BioRad. The following protease inhibitors were obtained from Sigma: PMSF, benzamidine, Na-tosyl-L-lysine chloromethyl ketone (TLCK), mammalian cell and tissue extracts cocktail 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, bestatin, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64), leupeptin, and pepstatin A. Calcium ionophore A23187 was from Calbiochem. Fluorescent reagents purchased from Molecular Probes included anti-rabbit IgG, anti-mouse IgG, anti-mouse IgM, and peanut agglutinin lectin (PNA) conjugated to Alexa Fluor 555 or 488. Peroxidase-conjugated anti-rabbit IgG (Sigma) and anti-mouse IgG (Jackson ImmunoResearch Laboratories) were used for Western blot analyses.
Specific antibodies directed against the different molecules analyzed in this study were obtained from several commercial and noncommercial sources as detailed herein. Monoclonal anti-TEX101 was generously provided by Dr. Yoshihiko Araki from Yamagata University (Yamagata, Japan). Dr. John Wilson from Michigan State University (East Lansing, MI) contributed the rabbit anti-HK1. Polyclonal anti-IZUMO was a gift from Dr. Masaru Okabe at Osaka University (Osaka, Japan). A specific polyclonal antiserum recognizing SPAM1 was obtained from Dr. Diana Myles and Dr. Paul Primakoff at the University of California, Davis. The antibody recognizing SLC2A3 was purchased from Alpha Diagnostic International. Dr. Kenji Kadomatsu from Nagoya University (Nagoya, Japan) generously provided the anti-BSG. Monoclonal antibodies reacting against FLOT1 and FLOT2 were purchased from BD Biosciences. Anti-CAV2 was obtained from Santa Cruz Biotechnology Inc. The monoclonal IgM that specifically recognizes the ganglioside GM3 was purchased from Seikagaku Corporation. Dr. Lonny Levin and Dr. Jochen Buck from the Department of Pharmacology, Weill Medical College of Cornell University (New York, NY) provided the antibody R2, which reacts against bicarbonate-dependent cyclase (ADCY10).
Mouse sperm were collected from CD1 retired male breeders (Charles River), euthanized in accord with Institutional Animal Care and Use Committee guidelines and following experimental protocols previously approved by the Animal Care Committee of the University of Massachusetts. For the sperm to swim out, the cauda epididymis from each animal was placed in 0.5 ml of modified Whitten-Hepes medium (WH) containing 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5.5 mM glucose, 1 mM Pyruvic acid, 4.8 mM L(þ)-lactic acid hemicalcium salt in 20 mM Hepes, pH 7.3, at 378C. After 10 min, sperm suspension was diluted into capacitating media or collected and centrifuged at 500 3 g for 5 min at room temperature (RT). For capacitation, 50 ll of the original suspension was diluted into 450 ll of capacitating media (WH supplemented with 20 mM NaHCO 3 and 3 mg/ml of bovine serum albumin [BSA], A-0281; Sigma) and incubated for 60 min as previously described [28] . To induce the acrosome reaction, capacitated sperm were treated with 3 lM calcium ionophore A23187 for 30 min. Total sperm extracts were prepared as detailed in Materials and Methods, separated using 10% (A) or 15% (B) acrylamide gels and analyzed by Western blot using different antibodies directed against the corresponding proteins. Arrowheads represent bands highlighted in Figure 2 .
FIG. 2. Distribution of proteins along the sucrose gradient. Sperm were extracted with 0.5% Triton X-100 and subjected to ultracentrifugation on a sucrose gradient as described in Materials and Methods. Nine fractions were collected from top to bottom and were analyzed by SDS-PAGE and Western blot using different antibodies. The molecular weights of the bands shown are in the right column. In the case of SPAM1 and ADCY10, bands shown are those marked with an arrowhead in Figure 1 . A nonraft protein (ADCY10) was included as control.
Sperm suspensions were centrifuged at 500 3 g for 10 min and washed with WH medium. The pellet was resuspended in 400 ll of TEN buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM edetic acid, pH 7.3) supplemented with 0.5% Triton X-100 and a protease inhibitor cocktail (1 mM PMSF, 1 mM NaF, 2 mM sodium orthovanadate, 20 lg/ml of leupeptin, 15 lg/ml of pepstatin, 0.8 mM aprotinin, 10 mM benzimidine, 3 lg/ml of TLCK, 1 mM AEBSF, 40 lM bestatin, and 14 lM E-64). This suspension was Dounce homogenized and sonicated with five brief bursts of 1 sec each. Samples were kept on ice for 5 min and then rotated at 48C for 45 min. Lysates were adjusted to 40% sucrose with the addition of 400 ll of 80% sucrose in TEN buffer and placed in the bottom of a 2-ml Beckman centrifuge tube. This suspension was gently overlaid with 800 ll of 30% sucrose in TEN buffer, followed by 400 ll of 5% sucrose in TEN buffer. The samples were then centrifuged at 200 000 3 g for 18 h in a TLS 55 swinging bucket rotor in a Beckman Optima-TLX ultracentrifuge. After centrifugation, 200-ll fractions were carefully collected from the top to the bottom of the gradient. Fractions were prepared for SDS-PAGE by the addition of 0.2 volumes of 53 nonreducing Laemmli buffer, boiled for 5 min, and kept frozen until use.
Total sperm extracts were obtained by cell suspension in nonreducing Laemmli buffer and by boiling for 5 min. Before running, samples were supplemented with 5% b-mercaptoethanol when required. The SDS-denaturing gels of different acrylamide concentrations (10% or 15%) were used depending on the protein under study. After electrophoresis, proteins were electroblotted to polyvinylidene fluoride membrane and blocked with 5% skimmed milk. All incubation and washing procedures were done with PBS supplemented with 0.1% Tween 20 (PBST). Membranes were blocked for 1 h at RT and then incubated with the different first antibodies overnight at 48C as previously described [29] . After washing three times for 5 min, the specific peroxidaseconjugated secondary antibody was added, and incubation was carried out for 1 h at RT. The membranes were washed with PBST, and immune complexes were located using ECL Plus and Kodak Biomax light films.
Sperm were fixed in suspension by the addition of fresh formaldehyde (prepared from paraformaldehyde [final concentration, 2%]) and incubation for 30 min at 48C. After washing two times with PBS, sperm were immobilized on slides and air dried. Cells were permeabilized with 0.1% Triton X-100 and 0.2% formaldehyde for 5 min at RT. After washing with PBS, blocking was done by 30min incubation with 1% BSA in PBS. Primary antibodies were diluted in PBS supplemented with 0.1% BSA and incubated overnight at 48C. After washing with PBS, cells were incubated with the respective Alexa Fluor-conjugated secondary antibody for 60 min at RT. When double staining was required, PNA coupled to Alexa Fluor 488 was included in the solution of the secondary antibody. After washing, cells were mounted using Slow Fade (Molecular Probes).
We previously identified a number of proteins using a combination of one-dimensional PAGE and tandem mass [20] . To confirm their association with the light-density fractions, antibodies against these proteins were obtained from different commercial and noncommercial sources as enumerated in Materials and Methods. Each of these antibodies was validated by Western blot of total sperm extracts as shown in Figure 1A , and the antibodies recognized proteins of the predicted molecular weight. In addition to the proteins identified previously [20] , we assayed for the presence of other proteins known to be enriched in membrane rafts in many cell types such as CAV2, FLOT1, and FLOT2. Antibodies against these proteins react with polypeptides of the expected molecular weight, indicating the presence of these proteins in mouse sperm (Fig. 1B) . We also tested antibodies against ADCY10, a nonraft protein.
The validated antibodies were then used to probe Western blots of sucrose gradient fractions obtained as described in Materials and Methods (Fig. 2) . Among the assayed proteins, it was possible to distinguish three different gradient distributions. Although all of them were found in the light fraction used previously for tandem mass spectrometry identification [20] , TEX101, SPAM1, FLOT1, and FLOT2 were present exclusively in the light fractions; IZUMO was more widespread and was also found in intermediate-density fractions; and a third set of proteins comprising SLC2A3, BSG, HK1, and CAV2 was found in most fractions, including the heavy ones. ADCY10 distributed exclusively in the heavy fractions.
A unique feature of sperm cells is the subdivision of the plasma membrane into well-defined regional domains with different compositions and functions [25] . In addition to the domains described in the Introduction, the cytosolic droplet that is present in a fraction of cauda epididymal mouse sperm constitutes another level of complexity. To analyze the specific distribution of the sperm DRM proteins in the different membrane regions, the already-validated antibodies were used to localize the respective proteins by immunofluorescence. As shown in Figure 3 , DRM proteins were found in the anterior head (IZUMO, CAV2, and FLOT2), midpiece (SLCA3, FLOT2, BSG, and SLC2A3), and principal piece (SLC2A3). TEX101, a protein that localized exclusively to the light buoyant sucrose gradient fractions, was present only in the cytoplasmic droplet.
In addition to the proteins enriched in membrane rafts, DRMs are enriched in gangliosides. Recently, fluorescently labeled cholera toxin has been used to address the presence of GM1 in sperm from different mammalian species [19, 21, 23, [30] [31] [32] [33] . Although GM1 is the best studied in sperm, other gangliosides are known to be enriched in membrane rafts [34] , and differential ganglioside distribution among these membrane microdomains has been reported [35] [36] [37] . In particular, GM3 appears to be the major ganglioside in the male reproductive system [38] , and several studies [39] [40] [41] demonstrate its abundance in bovine, ovine, and human sperm. However, localization of GM3 on sperm has not yet been analyzed, to our knowledge. To investigate the location of GM3 in mouse sperm, a specific anti-GM3 antibody was tested by immunofluorescence. This monoclonal antibody has been validated by Kotani et al. [42, 43] and was used in several studies [35, [44] [45] [46] [47] [48] . Anti-GM3 fluorescent signal was found to be restricted to the equatorial segment (Fig. 4) , whereas PNA signal exclusively stained the anterior acrosomal region. 4 . Detection of ganglioside GM3 in mouse sperm. Mouse sperm fixed and permeabilized as already described were incubated overnight with a monoclonal anti-GM3 antibody, followed by Alexa Fluor 555-conjugated secondary antibody (GM3). Cells were also stained with Alexa Fluor 488-conjugated PNA. No primary antibody controls were used (lower panels). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.
As previously stated, cholesterol content is relevant for the function of raft proteins and for sperm capacitation. To analyze if there was any relationship between these two events, we studied DRM proteins after incubating sperm under capacitating conditions. Most of the proteins did not change their respective immunofluorescence pattern upon incubation (data not shown). However, some proteins (IZUMO, CAV2, and FLOT2) and the ganglioside GM3 showed two different staining patterns in the capacitated population as shown in Figure 5 . In the case of CAV2 and GM3, some sperm displayed no signal; in contrast, FLOT2 and IZUMO showed a different staining pattern. Although these changes were observed in the capacitated population, in all cases they correlated with the loss of PNA staining, suggesting that the differential immunofluorescence patterns were due to the acrosome reaction and were not directly related to capacitation.
To further confirm this observation, the immunolocalization of FLOT2 and IZUMO was studied after inducing the acrosome reaction with the calcium ionophore A23187. As shown in Figure 6 , sperm that have lost PNA staining showed a new IZUMO localization. Although both FLOT2 and IZUMO displayed a different staining pattern in acrosome-reacted cells versus intact sperm, they did not colocalize (data not shown). While FLOT2 was restricted to the equatorial segment, IZUMO distributed into adjacent regions.
Capacitation has been associated with changes in the lipid composition of the sperm plasma membrane; in particular, cholesterol efflux induces signaling events in the sperm, including the capacitation-associated increase in tyrosine phosphorylation and the preparation of the sperm to undergo the acrosome reaction [1, 5] . Previous work from our group has used a proteomic analysis of low-density DRM-resident proteins in an effort to investigate whether cholesterol efflux regulates membrane rafts during sperm capacitation [20] . The use of detergent to relate protein association to rafts is the most widely used method to analyze the composition of membrane rafts and to identify putative resident proteins [9] [10] [11] [12] [13] [14] . However, this procedure has been questioned on the basis that detergent can induce the formation of membrane domains and fails to provide physiologically relevant information [8, 14] . Despite these criticisms, detergent separation can still be considered a first approach to determining components of membrane rafts. In particular, although proteins not belonging to rafts could be found in the low-density DRM fractions, it is FIG. 5. Differential immunofluorescence pattern of GM3, CAV2, IZUMO, and FLOT2 in capacitated sperm. Mouse sperm incubated under capacitating conditions were analyzed by immunofluorescence as described in Materials and Methods using anti-GM3, anti-CAV2, anti-IZUMO, and anti-FLOT2, followed by the respective Alexa Fluor 555-conjugated secondary antibody. Cells were also stained with Alexa Fluor 488-conjugated PNA to relate the staining patterns to the acrosomal status. Sperm that underwent the acrosome reaction correspond to those showing the new staining patterns (arrowheads). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.
generally believed that most proteins associated with membrane rafts will behave as detergent resistant [8] . In addition, an essential property of sperm cells is their highly compartmentalized structure [25] . Taking this into consideration, an alternative experimental approach is required to determine the original location of DRM proteins among the different sperm compartments.
In the present work, we chose a series of DRM-resident proteins to investigate their localization in sperm. These proteins were not restricted to one sperm region but were distributed among different sperm compartments; our findings regarding localization of these proteins are illustrated in Figure  7 . Some of these molecules (e.g., CAV2, GM3, IZUMO, and FLOT2) change their localization during the acrosome reaction. While CAV2 and GM3 appear to be lost, IZUMO and FLOT2 show a different pattern of localization in a fraction of the capacitated sperm population. The new patterns were found only in acrosome-reacted sperm, suggesting that the changes were due not to capacitation but to the acrosome reaction.
Sphingolipids are derivatives of the long-chain amino alcohols sphingosine and dihydrosphingosine. The sphingoid long-chain base is linked to a fatty acid molecule via an amide bond-forming ceramide. The addition of carbohydrates to this ceramide backbone leads to the formation of a great diversity of glycosphingolipids, including the gangliosides GM1 and GM3 that differ only in their oligosaccharide chains. Like cholesterol, sphingolipids are found to be enriched in membrane rafts; this behavior is due to the presence of two long saturated alkyl chains that can be organized and condensed by sterols to form the liquid-ordered phase of membrane rafts [49] . In sperm, other investigations on nonprotein raft markers have focused on GM1. Using cholera toxin, this ganglioside was shown to localize to the plasma membrane overlaying the acrosome in living sperm [32] . On the other hand, using biochemical methods, it has been demonstrated by biochemical methods that GM3 is the most abundant ganglioside in the male reproductive tract and in sperm [39] [40] [41] ; however, localization of GM3 in these cells was undetermined. In this study, we used previously validated antibodies against GM3 [42, 43] to determine the immunofluorescence pattern of this ganglioside in sperm and found it to be restricted to the equatorial segment.
Considering the method used in our original study [20] , the source of the proteins located in the light-density fractions could belong to more than one membrane. In addition to the aforementioned sperm compartments (e.g., head and tail), the subcellular structure of the sperm encompasses different membranes, including the plasma membrane, outer acrosomal and inner acrosomal membranes, and nuclear and mitochondrial membranes. Although results obtained from the proteomic analysis suggested the absence of mitochondria and nuclear membranes in the sperm light-density fractions, DRM proteins coming from the inner and outer acrosomal membranes were also found in these fractions. The results of this study are in agreement with this possibility and with the assorted origin of plasma membranes contributing to the light-density fractions. In the tail, localization of BSG, SLC2A3, and FLOT2 suggests that DRM can be found along the different sections comprising the sperm flagella. The results in this study provide some other insightful clues for the sperm head. IZUMO cannot be detected on live sperm and is only exposed when plasma membrane integrity is disrupted by freezing, permeabilization, or the acrosome reaction [50, 51] ; accordingly, it is hypothesized that this protein localizes to the inner acrosomal membrane. In this respect, the association of IZUMO with the light-density fraction provides evidence that the inner acrosomal membrane contributes to the total light buoyant DRM fractions. Even an ephemeral structure like the cytoplasmic droplet seems to preserve DRMs, as suggested by the highly restricted location of TEX101 in the light-density fractions and by the results of the immunofluorescence studies. These results confirm studies [37, 49] supporting the heterogeneity of DRMs with regard to their protein and lipid composition.
Pursuing our analysis on the putative involvement of DRMresident proteins in sperm function, the location of selected proteins associated with the light-density fractions was reanalyzed in sperm incubated under capacitating conditions. 7 . Schematic representation of the distribution of DRM proteins in sperm compartments depicting the results obtained using the antibodies directed against the different DRM-resident molecules analyzed in the study; HK1 localization as described previously [49] is also shown. As already noted in the text, IZUMO and FLOT2 demonstrate a different immunofluorescence pattern after the acrosome reaction (AR).
Although no relevant changes were noticed with capacitation, modifications in the immunofluorescence pattern of some proteins were observed in acrosome-reacted sperm. The acrosome reaction involves an extensive reorganization of the membranes located in the anterior head of the sperm cell. Multiple fusion points between the plasma and outer acrosomal membranes induce a fenestration process during which the components originally located in the apical portion of the sperm are lost [1] . After fusion, the inner acrosomal and plasma membranes become continuous, assembling a new sperm surface resulting from the mixing of their respective components. Caveolin 2, originally found in the anterior head of the sperm, could not be detected after the acrosome reaction, suggesting that this antigen is likely located at the plasma membrane covering the acrosomal cap and is consequently lost during exocytosis. In the case of GM3, although no signal was found on reacted sperm, the original localization of this ganglioside did not predict this behavior because the equatorial segment is excluded from the vesiculization process that takes place during the acrosome reaction [1] . Although the possible loss of GM3 cannot be ruled out, considering that anti-GM3 recognizes the glycoside portion of the GM3 molecule [42] , the lack of signal of acrosome-reacted sperm could also be due to the removal of the respective epitope by acrosomal glycosidases released during exocytosis [1] .
In contrast to CAV2 and GM3, the other two proteins analyzed, IZUMO and FLOT2, can still be detected by immunofluorescence in acrosome-reacted sperm, although they showed a different localization compared with the one originally displayed in nonreacted cells. In intact sperm, IZUMO was restricted to the dorsal portion of the sperm head; after the acrosome reaction, it distributed to a new region opposite to the anterior acrosome, including part of the postacrosomal region. Different IZUMO staining patterns have been previously observed [50] , but their connection with antigen relocation due to the acrosome reaction has not been analyzed in detail. Concerning FLOT2, the staining formerly observed in the midpiece did not change with the acrosome reaction, but the signal located in the apical portion of the head extended along the acrosomal domain, revealing the presence of FLOT2 in the equatorial segment in cells that underwent the acrosome reaction. In addition to FLOT2, several proteins that have been reported to relocate after the acrosome reaction such as SPAM1, CRISP1, and IZUMO are present in the light buoyant DRM fractions [51] [52] [53] . This common feature suggests that changes in localization could be related to these membrane substructures rather than to a specific aspect of the individual proteins.
Although both IZUMO and FLOT2 change their location after the acrosome reaction, they do not seem to colocalize. While FLOT2 is restricted to the equatorial segment, IZUMO can move farther, crossing to the ventral para-acrosomal sperm domains. These compartments are separated by a specialized structure that seems to be defined during epididymal maturation and involves tyrosine phosporylation [54] . Other proteins reported to redistribute after the acrosome reaction are localized to the equatorial segment and are not able to pass the postacrosomal boundary [52, 53] . This suggests that protein redistribution after the acrosome reaction is a regulated process that may comprise a particular mechanism for IZUMO. It is a matter to be resolved why only IZUMO among all the proteins that move after the acrosome reaction is able to cross the postacrosomal boundary and whether this has a functional significance considering its relevance in sperm-egg fusion [55] .
The membrane reorganization taking place during the acrosome reaction involves an additional change of high functional significance: the sperm becomes capable of fusing with the egg. Although IZUMO has been shown to be necessary for sperm-egg fusion [55] , the molecular mechanisms conferring fusogenic ability to sperm are not well understood. Membrane microdomains and their resident molecules have been implicated in processes requiring cell-cell fusion such as invasion [56, 57] . With respect to the molecules depicting a different inmmunofluorescence pattern after the acrosome reaction, while FLOT2 has been positively correlated with metastatic processes [58] , GM3 has been negatively associated with cell motility and invasiveness [59] . In the sperm, we can speculate that the presence of GM3 is associated with a nonfunctional equatorial segment and that GM3 modification or loss may be needed for the sperm to acquire the ability to fuse with the egg. Taking into consideration that IZUMO has a direct role in sperm-egg fusion as suggested by the null mutant phenotype [55] , the putative participation of those regions where IZUMO localized after the acrosome reaction in sperm-egg fusion should be reanalyzed. Global Expression Profiling in Epileptogenesis: Does It Add to the Confusion? Since the inception of global gene expression profiling platforms in the mid-1990s, there has been a significant increase in publications of differentially expressed genes in the process of epileptogenesis. In particular for mesial temporal lobe epilepsy, the presence of a latency period between the first manifestation of seizures to chronic epilepsy provides the opportunity for therapeutic interventions at the molecular biology level. Using global expression profiling techniques, approximately 2000 genes have been published demonstrating differential expression in mesial temporal epilepsy. The majority of these changes, however, are specific to laboratory or experimental conditions with only 53 genes demonstrating changes in more than two publications. To this end, we review the current status of gene expression profiling in epileptogenesis and suggest standard guidelines to be followed for greater accuracy and reproducibility of results. The understanding of epilepsy as a complex interaction between numerous excitatory and inhibitory neuronal connections influenced at the molecular level rightly brings about focus to global changes in the entire transcriptome in the epileptogenic process. Annotating the individual genomic changes at specific time points delivers only a snapshot of a much larger process. The development of global gene expression profiling platforms has revolutionized molecular biological research, allowing vast amounts of information to be identified, catalogued and measured. Genomic changes are seen in concert giving the potential for a broader understanding of the disease process.
The late 1990s heralded the development of several important technological advances in molecular biological research. DNA microarray and serial analysis of gene expression (SAGE) were introduced allowing comparisons of global gene expression utilizing differing conditions in laboratory experimentation. This has allowed the relatively new field of comparative genomics to flourish (43, 54) . With respect to the brain, patterns of gene expression have been reported as early as 1999 with comparative libraries generated between malignant glioma tissue and normal brain for SAGE, and between sleep-wake cycles for microarray (40, 55) . From this point there has been an explosion of genomic information with steadily increasing reports generated during development and aging (38, 94, 106) , malignant brain processes (37, 101) , neurodegenerative disorders (41, 58) , behavioral (12, 45) and anatomical studies (18) . Studies into epileptogenesis have also expanded since the first global gene expression studies into epilepsy in 2001 (29, 42) . For this reason we will review the published papers and discuss the usefulness of global transcriptome analyses with regards to epileptogenesis.
Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological and social consequences of this condition. An epileptic seizure is a transient occurrence of signs and/or symptoms caused by the abnormal excessive or synchronous neuronal activity in the brain (27) . It includes seizures occurring in the absence of a recognized cause as well as events occurring in patients with antecedent stable (non-progressing) central nervous system insults (unprovoked seizures), but excludes seizures occurring in a close temporal relationship with an acute systemic, toxic or metabolic insult, which is expected to be the underlying cause (provoked seizures) (28) .
Within the broad classification of epilepsy, mesial temporal lobe epilepsy (MTLE) is the most common location-related epilepsy. This may be lesional or non-lesional, with the great majority of non-lesional MTLE being associated pathologically with hippocampal sclerosis (HS). There remains much debate in the neurological world regarding HS as a cause or consequence of seizures. Pathological evidence has shown the presence of HS in brains of dementia patients without clinical epilepsy (19, 104) . In addition, MTLE patients with dual pathology may have HS that is: (i) a non-specific result of the primary epileptogenic lesion and not in itself epileptogenic; (ii) secondary to the primary epileptogenic lesions but also epileptogenic; or (iii) a primary HS that coexists with another epileptogenic lesions (109) . Proponents of the initiating hit theory of epilepsy argue that the progressive nature of MTLE demonstrates HS as a consequence of the seizure process. Typically a variable latent period between the initial precipitating incident and habitual unprovoked seizures is present, followed by a silent period between the first habitual unprovoked seizure and the onset of intractability. This strongly suggests that the pathology is progressive (5) . Worsening HS creates a cycle whereby increased seizure severity and frequency lead to further neuropathological changes and hence worsening clinical seizures. Several theories for the cause of HS have been proposed, with the most commonly quoted being febrile convulsions. Other theories include vascular injuries and developmental abnormalities. It is this process of progression to seizures of worsening severity with increasing frequency that is termed epileptogenesis (Figure 1 ).
HS was first described in 1825 by Bouchet and Cazauvieil (8) . It is a pathological diagnosis although advances in neuroimaging through magnetic resonance imaging had allowed the use of radiology as a supporting or diagnostic tool. The pathological diagnosis consists of atrophy of the hippocampal formation with loss of neurones and reactive gliosis in CA1, CA4 and the dentate gyrus. In CA4, there is a loss of polymorphic neurons and pyramidal neurons. CA3 shows some neuronal loss, but CA1 always shows significant neuronal loss that may be very severe. There is a variable degree of gliosis seen in these subregions and occasional vessel sclerosis has been described. Loss of granule cells in the dentate gyrus is variable, as is gliosis. Granule cell distribution is often disrupted and various patterns including dual lamination or dispersion are described. Sprouting of the mossy fiber system in the dentate gyrus is apparent with Timm's stain with many believing that this sprouting leads to aberrant reinnervation resulting in the hyperirritable lesion that constitutes the epileptogenic hippocampus. Within all these changes, CA2 is relatively spared (68) (Figure 2 ). Treatment of non-lesional MTLE continues to be a challenge for the epileptologist. Monodrug therapy is initially implemented however this may progress quickly to multidrug therapy because of continued poor seizure control. Targeted surgical excision of the epileptogenic zone in carefully selected medically refractory patients has been shown to improve outcome with 44% to 72% of patients remaining seizure free compared with 4.3% to 12% of patients on further trials of anti-epileptic medications (6, 108, 111) . Despite these figures, there remains a large proportion of epileptic patients who continue to suffer unpredictable recurrent seizures. Understanding the molecular biological changes underlying the process of epileptogenesis is crucial to the development of newer, more effective treatment regimes.
SAGE and DNA microarray generate large libraries of mRNA sequences enabling researchers to generate differential gene expression lists in disease states by statistical comparison of transcript frequencies between two or more conditions. Both originally described in 1995, the platforms have been extensively used in the study of human disease with millions of tags analyzed and compared with easily accessible online tag libraries. Massively parallel signature sequencing (MPSS) was developed in 2000 as an alternative quantitative open-ended platform and has risen in popularity as a research tool. A brief description of each platform follows and comparison of their relative advantages or disadvantages can be seen in Table 1 . 
SAGE as originally described is based on two basic assumptions: (i) that a short nucleotide sequence tag (9-10 base pairs) contains sufficient information to uniquely identify a given transcript; and (ii) concatenation or linking of the so-identified short sequence tags allows the efficient analysis of transcripts in a serial manner by the sequencing of multiple tags within a single clone (103) .
Practically, the genome of interest is interrogated from a defined four-base pair position, classically the NlaIII site. An anchoring enzyme binds to this defined complementary position and acquires adjacent short mRNA sequences (9-10 base pairs) that, based on the first assumption earlier, allows specific mapping of these transcripts to genomic libraries. Proportionate amplification of differ-ent transcripts gives a realistic quantification of gene expression for the particular experiment or disease condition (67, 103) .
SAGE has been utilized extensively in the study of human disease. In the first 5 years since its inception, close to five million tags had been analyzed using this method (102) . It has been used in cancer, cardiovascular and immunological studies (74, 76, 77) . Large online SAGE libraries are now freely available to the public, with access to raw SAGE sequence data, precomputed tag extractions and several modest analysis tools. SAGEmap currently contains over two million tags from 47 SAGE libraries and is currently available at http://www.ncbi.nlm.nih.gov/sage (56) .
In terms of data analysis, SAGE is extremely efficient with a complete genome investigation allowing both whole activated and inactivated pools of gene analysis in a quantitative and qualitative (20, 66, 67, 103) . Several possible key pitfalls have been identified in the SAGE technique. A high amount of starting mRNA is required that is often prohibitive and not feasible, especially in difficult to obtain human specimens or complex experimental procedures. In addition, the cost of SAGE is high and may preclude repeat experimentation to verify collected data. Although SAGE studies the entire genome, the use of a four-base pair anchoring enzyme relies on the fact that the majority of mRNA sequences contain an anchoring site on average every 256 base pair stretch (4 4 = 256). The reality is a small proportion of mRNA will not include this anchoring site, leading to a loss of some small fraction of mRNA from the analysis that is extremely difficult to quantify. Clearly the use of a 10-base pair sequence to map gene assignment may cause issues in terms of specificity and sensitivity. It is not uncommon for multiple genes to share the same tag or for a single gene to be mapped by multiple tags.
There have been several modifications to the original SAGE technique aimed at addressing the potential pitfalls. MicroSAGE and SAGE-Lite were developed to allow lower amounts of starting mRNA to be used allow significantly smaller amounts of human or animal specimens to be examined (76, 78) , whereas LongSAGE and Robust-LongSAGE were developed to improve tag or gene mapping by generating tags of 21 base pairs as opposed to 14 (35, 85, 105) .
Microarrays are ordered samples of DNA with each sample representing a particular gene. These arrays are then assayed for changes in gene expression following experimental treatment or in different disease states. It allows simultaneous monitoring of thousands of genes, thus providing a functional aspect to sequence information in a given sample (23) . There are several types of microarray technologies currently in use with studies using oligonucleotide microarray technology, the most common for high-throughput quantitative studies of RNA expression (47) . In this instance, RNA extracted from the material of interest is reverse transcribed to complementary DNA and incubated with a mixture of fluorescently labeled markers at set conditions on a microarray chip containing a predetermined set of genes. Quantification is then performed by computerized measurements of fluorescent intensities. Microarray is a relatively simple procedure to perform and is becoming a standard technology for research laboratories worldwide. They allow testing of thousands of genes simultaneously with quantification of differential expressions at a reasonable cost when compared with SAGE. However, microarray technology relies on prior knowledge of the genes to be identified with the possibility of selection bias during creation of the cDNA array. As a result, novel genes are not able to be identified. Second, the amount of starting material required was also prohibitively high in the initial stages. This has largely been addressed with a two-stage hybridization process or global RNA amplification prior to running the array (73, 100) .
MPSS was developed by Brenner et al in 2000 to address the variabilities, cost and scale of effort required by other high-throughput, genomic profiling methods. It utilizes the technology of DNA megacloning in combination with non-gel-based signature fluorescence sequencing to generate very large numbers of short read-length sequences (9) . This process of megacloning allows the in vitro cloning of fragments of cDNA onto microbeads as opposed to biological hosts. Millions of cDNA fragments can be cloned by hybridization of uniquely tagged molecules to microbeads carrying 10 6 complementary sequences to each oligonucleotide primer used (10) .
MPSS has been used to create large databases of gene expression in adult human tissues (50) . These have proved complementary to other database sets published using Affymetrix chip technology (89, 92, 93) . It has been used to document the transcriptome in human disease states including oncology (49, 61) , renal disease (30) and neuroembryology (11) .
Similar to SAGE, massively parallel signature processing does not require previous knowledge of any sequence to allow analysis. The only requirement is that the microbead library is constructed large enough to contain nearly all sequences from the library to be compared against. It is time efficient with simultaneous screening of all genes and counts virtually all mRNA molecules. The libraries created are more specific as MPSS generates a 17-nucleotide signature length (95% unique) as compared with a 14-nucleotide signature length (80% unique) in a conventional SAGE. MPSS also allows the creation of an extremely large data set of signature sequences (>1 000 000) by providing a depth of analysis that allows low abundance genes to be accurately quantified, something that SAGE has been criticized for missing with datasets being comprised of only 20 000 to 60 000 sequenced mRNAs (9, 83) .
Quantification of sequences is possible as MPSS generates a digital output. Differential expression comparisons can be formed with ease between samples with detection levels for statistical significance possible even with low abundance genes expressed at 30 to 40 copies per million. In contrast, hybridization techniques require replication of experiments, high abundance genes and large differences to provide adequate quantification (83) .
Reproducibility of the levels of gene expression is generally very good for signatures detected in all samples. However, cases with a zero signature count have been noted to demonstrate a significantly higher error rate than those with non-zero figures. It remains unknown as to whether this represents a true measurement or the absence of measurement. In cases with conflicting values (ie, one MPSS run claiming a value and the other claiming zero), the trends obtained by ignoring the zero measurements within runs of the same sample, are shown statistically to be closer to the ideal unbiased comparisons between two samples. As such, taking the maximum number of two runs has been suggested (83, 91) .
Seizure induction and epileptogenesis have been modeled extensively in animals. Epileptogenic substances such as alumina gel, penicillin, bicuculline, kainic acid, tetanus toxin, pentylenetetrazol and pilocarpine were directly injected to induce a primary focus of epileptogenic activity in the immediate area of placement with corresponding marked neuronal loss and gliosis, a process termed chemical kindling (60, 70) . This was more often than not accompanied by behavioral seizures including status and subsequent spontaneous seizures, and is also described as the "status epilepticus models." Other methods include the electrical kindling models first described by Goddard et al in 1967, whereby repeated stimulation of selected regions of the mesial temporal lobe structures result in behavioral seizures and the classical neuropathological changes seen in the human MTLE. The electrical kindling models also demonstrate seizures with progressive increase in frequency and severity following prolonged stimulation. A chronic increase in seizure susceptibility is seen; however, in contrast to the status epilepticus models, spontaneous seizures are unusual and not expected.
Status epilepticus models of epileptogenesis are widely used as a model of acute epileptogenesis following the application of chemical kindling. It becomes a useful model of chronic epileptogenesis with the appearance of spontaneous seizures at a time point distant to the initial convulsive event. This model demonstrates the classical progression of human temporal lobe epilepsy with an initial convulsive event leading to seizures and status epilepticus before a latency period of varying length and appearance spontaneous seizures following this. Advocates of this model point to the presence of a latency period as the period of opportunity, whereby interruption of the epileptogenic process during this time may bring about a stop in the progression of spontaneous seizures. The difficulty lies in measuring the duration of each animal's individual latency period and hence the optimal time point for intervention and study. The duration of latency is known to be related to the convulsant dose with higher doses yielding shorter onset latencies (39) . Current figures suggest a mean onset latency of around 40 days; however, shorter periods have been described (32) . As such, estimates of latency periods used in research studies vary from 8 to 50 days.
Mathern et al performed the only electrophysiological study looking into the time course of hippocampal interictal spike frequency after intrahippocampal kainite injection and correlated this with histopathological changes. Four stages were classified from this with: (i) the acute phase relating to the first 10 days after kainite-induced status epilepticus; (ii) the active phase from days 10 to 30; (iii) the latent phase relating to the latency period (days 30 to 90); and (iv) the chronic phase with the development of chronic hippocampal seizures (after 90 days). Interictal spike frequency was noted to drop during the latent phase and increase again during the chronic stage in concert with the development of behavioral seizures, neuronal loss and mossy fiber sprouting. (69) Status epilepticus models also demonstrate a varying clinical response of the animal subjects to differing doses of convulsive agents. A high mortality rate is not unexpected and severe neuronal loss is often encountered histologically. Initial seizures are unpredictable in their duration and severity and may be difficult to control. Acute antiseizure medications such as benzodiazepines are often needed to bring about a cessation of the status epilepticus state. It is clear, however, that the onset of spontaneous seizures is directly related to the duration of the status epilepticus state and at least 30 minutes duration of initial status epilepticus is required for manifesting an epileptogenic process (53) .
Kindling as reported by Goddard (34) refers to the repeated administration of subconvulsive electrical stimuli resulting in progressive development of seizure activity traditionally performed using daily stimulations. The term is therefore quite specific to low level electrical stimulation. Numerous alterations in stimulating paradigms have been used since Goddard's initial description of kindling and it is now well established as a chronic animal model of MTLE. It is accepted as a functional model of epilepsy where altered neuronal response develops in the absence of the gross morphological damage seen in many other epilepsy models.
The discovery of electrical kindling placed the process of epileptogenesis under greater control by the experimenter. The initial electrical kindling current stimulus was applied to a defined area and was, by definition, subconvulsive (33, 34) . With repeated application of the electrical stimulus once or twice a day for only seconds at a time, electrical afterdischarges were observed to appear and gradually lengthen, and recordable after-discharges were observed to broaden in origin from adjacent brain regions to more distant regions and eventually the opposite hemisphere. In addition to this electrical progression, a behavioral progression of seizure activity occurred commencing with focal seizure activity typically manifested as initial facial clonus contralateral to the side of stimulation after a short delay. Progression through a stepwise series of more overt and eventually generalized seizures follows. This behavioral progression has been assigned a grading system, from grade 1 to 5 by Racine (81) in his study of amygdala kindling in rats. An animal may be regarded as fully kindled after manifesting a number of grade 5 seizures.
There are many advantages of the kindling model for epilepsy research. Development of chronic epileptogenesis by a precise focal activation of the target brain site is readily achieved with no mortality and very little morbidity. The pattern of seizure propagation and generalization is readily monitored and interictal, ictal and postictal periods are easily manipulated. Furthermore, the absence of the classical neuropathological changes and placement of the stimulating electrode at an area distant from the anatomical area of interest (eg, amygdala kindling for investigation of hippocampal pathology) ensures genomic expression studies are as unadulterated as possible.
Kindling experiments, however, are relatively labor-intensive, requiring a two-stage electrode implantation-electrical kindling process usually spaced over 1 to 2 weeks. Stimulation paradigms are also spaced over minutes to days and may take months to reach the fully kindled state. The ideal animal model for human temporal lobe epilepsy would consist of similar pathology, a latent period after the initial insult appropriate for the species, a state of chronic hyperexcitability and the emergence of spontaneous seizures after a latent period (107) . The absence of a true latent period in electrical kindling is in opposition to this. Latency is seen from the first kindling stimulation to the eventual appearance of seizures; however, additional insults in the form of further electrical stimuli are being provided leading up to and merging with the evoked seizures. Electrical kindling also does not produce spontaneous seizures, unless prolonged kindling is performed. This is coined the "over-kindling" model (79) . In this model continued electrical stimulation is given beyond the standard stage-5 criterion of the fully kindled state. The number of additional stimulations is extremely variable with recent reports suggesting at least an additional 90 to 100 kindled seizures required in rats for recurrent spontaneous seizures to occur (71, 87) .
Similar to human changes there may be considerable variability in neuropathological changes. Status epilepticus models classically demonstrate concordant human histological changes of severe neuronal loss, granule cell dispersion, remodeling of mossy fiber structures and neurogenesis. The degree of neuronal loss is related to the concentration of inciting agent and duration of status epilepticus. Neurogenesis has been reported without corresponding cell death whereas granule cell dispersion may occur without evidence of neurogenesis (72, 98) .
Electrical kindling models typically do not demonstrate evidence of neuronal loss, the advantage of which is purported of enabling focused study into the effects of seizures alone without the changes associated with cell death.
An increasing number of laboratory and clinical studies involving MTLE have been published since the inception of global expression profiling platforms. Prior to this the focus of each laboratory was limited to one or a few genes selected by hypotheses generated from the available literature or laboratory experience, using conventional molecular biological techniques. These global expression studies are summarized in Table 2 .
The predominant profiling platform utilized in global expression profiling of epilepsy and epileptogenesis has been microarray. A Pubmed search utilizing the terms "microarray" and "epilepsy" or "seizures" revealed 49 reports from which there were 10 articles publishing results of global expression profiling in models of MTLE. Bibliographic review of these articles revealed an additional two articles giving a total of 12 reports.
The majority of microarray papers published with regard to epileptogenesis involve chemical kindling. These differ in terms of Tang) , directly compared the genomic expression changes with certain physiological and disease states. In this manner, several assertions were made. Elliott et al suggested support for the hypotheses of parallels existing between gene expression underlying development and epileptogenic plasticity during the latency period following comparisons with the adult and developing rats (4, 24, 95) .
Direct comparison of the genomic responses of brain in response to ischemic stroke, intracerebral hemorrhage, kainic acid induced seizures, hypoglycemia and hypoxia was made by Tang et al in 2002. Significantly, marked overlap of regulated genes was found throughout all disease states. In particular, all genes induced by kainic acid were also induced by ischaemia, hemorrhage or hypoglycemia. This suggests a multifactorial mechanism of injury for kainic acid induced seizures that mimics or utilizes the same pathways as those associated with ischaemia, hypoglycemia or hemorrhage (95) . Whether seizures themselves primarily induce these genomic changes or secondarily act through periods of ischemic or hypoglycemic during seizure episodes remain unclear. What is clear, however, is the similarities in histological changes seen in these conditions with marked neuronal loss, fibrillary gliosis with vacuolation and enlarged reactive astrocytes variously reported following severe hypoglycemia and ischemia, with or without associated status epilepticus (1, 13, 51, 52) .
Becker et al correlated temporal changes of genes associated with cellular stress and injury at 3-day post-status epilepticus, genes associated with cytoskeletal and synaptic reorganization at 14-day post-status epilepticus, and genes involved in neurotransmission pathways at the chronic epilepsy stage. This study also provided the first comparison with human hippocampal specimens with eighteen genes differentially expressed in both the chronic stage of pilocarpine induced epilepsy and medically refractory MTLE (4) .
Other experimental microarray studies investigated changes of growth factor signaling genes, transcription factors, angiogenesis signaling molecules, neuropeptides, neuronal plasticity and signal transduction using custom-made probe sets at varying time points. These are summarized in Table 2 (44, 63, 110) . The electrical kindling model was utilized by Lukasiuk et al to investigate the gene expression changes during specific phases of epileptogenesis with the hypothesis that remodeling of neuronal circuits underlying epilepsy is associated with altered gene expression during epileptogenesis. RNA was extracted from the hippocampus and temporal lobe at 1, 4 and 14 days following electrical kindling and hybridized with cDNA arrays containing approximately 5000 rat gene probes. There were 87 differentially expressed genes within the hippocampus with 37 genes regulated at 1 day, 12 at 4 days and 14 at 14 days. There was an overlap of 13 genes with the temporal lobe that demonstrated 208 differentially expressed genes in a similar time distribution as the hippocampus genes. Functional annotation of the regulated genes revealed genes involved in neuronal plasticity, gliosis, organization of the cytoskeleton or extracellular matrix, cell adhesion, signal transduction, regulation of cell cycle and metabolism (63) .
A literature search using the words "serial analysis of gene expression" and "epilepsy" and/or "seizures" produced four articles. Two articles were published from the same laboratory with one expanding on the initial report, whereas the other two studies involved human MTLE and the use of a genetically modified epileptic rat, the Ihara rat.
There is only one published SAGE report using the rat electrical kindling model of mesial temporal lobe. Unlike most electrical kindling models, marked neuronal cell loss and gliosis is seen histologically in the electrical kindling model utilized by Hendriksen et al with a latent period of between 1 and 2 weeks following electrical kindling of the angular bundle and the appearance of spontaneous seizures. As such, hippocampal harvest was performed 8 days after the final electrical stimulation, prior to the commencement of spontaneous seizures and during the process of epileptogenesis. Over 10 000 tags were analyzed in both stimulated and control groups resulting in SAGE libraries containing 5053 and 5919 different unique tags respectively. There were 92 differentially expressed genes, predominantly associated with ribosomal proteins, protein processing and axonal growth and glial proliferation (42) .
SAGE was also performed on hippocampi of the Ihara epileptic rat (IER). The IER is an animal model of MTLE that demonstrates progressive limbic seizures, eventually resulting in spontaneous generalized tonic-clonic seizures at 5 to 6 months of age. SAGE was performed on hippocampal tissue taken at 2 months of age and compared with the Wister rat. Over 7000 tags were analyzed with creation of a SAGE library for each group consisting of 2492 and 2141 genes, respectively. Eighty-one differentially expressed genes were identified, with genes associated with neurotransmission and intercellular component downregulated, and genes associated with protein synthesis, metabolism, membrane transport, the cytoskeleton and ion-channels upregulated (2).
Our laboratory has performed SAGE on adult male C57/BL6 mice following rapid electrical kindling of the amygdala. This model of MTLE is useful in allowing assessment of the effect of seizures during epileptogenesis without the confounding effects of neuronal loss and cell death (90) . RNA was extracted from hippocampi taken 3 h following the completion of kindling with around 28 000 tags analyzed in control and stimulated groups. In the control group, 13 213 unique tags were identified whereas 12 302 unique tags were identified in the stimulated group. There were 55 differentially expressed genes when comparing between the groups with functional classification denoting immune response, cellular metabolism, axonal growth and regeneration, signal transduction, ion transport, synaptic and neurotransmission and genes of unknown identity or function predominant (Wang et al, in preparation).
There has been a single published report using MPSS for gene expression studies during epileptogenesis. Potschka et al utilized the rat kindling model of epileptogenesis to characterize the genomic changes using MPSS. Seven thousand and four signatures with a minimal occurrence of two clones in each run were analyzed resulting in annotation of 5696 identified genes. Two hundred sixty-four differentially expressed genes were identified with 128 upregulated and 136 downregulated ones. The immediate early gene Homer 1A was most strongly induced at 2 h. Identification of this gene fueled further research into the possibilities of Homer 1A being an intrinsic anti-epileptogenic and anticonvulsant gene upregulated as a protective measure during epileptogenesis (80).
There remains a relative dearth of global gene expression studies involving human specimens. This is somewhat surprising given that mesial temporal lobectomy, hippocampectomy and amygdalectomy is a well-accepted treatment option for medically refractory MTLE in carefully selected cases giving a readily available source of hippocampal specimens (96) . Four studies have been reported utilizing microarray (3) and SAGE (1) of which only three dealt with MTLE patients.
Jamali et al analyzed the entorhinal cortex from the hippocampus of brains removed as a surgical treatment consisting of a standard anterior temporal lobectomy whereas Özbas-GerÇeker et al did not differentiate between hippocampal subregions. Both studies attempted to find control tissue in the form of autopsy hippocampal specimens; however, this obviously posed a significant problem in terms of reproducibility of "controls" not to mention the ethical dilemma. In an attempt to factor in a bias related to long-term exposure to anti-epileptic medications, Jamali et al also compared samples from autologous adjacent lateral temporal cortex that had been subjected to the same environment as the epileptic tissue.
Comparison of the entorhinal cortex with non-epileptic lateral temporal lobe revealed 16 regulated genes of which 10 were upregulated and six were downregulated. This study suggested dysregulation of the neurotransmission and complement systems within the entorhinal cortex as another pathway affected in the process of epileptogenesis (46) . On the other hand, the study by Özbas-GerÇeker et al revealed 143 differentially expressed genes that matched functionally to genes associated with basic metabolism, transcription regulation, protein synthesis and degradation, signal transduction, structural proteins, regeneration and synaptic plasticity and genes of unknown identity or function (74) .
Neither study attempted to perform a comparative approach using animal models with their human tissue that would add validity to any prior or future reports into this topic. On the other hand, the study by Becker et 
Proteomics is a technique that enables one to find proteins changed by the cells response to internal states, external stimulations or developmental cortex. The technique of the two-dimensional elec-trophoresis (2DE) allows global profiling of the proteomic changes in different disease states or conditions. Two studies into chemically induced epilepsy (pilocarpine) have investigated the proteomic profile following epileptogenesis in the whole hippocampi alone or in conjunction with the forebrain in rats. These suggested upregulation of pathogenic, neuroprotective and neurogenic responses with validation performed using Western blotting and immunohistochemistry (36, 59) .
A single study by Eun et al (25) attempted to analyze human temporal lobe specimens in patients with MTLE undergoing surgical resection by performing 2DE and Western blotting of the selected proteins. Control specimens were obtained from lateral temporal cortices of patients undergoing temporal lobe operations for tumors in the absence of seizures. This study reported significant changes in nine proteins with particular emphasis on proteins related to oxidative stresses; it also highlights the shortcomings in performing comparative studies in humans with control specimens coming from a variety of different diseases such as glioblastoma multiforme (7), sphenoid wing meningiomas (3) and malignant lymphomas (2) .
The advent of global expression profiling technology has led to an ever increasing amount of genomic information available. To the end of 2006, there are over 1100 published reports on microarray studies involving the brain and brain disorders, with nearly 100 SAGE studies of the same. For epilepsy studies the figures reveal 32 microarray and four SAGE studies (Figure 3 ).
Yet for all the additional knowledge that has been acquired, the outcomes of epilepsy treatment, particularly MTLE, remain in the main stagnant. There is as yet no active intervention to prevent progression of epilepsy either from the "initial insult" to the "latency period," or from the latency period to chronic epilepsy. The question follows then: what is the benefit of large-scale gene expression profiling? Is the accumulation of knowledge of benefit to the treating epileptologist or does it create more confusion in the treatment of this already complex condition?
The inherent problems with analyzing and applying data generated from global expression profiling stems from the obvious difference in epilepsy models, the profiling platform used, animal type and laboratory environment. Care must be taken in comparing genomic profiles generated from differing epilepsy models. Genomic reactions are clearly different between different species of the same animal (86) . These differences must be accentuated even more when comparing the different profiles in mice, rats and humans. Status epilepticus models of epileptogenesis possess the advantage of a clear-cut progression from initial insult to chronic epilepsy in contrast to electrical kindling where a predominantly time-dependent protocol is usually administered. The rapid electrical amygdala kindling model utilized in our laboratory allowed the effects of seizures in isolation to be investigated without the confounding effects of neuronal loss. In contrast, status epilepticus models of epileptogenesis demonstrate marked neuropathological changes and may manifest widespread systemic effects. The use of benzodiazepines to halt prolonged severe status epilepticus in these models may also adversely alter the transcriptome.
Tang's comparisons between kainic acid-induced seizures, hypoxia, hypoglycemia and hemorrhage suggest that the kainic acid-induced changes utilize the same pathological pathways as the other three conditions (95) . Indeed there are clear interrelations between all such conditions with prolonged seizures resulting in metabolic acidosis and an environment of relative hypoxia and hypoglycemia, and prolonged and severe hypoglycemia resulting in seizures in the generalized tonic-clonic form. The eventual histopathological outcome from these events culminates in varying degrees of neuronal loss, gliosis and even necrosis. If this is the case, it is difficult to proclaim that the genetic expression changes are actually specific to the process of epileptogenesis. Another study reported in abstract form (21) and cited in Lukasiuk et al's review revealed distinct differences in gene expression between the kainic acid and pilocarpine models of epileptogenesis, despite being studied in as exacting an environment of the same laboratory.
The pathology of HS deserves special mention at this point. It remains a point of controversy as to whether HS is the underlying cause, or end result of the clinical syndrome of MTLE. As a cause, the proposed theories underlying HS include childhood febrile convulsions, perinatal hypoxic events or vascular accidents, whereas as an end result it is suggested repeated seizures progressively inflict end-organ damage, particularly in the susceptible areas of the hippocampus described earlier. In a molecular biological sense, a causative action would allow quantification of gene expression changes of each cell type whereas as an outcome, genomic profiling would be representative of changes in cell population.
Confounding the arguments are the documented cases of normal hippocampal size and histology on positively electrophysiologically localized epileptogenic foci excised as a curative procedure (15) as well as autopsy specimens demonstrating histological changes consistent with HS in the absence of clinical seizures (48) .
The unique cerebral responses in different individuals must be kept in mind in all laboratory and translational research studies.
The number of published differentially expressed genes during epileptogenesis is fast approaching 2000. On review of the available data from these publications there is minimal conformity between the genes described. Lukasiuk et al has produced a list of common differentially expressed genes involving models of epileptogenesis, including traumatic brain injury studies (62) . From the near 2000 genes, only a small number (53) are noted to be differentially expressed in more than one study. However, if the gene profiles of traumatic brain injury studies are filtered out, only 38 genes remain differentially expressed across studies. In addition, a proportion of these common genes appear to be differentially expressed in conflicting directions.
In order to further assess the consistency of altered gene expression across the studies we annotated the list of commonality differentially expressed genes to include studies published to the end of 2006 as well as our currently unpublished SAGE data. Comparisons were performed using all available published lists as well as accessing complete gene lists made available through online downloads. Differences in epilepsy models and global expression profiling platforms were also recorded. There were 72 genes reported to be regulated during epileptogenesis in two or more studies. Thirty-nine genes demonstrated a consistent direction of change with 15 showing conflicting results. The direction of regulation could not be ascertained from the available literature in 20 genes. No genes were reported regulated in more than five papers with the majority (50) only regulated in two papers. The two genes consistently regulated in five reports were the rather ubiquitous glial fibrillary acidic protein and the S100related protein (Table 3 ).
Animal model The functional classification of these genes is also interesting to review. The gene ontology classification of cellular component, biological process and molecular function has been used for functional classification by the majority of authors whereas several have utilized functional groups described previously in the literature including immediate early genes/transcription factors, calcium homeostasis, intra/extracellular signaling, synaptic/vesicular, morphology, cell cycle/fate, injury/survival, metabolism and unknown. A list of the most prominent biological process regulated as described by each author is seen in Table 4 . It is clear that there is a wide range of biological processes involved in the epileptogenic process. These include basic cellular metabolism, regulation of transcription, protein processing, synaptic transmission, response to injury/cell death and regeneration and immune response.
The different time frames of the analysis of the gene expression also contribute to the differences to the profiling results. Transcriptional changes immediately following seizures will encompass the response to hyperthermia, mild hypoxia or hypoglycemia, and even direct trauma from any cerebral or truncal impacts. This contrasts with gene expression during the latent phase of epileptogenesis whereby a period of recovery with substrate replenishment and remodeling occurs, and during the chronic epileptic states seen in human studies. Numerous studies have demonstrated the transient nature of transcriptional changes within their own epileptogenesis model, let alone between different models (57, 63, 110) .
Lukasiuk et al have recently presented in abstract form and in a review article, an extension to their previous analysis of gene expression during epileptogenesis. Identification of highly represented functional classification as well as individual genes that appeared across data sets was performed with cell death and survival, neuronal plasticity and immune response as the most prominent. Seventy individual genes were identified to be consistently regulated across at least three studies using specific time-point analyses (64, 65) . Our data indicates only 22 genes consistently regulated across at least three studies. This difference is explained by the inclusion of animal models of traumatic head injuries in the analysis by Lukasiuk et al that we did not include. The justification for their inclusion is the initial hit theory whereby an early insult, be it ischemic, traumatic or infective, sets into motion a series of molecular changes that lead into the latency period before chronic epilepsy. However, recent figures suggest only 2% to 5% of traumatic brain injury cases will develop epilepsy (75) . Second, posttraumatic seizures are more commonly a result of cerebral contusions leading to scarring and altered neuronal circuitry rather than the classical MTLE syndrome. Filtering out the head injury papers from Lukasiuk et al's study leaves only four consistently regulated genes, three of which are included in our list of regulated genes (Gadd45a, Ctsd, Ctss, Zfp36). The last gene, zinc finger protein 36 (zfp36) is only reported differentially expressed in different arms of the same study by Becker et al and was excluded from our analysis. The complexity of neuronal structure and organization within the mesial temporal lobe regions may contribute to this issue. In particular, the anatomical subregions of the hippocampus demonstrate both histological and functional differences in their composition and response to certain conditions. Whereas CA1 and CA3 are considered integral to the transmission of input signals, CA2 is considered a transitional zone with no clear function and the dentate gyrus is considered the main input pathway into the hippocampus, receiving afferents itself from the entorhinal cortex via the perforant pathway (31) . Increasing interest in gene expression profiling between hippocampal subfields has suggested a molecular basis underlying the previously defined anatomic subregions of the hippocampus (112) . This may further improve understanding of the differing histopathological responses described earlier.
Few reports have attempted to stratify global gene expression profiles in terms of hippocampal subfields. Indeed, of the reviewed articles, only three reported comparisons between subregions of the brain. Reasons for this may include the large amounts of starting mRNA needed for global gene expression platforms in general, or the difficulty in isolating individual subfields. The use of stereo dissection or laser microdissection to accurately isolate different hippocampal subregions as performed by Becker et al, Datson et al and Torres-Munoz et al have challenged these notions. This technique has been described to allow direct microscopic identification of specific cell types without appreciable loss of their DNA or RNA. Furthermore microarray analysis was able to be performed on the limited amount of mRNA extracted using double round amplification to provide sufficient material (4, 17, 99) . Marked differences (>700 genes) were found in the hippocampal response to chronic glucocorticoid exposure between CA3 and the dentate gyrus; however, 79 genes reliably identified in individual subfields were virtually undetectable when looking at whole hippocampal specimens. The practice of analyzing gross or pooled hippocampal specimens fails to take into account these obvious differences and may skew ensuing results.
Although global expression profiling has the advantages of being time efficient in analyzing large portions of the transcriptome, there are limitations to the sensitivity and accuracy. It is well documented that SAGE may inconsistently detect low abundance transcripts leaving small genomic expression changes induced by the experimental model or disease undetected. This is evident in examining the epilepsy studies discussed above with each generated database containing 5000 to 14 000 unique tags only, a significant decrease from the estimated size of the entire hippocampal transcriptome being over 30 000 (16, 26) . Furthermore, the sequencing error of SAGE is estimated at an average of 1% per base because of the fact they utilize a single-pass sequence in data processing. This accumulates to roughly 10% per 100 base pairs, leading to lower correct tags counts and artificially inflate the tag counts of already established tags or establish a count of a tag that does not exist. Microarray studies have also been shown to demonstrate significant variations even under the same experimental conditions. As such, validation of global expression profiling results by independent methods, such as real-time polymerase chain reaction (PCR), Northern/Western blotting and immunohistochemical methods, is crucial. In our experience, a significant decrease in the number of differentially expressed genes is demonstrated when validating SAGE results using quantitative real-time polymerase chain reaction (qRT-PCR) with a custom-made, low-density array chip of 94 significant genes as indicated by SAGE (82). Following the changes into the proteomic stage will further validate results of global expression profiling. Translation into functional proteins represents the final pathway of genomic regulation. Quantifying the proteomic changes specimens through immunoperoxidase stainings and in situ hybridizations will not only complete the interrogation process, but also give insights into region-specific changes that may have been missed in interrogation of gross hippocampal, possibly negating the requirement for microdissection of hippocampal subregions (Figure 3) .
In terms of gene expression platforms, the laboratory conditions play a vital role in the reproducibility of results. It is not unusual for the same experiments performed on different days to produce differing results. Similarly, experiments performed by separate scientists on the same days will also often produce differing results. Subtle variations in the complementary tags used to interrogate the transcriptome can also cause biased results. Specifically, selection of cDNA probe sets on microarray cards or the use of modifications of the SAGE technique such as LongSAGE adds more potential for a varied result.
What then are the advantages of global expression profiling? The advent of these technologies in the mid-1990s heralded a new excitement into the possibilities of molecular biological research. It allowed researchers to access the diverse levels of biological processes in its entirety, from primary DNA sequences in coding and regulatory regions, to RNA expression in response to the physiological or diseased environment, to proteomic interactions and localizations. The sheer power of simultaneously interrogating thousands of genes enables the acquisition of global pictures of biological processes that would otherwise be out of reach utilizing traditional gene-to-gene approaches.
Prior to global expression profiling, laboratory research was being driven by theories and hypotheses developed from meticulously analyzed pathways or the occasional spontaneous brain wave. The formulation of these theories in the history of epilepsy research have resulted in much interest in the neural imbalance of excitatory/inhibitory control, or the involvement of ionic channels and aberrant neuronal synapses in the epileptogenic processes (97) . Indeed, numerous laboratories have published insightful papers documenting a net excitatory imbalance in MTLE models (14, 22, 84) . More recently two microarray studies revealed confirmatory findings with the downregulation of gamma-aminobutyric acid-associated receptors and the upregulation of glutamate-associated receptors in the kainite model of epileptogenesis as well as chronic human MTLE (3, 44) . What is not evident in the literature are the situations where there have been failed hypotheses resulting in a laboratory and scientific losses in terms of time, personnel and finance. The use of global profiling platforms to guide hypotheses may be a viable option to minimize these situations.
The advantage in global expression profiling is the development of a broad understanding of the mechanisms underlying the process. It is the accumulation of the supportive data gathered from functional classes that enables more robust hypotheses to be formed and tested. In turn this allows further focussed research into specific genes or functional classes as has already been performed by several laboratories (42, 80) . Novel treatment strategies may be identified from the identification of the involvement of functional groups with development of new pharmacological agents targeting the specific biological process.
The publication of comparable gene libraries produced using global expression profiling methods also allows indirect interrogation between epilepsy and other disorders. Identification of commonality genes across disorders may provide a greater understanding of the aetiological or consequential effect of a regulated gene/process. Detection of common pathways through which each disorder is propagated may allow cross-over treatment between disorders giving new insight and treatment options.
Global expression profiling during epileptogenesis allows a greater understanding of the broad mechanisms underlying the epileptic process. It demonstrates specific genomic changes operating in concert with the entire molecular environment, thereby opening the doors to further research into possible causative pathways and functions through gene ontology annotations of cellular components, biological processes and molecular functions. In addition, the availability of large-scale libraries produced using uniform protocols allows the comparisons of commonality genes within other disorders apart from epilepsy potentially identifying reactive general genomic changes, shared pathways of action or specific causative genes. There is also the potential for discovery of novel genes not previously described with SAGE resulting in new hypotheses and theories into the pathogenesis of epilepsy as well as the potential to guide new therapeutic treatments in epilepsy as well as other disorders, neural or non-neural.
However, the most exciting aspect of global gene expression profiling is the general starting point of the research study. Previously, investigations into specific genes were performed based on previous studies or individual hypotheses. The use of global expression profiling allows the entire genome to be studied with hypotheses developed following this. In this manner, future research hypotheses are generated from science rather than hypotheses leading to science, a situation that must lend itself to greater transparency in laboratory research.
It is vitally important that further research into epileptogenesis builds on prior knowledge and pitfalls. It is the opinion of our laboratory that the entire process of molecular transcription and translation be interrogated with validation of global expression profiling results utilizing qRT-PCR followed by proteomic profiling using any combination of Western blotting, immunoperoxidase staining and in situ hybridization. Focusing genomic profiles into specific anatomical regions may enable causative molecular pathways to be identified and allow greater understanding of the histological changes, particular in the situation of HS associated MTLE whereby disagreement remains over the cause or effect of the pathological changes. Fluorescence Competition Assay Measurements of Free Energy Changes for RNA Pseudoknots [Image: see text] RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 °C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1−stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson−Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures. In duplex part, optical melting result (-12.9 kcal/mol) of model duplex (5΄GAACAAACGG3΄/3΄AACUUGUUUGF5΄ in Table 1 ) made less favorable by 1.9 kcal/mol for 3΄ dangling AA (62) and 2.7 (=0.82+1.9) kcal/mol for 5΄FG/3΄GGC terminal overhangs, made more favorable by 2.36 kcal/mol for 5΄CG/3΄GC nearest neighbor parameter (39) and 2.1 (=0.82+1.3+0.0) kcal/mol for 5΄FC/3΄GGG terminal overhangs. Here 5΄FC/3΄G is approximated as the 0.82 kcal/mol of 5΄FG/3΄C (Table 1) Plots of reported vs T m for the BWYV pseudoknot (45) reveal the possibility that the transitions F→PK and PK→HP may have non-zero . Reported errors in values are ±15% (45), which does not allow a reliable estimate of . The data, however, indicates the possibility that for transitions F→PK, PK→HP, and HP→U can be on the order of -700, -330, and -20 calK -1 mol -1 , respectively. Points shown as diamonds are excluded from linear fit. Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined. nucleus are inhibited, and a number of viral nucleolar antigens accumulate in the nucleoli of infected cells (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) . Moreover, two cellular nucleolar antigens, UBF 1 and B23.1, have been shown to be sequestered into viral DNA replication centers where they affect viral DNA replication (11) (12) (13) . Thus, there is clear evidence that adenovirus infection has a substantial impact on the composition and function of the nucleolus. What is not clear is whether the sequestration of cellular nucleolar antigens is limited to a small number of proteins or widespread. In addition, it is unclear whether adenovirus primarily modulates the nucleolus for replicative advantage or whether the effects on the nucleolus are a side effect of cellular proteins being sequestered into virally induced structures.
Many other plant and mammalian viruses have also been shown to interact with the host cell nucleolus from human immunodeficiency virus to Kaposi sarcoma herpesvirus (14 -21) . We therefore wanted to use a systematic and unbiased approach to examine the fate of the nucleolus during a viral infection using an established model system.
The use of modern proteomics approaches to investigate viral infections is still relatively new, but recently several notable studies have been reported that illustrate the power and utility of these techniques (22) (23) (24) (25) (26) . Most of these have used 2D gel electrophoresis-based approaches with some success. For example, 2D electrophoresis was used to examine the total proteome of West Nile virus-infected cells, identifying about 100 different proteins, many of which were clearly upor down-regulated (27) . Much less common is the use of the more powerful quantitative MS coupled with stable isotope labeling by amino acids in cell culture (SILAC) technique to compare uninfected and infected cells. However, one study has investigated the composition of lipid rafts in hepatitis C virus-infected cells, identifying almost 1000 proteins (28) . Furthermore, quantitative MS/SILAC has been extensively used for proteomics analysis of the mammalian nucleolus under a range of conditions (29 -32) . Indeed, a key study examined the changes in composition of the nucleolus after inhibition of rRNA synthesis with actinomycin D (ActD), quantitating the effects on almost 500 proteins in the nucleolus (29) .
We wanted to further explore the potential of both 2D gel electrophoresis and quantitative MS/SILAC to examine the effects of adenoviral infection on the host cell. As stated, we have previously shown that nucleolar antigens UBF and B23 are visibly depleted from the nucleolus during adenovirus infection and play functional roles in viral DNA replication (11) (12) (13) . Thus, we would expect a proteomics approach to confirm these well established changes in nucleolar composition that are of functional relevance to the infection. This study not only identified proteins known to be affected by adenovirus infection, but it also identified a number of novel alterations to the nucleolar proteome that were subsequently examined by confocal microscopy for independent verification.
Cells and Viruses-HeLa cells were grown in Dulbecco's modified Eagle's medium ϩ Glutamax (Invitrogen) supplemented with 10% fetal calf serum, 100 g/ml streptomycin, and 100 IU/ml penicillin. Human adenovirus type 5 (Ad5) was propagated as described previously (33) and purified using a ViraBind adenovirus purification kit (Cellbiolabs). The HeLa cell line stably expressing GFP-tagged ribosomal protein RPL27 has been reported elsewhere (34) . For microscopy, HeLa cells were grown on glass coverslips in 6-well dishes.
SILAC Labeling of HeLa Cells and Nucleolar Isolation-Isolation of purified nucleoli from uninfected and infected cells was performed as described previously (29) . For both SILAC and standard 2D gel analysis, 10 8 HeLa cells were infected at a multiplicity of infection (m.o.i.) of 5 with Ad5 and harvested at 18 h postinfection.
For SILAC analysis, prior to infection cells were grown for five rounds of cell division in Dulbecco's modified Eagle's medium containing L-[ 13 C 6 , 15 N 4 ]arginine and L-[ 13 C 6 , 15 N 2 ]lysine (Cambridge Isotope Laboratories) supplemented with 10% dialyzed fetal calf serum (Biowest) to ensure all the cellular proteins were labeled to saturation. Prior to nucleolar isolation, equal numbers of uninfected, unlabeled cells and Ad5-infected, labeled cells were mixed. Nucleoli were then isolated from the mixed cell populations using a standard protocol (35) . Nucleoli were lysed by heating in lithium dodecyl sulfate sample buffer (Invitrogen), and nucleolar proteins were separated by SDS-PAGE on a 4 -12% precast gradient gel (Invitrogen). The gel was fixed, stained with colloidal Coomassie Blue (Invitrogen), and cut into six slices.
LC-MSMS Analysis-Peptides were isolated from each gel slice after in-gel digestion, desalted, and concentrated as described previously (32); separated by HPLC (Agilent) on a C 18 reverse phase column; and analyzed by a QSTAR XL hybrid quadrupole TOF mass spectrometer (Applied Biosystems). The peak lists were generated by the Analyst QS software, version 1.1 (Applied Biosystems). The MS data were searched against the NCBInr database (February 19, 2006 release, 3,230,559 sequences searched) for Homo sapiens using the MASCOT search engine, version 1.9 (Matrix Science). Variable modifications used were carboxymethyl (Cys), oxidation (Met) and phospho (Ser, Thr, and Tyr) as well as the appropriate SILAC modifica-tions. Trypsin specificity was used, two missed cleavages were allowed, and a mass tolerance of 0.5 Da was used for both precursor and fragment ions. Peptide charges of ϩ1, ϩ2, and ϩ3 were selected. Individual ions with MASCOT scores higher than 20 were used, making sure the "average peptide scores" of all identified proteins exceeded 20, a threshold commonly used for confident protein identification from tandem MS data (36) . Only bold red peptides were also considered, effectively removed duplicate homologous proteins from the results. Under these conditions, the estimated false positive rate is less than 5%, according to a previous analysis (37) . SILAC quantitation was done by the MSQuant software, which measures the averaged MS peak areas of the isotopic pairs. Only proteins with bold red peptides and combined scores higher than 50 were quantitated. Proteins with heavy/light isotopic ratios lower than 0.01 were discarded; they mostly represented environmental contaminants. The S.D. of a protein ratio represented the variations among the measured peptide ratios for the same protein. Functional classification of the identified proteins was performed using Proteincenter (Proxeon). The biological reproducibility was addressed by a parallel 2D gel-based approach and by microscopy analysis (see below).
2D Gel Electrophoresis-Nucleolar pellets were resuspended in 450 l of 7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromphenol blue, 0.5% (v/v) IPG buffer, pH 3-11 non-linear (GE Healthcare), and 1.2% (v/v) Destreak reagent (GE Healthcare) and loaded onto 24-cm Immobiline DryStrip gels (pH 3-11 non-linear) by passive rehydration for a minimum of 12 h. Following rehydration, the DryStrip gels were transferred to an Ettan IPGPhor 3 system (GE Healthcare), and isoelectric focusing was performed by applying 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10.5 h until a total of 64,000 V-h had been achieved. Following isoelectric focusing, strips were equilibrated in SDS equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromphenol blue) containing 1% (w/v) DTT for 15 min at room temperature followed by a further incubation in fresh SDS equilibration buffer containing 2.5% (w/v) iodoacetamide for 15 min at room temperature. Strips were then applied to 12.5% (w/v) SDS-PAGE gels and run at 5 mA/gel for 1 h, 8 mA/gel for an additional hour, and then at 20 watts/gel until completion using an Ettan DALT-6 separation unit (GE Healthcare). Gels were fixed for 1 h in 50% methanol and 10% acetic acid and stained overnight using SYPRO Ruby total protein stain (Invitrogen). Following destaining in 10% methanol and 7% acetic acid, the gels were imaged using a Typhoon 9400 Variable Mode Imager (GE Healthcare).
Spot Picking, Protein Processing, and MS-Selected protein spots were cut from the gel using the Investigator ProPic automated 2D spot picking robot and digested with trypsin using the ProGest automated digestion unit (both from PerkinElmer Life Sciences).
Mass spectra were recorded in positive ion reflector mode on an Applied Biosystems 4700 MALDI mass spectrometer. The instrument was calibrated before each run using the 4700 Calibration Mix 1 (Applied Biosystems), and in addition, individual MS spectra were internally calibrated using tryptic autolytic peaks (if present). MSMS analysis was calibrated on the fragmentation of Glu-fibrinopeptide. For MSMS analysis, the top five most intense, non-tryptic precursors were selected for fragmentation by collision-induced dissociation. Neither base-line subtraction nor smoothing was applied to recorded spectra.
MS and MSMS data were analyzed, and peak lists generated using GPS Explorer 3.5 (Applied Biosystems). MS peaks were selected between 800 and 4000 Da and filtered with a signal to noise ratio greater than 15 and to exclude masses derived from trypsin autolysis (842. 51, 1006.48, 1045.56, 2211.1, 2283.18, and 2299.18) . MSMS peaks were selected on the basis of a signal to noise ratio greater than 10 over a mass range of 50 to 20 Da below the precursor mass. A peak density filter was used with no more than 30 peaks per 200 Da and a maximum number of peaks of 65.
Data were analyzed using the MASCOT 1.9 search engine (Matrix Science) to search against the human (148,148 sequences) MSDB protein database (Release 20063108). Search parameters allowed for one missed tryptic cleavage site, the carbamidomethylation of cysteine, and the possible oxidation of methionine; precursor ion mass tolerance was 150 ppm, and fragment ion mass tolerance was 0.25 Da. All identified proteins have a MASCOT score greater than 64 (the default MASCOT threshold for such searches), corresponding to a statistically significant (p Ͻ 0.05) confident identification.
Antibodies and Plasmids-Antisera used were anti-adenovirus protein V ( Confocal Microscopy-HeLa cells grown on coverslips were either infected, mock infected, and/or transfected with expression plasmids encoding tagged proteins as indicated. At 18 h postinfection, cells were fixed for 5 min with 4% formaldehyde in PBS followed by washing in PBS before permeabilizing in 1% Triton X-100 in PBS for 5 min. After permeabilization, the cells were washed briefly with PBS before blocking for 30 min at room temperature in 10% FCS in PBS. The cells were then incubated with primary antibodies for 60 min, washed, and incubated with secondary antibodies. Cells were mounted with Vectashield plus DAPI to visualize the nuclear DNA. Images were taken using a Leica confocal microscope (TCS-SP2).
The rate of adenovirus replication depends on the serotype of virus, the cell type, and the m.o.i. For the prototype strain Ad5, infection of HeLa cells with a modest m.o.i. of about five infectious particles per cell results in detectable viral early gene expression by 4 h postinfection, detectable DNA replication by 10 h postinfection, production of structural proteins that will form progeny virions by about 18 h postinfection, and release of infectious particles from about 30 h onward. To ensure the isolation of intact nucleoli, we chose to examine the composition of the nucleoli at ϳ18 h postinfection when late structural gene expression would be just underway in some cells but when active viral replication would be well underway in virtually all the cells examined (for a review, see Ref. 49) .
In this study, we isolated nucleoli from infected and mock infected HeLa cells and compared the differences in the isolated proteins by quantitative proteomics. To test the suitability of this approach, we infected a HeLa cell line stably ex-pressing a GFP-tagged ribosomal protein RPL27 (34) with Ad5. RPL27-GFP was used as a nucleolar marker in live cells and in isolated nucleoli. At 18 h postinfection, the cells were harvested, and nucleoli were isolated using a standard protocol (29, 35) . As shown in supplemental Fig. 1 , live cell imaging revealed that the RPL27-GFP signal in the nucleolus of infected cells (supplemental Fig. 1C ) was similar to that in untreated cells (supplemental Fig. 1A ). The GFP signals in nucleoli isolated from untreated (supplemental Fig. 1B ) and infected (supplemental Fig. 1D ) cells were also similar, although the nucleoli from infected cells appeared slightly larger. Interestingly, adenovirus infection induced the appearance of RPL27-GFP in threadlike structures in the nucleoplasm. The significance of the accumulation of this ribosomal protein in these structures or the apparent slight enlargement of nucleoli is unknown. The threadlike GFP-containing structures were not observed in the isolated nucleolar preparation, suggesting that the preparation was not contaminated with nucleoplasmic materials and was suitable for future proteomics analysis.
SILAC Analysis of Virus-infected Cell Nucleoli-A total of 351 proteins were quantified by SILAC analysis comparing virally infected host cell nucleoli to uninfected nucleoli that were ranked according to whether there appeared to be depletion or enrichment in the infected cell nucleolus ( Fig. 1 and supplemental Tables 1 and 2). Functional classification (Fig.  1A) revealed that the distribution of the functional roles of the detected proteins was similar to that of the previously reported human nucleolar proteome (29) , indicating that the nucleoli isolated in this study were of purity comparable to the nucleolar preparations used in our previous proteomics studies. Interestingly, no viral proteins were detected in the nucleoli of the virally infected cells using adenoviral genome data in MASCOT searches, potentially reflecting a lack of abundance at that time point. Fig. 1B shows the changes in abundance of the detected proteins in isolated nucleoli after viral infection. Remarkably, a majority of the nucleolar proteins remained relatively unaffected by viral infection; very few proteins exhibited significant changes in levels (more than Ϯ1-fold). A closer comparison of the nucleolar responses to viral infection and with previously reported data (29) on the effects of transcription inhibition by ActD is shown in Fig. 1C . The SILAC -fold changes of the nucleolar levels of selected proteins after viral inhibition and ActD treatment were very different, indicating that the human nucleolus reacts to these two perturbations in highly distinct manners. In particular, the effects of viral infection on components of the rRNA transcription machinery were distinct from the effects of ActD treatment. The SILAC ratios of ribosomal proteins showed a mild decrease after viral infection (Fig. 1D ), whereas ActD treatment had a significant impact on a number of ribosomal proteins.
We next investigated the adenovirus-induced changes on nucleolar composition. Of the proteins shown to be depleted, UBF was the most affected in accordance with previous reports (11) . Protein B23.1 has also been shown to be sequestered into viral replication centers (12) , and this depletion was also detected by our high throughput proteomics analysis as an almost 2-fold depletion compared with uninfected cells. Accordingly, we concentrated on proteins that, like B23.1 and UBF, showed a 2-fold depletion or enrichment in the nucleoli of infected cells (Table I) . 2D Gel Analysis of Virally Infected Nucleoli-We repeated the isolation of nucleoli from uninfected and infected cells a further three times at 18 h postinfection. Western blotting confirmed the purity of the nucleolar fractions (data not shown). The nucleoli from these experiments were examined using 2D gel analysis, which showed that only a small number of proteins had clearly altered amounts at this time point. Fig.  2 shows a typical pair of gels, and seven proteins showing a change in quantity are marked. These spots were picked and sequenced as described under "Experimental Procedures." Their identities are listed in Table II .
Confocal Microscopy of Candidate Proteins Identified by SILAC-Because adenovirus induces significant changes in nuclear structure, it is possible that the nucleolar isolation protocol no longer reliably enriches intact nucleoli. Moreover, although many of the proteins identified could conceivably play a role in adenovirus infection, we wanted to independently verify any potential changes in the nucleolar proteome. This would have a further key benefit of prioritizing future work relating these changes to function. We therefore used a range of antisera and tagged expression constructs to examine the fate of a number of proteins identified as being enriched or depleted in the nucleolus. We concentrated on those proteins for which reagents and published data on subcellular location were already available so we could be confident of their locations in uninfected cells. We compared the location of target antigens relative to adenovirus DBP, a key component of the adenovirus DNA replication machinery that is expressed to high levels in the nucleoplasm but not the nucleolus in virus-infected cells. DBP has a distinct pattern of fluorescence and well defined localization relative to viral mRNA transcription and de novo viral DNA synthesis (50). Fig.  3 shows the effects of infection on those proteins apparently depleted from the nucleolus during viral infection. Two nucleolar antigens, hPOP1 and Nopp140, were clearly depleted from the nucleolus (Fig. 3, A and B) . hPOP1 was sequestered from the nucleolus into a mottled pattern within the nucleoplasm and was excluded from the DBP-rich centers (Fig. 3A) .
This table lists all the proteins identified as having a 2-fold enrichment or depletion in the infected cell nucleoli compared with the uninfected cell nucleoli. Each protein is listed with the gi number, the number of peptides identified, percent coverage a brief description of its function (if known), and a ratio of depletion (shown as a negative ratio) or enrichment (shown as a positive ratio Nopp140 protein showed some co-localization with viral DBP but also significant enrichment in centers adjacent to DBP, reminiscent of our previously published findings that UBF is sequestered into regions adjacent to DBP (11) . We therefore compared UBF with Nopp140 to confirm that some Nopp140 is apparently sequestered in a manner similar to that of UBF (Fig. 3B, bottom row) . The next group of proteins, hnRNPU isoform a, SFPQ, U2AF 65 , hnRNPA2/B1, histone H1c, and exportin 5 were not detectable in the nucleolus by immunofluorescence ( Fig. 1, C, D, E, and F, top rows) . However, they were consistently detected in the nucleolus using proteomics methods and are considered components of the nucleolar proteome (51) . In adenovirus-infected cells, they all showed a pattern of exclusion from the DBP-rich replication centers apart from hnRNPA2/B1, which did not show any striking change in its distribution. However, we noted that hnRNPU, SFPQ, and U2AF65 were localized adjacent to DBP-rich centers, whereas histone H1c was clearly distinct and separated from the DBP-rich centers (Fig. 3G) . Exportin 5 was also readily detectable in the cytoplasm of infected cells but not in uninfected cells (Fig. 3H) .
We also examined the localization of a number of proteins apparently enriched in the nucleolar fraction during infection (Fig. 4) . PP2C is typical of proteins for which we were unable to detect any consistent change in distribution in adenovirusinfected cells (Fig. 4A) . In a similar manner, we were unable to FIG. 2. Typical 2D gel analysis of uninfected cell nucleoli compared with adenovirus-infected cell nucleoli. Isolated nucleoli were subjected to separation in two dimensions prior to staining and visualization as described under "Experimental Procedures." Boxed proteins were consistently seen to vary in intensity in multiple experiments. Those spots were picked for sequencing. Note that spots 4 and 7 were apparently enriched in the nucleolar preparations after viral infection. detect significant consistent changes in the subcellular location of C-NAP1 and nucleoporin 210 (data not shown). However, for PIK3-p87 (Fig. 4B ) and ribosomal protein S15a (Fig.  4C) , we did see some enrichment in the nuclear compartment that could account for the apparent increase in detection in the nucleolar fractions analyzed.
Confocal Microscopy of Candidate Proteins Identified by 2D Gel Analysis-As with the SILAC data, we examined using immunofluorescence a number of candidate proteins identified by 2D gel analysis that were altered in abundance following infection. Fig. 5 shows that proteins RBM4 (Fig. 5A ) and eIF6 (Fig. 5B) were both nucleolar antigens in uninfected cells. On the other hand, RBM4 was apparently recruited from the nucleolus into the nucleoplasm but with exclusion from the DBP-rich centers. However, eIF6 became more diffuse with reduced nucleolar location but, notably, without specific exclusion from DBP-rich centers.
Confocal Microscopy of Proteins Identified by SILAC as Having No Change in Nucleolar Abundance at 18 h Postinfection-For completeness, we tested a protein that did not change its location at this time point postinfection. Nop132 is a nucleolar antigen that has not been previously investigated in the context of adenovirus infection. We used a FLAGtagged construct to examine its location in uninfected and infected cells. As can be seen from Fig. 6 , Nop132 maintained its apparent nucleolar location with no detectable depletion from the nucleolus to other compartments.
Proteome-We utilized both quantitative MS-based proteomics techniques and more traditional 2D gel analysis to provide the first detailed analysis of changes in the human nucleolar proteome after infection with a human pathogen. These data reveal novel nucleolar antigens that were affected by adenovirus infection and therefore identify new candidate proteins that may play either a direct or indirect role in viral infection. In addition, our data illustrate the benefits offered by confocal microscopy when assessing proteomics analysis of a subcellular fraction. We feel this combination of techniques is crucial for several reasons. First, viral infection may have unsuspected effects on the integrity of cellular compartments that could affect the fractionation process. Second, this combination enables targets identified by proteomics analysis to be prioritized for further in-depth analysis. Finally, a proportion of the proteins in our SILAC analysis were only identified by one peptide, and for these proteins further verification is important.
Changes in Nucleolar Proteome: Adenovirus Compared with Actinomycin D-Comparison with the response of the nucleolar proteome after transcriptional inhibition by ActD treatment (in which the flux of ϳ500 nucleolar proteins was characterized) was very revealing (29) . Comparison with our data set showed that the effect of adenovirus infection is very specific with only a limited range of proteins showing a relative change in abundance (e.g. compare our Fig. 1B with Fig.  4a of Andersen et al. (29) ). For example, although transcription inhibition by ActD causes depletion of ribosomal proteins of the 40 S subunit and, to a lesser extent, the 60 S subunit (29) , this pattern was not observed in viral infection (Fig. 1D) . This was interesting because adenovirus has been shown to eventually affect ribosomal RNA export (2) . However, our chosen time point of 18 h postinfection is the earliest time that such defects in rRNA export can be detected (2) . As such, it is probable that defects in processing or export of ribosomal proteins may be only just beginning at this time point and are therefore not yet readily detectable by our methods.
Thus, adenovirus infection induces a change in the nucleolar proteome that is distinct from the classical nucleolar disruption caused either by transcription inhibition or by other general cellular stresses. Viral infection in this case induces a much more limited and targeted effect on the nucleolar proteome. Moreover, the 2D approach reinforces the observation from SILAC analysis that adenovirus infection does not induce 
This table lists all the proteins identified as having a visual enrichment or depletion in the infected cell nucleoli compared with the uninfected cell nucleoli. Each protein is listed with the spot number as identified on the 2D gels in Fig. 2 , the accession number, the number of peptides identified, the percent coverage, and a brief description of its function. IRES, internal ribosome entry site. Fig. 1C (top panel) also reinforces the distinct effects of adenovirus infection compared with ActD treatment. Looking at rRNA transcription, upon ActD treatment, RNA polymerase I subunits and accessory transcription factors were similarly depleted from the nucleolus. However, in virally infected cells, nucleolar levels of the RNA polymerase subunits and transcription factor UBF were affected differently (Fig. 1C, bottom panel) . This is consistent with previous observations during adenovirus infection where UBF was depleted from the nucleolus whereas RNA polymerase I remained nucleolar as evidenced by immunofluorescence using antibodies specific for RNA polymerase I, in situ visualization of ongoing RNA synthesis, and S1 nuclease protection assay of rRNA synthesis initiation (11) . This suggests that adenovi- The second row of images shows the same indicated endogenous or expressed tagged protein in cells infected with adenovirus for 18 h. Viral infection was confirmed by anti-DBP serum. In B, a third row is shown in which cells were infected with adenovirus and the location of endogenous UBF was compared directly with that of endogenous Nopp140. A duplicate slide (not shown) confirmed that Ͼ99% of cells were infected in this experiment. In some cases, where compatible serum was available, a control nucleolar antigen, B23.1 or UBF, is also shown alongside the uninfected cell images. VSV, vesicular stomatitis virus; EGFP, enhanced GFP. rus induces a specific removal of UBF from the nucleolus, independently of other components of the rDNA transcription complex. This gives us confidence that our data reflect genuine changes in the nucleolar proteome because we know of no other way of inducing this pattern of changes on the rDNA transcription complex.
Comparison with Other Established Effects of Adenovirus on Nucleolus-The SILAC data point to depletion of UBF and B23.1 during viral infection, which reflects previously published data by ourselves and others that UBF and B23.1 are functionally sequestered upon adenovirus infection into viral DNA replication centers (11) (12) (13) . Another example is U2AF 65 , a splicing factor known to play a role in splicing of adenovirus transcripts (52, 53) . Finally, exportin 5 is known to export adenovirus VA RNA, competing with cellular microRNA export and potentially modulating cellular protein expression (54) . The identification of these proteins by SILAC/MS analysis further validates our approach because they are all well established functional partners in adenovirus infection. This gives us confidence that some, if not all, of the novel proteins Novel Proteins Depleted from Nucleolus on Infection-Of those proteins examined that show a clear nucleolar localization in uninfected cells, all had a dramatic redistribution in infected cells. For example, hPOP1 is normally involved in rRNA processing (42, 55) , and its mislocalization may reflect data showing that adenovirus eventually inhibits rRNA processing during infection (2) . Nopp140 mislocalization also fits with these observations because it is a chaperone for small nucleolar RNA (56, 57) . However, its co-localization with UBF and DBP implies that it may be an accessory factor in viral DNA replication during viral infection. Notably, in interphase cells, UBF and Nopp140 are co-localized and are known to be recruited together (56) . RBM4 has been reported to be either predominantly nuclear or predominantly nucleolar, depending on experimental conditions (43, 58 -60) . In our hands, the protein was nucleolar in Ͼ80% of cells. RBM4 has been implicated in the modulation of splice site selection, something of clear relevance to adenovirus infection, but it has also FIG. 4 . Distribution of proteins identified by SILAC as being enriched in nucleolus during viral infection. All the images are of a fixed focal plane ϳ0.3 m in depth, the DAPI stain is in blue in all cases, and the bar represents 10 m. A-C, in each case the top row of images is representative of the location of the indicated endogenous protein or expressed tagged fusion protein in Ͼ80% of cells examined. The second row of images shows the same indicated endogenous or expressed tagged protein in cells infected with adenovirus for 18 h. Viral infection was confirmed by anti-DBP serum. In B, the use of a cyan fluorescent protein (CFP) fusion protein precluded the use of DAPI as a nuclear marker, and so antiserum to U2AF65 was used instead. In A, UBF is also shown alongside the uninfected cell images as a marker for the nucleolus.
been suggested that this protein may inhibit cap-dependent translation while promoting internal ribosome entry site-mediated translation (43) . This too may be significant because at later times in infection adenovirus makes use of a ribosome shunting method to promote the translation of its structural proteins at the expense of cellular mRNA translation (61) . In a similar vein, ribosome assembly is regulated by eIF6, and it too may be used by adenoviruses to maximize the suitability of the infected cell for viral protein expression (41, 62) .
Many proteins in the current nucleolar proteome were not readily visualized in the nucleolus by fluorescence microscopy. However, our data show that even in these cases quantitative changes in nucleolar content are reflected in visible changes in protein distribution. For example, U2AF 65 , hnRNPU, and SFPQ (also known as PSF) are all known to be involved in mRNA metabolism, and at least U2AF 65 has been shown to play a role in adenovirus mRNA metabolism (38, 52, FIG. 5 . Distribution of proteins identified by 2D as being depleted from nucleolus during viral infection. All the images are of a fixed focal plane ϳ0.3 m in depth, the DAPI stain is in blue in all cases, and the bar represents 10 m. A and B, in each case the top row of images is representative of the location of the indicated endogenous protein or expressed tagged fusion protein in Ͼ80% of cells examined. The second row of images shows the same indicated endogenous or expressed tagged protein in cells infected with adenovirus for 18 h. Viral infection was confirmed by anti-DBP serum. In addition, as indicated in the images, a control nucleolar antigen (UBF or B23) is also shown in the uninfected cells.
FIG. 6. Distribution of proteins identified by SILAC as having no change in distribution. All the images are of a fixed focal plane ϳ0.3 m in depth, the DAPI stain is in blue in all cases, and the bar represents 10 m. As before, the top row of images is representative of the location of FLAG-tagged Nop132 fusion protein in Ͼ80% of cells examined. The second row of images shows the location of FLAG-tagged Nop132 in cells infected with adenovirus for 18 h. Viral infection was confirmed by anti-DBP serum. 63 ). As such, it seems reasonable to expect that these factors will be sequestered into regions adjacent to adenovirus DBP because these regions are known to be active in viral mRNA synthesis (50) . Indeed, U2AF 65 , hnRNPU, and SFPQ were all apparently sequestered into a similar pattern that would be consistent with a role for these proteins in viral mRNA metabolism. However, we do not see any particular sequestration into these regions for hnRNPA2/B1, which weakens the case hnRNPA2/B1 plays a role in viral mRNA metabolism, something which can only be determined through further experimentation.
Cellular Proteins Are Enriched in Nucleolus after Adenovirus Infection-That we detected cellular proteins enriched in the nucleolus was intriguing, and this was reflected in changes in distribution of p87 PIKAP and S15a by immunofluorescence (Fig. 4, B and C) . That ribosomal protein S15a is enriched in the nucleus/nucleolus during infection again may reflect eventual interference by adenovirus with aspects of rRNA maturation and/or export.
The enrichment of p87 PIKAP (also known as PIK3R6) is also interesting. This protein is a regulatory subunit of phosphoinositide 3-kinase ␥, which is part of the phosphoinositide 3-kinase signaling pathway. These pathways cover a very wide range of responses from cellular growth to cellular motility. However, activation of phosphoinositide 3-kinase ␥ is linked mainly to host-wide responses to injury or infection (64) . Indeed, it is clear that adenovirus does directly modulate the phosphoinositide 3-kinase pathway (65) .
A further set of proteins apparently enriched in the nucleolus were not detected by microscopy, possibly reflecting differences in sensitivity of proteomics methods compared with microscopy. Thus, a protein may be enriched in the nucleolus but still be below the level of detection by microscopic methods.
Proteins Unaffected by Adenovirus Infection-We also thought it a relevant control to examine a protein that did not apparently change in nucleolar abundance by 18 h postinfection. Nop132 is thought to help recruit dead box protein DDX47 to the nucleolus and therefore play a role in rRNA processing (48) . As can be seen in Fig. 6 , there is no apparent change in the pattern of distribution of Nop132 at the time examined.
Localization Patterns of Sequestered Cellular Antigens Are Heterogeneous-Interestingly, there was a diverse range of localization changes in response to infection. For example, eIF6 was diffuse throughout the infected nucleoplasm. Alternatively, hnRNPU, SFPQ, and U2AF 65 were adjacent to DBPrich centers, whereas histone H1c was cleared from the DBPrich centers and regions adjacent to the DBP centers. This suggests that exclusion of cell proteins from DBP-rich centers is not simply a nonspecific feature of the adenovirus-infected cell nucleoplasm. Moreover, hPOP1 was excluded from the DBP-rich centers but was clearly enriched in a mottled pattern distinct from Nopp140 and U2AF 65 for example. Thus, there is, as might be expected, enrichment of nucleolar antigens in distinct regions of the infected nucleoplasm. This suggests, not surprisingly, that distinct aspects of adenovirus replication benefit from different nucleolar antigens.
Comparison between SILAC and 2D Gel Analysis-Comparing 2D gel analysis and SILAC approaches revealed that the SILAC method provided more information. However, only one protein (hnRNPA2/B1) was identified by both methods, and the depletion of eIF6 and RBM4 was not detected by SILAC. This reflects two key points: first, the lower sensitivity of the 2D gel approach, and second, that for the SILAC analysis, we only divided the one-dimensional gel electrophoresis sample into six slices prior to analysis.
As with previous proteomics investigations of subcellular components, we anticipate that further refinement over time will be possible and will yield a more detailed picture of the effects of adenovirus on this important structure both at the 18-h postinfection time point analyzed here and other time points. Also of note is the lack of viral proteins identified by either method. In part, this is a consequence of the time point chosen as the expression of late adenovirus proteins that are known nucleolar antigens would have only just begun. Other explanations are inherent in the methodology; i.e. the viral proteins may dissociate from nucleoli under the conditions present in the nucleolar isolation protocol.
Several members of the cytoskeletal family were also highlighted by the 2D gel approach. This may well reflect the observation that adenovirus protease cleaves cytokeratin 18 during infection, leading to altered cytoskeletal structure (66, 67) .
Concluding Remarks-Our data show that adenovirus specifically targets aspects of the nucleolar proteome in contrast to nonspecific disruption by agents like ActD, providing the first high throughput insight into viral-nucleolar interactions. Moreover, by examining the nucleolar proteome, we were able to highlight misdistribution of antigens from all compartments of the cell. We believe this demonstrates the utility of proteomics approaches to generate meaningful data sets that will help us understand the role of the nucleolus in viral infection and to uncover previously unsuspected effects on the host cell during viral infection. Indeed, subcellular fractionation of this sort coupled with confocal microscopy provides a powerful new tool for understanding viral host cell interactions in a broad context and is a positive step toward a systematic understanding of virus host cell interactions. Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design. Neutralizing antibodies provide one arm of the adaptive immune response against the human immunodeficiency virus type 1 (HIV-1). Several reports demonstrated that the neutralizing antibody response exerts selective pressure during HIV-1 replication in vivo, which accounts in part for the extensive variation in the env gene observed soon after primary infection [1, 2] . Furthermore, selective pressure imposed by neutralizing antibodies has been demonstrated in a human trial where three neutralizing monoclonal antibodies (mAbs) administered during HAART treatment-interruption led to a reduction in viremia followed by selection of escape mutants [3, 4] . Passive transfer studies in macaques showed that the administration of HIV-1 neutralizing mAbs protects against vaginal or intravenous challenge with SIV-HIV-1 chimeric viruses (SHIV) [5, 6, 7, 8, 9] . In some models protection depended not only on viral neutralization but also on Fc-mediated antibody effector functions [10, 11] .
Given the predicted low-titer inoculum driving HIV-1 sexual transmission, a vaccine capable of eliciting antibodies that neutralize a broad spectrum of viral strains could potentially reduce or prevent infection. It has been anticipated that the identification of broadly neutralizing mAbs from HIV-1 infected individuals, and the characterization of their cognate epitopes will be instrumental in the design of immunogens capable of eliciting such a broad neutralizing response [12] . This idea has led to a major international cooperative effort within consortia of laboratories with complementary expertise in human immunology, structural biology and vaccine design [13, 14] .
HIV-1 is characterized by an extraordinary genetic diversity, reflected by the presence of several clades (subtypes), a fact that represents a significant impediment to vaccine development. Env is the most variable HIV-1 gene, with up to 35% sequence diversity among clades, 20% diversity within clades, and 10% diversity in a single infected individual [15, 16, 17] . Several conserved epitopes have been defined by a small panel of neutralizing mAbs isolated using different experimental approaches. One epitope that appears to be relatively conserved and overlaps with the CD4 binding site (CD4bs) on the surface Env glycoprotein gp120 is recognized by mAb b12, which is the most potent and broadly-reactive mAb of such specificity [18, 19, 20] . This site was recently shown to be a significant target of neutralizing antibodies present in the sera of selected patients [21, 22] . However, b12 was derived from a phage library in which heavy and light chains have been randomly reassorted, thus its relevance to naturally-occurring B cell responses in HIV-1 infection is unclear. A second epitope in a carbohydraterich region on the outer domain of gp120 is composed of glycans recognized by mAb 2G12, which shows an unusual interlocked VH domain-swapped dimer generating an extended and monovalent binding surface [23, 24, 25, 26] . The 2G12 epitope is not present in the majority of clade C isolates [27] , but, of more concern, no 2G12-like activity has been detected in the sera of HIV-1 infected individuals [21, 22] , suggesting that this type of neutralizing antibody may not be generally amenable to elicitation by B cells. A third target of the broadly-neutralizing mAb 4E10, and relatively broad mAbs 2F5 and Z13, is located on the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41 [28, 29, 30, 31] . As with 2G12, MPERspecific neutralizing antibodies are infrequently encountered in the sera of HIV-1-infected individuals [21, 32, 33] , potentially reducing their relevance to vaccine development strategies. Moreover, MPER-specific mAbs have features that suggest they are products of autoreactive B cells, casting doubts over the possibility of inducing such antibodies by vaccination [34] . A fourth target of neutralizing mAbs is the V3 loop, which is immunogenic but also a highly variable region involved in Env-coreceptor binding. Antibodies to the V3 loop, such as mAbs 447-52D and F425-B4E8, are mostly effective against neutralization sensitive (Tier-1) viruses and have generally a limited breadth of reactivity almost exclusively specific for clade B viruses, reflecting their provenance from clade B-infected individuals [11, 35, 36, 37] . A cluster of human V3 mAbs raised from B cells derived from non-B cladeinfected individuals showed variable neutralizing activity for two AG and two C clade primary isolates, although these were particularly neutralization sensitive [38] . Finally, the CD4-induced (CD4i) site has been described as a neutralization epitope primarily on Tier-1 isolates with limited accessibility on Tier-2 isolates [39] . Very recently, the isolation and partial characteriza-tion of two related MAbs from a clade A-infected donor that appear to have very broad and potent neutralization profiles was reported [40] . These MAbs recognize a discontinuous epitope only expressed on the native Env trimer, made up of segments of the CD4i, V2 and V3 loops. This publication is the first to provide new broadly neutralizing reagents from a non-clade B donor and provides proof-of-principle that new neutralization specificities remain to be discovered on HIV-1 Env.
Since the number of existing neutralizing mAbs with a relatively broad neutralization profile is very limited, and those described to date, with the exception of the Walker et al MAbs were isolated from clade B virus-infected donors, we undertook the task of isolating new human mAbs from HIV-1-infected donors. Our primary aims were: i) to isolate novel MAbs with cross-clade neutralizing activity from non-B clade donors; ii) to analyze the memory B cell repertoire in a representative panel of predominantly non-B clade-infected individuals. By using an improved memory B cell immortalization method [41] , combined with highthroughput parallel screening with a panel of recombinant Envbased antigens, we isolated a panel of 58 human mAbs which we have characterized with regard to epitope specificity and breadth of neutralization. We have more fully characterized three mAbs from this panel that demonstrated complementary and relatively broad cross-clade neutralizing profiles that target the gp120 CD4bs and the V3 crown, and the gp41 heptad repeat 1 (HR-1).
The study was approved by the ethical committee at Institute of Tropical Medicine and Queen Mary University. All participants gave written informed consent.
TZM-bl and HOS.CD4-R5 cells were obtained from the NIH-AIDS Research and Reference Reagent Program (ARRRP). 293T/17 cells were obtained from the ATCC. MAbs 2G12 [29] , 2F5, 4E10 [42] , b12 [18] , 3D6 [43] and 5F3 [29] were obtained from Polymun Scientific GmbH (Austria), as part of the Collaboration for AIDS Vaccine Discovery program.
The clade B and C HIV-1 Reference Panels of Env clones [44, 45] and the SF162 clone were obtained through the NIH-ARRRP. Other non-clade B isolates were provided by the Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC). HIV-1 subtype B clone JRFL was provided by Dennis Burton (Scripps Institute, La Jolla, US). Pseudoviruses were produced by co-transfecting HEK293T/17 cells with the envexpressing plasmids and the complementing viral-genome reporter gene vector, pNL4-3.Luc + .E 2 R + (kindly provided by John R. Mascola, VRC, NIAID, NIH, US).
Recombinant gp140 from HIV-1 isolates 92UG37, CA18, CN54, k530, UG21 and BR29 were provided by Simon Jeffs (Imperial College London, UK). Recombinant gp120 from HIV-1 isolates CM235, 93TH975, BaL and SF162 were obtained through the NIH-ARRRP, while gp160 from isolates MN and LAI were purchased by Prospec-Tany Technogene LTD (Israel). Recombinant YU2 gp120 and the two mutants D368R and I420R were provided by John R. Mascola and Richard Wyatt (VRC, NIAID, NIH, US). Recombinant soluble CD4 and recombinant gp120 from HIV-1 IIIB and CN54 were obtained from the CFAR, NIBSC (UK). The recombinant ectodomain of gp41 from isolate HxB2 (amino acids 541-682) was purchased by Vybion Inc. (US). HR-1 is a modified version of gp41 [46] exposing residues HLLQLTVWGIKQLQARILAVE (HxB2). 5HB was produced as described [47] . HR-1-FP comprises gp41-residues 512-594 (HxB2) including the fusion peptide and HR-1. HR-2 (HxB2) comprises gp41 residues 591-693 (HxB2) including the cys-loop region, HR-2 and MPER flanked by two coiled coil regions.
Patients were selected for inclusion into the study on the basis of the clade of their infecting virus (predominantly non-B clade) and on the ability of their plasma to neutralize a panel of HIV-1 primary isolates using two differerent assays. At the Institute for Tropical Medicine a panel of 4 primary A strains (VI191, 92UG37, VI820, VI 1031), 4 primary C strains (VI829, VI882, VI1144, VI1358) and 6 primary CRF02 strains (VI1090, VI2680, CI20, CA18, VI1380, VI2727) was used to select patients with .80% neutralization was measured at a 1/20 dilution of plasma. The plasma dilution was mixed with virus for 24 hours and absorbed to PHA/IL-2 stimulated freshly isolated PBMC for 1 hour. Virus-antibody mixture was then removed by extensive washing and p24 ELISA was performed after 14 days of culture to determine neutralization. At Queen Mary University of London, the TZMbl-based recombinant pseudovirus assay was employed with a selected panel of Tier 2-type clade A, B, C, CRF02_AG, _AE, F and BF viruses to measure .50% neutralization at a 1/20 dilution. MAbs were isolated using a previously described improved EBV immortalization method [41] . Culture supernatants were harvested 14 days after immortalization and assayed in parallel for binding to trimeric gp140 proteins (UG37, clade A and CN54, clade CRF07_BC) [48] , monomeric gp120 (CN54, CRF07_BC and IIIB, clade B) and gp41 recombinant ectodomain (HxB2, clade B). Those that were positive were then subcloned and grown up on a large scale for supernatant purification. The purity of all mAb preparations was assessed using a chromogenic LAL endotoxin assay (Genscript) and endotoxin levels were always ,0.05 EU/ml.
A standard ELISA was used to determine binding of mAbs to the panel of HIV-1 Envs. Briefly, ELISA plates were coated with each Env antigen, blocked with 10% FCS in PBS, incubated with human mAbs and washed. Bound mAbs were detected by incubation with AP-conjugated goat anti-human IgG (Southern Biotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. The relative affinities of mAbs binding to respective coated antigens were determined with ELISA by measuring the concentration of each mAb required to achieve 50% maximal binding at saturation (K 50 ). The ability of mAbs to inhibit binding of sCD4 to gp120 or gp140 was evaluated by ELISA. Serial dilutions of mAbs were pre-incubated with gp120 (or gp140) and added to plates pre-coated with sCD4. After 1 h plates were washed and incubated with sheep polyclonal antibody D7324 (Aalto Bio-Reagents) followed by washing, incubation with AP-conjugated rabbit anti-sheep IgG antibody (Abcam, Cambridge, UK), extensive washing and detection as above.
MAbs were purified on Protein G columns (GE Healthcare) and biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). The competition between unlabeled and biotinylated mAbs for binding to immobilized Env antigens was measured by ELISA. Briefly, unlabelled competitor mAbs were added at different concentrations. After 1 h biotinylated mAbs were added at a concentration corresponding to the 70-80% of the maximal OD level. After incubation for 1 h, plates were washed and bound biotinylated mAb was detected using APlabeled streptavidin (Jackson Immunoresearch). The percentage of inhibition was tested in triplicates and calculated as follow: (12[(ODsample-ODneg ctr)/(ODpos ctr -ODneg ctr)])6100.
Overlapping linear 15-mer and cyclized 15-mer peptides based on gp160 of HIV-1 UG037 and 93MW965 and the overlapping 15-mer peptides of gp41 strain SF162 were synthesized on polypropylene support (minicards), and were tested for reactivity with mAbs as described [49, 50] . Pepscan peptide binding analysis was carried out by an ELISA-based format in which the colored substrate was quantified with a charge-coupled device (CCD)camera and an image processing system. The values mostly ranged from 0 to 3000, a log scale similar to 1 to 3 of a standard 96-well plate ELISA-reader.
A single-cycle infectivity assay was used to measure the neutralization of luciferase-encoding virions pseudotyped with the desired HIV-1 Env proteins, as previously described [51] . Briefly, appropriate dilutions of the virion-containing culture supernatants were pre-incubated at 37uC for 1 h with mAbs at various concentrations. The virus-mAb mixtures were added to HOS-CD4-CCR5 cells and incubated for 3 days at 37uC. A similar protocol was used for supernatants screening using TZM-bl cells [45] . The cells were then lysed with Britelite reagent (Perkin-Elmer) and the relative light units in the cell lysates were determined on a luminometer microplate reader (Veritas, Turner Biosystems). The 50% inhibitory dose (IC50) was defined as the sample concentration at which relative luminescence units were reduced 50% compared to virus control wells. Some of the neutralization data for the reference mAbs b12, 2G12, 2F5 and 4E10 are taken from previous analyses [41, 42] carried out under identical standardized conditions to those used for the new mAbs reported here. A multi-cycle virus replication assay was used to measure the neutralization of replication competent luciferaseencoding viruses in 5.25.EGFP.Luc.M7 cells [52] . This cell line is a genetically engineered clone of CEMx174 that expresses multiple SIV and HIV-1 entry receptors (CD4, CCR5, CXCR4, GPR15/Bob) [53] . For the neutralization assay, 5000 tissue culture infectious dose 50 (TCID 50 ) of virus was incubated with multiple dilutions of test sample in triplicate for 1 hr at 37uC in a total volume of 150 ml in 96-well flat-bottom culture plates. A 100 ml suspension of cells (5610 5 cells/ml of growth medium containing 25 mg DEAE dextran/ml) was added to each well. Plates were incubated until approximately 10% of cells in virus control wells were positive for GFP expression by fluorescence microscopy (approximately 3 days). At this time, 100 ml of cell suspension was transferred to a 96-well white solid plate (Costar) for measurements of luminescence using the Britelite Luminescence Reporter Gene Assay System (PerkinElmer Life Sciences).
Plasma samples were obtained from patients entered into the Antwerp and London cohorts who were infected with a variety of HIV-1 clades. Patients were selected for MAb preparation on the basis of the clade of their infecting virus (predominantly non-B clade), and on the ability of their plasma to neutralize, to .80% at a 1/20 dilution, members of a selected panel of Tier 2-type clade A, B, C, CRF02_AG, AE, F and BF viruses in both a PBMCbased infectious primary isolate virus assay [54] and in the TZMbl-based recombinant pseudovirus assay [55] . In some cases only a restricted plasma neutralization analysis was possible due to limiting amounts of patient plasma. Twelve and nine infected donors from the London and Antwerp cohorts respectively were selected for B cell immortalization. Summarized clinical and virological data including HIV-1 clade, viremia, CD4 + T cell counts, therapy and breadth of neutralization are shown in Table S1 .
IgG + memory B cells were isolated from the selected donors and immortalized with EBV in the presence of CpG as described [41] . The immortalization efficiency of memory B cells from HIV-1 patients was significantly lower than that of non-HIV-1-infected donors (3% versus 20%, n = 21, p,0.001). This finding may be explained by the recent report that HIV-specific B cells are present within a population of ''exhausted'' memory B cells, characterized by the expression of inhibitory receptors and low levels of CD21 [56, 57] . To maximize detection of antibodies recognizing conserved features, cell culture supernatants were screened by parallel highthroughput ELISAs using five recombinant HIV-1 Env proteins, including trimeric gp140 [48] , monomeric gp120 and gp41 recombinant ectodomains. From the 21 donors interrogated we selected 58 B cell clones producing mAbs that bound to at least one of the screening antigens. The mAbs were purified and further characterized for binding specificity and neutralizing activity using an extended panel of recombinant Env proteins and pseudoviruses representative of several HIV-1 clades with diverse coreceptor usage, geographic origin and conformation.
The binding data for each mAb are displayed in Figure S1 and expressed as half-maximal binding concentrations at equilibrium (K 50 ), which approximate to the equilibrium dissociation constants [58] . Of the 58 mAbs, 37 bound to gp120 and 21 to gp41. Several gp120-specific and gp41-specific mAbs showed a broad pattern of reactivity with most recombinant proteins, although usually within a broad range of K 50 values. Overall, there was no relationship between the donor's HIV-1 clade and the clade specificity of the isolated mAbs.
The neutralizing activity of the mAbs was initially measured using 20 pseudotyped HIV-1 primary isolate envs characterized by different sensitivity to neutralization [44, 45] . The mAbs were tested at a fixed concentration (100 mg/ml) in a luciferase-based neutralization assay using HOS-CD4.R5 as target cells. Out of the 46 mAbs tested against the whole virus panel, 37 showed neutralizing activity on at least one isolate ( Figure 1A) . Three mAbs, HJ16, HGN194 and HK20, stood out for their breadth of neutralizing activity, neutralizing 10, 11 and 17 out of the 20 pseudoviruses, respectively ( Figure 1A) . These mAbs were further characterized in the same assay for their potency ( Figure 1B) . HJ16 showed high neutralizing activity, while HGN194 and HK20 were less potent. In addition, while most antibodies preferentially neutralized Tier-1 isolates, HJ16 preferentially neutralized Tier-2 isolates ( Figure 1A-B) .
Since it has been reported that HIV-1 neutralization is target cell type-sensitive [59, 60] , we next used TZM-bl as target cells and titrated the mAbs against a larger panel (n = 46) of predominantly (38/46) Tier-2 pseudoviruses (Table S2) . Generally the results obtained with the extended panel confirmed the previous results and showed that several mAbs had potent neutralizing activity, with IC 50 values ,20 ng/ml. A striking exception was HK20, which neutralized 17/20 pseudoviruses in the HOS-based assay, but only 3/46 pseudoviruses in the TZM-bl based assay.
We established the breadth of neutralization of these three novel mAbs by comparing them in the TZMbl assay with the five mAbs currently considered to be the most broadly reactive, namely b12, 2G12, 2F5, 4E10 and 447-52D. We used a large multi-clade panel comprising 10 Tier-1 and 82 Tier-2 isolates including clade B early-transmitted viruses [61] . The three new mAbs showed a pattern that was clearly distinct from that of previously described mAbs (Figure 2 ). In particular, HGN194 neutralized with high potency all Tier-1 viruses from clade A, B and C and 11% of Tier-2 viruses. By contrast HJ16 neutralized only 1 out of 10 Tier-1 viruses, but neutralized 39% of Tier-2 viruses. Thus HJ16 is comparable to b12 and 2F5 in terms of percentage of Tier-2 isolate neutralization and is superior to 2G12 and 447-52D. 4E10, as previously reported [27, 41] showed an extremely broad pattern of reactivity. Taken together the above results indicate that novel neutralizing mAbs can be isolated from memory B cells of nonclade B HIV-1-infected individuals, some of which display relatively broad neutralizing activity. Since one of our primary aims was to isolate and characterize novel MAbs with unusually broad and potent neutralization activity, we focused our attention on HJ16, HK20 and HGN194.
MAb HJ16, derived from a donor infected with clade C, has a unique neutralization profile with potent and selective neutralization of multiple Tier-2 pseudoviruses ( Figure 1B and Figure 2 ). When compared with the CD4bs-specific mAb b12 for gp120 binding, HJ16 showed similar binding curves ( Figure 3A ) and inhibited to a comparable extent the binding of gp120 to solid-phase sCD4 ( Figure 3B , IC 50 values of 1.57 and 1.16 mg/ml, respectively). However, cross-competition analysis of HJ16 and b12 for binding to gp120 revealed incomplete heterologous inhibition, with plateau values of approximately 80% ( Figure 3C-D) . These results suggest that HJ16 and b12 recognize related but non-overlapping CD4bsproximal epitopes. To further characterize HJ16 specificity we measured its binding to two YU2 gp120 mutants: the D368R mutant, which is not bound by CD4 or CD4bs-specific mAbs [21] and the I420R mutant, which is not recognized by CD4i-specific mAbs [62] . As already reported, b12 bound the I420R CD4i mutant, but failed to recognize the D368R CD4bs mutant. By contrast, HJ16 bound both mutants and indeed bound better to the D368R CD4bs mutant compared to the wild-type molecule ( Figure 3E-F) . Attempts to map the epitope by Pepscan analysis using overlapping linear peptides were unsuccessful (not shown), consistent with HJ16 recognition of a discontinuous epitope.
HJ16 and b12 neutralized 1/10 and 8/10 Tier-1 and 32/82 and 35/82 Tier-2 pseudoviruses, respectively ( Figure 2) . Interestingly, 22/32 Tier-2 isolates neutralized by HJ16 were not neutralized by b12, and reciprocally 24/35 Tier-2 isolates neutralized by b12 were not neutralized by HJ16. Another interesting finding is that HJ16 does not discriminate between clades as much as b12, 2G12 and 2F5 (e.g. b12 and 2G12 rarely neutralize clade A isolates while 2F5 and 2G12 rarely neutralize clade C isolates, Table S3 ). These results reveal a largely nonoverlapping pattern of reactivity of HJ16 and b12 and suggest that the combination of these two mAbs could be effective against a dominant fraction of HIV-1 isolates (57/82, i.e. 69%). Taken together these results are consistent with HJ16 recognition of a surface proximal to the CD4bs domain within gp120, but with an epitope completely distinct from that recognized by b12.
MAb HGN194, isolated from a donor infected with CRF02_AG clade, neutralized 11/20 pseudoviruses in the HOS-based assay and 19/92 pseudoviruses in the TZM-bl-based assay. Of note, HGN194 neutralized all Tier-1 isolates tested (10/10 neutralized) across clades A, B, C and recombinant AG and BC. Using Pepscan analysis with linear and cyclic peptide libraries of gp120 the epitope recognized by HGN194 was mapped to the sequence RRSVRIGPGQTF in the crown of the V3-loop ( Figure 4A) . Similarly, the epitope of 7 additional gp120specific mAbs was mapped to the same V3 region using different peptide libraries generated from the sequence of the isolate to which each mAb was mostly reactive ( Figure 4A) . The minimal sequence recognized by these mAbs ranged from 7 to 17 amino acids and, with a single exception, comprised the G(A)PGR/Q/K sequence, which is modeled to interact with CCR5 or CXCR4 during the viral entry process [63] . However, in contrast to HGN194 which neutralizes with high potency all Tier-1 isolates tested, the other V3-specific mAbs neutralized only a few Tier-1 isolates ( Figure 1A -B and Figure 2 ), similar to the activity of other recently characterized V3 loop-specific mAbs derived from non-B clade infected individuals [38] . We confirmed the neutralization activity of HGN194 in an M7 cell-based assay challenged with replication-competent HIV-1 viruses carrying a Luc reporter [52] and we observed that 3/5 clade B viruses were neutralized (Table S5) .
To better characterize the epitope recognized by HGN194 we performed a replacement scanning of each position with the 18 complementary amino acids. This analysis revealed only 3 positions (RRSVRIGPGQTF) where amino acid substitutions abrogated binding. Remarkably, only one mutation out of the 21 found in viral isolates (I to M in position 6) affected binding of HGN194 ( Figure 4B-C) . However, we observed that several HIV-1 isolates that were not neutralized by HGN194 encoded the same amino acid sequence shared by other HIV-1 isolates that were neutralized ( Figure 4B ). For instance, the epitope RKSVRIGPGQTF on 93MW965.26, which is strongly neutralized by HGN194 (i.e. IC50 ,0.02 mg/ml), is shared with Du422.1, ZM197M.PB7 and Du172.17, isolates not neutralized by HGN194. This implies that the HGN194 V3 epitope is unavailable for antibody recognition on the assembled trimer of these non-neutralized isolates.
Taken together these results indicate that HGN194 has unusual potency and breadth for a V3-specific monoclonal antibody. Of note, HGN194 appears to have a broader reactivity than 447-52D since it neutralizes all Tier-1 isolates and 11% of Tier-2 isolates, while 447-52D neutralizes 88% of Tier-1 and 4% of Tier-2 isolates (Figure 2 and Table S3 ). Another V3 mAb with unusual breadth of activity is F425-B4e8 [37] . We have not compared HGN194 with F425 here, but F425-B4e8 appears somewhat broader than 447-52D, neutralizing 1 clade C and 2 clade D pseudoviruses [37] . Further comparison between F425-B4e8 and HGN194 is difficult as different viruses and assays were used to determine neutralization breadth and potency. HK20, an HR-1 Specific mAb with Target Cell-Specific Neutralizing Activity HK20 was initially characterized as a gp41-specific mAb with broad neutralizing activity in the HOS-based assay but lacking robust activity in the TZM-bl assay. To map the epitope, we tested HK20 against all overlapping 15-mer peptides of the extracellular region of HIV-1 gp41. HK20 bound peptide QQHLLQLTVWGIKQL that overlaps the hydrophobic pocket sequence of HR-1 ( Figure 5A ). The specificity of HK20 was confirmed by immunoprecipitation of the 5-helix bundle (5HB) construct [47] and of a trimeric HR-1 construct that includes the gp41 fusion peptide, indicating that the fusion peptide does not interfere with HK20 binding ( Figure 5B ). In ELISA, HK20 bound to 5HB and HR-1 gp41 constructs with K 50 values of 210 and 95 ng/ml, respectively and also to the gp41 ectodomain, although with lower avidity (1.27 mg/ml) ( Figure 5C ), a finding that may be due to the partial unfolding of solid phase-bound gp41. Taken together, the above results indicate that HK20 recognizes a highly conserved site within the HR-1 region of gp41.
Since HR-1 occupies a restricted surface only transiently exposed during the HIV-1 entry process, we hypothesized that accessibility of the HK20 target epitope might be limited by the size of an IgG molecule. We therefore compared intact IgG and Fab fragments of HK20 for their capacity to neutralize a panel of 22 pseudoviruses in the HOS-based assay. The HK20 Fab fragments showed markedly increased breadth and potency compared to IgG, being able to neutralize 21/22 isolates with IC 50 values ranging from 0.01 to 5.7 mg/ml ( Figure 5D -E and Table S4 ). By contrast, HK20 IgG neutralized 19/22 isolates with IC 50 values ranging from 1.46 to 84 mg/ml. In addition, when the HK20 Fab fragment was tested in the TZM-bl assay it neutralized 10 out of 33 isolates with IC 50 values ranging between 0.4 and 8.8 mg/ml (Table S2) , thus suggesting that the lack of activity observed with HK20 IgG was possibly associated to a different susceptibility of this cell line to this type of entry inhibitors. Overall, these findings suggest that the reduced size of the Fab molecule allows increased access to the HR-1 region and imply that HR-1 accessibility is largely dependent on the target cell used. This is similar in concept to the enhanced neutralization profiles of Fab and ScFv specific for the CD4i surface [39] . Further studies using M7 cells [52] challenged with replication-competent HIV-1 viruses carrying a Luc reporter showed that HK20 exhibits broad neutralization consistent with the results from the HOS cell assay but unlike the TZM-bl assay, being able to neutralize 4 out 5 viruses tested (Table S5) .
The epitopes recognized by the 55 remaining mAbs were mapped using three experimental approaches: i) cross-competition against mAbs of known specificity or soluble CD4; ii) binding to gp120 mutants or gp41 constructs representing different conformational intermediates; iii) Pepscan analysis. The results of the analysis performed on the gp120-specific and gp41-specific mAbs are presented in Table 1 and Table 2 , and the data are summarized in a pie chart in Figure 6 .
Thirty-seven mAbs bound to gp120 targeting primarily the CD4bs and the V3 loop and to a lesser extent the CD4i site ( Table 1) . A first group of mAbs was assigned to the V3 loop based on cross-competition with two V3 loop specific mAbs (HR10 and HGA9). This group comprised 8 mAbs, which were already mapped to the V3 loop using the Pepscan analysis approach, and 7 additional mAbs that were not analyzed by peptide scanning (i.e. HGF9, HGT4, HGP21, HGP51, HGW48, HGD129 and HGP27). Four mAbs (HX44, HGP105, HGW7, HGY38) reacted with wt and CD4bs YU2 mutants but failed to bind the CD4i mutant, and therefore were assigned to the CD4i cluster. Notably, one mAb (HGP68) was mapped by Pepscan analysis to a novel epitope in the V2 loop (TVYALFYRLDIVP) and neutralized 4 Tier-1 isolates (Table S2) . Fifteen other mAbs were assigned tentatively or conclusively to the CD4bs based on their capacity to inhibit gp120-sCD4 binding. Most of these mAbs competed other CD4bs-specific mAbs, such as b12 and HJ16, while in some cases this assay could not be performed due to lack of epitope expression on the gp120 proteins used. Furthermore, this group of mAbs showed variable reactivities with the YU2 mutants. While most of them did not bind the D368R mutant similar to b12, others including HJ16, bound this mutant avidly. In addition, most of these new CD4bs-specific mAbs also showed decreased binding to the CD4i mutant, suggesting that they may span a broader, as yet undescribed region that includes elements of the CD4bs and CD4i sites. Finally, for 2 mAbs these analyses did not provide any relevant information.
Twenty-one mAbs bound to gp41 by targeting primarily the immunodominant C-C region, the HR-1 and the region recognized by 5F3 mAb [64] (Table 2) . MAbs HGK129, HGN146 and HGN35 were assigned to the C-C loop since they competed with 3D6 [43] , reacted with an HR2 construct and recognized synthetic peptides within this region. MAbs HGW17 and HGY25 were provisionally assigned to the C-C region since they competed with 3D6. These data are consistent with the immunodominance of the C-C region and with the notion that the antibody response against this region overlaps with the 3D6 epitope. Several mAbs competed with 5F3 and HK20, and bound both the HR-1 and 5HB constructs, suggesting that they may bind between the fusion peptide and the HR-1 region. HGB33 was provisionally assigned to the HR-1 region according to competition with HK20 and binding to the 5HB construct. Other mAbs bound specifically the 5HB construct but did not compete with any of the mAbs tested, thus indicating that the complete HR-1 coiled coil region exposed in 5HB harbors antibody epitopes available for B cell recognition in the gp41 pre-hairpin conformation [65, 66] . The concomitant binding to recombinant gp41 suggests that the latter may partially resemble the gp41 prefusion state. Of note, three gp41 binding mAbs (HGP40, HGP48 and HGY50) neither competed with any of the mAbs tested, nor bound to any constructs representing pre-hairpin conformations or native gp140 ( Figure S1 ), indicating that they recognize as yet uncharacterized regions that are only available in the recombinant gp41 protein. Interestingly, mAb HGF24 was assigned by Pepscan analysis to an epitope in the C-terminal region of HR-2 (TNLIYTLIEESQN), proximal to the 2F5 epitope, and neutralized 4 Tier-2 isolates (Table S2) . This mAb competed with HK20 for gp41 binding, in spite of their distinct cognate specificities (HR-2 and HR-1, respectively). This finding would be consistent with the proximity of HR-1 and HR2 in the six-helix bundle structure. Finally, although the gp41 protein used in the primary screening includes the 2F5 and 4E10 epitopes, no MPER specific mAbs were isolated, reinforcing the idea that this portion of gp41 is poorly immunogenic in humans [21, 22] In conclusion, regardless of their limited breadth of neutralization, this extended panel of human mAbs may represent a useful tool for understanding the molecular basis of Env recognition in humans.
Using an improved EBV immortalization method combined with a broad screening strategy we isolated from memory B cells of HIV-1 infected donors 58 mAbs that cover an extensive antigenic surface of Env. Of these, 37 neutralized at least one of the isolates tested and 3 mAbs in particular bound to the CD4bs, V3 crown and HR-1, showing considerable neutralizing breadth against a panel of HIV-1 pseudoviruses of different clades and spanning Tier-1 and Tier-2 isolates.
Several studies have questioned the existence of individual broadly neutralizing antibody specificities as part of a normal immune response to HIV-1 infection. In this respect, b12 was isolated from a phage library, while 2F5, 4E10 and 2G12, although isolated from memory B cells, do not appear to have a counterpart in human sera [22] . In a recent study, Nussenzweig and coworkers used trimeric gp140 to isolate antigen-binding memory B cells from which 500 antibody sequences were retrieved by single-cell PCR [62] . Surprisingly, although the B cell donors had broadly neutralizing serum activity, none of the mAbs isolated had this property. This raised the possibility that broad serum neutralizing activity results from multiple clonal responses each of unique epitope specificity but restricted breadth of viral strain recognition. However, an alternative interpretation might be that since available recombinant trimeric antigens vary in structure, the 'bait' used for B cell isolation was not optimal for enriching those secreting neutralizing antibodies. Our results indicate that monoclonal antibodies with a limited spectrum of neutralizing activity can be easily isolated, while those with a broad pattern of cross-clade reactivity are rare, in line with the study of Walker et al [40] and consistent with inferences from serum neutralization specificity mapping analyses [21] . In this respect our study demonstrates the feasibility of the EBV immortalization method, which is limited only by the size of the blood samples obtained (20-50 ml of peripheral blood). The characterization of the specificity and neutralizing activity of this new panel of human mAbs reveal some interesting features of the human antibody response to HIV-1 within a population of HIV-1-infected donors. First, most of the gp120-specific mAbs showed neutralizing activity (37 out of 58), indicating that they bind to sites available on the functional Env trimer. The selection of a highpercentage of trimer binding specificities may be a consequence our use of trimeric Env antigens during the screening of the EVB-B cell supernatants. The second aspect relates to gp41-specific antibodies, which, with a few remarkable exceptions, were not neutralizing. Interestingly the non-neutralizing antibodies bound gp41 in the context of the recombinant trimer, whereas the most effective neutralizing mAbs bound to gp41 fusion intermediates.
The three new neutralizing mAbs HJ16, HGN194 and HK20 show some interesting features. HJ16 showed a breadth of neutralizing activity comparable to, and generally complementary to b12, which aside from PG9 and PG16 [40] is currently the most potent of the relatively broad neutralizing antibodies available. HJ16 also showed selective neutralization of multiple Tier-2 isolates, making it particularly relevant as a template for vaccine design. HGN194 binds to an extremely conserved epitope in the V3 crown, and neutralizes all Tier-1, but only 11% of Tier-2 isolates tested. HGN194 showed broader activity than the well-characterized V3-specific mAb 447-52D against a panel of 92 pseudoviruses. The preference for Tier-1 isolates characteristic of V3-specific mAbs is consistent with the idea that the V3 loop is displayed to varying degrees in the context of the native trimeric Env protein of individual isolates [67, 68] and with the recent observation that sCD4 broadens neutralization of V3 mAbs [69] . The results obtained with HK20 are particularly intriguing, since this mAb has a broad pattern of neutralization observed with HOS but not TZMbl target cells. This mAb recognizes a highly conserved epitope in the HR-1 trimer and it is more effective as an Fab fragment as compared to intact IgG, a fact that is may be explained by the limited accessibility of the epitope, which is likely to be exposed only transiently on the cell surface during the viral entry process [70] . Intriguingly, a recent study indicates that in TZM-bl cells HIV-1 pseudoviruses penetrate the cytoplasm from within endosomes rather than from the cell surface [71] , a finding that might help explain the lack of activity of HK20 in the TZMbl assay if binding of this mAb is affected by the endosomal environment. Further studies on primary cells will be required to address the relative importance of these two entry pathways and to establish whether HK20 may be capable of exerting a broad and potent neutralizing activity in vivo. Preliminary studies using M7 cells [52] challenged with replication-competent HIV-1 viruses Cross competition with biotinylated mAbs of known specificity: +, 90-100% inhibition, +/2, 50-90% inhibition, 2, ,50% inhibition. Binding to different gp41 constructs by ELISA. Minimal linear epitopes using the Pepscan analysis and specificity assignment. C-C, C-C loop; 5HB, 5-helix bundle; FP, fusion peptideShown is also the number of isolates neutralized in the HOS-based and TZM-bl based assays. doi:10.1371/journal.pone.0008805.t002
carrying a Luc reporter showed that HK20 exhibits reasonably broad neutralization consistent with the results from the HOS cell assay, and similar to Fab HK20 in the TZM-bl cell assay. Whereas the TZM-bl assay is sensitive for certain antibodies such as CD4bs mAbs, some mAbs such as 2G12 to other epitopes are less sensitive to neutralization in this assay [72] , perhaps owing to the unnaturally high level of CCR5 expression on the TZM-bl cells [60] . In this respect, low coreceptor expression levels have been associated with delayed fusion kinetics and hence enhanced virus sensitivity to HR-1 binding inhibitors, such as T-20 [73] . Therefore, despite the advantages of high throughput with the TZM-bl assay, alternative neutralization assays might have closer relevance to the in vivo situation.
In conclusion, we have shown that in HIV-1-infected individuals a high proportion of HIV-specific B cells produce neutralizing antibodies but only a few possess neutralizing activity with relatively broad neutralization coverage. In the short term, some of these reagents will be tested for their ability, singly or in combination, to prevent HIV-1 or SIV transmission in a macaque challenge model as done previously with other mAbs [5, 6, 7, 8, 9] .
In the longer term, and combined with an atomic-level analysis of epitope specificity, some of these mAbs may facilitate the templatebased design of immunogens for the development of a vaccine able to induce neutralizing antibodies against the wide range of HIV-1 strains present in the global pandemic. Figure S1 Binding of gp120 and gp41-specific mAbs to a panel of 15 recombinant Env proteins from different clades. Found at: doi:10.1371/journal.pone.0008805.s001 (0.47 MB PDF) Figure 6 . Epitope specificity and neutralizing activity of the mAb panel. The pie chart shows the specificity of the 58 mAbs isolated from the 21 interrogated individuals. The fraction of antibodies with neutralizing activity against at least one isolate is indicated in parentheses. Partial CD4bs, incomplete inhibition of CD4 binding; HR-1/ FP, epitope located in between HR-1 and the fusion peptide as determined by 5F3 competition; HR-1/5HB, recognition of trimeric HR-1 and 5-helix bundle; 5HB, 5 helix bundle; C-C region, gp41 immunodominant region; HR-2, heptad-repeat 2; ND, not defined epitopes within gp120 or gp41. doi:10.1371/journal.pone.0008805.g006 Recode-2: new design, new search tools, and many more genes ‘Recoding’ is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. Although only a small proportion of genes utilize recoding in protein synthesis, accurate annotation of ‘recoded’ genes lags far behind annotation of ‘standard’ genes. In order to address this issue, provide a service to researchers in the field, and offer training data for developers of gene-annotation software, we have gathered together known cases of recoding within the Recode database. Recode-2 is an improved and updated version of the database. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. At present, the Recode-2 database stores information on approximately 1500 genes that are known to utilize recoding in their expression—a factor of approximately three increase over the previous version of the database. Recode-2 is available at http://recode.ucc.ie The term 'translational recoding' describes the utilization of non-standard decoding during protein synthesis and encompasses such processes as ribosomal frameshifting, codon redefinition, translational bypassing and StopGo (1) (2) (3) (4) (5) (6) (7) . What is often considered as a decoding error-e.g. a frameshifting error or mistranslation of a particular codon-may occasionally benefit the organism by increasing its fitness and survival. In such instances the propensity for the decoding 'error' may be selected for during evolution, leading to the formation of a particular sequence context that elevates the frequency of the 'error'. To discriminate such cases of programmed decoding 'misbehaviour' from promiscuous translational errors or translational noise, the term recoding is used. The position within an mRNA where a recoding event takes place is termed the 'recoding site'. Sequence elements responsible for increasing the efficiency of recoding events are termed 'recoding stimulatory signals', and a minimal sequence fragment that allows recoding to take place at the natural efficiency (i.e. relative to the level of standard decoding at the recoding site) is termed a 'recoding cassette'.
Recoding can benefit gene expression in a number of ways. It can regulate gene expression by being part of a sensor for particular cellular conditions. Prominent examples include ribosomal frameshifting in bacterial release factor 2 (RF2) and eukaryotic antizyme mRNAs. In both instances, ribosomal frameshifting is required for the production of the corresponding active full-length protein products. In the RF2 mRNA, the efficiency of frameshifting is negatively regulated by the cellular concentration of its product, RF2, providing an autoregulatory circuit for its biosynthesis (8) (9) (10) . In the antizyme mRNA, the efficiency of frameshifting is modulated by cellular levels of polyamines, whose concentration in turn is controlled by antizyme (11, 12) . Thus, this mechanism ensures the maintenance of antizyme production at the levels required to support physiologically appropriate concentrations of polyamines. Recoding can also be used for the diversification of protein products encoded by a single gene. An illustrative example is in bacterial dnaX mRNA, where frameshifting allows synthesis of two different protein subunits-sharing the same N-terminal part-from a single open reading frame (ORF) in its mRNA (13) (14) (15) . A presumed constant ratio of frameshifting in dnaX ensures a fixed stoichiometric balance between these two subunits (16) . This balance, then, is independent of the absolute levels of dnaX transcription and translational initiation on its mRNA. Similarly, in many viruses recoding is responsible for setting a ratio between protein products (such as those encoded by gag-pro-pol genes in retroviruses) produced from a single mRNA (17) . Recoding also provides RNA viruses with a mechanism for the translation of downstream ORFs on polycistronic RNAs [other mechanisms include leaky scanning, shunting, reinitiation, IRESs and the production of subgenomic RNAs (18) ] and may also be involved in global regulation mechanisms, such as mediating the switch between translation and replication on the same genomic RNA (19) . Finally, recoding provides a way for the incorporation of non-standard amino acids-e.g. amino acids that share their codons with termination signals (the most prominent example of which is selenocysteine, encoded by UGA) (20) (21) (22) . For further information on the diverse variety of recoding functions, see recent reviews (1, 3, 7, 23, 24) .
Recoding cassettes may be composed of a variety of diverse sequence elements. For example, primary nucleotide sequences may promote re-arrangements of tRNA molecules relative to their codons in mRNA inside the ribosome or affect recognition of tRNAs or release factors in the ribosomal A-site. On the other hand, many recoding signals act in the form of RNA secondary structures, such as simple stem-loops, or more complex pseudoknots, kissing stem-loops and other structures that involve interactions between considerably distant RNA regions (19, (25) (26) (27) (28) . Trans-acting RNA signals affecting ribosomal decoding through complementary interactions with ribosomal RNA (29-32), or through the nascent peptide acting within the ribosome exit tunnel (6, 33, 34) , are also known. Some recoding events-such as selenocysteine insertion-require the presence of additional specialized machinery such as selenocysteine tRNAs, selenocysteine-specific translation factors and several other components of the selenocysteine biosynthesis and insertion pathway (20, (35) (36) (37) . Recent reviews on stimulatory signals involved in the modulation of recoding events and molecular mechanisms of recoding provide further details (7, 25, 27, 38, 39) .
Despite considerable progress in the development of computational tools for the prediction of protein coding genes in sequenced genomes, the identification and annotation of recoded genes lags far behind. The hurdle lies not so much in the fact that recoded genes do not obey standard rules of genetic readout but, rather, in the considerable diversity of recoded genes and sequence elements responsible for recoding. Even among evolutionarily related genes, all utilizing recoding, the diversity of recoding signals can be considerable. An extreme example is when orthologous genes utilize recoding at different stages of gene expression to achieve the same goal. An example is in dnaX, where ribosomal frameshifting is employed by enterobacteria, but transcriptional slippage is used in Thermus thermophilus (40) . A similar situation occurs in bacterial insertion sequence (IS) elements, where a certain group of IS elements utilizes transcriptional slippage to produce ORFA-ORFB fusions, while many other IS elements utilize ribosomal frameshifting for the same purpose (41) . The diversity of recoding functions, combined with the wide spectrum of unrelated sequence elements involved in recoding, makes the design of a uniform model of recoding intractable. Nonetheless, in recent years, we have witnessed the development of specialized models and computational tools for the identification of particular subsets of recoding cassettes, or tools that are specific to recoding events in particular groups of homologous genes (42) (43) (44) (45) .
These developments, at least partially, were facilitated by the availability of a compiled dataset of known recoded genes collected together in the Recode database (http://recode.genetics.utah.edu), which was initially launched 9 years ago (46, 47) . To facilitate further development of computational tools for the prediction of recoded genes in the ever faster growing body of sequence data, as well as to provide bench researchers with upto-date information on recoding, an efficient means of Recode database population and annotation are now required. In this article, we describe the incarnation of the database, Recode-2. The major advances of Recode-2 (hosted in a new location http://recode.ucc.ie) over previous versions include a new web design allowing enhanced visualization of stimulatory signals, a uniform RecodeML format for the annotation of recoded genes, and a significantly larger number of entriesincluding many recently identified cases-that altogether have more than doubled the size of the database since its last published update.
The data are stored in a local PostgreSQL database that is queried by PHP scripts embedded in the web interface. The schema of the PostgreSQL database is shown in Figure 1 . The database stores information on individual genes that utilize recoding, the mechanisms and stimulatory signals involved, and references to the original literature sources that describe the recoding events. In order to facilitate the uniform annotation of recoding events, we have designed an XML-based format for the annotation of recoded genes, RecodeML. The document type definition for RecodeML is available at the Recode-2 web site at http://recode.ucc.ie/dtd The extensibility of the RecodeML format will allow incorporation of new annotation, if required, for newly discovered types of recoding, and the associated features, as they are being discovered. The database handles batch importation of properly designed RecodeML entries into the PostgreSQL database, thus facilitating rapid population of the database with new data.
The data in the database may be explored in two ways. They may be browsed by one of the three categories: kingdom (archaea, bacteria, eukaryotes and viruses), organism and type of recoding. The data may also be searched directly by key words that can be inserted into the search field. Searches that use regular expressions are allowed. The output of a database search is a list of Recode-2 entries in a short format that includes organism name, kingdom, genus, type of recoding event, status of Figure 2 shows an example of sequence annotation for the human oaz1 gene, alongside a diagram of a stimulatory RNA secondary structure, and the Recode-1 logo.
Unlike Recode-1, where all data on recoding events were introduced manually, Recode-2 also utilizes automated identification of recoding events by the recently developed computer programs ARFA (43) and OAF (44) , that are able to identify and annotate +1 frameshifting events in mRNAs of bacterial RF2s and eukaryotic antizyme (OAZs), respectively. However, a significant source of recoding events remains to be serendipitous discoveries by experimental studies that sometimes are complemented by more systematic studies of large groups of similar genes (51, 52) . Therefore, a large proportion of new data are still populated manually or semi-manually. To ease manual population of recoding events, a special form has been designed that is available in the database upon user registration. User registration needs to be approved by one of the database contributors. The novel data in the database include 249 RF2 mRNAs identified by ARFA, 152 events identified by OAF, 200 new selenoprotein genes (53) (54) (55) (56) and 200 new viral annotations (57) including the newly discovered frameshift cassettes in potyviruses (58), alphaviruses (59) and the Japanese encephalitis group of flaviviruses (60) .
The database will expand in accordance with the growth of available sequence information that will be scanned by one of the existing programs for recode annotation.
We also plan to continue developing tools for the automatic identification of recoding events from nucleotide sequences. As the field grows and the number of recoded genes progressively increases, it becomes harder to extract data from the relevant literature and a number of novel recoded genes may escape the database. Therefore, we encourage users and researchers in the field to submit their data directly to the Recode-2 database. We are also willing to provide help with the analysis of potential new recoding events. ViralORFeome: an integrated database to generate a versatile collection of viral ORFs Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such ‘ORFeome’ resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway® system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins. The number of viral genomic sequences available in public databases has increased exponentially, opening new perspectives to understand genetic basis and functional mechanisms that underlie virus replication, pathogenesis and evolution. In particular, this enabled to establish for each virus a list of potential regulatory and expressed sequences, a framework often referred as the 'parts list' of biological systems (1) . Current investigations aim at understanding how these viral components act upon each other and interact with host macromolecules to carry on viral replication and spreading. To reach such a system view of virus cycles, more functional analyses of viral components are necessary, especially in the field of virus-host molecular interactions (2, 3) . To address this question, a large collection of viral open reading frames (ORFs) established in a recombination-based cloning system allowing their mass transfer into various functional assays would be extremely helpful. Such ORF collections, often referred as 'ORFeomes', have been developed for human and few other organisms and represent great resources to explore protein functions in a large-scale setting (4) (5) (6) (7) (8) (9) . Recombination-based cloning technologies like the Gateway Õ system enable the mass cloning of polymerase chain reaction (PCR)-amplified ORFs into a 'donor' vector to create 'entry' clones. Once entry clones have been established, ORFs can be easily recombined into different 'destination' vectors that allow protein expression. So far, only few viral ORFeomes have been built and they are dedicated to a single virus, arguing that much more needs to be achieved (10) (11) (12) (13) .
Because of their small-sized genomes, most viruses exhibit a limited number of ORFs. Therefore, building a viral ORFeome collection covering a significant number of viral pathogens can appear like a manageable project. However, this eventually becomes a daunting task when considering virus strains and polymorphisms that affect viral proteins (Table 1) . Nonetheless, these variations cannot be neglected since they often determine virulence and/or host adaptation. For example, a unique amino acid mutation in the polymerase of poliovirus can alter its processivity, turning a highly pathogenic virus into an attenuated strain that can eventually be used as a vaccine (14) . Similarly, a Semliki Forest virus strain encoding an nsP2 protein, with a single amino acid mutation in its nuclear localization signal, is impaired for the control of type I interferon response and strongly attenuated in vivo (15) . Thus, specific care must be taken when cloning a virus ORFeome, since an accurate identification of the strain that is used as a template is absolutely critical, both in terms of genotype and phenotype. For this reason, wild-type virus strains corresponding to primary isolates will be generally preferred to culture-adapted laboratory strains that often exhibit an altered phenotype in vivo. Such biological samples are often difficult to obtain, since it requires access to patients, medical-care facilities and laboratories of an appropriate biosafety level. With such constraints to obtain suitable viral RNA or DNA templates, a collaborative effort between virology laboratories is needed to build a comprehensive viral ORFeome resource. This motivated the development of ViralORFeome 1.0, an open-access database and management system designed to assist academic laboratories in the development of a viral ORFeome collection using a recombination-based cloning technology.
ViralORFeome is a database for creating and managing viral ORF clones. It includes a web interface to search and display viral sequences and annotations downloaded from GenBank. When users have identified the viral ORF sequences that they want to clone, clicking on target sequences automatically creates virtual clones in ViralORFeome database together with suitable cloning primers. These primers are designed for PCR amplification and cloning of viral ORFs using the Gateway Õ system. To determine if 'entry' clones produced at the bench exhibit expected sequences, sequence traces can be uploaded in ViralORFeome, automatically aligned, and compared to corresponding virtual clones. Finally, ViralORFeome database allows users to manage their collection of viral ORF clones, keep track of expression plasmids derived from 'entry' clones and share plasmids with other laboratories. System architecture and data flow are depicted in Figure 1 .
The World Wide Web server for ViralORFeome is Apache (http://www.apache.org/) with Hypertext Preprocessor (PHP, http://www.php.net) and Asynchronous JavaScript and XML (AJAX) technologies. The relational database management system for ViralORFeome is PostgreSQL (http://www.postgresql.org/). ViralORFeome Entity-Relationship (ER) model was divided into five interconnected parts: 'Taxonomy', 'Genome', 'ORFeome', 'Interactome' and 'Users' schemes (Supplementary Figure S1 ).
ViralORFeome 1.0 is based on conventional sequence and virus databases. Virus and host organism classification was retrieved from the taxonomy database at National Center for Biotechnology Information [NCBI; ftp://ftp .ncbi.nih.gov/pub/taxonomy/; (16) ]. Complete viral CDS and associated annotations were downloaded from the CoreNucleotide division of GenBank [http://www.ncbi .nlm.nih.gov/sites/entrez?db=nucleotide; (17) ] using Entrez Programming Utilities (eUtils) at NCBI (see Supplementary Data). ICTV names were obtained from the most recent publication of the International Committee on Taxonomy of Viruses using Perl scripts (18) . Finally, host gene and protein sequence annotations were extracted from Ensembl database (19) .
ViralORFeome database can be accessed at: http://www .viralorfeome.com. When selecting 'Virus' option in the menu bar, ViralORFeome web interface allows users to navigate throughout viral sequences retrieved from GenBank by entering criteria such as virus species, taxon IDs or accession numbers (Supplementary Figure S2 ). ViralORFeome can also execute a BLASTn or BLASTp search among available viral sequences (20) . Virus genomes and coding sequences of interest are visualized with an integrated genome browser adapted from GBrowse that provides a synthetic view of genomic sequence features (21) . When users have identified a viral ORF sequence that they want to clone, clicking on this sequence automatically generates cloning primers and virtual clones in ViralORFeome database ( Figure 2 ). ORF-specific Gateway Õ primers are designed using the Oligonucleotide Selection Program (OSP) software adapted for recombinational cloning (22) . By default, primers are designed to clone full-length ORFs from ATG to STOP codon according to GenBank annotations. However, users can specify 5 0 -and 3 0 -coordinates to clone ORF fragments corresponding to specific domains or even upload manually designed primers. Once primers have been validated, virtual clones are generated and used as entry points in ViralORFeome to store and access all information relative to the viral ORF constructs (e.g. primers, sequencing traces, potential mutations, and comments). Importantly, when users want to clone some variant of an ORF that is not defined in GenBank, and therefore is not available in ViralORFeome, an interface allows for the creation of a 'variant' clone that is anchored to the most similar annotated sequence found by BLAST in the database (Supplementary Figure S3 ). This ensures a maximum of flexibility when viral sequences are known and characterized but not readily available in GenBank, a frequent situation when working with primary isolates. This also facilitates the management of mutants generated on purpose to study the role of specific residues in a protein's function.
In this recombination-based cloning pipeline, viral ORFs amplified by PCR or RT-PCR are cloned by in vitro recombination into donor vectors such as pDONR207 or pDONR223 (see Supplementary Data for detailed protocols). After transformation, a construct can be purified either from a mini-pool of bacteria colonies to keep the sequence diversity of the original template (e.g. when working with viral quasi-species) or from a single isolated colony. These two cloning strategies, referred as 1.0 and 2.0, respectively (5), are manageable using ViralORFeome interface (Figure 2 ).
To validate 'entry' constructs by sequencing, ViralORFeome automatically aligns uploaded sequence traces onto virtual clones using either an extended Libalign Perl tool or Phred and T-Coffee programs that build first a contig of sequence traces before the alignment is performed (23, 24) . Validated 'entry' constructs can be subsequently used to recombine viral ORFs into various Gateway Õ -compatible destination vectors to achieve protein expression. ViralORFeome allows users to keep track of the different expression constructs that have been established for each viral ORF.
Users can browse the set of viral ORF clones already established in ViralORFeome by selecting the 'clones' option in the menu bar. Queries can be performed by entering criteria such as virus species, GenBank protein accession numbers or ViralORFeome clone IDs. Viral ORF clones can also be searched by nucleotide or protein BLAST. Until now, three laboratories involved in the Infection-MAPping project (I-MAP) have used ViralORFeome interface and generated a collection of 528 viral ORFs cloned into pDONR vectors. Among them, 145 have already been stored in a dedicated repository for viral ORFeome resource (ORFeotheque; Hospices Civils de Lyon), and 134 are available upon request under material transfer agreement via ViralORFeome. This set of viral ORF clones can be searched selecting the 'ORFeotheque' option in the menu bar.
To validate the viral ORF clone collection built using ViralORFeome pipeline, we tested a subset of 66 ORFs (Figure 3a ORFs were expressed in fusion downstream of the red fluorescent protein Cherry and constructs transfected in human cells to achieve subcellular localization (Figure 3b ). We observed a specific localization pattern distinct from Cherry alone for most constructs, demonstrating that viral ORFs are properly expressed in this system. In addition, and even if the N-terminal tag was expected to alter the localization of proteins like viral envelopes, observed localization patterns were generally consistent with literature. For example, expression of nsP1 not only from Semliki Forest virus and Sindbis virus (25) but also Chikungunya virus induced characteristic filopodia-like extensions in transfected cells. The capsid of yellow fever virus (C) was found in the nucleus where it accumulated in a dot-like pattern, reminiscent of nucleoli, as reported for C of dengue and Japanese encephalitis viruses (26, 27) .
As numerous viral proteins are known to inhibit the host immune response, the 66 ORFs were also tested for their ability to block signaling downstream of two key antiviral cytokines: Interferon-b (IFN-b) and tumor necrosis factor-a (TNF-a). Each ORF was expressed in fusion downstream of the 3xFLAG tag and, using luciferase reporter constructs, tested for its ability to inhibit IFN-b or TNF-a signaling (Figure 3c and d) . Both pathways were inhibited by nsP2 from Chikungunya and Semliki forest viruses. This is consistent with previous reports that used mutant viruses or replicons to demonstrate that nsP2 from Old World Alphaviruses induces a transcriptional shutoff and Figure 2 . Building a viral ORF collection using ViralORFeome interface. Viral sequences and annotations from GenBank are visualized with a genome browser that provides a synthetic view of sequence features (1) . CDS are shown in blue and proteins in green. Users can design a new clone by clicking on a viral protein of interest (2) . By default, ViralORFeome will anchor cloning primers at the extremities of selected ORFs (Method 1), but user can specify 5 0 -and 3 0 -coordinates and clone ORF fragments corresponding to specific domains. Users can also upload manually designed primers (Method 2). ViralORFeome will automatically design Gateway Õ cloning primers (3) and after validation (4), a virtual clone is created in the database (5) . Users need to select between two cloning strategies, 1.0 ('in pool') or 2.0 ('individual clone'), before they can access a webpage where all information relative to the construct are stored (6) . This includes clone coordinates, primers, sequence and comments (upper panel), sequencing traces and alignments (middle panel), and available entry and destination vectors to achieve viral ORF expression (lower panel). When back to the genome browser (1), viral ORF clones are displayed in red (1.0 constructs) or purple (2.0 constructs). A CMV-Renilla plasmid was also co-transfected and used as an internal control for transfection efficiency and cell viability. After transfection, cells were incubated for 24 h in the presence of IFN-b (c) or TNF-a (d) to activate ISRE or NF-kB response elements, respectively. Cells in position A1 and B1 correspond to negative and positive controls that were respectively left untreated or stimulated with IFN-b or TNF-a. Relative luciferase activity was determined using a chemiluminescent substrate, and results expressed in relative percentage to positive control. Data show one representative experiment out of two. controls the host antiviral response (15, 28) . In contrast, nsP2 derived from a Sindbis virus infectious cDNA clone (29) did not localize in the nucleus and failed to block signaling. This supports previous reports showing that nsP2 from Alphaviruses must be nuclear to control the antiviral response (15) . We also confirmed that V proteins of measles and mumps viruses and V and W proteins of Nipah virus block IFN-b signaling (30) . Interestingly, the V protein of Tioman virus was unable to do so, suggesting that this virus infecting flying foxes (Pteropus genus) is not adapted to human cells. Altogether, observed localization patterns and functional assays validate the clone collection that was generated with ViralORFeome platform. Furthermore, these results illustrate how large collections of viral ORF clones established in a versatile cloning system provide access to reverse proteomic platforms and large-scale functional assays.
In conclusion, ViralORFeome is the first open-access database that provides an integrated set of bioinformatic tools to build a collection of viral ORFs clones in a versatile system suitable for reverse proteomic experiments. In this perspective, ViralORFeome was especially designed to handle the diversity of virus strains, variants and species. As shown here, our cloning pipeline has been validated using functional assays and a collection of 528 viral ORFs has been generated in the Gateway Õ system. As collaborative efforts between virology laboratories are required to establish an ORFeome collection covering most viral genera and species, we believe ViralORFeome will provide the community with a framework to sustain this global effort.
In the near future, our objective will be to motivate more laboratories to join this program. In addition, we would like to implement new modules allowing users to store and visualize all kind of functional data generated with viral ORFs from the collection. This virus clone collection was primarily developed to map virus-host protein-protein interactions using the yeast two-hybrid (Y2H) system as a collaborative effort between co-authors of this manuscript. Current storage and display of virushost interaction data obtained by Y2H screening of HCV proteins constitute a first attempt to reach this goal. The 'Interaction' option of the menu bar allows users to select HCV ORFs and access all interacting cellular cDNA clones identified by Y2H. Whereas filtered data sets have already been published (12) , ViralORFeome interface allows users to access Y2H raw data to filter these results according to their own quality criteria. Other functional data such as subcellular localization or interference with immune response pathways should be implemented.
Public access to the ViralORFeome is available at: http://www.viralorfeome.com. Registration is not necessary, and use of the database is free. Users who want to generate viral ORF clones that are tagged with their institutional name can create an account to register. Clinical review: Primary influenza viral pneumonia Primary influenza pneumonia has a high mortality rate during pandemics, not only in immunocompromised individuals and patients with underlying comorbid conditions, but also in young healthy adults. Clinicians should maintain a high index of suspicion for this diagnosis in patients presenting with influenza-like symptoms that progress quickly (2 to 5 days) to respiratory distress and extensive pulmonary involvement. The sensitivity of rapid diagnostic techniques in identifying infections with the pandemic 2009 H1N1v influenza strain is currently suboptimal. The most reliable real-time reverse transcriptase-polymerase chain reaction molecular testing is available in limited clinical settings. Despite 6 months of pandemic circulation, most novel H1N1v pandemic strains remain susceptible to oseltamivir. Ensuring an appropriate oxygenation and ventilation strategy, as well as prompt initiation of antiviral therapy, is essential in management. As the novel swine-origin influenza A (H1N1)v global pandemic is under way, the medical community has already experienced an increase in hospitalizations from influenzarelated complications in many geographic regions. Primary viral pneumonia is recognized as the most severe pulmonary manifestation of influenza. While uncommon during seasonal epidemics, the syndrome has been well documented during the H2N2 pandemic of 1957-1958 and is thought to be responsible for much of the mortality associated with the young healthy adult population during the 1918 H1N1 pandemic [1] . This paper reviews the clinical aspects of influenza and primary influenza pneumonia that may be of most interest to the practicing physician in the 2009 pandemic environment.
Seasonal influenza epidemics occur each year as a result of minor changes in the antigenic characteristics of the hemagglutinin and neuraminidase glycoproteins of the influenza viruses (antigenic drift) [2] . The morbidity and mortality associated with seasonal influenza outbreaks are significant, especially in older patients, who incur more than 90% of the influenza-related mortality each year [3] . Factors contributing to their increased vulnerability include a decline in cellmediated and humoral immune responses, a reduction in lung compliance and respiratory muscle strength, a diminished cough reflex associated with normal aging, the frequent presence of multiple comorbid conditions, nutritional deficiencies, and in the case of residents of long-term care facilities, greater exposure risk due to close living quarters and shared caregivers [4, 5] .
Influenza pandemics occur less frequently, as a result of major changes in the surface glycoproteins of the virus (antigenic shift). The emerging novel influenza strain then easily spreads into an immunologically susceptible population. Consequently, pandemics are characterized by a shift in mortality toward the otherwise young and healthy 18to 35-year-old adults, with relative sparing of older patients, as evidenced by epidemiological analyses of the 1918 influenza A pandemic [6] . This is likely due to the persistence of immunological memory in older patients after previous exposures to H1-type viruses similar to the pandemic strain [7, 8] . The virulence of the pandemic strain may also play a role, as demonstrated by recent experiments with the highly fatal 1918 influenza strain [9] .
Preliminary data from the 2009 H1N1 pandemic suggest a similar shift in age-related mortality. An analysis of 532 cases of 2009 pandemic H1N1 influenza A in the US, for example, has revealed that 60% of the cases occurred in patients not older than 18 years of age and that only 5% occurred in patients older than 50 years [10] . In the cohorts recently tested, the modest extent of immunological memory in older patients was confirmed by the presence of serum crossreactive antibodies to the pandemic H1N1 influenza A strain found in 33% of the adults older than 60 years of age versus 6% to 9% of the adults 18 to 64 years of age and none of the children [11] . Influenza attack rates during seasonal epidemics vary between 10% and 20% but can be much higher during pandemics. For example, an analysis of the pandemic 2009 H1N1 influenza A outbreak in La Gloria, Veracruz, found clinical attack rates of 29% in adults older than 15 years and 61% in children younger than 15 years of age [12] . However, these rates may be different in geographic areas of low population density.
Groups at high risk for severe disease and complications secondary to 2009 pandemic H1N1 influenza A include patients with underlying pulmonary (asthma) and cardiac comorbid conditions, some immunosuppressive states, pregnancy and post-partum states, diabetes mellitus, obesity [13, 14] , and, in children, prior neurological disabilities [15] . Severe primary H1N1 influenza pneumonia can also affect young adults without any underlying comorbidities [14] .
Person-to-person transmission occurs primarily through droplet spread via small particle-sized aerosols generated by coughing, sneezing, or talking [16] . Airborne transmission should be considered in those patients exposed to aerosolgenerating techniques, such as intubation or mechanical ventilation.
The incubation period is usually 24 to 48 hours. In the absence of antiviral treatment, viral shedding starts within 24 hours before the onset of symptoms and continues for approximately 5 days in healthy adults [17] . Viral shedding can last longer in children, patients with extensive comorbidities, older patients, patients who undergo mechanical ventilation, and immunocompromised hosts [18] [19] [20] . The infectious period can be significantly reduced by the use of antiviral medications within the first 48 to 96 hours of illness [20] .
After inhalation, the virus is deposited onto the respiratory tract epithelium, where it attaches to ciliated columnar epithelial cells via its surface hemagglutinin. Local host defenses, such as mucociliary clearance, or secretion of specific secretory IgA antibodies can remove some of the virus particles. However, if mucociliary clearance is impaired (as in smokers [21] or older patients [22] ) or secretory antiinfluenza IgA antibodies are absent (as in no antecedent exposure to the virus), infection continues unabated [23] . Respiratory epithelial cells are invaded, and viral replication occurs. Newer viruses then infect larger numbers of epithelial cells, shut off the synthesis of critical proteins, and ultimately lead to host cell death [24] .
In patients with uncomplicated influenza, bronchoscopy typically reveals diffuse inflammation and edema of the larynx, trachea, and bronchi, and biopsy may show cellular infiltration with lymphocytes and histocytes and desquamation of the ciliated columnar epithelium [25] . In patients with severe influenza infections that progress to primary viral pneumonia, the involvement of the respiratory tree is extensive, with necrotizing tracheobronchitis, ulceration and sloughing of the bronchial mucosa [26] , hyperemic alveolar capillaries with intra-alveolar hemorrhage, infiltration of alveolar spaces with fluid, fibrin, and cellular exudates, and lining of the alveoli with acellular hyaline membranes [1] . Autopsies from patients with primary influenza pneumonia confirmed bilateral severe hemorrhagic pneumonitis with interstitial inflammation, diffuse alveolar damage, and heavy viral loads observed in the periphery of the lungs.
The clinical features of uncomplicated influenza are virtually indistinguishable from those of other respiratory viral infections. Influenza is classically characterized by an abrupt onset of headache, high-grade fever, chills, dry cough, pharyngeal irritation, myalgias, malaise, and anorexia. The fever lasts an average of 3 days (range of 2 to 8 days). The cough, initially nonproductive and nonpurulent, may persist for weeks. Bronchial hyper-reactivity and small-airway dysfunction are often present in influenza virus infection. In the presence of asthma or structural lung disease, wheezing may be a prominent manifestation [24] . Vomiting and diarrhea, while rare in seasonal influenza, have been frequently reported in infections with the 2009 pandemic influenza A H1N1v strain [10] , particularly in children.
The clinical presentation of influenza in the immunocompromised host may be more subtle and manifest only as coryza; similarly, the classic fever symptom may be absent in the older patient, who may present only with lethargy, confusion, anorexia, and cough [27] . Influenza pneumonia and respiratory complications in patients with Th1 defects, such as HIV infection, are uncommon.
Pneumonia and the acute respiratory distress syndrome (ARDS) account for the majority of severe morbidity and mortality that accompany pandemic influenza infection [14] . Pneumonia may occur as a continuum of the acute influenza syndrome when caused by the virus alone (primary pneumonia) or as a mixed viral and bacterial infection after a delay of a few days (secondary pneumonia) [28] . Identifying patients who are more likely to develop severe complications from influenza pneumonia requires a high clinical vigilance. Commonly used pneumonia severity assessment tools, such as the Pneumonia Severity Index [29] or CURB65 [30] , are not useful in deciding which patients to hospitalize in the context of primary influenza pneumonia since these tools have not been developed and validated during a pandemic scenario. Thus, careful triage in the emergency department and early identification of young patients with decreased oxygen saturation, respiratory rate above 25, concomitant diarrhea, or hypotension are crucial. Elevated lactate dehydrogenase, creatine phosphokinase, and creatinine at hospital admission may also serve as prognostic indicators of severe disease [14] . C-reactive protein and procalcitonin are increased during this acute lung injury stage of early fibroproliferation.
The most ominous cases are those infections that progress rapidly to ARDS and multilobar alveolar opacification. These patients usually present with gradually increasing dyspnea and severe hypoxemia after an antecedent of 2 to 5 days of typical influenza symptoms [14] . The cough is usually productive of thin, often bloody, sputum with few cells. Hypoxemia increases progressively to the point of respiratory failure requiring intubation and mechanical ventilation, often after only one day of hospitalization [14] .
The radiological appearance of primary influenza pneumonia can be difficult to distinguish on chest x-ray from pulmonary edema, given the presence of perihiliar congestion and hazy opacification, at least in the lower lobes (Figure 1a,b) . Pleural effusions may also be present. Computed tomography scans ( Figure 2) can add further diagnostic insight and may be useful to differentiate primary viral pneumonia from bronchiolitis and interstitial pneumonias, which occur frequently in children and young adults but have a benign outcome. Concomitant myopericarditis should be excluded by echocardiography. Concurrent pulmonary emboli, as suggested by early case reports from hospitalized patients with pandemic influenza A H1N1v 2009 in the US [13] , may further contribute to clinical deterioration in some patients. However, the occurrence of concomitant pulmonary emboli has not been reproduced in other geographic regions so far.
Bacterial co-infection, though uncommonly reported in the early stages of the 2009 H1N1 pandemic, may be more prevalent than initially thought. A recent analysis of lung specimens from 77 fatal cases of pandemic H1N1v 2009 infection found a prevalence of concurrent bacterial pneumonia in 29% of these patients [31] . The most common coinfecting bacterial pathogens were pneumococcus, Staphylococcus aureus, and Streptococcus pyogenes, with a median duration of illness of 6 days [31] .
The real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) Swine Flu Panel for detection of pandemic H1N1 influenza, developed by the Centers for Disease Control and Prevention (Atlanta, GA, USA) and distributed to many laboratories in US and worldwide, is a reliable and timely method of diagnosing the pandemic strain [32, 33] . The viral culture, while the gold standard in influenza diagnostics, takes several days before the results are known [24] . The direct fluorescent antigen influenza test was recently reported to have a sensitivity of 93% compared with the rRT-PCR [34] , but the test requires considerable technical expertise in addition to a fluorescent microscope. The commonly used point-of-care rapid influenza tests provide results in less than 1 hour but are of only modest sensitivity for seasonal influenza viruses (63%) [35] and unacceptably insensitive for the detection of pandemic H1N1 influenza [35, 36] . Thus, for the majority of clinicians practicing during the 2009-2010 influenza pandemic, the access to a reliable and timely diagnostic modality may still be limited. As such, it is Chest x-rays of a patient with primary H1N1 (swine-origin influenza A) influenza pneumonia on day 1 (a) and day 6 (b) of hospitalization. reassuring to know that the patients presenting during influenza epidemics with both cough and fever within the first 48 hours of symptom onset are very likely to have actual influenza (79% positive predictive value) [37] .
The majority of patients with primary influenza pneumonia require ventilatory support. Mortality is high but can be decreased with an optimal protective ventilatory strategy (tidal volume of not more than 6 mL per kilogram of predicted body weight, with a plateau airway pressure goal of not more than 30 cm H 2 O), as shown in Acute Respiratory Distress Syndrome Network clinical trials; this strategy is therefore recommended in acute lung injury [38, 39] . Maintaining an adequate fluid balance is also important for survival in acute lung injury. The hemodynamic status should be optimized by appropriate repletion of intravascular volume deficits during the early systemic inflammatory stage [40] . Once acute lung injury has become established, a conservative fluid management protocol, which was associated with beneficial effects in clinical trials, should be considered [41, 42] . In severe refractory cases of primary influenza pneumonia, some patients require venovenous extracorporeal membrane oxygenation support and continuous renal replacement for acute renal failure. Antiviral treatment should be initiated as soon as possible, particularly in patients at high risk of complications. The majority of treatment benefits are derived when antivirals are initiated within the first 48 hours from onset of symptoms. Unfortunately, most patients with primary viral pneumonia receive oseltamivir after 3 to 8 days of influenza onset [14] . However, the experience with seasonal influenza suggests that a reduction in mortality for hospitalized patients has been documented even when oseltamivir was initiated after the first 48 hours following illness onset [43] . Thus, being out of the ideal therapeutic window should not be a reason to withhold antiviral treatment at any stage of active disease.
Both neuraminidase inhibitors (oseltamivir and zanamivir) are active against the novel H1N1v 2009 pandemic influenza A strain. The recommended adult dose for oseltamivir, considered the first-line therapy for H1N1 influenza infection, is 75 mg orally twice a day for a total of 5 days [44] . Dose adjustment may be required in the presence of reduced creatinine clearance, but the dosage should be maintained for patients undergoing continuous venovenous hemodialysis. A recent World Health Organization treatment guideline for pharmacological management of 2009 pandemic H1N1v influenza A recommends the consideration of higher doses of oseltamivir (150 mg twice a day) and longer duration of treatment for patients with severe influenza pneumonia or clinical deterioration [44] . Since hospitalized patients can shed influenza virus for prolonged periods of time, extending antiviral treatment beyond the first 5 days of treatment in cases of persistent influenza symptoms may be necessary. However, clear guidelines for these circumstances have not been established, and clinical trials examining the appropriate treatment dose and duration for severe H1N1 influenza in various patient populations are acutely needed.
Development of oseltamivir resistance in novel H1N1 influenza, though still exceedingly rare, has been reported from several countries [45] . It should be suspected in patients who remain symptomatic or have evidence of viral shedding despite a full treatment course of oseltamivir. Immunosuppression and prior exposure to oseltamivir, such as receipt of prolonged post-exposure prophylaxis, increase the risk for oseltamivir resistance [45] . Zanamivir remains an effective therapeutic option for these cases. Zanamavir is also indicated in the rare circumstance when an oral route for oseltamivir administration is not available for critically ill patients in the intensive care unit. The risk of bronchospam rarely associated with zanamivir, particularly in patients with underlying reactive airway disease, can be minimized by concurrent bronchodilator administration.
Adamantanes (amantadine and rimantadine) have no activity against the 2009 influenza A H1N1v pandemic strain. They are effective for seasonal H1N1 influenza strains, which are 100% resistant to oseltamivir. Therefore, for patients presenting with primary influenza pneumonia in geographic regions where seasonal H1N1 strains are circulating in addition to the novel H1N1 pandemic strain, amantadine or Computed tomography scan of the patient with primary H1N1 (swineorigin influenza A) influenza pneumonia whose chest x-rays appear in Figure 1 . rimantadine should be added to oseltamivir [46] . Rimantadine is also associated with immunomodulatory effects.
Patients presenting with severe influenza pneumonia who may have concurrent bacterial superinfection should also receive antibacterial agents effective against the most common etiologic pathogens, such as Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, according to published guidelines in the management of communityacquired pneumonia [47] .
Corticosteroids remain controversial in persistent ARDS and are not routinely recommended [48] . Further research is required to clarify their impact on outcome. Whether other adjunctive immunomodulatory therapies such as statins, chloroquine, and fibrates could prove useful in the context of an influenza pandemic [49] remains to be determined.
Primary influenza pneumonia caused by the 2009 pandemic influenza A H1N1v strain, though rare, carries a high mortality. The rapid progression from initial typical influenza symptoms to extensive pulmonary involvement, with acute lung injury, can occur both in patients with underlying respiratory or cardiac morbidities and in young healthy adults, especially if obese or pregnant. Prompt initiation of effective antiviral treatment, appropriate oxygenation and ventilation support, and antibacterial treatment in the case of concurrent bacterial pneumonia are critical for survival. The most reliable and timely diagnostic method for 2009 pandemic influenza A H1N1v infection is the rRT-PCR developed by the Centers for Disease Control and Prevention. Common 'point-of-care' rapid influenza tests are very insensitive. A negative test result in a patient with clinical symptoms compatible with influenza pneumonia does not accurately rule out influenza and should not be a deterrent to prompt oseltamivir treatment during this current pandemic. Further research is needed in order to identify the immunological dysfunction and determine the most effective dose and duration of oseltamivir as well as the role of potential adjunctive agents in the treatment of primary influenza pneumonia. Calculating the potential for within-flight transmission of influenza A (H1N1) BACKGROUND: Clearly air travel, by transporting infectious individuals from one geographic location to another, significantly affects the rate of spread of influenza A (H1N1). However, the possibility of within-flight transmission of H1N1 has not been evaluated; although it is known that smallpox, measles, tuberculosis, SARS and seasonal influenza can be transmitted during commercial flights. Here we present the first quantitative risk assessment to assess the potential for within-flight transmission of H1N1. METHODS: We model airborne transmission of infectious viral particles of H1N1 within a Boeing 747 using methodology from the field of quantitative microbial risk assessment. RESULTS: The risk of catching H1N1 will essentially be confined to passengers travelling in the same cabin as the source case. Not surprisingly, we find that the longer the flight the greater the number of infections that can be expected. We calculate that H1N1, even during long flights, poses a low to moderate within-flight transmission risk if the source case travels First Class. Specifically, 0-1 infections could occur during a 5 hour flight, 1-3 during an 11 hour flight and 2-5 during a 17 hour flight. However, within-flight transmission could be significant, particularly during long flights, if the source case travels in Economy Class. Specifically, two to five infections could occur during a 5 hour flight, 5-10 during an 11 hour flight and 7-17 during a 17 hour flight. If the aircraft is only partially loaded, under certain conditions more infections could occur in First Class than in Economy Class. During a 17 hour flight, a greater number of infections would occur in First Class than in Economy if the First Class Cabin is fully occupied, but Economy class is less than 30% full. CONCLUSIONS: Our results provide insights into the potential utility of air travel restrictions on controlling influenza pandemics in the winter of 2009/2010. They show travel by one infectious individual, rather than causing a single outbreak of H1N1, could cause several simultaneous outbreaks. These results imply that, during a pandemic, quarantining passengers who travel in Economy on long-haul flights could potentially be an important control strategy. Notably, our results show that quarantining passengers who travel First Class would be unlikely to be an effective control strategy. Clearly air travel, by transporting infectious individuals from the epicentre in Mexico to other geographic locations, significantly affected the rate of spread of influenza A (H1N1). However, the possibility of within-flight transmission of H1N1 has not been evaluated, although it is known that smallpox, measles, tuberculosis, SARS and seasonal influenza can be transmitted during commercial flights [1] . These pathogens can be transmitted both by direct contact with large respiratory droplets and by airborne transmission of small, aerosolized droplets [2] [3] [4] . Several recent empirical studies have attempted to determine which mode of transmission is the most important for influenza. Fabian et al. [2] conducted studies of seasonal strains to measure the concentration of influenza RNA viral particles exhaled by infected patients. In their study, infected patients exhaled into an Exhalair, a device that collects and counts viral particles in exhaled breath, and the concentration of RNA viral copies was measured in Teflon filters. It was found that 33% of infected humans exhaled droplets containing influenza. Lowen et al. [3] have experimentally determined that influenza can be transmitted amongst guinea pigs by droplet and/or aerosol spread. In these experiments the guinea pigs were inoculated intranasally and viral samples were collected from nasal washes of their upper and lower respiratory tracts. Viral growth was determined to occur in their lungs and nasal passages, establishing the guinea pig as a model host for droplet infection. Lowen et al. [3] also found airborne and droplet transmission could occur between animals by conducting experiments in which they placed infected and non-infected guinea pigs in the same cage, adjacent cages and separated cages at least 90 cm apart [3] . A similar and more recent experimental study using ferrets has shown that H1N1 can be transmitted by aerosolized droplets. Although several studies have shown that H1N1 appears to have similar infectivity as seasonal strains the importance of airborne transmission of H1N1 in humans has not yet been definitively established [4] [5] [6] . However, it should be noted that airborne transmission of seasonal strains of influenza during a flight, particularly if ventilation is poor, can be significant. As an extreme example, one individual infected with a strain of influenza A managed to infect 72% of passengers during a 3 hour flight on a plane without ventilation [7] . By assuming that H1N1 has a similar infectivity to that of seasonal strains, we calculated the expected number of H1N1 infections that one source case could cause during an international flight.
Typically, the number of infected individuals that can be expected during a large scale outbreak or epidemic is determined by calculating the basic reproduction number (R 0 ) [8] . R 0 is defined as the average number of secondary infections caused by one infectious individual in a population of susceptible individuals during the time in which they are infectious. Therefore, R 0 is a useful measure for predicting the number of infections that could be expected to occur during a large-scale outbreak or an epidemic. However, R 0 is not a useful measure for predicting the number of infections that one infectious individual could cause over a few hours in a confined space. In this case, the number of secondary infections is best predicted using methods from the field of quantitative microbial risk assessment. These are the methods that we have used in our analysis. Specifically, we have used the classic Wells-Riley equation [9] to estimate the number of passengers that could become infected with H1N1 during a commercial flight, assuming one infectious case is on board. The Wells-Riley equation was developed over 30 years ago [9] and is now the standard methodology for predicting (for infectious pathogens that have airborne transmission) the size of outbreaks within buildings and other enclosed environments.
The Wells-Riley equation is based on the number of exposed individuals, the respiratory rate of the source case, the length of exposure to the infectious droplets and the concentration of infectious viral particles over time.
Within an aircraft the concentration of infectious viral particles over time is determined by the ventilation rates, the volume of the cabins in the aircraft and the infectiousness of the index case. In the field of quantitative microbial risk assessment, infectiousness is quantified by measuring the number of infectious particles that an infected individual expels per litre of air. Infectivity is expressed in terms of infectious quanta; a measure defined by William Wells in 1934 [10] . Wells used the Poisson distribution to express the relationship between dosage and infection; he defined a quantum to be the number of infectious droplet nuclei required to infect 1 -1/e (that is, 63%) of exposed susceptible individuals. Quanta are now a standard measure for expressing the risk of infection with airborne pathogens and have been used extensively to characterize the infectivity of seasonal stains of influenza [2, 11, 12] . Rates of quanta production for influenza have been estimated by both direct and indirect means. Indirect infectivity estimates have been made using back calculation based on outbreaks in environments with little or no ventilation [12] . Specifically, Rudnick and Milton [12] used data from an influenza outbreak on a grounded plane with very limited ventilation and a single source case (see Moser et al. [7] ) to back calculate the rate of quantum generation for a highly infectious case of influenza. Direct measurements of quanta production rates have been made by determining the rate at which infectious individuals exhale RNA virus copies [2] . This is practically accomplished by measuring the RNA virus concentration in Teflon filters in conjunction with an individual's rate of respiration [2] .
During a flight, transmission of influenza due to direct contact is very limited because passengers remain seated, and therefore we only model airborne transmission. We used a sequential box model [5] to calculate the temporal concentration of infectious droplets (quanta) of H1N1 in a transatlantic airliner with four cabins: First Class, Upper Deck Business Class, Lower Deck Business Class and Economy Class. The sequential box model was developed in the 1980s by Ryan et al. [13] . It is a standard method for estimating the concentration of infectious particles in confined environments. The model calculates concentration based on volume of air in each cabin, rate of inflow of air from outside the aircraft, proportion of air recycled during the flight, efficiency of filters in purifying recycled air and strain infectivity. In all our calculations we assume that the index case is symptomatic. If the index case is asymptomatic they would expel very few infectious virions and, hence, would be very unlikely to cause any infections during a flight. Our sequential box model is specified by four equations, one equation for each cabin in the aircraft. The model assumes that the air in each cabin is well mixed. It enables us to track the concentration of infectious quanta of H1N1 (C) in each of the four cabins, over time t. The subscript to C signifies the cabin class: 1 = First Class; 2 = Upper Deck Business Class; 3 = Lower Deck Business Class; and 4 = Economy Class. The sequential box model and the airflow between cabins in the plane are shown diagrammatically in Figure 1 . The model is given in terms of a system of ordinary differential equations as:
, ,
, ,
, ,
The Sequential Box Model (SBM) that we used to determine the temporal concentration of infectious droplets of H1N1 in four cabins (C 1 , C 2 , C 3 and C 4 ) in a commercial airliner (specifically, in a Boeing 747) Figure 1 The Sequential Box Model (SBM) that we used to determine the temporal concentration of infectious droplets of H1N1 in four cabins (C 1 , C 2 , C 3 and C 4 ) in a commercial airliner (specifically, in a Boeing 747). Within the aircraft air diffuses between adjacent cabins (forward flow f i and backward flow b i ) and is removed and replaced by an air supply system. Air is replaced by outside air and by air that has been re-circulated through a HEPA filter.
We model four scenarios, with one infectious individual (index case) in each of the four cabins. The parameter q i represents the rate of production of infectious quanta of H1N1 in cabin i. The subscript to q signifies the cabin class. If there is no infectious individual in a cabin i then q i is set equal to zero. The infectivity of H1N1 is currently unknown. However, empirical data indicate that it appears to be equally (or slightly less) transmissible than seasonal strains of influenza [5, 6] . Therefore, in each scenario we varied the quanta production rate of the index case (q i ) from 50 to 128 quanta/hour, based on infectivity estimates for seasonal strains of influenza [2, 7, 12] .
The remaining parameters in the model characterize the environmental conditions in the plane. We parameterized the model to reflect the environmental conditions in a Boeing 747 airliner; parameter values were obtained from Boeing [14] . where N i represents the number of passengers in cabin i and ρ represents the volumetric breathing rate of the source case (ρ = 0.3 m 3 /h) [15] . We obtained estimates for C i (t), i = 1...4 by integrating our sequential box model.
We set the number of exposed individuals to reflect occupancy in a Boeing 747 operating at full capacity: 23 passengers in First Class; 40 in Upper Deck Business Class; 40 in Lower Deck Business Class; and 313 in Economy. We varied the length of exposure to the H1N1 virus (that is, flight duration) from 5 hours to 11 hours to 17 hours.
We calculate that the risk of catching H1N1 will, essentially, be confined to passengers travelling in the same cabin as the source case. We found that this result holds across the entire range of infectiousness (q) that we considered. If q is even lower (that is, the source case is even less infectious than we have assumed) transmission to other cabins would be even less likely. Since we have assumed that the air in each cabin is well mixed, individuals in the same cabin will have the same risk of infection.
Not surprisingly, we find that the longer the flight the greater the number of infections that may be expected ( Figure 2 ). Our results also show that the greater the infectivity of the source case, the greater the effect of flight duration on increasing the number of infections ( Figure  2 ). This occurs regardless of whether the source case is travelling in Economy or First Class; our results for Business Class are between those for First Class and Economy Class and are not shown.
We calculate that, because of the increased seating capacity of Economy Class, there is a higher probability (75%) that the infectious case will be travelling in Economy rather than in Business (10%) or First Class (5%). Therefore, in general, individuals travelling in Economy are always likely to be at greater risk of infection with H1N1 than individuals travelling in Business or First Class.
Our modelling clearly shows that the location of the source case will have a significant effect on the number of infections that occur during a flight ( Figure 2 ). If the plane is completely occupied, a single infectious individual in Economy Class will cause more infections that a source case in First Class ( Figure 2 ). We calculate that H1N1, even during long flights, poses a low to moderate withinflight transmission risk if the source case travels First Class. Specifically, 0-1 infections could occur during a 5 hour flight, 1-3 during an 11 hour flight and 2-5 during a 17 hour flight ( Figure 2 ). However, our results show that within-flight transmission could be significant, particularly during long flights, if the source case travels in Economy Class. Specifically, 2-5 infections could occur during a 5 hour flight, 5-10 during an 11 hour flight and 7-17 during a 17 hour flight (Figure 2 ). More infections would occur in the Economy Class than in Business or First Class because the majority of the passengers travel in Economy and that section has the smallest volume of air per passenger. However, if the aircraft is only partially loaded, under certain conditions, more infections could occur in First Class than in Economy Class. For example, we calculate that, during a 17 hour flight, a greater number of infec-
tions would occur in First Class than in Economy if the First Class Cabin is fully occupied but Economy class is less than 30% full.
Our modelling has shown (for the first time) that significant H1N1 transmission could be occurring during air travel. Our results have been obtained from a model parameterized for a Boeing 747. However, our results are generally applicable to other transatlantic aircraft with similar size cabins (in terms of seating capacity and volume) which may have different configurations. In order to obtain our results we only modelled the risk of acquiring H1N1 based on airborne infection. During a flight, trans-mission due to direct contact is very limited because passengers remain seated. If the virus is also transmitted by large respiratory droplets (through direct contact) then the risks of infection that we have calculated will be underestimates and passengers in close proximity to the source case will be at greatest risk. We recommend that more detailed models of within-aircraft transmission of H1N1 are developed in order to investigate these issues. These models could be used to investigate the potential effectiveness of interventions such as the use of facial masks for symptomatic individuals.
We have demonstrated how methodology from the field of quantitative microbial risk assessment can be used to
The expected number of H1N1 infections caused by a single infectious case as a function of flight duration and strain infectivity Infectivity of source case (infectious quanta per hour) assess the importance of air travel on the global spread of H1N1. We used infectivity estimates from studies of seasonal strains of influenza to parameterize our model as several studies have now shown that H1N1 appears to have similar infectivity to seasonal strains [5, 6] . We recommend, that in order to obtain further insights into the risks of acquiring influenza during air travel, the infectivity of all known strains of influenza (including H1N1) should be measured in terms of quanta/hour. In addition, we recommend estimating the infectivity of new strains of influenza as soon as they appear. As we have discussed in the introduction of this paper, there are already established methods for estimating infectivity. These methods measure infectivity in terms of infectious quanta by measuring virus RNA concentrations in exhaled respiratory droplets of infectious individuals [2] .
Several mathematical models for predicting the potential spread and magnitude of a global pandemic of H1N1 have recently been published [16, 17] . These large-scale models link H1N1 epidemics in different geographic regions by moving infectious individuals from place to place using air travel networks. They do not include the potential for transmission to occur during air travel. Based on our results, we suggest that hierarchical multi-scale network models are needed to more accurately predict the global dissemination of H1N1. Such models would expand on the current large-scale models based on air travel networks by linking them to small-scale models of H1N1 transmission within an aircraft. Such models would be hierarchical in nature as the small-scale models would be subcomponents of the large-scale models.
Our results have important implications for understanding and predicting the global dissemination of H1N1. They provide important insights into the potential utility of air travel restrictions on controlling influenza pandemics. We have shown that one infectious passenger could cause a significant number of H1N1 infections during long flights, particularly if they are travelling in Economy Class. These results imply that one individual travelling by plane, by infecting other travellers on the same flight, could cause multiple simultaneous outbreaks in different geographic locations rather than causing only one outbreak. Hence, quarantining passengers who travel in Economy Class on long-haul flights could potentially be an important control strategy in the winter of 2009/2010. Notably, our results show that quarantining passengers who travel First Class would be unlikely to be an effective control strategy.  Abstracts from the 11th International Congress of Behavioral Medicine  are efficacious in brief treatment settings, and our outcomes data supports this assertion. Limitations of the intervention include participant attrition and insufficient access to comprehensive mental health and substance use management resources. D. Further Recommendations: For many, maintaining treatment adherence is challenging. Implementing programs that offer unlimited sessions of mental health counseling and substance use treatment or harm-reduction strategies for people living with HIV could greatly increase treatment adherence. Additionally, public health policy could be modified with the aim of de-stigmatizing those living with HIV/AIDS. Effective legislation could include acts recognizing and legitimizing same sex couples. Comprehensive, accessible health care should be provided to anyone, regardless of socioeconomic status. These changes could help mitigate the shame reported by some people living with HIV/ AIDS, potentially leading to greater help seeking behaviors and lower transmission rates. Additionally, embracing HIV testing as a routine component of primary care could help to reduce HIV incidence and minimize stigma, potentially leading to a number of positive personal and public health outcomes, including increased treatment adherence. The impact of depression on treatment adherence in individuals with End Stage Renal Disease (ESRD) has been well-supported in the literature (Hailey & Moss, 2000) ; however, the influence of anxiety on adherence is less clear. It has been suggested that the relationship between anxiety and adherence may be indirect and influenced by other cognitive or psychosocial variables such as locus of control or knowledge of disease (Brownbridge & Fielding, 1994 , DiMatteo et al., 2000 . Anxiety has been related to adherence with antibiotic regimens (Cockburn et al., 1987) , and for patients with asthma (Baiardini et al., 2006) and COPD (Dowson et al., 2004) . A limitation of previous research on anxiety and adherence in ESRD is the failure to investigate the differential impact of anxiety on specific components of adherence (e.g., medication, appointment attendance, dietary guidelines). The goal of the present study was to conduct a preliminary investigation into the relationship of anxiety and renal disease knowledge to treatment adherence. Data includes 124 veterans with ESRD referred for pre-surgical psychological evaluations for renal transplantation. Renal disease knowledge was assessed in several subcategories of diet, medication, and laboratory tests. Adherence was assessed with respect to diet, medication, and appointment attendance. Both knowledge and adherence were rated by clinicians based on patient self-report and renal team staff-report. Patients were divided into "anxious" and "non-anxious" groups based on self-reported presence of anxiety symptoms. Data analysis on self-reported compliance revealed that there were no group differences with regard to renal disease knowledge; however, patients in the anxious group were rated as significantly more compliant with renal diet and appointment attendance than non-anxious patients (t > 2.20, p < .05). In addition, regression analyses indicated that anxiety and knowledge significantly predicted compliance with appointment attendance [F (1, 116) = 3.68, p = .05] and dietary adherence [F(4,115) = 5.95, p < .001].
Knowledge, but not anxiety, predicted medication adherence [F (3,114) = 3.63, p=.015]. Results for staff-reported compliance emphasized the role of disease knowledge over anxiety in predicting adherence. The present data support the need to consider medical adherence as a multifaceted concept by demonstrating that anxiety is related to some aspects of renal disease management. Results also highlight the need for more sophisticated models to explain the influence of anxiety on adherence in patients with ESRD. Limitations of the present study, implications, and suggestions for future research are presented. Background: Recent studies of health care referrals among the under five children with high fever have indicated that incomplete treatment due unfulfilled referrals was the major reason for the children dying in the homes. Despite the Zambian government acknowledging malaria and fever as leading cause of morbidity and mortality among the under five children, the community behaviour and practices on care-giving at household level, have not been well documented. Identification of reasons that lead to an increased number of children dying in the homes is understudied. To try and understand risks and benefits of home management of fever a community qualitative study was conducted. Methods: Participants consisted of 10 caregivers whose children were 5 years and below old and had died in the community, 10 parents of children 5 years and below, who were admitted in the children's hospital with severe fever, survived and discharged from the hospital and went back home and 10 participants, whose children had fever and survived within the community 14 days prior to the commencement of the study. Questions were devoted to knowledge and attitudes about health seeking behaviour at household level. Knowledge about disease transmission, treatment and prevention, perceived risks of failure to take the child to health facility for early treatment and completion of referrals, were investigated. Results: Of the 30 key informants who participated in the study, 10 informants were of caregivers of children died before seeing the medical specialist. Majority of children (67.8%) were between 1 and 24 months old. Over 87 and 90 percent respectively were living with biological parents at the time of death. Caretakers from the three groups feared of losing their children if admitted in the children hospital (54.3% and 58.9%). Fear that their children would be exposed to other disease, lack of knowledge about the disease and referral, and lack of medicines in health facilities characterized demotivation for early access of quality care. Fees and transportation resulted in delay accessing health care outside the home and unfulfilled referrals. Medication non-adherence affects health outcomes in chronic illness. Only 50% of hypertensive patients adhere to prescribed medication (World Health Organization, 2004) . Depression has been considered one of the main psychosocial predictors of medication non-adherence (DiMatteo, Lepper & Croghan, 2000) . The psychological mechanisms that can explain this relationship remain uncertain. Objective: To evaluate the role of depression as a medication nonadherence factor in hypertensive patients and the possible psychological mechanism involved in this effect. Methods: 204 hypertensive patients (67.6 yrs old, 36.8% men) attending Primary Health Care Centers were evaluated in their regular visit to the cardiovascular program. All patients had over 1 year of pharmacological treatment for hypertension and had blood pressure over 140/90 mmHg in the last health control visit. Study variables included depression (POMSdepression scale), medication beliefs (Specific Beliefs about Medicine Questionnaire), and adherence to prescribed antihypertensive medication (Hill -Bone Medication Adherence Scale). Results: Depressive patients had a higher level of specific medication concerns than non-depressive patients (t=3,351, p<,001) . Multivariate linear regression shows that medication non-adherence is predicted by depression (B=.172, p<.01), and medication beliefs have a mediating role on this effect. Mediation effects were studied using Baron and Kenny's Criteria (1986) : depression predicted medication beliefs (B=−.304, p<.01); medication beliefs predicted non-adherence (B=−.442, p<.01) , and the predictive role of depression on non-adherence decreased when medication adherence was included in the regression equation (B=.012, p=.863). Conclusion: Our results confirm, in a sample of Chilean hypertensive patients, the effect of depression and beliefs about medicine over medication adherence and suggest that concerns about medication should be included in anti-depression psychotherapy for hypertensive patients. Background: This study compares and contrasts patient and provider perceptions of challenges and opportunities for patients on antiretroviral therapy (ART) in India. Methods: HIV patients (2 groups, n=8 women, 8 men) and HIV providers (n =18) were recruited from the Post Graduate Institute for Medical Education & Research (PGIMER) in Chandigarh, India to participate in focus group discussions (FGDs) and key informant interviews (KIIs). KIIs included HIV peers, treatment providers, NGO stakeholders and researchers. FGDs included experienced ART users, those new to ART and HIV treatment providers; interviews and groups used semi-structured stem questions. Transcriptions were coded based on conceptual relevance; codes were expanded for common themes and grouped into categories. Results: Provider interviews and groups reflected disparate perceptions regarding the relative importance of specific issues with respect to ART adherence, e.g., patient knowledge, karma, use of alternative medications. Providers agreed, in order of importance, that education, illiteracy, depression and alcohol and drug use negatively affect ART adherence; disagreed on the influence of income, gender and karma. Patients and providers agreed on the impact of HIV knowledge and alternative therapies (homeopathy, ayurveda, unani, yoga, reiki, astrology) . Patients expressed concern about food insecurity, stigma, side effects, clinic wait time, distance from clinics and the frequency of visits required. Though medications are no-cost, patients noted the hidden costs of blood tests, travel to clinics, and days missed from work at clinics. For longer term patients, discontinuing medication following remission of symptoms occurred. Enabling migrant and traveling workers to obtain ART during time away from home, social support, increased patient morale, positive attitudes about medication and patient provider communication were identified as key elements in enhancing adherence. Conclusions: Preliminary findings highlight the need for a patient -provider collaborative approach to ART provision and enhancing adherence. Future initiatives to encourage providers and stakeholders to collectively explore existing policies for ART provision will be proposed. This study is supported by NIH grant no. R21NR011131. Post-operative adherence rates among heart transplant (HT) patients remain lower than expected when considering the potential consequences, and these rates tend to decrease as more time passes following surgery. In the initial period after transplant older patients have higher rates of mortality and morbidity, but in the long-term some studies have suggested that older patients have better adjustment and quality of life compared to younger patients. The purpose of this study was: 1) to identify age differences in long-term adjustment to HT across a range of psychosocial adjustment measures, and 2) to examine other demographic and psychosocial predictors of overall adherence behavior. HT patients (n=555, mean age=54) from multiple centers were assessed 5-10 years post-surgery for adherence behaviors, psychological factors and physical symptoms. Analysis of variance results showed that older adults (age≥60) had better adjustment on measures of quality of life, satisfaction with social support, negative affect and disease-related stress compared to younger adults. Linear multiple regression demonstrated that age and several psychosocial factors, including depressive cognitions, accounted for 25.7% of the variance in self-reported difficulty with adherence. Similar variables were significant in a logistic regression analysis predicting which patients would have difficulty or no difficulty with adherence. Clinical implications are discussed in terms of tailoring interventions to age groups for optimal postoperative adjustment.
Background: Despite the increase in the number of people using ART in South Africa, the use of traditional medicine in combination with ART becomes a challenge. This study explored the perceptions, knowledge and attitudes of patients, health workers and traditional healers about use of traditional medicine and ART. Methods: Data was collected through interviews with 14 participants: six patients on ART and TM; two doctors; 2 professional nurses and 4 traditional healers. Two focus group discussions, one in each research site were held with community health workers who work with HIV positive patients. Data was analysed using a thematic content analysis approach. Findings: When patients become sick they use traditional medicine first, then when all else fail they come to the health facility. Most patients start using traditional medicine before they are diagnosed with HIV. When they get sick the first point of entry seem to be the traditional healers. Most patients start using traditional medicine before they are diagnosed with HIV. Some people prefer using traditional medicine because they want privacy and to remain anonymous. Conclusion: This study has revealed that patients have a history of using traditional medicine for chronic conditions prior to their diagnosis with HIV, and continue using traditional medicine in conjunction with their ART. This indicates that traditional medicine is an integral part of the "treatment modality" for patients and is used in combination with conventional medicine. Learning Objectives: 1. Describe the types and sources of social support for Korean older adults. 2. Compare the received and desired social support by Korean older adults. Background: Korea is a fast-aging country experiencing changes in social and family values. While research on social support among older adults increases, understanding the context in which they perceive and expect social support is relatively limited. This qualitative study aims to illustrate how older adults in Seoul recognize received and desired social support in what context. Method: In-depth face-to-face interviews were conducted with 21 older adults in Seoul who were local senior center users. They were asked to describe the types and sources of received and desired social support; and the meaning of the gap between received and desired social support. Interview transcripts were analyzed using a grounded theory approach.
Results: While main sources of support perceived are spouses and adult children, local senior centers serve as supplementary source of emotional and instrumental support. Friends, neighbors, religious groups are also mentioned as social support sources. Although the respondents recognize the gap between received and desired emotional and financial support and are stressed by it, they could not identify a strategy to compromise the gap within their life circumstances. Instead, they either justify the gap as part of aging process or deny the need for support. Conclusion: As changes in family structure and economic challenges affect the social support dependency of older adults on their children, social ecological approaches to enhance support capacities from individual to system levels are requested. Local senior center is one example that can be fostered as a social support source in the community. Background: Quality of life (QOL) has been recognized as a meaningful outcome measure when assessing course of illness or evaluating effectiveness of interventions. Whether individuals with dementia can evaluate their own Quality of Life (QOL) has been debated. The debate evolves on whether patient ratings or proxy ratings be used or both? The purpose of this study was to determine concordance between the patient's selfreport on QOL and caregiver's appraisal on QOL of individual with dementia. Method: Sixty family caregivers who were living with and caring for person diagnosed with Alzheimer's disease and their care recipients participated in the study. The data is part of an ongoing intervention study to enhance resourceful skills in family caregivers. Patients have mild or moderate cognitive impairment. Quality of Life was measured using the 13-item Quality of Life for Alzheimer's Disease (QOL-AD: Logsdon et al, 2002) , developed for both patient and caregivers includes patient's and caregiver's appraisal of patient's physical condition, mood, interpersonal relationships, ability to participate in meaningful activities, finances and an overall assessment of self and life quality as a whole. Overall scores were computed separately for the patient and caregiver reports by summing the 13 items, for a total possible score ranging from 13 to 52, with higher scores indicating higher QOL. Data were analyzed using descriptive statistics, Pearson correlation and a single-measure intraclass correlation. Alpha Cronbach's coefficient for proxy QOL and patient's QOL were .71 and .85, respectively.
In Japan, there is little evidence about the effect of Cognitive Behavioral Therapy (CBT) for older adults compared to Europe and the United States. The purpose of this study was to develop CBT for older adults with depression which fit in with Japanese culture and research its effectiveness in outpatient care. By randomized clinical trial recruiting, 15 older adults were chosen. All the participants had a diagnosis of Major depression. The participants were randomly allocated to receive either Treatment as usual (TAU) group (n=10) or CBT group (n=5). We assessed the degree of depression before the treatments (baseline), at the 6th week and at the 12th week. Measures used in this study were the Zung Self-Rating Depression Scale (SDS), the Geriatric Depression Scale Japanese version (GDS), and the Montgomery Asberg Rating Scale for Depression -Japanese (MADRS-J).In CBT intervention, the psychoeducation about the typical cognitive characteristics of older adults' depression and the reminiscence based hearing of subjects' compliant were conducted in addition to basic CBT strategy. The results suggested that the score of SDS was significantly decreased in both CBT group and TAU group at 12th week (CBT; SDS baseline M=54.4, 12th week M=40.6, TAU; SDS baseline M= 58.5, 12th week M=46.7). The CBT developed in the present study was effective for improving depressive symptoms. However, the difference between CBT group and TAU group in the score of depression was nonsignificant. The results of GDS and MADRS-J showed the same tendency as SDS(CBT; GDS baseline M=10.8, 12th week M=8.6, MADRS-J baseline M=24.8, 12th week M=11.2, TAU; GDS baseline M=11.4, 12th week M=9.7, MADRS-J baseline M=25.2, 12th week M=10.6). The limitation of this study is that the number of CBT session, which was 6, might have not been enough to get the result which would make the differentiate it from TAU. Since CBT alone was effective for the patients to whom the medication of antidepressant had been stopped, the effectiveness of this treatment has been suggested. Future studies need long-term intervention of CBT in a large number of participants. The UNC Alumni Heart Study started in 1986 with data from college entry in 1964-66. We now have 46 years of postcollege follow-up and 12 waves of data collected during midlife from ages 40-60. The majority of the cohort were born in the 1940s and represent the beginning of the Baby Boom generation. We have a large variety of findings on the role of hostility and other MMPI based personality traits from college which have been shown to predict survival, CHD risk factors and hypertension and NEO Personality data from the decade of the 40s that are associated with behavioral risk indicators and life events.
Methods: We followed the occurrence of new care-needs certification for 3 years in 443 subjects who participated in the health examination and in the 395 non-participants we were able to interview during home visits (838 total subjects). Results: A total of 443 subjects participated in the examination. Home visiting for 449 older adults (of 904 non-participants) living in 8 of 16 districts in the village were conducted. We followed the occurrence of new care-needs certification for 3 years in 443 subjects who participated in the health examination and in the 395 non-participants we were able to interview during home visits (838 total subjects). Among the 838 subjects, there were 94 new certifications (11.2%) during the observation period. Starting in the second year, the occurrence rate of new care-needs certifications for non-participants was significantly higher than that of participants. The rate of care-needs certification over 3 years was significantly higher for non-participants (63/395, 16.0%) than for participants (31/443, 7.0%). Conclusion: Non-participants have a higher risk of care-needs certification. Projects aimed at understanding older adults are often performed during health examinations; however, it is necessary to investigate current data gathering methods for elderly patients who do not undergo these examinations.
CORRESPONDING AUTHOR: Emiko Saito, PhD, Tokyo Metropolitan University, Tokyo, 116-8551; saito@hs.tmu.ac.jp PSW-112d AGE-AND GENDER-RELATED DIFFERENCES IN THE PHYSICAL, COGNITIVE, AND PSYCHOLOGICAL FUNCTIONS OF ELDERLY LIVING AT HOME Shin Murata, Doctor Nishikyushu University, Saga, Japan.
To create basic data for the effective health guidance of elderly living at home, this study was conducted involving 291 adults aged 65 or older, with a comparison of physical, cognitive, and psychological functions between young-old and old-old adults, and by gender. Certain physical functions such as muscular strength of the extremities and the dexterity of digits seemed to be affected by gender. Overall, however, their physical functions were significantly affected by aging, and had decreased to a greater degree in old-old than young-old adults. Especially, it was suggested that balance while standing and the walking ability as well as cognitive functions were more significantly affected by aging than by gender. On the other hand, psychological aspects such as their subjective view of their own health and satisfaction with life showed no significant differences by gender or between age groups, suggesting their lower susceptibility to gender and aging. These findings demonstrated that both men and women showed a high risk of falling in the old-old adults, suggesting the importance of training to improve balance and walking ability as well as cognitive training in preventive care and health guidance for people in this age group.
Participants completed a self-administered questionnaire from a study that has been examining determinants of cigarette use among adolescents. We explored the relationship using a stepwise regression analyses and entered first demographics, then depressive symptoms variables and finally perceived beliefs about benefits associated with cigarette use. We also included in the model other determinants that have been previously associated with cigarette use as parental smoking and peer influences as control. Our findings suggest that negative affect (Beta=.116, t(566) = 2.607, p=.009) does predict current cigarette smoking. Once we entered perceived benefits, depressive symptoms continues to predict cigarette use (Beta=.108, t(566) = 2.456, p=.014). Perceived benefits on mood of cigarette smoking (Beta=.115, t(556) = 2.543, p=.014) also appear to predict this behavior. Our findings suggest that depressive symptoms influences cigarette use directly and indirectly through the perceived benefits youth perceive have that smoking cigarettes may improve their mood. We also report sex differences and implications of these findings to further understand cigarette use among adolescents. Background: Tobacco use is the leading preventable cause of death. Half of the regular smokers die prematurely of a tobaccorelated disease. Although the prevalence of youth smoking in some Western countries has been in steady decline, smoking prevalence in many developing countries is increasing. Thailand and Malaysia are in Southeast Asia, one of the critical regions for tobacco consumption. Objective: To compare the smoking behavior among adolescents in Thailand and Malaysia. Design/Methods: A population-based, national surveys were conducted among 1,694 adolescents with ages between 13 and 18 from Thailand (n=917) and Malaysia (n=777). Respondents were selected using multistage cluster sampling. Respondents were asked to complete self-administered questionnaires. Data were analyzed using descriptive statistics, Chi-Square tests and t tests.
Results: Approximately 5 percents of Thai and Malaysian adolescents were current smokers, with an additional 9.6 percents of Thai and 7.8 percents of Malaysian adolescents reported being a beginner smoker. On average, Thai smoker reported to have first smoked a whole cigarette at 14.6 years of age (SD=1.93), while Malaysian smoker at age 13.9 (SD=2.16). About half of Thai smoker (56.9%) reported that they bought cigarettes for them-selves and 31.5 percents got cigarettes from friends. In Malaysia, most of smoker (71.4%) reported that they bought cigarettes for themselves, only 19.8 percents got cigarettes from friends. Sixty seven percents of Thai adolescent smokers smoked factory-made cigarettes as their usual brand compare to 29.9 percents of Malaysian adolescent smokers (p<0.01). Eight percents of Thai adolescents and 13.6 percent Malaysian adolescents reported smoking hand-rolled cigarettes. Eighty four percents of Thai and 76.1 percent of Malaysian adolescent smokers reported that they tried to quit. Conclusions: Smoking prevalence of Thai adolescents was closed to that of Malaysian adolescents. Factory-made cigarette consumption is an important problem in Thai adolescents and needs to be targeted. Objectives: To monitor tobacco industry's marketing tactics pre and post implementation of pictorial health warnings in Malaysia. Methods: An observational study was conducted to monitor new marketing strategies and changes in product and packaging designs prior and post to implementation of pictorial health warnings on cigarette packs effective 1 January 2009. A survey instrument was used to collect data in selected point-of-sales in Penang, Malaysia. All new product and pack designs of the most popular cigarette brands were purchased and scrutinized for promotional features. Observations of point-of-sales displays were also made. Data were analyzed using qualitative methods. Results: Since the banning of advertising at point-of-sales the tobacco industry invested more resources into packaging design and point-of-sale display in order to communicate brand imagery and increase sales primarily targeting children and young people. With the implementation of pictorial health warnings and ban on descriptors, the industry continues to promote its products with introduction of a variety of innovative pack and product designs. Textual descriptors such as light and mild are replaced with varied colour tones (silver and blue) to shape consumer perceptions of risk and have the misleading effect as light and mild. Colours and pack designs are also used by the industry to dilute the effects of pictorial health warnings. Conclusions: Tobacco control policies have to be water-tight to prevent the tobacco industry from exploiting the loopholes in these policies. The Malaysian government should implement plain packaging which would increase the effectiveness of health warnings, reduce misconceptions about smoking risks and prevent the use of bran variants as a promotional tool. This will help reduce smoking uptake amongst children and young people.
Background: The smoking rate among adolescents has been decreasing in Japan. According to the National Survey, the current smoking rates of 7th grade students decreased from 7.5% (males) and 3.8% (females) in 1996 to 3.2% and 2.4% in 2004. In Kyoto, workshop-style tobacco-free classes based on theory of behavioral science have been conducted since 2002, in cooperation with a medical university, a medical association and the local government. Objective: During 2009, we conducted tobacco-free workshops for 3562 students at 26 municipal junior-high schools. In order to clarify the smoking status of these students, we investigated the perception of smoking and the relationship between smoking and other life-style habits. Subjects : There were 1644 students at 13 schools who agreed to participate in the survey. The response rate was 98.1%. Males: 820, Females: 721, not specified: 108. Methods: The questionnaire was anonymous and self-completed. We made sure that teachers would not check the questionnaire responses. Questions about lifestyle including smoking and drinking, were asked mainly in fourfold choice style. After analyzing the smoking status by school and sex, we examined the relationship between lifestyle and smoking. Results: The current smoking rate was less than 1% at half of the schools, but 3∼7% at 3 schools. Overall, 5.5% of the subjects had tried smoking, and 1.9% were current smokers. 52.9% were living with smokers; 46.7% had tried alcohol; 50.5% watched TV more than 3 hours a day and 4.4% shopped at convenience stores every day. Current smoking and previous smoking showed significant association with smoking by cohabiters, drinking experience, study time, attending school club activities and shopping at convenience stores. Discussion: The smoking prevalence of subjects was low compared with that in the National Survey, possibly because our subjects were not randomly selected and Kyoto has the lowest smoking prevalence among adults. This is the first report in Japan indicating that smoking habits may be related to shopping at convenience stores. The students are mainly buying cigarettes at convenience stores, where cigarettes companies place a large number of advertisements. Tobacco sales at convenience stores should be strictly monitored. The aim of this study was to explore the relationship between the age at cigarette smoking onset and the smoking level among adolescent current smokers. In 2007, one or two classes of 10th and 11th grade were randomly selected from the high schools sampled by PPS (probability proportional to size) sampling after all the schools in a large city in Korea were stratified by grade, gender, and region. A total of 743 current smokers among a sample of 4,602 students were analyzed with a self-administered questionnaire. The smokers were defined as those who had smoked at least one cigarette during past one month. The age of smoking onset was measured as the grade when the respondents first tried a cigarette even one or two puffs. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by multiple logistic regression analysis and they were adjusted for potential confounders. Compared with those who tried smoking first after entrance into high school, the OR of being a frequent smoker (i.e. those who smoked on 20 days or more during the past one month) among those who tried during middle school period was 2.40 , that of those who tried smoking during late elementary school period was 3.70 (CI 1.67∼8.17), that of those who tried smoking early elementary school period or earlier was 3.91 (CI: 1.74∼8.78). The OR of being a regular smoker (i.e. those who smoked everyday) among those who tried during middle school period was 1.77 (CI: 1.14-2.76), that of those who tried smoking during late elementary school period was 2.34 (CI: 1.14∼4.78), that of those who tried smoking early elementary school period or earlier was 4.83 (CI: 2.19∼10.69). The OR of being a heavy smoker (i.e. those who smoked one-half pack or more per day on the average) among those who tried during middle school period was 3.61 (CI: 1.84-7.08), that of those who tried smoking during late elementary school period was 6.67 (CI: 2.87∼15.50), that of those who tried smoking early elementary school period or earlier was 5.65 (CI: 2.34∼13.62). This study showed the importance of smoking prevention or delaying smoking onset among children and adolescents. Background: Maternal employment has been shown to influence health behaviors of children and adolescents such as overweight and obesity. The evidence related to adolescent smoking is limited as well as results using UK data. In this analysis, we assessed the influence of maternal employment in three periods of childhood on smoking of young people aged 16-21 years in British Household Panel Survey (BHPS). Methods: BHPS is annual panel survey that has started in 1991 and completed its 17th wave in 2007. Response rate to wave 1 was 74%, and response between waves 2 and 17 varied between 84%-89%. There are 3,615 young individuals with available data on smoking at age 16-21 and maternal employment data from age 0-16 years. Covariates, such as gender, maternal age, maternal education and marital status, household income or maternal smoking were used as explanatory variables in logistic regression analysis in STATA 10. Results: 29% of young adults aged 16-21 reported being current smoker. Approximately 40% of mothers worked at some point during age 0-4 (preschool age) of their child. This proportion increased to 59% at age 5-11 (primary school age) and 68% at age 12-16 (secondary school age). Children of mothers who were not employed were more likely to smoke (OR of smoking 1.18, 1.19 and 1.60 for 3 periods of exposure), although these effects were almost entirely explained when adjusted for maternal education and household income. Conclusions: The associations between maternal employment in childhood and young adults' smoking exist at least partly because of the generally higher social position and more stable family structure of households with working mothers. Maternal education and household income seem to be stronger social predictors of adolescent smoking than maternal employment status. 1 Kyoto Prefectural University of Medicine, Kyoto, Japan and 2 Kyoto First Red Cross Hospital, Kyoto, Japan.
Background: Many researchers have demonstrated that inappropriate drinking has various adverse effects. Japanese national survey have pointed out that people have not been sufficiently educated enough about the dangers of drinking, although the amount of alcohol consumed by Japanese people has been increasing. Objective: Since 2002, we have been asking detailed questions about the amount and the frequency of drinking by healthy people undergoing routine medical checkups and giving them advice based on their responses. We analyzed our findings in order to clarify the drinking status of these subjects and the effects of the intervention.
Subjects and Methods: Subjects were 3633 males who underwent medical checkups in both 2005 and 2007 (mean age 56.9± 11.2). Among these subjects, 959 were non-drinkers (ND), 1574 were light drinkers (LD: less than 140 g of alcohol /week), 943 (26.0%)were moderate drinkers (MD: 140-420 g/week), and 157(4.3%) were heavy drinkers (HD:over 420 g/week). We examined the relationship between the amount of alcohol intake and laboratory data by analysis of variance. We provided these subjects motivational support using medical data on the checkup day, and sent further advice 2 weeks later. We analyzed changes in the amount of alcohol intake and laboratory data 2 years later. Result: γ-GTP (IU/L) values in 2005 were ND (34.9±1.2), LD (44.5±1.2), MD (66.7±5.4), and HD (96.5±5.8). There were significant differences among the groups, except for that between ND and LD. The amount of alcohol intake was decreased in both MD&HD 2 years later. The γ-GTP, GPT, and TG values of 1100 subjects of MD or HD decreased significantly. Conclusions: One third of the subjects had inappropriate drinking behaviors, although they were sufficiently health-conscious to come for a medical checkup. After receiving motivational support, the amount of alcohol intake decreased and the laboratory data improved.
Many questions remain about the direct and indirect costs to families incurred during the course of cancer care. Despite availability, many patients fail to access co-pay assistance programs. The Cancer Support Community and Genentech piloted a national survey investigating psychosocial barriers to accessing co-pay assistance and the hidden costs of care. Methods: Cancer patients (n=79) diagnosed over 6 months ago or had active treatment within 2 years and caregivers (n=29) of patients who met the above criteria, completed a survey that included the Revised Impact of Event Scale to assess intrusive and avoidant ideation around the cost of care. Although the effect of smoking on lung cancer incidence is well known, it is not yet known if continued smoking after diagnosis affects prognosis. Methods We systematically searched for studies that measured the effect of quitting smoking after diagnosis of lung cancer on prognosis. Two researchers independently identified studies to include and extracted data. Estimates were combined using a random effects model and I2 statistic was calculated to examine heterogeneity. Using life tables, we modelled 5-year survival for non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using death rates for continuing smokers and quitters obtained from the review. Data sources We searched CINAHL (from 1981), EMBASE (from 1980), MEDLINE (from 1966), Web of science (from 1966) and CENTRAL (from 1977) to December 2008 and the reference lists of included studies. Results Continued smoking was associated with an increased risk of all-cause mortality and recurrence in early stage NSCLC ((HR 2.94 95% CI (1.15, 7.54) , HR 1.86 (1.01, 3.41) ) and with an increased risk of all-cause mortality, development of second primary and recurrence in limited stage SCLC ((HR 1.86 (1.33, 2.59) , HR 4.31 (1.09, 16.98) and HR 1.26 (1.06, 1.50) . Life table modelling estimated 41% 5-year survival in 65-year old NSCLC continuing smokers compared to 75% in quitters. In SCLC, an estimated 36% of continuing smokers survive for five years compared to 68% quitters. Conclusions There is evidence that smoking cessation after diagnosis of lung cancer is associated with improved prognosis. Assuming causality, the number of deaths prevented is larger than would be expected due to reduction of cardio-respiratory deaths after smoking cessation and it is likely the majority of the mortality gain is due to reduced cancer progression.
Results: Respondents (N=108) were ethnically diverse; 25% patients and 18% caregivers had household incomes less than $20 K. Patients reported high levels of intrusion (18.5±9.2) and avoidance (20.2±8.8). Caregiver levels were also high for intrusion (15.0± 10.0)and avoidance (17.8± 10.1). Based on clinical cut-offs,82% reported stress within moderate to severe levels. Due to care costs,74% patients depleted savings,44% downsized living accommodations; 32% caregivers worked extra jobs to help pay for care,60% reported extra stress at work. To reduce costs,49% patients delayed seeking psychological counseling. Among patients, increased intrusion correlated with having no one to help with insurance questions (p<.0001). Increased avoidance was associated with delay in seeking psychological counseling (p<.0001) and less awareness of co-pay assistance (p<.0001). 33% patients and 40% caregivers felt too overwhelmed to apply for financial help. Discussion: These data suggest that managing cancer costs is associated with clinically significant stress levels. Those with greatest need may not seek help, underscoring the need for programs that help families manage the stress related to the cost of care. The patient-provider relationship and specifically physicians' ability to communicate empathy greatly impacts chronic disease management. Empathetic communication is significant in navigating difficult circumstances like dealing with oncology patients. Unfortunately, research demonstrates that empathy declines in 75% of medical students as they advance in their training. There is growing recognition that medical education must address this decline in empathy and properly prepare physicians to work with diverse patients. However traditional educational techniques in India are unlikely to meet this need. Experiential learning may be used to address this problem. The purpose of this study was to investigate the effectiveness of faculty-supervised and selfinstructional listening micro skills training in increasing the responsiveness of medical students to the psychosocial needs of cancer patients and their families during medical student-patientfamily member interviews. This study employed a randomized controlled design. Two multiple baseline across subjects designs of four baselines each were used for the study.
Final-year medical students posted in clinical oncology were randomly assigned to conditions for training and to baselines within each condition. Training conditions were identical in informational content and time requirements. Self-instructional training incorporated two videotapes developed for the study. Through the use of graphs and t-tests, data from the training conditions were analyzed separately, comparatively, and on the basis of overall training effectiveness across all eight subjects. Overall, majority of the students felt that the intervention influenced their level of empathy (78%), should be a standard part of medical school curriculums (67%), and would impact all physicians (80%). Faculty-supervised training was found to be more effective than selfinstructional training in increasing responsiveness as measured by (a) observational data from videotaped interviews, (b) patient and family member ratings of interviews, and (c) number of psychosocial needs recognized on medical student dictation reports. Results based on t-tests indicated overall use of training was effective in increasing (a) appropriate use of four interview micro skills, (b) patient and family member interview ratings, and (c) number of psychosocial needs recognized on dictation reports. Findings suggest that experiential learning methods are well received, feasible, and effective in increasing empathy in medical students and should become a standard part of medical school curriculums.
The aim of this retrospective study was to assess the comparative frequency, gender, age, sub-site distribution and histologic differentiation of oral squamous cell carcinoma (OSCC) in Sistan & Baluchestan province, Iran. The medical records of 3070 patients with a histopathologic diagnosis of malignant neoplasm were reviewed during the 11-years period from 1995 to 2006.Available data suggested a steady increase in prevalence of OSCC during the last decades. .Of 3070 patients with malignancy, 217 cases had oral malignancy. The most common oral malignant neoplasia was OSCC (n=178, 82%) and then verroucose carcinoma (n=14, 6.4%).
Gables, FL. Hormonal treatment (HT) for advanced prostate cancer (APC) often results in side effects that can compromise quality of life (QOL). Limited work has evaluated the efficacy of psychosocial interventions among men undergoing treatment for APC. The current study evaluated the effects of a 10-week telephone-delivered and group-based Cognitive Behavioral Stress Management (CBSM) intervention on coping strategies, QOL and cortisol regulation among APC survivors undergoing HT. CBSM strategies included stress awareness, relaxation training, cognitive restructuring, and enhancing coping, social support and communication skills with an emphasis on managing treatment-related compromises in QOL. The HP condition involved delivery of health promotion information (e.g., benefits of proper diet, physical activity) with no therapeutic or interactive component. Participants (N=83) were randomized to either CBSM or a health promotion (HP) control condition and were assessed at two time points (T1; baseline, and T2; about 12 weeks post-randomization into the CBSM or HP conditions). The mean age was 70.0 years (SD=9.5) and the sample was ethnically diverse (66% White, 18% Black, 12% Hispanic, 4% Other). Participants had undergone an average of 19.1 months (SD=17.4) of HT. Measures included the Expanded PC Index Composite (EPIC) for treatment-related symptoms and QOL; the COPE Inventory (COPE); Measure of Current Status (MOCS) for perceived stress management skills and salivary cortisol. Analyses were conducted using repeated-measures group by time ANOVAs, controlling for relevant covariates. Participation in the CBSM intervention condition, relative to the HP control condition, was associated with improvements in disease-specific (i.e., urinary and bowel function) and general QOL, adaptive coping strategies, stress management skills and cortisol regulation (all p's<.05). Several mediation models were tested where coping and other stress management strategies were hypothesized to mediate the relationship between group condition and QOL. Both greater acceptance (p<.05) and less denial (p<.01) coping were found to be significant mediators of intervention-related improvements in QOL but did not mediate the effect of the intervention on cortisol regulation. These results suggest that participation in a telephone based CBSM intervention may be effective in improving general and disease-specific QOL, stress management skills and cortisol regulation. over time (p<.0001). Prostate cancer patients had significantly lower levels of anxiety, insomnia, fatigue and pain (ps<.001 or less) compared to patients with other types of cancer, but no significant differences were found across cancer stages over time. Patients having received chemotherapy were those displaying the greatest levels of insomnia and fatigue (ps<.05 or less), whereas patients treated by surgery only (no radiation or hormone therapy, or chemotherapy) were those displaying the lowest levels of anxiety and insomnia (ps<.001 or less) over time. In summary, cancer patients reported an overall decrease in depression, anxiety and insomnia symptoms, stable fatigue and an increase in pain over the 18-month period following surgery. Results also revealed some differences across cancer sites and treatment regimens received. Further longitudinal studies are needed in order to identify additional factors associated with changes in cancer-related symptoms over time.
CORRESPONDING AUTHOR: Claudia Trudel-Fitzgerald, BA, Groupe de recherche en psycho-oncologie, Centre de recherche de l'Hôtel-Dieu de Québec, Quebec, QC, G1R 2 J6; claudia.trudel-fitzgerald.1@ulaval.ca Physical activity (PA) and good dietary habits can moderate obesity while enhancing immune status, decreasing cancer risk. Innate immune cells, monocyte/macrophages, respond to and cooperate with other cells and components of the immune system to detect and eliminate abnormal cells. We report here the effect of physical exercise, with or without a high fat diet, on monocyte/macrophage numbers and activity in mice. Number and function of circulating blood monocytes from C57BL/6 mice were measured after 6 weeks of PA on a voluntary running wheel, compared with sedentary controls (SE) . PA Background: Cancer patients using complementary and alternative medicine (CAM) are generally found to exhibit more depressive symptoms compared to non-users. The available studies, however, have not investigated the association between depressive symptoms and use of specific types of CAM in breast cancer patients and often fail to adjust for potential confounders. Objectives: To explore the associations between use of 10 different types of CAM and depressive symptoms among breast cancer patients 3-4 months post-surgery. Methods: A total of 3343 Danish women diagnosed with early stage breast cancer (age 26 -70) provided questionnaire data 3-4 months post-surgery on CAM use since diagnosis, the types of CAM used, depressive symptoms (Beck Depression Inventory), physical function (SF-36), health behaviors, and Body Mass Index. Data on eligibility, disease status, treatment, and comorbidity were provided by the surgical departments. Information on socio-demographic factors and psychiatric history were obtained from national longitudinal registries. Results: Prevalence of overall CAM use was 40.1%. Multivariate negative binomial regression analyses showed that women using one or more types of CAM reported more depressive symptoms than non-users when adjusting for clinical, health-related, and socio-demographic variables (Ratio of Mean (RM) = 1.20; 95% CI = 1.13 -1.27; p < .001). When analyzing specific types of CAM, more depressive symptoms were found for women using dietary/nutrition supplements, massage, nutrition/exercise counseling, needle acupuncture, and relaxation/yoga (RM range: 1.11 -1.19; p <= .026 ). In contrast, use of herbal medicine, reflexology, healing, meditation, and kinesiology was unrelated to depressive symptoms. Conclusion: Use of some types of CAM may be seen as an attempt to cope with depressive symptoms. Alternatively, these types of CAM may exhibit a negative influence on mood. guidelines if they were male (24.5% vs 14.7%; χ2=10.3, df=1, p= 0.001), under 60 years (22.7% vs 16.0%; χ2=3.996; df=1, p=0.046), lived in the least deprived areas (21.5% vs 12.0%; χ2=8.795 df=1, p= 0.003), had a post-secondary education (22.9% vs 15.3%; χ2=5.563 df=1, p=0.018), and reported good heath (22.6% vs 10.4%; χ2= 17.993 df=1, p<0.001). Time since diagnosis, stage of disease, and disease status were not associated with physical activity. Among those not currently meeting guidelines, 59.9% thought that they ought to be more physically active. CONCLUSION: Overall levels of physical activity are low among men and women with cancer in the United Kingdom. However, the majority of insufficiently active participants showed awareness of the need to increase their activity, and may be receptive to interventions for promoting physical activity in this population. There is growing recognition that symptom communication is important in understanding how patients adjust to cancer and its treatment. This study examined two key aspects of communication: self efficacy for symptom communication and holding back from disclosing cancer related concerns. The sample included postmenopausal breast cancer survivors taking adjuvant hormonal therapy (N=35). Participants were on average 60.6 years old (SD 7.4) , mostly Caucasian (91%), and over half had at least a college education. Regression analyses were conducted to examine the degree to which self-efficacy for symptom communication and holding back predicted measures of symptoms and adjustment. These analyses controlled for demographic and medical variables that were significantly correlated with outcomes. Results indicated that holding back about discussing cancer related concerns, but not self-efficacy for symptom communication, was a significant predictor of menopausal symptoms, fatigue, intimacy, and functional wellbeing. Specifically, holding back was a significant predictor of physical menopausal symptoms (t(30) = 4.05, p=0.000), fatigue (t(31) = 3.47, p=0.002), intimacy (t(30) = −2.52, p=0.017), and functional well-being (t(31) = −3.77, p=0.001). Patients who reported a greater tendency to hold back from discussing their cancer related concerns had higher levels of menopausal symptoms (such as difficulty sleeping or a decrease in physical strength) and fatigue and lower levels of intimacy and functional well being. Taken together these findings suggest that holding back from disclosing cancerrelated concerns is a significant and consistent predictor of symptoms and adjustment to breast cancer in women taking adjuvant hormonal therapy to prevent recurrence. These findings raise the possibility that interventions designed to address symptom communication might be helpful to those patients who are prone to holding back from discussing their cancer-related concerns. To date, very little is known about the psychosocial predictors of depression among women with breast cancer (BCA). Even less is known about the psychosocial risk factors for depression across the maturational spectrum. Although premenstrual and postmenstrual women diagnosed with BCA share a substantially elevated risk of developing a major depression; the coping resources, life challenges, and cancer impact may differ markedly for younger and older women. The current study examined coping styles, social support quality, stressful life events, sleep quality, and sexual functioning in 103 women, ages 27 to 69, who had completed treatment for BCA within the previous five years. Women were sampled from across the U.S. and completed study measures electronically. The mean age of the sample was 46.71 (SD=9.88; median=46). The sample was generally well-educated with 94.6% of the sample having at least some college education. The majority of respondents were Caucasian (93.2%) leaving the proportion of minority respondents too small for meaningful analysis. Approximately 71% were employed either full or part time. The majority were married (70.9%) or cohabitating (4.9%). Consistent with previous reports, BCA staging and age were inversely and significantly related (r =−.225, p= .024) with younger women (<47 years) diagnosed with more advanced stage illness. Despite this differential level of life threat, current findings indicate that the emotional impact of BCA is generally equivalent across the maturational spectrum. Both younger and older BCA survivors endorsed significant levels of generalized psychological distress, as measured by the BSI, which was characterized primarily by rumination, anxiety, and hostility. Beck Depression Inventory-2 scores indicated that the sample, on average, experienced mild to moderate levels of depressive symptoms with younger women tending to be more symptomatic than the older women, although the difference did not reach significance (t= 1.68, p=.09). Sleep quality was also impaired across the sample with the disruption of sleep deprivation on daily activities more frequently found among younger women (χ2=15.82, p<.001). Data collection is complete and full results will be known well in advance of the conference. The differential experiences, resources, and needs of women struck by BCA at various stages in life are discussed. The identification of age-related risk factors for depression may inform early detection, intervention and prevention efforts. discharge from medical treatment. The data was analyzed with the content analysis, chi-square test, and correspondence analysis. A total of 11 attributes were extracted and categorized as follows: changes due to the cancer (N=24) and perceptions of the cancer (N=14). The attributes indicated by many of the participants were "having cancer brought my family closer together(N=14)", "I have met various people(N=12)", "cancer has been the precious experience of my life(N=11)", "I wonder why I got cancer(N=9)". Some attributes of positive changes due to the cancer and perceptions of the cancer were statistically significant differences among age. This study identified the important components of the impact of the cancer in Japanese childhood cancer patients after their discharge. Most patients thought that good things have come out of having had cancer. In addition, older patients have more diversified views of the cancer than younger patients because they have had many problems and benefits. We previously reported high prevalence of asthma (34% by parent report) in children in Puerto Rico (Nazario et al., 2004) . The purpose of this study was to describe the treatment, healthcare utilization, and morbidity of children with asthma in Puerto Rico. This phase of the study involved the administration of a parent interview, spirometry and physician examination, in order to learn more about medical care of children with asthma. Evaluations were conducted in 2001 on a sample of 300 5-10 year-old children enrolled in public schools in San Juan, including both children whose parents reported they had asthma (n=190) and presumed healthy children (n=110). The mean age was 6 years old (SD=.9 years) and 50.2% were females. Among those with asthma, 32% were prescribed anti-inflammatory medications, with 49% receiving only bronchodilators, and 18% not receiving any asthma medications. Using NHANES criteria, 46% had mild and 54% had moderate-severe asthma; of the latter, 13% of parents reported not having any medication prescriptions for their children. Children with asthma and those without did not differ on environmental risk factors or on forced vital capacity (FVC) measures. However, parents of children with asthma, compared to those without, reported they had significantly more symptoms of asthma in the last 14 days (p=.02) and missed more school in the last two months due to asthma (p=.02). Mean number of doctor visits, ED visits, and hospitalizations for asthma in the past year were 2.72, 1.64, and 0.42, respectively. Forty-one percent of parents of children with asthma reported that they did not have written instructions for taking medications; 96% were not told to use a peak flow meter; 82% did not have a written care plan for asthma care at school; and 72% did not having a written plan for environmental triggers. Of those prescribed nebulizer treatments, 51% reported not adhering to recommendations. Sixty-seven percent of those who were prescribed medication reported not adhering because of concerns regarding potential side effects. These results indicate that in 2001 children in San Juan were not being optimally treated for asthma. Given the high prevalence and morbidity of asthma in Puerto Rico, there is a need for educational efforts to improve treatment practices.
Background/aims: High levels of distress are reported in patients with Chronic Obstructive Pulmonary Disease (COPD) . Only a few studies looked into social support as a contributor and suggest that patients' perceptions of support are indicative for their wellbeing.
Research in other chronic diseases shows that overprotection (e.g. unnecessary help) and protective buffering (e.g. concealing worries) as reported by partners is often related to distress in patients. We studied overprotection and protective buffering in relation to distress in COPD patients, from a dyadic perspective. Methods: 103 couples were approached at a pulmonary rehabilitation center and an outpatient clinic. The analyses were performed using a sample of the 68 couples who consented (to take part in the study). We assessed distress with the Hopkins Symptom Checklist 25. Patients' and partners' perceptions of partner support were measured with a questionnaire developed by Buunk et al (1996) . Data were analysed using bivariate correlations and a stepwise regression analysis. Results: Mean age of patients was 63.8 (±9.5) and of partners 62.3 (±9.7). More male patients participated (57.4%) and more patients of the outpatient clinic (55.9%). Patients were quite distressed (M=44.3±11.4). Distress was predicted by patients' perceptions of protective buffering (R2=0.23, p<0.01), partners' report of overprotection (R2=0.10, p<0.01) and differences within a couple in their perception of overprotection (R2=0.08, p<0.01). Conclusions: Overprotection and protective buffering by partners explain a great deal of the variance in COPD patients' distress. Patient and partner perspectives on partner support, as well as disagreement between the couple, all seem to play an independent role in distress of patients.
Background: Research has shown that social support and certain coping strategies attenuated cardiovascular reactivity (CVR) to stressful circumstances. This hypothesis has not been tested in an immigrant population. The current study assessed the effects of perceived social support and problem-focused and emotionfocused coping on CVR to stress among Chinese immigrants in the New York City area. Method: First generation Chinese immigrants (N=150; Mean Age=53 years old; 86 Females, Average Length of Stay in the United States=20 years) were recruited from the New York Downtown Hospital in Chinatown. Following the study description and informed consent procedures, participants completed questionnaires which assessed social support and their use of problem focused and emotion focused coping strategies. Following an 8-minute adaptation period, participants recalled a stress-provoking event related to their immigration experience in a semi-structured interview format. Recovery was monitored for 20 minutes and participants were debriefed. Blood pressure and heart rate were monitored at 2 minute intervals during baseline and recovery, and 1 minute intervals during the interview. Result: Hierarchical multiple regression analysis revealed that the interaction perceived social support and emotion-focused coping is associated with low baseline SBP, β=-.18, t=-2.19, p<.03. However, perceived social support and emotion-focused coping is associated with higher SBP during provocation, β=.23, t=2.70, p<.01. Conclusion: Availability of social support is reassuring when coupled with the use of emotion-focused coping, and when the situation demands low coping resources (as during baseline). However, under conditions of stress where the demand for coping resources is high (e.g., during stress provocation), perceived social support and the use of emotion-focused coping does not help and may actually suggest an increase in CVR to stress. This may be related to the Chinese value of collectivism, which may manifest itself through an unwillingness to burden others with personal needs. Prospective studies demonstrate relations of subclinical depressive symptoms with enhanced risk for cardiovascular disease, yet little is known about underlying mechanisms. Depressive symptoms are also associated with unhealthy lifestyle behaviors and cardiovascular risk factors. We examined whether lifestyle factors mediated relations of depressive symptoms to cardiovascular risk factors in 205 healthy, community-dwelling older adults (55% male; mean age=66 years). Participants underwent a medical history, physical examination, blood chemistries, exercise treadmill testing with maximal oxygen consumption (Vo2max), an oral glucose tolerance test, clinical assessment of blood pressure and body mass index (BMI), completion of the Beck Depression Inventory (BDI), and assessment of smoking status and number of alcoholic drinks consumed per week. Primary outcome variables included systolic blood pressure, diastolic blood pressure, glucose levels at 0 and 120 minutes, waist circumference, triglycerides, and total, HDL, and LDL cholesterol. Potential mediators assessed were number of alcoholic drinks consumed per week, smoking status, Vo2max, and BMI. Multiple regression analyses adjusted for age, education, sex, race, and use of antihypertensives revealed a significant positive association between depression and triglyceride levels (b=24.2, p=.009), waist circumference (b=9.56, p<.001), and BMI (b=3.03, p=.001). Subsequent regression models testing for possible mediation indicated that the relation of BDI to triglycerides was mediated by BMI. Thus, in healthy older adults, depressive symptoms are associated with increased body mass, waist circumference, and triglyceride levels, and body mass mediates the relation of depressive symptoms to triglyceride levels. Obesity may be an important mechanism by which depressive symptoms influence cardiovascular risk in older adults. Patients with Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF) display high levels of psychological distress which may be associated with increased mortality, lower functional status, and poor self-care behaviors. The purpose of this study was to characterize the psychosocial, cognitive, physical, and demographic profiles of patients with COPD and IPF with the goal of better understanding similarities and differences among patients with these pulmonary disorders. Patients with COPD (N=16; Female=5) and IPF (N=15; Female=5) were recruited from The Ohio State University outpatient pulmonary clinics. Participants completed three visits over the course of twelve months and at each session completed a series of questionnaires and cognitive function tests. At baseline and twelve months, participants completed pulmonary evaluations. IPF patients were significantly older (M=69.3) than COPD patients (M= 58.7) and had better pulmonary functioning (FEV1 % predicted= 75.7% in IPF vs. 44.2% in COPD) . All analyses of psychological functioning and cognitive performance were conducted controlling for age. COPD patients indicated significantly more symptoms of depression (p<.01; CES-D) and anxiety (p<.05; BAI) than did IPF patients. COPD patients also reported less social support (p<.01, PSSS). In addition, COPD patients indicated significantly poorer emotional quality of life, as reflected in the Mental Component Score of the SF-36. However, no differences were observed between COPD patients and IPF patients on the Physical Component Score of the SF-36 or in symptoms of breathlessness, sleep quality, or cognitive performance. Thus, although perceptions of physical quality of life and breathlessness are similar among COPD and IPF patients, patients with COPD indicate significantly greater symptoms of emotional distress and poorer emotional quality of life than patients with IPF. Heightened distress among patients with COPD is important to consider in treatment of patients. In addition, further evaluation of disease-related processes and biological differences between COPD and IPF patients is needed to understand mechanisms that may explain the observed differences. Objective: This study investigated a reactivity hypothesis and a vagal brake hypothesis of cardiovascular reactivity and recovery in the expressive and suppressive hostility behavior patterns of coronary artery disease (CAD) patients. Method: 82 CAD patients (age=61.50±10.53; 13.40% were female) participated in this experimental study. Participants were separated into four groups using the Chinese Hostility Inventory_ Short Form: high expressive hostility group, high suppressive hostility group, high expressive with high suppressive hostility group, and control group. Heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured in two-minute intervals under baseline, anger recall task (Time 1, Time 2, Time3), and recovery. Results: After controlling the baseline level to examine reactivity, the results indicated that significant difference between four groups in SBP, DBP and HR; as well an interaction existed among groups and SBP under the anger recall task. Post-hoc analyses indicated that the high expressive hostility group had higher SBP and DBP than the control group in Time 2; the high suppressive hostility group had higher SBP than the control group in Time 3. After controlling the anger level to examine recovery, an interaction existed among groups and SBP, DBP and HR. Conclusion: This study verified the reactivity hypothesis in the expressive hostility group who had peak SBP and DBP reactivity in the beginning of the anger recall task; however, the suppressive hostility group demonstrated peak SBP reactivity at the end of the anger recall task. This study also extended Proges's vagal brake hypothesis in recovery. Hostility behavior patterns caused different cardiovascular reactivity and recovery that regulated by autonomic nervous system. These reactivity and recovery may predict different psychopathological mechanisms or prognosis pathways in CAD patients. Heart rate variability(HRV) has been related to cardiac mortality and morbidity in cardiac patients. This research is to study whether a 8-week course of interventional program could mediate ANS function in cardiac patients. Participants(N=39)were randomly assigned to either treatment group consist of eight sessions of cognitive behavior group program, psycho education, breathing retraining and HRV biofeedback(Stress Eraser). The ANS function (SNS) were measured pre-(week 0) and post-(week 8) sessions. 1 Graduate School of Human Sciences, WASEDA University, Tokorozawa, Japan; 2 Faculty of Human Sciences, WASEDA University, Tokorozawa, Japan; 3 National Center of Neurology and Psychiatry, Kodaira, Japan and 4 National Cerebral and Cardiovascular Center Hospital, Suita, Japan.
Introduction: Implantable cardioverter defibrillators (ICDs) are increasingly used for the prevention of sudden cardiac death in patients with life-threatening ventricular arrhythmias. Most ICD recipients are reported to experience some degree of psychosocial problems. Despite calls by international committees for improving cardiologists' recognition and treatment of psychosocial problems for patients with ICDs, little is known about cardiologists' beliefs for these problems in Asia. The purpose of this study was to identify cardiologists' beliefs about psychosocial problems for ICD recipients in Japan. Methods: Of all 337 licensed hospitals to implant ICDs in Japan, 145 (40.1%) participated in our mail survey. The subjects were cardiologists who frequently implanted ICDs in each hospital. The questionnaire consisted of 10 items designed to assess respondents' beliefs about psychosocial problems (i.e., "ICD recipients often have health care issues.") and prior experiences of psychiatric care (i.e., "How often have you prescribed psychotropic medications to patients diagnosed with mental disorders?"). Respondents rated each question on 4-point scales. Results: Most cardiologists (91-96%) identified "health care issues," "lifestyle issues," and "emotional well-being issues" as concerning factors that could affect the psychosocial problems of ICD recipients. On the other hand, many cardiologists (67-88%) did not provide mental health care such as pharmacotherapy, psychotherapy, counseling, and listening attentively. Conclusion: Our results indicate that most cardiologists do not provide mental health care for psychosocial problems of ICD recipients, though they are aware of these problems. Learning objectives 1. Describe the prevalence of abnormal weight among low-income Korean children 2. Explain behaviors associated with abnormal weight status. Background: A national school health screening program of Korea announces an increase of underweight in school while obesity increases in the country. Low-income children are vulnerable to such discrepancy and subsequent health outcomes. This study is intended to report the prevalence of abnormal weight status and associated behavioral and health characteristics of low-income Korean children. Methods: A total of 2,033 low-income elementary students were recruited from 106 Community Child Centers in Korea in 2006. Health examination and survey were conducted to detect behavioral characteristics and health conditions. Abnormal weight was determined by using the BMI-for-age and -sex chart of the national School Health Screening Program of Korea. Results: Higher prevalence of underweight(8.0%) and overweight/obesity(18.0%) is found among low-income elementary children. Differences in eating regularity and variety, smoking, and health education experience are found between abnormal and normal weight children, while previously established factors such as drinking soda and exercise level are not relevant in this study. Underweight children experience higher level of stress with academic performance (OR=3.757, p=0.005) than normal weight children, and overweight/obese children are more concerned about their appearance (OR=3.881, p<0.001). Conclusion: These findings highlight the importance of coordinated health promotion programs that integrate school health with services for low-income children. Periodical monitoring and nutritional service should concern preventing and intervening early in order to reduce abnormal weight problems. Research is needed to understand a more comprehensive picture of factors associated with both underweight and overweight/obesity among low-income Korean children. According to the NIMH (2004), a significant number of children suffer from untreated emotional and behavioral problems. This lack of service use has significant implications considering that mental health (MH) problems during childhood can lead to difficulties later in life. Despite increased efforts to tailor MH services to the needs of each consumer, ethnocultural disparities in access to and use of these services continue to abound (Yamada & Brekke, 2008) . Findings suggest that ethnic minority populations have a higher unmet need than Non-Hispanic Whites (e.g., Diala et al, 2002) . However, efforts to explain disparities in MH services have been limited. Researchers have identified several variables that influence individuals to utilize mental health services such as income, ethnicity, and attitudes (e.g., Cepeda-Benito & Short, 1998; Turner & Liew, in press) . Obasi & Leong (2009) hypothesize that it is also important to note that group difference exists, and it is imperative that future research investigate cultural variables (acculturation, traditions, etc.) . Previous research has found some ethnic minority groups use coping sources (i.e., friends, family) and practices (i.e., family, social, and religious) that are more consistent with their cultural beliefs instead of those more consistent with traditional MH services (e.g., Harrison et al., 2004; McMiller & Weisz, 1996) . The purpose of the current study is to examine to role of cultural identity, religiosity, perceived behavioral control (PBC), and attitudes on utilization of MH service among European Americans and non-European Americans. Participants were 150 caregivers (89% female) recruited from the Southwest United States. Mean age of participants was 34.4 years old. Participants completed the following measures a demographic questionnaire, the Parental Attitudes Toward Psychological Services Inventory (PATPSI; Turner, 2006) , and selfreported their cultural identity, religiosity, and likelihood of mental health service use. Results indicated that both attitudes and PBC were positively related to utilization, and participants' ratings of cultural identity and religiosity were not correlated with utilization. When examining group differences between European Americans and non-European Americans mean differences were only noted for perceived behavioral control. European Americans reported feeling more control over their ability to seek services if warranted. Finally, both perceived behavioral control and attitudes independently predicted utilization of mental health services.
Implications for future research and applications to families will be discussed. Background: Adult obesity is associated with poorer cognitive function and physical coordination in childhood. Hypothesis: Hearing impairment in childhood as a marker of childhood function is associated with adult obesity, and is a better marker for neurological function as it is less subject to social confounding and unlikely to influence exercise. Methods: Among 3288 male and 3527 female members of a longitudinal cohort born in Great Britain in 1970 associations with adult obesity for hearing impairment were assessed. BMI was measured at age 10 years and self-reported at age 34 years. Audiometry was conducted at age 10 years. The dependent variable in logistic regression was minor impairment with bilateral loss as a marker of systemic effects. Adjustment for potential confounding factors included social class, both parents living in the household, maternal education and pubertal development at age 10 years. Results: Among females, the adjusted odds ratios (95% CI) for associations observed with hearing impairment at age 10 years were 2.33 (1.36-3.98 ) for overweight/obesity; and at age 34 years they were 1.71 (1.00-2.92) for overweight and 2.73 (1.58-4.71 ) for obesity. Childhood BMI did not explain the association of childhood hearing impairment with adult BMI. There were no consistent associations among males for hearing impairment and BMI. Conclusion: The association between bilateral hearing impairment in childhood and subsequent adult-onset obesity might be due to early exposures such as psychosocial stress with chronic activation of glucocorticoid receptors, which may damage hearing through detrimental effects on the central nervous system (i.e. via interactions with estrogen receptors in the cochlea) and also result in obesity. Parenting involvement is important in children's management of T1D with the quality of involvement being a key factor. For example, negative or critical parenting has been associated with poorer adherence to T1D regimens and worse metabolic control. However, there is little research that has examined the psychosocial impact of non-supportive parenting on youth with T1D. Therefore, the current study examined the relationship of non-supportive parent behaviors and child self-efficacy, hypothesizing child depressive symptoms as a mediator. Baseline data from an RCT to promote adherence in preadolescents with T1D were used. For the current study, youth self-report was used; including measures of non-supportive parental behavior (Diabetes Family Behavior Checklist), depressive symptoms (Child Depression Inventory; CDI) and self-efficacy for diabetes care (Self-Efficacy for Diabetes Scale). Participants were 86 youth 8 to 11 years old. A mediation model was tested (Holmbeck, 2001) controlling for child age, insulin regimen, and ethnicity. None of the demographic variables were significant in the models. More nonsupportive parental behavior was associated with higher levels of depressive symptoms (F(1,77)=8.058, p< .01). Higher CDI scores were associated with lower self-efficacy (F(1,83)=15.316, p<.0001). With all variables entered into the regression equation (F(2,69)=7.201, p=.001), the association between non-supportive parental behavior and child self efficacy was no longer significant and was fully mediated by child depressive symptoms, as tested by Sobel's equation (z= -2.06). In sum, non-supportive parental behaviors appear to be related to increased child depressive symptoms, which in turn are related to lower self-efficacy for diabetes management. Clinical implications of these findings include encouraging positive parental involvement in children's diabetes management and decreasing negative or critical involvement, as well as assessing and treating depression in youth with T1D in order to improve diabetes self-efficacy and self care. Background: A negative social gradient in overweight among adolescents has been shown in developed countries but few studies have examined whether weight gain and the development of overweight differs among adolescents from different socioeconomic groups in a longitudinal study. Objective: To identify the possible association between parental socioeconomic position, weight change and the risk of developing overweight among adolescents between the ages 15 to 21. Design: Prospective cohort study conducted in Denmark with baseline examination in 1996 and follow-up questionnaire in 2003. A sample of 1,656 adolescents participated in both baseline and follow-up. Of these 1,402 had a body mass index (BMI=weight/ height2kg/m2) below 25 at baseline when adjusted for age and gender according to guidelines from International Obesity Taskforce, and were at risk of developing overweight during the study period. The main outcome measures were change in BMI and development of overweight (from BMI<25 to BMI>=25). Results: Average BMI increased from 21.3 to 22.7 for girls and from 20.6 to 23.6 for boys from age 15 to 21. An inverse social gradient in overweight was seen for girls at baseline and follow-up and for boys at follow-up. In the full population there was a tendency to a social gradient in the overall increase in BMI for girls, but not for boys. A total of 13.4 percent developed overweight during the follow-up period. Girls of lower socioeconomic position had a higher risk of developing overweight (OR's between 4.72; CI 1.31 to 17.04 and 2.03; CI 1.10-3.74) when compared to girls of high socioeconomic position. The risk of developing overweight or obesity during the follow-up period was not associated with parental socioeconomic position for boys. Conclusions: The levels of overweight and obesity among adolescents are high and continue to rise. Results from this study suggest that the social gradient in overweight becomes steeper for girls and emerge for boys in late adolescence (age span 15 to 21 years). Late adolescence seems to be an important window of opportunity in reducing the social inequality in overweight among Danish adolescents. Introduction: Adolescent sleep deprivation is an international health crisis affecting learning, high risk behaviors, and mood. Adolescent sleep is impacted by numerous factors; the family sleep environment being one of the most important and most amenable to change. There are currently no valid, reliable measures of family sleep environments. This research developed and validated the Howard Family Sleep Questionnaire (HFSQ) . We demonstrated that a) the HFSQ can predict sleep disorders b) gender interacts with family factors to predict the quality, duration and timing of sleep (still analyzing) and c) cultural impacts on the relationship between sleep and family factors (still analyzing). Methods: The HFSQ, the Pittsburg Sleep Quality Index (PSQI), the Epworth Sleepiness Scale (ESS), the Epworth-Billings Sleepiness scale (EBSS), and questionnaires assessing sleep behaviors were collected from 50 adolescents in various parts of Maryland. An additional 50 adolescents in Karachi, Pakistan were given the test packet. Results: In the Maryland sample, individuals identified with excessive daytime sleepiness (EDS) either by the ESS or the EBSS scored significantly higher on the HFSQ (ESS t (34) = -2.509, p = 0.017; EBSS t (34) = -2.028, p = .05). The HSFQ was significantly correlated with the PSQI total score (r = .376, p = .05) and the PSQI sleep disturbance index (r = .444, p = .001), while the ESS and EBSS scores were not significantly correlated with the HFSQ. Similar analyses on the Pakistan sample is about to begin. Conclusion: These results suggest that the family sleep environment can significantly predict excessive daytime sleepiness in adolescents. Additionally, the HFSQ gains construct validity by its significant relationship to the PSQI total score (i.e., poor sleepers) and PSQI sleep disturbance index. Hopefully, the HFSQ will be translated and validated in numerous cultures since there is a need for large scale international collaboration on this all important health topic. The objective of this study was to examine the effects of parental conflict on the health-related quality of life in a sample of Spanish children between 8 and 12 years. Participants were 1111 children with a mean age of 10,13 years (SD=1,29), 50,3% boys and 49,7% girls. The level of parental conflict was assessed by a fivepoint scale, in which children estimated the current relationship between his/her parents as very good, good, fair, poor, very poor. Quality of life was evaluated using the Child Health and Illness Profile-Child Edition that examines the children perceived health in five dimensions: satisfaction (with self and health), comfort (emotional and physical symptoms and limitations), resilience (positive activities that promote health), risk avoidance (risky behaviors that influence future health) and achievement (social expectations in school and with peers). Results show a positive and significant relation in all dimensions, indicating that the more conflictive is the relationship between parents, the worse the quality of life (satisfaction: F=26.52, p=.00; comfort: F=8.44, Parents of children with chronic illness typically report higher stress relative to those of healthy children; however, general parenting stress measures may not capture their unique challenges. The Pediatric Inventory for Parents (PIP) was developed to assess parents' stress in caring for a child with an illness. However, the extant psychometric data for the PIP is limited and has not been adequately examined within a large sample of parents of children and adolescents. The current study more thoroughly explores the psychometric properties of the PIP in Type 1 diabetes (T1D). Participants included 430 primary caregivers of children and adolescents with T1D who had participated in various research studies at a mid-atlantic children's hospital. Child ages ranged from 2.16-17.06 years (SD=3.49); illness duration ranged from .30-13.64 years (SD=2.76). All participants completed the PIP, which provides total frequency and difficulty scores and four domain scores (Communication, Emotional Functioning, Medical Care, Role Function). A substantial subsample completed self-report measures of theoretically associated constructs, including parent anxiety, depression, and family allocation of diabetes management responsibility. The internal consistency estimates for the total score (α = .97), and Frequency (α = .93) and Difficulty domains (α = .95), were very good. For the four domains, internal consistency ranged from adequate to good (α = .74-.90). The overall validity of the PIP was promising, as Frequency and Difficulty scores were significantly correlated with parental anxiety (r=.34-.53, p<.01) and depression (r=.29-.45, p<.01) scores across all domains. Greater parental responsibility for diabetes care was positively correlated with lower medical care stress (r= -.19, p<.05) . These data support the PIP as a valid and reliable measurement of pediatric parenting stress for primary caregivers of children and adolescents with T1D, indicating its utility in quantifying parenting stress in clinical and research endeavors with families of children with T1D and other chronic illnesses. , p=.00; resilience: F=30.45, p=.00; achievement: F =16.39, p=.00; risk avoidance: F=13.77, p=.00). Post hoc comparisons showed that significant differences were found between children who perceived the relationship between parents very good and those that considered it as good, fair, poor and very poor. However, there were no significant differences between the other groups. The results highlight the importance of the child's perception of a good family atmosphere and the absence of parental conflict for optimal health-related quality of life. Background: Studies have shown that children and adolescents who somatize are at high risk for presenting with underlying psychosocial problems. Objective: To investigate the relationship between somatization symptoms and psychosocial problems in children and adolescents receiving treatment in a pediatric Emergency Department (ED). Method: Three-hundred and fiftyeight patients (48.6% male, 51.4% female) were recruited from the ED at Childrens Hospital Los Angeles. Patients ranged from 8 to 18 years of age (M=12.8, SD=2.9). Investigators conducted an anonymous, cross-sectional, multi-informant (child and caregiver) study. Pearson's product moment correlation coefficient was used to analyze relationships between somatization (pain/weakness, gastrointestinal, and pseudoneurological symptoms) and psychosocial problems (attention, internalizing, externalizing, and total psychosocial problems). Results: Pain/weakness symptoms were significantly correlated with attention (r=.292, p<.01), externalizing (r=.242, p<.001), internalizing (r=.383, p<.001), and total psychosocial problems (r=.452, p<.001). Gastrointestinal symptoms also had a significant positive correlation with attention (r=.304, p<.001), externalizing (r=.242, p<.001), internalizing (r =.383, p<.001), and total psychosocial problems (r =.452, p<.001). Pesudoneurological symptoms also had a positive significant relationship with attention (r=.357, p<.001), externalizing (r=.211, p<.001), internalizing (r=.408, p<.001), and total psychosocial problems (r=.455, p<.001). Conclusion: All measures of somatization were associated with psychosocial problems. This demonstrates the importance of screening children and adolescents for somatization symptoms. The pediatric ED can be a useful setting to screen for somatization in order to identify children and adolescent who need referral for mental health assessment and treatment. Objective: To assess parental attributions of internal locus of control of adolescent obesity as a predictor of Expressed Emotion (EE), specifically Criticism, in parents of obese adolescents. Background: EE is a measure of family attitudes and feelings towards a symptomatic family member. One theory regarding EE, particularly the criticism facet, is that it stems from attributions of internal control to the afflicted family member. Significant correlations between EE and misattributions have been found in a number of disorders including schizophrenia, anxiety disorders and learning disabilities suggesting that correcting misattributions may in turn modify levels of EE, a positive predictor of relapse rates and a negative predictor of treatment compliance. Methods: Thirty four parent-adolescent dyads participating in a treatment study targeting adolescent overweight and obesity at Mount Sinai School of Medicine and at the University of Chicago participated in this study. Parents completed the Family Questionnaire to assess EE, and the Multidimensional Health Locus of Control Scale to assess attributions of their child's obesity. Data were collected at baseline assessment. Results: Linear regression analyses with parental BMI as a covariate indicated that parental internal locus of control regarding adolescent obesity was significant (beta=−.417, p=.039) as a unique negative predictor of the criticism factor of EE. Discussion: Contrary to our hypothesis, increased attributions of obesity to a locus of control internal to the child were associated with reduced parental criticism. Though contradictory to literature on attributions and EE in other disorders and conditions, our findings are consistent with data on parental obesity indicating that overweight parents tend to be less alarmed regarding their overweight child. Notably, the majority (92.6%) of parents in our sample were overweight or obese. Given the concordance of overweight or obese status between parents and offspring in our are domains of particular vulnerability. There have also been several reports of lower visual-motor drawing ability among children with complex congenital heart disease. Multiple medical and surgical variables have been recognized as impacting neurodevelopmental outcome for these children. Very few studies have specifically examined neurodevelopmental outcome for children with ventricular septal defects (VSDs), which are among the most common congenital heart problems with a rate of approximately one out of 500 births. This population is generally expected to have excellent medical outcome and typical development. As part of a larger study examining mid-term neurodevelopmental outcome for children VSDs surgically repaired in infancy, visualmotor ability was assessed among 31 children ages 3 to 16 years with a commonly used graphomotor drawing task. Whereas many areas of cognitive functioning, including full-scale IQ (M=95.6, SD=15.3, p=.12) were comparable to the general population (M= 100, SD=15), visual-motor ability was an area of weakness (M= 87.9, SD=14.2, p<.001) . Additional results showed that household income and the presence of a comorbid medical condition were significant predictors of visual-motor ability. Specifically, lower income was associated with lower visual-motor scores (p=.007). The presence of a comorbid medical condition was also associated with lower visual-motor ability (p=.019). Twenty-two additional patient and surgical factors were also examined, with no other variables statistically related to visual-motor ability. This study shows that the weaknesses in visual-motor ability observed among children with more complex, cyanotic congenital heart disease are also found among children with repaired VSD. Furthermore, surgical factors do not appear to affect visualmotor ability among children with VSD. This research aimed to investigate aspects of body image, stress and what coping strategies are used by adolescents in a stressful situation that involves body discomfort. A sample of 166 girls and 47 boys, aged 15-18 years, was recruited from public high schools in Ribeirão Preto, state of São Paulo, Brazil. The main outcome measures used were Figure Rating Scale, Lipp Stress Symptoms Inventory and The Ways of Coping Questionnaire. Descriptive analyses were performed. Correlational analyses tested the associations of body image, stress and coping with sex and BMI. Logistic regression analyses were conducted for each of the 8 coping strategies. The confidence intervals used were 95%. The results revealed that both boys and girls were dissatisfied with their body size and the majority overestimated it. The exception was the overweight adolescents that had significantly negative body distortion. Girls showed more body distortion than boys. The correlation between body dissatisfaction and body distortion was negative and moderate (r=− .65). A large proportion (55,86%) of adolescents are found to have significant stress symptoms. A greater proportion of females (61,40%) than males (36,20%) are found to have stress. Adolescents with stress had more probability using confrontive coping (OR=3, 14) , distancing (OR=2,09), seeking social support (OR=3,33), self-controlling (OR=2,06), accepting responsibility (OR=4,40) and escape-avoidance (OR=3,17) in a situation that involves body discomfort. Girls significantly employed more self-controlling than boys. In conclusion, negative body image perception and stress were observed in the present sample. Adolescents with stress used more coping strategies. University of Tsukuba, Tsukuba, Japan and 2 Ibaraki University, Japan.
Purpose: To clarify the efficacy of the revised life analytic counseling (LAC-R), a set of support skills that aims to teach students self health-care behaviors, on psychosomatic complaints and self-esteem in Japanese junior high school students. Method: A questionnaire was administered from September 2008 in October the first grade for the third grade of 30 public junior high schools in X Prefecture. The school nurse teachers at each school requested the cooperation of classroom teachers in the study. The questionnaires were distributed and collected by the classroom teachers. The purpose of the study was explained orally at the time of questionnaire administration and informed consent was obtained from students. LAC-R was then carried out for 3 months in 253 students. After LAC-R, the same questionnaire was administered and the differences in student responses before and after the intervention were analyzed. Using the psychosomatic complaint scale score as a dependent variable and grade level as an independent variable, ANOVA by time (pre-and post-intervention) was performed. Results: Data for 4367 boys and 4083 girls were analyzed. Significant differences in psychosomatic complaints and selfesteem scale score were observed between boys and girls (p<.001) and among different grade levels(p<.001). Moreover, for selfesteem scale score, the interaction between gender and grade level was significant (p=.003). Girls reported more psychosomatic complaints than boys, particularly in the higher grades. In addition, the self-esteem scale score for girls was lower than that for boys and decreased with increasing grade level. LAC-R was completed by 114 boys and 139 girls. In boys, self-esteem scale score increased after the LAC-R intervention (p=.041) Conclusion: A 3-month LAC-R intervention increased self-esteem in junior high school boys. Tottori University, Yonago City, Japan and 2 Tukuba University, Tukuba City, Japan.
This study used parent training (PT) for parents of children with Developmental Disorder. The usefulness of PT was evaluated parent's general health questionnaire (GHQ30 Japanese version) and the number of tasks achieved by the parents at their home setting. Total participants were 141 parents during five years. Each children were diagnosed as autism, asperger syndrome, ADHD or mental retardation. The five training components of the program were: lectures about behavior analytic theory, role playing, group discussion, homework and recording of behaviors about tasks, and feedback to parents from the training staff based on their recordkeeping. We carried out a lecture of applied behavior analysis in the first half of the each session and carried out group work in the latter half. The average scores of GHQ30 were improved. The change was significant. Regarding the behavioral modification of the children, 42.6-64.9% task achievement rate was obtained. The recent increase in the numbers of NEET young people (not in education, employment or training) is socially anxious problem in the many countries of the world. The purpose of the present study was to investigate the relationship between career indecision and the factors of ego identity. 4 types of mental situations (confusion, anxiety, hesitation and prudence) were constructed by the career indecision scales and 5 factors of adolescent sub-identity(group identity, individual identity, sex role identity, occupational identity and social identity) were constructed by the identity scales A questionnaire was administered to 408 university students, 182 males and 226 females in Japan. The findings by using multiple regression analysis showed that the total of career indecision were greatly influenced by the achievement of total ego identity (R square,.51). The confusion type of career indecision was closer relationship with the individual identity(β,−.352 p<.01). The anxiety types of career indecision was related to sex role identity (β,−.312 p<.01). The scales of occupational identity showed high relationship with total scores of the career indecision(β, −.402 p<.01) in male students, although the relationship between the occupational identity and the career indecision in female students was low(β,−.214 p<.01). The joint graduate school in science of school education, Hyogo university of teacher education, Simokume, Kato, Japan and 2 Joetsu university of Education, Joetsu, Japan.
In this study, we examined the effectveness of independent and interdependent group contingency to apply to the academic performance of students with developmental disorders enrolled in high school classes. In addition, the study also empirically addressed side effects with regard independent and interdependent group contingency. As a result, the application of independent and interdependent group contingency was effective in the overall occurrence of academic performance, including students with developmental disorders. On the other hand, when independent group contingency was applyed in writing test scene, some of students gave up the occurrence of academic behavior achieving the criteria to be reinforced. In addition, in the group learning situation, many students were found sabotage behavior after they achieve the criteria. We discussed the way of improbement on these issues and procedure to express positive side effect on group contingency. CORRESPONDING AUTHOR: Kazusa Wakabayashi, master's degree, The joint graduate school in science of school education, Hyogo university of teacher education, Simokume, Kato, 6731494; j195175m@gmail.com PSW-173 THE RELATIONSHIP BETWEEN THE TWICE-APPLICATION METHOD OF THE TREE-DRAWING TECHNIQUE AND EGO IDENTITY Sato Hideyuki, MD and Nagai Satoru, PhD Rissho University, Shinagawa-ku, Tokyo, Japan.
The tree-drawing technique is a projective technique that involves the drawing of a tree, and it is one of the most frequently used techniques in Japan. One modified procedure of the technique is "the twice-application method" of the tree drawing technique. The technique is administered by drawing a tree in the first picture and then immediately drawing a tree again in the second picture. It has been demonstrated that through this technique the subject expresses their self-image in two different projection levels-"social attitude" in the first picture, and "internal self-image" in the second. However, little recent studies have addressed the validity of the technique. Therefore, the present study aims to examine the relationship between the twice-application method of the treedrawing technique and ego identity. The participants were 277 university students who completed the twice-application method of the tree drawing technique and the ego identity status scales. In the present study, we scored the forms of the tree trunk drawing because the relationship between the indices in that element of the drawing and ego status has already been examined. The results of the ego identity status scales demonstrated that the subjects were distributed in identity statuses as follows: 15 identity achievements and 46 identity diffusions. The tree drawings of two groups were compared by chi-square tests. As a result of chi-square tests, we observed that identityachieved individuals who drew a trunk at the center in the first picture with consecutive lines also drew a trunk of medium thickness in the second picture. On the other hand, identitydiffused individuals who drew a trunk on the left side or the right side with a considerably wide trunk base in the first picture also drew a trunk with discontinuous lines and a thin trunk in the second picture. These results reveal that the uncertainty and the conflicts of their self-images were expressed in the twiceapplication method of the tree drawing technique. Adolescent smoking in China has become a major public health problem, with initiation rates on the rise, comparable to those in Western countries (Unger et al, 2001) . While many studies have documented the associations between peer influences and smoking behaviors in the U.S. (Hoffman et al, 2006 ) and among Chinese adolescents (Weiss et al., 2008) fewer studies have shown how these influences occur in relation to one's popularity and social status as measured by an individual's position in their social network. Furthermore, recent advances in social media channels now serve as an extension of one's social network and sphere of influence. Online social network sites such as Facebook (QQ in China) and mobile phone text messaging are rapidly gaining popularity. This study examines the interplay between these new media channels, peer influences, and smoking outcomes. Data were collected from 5,563 students from 24 professional and vocational high schools in Chengdu, the capital of Sichuan Province, China. Exploratory factor analysis of 12 Internet usage items revealed two types of online activity-social and non-social. Students' social status was measured by the number of "wellliked" nominations from classmates and the outcome variable was past-month smoking. Multilevel logistic models were constructed, controlling for school-level random effects and subject-level covariates such as age, gender, SES, friend smoking and friend media use. Results indicated that social Internet activity (gaming, chatting) was a risk factor for smoking (p=.01), while non-social Internet activity (news, forums, homework) had a protective effect (p<.01) among adolescents. High social status was positively associated with smoking (p<.01) and was found to significantly moderate the association between mobile phone usage and smoking (p=.04). Findings suggest that media usage may have differential effects on risky adolescent behaviors, which may have implications for designing tailored Web or mobile phone-based applications for health promotion interventions. Whereas race/ethnic disparities in health have been widely documented in pediatric populations, there is a dearth of information regarding disparities in quality of life (QOL) of children in the general population. Reflecting the child's wellbeing across multiple domains, QOL captures well the broad meaning of health espoused by WHO. This research therefore examined whether there are race/ethnic disparities in QOL. A multi-site study enrolled 4,824 5th grade children from one of the three major race/ethnic groups in the U.S. (Hispanic=38%, African American=36%, White=26%). Children reported their QOL with the PedsQL providing scores for their physical and psychosocial domains, as well as with instruments to measure selfworth and social well-being. Parents rated children's overall health status and provided demographic information on the household. Complex sampling analysis revealed non-White children reporting significantly lower QOL in all domains than White children. However, after controlling for SES differences (parent's education, income), these race/ethnic disparities remained only in the domains of school, self-worth, satisfaction with appearance, and social well-being. After also controlling for family contextual differences (family structure, English proficiency), disparities remained in the domain of social well-being only. Findings suggest that disparities across race/ethnicity in children are, mainly, products of sociodemographic differences, rather than being endemic to minority race/ethnic groups. As one of the first studies examining racial/ethnic disparities in QOL of children in the general population, this study provides a basis for further exploration of contextual influences on the QOL experienced by children. Correspondence education was introduced into Japan from the United States in 1946, and many working young people used it to learn. However, since many of the students today have mental or physical health problems, in this study we conducted a questionnaire survey by mail of 1984 students enrolled in correspondence high schools in two regions of Japan in order to determine the health status of the students and factors that affect it. 1083 students out of 1452 students (reply rate: 73.2%), who were included in the analysis, were between 15 and 29 years of age and were not being treated for any diseases and had replied to all of the items on a psychosomatic complaints scale (valid reply rate: 54.6%). The chi-square test was used to statistically analyze associations between psychosomatic complaints and both personal attributes and lifestyle habits according to gender, and the t-test was used for age and the self-esteem scale scores. We identified items which has a significant difference, and then performed a multiple logistic regression analysis. The results for factors that affected psychosomatic complaints among the males showed that low self-esteem (OR=0.90,95% CI=0.87-0.94,p<0.001), irregular or skipped breakfasts (OR= 1.91, p=0 .01), being a fussy eater (OR = 1.84,95%CI = 1.17-2.89,p = 0.008), and age (OR = 1.13,95% CI= 1.03-1.24,p=0.008) increased psychosomatic complaints. Among the females, low self-esteem (OR=0.89,95%CI=0.86-0.92,p<0.001), poor health status during puberty (OR=2.93,95% CI=1. 75-4.90,p<0 .001), being a fussy eater (OR=2.57, p<0.001) , and being a smoker (OR=1.95, p=0 .011) increased psychosomatic complaints. "Low self-esteem" and "being a fussy eater" increased psychosomatic complaints in both sexes. Thus, we would like to make a program which allows correspondence high school students themselves to reconsider their diets by increasing their self-esteem for the future investigation. BACKGROUND: US adolescent obesity increased from 5% to 18.1% between 1976-1980 and 2007-2008 . In addition to health complications (e.g., type 2 diabetes), adolescents who are obese are at risk for developing negative body image which is related to depression and is a risk factor for eating pathology. Having a supportive peer group is an essential part of healthy adolescent development, and for overweight adolescents, could be a protective factor against negative body image. PURPOSE: To examine whether the association between adolescent weight status and body image varies by peer support. METHODS: A US nationally-representative sample of 6,909 students in grades 6 -10 with over-sampling of African-American and Hispanic students completed the 2006 Health Behavior in School-aged Children survey. Body image, was assessed with 5 items (α = .87) asking for agreement or disagreement with statements about one's body. Weight status, determined from body mass index, was computed from selfreported height and weight and categorized using CDC 2000 growth charts. Peer support was computed as the average of 8 items (α = .67) asking about interactions with peers; higher scores indicated greater peer support. Linear regressions, separately by gender and controlling for age, race/ethnicity and socioeconomic status, were conducted with an interaction term (weight status x peer support). RESULTS: Adolescents' overweight status was related to poor body image in boys and girls. However, peer support moderated the relationship between weight status and body image for girls but not for boys. Overweight girls with higher peer support were more likely to have a positive body image (β=−0.69, p<0.000) compared to overweight girls with lower peer support (β=−.99, p<0.000). Overweight boys had lower body image compared to their normal weight peers, regardless of the level of peer support. CONCLUSION: Encouraging adolescent girls to develop strong healthy relationships with peers may prevent a negative body image. More research is needed to examine whether these findings vary by individual (e.g., race) or familial (e.g., presence of parental support) characteristics.
CORRESPONDING AUTHOR: Laura J. Caccavale, BA, Prevention Research Branch, Eunice Kennedy Shriver National Institute of Child Health & Human Development, Washington, DC, 20008; caccavalel@mail.nih.gov Few prospective studies about antibiotic use for mild acute respiratory infections (ARIs) have been conducted in community settings. This paper aims to assess knowledge of child-caregivers and actual antibiotic use for children under five, and to identify associated factors with antibiotic treatment for mild ARIs. Caregivers in 828 households in Bavi, Vietnam were interviewed using a structured questionnaire regarding the case management of childhood ARI and the selected children's most recent illness assessing both knowledge and practice. Then 823 children were followed for 28 days to collect information regarding symptoms and drug use. For management of non-febrile common colds, 85% of caregivers stated correctly that antibiotics are not required. For febrile colds and pneumonia, 45% and 47% of cases respectively would require antibiotics. Only 13% demonstrated correct, in accordance with standard guidelines, overall knowledge for all three situations. The symptoms of the most recent illness were consistent with mild ARI in 79% of the cases, and antibiotics were used in 71% of these. During the 28-day period, 62% of children had been given antibiotics. Out of all antibiotic courses recorded, 63% were used for mild ARIs. Half of the mild ARI episodes (528/1048) and 63% of the children with mild ARIs (392/623) were treated with antibiotics. Most of the unnecessary antibiotic treatment was recommended by healthcare providers (82%). Most of the children had been administered antibiotics for common colds although most caregivers believed that antibiotics were not required. Antibiotics were unnecessarily recommended at health facilities in the area. BACKGROUND: The benefits of physical activity (PA) for children and adolescents are numerous and include reduced risk for coronary heart disease, and improved mental health. Physical activity habits that are established during adolescence are likely to be sustained into adulthood. However, the prevalence of PA among adolescents is low, and even lower among overweight/obese (OT) adolescents, who are most in need of the benefits conferred by PA. Body image may mediate this relationship; overweight/obese adolescents are more likely to suffer from poor body image, which could be linked to less engagement in PA. PURPOSE: To examine the hypothesis that poor body image mediates the relationship between overweight/obesity and PA among adolescents. METHODS: Data are from the 2006 Health Behaviors in School-Age Children survey, a nationally representative sample of students in grades 6-10 during the 2006 school-year. Physical activity was measured by a question asking about the number of days respondents engaged in at least 60 minutes of PA over the last week. Body image was assessed with 5 items (α=.87) asking for agreement/disagreement with statements about one's body. Weight status, determined from body mass index, was computed from self-reported height and weight and categorized using CDC 2000 growth charts. Mediation analysis, stratified on gender and controlling for age, race/ethnicity and SES, was performed to determine whether body image mediated the relationship between weight status and PA. RESULTS: Overweight was negatively associated with physical activity only among boys whereas obesity was negatively associated with physical activity among boys and girls. Body image was a complete mediator of the association between weight status and PA among girls, and a partial mediator of the association of weight status with PA among boys. CONCLUSIONS: Mediation analyses demonstrate that body image is a significant mediator of the relationship between overweight/obesity and PA among boys and girls. Encouraging overweight and obese adolescents to develop positive body images may result in greater engagement in PA.
Many children experience compromised health status. Most obvious are chronic health conditions (CHC), but the majority of children only experience temporary health problems (e.g., infection, injury). How do these variations in health status affect quality of life (QL) in children? QL reflects the child's overall well-being across multiple domains. There has been no study examining the relationship between compromised health and QL in children in the general population who do not have a CHC, nor do we know how such variation interacts with race/ethnic status. Our hypotheses are that (1) variation in health status is positively associated with QL regardless of the presence of CHC and (2) Whites report higher QL than African Americans and Hispanics, regardless of health status. A multi-site study enrolled 4,824 5th grade children from one of the three major race/ethnic groups in the U.S. (Hispanic=38%, African American=36%, White=26%). Children reported their QL with the PedsQL providing scores for their physical and psychosocial domains, as well as on instruments to measure selfworth and social well-being. Parents rated their 5th graders' overall health status on the commonly used single item "In general, would you say your child's health is . . . (Excellent/Very Good/Good/Fair/Poor)?"and completed a screener, from which chronic health condition was classified. Results showed there was a reduction in each QL domain associated with each decrement in overall health status, even in the absence of a CHC. Moreover, African Americans and Hispanics report a lower QL than Whites, when in excellent health as well as at each decrement in health status. This demonstrates for the first time that even minor decrements in health status are associated with concomitant reduction in QL, not only in the physical domain, but also in the emotional and social domains. Several researchers have found that teenage pregnancy is associated with worst educational outcomes among adolescents and that girls who have a child early in life, are less likely to graduate from high school (Molina et al., 2004) . Other researchers have proposed that educational expectations do actually predict educational outcomes among youth. They have found that having higher educational expectations is associated with higher educational outcomes later in life (Mello, 2008) . However, researchers have not examined how educational expectations may be associated with adolescents' self-reported sexual behaviors. Some researchers suggest that there might be a self-selection process in which adolescents who do worst in school and do not have plans to continue further in their education may also engage in unprotected sexual behavior, putting them at higher risk of becoming pregnant. In the present study we examined the relationship between educational expectations and goals and different sexual behavior outcomes in a sample of 829 Mexican youth. Participants included 829 adolescents between the ages of 14 and 17 (mean 15.18, S.D.=0.68, 55,4% females) part of an HIV and STDs intervention program. We examine this relationship using time 1 data. Participants completed a self-reported questionnaire that asked about their sexual behavior and explore different psychosocial predictors of sexual behavior. Using a regression analyses we examined whether having plans to complete high school and continue their education predicted ever had sex, age in which girls and boys should have sex for the first time, plans to use condoms when having sex, and perceive that condoms may be effective to protect them of STDs and HIV. Our findings suggest that plans to finish high school predicts only perceptions that condoms may be effective to protect against HIV (t=2,06, p< 0.05), and not any of the other outcomes. Because we had very few participants who reported ever having sex (9,5%), we probably did not have enough power to find further differences. We explored how these variables may predict their sexual behavior at the 12 months follow-up, when 16% of participants reported ever having sex, and we found that having plans to attend college predicted not having sex among adolescents (Exp (B) = 0.358, p<0.01). Implications of these findings for intervention and practice are discussed. Purposes: Although many reports have suggested a role for the social skill in the treatment of depression, there has been little evidence that the social skill training is effective therapeutic strategies for treating it. The activation level are thought to be an important mediator of the social skill and the depression. Obata et al. (2010) found that the difficulty in the control of social anxiety lead to the inhibition of social activity and the increase of depressive mood in Japanese high school students. We hypothesized that the mechanism for exacerbation of depression may be explained by the behavioral activation and social skill, and these relationships differed with the level of social anxiety. Method: Questionnaires were conducted with 347 Japanese adolescents (16.0±0.6 years). Questionnaires were composed of Depression Self-Rating Scale for Children (DSRS; Birleson, 1981) , Assertiveness Skill Scale for High school students (ASS; Takaki et al., 2002) , and Behavioral Activation for Depression Scale (BADS; Martell et al., 2001) . Higher score group (n=50) and lower score group (n=50) were extracted in subjects on "social anxiety(reverse coded)" in ASS. Data analyses were tested using correlation analysis to examine relationship of between depression , social skill, and behavioral activation, using SPSS 16.0. Main Results: "Activation (reverse coded)" in BADS was significantly associated with "positive interpersonal entry" in ASS(higher score group: r=−.37, p<.01; lower score group: r=−.26, .n.s.). Conclusion: These results suggest that the control of social anxiety is an important procedure for behavioral activation on depression. Many people think that the children are not stress, in fact they can stress as the adult but their parent cannot notice or skip it. The objective is to study the association between state anxiety with emotional quotient and strength and difficulty of the secondary school student who also study in Sunday Buddhism and do not study by controlling some potential confounding. The self-report questionnaire was distribution to both type of schools to student age 11-16 years old. The tools of this study are Thai version of state-trait anxiety of Spielberger's; the Strength and Difficulty Questionnaire (SDQ); emotional quotient (EQ). The regression model used for analyzing and the data are centered to reduce the scaling problem. The 303 completed answer questionnaire was returned, the incomplete questionnaire was excluded from the study to reduce the effect of imputation. Mean age of sample is 13.5 years old, 42.24% is male, and 48.51% reported that they are also study in the Sunday Buddhism School. The model by stepwise selection presents that trait anxiety, pro-social, selfcontrol, ability to deal with problem, self-esteem, life-satisfaction and interaction between age and emotional problem, the adjusted R2 is 60.55% (p-value<0.0001). The increasing one unit of trait anxiety, self-control, self-esteem and life-satisfaction increases 0.52, 0.10, 0.11 and 0.13 standardize unit of state anxiety, the higher standardize score of ability to deal with problem decrease 0.07 standardize unit of state anxiety. The interaction of emotional and age reduce the standardize score of state anxiety. The higher emotional problem score increased 0.27 standardize unit of state anxiety while age reduce 0.01 unit of state anxiety (p-value<0.05). The gender and Sunday Buddhism school included into the model to control potential confounding factor, female has higher state anxiety score than male, the student in Sunday Buddhism School have lower state anxiety than non-student (p-value>0.05). The student with high score of self-control, self-esteem and lifesatisfaction have higher level of state anxiety which may cause from they are try to be the best which make them stress. The ability to deal with the problem can reduce state anxiety, the higher age (more maturity) reduce emotional problem and finally reduce state anxiety. The student who is good in some dimension of EQ may cause state anxiety. The mobilization of significant resources for global health coincides with the emergence of Global Health Initiatives (GHIs) and the rising importance of multilateral and private actors in global health; traditional actors in global health, such as the World Health Organization (WHO), have been joined by a variety of civil society and non-governmental organizations (NGOs), private firms, and private philanthropists. These new actors, which exist alongside traditional arrangements between sovereign states and UN bodies, signal a sea change in the institutional arrangements in global health as well as a change in the norms, expectations and rules relating to the relationships between these new philanthropists. It has become clear, however, that the new institutions in global health have often failed to adequately evaluate their investments and program implementation. With such enormous investments, it is critical to better understand these new institutions, evaluate their strategies to promote global health and document the successes and failures of these efforts. We propose that rigorous qualitative research methodologies can explore and describe how global health actors and their associated mechanisms interact with complex health systems to yield optimal Results: Qualitative methods, particularly case research, can overcome the three main weaknesses of quantitatively-oriented public health research models: underestimating complexity, failing to incorporate context and an inability to engage in the "why" questions. Qualitative methods can better evaluate if the activities funded by new global health actors are best aligned to meet the objective of delivering high value care to patients. Global health institutions can use this insight to bridge the knowledge-action gap in global health care delivery by translating the current understanding of medical interventions and health systems into evidence-based strategies for care delivery.
CORRESPONDING AUTHOR: Erin Sullivan, PhD, Global Health Delivery Project, Harvard University, Boston, MA, Ma; esulliva@hsph.harvard.edu tend to make more errors than others even within the exact same work environment, measures that consider individual characteristics are needed. This study aimed to elucidate the relationship between medical errors and the personality traits of nurses. We reported on this relationship before; this time, we studied a bigger sample size for greater accuracy. Methods: Medical errors and near-misses that had occurred in actual medical settings were analyzed, and "administration," which is one of the most common components of a nurse's job, was selected as the theme of this study. For this study, simulation software was developed that enabled nurses to experience all the human errors that could possibly occur in various settings under the broad category of "administration." The software was used to understand the reasons behind the subjects' tendency to commit medical errors. Furthermore, the Kretschmer Type Personality Inventory was administered to the nurses in order to determine their personality traits. This study was approved by the ethics committee of our university. Results and Conclusions: A relationship was found between the nurses' tendency to commit medical errors and their personality traits (self-repression, self-disclosure, steady-going nature, sensitive nature, unshakable belief, etc.) with a sufficient sample size. Clarifying the relationship between the personality traits of nurses and human errors may be useful for training. Also, this approach may help to prevent human errors by enabling the nurses themselves to become conscious of their actions and objectively aware of their own individual characteristics.
CORRESPONDING AUTHOR: Mayo Suzuki, PhD, Bunri University of Hospitality, Sayama-shi, 350-1336; mayo-suzuki@ bunri-c.ac.jp Purpose: The purpose of this study is to clarify the relationships between human errors and the job performance capability of nurses. Methods: The subjects were all of the newly hired nurses of five facilities. A simulated case experience software and the Uchida-Kraepelin Test were used. For data analysis, the subjects were separated into the groups based on the judgment results of their performance on the Uchida-Kraepelin Test. The groups were then analyzed for differences in software scoring and correct answer ratio. In the analysis, SPSS 12.0 J for Windows was used. This study protocol was approved by the Ethics Committee of Kyoto University. Results: The analysis subjects numbered 65 people. The average age was 24.9±4.7. There were 58 females (89.2%). Membership alignment in the groups via the Uchida-Kraepelin Test results were as follows: 72.3% for the typical group; 35.4%, 58.5%, and 55.4% respectively for the groups with no problems in movability, changeability, and durability; 72.3% for the group sufficient in efficiency and speed of work; and 67.7% for the group with no problem in degree of balancing in terms of temperament and behavior. The software scoring average was 64.3±29.7 points, and the correct answer ratio for each error was 36.9% to 83.1%. For nurses grouped in the job performance characteristic types of atypical nurses and nurses with problems in degree of balancing in terms of temperament and behavior, the software scoring was significantly low. Also, the correct answer ratio for each error was significantly low for nurses grouped in the job performance characteristic types of atypical nurses, nurses with problems in degree of balancing in terms of temperament and behavior, and nurses with problems in durability. Conclusion: Subjects possessing the job performance characteristics of "atypical type" and "problems in degree of balancing in terms of temperament and behavior" had significantly lower scores of the software than subjects not possessing these characteristics. Childhood obesity has rapidly become an epidemic in developed countries globally (WHO, 2000) . The development of effective treatment initiatives aimed to help obese youth adopt and maintain healthy lifestyles is prudent. The purpose of this study was to examine the effects of exercise intensity on obese adolescents' enjoyment, satisfaction and program adherence. Thirty obese adolescents (female=20, BMI≥95th percentile; 10-16 years of age) were randomly assigned to either a moderate (HRR=40-55%) or vigorous (HRR=60-75%) supervised 12-week exercise training program. Satisfaction was measured through self-reported questionnaires assessing satisfaction with outcome progress and current state. Enjoyment was measured using the PACES selfreport questionnaire. Adherence was assessed as a percentage of sessions attended. Repeated measures ANOVAs showed no significant time x group interaction effects (all ps>.05). Significant time effects in the expected direction were found for satisfaction with outcome progress (F(1, 26) = 17.62, η2=.40, p<.001), satisfaction with current state (F(1, 26) = 5.97, η2=.18, p<.05) and enjoyment (F(1, 26) = 9.4, η2=.27, p<.005) . No adherence differences were found between intensity groups (M for vigorous=86%, SD=.09; M moderate=85%, SD=.14). These data indicate that exercise intensity does not adversely affect satisfaction, enjoyment or adherence. Practical implications of these findings will be discussed. Proper nutrition is the foundation for athletic performance. This session introduces a theory based multi-level intervention spanning a convenience sample of 16 sports (n=250 athletes) at a Midwestern University. This program provides athletes and staff education about sport-related nutritional issues for performance and recovery. Emphasis of this program is placed on choosing healthy foods and making positive behavior modifications. Several methods of data collection were used for this project. All athletes were given a validated knowledge and dietary practices survey that was based on the health belief model, the EAT 26 survey which screens for disordered eating behaviors was distributed, and body composition was also determined using a Tanita scale. All data was entered into SPSS 16.0. Statistical analysis was conducted using t-tests and descriptive statistics. Changes in baseline knowledge, dietary practices, and body composition data will be reported and discussed. Formative evaluation which includes adherence to program guidelines and recommendations for program improvement will also be reported. Based on the data and information collected Project Ignite shows substantial promise as a model program for improving collegiate athlete dietary practices and knowledge about sport nutrition related issues.
CORRESPONDING AUTHOR: Amy Thompson, PhD, University of Toledo, Toledo, OH, 43606; amy.thompson4@utoledo.edu Background: Lifestyles that are associated with metabolic syndrome (MS) are affected by individual health locus of control (HLC). Objective: Association between HLC and the characteristics of MS were examined. Subjects and methods: Subjects were 200 people who underwent medical checkups. HLC was measured with 11 questions and subjects were asked about their motivation to improve lifestyle and the desire to receive health guidance. The MS components (i.e., blood pressure, waist circumference, glucose, triglycerides, and high-density lipoprotein cholesterol) were measured. The subjects were graded into 4 levels by the number of MS components (i.e.,0,1,2,3>). We reviewed the association between the levels of MS and HLC. Result: There were no significant differences in HLC between the levels of MS. In response to the item "I become sick because of luck of appropriate physical activity or diet", Level "3>" showed the highest tendency toward internals. In response to the item "I think I have reasons to be sick", Level "0" showed the highest tendency toward internals. Level "0" was significantly less willing to receive health guidance and had lower motivation to improve lifestyle than other levels. Among females, the higher the MS level was, the stronger the motivation to improve lifestyles. Among males, Level "2" had the highest motivation. Discussion: Those with Level "0" appear to make decisions themselves and they do not need to improve their lifestyles because they have no components of MS. However, our research showed that they have high awareness of not only preventing MS but also overall health. It is a problem that male with Level "3>" had lower motivation to improve lifestyle, possibly due to resignation or denial. We should raise awareness of overall health for the prevention of MS onset and encourage people who already have MS to make lifestyle changes. Introduction: In Finland, all men are liable to military service and over 80% of each age group complete it. Main meals complying nutrition recommendations of the Defence Forces are served but snacks are available for purchase during free time. Research on conscripts' eating habits is limited, suggesting they favor energydense and nutrient-poor foods such as fast food, pastries, soft drinks and confectionery. Aim: To study conscripts' eating habits before and during military service. Materials and Methods: In 2007, data on eating habits of 290 17-21-year old men were collected by self-administrated questionnaire at two garrisons (in the south and in the north) and at two arrivals (winter, summer) 1 month prior to beginning of military service and at 6 months of service. Three indexes were formed to describe main dimensions of eating habits: Fibre Index (FibI), Sugar Index (SI) and Fat Index (FatI). Results: At six months of service, the weekly consumption frequencies of rye bread (p=0.02) and fruit and berries (p=0.05) increased. This explained the increase in FibI (change 0.65±5.1, p=0.03). Weekly consumption frequencies of French fries (p= 0.001) and hamburgers and hot dogs (pp=0.005) decreased. However, this did not result in a significant temporal change in FatI. Finally, weekly consumptions of sweet pastries (p=0.002), desserts (p<0.001), sweets (p<0.001) and chocolate (p=0.01) increased. This resulted in a significant increase in SI (change 2.0±4.8, p<0.001). This change was greater (p<0.001) from summer to winter compared to winter from summer, and also higher (p=0.02) in the south than in the north. Conclusions: These results indicate that during military service men prefer eating both fibre and sugar containing foods. This may be due to an overall increase in energy intake. Still, the increase of sugary foods offers an opportunity to intervene also as seasonal and regional differences exist. treatment. Although 15 % showed negative attitudes to the treatment, their BMI and weight were reduced also. CONCLUSION: The cognitive-behavioral group therapy seems to be an adequate strategy to modify irrationals believes and eating behavior, which could derive in an excessive caloric intake and sedentary life. Research suggests that athletes do not have a higher prevalence of disordered eating behaviors compared to the general population however, their risk of sport related injury is increased when poor nutritional behaviors are present. Project Ignite is a nutrition education program designed to educate 250 collegiate athletes about eating for sport performance and avoiding injury associated with poor nutritional intake. Additionally, data such as body mass index, body-fat (measured with a Tanita scale), and knowledge and perceptions of sport nutrition was measured. All participants of this program completed the EAT 26 survey which has been found to be valid in measuring disordered eating. This presentation will outline 3 case studies of collegiate athletes with disordered eating. For confidentiality reasons all names of participants have been changed. Case studies will include 2 athletes with extremely low body-fat (4% for female; 2% male) and 1 athlete with extremely high body-fat (50% female). These case examples will discuss the need for health and nutrition policies that guide student-athlete participation in their respective sports when disordered eating is present. Moreover, there is a need for health education regarding proper nutrition for the athlete, coaches, team physicians, and athletic trainers. Further, many universities do not have polices that mandate when a collegiate athlete should be evaluated to determine the extent of any existing disordered eating behaviors or health conditions associated with their nutritional intake. When it is determined that an athlete has disordered eating behaviors or health risks there should be policies in place to determine player eligibility. The Live Healthy Work Well (LHWW) project examines whether access to life coaches, pharmacists, and other supports can improve health and prevent the loss of employment and independence due to diabetes-related complications. This poster summarizes treatment participant (n=128) usage and perceptions of intervention components. Data sources include: enrollment survey, service utilization records, satisfaction surveys, and voluntary focus groups. Six months into a one-year intervention period, 119 (93%) participants met with a life coach and 98 (77%) met with a pharmacist. Participants (n=97) responded to a survey assessing satisfaction and perceived effectiveness of the intervention model. Participants expressed satisfaction with life coaching and 91% (n= 68) of life coach comments were positive. Of pharmacist comments, 52% (n=66) were positive and 29% were negative. A second survey revealed that 85% (n=66) agreed/strongly agreed that coaching helped improve health, employment, and other areas of life; 75% agreed that goal setting helped them make positive changes and to continue to set goals; and 94% would recommend coaching to others. Sixty-eight percent (n=65) agreed/strongly agreed that pharmacists helped them find solutions to diabetesrelated problems and 69% agreed that sessions with a pharmacist were beneficial and would recommend pharmacist counseling to others. Treatment participants (n=47) attended voluntary focus groups which were audio recorded, transcribed, and coded to identify emergent themes. Participants were satisfied with support, scheduling, and goal setting with life coaches. Participants suggested that LHWW provide individualized approaches to pharmacist counseling, social supports and coordinated services. Scheduling with pharmacists and additional service providers was perceived as a barrier to program participation. Findings can be used to guide the development of future programs and health initiatives for employed adults with diabetes and results may be generalizable to employed diabetics across a range of ethnic groups including Asian and Pacific Islanders. measures 3 aspects of eating behavior, cognitive restraint (CR), uncontrolled eating (UE), and emotional eating (EE). Objective: To identify psychological factors that predict weight regain. Methods: Observation study after an intervention in which 32 obese female (mean age; 50±8 years) lost weight during an 8-week weight loss intervention program and were followed for a year. TFEQ-R18 was measured by dieticians 3 times at baseline and 8 weeks during the program and at 1 year after the program that included use of meal replacement drinks (diet's; SUNTORY WELLNESS LTD.) under the guidance of dieticians and counseling sessions by dieticians focused on skills of coping with hunger. Enrollment at Kyoto Medical Center occurred November, 2006 and intervention, January, 2007 Previous research has established demographic differences in metabolic control and chronic disease complications, yet few studies have examined differential demographic effects on psychosocial and disease care correlates of A1c. Group comparisons and regression analyses were conducted to investigate demographic differences in biological, psychosocial and disease care factors in youth with type 1 diabetes. Participants included 349 youth, aged 9 -17 years (79.9% Caucasian, 71.3% lived with two biological parents, M SES=46.24), recruited from endocrinology clinics in two major metropolitan areas. The 24-Hour Diabetes Care Interview was administered to assess specific disease care behaviors and a test of diabetes knowledge was used to measure parent and child disease information. Independent sample t-tests confirmed commonly reported ethnic differences in A1c, where African American youth had significantly higher A1c levels (M=8.84%) compared with Caucasian youth (M=8.25%). African American youth reported poorer disease care behaviors including fewer intensive insulin regimens, less frequent blood glucose monitoring and poorer dietary composition of carbohydrates and fats. Additionally, African American youth and parents had lower disease knowledge scores than Caucasian youth and parents. However, hierarchical multiple regression analyses that controlled for confounding influences of SES and parent marital status revealed most disease care differences attributed to ethnicity were better explained by SES. The only significant ethnic group difference that remained was a difference in diabetes knowledge; ethnicity explained an additional 8.3% variance and emerged as a significant predictor of diabetes knowledge (β=−.308, p<.001) after controlling for SES and marital status. Results may inform future interventions for youth at risk of poor metabolic control; lower SES families may benefit from careful attention to diabetes knowledge to facilitate successful implementation of diabetes care. This study examined the relation between abnormal eating behavior, body mass index (BMI), and body image dissatisfaction in 554 female university students (Mean age: 19.22, SD: 1.01). Measures included demographic data, the Body Image Dissatisfaction Scale (BIDS) (Yamatsuta & Nomura, 2007) , and the Abnormal Eating Behavior Scale (AEBS) (Yamatsuta, Nakai, & Nomura, 2009) . BIDS measures the degree of body image dissatisfaction. It contains three lower-rank factors (divided into high and low groups on the basis of one's mean factor score) including "dissatisfaction with body plumpness," "dissatisfaction with other people's opinions of one's own body," and "dissatisfaction with one's own face." AEBS comprises three lower-rank factors including "uncontrollable intake of excessive food," (e.g., binge eating) "purging behavior," and "control of food intake" (e.g., diet behavior). According to the BMI classification, based on that of the Japan Society for the Study of Obesity (1994), 102 subjects (18.41%) were thin, 406 (73.29%) were normal, and 46 (8.30%) were obese. First, we examined the relation between the BMI and the BIDS and AEBS factor scores; a single-factor analysis of variance of factor scores was conducted on each BMI classification. The results showed a significant main effect of BMI's class in all the factor scores excluding the "uncontrollable intake of excessive food" and "purging behavior" factor scores, and we can infer that the latter two factors have little relation to the BMI levels. Thus, abnormal eating behavior that resembles an eating disorder might have little relation to the actual body type. Second, we examined the relation between the "uncontrollable intake of excessive food," "purging behavior," and the BIDS factor scores; a t test on the former two factor scores from among the BIDS lower-rank factor group was conducted. The results indicated high scorers on these two factors in the high "dissatisfaction with body plumpness" group (t(531) = |5.08|, p<.01; t(531) = |2.14|, p<.05); hence, purging behavior might not be only a consciousness of other people's opinions of one's own body but also the problem of self. This work was supported by KAKENHI (20730455). Background: Co-Creating Health (CCH) is a national programme commissioned by The Health Foundation, (http://www.health.org. uk/) which aims to embed self management support within mainstream health services in order to achieve improvements in long term health (LTH) conditions patients activation. One of the ways CCH aims to increase activation is through the provision of a clinician and lay delivered self-management programme (SMP). The SMP is a condition specific programme for patients with either depression, pain, COPD or diabetes. The SMP comprises 7 weekly 3 hour sessions during which patients receive training in problem solving, relaxation and collaborative agenda setting, goal setting and follow up in clinical encounters. Aims: The aim was to determine whether patients report an increase in using a range of self-management skills after attending the SMP Methods: The Health Education Impact Questionnaire (hei-Q) is a generic eight scale questionnaire designed to measure selfmanagement skills/ outcomes associated with attending health education/self-management courses. Participants are asked to rate items on a 4 point likert scale ranging from "strongly disagree" (1) to "strongly agree" (4). Higher scores represent higher levels of self-management skills. Thirty patients completed the hei-Q prior to and 6 months after attending the SMP. Findings: After attending SMP patients reported a significant improvements in 4/8 subscales: Positive and active engagement in life; Emotional well-being; Constructive attitudes and approaches (all p=<0.01) and Skill and Technique Acquisition (p=0.03). Discussion: Patients reported higher levels of self-management skills after attending the SMP. Further research involving larger sample sizes will determine whether greater use of self-management skills has a positive impact on health-related quality of life.
Introduction: The Healthy ONES (Options for Nutrition Environments in Schools) study was conducted to determine the effects of changing public school nutrition environments on obesity rates in children. Intervention: The intervention was designed using systems theory, ecological models, and community based participatory research (CBPR) principals. The following environments were targeted for intervention: before and after school waiting areas, outdoor recreation areas, gyms/cafeterias, break rooms, front offices, classrooms, and administrative offices. Using the Institute for Healthcare Improvement's Plan-Do-Study-Act (PDSA) rapid improvement cycle process, we worked with stakeholder groups within each school to target small, temporary changes in each environment identified for change. Results: Environmental changes included: a "fruit at recess" program that eliminated outside food during morning recess and substituted cut fruit instead; jog-a-thon and prize walk fundraisers instead of cake walks and bake sales; using treasure chests with nonfood rewards in the classroom and principals' offices; adding vegetables to traditional lunch entrees; and cutting all fruit offered during school meals. In addition to extensive behavioral observation, we measured the heights and weights of 457 children (2nd/ 3rd graders) in year 1 and were able to measure 367 in year 2 (80% retention rate). Although not significant, obesity rates (> 95th% BMI) did not change for intervention schools (22% to 21%) and increased in control schools (21% to 24%). Year 3 measures will be done by October, 2010. Conclusion: Using a CBPR approach resulted in substantial changes in the school nutrition environment which may lead to meaningful changes in childhood obesity rates. Background: Scant efforts were made to investigate overweight trajectories and their links to multiple psychosocial adjustment problems during the period of pubertal transition. In this study we conducted a secondary analysis with data from the multisite Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Study of Early Child Care and Youth Development (SECCYD) with the application of state of the art analytic approaches of the group-based growth mixture and mixed-effect models. Methods: Longitudinal data consisted of repeated measures of weight, height, broad-band behavior problems (i.e. internalizing and externalizing problems) and narrow-band syndrome subscales (i.e. withdrawn, somatic complaints, anxious/depressed, social problems, thought problems, attention problems, delinquent behavior, and aggressive behavior) in 1,364 youths during the period from age nine to age fifteen. Results: Three trajectory patterns were identified from the whole cohort: never/rarely overweight (59.5%), late start/light overweight (12.1%), and chronically/heavy overweight (28.4%). After adjustments for gender, ethnicity, income-to-needs ratio based on federal poverty guidelines, and categorical timing of pubertal onset based on two dimensions of boys (genital and pubic hair) and girls (breast and pubic hair) development, youths with chronically/heavy overweight trajectory pattern had significantly worse scores of internalizing problems as well as syndrome subscales of somatic complaints, social problems and withdrawn than youths with never/rare overweight trajectory pattern. Conclusions: Study findings helped advance knowledge on the distinct developmental trajectory patterns of overweight and their associations with multiple behavior problems in adolescents during pubertal transition. Background: Experts affirmed that some health professionals are not taking the issue of obesity as seriously as they should, compromising the success of the treatment of obese people.
Objectives: This study tries to synthesize recent research that has explored the beliefs, attitudes and practices of health professionals about obesity. Method: The words "healthcare physicians, obesity, beliefs, attitudes, intervention, referral practices, counselling, general practitioners, advices, competency, knowledge and stigma" were entered in databases such as PubMed, Elsevier and PsycINFO. Twenty studies were found from 1999 trough 2009 and included in analyses. Results: Physicians are negative about their own role in obese treatment. Although they believed it was part of their job to advise obese patients on the health risks of obesity, the majority of doctors thought they had not made any difference in getting their patients to make long term changes in lifestyle. They are sceptical about the effectiveness of available interventions and only few doctors refer patients to behaviour therapy or to obesity surgery. Conclusions: This issue should be taken in account during physicians' education and in planning multidisciplinary teams of intervention in obesity, because physical doctors should be aware of how their own beliefs and attitudes will influence their behavior on the managing of obesity, compromising therefore the adherence and the successful in obesity treatment. Aims: The purpose of this study was to explore psychosocial and cultural values related to dietary and physical activity practices of Asians and Pacific Islanders (API) with type 2 diabetes. Methods: Content analysis was conducted with information collected from focus groups that totaled 15 men and women of API descent, ages 18-76, participating in "The Study of Cognitive Behavioral Interventions in Diabetes Self-Management," project. Focus group discussions were recorded and transcribed verbatim. Data was analyzed for categories and themes using the social ecological framework. NVivo 8 computer software was utilized for management of data. Results: Psychosocial themes related to dietary practices included depression, denial, happiness, self-control, and awareness of complications. Cultural values related to eating, such as upbringing, social events, food variety, food portions, and reciprocity were identified. Underlying medical and physiological conditions limited physical activities of some participants in the study.
Psychosocial and cultural values that influenced physical activities were motivation, family values and gender. Conclusions: Results support utilization of the social ecological framework when attempting to understand the health behavior of API. The families,environment, and the local culture greatly affected the dietary and physical activities of participants with type 2 diabetes. Empowerment with knowledge and support would allow individuals and their families to take ownership of their health care and thereby equipping them with tools to develop nutritionally and physically sound health habits to control diabetes. Depression and insulin resistance (IR) each are significant predictors of the development and progression of type 2 diabetes (T2DM), and are evident years in advance of the frank hyperglycemia of diabetes. Depression and insulin resistance (IR) each are significant predictors of the development and progression of type 2 diabetes (T2DM) and are evident years in advance of the frank hyperglycemia of diabetes. Depression is associated with factors that promote IR including inactivity, obesity, and hypercortisolemia. The purpose of the present study was to test the hypothesis of depression-associated insulin resistance (DAIR). Seventy-six persons (mean age 52±10 years, 76% female, 39% Caucasian) with a provisional diagnosis of T2DM (i.e., not taking antihyperglycemic medication and meeting ADA criteria per study OGTT) were enrolled. Major depressive disorder (MDD) was diagnosed using DSM-IV criteria. Depression symptom severity was assessed with the Beck Depression Inventory (BDI). Sixteen patients could be classified as depressed (i.e. DSM-IV MDD and and a BDI score ≥16) and 52 could be classified as non-depressed (i.e. no DSM-IV MDD and a BDI score <16). IR was derived from anthropometric measurements [body mass index (BMI)] and OGTT measurements [HOMA-IR, area under the curve for insulin (AUC-I)]. IR was uniformly higher but not statistically different in the depressed vs. nondepressed groups (BMI: 41.4±6.6 vs 37.5±8.4 kg*m-2, p=0.09; HOMA-IR: 8.0±5.6 vs 5.6±2.8,p=0.13; AUC-I: 180±123 vs 135 ±65 uU*mL-1*2 h, p=0.20). In regression analyses with baseline glucose as covariate, depression symptom severity was related to BMI (p=0.02) and trended toward prediction of HOMA-IR, and AUC-I (p=0.13 and p=0.10, respectively). These preliminary data support the plausibility of the DAIR hypothesis and the need for larger and more definitive studies. Background: The Laparoscopic Adjustable Gastric Banding has been considered the most effective intervention for morbid obesity, with higher success rates associated with this procedure. However, it appears that the results are not always kept for a long period of time, confirming the results of recent studies that found failure in 20% of bariatric surgeries. Objective: This study tries to understand factors related with failures, exploring obese expectations and their understanding of their role along this process. Method: Ten morbid obese were interviewed about the way they experienced their bariatric surgery, and the reasons and meanings of weight regain. Grounded theory was used to analyze data. Results: Speech elaboration is poor, emotional words are rare and impersonal mode is frequent. Main categories suggest a psychological "fight" between the individuals and food, a fight that sometimes they win with sacrifices, but other times they loose because their lack of control. Lifestyle changes needed to accomplish the objectives are perceived ambiguously between no responsibility in the treatment and the recognition of the need to be active on the process. Conclusions: Unsuccessfulness in bariatric surgery seems to be related with defeat in the fight with food which happens in moments of greater emotionality. This could be related with a lack of other coping strategies different from this one, contributing therefore to the regain of weight. psychosocial factors have been suggested to affect eating behavior, most previous studies were either in laboratory settings or with recalled self-reports. Therefore, valid relationships between food intake and psychosocial factors in daily life have not been fully understood. Recently, ecological momentary assessment (EMA), in which subjects record their momentary symptoms using portable computers as electronic diaries, has been used as an appropriate method avoiding recall bias. In addition, we have developed and validated personal digital assistant (PDA)based food diary with food photographs. The aim of this study was to investigate the relationships between food intake and psychosocial factors in healthy people in daily life, using EMA and the PDA-based food diary. Twenty subjects (age 23.6±4.2 yo) wore a watch-type computer for a week and recorded their momentary psychological stress, mood states and situational information into it several times a day including just before and after meals. They also recorded into the PDA-based food diary everything that they ate and drank. Multilevel modeling analyses revealed that energy intake when dining out was significantly more than that in the other situations (p=0.049) after controlling for meal types and that momentary depressive mood just before the meal was negatively associated with energy intake only for dinner (p=0.045). The results suggested possible effects of situational and psychological factor on energy intake with ecological validity and nutritional accuracy. Background: This study examined in adult Pennsylvanians from 1995 to 2005 trends in the prevalence and sociodemographic distributions of diabetes; and the associations of diabetes with selected risk factors over time. Methods: BRFSS data collected between 1995 and 2005 were used for this study. Key Measures: Diabetes prevalence was assessed by self-report of physician diagnosis. Obesity was assessed by body mass index (BMI) computed from self-report of height and weight. Analyses: Statelevel data for diabetes and associated obesity prevalence from 1995 to 2005 were reported by each year. The sociodemographic factors (age, sex, race/ethnicity, income, education) and the known disease risk factor obesity were reported for 1995-2005. Logistic regression modeling was used to examine associations between diabetes, sociodemographic and disease risk factors at two time points (1995 and 2005) (Hernandez-Pozo, Cerezo, Macias, et al, 2010) . 60 men and 60 women, ages 15 to 35, participated in the study, with BMI ranging from 15.05 to 38.28; 56 were underweight-normal (G1), and 64 were overweight-obese (G2). A behavioral measure for anxiety towards body weight and eating disorders consisted of a computerized discriminative 8 minutes test based on the emotional version of the Stroop task where thematic and neutral words were used as sample stimulus. Interference was computed by subtracting latencies to neutral words from latencies to thematic words, of the same length and lexical probability. Before and after the Stroop task, blood pressure and heart rate were monitored Additional self-report measures of eating disorders were recorded, by means of the Spanish version of the EAT26, CAR and SCOFF questionnaires. Results showed a significant difference between groups for RPSE (t=6.062, p<.0001) with higher scores for the heavier group, and for Beck index (t=5.159, p<.0001). Combining sex by BMI group significant differences were recorded for all three eating disorders questionnaires, with female heavier group scoring higher on CAR and SCOFF, and male lighter group scoring higher for EAT26. The puzzling finding was that no perceptual bias differences were registered for BMI groups by themselves nor combined by sex, that is, the only differences among them were in verbal self-reports. Heavier females exhibited the highest diastolic reactivity (F=8.05, df=3, p<.0001). We concluded that there is a gap between "talking about obesity and eating disorders" and the actual unconscious bias towards those themes, which might lead to postulate progressive stages of concerns about obesity and eating disorders, described by specific combinations of behavioral, physiological and verbal data. showed greater awareness than fathers. There is a significant correlation between the change in eating habits, the frequency of food intake, the avoidance and attraction to food. The majority of the parents indicated their children's weight as appropriate, which corresponded to their BMI, but they do not recognize the body image of their children. Only 40% of parents chose the images that corresponded to their children's BMI. The remaining parents chose images ranging from thinness to obesity. The BMI of the majority of the parents and children indicate that the parents and children have appropriate weight. Conclusions: Most parents recognize the importance of nutrition and physical activity and their own role in eating behaviour and physical exercise habits for their children. We examined the accuracy of self-reported energy intake (rEI) in low-income, urban minority school-aged children at risk for obesity and associated diabetes utilizing a relatively new, simple previously published prediction equation for identifying inaccurate reports of dietary energy intake. Participants included 614 nine year old boys (51%) and girls (49%). Three 24-hour dietary recalls were collected. Children's height, weight (used to calculate BMI) and %BF were measured. Physical fitness, reported family history of diabetes, and ethnicity were also collected. A previously published prediction equation was used to determine the validity of rEIs in these children to identify under-, plausible-, and overreporters. Additionally, we examined the question of whether there is a difference in reporting by sex, ethnicity, BMI and %BF. On average, 18% of the children were at-risk of being overweight, 43% were already overweight at baseline, yet these children reported consuming fewer calories on average than recommended guidelines. Additionally, reported caloric intake in this cohort was negatively associated with BMI and % body fat. Using the previously described methods 49% of participants were identified as under-reporters, while 39% and 12% were identified as plausible-and over-reporters, respectively. On average, children reported caloric intakes that were almost 100% of pER when the sedentary category was assigned; Inactivity and excessive energy intake are important contributors to obesity. With the rising rates of obesity and diabetes in children, accurate measures of EI are needed for better understanding of the relationship between energy intake and health outcomes There is increasing international concern about diets high in fat, sugar and salt that contribute to the burden of obesity, cardiovascular disease, diabetes and cancer. Nutritional labelling, especially of processed foods, is one population level intervention designed to achieve healthier food purchasing and consumption. A range of nutritional labels varying in information and format are being used but there is currently no consensus on the most effective labelling. A systematic literature review of the impact of nutritional labelling on behaviourally assessed food purchasing and consumption is in progress (Cochrane registration in progress). Pilot literature searches have identified 13 studies assessing behavioural outcomes and preliminary analyses conducted. Eight studies assessed the impact of labelling the calorie content of restaurant menu items and found no effect on purchasing from fast food restaurants but a reduction in calories purchased from non fast food restaurants and canteens. One study found more purchases of low fat options, labelled as such, from vending machines. Two studies assessed supermarkets purchases and, although finding no overall effect of nutritional labels, different effects of different labels were found. Specifically "traffic light" labels highlighting products with high levels of less healthy nutrients had no impact on purchasing compared to unlabelled products. In contrast, while "low trans fat" and "low calorie" labels resulted in more purchases of items thus labelled, "low fat" labels resulted in fewer purchases of items thus labelled. Finally, two related studies assessed the impact of "low fat" labels on consumption finding paradoxically greater consumption of products thus labelled with greater effects in those who were overweight. These results indicate that the impact of nutritional labels on food purchasing and consumption is modified by characteristics of the individual, of the food product labelled and of the label itself. The results further suggest that the purchase of healthier options will not necessarily result in healthier consumption. It is anticipated that the results of the full review will help to elucidate which labels most effectively promote healthy consumption across populations to inform the development of policy internationally. Accumulating scientific evidence demonstrates that e-health strategies such as dietary self-monitoring through the use of handheld computers are effective in assisting individuals to lose weight. However, it is unknown whether interpersonal coaching promotes the utilization of handheld computers to self-monitor health behaviors and whether increased utilization is associated with increased weight loss. The aim of this study was to determine whether interpersonal coaching leads to increased utilization of handheld computers for self-monitoring and weight loss in lower income multi-ethnic women. Participant women, aged 43±10 yrs among whom 85% were employed, 51% had a combined family income <$40,000, 61% were married/cohabitating, 41% were African-American, 31% White, and 28% Hispanic were randomized to either: a) self-paced use of handheld system (eSolutions, n=29); or b) use of the handheld system plus weekly interpersonal coaching sessions (eSolutions-Plus, n=25). At 12 weeks, tracking frequency was significantly higher in the eSolutions-Plus (mean: 36.6 days, SD: 20.9 days) compared to the eSolutions group (mean: 26.3 days, SD: 24.5 days, p<.05; mean tracking total: 31.6 days, SD: 23.6 days, range: 0-79 days). Regardless of group assignment, higher tracking frequency was also associated with greater weight loss (r=−.45, p<.001; mean total weight loss: −3.4 lbs, SD: 6.3 lbs, range: −18-12; e-Solutions: −3.3 lbs, SD: 7.2 lbs; eSolutions plus: −3.5 lbs, SD: 5.5 lbs; p=.919). To conclude, interpersonal coaching appears to foster the utilization of handheld computers for weight loss in lower income women. An upcoming hypothesis about the origins of type II diabetes indicates that it has roots in behaviour rather than diet and metabolism. However, little efforts have been made so far to test whether there are detectable behavioural differences in type II diabetics as compared to age matched healthy controls. Ultimatum Game and its versions have been used to assess economic behaviour such as rationality or fairness in distribution of reward. Prior studies have shown that endocrine status of an individual affects the offers made as well as acceptance levels in the ultimatum game. Individuals with low testosterone levels or high serotonin levels were shown to give as well as accept low offers which is economically more rational. Since both testosterone and serotonin are key players in type II diabetes, we examined whether the offers and acceptance levels of type II diabetics are different than age matched controls. A survey of 58 patients with type II diabetes and other metabolic syndrome disorders and 60 age matched healthy controls showed that the offers given by patients with type II diabetes, hypertension and cardiovascular diseases were significantly lower than the control. BMI was poorly correlated with the economic behaviour. Further in this sample, the ultimatum game offer was a better predictor of type II diabetes than BMI. Introduction: Persistent rumination about illness is considered as the leading symptom of health anxiety and hypochondriasis. Experimentally, this increased preoccupation with illness related information is reflected in prolonged attention allocation and better recognition memory for illness related stimuli. Yet, the cognitive-psychological mechanisms that underlie these alterations in cognition in health anxiety are poorly understood. In this respect, the ability to quickly and efficiently remove non-longer relevant information from working memory might be crucial. Method: Since a dimensional perspective of health anxiety is psychometrically and empirically superior to a dichotomous view (Ferguson, 2008) , the study was based on a sample of college students (N = 90) with varying levels of health anxiety. Cognitive, affective, perceptual, and behavioral aspects of health anxiety were assessed with the Multidimensional Inventory of Hypochondriacal Traits (Longley et al., 2005) . The ability to remove illness related information from working memory was measured with the modified Sternberg task (e.g., Oberauer, 2005) . In this task, illness related words (e.g., cancer, tumor) and matched neutral words (e.g., lamp, table) served as stimuli. The EZ-diffusion model (Wagenmakers, 2009 ) was used to simultaneously analyze response speed and accuracy data. Results: The ability to remove illness related words (but not neutral words) from working memory was negatively related to the behavioral facet of health anxiety (r = -.27), i.e., higher levels of hypochondriacal behavior were associated with less efficient inhibition of illness related words. Conclusion: Behavioral and interpersonal aspects of health anxiety (i.e., seeking for reassurance and social support) were found to be associated with deficits in the ability to remove illness related information from working memory. People with higher health anxiety might use behavioral strategies to distract from ongoing cognition about illness. Alternatively, certain behavioral strategies that have been acquired during childhood and adolescence may increase the salience of health threatening information leading to difficulties in their "forgetting".
The purpose of this study was to develop the Japanese Version of Body Image Concerns Inventory (J-BICI), which is a brief selfrating scale to assess a body dysmorphic concerns. We evaluated the factor structure, reliability and validity of J-BICI. Subjects were 1218 normal undergraduate (male=709, female=518) students. They completed the J-BICI and some other related questionnaires (Dysmorphic concerns Examination; DCE, Selfrating Depression Scale; SDS, Drive for Thinness and Bulimia subscale of Eating Disorder Inventory-91; abbreviated EDI-91, Japanese 32item short version of Padua Inventory; J-PI32). Factor analysis using promax lotation method was carried out and identified three interpretable factors. These factors were as follows: safety behavior for one's own appearance flaws (factor1), avoidant behavior from one's own appearance flaws (factor2), negative evaluation for one's own appearance (factor3). These factors had high degrees of internal consistency (α=.84-.91) and good test-retest reliability (r=.79-88, p<.01; 3 weeks interval). Female participants scored significantly higher on these three factors than male ones (factor1: t(1225) = −17.69, p<.001; factor2: t(1225) = −2.23, p<.05; factor3: t(1225)=−8.60, p<.001). In addition, there were significantly correlation and discrimination between J-BICI and other related questionnaires. In this study, it was shown that J-BICI had good reliability and validity as a measure of body dysmorphic concerns. Background: To investigate non-patient irritable bowel syndrome (IBS) change to IBS and to determine factors predictive of the onset of IBS, individual biological factors, psychological factors, behavioral factors, and environmental factors were examined. Methods: The subjects were 97 non-patient IBS (male=49, female= 48, average age:22.31 ±2.32), including 62 of the diarrheapredominant type and 35 of the constipation-predominant type selected from 1,409 university and technical college students by use of a questionnaire based on the Rome II diagnostic criteria. The subjects were followed for 7 years, and various characteristics and IBS symptoms were serially observed (28 times). The IBS incidence rate was calculated. Results: During the 7 years, 61 non-patient IBS (59.17%) changed to IBS: 42 diarrhea-predominant types and 19 constipationpredominant types. All IBS symptoms disappeared in 35 nonpatient IBS subjects (33.95%). According to quantification method II (discriminant analysis), seven factors (cognitive appraisal, stressor, stress coping style, three kinds of lifestyle habits and psychologically abuse) were adopted as a predictive model for IBS incidence and were confirmed as predictive of IBS. Conclusions: The results of this research show that non-patient IBS is a changeable state that can change into IBS or persons without symptoms. Most of the non-patient IBS subjects who became asymptomatic had had symptoms for six months or less. Furthermore, the longer a non-patient IBS subject had symptoms, the higher the risk of a change to IBS became. The findings suggest the usefulness of identifying and approaching non-patient IBS as early as possible to prevent the onset of IBS. It must be noted that the persons surveyed in the present study had only the diarrhea-predominant and constipation-predominant types. Therefore, the findings of the present study are limited only these two types. Further study including the mixed type is needed. While placebo controlled trials are common in the evaluation of novel drugs, they have been questioned because of the ethical problems of withholding effective treatment in (pain) patients. As alternative, comparator controlled trials ave been proposed. Both however, differ in the likelihood of providing effective treatment: 50% and 100%, resp. Methods: We investigated whether the likelihood of receiving drug or placebo determines the size of the drug response and the size of the placebo response (DR, PR) in treatment studies of irritable bowel syndrome (IBS). Results: In 92 published randomized placebo-controlled trials with more than 22.000 patients with IBS, 16 had a drug:placebo ratio of 2:1 or greater while the remaining were powered 1:1. Computing the overall DR and PR, both responses were lower with higher likelihood of receiving the drug (ratio 2:1 or higher), but the difference between DR and PR ("therapeutic gain") decreased. Ten of the 16 studies (4 with a 2:1 ratio, 3 with 3:1, and 3 with 4:1; total n=6484) were compared to 10 studies (n=3782) of similar size (>80 patients per study arm) and design (drug trials with novel compounds) based on a standardized patient definition (according to Rome criteria), and with comparable (dichotomous) endpoints. Again, the placebo response decreased with increasing likelihood of receiving a drug from 37.6% (1:1) to 29.1% (4:1). Similarly, the drug response dropped from 49.8% (1:1) to 42.4% (4:1) (significant decrease for DR (p=.035), but not for PR). Discrimination between DR and PR was best for 1:1 trials. Correlation between the drug response and the placebo response across all 20 trials was significant (Pearsons r=.781, p<.001), but was higher in 1:1 trials (n=10; r=.91, p<.001) than in trials with a higher drug-to-placebo ratio (n=10, r=.69, p=.027). Conclusion: The higher the likelihood of receiving active treatment (a drug as compared to a placebo), the lower the response rate to both placebo and the drug in IBS, at the expense of a poorer discrimination between DR and PR. This is contrary to respective data from depression trials. (Supported by a grant from DFG, En 50/25). Background: Much of public health research focuses on individual mechanisms of health (e.g., behavior, genetics), while few studies look at broader contexts of health behavior such as dyadic influences. Romantic partners and relationship factors can influence one's health and well being above and beyond individual factors. Methods: Preliminary analyses were conducted among 200 couples (N=400) from low income OB-GYN clinics in the U.S. Women and men were 18.7 and 21.3 years on average, and the average relationship length was 2.3 years. 77% of participants were Black or Hispanic. We assessed relationship satisfaction, mental health and physical health for both members of a couple. Dyadic analyses using multiple regression assessed actor and partner effects for both men and women. All analyses controlled for confounders. Results: Men and women shared predictors of relationship satisfaction, including greater relationship equity (Bm =.28, Bw=.44, ps<.05), less avoidant attachment (Bm=−.32, Bw=−.22, ps<.05), greater feelings of love (Bm=.31, Bw=.14, ps<.05), and less partner anxiety attachment (Bm=−.14, Bw=−.14, ps<.05). Among both men and women, relationship satisfaction related to less depression (Bm=−.38, Bw=−.52, ps<.05), less stress ps<.05) , and greater mental quality of life (Bm=.39, Bw=.37, ps<.05) . Relationship satisfaction related to greater physical quality of life for men (B=.18, p<.05), but not for women (B=.08, p = ns). Comparisons showed a stronger association between relationship satisfaction and depression for women (B=−.52 vs. −.38, p<.05 ) and physical quality of life for men (B=.18 vs. .08, p<.05) . No partner effects were significant for mental and physical outcomes. Conclusion: Relationship satisfaction was associated with better mental health outcomes for men and women, and better physical health outcomes for men. While relationship satisfaction had a greater influence on depression among women, it had a greater influence on the physical quality of life for men, suggesting that relationship factors may play a different role in mental and physical functioning across gender. Fear of childbirth in pregnant women has been associated with prior and future complicated deliveries, difficulties in the motherinfant relationship and risk of postpartum depression. The aim of this study was to investigate pre-and postpartum levels of childbirth fear in a cohort of pregnant women and examine factors associated with elevated fear. Three hundred sixty-one pregnant women (mean age=32, SD=4.5) completed questionnaires measuring: anxiety sensitivity, state-anxiety, fatigue, depression, social support, life events and parental self-efficacy in the third trimester of pregnancy. The Wijma Delivery Expectancy/Experience Questionnaire (W-DEQ) was used to assess childbirth fear at the preand postpartum assessment. Two-hundred sixty-three women returned their questionnaires at the 4-6 weeks postpartum assessment. Demographic variables (i.e. parity, education) and complications in the current pregnancy or in previous deliveries were also collected. Low levels of childbirth fear during pregnancy was reported by 25.8% (n=93) of the women, with 48.8% (n=176) moderately fearful and 25.5% (n=92) highly fearful. At the postpartum assessment 37.6% (n=99) reported low levels of fear, 44.1% (n=116) reported moderate fear levels and 18.3% (n=48) were highly fearful. Overall, fear levels in the postpartum decreased significantly by a mean of 6 points compared to the prepartum assessment (t (262) = 4.5, p<.001). The regression model examining prepartum childbirth fear showed that lower parental self-efficacy (p<0.001), higher state-anxiety (p<0.001), more life events (p=0.001) and greater anxiety sensitivity (p=0.003) were significant independent predictors of higher prepartum W-DEQ, explaining 44% of the variance. In addition to prepartum WEQ scores (p <0.001), postpartum childbirth fear was associated with greater anxiety sensitivity (p=0.001), lower parental self-efficacy (p=0.005) and delivery by caesarean section (p=0.014), together explaining 36% of the variance. Our findings underscore the importance of routine assessment of childbirth fear during pregnancy and highlights areas which can be targeted in order to manage childbirth fear. Women expressing fears may benefit from interventions aimed at improving self-efficacy and reducing stress to enhance the pregnancy and childbirth experience. Introduction: Recent studies show Female Sexual Dysfunction can be caused by an overemphasis on secondary self-analysis rather than direct experience of arousal. Training in mindfulness has been associated with decreases in self-judgment and enhanced body awareness. This controlled study assesses the impact of mindfulness training on reaction time for ratings of arousal to erotic stimuli and related psychological barriers to female sexual function. Methods: Fifty-six college students (female=29) participated in either a 15-week course with mindfulness "meditation labs" in addition to didactic class time (n=35) or an active control class (n=21) with a similar format (didactic and experiential laboratory sessions). All participants engaged in computerized neuropsychological performance batteries pre-and post-intervention where they rated erotic and non-erotic photographs in terms of arousal and valence. Scores and reaction times of ratings were recorded. Participants also completed the Five Factors of Mindfulness Questionnaire (Baer et al., 2006) , Mindful Attention Awareness Scale (Brown et al., 2003) , the Brief Symptom Inventory (Derogatis et al., 1983 ) and The Self Compassion Scale (Neff, 2003) at the beginning and end of the semester. Results: At baseline, female participants had a significantly slower reaction time in response to erotic photographs than males, t (52) = 2.5, p=.013. At post-intervention testing, females who participated in the meditation labs significantly faster reaction times, t (12) = 2.8, p=.016. This change was more than twice that of females in the control group (723.0 vs. 306.7 milliseconds). Scores related to selfjudgment also decreased significantly more in female meditators than controls, F (1,15)=4.8, p=.045. The faster reaction times were correlated with this decrease in self-judgment (r=.70, p=.012 ) as well as an increase in self-kindness (r=.84, p=.001). Additionally, female meditators showed improved scores in mindfulness, depression, anxiety, and combined clinical symptoms measures (all p<.05), but controls did not. Conclusions: These results suggest that mindfulness training is associated with improvements in many of the psychological barriers to female desire, arousal, and satisfaction. Most notably, it was associated with faster reaction times when reporting arousal that correlated with decreases in self-judgment. Female meditators were ostensibly enabled to react to erotic stimuli with decreased judgment and self-evaluation, thus becoming more engaged in the direct experience of their own arousal. These data strongly support mindfulness as a promising treatment for FSD. Purpose: The primary aim of this study was to evaluate the association between key psychosocial factors and aberrant crypt foci (ACF), a measure of colonic mucosa and perhaps a putative biomarker of colon cancer risk. We hypothesized that higher levels of depression, stress, and colon cancer worry (and lower levels of social support) would be associated with greater numbers of ACF among individuals at heightened risk for colon cancer. Sample and Methods: Participants were self-referred or referred by physicians from the Gastroenterology Clinic or Colon Cancer Prevention Program at the University of Connecticut Health Center for routine colonoscopy. A total of 93 individuals were consented (51% women; 49% men). Within two weeks prior to colonoscopy, participants completed measures assessing the psychosocial factors noted above. Individuals were also examined for ACF detection at colonoscopy. Regression beta weights were used to examine the association between the psychosocial variables and ACF. Results: Among women, higher levels of depression were associated with greater numbers of ACF (β=.731, p=.032) after controlling for age, race/ethnicity, stress, social support, and colon cancer worry. Among men, lower levels of social support were associated with greater numbers of ACF (β=-.398, p=.050) after controlling for age and race/ethnicity. These results suggest that although colon cancer affects men and women equally with regards to morbidity and mortality rates, there are important gender differences in how psychosocial factors impact colonic mucosa and perhaps colon cancer risk. Psychosocial interventions aimed at targeting these types of factors are warranted but need to consider the role of gender. Background: Gender differences in premature mortality rates and in the size of socioeconomic inequities in mortality vary across countries. We aimed to quantify the gender-socioeconomic status (SES) interaction on premature mortality and to analyse which psychosocial characteristics might explain the different gender patterns of socioeconomic inequalities in premature mortality in this respect in Hungary. Methods: Men (n=1130) and women (n=1529), aged 40-69 years, participants in the Hungarian Epidemiological Panel (2002) were followed for 3.5 years for total mortality. Cox-proportional hazard models were used to evaluate the association between several socioeconomic measures and death. Results: During the follow-up 99 men (8.8%) and 53 women (3.5%) died. The age-adjusted hazard ratios and the Rothman's synergy indexes showed that socioeconomic factors (education, blue collar work, personal and family income, subjective social status, lack of car and computer) were more deleterious in men compared to women. Adjustment for severe depression resulted in the highest decrease in the regression coefficients for the association between socioeconomic factors and male premature mortality. There was no indication that depression would mediate between low SES and mortality in women. Work stress measures might partly mediate the effect of socioeconomic deprivation on male mortality, while social support seems to counterbalance the negative aspects of socioeconomic deprivation among women. Conclusions: Middle-aged Hungarian men seem to be considerably more vulnerable to the chronic stress of material deprivation than women. This effect modification by gender might partly be explained by a stronger connection between low SES and depressive symptoms in men. While the effects on body image of unrealistically thin female fashion models have been extensively investigated, there is very little research on the effects of unrealistically muscular male models. We examined the impact of average-size and muscular male fashion models on men's and women's body image, and on perceived advertising effectiveness. A sample of 619 Australian university students (330 men and 289 women, aged 17-25 years) were systematically assigned to four image exposure conditions: no models, muscular models, average-slim models, and averagelarge models. In each condition, participants viewed eight glamour advertisements created for the purpose of the study, rated the effectiveness of each advertisement, and then completed standardised measures of body image state. Advertisements were matched across conditions for product and general style. Men and women rated average-size male models as equally effective in advertisements as muscular models. Amongst the men, those exposed to average-size models had more positive body image than those viewing no models, with no difference from those viewing muscular models. Similar results were found for women. These findings suggest that average-size male models can promote positive body image and appeal to consumers. Background: Mental health problems are often found to be more common among women than among men. We examined physical working conditions, psychosocial working conditions and familyrelated factors as explanations for gender differences in the use of psychoactive medication in an ageing employee cohort. Methods: Data on the explanatory factors were derived from mail surveys conducted in 2000-2002 among municipal employees of the City of Helsinki aged 40-60 (N=8960, response rate 67%). These data were linked with the prescription register of the Social Insurance Institution of Finland. Cox regression analysis was used to calculate hazard ratios (HR) with 95% confidence intervals (95% CI) for women as compared to men for the use of all psychoactive drugs (ATC codes in classes N05 and N06, excluding medication for dementia), antidepressants (N06A) and sleeping pills and sedatives (N05B and N05C) among younger (40-50 y) and older (>50 y employees) during three subsequent years. Results: Among younger women the risk for using psychoactive drugs was only slightly larger than in men (HR 1.24, 95% CI 1.01-1.54). Adjusting for physical working conditions or psychosocial working conditions somewhat increased the difference.
There was no association for gender with antidepressants or sleeping pills among the younger. Among the older, the HR for psychoactive drugs among women was 1.70 (95% CI 1.28-2.27), and it slightly increased after adjustment for physical working conditions. For antidepressant use, the female excess (HR 1.57, 95% CI 1.12-2.22) was slightly increased after adjusting for physical working conditions. The higher HR for the use of sleeping pills among women 2.28 (95% CI 1.50-3.47) was slightly increased after the adjustment for physical working conditions and slightly attenuated by adjustment for familyrelated factors. Discussion: The female excess in the use of psychoactive drugs was larger in the older than in the younger employees. Physical working conditions, psychosocial working conditions and family related factors in general did not explain the differences. On the contrary, adjusting for physical working conditions rather tended to increase the difference. Background: The importance of conducting genetic biobanking research about genetic factors that influence major chronic diseases is critical in disease management. Participants eligible for this line of research may not be aware of genetic biobanking research and reasons to participate. This disconnect between potential eligible participants and the importance of genetic biobank research may hinder progress in disease management. Qualitative studies are needed to identify factors that influence a multi-ethnic sample to participate in genetic biobanking research. Purpose: Project Diversity GATHER (Group discussions Around Themes on HEalthful genetic Research) examines health behaviors and factors that influence women's and men's participation in genetic biobanking research. A multi-ethnic sample was recruited to identify factors influencing research participation. Methodology: 85 women and 50 men from Texas completed an 89-item sociodemographic questionnaire and 60-minute non-ethnically or gendered matched focus group. Wegner's translation process model (1993) was used to indentify intra-and inter-cultural themes that influence participants' participation in research. Participants' characteristics were analyzed through descriptive analyses and qualitative themes categorized on 3 key focus group questions:1) research perceptions, 2) facilitating factors, and 3) barriers to The circadian clock is a self-sustained time-keeping system, which controls rhythmic behavioral, biochemical and physiological processes. Vital functions such as the timing of cell cycles or apoptosis depend on this intrinsic rhythm. In mammals, the cogwheels of this clock are contained in every single cell. These so-called clock genes control their own expression and the expression of clock-controlled genes via several feedback loops. One of these genes is Per1, a clock gene that disposes of a glucocorticoid-responsive element and is therefore most likely influenced by glucocorticoids. In humans, stress is associated with an increase in the glucocorticoid cortisol. Simultaneously, psychosocial stress is seen as a major factor in the etiology of numerous mental health problems. For this reason, we aimed to investigate first, the association of cortisol and hPER1 as well as hPER2, and second, the putative cortisol-mediated influence of acute and chronic psychosocial stress on the gene expression of the same two clock genes in thirty-one healthy men. Our findings suggest that first, cortisol strongly interacts with hPER1 and hPER2, and second, psychosocial stress influences the expression of hPER1 and hPER2. These results implicate a strong clinical relevance of stress on the human circadian rhythm. Most measures of motivation to seek genetic susceptibility testing were developed for the context of predictive genetic testing where mutations confer high risk for disease occurrence. It is unclear whether these measures will capture motives for seeking genetic susceptibility testing where gene variants are associated with small increases in disease risk. In a factorial study examining methods to convey low genetic risk for lung cancer to young smokers, we evaluated a 20-item measure of motives for or against genetic testing in 128 college smokers. Items were adapted from those used in other scales and unique items were generated by a panel of experts to assess the following domains: health behavior motivation/demotivation, altruism, uncertainty reduction, impact on perceived risk and worry, and curiosity about oneself and science. Eight items were correlated significantly with intentions to get tested (p<.05). Exploratory factor analysis of these eight items yielded two correlated factors (r =.22, p <.01): maintaining personal health and curiosity about self and technology. These items formed a reliable scale (alpha=.70) that correlated with testing intentions (r=.63, p<.001). We also asked participants open-ended questions to assess additional reasons for and against testing. Overall, themes paralleled those in our quantitative survey, though participants offered the novel point that they would not want to be tested because the awareness that would come from testing would lead to less enjoyment of life. Prior scales used to assess motivations for testing did not include the construct of curiosity about oneself and technology. This may be unique to intentions to seek new susceptibility tests. Understanding the role of curiosity in test uptake could be informative for this and other novel technologies where health utility has yet to be established. 1 Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 2 Social and Environmental Medicine, Kanazawa Medical University, Uchinada, Japan; 3 Faculty of Healthcare, Tokyo Healthcare Universit, Tokyo, Japan and 4 Ishikawa Health Service Association, Kanazawa, Japan.
Aims: A previous studies have reported that the long variant of the serotonin transporter-linked promoter region (5-HTTLPR) polymorphism is associated with problem solving style of stress coping among males (Wilhelm et al. 2007 ). However, the findings do not have been replicated. In addition, socioeconomic status, such as education, might interact with the genotype to determine stress coping style. This cross-sectional study aimed to examine the interactive effect of education and the 5-HTTLPR polymorphism on coping style in a sample of Japanese workers. Methods: A total of 241 (80%) out of all employees of a manufacturing factory in Japan completed a mailed questionnaire which included a scale of stress coping, the Brief Scale for Coping Profile (BSCP) (Kageyama et al., 2004) . Genomic DNA abstracted from leukocyte in their peripheral venous blood samples. The 5-HTTLPR polymorphism region was amplified using polymerase chain reaction (PCR), and classified into short/short (s/s) (n=153), short/long (s/l) (n=¬78), and long/long (l/l) (n=6), excluding those who had extra long variants (n=4). The respondents were also classified into high school graduates and university graduates. Analyses of covariance (ANCOVA) were applied to compare six subscales of BSCP among the three 5-HTTLPR groups, as well as test the interaction of the polymorphism and education, separately for men and women. Results: Compared to the s/s group, the s/l and l/l groups had a higher score of the subscale of "seeking help for solution" among male university graduates (p for trend=0.01); Subscale scores of "changing mood" were significantly different among three polymorphism groups, with a low score in the l/l group (p< 0.01). The interaction between the polymorphism and education was significant for the subscales of "seeking help for solution" and "changing mood" among men (p=0.08 and p=0.01, respectively). Discussion: The study replicated that long variant of 5-HTTLPR polymorphism was associated with problem solving (measured as "seeking help for solution" in this study) among men, while the l/ l group seems less likely to use "changing mood" coping. 5-HTTLPR polymorphism, education, and sex seem to interact to determine the problem solving stress coping. Int.J. Behav. Med. (2010) Background: Sociotropic cognition is a set of beliefs associated with social dependency and a high need for approval. Earlier studies with women found that high sociotropic cognition was associated with increased cardiovascular reactivity and sexual risk behavior. However, few studies have included men and the influence of sociotropy on other health risk behaviors has not been evaluated. Purpose: To examine the influence of need for approval, measured by sociotropic cognition, on health risk behaviors, including sexual risk, alcohol, drug, and tobacco use, and disordered eating in young men and women. Method: Participants were 236 college students who completed self-report measures of sociotropic cognition and health risk behaviors. It was hypothesized that higher sociotropic cognition would be associated with more alcohol, drug, and tobacco use, sexual risk behavior, and disordered eating. Multiple regression was used to examine these relationships. Results: Sociotropic cognition predicted sex-related alcohol expectancies (β=.16, t(201) = 2.24, p<.05) and negative consequences from drinking (β=.14, t(209) = 2.11, p<.05). Sociotropic cognition was associated with being pressured into having sex (t(227) = 2.19, p<.05), but not to recent intercourse or condom use. Sociotropic cognition predicted recent amphetamine (β=.21, t(229) = 3.26, p<.001) and inhalant use (β=.14, t(229) = 2.20, p<.05), but was negatively associated with alcohol (β=− .13, t(229) = -2.01, p<.05) and marijuana use (β=−.14, t(229) = -2.09, p<.05). There was no association with tobacco use. Sociotropic cognition predicted more body dissatisfaction (β=.32, t(225) = 5.09, p<.001) and more disordered eating behavior (β=.25, t(210) = 3.81, p<.001), and was not associated with BMI or physical activity. Conclusions: Sociotropic cognition appears to be an important correlate of interrelated health risk behaviors for men and women. Need for approval influences disordered eating behavior, drug use, problems with alcohol use, and some sexual risk behaviors. Cognitive and behavioral intervention strategies may be useful to address need for approval and its effect on these health risk behaviors. A meditational model was hypothesized. The three constructs of interest were significantly correlated (BMI and exercise avoidance, .47***; BMI and weight stigma, .50***; weight stigma and exercise avoidance, .51***). The primary model examined hypothesized that weight stigma would mediate the relationship between exercise avoidance and BMI. The model testing the relationship between the IV (BMI) and the DV (exercise avoidance) fit the data well. The models testing the relationship between the mediator (weight stigma) and the DV, and the relationship between the IV and the mediator, also fit the data well. The final model testing weight stigma's meditational role between BMI and exercise avoidance, found that weight stigma completely mediated the relationship between the IV and DV. The results indicated that as BMI increased exercise avoidance increased. Similar to other studies with college females, as BMI increased perceptions of weight stigma also increased. Not only were BMI, exercise avoidance, and weight stigma all related in college females, but weight stigma mediated the relationship between BMI and exercise avoidance, indicating that weight stigma may predict adverse health behaviors. Alternative models and implications will be discussed. Previous studies examining social determinants of health have found that individuals who tend to discount the future are more likely to engage in negative health behaviors such as smoking and substance use. Due to high rates and detrimental health consequences of negative health behaviors, it is important to identify factors influencing discounting rates. Personality dimensions and depressive symptoms have been identified as potential predictors of discounting rates, but past studies have utilized monetary discounting measures when examining health outcomes rather than health discounting measures. This study examined personal-ity and affective predictors of health discounting in a sample of 97 undergraduate students (41 women) who completed the following questionnaires as participants in a study for course credit: (1) a hypothetical health choice task (Chapman, 1996) , (2) Many studies have indicated that many people who experience psychological and interpersonal problems don't seek any help. Many researchers have tried to understand the reasons underlying people's decisions to seek or not to seek help. In these studies, measures of help-seeking intentions have been often used instead of measuring actual help-seeking behavior, because it is assumed that help-seeking intentions associate with actual help-seeking behavior. However, there is little evidence of this association. The purpose of this study is to examine whether help-seeking intentions can predict actual help-seeking behavior among Japanese university students. One hundred and eighty-five Japanese university students answered a questionnaire which included social support and help-seeking intentions (Time1). Four weeks later (Time2), participants answered another questionnaire which included psychological distress and actual help-seeking behavior. The correlation between help-seeking intentions at Time1 and actual help-seeking behavior at Time2 was moderate (r=.65 p<.01). The result of multiple regression analysis indicated that help-seeking intentions associated with actual help-seeking behavior (β=.42 p<.01). Social support at Time1 (β=.18 p<.05) and psychological distress at Time2 (β=.44 p<.01) also associated with actual help-seeking behavior. These variables accounted for 57.2% of the variance in actual help-seeking behavior. Thus, it was revealed that help-seeking intentions can predict actual helpseeking behavior among Japanese university students. Background: Suffering from infertility may cause significant stress both on a personal level as well as in the relationship. Expressive writing intervention (EWI) has been shown to be an efficient method for reducing psychological symptoms in both healthy and clinical populations. However, research using this method with infertile couples is limited. Our aim was to investigate the effects of EWI on infertility-related psychological symptoms in a small preliminary sample of patients undergoing In-Vitro Fertilization (IVF). Methods: 37 couples were included and 8 women were included without their partners (N=82). Participants were randomized to home-based EWI or a neutral-writing control group. Both groups completed repeated questionnaires measuring infertility-related stress, perceived relationship quality, and dispositional anxiety. Data were collected before the intervention, 3 weeks after the intervention and 6 weeks after the intervention. Despite strong intentions to improve their diet or engage in regular exercise, individuals frequently fail to achieve these health goals. According to Temporal Self-Regulation Theory (Hall & Fong, 2007) , executive function (including working memory and inhibitory control capacities) and prepotent behaviour (e.g., habits) may influence self-regulation abilities. The aim of this study was to replicate and extend initial evidence (Hall, Fong, Epp, & Elias, 2008) that, within the domains of diet and exercise, executive function moderates the intention-behaviour relationship. At baseline, university students and staff (N=123; ages 17-73; 33 men) completed an nback task (working memory), a Go/NoGo task (inhibitory control), and measures of dichotomous thinking and weight-cycling. They completed weekly measures of diet and exercise intentions and behaviours (including a modified version of the US National Cancer Institute Fruit and Vegetable screener over three weeks. Diet behaviours included eating five fruit and vegetables a day, low-fat foods, low-sugar foods, and a reduced-calorie diet. Regression analyses revealed interaction effects of executive function and intentions on reduced-calorie diet at Time 2 (β=.14, p<.05), and on fruit and vegetable intake and low-sugar intake at Times 2 and 3 (β's >.18, p's<.05). Intentions were more predictive of behaviour when executive functions was high than when it was low. Weight-cyclers (compared to those with no history of weight cycling) reported a higher dichotomous thinking style (t=2.09, p<.04) and exhibited lower levels of inhibitory control (t=2.14, p<.04). The findings support hypotheses that health goal achievement is influenced by self-regulation abilities with a significant biological basis. Research is needed to identify ways to increase these self-regulation capacities or develop alternative techniques for enabling individuals with low executive function to fulfil their health behaviour intentions. Background: Childhood sexual abuse has been associated with sexual risk behavior and alcohol abuse in adults. However, the relationship of other forms of childhood abuse and neglect to these health risk behaviors has been less studied. Aim: The purpose of this study was to examine the influence of childhood abuse on theoretical antecedents of health risk-taking, sexual risk behavior, and alcohol use. Methods: Participants (N=184) were recruited from a mid-sized northeast university and completed self-report questionnaires to assess childhood experiences with abuse and neglect. Outcome measures included alcohol use, drinking consequences, and sexual risk behavior. Theoretical antecedents to sexual risk behavior also were assessed, including attitudes, norms, self-efficacy, perceived behavioral control, and intentions to use condoms. Data were analyzed with hierarchical multiple regression. Tobacco use and physical inactivity are the leading causes of preventable death in the US, and persistent smokers tend to remain inactive. Most smokers who quit relapse within three months, suggesting that mainstream cessation assistance options do not work well for many smokers. Interventions using sustained, aerobic exercise as a smoking cessation technique have resulted in success rates comparable to conventional cessation methods. No trial to date has prescribed intermittent bouts of brief exercise in response to nicotine cravings, despite evidence suggesting its potential efficacy as a cessation tool. This randomized controlled trial attempts to replace smoking with exercise when cravings arise among generally healthy, inactive adult smokers who desire to quit. The first wave of participants (N=26; mean age 42.4[12.2] years; 50% female) were randomly assigned to a standard care Internet group (n=13) or an Internet+ exercise group (n=13) for the three-month intervention phase. The attrition rate (34.6%) was comparable to other smoking cessation trials and did not differ between groups. Self-reported quit rates among retained (29.4%) and total enrolled (19.2%) participants were higher than and comparable to, respectively, those attained by trials using traditional quit assist methods such as nicotine replacement therapy. Reduction rates among retained (49%) and total enrolled (37.4%) participants were substantial. Quit rates did not differ between groups, but reduction rates were twice as high among participants in the control (52%) vs. intervention (26%) group. From baseline to post-intervention, mean FTND scores ( Preliminary results from the first wave of participants in this lowcost, web-based trial are comparable to more resource-and timeconsuming cessation programs, suggesting that future iterations of this program may be quite promising. Further analyses will examine the potential roles of exercise adherence, selfmonitoring adherence, and self-efficacy, among other variables, in the smoking cessation and reduction rates associated with this program. With growing number of people diagnosed with diabetes, there is a lack of diabetic control among people with diabetes in Malaysia and we need to understand why this is. This study set out to explore the perspectives of patients' management of Type 2 diabetes and provide recommendations that aim to enhance adherence to treatment and help patients to improve their selfmanagement skills. In-depth interviews were carried out on a purposeful sample of patients and their health care professional (HCPs). Interviews were recorded, transcribed and audiotapes were analysed using NVivo software to identify emerging themes and code according to categories. Interviews were conducted in an Endocrinology clinic in Malaysia with 18 Malay patients (15-75 years, 9 males and 9 females) and 13 HCPs. Themes that emerged from interviews with patients' were problems with integrating the treatment regimen and difficulty developing coping skills to achieve the desired blood glucose level. Patients lacked knowledge about their medicines, dietary control, medicine taking, complexities of treatment, and side effects of medications. They needed to integrate many treatment requirements such as diet, medications, blood glucose monitoring and exercise into their daily routine posed problems. Other barriers such as ethnicity, beliefs (culture and financial) proved to be constraints in their ability to good control diabetes. Education and knowledge related to diabetes that influenced understanding of the disease were also reasons for non-adherence to treatment regimen. According to pharmacists, patients' ethnicity and education level played a substantial role in their attitudes toward diabetes management. Their beliefs, cultural and social needs shaped their attitudes in controlling or adherence to treatment management. Patients were willing to discuss their problems about self-managing diabetes if these barriers were addressed during consultations with the pharmacists. More positive approaches are needed in self-management of diabetes and health care professionals involved in the management of diabetes need to understand their patients' beliefs about their diabetes and their constraints to promote more awareness and to foster greater control of diabetes and improve health outcomes. Most major chronic diseases are caused by more than one behavioral risk factor and risk factors additively contribute to morbidity and mortality. Risk behaviors also tend to co-occur, but data on the prevalence of co-occurring mental and behavioral risk factors are scarce. Most prevalence reports focus on single risk factors. Better understanding the co-occurrence of risk factors, however, could help inform the feasibility and design of multiple risk factor (MRF) interventions, which potentially can be more cost-effective than delivering interventions targeted at single behaviors. We report on the co-occurrence of three primary disease risk factors (depression, low physical activity, and smoking) in a representative health plan population (n=66,901) in the U.S. Pacific Northwest. Data was collected during an online health risk assessment offered to all health plan members. Twothirds of respondents (66.6%) were female and 86% were white. Mean age was 47 years. Approximately 9% reported current smoking, 8.2% were moderate to severely depressed based on the PHQ-8, and 24.5% reported low physical activity per the IPAQ. While 33.1% of smokers had low physical activity and 18.8% reported significant depressive symptoms, the overall prevalence of all three behaviors was much lower. As a proportion of the entire sample, <2% (n=1089) met criteria for smoking and depression; 2.5% (n=1695) met criteria for smoking and low physical activity; and 3.2% (n=2137) met criteria for depression and low physical activity. Fewer (0.6%; n=405) met criteria for all three behaviors. The results highlight a challenge of MRF interventions. While many people report single risk factors, far fewer meet criteria for MRF's, even for common risk factors such as smoking, depression, and inactivity. Given this, MRF interventions may need to be easily disseminable across large populations in order to be cost-effective and have maximum impact. Implications for MRF treatment design will also be discussed. Introduction: The theoretical framework of Raghubir and Menon (2005) suggests that intention to behave in the future will be more favourable when individuals recall positive experiences and these experiences are easy to retrieve (such as when experiences occur on a regular basis). The aim of this study is to verify if perceived regularity in the adoption of behaviour adds to the prediction of intention, taking into account the constructs of the theory of planned behaviour (Ajzen, 1991) and constructs of other relevant social cognitive theories that aim to explain intention. Method: This is a descriptive correlational design in which 385 college students in Quebec (Canada) volunteered to complete a self-administered questionnaire measuring psychosocial constructs (intention, attitude, subjective norm, descriptive norm, perceived behavioural control, self-efficacy) regarding the consumption of at least five portions of vegetables and fruit every day during the next three months. Consumption of vegetables and fruit in the last seven days and the regularity of this consumption were also measured. Objectives: We previously showed that a single intervention session had large effects on the objectively assessed walking behavior of N=130 English adult volunteers (d=0.90), mediated by self-efficacy. The present research aims to establish (a) whether this effect is generalisable (when delivered by a different person (namely the second author), and (b) whether either of the motivational or volitional components of the intervention are alone sufficient to alter walking behaviour, or whether both components are required. Methods: Adult volunteers (n=35) randomly received one of three interventions: (i) a "combined" intervention containing motivational and volitional components in the same session, (ii) a "motivation first" intervention, where the motivational components were followed by volitional components a week later, and (iii) a "volition first" intervention, where the volitional components were followed by the motivational components a week later. Results: The "combined" intervention produced a large (d=1.06) and significant (p=0.036) increase in walking behavior, in contrast to both other interventions (time x groups interaction, p=0.003).
The "combined" intervention also produced a significant increase in self-efficacy, relative to the two other interventions.
Conclusions: This demonstrates generalisability of previous large intervention effects and suggests that neither the motivational nor volitional components appear to work effectively in isolation. Behavioral medicine , it is said, as Ebbinghous stated about psychology (( has along past but short history)). Behavioral medicine has been defined as interdisciplinary field that integrates behavioral & medical knowledge and applies that knowledge to health & disease. In Libya behavioral medicine is a pretty unknown field of knowledge and as has been defined here is not exist due to many and multiple factors, therefore it is not surprising that you would not find a teaching curriculum in medical schools for psychology or behavioral medicine at any university in Libya before 1988, we can say that the teaching of behavioral sciences started in 1988 in the faculty of medicine in Tripoli, University of Al-Fatah as apart of the community medicine course and is taught in the fourth year. in the faculty of medicine in Benghazi ,psychology was part of pre medical curriculum but behavioral medicine taught in the 4th year as apart of community medicine course. In this paper. the author highlight the historical development of behavioral medicine and behavioral sciences in Libyan medical schools and suggestion to include behavior medicine in the Libyan medical curriculum which should be a compulsory and not an optional subject. The main objective and the primary massage of this paper is to emphasize that modern and successful physicians needs two types of knowledge To date, there has been no shared language for describing the content, especially the 'active ingredients' of behavior change interventions; by contrast, biomedical interventions are precisely specified. This limits the possibility of replicating effective interventions, synthesising evidence, and understanding the causal mechanisms underlying behavior change. Our ultimate aim is to develop a reliable method of specifying behavior change techniques (BCTs), link them to relevant theory and specify the behaviors necessary to implement them. In several stages, we have generated lists of BCT labels based on a) systematic reviews of behavior change interventions b) systematic text-book search c) expert brainstorming. We have generated definitions a) from 9 textbooks, b) from dictionaries (including APA Dictionary of Psychology) and c) by reframing to specify the behavioral competencies required of the person implementing the BCT. Introduction: Non-communicable diseases are increasing in developing countries, exacerbated by growing urbanization. Contributory factors include limited access to affordable healthy food, readily available cheap unhealthy foods and limited scope for physical activity. Also significant are cultural beliefs and practices. This study examined the experiences and perceptions of people migrating from rural to urban areas in Cape Town, South Africa, and the impact these have had on the risk factors of noncommunicable diseases. Methods: The setting is an impoverished peri-urban township with a non-communicable disease prevention program that includes weekly health clubs. The program was initiated by a local university that also undertook the research. A qualitative approach was adopted, using a purposive sample of club members and nonclub members. Data was collected through in-depth interviews, participatory reflection and focus group discussions. Results: Significant changes in respondents' eating patterns and levels of physical activity were described, with accompanying increase in obesity. This was due to both socio-economic and environmental constraints. However, the respondents were not concerned about these changes. Despite many hardships, they were pleased with their new urban lifestyle. Furthermore, they approved of their weight gain as culturally it signified dignity and respect. Those attending health clubs found them both informative and socially and emotionally supportive. Conclusion: The study highlighted the complexity of the risk factors of non-communicable diseases, and the importance of developing prevention strategies that extend beyond the traditional lifestyle approach that focus on diet and exercise. , 1995) , and depressive symptoms among Japanese college students was examined. SC was defined as a function of both the number of self-aspects and the degree to which the self-aspects are differentiated from each other. Linville (1987) has suggested that high self-complexity serves as a buffer against the onset of depressive symptoms following negative events because it partly determines the impact of such events on thoughts and feelings about various self-aspects. Participants were randomly assigned to either the intervention group (n=20) or the control group (n=20). The intervention group received one session of psycho-education on SC and a paper-and-pencil exercise designed to calculate the number of their self-aspects (social roles, interpersonal relationships, activities, traits, etc.) and to evaluate each self-aspect using an adjective checklist. After the education and exercise, daily records of their self-aspects were assigned for one week. Selfreported questionnaires were completed three times in both groups (pre, post, and follow-up). The questionnaire was comprised of the Center for Epidemiologic Studies Depression Scale, the Positive and Negative Affect Schedule, and the Rosenberg Self-Esteem Scale. SC, P-SC, and N-SC, which were assessed using H, a statistical measure of dispersion that represents number of independent dimensions underlying a given trait-sorting, were measured by Linville's (1987) and Sato's (1999) trait sorting task. The Research Ethics Committee of the Hiroshima University Graduate School of Education reviewed and approved the study protocol. In the intervention group, the analysis of variance indicated that P-SC was significantly increased at the post-test and marginally increased at the follow-up test when compared to the pre-test. No significant differences in P-SC were found in the control group. Structured equation modeling revealed that in the intervention group, high P-SC at the post-test was related to lower levels of a depressed mood at the post-test and follow-up test. Results demonstrate that this program increases P-SC, which might ameliorate subsequent depressed mood among college students. Results: Based on the short questionnaire, the main lifestyle problems identified were: insufficient levels of leisure time physical activity, sedentary behaviour, and insufficient intake of fruit and vegetables.
Using information from both literature and FG interviews, vitality appears to consist of a mental and a physical component. The interviewees suggested to improve the mental component of vitality by means of relaxation exercises (eg. yoga); physical vitality could be improved by aerobic exercises involving aerobic endurance, strength and flexibility. Based on the information mentioned above, the intervention will consist of three visits to a Personal Vitality Coach combined with a Vitality Exercise Programme, consisting of supervised yoga and an aerobic workout, each once per week, and lasting 45 minutes. Moreover, fruit will be offered at the workplace to meet the recommended intake of fruit. Conclusion: The lifestyle intervention will be evaluated in a RCT among 698 older workers of two major academic hospitals in the Netherlands. The aim of our research is to study the psychical health state of the Romanian adults in three counties placed in the middle part of the country. We also have followed the lifestyle and the role of some psychological and social factors. The study is based on a complex questionnaire, which was completed by a representative sample among adult population. After our results depression is the most common psychical illness with 9,1% diagnosis, but in much higher rate exists depressive symptoms. According to Beck score, it was established a significant difference in the favour of female, elderly people, lower educational level, urban population. The percentage of suicide attempt among the studied population was 2,9%. According to WHO Positive life quality test, in two-third majority the adult population consider that it is psychically healthy. In our study it is underlined the role of religious feelings and the importance of health promotion and education to improve the psychical living standards of the population. Malaria is an excellent exemplar for a risk reduction intervention due to geographic fluctuations in its transmission range, lifethreatening consequences, and the vital need for adherence to personal protection measures. This study compared three versions of a web-based game targeting the malaria risk reduction decisionmaking of study abroad students. This population is in jeopardy due to the rapid expansion of programs in malaria endemic regions and the low level of acquired immunity and malaria knowledge among US citizens. Constructs from the self-regulation model and the cognitive theory of multimedia learning informed the creation and evaluation of this game. A three-condition experimental design was used to compare feedback strategies for game decisions: baseline corrective (right/wrong) feedback and explanatory feedback that is either provided automatically with each decision or only when players click on a link to access it. Primary outcome measures were malaria knowledge and player satisfaction. Study abroad email lists at seven Midwestern campuses were used to recruit participants without incentive. Of the 1428 participants who entered the study protocol and were randomly assigned to condition, 918 (64%) completed the pre-survey, 540 (38%) completed the game, and 482 (34%) completed the postsurvey. Only the automatically provided explanatory feedback condition produced a significantly higher knowledge score on the post-test when adjusted for pre-test score. There were no significant differences in player satisfaction between the three feedback conditions, while 65% of participants rated game satisfaction as 'very' to 'extremely' satisfying. Analysis of secondary outcome measures suggests that students prefer simplicity and efficiency within this interactive learning environment. Future research will examine the impact of a risk reduction game intervention on actual behavior. Introduction: Today it is important to maintain and promote mental health, since we are facing a lot of stress experiences. Acupuncture and moxibustion are closely related to behavioral medicine based on preventive medicine. Acupuncture and moxibustion medicine include diagnostic systems which enable us to detect sophisticated changes of the body and the mind as abnormalities of the meridians from a phase of pre-sickness. This study examined relationships between acupuncture and moxibustion medical assessments and perceived mental health states using subscores of Cornell Medical Index(CMI). The CMI used widely in the world was selected as the medical and psychiatric health questionnaire. Method: 235 healthy volunteer college students participated in this study. The cervical meridian test was introduced as a medical diagnosis of acupuncture and moxibustion which is easy to estimate objectively. Subjects were asked whether they felt pain, stretched feeling, dullness or malaise when they flex the neck backward, right side, left side, or forward , as well as perceived mental health using CMI. We compared to mental health related symptoms between individuals with and without perceived pain or stretching by the cervical meridian test. Results: Individuals with such feelings by flexing the neck forward and with such feelings by flexing the neck right and left sides tended to show higher most of scores on mental health related scores. Those with such feelings by flexing the neck backward tended to indicate higher scores on all subscores. Discussion: The cervical meridian test induced "pain"or "stretched feeling" and reflected perceived sensitively reflect mental health states. Acupuncture and moxibustion medical assessment can successfully be applied to mental health domain. Graduate school of Human Sciences, Waseda university, Tokorozawa, Saitama, Japan and 2 Faculty of Human Sciences, Waseda university, Tokorozawa, Saitama, Japan.
Background: Over-the-counter (OTC) medicines are increasingly used for self-medication, but such products are subject to misuse.
Although it is suggested that psychological factors may play a role in OTC drug misuse, the main factors that might be associated with one's manner of drug intake are unknown. Objective: The purpose of this study was to explore the illness perceptions and beliefs about taking OTC medicines and to investigate the differences between users and misusers of OTC medicines. Method: The subjects were 108 undergraduate students (age mean=20.27, S.D.=1.67). They retrospectively completed selfreport measures addressing (a) illness perceptions, (b) beliefs about taking OTC medicines, (c) how to take OTC medicines, (d) common cold symptoms, and (e) frequency of intake. Result: The analysis of covariance indicated that misuse of OTC medicines was related to an individual's perceptions that concern consequences of illness Breakups are usually distressing and generate different consequences (emotional, cognitive, behavioral and physiological) that can be associated to poor social relationships, physical/mental disorders and may eventually lead to suicide. The dissolution of a relationship implies a transitional crisis and is considered a relational loss that generates a grieving process. After breaking up with someone, the person must achieve mourning tasks, redefine the relationship, rebuild self-esteem and readjust to adaptive social patterns. A workshop was designed for vulnerable people, who had been detected with the Detection Questionnaire of Feelings, Emotions and Consequences of Loss (de la Serna, 2009), as a psychotherapeutic approach towards the mourning-task following the dissolution of a relationship. This study consisted of a preexperimental pretest-postest design with 7 participants, all female students aged between 18 and 26 in a public University in Mexico City. The workshop consisted of 10 weekly sessions in which theoretical information and cognitive-behavioral strategies (cognitive restructuring, training in social skills and problem solving) were taught in order to help participants understand their loss, work their grief and increase their self esteem and assertiveness in order to establish healthier social relationships. Three instruments were used for pre-post evaluation of the workshop and statistically significant differences (p≤.10) were obtained indicating its effectiveness. Mourning tasks were achieved with significant results in both sections of the detection questionnaire: feelings/emotions (t=2.012; p=.091) and consequences (t=4.621; p=.004). Significant differences (t=−3.746; p=.010) were also found in the Self Esteem Scale (Reidl, 2005) indicating an increase in participants' self esteem. Finally, assertive behavior also showed significant differences in the Assertion Inventory (Gambrill & Richey, 1975) with a decrease in the degree of discomfort (t=5.024; p=.002) and an increase in the probability responding assertively (t=3.533; p=.012). Background: Managing stress on a population basis is one of the promising approaches to enhancing health and reducing health care costs. This study are aims twofold. First, it is to translate and modify a transtheoretical model (TTM)-based stress management program developed originally for the US population for use in a different cultural context, Japan. Second, it is to report on 6-month outcomes of a randomized clinical trial (RCT) of this TTM-based stress management program from Pro-Change Behavior Systems, Inc., West Kingston, RI, using the internet. Methods: We translated and modified Pro-Change TTM-based stress management intervention program into Japanese and adapted it for use in a Japanese cultural setting. Stage-based expert systems were applied to reduce stress and enhance effective stress management behavior which is defined as setting aside approximately 20 min each day for a healthy activity such as talking with others, physical activity, or regular relaxation to manage stress. 456 university students were randomly allocated to either using the TTM self-help stage-based workbook for stress management followed by three individualized minimum reports at 0, 3 and 6 month) (i.e., workbook + feedback group) or the workbook followed by the same 3 individualized reports plus detailed prescriptions for processes of change (i.e., workbook + prescription group). Results: Results indicate that the stage-matched self-help learning for effective stress management behaviours followed by stagebased expert systems had significant effects on stress alleviation and progression of stage for stress management behaviours in both groups, irrespective of with and without individualized and tailored prescription for processes of change feedback reports. Conclusions: Stage-matched TTM expert systems for self-help stress management behaviour workbook can reduce stress for students irrespective of with and without individualized and tailored prescription for processes of change feedback reports. This study confirmed the long-term positive effects of this modified TTM-based stress management program in a different cultural context. Non-adherence to medication prescription is a serious problem in many chronic illnesses that require long-lasting treatment. There is considerable research showing that multiple factors such as patient's characteristics (e.g., age), medication related aspects (e.g., side effects) as well as environmental factors (e.g., availability) do affect adherence. In addition, the self-regulatory theory underlines the importance of subjective perceptions and beliefs guiding individual coping behaviour. The symposium addresses the interaction of illness perceptions and of treatment related beliefs with adherence to treatment from various perspectives. First, Nestoriuc et al. (1) (3) suggest that medication beliefs are associated with the intake of antihypertensive eye drops in patients with glaucoma, whereas illness perceptions do not additionally explain the degree of adherence. As both, the efficacy of a medication and patient adherence to the therapeutic regiment, influence the effectiveness of a treatment, it appears necessary to improve adherence in many illnesses, e. g. by psychological interventions. Petrie et al. (4) suggest a brief and economic intervention -a tailored text message programme -to change patients' illness perceptions with the aim to improve treatment adherence. Their controlled trial show promising results in asthma patients. Based on the presentations the symposium will offer a discussion about present evidence and perspectives regarding the role of patients' beliefs and perceptions on adherence. Background: While effective preventative medication is readily available in asthma, adherence is a major problem due to patients' beliefs about their illness and medication. Many non-adherent patients are reluctant to take their preventer inhaler when they are not experiencing symptoms and have concerns about their inhalers. Aim: We investigated whether a text message programme targeted at changing patients' illness perceptions would improve adherence in asthma patients. Methods: 212 patients aged between 16 and 45 on asthma medication were recruited from medication package inserts and health web groups. Participants were randomized to receive individually tailored text messages based on their belief profile over 18 weeks or no text messages. The type and number of text messages designed to promote beliefs associated with higher adherence. Adherence rates were assessed by phone calls to participants at 6, 12 and 18 weeks. Results: Overall adherence levels at baseline were low at 46% with only 12% taking over 80% of their prescribed doses. The data showed the targeted text group significantly improved adherence over the follow-up period compared to the control group with a relative average increase in adherence of 17%. The percentage taking over 80% of prescribed inhaler doses over the follow-up period increased by 20% in the intervention group compared to the control group. Conclusions: A targeted text message programme increases adherence to asthma preventer inhaler and may be useful for other illnesses where adherence is a major issue. Background: Non-adherence to immunosuppressant medications can contribute to medical complications in transplant patients. A recent pilot study showed that non-adherence is associated with liver transplant patients' beliefs that that the transplant has severe consequences and is distressing, as well as concerns about the harmful effects of medication. Aim: This study aimed to investigate associations between illness perceptions, medication beliefs, and adherence in liver transplant patients, extending previous work to a larger sample. Method: 193 liver transplant patients completed a postal questionnaire. Adherence was assessed using self reports (Immunosuppressant Therapy Adherence Instrument) and blood assays. Perceptions were assessed using the Brief Illness Perception Questionnaire, and the Beliefs about Medication Questionnaire. Results: 50% of the sample self-reported some form of nonadherence. Lower self-reported adherence was associated with lower education, lower beliefs about medication necessity, higher beliefs that medication was harmful, and had adverse effects, lower beliefs that immunosuppressants could prevent rejection, poorer understanding of the transplant, and higher distress about the condition. A higher percentage of drug test levels below the therapeutic range was associated with less time since transplant, and lower beliefs that medication could prevent rejection. Conclusion: This research provides further evidence that illness perceptions and medication beliefs are associated with non- Non-Adherence with intraocular pressure lowering eye drops is an important risk factor for going blind in glaucoma patients. Previous research evidenced a relationship between adherence to treatment and patients' illness and treatment related beliefs in a range of chronic illnesses. Objectives: The aim of this study was to investigate the associations between illness perceptions according to the "common sense model of self-regulation", medication beliefs, and adherence in subjects with glaucoma. Methods: 145 glaucoma patients with a mean age of 64 (SD= 12.7) completed a standardised questionnaire containing the revised Illness Perception Questionnaire [IPQ-R] , the Beliefs about Medicines Questionnaire [BMQ] , the Adherence to Refills and Medications Scale [ARMS] and a visual analogue scale on adherence [VAS-AD] . Clinical characteristics (e. g., ophthalmologic examination results) were recorded. Results: Unlike the expectation illness perceptions and disease severity indices did not correlate with adherence. Lower selfreported adherence was associated with the intensity of side effects and the medication related beliefs "General Harm", "General Benefit", "Specific Necessity", "Specific Concerns" [ARMS/ VAS-AD; p's<.05]. Multivariate analysis showed that the beliefs about medicines provide a useful model (R2=.14; F=3.9; p<.01) to predict eye drop taking behaviour with the two specific belief dimensions having the strongest predictive power (β=−.21/.21). Conclusions: This study suggests that beliefs about medicines seem to be useful predictors of adherence in glaucoma patients. By contrast the illness perceptions do not seem to affect adherence. The reasons for this result may lie in special characteristics of glaucoma and/ or the assessment methods used. The relevance of patients' beliefs will be discussed in the context of further factors impairing adherence in glaucoma patients. Individual treatment perceptions are relevant for the self-regulation of health and illness behavior. In people with chronic illnesses, beliefs about medicines have been shown to influence patients' treatment decisions, adherence and side effect reporting. However, to interpret these results epidemiologic data about medication beliefs is essential. In this study, a German translation of the Beliefs about Medicines Questionnaire (BMQ) was psychometrically evaluated. A representative sample of the German population (N= 2512) was screened for medication use, adherence, generic side effects, and beliefs about medicines. Reference scores for beliefs about medicines from the general population were calculated and compared for different drug classes. Moreover, to investigate criterion validity their influence on adherence was determined. A principal component analyses with oblique factors confirmed the original 6 factor structure of the BMQ. Further psychometric analyses revealed moderate to high internal consistencies and good construct validity. In a stepwise linear multiple regression, adherence was significantly predicted by participants' age, sex, current medication, side effects, and beliefs about medicines (adjusted r 2 =.25, p<.001). Specifically, beliefs about medicines explained an additional significant proportion of 20% of variance, after controlling for relevant demographic characteristics, medication intake and side effects. The German version of the BMQ is a reliable and valid self rating instrument for the assessment of peoples' perceptions of medication. Adherence in the general population is determined by sociodemographic characteristics, current medication intake, experienced side effects, and to a unique and substantial proportion by medication beliefs. Several behavioral intervention studies for coronary artery disease (CAD) have been published during the last decade in Belgium, Canada, Sweden and the United States. Many of the studies focused upon reducing CAD risk in patients already showing CAD manifestations. Hans-Christian Deter will examine the rationale and targets of the new German SPIRR-CAD trial, which is a combined psychodynamic and cognitive behavior group therapy intervention aimed at decreasing depressive symptoms, Type D behavior and CAD risk profile and events in CAD patients. Manfred Beutel will describe the diagnostic and therapeutic processes involved as well as the psychological quality management program used to insure a high quality of comparable intervention fidelity at the 10 centers collecting data in this SPIRR-CAD trial. Kristina Orth-Gomér will discuss the reduced mortality produced by psychosocial intervention in the Stockholm Women Intervention Trial for Coronary Heart Disease (SWITCHD) and how tailoring the intervention to the gender related needs of women may have contributed to the outcome. Neil Schneiderman will then describe biological and behavioral factors that appear to have been involved in those randomized controlled trials that have successfully used psychosocial interventions to reduce the recurrence of major adverse coronary events (MACE). University Medical Center, Berlin, Germany; 2 U Clinic Cologne, Cologne, Germany; 3 U Medicine Mainz, Mainz, Germany; 4 U Clinic Freiburg, Freiburg, Germany; 5 Hannover Medical School, Hannover, Germany; 6 Dresden U, Dresden, Germany; 7 Kerckhoff Reha Center, Bad Nauheim, Germany; 8 U Clinic Heidelberg, Heidelberg, Germany; 9 TU München, München, Germany; 10 U Clinic Dresden, Dresden, Germany; 11 Clinic Nord Nürnberg, Nürnberg, Germany and 12 U Clinic Göttingen and the SPIRR-CAD-study group, Göttingen, Germany.
Depressive symptoms are highly relevant in patients with (CAD). Unfortunately, recent psychotherapy trials showed only small effects on depression. However, combined treatment may be more efficacious than a behavioural or interpersonal treatment alone. In our new multicenter trial, funded by the German Research Foundation, we address negative affectivity and social inhibition in addition to depression to enhance treatment efficacy. Men and women (n=569, age 18-75 y) with HADS depression scores >7 hospitalized for any manifestation of CAD, are randomized into the intervention or control group. Initially, patients in intervention receive 3 sessions of supportive individual psychotherapy on a weekly basis. After reevaluation of depression 4 weeks after inclusion, patients with persisting symptoms of depression receive 25 sessions of combined psychodynamic and group cognitive behavior therapy (CBT) over 10 months. The control group receives one counselling session. Routine cardiac care is offered to both groups. Primary efficacy endpoint is change from baseline to year 1 in HADS depressive symptoms. Secondary endpoints include remission of depression, Type D pattern, health-related quality of life, cardiovascular risk profile, neuroendocrine and inflammatory activation, heart rate variability, cardiac events, and health care utilization, up to 24 months after inclusion. Preplanned subgroup analyses will assess gender, and genetic predictors of treatment success. Psychosomatic Clinic, U Medicine, Mainz, Germany; 2 Psychosomatics, U Clinic Cologne, Cologne, Germany; 3 Psychosomatics, U Medical Center, Berlin, Germany; 4 Psychosomatics, U Clinic Freiburg, Freiburg, Germany; 5 Psychosomatics, Medical School, Hannover, Germany; 6 Psychosomatics, U Clinic Dresden, Dresden, Germany; 7 Psychocardiology, Kerckhoff Reha Center, Bad Nauheim, Germany; 8 Gen. Internal and Psychosomatic Med, U Clinic Heidelberg, Heidelberg, Germany; 9 Psychosomatics, TU Munich, Munich, Germany; 10 Psychosomatics, U Clinic Dresden, Dresden, Germany; 11 Psychosomatics, Klinikum Nord Nürnberg, Nürnberg, Germany and 12 Psychosomatics, U Clinic Göttingen on behalf of SPIRR-CAD Study Group, Göttingen, Germany.
Psychotherapeutic interventions in the SPIRR-CAD trial are characterized by (1) a stepwise combination of individual and group treatment, (2) integration of behavioural skills and education into a psychodynamic group framework focusing on negative affect and social inhibition. In order to assure a high quality of comparable interventions at the 10 sites of the multicenter trial, systematic analyses of adherence and competence of therapists are performed on videotape recordings of all treatments. Based on rating manuals for psychodynamic and behavioral intervention, specific rating manuals were devised and raters were trained. Moderate to good interrater reliabilities were obtained between raters based on 18 randomly selected individual sessions (0.48 -0.69). Therapists are trained according to manual, undergo regular supervision and obtain written feedback on their performance. One out of two individual sessions is evaluated. Out of 25 group sessions, 3 are randomly selected from early, middle and termination phase.
According to preliminary results, adherence and competence are unrelated to the helping alliance as judged by patient and therapist. Implications for the identification of effective treatment components (e.g. therapist performance) are discussed. Stress at work has been shown to influence both incidence and progression of cardiovascular disease (CVD), but psychosocial interventions to reduce stress and CVD have produced inconsistent Results: Moreover, meta-analysis has indicated that psychosocial interventions have been more effective in reducing CVD in men than in women. One possible explanation is that men and women have different needs in terms of stress reduction. Thus, it appears that family problems constitute stronger emotional stressors and provoke more depressive feelings in women than men, and that women, even when working outside the home are more endangered by family stress than by work stress. Therefore, the Stockholm Women's Intervention Trial (SWITCHD) was specifically designed to meet the needs of women CVD patients in terms of tailored stress reduction. This randomized clinical trial decreased CVD mortality in women receiving the intervention compared to women receiving standard care, providing evidence that an intervention designed to satisfy the gender related needs of women may prolong life in women with CVD. Randomized controlled trials (RCT) that have used behavioral interventions designed to reduce morbidity and/or mortality in patients who have had major adverse coronary events (MACE) have reported positive, null and negative Results: In general RCT that have been successful used group based interventions, behavior change strategies, at least 20 sessions during the course of a year, and have followed patients for at least several years. Some successful studies (e.g., Stockholm Women's Intervention Trial for Coronary Heart Disease: SWITCHD) have employed relaxation training, suggesting that stress reduction is important. SWITCHD also found that the interaction between behavioral intervention and the use of statins also appeared to decrease mortality. And in research conducted in animals we have found that social affiliation can attenuate the development of atherosclerosis by decreasing inflammation and oxidative stress. Because patients with CAD typically are prescribed multiple medications, medication adherence may be a useful intervention target. Similarly, because most CAD patients are overweight, physically inactive, and may have metabolic syndrome, behavioral intervention strategies to increase weight loss and improve fitness could be useful for addressing these issues. This presentation will compare and contrast RCT in terms of the biological and behavioral bases of the intervention strategies used to treat CAD patients. The "hikikomori" (social withdrawal) describes a psychopathological and sociological phenomenon in which people, and especially younger generations, become completely withdrawn from society for six months or longer. The "hikikomori" was first reported in Japan, and had been thought as a culture-bounded psychopathology. But cases of "hikikomori" were reported also in Oman and Spain. Recently, a large-scale community-based epidemiology study in Japan, the World Mental Health Japan Survey, as well as other studies, reported that the prevalence, psychiatric comorbidity, and childhood risk factors of "hikikomiri". Also a qualitative study from Hong Kong reported that "hikikomiri" was a psychopathology with difficulties in interacting with others and it was not unique to the Japanese society. In this symposium, each speaker will present their recent research findings. The advances in epidemiology and qualitative research could provide a new perspective of the nature of "hikikomori" as a unique psychopathology developed in the context of the society. In Japanese, Hikikomori are people who voluntarily withdraw into social isolation for over six months . Etiology and epidemiology are poorly understood, and evidence for interventions is lacking. The purpose of this study is to describe onset and maintenance of hikikomori. Direct recruitment, snowballing and participant observation in on-line chat rooms were used to recruit participants and obtain data, which was then subject to grounded theory analysis. Of 168 participants several were unconnected with Japan, the oldest being 60 years old. Three major themes emerged of hikikomori as passive coping, trust and existence. Each theme comprised one or more categories, which in turn evidenced a number of different elements. Definitive characteristics of hikikomori were obtained from respondents through virtual participant observation. The emergent theoretical framework and the list are mutually supportive in the results obtained from this study, which suggested emotional pain exists in hikikomori in relation to human relationships. The results of the present study suggest that hikikomori is characterized by more diffuse features, including difficulties in coping with people or tasks, difficulties with trust, unhappiness about life and poor concept of time. There was no evidence of violent or aggressive behaviours.We conclude that Hikikomori is not unique to Japanese youth, but more widespread and diverse anomic coping style for dealing with social disaffection seen cross-culturally. Aims: Epidemiology of "hikikomori (acute social withdrawal)" in a community population is not clear, although it has been noted for the decade in Japan. The objective of this study is to clarify the prevalence of "hikikomori" and to examine the relation between "hikikomori" and psychiatric disorders. Methods: Face-to-face household survey was conducted of community residents (n=4,134). We defined "hikikomori" as a psychopathological phenomenon in which people become completely withdrawn from society for six months or longer. We asked all respondents whether they had any children currently experiencing "hikikomori". For respondents aged 20-49 years old (n=1,660), we asked whether they had ever experienced "hikikomori". Also respondents were asked another question if they had a child with "hikikomori". Results: A total of 1.2% had experienced "hikikomori" in their lifetime. Among them, 54.5% had also experienced a psychiatric (mood, anxiety, impulse control, or substance-related) disorder in their lifetime. A total of 78% of the "hikikomori" cases in our study reported that they had felt worried or irritable about their "hikikomori" situation. Most of the "hikikomori" cases did not feel the desire to go to work or attend school even if they still had such a duty. There was no case in this study who reported violent behaviors during "hikikomori". Respondents who experienced "hikikomori" had 6.1 times higher the risk of mood disorder. Among respondents, 0.5% currently had at least one child who had experienced "hikikomori". On the other hand, about 0.5% of respondents reported that they had a child who had or was experiencing "hikikomori". Discussion: The study suggests that "hikikomori" is common in the community population in Japan. Approximately 232,000 households are estimated to currently have a child with "hikikomori" in Japan. While psychiatric disorders were often comorbid with "hikikomori", a half of the cases seem to be the "primary hikikomori" without a comorbid psychiatric disorder. "Hikikomori", a form of social withdrawal among youths who retreat from social interaction for protracted periods of time, is widely acknowledged in Japan and some other countries. This study examines childhood adversities and family environments as childhood risk factors for "hikikomori" using the retrospective data derived from a population-based survey, the World Mental Health Japan. A random sample of residents aged 20 or older in nine communities in Japan was used, and interviews were conducted using the WHO Composite International Diagnostic Interview 3.0 (n=4,134, response rate=51%). The subjects of this study were a subsample who responded to the second part of the interview (Part 2), and were aged between 20-49 years (n=708). Multiple logistic regression was used to explore the association between the lifetime experience of "hikikomori" and childhood risk factors; social class, parental psychopathology, parents' child rearing style, and other adverse events during a respondent's childhood. The statistical model was adjusted for sex, age, and respondents' experience of common mental disorders using a sampling weight for Part 2. Among the 708 respondents, 15 experienced "hikikomori" in their lifetime (n=9.7 or 1.2% in a weighted analysis). Father's high educational level (OR=6.0, CI=1.6-22.9), mother's common mental disorders (OR=6.5, CI=1.1-38.0), and mother's panic disorders (OR=6.6, CI=1.1-39.1) were significantly and positively associated with the experience of "hikikomori" after controlling for respondents' sex, age, and history of mental disorders. Significant associations were not found for parents' child rearing styles or childhood adversities. Our findings suggest that "hikikomori" cases are more likely to occur in families where the parents have high levels of education. Maternal panic disorder may influence the development of "hikikomori" among children, possibly due to reactions of children to their mothers' disorder. Most observers agree that the category of hikikomori encompasses a wide range of problems and provocations. Furthermore, recent studies have found that a substantial portion of the hikikomori population reports symptoms of mental illness (Kawakami, forthcoming). Taking a cultural anthropological approach, this paper explores the social context that allows withdrawing from society to be a prevalent and viable coping mechanism in Japan. The paper draws on ethnographic, participant-observation data from three clinical facilities in the Tokyo area which treat hikikomori populations, and four special education programs which treat children who missed school for long periods due to learning disabilities and social maladjustment. The paper concludes by reviewing recent literature on mental health in Japan in order to hypothesize about whether Japanese psychiatry will move away from relying so heavily on families and towards psychopharmacological treatments as the dominant method and perceived "cure" for addressing psychopathology. Objective: To prospectively study the development of socioeconomic health differences in the Netherlands and to investigate possible explanations for socioeconomic variation in childhood health. Methods: The PIAMA study follows a birth cohort of 3,963 Dutch children during their first 8 years of life. Common childhood health problems, i.e. eczema, asthma symptoms, general health, frequent respiratory infections, overweight, and obesity were assessed yearly using questionnaires. Maternal educational level was used to indicate socioeconomic status. Possible explanatory lifestyle determinants (breastfeeding, smoking during pregnancy, smoking in the first 3 months and the use of day-care facilities) and biological determinants (maternal age at birth, birth weight and older siblings) were analyzed using General Estimating Equations (GEE). Results: Socioeconomic differences in a broad range of health problems are present early in life and persist during childhood. Socioeconomic differences in maternal age at birth, breastfeeding, and day-care center attendance contribute substantially to childhood socioeconomic health disparities. Conclusion: This study shows that health disparities already occur in the very early years of life, also in the Netherlands. The less healthy parental lifestyle among lower socioeconomic groups explains most of the observed socioeconomic health differences in childhood. Background: Several studies have shown that social capital in terms of social participation and trust are linked to better health. However, there are few studies on the effect of community interventions attempting to benefit from this. Methods: A large scale intervention by means of participatory methods, aiming at improving the physical and social environment in an inner city area characterised by a low-income and an ethnically diverse population was launched in a midsize city in southern Sweden. Interviews concerning environmental, sociodemographic factors (including social capital), and health (including mental health measured by GHQ12) were made with 630 randomly selected individuals at baseline. A follow-up with an identical interview was made with those who still lived in the area after three years. The response rate was 66%.
Results: Bridging social capital had increased significantly, but in a pattern determined by type of social capital, gender, educational level and ethnicity. Bridging social participation had increased among men, individuals born in Sweden and the low educated. Bridging trust on the other hand, increased among women, individuals born outside Sweden and the high educated. Selfrated health did not change in any group between baseline and follow-up. However, mental health improved significantly among individuals born outside Sweden. This seemed to be mediated by social capital components. Conclusion: Following the community-based intervention, bridging social capital increased among inhabitants who lived in the area for the whole three-year period in a pattern determined by gender, level of education and ethnicity. Mental health improved among individuals born outside Sweden, which may have been mediated by improvement of bridging social capital. Depression and obesity are the among the most common health problems globally and may share common social determinants. Social integration is suggested to protect adults from ill health; this study examines whether the protective effect of social integration is seen longitudinally and is present in the case of the cooccurrence of depression and central obesity. We used 3 sweeps of a prospective cohort study, the National Child Development Study, when the participants were aged 33, 42, and 45 (Men =3,043, Women=3,153). Co-occurrence of depression and central obesity was assessed at age 45, by the presence or absence of depression (scores over 2 on the depression subscale of the Clinical Interview Schedule) and central obesity (a waist to hip ratio greater or equal to 0.9 for men, 0.85 for women). Social integration was assessed at age 42 using information about cohabitation, employment status, and frequency of attendance at religious or socially organised groups. Psychological health, longstanding illness, and social position assessed at age 33 are treated as confounders and men and women were analysed separately. Findings using multinomial logistic regression showed the significance of employment as the key protective element of social integration among middle aged adults. Working full time at age 42 was less likely to be associated with depression at age 45 even along with central obesity, but not with central obesity alone. This effect was independent of the effects from the confounders and greater among men (Depression: OR=0.15, 95% CI=0.07-0.32; Comorbdity: OR=0.25, 95%CI=0.14-0.45). Although considered a confounder rather than an explanatory variable, it is worth noting that psychological ill health at age 33 was linked to depression at age 45 in men and women; however, the degree of the association is greater when central obesity is present, confirming the complex mechanism of co-occurrence of depression and central obesity. It appears employment, may protect middle aged adults against comorbid depression and central obesity through its relationship with depression. The four HOPE Projects build on over 16 years of community based research projects with low income women in rural counties in North Carolina. The Community-based participatory research process led us to develop interventions to address women's health within the context of the economic and social circumstances of their lives: poverty, unemployment, racism and lack of access to resources. HOPE (Health, Opportunities, Partnerships, Empowerment) Works (2004) (2005) (2006) (2007) (2008) (2009) ) was a successful obesity prevention intervention that drew upon development strategies including loan circles, microenterprise development, and American Indian talking circles. Community women were trained to organize and facilitate HOPE Circles of 8-12 women from their social networks. Circles met twice a month for 6 months: women set goals and supported each other to make health and social/economic improvements. Threads of HOPE (www.threadsofhopenc.org) is a spinoff from HOPE Works: it is a small business that produces sustainable conference bags, and a non-profit whose mission is to train low income women in business development and financial literacy. Seeds of HOPE (2009-2014) will disseminate the HOPE Works model through American Indian and African American churches and community health clinics. It will have a stronger emphasis on microenterprise development and financial literacy. HOPE Accounts for Women (2009) (2010) (2011) is funded by the American Recovery and Reinvestment Act through the National Institutes of Health. This innovative program uses the HOPE Works model but enhances the economic empowerment/financial literacy training by assisting low income women in saving money for starting a business or furthering education through a matched savings incentive. Data from the first 3 cohorts (159 Intervention, 118 Comparison) in the HOPE Works study show that 75% of women were African American and most were low income: outcome analysis demonstrated that HOPE Circle participants lowered BMI more than the comparison group with average weight loss difference of 4 pounds. Intervention women also increased hope, physical activity and fruit and vegetable intake. Introduction: It has been well documented that socioeconomicallydisadvantaged groups are more likely to be overweight/obese than their more-advantaged counterparts (this trend is seen more consistently among women). These differences may be due (in part) to socioeconomic differences in weight-control behaviors. Methods: Data was obtained from 1012 men and women aged 45 to 60 years residing in Brisbane, Australia (69.6% response rate). Data were collected by a postal questionnaire, which provided information on past and current weight-control behaviors. Socioeconomic position was characterised by highest attained education, household income and occupation. Results: Socioeconomically-disadvantaged participants were less likely to report engaging in weight-control in the past 12 months. Those with secondary school or lower education (OR=0.58, 95% C.I. 0.41-0.82), working in a blue-collar profession (OR= 0.36, 95% C.I 0.22-0.60), not in the work force (OR=0.62, 95% C.I. 0.41-0.94), or who belonged in the 1st quartile of equivalised household income (OR=0.64, 95% C.I. 0.41-0.98) were less likely to report trying to lose weight and/or avoid weight gain in the past 12 months. There were no significant differences between socioeconomic groups in engaging in potentially health-promoting or health-damaging weight-control behaviors. Conclusion: Socioeconomic differences in weight status may be due to differences in engaging in weight-control behaviors. However among those who do wish to maintain or lose weight, there are no socioeconomic differences in the nature of the strategies they employ. Background: Previous studies may have underestimated the contribution of unhealthy behaviours to social inequalities in mortality because of the assessment of health behaviours at only one point in time, resulting in a failure to capture long-term exposure.
Methods: This study used 4 repeated assessments of smoking, alcohol consumption, diet and physical activity over 24 years of follow-up to examine their role in mediating the association between socioeconomic position, measured using occupational position, and mortality from all-causes and specific causes in 9 590 men and women aged 35 to 55 years at baseline. Results: 654 participants died during the follow-up. Those with lowest occupational position had 1.60 times higher risk of death than those with highest position in analysis adjusted for sex and year of birth. For all-cause mortality, this association was attenuated by 42% (95% Confidence Interval (CI) 21%, 94%) when health behaviours assessed at baseline were entered into the model and by 72% (95% CI 42%, 154%) when health behaviours were entered as time dependent covariates. The corresponding attenuations were 29% (95% CI 11%, 54%) and 45% (95% CI 24%, 79%) for cardiovascular mortality and 61% (95% CI 16%, 425%) and 94% (95% CI 35%, 595%) for non-cancer noncardiovascular mortality. There was no association between occupational position and cancer mortality so no further analyses were undertaken. The difference between the baseline and longitudinal assessments was mostly due to an increased explanatory power of diet, physical activity and to a lesser extent, alcohol consumption. The role of smoking, the strongest mediator in these analyses, did not much differ when using baseline or repeat assessments. Conclusions: Health behaviours may be a more important explanatory mechanism for inequalities in mortality than previously estimated. According to South Africa's new constitution, access to health care is a fundamental right. In this paper we analyse the utilisation of ante-natal care services under the public health system of South Africa in order to inform policy concerned with equity of access about factors associated with the distribution of current services. We conceptualize access to care as covering three distinct but interacting dimensions of availability, affordability and acceptability. Each dimension reflects aspects of both the demand for and supply of services. We adopt WHO guidelines concerning the need for ante natal care (ANC) visits and explain variations in the number of visits among women giving birth in four selected communities, two urban and two rural.
The results indicate that more marginalised women (i.e., young, unmarried, low levels of education) and those with no previous deliveries were significantly less likely to have the WHO recommended number of 5 ANC visits. Further analysis revealed variations between facilities in the determinants of sufficient ANC visits. These findings show inequalities in utilisation which may suggest that inequities in access remain. Extant reviews of peer and social support interventions are disease-and approach-specific (e.g., use of community health workers in diabetes management). Because there is limited evidence within any one disease, such reviews do not provide strong evidence for peer support. In contrast, a more general review of peer support across a range of health behaviors can lead to more solid conclusions about its effectiveness. We conducted a systematic review of research on peer support for complex health behavior (e.g., cardiovascular risk reduction) across a variety of health problems. An initial PubMed search of papers published between 1/1/2001 and 8/31/2009 and using cognates of "coach," "promotora," "peer support," etc. identified 902 peer-reviewed articles. Of these, 595 articles were excluded as their titles indicated that they were not patient behavior programs (e.g. coaching for career growth among health professionals). The remaining 307 were reviewed to fit a broad definition of peer support: provided by a nonprofessional and addressing support for multiple health behaviors over time. Exclusion criteria consisted of: Interventions focused on isolated or single behaviors (e.g. cancer screening); interventions evaluating educational classes; and those without outcome data. The resulting 40 papers addressed: breastfeeding (6 papers); diabetes (5 papers), depression (5 papers), and asthma (4 papers), along with a variety of other health issues, and represented 8 different countries. Provisional rating of outcomes was: No evidence of benefit; Modest evidence of benefit (e.g., changes on self report measure of health behaviors or quality of life); and Strong evidence of benefit (e.g. statistically significant changes on objective clinical measure such as blood pressure or blood glucose, or on well validated psychological measure such as the CES-D). Of the 40 papers, 7 were scored as No, 15 as Modest, and 18 as Strong Evidence of Benefit. Further evaluation of effect sizes and weighting by sample sizes and adequacy of controls will be included in presentation at the Congress. This systematic review indicates that peer support is effective in promoting complex health behaviors across a variety of diseases and national settings. Background: Nepal is one of the least developed countries in the world. Poverty, illiteracy, poor public health system are tremendously impacting on majority of people's lives who are living in rural areas and limited access to primary health care services. The government of Nepal declared the free health care policy from early 2008 with a view to improve access to and utilization of health services for people who are poor, socially disadvantaged and marginalized communities. The new health policy has highlighted the needs to expand health services in rural areas where there is acute lack of human resources, irregular supplies of drugs and equipment and poor health infrastructure. The pro-poor health policy aims to narrow the wider gaps of disparities in health care by ensuring health equity and the rights of people to their health and life. Objective: The objective of this study is to explore the effects of pro-poor health care policy at the community levels. Methods: It was a cross-sectional study with primary focus on qualitative research methodology. The methods for data collection were focus group discussion and in-depth interviews with service providers in local health facilities, community groups, key informants and the beneficiaries. Besides, the data from health management information system at local health facilities were reviewed to analyze the trend of health service utilization over the period of two years. Results and Conclusions: The pro-poor health policy has been positively impacting on people's access to and utilization of health services. There is 15 to 20 % increment in health facility utilization by poor, socially disadvantaged and marginalized communities. There is significant level of community awareness of free health care policy and provision. The supplies of drugs and other logistics at health facilities are gradually improved in the rural areas where as human resources for health are still lacking and not adequate compared to the health care needs of the people. The pro-poor health policy has been instrumental in accessing and utilizing the services by the poorest of the poor as it is free of charge and available in local health facilities. Social support is thought to be a health protective resource, however, the underlying mechanisms are elusive. One possible mechanism is that social support has a buffering effect on the sympathetic stress response. The salivary enzyme alpha-amylase (sAA), reflecting the activity of the sympathetic nervous system (SNS), seems to be a promising indicator for stress research. The main purpose of the present study was to test the effect of perceived social support (PSS) on acute sAA stress reactivity. Thirty-three healthy, young male subjects participated in a computerized stress task consisting of arithmetic problems that had to be solved under time-pressure and social evaluation. Cortisol, sAA, salivary flow rate (SFR), heart rate (HR), and respiratory sinus arrhythmia (RSA) were assessed repeatedly, while perceived social support, chronic stress and rated stress reactivity were assessed using different questionnaires. Higher PSS was negatively correlated with the stress response of sAA (r=-.551, p<.01) and HR (r=−.344, p<.05), but was unrelated to cortisol, SFR, and RSA alterations. Furthermore, higher levels of PSS were negatively associated with ratings of chronic stress (r=−.384, p<.05) and habitual stress reactivity (r=−.372, p<.05). This is the first study examining the relationship between PSS and acute sAA stress reactivity. The results indicate that social support indeed buffers the effect of psychosocial stress on the SNS activity, and is further inversely associated with ratings of chronic stress and stress reactivity. In addition, our findings emphasize the relevance of sAA for stress and social support research. Music listening has been suggested to beneficially impact health via stress-reducing effects. However, the exact mechanisms through which music exerts its positive consequences on the body are poorly understood. It was the aim of the current study to address this gap in knowledge and to examine the underlying mechanisms of music effects across neuroendocrine, autonomous, cognitive, and emotional domains of the human stress response. Sixty healthy female volunteers (mean =25.27 years) were exposed to a standardized psychosocial stress test after having been randomly assigned to one of three different conditions prior to the stressor: 1) relaxing music ('Miserere', Allegri) (RM), 2) sound of rippling water (SW) and 3) rest without acoustic stimulation (R). Salivary cortisol and alpha-amylase (sAA), anticipatory cognitive appraisal, subjective stress perception and anxiety were repeatedly assessed in all subjects. We hypothesized that listening to music prior to the stressor, compared to SW or R would result in an attenuated stress reaction. The stressor caused significant changes in all measurements in all three groups over time. The three conditions significantly differed regarding cortisol responses (p=0.014), with highest values in the RM and lowest values in SW. sAA recovery delta showed a statistical trend (p= 0.060) in favor of the RM. Psychological measures did not significantly differ between groups during the experiment. Our findings indicate that music listening differentially impacts the psychobiological stress system. Listening to music prior to a psychological stressor increases rather than attenuates subsequent endocrine stress responses. In contrast, listening to water sound seems to result in an attenuated endocrine response to stress compared to no auditory stimulation. Listening to music seems to increase autonomic recovery more efficiently than listening to water sound or resting in silence. These findings bear potential to explain the effects of music on the human body. The aim was to study if health, reaction time, and the diurnal rhythm of cortisol were negatively affected when a group of shift workers changed their work schedule from ordinary night-day shift to "swing shift". This was tested on 19 healthy workers on a Norwegian oil rig in the North Sea. They worked two weeks offshore followed by 4 weeks off work. The ordinary schedule was to work 12-hour day shift during one work period (14 days), and 12-hour night shift on the next work period (14 nights)(fixed-shift). "Swing shift" involves night shift during the first week, then day shift the second week, for every working period. The advantage of swing shift is that the workers are readapted to a normal day-night rhythm when they start their 4 week off work period. The disadvantage is that the workers have to adjust their biological rhythms twice every work period instead of once every other work period. All participants worked ordinary night-day shift when baseline data were collected (questionnaires, saliva cortisol, and reaction time during work). After collection of baseline data the workers changed their work schedule to "swing shift", one year later the same data were collected. "Swing shift" did not give any negative health effects or any negative changes in reaction time during the day they shifted from night work to day work. During swing shift the cortisol rhythm went slowly back to a normal rhythm in the second week, but it was not fully returned to normal values when they returned home for the 4 weeks off period. When working swing shift the cortisol rhythms were readapted to normal values after one week at home. For personnel returning home directly from 14 consecutive night shifts, diurnal cortisol adaptation was not complete after one week at home. Low vagal function is related to several disorders. One possible underlying mechanism linking the vagus nerve and disorders is the HPA axis, although literature referring to this association is inconsistent. The main purpose of the present study was to examine the relation between vagal function and the biopsychological reactivity to acute psychosocial stress. Healthy male subjects (N =33) participated in a stress task (Montreal Imaging Stress Task, MIST; Dedovic et al., 2005) , while heart rate (HR), respiratory sinus arrhythmia (RSA), salivary cortisol and mood were assessed. Vagal function was determined using baseline, MIST-induced inhibition and Cold Face Test (CFT)-induced stimulation. The stress task induced a significant increase in cortisol and HR (both p<.001) and a decrease in RSA and mood (both p<.001). A linear regression model with CFT response predicted 17.9% of observed variance in cortisol AUCI (mood: 38.9%; tiredness: 19.8%) in response to the stressor. The results indicate that faster or stronger CFT response is associated with reduced cortisol increase, increased mood and wakefulness after acute stress. Our data support an inverse relationship between vagal function and the HPA axis. Emotional distress-induced up-regulation of Interleukin-6 (IL-6) has been found to be detrimental to health. As diabetic foot ulcers are characterized by chronic inflammation, it is plausible that emotional distress could delay the healing of such ulcers by upregulating and sustaining high ulcer tissue-specific levels of IL-6. Here we report a cross-sectional study of 41 patients with type 2 DM (76% male; mean age 55 yrs) and plantar neuropathic diabetic foot ulcers (University of Texas Classification: 82% grade 1A; 12% 1B; 3% 2A; and 3% 2B). The relationship between patient self reported generalized emotional distress, foot ulcer-specific emotional responses and ulcer biopsy IL-6 levels was investigated. Ulcer-specific IL-6 levels were determined via quantification of immunohistochemical wound biopsy tissue localization. Additionally, assessed was the physiologic marker of emotional distress, 9 am and 12noon serum cortisol (458.7±125.8 nmol/l and 625± 369.7 nmol/l, respectively). In the bivariate analysis, more severe generalized and foot-ulcer-specific emotional distress were each associated with higher levels of ulcer biopsy log-transformed IL-6 (raw mean: 608.3±545.5 cells/mm2): HADS-Anxiety (r=0.44; p= 0.031), PSS (r=0.44; p=0.034), PIN-Amputation Worry (r=0.54; p=0.007) and NeuroQoL-Interpersonal Burden (r=0.60; p= 0.004). No significant relationship was noted between serum cortisol and IL-6. These preliminary findings identify a strong link between subjective measures of emotional distress and wound specific IL-6 up-regulation. Whether this relationship contributes to non-healing of diabetic foot ulcers is under investigation. Chronic multi-symptom illnesses such as Fibromyalgia Syndrome (FMS) and Temporomandibular Disorders (TMD) are accompanied by complex interactions of cognitive, emotional, and physiological disturbances. Such conditions are complicated and draining to live with, and successful adaptation may depend on ability to self-regulate. Self-regulation involves capacity to exercise control and guide or alter reactions and behavior. Research indicates that self-regulatory strength is a limited source that can be depleted or fatigued, however, and patients with FMS and TMD may, when trying to adapt to the many demands of their illnesses, deplete or fatigue their self-regulatory resources. The current study aimed to show that patients with FMS and TMD, compared with healthy controls, are more vulnerable to selfregulatory fatigue as a consequence of their condition, and that self-regulatory fatigue has specific physiological markers. Research has found self-regulatory effort to lead to self-regulatory fatigue, indicated by less capacity to persist on a subsequent task. Patients (N=50) and matched controls (N=50) were exposed to an experimental self-regulation task, followed by a persistence task. Controls in the low self-regulation condition displayed continued high persistence as expected. However, patients in the same condition displayed similar persistence as patients and controls in the high self-regulatory condition, indicating that patients with FMS and TMD may in fact be suffering from chronic selfregulatory fatigue. Baseline heart rate variability, heart rate, blood glucose, and cortisol predicted persistence, more so for controls than for patients, and more so in the low versus high selfregulation condition. Impact of illness on self-regulatory effort was mediated by pain, but not by any other factors. The current study shows that patients with chronic multi-symptom illnesses likely suffer from chronic self-regulatory fatigue, and that selfregulatory capacity needs to be taken into account when aiming to treat and help these patients through behavioral interventions. Department of Health Sciences, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands and 3 University Centre for Sport, Exercise and Health, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands.
Objective: To investigate the appreciation of written education about pain neurophysiology in patients with fibromyalgia (FM) and its effects on illness perceptions and quality of life. Method: A booklet explaining pain neurophysiology (i.e. central sensitisation) was sent to participants with FM. Appreciation about the information in the booklet was assessed by 10 questions addressing perceived relevance (0-30) and perceived reassurance (0-30). Participants filled in the Revised Illness Perception Questionnaire (IPQ-R), the Pain Catastrophizing Scale (PCS) and the Fibromyalgia Impact Questionnaire (FIQ) at baseline (T0), after a 2 week control period (T1) and 6 weeks after the intervention (T2). Differences between T0, T1 and T2 were analysed using a repeated measures design. Pearson's correlation coefficients were used to reveal relationships between appreciation and changes in outcome. Results: Mean (SD) scores for relevance and reassurance were 21.6 (5.6) and 18.7 (5.7), respectively. Illness coherence, emotional representations, pain and fatigue changed significantly between T0 and T2, pain also before intervention. Correlations between appreciation and changes in illness perceptions, catastrophizing and quality of life ranged between r=.000 and r=.338. Conclusions: Although a majority of our subjects with FM appreciated the written information about pain neurophysiology, it did not have clinically relevant effects on illness perceptions, catastrophizing and quality of life. Appreciation of the information was not related to changes in outcome. Objective: Fibromyalgia patients are perceived by clinicians to be 'difficult' and not easy to manage. Injured athletes are considered as much less difficult to manage clinically. According to Leventhal's Common Sense Model these differences are related to the illness perceptions of these groups. The objective of this study, therefore, was: What are the illness perceptions of patients with fibromyalgia and injured athletes? Method: 196 fibromyalgia patients and 95 injured athletes participated in the study. In both groups the Illness Perception Questionnaire Revised, adapted for fibromyalgia and sport injuries, respectively, was used. The samples were recruited form physical therapy practices in the Netherlands. Results: The fibromyalgia group consisted of 172 (88%) female and 24 (12%) male patients with a mean age of 49 years (SD 11), familiar with the diagnosis since 7 years (SD 6). The athletes sample consisted of 31 (41%) female and 64 (59%) males; mean age was 30 (SD 12). The duration of the injury was in 47 athletes (49%) longer than 6 months; all were injured due to participating in a broad range of sports. Significant differences were found for illness identity, timeline acute chronic, timeline cyclical, consequences, treatment control and illness coherence. Fibromyalgia patients have a strong illness identity, perceive more symptoms (pain, fatigue, stiffness), have negative beliefs about the consequences of fibromyalgia, their faith in treatments is low and they do not understand their fibromyalgia and associated symptoms, they have a strong belief in the chronic and cyclical nature of the condition. Injured athletes have a weak illness identity associated with a high controllability; they see their injury as not chronic, and with minimal serious consequences. They understand the nature of their injury, given their high illness coherence and do not have a strong emotional representation. Conclusions: The illness perceptions in the two samples studied differ significantly, in a way that substantiates clinical impressions. These differences imply tailoring approaches by health care professionals. Assessing illness perceptions therefore seems useful for diagnostic and therapeutic decision making in patients with chronic conditions. Aim: To evaluate the effect of cognitive behavioral therapy (CBT) based stress management on FMS patients regarding pain, stress, and well-being. Study population and method: 48 women, 27-62 years of age were randomly allocated to two groups, an early treatment group and a waiting list group. A modified protocol for CBT originally designed for treating women after myocardial infarction was used with key components: education, self-monitoring, skills training, cognitive reconstructing and spiritual development. Brief relaxation was trained. The patients were treated in groups of 5-7 during three hours once week during half a year. During the next 6 months three "booster" sessions were held. Two experienced clinical psychologists with competence in CBT were responsible for the therapy. Outcome evaluation are based on Multidimensional Pain Inventory, the Type A behavior instrument, Vital exhaustion, Visual Analogue Scales measured pain, wellbeing and stress. Results: Perceived "life control" level increased during the intervention in the early treatment group from 3.1 to 3.5, but decreased in the waiting list group from 3.2 to 2.8. (p=0.01). There were similar but less pronounced differences for a number of other dimensions, but none was significant. Stress decreased from 53.7 to 35.9 mm in the early treatment group, but increased from 46.5 to 52.2 in the waiting list group (p=0.02). Pain and well-being showed similar trends but the differences were non-significant. Vital exhaustion decreased in the early treatment group from 24.9 to 22.0, but increased somewhat from 21.5 to 21.8 in the waiting list group (p=0.02). Stress behaviour decreased somewhat more in the early treatment group than in the waiting list group, Conclusions: CBT stress management given to female patients with fibromyalgia increased life control and decreased stress and vital exhaustion. The experience of pain was not significantly reduced. CBT has the potential to improve well-being among patients with cronic widespread pain. Background: Obesity is a leading cause of morbidity and mortality. Nearly ¼ of the UK population are obese, and by 2050 this is predicted to reach 60%. This has enormous cost implications for the NHS. Consequently, tackling obesity is a Government priority. An evidence-base exists of theoretically-informed behaviour change techniques for weight loss, however, in routine practice, doctors more commonly use theoretically-unfounded communication strategies (e.g. information giving). It is not known if the current focus on communication skills teaching during undergraduate training adequately prepares future doctors for this growing challenge. Objectives: To examine 1) what behaviour change techniques medical trainees use to facilitate behaviour change in obese patients and 2) how this impacts on patient behavioural intentions. Methods: Forty-eight students in their clinical years of a UK medical school were recruited to perform two simulated consultations each. Both consultations involved an obese patient where weight loss was indicated. Simulated patients (SPs) were used to standardise patient variables (e.g. barriers to behaviour change) and presentation of scenario order was counterbalanced. Following each consultation, students assessed the techniques they perceived themselves to have used. SPs rated the extent to which they intended to make behavioural changes. Anonymised transcripts of the audiotaped consultations were coded by independent assessors, blind to student and SP ratings, using a validated behaviour change taxonomy (Abraham & Michie, 2008) . Results: Students reported using a wide range of evidence-based techniques. In contrast, codings of observed communication behaviours were more limited, focusing on information-giving strategies. SPs behavioural intentions also varied and was explained with reference to students' communication. Treatment of fibromyalgia (FM), a chronic pain disease characterizes by widespread pain is regarded as challenging and the prognosis for recovery is poor. Psychological interventions are known to be effective in other pain disorders and therefore could be a promising treatment of FM. But only few qualitative reviews with divergent conclusions and no meta-analysis on this subject exist. The aims of the present analysis were to investigate the short-and long-term efficacy and treatment moderators of psychological interventions for fibromyalgia. A literature search identified 23 eligible studies with 30 psychological treatment conditions and 1396 patients. Meta-analytic integration resulted in a significant small effect size (g = 0.37, 95% confidence interval (CI): 0.27 -0.48) for short-term pain reduction and a significant small-to-medium effect sizes for long-term pain reduction over an average follow-up phase of 7.4 months (g = 0.47, 95% CI: 0.3 -0.65) for any psychological intervention. Psychological treatments also proved effective in reducing sleep problems (g = 0.46, 95 % CI: 0.28 -0.64) and depression (g = 0.3, 95 % CI: 0.17 -0.34). These effects remained stable at follow-up. Moderator analyses revealed cognitivebehavioural treatment to be significantly better then other psychological treatments in short-term pain reduction (g = 0.6, 95% CI: 0.46 -0.76) and cognitive-behavioural treatment and relaxation treatments in short-term sleep problems reduction (g = 0.68, 95% CI: 0.39 -0.97). Higher treatment dose was associated with better outcome. Publication-bias analyses demonstrated that the effect sizes were robust. It is concluded, the effects of psychological treatments for fibromyalgia are promising and comparable to those reported for other pain symptoms or drug treatment in FM. Psychological interventions of sufficient length should be included in FM treatment programmes.
Conclusions: It is suggested that current skills-based communication programmes inadequately prepare future doctors for the growing task of facilitating weight loss. Findings are discussed in relation to the social cognitive basis of behaviour change and communication training needs for health professionals. Clinicians are increasingly confronted with illness associated with health behaviors (e.g. diet and exercise). Helping patients to adopt healthier behaviors therefore is an important component of medical management and disease prevention. Communication training is a core feature of medical and nursing training. However, recent research reveals that communication training inadequately equips new health professionals for this growing clinical challenge and practitioners remain unconfident about how to discuss behavior change usefully with patients. The study aims to explore how doctors and nurses manage behavior change and identify what barriers operate to prevent effective behavior change management. Semi-structured interviews were conducted with a purposive sample of 14 medical and 14 nursing practitioners at a range of stages during their training (undergraduate and postgraduate). Interviews were transcribed verbatim and analysed thematically using principles of constant comparison to categorise emergent and recurring themes within and between transcripts. Thematic categories arising in initial interviews were explored subsequently and disconfirmatory evidence was sought until thematic saturation arose. Findings reveal that trainees and practitioners consider behavior change to be a fundamental part of medical practice, yet one they are not formally trained in. Participants had developed a range of strategies to encourage behavior change through experiential learning, but were unconvinced of their efficacy. Further barriers to implementing behavior change included the uncertainty over whose responsibility it is, the influence of patient authority within consultations, and desire to protect the doctor-patient relationship. Evidently current communication training is inadequate to equip health practitioners to motivate behavior change. Barriers arise from clinician-patient interactions as well as the context of health care services and have important implications for medical education. Background: Lifestyle-related diseases are increasing worldwide, and it is important to address lifestyle issues in primary health care. In order to facilitate this, a tool for lifestyle intervention was developed and implemented in primary health care in Sweden. Implementing new methods, guidelines or tools into routine care, however, is a slow and unpredictable process, and the factors that play a role in the adoption process are not yet fully understood. Objective: To identify key factors influencing the adoption of a lifestyle intervention tool being introduced in primary health care (PHC) in Sweden. Methods: A computer-based test for lifestyle intervention was implemented at six PHC units in Sweden. Two different implementation strategies, implicit or explicit, were used. Contextual factors and organizational climate were measured prior to implementation. Outcome in terms of staff referral rates to the lifestyle test was measured after six months of operation. A qualitative study where staff at the units participated in focus group interviews was performed. Interviews were analyzed using the method qualitative content analysis. Results: A positive organizational climate combined with an explicit implementation strategy did predict a positive implementation outcome when a lifestyle intervention tool was introduced in PHC. Adoption was positively influenced by positive expectations among staff, perceptions of the tool being compatible with existing routines and perceived advantages of the new tool. Organizational changes and staff shortage coinciding with implementation seemed to be obstacles for the adoption process. Conclusion: Organizational climate and staff expectations should be assessed in advance when a new tool is to be introduced in PHC. Explicit implementation strategy is preferable, and coincidence with major organizational changes should be avoided. The cervical cancer vaccine provides a safe and effective means of reducing the incidence and mortality rate from this disease. Given that uptake is less than optimal, despite the availability of no cost vaccination, there is a need to identify psychosocial factors associated with this preventive behaviour. Applying the Common Sense Model, we first identified the illness representations associated with the acceptance and non-acceptance of the cervical cancer vaccine among young Australian women aged 18-26. We then assessed whether information regarding the link between the viral causes of cervical cancer and the effect of the preventive vaccine can function as a facilitator of future vaccination behaviours. Participants (N=132) initially completed measures of vaccination status, intentions to be vaccinated, illness representations, perceived risk, perceived vaccine effectiveness and cervical cancer knowledge. Then we experimentally manipulated illness coherence relating to links between the Human Papillomavirus, cervical cancer and the vaccine, by randomly assigning participants to receive either a brief or detailed information condition, to ascertain the effect of this manipulation on vaccination intentions for non-vaccinated women. Higher levels of illness coherence, identity, and perceived vaccine effectiveness, having had a Pap smear in the past, and lower beliefs in the treatment cure/ controllability of cervical cancer were associated with vaccination uptake. Higher intentions to be vaccinated among non-vaccinated women were associated with higher levels of illness coherence and perceived risk for cervical cancer. Post-manipulation measures indicated that the detailed condition of the experimental manipulation resulted in significant increases in illness coherence. The identification of illness cognitions and the role of illness coherence influencing the acceptance of this vaccine adds to the paucity of information available on this underrepresented sample and provides direction for the design of future intervention strategies aimed at modifying illness cognitions to increase vaccine uptake. Although levels of consumption of dietary fibre (DF) are high in Australia, recent evidence indicates the need for increased intakes of the lesser known fibre form, resistant starch (RS), which has been implicated in the reduction of risk of serious diet-related health conditions. The purpose of the present study was to identify the kinds of health messages and the kinds of foods likely to be most acceptable to consumers. Data were obtained by means of a nationwide postal survey (N=849). Ratings of the importance of various RS health and functional claims and receptiveness to different foods as RS delivery vehicles were analysed according to the respondents' level of fibre engagement, as classified under the Precaution Adoption Process Model (PAPM) of Health Behaviour (Weinstein & Sandman, 1992) . Significant gender differences were found, with women found to be more fibre engaged and also more receptive than men to RS and its potential benefits. The pattern of association between fibre engagement and self-perceived risk of serious health conditions was consistent with previous observations of changes in risk perception at different PAPM stages. Participants' ratings revealed a general preference for healthy staples over indulgences as foods for the delivery of RS, with the margin in preference increasing markedly with increased dietary fibre engagement. This application of the PAPM illustrates its potential usefulness as a segmentation tool for the exploitation of community engagement with a relatively familiar health concept or behaviour in the promotion of a novel, related concept or behaviour. Unhealthy diet and inactive lifestyle reflect prevalent, correlated behaviors that heighten chronic disease risk. Intervening on more than one risk behavior simultaneously could be efficient, but the optimal goal framing remains unknown. MBC was designed to test which combination of two behaviors (one dietary, one activity) and two goal frames (increase healthy, decrease unhealthy) maximizes healthy behavior change across all four risk behaviors. N = 200 participants with all four risk behaviors [high saturated fat intake (Fat), low fruit and vegetable intake (FV), low physical activity (PA), high sedentary leisure screen time (Sed)] were randomized to one of four goal prescriptions: ↑FV↑PA; ↓Fat↑PA; ↑FV↓Sed; ↓Fat↓Sed. They used handheld technology and coaching to change one diet and one activity behavior simultaneously, while change in untargeted (collateral) behavior was also measured. The Familiarity hypothesis predicted best outcome from usual dieting behaviors (↓Fat↑PA). The Optimal Substitution hypothesis, based on behavioral economic theory, predicted best outcome from substituting healthful for unhealthful behaviors (↑FV↓SED). The Low Inhibitory Demand hypothesis, based on Baumeister's self-regulatory strength model, predicted best outcome from minimizing demand on self-control resources (↑FV↑PA). To scale all behaviors to a common metric, the primary outcome was healthy lifestyle change expressed as a z score reflecting improvement relative to the sample baseline for each behavior and averaged to yield a composite score. As predicted by behavioral economic theory, mixed linear models showed that ↑FV↓Sed maximized healthy lifestyle change compared to alternative interventions [t(345) = 3.3, p=.001], producing a sustained 1 s.d. improvement in saturated fat intake as well as in both targeted behaviors. The untargeted decrease in saturated fat intake complemented the targeted reduction in recreational screen time (r=.29, p=.04). Paralleling observed behavior changes, ↑FV↓Sed improved self-efficacy for changing FV (p<.05), Sed (p<.01), and, marginally, Fat (p=.09). Conversely, the traditional dieting regimen (↓Fat↑PA) produced less healthy lifestyle change than alternative prescriptions [t (345) = − 2.09, p=.04], yielding sustained 1 s.d. improvement only in saturated fat. Relatedly, ↓Fat↑PA failed to increase self-efficacy for targeted behaviors and decreased efficacy for FV and Sed (both p<.05). Increasing healthy low-rate eating behavior and decreasing unhealthy high-rate physical activity produced greater and unexpectedly sustained healthy lifestyle change than alternative prescriptions. Background: Research has demonstrated a strong link between the built environment, health outcomes & inequalities. Environmental elements can negatively impact lifestyle choices. The 'My Health Matters' project was designed to build partnership with members of the health economy to meet the challenge of increasing physical activity & healthy eating in three deprived areas of Stoke-on-Trent, UK. The project aims to develop & evaluate an intervention to reduce health inequalities by increasing physical activity & healthier eating as defined by the community. Results from phase I of the project will be reported including baseline mapping of the built environment using Geographical Information Systems (GIS) & a community survey. Methods: Environmental factors including convenience & proximity of physical activity spaces, greenspace & leisure facilities, neighbourhood connectivity/walkability, land use mix & population density, traffic, safety & crime, food outlets & restaurants, were measured using GIS mapping (calculated around each residential address). A community postal survey of randomly selected addresses (n=343) across the three areas was undertaken. The Neighbourhood Walkability Scale was used to assess residents' perceptions of their neighbourhoods. Perceived health (SF12), social capital, fruit & vegetable intake & levels of physical activity (International Physical Activity Questionnaire) were also measured. Results: GIS mapping illustrated a lack of local access to fresh food outlets but easy access to fast food outlets. 76-96% of residents were within 300 m walking distance of green space, but these spaces were of poor quality e.g. not functional. Physical activity facilities were within walking distance for some neighbourhoods but not for others. The community survey demonstrated that self-reported health was lower than average compared to England. Negative perceived characteristics of the neighbourhood included; aesthetics (e.g. lack of trees), The worldwide increasing prevalence of overweight and obesity is a cause for concern as the overweight-related morbidity, mortality and health care costs concurrently increase. The objective of our meta-analytic review is to critically examine the effectiveness of workplace physical activity and dietary behavior interventions on weight outcomes. Six databases were searched for English-written studies published between 1980 and November 2009. Two authors independently selected 43 studies which met inclusion criteria. Data could be extracted from 22 studies for meta-analysis. Methodological quality was assessed using a predefined checklist. The GRADE approach was used to determine the level of evidence for each pooled outcome measure. Results show there is moderate quality of evidence that workplace physical activity Introduction: Risk perceptions are a key component in many health behavior theories. However, such theories often do not differentiate between cognitive and affective risk perceptions (or the way in which they interact), and it is generally assumed that any increased risk perceptions of either type are associated with preventive behaviors. The present study aims to differentiate between cognitive (perceived risk) and affective (worry) risk perceptions and explore their direct and interactive relationships with two health-related behaviors. Methods: Combined data from the 2003 and 2007 Health Information National Trends Surveys were analyzed using logistic regression with perceived cancer risk, cancer worry, and their interaction independently predicting two dichotomous variables: a) fulfilling the 5-a-day fruit /vegetable consumption guidelines (n=7966) and b) engaging in any exercise in the past month (n=8069). Results: Neither perceived risk nor worry was significantly related to fruit/ vegetable consumption. However, their interaction was significant such that those who had both the highest perceived risk and worry were the least likely to fulfill the guidelines (OR: .77, p<.05). With respect to exercise, worry (OR=1.77, p<.01), but not perceived risk, was associated with exercise. More importantly, the interaction was again significant, such that those high in both perceived risk and worry were the least likely to report exercise (OR=.77, p=.01). Conclusion: Counter-intuitively, reporting both high perceived risk and high worry is associated with less exercise and produce consumption. This research may have important implications for interventions, which often do not differentiate between targeting cognitive and affective risk perceptions and may even strive to heighten both types of risk perceptions in efforts to prompt behavior change. In addition, this research may inform communication of risk information; optimal changes in health behaviors may be achieved when cognitive risk perceptions are lower, and affective risk perceptions are higher. Behavioral interventions have not yet been firmly established as being efficacious for reducing nonfatal or fatal cancer or coronary disease recurrence. Three separate randomized clinical trials using group based cognitive behavior therapy (CBT) however, have recently observed positive changes in disease endpoints in patients who had experienced breast cancer or adverse coronary disease events. Group CBT included 20-26 two hour sessions over one year. The trials all used strategies to reduce stress, improve mood, alter health behaviors and maintain adherence to medical treatment. These three studies all included women, used intentto-treat analyses and adjusted for relevant covariates. Andersen and colleagues randomized 227 women who had been surgically treated for regional breast cancer and found after a median of 11 years follow-up, a reduced risk of breast cancer recurrence (HR=.55, p=.034), breast cancer mortality (HR=.44; p=.016) and all-cause-mortality (HR=.51; p=.028). Orth-Gomér and collaborators randomized 237 women with acute coronary syndrome and found after a median of 7 years, a reduced risk of mortality (HR=.31; p=.009). Burell and colleagues randomized 362 women and men with acute coronary syndrome and found after a median of 8 years, a reduced risk of recurrent myocardial infarction (HR=.55; p=.007). Discussion will focus upon the implications of these results and the need for conceptual replication in large multicenter trials. A randomized clinical trial was designed to test the hypothesis that cancer patients coping with a cancer diagnosis but receiving a psychological intervention would have reduced risk for disease progression (1994) . Patients (N= 227) surgically treated for regional breast cancer participated. Before beginning adjuvant cancer therapies, patients were assessed with psychological and behavioral measures, had a health evaluation, and a 60 mL blood sample was drawn. Patients were randomized to Psychological Intervention or Assessment only arms. The intervention was psychologist led, conducted in small groups, and included strategies to reduce stress, improve mood, alter health behaviors, and maintain adherence to cancer treatment. Earlier papers (2004, 2007) showed that, compared to the Assessment arm, the Intervention arm improved across all of the latter secondary outcomes. T cell blastogenesis was also enhanced. An interim paper (2007) clarified treatment mechanisms in the Intervention arm. First, patients were satisfied with the intervention, but it was group cohesion that related to better outcomes. Second, offering patients a stress conceptualization (Selye) and teaching them multiple coping strategies related to better health. Third, three relationships between treatment utilization and outcomes are noted: 1) Relaxation training was associated with both distress reduction and symptom lowering. 2) Patients' use of assertive communication with health care providers was associated with better health outcomes. 3) Intervention strategy use was also associated with fewer signs/symptoms and cancer treatment toxicities. After a median of 11 years follow-up, recurrence occurred for 62 of 212 (29%) women and death for 54 of 227 (24%). Intent to treat hazard analyses showed, as predicted, Intervention arm patients had a Psychosocial factors are independently associated with increased risk of cardiovascular disease (CVD) morbidity and mortality, but the outcome effect of intervention for these factors on these endpoints has so far been uncertain. 362 men and women, aged 75 or less, discharged from hospital after a coronary heart disease (CHD) event within the past 12 months, were randomized to cognitive behavioral therapy (CBT) focused on stress management during one year (n=192), or to usual care (n=170). Median attendance rate to the intervention program was 85%. Risk factor and quality of life data were measured at baseline and after 6, 12, 18, 24 months. Hospital admission data and survival data were obtained from national registers. During 8 years of follow-up the intervention group had 41% less fatal and non-fatal first recurrent CVD rate, and 45% less recurrent acute myocardial infarction (AMI) rate than the reference group. The study was powered to examine the combined endpoint of fatal and nonfatal recurrent coronary event rate and found a positive significant difference between groups (HR 0.59, 95% CI 0.42-0.83, p=0.003). Subsequent analyses found a significantly reduced nonfatal recurrent acute myocardial infarction rate (HR 0.55, 95% CI 0.36-0.85, p=0.007), but no significant difference in allcause-mortality (HR 0.72, 95% CI 0.40-1.30, p=0.28). During the first 2 years of follow-up there were no significant group differences in quality of life, but the intervention group had a significantly stronger optimism about the future. The CBT stress intervention decreased the risk of recurrent CVD and recurrent AMI. A dose-response relationship between therapy attendance rate and risk was observed. This has implications for targeting and strengthening of secondary preventive programs in CHD patients. Psychosocial stress may accelerate atherosclerosis progression and worsen prognosis in women with coronary heart disease (CHD). In Stockholm women, we found marital stress to be a stronger predictor than job stress, but behavioral interventions to reduce such psychosocial stressors in women had not been identified. Based on previous findings, we implemented a cognitive behavior therapy (CBT) program for women and investigated its ability to improve survival in women coronary patients. Consecutive women patients, aged 75 years or younger, hospitalized for acute myocardial infarction, coronary artery bypass grafting, or percutaneous coronary intervention were randomized to group-based CBT or to usual care. Intervention groups of 4 to 8 women met for a total of 20 sessions that were spread over a year. We provided education about risk factors, relaxation training techniques, methods for self-monitoring and cognitive restructuring, with an emphasis on coping with stress exposure from family and work, self-care and compliance with medical advice. From randomization until end of follow-up (mean duration, 7.1 years), 25 women (20%) in the usual care and 8 women (7%) in the CBT condition died, yielding an almost three fold protective effect of the intervention (HR = 0.33; 95% CI, 0.15 to 0.74; P= 0.007). Controlling for relevant demographic and medical prognostic factors in multivariate models, confirmed the unadjusted results. CBT improved survival independently of medication with statins. Additionally, an interactive effect of statins with behavioral intervention was suggested. In women who received both treatments, only one woman died, (1.5 %) as compared to 15 out of 70 (22 %) women who received none of the treatments(p=0.007). We concluded that a group-based CBT program for women with coronary disease may prolong their lives. This effect may be independent of clinical prognostic factors. To evaluate correlates of sexual risk behaviors associated with HIV/AIDS infection among adolescents participating in 2005 Colorado Youth Risk Behavioral Survey, since previous studies have produced mixed findings. The State mandates abstinenceonly program across all public schools. Multinomial logistic regression was used to evaluate relationships among dependent variables and independent variables. We found no significant effect of having received in-school HIV/AIDS education on all outcome measures. Compared with females, males were more likely to initiate sex at a relatively younger age, report unprotected sex with multiple partners, and to drink alcohol before sexual intercourse. Efforts to control the HIV/AIDS epidemic among adolescents may need to focus on targeted interventions aimed at addressing gender-and racial/ethnic-specific risk exposures among this population group, including risk behaviors linked with lifetime sexual abuse. The need to re-examine the role of in-school HIV prevention programs to build adequate competencies among students, parents and community leaders is recommended. Irritable bowel syndrome is a highly prevalent functional gastrointestinal disorder (FGD) in western countries. Often, the disorder occurs in comorbidity with additional functional symptoms like chronic pelvic pain, fatigue or psychiatric disorders like anxiety and depression. Females suffer at least twice as often from FGD than men. Irritable bowel syndrome (IBS) is the most prevalent FGD and research shows a strong association between chronic stress and trauma in these patients. Due to the increasing knowledge on psychobiological mechanisms regulating adaptive processes to stress, research focuses on functional syndromes, which seem to be associated with maladaption of biological stress regulatory systems. This symposium refers to the different biological factors contributing to the etiology and maintenance of IBS. Besides prevalence rates on the high number of females with IBS, naturally occurring variation of female life conditions like menstrual cycle will be discussed with respect to the occurrence of IBS symptoms. Additionally, data on different biological systems like the HPA axis, pain perception, serotonergic pathways will be presented with reference to IBS and possible treatment approaches. Background & Aims: Stress has been considered as an important etiological factor in IBS. The physiological mechanisms between stress and gut disturbances, however, are incompletely understood. The current studies aimed to investigate the association between different stressors and functional gastrointestinal (FGI) complaints in female IBS patients and healthy controls. Methods: In an Internet-based survey conducted in a large number of students, the prevalence of FGI symptoms and perceived stress (chronic stress, coping strategies, stress reactivity) were measured. In a second trial basal and stimulated HPA axis activity was assessed in 57 women with IBS and 20 matched controls. IBS diagnosis was made according the Rome III criteria and psychiatric comorbidity was assessed using a clinical interview. Salivary morning cortisol and diurnal profile were obtained and a standardized psychosocial stress test (TSST) was applied. Cortisol and ACTH were measured before and within one hour following the stressor. Results: Two third of the online study sample (n=668) reported FGI complaints which were significantly predicted by female gender, high levels of chronic stress, dispositional stress reactivity, and use of maladaptive coping. The biological results indicated that female IBS patients have intact circadian rhythmicity of the HPA axis. However, women with diarrhea predominant IBS (IBS-D) exhibited substantially heightened cortisol at awakening and nearly no morning increase. In response to the laboratory stressor significantly blunted cortisol (p< 0.05) and slightly attenuated ACTH secretion was observed in patients compared to controls. In the recovery period ACTH levels were significantly lower in patients than in healthy subjects (p<0.05). Women with IBS perceived higher general and situation specific stress susceptibility. Conclusions: FGI syndromes, including IBS, seem to be common in apparently healthy subjects whereas female gender is a predictor. Several naturally occurring stressors are associated with the presence of FGI symptoms. In both studies psychological stress measures were overall increased in the IBS group and propose heightened stress susceptibility. Biological data indicate an association between basal HPA axis activity and predominant bowel habit. Furthermore, the downregulated HPA axis reactivity in women with IBS following the laboratory stressor (TSST) suggests an attenuated sensitivity of the endocrine system. The strong relationship between stress experience and the occurrence of IBS indicates that stress-reducing interventions may be beneficial in such individuals. Inst. of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Essen, Germany and 2 Institute of Diagnostic and Interventional Radiology and Neuroradiology, University Hospital Essen, Essen, Germany.
Background: In this set of fMRI studies, our goal was to establish experimental pain paradigms to address emotional and cognitive modulation of the behavioural as well as neural response to visceral pain. Method: The blood oxygen level-dependent (BOLD) response to painful rectal distensions was assessed at baseline, in a negative emotional context induced by psychological stress (emotional modulation study: carried out in 15 female IBS patients and 12 healthy women) and in a visceral placebo analgesia paradigm (cognitive modulation study: carried out in 36 healthy subjects). For visceral placebo analgesia, a deceptive study paradigm was used in which expectation of analgesia was manipulated by giving subjects different instructions regarding an intravenous infusion of either "a highly potent analgesic drug" or "an inert substance", when in reality all subjects received a placebo (saline) during painful rectal distensions. Results: Distensions delivered in a negative emotional context increased distension-induced discomfort and urge to defecate in IBS patients but not controls. At the same time, IBS patients demonstrated more pronounced stress-induced modulation of neural activation during rectal stimulation in the insula, midcingulate cortex, and ventrolateral prefrontal cortex. The placebo analgesia paradigm, carried out in healthy subjects, revealed that manipulation of expectation resulted in significantly decreased subjective pain ratings. Placebo responders demonstrated significantly greater activation of the dorsolateral prefrontal cortex during expectation of analgesia compared to non-responders. Discussion: Pain amplification and hypervigilance in IBS may be related to altered affective-cognitive modulation of the pain response.
CORRESPONDING AUTHOR: Sven Benson, PhD, Inst. of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Essen, 45122; sven.benson@uk-essen.de SS06c FOOD, MOOD, GENES, AND GENDER Paul Enck, PhD and Sibylle Klosterhalfen, PhD Psychosomatic Medicine, University Hospitals, Tuebingen, Germany.
The importance of serotonin in regulation of central (mood, hunger) and peripheral (intestinal) functions has long been established. Nutritive interventions however, e.g. the increase in dietary tryptophan -the metabolic precursor of serotonin -for the treatment of eating disorders (anorexia), mood disorders (depression), migraine, and the irritable bowel syndrome have not produced significant Results: The unbalanced population prevalence of these disorders in men and women allows concluding a contribution of gender to the efficacy such interventions. Shortterm depletion of tryptophan from food (called acute tryptophan depletion, ATD) has been shown to provoke the respective symptoms both in healthy volunteers as well as in patients, but seems to work predominantly in women. Therefore, gender differences must exist in the serotonin metabolism and its utilization in the brain and in the periphery. In addition, genetic predisposition has been shown to exist that determines -among others -the effectiveness of serotoninergic interventions in patients, with drugs as well as with nutrients. We will review the current evidence for the contribution of gender on food, mood, and genetic determinants of serotonin utilization. In the U.S. and other western industrial countries more women than men seek health care services for irritable bowel syndrome (IBS). In addition, the health care impact of IBS also appears to be greater in women as compared to men. Women with IBS commonly report other somatic and visceral symptoms. Muscle and joint pain and disturbed sleep patterns are among the most common and the most distressing. Gender differences exist in types of GI symptoms (women report greater problems with constipation while men more frequently report diarrhea) as well as other extra-intestinal symptoms. The role of menstrual cycle in IBS symptom reporting is based on observations that women with IBS frequently report cyclic changes in their symptom severity. Women with IBS often report other menstrual cycle linked conditions including migraine headache, dysmenorrhea and premenstrual dysphoria distress syndrome. Gender differences in IBS begin to emerge at puberty. In children, the rubric of recurrent abdominal pain (RAP) includes IBS, functional abdominal pain, and likely, dyspepsia. Prior to puberty there is an equal proportion of boys and girls with a diagnosis of RAP. The factors responsible for the divergence in prevalence after puberty are not clear. Studies suggest that both environmental (i.e., increased risk if a parent has IBS) and gender factors (i.e., changes in hormonal milieu) likely play a role. The incidence of IBS in both men and women appears to decrease in late middle age. The severity of GI symptoms, including abdominal pain, altered bowel habits, and bloating, varies across phases of the menstrual cycle as well as during the menopause transition in some women with or without IBS or other functional GI disorders. A limited number of studies indicate that premenses (days immediately preceding the onset of menses) and menses, compared with other cycle phases, are periods of increased GI symptom distress in women age 19-37. Women with IBS experience a greater impact of menstrual cycle on symptoms as compared to those without IBS. While not extensively studied, oral contraceptives appear to modestly reduce but not eliminate GI symptoms in those with IBS. This paper will present they hypothesis that declining or low ovarian hormone levels underlies the occurrence or exacerbations of abdominal pain/discomfort across the menstrual cycle and the perimenopause-early menopause transition in women with or without IBS. Increased availalability of data and recent developments in longitudinal modelling improve the possibilities to understand the long-term development of health. The symposium will present four papers exploring trajectories of health and health-related behavior -based on annual measurements over 9-18 years -in relation to retirement, sickness absence, and socioeconomic status. The two first papers show that retirement was associated with a substantial decrease in fatigue, depressive symptoms, and increase in adherence to medical treatment among individuals with chronic hypertension following retirement. Poor adherence before retirement was more common among individuals exposed to high work stress. The third paper shows that the association between diagnosis-specific sickness absences and sustained sub-optimal health varies by socioeconomic position. While the association with self-rated health was significantly stronger for mental disorders than for musculoskeletal disorders in the higher grades, the inverse was true in the lower grades. The fourth paper shows that weight continues to increase in the transition between midlife and old age, and that this increase is greater in the lower socioeconomic groups. As discussant, research director Archana Singh-Manoux will highlight the methodological possibilities and challenges in understanding the long-term development of health. Objectives: To determine, using longitudinal analyses, if retirement is followed by a change in the risk of incident chronic diseases, depressive symptoms, and fatigue. Design, Setting, and Participants: A prospective cohort study with multiple repeated annual surveys from seven years before through seven years after retirement in a large French occupational cohortthe GAZEL study -of 11,246 men and 2,858 women. Data were collected by questionnaire each year from 1989 through 2007 inclusive. Main Outcome Measures: Respiratory disease, diabetes, cardiovascular disease and stroke, mental fatigue, and physical fatigue, measured each year of the 15-year observation period, and depressive symptoms, measured at four time points. Results: Repeated-measures logistic regression with generalized estimating equations showed that the cumulative prevalence of respiratory diseases, diabetes, and cardiovascular disease and stroke increased with age, with no break in the trend around retirement. In contrast, we found retirement to be associated with a substantial decrease in the prevalence of both mental fatigue (odds ratio [OR] for fatigue one year after versus one year before the retirement 0.19; 95% CI 0.18 to 0.21) and physical fatigue (OR 0.27; CI 0.26 to 0.30). A slightly smaller decrease was observed in depressive symptoms (OR 0.60; CI 0.53 to 0.67). The decrease in fatigue around retirement was more pronounced among those with chronic disease before retirement.
Conclusions: Retirement appears not to change the risk of major chronic diseases, but it is associated with a substantial reduction in mental and physical fatigue and depressive symptoms, particularly among participants with chronic disease. Future research should investigate the generalisability of the findings to other countries and settings, examine the consequences of fatigue among older workers, as well as explore various possible determinants of such fatigue. Background: Retirement is a major social transition believed to have important consequences for health, but empirical evidence remains contradictory. The effects of retirement on the risk of specific diseases with major public health importance are unknown. Because poor adherence to treatment can increase the risk of severe medical complications, we examined if retirement is followed by a change in the use of antihypertensive medication among individuals with chronic hypertension. Methods: We linked an occupational cohort of 1820 retired employees in Finland (71% women) entitled to special reimbursement due to chronic hypertension before retirement to national health and prescription registers. The prescription register contains data on the exact dates and defined daily dosages of every purchase of prescription drugs. Annual use of antihypertensive medication was measured for each year during a period covering 4 years before and 4 years after the year of retirement. Results: Repeated-measures showed that adherence was significantly better in the years after retirement compared to the years before retirement. The ratio of mean number of days not covered by purchased prescribed drugs in the four years following, compared with the four years preceding, retirement was 0.74 (95% CI 0.67-0.81) after adjustment for sex, age at retirement, occupational status and year of retirement. This improvement in adherence was observed in both sexes, in all occupational grades and irrespective of type of retirement. However, the improvement in adherence following retirement was significantly greater among employees with high job strain before retirement compared to the others: the mean ratio of days not covered by prescribed drugs in the post-retirement years compared to the pre-retirement years 0.58 (95% CI 0.47-0.70) in the high strain group and 0.78 (95% CI 0.71-0.87) in the low strain group (test of interaction, p= 0.0004). Further adjustments for baseline health indicators had little effect on these associations. Conclusions: Repeated measurements provide evidence for a substantial and sustained increase in adherence to medical treatment among individuals with chronic hypertension following retirement. Poor adherence before retirement is more common among individuals exposed to high work stress. Because inadequate treatment of chronic hypertension increases the risk of severe medical complications, our findings suggest that the retirement-related relief of work-stress may decrease the risk of complications in high-risk populations. Background: Previous studies show a remarkably persistent association between sickness absence and future long-term selfrated health status for the majority of diagnostic categories for sickness absence but it is unclear whether this association differs by socioeconomic position. Methods. Prospective occupational cohort of 15,320 employees (73% men) aged 37 to 51, of whom 30% were in higher, 55% intermediate, and 15% in the lower occupational grades. Sickness absence records 1990-1992, including 13 diagnostic categories, were examined in relation to self-rated health measured annually for the years 1993-2006. Results: Among higher grade employees 9.5% had >30 days of sickness during the 3-year exposure window. The corresponding figure among intermediate and lower grade employees was 24% and 40%, respectively. Repeated-measures logistic regression analysis adjusted for age, sex, and chronic diseases showed higher, intermediate, and lower grade employees with >30 absence days, compared to those with no absences, to have 2.22 (95% CI 1.90-2.60), 2.13 (95% CI 1.95-2.33), and 1.86 (95% CI 1.58-2.20) times higher odds of suboptimal health over the 14 years of follow-up, respectively. Most of the 13 sickness absence diagnos-tic categories independently predicted an increased risk of sustained sub-optimal health in higher and intermediate grades.
However, few categories were predictive in the lower grades; mental disorders, diseases of the circulatory, genitourinary, and musculoskeletal systems. While the association with self-rated health was significantly stronger for mental disorders in the higher grades, there was no association with musculoskeletal disorders. Conclusions: The ability of diagnosis-specific sickness absences to predict sustained sub-optimal health differs by socioeconomic position. The remarkably persistent association between sickness absence and future long-term self-rated health status for the majority of diagnostic categories for sickness absence was observable only in higher and intermediate grades, while for lower grade this association was less ubiquitous. Objectives: To examine socioeconomic differences in trajectories of body mass index (BMI) and obesity between the ages of 45 and 65. Methods: 13 541 men and 4 652 women from the French GAZEL study reported their height and weight annually over 18 years, starting in 1990. Occupational position (managers, skilled and unskilled workers) at age 35 was the indicator of socioeconomic circumstances. Changes in Body Mass Index (BMI) and obesity between ages 45 and 50, 50 and 55, 55 and 60 and 60 and 65 were modelled using Linear Mixed Models (Generalized Estimating Equations). Results: BMI and obesity increased between the ages of 45 and 65, albeit at a slower pace at older ages. In men, BMI was higher among unskilled workers compared to managers at age 45, the difference increased from 0.83 kg/m2 (95% confidence interval (CI): 0.66, 0.99) at 45 years to 1.12 kg/m2 (95%CI: 0.90, 1.33) at 65 years. Estimated obesity rates were 3.37% and 7.54% in managers and unskilled workers at age 45 and 9.36% and 18.46% at age 65, the difference increasing by 4.93% (95%CI: 2.81, 7.13). A similar trend was observed in women. Conclusions: This study shows that weight continues to increase in the transition between midlife and old age; this increase is greater in the lower socioeconomic groups. Knowledge translation or exchange between health research, practice, and policy has received active attention recently. Experiences and models from the more individual level behavioral or clinical medicine need to be expanded to include complex organizational and political realities of more contextual health promotion and public health fields. The challenge of such expansion of models is increased all the more when moving from a national to a global perspective. The symposium will contribute to the latest international developments and discussions in bridging between research knowledge, practice and policy making both within health care and health promotion. Allan Best will present the results of a Canadian Institutes of Health Researchfunded meta-narrative review of conceptual models for knowledge to action, and a series of international workshops focused on "embedded" research or alternative organizational or network models to foster and support research applications to policy and practice. Arja R Aro will describe the experiences and challenges as well as present the 1st year evaluation results of the universitymunicipality contractual collaboration in health policy planning in Denmark. Edwin Fisher will describe models of peer support developed to support global promotion of peer support programs across differing cultures, populations, and health systems and then describe how that model is employed in global promotion efforts, focusing though at regional initiatives to demonstrate and advocate for peer support as a standard component of health care and prevention. The symposium will provide time for general discussion. A structural administrative reform in the Danish society in 2007 gave municipalities new responsibilities for preventive and health promotion activities. In this new situation a window of opportunity opened for knowledge exchange between academia, practice and municipal policy makers. Academia was expected to produce research evidence for applicable and cost effective interventions and policies. On the other hand, academia lacked contextual information necessary for tailored research evidence. Collaboration between these different stakeholders has proved to be challenging. The Danish model consists of 3 year contracts for junior researcher-practitioner positions, co-funded by the university and municipalities with the aim to carry out scientific projectbased research relevant to municipalities. The 1st year evaluation of the collaboration with 3 case municipalities has shown as major benefits: increased access and use of research-based working methods in the municipalities; image lift of the municipalities; concrete products such as usable research results and lectures delivered; and support in project implementation. On the other hand, problems or challenges experienced have been: different working traditions and time scales; translating complicated research results into immediate and applicable practical knowhow; lack of time and capacity for knowledge exchange; and sometimes lack of mutual respect and trust between stakeholders. All in all, collaboration and knowledge exchange has already in the 1st year lead to longer-term collaborative health policy planning; increased knowledge exchange; and also to an interest to start an evidence-based knowledge center within the area of health promotion. Challenges ahead lie in developing capacity building among all stakeholder groups; organizational barriers; development of 'service packages' including contextually tailored research information e.g. of health profiles of communities; implementation in practice and policy making as well as longterm follow-up of the implementation. The way we think about how health research, practice and policy inform and interact with each other dramatically shapes our efforts to improve health and health care. And the ways in which many of us involved in health system improvements think about these interactions have changed markedly over the last five years. Specifically, growing recognition of how systems dynamics and contextual factors affect implementation demands new models, more comprehensive comparative studies, and the development of new measures to evaluate key contextual factors. This paper reports on a Canadian Institutes of Health Researchfunded systematic review of conceptual models for knowledge translation that employed the metanarrative methodology devel-oped by Greenhalgh for a major UK National Health Services review of diffusion of innovations in health organizations. Key dimensions along which conceptual models vary will be used to frame a discussion of recent Canadian experiences with organizational models that foster and support research applications to policy and practice, and a series of preliminary international workshops focused on the need for prospective comparative case studies to derive generalizable lessons about how best to design systems support for knowledge to action. The research questions were: 1. What are the distinct research traditions that guide conceptual models for KTA? 2. What is the nature and strength of the theoretical principles that underpin each model? 3. What is the nature and strength of the evidence related to the application of conceptual model(s) in real-world situations? 4. What are the important features of each model that distinguish it from other models? 5. How does each model address contextual factors known to influence KTA processes and outcomes? 6. How has each tradition developed over time as early models have been refined by the addition of new research? 7. What criteria might guide selection and refinement of the best model in a specific decision context? The presentation will conclude with a summary of key issues and priorities for further development of conceptual models and evaluation tools for international studies examining knowledge to action systems in healthcare. Peer support is at once a) a very powerful influence on behavior and well being in general as well as in health, b) at least as old as primates, c) widely provided around the world through formal programs and informal channels, but d) not well articulated in the health literature, e) without a well defined state of the art, and, therefore, f) not recognized as an important part of health care, and g) not systematically available to all who may benefit from it. Peers for Progress of the American Academy of Family Physicians Foundation is dedicated to accelerating the availability of best practices in peer support around the world. A major challenge is how to develop a coherent model of peer support that is applicable across widely differing cultural, organizational, and national settings. "Standardization by function, not content" guided development of a coherent yet broadly applicable and flexible model. Through a WHO consultation with experts from 20 different countries, literature review, and work with 14 grantees representing 9 countries, four key functions of peer support have been identified: assistance in daily management, social and emotional support, linkage to clinical care, and ongoing availability of support. These are implemented in varied ways, by telephone, individual meetings, group meetings, etc. but have been found applicable across varied settings. A second challenge is how to promote best practices in something that is as old as primates and widely implemented around the world. This has led to emphasis on promoting a coherent model of support around the key functions described above, but, then, working with existing programs to encourage information and model sharing, knowledge management, and advocacy through networks and collaborations focused at the national and regional level. Eventually, a network of networks would link peer support programs around the world, facilitating their quality improvement, helping them to achieve recognition of peer support as a key component of care through their own health systems, and, thereby, increasing their ability to reach the millions they might benefit. In spite of promoting regular physical activity in healthy workplace, Thai employers who have been either risks of cardiovascular diseases or healthy are becoming less active physically. Identification of effective strategies to enhance regular physical activity is becoming a priority for health care providers. The Transtheoretical Model (TTM) has been successfully applied to promote physical activity in different countries, but has been limited testing in Thailand. The purpose of this study was to test the TTM constructs of stages of change, processes of change (POC), self-efficacy (SE), and decisional balance (Pros and Cons), for their applicability as determinants of engaging in regular physical activity (PA) among Thai employers in workplaces. This study used a descriptive design with an accidental sampling of 668 Thai employers working in two workplaces in Saraburi provinces, Thailand. All participants completed questionnaires measuring demographic, regular moderate-intensity PA, and variables from the TTM constructs. Data were analyzed by using mean, standard deviation, analysis of variance, and discriminant analysis. The results of this study indicated that the weekly moderateintensity PA, processes of change, self-efficacy, and pros consistently increased across 5 stages. Finally, the set of variables including TTM constructs and weekly moderate PA was able to correctly classify stage for 37.3% of subjects. The present study indicated that TTM may be useful to develop strategies to enhance regular PA among Thai Employers in workplaces. Background: There is a growing body of research that has demonstrated that conscientiousness is associated with longevity and health status. In parallel research, job strain (characterized by high job demands and low job control) has been found to be linked to increased cardiovascular disease risk. Previous work has focused on potential explanatory mechanisms such that conscientiousness has been found to impact on health behaviors. However, little research has explored the extent to which conscientiousness may moderate the job strain-health outcome relationship and has been overly reliant on "snapshot" retrospective measurements of key health behaviors. Therefore, using a multilevel daily diary design, this study investigated the impact of conscientiousness on health behaviors and daily stressors in individuals working in high compared to low strain jobs. Methods: 420 employees (193 male, 227 female) completed daily diaries over 4-weeks. Day-to-day within-person effects of daily stressors (or hassles) on health behaviors were examined, together with the influence of conscientiousness and job strain. Results: Using Hierarchical Multivariate Linear Modeling, the results showed in individuals working in high strain jobs, conscientiousness and its facets were associated with lower fat and sugar snack intake, and higher fruit and vegetable consumption. Moreover, the facets of conscientiousness were found to also significantly moderate the impact of daily stressors on a number of health behaviors. In contrast, few significant direct or moderated effects of conscientiousness were identified in individuals working in low strain jobs. Conclusions: Our results show that: i) conscientiousness and its facets may directly buffer the effects of high strain on daily health behaviors and moderate the impact of daily stressors on these behaviors and, ii) the influence of conscientiousness Non-response analysis of a nationwide mail survey from Finland Response rates in health oriented survey research are declining. The bias caused by this is unclear. Aim: The aim of the study was to compare all-cause and disease mortality and mortality related to external causes among participants and non-participants of a large mail survey in Finland. Study design: The study is based on the complete sample (N=67 644) of the Health and Social Support (HeSSup) prospective mail survey initiated in 1998 with a panel follow up survey in 2003. Individuals and Methods: Of the original sample 67588 individuals could be re-identified in registries comprising 33 446 women and 34 142 men The original sample represented the concurrent age cohorts of 20-24, 30-34, 40-44 and 50-54 years of age in Finland. The response rate of the initial survey was 40 %, (N=25 901). The participants were asked a signed consent for linkage of the survey data to national health and pension registers which was given by 92%. In 2007, the survey material was completed by Statistics Finland with data on mortality and by the Finnish Centre for Pensions with data on cumulative incidence of disability pensions and rehabilitation allowance from 1998 to 2006. The same data was anonymously delivered for non-respondents. The statistical analysis was performed with Cox proportional Hazard models. Results: Total mortality was significantly higher for nonrespondent compared to respondent women in age group 50-54 years and for non-respondent men in age groups 40-44 and 50-54 years. Violent mortality was higher for non-respondent men in age brackets 30-34 and 40-44 years but not for any age group of women. Disease mortality was higher for non-respondent women in age group 50-54 years but not for any age group of men. A significantly higher incidence of disability pensions among non-respondents was observed, but this was almost totally due to pensions related to mental disorders, especially among women. Conclusion: Non-response might bias health survey Results: However, the greatest implications probably influence how results can be generalized and not necessary results about particular risk and protective factors. Background: Socioeconomic differences in health are well established, and stable or increasing in Europe. Psychobiological factors may be mediators of these differences. The differences appear to occur as a gradient, rather than in distinct classes. Explanations offered for these differences may be classified into two possibly interacting categories: structural factors and individual factors. In this study we examine how individually acquired expectancies of being able or unable to handle the stressors and challenges of everyday life relate to individual health and to the socioeconomic gradient. According to the Cognitive Activation Theory of Stress (CATS) learned response outcome expectancies affect health through health behaviours and stress responses. Methods: A cross-sectional study of response outcome expectancies, socioeconomic status and health in the 2008 follow-up of a representative sample of the Swedish working population in 2003-2005 (N=11441) . Findings: Response expectancy variables (coping, helplessness, and hopelessness) showed clear and monotonous gradients over a Socioeconomic Status (SES) ladder. Multiple regression analysis revealed that coping was related to subjective SES, but could not be explained by age, gender, or objective SES. After controlling for age and gender, response outcome expectancies explained more variance (r2=0.162) in self-rated health than subjective and objective socioeconomic status (r2=0.078). Interpretation: The results indicate that there are systematic differences in coping across the socioeconomic ladder. These differences are independent of objective social status and are associated with health, suggesting that psychological and learning factors contribute to the socioeconomic gradient in health. Colorectal cancer is the third leading cancer in incidence and mortality among US adults, and these rates are higher in the African American community than among other subpopulations. Theory-based health communication interventions aim to increase awareness and screening for this disease. The Health Belief Model is one of the most used models on which behavior change interventions and their evaluations are based. Previous work has been conducted to develop quality assessments of the Health Belief Model constructs in both breast and more recently in colorectal cancer contexts. However, instrument development initiatives have not focused specifically on disparities-affected populations such as African Americans. We present adapted instruments to assess Health Belief Model constructs, that were adapted/enriched based on qualitative research (e.g., focus groups) among African Americans. Two brief scales assess barriers to colonoscopy and fecal occult blood test, specific to the healthcare system. Internal consistencies were modest (α=.33; α=.43, respectively) and may indicate multidimensionality. A third scale assesses additional areas of colorectal cancer knowledge (α=78), based on the qualitative research findings. Factor structures and other psychometric properties are reported. Scores on the instruments were associated with screening behaviors. This work is consistent with the recent special issue of Health Education and Behavior, on "Behavioral Constructs and Culture for Cancer Screening", in which it is suggested that traditional behavior change theories developed for mainstream US culture may need to be adapted when working with disparities-affected populations such as racial/ethnic minorities. The National Centre for Occupational rehabilitation, Rauland, Norway and 2 Uni Health, Bergen, Norway.
Background: Longitudinal studies are required to understand more about the complex RTW process after rehabilitation and to explore if there are different sick-leave patterns between different sociodemographic subgroups. The aim of this study was to examine if duration of sick-leave before rehabilitation and socio-demographic factors as gender, age, medical diagnosis, and occupation predict different sick-leave patterns or RTW outcomes in a 5-year follow-up period after a 4-weeks inpatient occupational rehabilitation program. Methods: The sample consisted of 584 patients (66% females, mean age 44 years [sd=9.3], long-term sick-listed; mean duration 9.3 months [sd=3.4] for psychological (47%), musculoskeletal (46%) and unspecific/other diagnosis (7%). Register data from The National Insurance Administration in Norway was analysed in an 8 state-model: working, graded sick-listed, sick-listed, medical rehabilitation, vocational rehabilitation, part-time disability pension, time-limited disability pension and permanent disability pension. Extended proportional hazards regression models, were applied to analyze socio-demographic prognostic factors for shifting between any of the 8 states during the 5 years follow up. Background: Evidence on the association between social status inconsistency (SI) and self-reported health is limited and mixed.
Moreover, only little research has been done to examine the pathway through which SI affects health. Therefore we analysed the mediating effect of effort-reward imbalance at work (ERI) on this association. Methods: Analyses were based on a random sample of 1111 male employees (25-64 years) from the south-west of Germany. Data were collected by a telephone interview in 2008/09. Information on socio-demographic variables, social status indicators (education, occupational grade and income), occupational strain (ERI), and physical as well as mental health (SF-12) were given. Results: SI was significantly associated to low mental health, both directly and, consistent to our hypothesis, indirectly via low rewards. As SI was also related to low effort, no association was found for the quotient of both scales (ERI) and mental health. The effect of SI on physical health was entirely mediated by low rewards without any direct effect. The inclusion CITATION ABSTRACT 10:48 AM-11:06 AM OS08-B WORKPLACE BULLYING AND MENTAL PROBLEMS Eero Lahelma, PhD, Tea Lallukka, PhD, Mikko Laaksonen, PhD, Ossi Rahkonen, PhD and Peppiina Saastamoinen, MSc Department of Public Health, University of Helsinki, Helsinki, Finland. Background Bullying is a common problem at workplaces with potentially long-lasting adverse consequences for the mental health of the bullied victims. We studied whether workplace bullying is prospectively associated with mental problems as indicated by antidepressant use among middleaged women and men. Methods The participants were municipal employees aged 40-60 from the Finnish Helsinki Health Study cohort baseline survey in 2000-02 (n=8960, response rate 67%). Workplace bullying was divided into those who reported being currently bullied and those who reported having been previously bullied. Data on antidepressant prescriptions from the Finnish Social Security Institute registers were available before the baseline survey and were followed up until the end of 2007. The prescription data were individually linked to the baseline survey data for those consenting for such linkage (78%). Hazard ratios (HR) adjusted for age were calculated using Poisson regression analysis, excluding those with antidepressant use 1.5 years prior to the baseline survey. Results At baseline 5% of both women and men reported being currently bullied, and 19% of women and 13% of men reported having previously been bullied. Compared to the never bullied (HR 1.00) the currently bullied were more likely use antidepressants during the follow up among women (HR 1.55, 95% CI 1.09-2.22) as well as men (HR 2.57, 95% CI 1.46-4.52). The previously bullied were also more likely to use antidepressants but somewhat less so among men. Conclusions Victims of workplace bullying are disproportionately prescribed antidepressants, suggesting that they run the risk of developing mental problems as a consequence of bullying. Workplace bullying is an occupational and public health issue of concern which should be tackled in order to safeguard employee mental health and well-being. of further risk factors (age, BMI) did not attenuate these results. Conclusions: SI is significantly associated to ill health. Moreover, results indicate that at least a part of this relationship is explained by low rewards at work, findings with implications for further research in job strain and health inequality. Background: Return to work (RTW) in acutely hand-injured patients is not only influenced by biomedical determinants, but work-related and psychosocial determinants may influence RTW as well. Aim of this study was to investigate which factors determine RTW. Methods: Patients with acute hand injuries (n=69) answered several questionnaires on biomedical, work-related and psychosocial factors. All patients were between 18 and 65 years, employed, had sustained an acute hand injury and were treated in one of the two participating rehabilitation centers. Results: Primary outcome measure was RTW, dichotomized into early (≤10 weeks) and late RTW (>10 weeks). The majority of the patients (55%) returned to work in the late stage. Six percent of the patients did not RTW within one year, and 4% failed to RTW. Accident location (Chi2:10.101; p=.001), job independence (U= 372.000; p =.021) and symptoms of Post-Traumatic Stress Disorder (S-PTSD) (U=257.000; p= .001) were univariately associated with RTW, and were entered in the logistic regression analysis. Both S-PTSD (having more symptoms) and accident location (accident sustained on the job) were found to statistically predict late RTW in hand-injured patients. Exploration of S-PTSD: As S-PTSD are predictors of RTW in patients with acute hand-injuries, it is relevant to investigate which patients develop S-PTSD, and which patients do not. Therefore, we investigated which factors predict S-PTSD in this population. Pain (r = .530; p < .001), satisfaction with hand function (r=.−.451; p<.001), aesthetics (r=−.320; p=.009), palliative coping style (r=.281; p=.022) and avoidant coping style (r=.283; p=.022) were univariately associated to S-PTSD. By performing a linear regression analysis, pain and aesthetics of the hand were found to statistically predict S-PTSD (adjusted R2: 33.9%). Conclusion: When patients sustained their hand injury on the job, attention should be paid to RTW during rehabilitation. Furthermore, patients should regularly be checked for S-PTSD, especially if the aesthetics of the hand are severely disturbed, or when patients experience pain. Most working adults with low back pain (LBP) continue to work despite pain, but few studies have assessed self-management strategies in this at-work population. The purpose of this study was to identify workplace challenges and self-management strategies reported by workers remaining at work despite recurrent or persistent LBP, to be used as a framework for the development of a workplace group intervention to prevent back disability. Workers with LBP (n=38) participated in 5 focus groups, and audio recordings of sessions were analyzed to assemble lists of common problems and coping strategies. A separate analysis provided a general categorization of major themes. Workplace pain problems fell within 4 domains: activity interference; negative self-perceptions; interpersonal challenges; and inflexibility of work. Self-management strategies consisted of: modifying work activities and routines; reducing pain symptoms; using cognitive strategies; and communicating pain effectively. Theme extraction identified 6 overarching themes: knowing your work setting; talking about pain; being prepared for a bad day; thoughts and emotions; keeping moving; and finding leeway. To retain workers with LBP, this qualitative investigation suggests future intervention efforts should focus on worker communication and cognitions related to pain, pacing of work, and employer efforts to provide leeway for altered job routines. The development of newborn screening is benefitted by technological advances enabling blood spot testing to identify a wider variety of conditions (Stewart 2006) . Introduction of these advances through programmes of research delivered in parallel to routine screening requires consideration of the ethical issues involved and appropriate mechanisms for gaining parental consent (Pollitt et al. 1997 , Chadwick et al. 1998 ). Ponder et al. (2008 argue that ethical review needs to be more flexible in its attitude to the provision of information and consent processes for some types of research. Exploration of parental views on consent practices is needed to inform the debate, and specifically consider whether more flexible research protocols are needed to fit with the public perception of research in this context. This study has been undertaken to explore the views of parents and future parents regarding information provision and consent processes for expanded newborn screening in the UK. Focus groups (n=29) and a web-survey (n=142) were employed to capture parental experiences of routine screening, and perceptions and attitudes to an expanded screening programme which would constitute research.
The results indicate that the provision of information and informed choice for routine screening is variable and generally unsatisfactory. Parents want guaranteed information provision with clear decision making powers and an awareness of the choice available to them. A paper-based information leaflet on expanded newborn screening information was reviewed by parents and found to be adequate for decision making (90%). Recommendations are made for how this information can be further developed. The difference between routine screening provision and expanded screening was not considered to be significant enough by participants to warrant formal written, informed consent. 71% of the sample deemed the informed choice model adopted within current screening provision to be sufficient, supporting use of existing procedures within a research remit. Compared to non-obese women, obese women are at higher risk for colorectal cancer (CRC), but also have lower rates of CRC screening, particularly colonoscopy. We used mixed methods to better understand these disparities. We conducted seven focus groups on CRC prevention with obese (BMI≥30) white and African American women (age≥50) who were not currently adherent to colorectal cancer (CRC) screening guidelines (N=31). Discussions and coding were guided by the Health Belief Model. Focus groups were followed by an online survey with 109 obese and 89 non-obese women. We used Chi-square to compare weight groups on screening barriers. Focus group revealed that women did not believe they were at risk for CRC and had poor knowledge of CRC prevention; they did not clearly understand the role of weight or screening in cancer prevention. Many were unaware of potential risk factors for CRC and were much more aware of potential screening barriers than benefits. Cost and time were the most frequently cited barriers to screening. Further investigation revealed that women's time and healthcare dollars were focused on more pressing co-morbidities. While most women relied on their physician to bring up important health issues, the internet was their most frequent source of prevention information. Survey data confirmed that obese women were more likely than non-obese women to agree they had too many other health costs (p=0.049) or more important health concerns that took precedence over colonoscopy (p=0.045). There were no significant differences by weight group in agreement with the more general cost and time barriers to colonoscopy. Furthermore, a higher proportion (p=0.03) of obese women (41.1%) agreed that screening tests were embarrassing compared to on-obese women (28.6%). Obese women were more than twice as likely as non-obese women to agree that they would not need a Purpose: To examine the preliminary efficacy of a culturally sensitive educational video in promoting mammography use among Chinese-American women (age ≥40) who had never or had not been screened in the past 12 months. Methods: 546 Chinese immigrant women were randomized to three groups: 1) viewed a culturally sensitive video; 2) viewed a generic video; and 3) read a fact sheet (control group). Videos, guided by the Health Belief Model, were presented in "soap opera" format, ending with physician recommendations. The cultural video particularly addressed Chinese culturally-based beliefs about cancer and was made in Chinese language. In contrast, the multiethnic generic video targeted common health beliefs about mammography, not specific to Chinese women, and was made in English with Chinese dubs.
Our outcome was self-reported mammography use six months post-intervention. Changes in key screening barriers (i.e., knowledge, cultural views, and health beliefs) pre-and post-intervention were evaluated using T-tests and linear regression models. Participants were successfully randomized. Thus, chi-square tests were used to examine group differences in screening outcomes. Results: The cultural video led to a significant 12 percentage point increase in mammography use relative to the control arm (41% vs. 29%, p=.02; baseline screening rate=0). The increased screening rate of the generic video group (38%) did not significantly differ from that of the control group. The cultural video also significantly increased mammography use among low-acculturated and never screened women relative to the control arm (39% vs. 26%, p=.03 and 29% vs. 15% p=.056, respectively); the generic video did not. Both videos had a greater impact on improving women's knowledge, cultural, and attitudinal barriers to mammography (p<.0001) than the control arm. Conclusions: A culturally sensitive video program is relatively efficacious in increasing mammography use over a standard intervention program for Chinese women, suggesting that cultural tailoring of intervention programs is needed to promote low screening rates of this growing immigrant population.
screening if they did not have symptoms (p=0.04). Treatment of obesity-related co-morbidities may take precedence over cancer prevention in primary care. Messages which focus on prevention and screening benefits may improve healthy behaviors in obese women. This study investigated whether differential framing of CRC risk and Fecal Occult Blood Test (FOBT) information impacted upon cancer attitudes and screening behaviour. N=660 people 50-71 years were assigned to one of 6 intervention groups. They completed a laboratory-based survey measuring cancer attitudes. After reading information about CRC risk statistics (positive or negative framing, two-level variable) and FOBT advantages, disadvantages, or both/balanced (three-level variable), attitudes were re-measured. Participants were given a card to request a free FOBT kit. Return of card within 7 days was measured as intention to screen, and return of completed kit within 35 days was measured as screening uptake. Results were analysed using Chi-square statistics and repeated measures ANOVA to examine the influence of information provision, in its different forms, on attitudes to CRC screening. From pre-intervention to post-intervention, risk perception scores increased (F(1,448) = 18.24;p<0.001), and optimistic bias decreased (F(1,654) = 14.50; p<0.001). Powerful others locus of control increased (F(1,654) = 11.22; p<0.001), as did Chance locus of control (F(1,654) = 29.25;p<0.001). There were no treatment effects for any of these variables. There was no significant difference for intention to screen between the CRC risk information frames or FOBT information conditions. For screening uptake, there was a significant difference between intervention groups (X 2 (5) = 13.167;p<0.05). Data partitioning indicated that there was no difference between FOBT uptake for the CRC positive risk information group by FOBT information condition. There were differences between CRC negative risk information group conditions (X 2 (2) = 11.316;p<0.01). Significantly more participants in the FOBT 'advantages' condition screened than those in the FOBT 'disadvantages' condition (x 2 (1)=3.85; p<0.05) or the 'balanced' condition (X 2 (1) = 11.001;p<.0001).
Overall, results suggest that providing information about the risk of CRC and the nature of the screening procedure can assist people to change their attitudes, regardless of the way this information is framed. Although framing the message around the screening test in terms of the likelihood of not being affected by CRC combined with the advantages of using the FOBT appears beneficial in a sample of people prepared to come to a laboratory testing session, future research is required to test the generalisability of the result. In the U.S. and other western industrial countries more women than men seek health care services for irritable bowel syndrome (IBS). In addition, the health care impact of IBS also appears to be greater in women as compared to men. Women with IBS commonly report other somatic and visceral symptoms. Muscle and joint pain and disturbed sleep patterns are among the most common and the most distressing. Gender differences exist in types of GI symptoms (women report greater problems with constipation while men more frequently report diarrhea) as well as other extra-intestinal symptoms. The role of menstrual cycle in IBS symptom reporting is based on observations that women with IBS frequently report cyclic changes in their symptom severity. Women with IBS often report other menstrual cycle linked conditions including migraine headache, dysmenorrhea and premenstrual dysphoria distress syndrome. Gender differences in IBS begin to emerge at puberty. In children, the rubric of recurrent abdominal pain (RAP) includes IBS, functional abdominal pain, and likely, dyspepsia. Prior to puberty there is an equal proportion of boys and girls with a diagnosis of RAP. The factors responsible for the divergence in prevalence after puberty are not clear. Studies suggest that both environmental (i.e., increased risk if a parent has IBS) and gender factors (i.e., changes in hormonal milieu) likely play a role. The incidence of IBS in both men and women appears to decrease in late middle age. The severity of GI symptoms, including abdominal pain, altered bowel habits, and bloating, varies across phases of the menstrual cycle as well as during the menopause transition in some women with or without IBS or other functional GI disorders. A limited number of studies indicate that premenses (days immediately preceding the onset of menses) and menses, compared with other cycle phases, are periods of increased GI symptom distress in women age 19-37. Women with IBS experience a greater impact of menstrual cycle on symptoms as compared to those without IBS. While not extensively studied, oral contraceptives appear to modestly reduce but not eliminate GI symptoms in those with IBS. This paper will present they hypothesis that declining or low ovarian hormone levels underlies the occurrence or exacerbations of abdominal pain/discomfort across the menstrual cycle and the perimenopause-early menopause transition in women with or without IBS. Objective: Premenstrual dysphoric disorder (PMDD) and the premenstrual syndrome (PMS) are psychiatric phenomena, which are associated with the menstrual cycle in women. Symptoms of PMS include mood changes, temper outbursts/anger, lack of concentration and abdominal pain. These symptoms also form the diagnostic criteria for psychiatric disorders, such as depression, pain disorder (e.g. chronic pelvic pain) or somatoform disorder, which are unrelated to the menstrual cycle. Our aim was to identify similarities and differences of PMS with these symptoms during different phases of the menstrual cycle.
Methods: A PMS questionnaire which was based on the DSM-IV-TR criteria for PMDD, a screening for somatic symptoms (SOMS, Rief 2008) , and the German version of the CES-D (Radloff, 1977) were presented online. In a prospective design, questionnaires were presented at baseline (t1) and at a second assessment (t2 Objective: To examine predictors of treatment outcome in IBSpatients who participated in a randomized controlled trial in primary care, where 149 IBS-patients were randomised to mebeverine hydrochloride (n=77) or mebeverine+cognitive behaviour therapy (CBT) (n=72). CBT offered additional benefit over mebeverine alone. Methods: Regression analyses were used to identify predictors of work and social adjustment (WASA) 12 months after treatment ended. The intervention groups were analysed separately in order to look at the separate effects in each group. Results: Lower levels of psychological distress (anxiety and depression) at baseline predicted a good outcome in the mebeverine group (β=0.388 (95%CI: 0.065-0.936), p-value= 0.025) but not in the mebeverine+CBT group. In the adjusted model for the mebeverine+CBT group less adaptive IBS related behavioural coping predicted a good outcome (β=0.285 (95%CI: 0.002-0.210), p-value=0.045). Conclusion: Different factors are associated with outcome depending on the treatment received. At assessment clinicians should assess patients coping styles and may want to consider recommending CBT to those patients with IBS in primary care who are engaging in unhelpful coping behaviour. Background: In Norway, the majority of medically certified sickness absence is granted for musculoskeletal complaints and mental disorders. The challenge for the general practitioners (GP)
is that the majority of these cases tend to be unspecific without any clear and significant objective findings. Traditional medical thinking requires a diagnosis, in order to find a specific and efficient therapy or to grant sickness certificate. The challenge is to find the proper diagnoses when the condition is unspecific, with many complaints, and a high level of comorbidity. The International Classification of Primary Care-second version (ICPC-2) is used in Norwegian primary health care. Aims: Our aims were to investigate if a dramatised case story (presented exactly the same way to, Norwegian GPs), representing a patient with unspecific and/or comorbid complaints get the same diagnosis and decision on sick leave. Method: Fifty-six GPs watched 10 case stories and diagnosed each case with ICPC-2 diagnosis and made decisions on whether to sick list or not. Results: Between 15 and 23 ICPC-2 diagnoses were given to the case stories as main diagnosis. Psychological diagnoses were most frequent, followed by musculoskeletal. Three to five different ICPC-2 organ chapters were used for each case. Decisions on sick leave yes/no and different type of insurance benefits varied greatly within each case story. Discussion: As expected, the Norwegian GPs did not agree on which diagnoses to use for these patients, and suggested a high number of diagnostic labels from the ICPC-2 system. This is, in our opinion, not an indication of lack of competence in the GPs but that the systems used today are unable to classify and describe these complaints. The high number of possible diagnoses offered by experienced GPs suggests that the conditions are composite, with a high level of comorbidity and the diagnostic system is unable to identify this. For this kind of patients there is no consensus on what kind of treatment to offer and we argue that the only available "treatment" today is sick leave. This increases the risk of long term sick leave, and permanent disability. Conclusion: Case stories with unspecific and comorbid complaints are diagnosed with a large variety of ICPC-2 diagnosis in general practice and they are frequently put on sick leave. Results: The majority of the women and men describe several adverse experiences during childhood. From a total of ten possible types of adversity, the mean for women was 5.05 (SD=2.63) and 2.63 (SD=2.18) for men. There was a high frequency of health risk behaviour and psychopathological symptoms for both samples, women reporting significantly more psychopathology and sexual risk behaviours (no significant differences in other risk behaviours). Conclusion: Detainees had a high frequency of adverse experiences during childhood, have high levels of psychopathology and several risk behaviours. The differences between women and men in what concerns adverse childhood experiences and psychopathological symptoms suggest more adverse childhood histories for incarcerated women. Those differences should be better explored in order to understand if a self-report effect or a fact of their lives. This data suggest a need to take into account the adverse histories because violent behaviour can function as a coping strategy to deal with negative affect and psychopathology related to prior victimization. Objectives: Although refugees are at an increased risk to suffer from mental disorders, as compared to other immigrants, little is known about possible differences in treatment response and service utilization. Methods: The current study includes data from three separate samples of Iraqi refugees and Arab immigrants. Prevalence of physician-diagnosed mental disorders along with treatment response and utilization data was collected from medical records or by surveys. Data was collected from 307 refugees and 326 immigrant controls. Results: The prevalence of depression was significantly greater in refugees as compared to immigrants (54% vs. 26%; p<.001). There were no differences between groups in diagnosed anxiety disorders. In addition, self reported complaints of depression (33% vs. 3.1%; p<.001), anxiety (20% vs. 4%; p<.001), and sleep difficulties (15% vs. 2%; p<.001) were more prevalent among refugees than immigrants. In general, treatment response was poor for both groups; however, for longer term patients, treatment response was significantly better (44% recovery vs. 15% recovery; p<.001) for immigrants than refugees. Typical common psychosomatic symptoms were more prevalent and associated with increased health care utilization in refugees as compared to immigrants. Conclusions: Results suggest that treatment offered to refugees is far from effective. There is a need to review health systems factors in order to promote cost-effective and timely mental health services for refugees, not least with the current influx of Iraqi refugees commonly suffering from mental and adjustment disorders. We found a positive relationship between PTSD and psychopatology, health symptom and negative with quality of life. War veterans presented a high percentage of extreme families particularly those with PTSD. We also found differences between veterans with and without PTSD on social support, family functioning and quality of life. Predictors of quality of life and mediation analysis were also performed. According to these preliminary results, psychological intervention, on this population, is necessary to help veterans and their families to better cope with war trauma. Objective: By recent estimates, 200.000 persons were imprisoned for political reasons in the German Democratic Republic (GDR) between 1949 and 1989. Former political prisoners suffer from the experience of persecution also in a long term. Up to 50% of those persons develop a post-traumatic stress disorder. Verbal disclosure (the revelation of the traumatic events) to the social environment has been shown to have positive effects on PTSD symptom severity. Little is known about whether this effect is moderated by the receiver of the disclosure. Method: A total of 157 former political prisoners (82% male) from the GDR (mean age: 65 years, from 39 to 86 years old) were assessed with a questionnaire. Sociodemografic data, intrafamilial communication about the traumatic imprisonment and symptoms of the posttraumatic stress disorder were assessed by self-reports. IES-R (with the subscales intrusion, avoidance and hyperarousal) was used for the assessment of PTSD symptoms. Results: PTSD symptom severity depends on who is the receiver of disclosure in the family. Persons who report that the actively disclosed their traumatic experience to their partners have lower intrusion (score in the subscale: 18.2. vs. 21.8; p=.02) and lower hyperarousal symptoms (score in the subscale: 20.4 vs. 24.5; p=.05).
The same applies to active disclosure to their children (score in the subscale intrusion: 17.8. vs. 21.4; p=.02 and score in the subscale hyperarousal: 19.5 vs. 24.5; p=.01). Disclosure to parents and to grandchildren is not connected with lower PTSD symptoms. Conclusion: The results provide evidence that the concrete receiver of the disclosure in the family should be taken more into consideration. Future research on effects of disclosure should observe that. Objective: To compare smoking rates among girls and young women in seven ASEAN countries (Cambodia, Indonesia, Lao PDR, Malaysia, Philippines, Thailand and Vietnam) and sociocultural factors associated with it. Methods: A regional study on smoking among females was conducted in 2007/08. A quasi-random sample of about 3,000 female respondents between the ages of 13 and 25 from secondary schools and colleges/universities from the capital city of each country together with one rural district was surveyed using a standardized self-administered structured questionnaire. Each country data set was processed and analyzed using SPSS version 12. Logistic regression analysis was used to determine sociocultural factors significantly associated with current smoking (in the last month).
Results: Ever and current smoking rates varied substantially between these countries, highest in the Philippines (38 vs. 20%) followed by Indonesia (20% vs. 7%), Malaysia (19% vs. 7%), Thailand (13% vs. 7%) and lowest in Lao PDR and Cambodia (2% vs. 1%). Socio-cultural factors such as parental and peer tobacco use and exposure to direct and indirect pro-tobacco advertising are important predictors of smoking in all countries. For example, Malaysian female students are four times more likely to smoke if their mothers smoke. Having close friends who smoke is another significant predictor (range between four to over ten times more likely to smoke). Indonesian and Philippines female students exposed to tobacco advertising and promotion are twice as likely to smoke compared to females without such exposure. The study also found that females with pro-smoking attitudes, positive image of smoking and a perception that smoking is the norm among people their age are more likely to smoke. Tobacco control policies in these countries are found to influence female smoking rates. Conclusion: To protect girls and women as well as boys and men, all ASEAN countries should implement the WHO Framework Convention on Tobacco Control, prioritizing raising of taxes on all tobacco products, introduction of comprehensive ban on tobacco advertising and promotion and prohibition of smoking in public places. BACKGROUND: For over twenty years, the tobacco industry has made marketing and distribution of tobacco products to youths a core of its marketing strategy in Nigeria. From the internal documents of the tobacco industry which was forced into public domain in the United States of America, it was revealed that the tobacco industry had deliberately marketed to young and underage persons in Nigeria. It was also revealed that British American Tobacco (BAT) had conducted several surveys to determine the rate of youth smoking in Nigeria. In one of such survey minors were referred to as YAUS (Young and Underage Smokers). METHODS: Experience Freshness was a recent promotion of the British America Tobacco in August 2008. This event was organized to promote the Pall Mall brand. It was organized in all the six geo political zones in the country and was heavy on young people. Invitation cards were secretly given out in secondary schools and colleges. It was a heavy all night smoking party.
Research and experience has showed various trick and strategies of the tobacco industries. It is therefore appropriate to implement laws that will curb every secret parties and advert used to promote its new product "Pall Mall" in developing countries especially Nigeria. There is also the need to build a strong alliance/network in high school thereby making them Students Action against Tobacco Smoking (SAATS). RESULTS: Increase awareness on the dangers of smoking and the deadly component in cigarettes thereby saying NO to the tobacco industries souvenirs, baseball cap, t-shirt and sponsorships. The method will also implement laws especially in Nigeria to curb both direct and indirect advertisement of the tobacco multinationals, thereby winning more adolescents for SAATS. CONCLUSION: Since the tobacco multinationals are recruiting more teenagers and youths everyday, thereby killing millions to make billions, efforts should be made in monitoring the tobacco industries to reduce both direct and indirect advertisement by the tobacco companies and engage more teenagers and youths in tobacco control activities. Background: Adolescents are a major target group of tobacco companies. Tobacco companies spend substantial amounts of money to promote smoking in adolescents. In Thailand, adolescents are considered the primary target for smoking prevention by the government. Objective: To determine the pattern of cigarette smoking among Thai adolescents over a period of three years. Design/Methods: A longitudinal study was conducted among adolescents between the ages of 13-18 sampled from 5 regions of Thailand using stratified multistage sampling. Wave 1 survey involved 1,000 adolescents was conducted during January to March 2005, wave 2 survey involved 962 adolescents was conducted during July to September 2006 and wave 3 survey involved 1,096 adolescents was conducted during January to March 2008. Self administered questionnaires were employed for data collection. Data were analyzed and compared using descriptive statistics. Results: Overall, smoking prevalence has increased from 11.3% in wave 1 to 13.7% in wave 2 and 18.3% in wave 3. Consistent with other Asian countries, smoking prevalence in young males were more than 10 times higher than young females. Among youth smokers, more than 70% smoked factory-made cigarette. The total amount of tobacco use per day increased from wave 1 to wave 3. In wave 1, no one reported that they smoked more than 20 cigarettes per day but in wave 2 and wave 3 surveys 6.9% of youth smokers reported that they smoked more than 20 cigarettes per day. In wave 1, approximately half (47.9%) of youth smokers reported that they obtained cigarette from friends with only 38% reported that they bought cigarette for themselves. The proportion of young smokers who reported that they bought cigarettes for themselves were significantly increased in wave 2 (61.3%) and wave 3 (68.2%) as compare to wave 1. More than half of young smokers reported that they tried to quit smoking within the past month. The proportion of youth smokers who reported that they never tried to quit smoking were slightly decreased from wave 1 as compare to wave 2 and wave 3 (20.7% in wave 1, 19.7% in wave 2 and 17.4% in wave 3). Conclusions: Cigarette smoking among Thai youth remains the important problem and needs further tobacco control policies to prevent and control tobacco consumption. There are high expectations that the communication of genetic risk information will motivate behaviour change. This symposium considers the theoretical and empirical basis for these expectations and the likelihood that they may be realized. Celeste Condit and Lijiang Shen present the results of three large population-based surveys in the US assessing public perceptions of the interaction between genes and behaviour.
Interpretations varied with whether genes and behaviour were conceived as health-damaging or health-protecting, suggesting a role for message framing in motivating behaviour change.
Winfried Rief presents the results of a study evaluating the impact of presenting genetic risk information to obese patients. Guided by Leventhal's Common-Sense Model, Linda Cameron presents the results of an internet-based experiment assessing whether information regarding links between physiological processes underlying genetic risk, disease development, and behavior enhances beliefs motivating protective behavior and alters willingness to buy tests. Finally, Theresa Marteau presents the results of a randomized controlled trial of using genotype to tailor nicotine replacement therapy in smokers attempting to stop, testing the hypothesis based on Leventhal's Common-Sense Model of Self-Regulation of Health and Illness that genetic risk information increases the use of medication as a coping response. The discussion will focus upon the understanding that these four studies contribute, both theoretically and empirically, to explaining and changing behaviour in the context of genetic risk information. Background: Public understanding of the relationship between health behaviors and genes is likely to affect the motivational impact of learning information about one's own genes. Extant research has featured difficulty measuring public understandings of this relationship. This essay explores public understanding of the relationship between genes and behavior, especially with regard to the mathematical relationships to risk contribution. It also contributes a psychometrically valid scale for measuring beliefs about gene-behavior relationships. Methods: Three population representative surveys (N=633, 658, 1218) were conducted using the Knowledge Networks panel platform. Results: Interpretations of risk vary depending on whether genes and behavior are conceived of as health-damaging (loss frame) or health-protecting (gain frame). In the loss frame, the majority of the population adopts an additive model of the relationship with approximately one third adopting an amplificative model. Scores on the nonmathematically based scale indicate higher belief in the existence of interaction than scores on the more concrete question format. Conclusions: The existence of different interpretations of genebehavior relationships based on gain/loss frame and abstract/ concrete modes indicates the need to select frame and mode carefully in both teaching and research. Research is needed to identify optimal configurations for teaching and presenting this relatively complex material. There is strong evidence for high heredity for obesity. Moreover, if obesity has developed, it is difficult to control weight, and unsuccessful and disappointing weight reduction programs are frequent. In this study we wanted to investigate whether information about the genetic background of obesity is helpful for affected people to develop realistic goals and reduce negative self-perceptions. After genetic screenings of 1000 obese people, we included 320 for a randomized clinical trial evaluating the effect of genetic information versus general information (without genetic content) in obesity. A subgroup of 16 participants was confirmed for functionally relevant MC4R mutations, and they received consultation with genetic information. Their results were analyzed in detail. The acceptance of a consultation including genetic information was high, and those people with a family history of obesity appreciated to receive genetic information in the consultation. People with MC4R mutations reported less feelings of shame and guilt and more realistic weight loss goals after the intervention. We conclude that consultation for obese people should include information about genetic backgrounds, especially if obesity-relevant genetic mutations are obvious, or if people have a family history of obesity. Increasingly, individuals can buy genetic tests to identify disease risks that can be reduced through behavior change. Given the challenges in understanding how behavior can reduce geneticallyconferred risk, there is a need to identify how to present information to optimize comprehension and adaptive behavior. Guided by the Common-Sense Model, we tested whether information about links between physiological processes underly-ing genetic risk, disease development, and behavior enhances beliefs motivating protective action and alters willingness to buy tests. We tested the impact of this information for genetic tests versus tests of a non-genetic biomarker, at higher versus lower risk levels, and when presenting risk information in graphic versus numeric formats. In an internet-based factorial design experiment, 782 adults responded to messages about a test for colorectal cancer risk. We varied messages by risk-action link information (provision or no provision of information describing how a low-fat diet reduces cancer risk given a positive result), risk increment (20%, 50%, or 80% risk given a positive result), risk format (numeric or graphic presentation of risk increments), and test type (genetic or enzyme test). ANOVAs revealed that providing risk-action link information enhanced beliefs of response efficacy (that low-fat diets reduce disease risk) and coherence (understanding how low-fat diets reduce risk), and lowered appraisals of anticipated risk with a low-fat diet (p's<.0001). These effects held across risk increments, risk formats, and test types. For genetic tests, provision of riskaction link information reduced the amount individuals were willing to pay for the test (p<.0001). Brief explanations of how action can reduce biomarker-detected risks promoted beliefs motivating protective action; paradoxically, by enhancing understanding of behavioral control over the risk, they reduced the value of genetic risk information. Despite growing interest in pharmacogenetics to tailor cessation treatment, the behavioural impact of genetic feedback is untested.
Communicating genetic information may increase medication adherence, but may also negatively impact motivation to quit if the initial attempt fails. We investigated the impact of genetic feedback on medication adherence and motivation to quit. We tested (1) whether adherence to NRT is greater following genetic (OPRM1 genotype) compared to phenotypic (heaviness of smoking) feedback, and (2) whether smokers who relapsed showed lower motivation to make another attempt following genetic compared to phenotypic feedback.
The study was an open label, parallel group randomised trial (ISRCTN: 14352545) conducted in primary care. Adult smokers (n=633) were prescribed NRT patch, randomised to a top-up dose of NRT based explicitly either on OPRM1 genotype or heaviness of smoking and followed for 6-months. Outcomes measures were: proportion of prescribed NRT consumed in the first 28 days; and motivation to make another quit attempt among those not abstinent at 6-month follow-up.
There was a significant effect of genetic feedback compared to phenotypic feedback on adherence at 7-day follow-up (75% vs 69%, p=0.040), and a marginally non-significant effect at 28-day follow-up (69% vs 64%, p=0.098). There were similar effects on abstinence at six-month follow-up (14% vs 8%, p= 0.018). Amongst those not abstinent at 6-month follow-up, there was no difference in motivation to make another quit attempt between trial arms (p=0.23). This is the first test of the behavioural impact of pharmacogenetic feedback. Genetic feedback had a measurable impact on shortterm medication adherence and abstinence at 6-month follow-up compared to phenotypic feedback, and appears to offer modest improvements to medication adherence to medication, and NRT effectiveness. This suggests potential for an important population impact. Prostate cancer is the most common form of cancer in men (other than skin cancer). The American Cancer Society estimates that 1 in 5 men will get prostate cancer sometime during his lifetime, and that 1 man in 35 will die of this disease. The society also estimates that more than 2 million men in the United States who have prostate cancer at some point are still alive today. Much of the current treatment for cancer (including surgery and hormone therapy) involves temporary or permanent impotence or urinary incontinence which would be distressing for all men but particularly for those who are invested in their sexual performance or being in control as signs of their masculinity. Men may therefore forego treatment or delay it, leading to increasing risk of complications from prostate cancer. Presenters in this symposium will look at the impact of male sex role norms (including emotional restriction, emphasis on sexual ability, and being independent) on diagnosis for prostate cancer as well as recovery from treatment. They will examine the kinds of responses men have to being diagnosed and treated for prostate cancer, the type of support they seek and find useful after treatment, and the role partners can play in helping men cope.
PROSTATE CANCER: MALE NORMS AND THE CONCEPT OF SUPPORT Betsy Bates, MA Antioch University, Santa Barbara, CA.
This presentation will summarize recent findings from the literature on psychological issues related to diagnosis, the seeking of psychological support, and threats to male identity posed by treatment options that can interfere with potency and continence. Qualitative studies, for example, point to a number of reasons why men may be diagnosed late, including a reluctance to appear weak (by reporting symptoms); embarrassment, guardedness and denial about vulnerability related to their bodies; powerlessness within the medical system, and the fear of a diagnosis that goes to the deepest core of masculinity, the prostate. A recent literature review highlighted clinically meaningful take-home messages, including the fact that men tend to either retreat inward following the diagnosis of cancer, keeping their feelings and worries to themselves, or rely heavily on a partner for emotional support. Studies contained in the review demonstrate that both men and women benefit equally from psychosocial support. When asked, men say that they want information rather than emotional support from psychosocial oncology teams; yet in online forums, men's supportive exchanges are clearly emotional, couched in dark humor and unifying battlefield terminology. The fact that men enthusiastically participate in emotional support offered at a distance (often anonymously) raises interesting questions about future directions for cancer support, which will be addressed. Also of significance is mens' highly consistent reliance on partners for support during cancer treatment, a finding that points to the possible unmet needs of men who are not in secure relationships during the diagnosis and treatment of prostate cancer. Although the psychosocial impact of cancer treatment varies across cancer patients, many experience some psychological distress following diagnosis. In addition to the emotional experience, men with prostate cancer often face myriad physical complications including impotence, urinary and bowel incontinence, and sleep disturbance affecting quality of life. The time immediately following treatment can be a critical period marked by increased feelings of fear, uncertainty, and loss of control. Such emotional responses may be experienced as inconsistent with psychological attributes often associated with traditional gender role norms, such as strength, independence, and emotional inexpressiveness. Evidence of the negative impact of suppressing emotions, and positive impact of expressing and processing emotions on psychological distress, physical health, and overall adjustment to cancer is growing. Drawing upon examples from the author's past and current research with men with prostate cancer, the relationship of gender-related influences on emotional approach coping efforts will be discussed within a stress and coping framework. Emotional approach coping (EAC) refers to strategies that involve acknowledging, understanding, and expressing emotions, and has been found to be associated with decreased distress and better adjustment in breast cancer patients, though little work has examined EAC in men with prostate cancer. Observations in healthy populations suggest that EAC varies by gender and has been linked to both better and poorer adjustment in men. Prior research in men with cancer found that gender role norms shape EAC processes, and that the interpersonal environment and age may be important contextual variables characterizing its utility in men with cancer. Current research examining the impact of masculine cancer threat, gender-linked personality factors, and gender role norms on health-related quality of life following treatment for prostate cancer will be highlighted. Management of type 1 diabetes (T1D) requires constant, daily attention to blood glucose levels, insulin administration, food intake, and physical activity in order to maintain near normal metabolic control. Caring for a child or adolescent with T1D can be demanding and research suggests that parents of children with diabetes are at greater risk for symptoms of depression, anxiety, and stress. The dynamic relationship between parent emotional functioning and daily diabetes responsibilities and care is not yet well understood and parent well-being may significantly influence critical child health behaviors and outcomes. This symposium provides a cohesive picture of the behavioral impact of parent emotional functioning on child health care in T1D. The symposium chair will highlight developmental differences and key risk and protective factors associated with parent well-being in a chronic illness population. Ongoing promising interventions, including interventions focusing on parenting strategies and support, parent-child communication, and internet-based support, will be discussed. The bidirectional impact of diabetes care and parent depression, anxiety, and wellbeing is a rich area for discussion and can inform future research.
, Parental anxiety appears to be a salient factor in T1D management in young children and treatment of parent anxiety may improve child health outcomes. The impact of anxiety on dietary management may be related to hypoglycemia fear and can provide a specific avenue for intervention. The direction of the relationship between anxiety and A1c is not presently known yet may be better understood with the completion of the RCT. Parental involvement is identified as a key factor in optimal management of pediatric diabetes. Further, research shows mothers are often primary care givers such that maternal psychological adjustment and its relation to children's disease care management are surprisingly understudied. Baseline data were analyzed from 179 mothers of generally middle-class young adolescents with T1D in a multi-site RCT. Mothers completed questionnaires and were interviewed about family functioning and diabetes management. On average, mothers were 42 years old and completed 2 years of post secondary education. Half were employed full time outside the home and 25% had no outside work. On average, children were 12.9 years old (range 11-14 years), were generally in fair metabolic control (HbA1c=8.7%) and had diabetes for about 5 years. A significant portion, 23% of the sample, had clinically elevated (mild to severe) depressive symptoms. Mothers who endorsed depressive symptoms (N=41) were matched on SES and compared to nondepressed maternal caregivers. More mothers with depressive symptoms were employed full time (60%) and were single parents (35%) than others (46% and 22 %, respectively). No significant group differences were found in maternal age or education, child age, HbA1c, or disease onset age. However, mothers who endorsed clinically elevated symptoms also reported more fear of hypoglycemia and lower self-efficacy for diabetes tasks, although youths' metabolic control did not differ between groups.
Mothers with higher depressive scores appear able to help their youth achieve comparable levels of metabolic control as those without significant symptoms. However, elevated depressive symptoms occurred more often in single mothers who worked full time suggesting these mothers may have less time for and help with disease care. As youths become more autonomous maternal fears of hypoglycemia may magnify and perceptions of selfefficacy may decline. Screening and identification of mothers with high depressive symptoms and referral for supportive counseling may help them cope better and help ensure long-term optimal youth disease care. Research has begun to highlight the negative effects of maternal depression and anxiety on youth with type 1 diabetes (T1D), including poorer family functioning, increased symptoms of depression, and poorer quality of life. However, researchers have typically used self-report measures of family functioning and child adaptation. The current study extends previous research by using observational methods to examine the relationship between maternal symptoms of depression and anxiety and specific parenting behaviors.
In this cross-sectional pilot study, we collected questionnaire (i.e. self-report measures of depression and anxiety), clinical (i.e., HbA1c), and observational data from adolescents with T1D (ages 10-16) and their mothers. Adolescents (n=30, 52% girls, mean age=12.6, 74% White) and their mothers engaged in a 15-minute discussion of diabetes-related stress, which was coded for parenting behaviors. In our sample, 23% of mothers were above the clinical cutoff for depression, and 13% for anxiety. Bivariate correlations indicated that maternal symptoms of anxiety were related to lower levels of observed child-centered behavior (displays of sensitivity) (r=−.50, p=.005) and listener responsiveness ( Psychological distress is a major factor in musculoskeletal pain.
As demonstrated in numerous studies, psychological distress and musculoskeletal pain commonly occur together; psychological distress is associated with a poor prognosis of musculoskeletal pain; and psychological distress is prognostic for a poor response to treatment. Research in this field is in need of better conceptual and theoretical underpinnings: the field is moving from empirical demonstrations of the impact of distress on pain towards better understanding of how psychological distress affects musculoskeletal pain and how psychological distress can be addressed during treatment. The overall goal of this symposium is to contribute to the theoretical understanding of the role of psychological distress in musculoskeletal pain, by summarizing recent developments in research and treatment.
Dr. Keefe will summarize the current state of science on pain coping in osteoarthritis patients. He will discuss the theoretical background of pain coping and then continue to critically review the research and intervention studies in this area. Dr. Crombez will review research on hypervigilance and attentional bias in chronic pain. He will discuss implications for the understanding of chronic pain and for future research. Dr. Steultjens will summarize current literature showing that psychological distress is a major predictor of poor outcome of multidiciplinary rehabilitation in chronic pain and fibromyalgia. He will discuss theoretical interpretations and practical implications of these findings. Dr. Denison will focus on recent treatment approaches aiming at improvement of function and reduction of distress in patients with musculoskeletal pain. Treatment builds on basic motor and behavior learning principles and cognitive-behavioral principles. This presentation provides an overview and update on the literature on pain coping in osteoarthritis patients. The presentation is divided into four sections. In the first section, the conceptual background for studies of pain coping in osteoarthritis is reviewed. In the second section, recent studies of pain coping are critically reviewed including studies on the role of pain catastrophizing, pain communication in osteoarthritis, pain coping in morbidly obese osteoarthritis patients, and brain correlates of pain coping. In the third section, interventions designed to enhance pain coping are highlighted, including studies of partner-assisted coping skills training, communication skills training, and combined pain coping skills training/lifestyle behavioral weight management. In the final section, the strengths and limitations of current research are summarized and important directions for future research described. Chronic widespread pain (CWP) and fibromyalgia (FM) are common syndromes characterized by widespread musculoskeletal pain with no obvious underlying pathology. Although multidisciplinary rehabilitation treatment has been shown to be effective in CWP and FM, on average effect sizes are small. One possible area of improvement is referring those patients for treatment in whom a clinically relevant treatment effect can be expected. To be able to do this, patient characteristics associated with treatment success need to be identified. This presentation will first provide results from a systematic review of current literature on predictors of improvement following multidisciplinary rehabilitation treatment. Four outcome domains were studied: pain, physical functioning, emotional functioning, and general improvement / quality of life. In general, the available evidence suggests that cognitive and emotional factors are predictive of treatment outcome. For both pain and physical functioning, poor treatment outcome was predicted by the presence of emotional problems, interpersonal distress, and depression. Apart from these cognitive and emotional factors, evidence for baseline pain intensity and impact of pain as predictors of treatment outcome were found. Additionally, data will be presented from a prospective cohort study on health outcomes in CWP and FM, for which analyses are currently in progress. Based on the findings from the systematic review and prospective cohort study, pathways for successful rehabilitation treatment in CWP and FM will be discussed. A majority of patients with musculoskeletal pain (MSP) are managed within primary health care (PHC). While physicians are involved in the initial assessment of patients' problems, physical therapists typically carry out interventions to reduce pain and improve function.
There is now ample evidence that psychosocial factors such as selfefficacy, fear of pain and/or movement/re-injury, catastrophizing, and social reinforcement contingencies contribute to the development of persistent pain and disability. In our research group at Uppsala University we have developed an integrated behavioral medicine and physical therapy treatment approach for patients with MSP that builds on basic motor and behavior learning principles and cognitivebehavioral principles. The approach comprises identification of problems and goals, monitoring of problems, functional behavioral analyses, basic and applied skills acquisition, generalization of skills, and relapse prevention. Evaluation of the approach involves clinical studies at both the individual level and group level and there are promising results at both short-term and long-term follow-up. Recent studies have focused on 1) targeting and tailoring of treatment based on screening of psychosocial factors and individual functional analyses, and 2) screening for psychosocial risk factors in telephone counseling. An emerging research area concerns implementation of the integrated approach in general clinical practice within PHC. Pilot work in our research group indicates that physical therapists may have greater difficulties in adopting this integrated approach than anticipated, lack of theoretical knowledge being one possible explanation. Further implementation efforts involve research as well as systematic integration of the approach in physical therapy curricula, the latter being done at two Swedish universities. Hypervigilance to pain and attentional bias are considered as key processes in understanding chronic pain, distress and suffering (Crombez et al., 2005; Pincus & Morley, 2001; Vlaeyen et al., 1995) .
Results are however contradictory. We performed a meta-analysis of attentional bias to pain-related information to address this issue, and in particular to investigate the role of pain-related fear, anxiety (trait and state) and depressive mood upon attentional bias to pain. Method: 61 studies (2739 participants) were identified using an emotional stroop paradigm, dot probe paradigm and exogeneous cueing paradigm. We coded methodological quality (internal and external), sample characteristics (chronic pain, experimental pain, procedural pain), and procedural characteristics. We obtained the original full dataset (80% response rate) to analyze the effects of our theory-based moderators. Results: The methodological quality of studies varied widely and is on average of moderate quality. Initial analyses revealed a significant attentional bias to pain-related information for whom pain was a current concern (d=0.127) in comparison with a control group (d=0.025). The effect was significant for the chronic pain group, the experimental pain group and the procedural pain group. Further analyses revealed that the attentional bias to sensory pain words was signficant, but not to affective pain words, stimuli related to antecedents of pain (e.g. injury or blood) or to stimuli related to consequences of pain (e.g. wheelchair). We are currently performing the moderator analysis focusing upon the role of pain-related fear, anxiety and depressive mood.
In conclusion, the methodological quality of attentional bias studies is moderate and can be improved. An attentional bias to sensory pain words exist in those who are concerned about pain. The implications for future research and for understandings of chronic pain, distress and suffering will be discussed. Objectives: People who drink in a problematic or risky manner comprise a larger group than people who are alcohol dependent, yet treatment for problem drinkers is much less prevalent in the U. S. Using technological solutions to bring interventions to problem drinkers promises to make appropriate treatment options more widely available. Interactive Voice Response (IVR) with speech recognition is an underused technology that is accessible to anyone with a telephone. IVR is being used to develop and evaluate an evidence-based automated treatment program based on Behavioral Self-control Training. This program is being tested in a randomized clinical trial for problem drinkers. Objectives: Stress and use of alcohol and drugs often vary from day to day. The goal of these studies was to investigate whether Interactive Voice Response (IVR) could serve as a useful technology for real-time assessments of stress as well as alcohol and drug use. Methods: Two samples are included. The first one is completed and includes 84 university students who were exposed to real-time IVR assessment three times daily during a total period of seven consecutive days. A second study is ongoing and will include 200 paroled clients to be followed daily during the first thirty days after release from prison. Half of the clients, randomised, will get automatic feedback on their results and simultaneously an e-mail report will be sent to the parole officer in order to provide a basis for further processing. Results and conclusions: In the first study, all the participating university students were followed throughout the week (100%). In total, stress and alcohol use was assessed in 1 567 out of 1 764 (89%) data collection calls. Preliminary data from the second study show the same high response rate, and 89% of all daily data collection calls have been answered. IVR methodology thus offers a promising new technology for daily assessments of stress and use of alcohol and drugs in both university students and paroled offenders. Background: The Influenza A/H1N1 outbreak was announced on April 24, 2009, and quickly made news headlines. This research investigated whether public anxiety about pandemic influenza was reflected in language use in web blogs, and whether the use of anxiety words on internet blogs was related to online informationseeking behaviour.
Methods: This research examined how word usage in web blogs (in particular anxiety, health, death and positive emotion words) differed between blogs that mentioned "swine flu" and control blogs published between April 24 and May 7, 2007. Web-based information-seeking behaviour was examined using the pattern of visits to H1N1-related Wikipedia pages from two weeks before to two weeks after the outbreak was announced. The relationship between information seeking and anxiety word use in web blogs was also investigated. Results: Blogs mentioning "swine flu" contained significantly higher levels of anxiety, health and death-related words, and lower levels of positive emotion words that control blogs. Use of anxiety words decreased steadily over the two week study period following the announcement of the H1N1 outbreak. The peak in Wikipedia visits to H1N1-related pages was three days after the peak in anxiety word use measured in web blogs. Lagged Pearson's correlations between language use and daily webpage visits three days later showed significant positive correlations between page views and use of anxiety words in web blogs (r=.76, p<.01).
Conclusions: The announcement of the H1N1 outbreak was associated with initial anxiety which decreased substantially over a two week time frame, and was found to be significantly associated with internet-based information-seeking behaviour. The analysis of web behaviour can provide a useful indicator of the level and changing pattern of public anxiety and informationseeking following an illness outbreak. Hepatitis C (HCV) is a blood borne virus: 75% of patients develop chronic infection (HCV+) and are at risk of cirrhosis, liver failure and cancer. While therapy can clear HCV in 50-80%, side effects may be debilitating. Disclosure of HCV+ status is at the discretion of the individual, but may be precipitated by the decision to seek treatment. The aim of this study was to determine whether patient characteristics were associated with patterns of disclosure in HCV+ people seeking treatment. Method: As part of routine care in a tertiary referral clinic, a database has been maintained which includes key demographic data and details of psychosocial assessments undertaken by a social worker. De-identified data was analysed from 2001 to 2008 inclusive.
Results: There were 548 complete records, mean age 42.5 years, 68% male. Most patients with a partner (married/de facto) disclosed to them (97%) but were less likely to disclose to anyone else, compared with other patients (p=0.0002). Women were more likely to disclose to a friend compared with men (p=0.0063). Younger patients were more likely to disclose to parents or siblings, while older patients were more likely to disclose to their children (Chi2 test for linear trend: p<0.0001). Of patients with dependent children (62%), single parents were less likely to disclose to children than others (p=0.027).
In multivariate analysis, patients were more likely to disclose to at least one person (other than a partner) if they were divorced, separated or widowed (OR 6. There is growing evidence that conscious types of perseverative cognition (PC), such as worry and rumination, can increase healthrelevant physiological parameters, and that PC may mediate the prolonged physiological effects of psychological stress. There are, however, several indications that part of PC is unconscious and may be responsible for a considerable part of daily prolonged physiological activity. For example, worry itself appears to be associated with prolonged physiological effects, both during nocturnal sleep and in daily life, up to two hours after worry episodes. The existence of unconscious PC (UPC) is likely because most cognitive processes are (partly or entirely) unconscious. Moreover, several studies using subliminal (under awareness threshold) negative emotional stimuli suggest that unconscious emotional cognition increase amongst others amygdala activity and skin conductance level. We hypothesized that subliminal negative stimuli also increase more substantial, healthrelated parameters such as blood pressure. Showing this would imply crucial support for the notion that UPC can have healthrelevant physiological effects. Eighty college students anticipating an examination in the next two weeks, were shown either (1) subliminal negative words, such as "failure" and "stupid", (2) subliminal neutral words, (3) supraliminal (above awareness threshold) negative words, or (4) neutral supraliminal words, during a neutral computer task. For subliminal stimulation as masking procedure was used. Blood pressure was measured continuously using a Portapres Model-2. An awareness check ascertained that no subject could consciously perceive the subliminal stimuli Results indicated that while negative words decreased systolic blood pressure marginally significantly across awareness conditions (F(1,77)=2.265, p<.10), they did so clearly and significantly in the subliminal condition (p<.05), but hardly and nonsignificantly in the subliminal condition. Diastolic effects were in the same direction but nonsignificant. High trait anxiety but not high trait worry augmented these differential effects of awareness of negative stimuli. The effects could not be explained by conscious PC or mood. Concluding, the results suggest that unconscious but not (or to a lesser extent) conscious negative emotional information can increase blood pressure. This lends credibility to the hypothesis that unconscious emotional processing (UPC) may have physiological consequences, and warrants further studies, including real life studies, using available measures of unconscious emotion, to examine robustness and magnitude of similar effects in real life. Many who develop posttraumatic stress disorder (PTSD) ultimately recover. Recovered persons may, however, display "biological vigilance," or physiological reactions to trauma cues. We examined this using measures correlated with aging-related morbidity and mortality: circulating levels of C-reactive protein and plasma interleukin-6 (IL-6), along with stimulated IL-6 production. Apparently healthy, postmenopausal women with divorce histories completed interviews to assess current and lifetime PTSD in response to past intimate partner violence, or other traumatic events. Women, all without current syndromal PTSD, were classified by history of PTSD diagnosis: none (PTSD-; n=33), subthreshold (PTSD+s; n=18), or syndromal (PTSD+; n=12). Women attended two research visits; for each, a blood draw was followed by questionnaires that were either benign (Baseline Visit 1), or trauma-and PTSD-related, plus a diagnostic interview (Visit 2 Trauma Assessment). Mixed effects models tested effects for PTSD History, Visit, and the PTSD History x Visit interaction. For circulating IL-6 levels only, there was a trend for a PTSD History x Visit interaction, F(2,59.4) = 2.89, p=.06. Interleukin-6 responses to the two visits differed for PTSD-and PTSD+ women (p=.03). For PTSD-women, IL-6 levels were higher at Baseline than Trauma Assessment (p=.05). In contrast, for PTSD+ women, IL-6 levels were higher at Trauma Assessment than Baseline, although this difference did not reach statistical significance (p=.16). Further, at Baseline only, there was a trend (p=.06) for lower IL-6 levels among PTSD+ and PTSD+ s women, compared to PTSD-women. These preliminary results, limited by sample size, require replication. Tentatively, however, the pattern suggests that women with past syndromal PTSD may be characterized by baseline IL-6 hypoarousal, along with plasma IL-6 increases in anticipation of confronting trauma reminders. Supported by NIH R21AG024902. About 4% of the German population experience chronic tinnitus, but only a minority suffers from it to an extent affecting quality of life. In order to explain tinnitus related distress, the neurophysiological model predicts associations of the tinnitus signal with an activation in limbic structures in distressed compared to nondistressed tinnitus patients. The aim of the study was to test this hypothesis by identifying neural correlates of tinnitus annoyance. High and low distressed chronic tinnitus patients and healthy controls were examined in an emotional sentence task using functional magnetic resonance imaging (fMRI). During the task, participants evaluated the personal relevance of tinnitus related, general distress related and neutral sentences. As a result high but not low distressed tinnitus patients showed activations in the Gyrus cinguli and Insula in reaction to tinnitus related vs. neutral sentences. Similar effects were found comparing distress related and neutral sentences. Additionally high distressed tinnitus patients showed an activation in the Gyrus cinguli in favor of tinnitus related compared to distress related sentences. The effect seems to be a specific reaction to tinnitus related and not to generally distressing stimuli. So the results are in line with the neurophysiological model. This paper analyzes the trend of the youth homicide rate (age groups 15-24 and 25-34) in Mexico in last 30 years and seeks to identify the socioeconomic variables that better explain the geographical variations of youth homicide rate in Mexico in recent years. The basic information for this study was obtained from official sources; trends of homicide rates by age groups 15-24 and 25-34 between 1979 and 2007 were analyzed; for each Mexican state, homicide rates by each age groups analyzed in triennial 2005-2007 were calculated; through the use of multiple regression analysis (stepwise method) the variables that better explained the interstate variations in the homicide rates were identified. The results show that the homicide rate in both age groups have diminished in last 30 years, however these rates are relatively high in the international context. Furthermore, although the male homicide rates are clearly higher in both age groups, this excess of male mortality has been notoriously reduced. Moreover impunity, drug trafficking, social exclusion and firearms possession are key elements to understand the geographic variations of the youth homicide mortality in Mexico in the analyzed triennial. In particular, drug trafficking has a great importance to analyze the variations of the homicide rate in 25-34 years old group. The found regression model equations explain more of 50% of the geographic variations of the homicide rate in both age groups. In this context, findings suggest that if Mexican government wants to reduce the geographical disparities in youth homicide rates, needs to develop new strategies to combat drug trafficking, establish programs to restrict firearm possession, and implement structural reforms to reduce poverty, social marginalization and socioeconomic disparities among states. The present paper seeks to establish the prevalence and type of violence among adolescents enrolled in public high schools and to analyze the social and cultural factors associated with aggressor's condition. This is a cross-sectional, analytic type study. The data were gathered by questionnaire applied to 661 students enrolled in public high schools, during the period from December of 2008 to June of 2009. In the sample selection approach -random and multi-stage -it was considered the entirety of state's high schools of the metropolitan area of Guadalajara, Mexico. According to obtained data, 48% of students were involved in some kind of violent event; 25% as victims, and 15% within a double role: eventually either as victim or aggressor, it was found that 8 out of 100 students were aggressors. Within the aggressors group around 30% were women and 70% were men. In the multivariate analysis for both genders, three are the variables that better explain aggressor's status: gender (OR 1.8), to be victimized for the family (OR 2.6) and to be involved in situations that unchain a police detention (OR 3). Conclusion: Before entering school the adolescent has blended a series of family experiences (interfamilial violence) and peer relationships (participation in gangs exhibiting criminal records) -that allow to forecast what could be the way of relating with schoolmates. Although the school becomes the new scenario where the adolescent has the opportunity to play a role already learned, in turn, this could become the field where the necessary transformation of risk behaviors and/or the learning of styles for social affront are mediated. In this paper are presented some cognitive dimensions related to couple violence studied in a group of women involved in it. Violence is a major social problem in the current life around the world. Family violence is the most common and continual type and couple violence has the highest prevalence. In any case it is a risk for health, it produces stress that for long periods of time affecting the functioning of the Immunological System. Violence as an aversive situation, is expected to have an avoidance or escape reaction. But as a world mean, it takes a woman near to 15 years to go out of a violent relationship. Staying in a violent partner relationship, is considered codependency with the aggressor. Multiple factors are involved for staying in a violent partner relationship: cultural, social and personal. Is it possible to identify the cognitive and social factors related to couple violence in women that are involved in a violent relationship? Could these factors explain women codependency with the aggressor?
To aim these objectives, it was developed a self report inventory based on cognitive, affective, and personality theories. 441 women that have being involved in a violent relationship and that look for governmental support programs answered the inventory. Their ages were from 18 to 62 years old. The Socioeconomic and educative levels were from low to middle. The responses were grouped into 6 dimensions: Strength for coping with the violent situation, Feelings of harassment or fear, Anticipation of consequences, Emotional dependency, Self efficacy, and Self confidence. These show elements of cognitive structure in women that suffer violence as a result of the social and cultural variables an o the violence experience. In the next research phase, it will be to identify the dimensions that show which dimensions produce differences between women that stay in the relationship and women that are out of it. These results will help to develop programs to support them. Background: Road traffic accidents (RTA) are an usual traumatic event. In case of serious accidents, behind physical injuries, victims can develop psychological symptoms and problems s (e.g. Acute Stress Disorder (ASD) and PTSD) that interfere with quality of life. Literature shows recent discussion on predictors of ASD and PTSD in these victims. Methods: 124 RTA victims (34 women and 90 male) were evaluated 6 days (T1) and 4 months (T2) after the accident. At T1 participants filled a demographic questionnaire that asked about risk perception, Standford Acute Stress Reaction Questionnaire , Peritraumatic Dissociative Experiences Questionnaire. At T2 participants filled a 17 items PTSD scale. Findings: At T1 64,5% had ASD. At T2 58,9% had PTSD and 21% had partial PTSD . PTSD symptoms were positively correlated with risk perception (rsp=.266, p<.01), dissociative experiences (rsp = .407, p <.01) and acute stress symptoms (rsp=.460, p<.01). In a regression analysis gender, risk perception, dissociative experiences and ASD symptoms accounted for 27,6% of PTSD symptoms variance. Women report more ASD symptoms and PTSD symptoms than men. Discussion: The participants in this study were victims all of victims of serious accident, and the number of subjects with PTSD is unusually high, suggesting the need for psychological evaluation and intervention. The predictors could be use to identify the ones that will be more disturbed after these events. Rapid urbanization has led to increasing violence, psychosocial and psychiatric morbidity, particularly in low income/poor communities. Such emerging violence amidst rising prosperity in Malaysia is posing serious challenges to behavioural medicine and healthcare. In-depth qualitative interviews were conducted with 25 purposively sampled young respondents from 12 low-cost housing neighbourhoods within Kuala Lumpur to explore their perceptions and experiences of violence, and health impact. The 25 multi-ethnic respondents, (12 men and 13 women) aged between 18-25 years, were residents of public low-cost high density housing where neighbourhood violence, viz. vandalism, street fights, robbery, drug dealing and homicide, was common. Perceptions of violence were varied and gender differentiated. Men tended to perceive crime as violence and serious violence to involve bleeding, hospitalization or death. Women regarded fights, injuring others and crime as violence and ranked murder and rape to be serious violence. Women and men were reluctant to intervene in domestic violence because they perceived it as a private matter although several respondents felt that a husband has no right to beat his wife. Men, more than women, had direct experience with violence. Some men had been robbed, threatened and beaten. Several men had perpetrated violence: selling drugs during teen years, involved in illegal motorcycle racing, and assaulting a neighbour. A couple of women had been robbed. None had suffered any physical injuries/disability due to violence or reported symptoms of severe psychological consequences viz. disturbed sleep, appetite and moods. Yet, women were more affected by high anxiety and pervasive fear of being victims of crime. Men were more concerned about safety of their families, especially female members. Girl child kidnapping, rape and murder happening in and around the neighbourhoods had created secondary traumatisation and strong sense of vulnerability to such violence among women. Women particularly experienced chronic stress in coping with the daily crime and violence in their neighbourhoods. Gendered experiences and impact of violence with regards to secondary traumatisation and chronic stress point to more sensitive and specific interventions, considering both behavioural and social factors, among low income/poor urban communities. Introduction: Usually, at the end of a lifestyle intervention trial, investigators present the predefined intervention strategy as well as the study Results: In practice, the intervention may not have been performed as intended. In the Health under Construction Study, 27 occupational health professionals (counselors) performed a six-month motivational interviewing-based lifestyle intervention for construction workers. The intervention was aimed at changing dietary, physical activity, and smoking behavior, and thereby lowering cardiovascular disease risk. The purpose of this process evaluation was to evaluate the counselors' adherence to the intervention protocol, their competence, and the associations between adherence, competence and body weight at follow up. Methods: The number of sessions and prescribed items discussed in the first counseling session were registered by the counselors. Counselors' competence in listening, supporting, motivating, and informing was rated by both participants and counselors. Adherence to motivational interviewing was determined by expert scoring of 19 audio-taped fragments. Associations between reach, dose, and competence, and body weight at follow-up were determined by linear regression analyses. Results: Two-thirds of all participants attended five or more sessions, and 38.5% attended all seven sessions. In 90.2% of all cases, the counselor discussed all four prescribed items in the first session. Adherence to motivational interviewing was concluded from only one audio-taped fragment. 86.3 percent of all participants agreed with the counselor being competent. Neither counselors' competence, nor number of sessions or items discussed, was significantly associated with body weight loss. Conclusions: Performing five sessions and discussing four prescribed items was feasible for the counselors, whereas adhering to motivational interviewing was not. Still, participants were positive about the counselors' competence and willing to attend the counseling sessions. Investigators are encouraged to report the evaluation of their intervention process, in order to improve future lifestyle interventions in research or in practice. Colorectal cancer is the third leading cancer in incidence and mortality among US adults, and these rates are higher in the African American community than among other subpopulations. Community-based approaches have grown in popularity, in an effort to fight these cancer disparities. Though church-based approaches have been included among these initiatives, few are "spiritually-based" in nature, using spiritual/religious themes and writings to support the core health message. Such an approach may increase the cultural relevance of a health communication, and perhaps even its efficacy. The efficacy of a Community Health Advisor-led intervention to increase awareness and screening for colorectal cancer was examined using a randomized controlled trial, in which a spiritually-based approach was compared against a non-spiritual (e.g., secular) comparison intervention of same structure and content. Trained Community Health Advisors led two educational sessions on colorectal cancer. Study participants (N =358) completed baseline, 1-month, and 12-month follow-up surveys. A 90% retention rate was achieved over the course of the study period. Within group analyses suggest that the spiritually-based intervention resulted in significant increases from baseline to one-month follow-up in colorectal cancer knowledge (p<.001), perceived benefits to screening (p<.001), and significant decreases in perceived barriers to screening (p<.001). Comparisons between the spiritually-based and non-spiritual intervention indicate that the spiritually-based intervention was more efficacious than the non-spiritual for increasing perceived benefits to screening (p<.001), while the non-spiritual was more effective for increasing knowledge (p<.001). Data are also examined among demographic subgroups such as men and women. Screening data at the 12-month followup are also reported. These findings suggest that the spirituallybased and non-spiritual approaches to health communication may have different impacts depending on the outcome and demographic subgroup being examined. Background: Humor in medicine is not a speciality at present but is gaining recognition as a clinical tool. Purpose: The purpose of this study is to review humor in a holistic manner and explore its various angles from its evolution to usage in the field of medicine. Method: A literature search was conducted using PubMed restricting to the statements of "Laughter Therapy" and "Humor and Laughter." Additional articles were sourced from reference list of relevant articles. These were critically analysed to identify use of humor in medicine. Results: This paper provides an overview of the field of humor focusing of what is known and not known about humor in medicine. This information serves as an important ingredient to reinforce the development of formal training in humor. Conclusions: Based on this review we argue that knowledge of humor should be imparted to care givers as a potential therapeutic tool in the field of medicine. Background: Evidence demonstrates self-management programs are an effective approach to assist patients with chronic diseases such as type 2 diabetes or cardiac conditions to modify their lifestyle for better managing their conditions. Using information technology (IT) has great potential to support self-management programs and assist patients to fulfill their goals in managing their conditions more efficiently and effectively. Examples of different types of technology used in self-management programs that have limited research support include: text messages, telephone followup, web-based programs, and other internet-assisted education. But little is known about the applicability and feasiability of different forms of technology for patients with chronic diseases such as those with type 2 diabetes and critical cardiac conditions. Furthermore, although there is some evidence of the benefits of using IT in supporting self-management programs, further research on the use of IT in such programs is recommended.
Objective: To develop and pilot test an integrated Cardiac-Diabetes Self-Management Program (CDSMP) incorporating telephone and text-message follow-up. Methods: A pilot study using randomised controlled trial is conducted in the coronary care unit (CCU) in a Brisbane metropolitan hospital in Australia to collect data on patients with type 2 diabetes admitted to CCU. The main outcomes included self-efficacy levels, knowledge, and quality of life. Results: Initial results reveal that patients with diabetes admitted to the CCU in the experimental group did improve their self-efficacy, and knowledge levels. Previous research has identified two important user reactions to Internet-based health behaviour interventions: users trust in an intervention and the extent to which users respond to the intervention as if it were a person (e.g. anthropomorphically). The aim of this study was to examine how these user reactions might be linked to users' engagement with five specific design features of the 'Internet Doctor', an Internet-based intervention to promote the self-care of cold and 'flu symptoms. The five design features were: social context and support; visual appearance; credibility; tailoring; and usability. Twenty six adults aged 18 to 63 participated in 'think-aloud' interviews. Inductive thematic analysis and techniques from grounded theory were used to identify recurring patterns within the data. Three user reactions were identified: users' trust of the 'Internet Doctor'; users' anthropomorphic responses to the 'Internet Doctor'; and users' preference for human involvement in their healthcare. These reactions appeared to be linked to differences in user engagement with four of the five design features (social context and support; visual appearance; credibility; and tailoring). For example, some users who trusted the 'Internet Doctor' expressed positive views of social context and support features (e.g. a question and answer format that was analogous to face-to-face dialogue; information about the 'real' doctor behind the advice) -using them as justification to trust the website's diagnosis and advice. In contrast, participants who expressed a desire for human interaction and were aware of their own anthropomorphic responses to the 'Internet Doctor' were uneasy about features that provided social context and support. Different user reactions were hence associated with different types of engagement with the same design feature. The implications of these findings for the optimal design of Internet-based health behaviour interventions will be discussed. Psychology, University of Evora, Evora, Portugal and 2 Psychology, Instituto Piaget -ISEIT, Almada, Portugal.
Objectives: The aim of this study is to investigate the influence of post-traumatic stress disorder (PTSD), peritraumatic dissociation, psychological distress and subjective health complaints in the psychological well-being of the emergency ambulance personnel. Methods: This is a cross-sectional study in which 250 ambulance personnel; both sexes completed a self-administered questionnaire, with measures of post-traumatic stress disorder, peritraumatic dissociation, psychological distress, subjective health complaints, psychological well-being and socio-demographic variables. Results: The results indicated that ambulance personnel have on average some PTSD symptoms, while 10% have a clinical diagnosis of PTSD, peritraumatic symptoms are also significant, as well as psychological distress and health complaints. Correlational analyses indicated a negative significant association between symptoms and psychological well-being. The peritraumatic dissociation, the subjective health complaints, psychological distress and sex were found to be predictors of the post-traumatic stress disorder. There were significant differences concerning the socio-demographic variables in relation to the dependent variables. Implications: The results of this study highlight relevant indicators of the need to develop and implement intervention programs to alleviate the psychological suffering, promoting the adjustment and the physical and psychological well-being in emergency ambulance personnel. Conclusions: This study presents a contribution to understanding the psychological consequences of daily exposure to traumatic incidents, and its impact on health and well-being of emergency ambulance personnel. Background: Nepal has different climate zones from ice freeze cold to hot in plain tarai regions. The maximum temperature is around 45-52o C. Implicit in all indices used for risk assessment in the prevention of heat stress is the assumption that workers are healthy and well hydrated; studies in Nepal cement, iron factory workers where workers are poorly hydrated, the level of protection offered by management strategies based primarily on environmental monitoring is compromised. Objectives: To investigate the hydration status of expatriate workers during summer in a range of works as large numbers of expatriate workers are employed as manual labourers in industries under extreme heat stress conditions where heat illness is a significant concern. The aim was to ascertain whether the generally inadequate hydration status, is also an issue in these workers and make practical recommendations for control. Methods: Studies were carried out at four sites to document the hydration status of exposed workers in different workplaces using urine specific gravity at three time points over two different work shifts. Results: Although the workers were found in general to be better hydrated than their other zone counterparts, a high proportion were still found to be inadequately hydrated both on presentation for work and throughout the shift. Workers take less drinking water and more alcohol, smoking. Conclusions: Interventions are required to ensure that workers in extreme heat stress conditions maintain adequate levels of hydration. Behavior change among workers to drink plenty of water, fluids and avoid alcohol, smoking, adequate sleep and rest. Failure to do so reduces the protection afforded by heat stress indices based on environmental monitoring. Regular Health assessment and behavior change programs in those areas is important for safety and reduce occupation health hazards as well. (2010) 17 (Suppl 1):S1-S329 S135
Background: Industrial Workers' health behaviour includes habits or actions related to physical exercise, nutrition, smoking, and drug or alcohol consumption. Unhealthy behaviour, and especially alcohol consumption, has been considered a source of accidents and injuries among construction workers. However, unhealthy behaviour can also be seen as a result of the safety and risk conditions of these jobs. Methods: The purpose of this paper is to contrast the role of unhealthy behaviour as a source or as an outcome of safety and risk in the industrial sector. Data was collected from 180 workers belonging to a Industrial and construction company. Two path models representing these two hypotheses were tested. The model in which unhealthy behaviour is an antecedent of injuries did not fit the data (Chi square=73.798, df=3, p<0.001).
Results: It supports the hypothesis of unhealthy behaviour as a result of safety and risk factors through the mediating effect of the experience of tension (Chi-square=4.507, df=2, p=.212). This model not only corroborates the stressful nature of exposure to risk and the absence of supervisors' safety response, but it also makes it possible to consider injuries as a cause of tension that, in turn, affects the employees' unhealthy behaviour. that included questions on the effort and reward at work, overcommitment, physical health, and a range of other social, demographic and behavioral indicators. All individuals were then followed for mortality through population mortality registers.
Mortality data are available in all 3 countries until the end of 2008. Cox regression modeling was used to estimate the association between ERI, overcommitment and all-cause mortality. Only men and women who were full-time working at the time of the interview were used for the analysis. Results: After controlling for age, sex and range of possible confounding variables, the effort-reward ratio and overcommitment were significantly related to the all-cause mortality. Adjustment for socioeconomic position did not substantially change the Results: We did not find strong evidence for any interaction between socioeconomic measures and work-related psychosocial indicators.
Conclusions: Stress at work expressed by effort-reward imbalance and overcommitment is related to all-cause mortality in these three middle-age Central and Eastern European populations. Throughout the course of a person's labour force participation, bouts of unemployment may be experienced. Studies have examined the relationship between single bouts of unemployment and psychological well-being as well as single and multiple bouts of unemployment with life satisfaction. This study examines the effect of multiple bouts of unemployment on psychological wellbeing. Specifically, whether people became sensitized, i.e. Psychological well-being becomes worse with each bout of unemployment, or adapted, i.e. Psychological well-being is worst with the first bout of unemployment and the impact of each additional period of unemployment is less than the first. Data came from the British Household Panel Survey, a large-scale longitudinal representative panel study. Psychological well-being was measured using the GHQ and participation in the labour force was self-reported; data were examined separately for males and females using multilevel modelling. The GHQ score at the first unemployment bout was used as the reference category. These scores were compared to scores from the period previous to the first unemployment bout, and at subsequent unemployment and re-employment bouts. Up to three unemployment and reemployment periods were allowed per individual. Results show that there are definite gender differences in reactions to unemployment. Among males and females, psychological wellbeing worsened significantly during the period up to the first unemployment bout (difference scores 0.89, p-value<0.0001 and 0.90, p-value<0.0001 for males and females, respectively). Psychological well-being significantly improved during the first re-employment stage for both males and females (difference scores 0.95, p-value<0.0001 and 1.04, p-value<0.0001 for males and females, respectively). Psychological well-being scores were significantly lower for the second unemployment bout compared to the first for males (0.74, p-value<0.05) but not for females (0.02, p-value>0.05). A test for linear trend was significant for both males and females; however differences between reactions became smaller with each unemployment period for males, but larger for females. There were differences in response to multiple bouts of unemployment between males and females in this study. Males experienced adaptation in that the response to the first bout of unemployment was significantly greater than the response to subsequent bouts (trend test p-value<0.0001). Females, however, experienced sensitization in that response increased with each bout of unemployment (trend test p-value<0.0001). The findings suggest that males are able to adjust to multiple unemployment bouts while females' psychological well-being becomes worse. A questionnaire survey regarding the factors affecting duration of untreated psychosis was conducted among 346 members of the association of family of psychosis patients in Mie Prefecture, Japan. Completed answers were returned from 138 members (118 parents, one spouse, 6 siblings and 13 patients themselves). Duration between the suspected first episode of psychosis and the first visit to 'physicians' (including primary care physicians, psychiatrists or others) was 18.5 months on average (SD 40.2). Forty-five subjects consulted family members or relatives first before visit to 'physicians'. It took 9.8 months on average (SD 11.2) for them to decide to visit, which were significantly shorter than the duration for subjects who consulted other than family members or relatives (22.8 months on average with 48.9 of SD) (p<0.05). Similarly, it took 40.2 months on average (SD 63.0) for 18 subjects who consulted teachers at school, which was significantly longer than the period for those who did not (15.1 months on average with 35.5 of SD) (p<0.05) . In case that reasons for visit to 'physicians' were physical symptoms (sleep disturbance etc), the mean span before the visit was 9.3 months (SD 12.1, 48subjects), which was significantly earlier than the visit because of other reasons (24.1 months on average, SD 50.1, 82 subjects) (p<0.05). Stigma or prejudice is supposed to be a major barrier to medical care, considering more than half of subjects (71) Background: Breastfeeding rates in the USA and UK are among the lowest in the world, yet breastfeeding is known to be the most healthy feeding method for infant and mother. Staff training is an essential element for organisations wishing to achieve the evidence based UNICEF Baby Friendly Initiative (BFI) and since it is known that those who volunteer for training are often the most competent, an objective training needs analysis is required. A breastfeeding training programme is being delivered to healthcare staff in the West Midlands, UK. Prior to the training staff undertake an online objective evaluation of their breastfeeding knowledge. Objective: To assess the levels of breastfeeding knowledge of healthcare staff working with mothers in England. To assess if training needs differed according to prior recent training in breastfeeding, years of service, and profession. Methods: Breastfeeding knowledge was measured before the training programme was undertaken. A web based assessment tool Coventry University Breastfeeding Assessment (CUBA) was used to assess healthcare staff in eight sub scales relating to breastfeeding knowledge and practice. Results: 248 healthcare staff completed the online assessment.
Overall, knowledge levels in relation to the sub scale breastfeeding difficulties and positioning and attachment were highest with 93.1% and 95.2% respectively achieving 50% or higher. Knowledge of who breastfeeds and the challenges to breastfeeding scored the lowest with 54.5% and 46.4% respectively scoring less than 50%. In six out of the eight categories 30% or more staff scored less than 50%. T tests showed scores relating to positioning and attachment, difficulties, challenges, support and total scores were significantly higher for those who had received training within the last year whereas no differences were found by length of service. Midwives had significantly higher mean scores for supporting breastfeeding and practices compared to health visitors. Integration of mental health and primary care services has made impressive strides in the past decade. Over the past three years, MH/PC integration has received unprecedented support within Veterans Affairs. This poster will detail how collaboration among primary care and mental health services at one VA has evolved into a successful integration program. The presentation/poster will include information about co-location, integration, and open-access as well as data regarding efficacy and model fidelity. Quantitative data will be presented regarding our universal suicide/homicide risk screening practice in primary care, our substance use co-integration initiative, and our integration project to address diabetes, HTN, and dyslipidemia. Qualitative data addressing primary care staff satisfaction with the program will also be presented. Four indicators of efficiency will be offered: (1) promptness of mental-health consultant availability, (2) market penetration (i.e., what percentage of patients seen are new patients), (3) brief visits to ensure rapid availability, and (4) efficient/ cost-effective utilization of specialty mental health resources (e.g., psychiatry, psychology, social-work clinics). With regard to efficiency/ cost-effectiveness, an integration program is considered more effective when more mild-moderate cases are treated in primary care, fewer referrals are sent to specialty mental health care and a greater proportion of specialty referrals are completed. These efficiency/cost-effectiveness markers offer an indication of whether a patient was appropriately triaged within primary care. This study examined the modifying role of psychosocial resources in the impact of life stress on psychosomatic symptoms in a 10-year follow-up period using a dynamic model of psychosocial resources in the stress coping framework. Subjects (N=1262) were participants in two follow-up surveys (1989/22yrs; 1999/32yrs) of a Finnish cohort study. The self-report measures included life stress (number of negative life events), psychosomatic symptoms (index of 17 somatic and mental complaints), and psychosocial resources (self-esteem, meaningfulness, locus of control). Analyses were done using change scores of life stress and psychosocial resources in structural equation modeling and regression analysis. Increase in life stress across 10 years was inversely related (standardized regression weight=-.25, p<.001) to the change in psychosocial resources. Further, increase in psychosocial resources was inversely related (-.38, p<.001) to psychosomatic symptoms at 32 years controlling for the baseline symptom level. The total effect of change in life stress on psychosomatic symptoms at follow-up was decomposed to a significant direct effect (.12, p<.001) and a significant indirect effect through changes in psychosocial resources (.10, p<.001), indicating a case of partial mediation. The resource change moderated the effect of increasing life stress on psychosomatic symptoms (p=.001), the effect being significantly smaller among those with increasing resources as compared to those with resource losses. The results lend support to the conservation of resources theory and emphasize the importance of dynamic conceptualization of psychosocial resources in the stress process: resource losses and gains form an important mediational path between life stress and health. The results also suggest that resource gains can compensate or buffer the negative effects of life stress on health even in the case of increasing life stress. The medical experience of intensive care unit (ICU) is associated with many negative conditions such as isolation, loneliness, immobility or dependence, which have significant impact on patient status. Therefore, it is not surprising that patients report worse quality of life in both physical status and depression, anxiety and discomfort once being discharged from the unit (Garcia et al, 2003) . Taking into account this fact and the importance of quality of life related to health, the aim of this study was to evaluate the differences in perception of quality of life reported by patients before and after admission to ICU. The study was based in 57 patients admitted to the ICU at General Hospital in Castellón. Of these patients, 70.5% were male and 29.5% female, with a mean age of 53.6 years. From this study, we excluded people who had any communicating problem, and also people who suffer a prior psychological disorder. The participants, whilst staying in ICU and after giving their consent, were administered various questionnaires. One of them was the Euro-Qol in its Spanish version (Badia et al, 1999 Background: Illness behavior in schizophrenia patients implies illness denial (lack of insight). Different studies of insight in schizophrenia have led to inconsistent findings and conflicting results alternatively stating that acknowledgement of one's mental illness is a detriment and a key to successful adaptation. Objective: This cross-sectional study investigates the relationship between illness denial in schizophrenia and its influence on frequency of hospitalization and relapse, as well as the way insight relates to other important variables, such as depression, quality of life and optimism, patient's social context and available support system in Romania
Methods: All patients diagnosed with chronic schizophrenia (DSM-IV-R), and having a history of relapse and admitted in a six month period were asked to participate in the study. Patients who gave consent (45 patients) were evaluated using the Quality of Life Enjoyment and Satisfaction Questionnaire (QLESQ-18), The Life Orientation Test-Revised (LOT-R), and the Perceived Stress Scale (PSS),. Results to PANSS item G12 were used for the assessment of insight. Admission charts and interview were used for the assessment of social and familial support. Results: The patients who had insight had also reduced optimism, a high rate of perceived stress, these being associated with a high frequency of hospital admissions and with a lower quality of life. 32 subjects, or 71%, had severe insight deficits or even lack of insight. These findings were associated with higher optimism, and a lower rate of perceived stress. Patients who benefited of support system and had an adequate social context had fewer hospital admissions. Conclusion: We could advance the premise that the incapacity to benefit from this higher insight is due to the poor quality of the supporting system in our country. It is vital to be able to connect the capacity of "being aware" that the patient has, with a proper supporting system and social context, in order to obtain a maximum of efficiency. Background and purpose: Despite a recent increase in the attention given to improving communication of chronic kidney disease (CKD) patients' decision-making (Murray, et al., 2009 ), understanding of patients view is still lacking in Japan. The purpose of this study was to describe Japanese dialysis patients' views about advantages and disadvantages of their dialysis treatments (CHD: in-center hemodialysis, CAPD: continuous ambulatory peritoneal dialysis, APD: automated PD). Methods: Forty-one participants, including 17 CHD, 17 CAPD, and 7 APD patients (mean age: 58.5 year old (SD=13.6), mean dialysis period: 43.5 months (SD=29.2)), were requested to undergo a semi-structured interview, and the data obtained were qualitatively analyzed. Results: The participants considered that their dialysis modalities had its advantages and disadvantages. In terms of advantage, "less trouble for the people around me" was the most common answer of all dialysis modalities. "Independence", "less hospital visit", and "flexibility" were advantages of CAPD and APD patients. On the other hand, CHD patients consider the advantages of the treatment were "professional care" and "dialysis-free days". As for disadvantage, "catheter care" and "making trouble for the people around me" were the common answers of CAPD and APD patients. For CHD patients, "making trouble for the people around me", "restriction of diet and fluid", and "feeling bad after carrying out dialysis" were common answers. Waseda University, Tokorozawa, Japan and 2 Tobu Maruyama Hospital, Satte, Japan.
In this descriptive study, we will demonstrate therapeutic processes of clinical cases of maladaptive patients with moderate mental retardation. Although behavioral interventions are one of the most effective treatments to such populations, the relationship often encounters difficulties and the therapy becomes unprogressive. It is partly because of their limitation of ability to use language which enables them to understand and express their thoughts, feelings and understandings. The aim of this descriptive study is to show some of the examples of inventive approaches which thought to be helpful to the progression of the therapies for the patients with a mental handicap. Method (process of treatment): The patients were male 20 s and female 30 s, both were unmarried and lived with their families. Three years of outpatient therapy, conducted once every two to three weeks, took place at the psychiatric hospital. Medication and behavioral interventions, including management of use of social welfare services and psycho-education, were given. In addition, the following approaches were developed in the course of treatment: (1) Psychiatrist and psychologist worked as a team to deal with the patients, and discussed about emotional burden that had been experienced.
(2) The patients were asked to come to therapy by themselves and with their families alternatively.
(3) Importance of attending work at the welfare facility was emphasized at each session and patients' refusals of work were disputed thoroughly. The patients gradually have become to hold back complaining and maladaptive behaviors decreased. Discussions: In order to maintain the therapy relationships and prevent drop out, three approaches listed above seemed useful. Approach (1) may enable therapists to manage negative feelings toward patient which are often underestimated in the case of patient with mental handicap. Approach (2) may give patients the room for privacy and it may also enable to observe and approach family problems at the same time. Approach (3) may set strong therapeutic framework which enables both patients and therapists to realize small progresses that have been achieved through course of therapy. Quantitative research and experimental study will be needed to specify the effect of these approaches. Clinical and behavioral research is commonly challenged by difficulties in recruitment and retention, particularly for many underrepresented or vulnerable populations, including HIVpositive individuals, minority individuals, and individuals with low socioeconomic status. Indeed, recruitment rates in recent published reports of behavioral interventions focused on health improvement in such populations range from as low as 13.5% to as high as 59%. Methods: We conducted post-intervention focus groups with 15 participants in the Making Our Mothers Stronger (MOMS) Project, a randomized behavioral intervention. MOMS participants were randomly assigned to either the experimental condition of a parenting skills intervention or to an attentioncontrol condition of a health related behaviors intervention. Two focus groups were conducted with participants from each intervention condition. A focus group guide ensured consistency between groups and covered participants' response to the MOMS Project as well as their perceptions and recommendations about recruitment and retention. Three coders developed a codebook using constant comparison to identify major themes and sub-themes. Results: 87% of the focus group participants were African-American; their mean age was 41 years and 53% were single/never married. Identified themes included approaches to and venues for recruitment success, the role of key staff, effective and ineffective incentives, the impact of group process on recruitment and retention, and program resources that are needed. Discussion: Specific recommendations that emerged from the focus groups include recruiting in trusted venues, employing a persistent and encouraging recruiter who is sensitive to stigma issues, ensuring acceptability of recruitment materials through field-testing, ensuring social benefits of the intervention are immediate, and providing programs that are highly salient and practical. Methods: Since the time interval between exposure to HIV and the development of AIDS is close to 10 years, it is likely that these young adults were exposed to HIV when they were teenagers; this has rendered many of them homeless and hopeless. A study should be conducted in Ibadan Nigeria for the homeless youths living with HIV. This study will determine the knowledge, attitudes and behaviours of homeless youths regarding HIV infection. Self administered semi structured questionnaire would be administered by 70% of the homeless youth with HIV. A focus group discussion will also be used to get responses from parents/ siblings of victims.
Results: This method will creatively educate, sensitize and enforce behavioral change among the homeless youth living with HIV. Percentage of homeless youth with HIV in Ibadan Nigeria would also be revealed, it will also reveal various means through which the victims are affected with HIV in Ibadan Nigeria. Conclusion: Educational, social and medical programs are needed to reduce the risk of infection in this population. There is need for more training to empower this group to take a leading role in the community to prevent the spread of HIV and make them Volunteers against HIV (VAH). This will reduce numbers of homeless youths living with HIV in Ibadan Nigeria. Previous work has shown that most, but not all, HIV-positive adults disclose their HIV status to their sexual partners. Factors associated with non-disclosure include stigma, fear of rejection, fear of violence, and privacy concerns. In work with populations at risk for HIV, psychopathy (a personality characteristic associated with antisocial behavior and egocentrism) has been associated with substance use and sexual risk behavior. Little work has explored associations between psychopathy, disclosure, and risk in HIV-positive adults. In the present study, HIV-positive men and women (N=310) were recruited from the waiting room of an infectious disease clinic. Participants completed a self-administered questionnaire assessing demographic information, disclosure of HIV status, psychopathy, and sexual risk behavior. The majority (74%) of participants disclosed their HIV status to all sex partners in the past 3 months. Individuals who did not disclose their HIV status to all partners scored significantly higher in psychopathy (M=24.00, SD=5.22) than those who had disclosed (M=21.02, SD=5.91; F=7.98, p<.001). Higher psychopathy scores were associated with higher rates of having sex after drinking (rho=0.20), having sex after using drugs (rho=0.24), the number of sexual partners (rho=0.13) and unprotected vaginal and anal sex acts in the past 3 months (rho=0.13; all ps<.05). Higher psychopathy scores were also associated with lower intentions to use condoms with future partners (rho=−0.22, p<.001). Findings suggest that psychopathy is associated with risk behavior among HIVpositive men and women. Disclosure of HIV status has the potential to help reduce the spread of HIV. Interventions focused on increasing disclosure need to take into account associations between psychopathy and non-disclosure. No studies were found that assessed the emotional impact beyond 18 months post-infection. Studies used various methods of appraisal: self-report, standardized measures or questionnaires of original design, and a small number of studies employed hospital chart reviews. For the purpose of this study, the research results were divided into two recovery stages: early stages (<3 months post infection), and later stages (>3 months post-infection). Results: Early study variables that emerged included "worry about infecting others," "elevated emotional distress," "changes in mood," "symptoms of acute stress disorder," "feelings of stigmatization," and "psychiatric complications," such as the presence of psychotic symptoms (possibly secondary to steroid toxicity). Later stage variables included "emotional distress", "depressed mood", and "posttraumatic stress symptoms." Evidence of psychological problems developing in the later stages was also found in a small proportion of survivors. Conclusion: The limited number of published studies available for review consistently reported high rates of emotional distress in patients in both the early stages and later stages of recovery up to 18 months post-infection, with worry about infecting others, feelings of stigmatization, and psychotic symptoms emerging earlier in the recovery process. The levels of psychological distress found in these studies are similar to those reported in other comparable chronic physical illnesses. SARS remains a new disease, and the course of its development, both physically and emotionally, is still unfolding as time progresses. Longer term follow-up to determine the course of SARS patients' psychological status will help to guide treatment interventions. This, in turn, will help to inform the needs for future emergent, possibly similar, viral infections. Background: Vitality is related to health and is characterised by a perceived energy level, feelings of fatigue and feeling fit. These subjective factors can be positively affected by physical activity. Since vigorous physical activity is strongly related to aerobic fitness (VO2max), it is hypothesised that VO2max is related also to vitality. Therefore, the aim of this study was to investigate the association between vitality and estimated VO2max in older workers. Methods: Participants (n=427) How should one measure the effect of interventions to reduce sick leave, which survey methodology and study designs should one use and what statistical methods should be applied? Various designs provide different information and quality of information, and this should be put in a perspective in the analyses and in the interpretation of the results from available data. Depending on how an outcome (e.g. return to work) is measured various statistical methods are natural. This presentation discusses the use of extended event history analyses in multi-state models for the analyses of sick leave data. Using multi-state models, one can exploit more of the data available in the evaluation of interventions to reduce sickness absence or the effect after rehabilitation programs. We show the advantage of multi state models uses two datasets as examples. The first data is from a randomised controlled trial (RCT) on 256 individuals with low back pain, while the second data is an uncontrolled study on 577 individuals with different diagnosis after an 8 week stay at a rehabilitation clinic. For the RCT we show that the intervention increases the number of days in work with 46 day during a period of three years, while for the uncontrolled study we find that 60 % of the participants return to work. Severity of each complaint included in the inventory is rated on a 4point scale (0=none, 1=some, 2=much, 3=severe). Each complaint is also scored for duration (number of days) during the last 30 days. The product of severity and duration can be used to obtain a total score (0 -90) indicating the degree of health problems (absence of positive health). The SHC inventory include 5 Subscales (flu, pain, allergy, anxiety and gastrointestinal) and a total health complain score is possible to get as well. The SHC is available in several languages, and psychometric properties were previously described for different language versions. The present work describe the adaptation process for the Spanish language and preliminary psychometrics. Method: The English version of the SHC inventory was translated to Spanish by two experts, the Spanish version were reviewed by a third colleague to check the accuracy of the translation. This version was backtranslated to English by an independent translator. Backtranslated version was contrasted with the English original by the two initial experts. The Spanish version was then piloted with a small group of health adults to check comprehension of the items and wording. No suggestions were made by pilot subjects. Then final version of the SHC inventory in Spanish was considered achieved. These steps were followed by a psychometric study (n=83). Evidences of reliability (internal consistency), validity (construct validity, CFA) and sensitivity to change (after an intervention) were assessed. Results and conclusions: Results will be described in detail in the poster. Good internal consistency was achieved for the inventory (>.70), structure of the questionnaire was confirmed, and inventory show change in scores after participation in a stress related intervention. The SHC inventory shows be a useful, reliable, valid and sensible measure to assess subjective health complains in Spanish adults, and is available to use in research in Spain. Purpose: A writing psychotherapy, "Role Lettering Therapy(RLT)" was developed in Japanese reformatory in 1983. The procedure is almost same as empty chair technique, but written language is used instead of spoken. Haruguchi (1987) indicated several original effects of RLT. That is, 1) clarification of emotions, 2) self counseling, 3) catharsis, 4) confrontation and acceptance, 5) acquisition of both sides' viewpoints, etc.
In empirical RLT studies, increase of self-esteem, "adult" and "nurturing parent" (in egogram)" was pointed out (Hosoi et al., 2002; Okamoto, 2006) . And in case studies, expressed emotions, insight, self-awareness were suggested as significant indicators of this therapy. But it is necessary so that qualitative studies with objective summary tool clarify long sentences. In this study, description codes for RLT are structured, and the applications are discussed. METHOD: Sentences extracted from 12 articles and 2 technical books about RLT were categorized by using KJ method. The codes were examined two steps of validity (correspondence, usability, frequency, clinical importance) by 3-5 researchers. And the reliability was confirmed by concordance rate of the coding by 7 researchers and graduate students. RESULT: As a result of classification, 15 codes for "letters to another" and 14 codes for "letter to self" were structured. The former consists of "anger/dissatisfaction", "dependence", "need/hope", "reflection/awareness", "care/thoughtfulness", "gratefulness", etc. The latter consists of "complaint/blame", "enmeshment", "advice/ opinion", "expectation/wish", "care", "comprehension/regard", etc. A rate of agreement in 2 people's cording (in arbitrary 3 in 7) is 82.2%, therefore, these cords need the use in more than 3 people. DISCUSSION: The codes are in practical use stage, and have following utilizations. a) Discovering patterns in letters, b) convert a description into variables, c) comparison of technique. Furthermore, it can be applied to RLT as a supporting tool for understanding. For example, ascertaining a fluctuation in the number of the cords leads to understanding of client's power of expression. ) and has used in many mental health surveys in the world. We only describe depression validity due to other data analysis of this study is still in processing. This study is a diagnostic study, conducted in 2009 at 5 psychiatric centers in 5 cities (Jakarta, Bandung, Yogjakarta, Surabaya and Makassar). The participants were minimum 18 years, were excluded if had severe dementia and mental retardation. All participants were administered by the MINI and followed by SCID as a reference test. The SCID interviewers were general doctor and who were following the postgraduate of psychiatry, the MINI interviewer were nursing student and nurse. Both of interviewers were blind about the respondent's diagnosis except for respondent from population. The data processing used computer with SPSS version 15 statistical program. There were 82 data analyzed in this study, consisted of 45 patients and 27 non patients from population, 41 males and 41 females. The sensitivity of depression is 0.66 and the specificity is 0.89. The positive predicted value is 0.33, the negative predictive value is 0.97 and positive likelihood ratio is 6. 3, negative likelihood ratio is 0. 37. The median duration of interview was 30 minutes for a completed MINI and 60 minutes for completed SCID. The validity of depression diagnosis in this study is good but it needs comprehensive interpretation. It seems that even though the sensitivity is above 0.50 ,the questions are more specific to diagnose depression. The positive predictive value is low due to lack of patients. The next analysis will be better by completed data from all sites. The experiences of the author, a psychologist, regarding clinical depression were analyzed in order to develop new research methods for behavioral medicine. The analysis focused on self-investigation, quantitative and qualitative approaches, and an evidence-based approach for the individual. In Period I (1994) (1995) (1996) (1997) (1998) (1999) (2000) , a female client was treated with cognitive behavior therapy (CBT) by a clinical psychology professor. In Period II (2000) (2001) (2002) (2003) (2004) (2005) , pharmacotherapy was conducted by a psychosomatician. In Period III (2005-present), CBT and holistic medical treatment were conducted by another psychosomatician. In the second year of Period III (Period III-2), to facilitate self-regulation, Self-Efficacy and Self-Evaluation Scales were developed with the following items: 1. Absorb fulfilling experiences, 2. Keep good memories, 3. Achieve the research goals, 4. Calmly express feelings, 5. Change one's mood to cope with or avoid stress, 6. Regulate drowsiness, 7. Assert own opinion, 8. Ward off unimportant things, 9. Keep appropriate distance from others, and 10. Keep good personal relationships. Items 1-6 were common to Periods III-2-III-5, whereas items 7-9 were used in Periods III-2 and III-3 and integrated into item 10 in Periods III-4 and III-5. In each session, the client rated her pre-practice self-efficacy and postpractice self-evaluation. Average self-efficacy and selfevaluation scores descended in the order of items 1, 2, 3, 5, 6, and 4. Self-efficacy and self-evaluation scores on items 7-9 increased from Period III-2 to Period III-3. Most of the problems occurred in the family and relatives area. The top three reasons for positive discrepancy of the scores (evaluation -efficacy) include fewer problems occurred, or there was less sensitivity to the problems, executed things in workplace or personal areas, and maintained good communications. Correlations among self-efficacy scores on items 1-3 and 5 were significant in Periods III-4 and III-5. These results suggest that the client became able to cope with the problems and stress. The significance of this research is discussed in relation to "clients as researchers." A limitation of self-reports for disease symptoms is recall bias including retrospective distortions of the respondents' experiences. To overcome this concern, ecological momentary assessment (EMA) and the day reconstruction method (DRM) have recently been used, but the validity of these methods has not been objectively examined. Therefore, the aim of this study was to investigate the validity of EMA and DRM by using physical activity level (PAL) as an objective external criterion. Twenty-two healthy undergraduates (age 21.9±2.6 yrs) wore a watch-type computer and recorded visual analog scales of fatigue and depressive mood at different EMA design; recorded momentary symptoms every two hours (time-based) and recorded the symptoms during the behavioral episode which was just finished when switching one behavior to another (episode-based). They also recorded the symptoms afterward according to the series of behavioral episodes they reconstructed (DRM) about the same days. PAL was obtained using an actigraph built into the watch-type computer. Multilevel modeling showed that the association between depressive mood recorded with time-based EMA and PAL averaged over 60 minutes around EMA recording was significantly negative (p= 0.002); however, depressive mood recorded with episode-based EMA and DRM had no association with PAL averaged over the period of corresponding episodes. As for fatigue, none of the methods showed significant association with averaged Pal. These results suggest that time-based EMA might have the validity against the objective PAL when assessing depressive mood in contrast to episode-based EMA and DRM. Though averaged PAL alone failed to confirm the validity of all three methods used in this study as assessment of fatigue, incorporating other PAL measures than a simple average might distinguish the validity of them and need to be investigated. National College of Nursing, Japan, Kiyose-shi, Japan; 2 School of Nursing, Bunri University of Hospitality, Sayama-shi, Japan and 3 Kubota Clinic, Sumida-ku, Japan.
Purpose: This study examined the factorial validity of the short version of the Self-Efficacy for Social Participation for People with Psychiatric Disabilities scale (SESP). To develop a short version to measure self-efficacy for social participation was possible to use the instrument easily for people with severe mental illness (SMI).
Methods: A total of 140 community-dwelling individuals with SMI completed an anonymous self-report questionnaire consisting of questions for background information and the original 27-item SESP on a 4-Likart scale. The first data analysis was an exploratory factor analysis (principal component method with varimax rotation). Then we mainly chose ten items which were big factor loading in the factor structure of the 27-item SESP. We assumed them the short version of SESP (SESP10). And we performed confirmatory factor analysis to inspect the factorial validity of SESP10. Results: From results of confirmatory factor analysis, SESP10 was kept structure of 4 interpretable factors: Trust for Social Self, Self Management, Social Adaptability, and Mutual Support. It became clear that SESP10 had enough constructive validity. In addition, Internal consistency of the total SESP10 was excellent (α=.91). Discussion: It was proved that SESP10 had a sufficient validity. It will be to review the predictive and criterion-related validity of SESP10 in future. The short version of the Self-Efficacy for Social Participation for People with Psychiatric Disabilities scale has potential as a reliable and valid scale for measurement of self-efficacy of people who are afflicted with severe mental illnesses and who live in the community. Fibromyalgia syndrome (FMS) is a disease of unknown pathogenesis characterized by chronic musculoskeletal pain. FMS also includes other symptoms like poor quality sleep, morning stiffness, fatigue, anxiety, depression and altered endocrinological responses, but findings are inconsistent. The aim of the present study was to investigate free salivary cortisol levels, urinary catecholamines (noradrenaline, adrenaline and dopamine), heart rate, self-reported pain, and psychological features in FMS patients compared with healthy controls. Twenty-nine female FMS patients and 29 age-matched healthy female controls were recruited. Questionnaires measuring pain levels, sleeping problems, perceived stress and personality were administered to the participants. Salivary cortisol samples were collected 8 times: in the afternoon when subjects arrived at the hospital, after stress provocation, in the evening, before they went to sleep, upon awakening, 30 and 60 minutes later, and during the afternoon of the second day. Urinary catecholamines were repeatedly measured from the corresponding 24-hours period. Results revealed significantly more pain, perceived stress, sleeping problems, anxiety and depression among FMS patients compared with controls. Patients with FMS had significantly lower cortisol levels during the day, most pronounced in the morning (Cortisol Awakening Response). Moreover, FMS patients had significant lower adrenaline and dopamine levels compared with healthy controls, but similar noradrenaline levels. Furthermore, FMS patients showed increased heart rate values and lower sleep quality. Results support the hypothesis of a dysfunction in the regulation of the stress systems in FMS patients, and increased pain perception and sleeping problems. Emerging evidence suggests that immersive Virtual Reality (VR) distraction is an efficacious intervention for procedural pain. VR distraction offers an alternative to pharmacological treatments that may cause adverse side effects. Although VR distraction has been demonstrated to be effective in laboratory and clinical settings, the method by which it works to combat pain remains unclear. McCaul and Malott (1984) hypothesize that pain processing requires conscious attention. A highly immersive distractor should leave less attention available for pain processing. This study examined whether a VR head-mounted display helmet increases the analgesic effects of interactive distraction. A head-mounted display occludes extraneous sights and sounds, helping a person feel 'present' in the virtual world, leaving less attention available for pain sensations originating outside of the virtual world. Seventy-three college students between the ages of 18 and 25 were exposed to cold pressor pain without any distraction intervention (baseline) and then two cold pressor trials during which they played a Nintendo® Wii videogame. They viewed the game on a 21" TV or through a VR head-mounted display unit. The study used a within-subjects repeated-measures design, with the two videogame distraction trials presented in counterbalanced order following baseline. LOG-10 transformation was conduced on all pain tolerance scores to correct for skew and kurtosis. Results showed that pain tolerance during the baseline trial (M=1.46, SD=.254) was significantly lower than pain tolerance during the helmet-on condition (M=1.72, SD=.329), t(72) = -11.24, p<.001, and the helmet-off condition (M=1.68, SD=.32), t(72) = -10.69, p<.001. Participants showed the greatest increases in pain tolerance in the helmet-on condition, t(72) = 2.82, p<.01. Further research is needed to determine whether these results could be replicated in a clinical setting. Persistent pain, sufficient to cause significant impairment in daily functioning affects up to 50 percent of older adults. Analgesics are the most common therapy used to manage pain, with opioids playing an increasingly important role in pharmacological treatment of chronic disabling pain in older adults. The efficacy of medication therapy needs to be balanced against potential side effects, including the risk of neurocognitive impairment. In this study we objectively examined neurocognitive effects of methadone in older adults (>65 years) suffering from daily chronic pain (osteo-arthritis). Older participants (mean age 76 years) were evaluated twice at baseline (prior to the start of medication) and again 24hours after initial dose and again after 5 and 21 days of medication. Subjective ratings of pain along with objective testing of memory, attention and concentration were measured. Physiologic measurement of medication effects (pupil size) were also obtained. Results indicate a slight decline in weekly pain ratings (NS) and a modest drop in pupil size (NS). Neurocognitive tests indicated no significant change from baseline on measures of memory (HVLT), concentration (D2), reaction time or global cognitive measures (MMSE). There was an improvement on a measure of working memory (p<.05) and a trend toward improvement on an attention task (p<.06). Global measures of functioning did not change. Taken together, results suggest that older adults suffering from chronic non-malignant pain do not experience significant decrements in cognitive functioning in response to methadone. This may be consistent with findings of improved cognition with improved pain control observed in younger samples. Aim of investigation: There is limited knowledge about vulnerability factors for pain in fibromyalgia (FM), but low education, hereditary influences and mood are known, while contextual triggers and personality factors are dubious. Our aim was to study how FM pain differed in two contexts. Methods: Fiftyfive female FM patients scored pain (VAS 0-100), demographic data (age, education, and single status), mood (depression/BDI and anxiety/STAI-T), Cluster C personality traits (avoidance, OCD, dependence by MCMI-I), sleep (VAS), TCA-use dummy and FM-duration. Main outcome was Pain-difference (VAS) between "pain measured in a doctor's office" and "pain measured alone at home" one hour later. Secondary outcomes were vulnerability and resilience factors for pain in those two contexts. Results: "Pain office" (VAS=50.6) versus "Pain home" (VAS=43.4) showed a significant difference=7.2 by a paired t-test [t = 2.4; df=54; p=0.022]. Generalized Linear Methods (GLM) formula end-model: "Pain office" ∼ Anxiety + Education showed that Anxiety (t=3.15, p<0.01) facilitated the "pain office" experience, while Education (t=−2.43, p<0.05) impeded it. Avoidance (t=2.66, p=0.01) and "poor sleep" (t=2.55, p<0.05) worsened the "home-context pain". Conclusions: Pain was perceived weaker at home than in the doctor-office. Avoidance and poor sleep were vulnerability factors for pain at home, while trait-anxiety worsened pain in the office. Education was a resilience factor against pain in the doctor-office, while no resilience factors were found at home except the home context itself. Symptoms of Irritable bowel syndrome (IBS) are often aggravated by stress, which alters colonic motility and visceral perception. We reported the possibility of improving IBS pathophysiology by passive abdominal muscle stretching as indicated by chromogranin A (CgA), a biochemical index of the activity of the sympathetic / adrenomedullary system. Although the degree to which patient intervention in the areas of exercise, and management of daily activity can improve symptoms of IBS through healthier lifestyle behaviors is unknown. Int.J. Behav. Med. (2010) 17 (Suppl 1):S1-S329 S157
The aim of this study was to determine the effects of intervention to non-patient with IBS on the short and long term outcomes, and the association between physical exercise and symptoms. The present case controlled study was conducted on 30 adult subjects with mild or moderate symptom of IBS who were allocated randomly to either the exercise (intervention) group (n=15) or the wait-listed control group (n=15). Four-week intervention consisted of components: self record, stretch of abdominal muscle, physical exercise of pelvis, and 15 min working. Measurements (symptom-limited daily life performance, psychological status, CgA, heart rate variability and regional cerebral oxy-hemoglobin change) were performed before and after intervention. Spearman rank correlations were used to assess the association between psycho-physiological data and symptom scores. Wilcoxon rank sum tests compared changes in scores versus their baseline values. After the intervention, tendency of improvements were found in symptom-limited performance of daily life and psychological status. The four-week intervention had immediate beneficial effects on physiological variables and some neurological factors in patients with IBS. The findings of this study could be used as a reference for intervention, education, and physical health policies in IBS. Fibromyalgia is one of the chronic pain syndrome most prevalent worldwide, but there are serious doubts about its origin and its causes yet. Its complexity difficults the diagnosis and, especially, the sick role experience of patients. In this context, showing a maladaptive illness behavior will produce a negative interference in daily lives of these people, increasing their subjective distress. To improve our knowledge about this topic, our pilot study compares the responses between a group of fibromyalgia patients (N=16) and a control group (N=13), all of them women, with a mean age of 44. All participants belonged to a local association (Association of Fibromyalgia of Onda The WHO International Classification of Functioning and Disability (ICF) has been widely accepted as a conceptual model of disablement, but further specification of behavioral factors is required. We proposed conceptualizing disability, or activity limitations, as behavior and integrating biomedical and behavioral determinants. We previously tested a model including constructs from ICF and Social Cognitive Theory (SCT) using structural equation modelling; it fitted better and explained more variance than ICF or SCT alone. The current study replicated that study with a new sample from the same population (orthopedic patients awaiting joint replacement), and extended the tests to after surgery. Two weeks before surgery, 342 orthopedic patients with chronic pain (most having arthritis) completed a questionnaire assessing impairment and activity limitations plus SCT variables (Self-Efficacy and Outcome Expectancy). One year after surgery, when both impairment and activity limitations were reduced, 228 completed the questionnaire again. We imposed the statistical models found in our previous study (before and after surgery) using structural equation modelling, with NNFI, CFI, RMSEA fit indices) to predict activity limitations. Before surgery, findings were very similar to our previous findings, with all models accounting for significant variance and fitting well, but the integrated model fitted better and accounted for more variance. (ICF: 35%; SCT: 60%; Integrated model: 64%). Each model showed stronger predictions after surgery, with the combined model accounting for most variance (ICF: 52%; SCT: 79%; Integrated model: 82%). On each occasion, all three predictor variables had significant coefficients. The integrated model provides a good explanation of disability in this population, superior to a biomedical or behavioral model alone, and identifies targets for intervention to reduce disability.
CORRESPONDING AUTHOR: Marie Johnston, PhD, Health Psychology, University of Aberdeen, Aberdeen, AB; m.johnston@ abdn.ac.uk
The purpose of this study was to conduct an in-depth exploration of potential mediators of change in children's physical activity and the feasibility of strategies targeting these potential mediators.
A qualitative study of 143 children aged 10-12 years including eight focus groups from eight schools and teachers, and one-onone interviews with 12 parents were conducted. Common mediators of physical activity were identified from the literature and behavioural theories and a semi-structured interview explored the potential mediators of change in children's physical activity. Thematic analysis identified key and emergent themes.
Several new potential mediators as well as previously identified mediators were identified (eg. fear of the unknown and competitiveness). Most of the potential strategies were seen to be feasible; however, differing views existed among children, parents and teachers.These findings will inform the development of effective strategies for promoting children's physical activity by identifying the most appropriate potential mediators to be targeted. The purpose of this study was to investigate whether degrees of self efficacy (SE) are associated with physical activities or psychological factors after self-monitoring intervention. The subjects found in this study consisted of 49 female college students. We put the subjects in three groups, according to ones degree of SE: high SE group, middle SE group and low SE group. We conducted an intervention study using group cognitive-behavioral intervention including the selfmonitoring method. We performed a psychological assessment in order to exam SE, self esteem and stress responses, while also applying experimental protocol in the pre, intervention and post period stages. ANOVA of exercise volume disclosed significant group × period interaction. The significant period effect was disclosed in exercise volume and total energy expenditure. In the post period, low SE group showed a significantly lower exercise volume than high SE group. Low SE group had a significantly lower exercise volume, total energy expenditure and step counts in the post period compared in the intervention period. It should be noted that this pilot study was conducted without a follow up period. In the future, we need to examine whether self-monitoring intervention effects physical activities in a follow up period. In conclusion, after we applied group cognitive-behavioral intervention including the self-monitoring method, we discovered that high SE promoted the habituation of exercise while also inhibiting the risk of rebound. Apathy was decreased in only the subjects categorized with low SE using group cognitivebehavioral intervention including the self-monitoring method. Spondyloarthropathies (SpA) consist of a group of chronic inflammatory diseases characterized by symptoms of back pain, stiffness, fatigue, and sleep disturbance. While regular exercise has been shown to be beneficial and is recommended as part of patient selfcare, little is known regarding intention to initiate, alter, or maintain exercise in this patient population. This study examined the distribution of exercise stages of change and the biopsychosocial factors which differentiate readiness to exercise among individuals with SpA. Demographic and clinical data (comorbid disease, disease duration), along with self-report standardized questionnaires assessing functional disability, disease activity, depressed mood, perceived stress, leisure time physical activity, exercise self-efficacy, exercise pros and cons and exercise stage of change were collected crosssectionally from a sample of 125 individuals with SpA. Data were analyzed by frequency analysis, ANOVA and chi-square. Fifty-two percent (52%) of the participants were in the action/maintenance stages for exercise adoption and 49% in the preaction stages (precontemplation=20.8%; contemplation=13.6%, preparation 13.6%). Patients in the preaction stages had more disease activity (p<.005), more functional disability (p<.001), higher perceived stress (pp=.008) and reported lower self-efficacy (p=.003) and less barriers for exercise (p=.008). Gender, age, socioeconomic status (i.e. education, income) and exercise pros did not differ across the stages. These findings suggest that strategies designed to improve exercise self-efficacy, reduce exercise barriers and emphasize the functional and emotional (i.e. stress reducing) health benefits associated with physical activity may be useful for patients with SpA who are less motivationally ready to engage in regular exercise. Regular leisure-time physical activity behavior is achieved by fewer than 50% of Americans. If it were possible to decrease the cognitive load associated with deciding to exercise, it might be easier to adhere to a regular exercise routine, as it becomes an automatic activity that does not require decision making; this type of behavior would be termed by theorists to be a "habit." However, empirical evidence is lacking as to whether those individuals who have successfully instituted a regular exercise routine have such aspects of "habit" in their behavior patterns. The present study reports the level of habit formation among regular exercisers using a survey format. The Self-Report Habit Inventory (SRHI) (Verplanken & Orbell, 2003) measures the subjective sense of habit formation. The present exploratory study surveyed a sample of individuals (n= 176) who attended a fitness club in a metropolitan area using the SRHI (with a 7-point Likert scale), as well as objective questions about regularity of behavior (frequency and constancy of time, place, and weekly and seasonal patterns).
Results indicated a median SRHI-average score of 5.17 (range: 1.25-7.00). Even though this sample was presumed to represent regular exercisers, it was notable that there was a substantial subsample whose scores on the SRHI were quite low. Therefore, a median split was calculated, and participants with low versus high scores were compared on the other (non-SRHI) survey items. Low-SRHI participants exercised significantly fewer days per week (p<0.001) and their exercise patterns were more likely to change seasonally than high-SRHI scorers (p<0.05). However, the groups did not differ in regularly of time (p=0.52), location (p=0.14), specific exercise behavior (p= 0.41), or weekly consistency of exercise (p=0.36). Correlational and Fisher's exact test analyses suggested that individuals who demonstrated constancy on one objective measure of habit were likely to score high on other objective measures of habit as well (ps<0.05). Findings suggest that subjective self-concept of "habit" may not strongly correspond to all objective measures of consistency of behavior, and that only some individuals performing regular exercise behavior do so through a habit formation processes. Background: Fuzzy Trace Theory (FTT) is a cognitivedevelopmental theory on representation and memory recently applied to decision making in health domains (Renya, 2007) . It posits two processes/representations: 'Gist', which is bottom-line meaning of information, vague and qualitative, as opposed to 'Verbatim' representation which is the exact surface form (literal), precise and quantitative. The paper applied this theory to examine differences in decision processes of novices and experts in biking. It was hypothesized that experts, who rely on gist processing and less on knowledge, will use fewer information dimensions, reach unequivocal decisions with starker separations among categories, yet make high-quality decisions (as compared to a gold standard). Methods: 90 participants, who were novices (up to a year), experienced (one-two years of experience) and expert bikers (two to five years) filled out a self-administered questionnaire comprised of knowledge, intuition, and nine scenarios depicting challenging biking situations. Two recognized experts' responses served as a gold standard. Findings: Significant differences were found between the groups, so that experts were higher in the knowledge and intuition from the experienced and novices. Experts also relied in their decisions on fewer dimensions of information, provided starker and unequivocal judgments, and made more similar decisions to the golden standards than experienced and novice bikers. Finally, their judgments were based more on intuition (=gist) than on knowledge.
Conclusion: Experts in sports think differently than novices, and are higher in their use of both knowledge and intuition. This study evaluated the effects of social facilitation and competiveness on exercise behavior among older adults participating in the Cybercycle Study. Social facilitation is defined as an increase in response due to the presence of others (Allport, 1924) . Several theories, such as the Self-Determination Theory (SDT) and the Cognitive Evaluation Theory (CET), have been developed to explain exercise behavior and the influences of intrinsic motivation on performance (Fredrick & Ryan, 1995) . Some studies have suggested that competitive environments fostered by social facilitation enhance performance on motor tasks (Zajonc, 1965) . It was hypothesized that a personality factor, such as competitiveness could influence the effect of social presence. Thirteen older adults (age=78.6) participating in the Cybercycle Study (a randomized clinical trial investigating the neuropsychological, physiological, and behavioral effects of a videogame-enhanced stationary bike) were assigned to ride an exergaming bike while competing against virtual riders. Participant performances during one month (frequency and intensity of rides) were compared with the previous month's performances when no other avatars had been present. Results show that with the introduction of on-screen riders, as competitiveness increased, ride frequency decreased (r= −.67; p=.01), while riding intensity increased (r=.55; p=.05). Social facilitation appears to interact with an exerciser's level of competitiveness such that it could enhance or detract from exercise goals, depending on the desired outcomes. This finding has interesting implications for prescribing various types of exercise according to personality style and desired exercise goals. Introduction: Data available in the literature show that children in daycare centres spend most of their time in sedentary activities that could have an incidence on their health. To the best of our knowledge, this is the first study to use a combination of theoretical frameworks (theory of planned behaviour, theory of interpersonal behaviour, process model on ease of retrieval) to identify psychosocial factors underlying the intention of daycare workers to make children aged 3 to 5 years move. It is important to study intention, because findings of meta-analyses indicate that this variable is the best determinant of behaviour.
Method: This study is based on a descriptive correlational design. Daycare centres in the City of Quebec (Canada) were chosen at random. Daycare workers were then invited to complete a questionnaire measuring their intention to make children move for at least two hours a day in the next 30 days, as well as the following variables: attitude, subjective norm, perceived behavioural control, self-efficacy, descriptive norm, past behaviour, habit, perceived moral obligation and perceived regularity at which they make the children move. The Cronbach alpha of each construct was satisfactory, with values ranging from .82 to .97. Multiple regression analysis was used to analyze the data. Findings: Preliminary findings obtained among 37 daycare workers from 15 daycare centres indicate that perceived behavioural control (b=.35), subjective norm (b=.26), perceived moral obligation (b=.23), attitude (b=.20) and the regularity with which daycare workers perceive they make children (b=.14) move explain 87% of the variance in intention F (5,31) = 51.23, p=.00. Conclusion. These preliminary findings indicate that a large part of the intention of daycare workers to make children move can be explained by factors in the theoretical frameworks selected. These factors could eventually be used to develop an intervention aimed at improving or consolidating the intention of daycare workers. However, the findings first need to be confirmed in a larger sample. It has been documented that global attitudes are very poor predictors for outcomes of singular behavioral responses toward a given psychological object (Ajzen and Cote, 2008) . In addition to the psychological object, we must also know its underlying factors; chiefly action, context, and time. When trying to assess an attitude toward a health behavior, it is important to specify as many of the details surrounding the health behavior as possible, otherwise the attitude may be too generalized, or global, to be predictive of actual engagement in the health behavior under question.
In this two-year multi-site, randomized trial, 75 participants (mean age=78.99; SD=8.49) were enrolled and assessed using the Theory of Planned Behavior (Ajzen, 2007) to help determine how personality, disposition toward health behaviors, and environment affect a person's choice to engage in cybercycling. However, many of the measures used in the Cybercycle Study help to assess only global attitudes towards exercising and health behaviors, and for this reason we decided to develop our own measure specifically tailored to the act of cybercycling, called the Cybercycling Attitudes Test (CAT). Our hypothesis is that the CAT would lead participants to take into account the previously mentioned factors of action, context, and time while completing the questionnaire, such that it would be more predictive than the other measures for successful engagement in cybercycling. We aim to demonstrate the reliability and validity of the CAT, as well as to offer suggestions for replication in future behavioral health research. We hope that our results will add to the literature in suggesting more stringent development criteria for future measures assessing health behavior attitudes. INTRODUCTION: Exercise has been shown to have a significant impact on cognitive function in older adults and it is thought to offset declines in cognitive performance with advancing age (Colcombe et al., 2004; Larson et al., 2006) . Exercise has also been shown to increase nutrient supply to capillaries, to decrease neuronal death and to promote an overall better cognitive health (Kramer et al., 2007) . Nevertheless, very little research has been done to clarify the impact of exercise on the brain using neuroimaging techniques such as electroencephalography (EEG). METHODS: The current study aims to establish normative EEG and neuropsychological data in a sample of adults before and after a single bout of exercise. The level of cognitive state will be measured before and after exercise using a neuropsychological battery focusing on executive functions (e.g., Stroop, Trails, etc). Participant's neurophysiological state will be measured using EEG. Resting amplitude variability in frequencies will be measured and analyzed. RESULTS: It is hypothesized that a single bout of exercise will enhance cognitive function, especially executive functions. It is expected that there will be a significant relationship between the performance on executive functions and the power of Alpha and Beta waves. This research will provide clarify regarding the neurophysiological and neuropsychological impact of exercise in normals and will be useful in additional research with impaired populations. Of particular concern, is Alzheimer's which is on the rise bringing devastating social and economic impacts (Brookmeyer, 2007) . It is hoped that the potential neurophysiological and neuropsychological benefits of interventions such as exercise can be further clarified and utilized in the fight against cognitive decline. BACKGROUND: Differences in characteristics between respondents and non-respondents to an objective physical activity assessement survey could indicate selection bias. The aim of this study was to identify the demographic and lifestyle characteristics of respondents to accelerometry in a field-based mail survey. METHODS: A cross-sectional mail survey was sent to 4,000 residents aged 20-69 years old and 50% male, randomly selected from the registry of residential addresses of four cities in Japan. There were 1,508 respondents (Responding subsample) to the initial questionnaire. Seven hundred eighty-six participants from the Responding subsample also agreed to wear an accelerometer for 7 days (Accelerometer subsample). Age and sex were compared between the Accelerometer subsample and all 3,214 Non-respondents (including those not responding to initial questionnaire). In addition, the Accelerometer subsample and the 722 respondents who participated only in the questionnaire survey but not in the accelerometry survey (Questionnaire Only subsample) were compared for demographic and physical activity differences using chi-square tests. RESULTS: The Accelerometer subsample included significantly more women and middle-to-older age individuals compared with all Non-respondents. Comparisons between Accelerometer and Questionnaire Only subsamples revealed that persons who reported walking>=150 minutes/week for any purposes (p<0.01) or walking for leisure (p<0.001) tended to participate in the accelerometry survey. CONCLUSIONS: The response pattern in this study reveals potential for selection bias in mail-based accelerometry surveillance studies. It is important to develop strategies to increase overall response rate and address this bias. Understanding which exercise prescriptions are best suited for increasing adherence and exercise levels in previously sedentary adults is critical for efforts to enhance wellbeing through exercise program recommendations. Improvements in psychological wellbeing have been found with short (15-minute) exercise bouts, and it is possible that a regimen involving short bouts of exercise in a day is superior to one involving a single, long bout of exercise in a day because: (1) the anxiety-reducing effects of any single exercise bout reportedly last 4 to 6 hours, and (2) adherence may be greater because participants find it easier to fit short bouts into their daily schedules. This study investigated changes in levels of exercise participation, mood, anxiety and perceived stress in 82 sedentary adults who were randomly assigned to one of three conditions: a prescription of twelve 15-minute sessions of walking exercise per week (15Minx12), a prescription of four 45-minute sessions of walking exercise per week (45Minx4), or a waitlist control condition. Measures of exercise participation and psychological wellbeing were assessed at baseline and [daily/weekly] over an eight-week intervention. Participants in the 15Minx12 group completed more walking minutes for every week of the intervention, and significantly more so, or bordering on significance, in three of the eight weeks (t's≥2.00, p's≤.05), relative to participants in the 45Minx4 group. Although group differences in psychological outcomes fell short of statistical significance, regression analyses demonstrated that, for some weeks, walking minutes was an independent predictor of psychological well-being. These findings support that a 15 minute twice daily prescription may yield greater exercise participation in previously sedentary adults, relative to a regime involving less frequent, longer bout sessions. This enhanced exercise participation may, in turn, promote psychological well-being. (Ajzen, 1985) . Selfefficacy is one type of control belief in the model, and personality factors may also play a role in predicting behavior (Ajzen & Cote, 2008) . This study examined self-efficacy, along with competitiveness and frustration intolerance as two personality factors that may influence older adults' exercise behavior. METHODS: Thirty-nine older adults completed the 3-month experimental exercise phase in Year 2 of the "Cybercycle Study," a randomized clinical trial investigating the neuropsychological, physiological, and behavioral effects of a videogame-enhanced stationary bike. Personality and behavioral measures were administered pre-intervention. Self-efficacy for sticking to exercise was measured by the Self-Efficacy for Physical Activities Scale (Sallis, 1988 Introduction: Sleep disturbance is a common complaint in various respiratory diseases (Lewis, 1999) . Although this area has been the topic of some research, certain respiratory disorders, such as sleep apnoea and chronic obstructive pulmonary disease (COPD), have received more attention than others. In addition, much of the research has been physiological in nature investigating sleep disturbance in terms of objective measures rather than assessing the sleep complaints of the individuals themselves. Aims: The aims of the present research were to describe in detail than in previous investigations the occurrence and nature of sleep problems and behaviours in patients diagnosed with respiratory diseases compared to healthy controls. Design: A cross-sectional questionnaire survey of the prevalence and range of sleep disorders was investigated in individuals diagnosed with respiratory diseases, these being asthma ( Background: Clinical research on insomnia has observed that many individuals with this sleep disorder exhibit a significant disparity between their subjective reports and objective measures of symptom severity. This subjective/objective disparity poses potential challenges for diagnosis and treatment of insomnia and adds complexity to our conceptualization of the disorder. Insomnia is most common, severe, and impairing in adults over 60 years of age (Lichstein, 2004) and additional impairments may be present in older adults with insomnia that is comorbid with other psychiatric or medical conditions. This population is also at particular risk for making inaccurate subjective impressions of their insomnia symptoms due to a higher endorsement of unrealistic sleep expectations or worry over the effect of compromised sleep on daytime functioning (Morin, 1993 Introduction: Shift workers' sleep is restricted to 4-5 h a day for several days in association with consecutive early morning shifts and night shifts. After sleep restriction workers try to recover by sleeping at least 8 h a night. We examined the consequences of this kind of sleep-wake pattern on the ability to self-monitor one's own cognitive performance.
Methods: A total of 20 healthy men (19-29 yr) participated in a laboratory study. 13 of them attended a sleep restriction (SR) condition including a baseline (BL) day (8 h in bed per night), five SR days (4 h in bed), and two recovery (RC) days (8 h in bed). Seven controls slept 7-8 h each night. Each participant had two 50 min multitask sessions daily. After an extensive training phase task difficulty was set individually. Before and after each task session the participants estimated their performance (% of all available scores). Results: Both performance and subjective performance estimates were affected by the group-by-day interaction effect (p<.001). In the SR group, these outcomes were at a lower level during the last three SR days than during the BL day (p<.01). In addition, the SR affected selectively the pre-task performance estimates and actual performance: the pre-task estimates decreased less than actual performance during the last two SR days (p<.05). The recovery days restored both the subjective performance estimates and actual performance to their baseline levels. In the control group, neither performance nor the subjective performance estimates showed a decrease over the experimental days. Conclusions: Cumulative sleep restriction makes one to prospectively underestimate the actual degree of cognitive impairment. This metacognitive effect of sleep restriction may play a role in an increased accident risk in shift work. Recovery sleep after sleep restriction seems to restore the relationship between subjective performance estimates and actual performance to its baseline level. Breast cancer screening uptake is lower in ethnic minority women compared to white women. In particular South Asian women are less respondent to invitations for screening but also have higher breast cancer risk. This study looked at beliefs about breast cancer awareness in South Asian Muslim women pre-mammography age (29-49 years).
The focus was to see how South Asian women conceptualised their beliefs in relation to healthy breast awareness and to determine the role of religion and body image in intention to be breast aware. Women (mean age 37 years) were recruited through mosques and community forums in the UK . They completed a brief questionnaire looking at constructs from the Health Belief Model (Champion, 1999) , body image and religiosity. Results showed that higher religiosity was associated with the following: greater perceived barriers to both breast cancer awareness and mammography screening, reduced confidence in breast self awareness, and less perceived benefits in mammography. In contrast positive body image was found to have a positive impact on breast health motivation, breast cancer awareness and finding out more about breast cancer. Good breast cancer awareness was associated with higher perceived benefits and lower barriers as expected.
Examination of the predictors of intention to self-examination showed that higher body image and educational level had a positive impact on confidence, although no direct relationship to intentions. In this study, we developed the Takeda Three Colors Combination Test (TTCC) as a simplified screening test for early detection of AD and examined its effectiveness. 166 senior persons participated in the research; 91 with mild AD and 75 healthy volunteers represented the control group. The TTCC, which is a colored cards configuration memory task, was examined for sensitivity and specificity as well as reliability and validity. The sensitivity of the TTCC in detecting mild AD was 84% with a specificity of 87%. The positive predictive value was 89%, the negative predictive value was 82% and the predictive accuracy was 86%. The odds ratio of the incorrect response to TTCC with control group as a standard was 32.0 (95%CI,13.1-78.1, p< 0.0001) for mild AD group. Including the results of the re-tests, the Phi coefficient indicated 0.76 (p<0.0001) and the consistency percentage between first and second trials was 88%. Spearman's rank correlation analysis, using MMSE as an external standard, revealed a significant correlation between MMSE and the TTCC results (ρ=0.58, p< 0.0001). Conducting the TTCC (including instruction and evaluation) was accomplished within 2 minutes for all subjects. No refusal or resistance to this test was observed among any of the subjects. The TTCC has proven to be of adequate sensitivity and specificity as a screening test for early detection of AD. Furthermore, administration time is short and requires no special training or skills. Thus, we believe the TTCC shows great potential for use as an AD screening test by a general practitioner in communities worldwide. Objective: To understand reasons that influence African-American participation in a study designed to increase CRC screening in primary care. Methods: As part of a larger randomized, controlled behavioral intervention, we administered a baseline telephone survey to 134 African-American primary care patients ages 50 to 79. The survey included sociodemographic background measures and perceptions about CRC and screening. We also asked consenting participants why they joined the study. The research team performed content analysis of responses to this question and tallied categories. Results: 75% of respondents were female and 25% male. Ages ranged from 50 to 79 with 72.4% of participants between ages 51-60. In this sample, 38% had a HS/GED education, 19% had a college degree or more, and 17% had a technical degree. Primary reasons for participation included: desire to support research (45%), contact by study staff (36%), and a desire to maintain health (34%).
Conclusions: As this was an African-American study sample, desire to support research as a primary participation reason is noteworthy. Contact by study staff as a major reason for participation is also significant. Importantly, respondents' desire to maintain their health was another principal reason cited for participation. These findings provide insight into reasons why African-American patients decide to participate in prevention research. We will add to these data through the analysis of responses from 896 individuals targeted for recruitment. We examined barriers to colorectal cancer (CRC) screening among urban and rural Pennsylvania Latino/Hispanic adults. A total of 82 Latinos/Hispanics engaged in one of eight focus groups stratified by sex and geography offered; and completed a 25-item survey eliciting their demographic information and CRC cancer screening behaviors. After the focus group, participants viewed a sevenminute American Cancer Society's published Spanish DVD entitled, "Get Tested for CRC. Here's How" and reassemble to answer questions about their preference and acceptability of the CRC screening options. Focus group data were audio taped, transcribed and grouped into thematic units using content analysis. We found substantial barriers by sex and geography, including urban residents received screenings during annual check-ups, while rural residents received screenings in response to symptoms. Of the 57 total barriers reported, four barriers were unique to urban Latino/Hispanic residents; and 13 barriers were unique to rural Latino/Hispanic residents. Low levels of health literacy, knowledge and awareness of CRC risk and screening were reported barriers across groups. The family unit and strong social support were also factors reported as influencing their CRC screening behavior. Participants identified barriers to CRC screening that fit into five categories: (a) physical environment, (b) structural factors, (c) sociocultural factors, (d) individual factors, and (e) physician-related barriers. Latino/Hispanic participants also identified potential strategies to overcome each reported barrier. These finding suggest that a targeted CRC screening intervention utilizing a physician-recommended home fecal immunochemical test with instructions is preferred among Latino/Hispanics over a non-targeted approach. African Americans are more likely to be diagnosed with, and to die from, colorectal cancer (CRC) than any other group. Approximately half of CRC deaths could be prevented if screening were consistently implemented. The Transtheoretical Model postulates that behavior change occurs over time with people moving through stages of readiness to adopt the behavior. The aims of this study were to: 1) examine distribution across stages of readiness to screen for CRC among African American primary care patients; and 2) determine whether demographic and health belief variables predict stage of readiness to screen. Baseline data were collected via telephone interviews from 311 patients who were due for screening and enrolled in a RCT testing efficacy of a computer-based intervention designed to increase CRC screening. Ordinal logistic regression models were used to examine demographic variables and Health Belief Model constructs (perceived risk, benefits, barriers and self-efficacy) as predictors of stage of readiness to screen for CRC. The sample was 56% male and a mean age of 58 years. For FOBT, 51.8% of patients were precontemplators, 37% were contemplators, and 11.3% were in preparation. oral health problems and their corresponding clinical signs (67 in total) were identified. The aim of this study is to present the content validity of the tool. Method. A dentist and three general practitioners in palliative care in Quebec (Canada) were invited to evaluate the relevance and sufficiency of each of the clinical signs identified. The relative importance of clinical signs was also assessed for certain oral health problems. As suggested by Polit, Tatano Beck and Owen (2007), an I-CVI of at least .75 was used to conclude about the relevance of a given clinical sign.
Findings. The I-CVI was satisfactory for 62 of 67 clinical signs. There was disagreement between the experts concerning the relevance of using pain/burning pain of mucosa and pain/ burning pain of tongue to detect both pseudomembranous candidiasis and erythematous candidiasis. Opinions were also shared concerning the use of tooth sensitivity to cold and that of nearby tissues as a clinical sign of pulpitis and dental abscess. Finally, the experts considered that some clinical signs were more important than others to detect erythematous candidiasis, hyperplasic candidiasis, mucositis/stomatite, infections of viral origin and xerostomia. Thus, it will be necessary to develop a scoring method that takes this aspect into account. immigrants. It appears that education is a more robust predictor of physiologic CRF compared to income. Given that education may reflect status prior to migration (Average Age at Migration=39 years), the interaction between status and physiologic indicators of health may be important to assess throughout the lifespan in future studies. It is also possible that Education has a higher range than income with almost 80% of the sample earning less than $30,000/year. status, and employment status, with using weights for the sampling design. Results: In the US sample, education, household income and SSS were significantly and inversely associated with psychological distress (p<0.05). In the Japanese sample, respondents with high education or low household income had a non-significant but slightly higher prevalence of psychological distress; furthermore, those with the highest or lowest level of SSS had a higher prevalence of psychological distress (p<0.05). A significant interaction between the country and education or SSS was observed (p<0.05).
Conclusions: While a linear association between any social class indicator and psychological distress was observed in the US, the association between subjective social class and psychological distress seems U shaped in Japan, probably reflecting the fact that those with high education or low income had a higher prevalence. Background. Despite a number of papers on the FCTC negotiation process and final text, there have not been attempts to test the usefulness of treaty process by quantifying its global impact on the adoption of domestic tobacco control policies. This paper suggests that the negotiation process provided a platform for increased communication and international learning through which tobacco control information spread to many countries; and that this simultaneous learning process accelerated the adoption of internationally promoted tobacco control policies by countries around the world.
Objective. The present study quantifies the impact of the FCTC negotiation process on the global diffusion of domestic tobacco control policies. Methods. Country characteristics, including income, population, region, democracy, tobacco production, smoking prevalence and network participation were analyzed to determine their effects on the frequency, type and strength of tobacco control policy adoption among WHO Member States. Bivariate analyses were conducted for each country characteristic and used to compare the frequency and strength of control policies adopted between pre-negotiation and negotiation periods. Multivariate regression analyses were performed to determine the predictive nature of these variables. Findings. The frequency of policy adoption escalated between 2002 and 2003, the years in which the FCTC negotiations were most intense. The strength of policies adopted also shifted significantly towards policies promoted by WHO. The average strength of policies adopted varied significantly with population size, region, democracy levels and network participation. All characteristics, with the exception of total and male smoking prevalence, were significantly associated with the number of policy types adopted.
Conclusion. This study suggests that investments in formal international legal processes can be effective and appropriate, even when the outcomes are unclear from the start. The FCTC negotiation process coincided with a rise in domestic policy adoption in the direction advocated by WHO. However, there remains the need to improve outreach and diffusion to lower-income countries in the area of tobacco control, and likely other areas of chronic disease control. Aims: The number of the unemployed has been increasing in Japan, as well as in other countries, due to the economic turn-down in 2008. The unemployed might have lower socioeconomic status (SES), thus have poor mental health, and difficulty in seeking help for mental health problems. This study aimed to clarify subjective social class, mental health, and help seeking behaviors among the employed who looked for a job, comparing with those currently employed. Methods: A community-based epidemiologic study of common mental disorders, the World Mental Health Japan 2002-2006 Survey, were conducted of random samples of residents in 11 municipalities (N=4,130), with using the WHO-CIDI 3.0. A subsample of the respondents (the part 2 sample, N=1,682) was asked to determine their social class, self-rated health, psychological distress (K6), 12-month DSM-IV mental disorders, and help-seeking behaviors for mental health problems. Analyses were conducted, comparing these variables between the currently unemployed who look for a job (N= 35) and the currently employed (N=997), adjusting for sex, age, and education, with considering the sampling weight. Results: The unemployed had significantly lower education and household income than the employed (p<0.05). The unemployed reported poorer physical and mental health status than the employed (p=0.08 and p=0.06, respectively), while there was no significant difference in prevalence of psychological distress or mental disorders. The proportion of those who sought help for mental health problems was not significantly different between the two groups; however, as a reason of the delay in seeking help, the unemployed more tended to concern "how much money the treatment would be cost"(p=0.07). Conclusions: The unemployed were at lower SES, had poor physical and mental health status, and delayed their help seeking because of their concern on treatment fee. Backgrounds and purpose Limited evidence has been available for regional socio-economic status (SES) and health in Asian countries. Whether regional SES influences individual cause-specific risk of death remained to be examined. The aim of this multilevel study is to evaluate whether regional SES influences cause-specific mortality independently from individual SES.
We used the data from the Japan Collaborative Cohort (JACC) Study in 1988-1990, including 22,638 men and 30,518 women who aged 40 to 64 with no medical history of cancer and cardiovascular diseases at baseline in 35 municipalities. They were followed up to determine mortality of various diseases by the end of 2005. All deaths were ascertained by death certificates from public health centers. The ICD 9th and 10th revisions were used to determine cause-specific mortality. Proportion of college graduates, per capita income, unemployment rate, per capita bank deposit, and proportion of households receiving welfare were used as indicators of regional SES. We adjusted individual SES (education level, occupation) and possible confounding variables. Gender-specific adjusted odds ratios for specific causes of deaths were estimated according to regional SES by multilevel logistic regression.
Regional SES indicators were associated with risk of mortality independently from individual SES. The effect of regional SES was statistically significant for cardiovascular disease, especially stroke among men, while it was apparent for respiratory diseases or external causes of death among women.
This multilevel study showed that SES of municipalities has independent influence on risk of mortality among individuals living in these areas. Not only individual but also regional SES could be social determinants of health. Introduction: Cardiovascular disease (CVD) is associated with high morbidity and mortality rates in the U.S. Negative associations between CVD and socioeconomic (SES) are well established. Socioeconomic status, generally defined by factors such as income, education and occupation, can be measured subjectively via-self report using a scale representing a ten rung ladder.
Recently, subjective SES has shown to be more predictive of health and cardiac outcomes than actual SES. Vital exhaustion (VE) is another risk factor associated with CVD and is characterized by feelings of excessive fatigue and demoralization, as well as in increase in irritability. To our knowledge, socioeconomic differences, both subjective and objective, with respect to VE, have not been explored. Therefore, the current study examined the associations between subjective SES and VE among cardiac patients. Death is a natural and inevitable phenomenon that everyone has to face it. Nevertheless, our society seems to refuse everything that could be related to illness, suffering and death. Daily we overhear news about unexpected deaths, which highlights the importance of being aware of one's death as a part of the cycle of life. The aim of this study was to analyze the main factors that could help to face the own death from young people's perspective. The sample was made up of 60 young people, aged between 18 and 27. All participants were evaluated by the "Helping to Die in Peace Questionnaire" (CAMP) by Bayés et al., (2000) , which is composed of two major items. The first one assesses the aspects that could help people to die in peace, answering to eleven sentences according to a 5 points scale. In relation to the second one, participants have to select the two most important factors when death is close, through the eleven previous statements.
Results showed the following factors as the most relevant to young people: "Being able to be close, communicate and strengthen my bonds with loved ones" (X=3,1; SD=0,8), "To think that my life has had some meaning" (X=3,0; SD=1,1), "To think that my death won't cause an unbearable burden to loved ones" (X=2,9; SD=1,1). On the other hand, the less important aspects were: "Believing in other life after death" (X=1,5; SD= 1,5), "To think that I may die at home" (X=1,8; SD=1,3), "To think that my dying process will be short in case of suffering" (X= 2,3; SD=1,4) and "Not to feel guilty about past personal conflicts" (X=2,3; SD=1,4). (2004). Results showed family satisfaction in descending order, so family members were more pleased in relation to "satisfaction with medical care to patients" (81,5%), "best care given to patients" (77,8%), "be called with some significant change in patients' condition" (75,9%) "care about the patients from hospital personnel" (74,1%), "understanding about patient's situation and procedures applied" (74,1%) and "honesty of the information provided" (70,9%). Lesser degree, they were satisfied in "explanations about patient's condition", "staff members attention to family" and "loneliness/isolation in the waiting area" (61-67%). Finally, the results pointed as the fewest satisfied aspects those connected with "comfort about visiting patients in ICU" (47,3%) "interest showed by staff members"(32,7%), "explanation about the equipment used" (25,9%) and "comfort of waiting room" (11,5%). These findings show that family members satisfaction to their ICU's experience was quite good in general. It is necessary taking into account those improvable aspects in order to provide them the best stay in our ICUs. BACKGROUND: Evidence on the association between social status inconsistency (SI) and self-reported health is limited and mixed. Moreover, only little research has been done to examine the pathway through which SI affects health. Therefore we analysed the mediating effect of effort-reward imbalance at work (ERI) on this association. METHODS: Analyses were based on a random sample of 1111 male employees (25-64 years) from the south-west of Germany. Data were collected by a telephone interview in 2008/09. Information on socio-demographic variables, social status indicators (education, occupational grade and income), occupational strain (ERI), and physical as well as mental health (SF-12) were given. RESULTS: SI was significantly associated to low mental health, both directly and, consistent to our hypothesis, indirectly via low rewards. As SI was also related to low effort, no association was found for the quotient of both scales (ERI) and mental health. The effect of SI on physical health was entirely mediated by low rewards without any direct effect. The inclusion of further risk factors (age, BMI) did not attenuate these Results: CONCLUSION: SI is significantly associated to ill health. Moreover, results indicate that at least a part of this relationship is explained by low rewards at work, findings with implications for further research in job strain and health inequality. Selection of a spouse reflects the cultural values of society. Spouse selection in the west is based primarily on falling-in-love whereas in India, selecting a spouse has been traditionally arranged. As a result of globalization, western influence in India is on the rise requiring a traditional society to modify its cultural norms. While arranged marriage is still prevalent in India, a new mode of marriage, love marriage, is becoming increasingly common. This transition introduces new social stressors, which may impact health. We hypothesize that traditional individuals will feel conflicted by the shift in cultural norms toward a more nontraditional system and possibly experience symptoms of depression. To test the hypothesis, a group of 350 college students in Mumbai, a major metropolis of India undergoing rapid cultural change in recent years were surveyed. We administered questionnaires measuring the influence exerted by tradition on mate Subjective socioeconomic status (SES) impacts health independently of objective SES. However, little is known about variables that contribute to perception of social standing other than objective SES, which is only modestly correlated with subjective SES. Spirituality also impacts on health and may synergistically interact with perceived SES to predict health. It is hypothesized that Subjective SES will correlate positively with Spirituality. And spirituality and subjective SES will predict health outcomes after controlling for objective SES. Urban college students (N=568; Mean Age: 19 years; 73.1% Female; Religion: 46.3% Christian; 37.3% Hindu; Other: 16.4%) in Mumbai, India completed questionnaires on Spiritual Well-Being (SWB) measuring Existential (EWB) and Religious Well-Being (RWB), somatic symptoms (CHIPS), subjective SES and objective SES. Results indicated that objective SES and subjective SES were positively but modestly correlated (r=.20, p<.001). There was a negative relationship between objective SES and SWB, RWB and CHIPS (r=−.11, p<.05; -.20, p<.001; -.10, p<.05, respectively) and between subjective SES and RWB and CHIPS (r=−.12, p<.005; -.20, p<.001, respectively). These data indicate that high SESperceived and actual -is associated with lower engagement in prayer and relationship with a higher spiritual power and with fewer somatic symptoms than low SES. There was no correlation found between objective SES and EWB and subjective SES and EWB were positively correlated (r=.20, p<.001) indicating that perceived social standing is associated with life satisfaction and stability. These data indicate the complexity of variables that contribute to material and spiritual well-being and its interaction with health where subjectively poor people engage more in religious practice and do not experience existential satisfaction and report more somatic symptoms. Discussions about medical information banks may influence decisions to participate in genetic biobanks. Project Diversity GATHER (Group discussions Around Themes on HEalthful genetic Research) examines demographics and health care perceptions that may affect willingness to participate in genetic biobanking. Participants (N=135, 63% female; mean age=46.67, SD=17.17, range =18-87) completed sociodemographic and psychosocial questionnaires and a one-hour focus group. Participants selfidentified as Black (49.2%), White (36.2%), Pacific Islander (2.3%), American Indian (2.3%), Asian (1.5%) and Other (8.5%); 19.3% of participants were Latino. Univariate and bivariate descriptive analyses and t-tests were performed where appropriate. Univariate and multivariate logistic regressions were applied to compare differences in (un)adjusted odds of who was willing to participate in biobanking vs. not controlling for confounds. Participants unwilling to participate in genetic biobanking had higher health care distrust than willing participants [t(126) = −3.14, p<.01]. Logistic regression models compared participants willing to participate in genetic biobankIng vs. not. Univariate logistic regressions noted significant relationships for race/ethnicity (OR=0.30, 95%CI=0.12-0.75), primary care physician status (OR=0.51, 95%CI=1.03-1.18), and health care system beliefs (OR=1.11, 95%CI=1.02-1.18), with gender, age, and education level yielding nonsignificant relationships. Multivariate analyses confirmed race/ethnicity (OR=0.25, 95%CI=0.09-0.70) and health care beliefs (OR=1.10, 95%CI=1.02-1.18) to be significant predictors. Participants characterized as racial/ethnic minorities or who are distrustful of the health care system may be less likely to participate in genetic biobanking. Our findings support previous research in this area and may help to explain why medical research often underrepresents racial/ ethnic minority groups. The relationship between health care distrust and unwillingness to participate in genetic biobanking highlights potential recruitment barriers in genetic testing. Clinicians and researchers may wish to assess and attend to distrust in patients for whom genetic testing is clinically indicated. Our purpose was to predict systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) during 24-hour ambulatory BP monitoring using respective diagnostic values of BP and HR, and measures of social support as predictors in a hierarchical regression analysis. The predicted periods of the ambulatory BP monitoring were 24-hours, morning, evening, and night. The participants were 95 healthy men with different BP levels from the Tampere Ambulatory Blood Pressure Study. The participants could be treated as one group, because the diagnostic BPs were normally distributed. The measures of social support were the total score of the Interview for Social Interaction (ISSI; Henderson, Byrne, & Duncan-Jones, 1981) and five subscales of it. The first sets in the hierarchical regression analysis had the respective diagnostic values of BP and HR as predictors. These were all significant predictors of daily cardiovascular measures as expected. In the second series of sets, the total score of ISSI and the subscale scores of it were added to the models, respectively. The prediction of ambulatory cardiovascular measures improved by up to 3% by adding the Adequacy of Social Integration (ADSI), a subscale of ISSI to the diagnostic SBPs. In conclusion, one specific measure of social support, the Adequacy of Social Integration (but not others) improved the prediction of cardiovascular measures in daily life. These results are in line with previous findings emphasizing the function of the quality of social support as a buffer in daily life. (Chaix et al. Hypertension, 2010) suggest that central adiposity and heart rate account for a sizable amount of the inverse association between individual education and SBP in a French cohort: body mass index (BMI) and waist circumference explained 28% and resting heart rate, 15% of this association. Psychological factors and health behaviors also helped account for the education-SBP association, but to a much smaller degree. We used data on a nationally representative U.S. sample of 15,701 young adult participants aged 24-32 years in Add Health, Wave IV, to determine whether a similar education-SBP association exists in a U.S. cohort and to examine several potential mediators of it. Individual education level was associated with SBP (p< 0.0001) as follows: Some high school or less, N=1212, SBP= 126.0+14.1(S.D.)mmHg; Graduated high school, N=2453, SBP= 126.6+13.9 mmHg; Some college or vocational tech, N=6710, SBP=124.7+13.8 mmHg; Bachelors degree, N=2942, SBP= 123.2+13.1 mmHg, and; Graduate school or more, N=1845, SBP=122.0+12.7 mmHg. When the education-SBP association was adjusted for BMI, the 4.0 mmHg difference in SBP between the lowest and highest education groups was reduced to 2.7 mmHg. Further adjustment for gender, race/ethnicity, physical activity, alcohol consumption, smoking, marital status, employment status, income, presence of financial strain and home ownership reduced the SBP difference to 0.6 mmHg. These findings replicate in a U.S. cohort the finding in a French cohort of a robust association between lower education level and increased SBP. In the larger Add Health sample, this association was nearly completely accounted for by BMI, race/ethnicity, gender, marital status and lifestyle characteristics. Introduction: Many patients fear upper gastrointestinal endoscopies. Natural anxiety may be aggravated by horror stories from friends or inappropriate remarks by endoscopy staff. Shavasan is probably the best-known relaxation exercise. Through relaxation of muscle tension, an anxiety free sate is reached. Objective: The study was to examine effects of relaxation in Shavasan posture in patients undergoing upper GI endoscopies. Method: Study was conducted on 63 consecutive patients between ages of 16 to 86 years. Patients were randomly assigned to two groups regardless of sex, age and underlying disease. Thirty one patients relaxed in shavasan, before and during procedure, while 32 patients did not. No patients were given sedation or local anesthetic spray. Blood pressure, heart and respiratory rate were recorded. Perception of procedure using a 5-point attitude scale was assessed. Results: Paired T-test was used to analyze the data. Test group results showed significant difference in three parameters i.e. blood pressure, systolic, heart and respiratory rate in the difference of values at the beginning and end of procedure. Control group did not show statistically significant difference in any parameters. However, data comparison in two groups showed statistically significantly difference in the heart and respiratory rate. More patients in test group reported decrease in distress. Conclusion: This preliminary study shows the effect of relaxation in Shavasan posture. Repeated practice is necessary to attain quick de-stressed state in distress. We suggest that this exercise could be applied to any other medical situations as well, which tend to generate undue psychological stress and anxiety. Problem: Coping resources can be of great assistance when one is placed in stressful situations because they influence whether or not the stressor is directly approached or avoided (Taylor & Stanton, 2007 ). This research proposes that self esteem, a key dispositional factor will reduce perceived stress for African Americans. Further, effective coping strategies may moderate the impact of self esteem on stress. This research hypothesizes that lower perceived stress will be predicted by higher self esteem and that effective coping strategies will increase this relationship. Procedure: 146 African American college students in small groups completed measures of self esteem, coping strategies and perceived stress. Results: As expected, low self esteem (r=−.29 p<.01; Rsquare= .09 β=−.29, t=−3.56 p<.01) predicted higher perceived stress. In addition, seeking social support(Rsquare=.15, β=.23 t=2.63 p<.01), an active coping strategy, and wishful thinking (Rsquare= .25, β =−.21 t=−2.78 p< .01), an avoidant coping strategy moderated the relationship between self esteem and perceived stress. Low social support seekers and high wishful thinkers showed stronger impacts of lower self esteem on increased stress. The coping strategy of seeking social support was not independently correlated with perceived stress but "wishful thinking" (r=.35 p<.01) was. Conclusion: This study found that low self esteem is directly related to increased perceived stress among African Americans. The inclination to seek social support reduces low self esteem individual's perception of a situation as stressful. However, wishful thinking increased the likelihood that an individual with low self esteem would perceive an environment as stressful. These findings suggest that cognitive-behavioral prevention programs that seek to alleviate stress among African Americans who have low self esteem should emphasize the importance of creating trusted social support systems and realistically assessing the magnitude of a problem in an effort to better manage one's stress responses. AIM: To explore the influence of occupational stress on mental health in Industrial worker. METHODS: A cross-sectional survey was conducted among 561 workers. The workers were invited to fill in a self-administered questionnaire exploring their socio-demographic characteristics, occupational stress levels, and 12-item general health questionnaire. A hierarchical multiple regression procedure was used to assess the effects of occupational stress on mental health. RESULTS: After controlling for age, educational level, marital status and years of offshore work, poor mental health was found to have a significant positive association with seven of the nine identified sources of occupational stress. They were: conflict between job and family/ social life, poor development of career and achievement at work, safety problems at work, management problems and poor relationship with others at work, poor physical environment of the work place, uncomfortable ergonomic factors at work, and poor organizational structure at work. All of these occupational stress sources together explained 19.9% of the total variance. CONCLUSIONS: The results confirmed that occupational stress was a major risk factor for poor mental health among industrial workers. Reducing or eliminating occupational stressors at work would benefit workers' mental health in developing country like Nepal. There should be some meditation program, behovior change and rest for workers to reduce the occupational Health Undergoing medical procedures, such as an amniocentesis, is stressful for pregnant women because they are often in a state of heightened anxiety about the well-being of their unborn child and experience uncertainness and possibly also increased physical discomfort. The aim of the pilot study was to evaluate the impact of an amniocentesis during pregnancy on cardiovascular stress response. Twelve healthy pregnant women in the second trimester of pregnancy (age: M=38.1, SD=2.8), underwent an amniocentesis during which heart rate (HR) and Respiratory Sinus Arrhythmia (RSA) were measured continuously, beginning 20 minutes before the examination and lasting until 30 minutes after termination of the examination. Additionally, a questionnaire was distributed to measure the participants' general control conviction in life. Analyses revealed a significant decrease of HR (p<.001) and a concomitant increase of RSA (p=.036). Furthermore, a general fatalistic control expectation attitude was associated with a smaller decrease in HR (r=.616, p=.077) and a smaller increase in RSA (r=−.735, p=.024) after the procedure. Our findings reveal a harmful anticipatory cardiovascular stress response in pregnant women prior to an invasive medical procedure with an anticipatory increase of HR primarily mediated by a decrease in cardiovagal activity as indexed by RSA. Furthermore, a fatalistic control conviction seems to dampen stress reactivity. Based on these findings, adjuvant stressprotective psychological intervention and counseling with special focus on control conviction should be considered for pregnant women undergoing medical procedures. Objective: Breathing control therapy may reduces emotion, but the neural underpinning of this effect is unknown. We induced emotion with aversive pictures in 20 subjects and using a breath control procedure to test a hypothesis that (a) Breathing control alters the visceral feedback as reflected in the insula activation (that reflects interoception), and (b) This leads to reduced fear as reflected in amygdala activation. Method: Subjects went through fMRI scanning while observing Neutral Pictures, Emotional Pictures and then practiced breathing control (Exp Group) or no breathing control (Control group) . Results: (a) Aversive pictures activated left amygdale and right insula in the control group. (b) The activations at the same areas were altered in the experimental group that practiced the breathing control procedure. Discussion: Intervention of emotion-related breathing can modulate left amygdala activation indicative of reduced fear and this effect cooccurred with altered activation at the right insula cortex indicative of altered interceptive feedback. Conclusion: Breathing control therapy involves altered activation of insula and amygdala. Mindfulness meditation is an ancient meditation technique with a modern name based on the concept of mindfulness, a relatively recent psychological construct. This form of meditation is rooted in Buddhist Vipassana and Zen practices, and involves momentby-moment, detached awareness and observation of the continually changing field of perception and its contents. Albeit the interest to verify the clinical efficacy of mindfulness practice has grown considerably in the last years, its specific psychobiological profile has been scarcely investigated, particularly with respect to the immune system, and this research does not seem to have been conducted, integratively, in clinical population. Thus, the present study was carried out to examine the effects of a mindfulness meditation program on immune parameters in a clinical sample. 16 patients with depression and anxiety symptoms participated in the study; these patients underwent a mindfulness meditation program for the period of eight weeks. For the quantification of immune parameters (leucocytes, immunoglobulins and complement), blood samples were taken before and at the end of the training program. T-test and Wilcoxon signed-rank test were performed as statistical analyses. After completing the mindfulness meditation program, patients displayed significantly lower levels of monocytes and IgM, and higher levels of C3 and C4, as compared with pre-training. Therefore, these results show that mindfulness meditation may exert a significant immunomodulatory action in anxious and depressed patients, which may hold clinical relevance. Future studies should confirm these findings, as well as fully assess their clinical implications. It is known that pro-inflammatory cytokine such as IL-1β plays an important role in behavioral and physiological alterations produced by exposure to immune stimulation or exposure to stressors. Similarities between the neurochemical, endocrine, and behavioral consequences of immune challenge and stressors have been described frequently but the impact of immune stimulation such as lipopolysaccharide (LPS) or stressors on central or peripheral IL-1β has not been systematically explored. To establish whether treatment of LPS or exposure to the foot shock stress (FS) leads to a changes in central or peripheral production of IL-1β and what types of cells express IL-1β in paraventricular nucleus (PVN) and hippocampus were investigated in rats. After treatment of LPS (100 ug/kg) or exposure to the FS (ten, 0.8-mA, foot shock for 5 sec each and 90 sec interval), fever were assessed and plasma, spleen, brains were collected to measure IL-1β levels and brains were perfused for immunohistochemical study. LPS produced widespread increase IL-1β in the brain, spleen and plasma, but FS produced significant increase in IL-1β only in the brain but not in spleen and plasma. In confocal immunofluorescence study, LPS and FS increased IL-1β immunoreactivities in the PVN and in the hippocampal CA1 and CA3 regions and the IL-1β immunoreactivities were colocalized with NeuN. Our results indicate that in brain both LPS and FS increase IL-1β but in periphery only LPS increases IL-1β and that IL-1β immunoreactivities on neuron may play an important role in the hypothalamus and the hippocampus regions. Heart rate variability (HRV) is largely regarded as a useful marker of cardiovascular functioning (Task Force, 1996) . Despite disparities in cardiovascular disease prevalence, especially hypertension, researchers have also suggested that African Americans (AA) actually display greater resting HRV compared to Whites (Li et al., 2009). One proposed explanation for the greater rates of hypertension in African Americans is a greater peripheral resistance in the systemic vasculature (Dorr et al., 2007) . In the present study, we sought to examine the relationships among HRV, and central (i.e. Cardiac Output|CO) and peripheral (i.e. Total Peripheral Resistance|TPR) hemodynamic measures. As part of a larger study, continuous measurements of HRV and hemodynamics were obtained for 34 AA participants (22 females, Mage=19.8, SD=2.7), during a five minute resting baseline period. Initial correlational analysis revealed a moderate association between resting HRV and resting CO(r=.29, p=.08). Analyses were then conducted separately for each gender. Interestingly, this association was stronger for AA females (r=.47, p=.02), but weaker and in the opposite direction for AA males (r=−.09, p=.79). Separate analyses also revealed a strong relationship between resting HRV and TPR in AA men (r=.60, p=.04), that was not observed for AA women (r=.13, p=.61). In follow-up regression analysis the effect for AA women was attenuated when BMI was added to the model; results for men were not diminished when controlling for age and BMI. These results suggest that important gender differences may modify the association between autonomic nervous system activity and hemodynamic parameters associated with cardiovascular disease. Sense of Coherence (SOC) scale measures the stress coping ability, viz comprehensibility, manageability, and meaningfulness. To investigate the relationships of SOC scores to perceived health status and job stress, self-administered questionnaires were distributed 1968 workers in some industries. Complete answers were recovered from 295 workers (14.9%, 192 males and 103 females) and used for the analysis(179 office clerks, 28 sales representatives, 21 technical engineers, and 67 others). Work stressors, psychological and somatic responses, social support, and job satisfaction were assessed by Brief Questionnaire for Job Stress (BQJS). Perceived health were evaluated by asking subjects to give 1-4 points to their own status (1 very healthy, 2 rather healthy, 3 not so healthy, 4 unhealthy). Ages were 49.3 years on average (SD11.4)for all respondents, and were 50.7 (SD 11.6) and 46.4(SD 10.7) for males and females, respectively. SOC scores were 56.8(SD6.1)on average, without significant differences between two genders (p>0.05). Multiple regression analysis indicated that, for both males and females, tension scores (BQJS) were inversely related to SOC scores, whereas were positively related to job load and stress by human relations assessed by BQJS (p<0.05); tension scores were also inversely related to supervisor's support and job satisfaction (BQJS). Similarly, in males, depression scores (BQJS) were related inversely to SOC, job satisfaction and supervisor's support, and positively to job load and stress by human relations (p<0.05). The similar relationships of depression scores to job load, supervisor's support and stress by human relations were observed in females (p<0.05). Perceived health was not significantly associated with SOC scores in the multiple regression analysis (p>0.05). It is thus suggested that SOC can reduce adverse responses to job stress(job load and stress by human relations). Objective: In recent years, salivary alpha-amylase (sAA) has been proposed as a reliable proxy for sympathetic activity. This study aimed at testing the association between sAA to a broad range of psychosocial factors, self rated health, cardiovascular risk factors and inflammatory markers in a normal population sample. Methods: 30 participants, all men between 50 and 54 years old, were randomly selected from a normal population based study. Saliva samples were collected at awakening, 30 minutes after awakening and just before going to bed. sAA was measured by a calorimetric method using Phadebas amylase test. Linear regression models were used to test associations between sAA levels and a broad spectrum of psychosocial factors (e.g. depressive symptamology, vital exhaustion, mastery and sense of coherence) self rated health and inflammatory markers (e.g. C-reactive protein). Adjustments were made for physical exercise, smoking, blood lipids and time point when sample was collected. Results: sAA levels at awakening were positively associated with depressive symptamology (p=0.046), vital exhaustion (p=0.025) and negatively associated with sense of coherence (p=0.034). It was further associated positively associated with levels of C-reactive protein (p=0.024) and negatively associated with self reported general health (p=0.010). Samples taken just before going to bed were showing similar results, whereas samples taken 30 minutes after awakening only showed a few significant associations. Conclusions: The associations found give further support for the use of salivary alpha amylase as a psychoneuroendocrinological biomarker. Assessment just after awakening or just before going to bed seems to be more reliable than samples 30 minutes after awakening. Background: Depression symptom severity contributes independently to risk for recurrent cardiac events, and inflammation has been suggested as a mechanism by which this risk is conferred. Evidence regarding the directionality of the depressioninflammation relationship is equivocal however, and indicative of possible complex and bidirectional associations. Methods: At the time of their index event (baseline), and then again 1-and 3months later, 163 post-Acute Coronary Syndrome (ACS) patients completed the Beck Depression Inventory (BDI), a measure of depression symptom severity with cognitive and somatic components. C-Reactive Protein (CRP) level was also assessed at these visits, as were known correlates of depression and CRP (i.e., age, sex, body mass index, education, raceethnicity, mean arterial pressure [MAP], smoking status, history of rheumatic disease, and history of diabetes). Path analyses were conducted to evaluate the directionality of the depression-CRP relation and whether directionality varied by sex and/or specific symptom dimensions of depression. Results: Baseline total depression symptom severity predicted an increase in CRP from baseline to 1-month (β=0.23, p<0.001) controlling for all covariates, as did baseline cognitive-affective depression symptom severity (β=0.28, p=0.02). In contrast, baseline somaticvegetative depression symptom severity did not predict an increase in CRP (β=−0.01, p=0.92). CRP did not significantly predict 1-or 3-month change in total, cognitive-affective, or somatic-vegetative depression symptom severity. Results did not differ for men and women. Conclusion: Cognitive-affective and total depression symptom severity at the time of a cardiac event predicted 1-month increases in CRP for both men and women, but there was no evidence that earlier CRP predicts later depression symptoms. This study examined the impact of social network variables on diurnal rhythm of cortisol levels among a group of 84 older people (45 men) whose ages ranged from 59 yrs to 86 yrs (mean= 73.02 yrs). Eight saliva samples were collected from the participants for two days at specific times after waking: immediately, 15 minutes, 30 minutes, 45 minutes, 3 hours, 6 hours, 9 hours, and 12 hours. Cortisol data of the two days were aggregated for analysis. The two components of diurnal rhythm, namely, awakening response and diurnal decline were examined separately. For the cortisol awakening response, two separate indices reflecting the mean level of secretion and the rise from immediately to 45 minutes post-awakening, AUCG and AUCI, were computed using the trapezoid formula. Similar procedure was applied to arrive at two indices of decline: AUCG-d and AUCI-d, from the peak level in the morning to that of 12 hours post-awakening. The relation of each of the two indices within each diurnal component to the availability, use and development of social network was examined in a multiple regression analysis while controlling for the effect of gender, age, socioeconomic status, waking time, humor, and self-esteem. Results indicated that higher scores in social network development were associated with a steeper rise in the morning (Beta=.32, t=2.24, p<.05) and a faster decline over the day (Beta=.34, t=2.38, p<.05). Participants having higher scores on this variable exhibited a stronger motivation to develop and extend their social relationships. Its effect on cortisol might stem from the positivity underlying the intention, which is consistent with what prior data suggest. None of the other variables included in the regression analysis had significant impact on cortisol levels. Findings of the present study suggest that the hypothalamic-pituitary-adrenocortical (HPA) axis is the major pathway whereby positive psychosocial factors exert their health effects in the aging population.
CORRESPONDING AUTHOR: Julian C. Lai, PhD, Applied Social Studies, CIty University of Hong Kong, Hong Kong, n/a; ssjulwin@cityu.edu.hk PST-179c SALIVARY IL-6 AND CRP INCREASE IN RESPONSE TO ACUTE PSYCHOSOCIAL STRESS Shuhei Izawa, PhD, 1,2 Nagisa Sugaya, MA, 2 Kenta Kimura, PhD, 3 Namiko Ogawa, MA, 2 Kosuke C. Yamada, PhD, 4,2 Kentaro Shirotsuki, PhD, 5 Ikuyo Mikami, BA, 2 Kanako Hirata, BA, 2 Yuichiro Nagano, PhD 6 and Shinobu Nomura, PhD 2 1 National Institute of Occupational Safety and Health, Japan, Kawasaki, Japan; 2 Waseda University, Tokorozawa, Japan; 3 Nagoya University, Nagoya, Japan; 4 National Institute of Advanced Industrial Science and Technology, Koto-ku, Japan; 5 Tokai Gakuin University, Kakamigahara, Japan and 6 Bunkyo Gakuin University, Fujimino, Japan.
Although inflammatory activity is mostly measured in the blood, recent studies suggested that some inflammatory activity could be measured in the saliva. This study investigated the effects of acute psychosocial stress on two salivary inflammatory biomarkers, Interleukin-6 (IL-6) and C-Reactive Protein (CRP). Fifty-seven male and female students (mean age 21.4 yrs.) were subjected to the psychosocial stress test "Trier Social Stress Test" (TSST), in which the participants were asked to deliver a speech and perform a mental arithmetic in front of two audiences. Saliva samples were taken before, during, and after TSST. Salivary IL-6 and CRP concentrations significantly increased by an average of 71% and 56%, respectively, compared to each baseline value. Both concentrations peaked during stress tasks and IL-6 and CRP levels remained elevated for 20 and 10 minutes, respectively, after the cessation of stress tasks. IL-6 response was moderately correlated to CRP response (r=.305, p<.05). These results indicated that both salivary inflammatory biomarkers are stresssensitive and could be applied for stress research. It has been reported that unfavourable prenatal conditions -such as prenatal maternal stress, drugs, tobacco smoking or medical complications -affect the intrauterine development of the fetus, and may be associated with an increased risk for physical and mental disorders in the offspring. Severe maternal stress during pregnancy affects the offspring's hypothalamic-pituitary-adrenal axis (HPA) and may result in longterm diseases like metabolic syndrome, type 2 diabetes, hypertension or psychiatric disorders. This phenomenon is called 'prenatal programming'. We here investigated 'prenatal adversity' as a potential risk factor in the etiophathogenesis of Borderline Personality Disorder. We assessed whether patients with Borderline Personality Disorder experienced more pre-, peri-and postnatal adverse life events than healthy controls, and tested the predictive value of these factors. One hundred patients with a primary DSM-IV diagnosis of Borderline Personality Disorder and 100 healthy controls underwent semi-structured interviews about the course of pregnancy, severe maternal stress, birth complications and childhood trauma. Further information was gathered from birth records, self-rating questionnaires and from close relatives of the participants. Statistical analyses revealed that mothers of patients with Borderline Personality Disorder experienced significantly more adverse life events during pregnancy compared to mothers of healthy controls (e.g. traumatic stress p=.029, low social support p=.007, severe familial conflicts p=.008 or medical complications p=.017). Logistic regression analyses revealed that prenatal risk factors explain 30.9% of variance in Borderline Personality Disorder. In particular tobacco smoking (p=.003) and medical complications (p =.006) seem to mark important risk factors. Furthermore patients with a history of early adversity show significantly more somatic comorbidities than controls (p<.001). Our findings suggest that pre-and perinatal adversity constitute important risk factors in the etiopathogenesis of Borderline Personality Disorder. Background: Social conditions during childhood have been mentioned as a possible confounder in the relationship between job strain and myocardial infarction risk. However, stress theory also suggests that early experiences may modify the individual's vulnerability to later stress, for instance through learned helplessness or hopelessness. Methods: In a prospective population-based cohort (effective n= 771; 72%), we examined the association between on the one hand exposure to an adverse social environment in adolescence, measured at age 16, and job strain measured with the Demand-Control Questionnaire (DCQ) at age 43, and on the other hand allostatic load at age 43. Adversity in adolescence was operationalised as an index comprising residential mobility and crowding, parental loss, parental unemployment, and parental physical and mental illness (including substance abuse). Allostatic load was operationalised as an index comprising body fat, blood pressure, inflammatory markers, glucose metabolism, blood lipids, and cortisol area under curve. Results: Adversity in adolescence was associated with higher adult allostatic load in women (β=0.170, p=0.001). There was also a significant interaction between adversity in adolescence and job strain in the whole cohort (β=0.081, p=0.026), indicating that the ability to cope with the demands in working life may be negatively affected by exposures in early life. Conclusion: Exposure to an adverse social environment in adolescence was associated with increased vulnerability to job strain in mid-life, indicating that sensitivity to stress and social inequalities in health may both be partially determined by material factors in early life. Meta analyses have shown that religiousness, reflected in behavior such as frequently attending church, has documented health advantages. However, this has not been examined in HIV, nor has it been determined whether advantages of church attendance may be independent of spirituality. This study examined whether frequent church attendance is related to disease progression markers (CD4 and Viral Load (VL)) over time in people with HIV, and whether these effects may be independent of spirituality. Methods: One hundred diverse participants with HIV (men, women, African American, Caucasian, and Hispanic) answered questions about church attendance and spirituality as part of a psychosocial battery. Participants were followed every 6 months for CD4 and VL changes over 4 years. Hierarchical Linear Modeling assessed whether frequency of church attendance was associated with slope changes in CD4 or VL (log) over time controlling for baseline CD4, VL, age, gender, ethnicity, education, and spirituality. Results: Church attendance was significantly associated with slower CD4 decline (t=3.52 p =.001) but not VL. Note that the impact of church attendance was significant even above spirituality. Conclusions: Church attendance predicts slower CD4 decline in people with HIV, and these effects seem to be independent of spirituality. A discussion of why there may be benefits independent of spirituality will be discussed. Objective: To investigate the behavioral and emotional status of the migrated children one year after they were victimized from earthquake in Wenchuan. Method: Using Achenback Child Behavior Checklist (CBCL), the Screen for Child Anxiety Related Emotional Disorders (SCARED) and Depression Self-rating Scale for Children (DSRSC), we investigated the behavioral, anxious and depressive dimensions of 513 victimized children as pupils studying in Rizhao, Shandong province ,to which they were migrated from Wenchuan, Sichuan province. The controls were local children (n=475) coming from the same schools as the victimized subjects. Results: We got 487 answered files that were validated. There were significant differences between the victimized and migrated children and the local children in CBCl's total scores, and its withdrawal, somatization complaining and anxiety-depression scores, as well as its factors such as social problem, attentional problem, attack behavior, internalizing behavior, social communication and school scores. On the SCARED and DSRSC, the two groups were significantly different in total scores of anxiety and depression as well as in such factors as somatization, generalized anxiety, separated anxiety. Conclusion: Though migrated out of the earthquake area, the victimised children still suffer obviously from behavioral problems, and anxious and depressive emotion one year after the earthquake, making it necessary to continue psycho-behavioral intervention in them. Key words: earthquake; children; behavior; anxiety; depression. Kurume University, Kurume, Japan; 2 Kurume University, Kurume, Japan and 3 Beppu University, Beppu, Japan.
Background: Pressman (2005) found that high levels of momentary or experiences of loneliness were associated with elevated morning and evening cortisol. Recently, it is thought that the salivary cortisol awakening response (CAR) can serve as a reliable marker of HPA axis to respond to stress. The CAR is greater in people under chronic work stress and on the work day than the weekend. We recently demonstrated that the difference on this weekday and weekends is an adequate responsiveness to adapt to stress. However, there are no studies that investigate the relationship between the CAR and loneliness on the work days and the weekends in women. The present study was to investigate the relationship between the CAR and loneliness on the work days and the weekends in women. Methods: The participants were healthy 90 women (20-50 years old). Loneliness was evaluated using the revised UCLA loneliness scale (Russell et al., 1980) and participants were classified into high (average+1SD) (n=13) or low loneliness groups (average -1SD) (n=13). The saliva samples were collected over the work days and the weekends. Participants were instructed to collect saliva on six occasions each day: before sleep, immediately on awakening, 30 minutes after awakening, and then as near as possible to 10:00 am, 12:00 pm, and 3:00 pm. Results: CAR was larger on weekends in the high loneliness group than the low loneliness group. However, there were no significant differences in CAR on workdays between subjects with higher loneliness level and with lower one. In addition, there were significant differences in CAR on low loneliness group between workdays and weekends but not in high loneliness group. Discussion: Loneliness was associated with a promoted CAR, especially on weekends. These results indicated that the CAR and cortisol levels on high loneliness people showed no significant differences between work days and weekends due to allsotaic load that the HPA system is always activated. Background: It is a common stereotype, that women experience decrease in memory function during pregnancy. Previous studies about memory changes associated with pregnancy showed inconsistent Results: Working memory is a type of a short-term memory that is responsible for monitoring, keeping and applying information. A decrease of working memory function may lead to unexpected mistakes, absentmindedness and as a result to anxiety stress and decrease in self-esteem. Current research assesses the changes in working memory of healthy pregnant women. We also evaluate the influence of other variables such as depression, sleep changes, and negative self-beliefs on working memory. Materials and methods: Healthy pregnant women going for routine prenatal care were enrolled in the study. Overall 25 women were approached and agreed for participation. Control was matched for age and consisted of 10 non-pregnant women. Validated neurocognitive tests battery (Symbol-Digit Modalities Test and E-prime computer based test) was used to evaluate working memory function. Results: There was no significant difference between the groups in scores of memory encoding and retrieval. Depression and insomnia were not associated with changes of the working memory. Negative self-perception on Symbol-Digit Modalities Test was associated with decrease in working memory function (r=−.43). Conclusion: We failed to prove that pregnancy causes changes in the working memory. Self-perception reduces memory performance in both control and experimental groups. The prevalence of overweight and obesity has reached epidemic proportions, also in the Netherlands. Weight gain prevention interventions have shown to be feasible within the occupational health services, but are hardly being implemented by occupational physicians (OPs) due to a lack of methods and materials. The aim of this study is to 1) develop, 2) evaluate, and 3) implement a weight gain prevention guideline to be used by OPs. Following the standard template of the Netherlands Society of Occupational Medicine and using the Intervention Mapping protocol, a draft guideline was developed based on literature, interviews with employers, OPs and employees, input from an external project group consisting of OPs and lifestyle experts, and input from independent lifestyle experts. Based on all sources mentioned above, the developed guideline consists of clear cut recommendations for OPs to 1) advise employers to promote employees' physical activity and diet and 2) counsel employees to adopt a physically active and healthy diet behaviour. Tools to implement recommendations consist of a minimal intervention strategy to counsel employees, and a scan to assess the obesogenic environment. The guideline is now being evaluated in a RCT among 30 OPs and 350 employees. If proven effective, this weight gain prevention guideline will be implemented on a larger scale within the occupational health services in the Netherlands. Background: Health policy in the UK and the USA supports the design of health services for people with long term conditions (LTC) to be conducive to self-management support. The Wagner's chronic care model suggests that self-management support needs to be embedded in the system. Coote suggests that co-production can be applied to health care. It is a model in which true partnership is based on the active input of recipients in the provision of their own care however it is at best 'too flexible' to guide practice. Aims: To define the key observable behaviours that clinicians and patients require for co-productive interactions in LTC consultations. Methods: A convenient sample of eleven UK and US experts in self-management support and communication skills were interviewed for an hour using a semi-structured format. The transcripts were analyzed thematically. Results: Clinicians and patients need to enter in a partnership, recognizing mutual expertise within a long term relationship; consultations should include discussions about self-management strategies used to develop a highly personalized solution; shared decision making should develop e.g. over health priorities. This means negotiating an agenda, setting goals and agreeing in a followup; the development of rapport is important to engage patients in behaviour change and co-production of health requires a system that facilitates joint decision making. The final model will help to develop a measurement tool using the Roter Interaction Analysis System to assess co-production of health in video-recordings of real life clinician-patient consultations across the UK.
Discussion: This study adds to the understanding of clinical practices and communication behaviours in consultations for people with LTC. The results will be submitted to a further e-Delphi consultation process with the same experts (n=11) and also a wider group of health care professionals to reach a reasonable level of agreement and to confirm the model. The Family Health Program (FHP) was implemented across Brazil in 1994 to deliver primary healthcare services, such as diarrhea treatment and prevention, through health teams that include physicians, nurses, nurse assistants and community health workers (CHWs). Several studies have pointed to the impacts of the FHP on improving child survival in Brazil. Few studies, however, have examined the mechanisms underlying the successful delivery of services in the community, particularly from the perspectives of the health professionals. The goal of this study, therefore, was to evaluate the perceptions of FHP professionals of Vespasiano, Brazil on training, workplace challenges, and practices for child health. This cross-sectional study involved interviews with 77 professionals using a mixed-methods approach. Data was doubleentered and analyzed in Epi Info version 3.5.1. Open-ended responses were stratified by professional category, translated from Portuguese to English, and analyzed using a thematic coding approach. The results show that 92% of professionals received training in their first 12 months in the program but the majority found the training was limited in quantity, content, methods of delivery, and overall quality. Challenges in the workplace include insufficient government support, poor infrastructure, and lack of resources. In spite of these limitations, over 70% of CHWs reported promoting important health messages to caretakers and 87% of professionals were satisfied with the FHP child diarrhea services. In addition, the majority of professionals perceived the FHP is created for the needs of the community (83%) and is accepted by the users (81%). Translation of this research will be important for policies and program developments to improve on the limitations and promote the strengths of the FHP in order to advance Brazil's healthcare model to address leading causes of disease among the Brazilian pediatric population Aims: Childhood maltreatment had been considered a risk factor for psychopathology and health problems, including health complaints, health risk behaviors and diseases. Studies usually use retrospective methodologies and clinical samples. However, some authors have been discussing the validity of the associations these studies usually found suggesting that recall bias can explain the relation between maltreatment and symptoms. The aim of this study was to characterize current psychological and health functioning in young adults that were victims of maltreatment as children and compare the self report with official records from Child Protection Services. Method: Official records of 109 young adults that were identified as victims by protection services during childhood (55 female and 54 male) were compared with self-reported maltreatment history data using Childhood History Questionnaire. Participants also filled Brief Symptoms Inventory and Rotterdam Symptoms Checklist. Results: Subjects that report more adverse experiences have more psychopathology (r=.48; p=.000) and health complaints (r=.36; p=.000). Some subjects (21%) do not report any maltreatment experience, 36% report only some of the experiences they were victims and 44% do accurate reports. These groups are not different in relation to psychopathology (F(2, 104) = .618; p=.541) or health complaints (χ2(2) = 4.40; p=.111) Conclusion: Self-report false negatives for adverse childhood experiences are very high and most of the subject's don't report all the experiences they were victims. Some authors had suggested that the relation between reported childhood experiences and symptoms could be interpreted as a record bias for participants that are symptomatic in the evaluation moment, but this data does not confirm this hypothesis since there were no symptoms differences between the groups. Reasons for not report and relation between adversity and health should be better understood in future research. Research documents the impact of social environments on health related communication and behavior, and actual health. However, one aspect of the environment along with its related area of scientific investigation and professional application, all with clear implications for health issues, has received less attention than is warranted. Specifically, we are referring to Interpersonal conflict as described by Contemporary Social Conflict Theory (CSCT), which is not well known or understood. The result is that Its implications for health issues are underestimated or ignored. Conventional views equate conflict with behavior (arguing/fighting), view it as "bad," and advocate avoidance as a response to prevent violence. CSCT views conflict as a cognitive process based on beliefs concerning needs and the expectations for satisfying them. Conflict, defined as "perceived divergence of interest" is regarded as inevitable, ubiquitous and necessary for change. For CSCT, contention (fighting) is one of five response modalities -accommodation, avoidance, compromise, and problem solvingare the other "Conflict Management Styles (CMS's), each with predictable consequences. Conventional views of conflict limit us to consideration of the contentious CMS which has implications for health ranging from covert effects of stress from using this CMS on a regular basis to overt effects of violence that can result from this CMS. However, other CMS's have implications for health outcomes, raising a variety of research questions, e.g., who gets better treatment, the demanding "contentious" patient or the compliant "accommodating" patient? Does an avoidant CMS result in lessened ability to address important health care issues with family or healthcare providers? Does a problem solving CMS result in proactive health behavior, better communication with HCP's, and higher rates of compliance? The purpose of this poster is to explicate the principles and practices of CSCT and suggest their potential practical use -especially those involving decision making activities about treatment. Purpose: A mantram (sacred word) intervention has demonstrated significant reductions in PTSD symptom severity when applied as a complementary treatment for Veterans with military-related trauma compared to usual care controls. Little is known, however, about mechanisms for this improvement. Prior studies of the mantram intervention have demonstrated increased spiritual wellbeing (SWB) and decreased psychological distress attributable to this intervention. Therefore, we hypothesized that increases in SWB may mediate reductions in PTSD symptom severity. Methods: Path analysis was conducted on data from a randomized clinical trial in which 66 Veterans with chronic military-related PTSD completed a 6-week (90-minutes/week) mantram group intervention and were compared to 70 usual care controls. The dependent variable was the pre-to post-treatment change scores on the PTSD Checklist (PCL). The mediator variable was the Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being scale (FACIT-SWB). The sample was 97% male; 59% Caucasian, 24% African American, 7% Hispanic and 10% other. They ranged in age from 25 to 83 (M=57, SD=9.26), averaged 14 years (SD = 2.1) of education and experienced an average of 13 months (SD=10.0) of combat. Results: The hypothesis that SWB partially mediates reductions in PTSD symptom severity was supported. The mantram intervention predicted post-treatment changes in PCL (b=3.71, p<.05). There was a significant and positive, indirect effect in the path from the mantram intervention to SWB (b=6.37, p<.05) and a significant, negative effect from SWB to PCL (b=.32, p<.0002). Similar results were found using the Baron and Kenny statistical approach. The Sobel test was significant at 2.55 (p<.01). Conclusion: Findings suggest that one potential mechanism by which the mantram intervention reduces PTSD symptom severity in Veterans is by promoting beliefs about meaning and/or purpose in life and feelings of peace and harmony. Spiritual wellbeing may be an important factor to consider when treating PTSD. Only recently researchers became interested in studying the physical health consequences in adulthood that result from physically abusive experiences during childhood, but now is a promising area of research. The state of the art about the topic is quite interesting and researchers deal with many challenges. One of them is the difficulty to establish a clear relationship between the experience of physical violence and health variables, mainly due to methodological issues. In order to overcome this concern some epidemiological guidelines were defined, namely: major criteria (temporal relationship; biological plausibility; consistency), alternative explanations (confounding variables), other considerations (dose-response relationships; strength of association; and cessation of exposure). Regarding these criteria, we proceeded to a systematic review of nineteen studies about the long-term physical health impact of child physical abuse in community samples. These researches included at least one of seven health categories: sexual risk-behaviors, substance dependence, alcohol consumption, drug consumption, nicotine consumption, current general health and physical health condition. Our review revealed that results are inconclusive and mixed for all categorical groups. However, it seems to be a positive association between "being victim of child physical abuse" and "sexual riskbehaviors" and "alcohol consumption" in men; "physical health condition" in women; and "drug consumption" in mixed samples. Based on our results, we cannot draw an unambiguous conclusion between these variables and therefore this issue should be further analyzed. Sleep deprivation is a significant problem in the modern workplace and has negative consequences for employee health. This trial assessed the effects of two mental imagery techniques, one inducing implementation intentions so as to close the intention-to-action gap in sleep promoting behaviours and the other inducing relaxation so as to reduce arousal prior to going to sleep. We recruited 72 employees from local businesses who reported difficulty sleeping. Following screening, randomization and a training session, they were asked to practice brief imagery exercises involving either arousal reduction imagery, implementation intention imagery (i.e., specifying context and action), a combination of the two, or imagery of their normal night routine (control) twice a day over 21 days. They completed an online, daily diary of sleep behaviours and, at baseline and day 21, an online questionnaire that included the Pittsburgh Sleep Quality Index (PSQI), Pre-Sleep Arousal scale, Sleep Hygiene Index, Dysfunctional Sleep Beliefs Scale, and measures of state anxiety and sleep-related planning. Repeated measures ANOVAs showed that, relative to the groups not using implementation intention imagery, the implementation intention groups had a greater reduction in negative sleep habits (p<.01) and greater improvements in the sleep-related actions targeted in the imagery exercise (p<.01). Increases in sleep motivation (p<.001) and response efficacy for sleep behaviours (p<.001) were found across all groups, along with decreases in anxiety (p<.001), dysfunctional sleep beliefs (p<.001) and pre-sleep arousal (p<.001). Improvement in all groups was also shown for total PSQI scores (p<.001) and level of detail in plans for positive sleep behaviour (p<.001). Mixed modeling analyses of the daily data indicated that those using implementation intention imagery had faster times of falling asleep (p<.01) and earlier times of lights out (p<.01) compared to those not using this imagery. These brief, low-cost techniques appear to provide a promising alternative to the use of medication for the improvement of sleep in the general population. The National Centre for Occupational rehabilitation, Rauland, Norway and 2 Uni Health, Bergen, Norway.
Background: RTW is a complex process, where the individual may be in multiple and recurrent states. The aim of this study was to describe the evolving RTW-process in a 5-year follow-up period after a 4-weeks inpatient occupational rehabilitation program. Methods: The sample consisted of 584 patients (66% females, mean age 44 years [sd=9.3], long-term sick-listed; mean duration 9.3 months [sd=3.4] for psychological (47%) and musculoskeletal (46%) diagnosis. Register data from The National Insurance Administration in Norway over a 5-year follow-up period was analysed. Outcome was measured in an 8 state-model: working, part-time sick-listed, sick-listed, medical rehabilitation, vocational rehabilitation, part-time disability pension, time-limited disability pension and permanent disability pension. Extended statistical tools for multistate models were used to calculate transition probabilities between the different states. Results: During the 5-year follow-up period the patients had on average 4.6 [range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] shifts between the 8 different states. During the first 30 days after rehabilitation 9% returned to full time work, 23% were graded sick-listed, 45% were full-time sicklisted, 15% were on medical rehabilitation, 6% on vocational rehabilitation and 2% on graded disability pension. After 5 years 58% had returned to full-time work, 0% was sick-listed or on medical rehabilitation, 10% were on vocational rehabilitation, 14% on part-time disability pension, 5 % on time-limited disability pension and 13% received permanent disability pension. Conclusions: The RTW-process for long-term sick-listed individuals is long and complex. Despite poor RTW during the first 30 days after occupational rehabilitation, the long-term RTWprospects seem advantageous. It is of interest to examine what role environmental and legal system factors and of the work-place and health-care providers play in this prolonged RTW-course. Background: the influence of self-efficacy in the RTW process has been emphasized; however, a comprehensive understanding of the role of self-efficacy on RTW behavior is still lacking. Aim of the study is to investigate the association between self-efficacy for RTW (SERTW) and: a. the intention to change, and b. RTW status, in a cohort of injured workers with work-related musculoskeletal conditions. Methods: A baseline telephone interview was conducted in 632 claimants one-month post-injury and 446 claimants (six-month post-injury). Return-to-Work Self-Efficacy (RTWSE) was measured with a validated questionnaire consisting of three subscales: RTWSE with regard to 1. Controlling pain at work; 2. Obtaining help from supervisor and 3. Obtaining help from co-workers. Intention to change was assessed with the validated Readiness for Return-To-Work (RRTW) Scale, reflecting closely the five stages described in the Readiness for RTW model [1, 3] . RTW status was measured based on the workers' responses to the question whether they returned to work at one-and six-month follow-up. ANOVA tests were conducted to compare relationships for each of the three RTWSE subscales with the stages of change and the RTW status. Results: with regard to intention to change all univariate F-tests were significant at both measures in time, except for the co-worker RTWSE subscale at one-month follow-up. RTWSE is at its peak in workers who are actively preparing to return to work, however, it decreases significantly for people who start working again on all three RTWSE subscales. Comparing RTWSE with RTW status significant differences were found only for the Pain SERTW subscale at both time measures. Workers who returned to work showed higher Pain RTWSE scores compared to those who were still sick-listed. Conclusion: a strong association between SE-RTW and Readiness for RTW stages was found, however the association between SE-RTW and RTW status was only strong for RTWSE. Our findings suggest that self-efficacy is more strongly associated with intention/motivation for the behavior (internalized processes of behaviour), than strictly behavioural ones. Message: 1. Self-efficacy for RTW is significantly associated to the intention to change but not to the actual behavior, i.e. RTW status. 2. Self-efficacy for RTW changes over time during RTW. It is at its peak in workers who are actively preparing to return to work, however it decreases significantly for people who start working again. 3. Our findings suggest that self-efficacy is more strongly associated with intention or motivation for the behavior, than strictly behavioural ones. Background: Smoking and work disability are major public health and welfare problems. The health consequences of smoking are well-documented but less is known about the independent effect of smoking on disability pension of various diagnoses. The aim of this study was to examine the impact of smoking on subsequent disability pensions among middle-aged public sector employees. Methods: Baseline survey was collected in 2000-2002 among middle-aged employees of the City of Helsinki (n=6373, 80% women). Data on disability pensions were obtained from the Finnish Centre for Pensions (2000) (2001) (2002) (2003) (2004) and were linked to the questionnaire data. Cox regression analysis was used to calculate hazard ratios (HR) for disability pension. To examine the independent effect of smoking age, socioeconomic position, working conditions, alcohol drinking, physical activity and body mass index were adjusted for. Results: There were 232 (4%) disability pension events during the follow-up. Age adjusted smoking strongly predicted disability pensions (HRs 1.5-1.6) among women and men. Risk for disability pension increased with number of smoked cigarettes. Especially strong was the effect among those who smoked more than 20 cigarettes per day. Adjusting for confounders attenuated the effect but it remained.
To support employees quitting smoking may prevent work disability pensions. Background: Refugees are at a dramatically increased risk to suffer from mental & somatic disorders. Research has focused on pre-displacement trauma. However, many refugees find themselves unemployed or underemployed in their new country. Employment is known to be critical for social integration & well-being. This study focuses on the relationship between postdisplacement skills utilization among Iraqi refugees and health. Methodology: Iraqis throughout the world were contacted using web-pages commonly used by Iraqis and by emails. Inclusion criteria were bachelor or above and having left Iraq as refugees 1990 or later. Potential participants were referred to a Monkey Survey Link, & following consenting to the study, they responded to a survey with 34 questions concerning socio-demographic, employment, stress, psychosomatic, mental & somatic complaints. All respondents (n=408) were classified according to employment status; Unemployed (G1, 42% of total), Professional working at their skills level (G2, 27%), & Professionals working below their skills level (G3, 31%), representing 65 countries. Significant group differences are presented. WSU HIC approved the study. Results: Participants in the G1 group rated their health the highest (Excellent to Good, 69%), versus 80% in G3, and 86% in G2. Stress, sleep difficulties, depression and psychosomatic symptoms were the highest in G1, intermediate in G3, & the lowest in G2. 90% of G2 enjoyed their work significantly more as compared to G3 (10%). The major barriers to getting a job were: language, financial limitation for training, & insufficient professional skills. Logistic regressions showed that G1 rated their health better versus unemployed (OR 2.6; 95% C.I. 1.4-5.2). There were no differences between G2 and G3. Conclusions: Professional refugees allowed working at their skills level, fair better than underemployed following forced displacement. Results have implications for refugee policy. Background: Diverging return to work (RTW) interventions can be found in literature, designed for specific populations. However, no such intervention exists for patients with acute hand injuries, while these injuries can be devastating and severely influence a person's ability to work. Regular rehabilitation interventions' focus is on functional recovery of the hand, including tasks necessary to perform the job. However, also psychosocial and work-related factors influence RTW strongly. Therefore, we developed a psychosocial intervention for patients with acute hand injuries, aimed at to facilitate RTW in this population. Methods: A combination of qualitative research techniques was used, comprising a mixture of literature study, survey, individual and focus group interviews with patients, rehabilitation practitioners and work counseling experts, and translation methodology.
Results: Some preliminary ideas about the nature of the intervention came to fore. The RTW intervention should: 1) take into account the acute character of the injury; 2) be short of duration; 3) be non-invasive, as most patients do not consider themselves sick; 4) be aimed at RTW; 5) reduce costs and therefore group sessions are favored. Solution-Focused Therapy (SFT) met most of the aforementioned criteria. However, as SFT originates in family therapy, its principles and practices had to be translated to the problems of patients with acute hand injuries targeted on RTW. SFT therapists use a number of specific questioning techniques that invite clients to co-construct a vision of a preferred future and draw on their past successes, strengths, and resources to make that vision a reality. Examples of questioning techniques are the miracle question, scaling questions and coping questions. The SFT treatment ingredients were incorporated in a new intervention, consisting of one individual meeting, and four group meetings. In every group meeting one topic is discussed in a SF manner. Background: The association between organizational justice and workplace bullying has not yet been investigated, while the workplace atmosphere is supposed to responsible for it. The objective of the study was to explore the crosssectional association of organizational justice, as well as job strain (the ratio of job demands to job control) and effortreward imbalance, with workplace bullying in a sample of civil servants in Japan. Methods: The study sample consisted of 807 males and 735 females of the civil servants in the Kanto region in Japan (response rate, 46.7%). They were asked to fill in a selfadministered questionnaire including scales of workplace bullying, organizational justice (interactional justice and procedure justice), job demands and job control, effortreward imbalance (ERI), supervisor and coworker support, and other covariates (sex, age, marital status, occupational status, employment contract, work shift, overtime in the past month, unit size, and sex ratio of the unit). Workplace bullying was measured by the Negative Acts Questionnaire-Revised, and was defined as those experiencing at least one of 22 negative acts weekly or more frequently during the past six months. Each job stressor variable was categorized into the tertiles. Odds ratio of workplace bullying associated with each job stressor was estimated by using logistic regression analysis, using the lowest tertile as the reference, adjusting for the covariates. Results: After adjustment for the covariates, high interactional justice (odds ratio 0.23, 95%Cl 0.14-0.39) and high procedure justice (odds ratio 0.21, 95%Cl 0.13-0.34) were negatively associated with workplace bullying. High job strain, high effortreward imbalance, low supervisor support, and low coworker support were also significantly associated with workplace bullying in an expected direction (p<0.05). Conclusion: Low organizational justice may be a risk factor for workplace bullying among Japanese civil servants. The study also confirmed that job strain, ERI, and low worksite social support were associated with bullying. To investigate 1) whether socioeconomic state such as income, education attainment, and occupational class are associated with employees' depression, and 2) whether occupational stress would explain the associations in Japanese working populations, we developed a cohort of employees of 3 different manufacturing workplaces and sent a self-administered questionnaire as the baseline data. A total of 3767 employees responded to the survey (response rates 75-96%). Associations of income level (lower or higher than ¥5,000,000 of annual household income), educational attainment (lower or higher than the level of high school education) and occupational class (managerial or not) with depression were examined. Based on a recent validation test among Japanese workers using the Mini International Neuropsychiatric Interview, depression was defined as a dichotomize variable with a cut point of the CES-D scores 19 or above. Occupational stress was evaluated using Japanese versions of demand-control questionnaire and effort-reward questionnaire. Using the sex-specific median values, we produced a quadrant scheme with 4 exposure categories, with low job demand and high job control representing a relax job, high job demand and high job control representing an active job, low job demand and low job control representing a passive job, and high job demand and low job control representing a strain job. According to Siegrist, we defined a ratio of the scales 'effort' and 'rewards', weighted for number of items where a value>1 as the critical 'high cost / low gain' condition. Univariate analyses showed that depression was prevalent among the lower socioeconomic groups than the counterpart groups. Strain and passive jobs were prevalent among lower socioeconomic groups, whereas effort-reward imbalance tended to be prevalent among higher socioeconomic groups. Multivariate analyses revealed that socioeconomic indices were not associated with depression but the occupational stressors were significantly associated with depression. The association between occupational class and depression appeared to be explained by occupational stress, in particular, low job control.
In conclusion, the current cross-sectional analyses in our Japanese sample did not reveal independent associations between socioeconomic state of interests and depression. Although the two occupational stressors were associated with socioeconomic indices differently, they were consistently associated with depression. Background: There is increasing evidence that chronic diseases are associated with work stress. The mechanisms underlying these associations remain porly understood, but disturbances in cardiac autonomic control may be involved. Aims: To investigate associations between work stress, in terms of job strain and job control, and heart rate variability (HRV) over the working day. Methods: Job strain and control was measured by the Whitehall II Study job strain questionnaire, which was developed to test Karasek's job strain hypothesis of the demand-control model. HRV was measured using spectral analysis from Actiheart recordings obtained over the working day from 169 women working in Budapest, Hungary. Results: Linear regression analysis adjusted for age, educational level, BMI, social support, smoking status, alcohol consumption, physical activity, sleep quality, working hours was used to determine the relation between job strain and job control and HRV. We found a significant positive relationship between job control and high frequency to low frequency ratio (HF/LF ratio) adjusted for covariates (p < 0.05) and a positive tendency with high frequency power during work (p < 0.1). Heart rate variability (high frequency power) after work was negatively associated with job strain (p<0,05). Conclusions: The results suggest that low job control and high job strain may contribute to the development of chronic disease through the dysregulation of highly integrated physiological systems. The findings implicate autonomic deregulation under job strain conditions, providing a powerful explanation of heart disease, as well a potential explanation of other stress-related chronic diseases. However, further studies are needed to explicitly demonstrate how the cardiac regulation measured by HRV serves as the cardiovascular disease pathway for work stress. Background: Nurses are frequently exposed to work-related stressful situations. This often makes it difficult for hospitals to recruit nurses and to retain experienced staff. Current research has found that new nurses may be particularly vulnerable to job stress, and that stress levels among nurses are inversely related to job satisfaction. Purpose: To determine if nurse resident's (NRs) sense of group cohesion and organizational commitment serve as protective factors against the negative effects of preexisting trauma and current stress on job satisfaction, burnout, and fatigue. Methods: A longitudinal study of 178 nurse residents (15 male, 163 female) from Childrens Hospital Los Angeles was conducted across three time points. Levels of preexisting trauma, current stress, compassion satisfaction, burnout, compassion fatigue, job satisfaction, group cohesion and organizational commitment were measured. A range of regression models was developed to examine the relationships between the predictor variables (pre-existing stress and current stress), the moderating variables (group cohesion and organizational commitment), and outcome variables of interest (job satisfaction, fatigue, burnout, and compassion satisfaction). Results: Higher organizational commitment scores provided a 'protective' effect against pre-existing trauma with respect to compassion satisfaction (R 2 =.35, p<.0001). Group cohesion was found to be a predictor of job satisfaction, and was found to have protective effects for job satisfaction from pre-existing trauma and current stress (R 2 =.25, p<.0001); it also provided direct 'protection' against burnout, which was affected by current stress (R 2 =.24, p<0.0001). Pre-existing trauma was a strong predictor of fatigue, and accounted for 50% of observed variance in fatigue scores with (R 2 =.50, p<0.0001). Conclusion: Results from the study suggest that group cohesion and organizational commitment serve as protective factors against the negative effects of pre-existing trauma and current stress. The findings have implications for hospital-based interventions structured to address nurse stress, while enhancing group cohesion and organizational commitment. Nursing, Chang Gung Institiute of Technology, Tao-yuan, Taiwan and 2 Nursing, Li Shin Hospital, Tao-yuan, Taiwan.
The purpose of this study was to explore the effects of empowering in-service training program on health care problems among nurse aides in long term care facilities. A non-equivalent control group design was conducted. Purposeful sampling was employed. A total of 104 nurse aides subjects were recruited from Tao-yuan area, and divided to experimental and control groups. The empowering inservice training program involved the curriculum content which include the cognition of workplace difficulties, social interaction and communicates, the pressure manages and refuses technique etc. Both experimental and control groups took pre-test as baseline, and only the experimental group obtained 6-weeks classes regarding to the prevention of smoking. The experimental group took post-test immediately after intervention in order to evaluate the instant effect of teaching programs. Data was analyzed by using descriptive statistics and t-test. The results showed as following:1. In the task pressure part, there was a significance difference in experimental group after the empowering in-service training program; 2. In the effect of job satisfaction aspect, the average scores had increased but there was no significant statistical significance after the training program; 3. From the quality of working life perspective, the average scores had increased but there was no significant statistical significance after the training program, however, there was a significant clinic explanation on the intervention. These results could provider information as supplementary for aboriginal nurse aids in-service in long-term care facilities, and to increase their job satisfaction. Behavioral Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; 2 School of Psychological Science, Health Sciences University of Hokkaido, Sapporo, Japan and Objective: To examine a possible relationship between obesity, job stress, eating behavior, physical activity, and social skill in Japanese part-timers. Method: Questionnaires on job stress (Brief Job Stress Questionnaire; Shimomitsu et al., 1998) , eating behavior (Eating Behavior Scale; Sakata, 1997), self-efficacy in controlling situational appetite (Situational Appetite Measure; Shimai et al., 2000) , physical activity (Physical Activity Assessment Scale; Wakui & Suzuki, 1997) , and social skill (W-KiSS-16; Tanaka, 2007) were conducted with 254 part-timers aged 19-72 years (46.96±11.41 years) in a food industry in Japan. The relation between obesity, job stress, eating behavior, physical activity and social skills were analyzed between 52 obese subjects (BMI≥25.0 kg/m2), 185 normal range subjects (18.5 kg/m2≤BMI<25.0 kg/m2), and 17 underweight subjects (BMI<18.5 kg/m2). Statistical analyses included correlation analysis and multivariate regression analysis. Result: In Obese subjects, eating behavior was significantly associated with job stress response of physical stress response (β=.42, p<.001), self-efficacy in controlling situational appetite of reward (β=−.37, p<.01), relax (β=−.25, p<.05), and physical activity was significantly associated with job stress response of fatigue (β=−.46, p<.001). In normal range subjects, eating behavior was significantly associated with job stress response of physical stress response (β=.17, p<.01), self-efficacy in controlling situational appetite of negative feeling (β=−.23, p<.01), relax (β=−.30, p<.001), and physical activity was significantly associated with job stress factor of qualitative workload (β=.16, p<.05), family and friend support (β=−.19, p<.05). Conclusion: The present study suggests that obese part-timers tend to be in a stressful state in the workplace. Such stressful conditions may eat much and contribute to obesity. Stress management might be necessary in workplace for prevention and treatment of obesity among part-timers. Objectives: During six years from 2003 to 2008, the number of employees of information services increased from 567,000 to 858,000 in Japan. Their health problems have increased over the last 10 years. In the information services, it was reported that the stress factors of their work environment was associated with their health than their personal relationships on business and their life event. The purpose of this study was to identify the association between the level of fatigue accumulation and the working conditions in Japanese system engineers of information services.
Methods: We conducted an anonymous self-report questionnaire in 160 system engineers of information services in 2008. A checklist of the level of fatigue accumulation developed by the Ministry of Health, Labor and Welfare in Japan was used. Items of questionnaire were degree of a burden of working hours, work contents, eye strains, sleeping hours and situations of daily life. A description of entry by voluntary agreement was attached to questionnaires. We obtained approval from respondents because of submitting their questionnaires. Results: Responses were obtained from 120 people (75%). One hundred (83%) people for men and they had most 30 s in 59 people (49%). The number of people with 45 hours or more overtime work in month was 26 people (22%). The number of low risk group of fatigue accumulation was 53 people (44%) and highrisk group was 67 people (56%). The high risk group had a significantly higher percentage of 60 minutes or more of commuting time, seconded staff and 45 hours or more of overtime hours than low risk group. The high risk group had a significantly higher percentage of the burdens in working hours, amount of work, difficulty of work, time pressure on the appointed date of delivery, troubles after the delivery of the order, communication problems, uncomfortable setting, eyes or neck pain and fatigue. Conclusion: This study revealed that the level of fatigue accumulation of Japanese system engineers related their work environment. It was suggested that nurses in business setting need to approach the level of fatigue accumulation depending on characteristics of the work condition and environment of system engineers. Objectives: The aim of this longitudinal study is to evaluate wellness effects of a tailored problem solving process intervention program for health care employees. Methods: Socio demographic variables, work conditions, higher order goal facilitation as well as health related outcomes were assessed from the experimental group (N=461) as from the control group (N=246; Total: N=707) at a first measurement moment (T1) and a second T2 (3 years later). Regression analyses were used to explore the changes between T1 and T2 and the effects on health related outcome variables. Results: Generally, the results indicate that positive changes in work conditions and higher order goals significantly affect health and wellbeing of health care employees positively. The effects are stronger in the experimental health care centres compared to the effects in the total sample group. S194
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Conclusions: These findings suggest that a tailored problem solving process intervention based on motivational regulation techniques, such as goal setting, action planning and training in communication-and cooperation skills positively affect quality of work and wellbeing in health care employees. Recovery from work stress is crucial to avoid stress-related illhealth. The aim of this longitudinal study was to investigate whether processes that prolong the mental exposure to work stressors, e.g. overcommitment (OC), along with psychosocial work conditions, predicts fatigue and poor next-day recovery six months later. 160 women working in dental care completed selfratings in questionnaires at two time points, six months apart. Psychosocial work conditions and OC were used to predict fatigue and poor next-day recovery respectively, in multiple regression analyses. Results show that having children living at home, high job demands and high OC were associated with fatigue six months later. Job demands and OC, but not having children at home, predicted poor next-day recovery. Sobel's test and bootstrapping procedure indicated that OC partly mediated the relationship between job demands and fatigue and between job demands and next-day recovery. These findings highlight the importance of considering OC, e.g. inability towithdraw from work, in relation to fatigue and recovery from work and that such factors should be included in interventions targeting workrelated stress. RN/LPN reported less job strain overall (7 out of 85 nurses), limiting the power to detect potential disparities. There were no differences regarding social support at work in either occupation. Conclusion: NHB nurses, compared to NHW nurses, were six times more likely to report job strain. The effect was higher among CNA. There are several possible reasons for these differences, including confounding by an unmeasured variable such as skill level or differential self-report by race. These differences may also reflect unmeasured organizational and interpersonal behaviors, including discrimination based on race. Further research should consider these alternative explanations for racial disparities in selfreported job strain. Following the first screening, 92 men and 63 women were determined as highly depressive. Among them, 87 men and 63 women were interviewed using CIDI-SS-RR as a second screening. Finally, 20 men and 12 women were detected as ones to be recommended for medical care. Highly depressive men by the first screening were more likely to live alone (p<0.01), and had more job demand (p<0.05), more physical loads (p<0.05), less friendly work place (p<0.05), less job satisfaction (p<0.01) than non-highly depressive men. Highly depressive women by the first screening had more job demand (p<0.05), less job control (p<0.05), and less job satisfaction (p<0.05) more than non-highly depressive women. Men that were recommended for medical care were more likely to live alone (p<0.05) and had more physical loads (p<0.05), less job satisfaction (p<0.01), and less friendly work place (p<0.01) than non-highly depressive men by the first screening. Women that were recommended medical care had more job demand (p<0.05) than non-highly depressive women by the first screening. Depressiveness of 18 men and 14 women increased in 2008. There was the trend to increase in physical loads and decrease in work place friendship or job satisfaction among these men, and that to increase in physical loads and decrease in job control or job satisfaction among these women. Conclusion: These results will support our previous report, in which job involved factors may relate to depressive mood of workers, and these factors may relates to increase depressive mood. OBJECTIVES: To examine the effect of web-based mental health check system for workers (MENTAL-ROSAI hereinafter referred to as the "M-R"). M-R provides self-check questionnaires on stress symptoms and work and lifestyle related stressors, and a computer-tailored personal report based on the check results via internet. This study tested the effect of individually computertailored messages designed to maintain mental wellness and reduce work and lifestyle related stressors.
METHODS: Employees of a Japanese IT company were surveyed using M-R at baseline, and 317 males, who were not under treatment for mental illness and having few depressive symptoms (CES-D<16), were randomly assigned to Group C in which the participants receive a simple report containing scores and charts only or Group M in which participants receive individually tailored stress-management messages added to the simple report. The change in stress symptoms and work and lifestyle related stressors were examined on 206 participants who answered both 2-and 4-month follow-up surveys using M-R. At 2-month survey, those participants exhibited higher depressive symptoms (CES-D≧16) received an additional message from a medical doctor. RESULTS: At 2 months, maintenance of mental wellness and improvement of lifestyle were observed in 7% more participants in the Group M, but the difference was not statistically significant. Of those who scored over 16 on CES-D at 2 months and received a doctor's message, the CES-D score significantly decreased at 4 months in Group M only (p<.01). Somatic stress symptoms also tended to decrease at 2 months in Group M and it maintained up to 4 months. Significantly more participants in Group M (79%) than Group C (68%) maintained mental wellness all through 3 surveys (p<.001). No significant change in work and lifestyle related stressor was observed. CONCLUSIONS: Adding tailored messages to the simple report of M-R was more effective in maintaining mental wellness and improving depressive symptoms. However, to support improvement in work or lifestyle related stressors, complementing the concise report of M-R by extending our center's health care services may be needed. Background and purpose. More than thirty thousand people commit suicide in Japan every year from 1998. Therefore, preventing suicides is one of important issues to be addressed in Japan. Our prefectural government also launched a project of screening for depression among workers in local area from 2007 in order to prevent suicides. Methods and Results: For the project, we performed screening of depression for 475 male and 284 female workers, who work in some small enterprises, during their annual medical checkup in 2007. They answered self-reported question-S196 Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329 naire including items screening for depression, living alone or not, work arrangement, working time, job demand, physical loads, job control, friendly work place or not, and job satisfaction. Following the first screening, 203 men and 129 women were determined as highly depressive. Among them, 138 men and 97 women were interviewed using CIDI-SS-RR as a second screening. Finally, 21 men and 22 women were detected as ones to be recommended for medical care. Highly depressive men by the first screening were more likely to live alone (p<0.05), worked longer (p<0.05), and had more job demand (p<0.01), more physical loads (p<0.01), less friendly work place (p<0.01), less job satisfaction (p<0.01), and lower job control (p<0.05) than non-highly depressive men. Highly depressive women by the first screening were younger (p<0.05) and had more job demand (p<0.01), more physical loads (p<0.05), less friendly work place (p<0.01), and less job satisfaction (p<0.01) more than non-highly depressive women. Men that were recommended for medical care were more likely to live alone (p<0.05) and had more job demand (p<0.01), more physical loads (p<0.05), less job satisfaction (p<0.01), and less friendly work place (p<0.01) than non-highly depressive men in the first screening. Women that were recommended for medical care were younger (p<0.01) and had more job demand (p<0.01), more physical loads (p<0.01), and less job satisfaction than non-highly depressive women in the first screening. Conclusion: These results suggest that job involved factors relate to a depressive mood of workers. Results: The majority of the ambulance workers had experienced critical incidents in their work. Over time there was a decrease in PTSD symptoms and peritraumatic dissociation, an increase of health complaints, and a significant improvement in psychological well-being. However, correlational analyses indicated a negative significant association between symptoms and psychological wellbeing at the two time points. The predictors of post-traumatic stress disorder were traumatic incident-related, peritraumatic dissociation, psychological distress and subjective health complaints at both assessments. Conclusions: Ambulance personnel are at risk to develop health symptoms due to work related stressors. However, the results suggest that shifts in symptomatology may be associated with the time of the year in which the assessments took place. Further research should explore whether these symptoms increase in peak times of critical incidents over the year. Poor or insufficient sleep is a risk factor of accidents and several illnesses including cardiovascular diseases. We aimed to clarify aspects of shift arrangements which are important for sleep in shift work. The participants were 305 (248 men) aged 25-60 yrs. shift workers in a Finnish airline company. Sleep length, insomnia symptoms and work arrangements were self-rated by the participants. The considered aspects of shifts were: frequency of night shifts, the number of consecutive night shifts, mean and the maximum shift length, frequency of shifts starting before 06:00, quick returns (less than 11 hours between shifts), less than 28 hours of free time after the last nigh shift, a single free day between shift periods, and the regularity of shift system with a question on the direction of rotation (backward vs. forward). Sex and age were included as covariates in all analyses and additional adjustments were done for education and health habits (alcohol use, physical activity, and smoking).
The number of years in shift work and possibility to influence the shift schedule were significant predictors of insomnia (p<.001) but were unrelated to sleep length. Insomnia was related to a high number of consecutive night shifts (p=.006), less than 28 hours of free time after the last nigh shift (p=.013), and backward rotating shift system (p=.019). Shorter sleep was associated with early starting morning shifts (p=.030) and a high number of single free days between two shift periods (p=.047) and marginally with the maximum length of shift (p =.067). Quick returns and early morning shifts were marginally significantly associated with insomnia (p-values .091, .069). Additional adjustments for education and life habits did not change the Results: The present results suggest that the sleep of shift workers would benefit from employee control over working times and shift systems that properly take into account the extra need for recovery in shift work. Objectives: Karasek's Job Content Questionnaire (JCQ) has been adopted to assess psychosocial work environment according to the Demand-Control-Support (DCS) model. Meanwhile, for instance, the Demand-Control-Support Questionnaire (DCSQ) is also used in Scandinavian countries. We translated it into Japanese, i.e., the Japanese-translated DCSQ (J-DCSQ), and investigated its reliability and validity. Methods: The subjects were volunteer 239 workers in 19 nursery schools in Japan. Psychological demands (PD: calculated with 5 items), decision latitude (DL: 6 items), and social support (SS: 6 items) were assessed with the J-DCSQ. One-week test-retest reliability and internal consistency were estimated by means of intra-class correlation coefficient (ICC) and Cronbach's alpha, respectively. Concurrent validity was evaluated with Pearson's correlation coefficient for which the Japanese-version JCQ (J-JCQ) was used as the reference. Factorial validity was evaluated with factor analysis. Results: ICCs were 0.80, 0.83, and 0.81 for PD, DL, and SS, respectively. Correspondingly, Cronbach's coefficient alphas and Pearson's correlation coefficients were 0.65, 0.63, 0.87 and 0.72, 0.61, 0.57 for PD, DL, SS. Factor analysis suggested a 4-factor construction which explained the variance as much as 50%. The first factor was constructed of 4 DL items. The second factor included the whole of SS items. The third factor was composed of 3 PD items and 2 DL items. The last factor was constituted of 3 DL items and 1 PD item. Conclusions: The J-DCSQ exhibited as equivalent reliability as the J-JCQ. The present findings suggested adequate concurrent validity of the J-DCSQ. Factor analysis did not present factor structure of the J-DCSQ as theoretically hypothesized. Previous studies also reported similar factor structure of the J-JCQ. Further studies are needed to better identify psychosocial properties among larger samples. Although previous studies revealed that long working hours predicted fatigue and depressive symptoms, little is known about how these relationships vary among different industries or occupations. This study examined the effect of working hours on fatigue and depressive symptoms among particular industries and occupations. Longitudinal data on 1,588 Japanese daytime workers (575 women and 1,013 men) were used to estimate the effect of working hours on fatigue and depressive symptoms. Working hours per week at baseline was divided into three categories: "less than 60 h" (N=1,346, 84.8%), "61-65 h" (N=87, 5.5%), and "66 h or more" (N=155, 9.8%). Participants completed the Self-Diagnosis Check List for Assessment of Worker's Accumulated Fatigue developed by the Japan Ministry of Health, Labour, and Welfare, and the Center for Epidemiologic Studies Depression Scale at 1-year follow-up. Industries were divided into 13 categories based on the International Standard Industrial Classification of All Economic Activities, Rev. 4 by the United Nations.
Occupations were divided into 6 categories based on the International Standard Classification of Occupations-88 by the International Labour Office. Analyses of variance were carried out separately for industries and occupations. Multiple comparisons with Bonferroni method indicated that in the manufacturing industry and professionals, the "66 h or more" group had a higher level of fatigue than the "less than 60 h" group (P<0.05). In the human health and social work activities industry, the "61-65 h" and "66 h or more" groups had a higher level of fatigue than the "less than 60 h" group (P<0.05). No significant association was found between working hours and depressive symptoms in any industry or occupation. Although reducing the number of working hours is essential to ameliorate workers' fatigue, special attention should be paid to workers in manufacturing or human health and social work activities industries and professionals who work long hours. Especially, workers employed in the human health and social work activities industry who worked at least 61 hours per week, showed higher scores of fatigue. This suggests that the association between working hours and fatigue is varied among different industrial sectors. Psychosocial work conditions including high demands, lack of control and support have been linked to poor health. Yet, the influence of individual factors such as general mental ability (GMA) remains to be examined. The present study set out to investigate how childhood mental ability and psychosocial work characteristics relate to different health indicators in a cohort of working women(n=271) and men (n=291). Specifically, childhood GMA and self-reports of job demands, job control and social support were linked to two positive health indicators (sense of coherence and self-rated health) and two negative health indicators (musculoskeletal problems and anxiety in midlife). Considering the gendered labor market and variations in health patterns between women and men, gender specific analyses were performed. Results revealed no linkages between childhood GMA and the health indicators included. Further, there were no significant interactions between GMA and the psychosocial factors. The overall impact of occupational level was low and controlling for occupational level did not change the results much. These findings are likely to result from the study cohort being fairly homogeneous and the women and men being in good health. Objectives: Few studies have been devoted to exploring hospital physicians' work stresses to their quality of work lives. This study was aimed to explore the relationship between work stress and the quality of work lives, especially under the circumstances of given work stress, the moderating effects of labor activities on the work stress and quality of work lives. Methods: Developed, structured and validated questionnaire was released by mails to hospital physician executives in the period of Aug-Nov 2009 and 737 respondents returned in this study with a response rate of 32.83%. Measures included work stressors as financial-oriented and norm-oriented, and work lives as work and career satisfaction, health status and illness, and substance uses. Labor activities were measured as body labors and leisure activities. Personal characteristics and work status of hospital physician executives were also collected. Descriptive analyses, factor analyses, and multiple regressions were performed. Results: Controlling the personal characteristics and work status of hospital physician executives, it was found that perceived financial-oriented and norm-oriented stresses by hospital physician executives had lower work satisfaction and career satisfaction, more illness symptoms, and worse perceived health status; however, more labor activities had positive effects on quality of work lives. Especially, body labors could moderate financialoriented stress to better career and work satisfaction, better perceived health status, less muscular illness, and reduced sleeping disorders. Also, leisure activities moderated norm-oriented stress to reduced nose and throat illness and moderated financialoriented stress to higher alcohol assumptions. Conclusions and implications: We verified the negative relationships of work stress to quality of work lives for hospital physician executives. Moreover, labor activities played important roles in moderating the work stress to work lives. Under the given work stresses in the health care work environments, hospital administrators could recognize the values of body labors and leisure activities into health promotion programs for their health care professionals to create healthy work environments. Cardiovascular reactivity has been intensively studied for some decades as a potential risk factor or risk marker of cardiovascular disorders and diseases. Among other end-points hypertension, coronary heart disease, and stroke have been possible to predict in research using reactivity measures. However, these predictions have not always been successful. Progress needs to be made. One of the possibilities is using cardiovascular recovery in addition to reactivity in the prediction of pathological processes of hemodynamics and neuroendocrinology as well as direct disorders and diseases. New steps towards applying the research results in clinical practice are necessary to realize the promises of cardiovascular behavioral research. This symposium is one step in this direction. In this symposium researchers will present papers regarding different aspects of cardiovascular regulation, reactivity, and recovery. The symposium starts with Dr Gellman who has comprehensively studied the relationship among hostility, metabolic syndrome, and cardiac structure & function. Dr. Gellman's research is relevant for many of the most common life style factors. The second paper will be Dr. Hamer's presentation regarding psychophysiological responses and their relationship with sub-clinical coronary heart disease. This topic will help particularly in the planning of coronary heart disease prevention. Third, Dr. Tuomisto will illuminate different patterns of cardiovascular reactivity and recovery in intra-arterial blood pressure of normotensive and hypertensive individuals. The results may promote research in the prediction of cardiovascular risks. The last presentation will be Dr. Zanstra's study of challenge appraisals' role in the prediction of a myocardial reaction pattern in anticipation of and reactivity to a naturalistic stressor. The study has interest especially for those who do research in real world circumstances. All these presentations will have both indirect and direct relevance for clinical research in the prediction and prevention of cardiovascular diseases in addition to more basic theoretical issues. Acute psychophysiological stress testing, involving measurement of cardiovascular and biological responses to laboratory-induced mental stress, is an important tool to investigate mechanisms that might account for the association between psychosocial stress and cardiovascular diseases (CVD). The present study was designed to examine associations of disturbed psychophysiological responses with sub-clinical atherosclerosis. Participants were 543 healthy men and women (mean age=62.9±5.7 yrs), without history or objective signs of CVD, drawn from the Whitehall II epidemiological cohort. Various cardiovascular (beat-to-beat blood pressure, heart rate variability) and biological measures (inflammatory and haemostatic markers, salivary cortisol) were assessed during and following mental stressors, consisting of a 5-min Stroop task and a 5-min mirror tracing task. Coronary artery calcium (CAC), a measure of subclinical coronary atherosclerosis, was measured using electron beam computed tomography. The tasks induced significant increases in blood pressure, cortisol, interleukin-6, and fibrinogen. Cortisol responders were at higher risk of having clinically relevant levels of CAC (odds ratio=2.17, 95% CI, 1.37 -3.41) after adjustments for age, gender, baseline cortisol, social status, depressive symptoms, and conventional risk factors. Cardiovascular responses were not associated with CAC. Basal levels of inflammatory markers were associated with CAC although the stress responses were not. These data support the notion that cortisol stress reactivity, an index of hypothalamic pituitary adrenal function, is one of the possible mechanisms through which psychosocial stress may influence the risk of CHD. Cardiovascular reactivity to behavioral stress tasks has been shown to be associated with pathological hemodynamic and neuroendocrinological processes. Later, recovery from such stress has been studied for similar reasons. Our purpose was to study cardiovascular reactivity from a baseline condition, a relaxation to behavioral stress tasks and recovery from the tasks to a relaxation condition after the tasks. As data, we used the Tampere Ambulatory Hypertension Study data bank of intraarterial blood pressure (BP) from the 1990's. The participants were 95 newly detected, WHO-classified, age-matched normotensive (NT; n=33), borderline hypertensive (BHT; n=30) and hypertensive (HT; n=32) men recruited through routine health examinations. They underwent a 5-minute cue-controlled relaxation (R1) followed by 8 standardized behavioral challenges: a video game, arithmetic, social problem solving, imagery, a habituation test, a comic film, and the cold pressor test. After the tasks, a 10-minute relaxation period (R2) was started. Systolic and diastolic BP (SBP, DBP) and heart rate (HR) and were analyzed. First, in a mixed-design ANOVA, Time factor, R1 (the reactivity baseline) and R2 (the recovery baseline) was studied together with the groups. The Time x Group interactions did not differ for SBP, DBP and HR: Fs(2, 92) < 1.04. However, the direction of recovery was different for BPs and HR. During the recovery baseline, HR was lower than during the reactivity baseline. The reverse was true for both SBP and DBP. Second, R2 variables were compared in ANCOVA using R1 variables as covariates. Following main effects were obtained: SBP, F(2, 91) = 3.14, p<.05 and DBP, F(2, 91) = 5.98, p<.005. In Bonferroni controlled group contrasts, the recovery baseline measures showed differences between the hypertensive groups in DBP (p<.05, 95% confidence interval=0.8 -6.59) while reactivity measures had shown DBP differences between these groups only in the cold pressor test. In conclusion, intra-arterial BP and S202
Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329 HR in NT and newly detected HT groups showed different patterns in reactivity respective recovery. These results are in accordance of some earlier research and show that optimizing the use of cardiovascular data requires that these different patterns are taken into account in the predictions of health risks. The current study examined the changes in parameters underlying blood pressure in relation to anticipation, reactivity and recovery to stress. Further, it was examined how these vascular and myocardial parameters relate to cognitive appraisal in a real-life stressful situation. 24 men aged 19-28 (mean age: 23.5; SD: 2.5) participated in this study. Ambulatory blood pressure was recorded on a beat-to-beat basis before and during performance of a real life speech. Vascular and myocardial hemodynamic parameters (total peripheral resistance (TPR) and cardiac output (CO), respectively) were derived from the blood pressure waveform. Participants' appraisals were obtained prior to the stressor. Evidence was obtained suggesting a myocardial reaction pattern during the stressor. This finding is consistent with the hypotheses as the stressor is an active coping task. The anticipation was hypothesized to be a relatively passive situation and therefore expected to engender a vascular response. However, the reaction pattern during anticipation was myocardial. Finally, recovery was slower in the vascular than in the myocardial variables. Multilevel regression models (MLwiN v2.2) were used to analyze the relationships between appraisal and CO and TPR. During the anticipation period prior to the stressor, increased challenge was associated with decreased vascular resistance (Z=2.70 p<.01) and increased myocardial reactivity (Z=2.43 p<.01). During the stressor increases in challenge were associated with further increases in myocardial responding (Z=2.03 p<.05) but relationships between appraisal and vascular resistance were not significant (Z=1.82, n.s.). This study is among the first to examine the hemodynamic reactivity and recovery patterns to a real life stressor in conjunction with stressor appraisals. Furthermore, our findings suggest that challenge appraisals to real-life stressors contribute to an, arguably healthier, myocardial reaction pattern. This ambulatory study was followed up by a study aimed at changing appraisals and appraisal related hemodynamic reactivity patterns. With advances in visual media technologies, health communications and interventions can now incorporate a variety of sophisticated images and pictures. Optimal use of these technologies requires that we advance our understanding of how individuals respond to health imagery, particularly since information processes for imagery differ markedly from those involved with linguistic information. This symposium reviews recent research on techniques using visual images to motivate behavior change. The speakers represent multidisciplinary teams of experts in psychology, sociology, communications, graphics, and information technology. Gerry Humphris will present research on using graphic warning labels on cigarette packets to motivate smoking cessation. Using data from a large community survey, his team found that images evoking disgust responses are potent triggers of motivations to quit. Gareth Hollands will present research on an intervention in which aversive images are used to condition responses that reduce motivations to eat unhealthy foods. This technique, based on implicit cognition and evaluative conditioning principles, altered implicit attitudes about foods and increased healthy food choices. Susan Persky will present research in which immersive virtual environment (IVE) technologies are used to motivate protective behavior. The studies demonstrate how IVEs can instill experiential knowledge of behavioral consequences and enhance understanding of complex concepts such as genebehavior interactions. Brian Williams will present research on a framework for developing theoretically-guided interventions involving visual media. The four-stage process was tested in the development of an intervention incorporating 3D animation about links between obesity and arteriosclerosis. Throughout the symposium, discussion will address how the research informs theory on visual imagery processes in health behavior as well as the use of imagery in interventions. Background: A growing body of research suggests that communicating associations between aversive health-related images and a given behaviour may diminish the likelihood of that behaviour. However, there is limited experimental evidence of this relationship. The current study examines the behavioural and attitudinal effects of communicating images of energy-dense snacks, paired with images of the body indicative of health risk.
Methods: Participants (n=132) were randomly allocated to either a conditioning procedure pairing images of snack foods with images related to cardiovascular disease, or a control condition featuring images of snack foods alone. They completed a measure of implicit attitudes (pre and post-intervention) and two behavioural choice tests in which they chose between fruit or snack products (post-intervention). Results: The conditioning intervention made implicit attitudes towards energy-dense snacks more negative (F(1,129) Background: Health promotion interventions frequently employ visual images to influence knowledge, beliefs, attitudes, and thus behaviour. However, such interventions are often highly pragmatic and lack clear theoretical underpinning. The stages of development are often also poorly documented. Objectives: To develop a visual intervention informed by behavioural theory and concepts in order to increase understanding of arteriosclerosis and intentions to behaviour change. Methods: A multidisciplinary group applied the first two stages of the MRC Framework for the Development and Evaluation of Complex Interventions. This was operationalised in 4 subphases. 1. Establishing a theoretical basis: the creation of conceptual content 2. Establishing structure: creating a visual narrative 3. Establishing the "look": visual rendering of narrative and concepts 4. Establishing interpretation and impact: creating evidence Initial testing of the animation included in-depth interviews with 15 patients and 5 health professionals. Understanding of the animation was explored, followed by emotional reaction, acceptability and suggestions for improvement. Interviews were recorded, transcribed and analysed using the "Framework" method. Results: Phases 1 to 3 established an animation of the body and heart that sought to increase knowledge, perceived severity, susceptibility, self-efficacy and coherence. In phase 4 testing all but one participant provided an accurate and unprompted interpretation of the animation. Increases in knowledge, perceived susceptibility, self-efficacy and coherence were reported. Numerous participants indicated that the animation moved illness representations from the abstract to the concrete. An increase in anxiety and worry was reported but regarded as acceptable by all participants. Conclusions: We have established a detailed stepwise process to develop and document the creation of visual-based, theory-informed behavioural interventions. The digital animation appears acceptable to staff and patients, and to influence intended behavioural concepts. These features could heighten many types of health messages conveyed via visual imagery. For example, a major purpose for invoking imagery is to demonstrate consequences of one's behavior (e.g., illustrating detrimental effects of smoking). These consequences can be experienced firsthand in an IVE, and an interactive component could include practicing and building efficacy for cessation behaviors while observing how these behaviors mitigate health consequences. Indeed, an imagery-based IVE for smoking cessation was recently created by Canadian researchers. Another use of imagery involves conveying health concepts, often via metaphor (e.g., a ladder as a metaphor for varying risk levels). In IVEs individuals can directly experience a visual metaphor. They might also manipulate situational variables to gain nuanced understanding of the processes a metaphor conveys. Our group created and tested immersive visual metaphors based on an elevator and a bridge for conveying genetics concepts. Going forward, IVEs are poised to become an innovative medium for imagery provision and a vital research tool for exploring issues around image effectiveness. Recent research has highlighted the possibility that the emotion of disgust can act as a significant driver to initiate behaviour change as a separate or even additional component to traditional fear appeal approaches. Pictorial images may be particularly effective in generating disgust emotion. Therefore the aim of this investigation was to test the specific image qualities associated with reported disgust and rating of potential success in promoting behaviour change. Data were obtained from a public consultation exercise (Department of Health, England and Wales, 2006) that requested, through a web-based answering system, participant preferences of 42 images that might be effective to encourage tobacco cessation in smokers. Initial results indicated preferences for effectiveness of cessation message were rank ordered according to the level of disgust emotion generated, i.e. images that might persuade smokers to quit tobacco consumption appeared revolting. To confirm that disgust emotion generated by the UK tobacco packet images was associated to the public's selection of possible effective images; three cross-sectional opinion surveys were conducted. These included students from medicine and psychology disciplines, and a section of the public. The web opinion consultation database was utilised for secondary analysis. A total of 291 participants were involved in the three convenience surveys and 19812 participants gave complete replies to the public consultation website. The pictorial Disgust Sensitivity Scale was used to assess the individual's rating of every image on a five category rating ranging from 'extremely disgusting' to 'not disgusting'. Significant correlations (ranging from 0.91 to 0.94) existed between the image rank order aggregated preference ratings from the original public consultation and the average final score of the disgust ratings for specific items for the three groups. The correlations were very similar regardless of smoking status of respondents (in both public preference and opinion surveys). The emotion of disgust may be a possible intervening variable to explain the initial reactions to health promotion materials and smoking cessation. Future research needs to explore the strength of disgust emotions and fear generated by images of this nature with actual behaviour change and other intervening constructs. Over the last decade, the technique of using ambulatory saliva sampling has become increasingly popular in field research and clinical studies. The non-invasive method is easy to administer and analyze, and therefore allows implementation in large scale study designs. However, this large interest in use of saliva cortisol measurement is paralleled with frustrations on opposing Results: This symposium is based on a critical evaluation of existing literature on salivary cortisol, aiming to evaluate the utility of salivary cortisol as a biomarker in various settings and how we can understand cortisol reactivity using evidence of experiences from different study designs. The work is compiled by the Scandinavian Stress and Cortisol Network, a network financed by the Swedish Research Council, and one main question asked was: is it possible that different results of studies involving cortisol assessments are functions of differences in the theoretical assumptions made and methods used? In particular the symposium will focus on how the many different ways of evaluating levels and dynamics of salivary cortisol (i.e. with regards to time points of assessment and different algorithms based on multiple time points) may have an impact on the interpretation of cortisol measurements in various contexts. Salivary cortisol has been studied in relation to the following topics: demographic variables, psychosocial work environment, psychological resources (e.g. mastery) and outcomes (eg burnout), sleep quality, biological markers (markers of cardiovascular risk, inflammation and metabolism) and somatic outcome. The following topics shall be covered at the symposium; where all data shall refer to how results of analyses can differ depend on methods used. Over the last decade, the technique of using ambulatory saliva sampling has become increasingly popular in field research and clinical studies. The non-invasive method is easy to administer and analyze, and therefore allows implementation in large scale study designs. However, this large interest in use of saliva cortisol measurement is paralleled with frustrations on opposing Results: This presentation is based on a critical evaluation of existing literature on salivary cortisol, aiming to evaluate the utility of various measures of salivary cortisol as a biomarker in relation to psychosocial work stressors. Various measures of salivary cortisol were studied in relation to psychosocial work stressors. Twenty-seven papers including psychosocial work stress in terms of the Job Demand-Control-Support model or the Effort-Reward Imbalance model were reviewed. The presentation focuses on how salivary cortisol relates to work stressors, and discusses how these results may vary depending on measurement and data analysis.
This presentation is based on a literature search using PubMed, aiming to evaluate the utility of various measures of salivary cortisol as a biomarker in relation to cardiovascular risk factors. In particular, the presentation focuses on a discussion on how these results may vary depending on measurement and data analysis. Background: The altered cortisol is sometimes regarded as a warning reaction essential part of the survival strategy of living organisms. An often asked question is "Is it unhealthy to have an increased or decreased cortisol awakening response?" In particular, correlations between altered cortisol levels and cardiovascular risk factors have been discussed. The anti-inflammatory properties of exogenous cortisol are well described. However, empirical data on endogenous cortisol and its correlations with inflammatory markers are few. This presentation is based on a literature search using PubMed, aiming to evaluate the utility of various measures of salivary cortisol and its correlation with various inflammatory markers such as interleukines, acute phase proteins, and growth factors. The presentation focuses on a discussion on how these results may vary depending on measurement and data analysis.
In particular, the results would be discussed in the light of a possible down-regulation of glucocorticoid receptors. Insomnia, a type of sleep disruption, is an extremely common and troublesome condition associated with cancer. The diagnosis of cancer represents a threat to survival; consequently, both patients and physicians believe that transient sleep dysfunction in response is normal. Currently, relatively little is known about the etiology and evolution of insomnia in cancer patients. Emerging evidence suggests that a significant minority of cancer patients develop a chronic persistent form of insomnia as a result of the diagnosis and treatment of their cancer. Chronic insomnia is a substantial problem; in the general population insomnia is a risk factor for additional morbidity (e.g., depression, fatigue, heart disease). This symposium will first discuss the prevalence of insomnia and its progression over an 18-month period in a large sample of newly diagnosed cancer patients. The second presentation will describe the sleep architecture of cancer patients undergoing chemotherapy. Polysomnographic sleep findings revealing the prevalence of slow wave sleep (SWS) in cancer patients before treatment, following treatment, and at later follow-up will be discussed. Finally, results from a national randomized clinical trial of YOCAS ® Yoga will show how a brief, community-based intervention of 4 weeks' duration can significantly reduce insomnia and use of sleep medication in cancer survivors, suggesting that persistent insomnia responds quickly to a behavioral exercise intervention and can be treated easily and effectively. The goal of this symposium is to disseminate exciting recent findings in the areas of sleep and cancer and to generate new ideas and an awareness of chronic insomnia in cancer patients so that its negative effects can be addressed. The goal of this large scale epidemiological study was to assess the prevalence and incidence of insomnia comorbid with cancer over an 18-month period. All patients scheduled to undergo surgery after a first diagnosis of non-metastatic cancer were solicited at their pre-operative visit. Among the 3196 patients approached, 1681 were found eligible and 962 (57%) accepted to participate. The participants completed a semi-structured interview for insomnia at baseline (T1), 2 (T2), 6 (T3), 10 (T4), 14 (T5) and 18 months (T6). The prevalence of the insomnia syndrome (T1: 28%; T2: 26%; T3: 25%; T4: 23%; T5: 21%; T6: 22%) decreased progressively and significantly over the 18-month period, F(5,3725) = 7.58, p<.01. Similarly, the prevalence of insomnia symptoms (including patients with an insomnia syndrome) decreased steadily and significantly over time (T1: 60%; T2: 48%; 46%; T3: 46%; T4: 41%; T5: 38%; T6: 37%), F(53725) = 43.77, p<.01. Simple effects indicated that the decrease was significant between T1 and T2 and between T3 and T4 (ps<.01). Prevalence rates of insomnia symptoms were the highest in breast cancer patients (42-69%) and the lowest in prostate cancer patients (25-38%). The overall reduction in prevalence rates was reproduced for each cancer site except for prostate cancer patients in which the decline between T1 and T4, was followed by a resurgence of insomnia symptoms. Based on mixed models analyses, the incidence of insomnia among good sleepers at the previous time assessment was 20% at T2, 22% at T3, 17% at T4, 17% at T5, and 14% at T6. Insomnia is highly prevalent in cancer patients, particularly at the time of cancer surgery and then decreases progressively over time. Despite this overall pattern, insomnia symptoms develop in a significant proportion of patients during the cancer care trajectory, thus suggesting distinct patterns of evolution across patients. Background: Sleep disturbance is prevalent among cancer patients undergoing chemotherapy. Our previous work showed that nearly 80% self-report disturbed sleep during treatment. However, little objective evidence has been gathered on the patterns of sleep before and following chemotherapy. Method: 26 Patients scheduled to receive chemotherapy were recruited. Sleep architecture was assessed by in-lab polysomnography (PSG) for two consecutive nights prior to first chemotherapy (T1), approximately two weeks following the patient's last chemotherapy or radiation treatment (T2) and three months following the last treatment. Most subjects (20) Research about medicine use among adolescents is scarce. However, studies have shown that medicine use for common complaints is widespread among adolescents although there are major differences across countries. Medicine use is a public health concern. First, medicines are toxic and have adverse side effects. Second, medicine use in childhood and adolescence tracks into adulthood. Third, among adults medicine use is the most common response to ill health. Therefore, it is important to identify the main determinants of medicine use in adolescence. A few studies have suggested that adolescents' medicine use is a response to a range of stressors, e.g. exposure to bullying, belonging to a lower socio-economic group or an ethnic minority, and poor self-rated health. The objective of this symposium is to present a scientific rationale for the study of adolescents' medicine use, to provide an overview of recent empirical studies of adolescents' medicine use, to discuss methodological challenges in this area of interest, and to present new empirical findings from the international Health Behaviour in School-aged Children (HBSC) study. Adolescent mental health has been related to a variety of physical health problems and adolescents can have considerable independence in their use of medicines to treat acute health problems. The relationship between adolescent mental health and medicine use (MU) has not received sufficient attention. The present analyses examine relationships of adolescent mental health and MU and whether mental health contributes to MU beyond the effect of experiencing symptoms of those health problems. Self-reported mental health, MU for four common health problems (headache, stomachache, difficulties sleeping, and nervousness) and symptoms for common health problems were assessed in a nationallyrepresentative sample of 7,611 U.S. students in grades 6 through 10 and in 26,111 11-, 13-, and 15-year-old students in six countries participating in the Health Behavior in School-Aged Children study: Austria, Luxembourg, Germany, Switzerland, Macedonia, and Scotland. A brief measure of depression was included in the US HBSC survey and a broader mental health index (KIDSCREEN) was included in the surveys of the six European nations. In US adolescents, depression predicted MU for each of the four health problems but when both depression and symptoms were in the regression models depression added to the prediction for difficulties sleeping (p=.0071) and nervousness (p<.0001) only. These findings were replicated in the European sample; using hierarchical linear modeling, there no significant effects due to country. MU for each of the four health problems was predicted by mental health; however, MU for headache and stomachache were no longer significant when symptoms were added to the models. Adolescents with poorer mental/psychological health are more likely to experience symptoms of common health problems and to take medicine for these problems. However, self-reported symptoms may partially mediate the relationship between adolescent mental health and MU. First methodological challenges and opportunities in the study of adolescents' medicine use will be discussed for the constituting parts 'adolescents' and 'medicine use'. These parts will then be combined in the context of the study of ADHD medication use in adolescents. The very word 'adolescence' draws attention to the developmental perspective of any study involving adolescents. Advances in developmental neuroscience can help decide on age boundaries. However, the developmental perspective requires more than age boundaries due to the social and cultural aspects and expectations in this transitional phase, particularly in multi-national adolescents' medicine use studies. Consequences of this developmental perspective for data collection and data analysis will be discussed. With regard to medication use, prescription medication and overthe-counter medication are often distinguished. Another distinction is on-label, off-label and unlicensed medication. A special issue in adolescence is the study of compliance since adolescents seem more reluctant to take medicine according to instructions. Over-the-counter drugs and the use of illegal drugs also require specific attention when we study adolescents' medicine use, as does the difference between incidental and frequent or prolonged medicine use. These differences have consequences for data collection and data analysis. Adolescents' use of ADHD medication is usually on-prescription. Methodological issues in the study of prolonged monitoring of adolescents' ADHD medication use as hindered by factors S210 Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329
belonging to the developmental perspective and as supported by technological opportunities, will be discussed. Finally, some issues and opportunities in the study of non-prescribed ADHD medication will be presented. Objective: To examine the association between menarche and use of medicine for common complaints (headache, stomach ache, difficulties in getting to sleep, nervousness) in a national representative sample of 11-and 13-year-old girls. Methods: This cross-sectional school survey included all girls in the fifth (mean age 11.6 years) and seventh (mean age 13.6 years) grade in a random sample of Danish schools, n=2,423. Medicine use was measured by self-reports. We used logistic regression analysis with menarche as the independent variable and medicine use as the dependent variable, adjusted for social class and prevalence of the symptom for which the medicine was used. The dual pathway model (DPM) of bulimic pathology represents an integrative etiological theory that contemplates the mediational mechanisms by which the risk factors might work together to promote bulimic behavior (BB).The internalization of the thin ideal contributes to body dissatisfaction (BD); increased BD, in turn, fosters dieting and negative affect (depression and stress, in this study), which increase the risk for BB. Individuals may initiate BB because of either extreme dieting or chronic negative affect. In order to gain a better comprehension of BB and the main risk factors (thin ideal, body dissatisfaction, restrained dieting, and negative affect) two structural models were built and compared by sex (the DPM of women vs. the DPM of men) in order to detect which one of the group's models: a) shows more risk factors for BB, and which one of them b) provides a better understanding (higher explained variance) of the predictive role of the risk factors for BB. The total sample was formed by N=196 college students, comprised of women (121) There is no doubt that nutrition affects athletic performance. Unfortunately, few collegiate sports programs include a comprehensive nutrition component. The purpose of this study was to determine stability-reliability of a 29 item instrument created to provide baseline data that would be used to develop a multi-level nutrition program for collegiate student-athletes. This survey also included demographic questions and was based on the Health Belief Model. Questions included measures of self-efficacy, perceived susceptibility, benefits and barriers. In addition, the Stages of Change Model was used to assess current nutritional activities. The survey was administered to a convenience sample of 41 collegiate-athletes and yielded 41 matched pairs used for analysis. A single item measuring Stages of Change resulted in a moderate correlation (r=.6), and 5 items on self-efficacy ranged from r=.33 -.68, for an overall moderate correlation (r=.6). There were 6 items that assessed benefits of receiving sport nutrition education (range r=.2-1.0) and 7 items that measured barriers to healthy eating (range r=. .4-1.0). These items yielded a total correlation score of .6 and .7 respectively. There were 15 items that measured nutritional knowledge. Because these items were dichotomous, a Kuder Richardson 20 (KR 20) test was used. Correlation coefficients for this subscale ranged from .03 to 1.0, for an overall correlation coefficient of .52. Items with correlation scores less than .06 were modified to yield more consistent responses and thus this survey shows promise as a potential valid and reliable assessment of collegiate athletes' knowledge, perceptions, and dietary practices. This information is essential when designing nutrition education programs and interventions for this population. In total institutions such as the military, meal times serve as release from daily routines. In Finland, all men are called for military service and 80% complete. Main meals are served as part of the service, but snacks are available for purchase during free time. The aim of the study was to analyse young men's attitudes towards health and eating before and at military service and their associations with eating patterns. A total of 290 men (age 18-21) participating in the DefenceNutri project filled in questionnaires before service and at 6th month of service. Four sub-indexes of Health and Taste Attitude scale (HTAS) were measured: General health interest (GHI); Craving for sweet foods (CSF); Using food as a reward (REW); and Pleasure (PLE), each with 4-6 statements rated from strongly disagree to strongly agree. The food consumption was measured as the number of days/week 6 sweet foods (Sweet food index) and 6 fatty foods (Fatty foods index) were consumed. Changes of HTAS indexes were analysed with paired sample t-test and associations with eating patterns with ANOVA.
GHI remained stable throughout the follow-up. CSF (3.0-3.6,p< 0.000), REW (3.3-4.0,p<0.000), and PLE (3.9-4.1,p<0.000) increased. Before entering military service eating of sweet foods was associated negatively with GHI (p=0.033) and positively with CSF (p<0.000) and REW (p<0.000); and eating of fatty foods negatively with GHI (p=0.003) and positively with REW (p= 0.001). At the end of 6th month of service eating of sweet foods was explained again by GHI (p=0.002), CSF (p=0.001) and REW (p=0.021), but eating of fatty foods was not associated any of the attitude scales. PLE was not associated with eating of fatty or sweet foods in neither measurement.
Military service increases men's tendency to view food as a source of reward and pleasure, and to crave for sweet foods. In civilian life, health pursuit decreases eating of sweet and fatty foods but during service only of sweet foods. During service eating of fatty foods is not related to pleasure pursuit. Introduction: Screening for type 2 diabetes is increasingly being offered in general practice. Once the diagnosis is made healthcare services are likely to offer health promotional and preventive initiatives. Preventive and treatment recommendations are fully developed, but how we motivate people to make use of these initiatives and offer the right ones remains less clear. Aim: To investigate socio-economic and disease-related predictors for participating in the "Ready to Act" program dealing with support to action competence for living with dysglycaemia. We hypothesize that the possibility of participating in this particular program will increase in accordance with a higher level of education, cohabitant and no competing diseases. Design: A cohort study was embedded in the randomized controlled trial of a primary care/general practice screening and intervention programme. The study population (n=320) were adults with screendetected prediabetes or with type 2 diabetes recruited by a mailed invitation after the screening procedure. The "Ready to Act" program, which consisted of 2 individual interviews and 8 group meetings delivered over 12 weeks, was offered to the intervention group by a multidisciplinary team of health professionals, as a supplement to intensive treatment in general practice. In total 45% (n=144) accepted to participate in the program and 38% (n=123) fulfilled.
Study plan: A comparison between those who accepted the program and those who did not will be conducted according to the following outcomes: socio-economic: age, sex, cohabitant and education and disease-related: diagnosis, duration of diagnosis, comorbidity (cancer, CHD, COPD). Perspective: The evidence of predictors for participation in health promoting programs is of great value for the future planning of target group differentiated health service initiatives. Many working adults with diabetes face major barriers to effective disease self management. The Hawaii Demonstration to Maintain Independence and Employment was a randomized controlled trial providing treatment group participants with individual life coaching and a pharmacist counseling while tracking health and employment outcomes. Self-reported diabetes self-efficacy, quality of life, functioning, hours worked, work productivity, and use of government services data were obtained at baseline, 6, and 12 months. Additional health information was obtained from healthcare providers. Generalized linear models for repeated measures are being used to evaluate the effect of the intervention. Surveys and focus groups were used to obtain participant perceptions about the intervention. 190 employed individuals diagnosed with diabetes or with indication of uncontrolled blood sugar were enrolled. Preliminary results show that body mass index and diabetes self-efficacy changes were significantly improved in the treatment group (p<0.05). Treatment group participants reported greater work productivity improvements. Diabetes management improvements were frequently attributed to increased accountability and goal setting with a life coach, as well as nutrition and diabetes education provided by pharmacists. This presentation will discuss how participant engagement, perceptions, and intervention components may have influenced employment and health indicators. A diabetes management intervention was well received by participants and showed the potential to delay functional decline and reliance on government assistance programs. Findings have implications for employers, health care providers, and professionals using early intervention models intended to prevent disability and support the ongoing employment of individuals with chronic conditions. The use of Life Coaching and Pharmacists as counselors is an emerging service delivery approach. Further RCT research is warranted to determine the viability of this model for chronic disease management. Cost effective analysis would also be valuable. Depression is a serious problem affecting 25% of persons with diabetes, and the risk of depression is doubled in women compared to men with diabetes. Other moods (e.g., anxiety, anger) often accompany depression and negatively impact diabetes outcomes. We tested the feasibility of the Study of Women's Emotions and Evaluation of a Psychoeducational (SWEEP) program to assess the effectiveness of group cognitive behavioral therapy (CBT) for treating depression and dysphoric symptoms in women with type 2 diabetes. Seventy four women with diabetes and co-morbid depression were randomized to the SWEEP program (n=34) or usual care (n=36). The treatment included 8 weekly sessions targeted at depression, anxiety, and anger. Sixty women (81%) completed baseline, 3 and 6 month measurements. Findings indicated a greater decrease in depression (CES-D) for the treatment (T1=26.8, T2=15.2, T3=12.6) as compared to the control group (T1=28.9, T2=23.3, T3=21.5), p=.03. Similarly, there was a greater decrease in trait anxiety (STAI) (T1=49.9, T2=41.2, T3=36.8) for the treatment group as compared to the control group (T1=50.5, T2=46.6, T3=45.3), p=.01. For anger (STAXI), although there were no significant differences between the treatment (T1=20.0, T2=17.3, T3=15.4) and the control conditions at any point (T1=18.0, T2=16.2, T3=16.0), there was a significant decrease in anger scores overall, p<.001. There was a clinically significant decrease in HBA1C in the treatment group from baseline to 3 months (T1=7.6, T2=7.2) however, it was not statistically significant and the improvement was not sustained (T3=7.4). There were minimal changes in HBA1c in the control group (T1=7.9, T2=7.9, T3=7.8). Overall, the SWEEP program significantly improved depression and anxiety outcomes in depressed women with type 2 diabetes. There were also favorable effects on anger and HBA1C. These data provide the first demonstration that depression, a common co-morbidity in women with type 2 diabetes, can be effectively treated in a group setting using CBT techniques. Introduction: More than 60% of male construction workers in the Netherlands are overweight or obese and more than a quarter is at risk for cardiovascular disease (CVD). The aim of this study was to evaluate the effectiveness of a motivational interviewing-based lifestyle intervention in this population. Methods: 816 Male construction workers with an elevated CVD risk were individually randomized into the intervention or control group. The 6-month intervention consisted of three face-to-face and four telephone contacts with an occupational health professional, in which behavior change was discussed in the style of motivational interviewing. Participants chose to aim at either energy balance or smoking behavior. The control group received usual care. At 6 and 12 months, dietary intake, physical activity, and smoking behavior were assessed by means of a questionnaire. Moreover, body weight, cholesterol, blood pressure, and HbA1c were measured according to a standardized protocol. In order to determine the intervention effects, linear and logistic regression analyses were performed in the total study population as well as in the energy balance and the smoking subgroups. Results: The intervention had a statistically significant beneficial effect on fruit intake (pieces per week: β=1.7, 95%CI 0.6; 2.9) and smoking at 6 months (OR=0.3, 95%CI 0.1; 0.7), and snack intake (pieces per week) at 6 (β=−1.9, 95%CI -3.7; -0.02) and 12 months (β -1.9, 95%CI -3.6; -0.03). No intervention effect was found for physical activity. In the energy balance subgroup, a significant intervention effect was found for body weight at 6 months (β=−2.1, 95%CI -2.9; -1.3), which was sustained until 12 months (β=−2.2, 95%CI -3. Although weight loss programs tend to produce clinically meaningful though modest weight loss, most individuals steadily regain their weight. The current study compared the efficacy of a standard behavioral weight loss maintenance intervention (SBWLM) to a novel 12-week mindfulness-based intervention plus coaching tailored to WLM (Enhancing Mindfulness for the Prevention Of WEight Regain: EMPOWER). Community members who had lost a minimum of 9% of their body weight in the previous three years were recruited and randomized. Both groups received identical information about nutrition, physical activity, the importance of stress management, and values and goal setting. The EMPOWER group, however, was presented the information in the context of learning mindfulness skills to facilitate change. EMPOWER participants were also taught meditation, mindful eating, and mindfulness strategies to reduce stress-related reactivity while SBWLM participants spent equivalent time reading and discussing relevant material. Both groups were carefully structured to ensure parity in numerous factors (e.g., time invested, peer and interventionist support, positive expectancy). Mixed models determined that incremental weight regain rates did not differ by group. On average, both groups maintained their baseline weight loss 16 months later, one year post intervention (EMPOWER: ↓ 1.5 kg; SBWM: ↑ 0.9 kg). Weight regain rates were equivalent to or lower than those reported in other large scale studies (e.g., Svetkey et al., 2008 , Dansinger et al., 2007 . Only the EMPOWER group reported increases in intuitive eating (p<.0001), a trait previously associated with better physical health (Hawks, et al., 2005) and lower eating pathology (Tylka & Wilcox, 2006) . Further analyses will determine whether metabolic and inflammatory biomarkers were impacted differentially by group. These data support the use of the EMPOWER program as a novel and effective approach for supporting individuals in the maintenance of weight loss. The metabolic syndrome disorders (MSD) including type 2 diabetes, hypertension, hypercholesterolemia, atherosclerosis and cardiovascular disease are known to be triggered by lifestyle change. We state and support a hypothesis here that changes in behavior rather than changes in diet and metabolism are central to MSD. We provide and discuss evidence in support of the following sequence of arguments. (i) Diet, thriftiness and obesity centered paradigm is inadequate to explain most of the physiological and immunological changes associated with metabolic syndrome disorders (MSD). (ii) Comparative studies show that relative obesity rather than absolute obesity is strongly associated with various parameters of MSD and further perception of calories without actual change in intake of calories can induce physiological changes. (iii) Alternative behavioral strategies co-exist in animal populations which are associated with characteristic metabolic states and some of these states show close resemblance with MSD in humans. (iv) Obesity and MSD are strongly and cross culturally associated with certain behavioral traits. (v) Deficiencies of certain behaviors that evolved with hunter gatherer life as adaptive behaviors but that are deficient in modern life lead to corresponding dysfunctions of neuro-endocrine mechanisms. Through a meta-analysis study we show that deficiency of physical aggression is strongly associated with MSD as seen by neuro-endocrine mechanisms as well as epidemiological data. This raises a possibility that the effects of behavioral deficiencies may be reversed by behavioral supplementation. (vi) On a pilot scale, behavioral interventions substantially ameliorated many components of MSD without significantly changing body weights. All the evidence collec-tively indicates that MSD originates due to behavioral deficiencies rather than metabolic thriftiness and therefore behavioral supplementation may be effective in reversing the chronic conditions. 1 Lifestyle and Participation, National Institute for Health and Welfare THL, FI-00271 Helsinki, Finland; 2 Department of Health, University of Tampere, Tampere, Finland and 3 Department of Sociology, University of Helsinki, Helsinki, Finland.
Introduction: All eligible young male citizens in Finland are conscripted in the Defence Forces (DF) for 6-11 months. DF has been stressing physical training but is currently emphasizing healthy nutrition increasingly for physical performance, too, as weight gain is a cohort trend. In 2007 THL and DF started a 3-year intervention study (DefenceNutri) to find instruments to improve the conscripts' diet, including a control ( At 6 months SBP was lowered in average by 2.1 hgmm (p<.001), DBP and LDL were kept maintained, but triglycerides and fasting glucose were increased by 0.4 mmol/l (p<.001), respectively. Low-educated subjects had somewhat more detrimental characteristics at start, but differences between educational groups largely levelled-off during the service. Conclusions: While the DF provided nutrition already follows guidelines there is a need for supply/demand targeted nutrition intervention related especially to saturated fat and sugar containing snacks among conscripts. OBJECTIVE: Two hundred and fifteen industrial workers suffering from occupational skin diseases (OSD) have attended a 6months combined dermatological and educational prevention program with an education and counseling scheme as well as an intervention The aim of this program, conducted from 2007-2009, was to enable the affected worker to remain at work without suffering from major OSD.
METHODS: To assess the sustainability of this interdisciplinary medical, behavior and educational training program, the intervention group (IG, N=215) and a control group (CG, Industrial worker with OSD who solely received dermatological treatment, N=85) were followed up 9 month and 2 years after their individual project participation by a standardized questionnaire. A subcohort of the intervention group (IG(2007,) During the study period, there was no change in job stress (JCQ) or effort-reward imbalance (ERI). Compared to baseline, in both intervention groups there was significant decrease in stress level (PSS10, WFC), a reduction in symptom scores (STAIT, PHQ15) and an increase in well being (WWB5, LM). Overcommitment decreased only in the long intervention group. Employees working in the human sector (teachers, social workers, health care providers) benefited more, than industrial workers or those working in law enforcement. Our results confirm the benefits of the stress management interventions at the workplace. The short, 4 lesson program was found nearly as effective as the longer 16 lesson one. These results suggest the feasibility of short interventions, and the importance of focusing on overcommitment, a key factor in work-related stress. Further analysis are needed to clarify the magnitude of the beneficial effects in connection with personal and work stress characteristics of the participants.
Research supported by the Hungarian Ministry of Health. , and WHO Well-being (WWB5) were also taken. 283 persons participated in a two-day stress management training where communication skills, assertiveness, empathy and various coping strategies were taught. 191 employees took part in a short program of 4 hours. At 3 month follow up 73% of the participants (N=229 and 117) completed our questionnaire. Results: Controlled for age and gender, overcommitment in work showed strong correlation with effort-reward imbalance, perceived stress, anxiety, WHO well-being, work-related work-family conflict.
Overcommitment decreased significantly after intervention in the two-day training group (p<0,001) and showed no significant change in the short program group. In the two-day training group we found significant decrease in perceived stress (p<0,001), anxiety (p<0,001), work-related work-family conflict (p<0,01), subjective health complaints (p<0,001) and significant increase in WHO well-being (p<0,01) among employees with high baseline overcommitment score (N=132). Among participants with low overcommitment scores (N=97) there were no significant changes in these parameters. Conclusion: Stress-management trainings can be effective to decrease overcommitment at work and are more beneficial for participants with higher overcommitment. Further investigations are needed to clarify the role of overcommitment in work-related stress and quality of life.
Research supported by Hungarian Ministry of Health. Ilmarinen has developed the Work Ability Index (WAI) in 1991 as a tool for evaluation of the concept of work ability. According to the results obtained by this author and his colleagues at the Finnish Institute of Occupational Health through a longitudinal study of 11 years, the WAI is presented as a good predictor of early retirement due to disability and validated support for preventive interventions or rehabilitation. Promote the ability to work in the labour force is even more important to allow individuals to maintain their health and functional capacity in the reform period.
Considering the foregoing, the present study aims to evaluate the work ability index in Portuguese workers in various professions and see how the ability to work is linked to demographic characteristics. A cross-sectional study was carried out, including 1955 workers (40,3% males) from chemical and metal-mechanic industry, nurses, teachers and public administration. Subjects responded to self-administered questionnaires evaluating demographic characteristics and work ability index. The causal relationship between the sociodemographic characteristics and the WAI was quantified by calculating the odds ratio and confidence intervals of 95%.
In conclusion, it was found that the vast majority of workers analyzed present an ability to work fairly well. And consistent with the literature, it appears that the younger the better their individual work capacity. Similarly, this study shows that male workers and married workers have a better capacity for work compared to female workers and unmarried ones. It is an instrument of great importance to occupational health, in that it permits evaluation of the working capacity of the individual, the perception of the current state of work ability compared to his best, allowed the identification of areas of health injured and potentiate loss of working capacity and thus corrective measures, allows the evaluation of the effectiveness of these measures in overall capacity for work. Background and Aim: In chronic low back pain (CLBP) there is a high degree of comorbidity, and the prevalence of comorbid psychiatric disorders is between 40%-100%. The aim of this study was to assess the prevalence of psychiatric comorbidity in a population of CLBP patients, using a psychiatric diagnostic interview. Methods: 400 patients sick listed between 2 and 10 months for unspecific LBP were included in the study. All were recruited as part of an ongoing trial in secondary care, and were assessed with the Mini-International Neuropsychiatric Interview (MINI), which is a short structured diagnostic interview for DSM-IV and ICD-10 psychiatric disorders. Results: The prevalence of current psychiatric disorders was 30%. The diagnoses included a wide range of psychiatric disorders, with the most common being major depressive disorders (4%), substance abuse (4.3%), anxiety disorders (10%), and somatoform disorders (18%). There were no gender differences in prevalence of psychiatric disorders, and no differences in mean duration of back pain between those with and without a psychiatric disorder. Discussion: The results show that CLBP patients present with more psychiatric disorders than the general population, but less compared to previous studies of psychiatric comorbidity in CLBP. The difference could be a result of different compensational systems, different settings were the interviews were conducted, or different characteristics of the populations in the comparative studies. Conclusion: In a large population of CLBP patients, 30% fulfilled the criteria for at least one current psychiatric disorder when measured with a diagnostic interview. The results imply that screening LBP patients for psychopathology in secondary care is important and should involve a wide range of psychiatric disorders since psychopathology might have consequences for prognosis and treatment. Objective: To predict the two-year course of activity limitations in patients with early knee and/or hip osteoarthritis. Methods: The CHECK cohort comprising participants (N=1002) with early osteoarthritis-related knee and/or hip symptoms was followed for two years. Participants completed questionnaires, and underwent physical, laboratory and radiographic examination. Regression models were used to examine whether baseline variables predicted the course of activity limitations as measured with the WOMAC. Analyses were performed separately for participants with knee symptoms and participants with hip symptoms.
Results: After two-years of follow-up activity limitations slightly decreased in participants with knee symptoms, and were not significantly changed in participants with hip symptoms. Large between subject variation was observed in WOMAC change scores.
In participants with knee symptoms, few activity limitations at baseline, non-western or Indonesian ethnicity, low educational level, bilateral hip pain, high comorbidity count, high BMI, pain on palpation of the joint line, high bodily pain and poor general health perception were associated with a two-year increase in activity limitations. In participants with hip symptoms, few activity limitations at baseline, low educational level, bilateral hip pain, high comorbidity count, no paid employment, few physical activity during leisure, restricted active hip flexion, poor general health perception, and frequent use of the pain coping strategy transformation were associated with a two-year increase in activity limitations. Conclusion: After two years of follow-up large between subject variation was observed in the course of activity limitations. Risk factors for an unfavorable course of activity limitations are identifiable already at an early stage of the disease. Medically unexplained symptoms (MUS) are an economic and humanitarian burden. Amongst them, pain complaints without organic pathology are the most prevalent. Theoretically, activated illness-related memory may cause reporting of symptoms by changing perception and interpretation of bodily signals. In the current study, we used a subliminal priming technique to test whether activating memory related to illness without conscious awareness leads to increased reporting of pain. The subliminal priming task consisted of a simple computer task during which words were shown for a short duration (34 ms). Participants were divided over four different conditions, with prime words describing either (a) health complaints, to activate an illnessrelated memory, or three control categories: (b) neutral words, (c) words describing bodily sensations and (d) negative valence words. The latter two conditions were added to test whether reduced pain tolerance would already be observed after the semantic activation of these two components or correlates of health complaints. For pain stimulation, we used a cold pressor task (CPT). We hypothesized that participants primed with health complaint words would show less pain tolerance (PT) compared to participants primed with neutral words. Participants were healthy students (n=66; mean age=21.57 years, SD=3.45; 84.8% female). Participants primed with health complaint words (n=15; mean PT=77.9 s, SD=79.7) showed reduced pain tolerance compared to participants primed with neutral words (n=17; mean PT=127.2 s, SD=92.5), t(30)=-1.963, p=.027 (one-tailed). Participants primed with either sensation words (n=17; mean PT=95.9 s, SD=75.3) or negative valence words (n=17; mean PT=95.4 s, SD=77.8) did not significantly differ in pain tolerance from participants primed with neutral words, respectively t(32)=-0.581, p=.282 (one-tailed) & t(32)=-0.859, p=.197 (one-tailed). Thus, pain complaints can be involuntary produced by activated illness-related memory. This memory is thought to be chronically over-activated in MUS patients. This would explain why they have complaints without observable bodily pathology and thus without a medical explanation.
Background: Catastrophizing is an integral concept in the fearavoidance model of chronic pain, which has only recently been applied to headache disorders. Thorn (2007) found that behavioral treatment decreases pain-related catastrophizing. Holroyd (2007) found that lower levels of catastrophizing were associated with lower levels of disability. The relationship between changes in catastrophizing and changes in disability in the treatment of headache disorders has not been examined. Methods: After receiving one month of optimal acute therapy, 232 severe migraine sufferers (79% female) were randomized into a 2 Introduction: In the rehabilitation of non-specific chronic low back pain (CLBP) patients the emphasis has shifted from biomedical to bio-psychosocial rehabilitation. These rehabilitation programs reduce perceived disability. However it is unclear which determinants contribute to this reduction. Change in psychophysical capacity, calculated as the ratio between physical capacity and perceived effort, may be a determinant of change in perceived disability. Aim: The aim of this historical cohort study was to identify determinants for change in perceived disability measured with the Roland Morris Disability Questionnaire (RMDQ) in patients with non-specific CLBP after a cognitive somatic rehabilitation program. Method: Data of 84 outpatients with non-specific CLBP who participated in a rehabilitation program were gathered. Aerobic capacity, physical lifting capacity, perceived lifting effort, psychophysical capacity (psychophysical static leg lift, psychophysical static trunk lift, psychophysical dynamic lifting capacity), and perceived disability (RMDQ) were assessed. Associations between change in RMDQ and potential determinants were calculated. Variables significantly changed with the change in RMDQ were entered in a multivariate linear regression analysis (backward). Results: Change in psychophysical static trunk lift (r=−0.51) and psychophysical dynamic lifting capacity (r=−0.53) and psychophysical static leg lift capacity (r=−0.23) were significantly associated with change in RMDQ. The RMDQ score at baseline (β=−0.438), change in psychophysical dynamic lifting capacity (β=−0.109), psychophysical static trunk lift capacity (β=−0.038), psychophysical static leg lift capacity (β=−0.012) and static leg lift capacity (β =0.007) all contributed significantly to the regression model (r2=52%). Conclusion: Improvements in psychophysical lifting capacity are determinants for a reduction of perceived disability after rehabilitation in non-specific CLBP patients. Aims: Because there is little information on interventions for Asians and Pacific Islanders (API) the purpose of this study was to 1) examine the effects of cognitive behavioral intervention (CBI) on improving health, psychosocial, and behavioral outcomes in API with type 2 diabetes; and to 2) determine variations between the groups. Methods: API participants who met the inclusion and exclusion criteria were enrolled and randomized into two groups. Groups met for 6 successive weekly meetings that averaged 2 hours, with group sizes ranging between 2-6 participants and their significant others. Participants in the CBI group were provided 6 modules on mood management, relaxation, biofeedback, cognitive restructuring, values clarification, and cultural responses to life styles. The diabetes educational/support (DES) group participants were provided modules of a refresher course on diabetes education topics and allowed to express their issues and needs. They all completed a demographic form, and various measures on depression, quality of life, self efficacy, self-care activities, health beliefs and adherence measure. Results: The 207 participants between the ages of 18-76 years had a mean age of 58 years. Three-fourths of the participants were Asians and one fourth primarily PIs. Pre and post intervention data indicated that the CBI group on the CESD scored 1.95 units lower than the DES group (p<0.02), a 19.3% decrease relative to the baseline average. For the SF-36 subscale on emotional well being, the CBI group scored 3.92 units higher than the DES group (p< 0.0457), a 4.9% increase relative to the baseline average. On the Fatigue subscale, the CBI group scored 4.72 units higher than the DES group (p<0.03), a 7.9% increase relative to the baseline average. Conclusions: Initial intervention results indicate efficacy of CBI for depression and some quality of life measures. The important long term effects of CBI will be assessed with the soon to be completed one-year follow up data. The 23rd of April, the Mexican population woke up to an unfamiliar influenza epidemiological alarm. The Mexican government implemented vigorous pandemic control measures. The objective of the present study was to describe the psycho-social reactions to the epidemic, risk perception behaviors and compliance with the recommended control measures. An online questionnaire that was posted at the National University's Web page in May 2009, was answered by 13,690 students and employees. Knowledge, beliefs, perceptions, attitudes and behaviors in regard to both, the epidemic and the contingency, were studied. Results: 23% of the population believed that the epidemic was not real but in fact political manipulation, while 34% thought it was a new influenza strain. Worry was the most commonly reported feeling in the academic employees (74%), while students felt mainly confused or anxious (40%). The measurement of the chronological tendency of these feelings showed that at the beginning the population was mainly worried and anxious, then they felt confused, isolated and depressed, and finally they felt discriminated. Information was the most widely expressed need during the contingency and the University Web page was the most trusted source. 55% thought that the epidemic was more severe in Mexico than in other countries. The higher the perceived severity of the problem, the higher the acceptance of vigorous population control measures. Those that personally knew someone affected by the influenza A H1N1 virus were more likely to have sought the seasonal flu vaccine (OR 2.1, p<0.0001). Contingency had the following social impacts on daily life: impossibility to assist to public entertainment places which were closed (68%), interruption of academic work (60%), increased time with the family (50%), more frequent hand washing (91%) and mask wearing (66%). Immediate behavioral research must be considered within epidemic preparedness plans. People with psychosis are at increased risk of weight gain, heart disease and diabetes. While part of this risk is attributable to medication, the impact of psychosis on life style also plays a role. We aimed to design and test an intervention to encourage exercise and healthy eating in this group. Our development work included a systematic review of previous randomised controlled trials of healthy living interventions, to determine which aspects of interventions had been successful previously. We interviewed service-users and case-managers to determine preferences, accessibility and feasibility of different models. We performed a systematic synthesis of the outputs of these exercises, and used this to design a culturally sensitive intervention incorporating theory-based intervention techniques. Our systematic review suggested that our intervention should last longer than a few months, and that it should include some structured activities. Some service users expressed a preference for group activities while others preferred to meet with a therapist on a one-to-one basis. Our intervention employs a mix of one-to-one sessions with a trained support-time-recovery (STR) worker, and the opportunity to join activities and groups facilitated by exservice-users. It starts by eliciting service users' beliefs and knowledge about their weight gain, their motivations to change and their goals. Service users and STR workers then collaboratively develop action plans with cues for action, strategies for overcoming barriers, regular reviews and feedback. The intervention is currently being piloted. The potential strengths and limitations of our development process will be discussed. Purpose: Sleep problems and obesity are prevalent public health problems. However, the contribution of sleep problems to weight gain is poorly understood. We examined sleep problems as predictors of subsequent weight gain. Methods: Data were derived from baseline (2000-2002, response rate 67%) and follow-up (2007, response rate 83%) postal questionnaires surveys (n=7330, aged 40-60 years). At baseline, all participants were employed by the City of Helsinki, Finland. The follow-up questionnaire surveys were mailed to all those who returned the baseline questionnaire. Weights and heights were self-reported. Those who had gained at least five kilograms during the follow-up were examined as reporting major weight gain. Sleep problems were measured by problems maintaining and initiating sleep, and non-restorative sleep during the previous month. Logistic regression models were fitted, adjusting for age, baseline body mass index, socio-demographic background, health behaviours, health status, sleep duration, and shift work. Results: Altogether 26% of women and 24% of men had gained at least five kilograms during the follow-up. Sleep problems at least three times a week (20% of women and 17% of men) were associated with major weight gain among women after adjusting for age (OR 1.41; CI 95% 1.13-1.75). The adjustments for baseline body mass index, marital status, education, heavy drinking, current smoking, physical activity, limiting long standing illness, sleep duration, and shift work had practically no effect on the association. However, adjustment for baseline mental disorders attenuated the association. Among men, none of the associations were statistically significant. Conclusions: Sleep problems remained associated with weight gain among women after pertinent risk factors have been accounted for. Associations among men and the effects of co-occurrence of mental health problems need to be confirmed in further studies. Promoting better sleep and preventing sleep problems might help prevent weight gain and subsequent health risks. Tinnitus can lead to a substantial decline in psychological wellbeing. Existing treatments focus mainly on chronic tinnitus-sufferers, but treatment in the acute phase seems also desirable to prevent a decompensation. The aim of this study was to develop a self-help program for acute tinnitus-sufferers and to evaluate its efficacy. Participants were 18 to 75 years old and had suffered a minimum of two weeks and a maximum of 6 months from subjective tinnitus.
Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329
They were randomly assigned to either a CBT-based self-help treatment (internet-or paper-based, n=68) or a control group (n=38). Some basic information about tinnitus was given to members of the control group, none of which was relevant to treatment. Tinnitus distress and coping abilities were measured as main outcomes. Assessments were performed via self-report inventories at entry and after 3 months. Results of a one-way repeated-measures ANOVA showed that overall tinnitus distress improved in both groups over time (F (104) = 40.27, p=.000, Eta2=.28). In addition, the intervention condition led to more extended improvement than the control condition (F (104) = 12.56, p=.001, Eta2=.11). In both groups use of helpful self-instructions became more likely over time (F(99) = 5.86, p=.017, Eta2=.06) while unhelpful self-instructions decreased (F (99) = 15.59, p=.000, Eta2=.17). Between-group comparison showed that participants in the self-help program reported more helpful self-instructions (F(99) = 5.66, p.=.019, Eta2=.05) and fewer unhelpful self-instructions (F (99) = 5.78, p=.018, Eta2=.06) than controls. Post-hoc tests revealed no differences between internet-based and paper-based intervention and the control condition. The results indicate that the self-help manual leads to additional improvement regarding tinnitus distress and enhances copingabilities. To further investigate the long-term effects of the program on tinnitus distress a 6-month follow-up measurement is planned. Fatigue is a symptom common to many chronic illnesses. It is also an unpleasant side-effect of medical interventions such as curative treatment for cancer. Even when fatigue is not one of the key presenting symptoms, it if often described by patients as one of the most disabling and difficult to manage. This symposium will present a series of treatment trials which have looked at cognitive behavioural therapy (CBT) treatment packages for fatigue in three different patient groups, cancer, multiple sclerosis (MS) and chronic fatigue syndrome (CFS). Four key questions will be addressed during the symposium talks and discussion 1. Is CBT an effective treatment for fatigue across conditions? 2. Can CBT for fatigue be offered in a more cost-effective and potentially widely available web-based format? 3. What are the key mediators of CBT in the treatment of fatigue? 4. How can our understanding of mechanisms of change in CBT help maximise our treatment effects? The first talk presents data from a randomised controlled trial of CBT versus a brief nursing intervention for the reduction of fatigue during curative cancer treatment. CBT is shown to be the only effective treatment. Interestingly, increases in physical activity do not mediate the treatment effect. The second study shows that therapist driven CBT is useful for the treatment of fatigue in MS. Mediation analysis suggests that the key mediator of change in fatigue is change in patients' negative beliefs about fatigue. The third study discusses the development of a web-based intervention to treat MS fatigue. Data from a pilot RCT suggests that this approach can substantially reduce fatigue and is economical, but the effect size is smaller than therapist delivered CBT. The final presentation looks at both mediators and predictors of the effectiveness of CBT in a sample of patients undergoing treatment at a specialised fatigue clinic. Catastrophising about symptoms is the key treatment mediator, while extreme behavioural or cognitive avoidance predicts worse treatment outcome. The symposium discussion will focus on the best way to package CBT for different patient groups. How the findings from these talks can help us understand models of fatigue and treatment processes will also be addressed.
Medicine, London, United Kingdom. Background
The majority of patients with multiple sclerosis (MS) report fatigue that is often chronic and interferes with daily life. Although fatigue in MS is poorly understood, cognitions and behaviours in relation to symptoms and fatigue appear to play a role in the maintenance of MS fatigue. An intervention was developed on the basis of a cognitive-behavioural model of MS fatigue. The efficacy of this cognitive behaviour therapy (CBT) was tested in a randomised controlled trial. CBT resulted in a greater reduction in MS fatigue than relaxation training directly following treatment. In this study the data of this RCT were reanalysed to address the following questions: 1) Does CBT lead to significant change in the cognitions and behaviours hypothesised to perpetuate MS fatigue when compared to relaxation therapy? 2) Do any of these cognitive-behavioural variables mediate the effect of CBT on MS fatigue? Method 70 patients were randomised to CBT (n=35) or relaxation training (n=35). Patients completed the Cognitive and Behavioural Responses to Symptoms Questionnaire, the Brief Illness Perception Questionnaire (B-IPQ) which was modified to measure beliefs about fatigue, and the Fatigue Scale, pre-and post-therapy. Multiple mediation analysis using bootstrapping was used to determine which of the variables mediated the change in fatigue brought on by CBT.
There was a significant change in several cognitive variables directly following CBT. Patients focused less on MS symptoms and their interpretation of MS symptoms became less negative (embarrassment, catastrophising and damage beliefs decreased). Also, the overall perception of fatigue, measured by the B-IPQ, became more positive. Mediation analysis showed that the change in fatigue severity in the CBT group was fully mediated by the change in the perception of fatigue. Change in anxiety also co-variated with the reduction in fatigue. Discussion Changing beliefs about fatigue seems an important factor in CBT for MS fatigue. The change in fatigue is also closely related to a reduction in anxiety. The results of the present study are comparable to those investigating the effects of CBT for chronic fatigue in other conditions. Background: Fatigue is a common, distressing and disabling symptom associated with multiple sclerosis (MS). Therapist delivered, manualised cognitive behavioural therapy (CBT) has been shown to be effective in treating MS fatigue. The purpose of this study was to develop and pilot the efficacy and costeffectiveness of a web-based version of this CBT package. Methods: The MS-Invigor8 programme was developed over 9 months by a team of software developers, psychologists and people with MS. 40 patients with MS fatigue were then randomised to the web intervention in conjunction with 3 therapy phone calls (n=23) or a wait list control group (n=17). Patients were assessed using standardised self-report measures of fatigue severity and impact prerandomisation and 10 weeks later. They also completed measures of health care use and quality of life for the economics analysis. Patients in the intervention group were interviewed to see how they had experienced the treatment approach. Results: Intention-to-treat analysis showed that patients randomised to MS-Invigor8 reported significantly greater reductions on the Fatigue Scale (F(3,36) = 16.59, p<.001) and the Modified Fatigue Impact Scale (F(3,34) = 17.15, p<.001) at 10 weeks than those on the wait list. The QALY gain based on the EQ-5D was 0.015 higher in the intervention group (p=0.038), but there were no differences in service SS17b  HOW IMPORTANT IS INCREASING PHYSICAL ACTIVITY AIMING TO REDUCE  FATIGUE DURING CURATIVE CANCER TREATMENT? A RANDOMISED  CONTROLLED TRIAL OF A BRIEF NURSING INTERVENTION AND COGNITIVE  BEHAVIOUR THERAPY The assumption is that physical activity is essential to reduce fatigue. However, the mediating role of physical activity in fatigue interventions during cancer treatment has never been demonstrated.
Our aim was to evaluate two interventions during curative cancer treatment in a RCT and to determine the role of physical activity. Patients (N=220) with various malignancies receiving cancer treatment with curative intent were assessed before (T1) and two months after cancer treatment (T2) and randomized to a brief nursing intervention (BNI), cognitive behaviour therapy (CBT) or usual care (UC). Fatigue was measured with the Checklist Individual Strength, physical activity with actigraphy and questionnaires.
The BNI (2 sessions) focused solely on increasing and maintaining physical activity during cancer treatment. The CBT (6 sessions) focused additionally on regulating the sleep-wake cycle, accepting the consequences of cancer and treatment, managing the experience of cancer in relation with others, and challenging to make future plans. The CBT group was significantly less fatigued after cancer treatment compared to the UC, but no difference was found between BNI and UC (ANCOVA). Mediation analysis showed that none of the physical activity measures mediated the reduction in fatigue realised by CBT. Conclusion: CBT proved to be effective in reducing fatigue during cancer treatment. Physical activity had no mediating role in the effect of CBT. Thus, our findings indicate that with CBT it was possible to realize a significant reduction in fatigue without a lasting increase in physical activity. Funding provided by the Dutch Cancer Society.
use between the groups. The mean number of sessions completed in the intervention group was 5 out of 8. Qualitative data suggested that most patients found the programme and telephone sessions helpful, had made lifestyle modifications to manage their fatigue, felt they were more in control of their fatigue and had greater understanding of this symptom. Modifications were suggested to further improve the programme. Discussion: This pilot study suggests that computerised CBT may be an effective, accessible and acceptable intervention for managing fatigue in MS. The improvements in quality of life and the relatively low cost of the intervention highlight the potential for it to be a costeffective way of offering CBT to this group. Background: Cognitive behaviour therapy (CBT) is an effective treatment for chronic fatigue syndrome (CFS). The process of change in cognitive behaviour therapy for chronic fatigue syndrome (CFS) is potentially complex. Methods: First we examined whether cognitive behavioural responses changed after a course of CBT. Second, we investigated which cognitive and behavioural responses were important in mediating change in the treatment of CFS/ME and examined factors which predicted treatment outcome in a specialist clinic in the UK. Findings: Three hundred and fifty six patients entered the study and commenced CBT. Change in catastrophic thinking mediated change in social adjustment in CFS/ME. Extreme behavioural and cognitive avoidance predicted worse social adjustment and fatigue. Discussion: Implications of these findings for understanding treatment process and mechanisms of change in a range of disorders will be discussed. Investigators in these countries are developing behavioral interventions, grounded in the Preventive Health Model (PHM),that can be used to increase CRC screening. In Australia, the Adelaide Colorectal Cancer Collaborative has developed a web-based, interactive decision support tool that utilizes empirical evidence about the predictors of screening to deliver tailored information designed to improve readiness to screen. In several Canadian provinces, the Institute for Health Research Team is carrying out pilot studies of invitation letter promotions related to stool blood testing and colonoscopy, initiating tailored-navigation intervention research, and developing CRC screening management software useful for multiple cancer screening programs. In the United States at the Kimmel Cancer Center of Thomas Jefferson University, preferencebased tailored navigation methods are being tested in diverse populations served by primary care practices. In each setting, developmental research had has been completed and randomized controlled trials of PHM-based interventions are underway. The symposium will elaborate the theoretical foundation for the international research initiatives; describe tailored navigation interventions and provide data on implementation results; and summarize lessons learned to date. Background: Colorectal cancer (CRC) screening is underutilized. Effective methods to increase screening use are needed. This study sought to determine the impact of tailored navigation on CRC screening in primary care. Methods: The study included 154 primary care practice patients who were 50 or more years of age, were eligible for CRC screening, and had an office visit within two years prior to study initiation. Baseline telephone survey data were collected on participant sociodemographic characteristics, psychosocial factors, and screening test (fecal occult blood test (FOBT) or colonoscopy) decision stage. By comparing decision stage data, we identified that test with the highest decision stage (i.e., preferred screening test). Participants who preferred FOBT were sent an FOBT kit and a reminder. Those preferring colonoscopy were sent colonoscopy instructions. After this mailing, a study patient navigator made a telephone call to guide participants towards screening. Six-month endpoint survey and medical records data were obtained. Univariable and multivariable analyses were performed to identify predictors of screening and of change in preferred screening test decision stage. Results: At endpoint, 63 (41%) study participants had screened. From baseline to endpoint, overall screening preference increased for 75 (63%) participants. Age and perceived salience and coherence (i.e., screening is important and sensible) were positive, significant predictors of screening use (p=0.02 and p=0.05, respectively); while only age predicted change in overall screening preference (p=0.03). In 2007, Ontario (population, 12.9 million) launched a provincewide CRC screening program, ColonCancerCheck, offering FOBT to average risk individuals and colonoscopy to individuals with first degree family members affected by CRC. Also in 2007, the Alberta Colorectal Cancer Screening Program was launched, an initiative recommending all provincial residents be tested annually with FOBT (beginning at age 50) while in 2008, the Forzani and MacPhail Colon Cancer Screening Centre, opened in Calgary ( Alberta) the only non-hospital Canadian colon cancer screening facility. In Canada, only 40% report being up-to-date CRC screening using FOBT or endoscopy, with FOBT compliance especially suboptimal. The Canadian Institute for Health Research Team in Population-based Colorectal Cancer Screening was accordingly funded to investigate CRC screening activities in several provinces and to originate multiple activities, notably focus groups aimed at ascertaining optimal methods for promoting FOBT-CRC and Targeted and Tailored-Navigated Intervention Trials that directly test specific approaches to increasing screening uptake. In this presentation, a range of findings will be shared, as related to regionally-diverse Focus Group Studies, pilot studies of invitation letter promotions on a provincial basis (Ontario) and to individuals awaiting colonoscopies (Alberta); and Tailored-Navigation interventions directed at attendees of a large primary care practice where patient navigators undertake promotion/ information related to screening promotion. Findings will also be shared from efforts in Saskatchewan, Canada to develop CRC screening management software. In 2006, the Australian government implemented the National Bowel Cancer Screening Program. Invitations were sent to people turning 55 and 65 years. They offered the opportunity to screen for Bowel Cancer with the Faecal Immunochemical Test. Participation rate was 39.7%. This less than optimal uptake highlights the size of public health challenge. How do we optimize participation rates utilizing publicly delivered rather than clinician-based interventions?
The Adelaide Colorectal Cancer Collaborative has undertaken trials designed to identify solutions. Results indicate improved participation associated with use of an advance notification letter, reminder letters, medical practitioner endorsement (including personalized letters), the removal of dietary restrictions from the requirements of testing, and minimally "distasteful" sampling technology. Recent behavioral studies have further highlighted the importance of addressing attitudinal and cognitive barriers. These studies indicate that the Australian population is influenced by the way messages are framed, confidence in their capacity to use the kit (self-efficacy), perceived salience and coherence of screening, cancer worries, response efficacy (i.e., belief in the effectiveness of screening), social influences and perceived susceptibility to bowel cancer.
The collaborative has also developed a web-based, interactive decision support tool that utilizes empirical evidence about the predictors of screening to deliver tailored information designed to improve readiness to screen. Pilot data highlight the potential utility of this approach. They document the prevalence of internet use and acceptability in the target age group, establish the validity of the social cognitive variables included in the decision support tool as correlates of Australians' readiness to screen for bowel cancer, and demonstrate within the laboratory, the greater effectiveness of tailored compared with generic messages. Medically unexplained physical symptoms are a major challenge not only for the general health care system, but also for the development of reliable and valid classification approaches and effective interventions. A revision of the classification of somatoform disorders seems necessary, although there is continuing discussion what should be revised in detail. Joel Dimsdale, the speaker of the DSM-V work group, will present the latest DSM-V proposal for the classification of somatic symptoms causing significant psychological distress, and empirical data supporting this new classification approach. Per Fink and colleagues will provide data from Denmark indicating one new approach to tackle diagnostic problems of somatoform disorders, and its empirical validation. New frontiers of interventions are reported by A. Schröder who focus on functional somatic symptoms. The presentation of Arthur Barsky will finally address the different nature of somatic symptoms, and especially the distinction between nonpathological bodily discomfort versus psychopathological features that need to be diagnosed. The symposium will result in a discussion of future options for classification and treatment of this prevalent and costly medical condition. Medically unexplained somatic symptoms may result from many sources other than psychiatric disorder, and somatization therefore is not by definition psychopathological. Such symptoms are a final common pathway for the expression of situational, intrapsychic, behavioral, interpersonal and sociocultural distress. Symptoms without a serious medical cause are a nearly universal human experience, arising in the daily lives of perfectly healthy people. Some are a non-verbal interpersonal communication in which the individual seeks the acknowledgment from others that they are in distress and suffering, hoping thereby to secure special attention, support, and consideration. For some patients, a lifetime of medically unexplained complaints can be best conceptualized as a stable personality trait, one that has been associated with other enduring personality characteristics. Somatization can also be understood as a form of illness and sick role behaviornot something an individual has, but rather something he/she does. Alternatively, emotional distress, particularly when combined with certain early childhood experiences of trauma, adversity or deprivation, can generate somatic symptoms. Preoccupied and fearful attachment styles that are learned in childhood can also lead to somatization in adulthood. The presentation of medically unexplained symptoms is also subject to socio-cultural influences. Cultural norms and values encourage the expression of distress in certain forms and discourage it in others; these cultural forces shape the experience and reporting of bodily symptoms by influencing how they are identified, interpreted, described and reported. Finally, the search for neurobiological mechanisms of somatization is under way, implicating the hyperresponsivity of centrally acting proinflammatory cytokines, dysregulation of central and peripheral serotonergic systems, and dysregulation of the hypothalamic-pituitary-adrenal axis. It is only when these medically unexplained symptoms are recurrent, especially distressing, severe, prolonged, and disabling that they may be considered psychopathological. Background: Functional somatic syndromes (FSS) such as fibromyalgia or irritable bowel syndrome are highly prevalent and may be persistent and disabling for patients and costly to services. We assessed the efficacy of a cognitive behavioral group treatment (STreSS) for patients with a variety of severe FSS in improving physical health. Methods: Patients aged 20-45 year with chronic, disabling FSS or somatoform disorders who met criteria for the unifying diagnosis of severe, multi-organ Bodily distress syndrome were eligible for this randomized trial at a General University Hospital. 120 patients were randomly assigned by means of a computer-assisted block protocol to a cognitive behavioral group treatment (54 patients) or to enhanced usual medical care (66 patients). The primary outcome was the difference in change from baseline to 16 months in the SF-36 Perceived Physical Health (PPH) score between the two groups. Analysis was by intention to treat. Results: 44 patients in the STreSS group and 50 in the comparison group completed follow-up at 16 months. By use of repeated measures mixed effects modeling and multiple imputation, all 120 participants were included in the analysis. The adjusted mean difference in PPH score change from baseline to 16 months was 4.1 (95 % CI 1.5-6.7, p=0.002), a moderate effect size (0.51; [0.19-0.83]). The number needed to treat to achieve one additional treatment response was 5 (3-53, p=0.03). Conclusion: Specialized Treatment for Severe Bodily Distress Syndromes (STreSS) provides a promising model for the management of patients with various severe FSS. Our approach may be a feasible alternative to the current organization of care in many different medical specialties.
basing the diagnosis on negative rather than positive manifestations. Patients regard the terminology as offensive. It is difficult to understand the prevalence and treatment of such disorders as long as a hierarchical decision model prevents diagnosing these disorders when other psychiatric disorders are present. The workgroup feels that the hallmark of such disorders is the presence of somatic symptoms associated with significant distress and/or dysfunction. These disorders typically present first in nonpsychiatric settings. Somatic symptoms are common in every day life & may be initiated, exacerbated or maintained by combinations of biological, psychological and social factors. Somatic symptom disorders can accompany diverse general medical as well as psychiatric diagnoses. When criteria are fulfilled, for example, for major affective disorder and for complex somatic symptom disorder, both diagnoses should be coded.
Major changes are summarized below: 1. move "Psychological Factors Affecting a Medical Condition" from "other factors that may be of interest." 2. introduce a new diagnosis "Complex Somatic Symptom Disorder." This disorder is characterized by multiple somatic symptoms, as well as misattributions, excessive concern or preoccupation with symptoms & illness. The workgroup feels that this rubric accurately describes disorders such as somatization disorder, hypochondriasis, undifferentiated somatoform disorder, and pain disorder. 3. The workgroup proposes changes to criteria for conversion disorders. Most notably, we recommend removing the requirement for psychological factors to be associated with symptom onset. This matter is difficult to establish. There is currently discussion regarding whether conversion disorder is most usefully aligned with somatic symptom disorders or anxiety disorders. Background: Chlamydia is a common sexually transmitted infection and is often asymptomatic, so early treatment can be difficult. The National Chlamydia Screening Programme aimed to increase screening of sexually active 15-24 year olds. Half of PCT's are under-performing against the Department of Health's target for all Primary Care Trusts (PCTs) to screen 18% of this population. Therefore there is a need to improve opportunistic screening in GP practices. As part of a wider project to develop an intervention this study assesses individual staff attitudes and intentions to screening, and informs the content of the intervention. Method: A theory based survey was developed from previous qualitative and quantitative data from practice staff. The survey was administered to clinical and non-clinical staff in GP surgeries; of which 466 had been returned Findings: Intention to offer Chlamydia screening was high (mean 5.50, SD=1.07). Subjective norms and PBC scores were also high (mean 6.01, SD=1.16; mean 6.24, SD=.82 respectively), with attitude similarly positive (5.97 (SD=.75). Linear regression analysis showed that intention significantly predicted approximately 13% variation in behaviour in clinical staff (F=34.708, AdjR2=.131, p<.001) but was insignificant for non-clinical staff. Attitude and subjective norms were the most significant predictors of intention (p<.005) whilst PBC did not significantly predict intention. Discussion: Intention to offer screens is directly related to actual behaviour, and those who have more positive attitudes and perceive their colleagues to be more approving are more likely to offer screens. The lack of predictive power with non-clinical staff may reflect their view that offering information is beyond their role, and thus it is important to raise awareness of the value of doing so. The challenge of increasing numbers of elderly in need of mental health services in the developing world will not be adequately met without effective undergraduate education for all health professionals caring for elderly patients. There is a need for continuing professional training to keep the skills, attitudes and performance of care providers appropriate and update for recognizing and treating old age mental health disorders. There is also a need for more research on effective training approaches. Keeping this in mind, the present study sought to investigate the level of psychogeriatric knowledge and attitudes towards care of the elderly among primary care personnel of Orissa, India and an assessment of psycho-geriatric training needs in primary health care.
The specific aims of the study were to explore the present knowledge of geriatric psychiatry among primary care physicians, S232 Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329 nurses and social workers and compare it with that of psychiatry residents and nurses and to explore the attitudes of the same groups toward the elderly. It was expected that the study results would provide relevant inputs towards developing continuing professional education methods for primary care professionals and to enable them to care for old age mental problems The participants of this study were primary care health workers, nurses and physicians. The reference group for comparison comprised psychiatry residents and nurses. All were administered knowledge and attitude questionnaire. Background questionnaire covering previous psycho-geriatric education, work experience and need for further training was collected and the data were assessed as group mean comparisons. Psycho-geriatric knowledge was measured by a 40-item knowledge test on psychopharmacology, clinical syndromes, assessment, old age and psychotherapy. The attitudes were measured by a 30-item Likert-type questionnaire. The geriatric psychiatry knowledge level and the reported attitudes toward care of the elderly of the primary care groups were significantly lower than the reference group. The discrepancy was attributed to lack of adequate training and undergraduate education.
Most of the participants felt that they could benefit from suitable tailor-made continuous multi-professional education. The focus areas for training were clinical assessment and psychopharmacology. The multi professional approach was favored in order to optimize the benefits of care for elderly patients with multiple problems. There is a need for continuing professional education in old age mental health for primary care personnel and its incorporation into extant primary health care. Resources provided by guidelines and modern technology should be available for professionals working with elderly. Little is known about the impact of self-efficacy (SE) in changing specific diabetes self-management (DSM) behaviors on patient adherence to a DSM regimen, compared to general SE in DSM. We used data from the American Association of Diabetes Educators Outcome System, implemented in eight DSM education programs in western Pennsylvania, to examine the bivariate relationship between adherence to the DSM regimen and SE in changing specific behaviors and general SE in DSM among patients with type 2 diabetes. DSM regimen adherence was measured as the frequency with which patients followed their recommended DSM regimen, including medication taking, diet, exercise, and self-monitoring of blood glucose [SMBG] ). Behavior-specific SE was measured by asking patients their level of confidence in making changes in these four DSM behaviors. Higher scores indicated greater adherence or confidence. General SE in DSM was measured by asking "How sure are you that you can manage your diabetes?" with four responses (A lot/ some/ a little/ not at all). We used correlation and simple linear regression to analyze the data. The sample (N=709) was 88.3% white and 53.8% female, with a mean age of 58.2 years and an average of 7.9 years since diagnosis of diabetes. We observed a stronger relationship with SE in changing diet (r =.29, p<.001) and exercise (r=.21, p<.05) and the corresponding adherence behaviors than we found between DSM self-efficacy and adherence to diet (r=.14, p<.001) and exercise (r=.10, p<.05). These associations remained significant after controlling for age, gender, ethnicity, education, family history, and time since diagnosis. Medication adherence was weakly correlated with SE in DSM (r=.09, p=.02) but not medication-specific SE (r=.25, p=.08). No associations were found between general or specific SE measures and SMBG (ps>.05). Further evidence is needed to confirm our results in a research setting. Our findings suggest that promoting patients' confidence in managing their diabetes, especially with respect to making changes in diet and exercise could improve patient adherence to their recommended diabetes self-management regimen. Background: Training in self management support (SMS) for long term conditions (LTC) were offered to 367 clinicians as a part of The Health Foundation's Co Creating Health Initiative. We applied the Self Determination Theory to explain how SMS behaviours are formed and sustained. Aims: Explore the relationship between motivational regulation to support self management and clinicians' practices in SMS. Method: We obtained 78 responses . To assess the use of SMS practices we applied the Practices in Self Management Support questionnaire (PSMS) developed by the authors. The questionnaire comprises 3 subscales: Clinical Self Management Support (CSMS), Patient Centeredness (PC) and Organization of Services to Support Self Management (OSMS). We adapted the Self-Regulation Background and purpose: Stress-related illness with psychosocial antecedents has increased globally. Patients seeking help in primary care often exhibit a wide variety of problems with both somatic and psychological symptoms. The role of primary care has also changed in relation to psychiatry in that it now constitutes the first-order treatment for psychiatric disorders. A biopsychosocial perspective, increased knowledge of behavioural medicine and improved inter-professional collaboration all offer a viable response to this situation. Inter-professional courses at the advanced undergraduate level provide opportunities for interprofessional learning, a learning about, and with one another. At Karolinska Institutet, a cross-program elective course in applied behavioural medicine has been offered since the fall of 2006, first for medical and psychology students, and since 2008 also for physiotherapy students. This presentation describes the course structure and presents evaluation results from the five-week interprofessional course between 2006 and 2009. Results and discussion: In 2006 and 2007 the course was held with medicine and psychology students; in 2008, only physiother-apy and clinical psychology students were recruited. Medical students did not apply due to new program changes. All three groups have been very satisfied with the course, experiencing it as an important complement to their undergraduate education. It has been rewarding to train themselves to communicate and interact around the patient's problems from each separate perspective. Individual professional roles have become clearer, and all indicate their conviction of the need for inter-professional collaboration. Evaluation of attitudes toward other professional groups showed that only psychology students changed their perceptions of physicians and physiotherapists during the course. Conclusion: Inter-professional courses at the advanced undergraduate level offer a useful meeting between the different professions and can therefore be an important step in applying bio-psychosocial model to work in practice. Results will also be presented from the 2009-10 courses where registered and professionally active physiotherapists, doctors and psychologists have also been recruited as course participants alongside the undergraduate students. Disadvantaged children and adolescents need more supportive care, and most studies show that parenting plays a significant role in explaining mental health among them. This research is to evaluate connections between supportive behavior and the need of mental care for disadvantaged boys and girls in Taiwan. By collecting the data of thirty ordinary families and thirty disadvantaged families, parenting and psychological conditions of children are categorized and compared. Major parenting behavior produces results similar to or lower in quality than those acquired from ordinary families; however, according to the analysis of scale, disadvantaged boys and girls face more conflicts and difficulties in mental condition. Even though parenting affects the mental health of young kids, it seems that other factors must be considered. Findings provide empirical support for further research and practice in supportive parenting. Additionally, the attitude of children and adolescents on feeling supportive behavior is also a key aspect to understand their mental health. Introduction: A child's HSCT is distressing for both parents and children. The child experiences side effects to treatment, infections, Graft versus Host Disease under isolation conditions. A psychosocial intervention study aimed at reducing parents and child's level of distress identified a group of parents with an immediate, extended need for psychosocial intervention.
Patients: Parents to 25 HSCT children were included in a psychosocial intervention study during the child's admission.
The intervention included daily psychosocial support. Methods: Case study combined interviews and observational study.
Results: Parents to 8 of the 25 children had complex psychosocial problems which impaired their ability to support and care for their child during hospitalisation and after discharge. The parent's were characterised by: 1) low level income 2) low level education 3) low level support 4) marginal relationship to the work market 5) physical illness 6) psychiatric illness 7) nonwestern ethnicity and 8) Danish as a second language. Parents of the 8 children had 4 or more of the 8 characteristics, which place them at risk of social exclusion. These parents needed individual interventions such as 1) help to understand the child's HSCT treatment 2) the child's reactions 3) extended emotional support 4) help to facilitate communicate with the welfare system 5) additional economical support and 5) additional help to care for the child. Conclusion: Parents' prior psychosocial situation in conjunction with the strain of caring for a HSCT child exceeds the parent's resources, being the reason why psychosocial interventions are required so the child's health is not placed at an additional risk. Parents of HSCT children at risk of social exclusion experience difficulties with manoeuvring within the welfare-based system, which increases their risk of social exclusion.
Psychosocial screening and interventions to parents of HSCT children is recommended. Parents reported additional parenting practices, education level and employment status. Logistic regression was used in analyses. Results: Reporting simultaneously high amount of parenting practices and parental care compared to few practices and no parental care was among children associated with a higher probability of frequent nutrient-dense food intake, having a regular meal pattern, sleeping at least 10 hours per night, and having less than 2 hours daily screen time. The odds ratio for less screen time was highest when reporting a high amount of practices and no parental care. The combination few practices and parental care increased the odds for higher frequent intake of nutrient-dense food. No associations were found between parenting practices, parental care and intake of energy-rich food. Conclusion: A high amount of parenting practices concerning daily routines and simultaneously parental care seem to be associated with more favorable health behaviours in children. Background: A higher prevalence of disruptive behavior is found in boys than in girls. The causes and pathways to these problems, such as socio-economic deprivation and maternal depression, remain unclear, particularly in relation to potentially different pathways by gender and at younger ages. The objective of this paper is to examine the relationship between maternal depression and disruptive behavior and potential pathways via socio-economic circumstance and parenting behaviours in boys and girls in early childhood. Data used were from the UK Millennium Cohort Study (MCS) made up of 18,552 families recruited between 2000-2001 when the cohort children were aged 9 months old, with follow ups at ages 3 years old (n=15,590) and 5 years old (n=15246). Findings: At aged 3 12% of boys and 10% of girls had a high conduct score; whilst 11% of boys and 7% of girls had a high hyperactivity score. Boys with a depressed mother had a 4.5 times higher odds (unadjusted) of having a high conduct score (p<0.0001) and a two and half times higher odds (unadjusted) of a high hyperactivity score (p<0.0001) compared with boys whose mothers were not depressed. Girls with a depressed mother had a 4 times higher odds (unadjusted) of a high conduct score (p <0.0001) and a three-fold higher odds (unadjusted) of a high hyperactivity score (p<0.0001) compared with girls whose mothers were not depressed. The relationship was tested using logistic regression models controlling for child and parent characteristics, socio-economic circumstance and parenting behaviors. The odds for both boys and girls of depressed mother having a high conduct score remained significantly higher compared to boys and girls of non-depressed mothers but were attenuated to two-fold higher odds. The odds of boys with a depressed mother having a high hyperactivity score remained significantly higher compared to boys of nondepressed mothers but decreased to a one and half times higher odds, whilst for girls the relationship was no longer significant. Conclusion: The relationship between maternal depression and a high conduct and hyperactivity score in 3 year old boys and a high conduct score in girls is not wholly accounted for by socio-economic circumstance or parenting behaviors and therefore other mechanisms are likely to be additionally important. Background: Mothers of young children are increasingly combining paid work with childrearing. Empirical evidence on the effects of maternal employment on children is contradictory and has little work has considered the impact of maternal employment within the context of the employment patterns of both parents.
Objectives and methods: This study examines the effects of parental employment in the early years on child socio-emotional behavior at age 5 in a recent birth cohort study in the United Kingdom. Participants of the Millennium Cohort Study were born between September 2000 and January 2002 (n=18,819). Data on parental employment across three sweeps of data collection (infancy, age 3 and age 5) were used to investigate: (i) whether children whose mothers were in paid work during their first five years were more likely than children whose mothers were at home full-time to display adverse behavioral symptoms at age 5, independent of maternal education, mental health or economic position; (ii) whether effects of maternal employment on child socio-emotional development were cumulative in nature, or whether children were more sensitive to the effects of maternal employment during their first year; and (iii) the effects of different types of parental work arrangements on child socio-emotional behavior at age 5. Child socio-emotional behavior was measured using the Strengths and Difficulties Questionnaire with clinically relevant cut-points for problem behaviors. Results: No evidence of detrimental effects of maternal employment in the early years on subsequent child socio-emotional behavior was seen. There were significant gender differences in the effects of parental work arrangements on behavioral outcomes. Girls whose mothers were not in paid work during their first five years were 77% (95% CI= 1.21-2.57) more likely to have behavioral difficulties at age 5 than girls whose mothers were in paid work throughout their early years, independent of maternal characteristics and household income. For boys this was not the case, but boys in two-parent households in which their father was not in paid work for at least one period during their first five years were at an increased risk for behavioral problems at age 5. The most beneficial working arrangement for both girls and boys was that in which both mothers and fathers were present in the household and in paid work, independent of parental educational attainment and household income. The purpose of this study is to develop a cognitive-behavior group intervention program to reduce the hostility levels of coronary heart disease (CHD) patients and to examine its effect on blood coagulation function. A case control study with matched age, sex and education, based on a two group pre-and post-test design was adopted. Participants of the experimental group were 9 CHD patients (mean age=56.33±10.45, male 67%) who attended a weekly two-and-a-half hour session and did homework for two months. The intervention foci included psycho-education, biofeedback-assistant relaxation training, and cognitive-behavior strategies to reduce the levels of hostility affect, hostility cognition, and hostility behavior. Participants of the control group were 13 CHD patients (mean age=59.23±6.80, male 69%) who did not receive any psychological treatment until they finished the three-month waiting status. After intervention, participants showed a significant reduction in hostility level (t=4.27, p<.01) and respiration rate (t=3.06, p<.01), and a significant increase in prothrombin time (t=−4.75, p<.001). The results of the repeated measure of ANOVA indicated that the experimental group revealed a significantly higher reduction of respiration rate (F= 8.76, p<.01), and a marginally higher increase of prothrombin time (F=−3.66, p=.06) than the waiting control group. In conclusion, the results of this study imply that psychological intervention is efficient to reduce the biological risk of disease recurrence for CHD patients. Complementary therapies are popular and frequently used by patients with CVD. Survey data indicate that herbal therapies and mind-body therapies are most commonly used. One such treatment option is qigong. Several reviews have claimed that qigong has therapeutic effects on blood pressure in patients with hypertension. However, these reviews are non-systematic and therefore open to bias. To systematically assess the clinical evidence of qigong for hypertension. Databases were searched up to December 2009. All randomised clinical trials testing qigong in patients with hypertension of any origin and assessing clinically relevant outcomes were considered. Trials using any type of control intervention were included. The selection of studies, data extraction and quality assessment were performed independently by at least two reviewers. Methodological quality was evaluated using the Jadad score. 12 RCTs could be included. Seven of these RCTs tested qigong in combination with antihypertensive drugs compared with antihypertensive drugs alone. The meta-analysis of four trials reporting adequate data suggested beneficial effects in favour of qigong (weighted mean difference, systolic blood pressure (mmHg) -12.06, 95% confidence interval (CI) -17.12 to -7.00; diastolic blood pressure -8.46, 95% CI -12.55 to -4.37). Qigong alone was compared with waiting list control in 2 RCTs and was found to significantly reduce systolic blood pressure (weighted mean difference (mmHg) -18.46, 95% CI -23.07 to -13.86). In further three RCTs comparisons made were: qigong combined with antihypertensive drugs versus muscle relaxation combined with antihypertensive drugs; qigong as a sole treatment versus exercise and/or antihypertensive drugs, all reported positive results in at least some of the relevant outcome measures. The methodological quality of the original studies was low. There is some encouraging evidence to suggest that qigong is effective for lowering systolic blood pressure. However, the conclusiveness of these findings is limited. Rigorously designed trials seem warranted to confirm these results. Background: One in five people suffer from depression after myocardial infarction (MI) or heart attack. Depression impedes important indicators of recovery including Quality of Life (QOL) and employment outcomes. Yet depression continues to remain under-recognised and under-treated in cardiac patients. We are conducting a randomised controlled trial of a state-of-the-art, telephone-delivered counselling and educational program (MOOD-CARE) for MI patients diagnosed with depression and investigating its effectiveness in relation to mental and physical functioning and employment outcomes. Method: Newly diagnosed MI patients admitted to five hospitals in Australia are being screened for depression using the Patient Health Questionnaire (PHQ-9) (n=1619). Those with PHQ9=5-19 (mild-moderately severe depression) at discharge who are still symptomatic 2 weeks later will be randomly assigned to a Usual Care (UC) or Intervention (MOOD-CARE) group, stratified by current DSM-IV diagnosis of depression versus subthreshold depression. MOOD-CARE includes up to 10 telephone counselling sessions over 6 months, delivered by clinical psychologists using Cognitive Behaviour Therapy (CBT) and coronary heart disease (CHD) risk factor management.
Results: Presentation will describe study methods, baseline recruitment and related findings. Conclusion: MOOD-CARE has the potential to improve psychosocial, vocational and health outcomes of depressed MI patients. The study will provide valuable clinical, vocational and economic information about the value of the program and its potential translation into the health system.
Colombia, it is the third leading cause of cardiovascular death. Cardiac rehabilitation programs aimed at individuals with CHF are designed to decrease morbidity and mortality and improve quality of life (QOL). Little data is available on the impact of cardiac rehabilitation programs on QOL in CHF patients in Colombia. The SF-36, a self-report instrument validated in Spanish with 8 subscales and a question regarding Health Expectations, was used to measure QOL in 35 CHF patients participating in a cardiac rehabilitation program at St. Vincent de Paul Hospital in Medellín, Colombia at baseline, 3-month, and 6-month follow-ups. The rehabilitation program consisted of 12-session treatment protocol including exercise, occupational therapy, and psycho-education. One-way repeated measures ANOVAs were conducted for each QOL sub-scale. Results indicated that CHF patients participating in cardiac rehabilitation significantly improved their Physical Performance sub-scale scores and Health Expectations score over time (p<0.001 and p<0.01 respectively). Cardiac rehabilitation CHF patients also tended to have improved General Health subscale scores and Physical Functioning sub-scale scores over time (p=0.05 and p=0.09 respectively). Scores on the remaining SF-36 sub-scales (Pain, Emotional Performance, Social Functioning, Mental Health, and Vitality) did not vary over time (p = NS). A cardiac rehabilitation directed at CHF patients appears to improve specific physical health-related domains of QOL, although future research involving a control group would strengthen these Results:
The findings suggest the need for psychologists, nurses, and other health professionals to integrate psychosocial aspects into existing cardiac rehabilitation program in order to potentially improve other domains of QOL in Colombian patients with CHF. Delivery of the regular and long-term patient support required to improve diabetes self-management remains a challenge for health systems. This study is evaluating the health outcomes and costeffectiveness of a program using an automated interactive telephone system, the Diabetes Telephone Linked Care (TLC) Australia, designed to promote and support behaviour change and disease management.
Participants (n=120) were adults from Brisbane, Australia, aged 18 to 70, diagnosed with type 2 diabetes at least 3 months prior, with sub-optimal glycemic control (Haemoglobin A1C (HbA1c) ≥ 7.5%). Participants (79 males, mean age=57.47 years, SD= 8.26) were randomised to a usual care group (n=60) or to the intervention group. For 6 months, participants in the latter group upload their past week's blood glucose levels to the TLC system's database via a cell phone link prior to calling the system weekly to "converse" on one or more of the following topics: blood glucose monitoring, nutrition, physical activity and medication taking. Primary outcomes are HbA1C and quality of life at 6-and 12-month follow-up. Secondary outcomes include self-care behaviours and measures of depression and social support. Baseline HbA1C ranges from 7.5 to 15.0% (M=8.8, SD=1.4). Participants' average BMI is 33.62 (SD=6.89); 56.7% meet the Australian physical activity recommendations; 82.5% and 65.8% have a normal score on the HADS depression and anxiety scales respectively. The average duration of a call to the TLC system is 10.77 (SD=1.60) minutes and to date participants have made on average 81.7% of their weekly calls. The presentation will include 6-month follow-up Results: This study is examining the effectiveness of a telehealth program targeting essential diabetes self-care behaviours and holds potential for addressing barriers faced in the delivery of long-term diabetes self-management support. Background: Interventions involving peer support offer a promising approach for providing diabetes self-management support (DSMS) that is ongoing, culturally responsive, and low cost. Purpose: The objectives of this study were (1) to pilot a program training peer leaders (PLs) to facilitate ongoing, empowermentbased, DSMS interventions, (2) to determine the feasibility of graduating PLs who achieve the pre-established competency criteria, and (3) to assess program satisfaction. Methods: The Peer Leader Training (PLT) pilot program recruited eight African-American adults. The 12-week program utilized a wide variety of instructional methods and addressed three major components including self-management education, communication skills, and facilitation skills. To graduate, participants were required to meet the pre-established criteria for four competency domains including diabetes-related knowledge, active listening skills, empowerment-based facilitation skills, and self-efficacy. Participants were given three attempts to pass all four domains. We also assessed program satisfaction and perceived efficacy of training using qualitative (i.e., focus group) and quantitative approaches. Results: All participants achieved the criteria for all four competency domains. On the first attempt 75%, 75%, 63%, and 75% passed the diabetes knowledge, empowerment-based facilitation skills, active listening skills, and self-efficacy, respectively. Participants who failed at least one competency domain on the first attempt successfully passed on the second attempt. Participants were generally satisfied with the program length, training session length, balance between content and skills development, and preparation for leading group and individual-based support activities. Conclusion: Findings suggest that it is feasible to train and to graduate PLs with the diabetes-related knowledge, communication skills, and facilitation skills needed to lead ongoing DSMS interventions. Subsequent research should examine the impact of this peer-led DSMS model on improving patients' diabetes-related health outcomes. Background: Obesity continues to present a major public health concern (NHS, 2008). This study aimed to explore the relationship between body mass index (BMI) and the affective states: happiness, anxiety and depression, alongside self efficacy and eating behaviours. It further aimed to identify if these components influence overeating behaviour, a factor that is empirically linked to a high BMI (Macht, 2008). Method: Data was collected from 104 adults (76% female), mean age 38.48 (SD=12.47) years, who completed questionnaires which measured happiness, depression, anxiety, generalised self efficacy, dietary restraint, emotional eating and binge eating. Height and weight were also taken to calculate BMI, which ranged from 16.41-44.38 with a mean BMI of 26.92 (SD=5.98). Results: Findings confirm that there are significant relationships between personal control, affective states, eating behaviours and BMI. Generalised self efficacy and happiness were revealed as predominant psychological factors, correlating with all variables (p<.001). Controlling for age and gender, generalised self efficacy explained the variance in emotional eating by 16%, binge eating by 12% and BMI by 19%. Dietary restraint accounted for 66% of the variance in binge eating behaviour and was also the highest predictor of BMI (R 2 =.45), with depression explaining a further 9%. Finally, investigating the influence of emotional eating, negative affect (R 2 =.32) and dietary restraint (R 2 =.07) were revealed to be the most significant predictors. Conclusion: Evidence presented here highlights the importance for behavioural medicine to consider the influence of self efficacy and affect when tailoring weight loss interventions, while also exercising caution over the negative influence of dieting behaviour. International Health Institute, Brown University, Providence, RI.
Overview: The global challenge of translational behavioral research requires acute attention to the cultural context into which interventions are translated and awareness of the culturallyspecific needs of targeted patients. Formative qualitative research is often used to translate interventions developed for one population to another. We review the role of qualitative methods in "Diabetes Care in American Samoa" a clinical trial of a CHWdelivered intervention. Methods: Formative research included 14 individual interviews (IDI) with medical clinic staff, 6 focus groups (FG) with 39 diabetes patients and 10 cognitive interviews (CI), also with diabetes patients. IDIs and FGs inquired on barriers or facilitators of diabetes care and sought input on planned intervention components. CIs explored the effectiveness and cultural relevancy of a subset of quantitative survey items. NVivo software was used for data management; relevant themes were identified and applied to the Precede-Proceed model to adapt intervention materials. Results: Analysis identified barriers to diabetes self care including preferences for high fat foods, consumption of large quantities at social gatherings, and social expectations to eat large amounts. Facilitators included extended family networks, respect for doctors, and strong religious faith used in support of self care. CIs confirmed linguistic translations, with accommodations for relevant cultural examples within items. The research practice of identifying barriers and facilitators of healthy behavior does not view those cultural elements the way participants do. Therefore, we have also sought to understand how participants see these same behaviors, including their variability. Conclusions: Identifying the cultural meaning of self care-related behaviors is important to designing interventions to address them; qualitative methods are extremely valuable for identifying key cultural features on which to adapt intervention delivery models, materials and measures. This paper uses longitudinal data from the International Tobacco Control Policy Evaluation (ITC) China Survey to examine prospective predictors of smoking cessation among adult smokers in China, and compares them with those found in previous research in two Southeast Asian countries (Malaysia and Thailand) and four Western countries (Australia, Canada, the UK and USA) that are also part of the broader ITC project. In April 2006 a total of 4732 adult smokers were first surveyed in Beijing and other 5 cities in China. Of these, 3863 were successfully followed up in the second Wave in late 2007 (with a retention rate of 81.6%). Baseline measures of sociodemographics, dependence and interest in quitting were used prospectively to predict both making quit attempts and quit success.
Overall, 979 out of the 3863 (25.3%) Chinese smokers reported having made at least one quit attempt between Waves 1 and 2; of these, 212 (21.7%) were still stopped at Wave 2. Independent predictors of making quit attempts included having higher quitting self-efficacy, previous quit attempts, some intention to quit, disagreeing that s/he enjoyed smoking too much to quit, and having very negative opinion of smoking. Independent predictors of quit success among those who attempted were having longer previous abstinence from smoking (7 months Background: This study represents the first longitudinal analysis effort identifying developmental trajectories of cigarette use as well as risk factors associated with the distinct developmental courses of smoking in Chinese early adolescents from age 12 to 16. Methods: Analysis was conducted with secondary data from a longitudinal, prospective cohort, which consisted of 3,521 Chinese adolescents randomly selected from 4 rural and 7 urban middle schools in Wuhan, China. A group-based growth mixture modeling approach was adopted to identify the developmental trajectories of cigarette use. Multi-layered intrapersonal (e.g. attitudes toward smoking, grade point average, subjective academic performance, school bonding, and depressive symptoms) and inter-personal (e.g. parental smoking, perceived parental disapproval of smoking, parent-child relationships, family disharmony, perceived norms of peer smoking, good friend smoking, troubles with teachers) risk factors selected from an ecological perspective were prospectively linked to the identified patterns of smoking trajectory. Results: Three trajectory patterns were identified from the whole cohort: non/experimental smokers (53.8%), light/occasional smokers (43.9%), and heavy/regular smokers (2.3%). After adjustments for gender, urban residence, and family socioeconomic status, adolescents with higher levels of problems in parent-child relationships and family disharmony, higher perceived norms of peer smoking, higher proportion of good friend smoking, having more troubles with teachers, poorer academic performance and reporting more frequent depressive symptoms were significantly more likely to be in the trajectory groups of either light/occasional smokers or heavy/regular smokers than in the group of non/experimental smokers. The probability of being in the heavy/regular smoking trajectory group was positively and significantly related to parental smoking and lack of school bonding. Conclusions: Study findings helped advance knowledge on the distinct developmental courses of smoking behavior and their associations with multi-layered risk factors in Chinese early adolescents. more), and having greater interest in quitting (planning to quit within 1 month). Compared to other countries fewer adult smokers in China attempt to quit and predictors vary: with measure of nicotine dependence less predictive and, like in Malaysia and Thailand, interest in quitting more predictive of success. These findings indicate that existing knowledge from Western countries about smoking cessation are not necessarily readily generalizable to China, which has different social-economic conditions and tobacco control environment. Separation of the "expected" from the "true" drug effect in placebocontrolled drug trials can be achieved by informing prior to testing half of the subjects in each group (drug, placebo) correctly that they receive the drug or placebo, while the others are misinformed (deception) about the drug they received; this is called the "balanced placebo design" (BPD). A half-BPD provides no drug but only placebo. Methods: Sixty-four healthy subjects (32:32 males:females, 24 years) balanced for smokers and never-smokers were made to believe that a chewing gum they received may or may not contain 4 mg nicotine to improve vigilance and reaction time, while in fact all received placebo. The experimenter conducting the study was also made to believe this was a fully BPD to maintain double-blindness. Immediately prior to neurocognitive function testing (N-back tasks with three levels of difficulties and two consecutive runs) they were informed that they belong to the nicotine group or to the placebo group. Dependent measures were reaction time (RT, ms) and number of false go (GO) and no-go (NGO) responses. Results: A significant (p=.028) interaction of all five factors (within: level of difficulty, runs; between: information, smoking status, gender) and age as covariate was found indicating that the information to receive nicotine shortened RT in male, but not in female smokers, while in nonsmokers, the information prolonged RT, specifically in males. The placebo information produced no differences in RT in smokers/ nonsmokers and in males/females. The differential effects on RT were more pronounced with a high cognitive demand, and during the second run. No effect of any of the factors was found for the number of GO and NGO errors. Conclusion: The known effects of nicotine on RT can be mimicked by the belief to receive nicotine, but males are more prone then females to respond to such information. Tobacco dependence is a leading cause of increased morbidity and mortality and current tobacco smoking is estimated will cause about 450 million deaths worldwide during the next 50 years. Tobacco dependence has also dramatically impacted Asian countries and subsequently Asian Americans who migrated to the United States. Korean male immigrants in the US have been known for the highest smoking prevalence and highest smoking-attributable cancer death fractions. The study is designed to test the applicability of Theory of Planned Behavior to Korean Americans' smoking cessation. Its main hypothesis is that perceived risks and benefits of quitting, social norms for quitting, self-efficacy in smoking cessation, and nicotine dependence will predict behavioral intentions to quit smoking among Korean American smokers. A nationwide sample of Korean American smokers is currently recruited into this one-time telephone survey. Subjects are randomly selected from the online website InfoSpace.com by their Korean surname such as "Kim", "Lee", "Choi", etc. The sample size of each city was weighted by the Korean population in the 2000 U.S. Census data. Three cities such as Duluth, GA that lately have the large influx of Korean Americans were added to the list of cities. Preliminary findings with 181 subjects (150 males and 31 females) revealed that gender moderates the relationships between perceived risks of quitting and behavioral intentions to quit smoking in Korean American smokers. Perceived family social norm for quitting was the only significant factor associated with Korean male smokers' behavioral intentions to quit smoking, whereas, perceived risks of quitting, and perceived family and friend social norms for quitting were the factors associated with Korean female smokers' behavioral intentions to quit smoking. However, perceived risks of quitting showed the strongest relationship. Data collection will be continued till May 31, 2010 and final results will be presented. Background: There are over 100,000 people in the USA and 10,000 in the UK awaiting organ transplants. Over 90% of the general population state that they are in favour of organ donation, however only 26% of the UK population are registered on the organ donor register. There is an urgent need to identify barriers, and find ways of increasing the number of organ donors.
Objectives: This research tested the role of emotional barriers to organ donation such as the "ick factor" (a basic disgust reaction to The negative reinforcement explanation for smoking comprises a set of ideas derived from popular self-help books which aim to change the way smokers perceive the benefits of smoking and the post-quit withdrawal discomfort. Acceptance of these ideas is hypothesised to facilitate smoking cessation, possibly through a reduction in post-cessation urges to smoke. Aims To assess the impact of a cognitive intervention aimed at communicating the negative reinforcement explanation for smoking upon participants' reported urges to smoke measured at one-week post cessation. Design A two group, cluster-randomised controlled trial (ISRCTN: 65854845) was conducted in which groups of smokers received either the experimental intervention, comprised primarily of a 20 minute presentation accompanied by group discussion and a self-monitoring task, or a control intervention which involved watching a video on the health risks of smoking. Both interventions were offered in addition to 'standard care'. The primary outcome was participants reported urges to smoke at one week post-cessation. Potential cognitive mediators assessed were participants' levels of acceptance of the negative reinforcement explanation for smoking, positive outcome expectations for the affective benefits of smoking and self-efficacy. Participants (n=205) were volunteers drawn from smokers attending for treatment at a specialist smoking cessation clinic (SSCC).
The intervention failed to have a significant effect on any of the proposed cognitive mediators. There was also no significant difference between the groups on post-cessation urges to smoke (Adjusted expt. group mean = 2.78, Control group mean =3.00, (F(1,100) = 1.17, p=.28).
The experimental intervention based on a set of ideas commonly communicated in popular selfhelp titles, failed to alter smokers' cognitions or lead to a reduction in post-cessation urges to smoke. The intervention may have lacked sufficient power to bring about the intended cognitive shift or these highly conditioned beliefs about smoking may be resistant to change by socialcognitive means alone. Implications for smoking cessation treatments will be discussed.
the idea of organ donation). In addition we tested the potential role of manipulating anticipated regret to increase intention to donate in people who are not yet registered as organ donors Methods: In 3 Experiments involving 635 members of the UK general public, participants were invited to complete questionnaire measures tapping the emotional factors such as "ick", the desire to retain body integrity after death and medical mistrust. Registered organ donors were compared with non-donors. Non-donors were randomly allocated to an anticipated regret manipulation (2 questions) versus a control condition, and the impact on intention to donate was tested. Results: In all 3 experiments non-donors scored significantly higher than donors on the ick factor and bodily integrity scales. Traditional rational/cognitive factors such as knowledge, attitude and subjective norm failed to distinguish doors from non-donors. For non-donors, the anticipated regret manipulation led to a significant increase in intention to register as an organ donor in future. Conclusions: Negative emotional factors are important barriers for people becoming organ donors. In particular a basic disgust reaction when contemplating donation and the desire to retain bodily integrity after death are key areas to target to test if they are changeable. In addition, a simple anticipated regret manipulation has the potential to significantly increase donation rates. Background: Malaysia's organ and tissue donation rates are amongst the lowest in the world. Having the three ethnic groups (namely the Malays, Chinese and Indian) with the most pronounced cultural and religious prohibitions to organ donation, the shortage of organ donations continues to raise extreme concern. As evident, the rate of procurement (per million population) in Malaysia was only 1.01 in year 2006. From year 1997 until 2006, only a total of 162 organ procurements were carried out. Its rich ethnic diversity allow for a comprehensive understanding of a variety of factors associated to organ donation. Methods: A cross-sectional, population-based, computer-assisted telephone interview exploring multi-ethnic participants' knowledge, attitudes, practices and behavioral on deceased organ donation and transplantation was conducted during February to April 2009. Results: Although only 5.5% of the total participants (N=1174) reported that they have registered to be organ donor, a further 35.2% of those who have not register for organ donation indicated willingness to donate one's own. In particular, Malays (20.7%) indicate lower willingness to donate organs compared to the Chinese (36.6%) and Indians (51.4%) (P<0.001). In multivariate logistic regression analysis, willingness to donate one's own organ was associated with knowledge score (OR=1.17, 95% CI=1.13-1.22), attitude score (OR=1.17, 95% CI=1.05-1.31), secondary school education (OR= 1.46, 95% CI=1.05-2.02) and Malay ethnicity (OR=0.18, 95% CI= 0.03-0.94). Significant socio-demographic disparities with respect to knowledge and attitudes scores were observed. Study showed marked ethnic differences in attitudes towards organ donation. Some knowledge inaccuracies and misconception identified in this study need particular attention in future educational campaign. Conclusion: Findings assist organ donation and transplantation organizations to reach out the diverse socio-demographic and ethnic communities with cultural-specific information about organ donation. The data also highlight the importance of educational efforts to enhance knowledge and cultivate supportive attitudes toward organ donation. The use of personal financial incentives (PFI) to encourage people to change their behaviour is an emerging model of health intervention being trialled internationally. Recent reviews conclude that PFI can be efective in promoting behaviour change, but this is tempered by provisos. Women who smoke during pregnancy, particularly women of low SES, have been resistant to traditional smoking cessation initiatives. Smoking in pregnancy leads to significantly poorer health outcomes for mothers and their babies. There is an urgent need to develop effective smoking cessation interventions that can readily be adopted into routine antenatal care. This paper reports on the acceptability of PFI for reducing smoking in pregnant women among both patients and antenatal care providers.
Bangkok, Thailand; 3 Faculty of Nursing, Burapa University, Chonburi, Thailand; 4 Pamok Hospital, Angthong, Thailand and 5 Pamok District Public Health Office, Angthong, Thailand.
Background: To assess situations, problems, needs, and ways of life of the diabetic people in each community, which tailored by culture, religion, socioeconomic, health care service system, and political system is crucial before mobilizing stake-holders and changing behaviors of these diabetes patients, family and community. Method:
The integration of Sufficiency Economy(SE), Innovative Care for Chronic Conditions(ICCC) and Community-Based Participation Research(CPBR) was employed to five communities of Pamok district, Angthong province. Five stake-holders(diabetes patients, diabetes risk group, health care volunteers, health officers, and local administrators) were invited to participate in this study. The process in each community consisted of focus group discussion using Appreciation Influence Control(AIC), semi-formal interview, and informal interview. Results: The similarity of diabetes problem situations in each stake-holder were; less knowledge or no awareness on diabetes, unhealthy food consumption, lack of exercise, and non-adherence treatment. The likeness vision of diabetes in each community were; self care management, empowerment health volunteer, health promotion activities exercise, local wisdom of herb and alternative medicine, and integrate all stake-holder together. After discussing and priority setting of the health promotion project for diabetes and risk group under concepts of SE, ICCC, and CBPR, five different health promotion projects were initiated. Conclusion: Community-based participation research was an apparatus to build relationship and trust among the stake-holders and researchers which were the preliminary keys to raise awareness, responsibility, and empowerment to develop health promotion behaviors to reduce complication and prevention diabetes in the community. Diabetes mellitus is currently a significant health problem in Thailand and in many countries. To solve this problem, health care providers have to provide not only curative care, but also, prevention, promotion and rehabilitation services. Risk communication is a crucial process to help the people to maintain and improve their health through understanding risk issues and theirs context. The different perceptions on risks and their relevant factors among stakeholders make it difficult to implement effective risk communication.
This study aims at developing a model on risk communication for preventing and controlling diabetes mellitus. A six-month study was conducted in March to September 2008. Literature reviewing from both national and international articles provided a conceptual framework and a process for developing risk communication model which was preparing, assessing and working with the community. Stakeholders were approached to involve in all steps of the study. The setting of the study was in Mugdhaharn Province, Thailand. The conceptual framework of the risk communication was developed from literature review comprising two major components: 1) risk assessment and 2) risk management. In relation to risk assessment, qualitative and quantitative techniques were applied. The target groups of risk assessment were people at risk of diabetes mellitus, family members, and related persons. The issues to be assessed covered risk factors, risk perception, strengths and weaknesses of self care and risk contexts. The results of the risk assessment led to the guidelines and strategies for coping with the risk factors. Regarding the risk management, the guideline and strategies were initially implemented by focusing on the engagement of the stakeholders which resulted in learning and better understanding among related people. The proposed model reflecting two significant components and its process was accepted by stakeholders and further implemented by local health care team in the study area. HIV prevention programs with adolescents use different strategies with varying degrees of success. Behavioral programs have been effective in reducing risk factors, although data on the most effective components are needed. This symposium is addressed from an international point of view different experiences of program evaluation focused specifically on HIV, applying experimental methodology, with high-risk and general population, and combining the evaluation of empirical studies, the quantitative review and proposed new challenges.
In the first presentation, by Espada et al, a behavioral program implemented by experts and peers is evaluated. Griffin will present a proposal for joint intervention on substance abuse and HIV transmission. Orgiles et al. address HIV prevention with young people under the stressor of the divorce of their parents. Huedo-Medina will show a meta-analytic review of the effectiveness of prevention programs with adolescents. It thus seeks to answer outstanding questions about HIV prevention with adolescents, the degree of the overall effectiveness of behavioral programs, the better alternatives to applicators, the relationship between HIV prevention and other risk behaviors, and intervention with high risk populations served shortly.
The main objective of this work was to provide an updated review of the efficacy of behavioral interventions to reduce sexual risk of HIV among adolescents. We searched electronic databases, leading public health journals, and the document depository held by the Synthesis of HIV/AIDS Risk Reduction Project. Studies that fulfilled the selection criteria and were available as of December 31, 2008 were included. Studies were included if they investigated any behavioral intervention advocating sexual risk reduction for HIV prevention, sampled adolescents (age range, 11-19 years), measured a behavioral outcome relevant to sexual risk, and provided sufficient information to calculate effect sizes. Data from 67 studies and 98 interventions (N = 51,240 participants) were included. Independent raters coded participant and intervention characteristics as well as methodological features. Weighted mean effect sizes, using both fixedand random-effects models, were calculated; positive effect sizes indicated greater risk reduction.
Relative to controls, interventions succeeded at reducing incident STIs, increasing condom use, reducing or delaying penetrative sex, and increasing skills to negotiate safer sex and to acquire prophylactic protection. Initial risk reduction varied depending on sample and intervention characteristics but did not decay over time. Comprehensive behavioral interventions reduce risky sexual behavior and prevent transmission of STIs. Interventions are most successful when administered in larger doses. School-based drug abuse prevention programs for adolescents are an efficient way to deliver prevention services to young people before they become regular substance users or abusers. Contemporary prevention programs focus on social refusal skills training along with training in more generic "life skills" using cognitive, behavioral, and affective skills training techniques. This type of programming teaches principles of cognitive reframing, behavioral self-control, coping, and relaxation skills, along with how to apply these skills outside of the classroom in high risk situations. This presentation will review the concepts and empirical findings from the "Life Skills Training" prevention program, including short and long-term effects on related risk behaviors. Life Skills Training (LST) is a theoretically-driven evidence-based drug abuse prevention program for middle school students that emphasizes social resistance skills as well as more generic skills such as communication, assertiveness, decision-making, goal-setting, selfcontrol, and coping skills. Over 30 peer-reviewed articles with analyses of 18 separate cohorts of students have demonstrated that the LST program is effective with a variety of populations, producing behavioral effects on alcohol, tobacco, illicit drug use, as well as aggression and delinquency. Because LST targets etiologic factors common to both drug abuse and HIV risk and teaches skills that have a broad application, we examined the extent to which the intervention may reduce later HIV risk behavior. This presentation reviews findings demonstrating that the intervention reduced drug use and HIV risk behaviors in a sample of 2,042 predominantly white, suburban young adults who had received the preventive intervention in junior high school. Additional findings from a sample of 2,122 predominantly minority young adults will be presented demonstrating that several key skills measures (e.g., cognitive and behavioral self-control skills) that are targeted by the intervention predict various forms of substance use and HIV risk behavior in a similar fashion. These findings are presented in order to frame a discussion on the opportunities for developing competence-enhancement prevention programs to address both substance use and HIV risk behaviors, along with the challenges associated with addressing multiple risk behaviors in a single intervention. One goal of the presentation is to suggest that broad-based competence enhancement prevention programs may be an efficient and effective way to prevent a variety of negative outcomes during adolescence. A quasi-experimental design with three experimental conditions was carried out: program implemented by experts, program implemented by peers, and a waiting-list control group. Each participating center was randomly assigned to one condition. The intervention consisted of 5 sessions of one hour with the following components: information and cognitive restructuring, social skills training, problem solving training, and covert behavioral rehearsal. Three measures were conducted: pretest, posttest and 12-months follow-up. After the implementation of the program have shown improvement in the degree of knowledge and a significant attitudinal change. Comparisons between groups indicated that significant differences exist between the programs implemented by experts vs. the control group, and the program applied by peers. The effect size comparing the two types of applications was low. It shows how an intervention based on cognitive behavioral techniques succeeded in improving the level of knowledge about HIV / AIDS and change attitudes towards the disease, risk behavior and condom use. The program implemented by experts who performs better when implemented in pairs.
Jac van der Klink, PhD, MD Health Sciences, University Medical Centre Groningen, Groningen, Netherlands.
Job control (autonomy and decision latitude) is an important aspect of stress theories. In the Job-demand-control model of Karasek and Theorell it is the single important factor that compensates for high demands. In the more recent Job-demand-resources model control is an important factor besides other resources.
The level of control that employees experience -a powerful resource -can be threatened at three levels: 1) the function in itself can, regarding organizational structure and task embedding, offer too little possibilities of decision latitude and be at a 'wrong' level of control, 2) the function is all right but the department has a supervisor who cannot delegate 'job control' or decision latitude 3) 1 en 2 are well but the employee himself cannot see and use the decision latitude and autonomy that is there. Stress management programms or other interventions are seldom targeted as a result of a thorough (organisational) diagnostic proces. Most of the time interventions are mainly indicated by the specific orientation of the provider and most of the interventions are entirely focused on the individual level. In this symposium we will present a study in which the explained variance on the different organisational levels will be presented. Besides that, we will present the present state of knowledge on the three levels of intervention: function redesign, supervisor focused interventions and individual focused interventions. Recent trends in workplace intervention include comprehensive and integrative approaches. An important element in these interventions is the participatory approach, which implies control and empowerment for those involved. Greater participation in the process was found to be associated with increased levels of job control and communication and consequently reduced symptoms and time loss at work. In a cluster randomized controlled trial, the effect of a participatory intervention for workplace improvement on workers' mental health and productivity was evaluated. Eleven assembly lines in a medium-sized company producing electrical devices were randomly allocated to six intervention and five control lines (47 and 50 workers, respectively). The team-based, problemsolving intervention was based on active employee involvement, shared work-related goals, and action planning to improve the work environment for stress reduction. The intervention focused on environment improvement or job redesign rather than behavioral change in symptoms of ill health. A participatory approach has several principles which include 1); to build on local practice (starting from real problems of the enterprise that exist at the site where employees work and need improvement), 2) to focus on achievements (good practices already available), 3) to link working conditions with other management goals (ie, productivity), 4) to encourage exchange of experience, 5) to promote employee involvement, and 6) to use learning-by-doing. General Health Questionnaire scores significantly deteriorated in the control lines, whereas the score remained at the same level in the intervention lines. Health and Work Performance Questionnaire scores increased in the intervention lines, but decreased in the control lines, yielding a significant intervention effect. The process evaluation revealed that job control was improved in some lines which was supposed to bring about beneficial effect. For example, in a line where a variety of small parts were dealt with and the tasks had not been standardized, the workers made themselves a plan and the small units they worked on were adjusted according to the specific task. The realization of workplace intervention requires the understanding and cooperation of employers or administrators, because these measures depend largely on organizational redesigns.
Researchers are required to ensure that administrators and other key stakeholders have a clear understanding of their roles and responsibilities, and for the workers, to encourage them to sustain the autonomous activities for workplace improvement. et al., 1994; Theorell et al., 2001; Kawakami et al., 1997) and three randomized controlled trials (RCTs) (Kawakami et al, 2005 (Kawakami et al, & 2006 Takao et al 2007) . Studies were conducted in Japan, except for one from Sweden. Data were entered into and analyzed by STATA.
Results: In all studies except one, supervisor training included provision of basic knowledge of psychosocial factors at work; In most studies from Japan the training focused on providing skills to communicate with subordinates (such as "active listening" skills); in one study from Sweden, they put more focus on redesign and improvement of psychosocial work environment. A onetime session was applied for most studies; multiple sessions with two-week intervals during one year were provided in one study. One RCT used a small group discussion; two RCTs used a web-based training method. Most studies used three-month follow-up. A significant overall effect of supervisor training was found on mental health among subordinate workers (Effect size, -0.13; 95%CI, -0.26 to 0.00; p=0.05). A marginally significant effect was observed on job control (Effect size, 0.100; 95%CI, -0.01 to 0.21; p= 0.06). No significant effect was observed on job demands, supervisor or coworker support, or work performance (p>0.05). Conclusions: The meta-analysis indicated the effect of supervisor training on mental health among their subordinate workers, while the effect size was small. Supervisor training may enhance worker mental health through improving their job control. Supervisor training does not seem to have an effect on increasing work performance.
istics and coping behaviour. The explained variance at the departmental and the occupational levels on mental health, work characteristics and coping was assessed using Anova's. Results: 90 employees working in departments which were headed by one supervisor, were eligible for this study. The variance explained by function was 38% for autonomy, 36% for skill discretion, 14% for job demands, 27% for support by supervisor, 10% for coworker support, 17% for distress and for anxiety, 22% for depression, 22% for active coping, 10% for passive and 15% for distraction. The variance explained by departmetn (supervisor level) was 31% for autonomy, 29% for skill discretion, 28 % for job demands, 39% for support by supervisor, 31% for coworker support, 14% for distress, 16% for anxiety, 9 % for depression, 20% for active coping, 20% for passive and 25% for distraction. The present paper deals with predictors of the duration to full return to work of employees on sick leave due to psychological complaints. Research we recently conducted revealed that two factors may be predictive for the duration to full return to work (RTW). These factors are early partial RTW and work related selfefficacy. This conclusion is based on three individual focused intervention studies that have been conducted in recent years. The first study was a randomized controlled study with two experimental interventions and one no treatment condition. One hundred self-employed were randomly assigned to one of the conditions. Psychological complaints, professional efficacy and other variables were assessed before, after 4 months and after 10 months. Partial and full RTW was assessed continuously. The second study was a cluster randomized comparative intervention study with an experimental condition and a care as usual condition. One hundred sixty eight employees absent form work due to psychological complaints enrolled in the study. Psychological complaints, work related self-efficacy and other variables were assessed at three times, before, after 6 months and after 12 months. Partial and full RTW were assessed continuously. The third study was a longitudinal cohort study with 177 employees absent form work due to psychological complaints. Partial and full RTW were assessed continuously. Further, psychological complaints, work related self-efficacy and other variables were assessed at a three months time interval during one year. Both partial and work related self-efficacy appeared to be strong predictors of full RTW. This result fits the theoretical notion of graded activity as part of an individual focused treatment and the concept of self-efficacy as a moderating or facilitating factor in restoring an individual's work capacity after sick leave due to work related psychological complaints. Ethnic/racial disparities in early childhood health and development are evident in the US and UK. To date little cross-national comparative work has been carried out in this area, but with the availability of similar data collections in the US and UK such comparative work is now possible. Data from two nationally representative population based surveys, the Early Childhood Longitudinal Study in the US and the Millennium Cohort Study in the UK allow questions on the origins of ethnic/racial disparities to be addressed. This symposium examines how racial/ethnic disparities in childhood health and development vary across groups and national contexts. Presentations consider the underlying pathways to a diverse set of child health and developmental markers: growth, socioemotional difficulties, mental and motor development, infant feeding and obesity. Paper 1 given by Prof Sacker will examine whether weight at birth is important for subsequent child growth and development and how patterns might vary across ethnic groups. Paper 2 given by Dr Davis-Kean examines relationships between the development of early motor skills and later mental and motor development. Paper 3 given by Dr Kelly considers whether experience of racism translates into poor developmental outcomes in childhood, and paper 4 given by Dr Simonton examines the links between unwanted pregnancy and the likelihood of breastfeeding initiation across ethnic/racial groups. Important to our understanding of ethnic/racial disparities are factors relating to the drivers for migration to the US and UK and national policy contexts for health and social care and these will be discussed. This symposium explores issues on ethnic/racial disparities in an interdisciplinary manner from the joint perspectives of psychology, epidemiology and sociology. There are clear differences in birth weight by ethnic group, but whether these disparities are replicated in later markers of physical development is unknown. We examine evidence for ethnic disparities in children's height at 3 and 5 years. Data are from the UK Millennium Cohort Study, constructed to over-represent children from ethnic minority, giving us reasonable sample sizes for Indian, Pakistani, Bangladeshi, Black African and Black Caribbean groups. Mean birth weight of ethnic minority children was significantly lower than that of the ethnic majority (range 3.06 -3.34 kg versus 3.41 kg). By contrast, ethnic minority children were taller than the White majority at ages 3 and 5, significantly so in the case of Pakistani, Black Caribbean and Black African children (by 0.5 cm, 1.4 cm. and 3.5 cm at 5 years). Controlling for parental height to take account of intergenerational effects did not affect height differentials. Two mechanisms were hypothesised: (a) a poorer intrauterine environment given the short stature of some minority children's mothers resulted in catch-up postnatal growth and (b) conditions during the parents' childhood led to a reduced capacity to reach their own height potential. A reparameterization of parent heights showed that mother's height contributed more to predicting child height than mean parental height. Adding birth weight to the model showed that height was positively related to birth weight and attenuated the extra contribution from mothers' heights to non significance. The interaction of mean parental height with ethnicity was significant in models at age 3 and age 5. For all ethnic minority groups, mean parental height was more weakly related to child height than in White majority families, although only significantly different in the largest Pakistani group. In conclusion, we found evidence of catch-up growth in ethnic minority children and that ethnic minority parents had not reached their height potential. There was no suggestion that the lower birth weight of ethnic minority children has adverse consequences for their growth in the preschool years. Background: Previous work has shown that experience of racism is related to poor health outcomes in adults, but little is known about whether such experiences translate into developmental outcomes in children. Objectives: 1. to examine the relationship between parental experience of racism and markers of child development 2. to assess whether family socioeconomic position and neighbourhood level disadvantage explain observed relationships. Methods: Data from the UK Millennium cohort study on ethnic minority participants of Indian, Pakistani, Bangladeshi, Caribbean and African descent were used (N=2157). Questions on parental interpersonal experience of racism, and the perception of racist attacks in the residential neighbourhood were asked when cohort members were aged 5 years. A summary 'objective' score for residential perception of racism was created. Markers of child development at age 5 were obesity and overweight, socioemotional difficulties, and cognitive ability scores. Results: 43% of parents reported any experience of interpersonal racism, and 12% reported that racist attacks or insults were common in their residential neighbourhood. It appeared that parental reports of interpersonal racism were not strongly associated with poor developmental outcomes. Conversely, perception of racism in the area of residence was significantly associated with socioemotional difficulties (coeff 1.4, p<0.001) and spatial ability (coeff -1.4, p<0.01). A trend was observed between the objective marker of area racism and an increased risk of obesity and lower verbal and non-verbal ability scores. The objective measure of area racism was found to be associated with lower spatial ability score. Statistical adjustment for socioeconomic factors attenuated associations, but area and objective measures remained independently associated with socioemotional difficulties and spatial ability. Conclusions: Parental perceptions of racism are linked to poor developmental outcomes in children. Economic disadvantage among ethnic minority groups appears to partly explain some of these relationships. Recent research has shown that motor skills in children may be an important predictor in achievement in the elementary school years (Grissmer, et al, 2009) . In this study we test whether early gross motor skills and early mental skills in infants and toddlers are predicted by early motor movements such as pulling up or walking with the assistance of other. These early skills may be important for indicating brain development that may be promoting later motor development and problem solving. In order to examine our model we use the first wave (9 months old) of the Early Childhood Longitudinal Study-Birth Cohort (ECLS-B), which is a nationally representative sample of children born in the US in 2001. For the analyses, 10, 680 children were examined. We controlled for child's age, being foreign born, multiple births, low birth weight, highest parental education in the home, and household income. We examined whether children were breastfeed as a parental nutrition indicator, and age child pulled themselves to standing, age of walking with help as child as indicators of early motor skills/early brain development.
Using hierarchical regressions we find that low birth weight, age of pulling oneself to a standing position and walking with help are negative predictors of early gross motor skills (B=−.07,−.07, −.02 respectively). Being Black, is a positive predictor of gross motor skills (.03). These variables with controls account for 70% of the variance in gross motor skills. For early mental skills, being Black and household income is associated with a .02 positive increase and age walking with help (.06) was also positive. Being Asian (-02), low birth weight (-.04), and ever breastfeed (-.02), are negative predictors. Similar levels of variance are accounted for (.70) with children's age being the largest predictor of both gross motor and mental skills (.82, .84, respectively) . Though the effects are small, there is some indication that early motor and mental skills may be related to early breastfeeding and physical motor skills. Automobile-oriented urban design is linked with increasingly sedentary lifestyles, which are associated with an array of chronic diseases as people age. Yet little work has occurred on the relationships among built environment features and active transport and other physical activity (PA) forms among older adults. 709 adults ages 66 yrs and above living in Seattle or Baltimore regions were recruited from GIS-enumerated neighborhoods identified as high or low affluent and high or low 'walkable' based on land use mix, street connectivity, and other features. Variables included reported active transport (walking/bicycling for errands), general PA (via accelerometry and self-report), and reported BMI and mobility impairments. Persons in less affluent neighborhoods reported generally less PA and higher BMI. After controlling for individual and neighborhood-level sociodemographic and regional factors, walkability was positively associated with active transport (p< .0001), total PA (p= .036), leisure walking/cycling/jogging (p < .008), accelerometry-derived minutes/day of moderate or vigorous PA (p=.056), and lower BMI (p= .02). A significant walkability-mobility impairment interaction was observed for active transport only; as mobility increased, differences in minutes/week of active transport among those living in low vs. high walkable neighborhoods increased (p=.001). Persons in the lowest mobility tertile living in high walkable neighborhoods had active transport levels comparable to persons in the two higher mobility tertiles but living in less walkable neighborhoods (means=35 mins/wk vs. 27 mins/wk, respectively, p>.13). Results suggest the potential importance of environmental features in facilitating active transport and other healthful patterns among older adults across mobility levels, with potential benefits related to both health and independence as people age. Objective: The project's aim was to analyze built environment and parental perception factors that influence youth travel mode to school. Methods: Data from a sample of Atlanta youth ages 5-18 was used along with objective measures of urban form surrounding home and school and along the route from home to school. The large sample size (n=5890 trips) allowed stratified analysis by age group (5-10, 11-15, 16-18) and distance to school (0-1.5 miles, 1.5-3 miles, over 3 miles), while controlling for sociodemographic factors known to influence travel. Results: Using a nested logit model of travel mode choice, we observed more sidewalks, higher residential densities and interconnected street networks along the route to school and higher parental perception of neighborhood school quality were positive, significant (p<=.05) predictors of higher probabilities of walking to school for shorter distances (0-1.5 miles) and younger children (ages 5-10). Further analysis indicates small marginal changes can substantially improve probabilities of selecting walking -the probability of walking after the increase in sidewalk coverage to 60th percentile is 118.44% of the probability of walking at median sidewalk coverage. However, it also appeared that the influence of urban form can be overwhelmed if other factors (parental perceptions of school quality, traffic safety and crime, or even other urban form characteristics) are not supportive. Land use mix, a consistently significant predictor of adult travel behavior, was consistently not associated with youth school trips. Discussion: Travel mode may be predetermined by parents or constrained by other circumstances, and it may only be under certain circumstances -youth located in communities safe from crime and traffic that are close to school -where urban form may influence travel mode. There are indications that school quality may have an indirect impact on physical activity through its impact on neighborhood school choice. Objective: To apply an analysis of urban form and physical activity relationships to a land use planning tool in King County WA (Seattle region). Method: Regional travel survey data was used to measure active transportation (number and miles of bicycle or walk trips) in King County (n=2699 households); objectively measured data from the Neighborhood Quality of Life Study (NQLS, n=1200 households) was used to measure daily minutes of moderate and vigorous physical activity (PA). Urban form was measured at the 1 km network buffer level around each household using GIS parcel data. A multiple regression model which controlled for sociodemographic factors was used to estimate coefficients, which were subsequently incorporated into I-PLACE3S, a sketch planning tool developed by the California Energy Commission. All statistically significant variables (p<=.05) were integrated into I-PLACE3S; urban form variables of particular policy relevance that did not attain p<=.05 were included in the final models for incorporation into I-PLACE3S if they were logically signed. Results: Residential density, retail density, and presence of a park within the buffer were significantly related (p<=.05) to PA. Intersection density approached but did not attain statistical significance on PA, and was left in the model for incorporation into I-PLACE3S. Of the urban form variables, intersection density had the strongest association to active transportation trips and miles (β=.022 and β=.023, respectively; p<.01). Retail density was also significantly related to active transportation trips and miles (β=1.876 and β=2.888, respectively; p<.01). The number of retail/food-related parcels significantly increased the number of nonmotorized trips, but did not significantly change number of miles. In addition to economic factors, globalization has an impact on cultural, social, and psychological aspects of functioning. These changes require adaptation and are often a source of stress. The current study assessed the stress experienced due to globalization. Undergraduate participants from St. Xavier's College in Mumbai, India (N=56; Mean Age=19 years; 3 Men; 50% Christian & 28% Hindu) reported on the impact of globalization on their lives and on the culture and the society where they lived. Two independent raters identified common themes and three additional independent raters reviewed the themes for overlapping content and modified the themes listed until they had consensus among them on 32 themes that described the experience of globalization. Antenatal care (ANC) has been recognised as a way to improve health outcomes for pregnant women and their babies. Only 29% of pregnant women receive the recommended four antenatal visits in Nepal and reasons for such low utilisation of ANC are poorly understood. The main aims of this paper are to explore the mother-in-law's role in ANC uptake and decision-making about using ANC services in Nepal.
In-depth interviews were conducted with 30 purposively selected prenatal or postnatal mothers (half users, half non-users of ANC), 10 husbands and 10 mothers-in-law in two communities.
Our findings suggest that mothers-in-law are pivotal family members who often make decisions about ANC check ups for their daughters-in-law. Sometimes this influence is positive, encouraging women to seek ANC, but more often it is negative. A number of mothers-in-law had not used ANC themselves and did not see any benefits. The main factor leading mothers-in-law not to support/ encourage ANC check ups were workload for pregnant women, mothers-in-law's perceptions of benefits deriving from ANC, their control over resources, their own past experiences and power relations between mothers-in-law and daughters-in-law. Mothers-in-law have a strong influence on the use of ANC. Health promotion and education interventions to improve the use of ANC should target both women and their family members, particularly mothers-in-law. Objective: To explore the perceptions of healthcare providers on antibiotic use and resistance development in relation to environmental factors i.e. physical, natural, social and behavioural factors. Methods: A qualitative interview study was conducted using faceto-face, semi-structured interviews among registered allopathic doctors, veterinarians and drug dispensers in Orissa, India. The interview transcripts were analyzed using content analysis. Results: The main findings of this study relate to two themes: 'Interrelationship between antibiotic use, resistance development and environmental factors' and 'Antibiotic handling contributing to the development and spread of resistance'. Changes in the natural and physical environment i.e. climate variability, physiography and population growth; the socioeconomic environment affecting healthseeking behaviour and noncompliance with medication; a lack of healthcare facilities and poor professional attitudes; and ineffective law enforcement on medicine dispensing, manufacturing and disposal were viewed as possible contributors to resistance development. Conclusion and implication: Our study indicates that although behavioural and social environmental factors are major contributors to resistance development, changes in the physical and natural environment exacerbate these problems, and suggests further quantitative studies. The study also indicates a lack of information about and awareness of what constitutes prudent use of antibiotics and thus emphasizes the need for comprehensive action including information, education, dissemination and proper implementation and enforcement of legislation at all levels of drug delivery and disposal in order to rationalize antibiotic use. For SBP this interaction was significant for Chinese (p < .04) but not for Malays and Indians. TA also interacted with positive emotions such that for males with high TA higher levels of positive emotions were associated with increased SBP (p <.05) whereas this was not true for males with low TA or for females. In addition, Indian males with high TA showed reduced HR with higher levels of positive emotion whereas this was not true for those with low TA (p < .01). This interaction was not found for Chinese or Malays. Conclusions: These data provide further evidence for the interaction of TA with negative emotions in cardiovascular reactivity and also obtained ethnic and gender differences in this relationship. However, the fact that TA also interacts with positive emotions in its effects on cardiovascular parameters suggests that individuals with high TA, particularly if they are Chinese and/or male, may be more generally reactive to emotional experiences, irrespective of valance. Theory: Memory dysfunction is correlated with reduced adherence in various patients groups. However, non-adherence is not only caused by memory dysfunction, as adherence is a complex behavioural regimen including planning, forming intentions and implementing actions. Neurocognitive executive functioning is a precondition for these action-plans and therefore serves as a base for adherence. Based on a model of executive function in medical illnesses this study investigates the link between executive function and adherence to medication in heart failure patients. Method: Executive function in patients (N=130) with heart failure is tested using Trail-Making test, Go/noGo-, Visual Set-Shifting-and n-Back task; other neurocognitive tests include digit-span (forward/backward), word recall (short/long-term), alertness and focussed attention. Self-reported adherence is assessed using the Medication Adherence Rating Scale adjusted for social desirability. As possible confounders age, years of education, beliefs about medicines, illness perception, and depression are analyzed. To define health status quality of life, NYHA-class and relevant biological health markers are measured. Results: Preliminary results indicate that non-adherent patients showed worse performance on Trail-Making test and n-Back task (p<.05). Results from multiple regression analyses are presented and a neurocognitive model of adherence is introduced. Discussion: The role of executive function on adherence to medication has not been investigated yet. As medication taking involves complex regimens, executive dysfunction could explain non-adherent behaviour. Patients with executive dysfunction may benefit from an early screening and guided medication taking procedure to overcome this barrier. This study aimed to identify distinctive trajectories for pain/ disability and posttraumatic stress disorder (PTSD) symptoms following whiplash injury and to examine the effect of injury compensation claim lodgement on the trajectories. In a prospective study, 155 individuals with whiplash were assessed at <1 month, 3, 6 and 12 months post injury. Outcomes at each time point were: Neck Disability Index (NDI) and the Posttraumatic Stress Diagnostic Scale (PDS). Group based trajectory analytical techniques were used to identify outcome profiles. The analyses were then repeated after including third party compensation claim lodgment as a binary time-changing covariate. Three distinct NDI trajectories were determined: 1) Mild: mild or negligible pain/ disability for the entire 12 months (45%), 2) Moderate: initial moderate pain/disability that decreased to mild levels by 3 months (39%) and 3) Chronic-severe: severe pain/disability persisting at moderate/severe levels for 12 months (16%). Three distinct PTSD trajectories were also identified: 1) Resilient: mild symptoms throughout (40%), 2) Recovering: initial moderate symptoms declining to mild levels by 3 months (43%) and 3) Chronic moderate-severe: persistent moderate/severe symptoms throughout 12 months (17% Rebelliousness is about feeling compelled to do something contrary to that required by some external agency. More than hostility (a known psychological risk indicator for heart disease), rebelliousness might be strongly related to health and heart disease via rebellious persons opposing and obstructing health campaigns aimed at modifying unhealthy behaviours. We set out to examine, firstly, the psychometric properties of a rebelliousness question-naire and, secondly, the extent to which rebelliousness and hostility compete in the prediction of incident heart disease and in the contribution to socioeconomic differences in health. Using Dutch data on older men and women (n=4,485), we found moderate reliability (Cronbach's α ranging between 0.50 and 0.60) and moderate (construct) validity, as indicated by rebelliousness being (moderately) associated with low control beliefs, hostility, and unhealthy behaviours. Using longitudinal data (n> 2,000), we will further report on the extent to which rebelliousness is more common (than hostility) in lower socioeconomic status groups, the extent to which rebelliousness is more common (than hostility) in the etiology of heart disease, and the extent to which rebelliousness more strongly (than hostility) affects heart disease through poor health behaviours. The findings add to the body of evidence on the psychological pathways involved in how socioeconomic structure gets under the skin. Epidemiology, Ulm University, Ulm, Germany; 2 Bethesda Hospital, Ulm, Germany and 3 Robert-Bosch Hospital, Stuttgart, Germany.
Physical activity is a powerful predictor of health in older ages. However, in most population-based studies on this topic physical activity has been measured retrospectively with the help of questionnaires. Accordingly, bias may occur. The present investigation applies an ambulatory objective measurement of physical activity which helps to avoid bias. The baseline screening of a prospective study is currently conducted in a random sample of the older population in the greater Ulm area, Germany. It is planned to investigate 1500 men and women aged 65 to 90 years. Recently, about 1000 are included. Extensive information on cognitive and physical functioning, chronic disease, quality of life and socio-demographic variables is derived from well-tested questionnaires and tests. Information on medication and falls is assessed prospectively. Lung function is measured according to international standards. Blood samples are taken to determine biomarkers. Furthermore, physical activity is assessed ambulatory with the help of accelerometers.The accelerometer information is combined with an activity diary. First preliminary results show associations between physical activity as measured by the accelerometers and health. Individuals with type II diabetes are significantly less active as compared to elderly without this disease even after adjustment for age and gender. Over a period of at least 5 days subjects with diabetes are on an average one hour per day longer inactive as compared to the healthy group. Further analyses will include a number of additional variables presumably influencing these associations like depression, lung function, and cognitive as well as physical functioning. First preliminary results of the IMCA-ActiFe study indicate the validity of objective measurement of physical activity and show associations of inactivity with type II diabetes. For the future a more clear and detailed picture of daily activity among elderly can be expected from accelerometer measurement. Accordingly, interventions better tailored to individual needs can be developed. Background: Despite the evidence that providing care to a person with dementia has adverse health consequences on caregivers, there are individual variations in their responses to the caregiving role. Approximately 10.4 million family caregivers of people with Alzheimer's and other dementias provide care to their loved ones in the community. The purpose of this study was to examine the differences between highly resourceful and less resourceful family caregivers on role reward, preparedness for caregiving, anxiety and depression. Method: A comparative descriptive design was used on baseline data of 84 family caregivers who were living with and caring for person diagnosed with Alzheimer's disease. The data is part of an ongoing intervention study to enhance resourceful skills in family caregivers. Instruments: Resourcefulness was with the 36-item Self -Control Schedule (SCS: Rosenbaum, 1980) . Depression was measured with the 20-item CESD scale. Preparedness was measured using the 8-item Family Preparedness Scale (Archbold & Stewart, 1994) . Anxiety was measured using the Anxiety Scale on the State Trait Anxiety Inventory (Speilberger et al, 1970) . Role reward was measured with Family Role reward Scale (Archbold & Stewart, 1994) , a 15-item Likert-type tool with a 5 point scale. Descriptive statistics and t-test were used to analyze the data. Findings: The results showed that family caregivers with low SCS scores (less than 25) were significantly less prepared (t=3.03, df= 83, p<.004), more anxious (t-3.055, df=83, p,.004) and reported less role reward(t=3.59, df=83, p<.001) in caregiving than those who were highly resourceful. There were no difference in depression, and appraisal of behavior of behavior problems between groups. The implication is to assess and enhance caregivers' resourcefulness on those caregivers with low resourceful skills. Background: The German elderly population experienced more traumatic experiences across their life-span than the younger cohorts. Even sixty years after the WW II, this cohort is significantly more often affected by posttraumatic symptomatology compared to the younger cohorts (Maercker et al., 2008) . The present study is addressing prevalence rates of posttraumatic symptomatology and it's comorbidity with depression and somatization. Methods: The study examines PTSD according to DSM-IV, partial PTSD, depression and somatisation in a randomly selected sample of the German general population aged 60 to 85 years (N=1.659) using self rating instruments (PHQ, PDS). Results: One-month prevalence rates were 2.8% for DSM-IV PTSD, another 11.9% fulfilled the criteria of partial PTSD. This points out, that sub threshold symptomatology plays a major role in the elderly. 11.5% of the persons affected by PTSD symptomatology fulfil the criteria of a somatoform syndrome, 8.5% fulfil the criteria of Major Depression and 10.4% fulfil the criteria of other depressive syndromes according the Patient Health Questionnaire. 5.6% report both -depression and somatoform disorder. Discussion: Even sixty years after WW II, posttraumatic symptomatology is more common in the elderly in Germany compared to the younger cohorts. It is often accompanied by other comorbid mental disorders like depression or somatisation. Implications of these findings for health and health care in the elderly will be discussed.
CORRESPONDING AUTHOR: Heide Glaesmer, Dr, University of Leipzig, Leipzig, 04103; Heide.Glaesmer@medizin.uni-leipzig.de Objective: To explore how African American youth cope with the diagnosis and treatment of parental breast cancer and to identify culturally sensitive ways to recruit and sustain participation of this vulnerable population in intervention programs. Methods: Three qualitative focus groups which were part of a larger study were conducted with 12 African American youth between the ages of 11 and 18 currently coping with parental breast cancer from the northeastern part of the United States. Interviews were audiotaped and transcribed verbatim, and analyzed using content analysis. Results: African American youth described fear and uncertainty about the mortality of their parent, their unpredictable future, and discomfort in negotiating breast cancer's relationship with the entire family. Four primary themes emerged which were coping with cancer, surviving cancer too, changes in family function, and growth through pain. African American youth described feeling overlooked by their families and oncology staff treating their parents, often being in the role of protecting their parents physically and emotionally. Conclusions: This study suggests that clinicians can improve the care of African American breast cancer patients and their adolescent children by being more family-centered. Adolescents need more developmentally appropriate preparation for the family changes likely to occur when a parent is diagnosed and treated for breast cancer. Developing a support group comprised of other youth coping with parental breast cancer from diagnosis throughout treatment was described as a preferred intervention to promote a shared understanding in order to overcome feelings of isolation, worry, and fear. Background: Previous studies suggest that use of complementary and alternative medicine (CAM) may have a beneficial influence on emotional well-being of cancer patients. Most existing studies, however, are based on small non-representative samples and have not differentiated between different types of cancer and specific types of CAM. Objective: To investigate if use of specific types of CAM measured 3 months after surgery predicted the level of depressive symptoms at follow-up 15-16 months after surgery when controlling for baseline depression and sociodemographic-, health-, disease-, and treatment related confounders.
Methods: In a nationwide cohort of 3343 Danish women (age 26-70) (response rate: 68%), use of CAM since diagnosis, depressive symptoms (Beck's Depression Inventory), and health-related variables were assessed at baseline 3 months post-surgery. Depressive symptoms were re-assessed at follow-up 15 months post-surgery (response rate for disease free women: 94%). Sociodemographic-, and clinical variables and data on comorbidity were obtained from the surgical departments and national longitudinal registries. Results: Overall CAM use was associated with more depressive symptoms at follow-up. This was due to users of dietary or vitamin supplements (N =895) at baseline exhibiting more depressive symptoms at follow-up, compared to non-users in a negative binomial regression analysis fully adjusted for baseline depression and potential confounders (ratio of mean (RM) = 1.11; 95% CI=1.04 -1.19; p=.003). Conclusion: In this prospective study use of dietary or vitamin supplements showed to be a risk factor of depressive symptoms. It remains to be explored if this finding may be explained by a hidden vulnerability for depressive symptoms among women using dietary or vitamin supplements, by interaction effects with conventional treatment, or by a direct negative influence of dietary or vitamin supplements. Learning objective 1: To gain knowledge of the association between use of CAM and changes in QoL in breast cancer patients. Learning objective 2: To gain knowledge of the association between specific types of CAM use and changes in QoL in breast cancer patients. Increasingly, breast cancer(BC) is managed as a chronic disease with persistent physical and psychosocial demands. Yet,little research has focused on the long-term psychosocial needs of women across the care continuum. The Cancer Support Community(CSC) fielded a national survey to better understand the current psychosocial needs of BC survivors. Methods: CSC conducted an online survey of 1,004 breast cancer survivors,0 to 5 years post-diagnosis. Questions were developed with input from a National Advisory Council of interdisciplinary experts representing advocacy, oncology,psychology,policy,industry and managed care. Results: 87% participants were white,with a mean age of 54. 45% graduated from college and 14% had a graduate degree. 75% had stage II cancer or less and 13% had a BC recurrence. 81% survivors reported some form of physical, emotional or spiritual distress with 67% reporting emotional distress. 64% women indicated fear of recurrence,58% physical changes,55% anxiety,47% depression,46% financial worries as the leading contributors to distress. Regarding their immediate needs, women wanted support around fears of recurrence (62%);making healthy lifestyle choices (60%);financial matters (42%);and reducing anxiety (41%). 86% believe emotional support is important to overall health,while only 52% had someone from their medical team address their emotional needs. 37% respondents did not receive a referral for support services. Of those that did receive a referral,30% did not follow up on any referrals. To reduce the cost of breast cancer,38% of respondents delayed seeking counseling or support. 64% would be likely to seek psychosocial support if it were free of charge. Discussion: Women with breast cancer recognize the importance of emotional support as part of survivorship care,yet few survivors access the care they need. As part of the M.A.P. Project,these data will inform the development of a Cancer Survivor Registry to identify behavioral trends and gaps in psychosocial care across the survivorship continuum. Background: Several studies have reported beneficial effects of expressive writing intervention (EWI) on emotional distress in patient populations. However, moderating variables are often involved and possible deleterious effects have received little attention Objectives: To explore whether the cognitive strategy of temporal comparison (i.e. comparing present life with past life) moderates the effect of EWI on negative affect (NA). Methods: A population based sample of women completing adjuvant treatment for breast cancer were randomized to a EWI group (n=253) writing about the most traumatic experience or to a control group (n=254) writing about daily activities. Both groups completed three home-based writing sessions one week apart. Temporal comparison was an evaluation of "Compared to previously, my life now is…" on a 5-point Likert scale ranging from "much worse" to "much better". NA was measured by the POMS. Both variables were assessed before randomization, 3 months, and 9 months after the first writing session. Results: The EWI and control group did not differ in use of comparisons or in NA at baseline. Repeated-measures ANOVAs showed no main effects of EWI on NA (F=0.48, ns). When temporal comparison was included in the model, a significant effect of EWI on NA was found (F=3.53, p<0.001), together with an interaction between EWI and temporal comparison (F=2.13, p<0.05). Conclusion: The results showed that EWI-women who at baseline evaluated their present life as better than previous periods increased in negative affect, whereas in the control condition, these women showed stable or reduced negative affect. This suggests that EWI could have an adverse effect in individuals who are not settled with their past. Hence, future studies may consider prescreening to optimize outcomes of EWI in patient populations. Introduction: Socioeconomic differences in weight status are well documented. These inequalities may be partly due to differences in takeaway food consumption patterns as frequent fast-food consumption is associated with increased risk of weight gain. However, the findings of studies examining socioeconomic position (SEP) and fast-food consumption are inconsistent. The current study examines SEP and takeaway food consumption behaviours. Method: A cross-sectional postal survey was conducted among 1500 adults aged 25 and 64 years (response rate=63.6%). SEP was ascertained from participants' highest achieved education level, and takeaway consumption was measured using a detailed food frequency questionnaire of 22 takeaway food and beverage items. Socioeconomic differences in the frequency (whether ate≥2 times per week) and types of takeaway food consumed were determined by logistic regression. Results: About 42% of men and 33% women consumed takeaway food at least weekly. There were no education differences in the frequency of takeaway food consumption between the highest and lowest education groups (men OR 1.48; 95% CI 0.68, 3.18; women OR 1.39; 95% CI 0.69, 2.80) . However, lower educated men and women were more likely to make unhealthy takeaway choices though this difference only reached statistical significance among women (OR 2.87; 95% CI 1.51, 5.45) . Conclusion: Takeaway food consumption may be an important contributor to dietary intakes among adults and may contribute to socioeconomic inequalities in dietary intakes and subsequently inequalities in overweight/obesity and chronic diseases. Poor mental health is more prevalent among diabetic patients and may be a risk factor for worse prognosis. Worsening glucose metabolism appears to contribute to patient's level of distress and may be an important factor in explaining future prognosis. We examined associations between psychological distress, glucose metabolism and death in adults with and without diabetes. In a representative, prospective study of 18,525 adults (4.1% with physician-diagnosed diabetes) we measured levels of psychological distress using the 12-item General Health Questionnaire (GHQ-12) and blood was collected for the assessment of glycated haemoglobin, a marker of glucose metabolism. The main outcome was all cause death. Psychological distress, that was defined as GHQ-12 score of ≥4, was apparent in 19.5% and 13.9% of participants with and without diabetes, respectively. Distress was associated with greater cardiovascular morbidity although not with impaired glucose metabolism in diabetics. Among diabetics there were 187 deaths over an average of 6.0 years of follow up. Distress was associated with higher risk of death (multivariate adjusted hazard ratio=1.95, 95% CI, 1.34 -2.84) after adjustment for glucose control, health behaviours, and cardiovascular comorbidity. The synergistic coexistence of diabetes and distress was associated with an elevated risk of all cause death, beyond that due to having either diabetes or distress alone. In summary, psychological distress is an independent risk factor for death in diabetic patients, but this association was not explained through impaired glucose metabolism. Early screening for distress may have important implications for prevention and treatment in diabetics. Background: Research suggests that men may underestimate both their weight and the health risks associated with their weight. However, there is only limited understanding of the health beliefs and health behaviours of obese men. This qualitative study of Australian men with obesity (BMI over 30) aimed to explore their beliefs about their weight, health and wellbeing; their attitudes towards weight loss; and the strategies that they employed to lose weight or improve their health. Method: In-depth semi-structured telephone interviews were conducted with a community sample of men with a body mass index of 30 or more. A range of community engagement strategies were used to encourage men to take part in the study from different socioeconomic and geographical backgrounds. Data were analysed using a descriptive thematic approach. Results: Thirty-six men participated. Most men stated that they were not aware of their weight gain and that their weight had increased slowly and steadily over a number of years. Often a significant life event, for example a health scare, motivated men to recognise that their weight was a problem and that they needed to lose weight. Men often blamed themselves for their obesity. They believed that their obesity was primarily caused by a lack of activity rather than their eating behaviours. Most strongly believed that it was their personal responsibility to lose weight and felt confident that they could achieve weight loss without the support of family members or health professionals. However, they wanted to be engaged in strategies that focused on improving their health and fitness, rather than weight loss. They felt that this was a more empowering approach and the most effective way to lose weight and keep it off. Perceived barriers to successful weight loss included commitment to work and family; social pressures to maintain unhealthy behaviours; and the costs of interventions such as gym memberships, personal trainers and commercial diets. Conclusion: The information provided in this study informs how we can tailor medical and public health strategies for obese men that resonate with their lived experiences. It also highlights the importance of social and contextual factors in understanding the experiences of obese adults. Background: Previous studies of public perceptions of obesity interventions have been quantitative and based on general population surveys. This study aims to explore the opinions and attitudes of obese individuals towards obesity interventions aimed at the population and individual level in Australia. Methods: Qualitative methods using in-depth semi-structured telephone interviews with a community sample of obese adults (Body Mass Index≥30). Theoretical, purposive and strategic sampling techniques were used to ensure a broad representation of individuals from different demographic and geographical backgrounds. Participants were asked about their attitudes towards three population based interventions (regulation, public health campaigns, and social marketing) and three individual interventions (tailored fitness programs; commercial dieting; and bariatric surgery), and the effectiveness of these interventions. Results: One hundred and forty two individuals (19-75 years) were interviewed. Participants strongly supported non-commercial interventions that were focused on encouraging individuals to make healthy lifestyle changes (regulation, physical activity programs, and public health campaigns). There was less support for interventions perceived to be invasive or high risk (gastric band surgery), stigmatising (social marketing) or commercially motivated (commercial diets and gastric band surgery). Personal experiences with weight and weight loss attempts strongly influenced attitudes. Conclusion: Obese adults support non-commercial, nonstigmatising interventions which are designed to improve lifestyles, rather than promote weight loss. While it is well established that depression is frequently comorbid with diabetes, it is still unclear whether screening for depression has utility. In this study the aims was to assess the impact of screening for depression in a hospital outpatient diabetes clinic by comparing the outcomes in patients with diabetes and depression following random allocation to three different management options. 1070 outpatients with type 1 and 2 diabetes were screening using the CES-D. Those that screened positive had a diagnosis of a major depressive episode (MDE) confirmed using the CIDI. Thus 178 patients with MDE were randomly allocated to direct referral to the hospital psychology service, information and a letter for patients to give to their primary care practitioner or usual care. Patients were assessed and then followed up six-months later to assess depression and diabetes outcomes. Patients in both the direct referral and the primary care conditions improved significantly on measures of depression compared to those in the usual care condition. However, only those patients in the primary care condition also improved significantly on a measure of diabetes control, HbA1. These findings suggest that screening for depression following by primary care is valuable in the management of diabetes. The lack of a well characterized state of the art in peer support interventions impedes their routine inclusion as standard and funded components of health care. A major challenge to efforts to characterize peer support is, in turn, the extent to which it is culturally contingent, that is, how characteristics of support are dependent on differing cultural, social, and organizational standards, norms or values. What is a sensitive offer of help in one culture may be intrusive in another or too reserved and thereby indicative of disinterest in a third. Additionally, varying attitudes toward health, fate, life and death as well as cultural contingencies surrounding key health behaviors such as diet add complexity to efforts to promote peer support across different countries. To address these challenges, Peers for Progress, a global program to promote peer support in health care, has followed a strategy of "standardization by function, not content." This identifies key functions of peer support across cultures and settings which, at the same time, allow for considerable flexibility in content of local implementation. Peers for Progress and its grantees have identified as key functions of peer support 1) assistance in daily management, 2) social and emotional support, 3) linkage to clinical care, and 4) ongoing availability of support. This symposium will explore how this approach has been realized in peer support programs in Australia, China, Thailand and Uganda. From Australia, Dr. Oldenburg will describe extension of support groups that Diabetes Australia-Victoria has established for over a decade to improve daily management, social and emotional support, and linkage to clinical care. From Hong Kong, Dr. Chan will describe "Pearl," a program that integrates peer support within "Jade," a program to promote algorithm based, systematic clinical care for diabetes in primary care. From Uganda, Dr. Baumann will describe challenges faced and successes of a peer support program in small, severely under-resourced villages. Finally, Dr. Boothroyd will describe how these and the other eleven project of Peers for Progress have increased understanding of important features of the four key functions of peer support, identified important aspects of implementation, and addressed programmatic, cultural and organizational challenges. Across all of these, identification of common functions guides program development that reflects generalized knowledge while allowing for tailoring to the strengths and characteristics of populations, cultures, and settings. Diabetes is a killing and costly disease although many of its complications are preventable and highly treatable. The Joint Asia Diabetes Evaluation (JADE) Program is the first web-based program incorporating a comprehensive risk engine, care protocols, clinical decision and self management support to improve ambulatory diabetes care. It uses state-of-the-art information technology to facilitate healthcare professionals to create a diabetes registry. The JADE e-portal also incorporates an interactive risk engine which stratifies patients into different risk levels based on clinical and biochemical data measured annually. With this risk stratification, the JADE e-portal recommends a care protocol tailored to these risk levels with decision support triggered by various risk factors. On top of 'High-Tech' accessories, quality diabetes care requires informed-decisions of motivated care providers and diabetes patients. We started the Peer Support, Empowerment And Remote Communication Linked By Information Technology (PEARL) Study in early 2009 in Hong Kong to use peer support and information technology to facilitate care providers to implement structured care and empower diabetes patients acquire selfmanagement skills. PEARL is a multi-component program making use of peer support, JADE Program and the Australasian Telephone Linked Care (TLC) system. TLC system utilizes an automatic, interactive, computer-controlled telephone system to enhance knowledge on diabetes self-management The PEARL Program involves trained mentors who are themselves diabetes patients with good self care and are motivated to support their peers. Diabetes patients receiving structured care in Hong Kong through the JADE Program will be recruited as mentees. They will receive peer support by the mentors (1 mentor to 10 diabetes patients or mentees) through regular phone calls and sharing session. They will also be reminded to use the TLC for knowledge enhancement and motivational support. Effects of various components of peer support on the patients' disease control as well as user acceptability and cost-effectiveness of these programs will be examined. We believe the combined use of a registry, structured care protocol and peer support will make quality diabetes care more accessible, sustainable and affordable, especially in resource-scare areas. Diabetes Australia-Victoria (DA-Vic) is a very well established NGO that has sustained a community network (ComNet) of support groups for people with diabetes for more than a decade. They aim to convey expert information about diabetes and to provide social support to members. Building on this experience S266 Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329 over many years, the Australasian Peers for Progress Diabetes Program (PfP-DP) aims to implement and evaluate a peer-led group program to improve daily management, social and emotional support, and linkage to clinical care for people with type 2 diabetes (T2DM). Participants will be recruited from the membership of (1) the National Diabetes Services Scheme, an Australian government initiative for people with diabetes to obtain subsidized consumables, and (2) DA-Vic membership of people with T2DM. The intervention trial involves 12 groups of 8-12 people led by lay peer leader(s), who are receiving training in group facilitation, communication, goal setting and motivational skills. Groups will meet monthly for 12 months, during which time group participants will review difficulties with their diabetes care, successes and achievements. The control (waitlist) arm comprises 12 "virtual" groups of similar composition who do not meet and receive their usual care until after the first 12 months. All study participants, and peer leaders, receive 1-day of basic diabetes self-management education prior to intervention groups commencing. Primary outcome is based on cardiovascular risk score (using the UKPDS equations) at baseline, 12 and 18 month follow up. Secondary outcomes include self-care behaviors, psychosocial functioning and quality of life. The program will also undergo an economic evaluation. The program partnership with an NGO and program delivery by volunteer, lay peer leaders trained to support behavior change in people with T2DM could enhance program transfer to other countries within the Asia Pacific region, where lay workers and community health workers are already integral to delivery of health services. The purpose of this 3-month pilot intervention was to test the feasibility of a peer support program for persons with type 2 diabetes in a resource-poor setting. Using a pre-post-intervention design, data were collected on A1C, perceptions of social support, coping, and diabetes self-care behaviors. Outcomes were improved daily self-care behaviors, social and emotional support, A1C values, and linkages with healthcare providers. Peer Champions (n=19) were patients who attended the Mityana diabetes clinic, could speak and read English, and agreed to contact another patient with diabetes (Partner) at least weekly. Champions received education in diabetes self-care and in providing supportive communication to peer Partners. Peer Partners (n=27) were patients with diabetes who attended the Mityana Clinic. Champions and Partners met at a community meeting where diabetes self-care education was again provided, and participants were assigned to pairs or triads. Telephones provided were linked to a pre-paid network so that calls were free if they were to another peer or champion, or to a healthcare provider. Participants who completed the intervention (28 females, 13 males) had a mean age of 55 years (range=32 to 74) and a mean duration of diabetes of 6.5 years. A post-intervention meeting was held with 16 Champions and 25 Partners to complete measurements and share experiences. Sixty-six percent of participants reported peer contact at least weekly. There was no change in scores on a 5-item diabetes coping scale, a 14-item barriers scale, physical activity or missing medications. Perceptions of social support from family and friends and a single item rating on "confidence in managing diabetes" significantly decreased (p=.03, and p=.02 respectively). There was improvement in eating behaviors (p<.001) and 77.8% reported that they contacted the clinic and healthcare providers more often than prior to the intervention. The most striking finding was a reduction (p<.05) in mean A1C from 9.8% to 6.8% (by IFCC standards). Eighty percent of participants indicated that the most helpful aspects of peer support were 'advice about taking care of diabetes' and 'encouragement to contact the clinic'. Participants especially liked using pre-paid telephones to contact healthcare providers and peers and receiving written material about diabetes self-care; however, most would have preferred materials in the local language and 92% reported difficulty reading because of poor eyesight. In conclusion, both providers and peers were actively engaged in a program that resulted in improved glycemic control and linkages with healthcare providers; other results may be explained by the resource-poor setting and unique cultural factors. In addition to the programs from Australia, China and Uganda described in this symposium, Peers for Progress has funded 11 other projects representing Argentina, Cameroon, England, South Africa, Thailand, and the United States. Observations from these provide opportunity for identifying ways in which generalizable key functions of peer support (assistance in daily management, social and emotional support, linkage to clinical care, and ongoing support) are represented in programs as well as ways in which programs are tailored to local characteristics or strengths. In particular, the experiences of individual projects have led not to major changes in the four key functions but to a) enhanced understanding of several of them, b) recognition of important implementation considerations, and c) attention to challenges in implementation. Enhanced understanding of the important of the functions has included recognition of the importance of attention to medications and adherence in a disease like diabetes, emotional and social support, and assisting individuals in navigating the health care system. Implementation considerations include recognizing varied modes of delivery (face-to-face, individual, group, telephone, etc.), the needs not just of individuals but of their family and caregivers, and the need of support providers for their own ongoing encouragement and support. In implementation, recognition has also grown regarding the importance of integration of understanding of the role of peer support across members of the health care team so that everyone delivers a solid and common message though they may be using different "languages." Challenges have included the widespreaqd and substantial barriers individuals face in transportation, infrastructure (e.g., electricity), and employment, difficulties in negotiating and establishing organizational partnerships, encouraging among peer supporters a stance of "peerage" rather than teaching, and cultural challenges such as those related to roles of women, roles of patients vis a vis professionals, etc. The four functions of peer support that Peer for Progress has emphasized provide important structure to thinking about peer support across national lines, but, nevertheless, leave much room for attention to the kinds of needs and challenges which successful implementation in individual settings must address. Insomnia is one of the most common complaints in the general population, and is associated with a range of adverse consequences and conditions. In the four presentations in this symposium, we focus on various aspects of insomnia at different stages throughout the life-span. Beginning with a longitudinal study on children with chronic illness, Dr Mari Hysing will present data on the chronicity of insomnia, as well as on behavioral and emotional correlates in this population. Dr Paula Salo will then present data from a Finish cohort examining the effect of insomnia on a range of depression-related outcomes. Dr Arnstein Mykletun will then present data on the association between individual and combined anxiety/depressive symptoms and insomnia, and cause specific mortality over a 3-6 year period. Finally, Dr. Børge Sivertsen will explore the consequences of chronic use of sleep medication among the elderly. Background: Children with a chronic illness have an increased rate of sleep problems and this has previously been related to emotional problems. Less is known about what specific aspects of emotional problems that contribute to sleep problems, and if sleep can predict the development of emotional problems in children with a chronic illness. Aim: The aim of this study was to assess the relation between sleep problems and emotional problems in general and more specifically; anxiety, perfectionism and obsessive compulsive symptoms in children with a chronic illness. Further, we want to examine sleep problems as a predictor of emotional problems from the age of 7-9 to 11-13. Methods: Using data from a longitudinal total population study in Norway, The Bergen Child Study, data on sleep problems, chronic illness, emotional and behavioral problems (SDQ; Strength and Difficulties Questionnaire) and anxiety (SCARED) were assessed at ages 7-9 and 11-13. Results: 496 children with a chronic illness in the age group 11-13 participated in the Bergen Child study, including a 295 of 4025 (7.3%) in the longitudinal sample. Children with a chronic illness had a higher rate of sleep problems and anxiety symptoms than their peers (p<0.001). In children with a chronic illness sleep problems were related to anxiety symptoms both based on parent and child report. Sleep problems were independent risk factors of later emotional problems (odd ratio=3.5), also when adjusting for emotional problems at time 1 (odd ratio=3.0). Conclusions: Emotional problems in general and anxiety more specifically, were related to sleep problems in children with chronic illness. Sleep problems predicted the development of emotional problems over time. Based on the results screening for anxiety is recommended as a part of sleep assessment in children with a chronic illness. Background: Depression is reported to be associated with increased mortality, although underlying mechanisms are uncertain. Increased cardiovascular (CVD) mortality and suicide is reported in depression, but the effect of depression on other causes of death is uncertain. The few studies of anxiety or insomnia in relation to mortality have produced inconclusive Results: Aims To investigate associations between individual and combined anxiety/ depression symptom loads (HADS) and insomnia, and cause specific mortality over a 3-6 year period. Method: We utilized a unique link between a large population survey (HUNT-2, n=61,349) and a comprehensive mortality database.
Results: Case-level depression was associated with increased mortality (Hazard Ratio=1.52, 95% CI 1.35-1.72) comparable to that of smoking (Hazard Ratio=1.59, 95% CI 1.44-1.75), and which was only partly explained by somatic symptoms/conditions. Depression increased CVD mortality as much as other causes of death. Anxiety comorbid with depression lowered mortality compared to depression alone (anxiety by depression interaction p=.017). The association between anxiety symptom load and mortality was U-shaped. There was no effect of insomnia on mortality beyond confounding. Conclusions: Depression is an important and independent risk factor for mortality across common causes of death. Comorbid anxiety reduced mortality compared to depression alone. The relationship between anxiety symptoms and mortality was more complex with a U-shape and highest mortality in those with the lowest anxiety symptom loads. Declaration of interest None. Objective: To examine polysomnographic sleep parameters as well as the prevalence of sleep apnoea and periodic limb movement disorder (PLMD) in older chronic hypnotic users compared to aged-matched drug-free patients complaining of insomnia and good sleepers. Methods: Polysomnographic data were collected at a university-based outpatient clinic for adults and elderly. 19 patients using sleep medications on a daily basis (5-7 days per week) for at least one year were compared with 64 drug-free patients complaining of insomnia and 26 good sleepers. Mean age was 63.8 years (SD=7.0). Outcome measures were polysomnographic sleep parameters (total sleep time, wake after sleep onset, sleep onset latency, sleep efficiency, slow-wave sleep), and prevalence estimates of sleep apnoea and PLMD. Results: The chronic hypnotic users spent more time awake and had reduced sleep efficiency compared to good sleepers. The amount of slowwave sleep was significantly lower among chronic hypnotic users compared to the other two groups. There were no differences between the chronic hypnotic users and drug-free patients complaining of insomnia on any of the other PSG-parameters. The prevalence rate of sleep apnoea (AHI>10) was 42% both among the drug-free patients complaining of insomnia and chronic users of sleep medications, compared to 12% in the good sleepers group. There were no significant group differences in the prevalence of PLMD between the three groups (37%-39%).
Conclusions: This study suggests that the sleep of chronic users of sleep medications is no better than that of drug-free patients complaining of insomnia. It is disturbing that 42% of the patients treated pharmacologically for insomnia also suffers from sleep apnoea. We suggest careful sleep assessment as a prerequisite for long-term prescription of sleep medications. Four unhealthy behaviors are especially prevalent. in industrialized cultures: eating a high-fat diet, consuming few fruits and vegetables, extensive television viewing, and low moderatevigorous physical activity. Public health guidelines advise correcting all of these unhealthy behaviors, but little guidance is available about the best approach. To address that question, we conducted a head-to-head comparative effectiveness randomized clinical trial (n=200) of four diet and activity interventions, all of which entailed guideline-recommended behavior change. In each condition, participants modified one diet and one activity behavior, while effects on all four behaviors were evaluated as an aggregated healthy lifestyle score. Results indicated that the conventional intervention practice of increasing physical activity and decreasing fat intake produced the least improvement in overall healthy lifestyle. Conversely, a novel approach suggested by behavioral economic theory (increase fruits/ vegetables and decrease recreational screen time) maximized acrossthe-board healthy lifestyle change. To fulfill its full potential, however, comparative effectiveness research needs to extend beyond determining only what works best for the average patient. In the present study, for example, traditional dieting (decrease fat, increase physical activity) minimized overall healthy lifestyle change for females but maximized it for males, suggesting that gender-tailored guidance might be needed. Comparative effectiveness research holds potential to undergird personalized, evidence-based behavioral medicine. The aim is for intervention decisions to be guided by evidence that conveys what works best for particular types of individuals or communities and in different settings and circumstances. Depression is a very common and disabling condition. Prevalence rates of major depressive disorder (MDD) have been estimated to be a high as 25%, with point prevalence rates estimated at up to 45% for patients with minor depression (mDD) or elevated depressive symptoms. Moreover, depression in cardiac patients has been associated with greater than a 2-fold increase in risk for adverse clinical events, including death. Although effective treatments for depression are available, the optimal ways to manage depression in cardiac patients are not known. This presentation will briefly review the prior randomized clinical trials for treating depression using cognitive behavioral therapy (CBT) and pharmacotherapy. Recent studies of exercise in treating depression also will be reviewed, and data examining the comparative efficacy of exercise, medication and CBT will be examined. The added physical and mental health benefits of exercise training in treating depression in cardiac patients will be highlighted and directions for future research in the area will be discussed. Older people are the least active segment of the population. This contributes to their high level of disease (including cardiovascular disease), disability and healthcare usage. Practical and effective interventions are needed to increase activity in this population. A brief behavioural change intervention consisting of goal setting, planning, self monitoring delivered with or without a pedometer was compared with no treatment in a 3 arm prospective study of 204 sedentary community dwelling women aged ≥70 years (average age 77 years). The primary outcome was daily activity counts assessed by accelerometer over 7 days prior to treatment, at 3 months and at 6 months. Secondary outcomes included lower limb function, health related quality of life, anxiety, depression and falls. 179 women completed the trial with most dropouts from the behaviour change alone condition (15/68). Over the first 3 months activity increased reliably more in the intervention groups than the control (which did not change). Pedometers did not add to the effects of the behaviour change techniques. The increase in activity was not maintained at the 6 month assessment. The secondary outcomes were not affected by the intervention. The psychological and behavioural processes mediating the change in activity, and the failure to sustain it, will be discussed. Heart failure (HF) affects millions of individuals and is increasing in prevalence each year as the population ages. Approximately one third of HF patients experience clinical depression symptoms, which are further associated with elevated risk of cardiovascular hospitalization and mortality. However, depression treatments such as cognitive-behavioral therapy (CBT) do not decrease morbidity/mortality in patients with coronary heart disease. This is not surprising since recent evidence suggests somatic but not cognitive/affective depressive symptoms have a greater association with cardiovascular-related mortality and events. Somatic symptoms such as fatigue and sleep disturbances are common among HF patients that may lead to physical inactivity and create a spiraling decline in physical and cardiac function. Indeed, physical inactivity is associated with reduced quality of life and greater morbidity and mortality in HF. Meanwhile, Tai Chi practice is observed to increase vigor, and reduce tension, anger and fatigue. Tai Chi emphasizes a holistic approach, both mind and body. It is a meditative exercise originating from China, and with its moderate intensity and relative ease of performance it is well-suited even for those with significant physical impairments. The present study consists of HF patients (n=29 completers; 43-83 years old; mean age=72.9, SD=5.1) that had Tai chi training (n=19) twice per week for 12 weeks or were in a waitlist control group (n = 10). At baseline and after the 12-week intervention period, patients completed the Beck Depression Index (BDI), subcategorized into somatic (BDI-s) and cognitive (BDI-c) symptoms, and the Multidimensional Fatigue Symptom Inventory -Short Form (MFSI-SF). All subjects also were measured pre-and post-intervention period for physical function, in a 6-min walk task. Compared to controls, HF patients in the Tai Qigong is a traditional Chinese energy medicine practice combining breathing, movement, and meditation. Qigong therapy is becoming a popular adjunctive treatment for type 2 diabetes. Previous studies have suggested benefits of Qigong practice for this disease; however, its effectiveness has never been rigorously evaluated in adults diagnosed with type 2 diabetes. We conducted a randomized, controlled 3-arm clinical trial in comparing 12weeks of Qigong, Progressive Resistance Training (PRT), or usual care in 32 adults with type 2 diabetes. Outcome measures included fasting glucose, hemoglobin A1c, insulin, C-peptide, BMI, HOMA-IR, Perceived Stress Scale (PSS) and Beck's depression Index (BDI) before, during and at the end of the interventions. We found that body weight increased in the usual care group, but S272 Int.J. Behav. Med. (2010) 17(Suppl 1):S1-S329 decreased in both Qigong and PRT groups. Additionally, the Qigong group showed significant reductions in fasting glucose (P=0.003) and PSS (P=0.045) and also demonstrated trends for improved insulin resistance, as measured by the HOMA-IR Index. In contrast, the PRT group showed a significant reduction in depression scores with an increase in fasting glucose and insulin resistance, which also increased in the usual care group (NS). In this presentation, I will discuss three key themes in conducting comparative efficacy clinical trials: 1) understanding the different schools of Qigong; 2) choosing the best control group for Qigong intervention; and 3) choosing an appropriate patient population with type 2 diabetes for Qigong intervention. Complementary therapies are popular and frequently used by patients with CVD. Survey data indicate that herbal therapies and mind-body therapies are most commonly used. One such treatment option is qigong. Several reviews have claimed that qigong has therapeutic effects on blood pressure in patients with hypertension. However, these reviews are non-systematic and therefore open to bias. To systematically assess the clinical evidence of qigong for hypertension. Databases were searched up to December 2009. All randomized clinical trials testing qigong in patients with hypertension of any origin and assessing clinically relevant outcomes were considered. Trials using any type of control intervention were included. The selection of studies, data extraction and quality assessment were performed independently by at least two reviewers. Methodological quality was evaluated using the Jadad score. 12 RCTs could be included. Seven of these RCTs tested qigong in combination with antihypertensive drugs compared with antihypertensive drugs alone. The meta-analysis of four trials reporting adequate data suggested beneficial effects in favour of qigong (weighted mean difference, systolic blood pressure (mmHg) -12.06, 95% confidence interval (CI) -17.12 to -7.00; diastolic blood pressure -8.46, 95% CI -12.55 to -4.37). Qigong alone was compared with waiting list control in 2 RCTs and was found to significantly reduce systolic blood pressure (weighted mean difference (mmHg) -18.46, 95% CI -23.07 to -13.86). In further three RCTs comparisons made were: qigong combined with antihypertensive drugs versus muscle relaxation combined with antihypertensive drugs; qigong as a sole treatment versus exercise and/or antihypertensive drugs, all reported positive results in at least some of the relevant outcome measures. The methodological quality of the original studies was low. There is some encouraging evidence to suggest that qigong is effective for lowering systolic blood pressure. However, the conclusiveness of these findings is limited. Rigorously designed trials seem warranted to confirm these results. After-school activities provide valuable opportunities for health promotion activities that do not interfere with the regular school day, especially in minority populations with higher rates of obesity and type 2 diabetes. The current study is an evaluation of an after-school health education and physical activity program conducted in 8 elementary schools along the U.S.-Mexico border in El Paso, Texas.
The intervention was a 10-12 week (twice a week) after-school program including a pilot (with 2 experimental schools) and main intervention (6 schools each including control and experimental groups). Each session consisted of health education taught by a Community Health Worker, based on the bilingual BienEstar ('Well-Being') curriculum, and physical activity based on the Coordinated Approach to Children's Health (CATCH) curriculum. Main outcome variables were Body Mass Index (BMI), aerobic capacity health knowledge and healthy intentions. Participants (n=1,103) were predominantly socio-economically disadvantaged Hispanic 3rd to 5th graders. At baseline, 21.6% of children were overweight and another 25.6% obese. The intervention was successful in recruitment, implementation and retention of participants. The pilot study (n=172) found a significant BMI reduction and increased aerobic capacity. In the main intervention, experimental group participants (n=323) reduced their BMI, but this reduction was not significantly larger than the control group (n=608). Intervention group participants significantly increased their healthy nutrition intentions compared to control group participants, but not health knowledge. While the findings in regards to changes in health indicators were modest, all findings were in the expected direction. The current project provides evidence for a strong need and desire to participate in projects that are accessible and address primary prevention of chronic diseases within a population that has a high prevalence of risk factors and limited access to care.
( Background: Maternal employment has been shown to influence various child/adolescent outcomes such as cognitive outcomes, educational achievement, behavioural problems, overweight, selfrated health or child fatalities. There is, however, only limited number of studies using UK longitudinal data, and limited number of data using health measures as study outcomes. The aim of this study is to cover these gaps by looking at influence of maternal employment in three periods of childhood on health outcomes among young people aged 16-21 years in British Household Panel Survey (BHPS). Methods: BHPS is annual panel survey that has started in 1991 with currently completed 17th wave of data. Response rate to wave 1 was 74%, and response between waves 2 and 17 varied between 84%-89%. There are 3,696 individuals for whom selfrated health and/or GHQ-12 at age 16-21 (study outcomes) and maternal employment data prior to age 16 years are available.
Other variables, such as gender, maternal age, maternal education and marital status, household income or maternal smoking were used as additional explanatory variables. Regression modeling in STATA 10 was used to estimate the associations between study outcomes and maternal employment. Results: 19% of young adults aged 16-21 reported poor self-rated health, and mean GHQ-12 was 10.1. Approximately 40% of mothers worked at some point during age 0-4 of their child. This proportion increased to 59% at age 5-11 and 68% at age 12-16. Children of mothers who were not employed reported OR of poor SRH 1.48, 1.11 and 1.28 for 3 periods of exposure, however these effects were mostly explained when adjusted for maternal education and household income. The differences in GHQ-12 by maternal employment were similarly reduced by indicators of social position. Conclusions: The associations between maternal employment in childhood and young adults' health exist at least partly because of the generally higher social position and more stable family structure of households with working mothers. The design of invitation and reminder letters to screen for cancer has been influenced by studies comparing the psychological and attitudinal differences between participants and non-participants. Participation in cancer screening has been thought to follow the continuum of resistance model, which assumes the same factors influence early and late participation. This study investigated the validity of the continuum of resistance model for explaining participation in colorectal cancer (CRC) screening. People aged 50 to 74 years, recruited from the electoral roll who had completed a baseline survey and were in the early stages of readiness to screen (n =376) were subsequently invited to complete a faecal occult blood screening test. Multivariate analyses examined the predictors of early participation (kit returned in weeks 1-6) and late participation (kit returned in weeks 7 -12). Results confirmed that different factors influenced early and late participation. Early participation was associated with employment status and barriers to screening, with retired people more likely to screen early than employed people (relative risk RR=1. Colorectal cancer (CRC) is a major public health problem. It is the most commonly diagnosed non-cutaneous cancer in Australia, and the second most common cause of cancer related death. The Australian government has introduced mass population screening by faecal occult blood test (FOBT) to reduce CRC incidence and mortality. However, participation in this program has been suboptimal.
Research shows that cancer decision aids increase knowledge about the benefit associated with screening and decrease levels of decisional conflict. This subsequently leads to better behavioural outcomes including participation in cancer screening programmes. The purpose of this study then, was to investigate the utility of an internet delivered CRC decisional support aid. Specific aims were to examine (1) whether changes in knowledge and attitudes after exposure to the PDS were moderated by the perceived usability and acceptability of the tool, and (2) test the degree to which perceived usability was dependent on "technophobia" as measured by computer anxiety and computer self-efficacy. Little is known about how breast cancer and beliefs about mammography relate to psychological distress assessed at the time of mammography. This study examined perceived breast cancer risk and perceived benefits and barriers to mammography in breast cancer survivors (N=64) and women with no cancer history (N =70). A key goal of this study was to examine associations between perceived risk and anxiety, depression, and mammography specific distress. In the breast imaging clinic and immediately prior to undergoing mammography, breast cancer survivors rated their perceived risk of breast cancer recurrence (M=14, SD=6) and beliefs about mammography. Women with no cancer history rated their perceived risk of developing breast cancer (M =12, SD=6). Breast cancer survivors reported higher perceived risk (p=.05) and lower perceived benefits of mammography (p = .01) compared to women with no cancer history. Perceived barriers did not differ across groups. Immediately prior to mammography, women also rated their mammography specific distress, anxiety, and depression. Levels of anxiety and depression did not differ across groups, but breast cancer survivors reported higher mammography specific distress (p<.001). Multiple linear regression analyses showed that anxiety and mammography specific distress were positively associated with perceived risk in breast cancer survivors, but these associations were not found in women with no cancer history Background: Although we have some knowledge on general practitioners' (GP) practices on sick-listing in general, there is scarce knowledge about how they make their assessments especially in the most complex cases of medically unexplained conditions. Aims: The aim of this study was to explore on considerations made by GPs when dealing with sick-listing of patients suffering from medically unexplained conditions. Materials and Methods: Data were drawn from focus-group interviews with 48 general practitioners (GP). The GPs were recruited as they participated on a course in Social Medicine. We established groups with variation regarding age, gender, nationality and work experience. Seventeen women and 31 men (15 with other nationalities) aged 32-65 accepted to participate in the interviews. The doctors had worked in general practice from 1 to 34 years. Results: Sick-listing patients with medically unexplained disorders was regarded as a very challenging task by many GPs. Trust in the patient's own story and his self-judgement is deemed crucial, but many miss hard evidence of illness and loss of function. Several factors which may influence the decision making regarding sickleave were identified: To which extent the patient manage to present his story, extensive prior knowledge of the patient, but also properties within the doctor himself: own experience as a patient, tendency to avoid conflicts and others. Strategies in dealing with sick-listing would be to initially comply with the patient's request in order to build alliance and then start to motivate for a return to work. Confrontation when they disagreed strongly on a sick note was another strategy mentioned, but at the same time risking that the patient would leave them for a more cooperative colleague. Many judged themselves as having no real power in the decisionmaking concerning sick-listing, evoking frustration in some. The LSMS data is used to estimate health and consumption spending as well as examine certain health access variables. We assessed health-related impacts on household poverty using the methodology of van Doorslaer et al. (2006) . Similarly, we constructed estimates of the proportion of the Guatemalan population incurring catastrophic levels of health expenditures to assess the financial risk of ill health using the methodology of Xu et al. (2003) . We defined catastrophic health spending (on both inpatient and outpatient) as a level of health spending that exceeds 40% of a household's ability to pay, although other thresholds are obviously possible. Results: There is both a high level and inequitable distribution of financial burden due to ill health in Guatemala. This is primarily due to low levels of insurance coverage and a heavy concentration of the uninsured among the less well off and rural populations as well as poor quality and low levels of access to public services for the poor, stagnant or declining government spending on health and increasing privatization that likely has resulted in rising treatment costs.
Conclusions: High levels of catastrophic health spending and poverty co-exist with significant economic inequality and poverty in Guatemala. With health system characteristics and an economic structure similar to many other developing countries, international experience provides useful lessons to help Guatemala devise innovative financing and payment mechanisms to address these concerns. The aim of this research is to demonstrate why in global governance Pareto principle from an economic perspective and liberty principle from a political perspective are incompatible. Impossibility Theorem is applied to analyze the international relations among players with different preferences. The theoretical proposition of incompatibility is followed by the example of conditional foreign aid, with distinction of indirect liberty and direct control. Two rival hypotheses, global Pareto condition and national liberty condition, are tested in the empirical evaluation of the foreign aid conditional on the Washington Consensus from 1989 to 1999. First, small-N design is to compare the pre-treatment and post-treatment situations in 3 representative countries, as the non-complier, partial-complier and complete-complier of the Washington Consensus. Second, large-N regressions are to estimate the effect of foreign aid on social developments measured by health outcomes: (1) foreign aid without controlling for the adoption level of the Washington Consensus, which reflects in liberty principle; (2) foreign aid after controlling for the adoption of the Washington Consensus, which reflects in Pareto principle. Furthermore, responding to the criticism of "what causes what" in the relationship of underdevelopment and foreign aid, oil price and oil reserve are introduced as instrumental variable (IV) into the model to isolate the causation of foreign aid on social developments, especially health outcomes. Finally, the concept of overlapping consensus from John Rawls is incorporated to interpret why partial-compliers are better off than both non-compliers and complete-compliers given conditionality of foreign aid. The strategy is to not only work within the domain of the impossibility theorem with empirical evidences but also step out to examine the assumptions that constrain the outcome through the inspiration of overlapping consensus. [Objectives] Depression is a heterogeneous heritable psychological trait, also influenced by environmental factors. Previous studies have found the associations between major depression and gene polymorphisms such as serotonin transporter gene linked polymorphic region (5-HTTLPR). Since fatty acid (FA) metabolism appear to contribute to the pathoaetiology of affective disorders, we examined the associations between depressive symptoms and genetic variation in FA metabolism-related genes.
[Methods] A cross-sectional study was conducted on 166 female workers of a hospital and nursing homes. Depressive symptoms and subjective psychological stress were assessed by the Center for Epidemiologic Studies Depression (CES-D) scale and visual analogue scale, respectively. Selected gene polymorphisms associated with FA metabolism as well as 5-HTTLPR were analysed.
[Results] Linear regression analysis was performed in which CES-D scores served as a dependent variable, and subjective stress and gene polymorphisms as independent variables: consequently, SS carriers of the 5-HTTLPR gene showed significant higher depressive symptoms in comparison with LL/SL carriers, even after controlling for confounders (F=27.6, standardized beta=2.2, p<0.05). Regression analysis also confirmed some other gene polymorphisms significantly associated with depressive symptoms.
[Conclusion] Some gene polymorphisms associated with FA metabolism may affect depressive symptoms as the 5-HTTLPR does.
Due to considerable heterogeneity (Q=131,10; p<0.0001), a random effects model was applied. The overall effect size found for the association between depression and IVF-outcome was statistically significant, with a higher number of depressive symptoms being associated with reduced pregnancy chances (ESr=−0,06, failsafe N=114, p<0.05 Growing evidence shows that that elevated stress during pregnancy may increase the risk of preterm delivery and other pregnancy and birth complications. Neuroendocrine, immune, inflammatory, and cardiovascular factors have been suggested as possible mediators of the relationship between stress and birth outcomes, and recent data suggest that stress-related changes in cytokine production and inflammation may contribute to these effects. Work in our laboratory has focused on quantifying the effects of perceived stress and social support on pregnancy outcome and the degree to which neural-immune interactions may be involved in these effects. Initial work in the lab focused on establishing connections between maternal prenatal stress and low social support and alterations in cytokine balance in the circulation and cytokine production by stimulated peripheral lymphocytes. This work indicated that indeed, elevated stress and lower social support was related to increases in circulating inflammatory mediators, reduction in anti-inflammatory cytokines, and similar changes in the production of cytokines in vitro by mitogenstimulated peripheral blood lymphocytes. Additional studies began to connect these findings to the likelihood of premature delivery and other complications of pregnancy, and have started to demonstrate that indeed, increased stress and distress early in pregnancy is related not only to elevations in inflammatory mediators in the prenatal period, but is also associated with higher frequency of pregnancy and birth complications. Most recently, our work has focused on the role of maternal ethnicity and acculturation in the impact of stress on pregnancy, and on the role of catecholamines in these relationships. Elevated proinflammatory cytokines were lower levels of support and higher stress throughout pregnancy, and higher social support was associated with higher levels of anti-inflammatory cytokines late in pregnancy. Moreover, elevated inflammatory markers were related to higher pregnancy-specific distress early in gestation, as well as lower 1-minute APGAR scores, lower gestational age at birth, and increased occurrence of birth complications including meconium aspiration, nuchal cord, and asphyxia. Together, these studies indicate that psychosocial factors are related to increases in inflammatory mediators and subsequently, increased risk of pregnancy complications and untoward birth outcomes. , and African American (AA) (n=1521) females in grades 9 -12 were examined. FSI history was assessed by asking the following yes/no question: "Have you ever been physically forced to have sexual intercourse when you did not want to?" The outcome variable, suicidality, was measured by a single yes/no item: "During the past 12 months, did you ever seriously consider attempting suicide?" Alcohol use, was assessed by asking, "During the past 30 days, on how many days did you have 1 or more drinks of alcohol?" Responses were dichotomized as "yes/no" to having one or more drinks of alcohol on at least 1 day in the past 30 days. , and a range of other covariates. 4 domains of change were coded into three categories: good change, no change, bad change. The analyses were then conducted for single domains and for a sum score indicating the overall rating of change. The CAGE questionnaire score, the score of drinking-related problems and mean annual intake of alcohol were used as the outcomes, and regression modelling was used to analyze the association between the exposures and the outcomes taking range of covariates into the account. The analysis was done separately for men and women. Results: After controlling for age, sex and range of possible confounding variables, the persons who rated the changes as negative have more alcohol-related problems and alcohol abuse (odds ratios of a high CAGE score 1.30 for men and 1.76 for women for those reporting negative change compared to those reporting no change). The amount of consumed alcohol is significantly higher among the "winners" (those who rate the changes as good), followed by loosers with those rating changes as neutral drinking the least. Conclusions: While the alcohol-related problems and alcohol abuse are more likely among those who perceive the social and economic changes as negative, the amount of consumed alcohol is significantly higher among the "winners" of social change. There was a large reduction in the price of alcohol in 2004 in Finland due to a reduction in alcohol taxes by one-third, on the average, and due to the abolition of travellers' duty free allowances from the EU. The present study investigated the effects of this reduction in alcohol prices on alcohol-related and all-cause mortality, alcohol-related hospitalisation, and interpersonal violence. This study used several population-based register data and statistical Methods: Time series intervention analysis modelling was applied to monthly aggregations of deaths and hospitalisation in 1996-2006 for men and women aged 15 years and over. Socioeconomic differentials in alcohol-related mortality and interpersonal violence data were analysed with a before/after design. Alcohol-related mortality was defined using information on both underlying and contributory causes of death. Hospitalisation related to alcohol was defined as those with a reference to alcohol in the primary diagnosis.
The results of this study indicated that alcohol-related deaths increased substantially among persons aged 50-69 years after the price reduction when trends and seasonal variation were taken into account. The increase was mainly due to liver diseases. In contrast to alcohol-related mortality, mortality due to cardiovascular diseases and all-cause mortality decreased considerably among men and women aged over 69 years. Among persons aged 30-59 years, the increase in absolute terms was largest among the unemployed or early-age pensioners and those with low education, social class, or income. The employed and persons aged below the age 35 did not suffer from increased alcohol-related mortality. Alcohol-related hospitalisation rates in both chronic and acute causes for men increased among those below the age 70, and for women in the 50-69 year-olds. The increase was mainly due to mental and behavioural disorders due to alcohol. The rates in interpersonal violence did not increase. These findings suggest that reduction in alcohol prices may lead to a substantial increase in alcohol-related mortality and morbidity. However, large population group differences were observed. Those in the older age groups may even benefit from cheaper alcohol in terms of decreased rates of CVD mortality. Moreover, reductions in alcohol prices do not necessarily affect interpersonal violence. The population group differences in the effects of price changes should be acknowledged, and therefore, the actions of policy should be focused on the population subgroups which are primarily responsive to the price reduction. Objective: To determine the effect of adherence within and after an exercise therapy treatment period on patients' outcome on pain, physical function and patient self-perceived effect in patients with osteoarthritis (OA) of the hip and/or knee. Methods: One hundred fifty patients with OA of the hip and/or knee receiving exercise therapy were followed 60 months. Data were obtained from a randomized controlled trial, with assessments at baseline, 3, 15, and 60 months follow-up. Exercise adherence was defined as the extent to which a person's behaviordoing home exercises, home activities and being more physically active -corresponds with agreed recommendations by the patient's physical therapist. The association between exercise adherence and patients' outcome on pain, physical function and selfperceived effect was examined using generalized estimating equations (GEE) analyses. Results: Adherence to recommended home exercises and being more physically active was significantly associated with better treatment outcome on pain, self-reported physical function, physical performance and self-perceived effect. The association between adherence and outcome was consistent over time. Adherence to home activities was only associated with better self-perceived effeCT. Conclusion: Better adherence to recommended home exercises as well as being more physically active improves the long-term effectiveness of exercise therapy in patients with osteoarthritis of the hip and/or knee. Both within and after the treatment period better adherence is associated with better patients' outcome on pain, physical function, and self-perceived effect. Since exercise adherence declines over time, future research should focus on how exercise behavior can be stimulated and maintained in the long-term. Persistent unexplained pain in the face and surrounding tissues are common presentations to dental and medical services. Routine dental treatment of chronic oro-facial pain (OFP) primarily involves using splints to correct malocclusion or teeth-clenching/ grinding. However this mechanistic approach is ineffective. Moreover, such treatments can result in physical and psychological iatrogenesis. In contrast, a growing evidence-base recognises the effectiveness of psychological interventions for OFP, suggesting a role for psychological services within dentistry. In order to translate research into practice, further understanding is needed of patients' and physicians' current experience of OFP and its management. Semi-structured interviews were conducted with a purposive sample of 25 patients and 35 clinicians (comprising medical and dental practitioners from a range of primary and secondary care services). Audiotaped interviews were transcribed and analysed thematically using principles of constant comparison to categorise emergent and recurring themes within and between transcripts. Thematic categories arising in initial interviews were explored subsequently and disconfirmatory evidence sought until thematic saturation arose. Similar themes emerged from both patients and physicians. Whilst both sets of participants recognised the role that psychological factors could play in the development and maintenance of OFP, management and self-management strategies were largely limited to biomedical interventions. Achieving a diagnosis proved problematic but functional for both parties. Frustration at the current inadequacy of OFP management often led to conflict with (or disengagement from) the clinicianpatient relationship. Current management of OFP is ineffective and unsatisfactory for both patients and clinicians and impacts on their relationship. A key barrier to implementing psychological interventions for OFP is the ineffective communication between physicians and patients, and between medical and dental practitioners. Objectives: Reaching the rehabilitation goals the doctor and the patient have agreed on appears to be important, and there is presumed to be a correlation between goal attainment and patient satisfaction. The main goal of the study was the detailed assessment of patient satisfaction and the investigation of the correlation between patient satisfaction and the setting and attainment of goals.
Methods: The cross-sectional questionnaire-based assessment was performed anonymously by 276 patients in psychosomatic rehabilitation at the end of their inpatient stay. In this way patient satisfaction, sociodemographic, disease-related and outcome parameters as well as rehabilitation goals at the beginning of rehabilitation and their attainment at the end were assessed in a standardized way. Results: The primary goals set by patients were related to symptom reduction and stress coping. Goal attainment scales based on factor analysis showed a high degree of goal attainment with respect to "life style and priorities" and "psychotherapeutic goals". Patient satisfaction correlated most significantly with the goal attainment level at the end of rehabilitation (r=.62), followed closely by the subscale "attainment of psychotherapeutic goals" (r =.57). With regards to the number of attained or non-attained goals it was found, that both had a significant relationship with patient satisfaction, but the non-attaining of goals (r=−.55) was more closely related with patient satisfaction than the attainment (r=.40). Discussion: The results confirm the central role of goal setting and goal attainment in rehabilitation. The explicit consideration of goal setting in the rehabilitation process as well as the assessment of goal attainment appears to be unavoidable. Evidence-based practice (EBP) can be defined as accountable health care policy that conscientiously funds those interventions whose effectiveness is supported by research. As applied to particular individuals, communities or populations, EBP uses best available evidence to make decisions about how to promote health. The EBP process integrates three domains of information:
(1) best available evidence; (2) client characteristics, including values and preferences; and (3) resources. The process proceeds through a step sequence that includes appraising the quality and contextual relevance of research evidence.
As the major health professions have embraced evidence-based practice globally, practice and policy guidelines have increasingly become based upon systematic evidence reviews. Dissemination of systematic evidence reviews has become a worldwide enterprise supported by the Cochrane and Campbell Collaborations. However, a number of challenges surround the uptake of evidencebased practices, especially when interventions are introduced into cultures very different than those from which they originated. After Bonnie Spring introduces the topic, speakers will present lessons learned when implementing globally in diverse real-world settings behavioral medicine interventions that were validated in an Anglo-European context. Ross Brownson will discuss findings derived from introducing public health physical activity campaigns in South America. Geoffrey Setswe will discuss implications for intervention of social aspects of HIV/AIDS and health across the African continent. Brian Oldenburg will share observations based on the spread of diabetes interventions between different countries, cultures, and populations. Discussant, Stephen Weiss will synopsize lessons learned about the process of engaging in evidence-based practice when the relevance of the available research is unclear. Presenters will address three issues: 1) How can an evidence-based intervention best be tailored to a local individual or community? 2) How should an investigator evaluate both the adequacy of contextualization and the success with which active intervention ingredients were preserved? 3) What does treatment fidelity mean when interventions are to be implemented in diverse cultural contexts? Introduction: Many HIV prevention interventions have been tested for efficacy or effectiveness. However, in almost three decades of the HIV/AIDS epidemic, we are still not sure which interventions work best in preventing HIV transmission in different communities and cultural settings. This presentation provides evidence of HIV prevention interventions that work and also presents lessons learned about applying them in different contexts. Methods: A systematic review of randomized controlled trials (RCTs) testing HIV prevention interventions for evidence of efficacy or effectiveness was conducted. The interventions were classified as biomedical, behavioral and structural. The evidence provided by RCTs was graded in four different levels: best (80% or more), good (60-79%), promising (30-59%) and poor or no evidence (<30%). We calculated the effect size to show the difference between treated and control conditions. Findings and Discussion: Using the grading system, male and female condoms and PMTCT were classified as best evidence HIV prevention interventions because they had achieved more 80% level of efficacy or effectiveness. ART, male circumcision, HIV Counseling and Testing for people who test positive were classified as good evidence HIV prevention interventions as they had achieved between 60 and 79% level of efficacy or effectiveness. Treatment of STI, a microbicide called PRO2000, the RV144 Thailand vaccine trial and a structural community RCT that reduced intimate partner violence were classified as promising evidence HIV prevention interventions as they had achieved between 30 and 59% level of efficacy or effectiveness. All other interventions that obtained less than 30% effectiveness were classified as poor or no evidence. We will present the effect size of these interventions. Conclusion: There is no "magic bullet" for HIV prevention, particularly as applied in different cultural settings. The lesson learned is that the acceptability of intervention strategies is not the same across most cultures and individuals. A community should choose to use a combination of interventions that best suits their context from among best, good or promising practices. (2) How to extend the reach of such programs to low and middle income countries, as the current trial evidence based in such countries is currently very limited? This presentation considers the development, implementation and evaluation of such programs in Finland and Australia, as well as the recent uptake of lifestyle-related interventions for reducing diabetes risk in other countries. The GOAL study in Finland involved the "real world" evaluation of a moderate-intensity group-based lifestyle change counselling program to prevent type 2 diabetes, adapted from the original Finnish Diabetes Prevention Study (DPS). Drawing on evidence from the Finnish DPS and the short-term results from the GOAL implementation trial, the Australian Diabetes Prevention Program (DPP), adapted from the GOAL program, achieved similar positive behavioral and clinical outcomes. Currently, more routinized programs, that are building on the evidence from these 'real world' implementation trials, are being implemented and evaluated in Finland and Australia. As key components of the Finnish GOAL program have become more refined over time, successive versions of the program in different countries are aiming to improve the 'fit' between characteristics of the interventions, the adopters, and the implementation environments. Core components of the GOAL program are now also being adapted for use in programs that are currently being evaluated in Malaysia, India and South Africa. There is an urgent need for more collaborative and coordinated approaches that compare and contrast methods for adapting and spreading effective intervention programs between settings, regions, and countries. Adults diagnosed with cancer are often faced with multiple treatment options. Many want to participate with their physicians in the process of deciding which treatment is best for them. This symposium features four projects that are testing various applications of the SCOPED process in different settings, using different delivery models and intervention components. SCOPED is a six step process for helping patients formulate and analyze issues, synthesize insight, and translate the insight into action. The steps include documenting questions and answers about the Situation, Choices, Objectives, People, Evaluation, and Decisions. Each project will present data about using the SCOPED process to inform and involve adults making cancer treatment decisions. Introduction: The 'Better Cancer Care' campaign launched by the Scottish Government promotes partnership between patients and healthcare professionals. The aim of this study in progress is to evaluate decision navigation using the SCOPED question-listing and note taking process within NHS Scotland. Methods: Newly diagnosed breast and prostate cancer patients attending the Edinburgh Cancer Centre are invited to participate. Patients are randomized to intervention or control. Decision self efficacy (DSE) is measured in both groups (scale of 0 to10) at baseline (T1) and after the consultation (T3). DSE is additionally measured in the intervention group after the decision navigation session (T2). Intervention patients also receive a service evaluation form at T2. Preliminary Analysis: Prostate: N=63 age range 56-78 years. As of November 2009, there are no differences between groups at T1 on the DSE x=8.39 in the intervention group and x=8.3 in the control group (p=.82). At T3 there is a significant difference between groups x=9.0 in the intervention group and x=8.3 in the control group (t(51) = 2.059, p=.045). 90% of the intervention group (n=24) found decision navigation 'very helpful' and 10% (n=3) 'somewhat helpful'. Breast: N=40 age range 41-81 years. 79% of the intervention group (n=15) found decision navigation 'very helpful' and 21% (n=4) 'somewhat helpful' at T2. The intervention and control group do not differ significantly on the DSE. Feedback from a subset of patients (n=9) stated that the intervention was "supportive" "helped to structure thoughts" and "reason through the decision process". Discussion: It is premature to draw conclusions from these preliminary data. Eventual differences in impact of decision navigation between breast and prostate patients may be attributable to the greater degree of choice left to prostate patients. In contrast, breast cancer patients are strongly advised to follow set treatment guidelines for adjuvant therapy. Introduction: Open to Options, a pilot project of ENACCT and CSC provides one-on-one support to hematological cancer patients in Philadelphia, PA, San Francisco/East Bay, CA, and Cincinnati, OH. Methods: Trained CSC facilitators use the SCOPED process to prompt patients to list questions for their providers. Patients complete pre and post session surveys, and follow-up evaluations at 30 and 90 days. The surveys measure distress, anxiety, question self-efficacy, satisfaction and (for a subset), patient use of the question list during a health care provider visit. Results: Three facilitators have used the SCOPED question-listing process with 42 patients. Among patients, there was a significant decrease in distress (p=0.044) and anxiety (p=0.045) and a significant increase in the patient's question self-efficacy (p< 0.001). To date, 26 have made a treatment decision, and 4 (15%) of these chose treatment through a clinical trial, which is higher than the national participation rate of 3-5%. Thirteen of 25 respondents reported discussing cancer clinical trials with their oncologist. Satisfaction among participants was high post-intervention (mean= 8.6 out of a maximum of 10) and remained high at 30 (mean=8.5) and 90 (mean=8.7) days follow-up. Fifteen of 16 patients surveyed (94%) reported bringing their question list to their provider appointment, 13 (81%) thought the question list contributed to a more productive appointment, and 13 (81%) reported their oncologist answered most of their questions. Participants reported the question list: "gave structure to the meeting like an agenda"; "clarified my thoughts"; and "was empowering." Discussion: Preliminary findings suggest use of the SCOPED process for question-listing among blood cancer patients in a community-based setting may be associated with improvements on psychosocial measures and with enhanced enrollment in clinical trials.
CORRESPONDING AUTHOR: Melissa F. Miller, PhD, MPH, Cancer Support Community, Fairfax, VA, 22033; melissa@cancersupportcommunity.org in 74 female and male participants, who were grouped according to gender role on the basis of the Bem Sex Role Inventory. Using sex, gender and psychological traits (catastrophizing, fear of pain), it was found that sex, but not gender, strongly predicted pain responsivity in nearly all pain measures and fear of pain was a mediator. The fear avoidance model postulates that in chronic low back pain (CLBP) a fear of movement is acquired in the acute phase, which leads to subsequent avoidance of physical activity and thereby drives the chronification of the pain syndrome. In an event-related fMRI study we investigated neural correlates of the hypothesized fear of movement. 60 women (30 CLBP patients, 15 healthy controls and 15 women with spider phobia, mean age: 46.8±9.8 years) participated. The CLBP patients were divided on the basis of the Tampa Scale of Kinesiophobia (TSK) into a high and low fear-avoidant group. They viewed 120 photographs depicting neutral and painful movements, generally fear-inducing and neutral objects from the International Affective Picture System (IAPS) and spiders while functional MRI data were acquired. For CLBP patients activations were found in brain areas commonly implicated in emotional processing (cingulate cortex, orbitofrontal cortex) for the contrast of painful > neutral movements. However, in a random effects analysis, CLBP patients with high fear avoidance did not differ from those with low fear avoidance or controls with regard to this contrast. For the contrast fear-inducing IAPS pictures > neutral pictures, CLBP patients with high fear avoidance exhibited an activation in the left anterior insula, which was not found in those with low fear avoidance. The spider phobics showed extensive bilateral activation in the cingulate cortex and the insulae for the contrast spiders > neutral pictures and the random effects analysis revealed marked differences in these areas between the spider phobics and the healthy controls.
On the basis of the fear avoidance model we expected group differences between CLBP patients with high and low fear avoidance as well as between CLBP with high fear avoidance and controls. Regarding neural correlates such no such differences were found, thereby casting doubt on the concept of fear avoidance as a process primarily driven by fear or phobic fear. We examined the hypotheses that sex and gender, assessed by the Bem Sex Role Inventory, modulate responsiveness to experimentally induced pressure pain. Furthermore various psychological variables assumed to be potential mediators were assessed. 74 students took part in the experiment. Pain threshold (PT), intensity (PI) and unpleasantness (PU) of a standard pressure stimulus were determined as well as sensory and affective pain (SP, AP). Analysis of variance revealed a significant main effect of sex for nearly all pain variables. Women exhibited a lower PT, rated pain to be more severe and unpleasant, and reported more SP and AP. Against our hypothesis gender role did not influence pain behaviour. Fear of pain (FPQ / PASS) and catastrophizing (PCS) showed significant correlations to all pain parameters except PT. The variable depressive symptoms was almost without influence whereas coping style modulated pain responsiveness. Gender Role Expectations of Pain (GREP) correlated only in a few cases with pain responses. Using sex and gender and all significant psychological variables as predictors in multiple regression analyses showed that essentially sex and fear of pain significantly predicted pain. PT was the only parameter that was not influenced by psychological variables. In summary, our study corroborated a distinct effect of sex regarding mechanical pain stimuli, showing women to be more responsive. In contrast the expected effect of gender role was not confirmed. Fear of pain seems to mediate the effect of sex on pain responsiveness, at least partially. Chronic fatigue syndrome (CFS) is a debilitating disorder for which no single etiological factor has been identified. Stress has been suggested to play a role in the manifestation and maintenance of CFS. Various studies haven shown associations between different aspects of stress (e.g. childhood trauma, maladaptive coping, adult chronic stress) and fatigue. However, no studies have simultaneously measured and analyzed multiple characteristics of stress in one single study of CFS. In the current population-based study, 501 participants took part and provided information on early life stress, personality characteristics, chronic stress, coping, cortisol as biological stress marker, and CFS symptoms. Data were analyzed using structural equation modelling (SEM, LISREL). After comparing various theoretically sound models, the strongest model focused on the combined effect of childhood trauma and personality features. This model shows a strong effect of maladaptive personality features on CFS symptoms, in part mediated by adult chronic stress and coping. The impact of childhood trauma was significant, but the effect was lower than that of personality. SEM analysis indicates a good fit of this model (Chi2=855.19, p= 0.0, GFI=.88; AGFI=.85, CFI=.95, SRMR=.05; RMSEA=.071, p (RMSEA) = 0.0). Our findings indicate the importance of stress in CFS. We have combined various aspects of stress in our study and were able to test a model encompassing a broad variety of stress characteristics. Future studies should include longitudinal designs in order to test causal relationships. It seems critical to devote research resources to a detailed understanding of the processes that lead from stress to CFS in order to improve current treatment strategies. Background: Attrition and workload are serious issues for faculty members, faculty associations and university administration. Despite the debates about methods for measuring workload, there is a high level of agreement that faculty members devote large amounts of time to their work, causing high levels of stress, and affecting their well-being and work commitment and. This study explores relatively underexamined questions related to factors affecting faculty members and their intention to leave their jobs, such as work demands, control over work, work-related rewards and stress pertinent to work in academic settings. Methods: The faculty workload study was carried out using an online survey that completed 637 faculty members. The data on faculty intentions to quit, workload as an indicator of work demands, control over work, rewards, and work-related stress specific to the university setting were collected with a survey developed for this study. This study also collected open-ended, qualitative data that are included in the report, and compared this data with that from similar Canadian surveys, i.e., the Labour Force Survey (LFS) and the Work and Lifelong Learning Survey (WALL). Findings: The study shows a high faculty workload, as demonstrated by the large number of work hours, significantly exceeding the national average found in the LFS and the WALL surveys (41% of faculty work more than 60 hours per week), and by the large number of courses taught during the academic year (24% of faculty teach five to eight courses). The results show significant bivariate associations between faculty's desire to leave their jobs with the majority of indicators related to workload, control over work, rewards, and work-related stress. Multiple logistic regression analysis, controlled for relevant demographic factors (age, sex and years of service), shows that faculty who perceive low control over work demonstrate the strongest likelihood of leaving their jobs (adjusted odds, AO= 3.775), followed by those who perceive lack of rewards (AO= 2.402), high work-related stress (AO = 2.363), and higher workload (AO=2.278). Conclusions: Similarly to the growing number of studies demonstrating that the control-demand and effort-reward models complement each other, this study identifies the important roles of both models of work-related stress, but suggests that among faculty members, control over work plays a highly important role in their intention to quit.
CORRESPONDING AUTHOR: Milosh M. Raykov, PhD, University of Toronto, OISE, Toronto, ON, M5S 1 V6; milosh. raykov@utoronto.ca Predictors for specific stress responses (BPS anger, guilt and pressure) were the same and in the same direction. In addition, co-worker social support predicted stress responses of guilt (β= −.19, p=.04) and anger (β=−.19, p=.04). The total variance explained by these predictors in nurses' stress responses ranged from 15 to 27%. Some professional variables (night shift work and daily work load) and family variables (number of children at home) were also significant predictors of reported stress (variance explained ranging from 4 to 8%). Findings support the high incidence of distress in Portuguese nurses reported in other studies. Furthermore, the data points to the need to consider the family-home spill over in occupational health intervention programs. Coping resources and co-worker social support constitute key variables in reducing work stress among this population. OBJECTIVE: The objective of this study was to evaluate the relative contribution of individual characteristics, lifestyle factors, work-related risk factors, and work ability on the occurrence of short (<2 weeks), moderate (2-12 weeks), and long (>12 weeks) durations of sickness absence. METHODS: Altogether 5867 cement construction workers with complete sick leave registration were followed from the day of their medical examination in 2007 until the end of 2009. The main outcome of the study was the duration of sickness absence, as registered by an occupational health service. Independent variables consisted of individual characteristics, lifestyle factors, workrelated factors, and the work ability index. We used Poisson regression analyses with repeated occurrence of sick leave to calculate rate ratios (RR) and 95% confidence intervals of independent variables for the three categories of sick leave duration. RESULTS: Predictors for sick leave lasting 2-12 weeks and >12 weeks were: older age, obesity, smoking, manual materials handling, lack of job control, lung restriction, and a less than excellent work ability. For most predictors, higher RR values were observed with a longer duration of sickness absence. Obesity, smoking, manual materials handling, and lack of job control remained important risk factors for moderate and long durations of sick leave after adjusting for the strong effects of work ability on sickness absence. The highest population-attributable fractions were observed for: age over 50 years (18%), manual materials handling (20%), and good (18%), moderate (28%), and poor (2%) work ability. CONCLUSION: This study suggests that a variety of preventive measures targeted at smoking, obesity, physical load, psychosocial work factors as well as work ability will contribute to a reduction in the occurrence of sick leave. Background: Women generally show higher frequencies of DSM IV symptoms of PTSD, depression, and anxiety than men. Yet, widowers have relatively higher excess mortality rate after the loss than widows compared to non-bereaved counterparts. When nonbereaved controls are not taken into account, widowers and widows show similar frequencies of symptoms and gender differences in coping with spousal bereavement appear to be relatively stable across previous studies. Objectives: The aim was to investigate the importance of gender in relationship to other risk factors for poor bereavement outcome, using an integrative framework to enable the relative impact of different variables to be established. Methods: 296 elderly bereaved people (Mean=73 years, 38% men) were examined at 2, 6, 13, and 18 months post loss using self-report scales measuring posttraumatic stress, coping style, crisis support, and personality factors. Results: A selection of major risk factors was entered into a hierarchical linear regression. The model predicted 49% of the variance in long-term traumatic stress (18 months). When conducted for widows and widowers separately, the predictive power of the model rose to 62% for the widows and 58% for the widowers. For the widows, emotional coping, emotional loneliness, and satisfaction with social support predicted 62% of the variance. When early traumatic distress (2 months) was entered at the last step of the model, the predictive power of the model remained unchanged. For the widowers, sense of peace and satisfaction with social support predicted 40% of the variance in long-term traumatic distress. When early distress was entered, the predictive power of the model rose to 58%. Conclusion: Stable, interpersonal traits such as emotional coping style may predict bereavement outcome well for widows, while strong early distress may be a better predictor for widowers. Frequently, widowers are excluded or combined with widows in research. These results indicate that false conclusions will be reached, particularly regarding widower's reactions, if this is done. Age-related subclinical brain pathology is associated with poor health outcomes, though relations with health related quality of life (HRQoL) are poorly understood. This study investigated sex differences in relations of subclinical cerebrovascular disease (SCD), brain atrophy (BA) and HRQoL in 154 healthy older adults (50.6% male; mean age=66 years) free of major medical, neurologic, and psychiatric disease. Participants completed the Medical Outcomes Survey Short Form-36 (SF-36) and underwent brain magnetic resonance imaging (MRI) rated by a neuroradiologist for markers of SCD (i.e. periventricular and deep white matter hyperintensities, number of silent infarcts) and BA (i.e. ventricular enlargement, sulcal widening). We create two rank-sum variables (SCD, BA) to serve as primary predictor variables. Multiple regression analyses (adjusted for age, race, education, blood pressure, body mass index, smoking, and Beck Depression Inventory scores) examined the interaction of sex with SCD and BA on HRQoL. Results revealed that for women, SCD was negatively related to the Mental Component Score of the SF-36 (MCS; rsq=−.09, b=−4.26, p=.02). For men, BA was negatively related to the Physical Component Score of the SF-36 (PCS; rsq=.06, b=−1.58, p=.04). Results suggest that deleterious changes in brain structure (particularly SCD) are related to poorer outcomes in mental health dimensions of older women's quality of life. Conversely, in men, brain atrophy relates to poorer physical function. Thus, there are sex differences in the relations of subclinical brain pathology to HRQoL in older adults. OBJECTIVES: Objective of this study was to investigate relationship between self-reported functional disabilities and measured physical performance among aged homecare clients DESIGN: Baseline measurement of a physical performance intervention SETTING: Health centers in six semi-urban and rural municipalities in Finland PARTICIPANTS: One hundred and seventy-one aged (>mean age 82 yrs.) home care clients MEASUREMENTS: Functional balance was measured using the Berg Balance Scale (BBS), physical performance by the Short Physical Performance Battery (SPPB), the 10-meter walking time test, the hand grip strength test and self-reported disability in activities of daily living (ADLs) and instrumental activities of daily living (IADLs) by Euro-Reves. RESULTS: Significant correlations were found between the number of functional limitations and the measured performance status. Self reported disabilities were strongly related to the measures of physical performance and in the hierarchical regression model these variables explained 40% of the variance of the functional limitations CONCLUSIONS: Self-reported difficulties in the ADL and IADL activities were in line with previous studies indicating that the progression of physical disability follows a hierarchical pattern, starting with difficulties in mobility, progressing to difficulties with IADLs, and, finally, culminating in problems with ADLs. A threshold of physical performance on/above which people seem to report no/minimal disability was found. However, even a high performance status does not entirely protect a person from having multiple disabilities. Present study indicated that physical performance measures are independent predictors of functional disability and therefore interventions should directly address factors associated with physical performance in order to enhance autonomy of aged persons. Background: Frailty is a geriatric concept that represents a lack of reserve functional capacity in older people. It has practical clinical implications because it constitutes a state of health that is still modifiable but is on the threshold to chronic irreversible health problems. In this study we analysed the development of frailty over a ten-year follow-up period in older Dutch men and women from different socioeconomic groups. Methods: Data came from a nationally representative cohort of 1,509 men and women aged 55-85 years. Frailty was measured with the following markers: physical activity, and grip strength, BMI, cognitive functioning, vision and hearing problems. A maximum of 4 observations of the degree of frailty were acquired over a ten-year follow-up period. Indicators of socioeconomic position were education level and household income. We conducted several types of linear mixed model analyses to assess socioeconomic inequalities in the development of frailty, including complete case analyses, and with frailty imputed for those who died during follow-up. Results: Adjusted for age, sex, and a partner being present inside the house or not, primary educated had higher odds of developing frailty than tertiary educated men and women (OR=1.76; 95%CI: 1.05-2.97), and the lowest income groups had a higher odds than the highest income groups (OR=1.90; 95%CI: 1.20-3.02). Interactions of education and income with time of frailty measurement suggested that the association of education and income with frailty increased during follow-up. Analyses with imputations for missing observations due to deaths yielded similar results. Conclusion: Having a high socioeconomic position is still protective against one of the core geriatric health problems; frailty. This suggests that differences in vulnerability late in life may account for socioeconomic inequalities in health at even later stages of life, but also that reversing these inequalities is partly feasible. The purpose of this study is to assess cognitive function in a random population undergoing regular eye exams at a Family Practice Eye Service. The goal of the study is to identify individuals at risk for Mild Cognitive Impairment (MCI) in order to help develop early treatment for the prevention of Alzheimer's disease and other mental disorders. Study aims:1) To determine the predictors of mild cognitive impairments in older adults of diverse background attending a Family Practice Eye Service; 2) To evaluate ethnic group variations; and 3) To evaluate a standardized computerized screening battery to detect mild cognitive impairment on a random sample of older adults. Study Hypothesis: A great number of elderly patients attending a community Eye Clinic will present significant Mild Cognitive Impairment (MCI) as measured by the Stroop Test, Reaction Time Test (RTT), Shifting Attention Test (SAT), and Continuous Performance Test (CPT) from the CNS Vital Sign Computerized Battery Neuropsychological Assessment. Study Design: Feasibility study and cross-sectional. Outcomes: Demographical, cognitive, mood and visual assessments (one time; cross-sectional). Statistical Plan: Descriptive statistics (proportion, range, means, and SDs), correlations, test of mean differences. Results: Hypothesis is supported by cognitive assessment analysis of 143 research participants. These results support that 55% of the older adults attending the community Eye Clinic showed at least one abnormal score in one of the cognitive tests, indicative of mild cognitive impairment or cognitive impairment. Further data and results will be discussed. Little is known about the long-term effects of group intervention programs targeting physical exercise. This paper reports on the effectiveness of MoVo-LISA, a theory-based (MoVo-concept) standardized intervention program. Participants are taught cognitive-behavioral strategies of goal-setting, action planning, barrier management and self-monitoring. Methods: N=220 in-patients of an orthopedic rehabilitation clinic were assigned (quasi-experimental design) to the control group (usual care) or the intervention group. Assessments were conducted at five time points. Results: At 12 month follow-up, level of exercise in the intervention group was 28.5 min/week higher than in the UCG (p=.05). Moreover, 50% of the IG was exercising for at least 60 min/week, but only 33% of the UCG (p=.01). Results also revealed significant intervention effects on cognitive mediator variables: the IG reported enhanced self-efficacy and more positive balance of outcome expectations at six months as well as stronger goal intentions, more elaborated implementation intentions, and optimized strategies of intention shielding at 12 months follow-up. Conclusions: Results provide evidence that intervention programs based on the MoVo concept lead to long-term improvement in exercise behavior and underlying cognitive factors. Aim of study: This community-based exploratory study investigated the results of supervised low technology training on function, physical activity, and falls in older people with functional limitations. Methods: Home-dwelling people from four municipalities (n= 123, age 75-95 yrs) were included. Supervised balance and strength training was performed at home for 16 weeks. External load was 65% of 1RM. The instructors used the principles of positive reinforcement to enhance motivation for regular physical activity. After the 16 weeks contact was established between volunteer instructors and participants who wanted to continue exercising. Assessments were performed by health professionals who did not train the participants. Function (static balance (SPPB) and Senior Fitness test) was assessed at baseline and after 16, 32 and 52 weeks. Self reported data were collected at baseline and after 52 weeks. Results: At baseline 86% used walking aid, 89% had balance problems and 62 % had fallen during the previous year, and 3% met the physical activity recommendations. Seventy-six % completed the intervention. Those who were able to rise from a chair without using the arms (83%) improved in static balance, 30 s-chair-stand, 30 s-arm-curl and 8-feet-Up&Go on average 16%, 50%, 34% and 16%, respectively (p<0.001). Improvements were preserved at 52 weeks except for the 30 s-chair-stand, but these results were better compared to baseline (p<0.01). No changes were found in participants who needed the arms to rise from a chair, and drop out was high in this group (60%). At 52 weeks the participants were more physically active with 55% exercising in groups or with an exercise-friend (p<0.0001). Objectives: Traumatic Brain Injury (TBI) is among the most common causes of brain damage in the United States and results in the disturbance of cognitive, behavioral, emotional, and physical functioning that leads to functional impairments, disability, and decreased quality of life. However, there are very few studies to date that have sought to determine the relationship between objective functional status evaluations and subjective QoL measures in individuals after TBI. The purpose of this study was to explore the relationship between functional status and QoL over time controlling for demographics and injury severity after severe TBI. Methods: This prospective study used longitudinal data collected from patient questionnaires and from medical records of subjects with severe TBI (N=46). In addition to demographic and injury related data, patient functional status was measured by Glasgow Outcome Scale (GOS), Disability Rating Scale (DRS) and Functional Independence Measure (FIM). Patient QoL was evaluated using the Modified Perceived QoL questionnaire. Data was collected regarding 6 month and 12 month for both functional status and QoL. The baseline QoL was recalled by patients after TBI. A mixed effects model for repeated measures was performed to examine the relationship between each measure of functional status and QoL overall in patients 6 and 12 months after TBI while controlling for age, gender, injury severity and QoL at baseline respectively. Results: Overall, QoL measure significantly improved over time (P<0.05). Baseline overall QoL and GOS were both significantly and positively correlated with the overall post-TBI trajectory QoL score (P<0.01). DRS was significantly and inversely associated with overall post-TBI trajectory QoL score (P<0.01). There is no relationship between FIM and overall post-TBI trajectory QoL score (P>0.10). Age, gender and injury severity were not significantly related with overall post-TBI QoL scores (P>0.05). Conclusions: As a subjective measure of TBI outcome, the QoL of patients after TBI continues to improve over time. Baseline QoL before TBI and objective global function measures (GOS and DRS) are strongly associated with long term QoL for TBI patients. For frail elderly persons, exercise is essential to preserve functional fitness and quality of life (QOL). Municipalities in Japan offer elderly persons interventions consisting of groupbased exercise sessions and behavioral counseling; however, for a variety of reasons, many individuals do not participate in the interventions. Some are physically frail, some have limited means of transportation and others are self-conscious about exercising with friends. Therefore, it is important to confirm the effect of low-frequency exercise participation compared with that of highfrequency. Thirty-nine participants, aged 67-85 years, participated in this study. All were identified as frail elderly persons according to the criteria of the Ministry of Health, Labor and Welfare, Japan. The participants were divided into two groups that were assigned to different intervention schedules. The high-frequency group (n= 14; 68-83 years) met 12 times over a 3-month period, while the low-frequency group (n=25; 67-85 years) met 6 times over a 3month period. Each 90-minute session consisted of group-based exercises and behavioral counseling. The exercises, which the participants were encouraged to practice daily at home, included 4 stretching and 6 strengthening exercises. To encourage exercise, 6 behavioral counseling sessions were offered in which illustrated stories or four-panel cartoons were used. After 3 months, members of both groups showed significant improvement in functional fitness as well as in 5 of 8 dimensions of QOL measured by the SF-36. Improvement in the social dimension of QOL was significantly higher in the high-frequency group than in the lowfrequency group. Thus, both low-and high-frequency interventions offer significant positive effects for frail elderly persons. If elderly individuals need a great amount of socialization, a highfrequency intervention is recommended. Although physical activity (PA) provides multiple benefits for breast cancer survivors (BCS), most do not exercise regularly. Identifying mediators of PA maintenance is important for assisting BCS in adopting sustainable PA behavior change. Because we have reported continued improvements in PA 3 months after completing a PA behavior change intervention for BCS, our study aim was to determine if the change in social cognitive theory (SCT) constructs during the intervention mediated the effect of our intervention on PA. We randomized 41 BCS to receive a SCTbased 3-month PA behavior change intervention (INT; n=21) or usual care (UC; n=20). The INT group received 12 supervised aerobic exercise sessions tapered to home-based exercise. Behavioral support was provided in 6 discussion groups and 3 individual face-to-face counseling sessions. The UC group received written materials. PA (i.e., accelerometer) and SCT constructs (i.e., survey) were assessed at baseline, immediately post-intervention (i.e., 3 months), and 3 months post-intervention (i.e., 6 months). The mediation of the intervention effect on PA at 6 months by changes in SCT constructs from baseline to 3 months was examined using the Freedman and Schatzkin approach. Compared to UC, the INT group reported improved barriers interference (mean difference=−7.8; 95% CI=−15.1 to -.4; d=−.67; p=.04) and PA enjoyment (mean difference=.7; 95% CI=0 to 1.5; d=.61; p =.06). Barriers self-efficacy d was .28 (p =.38). Barriers interference and self-efficacy mediated 39% (p=.004) and 19% (p=.02) of the intervention effect on sustained adherence, respectively. In conclusion, only barrier interference and barriers self-efficacy significantly mediated the effect of the intervention on PA 3 months after intervention completion. The increase in enjoyment did not explain sustained PA improvement. Our results suggest that reducing barriers may partially explain the ability of our intervention to demonstrate longer term PA adherence. Treating pediatric obesity is essential to reduce adult morbidity and mortality and improve the health of obese youth. We report 6month nutrition data from a 2-year multidisciplinary weight management program for obese adolescents, the T.E.E.N.S. Program (N=173 at baseline, N=66 at 6 months). At baseline, participants were 76% African American, 65% female, with a mean age of 13.7. Mean BMI percentile was 99.1 (mean BMI z-score=2.5) at baseline. Repeated measures MANOVAs were used to examine differences from baseline (T1) to 6-month (T2) followup on a 24-hour dietary recall measure. Changes in anthropometrics and blood lipid parameters were also evaluated. At 6months, participants significantly reduced their intake of calories, fat, sodium, and sugar (p<.05); no changes in calcium or fiber intake were found (p>.05). Gender stratified models showed that boys had greater increases in fruit and vegetable and vitamin D intake compared to girls (p<.05). Significant improvements in body mass index and lipid profiles were found in the total sample (p<.05). Multiple regression analysis suggested that caloric reduction from baseline to 6-month was associated with decreases in BMI z-score (β=−0.11, p<.05). However, when parental compliance with nutrition visits was included in the regression, change in calories was no longer significant. Parental compliance became the only significant predictor of 6-month BMI z-score, while controlling for baseline BMI z-score (β=−0.16, p<.01). Findings suggest that promoting intake of essential nutrients may be more challenging than reducing excessive intake. Additional gender specific strategies for dietary modification may be warranted. Further, increased parental participation in adolescent weight management programs is also recommended to enhance weight loss. Regular physical activity (PA) is a challenge for obese persons. It is even more challenging for obese persons with either depression or pain, but the impact of these conditions may differ. The present study examined the effects of depression and pain on perception of barriers to PA, confidence about engaging in PA, and current weekly PA in a group of overweight/obese male veterans. Study participants completed the Barriers to Being Active Quiz, Exercise Self-efficacy Scale, and the International Physical Activity Questionnaire. The study sample included 101 male Veterans with BMI≥25. The mean age was 61.2 years. Of the sample, 62.4 % reported a painful medical condition (i.e., either arthritic joint pain or back pain) and 48.5% reported a clinically significant Patient Health Questionnaire-9 depression score≥15. Reporting a pain condition was marginally associated with depression (p=.064). Of the study sample, 85.9% reported weekly PA, ranging from walking to vigorous activity. Amount of activity was brief; only 27.7% reported PA≥1 hour. Two factor ANOVAs assessed the contributions of both pain and depression on barriers to PA and exercise self-efficacy. Results revealed a main effect for depression on three of seven perceived barriers to PA: lack of energy, motivation, and skill level. In contrast, a main effect for pain was revealed for one barrier, fear of injury, as well as for exercise selfefficacy (p's<.05). Veterans with pain (but not depression) were less likely to report engaging in weekly PA (X2=4.0, p<.05). Results suggest that although frequently comorbid in obese Veterans, depression and pain are associated with different perceived PA challenges. Depressed Veterans are more likely to report energy and motivational barriers to PA, while Veterans with pain are specifically concerned about hurting themselves during exercise and are less confident that they could actually engage in exercise under challenging conditions. In turn, veterans with pain are less likely to engage in any weekly activity. Aim: We examined whether experience of health care discrimination was associated with weight gain in normal weight, moderate obese and severe obese individuals. Methods: Men and women with normal weight (BMI 18.5-24.9), moderate obesity (BMI 30-34.9) and severe obesity (BMI>35) were identified from a yearly population based national survey investigating living conditions. Data on height and weight were available from the years between 1996 and 2006. A questionnaire about experiences of previous health care discrimination and current weight and height was mailed to 5018 individuals in 2008.
Multiple linear regression analyses were performed investigating the association between weight gain and health care discrimination controlling for a range of socio-demographic and psychological variables. Results: Totally 2633 individuals had complete data on all variables used. The average weight gain in the different weight groups according to sex was: 2.5 kg (SD=6.1) for normal weight women, 2.4 kg (SD=10.0) for moderate obese women, -0.3 kg (SD=15.7) for severe obese women, 3.7 kg (SD=7.4) for normal weight men, 1.4 kg (SD=8.2) for moderate obese men and -1.3 kg (SD=14.4) for severe obese men. Health care discrimination was not associated with weight gain in men (-0.3 kg 95%CI -1.8; 1.1). This was regardless of weight status. In severe obese women, experience of health care discrimination was associated with an average weight gain of 6.3 kg (95%CI 1.0; 11.6). No association was found between health care discrimination and weight gain in normal weight women ( Background: As a society we are increasingly fixated on the consequences of 'being fat'. Public responses have focused on alerting individuals to the risks associated with obesity and the urgent need for intervention and self-regulation. However, there is limited understanding of the impact of these messages on obese adults. This study sought to explore obese individuals' perceptions and attitudes of population based obesity messaging in Australia, how they applied these messages to themselves and how personal and social contexts and experiences influenced these perceptions. Method: We adopted a qualitative approach which utilised indepth semi-structured telephone interviews with a community sample of obese adults (Body Mass Index 30 and over). Theoretical, purposive and strategic sampling techniques were used to ensure a broad representation of individuals from different demographic and geographical backgrounds. We analysed the data using a descriptive thematic approach.
Results: One hundred and forty two adults participated in this study. Participants' attitudes were strongly influenced by personal and contextual factors, including personal experiences with weight and weight loss attempts. Participants felt that messages were overly focused on the physical rather than emotional health consequences of obesity i.e. the risk of getting cancer, or dying from obesity. Many, particularly those who were severely obese, described feeling stigmatised and blamed by the simplicity of messages and the lack of realistic solutions available to them. Whilst they trusted government based messages, they acted upon the solutions provided by the commercial weight loss industry. Participants supported non-stigmatising messages from noncommercial sources that were focused on encouraging individuals to make healthy lifestyle changes, rather than promoting weight loss. Conclusion: This study is the first step in exploring the ways in which we understand how obesity messages resonate with obese individuals in Australia and influence their weight loss behaviours. However, further qualitative and quantitative research is needed before we fully understand the impact of obesity messaging on the health beliefs, behaviours and wellbeing of obese adults. Objective: Bariatric surgery has been indicated as a preferential treatment for morbid obesity and is associated with a set of physical and psychological improvements. The aim of our study was to characterize the current functioning and the physical and psychological health problems of bariatric surgery candidates in the surgery moment, in a follow-up of 6 and 12 months. Methods: 30 obese subjects filled Childhood History Questionnaire, Brief Symptoms Inventory, Rotterdam Symptoms Checklist, Health Questionnaire and Ways of Coping Questionnaire at the surgery, in a follow-up of 6 and 12 months. Results: The number of reported adverse childhood experiences does not change over time and the prevalence was low. Preliminary results indicate that at the time of surgery, participants reported more passive coping, more problems and physical health complaints than in the 6 months follow-up. 12 months after, there is an increase of psychopathology, passive coping and physical complaints. Conclusion: The prevalence of adverse childhood experiences is lower when compared with other studies with this population. There is an improvement on current functioning and quality of life after 6 months, however, these gains are going to be blurring the end of 12 months. Obese seem to use more passive coping strategies and do not actively involve in solving their problems.
These results reinforce the importance of developing interventions aimed at behavioural change allowing the empowerment of patients and learning more active coping strategies. Moreover, it is necessary to follow these subjects longitudinally allowing greater and better understanding of its evolution during treatment. Individuals diagnosed with a head and neck cancer (HNC) are at substantial risk of experiencing disfigurements and dysfunctions as a result of the disease process and medical treatments. Consequently, it is not surprising that a substantial number of studies have revealed that HNC patients are susceptible to experiencing clinical levels of anxiety (including PTSD) and depressive disorders. Surprisingly however, there is a paucity of psychological studies targeting the management of stress and depressive disorders in this population. Similarly, there are only a very limited number of studies that have evaluated the efficacy of psychotherapy programs tailored for other types of oncology populations (including breast, prostate, and gastro-intestinal cancers) specifically suffering from clinically elevated anxiety or mood disturbances. Cognitive Behavioural Therapy (CBT) has been shown to be helpful in managing anxiety and depression in various psychiatric and health populations. Several studies have found the early application of a brief version of CBT, to be a useful treatment in reducing severe acute stress, as well as preventing the development of chronic psychological disorders in survivors following non-cancer traumatic events. Accordingly, the aim of the present RCT study (still in progress) was to test an early, brief (6-session) CBT program in reducing clinically elevated posttraumatic stress and depressive responses in newly diagnosed HNC patients undergoing radiotherapy. The efficacy of the CBT program was compared to a 6-session Supportive Counselling (SC) intervention. Participant inclusion criteria comprised adult individuals (aged 18 -70 years) who met subthreshold or full diagnostic criteria for cancer-related PTSD and/or comorbid anxiety and depression disorders, and who had been prescribed to receive radiotherapy as part of their medical treatment for their HNC. To date, 25 patients have been randomized to receive either the CBT or SC intervention, and 16 patients have completed the therapy program. The results to date indicate that the CBT program is effective in reducing both posttraumatic stress and depressive symptoms, as well as enhancing social well-being. Findings will be discussed in context of the utility of this early brief intervention in treating acute stress and preventing chronic psychopathology in traumatized cancer patients. Discussion will also focus on logistical considerations in conducting psychotherapy concurrent with medical treatments in oncology populations. There is increasing recognition of mindfulness and mindfulness training as a way to decrease stress and increase psychological functioning. The aims of this study were to examine the effects of mindfulness stress reduction training on perceived stress, psychological well-being and levels of cortisol, and to examine if changes in mindfulness mediate intervention effects on these outcomes. Seventy women and one man with a previous cancer diagnosis (mean age 51.8 years, SD=9.86) were randomized into an intervention group or a wait-list control group. The intervention consisted of an 8-week mindfulness-training course. A repeated measures MANCOVA showed that compared to participants in the control group, participants in the mindfulness-training group had significantly decreased perceived stress, post-traumatic avoidance symptoms, and increased coping self-efficacy at 6-month followup (F16, 51=1.32, p<0.05). Those who participated in the intervention reported significant increase in mindfulness when compared to controls (F10, 57=2.34, p<0.05). The increase in mindfulness mediated the effects of the intervention on perceived stress, and coping self-efficacy. The results indicated and intervention effect on awakening cortisol. Among those with initial low levels of cortisol, there was an increase from baseline to 3-month follow-up, and among those with initial high levels there was a decreased level of cortisol at 3-month follow-up. There was an association between changes in stress symptoms and cortisol levels. This study indicates that increased levels of mindfulness can potentially explain reduction of stress symptoms resulting from mindfulness stress reduction training. The results indicated a normalization of cortisol levels as a result of the intervention. The importance of these findings for future research in the field of mindfulness is discussed. Objective: To test the effects of an Expressive Writing Intervention (EWI) on subsequent self-reported health of women treated for early stage breast cancer and to examine repressive coping as a moderator. Methods: Breast cancer patients (n=507; mean age 53.6) were recruited following completion of adjuvant treatment randomly assigned to 3 weekly 20 min home-based writing exercises involving either: 1) emotional disclosure (EWI) about one of their most traumatic experiences, or 2) a control topic (CTRL) of how they spend their time. The primary outcome was self-reported physical health (Patient Health Questionnaire (PHQ)). Repressive copers (n =161) were identified at baseline by having high defensiveness (Marlowe Crowne Social Desirability Scale) and low negative affectivity (Taylor Manifest Anxiety Scale). Results: A repeated measures ANOVA showed no effect of EWI (time x group interaction) (p=.31) but did reveal a significant time x group x repressive coping interaction (p=.024). Repressive copers in the EWI group had improved PHQ-scores at follow-up (9 mo), while PHQ-scores of repressive copers in CTRL worsened. The results continued to be statistically significant (p=.045) with potential confounders included in the model. Conclusions: An expressive writing intervention may have positive effects on health outcomes among breast cancer patients high in repressive coping, suggesting the potential utility of targeting the intervention to such individuals in future studies. The 'teachable moment' has long being considered a cornerstone of public health intervention planning. The Teachable Moment (TM) model suggests that there are certain times when an individual is especially ready to change behavior and thus, open to receiving messages about behavior change. According to the TM model, experiences that jointly (1) increase risk perceptions, (2) prompt concordant emotional responses, and (3) impact selfconcept may offer a powerful motivational context for promoting behavior change. Further, tailoring smoking cessation interventions on TM components may increase the salience of health messages. In the present study, TM model was used to examine the desire to quit smoking among family members of lung cancer patients. Further, the relationship between desire to quit and engagement with and reactions to self-help smoking cessation materials was examined. Participants were family members of lung cancer patients recruited for a randomized controlled trial testing a counseling intervention for smoking cessation. The main independent variable was lung cancer diagnosis in a family member and the main dependent variable was intention to quit smoking. Study results indicated that components of the TM model were related to desire to quit in family members of lung cancer patients. Specifically, increased perceived risk, negative self image, and high subjective norm for quitting were related to high desire to quit. In a final path analysis model that controlled for age, sex, ethnicity, education and smoking rate, the construct of perceived individual risk of cancer was identified as a mediator of the relationship between having a family member with a diagnosis of lung cancer and quitting intention. Findings on engagement with and reactions to materials were mixed. Engagement with materials was not related to baseline desire to quit, but was related to positive reactions to the materials. Those with a higher desire to quit were more likely to report that the information in the tailored booklet applied to them, and was new to them, interesting, trustworthy, and moving. Finally, family members with a higher desire to quit were more likely to say that the information in the booklet make them want to quit smoking. Evidence from this initial study indicates that the TM model provides a strong conceptual framework for (1) identifying specific determinants of desire to quit among family members of lung cancer patients; and (2) developing effective tailored selfhelp materials for this population. These data suggest that the concept of the teachable moment may be adopted and adapted for the smoking cessation in the oncology context in Indian set up. Purpose: Worldwide, the number of cancer survivors is increasing. In the Danish population of 5.4 million people, more than 300 000 are cancer survivors. Given the range of physical, psychological and social late effects of cancer and its primary treatment many survivors need tailored rehabilitation interventions. This randomized study evaluates the short term effect of a multi-focused, psychosocial 6-day residential rehabilitation course at the Dallund Rehabilitation Centre in Denmark. Methods: Breast, prostate or colorectal cancer survivors were randomly assigned to the rehabilitation course (IG) or a standard care control group (CG). Outcome measures included self-reported quality of life (qol), psychological mood and mental adjustment styles and were assessed at baseline, 1-, 6-and 12-month follow up. Multiple linear regression was used in preliminary analyses to compare differences between IG and CG. Global qol at baseline, cancer diagnosis, age, gender and education were examined as confounders. Results: The preliminary analyses included 352 patients (IG n= 165 and CG n=187). A total of 188 patients had breast cancer, 94 had colorectal cancer and 70 had prostate cancer. Participants were on average 61 years old and 228 were women. No effect of the intervention was found on change in global qol at 1-month follow up (β=0.40; 95% CI -2.95-3.74). Conclusion: This is the first study to evaluate a residential rehabilitation course among cancer patients in a randomized design. Preliminary analyses could not confirm an effect of the intervention on qlobal qol. This might be due to methodological limitations of the study. However, the effect of the intervention on the different domains of quality of life and other outcome measures at the 6-and 12-month follow up taking into account clinical variables still needs to be examined. Prenatal stress seem to have a significant impacts on the biological and psychological development in the offspring. In particular, prenatal stress is associated with long-term disturbances in HPA axis function.A sample of 115 10-year old children was examined in a cross-sectional study design. The experimental group (n=43) was characterized by a glucocorticoid treatment of their mothers due to high risk of preterm delivery during the second trimester. Control group A (n=35) consisted of children who were at risk of being born prematurely but did not receive glucocorticoid treatment. In control group B (n=37) children were included born after complication-free pregnancy. A standardized psychosocial stress test for children (TSST-C) was applied. All children were healthy and showed no disparities in their level of development. Salivary cortisol and heart rate were measured during the experiment as well as psychological parameters using self-report questionnaires (EWL-KJ and PASA). The TSST-C induced a marginally significant increase of salivary cortisol (F(4.814,269.601) = 1.939, p=.091). Significant higher cortisol levels were observed in children of the experimental group as compared to children of control group B. Referring to the data for the heart rate in response to the TSST-C a trend towards significance was revealed (F(15.069, 836.313) = 1.505, p=.096). lower heart rates were found in The experimental group showed lower heart rates than the control groups A and B. Furthermore, stress appraisal and mood significantly differed between the three groups.Even though all children were comparable concerning their level of education and mental abilities, we found subtle differences in their psychobiological stress reactivity. The prenatally stressed offspring demonstrated a stronger glucocorticoid response in reaction to challenging situations than controls. In addition, the results revealed long term effects of prenatal glucocorticoids on psychological and on autonomic reactivity to a standardized psychosocial stress test. Background: Previous work suggests that family conflict impacts adversely on child development. But it is not clear to what extent these relationships might hold in a contemporary population setting. Objectives: 1. to examine the extent to which markers of parental conflict are associated with child development 2. to assess the influence of socioeconomic and psychosocial factors in observed relationships. Methods: Data from the UK Millennium cohort study were used (N=9144). Questions on parental conflict -whether they felt they were on the brink of separation and on the experience of physical force/violence in the relationship were answered by both parents when cohort members were age 9 months, 3 and 5 years. Markers of child development at age 5 were height, socioemotional difficulties, and verbal ability. Results: Girls and boys whose mothers felt they were on the brink of separation from their partner were shorter (girls 109.0 vs 110.3, boys 110.9 vs 111.3 cm), more likely to have socioemotional difficulties (girls 9.7 vs 4.1, boys 15.7 vs 6.4%) and lower verbal test scores (girls 55.5 vs 57.1, boys 52.9 vs 56.5) compared with children where this was not the case. For girls height differences remain statistically different on adjustment for socioeconomic and psychosocial factors, whilst differences in socioemotional difficulties and verbal ability were rendered statistically nonsignificant on adjustment for psychosocial factors. For boys differences in the likelihood of socioemotional difficulties and verbal ability were reduced on adjustment for socioeconomic and psychosocial factors but remained statistically significant. Conclusion: Children living in homes in which there was likely family conflict had poorer developmental outcomes compared with children living in more favourable circumstances. Gender differences in factors that explained observed patterns were apparent. Objective: The aim of the study was to ascertain the prevalence of multiple pains (MP) and detect psychosocial risk factors assessed one year prior. Method MP was defined by the number of sites for which pain was reported to occur at least "sometimes" in a sample of 2219 German youths (10-17 years).
Results: More than one site of pain was reported by 37% of the children. Girls were much more prone to MP than boys. A regression model revealed that prior MP, age, gender, internalizing / externalizing symptoms, dysfunctional coping, and watching TV explained 33% of the variance: The contribution of the psychosocial factors to the model was small. Separate analyses for boys and girls displayed different risk factor profiles and a higher degree of predictability in girls. Conclusion: It is assumed that the variable MP represents a dispositional, rather stable trait conceived as pain vulnerability. Most lifecourse research concentrates on how early life factors influence adult health through processes enacting within and across generations. Our paper makes the case for the inclusion of adolescence as a distinct stage in life course studies. We introduce a model to capture the most important pathways and mechanisms working over adolescence to develop adult health inequalities, and review and include relevant literature to illustrate the evidence for these pathways and mechanisms.
For each of the four pathways: the health-, the health behaviour-, the relational-and the educational pathway, the paper investigates whether indicators are socially patterned in adolescence, whether they track into adulthood, and whether any socially differential tracking or -vulnerability has been investigated. The paper focuses on examples of indicators from each pathway that is prevalent and/or have serious implications for later life: pains, overweight, depression, low physical activity, low fruit/vegetable intake, smoking, alcohol consumption (behavioural indicators), lone parenting, bullying victimization (relational indicators) and education.
Most health indicators are socially patterned in adolescence and track into adulthood, with higher risks of adverse outcomes among individuals from lower socioeconomic positions. Adolescent health behaviours track into adulthood. Smoking, physical activity and especially fruit and vegetable intake are socially patterned, while evidence for social patterning of alcohol-use is less consistent. Lone parenthood and bullying are socially patterned and track over time, and there are indications of a socially differential vulnerability to the effects of these types of relational strain.
Very little research has investigated the social patterning over time or studied social vulnerability of most of the above indicators from adolescence to adulthood. However, all four mechanisms: socially differential exposure, tracking, socially differential tracking and socially differential vulnerability seem to be active in establishing social differences in adult educational attainment. In recent years, there has been increased attention to the impact of trauma on children and youth. Evidence suggests that devastating natural disasters (such as hurricanes and bushfires) (La Greca et al. 1996; Yelland et al., in press), injuries resulting from motor vehicle and other types of accidents (Meiser-Stedman et al., 2008) , and life-threatening medical conditions such as cancer (Phipps, Jurbergs & Long, 2009 ) are associated with the development posttraumatic stress (PTS) symptoms and disorder (PTSD) in children and adolescents. Although existing research has focused on mental health reactions of child trauma victims, very little attention has been devoted to the health effects of traumatic events. This is especially surprising, in that stress has long been known to affect immune functioning and susceptibility to disease. Nevertheless, there has been recent interest in the health effects of children's trauma exposure (Fairbank & Fairbank, 2009) , and this symposium will address this issue. The three papers in the symposium address the impact of traumatic stressors on child health. Each of the papers in the symposium focuses on different traumatic stressors and their potential health effects. The first paper by Kenardy, Anderson, and Le Brocquehus focuses on the predictive value of posttraumatic stress on health outcomes in a large sample of children with mild to severe traumatic brain injury. The second paper by La Greca and colleagues will present two recent studies that examined the impact of destructive natural disasters (hurricanes) on children's somatic symptoms. Finally, the third presentation by Llabre and Hadi presents data on two studies of the psychological and health consequences of war exposure on children. All authors will discuss the implications of their findings for assessing children affected by traumatic events. Hierarchical regressions revealed that hurricane experiences accounted for 7.2% of the variance in somatic symptoms at Time 2 (p=.001). Time 1 PTSD also predicted Time 2 somatic symptoms (p=.001), but hurricane experiences were not significant when PTSD symptoms were included. Major life events added to the prediction of Time 2 somatic symptoms (p<.001). Study 2 included 328 children (50% girls; M age=8.72 years) who completed similar measures 9 months post-disaster. Using regression to predict somatic symptoms, demographics were entered first (8% of the variance, p<.01); then hurricane-exposure (14% of the variance, p<.001); and finally children's PTSD symptoms (16% of the variance, p<.001). In the final step, only PTSD symptoms, child's age (β=−.15) and stressors occurring during the hurricane (β=.11) remained significant. These studies are the first to examine health outcomes in children exposed to natural disasters. Children exposed to destructive disasters and who experience disaster-related stressors report significant somatic symptoms 9 and 21 months postdisaster. Findings suggest that children exposed to natural disasters may be vulnerable in terms of their physical health as well as mental health. Further attention the health status of children exposed to natural disasters is important in to address. Two studies, conducted in the Middle East, document the psychological and health consequences of war exposure in childhood. One study, conducted in Lebanon after the Lebanese-Israeli war in the summer of 2006, included a population-based sample of over 6000 children in grades 1 through 12 who were assessed for multiple exposures, prior exposure, sociodemographic characteristics, and symptoms of posttraumatic stress (PTS). The prevalence of PTS symptoms was estimated at 27.7% in children who were in grades 1 to 5 and at 26.4% in children and adolescents in grades 6 to 12. Specific symptom clusters had higher prevalence. Between 41-53% (depending on age) reported difficulty in falling and staying asleep. Older age was associated with increased symptoms. Past and concurrent exposures to warrelated events were the strongest predictors of PTS symptoms. The other study included 151 Kuwaiti children who were 7-10 years old during the first Gulf war, selected for participation based on their level of exposure to war-related events. Participants were assessed two years after the war and again as young adults (19-23 years old). As adults they reported on their general health, medical diagnoses, and sleep habits. At both assessments they reported on symptoms of posttraumatic stress, depression, and anxiety. A structural model supported the notion that psychological distress mediates the effect of war exposure on self-rated health, quality of sleep, and obesity. The session will conclude with a brief discussion with all presenters participating Research on Iraq War veterans who have experienced traumatic brain injuries has found that posttraumatic stress mediated the effect of the brain injuries on health outcomes. The aim of this paper is to explore the predictive value of posttraumatic stress on health outcomes in children with mild to severe traumatic brain injury (TBI). 204 children aged 6 to 14 years were recruited after admission to hospital for TBI. Participants were followed before 2 months and at 3, 6, 12, and 18 months post injury. Posttraumatic stress was identified via a structured clinical interview (CAPS-CA). Health outcomes were assessed using the Child Health Questionnaire (CHQ). The presence of PTSD was found to be unrelated to the severity of the TBI. After accounting for the severity of the TBI, PTSD was associated with longer recovery times over the 18 months as assessed by physical outcomes. Of greater concern PTSD was associated with a lack of recovery as assessed by psychosocial outcomes. This has significant implications for the assessment and rehabilitation of pediatric TBI. Subjective health complaints, modern health worries and coping. Camilla Ihlebaek Terms like functional somatic illnesses, unexplained medical symptoms, and somatization disorders have been used on a variety of combinations of multiple symptoms, with diagnostic labels such as fibromyalgia, irritable bowel syndrome, multiple chemical sensitivity, chronic fatigue syndrome, and chronic whiplash syndrome. These conditions are described by common subjective health complaints such as musculoskeletal pain, gastrointestinal discomfort, tiredness, fatigue, allergic and respiratory complaints, depression, and anxiety. Subjective health complaints are common in the general population. For most people the complaints are minor everyday nuisance, however for some it becomes substantial and is given a diagnostic label and professional help is needed. An important question is why and to whom this happens. A possible mechanism for such multiple symptoms could be somatic and cognitive sensitization. Furthermore, two cognitive mechanisms that seem to be closely linked to the process of sensitization are worrying and coping. There is an increasing interest of health in media and in the population. This might lead to worries concerning features of modern life and its affect on health. Worrying could play a role by enhancing biased memory and interest of health related information, leading to sensitization. Therefore, modern health worries could be associated with health complaints and medically unexplained symptoms. Individuals that uses active problem solving coping strategies have been found to have lower levels of subjective health complaints than others in stressful work situations. An essential condition for coping to occur is that the individual expects a positive result of the handling of a stressful situation, i.e. positive response outcome expectancy. The result of no control of the outcome would be learned helplessness and negative response outcome expectancy could be defined as hopelessness. Both hopelessness and helplessness may lead to depression and multiple subjective health complaints. In this symposium the relationships between subjective health complaints, modern health worries and coping will be discussed. Background: Worries about environmental and technical changes affecting people's health have been shown be associated with the use of health care services, health behaviours, mood and reporting of physical symptoms. Aim: In a representative sample of the German population we examined the relationship of modern health worries to demographic factors. depression, symptom reporting and health-related quality of life. Methods: 2,524 participants of a representative sample of the German completed the Modern Health Worries Scale, Depression and Somatization Module of the Patient Health Questionnaire, as well as the SF-12 health-related quality of life scale. Socioeconomic status and demographic information were also gathered.
Results: Higher levels of modern health worries were found in females but were not associated with income, age or education. Higher levels of modern health worries were significantly associated with depression, symptom reporting and lower healthrelated quality of life. Hierarchical regression analyses show depression to mediate the relationship between modern health worries and symptom reporting. Conclusions: In a large general population modern health worries were higher in females but unrelated to other demographic factors. Depression seems to play a more important role than modern health worries in explaining symptom reporting. Background: 3 workshops were offered to 367 clinicians working with patients with long term conditions (LTC) (diabetes, COPD, depression and chronic musculoskeletal pain) as a part of The Health Foundation's Co Creating Health Initiative. Workshops teach techniques that impact on the clinician-patient relationship and support patient self-management. The ADP is co delivered by clinical and a lay tutor who has a LTC. Aims: Assess the overall effectiveness of the ADP in improving clinicians' self management support practices. Method: 78 clinicians responded, before and after attending the ADP. Self management support (SMS) practices was assessed by the Practices in Self Management Support questionnaire (PSMS) developed by the authors. The questionnaire comprises 3 subscales assessing Clinical Self Management Support (CSMS), Patient Centeredness (PC) and Organization of Services to Support Self Management (OSMS). We also interviewed 14 completers asking them about their experience of ADP. Results: Paired T Test results showed that after completing ADP clinicians significantly increased PC practices (t=−1.9; df=75; p= 0.05). CSMS and OSMS did not change.
Interview data showed that the most useful practices were agenda setting, goal setting, exploring ambivalence and assessing patient's confidence to change. Time restrictions and lack of systems in place to support self management on organizational level are considered the biggest barriers for SMS. Conclusion: ADP training effectively builds clinicians' SMS practices. Further research is necessary to establish whether applying those practices in clinical consultations influence patients' engagement in self managing their long term condition. A husband's neglecting/abandoning her if diagnosed with breast cancer is a woman's greatest fear and barrier to screening/early detection, a major reason for rising breast cancer morbidity and mortality in Malaysia. Thus, MENCARE!, focusing on men advocates to empower women for breast cancer screening, has been implemented. MENCARE! trains men working in public and private corporations to provide support for women's breast cancer screening. Project effectiveness was evaluated by assessing changes in policies and procedures in one public agency and changes in men's knowledge, attitudes on breast cancer, and actions to support women's breast cancer screening, using multistage pre & post advocacy evaluation design with mixed Methods: One month and one year post-training evaluation of 75 male advocates through questionnaire surveys and 3 focus group discussion (FDG) were conducted. In-depth interviews were conducted with 5 management personnel pre and one-year post advocacy. Organisationally, while existing health package within the agency still did not include breast cancer screening, it would support all cancer treatment 100% at the one year post-advocacy. The flexible time-off procedure for male staff needing to support spouse/family for breast cancer screening continued a year later. "Many of them have gone for screening" was observed by one personnel interviewed one year post-advocacy. Request for a mobile mammography clinic on-site was made by management a year later. Individually, the training increased male staff's knowledge of breast health, cancer and screening in certain though not all aspects. The training increased their willingness to be involved in all types of support for women: to discuss/motivate women to get early and complete treatment, to prepare types and levels of support. Yet, men expressed some problems: lack of awareness/ exposure to what is commonly regarded as a women's disease/ problem; embarrassed to talk about women's breasts; lack of screening facilities and medical insurance, especially in rural areas; and men's busy work schedule. Buying in of public or private corporations and targeting male support at the work-place appears strategic in addressing women's delayed or low uptake of breast cancer screening through changes in organisational policies and procedures as well as individual changes in knowledge, attitudes and support actions. Once a health intervention is proven effective at a technical level, the steps required to generate a delivery system that produces positive health outcomes are nontrivial and poorly understood. For governments, the political environment often precludes experimentation or data-driven decision-making at the program level. Particularly in HIV, the political processes often fail to serve those most at risk, the socially marginalized. We explore the Bill and Melinda Gates' Foundation's novel approach of building a large-scale HIV prevention program in India, called the Avahan Initiative, through partnerships with NGOs, small-scale delivery experiments and rigorous data collection, to then transition to the government, to understand its effectiveness as a strategy to yield scientific programming and policy nationally.
In 2009, we interviewed over 70 stakeholders, including key decision-makers at the government, Avahan, and implementing NGOs. Fieldwork also included site visits to various programs and interactions with community members. Data included costing and organizational level metrics. Commonly mentioned distinctions between Avahan's approach and that of other aid organization and the public sector, include: depth and application of data, capacity building of local NGOs, financing mechanisms, and intensiveness of technical assistance. Avahan's decisions around how to interact with other key players in HIV in India featured prominently in discussions around sustainability. Many of Avahan's strategies, metrics and programmatic structures have been adopted as national guidelines. Assessments of the feasibility and logic of the transition strategy varied widely across stakeholders. Though it is premature to assess the success of Avahan's transition strategy, our research suggests that it achieved and maintained an effective HIV prevention program and has influenced governmental policy significantly. Large-scale NGOs may be well positioned to conduct iterative delivery experiments and ensure that governments adopt and promote robust public health models. Background: Secondhand smoke (SHS) is a major preventable cause of death and disease. Many countries have enacted laws banning smoking in public places as part of their strategy to decrease the ill effects of smoking. In some countries this strategy appears to be successful in reducing smoking and in reducing levels of non-smoker's exposure to SHS. In recent years some countries have banned smoking in pubs and bars, the last public venues where it was still allowed. Subsequent studies measuring SHS have shown dramatic drop in SHS following the ban. In 1983, a law banning smoking in public places in Israel was enacted and amendments to the basic law were added over the years. The latest amendment came into effect in 2007, adding pubs and bars to the list of public places in which it is prohibited to smoke. Objective: To measure the adherence of bars and pubs with the law banning smoking in them, and to identify variables that may predict adherence to the law. Methods: This study includes data collection from two sources. (1) Passive airborne nicotine monitoring devices used to measure indoor SHS in pubs and bars in about 20 Jewish-Israeli cities.
(2) Face-to-face interviews with bar owners were conducted to identify variables predicting implementation of the law. Results: Two years after the Israeli smoke-free law has come into effect levels of SHS in most of the pubs and bars remains relatively high, however, in some bars the ban is implemented.
Smoking and SHS is higher in bars and pubs in cities where the city-hall does not enforce the law by sending inspectors to inspect bars and pubs and give fines. Bar owners respond directly to the chances of receiving a fine. Conclusions: Although Israeli non-smoking legislation is progressive, enforcement is lacking, and smoking in public places still exists. Law enforcement is the responsibility of the local authorities, however, not enough has been done to enforce the law and city-halls should be encouraged to enforce the law more successfully as this has a profound effect on SHS in the pubs. Background: Stress is a consequence of different types of stressors that all separately are associated with IHD. It has not been investigated whether the impact of stress on IHD is similar for all kinds of stressors or whether accumulation of stressors increases the risk of IHD/MI.
Objective: To study the effect of individual and cumulative psychosocial stressors on the risk of incident IHD and MI among men and women. Material: In 1991-3 a random sample of 9,599 participants, 57 % women, from the Copenhagen City Heart Study answered a range of questions on major life events (LE) in childhood, adulthood and at work, vital exhaustion, lack of social network and daily use of psychotropic medication and were followed in a nationwide hospital discharge register until 2007. Results: During follow-up 257 experienced an event of MI and 714 an event of IHD. The hazard ratio of IHD among those exposed to life events both in adulthood and at work was HR 1.25 (CI 95% 0.9-1.7), whereas no association was found between life events in childhood, adulthood or work-related, respectively, nor for all three types of LE accumulated. The association between Vital Exhaustion ≥10 and IHD was HR 1.79 (1.3-2.4), and people lacking a close confident had a HR of IHD of 1.73 (1.0-2.9). We found no association with MI. By means of logistic regression we found an association between accumulated LE in child-, adulthood and work-related and Vital Exhaustion ≥10 with an OR 11.9 (CI 95% 7.8-18). The association between accumulated LE and lack of a close confident was OR 4.52 (2.2-9.2). We were unable to elucidate any gender difference. Conclusion: In this population-based cohort study we found that neither Individual nor accumulated LE are associated with IHD/MI, but are associated with other psychosocial risk factors of IHD/MI. Objectives: To test if health beliefs mediate the relation between family history of coronary heart disease (CHD) and 10-year CHD incidence in a large population-based sample of Canadians (n= 2,688), controlling for established CHD risk factors. Background: CHD is the leading cause of death in the United States today, with family history (FH) long known as an important risk factor. We tested the hypothesis that those who believe that CHD is not preventable, possibly because they have a positive FH, do not alter their health behavior (in this case exercise) to be cardioprotective. Methods: Cox proportional hazards models were used to estimate the adjusted hazard ratios (HR) of FH, preventability belief, and exercise for incident CHD. We first constructed a baseline Cox proportional hazards regression model of the association between incident CHD and FH controlling for age at baseline and Framingham risk score (an index of standard CHD risk factors). We then constructed a second Cox proportional hazards regression model to test if preventability belief was related to FH and a third model to test if belief could be viewed as a mediator of the FH-CHD relation. Finally we evaluated if the preventability belief-CHD relation was itself mediated by exercise .Results: Preventability belief was correlated with family history for both genders (β=.724, 95% confidence interval [CI] .592-.885, p<.005), with those that had a family history of heart disease less likely to believe that heart disease can be prevented. Preventability belief was also found to be a mediator between family history and CHD, but for women only (β=2.51, 95% CI 1.65-3.80, p<.001). Finally exercise mediated this relation, again for women only (β=− .868, HR=.420, 95% CI (HR) 0.770 -0.907, p<.001) Conclusion: Health belief in the preventability of CHD was found to be an important mediator in the relation between family history of CHD and incident CHD in women because of its effect on exercise. Heart failure (HF) affects millions of individuals and is increasing in prevalence. Close to a third of HF patients exhibit clinical levels of depressive symptoms. In turn, depression is associated with greater morbidity and mortality in HF. The mechanisms linking depression and worse outcome in HF are unclear. One suggested pathway links depression with heightened inflammation that may then be related to undesirable cardiac remodeling in HF. However, there have been mixed results in the literature connecting depression with inflammation in cardiac patients. Methods: The present study attempts to further delineate relations between depression and inflammatory markers in HF patients by using an exercise task to unmask group differences. Further, components of depression were examined separately to determine if inflammatory markers were differentially associated with somatic or cognitive depression symptoms. 40 NYHA II-IV HF patients and 35 healthy controls performed a moderate 20-min bicycle exercise task and completed the Beck Depression Inventory (BDI), sub-categorized into somatic (BDI-s) and cognitive (BDI-c) symptoms. Plasma levels of IL-6 were measured at baseline, 10 min and 30 min after exercise. Repeated measures ANCOVA were performed, controlling for age, gender, body mass index and physical function. Results: Both HF patients and controls with elevated BDI-s exhibited higher plasma levels of IL-6 at baseline, which remained elevated until 30 min post-exercise task (time x BDI-s effect, F(8, 65)=4.16, p=.045). Meanwhile, those with lower BDI-s had reduced IL-6 levels across the entire period. In contrast, elevated BDI-c symptoms were associated with low IL-6 at baseline in both HF and non-HF groups, but then an increase in IL-6 in the non-HF controls by 30 min post-exercise (group x time x BDI-c effect, F(8,65) = 7.6, p=.007). Conclusions: Elevated IL-6 levels until 30-min post task were associated with heightened somatic symptoms of depression, whereas HF patients with elevated cognitive symptoms had lower levels of IL-6 through 30 minpost exercise. By further understanding the components of depression associated with elevated inflammatory markers, interventions may be developed to specifically address symptoms of depression that may worsen cardiac outcomes. Psychosocial factors are independently related to cardiovascular disease outcomes, but less is known about the effect of these factors on stroke risk. The purpose of this study was to determine the effect of multiple psychosocial factors on stroke mortality in older adults. Participants included 3,656 adults (59.9% African American, 40.1% non-Hispanic white; 61.2% female) with a mean (SD) age of 77.0 (6.2) years from the Chicago Health and Aging Project (CHAP). CHAP is an ongoing, longitudinal study of risk for Alzheimer's Disease and other chronic conditions of the elderly. Mortality was ascertained through 12/31/05 and verified by data from the National Death Index; over 7.7 years of followup, 76 fatal strokes were identified. Prior principal component analysis on nine psychosocial questionnaires revealed three primary factors related to distress (depression, neuroticism, perceived stress, life satisfaction), social connectivity (social engagement, spirituality, social networks), and unfair treatment (hostility, perceived discrimination). Factor scores were computed by summing individual z-scores for each questionnaire loading on the factor. Cox models were conducted for each factor score adjusting for age, race, sex, education, systolic blood pressure, body mass index, physical activity, smoking and chronic conditions. Each 1-point higher distress score related to a significant 10% increase in stroke mortality [HR=1.10; 95% CI=1.03-1.18; p<.008]. Results were unchanged further controlling for history of stroke. There were no main effects of social connectivity and unfair treatment on stroke mortality. In this cohort of elderly African American and non-Hispanic white adults, higher levels of distress contributed significantly to greater risk of dying from stroke over nearly 8 years of follow-up. Additional research is needed to identify mechanisms underlying the association between distress and stroke mortality. There is an increasing research interest on the positive psychosocial factors that are assumed to maintain and enhance health. However, there is discussion whether positive and negative constructs are separate or bipolar opposites of the same phenomenon. High sense of coherence (SOC) refers to a dispositional orientation that includes flexible and adaptive coping with stressors. We examine the relationships between SOC and cardiovascular disease (CVD) and compare these results with a negative construct, depressive symptoms. Participants included men (N=3850) and women (N=4083) aged 25-74 years who had participated in risk factor surveys (The FINRISK Study) conducted in 1992 and 1997. SOC was measures with a 13-item scale. Depressive symptoms were measured with the 21-item Beck Depression Inventory (BDI) and had correlation of -0.60 with SOC. Incident CVD and IHD were derived from hospital records/death certificates. Subjects with a history of CVD or IHD at baseline were excluded. During a ten-to 15-year follow-up there were 397 nonfatal and fatal CVD and 219 IHD events. The 2nd SOC tertile had a higher risk of CVD (RR 1.32; 95% CI 1.03-1.69) than the highest SOC tertile after adjustment for age, gender, education, smoking, body mass index, blood pressure, cholesterol and alcohol consumption. The highest BDI tertile (RR 1.31; 1.01-1.70) had a higher risk for CVD compared to the lowest BDI tertile after adjustment for age, gender, education, and smoking. The second SOC tertile had a higher risk of CVD (RR 1.34; 1.04-1.72) in the age-adjusted model but BDI tertiles in the same model did not differ significantly. Neither SOC nor BDI were related to IHD events. Sense of coherence was related to CVD events but not to IHD events. However, the association was not linear as was the case with depressive symptoms. Further studies on psychosocial factors in CVD could benefit from parallel comparisons of positive and negative constructs. Objective: Besides general accepted complications, diabetesrelated symptom distress is an important outcome measure to understand the total burden of diabetes. Besides, diabetes-distress severity contributes to the variation in different aspects of quality of life (QOL). We investigated the longitudinal relationship of diabetes-related symptom distress among elderly with type 2 diabetes over a 7-year period.
Research design and methods: The Hoorn Study is a prospective population based cohort study (2000/01 and 2007/08) among elderly in the region of Hoorn, The Netherlands. Complete data on glucose metabolism and diabetes-related symptom distress for both measurements were available for 34 subjects. Changes in total diabetes-related symptom distress and 8 subgroups (psychological fatigue, cognitive, neuropathic pain, sensoric, cardiovascular, ophthalmological, hypoglycaemic and hyperglycaemic distress) were assed by the validated Type 2 Diabetes Symptom Checklist (DSC-R). Results: Crude analyses showed an increase in total symptom distress (range 0-100) over time, especially in the subgroup psychological fatigue (mean difference 7.90 (95% CI; -0.66 -16.47) p, 0.069). However, these results were not significant. Conclusion: Due to missing power we were not able to detect significant differences over time in symptom-distress. Further research is needed in larger sample size with inclusion of potential and reasonable influences of co-morbidities and diabetestreatment. Introduction: Food insecurity is the limited/uncertain availability, access to or ability to acquire nutritionally-adequate, culturallyrelevant and safe foods. Adults suffering from food insecurity are at risk of inadequate nutrient intakes or, paradoxically, overweight/ obesity and the development of chronic disease. Despite the global financial crisis and rising costs of living, there are few studies investigating the potential dietary consequences of food insecurity among the Australian population. This study examined whether food insecurity was associated with weight status and poorer intakes of fruits, vegetable and takeaway foods among adults residing in socioeconomically-disadvantaged urbanised areas. Methods: In this cross-sectional study, a random sample of residents (n=1000) were selected from the most disadvantaged suburbs of Brisbane city (response rate 51%). Data were collected by postal questionnaire which ascertained information on sociodemographic information, household food security status, height, weight, fruit and vegetable intakes and takeaway consumption. Data were analysed using chi-square and logistic regression. Results: The overall prevalence of food insecurity was 31%. Food insecurity was not associated with weight status among men or women. Associations between food security status and potential dietary consequences differed for men and women. Among women, food security was not associated with intakes of fruit, vegetable or takeaway consumption. Contrastingly, among men food security was associated with vegetable intakes and consumption of takeaway food: men reporting food insecurity had lower intakes of vegetables and were more likely to consume takeaway foods compared to those that were food secure. Conclusion: Food security is an important public health issue in Australia and has potential dietary consequences that may adversely affect the health of food-insecure groups, most notably men residing in food-insecure households. Background: Pakistani immigrants living in Oslo, Norway, have a high prevalence of Type 2 diabetes (T2D), but there are few data on risk factors for the disease. Aim: To study the distribution of, and association between, physiological and psychological risk factors for T2D in a group of Pakistani immigrant women living in Oslo. Methods: Female Pakistani immigrants (n=198, aged 25 to 63 years) with increased risk of, or newly diagnosed T2D were interviewed to give information on diet, physical activity, and demographic and psychological data. Anthropometric and blood data were also obtained. Additionally, an oral glucose tolerance test (OGTT) was performed, and maximum heart rate and level of physical activity were recorded, using SenseWear Armband. Results: Ninety eight % had body mass index (BMI) values higher than the IDF recommendation for South-Asians (BMI>23 kg/m2) and 39 % were obese (BMI≥30). Impaired glucose tolerance (IGT) was found in 37.4 % and T2D in 12.6 %. The participants had low energy expenditure, despite acceptable number of steps. According to the "Findrisk" and "Indian risk scores", 29 % and 89 %, respectively, had a high risk of T2D. Subjects with the metabolic syndrome reported more pain, reduced physical functioning and more subjective health complaints than those without the syndrome.
Conclusions: T2D risk factors are prevalent in young, female Pakistani immigrants in Oslo, Norway. It is not known whether the positive effect of breast feeding on child physical health extends into adult psychological adjustment. We examined changes in the social distribution of breast feeding and its effect on adult psychological well-being through the pathway of childhood psychological health by using the available cases from the National Child Developmental Study (NCDS, N= 7,750, born in 1958 ) and the 1970 British Cohort Study (BCS70, N=6,492 born in 1970 . Psychological well-being was defined in terms of measures of self-efficacy and psychological ill-health. Childhood adversity was measured by a dichotomised index created after tabulating information about parenthood, mother's age, mother's education, and presence of older siblings. Childhood psychosocial adjustment was measured by the Bristol Social Adjustment Guides for the NCDS and the Rutter scale graded by a teacher for the BCS70. We used logistic regression to test if there was an effect of breastfeeding on adult psychological well-being over and above the effects of adversity and psychosocial adjustment in childhood. The effect of breastfeeding appears to be gender specific. Findings showed that the magnitude of the effect of breastfeeding on psychological well-being was larger in women than in men. After accounting for the effect of childhood social adversity, breastfeed- Hormonal methods of contraception are highly effective, but in some users unscheduled vaginal bleeding occurs. If this is judged unacceptable it can lead to discontinuation, and hence risk of unwanted pregnancy and wasted health care resource. Data from a trial (where test drug had no consistent effect on bleeding) will be utilised to explore acceptability to women of differing patterns of unscheduled bleeding. Methods: Further analysis of data from a double-blind RCT (funded by NICHD), comparing intermittently administered antiprogestin CBD2914 with placebo, involving 136 new users of an hormonal intrauterine contraceptive implant. Six month follow-up comprised daily bleeding diaries and questionnaires at 3 points. Results: Days bleeding/spotting decreased markedly across the study, from 65% of days in the first three weeks to 32% in the last block (p<0.001). Side effects most commonly reported at 6 months were irregular bleeding (40%), mood swings (22%), bloating (21%) and skin problems (20%). By study end, 4 women had had removal of implant, and 10 were considering this in the near future, with main reasons given as weight gain (2 & 2), and bleeding (0, 2). Pattern of bleeding was reported as worrying by 40%, and more inconvenient than before by 41%. Total days bleeding was more strongly associated with 'worrying' than 'more inconvenient', which might be more strongly related to pattern, or individual propensity/tolerance, given that at baseline 26% of women reported having previously stopped a method of contraception for irregular bleeding. Total days bleeding was most strongly associated with reporting, as more problematic than before insertion, bleeding outside menstrual period, too many days bleeding, interruption to sex life, interruption to daily life, irregular periods, extra washing, and 'lose too much blood' (p<0.001). Further descriptive analyses will be presented. Conclusion: Better understanding is needed of women's judgements regarding unscheduled bleeding, and other side-effects, so as to discuss concerns and develop appropriate interventions. Although gender-role related personality traits agency and communion are positively related to psychological adjustment and physical health in both sexes, they have rarely been studied in the context of health behavior interventions. However, both might contribute to health goal attainment, potentially interplaying with domain-specific psychosocial factors: Agency, mediated by health-related self-efficacy, and communion, moderated by available social support for the goal at hand. We tested these hypotheses among the participants (N =385; women 73.2%, men 26.8%) of the GOAL Lifestyle Implementation Trial to prevent type 2 diabetes. Health-related self-efficacy was measured at baseline (T1) and after the intensive phase of the intervention at 3 months (T2). Perceived social support received during the intervention was measured at T2. Waist circumference, indicator of abdominal overweight, was measured at T1, at one-year (T3) and three-year (T4) follow-ups. Agency and communion were measured at T4 with the Personal Attributes Questionnaire (PAQ). Among women, higher agency was associated with a greater likelihood of at least 5% waist circumference reduction (WCR) by T3 (OR=2.70, CI 95% 1.19-6.15); among men, the association was reversed (but only marginally significant p=.057). Further analyses in women's sample showed that agency and increase in self-efficacy independently predicted T1-T3 WCR (both p<.05).
High communion was associated with WCR when social support was high (p-value for interaction term=.034). T1-T4 WCR was only predicted by T1-T2 self-efficacy increase (β=−.17, p=.024). One plausible explanation of the results is that agentic women are more successful at weight loss because they are less affected by feminine self-sacrificing role demands. However, also communal women, sensitive to their social environment, succeed well when backed up by social support for their personal goal pursuit. African-Americans experience disparately high rates of HIV and other sexually transmitted infections (STI); these rates are increasing among rural drug-using communities. However, sexual risk reduction interventions for drug users have traditionally focused on urban populations. We used qualitative techniques to understand rural African-American cocaine users' perspectives on sexual risk and HIV/STI so that we could tailor an existing intervention to this underserved population. Using purposive sampling, 40 African-American cocaine users from two rural towns were recruited to participate in four focus groups, which were audio-taped and transcribed verbatim. Three investigators coded the data using content analysis and constant comparison. Three major themes emerged: HIV knowledge and beliefs, selling & buying sex, and perceptions of condom use. Participants held many inaccurate beliefs about HIV, including how to identify high-risk partners. Attitudes about HIV testing varied by gender, with women being more accepting; however, most were doubtful about effective HIV treatments. Most participants had complex beliefs about trading sex, with "survival sex" seen as acceptable but "tricking for drugs" as unacceptable, despite reports that both types were common. Both men and women were socially classified as "dirty" or "nasty" based in large part on sexual trading behaviors. Participants viewed condoms as protective against HIV and STI, but also reported strong resistance to condom use by both men and women. They reported using hygiene as a proxy for gauging a potential partner's risk and therefore the importance of condom use. Significant knowledge deficits, high-risk beliefs, and social pressures appear to converge on rural African-American cocaine users and contribute to increased HIV and STI rates. These findings were incorporated into a social cognitive theory-based intervention to address these challenges and reduce risk behaviors. The growing amount of youth in the United States living with HIV/AIDS is a persistent problem. Not only has the number of newly infected 15 to 29 year olds consistently increased over the last decade, the emergence of antiretroviral therapy has meant that those born with HIV are growing into adolescence and beyond.
Consequently, HIV/AIDS is now being treated more like a chronic illness than a terminal disease. While much focus has been paid to identifying the specific factors that place individuals at risk for acquiring or transmitting HIV, little attention has been directed toward those behaviors that help promote health in the existing population of young people living with HIV (YPLH). These health protective behaviors can assist in prolonging lives. The preliminary data being presented here includes ninety-one participants who have completed their initial visits of a study assessing coping styles as predictors of adherence in urban YPLH. The sample was recruited from two clinics that serve adolescents with perinatal and risk-acquired HIV. The youth range from age 13 -22 years. Specific health protective behaviors were identified that either help promote immune functioning or delay disease progression in YPLH. Study participants responded to questions regarding their diet/nutrition, exercise, sleep habits and taking of multivitamins/supplements. Results highlighted some health protective behavior areas of promise and need. Most respondents reported eating vegetables and fruit "some days" of the week (71.4 % and 76.9 % respectively). In the month prior to baseline, Male injecting drug users (IDU) play a key role in the spread of HIV in mainland China. They face double risk of HIV acquisition through drug using as well as sexual risk behaviors. They also may form a ëbridgeí for transmission of HIV between high risk and low-risk populations such as their non-drug-using female sex partners. Better understanding of the underlying factors of their sexual behaviors is of critical public health importance. The present study examined factors in association with condom use intention among Chinese male IDU, using the Health Action Process Approach (HAPA) as a basic conceptual framework. An anonymous crosssectional survey, measuring background characteristics and specific constructs of the motivational phase of HAPA (conditional sexual risk perceptions (acquisition as well as transmission to others), self-efficacy, outcome expectancies, and behavioral intention), was conducted. Data obtained from 243 non-institutionalized Chinese male IDU with an exclusive female regular sex partner (RSP) in mainland China were reported. Of the respondents, the mean age was 35.2 (SD=5.7). Seventy percent received junior high education or less, 51% were currently married, 54.7% injected drugs for 5 years or more, 13.6% had shared syringes or injection paraphernalia with others and 85.6% did not use condoms consistently with RSP in the last 6 months. In line with the HAPA, perceived risk of contracting HIV from RSP (Beta=0.23, p<0.05), condom use negative outcome expectancies (Beta=-0.37, p<0.001), and condom use self efficacy (Beta=0.21, p<0.001) predicted intention to use condoms with RSP. Neither perceived risk of transmitting HIV to RSP or condom use positive outcome expectancies were significant factors. For the first time, support for the use of HAPA to understand condom use behaviors among Chinese male IDU was found. Study findings inform future design of theorybased intervention programs.
only 32% reported eating fast food one or more times a week. More than half of the youth (50.6%) had at least 20 minutes of exercise less than 3 days a week. While a third of the group (33%/ 30 pts) walked briskly for at least 10 minutes daily. On the other hand, almost all participants reported eating candy, chips or cookies "some days" (52.7%) or "every day" (41.8%). Similarly, 73.7 % had less than 4 days of exercise in the prior month and 43% received less than the recommended amount of sleep. Health protective behaviors are an important aspect of managing care for urban YPLH. Introduction: The worldwide scale-up of antiretroviral treatment (ART) in recent years (>4 million under treatment in 2009) occurred against a backdrop of strong economic growth. Still, 5 million people needing ART remain uncovered. Further, this progress is threatened by the global financial crisis making it urgent to estimate how funding changes will affect ART programs. Methods: We quantify the impact of ART funding changes by modeling their effect on ART coverage and incorporating feedback from coverage to mortality, and from coverage to HIV incidence using a behavioral sub-model. We analyze the changes in ART coverage, HIV incidence, and mortality for several funding scenarios to make concrete policy recommendations. We apply our model to South Africa (SA) using published estimates of current ART coverage, ART effectiveness, HIV incidence, HIV prevalence, HIV transmission probabilities and the annual number of sexual partners. Results: Universal ART coverage (UC) can be achieved in SA within a few years if funding increases every year at the rate it increased from 2007-08. In contrast, only maintaining current funding levels will result in declining coverage. Surprisingly, even a 1/3rd funding decrease over 3 years followed by increases at the 2007-08 rate delays achieving UC by 10 years. Increased prevention effectiveness can substantially reduce time to achieving UC. In contrast, increases in the CD4 count threshold for treatment eligibility could make UC very hard to achieve. Discussion: Only continued increases in ART funding in coming years can ensure that ART coverage levels can be increased in the future. Policy choices, such as investment in prevention and ART eligibility criteria, will be crucial in ensuring the future success of the ART roll-out. Purpose: A cancer diagnosis often involves distress which may in the long term develop into clinical depression. Screening instruments identifying highly distressed patients are needed. In Denmark, no validated instrument has been developed to identify cancer patients in need of psychosocial support. We are thus examining the psychometric properties of a Danish version of the single-item Distress Thermometer. Methods: A total of 434 women newly diagnosed with breast cancer (within 1-2 days) were invited to participate in this study. A total of 362 accepted (83%). The accuracy of the single item Distress Thermometer is compared to the longer validated scales of Hospital Anxiety and Depression Scale (HADS) and Impact of Event Scale (IES) using Receiver Operating Characteristic curve analyses. Results: Preliminary results show that 72% are distressed according to a cut-score of ≥4 on the Distress Thermometer (11-point scale, 0-10) with a mean of 5.4 and a standard deviation of 3.1. We plan to present results on the accuracy of the Distress Thermometer compared to the HADS and IES. Psychometric properties including sensitivity, specificity, positive and negative predictive value of the Distress Thermometer will be presented. Also, a cut-point for clinical use will be discussed. Conclusion: We expect that the psychometric properties of the Distress Thermometer will be satisfactory and that we will be able to determine an optimal cut-point. The results will be important for improved handling of psychosocial problems among breast cancer patients in Denmark. By targeting psychosocial support and rehabilitation to those who are most distressed we may be able to tailor the support and hence prevent that some patients develop clinical depression. BACKGROUND: Treatment-related pain is an important issue in breast cancer rehabilitation. As most studies in the area are crosssectional, relatively little is known about possible predictors. AIM: To explore the prevalence of pain and the role of depression as predictor in a nationwide prospective study of women treated for breast cancer. METHODS: The study cohort consisted of 3343 Danish women (aged 26-70) treated for primary breast cancer according to standardized guidelines. A questionnaire package including Beck's Depression Inventory was mailed out 3-(baseline) and 15-months post-surgery (follow-up). The response rate at follow-up was 94%. Probable cases of major depression (MD) were identified according to the manual (cutoff>=17). Frequency and severity of treatment-related pain in breast and arm or shoulder was assessed by 4 items at follow-up (range: "no pain" to "all the time" and "no pain" to "extremely severe"). DBCG and the surgical departments provided data on eligibility, clinical variables and comorbidity. Data on psychiatric history, demographic-, and socioeconomic factors were obtained from national registries. RESULTS: At follow-up, 73% reported pain during the recent month and 33% had pain "almost every day" or more often. Twenty-eight percent were "somewhat" or more affected by pain and 11% were "very" or "extremely" affected. Women with MD at baseline were more likely to experience pain "almost every day" or more often at follow-up (OR=2.63; (95% CI, 2.12-3.27)) and more likely to be affected "somewhat" or more by pain (OR=3.49; (95% CI, 2.80-4.35)). When adjusted for potential confounders (demographic-, socio-economic-, clinical-, health status-, and health behavior variables), the predictive power of MD persisted (OR=1.93; (95% CI, 1.50-2.48), and OR=2.31; (95% CI, 1.79-2.99)). CONCLUSION: The prevalence of treatment-related pain was substantial with nearly 3 out of 4 experiencing pain 15 months post-surgery and 1 out of 9 reporting to be very or extremely affected. Depression was found to be an important independent predictor of pain. AIM: Relative to other oncology populations, the brain tumor (BT) population is unique given that both benign and malignant BTs can be life-threatening and can cause severe neurocognitive and psychosocial disturbances. No study to date however, has investigated the effects of BT-related posttraumatic stress (PTSS) on neurocognitive functioning and overall quality of life in adult BT patients. The aim of the present study was to address this question. METHOD: Utilizing a longitudinal design, adult individuals diagnosed with a primary BT were assessed on average, within 6-weeks prior to starting radiotherapy (T1), and re-assessed at 3 months post-radiotherapy (T2). At both assessments, participants were administered a standardized battery of tests measuring BT-related PTSS, mood, quality of life, memory and executive functioning. RESULTS: To date, 64 patients (36 females, 28 males) have been re-assessed at T2. At both T1 and T2, 14% of the sample reported clinically elevated levels of PTSS pertaining to their BT experience; although, the effects of PTSS fluctuated over time. Whereas 40% of individuals who reported elevated PTSS prior to commencing radiotherapy continued to report elevated PTSS at T2, 60% of patients with elevated PTSS at T2 did not experience elevated PTSS at T1. Prior to radiotherapy (T1), individuals with elevated PTSS were found to have significantly reduced functional well-being, elevated BT symptoms and social constraints, as well as heightened depressive, anger, fatigue and confusion symptoms (p's<.05) compared to patients with low PTSS at T1. Individuals with elevated PTSS also evidenced significantly reduced scores on working memory and executive tests (p's<.05) compared to individuals with low PTSS at T1. Similarly, individuals with elevated PTSS at T2 reported significantly reduced functional well being, vigor, and overall quality of life, as well as elevated BT symptoms, depression, anger, confusion, and social constraints (p's<.05) compared to individuals with low PTSS, 3-months post-radiotherapy. Individuals with high PTSS at T2 also performed significantly worse on working memory and executive tests (p's<.05). DISCUSSION:
The findings suggest that approximately one in seven individuals diagnosed with a primary BT are at risk of suffering from PTSS as a result of their tumor experience. Notably, these results indicate that PTSS compromises neurocognitive and psychosocial functioning including overall quality of life in BT patients undergoing radiation therapy. The findings will be discussed in context of developing and implementing appropriate counseling and rehabilitation services to address the unique needs of BT survivors. Background: Oestrogen has been shown to influence cognitive functioning, and recent studies have focused on the effects of endocrine therapy on cognitive functioning in breast cancer patients. The current results, however, have been inconclusive. Objectives: To examine the effects of endocrine therapy (tamoxifen) on cognitive functioning in breast cancer patients. Methods: 20 patients receiving adjuvant chemotherapy followed by tamoxifen and 12 patients receiving chemotherapy only were assessed with neuropsychological tests before and again 4-6 weeks after completion of the chemotherapy. Results: Group differences were only found for one out of 21 neuropsychological tests. When controlling for age, patients treated with both chemotherapy and tamoxifen scored significantly lower on the letter-number sequence test (WAIS-III LN) measuring working memory (P=0.013) than patients treated with chemotherapy only. Of the 21 tests, 5 showed differences corresponding to an effect size>0.3 (Cohen's d). These 5 tests were WAIS-III LN (letter-number sequence), WAIS-III A (Arithmetic), TMA (Trail-making A), RAVL (Rey Auditory Verbal learning) and WF-N-words (Word Fluency N-words) representing the cognitive domains of working memory, processing speed and verbal fluency. Conclusion: While we cannot conclude that tamoxifen causes cognitive impairment in breast cancer patients, our results do not exclude the possibility. Our finding that 25% of the neuropsychological tests showed effect sizes greater than 0.3 are in agreement with results of previous studies. The small samples and subsequent low statistical power of existing studies suggest the need for largescale studies. If tamoxifen causes cognitive decline in breast cancer patients, this knowledge could be relevant for the patients' choice of treatment. Introduction: This study examined models of clinical survivorship care and outreach, organizational factors, and center evolution over time in the eight participating centers in the Lance Armstrong Foundation LIVESTRONG Centers of Excellence (COE) Network, to provide a set of recommendations to aid other institutions in the utilization of "best practices and processes" in the development of successful comprehensive cancer survivorship programs. Method: Guided by the Chronic Care Model this mixed methods study used both quantitative and qualitative approaches to characterize institutional barriers, models of care, community outreach and education among the COEs. The three phase approach included document review, in-depth key informant telephone interviews, quantitative surveys and site visits over a one year time period. Result: Findings identified several overarching themes, lessons, and recommendations for developing and sustaining survivorship care: one size does not fit all-each program is unique with different organizational and environmental factors that inhibit and facilitate progress ; developing survivorship models of care is an ongoing process of trial and error; leadership and institutional commitment are essential to provided needed resources and support; lack of awareness of the need for survivorship care and resistance to change are critical barriers to address in order to institute survivorship care in oncology practice; and establishing community-based centers has the potential to advance the field of survivorship beyond comprehensive cancer centers. Background: Comparatively, a higher rate of Arab Children in Israel are victims of car accidents compared to Jewish children, this may be a result of low compliance with the law regarding child restraint in cars among Arabs. One possible explanation for the low use of car restraints may be fatalistic beliefs, common in the Arab community. These beliefs suggest that health is a result of luck and beyond an individual's control. Objectives: To asses levels of fatalistic beliefs and their association with child restraint in motor vehicles among Arabs in Israel. Methods: A cross sectional observational study of child restraint in motor vehicles was conducted. Four hundred cars with 835 children under the age of 8 in them were observed while entering a parking lot. The drivers were interviewed regarding beliefs and attitudes towards child restraint and in addition their fatalistic beliefs towards road accidents and fatalistic beliefs in general. The observations were conducted within an Arab town and seven Arab villages in the northern part of Israel. Results: Of the 400 drivers, 63% were restrained while driving the car while only 32% of the children in the cars were restrained. A higher percent of the children under one year old were restrained (64%) compared to 41% of the children aged 1to 3 and 9% of children aged 3-8. Around 60% of the drivers reported high fatalistic beliefs. High fatalist beliefs were associated with lower education and were more frequent among women. After adjustment for gender, age of driver, education, number of children and driver's restraint, fatalism was associated with child restraint. Those with high fatalistic beliefs restrained their children more often. Conclusion: High levels of fatalism may inhibit people from use of child restraint in motor vehicle among Arabs in Israel. Targeting these beliefs may increase levels of compliance with the law. Purpose: The lack of physical activity (PA) is associated with obesity among school-aged children. Although parental support has been found to increase children's PA, few studies tested predictors and effects of parental support for PA among young Hispanic children. The main purpose of this study is to examine the effect of a multi-component intervention program on parental support for PA among predominantly Hispanic children from K to 2nd grade, accounting for parents' characteristics such as acculturation, education, and age. Design and setting: Quasi-experimental research design was used to evaluate a multi-component intervention program, in which five waves of data were collected from two sites in West Texas. Subjects. Data are collected from predominantly Hispanic and low-income parents/guardians (N=442) and their children of 5 to 9 years (N=848). Measures: Children's anthropometric measures were obtained, and their overweight status was determined based on their age-andgender adjusted BMI. A demographic questionnaire, an acculturation scale (Brief ARSMA II), and a parent survey were used among parents who reported on home environmental factors (e.g., whether there is a TV in the study child's bedroom) and children's behaviors (e.g., physical activity, TV viewing, sweetened beverage intake, family meals). Analysis: Descriptive statistics were obtained, and random coefficient regression was used to test the hypotheses. Results: Intervention increases parental support for PA (intercept). Time alone does not influence parental support (slope). The more acculturated parents provide more parental support (intercept, main effect). More acculturated parents are less influenced by the intervention (negative interaction between intervention and acculturation level), but not enough to counterbalance the effect of the intervention. Also, parents who have higher level of education are more supportive of PA, and older parents give less support for PA. Children's age and gender are not significant covariates, and neither is the site where the intervention is delivered. Conclusion…. More acculturated parents are more likely to provide support for PA. Intervention is important in raising parents' awareness of problems of children's obesity and mobilizing parents to provide more support for PA. Issues: Like many developing countries, Nepal has facing difficult problem to youth knowledge, awareness, behavior and attitude on their reproductive health and sexuality.. But, on the other hand, today's nepalese young girls and boys are more tolerant toward premarital sexual relations. The youth reproductive health services is then directed to assist youth to possess knowledge, awareness, behavior and attitude of responsible reproductive health through promotion by information, education, and communication, counseling, services for youths particularly those with particular problems, and supporting positive activities for youths. Methods: This paper aims to examine the result of the youth reproductive health services in nepal through explore the realty of their knowledge, awareness, behavior, and attitude of sexual and reproductive health from 2007 to mid 2009. To examine this reality this paper using the data from the Survey of High Risk Behavior of Adolescent Including Sexuality and HIV/AIDS Prevention with Implication on Reproductive Health which interviewed 1956 youths in 5 districts; 310 school and colleges. The data were edited and anaysed by EPI info software program. Results: The summary of the result found that the reality of the knowledge, awareness, behavior, and attitude of sexual and reproductive health is still low. For the knowledge of reproductive health: most (53,6 percent) do not know basic meaning of reproduction, 78 percent of female in rural do not know the cycle of menstruation period, 64.8 percent never discussed reproductive health and even, 51 percent never discussed it with family, and 64.6 percent didn't getting family planning lesson. But, on the other hand, there are 49.1 percent kissing, 45.8 percent embracing/ hugging, and 2.6 percent having sexual intercourse during dating. This realty implicated on enhancing effort to create and promote more attractive program in informing reproductive health information and launching friendly service concern to the young girls and boys. Context and objectives: With over 80% of those currently living with AIDS aged between 15 and 24 and 75% of these youth living in sub-Saharan Africa, young people in sub-Saharan Africa have been worst affected by the AIDS pandemic. Since HIV in Togothe most common cause of death, representing 17% of all deathsis spread primarily through unprotected sex, safe sex practices such as condom use can reduce HIV spread significantly. We examined the efficacy of a behavioral intervention to promote condom use among Togolese young people at high risk for HIV infection. Methods: We randomized -without known HIV infection -446 Togolese people aged 16 to 24 years consulting 3 sexually transmitted disease clinics in Lomé, who had recently had unprotected sex, to a 1-hour behavioral intervention or a didactic control condition. At baseline and 6 months, participants underwent interviews about their sexual behaviors and consistent condom use and testing for HIV. Results: We observed a 51% decline in HIV infection incidence (P=.03) in the intervention group. Incidence density for the intervention versus control groups was 1.6 versus 3.27 per 100 person-years (P<.001). Participants in the intervention group reported using condoms more consistently in the 30 days preceding the 6-month assessment (unadjusted analysis, intervention, 74.2% vs control, 48.7%). Participants in the intervention group reported using condoms more consistently over the entire 6-month period (adjusted odds ratio, 2.30; 95% CI, 1.51-3.50; P=.001). Conclusions: This brief behavioral intervention shows promise in reducing HIV risk behaviors among Togolese young people and may be transferable to other sub-Saharan African countries. Multinomial logistic regression was used to evaluate relationships among dependent variables and independent variables. We found no significant effect of having received in-school HIV/AIDS education on all outcome measures. Compared with females, males were more likely to initiate sex at a relatively younger age, report unprotected sex with multiple partners, and to drink alcohol before sexual intercourse. Efforts to control the HIV/AIDS epidemic among adolescents may need to focus on targeted interventions aimed at addressing gender-and racial/ethnic-specific risk exposures among this population group, including risk behaviors linked with lifetime sexual abuse. The need to re-examine the role of inschool HIV prevention programs to build adequate competencies among students, parents and community leaders is recommended. STUDY DESIGN AND DATA SOURCE: We used cross-sectional data from the 2005 Colorado Youth Behavioral Risk survey (YRBS). Details of the survey design and psychometric validity of questionnaire items are found in other publications. Briefly, the YRBS is a self-reported paper-based survey of high school students conducted every two years by the Centers for Disease Control and Prevention (CDC) in collaboration with Colorado local implementing partners. The survey monitors risk behaviors and other characteristics associated with mortality and morbidity among students in grades 9 through 12. In 2005, the survey was administered to a representative sample of 1,498 students from 29 public high schools throughout the state, and the overall response rate was 60%. Surveyed schools and classes were selected systematically with probability proportional to enrollment based on grade levels. OUTCOME VARIABLES: Sexual Risk Behavior was a threelevel variable created from items suggested in previous studies, and categorized as: no risk (never had sexual intercourse or engaged in any sexual risk behavior at the time of survey), low risk (had sex with one partner, used condom and did not drink alcohol in the last sexual intercourse, and used a method of contraception) and high risk (had sex with multiple partners, did not use condoms and/or drank alcohol before the last sexual intercourse, and used no method of contraception). Age at first sexual intercourse was categorized based on respondents' age at their first sexual intercourse as: no sexual intercourse (participants reported as not having ever had sexual intercourse at the time of survey irrespective of age), young adolescents (11-14 years), and older adolescents (≥15 years). Number of current sexual partners was assessed based on the reported number of sexual partners in the 3 months preceding the survey and categorized as following: never had sexual intercourse, had 1 partner, and had ≥2 sexual partners. These variables were selected because of their known association with transmission of HIV infections (Mullings, Marquart & Brewer, 2000) . COVARIATES: HIV/AIDS education, substance use score, physically forced sex, alcohol use. Potential confounding variables selected include age, gender, number of hours watching TV, feeling sadness or hopelessness, and cigarette smoking because of their documented relationships with sexual risk behavior. STATISTICAL ANALYSIS: Characteristics of respondents participating in the survey were compared according to race and gender using Rao-Scott χ2 tests for categorical variables. To further assess the relationship between independent and outcome variables, all independent variables that attained a statistical significance of P≤.25 in bivariate analyses were entered into multinomial logistic regression models in a manual stepwise fashion, starting with those with the lowest P-value. This method allows dependent variable to have more than two categories, thereby providing a single estimate of odds ratio (OR) for the association between the independent variables and each combination of the dependent variables, while controlling for potential confounders. Odds ratios (OR) were calculated and used as estimates of risk ratios. The likelihood ratio test was used to compare models to determine which variables were retained in multivariate models at a significance level α=.05. Age and grade level were not included in the same models because of potential collinearity. All analyses were performed with SAS 9.1.3 (SAS Institute, Cary, NC) and the survey weight variable was used to account for the complex sampling design of the survey to provide estimates representative of the high school student population across the state. RESULTS: Nearly 64% of respondents were non-Hispanic whites, Hispanics 24% while Blacks and other racial/ethnic groups represented 12%. These proportions mirror the approximate distribution of racial groups across the state. Both genders are equally represented in this sample with older adolescents (ages 15-18 years) being the predominant group. Nearly 85% of students reported receiving voluntary in-school HIV/AIDS education. In adjusted analyses, predictors significantly associated with high risk sexual behaviors included being male, Hispanic, and having had physically forced sex. Likewise, light and binge drinking, smoking, and illegal substance use showed significant effects on sexual risk behaviors. The highest risk for early sexual debut was observed among Blacks, and Hispanics. Similarly, there was an 8-fold increase in the odds of early sexual debut among adolescents reporting physically forced sex. A dose-response relationship was observed between illegal substance use and age of sexual debut, with the strongest effect among young adolescents. Likewise, current or past smoking was associated with increased odds of early sexual debut. Statistically significant correlates of having one or more sexual partners included being Latino and high user of illegal substances, reporting forced sex, drinking alcohol, and current smoking. Associations appeared more marked among respondents using two or more drugs and having ≥2 sexual partners.  IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1(−/−) mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1(−/−) mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection. West Nile virus (WNV) is a neurotropic flavivirus and is an emerging public health threat. Infection with WNV now constitutes the leading cause of mosquito-borne and epidemic encephalitis in humans in the United States [1] . WNV is enveloped and contains a single strand positive sense RNA genome of approximately 11 kb in length that encodes three structural (C, prM/M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). It cycles enzootically between birds and Culex mosquitoes, with humans infected as dead-end hosts. WNV infection has been modeled in inbred mice wherein infection and pathogenesis recapitulate many of the features of human infection (reviewed in [2] ). Following subcutaneous inoculation, WNV replicates in dendritic cells (DCs) at the portal of entry and in the draining lymph node. A primary viremia develops and virus spreads to visceral organs including the spleen, where further amplification occurs, leading to central nervous system (CNS) dissemination and encephalitis. In humans, WNV causes an acute febrile illness that can progress to severe and sometimes lethal neuroinvasive disease, especially in the elderly and immunocompromised [3] . However, healthy young adults are also afflicted with severe neurological disease [4, 5, 6] , indicating that virulence can occur independently of immune deficiencies or aging.
Intracellular innate immune defenses and the actions of type I interferon (IFN) provide a first-line of defense against virus infection and are essential for the control of WNV replication, dissemination, and neurovirulence [7] . Innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (PAMP) motifs within viral products by intracellular pathogen recognition receptor (PRR) proteins in infected cells. PRR signaling directs downstream activation of latent transcription factors, including NF-kB, interferon regulatory factor (IRF)-3 and IRF-7, in a cell type-specific manner to induce antiviral response programs that include expression of proinflammatory cytokines, chemokines, type I IFN, and interferon stimulated genes (ISGs) [7, 8, 9, 10] . The ISG products induced through autocrine and paracrine actions of IFN confer antiviral activity by limiting virus replication and cell-to-cell virus spread. Modulation of IFN signaling has been identified as a virulence feature of pathogenic strains of WNV [11, 12] .
The RLRs, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation antigen 5 (MDA5) [13, 14, 15, 16] , are PRRs that play critical roles in triggering immune defenses against RNA virus infection, including WNV. RIG-I and MDA5 are cytosolic RNA helicases that contain an amino terminal tandem caspase activation and recruitment domain (CARD). Upon engaging RNA substrates, the RLRs undergo a conformational change and bind to the mitochondrial associated protein, interferon promoter stimulator-1 (IPS-1) through a CARD-CARD interaction, leading to IPS-1-dependent signaling of IFN production and expression of immune response genes [17, 18] . RLR signaling and IPS-1 function have an essential role in triggering IFN defenses during WNV infection of mouse embryo fibroblasts (MEFs) and human cell lines in vitro. Cells lacking either RIG-I or MDA5 were attenuated in their ability to generate an effective innate immune response to infection, whereas cells lacking both RIG-I and MDA5 or those deficient in IPS-1 alone were unable to respond to infection with WNV and related flaviviruses [19, 20, 21, 22] . Recent studies examined the role of another class of pattern recognition receptors, Toll like receptor (TLR)3 and TLR7, and show that these receptors are also important PRRs of WNV infection, as they play a role in signaling IFN production and an inflammatory response upon viral ligand recognition [23, 24, 25] . TLR3 has been shown to contribute to both enhancement and protection of CNS inflammation and neurovirulence of WNV in vivo [23, 24] , while TLR7-dependent signaling was shown to be essential for directing proper immune cell homing to sites of WNV infection during the adaptive immune response in vivo [25] .
Type I IFN, a major product of PRR signaling, has been shown to link innate and adaptive immune responses. However, the specific PRR pathways that mediate this during acute WNV infection have not been delineated nor has the RLR pathway been evaluated in this context. The quantity and quality of the innate and adaptive immune responses after infection must be carefully regulated to avoid aberrant inflammation and immunopathogenesis. Regulatory T (T reg ) cells and inflammatory dendritic cell (DC) subsets regulate inflammation during acute virus infection through T cell suppression and by modulating the trafficking and inflammatory cytokine production of immune cells into infected tissues [26, 27, 28] . Thus, the level of local and peripheral T reg cells, and the composition of local DC subsets that develop during WNV infection may determine immune control and WNV disease.
Here, we assessed the role of RLR signaling and IPS-1 in WNV infection and immunity. Our studies define IPS-1 as an essential modulator of immunity in vivo and demonstrate that IPS-1-dependent signaling orchestrates an innate/adaptive immune interface that regulates immune responses to effectively control WNV infection.
WNV infection of primary embryonic fibroblasts recovered from RIG-I 2/2 mice revealed that RIG-I was important in eliciting innate antiviral immune defenses early during infection, whereas MDA5 was important for enhancing and sustaining this response [21] . We further evaluated WNV infection of RIG-I 2/2 or MDA5 2/2 mice and confirmed that RIG-I serves a dominant role among the RLRs for the acute induction of innate immune defenses and protection against WNV infection in vivo (data not shown). Since the RLRs signal innate defenses through the IPS-1 adaptor protein [29] , we also examined the role of IPS-1 in protection against WNV infection upon a sub-lethal virus challenge of wild type and IPS-1 2/2 mice. IPS-1 2/2 mice were highly susceptible to WNV infection and exhibited 100% mortality with an average survival time (AST) of 7.3 days as compared to wild type mice (38.5% mortality with an AST of 13.2 days; p,0.0001; Fig 1A) . Thus, RIG-I and IPS-1-dependent signaling are essential for protection against WNV infection.
To define the role of IPS-1 in controlling WNV in vivo, wild type and IPS-1 2/2 mice were infected subcutaneously (s.c.) with 100 PFU of WN-TX and viral burden within peripheral tissues and the CNS was measured over time post-infection (pi). IPS-1 2/2 mice exhibited increased viremia compared to wild type mice (45.7 fold enhancement at day 1 pi, P,0.05) throughout the course of infection (Fig 1B) . Similarly, viral loads in the spleen were elevated in the infected IPS-1 2/2 mice (Fig 1C) . WNV infection of IPS-1 2/2 mice displayed an expanded tissue tropism as infectious virus was found in the kidneys, a tissue that is not normally permissive to infection in wild type mice ( Fig 1D) . WNV is typically detected in the CNS of wild type mice after s.c. challenge between 4 and 8 days pi [2] . Consistent with this time course, infected wild type mice exhibited detectable viral loads (average viral titer of 10 1.8 pfu/gram of tissue) in the brain by day 6 p.i., although virus was not detected in the spinal cord (Fig 1E  and F ). In contrast, WNV spread to the brain ( Fig 1E) and spinal cord of IPS-1 2/2 mice (Fig 1F) by day 2 pi, with viral loads rising through day 6 pi. Together these results indicate that IPS-1, likely
West Nile virus (WNV) is a mosquito-transmitted RNA virus that has emerged in the Western hemisphere and is now the leading cause of arboviral encephalitis in the United States. However, the virus/host interface that controls WNV pathogenesis is not well understood. Previous studies have established that the innate immune response and interferon (IFN) defenses are essential for controlling virus replication and dissemination. In this study, we assessed the importance of the RIG-I like receptor (RLR) signaling pathway in WNV pathogenesis through analysis of mice lacking IPS-1, the central adaptor molecule of RLR signaling. Our studies revealed that IPS-1 is essential for protection against WNV infection and that it regulates processes that control virus replication and triggering of innate immune defenses. We found that IPS-1 plays an important role in establishing adaptive immunity through an innate/adaptive interface that elicits effective antibody responses and controls the expansion of regulatory T cells. Thus, RLRs are essential for pathogen recognition of WNV infection and their signaling programs help orchestrate immune response maturation, regulation of inflammation, and immune homeostasis that define the outcome of WNV infection.
through RLR signaling of innate immune defenses, limits WNV replication, viremia, and peripheral spread, and is essential for the control of viral invasion of the CNS.
Myeloid cells, including tissue and lymphoid DC and macrophages (Mw), are among the first cells to encounter WNV during infection and thus function to restrict the spread of virus to distant tissues and the CNS [2] . To define the role of IPS-1 in controlling virus replication and innate immunity in myeloid cells, we analyzed WNV infection and host responses in primary bone marrow-derived DC and Mw recovered from wild type and IPS-1 2/2 mice. DC and Mw were infected at an MOI of 1.0 (relative to viral plaque assay quantification of BHK-21 cells; see Methods) and evaluated for virus replication, IFN induction, and innate immune triggering of ISG expression (Fig 2) . IPS-1 2/2 DCs sustained significantly higher WNV replication at 36 and 48 hours pi compared to wild type infected cells (Fig 2A) . WNV infection of wild type DCs induced IFN-b secretion but this response was completely abolished in IPS-1 2/2 DCs (Fig 2B) . The lack of IFN-b induction in IPS-1 2/2 DCs correlated with a lack of ISG expression including RIG-I, MDA5, and STAT-1 ( Fig 2C) . In addition, expression of ISG54 and ISG49, which are direct IRF-3 target genes [30, 31] , were not induced during WNV infection of IPS-1 2/2 DCs (Fig 2C) . Moreover, ISG56, another IRF-3 target gene [31] , was induced late during infection and to lower levels as compared to ISG54 and ISG49 in wild type, infected DCs. WNV infection of IPS-1 2/2 Mw resulted in significantly higher virus replication between 24 and 48 hours pi as compared to infected wild type cells ( Fig 2D) . Whereas wild type infected Mw expressed IFN-b, this response was completely abolished in IPS-1 2/2 Mw (Fig 2F) . We also observed a differential expression of ISGs and IRF-3target genes within WNV-infected Mw. RIG-I, MDA5, and STAT-1 were not induced in IPS-1 2/2 Mw, whereas, ISG56, ISG49, and PKR were expressed at reduced levels and with delayed kinetics. These data establish that IPS-1-dependent RLR signaling is the major innate immune signaling pathway that controls virus replication in conventional DCs and Mw.
The RLR signaling pathway triggers the innate immune response to WNV infection in primary cortical neurons Neurons represent the target cell of WNV infection in the CNS and their death after infection is a key factor in pathogenesis and neurological sequelae [32, 33] . To define the role of RLR signaling in restricting virus replication in neurons, primary cortical neurons were generated from wild type and IPS-1 2/2 mice. Cells were infected at an MOI of 1.0 with WN-TX and virus yield, IFN-b induction, and ISG expression were evaluated. In the absence of IPS-1, WNV replicated faster and to higher levels resulting in a 2.2 and 4.2-fold (p,0.05) increase in viral production at 24 hrs and 48 pi, respectively as compared to infected wild type neuronal cells ( Fig 3A) . This relatively modest virologic effect in neurons compared to that observed in IPS-1 2/2 DC and Mw was expected, as IFN-a or -b pre-treatment only inhibits WNV infection in cortical neurons to a maximum of 5 to 8-fold [12] , suggesting that the IFN response is comparably less potent in neurons. IFN-b expression was induced to lower levels in IPS-1 2/2 neurons compared to wild type infected neurons at 24 (10-fold, p,0.05) and 36 hours pi (5-fold, p,0.05) despite the higher levels of virus replication (Fig 3A and 3B) . Expression of ISGs, (including RIG-I and MDA5) and IRF-3 target genes (including ISG56 and ISG49) followed this pattern and were dependent on IPS-1 for rapid and high level expression ( Fig 3C) . The presence of IFN-b and ISG transcripts in IPS-1 2/2 cells at 48 hrs pi is consistent with the finding that TLR3 has an independent and subordinate role in triggering innate immune responses in cortical neurons at later time points after WNV infection [23] . These results demonstrate that the RLR signaling pathway controls virus replication and induces innate immune responses against WNV infection in cortical neurons.
Neuronal destruction and CNS inflammation are enhanced in WNV infected IPS-1 2/2 mice To determine the role of the RLR pathway in protection of neurons against WNV pathogenesis in vivo, we conducted histological analysis of brain tissue from wild type and IPS-1 2/2 mice infected with WN-TX ( Fig 4A) . Analysis of brain sections from infected wild type mice revealed little or no inflammation or neuronal damage, with sparse and focal cell infiltrates restricted to the hippocampus and cerebral cortex on day 6 pi. By day 10 pi (a timepoint in wild type mice in which peak virus replication in the CNS occurs [34] ) cellular infiltrates were present in the parenchyma and neuronal destruction was observed throughout the cortex and hippocampus. In contrast, brain sections from infected IPS-1 2/2 mice recovered on day 6 pi displayed extensive injury to neurons in the cortex and granular neurons of the hippocampus. Damaged neurons appeared pyknotic with vacuolation, degeneration and cell dropout. Somewhat surprisingly, we observed extensive inflammation in the brains from infected IPS-1 2/2 mice within the cortex, hippocampus, and cerebellum (data not shown) displaying prominent perivascular and parenchymal immune cell infiltrates.
To evaluate the composition and antigen-specificity of the inflammatory cells within the brains of WNV-infected mice, lymphocytes were isolated from infected brains on day 6 pi and were characterized from pools (n = 5) of wild type and IPS-1 2/2 infected mice. Brains from IPS-1 2/2 infected mice showed an 2.9fold increase in the total number of immune cells as compared to wild type infected mice (Fig 4B) , and this was associated with an increase in absolute numbers of infiltrating CD4+ and CD8+ T cells ( Fig 4C) . Among the brain CD8+ T cells isolated from IPS-1 2/2 mice, there was a remarkable 27-fold increase in the number of antigen-specific cells that expressed IFN-c after treatment with an immundominant NS4B peptide ( Fig 4D) [35, 36] . Analysis of microglia/Mw cells, based on relative surface expression of CD45 and CD11b [37] , revealed increased numbers of microglial cells (CD45+ lo /CD11b+) and infiltrating macrophages (CD45+ hi / CD11b+) within the brains of infected IPS-1 2/2 mice when compared to wild type mice (Fig 4E) . Similar findings were observed in the spinal cords from infected IPS-1 2/2 mice (data not shown). Combined with the histological analysis, these results demonstrate that in the absence of IPS-1, WNV infection induces a strong inflammatory response in the CNS. While this response is likely associated with increased viral loads, the failure of this increased inflammatory response to elicit protection or control CNS pathology, in the absence of IPS-1, suggests a role for the RLR signaling pathway as a regulatory program that controls the quality of the inflammatory response to WNV infection.
To further characterize how IPS-1 modulates the inflammatory response to WNV infection, we measured levels of systemic type I IFN, proinflammatory cytokines, and chemokines present in the serum of WNV-infected mice at 1 and 4 days pi. Paradoxically, a trend towards more rapid induction and increased levels of type I IFN were observed in the serum of IPS-1 2/2 mice compared to wild type mice (Fig 5A) . We note that in this case type I IFN was detected and quantified through a mouse-specific type I IFN bioassay, which does not differentiate between the IFN-a or -b species. This result is consistent with our recent studies showing that serum type I IFN levels accumulate during WNV infection in an IRF-7-dependent but IRF-3-independent manner [8, 9] . In this case IFN-a species are likely accumulating through a TLR7dependent signaling pathway involving plasmacytoid DCs, which do not require the RLR pathway for IFN production [38] . More recently, Town et al. assessed the role of TLR7 and MyD88 2/2 during WNV infection and found that mice lacking MyD88 produced elevated levels of systemic IFN during WNV infection [25] . Thus, during WNV infection systemic IFN is regulated through RLR-dependent and independent processes. Correspondingly, when compared to wild type mice, IPS-1 2/2 infected animals, which show higher viremia (Fig 1B) produced increased serum levels of proinflammatory cytokines and chemokines in response to WNV infection. Elevated levels of systemic IL-6, TNFa, CXCL10, and IFN-c were observed at 1 and/or 4 days pi in IPS-1 2/2 mice (Fig 5B) . Serum cytokine levels were also compared between uninfected wild type and IPS-1 2/2 mice and showed no differences in basal cytokine expression (data not shown). These results demonstrate that in the absence of IPS-1, greater proinflammatory cytokine and chemokine responses are induced during WNV infection.
Altered WNV-specific antibody profiles in IPS-1 2/2 mice WNV-specific antibody responses are essential for suppressing viremia and virus dissemination and limiting lethal WNV infection [39, 40] . To determine if a deficiency in IPS-1 modulated the quality and quantity of the humoral immune response, we characterized the antibody profile in sera during WNV infection. In wild type mice, neutralizing virus-specific IgM antibodies are typically detectable by day 4 pi with WNV and production of neutralizing virus-specific IgG antibodies follow between days 6 and 8 pi [40] . A time course analysis in wild type and IPS-1 2/2 infected mice showed that between 4 and 6 days pi, WNV-infected IPS-1 2/2 mice exhibited significantly higher levels of virus-specific IgM, IgG, and IgG subclasses as compared to infected wild type mice ( Table 1) . WNV-specific IgG1 antibodies were detected at low levels on day 6 pi in sera from wild type and IPS-1 2/2 mice. Additionally, we observed a ,72.9-fold increase in WNV-specific IgG2a levels in infected IPS-1 2/2 as compared to wild type mice on day 6 pi and ,2.2-fold increase on day 8 pi. Assessment of the virus-specific antibody responses through a PRNT assay revealed that neutralization titers in sera from wild type mice increased dramatically between 6 and 8 days pi. Sera from IPS-1 2/2 infected mice exhibited a modest increase in neutralization titer to 1:1280, despite having much higher levels of virusspecific antibodies. This difference translated into a serum neutralization index that was ,39-fold lower on day 6 pi in the infected IPS-1 2/2 mice compared to wild type mice. These results demonstrate that the humoral responses in WNV-infected IPS-1 2/2 mice are distinct from responses in wild type infected mice. Thus, RLR signaling and IPS-1 actions likely contribute to regulatory processes that govern the levels, IgG class switching, and neutralizing capacity of antibodies generated in response to WNV infection.
To characterize the immune parameters associating with the dysregulated inflammatory and humoral responses in WNV infected IPS-1 2/2 mice, we analyzed the immune cell composition in draining lymph node and spleen tissues. Wild type and IPS-1 2/2 mice were challenged with diluent alone or with WN-TX, and draining popliteal lymph node (DLN) and the spleen were harvested at 1 and 6 days pi, respectively. Analysis of the popliteal DLN provides insight into how IPS-1 modulates the inflammatory response immediately after infection whereas assessment of the spleen elucidates characteristics of the adaptive immune response prior to the infection endpoint. Immune cells were isolated from the popliteal DLN and were characterized by flow cytometry (Fig 6) . Analysis of CD8a DC subsets, which are phenotypically the major antigen presenting cells within lymphoid tissues and are implicated in eliciting virus-specific CD8 T cell in response to acute WNV infection [41] , showed that infected wild type and IPS-1 2/2 mice exhibited similar increases in the numbers of CD8a + and CD8a 2 DCs compared to mock-infected mice (Fig 6A, B) . However, a significant increase (,3-fold; p,0.05) of a proinflammatory DC subset, characterized as CD11c + CD11b hi Ly6C + , was observed within the popliteal DLNs of IPS-1 2/2 infected mice (Fig 6C) . This DC subset is monocytederived and typically recruited to sites of acute inflammation where they propagate the inflammatory response [42] . We found that these DC subsets were not significantly expanded and showed no differences in their recruitment to the DLN in either wild type or IPS-1 2/2 infected mice at 12 hours pi (data now shown). Thus, as early as 24 hours pi, an elevated cellular inflammatory response is initiated in the IPS-1 2/2 mice. In contrast, similar increases in plasmacytoid DCs were observed within infected wild type and IPS-1 2/2 infected mice (Fig 6D) , demonstrating that an absence of IPS-1 did not directly affect expansion and/or recruitment of this DC subset. Within the popliteal DLNs, mock-infected IPS-1 2/2 mice compared to wild type mice generally showed elevated numbers of B cells, CD4+ T cells (p,0.05), and CD8+ T cells (Fig 6E, F, and G) . 
We further analyzed the lymphocyte composition of the spleen on day 6 after WNV infection (Fig 7) . Gross pathologic analysis revealed that WNV infection of IPS-1 2/2 mice results in massive splenomegaly whereas infection of wild type mice induces only a slight increase in spleen size (Fig 7A) . Cell analysis revealed increased numbers of total lymphocytes in the spleens of infected IPS-1 2/2 mice as compared to wild type mice (Fig 7B) .
Regulatory T (T reg ) cells have recently been shown to contribute to the dampening of inflammation and adaptive immune responses during acute virus infections [26, 43, 44] . Moreover, a reduction in the number of circulating T reg in mice leads to enhanced lethality after WNV infection [45] . A time course analysis of T regs in wild type mice revealed that WNV infection results in a significant increase in the numbers of FoxP3+ T cells as compared to mock-infected mice beginning on day 4 and peaking by day 6 pi (Fig 7C) , indicating the expansion of T regs during acute WNV infection. Despite their marked increase in viral load, the infected IPS-1 2/2 mice did not display an increase in the numbers of FoxP3+ T cells at any timepoint analyzed. Thus, IPS-1 signaling directly or indirectly impacts T reg proliferation and does so independently of viral load.
We also observed that spleens from infected IPS-1 2/2 mice exhibited significantly increased numbers of CD8+ T cells in comparison to those from infected wild type mice, whereas the expansion of splenic CD4+ T cells in wild type and IPS-1 2/2 mice were not different (Fig 7D) . The spleens from WNV-infected IPS-1 2/2 mice showed significantly higher numbers (3.9-fold; p,0.05) of WNV-specific CD8+ T cells producing IFNc.
To further define the phenotype associated with WNV-induced splenomegaly in IPS-1 2/2 mice, we also assessed the numbers of NK cells and neutrophils. Spleens from infected IPS-1 2/2 mice contained greater numbers of NK cells (CD4 2 CD8 2 NK1.1 + cells), NKT cells (CD4+/CD8+/NK1.1+ cells) and neutrophils (CD11b + Gr1 + cells) (Fig 7E) . Although WNVinfected wild type mice infected displayed slight increases in the absolute numbers of these specific cell types, a deficiency of IPS-1 resulted in a more marked enhancement of these immune cell populations.
In this study, we establish a major role for RLR signaling in protection from WNV pathogenesis, and demonstrate that IPS-1 is critical for the control of WNV infection in vivo. Our studies indicate that IPS-1-dependent RLR signaling functions to establish balanced, effective, and protective innate and adaptive immune responses, and that IPS-1 links adaptive immune regulation with the innate immunity triggered by RLR signaling during WNV infection. In the absence of IPS-1 in vitro, innate immune defense programs of myeloid DCs and macrophages were ablated or severely attenuated. Moreover, in vivo analysis of infected IPS-1 2/ 2 mice showed altered IgG and IgM antibody responses with diminished virus neutralization activity. The inflammatory response to WNV infection is regulated by IPS-1-dependent processes such that a deficiency of IPS-1 resulted in elevated proinflammatory cytokine and chemokines and increased numbers of inflammatory DCs, antigen-specific T cells, natural killer cells, and neutrophils in lymphoid organs, and activated macrophage/ microglial cells within the CNS. The dysregulated inflammatory response to WNV infection in IPS-1 2/2 mice was associated with a reduction in the numbers of T reg cells and their failure to expand during acute infection. These observations demonstrate the critical role of IPS-1 in mediating RLR signaling of innate antiviral immunity against WNV infection, and reveal novel features of IPS-1 function in regulating immune homeostasis, inflammation, and adaptive immunity to infection.
Although infection of primary DCs, macrophages, and neuronal cells failed to induce type I IFN upon WNV infection, WNVinfected IPS-1 2/2 mice showed enhanced systemic type I IFN responses. This finding agrees with previous studies that indicate both IPS-1-dependent and -independent pathways contribute to the systemic type I IFN production in vivo [8, 9, 23, 25] . Most importantly, the enhanced tissue tropism and rapid viral entry into the CNS observed in the IPS-1 2/2 mice is not affected by the elevated systemic IFN responses. This suggests that local tissuespecific and intracellular responses triggered by RLR-dependent signaling are more essential for reducing viral burden and dissemination. One possible explanation is that a distinct set of RLR-responsive genes function to control virus replication at the site of infection. This could explain, in part, the elevated levels of virus replication, enhanced tissue tropism and cell-to-cell spread in mice or cells deficient in IRF3 or IRF-7, each of which are downstream transcription factors of RLR signaling [8, 9, 10] . Additionally, it is likely that pDCs, which are specialized dendritic cells for producing systemic type I IFN during a viral infection [46] , are likely contributing to the IFN responses observed during WNV infection. Studies by Silva et al. have shown that WNV triggers IFN induction in pDCs through a replication-independent manner [47] . Interestingly, within the DLN, we observed similar expansion of pDCs between wild type and IPS-1 2/2 infected mice, yet at the same timepoint (24 hours pi), elevated systemic type I IFN responses were observed in IPS-1 2/2 mice. This suggests two possibilities: 1) splenic pDCs or circulating pDCs are likely responding to the high levels virus in the serum from the IPS-1 2/2 infected mice to produce IFN at 24 hours pi and/or 2) various other cell types that express TLR3 and/or TLR7 are responding to WNV infection and contributing to systemic IFN responses. Taken together, these studies indicate that RLR signaling and the actions of IRF-3/7 are important in triggering IFN production and ISG expression to limit WNV replication and spread, and that TLR signaling may impart additional, RLRindependent defenses that regulate immunity against WNV infection. The production of and response to type-I IFN is a major linkage point between innate and adaptive immunity, as IFN-a and IFN-b sustain B cell activation and differentiation [48, 49, 50] , expand antigen-specific CD8+ T cells [51, 52] , CD4+ T cells [53] , and activation of NK cells [54] . One of the most intriguing aspects of this study was the global alteration of the immune response elicited in the IPS-1 2/2 mice, indicating that RLR signaling couples innate immunity with regulation of the adaptive immune response. Infection of IPS-1 2/2 mice exhibited increased IgM and IgG WNV-specific antibodies, enhanced WNV-specific CD8+T cell response, and increased expansion of neutrophils, NK cells and NK-T cells. One trivial explanation for these differences is that there is an increased antigen load in the absence of IPS-1 and, as a result, enhanced virus-specific (e.g. CD8+ T cells, IgG and IgM antibodies) and nonspecific (e.g. Neutrophils, NK cells) responses. However, there are several key findings from this study that argue against these responses simply being attributed to higher antigen load: (1) In the absence of IPS-1, the CD4 and CD8 T cells, which are protective against WNV infection [34, 35, 36, 55, 56, 57, 58] , were significantly enhanced in the peripheral and CNS compartments but failed protect against infection. One explanation for this observation is that the expansion and migration of CD4 and CD8 T cells to different tissues was itself uncontrolled, resulting in T cell-mediated pathology rather than T cell-mediated protection.
(2) While the quantity of virus-specific IgM and IgG antibody responses were greatly enhanced in the absence of IPS-1, there was a marked reduction in antibody quality in terms of neutralization capacity. In contrast deficiencies in TLR3 or MyD88 (data not shown) did not alter virus-specific antibody responses and neutralization capacities. Collectively, these findings suggest that RLR-dependent signaling coordinates effective antibody responses during WNV infection through as yet undefined pathway. (3) While systemic IFN responses provide a link between innate and adaptive immune responses, our studies suggest that the PRR signaling pathways (RLR-dependent vsindependent) and levels of IFN production in combination with production other proinflammatory cytokines or chemokines regulate the quantity and quality of the immune response during virus infection. Thus, in the absence of IPS-1 signaling, infected conventional DCs or Mw, two integral cell types in establishing adaptive immunity, likely do not produce IFN or the normal array and level of proinflammatory cytokines/ chemokines. Instead, IFN and other mediators may be strictly produced by infected or bystander cells during WNV infection, occurring with altered kinetics and magnitude, through TLR-dependent pathways, such as TLR3 and/or TLR7 [23, 25] . (4) In the absence of IPS-1, the enhanced expansion of Ly6C+ ''inflammatory'' DCs failed to limit early WNV replication and dissemination. This inflammatory DC subset migrates to sites of infection, secretes pro-inflammatory cytokines, and promotes CD8+ T cell expansion during a secondary virus infection, suggesting it sustains the effector T cell response [59] . Our data indicate that Ly6C+ DC recruitment and/or expansion is governed by IPS-1-dependent events of RLR signaling. Thus, the aberrant recruitment/expansion of these inflammatory DCs may contribute to immunopathogenesis and limit development of an effective immune response to control WNV virus infection. (5) The lack of T reg expansion during WNV infection correlated with altered IFN levels, increased proinflammatory cytokines and chemokine levels, and an increased number and distribution of antigen-specific CD8+ T cells. These observations implicate an indirect or direct role for IPS-1 in regulating T reg levels during WNV infection, and provide evidence that links a lack of T reg expansion to immune dysregulation.
While their importance in autoimmunity is established [60] , recent studies have implicated an integral role for T regs in controlling inflammation and adaptive immune responses during acute virus infections [26, 43, 44] . During acute infection T regs function to locally contain and control the immune response with the dual outcome of suppressing viral dissemination while decreasing the likelihood of immune-mediated pathology. In support of this model, infection studies with herpes simplex virus (HSV) and mouse hepatitis virus (MHV), two well established models of viral encephalitis, have demonstrated the importance of T regs in limiting proinflammatory cytokine and chemokine responses to protect the CNS and enhance survival [26, 43] . Recent work also implicates T regs in the control of WNV pathogenesis, wherein peripheral expansion of T regs was associated with asymptomatic infection among WNV-infected blood donors but reduced T reg levels associated with WNV disease [45] . Furthermore, these studies revealed that the conditional depletion of T reg cells in mice results in enhancement of WNV virulence and expansion of antigen-specific CD8 T cells. Interestingly, from our studies, type I IFN does not appear to be the major contributor to T reg expansion during WNV infection, as T regs failed to expand in the IPS-1 2/2 infected mice despite their elevated levels of systemic type IFN. These observations suggest that RLR signaling through IPS-1 provides essential signals that directly or indirectly impart the expansion of T regs during WNV infection.
We propose that IPS-1 coordinates an innate/adaptive immune interface wherein IPS-1-signaling after RLR engagement regulates the quantity, quality, and balance of the subsequent immune response. The integrity of the innate/adaptive immune interface is central to the eliminating virus but also restricting immunopathogenesis and inflammation during infection. RLR signaling is essential for triggering the innate immune response to RNA viruses that cause human disease, including the influenza viruses, respiratory syncytial virus and other paramyxoviruses, picornaviruses, reoviruses, flaviviruses, and hepatitis C virus [14, 19, 22] . Thus, in addition to WNV, IPS-1-dependent RLR signaling will likely have a broad impact for the control of inflammation, immune response quality, and viral disease.
BHK21 and L929 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 1 mM sodium pyruvate, antibiotic-antimycotic solution, and 16 nonessential amino acids (complete DMEM). WNV strain TX 2002-HC (WN-TX) was isolated by as previously described [11] . Working stocks of WN-TX were generated by a single round of amplification on Vero-E6 (ccl-81; ATCC) cells, and supernatants were collected, aliquoted, and stored at 280uC. Virus stocks were titered by a standard plaque assay on BHK21 cells as previously described [40] .
IPS-1 2/2 (C57BL/66129Sv/Ev) and their wild type littermate control mice have been published [38, 61] and were obtained as a generous gift from Dr. S. Akira (Osaka University, Osaka, Japan). Mice were genotyped and bred under pathogen-free conditions in the animal facility at the University of Washington. Experiments were performed with approval from the University of Washington Institutional Animal Care and Use Committee. The methods for mice use and care were performed in accordance with the University of Washington Institutional Animal Care and Use Committee guidelines. Age-matched six to twelve week old mice were inoculated subcutaneously (s.c.) in the left rear footpad with 100 PFU of WN-TX in a 10 ml inoculum diluted in Hanks balanced salt solution (HBSS) supplemented with 1% heatinactivated FBS. Mice were monitored daily for morbidity and mortality.
For in vivo virus replication studies, infected mice were euthanized, bled, and perfused with 20 ml of phosphate-buffered saline (PBS). Whole brain, spinal cord, kidney, and spleen were removed, weighed, homogenized in 500ul of PBS, and titered by plaque assay.
Bone-marrow derived DC and Mw were generated as described previously [9] . Briefly, bone marrow cells from wild type and congenic deficient mice were isolated and cultured for 7 days in either RPMI-1640 supplemented with granulocyte-macrophagecolony stimulating factor, and interleukin-4 (Peprotech) to generate myeloid DC or in DMEM supplemented with macrophage colony stimulating factor (Peprotech) to generate Mw. On day 7, DC or Mw were infected with WN-TX at an MOI of 1.0 and at 12, 24, 36, and 48 hours post-infection (hpi), supernatants were collected for titration of viral burden by plaque assay on BHK21 cells and levels of IFN-b (described below). Cells were collected in parallel for western blot analysis. Cortical neurons were isolated from 15-day-old embryonic mice and cultured as described previously [62] . On day 6 of culture, neurons were infected with WN-TX at an MOI of 1.0 and at 12, 24, 36, and 48 hpi, supernatants were collected for virus titration by plaque assay on BHK21 cells and cells were collected for RNA analysis by RT-qPCR (described below).
Cells were lysed in modified RIPA buffer (10mM Tris [pH 7.5], 150mM NaCl, 0.5% sodium deoxycholate, and 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail II (Calbiochem). Protein extracts (25 mg) were analyzed by immunoblotting as described previously [11] . The following primary antibodies were used to probe blots: mouse anti-WNV from the Center for Disease Control; rabbit anti-ISG56, rabbit anti-ISG54, rabbit anti-ISG49, kindly provided by Dr. G. Sen; mouse anti-PKR from Santa Cruz; rabbit anti-RIG-I and rabbit anti-MDA5 from IBL; mouse anti-tubulin from Sigma; and rabbit anti-STAT-1 from Cell signaling. Secondary antibodies included peroxidase-conjugated goat anti-rabbit, goat anti-mouse, donkey anti-rabbit, and donkey anti-mouse were from Jackson Immunoresearch.
For analysis of viremia, serum was separated (BD Microtainer tube SST) and RNA was extracted as previously described [8] . WNV RNA copy number was measured by RT-quantitative PCR (RT-qPCR) as previously described [63] . For cultured cells, total RNA was extracted using the RNeasy kit (Qiagen), DNase treated (Ambion) and evaluated for ISG49, ISG56, IFN-b, RIG-I, and MDA5 RNA expression by one-step SYBR Green RT-qPCR. Specific primer sets for ISG-49, ISG-56, RIG-I, and IFN-b have been described previously [30, 64] . Primer sets for MDA5 are: 59-GTGGTCGAGCCAGAGCTGAT and 39-TGTCTCATG-TTCGATAACTCCTGAA.
IFN-a and -b were measured in sera using a biological assay as previously described [65] . Briefly, L929 cells were seeded at 3610 4 cells/well in a 96 well plate one day prior to the addition of interferon standards or experimental samples. Mouse sera (diluted 1:10 in L929 media) were treated with UV light for 20 minutes to eliminate residual virus. Duplicate sera samples were then added to the 96-well plates in two-fold dilutions along with a murine IFNb standard. The following day, EMCV challenge virus was added to the cells in 50 ml/well at an MOI of 5.0. Twenty-four hours later, cytopathic effect was measured by a blinded scorer and IFN levels in the sera was calculated based on the IFN standard. IFN-b in cell culture supernatants was analyzed using mouse-specific ELISA kits from PBL Biomedical Laboratories according to the manufacturer's protocol.
WNV-specific IgM, total IgG, IgG1, and IgG2a levels were determined by an ELISA using purified recombinant E protein as previously described [55] . The neutralization titer of serum antibody was determined by using a previously described plaque reduction neutralization assay [40] . Briefly, sera samples from mock or WN-TX infected mice were diluted in DMEM followed by incubation at 56uC for 30 minutes to inactivate virus and complement factors. Sera were further diluted in two-fold increments and incubated with 100 PFU of WN-TX at 37uC for 1 hour. Standard plaque assays were performed on BHK21 cells and the dilution at which 50% of plaques were neutralized was determined by comparing the number of plaques formed from WNV-infected sera samples to mock infected sera samples.
Cytokine/chemokine analysis WNV infected sera were analyzed for the presence and levels of TNF-a, IFN-c, CXCL10 (IP-10), and IL-6 by a mouse-specific cytokine/chemokine Milliplex ELISA (Millipore).
Mock-infected or WNV-infected mice were exsanguinated and perfused with PBS, 4% paraformaldehyde, pH 7.3. Brains were embedded in paraffin and 10-mm sections were prepared and stained with hematoxylin and eosin (H&E) by the UW histology pathology laboratory. Sections were analyzed using a Nikon Eclipse E600 microscope (UW Keck microscope facility).
Draining lymph nodes from mice were isolated and digested with collagenase (Roche) and type I DNase in serum-free RPMI media at 37uC for 40 minutes with mechanical disruption. Cells were then incubated with RPMI media containing 10% FBS with EDTA and HEPES for 10 minutes at room temperature, pelleted, and resuspended in PBS containing 2% FBS and 0.1% sodium azide (FACS Staining buffer). Splenocytes were isolated, washed, and re-suspended in RPMI 1640 containing 10% FBS before in vitro stimulation. Cells were washed twice before FACS staining. For isolation of CNS immune cells, mice were euthanized and perfused extensively with PBS to remove residual intravascular leukocytes. Brains and spinal cords from 5 mice per experimental group were isolated and pooled. Tissues were minced in RPMI media, triturated, and digested with Liberase (Roche) and type I DNase in serum-free RPMI media at 37uC for 45 min. Immune cells were isolated after gradient centrifugation from a 37/70% Percoll interface and washed twice with FACS staining buffer. Immune cells were stained with antibodies specific to CD11c, CD11b, B220, CD3, CD25, CD4, CD8, NK1.1, Gr-1, siglec H, and CD45 (all reagents from eBiosciences). Intracellular FoxP3 staining was performed as previously described [26] . Intracellular IFN-c staining was performed on splenocytes and CNS immune cells as previous described [35, 36] . Briefly, lymphocytes were stimulated with 1 mg/ml of the WNV NS4B peptide (SSVWNAT-TAI) for 4 h at 37uC. Cells were washed and stained for cell surface markers followed by permeabilization-fixation using the Cytofix-Cytoperm Kit (BD-PharMingen) and stained with a Pacific-Blue conjugated IFN-c antibody (eBiosciences) at 4uC for 30 min, washed and analyzed by flow cytometry. Flow cytometry was performed on a BD LSRII machine using BD FACSDiva software. Cell analysis was performed on FlowJo (v.8.7.2) software.
For in vitro studies and immune cell analysis an unpaired student T-test was used to determine statistical differences. For in vivo viral burden analysis, Mann-Whitney analysis was used to determine statistical differences. Kaplan-Meier survival curves were analyzed by the log-rank test. A p-value #0.05 was considered significant. All data were analyzed using Prism software (GraphPad Prism5). Severe malaria - a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report BACKGROUND: Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here. CASE PRESENTATION: A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent. CONCLUSIONS: The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans. The underlying pathophysiology of malaria is not well understood despite considerable and prolonged international research effort. This effort is largely driven by the need to reduce the impact of Plasmodium falciparum on human life, particularly in African children [1, 2] . Part of the difficulty in studying the pathophysiology of P. falciparum malaria is the lack of a permissive animal model and a comparator disease in man. With the recent discovery of severe malaria, caused by the zoonotic parasite Plasmodium knowlesi in the human population of Southeast Asia [3, 4] , it is now possible to link findings in primates and humans through this common agent.
In the natural primate hosts (long and pig-tailed macaques), P. knowlesi causes asymptomatic low-grade parasitism. In contrast, hyperparasitaemia and death ensue in the well-used Rhesus experimental model [5] . Consequently, there is much information on experimental P. knowlesi malaria, including organ pathology in various animal models [6] [7] [8] . Natural P. knowlesi infections of humans were formerly missed because of morphological similarity with Plasmodium malariae [9, 10] . Now clinical descriptions of knowlesi malaria encompass a spectrum of disease ranging from uncomplicated to fatal malaria [3, 4] .
The pathophysiology of severe knowlesi malaria in humans is undescribed, but nonetheless important for several reasons. First, to provide improved guidelines for the diagnosis, treatment and management of severe knowlesi cases. Secondly, when severe knowlesi infection is compared with severe falciparum malaria, this can help clarify determinants of severe disease. Thirdly, it is possible that we are observing a P. knowlesi vertebrate host switch from Southeast Asian macaques to a fullblown emergence into the human population [11, 12] . Therefore, it is important to understand properly the disease caused by P. knowlesi to assist strategies to reduce health-impacts if human-to-human transmission of this virulent pathogen is established.
The clinical, laboratory and, for the first time, postmortem findings in a fatal case of knowlesi malaria are reported here. The histopathology in various organs is explained within the context of P. knowlesi biology and comparisons with the salient features made with what is known of the pathophysiology of severe falciparum malaria [13] [14] [15] [16] .
A forty year-old male was brought to the Queen Elizabeth Hospital, Kota Kinabalu, Sabah (8.30 am) in a state of collapse. He was unable to give a history himself or to stand. On examination his blood pressure was unrecordable and oxygen saturations were recorded as low.
The patient had no past medical history. He had spent two weeks in the jungles of Borneo before leaving for an urban setting. Ten days after leaving the jungle he experienced the onset of fever and body aches. Two days later, he sought treatment at a government outpatient clinic continuing to complain of fever and myalgia. A specific diagnosis was not made and he was able to work although he developed rashes the next day. He remained unwell for the next two days, when he presented in a state of collapse and had developed abdominal pain.
Resuscitation measures were begun and the patient was immediately intubated, given adrenaline/atropine and sodium bicarbonate. On examination and after resuscitation measures his vital signs were: symmetrical air entry into lungs, BP 58/44 mm Hg, pulse rate 40-50 per minute with poor peripheral perfusion and cyanosis. Heart sounds were normal. He had generalized petechiae and his abdomen was tense and distended.
Resuscitation measures continued for one hour, during which time "coffee grounds" were observed in the nasogastric aspirate. The patient became asystolic after one hour and although cardiopulmonary resuscitation was given for a further 20 minutes, there was no response. The patient was pronounced dead two hours after admission.
Dengue haemorrhagic shock was suspected and a post-mortem examination was performed approximately 24 hours later.
Laboratory results are summarized in Table 1 . The patient was not anemic, but was thrombocytopenic and had an eosinophilia. He was also hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values. Serum creatinine was not available. A blood sample taken 24 hours post-mortem showed >10% of erythrocytes infected with predominantly pigmented parasites ( Figure 1a ). Heavily pigmented monocytes were also present ( Figure 1b ). Plasmodium knowlesi, as a single species infection, was confirmed by nested-PCR [9] . Post-mortem dengue serology was negative (Table 1 ) and dengue, respiratory syncytial virus and enterovirus were not isolated in organ samples.
External examination showed a well-nourished adult male. The conjunctivae showed tinges of jaundice and the right eye had subconjunctival haemorrhages. There were multiple petechial haemorrhages on the body and venepuncture sites were associated with marked bruising. Coffee ground material was noted in the mouth. Internal examination revealed no tissue oedema or excess fluid in the body cavities.
The external surfaces of the cerebrum were dusky. The cut sections showed multiple petechial haemorrhages. The cerebellum also showed petechial haemorrhages externally and on multiple cut sections (Figure 2a and 2b). The brain stem and upper spinal chord were grossly normal. Both lungs were heavy (weighing on the right 720 g and left 690 g) and cut sections were congested Figure 1 Thin blood film showing mostly late trophozoites of P. knowlesi in poorly defined erythrocytes (1a) and heavily pigmented monocyte (1b). Note that the blood film was prepared 24 hours post mortem and shows red cell ghosts, particularly among parasite infected red blood cells.
and 'beefy' in appearance. Petechial haemorrhages were present on the endocardium with extensive subendocardial haemorrhages involving the left ventricular wall. The haemorrhages were most prominent at the apex of the heart. The liver (2640 g) and spleen (340 g) were markedly enlarged. The cut surfaces of the spleen were soft and friable. The gallbladder, pancreas and kidneys were grossly normal.
Haematoxylin and eosin stained sections from various organs were available for examination. Parasitized red blood cells (PRBC) were abundant although parasite bodies were obscured by haemozoin (malaria) pigment. Chemical removal of pigment and oil immersion (×1,000) magnification revealed trophozoites that were discernibly bigger than those of P. falciparum. Immunohistochemistry stained sections from the brain were Plasmodium anti-aldolase positive ( Figure 3 ) and negative for P. falciparumspecific staining [17] .
Many petechial haemorrhages (up to 600 μm diameter) arising from the rupture of the small vessels of the cerebrum and cerebellum were observed (Figures 4a  and 4b ) Sequestration of PRBC was evident within small blood vessels (Figures 4c and 4d ). Congested larger vessels and areas of haemorrhage showed considerable amounts of malaria pigment (Figures 4e and 4f ). Clumps of platelets or evidence of thrombi in vessels were not seen. There was no evidence of vasculitis or perivascular chronic inflammatory reaction in the brain or any other organ examined (heart, kidney, liver, adrenal gland and spleen). There was no evidence of perivascular or diffuse parenchymal oedema in the brain. Diffuse astrocytosis or microgliosis was not observed, nor was there evidence for acute gliotic reactions about the haemorrhages. There was no aggregation of polymorphs in the vessels, no perivascular inflammation, nor generalized encephalitis. There was no diffuse thrombotic microangiopathy, but within one haemorrhage there was probably some fibrin at the site of the vessel. Immunohistochemistry of sections from the brain was negative for CD54 (which stains intercellular adhesion molecule-1, ICAM-1). Figure 2 Gross appearance of the brain. The outer surface appears dusky with petechial haemorrhages seen on the outer surface of the cerebellum (2a). Cut section of the cerebellum with multiple petechial haemorrhages seen within the cerebellar grey matter (2b).
Plasmodium-specific anti-aldolase immunohistochemistry stained sections from the brain. Parasites appear red.
Although sections from the spleen showed some autolysis, expansion of the red pulp and atrophy of the white pulp was noted. Germinal centers were not observed. Abundant pigment-containing macrophages and some haemophaghocytosis was evident in the red pulp and parasitized red cells were plentiful (Figure 5a ). There was no necrosis or fibrin deposition in the spleen.
There were many PRBC's in the liver sinusoids with haemozoin pigment in Kupffer cells and evidence of haemophagocytosis. The portal tracts and sinusoids had moderate chronic lymphoplasmacytic inflammation. Overall the liver was non-cirrhotic but with severe macrovesicular steatosis. No cholestasis, regional necrosis or thrombotic microangiopathy was observed (Figure 5b) .
The renal cortex showed dilated and congested blood vessels. Many PRBC were observed within glomerular capillaries with pigment deposition in the mesangium. There was no evidence of thrombotic microangiopathy (disseminated intravascular coagulation, DIC). The tubules showed acute tubular necrosis and regeneration. There were a small number of eosinophilic intra-tubular casts (Figure 5c) .
Sequestration of PRBC's was evident in the small vessels of the heart (Figure 5d ). Endothelial cells were prominent as sometimes observed in patients with sepsis whose cells are responding to generalized stimuli [18] . There was no evidence of myocarditis and the heart muscle fibers appeared normal. There was focal petechial haemorrhage in the subendocardium, which may relate to resuscitation, or be secondary to malaria.
The adrenal gland appeared active with eosinophilic cytoplasm in the fasciculata layer with no evidence of PRBC sequestration or of parenchymal haemorrhage. Samples of lung, intestine or bone marrow were not available for histopathology examination.
The overall picture was one of systemic malaria infection with multi-organ damage, particularly in the brain where there was much vascular rupture and petechial haemorrhaging.
Fatal human knowlesi malaria has hitherto not been reported at post-mortem, so this report may increase our understanding of severe and fatal malaria whatever species cause these syndromes. Interestingly, vivax malaria is also becoming recognized as causing severe and sometimes fatal infection in a significant proportion of individuals, suggesting re-evaluation of the dogma that falciparum infection may be the only important cause of fatal disease [19] [20] [21] .
The WHO classification for severe falciparum malaria would have included this individual because he had several qualifying features including hyperparasitaemia (~10% infected erythrocytes), renal impairment, jaundice and ARDS, although coma was not prominent in the history [3, 4, 22] . Commonly, dengue haemorrhagic fever can cause some of these signs, but was excluded by investigations. Co-infection of knowlesi with falciparum was also excluded by specific PCR and immunohistochemical examination.
The beefy appearance of lung tissue in this case is consistent with respiratory distress syndrome, but could not be confirmed histologically. Changes in the kidney of some recovering areas of acute tubular injury are consistent with the observation of renal impairment reported in other severe cases of knowlesi malaria [3] and an underlying process of acute tubular necrosis related to systemic shock. There was disorganized white pulp in spleen perhaps as part of a wider stress response [18] .
Despite the absence of antecedent established coma, cerebral pathology in this case is very similar to that observed in fatal falciparum infections, confirming that it was important to exclude this co-infection. Although there was obvious accumulation/sequestration of infected erythrocytes in capillaries and venules, there were also some differences between these appearances and those seen with fatal falciparum infections. For example, there was no platelet clumping, no notable thrombi, and uninfected erythrocytes were also interspersed with infected cells. Neither was up-regulation of ICAM-1 expression detected in endothelial cells, suggesting that the mechanism of sequestration/accumulation of infected erythrocytes in knowlesi infections needs further investigation. In falciparum cerebral malaria, display of ICAM-1 by up regulated endothelial cells mediates adhesion to parasitized red cells [19] .
Hyperparasitaemia is a marker of severe falciparum malaria. It is also apparent in severe knowlesi infections [3] , but is less commonly observed with severe vivax malaria. The 24 hr replicative cycle of asexual knowlesi may contribute to the rapidity with which hyperparasitaemia and clinical complications ensue. This shorter cycle compared with falciparum and vivax infections may overcome the reduced multiplicative capacity associated with the fewer merozoites generated by P. knowlesi (up to 16 per mature schizont) compared with P. falciparum (up to 36). Although up to 24 merozoites per schizont have been observed in Plasmodium vivax, parasitaemia is restricted to reticulocytes by limitation of invasion pathways in this species, rather than merozoite number.
Hyperparasitaemia in falciparum malaria is often explained by protection of late stage parasites from the filtering action of the spleen. Unlike P. falciparum infections, all asexual developmental stages are seen in the peripheral circulation of knowlesi-infected patients [10] . Hyperparasitaemia in knowlesi malaria may not be modulated by splenic clearance in a similar manner as suggested for other non-sequestering human infections.
As with many fatal cases of falciparum malaria, malaria pigment was evident in blood films and was present in circulating leucocytes (~40%), tissue sections and organ specific macrophages. Reticuloendothelial changes in both liver and spleen were associated with pigment, accumulation of red cells and laden macrophages, and some inflammation in liver portal tracts was observed. Circulating pigment has been variably implicated as an indicator of poor prognosis for falciparum malaria [23] [24] [25] and there is evidence to support pigmentinduced immuno-suppression, particularly of pigmentladen macrophages and monocytes [26] [27] [28] . It may be that the associations between parasitaemia, pigmentaemia and disease severity are more quantifiable in knowlesi compared with falciparum infections, where pigmented parasites are sequestered from peripheral blood samples and, therefore, unreliably quantifiable. In vivax malaria, pigment in parasites often appears more dispersed and it is rarely reported as a correlate of disease [29] .
Among the malaria parasites of humans, cytoadherence is purportedly unique to P. falciparum, resulting in sequestration of all but early trophozoite stages in falciparum malaria. Cytoadherence is often implicated in malaria pathology and is mediated by the expression of variable var gene products (of the PfEMP-1 family) at the surface of infected host red blood cells with concomitant expression of post-capillary endothelial cell receptors [15, [30] [31] [32] . Although not easily quantifiable, partial sequestration was observed in this fatal case of knowlesi malaria as evidenced by an abundance of large pigment bearing parasites on the blood film obtained post-mortem and as accumulations in the microvasculature. We hypothesize that partial sequestration may be due to P. knowlesi infected cell agglutination mediated by variant surface antigens of P. knowlesi SICA var genes (orthologues of the PfEMP-1 family) [33, 34] . Importantly, in this fatal case of P. knowlesi, up-regulation of ICAM-1 was not detected, nor was there evidence for cytoadherence as parasitized cells were not marginalized and were interspersed with uninfected erythrocytes in smaller vessels.
Cerebral malaria and severe acute anaemia are often peculiar to falciparum infection while organ, respiratory and metabolic dysfunction are common with severe knowlesi, vivax and other forms of severe falciparum infection [3, 4, 19, [35] [36] [37] [38] [39] [40] . Although late stages of parasites may be visible in the blood in falciparum infections, they are rare and associated with a poor prognosis. Testing the relative contribution of virulence factors to the development of severe malaria including, cytoadherence, hyperparasitaemia, cerebral malaria, organ failure, metabolic and respiratory dysfunction and anaemia was previously difficult without comparative information from severe malaria caused by another species [16, [41] [42] [43] [44] . Severe and fatal cases of knowlesi malaria will add much needed perspective to what is known of malaria pathophysiology.
The need to develop knowlesi-specific diagnosis, treatment and management guidelines is urgent. A recent prospective clinical study in Sarawak Malaysian Borneo revealed that approximately 1:10 patients infected with P. knowlesi present with or develop severe symptoms and 1-2% of cases are fatal [4] . Complications in survivors included ARDS, liver or renal dysfunction, hypotension with or without parasitaemia >100,000/uL. Complications in all fatal knowlesi cases had either clinical or laboratory evidence of abdominal pain, combined hepatorenal dysfunction and hyperparasitaemia [3, 4] . There is a prospect that P. knowlesi may emerge as a human pathogen beyond its current zoonotic manifestations [11, 12] , specific surveillance, control and clinical guidelines are necessary to contain potential larger outbreaks.
Approval to use this case for educational purposes was obtained from the Deputy Director for Health, Sabah State Department of Health, Ministry of Health, Malaysia. Mathematical Modeling of the Effectiveness of Facemasks in Reducing the Spread of Novel Influenza A (H1N1) On June 11, 2009, the World Health Organization declared the outbreak of novel influenza A (H1N1) a pandemic. With limited supplies of antivirals and vaccines, countries and individuals are looking at other ways to reduce the spread of pandemic (H1N1) 2009, particularly options that are cost effective and relatively easy to implement. Recent experiences with the 2003 SARS and 2009 H1N1 epidemics have shown that people are willing to wear facemasks to protect themselves against infection; however, little research has been done to quantify the impact of using facemasks in reducing the spread of disease. We construct and analyze a mathematical model for a population in which some people wear facemasks during the pandemic and quantify impact of these masks on the spread of influenza. To estimate the parameter values used for the effectiveness of facemasks, we used available data from studies on N95 respirators and surgical facemasks. The results show that if N95 respirators are only 20% effective in reducing susceptibility and infectivity, only 10% of the population would have to wear them to reduce the number of influenza A (H1N1) cases by 20%. We can conclude from our model that, if worn properly, facemasks are an effective intervention strategy in reducing the spread of pandemic (H1N1) 2009. Novel influenza A (H1N1) (hereafter referred to as pandemic (H1N1) 2009 in keeping with the World Health Organization (WHO) nomenclature) is a new flu virus of swine, avian, and human origin that was first identified in mid-April 2009 in Mexico and the United States [1] . The virus soon spread to the rest of the world and on June 11, 2009 the WHO declared novel influenza A (H1N1) a pandemic. The virus continues to spread, with most countries reporting cases of pandemic (H1N1) 2009 [1] . Even though the WHO's declaration of a phase six pandemic alert level does not explicitly refer to the severity of the disease, as many people contracting the virus recover without medical treatment, the number of deaths continues to rise [1] . The rapid spread of influenza, due to its short incubation period and lack of strainspecific vaccine, pose a challenge to the implementation of effective mitigation strategies during the expected reemergence of pandemic (H1N1) 2009 in the fall/winter flu season. Every year approximately 36,000 people die from seasonal influenza or flurelated causes in the U.S. [2] . However, the number of casualties may increase with a new and more virulent strains of influenza, such as the pandemic (H1N1) 2009.
The emergence of an unexpected or new strain of influenza means there are no prepared vaccines and the existing antivirals may be ineffective in combating the spread of infection. Vaccination is typically the first line of defense against influenza viruses [3] . The entire vaccine production process takes at least six months to complete [4] and although a pandemic (H1N1) 2009 vaccine became available in the U.S. in October 2009, there are severe shortages in the amount of vaccines available. Another concern is that the currently circulating H1N1 strain could mutate, making the vaccine ineffective or less effective.
In the recent pandemic (H1N1) 2009 outbreak, non-pharmaceutical interventions such as school closings and thermal screenings at airports were implemented to slow the spread of disease [5, 6] . Other common non-pharmecuetical interventions include quarantine, isolation, travel restrictions, closing of public places, fear-based self quarantine, and cancellation of events. These interventions all have economic costs to individuals and society related to lost work, increased school absenteeism, and decreased business revenues.
Another non-pharmaceutical option is the use of facemasks. In the 2003 SARS outbreak many individuals used facemasks to reduce their chances of contracting infection. In Hong Kong 76% of the residents reported using masks during the 2003 SARS epidemic [7] . Even though individuals have taken upon themselves to wear facemasks during disease outbreaks, little research has been done to quantify the impact of the use of facemasks during an epidemic. Mathematical models of the spread of infectious disease can be useful in assessing the impact of facemasks on reducing the spread of a disease, specifically pandemic (H1N1) 2009.
Pandemic (H1N1) 2009 spreads through person-to-person contact, airborne particles, coughing and sneezing, and by fomites [1] , therefore, the use of facemasks is a logical line of defense. The Centers for Disease Control and Prevention (CDC) have interim recommendations on the use of facemasks and respirators for the current pandemic (H1N1) 2009 virus. The CDC defines the term facemask as a disposable mask cleared by the U.S. Food and Drug Administration (FDA) for use as a medical device, such as surgical masks. Surgical masks are designed to help stop droplets from being spread by the person wearing the mask, not to protect against breathing in very small particle aerosols that may contain viruses [8] . We will use of the term 'respirator' for an N95 or higher filtering facepiece respirator certified by the CDC/National Institute for Occupational Safety and Health (NIOSH); a respirator is designed to protect the person wearing the mask against breathing in very small particles that may contain viruses [8] . The CDC states that the effectiveness of the use of facemasks and respirators in various settings is unknown and do not generally recommend the use of facemasks or respirators in home or community settings nor in non-medical occupational settings [8] . In certain circumstances the CDC recommends the use of masks for individuals who are at high risk of infection and cannot avoid situations with potential exposure to the disease [8] .
There have been a handful of studies that have analyzed the effectiveness of facemasks against nanoparticles in the size range of viruses using manikin-based protocol in which the masks were sealed on the manikin's face so that no leakage would occur [9] [10] [11] . All three studies show similar results in penetration percentage for the N95 respirator. The high fit N95 respirator had penetration percentages from about 0.5% to 2.5% at 30 l/ min and from about 0.5% to 5% at 85 l/min [9] [10] [11] . The low fit N95 respirator had penetration percentages from about 1.5% to 3.5% at 30 l/min and from about 1.5% to 6% at 85 l/min [9] [10] [11] . The surgical masks tested in Balazy et al.'s [10] study show a much greater penetration percentage. At 30 l/min one model of surgical mask (SM1) allowed 20-80% of particles to penetrate the mask, while another model (SM2) allowed 2-15% [10] . At 85 l/min SM1 allowed penetration of 30-85% of particles while SM2 allowed 5-21% [10] . The N95 respirator in a sealed manikin test seems to be fairly effective against nanoparticles, almost holding up to its 95% certification. The surgical masks are not as effective, allowing a much greater percentage of particles to pass through to the wearer even when sealed tightly to a manikin.
Unfortunately, this type of testing does not provide an accurate estimate of the level of protection for everyday use of a mask by a person. While these studies provide data on the actual protection of masks against nanoparticles in a perfect setting, it does not take into consideration that a mask will not be completely sealed on an individual nor will it fit perfectly. Furthermore, one must consider that an individual will not always be wearing the mask, for example, a mask will be taken off to eat and sleep, or possibly because it becomes uncomfortable to wear.
Lee et al. [12] performed a study on N95 respirators and surgical masks using human subjects. The challenge aerosol used was NaCl, with particles in the size range of bacteria and viruses (.04-1.3mm). They tested four models of N95 respirators: 1) high protection level, 2) medium protection level, 3) exhalation valve, and 4) exhalation without valve and three models of surgical masks: 1) high protection level, 2) medium protection level, and 3) low protection level. The results from the study showed that the lowest protection offered from N95 respirators is when particles are in the size range of 0.08-0.2mm and for surgical masks when particles are in the size range of 0.04-0.32mm. The size range of influenza virus is in the range of 0.08-0.12mm, which falls into both masks most penetrating particle size range. The N95 respirator was found to be 21.5% effective and the surgical mask was 2.4% effective in protecting against nanoparticles. The N95 respirator provides approximately nine times greater protection than a surgical mask and is clearly a better option in protecting against infection.
A University of Michigan School of Public Health study led by Dr. Allison Aiello [13] is evaluating the effectiveness of handwashing and facemasks in preventing influenza from spreading. The study, called M-FLU, conducted a randomized cluster intervention trial among students living in dorm housing. The students were randomly separated into two intervention groups, one wearing masks and practicing hand hygiene, one just wearing masks, and also in a control group. The study was carried out over the 2006-2007 influenza season, which was a mild season. The study found that facemasks and hand hygiene were correlated with a 35-51% reduction in influenza-like illness [13] .
There are many factors that influence people's willingness to wear a mask. In a study by Tang and Wong [14] a total of 1,329 . The arrows that connect the boxed groups represent the movement of individuals from one group to an adjacent one. Nonmask wearing susceptible individuals (S) can either become exposed (E) or susceptible wearing a mask (S m ). Non-mask wearing exposed individuals (E) can either become infectious non-mask wearing (I) or mask wearing exposed (E m ). Non-mask wearing infectious individuals (I) can either recover (R), die (D), or become infectious wearing a mask (I m ). Mask wearing susceptible individual (S m ) can either become an exposed mask wearer (E m ) or a non-mask wearing susceptible (S). Mask wearing exposed individuals (E m ) can either become an infectious mask wearer (I m ) or a non-mask wearing exposed individual (E). A mask wearing infectious individual (I m ) can either recover (R), die (D), or stop wearing the mask while they are still infectious (I). doi:10.1371/journal.pone.0009018.g001
adult Chinese residing in Hong Kong were surveyed on their use of facemasks during the 2003 SARS epidemic. Overall 61.2% of the respondents reported the consistent use of facemasks to prevent contracting the disease. The study found that women in the age group 50-59 and married respondents were more likely to wear facemasks, suggesting that the aesthetics of wearing a facemask may be a concern. Also, the study found that individuals who had a university education or earned more than US$5,000 per month were more likely to wear a mask. Tang and Wong also showed that perceived susceptibility, cues to action, and perceived benefits, were significant predictors in whether or not an individual consistently wore a mask.
Following the approached developed in [15] , the population is divided into two subgroups: a mask wearing group (subscript m) and a non-mask wearing group. People move back and forth between the mask and non-mask groups based on the number of individuals infected with pandemic (H1N1) 2009. Individuals in each activity group are characterized by their epidemiological status: susceptible, denoted by S and S m , exposed, denoted by E and E m (i.e., people who are infected but not yet fully contagious), and infectious individuals, I and I m . Definitions of the eight epidemiological classes are summarized in Table 1 and the transfers are shown diagrammatically in Figure 1 . Because we are evaluating the effectiveness of masks in a single influenza period, we use a closed system with no migration in or out, and births and natural deaths are not included in the model.
As seen in Figure 1 , the transfer rates of people from the exposed classes, E and E m , to the infectious classes, I and I m , are vE and vE m . Infectious individuals can move to group D, at rate mI and mI m , when they die from infection or to group R, at rate dI and dI m , upon recovery. The mean times in the infectious classes, I and I m , are 1=(dzm). Hence, the infectious fraction d=(mzd) recovers and the infectious fraction m=(mzd) dies as a consequence of this disease. We assume that there is homogeneous mixing between groups and that contact activity levels remain normal throughout the epidemic. We define t 0 as the beginning of the epidemic. Movement of individuals between mask and non-mask groups depends upon the number of pandemic (H1N1) 2009 cases in the population. A specified percentage of the population starts wearing masks as the number of infected people increases.
We define Q Sm S, Q Em E, and Q Im I to be the transfer rates from the S, E, and I classes to the S m , E m , and I m classes, respectively, similarly Q S S m , Q E E m , and Q I I m are the transfer rates from the S m , E m , and I m classes to the S, E, and I classes, respectively.
The rate coefficients are modeled by step-functions of the number of infectious individuals:
for i = S, E, I, S m , E m , and I m . Here the parameters a and b are positive constants that determine the rate of movement and t is the number of pandemic (H1N1) 2009 cases that determines when masks are implemented. For i = S, E, and I, b i is set at 0.1 or 10% of the population.
Using the transfer diagrams in Figure 1 we obtain the following system of differential equations:
Here l (non-mask group) and l m (mask group) are the forces of infection and lS and l m S m are the transfer rates from the susceptible classes, S and S m , to the exposed classes, E and E m . The infection rates, l and l m , incorporate the probability of transmission per contact, b, the reduced infectiousness due to incubation, a, the reduced number of contacts because of symptomatic infection, h, and 1{g j , (j = s or i), which accounts for the effectiveness of the mask in reducing either susceptibility (g s ) or infectivity (g i ). The transmissibility, b, is defined as the Effective Reproduction Number < con for N95 respirators. Notice that < con decreases as a higher percentage of people wear masks as well as when masks are more effective. < con is greatly reduced when 50% of the population wears masks and masks are 50% effective. doi:10.1371/journal.pone.0009018.t005
susceptibility of the population multiplied by the infectivity of the disease multiplied by the average number of contacts an individual has per day. The definitions of the parameters are summarized in Table 2 . The forces of infection for the non-mask group and mask group are shown by:
where r~N{(1{h)(IzI m ) and N is the total population 
The effective reproduction number, < eff , is the average number of secondary cases produced by a typical infectious individual Effective Reproduction Number, < con , for surgical masks. Notice that < con decreases as a higher percentage of people wear masks as well as when masks are more effective. However, < con is not greatly reduced even when 50% of the population wears masks and masks are 50% effective. doi:10.1371/journal.pone.0009018.t006
during the infectious period [16, 17] . The effectiveness of intervention strategies are often measured by their ability to reduce the spread of a disease in a given population. In an epidemic model the magnitude of the effective reproduction number, < eff , determines whether or not an epidemic occurs and its severity [15] . When < eff w1, the number of infections grow and an epidemic occurs, however when < eff v1, the number of infections does not increase and there is no epidemic outbreak [15] . Without any interventions the model has an initial effective reproduction number (uncontrolled) < unc given by:
This < unc is the product of the average number of people infected per unit time b and the weighted sum of the average infectious period 1=(mzd) plus the average incubation period 1=v.
The 'next-generation operator' approach [17] is used to find an expression for the effective reproduction number (controlled) < con for our epidemic model when masks are used as an intervention strategy. The computation is done by linearizing the system of equations (2) around the disease-free equilibrium (DFE). The DFE has E, E m , I, and I m equal to zero with S 0 , S 0 m , and R 0 positive. Since there is no immunity from previous infection or vaccination R 0 is also equal to zero. The resulting fourdimensional linearized system is of the form dX dt~( The effective reproduction number < con is the largest eigenvalue of the matrix FV {1 [17] . Hence < con is the only non-zero eigenvalue of the matrix FV {1 and is given by the expression: Figure 2 with respirators is also seen here: as the masks effectiveness is higher the number of cumulative cases decreases and the number of cases also decreases if a higher percentage of people wear masks. However, the difference in the number of cumulative cases is not nearly as large when surgical masks are worn; this is due to their lower effectiveness. doi:10.1371/journal.pone.0009018.g003
where c 1~QEm zv, c 2~QE zv, c 3~QIm zdzm, c 4~QI zdzm, and s~S 0 zS 0 m . We use equations 4 and 7 to define the effective reproduction number for the model as:
where t is the threshold number of infected individuals at which masks start to be used.
The epidemiology of pandemic (H1N1) 2009 is not accurately known since it continues to spread across the world. The parameter values shown in Table 2 were chosen based on the best available data. The incubation period for pandemic (H1N1) 2009 has been reported to be 2-10 days with a mean of 6 days [18] . The mean time in the exposed classes E and E m corresponding to the incubation period has been assumed to be 6 days, making the transfer rate to the infectious classes, I and I m , constant at v = 1/6. The infectious period is believed to be between four and seven days, with an average of five days [19, 20] . Thus, the baseline value for the recovery rate is constant at d = 1/5. The fatality rate of the pandemic (H1N1) 2009 is thought to be in the range of 0.3%-1.5%, with a mean of 0.46% [21] [22] [23] . The case fatality rate for our model is m=(dzm), setting this equal to 0.0046 results in m~0:0046d=0:9954~0:001.
The current estimates on the transmission of pandemic (H1N1) 2009 are that one infected person may typically infects one to two people [24] [25] [26] . The transmissibility, b, is the product of the susceptibility of the population, the infectivity of the disease, and the number of contacts an individual has in a day [27, 28] . The susceptibility of the population is set to one, as it is believed few people are immune to pandemic (H1N1) 2009, and the number of contacts an individual has per day is assumed to be 16 [29] . The infectivity is found by 1:8=(16( a v z h mzd )), so that R 0 = 1.8 in a completely susceptible population and the infectivity is .0141. So b~0:23 gives the transmission rate, the fraction of contacts per day that is sufficient for the transmission of pandemic (H1N1) 2009. The baseline population size N for the model is set at one million people and all are initially in the susceptible class S. The initial infected fraction, I/N, is set at 0.00001 so that when N = 1000000, I = 10. The model scales linearly so that the initial population size N and the initial number of infected individuals I are both scaled up or down by the same factor. We assume that individuals will start wearing masks after 100 people are infected, Figure 4 . Sensitivity to < unc < unc . The number of cumulative cases is very sensitive to the value of the uncontrolled effective reproduction number (< unc ). Higher values of < unc result in a larger number of cumulative cases. A large difference in the number of cases is seen when the < unc is equal to 1.83 and when < unc is equal to 1.7; for such a slight difference in < unc the difference in the number of cases is quite large. doi:10.1371/journal.pone.0009018.g004 (6) once there is enough number of cases in a community to convince people to start wearing masks. We analyzed the impact of masks when 10%, 25%, and 50% of the population wear them.
Using the studies published on the effectiveness of masks we determined the baseline values for the effectiveness of N95 respirators to be g s = 0.2 and g i = 0.5 and for the surgical masks g s = 0.02 and g i = 0.05 [12] . The effectiveness of masks in decreasing the infectivity of a sick individual is greater because the mask contains the virus particles, preventing them from becoming airborne, and therefore preventing the contamination of surrounding surfaces as well as people [30] .
Although it is possible that some sick individuals may change their behavior due to the symptoms [15] , we assume that sick individuals will not change their behavior and continue to have the same number of daily contacts as a healthy individual. Therefore, we set the baseline value for the reduced number of contacts due to illness h at 1, as people usually do not greatly alter their daily behavior during the incubation period. Individuals in the exposed classes, E and E m , are thought to be 50% less infectious due to incubation than those in the infected classes, I and I m , so we set a = 0.5 [19, 31] .
We analyzed two scenarios: one in which the N95 respirator is worn and one in which surgical masks are worn; for both types of masks we considered three different variations in mask effectiveness. Each case is evaluated with 10%, 25%, and 50% of susceptible and exposed individuals wearing masks, while in each case the fraction of infectious individuals wearing masks is slightly larger. When 10%, 25%, and 50% of susceptible and exposed individuals are wearing masks the fraction of infectious individuals wearing masks is 30%, 40%, and 50%, respectively. All simulations assume that in a population of one million there are initially 10 infected individuals reported and everyone else is susceptible. Mask start being used when there have been 100 reported cases of pandemic (H1N1) 2009.
The numerical results for the percentage of pandemic (H1N1) 2009 cases are shown in Table 3 for the N95 respirator and in Table 4 for surgical masks. The effective reproduction numbers for each case are shown in Table 5 for N95 respirators and in Table 6 for surgical masks. The cumulative number of pandemic (H1N1) 2009 cases can be seen graphically for the varying mask effectiveness and the different fractions of individuals wearing masks in Figure 2 and in Figure 3 for N95 respirators and surgical masks, respectively. Table 3 and Table 4 show that when masks are not used, then the total percentage of the population who will be infected is 74.61% in a population of 1 million people. With the implementation of N95 respirators Table 3 exhibits a reduction in the cumulative number of cases of almost 200,000, or a 19% decrease, when 10% of the population wears masks and they are 20% effective. Table 5 shows the implementation of the N95 Respirators' impact on the effective reproduction number < con ; it is reduced from 1.83 to 1.66 when masks are 20% effective in reducing both susceptibility and infectivity and 10% of the population is wearing masks. When effectiveness is increased to 50% < con is reduced even further to 1.4. As the fraction of the population wearing N95 respirators increases, < con is reduced Table 4 shows that surgical masks do not have as large of an impact in reducing the cumulative number of cases as does the N95 respirator. Table 6 displays the effective reproduction number < con when surgical masks are implemented. The lowest value surgical masks reduce < con to is 1.77.
In Figure 2 the effectiveness of the N95 respirator in reducing the spread of pandemic (H1N1) 2009 is significant. As the percentage of the population wearing masks increases the number of cumulative cases decreases and when the mask effectiveness is greater, the number of cases is also greatly reduced. The impact of surgical masks is not as large as seen graphically in Figure 3 , the reduction in the cumulative number of cases is relatively small compared to that of the N95 respirator. If mask effectiveness is 5% and 50% of the population wears surgical masks the reduction in the number of cumulative cases is 6%.
Even though the parameter values were estimated from epidemiological data, there is still some uncertainty in their values. Since pandemic (H1N1) 2009 is a new virus, there is a wide range of estimated values for the parameters. In our model we chose the averages for our baseline parameters, here we look at a range of parameters and how changing a specific one effects the outcome of the model. This sensitivity analysis examines the effects of changes in the reproduction number (< unc ), mask effectiveness (g s and g i ), index cases (I=N), fraction of population wearing masks (Q i ), number of initially infected at which masks are implemented (t), as well as the effect of which epidemiological group wears masks (S or I). Unless otherwise stated the other parameters are fixed at their baselines values found in Table 2 .
Effective reproduction number. The effective reproduction number < unc determines the average number of secondary cases resulting from one typical infectious individual during the infectious period without the implementation of facemasks. Since there is a delay in the implementation of facemasks the initial growth of the epidemic is affected by < unc . The estimates of < unc for pandemic (H1N1) 2009 vary widely, the common range is assumed to be between 1.2 and 2.2. As the value of < unc increases the number of pandemic (H1N1) 2009 cases increases significantly as shown graphically in Figure 4 .
Mask effectiveness. The effectiveness of the mask greatly affects the number of cumulative cases. The higher the effectiveness the fewer number of cases (shown in the Results section). The effectiveness of the masks not only depends upon the type of mask and quality but also proper usage.
Index cases. The number of initially infected individuals can have a major impact on the size of the epidemic. In Figure 5 we vary the number of initially infected individuals in the population.
Fraction of population wearing masks. We consider variations in the percentage of the population that wears masks. We look at the effect of 10%, 25% and 50% of the population wearing masks. The model shows that the higher the percentage of the population wearing masks the fewer the number of cumulative cases, this is shown in Figure 6 . Implementation of masks. The epidemic is sensitive to the delay in the implementation of masks as seen in Figure 7 . We look at the cumulative number of pandemic (H1N1) 2009 cases for the N95 respirator when 10% of the population is wearing masks. Figure 7 shows that the earlier masks are implemented, the bigger the reduction in the cumulative number of cases.
Who wears masks. The model is sensitive to who wears masks. Here we look at the effect if only infected individuals wear masks and if only susceptible and exposed individuals would wear masks. Figure 8 shows that it is important for both infected, as well as susceptible and exposed individuals, to wear masks.
The standard mitigation strategies used for influenza viruses are vaccines and antivirals. However, in the case of a novel virus these may not be readily available and other mitigation strategies will be needed. As seen during the 2003 SARS outbreak and the current pandemic (H1N1) 2009 people are willing to wear facemasks to reduce the spread of disease. We used a mathematical model to examine the possible impact of N95 respirators and surgical masks on reducing the spread of pandemic (H1N1) 2009. When modeled with a low mask effectiveness and a small fraction of the population wearing masks, the implementation of facemasks still has a relatively large impact on the size of the pandemic (H1N1) 2009.
The numerical simulation results in the results section show that without any interventions, we predict that a large percentage of the population will be infected with pandemic (H1N1) 2009 influenza strain. This result is not surprising as the population is 100% susceptible and the effective reproduction number < unc is 1.83, which is higher than that of typical seasonal influenza. In reality, the R unc may be lower due to heterogeneous mixing patterns, pre-existing immunity, and other interventions in place. With 10% of the population wearing N95 respirators with effectiveness at 20% in reducing both susceptibility and infectivity there is a 19% reduction in the cumulative number of cases. With the same mask effectiveness but 25% of the population wearing N95 respirators, the total number of pandemic (H1N1) 2009 cases is reduced by almost 30% and with 50% of the population wearing masks, it results in over a 36% reduction in the number of cases.
The effectiveness of surgical masks is low, therefore the impact of wearing them during an epidemic is not significant. Even at 50% effectiveness in reducing both susceptibility and infectivity and with 50% of the population wearing surgical masks only a 6% reduction in the number of cumulative cases is seen.
The sooner an epidemic is recognized and masks are implemented, the bigger the reduction in the number of cases will be. As seen in the results section the epidemic is sensitive to the delay in implementing masks. The difference in the total number of pandemic (H1N1) 2009 cases when masks are implemented at 100 infected individuals and 1,000 infected individuals is over 7%.
The implementation of neither N95 respirators nor surgical masks lowered the effective reproduction number < unc below one. However, N95 respirators greatly decreased < unc , in some scenarios very close to one. While facemasks will not stop the pandemic (H1N1) 2009, they could greatly reduce its severity and allow for more time to develop effective vaccines and antivirals.
There are currently more trials being conducted on the effectiveness of surgical masks and N95 respirators [32] , which will allow us to refine the assumptions made in the model. However, it must be noted that in order for masks to be effective they must be: (1) available, (2) affordable, (3) worn properly, (4) replaced or sanitized daily, and (5) N95 respirators should be fit-tested. Only 10% of the population would have to wear masks in order to reduce the percentage of cases by 20%. Facemasks are inexpensive, relatively easy to implement, and would not cause a large economic burden to society. Masks are a powerful tool and can be used by countries with limited supplies of antiviral drugs and vaccines. In addition, economically feasible preventative global mitigations will benefit the world as a whole. We can conclude from our model that N95 respirators if worn properly are an effective intervention strategy in reducing the spread of the pandemic (H1N1) 2009. Figure 8 . Sensitivity to Who Wears Masks. In order to achieve the greatest possible reduction in the cumulative number of cases both infectious individuals and susceptible and exposed individuals should wear masks. If only infectious individuals wear masks the number of cases is not significantly reduced. doi:10.1371/journal.pone.0009018.g008 Correcting the Actual Reproduction Number: A Simple Method to Estimate R(0) from Early Epidemic Growth Data The basic reproduction number, R(0), a summary measure of the transmission potential of an infectious disease, is estimated from early epidemic growth rate, but a likelihood-based method for the estimation has yet to be developed. The present study corrects the concept of the actual reproduction number, offering a simple framework for estimating R(0) without assuming exponential growth of cases. The proposed method is applied to the HIV epidemic in European countries, yielding R(0) values ranging from 3.60 to 3.74, consistent with those based on the Euler-Lotka equation. The method also permits calculating the expected value of R(0) using a spreadsheet. The basic reproduction number, R 0 , of an infectious disease is the average number of secondary cases generated by a single primary case in a fully susceptible population [1] . R 0 is the most widely used epidemiological measurement of the transmission potential in a given population. Statistical estimation of R 0 has been performed for various infectious diseases [2, 3] , aiming towards understanding the dynamics of transmission and evolution, and designing effective public health OPEN ACCESS intervention strategies. In particular, R 0 has been used for determining the minimum coverage of immunization, because the threshold condition to prevent a major epidemic in a randomly-mixing population is given by 1-1/R 0 [4] . In addition, R 0 gives an estimate of the so-called final size, i.e., the proportion of the population that will experience infection by the end of an epidemic [5, 6] .
Methodological discussions concerning the statistical inference of R 0 are still in progress, and it is recognized that the estimate is very sensitive to dispersal (or underlying epidemiological assumptions) of the progression of a disease [7] . When one estimates R 0 using epidemic data of an emerging (or exotic) disease, the exponential growth rate, r, of infections during the initial phase of the epidemic is used [8, 9] . Assuming that the generation time, i.e., the time from infection of a primary case to infection of a secondary case generated by the primary case [10] , is known (or separately estimated from other empirical data), the growth rate r is transformed to R 0 using that knowledge (see below). That is, the conventional estimation technique has required two statistical steps, namely, first estimate r and then convert r to R 0 .
The estimation method can be illustrated by employing a simple renewal process which adheres to the original definition of R 0 [1] . Let j(t) be the number of new infections (i.e., incidence) at calendar time t. Supposing that each infected individual on average generates secondary cases at a rate A() at time  since infection (where  is referred to as the "infection-age" hereafter), j(t) is written as:
Since R 0 represents the total number of secondary cases that a primary case generates during the entire course of infection, the estimate is given by ( [11] ):
When j(t) follows an exponential growth path, it is easy to extract the integral of A() from equation (1) . Supposing that the incidence grows exponentially at a rate r, we have j(t) = kexp(rt)
where k is a constant, and moreover, j(t − ) = kexp(rt)exp(−r). This simplifies (1) to the so-called Euler-Lotka equation:
Since the density function of the generation time, g(), represents the frequency of secondary transmissions relative to infection-age , we can write g() as:
Replacing A() in the right-hand side of (3) by that of (4), an estimator of R 0 is obtained [9] :
The estimation of R 0 is achieved by measuring the exponential growth rate r from the incidence data and also by assuming that g() is known (or separately estimated from empirical observation such as contact tracing data [12, 13] ). It should be noted that the above mentioned framework has not been given a likelihood-based method for estimating R 0 (i.e., a likelihood function used for fitting a statistical model to data, and providing estimates for R 0 , has been missing). Moreover, equation (5) may not be easily used by non-experts, e.g., an epidemiologist who wishes to estimate R 0 using her/his own data.
The purpose of the present study is to offer an improved framework for estimating R 0 from early epidemic growth data, which may be slightly more tractable among non-experts as compared with the above mentioned estimator (5) . A likelihood-based approach is proposed to permit derivation of the uncertainty bounds of R 0 . For an exposition of the proposed method, the incidence data of the HIV epidemic in Europe is explored.
In addition to R 0 , a different measurement of the transmission potential using widely available epidemiological data, the actual reproduction number, R a , has been proposed for HIV/AIDS [14] . The concept of R a is much simpler than R 0 in that R a is defined as a product of the mean duration of infectiousness and the ratio of incidence to prevalence [15] . The prevalence p(t) at calendar time t is written as:
where () is the survivorship of infectiousness, or probability of being infectious, at infection-age .
Letting () be the transmission rate, which depends primarily on the frequency of contact and infectiousness at infection-age , the above mentioned A(), the rate of secondary transmission per single primary case at , under an assumption of a Kermack and McKendrick type model, is decomposed as ( [16] ):
The actual reproduction number R a is written as:
where D is the mean generation time (or what was previously described as the mean duration of infectiousness [14, 15] ). If the transmission rate () is constant  (i.e., independent of infection-age), R a = D. Moreover, from equation (2), R 0 is given by: Figure 1 . The relative frequency of secondary transmissions of HIV as a function of the time since infection. A step function is employed to approximately model the frequency of secondary transmissions relative to infection-age. For d 1 years shortly after infection, the frequency g 1 is very high. Subsequently, for d 2 years (i.e., during the asymptomatic period), g 2 is persistently low, followed by a time period with high infectiousness g 3 for d 3 years until death or no secondary transmission due to AIDS. Following a statistical study [20] , d 1 , R a coincides with R 0 as long as () is constant. Nevertheless, in many instances, the contact frequency and infectiousness (which may be partly reflected, for example, in the viral load distribution of the infected host) vary as a function of infection-age . The variation in () is particularly the case for HIV infection. Thus, although the usefulness of the incidence-to-prevalence ratio and R a has been emphasized to have an application in HIV/AIDS [15] , R a tends to yield a biased estimate (if R a is regarded as a proxy for R 0 ), and the estimate of R a is not as robust as that is obtained with equation (5) to objectively interpret the transmission potential [17, 18] .
Here, the above mentioned negative aspect of R a is reconsidered by correcting the definition of R a . The disease of interest in the present study is HIV. The frequency of secondary transmissions relative to infection-age  (i.e., the generation time distribution), approximated by a step function, is shown in Figure 1 . As has been known for some time [19] , the frequency of secondary transmissions is very high shortly after infection (for a duration d 1 = 0.24 years), followed by a long asymptomatic period with a low frequency of secondary transmissions (for d 2 = 8.38 years) [20] . Subsequently, the frequency rises sharply again resulting in a substantial number of secondary cases for a duration d 3 = 0.75 years until death or until the infected individual ceases risky sexual contact due to AIDS [20] [21] [22] . Assuming that the contact frequency does not vary as a function of infection-age, g 1 , g 2 and g 3 have been estimated at 1.30, 0.05 and 0.36 per year [20] . Here, g() is the density function of the generation time, with a mean of 3.79 years.
Here the concept of R a is corrected. The equation (8) is rewritten as:
The numerator represents the number of new infections at calendar time t, while the denominator was originally intended to represent "the total number of infectious individuals" who can potentially be primary cases with an equal chance at time t. Nevertheless, in order that the estimator of the actual reproduction number coincides with that of R 0 , the concept of the denominator is better replaced by "the total number of effective contacts (which can potentially lead to secondary transmissions with an equal probability)". Therefore, the corrected R a is better written as:
where g(), in the renewal equation with the Kermack and McKendrick type assumption, is written as the normalized density of secondary transmissions, i.e.,:
Replacing g() in the right-hand side of (11) by that of (12), we get:
Thus, the estimator of corrected R a in (11) is identical to that of R 0 . In other words, R 0 can be estimated from the incidence data and the generation time without assuming exponential growth of cases during the early phase of an epidemic. It should be noted that the ratio of prevalence to mean generation time p(t)/D in the denominator of the right-hand side of (10) has been replaced by "the total number of effective contacts that have equal potential to generate secondary cases".
Here the epidemic data of HIV/AIDS in three European countries: France, the Western part of Germany (i.e., the former Western Germany) and the United Kingdom (UK) are investigated [23] . Figure 2A shows the yearly incidence in these countries from 1976-2000. During the observation period, a total of 23,243, 13,126 and 11,491 AIDS cases were diagnosed in France, Western Germany and the UK, respectively. Although the time of HIV infection is not directly observable, the HIV incidence has been estimated by employing a back-calculation technique and using the AIDS incidence and the incubation period distribution of AIDS [23] . The present study does not discuss the details of back-calculation, but explanatory guides can be found elsewhere [24] [25] [26] . Figure 2B shows the enlarged HIV incidence curve during the initial phase of an epidemic. The peak incidence was observed in 1982 for Western Germany and 1983 for France and the UK. In the following, the time period from 1976 up to one year prior to the peak incidence is taken as the early growth phase. For the purpose of an exposition of the proposed method, the HIV incidence is assumed to have been in a single homogeneously-mixing population. 
R 0 is estimated using two different methods, one employing the estimator (5) and another using the corrected R a . For the former approach, the exponential growth rate is estimated via a pure birth process [27] . Given that the cumulative incidence from year 0 to t − 1 is observed, the conditional likelihood of observing the cumulative incidence J t cases in year t is proportional to ( [28] ):
from which the maximum likelihood estimate and the 95% confidence intervals (CI) of r (per year) are obtained. Given a fixed generation time distribution g(), the uncertainty bound of R 0 mirrors the uncertainty in the estimate of r. The translation of r into R 0 via (5) is made by using g() in Figure 1 .
The latter method, proposed in the present study, employs the estimator of corrected R a in (11) . Since the data are yearly, the equation (11) needs to be rewritten in discrete-time: (15) where the discrete density function of the generation time, g s is assumed to be given by g s = G(s + 1) − G(s), where G(s) is the cumulative distribution function of the generation time of length s, but g 0 is calculated as a normalized yearly average, because d 1 is as short as 0.24 years.
The likelihood of estimating R 0 with (15) is proposed as follows. First, the inverse of both sides of (15) is taken: The numerator of the right-hand side indicates the total number of effective contacts made by potential primary cases in year t that have an equal probability of resulting in a secondary transmission, and the denominator is the number of secondary cases in year t. Given that all the potential contacts made by primary cases (black circles) are known using the incidence data and the generation time distribution, the probability that each potential contact resulted in a secondary transmission is given by 1/R 0 . 1 The intrinsic growth rate during the exponential growth phase; 2 the basic reproduction number estimated using equation (5); 3 the basic reproduction number estimated using equation (17); the 95% confidence intervals are shown in parentheses.
The right-hand side of equation (16) is interpreted as follows. Figure 3A shows a transmission tree, i.e., a representation of who infected whom, where each primary case on average generates two secondary cases. A transmission tree of this kind is usually unobserved unless rigorous contact-tracing with microbiological examination is implemented. Thus, a likelihood-based approach to reconstructing the tree is considered ( Figure 3B ) [29] [30] [31] . Given the total number of effective contacts that have equal potential for resulting in secondary transmission, the probability of a single effective contact resulting in a secondary transmission (or the probability that a secondary case is linked to an effective contact made by a single primary case in year t) is given by 1/R 0 . This is a simple binomial sampling process. In other words, the likelihood function for estimating R 0 is: (17) where T is the most recent time point of observation within an early (linear) epidemic growth stage. The maximum likelihood estimate of R 0 is obtained by minimizing the negative logarithm of (17) , and the 95% CI are derived from the profile likelihood. Table 1 shows the estimates of r and R 0 for HIV in France, Western Germany and the UK. The maximum likelihood estimates of r ranged from 1.15 to 2.15 per year with the smallest estimate in France and the highest in Western Germany. The 95% CI for Western Germany did not overlap with those of the other two countries, reflecting the steep rise in incidence in this region in Figure 2B . Based on the exponential growth assumption in (5), the estimate of R 0 ranged from 3.65-4.08. Again, Western Germany yielded the highest estimate without an overlap of the uncertainty bound with the other two countries. The maximum likelihood estimate of R 0 based on the proposed new method ranged from 3.59 to 3.74. Not only were the qualitative patterns for the expected values of R 0 consistent with those based on an exponential growth assumption, but the 95% CI also broadly overlapped with those based on the other method. In particular, although R 0 in Western Germany using the proposed method is smaller than that based on an exponential growth assumption, the 95% CIs of the two methods overlapped. The 95% CI based on the proposed method appeared to be wider than those based on exponential growth assumption. Since HIV is mainly transmitted via sexual contact, the above mentioned estimate may vary with the mixing pattern and contact frequency (thus, there is no general disease-specific R 0 , especially for HIV/AIDS). At least, compared with a previous estimate of R 0 as ranging from 13.9 to 54.5 in the USA, based on an exponential growth assumption that adopted a mean infectious period of 10 years [32] , R 0 in the present study appeared to be much smaller using a precise estimate of the generation time distribution.
The present study proposed the use of the corrected actual reproduction number, R a , for statistical inference of R 0 based on incidence and known relative frequency of secondary transmissions (i.e., the generation time distribution) during the early growth phase of an epidemic. The previously available method using (5) forced us to adopt an exponential growth assumption, and moreover, an additional step towards the estimation of r (i.e., the translation of r to R 0 ) was required [9] . The proposed method does not necessarily require an exponential growth assumption and provides a "short-cut" to estimate R 0 from incidence data. The simple likelihood function employing a binomial distribution was also proposed to yield an appropriate uncertainty bound of R 0 . It should be noted that given the knowledge of g s and readily available incidence data, equation (15) permits calculation of the expected value of R 0 without likelihood. Such a calculation can be attained using any spreadsheet.
The usefulness of the actual reproduction number, calculated as a product of the mean generation time and the incidence-to-prevalence ratio, has been previously emphasized in assessing the epidemiological time course of an epidemic [14, 15] . However, it appears that R a does not precisely capture the secondary transmission if the transmission rate () varies with infection-age  [18] , and moreover, the cohort-and period-reproduction numbers directly derived from the renewal equation have been suggested to be more accurate figures in capturing the underlying transmission dynamics [17, 33] . In the present study, replacing the denominator (i.e., prevalence) of R a by the total number of potential contacts, it was shown that the R 0 derived from the renewal equation coincides with the corrected actual reproduction number, R a , and also that the likelihood can be quite easily derived. The corrected R a does not require prevalence data, and uses only the incidence data and the generation time distribution.
Many future tasks remain, however. Most importantly, the estimation of R 0 from early epidemic growth data for a heterogeneously-mixing population is called for. R 0 in the present study can even be interpreted as R 0 for a heterogeneously-mixing population (i.e., the dominant eigenvalue of the nextgeneration matrix), provided that the early growth rate is the same among sub-populations (though it is not the case if the growth rate varies across sub-populations) [34, 35] . Analyzing heterogeneous transmission among approximately-aggregated discrete groups, the estimate of the next-generation matrix would give more detailed insights into the epidemic dynamics, including the most important target host for intervention [36] . As discussed in a modeling study in this special issue of the International Journal of Environmental Research and Public Health [37] , understanding the implications of sexual partnerships and their variations as a function of calendar time as well as infection-age is also of utmost importance. As the next step for a similar estimation framework, methods for estimating robust R 0 and the next-generation matrix from structured data (i.e., data stratified by age-and/or risk-groups) will be useful. Despite the future challenges, I believe the present study at least satisfies a need to offer a likelihood-based approach to estimate R 0 from early epidemic growth data, while being easily tractable and calculable among general epidemiologists. Using Satellite Images of Environmental Changes to Predict Infectious Disease Outbreaks Recent events clearly illustrate a continued vulnerability of large populations to infectious diseases, which is related to our changing human-constructed and natural environments. A single person with multidrug-resistant tuberculosis in 2007 provided a wake-up call to the United States and global public health infrastructure, as the health professionals and the public realized that today’s ease of airline travel can potentially expose hundreds of persons to an untreatable disease associated with an infectious agent. Ease of travel, population increase, population displacement, pollution, agricultural activity, changing socioeconomic structures, and international conflicts worldwide have each contributed to infectious disease events. Today, however, nothing is larger in scale, has more potential for long-term effects, and is more uncertain than the effects of climate change on infectious disease outbreaks, epidemics, and pandemics. We discuss advances in our ability to predict these events and, in particular, the critical role that satellite imaging could play in mounting an effective response. A tmospheric chemists and climate modelers have little doubt that the earth's climate is changing. Concomitant with rising carbon dioxide levels and temperatures, severe weather events are increasing, which can lead to sub-stantial rises in sea level, flooding, increased droughts, and forest fires (1) . In recent decades, infectious diseases have resurged, and previously unrecognized agents of disease have been characterized (2) . Evidence is accruing that these phenomena may in part be linked to environmental change (3) . Several questions have emerged from events that have occurred over the past 20 years: was cryptosporidiosis inevitable in Milwaukee, Wisconsin, USA, in 1993, and was Escherichia coli O157 infection inevitable in Walkerton, Ontario, Canada, in 2000? Both events were preceded by heavy rains; had highly concentrated sources of pathogens in the form of untreated sewage and animal waste, respectively; and had vulnerable infrastructure. Although the situations were perhaps more complex, could we have predicted epidemic cholera in South America in 1991 after a 100-year absence and the emergence of a new strain of potentially pandemic cholera in India in 1992?
A considerable body of knowledge has accumulated over the past decade or so about the relationships between environment and disease, yet far more information and resources are needed if we are to develop effective early warning systems through environmental surveillance and modeling as well as appropriate emergency response. In the United States, we face a crisis in funding that not only affects basic and applied research in this field but also undermines our ability to deploy remote sensing technologies that provide the most promising means for monitoring our environment. Using examples of waterborne and vectorborne disease, we will discuss how remote sensing technology can be used for disease prediction. We will then examine the lessons learned from these examples and provide recommendations for future modeling. 
Water and climate go hand in hand, with precipitation and extreme events known to be associated with waterborne outbreaks (4) . Flooding is the most frequent natural weather disaster (30%-46% of natural disasters in [2004] [2005] , affecting >70 million persons worldwide each year (data for 2005 [5] ).
The most common illnesses associated with floods described in the literature are diarrhea, cholera, typhoid, hepatitis (jaundice), and leptospirosis. Unusual illnesses such as tetanus have also been reported. The etiologic agents identified include Cryptosporidium spp., hepatitis A virus, hepatitis E virus, Leptospira spp., Salmonella spp., and Vibrio spp. Severe outbreaks of cholera, in particular, have been directly associated with flooding in Africa and in West Bengal, India (6,7).
A rise in sea level, combined with increasingly severe weather events, is likely to make flooding events commonplace worldwide. The Climate Change 2001 Synthesis Report from the Intergovernmental Panel on Climate Change (8) suggests that the average annual numbers of persons affected by coastal storm surges will increase from <50 million at present sea levels to ≈250 million by the 2080s, assuming a 40-cm rise in sea level. Even with enhanced protection through engineering interventions, this number is anticipated to reach ≈100 million persons. The initial proportion of deaths from these events is huge, but without extreme vigilance and better monitoring and response, major epidemic waterborne diseases will continue to occur. Factors that promote waterborne diseaseovercrowding, lack of sanitation, lack of clean water, certain domestic animal practices, waste disposal-are exacerbated by flooding.
Effective prediction depends on many factors, not just the prediction of an event. Cholera may be the most studied and best understood of the waterborne diseases and, perhaps in hindsight, we could have predicted the occurrence of cholera in South America in 1991 (9) . Models for cholera prediction, although country specific, are constantly improving. For example, considerable work has gone into predicting outbreaks of cholera in Bangladesh. Remote imaging technologies developed by the US National Aeronautics and Space Administration have been used to relate sea surface temperature, sea surface height, and chlorophyll A levels to cholera outbreaks ( Figure 1 ) (R.R. Colwell and J. Calkins, unpub. data). This process used a composite environmental model that demonstrated a remarkable similarity between predicted rates based on these 3 parameters and actual cholera incidence. These data are far from perfect and considerable uncertainty still remains. For example, rates of cholera were much higher than predicted in January 1998 and January 1999, yet many of the predicted peaks closely aligned with actual incidence. Because the model is constantly being improved and the satellite data are becoming increasingly accurate through ground truthing (real-time collection of information on location), we believe that satellite imaging provides tremendous promise for prediction of cholera, weeks and even months in advance of an epidemic.
Knowing when an outbreak is likely to occur can inform public health workers to stress basic hygiene and sanitation and to implement simple mitigation efforts such as filtration of water with sari cloth, which in some areas is credited with reducing deaths from cholera by >50% (10) . Although remote sensing technology is currently still a research tool, the example of cholera prediction through its use provides a compelling argument to maintain and adequately fund our satellite programs; unless this is done, this extraordinary effort at disease prediction will fail.
Some of the critical needs that must be met to predict the effect of environmental change on waterborne disease include the following: 1) better knowledge of disease incidence and pathogen excretion; 2) better characterization of the pathogens in sources (e.g., combined sewer overflows, septic tanks) and these sources' vulnerabilities to climate change; 3) better monitoring of sewage indicators to gather source, transport, and exposure information (event monitoring); 4) improved understanding of sediments and other pathogen reservoirs; 5) more quantitative data for risk assessment; and 6) better health surveillance data. In turn, this information can be used to better use ground truthing in combination with remote sensing technologies as predictors of waterborne disease outbreaks.
Other emerging and reemerging infectious diseases also are environmentally driven. Many are zoonotic, vector- borne, or both, and have complex life histories that make predicting disease emergence or reemergence particularly difficult. An insect or rodent vector can make it almost inevitable that a pathogen will be globally transported by plane or boat. With environmental change, disease range, prevalence, and seasonality may change in direct relationship to the vector or animal host. Therefore, to understand the life cycle of a pathogen and the risks of disease emergence, all stages of that life cycle and the life cycles of its intermediate hosts must be considered.
To date, predicting vector-borne diseases has proved to be complex. Although climate change and other environmental stressors are major components, separation from human factors is difficult. Climate change undoubtedly affects the distribution of disease, but changes in human behavior that increase exposure risk are also critical factors. Šumilo et al. (11) reported that climatic variables explain only 55% of spatial variation in tick-borne encephalitis in the Baltic States, which have seen an increase in disease incidence over the past 3 decades. These authors report that changes in predation pressure on intermediate hosts and shifting socioeconomic conditions that increase or decrease peoples' visits to forests (for recreation, work, or berry and mushroom harvesting) are important factors in disease distribution (12) .
Effective modeling of future risk for vector-borne disease outbreaks needs to take into account human behavior that increases exposure, as well as other factors that effect the ecology of the vectors, such as predation pressure and habitat change. Coupled with remote sensing technologies that monitor environmental and climatic changes, human observations of population movement and distribution will be necessary.
Malaria also presents a challenge. This disease continues to devastate sub-Saharan Africa and other parts of the developing world. Substantial resources over the past several decades have gone toward eradication, vaccination, treatment, and, more recently, prediction of malaria outbreaks. Satellite imaging has been used to predict the distribution of 5 of the 6 Anopheles gambiae complex species that are responsible for much of the malaria transmission in Africa (13) . However, human factors again make accurate prediction of disease events complex. Prediction of a disease event is complicated by host immunity effects, which can result in cycles of infection that would appear to bear no relationship to environmental variables. To predict malaria outbreaks, remote sensing technologies need to be coupled with a better understanding of how specific populations are effected by host immunity, which could allow population susceptibility at any given time to be estimated.
Although considerable uncertainty exists in disease prediction through remote sensing technology, particularly for vector-borne disease as discussed above, satellite technology has been applied with some success to predictive modeling for cases of hantavirus pulmonary syndrome (HPS). The 1993 outbreak of HPS in the southwestern United States was believed to be linked to environmental conditions and, in particular, to abnormally high rainfall that resulted in increased vegetation with a subsequent explosion in the rodent populations. Several research groups have subsequently modeled conditions that led to an HPS outbreak, with mixed success. Engelthaler et al. (14) looked at 10 years of data on monthly precipitation and daily ambient temperature in the Southwest region (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) in relation to HPS cases (1993) (1994) (1995) . They found that cases tended to cluster seasonally and temporally by biome type and elevation and only indirectly demonstrated a possible association between the 1992/1993 El Niño precipitation events and HPS. Glass et al. (15, 16) were also unable to make a definitive link with precipitation events in their analyses of HPS in the southwestern United States. They did, however, find a relationship between Landsat Thermatic Mapper (LTM) images recorded by satellite in 1992 and HPS risk the following year. LTM generates numbers that represent reflected light in 6 bands, 2 of which were associated with decreased risk and 1, in the mid-infrared range, with increased risk. The authors admit that considerable ground truthing is necessary to relate satellite imagery to the environmental variables being measured (i.e., vegetation, soil type, soil moisture) and their relation to rodent population dynamics.
However, this work does demonstrate the utility of remote satellite imaging and the increasingly important role it can and should play in disease prediction. In 2006, Glass et al. (17) reported strong predictive strength from logistic regression modeling of LTM imagery from 1 year, when estimating risk of HPS the following year, for the years 1992-2005. Their risk analysis for 2006, based on Landsat imagery for 2005, when precipitation levels increased dramatically over prior drought years, suggested an increased risk for HPS, particularly in northern New Mexico and southern Colorado. This prediction was unfortunately borne out in the early part of 2006 when 9 cases of HPS occurred within the first 3 months, 6 of those cases in New Mexico and Arizona. However, the anticipated threat to Colorado did not occur, with a fairly typical number of 6 cases, compared with a total of 11 cases for the state in 2005 (18) .
However, these results are not necessarily a failure of prediction. In fact, they may illustrate that an early warning system serves to reduce exposure of persons to the deer mice habitat. For example, USA Today highlighted HPS risks with a June 8, 2006, article titled "Officials warn of increased threat of hantavirus" (www.usatoday.com/news/ health/2006-06-08-hantavirus-x.htm). The role of the popular press is hard to quantify but undoubtedly does have an effect on human behavior patterns. Many health departments in the western states produce health advisories warning the public about the risks of exposure to the virus through inhalation of dust contaminated with rodent urine, feces, or saliva. The popular press may serve an important role in increasing awareness of a heightened health risk, which, in turn, promotes greater compliance with health advisories.
The scientific community has a relative consensus that epidemic and pandemic disease risks will be exacerbated by environmental changes that destabilize weather patterns, change distribution of vectors, and increase transport and transmission risk. Predictive modeling may lead to improved understanding and potentially prevent future epidemic and pandemic disease. Many respiratory infections are well known as highly climate dependent or seasonal. Although we are not yet able to predict their incidence with great precision, we may well be able to do this in the future. Meningococcal meningitis (caused by Neisseria meningitidis) in Africa is probably the best known example. In the disease-endemic so-called meningitis belt (an area running across sub-Saharan Africa from Senegal to Ethiopia), this is classically a dry season disease, which ceases with the beginning of the rainy season, likely as a result of changes in host susceptibility (19) . Many other infectious diseases show strong seasonality or association with climatic conditions (20) . Perhaps one of the most interesting is influenza, which is thought of as a wintertime disease in temperate climates but shows both winter and summer peaks in subtropical and tropical regions (21) . Although the reasons for seasonality are often poorly understood, the close dependence of such diseases on climatic conditions suggests that these, too, are likely to be amenable to prediction by modeling and remote sensing (22) .
When we consider influenza, it is hard not to think about the future risks from pandemic influenza. Public health agencies in the United States and around the world are focusing on influenza preparedness, notably concerning influenza virus A subtype H5N1, which has captured attention because it causes severe disease and death in humans but as yet has demonstrated only very limited and inefficient human-to-human transmission. The severity of the disease raises images of the 1918 influenza epidemic on an unimaginably vast scale if the virus were to adapt to more efficient human-to-human transmission. Can predictive modeling using satellite or other imaging of environmental variables help in prediction of future influenza pandemics? Xiangming Xiao at the University of New Hampshire was funded in 2006 by the National Institutes for Health to lead a multidisciplinary and multi-institutional team to use remote satellite imaging to track avian flu. Xiao et al. have used satellite image-derived vegetation indices to map paddy rice agriculture in southern Asia (23) . They believe that a similar approach can be used in conjunction with the more traditional approach of analyzing bird migration patterns and poultry production (24, 25) to map potential hot spots of virus transmission (26) .
An interesting question is why did we not see disease epidemics in Indonesia, following the devastating tsunami disaster of December 2004? Could rapid public health intervention be credited with minimizing spread of disease? In the case of Aceh Province, many communities reported diarrhea as the main cause of illness (in 85% of children <5 years of age), but no increases in deaths were reported, and no outbreaks of cholera or other potentially epidemic diseases occurred (27) . Given the massive scale of the disaster, was this likely? In some towns, more than two thirds of the population died at the time of impact, almost 100% of homes were destroyed, and 100% of the population lacked access to clean water and sanitation (27) . To a large extent, the Australian army and other groups are to be credited with rapidly deploying environmental health teams to swiftly implement public health measures, including provision of safe drinking water, proper sanitary facilities, and mosquito control measures (28) . Widespread fecal pollution of the surface waters was shown, yet the saltiness of the potable water supply after the disaster made much of the water unpalatable. Wells were vulnerable, perhaps to other etiologic agents of fecal origin including viruses and Shigella spp., with greater probability of infection than Vibrio spp., thus leading to the widespread diarrhea.
The most important lesson from the Asian tsunami is that disease epidemics can be prevented by public health intervention. Unfortunately, most flooding events, and other conditions that promote infectious disease epidemics, do not receive the same global media attention. A tsunami captures the imagination of the world in a way that weeks of rainfall in the Sudan or a rise in sea surface temperature cannot. However, if climatologic data can be used to predict future disease outbreaks, public health interventions can be mobilized in a more timely and proactive manner.
A continuing concern is the conditions that result in newly emergent virulent strains of pathogens. Faruque et al. have provided molecular evidence that V. cholerae O139 strains are derived from O1 strains through genetic modification (29) . In addition, Chakraborty et al. in Kolkata have seen the presence and expression of virulence genes in several environmental strains of V. cholerae cul-tured from surface waters (30) . Recently, E. coli O157 has been isolated from the Ganges River in India for the first time (31) . Indications are that it is metabolically different from E. coli O157 isolated from other parts of the world, but the conditions that have led to these differences are as yet unclear. From the above studies, risk for transmission of virulence genes is likely to be high, but studies of conditions promoting transmission and approaches to modeling resultant disease risks are in their infancy. New epidemic strains could potentially occur through mutation of existing epidemic strains or through gene transfer. Environmental stressors such as chemical contaminants are thought to accelerate both mutation rates and gene transfer (32) . Thus, the degree of chemical pollution may need to be a component of disease models (in addition to other stressors).
The scientific community is a long way from incorporating environment-gene interactions into predictive models and clarifying the risks posed to human society from emerging diseases. However, investigation of these parts of the pathogen's ecology should remain on the national research agenda as we move forward with developing predictive models of disease outbreaks.
Current modeling of infectious diseases is by necessity retrospective. Environmental parameters measured by remote satellite imaging show the greatest promise for providing global coverage of changing environmental conditions. With current imaging technologies, we can measure sea surface temperature, sea surface height, chlorophyll A levels, and a variety of vegetation and soil indices, in addition to many other physical, biologic, and chemical parameters of the earth's surface and atmosphere. A variety of these parameters can be incorporated in complex mathematical models, together with biotic and ecologic variables of the pathogen and host life cycles, to correlate environment with outbreaks of disease ( Figure 2 ). However, we are still far from being able to accurately predict future disease events on the basis of existing environmental conditions. Successful predictive modeling of disease and the establishment of early warning systems have reached a critical junction in development. As we improve our understanding of the biology and ecology of the pathogen, vectors, and hosts, our ability to accurately link environmental variables, particularly those related to climate change, will improve. What has become clear over the past few years is that satellite imaging can play a critical role in disease prediction and, therefore, inform our response to future outbreaks.
We conclude that infectious disease events may be closely linked to environmental and global change. Satellite imaging may be critical for effective disease prediction and thus future mitigation of epidemic and pandemic diseases. We cannot stress too strongly our belief that a strong global satellite program is essential for future disease prediction. Ground truthing (real-time measurements of soil, water, and other environmental parameters)
and ecology Pathogen biology and ecology (e.g., biofilm mode of growth, association with plankton, plasmid biology)
Human host biology and ecology (e.g., population susceptibility/immunity, exposure patterns) Figure 2 . Components of a predictive model of infectious disease based on satellite imaging to assess environmental change. SST, sea surface temperature; SSH, sea surface height. Clinical Assessment and Improved Diagnosis of Bocavirus-induced Wheezing in Children, Finland Human bocavirus (HBoV) is a widespread respiratory virus. To improve diagnostic methods, we conducted immunoglobulin (Ig) G and IgM enzyme immunoassays with recombinant virus–like particles of HBoV as antigen. Acute-phase and follow-up serum samples from 258 wheezing children and single serum samples from 115 healthy adults in Finland were examined. Our assays had a sensitivity of 97% and a specificity of 99.5%. Of adults, 96% had immunity; none had an acute infection. Of 48 children with serologically diagnosed acute HBoV infections, 45 were viremic and 35 had virus in nasopharyngeal aspirates (NPAs). Of 39 HBoV NPA PCR–positive children co-infected with another virus, 64% had a serologically verified HBoV infection. HBoV caused illness of longer duration than rhinovirus and of equal severity to that of respiratory syncytial virus. Among children with bronchiolitis, >25% had acute HBoV infections. Accurate HBoV diagnosis requires serologic analysis or PCR of serum; PCR of NPAs alone is insufficient. covered during sequencing of respiratory tract samples from children. It has been detected worldwide in the nasopharyngeal tract, mainly in small children with lower respiratory tract infections (1, 2) . HBoV has been associated with upper and lower respiratory tract infections and shown to be a cause of pneumonia in children (3) (4) (5) (6) (7) (8) . Prolonged shedding of virus has been reported; >26% of children shed virus for 2 months, 4% for 3 months, and 2% for 4 months (9) . Diagnosis of HBoV respiratory tract infections has been PCR based, leading to overrepresentation of HBoV co-infections with other respiratory viruses (9) (10) (11) .
Along with others, we have shown that respiratory infections with HBoV elicit B-cell immune responses (11) (12) (13) (14) (15) and can be diagnosed serologically by using prokaryotic virus protein 2 (VP2) antigens in immunoblots (11) . We report production in insect cells of VP2 of virus-like particles (VLPs) and their use in enzyme immunoassays (EIAs) for detection of HBoV-specific immunoglobulin (Ig) M and IgG in paired serum samples of pediatric patients with acute wheezing and in single serum samples of young healthy adults. Serologic results were compared with those of HBoV quantitative PCR (qPCR) of nasopharyngeal aspirates (NPAs) and paired serum samples of 258 children with complete sample sets. Clinical signs and symptoms of wheezing children with serologically verified acute HBoV infections with or without other respiratory virus infections (15 other viruses studied [10] ) were compared with those of children infected with respiratory syncytial virus (RSV) or rhinovirus.
Acute-phase (at the time of admission) and convalescent-phase (2 weeks later) serum samples and NPA samples at the time of admission were obtained from 259 children (age range 3 months to 15 years, median 1.6 years) with acute expiratory wheezing (10, 16) . These children were tested by NPA PCR for 16 respiratory viruses (10) ; 117 of these children were tested by HBoV IgM and IgG immunoblots and HBoV serum qPCR (10, 11) . All remaining serum samples, except 1 convalescent-phase serum sample that Clinical Assessment and Improved Diagnosis of Bocavirus-induced Wheezing in Children, Finland was depleted, were tested by HBoV qPCR specific for the nucleoprotein 1 gene as described (11) ; all serum samples were tested by EIA. For 93 of these 258 children, follow-up serum samples were obtained 5-8 years later. In addition, 115 serum samples from healthy medical students were collected after informed consent was obtained. The study was reviewed and approved by the Ethics Committees of Turku and Helsinki University hospitals.
The putative major virus capsid protein VP2 gene (nt 3443-5071) of the HBoV St 2 isolate (GenBank accession no. DQ000496) was cloned into a baculovirus vector pAcSG2 (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) by standard procedures and confirmed by sequencing. The VP2-containing vector was transfected into Sf9 insect cells by using FuGENE 6 Transfection reagent (Roche, Basel, Switzerland). Two million adherent cells in T25 bottles were transfected in 1 mL of Insect Express media (Lonza, Basel, Switzerland) with a mixture of 2 µg plasmid, 250 ng linearized baculoGold DNA (Becton Dickinson Biosciences), and 15 µL FuGENE reagent. Fresh cells were infected 3 times every third day by using virus medium collected from the previous infection. VP2containing Sf9 cells were harvested on day 3, and cell pellets were resuspended in phosphate-buffered saline (PBS), pH 7.5, at a concentration of 2.1 × 10 7 cells/mL. Protease inhibitor (complete EDTA-free; Roche) was added (≈75 µL/mL), and cells were lysed by sonication (4 × 20 s). After subsequent centrifugation at 13,200 rpm for 3 min, VLPs were purified by 48-h CsCl gradient ultracentrifugation at 24,200 rpm (L-70 Ultracentrifuge; Beckman, Fullerton, CA, USA) at 4°C after fraction collection and dialysis against PBS. The product was concentrated in columns (Amicon Ultra-15 50,000 MWCO; Millipore, Billerica, MA, USA). Expressed HBoV VP2 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of ≈60 kDa and by electron microscopy to be spherical symmetric parvovirus-like particles ≈20 nm in diameter ( Figure 1 ). Before use as antigen, VLPs were biotinylated as described (17) .
The HBoV IgG EIA was conducted as described for parvovirus B19 (18) except that biotinylated VLPs were used at concentrations of 60 ng/well. Cutoff values were calculated from IgG EIA absorbances of the first serum sample obtained at admission of 34 children who had no IgM or maternal (waning) IgG against HBoV and showed a >4-fold increase of IgG against HBoV during follow-up testing. Cutoff absorbances for negative and positive IgG EIA results were 0.154 (mean + 3 SD) and 0.188 (mean + 4 SD), respectively.
For IgM EIA, a µ-capture format was used (19) . Serum samples diluted 1:200 in PBS and 0.05% Tween (PBST) were applied in duplicate into wells of plates coated with goat anti-human IgM (Cappel/ICN Biomedicals, Costa Mesa, CA, USA) for 60 min at room temperature. After being rinsed 5 times with PBST, biotinylated HBoV VLPs were applied at a concentration of 25 ng/well and incubated for 45 min at 37°C. Bound antigen was visualized by using horseradish peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) at 1:12,000 in PBST plus 0.5% bovine serum albumin for 45 min at 37°C, followed by ophenylenediamine dihydrochloride (Dako) and H 2 O 2 for 15 min at 37°C. Cutoff values were calculated from IgM EIA absorbances of 5-year follow-up samples of 61 children who were IgG positive 5 years earlier. Cutoff absorbances for negative and positive IgM EIA results were 0.136 (mean + 3 SD) and 0.167 (mean + 4 SD), respectively. Parvovirus B19 serologic analysis was conducted by using commercial (Biotrin, Dublin, Ireland) and in-house EIAs (18, 20) .
Because most continuous data were skewed (by Kolmogorov-Smirnov test), they were analyzed by using regression analysis and generalized linear models after logarithmic transformation. Logistic regression analysis was used for categoric data. Statistical analyses were conducted by using SAS/STAT(r) software version 9.1.3 SP4 (SAS Institute Inc., Cary, NC, USA).
Complete sets of HBoV qPCR results for NPAs and serum samples were available for 258 children ( dren who showed viremia in the first sample (62%) or both samples (87%) had a high (>10 4 copies/mL) HBoV-DNA load in NPAs; most children who showed viremia only in the second sample (58%) had DNA-negative NPAs. These findings suggest a very recent infection. HBoV DNA loads in serum samples ranged from 112 copies/mL to 600,000 copies/mL and did not correlate with NPA DNA loads. Forty-nine (19%) of 258 children had NPAs that were PCR positive, but only 34 (13%) had both NPAs and serum samples that were PCR positive; 194 (75%) children had negative PCR results for both sample types (Table 1) . Of long-term follow-up serum samples, only 1/93 was PCR positive; this sample was from a child who was seronegative 5 years earlier and seroconverted during follow-up. All 115 adult serum samples were HBoV PCR negative.
Of 258 wheezing children, 111 (43%) had serologic evidence of past infection; 48 (19%) of acute primary HBoV infection (Table 1; Figure 2 ). Of the latter group, 32 had detectable IgM with either IgG conversion (27/48) or a diagnostic increase (5/48); a total of 15 had IgM with no IgG (5/48) or with a constant IgG (10/48) absorbance value. A girl 1 year of age showed seroconversion in a convalescent-phase serum sample weakly positive for IgG but she had no IgM in either sample (Table 1 ). All other children who showed seroconversion or an increase in IgG were IgM positive. Incidence rates of serologically verified, acute, primary HBoV infection among wheezing children varied with age, with a peak of 28% in children 1-<2 years of age (Table 2) . Prevalence of HBoV immunity increased with age, reaching 100% at 7 years of age (Table 2 ). This study included 27 (10%) of 258 children <6 months of age (median 4.8 months, mean 4.7 months). Eight (30%) of 27 children were IgG positive; IgG in 7 of these children was presumably of maternal origin. One infant (3.7%), a 3.6-month-old boy, had a serologically verified acute HBoV infection. (10) was not included in this comparison because of depletion of the convalescent-phase serum sample before the PCR (NPA and acute-phase serum samples were PCR negative; both serum samples were IgG positive and IgM negative, indicating immunity). Comparison of qPCR and EIA results for 258 children is shown in Table 1 . Among 49 children who showed viremia, 45 (92%) had a serodiagnosis of HBoV infection: i.e., had IgM or an increase in IgG. Of the remaining 4 viremic children, 1 was seropositive and 3 were seronegative; 2 of the seronegative children showed viremia in a second sample, possibly indicating a very acute infection (their NPA samples at the time of admission had been PCR negative). Among 209 nonviremic children, 206 (99%) showed nondiagnostic serologic results (Table 1) . Only 3 (1.4%) of 209 children had a serodiagnosis: the 1-year-old girl with an apparent false seroconversion described above and 2 children with PCR-positive NPAs and a positive result for IgM but a constant IgG level, which suggests a subacute infection.
Of 49 (19%) of 258 HBoV NPA PCR-positive children, 35 (71%) had a serodiagnosis, 33 of whom were also viremic ( Table 1 ). Of 28 patients with a high load of HBoV DNA in NPAs, 27 (96%) had an HBoV serodiagnosis, compared with only 8 (38%) of 21 with a low DNA load. Conversely, among 209 children without HBoV DNA in NPAs, 13 (6%) had a serodiagnosis, of whom 12 were also viremic.
Among 34 (13%) of 258 children who were HBoV PCR positive by both NPAs and serum samples, 33 (97%) had a serodiagnosis; the remaining child (a girl 6 months of age) was seronegative and viremic only in the second sample, which indicated a very acute infection. In contrast, of 194 (75%) of 258 children who were HBoV PCR negative in both NPAs and serum, only 1 (0.5%) had a serodiagnosis (the IgM-negative 1-year-old girl). Conversely, of 48 (19%) of 258 children who had a serologically diagnosed acute HBoV infection, 35 (73%) were PCR positive for NPAs, 45 (94%) were viremic, and 47 (98%) were PCR positive for NPAs or serum (Table 1) . If one considers a positive PCR result for serum as the standard for diagnosis (n = 258), our EIA had a sensitivity of 92%, a specificity of 99%, and a positive predictive value of 94%. If PCR positivity for NPAs and serum is the standard for diagnosis (n = 128), the sensitivity is 97%, the specificity is 99.5%, and the positive predictive value is 97%.
All 258 children had been tested for 16 respiratory viruses (10) Serodiagnostic findings for infection with parvovirus B19 (IgM positive or low epitope-type specificity index) (18, 20) were not observed among children with a serodiagnosis of HBoV infection. Among other children, 3 were IgM positive for parvovirus B19, of whom 1 was seronegative for HBoV and 2 were seropositive for HBoV.
The 258 children tested for 16 viruses were analyzed for clinical characteristics. Median age of 46 children with acute HBoV infection diagnosed by serologic analysis and PCR of serum was 1.3 years (range 0.3-6.1 years), median age of 91 of 258 nonexposed seronegative children was 1.1 years (range 0.2-4.2 years), and median age of 110 of 258 children with HBoV immunity was 2.8 years (range 0.5-5.2 years) (p<0.0001).
Clinical data were compared among children infected only with HBoV (n = 12), rhinovirus (n = 56), RSV (n = 36), and HBoV and any other virus (n = 34) (Table  3) Table 1 and text). †Five infants had maternal antibodies (defined as waning immunoglobulin G absorbance values in paired serum samples). Two other infants became seronegative and were considered virus negative.
= 0.0012), and longer duration of cough before admission (p<0.0001) for patients with acute HBoV infections than for patients with rhinovirus infections. However, children infected with rhinovirus had a higher leukocyte count at the time of admission (p = 0.0002). When compared with patients infected with RSV, patients infected with HBoV showed longer duration of cough before admission (p = 0.019). Differences in clinical variables were not observed for rhinovirus or RSV, whether in children with single infections or those co-infected with HBoV. We found no differences between children co-infected with HBoV and 1 or 2 other viruses. Nonrespiratory symptoms, including diarrhea, were rare (Table 3) .
Acute HBoV infection was found in children with bronchiolitis. Among children <2 years of age, acute HBoV infection was detected in 26 (27%) of 95 children having their first wheezing episode and in 35 (25%) of 141 children having their first or recurrent wheezing episode, excluding asthmatic children (defined as children considered for initiation of daily long-term control therapy according to the recent US guidelines for diagnosis and management of asthma [21] ). Children with HBoV and RSV single infections showed a similar overall severity of illness (median 7, range 4-10 on a scale of 0-12), whereas acute otitis media (AOM) was more frequent among children with RSV single infections (p = 0.0005) ( ‡A child with 1 PCR-positive nasopharygeal aspirate and a PCR-negative serum sample was classified as having a subacute HBoV infection. In Table 1 , he was 1 of 3 nonviremic children with a serodiagnosis for HBoV infection. §Unadjusted. ¶Adjusted for age.
HBoV infections have been commonly diagnosed by PCR of respiratory tract samples. Only a few serologic studies have been reported; these studies have addressed mainly epidemiologic issues (11) (12) (13) (14) (15) . However, Endo et al. documented seroconversions by immunofluorescent analysis in 4 HBoV PCR-positive patients (12) , and Lindner et al. detected IgM against HBoV by EIA in 12 patients, 10 of whom were viremic children (15) . In a study of 117 wheezing children, we showed by using immunoblotting and prokaryotically expressed HBoV VP2 capsid antigens that HBoV infections can be diagnosed serologically (11) . We also showed that the unique region in VP1 is far less immunogenic than the major virus capsid protein VP2. We have now expressed HBoV VP2 VLPs in insect cells for use in IgM and IgG EIAs that are superior to immunoblots in diagnostic performance. We diagnosed acute primary HBoV infections in 48 (19%) of 258 children with expiratory wheezing. Consistent with other reports, no crossreactivity between 2 human pathogenic parvoviruses (B19 and HBoV), was detected (11) (12) (13) 15) . Prevalence rates of immunity increased with age from 5% in infants to >64% in children 2-4 years of age and continued to increase until a maximum of 100% was reached in children 7 years of age. Seroprevalence among young adults was 96%. Furthermore, IgG levels of adults were as diverse as those of children ( Figure 2 ). These results contrast sharply with our previous immunoblot results with denatured VP2 antigen (11) , which showed decreased seropositivity among children >2 years of age. This difference is likely caused by a time-related conformational dependence of the antibody, similar to immunity to B19 virus (18, 20, 22, 23) . In other HBoV seroprevalence studies, similar rates were reported (12, 13, 15) , which validate the accuracy of our results.
HBoV infection has been shown (by PCR of NPA samples) to be most prevalent in children 6 months to 3 years of age; adults are less affected (8, (24) (25) (26) (27) (28) (29) (30) . Consistent with this finding, the incidence of serologically verified acute HBoV infections in our study was highest (28%) during the second year of life; median age of children with acute HBoV infections was 1.3 years. Only 2 children were infected at >4 years of age; 1 of them was seronegative. Children <6 months of age might be protected from infection by maternal antibodies. Rapidly decreasing seroprevalence rates from ≈90% in infants <3 months of age to ≈5% in infants 6 months of age were reported (12, 13) . In our study, 26% of 27 children <6 months of age (including 1 infant <3 months of age) had maternal antibodies, and only 1 child in this age group had an acute HBoV infection.
When we compared serologic results with those of PCR, we found a profound difference between NPA PCR results and serum PCR results. Although results of serodiagnosis were identical to results of serum analysis by PCR, only 71% of NPA-positive children and 6% of NPA-negative children had an HBoV serodiagnosis. However, all but 1 (96%) of the children with a high load of HBoV DNA in NPAs had a serodiagnosis, compared with only 38% of those with a low DNA load. This finding supports the view that a low HBoV DNA load is not evidence of acute primary infection (10, 11, 31, 32) . Studies of consecutive NPA samples have shown that HBoV DNA can persist in the nasopharynx for several months (9, 33) . We also noted that 22% of children with HBoV DNA in their first serum sample continued to show viremia (with 2 logs less DNA) in the second serum sample obtained an average of 19 days later. That HBoV does not often persist in serum indicates that regardless of its magnitude, viremia is an excellent marker of acute HBoV infection.
The clinical role of codetection of HBoV and other viruses in NPAs has been questioned. It is not easy to determine whether such co-infections are sequential infections or simultaneous viral infections. Serologic analysis is a more precise approach for diagnosis of HBoV infections. When compared with PCR-positive results in serum and NPAs, diagnostic sensitivity and specificity of our antibody EIAs were as high as 97% and 99.5%, respectively; positive predictive value was 97%. We showed by using EIAs that among wheezing children, >60% of co-infections in children with HBoV NPA PCR-positive results, particularly in children with a high HBoV DNA load, are acute primary HBoV infections and should be considered in the diagnosis of respiratory disease. No differences in occurrence of co-infections were observed in children with a serologically confirmed diagnosis of infection with HBoV compared with children positive for HBoV by only PCR of NPAs.
Several groups have compared clinical features of HBoV infections with those of other respiratory virus infections. In those studies, diagnoses of infection with HBoV were based only on PCR positivity of NPAs, which as we showed, is not an ideal marker for detection of acute HBoV infection. We assessed clinical findings of our patients with serologically verified acute HBoV infections. Comparison of HBoV-induced wheezing with that induced by rhinovirus is notable because rhinovirus is commonly associated with wheezing in older children and has been recognized as a risk factor for recurrent wheezing and asthma in young children (34) (35) (36) . Also notable is a comparison of HBoV and RSV because RSV is the dominant cause of bronchiolitis in infants (37, 38) . Our data showed that wheezing induced by RSV occurred at the youngest age (median 10 months), followed by that induced by HBoV (17 months), and rhinovirus (25 months). Age-adjusted comparisons showed that HBoVinfected children were hospitalized longer than rhinovirusinfected children. Illnesses after HBoV infection lasted longer than illnesses after rhinovirus infection. However, we did not demonstrate that co-infection with HBoV would increase illness duration or severity, as has been reported for rhinovirus-and RSV-induced bronchiolitis (39) .
Children with HBoV co-infections seemed to have more AOM (47%) than those with single HBoV infections (33%), but this difference could be explained by inclusion of 7 RSV-positive children in this group. The highest rate of AOM (72%) was in children with RSV-induced wheezing. Alper et al. reported differences in frequencies of various respiratory viruses associated with AOM (HBoV was not included), but statistical significance was not achieved (40) . We found a difference in the frequency of AOM between children with RSV-induced wheezing and those with HBoV-induced wheezing.
Serologically confirmed primary HBoV infections detected in 12 symptomatic children with no signs of other respiratory virus infections (by PCR, culture, antigen detection, or serologic analysis) demonstrate that HBoV is a cause of acute wheezing in young children. Moreover, the fact that acute HBoV infection was detected in 27% of hospitalized children who were <2 years of age when they had their first episode of wheezing indicates that HBoV is a causative agent of bronchiolitis with clinical severity comparable with that of RSV. HBoV respiratory infections can be diagnosed with moderate accuracy by qPCR of NPAs. However, the most reliable methods for diagnosis of acute symptomatic HBoV infection are PCR of serum samples and serologic analysis for IgM and IgG. Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4(+ )T cells HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane. While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4(+ )T cells that are natural targets of HIV-1 is poorly understood. Here we show that in primary CD4(+ )T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes. Our data strengthen evidence that Gag-Env interaction is important in envelope association with lipid rafts containing GPI-anchored proteins for efficient assembly onto mature virions resulting in productive infection of primary CD4(+ )T cells. glycoprotein while abundant incorporation of raft lipid components such as ganglioside GM1, glycosylphosphatidylinositol (GPI)-anchored proteins Thy-1 and CD59 strongly suggest that HIV-1 specifically buds from rafts [5, 11] . A stable interaction between intracellular Pr55Gag and the gp41 cytoplasmic domain of envelope [12] was shown to be important for envelope association with detergent resistant membranes, incorporation onto virions and infectivity [13, 14] . The precise sequence by which envelope utilizes cellular machinery in migrating towards the site of viral assembly is not clearly understood. Glycoproteins of several enveloped viruses, have been found to contain lipid moieties [15, 16] and has generated notion on the importance of lipid rafts as a docking site for the assembly of enveloped viruses [17] [18] [19] [20] [21] [22] . Association of HIV-1 envelope with polarized lipid raft markers GM1 and CD59 was shown to influence transmission between T cells [10] . Gag has been shown to play an important role in envelope assembly onto virions, notably by interaction of its p17 matrix domain with gp41 cytoplasmic domain of envelope [14, [23] [24] [25] . While HIV Gag intrinsically associates with detergent resistant membranes (DRMs) [5, 26] , influenza virus M1 protein transport to DRMs depends on coexpression of HA and NA glycoproteins [27] . Likewise, the association of Sendai virus M protein in DRM is dependent on expression of F or HN protein [28] , while the Rous sarcoma virus M protein requires expression with the F protein for DRM association [29] . In contrast, the Measles virus M protein has been shown to associate DRMs intrinsically independent of other viral proteins [30] . Motifs in gp41 cytoplasmic domain regulating association of HIV-1 envelope protein with DRM [8] and this phenomenon is Gag dependent in a T cell line [13] was previously reported. However, it was not known whether this phenomenon is cell type-dependent and if it differs between cell lines and those that are natural targets in vivo. Moreover, whether DRM association of envelope corroborates with their ability to traffic to classical lipid rafts was also not known. In the present study we investigated the role of Gag in intracellular transport of HIV-1 envelope into well defined GPI-anchored proteins such as CD59 and GM1 (monosialotetrahexosylganglioside) and its relevance of envelope assembly onto budding virions in primary CD4 + T cells. Precisely, we examined whether a point mutation (L30E) in matrix domain of Gag known for disrupting Env incorporation [23, 31] affects envelope trafficking to CD59+ compartment in primary CD4 + T cells and if this phenomenon has any association with cellfree infectivity in primary CD4 + T cells.
We first examined whether a point mutation (L30E) in matrix domain of Gag previously reported to abrogate envelope incorporation, infectivity [23] and DRM association [13] in cell lines affect infectivity and modulate envelope association with CD59-enriched compartment in primary CD4 + T cells which are predominantly the natural targets in vivo during entire course of HIV-1 infection. CD59 marker was selected as it is linked with phosphatidylinositol and segregates into rafts. Primary CD4 + T cells were purified by negative selection from whole blood using RosetteSep® Kit (Stem Cell Technologies Inc.) following manufacturer's protocol. Briefly, CD4 + T Cell Enrichment Cocktail was added at a final concentration of 50 μl/ml of whole blood and incubated at room temperature for 20 min. The mixture was diluted with RPMI/2% Fetal Bovine Serum (GIBCO, Inc), layered onto Ficoll Hypaque (Sigma, Inc) and centrifuged for 20 min at 1200 × g at room temperature. The enriched CD4 + T cells in the interface of density medium and plasma yielded approximately 98% purity; subsequently stimulated with phytohemagglutinin (0.5 ug/ml) and interleukin-2 (10-20 U/ml) before infection with virus stocks. Primary CD4 + T cells were infected with equal infectivity titers of VSV-G pseudotyped pNL4.3 WT and pNL4.3 L30E viruses and the cell lysates made from CD4 + T cells infected with wild type and L30E expressing equal amounts of p24 and gp41 were incubated with anti-gp41 monoclonal antibodies to immuno-precipitate Env. Briefly, CD4 + T cells were lyzed with 1% Triton X-100 in PBS (pH 7.4), and incubated with 1:1 mixture of gp41 monoclonal antibodies 2F5 and 4E10 (1:1000 dilution) for 4 hours at 4°C followed by additional 1 hour incubation with Protein A/G Figure 1 A. L30E Gag confers defective association of Env with CD59-enriched membranes in primary CD4 + T cells. Primary CD4 + T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were lyzed and intracellular p55, gp41 and CD59 contents were measured in total cell lysates (as shown here in upper panels). Cell lysates expressing comparable p55 and gp41 were immunoprecipitated with gp41 antibodies 2F5 and 4E10 as described in the Materials and Methods resolved in 12.5% SDS-PAGE and electrophoretically transferred onto PVDF membrane. The level of CD59 co-immunoprecipitated with envelope was detected by Western blotting using monoclonal anti-CD59 antibody. B. Defective incorporation of HIV-1 envelope proteins onto virus particles due to L30E Gag substitution. Equal amounts of cell free virus particles were resolved in SDS-PAGE and Western blotting done by monoclonal antibodies to gp41 and p24 as described in the text. Note that with L30E gag mutation, envelope incorporation onto virions is severely abrogated.
beads (Pierce Inc). Lysates were further washed with cold PBS and were resolved in 12% SDS-PAGE under denaturing conditions. Equal amounts of immunoprecipitated materials were subjected to 12% SDS-PAGE under denaturing conditions [32] , transferred onto PVDF membranes and subsequently Western blot assay done with anti-human CD59 antibody (at 1: 1000 dilution) to assess the ability of Env association with CD59enriched compartment. As shown in Figure 1A , due to L30E mutation in Gag, Env failed to recruit CD59 in contrast to pNL4.3 wild type. Our data indicate that the mutation in Gag MA region (L30E) restricts the interaction between Gag and Env resulting in down modulation of envelope trafficking to GPI-anchored membranes in primary CD4 + T cells such as CD59. Moreover, in order to assess the effect of L30E mutation in p17 gag on HIV-1 envelope incorporation on virions in primary CD4 + T cells, cell-free virus pellets were obtained by centrifugation as described previously [13] . Equal amounts of virus particles (p24) were resolved in SDS-PAGE under denaturing conditions followed by Western blotting using monoclonal antibodies to p24 (183-H12-5C) and gp41 (1:1 of 2F5 and 4E10). As shown in Figure  1B , L30E substitution in p17 Gag was found to drastically affect envelope incorporation onto virus particles in CD4 + T cells as expected.
We next assessed if there is any correlation between disruptions of envelope association with CD59+ compartment in primary CD4 + T cells and its association with DRM of the same cell type. The DRM assay was Figure 2 Envelope association with DRM in primary CD4 + T cells. A. CD4 + T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm 3 ) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39] . carried out essentially as described previously [8] . Briefly, primary CD4 + T cells infected with VSV-G pseudotyped pNL4.3 WT and pNL4.3 (L30E) were lyzed with cold 0.5% Triton-X-100 and fractionated through sucrose density gradient centrifugation in an ultracentrifuge (Beckman Coulter Inc) at 1,00,000 × g for 8-12 hours at 4°C. Ten equal fractions were collected and were examined for presence of gp160, CD59 and GM-1 ganglioside by Western blot using human anti-gp41 monoclonal antibody (2F5) [31] and 4E10 [33] , mouse anti-human CD59 (BD Biosciences Inc) and cholera toxin conjugated to horse radish peroxidase (HRP) (Sigma, Inc.) respectively. As shown in Figure 2 , L30E mutation in Gag restricted envelope association with DRM fractions of CD4 + T cells in contrast to the wild type. Our results were further substantiated by the presence of both CD59 and GM1 in DRM fractions under same experimental conditions.
In summary, we show the modulatory role of Gag on envelope association with lipid enriched micro domain that play an important role in transmission by modulating envelope assembly onto virus particles in primary CD4 + T cells. Despite its close proximity, DRMs isolated from cells may not necessarily represent the preexisting rafts in living cells. [34, 35] . Hence, GPI anchored proteins because of their raftofilcity often are regarded as best choice for studying raft association and targeting [36] . In our present study, in addition to investigate role of gag in envelope association with DRM, we attempted to look into GPI-anchored proteins for their high affinity towards lipid raft association [35, 37] . The lipidbinding B subunit of cholera toxin (CTxB), recognizing GM1 at cell surface in plasma membrane, was used to study the envelope transport into lipid rafts. Like other GPI-anchored proteins, these markers were also known to be associated with DRM [38] . Our data showed that abrogation of Gag-Env interaction down modulated envelope transport into CD59 positive compartment in primary CD4 + T cells and also failed to associate with DRM fractions. While viral precursors Gag and Gag-Pol are synthesized by polysomes in cellular cytoplasm, the oligomeric envelope protein is synthesized in endoplasmic reticulum and post-translationally modulated in the Golgi apparatus, traverses into the secretory pathway towards assembling onto budding virions. We envisage that Gag by acting as cargo transport intermediate carry envelope protein through trans-golgi route sorting into domains of lipid rich plasma membrane enable them assemble onto budding virions in primary CD4 + T cells.
DRM: detergent resistant membrane; GM1: monosialotetrahexosylganglioside; GPI: glycosylphosphatidylinositol; GHOST: human osteosarcoma cells expressing CD4 and green fluorescent protein; CTxB: cholera toxin-b subunit. Two novel HLA-A*0201 T-cell epitopes in avian H5N1 viral nucleoprotein induced specific immune responses in HHD mice The influenza A nucleoprotein (NP) is an attractive target for avian flu vaccine development because of its high conversancy in the evolutionary chain of the virus. Here we identified two novel HLA-A*0201 restricted NP epitopes, named H5N1 NP373-381 AMDSNTLEL (NP373) and NP458-466 FQGRGVFEL (NP458), using computational bioinformatic analysis. The NP peptides showed a high binding affinity to HLA-A*0201 on T2 cells, and were able to induce the activation of the cytotoxic T cells in the human peripheral blood mononuclear cells. We examined the potential of using NP373 and NP458 peptide sequences supplemented with a single-chain trimer as potential DNA vaccine candidates in an HHD transgenic mouse model. A gene gun delivery system was used for administrating the vaccine candidates into the animals. The results from cytotoxicity and ELISPOT assays indicated that a significant amount of IFN-γ was secreted by the T cells of the vaccinated mice, and the T cells were able to eliminate the corresponding peptide-loaded T2 cells. The discovery of these novel immunogenic NP peptides provides valuable information for avian flu vaccine design and construction. The transmission of a pathogenic avian H5N1 virus from poultry to humans in Hong Kong and other Asian countries has caused a number of human deaths [13, 17, 19] . Confirmed cases of H5N1 transmission have also been reported in various African and European countries 1 and all incidences have caused an enormous economic loss in the poultry industries worldwide. The threat of an H5N1 pandemic is becoming a major problem not only in Asian countries but countries worldwide. The urgency of developing a strategy to combat the H5N1 virus is an imperative task for most health authorities, and one possible solution is to develop a human vaccine against the H5N1 virus.
The production of a neutralizing antibody is the primary step to combat viral infection. Another important mechanism for virus elimination is the initiation of a cell-mediated immune response involving the induction of effector CD8+ cytotoxic T lymphocytes (CTL). Conserved components within the influenza A viral genome are good targets for inducing CTL responses because they can withstand viral mutations and have the capability to induce cross-reactive immune response among different subtypes and variations [14, 15, 24] .
Nucleoprotein (NP) generally binds to the viral RNA and is synthesized in large amounts during viral replication within the infected cells [26] . In the influenza A virus, NP is a comparatively conserved protein, and there is no recorded evolution in the viral strains of most birds over the past 60 years. [26] . Therefore, because of its high conservancy during the evolution of the virus, NP is an attractive target for inducing T-cell immunity [10, 26] . One of the promising approaches involves a direct DNA vaccination to stimulate T-cell immunity against NP. Some DNA vaccines and recombinant viral vaccines such as those constituting an adenovirus encoding the whole NP gene, are found effective in eliciting the T-cell immunity [6, 7, 14, 23] .
We previously showed that a single-chain trimer (SCT) system is a promising system for use in the construction of DNA vaccines [2] . Here, we identified two novel HLA-A*0201-restricted H5N1-NP-protein peptides and a modified SCT system was used in vaccine construction. The efficacy of the constructed vaccine candidates was investigated in a special genetically modified HHD transgenic mouse model. Our results suggest that the H5N1 NP373-381 AMDSNTLEL and NP458-466 FQGRGVFEL peptides might be promising candidates for use in H5N1 vaccine construction.
H5N1 NP peptides from the H5N1-Thailandhuman-2004 strain were predicted by the HLApeptide-binding prediction program, SYFPEITHI 2 . Nine 9-mer potential peptides were synthesized by solid-phase strategies. They were named NP48 KLSD-YEGRL, NP55 RLIQNSITI, NP158 GMDPRMCSL, NP189 MVMELIRMI, NP256 LIFLARSAL, NP275 CLPACVYGL, NP357 QLSTRGVQI, NP373 AMDSNTLEL, and NP458 FQGRGVFEL. A peptide previously identified (N220 LALLLLDRL) was used as a positive control [2] .
HHD transgenic H-2D b-/-b2m -/double knockout mice (6-8 weeks old) purchased from the Institute Pasteur in France were bred and maintained under pathogen-free conditions (Animal & Plant Care Facility, HKUST, Hong Kong, China). HHD mice were originally derived from C57BL mice with the mouse b 2 -microglobulin and MHC I knock-out, and HLA-A*0201 knock-in expressing the chimeric HLA class-I molecules, which were composed of the human b 2 -microglobulin, HLA-A*0201 a-1 and a-2 domains and the a-3 domain of H-2D b [21] .
Recombinant NP was expressed from pET16b in BL21-condon plus (DE3)-RIL cells (Stratagene, La Jolla, CA, USA). Soluble NP was purified by a His-Bind Kit (Novagen, San Diego, CA, USA) and purified proteins were analyzed and confirmed by SDS-PAGE. Western blotting analysis was performed using an anti-influenza A NP antibody (Chemicon, Billerica, MA, USA).
T2 cells (174 · CEM.T2, ATCC, Manassas, VA, USA) were identified as HLA-A*0201 positive, and they were transporters for the antigen-processingdeficient cells. The cells were maintained in IMDM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% FBS (Invitrogen), 100 U/mL penicillin and 100 lg/mL streptomycin (Invitrogen) at 37°C with 5% carbon dioxide. In T2-cell binding assays, the cells (1 · 10 5 ) were pulsed with each of the target NP peptides (10 lg/mL), respectively, in serum-free medium containing the human b 2 -microglobulin (5 lg/mL) for 2 h. The cells were washed with 1· PBS, stained with an FITC-conjugated anti-human HLA-A2 antibody (BD Pharmingen, San Diego, CA, USA), and finally subjected to flow cytometrical analysis (BD FASC Aria II, BD Bioscience, San Jose, CA, USA). The fluorescent index was calculated by the formula as follows: FI = [MFI (T2 + peptide)/MFI (T2 only)] À 1. The flu peptide, GILGFVFTL, was used as a positive control [1] . 
Intracellular cytokine staining was performed as previously described with minor modifications [28] . Briefly, primed T cells were incubated with peptideloaded T2 cells in the presence of Brefeldin A (eBioscience, San Diego, CA, USA) for 8 h. The cells were stained with a PE-conjugated anti-human CD8 antibody (eBioscience) at 4°C for 30 min. After washing with 1· PBS, the cells were fixed and permeabilized with a permeabilization buffer (eBioscience) according to the manufacturer's instructions. The cells were finally stained with an FITC-conjugated anti-human IFN-c antibody at 4°C for 30 min in the dark, and the fluorescent signals were detected by flow cytometrical analysis (BD FASC Aria II, BD Bioscience). T2 cells loaded with previously identified non-responsive peptide (N160 LQLPQGTTL), were used as a negative experimental control [2] .
Plasmids including NP158HHDpBudCE4.1, NP-189HHDpBudCE4.1, NP373HHDpBudCE4.1, and NP458HHDpBudCE4.1 were prepared as follows. In brief, the single-chain trimer gene (HHD) was constructed by connecting the leader sequence, the human b 2 -microglobulin, the human HLA-A*0201 a-1, a-2 domains, and the mouse H-2D b a-3 domain.
It was subsequently cloned into a pBudCE4.1 vector (Invitrogen). Plasmids encoding different peptide sequences were constructed by adding the corresponding DNA fragments after the leader sequence of the HHD using PCR with the following primers: sense (NP158: 5 0 -ATCGACCGGTCTATATGCTGG AATGGACCCTCGGATGTGTTCCCTGGGAGGA GGTGCTAG-3 0 , NP189: 5 0 -ATCGACCGGTCTATA TGCTATGGTGATGGAGCTGATCAGAATGATCG GAGGAGGTGCTAG-3 0 , NP373: 5 0 -ATCGACCGG TCTATATGCTGCCATGGACAGCAACACCCTGG AGCTGGGAGGAGGTGCTAG-3 0 , NP458: 5 0 -ATC GACCGGTCTATATGCTTTCCAGGGACGGGGA GTGTTCGAGCTGGGAGGAGGTGCTAG-3 0 ) and antisense (5 0 -ATCGCTCGAGTCACGCTTTACAAT C-3 0 ) primers. The constructed fragments were cloned into HHDpBudCE4.1 at Age I and Xho I sites. The N220HHDpBudCE4.1 expressing an identified HLA-A*0201 epitope (N220) was used as a control plasmid [2] .
Animal vaccination was carried out by a gene gun delivery system and the preparation of DNA-coated gold particle cartridges was performed as previously described [2] . Briefly, cartridges were prepared by precipitating the plasmid DNA on 1 lm gold particles (Bio-Rad, Hercules, CA, USA) in 0.05 M spermidine (Sigma-Aldrich Corp) and 1 M CaCl 2 . The microcarrier/DNA suspension mixed with polyvinylpyrrolidone (5 mg/mL, Sigma-Aldrich Corp) was coated into the plastic tubing. The coated tubing was cut into 0.5 inch cartridges, and each cartridge contained 1 lg of DNA. DNA-coated particles were delivered to the shaved abdominal region of the HHD mice using a Helios gene gun (Bio-Rad, Hercules, CA, USA) with a discharge pressure of 400 psi. The mice received DNA (1 lg) for each vaccination. All animal groups (three mice per group) received a total of three vaccinations at 1-week intervals.
The cytotoxicity assay was performed using a Delfia EuTDA cytotoxicity detection kit (Perkin-Elmer, Waltham, MA, USA). Mice were sacrificed 1 week after the last vaccination for splenocyte extraction. Splenocytes were cultured in an IMDM medium (Invitrogen) supplemented with 5% FBS (Invitrogen), the corresponding target peptides (10 lg/mL), and IL-2 (10 U/mL, eBioscience) in 12-well plates (BD Bioscience) at 37°C for 5 days. They were seeded with the TDA-labeled, peptide-loaded T2 cells HLA-A2 T-cell epitopes in H5N1 nucleoprotein Vet. Res. (2010) 41:24 in ratios of 50:1, 10:1, and 5:1, respectively. The mice immunized with N220HHDpBudCE4.1 were used as an experiment control. The splenocytes seeded with N220-loaded T2 cells were used as a positive control while those seeded with N160-loaded T2 cells served as a negative control [2] . After incubating at 37°C for 1 h, the culture medium was collected and the fluorescent signals emitted by the dead peptide-loaded T2 cells were detected with the cytotoxicity reagents according to the procedures stated by the manufacturer. The cytotoxicity was determined by the following equation:
Two controls were set up for the cytotoxicity assay: a maximum release was measured from the peptide-loaded T2 cells lyzed by a lysis buffer, and the spontaneous release was measured as a natural release from the peptide-loaded T2 cells.
An ELISPOT assay was performed as previously described with minor modifications [2] . Briefly, a 96-well nitrocellulose plate (MultiScreen-HA, Millipore, Billerica, MA, USA) was coated with an anti-mouse IFN-c antibody (5 lg/mL, eBioscience) at 4°C for 20 h followed by blocking with an IMDM medium (Invitrogen) supplemented with 5% FBS (Invitrogen) at 37°C for 2 h. Splenocytes (1 · 10 6 ) were seeded into the four wells of a 96-well nitrocellulose plate, and the corresponding target peptides (10 lg/mL) were added. The plate was incubated at 37°C for 24 h. The mice immunized with N220HHDpBudCE4.1 were used as experiment controls. The splenocytes with N220 peptide added were used as positive controls while those added with N160 peptide served as negative controls [2] . Subsequently, the plate was washed with 1· PBS containing 0.01% Tween 20 and incubated with a biotinylated anti-mouse IFN-c antibody (0.5 lg/mL, eBioscience) at room temperature for 1 h. After washing, a streptavidin-AP solution (1 lg/mL, Sigma-Aldrich Corp) was added and the plate was incubated at room temperature for 30 min. After a final wash, spots were developed by adding a BCIP/NBT solution (Invitrogen) and the visible spots were counted by an ELISPOT reader (Thermo Labsystems, Franklin, MA, USA).
Two controls for each group of splenocytes were set up for the ELISPOT assay. Splenocytes without the addition of peptides were used as a negative control while the splenocytes with staphylococcal enterotoxin B (Sigma-Aldrich Corp) added were used as a positive control.
Statistical analysis was expressed as means ± standard error (SE). A comparison between the individual readings was performed using the student's t-test. p values < 0.05 were regarded as statistically significant.
In order to search for the immunogenic NP peptide sequence, nine potential peptides were predicted using SYFPEITHI software. Various assays were used to determine the immunogenicity of the peptides and the desirable ones (i.e., those with both the highest binding affinity to HLA-A*0201 positive cells and the ability to stimulate human CD8+ T cells) were selected for further in vivo experimentation. In order to determine their binding affinity to HLA-A*0201 positive cells, the peptides were synthesized and a T2-cell binding assay was performed. T2 cells normally express unstable HLA molecules on the cell surface and therefore if a peptide is able to fit into the peptidebinding groove of an HLA molecule, it will form a more stable structural complex with the HLA molecule in the presence of b 2 -microglobulin. This complex can be detected with an anti-human HLA-A2 antibody via flow cytometry. In our assay, a flu peptide (GILGFVFTL), which is an HLA-A*0201 epitope, was used as a positive control for the experiment. The results indicate that the NP373 (AMDSNT-LEL), NP189 (MVMELIRMI), and NP458 (FQGRGVFEL) showed the highest scores in the T2-cell binding assay (Fig. 1) .
The potential of the nine peptides in stimulating the human CD8+ T cells was also determined. Two groups of CD8+ T cells isolated from PBMC of two healthy donors, respectively, were primed with autologous mature NP-loaded DC three times. Intracellular cytokine staining was performed to detect the release of IFN-c from the CD8+ T cells, and the peptide-loaded T2 cells were used as the target cells. NP once processed by DC, will combine with the HLA molecules and eventually become presented on the cell surface for the priming of the CD8+ T cells. The primed CD8+ T cells could therefore recognize the peptide-MHC complex on the T2 cells if the target peptides are processed and presented on the DC surface. The results from intracellular cytokine staining showed that the highest number of CD8+ IFN-c-secreting T cells was produced after the stimulation with the NP189 (MVME-LIRMI), NP373 (AMDSNTLEL), and NP458 (FQGRGVFEL) peptides, and the lowest number of the stimulated T cells was observed after stimulation with the NP158 (GMDPRMCSL) peptide. The N160 (LQLPQGTTL) peptideloaded T2 cells used as a negative control did not show any significant changes (Fig. 2) [2] .
From the results of the two assays, we selected four target peptides, NP158 (GMD-PRMCSL), NP189 (MVMELIRMI), NP373 (AMDSNTLEL), and NP458 (FQGRGVFEL) for animal experimentation. Based on our findings, three of the peptides showed the highest binding affinity to the HLA-A2 molecules and they were able to activate the highest number of CD8+ T cells. Since NP158 (GMDPR-MCSL) exhibited a comparatively low ability to stimulate T cells against the peptide-loaded T2 cells and induced only a small amount of the IFN-c release, it was included in the sample peptides as a control for checking the consistency of the results previously shown by the in vitro T-cell stimulation experiment.
An animal experiment was performed to test the immunogenicity of the target peptides in vivo. DNA corresponding to the target peptides was cloned into the HHD gene to produce a DNA vaccine candidate containing the peptide-b 2 -microglobulin-MHC class-I heavy chain complex that could be expressed on the cell surface and trigger the immune system. Mice received a total of three administrations and were sacrificed 1 week after the last injection. Splenocytes were collected for the cytotoxicity and ELISPOT assays. Cytotoxicity was measured by the amount of TDA released from the target cells. The target cells (T2 cells) were loaded with the target peptides. Therefore, the peptide-loaded T2 cells would be killed if the corresponding peptides were immunogenic to the mice. The results from the cytotoxicity assay showed that the highest percentage of cytotoxic response was triggered in mice immunized with plasmids encoding NP373 and NP458, and the effect was similar to that produced by the positive control (N220). During an 1 h incubation period, NP373 exhibited a 14% and 10% killing efficiency with the effector cell-to-target cell ratios of 50:1 and 10:1, respectively, and NP458 exhibited a 13% and 9% killing efficiency with the effector cell-to-target cell ratios of 50:1 and 10:1, respectively. However, the killing efficiency of T cells triggered by the NP189 was comparatively low (7%) with the effector cellto-target cell ratios of 50:1. The result produced by NP158 was significantly lower, with only a 3.7% killing efficiency at an effector cell-totarget cell ratio of 50:1 (Fig. 3 ).
An ELISPOTassay was used to determine the splenocyte recognition of the corresponding peptides via IFN-c release after animal immunization. The largest number of splenocytes (appeared as spots) was produced in mice after immunization with NP373 and NP458 containing the SCT-DNA constructs (79 and 72 spots, respectively). In contrast, only 14 and 4 spots were observed in animals immunized with NP189 and NP158 containing the SCT-DNA constructs, respectively. The results were very similar to the negative control (5 spots only). The immunogenicity triggered by NP189 in mice was comparatively low when compared with the results of the T2-cell binding assay and the intracellular cytokine staining. Nonetheless, the results indicate that the immunogenicity of NP373 and NP458 was more than 18 times higher than that induced by NP158 (Fig. 4) .
The prediction process was repeated and the prediction score indicated that NP373 and NP485 may be able to bind to a variety of other HLA types, such as HLA-A1, A11, A24, A26, B7, B8, B14, B15, B27, B37, B38, B39, B40, B44, and B47 (Tab. I).
HLA-A*0201 is a global common HLA type, which occurs at high frequencies in populations of northern and southeast Asia and North America [18] . In this study, we selected nine peptides that might be potential vaccine candidates for human influenza A using the HLA-peptidebinding prediction program. Two novel epitopes for HLA-A2 specific H5N1 NP were found, namely NP373-381 AMDSNTLEL (NP373) and NP458-466 FQGRGVFEL (NP458). We further repeated the prediction process to investigate the binding of the two identified epitopes to other HLA types (Tab. I). We found that these two epitopes might be able to bind to other HLA types, and therefore further experimentation may be required to confirm the prediction. An ideal vaccine capable of counteracting infectious viruses should be able to trigger both neutralizing antibody production and the cytotoxic T-cell response which are critical processes in eliminating the virus during a viral pathogenesis. As previously mentioned, current studies of H5N1 vaccines have mainly focused on the generation of neutralizing antibodies [3, 11] . Another research stream involves the study of cell-mediated immunity against H5N1. Epitopes have been found in different internal proteins of influenza virus and with different HLA types [9, 20, 22, 25] .
Amongst the identified epitopes, all NP epitopes are categorized as HLA-B types. We decided to examine the NP epitope binding specificity of HLA-A*0201 to CD8+ T cells. A desirable peptide sequence for inducing cytotoxic T-cell response should be presented as an endogenous peptide epitope through proteasome digestion and have a high binding affinity towards human MHC class-I molecules. Therefore, apart from the T2-cell binding assay, the stimulating ability of the predicted peptides was also accessed via an in vitro stimulation using human PBMC. In vitro stimulation was carried out by priming the T cells with the naturally processed NP peptides from autologous DC. Our results showed that the NP189 peptide stimulated the strongest immune response amongst the four peptides used. The binding affinity of NP158 in the T2-cell binding assay was relatively high but no significant change in the immune response was observed during in vitro administration of human PBMC, and the result produced was similar to the negative control. This suggests that the peptide prediction software can only give a computational estimation of immunogenicity of potential vaccine candidates. Hence, the predicted epitopes may not truly reflect experimental conditions in vitro nor mimic the real state in vivo. Natural processing and peptide presentation on the HLA molecule significantly contribute to the magnitude of the immune response [5, 8] . In our experiment, the number of CD8+ T cells detected via intracellular cytokine staining was not as high as expected compared to the whole pool of CD8+ T cells. In normal natural processing, all epitopes on NP-loaded DC are presented to the CD8+ T cells, based on the cross-presentation characteristics of the DC [4, 16, 27] . Therefore all activated CD8+ T cells should be responsive to the immunogenic epitopes presented on NP. In our experiment, our target cells were loaded with only a single type of the target peptide which may explain why such a low percentage of cell activation caused by the corresponding peptide-specific CD8+ T cells is observed.
We used an SCT approach to construct our peptide vaccine candidates. With this method, peptides are able to specifically bind to the peptide-binding cleft created by the HLA-A*0201 a-1 and a-2 domains, and this phenomenon can imitate the natural binding of peptides onto the MHC class-I molecules during normal antigen processing [12] . Also the SCT system ensures the stability of the MHC I-peptide complex after translation [29] , and a more precise and direct priming of the professional antigen presenting cells to the cytotoxic T cells is achieved. Hence the stimulating efficiency of the peptide vaccine can be greatly maximized [2] .
The selected peptides were tested for their cytotoxic T cell stimulating ability in vivo using a HHD transgenic mouse model. HHD is a special mouse type which lacks mouse b 2 -microglobulin and MHC I, but contains human b 2 -microglobulin and HLA-A*0201 a-1, a-2, as well as a mouse a-3 fusion gene. This genetically modified mouse immune system can impersonate a human system's immune response to stimulation restricted to the reaction between HLA-A*0201 and T-cell receptors only, eliminating the background caused by mouse MHC class-I molecules [21] . In our experiment, cytotoxicity and ELISPOT assays showed that the NP373-381 AMDSNT-LEL and NP458-466 FQGRGVFEL are the most immunogenic peptides. The reactivity of the two peptides was consistent with all the tests during the process of epitope identification. Another target peptide, NP189, although it showed good responses in the T2-cell binding assay and in vitro stimulation of human CD8+ T cells, it induced a weak response in animal experiments. This is another indication that the in vitro system does not reflect the in vivo situation. Nonetheless, in vitro experiments are essential for acquiring basic information to verify the characteristics of these potential peptides and discovered immunogenic peptides, since the testing of the peptides cannot be performed in clinical trials without extensive investigation. The T2-cell binding assay is a good methodology for examining the potential binding affinity of the target peptides to the groove of HLA-A*0201 molecules. The antigen processing and presentation, and the magnitude of the CD8+ T-cell responses corresponding to the stimulants can be investigated via in vitro stimulation of the CD8+ T cells. We believe that the HHD transgenic mouse model is an appropriate animal model for investigating the stimulating effects of the peptide-HLA complex to the corresponding T-cell responses.
In summary, we identified two novel HLA-A*0201 T-cell epitopes, NP373 and NP458, in the H5N1 viral NP. Our results indicate that these two epitopes are capable of inducing a strong immune response, and that the SCT system can improve the immune responses. The finding of these novel cytotoxic T-cell epitopes presented provides significant information for the development of a human H5N1 vaccine. In the future, experiments using a mixture of SCT-DNA constructs encoding H5N1 NP epitopes instead of using one type of SCT-DNA construct encoding a single peptide are necessary to investigate whether there are any improvements for the immunogenicity. Anthropogenic factors and the risk of highly pathogenic avian influenza H5N1: prospects from a spatial-based model Beginning in 2003, highly pathogenic avian influenza (HPAI) H5N1 virus spread across Southeast Asia, causing unprecedented epidemics. Thailand was massively infected in 2004 and 2005 and continues today to experience sporadic outbreaks. While research findings suggest that the spread of HPAI H5N1 is influenced primarily by trade patterns, identifying the anthropogenic risk factors involved remains a challenge. In this study, we investigated which anthropogenic factors played a role in the risk of HPAI in Thailand using outbreak data from the “second wave” of the epidemic (3 July 2004 to 5 May 2005) in the country. We first performed a spatial analysis of the relative risk of HPAI H5N1 at the subdistrict level based on a hierarchical Bayesian model. We observed a strong spatial heterogeneity of the relative risk. We then tested a set of potential risk factors in a multivariable linear model. The results confirmed the role of free-grazing ducks and rice-cropping intensity but showed a weak association with fighting cock density. The results also revealed a set of anthropogenic factors significantly linked with the risk of HPAI. High risk was associated strongly with densely populated areas, short distances to a highway junction, and short distances to large cities. These findings highlight a new explanatory pattern for the risk of HPAI and indicate that, in addition to agro-environmental factors, anthropogenic factors play an important role in the spread of H5N1. To limit the spread of future outbreaks, efforts to control the movement of poultry products must be sustained. (Received 23 July 2009; accepted 11 December 2009) Abstract -Beginning in 2003, highly pathogenic avian influenza (HPAI) H5N1 virus spread across Southeast Asia, causing unprecedented epidemics. Thailand was massively infected in 2004 and 2005 and continues today to experience sporadic outbreaks. While research findings suggest that the spread of HPAI H5N1 is influenced primarily by trade patterns, identifying the anthropogenic risk factors involved remains a challenge. In this study, we investigated which anthropogenic factors played a role in the risk of HPAI in Thailand using outbreak data from the ''second wave'' of the epidemic (3 July 2004 to 5 May 2005 in the country. We first performed a spatial analysis of the relative risk of HPAI H5N1 at the subdistrict level based on a hierarchical Bayesian model. We observed a strong spatial heterogeneity of the relative risk. We then tested a set of potential risk factors in a multivariable linear model. The results confirmed the role of freegrazing ducks and rice-cropping intensity but showed a weak association with fighting cock density. The results also revealed a set of anthropogenic factors significantly linked with the risk of HPAI. High risk was associated strongly with densely populated areas, short distances to a highway junction, and short distances to large cities. These findings highlight a new explanatory pattern for the risk of HPAI and indicate that, in addition to agro-environmental factors, anthropogenic factors play an important role in the spread of H5N1. To limit the spread of future outbreaks, efforts to control the movement of poultry products must be sustained.
avian influenza / epidemiology / poultry farming / spatial analysis / Thailand
After emerging in southern China in the mid-1990s, the highly pathogenic avian influenza (HPAI) H5N1 virus spread across east and Southeast Asia, causing unprecedented epidemics in 2003-2004 [13] . As of 24 September 2009, the virus has caused 442 human cases, with 262 deaths worldwide 1 . Controlling the spread of H5N1 disease in poultry may contribute to the reduction of risk for humans [27] by preventing the emergence of a viral form with efficient human-to-human transmission capable of triggering a global pandemic [13] . Determining the factors involved in the spread of H5N1 in poultry and producing risk maps are critical to disease control as they would enable control measures to be targeted and surveillance in ''high-risk'' areas to be strengthened. The HPAI H5N1 virus is now well established in the poultry population in Asia, where the virus has been able to maintain itself and spread as well as periodically re-emerge. The main pathways that have been identified for the spread of H5N1 are the migration and trade of wild birds and the transport of poultry and poultry products [12] . However, the respective roles of these pathways at the global or national scale are still unclear [12, 18] . The persistence of HPAI H5N1 virus in Southeast Asia has been linked to a specific agro-ecosystem [9] that associates freegrazing ducks with rice cultivation. A separate study found that the risk of HPAI outbreaks was reduced in areas with agricultural activities other than rice farming [10] . Free-grazing ducks form a reservoir of HPAI H5N1 in Asia [29] and may contribute to the spread of the virus when they are moved among rice fields which also constitute a habitat for wild waterfowl [8] . In addition to free-grazing ducks, it is likely that backyard poultry raised in low biosecurity systems and fighting cocks are involved in the diffusion of the virus [8] ; however, their roles are still unclear. Research to date suggests that the spread of HPAI H5N1 is influenced primarily by human activities related to poultry production and poultry trading [18] , however, little is known about the underlying processes involved. Live poultry markets probably play a role in the maintenance of the virus in Asia [26, 28] and the movement of poultry within trade chains may have facilitated the spread of the HPAI H5N1 virus.
In Southeast Asia, Thailand was affected by HPAI H5N1 early, with the first official report of poultry and human outbreaks on 23 January 2004. By the end of January 2004, 32 provinces throughout the north and several in the south experienced outbreaks in many types of poultry. The disease caused 17 human cases from January 2004 to June 2005 2 . The epidemic peaked during a ''second wave'' with 1 717 outbreaks in poultry. Beginning in early 2004, Thai authorities implemented a control strategy based on the prohibition of vaccination and the use of preemptive culling. Approximately 60 million poultry were culled during the first wave, with stamping-out measures applied inside a 5-km radius around an outbreak. From July 2004, culling was restricted to suspected farms or villages; 3 million poultry were destroyed during the second wave of outbreaks. The movement of poultry and poultry products was also restricted around infected areas and fighting cocks and free-grazing ducks were targeted by control measures. Apart from these control strategies, the Thai authorities strengthened the surveillance of HPAI. In addition to the passive surveillance system and routine laboratory surveillance, the Government implemented an intensive survey known as the ''X-ray campaign'' in October 2004 with 990 000 volunteers conducting door-to-door surveys [23] to check poultry in every house nationwide. From mid-2005, the number of outbreaks in poultry decreased substantially but the occurrence of poultry outbreaks in two provinces of Thailand in late 2008 indicates that the threat of HPAI in Thailand remains present.
The fact that surveillance was strengthened in response to the large HPAI epidemic makes Thailand a prime place to analyse which factors play an important role in the spread of the disease. A set of environmental risk factors was identified in Thailand [8] , but aside from the findings of Tiensin et al. [24] , little has been learned about anthropogenic risk factors in the country. Some aspects of the role of human activities in HPAI risk were recently reported from Vietnam [19] . The discovery of HPAI H5N1 in Thai poultry markets in 2006 and 2007 [2] suggests that the HPAI virus has continued to spread among poultry through trade activities despite the presence of control measures. Apart from the duck-rice agro-ecosystem which has been shown to be a source of infection, the role of humans in the spread of HPAI H5N1 has not yet been fully investigated. The risk of HPAI varies spatially according to the anthropogenic characteristics of the different geographical areas of interest, each characterized by a variety of human activities such as poultry farming practices, trade activities and market rules, land use and agro-ecosystems, and veterinary services structure and control. The objective of the present work was to study, through a spatial approach, the risk factors of HPAI H5N1 linked to human activities. It complements previous work by identifying the high-risk areas of HPAI H5N1 in Thailand and by determining which anthropogenic factors are associated with an increased risk.
The subdistrict (an administrative unit covering an average area of 69.7 km 2 , called ''Tambon'' in Thaï) was used as the spatial scale of interest for the study. Out of the 7 408 subdistricts in Thailand, we excluded from the analysis those located on islands since we assumed that they played a minor role in disease distribution. This resulted in a geo-database of 7 366 subdistricts which we considered as the statistical units for the analysis.
Epidemiological data relevant to HPAI H5N1 outbreaks in poultry were provided by the Avian Influenza Control Center, Department of Livestock Development (DLD, Bangkok, Thailand), a unit in charge of surveillance and monitoring of avian influenza (AI) in poultry. Since January 2004, DLD has been recording information on all poultry outbreaks confirmed by a diagnostic test. Tests were carried out by diagnostic laboratories on sick or dead poultry or cloacal samples using reverse-transcriptase polymerase chain reaction and virus isolation [22] . We restricted the study to outbreaks in chickens and ducks which occurred during the second wave of HPAI because at that point both the passive and active components of the surveillance system were fully implemented to detect the disease. The data included records of 1 717 laboratory-confirmed cases (flocks of affected poultry) dating from 3 July 2004 to 5 May 2005. The background susceptible population was calculated for each subdistrict based on the poultry census data that DLD collected during the X-ray survey of February 2005. This was used to model the relative risk. We computed several anthropogenic factors to investigate the role of humans in disease spread since poultry product trading activities operate at different geographic scales. To take into account these different commercial activities, we built anthropogenic indicators based on the road network and human population settlements. Using the 2004 human population census database of the Thailand Department of Provincial Administration 3 , we calculated the human population density in subdistricts to study its association with the relative risk of HPAI, previous papers having found differing results regarding the effect of human population density on HPAI [8, 24] . In addition, we computed the distance from the subdistrict to major cities (defined as having a population of 100 000 or more). We believed that major cities may have played a role in disease spread due to the intensity of poultry trade in the areas surrounding them. Information on the road network (primary and secondary roads, highways) was obtained from the Ministry of Transport, Bangkok, Thailand. This information made it possible to compute the road density per subdistrict (grouping primary and secondary roads) which was taken as an indicator of the intensity of the local trade of poultry products within a subdistrict. We suspected that highways played a role not only in the long-distance spread of the virus through the dispersal of infected materials, but also in the short-distance spread to subdistricts located in their vicinity. Therefore, we introduced the distance of a subdistrict to the closest highway as an explanatory variable. Finally, we computed the distance to the closest highway junction, which was assumed to function as a ''dissemination node'' for the HPAI virus. We assumed that if the virus was transported mainly through the road network, the subdistricts located close to a highway junction were more likely to come in contact with the virus than those located further away. To take into account environmental risk factors, we used topographic data (altitude, hydrology) which were obtained from the 1996 digital database of the Thailand Environment Research Institute 4 (Bangkok). Research has shown that rice-cropping intensity is a relevant risk factor in Thailand and other Asian countries [9] . Therefore, we included maps of rice-cropping intensity based on MODIS sensor images processed from satellite-based mapping algorithms developed by Xiao et al. [30] . Using data from the DLD poultry census, we also estimated for each subdistrict the density of animals and households raising poultry for different types of poultry: native chickens, fighting cocks, broiler and layer chickens, free-grazing ducks, and broiler and layer ducks.
All data were integrated into a geographical database and geoprocessing was carried out with the Spatial Analyst extension of ArcGIS software v. 9 
Disease modelling and mapping was performed for the whole of Thailand at the subdistrict level. We ran two parallel models (one for chickens, one for ducks) since we assumed that the respective spatial patterns for chickens and ducks were different. We aimed to produce disease maps based on ''relative risk'', which was taken to be a ratio of the risk of HPAI in a given subdistrict to the average risk nationwide. The latter was estimated from the overall number of cases and the poultry farm population in Thailand. Due to the widely varying number of poultry farms in each subdistrict, and because of spatial dependency between the subdistricts [17] , we applied the hierarchical Bayesian approach described by Besag et al. [3] to the HPAI H5N1 data. This method made it possible to compute area-specific relative risk estimates [17] while considering spatial interactions through a spatial smoothing based on a Gaussian auto-regressive model [3] . We used a first order spatial interaction neighbourhood based on the contiguity between the spatial units. The original method in Besag et al. [3] uses a Poisson distribution to model the occurrence of cases, which is appropriate for rare, non-contagious diseases such as cancers in humans [15] or bovine spongiform encephalopathy in cattle [1] . However, due to the contagiousness of HPAI within each spatial unit, applying this method to HPAI would result in overdispersion compared to the Poisson distribution. We handled this problem by modelling the locally observed number of cases using a negative binomial distribution [14] , an approach that has been used to model influenza by Fraser et al. [7] . Monte Carlo Markov Chain (MCMC) simulations were used to estimate the parameters of the model, including the estimate of relative risk for each spatial unit [16] . The estimation was performed using LinBugs [21] , with 1 million iterations, each producing a random simulation of the relative risk for all of the statistical units (e.g. subdistricts). Geweke and Heidelberger-Welch tests were used to assess the convergence of the models [5] . Considering a long safety burn-in period, parameters were estimated from a subset of 3 000 of the random simulations (with a systematic step of 1 over 3 to overcome auto-correlation problems). From this subset of 3 000 simulations, we computed credible intervals containing 95% of the values of relative risk. We tested the link between the relative risk values in subdistricts on chickens and ducks using a Spearman rank order correlation test. The relative risk was mapped for chickens and ducks using ArcGIS software v.9.1 (ESRI Inc.). Maps made it possible to identify groups of subdistricts with either a significantly high or a significantly low risk of HPAIinfected flocks compared to the rest of the country.
We aimed to identify the factors associated with the spatial risk of HPAI. To do so, we constructed a linear model with fixed effects. We used the logarithm of the relative risk estimated through the Bayesian approach (which modelled the exponential of relative risk values through a Gaussian distribution in each statistical unit, as mentioned by Besag et al. [3] ) as the dependent variable. Separate models of HPAI outbreaks were constructed for the chicken and duck populations. Each of the two models contained 18 variables which included environmental, poultry farming, and anthropogenic factors. Multicollinearity was investigated by checking the standard errors of regression coefficients and the variance inflation factors (VIF) [6] . The density of native chickens and the density of farms with native chickens were found to be positively correlated; consequently, only the former was introduced into the analysis. Multicollinearity was finally assumed not to cause any serious problem in the model (VIF values < 5.1) [6] .
Since we expected some non-linear relationships, continuous variables were transformed into categorical data before they were entered into the model. Four categories were chosen for each variable with the exception of free-grazing ducks (density of animals and density of farms) whose distribution allowed only 3 categories. We selected the thresholds that simultaneously fitted the non-linear relationships and had a sufficient number of statistical units per category. Finally, we added the level of relative risk in the duck population as a covariate in the chicken model and vice versa for the duck model. For each species, this thereby made it possible to adjust the analysis to account for the level of disease occurrence in the other species. The multivariate analysis was carried out using a stepwise backward elimination process; the significance of each variable in the full model was assessed in turn, with the least significant variable deleted and the process repeated until all of the remaining variables were significant at p value 0.05 (Fisher test) [6] . From the effect estimates computed in the final linear model, we deduced values of risk ratios (RR) and their confidence intervals (95%) for the different variables. For each variable, the reference category was defined as the category expected to be at the lowest level of risk based on our hypothesis and findings of previous studies in Thailand [8, 24] . Although spatial dependence already had been taken into account through the spatial contiguity in the Bayesian approach, we looked for any remaining spatial autocorrelation in the linear models. The semi-variograms of model residuals showed that autocorrelation may have played a role only in a very short-distance range (2 250 m). Computing the distances between each subdistrict to the next nearest subdistrict, we found that only a small portion of subdistricts (< 5%) actually had a chance of being influenced by their neighbours within that range. Thus the likelihood that spatial autocorrelation affected the results was assumed to be low. The statistical analysis was performed using R software v.2.9.2 5 . Figure 1 shows the geographical distribution of HPAI outbreaks in chicken and duck flocks during this time period. Figure 2 presents the spatially smoothed relative risk maps for chickens and ducks and shows that the two maps resemble each other fairly closely (Spearman rho = 0.91, p < 1eÀ16). The maps visually confirm the presence of a ''hot spot'' of HPAI risk in the central plain of Thailand where the relative risk was significantly higher than the national average (relative risk > 10 for both chickens and ducks). For ducks, however, the high-risk area tended to extend further across the western part of the central plain of Thailand. In contrast, the extreme south of Thailand appeared to be a high-risk area for chickens, with values of relative risk significantly > 10. On the contrary, some areas were especially low-risk for both chickens and ducks despite the occurrence of outbreaks (relative risk significantly < 0.5), as in northeastern Thailand and in the middle part of the peninsula. Northern Thailand had low values of relative risk (significantly < 0.5) only for chickens.
We focussed on the RR values and highlighted the variables with high or very low RR. The estimated effects of the environmental, poultry farming and anthropogenic risk factors are displayed in Tables Ia, Ib and Ic. The level of relative risk of HPAI for ducks was the main risk factor associated with the relative risk for chickens, and vice versa. Apart from this, the mean number of rice crops per year was the most relevant risk factor for the relative risk of HPAI for both chickens and ducks. A low average altitude in a subdistrict ( 50 m) was also found to be a risk factor of HPAI for chickens and ducks, while medium altitude was associated to RR below 1. A high density of free-grazing ducks appeared to be one of the main risk factors for HPAI. For chickens and ducks, the HPAI risk was connected more closely to animal density than to the density of farms or households with poultry. Areas with a high density of broiler and layer ducks were associated with a strong increase in the relative risk for both chickens and ducks (RR > 4 for subdistricts with more than 80 ducks per km 2 ). A high density of broiler and layer chickens (> 500 chickens per km 2 ) was found to be associated with a high risk of HPAI (RR > 3) only in the duck model. Our results demonstrated a relationship between a high density of native chickens and a low risk of HPAI H5N1 for both chickens and ducks. To a lesser extent, a high density of fighting cocks was found to be associated with an increase in the HPAI risk for both species. The role of several anthropogenic factors related to proximity to main transportation axes and major cities (Fig. 3 ) still remained strong after adjusting for the effects of the other variables. We found that a high HPAI risk was strongly associated with highly-populated areas, short distances to the highway junction (< 20 km), and a high density of roads in a subdistrict. Moreover, the HPAI risk decreased when the distance radius to major cities (with a human population of over 100 000) increased. To a lesser extent, a very short distance to the closest highway (< 5 km) was also associated significantly with a higher HPAI risk for chickens and ducks.
We used Bayesian spatial analysis to characterise HPAI risk areas in Thailand during the second wave of the HPAI H5N1 epidemic and to explore the link between anthropogenic factors and the relative risk of HPAI. We focussed on risk factors that contributed to the spread of HPAI H5N1. This analysis shows that, when adjusted for the effects of environmental and poultry variables, several anthropogenic factors were significantly associated with an increased risk of HPAI in both chicken and duck populations.
First, we generated maps of the relative risk of HPAI H5N1 for chicken and duck flocks, and showed that the spatial pattern for chickens and ducks was similar. This indicated that chickens and ducks either infected each other or shared the same spatial source of infection. The results of the multivariate analysis suggest that both explanations may be valid. When, for example, we considered chickens, we found that the relative risk in ducks and several other risk factors were significant when adjusted for each other in the final model. Furthermore, the model for chickens and the model for ducks indicated a common set of risk factors. Second, our results were consistent with previous studies on ecological risk factors of HPAI in Thailand [8, 9] . However, adding on to previous work, our analysis made it possible to identify classes of values associated with higher risk, which provides greater detail regarding the possible role of ecological risk factors. A high HPAI risk was associated with a high density of free-grazing ducks (> 10 ducks per km 2 ), more than one rice crop per year, and a short distance to a river ( 2 km). Altitude may be considered as an indicator of other unmeasured environmental variables related to HPAI risk. Subdistricts with a low average altitude ( 50 m) were associated with a high risk of HPAI. The mixture of wetlands, ponds, irrigation networks and agriculture in these areas combined with intensive land use [9] , may have constituted a favourable environment for the HPAI H5N1 virus. In contrast, subdistricts with a medium altitude (50.01-400 m) have higher slopes and a land cover dominated by forests and permanent vegetation [9] . Medium average altitude in subdistricts associated with low RR was found to constitute a kind of protective factor regarding HPAI risk. We also provide new insight into the role of factors related to poultry farming in the spread of HPAI. Like Tiensin et al. [24] , we found no indication that native chickens represent an increased HPAI risk despite the fact that these chickens are raised in low biosecurity systems and were affected massively by the disease (920 out of the 1 158 chicken flocks infected during the second wave). Conversely, a high density of native chickens (100-300 and > 300 native chickens/km 2 ) was associated with RR significantly below 1. In Thailand, wet markets have always been rare [2] and native chickens mainly are raised for family consumption using little input and involving little trading activity. This may have resulted in a protective effect against HPAI in subdistricts with a high density of native chickens. These subdistricts probably were less exposed to the virus because they were not connected to trading chains which potentially spread the disease. In addition, the pre-emptive culling, which focussed in the beginning on native chickens around an outbreak, may have contributed to containing the spread within these subdistricts. Fighting cocks are believed to have worsened the HPAI situation in other Asian countries [29] and in Thailand [22] . The association we found between high densities of fighting cocks and HPAI risk was significant but weak, as did Gilbert et al. [8] . In Thailand, fighting cocks were also targeted when control measures were implemented in 2004, with a prohibition on cockfighting, compulsory registration, and disease monitoring. Given their high monetary and cultural value, roosters receive very special attention from their owners, who may have changed their practices early to protect their poultry from the disease. Together, these two elements may have resulted in a decreased effect of fighting cock abundance on the HPAI risk.
Third, we identified a new set of significant risk factors that help to refine the current understanding of the HPAI H5N1 epidemic in Thailand. Previous studies have suggested that broiler and layer ducks do not constitute a risk factor for HPAI risk in Thailand [8, 9] . In contrast, we found a significantly increased risk of HPAI, both in chicken and duck flocks, in areas with a high density of broiler and layer ducks. During the period studied, only a quarter of the total number of broiler and layer ducks were raised in closed facilities with high biosecurity systems [20] . Since it has been proven that farm duck breeds can shed the H5N1 virus with minimal clinical signs [11] , our results suggest that farm ducks may also have played the role of silent carriers during the second wave of the epidemic, contributing to the spread of the disease. In addition, an increased risk in duck flocks was shown for subdistricts with a high density of broiler and layer chickens (> 500 chicken/km 2 ). In Thailand, broiler and layer chicken production range from large-scale industrial farms to small, family-run operations [24] . The latter refer to small or medium-scale businesses with links to several middlemen or companies for the transformation and transportation of both farm inputs and outputs (feed, wastes, poultry products. . .) [23] . During the second wave of the epidemic, it is possible that biosecurity rules were not applied fully throughout these complex poultry production chains, thus resulting in the spread of H5N1 in subdistricts with a high density of broiler and layer chickens.
Furthermore, we identified several statistically significant relationships between indicators of human activity and the relative risk for HPAI in chicken and duck populations. Human population density was the only anthropogenic risk factor thus far identified in previous research in Thailand. On the contrary to Tiensin et al. [24] , but in accordance with Gilbert et al. [8, 9] , our study found a progressive increase of HPAI risk with an increase of human population density for both chickens and ducks. In addition, we showed that areas located within a short distance radius around major cities and highway junctions constitute ''hot spots'' for HPAI risk. Cities are characterised by highly intense poultry trade activities involving live poultry markets, food markets, slaughterhouses, and poultry plants. This intensity may have resulted in increased possibilities of virus introduction and spread in surrounding areas. If the HPAI virus was transported through the road networks, the subdistricts located a short distance from the highway junction were more likely to be in contact with the virus than those situated further away. Highway junctions thus may have functioned as ''dissemination nodes'' for the HPAI H5N1 virus. A significant association was identified between a high HPAI risk and proximity to the closest highway. This may underline the role of the movement of poultry and poultry products during the second wave of the epidemic, not only in the long-distance spread of the HPAI virus, but also in the short distance dissemination in the areas surrounding the highways. Free-grazing ducks that are moved along the central plain for hundreds of kilometers and are known to be reservoirs of HPAI may have contributed to the spread of the disease [8, 20] . Other types of poultry also were moved from farms to slaughterhouses, markets, or fighting arenas, while farm inputs and poultry products such as eggs, meat, litter, and poultry manure were transported along collection circuits. The movement of live poultry, people, and infected material may have resulted in the spread of the virus between houses through direct or indirect transmission. Subdistricts with a high road density were associated with an increased risk of HPAI H5N1. Local poultry product and by-product business activities involve frequent contacts which revolve around road networks. Once the HPAI virus was introduced into a subdistrict, a dense road network may have facilitated its local spread.
Thus, while the spatial pattern of relative risk is known to be largely associated with an abundance of free-grazing ducks and rice-cropping intensity, we found that several indicators of human activities were also associated with HPAI risk in Thailand during the second wave of the epidemic. This suggests that in addition to the ''rice paddy-free grazing duck system'', major cities, the highway network, and local road networks may also have played key roles in the spread of HPAI in Thailand. Through the transportation of potentially infected live poultry or contaminated poultry products, highways may have contributed to both the long distance and the local spread of the HPAI H5N1 virus. Local road networks were possibly involved in short-distance spread. In addition, major cities and highway junctions may have functioned as ''dissemination nodes'' for the HPAI H5N1 virus through the intense traffic of poultry products and poultry trading activities in their surrounding areas. Moreover, our results suggest that activities related to layer and broiler ducks may have played a more significant role than previously thought, as well as to a lesser extent -layer and broiler chickens.
To tackle the outbreaks, Thai authorities implemented different control measures which evolved over time, but the initial plan aimed to control poultry product movement countrywide. Beginning in July 2004, in addition to pre-emptive culling, the movement of poultry and avian products was restricted within a 5-km radius of an infected flock; these restrictions were extended during the second wave. The whole country was also zoned into 5 areas, and poultry movements were strictly controlled through 32 check points between zones [4] . This helped to contain the disease from spreading countrywide. In addition to this set of control measures, and because free-grazing ducks were suspected of being H5N1 HPAI reservoirs, the Thai Government encouraged duck producers to change their practices from a free-grazing to a housed system. However, farmers were not able to change their practices in a short period of time [23] . In 2005, ducks were still allowed to graze in paddy fields, but the DLD prohibited long-distance movements.The free-ranging practice became illegal in March 2006, obliging farmers to house every duck flock [23] . However, by this time the epidemic was already under control: the number of outbreaks had dropped from 1 717 during the second wave to 75 during the third wave (1 July 2005 to 9 November 2005). Thus, while the housing of all free-grazing ducks took time to achieve, restrictions on the long-distance movements of free-grazing ducks had already contributed largely to limiting HPAI spread in Thailand.
The H5N1 virus may now be well established in different Southeast Asian countries. Despite the implementation of control measures, it is probable that these countries will continue to face new outbreaks in poultry. The conditions under which the virus maintains itself in the environment are not well known. It is difficult to prevent virus re-emergence in possible local persistence spots, or the periodic reintroduction of the virus [25] . Controlling the disease within the poultry population is a critical issue for both the public health and agricultural economic systems. The restructuring of poultry production from open-housed to closed systems has started in Thailand but the process will take time and considerable cooperative effort. Therefore, to limit the number and size of future outbreaks in the poultry population, the focus of efforts should be on controlling the movement of both live poultry and avian products. Systematic analysis, comparison, and integration of disease based human genetic association data and mouse genetic phenotypic information BACKGROUND: The genetic contributions to human common disorders and mouse genetic models of disease are complex and often overlapping. In common human diseases, unlike classical Mendelian disorders, genetic factors generally have small effect sizes, are multifactorial, and are highly pleiotropic. Likewise, mouse genetic models of disease often have pleiotropic and overlapping phenotypes. Moreover, phenotypic descriptions in the literature in both human and mouse are often poorly characterized and difficult to compare directly. METHODS: In this report, human genetic association results from the literature are summarized with regard to replication, disease phenotype, and gene specific results; and organized in the context of a systematic disease ontology. Similarly summarized mouse genetic disease models are organized within the Mammalian Phenotype ontology. Human and mouse disease and phenotype based gene sets are identified. These disease gene sets are then compared individually and in large groups through dendrogram analysis and hierarchical clustering analysis. RESULTS: Human disease and mouse phenotype gene sets are shown to group into disease and phenotypically relevant groups at both a coarse and fine level based on gene sharing. CONCLUSION: This analysis provides a systematic and global perspective on the genetics of common human disease as compared to itself and in the context of mouse genetic models of disease. Common complex diseases such as cardiovascular disease, cancer, and autoimmune disorders; metabolic conditions such as diabetes and obesity, as well as neurological and psychiatric disorders make up a majority of health morbidity and mortality in developed countries. The specific genetic contributions to disease etiology and relationships to environmental factors in common disorders are unclear; complicated by many factors such as gene-gene interactions, the balance between susceptibility and protective alleles, copy number variation, low relative risk contributed by each gene, and a myriad of complex environmental inputs.
Genetic association studies using a candidate gene approach and more recently whole genome association studies (GWAS) have produced a large and rapidly increasing amount of information on the genetics of common disease. In parallel, mouse genetic models for human disease have provided a wealth of genetic and phenotypic information. While not always perfect models for human common complex disorders, the genetic purity and experimental flexibility of mouse disease models have produced valuable insights relevant to human disease.
Gene nomenclature standardization [1] , database efforts [2] [3] [4] , and phenotype ontology projects [5] in both human and mouse over the past decade have provided the foundation for integration of information on genetic contributions to disease and phenotypes. This allows the opportunity for systematic comparison and higher order systems analysis of disease and phenotypic information. In this report, we summarize and integrate large scale information on human genetic association information and mouse genetically determined phenotypic information with the goal of identifying fundamental relationships in human disease and mouse models of human disease.
The Genetic Association Database [2] (GAD) http:// geneticassociationdb.nih.gov is an archive of summary data of published human genetic association studies of many common disease types. GAD is primarily focused on archiving information on common complex human disease rather than rare Mendelian disorders as found in the Online Mendelian Inheritance in Man (OMIM) [6] . GAD contains curated information on candidate gene studies and more recently on genome wide association studies. It builds on the curation of the CDC HuGENet info literature database [3] in part by adding molecular and ontological annotation creating a bridge between epidemiological and molecular information. This allows the large-scale integration of disease based genetic association information with genomic and molecular information as well as with the software tools and computational approaches and that use genomic information [7] [8] [9] [10] [11] [12] . This report is a summary and analysis of the genes and diseases with positive associations in the Genetic Association Database with regard to replication, comparisons between diseases, and within broad phenotypic disease classes. Although GAD contains information on gene variation, this report is at the gene level only and does not consider specific gene variation or genetic polymorphism.
The Genetic Association Database (GAD) currently contains approximately 40,000 individual gene records of genetic association studies taken from over 23,000 independent publications. Importantly, a large number (11, 568) of the records in GAD have a designation of whether the gene of record was reported to be associated (Y) or was not (N) associated with the disease phenotype for that specific record. Many records, for various reasons, do not have such a designation. In addition, a portion of the database records have been annotated with standardized disease phenotype keywords from the MeSH http://www.nlm.nih.gov/mesh/ vocabulary. The GAD summations shown below are a subset of the records in GAD. They only include those records that are both; a) positively associated with a disease phenotype, and b) have a MeSH disease phenotype annotation. This represents a subset of 10,324 records having both positive associations to disease and records with MeSH annotations. Records designated as not associated (N) with a disease phenotype and those without MeSH disease annotation are not considered at this time in this report.
The mouse phenotypic information described here was obtained from the Mouse Genome Informatics (MGI) database [4] http://www.informatics.jax.org/ Phenotypes, Alleles and Disease Models section. The file used for mouse phenotypic information (see methods) is comprised of 5011 unique genes and 5142 unique phenotypic terms derived from information from specific gene mutations in multiple mouse strains. The mouse phenotypic information had been annotated to the mouse gene mutation records using Mammalian Phenotype terms and codes in the mouse phenotype database as a component of the Mouse Phenotyping Project [5, 13] .
Quantitation of how often a disease phenotype was positively associated with a gene was performed as follows. GAD records having both recorded positive associations and annotated MeSH disease keywords were extracted and stored in a database according to their relationships. Using a perl script, the number of times of co-occurrence of a MeSH disease keyword was positively associated with a specific gene was recorded as found in the GAD database. These counts were sorted in declining order for each unique gene grouped by the disease MeSH term with which they are associated.
The mouse phenotypic information described here was obtained from the Mouse Genome Informatics (MGI) http://www.informatics.jax.org/; Phenotypes, Alleles and Disease Models section; ftp://ftp.informatics.jax.org/pub/ reports/index.html#pheno Using these three files downloaded on 4-4-2008ftp:// ftp.informatics.jax.org/pub/reports/MPheno_OBO.ontologyftp://ftp.informatics.jax.org/pub/reports/MGI_Pheno-typicAllele.rptftp://ftp.informatics.jax.org/pub/reports/ MGI_PhenoGenoMP.rpt
The mouse phenotype files were extracted using a perl script annotating each gene with the phenotype term associated with each Mammalian Phenotype (MP) code.
Individual GAD primary gene sets were analyzed using Venny [14] http://bioinfogp.cnb.csic.es/tools/venny/index. html. Pathway Venn Diagram comparisons were performed by placing individual GAD primary gene sets into WebGestalt [15] http://bioinfo.vanderbilt.edu/webgestalt/ to identify KEGG pathways, then placing the resulting pathway names into Venny.
Relationships between diseases were identified by a unique method similar to phyologenetic classification. First the distance between the diseases were calculated by pairwise comparison of the diseases by finding the common genes between the pairs and dividing it by the smallest group of the pair. This number was then subtracted from 1. This step was done because if two lists are identical (100% match) then the resultant distance should be 0. This is represented in the formula:
Where: C k : Genes in each disease set (where k = i, j); N(C k ): Number of genes in each disease set (where k = i, j); d ij is the pairwise distance; i, j: index of genes in each disease set where; i = 1, 2, 3, ........., n; j = 1, 2, 3, ........., m
The disease relationships were calculated from the distance matrix using the Fitch program from the Phylip package [16] . It calculates the relationships based on the Fitch and Margoliash method of constructing the phylogenetic trees [17] using the following formula (from the Phylip manual):
where D is the observed distance between gene sets i and j and d is the expected distance, computed as the sum of the lengths of the segments of the tree from gene set i to gene set j. The quantity n is the number of times each distance has been replicated. In simple cases n is taken to be one. If n is chosen more than 1, the distance is then assumed to be a mean of those replicates. The power P is what distinguished between the Fitch and Neighbor-Joining methods. For the Fitch-Margoliash method P is 2.0 and for Neighbor-Joining method it is 0.0. As running Fitch took a long time when the gene-set size was huge (weeks for the human gene-sets and months for the mouse gene-sets), Neighbor-Joining method was used to create the replicate dendrograms (not shown) after randomizing the input order for greater confidence. The resulting coefficient matrix files were displayed using the Phylodraw graphics program [18] .
Ward's minimum variance method [19] was used to find the distance between two diseases. The distance between the clusters is the ANOVA sum of squares between the two clusters added up over all the variables. At each generation, the within-cluster sum of squares is minimized over all partitions obtainable by merging two clusters from the previous generation. Ward's method joins clusters to maximize the likelihood at each level of the hierarchy under the assumptions of multivariate normal mixtures, spherical covariance matrices, and equal sampling probabilities. Distance for Ward's method is:
where N K is the number of observations in C K (which is the Kth cluster, subset of {1, 2, ..., n) where n is the number of observations). x k is the mean vector for cluster C K .
Each record in GAD represents a specific gene from a unique publication of a human population based genetic association study and is categorized into one of 24 general disease classes corresponding to broad MeSH disease or disease phenotypic groupings. Table 1 is a summary of the number of positively associated human genes in each MeSH human disease class. As represented by these disease classes the GAD database covers a broad selection of diseases falling into major disease classes including; aging studies, cancer, immune disorders, psychiatric diseases, metabolic conditions, pharmacogenomic studies, and studies of chemical dependency, among others. Similarly, each record in the phenotype files from the MGI phenotype database represents a unique mouse gene specific genetic model. Table 2 shows the general categories represented by the mouse phenotype summary files and the number of mouse genes found in each top level phenotype class. The mouse files contain a greater number of intermediate developmental and morphological phenotypes (e.g. insulin resistance, absent CD4+ T cells, abnormal spatial learning) while the human files tend to comprise a greater number of end stage clinical disease phenotypes (e.g. Type 2 Diabetes, multiple sclerosis, autism). Table 3 introduces examples of human genes from fundamental biological pathways that have been consistently associated with major disease phenotypes highlighting the sometimes-broad pleiotropic effects that major regulatory molecules have on multiple disease phenotypes. Genes such as NOS3, nitric oxide synthase 3, regulating nitrous oxide production; HLA-DQB1, the MHC class II molecule DQ beta 1, involved in antigen presentation; ACE, angiotensin I converting enzyme, central to the renin-angiotensin system and PPARG, peroxisome proliferator-activated receptor gamma, regulating transcription in pathways important in lipid metabolism are examples of genes that affect multiple tissues and different organ systems through the complex course of disease progression. Importantly, all the mouse orthologs of the human genes in Table 3 have experimentally determined phenotypes that are similar or broadly overlapping with human clinical disease phenotypes (see below).
The majority of this report is built upon large nonredundant general summary lists for both human and mouse, shown below. These lists take two complimentary forms in both human and mouse. The first sets are GENE-to-Disease/Phenotype lists. These are non-redundant lists of genes showing the diseases or phenotypes that have been associated with each gene (Table 4  human, table 5 mouse, and table 6 human-mouse). The second sets of basic lists are DISEASE/PHENOTYPE-to-Gene lists. These are non redundant lists of diseases or phenotypes with the genes that have been associated with that disease or phenotype (Table 7 human and  table 8 mouse) . Table 4 shows examples of selected genes in each row that have been positively associated with specific disease phenotype keywords. Each human gene symbol is followed by a specific MeSH disease term and the number of times that gene has been positively associated with the term, in declining order. A major feature of Table 4 is that individual genes have been positively associated with sometimes overlapping disease phenotypes over a broad range from more frequently to less frequently. Table 4 is a small representative subset, truncated in the number of genes (rows) and the number of MeSH terms (columns). The complete list of 1,584 human genes with additional information can be found in Table S1a [20] . An interactive version of the same list can be found in Table S1b [21] .
Quite often the resulting list of phenotypes associated with a specific gene may include the major disease phenotype followed by specific sub-phenotypes of the disease that contribute distinct aspects to the overall clinical disease phenotype. For example, IL13 has been associated with asthma at least 11 times as well as to the asthma sub-phenotype immediate hypersensitivity 4 times. Similarly, the gene CFH has been associated with macular degeneration at least 19 times, as well as to the endophenotype of macular degeneration, choroidal neovascularization 3 times. Although replication in genetic association studies has been widely debated [22] , consistent replication by independent groups, although sometimes with both modest risk and significance values [23] , suggests a fundamental measure of scientific validity. This is true for both candidate gene as well as GWAS studies.
In other cases, individual genes have been associated with independent but related disorders that may share fundamental biological pathways in disease etiology, such as HLA-DQB1, CTLA4, and PTPN22 as in the case of autoimmune disorders. This gene overlap emphasizes the fundamental, often step-wise biochemical role each gene plays in shared disease etiology [24] [25] [26] [27] . That is, HLA-DQB1 in antigen presentation, CTLA4 in regulation of the expansion of T cell subsets, and PTPN22 in T cell receptor signaling, all contributing to immunological aberrations and progression to clinical disease, as in rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. In other cases, the same gene has been associated with quite different clinical phenotypes, suggesting sharing of complex biological mechanisms at a more underlying level. For example, the gene CFTR, widely recognized as the cause of cystic fibrosis, has been consistently associated with pancreatitis, may be implicated in chronic rhinitis [28] , and may play a protective role in gastrointestinal disorders [29] .
Tables 5 and S2 are the mouse equivalents of the human GENE-to-Disease/Phenotype lists (tables 4 and S1 for human). These were developed from the mouse phenotype table of genes with mouse phenotype ontological codes ftp://ftp.informatics.jax.org/pub/reports/ index.html#pheno, downloaded on 4-4-08. To build tables 5 and S2, the matching phenotypic terms were exchanged for each Mammalian Phenotype code (MP:#). This resulted in the mouse GENE-to-Disease/Phenotype tables (tables 5 and S2) similar in structure to human GENE-to-Disease/Phenotype tables (tables 4 and S1). Unlike the human tables, the mouse GENE-to-Disease/ Phenotype tables come from individual mouse experimental knockout or other genetic studies. They are not based on population based epidemiological studies. They also do not have the quantitative aspect of the human tables with publication frequency counts tagged to each record. In addition, although they include a wide variety of physiological, neurological, and behavioral phenotypes, they do emphasize developmental studies and observational morphological phenotypes common in mouse knockout studies. Table 5 is a small representative subset, truncated in the number of genes (rows) and the number of Phenotype terms (columns). The complete list of 5011 mouse genes with annotated phenotypes and additional information can be found in Table S2a[ 30] . An interactive version of the same list can be found in Table S2b [31].
We can now compare these tables directly, thereby allowing gene-by-gene comparison of human disease phenotypes and mouse genetic phenotypes. Tables 6 and S3 are comparisons of the genes that overlap between the human and mouse gene lists (Table S1 and  Table S2 ) showing mouse gene symbols and their human orthologs. Table 6 is a small subset of selected gene-phenotype cross species comparisons. Even though in some cases the human studies have not been replicated, there is often a striking concordance between human disease phenotypes and mouse genetically determined phenotypes. For example, the human gene inhibin alpha (INHA) has been associated with premature ovarian failure [32] , and shows mouse phenotypes of abnormal ovarian follicle morphology, female infertility, and ovarian hemorrhage [33] , among other phenotypes relevant to human disease. Similarly, in humans the engrailed homeobox 2 gene (EN2) has been associated with autistic disorder [34] while the comparison to mouse En2 has genetic mutations involved in abnormal social integration, spatial learning, and social/consecutive interaction, among others [35] . Importantly, the few mouse studies highlighted above, and many found in the main table S3, were published after the corresponding human genetic population based epidemiological studies. Given concerns of false positives and publication bias in human genetic association studies, direct comparisons to related mouse phenotypes may provide supporting evidence that a given gene may be relevant to a specific human disease phenotype. Table S3 [36] is a full listing of the 1104 shared genes between the human disease and mouse phenotype summaries.
The second type of main summary tables are DISEASE/ PHENOTYPE-to-Gene lists. Disease/Phenotype gene summaries are essentially transposed versions of the GENE-to-Disease/Phenotype summaries (Tables S1 & S2) that allow different types of comparisons. These are non-redundant lists of phenotype keywords, MeSH disease terms in the case of human and Mammalian Phenotype Terms (MP) in the case of mouse, followed by the genes associated or annotated to those disease phenotype keywords. Table 7 shows examples of selected human disease phenotypes in each row positively associated with specific human genes for 8 major MeSH disease classes including cardiovascular, digestive system diseases, diseases of environmental origin, immune system diseases, mental disorders, nervous system diseases, nutritional and metabolic diseases, and eye diseases. Each Mesh phenotype term is followed by the number of times that a specific disease term has been positively associated with a particular gene in each row, in decreasing order. Table 7 is a small representative set, truncated in the number of disease phenotypes (rows) and the number of genes (columns). The complete list of 1,318 MeSH disease phenotype terms with additional information can be found in Table S4a[ 37] . An interactive version of the complete list can be found in Table S4b [38].
Tables 8 and S5 constitute the mouse DISEASE/PHE-NOTYPE-to-Gene summaries. Table 8 consists of selected mouse phenotypes which fall into similar general classes of the human table 7 followed by 6 representative genes that have been assigned to the appropriate phenotypic term due to a specific mouse genetic model. Unlike the human Disease/Phenotype-to- (7) BREAST NEOPLASMS (7) MYOCARDIAL (7) Celiac Disease (6) Tuberculosis, Pulmonary (5) 1493 CTLA4 Diabetes Mellitus, Type 1(28) Graves Disease (21) Thyroiditis, Autoimmune(10) Autoimmune Diseases (8) 183
AGT Hypertension (24) Coronary Disease (6) Diabetic Nephropathies (5) Myocardial Infarction (5) 1814 DRD3 Schizophrenia (24) Dyskinesia, Drug-Induced (6) Psychotic Disorders (5) Alcoholism (2) 4846 NOS3
Hypertension (20) Myocardial Infarction (18) Coronary Artery Disease (15) 
Cardiomyopathy, Dilated (2) 5468 PPARG Diabetes Mellitus, Type 2 (18) Obesity (11) Diabetes Mellitus (6) Insulin Resistance (4) 2784 GNB3
Hypertension (18) Insulin Resistance(4) Diabetes Mellitus, Type 2(3) Obesity (3) 1815 DRD4 Attention Def. Dis. with Hyperact. (17) Schizophrenia (8) Substance-Related Disorders (4) Mood Disorders (4) 1813 DRD2
Alcoholism (17) Schizophrenia (14) Personality Disorders (2) Depressive Disorder (2) 155 ADRB3
Obesity (17 Table 8 is also a small representative set, truncated in the number of disease phenotypes (rows) and the number of genes (columns). The complete list of 5,142 mouse phenotype terms with their corresponding Mammalian PhenoCode designations can be found in Table S5a[ 39] . An interactive version of the complete list can be found in Table S5b[40] .
The purpose of this project is not simply to generate lists and information. It is to provide a distillation of disease and phenotype information that can be used in dissecting the complexities of human disease and mouse biology. Now that we have generated GENE-to-disease/ phenotype summaries and DISEASE/PHENOTYPE-togene summaries for both mouse and human, they can be used for systematic analysis, comparison, and integrating of orthologous data with the goal of providing higher order interpretations of human disease and mouse genetically determined phenotypes.
Gene sets have been defined simply as groups of genes that share common biological function, chromosomal location, or regulation [41] . Gene sets are used in highthroughput systematic analysis of microarray data using a priori knowledge. Unlike previously defined gene sets based on biological pathways or differentially expressed genes [41] , GAD disease gene sets are unique in that they are composed of genes that have been previously shown to be both polymorphic and have been determined to be genetically positively associated with a specific disease phenotype in a human population based genetic association study. Similarly, Table S5a [39] the mouse DISEASE/PHENOTYPE-to-Gene list is used as a source for gene sets for mouse phenotypes (MP gene sets) comprised of unique gene based mouse genetic models. These gene set files are currently the largest set of gene set files publicly available and the only gene sets files where each gene is based on direct human or mouse genetic studies.
One aspect of common complex disease is that the development of disease and disease phenotypes quite often present along a broad spectrum of symptoms and share clinical characteristics, endo-phenotypes, or quantitative traits with closely related disorders [25] . This is evident in gene sharing, as mentioned above, and equally in the overlap of biological pathways between related disorders. Using GAD disease gene sets, Venn diagram comparisons among related disorders shows modest gene sharing. However, when gene sets are then placed into biological pathways and compared by Venn analysis, there is a marked increase in the overlap in pathways between related disorders. This was not found in gene sets from unrelated disorders. For example, major autoimmune disorders quite often share endophenotypes of lymphoproliferation, autoantibody production, and alterations in apoptosis, as well as other immune cellular and biochemical aberrations. As shown in Figure 1a , genes that have been positively associated with type 1 diabetes, rheumatoid arthritis, and Crohn's disease show a modest overlap. However, when individual gene sets are fitted into biological pathways, then compared for overlap of pathway membership, there is a striking increase in the overlap at the pathway level. This is true in a comparison of gene and pathways for type 2 diabetes, insulin resistance, and obesity as well (Figure 1b ). This pattern of major pathway overlap does not seem to occur between unrelated disorders, such as insulin resistance, rheumatoid arthritis and bipolar disorder (Figure 1c ). This disease related sharing at the pathway level suggests common regulatory mechanisms between these disorders and that the original positive associations are not necessarily due to random chance alone.
Dendrogram analysis of human disease gene sets As archival information grows, analysis of complex molecular and genetic datasets using clustering or network approaches has become increasingly more useful [13, [42] [43] [44] [45] . Therefore, in addition to comparisons between individual diseases using human and mouse gene sets, we analyzed large gene groups using dendrogram and clustering approaches based on gene sharing between gene sets. Figure 2 shows a broad based dendrogram comparison based on gene sharing between 480 GAD disease gene sets, using gene sets each containing at least 3 genes. A striking feature of this analysis is that at a coarse level, major disease groups cluster together in space demonstrating shared genes between major clinically important disease groups. Disease domains are represented by groups such as cardiovascular disorders, metabolic disorders, cancer, immune and inflammatory disorders, vision, and chemical dependency. At finer detail within a specific broader group, it becomes clear that individual diseases with overlapping 
Heart Failure ADRA2C (5) phenotypes are found close in space, such as asthma, allergic rhinitis, and atopic dermatitis. This overlap due to gene sharing recapitulates an overlap in clinical characteristics between these related disorders. Similarly, phenotypes within the metabolic group related to diabetes are closely aligned in space including; insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia. This close apposition of related disease phenotypes and sub-phenotypes at both a coarse and fine level is a consistent feature of the overall display. The human gene sets used in creating this tree diagram can be found in Table S6[ 46] . It is important to emphasize that this display and the distance relationships between diseases are calculated through an unbiased gene-sharing algorithm independent of disease phenotype labels and not as a result of an imposed logical hierarchy or an ontological annotation system. This grouping of major disease phenotypes based solely on gene sharing provides supporting evidence that the underlying disease based gene sets may have a fundamental relevance to disease and may not be reported in the literature by chance alone. Dendrogram analysis of mouse phenotypic gene sets Figure 3 is a similar dendrogram to the human tree using 1056 mouse phenotypic gene sets, using gene sets each containing at least 10 genes. This was produced using the same gene sharing algorithm as for the human gene sets in Figure 2 . As with the human dendrogram, the mouse tree displays informative groupings at both a coarse and fine level. This tree groups into major groupings nominally assigned as brain development and brain function, embryonic development, cardiovascular, reproduction, inflammation, renal function, bone development, metabolism, and skin/hair development. The identification of major groupings emphasizing developmental processes reflects the emphasis of gene knockouts and developmental models resulting in observable morphological traits and less so with regard to end stage clinical diseases as in the human dendrogram. Like the human dendrogram ( Figure 2 ) discrete major functional groupings in the mouse dendrogram suggests that individual experimental observations are not random. Fundamental complex processes such as metabolism, cardiovascular phenomena, and developmental processes are integrated by extensive sharing of related pliotropic genes. Moreover, like the human tree, fine structure in the mouse tree shows related mouse phenotypes are closely positioned in space. For example, in the metabolism major grouping, the individual phenotypes of body mass, adipose phenotypes, and weight gain are closely positioned. Similarly, in the brain function group, the behavioral phenotypes of anxiety, exploration, and responses to novel objects are found next to one another. This pattern is a fundamental feature of this tree. Like the human tree, the mouse dendrogram shown here is based solely on a gene sharing algorithm using genes assigned to individual phenotypes. It is not based on an imposed predetermined hierarchy or ontology. Importantly, unlike the human tree, the information contained in the mouse tree is derived from individual independent mouse genetic studies and phenotypic observations and not from large case controlled population based epidemiological studies. Controversial issues such as publication bias or study size which confound human genetic association studies are not as relevant here in the context of studies of experimentally determined individual mouse gene knockouts and related studies. The mouse gene sets used in creating this tree diagram can be found in Table S7[ 47] . Hierarchical clustering of human and mouse gene sets Hierarchical clustering has become a common tool in the analysis of large molecular data sets [48] allowing identification of similar patterns in a scalable fashion from the whole experiment down to a level of fine structure. To provide further evidence of disease relevance and biological content contained in both the human and mouse gene sets hierarchical clustering was performed on both human and mouse. Four hundred and eighty human gene sets were clustered producing 46 major disease clusters. In the mouse, clustering was performed on 2067 mouse phenotype gene sets, using gene sets containing at least 3 genes. This resulted in 165 major subgroups of functional phenotypic specificity. Hierarchical clustering is shown for human [Additional file 1 and Additional file 2] and for mouse [Additional file 3 and Additional file 4]. Like the human and mouse dendograms, this hierarchical clustering showed functional disease grouping at both a coarse group level and at a fine level within major phenotypic groupings. These clusters in both human and mouse falling into closely defined broad functional groups as well as closely related clinical, physiological, and developmental phenotypes demonstrates a general pattern of relevance to disease in their original underlying genetic associations. As in the dendrogram displays, this suggests that the genes nominally positively associated to these disorders, drawn from the medical literature, are not pervasively randomly assigned or due to a widespread pattern of random false positives associations.
This report describes a summary of the positive genetic associations to disease phenotypes found in the Genetic Association Database as well as a summary of mouse genetically determined phenotypes from the MGI phenotypes database. The genes and disease lists described here were derived from a broad literature mining approach. We have shown disease relevance in three distinct ways; a) in comparing individual gene lists and pathways, b) comparing between species and, c) in broad based comparative analysis utilizing complex systems approaches. Moreover, we identify disease based genes sets for 1,317 human disease phenotypes as well as 5,142 mouse experimentally determined phenotypes. These resources are the largest gene set files currently publicly available and the only gene set files derived from population based human epidemiological genetic studies and mouse genetic models of disease.
Each individual GAD disease gene set (i.e. a single disease term followed by a string of genes) or mouse phenotype gene set becomes a candidate for a number of uses and applications including: a) contributing to complex (additive, multiplicative, gene-environment) statistical models for any given disease phenotype [49] [50] [51] [52] [53] ; b) use in comparative analysis of disease between disease phenotypes; c) use in interrogating other related data types, such as microarray (see below), proteomic, or SNP data [54] [55] [56] ; and d) integration into annotation engines [57] or genome browsers Table S7 [47]. [58] or other analytical software to add disease information in comparative genomic analysis. In a sense, each individual human or mouse disease/phenotype gene set becomes a unique hypothesis, testable in a variety of ways. Increasingly, combinations of genes may have important predictive value as combinatorial biomarkers in predicting disease risk as opposed to single candidate genes [59, 60] .
In addition, in an ongoing parallel set of experiments, using a Gene Set Analysis (GSA) approach using the web tool Disease/Phenotype web-PAGE, in the analysis of orthologous microarray data (De S, Zhang Y, Garner JR, Wang SA, Becker KG: Disease and phenotype gene set analysis of disease based gene expression, unpublished), both the human and mouse disease/phenotype gene sets defined above demonstrate striking disease specificity in PAGE [61] gene set analysis of previously published microarray based gene expression studies from numerous independent laboratories in both a species specific and cross species manner. This was true when studying gene expression studies of type 2 diabetes, obesity, myocardial infarction and sepsis, among others, providing further evidence of the disease and clinical relevance of both the human and mouse gene sets.
This approach is limited in a number of ways. In particular, the GAD database compares the results of human population based epidemiological studies performed using different sample sizes, populations, statistical models, and at different times over approximately the last 16 years. In addition, the GAD database draws on association studies of broad quality with different degrees of detail provided. Although all human genetic association studies discussed here have been individually determined to be positively associated with a disease or phenotype in a peer reviewed journal, we make no assertion that any individual study is correct and we recognize the controversy in the genetics community regarding statistical and biological significance of genetic association studies. Moreover, although the GAD database contains information on polymorphism and variation, and each GAD record is fundamentally based on polymorphism, this report does not consider variation or polymorphism in the summaries shown. Likewise, mouse genetic models in many cases are weighted to gene knockouts which may not be necessarily be directly representative of multifactorial human common complex disease.
However, even with these limitations, we believe valuable insights can be gained from broad based literature assessments of the genetic contribution in human common complex disease and in mouse phenotypic biology. More importantly, this suggests greater opportunities for systematic mining and analysis of published data and in cross comparison of archival molecular databases in both human and animal models of disease with regard to genetic variation, population comparisons, and integration with many different types of orthologous information. Pneumonitis as A Consequence of (Peg)Interferon-Ribavirin Combination Therapy for Hepatitis C: a Review of the Literature Combination of peginterferon and ribavirin is the current therapy for chronic hepatitis C infection (HCV). Interstitial pneumonitis is a rare side-effect of HCV therapy and is an important cause of dose reduction or discontinuation, impairing success of antiviral therapy. We performed a review of the literature in order to present diagnostic modalities and possible treatments for pneumonitis and to offer guidelines. We searched for cases where pneumonitis as a side-effect of HCV treatment was documented. First we performed a literature search via PubMed and Web of Science interface and second we searched three drug toxicity databases. We systematically analyzed all case reports with respect to clinical manifestations, type of treatment, and outcome. A literature search revealed 19 articles, containing 25 case descriptions, while we traced 33 cases from the drug toxicity databases. Pneumonitis presented with any of the combination of fever, dyspnea, and cough and can arise with any type of (conventional or pegylated) interferon. Mortality secondary to pneumonitis was seen in 7% of cases, exclusively with peginterferon α-2b. In most cases therapy was discontinued and steroids were started. Interferon-induced pneumonitis during HCV treatment is a severe complication and should be recognized in order to prevent further pulmonary damage and/or death. Hepatitis C (HCV) persists in up to 85% of patients and may result in liver cirrhosis and hepatocellular carcinoma [1] . The combination of peginterferon and ribavirin is the mainstay of treatment. Eradication of the virus can be achieved in up to 90% of chronic HCV patients, depending on a number of host-and virus-related factors [1, 2] . Successful treatment results in resolution of hepatic necroinflammation and regression of fibrosis.
Though potentially successful, peginterferon and ribavirin are known to cause various side-effects in HCV patients. Most individuals suffer from some side-effects such as flu-like symptoms, myalgia, fatigue, gastrointestinal disturbances, psychiatric disorders, and hematological abnormalities such as anemia and leukopenia [3] [4] [5] .
These adverse effects are relatively common and can be managed with supportive care. There are also less wellknown side-effects of antiviral treatment that may hamper successful eradication of the virus. Knowledge of the extensive gamut of side-effects of combination therapy is critical for the adequate management of side-effects.
Pulmonary toxicity in patients undergoing HCV combination treatment is rare, and may include interstitial pneumonitis, sarcoidosis, pleuritis, bronchiolitis obliterans organizing pneumonia (BOOP), and exacerbation of asthma [6, 7] . Pneumonitis occurs only rarely as a side-effect of HCV combination therapy and can arise at any stage of the treatment. Most commonly it presents with cough, which is difficult to differentiate from the ubiquitous cough that may occurs as a common side-effect of combination treatment.
Interferon is the agent that is thought to be associated with pneumonitis in these patients. This paper aims to review the salient issues of pneumonitis in the setting of HCV combination treatment and aims to offer guidelines for diagnosis and treatment of pneumonitis.
A 51-year-old male was seen in August 2005 because of HCV infection. He had been using intravenous (i.v.) drugs until 1985 and his medical history revealed multiple total hip replacements and revisions because of hip necrosis attributed to i.v. drug-related blood-borne infections. Liver function tests were abnormal: alanine aminotransferase (ALAT) 237 IU/L (normal \45 IU/L), aspartate aminotransferase (ASAT) 235 IU/L (normal \50 IU/L), c-glutamyl transpeptidase 241 IU/L (normal \55 IU/L), and bilirubin 19 lmol/L (normal \10 lmol/L). The patient was infected with genotype 3 HCV virus with high viral load ([5 9 10 5 IU/ml). A liver biopsy was performed and showed moderate necroinflammation with portoportal fibrosis (METAVIR; grade A2, score F2). In view of this advanced stage we offered the patient a 24-week treatment for HCV consisting of peginterferon a-2b (Pegintron Ò ; 150 lg s.c. weekly) in combination with ribavirin (Rebetol Ò ; 400 mg twice daily).
Four weeks after starting treatment he consulted us for a dry cough. Chest X-ray at that time showed no abnormalities. Although dry, nonproductive cough is usually caused by ribavirin, it typically clears after stopping the drug. The dry cough, however, persisted after end of treatment, being the reason to consult a chest physician. Physical examination revealed bibasal crepitations. A new chest radiograph showed bilaterally a diffuse, interstitial pattern ( Fig. 1a) and chest high-resolution computed tomography (HRCT) demonstrated bilateral ground-glass opacities in central and upper zones (Fig. 1b) . Pulmonary function tests indicated a restriction and a diminished diffusing capacity. Maximal incremental cycle ergometry showed decreased breathing reserve (0 l/min), high breathing frequency (65/ min), and exercise-induced hypoxemia caused by an oxygen uptake problem, compatible with interstitial pulmonary disease. Results from bronchoalveolar lavage supported a diagnosis of drug-induced interstitial pneumonitis (cell count 1.68 9 10 9 /L, 73% lymphocytes, CD4/CD8 ratio 0.5). The results of cultures and stains of the bronchoalveolar lavage specimens were negative for bacteria, fungi, acid-fast bacteria, cytomegalovirus, herpes simplex virus, and malignant cells. There were no signs of infection (blood culture was negative).
As after 6 weeks the cough persisted, steroids up to 40 mg daily were started, which resulted in slow amelioration of symptoms, normalization of pulmonary function tests, and disappearance of the ground-glass effect on HRCT. One year after end of treatment the patient is well, without evidence of recurrence of hepatitis C.
First we performed a literature search for articles on pneumonitis as a side-effect of HCV treatment in order to obtain a comprehensive overview of this particular side-effect. We took advantage of the PubMed and Web of Science interface (http://www.ncbi.nlm.entrez and http://apps.isiknowledge. com) and searched with the following keywords: interferon, interstitial, pneumonitis, and hepatitis C, for the period 1990-2008. Articles written in English, French or German were included in the analysis. Furthermore, we searched the three drug toxicity databases. We included the Netherlands Pharmacovigilance Center (www.lareb.nl) database and the Fig. 1 a Chest X-ray with diffuse, bilateral interstitial pattern. b High-resolution computer tomography image showing bilateral ground-glass opacities in central zone Drug-Induced Lung Diseases database (www.pneumotox. com), and lastly we performed an exhaustive search of the database of the World Health Organization (WHO) via the Uppsala monitoring center (http://www.who-umc.org). We used as keywords interferon and pneumonitis. The WHO database is a collection of data about adverse drug reactions from around the world, especially from countries that are members of the WHO, and the generation of signals of drugs which might possibly have problematic side-effects.
Inclusion criteria were cases that developed interstitial pneumonitis in the setting of HCV treatment, written in English, French or German. Exclusion criteria were other pulmonary diseases, e.g., sarcoidosis, exacerbation of asthma, BOOP, acute respiratory distress syndrome (ARDS), infectious pneumonia, liver transplanted patients, HIV co-infected patients, and interferon beta treatment. Patients who developed interstitial pneumonitis during interferon therapy with a disease other then hepatitis C were also excluded. The references of the traced articles were scrutinized for additional articles.
Initial analysis yielded a total of 291 articles. The abstracts of this set of articles were scrutinized for cases that developed (interstitial) pneumonitis in the setting of HCV treatment. Subsequently data were retrieved with special attention to the following items: demographics (ethnicity, gender, and age), dosage and type of (peg)interferon, concomitant use of ribavirin (dosage), symptoms, interstitial pneumonitis, onset, symptoms, and outcome on follow-up.
We retrieved 19 articles, which contained detailed clinical descriptions of 25 cases of interstitial pneumonitis during or after HCV treatment. Our own case was added to the analysis. Articles were published in the time frame 1988-2008.
A total of 25 cases, 12 (48%) males and 13 (52%) females, with mean age of 55.2 years, developed an interstitial pneumonitis. Ethnicity was mentioned in seven cases, being Caucasian (five) and Asian (two). Tables 1 and 2 present the demographic characteristics of all cases.
Fourteen patients were treated with conventional interferon, while 11 patients developed pneumonitis during or after treatment with peginterferon and ribavirin.
Interferon a-2b had been used in eight patients (62%), while interferon a-2a and lymphoblastoid interferon (6 MU q.d. 9 10 days, then 6 MU t.i.w.) was used in one single case. Interferon type was not specified in three cases. Dosages regimes varied with type of interferon and are presented in Table 2 . Onset of symptoms of interstitial pneumonitis ranged from 20 days to 23 weeks of therapy. Symptoms included cough, dyspnea, and fever.
Peginterferon a-2b was used in eight patients (67%), while peginterferon a-2a was used in four patients (33%). Consensus interferon (Infergen, 9 lg q.d.) was given in a single case. Dosage regimes varied from 100 to 180 lg/week, depending on type of peginterferon, and from 400 to 1,200 mg/day for ribavirin. In two cases Amantadine was added to the treatment regimen.
Onset of symptoms of interstitial pneumonitis ranged from 2 to 12 weeks of therapy.
In all interferon cases therapy was discontinued, and five of these cases resolved without treatment [8] [9] [10] . Eight patients needed to be treated with various steroids. Interstitial pneumonitis in a 72-year-old female patient was treated with 30 mg/day prednisolone, and she was then maintained on a regimen of intermittent pulse therapy with 100 mg/day; a 56-year-old male patient was started with 2 g/day methylprednisolone for 3 days and 40 mg/day prednisolone for 2 days [10] . Prednisolone (60 mg/day) and 100 mg/day azathioprine were given after a relapse of interstitial pneumonitis that was initially treated with 30 mg/day prednisolone [11] .
In one case of therapy with methylprednisolone pulse therapy, 1 g/day i.v. for 3 days was given, followed by 40 mg/day oral prednisolone twice over 2 weeks; the other cases in that article received 50 mg/day oral prednisone [12] . Therapy for one case was not specified [13] .
In eight cases combination therapy was discontinued and steroid therapy was initiated. While dosage and length of steroid treatment was highly variable, most authors started at a relatively high dosage. Intravenous therapy with 180 mg/day methylprednisolone was started in one case [14] , while other authors started with 125 mg/day prednisolone [15, 16] . Others started patients on oral steroid therapy (40 mg) [6, 17, this study], while two authors initiated inhalation steroids [18, 19] .
Interstitial lung disease resolved in one case with oral antibiotics, given under presumptive diagnosis of community-acquired pneumonia. She recovered without sequel [7] . In four combination-therapy cases the dosage of steroid therapy was not defined [20] [21] [22] [23] .
We identified 60 pneumonitis cases in association with (peg)interferon a from the WHO database. In 33 cases the indication of (peg)interferon (and ribavirin) combination treatment was HCV. There were 16 (48%) males and 15 (45%) females in this cohort, with mean age of 53 years. We failed to retrieve cases that met the inclusion criteria from the Netherlands Pharmacovigilance Center and Drug-Induced Lung Diseases databases. Interferon monotherapy was used in three (9%) patients. Interferon a-2b in combination with ribavirin was used in eight patients (24%), while interferon a-2a in combination with ribavirin was used in a single case.
Peginterferon a-2b in combination with ribavirin had been used in 6 patients (18%), while in 13 patients (39%) combination therapy with peginterferon a-2a was used. In two cases type of interferon and ribavirin was not described.
Dosages regimes varied from 80 to 180 lg/week, depending on type of peginterferon, in combination with ribavirin (800-1,200 mg/day). Onset of symptoms of interstitial pneumonitis ranged from 23 days to 10 months of therapy. Four patients recovered and one patient died after drug-induced pneumonitis; outcomes of the remaining patients were not described. There was no description on treatment regimen for pneumonitis.
Collectively, the cohort consisted of 25 patients from literature cohort and 33 patients from the drug toxicity database. Four patients (7%) from these two cohorts died following development of interstitial pneumonitis. All patients had been treated with peginterferon a-2b.
Death in one case was due to multiple organ failure [14] , in another case due to cerebral edema [15] , and in one case because of development of acute cholestatic hepatitis [16] . The cause of death of one case from the WHO database was not described.
Interstitial pneumonitis occurs only rarely as a side-effect of HCV combination treatment and often leads to discontinuation of therapy, which has great implications for patients. In this article we presented our case of pneumonitis during combination therapy and performed a review in order to generate guidelines to manage symptoms and treatment. Given the paucity of reports with interstitial pneumonitis after ribavirin monotherapy, we suspect that interferon is the culprit. The most common presenting symptom of pneumonitis is any combination of dry cough, dyspnea, fever, and fine inspiratory crackles noted on examination. Hemoptysis, wheezing, and signs of consolidation are rare. Onset of pneumonitis can be at any stage of HCV treatment, supporting the idiosyncratic nature of this side-effect.
Chest radiographs usually show bilateral patchy infiltrates or opacifications, and thin-section CT scans show bilateral patchy consolidation as well as ground-glass attenuation [24] .
In most cases, symptoms of pneumonitis are reversible after cessation of treatment with (peg)interferon and ribavirin, again in support of drug-induced interstitial pneumonitis.
There is no consensus regarding treatment of interstitial pneumonitis induced by (peg)interferon and ribavirin. Upon review of the literature, three options are possible: first, to stop combination treatment of HCV and wait until the disease resolves, which was done in a limited number of cases [7] [8] [9] [10] ; second, to give steroids, although dosage and route of administration regimes vary widely [6, 10-18, this study]; and thirdly, in therapy-resistant or relapsing cases, adding azathioprine to steroids may be beneficial in order to resolve the interstitial pneumonitis [11] .
The relatively high mortality rate in our series suggests that a more aggressive approach is warranted, and favors the early administration of steroids.
One remarkable finding is that mortality occurred exclusively in patients with peginterferon-induced pneumonitis in comparison with conventional interferon (7% versus 0%). The reason for this is unclear, but (peg)interferon has been associated with other pulmonary toxicity such as sarcoidosis, pleuritis, BOOP, and exacerbation of asthma.
Patients who died in the combination-therapy group were relatively young and had no relevant pulmonary or other severe diseases in their medical history. The doses of peginterferon and ribavirin varied but ranged within limits offered by treatment guidelines [25] .
The mortality, only observed in patients treated with peginterferon and ribavirin combination therapy, raises the issue of whether the peg molecule increases the severity of pneumonitis once it arises. Interferon toxicity is generally dose and duration dependent [26] , which leads us to speculate that pulmonary toxicity may occur more severely with long-acting peginterferon; however, we saw no effect of dosage on the occurrence of pneumonitis. Patients died due to different causes (e.g., hypoxia-induced cerebral edema, acute cholestatic hepatitis, and multi-organ failure), all were induced by complications after initially interstitial pneumonitis. One alternative explanation for the increased mortality might be that all patients with peginterferon were also treated with ribavirin, although ribavirin per se is not associated with pulmonary toxicity.
Our data suggest no significant difference between interferon a-2a or 2b, which suggest it is not due to the interferon molecule. In most interferon monotherapy cases of interstitial pneumonitis in HCV treatment in Japan the use of Sho-Saiko-to, a herbal medicine, led to pneumonitis. Sho-Saiko-to has been approved by the Japanese Ministry of Health and Welfare and has often been administered in chronic viral liver disease [27] .
The mechanism of this side-effect, probably caused by interferon, remains unclear and several pathophysiological mechanisms have been proposed, centering on the known immunomodulatory activity of interferon [28] . Interferon has direct antiviral and immunomodulatory effect, including cytokine reduction, increased natural killer cell function, and enhanced cellular expression of major histocompatibility class 1 antigens [28] [29] [30] . It is plausible that interferon triggers a lung-specific immune-mediated response resulting in interstitial pneumonitis, similar to other autoimmune diseases.
Failure to recognize interferon-associated pulmonary toxicity may result in persistence of pulmonary damage. Mortality was seen only in patients treated with pegylated interferon combination therapy. Therefore, clinicians should be aware of development of interstitial pneumonitis in chronic hepatitis C patients who develop pulmonary symptoms such as cough or dyspnea. The threshold for obtaining chest X-ray or pulmonary HRCT scan in these patients should be low.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. HIV-related restrictions on entry, residence and stay in the WHO European Region: a survey BACKGROUND: Back in 1987, the World Health Organization (WHO) concluded that the screening of international travellers was an ineffective way to prevent the spread of HIV. However, some countries still restrict the entrance and/or residency of foreigners with an HIV infection. HIV-related travel restrictions have serious implications for individual and public health, and violate internationally recognized human rights. In this study, we reviewed the current situation regarding HIV-related travel restrictions in the 53 countries of the WHO European Region. METHODS: We retrieved the country-specific information chiefly from the Global Database on HIV Related Travel Restrictions at hivtravel.org. We simplified and standardized the database information to enable us to create an overview and compare countries. Where data was outdated, unclear or contradictory, we contacted WHO HIV focal points in the countries or appropriate non-governmental organizations. The United States Bureau of Consular Affairs website was also used to confirm and complement these data. RESULTS: Our review revealed that there are no entry restrictions for people living with HIV in 51 countries in the WHO European Region. In 11 countries, foreigners living with HIV applying for long-term stays will not be granted a visa. These countries are: Andorra, Armenia, Cyprus (denies access for non-European Union citizens), Hungary, Kazakhstan, Moldova, the Russian Federation, Tajikistan, Turkmenistan, Ukraine and Uzbekistan. In Uzbekistan, an HIV-positive foreigner cannot even enter the country, and in Georgia, we were not able to determine whether there were any HIV-related travel restrictions due to a lack of information. CONCLUSIONS: In 32% of the countries in the European Region, either there are some kind of HIV-related travel restrictions or we were unable to determine if such restrictions are in force. Most of these countries defend restrictions as being justified by public health concerns. However, there is no evidence that denying HIV-positive foreigners access to a country is effective in protecting public health. Governments should revise legislation on HIV-related travel restrictions. In the meantime, a joint effort is needed to draw attention to the continuing discrimination and stigmatization of people living with HIV that takes place in those European Region countries where such laws and policies are still in force. We read the article, "Fear of foreigners: HIV-related restrictions on entry, stay and residence" [1] , in this journal with great interest. In their contribution to the debate over HIV-related travel restrictions, Amon and Todrys stress the urgency on this issue, which affects not only the lives of people living with HIV (PLHIV) all over the world, but also the wellbeing of the communities in which they live. HIV-related travel restrictions not only violate the fundamental rights of PLHIV, but they also impede HIV prevention, care and treatment efforts among all people.
The United Nations Human Rights Committee has stated, "Liberty of movement is an indispensable condition for the free development of a person" [2] . Earlier, the Office of the High Commissioner for Human Rights stated that:
The [International] Covenant [on Civil and Political Rights] does not recognize the right of aliens to enter or reside in the territory of a State party. It is in principle a matter for the State to decide who it will admit to its territory. However, in certain circumstances an alien may enjoy the protection of the Covenant even in relation to entry or residence, for example, when considerations of non-discrimination, prohibition of inhuman treatment and respect for family life arise [3] .
Governments do, of course, have the right to control entry to their borders and have a certain margin of appreciation to justify differential treatment compatible with international human rights law. But the measures must pursue a legitimate aim and need to be proportional to the achievement of this aim [4] .
Back in 1987, the World Health Organization (WHO) concluded that the screening of international travellers was an ineffective way to prevent the spread of HIV [5] . In 2002, Member States of the WHO European Region resolved "to develop a supportive social and legal environment for groups at risk, especially sex workers, and for people living with HIV/AIDS and to fight social and legal exclusion, including travel restrictions" [6] .
Since then, travel restrictions connected with communicable diseases in general and HIV in particular have often been discussed [7] [8] [9] , including recently in conjunction with the 2009 outbreak of influenza virus A (H1N1). Together with international organizations, such as the International AIDS Society (IAS) [10] , the International Organization for Migration and the Joint United Nations Programme on HIV/AIDS (UNAIDS) [11] , Amon and Todrys emphasize how HIV-related travel restrictions have serious implications for individual and public health and violate internationally recognized human rights.
This important discussion prompted us to review the current situation in the 53 countries of the WHO European Region, given that restrictions on entry, residence and stay affect a wide range of PLHIV, including not only students and employees, but also members of vulnerable groups, such as refugees, asylum seekers and other migrants.
In this study, which we carried out in April and May 2009, our concern was to map formal entry and residence restrictions that required an HIV test or a medical certificate of HIV status. It should be noted that in practice, however, some of the countries did not apply the rules that were legally valid at this time. We also reviewed whether people can be denied entry when applying for long-term stay (but not residence) or be deported if authorities obtain evidence of HIV infection.
To obtain a valid, up-to-date overview of HIV-related travel restrictions in the European Region, we collected data from a variety of sources. We retrieved the information chiefly from the Global Database on HIV Related Travel Restrictions at hivtravel.org [12] , an initiative of the German AIDS Federation, the European AIDS Treatment Group (EATG) and the IAS. The information in this database is based on replies to a structured self-administered questionnaire from German embassies abroad and foreign embassies in Germany between November 2007 and June 2008.
We simplified and standardized the database information to enable us to create an overview and compare countries. Where data was outdated, unclear or contradictory, we searched the websites of foreign ministries in the countries and contacted WHO HIV focal points in the countries or appropriate non-governmental organizations (NGOs), such as the Eurasian Harm Reduction Network and the Hungarian Civil Liberties Union.
We also used the United States Bureau of Consular Affairs website [13] to confirm and complement these data. Most of the information provided by the focal points and NGOs was clear, sufficient and based on national laws and regulations. However, in some instances, the information was vague, and several communications were sometimes necessary to clarify unresolved questions.
For 11 of the 53 countries (Armenia, Belarus, Bulgaria, Cyprus, Georgia, Hungary, Israel, Moldova, Tajikistan, Ukraine and Uzbekistan), publicly available information did not provide a sufficient or clear picture of HIVrelated travel restrictions. In these cases, we contacted focal points and NGOs, receiving replies from every country except Israel.
The resulting information and our initial review of the hivtravel.org database revealed that there are no entry restrictions for PLHIV in 51 countries (see Table S1 , Additional file 1 and Figure 1 ). In Uzbekistan, however, the law mandates that visitors carry a certificate attesting that they are not infected with HIV. Foreigners from countries requiring visas to enter or stay in Uzbekistan will not be issued a visa to enter the country if they are found to be HIV positive. In Georgia, the situation for PLHIV wishing to enter the country is uncertain due to unclear information. In 36 countries, there are also no HIV-related restrictions for long-term visits (see Figure 2 ). In Georgia, the policy on long-term visits is unclear. In eight countries (Belarus, Moldova, Poland, the Russian Federation, Tajikistan, Turkmenistan, Ukraine and Uzbekistan), an HIV test is required for all foreigners wishing to stay for more than three months. In three of these countries (Republic of Moldova, the Russian Federation and Turkmenistan), this requirement also applies to students and employees. In the Russian Federation, an HIV test is not required for citizens of countries in the Commonwealth of Independent States, who do not need visas for longterm stays.
Andorra will not grant residency or work permits to PLHIV (See Figure 3) . In Hungary, an HIV test is required of all foreigners wishing to stay for more than one year. In Kazakhstan, an HIV test is required for foreigners staying for more than 30 days. In Cyprus, people who are not citizens of the European Union must present an HIV test to apply for a work or study permit, which will be denied if the test is positive. In Slovakia, an HIV test is also required for foreigners applying for residence or a work permit. In the German state of Bavaria, an HIV test can be required for people staying for more than 180 days, while in the states of Saxony and New Brandenburg, there is mandatory HIV testing for asylum seekers.
In Armenia, the situation for long-term visitors is complex. A negative HIV certificate is required from all foreigners applying for visas. Until 14 July 2009, foreign PLHIV already in the country were subject to deportation. On that date, a new law came into force, specifying that foreigners would not be deported if found to be HIV positive. Yet a foreigner applying for a visa still has to present a negative HIV test. Armenia is working to change these regulations.
And finally, while we did not find sufficient information on requirements for long-term stays in Israel, there are indications that foreigners in general do not need to Of the 17 countries requiring an HIV test or certificate for applying for long-term stays, 11 countries (69%) will deny a foreigner holding a positive HIV test entry into the country. In addition to Cyprus, which denies access to non-EU citizens, these countries are Andorra, Armenia, Hungary, Kazakhstan, Moldova, the Russian Federation (for citizens outside the Commonwealth of Independent States), Tajikistan, Turkmenistan, Ukraine and Uzbekistan.
Our research shows that only 36 out of 53 countries have no travel restrictions of any kind for PLHIV. This means that in 32% of the countries in the European Region, either there are some kind of HIV-related travel restrictions (as defined in this paper) or we were unable to determine if such restrictions are in force.
Although most countries with HIV-related travel restrictions defend them as being justified by public health concerns, the WHO Regional Office for Europe has explicitly rejected this claim [6] . Not only do HIVrelated travel restrictions tend to be ineffective and lead to a false sense of protection -a country's nationals can just as easily contract the virus abroad and spread it at home, for example -but they also contribute to and reinforce the discrimination and stigmatization to which PLHIV are subjected. Further, people facing restrictive measures at entry may hide their status and avoid HIV testing and even health care services in general. Further, the European Union HIV/AIDS Civil Society Forum has called for the elimination of all HIV-related travel restrictions in Europe by 2010 [14] .
The Office of the United Nations High Commissioner for Human Rights and UNAIDS, for example, have unequivocally stated that "any restrictions on these rights [to liberty of movement and choice of residence] based on suspected or real HIV status alone, including HIV screening of international travelers, are discriminatory and cannot be justified by public health concerns" [15] because while HIV is infectious, it cannot be transmitted through casual contact [16] . Those countries without HIV-related entry, stay, and residence restrictions have not reported any negative public health consequences [17] .
Additional considerations arise with respect to travel within the 27 countries of the European Union because free movement of people within the EU is one of its founding principles, a principle acknowledged not only in its founding and subsequent treaties, but also in the European Convention of Human Rights. For example, Council Directive 2004/38/EC [18] states that:
Without prejudice to the provisions on travel documents applicable to national border controls, all Union citizens with a valid identity card or passport ... shall have the right to leave the territory of a Member State to travel to another Member State [Article 4.1].
And similarly:
Without prejudice to the provisions on travel documents applicable to national border controls, For example, travel restrictions can be used to limit the spread of highly contagious diseases, such as cholera or acute respiratory syndrome (SARS), but such measures tend to be short-term and are most likely not very effective. Even in these cases, authorities must still consider human rights and the broad social, economic and public health consequences of initiating travel restrictions of any kind.
In general, WHO does not support travel restrictions in relation to communicable diseases, and the recent case of influenza A (H1N1) was no exception [19] . According to the International Health Regulations [20] , a binding document signed by all WHO Member States, national health measures for travellers must not be more restrictive of international traffic, or more invasive or intrusive to the individual, than available alternatives that provide an appropriate level of health protection. If such measures are implemented, they should be justified by scientific principles, available scientific evidence or WHO advice. In the case of HIV, there is no evidence that denying HIV-positive foreigners access to a country is effective in protecting public health.
In contrast to HIV, the highly contagious diseases that we have mentioned have short incubation periods and are transmitted through casual contact. While HIV transmission is mostly due to risk behaviours like sharing needles or unsafe sex, these diseases are transmitted much more readily, through droplets in the air or contaminated food or water. In the light of these differences, as well as the potential for discrimination and stigmatization, the public health justification for HIV-related travel restrictions is inadequate and even irrational. Triple Combination of Amantadine, Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21(st) century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza. Globally, influenza viruses cause substantial morbidity and mortality in humans and economic dislocation in afflicted nations. Each year in the United States, seasonal influenza virus infection result in an estimated 36,000 deaths and 200,000 hospitalizations [1] . Antiviral drugs are an important means to mitigate the impact of the yearly influenza epidemics and potential pandemics. Currently, two classes of antiviral drugs have been approved for the prevention and treatment of influenza infection -the M2 channel inhibitors (aminoadamantanes; amantadine and rimantadine) and the neuraminidase inhibitors (NAIs; oseltamivir and zanamivir). However, the prevalence of drug-resistant strains, which has been reported for both classes of antiviral drugs for seasonal influenza [2, 3] , could undermine their clinical benefit when utilized as monotherapy. Indeed, in 2009, the Centers for Disease Control and Prevention (CDC) reported that 100% of the seasonal H3N2 virus isolate tested were resistant to the adamantanes, and 99.6% of the seasonal H1N1 viruses tested were resistant to oseltamivir [4] .
In April, 2009, a novel H1N1 virus of swine-origin capable of human-to-human transmission likely emerged in Mexico and was first isolated from patients enrolled in two separate surveillance activities in Southern California [5] . The emergence and spread of 2009 H1N1 to over 168 countries has led U.S. officials [6] and the World Health Organization (WHO) [7] to declare a public health emergency; on June 11, 2009 the WHO raised the influenza pandemic alert from phase 5 to phase 6, the official declaration of a pandemic [8] . Early published results from the CDC showed that 2009 H1N1 bears the amantadine-resistance associated S31N mutation in the M2 ion channel, but remains susceptible to oseltamivir and zanamivir [9] . More recently, however, the CDC has reported that ten 2009 H1N1 isolates tested in the United States have been found to be resistant to oseltamivir, raising the concern that dually resistant viruses may become prevalent [4] .
In an earlier study, we explored the in vitro antiviral activity and synergy of single, double, and triple combinations of amantadine, ribavirin and oseltamivir against a panel of influenza A viruses that were susceptible to these drugs [10] . Our hypothesis was that a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action, and which act at different stages in the viral life cycle, could result in synergistic antiviral activity. Our results showed that these drugs did indeed act synergistically, with the triple combination showing significantly greater synergy than any of the double combinations evaluated. Furthermore, we found that the synergy of the TCAD regimen was maintained across multiple seasonal and avian influenza A strains, including the three major subtypes -A/H1N1, A/H3N2, and the avian A/H5N1 -that currently cause significant morbidity and mortality in humans.
In this study, we sought to evaluate the activity and synergy of the TCAD regimen against influenza viruses which were resistant to amantadine or oseltamivir. Our data showed that against amantadine-resistant viruses -both seasonal and 2009 H1N1and oseltamivir-resistant seasonal viruses, the TCAD regimen was strongly synergistic, and the synergy of the TCAD regimen was greater than the synergy of any double combination. Surprisingly, we found that amantadine and oseltamivir contributed to the synergy of the TCAD regimen at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our findings highlight the utility of the TCAD regimen as a potential approach to address the current limitations of antiviral potency and drug resistance, and as a viable broad-spectrum therapeutic option for serious influenza virus infection.
Oseltamivir carboxylate, the active metabolite of oseltamivir, was obtained from Charnwood Molecular (Loughborough, U.K.) through synthesis via the NBoc-protected acid from oseltamivir phosphate. Amantadine was obtained from Moehs Catalana (Barcelona, Spain). Ribavirin and rimantadine were purchased from Sigma-Aldrich (St. Louis, MO). Peramivir was obtained from Jubilant Chemsys LTD (Uttar Pradesh, India) as free base through NBoc synthesis, and purity was confirmed using NMR and chiral HPLC. Zanamivir was obtained from Haorui Pharma-Chem, Inc. (Edison, NJ). 
Cells were passaged in minimal essential medium containing 5% fetal bovine serum (Hyclone Laboratories, Logan, UT). During antiviral evaluations, the serum was removed and the medium was supplemented with gentamicin (50 mg/mL), porcine trypsin (10 units/mL) and EDTA (1 mg/mL).
To obtain single agent concentration-response curves, individual drugs were added to MDCK cells in 96-well microplates (8610 4 cells/well) using three wells for each concentration used. The compounds were added at the following concentrations: oseltamivir carboxylate, zanamivir, and peramivir at 0, 0.000032, 0.0001, 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0 and 100 mg/mL; amantadine, rimantadine, and ribavirin at 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10, 32 and 100 mg/mL. Untreated wells of infected cells (virus controls), uninfected cells (cell controls), and drug cytotoxicity controls (cells and drugs only, using the same dilution range for each drug as the test wells) were included on each test plate. At three days after infection, the virus control wells exhibited 100% cytopathology. The 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (TC 50 ) was determined for each drug as outlined below.
For double and triple combination studies, each drug was tested in triplicate at five or six concentrations (including no drug), in which the high concentration for each drug was set to approximate the EC 50 of the drug as a single agent. The concentrations of each drug used in double and triple combinations against each virus are provided in Table S1 . The cytotoxicity of the double and triple drug combinations were determined using the same experimental format in three separate experiments, and using the same concentration ranges as outlined in Table S1 .
The extent of viral cytopathology in each well was determined by staining with Neutral Red (NR) as detailed elsewhere [11] . Briefly, the cells were stained with 0.011% NR diluted in MEM to determine cell viability. Two hours later the plates were processed for quantification of NR uptake into viable cells. The amount of NR taken up by cells was determined spectrophotometrically.
EC 50 , TC 50 , and Synergy Calculations EC 50 , TC 50 , and synergy calculations were done as described previously [10] . Briefly, EC 50 and TC 50 calculations for single agents were made by normalizing the NR data for each well against the cell and virus controls, which was assumed to represent 100% cell viability and 0% cell viability, respectively. Normalized data were plotted as percent cell viability versus compound concentration. The data points were then fitted using fourparameter curve fitting in Graphpad Prism (Graphpad Software, La Jolla, CA) to derive the EC 50 and TC 50 . Statistical comparisons between best-fit EC 50 values for any two curves were performed in Prism using the extra sum-of-squares F test; differences in EC 50 values between two curves with a P-value of ,0.05 were considered significant.
Synergy was calculated using the MacSynergy II software developed by Prichard and Shipman, which was modified to accommodate a three-drug combination [12] and is similar to that reported previously describing this approach [13] . The theoretical additive interactions were calculated from the concentrationresponse curves of each drug as a single agent. This calculated additive surface was then subtracted from the observed, experimental surface to reveal regions that deviate from the calculated additive effects. Purely additive interactions are represented graphically as areas in grey, indicating that they do not differ from the calculated additive effects. Synergistic interactions result in greater inhibition than the calculated inhibition, and are represented as areas in blue. Conversely, antagonism is represented as areas in red. Synergy plots are shown as the percent inhibition above or below expected (calculated additive inhibition), and are presented as the mean of three replicates at a level of 95% confidence, which eliminates insignificant deviations from the additive surface.
Synergy volume for each double and triple combination was also calculated, which represents the sum of the synergy or antagonism across all concentrations of a combination. Synergy volumes are presented as a quantitative measure of the overall interaction of the drugs within a combination. As determined by cytopathic effect (Neutral Red assay), synergy volumes .100 mg/ mL 2 % for double combinations or .100 mg/mL 3 % for triple combinations are considered to be strongly synergistic. Conversely, combinations with synergy volumes ,2100 mg/mL 2 % or mg/ mL 3 % are considered to be strongly antagonistic.
The synergy of the cytotoxic effects of double and triple drug combinations were calculated in a similar manner.
Between three and nine independent experiments were conducted using identical dosing ranges for amantadine, ribavirin, and oseltamivir carboxylate against each virus. The experiments used a 3-replicate plate format, for a total of 9 to 27 observations for each condition. The data from the independent experiments were merged and subjected to statistical analysis using the random effects model. The raw data were imported into the program R and were normalized to the virus and cell controls as described above, and synergy (percent inhibition above calculated) is calculated as above. For synergy volume estimates, random effects models were applied to the data to account for variation between replicate measures within experiments. Therefore, the models took the form as given by:
where Y ij is the measure of the j th replicate in experiment i; b 0 is the grand mean; u 0i ,N(0,t 2 ); and e ij ,N(0,s 2 ). As the observations in the data are not independent, this model allowed for proper estimation of standard errors. The standard errors were then used to determine the statistical significance of synergy volume.
The inhibitory quotient (IQ) is defined herein as the ratio of the expected average total (free and protein-bound) plasma concentration (C ave ) of each drug at recommended dosage to the EC 50 (C ave /EC 50 ). Thus, an IQ of 1 or greater means that the achievable total plasma concentration of the drug is equal to or greater than the in vitro 50% effective concentration, and the higher the IQ translates to a greater predicted efficacy. The C ave of each drug was determined by pharmacokinetic modeling using a non-compartmental model in the progam WinNonLin. The recommended doses, along with the pharmacokinetic parameters, used for modeling were obtained from the package inserts for amantadine [14] , oseltamivir [15] , and ribavirin [16] , and the references therein. For ribavirin, since the plasma concentration does not achieve steady-state until ,4 weeks after the start of dosing, the C ave was determined for the first 10-day window. Based on these parameters, the expected C ave for amantadine was determined to be 0.43 mg/mL based on the recommended dosage of 100 mg twice daily for the treatment of influenza infection; the expected C ave for oseltamivir was determined to be 0.3 mg/mL based on the recommended dosage of 75 mg twice daily for the treatment of influenza infection; and the expected C ave of for ribavirin was determined to be 1.3 mg/mL after 10 days of treatment based on the recommended dosage of 600 mg twice daily for the treatment of hepatitis C infection. To determine the IQ of the triple combination, amantadine, ribavirin, and oseltamivir carboxylate were tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. The ratio of drugs in the TCAD regimen was based on the expected C ave of each drug. A dilution curve of TCAD regimen was created by first preparing a solution of all three drugs at 100-fold the C ave of each drug (43 mg/ mL amantadine, 30 mg/mL oseltamivir carboxylate, and 130 mg/ mL ribavirin), and then serially diluting this solution in 0.5-log 10 increments. In this manner, the EC 50 of TCAD regimen as a fixed dose combination was determined as ratio of the C ave and expressed in units of fold-C ave .
The susceptibility of three 2009 H1N1 influenza strains -A/ California/04/09 (CA04), A/California/05/09 (CA05) and A/ California/10/09 (CA10) -to each of six antiviral drugs (amantadine, rimantadine, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin) as single agents was determined by measuring the inhibition of virus-induced CPE in MDCK cells. Against the three strains, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin produced concentration-dependent inhibition of cytopathic effect (data not shown). Amantadine was active only at higher concentrations (EC 50 of 16-20 mg/mL; 85-106 mM) (Table S2) , which represents a .250-fold reduction in susceptibility compared to the published values for a wild-type virus [17] . Rimantadine did not produce inhibition up to the 50% cytotoxic concentration (EC 50 .12 mg/mL; .55 mM). The EC 50 values for the six drugs as single agents against all three strains are summarized in Table S2 , and confirm that the three virus strains remain susceptible to oseltamivir carboxylate, zanamivir, peramivir, and ribavirin. These results are consistent with the results previously published that demonstrated that 2009 H1N1 contained a mutation (S31N) in the M2 channel that has been associated with resistance to adamantanes, but remained susceptible to NAIs [18] .
We next assessed the synergistic activity of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate over a range of concentrations of each drug against each 2009 H1N1 isolate. A quantitative measure of the total synergy (or antagonism) of a drug combination can be expressed in terms of synergy volumes, which represents the cumulative synergy and antagonism across all concentrations for all the drugs in a combination. Based on the empirically determined criterion of synergy volume .100 mg/mL 2 % using the lower confidence interval, all three double combinations were additive against the 2009 H1N1viruses (Table 1) . By contrast, the TCAD regimen was synergistic against all three viruses over multiple concentrations of all three drugs, with the synergy occurring at 0.1 mg/mL and above for amantadine, 0.32 mg/mL and above for ribavirin, and 0.0032 mg/mL and above for oseltamivir carboxylate (Table 1) . These data show that the synergy of the TCAD regimen occurred at clinically achievable concentrations for all three drugs, given that the expected average plasma concentrations based on the recommended doses are 0.43 mg/mL for amantadine, 1.3 mg/mL for ribavirin, and 0.3 mg/mL for oseltamivir carboxylate. Furthermore, the synergy of the TCAD regimen was greater than the synergy of any double combination tested for all three 2009 H1N1 strains. Figure 1B -1D shows the synergy of the TCAD regiment as a function of increasing concentrations of amantadine, ribavirin, or oseltamivir carboxylate as the third drug, representing the contribution of each drug to the synergy of the double combination without the third drug. These data reveal that the addition of each drug as the third drug to the double combinations resulted in a concentration-dependent increase in synergy, indicative that each drug contributed to the synergy of the TCAD regimen.
Importantly, despite the fact that amantadine had no significant antiviral activity as a single agent below 3.2 mg/mL (data not shown), we find that the amantadine contributed to the activity of the TCAD regimen at clinically achievable concentrations (0.43 mg/mL). Statistical analysis of the variability across all replicates from the six experiments for each virus revealed that amantadine made a significant contribution to the synergy of the TCAD regimen at concentrations 0.1 mg/mL and 0.32 mg/mL and above against CA05 and CA10, respectively, compared to the double combination of ribavirin/oseltamivir carboxylate without amantadine ( Figure 1B ). For CA04, while only the 3.2 mg/mL amantadine concentration had statistically significant greater synergy volume than the double combination without amantadine, there was a trend toward increasing synergy volume starting at 0.32 mg/mL. Thus, amantadine contributed to the activity of the TCAD regimen at concentrations where it was inactive as a single agent, and at concentrations that were clinically achievable.
Synergy plots, which reveal the extent of synergy at each concentration of each drug in the combinations, are shown in Figures S1 for CA05. The data are presented as contour plots, in which regions where inhibition is greater (synergy) or less (antagonism) than expected are identified by subtracting the theoretical additive inhibition from the observed inhibition. Synergy plots for TCAD regimen showed a concentrationdependent increase in synergy with respect to amantadine ( Figure S1B , top plane). At the highest concentration tested (3.2 mg/mL), synergy was observed over a wide range of concentrations of ribavirin and oseltamivir carboxylate tested. At lower concentrations of amantadine, synergy occurred at 1 mg/mL ribavirin and higher, and at 0.0032 mg/mL oseltamivir carboxylate and higher. No significant antagonism was observed at any dose of any drug in the double combinations or the TCAD regimen. Similar patterns of synergy were observed with double and triple combinations of these antiviral drugs against CA04 and CA10 (data not shown).
One notable consequence of synergy is that the antiviral activities of each drug in the combination is enhanced compared to the activities of the drugs as single agents. To demonstrate this, we compared the antiviral activity of each of the three drugsamantadine, ribavirin, and oseltamivir carboxylate -as single agents and in the presence of fixed concentrations of the second and third drugs against CA10 replication. For each drug, the EC 50 was reduced in triple combination compared to the EC 50 as a single agent, indicative that each drug was active at lower concentrations (greater potency). For example, the EC 50 for amantadine as a single agent was reduced by 3.2-fold in combination with 1 mg/mL ribavirin and 0.0032 mg/mL oseltamivir carboxylate; the EC 50 for ribavirin as a single agent was reduced by 2.7-fold in combination with 0.0032 mg/mL oseltamivir carboxylate and 1 mg/mL amantadine; and the EC 50 for oseltamivir carboxylate as a single agent was reduced 16.2-fold in combination with 1 mg/mL ribavirin and 1 mg/mL amantadine ( Table 2) . For all three drugs, the reduction in EC 50 values observed in the triple combination compared to the single agent was statistically significant (P,0.05), indicative that each drug had greater antiviral potency and was effective at lower concentrations. In addition, the EC 50 of all three drugs in triple combination were reduced 1.5-to 6.5-fold compared to the EC 50 in double combinations, indicative of that the antiviral activity of the drugs in triple combination were enhanced compared to double combinations. Importantly, the data presented here do not represent the maximum reductions in EC 50 values for the three drugs. Due to the dynamic range of the assay, we were only able to obtain precise dose-response curves for each drug at fixed concentrations of the second and third drugs which were well below their EC 50 values and well below concentrations where maximum synergy occurred. At higher concentrations, the antiviral activity of the second and third drug contributed significantly to the inhibition, which decreased the linear range of the assay and reduced the accuracy of the curve-fitting (data not shown). A comprehensive assessment of the interaction of two or three drugs in combination requires the evaluation of multiple concentrations of each drug in order to quantify synergy over the entire dosing range, as was done in the section above.
We also evaluated the interactions of double combinations of neuraminidase inhibitors (NAIs) against 2009 H1N1. As shown in Table 1 , the synergy volumes for the zanamivir/oseltamivir carboxylate were 2246116 mg/mL 2 %, 2155689 mg/mL 2 %, and 2197698 mg/mL 2 % against CA04, CA05, and CA10, respectively. These values suggest that the zanamivir/oseltamivir carboxylate combination was additive against these viruses. For the zanamivir/peramivir combination, synergy volumes were 2356112 mg/mL 2 %, 21976108 mg/mL 2 % and 2239693 mg/ mL 2 % against CA04, CA05, and CA10, respectively, indicative of additivity to moderate antagonism. Consistent with these values, the synergy plots for the NAI double combinations against CA05 revealed regions of antagonism (red areas), which occurred at higher concentrations of zanamivir (0.01-0.1 mg/mL) and at variable concentrations of the second NAI (oseltamivir or peramivir, Figure S2 ). Similar results were found with the other two 2009 H1N1 strains. Furthermore, evaluation of the EC 50 of each NAI in combination with a fixed concentration of a second NAI revealed that the antiviral activity of each drug was not enhanced in combination with a second drug (data not shown). The observation that double combinations of neuraminidase inhibitors were not synergistic is consistent with the fact that all three drugs are known to target the same enzyme, and all bind in the same substrate binding pocket in a similar manner [19] .
As an extension of these studies we assessed the contribution of amantadine to the synergy of the TCAD regimen against . As a single agent, amantadine had no activity against these viruses at concentrations up to the highest concentration tested (32 mg/mL; data not shown). As shown in Figure 2 , against NC V27A and WI S31N, amantadine contributed to the synergy of the TCAD regimen in a concentration-dependent manner. The synergy of the TCAD regimen in the presence of amantadine was greater than the synergy of the ribavirin/oseltamivir carboxylate double combination without amantadine, and the increase in the synergy of the TCAD regimen was observed at 0.32 mg/mL amantadine for NC V27A and 1 mg/mL amantadine for WI S31N, when significance was evaluated at the level of ,0.05. Against DK A30T, however, amantadine did not make a contribution to the synergy of the TCAD regimen within the concentration range tested (0.1-3.2 mg/mL), and the synergy of the TCAD regimen was not greater than the synergy of the ribavirin/oseltamivir carboxylate double combination. Whether the lack of contribution from amantadine against the DK A30T virus was due to the specific subtype (H5N1), the M2 mutation (A30T), or the combination of both remain to be determined. However, it should be noted that while amantadine made no contribution to the activity of the TCAD regimen against DK A30T, both ribavirin and oseltamivir remain active and their interactions were additive, indicative that two out of three drugs contribute to the activity of the TCAD regimen against this virus (data not shown).
Given that a large percentage of seasonal influenza circulating viruses are resistant to oseltamivir, we next assessed the activity and synergy of the TCAD regimen against oseltamivir-resistant viruses to evaluate the spectrum of activity of the TCAD regimen, and to determine whether oseltamivir contributed to the activity of the TCAD regimen against oseltamivir-resistant viruses. Two H1N1 oseltamivir-resistant viruses were used, both of which bear the H274Y substitution in NA which has been demonstrated to confer resistance to oseltamivir [20] : A/Mississippi/3/01 (MS H274Y) and A/Hawaii/21/07 (HI H274Y). As a single agent, oseltamivir carboxylate had no antiviral activity against either virus below 1 mg/mL (data not shown). The synergy volumes of double and triple combinations of amantadine, ribavirin, and oseltamivir were determined against both viruses, and the data are presented in Figure 3 . As double combinations, amantadine/ oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/ oseltamivir carboxylate were all additive ( Figure 3A) . As was seen with the 2009 H1N1 viruses, the TCAD regimen was synergistic, and the synergy volume was greater than the synergy volume of any double combination, with each drug making a contribution to the synergy of the TCAD regimen against the oseltamivir-resistant viruses ( Figure 3B-D) . Importantly, oseltamivir carboxylate contributed to the synergy of the TCAD regimen starting at 0.1 mg/mL against MS H274Y (P,0.05) and at 0.32 mg/mL against HI H274Y (P,0.01). At these concentrations, which are achievable clinically, oseltamivir carboxylate is not active as a single agent against these resistant strains. Synergy plots for the TCAD regimen against MS H274Y and HI H274Y are provided in Figure S3 , and reveal increasing synergy with increasing concentrations of oseltamivir carboxylate. At the highest concentration of oseltamivir carboxylate tested (3.2 mg/mL), synergy occurred over wide concentrations of ribavirin and amantadine. At lower concentrations of oseltamivir carboxylate, synergy occurred at higher concentrations of amantadine (0.1 mg/mL or higher), and at wide concentrations of ribavirin. No significant antagonism was detected at any concentrations of the three drugs. We also determined the EC 50 of oseltamivir carboxylate as a single agent and in double and triple combinations against both oseltamivir-resistant viruses. As summarized in Table 3 , the EC 50 of oseltamivir carboxylate as a single agent was 74 mg/mL against MS H274Y and 15 mg/mL against HI H274Y. These represent 1480-and 300-fold reductions in susceptibility compared to the published values for a wild-type virus [11] . The EC 50 of oseltamivir carboxylate was not reduced in double combination with 1mg/mL ribavirin or 0.032 mg/mL amantadine against MS H274Y, and was reduced by only 1.7-and 1.4-fold, respectively, against HI H274Y. However, in triple combination with ribavirin and amantadine at the same concentrations used in double combination, the EC 50 of oseltamivir carboxylate was reduced by 21-fold against MS H274Y and by 5.8-fold against HI H274Y.
The TC 50 of amantadine as a single agent was 37-40 mg/mL, whereas the TC 50 of ribavirin and oseltamivir carboxylate as single agents were .100 mg/mL (Table S2) . These values are more than 10-fold higher than the highest concentration of each drug used in the combination experiments (Table S1 ). Furthermore, synergy analysis of the double and triple combinations revealed no synergistic cytotoxicity for any double combination or the TCAD regimen within the concentration ranges tested (data not shown). For example, cells treated with the TCAD regimen at the highest concentrations tested for all three drugs used in the combination experiments (3.2 mg/mL amantadine, 10 mg/mL ribavirin, and 3.2 mg/mL oseltamivir carboxylate) exhibited 97% viability, which was not statistically different than the cell control (P = 0.47 as determined by Student's t-test, Figure S4 ).
One indicator of the expected clinical antiviral activity is the inhibitory quotient (IQ), defined herein as the ratio between the average total plasma concentration (C ave ) and the 50% effective inhibitory concentration (EC 50 ). In order to predict the effectiveness of the TCAD regimen against the circulating influenza strains in the clinical setting, we calculated and compared the IQs for amantadine, ribavirin, and oseltamivir carboxylate as single agents and the TCAD regimen against susceptible and resistant influenza viral strains (including 2009 H1N1). To determine the IQ of the TCAD regimen, amantadine, ribavirin, and oseltamivir carboxylate was tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. A dilution curve of the TCAD regimen was created by first preparing a solution of all three drugs at 100-fold the C ave of each drug (43 mg/mL amantadine, 130 mg/mL ribavirin, 30 mg/mL oseltamivir carboxylate), and then serially diluting this solution in half-log 10 increments. In this manner, the EC 50 of the TCAD regimen was determined as a ratio of the C ave and expressed in units of fold change from C ave .
As representatives of the current circulating strains, we used A/ New Caledonia/20/99 (H1N1) (NC20) as the susceptible virus (susceptible to amantadine, ribavirin, and oseltamivir carboxylate), CA10 as the amantadine-resistant virus, and MS H274Y as the oseltamivir-resistant virus. Concentration-response curves for amantadine, ribavirin, oseltamivir carboxylate, and TCAD were generated against all three viruses, and the EC 50 s and IQs are summarized in Table 4 . Amantadine was effective at inhibiting NC20 and MS H274Y in vitro, resulting in IQs of 1.95 and 7.17, respectively. However, the IQ for amantadine was reduced to 0.02 when tested against CA10, indicative that an in vitro concentration representing the achievable plasma concentration at the recommended dose was not adequate to inhibit the amantadineresistant virus in vitro. An IQ of 0.02 represents a 100-fold reduction compared to NC20 and a 350-fold reduction compared to MS H274Y. Similarly, oseltamivir carboxylate was effective at inhibiting NC20 and CA10 in vitro resulting in IQs of 1.5 and 5.0, respectively, but not MS H274Y (IQ = 0.004). Similar to amantadine against the amantadine-resistant virus, the IQ for oseltamivir was reduced 350-to 2300-fold against the oseltamivirresistant virus compared to susceptible viruses. The IQs for ribavirin were uniformly low and below 1 for all three viruses (0.23 to 0.42). On the other hand, the IQs for the TCAD regimen as a fixed dose combination were consistently high against all three viruses (8.33 to 17.24), varying by no more than 2-fold between susceptible and resistant viruses. These data suggest that the TCAD regimen may have broad utility against all circulating influenza strains, including strains that are resistant to either amantadine or oseltamivir.
Given that virtually all seasonal H3N2 and 2009 H1N1 strains are resistant to amantadine, and virtually all currently circulating seasonal H1N1 strains are resistant to oseltamivir, the pharmacologic rationale for the development of a triple combination antiviral drug (TCAD) composed of amantadine, ribavirin, oseltamivir is that at least two, and possibly three drugs, in the TCAD regimen will be active against all of these viruses. A number of studies have evaluated double combinations of antivirals [21] [22] [23] [24] [25] [26] [27] against influenza A infection in vitro, and Hayden et al. have tested a triple combination of two antivirals with human interferon a [28] . However, there have been few reports on the effects of drug combinations on resistant influenza viruses [26, 27, 29] . Recently, Smee et al. evaluated the effects of double combinations of amantadine, ribavirin, and oseltamivir against the same amantadine-resistant H5N1 virus used in this study (A/Duck/MN/1525/81) [26] . The authors found that the presence of amantadine in double combinations did not provide added benefit over the second drug alone, either in cell culture or in mouse models.
In the present study, we examined the efficacy and synergy of the TCAD regimen against viruses which were resistant to oseltamivir or amantadine, including 2009 H1N1. Consistent with the previous findings [9] , we found that the 2009 H1N1 strains were susceptible to NAIs (oseltamivir carboxylate, zanamivir, and peramivir) and ribavirin, but were resistant to adamantanes (amantadine and rimantadine). Surprisingly, we found that amantadine as a single agent retained partial activity against these viruses (Table S2) , albeit the activity was reduced by 100-fold compared to a susceptible virus, whereas rimantadine had no activity below the 50% cytotoxic concentration. This observation suggests that phenotypic testing, in addition to determination of the genotype, may be necessary in order to fully understand the susceptibility profile of a novel virus and may have important implications in guiding the choice of antivirals for use in combinations.
Against the 2009 H1N1 strains, the interactions of oseltamivir carboxylate, peramivir, and zanamivir in double combinations ranged from additive to moderately antagonistic, indicative that the activity of these drugs was not enhanced in combination compared to their activity as single agents. These results suggest that double combinations of NAIs may not provide any added benefit over the drugs as single agents. Given that all NAIs bind in the same substrate binding pocket in NA, the use of these drugs in combination in the absence of enhanced activity raises the risk of selecting for a single mutation that could confer resistance to both neuraminidase inhibitors simultaneously. Indeed, cross-resistance to oseltamivir and zanamivir resulting from a single amino acid change has been documented for seasonal influenza A and B viruses [30] . If this were to occur in the 2009 H1N1 background, the resulting virus would be resistant to all approved anti-influenza drugs.
In total, we tested the activity and synergy of the TCAD regimen against six amantadine-resistant viruses, including three strains of 2009 H1N1, and two oseltamivir-resistant viruses. The viruses tested in this study come from the three subtypes that cause significant morbidity and mortality in humans (H1N1, H3N2, and H5N1), and include seasonal, avian, and pandemic strains. The double combinations of amantadine/oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/oseltamivir carboxylate were additive against 2009 H1N1, and ranged from additive to moderately synergistic against the other viruses (data not shown). In contrast, with the exception of the duck H5N1 virus, we found that the TCAD regimen was synergistic at clinically achievable concentrations of all three drugs, and that the synergy of the TCAD regimen was greater than that of any double antiviral drug combination. These data suggest that the TCAD regimen may have broad-spectrum antiviral activity against circulating influenza A viruses, including strains that are resistant to either classes of antivirals. To date, most influenza A strains in circulation (,99%) are resistant to either the adamantanes or oseltamivir, and not to both [31] , and thus are expected to be susceptible to the TCAD regimen. Currently, rapid diagnostic tests are not available to determine the susceptibility profile of influenza viruses in real time, and thus clinicians do not often have the necessary information with which to guide appropriate antiviral use. The availability of a broad-spectrum antiviral therapy that would be effective against the majority of circulating strains regardless of the susceptibility would be of high clinical utility.
Importantly, we found that amantadine and oseltamivir contributed to the synergy of the TCAD regimen against amantadine-resistant and oseltamivir-resistant viruses. The contributions from both drugs to the synergy of the TCAD regimen were significant at clinically achievable concentrations where they had little or no antiviral activity as a single agent. For instance, a comparison of the synergy volume of the TCAD regimen at 0.32 mg/mL amantadine to the synergy volume of the ribavirin/ oseltamivir carboxylate double combination (no amantadine) revealed that amantadine contributed 39%, 24%, and 44% to the total synergy of the TCAD regimen against CA04, CA05, and CA10, respectively. Similarly, against the oseltamivir-resistant viruses, oseltamivir carboxylate at 0.32 mg/mL contributed 76% and 83% to the total synergy of the TCAD regimen against MS H274Y and HI H274Y, respectively. Thus, all three drugs contributed to the synergy and activity of the TCAD regimen against amantadine-and oseltamivir-resistant viruses, and the activities of amantadine and oseltamivir were restored in the context of the TCAD regimen against influenza strains that were resistant to these drugs, thereby maximizing the clinical utility of these drugs. The mechanism(s) by which amantadine and oseltamivir carboxylate contribute to the synergy of the TCAD regimen against resistant strains is unclear. The interactions between M2, HA, and NA on the surface of the influenza particle are complex and not well understood, and a number of studies have demonstrated that HA-M2 and HA-NA interactions can affect the susceptibility to amantadine and oseltamivir, respectively [32, 33] . Furthermore, amantadine has been demonstrated to exert antiviral activity via interactions with HA at higher concentrations [34, 35] . It is conceivable that, as the result of protein-protein interactions between M2, HA, and NA, the binding of a drug at one site may affect the conformation and therefore affinity for another drug at another site. The mechanism by which ribavirin contributes to the synergy of the TCAD regimen is also unclear. Ribavirin has been documented to act through multiple mechanisms affecting both virus replication and host immune response [36] [37] [38] , and it remains to be elucidated which of these mechanisms are responsible for the synergy with amantadine and oseltamivir.
Finally, we evaluated the activity and inhibitory quotient (IQ) of TCAD against susceptible and resistant viruses representing the currently circulating strains. While the correlation between IQ and clinical efficacy has not been demonstrated for influenza, it is valuable to construct a relative ranking of the IQ of different antiviral regimens against susceptible and resistant viruses in order to assess the spectrum of their activity. When tested against a seasonal susceptible H1N1 virus, an amantadine-resistant 2009 H1N1, and a seasonal oseltamivir-resistant H1N1 virus, TCAD was uniformly active against all three viruses with significantly high IQs (8.33 to 17.24; Table 4 ). This suggests that TCAD may have broad antiviral activity against all currently circulating influenza strains and may have good efficacy in the clinical setting against these strains.
Our data suggests that a triple combination antiviral drug (TCAD) composed of amantadine, ribavirin, and oseltamivir may be an effective and viable therapeutic option for the treatment of pandemic and seasonal influenza infection. The body of data presented in this report validates the TCAD hypothesis, which states that for any given susceptible or resistant circulating influenza virus, at least two, and in some cases all three, drugs in TCAD will be active. Furthermore, the TCAD regimen appears to overcome baseline drug resistance and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza. The safety, pharmacokinetics, distribution, and metabolism of amantadine, ribavirin, and oseltamivir as single agents are well understood, and it is not expected that co-administration of the three drugs will result in substantially increased risk to patients compared to the administration of the individual drugs. In addition, all three double combinations have been tested in humans without adverse effects, including amantadine plus oseltamivir [39] , amantadine plus ribavirin [40] , and ribavirin plus oseltamivir [41] . Clinical trials to assess the efficacy and safety of TCAD for the treatment of influenza have been initiated, and will provide important data on the use of TCAD against both pandemic and seasonal influenza.
Table S1 Concentration ranges (mg/mL) of each drug tested in double and triple combinations against different influenza A viruses. Drugs were titrated at half log 10 Figure S1 Synergy plot of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate against 2009 H1N1 A/California/05/09 (CA05) replication as determined by Neutral Red assay in MDCK cells. Calculated additive interactions were subtracted from the experimentally determined inhibition to reveal regions of synergy (inhibition above expected) or antagonism (inhibition below expected). Values were derived from mean triplicate data and presented at 95% confidence. This experiment was repeated a total of six times with similar results. Blue areas indicate concentrations of each drug that are synergistic, gray areas indicate concentrations that are additive, and red areas indicate concentrations that are antagonistic. The intensity of the color (blue or red) corresponds to percent inhibition above or below expected. Figure S4 Viability of MDCK cells treated with the TCAD regimen. MDCK cells were incubated with the TCAD regimen at the highest concentrations of all three drugs used in the synergy experiments (3.2 mg/mL amantadine, 10 mg/mL ribavirin, and 3.2 mg/mL oseltamivir carboxylate), and cell viability was determined by Neutral Red assay after 72 hours. Values are the mean of nine replicates from three experiments, with standard deviations. The difference in viability between the TCAD treated cells and the cell controls was not statistically significant (P = 0.47, Student's t-test). Found at: doi:10.1371/journal.pone.0009332.s006 (1.00 MB TIF) Successful treatment of HIV-associated multicentric Castleman's disease and multiple organ failure with rituximab and supportive care: a case report INTRODUCTION: Multicentric Castleman's Disease (MCD), a lymphoproliferative disorder associated with Human Herpes Virus-8 (HHV-8) infection, is increasing in incidence amongst HIV patients. This condition is associated with lymphadenopathy, polyclonal gammopathy, hepato-splenomegaly and systemic symptoms. A number of small studies have demonstrated the efficacy of the anti-CD20 monoclonal antibody, rituximab, in treating this condition. CASE PRESENTATION: We report the case of a 46 year old Zambian woman who presented with pyrexia, diarrhoea and vomiting, confusion, lymphadenopathy, and renal failure. She rapidly developed multiple organ failure following the initiation of treatment of MCD with rituximab. Following admission to intensive care (ICU), she received prompt multi-organ support. After 21 days on the ICU she returned to the haematology medical ward, and was discharged in remission from her disease after 149 days in hospital. CONCLUSION: Rituximab, the efficacy of which has thus far been examined predominantly in patients outside the ICU, in conjunction with extensive organ support was effective treatment for MCD with associated multiple organ failure. There is, to our knowledge, only one other published report of its successful use in an ICU setting, where it was combined with cyclophosphamide, adriamycin and prednisolone. Reports such as ours support the notion that critically unwell patients with HIV and haematological disease can benefit from intensive care. The spectrum of HIV-associated disease on the ICU has changed markedly since the widespread adoption of combination antiretroviral therapy (Highly Active Antiretroviral Therapy, HAART) in the late 1990s. Whilst the incidence of opportunistic infections has decreased, that of several neoplasms, including Multi-centric Castleman's Disease (MCD) and Hodgkin's lymphoma is increasing. MCD is a lympho-proliferative disorder associated with Human Herpes Virus-8 (HHV-8) infection, characterised by fever, lethargy, anaemia and lymphadenopathy. Lymph node histology typically reveals angiofollicular hyperplasia and plasma cell infiltration. There is as yet no accepted therapeutic gold standard for MCD. Initial treatment approaches involved chemotherapy with agents such as vinblastine, etoposide and doxorubicin plus corticosteroids. More recently the anti-CD20 monoclonal antibody, rituximab, has been used. This targets HHV-8-infected plasmablasts, which coexpress the B cell antigen CD20. Small case series of patients with MCD have shown rituximab to be an effective therapy in patients that do not require organ support on the intensive care unit [1] [2] [3] .
A 46 year old Zambian woman was referred from another hospital with a 4 week history of fevers, night sweats, vomiting, diarrhoea, and renal impairment. She had been diagnosed HIV positive in 2005, and started on HAART one year later. She had previously been treated for Herpes simplex virus infection, Cytomegalovirus pneumonitis, and Pneumocystis jirovecii pneumonia (PCP). At referral her blood CD4 count was 480 × 10 6 / L (range in HIV negative populations, 400-1500 × 10 6 / L); and she had an undetectable plasma HIV load. On arrival at our centre, she was confused, and had obvious pitting oedema of both lower limbs, widespread lymphadenopathy, and hepato-splenomegaly. Investigations (Table 1) revealed anaemia, leucocytosis, thrombocytopaenia, and acute renal and liver dysfunction.
A CT scan showed hepato-splenomegaly and gross lymphadenopathy involving the thorax, abdomen and pelvis ( Figure 1 ). Inguinal lymph node excision biopsy confirmed the clinical suspicion of Multi-centric Castleman's disease (MCD) (Figure 2 ). Rituximab (375 mg/m 2 ) together with hydrocortisone and rasburicase, was administered as specific treatment. She developed rapidly progressive metabolic acidosis, oliguria, and rising serum creatinine and was admitted to the ICU for haemofiltration. Antiretroviral therapy was continued on the ICU with ritonavir-boosted lopinavir and saquinavir. Abacavir and lamivudine, which the patient was already taking, were stopped because of their association with lactic acidosis and hepatic steatosis.
Following admission to ICU she rapidly became hypotensive, hypoglycaemic, coagulopathic and more anaemic. A possible basis for this could have been the systemic manifestations of a "cytokine storm" associated with MCD; increased expression of IL-6 is typical of MCD. Vasopressor and inotropic support with noradrenaline and dobutamine was required to maintain an adequate mean arterial pressure (MAP). Because of rapidly escalating requirements for noradrenaline she received a continuous infusion of hydrocortisone (10 mg/h) as per local departmental protocol, to treat probable relative adrenal insufficiency. Empirical antibiotics and antifungal agents were given to treat sepsis as a potential cause for ensuing multi-organ dysfunction, and she required a continuous infusion of 20% dextrose for refractory hypoglycaemia. To treat her acute renal failure and profound metabolic acidosis (serum lactate of 18.5 mmol/L), haemofiltration was undertaken with large volume 5 litre cycles (~90 ml/kg/hour) of lactatefree replacement fluid. This strategy was adopted to target early shock reversal and removal of IL-6, increased expression of which is a hallmark feature of MCD.
The chest radiograph progressed over four days to bilateral diffuse patchy consolidation (Figure 3 ), associated with greatly increased oxygen requirements (FiO2 0.8), and consistent with a diagnosis of acute respiratory distress syndrome (ARDS). The patient became drowsy and hypercapnic. Her trachea was therefore intubated, and mechanical ventilation was commenced.
She developed epistaxis and bleeding from insertion sites of arterial, central venous, and haemofiltration catheters. She had a positive direct Coombs' test consistent with autoimmune haemolytic anaemia (AIHA), a recognised association of MCD. In addition she had elevated prothrombin (PT) and activated partial thromboplastin times (APTT), reduced platelets and reduced serum fibrinogen consistent with disseminated intravascular coagulation (DIC). She received methylprednisolone, folinic acid, and red cell concentrate to treat anaemia; plus cryoprecipitate, fresh frozen plasma, vitamin K, and platelets for DIC. In addition to this extensive physiological support, her Castleman's disease was treated with weekly infusions of the anti-CD20 monoclonal antibody, rituximab, for four weeks.
From day 10 there was evidence of clinical improvement. She had a tracheostomy in the second week of her ICU stay, and she was slowly weaned from inotropic/vasopressor, ventilatory, and finally renal support. At day 21 of her ICU admission, she was discharged to the ward to complete her treatment with rituximab, and to continue rehabilitation from global muscle weakness, and reduce dependence on her tracheostomy. The patient was discharged home, in remission from her disease, after 149 days in hospital. When last seen in clinic she remained in remission and living independently 14 months from her treatment.
Survival of patients with HIV admitted to the ICU has improved substantially in the last 10 years, with HIV patients now being able to expect a similar chance of survival through to hospital discharge as general medical patients admitted to the ICU. The basis for this improvement is likely to be multifactorial, reflecting better understanding of HIV-associated disease, the availability of new combination antiretroviral therapy, the improved care of HIV patients both outside and within the ICU, and protective ventilation strategies for HIV patients with respiratory failure and acute lung injury.
Prognostic factors previously shown to be associated with a poor outcome in HIV patients admitted to ICU are low CD4 count and advanced HIV disease stage, acute illness severity (SAPS I: simplified acute Table 1 Laboratory investigations on admission to Royal Free Hospital Haematology unit physiology score I; or APACHE II: acute physiology & chronic health evaluation II), and the need for, and duration of, mechanical ventilation whilst on the ICU [4] . In addition, a haematological malignancy also independently further confers a poor prognosis. Such patients typically have severely impaired host defences and the undertaking of invasive procedures, such as endotracheal intubation and central venous cannulation, carries a major risk of infection.
Cornet et al [5] reported an ICU mortality rate of 60% for haemato-oncological patients compared with a rate of 27% for general critically ill patients. They also highlighted the poor long-term prognosis, with a 1 year mortality of 88% for haemato-oncological patients. However, such patients may not fare so badly on ICU if organ support facilitates administration of a specific therapy for a treatable condition. Benoit et al [6] have recently described successful outcomes in severely ill patients with haematological malignancies who receive intravenous chemotherapy in intensive care. Hence admission to ICU for specific therapy can be lifesaving.
The gold standard therapy for HIV-associated MCD is yet to be established. Vinblastine, etoposide and doxorubicin plus corticosteroids have all been used previously. Etoposide has been shown to be effective with resolution of systemic symptoms during its administration. However it has not been associated with prolonged remission. Interruption of chemotherapy usually results in clinical recurrence and most patients remain chemotherapy-dependent for life. In addition its use can be associated with cytopaenias. The use of etoposide would have been considered as adjuvant therapy in our case had the MCD not responded to rituximab monotherapy. In contrast rituximab is well tolerated and has been shown to be effective in case reports and series [1] [2] [3] . The role of rituximab in therapy for critically ill patients is less well established. In previously published reports, those admitted to ICU did not survive. Recently, however, a successful outcome of rituximab, in conjunction with cyclophosphamide, adriamycin and prednisolone, in the ICU setting, has been described [7] .
In our patient, the clinical presentation was consistent with a systemic illness with a large inflammatory/infective component. The patient was originally from a country with endemic mycobacterial and fungal infection. Investigations therefore were undertaken to exclude a variety of possible causes -including disseminated mycobacterial and fungal disease, as well as lymphomalike conditions, such as MCD. Prompt diagnosis was made following lymph node biopsy. The decision to biopsy an inguinal lymph mode, rather than a cervical node was based on the clinical findings of a large and easily palpable inguinal lymph node mass. Although small volume bilateral cervical lymphadenopathy was present, this was far less clearly abnormal than the groin lymph nodes. A particular issue in HIV patients is the presence of persistent generalised lymphadenopathy, which represents an immune response directed against HIV. This typically results in findings similar to the cervical adenopathy present in this patient; and therefore neck biopsy may have in fact slowed the diagnostic process. Antiretroviral therapy was continued, but modified on the ICU. Notably the patient had been using antiretroviral therapy (HAART) for two years prior to her presentation to our service. She had had a persistently undetectable plasma HIV load (<50 copies/mL). Her nucleoside reverse transcriptase inhibitors (abacavir and lamivudine) were stopped because of their association with lactic acidosis and hepatic steatosis. Although it would be surprising for this to occur after such a prolonged time on these drugs, it was important to minimise any possible mitochondrial toxicity that might have resulted from these agents; and which in turn could have contributed to acidosis. Monotherapy with ritonavir-boosted protease inhibitors is a useful treatment option in patients who are intolerant or resistant to other agents [8] . The approach is particularly successful in patients who have already suppressed their plasma HIV load, such as in this case.
Her condition progressed rapidly to one of multiorgan failure requiring a high level of support on the ICU. Supportive strategies included mechanical ventilation for acute respiratory distress syndrome; circulatory support with inotropes/vasopressors and corticosteroids; and high volume haemofiltration for renal failure and severe metabolic acidosis. Corticosteroids were used empirically to treat probable underlying relative adrenal insufficiency. This occurs in up to 25% of critically ill patients with sepsis. Surviving Sepsis guidelines [9] published in 2004 do not suggest mandatory testing (by adrenocorticotrophic hormone: ACTH stimulation test) unless there is strong suspicion of undiagnosed primary adrenal insufficiency. The 10 mg/h infusion used in our patient was in line with the recommended 24 hour total dose of 200-300 mg. High volume haemofiltration was undertaken to target early shock reversal and removal of IL-6, increased expression of which is a hallmark feature of MCD. In animal studies, such a strategy is associated with improved haemodynamics and gas exchange, reduced immuno-paresis and increased survival [10] . The length of hospital stay post ICU discharge for our patient largely reflects the need for weaning from respiratory support and rehabilitation. Patients such as ours often experience profound muscle weakness (ICUacquired weakness) after mechanical ventilation on intensive care, and require intensive physiotherapy, as well as nutritional, psychological and social support.
Our patient's successful outcome can be attributed to relatively simple treatment for MCD: that is rituximab; and extensive multi-organ care support on the ICU. Rituximab is a newly-established therapy for MCD [1] . Its efficacy has thus far been chiefly demonstrated in limited studies of patients outside of the intensive care setting. Recent literature suggests ICU admission and support can be beneficial for haemato-oncological patients to facilitate specific chemotherapy [6] . Consistent with this notion, our report illustrates the benefit of ICU support during rituximab treatment for HIV associated MCD.
Written informed consent was obtained from our patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Abbreviations ACTH: adrenocorticotrophic hormone; AIHA: autoimmune haemolytic anaemia; APACHE II: acute physiology and chronic health evaluation II; APTT: activated partial thromboplastin time; ARDS: acute respiratory distress syndrome; CNS: central nervous system; DIC: disseminated intravascular coagulation; FiO 2 : fraction of inspired oxygen; HAART: highly active antiretroviral therapy; HHV-8: human herpes virus-8; HIV: human immunodeficiency virus; ICU: intensive care unit; MAP: mean arterial pressure; MCD: Multicentric Castleman's Disease; PT: prothrombin time; SAPS I: simplified acute physiology score I. Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10(9) CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID(50) of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10(9) CFU of p3D-NT56/S. cho were protected in 9 days. Short interfering RNA (siRNA) are small, double-stranded RNA molecules that direct the degradation of complementary messenger RNA via a cellular process known as RNA interference. It is widely believed that RNA interference is an evolutionarily conserved mechanism within eukaryotes and that its functions include endogenous gene regulation, viral defense and the maintenance of genomic stability [14] . Because of the rapidity and specificity of RNAi, this technology could complement and improve the traditional tools available to control important animal pathogens.
Foot-and-mouth disease (FMD) is an extremely contagious disease that affects more than 33 species of cloven-hoofed animals including This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted use, distribution, and reproduction in any noncommercial medium, provided the original work is properly cited.
cattle, swine, and other livestock [36] . The disease has been known for five centuries and its causative agent, the FMD virus (FMDV), belongs to the genus Aphthovirus of the family Picornaviridae [1] . The FMDV genome is composed of a positive-sense, single-stranded RNA molecule of about 8500 nucleotides that contains a unique open reading frame. Like other RNA viruses, FMDV is antigenically variable and undergoes rapid mutation. There are seven distinguishable serological types of FMDV, namely O, A, C, Asia I, SAT1, SAT2, and SAT3. Current FMD vaccines based on inactivated virus are effective in preventing the disease, but carry the risks of incomplete inactivation and viral escape from vaccine production laboratories [26] , and they fail to induce an immune response in a short period. Thus, the development of emergency antiviral strategies is necessary in order to stem outbreaks of FMD.
As an antiviral technology, RNAi has already been widely researched for use with FMDV [5, 24, 33] . However, establishing RNAi as a viable approach to prevent FMDV requires resolving at least one major issue [17, 20] : the high genetic variability of FMD viruses. RNAi directed toward specific gene sequences of certain FMDV strains may face risks, especially in the event of an emergent FMD outbreak, because no information about the serotype or genotype of the isolated pathogen would be available while early protection is needed. Therefore, it may be necessary to design several siRNA that focus on the conserved regions of the viral genome [16, 17, 42, 46] . Previously, we showed that siRNA generated in vitro can effectively inhibit the replication of FMDV in either a specific or cross-inhibitory manner [28] . Another crucial issue that needs to be addressed is the optimal vector for delivery of siRNA-expressing cassettes. Chen et al. [6] demonstrated that treatment with recombinant, replication-defective human adenovirus type 5 expressing short-hairpin RNA significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. However, adenovirus VA1 non-coding RNA is able to inhibit the biogenesis of siRNA and microRNA [30] . This finding suggests that novel siRNA delivery systems would be necessary.
Salmonella, a member of enteric bacilli, is one of the most severe etiological agents of food-borne diseases. It is widely accepted that recombinant live vaccines using attenuated Salmonella as a vector to deliver passenger antigen induce immune responses not only against salmonella infection, but also against passenger pathogenic infection [8] . Live-attenuated Salmonella has been shown to deliver DNA vaccines [9, 35, 41, 47] . Early in the 1970s, Fang et al. screened out an attenuated Salmonella choleraesuis C500, which was a licensed live vaccine against swine paratyphoid [13] . Liu et al. proved that the attenuated S. choleraesuis C500 carrying an oral DNA vaccine induced an immune response against FMDV [29] .
In the present study, we constructed several siRNA-expressing plasmids targeted to conserved sequences within the coding regions of viral polymerase protein 3D, capsid protein VP4 and nonstructural (NP) protein 2B of the FMDV genome. Some of the plasmids inhibited several isolates of serotype O and serotype Asia I FMDV in baby hamster kidney (BHK-21) cells and suckling mice. Furthermore, we reported that siRNA directed against the polymerase protein 3D of FMDV and delivered by attenuated S. choleraesuis were capable of inhibiting virus replication in guinea pigs and swine.
The attenuated vaccine strain S. choleraesuis C500 was obtained from Nanjing Biological Pharmaceutical Co., Ltd. Attenuated Salmonella typhimurium LB5010 was kindly provided by Prof. Aoquan Wang (Institute of Microbiology, Chinese Academy of Sciences). The plasmid pU6, which has the mouse U6 promoter (P U6 ), was constructed and maintained in our laboratory.
Suckling mice (C57BL/6), 2 to 3 days old and weighing 3 to 4 g, were maintained by Bio-pharmacy, Jinyu Group Co., Ltd. Male and female guinea pigs weighing 250 to 300 g and large white swine, Vet. Res. (2010) 41:30 2-to 3-months-old and weighing 40 to 50 kg, were used to perform viral challenge. All of the animals were housed in disease-secure isolation facilities in an FMDV-free area, and had no previous FMD contact as confirmed by the absence of detectable anti-FMDV antibodies in their sera.
Human kidney cells (AD-293) were used to grow recombinant replication-defective human adenoviruses (rAd5) and determine virus titers. BHK-21 cells were used to grow FMDVand determine virus infectivity. Both of the cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (pH 7.4). Cultures were incubated at 37°C with 5% CO 2 . Two FMDV isolates of serotype O (HKN/2002 (GenBank accession number AY317098) and CHA/ 99 (GenBank accession number AJ539138)) and two FMDV isolates of serotype Asia I (YNBS/58 (GenBank accession number AY390432) and Jiangsu/ 2005 (GenBank accession number EF149009)) were used for viral challenge.
Recombinant replication-defective human adenoviruses (Ad5-POL and Ad5-LacZ) were constructed and maintained in our laboratory [6] . Ad5-POL expressed siRNA targeting the FMDV 3D sequence corresponding to nucleotides (nt) 1225-1280 of HNK/2002 3D. As a control for nonspecific effects of rAd5, Ad5-LacZ expressed siRNA corresponding to nt 1353 to 1435 of the b-galactosidase gene of Escherichia coli, which has no homology to the HKN/2002 genome as confirmed by sequence analysis. The sequence of Ad5-POL encoding the FMDV 3D-specific siRNA was 5 0 -GAGGCTATCCTCTC CTTTGCACGCCGTGGGACCATACAGGAGAAG TTGATCTCCGT-3 0 (sense), and that of Ad5-LacZ encoding the E. coli b-galactosidase-specific siRNA was 5 0 -GAGTGTGATCATCTGGTCGCTGGGGA ATGAATCAGGCCACGGCGCTAATCACGACGC GCTGTATCGCTGGATCAAATCTGTCG-3 0 (sense).
As a general strategy for constructing siRNAexpressing plasmids, inverted repeats targeting the sequence were cloned into plasmid pU6 at the EcoRI/HindIII sites, under the control of the U6 promoter and the termination signals of 4 or 5 thymidines. Plasmids p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25 contained inverted repeats corresponding to the nucleotide sequences of the FMDV genome, as described below.
As negative controls for nonspecific effects, plasmid pNTH21 and pLacZ contained inverted repeats of 21 nt and 83 nt, respectively, which have no homology to the FMDV genome, as confirmed by sequence analysis.
The targets of interest were essential during the life cycle of FMDV and relatively conserved in their nucleotide sequences. The reference sequences of the conserved regions of the FMDV genome were obtained from the National Center for Biotechnology Information (NCBI) website 1 and compared by nucleotide (nt) BLAST. The sequence of 3D-NT19 was 5 0 -GTTCTTGGTCACTCCATAA-3 0 , corresponding to nt 1123-1141 of the HKN/2002 3D gene; the sequence of 3D-NT56 was 5 0 -GAG GCTATCCTCTCCTTTGCACGCCGTGGGACCAT ACAGGAGAAGTTGATCTCCGT-3 0 , corresponding to nt 1225-1280 of the HKN/2002 3D gene; the sequence of VP4-NT65 was 5 0 -TCAGGCAA CACTGGAAGCATCATTAACAACTACTACATGC AGCAGTACCAGAACTCCATGGACAC-3 0 , corresponding to nt 43-107 of the YNBS/58 VP4 gene; the sequence of VP4-NT19 was 5 0 -ACAACTACTA CATGCAGCA-3 0 , corresponding to nt 68-86 of the YNBS/58 VP4 gene; the sequence of 2B-NT25 was 5 0 -CCAGATGCAGGAGGACATGTCAACA-3 0 , corresponding to nt 57-81 of the CHA/99 2B gene.
Virus infectivity was determined by serial dilution of the sample on BHK-21 cells grown in 96-well plates and the virus titer was calculated as a tissue culture infective dose by the Reed-Muench method [38] . A viral suspension titrated at 10 5 to 10 7 TCID 50 per 0.1 mL was used for viral challenge. BHK-21 cells were seeded in 96-well plates in DMEM 24 h before transfection. The cells (about 95% confluent) were transiently transfected with 0.1 lg of a single plasmid expressing 3D, VP4 or 2B specific siRNA. Five hours after transfection, the transfection complex was removed, cells were washed twice with DMEM, and 100 TCID 50 of FMDV per 0.1 mL was added to each well. After 1 h of adsorption, the inoculum was removed and cells were washed twice with DMEM. The infection then proceeded in DMEM supplemented with 10% fetal bovine serum. Cell supernatants were collected at designated time points, and the virus titers were determined three times on BHK-21 cells. Statistical analysis was performed with the Microsoft Excel program (Microsoft, Redmond, WA, USA).
Suckling mice (8 groups of 20 each) were subcutaneously injected in the neck with 100 lg of plasmids in 100 lL of phosphate-buffered saline (PBS). After 6 h, the suckling mice were challenged with 20 LD 50 of FMDV per 0.1 mL via subcutaneous injection into the neck near the site that received the injected DNA. The mice were then observed for 5 days post-challenge (dpc). Statistical analysis was performed with a log rank test 2 .
In order to prepare the recombinants (p3D-NT19/ S. cho, pLacZ/S. cho and p3D-NT56/S. cho), the attenuated S. typhimurium LB5010, an intermediate host cell, was first transformed with plasmids (pLacZ, p3D-NT56 and p3D-NT19) using a standard CaCl 2 method. Then, the plasmids were extracted and transferred to a terminal host cell-attenuated S. choleraesuis C500 by electroporation. The preparation of cells for electroporation was as follows: C500 cells were cultured in 3 mL LB medium at 200 rpm overnight at 37°C, and then were inoculated into 25 mL liquid LB medium at 1% (v/v) at 37°C, again with shaking at 200 rpm. Bacteria were grown to an optical density (OD) of 0.6-0.7, chilled and then collected by centrifugation at 4 000 rpm for 10 min at 4°C. The cells were then washed with 10% glycerol three times. The cells were re-suspended in 10% glycerol at a concentration of 3 · 10 10 cells/mL, frozen in dry ice, and stored at À70°C. A 200 lL aliquot of the cells was thawed and used for transformation. One microgram DNA (the control setup with no DNA) was mixed with cold cell suspension and then transferred to a chilled cuvette. Transformation was carried out with a Bio-Rad Gene Pulser at 2.0 kV, 25 lF, 200 X at 0-4°C. Transformed competent cells were transferred to a tube, and 400 lL of media were added prior to incubating the cultures for 1.5 h at 50 rpm at 37°C. Then, 80 lL of the cultures were transferred onto solid LB medium containing 100 lL/mL ampicillin. Recombinant bacteria were identified via PCR. The presence of the recombinant plasmid was confirmed by digestion with XhoI. Serological characteristics of recombinant S. choleraesuis (rS. cho) were tested using a congregation reaction on a glass plate to confirm its immunogenicity.
Live bacteria were prepared by culturing cells in 25 mL liquid LB medium at 37°C for 18 h followed by inoculation of 2.5 mL cultures into 25 mL liquid LB medium to produce 2 · 10 9 -5 · 10 9 CFU/mL. Live bacteria counting was performed by serial dilution of cultures followed by spread plating. Serotype O FMDV strain HKN/ 2002 passaged five times in suckling mice was used to challenge guinea pigs, and the virus passaged three times in swine was used for swine experiments. The viruses were titrated on guinea pigs and swine. The dose of FMDV used was determined through four 10-fold serial dilutions of virus (i.e. 10 À4 , 10 À5 , 10 À6 , and 10 À7 ) in PBS. Guinea pigs (4 groups of 6 animals each) were inoculated intradermally in the left rear foot with 0.1 mL of serially diluted viruses. According to the method described by Chen et al. [6] , the swine (4 groups of 4 each) were inoculated in the neck region by intramuscular injection of each animal with 2 mL of serially diluted virus. All of the animals were then monitored for the major clinical sign of FMD, the appearance of vesicles on the mouth or feet. The 50% animal infective dose (ID 50 ) was estimated using the Reed-Muench method [38] . Statistical analysis was performed with a log rank test 2 .
Guinea pigs in several groups of five each were inoculated by intramuscular injection with 1.0 · 10 9 CFU of recombinant attenuated S. choleraesuis in 0.1 mL of saline. Thirty-six hours after inoculation, FMDV challenge was carried out via intradermal injection of each animal with 0.1 mL of guinea pig infectious dose 50 ID 50 of HKN/2002 in the left rear foot, as was done for virus titration.
In a parallel experiment, animals were treated with 1.0 · 10 6 PFU of rAd5 and challenged 36 h later with 50 ID 50 of HKN/2002. To test whether the combined inoculations of rAd5 and rS. cho protected animals, some groups were injected intramuscularly with the p3D-NT56/S. cho+Ad5-POL mixture of 1.0 · 10 9 CFU and 1.0 · 10 6 PFU, respectively; then, after 36 h, they were challenged with 50 ID 50 of HKN/2002. 
Eighteen swine were divided into 5 groups of 3 or 4 animals each. The animals in each group were cohoused in a separate room. All groups were inoculated via intramuscular injection in the neck area. Group 1 was a mock control group inoculated with 2 mL of saline. As negative controls, groups 2 and 3 were inoculated with 5 · 10 9 CFU of S. choleraesuis C500 or LacZ/S. cho in 2 mL of saline, respectively. Group 4 was inoculated with 5 · 10 9 CFU of p3D-NT56/S. cho in 2 mL of saline. Group 5 was treated with a high dose of p3D-NT56/S. cho containing 5 · 10 10 CFU in 2 mL of saline. After 24 h, all animals were challenged via intramuscular injection with 100 ID 50 of HKN/2002 in 2 mL of PBS in the neck area. To avoid overexposure to the challenge virus, animals that developed disease were moved to another room, and then observation proceeded. After challenge, the animals were examined daily for clinical signs of FMD, including an increase in body temperature (above 40°C) and the appearance of vesicles on the mouth and feet. The lesion score was determined at various time points post-challenge by determining the number of digits plus mouth with vesicles for each animal. The observations were terminated on day 14 post-challenge, when the animals were humanely killed.
Blood and serum samples were collected at days 7 and 14 after challenge in swine experiments. To assess the neutralizing antibody response in the swine, plaque reduction neutralization assays were performed as described previously [31] . Neutralizing titers were reported as the highest serum dilution causing a 50% reduction in the number of HKN/ 2002 plaques on BHK-21 cells. The presence of antibodies against viral NS protein 3ABC of FMDV in the sera was detected using a solid-phase blocking enzyme-linked immunosorbent assay (SPB-ELISA) according to the procedures described by Chenard et al. [7] . Briefly, ELISA microtiter test plates were coated overnight at 4°C with recombinant 3ABC antigen expressed in E. coli [45] . After five washes with PBS containing 0.05% Tween 80 (PBST), each well of the plates was filled with 100 lL of test or reference serum diluted 1:2 in PBST and incubated at 37°C for 60 min. The serum was then removed, and the wells of the plates were washed as described above. After rewashing, 100 lL of anti-3ABC immunoglobulin-horseradish peroxidase conjugate was added, and the incubation proceeded at 37°C for 30 min. The conjugate was then removed, the wells of the plates were washed again, and 100 lL of a commercially ready-to-use tetramethyl benzidine chromogen substrate was added to the plates. After incubation at 37°C for 10 min, the reaction was stopped by adding 50 lL of 2.0 M H 2 SO 4 per well, and the OD at 450 nm was measured by using an ELISA reader. The sera were considered positive if the OD was reduced by ! 30% compared to a standard negative serum corrected for background signal by subtracting the OD of the high positive reference serum. Statistical analysis was performed with the Microsoft Excel program.
To test the antiviral activity of siRNA, BHK-21 cells cultured in 96-well plates were transfected with siRNA-expressing plasmids individually. As controls, the cells were either not treated or treated with plasmid pNTH21 or pLacZ. After 24 h, the cells were infected with 100 TCID 50 of CHA/99, HNK/2002, YNBS/ 58 or Jiangsu/2005. The cells were observed continuously under the microscope. Microscopy examination revealed that the cells treated with p3D-NT56 or p2B-NT25 had delayed cytopathic effect (CPE) development when challenged with either serotype O or Asia I of FMDV. However, the control cells, either blank or transfected with the control plasmids, showed an extensive CPE within 24 h post infection (p.i.). Furthermore, we investigated the effect of plasmid-mediated RNAi on FMDV replication by measuring the TCID 50 of supernatants of lysed cells, which were collected at 12, 24 and 48 h after viral challenge. Consistent with the observation, p3D-NT56 and p2B-NT25 showed a wider cross-inhibitory effect than the other plasmids, and these 2 plasmids inhibited 4 FMDV isolates (Fig. 1) . The supernatant TCID 50 for CHA/99, HNK/2002, YNBS/58 and Jiangsu/2005 of the cells treated with p3D-NT56 or p2B-NT25 was reduced by 30-to 300-fold at 48 h post infection (Fig. 1 ) compared to the control groups.
To further examine the anti-FMDV potential of p3D-NT56 or p2B-NT25 in vivo, we challenged suckling mice pretreated by subcutaneous injection of plasmids with FMDV strains HKN/2002 or YNBS/58. All PBS-treated mice died within 60 h after either HNK/2002 or YNBS/58 challenge (Fig. 2) . The animals treated with control plasmids were also not protected at all, although death in some groups was delayed slightly (p = 0.47, p = 0.157) (Fig. 2) . In HKN/2002 challenge experiments, 20% of the animals treated with either p3D-NT56 or p2B-NT25 were protected ( Fig. 2A) . The difference between the survival of mice treated with either p3D-NT56 or p2B-NT25 and the PBS control was statistically significant (p < 0.0001). In the case of challenge with YNBS/58, 8 of 20 mice treated with p3D-NT56 and 7 of 20 mice treated with p2B-NT25 survived a viral challenge of 20 LD 50 (Fig. 2B) , and a significant difference in mouse survival was seen between mice treated with either p3D-NT56 or p2B-NT25 and controls (p < 0.0001). Furthermore, in both HNK/2002 and YNBS/58 challenge assays, the time of 50% death of mice was delayed for 9 to 24 h in groups treated with FMDV-specific siRNAexpressing plasmids as compared with control groups. 
The data obtained in the cell culture and suckling mice assays demonstrated that siRNA targeted to conserved regions of the FMDV genome could be effective against FMDV in either a specific or a cross-inhibitory manner. The potential of RNAi as an antiviral strategy against FMDV in relevant animal systems is of great interest. The fact that attenuated Salmonella is widely used as a DNA vaccine vector made us choose it as the vector for siRNA delivery. To test the anti-FMDV potential of rS. cho, we challenged guinea pigs pretreated by intramuscular injection of rS. cho constructs with FMDV HKN/2002. All saline-treated animals developed the major clinical sign of FMD, the appearance of vesicles on the feet, within 48 h of challenge (Tab. I). Guinea pigs treated with pLacZ/S. cho were not protected at all, though the clinical signs were delayed in some animals, as shown in Table I . However, 4 of 5 guinea pigs pretreated with 10 9 CFU of p3D-NT56/S. cho and challenged with 50 ID 50 of HKN/2002 developed none of the clinical signs such as vesicles or high fever. These results indicated that S. cho-delivered siRNA exerted in vivo inhibitory activity against FMDV in guinea pigs.
We further evaluated the potential of treatments with an FMDV-specific rS. cho and rAd5 mixture in guinea pigs. Three of the five animals in the group treated with the p3D-NT56/S. cho+Ad5-POL mixture were completely protected as determined by the absence of vesicles on their feet (Tab. I). No significant increase in antiviral effect was achieved in this group when compared to the group treated with p3D-NT56/S. cho alone (Tab. I). This indicated that a simple combination of two vectors could not improve the protection of animals.
Based on the experiments in guinea pigs, we evaluated the antiviral activity of p3D-NT56/S. cho in swine. Animals were intramuscularly inoculated with 5 · 10 9 CFU of p3D-NT56/S. cho and challenged 24 h later with 100 ID 50 of HKN/2002. All of the saline-treated swine developed acute signs of FMD, including vesicles and fever (Fig. 3A) . Animal 1-48 had vesicles as early as 3 dpc, and its symptoms were extraordinarily severe. In comparison with this group, animals inoculated with either pLacZ/ S. cho or S. cho showed no signs of significant antiviral activity (Figs. 3B and 3C) . However, p3D-NT56/S. cho conferred marked antiviral activity that could be seen for more than 9 days of observation (Fig. 3D) . Animal 2-7 in this group developed a fever but no vesicles during the observation period. Although animals 5-6, 3-3 and 1-1 developed vesicles, they were fewer and developed much later. In another group inoculated with 5 · 10 10 CFU of p3D-NT56/S. cho, none of the animals were protected (Fig. 3E ), but animal 7-1 showed delayed disease onset and milder disease than the control animals. Serum samples were collected at 0, 7, and 14 dpc and tested for the presence of neutralizing antibodies and antibodies against viral NS protein 3ABC of FMDV. No animals had marked FMDV-specific antibodies at 0 dpc, including anti-3ABC antibodies (Tab. II). Animals in the control groups treated with saline, S. cho or pLacZ/S. cho developed a high neutralizing antibody response at 7 and 14 dpc, suggesting that these animals were exposed to a very large amount of FMDV (Tab. II). A very low neutralizing antibody response was detected at 7 dpc in animals (2-7, 5-6, 3-3, 1-1, 7-1 and 1-34) that were inoculated with p3D-NT56/S. cho, suggesting that these animals effectively suppressed virus replication. A key distinguishing feature of infected animals is the induction of antibodies against the polyprotein 3ABC of FMDV [45] . Using an LPB-ELISA, we showed that the animals developing FMD had significantly higher levels of antibodies against 3ABC as compared to the disease-free animals (Tab. II). These results of serological analysis were consistent with the observation of clinical signs.
Strategies aimed at conferring rapid and efficient protection against FMDV have to face one main challenging factor: the rapid, acute infection caused by this virus, which makes the absence of sufficient amounts of antibodies or other interfering factors essential for protection. The traditional emergency vaccines based on virus inactivation could be effective in preventing disease within 4 to 5 days post-vaccination, due to a critical role for innate immune defenses [2, 39] . However, signs of disease can appear as early as 2 days post-exposure. Thus, it is necessary to develop antiviral strategies capable of inducing early protection in the face of an outbreak that threatens to spread widely. Recently, we and several other groups demonstrated that RNAi can be potentially used as a therapeutic Table I or prophylactic mechanism against FMDV [6, 24, 33] . However, to be effective against RNA viruses such as FMDV, which are highly antigenically variable, the siRNA must be targeted to conserved regions. Here, we provide evidence that siRNA targeted to conserved regions of the FMDV genome could effectively inhibit the virus replication of multiple FMDV serotypes. In this study, three genes, namely 3D, VP4 and 2B, were selected as targets for three reasons. First, it is well known that different siR-NA have different efficiencies in inducing RNAi [21] [22] [23] . Second, the proteins encoded by these genes have essential functions in different phases of the viral replication cycle. The RNA-dependent RNA polymerase (3D) is a key enzyme in viral RNA replication [34, 44] ; VP4 is a structural protein, located inside the mature virus particle, and is involved in conversion of provirions to mature virions [1] ; and 2B is involved in membrane rearrangements required for viral RNA replication and capsid assembly [19] . Third, we compared 14 reported FMDV isolates of serotype O and serotype Asia I. The analysis showed that the sequences in these regions are relatively conserved between different FMDV serotypes. Efficient delivery of siRNA into cells or organs in vivo remains a major bottleneck in antiviral therapy. A number of methods for delivering siRNA in vivo have been tested [3, 15, 27, 49] . Liposome formulations are generally used [3, 15, 49] , but a simple intranasal administration of naked siRNA has been shown to be effective against respiratory viruses [3] . Chen et al. [6] used adenoviruses as the vectors for the delivery of duplex RNA to the target tissue, and total RNA were extracted from five tissues (oropharynx, lung, liver, muscle, and epidermis of the foot) of guinea pigs treated with rAd5 and assayed for rAd5 GFP mRNA. The vast majority of rAd5 was found in the liver. In cloven-hoofed animals, the oropharynx has been identified as the major site of FMDV replication during acute and persistent infection [37] . Brown et al. [4] indicated that the FMDV RNA was localized in the tonsil, pharynx and the tracheo-bronchial lymph node. The different tissue distribution of rAd5 and FMDV is probably related to antiviral activity of rAd5 in vivo. Our overall goal was to find a siRNA vector that can spread through the similar infection and transmission tract as FMDV. Attenuated Salmonella has become popular for expressing foreign antigens as a vaccine strain or as a delivery vector of DNA vaccines [10, 12] . Gray et al. [18] found that S. choleraesuis was most often recovered from the lymph node, tonsil, lung and alimentary tract of swine inoculated with S. choleraesuis. These studies aroused our interest in evaluating the potential of Salmonella as a vector for siRNA delivery. We found that 80% of guinea pigs were completely protected when inoculated with 10 9 CFU of p3D-NT56/rS. cho, and that rS. cho also conferred marked antiviral activity in swine. Surprisingly, the antiviral potential of RNAi was evidently impaired in animals treated with a high dose of rS. cho. Srinivasan et al. [43] demonstrated that variation in the initial dose of infection with Salmonella had a profound effect on the response of Salmonella flagellin-specific CD4 T cells in vivo, and that low-dose infection could evade Salmonella flagellin-specific T cell activation completely. Mercado et al. [32] reported that a 25-fold dilution in bacterial challenge dose did not significantly alter the size or kinetics of the Listeria LLO 91-99 -specific CD8 T cell response. The low-dose effect may represent a general mechanism to increase virulence. The immune evasion could probably make the lower dose salmonella more efficient as a vector to carry siRNA.
Because Ad5 lacking E1A and E1B had been used for delivery of RNAi by several groups [6, 40, 48] , we evaluated the antiviral potential of combination inoculations of rS. cho and rAd5 in guinea pigs. The results indicate that no significant increase in antiviral effect was achieved in the group treated with the p3D-NT56/S. cho+Ad5-POL mixture, as compared to the group treated with p3D-NT56/S. cho alone. Kim et al. [25] attempted to improve the antiviral effect by use of a mixture of two adenovirus constructs, by giving two injections of shRNA prior to FMDV challenge and by giving injections after FMDV challenge. The high survival rate was maintained by treatment with adenoviruses post-challenge. These results could encourage us to seek more efficient methods of treatment.
In conclusion, a licensed strain of S. choleraesuis has a potential as an RNAi delivery system. This strain is very safe for swine, relatively inexpensive to manufacture and well suited for large-scale administration. However, it remains unclear the attenuated Salmonella how to deliver siRNA in vivo. Future studies relevant to cytokine and distribution of siRNA are necessary to examine this issue in more detail. Community acquired methicillin-resistant Staphylococcus aureus pneumonia leading to rhabdomyolysis: a case report Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is considered an underreported entity in India. In this case report, the authors describe a thirty-five year old immunocompetent male presenting with severe respiratory distress requiring intubation. On further work up, a CT thorax showed features consistent with necrotizing pneumonia. The morphology and sensitivity pattern of the organism found in the bronchoalveolar lavage fluid and blood culture were consistent with MRSA. The patient's stay in the hospital was complicated by acute renal failure due to rhabdomyolysis with CPK levels of 9995 U/L. The patient was started on dialysis and improved there after. This case brings to light that CA-MRSA is becoming a problem in developing nations where antibiotics are frequently used empirically with little laboratory guidance. It also is a rare reporting of rhabdomyolysis due to CA-MRSA. CA-MRSA commonly causes skin and soft tissue infections. In the current case report, we describe community acquired pneumonia due to MRSA which is until now infrequently seen. Further, this patient's clinical course was complicated by rhabdomyolysis which is even more uncommon.
A 35 year old South Indian male presented to the emergency department in a drowsy state with a history of high grade fever for 5 days and cough with mucoid expectoration for 3 days. The patient gave history of a thorn prick on his right hand a few days previously. He was not a know case of diabetes, hypertension or previous tuberculosis. He was a non-smoker and non alcoholic. He and his relatives denied any illicit intravenous drug use. There was no history of surgery or hospitalization in the past 10 years and no history of trauma, other than the thorn prick.
On examination, in addition to drowsiness, the patient was found to be febrile and tachypneic. An abscess was present over the thenar eminence of the right hand which corresponded to the site of the thorn prick [ Figure 1 ]. Systemic examination was significant for bilateral crackles over the lung fields.
His Arterial Blood Gas analysis (ABG) showed hypoxemia with PO2/FIO2 < 200. Chest x-rays showed bilateral diffuse non-homogenous opacities [ Figure 2 ], following which a diagnosis of community acquired pneumonia was made. His cardiac functions, as assessed by a surface 2D echocardiogram (2D-echo) showed normal systolic and diastolic functions. Hence, with this acute presentation, bilateral infiltrate, normal cardiac functions and PO2/FIO2 < 200, the patient was diagnosed to have acute respiratory distress syndrome (ARDS).
The patient, who was subsequently intubated in view of falling oxygen saturations was monitored in the intensive care unit and started empirically on intravenous piperacillin and tazobactum (according to the hospital policy on empirical antibiotics for severe community acquired pneumonia based on the local pattern of infecting organisms and sensitivity pattern).
His admission labs [ Table 1 ] showed his total leukocyte counts were 2500 cells/mm3 with a polymorphic predominance, and tests for HIV as well as HbsAg were negative.
Two surface 2D echos and one transesophageal echo showed no evidence of infective endocarditis. Following up with the diagnosis of bilateral bronchopneunomia with ARDS, a CT thorax was ordered for the patient, which revealed necrotizing pneumonia [ Figure 3 ]. Serial Chest X-Rays were taken there after to monitor the patient.
The bronchoalveolar lavage (done prior to intubation) and two blood cultures (from two different sites drawn at two different times) were positive for MRSA (sensitive only to vancomycin and linezolide). The patient's antibiotics were changed to vancomycin and meropenam (as per glomerular filtration rate calculation using Cockroft and Gault formula). Meropenem was added as an empirical gram negative cover.
On day 12 of hospitalization, the patient developed a decreased urine output, with a BUN (blood urea nitrogen) of 114 mg/dl, serum creatinine of 5.8 mg/dl and CPK (creatine phosphokinase) of 9995 U/L. The patient's urine was positive for myoglobin and a diagnosis of rhabdomyolysis was made. Hemodialysis was initiated. Vancomycin was changed to Linezolide 600 mg twice a day and continued for 6 weeks till two cultures were negative.
Following this, the patient improved and was discharged with a serum creatinine of 3.7 and with advice regarding continuing hemodialysis on an outpatient basis, respiratory rehabilitation and physiotherapy and tracheostomy care.
Methicillin resistant Staphylococcus aureus (MRSA) is growing in prevalence in the Indian scenario. Studies form different centers around the country estimate the prevalence to be between 20 and 40% and sometimes even higher [1, 2] .
While MRSA has traditionally been acquired nosocomially, the occurrence of community acquired MRSA infection has recently begin to pose a threat to health care. By definition, hospital acquired infections are diagnosed when they develop after 48 hours of hospitalization and community acquired infections when they develop before that period.
One study in India, suggests that the incidence of CA-MRSA is low enough to consider it unwarranted to treat community acquired infections with antibiotics that cover MRSA [3] .
CA MRSA have been found to cause skin and soft tissue infections more commonly then when compared to HA (hospital acquired)-MRSA [4] . They are also associated with certain exotoxin genes (like the Panton Velentine leukocidin gene) not usually seen in nosocoimally acquired stains [5] . In a study comparing invasive CA-MRSA with those causing skin and soft tissue infections, the latter was more commonly seen in male patients with a history of underlying conditions (immunosuppressive therapy, emphysema/COPD, injection drug use and smoking) [6] .
Rhabdomyolysis is described as the dissolution and disintegration of striated muscle, that can result in an acute, potentially fatal clinical syndrome. Acute renal failure occurs as a result of renal vasoconstriction, heme-protein induced toxicity and intraluminal cast formation. However, with out the presence of aciduria and hypovolemia along with the heme-protein, it would not have its nephrotoxic effect [7] .
Infections form a part of the long list of etiologies of rhabdomyolysis. Respiratory infection appears to the chief contributor among the infectious causes of rhabdomyolysis [8] Rhabdomyolysis in CA-pneumonia is more commonly associated with Legionella, Influenza virus, Streptococcus pneumoniae, Chlamydia psttaci and Mycoplasma [9] . Our patient had a thorn prick in his hand which turned into an abscess. This could have been the source for Staphylococcus aureus infection. He was not hospitalized or hadn't visited the hospital in the previous several years. In addition, the two blood cultures drawn at two different sites under sterile precautions at the time of admission were positive for MRSA and the bronchoalveolar lavage taken just prior to intubation showed MRSA. Hence his infection was community acquired and not hospital acquired.
In a study of 41 patients with community-acquired pneumonia admitted in the ICU, the 29% who had had an elevated CPK (more than 1000 U/l) had higher mortality when compared to the 71% without elevated CPK, though the initial severity and renal impairment was the same for the two groups. This suggests that the presence of rhabdomyolysis is a bad prognostic factor irrespective of the presence or absence of renal impairment [10] .
The diagnosis of rhabdomyolysis mandates measures to prevent renal impairment. The patient should be adequately hydrated and monitored for urine output, electrolyte balance and acid-base status. The onset of oligouric renal failure, persistent electrolyte and pH disturbance and other related complications requires the initiation of dialysis.
Patients with severe community acquired pneumonia should not be excluded from suffering from CA-MRSA due to the growing problem of drug resistant bacteria beyond the confines of the hospital. In addition, respiratory infections are a common cause of rhabdomyolysis, which should be considered with a high index of suspicion in order to initiate timely intervention.
CA-MRSA: community acquired -methicillin resistant Staphylococcus aureus; ARDS: acute respiratory distress syndrome; ABG: arterial blood gas (analysis); Echo: echocardiogram; HIV: human immunodeficiency virus; CT: computerized tomography; BUN: blood urea nitrogen; CPK: creatine phosphokinase.
Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Binding of Herpes Simplex Virus Type-1 Virions Leads to the Induction of Intracellular Signalling in the Absence of Virus Entry The envelope of HSV-1 contains a number of glycoproteins, four of which are essential for virus entry. Virus particles lacking gB, gD, gH or gL are entry-defective, although these viruses retain the ability to bind to the plasma membrane via the remaining glycoproteins. Soluble forms of gD have been shown to trigger the nuclear translocation of the NF-κB transcriptional complex in addition to stimulating the production of Type I interferon. By taking advantage of the entry-defective phenotype of glycoprotein-deficient HSV-1 virus particles, the results presented here show that binding of virions to cellular receptors on the plasma membrane is sufficient to stimulate a change in cellular gene expression. Preliminary microarray studies, validated by quantitative real-time PCR, identified the differential expression of cellular genes associated with the NF-κB, PI3K/Akt, Jak/Stat and related Jak/Src pathways by virions lacking gB or gH but not gD. Gene induction occurred at a few particles per cell, corresponding to physiological conditions during primary infection. Reporter assay studies determined that NF-κB transcriptional activity is stimulated within an hour of HSV-1 binding, peaks between two and three hours post-binding and declines to background levels by five hours after induction. The immediate, transient nature of these signalling events suggests that HSV-1 glycoproteins, particularly gD, may alter the cellular environment pre-entry so as to condition the cell for viral replication. Subjugation of the intracellular environment by viruses is essential to ensure the effective expression and replication of the viral genome to allow production of progeny virions. One such viral strategy involves hijacking signalling pathways that ultimately control host gene transcription. Interactions between intracellular viral proteins and cellular kinases responsible for signal transduction are a means by which to achieve this. However, there is growing evidence to support the premise that glycoproteins on the surface of virus particles may trigger intracellular signalling pathways by interacting with their cognate receptors on the host cell membrane [1] .
Binding of HSV-1 to permissive cells occurs through viral glycoproteins on the viral envelope interacting with specific receptors on the cell surface, triggering fusion of the plasma membrane with the outer envelope. Five of these glycoproteins are known to be involved in virion binding to the cell surface: gB, gC, gD and the heterodimer gH-L. Of the five, only gC is dispensable for allowing productive infection as deletion of gB, gD or gH-L results in an entry-defective phenotype [2] [3] . gD is known to bind independently to HvEM, nectin-1 and nectin-2, whereas gH interacts with the avb3 integrin, with the paired immunoglobulin-like receptor, PILRa, acting as a receptor for gB [2] [4] [5] .
One of the first studies to examine whether HSV-1 glycoproteins play a role in the induction of signalling found that gD was able to block Fas-mediated apoptosis [6] . U937 monocytoid cells were rendered resistant to apoptosis after infection with UVinactivated HSV virions, when co-cultured with a stably transfected HSV-1 gD-expressing cell line or after treatment with soluble gD. Inhibition of NF-KB signalling by the introduction of a dominant-negative NF-kB repressor abolished protection from gD-induced Fas-mediated apoptosis. More specifically, the interaction of gD with one of the gD receptors, HVEM, was involved in preventing apoptosis induction [1] .
There are reports showing that HSV-1 triggered the translocation of NF-kB by six hours post-infection [7] . However, it has come to light that HSV-1 may induce two distinct phases of NF-kB activity. The initial phase is transient, lasting only two hours, and occurred shortly after viral adsorption by both wild-type and UV-inactivated virus particles. This correlates with evidence showing that supernatant taken from gD-expressing cells may also cause an increase in NF-KB binding activity in monocytoid cells by 30 minutes post-treatment [6] . A second phase relied on de novo viral protein synthesis as it was stimulated only by replication-competent HSV-1 and not by UV-inactivated virions [8] . During this second phase, NF-kB complexes were shown to have associated with their consensus sequence found in the promoter region that drives ICP0 expression [8] .
In comparison to wild-type gD, a mutated form of gD that was unable to bind to HVEM did not stimulate NF-KB activity in cocultured monocytoid cells [9] . HEp-2 cells, which do not express HVEM, lacked detectable NF-kB signalling after infection with UV-inactivated HSV-1, implying that gD-mediated NF-kB stimulation may be dependent on expression of HVEM [9] .
Soluble glycoproteins from beta-and gammaherpesviruses can also stimulate intracellular signalling pathways; gB of CMV has been shown to stimulate the differential expression of cellular transcription factors and interferon-stimulated genes (ISGs), and soluble gp350 of EBV can trigger NF-kB activation [10] [11] [12] .
The treatment of cells with soluble glycoproteins or UVinactivated virus has provided strong evidence that HSV-1 binding to the plasma membrane triggers signal transduction in the host cell as a prelude to infection, but interpretation of these experiments is not straightforward. The use of soluble glycoproteins does not mimic the binding of individual virus particles and the physiological significance of the results is difficult to assess. The use of UV-inactivated particles in signalling studies simulates infection but these particles are able to enter cells and deliver virion proteins to the cytoplasm. Experiments with UV-inactivated particles do not therefore distinguish between events arising from virus binding from those resulting from virus entry.
The main focus in these studies was to examine signalling events using virus particles that are capable of binding to the cell surface but are incapable of entry due to the absence of one of the three essential glycoproteins, gB, gD or gH [13] [14] [15] [16] , and to find whether signalling occurs at particle concentrations that would mirror physiological conditions.
All cells other than Human Foreskin Fibroblasts (HFF) (ATCC cell line CRL-2522) were grown in Glasgow Modified Eagles Medium supplemented with 10% foetal bovine serum, 4 mM glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin. HFF cells were grown in Dulbecco modified Eagles medium supplemented as described above but with the addition of 1x MEM non-essential amino acids.
Working stocks of HSV-1 SC16 were grown on HaCaT cells and assayed on Vero cells [17] . Mutant viruses lacking functional genes for gH (SCgHZ) [14] , gB (HFEMdUL27-lacZ) [18] or gD (SC16gDdelZ) [18] were grown and assayed on helper cell lines expressing the corresponding glycoprotein: CR1 cells, expressing gH [19] ; D6 cells expressing gB [13] ; and VD60 cells expressing gD [18] . All stocks were grown using an MOI of 0.1.
Purified preparations of WT virus, or of virions lacking individual glycoproteins, were produced as previously described [20] . Purified preparations were assayed for particle numbers using electron microscopy [21] and for infectivity by plaque assay on Vero cells or, in the case of glycoprotein-deficient virions, on the relevant helper cell line. Preparations of WT virions had a particle to infectivity ratio of 3610 1 . Preparations of gHnegative, gD-negative and gB negative virions had particle to infectivity ratios of 2610 10 , 5610 6 and 3610 6 respectively. The maximum virion dose of entry-incompetent virions used in the experiments described in this paper was 1000 particles per cell and it therefore follows that in the maximum infectivity of a preparation of an entry-defective virus (i.e. using gB-negative virions) would be less than one cell per thousand. Virus preparations were deglycosylated with 500 U of PNGase F for 18 hrs at 37uC in the absence of denaturing buffer [22] . Removal of N-linked sugars was confirmed by a shift in the electrophoretic mobility of gD on a protein denaturing gel.
Human foreskin fibroblast cells (HFF) were seeded at 1.5610 6 in a 175 2 cm flask and grown to 90% confluence then growtharrested in medium supplemented with only 100 U/ml Penicillin-G and 100 mg/ml Streptomycin for five days. Real-time PCR experiments were conducted with growth-arrested HFFs that were inoculated in triplicate with 1000 particles/cell of entry-defective HSV-1 in warm, serum-free medium, with parallel mock-infected cells that received an equivalent volume of PBS in warm serumfree medium. Inoculations took place at 37uC. At the indicated times after the addition of virus, the medium was removed, the cells were lysed with TRI Reagent (Sigma) and total RNA was immediately purified. For each entry-defective HSV-1 mutant, three independent biological replicates were carried out. Microarray experiments were conducted under the same conditions using wild-type HSV-1 (SC16) at an MOI of 20.
Total RNA was isolated from TRI reagent lysates as per the manufacturer's instructions. Contaminating genomic DNA was digested with 2 U DNase I (Invitrogen) and RNA was re-purified with TRI reagent. cDNA was derived from 30 mg total RNA using anchored oligo (dT)20 primers (Invitrogen) as described in the manufacturer's instructions. For each entry-defective HSV-1 mutant, the cDNA from three independent infections were used in technical triplicates for real-time PCR reactions.
Microarray analysis was carried out using the Signal Transduction PathwayFinder cDNA array as per the manufacturer's instructions (Superarray). Nylon cDNA microarrays membranes were pre-hybridised with supplied sheared salmon sperm DNA at 60uC for one to two hours. The pre-hybridisation solution was replaced with the biotin-dUTP labelled cDNA probe, which was diluted in sheared salmon sperm DNA. The solution was left to hybridise at 60uC overnight. The following morning the cDNA probe was discarded and the membrane was washed twice then blocked in Solution Q, as provided by the manufacturer. After 40 minutes, the blocking solution was discarded and the membrane was incubated with a binding buffer, Solution F. After washing, the chemiluminescent substrate was incubated with the membrane for two to five minutes at room temperature. The microarray membrane was then exposed to X-ray film. Analysis was performed using GEArray Expression Analysis Suite as provided by the manufacturer (Superarray). All the data sets used in the microarray analysis are available in Table S1 .
cDNA derived from cells that were mock-infected or inoculated with 1000 particles/cell of entry-defective DgB, DgD and DgH virions was used for real-time PCR analysis. Primers were designed, using the Primer3 software [23] , to anneal at 60uC, with a minimum product melting temperature of 80uC (Table S2) . Reactions were set-up with cDNA corresponding to 100 ng total RNA, primers (70 nM), and 10 ml SYBR Green PCR master mix (Abgene) made to a total volume of 20 ml with nuclease-free dH 2 O. A Corbett Rotorgene 3000 was used to determine the levels of SYBR green fluorescence over 40 cycles. Samples were denatured for 10 minutes at 95uC, annealed at 60uC for 30 seconds, with extension of the primer product at 72uC for 40 seconds then a final 80uC step for 20 seconds that removes background fluorescence from primer-dimers. A melt curve analysis was produced at the end of the cycling to ensure the specificity of PCR product amplification and associated SYBR green fluorescence. Relative gene expression levels between mockinoculated and inoculated cells were calculated using the Pfaffl method with potential variations in cDNA quantity between samples normalised to the transcript for ribosomal protein L13a (RPL13a) [24] . Serial dilutions of PCR product across 8 logs were used to determine the efficiency of PCR amplification for each primer set under the above conditions so that the relative quantitation could be adjusted as defined by the Pfaffl method.
A relative change in expression of two-fold was set as a threshold for determining whether differential expression of a gene had occurred. The p values associated with the fold-change in expression were calculated using a Students t-test on the realtime PCR C T values comparing triplicate mock-inoculated and HSV inoculated cells.
An NF-kB reporter assay system was designed using a pGL4-NFkB construct (Promega). Quadruplicate repeats of the NF-kB binding consensus sequence 59-GGGAATTTCC-39 were excised from a p-NF-kB-Luc (Gift from Dr. Heike Laman, University of Cambridge) vector using KpnI and HindIII and ligated into the pGL4.20 vector multiple cloning site using the same restriction sites. pGL4.20 was chosen as it is optimized for more efficient expression in mammalian cells, compared to previous generations of luciferase constructs, and contains fewer transcription factor consensus sequences such that background luciferase expression is reduced. HFFs were electroporated in batches of 6610 5 cells per cuvet (Amaxa) with 2 mg of pGL4-NF-kB, pooled together and aliquoted at 10 5 cells per well of a 24-well plate. After 48-hours recovery in supplemented media, the transfected HFFs were serum-starved for five days. HFFs were inoculated with 1000 particles/cell of DgH HSV-1 and were lysed at the indicated time points in 100 ml passive lysis buffer (Promega) and 20 ml of lysate were examined for luciferase activity using 50 ml assay buffer (Promega).
Western Blotting gD and the major tegument protein, VP16, were detected by immunoblotting using the monoclonal antibodies LP2 and LP1, respectively [25] [26] . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as described previously [27] .
Signalling-specific microarrays were chosen as an initial route to identifying signalling pathways that may be stimulated early in HSV-1 infection, and more precisely, by virion binding. The Signal Transduction PathwayFinder cDNA array from Superarray incorporates 96 targets covering 19 associated signalling pathways, in addition to two negative controls corresponding to pUC18 plasmid DNA and a 'blank' spot, as well as four positive controls representing the housekeeping genes for GAPDH, beta actin, cyclophilin A and ribosomal protein L13a.
Serum-starved HFFs were inoculated with 1000 particles/cell of DgB, DgD or DgH entry-defective HSV-1 virions. Total RNA was isolated immediately after inoculation, corresponding to 0 hpi, and 6 hpi. Changes in gene expression associated with the activation of signalling pathways were identified by comparison to the transcript abundance at 0 hpi. The results from duplicate experiments indicated that downstream gene targets of a number of signalling pathways are differentially expressed above the twofold threshold after virion binding (Table 1) . These preliminary results were then used as the basis of quantitative PCR studies.
Using the same methodology, serum-starved HFF cells were exposed to 1000 particles per cell of virions lacking either gD, gB or gH and RNA was extracted for cDNA synthesis immediately (0 h) or after 6 h. Table 2 gives the results of three independent experiments for those genes that were differentially expressed between 0 h and 6 h post treatment and is compared to the initial microarray results in Table 3 . It is striking that in virtually every instance where gene expression is modified by virion binding, gD appears to be the key effector. Virions lacking gH or gB had were capable of altering cellular gene expression while those lacking gD had no significant effect. The results can be summarised as follows.
The JAK/STAT and related JAK/Src pathway show a similar response to that seen for NF-kB. Of the JAK/STAT-responsive genes, only NOS2A is regulated by more than one pathway, with DgB and DgH virions inducing their transcription and DgD particles having no significant effect ( Table 2) . Although the JAK/ Src pathway has only two targets on the signalling microarraysupon which the real-time PCR experiments were based -BCL2L1 is unique to the pathway, with BCL2 expression regulated by multiple signalling routes. Interestingly, while DgB and DgH virions up-regulated BCL2 and BCL2L1, DgD virus particles appeared to down-regulate both targets by a significant amount.
Induction of this pathway may be responsible for the upregulation of CCND1, MYC, BCL2 and MMP7 after inoculation with DgB and DgH but not DgD HSV-1 ( Table 2 ). Differential expression of CCND1 and MYC can be regulated by the Wnt and PI3K signalling pathways, with MYC also being controlled by Protein Kinase C. The up-regulation of MMP7, which is the only target unique to the PI3K pathway that is differentially expressed, highlights the potential crosstalk between signalling pathways and cannot exclude the involvement of PI3 kinase signalling as a pathway leading to transcription of all four cellular genes.
Both DgB and DgH virions are capable of stimulating an NF-kB response (Table 2) , a result that correlates with the preliminary microarray data (Table 1) . Transcripts for both BIRC-2 and BIRC-3 are increased above the two-fold threshold after inoculation with DgB and DgH HSV-1 but not DgD. There appears to be no change in BIRC-1 expression after inoculation with any of the three entry-defective mutants. Components of the NF-kB transcriptional complex, NFKB1 and REL, showed similar levels of increased transcription after binding by DgB and DgH virions, a response that was absent in cells stimulated with gDdeficient virions. Two markers of inflammation, CCL2 and PECAM1, also showed a significant change in gene expression at 6 hpi with entry-defective HSV-1 lacking gB or gH but not gD.
The downstream signalling appears specific as not all NF-kB targets were up-regulated. The NF-kB repressors, NFKBIA and NFKBIB, were only up-regulated by DgB virion binding ( Table 2) .
To confirm that virion binding induces NF-kB and to examine the kinetics, an NF-kB promoter-driven luciferase construct was transfected into HFF cells and cells were treated with DgH virions (1000 particles per cell) or were mock-treated. Cells were harvested every hour until 9 hr post treatment, lysed and assayed for luciferase activity in triplicate. Figure 1(a) shows the fold-change in luciferase activity of those HFFs inoculated with DgH in comparison to mock-inoculated cells at the same time point. Biological duplicates of the inoculations were performed with the lysates used in triplicate reporter assays. At 1 hpi, there is an average of 2.4 fold change in luciferase activity from the NF-kB reporter, rising to 3.5 at 2 hpi and is maintained through to 3 hpi whereby it drops to 1.8 at 4 hpi, which correlates with the half-life of luciferase [28] . By 5 hpi, and until 9 hpi, luciferase activity drops to baseline levels at, or near, 1-fold change. Error bars represent the standard error across the six readings taken from technical triplicates on biological duplicates. As induction of the NF-kB reporter construct occurred within one hour of inoculation with DgH virions and peaked at around two-and-a-half hours post-inoculation, then the transcripts previ- HFFs were stimulated with 1000 particles/cell of DgB, DgD or DgH HSV-1 for six hours and a cDNA microarray corresponding to targets of 19 signalling pathways was used to detect changes in cellular gene expression when compared to mock-infected. Infections and mock infections were carried out in duplicate. A number of signalling pathways were shown to be stimulated by one or more of the entry-defective mutants. These data were used to independently verify changes in expression using real-time PCR. doi:10.1371/journal.pone.0009560.t001 ously shown in Table 2 to be up-regulated as a result of DgH virions binding were examined for changes in transcript levels at two-and-a-half hours after inoculation with DgH. Figure 1 (b) compares expression levels at two-and-a-half and six hours postinoculation. Transcripts for the NF-kB-responsive chemokine CCL2 were up-regulated to a greater degree at two-and-a-half hours than at six hours post-inoculation. Of the NF-kB-responsive genes, REL transcription was induced to a higher level whereas PECAM1 transcript abundance was reduced in comparison to six hours. Although JAK/STAT-responsive targets were not differentially expressed by two-and-a-half hours, the JAK/Src targets BCL2 and BCL2L1 were up-regulated above the two-fold arbitrary threshold to similar transcript levels that were seen at six hours post-inoculation.
The initial microarray screen was uninformative with respect to interferon signalling, but this was of interest because soluble gD has been reported to induce IFN [29] , while, in contrast, ISG induction is reported to require virion entry but not de novo protein synthesis [30] . Primers were designed to Type I interferons, a (IFNA1) and H (IFNB1), and the newly designated Type III interferon l (IL29). Human fibroblasts do not produce interferon gamma. Additionally, the cDNA preparation was used to identify any change in the abundance of ISGs. Both DgB and DgH virions were able to stimulate the up-regulation of IFN-a expression 2.81 fold and 2.34 fold, respectively, with a high degree of statistical significance (p#0.05) ( Table 4 ). HSV-1 particles lacking gD were unable to do so, with a fold change of 21.11 and a p = 0.15 (Table 4) .
IFN-H transcript levels were up-regulated 2.22 fold by DgB virions and 2.06 fold (p = 0.01) by DgD virions, again with a high degree of statistical significance. Inoculation with virus particles lacking gH led to a 21.07 fold change (p = 0.44) in IFN-H expression when compared to mock-infected cultures. All three entry-defective mutants were unable to modulate the expression of IFN-l above the arbitrary two-fold threshold.
The glycosylation status of IFN-inducing viral glycoproteins is a potential determinant of induction efficacy [22] [31] . HSV-1 gD has three potential N-glycosylation sites but lacks O-linked glycosylation. DgH virions, that retain expression of gD, were treated with the endoglycosidase, PNGase F, which removes Nlinked sugars. A long incubation was required as the denaturing buffer was excluded from the reaction to avoid conformational changes that might abrogate virion binding. As previously shown, wild-type virions treated in a similar manner retained infectivity ( Figure S1 ) [22] .
HFF were inoculated as above with DgH virions that had been incubated for 18 hrs in the presence or absence of PNGase. Glycosylated and deglycosylated DgH virions stimulated a 2.23 and 2.28 fold increase in IFN-a transcription, respectively. These results suggest that glycosylation has no impact on the interferogenic properties of DgH virions, unlike the glycoproteins of MHV and TGEV, which appear to stimulate Type I IFN through There was a degree of correlation between the change in expression determined by microarray studies and those confirmed by real-time PCR, particularly for genes under the control of NF-kB. Note: rel and nfkbib were not present on the original signalling-specific microarray, but were included in the real-time analysis, as they are known to be up-regulated as part of the NF-kB feedback mechanism. doi:10.1371/journal.pone.0009560.t003 a lectin-like action, HSV-1 gD may trigger IFN up-regulation through a different mechanism, possibly analogous to that used to stimulate other signalling pathways. Five interferon-stimulated genes were chosen to examine whether they were differentially expressed by virion binding. IFN-induced protein 54 (ISG54) transcripts are routinely used as a marker of ISG induction. Binding by DgB, DgD or DgH virions was insufficient to cause changes in the transcript abundance of interferon regulatory factor-1, -3, -7, -9 or ISG54 (Table 4) .
To ensure that these observations could not result from a background of infection by competent virus, parallel cultures were infected with wild type virus at an MOI of 0.01, a level of infection some tenfold higher than would result from the 'worst case' scenario using preparations of entry defective virions (see Methods). These cultures exhibited no induction of IFN or ISGs ( Table 4 ).
All the experimental work described above was performed using treatments with 1000 virus particles per cell, a condition that would be unlikely to pertain in natural primary infections. To establish whether much lower virion numbers would be effective, HFFs were serum-starved for five days then mock-inoculated or inoculated with 1, 10, 100 or 1000 particles per cell of DgH HSV-1 for six hours. Infections were carried out in duplicate with mockinoculated controls that received an equivalent volume of PBS. Total RNA was purified and reverse transcribed, with the cDNA from each duplicate inoculation used in triplicate for real-time PCR.
The NF-kB responsive genes NFKB1, REL and CCL2, in addition to the JAK/STAT target NOS2A, were used as they had previously been shown to be differentially expressed after inoculation with 1000 particles/cell of DgH HSV-1. Recalling that the more abundant a transcript the fewer number of cycles it takes to cross a threshold set in the exponential phase of PCR product accumulation (the C T value), Figure 2 plots the DC T value on the y-axis, (i.e. the C T value for the gene of interest after normalisation against the RPL13) against the multiplicity of infection on the x-axis. As the multiplicity of DgH increases the C T value decreases, indicating an increase in the abundance of transcripts for all three NF-kB targets (Figure 2(a) ). A logarithmic regression analysis across three logs of multiplicity show a high correlation between the four C T values for each transcript with r 2 .0.98. Similarly for the JAK/STAT responsive target NOS2A there are almost two cycles of a difference between the level of transcripts after infection with 1 particle/cell compared to 1000 particles/cell (Figure 2(b) ). This larger response than those for NF-kB responsive genes is indicative of the different level of induction shown with previous real-time PCR analysis of these transcripts ( Table 2) .
Arrows in Figure 2 indicate the C T value for each of the four targets examined. Inoculation with 1 particle cell gave no detectable increase in transcript abundance but an increase was observed using 10 particles per cell and the dose response curves strongly suggest that lower doses have an effect. Interpreting these data in the context of infection is not straightforward as particle:infectivity ratios for herpes simplex virus are, at best, 20 or more. On this basis a single infectious unit per cell is clearly sufficient to increase transcript abundance, indicating that signalling occurs at 'physiological' multiplicities of infection.
Evidence from previous studies using soluble HSV-1 glycoprotein and UV-inactivated virus suggested that binding of HSV-1 virions to the cell surface might be sufficient to stimulate intracellular signalling pathways. We undertook preliminary microarray studies with entry-defective HSV-1 virions and identified a number of pathways that were stimulated at early time points during infection.
Microarray experiments are inevitably vulnerable to false positives and negatives due to non-specific binding of labelled cDNA probes, necessitating a more robust follow-up with highly sensitive methods. Due to such confounders, these data were used exclusively as a guide to select, for real-time PCR, the transcriptional targets of intracellular signalling pathways stimulated after inoculation with entry-defective HSV-1.
Gene targets associated with the NF-kB, JAK/STAT/Src, and PI3K/Akt pathways were shown to be differentially expressed after inoculation with glycoprotein-deficient virions, with the majority of signalling events being associated with the presence of gD on the envelope. Nineteen other signalling pathways present on the preliminary microarray experiments, and confirmed by real-time PCR, were not stimulated (data not shown). These results are compatible with our model, shown in Fig. 3 .
Real-time PCR confirmed that changes in transcription associated with the NF-kB, JAK/STAT, JAK/Src and PI3K pathways were modulated as a result of virion binding, all of which required gD on the envelope surface To demonstrate that signalling occurred at physiologically relevant multiplicities of infection, HFFs were inoculated with either 1000, 100, 10 or 1 particles per cell of entry-defective HSV-1. Changes in gene transcription occurred in a dose-dependent manner and were detectable at 10 virus particles per cell. Given the particle:infectivity ratios usually quoted for HSV1, we argue that this corresponds to physiological conditions (i.e. less than one infectious unit per cell), but it is impossible to know the circumstances that pertain in vivo when a single cell becomes infected. What seems almost certain is that an infected cell will normally present an uninfected neighbouring cell with 10 or more progeny particles.
HSV-1, as well as beta-and gammaherpesviruses, are capable of stimulating the NF-kB pathway in a bi-phasic manner, with our data supporting that an early, transient induction is reliant on virions expressing gD [8] [32] [33] . Suppression of NF-kB activity is via negative feedback up-regulation of the inhibitor IkBa (nfkbia), which was also stimulated by the binding of entry-defective HSV-1 virions. The triggering of early NF-kB transcriptional activity was most likely through the coupling of gD on entry-defective virions to the TNF superfamily receptor HvEM [1] . In doing so, not only does the initial activation of this pathway allow for the subsequent sequestration of the NF-kB p65 subunit to the ICP0 promoter, but is crucial for immediate-early gene transcription and subsequent HSV-1 replication [8] . Intracellular signalling induced by soluble gD can protect against Fas-mediated apoptosis with inhibition of NF-kB signalling leading to a loss of this protection [6] . Infection with UV-inactivated virions also led to an increase in the expression of the anti-apoptotic protein c-IAP2 (birc3), which we have demonstrated to be up-regulated after inoculation with entry-defective virions containing gD. Additional studies have supported the anti-apoptotic role for NF-kB during HSV-1 infection yet there are conflicting data that demonstrate possible pro-apoptotic activity [34] [35] . This inconsistency may be due to differing cell types used in those studies. Primary human foreskin fibroblasts have been shown to be resistant to apoptosis after infection with recombinant HSV-1 that is unable to express ICP4 or ICP27 whereas infection with either virus has been shown to cause apoptosis in transformed cell lines [36] .
Aspects of the HSV-1 life cycle, such as stimulating the progression of the cell cycle in the absence of serum, may be sufficient to induce a stress response and trigger apoptosis. Both bcl2 (Bcl-2) and bcl2l1 (Bcl-xl) belong to the Bcl-2 family of apoptosis regulators that provide cellular protection from a range of harmful stimuli such as cytokine deprivation, UV-and cirradiation [37] . Bcl-2 and Bcl-xl are found in the outer mitochondrial membrane and are thought to suppress apoptosis by blocking mitochondrial outer-membrane permeabilisation through the sequestration of pro-apoptotic Bcl2 family members [38] . Given the up-regulation of four anti-apoptotic genes, birc2, birc3, bcl2 and bcl2l1, through the activation of multiple signalling pathways by entry-defective HSV-1, this establishes a role for gD binding in shifting the intracellular environment towards a more anti-apoptotic stance. . Model for glycoprotein-receptor interactions in the induction of intracellular signalling pathways by HSV-1. Glycoprotein D acts as the main signalling molecule on the surface of the HSV-1 envelope. gH interacts with a v b 3 integrins to potentially trigger the production of IFN-b, which is known to involve IRF-3 and 7 [48] . Binding by gD to HvEM may lead to the activation of TRAF molecules, which in turn stimulate the NF-kB signaling cascade. This pathway up-regulates a number of cellular genes in addition to augmenting early viral gene expression. NF-kBresponsive genes, birc2 and birc3, have an anti-apoptotic role, but paradoxically, inflammatory mediators such as ccl2 are also up-regulated. gDinduced signalling of the Jak/Stat and Jak/Src pathways also results in the differential expression of genes associated with anti-apoptosis and inflammation. The up-regulation of c-Myc could lead to a corresponding increase in cdk2, which has a role in promoting DNA replication and gene transcription during infection. It should be noted that most signalling cascades have been elucidated in non-fibroblast cells lines, so the role of specific kinases may vary in HFFs. doi:10.1371/journal.pone.0009560.g003
It is less apparent as to the biological relevance of an innate immune response stimulated through HSV-1 binding. It may be that there is a ''cost'' associated with altering the intracellular environment, which leads to the differential expression of cytokines, such as ccl2, that are under similar transcriptional regulation as those host factors that are favourable for virus replication.
Signalling by secreted Type I IFNs occurs through the Jak/Stat pathway results in the expression of various ISGs; a response that is also triggered by virus entry [39] . Nevertheless, productive infection with HSV-1 can down-regulate the triggered ISG response, allowing viral replication to continue unhindered [40] . Despite our evidence that gD binding by entry-defective virions can induce IFN-a mRNA expression, independent of gD glycosylation status, these data also fit with published observations that binding by HSV-1 is insufficient to cause the up-regulation of interferon-stimulated genes [30] . The up-regulation of IFN-b, albeit it through a different mechanism, is suggestive of a previously unidentified role for gH in eliciting a change in host gene expression. An entry-defective HSV-1 mutant lacking gB that also contains an RGE rather than the integrin binding RGD motif of gH has been constructed and future studies may further elicit the role of gH in interferon stimulation.
The methodology used here required the serum-starvation of primary human fibroblasts for five days. In the absence of serum, primary fibroblasts rapidly enter a quiescent state. As a DNA virus that requires host nuclear factors to replicate its genome, it is therefore not surprising that HSV-1 would stimulate cells from a G0 state into one that would favour DNA replication and possibly promote the transcription of viral genes.
Quiescent cells in vitro have very low levels expression of the transcription factor c-Myc. Its up-regulation is rapidly induced after mitogenic stimulation or the introduction of serum and increased expression of c-Myc is consistent with the advancement of cellular proliferation [41] . Control of myc transcription can be influenced by a number of pathways, including PI3K/Akt signalling, which was shown here to occur as a result of binding by gD. A central role for c-Myc in promoting cell-cycle progression is evident from the genes that it can up-regulate such as eIF2 [42] . Progression of the cell cycle depends on the additional activity of cyclin-dependent kinases. By interacting with the promoters for genes encoding cyclins and cyclin-dependent kinases, c-Myc can influence the advancement on the cell cycle into the G1/S phase [43] .
Cyclin-dependent kinase 2 (CDK2) is one such downstream target of c-Myc activity, as well as the Androgen pathway, highlighting the signalling cross-talk that may occur [44] . CDK2 is involved in the progression of the cell cycle from G1 through to S phase. Transient activation of CDK2 was shown to occur early in HSV-2 infection at two hours post-infection, and is crucial in early HSV-1 infection [45] [46] . Kinase action by the cyclin A/CDK2 complex liberates the bound transcription factor E2F from Rb, a transcription factor that has previously been shown to be active during HSV-1 infection [47] .
Epithelial cells at the initial site of HSV-1 infection in vivo are likely to be in a resting state, necessitating the virus to evolve a preentry signalling mechanism by which to stimulate the cell to provide host factors that are necessary for viral replication. We have demonstrated that signalling induced by HSV-1 glycoproteins, primarily gD, has the potential to: activate cellular transcription factors that augment viral gene transcription, differentially express a number of cellular genes so as to condition the cell for optimal replication or, alternatively, signal transduction may occur as a secondary effect to the appropriation of cellular receptors to achieve viral entry.  Seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort study in Berlin, Germany, 2006/07 BACKGROUND: Influenza immunisation for healthcare workers is encouraged to protect their often vulnerable patients but also due to a perceived higher risk for influenza. We aimed to compare the risk of influenza infection in healthcare workers in acute hospital care with that in non-healthcare workers over the same season. METHODS: We conducted a prospective, multicentre cohort study during the 2006/07 influenza season in Berlin, Germany. Recruited participants gave serum samples before and after the season, and completed questionnaires to determine their relevant exposures and possible confounding factors. The main outcome measure was serologically confirmed influenza infection (SCII), defined as a fourfold or greater rise in haemagglutination inhibition antibody titres to a circulating strain of influenza (with post-season titre at least 1:40). Weekly mobile phone text messages were used to prompt participants to report respiratory illnesses during the influenza season. A logistic regression model was used to assess the influence of potential risk factors. RESULTS: We recruited 250 hospital healthcare workers (mean age 35.7 years) and 486 non-healthcare workers (mean age 39.2 years) from administrative centres, blood donors and colleges. Overall SCII attack rate was 10.6%. Being a healthcare worker was not a risk factor for SCII (relative risk 1.1, p = 0.70). The final multivariate model had three significant factors: living with children (odds ratio [OR] 3.7, p = 0.005), immunization (OR 0.50, p = 0.02), and - among persons living in households without children - ownership of a car (OR 3.0, p = 0.02). Living with three or more children (OR 13.8, p < 0.01) was a greater risk than living with one or two children (OR 5.3, p = 0.02). 30% of participants with SCII reported no respiratory illness. Healthcare workers were at slightly higher risk of reporting any respiratory infection than controls (adjusted OR 1.3, p = 0.04, n = 850). CONCLUSIONS: Our results suggest that healthcare workers in hospitals do not have a higher risk of influenza than non-healthcare workers, although their risk of any respiratory infection is slightly raised. Household contacts seem to be more important than exposure to patients. Car ownership is a surprise finding which needs further exploration. Asymptomatic infections are common, accounting for around a third of serologically confirmed infections. The German standing commission for immunisation, along with other authorities [1] [2] [3] currently recommends that healthcare workers (HCWs) be vaccinated against seasonal influenza. Two reasons are cited: firstly that HCWs can be a source of infection for vulnerable people under their care [1] [2] [3] , and secondly that HCWs are at increased risk for contracting influenza [1] . Vaccination of HCWs should theoretically reduce the risk of influenza infection both in themselves and their patients. There is evidence to support the first reason for vaccination, the protection of patients. HCWs can transmit influenza to those under their care during both outbreaks and non-outbreak situations [4] [5] [6] [7] . Low vaccination rates in HCWs have been associated with nosocomial outbreaks [4, 8] , and higher vaccination rates with reduced nosocomial influenza incidences [7] .
HCWs may transmit influenza to those under their care, but is there evidence that their occupational exposures (to patients, relatives, colleagues and the hospital environment) confer an increased risk of influenza compared to the general population? Influenza serological attack rates in HCWs of 23% (single season, [9] ) and 14% (an average of two seasons, [10] ) have been documented. However, as serological attack rates of influenza may vary considerably from season to season as well as from location to location, without the inclusion of a comparison group of non-HCWs neither study could demonstrate an increased risk.
Another aspect of influenza in HCWs is that many HCWs argue that they withdraw from work when they become ill with influenza-like illness to reduce their risk of transmitting influenza to their patients. However, not all serologically diagnosed influenza infections experience an influenza-like illness, and a proportion will be asymptomatic. The frequency of asymptomatic influenza infection in HCWs has been assessed in volunteer studies (33%, [11] ), a cohort study (28%, [9] ) and one randomised controlled study (42%, [10] ).
The objective of this study was to address the question of whether HCWs in the acute care hospital setting have a higher risk of serologically confirmed influenza infections (SCII) than non-HCWs, and to assess the proportion of individuals with SCII who experience either any respiratory symptoms or an influenza-like illness.
We conducted our study in people living or working in Berlin during the influenza season of 2006/07, using a multicentre, prospective cohort design.
There were 11 study sites: three hospitals, two administrative centres (the Robert Koch Institute and Vivantes Healthcare administrative centre), four blood-donation centres and two colleges. HCWs and some non-HCWs were sought from the hospitals, but only non-HCWs were sought from all other sites.
We recruited participants through occupational health services in the hospitals and one administrative centre; through direct recruitment at blood donation centres; and through active recruitment during site visits at the other study sites (non-healthcare workers from one administrative centre and two colleges).
On recruitment before the influenza season, consenting participants gave a single serum sample and completed an exposure questionnaire. Details recorded included age, sex, type of employment, risk factors for influenza, smoking status, and vaccination status. Where participants had been vaccinated fewer than 14 days before the sample was taken, or were found to have been vaccinated shortly after the initial sample was taken, a second sample, taken at least 14 days after vaccination, was sought through direct recall or through occupational health.
Blood samples were refrigerated, then transported within three days to the national reference laboratory for influenza at the Robert Koch Institute in Berlin, where they were centrifuged and frozen.
HCW in our context were defined as people working on a daily basis with unwell patients in an acute hospital setting, including nurses (trainee and qualified) and doctors. Non-HCW were those working or studying at the study sites, or attending the blood donation centres, who did not fit the definition of HCW.
Exclusions (applied to both HCWs and non-HCWs) were: people with patient contact in the community (such as community doctors and nurses, dentists, and pharmacists); people working in care homes; laboratory workers who had contact with respiratory samples or with influenza virus; people who planned to be away from Berlin for more than two weeks during the projected season (January to April); and people who did not wish to be contacted weekly by mobile phone Short Message System (SMS) or email.
For the analysis of serologically-confirmed infections, we excluded vaccinated individuals where the baseline serum sample was taken fewer than 14 days after vaccination, or where a later second sample was not obtainable (see recruitment above). These participants were excluded from the serological analyses as any titre rise could have been caused by vaccination and not infection.
Because pigs can carry influenza viruses, and participants with pig contact were associated with SCII, participants reporting contact with pigs were excluded.
In order to document weekly occurrences of respiratory infections during the influenza season, we contacted all participants weekly through SMS or through email, asking them if they had experienced a "new" respiratory infection during the previous 7 days. Where participants answered "yes", they were contacted by telephone and details of their infection were obtained using an illness questionnaire.
Weekly surveillance for respiratory infections covered the period from January 13, 2007 to March 30, 2007, a period chosen to coincide with the influenza season. This strategy was employed in order to maximise the predictive value of a positive answer.
After the influenza season, participants were recalled by SMS or email. They gave a second serum sample and completed a further questionnaire, including repeat questions on vaccination status and employment type; number of patient contacts on a typical day between January 13 and April 6, 2007; broad age classification of patient contacts (adults (>17 years) and children (<18 years); clinical specialty and usage of facemasks (for HCWs); daily professional and household contacts; use of public transport and car ownership; contact with pigs (for veterinary students); and vaccination in previous years. Participants were asked again if they had had respiratory infections over the period January 13, 2007 to 6 th April 2007, extending the period of surveillance to one week after the last weekly SMS was sent.
Contact was defined as either touching or having a two way conversation with someone close by, or (for patient contact only) examining or giving care to a patient. Participants were asked to estimate the number of contacts made during a typical day in household and work settings.
The paired blood samples were defrosted and antibody titres were determined on the same day using the haemagglutination inhibition test to determine infection in any of the two A subtypes or the two B lineages. We tested for antibody to the following strains: Titres of below 10 were assigned the value of 5 in order to allow calculation of the titre rise. SCII was defined as a fourfold or greater titre rise between preand post-season samples, with a postseason titre of at least 40. As A/Wisconsin/67/2005 (H3N2) and A/ California/07/2004 (H3N2) were closely related a titre rise to either of these strains was considered as a single SCII due to A/H3N2.
The primary outcome was evidence of SCII by any of the above strains.
The clinical outcomes were influenza-like-illness (ILI), defined as an illness with an acute onset, self-reported fever, cough, and head or body pains; and acute respiratory infection (ARI), defined as any reported infection with coryza (nasal discharge) or cough. Clinical outcomes were based only on completed illness questionnaires or postseason illness reports, not on SMS or email replies, the latter being used only as the prompt for collection of illness data.
Where illness was reported over more than one week, symptoms for each week were combined to produce a single illness episode. To produce an epidemic curve of SCII with any ILI, or in its absence, another ARI, the respective dates of illness onset were plotted. Where more than one illness episode was reported, we used the onset date of the episode closest to the peak, on the assumption that this episode was the most likely to be due to influenza virus.
We undertook bivariate analyses for all binary exposure variables and calculated risk ratios (RR), their 95% confidence interval and p-values. For the comparison of the exposure groups (HCW vs. non-HCW) continuous variables were analysed using the Kruskal-Wallis test. For the analysis of the association with SCII continuous variables were explored by grouping them in categories. We regarded a p-value of less than 0.05 as statistically significant. We then constructed a multivariate logistic regression model (logistic command, STATA [StataCorp. 2007. Stata statistical software: Release 10. College Station, TX: Statacorp LP]) using variables which were associated with the outcome with a p-value of less than 0.1 in the bivariate analysis. Variables with p values between 0.1 and 0.2, along with healthcare worker status, were also tested in the final model.
In order to determine whether the site of recruitment had any group-level effects on the model, we constructed a random-effects logistic regression model with the same variables as the standard model plus study site as the grouping variable, and a likelihood ratio test for the proportion of variance attributable to the group level (rho) was performed (xtlogit, STATA).
The data protection protocol was approved by both state and national data protection offices in Berlin, and shared with all study partners. Ethical approval for the study was obtained from the University of Berlin, faculty of medicine (Charité) ethics committee.
We recruited 1044 participants, of which 736 (71%) were included in the analysis with SCII as outcome, and 866 (93%) for the clinical outcome analysis ( Figure 1 ).
Those not included in the study were of younger age (38 vs 34, p < 0.01 Kruskall-Wallis) and were more likely to be HCW at recruitment (42% vs 34%, p < 0.01), but there were no significant differences in sex, current immunisation, smoking status, car ownership or use of public transport in excluded versus non-excluded participants.
Of the 736 participants included in the analysis with the outcome SCII 250 (34%) were HCWs and 486 (66%) were non-HCWs. Most participants (71%) were female. The age distribution was bimodal, with peaks in the age groups 20-29 years and 40-49 years both in HCWs and non-HCWs.
Of the 250 healthcare workers, 41 (16%) were doctors, 97 (39%) were trainee nurses and 112 (45%) were qualified nurses. Of the 486 non-healthcare workers, 178 (36%) were administrative or information technology staff, 107 (22%) scientific staff, 45 (9%) students, 6 (1%) teaching or lecturing staff, 25 (5%) in manual or technical roles, 21 (4%) in retail or service, 9 (2%) veterinary staff, and 7 (1%) in other occupations. In 88/486 (18%) the occupation was not stated or unclear. Table 1 compares study characteristics in the exposure group (HCW) and control group (non-HCW). HCWs were significantly younger than controls (35.7 versus 39.2, Kruskal-Wallis test, p < 0.01). They were also significantly more likely to be current smokers, female, vaccinated in the current season, and to own a car.
Healthcare workers had significantly more non-patient contacts at work (mean 14.3 versus 13.8, p = 0.021) but fewer contacts at home (mean 2.7 versus 3.5, P < 0.01) There were no significant differences in immunisation in the previous three seasons, or in household exposure to children. HCWs were significantly less likely to have a titer of <40 against A/California/07/2004, the A/ H3N2-strain of the previous season.
In total there were 82 titer rises among 78 participants. Of these, 13 (16%) were due to A/New Caledonia/20/ 1999 (H1N1), 64 (78%) due to either of the two A/ H3N2-strains tested, two (2%) due to B/Malaysia/2506/ 2004 and three (4%) due to B/Jiangsu/10/2003. There were four double-infections, three with H3N2/H1N1 coinfections and one with titre rises to both B strains. Table 2 shows SCII and reported infections by healthcare worker status. The overall attack rate for SCII was 10.6% (78/736). Of the 78 people with evidence of SCII, 23 (30%) reported neither ARI nor ILI, 33 (42%) reported at least one ARI but no ILI, and 22 (28%) reported at least one ILI.
HCWs did not have a significantly higher risk of SCII than non-HCWs (RR 1.09, p = 0.70). In addition, neither working as a nurse (RR = 0.94, p = 0.82) nor as a doctor was significant (RR = 1.76, p = 0.13). There was also no significant difference in the risk of SCII between HCWs and controls after stratification by vaccination status, car ownership, having children, or regular use of public transport.
There were three exposures with p-values below 0.1: vaccination, having a car in the household and having three or more children at home (Table 3) . Household contacts increased the unadjusted risk of SCII ( Figure  2 ). Attack rates were lowest in those living alone (4%), intermediate in those living with adults but no children (10%) or one or two children (12%), and highest in those with three or more children in the household (24%). In a stratified analysis, the effect of car ownership was significant when participants did not live with children (RR = 2.77, p = 0.01), but not for participants who lived in a household with children (RR = 0.98, p = 1.00).
Variables significantly associated with SCII were the type of household contact environment, car ownership, and vaccination status (Table 4 ). Living with children was associated with an increased risk of SCII, with an overall odds ratio of 3.7 (p < 0.01, separate model without stratification of household contacts). Three or more children in the household (OR13.8, p < 0.01) were a greater risk than one or two children (OR 5.3, p = 0.02).
Household car ownership was a significant risk factor only among persons living in households without children and had an odds ratio for SCII of 3.0 (p = 0.02), while owning a car in households with children was not statistically significant (OR = 0.95; p = 0.94) and so was not included in the final model.
Immunisation against influenza was associated with an OR of 0.50 (p = 0.02). Vaccine effectiveness against SCII was therefore 50% (95%CI 12-71%). Effectiveness against SCII with a reported ILI was higher at 73% (95%CI 6-92%; p = 0.04). Other variables were added to the multivariate model, but were found not to be significant at the p < 0.2 level. These were being a doctor; having 7 or more child patient contacts; and being a healthcare worker. Addition of a group-level variance term for recruitment site to the random-effects model was not statistically significant (p(rho = 0) > = 0.498).
The four variables with a p-value < 0.2 in the bivariate analysis were age below 51 (p = 0.02), female sex (p = 0.03), HCW (p = 0.03) and smoking (p = 0.12). In the multivariate model (n = 850) all except female sex (p = 0.054) were significant, and the effect of smoking became statistically significant. Age below 51 (OR 1.44, p = 0.04), female sex (OR 1.36, p = 0.05) and HCW status (OR 1.34, p = 0.04) had an OR above 1, and smoking had an OR below 1 (OR 0.72, p = 0.04). Figure 3 shows the distribution of dates of onset for all SCII where an episode of ARI or ILI was reported.
The peak seen in week 9 corresponded with the peak number of positive influenza tests from Berlin patients performed at the national reference laboratory for influenza, from patient samples collected through the German influenza sentinel surveillance system [12] .
We found no significant association between being a healthcare worker in acute hospital care and serogically confirmed influenza infection. Instead we identified household contacts, in particular children, and car ownership, as important risk factors for SCII. 30% of participants with SCII reported no symptoms, and only around one-quarter of those reporting influenza-like illness also had SCII.
We did not demonstrate a strongly increased risk of influenza infection in HCWs in acute hospital care. As HCWs do appear to have an increased risk for ARI (OR = 1.3) it would be plausible to expect an effect of similar order for influenza infections. Although study limitations might have led to a failure to detect a true increase, it is unlikely that we have missed a large difference in the risk of influenza. Why might HCWs in hospital not have a higher risk of influenza? The prevalence of infectious influenza in patients may have been low, either due to the absence of influenza patients or because patients admitted later in the course of illness might have been less infectious. Also, infection control measures such as use of personal protective equipment and individual or cohort isolation might have reduced the risks of infection in HCWs. Lastly, prior immunity may have played a role. HCWs were less likely than non-HCWs to be susceptible (preseason titer of less than 40) to A/California/07/2004 (H3N2), which was the A/H3N2 strain of the previous season and related to A/Wisconsin/67/2005 (H3N2), the dominant strain in the 2006/07 season.
As most influenza patients admitted to hospitals are children or older people, one might expect to find an increased risk for SCII in HCWs who work with these patient groups. Our finding of an elevated odds ratio for HCWs with more frequent contact with child patients, albeit not significant in this setting, would be consistent with this supposition. Another study focusing on this hypothesis is needed to investigate it further.
Household contacts, and in particular children in the home, were the main significant risk factors identified in Table 4 Logistic regression model for persons with serologically confirmed influenza infection as outcome (n = 727) Exposure
Number Figure 3 Cases of serologically confirmed influenza infection reporting acute respiratory illness (ARI; blue) and influenza-like illness (ILI; red), by date of onset of reported illness (n = 53). Green line (on secondary y-axis): smoothed line of influenza cases in Berlin identified at the national reference laboratory for influenza from samples collected within the German sentinel surveillance system [12] .
this study. This finding is similar to that in another droplet-transmitted infection, meningitis, where household contacts constitute by far the most important risk group [13] . We found a strong dose-response relationship for child household contacts. The role of children as the main sources of influenza transmission has been suggested in several studies [14] [15] [16] [17] . Adult household contacts may play a role analogous, but possibly less marked, to that of children. Whilst public transport usage was not associated with SCII, car ownership was a significant risk factor, albeit only in households without children. This was unexpected, as car ownership was only included as a counterpart question to public transport usage. For this reason, further details on passengers, frequency or duration of car use were not obtained in the study.
Possession of a car has not previously been recognised as a risk factor for influenza acquisition. However, sharing a car does involve prolonged close contact in an enclosed space and has been linked to transmission of one airborne pathogen [18] . In one observational study on risk factors for SARS in China "using a taxi more than once a week" was identified as independent risk factor for SARS infection with an increased odds ratio close to significance (p = 0.07). Furthermore, this variable was kept in the multivariate model while "riding a bus" and "taking the subway" (who were significant in bivariate analysis) were not [19] . Ownership of a car may also be a marker for having a greater number of social contacts, perhaps including children. Car users are also exposed to air pollution from other vehicles, and this may predispose users to respiratory infections. Despite the unexpected nature of this finding, it is possible that car usage is indeed a risk factor and should be explored through further studies.
We did not find an increased risk of influenza in the 58% of participants who were regular users of public transport. Public transport usage has been cited as a possible risk factor for influenza infection, particularly in the context of pandemic planning [20] . In a recent international survey of precautionary behaviour for pandemic influenza, 75% of respondents said that they would avoid public transport [21] . Both rail and air travel have been associated with respiratory infection transmission [22, 23] . Whilst it is possible to contract influenza on public transport, our results suggest that, at least in Berlin, using public transport does not increase a person's risk of influenza. In Berlin, crowding on public transport is infrequent and not extreme, so this result may not be generalisable to cities with different conditions.
Vaccine uptake was relatively high both in HCWs (42%) and non-HCWs (32%). Reasons for this high HCW uptake include the participation of a hospital from former East Germany where vaccination uptake in hospitals is still found to be higher than in West German hospitals. In non-HCWs, the participation of a federal public health institute where influenza vaccination is freely offered to employees may account for the high vaccination rate. High vaccination uptake limited the number of SCII and reduced the study power. Vaccination effectiveness was 50% against SCII and reached 73% for a more severe outcome, SCII with reported ILI.
Only 28% of participants with SCII reported an ILI, the majority reporting no symptoms or more minor respiratory illness. The proportion of asymptomatic infections (30% or more) was substantial and similar to that reported by Elder (28%, [9] and consistent with an estimated proportion of 33% obtained from pooled challenge studies [10] . In addition, only 26% of persons with an ILI had evidence of an influenza infection. Thus, the ILI-syndrome was a poor marker for influenza infections. Unvaccinated HCWs who intend to withdraw from work when they become ill with influenza-typical symptoms may overlook many (symptomatic or asymptomatic) influenza infections, putting patients at unnecessary risk.
The association of HCW status and ARI may reflect the fact that HCWs communicate with or care for a large number of people including patients, colleagues, and relatives, and are thus exposed to a wide variety of respiratory pathogens. The reduced risk for ARI in people aged over 50 years might be explained by their cumulative immune experience or a lower contact rate. The apparent protective effect of smoking could be due to lower ascertainment of ARI in this group, where a background of smoking-related symptoms may have masked the onset of an additional, mild infectious respiratory illness. Conversely, respiratory symptoms due to non-infectious causes may be over-reported in non-smokers.
What significance does this study have for the current H1N1v pandemic? As pandemic influenza should result in a higher attack rate than seasonal influenza, the prevalence of influenza in hospital inpatients and staff is likely to be higher than for seasonal influenza, so increasing the risk of exposure and infection for HCWs compared to that described here. For the same reasons, the risk in HCWs and non-HCWs from their household contacts would also be higher than for seasonal influenza. Therefore even if a repeat study during pandemic conditions identified a significant occupational risk for HCWs, household exposures might still be more strongly associated with influenza infection.
This study was subject to a number of limitations. As recruiting at the hospital and one administrative study site was done through the occupational health departments, only a small proportion (fewer than 10%) of the HCWs at these sites were enrolled in the study. Selection bias could have concealed any actual relationship between HCWs status and influenza infection, if HCWs with a lower risk of influenza than their non-recruited colleagues were to have enrolled, or if non-HCWs with a higher risk of influenza enrolled. It is possible that HCWs with a higher number of patient contacts and, therefore in theory a higher risk of influenza, would be less likely to participate due to pressure of work. Alternatively, HCWs who were concerned about their higher risk of influenza may have protected themselves better and may have been more likely than other HCWs to participate, which would tend to decrease the relative risk in this group.
Loss of recruited participants, mostly due to losses to follow up (despite repeated attempts to contact) and to the timing of vaccination, reduced the effective power of the study and may have worsened selection bias, although excluded participants did not differ significantly with respect to variables found to influence the outcome.
Although there were differences between the two comparison groups with respect to sex, recent immunisation, smoking and use of public transport, the logistic regression methods should have adjusted for these where they had an effect on the outcome (in particular immunisation). The lower average age and higher proportion of females in the HCW group is likely to be due to recruitment among trainee nurses in the larger hospitals.
Serological testing for influenza infection alone, rather than molecular testing methods, may have underestimated the true number of influenza infections [24] . This, along with the relatively high vaccination uptake, will have reduced the number of SCII and thus the effective study power. With an attack rate of 10% in non-healthcare workers, the sample size of 736 analysed would not have been sufficient to detect a relative risk below 1.8 (80% power, 5% significance).
We explored the possibility of influenza clustering due to site-level group influences or localized influenza outbreaks. Analysis of symptom onset dates revealed no evidence of separate site-specific outbreaks, but instead the epidemic curves at each study site followed the overall trend. In addition the multilevel analysis suggested that there were no significant group-level influences on the outcome.
Our study results suggest that HCWs in an acute hospital care setting are at no higher risk of influenza than the general public, or that if they are, the increased risk is modest. Household contacts, particularly children, play an important role with individuals with three or more children being at highest risk. Use of public transport does not increase the risk of influenza, whereas (in the absence of household children) car ownership does seem to significantly increase the risk for influenza infection, although the mechanism is unclear. Further research would help to clarify the role of household contacts of different age groups, the relevance of car ownership, and whether subgroups of hospital HCWsor HCWs in other settings, such as in primary care,are at increased risk for influenza infection. Finally, the ILI-syndrome is a poor marker for influenza infection, suggesting that HCWs cannot rely on this syndrome if they wish to confidently protect their patients but, should instead be vaccinated. Predicting Drug-Target Interaction Networks Based on Functional Groups and Biological Features BACKGROUND: Study of drug-target interaction networks is an important topic for drug development. It is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. As a complement, the in silico prediction methods can provide us with very useful information in a timely manner. METHODS/PRINCIPAL FINDINGS: To realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. The optimal feature selection procedures are adopted by means of the mRMR (Maximum Relevance Minimum Redundancy) method. Instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, G-protein- coupled receptors and nuclear receptors. Thus, four independent predictors are established using the Nearest Neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. As a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are 85.48%, 80.78%, 78.49%, and 85.66%, respectively. CONCLUSION/SIGNIFICANCE: Our results indicate that the network prediction system thus established is quite promising and encouraging. Identification of drug-target interaction networks is an essential step in the drug discovery pipeline [1] . The emergence of molecular medicine and the completion of the human genome project provide more opportunity to discover unknown target proteins of drugs. Many efforts have been made to discover new drugs in the past few years. However, the number of new drug approvals remains quite low (around only 30 per year). This is partially because many compounds or drug candidates have to be withdrawn owing to unacceptable toxicity. Such failures have wasted a lot of money. It would be beneficial to develop computational methods for predicting the sensitivity and toxicity before a drug candidate was synthesized [2, 3, 4] . However, a number of problems need to be overcome in order to find out the exact effects of a drug. Firstly, drugs could have numerous effects including positive and negative effects, and it is hard to find out and elucidate the possible effects; secondly, different people would have completely different responses to a drug even though the same gene products are only slightly different [5, 6, 7, 8] ; thirdly, it is very hard to trace the drug effects since the biological interaction pathways are extremely complicated in human beings. Therefore, it would be very helpful for drug development if the interactions between drugs and target proteins could be predicted more accurately and the underlying mechanisms could be better understood.
Several computational approaches have been developed for analyzing and predicting drug-protein interactions. The most commonly used are docking simulations [9, 10, 11, 12] , literature text mining [13] , and combining chemical structure, genomic sequence, and 3D structure information [14] , among others (see, e.g., [15, 16, 17] ).
Machine learning and data mining methods have been widely used in the computational biology and bioinformatics area. Many researchers have made lots of efforts to develop useful algorithms and softwares to investigate various drug-related biological problems, such as HIV protease cleavage site prediction [18, 19] , identification of GPCR (G protein-coupled receptors) type [20, 21] , protein signal peptide prediction [22] , protein subcellular location prediction [23, 24, 25] , analysis of specificity of GalNAc-transferase protein [26] , identification of protease type [27, 28] , membrane protein type prediction [29, 30, 31, 32] , and a series of relevant webserver predictors as summarized in a recent review [33] .
Here we propose a predictor for drug-target interactions based on the Nearest Neighbor algorithm [34] . Since biochemical and physicochemical features [35] are important for characterizing proteins, in this study they are used to represent proteins as done by many previous investigators (see, e.g., [36, 37, 38] . To improve the predictor's performance, minimum Redundancy Maximum Relevance (mRMR) algorithm [39] is used to rank the features. Meanwhile, the Incremental Feature Selection and Forward Feature Selection are applied for feature selection. The protein targets for drugs are divided into enzymes, ion channels [40, 41, 42, 43] , GPCRs [44, 45] , and nuclear receptors [14] in this study. Finally, four predictors for predicting the interactions of drugs with each of the four protein families are developed in hopes that they can help provide useful information for drug design.
In addition to the dataset used by Yamanishi et al. [14] , information about drug compounds and genes can be obtained from KEGG [46, 47] by the FTP operations: ftp://ftp.genome.jp/ pub/kegg/ligand/drug/drug for the drugs, and ftp://ftp.genome. jp/pub/kegg/genes/fasta/gene.pep for the genes. After excluding the drug-target pairs that lack experimental information, we finally obtained a total of 4,797 drug-target pairs, of which 2,719 for enzymes, 1,372 for ion channels, 630 for GPCRs, and 82 for nuclear receptors. All these datasets were used as the positive datasets in the current study.
The corresponding negative datasets were derived from the above positive datasets via the following steps: (1) separate the pairs in the above positive dataset into single drugs and proteins;
(2) re-couple these singles into pairs in a way that none of them occurs in the corresponding positive dataset; (3) randomly picked the negative pairs thus formed until they reached the number two times as many as the positive pairs.
The drug-target benchmark datasets thus obtained for enzymes, ion-channels, GPCRs, and nuclear receptors are given in Online Supporting Information S1, S2, S3, and S4, respectively.
Representing drugs with chemical functional groups composition. The number of drugs is extremely large. However, most of them are small organic molecules and are composed of some fixed small structures, called functional groups. Since functional groups usually represent the characteristics of a compound as well as its reaction mechanism with other molecules, features derived from its functional groups could be very effective in characterizing a drug. Moreover, the number of common functional groups is quite small, and hence it is possible to use the functional group composition to uniquely represent a drug [48] . A number of functional groups are available in nature, and we selected the following 28 common groups for the current study: (1) alcohol, (2) aldehyde, (3) amide, (4) amine, (5) hydroxamic acid, (6) phosphorus, (7) carboxylate, (8) methyl, (9) ester, (10) ether, (11) imine, (12) ketone, (13) nitro, (14) halogen, (15) thiol, (16) sulfonic acid, (17) sulfone, (18) sulfonamide, (19) sulfoxide, (20) sulfide, (21) a_5c_ring, (22) ar_6c_ring, (23) non_ar_5c_ring, (24) non_ar_6c_ring, (25) hetero ar_6_ring, (26) hetero non_ar_5_ring, (27) hetero non_ar_6_ring, and (28) hetero ar_5_ring. Thus, following the same treatment as in [23] , a drug compound can now be formulated as a 28-D (dimensional) vector given below:
where g i (i~1, 2, Á Á Á , 28) is the occurrence frequency of the i-th functional group in the drug D, and T the matrix transpose operator.
Representing target proteins with pseudo amino acid composition by incorporating biochemical and physicochemical features. Now the problem is how to effectively represent a target protein. Two kinds of representations are generally used in this regard: the sequential representation and the non-sequential representation. The most typical sequential representation for a protein sample is its entire amino acid sequence, which can contain the most complete information of a protein.
To deal with this model, the sequence-similarity-searchbased tools, such as BLAST [49] , are usually used to find the desired results. Unfortunately, this kind of approach failed to work when the query protein did not have significant homology to the proteins in the training dataset. Thus, various non-sequential representations or discrete models were proposed. The simplest discrete model was based on the amino acid composition (AAC) (see, e.g., [50] ). However, if using the AAC model to represent a protein, all its sequence-order information will be lost. To avoid completely losing the sequence-order information, the pseudo amino acid composition (Pse-AAC) was proposed [36] to represent the sample of a protein. The PseAAC can be used to represent a protein sequence with a discrete model yet without completely losing its sequence-order information. For further information about PseAAC, see the web-page by clicking the link http://en. wikipedia.org/wiki/Pseudo_amino_acid_composition. Ever since the concept of PseAAC was introduced, it has been widely used to study various problems in proteins and protein-related systems (see, e.g., [37, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66] ). Meanwhile, many different forms of discrete models were also proposed (see, e.g., [20, 30, 32, 51, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82] ). However, regardless of how much different these models are, they just belong to different forms of PseAAC, as elucidated in a recent comprehensive review [83] . Here, we are to propose a different PseAAC to represent drug-targeted proteins in terms of their biochemical and physicochemical features [84] . Six different types of features were considered: (1) hydrophobicity, (2) polarizability, (3) polarity, (4) secondary structure, (5) normalized van der Waals volume, and (6) solvent accessibility.
Each amino acid residue in a protein sequence can be represented by a set of different states according to its features. For instance, its hydrophobicity feature can be marked by one of the following three states: ''polar'', ''neutral'', or ''hydrophobic'' [85] ; its solvent accessibility feature by one of the two: ''buried'' or ''exposed to solvent'', as predicted by PredAcc [35] ; its secondary structure feature by one of the three: ''helix'', ''sheet'', or ''coil'', as predicted by the method in [86] ; and so forth.
Thus, a protein sequence can be translated to a series of codes according to the biochemical and physicochemical properties of its constituent amino acid residues. For example, if using ''P'', ''N'' and ''H'' to represent the three states of hydrophobicity: ''polar'', ''neutral'', and ''hydrophobic'', the protein sequence ''DMAEIMSDKP-QAGML'' can be translated to ''PHNPHHNPPNPNNHH'' according to the codes of the hydrophobic property feature. The encoded sequences thus obtained would have different length for proteins of different sizes, which will make the prediction engine difficult to handle.
To make the feature-encoded sequence to be a vector with a fixed number of dimensions, three properties of a sequence was used: composition (C), transition (T), and distribution (D). C represents the global composition of each letter in the sequence; T, the frequency of a code letter changing from one to another; D, the distribution pattern of the code letters along the sequence, measuring the percentage of the sequence length within which the first, 25%, 50%, 75%, and 100% of the amino acids of each code letter is located. Take the above hydrophobic property sequence as an example: its C feature is 5/15 = 33.3% for all of P, H, and N, while the T feature is 2/10 = 20%, 3/10 = 30% and 5/10 = 50% for the changes between H and P, N and H, N and P, respectively. The measurement of feature D is a little more complicated. For the letter H, the first, 25%, 50%, 75% and 100% of Hs in the sequence is located at the position of 2, 5, 6, 14, and 15. Thus its D feature is ( , with a total of 21 components. Likewise, for the sequences encoded by the other four biochemical properties, each is also corresponding to 21 components. But for the sequence encoded by the solvent accessibility with only two states (''buried'' or ''exposed to solvent''), the encoded sequence is corresponding to only 14 components. Finally, by adding the 20 components of AAC [87] into the vector concerned, the total number of components thus obtained for a given protein is 5|21z20z14~139; i.e., the protein can be formulated as a 139-D vector given by
where p i (i~1, 2, Á Á Á , 139) is the i-th component of the protein P.
Of the 139 components, 119 are derived according to the codes of the above six biochemical and physicochemical features, and 20 are the AAC components of P.
With all samples represented by a feature vector, now it is possible for us to construct our predictor using the machine learning approach. The NN (Nearest Neighbor) algorithm is quite popular in pattern recognition community owing to its good performance and simple-to-use feature. According to the NN rule [88] , the query sample should be assigned to the subset represented by its nearest neighbor. In this study, if the drug-target pair with the shortest distance is a positive sample, meaning that they can interact with each other, the sample for test is seen as a positive drug-target pair. Otherwise, the test sample is seen as a negative one.
There are many different definitions to measure the ''nearness'' for the NN algorithm, such as Euclidean distance, Hamming distance [89] , and Mahalanobis distance [50, 90, 91] . In the current study, the following equation was adopted to measure the nearness between samples V x and V y
where V x : V y is the dot product of the two vectors, and V x k k and V y their modulus, respectively. When V x :V y we have D(V x ,V y )~0, indicating the ''distance'' between these two sample vectors is zero and hence they have perfect or 100% similarity.
After constructing the drug-target interaction predictor, we have to evaluate its performance. In statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (K-fold cross-validation) test, and jackknife test [92] . However, as elucidated by [24] and demonstrated by Eq.50 in [93] , among the three cross-validation methods, the jackknife test is deemed the most objective that can always yield a unique result for a given benchmark dataset, and hence has been increasingly used and widely recognized by investigators to examine the accuracy of various predictors (see, e.g. [51, 53, 54, 55, 56, 57, 59, 62, 63, 64, 94, 95, 96] ).'' Accordingly, in this study the jackknife cross-validation was adopted to calculate the success prediction rates as well.
Although we've constructed the drug-target predictor based on the original feature set described above, it is possible to improve its performance with a better feature set. Apparently, not every feature in the feature set is equally relevant to the drug-target interaction. What's more, features may not be independent with each other. The ''bad'' will have negative impact on the accuracy and efficiency of the predictor, so it is possible to do the feature selection process to construct a more compact and effective feature set. The first step is using Maximum Relevance Minimum Redundancy (mRMR) [36] to do feature evaluation. Maximum Relevance Minimum Redundancy (mRMR) [39] was firstly developed for analysis of microarray data. It ranks each feature according to its relevance to the target and redundancy to other features. The better a feature is deemed to be, the higher the rank it will be assigned to. Mutual information (MI), denoted by I to indicate the dependence of two features used to quantify the relevance and redundancy. MI is defined as following:
Based on MI, we can quantify relevance (D) and redundancy (R) as:
where f candidate is the feature to be calculated, and c is the target variable. By combining the above two equations to maximize relevance and minimize redundancy, the following mRMR function is constructed: 
where V s and V t are the already-selected feature set and to-beselected feature set, respectively, and m and n are the sizes of these two feature sets, respectively. The earlier a feature is selected, the better it would be though of. Finally, we can get an ordered feature list with a rank for every feature to indicate its importance in the feature set. In our study, the mRMR program is obtained from: http://research. janelia.org/peng/proj/mRMR/index.htm.
To calculate MI, the joint probabilistic density and the marginal probabilistic densities of the two vectors were used. A parameter t is introduced here to deal with these variables. Suppose mean to be the average value of one feature in all samples, and std to be the standard deviation, the feature of each sample would be classified into one of the three groups according to the boundaries: mean+(t : std). In our study, t was assigned to be 1.
As mentioned above, the importance of each feature is rated according to its rank in the mRMR analysis. The next step is to determine which features should be selected as the optimal feature set for our drug-target predictor. Here the IFS (Incremental Feature Selection) procedure is used to solve the problem. Each feature in the mRMR feature list was added one by one, and N different feature sets are obtained if the total feature number is N, while the i-th feature set is:
Based on each of the N feature sets, an NN algorithm predictor was constructed and tested with the jackknife cross-validation test. With all the N overall accurate rates calculated, we could draw an IFS curve with the index i to be the x-axis and the corresponding overall accurate rate to be the y-axis. Thus, S opt~f f 1 , f 2 , :::, f n g is regarded as the optimal feature set if the curve reach its peak where the value of its x-axis is nƒN.
Because four independent predictors are needed for the four different classes of drug-target pairs, the IFS analysis procedure will be processed four times with each for a specific predictor.
To refine feature selection, the FFS (Forward Feature Selection) procedure based on the result of IFS was used. FFS is a feature selection method based on IFS results which tries every feature in the candidate feature set and adds the feature that achieves the highest prediction accuracy into the already-selected feature set in each goes. Suppose the IFS curve reaches its peak with apex as its x-axis, the initial FFS-selected feature set was constructed as:
More features in FFS-to-be-selected feature set would be added into the FFS-selected feature set one by one. The FFS-to-beselected feature set with M features covers the features with mRMR ranks between k+1 and k+1+M, where M is a user-defined positive integer smaller than N{k with N to be the size of the original feature set. In each round of FFS, each feature in FFS-tobe-selected feature set would be taken out and added to the FFSselected feature set. Each predictor based on each new FFSselected feature set would be tested, and the feature set obtained the highest overall accurate rate would be used as the new FFSselected feature set. This process would be run for M times, until the FFS-to-be-selected feature set becomes a null set. An FFS curve similar to the IFS curve could be drawn with x-axis as the index and y-axis as the overall accurate rate.
In this study, FFS was run for each of the four benchmark datasets based on the corresponding IFS result. M for all these processes was set to 50, while k for each FFS was set to be the index of the point with the first maximum value (i.e. the maximum point with the smallest index) in the corresponding IFS curve.
To improve performance of the predictor of drug-target interaction, feature selection process was carried out. The first step of feature selection is feature evaluation. In this study, mRMR was used to evaluate every feature in original feature set. Listed in Online Supporting Information S5 are two kinds of outputs: the first one is the MaxRel list which shows ranks of features for their relevance to the target; the second is mRMR list showing the mRMR ranks according to the feature order satisfying Eq. 3. In this study, only the mRMR list was used as the results of feature evaluation. Since there are four groups of samples, mRMR was run four times with each for one of them.
With the four mRMR lists, IFS was processed for each of the four sample groups, generating four IFS curves. Based on these results, we set k in FFS to be 16, 15, 14 and 19 for the data of enzymes, ion channels, GPCRs and nuclear receptors, respectively. Each of these figures is the index of the point of the first maximum value in the corresponding IFS curve. Shown in Fig. 1 are the four IFS curves with their corresponding FFS curves. The peaks of the four FFS curves finally reach the overall success rates of 85.48% with 32 features, 80.78% with 37 features, 78.49% with 30 features, and 85.66% with 32 features for enzyme group, ion channel group, GPCR group and nuclear receptor groups, respectively.
Features selected by mRMR+FFS for the four different groups are quite different from each other, showing the intrinsic differences between them. Although there are more features for target than those for drug in the original feature set, more drug features were selected, showing the important role of drugs. Many of the selected target features are for protein secondary structure, especially for enzyme group (half of selected target features are for this). All types of features are selected in at least one group, showing that all biochemical and physicochemical features have their irreplaceable positions in drug-target interaction process.
For the details of the optimal feature-set outputs by FFS for the four benchmark datasets, see the Online Supporting Information S6.
For the specificity and promiscuity, we divided the drug-protein interactions into four groups according to the targets of drugs: enzymes, ion channels, GPCRs, and nuclear receptors. We used all the known drugs and target proteins in the gold standard data as training data to predict the potential interactions between all human proteins annotated as members of the four classes in KEGG genes and all compounds in KEGG ligands.
Enzyme recognition is the primary event involved in the interaction of proteins with other proteins and with small molecules such as metabolites and therapeutics. Predicting drugenzyme interactions has direct application for completing genome annotations, finding enzymes for synthetic chemistry, and predicting drug specificity, promiscuity and pharmacology. It is suggested that the secondary structure information plays the major role in determining the drug-enzyme interactions activity. For example, cytochrome P450 (CYP) induction-mediated interaction is one of the major concerns in clinical practice and for the pharmaceutical industry [97] . Induction of CYP1A enzymes with a specific structure-stable state may activate some xenobiotics to their reactive metabolites, leading to toxicity [98, 99] . Amino acid composition and hydrophobicity also contribute considerably to these interactions. An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) have an influence on the antihypertensive response, particularly when using ACE inhibitors (ACEI) [100] , mirroring that the amino acid composition did contribute to the interactions. Hydrophobicity plays a role in determining the coefficients of drug-enzyme interaction energy with the application to drug screening as well as in silico target protein screening [101, 102] .
The G-protein coupled receptor (GPCR) superfamily, which is comprised of estimated 600-1,000 members, is another largest known class of molecular targets with varieties of physiological activities and proven therapeutic value [103] . They are integral membrane proteins sharing a common global topology that consists of seven transmembrane alpha helices, intracellular Cterminal, an extracellular N-terminal, three intracellular loops and three extracellular loops [33, 44] . It is suggested that secondary structure and polarity would play a major role in determining the drug-GPCRs interactions activity. Small secondary structures such as helices and loops are identified as entities potentially involved in stabilizing interactions with ligands [33] . These motifs were situated mainly in the apical region of transmembrane segments and included a few extracellular residues [104] . Crystal structures of engineered human beta 2-adrenergic receptors (ARs) in complex with an inverse agonist ligand, carazolol, provide threedimensional snapshots of an important G protein-coupled receptor (GPCR) with a beta-sheet structure and forms part of the chromophore-binding site [105] . GLIDA provides interaction data between GPCRs and their ligands, along with chemical information on the ligands, as well as biological information regarding GPCRs [106] . Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane, the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as ''polarity'', the close packing of side-chains throughout the transmembrane domain. When more experimental 3D structures become available for GPCRs in the future, this will help building reliable models for a wider range of GPCRs that would be suitable for docking studies. Joint use of ligand-based chemogenomic and docking would certainly improve the prediction.
Ion channels are a large superfamily of membrane proteins that pass ions across membranes and are critical to diverse physiological functions in both excitable and nonexcitable cells and underlie many diseases. As a result, they are an important target class which is proven to be highly ''druggable''. According to our analysis, secondary structure and polarity play the major role in determining the drug-ion channels interactions activity. Secondary structure controls the membrane potential and interrogates ion channels in different conformational states. The drug-ion channels interaction needs gated state where they can switch conformation between a closed and an open state [42, 43] . Simulations on model nanopores reveal that a narrow hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small increase in pore radius or an increase in polarity [107, 108] . Nowadays, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction [109] .
Nuclear receptors (NR) are ligand-activated transcription factors that regulate the activation of a variety of important target genes, which are the most important drug targets in terms of potential therapeutic application. According to our results, secondary structure and polarizability play the major role in determining the drug-NRs interactions. The conservative motif of the NR is typically described as three stacked alpha-helical sheets. The helices that make up the ''front'' and ''back'' sheets are aligned parallel to one another. The helices in the middle sheet run across the two outer sheets and only occupy the space in the upper portion of the domain. The space in the lower part of the domain is relatively void of protein, and for most NRs, this creates an internal cavity for small-molecule ligands [110] . Hydrogen bonds with polarizability activity play a crucial role in protein-drug interactions (see, e.g., [11] ). Our approaches and the results thus obtained could be used to demonstrate how nuclear hormone receptors form a network of direct interactions. And this network increases in complexity to describe the interactions with target genes as well as small molecules known to bind a receptor, enzyme, or transporter.
A comprehensive drug-target interaction network system has been established that contains four classifiers for predicting the drugable interaction of compounds with enzymes, ion-channels, GPCRs, and nuclear receptors, respectively. It is anticipated that the network predictor system may become a very useful tool for drug development. Particularly it may help us find new or potential drug-target interactions.
Online Supporting Information S1 The benchmark dataset for the drug-target enzyme interaction system. It contains 8,157 genedrug pair samples, of which 2,719 are positive and 5,438 negative. The 1st column of the table indicates the nature of samples with 1 for positive and 2 for negative; the 2nd column shows the code of target gene; and the 3rd column shows the code of drug. All the detailed information for the genes and drugs listed here can be found in  An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of −1 ribosomal frameshifting An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs. During translation, the ribosomes have to maintain correct reading frame to faithfully decode the in-frame messenger RNA (mRNA) sequences into ordered amino acid sequences. However, specific information within mRNAs can trigger the slippage of reading frames for elongating ribosomes, and thus lead to the frameshifting events (1) . In one case, the ribosomes are induced to move backward one base in the 5 0 direction in response to specific signals within mRNAs, and then continue translation in the new À1 reading frame (2) . Such a À1 programmed ribosomal frameshifting (PRF) is adopted by a variety of viruses to maintain a specific ratio between structural and enzymatic proteins (3) (4) (5) (6) (7) , and examples of À1 PRF in cellular genes are also reported (8) (9) (10) . The mechanism for the induction of À1 PRF by a stimulator is a delicate process and may involve multiple factors. Efficient eukaryotic À1 PRF requires two in cis RNA elements within the mRNA (11, 12) : a heptanucleotide slippery site of XXXYYYZ sequences, where the frameshift occurs, and a downstream RNA structure connected by a spacer. The optimal distance between stimulator and slippery site, separated by the spacer, will then position the A-and P-site tRNAs on the slippery site (13) (14) (15) . With the sequence feature of X being any three identical nucleotides, Y being either AAA or UUU, and Z not being a G, these cooperative RNA elements can increase the probability of a ribosome to slip with A-and P-site tRNAs in the 5 0 direction by one base along the mRNA (from X XXY YYZ to XXX YYY Z) (15) .
The downstream RNA stimulator is usually a hairpintype RNA pseudoknot in which nucleotides from a hairpin loop form base pairs with a single-stranded region outside the hairpin. It leads to a quasi-continuous RNA double-helical structure, with a topology featuring two helical stems of the base-pairing region (stems 1 and 2) connected by two single-stranded loops (loops 1 and 2) (16) . It is thought that the resistance of a stimulator against deformation by the ribosome helicase activity can cause the marching ribosome to pause. However, RNA hairpin not capable of stimulating À1 PRF also causes the ribosome to pause (13, 14, 17) . As unwinding of stem 1 by ribosome will require rotation of the rest of the pesudoknot, torsional restraint hypothesis suggests that pseudoknot topology can restrain loop rotation during *To whom correspondence should be addressed. Tel: +886 4 22840468 (ext. 218); Fax: +886 4 22853487; Email: kychang@dragon.nchu.edu.tw the unwinding of stem 1, and thus interferes with this process (18) . Interestingly, only a subset of RNA pseudoknots can stimulate À1 PRF and non-pseudoknot RNA elements have been characterized to induce À1 PRF (19) (20) (21) , implicating the existence of uncharacterized determinants. In addition, the involvement of specific interactions between pseudoknot stimulator and ribosomal components during the unwinding process cannot be ruled out (22, 23) .
Based on the length of stem 1, the RNA pseudoknots capable of stimulating À1 PRF can be classified into two groups. One group of pseudoknot requires a long stem 1 to stimulate À1 PRF efficiently and can be represented by the infectious bronchitis virus pseudoknot (IBV-PK), which contains 11 bp in stem 1. Deviation from the optimal 11-12 bp in stem 1 will lead to the impairment of IBV-PK frameshift efficiency (24) . However, no high-resolution structural information is available for this class of pseudoknot. In contrast, the other group of pseudoknots, which includes mouse mammary tumor virus pseudoknot (MMTV-PK), beet western yellow virus (BWYV-PK) and simian retrovirus pseudoknot (SRV-PK), has a short stem 1 of <7 bp in length (16) . The analysis of high-resolution structures of several short stem 1 possessing pseudoknots indicated that they all share a common structural feature, involving tertiary loop-helix interactions (between stem 1 and loop 2) close to the helical junction (22, (25) (26) (27) (28) . The requirement of these tertiary interactions in À1 PRF activity has been verified by mutagenesis analysis (29) (30) (31) (32) . However, the relative small size of this group of pseudoknots makes it difficult to rationalize the results of extensive mutagenesis analysis because a mutant may not necessarily still form a pseudoknot. Furthermore, NMR structures of a luteoviral pseudoknot and its poorly functional variant suggest that both pseudoknots possess essentially identical global structure (27, 28) . Therefore, how structurally similar pseudoknots produce distinct À1 PRF efficiency remains an open question.
Here, we demonstrate that an RNA pseudoknot, hTPK-DU177 (DU177), which is derived from human telomerase RNA (33, 34) , can function as an efficient À1 PRF stimulator. Because well-characterized À1 PRF pseudoknots containing short stem 1 all possess relative short loop 1 (22, (25) (26) (27) (28) , information for the contribution of loop 1-stem 2 interactions to À1 PRF activity of a pseudoknot is limited (18) . Therefore, the long loop 1 of DU177 and its extensive stem-loop interactions with the stem 2 make this well-defined pseudoknot an excellent model to explore this issue. Using DU177 and its variants, we show that the À1 PRF efficiency of DU177 can be impaired by the disruption of specific base-triple interactions flanking the helical junction region. In contrast, a DU177 mutant, lacking most of the triple interactions identified in DU177, has essentially no frameshift activity but still adopts a pseudoknot conformation. Furthermore, the triplex in DU177 is then mimicked and constructed intermolecularly (35) , and is used to decouple triplex formation from pseudoknot topology to resolve their roles in À1 PRF activity. Therefore, our work provides a platform to characterize the contributions of two structural determinants of a À1 PRF pseudoknot. Finally, the results from this work also imply that a non-coding RNA may act in-trans to stimulate À1 PRF on targeted mRNAs by specific intermolecular triplex formation.
The p2luc reporter was a kind gift from Professor John Atkins at the University of Utah (36) , and the pRL-SV40 vector was purchased from Promega. Oligonucleotides containing the slippery sequence (TTTAAAC), spacer (GGGTT) and the stimulator sequences were chemically synthesized. They were amplified by forward and reverse primers containing BsrGI and BsaAI sites, respectively, and ligated into the BsrGI/BsaAI sites (1392/1426) of pRL-SV40 vectors treated with restriction enzymes. Oligonucleotides containing DU177, hTR-5 0 hp, hTR5 0 hp-L1c and hTR174T5 0 hp sequences were also inserted into p2luc reporter in a similar way, except for the use of SalI and BamHI restriction sites instead. A À1frame stop codon was inserted into the DU177-containing p2luc reporter, thus generating a premature À1-frame protein product, to facilitate the À1 PRF assays of DU177-related variants in vitro (37) . All of the mutants were constructed using the quick-change mutagenesis kit from Stratgene, and the identities of all cloned and mutated genes were confirmed by DNA sequencing analysis.
RNA synthesis and enzymatic structure probing RNA transcripts were generated by in vitro transcription using T7 RNA polymerase. The purified RNAs of desired length were then dephosphorylated by calf intestine alkaline phosphatase, 5 0 -end labeled with [g-32 P] ATP using T4 polynucleotide kinase, and then separated by a 20% sequencing gel for recovery. All the RNase protection experiments were performed with 50 000 to 70 000 cpm of 5 0 -end labeled RNA in the presence of RNase cleavage buffer (30 mM Tris-HCl, pH 7.5; 3 mM EDTA; and 100 mM LiCl) for each reaction, and 10 mM MgCl 2 were included in the same buffer for RNase V1 experiments. Before the addition of probing enzymes, the RNAs were denatured by heating at 65 C for 5 min and followed by slow cooling to 20 C for structural mapping. Finally, 0.02-0.5 U of RNase T2 (USB) or 0.02-0.5 U of RNase V1 (Amersham Pharmacia) was added to each reaction. The hydrolysis RNA ladders were obtained by incubation of RNA in the hydrolysis buffer at 100 C for 2 min, and parallel RNA sequencing products were obtained by the treatment of unfolded RNA with RNases T1 or A. They were used as markers for the assignment of guanines and pyrimidines, respectively. All reactions were incubated at 20 C for 10 min unless specified. The reactions were terminated by addition of gel loading dye, and the cleavage products were resolved by a 20% denaturing gel and visualized by phosphorimagery.
The purified RNAs were analyzed on a 20% nondenatured polyacrylamide gel (29:1 acryl:bisacryl ratio) in the 1Â TBE (Tris-Borate-EDTA) buffer. The gel was run at a constant voltage of 150 V for 8 h at room temperature. The results were visualized by the staining of ethidium bromide.
The capped reporter mRNAs were prepared by the mMESSAGE mMACHINE high-yield capped RNA transcription kit (Ambion) by following the manufacturer's instructions. The reticulocyte lysate system (Progema) was used to generate the shifted and non-shifted protein products. In each assay, a total of 5 ml reaction containing 250 ng of capped reporter mRNA, 2.5 ml of reticulocyte lysate and 0.2 ml of 10mCi/ml 35 S-labeled methionine (NEN) was incubated at 30 C for 1.5 h. The samples were then resolved by 12% SDS polyacrylamide gels and exposed to phosphorimager screen for quantification after drying. The À1 PRF efficiency was calculated by dividing the counts of the shifted product by the sum of the counts for both shifted and non-shifted products, with calibration of the methionine residue number in each product.
Both major-groove and minor-groove triple interactions are required for the Z1 PRF activity of DU177
A comparison between DU177 and several representative À1 PRF pseudoknot stimulators ( Figure 1A -F) revealed a consensus AACAA sequence in the loop 2 of three short stem 1-containing pseudoknots ( Figure 1C -E). Furthermore, both PEMV-1 PK and DU177 contain an unusual Hoogsteen AU base pair, formed between loops 1 and 2, to bridge the helical junction. We thus examined if DU177 could function as a À1 PRF stimulator when it is positioned downstream of a slippery sequence. To this end, the À1 PRF activity of DU177 was measured and compared with those of several well-characterized pseudoknot stimulators available in our laboratory. The results, shown in Figure 1G , indicate that DU177 can act as an efficient À1 PRF stimulator in vitro. The À1 PRF efficiency stimulated by DU177 was comparable with that of the minimal IBV pseudoknot (IBVm-PK) and was in the range of 50%. This value was higher than those of MMTV-PK (24%) and the other two AACAA-containing pseudoknots, BWYV-PK and SRV-PK (21% and 34%, respectively). In addition, DU177 possessed substantial À1 PRF activity in vivo (data not shown). Therefore, the high À1 PRF activity and the well-resolved structure of DU177 (33) . The nucleotides in DU177 are numbered according to those in ref. (33) . The common AACAA sequences are highlighted by gray shadow, the unusual Hoogsteen AU base pairs are boxed, and the adenines stacking and tertiary interactions are represented by filled circles and dotted lines, respectively. (G) The 12% SDS-PAGE analysis of the À1 PRF assays of several viral RNA pseudoknots, including a minimum IBV-PK (IBV m -PK), MMTV-PK, SRV-PK, BWYV-PK and DU177 RNA (22, 25, 29, 32, 33) . Each pseudoknot was incorporated into a pRL-SV40-based reporter, assayed as described in the 'Materials and Methods', and the translated proteins corresponding to the 0-frame and À1 frame products were labeled as indicated. Please note that a negative control without insertion of a pseudoknot only showed background À1 PRF activity (data not shown).
make it a good model molecule for dissecting the structural determinants within a pseudoknot for the stimulation of À1 PRF. A structural feature found in several À1 PRF pseudoknot stimulators, which involved stacking of the loop 2 adenines in the minor groove of stem 1 in a pseudoknot, was also identified in DU177 (33) . The residues 166-168 of DU177 were thus mutated, and the resulting mutants (C166U, A167C, A168U and L2-UU in Figure 2A ) still possessed substantial À1 PRF activities ranging from 50% to 30% ( Figure 2B, lanes 2-5) . Similarly, mutagenesis on loop 1 nucleotides 103 and 105 generated mutant L1-ACA with a DU177-like À1 PRF activity (compare lane 1 with lane 6 in Figure 2B ). Therefore, the À1 PRF activity of DU177 is not very sensitive to nucleotide identity in the 3 0 portion of its loop 1.
As base-triple interactions were shown to be important for À1 PRF activity in several pseudoknots (22, (25) (26) (27) (28) 32) , we disrupted the three major-groove triples and the two minor-groove triples by making mutations on loops 1 and 2 of DU177, respectively. As shown in Figure 2B (lanes 7-9), the À1 PRF efficiencies of both mutants (L1-CCC and L2-GU) were reduced dramatically to below 5%, whereas the CCC/GU mutant, with all five base triples impaired, lost its À1 PRF activity to essentially 0%. The A173G mutant was then made to destroy the crucial Hoogsteen AU base pair that helps in anchoring the interhelical triplex. Similar to what was observed in CCC/GU mutant, this single mutation wiped out À1 PRF activity completely ( Figure 2B , lane 10) and is consistent with the involvement of triplex formation in the stimulation of À1 PRF activity of DU177.
To examine if the mutants with disrupted base-triple interactions still adopt the pseudoknot conformation, non-denatured gel electrophoresis and enzymatic structure probing experiments were performed on CCC/GU RNA, and the results were compared with those of DU177 pseudoknot. Analysis of non-denatured gel confirmed that both RNA constructs adopt a major conformation ( Figure 3A ). However, distinct gel mobility of these two RNAs suggested that they might have different conformations. The enzymatic probing data are shown and summarized in Figure 3B -E. We used DU177 RNA as the standard for comparison because its NMR structure is well defined (33) . For both DU177 and CCC/GU RNAs, the distributions of cleavage patterns by ribonuclease T2, the probe for single-stranded region, are in agreement with the formation of the two loop regions. In addition, the distributions of cleavage patterns by RNase V1, the probe for duplex and stacked conformations, can be localized to the two predicted stem regions for both RNAs. Specifically, both RNAs possess signature V1 cleavage for the quasi-continuous helical region corresponding to the 3 0 -portion of stem 1 and the 5 0 -portion of stem 2 ( Figure 3B,D) . Therefore, these probing data indicate that CCC/GU RNA also adopts a pseudoknot conformation and suggest that the other mutants containing more base triples should also adopt the pseudoknot conformations. In the related UV-melting and singlemolecule mechanical unfolding experiments for DU177 and CCC/GU RNAs, transitions corresponding to the melting or mechanical unfolding of stems 1 and 2 were also observed and are consistent with the probing data (33, 38) . In addition, an extra transition corresponding to the disruption of tertiary interaction network was also reported in single-molecule unfolding studies and can only be observed in DU177 RNA (38) . Taken together with the mutagenesis studies above, the results of these experiments suggest that DU177 RNA can be converted into a À1 PRF-incompetent pseudoknot by disruption of the triplex structure spanning the helical junction.
Stem-loop interactions and their relative position to the helical junction of pseudoknot affect the Z1 PRF efficiency of DU177
To dissect the contribution of individual base-triple interaction in À1 PRF activity of DU177, we generated mutations in loops 1 and 2 to disrupt specific stem-loop Please note that a p2luc reporter, with a stop codon inserted into the À1 frame of the N-terminal region of firefly luciferase, was used in these experiments and will thus generate a premature À1 frame protein product.
interactions (see Figure 4A for mutation scheme). As shown in Figure 4B , a dramatic reduction of À1 PRF activity to $10% can be observed for mutants U100C, U101C, A171G and A172U, whereas mutant U102C possesses a À1 PRF efficiency of 33% ( Figure 4B , lanes 1-5). Therefore, the stem-loop interactions flanking the helical junction, contributed by the first two base triples of both stems, are crucial for efficient The enzymatic cleavage results were resolved in a 20% sequencing gel, with the first and eighth wells as alkaline hydrolysis ladder and control, respectively, whereas the last two wells represent guanine and pyrimidine assignment markers. The cleavage results after RNase T2 and V1 treatments, with increment of RNase concentration, are shown in wells 2-4 and 5-7, respectively. In addition, the assigned residues and the corresponding stem/loop regions are listed on the right and left sides of the gel, respectively. Please note that the residues different from each other are boxed for comparison between both RNAs. Finally, the extent of cleavage for each probe is defined as major or minor cut, and summarized in (B) and (D), with gray rhombuses representing RNase T2 cleavage and filled triangles representing RNase V1 cleavage. They are presented along the predicted secondary structures of both RNAs, with the five mutated loop nucleotides in CCC/GU RNA typed in gray. À1 PRF activity. In contrast, the stem-loop interactions from the distal third major-groove triple (U113-A176ÁU102) are less important. During the progress of this work, an extra major-groove base-triple was proposed in the refined DU177 structure (34) . It locates next to the distal third triple, and its formation will be blocked in the L1-ACA mutant created in Figure 2 . Both DU177 and L1-ACA having similar À1PRF efficiencies (compare lane 1 with lane 6 in Figure 2B ) further support the idea that the contribution of a major-groove triple in À1 PRF activity of DU177 also depends on its relative position to the helical junction.
We further mutated the stem U-A base pair to the C-G base pair for every U-AÁU major-groove triple (mutants 174W, 175W and 176W in Figure 4A ). As shown in Figure  4B , each of these mutants possesses lower À1 PRF activity than that of DU177 ( Figure 4B, lanes 6, 8 and 10) . The frameshift activities range from 49% for 176W to 22 and 14% for 175W and 174W, respectively. Therefore, mutation on the stem base pair of the distal third major-groove triple, 176W, has less impact on À1 PRF activity than those of the two junction-flanking major-groove triples. Finally, mutating the U of C-G Á U triple (174W or 175W) to the C, thus forming a C-G Á C triple (174T or 175T), can restore frameshift efficiency to the level of DU177. Interestingly, the U-A Á U triple, with a weaker U-A base pair in the stem, seems to have higher À1 PRF activity than the C-G Á U base-triple containing a stronger stem C-G base pair. Taken together, these data strongly suggest that the stem-loop interactions in the major-groove triples and their positions relative to the helical junction play important roles in the À1 PRF activity of DU177.
A triplex formed intermolecularly to mimic triplex structure in DU177 can stimulate Z1 PRF Previous studies have shown that the tertiary interactions in human telomerase RNA pseudoknot region can be maintained in a two-piece assembly (35) . Indeed, UV-melting experiments on a bimolecular pseudoknot have indicated that the triplex formation is independent of loop closure embedded in the pseudoknot topology (33) . Because of the dominant role of triplex formation in the À1 PRF activity of DU177 revealed above, we used a similar bimolecular pesudoknot approach to examine the contribution of pseudoknot topology to the À1 PRF activity of DU177. In this approach, the 5 0 -hairpin portion of DU177 was used to replace the DU177 pseudoknot in the reporter mRNA to form the hTR-5 0 hp construct, and an in-trans hTR3 0 ss RNA corresponding to the loop 2 and the 3 0 portion of stem 2 in DU177 was constructed separately ( Figure 5A ). Based on the results from previous UV-melting experiments (33) , the association of hTR3 0 ss RNA with hTR-5 0 hp construct will restore stem 2 and all the triple interactions corresponding to those in the DU177. The bimolecular pseudoknot construct will thus resemble the unimolecular DU177, except for it missing the covalent linkage that connects stem 1 and loop 2. The results shown in Figure 5B indicate that, upon the addition of hTR3 0 ss RNA, the À1 PRF efficiency of 5 0 -hairpin-containing mRNA reporter (hTR-5 0 hp) changes from 0.25 to 5% in a dosage-dependent way. In contrast, a control mRNA reporter, hTR5 0 hp-L1c, designed to disrupt a potential intermolecular major-groove base-triple interactions, does not respond to even a higher dosage of hTR3 0 ss RNA. Similarly, in-trans oligo-nucleotides (171G3 0 ss and 172U3 0 ss RNA), designed to disrupt potential intermolecular minor-groove base-triple interactions, cannot induce significant À1 PRF activity on hTR5 0 hp mRNA reporter, either ( Figure 5C ). Finally, we also examined if an intermolecular U-A Á U major-groove base-triple interaction can be replaced by an intermolecular C-G Á C triple. As shown in Figure 6 , 174T3 0 ss RNA, designed to form a C-G Á C triple with 174T5 0 hp mRNA reporter, can stimulate significant À1 PRF activity on 174T5 0 hp reporter only, but not on hTR5 0 hp or hTR5 0 hp-L1c reporter. Therefore, an intermolecular triplex mimicking the triplex structure spanning the helical junction of DU177, although it does not possess a completed pseudoknot topology, can stimulate À1 PRF activity. Together with the observation that the removal of the continuing triplex structure converts DU177 into a À1 PRF incompetent pseudoknot, these experimental results argue that both the pseudoknot topology and the triplex structure are required for high-efficiency of À1 PRF, with the pseudoknot topology playing an enhancer-like role to further increase À1 PRF efficiency.
Mutational analysis indicate that mutants with junctionflanking C-GÁU or C-GÁC triple do not have higher À1 PRF activity than those of DU177 with U-AÁU triple occupying the same position. This is intriguing as one extra hydrogen bond is contributed by the stem C-G base pairing in the process of the U-AÁU to C-GÁU conversion. However, one stem-loop interbase hydrogen bond is also lost at the same time. In contrast to the C-GÁU triple, the C-GÁC triple can possess more than one Hoogsteen interaction depending on the pH condition (39) , and the À1 PRF efficiency may thus be restored to the level of U-AÁU triple. Therefore, it suggests the important role of tertiary stem-loop interactions in modulating the À1 PRF activity of DU177.
As demonstrated by the observation that disruption of the adenine stacking between loop 2 and stem 1 of DU177 also affects the À1 PRF efficiency of DU177, there may be more than one way to interfere with the helicase activity of a translocating ribosome. The studies on À1 PRF pseudoknots containing short stem 1, such as BWYV-PK and ScYLV-PK, indicated that specific minor-groove base triples involving S1 and L2 are important for À1 PRF activity, and the impact on frameshift efficiency depends on their relative position to the helical junction (27, 28, 30, 31) . Particularly, it has been shown that junction-flanking tertiary interactions between the 3 0 -nucleotide of loop 2 and base pairing in stem 1 can organize an interhelical interaction network that may act as a kinetic barrier during the unfolding of pseudoknot by ribosome (28) . In contrast, the roles of loop 1 and stem 2 in a À1 PRF pseudoknot stimulator are less addressed as both BWYV-PK and ScYLV-PK have short loop 1 and stem 2 (18) . In this work, we reveal that the major-groove The nucleotides that will interfere with base triple formation in the bimolecular construct are typed in gray. Please note that the slippery site and spacer are not shown in the drawing, and a p2luc reporter, with a full-length firefly luciferase generated as the À1 frame product, was used in this experiment. (B) Results of À1 PRF assays of intermolecular major-groove base triple analysis. (C) Results of À1 PRF assays of intermolecular minor-groove base triple analysis. The designated asterisk indicates the estimated molecular ratios between the in-trans RNA (hTR3 0 ss, 171G3 0 ss and 172U3 0 ss) and the mRNA reporter (hTR5 0 hp and hTR5 0 hpL1c), and the translated proteins corresponding to the 0-frame and À1 frame products are also labeled.
base-triple interactions closer to the helical junction are more important than the distal ones for À1 PRF activity of DU177 ( Figures 2B and 4B) . Therefore, the relative position toward the helical junction is important for both major-and minor-groove base-triple in determining the contribution of a base-triple to the À1 PRF stimulation activity of a pseudoknot. This position-dependent effect can be rationalized by the stabilization of stem 2 by stem-loop interactions, and the anchoring of stem 2 into stem 1 via the junction bridging triplex formation. Such interactions will provide extra restraints during the unwinding of stem 1 by ribosome and are consistent with the torsional restraint hypothesis (18) . Both the helical geometry of a pseudoknot and its ability to against deformation by a ribosome can be affected by the stem-loop interaction in a positiondependent way, too. Indeed, modulation of frameshift efficiency by the modification of junction geometry via tertiary interactions has been observed in the helical junction of MMTV-PK and BWYV-PK (29, 30) . In particular, the DU177 pseudoknot also adopts a bent conformation around the helical junction (33, 34) , and the tertiary stem-loop interactions contributed by the junction-flanking base triples may thus play a unique role to facilitate this bending process. Alternatively, the single-molecule unfolding analysis of a set of DU177 variants, with À1 PRF efficiency ranging from 50 to 0%, reveals a correlation between pseudoknot mechanical stability and frameshift efficiency (38) . Building upon this foundation, the position effect of stem-loop interaction on the mechanical stability of a pseudoknot can be analyzed further in the future.
The roles of loop-closure constraint within a Z1 PRF pseudoknot stimulator
The stimulation of À1 PRF activity by a bimolecular pseudoknot mimicking DU177 suggests an enhancement role for the pseudoknot topology in À1 PRF activity, particularly for those pseudoknots possessing a short stem 1 (22, (25) (26) (27) (28) 32) . In these cases, the pseudoknot topology may just serve to hold the structural determinants of À1 PRF activity together within the same molecule. With loops crossing the grooves of stems under the pseudoknot topology, an interlocked helical junction can be efficiently generated by the junction-flanking stem-loop interactions, and thus restrict the rotational flexibility between stems 2 and 1 as proposed in the torsional restraint hypothesis (18) . In addition, the loop 2 to stem 1 adenine stacking interactions described above can be put in place easily. Alternatively, the fact that most À1 PRF competent pseudoknots possess a short loop 1, thus creating strains (40) , argues for a more active role for the loop-closure constraint imposed by a pseudoknot. In fact, the bimolecular pseudoknot construct still maintains one of the two constrained loops seen in DU177, and the intact DU177 indeed has a much higher À1 PRF efficiency than that of the bimolecular pseudoknot. This can explain why removing one loop-closure constraint from DU177 can reduce its À1 PRF efficiency dramatically, and thus underscores the importance of pseudoknot topology in À1 PRF. In agreement with this work, designed RNA-DNA hybrids, with loop-loop interaction between mRNA and in-trans DNA hairpin to mimick rotational restraints imposed by pseudoknot topology, have been shown to stimulate À1 PRF significantly (18) .
It is intriguing that an RNA motif derived from the RNA component of telomerase can act as a À1 PRF stimulator. This could be explained by the existence of a common structural feature such as a specific RNA-protein contact, which is needed for a telomerase as well as a À1 PRF stimulator to function properly. Interestingly, telomerase RNA subunit has been identified in Marek's disease virus (41) . Perhaps, different viruses might pick up this motif during evolution and modified it to fit replication purpose, including the tuning of À1 PRF efficiency through mutation (30) . Nevertheless, it is informative to note that specific 2 0 OH groups protruding from the triple helix of DU177 have been shown to contact with the protein component of telomerase (42) . Therefore, the bimolecular pseudoknot provides a workable platform to explore the possibility of specific stimulator-ribosomal helicase interaction during À1 PRF as suggested previously (22) . In addition, it should be useful in probing the geometry of the mRNA entry channel and the helicase activity of eukaryotic ribosomes. Finally, the regulation of À1 PRF in-trans shown here raises the possibility that Figure 6 . An intermolecular C-GC major-groove base-triple interaction can replace an intermolecular U-AU triple. The C-GÁC majorgroove base-triple interaction reconstituted in the bimolecular pseudoknot construct is typed in gray, and the other designations are the same as those in Figure 5 .
non-coding RNA may use a similar mechanism to regulate gene expression although the À1 PRF efficiency observed here is not dramatic compared with the reported examples (20, 21) . However, further enhancement of frameshift efficiency for application purpose is possible, such as that demonstrated by the use of modified oligonucleotides (20) . Early Days of Food and Environmental Virology In July 1962, the author joined the Food Research Institute (FRI), then at the University of Chicago, to become its food virologist. There was a limited record of waterborne viral disease outbreaks at the time; recorded data on foodborne outbreaks were fewer still. Laboratory environmental (water and wastewater) virology was in its infancy, and food virology was in gestation. Detection of viruses was most often attempted by inoculation of primary primate cell cultures, with observation for plaque formation or cytopathic effects. Focus was initially on enteroviruses and reoviruses. Environmental and food samples had to be liquefied if not already in liquid form; clarified to remove solids, bacteria, and fungi; and concentrated to a volume that could be tested in cell culture. Cytotoxicity was also a concern. Studies at the FRI and some other laboratories addressed all of these challenges. The FRI group was the World Health Organization’s Collaborating Center for Food Virology for many years. Other topics studied were virus inactivation as functions of temperature, time, matrix, disinfectants, and microbial action; peroral and ex-vivo infectivity; and the suitability of various virus surrogates for environmental monitoring and inactivation experiments. Detection of noroviruses and hepatitis A virus required molecular methods, most often RT-PCR. When it was found that inactivated virus often gave the same RT-PCR signal as that of infectious virus, sample treatments were sought, which would prevent false-positive test results. Many laboratories around the world have taken up food and environmental virology since 1962, with the result that a dedicated journal has been launched. I am delighted to have this opportunity to share some reminiscences of the evolution of food and environmental virology. Although this is largely a first-person account and subject to the problems of a failing memory, I console myself with the thought that most of what is known of evolution has been learned from old fossils. My research in environmental virology began in 1962. Only virology is addressed in this narrative, although our group worked with other intestinal pathogens (bacteria, protozoa, etc.) at times. My research career has led me to view the world as though peering outward through the anal orifice-''reverse proctoscopy'' has contributed significantly to addressing problems in unusual ways.
The article is divided into arbitrary topic areas because putting the entire record together in chronological order would have been hopelessly confusing. I hope the reader finds some coherence in the scheme that I have chosen.
Hepatitis A (HA) was formerly called epidemic jaundice and then infectious hepatitis. A waterborne outbreak was reported in the UK as early as 1896 (Plowright 1896) . A small outbreak of poliomyelitis that occurred in 1914 in the UK was also recorded (Jubb 1915) . Understanding of the nature of viruses was very limited in those early times, and so laboratory testing was not an option and epidemiologic investigations were problematic. By 1967, I was able to compile a total of 36 reported foodborne outbreaks of HA and 10 foodborne outbreaks of poliomyelitis (Cliver 1967c) . The predominant vehicle for HA was shellfish, as is apparently still the case in many countries. The predominant vehicle for polio was raw milk-this may well have been due to using fecally soiled hands to milk cows and failure to pasteurize the milk, but it is also possible that there was some bias because milk was the vehicle for so many other diseases. Details of these outbreaks and further source references have been compiled elsewhere (Cliver 1967c (Cliver , 1983 . The possible viral causation of ''foodborne gastroenteritis of unknown etiology'' was also recognized early (Cliver 1969) . After World War II, it appeared that water virology progressed faster than food virology, based on the study by the US Public Health Service and others (Berg 1967; Metcalf et al. 1995) .
Universities I was born in 1935 and raised in Berwyn, Illinois-a Czech-speaking, close-in suburb of Chicago. Though my upbringing was urban, summer exposures to small-scale dairy farming in central Wisconsin attracted me. I chose to study Dairy Husbandry at Purdue University (BS 1956 , MS 1957 ) and was exposed to laboratory research there. After 6 months of active duty for training as an Army officer, I began doctoral studies in Dairy Science at the Ohio State University in January of 1958. My study was intended to address the transfer of immunity from the cow to the calf in colostrum-the first milk produced after the cow gives birth. Fortuitously, I was assigned to a laboratory in the School of Veterinary Medicine that had begun studying porcine enteroviruses, supported by the Office of Naval Research. The leader of the group was Edward H. Bohl, DVM, PhD, who was willing to expand his studies to include bovine enteroviruses. My mentor in the laboratory was Louis Kasza, DVM, who had left Hungary during the 1956 revolution. His knowledge and laboratory skills were excellent, but his spoken English was somewhat challenging. I had grown up in a community where English was the second language, so I had relatively little problem learning from him. He taught me what were then state-ofthe-art laboratory methods in cell culture and virology. We later became roommates; and still later, he was ''best man'' at my wedding (Kasza 2003) .
Very little that was needed for this study was available from catalogs at the time. In order to study bovine enteroviruses, it was understood that I must produce primary bovine kidney cell cultures. Balanced salt solutions (Hanks' and Earle's) were produced from individual salts, glucose, and deionized water. Medium was either of the salt solutions plus 0.5% lactalbumin enzymatic hydrolyzate. These were sterilized by autoclaving. Growth medium required addition of 5% bovine serum, and maintenance medium had 2% bovine serum. Bovine serum was produced by collecting blood at a slaughterhouse, allowing it to coagulate, and sterilizing the expelled serum by filtration through an asbestos-mat Seitz filter. The kidney to be cultured was collected as aseptically as possible from a freshly slaughtered animal. Only the cortex of the kidney was suitable for culture; since the bovine kidney is lobular, it was necessary to dissect the cortex individually off of each lobe of the kidney. These pieces were minced and mixed with trypsin solution, in an Erlenmeyer flask with a magnetic stir bar. Periodically, cells that had been freed were harvested and sedimented gently in a centrifuge; fresh trypsin solution was added to the flask until enough cells had been harvested to produce a week's cultures. Cultures were grown principally in 4-ounce (*100 ml) prescription bottles closed with white rubber stoppers (Cliver and Bohl 1962a) . These were highly compatible with the cells, but required rigorous cleaning if they were used more than once. Unevenness in the glass limited visibility with the microscope and caused at least some problems with distribution of the cells when the culture was seeded and with distribution of virus inoculum during plaque assays. Semisolid overlays for plaquing combined double-strength growth medium with double-strength agar solution and 0.5% neutral red. When the agar had solidified, the cultures were incubated cell-sideup in darkness at 37°C and observed for plaque formation at approximately 2-day intervals. Further details of how individual enterovirus strains were isolated and how quantitative neutralization tests were performed have been published elsewhere (Cliver and Bohl 1962a, b) .
My PhD was awarded in March of 1960, and I stayed the rest of that year doing postdoctoral amplifications of our findings. After a 6-month hiatus, I joined the U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland, as a National Academy of Sciences-National Research Council Resident Research Associate. During a year there, I learned the then-accepted procedures for working with highly pathogenic agents; most of these procedures are now obsolete. My own research concerned the kinetics of neutralization of Semliki Forest virus by rabbit antiserum, as measured in primary cultures of chicken embryo fibroblasts. Chicken embryos (9-10 days incubated) to be cultured were received in the shell and were decapitated, minced, and trypsinized more or less as described above. Although plastic cell-culture flasks had become available, their failure rate was so high in those early years that they were not permitted for use at Fort Detrick, for fear of contaminating an entire incubator with leaked virus suspension. Soft-glass prescription bottles continued to be the vessel of choice: at least they were only used once. I was not encouraged to publish my research findings, at least partly because Fort Detrick was a relatively secretive establishment. Meanwhile, the fact that my wife and I were of disparate races had made our life in Frederick unpleasant, and so we determined to move on. Up to this point, I had decided that I would like to continue in virology, not necessarily with veterinary applications.
While I was exploring other opportunities, I was invited to meet two visiting faculty members (Dr. Merlin S. Bergdoll and Dr. Hiroshi Sugiyama) from the Food Research Institute (FRI) of the University of Chicago (UC), who were consultants at Fort Detrick. I learned that the founder and director of FRI, Dr. Gail M. Dack, had decided to add viruses to the foodborne pathogens being studied at FRI. I was fortunate to be interviewed and promptly offered a position as ''Research Associate (Instructor),'' beginning July 1, 1962. The job description was necessarily vague, as no one knew what a food virologist was at the time-least of all me. History was also difficult to explore, in that there was no Internet and all the needed information was recorded in books, on cards, and on pieces of paper, not all of which were readily available at any given time.
We began by trying to obtain suitable human placentas for culture, from the obstetrical facility at the Chicago Lying-in Hospital, which was part of the UC Medical School. After several culture failures, I was told that these placentas were inevitably slathered with tincture of green soap. Colleagues on the medical school faculty had already cornered the supply of ''clean'' placentas from voluntary hysterectomies. The alternative was monkey kidney. Dr. Bergdoll maintained a colony of approximately 100 rhesus monkeys (Macaca mulatta), which were used in detecting staphylococcal enterotoxins and determining the mode of enterotoxin pathogenesis. After a few experiments, an animal would become refractory to the enterotoxin and of no further use to that project. We arranged to purchase these ''alumni'' as kidney donors. We then learned from experience that the monkey house had a high population of airborne yeasts, which would contaminate the kidneys collected in the animal facility and were resistant to our antibiotics. We had to collect the kidneys with their capsules intact and carry them back to the laboratory in a beaker of Dakin's solution (a strong, buffered solution of hypochlorite). At the laboratory, sterile saline was used to rinse away the hypochlorite before the capsules were opened. The kidneys were relatively small and costly, and so we tried culturing the medullas along with the corticesit did not work. Thereafter, the cortices were dissected off of the kidneys and used as the sole source of tissue for primary culture. One of the medical school faculty members routinely harvested the primary cultures and made secondary cultures to amplify the supply obtained from one monkey. Our trials of this method did not yield the desired results, and so we simply digested the tissue as extensively as possible, obtaining enough cells to produce 200-240 of 25-cm 2 cultures (in styrene flasks) from one monkey. Cultures were also grown in 16 9 150 mm test tubes (in slanted racks that confined the medium and the cells near the butt of the tube) and in Leighton tubes, which contained a coverslip near the butt end, on which the cells were grown to provide superior microscopic imaging. Our methods were later described in detail (Cliver and Herrmann 1969) , as well as a machine for changing the medium in the cultures during their outgrowth period (Cliver 1973a) . Our research at UC was funded by food industry donations to FRI and a small grant from the US Public Health Service. My application to renew the grant was unsuccessful.
In 1966, on the occasion of Dr. Dack's retirement as Director, UC evicted the entire FRI on the grounds that our research was too applied to merit space on their campus. Fortunately, we were invited to join the University of Wisconsin (UW) at Madison, under the directorship of Dr. E. M. Foster. The facilities into which we moved could best be described as a work in progress.
The evolution of tissue/cell culture was very much a work in progress at this time. Explants had earlier been embedded in plasma clots and maintained with various fluid media while cells migrated outward in a single layer that could be viewed with a microscope. Primary cell cultures were prepared by digesting animal tissue to component cells, using trypsin or other enzymes, often enhanced with versene (ethylene-diamine-tetracetate) as a chelator. The cells thus freed were washed and planted in sealed glass vessels in medium typically based on either Hanks' or Earle's balanced salt solution-these had a physiologically balanced content of cations (sodium, potassium, calcium, and magnesium) with chloride ions and a phosphate buffer system, plus glucose. Adjustment of pH was done with varying levels of sodium bicarbonate: Earle's solution was formulated for higher levels of bicarbonate, which was useful as cell populations built to a level where their Food Environ Virol (2010) 2:1-23 3 metabolic acid needed more neutralization. Cell culture vessels (e.g., flasks, bottles, tubes) were sealed because the bicarbonate buffer equilibrated with CO 2 in the vessel's airspace: if the CO 2 escaped, then the sodium bicarbonate became sodium hydroxide, and the pH climbed and killed the cells. When incubators were invented that maintained a 5% CO 2 atmosphere in their interiors, cells could be grown in unsealed containers (e.g., Petri plates), but there were (are) always risks that the controls would fail, resulting in the death of the all of the cultures. Various media with CO 2 -free buffer systems have been devised, but CO 2 has not yet been entirely replaced. One formulation substituted galactose for glucose so as to inhibit acid production by the cultured cells. Nitrogen sources could be as simple as enzymatic hydrolyzate of bovine lactalbumin, which worked well with various primary cultures and was inexpensive and autoclavable. Medium 199 was a pioneer synthetic medium that contained virtually all the known chemical constituents of mammalian tissue, including nucleic acid bases; most of its ingredients are not known to be required by cells in vitro, and it was a huge project to compound from individual chemicals, but it is still used to some extent now that it can be bought from catalogs. A turning point was the research by Harry Eagle to determine the specific nutritional needs (amino acids and vitamins) of a selected cell line. He devised Basal Medium Eagle in a version for his line of HeLa cells, and another for his line of L-cells. The next step was the development of Minimum [sic] Essential Medium, which would meet the needs of a variety of cell lines. An additional solution of ''Nonessential Amino Acids'' was devised for planting new cultures and for lines that had special needs. Some researchers use these nonessential additives routinely. Another persistent component of cell culture media is animal blood serum-often, but not always, derived from fetal calves at present. Serum-free media have been developed, but the majority of medium formulations still include serum. The degree to which bovine serum enhances growth of cells of other animal species is remarkable.
The transition between primary cultures and established cell lines is large. Once animal tissue (often kidney cortex, as mentioned above) is broken down and the dispersed cells planted in a culture vessel, some of the cells settle to the glass or plastic surface, attach, spread, and multiply until a confluent monolayer of cells is formed. This is almost entirely fortuitous-the selection of cells that attach to the growing surface (those that do not are discarded at the first medium change) and multiply, and why they ideally stop multiplying when the monolayer is complete (the so-called contact inhibition) are beyond the control of the scientist, although the preparation of growing surfaces for this purpose is now well understood. In the case of the monkey species from which kidney cultures played such a key role in poliomyelitis research, it is said that poliovirus did not infect upon direct injection into the kidney of a living monkey. One can harvest cells from primary culture in a number of ways, dilute them in fresh growth medium, and plant a larger number of secondary cultures with a high probability of success. However, subsequent passages fail more often than not, and so the establishment of a durable cell line requires considerable tenacity. Primary cell cultures were considered better for production of vaccines, even though primary monkey kidney cells often harbored adventitious simian viruses that might threaten the vaccinee, because established lines were suspected of having undergone a malignant transformation. Primary cells were also widely used in food and environmental virology because they often had a wider virus-susceptibility spectrum and a greater tolerance for toxic substances in field samples than did available established lines. ''Diploid'' cell lines were developed which were said to preserve the karyotype of the source species: these tended to grow slowly and sometimes develop karyotypic abnormalities. Established lines, such as the venerable HeLa, have recognized identity, but they too are affected by selective pressures during repeated passage in any given laboratory, whereby they may acquire an identity that varies from what would be obtained under the same name, say, from the American Type Culture Collection.
In many applications of cell cultures to virology, all that was required was scrupulous aseptic technique. However, recovery of viruses from foods and from environmental samples inevitably entails elimination or suppression of contaminating bacteria and perhaps other microbes. Antibiotics often serve this purpose; treatment of the sample extract with chloroform is often feasible, in that most enteric viruses are un-enveloped and not damaged by some of the organic solvents. Membrane filters have now evolved to the point that they can serve to remove bacteria, mold spores, yeasts, etc. very reliably.
Increasingly, in recent times, cultures of tissues other than kidney have given rise to lines that have special applications. And, as will be described later, explant cultures may serve special purposes, where the in vivo organization of the tissue is significant to the investigation.
At UC, we had cell cultures and a variety of viruses obtained from the Viral and Rickettsial Registry of the American Type Culture Collection and from colleagues. The focus was on enteroviruses, but our first published method for virus detection in cell culture was done with reovirus type 3 (RE3) (Gibbs and Cliver 1965) . Reoviruses were newly identified and perceived as a threat to human health at the time. The method of detection in cell culture was based on staining the infected cells (on a coverslip from a Leighton tube) with acridine orange, which associates with nucleic acids and fluoresces red-orange with single-stranded nucleic acids and yellow-green with double-stranded nucleic acids. RE3 infection tended to cause the host cells to spread to larger-than-usual proportions in the monolayer; viral inclusions formed in the cytoplasm and fluoresced yellow-green (because reoviral RNA is double-stranded) against the orange fluorescence of the rest of the cytoplasm. With careful scanning on a fluorescence microscope, a single infected cell could be detected on a coverslip. The principal drawback of the method was tedium. Some preliminary concentration methods were also explored: adsorption to and elution from bovine RBC, dialysis against polyethylene glycol, and preparative ultracentrifugation. Adsorption to bovine RBC is unique to RE3; the other methods found broader applications and will be discussed in the next section.
Detection of viruses by inoculation of cell cultures may be based on plaque formation or production of cytopathic effects (CPE); other things being approximately equal, the two methods are equally sensitive (Kostenbader and Cliver 1973) . Limitations are the susceptibility of the inoculated cells to the virus that happens to be present, and the volume of inoculum that can be tested in single culture. Where more than one cell culture type was needed to cover the spectrum of viruses of concern, we tried mixing two types of cells in a single culture but later learned that the inoculum could be harvested from a cell culture of one type, after an appropriate adsorption period, and transferred to a culture of another type with little loss of infectivity, and that much larger volumes of inoculum than usual could be tested per cell culture flask, under the right conditions, by the CPE method Cliver 1977, 1986) .
Viruses are very small and are likely to occur in the environment in large volumes of water or in problematic food matrices. The default detection method was to infect susceptible cell cultures, which have a limited capacity for sample volume, a low tolerance for food residues, and the high susceptibility to contaminating bacteria and molds. We and others faced the challenge of concentrating virus from large volumes of fluid suspension, perhaps after separating the virus from a solid food sample.
In the early 1960s, working-class preparative ultracentrifuges were becoming available, and we were able to get one. This represented a ''brute-force'' approach to virus concentration, with as much as 100,0009g to sediment the virus. The forces were massive, but there was a tendency for the sediment to stir back into the supernatant as the rotor decelerated, even with the brake off. We determined that a 2% solution of gelatin was solid at 4°C and liquid at 20°C. This was used to install a 0.1-ml ''trap'' at the point in the tube farthest from the axis of rotation. The rotor and sample were pre-chilled, so that the trap remained solid until the end of the run. Then, the cap was removed from the tube and the supernatant poured off; the trap was liquefied and collected in a small volume of sterile diluent. This was quantitatively efficient, but the volumes of virus suspension that could be processed in a 1-2-h run were relatively limited (Cliver and Yeatman 1965; Gibbs and Cliver 1965) . There was also the problem of an occasional ''catastrophic disassembly event''-the ultracentrifuge manufacturer's euphemism for disintegration of the rotor at speed. This happened twice in our ultracentrifuge; although no one was hurt, cleaning, disinfection, and reconstruction were very problematic.
We also tested a procedure whereby the virus suspension was sealed in dialysis tubing and immersed in a 50% solution of polyethylene glycol (PEG). This could be done with virtually any virus and any number of samples (PEG was inexpensive), but harvesting the samples typically awaited the following day (Cliver 1967a; Gibbs and Cliver 1965) . There was the additional problem that if PEG contaminated the concentrated sample, the sample would intoxicate the inoculated cell culture. It was necessary to rinse the outside of the dialysis tubing carefully before opening it; we also devised a wringer apparatus that would force the concentrated sample to the open top of the dialysis tube for aseptic collection.
Eventually, we undertook to combine PEG dialysis with ultracentrifugation. A wide-mouth funnel was inserted into the ultracentrifuge tube, and a cast cylinder of PEG was inserted into a dialysis tube that extended from almost the bottom of the ultracentrifuge tube to the top of the funnel. When a 100-ml sample was poured into the funnel, excess fluid was imbibed by the PEG inside the dialysis tube until the total volume could be contained by the ultracentrifuge tube. At this point, the funnel and the PEG-containing dialysis tube were removed, and the ultracentrifuge tube was capped and placed in the rotor (Herrmann and Cliver 1968b) . This approach was not compatible with use of the trap described above.
Another important development at that time was the introduction of cellulose-ester membrane filters, which could be stored in the dry state and sterilized by Food Environ Virol (2010) 2:1-23 5 autoclaving. Cellulose nitrate (gun cotton) had long been in use as a component of smokeless gunpowder-no accidents seem to have occurred with the filters. Some of the filters were said to comprise pure cellulose nitrate, while others were composed of mixed esters of cellulose, with nitrates predominating. These represented a huge advantage from the standpoints of convenience and reliability. They were produced in a great array of nominal pore sizes, based on the size particles that would pass through and on the pressure required to force air through water-wet filter. The pores themselves were not uniform, and the filters resembled a sponge 150 lm thick, but their ability to retain or reject particles larger than their nominal pore size was phenomenal. A distinguished diagnostic virologist, G. D. Hsiung, soon put this rejection capability to work in classifying unknown viruses (Hsiung 1965; Hsiung and Henderson 1964) . According to her scheme, small viruses would pass a nominal 50-nm filter, medium viruses would pass a nominal 100-nm filter but not the 50 nm filter, and large viruses were completely retained by a filter of 100-nm porosity. She noted that these properties were qualitative, in that small viruses, for example, would be found in the filtrate of the 50-nm filter, but a large proportion of the viral particles were retained in the filter matrix. We were able to show that adding a small proportion of animal serum to the virus suspension obviated such retention (Cliver 1965a) .
Given that the affinity of the membranes was such that they might retain more than 99% of the virus, it occurred to me that this might afford a basis for concentration of viruses from fluid suspension, assuming that means could be found to elute the virus from the filter matrix. We used an eluent of 30% agamma chicken serum in phosphate-buffered saline (PBS) and reported a 50% probability of detecting coxsackievirus A9 (CA9) in tap water at the level of 2 PFU per liter (Cliver 1967b) . These results were summarized at a 1965 symposium (Transmission of Viruses by the Water Route) convened by Dr. Gerald Berg in Cincinnati and hosted by the Federal Water Pollution Control Administration, falling within the US Department of Interior. I coined the term ''membrane chromatography'' for this process: I had done column chromatography previously, and I envisioned the membrane as a cylinder with an extreme height-to-diameter ratio. More recently, ''virusadsorption-elution'' has been contracted to ''viradel.'' Advance notice of our study resulted in several discussants mentioning experiments that basically corroborated our report. At that point in my career, I did not realize that publication in a proceedings volume was not equivalent to publication in a refereed journal; worse yet, the proceedings volume did not appear until 1967 (Berg 1967) . I also suffered from inability to think big-since that time, many innovations have been added, some of which have not stood the test of time, commercial versions have been developed, and adsorption-elution has become the basis for large-scale official water testing methods of the US Environmental Protection Agency (EPA-EPA 2001) . I had little input to these developments.
Another procedure for concentrating enteric viruses from dilute suspension-the aqueous polymer two phase system-was also being studied seriously during the 1960s (Philipson et al. 1960) . Two groups reported their studies of this method at the 1965 symposium (Lund and Hedstrom 1967; Shuval et al. 1967 )), and one reported further study in a journal (Lund and Hedstrom 1966) . The method entailed adding polyethylene glycol 6000 and sodium dextran sulfate to the sample; the thoroughly mixed suspension was placed in a cold room to separate for 18-24 h, and the small, lower phase (formed by the sodium dextran sulfate) was harvested and tested for virus. We tested the procedure with seven types of enteroviruses plus influenza A virus and found that the sodium dextran sulfate was apparently inhibitory to coxsackievirus B2, echovirus 6 (EC6), and influenza A virus . The last of these was probably of no consequence, but the inhibitory action of the sodium dextran sulfate seemed likely severely to bias surveys of water or wastewater such as had been reported previously (Lund and Hedstrom 1967) . We later showed that substituting dextran T-500 for the sodium dextran sulfate mitigated the inhibition problem (Grindrod and Cliver 1970) ; however, the method was relatively ponderous and slow; so it found limited use. It eventually developed that viruses could be precipitated with polyethylene glycol, without dextran for phase separation (Lewis and Metcalf 1988 ).
We later did some study on antibody capture (Deng et al. 1994 ) and on immunomagnetic concentration of hepatitis A virus (HAV) López-Sabater et al. 1997 ) and were able to show that the urea-argininephosphate buffer that had been used successfully to elute human viruses adsorbed to filters was also applicable to phages (Jothikumar and Cliver 1997) .
Very early in my tenure with FRI, I was invited to review knowledge of irradiation of viruses, as it might pertain to food preservation (Cliver 1965b) . Some irradiation-inactivation results were presented at the 1965 water symposium, but these were not for viruses that were likely to be foodborne (Sharp 1967) . Picornaviruses and caliciviruses present extremely small targets and are thus likely to require large doses of ionizing irradiation to achieve substantial degrees of inactivation (Kaplan and Moses 1964) . Predictability of ''kills'' is further complicated by the tendency of viruses to aggregate, whereby an infectious unit may comprise two or more virions, all of which must be inactivated before the infectivity of the unit is lost Chang 1967) . Studies we did with support from the US Atomic Energy Commission seemed to indicate that a low dose of 60 Co gamma-rays had induced both a host range and an antigenic mutation in CA9 (Cliver 1968 ), but the results were not repeatable. We later learned that CA9 was rather idiosyncratic among enteroviruses (Herrmann and Cliver 1973a ). More extensive virus irradiation studies were conducted in the Virology Branch of the US Food and Drug Administration (FDA- Sullivan et al. 1971) , who later reported that radiation sensitivity was lower in ground beef than in cell culture medium (Sullivan et al. 1973) .
Irradiation of shellfish was considered as a means to control bacterial contaminants (Licciardello et al. 1989 )-Vibrio parahaemolyticus should be a special concern, as its presence is not related to fecal contamination of the growing water. In this connection, the irradiation process was examined as to its probable effect on contaminating enteric viruses. This was a time when aquarium studies of virus uptake and depuration of shellfish were rare (Power and Collins 1989) , and so it was an accepted practice to inject virus into the soft tissue (no specific organ) of shellfish for irradiation. A first such study was conducted in the 60 Co irradiation facility of the University of Lowell, Massachusetts, with HAV and rotavirus in hard-shell clams (Mercenaria mercenaria) and eastern oysters (Crassostrea virginica) (Mallett et al. 1991) . Viruses produced at the Baylor College of Medicine were inoculated into the shellfish at the University of Lowell (no details given), irradiated, shucked, and shipped back to Baylor for assay. The virus extraction process was not described. Decimal reduction (D 10 ) values of 2.0 kGy were reported for HAV, and 2.4 kGy for rotavirus; irradiation of live, un-inoculated shellfish at these doses was said to have minimal adverse effects on their viability and their palatability after various styles of cooking.
We were offered a small grant by the International Atomic Energy Agency to look further into this process. With this and limited support from other sources, we planned to produce the viruses in Madison and send them to Lowell to be inoculated into the shellfish and irradiated as was done in the earlier study, with the soft tissue shipped back frozen for assay in our laboratory. Extraction of the virus was to be done by our ''Cat-Floc'' method (Kostenbader and Cliver 1981) . Because of the high cost of irradiation, we mixed poliovirus 1 (PO1) with HAV, so that they could be inoculated and irradiated together. When the virus mixtures were inoculated onto FRhK-4 cell monolayers, PO1 plaques were seen within 5 days; however, if the mixture was treated with anti-PO1 antiserum before inoculation, then HAV plaques were seen at approximately 16 days of incubation, and the PO1 was not expressed. This also worked with the shellfish extracts-to our knowledge, this approach has not been used by others, although mutually exclusive host systems have permitted inactivation studies on mixtures of two animal viruses and two phages (Olivieri et al. 1983 ). The first shipment of viruses was lost in transit to Lowell, so another had to be sent. Eventually, the samples arrived back in Madison for extraction and assay. Calculated D 10 values for viruses in clams (PO1, 5.43 kGy; HAV, 5.95 kGy) were higher than those reported by others (Fig. 1) , perhaps due to matrix effects and the different extraction procedure. Unfortunately, the laboratorian (Kenneth D. Kostenbader, Jr.) developed a fatal illness, and the oyster samples were not assayed, nor have the results presented here been published elsewhere.
James Mosley, then chief of the Hepatitis Unit at the Communicable Disease Center (now Centers for Disease Control and Prevention-CDC), stated in 1965 that infectious hepatitis. (now hepatitis A-HA) was the only viral disease for which there was expert consensus in favor of waterborne transmission, although he also considered the possibility of waterborne poliomyelitis (Mosley 1967) . He tabulated 50 waterborne HA outbreaks worldwide; he also stated that 14 foodborne HA outbreaks had been recorded in the USA from 1952 to 1964. In those years, diagnosis was based on clinical signs and on serum transaminase levels-there was no direct test for HA (Cliver 1966) . Another problem in investigating common-source outbreaks of HA is the long incubation period (15-50 days, median 28 days), which challenges the victims to remember what they have eaten and the epidemiologists to create a coherent record of events. Once the frequent association of HA with raw shellfish consumption was recognized, it became routine to ask about shellfish consumption when HA was diagnosed: this may have imposed some bias against identification of other potential vehicles. By the publication of the WHO Manual on Food Virology in 1983, we were able to tabulate 153 foodborne HA outbreaks recorded between 1943 and 1982, as well as 117 waterborne HA outbreaks between 1895 and 1980 (Cliver 1983) .
Research with HAV in our laboratory awaited the development by others of means to do the investigations we desired. Although HAV had been propagated in cell culture as early as 1979 (Provost and Hilleman 1979) , it Food Environ Virol (2010) 2:1-23 7 was not until 1972 that a combination of an HAV strain and a cell culture strain that resulted in cytopathic effects and plaque formation reported (Cromeans et al. 1987) . A good deal of study on foodborne HAV had been done in the early 1990s (Cromeans et al. 1994 ). Our eventual contributions included development of an immunomagnetic method for detecting HAV in oysters (López-Sabater et al. 1997) , disinfection studies with ClO 2 (Mariam and Cliver 2000a) , and demonstration that HAV is only *90% inactivated by pasteurization in raw milk (Mariam and Cliver 2000b) .
It was clear from the outset that food matrices presented special problems with respect to virus detection. Quite small quantities of virus needed to be separated from the solid phase of the food sample (and any incident microflora), concentrated to a small volume of fluid, and inoculated into a susceptible cell culture. There were no concerns at that time regarding PCR inhibitors, but many other complications. Shellfish were an early concern because of their association with outbreaks of HA (Koff et al. 1967; Mosley 1967) . T. G. Metcalf, then at the University of New Hampshire, pioneered laboratory studies on the association of enteric viruses with oysters (Metcalf and Stiles 1965) . He made several further contributions to environmental and shellfish virology there and after his move to the Baylor College of Medicine (Atmar et al. 1995 (Atmar et al. , 1996 Stiles 1967, 1968; Metcalf et al. 1979 Metcalf et al. , 1980a Metcalf et al. , b, 1995 .
Because our group was in Chicago and then Madison, far from the sea, our attention was often directed to foods other than shellfish. We considered that foods might be liquids, or solids that could be contaminated in depth, or solids with superficial contamination limited to a relatively impermeable surface. The most challenging of these would be solids that could be contaminated in depth-our first model system comprised cottage cheese and CA9 (Herrmann and Cliver 1968a). An inoculated, 25-g sample was slurried with 100 ml glycine-NaOH buffer, treated with Freon TF (1,1,2-trichloro-1,2,2,-trifluoroethane) and bentonite, clarified by centrifugation, and concentrated by a two-stage (PEG dialysis followed by ultracentrifugation) process that yielded 0.5 ml for testing in cell culture. Virus recoveries from the samples inoculated with 50 PFU or less were roughly 50%, as determined by the plaque technique. In addition to cottage cheese, the method was then adapted and tested with beef with gravy, carrots, chicken pot pie, chocolate éclairs, clams, ground beef, peanut-butter sandwiches, potato salad, and strawberries. The beef with gravy and the peanut-butter sandwiches had been freeze-dried to be fed to astronauts early in the US space program (Herrmann and Cliver 1968b). Viruses inoculated included CA9, coxsackievirus B3 (CB3), and EC6; poliovirus 2 was used in experiments on recovery of antibody-neutralized virus. Not surprisingly, optimal extraction methods varied among foods, with bentonite omitted and serum substituted for low-protein foods. Recoveries of EC6 were somewhat less than those for the coxsackieviruses. Overall, it was found that a 25-g food sample must contain at least 3-4 PFU for a 50% probability of detecting virus. We supposed that viral contamination of tomatoes would be largely limited to the surface, so we devised an apparatus that would dislodge virus from the tomato surface without suspending the food solids in the extract . Surface dirt, fecal solids from the model contaminant, and a variety of bacteria appeared in the washings and had to be dealt with before the concentration step; recoveries of CB3 were roughly 49%, with a 50% endpoint, for a positive test result, of slightly under 2 PFU per tomato.
At the request of the Calgon Company of Pittsburgh, we tested a product called Cat-Floc (a polydimethyldiallyl ammonium chloride, MW *500,000) for virus removal when used as a primary coagulant or coagulant aid in treatment of drinking water and wastewater (Cliver 1971) . Results were encouraging, and we were left with a great deal of the product when the trials ended. Oysters (Crassostrea virginica and Ostrea edulis) were experimentally inoculated with enteroviruses and minced with scissors, then stirred with 100 ml PBS to which was added 2 ml of a 1% Cat-Floc solution; after 5 min of stirring and 15 min of settling, the suspension was filtered by pressing in a potato ricer (Kostenbader and Cliver 1972) . The extract could be concentrated by ultracentrifugation or ultrafiltration. Quantitative virus recoveries exceeded 80%, but it was shown that poliovirus neutralized with coproantibody was not reactivated by this process. CA9, coxsackievirus B2, and EC6, which had been inhibited in previous studies with polymers Cliver 1969, 1970) , were not affected by the Cat-Floc. A number of other groups have since used Cat-Floc in extracting viruses from mollusks, crustacea, and estuarine sediments (Johnson et al. 1981; Landry et al. 1982; Richards et al. 1982; Seidel et al. 1983; Wait and Sobsey 1983) .
A filter called ACG/B (activated carbon particles in a matrix of cellulose and glass fibers, H. R. Reeve-Angel, Clifton, N.J.) was tested with and without Cat-Floc for recovery of CB1, EC6, and PO1 from a variety of foods (Kostenbader and Cliver 1973) . Meat products included ground beef and frankfurter sausages; chicken salad served as a representative mixed meat product; fresh vegetables included lettuce and carrots; flour products were creamfilled cakes and bread rolls; seafoods comprised oysters (Crassostrea virginica) and clams (Mercenaria mercenaria); dairy products included creamed-curd cottage cheese and cheddar cheese. Filtrates could be filter-sterilized (0.20 lm porosity) and concentrated before testing in cell culture; recoveries of at least 80% were recorded with optimal selection of procedures. In a later version, the Whatman GF/F glass fiber filter was substituted and a great number (seven dry-form and 13 liquid) of polyelectrolytes were compared for recovery of PO1 from ground beef (Kostenbader and Cliver 1981) . Cat-Floc T performed best among the liquids (88% recovery), and Cation 105C (ICI America, Wilmington, Del.) was the best of the dry-form flocculants (107% recovery). Cat-Floc was also found useful in the recovery of reovirus 1 from ground beef (C73% recovery) and oysters (C. virginica, C50% recovery). Reviews comparing the various extraction and detection methods as applied to different foods were published in the Journal of Food Protection (Cliver et al. 1983a, b) and in various editions of the Compendium of Methods for the Microbiological Examination of Foods (Cliver 1976; Cliver et al. , 1992 Richards and Cliver 2001) .
The first WHO support for our food virology study was received at UW in 1967; it came from the Veterinary Public Health (VPH) division, which had primary responsibility for food safety at that time. Our principal contact at the time was Dr. Z. Matyáš, who was then the Food Hygienist in VPH. In September of 1969, I was invited to chair a small informal consultation on virus transmission via foods at the WHO in Geneva; most of those present studied animal viruses that might occur in foods, rather than viral pathogens of humans. I was appointed a WHO consultant on virus transmission through foods on that occasion-a designation that may still survive, but is not active. Further informal consultations were held in Geneva and in Brno, Czechoslovakia, over the next few years. In addition to nominal support for our research in food virology, WHO wanted an international system of information sharing, based eventually in our group. We were the Data Collection Centre for Food-borne Virus Disease and Research on Viruses in Foods, World Health Organization Food Virology Programme, from 1971 to 1975, after which we were designated the World Health Organization Collaborating Centre on Food Virology. What evolved was a three-part program: (1) the Data Collection comprised edge-punch cards, preprinted in the UK, on which bibliographic information and abstracts of pertinent publications were recorded with a typewriter; (2) a Request for Specific Information form was designed and made available to investigators worldwide, to be completed and mailed to Madison for response from the Data Collection; and (3) a List of Food Virologists, compiled largely from authors of articles in the Data Collection. Authors were contacted by mail at their address of record and asked to provide complete contact information and a brief (B25 words) description of their study. When as many responses had been received as seemed likely to come, a List was compiled and mailed to everyone on it. New Lists were undertaken at roughly 2-year intervals. This was a time when there were no computers, word processors, or Food Environ Virol (2010) 2:1-23 9
Internet available, and so everything was done on paper and distributed by post. We issued an Information Alert, listing recent, pertinent publications, to members of the List at least annually. The edge-punch cards that were the Data Collection were roughly 12.5 9 40 cm, with holes along two edges that could be coded and opened to the outside of the card, for sorting with a long needle; obviously, the system of classification was limited in precision and scope. All the same, a few aspiring food (and water) virologists did avail themselves of the service, and we responded as best we could with our limited personnel. Details of these efforts appear in the WHO Manual on Food Virology (Cliver 1983 
Not all of the methods we developed were applicable only to food and environmental virology. Because of our chronically impecunious situation, we tried to devise cell culture methods that saved costs and labor (Cliver 1973a; Cliver and Herrmann 1969) . We determined to what extent the purchased cell cultures could substitute for those grown in one's own laboratory .
The ability of membrane filters to adsorb virus efficiently afforded a rapid method to estimate the specificity of radioactive labeling of virus-nuclide that was not virus associated was found in the filtrate (Herrmann and Cliver 1973c) . We also devised an electrophoretic method for collecting viral particles on a polycarbonate membrane so that the particles could be enumerated by scanning electron microscopy (Heinz et al. 1986 ).
Some of our earliest studies on recovering viruses from foods had been sponsored by the US Air Force School of Aerospace Medicine (Herrmann and Cliver 1968a) , which explains the inclusion of two freeze-dried foods that had been developed for astronauts (Herrmann and Cliver 1968b ). Subsequently, we were contacted by the life support groups of the US National Aeronautics and Space Administration (NASA) regarding both astronaut foods and the spacecraft water supply. Electricity aboard spacecraft was generated by hydrogen-oxygen fuel cells, so that the water produced as a byproduct was available for drinking and for rehydration of foods that had been freeze-dried to save weight. The food studies were largely precautionary-NASA had a great many ''what-if'' concerns at that time. Low-moisture foods studied included bacon squares, beef bites, cheese sandwiches, spaghetti with meat sauce, and banana pudding . Viruses tested included influenza virus type A (strain PR8), parainfluenza virus type 3 (strain SF-4), reovirus type 1 (strain Lang), EC6 (strain D'Amori), and various polioviruses (some in the feces of infants who had received the trivalent oral polio vaccine). The influenza, parainfluenza, and reoviruses persisted for B3 days in inoculated low-moisture foods; whereas the enteroviruses persisted [2 weeks at room temperature, and [2 months in the refrigerator. Various other temperature-storage regimes were also evaluated; poliovirus of fecal or cell-culture origin behaved similarly. Fecal poliovirus was inactivated 10 -2 during freeze drying in cream-style sweetcorn, but the residual virus persisted with little loss during 15 weeks' storage at 5°C. The water-system studies were inspired by a technological problem. Although the water produced by the hydrogen-oxygen fuel cells had\1 ppm total solids, it was mixed with other water (such as condensate from inside the space suits) and stored in a reservoir lined with a polymer bladder. Evidently, the ultrapure water leached enough solute (either plasticizer or unreacted monomer) to support the growth of the so-called distilled-water bacteria (e.g., Pseudomonas aeruginosa). These created a nuisance; and since check valves were unreliable in a weightless environment, other potential modes of water contamination were envisioned. NASA had commissioned development of an electrolytic silver-ion generator that was intended to decontaminate the water; it was demonstrably capable of generating silver ions in water, but the antimicrobial effectiveness of these ions had not been tested. We were asked to determine the effectiveness of very low silver-ion levels against both distilled-water and pathogenic bacteria, as well as viruses. This required a team, in that we needed help measuring the low levels of silver ions and a more experienced bacteriologist than I. Silver-ion levels in the working range of 50-250 ppb were estimated by a technique called neutron activation analysis (NAA) by Dr. Wesley K. Foell of the UW Department of Nuclear Engineering. Quantification of silver by NAA turned out to be something of an art form; we were not confident enough of the measurements to submit our findings for journal publication. Bacteriological results, reported by Dr. John M. Goepfert of the UW FRI, will not be described here, except to say that substantial kills were obtained at the higher end of the silver-ion working range. Among the viruses tested, only vaccinia appeared to be completely resistant to silver ions. The viruses tested, in order of increasing silver sensitivity, were influenza type A, several enteroviruses, reovirus type 1, and rhinovirus type 1A. The time dimension for inactivation of these viruses ranged from days to minutes. The rate of inactivation was similar, whether the silver ions had been added by an electrolytic generator or as a soluble salt. The concentration of silver (in the range of 50-250 ppb) was not always the principal rate-limiting factor in inactivation of the virus. We were not able to measure the uptake of silver by the virus particles. Extremely pure water was not necessary for viruses to be inactivated by silver ions. However, feces (\1 ppm, or a dialyzable component of feces) were extremely effective in preventing silver inactivation of enteroviruses and, to a lesser extent, reovirus. Respiratory mucus did not show this sparing effect.
Environmental virology began with water and wastewater. Although our domain was supposed to be foodborne viruses, there was much to be learned from the study in progress regarding detection and inactivation of viruses in water and wastewater. As mentioned above, we were requested to do some evaluations of a polycation coagulant (Cat-Floc) in controlling viruses in this context. The product showed promise (Cliver 1971) ; however, our application of the product to extraction of viruses from food samples was probably a mixed blessing to the manufacturer-they wanted to sell their product in large containers, and laboratory applications in food virology mostly produced requests for small volumes.
We were invited to join an interdisciplinary group at UW called the Small Scale Waste Management Program (SSWMP) that was studying on-site wastewater treatment, with a focus on septic tanks and the transport of contaminants through soil in septic tank effluents. We did both model studies of soil transport in laboratory columns Green and Cliver 1975) , and transport studies in the field (Alhajjar et al. 1988; Cliver 1984) . Field studies were done at homes where SSWMP had already installed groundwater sampling wells, so as to be able to study transport of contaminants from septic tank soil fields (Stramer 1984) . Inoculum was obtained by offering unlimited supplies of disposable diapers to mothers whose infants were receiving the oral polio vaccine, if the mothers would freeze and return any diapers with feces in them. Our laboratory thawed and assayed the feces. When at least 100 g of high-titer feces were accumulated, these were flushed down a toilet at a study-site home, and samples were taken from the septic tank and groundwater over time.
The poliovirus tended to accumulate in the sludge in the bottom of the tank, with periodic eruptions that caused the virus to leave the tank with the supernatant and, in time, be detectable in groundwater down-gradient from the soil absorption field. We also studied methods of disinfection for the material that is pumped out of the septic tank at intervals (Stramer and Cliver 1984) and modeled the mixing of human (septic tank effluent) and animal wastes (manure slurry) for disposal to land (Snowdon et al. 1989a, b) .
Over time, we had various opportunities to study viral disinfection of urban wastewater effluents; association of viruses with wastewater solids was considered (Cliver 1975 ). An early project used a mixture of two animal viruses and two bacteriophages to determine how each was inactivated by chlorine dioxide (Olivieri et al. 1983 ). We worked with Milwaukee, Wisconsin on disinfection of its effluents for discharge to Lake Michigan (Warriner et al. 1985) . We helped the Madison (Wisconsin) Metropolitan Sanitary District with a pilot study to show that UV would inactivate viruses in their treated effluent (unpublished) and later worked with them on seasonal antiviral disinfection of their effluent when the full system was in place (Buyong et al. 1993) .
We worked with the Committee on the Challenges of Modern Society (the civilian branch of the North Atlantic Treaty Organization) to compile an international review of the microbiology, including virology, of drinking water in industrialized nations (Cliver and Newman 1984) . The article, written by 50 scientists in 11 countries, was eventually published as a special edition of a journal (Cliver and Newman 1987) . Biodegradation Early on, I had the idea that, since viruses are not known to autolyze when they lose infectivity, they may be subject to enzymatic or biological degradation in the environment or else they would accumulate at high levels over millennia. Accumulation of vast numbers of viruses in marine water has been reported (Chen et al. 2001 ), but these are not necessarily agents of human infection, nor are they necessarily infectious as detected. We were able to show that some proteases and some microbes would attack enteroviruses (Cliver and Herrmann 1972) ; CA9 was especially susceptible to enzyme attack (Herrmann and Cliver 1973a) . By differential radionuclide labeling, we demonstrated that CA9 and PO1 in lake water were inactivated by Pseudomonas aeruginosa, which selectively used their coat protein, but not the RNA, as substrate (Herrmann et al. 1974) .
Later, as part of the SSWMP program, we were asked to investigate the possibility that septic tank effluent could safely be mixed with animal manure slurry on farms, for eventual disposal to land. After a review of the literature (Snowdon et al. 1989b ), we reported some preliminary studies that indicated the manure slurry had some antiviral effect (Snowdon et al. 1989a ). More detailed study showed that bacteria from swine manure, as well as in a waste mixture of septic tank effluent and swine manure slurry, were capable of inactivating poliovirus (Deng and Cliver 1992) . Further studies showed that bacteria in either dairy or swine manure could inactivate HAV (Deng and Cliver 1995b) . Specific bacteria, isolated in pure culture, were shown to attack HAV (Deng and Cliver 1995a) ; some of the active substances were characterized as specific proteases, but others were of quite low molecular weight and unfortunately were not studied further.
The hope is that enteric viruses will lose infectivity between when they are shed and when someone else ingests them (Rzezutka and Cook 2004) . In addition to the NASA studies described above, we considered foods with a rich bacterial flora that might cause biodegradation of contaminating virus. Lynt had reported that spoilage of several foods held at room temperature had little effect on the persistence of inoculated enteroviruses (Lynt 1966) . Because CA9 had been found particularly vulnerable to proteases (Herrmann and Cliver 1973a) , it was chosen as the model contaminant in a study of sausage fermentation and of microbial spoilage of ground beef (Herrmann and Cliver 1973b) . Although proteolysis (putrefaction) was very evident in the ground beef, less than one log of inactivation was recorded by 8 days at either 4 or 23°C; however, substantial reductions were seen at 14 days. Earlier, Kalitina had reported that group B coxsackieviruses persisted equally well in ground beef that had or had not been autoclaved (Kalitina 1966) . CA9 was 84% inactivated during 24 h of Lactibacillus fermentation at 30°C in making Thuringer sausage (pH reduced from 6.0 to 4.8); subsequent heating of the sausage for 6 h at 49°C left just 0.1% of the original inoculum infectious (Herrmann and Cliver 1973b) . Kalitina had also reported that cottage cheese fermentation did not affect enterovirus persistence (Kalitina 1969) . We studied the various stages involved in making cheddar cheese and found that pasteurization of the milk caused a million-fold inactivation of PO1; however that if the virus was added with the starter culture, then there was about 98% inactivation during cheesemaking and little additional virus loss during 7 months of storage of the product at 4°C (Cliver 1973b) . Influenza A and vesicular stomatitis viruses, on the other hand, were undetectable in the pressed curd at the end of the cheesemaking process ([5-log inactivation) .
We undertook to create a mathematical model-outside the context of real foods-of the effects of pH 3, 5, 7, 9), temperature (2 & 30°C), time (days-weeks), and specific salts on the stability of enteroviruses (Salo and Cliver 1976) . PO1 was inactivated faster at any pH at 30°C than at any pH at 2°C. At pH 3, glycine-based buffer was somewhat more antiviral than phosphate-based buffer, whereas the reverse was true at pH 9. NaCl and other chloride salts accelerated PO1 inactivation at pH 3, but NaCl was much less effective at pH 4.5-7. In this era before the advent of ''molecular'' techniques, methods of demonstrating virion degradation were relatively unsophisticated. Loss of RNA infectivity appeared to accompany loss of infectivity of the virion, except at pH 3 in the presence of MgCl 2 . Susceptibility of the virus to RNase or to chymotrypsin was tested with radioactively labeled virus ( 32 P in the RNA or 14 C-leucine in the capsid) and trichloracetic acid (TCA) precipitation. TCA precipitates large molecules nonspecifically; so if the radionuclide of interest was soluble in the presence of TCA, the molecule from which it derived had been degraded. RNA hydrolysis was found to have occurred in PO1 at pH 5 and 7; and the viral RNA became susceptible to RNase in virus inactivated at pH 3, 5, 6, and 7. Only virus inactivated at pH 3 became sensitive to chymotrypsin. Echovirus 7 (EC7) has the fortuitous property of agglutinating human RBC (we used blood group O); although this may have nothing to do with infectivity, it seems similar to the blood group antigen affinities of norovirus (Marionneau et al. 2002) . The hemagglutinins of EC7 were destroyed during inactivation at pH 3, 4, 5, and 6; loss of hemagglutinin seemed precede loss of infectivity at pH 6 (Salo and Cliver 1976) .
Additional methods of characterizing modes of virus inactivation were applied in studies of inactivation of CA9, EC7, and PO1 by ascorbic acid and sodium bisulfite (NaHSO 3 ) (Salo and Cliver 1978) . These studies had been prompted by Lynt's perception that sodium bisulfite in cole slaw had antiviral activity (Lynt 1966) . All the three viruses were susceptible to these food additives at levels of 0.1 M and sometimes much less (Salo and Cliver 1978) . Methods of characterizing virus degradation during inactivation included: loss of hemagglutinating activity (EC7 only); differential adsorption to cellulose nitrate (Millipore GSWP) and cellulose acetate (Gelman GA-8) filters; differential attachment to primate (HeLa) and non-primate (MDBK-bovine) cells; RNA infectivity of whole virus or cold phenol-extracted RNA as facilitated by DEAE-dextran; and sedimentation in sucrose density gradients. When whole virus was apparently inactivated, in that it could no longer initiate plaques, it was often infectious when inoculated together with DEAE-dextran, which showed that the viral RNA was intact. The native viruses were adsorbed by cellulose nitrate but not cellulose acetate filters, as reported previously (Herrmann and Cliver 1973c) ; however, PO1 labeled with 32 P-RNA and inactivated by 25 mM NaHSO 3 gradually developed affinity for the cellulose acetate membrane, as measured by retention of virus-associated radionuclide. 14 C-leucine labeled PO1 showed low nonspecific attachment for MDBK (bovine) cells: after inactivation, the virus had no more affinity for HeLa (human) cells than for MDBK. Much 32 P in PO1 inactivated by ascorbate no longer banded as native virus fraction in the sucrose density-gradient experiments; in one instance, EC7 (which has the same sedimentation profile) was mixed with the PO1 before centrifugation. EC7 hemagglutination identified the fractions that would contain native PO1. Some of these means of characterizing inactivated virus might be adaptable for use with molecular methods.
Konowalchuk and Speirs had reported that viruses were inactivated in vitro by a variety of fruits and fruit juices (Konowalchuk and Speirs 1976a, b; Konowalchuk and Speirs 1978a, b) . Inactivation by apple juice was said to be irreversible (Konowalchuk and Speirs 1978a) . We focused on the effects of Concord grape juice, which inactivated CB3, EC6, and PO1 but not parainfluenza virus type 3 . Filtration of 32 P-labeled, inactivated PO1 through a 50-nm porosity polycarbonate membrane ruled out aggregation as a cause of titer loss. The inactivated virus showed limited attachment to homologous host cells but did not infect them. Treatment with polyethylene glycol (m.w. 20,000) reactivated the inactivated virus, as did human blood serum, indicating that the capsid had not been permanently modified. We had fed infant pigs human foods in an effort to make their GI tracts as nearly analogous to humans' as possible. We collected contents of stomach, duodenum, jejunum, ileum, cecum, and descending colon, and reacted these with grape-juice inactivated PO1 for 30 min at room temperature; the treated virus was reactivated 42-82%, depending on which GI contents were used. In addition to native virus, we used coproantibody-neutralized PO1 as a control; reactivation occurred with stomach, duodenum, and cecum contents, but not the material from other segments of the GI tract. It is unfortunate that the apparent reactivation of coproantibody-neutralized virus, which would have occurred upon ingestion, was not given more weight (not mentioned in the title, and only briefly in the abstract), as this finding may well have had greater public-health significance than the grape-juice results.
Enteroviruses attracted attention to coproantibody (presumably IgA) because they are shed for prolonged periods (often weeks) and are neutralized by coproantibody during the later part of the shedding period. Our first interest had been in how coproantibody might interfere with virus detection in cell cultures; the study just described was perhaps the first to show that coproantibody-neutralized virus is quite likely infectious if ingested. After extraction from potato salad by our early Freon method, PO2 was completely reactivated if it had been neutralized with coproantibody, but it was only partly reactivated if neutralized with hyperimmune rabbit serum (presumably IgG) (Herrmann and Cliver 1968b) . Some laboratories have been reluctant to use Freon, for various reasons. We later showed that coproantibody-neutralized PO1 extracted from ground beef could be reactivated by treatment with pancreatin (an extract of bovine pancreas containing a variety of proteases and other enzymes); the resulting extract could be tested as much as 35 ml per 25-cm 2 cell culture, and could be passed among different types of cell cultures, to detect cytopathic viruses (Kostenbader and Cliver 1986) .
We participated in a cooperative study on ClO 2 inactivation of enteric viruses in water mentioned earlier (Olivieri et al. 1983 ). The primary target was PO1; but we included a porcine enterovirus, an RNA bacteriophage (f2), and a DNA bacteriophage (/X174). Because these had mutually exclusive host ranges, all the four were included in the reaction mixture and assayed individually in the samples. Most similar to the PO1 in inactivation was the /X174; least similar to the PO1 was the porcine enterovirus, which proved much more sensitive than any of the other three to ClO 2 : see Surrogates, next section. RNA infectivity of the PO1 was apparently little affected by ClO 2 . HAV in tap water and in strawberry wash water was inactivated by ClO 2 at 4 ppm; ClO 2 was less efficient at inactivating HAV in experimentally contaminated strawberries, which were better disinfected by heat treatment (71.7°C, 60 min) after the strawberries had been made into puree (Mariam and Cliver 2000a) .
The two principal legal pasteurization methods for milk in the USA are the low-temperature, long-time method (LTLT: 63°C, 30 min) and the more common high-temperature, short-time method (HTST: 72°C, 15 s). We found that HAV was somewhat more susceptible to LTLT pasteurization than to HTST pasteurization in both homogenized, pasteurized whole milk and in raw whole milk. Inactivation was 94% by LTLT and 27% by HTST in raw milk (Mariam and Cliver 2000b) . This study was inspired by an earlier Michigan outbreak of HA in a religious group that did not use electricity or machinery. They milked their cows by hand (with highly inadequate hand-washing facilities) and sold it to a neighboring cheese plant; these data indicated that the cheese produced after the milk was pasteurized would not have been safe, in contrast to our earlier result with poliovirus (Cliver 1973b) .
RT-PCR was known to detect inactivated viruses in water and wastewater (Sobsey et al. 1998) ; the same was likely to be true for inactivated viruses in food. We found that a combination of proteinase K and RNase would eliminate most false-positive RT-PCR results with feline calicivirus (FCV-at that time, the best available surrogate for human norovirus), HAV, and PO1 inactivated by chlorine, heat (72°C), and UV . The treatment did not prevent RT-PCR detection of viruses inactivated at 37°C over long periods of time, as might occur in the environment; although the capsid protected the RNA, it was unable to attach to host cell receptors (Nuanualsuwan and Cliver 2003a) . We compared the infectivity of RNA in PO1 inactivated by chlorine, heat, and UV and found that the rates of inactivation for RNA and whole virus were similar with chlorine and UV inactivation but that RNA infectivity persisted during inactivation at 72°C (Nuanualsuwan and Cliver 2003b) . Several laboratories are now studying other methods to reduce false RT-PCR positive results with inactivated virus (Rodriguez et al. 2009 ).
Presently, model agents such as murine norovirus are being used experimentally to predict the persistence of human noroviruses in food and the environment, in that cultivation of human noroviruses in laboratory cell cultures has not lent itself to such experiments (Duizer et al. 2004 ). However, the challenges of detecting viruses in food and water, even when host systems were available, have always evoked wishes that surrogates could be found that would obviate testing field samples for the presence of viruses themselves. Both bacterial ''indicators'' and other viruses, including bacteriophages, have been considered. I will not try to survey the vast literature on this subject, but only give an overview of our activities. In that we were in a landlocked location, we welcomed the opportunity to join a collaboration looking at bacterial indicators and Gulf Coast oysters (Fugate et al. 1975) . EC6, PO1, and poliovirus 3 were found in the oysters, with no apparent correlation with coliform MPN, E. coli MPN, aerobic plate count, or the presence of Pseudomonas aeruginosa.
Concern about viral contamination of groundwater by on-site wastewater treatment systems led to consideration of coliphages as indicators (Johnson and Cliver 1986; Snowdon and Cliver 1989) . In field studies with poliovirus introduced to groundwater via septic tanks, indicator bacteria (total coliforms, fecal coliforms, fecal streptococci) were not consistently present in groundwater samples in which the virus occurred (Alhajjar et al. 1988; Stramer 1984) . Since human viruses cannot multiply in the environment, it was important to show that candidate indicator coliphages could not multiply in the environment, either. We focused on FRNA (''male-specific'') coliphages, which are similar in size to the small enteric viruses and were detected in about half of septic tanks from various areas of Wisconsin (Woody and Cliver 1994) . These were not found in groundwater samples, even if taken directly under the soil infiltration field. Phage Qb was used as a model: it was found that F-pilus synthesis by host cells did not occur at temperatures below 25°C-a temperature not encountered in Wisconsin groundwater nor in many Wisconsin surface waters (Woody and Cliver 1995) . Also, the host E. coli cells had to be actively growing (log phase) to support Qb replication, which is less likely in the environment than in permissive conditions in the laboratory. Other constraints that were identified included competition from insusceptible bacteria with the host cells, which needed to be present at at least 10 4 CFU/ml to support Qb replication, competition from other phages, lack of nutrients in environmental waters, etc. (Woody and Cliver 1997) . We did describe a convenient viradel method for detecting waterborne coliphages (Jothikumar and Cliver 1997) , and a fluorescent plaque assay for coliphages from environmental samples .
The comparative study of ClO 2 disinfection was discussed in the previous section; the similarity of inactivation of PO1 and /X174 was apparently fortuitous, in that the porcine enterovirus, which resembled PO1 biologically, lost infectivity much more rapidly than PO1 and either bacteriophage (Olivieri et al. 1983) . We compared an FRNA coliphage (MS2) and a small DNA coliphage (/X174) with HAV under various conditions of inactivation: MS2 inactivation was fairly similar to that of HAV during heating in water and milk, especially at 72°C (Mariam and Cliver 2000b) . Both phages were more susceptible than HAV to drying and to ClO 2 disinfection. A UV disinfection study yielded decimal inactivation doses for FCV, HAV, PO1, MS2, and /X174 of 47.85, 36.50, 24.10, 23.04, and 15 .48 mW s/cm 2 , respectively .
The majority of our virology studies, especially with food, were necessarily performed in the laboratory. All the same, there was a constant urge to look for viruses in the real world of the food chain. The FRI was sponsored by many major food companies, and so it seemed reasonable to approach them first for permission to enter and sample. Few were willing, and many constraints came with the permissions we did receive. Seven plants were eventually sampled, each processing a different group of foods (Kostenbader and Cliver 1977) . In order to deal with this diversity, we developed a sampling plan that was adaptable to all. In order to monitor inputs, we chose to sample: (1) raw materials, (2) water used in processing, and (3) plant personnel. Outputs of the following types were sampled: (1) finished product, (2) by-products, (3) wastes, (4) plant personnel, and (5) wastewater. All the samples were to be tested for ability to produce CPE or plaques in cell cultures. Because of the diversity of samples in prospect, the following types of cell cultures were used as appropriate: primary monkey (Macaca mulatta) kidney, primary swine embryo kidney, chicken embryo fibroblast, HeLa, Vero (Cercopithecus aethiops), and Madin-Darby bovine kidney. Two methods mentioned earlier were quantitatively validated: one entailed inoculation of as much as 1.4 ml of sample per square centimeter of cell monolayer, with subsequent observation for CPE; the second involved testing the same sample in more than one type of cell culture by incubating the inoculum in the first culture for 20 h at 37°C and then transferring the inoculum to another type of cell culture. Both practices were shown to result in minimal loss of viral infectivity, even when the first inoculated culture was an insusceptible cell type, sometimes infected with a competing virus. Processing methods for diverse samples were also validated.
Plant A manufactured groceries. Samples tested included isomerose, gelatin, well water, wastewater, cheesecake mix, breakfast drink mix, freezer ''pops,'' and strawberry preserves; no virus was detected.
Plant B dried potatoes. Samples tested included raw well water; whole raw potatoes; stool samples from four workers who handled raw potatoes; dried potato granules, flakes, and slices; wastewater sludge from primary and secondary treatment; mud washed from the potatoes; floor sweepings; and stool samples from three workers who handled final product. No virus was detected.
Plant C slaughtered and processed swine. Samples tested with negative results included swine blood, raw well water, wastewater from sewer lines serving a spice-kitchen workers' locker room and a kill-floor workers' locker room, supernatant fluid from process wastewater, and packaged luncheon meat. The rate of intestinal infection of animals at the time of slaughter was found to be quite high. All the 10 fecal samples taken from holding pen floors on Monday, when the animals had been together for three days were positive. Three of these viruses were identified as reoviruses and the remaining seven were enteroviruses, of which six replicated only in swine cells, but one also replicated in monkey cells. A small, heat-stable agent that could not be sustained beyond two passages in swine cells was detected in sediment from process wastewater, in de-watered primary sludge, and in raw and cooked meat scraps. Follow-up sampling of swine feces and intestinal contents at the plant revealed a high incidence of enteroviruses, some of which replicated only in swine cells, while others replicated in both swine and monkey cells.
Plant D shelled eggs and froze liquid egg products. Negative test results were obtained from all the samples: eggs in the shell, washings from the shells, liquid whole eggs, liquid egg whites, ''leakers,'' spent shells, and process wastewater.
Plant E slaughtered cattle and swine. Samples taken there-contents of cattle and swine large intestines, swine blood, bologna, raw beef scraps, raw pork scraps, cooked mixed scraps, dried mixed scraps, and samples from the combined process wastewater and sanitary sewage treatment system (raw, digested, and dried sludge and aerated effluent)-failed to reveal virus. This plant was more than 1000 miles (*1600 km) from plant C, and climatic conditions were very different.
Plant F produced frozen ground beef patties from chunks of boned beef. Final product from three different days' operations was tested. Extracts of one of these samples repeatedly produced CPE in bovine cell cultures, but was not characterized because it could not be carried beyond the second passage.
Plant G slaughtered and processed chickens. Samples of chicken blood and intestines, raw well water, diced chicken meat, plant solid waste, and wastewater from the sanitary sewer line gave negative test results.
We did a second sampling series that was directed especially to food processing personnel in Plant C and eight other establishments. Samples were collected from the sanitary sewer lines that served personnel toilets, either as grab samples or with swabs suspended in the line for a week. None of these was shown to contain the virus.
We also tested 10 retail samples of each of six foods from a total of five different stores. Samples included ground beef, luncheon meat, lettuce, poultry pot pies, delicatessen salads, and tomatoes. Many of these food samples were tested repeatedly, but no virus was found.
Of the viruses that we were capable of detecting at that time, none was found that was of apparently human origin, either in the processing facilities or the final products. The swine viruses detected in plant C were probably of no significance to human health (Kostenbader and Cliver 1977) .
One other ''practical'' study will be described here (Cliver and Kostenbader 1984) . The topic was disinfection of fingers contaminated with virus in feces. Many such studies have since been done (Ansari et al. 1988 (Ansari et al. , 1989 Bidawid et al. 2000 Bidawid et al. , 2004 Mbithi et al. 1992 Mbithi et al. , 1993 Sattar and Ansari 2002) . Our study was distinguished by the use of virus shed in feces in the course of infection, inclusion of a number of brand-name disinfectants, application of glass coverslips as a receiving surface, and evaluation of disposable plastic gloves as a virus barrier. Some of the fecal material came from pigs infected with a swine enterovirus; other samples came from a child shedding poliovirus after oral vaccination; all were frozen at -20°C until used experimentally. Fingers (principally those of the present author) of hands that had been thoroughly washed with soap and water were pressed lightly onto the fecal surface and, in baseline studies, touched to sterile glass coverslips. This permitted precise gravimetric determination of the quantity of fecal material deposited; food surfaces, faucet handles, bar soap, towels, etc. were also touched, but the feces were ultimately deposited on coverslips to allow quantification. Some of the experiments were performed on shaved swine skin as well. When the virus was recovered from the coverslip and assayed by the plaque technique, the weight determination enabled estimation of how much virus had been removed by cleansing and how much had probably been inactivated. A highly alkaline (pH 8.8) hand soap was found to be strongly antiviral, and a comparable alkaline buffer was found to be equally effective. In general, disinfectants that were well tolerated by human skin were relatively ineffective. Disposable plastic gloves were shown both to prevent contamination of clean fingers touching virus-containing feces and to prevent contamination of other surfaces contacted by fecally contaminated fingers.
Cell cultures provided a powerful tool for food and environmental virology, as well as many other aspects of virology. Still, questions continued to arise that could not be answered by cell-culture experiments alone. Two that we undertook to address were: (1) How much virus comprises a peroral infectious dose? (2) What happens when ingested virus encounters receptors in the intestinal mucosa?
Peroral infectivity trials had been done with human volunteers (Gary et al. 1987; Grohmann et al. 1981; Schiff et al. 1984 ), but we felt that they included too many uncontrolled variables. With support (1975) (1976) (1977) (1978) from the US EPA, we undertook peroral infectivity studies using young swine as experimental subjects. The Abstract of the final report to EPA (Cliver 1980) says:
''This study was designed to examine the relationship of waterborne enteroviruses to infections and disease. Young weanling swine and their homologous enteroviruses were chosen as the model system: The porcine digestive tract is like that of man, but pigs can be handled under more closely standardized conditions than humans or other primates. Porcine enteroviruses resemble those of man in every way, but they infect swine so specifically that handling the most virulent of the porcine agents is apparently no threat to the health of research personnel. Known quantities (as measured by the plaque technique in tissue cultures) of two enteroviruses were administered in 5 ml of drinking water in such a way that the subjects were obliged to swallow all of it. The host's body was found to be about 1000 times (600-750 for one virus and 1800-2500 for the other) less likely than the tissue cultures to be infected by a given quantity of enterovirus. The ratio did not depend on whether the animals were fed just before challenge. The probability of infection was cumulative with iterated small doses: this indicated that there was, in the strict sense, no minimum infectious dose. None of the infected animals became ill, despite the reported virulence of the challenge viruses. Chlorine treatment of a concentrated virus suspension, which reduced infectivity to a level detectable by cytopathic effect but not plaque formation in tissue culture, left enough virus to infect one of five challenged subjects. Neither of two colostrum-deprived pigs, challenged by stomach tube with 20 plaque-forming units of enterovirus at 1 h of age, became infected.''
Inevitably, there were complications: groups of 10 pigs were purchased from University and commercial swine farms and left with their dams for 2-8 days to ensure that they got as much passive immunity from colostrum as possible. Then they were moved to a ''fostering unit,'' developed by the UW Department of Meat and Animal Science, in the FRI animal quarters. This was a box, inside of which were a slanted false floor, thermostatically controlled ventilation system, and dispensing troughs for milk replacer; observation and access were permitted by hinged acrylic panels at the top. The principal purpose of the fostering unit was removing the pigs from potential sources of enterovirus infection long enough to complete testing of the initial fecal specimens before the animals were put into isolators. Even with the colostrum for passive immunity and various other precautions, diarrheal illness occurred frequently, and it was not always possible to keep the pigs healthy and normal (or at times, even alive) to the age of 3 weeks. At approximately 3 weeks, assuming the animals were healthy and had been found not to harbor adventitious enteroviruses, eight of them were moved to individual isolators. Any remaining pigs became donors for kidney cell cultures. The isolators had acrylic fronts and HEPA-filtered air supplies, to preclude non-experimental sources of infection. The animals were fed two meals per day of food produced for human consumption, plus water ad lib. Feeding and watering, as well as emptying the litter trays from beneath the false floors, were done with aseptic precautions. Cages were entered in numerical order, and the pig in cage 8 always served as an unchallenged control. Despite our precautions, on three occasions, 14-day fecal specimens were positive from pigs that had not been challenged, though never from the pig in cage 8. Since the corresponding 7-day specimens had been negative, we surmised that the pigs had become infected while in the isolators, but were never able to determine how this could have happened. In addition to these technical challenges, we learned that well-fed pigs quickly become large and intractable. All suspect results were discarded, and a succinct report of the research was published in the Journal of Food Protection (Cliver 1981) . This was a one-of-a-kind study; we believe that the results (particularly regarding split doses) are significant to the question of peroral infectivity of viruses.
Another study to be described here addressed what I chose to call ''ex-vivo infectivity.'' We undertook to maintain explants of intestinal mucosa in their native functional state outside the donor's body, so as to observe early events in enterovirus infection. A number of such studies had been done before ours, using explant cultures from swine, cattle, feline, and human fetal intestines (Bridger et al. 1978; Derbyshire and Collins 1971; Dolin et al. 1971 Dolin et al. , 1972 Hoshino and Scott 1980; Wyatt et al. 1973) ; we hoped to develop additional, useful information. Our first experiments were done with 2-3-mm square explants from the distal ileums of 22-30-cm fetal pigs (Jensen and Cliver 1984) . Five areas per Petri plate had been scratched to facilitate attachment; the prepared explants were placed mucosal-side-up in these areas and incubated at 37°C with MEM plus fetal calf serum and antibiotics. Porcine enterovirus 3 had been photosensitized by replication in the presence of neutral red (Wilson and Cooper 1965) : a strong pulse of visible light from a fluorescent lamp reduced the titer by 5 logs. The virus and the explants were held in the dark and handled by the light of a photographer's red safe light. A dissecting microscope showed apparently normal villous structure during the first 4 days, with significant deterioration thereafter; net yields of light-resistant progeny virus ranged from 1 to 3 logs by day 2 or 4.
In a later study, we paid closer attention to maintaining normal organization of the villous mucosa (Heinz et al. 1987) . Intestinal tissue was collected from female Yorkshire pigs, either 4-6 weeks or 9-11 months old and prepared in 4-cm 2 explants; histology was monitored by light microscopy and scanning and transmission electron microscopy. Comparison of various medium formulations led to the selection of CMRL-1066, supplemented with insulin and cortisone, which would maintain apparently normal villous organization for 48 h. Explants were inoculated with either coxsackievirus B5 (CB5), which is infectious for swine, or with PO1, which is not.
Only 24 h at 37°C were allowed, to ensure that the explants were as normal as they looked. Retention and replication of the two viruses were compared in explants of absorptive and lymphoid mucosa from young and adult animals. Retention was limited, but favored CB5 in all cases and was greater in absorptive tissue than in lymphoid tissue; age differences were minimal. Replication of CB5 was also limited, but statistically significant and greatest in absorptive tissue from young animals and least in lymphoid tissue from young animals; yields from adult absorptive and lymphoid tissue were intermediate between these and approximately equal. We then focused more closely on the earliest interactions between these viruses and explants (Heinz and Cliver 1988) . Tritiated CB5 and PO1 were incubated with explants for 6 h at 6°C to measure and attachment or 1 h at 37°C to measure penetration, followed by liquid scintillation counting and autoradiography. Results at 6°C were anomalous, suggesting that this was not a valid temperature for measuring virus attachment to explant cultures. Retention at 37°C was apparently greater in adult absorptive and lymphoid tissue, but more specific in young lymphoid tissue, where the ratio of CB5:PO1 was 4.3. It was seen that only a small proportion of CB5 associated with the explants, and penetration was principally into the epithelial cells along the upper third of the villi (those at or approaching senescence) and/or the lamina propria. Virus that associated with enterocytes further down the villi or in the crypts apparently did not penetrate the cells. It seemed that, since the enterocytes into which the virus penetrated were nearing the end of their functional lives, this might to some extent explain the typical absence of diarrhea in enterovirus infections. The findings might be quite different if the experimental system comprised porcine ileal explants inoculated with porcine norovirus.
Though most of our studies since 1962 were directed to getting viruses out of environmental samples and into cell cultures for detection, it became clear that cell cultures were not the only means of virus detection and, in some instances, were of no use whatever in this application. A number of virus-detection methods based on enzymelinked immunosorbent assay (ELISA) had been described by 1980 for use in rapid clinical diagnosis (Deng and Cliver 1984) . Each of these used selected antibodies for individual viruses. In that pooled human immune serum globulin (HISG) contains antibodies reflecting the combined immunologic experience of the donor population, we considered how HISG might be used in a broad-spectrum virus detection method. Five human enteroviruses, reovirus 1, and two porcine enteroviruses were selected for testing by a method modified from that of Herrmann et al. (Herrmann et al. 1979) . Wells in a 96-well, polystyrene microtiter plate were pre-coated with poly-L-lysine. Virus suspensions (various concentrations) were incubated at 4°C for 20-24 h in these wells, after which the wells were washed with PBS ? Tween-20 and bovine serum albumin. HISG that had been absorbed with host cell antigens was added to the wells and incubated for 30 min at 37°C, after which the wells were washed again. Goat anti-human IgG labeled with peroxidase was incubated in each well for 2 h at 37°C, and the wells were washed again and reacted with 2,2 0 -azino-di-3-ethyl-benzthiazoline-6-sulfonate-hydrogen peroxide substrate for 20 min at room temperature and stopped with hydrofluoric acid. Optical densities were read at 410 nm in a microplate reader. With a positive:negative ratio of 2 as the cut-off, the human enteroviruses and reovirus were detected at levels of 10 4 -10 6 infectious units (PFU or MPNCU) per well; no signal was obtained with the porcine enteroviruses. In that the reaction was not typespecific, positive signals from two different viruses in the same well augmented each other. We did not pursue this method further because of its limited sensitivity, but there might well be ways of applying HISG for broad-spectrum immunomagnetic capture before RT-PCR testing.
After the FRI installed a PCR facility, we did apply antigen capture in tubes for RT-PCR detection of HAV in various inoculated wastewaters and in 60 Co-irradiated oysters and clams (Deng et al. 1994 ) and then immunomagnetic capture for HAV detection in oysters (López-Sabater et al. 1997) . We were obliged to install our own, much less elegant PCR facility after moving to UCD. HAV inoculated into water and sewage samples was concentrated by the viradel procedure (elution with urea-arginine phosphate buffer), concentrated specifically by immunomagnetic capture, and detected by RT-PCR ).
Later, we took up the issue (discussed earlier) of detection by RT-PCR of inactivated virus. Although our work yielded only a partial solution , the study has been taken up by others and progress will surely be made. We were able to characterize some of the changes in the virion that occurred with inactivation, in the hope that this information may point to further modes of attack (Nuanualsuwan and Cliver 2003a, b) .
A further confounding factor is that the number of viral particles and of genomes detectable by RT-PCR greatly exceeds the number of infectious units measured by plaque formation or cytopathology (TCD 50 or MPNCU). Isolation of more than one type of poliovirus from a plaque in a culture that received mixed inoculum has been reported by Teunis et al. (2005) , but this represents a relatively small potential disparity compared to the numbers of virions often shown to be present in a suspension. Direct particle counts by scanning electron microscopy gave mean particle:PFU ratios of 448 for a vaccine strain of PO1, and 38 for an ostensibly virulent strain of CB5 (Heinz et al. 1986 ).
In an antigen-capture RT-PCR study, a cDNA-RNA technique yielded an estimate of 79 HAV particles:PFU; the estimated limit of detection was 0.053 PFU, the equivalent of four particles (Deng et al. 1994) . These degenerate ratios are more likely the result of inefficiency in the initiation of infection at the cell level than of a large proportion of defective or noninfectious virions.
Surely, one of the most fascinating events in our field has been the demonstration of prion diseases (transmissible spongiform encephalopathies) as an international threat to human health. Our group had no prospect of acquiring the safety facilities required to work with these agents, but I did have the good fortune to serve on the US Food and Drug Administration Transmissible Spongiform Encephalopathies Advisory Committee from 1998 to 2002. We advised about regulatory approaches to preventing prion-disease transmission, particularly in the context of human healthcare (e.g., blood transfusion and blood product processing and distribution). I also served on the National Academies Institute of Medicine, Committee on Transmissible Spongiform Encephalopathies: Assessment of Relevant Science during 2002 and 2003; we developed a book-length set of recommendations for future TSE research in the USA (Erdtmann and Sivitz 2004 )-its effects on the course of public health research are uncertain.
At least some of the regulatory approaches to excluding bovine spongiform encephalopathy from transmission via food to humans were predicated on detecting prohibited bovine tissues in animal-origin foodstuffs. We were able to contribute to the selection of detection methods (Hajmeer et al. 2003) and their application to ''advanced meat recovery'' products (Hajmeer et al. 2006) . The methods used immunological procedures to detect banned tissue: the latter was a method for detecting abnormal prions, minus the proteinase K that would remove normal prions from the sample.
The years from 1962 to 2007, when I retired, witnessed enormous progress in the field of food and environmental virology: I never thought there would be a journal devoted to this field. My group made several contributions, though much of the time we were handicapped by severe lack of funding. A presentation I gave some years ago was entitled, ''Viruses around Us, or How We Are Identifying and Solving the World's Environmental Virology Problems on Practically No Money at All.'' This must have been due partly to my lack of salesmanship, in that most granting agencies, most of the time, did not regard foodborne and waterborne viruses as a major threat to human health. Some other university researchers did better than I. Intramural research was sustained by EPA and less so (over time) by FDA. The Canadian government had an on-andoff virology program: when their virologists were in a hiatus, they discovered that the shiga-like toxin of E. coli was toxic to the Vero cells they had been using in virus research Speirs et al. 1977) . We studied other topics at times, as well, and some of the virology studies were abandoned before reaching maturity. I wish we had accomplished more and trained more students, but we did what we could and enjoyed almost every minute of it. I wish those who are now carrying on the research as much pleasure as I have had from it.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Mitigation Approaches to Combat the Flu Pandemic Management of flu pandemic is a perpetual challenge for the medical fraternity since time immemorial. Animal to human transmission has been observed thrice in the last century within an average range of 11-39 years of antigenic recycling. The recent outbreak of influenza A (H1N1, also termed as swine flu), first reported in Mexico on April 26, 2009, occurred in the forty first year since last reported flu pandemic (July 1968). Within less than 50 days, it has assumed pandemic proportions (phase VI) affecting over 76 countries with 163 deaths/35,928 cases (as on 15(th) June 2009). It indicated the re-emergence of genetically reassorted virus having strains endemic to humans, swine and avian (H5N1). The World Health Organisation (WHO) member states have already pulled up their socks and geared up to combat such criticalities. Earlier outbreaks of avian flu (H5N1) in different countries led WHO to develop pandemic preparedness strategies with national/regional plans on pandemic preparedness. Numerous factors related to climatic conditions, socio-economic strata, governance and sharing of information/logistics at all levels have been considered critical indicators in monitoring the dynamics of escalation towards a pandemic situation. The National Disaster Management Authority (NDMA), Government of India, with the active cooperation of UN agencies and other stakeholders/experts has formulated a concept paper on role of nonhealth service providers during pandemics in April 2008 and released national guidelines - management of biological disasters in July 2008. These guidelines enumerate that the success of medical management endeavors like pharmaceutical (anti-viral Oseltamivir and Zanamivir therapies), nonpharmaceutical interventions and vaccination development etc., largely depends on level of resistance offered by mutagenic viral strain and rationale use of pharmaco therapeutic interventions. This article describes the mitigation approach to combat flu pandemic with its effective implementation at national, state and local levels.  NSs Encoded by Groundnut Bud Necrosis Virus Is a Bifunctional Enzyme Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism. The genus Tospovirus of the family Bunyaviridae is unique in infecting plants whereas the other members of this family infect only animals. Tospoviruses are transmitted by thrips in a persistent manner. They are quasi spherical enveloped viruses of 80-120 nm diameter with tripartite single stranded RNA genomes. The largest of the three single stranded RNA molecules, L RNA, codes for RNA dependent RNA polymerase in the virion complementary sense. The middle (M) RNA encodes the precursor for glycoproteins G1 and G2 in the virion complementary sense and the non structural protein, NSm in the virion sense orientation. The small (S) RNA codes for the nonstructural protein, NSs in the virion sense and nucleocapsid protein N in the virion complementary sense [2] . Using a green fluorescent protein based transient suppression assay, it was shown that NSs from Tomato spotted wilt virus (TSWV) (type member of Tospovirus) could function as a suppressor of post transcriptional gene silencing (PTGS) [3] . NSs is also associated with the symptom severity [4] and is known to form the paracrystalline array in the cytoplasm of infected plant and thrips [5] . However, biochemical characterization of NSs has not been reported so far. Among Tospoviruses reported from India, Groundnut bud necrosis virus (GBNV) also called Peanut bud necrosis virus (PBNV) has been well characterized [6, 7, 8, 9, 10] . The N and NSm genes of several isolates from different locations in India infecting Leguminosae and Solanaceae plants have been sequenced and shown to be strains of GBNV [8, 9, 11, 12, 13, 14] . We have earlier purified and characterized a strain of GBNV infecting tomato in Karnataka GBNV-To (K) [1] . In the present study, we have cloned the NSs gene of GBNV-To (K) and overexpressed it in E.coli. The amino acid sequence of NSs protein contains Walker A (GxxxxGKT) and Walker B (DExx) motifs. The proteins which posses the Walker A and Walker B motifs exhibit RNA/DNA stimulated NTPase activity [15] . Some of the viral encoded non structural proteins such as NS3 protein from West Nile flavivirus, Hepatitis C virus, Yellow fever virus and Dengue virus type 2 [16, 17, 18] , which have these motifs are shown to exhibit nucleic acid stimulated NTPase activity. However, none of the viral encoded proteins of the Bunyaviridae family have been shown to exhibit the NTPase activity. Therefore, it was of interest to examine, if the NSs possessed the NTPase activity. The rNSs exhibited poly(A)stimulated metal ion dependent NTPase/dATPase activity. Further it was shown to exhibit ATP independent 59 DNA/RNA phosphatase activity. Mutation of K189 to A in the Walker motif A resulted in the complete loss of ATPase activity but not the 59 phophatase activity, whereas mutation of D159 to A in the Walker motif B resulted in the partial loss of both the activities. Thus, for the first time we demonstrate that NSs is a bifunctional enzyme and it might play a pivotal role at different stages of viral life cycle.
The fine chemicals used for biochemical and molecular biological work were purchased from Sigma, Calbiochem and Novagen. Restriction endonucleases and DNA modification enzymes and polymerases were purchased from New England Biolabs and MBI Fermentas. Radioactive isotopes were purchased from PerkinElmer Life Sciences. All the other chemicals used were of analytical grade.
The NSs gene of GBNV-To (K) was amplified by RT-PCR using partially purified viral RNA as template and specific sense and antisense primers, designed on the basis of the genomic sequence of S RNA of GBNV (accession number U27809, table 1). The PCR product was digested with the restriction enzymes NheI and XhoI (purchased from MBI Fermantas) and ligated into the corresponding sites of plasmid pRSET-C (purchased from Invitrogen). The plasmid was named pRSETC-NSs and propagated in E. coli. The NSs coding sequence was confirmed by DNA sequencing. The recombinant NSs clone was transformed into C43 (DE3) E.coli cells and over-expressed by induction with 0.3 mM IPTG (purchased from Sigma) for 3 hours at 37uC. The cell pellet was resuspended in buffer A (20 mM Tris-HCl pH 8.0 and 300 mM NaCl), sonicated and centrifuged at 12000 rpm for 15 min. The supernatant and pellet fractions were analyzed by 12% SDS-PAGE. The rNSs protein was purified from the soluble fraction.
The rNSs with its N-terminal histidine tag was purified by the Ni-NTA chromatography. C43 E.coli cells harboring the pRSETC-NSs clone were cultured in LB medium (2L) containing ampicillin (50 mg/ml) at 37uC. The expression of rNSs was induced, when the O.D. at 600 nm reached 0.6, by the addition of IPTG (0.3 mM) and the cells were cultured further for 4 hours at 37uC. The cells were harvested, re-suspended in buffer A and sonicated till the suspension became optically clear. The cell lysate was then spun down at 10000 rpm for 10 minutes. To the supernatant, Ni-NTA resin (500 ml/500 ml of culture) was added and left for 2 hours in an end-to-end rotor for binding. The beads were allowed to settle and the unbound supernatant discarded. The beads were washed once with buffer A and then twice with buffer A containing 20 mM imidazole. The rNSs was eluted with 5 ml buffer A containing 250 mM imidazole pH 8.0. The eluted fractions were analyzed by SDS-PAGE [19] . The fractions containing rNSs were pooled and the protein was dialyzed against 20 mM Tris-HCl pH 8.0 buffer containing 100 mM NaCl. The protein obtained by this method was reasonably pure and was suitable for further characterization. The molecular mass of the purified rNSs was determined by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometer.
The site directed mutagenesis was carried out by PCR based method [20] using appropriate sense and anti sense primers with desired changes. The oligonucleotide primers used for site-directed mutagenesis (SDM) were custom made from Sigma. Mutagenic DNA primers (table 1) were designed to create alanine substitution at residues K189 and D159 in GxxxxGKT and DExx motifs of rNSs respectively. The presence of the desired mutations was screened initially by the gain or loss of a restriction site and was confirmed by DNA sequencing.
The far UV CD spectrum of rNSs protein was recorded using a Jasco-815 spectropolarimeter. The ellipticity was monitored at 25uC from 200 nm to 250 nm using 0.2 mg/ml of the purified protein in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. A scan speed of 50 nm/min, 0.2 cm path length cuvette, band width of 1 nm and response time of 1 s were used. The average of three spectra was taken and corrected with buffer blanks. The molar ellipticity was calculated using the software provided by the manufacturer.
The thermal stability of rNSs was monitored in a Jasco-815 spectropolarimeter fitted with Peltier thermal control system (PTC-423S). 0.2 mg/ml of the purified protein in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl was heated from 10uC to 100uC. The change in molar ellipticity at 222 nm was monitored as a function of temperature to obtain the thermal melting curve. The ellipticity was measured at 222 nm using 0.2 mg/ml protein in a 0.1 cm path length cuvette with band width of 1 nm and response time of 1 sec. The temperature was raised at a rate of 2uC/minute from 10-100uC.
The fluorescence spectra were recorded in a PerkinElmer LS5S luminescence spectrometer. The intrinsic fluorescence spectrum was monitored from 300-400 nm upon excitation at 280 nm in a 0.5 cm path length cuvette. The concentration of the protein used was 0.1 mg/ml. 0.5 mg of bovine serum albumin per ml, in the presence or absence of 0.2 mg per ml poly(A) for 30 min. and the reaction was stopped by the addition of 100 mM EDTA (1 ml). The total volume of the reaction mixture was 10 ml. For kinetic studies, initially a stock of cold NTPs containing 0.008 mCi of 32 P labelled respective NTP was prepared and the substrate concentration was varied from 100-1000 mM for ATP and 10-80 mM for CTP, GTP, UTP and 2.5-20 mM for dATP. The reaction mixture was incubated with 2 mg of rNSs protein in the assay buffer. To determine the kinetic parameters of D159A mutant NSs, 10-70 mM ATP was used with 2 mg of mutant protein. Three independent sets of experiments were carried out to determine the kinetic parameters. Substrate hydrolysis was measured by Imagegauge software (Fujifilm) and the enzyme activity (v) was calculated using the formula given below:
where Fc, Fractional Cleavagẽ ½Intensity of product released=Intensity of substrate zintensity of product released::
The Pi released or a-NDP/dADP released was measured when [c-32 P] ATP/GTP or [a-32 P] NTP/dATP was used as tracer respectively.
The K M and k cat values were calculated from the Lineweaver-Burk plot (1/v vs 1/s).
Reaction products of NTPase/dATPase and phosphatase assays were analyzed by thin layer chromatography using polyethyleneimine-cellulose (PEI cellulose F) plates (purchased from Merck). For this, the chromatographic chamber was saturated with developing solution of formic acid (1 M), EDTA (1 mM) and LiCl 2 (0.5 M) for two hours. 0.5 ml reaction mixture was spotted on the PEI cellulose plate and the plate was kept in saturated chromatographic chamber for developing the chromatogram. The intensity of the spots was measured using a phosphor imager.
The substrates for the phosphatase reaction were 59 end labeled using [c-32 P] ATP and T4 polynucleotide kinase according to manufacturer's protocol. Free nucleotides were removed from the end labelled substrate using Sephadex G25 spun column. rNSs (1.5 mg) was incubated at 25uC for 30 min with 59 labeled ssDNA (K189A sense primer, 0.5 nM), ssRNA {poly(A), 4 nM}, dsDNA (1 nM) and dsRNA (0.2-1 nM) in 50 mM MOPS buffer pH 7.0 containing 2 mM magnesium chloride, 2 mM dithiothreitol, 0.5 mg of bovine serum albumin per ml. The reaction was stopped by addition of 100 mM EDTA (1 ml) in a total volume of 10 ml and release of Pi was measured as described above.
Preparation of dsDNA and dsRNA substrates for 59 phosphatase activity dsDNA was made by annealing of sense and antisense primers (D159A). Resulting annealed product was end labelled using [c-32 P] ATP as described above. Further, dsDNA was gel eluted from the mixture of dsDNA and ssDNA. dsRNA was generated by annealing of in vitro transcribed sense and antisense 1 kb Sesbania mosaic virus RNA transcripts (kind gift from K. Govind). dsRNA was separated from ssRNA on 1% Agarose gel and dsRNA was gel eluted. The concentration of dsDNA/dsRNA was determined by measuring the O.D. at 260 nm in a spectrophotometer.
Purified rNSs (2 mg) was incubated with polyclonal antibodies raised in rabbit against rNSs at 4uC for two hours. The rNSsantibody complex was precipitated by the addition of protein A sepharose beads. The ternary complex was separated by centrifuging the mixture at 5,000 rpm for two minutes and the supernatant was tested for ATPase activity.
In order to establish the role of NSs during the viral life cycle, bioinformatic analysis was performed to identify the motifs present. Supplementary Fig. S1 . shows the putative amino acid sequence of GBNV-To (K) NSs along with secondary structure prediction. The protein was predicted to have 27% a helix, 25% b strand and 48% random coil. Motif search using the expasy server showed the presence of Walker motif A and B (Fig. S1 ). It may be noted that, in most ATPases Walker A motif precedes Walker B motif. However, in the NSs protein, the two motifs are reversed. Multiple sequence alignment of GBNV-(To) K NSs protein with six different tospoviruses representing different species revealed that glycine (G188) and lysine (K189) residues of Walker motif A are very well conserved across the Tospovirus genus (Fig. 1) . In TSWV and Impatiens necrotic spot virus (INSV), which show the lowest percent identity with PBNV (19% and 17%, respectively) also these residues are conserved, even though the canonical Walker A (GxxxxGKT) motif is not present in these two viruses. On the other hand, walker motif B (DExx) is conserved only in PBNV, Watermelon silver mottle virus (WSMV) and Capsicum chlorosis virus (CCV) (Fig. 1 ).
The GBNV-(To) K NSs gene was cloned into pRSET-C vector and overexpressed, as described in the materials and methods section, in C43 (DE3) E.coli cells. rNSs was purified from the soluble fraction by Ni-NTA affinity chromatography and the purity of the protein was checked by SDS-PAGE analysis. A single band corresponding to the expected molecular mass of 52 kDa was observed in the eluted fractions ( Fig. 2A lane 1-3) . The yield of the purified protein was 7-10 mg from 500 ml culture. The molecular mass of rNSs was confirmed by mass spectrometric analysis. The molecular mass thus obtained was 51810 Da, which agreed with that predicted from the sequence of histidine-tagged rNSs (52 kDa).
The Circular dichroism spectra analysis of rNSs showed a minimum in the range of 215-223 nm (Fig. 2B) . The intrinsic fluorescence emission spectrum showed a maximum at 338 nm upon excitation at 280 nm (Fig. 2C) . rNSs contains five tryptophans and eleven tyrosines which are buried as is apparent from the emission fluorescence maximum at 338 nm. These results demonstrate that rNSs is in a folded conformation. The thermal stability of rNSs monitored by CD spectroscopy revealed that it is a stable protein with a Tm of 65uC (Fig. 2D ).
ATPase activity was assayed as described in the methods section using [c-32 P] ATP as the substrate. The radiolabelled Pi released was monitored by TLC on PEI cellulose plates. As mentioned earlier, proteins which posses the Walker A and Walker B motifs are known to exhibit the RNA/DNA stimulated NTPase activity [15] . Therefore, purified rNSs (0.4-2 mg) was used to determine the ATPase activity in the absence and presence of Poly (A). As shown in Fig. 3 , rNSs could hydrolyze [c-32 P] ATP in a concentrationdependent manner. The activity was higher in the presence of poly(A) (Fig. 3B ). The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase which was used as the positive control ( Fig. 3A and B, lane 6). In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, did not show the release of phosphate ( Fig. 3A and B, lane 8), suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate was detected following immunodepletion of rNSs, confirming that the activity was inherent to the NSs protein ( Fig. 3A and B, lane 7). The reaction was completely inhibited by 10 mM EDTA ( Fig. 3A and B, lane 9) suggesting that the reaction is metal-ion dependent. The amount of product (phosphate) released was quantitated as described in the methods section. There was a 10-fold increase in the activity in the presence of poly(A) as compared to that without poly(A) (Fig. 3C) . The reaction was linear in the presence of poly(A) upto 1.6 mg of rNSs (Fig. 3C ). Since the ATPase activity of rNSs was much higher in the presence of poly(A), all further standardization of reaction conditions was carried out in the presence of poly(A). The product release was linear up to 30 mins (Fig. S2A ) and the ATPase activity of rNSs was enhanced by the addition of MgCl 2 . Maximum activity was observed at 2.5 mM of MgCl 2 (Fig. S2B) . Addition of increasing concentrations of EDTA lead to inhibition of activity and the ATP cleavage was completely inhibited at 5 mM EDTA (Fig. S2C) . The pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig. S2D ) and the optimum temperature of the reaction was 25uC (data not shown).
The specificity of cleavage of nucleotide triphosphate by rNSs was examined using [c-32 P] labelled ATP and GTP or [a-32 P] CTP, UTP and dATP as substrates. The activity was monitored by TLC as described earlier. The amount of product (NDP or Pi) released was quantitated and the substrate saturation curves as well as Lineweaver-Burk plots were drawn for each of the substrates as described in material and methods section. Fig. 4A depicts the substrate saturation curve with ATP as the substrate. Inset to Fig. 4A shows the Lineweaver-Burk plot of the same data. The K M and k cat values were calculated to be 515.2 mM and 10.8610 23 sec 21 respectively. Similar experiments were performed with the other NTPs. rNSs was able to hydrolyze all the NTPs and the K M and k cat values obtained are presented in table 2. Although the k cat value was the highest with ATP, the K M was also increased by an order of magnitude compared to other NTPs.
It was of interest to examine whether rNSs could cleave dATP. As shown in Fig. 4B , rNSs could also use dATP as substrate and the kinetic parameters obtained from the Lineweaver-Burk plot (Fig. 4B inset) are also presented in table 2. The k cat for dATP was approximately 10 times lower than that of ATP, although the K M (15.5 mM) was much lower. The specificity of rNSs was also tested by using the ATP analog, adenosine 59 (b, c imido) triphosphate. 5 mM concentration of this ATP analog resulted in considerable decrease in the activity (Fig. 4C lane 4 and 5) . Further increase in the concentration of the analog abolished the product formation completely (Fig. 4C lane 6 to lane 9) .
The results presented thus far clearly demonstrate that rNSs exhibits poly(A) stimulated NTPase/dATPase activities. As mentioned earlier, rNSs has sequence motifs (Walker A and Walker B) that are typical of NTP binding proteins (Fig. 1) . It was of interest to examine the role of these motifs in the NTP/dATPase function of rNSs. It was shown earlier that the lysine residue of Walker motif A is crucial for NTP binding whereas aspartate and glutamate of Walker motif B coordinate the Mg 2+ ion [21] . Therefore, K189 and D159 of the Walker A (GxxxxGKT) and B (DExx) motifs respectively in rNSs were mutated to alanine by site directed mutagenesis as described in the methods section. The K189A and D159A rNSs mutants were over-expressed and purified by Ni-NTA chromatography as described for the wild type rNSs. The purified mutant proteins were analysed on a 12% SDS-PAGE and the proteins were nearly homogeneous. The ATPase activities of the mutants were tested using 0.4, 0.8 and 1.2 mg of the protein along with the wild type rNSs as the control. As shown in Fig. 5A , the K189A rNSs mutant was completely inactive whereas the D159A mutant was partially active. Kinetic analysis of D159A mutant NSs (Fig. 5B ) revealed that the k cat was reduced by 28 fold (table 2) compared to wild type rNSs.
The ATPase activity of rNSs is stimulated by poly(A). It was of interest to examine the activity with 59 phosphorylated poly(A).
For this, poly(A) was 59 end labelled using T4 polynucleotide kinase (MBI fermentas) and [c-32 P] ATP according to the manufacturer's protocol. The 59 end labelled poly(A) was purified using Sephadex G25 column and run on 6% native PAGE to confirm that the contaminating free [c-32 P] ATP was completely removed. The 59 labelled purified poly(A) free of [c-32 P] ATP was used as substrate in a reaction containing 1 mg of rNSs and the 59 phosphatase activity was assayed as described in the methods section. As shown in Fig. 6A (lanes 2-4) , rNSs could cleave the 59 phosphate from end labelled poly(A) in the absence of ATP and the reaction was inhibited by the addition of 1 mM ATP (lane 5-7). However, control ATPase (Sigma) previously tested to be active for the hydrolysis of ATP, was unable to cleave the 59 phosphate The CD spectrum of rNSs (0.2 mg/ml) was recorded using Jasco-815 spectropolarimeter. The molar ellipticity was calculated using subunit mass of 52 kDa. (C) Fluorescence spectrum of rNSs. The fluorescence spectrum of rNSs (0.1 mg/ml) was recorded using Perkin Elmer LS5S luminescence spectrometer after excitation at 280 nm. (D) Thermal melting of rNSs. The molar ellipticity (Y-axis) of rNSs was measured at 222 nm, as a function of temperature (X-axis) and plotted as shown. doi:10.1371/journal.pone.0009757.g002 from the end labelled poly(A) (Fig. 6A lane 8) . These results suggest that rNSs indeed possesses a novel 59 phosphatase activity as well. AMP inhibits the 59 monophosphatase activity but not ATPase activity. Therefore the rNSs was incubated with increasing concentrations of AMP (1, 2 and 3 mM) and the 59 phosphatase activity was monitored (Fig. 6B lane 3-5) . The 59 phosphatase activity was completely inhibited by 3 mM AMP (Fig. 6B lane 4) . Further, immunodepleted rNSs (Fig. 6B, lane 7) or heat denatured rNSs (Fig. 6B, lane 8) did not cleave the end labelled poly(A) confirming that the cleavage was indeed due to the addition of rNSs. 59 labelled poly(A) was treated similarly with Calf intestinal alkaline phosphatase (MBI Fermentas) and was used as positive control (Fig. 6B lane 6) . siRNA (siblue, purchased from Dharamacon), ssDNA (D159A sense primer), dsDNA (D159A sense and antisense primers annealed) were also 59 end labelled and used as substrates for 59 phosphatase activity of rNSs. Interestingly, rNSs could cleave the 59 phosphate from end labeled dsDNA and ssDNA (Fig. 7A) but not from 59-end labeled siRNA (Fig. 7C) . ATP inhibited the 59 phosphatase activity even when 59 end labelled dsDNA or ssDNA were used as substrate (Fig. 7 B) . siRNA failed to act as substrate probably because its 59 end is recessed. To test this possibility, dsRNA prepared by annealing sense and antisense transcript as described in methods section, was used as substrate and indeed rNSs could remove the 59 phosphate from dsRNA (Fig. 7D) . Next, we wanted to examine whether active site residues for ATPase and phosphatase activity are the same or different. Therefore, the D159A and K189A mutants described earlier were purified and their 59 phosphatase activities were measured with 59 end labelled poly(A) as substrate along with the wild type enzyme as control. The fractional cleavage by these three proteins (1 mg) at saturating concentration of poly(A) (3 nM) is shown in Fig. 6C . As apparent, D159A mutant showed 20% decrease in activity whereas K189A mutant was as active as the wild type enzyme. These results demonstrate that K189 is not an essential residue for 59 phosphatase function whereas it is absolutely important for ATPase function.
The results presented in this paper demonstrate for the first time that NSs of GBNV-To (K) is a bifunctional enzyme. Bioinformatic analysis revealed the presence of Walker motif A and B. However, the Walker motif B precedes the motif A in this protein (Fig. 1) . It was of interest to examine if rNSs could bind to NTPs and cleave them. As shown in Fig. 3 , rNSs exhibited the Poly(A) stimulated ATPase activity. The activity was tenfold higher in the presence of poly(A) compared to that without poly(A). None of the NSs proteins from bunyaviridae family have been shown to possess this enzymatic activity. The product was not formed when immunodepleted rNSs was used, which ruled out the possibility of any contaminating bacterial protein (ATPase) co-purifying with rNSs. Further, rNSs could hydrolyze other NTPs and dATP ( Fig. 5 and table 1) and the ATPase activity of rNSs was inhibited by an ATP analog {adenosine 59-(b, c imido) triphosphate} (Fig. 4C) . These results suggested that NTPase/dATPase activity is an intrinsic property of rNSs. On the basis of structural data, it has been suggested that the binding of nucleic acid induces conformational changes in the protein, which, in turn, stabilizes the bound ATP molecules in a conformation that is required for rapid hydrolysis [22, 23] . It is therefore reasonable to suggest that the observed stimulatory effect of poly(A) on GBNV NSs ATPase activity may reflect similar conformational changes. Point mutation in Walker motif B (D159A) reduced the NTPase activity (Fig. 5) . NTPase activity is metal ion dependent. The D159, like in other NTP binding proteins, is probably responsible for co-ordination of the metal ion and therefore mutation of this residue affects the NTPase activity. However, the mutation of K189 to A abolished the NTPase activity completely. The conserved lysine residue in the Walker motif A is suggested to be important for interaction with b, c phosphate of NTP [21] . Therefore, mutation of this residue renders the protein inactive. The lysine residue in the GKT motif is shown to be critical for ATP hydrolysis in many other ATPases as well. For example, the mutation of lysine to glutamine in eIF-4A protein resulted in severely reduced ATP binding, and complete loss of ATPase activity [24] . Gross and Shuman [25] reported that an alanine substitution at the lysine residue of the GKT motif of NPH II decreased its NTPase activity by 1/20 of that of the wild type protein in the presence of RNA or DNA cofactors. The difference in the mutational effects at the same site in eIF-4A and NPHII could be a result of the nature of each protein and sensitivity of the specific experiments. Similar mutation has been performed with NS3 protein of Dengue virus type 2 [26] and HCV, which abolished the ATPase activity [27] .
In addition to NTPase and dATPase activity, rNSs also exhibited the nucleic acid 59 phosphatase activity (Fig. 6A ) that was inhibited by AMP (Fig. 6B) . The rNSs could remove the 59 Phosphate from ssRNA (Fig. 6A) , ssDNA, dsDNA and dsRNA (Fig. 7A, B and D) . However, it failed to remove the phosphate from siRNA in which the 59 end is recessed (Fig. 7C) . The 59 a Phosphatase activity of rNSs was inhibited by ATP. It is possible that ATP acts as the competitor for the binding of the substrate. Interestingly, K189A mutant which was completely inactive as far as the ATPase activity was concerned, was fully active and could remove the 59 phosphate from 59 end of the nucleic acid (Fig. 6C) . As mentioned earlier, the lysine in the GKT motif is involved in the binding of b, c phosphate for positioning and cleavage of c phosphate. However, the rNSs removes the a phosphate from the 59 end labelled nucleic acid. Therefore, the mutation K189 to A did not result in loss of 59 phosphatase activity. In the D159A mutant, only a partial decrease (20%) in activity was observed for 59 phosphatase activity suggesting that this reaction could also be metal ion dependent.
Similar kind of poly(A) stimulated NTPase/dATPase activity has been observed in the case of NS3 protein from West Nile [16, 30, 31] . Most of these proteins also exhibit the nucleic acid unwinding activity [32, 33, 34, 35, 36] . However, none of the plant viral proteins have been shown to exhibit such activities.
There is only one report on the in vivo function of NSs from Tomato spotted wilt virus, the type member of Tospovirus genus, as a suppressor of PTGS [3] . There has been no further work on this protein. RNA silencing or PTGS is a major strategy by which plants mount defense responses against molecular parasites such as viruses [37, 38] . The RNA silencing pathway involves the initial processing of viral dsRNA into small interfering RNA (siRNA) duplexes of length 21-25 nt by DICER or its homologs. One of the strands of the siRNA incorporated into the RNA induced silencing complex (RISC) specifically targets viral mRNA with a complementary sequence for degradation [39] . Viruses evade such a defense response by expressing viral suppressors of RNA silencing (VSRs). VSRs encoded by viruses of different families and genera [40, 41] share no homology at the level of primary sequence and the mechanisms by which they inactivate the PTGS are different. The results presented in this manuscript implicate that NSs might act as a suppressor of PTGS by removing 59 phosphate from dsRNA, the substrate for DICER. It has been observed that DICER does not recognize the dephosphorylated dsRNA [42] and therefore removal of the 59 phosphate by NSs could eventually block the PTGS pathway. Interestingly, even in some animal viruses dephosphorylated viral RNA fails to induce the Interferon pathway [43, 44] thereby suppressing the host defense mechanism. Further, ATP is required for the PTGS pathway. The ATPase activity of NSs might result in the suppression of this pathway. The ATPase and 59 phosphatase function of NSs could also be of functional importance in the replication and transcription of the virus. 
We thank our colleagues for the valuable suggestions during the preparation of this manuscript. We thank K. Govind for providing the ssRNA transcripts to generate dsRNA, J.N.Kalyani for her help in determining the kinetic parameters and B.Chiranjivee for the kind gift of purified RecoP5I protein.  Mortality among patients with tuberculosis requiring intensive care: a retrospective cohort study BACKGROUND: To describe the characteristics of patients with tuberculosis (TB) requiring intensive care and to identify the factors that predicts in-hospital mortality in a city of a developing country with intermediate-to-high TB endemicity. METHODS: We conducted a retrospective, cohort study, between November 2005 and November 2007. The patients with TB requiring intensive care were included. Predictors of mortality were assessed. The primary outcome was the in-hospital mortality. RESULTS: During the study period, 67 patients with TB required intensive care. Of them, 62 (92.5%) had acute respiratory failure and required mechanical ventilation. Forty-four (65.7%) patients died. Coinfection with human immunodeficiency virus was present in 46 (68.7%) patients. Early intensive care unit admission and ventilator-associated pneumonia were independently associated with the in-hospital mortality. CONCLUSIONS: In this study we found a high mortality rate in TB patients requiring intensive care, especially in those with an early ICU admission. Across the world tuberculosis (TB) remains an important public health problem, especially in developing countries. One third of the world's population is infected with Mycobacterium tuberculosis. Brazil is ranking 15 th among the 22 high-burden countries that collectively account for 80% of TB cases globally. The incidence of TB was of 50 cases/100,000 population/yr in 2006, and recently reached approximately 100 cases/ 100,000 population in the city of Porto Alegre (southern Brazil) [1] . Every year, almost 2 million people die of TB, most of them in low-and middle-income countries. The annual death rate from TB in Brazil was estimated at 4.0/100,000 population/yr in 2006 [2] .
Despite the availability of curative therapy, a large proportion of patients with TB are being hospitalized. Inhospital mortality rates remain high, particularly among patients with TB requiring intensive care unit (ICU) admission. These cases represent 1-3% of all patients with TB [3] . Acute respiratory failure (ARF) caused by TB necessitating mechanical ventilation (MV) has been associated with mortality rates between 25.9% and 81% [3] [4] [5] [6] . Furthermore, such patients have a prognosis significantly worse than patients with nontuberculous pneumonia requiring MV [6] .
Some studies reported a few factors that contribute to mortality among critically ill patients with TB. Disseminated disease, usually in the setting of human immunodeficiency virus (HIV) infection, has been recognized as an important predictor of death. Other factors that can influence mortality rates are extensive fibrocavitary disease and consolidations on chest radiographs. Acute respiratory distress syndrome (ARDS), sepsis and multiple organ failure (MOF) also carry a very high mortality [5] [6] [7] .
The purpose of this study was to describe the characteristics of patients with TB requiring intensive care, and to identify the factors that predict in-hospital mortality in a city of a developing country with intermediate-to-high TB endemicity.
This study was conducted in Porto Alegre, Brazil, from November 2005 through November 2007. Adult patients with TB that were admitted to the ICU of the Clinics Hospital of Porto Alegre (HCPA) were identified retrospectively. The HCPA is a general, tertiary care, university-affiliated hospital with 750 beds (34 adult ICU beds) and is a reference center for HIV in southern Brazil. Porto Alegre is the city with the highest acquired immunodeficiency syndrome (AIDS) incidence in Brazil (68.7 cases/100.000 population) [1]. In our hospital, in 2008 for example, we had approximately 29.000 hospitalizations per year, with 185 cases of pulmonary TB. In addition, the number of outpatient visits was 551.968, but we do not have data on TB treatment, because our hospital does not provide TB treatment for outpatients; in Brazil, patients are treated in public outpatient health care services. The medical records of patients were reviewed, and predictors of mortality were assessed. The protocol was submitted to the Ethics Committee at HCPA and an approval was obtained. A waiver of consent was approved and the investigators assigned a confidentiality term.
Pulmonary TB is diagnosed according to the following criteria established in the Brazilian Guidelines for Tuberculosis [8] The following data were collected in a standardized questionnaire: demographic data (sex, age, race, years of schooling), smoking status, alcoholism, injection drug use, clinical form of TB, symptoms at admission, methods of diagnostic, presence of comorbidities, prior TB treatment, drug regimen, interval from hospital admission until initiation of treatment, delayed treatment (failure to initiate treatment within the first 24 hours after admission to hospital), reasons for ICU admission, interval from hospital admission until ICU admission (early ICU [admitted directly or transferred to an ICU within 4 days of admission] and late ICU [admitted to an ICU after 4 days of hospitalization]), ARF, ventilator-associated pneumonia (VAP), Acute Physiologic and Chronic Health Evaluation (APACHE) II scores, presence of organ failures, ARDS, sepsis, septic shock, length of hospital and ICU stay, length of mechanical ventilation, laboratory investigations (white cell count, hemoglobin, coagulation profile, liver and renal function parameters, albumin, C-reactive protein, blood gas analysis), Glasgow coma score, urine output, use of vasoactive drugs, hospitalization outcome (death or discharge), and outcome after discharge (cure, dropout, death). Data after discharge were obtained from SINAN (National System of Information on Notifiable Diseases). SINAN is a database from the Brazilian government which stores information concerning all notifiable infectious and contagious diseases. The duration of follow-up period was one year.
A diagnosis of respiratory failure was made after the determination of arterial oxygen pressure (PaO 2 ) level of less than 60 mmHg or arterial oxygen saturation (SaO 2 ) < 90%, with or without elevation of arterial PCO 2 . ARDS was diagnosed based on the criteria defined in the American-European consensus conference [9] . For the diagnosis of organ failure, the criteria by Knaus [10] were used. Multiple organ failure (MOF) was defined as the failure of more than one organ. Ventilator-associated pneumonia (VAP) was diagnosed based on the American Thoracic Society (ATS) criteria [11] . Anemia was defined by hemoglobin levels < 13.5 g/dL for males and < 12.0 g/dL for females. Hypoalbuminemia was considered when serum albumin levels were less than 3.5 g/dL.
Data analysis was performed using SPSS 14.0 (Statistical Package for the Social Sciences, Chicago, Illinois). Data were presented as number of cases, mean ± standard deviation (SD), or median with interquartile range. Categorical comparisons were performed by chi-square test using Yates's correction if indicated or by Fisher's exact test. Continuous variables were compared using the t-test or Wilcoxon test. Variables with p < 0.20 in the univariate analysis were analyzed in the multivariate analyses. We have chosen this widespread adopted cutoff because using a more traditional level (P < 0.05) might fail to identify variables known to be important. In addition, confusion variables can affect estimates even when their level of significance does not reach 0.05. Kaplan-Meier survival curves were used to analyze the survival of patients. Those factors significantly associated with survival were further analyzed with the Cox proportional hazard model (method forward) adjusted by sex and age. Hazard ratio (HR) with 95% confidence interval (CI) was used to report the results. A two-sided p value < 0.05 was considered significant for all analyses.
Between November 2005 and November 2007, 67 patients with TB required intensive care in our hospital.
Of them, 62 (92.5%) had ARF and required MV. The mean age of all patients was 43.2 ± 14.1 years and males slightly outnumbered female patients (55.2% vs. 44.8%). A history of previous TB was present in only 6 patients (9%). Comorbid illnesses were identified in 58 (86.6%) patients, with coinfection with HIV being the most common, present in 46 (68.7%) patients. The median CD4/ mm 3 count of these patients was 83 (range: 34 -145), and only 6 (13.0%) were receiving highly active anti-retroviral therapy (HAART) at the time of hospitalization. A history of alcoholism was present in 25 (37.3%) patients and 20 (30%) were current smokers. Demographic and clinical characteristics did not differ between the survivors and those that died (Table 1) .
Twenty four (35.8%) patients had pulmonary disease only, 21 (31.3%) had extrapulmonary disease only, and 22 (32.8%) had association of pulmonary and extrapulmonary disease. The most common symptoms were fever (44.8%), loss of weight (38.8%), cough (29.9%), and neurologic ones (22.4%). Reticular infiltrate (35.8%) was the most common radiographic finding, followed by consolidation (23.9%). Forty six patients collected a sputum sample, of whom 25 (54.3%) were sputum-smear positive. Mycobacterial cultures were positive in 35 (52.2%) patients (13 bronchoalveolar lavage, 8 pleural effusion, 3 spontaneous sputum, 3 bone marrow, 2 cerebrospinal fluid, 2 blood, 1 induced sputum, 1 fine needle aspiration of lymphonode, 1 ascitic fluid, 1 urine). In 9 (13.4%), TB diagnosis was based on clinical, epidemiologic and radiographic findings. The median interval from hospital admission until initiation of treatment was 3 days (range: 1 -8).
The primary cause for ICU admission was ARF in 42 (62.7%) patients. Other reasons were cardiopulmonary arrest (10.4%), septic shock (7.5%), sepsis (6.0%), and altered sensorium (6.0%). The mean APACHE II score was 22.8 ± 6.8. Among ICU admissions, 18.9% and 81.1% were classified as early and late, respectively. At ICU admission, most of the patients had anemia (92.5%), with more than a half (52.2%) of these having hemoglobin levels < 9 g/dL. The white blood cell (WBC) count was less than 4000 mm -3 in 14 patients (20.9%) and more than 12000 mm -3 in 13 patients (19.4%). Hypoalbuminemia was present in 45 (67.2%) patients. Mean serum albumin level was 2.2 ± 0.8 g/dL.
Although only 6.0% and 7.5% of patients had sepsis and septic shock, respectively, most of the patients developed sepsis (95.5%) and septic shock (83.6%) during ICU stay. Only 9 (13.4%) patients developed ARDS. During the course of ICU stay, 20 (29.9%) patients were diagnosed with VAP, and over 80% of the patients had MOF. Almost all patients (95.5%) developed acute renal failure. Out of these patients, 29.9% required dialysis. The median duration of hospitalization was 25 days (range: 12.8 -50), and the median ICU stay was 10 days (range: 3 -16.8).
Overall, 44 (65.7%) patients died, of whom 38 (56.7%) died in ICU and 6 (8.9%) died after been transferred to the ward. The Kaplan-Meier survival curve is showed in Figure 1 . Of the 23 patients who were discharged from the hospital, 17 (73.9%) were cured, 2 (8.7%) abandoned the treatment after 1 month, and 4 (17.4%) died after a mean of 3.7 ± 2.5 months. As shown in Table 2 , early ICU admission was associated with mortality (p = 0.004). Early ICU admission was also associated with delayed diagnosis (p = 0.001) and acute renal failure (p = 0.030). Miliary radiologic pattern and smear-positive sputum were protective factors (p = 0.016 and 0.030, respectively). Sixty three (94%) patients used rifampicin, isoniazid and pirazynamid; 3 (4.5%) used streptomycin, isoniazid and ethambutol; 1(1.5%) used rifampicin, isoniazid and ethambutol. Only 3 patients did not receive rifampicin-based regimen because of hepatotoxicity. Nonsurvivors used more rifampicin-based regimens compared to survivors (p = 0.037). Neither hypoalbuminemia (p = 0.111) nor hepatic dysfunction (p = 0.999) had effect on mortality. We have found no association between diagnosis of HIV and mortality (p = 0.999), regardless of level of immunosuppression and use of HAART. Although not statistically significant, a higher mortality rate was found in patients with acute renal failure (p = 0.133). VAP was more common in survivors than in nonsurvivors (p = 0.041). The mean duration of mechanical ventilation was not statistically different between survivors and non-survivors (p = 0.228). All predictors with p values ≤ 0.20 in the univariate analysis were retained in the Cox-proportional hazard model. The variables alcoholism, corticosteroid use, desnutrition, early ICU admission, miliary radiological pattern, WBC > 12000 mm -3 , rifampicin-based regimens, hepatotoxicity, septic shock, VAP and acute renal failure, adjusted by sex and age, were introduced into the forward conditional Cox regression model, which showed that early ICU admission (HR 4.93, 95% CI 2.44 -9.97, p < 0.001) and VAP (HR 0.26-0.12, 95% CI 0.12 -0.59, p = 0.001) were factors affecting the in-hospital mortality rate (Figure 2 ).
In this study, we aimed to evaluate, on a retrospective basis, the risk factors associated with mortality among patients with TB requiring intensive care. Overall, we found a high in-hospital mortality rate (65.7%) in patients with TB admitted to the ICU. Using multivariate analysis, we identified early ICU admission as the most important factor independently associated with patient mortality. In addition, VAP was as a protective factor for mortality.
Our results are consistent with previous epidemiological data. Other studies have shown mortality rates up to approximately 80% in TB patients with ARF requiring MV [3] [4] [5] [6] 12] . In accordance with our findings, another study demonstrated an ICU mortality rate of 61% and an in-hospital mortality rate of 65.9% [5] . Early ICU admission was independently associated with higher mortality in multivariate analysis. This finding is consistent with a previous study [13] , and may be due to the Data are presented as mean ± SD, n (%), or median (interquartile range). WBC: white blood cell. ARDS: acute respiratory distress syndrome. more severe disease in these patients. Actually, this appears to be a plausible explanation as they have a higher incidence of acute renal failure compared to patients with a late ICU admission. Delayed treatment was also more frequent in this group, which confirms earlier observations [13, 14] . Another factor that contributes to the failure to initiate treatment within 24 hours after admission is the less classic presentation. Indeed, atypical features as sputum-smear negativity have been suggested to be associated with delays in both diagnosis and treatment and with mortality [13, 15, 16] . Our study showed that sputum smear-positivity and a miliary radiographic pattern were associated with a reduced risk of death. Surprisingly, we found in univariate and multivariate analysis that VAP was a further protective factor for mortality, in contrast with prior reports [4, 17] .
Although not statistically significant, the mean duration of mechanical ventilation was longer in survivors compared with nonsurvivors, which could explain the higher incidence of VAP in the formers. Our study has some methodological limitations. First, the information was obtained retrospectively from chart review and probably was not as complete and accurate as when data collection is done prospectively. Certain important factors that have been suggested as risk factors for mortality could not be included. Second, it must be considered that this is a single center study with small sample size. Despite these limitations, our results provide important implications for similar demographic areas and clinical settings.
In the present study, we observed a high incidence of acute renal failure. In univariate analysis, we found that acute renal failure was associated with death. Acute renal failure is a common condition that affects many ICU patients and is a well-recognized risk factor for mortality [18, 19] . However, the results reported across studies in TB patients with acute renal failure are inconsistent. Although acute renal failure was identified as an independent predictor of death in some studies [4, 6, 20] , others [3, 12] did not find a significant impact of such complication on the mortality of critically ill TB patients.
Previous studies have shown a critical course in patients with miliary TB and in HIV-infected patients with TB [6, 21] . However, we did not find an association between a diagnosis of HIV and mortality in ICU patients. In fact, mortality in patients with TB-HIV coinfection seems to be related to the patient's overall degree of immunosuppression [21] . In spite of the free access to HAART in Brazil and the high level of immunosuppression of our study population, only 13% of patients were receiving HAART at the time of hospitalization, which is probably related to poor or no adherence to treatment. Even so, CD4/mm 3 count was not a predictor of death in these patients. Furthermore, serum albumin levels, reported by others as good predictors of CD4/mm 3 lymphocyte counts in patients with TB [22] , were not associated with mortality in our study. The lack of association between mortality and the HIV epidemic could be explained by the fact that our non-HIV population had a high proportion of comorbidities and were also too ill with high mortality rate.
We found that patients who used rifampicin-based regimens had a higher mortality rate, even though not confirmed in multivariate analysis. One hypothesis to explain such observation is the possibility of multidrugresistant tuberculosis (MDR-TB). However, culture and sensitivity test to antituberculosis drugs were not routinely performed, so we could not estimate the prevalence of MDR-TB in our sample. On the other hand, uncertain enteric absorption in critically ill patients [23] and subtherapeutic serum levels of rifampicin, especially in HIV-positive population [24, 25] , have been reported [26] [27] [28] . Drug interactions among antituberculosis drugs and HAART are also important factors to be considered. In addition, low serum albumin levels could impair drug absorption and was associated with low rifampicin and ethambutol concentrations [29] . Whereas the great majority of our study sample is composed by HIV-positive patients and hypoalbuminemia was a prevalent finding, it is possible that some patients had low serum rifampicin levels that went undetected.
To our knowledge this is the first study in Brazil that described TB cases and their outcomes in patients requiring intensive care. While effective treatment is available on an outpatient basis, TB hospitalization and mortality rates are substantially high in our center. Patients with an early ICU admission have the highest risk of death. Some factors were significant only in univariate analysis, such as acute renal failure, miliary radiological pattern, smear-positive sputum and rifampicinbased regimens. The small sample size may have lacked sufficient statistical power to reveal an association with some of the factors. These results, if confirmed in a larger prospective study, could contribute to a better understanding of characteristics associated with mortality.
In conclusion, in this study we found a high mortality rate in TB patients requiring intensive care, especially in those with an early ICU admission. Nevertheless, additional studies are necessary to confirm this data, taking into account other possible risk factors for mortality like MDR-TB and low serum levels of antituberculosis drugs, potentially improving the care of critically ill TB patients. Comparative Efficacy of Hemagglutinin, Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge. Since 1997, the highly pathogenic avian influenza A H5N1 viral strain has caused severe disease in poultry and wild birds. Although H5N1 has not spread widely in humans, sporadic infections have been seen throughout countries of eastern Asia, the Middle East and Africa. To date, there have been more than 445 confirmed human cases of H5N1, with 263 deaths (59% mortality rate) reported by the World Health Organization (http://www.who.int/csr/disease/ avian_influenza/country/cases_table_2009_12_11/en/index.html). In almost all cases, those infected with H5N1 had physical contact with infected birds. While the primary mode of transmission may be animal-to-human, the concern remains that this virus may evolve into a strain capable of human-to-human transmission. Vaccination offers a practical and effective measure for controlling the spread of this highly pathogenic virus. The threat posed by emerging strains of influenza is unpredictable and varies among countries, as evidenced by the recent swine origin H1N1 pandemic, highlighting the need for improved vaccines that can confer broad protection against multiple viral strains and various influenza A subtypes.
While the hemagglutinin (HA) surface protein is conventionally the primary target of strain-specific influenza DNA vaccines, conserved viral epitopes have the potential to induce immunity against diverse influenza strains. Two highly conserved influenza viral proteins, NP and M2, have been widely targeted as possible broadly protective vaccine candidates [1] [2] [3] [4] [5] [6] [7] [8] [9] . The main function of the nucleoprotein is encapsidation of the viral genome to form a ribonucleoprotein particle for transcription and packaging. NP also interacts with other viral proteins (PB1, PB2, and M1) and cellular proteins (Importin a, F-actin, CRM1/exportin1) for viral transcription control and nuclear transportation control [10] . M2 is responsible for protein translocation, and is expressed at a high density in the plasma membrane of infected cells in tetramer forms. This ion channel protein is also a target for the antiviral drugs amantidine and rimantadine, which control viral replication and have been used for influenza prophylaxis and treatment [11] .
In mice, DNA/rAd5 vaccination with NP and M2 from the H1N1 PR/8 strain induced both humoral and cellular immune responses that protected against lethal H5N1 challenges [12] . However, the mouse model is not ideal for the evaluation of H5N1 infection and vaccines due to differences in HA receptor specificity and distribution, influenza pathogenicity, as well as clinical symptomatology [13] [14] [15] [16] [17] . Infection in the ferret shows greater similarity to human infection in terms of anatomical distribution and disease. Outbred ferrets exhibit severe lethargy, fever, weight loss, and transient lymphopenia, as well as viral replication in the upper and lower respiratory tract, brain, and multiple systemic organs after infection with various strains of H5N1 virus. Thus, this model is widely considered to be more reflective of human influenza infection [13, 18, 19] . While we continued to evaluate the protective efficacy of NP and M2 in the mouse model, we extended our investigation into the ferret model in this study.
Previous studies have investigated the protective efficacy of these conserved epitopes against lethal influenza challenge in mice and ferrets [7, 20, 21] . DNA vaccination with NP in combination with M2 formulated with Vaxfectin has been shown to protect mice against heterosubtypic challenge with H3N2 and H1N1 viruses [7] . Vaxfectin-formulated DNA vaccine encoding NP+M2 protected mice, but against high challenge doses of H5N1 virus in ferrets it only delayed time to illness and death [20] . However, triple prime with rAd boost regimens of NP in combination with M2 have been shown to protect ferrets against the H5N1 virus, albeit at a relatively low challenge dose [21] . In this study, we evaluated protective immunity induced by NP and M2 alone or in combination with HA in a triple prime, rAd boost regimen against high dose lethal H5N1 challenge. Initially, we tested the ability of DNA immunization with HA alone, NP alone, HA+NP, and HA+NP+M2 to protect against high doses of lethal H5N1 challenge in mice. We then assessed the ability of NP and M2, alone or in combination, to protect ferrets against a high challenge dose of lethal H5N1 virus, when delivered in a triple DNA prime, rAd5 boost regimen. We compared these groups to ferrets immunized with HA alone or in combination with NP+M2.
Plasmids encoding HA (A/Thailand/1(KAN-1)/2004, Gen-Bank AY555150), NP (A/Thailand/1(KAN-1)/2004, GenBank AAV35112 and A/PR/8/34, GenBank AAM75159), and M2 (A/Thailand/1(KAN-1)/2004, GenBank AAV35111) were synthesized using human-preferred codons and constructed in a CMV/R backbone by GeneArt (Regensburg, Germany) as previously described [22] . Gene expression was confirmed using 293T (Invitrogen, Carlsbad, CA) transfected cells by Western blot analysis.
Three separate replication-defective rAd serotype 5 vectors expressing HA (KAN-1), NP (KAN-1), and M2 (KAN-1) genes were produced as previously described [23] . Briefly, the genes were inserted into the GV11 adenoviral vector system (GenVec, Gaithersburg, MD), which is based on human serotype 5 and contains deletions of the E1 and E4 regions and part of the E3 region, rendering it replication-defective. The vectors used were as described elsewhere [23, 24] . The vector stocks were serially passaged in complementing mammalian cells (293-ORF6) to generate high-titer stocks of replication-defective adenoviruses [25, 26] . Absence of replication-competent adenovirus was confirmed by product-release assays. Gene expression in A549 (American Type Culture Collection, Manassas, VA) cells was confirmed by Western blot analysis.
Prior to animal immunization, plasmids and adenoviruses encoding various influenza viral genes were tested for their expression in 293T and A549 cells. Plasmids encoding HA protein of A/Thailand/1/KAN-1/2004, NP proteins of A/PR/8/34 and A/Thailand/1/KAN-1/2004, and M2 protein of A/Thailand/1/ KAN-1/2004 were transferred into 293T cells using the calcium phosphate-mediated ProFectionH Mammalian Transfection system (Promega, Madison, WI). Adenoviruses encoding HA (KAN-1), NP (KAN-1) and M2 (KAN-1) were transfected into A549 cells for 48 hours followed by a change of media. Cell lysates were collected 48 hours post-transfection and subjected to Western blot analysis by mouse monoclonal antibodies against HA (KAN-1), NP (KAN-1), NP (PR/8), and by ferret anti-serum raised against M2 (KAN-1). Specific bands of the predicted size of proteins were detected by comparison to a known vector control.
The highly virulent A/Vietnam/1203/2004 (H5N1) virus, isolated from a human with a fatal case of influenza [19] , was used in this study. The virus stock was propagated in the allantoic cavities of 10-day-old embryonated hens' eggs following incubation at 37uC for 24 to 29 hours. Allantoic fluid from multiple eggs was pooled, clarified by centrifugation, divided into aliquots, and stored at 270uC. The 50% egg infectious dose (EID 50 ) titers were determined by serial titration of viruses in eggs and calculated by the Reed and Muench method [27] . The 50% lethal dose (LD 50 ) was determined as previously described [19] . All research with HPAI virus was conducted under Biosafety Level 3 containment, including enhancements required by the U.S. Department of Agriculture and the Select Agent Program [28] .
All animal research was conducted under the guidance of the Centers for Disease Control and Prevention's Institutional Animal Care and Use Committee in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Female BALB/c mice, 6-8 weeks old (Jackson Labs, Bar Harbor, ME), were immunized as previously described [22] with HA from A/Thailand/1(KAN-1)/2004, NP from A/PR/8/34, and M2 from A/Thailand/1(KAN-1)/2004. Briefly, mice (10 animals for each group of HA alone, HA+NP, and HA+NP+M2; and 5 animals for the NP alone and vector control) were immunized three times with a total of 15 mg plasmid DNA in 100 ml of PBS (pH 7.4) intramuscularly at 0, 3, and 6 weeks. For the single HA or NP plasmid group, each group received 5 mg DNA for each plasmid with 10 mg of control vector as filler DNA (total 15 mg). For the 2 plasmid combination group (HA+NP), 5 mg of each plasmid plus 5 mg control vector was used. For the 3 plasmid combination group (HA+NP+M2), 5 mg of each plasmid DNA was used as total DNA remained the same (15 mg). Serum was collected 10 days after the last DNA vaccination.
Viral challenge experiments were performed 3 weeks after the last immunization. All challenged animals were exposed under CO 2 anesthesia to an intranasal viral inoculum of 100 LD 50 of A/ Vietnam/1203/2004 virus. Body weight and survival were monitored for 21 days. The viral challenge experiments were conducted at the Centers for Disease Control and Prevention (Atlanta, GA) as previously described [17] .
Male Fitch ferrets, 6-12 months of age (Triple F Farms, Sayre, PA), that were serologically negative by hemagglutinin inhibition (HAI) assay for currently circulating influenza viruses, were used in this study. All HA, NP and M2 genes are from A/Thailand/1(KAN-1)/2004. The numbers of animals used in our studies were as follows: (a) 4 animals for each group of NP and M2 alone, (b) 5 animals for the NP+M2 and the negative control group, (c) in another experiment, 4 animals for all three groups: HA alone, HA+NP+M2 and the vector control. The ferrets were immunized three times with a total of 250 mg plasmid DNA in 500 ml of PBS (pH 7.4) intramuscularly in the quadriceps muscle at 0, 3, and 6 weeks. For the single component plasmid group, each animal received 83 mg DNA for each plasmid with 167 mg of control vector as filler DNA (total 250 mg). For the 2 plasmid combination group (e.g. NP+M2), 83 mg of each NP and M2 plasmid with 83 mg control vector was used (total 250 mg). For the 3 plasmid combination group (HA+NP+M2), 83 mg of each three plasmid DNA was used as total DNA remained the same (total 250 mg). At week 9, the ferrets were immunized intramuscularly with 10 10 particles of recombinant adenoviruses expressing HA, NP, M2, or in different combinations similar to their DNA immunization combinations. Serum was collected 10 days after the last vaccination. The DNA and adenovirus immunizations were conducted at BioQual, Inc. (Rockville, MD). Nine to ten weeks after the adenovirus boost, the immunized ferrets were challenged with A/Vietnam/1203/2004 virus, which has the identical NP and M2 amino acid sequence as that of the immunized strain A/Thailand/1(KAN-1)/2004, at the Centers for Disease Control and Prevention (Atlanta, GA) as previously described [19] . Briefly, 2 days prior to infection, baseline serum, body temperature, and weight measurements of the ferrets were obtained. After the ferrets were anesthetized by an intramuscular injection of ketamine hydrochloride (24 mg/kg), xylazine (2 mg/kg) and atropine (0.05 mg/kg) cocktail, they were inoculated intranasally with 10 7 EID 50 of virus in 1 ml of PBS. The ferrets were monitored for changes in body temperature and weight and the presence of the following clinical symptoms: sneezing, lethargy, anorexia, nasal or ocular discharge, dyspnea, diarrhea, and neurological dysfunction. Body temperatures were measured using an implantable subcutaneous temperature transponder (BioMedic Data Systems, Inc., Seaford, DE). Viral titers were measured in nasal washes collected on days 3, 5, and 7 post-infection from anesthetized ferrets as previously described [6] . The nasal washes were immediately frozen on dry ice and stored at 270uC until they were processed. Viral titers in the nasal washes were determined in eggs as described above. Any ferret that lost more than 25% of its body weight or exhibited neurological dysfunction was euthanized and submitted to postmortem examination. Body weight, clinical symptoms, signs of morbidity, and survival were monitored for 7 or up to 13 days. The statistical significance of differences in survival between groups was determined using a log-rank test.
The ELISA assay used in this study was previously described in detail [22, 29, 30] . Briefly, ELISA plates were coated with antigens (100 ml/well) of the following: End-point titers were determined by linear regression analysis of the absorbance values (OD 450) as previously described [32] [33] [34] , with R 2 .0.9 obtained from a series of three-fold dilutions, as the cut-off value was set as 0.3.
A microneutralization assay to detect humoral neutralization responses against influenza was performed as previously described [22, 35] . For mice, two-fold dilutions of heat-inactivated sera were tested for the presence of antibodies that neutralized the infectivity of 100 TCID50 (50% tissue culture infectious dose) of H5N1 viruses on MDCK cell monolayers using two wells per dilution on a 96-well plate as described [35] . After 2 days of incubation, cells were fixed, and ELISA was performed to detect the presence of viral nucleoprotein (NP) and determine the neutralization activity. For ferrets, neutralizing antibody activity was analyzed in an MN assay based on the methods of the WHO Global Influenza Program [36] . Sera were treated with receptor-destroying enzyme by diluting one part serum with three parts enzyme and incubated overnight in a 37uC water bath and heat-inactivated as described for the HAI assay. Virus strains used for the MN assay are lowpathogenic, H5N1-PR8 re-assortants, obtained from Ruben Donis at the CDC Influenza Branch Hemagglutination inhibition (HAI) assay of ferret sera HAI assays were performed using four hemagglutinin units of virus and 1% horse RBC as previously described [37] [38] [39] [40] . Ferret sera were treated with receptor-destroying enzyme by diluting one part serum with three parts enzyme and incubated overnight in a 37uC water bath. The enzyme was inactivated by 30 min. incubation at 56uC followed by addition of six parts PBS for a final dilution of 1/10. Virus strains used for the HAI assay were low-pathogenic, H5N1-PR8 re-assortants obtained from Ruben Donis at the CDC Influenza Branch (Atlanta, GA): Clade 1, A/ Vietnam/1203/2004(H5N1)/PR8-IBCDC-RG and Clade 2.1, A/Indo/5/2005(H5N1)/PR8-IBCDC-RG2. Seed stocks of the re-assortant strains were obtained and expanded at BIOQUAL in 10-day-old embryonated chicken eggs. Virus strains used for the HAI assay were identical to the low pathogenic re-assortants listed for the MN assay.
The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [22, 29, 31, 41] . This assay has been developed as a safer, highly sensitive alternative to HAI and MN assays that can be applied in a high-throughput format for influenza vaccine evaluation [42] [43] [44] . Briefly, HA-pseudotyped lentiviral vectors encoding luciferase were first titrated by serial dilution. The concentration of virus giving 25% maximum activity was then incubated with the indicated amounts of mouse anti-serum before being added to 293A cells. Plates were washed and replaced with fresh media 14-16 hours later. Luciferase activity was measured after 48 hours as previously described [45] using mammalian cell lysis buffer and luciferase assay reagent (Promega, Madison, WI) according to the manufacturer's protocol.
Linear regression was utilized to determine the end-point titers of the antibodies against different antigens. In addition, the survival differences between animal groups were tested by log-rank test using GraphPad Prism software (San Diego, CA). End-point antibody titers in log 10 scale of different groups were compared by one-way Analysis of Variance (ANOVA). If this was significant at alpha = .05, we proceeded to look at pairwise comparisons using t-tests. For four or more groups (mice studies), the p-values from the pairwise tests were compared to a Bonferroni-adjused threshold of .05/6 = .008, where 6 comparisons were being made between each set of 4 groups with relevant immunogens. For three groups (ferret studies), we used instead Fisher's Least Significant Difference method to maintain a threshold of alpha = .05 for the pairwise tests following a significant ANOVA.
Prior to animal immunizations, expression of specific influenza viral genes was confirmed in 293T cells ( Figure 1A Combinatorial DNA vaccination with HA, NP, and M2 followed by viral challenge in mice
We evaluated different DNA immunogens [HA (KAN-1) alone; HA (KAN-1) with NP (PR/8); HA (KAN-1) with NP (PR/8) and M2 (KAN-1); or NP (PR/8)] for their ability to elicit protective immunity against A/Vietnam/1203/2004 (H5N1) using the mouse challenge model. Study cohorts consisted of 10 female BALB/c mice for the HA alone, HA+NP, and HA+NP+M2 groups, and 5 animals for the NP alone and vector control groups. Serum was collected 10 days after the last DNA vaccination, and end-point ELISA titers were evaluated after DNA immunization ( Figure 2 ). All HA-containing groups showed an increase in HA ELISA titer compared to the control cohort (p,0.0013) (Figure 2 , left panel). As might be expected, these levels appeared to decrease with the number of gene products in the DNA vaccine, although significant differences were not observed amongst the HAcontaining groups.
In analyzing antibody responses against NP protein (Figure 2 , middle panel), the result of the ANOVA was a borderline p-value of 0.0504. Pairwise comparisons between the HA alone and HA+NP group were similar (p = .0080 with a Bonferroni-adjusted threshold of .05/6 = .0083). However, when analyzing antibody responses against M2 protein, DNA vaccination with M2 in combination with both HA and NP elicited a significant humoral response compared to controls (p,0.0001) (Figure 2, right panel) . This result suggests that M2 is immunogenic, although immunization with M2 alone was not included in this study. To define the neutralizing antibody responses, mouse sera were pooled by groups and pseudotype neutralization, and microneutralization assays were performed. These analyses revealed neutralization activity in the HA group, with lower titers in animals immunized with HA+NP and lowest responses in those vaccinated with HA+NP+M2 (Table 1) . However, minimal neutralization was evident in NP-only immunized animals. HAI assays were not performed on the mouse sera.
To determine the efficacy of these alternative DNA immunizations, mice were challenged with the HPAI A/Vietnam/1203/ 2004 virus. Mice were challenged intranasally with 100 LD 50 of A/Vietnam/1203/2004 virus, providing a stringent evaluation of protective efficacy. All animals in the control and NP alone groups died within 6 days after viral challenge, whereas animals immunized with HA alone, HA+NP and HA+NP+M2 showed survival rates of 100, 90 and 70%, respectively ( Figure 3A ); these survival rates were not statistically significantly different. As expected, the HA alone group showed the least amount of body weight loss, while groups HA+NP and HA+NP+M2 showed similar weight loss patterns ( Figure 3B ). In contrast, the control and NP groups that showed no immune protection demonstrated severe weight loss ( Figure 3B ). This finding suggests that NP DNA immunization does not confer protection against H5N1 viral challenge at the doses used here.
To determine the comparative efficacy of alternative gene-based vaccines in ferrets, we immunized ferrets with these gene products in different combinations in a triple DNA inmmunization. In this experiment, animals received a recombinant adenovirus serotype 5 (rAd5) boost in order to increase the immunogenicity of DNA priming. Ferrets were immunized with HA alone (n = 4); HA+NP+M2 (n = 4); NP alone (n = 4); NP+M2 (n = 5); M2 alone (n = 4); or empty vector controls (n = 4+5). ELISA titers to HA, NP and M2 were determined after DNA immunization (Figure 4 , white bars) and after the rAd5 boost (Figure 4 , black bars). As expected, after DNA immunization, the HA alone group elicited Each immunized group was compared to controls as well as other groups containing relevant immunogens. ANOVA tests were significant for the responses against HA and M2, but not against NP. For HA and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.05/6 = 0.0083 (Bonferroni Correction) were considered significant for these pairwise comparisons. A single asterisk (*) represents a p-value between 0.008 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. All HA-containing groups showed significant antibody responses against HA compared to controls (p,0.0013), but did not differ significantly among themselves. Differences between NP-immunized groups were at the border of statistical significance by ANOVA (p = 0.0504), as was the comparison between HA+NP and HA groups (p = 0.008) when adjusted for multiple comparisons. The only M2-containing group, HA+NP+M2, elicited a significant humoral response against M2 protein compared to control (p,0.0001). doi:10.1371/journal.pone.0009812.g002 significant anti-HA immunity that increased after rAd5 HA boost ( Figure 4A , left panel) compared to controls (p,0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (p = 0.0006) and anti-M2 responses (p,0.0001) after the rAd boost ( Figure 4A , middle and right panels) when compared to controls. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (p,0.0001) ( Figure 4B , middle panel) compared to controls. Significant anti-M2 humoral responses were detected in animals immunized with NP+M2 post-rAd boost (p = 0.0005), but not in the M2 alone group ( Figure 4B , right panel) relative to controls. Anti-M2 humoral responses were not detectable in most cases, except after DNA/rAd5 immunization with M2 in combination with NP.
The ability of the HA antibodies from immunized ferrets to neutralize H5N1 virus was analyzed with three assays: a pseudotyped lentiviral vector neutralizing assay, an HA inhibition assay, and a microneutralization assay (Table 1 ; Ferrets). Only HA-containing groups showed substantial neutralizing antibody titer responses, while NP, M2, and NP+M2 groups showed no neutralization in each assay.
At least nine weeks after the rAd5 boost, ferrets were challenged with a high dose of A/Vietnam/1203/2004 (H5N1) virus. All groups that lacked HA, including NP alone, NP+M2, and M2 alone, developed severe disease, manifested by significant weight loss and neurological dysfunction less than seven days after the viral challenge, similar to the clinical symptoms observed in the control group ( Figure 5A, left panel) . The animals were euthanized due to severity of symptoms. The body weight loss among these groups was very similar ( Figure 5B, left panel) . In contrast, the HA and HA+NP+M2 groups were completely protected from lethality after a high dose of influenza virus challenge compared to no survival in empty vector immunized controls ( Figure 5A, right panel) . Three animals showed very mild clinical signs such as slight temperature elevation or weight loss three days after the viral challenge, but these symptoms disappeared within two days. Body weight of the two groups remained steady post-viral challenge in HA-immunized ferrets ( Figure 5B , right panel), in contrast to the controls. Viral titers from the nasal washes confirmed the antiviral effects of HAbut not NP-containing vaccines (data not shown). Control animals showed peak viral titers at day 3, and subsequently died by day 6. Both HA and HA+NP+M2 groups showed moderate viral titers at day 3, followed by quick clearance with no viral titers detectable at day 5 and day 7 (data not shown). Survival, body weight, and viral titers are indistinguishable between the HA and HA+NP+M2 groups after viral challenge, suggesting that the NP and M2 did not contribute to immune protection in ferrets. HA immune responses alone were necessary and sufficient to protect ferrets from the lethal effects of infection under these challenge conditions.
The highly conserved viral genes NP and M2 have become a focus for the development of broad, cross-protective or ''universal'' influenza vaccines. In mice, several studies have shown that genebased immunization with NP and M2 induce strong humoral and cellular responses, and protect against lethal H5N1 challenges [1, 4, 6, 9, 22, 35, [46] [47] [48] . In this study, mice were immunized with DNA vaccines encoding HA alone, NP alone, HA+NP, and HA+NP+M2. All HA-containing groups and the M2-containing group generated significant antibody responses against HA and M2 proteins, respectively. However, only the HA+NP group elicited a response against NP protein, and that was marginally significant at best. Moreover, while all HA-containing groups were protected against lethal H5N1 challenge with a survival rate of at least 70%, immunization with NP alone did not protect mice from lethal H5N1 challenge (Figure 3) . Neutralization was determined by lentiviral inhibition assay, hemagglutinin inhibition assay, and microneutralization assay. Sera from the indicated mouse and ferret immunizations with the indicated viral antigens by DNA alone or DNA/rAd5 before the viral challenge were evaluated by pseudotyped lentiviral inhibition, hemagglutinin inhibition (HAI), and microneutralization assays (MN). UD represents serum samples with undetectable neutralization activities even at the lowest dilutions, while NA represents samples that were not available, and therefore not assessed. In both mice and ferrets, only HA-containing groups stimulated strong humoral responses. doi:10.1371/journal.pone.0009812.t001
Previous studies with NP immunization have yielded mixed results, likely dependent on the mode of gene-based vaccination and on the dose of challenge virus. Though the first studies using NP DNA immunization conferred protection against lethal influenza challenge [5, 48] , in retrospect, this result may be seen with relatively low challenge doses. In a more recent study, a challenge dose that overcame NP DNA vaccine protection was similar to the amount used here [2] . NP immunization with DNA/ rAd derived from H1N1 strain A/PR/8 resulted in protection in mice against challenge with a heterologous virus strain, H5N1 A/ HK/156, but no protection was seen against a more virulent strain, A/HK/483 [1] . It appears that protection afforded by immunization with NP in mice diminishes markedly as the dose and virulence of the challenge virus increase. However, DNA vaccination with NP in combination with M2 has been shown to protect mice in both Vaxfectin formulations and rAd-boost regimens [7, 20, 21] . While we did not investigate M2 alone in mice, a previous study has shown that vaccination with M2 alone is capable of protecting mice against heterologous strains of influenza virus challenge, including H5N1 [9] .
Despite showing no neutralizing antibody responses, mice immunized with HA+NP+M2 were fully protected against lethal challenge. Lalor et al. showed a similar result in which a Vaxfectin-formulated DNA vaccine encoding H5+NP+M2 consensus genes protected mice against H5N1 challenge, despite low HAI responses. This is suggestive of other useful mechanisms of protection, such as cytotoxic T-lymphocytes, although this was not assessed in our study. A previous study with DNA/rAd5 immunization has also suggested that cellular immunity may contribute to protection in this model [12] .
While the mouse model has been used for immunogenicity studies, the murine disease does not have the same pathogenicity as human infection and is not ideal for H5N1 infection studies. These differences may be due to species changes in HA receptor specificity and distribution, as well as differential immunopathogenicity [13] [14] [15] [16] [17] . In contrast, it is generally accepted that ferrets exhibit pathology more similar to humans after H5N1 infection, including severe lethargy, fever, weight loss, transient lymphopenia, and viral replication in the upper/lower respiratory tracts and multiple systemic organs [13] . Furthermore, human isolates of influenza virus have been shown to attach and infect ferret airways [49, 50] , indicating that humans and ferrets share similar HA receptor specificity [51] .
Ferrets were immunized with HA alone, NP alone, M2 alone, NP+M2, HA+NP+M2, and control using gene-based vaccines delivered in DNA/rAd5 vectors. Since there are no established There was no statistical difference between the control group and groups immunized with NP, NP+M2, or M2. Both the HA and HA+NP+M2 groups showed 100% survival (right panel), whereas the vector control group showed 0% survival after the viral challenge. There was no statistically significant difference between the HA and HA+NP+M2 groups (p = 1.00), but there was a significant difference between these groups and the control (p = 0.008), by a log-rank test. (B) Body weights of the ferrets were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weight (left panel). Ferrets immunized with HA and HA+NP+M2 groups showed no weight loss, while the control group ferrets showed rapid weight loss (right panel). The survival and initial animal numbers in each group on the last day of body weight data collection are indicated next to the curve labels. The survival percentage for each group was analyzed statistically by a log-rank test. doi:10.1371/journal.pone.0009812.g005 M2(KAN-1) antigens. Each bar represents the group mean for the end-point titers of total IgG and IgM, determined in duplicate by series dilution of ELISA assay with the error bars indicating the standard deviation. ANOVA tests were significant for only the responses against HA at the first time point, and for all three antigens after the rAd5 boost. For HA and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.05 are indicated. * represents a p-value between 0.05 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. As expected, the HA alone group elicited significant anti-HA immunity that increased after rAd5 HA boost (left panel) compared to controls (p,0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (p = 0.0006) and anti-M2 responses (p,0.0001) after the rAd boost (middle and right panels). (B) Sera from the NP, M2, NP+M2 or vector controls were collected 14 days after the third DNA immunization (white bars), and the sera from the same animals were also collected 14 days after the recombinant adenovirus boost (solid bars). ANOVA tests were not significant for any of the antigens at the first time point, and for NP and M2 after the rAd5 boost. For NP and M2, significant pairs of groups are noted on the graph. Only p-values less than 0.008 are indicated. * represents a p-value between 0.008 and 0.001, while ** indicates ,0.001, and *** indicates ,0.0001. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (p,0.0001) (middle panel). Significant anti-M2 humoral responses were detected in animals immunized with NP+M2 post-rAd boost (p = 0.0005), but not in the M2 alone group (right panel). doi:10.1371/journal.pone.0009812.g004 assays available to measure cellular responses against HA, NP and M2 in ferrets, the efficacy of these immunogens was evaluated based on the measurement of neutralizing antibody titers through microneutralization and HA-inhibition assays. When HA and NP were present, significant humoral responses were stimulated against these proteins. However, M2 antibodies were only stimulated when M2 was in combination with NP, suggesting possible immune synergy of the two gene products. Similar adjuvant effects of NP in the anti-M2 response have recently been reported in mice [21] . Each group was challenged with a lethal strain of HPAI H5N1, and only vaccines containing HA conferred protection while NP, M2, and NP+M2 groups did not survive.
Lalor et al. showed NP+M2 to be protective in mice, but only when formulated with Vaxfectin and in a vaccine dose 6.6 times greater than that used in this study. However, in ferrets, in the absence of HA, this combination only resulted in delayed illness and death [20] . Price et al. showed that NP+M2 protects ferrets against H5N1 in a triple-prime rAd boost regimen similar to the one used here, but this difference in results may be due to the higher dose of DNA in the primary immunization (ten-fold greater than the present study) as well as a much lower viral challenge dose (56LD 50 [21] compared to 3610 5 6LD 50 in this study). However, due to differences in experimental parameters such as the immunization regimen and assay standardization, direct comparisons of NP and M2 immune responses between studies are difficult. In addition, different vaccinations may alter various antibody and cellular immune responses which may affect the protective immunity in various animal models. Nonetheless, the evidence suggests while NP or NP+M2 may provide moderate levels of protection against low dose viral challenges in ferrets, they are insufficient against high challenge doses of HPAI. On the other hand, HA elicits effective immune protection even against very high HPAI viral challenge doses. Although vaccines encoding NP or M2 alone are not required for protection against H5N1, they could potentially augment HA-encoded vaccines, particularly when there is a mismatch between the vaccine and viral HA proteins. Studies in mice have shown that M2 antibodies may help to reduce viral replication [8, 9, 52, 53] , while studies in ferrets have shown M2 to be associated with reductions in viral recovery [20, 54] . However, based on our H5N1 challenge results in ferrets, HA DNA immunization is superior to NP and M2 DNA immunization in terms of protection. These highly conserved viral genes may require combinatorial vaccination with HA to be suitable candidates for universal influenza vaccines. The Impact of Contact Tracing in Clustered Populations The tracing of potentially infectious contacts has become an important part of the control strategy for many infectious diseases, from early cases of novel infections to endemic sexually transmitted infections. Here, we make use of mathematical models to consider the case of partner notification for sexually transmitted infection, however these models are sufficiently simple to allow more general conclusions to be drawn. We show that, when contact network structure is considered in addition to contact tracing, standard “mass action” models are generally inadequate. To consider the impact of mutual contacts (specifically clustering) we develop an improvement to existing pairwise network models, which we use to demonstrate that ceteris paribus, clustering improves the efficacy of contact tracing for a large region of parameter space. This result is sometimes reversed, however, for the case of highly effective contact tracing. We also develop stochastic simulations for comparison, using simple re-wiring methods that allow the generation of appropriate comparator networks. In this way we contribute to the general theory of network-based interventions against infectious disease. Modelling has become a central tool in understanding the epidemiology of infectious disease, and designing control strategies. One control method, contact tracing, has been considered in a large number of disease contexts. These include the 2003 SARS pandemic [1, 2] , the 2001 UK FMD epidemic [3] [4] [5] [6] , contingency planning for deliberate release of smallpox [7, 8] , and control of sexually transmitted infections [9] [10] [11] . A particular benefit of tracing is that it allows targeting of control, at the cost of effort spent on finding the individuals at risk.
Since contact tracing takes place as a process over the network of interactions between hosts, it is natural to consider networkbased models of this process. Theoretical work has so far dealt with contact tracing as a branching process [12] , through modifications to mean-field equations [13] , pairwise approximations [14] and simulation [15] . This work means that the implications of heterogeneous numbers of contacts (and related network properties such as assortativity) for the efficacy of contact tracing are reasonably well understood.
For the case of clustering, due to the analytical challenge posed by the existence of short closed loops in the contact network, it has generally been more difficult to make similar progress. Existing theoretical work has therefore either been restricted to the 'limiting case' of clump structured populations, with all clustering due to completely connected cliques [16] , or else simulation on exemplar networks [13, 14, 17] .
In this work, we derive an improved triple closure for clustered pairwise models that removes two significant problems with existing closure regimes, and use this to make a systematic investigation of the impact of clustering on the efficacy of contact tracing, keeping other network and epidemiological parameters constant as appropriate. We find that, for many parameter choices, there are intuitive explanations, borne out by modelling, for the increased impact of contact tracing as clustering increases. This is not, however, a completely general result, meaning that the full implications of clustering for the efficacy of contact tracing are subtle and should be the subject of case by case investigation.
We perform our analysis within the SIS paradigm, meaning that while some of our terminology will be general to all infectious disease epidemiology, other statements will be geared towards the modelling of sexually transmitted infections where recovery/ treatment does not confer lasting immunity.
The dynamics underpinning our model are shown schematically in Figure 1 . Individuals are either susceptible (S), infectious (I) or traced (T) and move between these compartments due to four processes: infection; treatment; tracing; and stopping tracing. This paradigm is suitable for the consideration of sexually transmitted diseases, where infectious individuals can transmit infection to contacts, then seek treatment, which clears the pathogen and stops transmission but leaves the individual susceptible. It also involves the process of contact tracing, which we use as a general term that includes both partner notification and efforts by public-health workers to track down potentially infected individuals.
The four processes described so far separate into two categories: those that happen at an individual level, and contact processes. Seeking treatment and the cessation of tracing take place in the population at rates proportional to a number of individuals, and so fall into the former category. Using square brackets around a quantity to indicate its expected number in the population (so that quantities in square brackets are extensive expected numbers rather than intensive proportions) we take treatment to happen at a rate g½I, where g is the treatment rate constant, and the cessation of efforts to trace a treated individual's contacts to happen at rate g T ½T, where g T is the rate constant associated with the end of tracing.
Infection and contact tracing, on the other hand, are contact processes, and so take place at a rate proportional to a number of partnerships in the population. The full set of partnership links can be thought of as forming a network, through which contact processes spread. For infection, the rate is t½S/I, where the term in brackets is the number of susceptible-infectious pairs in the population and t is the transmission rate constant, and for tracing, the rate is r½I/T, where the term in brackets is the number of infectious-traced pairs in the population and r is the tracing rate constant. We have introduced here a notation in which a arrow is drawn from a state that transmits across the link to the state that is affected by the transmission, which will become important when we consider triples in addition to pairs.
To consider the impact of network structure, in particular clustering, on the efficacy of contact tracing, we consider a scenario in which an infection with underlying SIS dynamics is at its endemic equilibrium, and then contact tracing is introduced and the numbers infectious measured over time. This requires a dynamical model, and so we now turn to two complementary methods that we use to study the system in question: ODE-based models and stochastic simulation.
Models based on ordinary differential equations (ODEs) are widely used in infectious disease modelling. We present here a series of ODE systems that can be used in the context of network models, starting with mean-field approaches, and moving on to pairwise models. We have found that, for application to contact tracing, mean-field models and existing pairwise closures are inadequate and so we develop an improved pairwise model to study this system.
Mean-field models. For SIS dynamics with transmission rate t across a network link and treatment rate g on a large network, the expected numbers of susceptible and infectious individuals evolve according to the following exact, but unclosed, set of equations.
In our notation, ½A refers to the number of individuals in state A, ½A{B and ½A/B to the number of pairs with one individual in state A and one in state B, and a directed arrow on the right hand side of a differential equation denotes the direction of transmission for a contact process. 
There are multiple ways to control infectious diseasesvaccination and drugs such as antibiotics or anti-virals form part of the pharmaceutical approach, however another route is to stop people infecting each other. This can be done either through general efforts to reduce epidemiologically relevant contacts, or through a more targeted attempt to trace the contacts of known cases who can then be isolated or treated. The impact of this kind of contact tracing is a priori likely to depend strongly on the network of contacts linking people together. In this paper, we develop new mathematical and computational techniques to model the impact of clustering: the probability that any two contacts of a given individual are also linked to each other in the network, creating triangles. Often, and for intuitively understandable reasons, the presence of clustering increases the efficacy of contact tracing, however we show that in the regime of highly effective contact tracing sometimes the opposite is true.
To produce a mean-field model, we use a low-level closure that approximates pairs in terms of individuals.
where N is the number of nodes in the network, and n is the number of links per node. For SIR dynamics, improvements of this scheme are possible that have a factor of n{1 in the numerator of (2) in the place of n, representing the fact that after the first infection, each infected individual in an unclustered network will have one fewer link due to the individual they were infected by. For clustered networks, SIS dynamics and contact tracing, all of which we are considering here, it is not clear that a similar argument can be used and so we keep the factor of n. Pairwise models. In pairwise models, rather than using assumptions like (2), equations for the pair-level variables that appear on the right-hand side of (1) are written down, leading to triple-level variables that are then closed in terms of pairs.
The starting point for our analysis is the standard pairwise model for SIS dynamics [18] , with transmission rate t across a network link and treatment rate g. This consists of the unclosed equations (1) above, together with the following equations for pairs.
Here, ½A{B/C is the number of nodes of type B connected to both an A and a C, which may or may not be connected themselves.
We have continued use of the notation in which a directed arrow on the right hand side of a differential equation denotes the direction of transmission for a contact process, as explained above. The equations (3) are, like (1), exact, but to produce an integrable system it is necessary to introduce a system of spatial closure. The standard approximation for a population of size N, with exactly n links per node and a clustering coefficient of w-defined as the ratio of triangles to triples in the network-was derived in [19] and is:
For clarity about the definition of w, where the network adjacency matrix is G~(G ij ), then
There are two problems with the approximation (4) that are particularly relevant for the question of contact tracing. The first of these is that we would like to preserve the pair-level relation P b ½A{b~n½A. For the dynamical system (3), this pair-level relation will be preserved over time provided the following triple-level equation holds:
Equation (6) above holds for the standard closure for unclustered networks, but fails to be satisfied for non-zero clustering. The second problem with the standard closure is the question of how triangles of three infected individuals behave during the early asymptotic stage of an epidemic, where all dynamical variables are governed by the proportion of the population that is infectious, (½I=N)%1. While for pure SIS dynamics these triples are not dynamically important, when we come to consider contact tracing similar terms will become relevant. Under (4) and assuming the prevalence of infection is very low, the proportion of unclosed triples composed of three infected individuals is proportional to ½I=N as expected. However, under (4), the proportion of triangles where all three individuals are infected is not small (and does not scale with ½I=N); clearly, this is inconsistent and should be rectified in any improved closure. Motivated by these two considerations, we propose an alternative that respects (6) and has appropriate polynomial dependence on ½I=N during the early epidemic.
This closure breaks the standard symmetry between A and C, however if contact processes are consistently identified on the right hand side of ODE systems like (3) using arrows, then this is not conceptually problematic.
For the rest of this paper, we call pairwise models based on the closure (7) improved pairwise models; while pairwise models based on (4) are called standard pairwise models.
Full pairwise system. Putting together all four processes for our model with tracing, our pairwise system consists of the following exact equations together with the closure approximation (7) 
We will also consider, for comparison, these equations closed using (4), and mean-field models.
A complementary approach to pairwise models comes from individual-based, stochastic simulation where an explicit network is generated and dynamical processes are simulated using Monte Carlo methods.
In order to provide a good comparison between pairwise models and simulation, we generate explicit networks that are designed to introduce structure to the population along the lines that we have been considering, by introducing finite neighbourhood size and clustering, without introducing other significant structural features. This enables us to test results derived using pairwise equations against stochastic results. It also complements our general approach of looking at the implications of finite neighbourhood size and clustering ceteris paribus, as an aid to intuitive understanding of the impact of population structure on disease and intervention dynamics.
While other methods exist to generate networks with significant clustering coefficients, such as [20] [21] [22] , and some special clustered networks have the attractive property of begin easily generated and analysed [23, 24] , we use simple rewiring methods that are easily described and whose implications for global network structure can be readily understood, but which limit us to a smaller region of network parameter space. Most importantly, we find that giant component sizes for networks generated using our methods typically exceed 99%.
Creation of a homogeneous random network. In order to create a homogeneous random network, we firstly generate a onedimensional ring with k -th nearest neighbour links. Since we consider networks where n is even, we set k~n=2, and then make five cycles through every node i, and for each of that node's links ½i{j, swap with a random link ½k{l as below,
This generates a homogeneous random network free from dynamically relevant biases. Increasing clustering. In order to increase the clustering coefficient for a network, whilst keeping degree distribution constant, we use a new rewiring method that we call the 'big V'. This means making the following network re-wiring for a 'V' of nodes A{a{O{b{B as below,
provided the rewiring does not reduce the clustering coefficient overall. Clearly, such a rewiring does not modify the link distribution, but does increase the clustering coefficient. Empirically, we find that at low neighbourhood sizes, this method generates networks with clustering parameters up to w~0:3 before running out of possible rewirings. Whether alternative methods could yield larger values of w without either a significant reduction in the giant component size or other dynamically relevant biases remains an interesting question, however the levels of clustering given by this rewiring are sufficient to demonstrate the qualitative epidemiological effects in which we are interested. Other recent work making use of this rewiring includes [25, 26] .
Stochastic dynamics. We simulate SIS dynamics with tracing on a network using a standard continuous-time algorithm [27] . The implementation of such algorithms, and the differences between them and discrete-time equivalents, in the context of epidemic models is discussed in [28, Chapter 6] . Since the two contact processes involved (infection and tracing) both involve the explicit network, our model is essentially individual based.
For our baseline network parameters, we set n~4 to determine the effects of finite neighbourhood size and clustering. We also take the network size to be N~10 5 to produce little variability due to stochastic effects after the initial stages of an epidemic. Our main aim is to measure the effects of clustering, w, and this is varied between 0 and 0:5. The recovery rate, g, can be formally set to 1 through non-dimensionalisation, and we set g T~1 0 3 to achieve separation of timescales. Our epidemiological motivation for this separation is the expected difference in the time from infection to detection and treatment, and the time taken to notify sexual partners. For emerging respiratory infections, such a separation of timescales would, of course, not exist.
The other dynamical rates, t and r are fixed indirectly. For the tracing rate, r, we vary the proportion of contacts successfully traced, e~r rzg T , between 0 and 1, which then determines r. For the infection transmission rate, t, we need methods for fitting to a given endemic equilibrium, in both stochastic and ODE contexts. Pairwise transmission fitting. In the case of fitting to an endemic state, we solve the algebraic equations generated by setting
in equations (8), giving a transmission rate t à that yields the default endemic equilibrium, I Ã~0 :5. Stochastic transmission fitting. For computational efficiency, we use the following method to find the transmission rate t à needed to sustain a given endemic prevalence I à at constant treatment rate g:
1. Each individual is set as infectious with probability I Ã (and conversely, the probability of being set susceptible is 1{I Ã ). 2. A random ½S{I pair is chosen, and the susceptible individual is infected. 3. A random infectious individual is placed into the susceptible class. 4. Steps 2 and 3 are repeated until spatial structure is equilibrated, and then averages ½I and ½S{I of the number of infectious individuals and susceptible-infectious pairs are taken over a further set of iterations of 2 and 3. 5. The transmission rate is then given by t Ã~g ½I ½S{I .
While this method is not simply described, it is accurate and, most importantly, computationally efficient.
Dynamics in the absence of tracing Figure 2 shows the comparison of stochastic simulation on networks of the type we have been considering with both mean-field SIS, standard pairwise, improved pairwise, and also the triplewise model of [29] . This demonstrates good agreement between simulation and network ODE models, but poor agreement with the mean-field model. The inclusion of the triplewise model shows that disagreements between pairwise models and simulation in the clustered network are largely due to higher order structure, however these effects are nowhere near as large as the differences between mean-field and pairwise models. Since triplewise models involve a massive increase in computational burden, we do not consider that in this case their use is justified.
The results of Figure 2 were obtained by fitting the improved pairwise model to a given endemic state, I Ã~0 :5. The impact of this fitting on the transmission rate and number of ½I{I pairs, while varying the clustering coefficient w, is shown in Figure 2 , panes C and D.
The need to incorporate network structure into models that involve contact tracing is shown by Figure 3 . Panes A and B show predictions of prevalence over time for several models, which demonstrate that while both pairwise approaches are in good agreement with simulation, the failure of the mean-field model is dramatic-and similarly large failures can be observed in several other regions of parameter space.
For the case of a clustered network in Pane B, the agreement between pairwise models and simulation becomes slightly worse than for the unclustered network results of Pane A, with the improved pairwise model providing a closer fit. Most importantly, the improved pairwise model is in qualitative disagreement with simulation-while both mean-field and standard pairwise models predict a peak in infection before reaching the endemic state, which is not seen in either the improved pairwise model or simulation. We therefore use the results of Panes A and B to rule out the use of mean-field and standard pairwise models. This leaves the improved pairwise model, which we systematically compare to simulation in Panes C and D. Since both the improved pairwise model and simulation depend on underlying parameters in the same way, they form a complementary pair of approaches to the study of contact tracing in clustered populations. The only exception to this is the case of low prevalence of infection, where stochastic effects become important and the stochastic model predicts extinction at higher transmission rates than the pairwise model. 
We consider the effects of clustering on the efficacy of contact tracing using pairwise models by starting the system at the endemic state in the absence of any contact tracing. We then introduce tracing at a success probability e, and allow the system to evolve away from the endemic state for 0.1 and one generations (time periods 1=(10g) and 1=g respectively, corresponding to policy evaluation after a number of months and a number of years for an endemic STI) and measure the numbers infectious. This gives the results in Figure 4 , which show that clustering increases the efficacy of contact tracing at a given success rate at one infectious generation, but not at 0.1 generations, depending on the actual tracing success rate. Pane C shows this variety of responses, where clustering is more effective for large success rates at this small time-the very large rates require still smaller times to demonstrate this effect, since after 0.1 disease generations they have passed into the regime where clustering leads to less effective tracing.
The results shown in Panes C and D of Figure 2 provide a guide to intuition to explain these results. Clustering increases the number of ½I{I pairs present at a given endemic state, and contact tracing can be viewed as hyper-parasitism on the network of infected individuals. This means that clustering can be expected to enhance the efficacy of contact tracing by increasing the neighbourhood size of the infected sub-network. On the other hand, to explain a constant level of endemic infection as clustering is increased, a larger underlying rate of transmission must be present, which will undermine tracing as an individual left untouched by a wave of tracing will reinfect their immediate neighbourhood more quickly. Exactly which parameter choices allow either effect to dominate is not clear, except that lower levels of tracing success always cause clustering to increase the efficacy of tracing. Otherwise, it appears that the impact of clustering on contact tracing needs to be evaluated on a case-bycase basis.
To see the dynamics of infection that generate the results in Figure 4 , we plot stochastic and improved pairwise temporal and parametric dynamics for two exemplar values of contact tracing success, e, in Figure 5 . For e~0:15, we see in Pane A that the system settles over time to a different endemic state, and in Pane C that this involves consistently lower levels of infection in the clustered system than the unclustered system, meaning that clustering has enhanced the efficacy of contact tracing.
By contrast, for e~0:65, we see in Pane B that contact tracing drives infection to extinction, and from Pane D that this involves firstly higher levels of infection in the clustered system and then lower levels of infection for both the pairwise and stochastic models. We see a final reversal of the impact of clustering in Pane B, which is present in only the pairwise system: at longer times clustering again reduces the efficacy of contact tracing. At this point, stochastic variability in simulations has become highly significant and so we would not expect the two models to agree, since the pairwise equations should only hold in the limit where stochastic effects are negligible. To simulate in this regime would require extremely large population sizes, perhaps beyond what would ever be considered for realistic human scenarios.
We have provided an intuitive and general framework in which to study the impact of network clustering on the efficacy of contact tracing in the control of infectious disease. This has produced three major results.
Firstly, the effects of contact tracing often cannot be accurately captured by mean-field models, necessitating a modelling approach that incorporates network structure.
Secondly, we have demonstrated that due to the increased number of infectious-infectious pairs seen in clustered networks at a given pathogen burden, contact tracing at a fixed, relatively low success rate will be more effective at larger levels of clustering than at the same fixed success rate without clustering.
Finally, we have demonstrated that this increased efficacy is not completely general, and is reversed for large tracing success rates at certain times. This demonstrates the need to be cautious in the consideration of the epidemiological effects of a network property as subtle as clustering-unfortunately it is not possible to obtain a general 'rule of thumb' for its impact.
Our approach has been to consider the impact of clustering on a network with fixed, finite neighbourhood size, in the absence of other known important dynamical effects such as risk structure and assortativity. The complexity of even our simplified problem provides justification for our approach, however it would be of significant interest to see how these quantities interact with each other. The full impact of higher order structure than triangles is also, as suggested by our stochastic results, likely to be important.
Another important difference may manifest itself if we were to consider a disease with long-lasting immunity, obeying SIR dynamics, or other compartmental structure, including complex intervention strategies and comparable tracing and recovery timescales. Our preliminary work in this direction suggests that our novel result about clustering reducing contact tracing efficacy can be extremely significant in other contexts, however a full consideration of this would take us significantly beyond the aims of the present work.  Serological Profiling of a Candida albicans Protein Microarray Reveals Permanent Host-Pathogen Interplay and Stage-Specific Responses during Candidemia Candida albicans in the immunocompetent host is a benign member of the human microbiota. Though, when host physiology is disrupted, this commensal-host interaction can degenerate and lead to an opportunistic infection. Relatively little is known regarding the dynamics of C. albicans colonization and pathogenesis. We developed a C. albicans cell surface protein microarray to profile the immunoglobulin G response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. This report also identifies cell surface antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Interestingly, a large proportion of these cell surface antigens are involved in either oxidative stress or drug resistance. In addition, we identified 33 antigenic proteins that are enriched in convalescent sera of the candidemia patients. Intriguingly, we found within this subset an increase in antigens associated with heme-associated iron acquisition. These findings have important implications for the mechanisms of C. albicans colonization as well as the development of systemic infection. The yeast Candida albicans exists in a dichotomist relationship with the human host. C. albicans is frequently found as a commensal organism on the human skin, gastrointestinal (GI) tract and the vulvovaginal tract [1] . Close to 60% of healthy individuals carry C. albicans as a commensal in the oral cavity. Colonic and rectal colonization is even higher, ranging from 45% to 75% among patient groups. Alterations in the host immunity, physiology, or normal microflora rather than the acquisition of novel or hypervirulent factors associated with C. albicans, are suggested to lead to the development of candidiasis [2] . Both neutrophils and mucosal integrity of the GI tract, are critical in preventing hematogenously disseminated candidiasis [3] . The development of candidemia can begin with the translocation of C. albicans into the bloodstream from initial commensal GI colonization or the shedding from developing biofilms on indwelling catheters [4, 5] . Fungal cells that evade the host immune system can spread to deep organ systems leading to hematogenously disseminated candidiasis, which has an estimated mortality rate of 40%, even with the use of antifungal drugs [2] .
Information on in vivo gene expression would provide insight into how C. albicans interacts with host cells during the transition from commensal colonization to an opportunistic pathogen in the immunocompromised host. However, in vivo transcription profiling of C. albicans during commensal colonization or candidemia is technically challenging [6] . Instead, several genome-wide transcriptional analyses of C. albicans responses to host cells have been performed using ex vivo and in vivo infection models. These include phagocytosis of C. albicans cells by neutrophils [7] and macrophages [8] , exposure to human blood, plasma, and blood cells [9, 10] , as well as invasion of perfused pig liver and reconstituted human epithelium [11, 12] . Genes that are associated with morphological changes, metabolic adaptation, and oxidative stress are the major responses of C. albicans to host cells identified in these studies. The changes in gene expression identified in these in vitro model systems possibly reflect tissue-or stage-specific expression during an infection in patients. Profiling of antibody responses during infection in patients offers an alternative approach that can overcome technical challenges of in vivo transcription profiling. An antibody-based approach has been used to identify C. albicans gene expression during thrush in individuals with HIV [13] .
Currently the isolation of C. albicans from blood cultures is the standard method for the diagnosis of candidemia. Nevertheless, blood cultures may only become positive late in infection, and in one study up to 50% of all autopsy-proven cases of candidemia were reported as negative in blood cultures [14] . Thus, the ability to rapidly and easily diagnose candidiasis is urgently needed. An alternative approach to microbiological confirmation of C. albicans infection is serological diagnosis. An immunoproteomic approach using two-dimensional electrophoresis followed by quantitative Western blotting and mass spectrometry has been used to profile serologic response to peptides from cell surface extracts in candidemia [15] [16] [17] . A significant proportion of antigens identified were glycolytic enzymes and heat shock proteins. An antigenic multiplex consisting of the peptides Bgl2, Eno1, Pgk1, Met6, Gap1, and Fba1 provides 87% sensitivity and 74% specificity when distinguishing patients with candidemia from uninfected hospital patients [17] . However, this approach has several limitations; only the most abundant and soluble proteins can be resolved on the immunoblot, there is a lack of reproducibility of cell wall preparations, and most importantly, there is the inability to account for various stage-and tissuespecific gene expressions from the cultured cells. These limitations can be addressed by using a protein microarray to profile antibody responses [18] [19] [20] [21] .
To investigate the establishment of the humoral immunity during commensal sensitization, as well as the adaptive immune response to candidemia, we have developed a C. albicans cell surface protein microarray. Our rationale in developing a cell surface protein microarray is that the cell surface of C. albicans is the immediate target of the human immune system when C. albicans cells enter the bloodstream. Cell surface proteins play important roles in host interaction, and many of them are known virulence factors. In addition, a recent study showed that there is a significant expansion of cell wall, secreted and transporter gene families in pathogenic Candida species in comparison to nonpathogenic yeasts [22] . In this study, profiling of serological response on the protein microarray with sera from candidemia patients, blood-culture negative hospital patients and healthy individuals lead to the identification of serological signature specific for acute and convalescent stages of candidemia.
Intriguingly, large proportions of the identified antigens are involved in oxidative stress, drug resistance and iron acquisition. Furthermore, strong IgG response to many proteins known to be induced and/or required for C. albicans invasion of epithelial and endothelial cells is observed in both candidemia patients and noncandidemia controls, including all healthy individuals. Our findings provide new insights into commensal colonization and pathogenesis of C. albicans, as well as the characterization of potential serodiagnostic antigens and vaccine candidates.
Hospital patient sera were collected from Shands Hospital at the University of Florida (UF) (SH-UF) from January 2004 to December 2006. We collected sera from 21 patients with candidemia where the etiological agent was C. albicans. The median time from the date of positive culture to serum collection was two days. The study population was classified by age, gender, underlying disease, portal of entry, antifungal received, and outcome of stay (Table S1) . A subset of the candidemia patients was followed through acute infection (days 0-14) to early convalescent (week 4) and mid convalescent (week 12) infection. We also used sera from 12 hospital patients and 50 healthy individuals who had no evidence of candidiasis as our negative control groups.
C. albicans cell surface protein microarray construction and hybridization C. albicans cell surface proteins were chosen for the protein microarray because they interact directly with the host and thus are likely important for colonization and infection, as well as likely targets for the host immune system. Furthermore, many of their protein expression levels are regulated in response to extracelluar signals, such as stress, nutrients, host factors, or changes in environment. Known antigenic proteins are also included as controls (Bgl2, Eno1, Pgk1, Gap1, Cdc19, Tkl1, Hsp90, and members of the Hsp70 family) [15, 17] . The collection contains 451 His-and HA-tagged peptides (Table S2 ) that represent 363 different proteins, since ORFs .3,000 bps were cloned into two or more segments. All tagged proteins were confirmed individually by western blot and again on the protein microarray.
We have used the C. albicans cell surface microarray to evaluate the antibody profile of patients with candidemia against healthy individuals and uninfected hospital patients to determine relevant cell surface antigens that correlate with infection. Arrays were probed with a collection of sera consisting of different stages of candidemia: acute, early convalescent (approximately 4 weeks after onset of infection) and mid convalescent (approximately 12 weeks after onset of infection), as well as uninfected hospital patients and healthy individuals. Figure S1 shows a representative image of the microarray hybridized with the serum of an acute candidemia patient. All hybridizations in this study were done under the same conditions and dilutions with protein microarrays printed from the same batch. Their serological reactivity is shown as a heatmap where the antigens are sorted by increasing normalized global mean intensity, with bright green having the weakest intensity, red being the strongest, and black in between ( Figure S2 ). An examination of the IgG response to the entire C. albicans cell surface protein microarray showed that the mean global signal intensity was similar among different groups (data not shown), although antigenic profiles are not identical between individuals.
Candida albicans has both a benign and pathogenic association with the human host. Previous to this study, little was known in regard to how the host humoral system responds to the commensal colonization of C. albicans, as well as the development of hematogenously disseminated candidiasis. We show using a C. albicans cell surface protein microarray that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans, and undergoes stage-specific antibody responses as the yeast transitions from a benign microbe to an opportunistic fungal pathogen. Also identified were serological signatures specific for acute and convalescent stages of candidemia. Our findings provide new insight in the characterization of potential serodiagnostic antigens and vaccine candidates to the opportunistic pathogen C. albicans.
We were interested in determining the most seroprevalent antibodies in the acute candidemia patients and how their humoral response compared against the negative control groups. Antigens to the most seroprevalent antibodies were defined as serodominant antigens and characterized as having mean antigen reactivity 2-fold greater than the in vitro transcription/translation reaction mixture containing no vector. The top-forty serodominant antigens in the candidemia patients consisted of many previously characterized antigenic peptides such as Bgl2 [17] , Tkl1 [15] , Hwp1 [13, 23] , Eft2 [15] , and Cdc24 [13] (Table 1) . Also among the top-forty serodominant antigens were many previously identified virulenceassociated and/or hyphal-regulated proteins (eg. Int1, Hwp1, Als1, Als3, Als5, Ece1, Hyr1, Cdc24, and Utr2) ( Table 1 ) [24] [25] [26] [27] [28] [29] [30] [31] [32] . Interestingly, this serological response of acute candidemia patients was shared with both uninfected hospital patients and healthy individuals. The mean signal intensity to the top-forty serodominant antigens was 8,825 in acute candidemia patients, 8,837 in uninfected hospital patients, and 10,790 in healthy individuals. A two-way hierarchical cluster analysis of the top-forty serodominant antigens shows that the serum specimens of both the positive and negative candidemia groups were randomly dispersed throughout the hierarchical tree ( Figure 1A ). To further confirm that the topforty serodominant antigenic signatures are shared among acute candidemia patients, the uninfected hospital patients and healthy individuals, principal component analysis (PCA) was used to generate a three-dimensional projection of the data ( Figure 1B , 1C and 1D). The PCA shows that a large proportion of both the positive and negative acute candidemia sera are clustered together. These analyses suggest that IgG levels to the top-forty serodominant antigens are similar in both the negative control groups and acute candidemia sera. Since many of the top-forty antigens are either important for or induced during the invasion of epithelial or endothelial cells [11, 33] , their expression in healthy people, inferred from the presence of their antibodies, indicates the existence of a permanent host-pathogen interplay in immunocompetent individuals.
To determine stage-specific biomarkers of acute candidemia, the normalized serological expression of acute candidemia patients were compared against the humoral reactivity of the uninfected hospital patients and healthy individuals. Serodiagnostic antigens were defined as having an IgG response significantly greater in acute candidemia patients (days 0-14) as compared to the negative control groups with Benjamini and Hochberg (BH) adjusted Cyber-T p-values ,0.05. Thirteen antigens met this requirement (Table 2) . Moreover, among the proteins identified as serodiagnostic markers, proteins involved in oxidative stress response appeared to be enriched over other functional categories. Sln1 and Nik1 are two out of three histidine kinases on the cell surface protein microarray and they are both identified as serodiagnostic antigens. Sln1 and Nik1 are sensors for the high-osmolarity glycerol (HOG) pathway, a mitogen-activated protein kinase cascade responsible for osmotic and oxidative stress adaptation in C. albicans [34, 35] . In addition, the expression levels of CDR4, RAS2, and ALS9 are up-regulated during oxidative stress [35] . Another functional group over-represented among the serodiagnostic antigens are transporters associated with drug resistance (Cdr1, Cdr4, and Yor1) [36] .
The 13-serodiagnostic antigens were also evaluated with a twoway hierarchical cluster analysis on candidemia positive and negative sera. Interestingly, the sera clustered into two distinct groups based on their responses to the 13 antigens ( Figure 2A ). Cluster I contained 10 candidemia sera and only one uninfected hospital patient. Cluster II contained all 50 healthy individuals, 11 of the 12 hospital patients, and 8 acute candidemia sera ( Figure 2A ). To further confirm that the antigenic signatures identified during the acute phase of candidemia differed from the negative control groups, PCA was used to create a threedimensional projection of the data ( Figure 2B , 2C, and 2D). In agreement with the two-way hierarchical cluster analysis, two distinct groups were observed ( Figure 2B and 2C) . Also, the PCA of the negative control groups showed individuals are clustered together with the exception of one outlying uninfected hospital patient found clustered with the acute candidemia patients ( Figure 2C and 2D). These data provide further support of the antigenic signature of patients during the acute phase of candidemia. Multiple linear regression models determined that the antigenic profiles of acute candidemia patients were not related to various risk factors (i.e. age, gender, course of treatment, coexisting disease, and recovery/fatality) (data not shown). However, this determination is limited by the small sample size of our study.
Multiple independent serodiagnostic antigens can dramatically improve the sensitivity and accuracy of serodiagnostic tests [37] .
To establish a collection of antigens that could be used as a multiplex set to accurately distinguish candidemia cases from controls, we studied the discriminatory power of different sets of proteins using receiver operating characteristic (ROC) curves. First, ROC curves were generated for individual serodiagnostic antigens and the area under the ROC curves (AUC) for each antigen is listed in Table 2 . The top-five cell surface proteins all have an AUC greater than 0.76, with CDR1 (3) (AUC 0.87, BH adjusted Cyber-T p-value ,1.04e-7) giving the best single antigen discrimination ( Table 2 ). The 13 th antigen has an AUC of 0.630, which still exceeds the upper 95% confidence interval for random expectations for the AUC. To extend the analysis to combinations of antigens, we used kernel methods and support vector machines to build linear and nonlinear classifiers. As inputs to the classifier, we used the highest-ranking AUC antigens in combinations of 2, 5, 10, 11, 12 and 13 proteins and the results were validated with 10 runs of three-fold cross-validation ( Figure 2E ). Increasing the antigen number from 2 to 5, and 5 to 10 produced improvements in the classifier. But as the antigens increased to 13, a reduction in accuracy was observed. Using the ten most significant diagnostic antigens (in rank order: Cdr1 (3), Cfl91, Cdr4 (3), Als9 (2), Cdc19, Nik1 (2), Chs8 (2), Rta4, Sln1 (2), and Trk1 (2)), the classifier predicts 83% (95% CI, 76-89%) sensitivity, 72% (95% CI, 68-76%) specificity, and 74% (95% CI, 72-76%) accuracy in diagnosis of acute phase candidemia from the negative controls (healthy individuals and uninfected hospital patients) ( Table 3) .
We were next interested in identifying antigens that are significantly different between the early/mid convalescent candidemia patients (weeks 4 and 12 of the infection, respectively) and the negative control groups. The convalescent patient sera consisted of three patients whose serum was drawn under all three disease phases (acute phase, early and mid convalescent phases), 4 patients who had blood drawn at the acute and early convalescent phases, and 3 patients whose blood was drawn only at the early convalescent phase. Using BH adjusted Cyber-T p-values ,0.05, we identified 33 antigens, 11 of which are from the 13 diagnostic antigens for the acute phase of infection (Table 4 ). Among the identified convalescent biomarkers were marked expansions in proteins involved in iron acquisition (Rbt5, Csa1, Flc1, and Cfl91) ( Table 4 ). Cfl91 is a putative ferric reductase similar to Fre10, which is required for the release of iron from transferrin and the reduction to ferrous iron [38] . The protein Flc1 has been identified as having heme uptake activity [39] whereas, both Rbt5 and Csa1 have been implicated as receptors of hemoglobin whose function is to deliver the hemoglobin by endocystosis to the vacuole where iron is released by acidification [40, 41] . The remainders of the identified proteins have roles in cell wall biogenesis, membrane lipid organization, and drug resistance. We next evaluated antibody response to the 33 antigens in the acute, convalescent candidemia patients and the negative control groups by two-way hierarchical cluster analysis. The individuals in Cluster II were the same as those identified previously with 13 serodiagnostic antigens (Figure 2A and 3A ) with the addition of one convalescent candidemia patient whose only sera was drawn during week 4 of the infection. Individuals in Cluster I consisted of candidemia patients with the exception of the one uninfected hospital patient from Figure 2A . Three of the candidemia patients' acute and convalescent profiles were all found in Cluster I, whereas four candidemia patients' profiles converted from Cluster II to I during the convalescence phase of the disease. In addition, the remaining two-candidemia patients whose only blood draws were during week 4 also grouped in Cluster I ( Figure 3A ). This conversion of the antigenic profile from the negative control groups (Cluster II) to the antigenic profile consistent with candidemia (Cluster I), indicates an adaptive immune response to C. albicans that is different from commensal sensitization. Again, PCA was used to further confirm that the antigenic signatures identified during the convalescent phase of candidemia differed from the negative control groups ( Figure 3B , 3C and 3D). ROC curves were generated to assess the ability to separate the control and convalescent candidemia. AUC was determined for each of the 33-serodiagnostic antigens and listed in Table 4 in decreasing order. The top-five ORFs all have an AUC greater than 0.94. We then used SVMs to build multiplex classifiers with 2, 5, and 10 antigens with the highest-ranking AUC from Table 4 . The results were validated with 10 runs of three-fold cross-validation ( Figure 3E ). Increasing the antigen number from 2 to 5 maintained the diagnostic accuracy in the classifier and a reduction in accuracy occurred as the antigens increased to 10 due to overfitting. The top-five serodiagnostic antigens are associated with xenobiotic-transporting activity (Cdr4 and Yor1) [36] , phospholipid-transporting activity (Drs23), a putative ferric reductase (Cfl91), and a mucin-like cell wall protein (Ipf25023) ( Table 4 ). Using the top-five antigens, the classifier predicts 93% (95% CI, 89-96%) sensitivity, 96% (95% CI, 95-96%) specificity, and 95% (95% CI, 94-96%) accuracy in the differentiation of early/mid convalescent phase candidemia from the negative controls (healthy individuals and uninfected hospital patients) ( Table 3) .
Having identified 33 antigens that are correlative with convalescent candidemia in comparison to the negative control groups, we next wanted to determine the temporal change in IgG response to these 33 antigens during the transition from acute infection (AI), to early convalescent (EC), and mid convalescent (MC). A two-way hierarchical cluster analyses was performed on differential IgG responses to the 33 antigens in 3 patients with AI, EC and MC sera, and 4 patients with only AI and EC sera ( Figure  S3) . A one tailed t-test was carried out to look for differences where the EC antigen intensity is significantly greater than the AI antigen intensity, possibly indicating the selection of a protective antibody response. We observed a significant increase in the IgG response from AI to EC in the following antigens, which are ranked according to their p-values: Apc5 (2) 
In this study, we have developed a C. albicans cell surface protein microarray and profiled host humoral responses during conmmensal colonization and during the progression of candidemia. Thirteen novel serodiagnostic antigens were identified for differentiating acute candidemia from commensal sensitization and 33 antigens were found to discriminate convalescent candidemia from non-candidemia controls. The sensitivity and specificity for the identification of acute candidemia determined by the top 10 antigens from the set of 13 serodiagnostic markers are comparable to that obtained using the method of 2D-PAGE and immunoblots [17] . When using the top 5 antigens from the set of 33, both sensitivity and specificity are dramatically improved for convalescent candidemia. Pitarch et al. reported that the anti-Bgl2p IgG antibody levels mainly define the proteomic signature for candidemia patients [17] . In this study, Bgl2 is on the list of 33 diagnostic antigens from convalescent sera. Although it is classified as a serodominant antigen by acute candidemia sera, the BHadjusted p-value of Bgl2 (0.116) is just above cutoff (0.05) to be considered as diagnostic by our definition, and the mean anti-Bgl2 antibodies in acute candidemia is higher than the mean in noncandidemia controls. Bgl2 is a glycoprotein and the glycan moieties on other b-1,3-glucanosyltransferases seem to contribute to antigenicity. Since our Bgl2 is expressed in vitro without any glycosylation, its antigenicity is likely different from the Bgl2 produced by C. albicans used in the 2D-PAGE immunoblots. The previously identified immunogenic heat shock protein 90 (Hsp90) is also one of 33 biomarkers for convalescent candidemia identified from this study. Hsp90 has been shown to elicit a protective humoral response [42, 43] and its antibodies are known to associate with patients that recover from candidiasis. The use of protein microarray technology allowed us to identify new diagnostic antigens that were missed by previous studies. The use of 2-D PAGE to accurately identify and separate clinical markers of candidemia from commensal sensitization is limited by the range in protein abundance and various properties associated with peptides such as their mass, isoelectric point, hydrophobicity, and post-translational modification, as well as the semi-quantitative nature of a Western [18] . Using a C. albicans cell surface protein microarray helped us overcome many of the technical difficulties found with traditional proteomics, since the expression level of recombinant-derived proteins vary by only a single log and the use of fluorescent-labeled antibodies allows for greater linearity, precision, and sensitivity in the quantitative measurement of the humoral response to C. albicans. One of the most beneficial aspects in the use of the protein microarray assay is its ability to detect significant differences in the IgG response that under traditional immunoblot conditions would be below the detectable threshold. However, a potential limitation to our study is that the microarray is based on recombinant peptides. Because of the cell free nature of our in vitro translated peptides, potential epitopes may have been lost due to miss folding and a lack of glycosylation, both of which may affect the conformational structure of the native protein. On the other hand, the removal of posttranslational modifications, such as glycosylation, from the peptides may have revealed hidden peptide epitopes only seen during a strong host immune response. A large collection of peptide epitopes may increase the specificity in diagnosis of infection. In support of this, our study has identified many new clinical biomarkers that are associated with differing states of interactions with the host as well as the characterization of potential new targets for therapeutics and vaccine candidates. To our knowledge, this is the first study using a protein microarray to analyze the serological response to an organism that is capable of existing as both commensal flora and an opportunistic pathogen in the human population.
Commensal colonization of C. albicans is common in humans and attenuated host immunity is a perquisite for the transition from commensal colonization to infection. Historically, it was believed that C. albicans switched from a commensal to a pathogen using distinct pathogen-associated genetic programs when the host immune status was altered. An intriguing review challenges this notion, Hube postulates that C. albicans exists in a permanent hostpathogen interplay where overgrowth and invasion is only observed under immunocompromising conditions [44] . The review puts forth two-models of a permanent infection strategy: (1) constitutive gene expression where attenuated immunity induces little or no change in the pathogenic profile of C. albicans or (2) a variable transcriptional profile where C. albicans expression is dependent on the stage-and tissue-specific interactions with the host. Our study indicates the existence of permanent hostpathogen interplay with variable gene expression over the course of infection. The serological response to the entire C. albicans cell surface protein microarray detected considerable homogeneity as well as differences in the patterns of antigens recognized among patients and healthy individuals. The majority of healthy individuals and uninfected hospital patients have moderate to strong IgG responses to many C. albicans cell surface proteins that have long been associated with virulence or hyphal-regulation (a hallmark of virulence in itself). In agreement with our protein microarray data, Naglik et al. observed similar levels of IgG titers to the hyphal wall protein Hwp1 in patients with oral candidiasis and asymptomatic mucosal infections as well as healthy culturenegative controls [23] . These serodominant cross-reactive antigens include adhesins such as Als1, Als3, Als5, Hwp1 and Int1 and hyphal-regulated genes such as Als3, Hwp1, Ece1, Hyr1, and Cdc24. Both functional groups are known to be important for invasion and virulence [45] . Among the identified serodominant antigens are many previously characterized immunogenic peptides such as Bgl2 [17] , Tkl1 [15] , Hwp1 [13, 23] , Eft2 [15] , and Cdc24 [13] . Intriguingly, the average signal intensities to the top-forty serodominant antigens are higher in the healthy individuals than the uninfected hospital patients and acute candidiasis patients (10,380 vs. 8,837 and 8,825, respectively). It is interesting to speculate whether the healthy individuals' IgG response limits colonization and overgrowth since many of the serodominant antigens are against adhesins. In particular is the strong humoral response to the integrin-like protein, Int1, which may play dual roles in limiting both intestinal colonization of the cecum and systemic invasion of deep tissue organs [46, 47] . Another interesting serodominant antibody response is to the protein Ece1, which has been shown to promote adhesion and is important for GI colonization [48] . ECE1 transcription is highly expressed during GI colonization and invasion of host tissue [33, 48] . However, one can not discount that the high IgG titer of colonized individuals may be due to a previous superficial infections such candidal vaginitis [49, 50] .
The microenvironmental conditions during commensal colonization of the host may also play a role in the induction of the IgG response to certain cell surface proteins. Previous studies have evaluated characteristics common to the GI and/or vulvovaginal tract such as blood, hypoxia, iron restriction and weak acid as modifiers of gene expression [9, [51] [52] [53] . Intriguingly, the expressions of these genes share common features to the identified serodominant antibodies. Interestingly, genes transcriptionally upregulated in blood (Als1, Als3, Hwp1, Ece1, Hyr1, and Bgl2) were serodominant and cross-reactive with both positive and negative candidiasis individuals, as were genes up-regulated under hypoxic conditions (Als1, Als3, Hwp1, Rbt5, Utr2, and Tos1), iron restriction (Int1, Rbt5, and Fet35), and weak acid (Crp1, Fet35, and Ipf9655) ( Table 1) . Furthermore, some of the serodominant antigens (i.e. Als3, Ece1, Hwp1, and Rbt5) have been shown to be induced during the invasion of epithelial or endothelial cells [11, 33] . Therefore, the expression of the serodominant antigens in healthy individuals indicates the existence of permanent hostpathogen interplay during commensal colonization. In addition, the presence of serodominant IgGs in all 50 healthy individuals suggests that commensal colonization is much more prevalent than previously reported. One of the most challenging tasks in characterizing serodiagnostic antigens from C. albicans is the identification of discriminating peptides that can differentiate between commensal colonization and candidemia with high sensitivity and specificity. By profiling antibody response from patients with varying stages of candidemia against healthy individuals and candidemia-negative hospital patients, we have identified 13 diagnostic antigens for acute phase of candidemia and 33 for the early/mid convalescent candidemia. The serologic signature in candidemia patients likely reflects an alteration in the level of those proteins due to a change either in transcription and/or protein stability. Stage-and tissuespecific gene expression during the course of systemic infection is expected as C. albicans cells transition through differing microenvironments of the host. Among the 13 diagnostic antigens for acute candidemia, three are associated with drug resistance (Cdr1, Cdr4, and Yor1) [36] . The exposure to antifungal drugs in patients undergoing acute candidemia may have acted as an additional environmental stress that stimulates the expression of these antifungal drug transporters [54] . Intriguingly, two out of the 13 biomarkers are the osmosensors Sln1 and Nik1 for the HOG pathway that is responsible for osmotic and oxidative stress adaptation in C. albicans [34, 35] . The host-pathogen interaction commonly associated with oxidative stress is typically seen during phagocytosis by neutrophils, the initiating immune response to C. albicans overgrowth and infection. Furthermore, a study of global transcriptional responses to oxidative stress observed an increase in the transcriptional expression of CDR4 (4.1-fold), RAS2 (2.5-fold) and ALS9 (1.5-fold) [35] . Taken together, our data indicates a strong correlation between the IgG response to oxidative stressrelated cell surface proteins and the initial cell-mediated immune response during acute candidemia. In further agreement, previous studies have shown that oxidative stress functions are primarily induced when C. albicans is initially exposed to human blood or following phagocytosis by neutrophils and granulocytes [7, 9, 10, 55] . The 33 convalescent diagnostic antigens include proteins involved in iron acquisition, cell wall biogenesis, membrane lipid organization, and drug resistance. Of particular interest is the dramatic increase in antibodies to proteins for iron acquisition (Cfl91, ferric reductase; Rbt5 and Csa1, hemoglobin receptors; and Flc1, heme uptake). Iron is an essential nutrient for C. albicans. Circulating iron in serum is bound to transferrin and ferric reductases are required in the acquisition of iron from transferrin. Interestingly, Cfl91 is found as a biomarker for both acute and convalescent candidemia patients. Of particular interest is the increase antibody response to hemoglobin and heme-related proteins as these molecules are normally sequestered in erythrocytes [56] . The proteins Rbt5, Csa1 and Flc1 are required for iron acquisition from hemoglobin or heme [39, 40] and are diagnostic antigens only for convalescent candidemia. Thus, it is interesting to speculate whether free hemoglobin becomes a by-product of lysed erythrocytes after post-operative surgery or other invasive clinical procedures. Nevertheless, the data from this study should provide critical information for the development of diagnostic antigenic profiles for patients at risk for candidemia and for the assessment of progression of hematogenously disseminated candidiasis. Future studies will need to be done to determine whether serological differences exist between superficial and systemic infections, as well as commensal sensitization. The development of the antigenic profiles over the course of candidiasis (acute infection, early convalescence, and mid convalescence) may also provide insight into a protective humoral response against C. albicans. Even though previous sensitization to commensal colonization does not limit mortality or even morbidity in patients, experimental studies have identified protective antibodies against hematogenously disseminated candidiasis, such as heat shock protein 90 (Hsp90) or b-mannan [57] [58] [59] [60] . Future studies will need to address whether the serodiagnostic antigens identified in this study could provide protection from hematogenously disseminated candidiasis. Of particular interest are the convalescent serodiagnostic antigens where the EC antigen intensity is significantly greater than the AI antigen intensity, which may possibly indicate the selection of a protective antibody response.
Human sera from candidemia patients and hospitalized patients were collected from SH-UF under protocols approved and created by the UF Institutional Review Board. Sera from healthy individuals were obtained from volunteers at the General Clinical Research Center at the University of California, Irvine. Written, informed consent was obtained from participants.
Candidemia was defined as the recovery of C. albicans from blood cultures. Sera from candidemia patients and hospitalized patients (no clinical or microbiological evidence of candidemia) were collected from SH-UF as previously published [61] . Briefly, patients at SH-UF were identified on the day blood cultures were positive for C. albicans. The Infectious Diseases Consultation Service at SH-UF identified controls. Sera were collected and stored at 270uC in the repository at the UF Mycology Research Unit. For patients with candidemia, sera were obtained from the earliest possible date on or after the date that the first positive cultures were drawn. In all cases, this was within 7 days of the first positive culture (acute-phase sera). For ten patients with candidemia, sera were also recovered 4 to 12 weeks after the date on which the first positive cultures were drawn (convalescent-phase sera).
Cell surface proteins were selected from the Candida Genome Database (CGD) using keywords such as ''cell surface'', ''plasma membrane'', and ''cell wall''. The CGD annotation of cell surface proteins is based on published experiments [32, [62] [63] [64] [65] [66] , functionbased prediction of cellular localization, and sequence prediction. Known antigenic proteins are also included as controls (Bgl2, Eno1, Pgk1, Gap1, Cdc19, Tkl1, Hsp90, and members of the Hsp70 family) [15, 17] . Coding regions of the genes were PCR amplified from the clinical isolate SC5314 of C. albicans with primers listed in Table S2 , and cloned into a pXT7 expression vector with a HA-tag at the N-terminus and His-tag at the Cterminus by homologous recombination in E. coli as described [67] . Protein expression was carried out using an E. coli based cell- Figure 3 . Discrimination of convalescent candidemia patients from the study population. (A) Two-way hierarchical cluster analyses of the 33 differentially expressed anti-C. albicans cell surface antibodies from early/mid convalescent candidemia sera. The heatmap is organized with antigens, in rows, and acute candidemia patients (n = 18), early and mid convalescent patients (n = 10) and negative control groups (hospital patients (n = 12) and healthy individuals (n = 50)) in columns. The colorized scale ranks the antigens with red being the strongest, bright green the weakest, and black in between. (B, C & D) Principal component analyses of serum anti-C. albicans cell surface IgG antibody expression profiles that discriminate between convalescent candidemia patients and each negative control group (hospital patients and health individuals). Each circle denotes the anti-C. albicans cell surface antibody profile of asingle serum specimen. Samples are color coded as the following acute candidemia patients (red), convalescent candidemia patients (brown), healthy individuals (green), and hospital patients (blue). (E) The graph shows the ROC curves generated using different sets of serodiagnostic antigens. doi:10.1371/journal.ppat.1000827.g003 free in vitro transcription/translation system (RTS 100 E. coli HY kit, Roche). The protein microarray was made by printing the peptides onto nitrocellulose-coated FAST glass slides (Schleicher & Schuell) using the OmniGrid 100 (GeneMachines) in the UCI Microarray Facility. Each peptide was printed in duplicate and showed homogenous spot morphology as well as low background. Internal controls consisting of buffer alone and a reaction mixture with no DNA were also printed onto the FAST slides. After the addition of the plasma samples the microarray was incubated with a biotin-conjugated donkey anti-human IgG Fc c fragment specific secondary antibody (Jackson Immunoresearch). The secondary antibody was then removed and the microarray was incubated with Streptavidin: SureLight H P-3 (Columbia Biosciences). Details concerning microarray construction and controls, antibody profiling, data normalization, as well as the reproducibility and validity of the microarray are given in the Text S1.
All analysis was performed using the R statistical environment (http://www.r-project.org). It has been noted in the literature that data derived from microarray platforms is heteroskedatic [68] [69] [70] . This mean-variance dependence has been observed in the arrays presented in this manuscript [71, 72] . In order to stabilize the variance, the vsn method [73] implemented as part of the Bioconductor suite (www.bioconductor.org) was applied to the quantified array intensities. In addition to removing heteroskedacity, this procedure corrects for non-specific noise effects by finding maximum likelihood shifting and scaling parameters for each array such that the variances of a large number (default setting used: 85%) of the spots on the array are minimized. In other words, the method assumes that variance in binding for the vast majority of the proteins on the array are due to noise rather than true differential immunological response. In essence, 85% of the spots on the array are used as controls for sample-by-sample normalization. This calibration method has been shown to be effective on a number of platforms [74] [75] [76] . A simple ranking normalization where all of the proteins are ordered for each sample by binding intensity and assigning the integer rank was performed as well with similar results (results not shown). Finally, VSN normalized data is retransformed with the 'sinh' function to allow visualization and discussion at an approximate raw scale.
Diagnostic biomarkers between groups were determined using a Bayes regularized t-test adapted from Cyber-T for protein arrays [69, 77] . To account for multiple testing conditions, the Benjamini and Hochberg (BH) method was used to control the false discovery rate [78] . Statistical analyses were performed with R 2.0 (www.rproject.org) and STATA (version 10.0, StataCorp). Multiple antigen classifiers were constructed using linear and non-linear Support Vector Machines (SVMs) using the ''e1071'' R package. To prevent overfitting and show the generalization of the classification method, 10 repeats of three-fold cross-validation were performed. In this methodology, the data is split into 3 classstratified subsets. For each subset, a classifier is trained using the remaining two-thirds of the data. The classifier is then evaluated on the one-third of the data not used for training. This process is repeated for each split and for 10 different splits, yielding 30 evaluation measures. The ROCR package was used to construct receiver-operating-characteristic curves and perform sensitivity and specificity analyses. Blast2Go (www.blast2go.org) was used for gene ontology annotation and enrichment analysis. To confirm that the identified antigens were accurate, their vectors were resequenced. The Tables S3 and S4 list the statistical data of acute and convalescent candidemia patients, respectively.
Detailed information for the genes/proteins from this study can be found at the Candida Genome 
Text S1 Supplemental Experimental Procedures and Supplemental References Found at: doi:10.1371/journal.ppat.1000827.s001 (0.08 MB DOC) Figure S1 C. albicans cell surface protein microarray. Representative image of the cell surface protein microarray of C. albicans hybridized with the sera of an acute candidemia patient. The array consisted of sixteen subsets. Each of the C. albicans cell surface peptides were printed in duplicate. The yellow box indicates a duplicated print of buffer alone and the red box shows a duplicate print of reaction mixture with no DNA. Found at: doi:10.1371/journal.ppat.1000827.s002 (0.13 MB PDF) Figure S2 Global expression profile of C. albicans cell surface antigens. Heatmap of the entire C. albicans cell surface protein microarray probed with a collection of acute candidemia patients (n = 18), early and mid convalescent candidemia patients (n = 10), uninfected hospital patients (n = 12) and healthy individuals (n = 50). The antigens are in columns and are sorted by normalized mean intensity. The colorized scale ranks the antigens with red being the strongest, bright green the weakest, and black in between. Found at: doi:10.1371/journal.ppat.1000827.s003 (0.22 MB PDF) Figure S3 Development of the antigenic profile overtime in candidiasis patients. Two-way hierarchical cluster analyses of differential IgG response to the 33 convalescent serodiagnostic antigens (rows) and serum specimens (columns) from candidemia patients. The patients are ordered from left to right starting with the acute infection (AI) phase, early convalescent (EC), and mid convalescent (MC). The colorized scale ranks the antigens with red being the strongest, bright green the weakest, and black in between. Cell surface proteins that showed a significant increase in IgG response from AI to EC are labeled red (p-value #0.05).  DNA Vaccines: Developing New Strategies against Cancer Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed. Spontaneous tumour regression has followed bacterial, fungal, viral, and protozoal infections. Intratumoral infections may reactivate defensive functions, causing tumour regression.
These phenomena inspired the development of numerous rudimentary cancer immunotherapies, starting with nonspecific immunostimulatory approaches first used by William Coley [1] and leading to the concept of therapeutic vaccination against cancer. The recent identification and characterization of genes coding for tumour antigens (Ag) has enabled the design of antigen-specific cancer vaccines based on plasmid DNA and recombinant viral vectors. Gene therapy can be used to manipulate the immune system to help body's natural defences to recognise and target cancer cells.
In the last few years, it is estimated that in Europe there were almost 3 000 000 cancer cases diagnosed (excluding non melanoma skin cancers) and more than 1 500 000 deaths from cancer each year [2] . Standard therapeutic procedures currently in practice, including surgery, radiation, and chemotherapy have not greatly impacted the spread and recurrence of progressive malignancies [3] , reducing the ability of the immune system to provoke "spontaneous" regressions. Newer strategies are needed to improve upon the current treatment success rate.
Historically, Wolff and colleagues [4] first demonstrated that long-term gene expression in mouse skeletal muscle could be achieved with direct intramuscular injection of 2 Journal of Biomedicine and Biotechnology plasmid DNA. This and other early studies, demonstrating the feasibility of direct intramuscular gene transfer for DNA vaccination purpose, propelled the first vaccination studies utilizing plasmid DNA in protection scenarios involving influenza [5] and HIV-1 [6] . Cellular and humoral immune responses have been demonstrated after the injection of naked plasmid DNA vaccines into the dermis or muscle tissue of mice [5, 7] . Such responses have induced protection in preclinical models of infectious disease and malignancies (for review, see Donnelly et al. [8] ).
The DNA vaccine is a prime example of a modern genetic vaccine. The use of naked plasmid DNA as vaccine to elicit the immune system against disease provides a variety of practical benefits for large-scale vaccine production that are not as easily manageable with other forms of vaccines including recombinant protein or whole tumor cells [9, 10] .
The effectiveness to screen for antigens rapidly and to design specific types of expression constructs has made the study of DNA vaccines a valuable field for immunotherapy of cancer.
New technologies including gene-expression profiling has increased the list of candidate tumor antigens. Investigators have focused on targets which are either tumourspecific, including idiotypic antigens of B-cell tumours [11] or tumour-associated antigens [12] that are also expressed by the normal cell of origin [13] and that include the so-called cancer-testis antigens [14] .
Examples under intensive investigation are the antigens of melanoma (http://www.cancer.gov/cancertopics/types/ melanoma), prostate cancer (http://www.cancer.gov/cancer topics/types/prostate), and other epithelial cancers (http:// www.cancer.gov/cancertopics/types/skin).
DNA vaccines offer the opportunity to incorporate additional genes encoding molecules aimed at overcoming the weak immunogenicity of tumor antigens and the patients' tolerized immune repertoire.
This paper briefly summarizes findings and key technologies that have contributed to the rapid progress of DNA vaccines (mode of action, design, and optimization of DNA vaccines) as well as the state of the art of some of the more encouraging clinical studies using or against tumor antigens.
Scientists have identified a large number of cancer-associated antigens, many of which are now being used to perform cancer treatment vaccines both in basic research and in clinical trials. The list of candidate tumor antigens grows daily, largely because of expanding genetic technology including human genome sequencing and gene-expression profiling. Tumor antigens have been classified into two broad categories: tumor-specific shared antigens and tumorspecific unique antigens [15] . Shared antigens or tumorassociated antigens (TAAs) are expressed by more than one type of tumor cells. A number of TAA are also expressed on normal tissues, albeit in different amounts. As reported in the official National Cancer Institute website (NCI, http://www.cancer.gov/), representative examples of such shared antigens are the cancer-testis antigens [14] , human epidermal growth factor receptor 2 (HER2)/neu protein (reviewed in [16] ), and carcinoembryonic antigen (CEA) [17] . Unique tumor antigens result from mutations induced through physical or chemical carcinogens; they are therefore expressed only by individual tumors. Tumor-specific unique antigens encompass melanocyte/melanoma differentiation antigens, such as tyrosinase [18] , MART1 [19] and gp100 [20] , prostate-specific antigen (PSA) (reviewed in [21] ) and Idiotype (Id) antibodies [11] . Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and delivery strategies to achieve positive clinical results. An important dilemma for vaccination against overexpressed tumor-associated antigens is how to induce effective immunity against the chosen target without leading to damaging autoimmunity. The precision offered by DNA vaccines will induce focused immunity against selected antigens, and, as they become more powerful, targets will have to be selected carefully to avoid autoimmunity. Recently, an NCI pilot prioritization study produced a well-vetted, priority-ranked list of cancer antigens [22] . Antigen prioritization involved developing a list of "ideal" cancer antigen criteria/characteristics and assigning relative weights to those criteria using pairwise comparisons. The result of criteria weighting was as follows: (a) therapeutic function, (b) immunogenicity, (c) role of the antigen in oncogenicity, (d) specificity, (e) expression level and percent of antigen-positive cells, (f) stem cell expression, (g) number of patients with antigen-positive cancers, (h) number of antigenic epitopes, and (i) cellular location of antigen expression [22] . Such an effort to prioritize cancer antigens represents the logical next step in attempting to focus translational efforts on cancer vaccine regimens with the highest potential for success.
A biological issue limiting the efficacy of cancer vaccines is the low immunogenicity of cancer antigens. Strategies to enhance antigen immunogenicity are discussed in a later section.
DNA vaccines are simple vehicles for in vivo transfection and antigen production. A DNA vaccine is composed of a plasmid DNA that encodes the antigen of interest under the control of a mammalian promoter (i.e., CMV-intA, CMV immediate/early promoter, and its adjacent intron A sequence) and can be easily produced in the bacteria [23] .
The optimized gene sequence of interest is delivered to the skin (intradermally), subcutaneum or to the muscle by one of several delivery methods. Using the host cellular machinery, the plasmid enters the nucleus of transfected local cells (such as myocytes or keratinocytes), including resident antigen presenting cells (APCs). Here, gene expression from plasmid is followed by generation of foreign antigens. Although the elucidation of all immunological components involved following DNA immunization has not been entirely achieved, the mode of action of plasmid DNA vaccines appears twofold. DNA plasmids, which are derived from bacteria, stimulate the innate immune system by interacting Journal of Biomedicine and Biotechnology 3 with Toll-like receptor 9 (TLR9) [24] ), a receptor found on APCs, although the differential expression of TLR9 in mice and primate immune cells makes more complex their role as adjuvants in primates. This nonspecific immune response augments the antigen-specific immune response, where the direct and indirect presentations of antigen to APCs are involved. Two overarching models have been proposed. The antigen encoded by the plasmid is produced in host cells, either in professional APCs leading to direct priming of immune responses or in nonprofessional cells from where the antigen can be transferred to APCs leading to crosspriming.
A series of studies intended to determine how such vaccines could work investigated the source of Ag presentation, the immunological properties of the DNA itself, and the role of cytokines in eliciting the immune responses.
Early studies showed that DNA delivery method affected the cell types that were transfected. Gene Gun (bombardment of the epidermis with plasmid coated onto gold microbeads) tended to directly transfect epidermal keratinocytes and also Langerhans cells, which were shown to migrate rapidly to regional lymph nodes [25] . In this case, professional APCs were transfected directly and behave as the source of Ag presentation.
Alternatively, intramuscular injection of plasmid predominantly led to transfection of myocytes. Myocytes lack expression of major histocompatibility complex class (MHC) II and costimulatory molecules and thus would not be expected to prime T lymphocytes directly. Instead, immune priming likely occurs by dendritic cells (DCs) [26, 27] that presumably migrate to the site of DNA inoculation in response to inflammatory or chemotactic signals following vaccination [28, 29] . These DCs are thought to present antigen by cross-presentation of extracellular antigen or following direct transfection of plasmid DNA [26, 30] .
Thus, in terms of induction of immunity, there is an influence of the site and procedure used for injection, with muscle and skin cells clearly able to act as antigen depots but unable to prime the immune response. It is likely that cross-presentation from these sites to APCs is the major route to priming [26] , but there is also evidence for direct transfection of APCs, especially when delivery is to skin sites through a gene gun [25] . The host-synthesized antigen is then processed and presented by APCs in the context of both MHC I and MHC II.
Antigen-loaded APCs travel to the draining lymph nodes, where they present peptide antigens to naïve T cells, thereby eliciting both humoral and cellular immune responses. Although plasmid DNA vaccines vectors can induce antibody and CD4 + T cell helper responses, they are particularly suited to induce CD8 + T cell responses because they express antigens intracellularly, introducing them directly into the MHC I antigen processing and presentation pathway [31] .
Whatever, the process that conveys antigens to the APC seems highly efficient since DNA vaccines, that produce only very low levels of antigen, can induce all arms of the immune response [32] .
One lesson learned in the last years is that the development of plasmid DNA as cancer vaccine raises key issues such as the need to break immunological tolerance, gradual loss of MHC and antigen in tumour cells, regulatory T cells that could negatively influence the induction of antitumour responses, systemic defects in dendritic cells, secretion of immunosuppressive cytokines, resistance to apoptosis [33, 34] as is discussed elsewhere.
The use of DNA vectors represents an important platform for clinical applications, in which large-scale vaccine production is not easily manageable with other forms of vaccine including recombinant protein, whole tumor cells, or viral vectors [35] .
Although viral mediated gene transfer by genetically modified lentiviruses, adenoviruses, adeno-associated viruses, and retroviruses is advantageous because of its high transfection efficiency and stability [36] , the largest hurdles using viral vectors are to overcome the immunogenicity of the viral packaging proteins. Furthermore, viral methods are disadvantageous because of their high expense, toxic side effects, limits on transgene size, and potential for insertional mutagenesis [37] .
On the one hand, nonviral vectors are highly flexible, are capable of encoding a number of immunological components, are associated with a lower cytotoxicity, are relatively more stable, and are potentially more cost-effective for manufacture and storage (Table 1) .
Their safety in terms of adverse reactions after injection has been demonstrated in animal models [38, 39] as well as in human clinical trials.
The first clinical trail, initiated to monitor the safety and efficacy of a DNA vaccine against HIV-1 infection [40] , demonstrated that DNA plasmid vaccines were safe and were capable of inducing detectable immune cellular and antibody responses [40] [41] [42] .
The simple plasmid backbone coupled with the technology of gene manipulation allows incorporation of genes, which are then expressed by cells transfected in vivo. Although the transfection process is inefficient and varies with the target tissue and means of delivery, sufficient DNA is generally taken up to prime the immune response [32] .
DNA vaccines are free of the problems associated with producing recombinant protein vaccines, and they are also safer than live attenuated which can cause pathogenic infection in vivo. Additionally, studies with DNA vaccines have shown that even after multiple immunizations, anti-DNA antibodies are not produced [43] .
The ability to introduce antigen to the host immune system, thus enabling it to elicit strong Th1 type CD4 + T cells and CD8 + cytotoxic T cells, is a unique feature of DNA vaccines which makes them distinct from conventional protein or peptide vaccines. Because of this feature, they can readily induce humoral as well cellular immune responses [44] .
Plasmid-based gene transfer can also deliver oligonucleotides that can alter gene splicing or gene expression, for example, siRNA [35, 45] . 
Despite immunogenicity of DNA vaccines has been well established in animal models, low immunogenicity has been the major deterrent towards the development of DNA vaccines in large animal models and human. In order to overcome this hurdle, several approaches have been investigated including plasmid design, immunomodulatory molecules, delivery techniques, and prime-boost strategy ( Table 2) .
Early in the development of DNA vaccines, it became clear that maximizing the expression of the encoded Ag was critical to the induction of potent immune responses. Strong viral promoters, such as CMV-intA, are generally favoured over regulated or endogenous eukaryotic promoters [70] . Furthermore nuclear targeting sequence (NTS) could be introduced to increase the efficiency of nuclear plasmid uptake from cytoplasm after intramuscular injection [48, 71] . The utilization of codon-optimized sequences instead of the wild-type coding sequences is a general and potent method to improve vaccination. An optimal coding sequence is determined back from the amino acid sequence of the antigen by algorithms that take into account the abundance of specific tRNAs in the cytosol of human cells and the predicted structure of the mRNA. Thereafter the selected gene sequence is constructed in vitro using synthetic oligonucleotides. Adverse rare codons are avoided and secondary structures in the mRNA are minimized. Thereby, the synthetic gene is optimal for expression and consequently for the induction of an immune response [72] .
The flexibility of plasmid design coupled with the technology of gene manipulation allows also "gene optimization." Indeed, the variable regions of the heavy (V H ) and light chain (V L ) of the tumor immunoglobulin, specific for the B-cell malignancies, can be readily cloned and combined into single-chain variable fragment (scFv) format, encoding a single polypeptide consisting of V H and V L genes linked together in frame by a short 15-amino acid linker [73] .
As already discussed, the backbone of bacterial DNA includes cytosine-phosphate-guanine (CpG) unmethylated regions as sequence motifs that stimulate innate immunity, creating an inflammatory milieu for triggering the adaptive immune response [74] . The role of CpG motifs as adjuvants of immune response to DNA vaccines is well documented in mice [75] . Preclinical studies showed that the addition of CpG motifs in the plasmid can result in the induction of proinflammatory cytokines, for example, IL-12 or IFN-I [75] . CpGs are recognized by TLR9, a receptor found on APCs, helping cytotoxic T-lymphocyte (CTL) differentiation and priming. The coadministration of genes encoding ligands for Toll-like receptors (TLRs) or their signaling molecules has been shown to improve the immunogenicity of DNA vaccines [66, 76] .
Engineering DNA vaccine design for maximizing epitope-specific immunity has allowed epitope enhancement by sequence modification. The recent molecular understanding of the immune response is leading to new strategies to induce more effective immune responses. Self-tolerance might lead to deletion of T cells specific for the most effective epitopes, leaving only low-avidity T cells [77, 78] . Therefore, not all sequences are optimal antigenic epitopes. A process termed epitope enhancement is expected to make the sequences of many epitopes of cancer more immunogenic [79] . Epitope sequences can be modified to increase the affinity of the epitope peptide for the MHC molecule. The knowledge of sequence motifs for peptide binding is the key to improve the primary and/or secondary "anchor residues" that provide much of the specificity of binding to the MHC molecule [80, 81] . This strategy can greatly increase the potency of a vaccine and can convert a subdominant epitope into a dominant one by making it more competitive for available MHC molecules, thereby increasing the level of specific peptide-MHC complexes on the antigen presenting cell surface [82] . Epitope enhancement has been used to increase the affinity for both MHC class I and class II molecules (reviewed in [83] ).
To enhance the immunogenicity of DNA, vaccines encoding immunostimulatory RNA, such as double-stranded RNA or replicon RNA, were also generated [84] . [46] [47] [48] intradermal/EP prostate cancer; colon cancer [49, 50] gene gun cervical cancer [51, 52] tattoo perforating needle melanoma [53] intratumor melanoma; renal carcinoma [54, 55] high-pressure liquid delivery B-cell lymphoma; [50, 56] Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspect that can be easily handled in the DNA vaccine technology. If an antibody response is the goal, it is clearly desirable to direct antigen expression to the endoplasmic reticulum (ER), in which folding and secretion can occur. An appropriate leader (signal) sequence can achieve this. (reviewed in [47] ). For induction of CTLs, addition of genes encoding molecules such as ubiquitin, aimed to enhance degradation and peptide production in the proteasome, can be effective (reviewed in [85] ). Similarly, targeting expression to different subcellular pathways such as the endosome or lysosome can amplify CD4 + T cell responses [85] . Thus, DNA vaccines can be designed to induce an appropriate effector pathway, including antibody against cell-surface antigens, or CTL response against intracellular antigens expressed only as MHC class I-associated peptides. Since tumor antigens are often weakly immunogenic and the immune repertoire in patients may have been tolerized, the central question is whether DNA vaccines can activate and maintain the high level of immunity required to suppress cancer cell growth.
The pivotal position of CD4 T helper (Th) cells in helping B cells to produce antibody and control induction and maintenance of CD8 T cells [86] has led some investigators to focus on their importance in responses to DNA vaccination. By selecting genes encoding microbial proteins fused to the tumor antigen sequence, it was possible to activate Th cells and to dramatically amplify immunity against tumor cells [87] . As discussed in the following section, DNA vaccines offer the opportunity to activate Th cells and transform weak and ineffective immunity to a powerful antitumor attack [88] .
Even though specific antibody and CTL responses could be induced in clinical trials with naked DNA vaccines, by the intramuscular or intradermal route, high doses of DNA were necessary to elicit detectable immune responses [89, 90] . Large quantities, that is, 5-10 mg, are required to induce only modest immunogenicity [91] .
Modifying the microenvironment of the vaccinated site by coadministration of genetic, that is, DNA plasmids coding for immunostimulatory molecules, protein, or chemical adjuvants, improves the low immunogenicity of DNA vaccines [31] .
Progress has been made in developing improved techniques for encapsulating plasmid DNA (liposomes, polymers, and microparticles) although few of these formulations have been shown to elicit immune responses that are superior to those elicited by simple intramuscular plasmid DNA, still disappointing in human clinical trials [92] .
Considering the ease in design and construction of plasmid DNA used to target a particular neoplasm, biological adjuvants can be tailored and encoded within the same DNA vector as well [35] . A vast array of molecules able to modulate immune responses can be delivered (Table 3) . They include chemokines to attract APC [93] , activating cytokines [94, 95] , costimulatory molecules, APC-targeting antibodies, and molecules to manipulate antigen presentation and/or processing [96] .
One of the common cytokines employed in plasmid DNA vaccine is granulocyte-macrophage colony-stimulating factor (GM-CSF), a molecule able to enhance immune responses by inducing proliferation, maturation, and migration of DCs as well as expansion and differentiation of B and T lymphocytes [62] .
In addition to codelivery, DNA vaccines allow fusion of genes encoding activating molecules to the antigen-encoding sequence. This is an advantage, and fusion genes can create single vaccines capable of multiple functions.
Biragyn and colleagues showed that the efficiency of DNA vaccination in vivo could be greatly increased by encoding a fusion protein consisting of scFv fused to a proinflammatory chemokine moiety that facilitates targeting of APCs for chemokine receptor-mediated binding, uptake, and processing of scFv antigen for subsequent presentation to CD4 + or CD8 + T cells, or both [63] . In two independent models, vaccination with DNA constructs encoding a fusion protein consisting of scFv fused to the monocyte chemotactic protein 3 (MCP-3) or the interferon inducible protein 10 (IP-10a) generated superior protection against a large tumor challenge (20 times the minimum lethal dose), as compared with the best available protein vaccines [63] .
Additional strategies to activate effective immunity against poorly immunogenic tumor antigens employ the "DNA fusion genes vaccines" to activate T cell help for antitumor responses. The CD4 + Th cell, as pivotal cell of the immune response able to induce high levels of immunity and the maintenance of the response, has been extensively studied by Stevenson and coworkers [97] . The requirement for foreign sequences to induce Th for the B-cell response and to help the CTL response has been known for many years [98, 99] . Since Th cells control responses to vaccination, it is quite obvious that self-antigens, which do not contain epitopes likely to be recognized by available Th cells, are incapable of inducing immunity. A strategy to activate Th cells for inducing antitumor immunity is to engage a repertoire against nontolerized antigens. The use of xenogeneic antigen to break tolerance is likely due to the presence of some foreign sequences in the xenogeneic antigen that are able to activate Th cells [100] . Focusing on the antimicrobial repertoire, the principle has been applied to realize the DNA fusion gene vaccines encoding the tumor antigen linked to an antigen derived from tetanus toxin. Fusion of the Fragment C (FrC) of tetanus toxin amplified the immune response against a range of tumor antigens, leading to suppression of tumor growth [87] . Clinical trials by using these approaches to breaking self-tolerance for therapeutic purposes in patients with lymphoma and prostate carcinoma are discussed elsewhere.
It is increasingly apparent that the immunogenicity of DNA vaccines greatly depends upon the delivery methods used for immunization [101] .
In a melanoma mouse model, DNA vaccination was administered together with intratumoral delivery of antiangiogenic plasmids, encoding angiostatin, and endostatin. Combined melanoma vaccination resulted in 57% tumorfree survival over 90 days after challenge [54] . In a modest proportion of patients with malignant disease, intratumoral injection of DNA led to regression of tumor at distant sites [102] .
The recent studies have confirmed that physical methods are superior over other delivery methods that administer DNA in various chemical solutions [103, 104] .
Biolistic gene gun delivery involves adhering naked DNA to gold beads and shooting the particles through a highpressured instrument. This system delivers DNA directly into skin and Langerhans cells in a highly efficient process. Gene gun immunization has been shown to induce a greater CD8 + T cell response as well as to require less vaccine to achieve tumor immunity [51] .
A promising strategy is electroporation (EP), which in primates increases not only the level but also the breadth of response [105] , overcoming the difficulty in translating the effectiveness of DNA vaccination from preclinical rodents to large animals, including human subjects [106] .
Electroporation-based DNA delivery technology dramatically enhances cellular uptake of DNA vaccines. EP itself works as an adjuvant to enhance the necessary "danger signals" that become detectable by the immune system. The tissue damage caused by the application of EP causes inflammation and recruits DCs, macrophages, and lymphocytes to the injection site [107, 108] inducing significant immune responses, including antibody and T-cell responses. Moreover, it is tolerable without anesthetic and does not induce unwanted immune responses against the delivery mechanism, therefore it can be used for repeat administrations.
A newly developed intradermal DNA delivery is the tattoo technology. The tattoo device has a cartridge of nine fine metal perforating needle that oscillate at a constant high frequency and puncture the skin, leading to DNA transfer to skin-associated cells. The expression of reporter genes results in robust T-cell responses [109] . Recently tattooimmunization was applied in a phase I study to assess the toxicity and efficacy of inducing tumor-specific T-cell immunity against melanoma [53] .
Vaccination schedules based on combined prime-boost regimens using different vector systems to deliver the desired antigen (i.e., heterologous primeboost immunization regimen) appear to be a successful improvement in DNA vaccine platform.
Actually, prime-boost regimens have shown promise in eliciting greater immune response in humans compared with DNA vaccination alone [101] .
The DNA-prime-viral vector-boost approach focuses on the induction of T-cell immune responses. In this approach, homologous boost immunization carries the equivalent antigen than the previous immunization. Viral vectors that have been tested as booster vaccine include adenovirus, vaccinia virus, fowlpox [110, 111] as well as recombinant vesicular stomatitis virus [112] .
Likewise, the DNA-prime-protein-boost approach employs recombinant protein antigens that match with the antigens used in DNA prime immunization [68, 113, 114] . This strategy aims to develop balanced humoral and cellmediated immune responses with a focus on eliciting high quality protective antibody responses.
The heterologous prime-boost vaccination regimen exploits the ability of the immune system to generate a large number of secondary antigen-specific T cells. Following a priming immunization, a proportion of the antigenspecific T cell population transforms into antigen-specific memory T cells, which have the ability to expand rapidly upon encounter with the same antigen a second-time round.
Since the priming and boosting vectors are different, this strategy allows for greater expansion of the disease antigen-specific T cell populations [115] . To date, heterologous prime-boost regimens are among the most potent strategies to induce cellular immune responses. Compared to homologous prime-boost approach with the same DNA vaccine, boosting a primary response with a heterologous vector will result in 4-10-fold higher T cell responses [116] [117] [118] .
On the one end, a combination of DNA vaccines with EP in a homologous prime-boost approach could generate antibody responses comparable to those that are induced by protein in Complete Freund Adjuvant, and also amplified CTL responses [46] . EP may provide a prime-boost combination equivalent to that observed using viral vectors, and it is now undergoing testing in the clinic using a DNA vaccine for patients with prostate cancer. Repeated EP has been accepted by patients without the need for general or local anaesthesia and with no apparent long-term ill effects [47] .
Immune suppression is a feature of the tumor microenvironment and a barrier to tumor immune therapy. The microenvironment of tumors is established through the activity of both myeloid and lymphoid regulatory cells, as well as through the production of immune-suppressive factors by malignant cells themselves.
Many tumor-infiltrating macrophages, referred to myeloid-derived suppressor cells (MDSCs), have an immune-suppressive phenotype [119] . These macrophages are abundant in many tumors arising in both humans and mice and can exert powerful anti-inflammatory effects. In addition to MDSCs, regulatory T cells (Tregs) also heavily infiltrate many tumors [120] . These cells, characterized by the expression of the transcription factor FoxP3 as well as CD4 and CD25, play a key role in the regulation of adaptive immunity. Tregs can suppress immune responses through the secretion of suppressive cytokines like TGF-β and IL-35 [120, 121] . Tregs are a potential barrier to developing productive immune therapies for cancer, and they represent an attractive target for enhancing antitumor immunity.
Cancer immunotherapy is designed to specifically target cancer types using components of the immune system. Therefore DNA vaccines are also faced many obstacles that include breaking peripheral T cell tolerance against tumor self-antigens, to elicit appropriate immune reactions, as well as overcoming tumor-derived immunosuppressive networks and evasion tactics. Evasive mechanisms adopted by malignant cells to prevent immune cell function are numerous and lead to the clonal expansion of non-immunogenic tumor cells, by loss of tumor antigen, and to the apoptosis prevention [35] .
Tumour cells can downregulate expression of MHC and target antigens and often secrete immunosuppressive molecules to defend themselves against attack [122] . Tumours can create a tolerogenic environment which spreads to draining lymph nodes and can enhance regulatory Tcell activity. The hurdles to successful reversal of tolerance and induction of effective immunity are becoming clear and vaccines must incorporate elements to overcome them [123] .
Furthermore cancer cells secrete soluble factors in the tumor microenviroment, for example, VEGF, IL-10, and TGF-β, that affect the maturation, differentiation, and activity of APCs as DCs [124] , interfering with immune cells maturation and effector properties. The tumour microenvironment may drive tumour growth and even selectively support a subset of tumour cells, the cancer stem cells (CSCs).
The DNA vaccination platform can be capable of suppressing the progression of already established tumor by targeting those secreted soluble factors in the tumor microenvironment [125] , reversing immunological attenuation mechanisms and improving DNA vaccine potency.
The concept of combining cancer vaccination with angiogenesis inhibition is appealing, due to favorable safety profile of both approaches, as well as possible biological synergies [54] . DNA vaccination in mice against the VEGF receptor, FLK-1, abrogated the tumor vasculature and protected DNA vaccinated animals from tumor challenge in prophylactic approach [126] . Expression of the plateletderived growth factor receptor (PDGFR) in stromal cells directly correlates with advanced stage disease in human colorectal cancer. DNA vaccine against PDGFRβ suppressed 8 Journal of Biomedicine and Biotechnology growth and dissemination of human colorectal cancer cells injected into mice [127] .
In vivo coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand (Flt3L) with the plasmid DNA augments the immunogenicity of the vaccine, mobilizing and activating large numbers of DCs at the site of inoculation [128] .
Consistent with the concept that most effective cancer therapies are multimodal, combining Treg depletion with active cancer immunotherapeutic interventions is an attractive prospect, supported by abundant data in mice [129] [130] [131] [132] and by preliminary human trials [133] [134] [135] . Lastly, additional strategies aimed to altering regulatory T cell function in cancer immunotherapy, including blocking Treg trafficking, differentiation, and/or function and reducing effector cell susceptibility to suppression, have already proven successful in preliminary studies [136] [137] [138] .
The goals of the various clinical trials were to demonstrate the safety and tolerability of the candidate vaccines, and to explore the efficacy of DNA vaccines in humans. Injection of the plasmid DNA construct is tolerated well in terms of safety in the patient population and rarely involves systemic toxicities. DNA vaccines that are currently being tested do not show relevant levels of integration into host cellular DNA [139, 140] . Besides, preclinical studies in nonhuman primates as well as early studies in humans did not detect increases in antinuclear or anti-DNA antibodies. Participants in human trials of DNA vaccines are followed for possible signs and symptoms of autoimmunity induced by DNA vaccination giving no convincing evidence of autoimmunity developing in association with a DNA vaccine [40, 42, 141] .
The earliest Phase I clinical trial for a DNA vaccine was of an HIV-1 candidate tested in individuals infected by HIV-1, followed by studies in volunteers who were not infected by HIV-1 [40] . Other prophylactic and therapeutic DNA vaccine trials followed, including trials that tested DNA vaccines against cancer influenza, malaria, hepatitis B, and other HIV-1 candidates [24, 41, 42] . These trials demonstrated that the DNA vaccine platform is well tolerated and safe, as no adverse events were reported and all studies went to completion.
The evident safety of DNA vaccines has led to a relaxation of the requirements for approval by both the United States Food and Drug Administration and the national competent regulatory authorities in Europe. This is why many clinical studies tend to melt the first phase with the second phase. Then the issue has become the efficacy rather than toxicity.
Since tumour antigens are generally weakly immunogenic, they often induce a low level of spontaneous immunity or, in other cases, the spontaneous response can lead to tolerance [47] . The molecular precision offered by genebased vaccines, together with the facility to include additional genes to direct and amplify immunity, could lead to an efficient methods to use the immune system against cancer.
The inclusion of FrC sequence, or other nonself antigens, activates T-cell help to reverse tolerance and induces high levels of immunity [47, 64, 65] . To further increase immunogenicity of DNA vaccine, the use of molecular adjuvants such as cytokines and immunomodulatory molecules has been extensively employed in clinical trials [56, [58] [59] [60] [61] [62] [63] 67] .
Clinical trials conducted over the last few years have led promising results, particularly when DNA vaccines were used in combination with other form of vaccines, as demonstrated in prostate and liver cancer clinical trials [58] [59] [60] 68] . Delivery of gene-based vaccines by physical methods, that is, electroporation and gene gun, has demonstrated to amplify the immune responses induced by therapeutic vaccines against cancer [46, 47, 64] .
In the following section, an overview of various types of clinical trials will be given to highlight the issue for usage of plasmid DNA in humans. Table 4 provides a brief summary of clinical trials discussed in this review.
6.1. Lymphoma. DNA vaccination is an attractive and effective approach for active therapeutic vaccination against Bcell malignancies given the ease of production compared to Id protein vaccines.
Patient-specific DNA vaccines for therapy of B cell lymphomas and multiple myelomas based on scFv encoding a chimeric immunoglobulin molecule consisting of V H and V L genes derived from each patients's tumor were shown to be effective in animal models [47, 73] .
The first phase I/II trial of idiotypic vaccination for follicular B-cell lymphoma using a genetic approach [142] was conducted by Hawkins and colleagues. Vaccines encoding individual DNA idiotypic scFv fused to TTFrC were delivered as naked DNA by i.m. injection in patients with follicular lymphoma in clinical remission following chemotherapy, and plasmid DNA were able to develop cellular or/and humoral antiidiotype immune responses in 38% of patients over a period of several months [47] .
In a second study of vaccine therapy for B-cell lymphoma, the patient's tumor scFv was linked to the IgG2a and κ mouse immunoglobulin heavy-and light-chain constant regions chains, respectively. In this phase, I/II trial patients in remission after chemotherapy received two series of i.m. DNA vaccinations and at the end of the second vaccination, 50% of patients exhibited humoral and/or T-cell anti-Id responses; yet, these were cross-reactive with Id proteins from other patient's tumors. Subsequently, a third series of vaccinations was carried out using human GM-CSF DNA mixed with Id DNA: humoral or T-cell responses were boosted in some cases [56] .
After the important insights provided in preclinical studies [49] , the department of Oncology of the University Hospital of Uppsala is recruiting participants for a phase I/II trial, where intradermal EP (DERMA VAX) will be used as a delivery system. This study will assess the feasibility and safety of vaccination with increasing doses of xenogenic DNA coding for the Rhesus Prostate Specific Antigen (rhPSA), a protein that is 89% homologous to human PSA, administered in patients with relapsed prostate cancer.
A phase I/II, dose escalation, DNA vaccination trial with plasmid DNA, which carries prostate-specific membrane antigen (PSMA), fused to a domain (DOM1) of Fragment C of tetanus toxin, delivered either by i.m. or by i.m. followed by EP, was performed in patients with recurrent prostate cancer. The epitope used in this study, PSMA 27 is a short stretch of 9 amino acids tumor-derived epitope belonging to the PSMA. Preliminary analysis of CD8 + Tcell reactivity against the prostate-specific membrane antigen target peptide indicated significant responses in 3 out of 3 patients and CD4 + T-cell responses against the DOM1. These data validated EP as a potent method for stimulating humoral responses induced by DNA vaccination in humans [46, 47, 64] .
Results of a phase I/II trial, conducted with DNA vaccine encoding human prostatic acid phosphatase (PAP) coadministered intradermally with GM-CSF, in prostate cancer patients (stage D0) are associated with an increased PSA doubling time (PSADT), 6.5 months pretreatment versus 9.3 months in the 1 year posttreatment [61] . A longer PSADT is associated with an extremely low risk of death from prostate cancer. Besides, 14% of patients developed PAP-specific IFN gamma-secreting CD8 + T-cells immediately after the treatment course, and 41% of patients developed PAPspecific CD4 + and/or CD8 + T-cell proliferation, confirming the preclinical studies [143] .
Todorova and colleagues [58] enhanced the DNA vaccine efficacy by heterologous prime-boost regimen in a Phase I/II study. Prostate cancer patients were prime-boosted with alternate injections of recombinant adenoviral vector expressing PSMA and plasmid DNA encoding PSMA and CD86 alongside receiving GM-CSF proteins as adjuvants. After 36-month observation period from the first vaccine injection, 86% of participants developed anti-PSMA antibody.
6.3. Melanoma. DNA vaccine platform is a promising therapeutic approach also for the treatment of malignant melanoma, as demonstrated by already completed and ongoing clinical trials.
In stage IV melanoma patients, a phase I/II pilot study of intranodal delivery of Synchrotope MA2M plasmid DNA vaccine induced both humoral and CTL responses against cells expressing tumor two melanoma-associated antigens [144] . Synchrotope MA2M plasmid is a bivalent DNA vaccine encoding epitopes for both Melan-A (MART-1) and tyrosinase with potential antineoplastic activity.
The same approach was used in a improved trial conducted with the Synchrovax SEM plasmid DNA vaccine containing a plasmid pSEM that encodes 4 peptide epitope sequences, Melan-A (26-35), Melan-A (31-96), tyrosinase (1) (2) (3) (4) (5) (6) (7) (8) (9) , and tyrosinase (369-377), resulting in antigen-specific immunity even though not induce regression of established disease [145] .
DNA plasmids encoding the gp100 nonmutated melanoma-melanocyte antigen alone were administered in patients with metastatic melanoma. Rosenberg et al. showed that neither intramuscular nor intradermal injection was capable of raising cellular immune reactivity or a significant incidence of antitumor effects [57] . Increasing results were obtained in a phase II study with interleukin-2 cytokines as adjuvant used in combination with same vaccination protocols (ClinicalTrials.gov Identifier: NCT00019448) and in a phase II trial with human GM-CSF plasmid DNA in conjunction with a multipeptide vaccine encoding gp100 and tyrosinase peptidse [62] .
6.4. Cervical Cancer. Current vaccination strategies are based on the induction of neutralizing antibodies against the major and minor capsid proteins, L1 and L2, of human papillomavirus, and Gardasil is only effective against a subset of HPV genotypes [146] . Further therapeutic interventions for early-stage and late-stage cervical cancers or HPV-related disease are uneffective.
DNA plasmid platform could represent an ideal vaccine against HPV infections since it could generate both humoral immune response to prevent new infections as well as cellmediated immunity to eliminate established infection [146] .
A recent phase I/II clinical trial in patients with high-grade squamous intraepithelial lesion associated with HPV16 provided DNA plasmid expressing a mutated nonfunctional E7 incapable of binding retinoblastoma protein, with no transforming activity, linked to HSP70. A signal sequence was also attached to the hybrid antigen which results in secretion of the linked E7 antigen [67] . E7 HPV antigen as well as E6 antigen are essential for transformation and are coexpressed in HPV-associated lesions hence they represent ideal targets for the development of HPV therapeutic vaccines.
In preclinical studies, mice were successfully DNA-based immunized [147] . In a prime-boost approach, coadministration of plasmids DNA encoding murine alpha fetoprotein (AFP) and murine GM-CSF was followed by boosting with an AFP-expressing nonreplicating adenoviral vector [60] leading to tumor protective immunity.
The early studies were applied in a phase I/II clinical trial in patients with HLA-A * 0201-expressing stage II-IVA hepatocellular carcinoma. Vaccine therapy, comprising AFP and sargramostim (GM-CSF) plasmids DNA, followed by AFP adenoviral vector boost determined the dose-limiting toxicity and maximum tolerated dose of adjuvant vaccination (ClinicalTrials.gov Identifier: NCT00093548).
6.6. Breast Cancer. Since HER-2/neu (HER2) oncogenic protein is a tumor antigen in patients with breast and ovarian cancer, several vaccine strategies have been developed and are being evaluated for safety and immunogenicity in phases I and II clinical trials (ClinicalTrials.gov). Patients whose tumors overexpress the antigen have both detectable antibody and T-cell immunity directed against HER2. Likewise preclinical studies suggest that the HER2 protein, particularly the intracellular domain (ICD), is a tumor rejection antigen [148] . Salazar and colleagues are studying the immune response in patients overexpressessing HER2 epitope who have undergone vaccine therapy in a heterologous prime-boost regimen (ClinicalTrials.gov identifier: NCT00363012). After vaccination with a plasmid encoding HER2 ICD in patients with advanced stage HER2 overexpressing breast and ovarian cancers patients receive HER2 ICD protein treatment intradermally at 6 months postvaccination with the pNGVL3-hICD vaccine. The injection site is biopsied and examined for infiltrating T-cell and antigen-presenting cell populations and blood samples are examined for the presence of memory markers to demonstrate the development of HER2 ICD memory immunity.
Plasmid DNA is a new generation biotechnology product that is just beginning to enter the marketplace. Progress in the application of DNA vaccines as an immunization protocol is evident from the increasing number of such vaccines under evaluation in clinical trials and by the recent approval of several DNA vaccine products for veterinary applications.
The goal of DNA vaccination will be the development of effective immunization strategies against previously established tumors. Because of tolerance to tumour antigens, efforts are ongoing to optimize the DNA vaccine technology platform. Strategies to improve antigen expression, inclusion of adjuvants in the formulation, or as immune modulators to improve the immunogenicity, and the use of next-generation delivery methods are under intensive investigation. Current effort to prioritize cancer antigens represents the logical next step in attempting to focus translational efforts on the most promising cancer antigens into vaccines for cancer treatment or prevention. It is likely that these vaccines will have to be combined with other treatment modalities. It has become appreciated that vaccine approaches may enhance subsequent responses to radiotherapy and that certain chemotherapies actually enhance responses to vaccines. Accordingly, several late-stage clinical trials are already evaluating the benefit of vaccination in addition to conventional chemotherapy. One attractive setting is in patients during complete remission after standard adjuvant treatment (chemotherapy, radiotherapy, etc., or a combination) to whom vaccination can be given after immunological recovery [149] . Combining immunotherapy with conventional chemotherapy, antiangiogenic therapy, and other approaches could yield synergistic or additive therapeutic results.
There is still much to do in terms of optimizing vaccine design, activation and selecting appropriate target antigens, improving immune recruitment, and delivery technology. Nevertheless, in the next years an increasing number of DNA vaccines will enter more advanced phases of human studies, aimed to establish their efficacy as real clinical products. Therapeutic regimens composed of optimal vaccine formulations with combinations of immunotherapy agents and delivery strategies could offer hope to patients suffering from incurable cancer that current standard therapies cannot provide alone. TLR agonist–Stat3 siRNA conjugates: cell-specific gene silencing and enhanced antitumor immune responses Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment. siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.
RNA interference provides compelling opportunities to control gene expression in cells and siRNAs therefore represent a family of new drugs with broad potential for the treatment of diverse human diseases. Several recent studies have demonstrated the feasibility of in vivo siRNA delivery, leading to therapeutic effects in mouse models 1 , 2 , 3 , 4 and also in nonhuman-primates 5 , 6 . Nevertheless, efficient in vivo targeted delivery of siRNA into specific cell types, especially those of immune origin, which are important constituents of the tumor microenvironment and active players in promoting tumor progression, remains to be fully explored. One promising approach for targeted delivery of siRNA is the use of aptamers, which are oligonucleotide-based ligands that bind to specific receptors, such as those on tumor cells 2 .
The immune system can serve as extrinsic tumor suppressor 7 , 8 , 9 . However, the tumor microenvironment is characterized by lack of tumor-specific CD8 + T cells and an excess of regulatory T cells and myeloid-derived suppressor cells (MDSC) that promote tumor immune evasion 10 , 11 . Myeloid cells and other immune cells in the tumor microenvironment also produce growth factors and angiogenic/metastatic factors critical for tumor progression 12 . The orchestration of these processes in the tumor microenvironment appears to be highly dependent on the oncogenic transcription factor, Stat3 13 , 14 , 15 , 16 , 17 . In particular, we and others have recently demonstrated a role of Stat3 in mediating tumor immune evasion 18 , 10 , 17 . Activated Stat3 in myeloid cells inhibits expression of a large number of immunostimulatory molecules related to Th1-type responses, while promoting production of several key immunosuppressive factors 17 , 19 , 20 as well as angiogenic factors 12 . In addition, by mediating signaling of certain cytokines and growth factors, notably IL-6, Stat3 activation in myeloid cells activates Stat3 in tumor cells, enhancing tumor cell proliferation and survival 21 -24 . Because Stat3 also restrains TLR-mediated Th1 immune responses 10 , 25 , 17 , we reasoned that simultaneously silencing Stat3 by siRNA and triggering TLRs by their agonists could effectively shift the tumor microenvironment from prooncogenic to anti-tumor. A recent study using polymer-mediated in vivo transfection of 5′triphophate-Bcl2 siRNA has demonstrated the benefits of simultaneously inducing antitumor immunity and silencing a pro-oncogenic gene 4 .
In this study, we explored a strategy of linking siRNAs to synthetic oligonucleotide agonists for endosomal TLRs, which include TLR3, TLR7, TLR8 and TLR9 26 , 27 , 28 , for targeted delivery of siRNA into immune cells, together with TLR-dependent activation of antitumor immune responses. The endosomal location of the oligonucleotide-binding TLRs might be advantageous in facilitating the siRNA component to reach the cytosol for efficient gene silencing in cells selectively expressing the cognate TLR. In order to model this concept, we chose TLR9-specific oligodeoxynucleotides containing an unmethylated CpG-motif (CpG ODN), because they are already in clinical testing 29 . Additionally, CpG ODNs are efficiently internalized by various antigen-presenting cells, such as dendritic cells (DCs), macrophages and B cells, and their binding to TLR9 can initiate a cascade of innate and adaptive immune responses 30 , 28 , 29 . Myeloid cells and B cells are also critical components of the tumor microenvironment that actively promote oncogenesis 12 , 17 , 19 , 21 , 22 . By linking the  single-stranded CpG ODN with double-stranded siRNA, we have created a single synthetic  molecule capable of delivering siRNA into myeloid and B cells, silencing an immune  checkpoint and/or oncogenic gene, and activating TLR, leading to therapeutic antitumor  immune responses. 
Synthesis of the antisense strand of the siRNA (27mer) was followed by CpG1668 ODN synthesis 31 , 32 , producing a single stranded oligonucleotide connected through a carbon chain linker (Fig. 1a) . A 25/27mer siRNA was chosen over the conventional 21mer duplex to allow uncoupling of the siRNA from the CpG sequence by the Dicer enzyme once inside the cell. The asymmetric 25/27mer siRNA was optimized for specific processing by Dicer and was more potent in target gene silencing 33 , 34 . Adding either CpG1688 alone or CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resulted in a similar increase in expression of co-stimulatory CD40 and CD80 molecules, suggesting that CpG-Stat3 siRNA conjugate retains its capacity to activate TLR9 (Fig. 1b) . In addition, the immunostimulatory properties of CpG-siRNA conjugates do not differ from the effect of CpG alone as measured by production of inflammatory cytokines in primary cells and NF-κB/AP1 activation in a stable macrophage cell line ( Supplementary Fig. 1 ). To assess whether linking siRNA with CpG moiety would still allow Dicer processing, we compared in vitro Dicer activity on CpG-Stat3 siRNA substrate versus 25/27mer Stat3 siRNA. Both the CpG-Stat3 siRNA and Stat3 siRNA were processed to 21mer siRNA by recombinant Dicer (Fig. 1c) . Finally, to determine whether the CpG-Stat3 siRNA retains gene silencing function, the chimeric molecule was transfected into cells using Lipofectamine. Results from this experiment indicated that linking CpG ODN to siRNA did not interfere with Stat3 gene silencing (55% and 49% at protein level, respectively, as measured by densitometry) (Fig. 1d ).
To determine the specificity and efficiency of CpG-siRNA uptake, freshly prepared mouse splenocytes were incubated with CpG-linked Stat3 siRNA or an unconjugated Stat3 siRNA, in the absence of any transfection reagent(s). Both the CpG-Stat3 siRNA and unconjugated Stat3 siRNA were labeled with fluorescein isothiocyanate (FITC). Fluorescein-positive DCs, macrophages, B cells, granulocytes and T cells were assessed by flow cytometric analysis, which indicated that the chimeric CpG-Stat3 siRNA was efficiently taken up by both plasmacytoid (CD11c + B220 + ) and conventional (CD11c + B220 − ) splenic DCs, macrophages (F4/80 + Gr1 − ) and B cells (B220 + CD11c − ), but only minimally by splenic granulocytes (Gr1 + F4/80 − ) or T cells (CD3 + ) (Fig. 2a, Supplementary Fig. 2 and Supplementary Table 1 ). This uptake pattern reflects the known distribution of TLR9 expression in murine leukocyte subsets 35 , 26 . Intracellular staining of TLR9 in fixed splenocytes by flow cytometry CD11c + DCs confirmed TLR9 expression (Fig. 2a, bottom right) . Unconjugated Stat3 siRNA was not efficiently incorporated into DCs even after 24 h incubation, demonstrating that linkage to the TLR9 ligand facilitates siRNA uptake (Fig. 2a, bottom left) .
We further evaluated CpG-Stat3 siRNA-FITC uptake by DC 2.4 mouse DCs. Flow cytometry and fluorescent microscopy indicated that without transfection reagents, the CpG-Stat3 siRNA-FITC was internalized by DC 2.4 cells, with kinetics similar to that CpG-ODN alone ( Fig. 2b -two top rows and Supplementary Fig. 3 ) and reported previously 36 . By 60 min, greater than 80% DC 2.4 cells were positive for uptake of the conjugate, which was confirmed by confocal microscopic analysis. The uptake of the CpG-Stat3 siRNA-FITC was dose dependent (Fig. 2b, bottom row) .
Confocal microscopic analyses further showed that at one hour after incubation, the CpG-Stat3 siRNA colocalized with TLR9 within perinuclear endocytic vesicles (Fig. 2c , two top rows; and Supplementary Fig. 4 ). This colocalization diminished at 2 and 4 h after CpG-siRNA treatment (Fig. 2c , two top rows. Previous studies have demonstrated that binding of the Dicer nuclease to the siRNA oligonucleotide is required for further siRNA processing to shorter 21mer fragments that mediate RISC complex-dependent mRNA degradation 37 . We observed transient colocalization of the CpG-Stat3 siRNA with Dicer within 2 h after adding the oligonucleotide to cultured DCs (Fig. 2c , two bottom rows and Supplementary Fig. 4 ). The colocalization of CpG-Stat3-siRNA and Dicer became weaker by 4 h (Fig. 2c ) and undetectable after 24 h (data not shown).
Gene silencing effects of the CpG-Stat3 siRNA in DC2.4 cells were determined by quantitative real-time PCR analysis of the Stat3 mRNA (Fig. 2d ). Maximum effect on Stat3 silencing was observed at relatively high concentration of CpG-Stat3 siRNA (1 μM) in serum-containing cell culture medium. GpC-conjugated Stat3 siRNA, which binds but does not activate TLR9 36 , failed to silence Stat3, suggesting a possible requirement of TLR9 activation for the CpG-siRNA to be processed. Further experiments demonstrated that in TLR9 −/− myeloid cells and DCs, while cellular uptake of CpG-Stat3 siRNA was normal ( Supplementary Fig. 5 ), the gene silencing effect of CpG-siRNA was impaired (Fig 2e) . We also confirmed the gene silencing effects using electrophoretic mobility shift assays (EMSA) to detect Stat3 DNA-binding activity in DC2.4 cells, which was induced by IL-10 stimulation (Fig. 2f ). Note that, as indicated above, stimulation using CpG itself also resulted in Stat3 activation 25 , 38 (Fig. 2f) , thereby complicating the EMSA analysis for detection of Stat3 silencing. None-the-less, at higher concentrations, CpG-Stat3 siRNA diminished Stat3 DNA-binding activity relative to the conjugate scrambled RNA controls. To demonstrate the generality of this approach to gene silencing, we used TLR9-positive mouse A20 B cell lymphoma cells to test CpG-siRNA internalization ( Supplementary Fig.  6a ), and gene silencing, for which we tested a chimeric conjugate linking CpG1668 ODN with a Dicer substrate siRNA specific for firefly luciferase (Luc) in inhibiting luciferase activity in A20-Luc cells ( Supplementary Fig. 6 b) .
Biodistribution experiments in naive tumor-free mice confirmed that CpG-Stat3 siRNA is specifically internalized by resident macrophages in different tissues as well as DCs and B cells in lymph nodes ( Supplementary Fig. 7) . We also assessed the uptake of the CpG-Stat3 siRNA by macrophages and DCs in tumor-bearing mice. C57BL/6 mice with B16 tumors were injected peritumorally with CpG-Stat3 siRNA (FITC) at 0.78 nmol (20 μg)/injection. Immunofluorescent staining showed myeloid cells positive for CpG-Stat3 siRNA accumulated at the injection site (Fig. 3a) . Flow cytometry also indicated the presence of CpG-Stat3 siRNA in tumor-associated myeloid cells ( Supplementary Fig. 8a ). Furthermore, in vivo intravital two-photon microscopy showed the presence of FITC-positive cells in tumor draining lymph node (TDLN), as early as 1 h after injection of the labeled construct ( Fig. 3b, Supplementary Fig. 8b and Supplementary Fig. 9 ), but not in the contralateral lymph nodes (Supplementary Fig. 9 ). Additionally, high resolution imaging by intravital two-photon microscopy revealed an increased number of FITC-positive cells in TDLNs, as well as an accumulation of FITC-labeled CpG-Stat3 siRNAs in perinuclear endocytic vesicles (Fig. 3b, bottom right) , which was also observed in cultured DCs (Fig. 2c) .
We next evaluated the gene silencing and antitumor effects of CpG-Stat3 siRNAs in vivo. Peritumoral injections of the CpG-Stat3 siRNA resulted in relatively effective gene silencing in DCs, macrophages and B cells in TDLNs, compared to control CpG-Luc siRNA, as measured by quantitative real-time PCR (Fig. 3c ). Stat3 inhibition in CD11c + DCs isolated from TDLNs was confirmed at protein level ( Fig. 3d) . Furthermore, quantitative real-time PCR and Western blotting indicated Stat3 silencing in the total TDLNs (Supplementary Fig.  10 ). We also used CpG-Luc siRNA conjugate to confirm that CpG-siRNA conjugates are able to reduce protein expression specifically within myeloid cells in vivo. Mice overexpressing firefly luciferase under control of the β-actin promoter 39 were challenged with tumor cells, followed by repeated peritumoral injections of CpG-Luc siRNA. Results from these experiments indicated effective inhibition of luciferase activity in CD11b + myeloid cells but not in CD4 + lymphocytes within TDLNs (Fig. 3e ).
Both DCs and macrophages in the tumor microenvironment are known to promote immune tolerance 11 , 17 . Our previous work demonstrated that Stat3 is persistently-activated in myeloid cells in the tumor milieu and that genetic ablation of Stat3 in the myeloid compartment elicits potent antitumor immunity 10 . Furthermore, both CpG and LPS treatment activates Stat3 38 , 25 , 40 , which acts as a negative feedback mechanism to constrain Th1 immune responses. Therefore, we assessed whether the CpG-Stat3-siRNA conjugates could reverse the immunosuppressive effects imposed by the tumor-microenvironment and at the same time allow effective antitumor immunity induced by TLR9 triggering. Local treatment with CpG-Stat3 siRNA oligonucleotides inhibited growth of subcutaneously growing B16 melanoma (3-5 mm in diameter at the initial treatment). In contrast, treatment with unconjugated CpG-ODN plus Stat3 siRNA, or CpG-scrambled RNA construct, or GpC-Stat3 siRNA had significantly less antitumor effects (Fig. 4a) . The antitumor effects of CpG-Stat3 siRNA were confirmed by using two alternative Stat3 siRNA sequences (data not shown). To directly show that the antitumor effects induced by CpG-Stat3 siRNA were mainly mediated by immune cells, we performed in vivo experiments with antibodymediated depletion of CD8/CD4 T cells and NK cells. Without CD8 + and CD4 + immune cell populations (possibly also the cross-priming CD11c + CD8 + DCs), the effects of CpG-Stat3 siRNA treatment were partially reduced (Fig. 4b) . The therapeutic effects of CpG-Stat3 siRNA were abrogated in the absence of NK cells (Fig. 4b) .
We confirmed that local treatment with CpG-Stat3 siRNA can reduce growth of other tumors independently of their genetic background. CpG-Stat3 siRNA oligonucleotides inhibited growth of both a poorly immunogenic variant of K1735 melanoma, C4 41 , and CT26 colon carcinomas in C3H and BALB/c mice, respectively (Fig. 4c,d) . Furthermore, CpG-Stat3 siRNA treatment of the carcinoembryonic antigen (CEA) transgenic C57BL/6 mice bearing MC38 colon carcinomas expressing CEA led to tumor regression in some mice (Fig. 4e) . To assess in vivo effects of the CpG-Stat3 siRNA on tumor cells, we stained B16 tumor tissues with fluorescent antibody specific to activated caspase-3. Data from these analysis showed that B16 tumors received CpG-Stat3 siRNA had undergone more extensive apoptosis relative to the other three treatment groups ( Supplementary Fig. 11 ). We also investigated the possibility that CpG-Stat3 siRNA could be systemically delivered to achieve gene silencing and antitumor effects. Intravenous injection (i.v.) of CpG-Stat3 siRNA (0.78 nmol) reduced Stat3 expression in DCs within TDLNs relative to CpGscrambled RNA (Fig. 4f) . Systemic delivery of CpG-Stat3 siRNA to inhibit metastasis was tested in an experimental B16 lung metastasis model. Relatively small amounts of the oligonucleotide (< 1 mg/kg) were used for the systemic injection, which led to significant reduction in the number of lung metastasis (Fig. 4g) . Lower antitumor effect due to CpGscrambled RNA and CpG ODN alone was also observed. Thus, maximal antitumor effects required conjugation of the CpG TLR9 ligand with a functional Stat3 siRNA.
To further assess the role of immune modulation in the observed antitumor effects mediated by CpG-Stat3 siRNA conjugate treatment, we analyzed changes in Th1 cytokine/ chemokines and co-stimulatory molecule expression by DCs in the TDLNs. Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines 10 , 20 , 42 , 43 , which can be greatly amplified by CpG treatment 25 . As shown in Fig. 5a , injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation 10 , 20 , 42 , 43 . It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance 44 , which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in tumor-associated DCs 10 . We therefore analyzed expression of co-stimulatory molecules by DCs enriched from TDLNs. Results from these analyses indicated that CpG-Stat3 siRNA reduced the number of the DCs with low expression of costimulatory molecules, including MHC class II, CD80 and CD40, which was accompanied by a modest increase in expression of these co-stimulatory molecules ( Supplementary Fig.  12 ). Stat3 ablation in myeloid cells followed by local treatment has been shown to induce potent antitumor innate immune responses that involve neutrophils 10 . We therefore assessed whether CpG-Stat3 siRNA conjugate treatment could lead to neutrophil-associated tumor cell apoptosis. Co-staining B16 tumor tissue sections with antibodies specific to activated caspase-3 and neutrophils revealed that CpG-Stat3 siRNA treatment-induced massive tumor cell apoptosis (activated caspase 3-positive), which was associated with an increase in tumor-infiltrating neutrophils (Fig. 5b ). An increase in neutrophils in tumor after CpG-Stat3 siRNA treatment was further confirmed by flow cytometry (Fig. 5c) .
The ratio of effector to regulatory T cells within the tumor microenvironment is considered indicative of the effect of adaptive immune responses on tumor progression and metastasis 45 . We investigated the numbers of tumor infiltrating T cell populations in subcutaneously growing B16 tumors treated locally for 2 weeks with CpG-Stat3 siRNA, CpG-scrambled RNA control or PBS only. Although CpG-Stat3 siRNA treatment did not induce significant changes in overall CD4 + T cell numbers within the tumors, as shown by flow cytometric analysis (Fig. 6a) , the percentage of CD4 + /FoxP3 + Treg cells within all CD4 + T cells dropped from approximately 60% to 25% after peritumoral injections of CpG-Stat3 siRNA (Fig. 6b) . We observed an increase in the infiltration of total CD8 + T cells in the tumor stroma, although CpG-scrambled RNA control treatment also led to some recruitment of CD8 + T cells (Fig. 6c) . These effects probably result from TLR9-mediated immunostimulation of tumor-infiltrating antigen presenting cells. Fluorescent immunostaining of frozen tumor tissues with anti-CD8 antibody confirmed data generated by the flow cytometric analysis that CpG-Stat3 siRNA treatment caused increased CD8 + T cell infiltration in tumors (data not shown). Activation of tumor antigen-specific CD8 + T cells is believed to be critical for immune-mediated antitumor effects. We therefore examined the ability of CpG-Stat3 siRNA treatment to generate CD8 + T cells specific for the B16 tumor antigen, TRP2. ELISPOT assays to determine IFNγ production by T cells isolated from TDLNs in response to recall stimulation with TRP2 peptide antigen indicated that in vivo CpG-Stat3 siRNA administration indeed induced antigen-specific CD8 + T cells (Fig. 6d ).
We have developed a new strategy for targeted siRNA delivery together with immune activation by covalently linking TLR oligonucleotide agonists to siRNAs. These conjugates encompass three activities in a single molecule: targeting to immune cells, which include DCs, macrophages, and B cells, TLR activation and immune checkpoint silencing. In addition to TLR9, several other intracellular TLRs, such as TLR3, TLR7 and TLR8, also recognize/interact with nucleic acids, suggesting a broad application of this approach using various ligands for these TLRs to deliver siRNAs into different immune cells. TLRs are important for stimulating DC maturation, antigen uptake and presentation, leading to CTL activation and CD4 + T helper cell differentiation. Therefore, TLR agonist-siRNA approaches can further stimulate desired immune responses for treating diseases such as cancer and infections. Although it has been established that binding to TLR9 is necessary for CpG-mediated immune activation, it remains to be fully explored how CpG ODN enter cells 36 . Our results indicated that cellular uptake of both CpG ODN and the CpG-siRNA constructs can occur in the absence of TLR9. However, TLR9, at least in mouse cells, is essential for CpG-siRNA-mediated gene silencing. While the exact underlying mechanism(s) remains to be determined, it is possible that triggering TLR9 could effect either endosomal release of CpG-siRNA into the cytoplasm, or/and its intracellular processing.
Although TLR9 is expressed in different types of mouse DCs, its expression is more selective in humans. Nevertheless, while the highest levels of constitutive TLR9 expression is observed on human plasmacytoid DCs and B cells, it is also expressed at lower levels on human monocytes and macrophages 26 . These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity 27 , 29 , 46 . Furthermore, it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor effects of siRNA by stimulating antitumor immune responses, likely through intracellular RNA sensors such as RIG-I or MDA-5 4 . It is therefore conceivable to incorporate triphosphate to the CpG-siRNA to further amplify antitumor immunity. In addition, a critical role of tumor stromal macrophages and B cells in promoting tumor development has been well documented 47 , 48 . Importantly, Stat3 and several other molecules produced by the tumor myeloid population, and possibly tumor-associated B cells, are critical for tumor immunosuppression 17 , and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival 12 , 21 , 22 , 23 . In addition to Stat3, other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies. Therefore, being able to target the tumor stromal myeloid cells/B cells through CpG-siRNA conjugate molecules is highly desirable for cancer therapies. In addition to normal immune cells, several types of tumor cells, including those of B cell origin, and some solid tumor cells, are also positive for TLR9 49 . Our preliminary results suggested the feasibility of CpG-siRNA approach to silence genes in TLR9 + tumor cells. For example, treating human TLR9 + tumors in NOD/SCID/IL-2RγKO mice with CpG-Stat3 siRNA resulted in tumor cell apoptosis and tumor growth inhibition (Kortylewski and Yu, unpublished data). More experiments remain to be performed to optimize this approach for potential clinical use.
Our results indicated that the gene silencing effects by CpG-siRNA in cultured cells requires high concentrations of the conjugates and are suboptimal relative to in vivo treatment. Work is underway to determine the possible cause(s) of this difference, which might include serum-associated degradation of CpG-siRNAs or reduction of the overall silencing effect in rapidly dividing cell populations. It is possible that in vivo repeated treatments allow accumulative gene silencing effects, and the crosstalk between various cells in the tumor microenvironment could lead to secondary inhibitory effects on Stat3 activity 23 . The halflife of the constructs at present is limited. Although the CpG ODN in the construct is phosphothioated, which should resist serum degradation, the siRNA in the chimeric construct is unmodified and negatively charged. Chemically modifying the siRNA to prolong serum stability and to neutralize the negative charge of the siRNA to facilitate endosomal release may improve the efficacy of TLR agonist-siRNA approach. Although more studies are necessary to further understand the mechanism(s), and to improve CpG-siRNA gene silencing effects, our results raise the possibility to use oligonucleotide TLR agonists for siRNA delivery into tumor-associated myeloid cells and B cells, and possibly certain tumor cells, to inhibit expression of tumor-promoting/immunosuppressive molecules while activating TLR(s) for immune activation.
Murine B16 melanoma, CT26 colon carcinoma and A20 B cell lymphoma lines were purchased from American Type Culture Collection. Mouse dendritic DC2.4 cells were originally from Dr. Kenneth Rock (University of Massachusetts Medical School, MA). Highly metastatic clone of K1735 melanoma (C4) was kindly provided by Drs. S. Huang and J. Fidler of M. D. Anderson Cancer Center (Houston, TX). The stably transduced A20-Luc cell line was provided by Dr. Defu Zheng (City of Hope, Duarte, CA). The generation of transgenic C57BL/6.CEA mice and MC-38.CEA cell line was previously described 50 .
The phosphothioated oligodeoxyribonucleotide(ODN) and antisense strands (AS) of siRNAs were linked using 6 units of C3 carbon chain linker, (CH 2 ) 3 The sequence of firefly luciferase-specific 25/27mer siRNA (Luc1 R 25D/27), used for the CpG1668-Luc siRNA conjugate molecule, was published 34 . The correct formation of siRNA duplex was confirmed by in vitro Dicer cleavage assays. 0.5 μg of each ODN-siRNA construct was subjected to processing by 1U of Dicer (Ambion) in 37°C for 1.5 h, resolved with 15% polyacrylamide/7.5M urea gel and results of the dicing reaction were visualized with SYBR Gold staining (Invitrogen).
Total RNA was extracted from cultured or primary cells using RNeasy kit (Qiagen). After cDNA synthesis using iScript cDNA Synthesis kit (Bio-Rad), samples were analyzed using pairs of primers specific for Stat3, TNF, IL-6, IP-10, RANTES, p35/IL-12, p40/IL-12 and GAPDH mRNAs (SuperArray Bioscience Corporation). Sequence-specific amplification was detected by fluorescent signal of SYBR Green (Bio-Rad) by using Chromo4 Real-time PCR Detector (Bio-Rad).
B16 cells were transiently transfected with 15 nM CpG-linked or uncoupled Stat3 siRNA and scrambled RNA using Lipofectamine 2000 (Invitrogen). 48 h later cells were lysed and used for Western blot analysis.
EMSA and Western blot analysis to detect Stat3 DNA-binding and protein expression were performed as described previously 18 . The protein levels of Stat3 detected by Western blotting were later quantified by densitometry using AlphaEase FC software (Alpha Innotech).
Luc + mice treated with CpG-Luc siRNA were lysed and luciferase activities were determined using the Luciferase Assay System (Promega) after normalization to the protein content of the sample.
Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with established institutional guidance and approved protocols from Research Animal Care Committees of the City of Hope. For s.c. tumor challenge, we injected 1 × 10 5 B16, C4 or CT26 tumor cells into 7-8 weeks C57BL/6, C57BL/6.CEA, C3H or Balb/C mice, respectively. After tumors reached average size of ca. 5 mm, mice were injected peritumorally with 0.78 nmol of CpG1668 ODN alone, in combination with Stat3 siRNA or CpG/GpC ODN linked to various double stranded RNA (dsRNA) sequences described above. Tumor growth was monitored every other day. For the analysis of cellular and molecular mechanisms of CpG/GpC-dsRNAs effects, mice were killed after 2-3 weeks of treatment. For experiments on silencing of luciferase activity in vivo, Luc + mice (originally kindly provided by Dr. Christopher H. Contag, Stanford University School of Medicine, CA) were challenged with tumor and treated with 3 daily injections of CpG-Luc siRNA or CpG-scrambled RNA. Lymph nodes as well as tumor specimens were harvested. For B16 lung metastasis model, mice were challenged with 0.5 × 10 5 B16 tumor cells (i.v.) and two d later, after tumors were established, were treated systemically with 0.78 nmol (ca. 1 mg/kg) of CpG1668 ODN or various CpG-dsRNAs. Intravenous injections were repeated every other day for two weeks. Lungs were harvested, fixed and the number of B16 tumor colonies was manually counted. The level of Stat3 silencing was assessed by real-time PCR in DCs isolated from tumor-draining inguinal (for s.c. tumor model) or cervical (for metastasis model) lymph nodes. For immune cell depletion, mice were pretreated with anti-CD8 plus anti-CD4 antibodies (clone 2.43 and GK1.5, respectively, depleting 99% and 98% of CD8 and CD4 cells, respectively) or anti-asialo-GM1 antibodies (Wako, depleting min. 79% of NK cells) before tumor inoculation then injected weekly.
We prepared single-cell suspensions of spleen, lymph node or tumor tissues by mechanic dispersion followed by collagenase D/DNase I treatment as described before 10 . For extracellular staining of immune markers 1 × 10 6 of freshly prepared cells suspended in PBS/2% FCS/0.1% w/v sodium azide was preincubated with FcγIII/IIR-specific antibody to block non-specific binding and stained with different combinations of fluorochrome-coupled antibodies to CD11c, I-A b (MHCII), CD40, CD80, B220, CD11b, Gr1, CD3, CD8 or CD4 (BD Biosciences). Prior to intracellular staining with antibodies to TLR9 (eBioscience), Dicer (Santa Cruz) or FoxP3 (eBioscience), various immune cell subsets were fixed in paraformaldehyde and permeated in methanol. Fluorescence data were collected on FACScalibur (Beckton Dickinson) and analyzed using FlowJo software (Tree Star).
ELISPOT assay 5 × 10 5 cells isolated form TDLNs of CpG-or CpG-siRNAs-treated mice, were seeded into each well of 96-well filtration plate in the presence or absence of 10 μg/ml of TRP2 peptide. After 24 h of incubation at 37°C, peptide-specific IFNγ-positive spots were detected according to manufacturer's procedure (Cell Sciences), scanned and quantified using Immunospot Analyzer from Cellular Technology Ltd.
For immunofluorescent staining, we fixed the flash-frozen tumor specimens in formaldehyde and permeabilized with methanol before antibody staining. For confocal microscopy, cultured cells were fixed with 2% paraformadehyde for 20 m, permeabilized in PBS/0.1 % Triton X-100/1 mM MgCl 2 , and 0.1 mM CaCl 2 for 5 m and quenched in 50 mM NH 4 Cl for 5 m before blocking in 1% BSA for 1 h. Samples were stained with antibodies to CD11b (BD Biosciences), neutrophils (7/4, Cedarlane), active caspase-3 (Cell Signaling), TLR9 (eBiosciences), Dicer (Santa Cruz) and detected with Alexa488-or Alexa555-coupled secondary antibodies (Invitrogen). After staining the nuclei with DAPI (Vector) or Hoechst 33342, slides were mounted and analyzed by fluorescent or confocal microscopy. The confocal imaging was carried out using a 63x water immersion objective on cLSM510Meta confocal microscope (Zeiss). For intravital two-photon imaging, B16 tumor-bearing mice received single intratumoral injection of 0.78 nmol FITC-labeled CpG-Stat3 siRNA, followed by retroorbital injection of dextran-rhodamine (Invitrogen) and Hoechst 33342 prior to imaging 1 h later. Mice were anesthetized and intravital two-photon microscopy was carried out using equipment and software from Ultima Multiphoton Microscopy Systems.
Unpaired t-test with equal or unequal variance (specifically for the analysis of cytokine expression in Fig. 5a ) was used to calculate two-tailed P value to estimate statistical significance of differences between two treatment groups in the whole study. One-way ANOVA followed by Newman-Keuls test was applied for comparison of multiple treatment groups. Two-way ANOVA plus Bonferroni posttest were applied to assess statistical significance of differences in tumor growth kinetics between multiple treatment groups. Statistically significant P values were indicated in figures and/or legends and labeled as with 1×10 5 B16 cells, n = 5-6. Statistically significant differences between CpG-Stat3 siRNA-and CpG-scrambled RNA-treated groups are indicated by asterisks. Similar results were obtained in 3 independent experiments. (b) Tumor growth inhibition by CpG-Stat3 siRNA involves NK cell-and T cell-mediated immunity. Mice with established B16 tumors were depleted of NK cell or CD4/CD8 lymphocytes prior to CpG-Stat3 siRNA treatment;
shown are means ± SEM, P < 0.0001 (from two-way ANOVA test). (c, d) Local treatment with CpG-Stat3 siRNA reduces growth of C4 melanoma (c) and CT26 colon carcinoma (d).
Tumor cells were injected (s.c.) into C3H or BALB/c mice, respectively. Mice with established tumors were treated by peritumoral injections of CpG-Stat3 siRNA, CpG-Luc siRNA, CpG alone or PBS every other day, starting 7d after challenge with 1×10 5 tumor cells. Statistically significant differences between the CpG-siRNA-treated groups are indicated by asterisks. (e) C57BL/6.CEA mice were challenged s.c. with 1×10 5 of MC38.CEA cells and treated as described above using CpG-Stat3 siRNA or CpG-Luc siRNA starting from d 11 after tumor challenge, P < 0.0001 by two-way ANOVA (n = 4 for each group). (f) Systemic treatment with CpG-Stat3 siRNA reduces Stat3 expression in DCs within TDLNs (cervical). Samples pooled from 6 mice/group were analyzed by real-time PCR. Shown is the average level of Stat3 expression in CpG-Stat3 siRNA-treated mice from one of two independent experiments analyzed in triplicates ± SEM, relative to control CpGscrambled RNA, which was set as 100%.  Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA BACKGROUND: Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates. METHODOLOGY/PRINCIPAL FINDINGS: Sequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice. CONCLUSIONS/SIGNIFICANCE: These data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus. Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks among livestock and humans in sub-Saharan African countries [1] . RVFV infection results in high mortality and abortion rates in domestic ruminants with severe hepatic diseases. It also causes an acute febrile myalgic syndrome, a hemorrhagic syndrome, ocular disease, and encephalitis in humans [2, 3] . Transmission of RVFV to humans is primarily mosquito-borne or due to direct contact with infected animal blood, tissues or products of abortion. Since the late 1970s, several major outbreaks of Rift Valley fever have occurred outside of sub-Saharan Africa, e.g., in Egypt [4] , Madagascar [5] , Saudi Arabia, and Yemen [2, 6] . The most recent outbreak was reported in Kenya and resulted in a high reported case-fatality ratio in infected humans [7] .
RVFV has a single-stranded, tripartite RNA genome composed of the L, M, and S segments. The L segment is of negative polarity encoding the RNA-dependent RNA polymerase (L). The anti-viralsense M segment encodes two envelope glycoproteins, Gn and Gc, and two accessory proteins,14-kDa NSm that suppresses virusinduced apoptosis [8] and the 78-kDa protein. The S segment uses an ambisense strategy for gene expression; a nonstructural protein, NSs, is translated from viral-sense mRNA, while N protein is expressed from anti-viral-sense mRNA [1] . N protein and L protein are essential for viral RNA synthesis [1] and NSs protein suppresses host innate immune functions by suppressing host gene expression [9] , including interferon-b [10] , and promoting PKR degradation [11, 12] . Experiments using reassortant viruses between an attenuated MP-12 strain of RVFV and wild-type (wt) RVFV suggested that RVFV virulence characteristics in the mouse are under polygenic control [13] . Further studies using reassortant viruses between the wt ZH548 strain of RVFV and an attenuated RVFV isolate clone 13, a plaque clone variant of the wt 74HB59 strain carrying a deletion of 69% of the NSs gene [14] , suggested that RVFV mouse virulence is controlled by the S segment [15] . The importance of NSs on viral virulence was confirmed by using a wt RVFV lacking the entire NSs gene [16, 17] . Studies using the wt RVFV lacking the NSm gene showed that NSm also affected virus virulence in rats [18] . In contrast, how Gn and Gc envelope proteins and L protein contribute to viral virulence is unknown.
The present study revealed that wt RVFV ZH501 stock, which was originally isolated from a patient during the 1977 outbreak of RVFV in Egypt, was made up of two major virus populations with single nucleotide substitution within the Gn gene and that a single nucleotide difference in the Gn gene of wt RVFV substantially affected viral mouse virulence. Furthermore, we observed a remarkably rapid emergence and accumulation of the virulenttype virus in the mice that had been inoculated with a low virulenttype RVFV.
Sequence analysis of the RT-PCR products of intracellular ZH501-specific RNAs showed sequence homogeneity in all three RNA segments, except for nucleotide 847 of the anti-viral sense M segment RNA (M847), which consisted of a mixture of A and G residues (Fig. 1) . Of 35 independent, cloned RT-PCR products of a region including M847, 18 clones and 17 clones had A and G, respectively, at M847, which suggested to us that the ZH501 virus stock consisted of roughly a 1-to-1 mixture of two major viral subpopulations, one carrying A and the other carrying G at M847. The virus carrying A residue at M847 encodes glutamic acid at amino acid 277 (Glu277) of the Gn envelope protein, while that carrying the G residue encodes glycine (Gly277). Sequence analysis of the cloned PCR products also showed other sequence heterogeneities, including silent mutations and those that altered amino acid sequences, within the M segment (data not shown), revealing a quasispecies nature of the ZH501 virus stock. None of these other nucleotide heterogeneities were shared among clones (data not shown).
To know the effects of the nucleotide difference at M847 on biological properties of the virus, we have recovered ZH501 carrying A at M847 (rZH501-M847-A) and that carrying G (rZH501-M847-G) at the corresponding site using a reverse genetics system of ZH501, in which endogenously expressed T7 polymerease in BHK/T7-9 cells drives expression of viral RNAs and proteins from the transfected plasmids. Sequence analysis of the entire L, M and S RNA segments of rZH501-M847-A and rZH501-M847-G revealed that both viruses had the expected primary sequences.
No substantial differences in the plaque sizes and morphologies were detected among ZH501, rZH501-M847-G and rZH501-M847-A in both Vero cells and MRC-5 cells (data not shown). ZH501, rZH501-M847-G and rZH501-M847-A showed similar replication kinetics in VeroE6 cells, mouse 3T3 cells and mouse macrophage-derived J774.1 cells (Fig. 2) , while titers of rZH501-M847-G were roughly 10 times higher than those of rZH501-M847-A in human lung fibroblast MRC-5 cells from 24 h to 72 h p.i. (Fig. 2) . Also there was a trend that CPE induced by rZH501-M847-A was less prominent CPE than that induced by rZH501-M847-G or ZH501 at a given time in all four cell lines (Fig. 2) . Unpaired-t-tests of three independent experiments examining the replication kinetics of rZH501-M847-G and rZH501-M847-A in MRC-5 cells from 0 h to 72 h p.i. revealed that the titer of rZH501-M847-G was a significantly higher (P,0.01) than that of rZH501-M847-A at 72 h p.i. (Fig. S1 ).
We examined the virulence of rZH501-M847-G, rZH501-M847-A and ZH501 by intraperitoneal (i.p.) inoculation of 10 0 , 10 1 , 10 2 , 10 3 , 10 4 or 10 5 plaque-forming units (PFU) of each virus into five 5-week-old female CD-1 mice. Hanks' balanced salt solution (HBSS) was inoculated into control mice. These mice were observed daily for 28 days p.i. (Fig. 3) . None of the HBSSinoculated mice died, while inoculation of 10 2 to 10 5 PFU of ZH501 resulted in the death of all of the mice within 13 days p.i., and one and three mice survived after inoculation of 10 1 and 10 0 PFU, respectively. All mice that were inoculated with 10 2 to 10 5 PFU of rZH501-M847-A died within 9 days p.i., and infection with 10 1 and 10 0 PFU resulted in the survival of one and four mice, respectively. To our surprise, none of the rZH501-M847-G infected mice died after inoculation of 10 0 to 10 2 PFU, four out of five mice survived after inoculation of 10 4 and 10 3 PFU, and one mouse survived after infection with 10 5 PFU. These data demonstrated that ZH501 and rZH501-M847-A were highly virulent to mice, whereas rZH501-M847-G had reduced mouse virulence.
To know the kinetics and titers of virus replication in various organs, mice were inoculated i.p. with 10 5 PFU of rZH501-M847-G, rZH501-M847-A, ZH501 and a mixture of 10 5 PFU of rZH501-M847-G and the same titer of rZH501-M847-A. Virus titers in livers, spleens, kidneys, sera and brains were determined from 1 to 7 days p.i.; two to sixteen mice were used for each time point (Fig. 4) . The mice infected with ZH501, rZH501-M847-A, and the mixture of rZH501-M847-G and rZH501-M847A exhibited rapid, efficient virus replications in the liver early in infection, high titers of viremia from 1 to 5 days p.i. and a gradual increase of efficient virus replication in the brain, where the virus titers exceeded 10 6 PFU/g at day 6 p.i. Efficient virus replication also occurred in the spleens and the kidneys, and all mice died by day 8 p.i. The mice infected with rZH501-M847-G also showed rapid virus replication in the liver, and yet the maximum liver virus titer was approximately 10 times lower than that in the mice infected with rZH501-M847-A, ZH501 and the mixture of rZH501-M847-G and rZH501-M847-A. Also rZH501-M847-G titers in the sera, kidneys, spleens and brains were substantially lower than the virus titers in the corresponding organs of mice that were infected with rZH501-M847-A, ZH501 and the mixture of rZH501-M847-G and rZH501-M847-A. Unpaired-t-test results showed that virus titers at days 1 to 3 in the serum and the liver, days 2 to 4 in the spleen, days 2 to 5 in the kidney and days 2 to 6 in the brain of rZH501-M847-G-infected mice were statistically lower (p,0.01) than the titers in the corresponding organs of rZH501-M847-A-infected mice.
We next examined serum-neutralizing antibody titers in the infected mice; ZH501 was used for the neutralizing antibody assay. For each virus group, 19 to 23 mice were used. As shown in Fig. 5 , all mice that were inoculated i.p with 10 5 PFU of rZH501-M847-A, ZH501 or a mixture of 10 5 PFU of rZH501-M847-G and the same titer of rZH501-M847-A had less than detectable levels of neutralizing antibodies until day 3 p.i. In contrast, rZH501-M847-G-infected mice showed low, but detectable levels of neutralizing antibodies early in infection; moreover, a low titer of neutralizing antibody was detected as early as 1 day p.i. in some of rZH501-M847-G-infected mice. Although neutralizing antibody titers increased in infected mice after day 5 p.i., there was a trend that rZH501-M847-G-infected mice had higher neutralizing antibody titers than did other infected mice. No neutralizing antibodies were detected in sham-infected mice (data not shown).
Cross-neutralization analysis of rZH501-M847-A, rZH501-M847-G and ZH501
We performed cross-neutralization assays to determine how efficiently antiserum from rZH501-M847-G-infected mice and that from rZH501-M847-A-infected mice could neutralize rZH501-M847-G, rZH501-M847-A, and ZH501. Anti-rZH501-M847-G serum demonstrating an 80% neutralizing antibody titer (PRNT 80 titer) of 1:640 to ZH501 and anti-rZH501-M847-A serum demonstrating a PRNT 80 titer of 1:80 to ZH501 were obtained from rZH501-M847-G-infected mice and rZH501-M847-A-infected mice, respectively. We used as controls, diluent, normal mouse serum and convalescent goat anti-ZH501 serum showing a PRNT 80 titer of 1:5,120 to ZH501. In this assay, virus titers were determined after overnight incubation of 100 ml of approximately 5.0 log 10 PFU/ml of rZH501-M847-G, rZH501-M847-A, and ZH501 with the same volume of undiluted antiserum or diluent at 4uC (Table 1) . Goat anti-ZH501 serum efficiently neutralized all viruses, while incubation of rZH501-M847-A and rZH501-M847-G with the normal mouse serum resulted in small reductions of the virus titers. Anti-rZH501-M847-G serum and anti-rZH501-M847-G serum neutralized the three viruses at different efficiencies. The differences between the virus titers (in log 10 PFU/ml) after incubation with each antiserum and those after incubation with normal serum are shown within parentheses in Table 1 . The anti-rZH501-M847-G serum reduced the titer of rZH501-M847-G by 1.63 log 10 PFU/ml, while it reduced the titer of rZH501-M847-A and ZH501 by 0.82 and 0.52 log 10 PFU/ml, respectively. The anti-rZH501-M847-A serum reduced the titers of rZH501-M847-A, rZH501-M847-G and ZH501 by 1.60, 0.7, and 1.0 log 10 PFU/ml, respectively. These data demonstrated that mouse antiserum neutralized the homologous virus to a similar extent, regardless of the PRNT 80 titers, that antiserum neutralized the heterologous virus equally but to a lesser degree than the homologous virus, and that anti-rZH501-M847-A serum was able to neutralize the titer of ZH501 to a slightly greater degree than anti-rZH501-M847-G serum; however, neither serum neutralized ZH501 equal to the neutralization of their homologous viruses.
To further understand the pathogenesis of rZH501-M847-G and rZH501-M847-A, we performed histopathological and IHC analyses of various organs of mice inoculated i.p. with 10 5 PFU of rZH501-M847-G, rZH501-M847-A, ZH501 or a mixture of 10 5 PFU of rZH501-M847-G and the same titer of rZH501-M847-A at various times p.i. (Figs. 6 and S2).
The livers of rZH501-M847-A-infected mice showed multifocal hepatocellular necrosis with prominent aggregating mononuclear inflammatory cells and sparsely scattered viral antigens throughout the liver lobe as early as day 2 p.i. On day 3 p.i., most of the hepatocytes were necrotic and positive for viral antigens (Fig. 6B) . Also, severe hemorrhages were noted throughout the lobe. After day 4 p.i., the livers from mice examined showed less severe hepatocellular damage than livers taken earlier, and the livers examined on day 6 p.i. showed near complete resolution. ZH501infected mice presented with similar lesions and distribution of viral antigens, and yet the progress of the lesions was somewhat slower than those in rZH501-M847-A-infected mice (Fig. 6A) . Although rZH501-M847-G-infected mice had focal-to-multifocal hepatocellular necrosis from days 2 to 4 p.i (Fig. 6C) , they exhibited mild hepatic lesions compared to ZH501-infected mice and rZH501-M847-A-infected mice. Among mice inoculated with the mixture of rZH501-M847-G and rZH501-M847-A, one showed severe hepatic lesions like those of rZH501-M847-Ainfected mice, while others had mild hepatic lesions like that of rZH501-M847-G-infected mice (data not shown).
The spleens of rZH501-M847-A-infected mice (Fig. 6E ), ZH501-infected mice (Fig. 6D ) and mice inoculated with the mixture of rZH501-M847-G and rZH501-M847-A (data not shown) were characterized by a depletion of lymphocytes in the white pulp from day 2 p.i. to day 5 or 6 p.i., with maximum All infected mice, except for rZH501-M847-G-infected mice, had moderate, non-suppurative encephalitis throughout their brains from day 5 p.i. (Fig. S2A ). Viral antigens were detected as early as day 2 p.i. in the endothelial cells of some mice. After day 5 p.i., viral antigens were detected in neurons at the thalamus, and spread to neurons of the surrounding areas (Fig. S2A, inset) .
The lesions were diagnosed as mild meningitis or mild encephalitis in the brains of rZH501-M847-G-infected mice (Fig. S2E) , and viral antigen was detected throughout the brains after day 7 p.i.
Kidneys of some of rZH501-M847-A-infected mice and the mice infected with the mixture of rZH501-M847-G and rZH501-M847-A showed pyknotic cells in glomeruli on day 3 p.i (Fig.  S2B ). Most of the infected mice had viral antigens in the smooth muscle of interlobular and arcuate arteries from day 2 p.i., and in the mesangium of glomeruli on days 3 and 4 p.i (Fig. S2C, D) . Although no pathology was detected in the kidneys of the ZH501- Five-week-old, female CD-1 mice were inoculated i.p. with 10 5 PFU of ZH501, rZH501-M847-A, rZH501-M847-G or a mixture of rZH501-M847-A and rZH501-M847-G. At 1 through 7 days p.i., serum, liver, spleen, kidney and brain were collected from infected mice, and virus titers were individually determined by plaque assay. Two to sixteen mice were used for each time point. In some cases, samples were collected from dead mice. They were: ZH501-infected mice-2 at day 3 p.i., 1 at 5 day p.i. and 1 at day 6 p.i.; rZH501-M847-G-infected mice-1 at day 7 p.i.; rZH501-M847-A-infected mice-2 at days 2 and 4 p.i., 5 at day 3 p.i., 3 at day 5 p.i., and 4 at day 6 p.i.; and rZH501-M847-G/rZH501-M847-A-co-infected mice-8 at day 3 p.i., 7 at day 6 p.i., and 2 at day 6 p.i. doi:10.1371/journal.pone.0009986.g004 infected mice, viral antigens were detected from day 3 in the mesangium of glomeruli and in the smooth muscle of interlobular and arcuate arteries from days 3 to 7 p.i. The kidneys of rZH501-M847-G-infected mice showed no pathology. Some of the infected mice exhibited viral antigens from days 5 to 7 day p.i. in the smooth muscle of interlobular and arcuate arteries (Fig. S2H) , whereas no viral antigens were detected in the glomeruli (Fig.  S2G ).
In summary, there were rough correlations between the presence of viral antigens and viral titers in the given organs, and rZH501-M847-G-infected mice had delayed development of lesions or showed less severe lesions than did other infected mouse groups.
We next examined whether the viruses that accumulated in infected mice retained the input-virus genome sequence at M847. To this end, we took advantage of the presence of a naturally occurring Sac I site (GAGCTC), which includes the A residue (underlined) at M847 of rZH501-M847-A. Due to single nucleotide substitution, the corresponding region of rZH501-M847-G lacked this restriction enzyme site. Mice were infected i.p. with 10 5 PFU of rZH501-M847-G or rZH501-M847-A (n = 9 for each group). Organs were isolated at various times p.i., and RT-PCR products that included M847 were subjected to Sac I digestion. For each virus group, we collected 3 livers at 4 day p.i., 3 spleens, 3 kidneys and 3 brains at day 6 p.i., and 3 brains at day 7 p.i. RT-PCR products of the expected size were obtained from all samples of rZH501-M847-A-infected mice, and all were fully susceptible to Sac I digestion (Fig. 7) , demonstrating that replicating rZH501-M847-A retained A residue at M847 in the infected mice. We were unable to obtain RT-PCR products from one spleen sample at day 6 p.i., one kidney sample at day 6 p.i., one brain sample at day 6 p.i., and one brain sample at day 7 p.i. from rZH501-M847-G-infected mice (data not shown), probably due to low virus titers in these samples. Two liver samples at day 4 p.i., were resistant to Sac I digestion (Fig. 7) . Unexpectedly, the RT-PCR products of one liver sample were a mixture of Sac Isusceptible PCR product and Sac I-resistant products (Fig. 7) and all other RT-PCR products, including two spleen samples at day 6 p.i., two brain samples at day 6 p.i., and two brains at day 7 p.i. (data not shown) were susceptible to the Sac I digestion (Fig. 7) . These data strongly suggested an accumulation of rZH501-M847-A-type virus carrying A residue at M847, in rZH501-M847-G infected mice at 4 days p.i. in livers and 6 days p.i. in spleens and brains.
To learn how quickly rZH501-M847-A-type virus accumulated in various organs of rZH501-M847-G-infected mice, we inoculated 25 mice i.p. with 10 5 PFU of rZH501-M847-G, and livers, spleens and brains were isolated every day through day 8 p.i. To eliminate the possible contamination by rZH501-M847-A in the samples, rZH501-M847-A was not used in the BSL-4 laboratory during the experiments. For each sample, the virus titers, production of RT-PCR products and susceptibilities of the RT-PCR products to Sac I digestion are summarized in Fig. 8A and the representative data concerning the Sac I digestion of the RT-PCR products are shown in Fig. 8B . Most samples carrying detectable levels of infectious viruses and many samples in which virus titers were below the detection limit yielded the RT-PCR products. Accumulation of rZH501-M847-A-type virus was detected in the liver of a single mouse as early as 2 days p.i. and rZH501-M847-A-type virus became the major virus population in livers, spleens and brains from day 5 to 8 p.i. We performed sequence analysis of some of the cloned RT-PCR products to firmly establish the accumulation of rZH501-M847-A-type virus in rZH501-M847-G-infected mice; the numbers of the clones used for sequence analysis and the samples were: 20 clones from mouse 8b brain, 10 clones from mouse 6c spleen, 10 clones from mouse 7c brain, and 10 clones from mouse 7c spleen (see Fig. 8A ). Consistent with the data of Sac I digestion of the RT-PCR products of these samples, all clones had A at M847. These data unambiguously demonstrated that rZH501-M847-Atype virus became the major virus population in rZH501-M847-G-infected mice. To know whether the phenomenon of emergence of a major population of rZH501-M847-A-type virus after infection with rZH501-M847-G-type virus also occurred in cell culture, rZH501-M847-G was independently passaged five times at a multiplicity of infection (moi) of 0.01 and 1 in both VeroE6 cells and MRC-5 cells. RT-PCR products, including M847, were generated from intracellular RNAs, extracted from cells infected with viruses at passage levels 1 and 5, and subjected to Sac I digestion. We did not observe the accumulation of rZH501-M847A-type virus after 5 passages of rZH501-M847-G in both cell types (data not shown), demonstrating that the rZH501-M847-A-type virus did not emerge after passages of rZH501-M847-G in cultured cells but it became the major virus population in mice that were infected with rZH501-M847-G.
Finally, we examined mouse virulence of rZH501-M847-Atype virus that had accumulated in rZH501-M847-G-infected mice by i.p. inoculation of 10 1 , 10 2 , 10 3 , 10 4 or 10 5 PFU of the brain homogenates of 8b mouse (see Fig. 8A ) into mice (n = 5 for each dilution); RT-PCR analysis of mouse 8b brain extracts revealed that rZH501-M847-A-type virus was the major virus population, and sequence analysis of all 20 cloned RT-PCR products of this brain extract showed that M847 was A. Inoculation of 10 4 or 10 5 PFU of the recovered virus resulted in the death of all mice within 7 days p.i., while 2 mice, 3 mice and 4 mice survived after inoculation of 10 3 PFU, 10 2 PFU and 10 1 PFU of the virus, respectively (Fig. 9 ). The recovered viruses had a higher mouse virulence than did rZH501-M847-G, while they were not as virulent as rZH501-M847-A or ZH501 (Fig. 3) .
A past report using reassortant viruses between an attenuated MP-12 strain and wt RVFV suggested that RVFV virulence in mouse is under polygenic control [13] . Past studies revealed that the NSs gene, encoded in the S segment, and the NSm gene, encoded in the M segment, both contribute to viral virulence [16, 17, 18] . We presented here our findings that a single nucleotide difference in M847 of the Gn gene of ZH501 substantially affected virulence in mice, a result that further supports the notion that RVFV virulence is under polygenic control [13] .
Roughly a 1-to-1 mixture of the rZH501-M847-G-type virus and rZH501-M847-A-type virus existed in the ZH501 virus stock. Because the original ZH501 sample from a patient in the 1977 RVFV outbreak was not available, it is unclear whether high titers of both rZH501-M847-G-type virus and rZH501-M847-A-type virus replicated in the patient. We found that the rZH501-M847-A-type virus became a major viral population in mice inoculated with rZH501-M847-G, while viruses accumulated in mice inoculated with rZH501-M847-A were the rZH501-M847-A-type virus. Additionally, 38 of 39 isolates from wt RVFV thus far reported were found to have A at M847 and carry Glu277 [19] , which is also encoded by rZH501-M847-A. These data suggest that the rZH501-M847-A-type virus probably represented the major virus population in the patient. However, it is possible that rZH501-M847-G-type virus existed as an RVFV quasispecies in the patient and affected the severity of the disease; quasispecies have cooperative interactions to control viral pathogenesis [20] . Consistent with this notion, we found a lesser virulence in a virus sample consisting of a major population of rZH501-M847-A-type virus and a minor population of rZH501-M847-G (Fig. 8A ) and obtained from a rZH501-M847-G-infected mouse brain at 8 days p.i., than did rZH501-M847-A or ZH501 (Figs. 3 and 9) . Although the accumulation of rZH501-M847A-type virus did not occur even after 5 passages of rZH501-M847-G in VeroE6 cells and MRC-5 cells, we observed that rZH501-M847-G replicated to higher titers than rZH501-M847-A in cultured cells (Figs. 2 and S1). This trend in virus replication in vitro pointed to the possibility that the rZH501-M847-G-type virus that was present as a minor virus population in the patient could have amplified in cell culture to become a major viral subpopulation in the ZH501 virus stock that was generated in vitro.
The kinetics of virus accumulation in various organs in mice inoculated with ZH501, rZH501-M847-A or rZH501-M847-G showed that RVFV replicates in the liver early in infection. The efficient replication of rZH501-M847-A and ZH501 in the liver and in other initial target organs probably induced high viremia titers that facilitated the spread of the virus to other organs, including the spleen, kidneys and brain ultimately leading to death of the animals. The virus titers in the liver of rZH501-M847-G-infected mice at 2 days p.i. were roughly ten times lower than those in the liver of rZH501-M847-A-infected mice (Fig. 4) . Likewise, virus titers in other organs and serum of the rZH501-M847-G-infected mice were substantially lower than those of the corresponding organs of rZH501-M847-A-infected mice. The present data imply that the level of virus titers in the initial target organ, e.g., the liver, at 2 to 3 days p.i. may determine the severity and ultimate outcome of RVFV infection in mice.
Although we have demonstrated the importance of single nucleotide substitution at M847 of Gn protein for wt RVFV mouse virulence, further studies are required to determine how this single nucleotide substitution affected RVFV's virulence. Past studies showed that the gain-of-positive-charge mutations in the flavivirus E protein [21, 22, 23] and the alphavirus E2 protein [24] can enhance binding to negatively charged glycosaminoglycans (GAG) and increase the efficiency of virus uptake into cultured cells. However, the virulence of the variant viruses in mice is reduced, as these variant viruses are rapidly cleared from the circulatory system [22, 24, 25, 26] . Probably, the gain-of-positivecharge mutations in the envelope protein facilitates viral adherence to GAGs present in blood cells and subsequent reticuloendothelial system-mediated virus elimination from the circulation. Likewise, rZH501-M847-A, which had a negatively charged Glu277 of the Gn envelope protein, was highly virulent for mice, while rZH501-M847-G carrying an uncharged Gly277 was substantially less virulent (Fig. 3) . Furthermore, rZH501-M847-A-infected mice showed higher viremia titers than rZH501-M847-G-infected mice at 1-3 days p.i. (Fig. 4) , but rZH501-M847-G replicated at a higher titer than did rZH501-M847-A in MRC-5 cells (Fig. 2) . Accordingly, it is possible that rZH501-M847-G binds efficiently to GAGs in the circulation, resulting in low levels of systemic infection, whereas rZH501-M847-A carrying Glu277 binds to GAGs less efficiently, resulting in high titers of viremia, efficiently establishes a systemic infection that involves the CNS, and kills the infected mice. The attenuated virus, MP-12, developed by serial passage of the wt RVFV ZH548 strain in the presence of 5-fluorouracil [27] , carries A at M847 and encodes Glu277, suggesting that the presence of Glu277 in Gn does not always make RVFV virulent. The amino acid sequence of the M segment of MP-12 differed from that of rZH501-M847-A by 5 amino acids. We suspect that a combination of some or all of these mutated amino acids in the M segment contributed to the attenuation of MP-12.
The present study showed the importance of the A nucleotide at M847 of wt RVFV in mouse virulence, whereas it is unclear whether this nucleotide also affects viral virulence in other animals. Like rZH501-M847-G, one wt RVFV isolate 763/70, which was obtained from an aborted bovine fetus in Zimbabwe in 1970 [28] , has G at M847 and carries Gly 277 [19] , while all the rest of the wt RVFV isolates thus far sequenced carry A at M847 and encode Glu277 [19] . No information is available as to the titers of 763/70 in different tissues of the aborted fetus and the viremia status of its mother. Although we reported that pregnant cows inoculated with ZH501 become febrile, have 2.3 to 4.0 log 10 PFU/ml of viremia, and undergo abortion [29] , the relationship between level of viremia and abortion in cattle has not been carefully examined yet. We observed that very low viremia can result in abortion in experimentally infected sheep with ZH501 (J. Morrill, unpublished data), and, hence, we speculate that placental tissue in sheep and probably cattle may be exquisitely sensitive to infection, and low titers of viremia can infect the placenta at very low levels and gain access to the fetus. Virulence of 763/70 in the bovine has not been experimentally tested and 763/70 differs from ZH501 at other genome regions. Accordingly, the role of G at M847 of 763/70 in virus virulence for bovine is unclear. Experimental infection of rZH501-M847-A and rZH501-M847-G into domestic animals will show whether the importance of the A nucleotide at M847 in viral virulence is limited to the mouse model. Figure 7 . Sac I digestion of the RT-PCR products. Five-week-old female CD-1 mice were infected i.p. with 10 5 PFU of rZH501-M847-G or rZH501-M847-A. Total RNAs from 3 livers at 4 day p.i., 3 spleens at day 6 p.i., 3 kidneys at day 6 p.i., 3 brains at day 6 p.i., and 3 brains at day 7 p.i. were collected and subjected to RT-PCR analysis. In the samples where RT-PCR products were obtained, RT-PCR products were digested with Sac I and subjected to agarose gel electrophoresis. As controls, PCR products that were obtained from plasmid encoding M segment of rZH501-M847-A were digested with Sac I or incubated in the absence of Sac I (Control). doi:10.1371/journal.pone.0009986.g007 Figure 8 . Accumulation of viruses carrying the genotype of rZH501-M847-A in rZH501-M847-G-infected mice. Five-week-old female CD-1 mice were infected i.p. with 10 5 PFU of rZH501-M847-G and liver, spleen and brain were collected every day from day 1 to day 8 p.i. Virus titer in each sample was determined by a plaque assay. RT-PCR products were obtained from total RNAs in these samples and then subjected to Sac I digestion. (A) Color and number in each box represent the genotype of major viruses and the virus titer in Log 10 PFU/g of tissue, respectively, in each mouse. Color legend: G = genotype of rZH501-M847-G. A = genotype of rZH501-M847-A. G = A = a mixture of similar amounts of rZH501-M847-G genotype and rZH501-M847-A genotype. G.A = viruses carrying the rZH501-M847-G genotype were more abundant than those carrying the rZH501-M847-A genotype. A.G = viruses carrying the rZH501-M847-A genotype were more abundant than those carrying the rZH501-M847-G genotype. Not decided = the major genotype of viruses was not determined due to weak RT-PCR signals. Not detected = RT-PCR products were not obtained. (B) Agarose gel electrophoresis of the RT-PCR products that underwent SacI digestion. Only representative samples are shown. The mouse identification number in the gel is shown in (A). Control = PCR products that were obtained from plasmid encoding M segment of rZH501-M847-A were digested with Sac I or incubated in the absence of Sac I. N = Not detected. W = Not decided. doi:10.1371/journal.pone.0009986.g008
A remarkably rapid emergence and accumulation of rZH501-M847-A-type virus occurred in the mice inoculated with rZH501-M847-G (Figs. 7 and 8 ). In the past, an experimental infection using swine influenza virus resulted in a similarly rapid emergence of a variant virus in infected hosts during single infections [30] . There are other examples of this type of quasispecies emergence and fixation in vivo [31, 32, 33] . Although rZH501-M847-G was rescued from cDNAs and direct sequencing of the M segment of rZH501-M847-G did not show the presence of rZH501-M847-Atype in the inoculum (data not shown), rZH501-M847-A-type viruses might have been generated during T7 polymerasemediated primary transcription from the plasmid or during M segment RNA replication and existed as one of a minor virus population of ZH501-M847-G quasispecies in the inoculum. Alternatively, rZH501-M847-A-type virus was generated by a point mutation at M847 of the M segment in the rZH501-M847-G-infected mice and became a major virus population as early as 2 days p.i. As discussed above, we speculate that rZH501-M847-A may bind to GAGs less efficiently than does rZH501-M847-G. This putative phenotype of rZH501-M847-A-type virus might have prevented the efficient elimination of the virus from reticuloendothelial systems, and facilitated continuous replication and systemic infection.
We noted that neutralizing antibodies were rapidly elicited in rZH501-M847-G-infected mice within 3 days p.i. (Fig. 5) , during which time most of the viruses retained the genotype of rZH501-M847-G (Fig. 8) . Because the importance of neutralizing antibodies for the protection of animals from RVFV infection is well documented [34, 35, 36, 37, 38, 39, 40, 41, 42] , a rapid and efficient generation of neutralizing antibodies probably contributed to low titers of viremia and prevention of systemic infection in rZH501-M847-G-infected mice. It is interesting to note that the antiserum neutralized the heterologous virus equally but to a lesser degree than the homologous virus in the cross neutralization assay (Table 1) . These results were consistent with a report that the region including Glu277 or Gly277 of Gn protein is a major neutralizing epitope site [43] . It is likely that in the rZH501-M847-G-infected mice, rZH501-M847-A-type virus emerged partly because this virus was less susceptible to rapidly generated neutralizing antibodies directed against rZH501-M847-G. We further suspect that in the rZH501-M847-G-infected mice, newly emerged rZH501-M847-A-type virus failed to continuously replicate efficiently after 5 days p.i. in various organs primarily due to an increase in the titers of neutralizing antibodies, resulting in the survival of the mice. The virus titers in rZH501-M847-Ainoculated mice were higher than those inoculated with rZH501-M847-G in many organs during the first 4-5 days p.i., while the neutralizing antibody titers tend to be higher in rZH501-M847-Ginoculated mice than in rZH501-M847-A-inoculated mice (Fig. 5) . These data suggest that rZH501-M847-A replication might have induced immunosuppression. This possibility was consistent with the data that lymphocytes were depleted in the white pulp of mice inoculated with rZH501-M847-A, but not those inoculated with rZH501-M847-G, a finding probably due to apoptosis (Fig. 6 ).
The ZH501 strain of wt RVFV was obtained from the Special Pathogens Branch at the Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia). RVFV ZH501 was originally isolated from a patient during the 1977 outbreak of RVFV in Egypt by inoculating the human specimen into suckling mice brain. After amplification of the virus in the suckling mice brain one more time, the ZH501 virus stock was generated after two passages in FRhL cells and two passages in VeroE6 cells. The Vero cell line and MRC-5 cell line were used for a viral plaque assay as described previously [44] . All of the experiments that used virulent ZH501 were performed in The Robert E. Shope, MD BSL-4 Laboratory in the John Sealy Pavilion for Infectious Diseases Research at The University of Texas Medical Branch at Galveston, Texas.
Standard molecular biological techniques were used to construct plasmids. Total RNA of VeroE6 cells infected with RVFV ZH501, which was obtained from Stuart Nichol at the CDC, was used for first-strand cDNA synthesis. ZH501 virus stock at CDC [16] and our ZH501 stock underwent the same passage history. PCR fragments of full-length, antiviral-sense ZH501 S and L-segments were cloned into a pProT7 plasmid [45] , designated as pProT7-wS(+) and pProT7-wL(+), respectively. The ZH501 Msegment carrying G and A at nucleotide 847 of anti-viral-sense was cloned into a pProT7 plasmid, designated as pProT7-wM(+)-M847-G and pProT7-wM(+)-M847-A, respectively. The plasmids expressing ZH501 N or L proteins under the control of T7 RNA polymerase were reported previously [46] . The ORF of ZH501 glycoproteins, which carry G at nucleotide 847 of anti-viral-sense M were cloned into the pCAGGS plasmid under chicken b-actin promoter, designated as pCAGGS-G. All of the constructs were confirmed to have the expected sequences.
Virus rescue rZH501-M847-G, a recombinant ZH501 carrying G at nucleotide 847 of anti-viral-sense M, was recovered by cotransfection of pProT7-wS(+), pProT7-wM(+)-M847-G, pProT7-wL(+), pT7-IRES-N, pT7-IRES-L and pCAGGS-G into BHK/ T7-9 cells by TransIT-LT1 (Mirus, Madison, WI), as described previously [45] . rZH501-M847-A was recovered by co-transfec- Figure 9 . The virulence of rZH501-M847-A-type virus obtained from the brain of rZH501-M847-G mouse. Five 5-week-old female CD-1 mice were inoculated i.p. with 10 1 , 10 2 , 10 3 , 10 4 or 10 5 PFU of the brain homogenates from the 8b mouse (Fig. 8A ). Control mice were inoculated with HBSS. Mortality was recorded daily for 28 days p.i. No mice died after 9 days p.i. doi:10.1371/journal.pone.0009986.g009 tion of pProT7-wS(+), pProT7-wM(+)-M847-A, pProT7-wL(+), pT7-IRES-N, and pT7-IRES-L into BHK/T7-9 cells. The culture medium was replaced with fresh medium at 24 h post transfection. At 5 days post transfection, the culture supernatants were collected, clarified and then inoculated into VeroE6 cells. The supernatant of infected VeroE6 cells at 3 days p.i. was collected and used for titration of virus infectivity by plaque assay and for the subsequent experiments. Sequence analysis of M segment RNA confirmed the presence of the expected nucleotide at M847 in rZH501-M847-G and rZH501-M847-A.
Intracellular viral RNAs of infected MRC-5 cells served as the source of sequence analysis. Most of the sequences of the L, M and S RNA segments of rescued rZH501-M847-A and rZH501-M847-A were determined by direct sequencing analysis of the virus-specific RT-PCR products. For sequencing of the 59-end of viral RNAs, free 59-phosphates in intracellular RNAs were removed by calf intestinal alkaline phosphatase and then the samples were treated with tobacco acid pyrophosphatase to remove the cap structure. After ligation of the RNA adaptor to uncapped RNA by using T4 RNA ligase, cDNA was synthesized by employing an RLM-RACE kit (Ambion, Austin, TX) using random primers. Amplicons were obtained by using a virusspecific and an adaptor-specific primer and subjected to sequencing analysis. For sequencing of the 39-end of viral RNAs, a poly A tail was added to viral RNAs by using a Poly(A) Polymerase Tailing Kit (Epicenter biotechnology, Madison, WI). First-strand cDNA was synthesized from RNA containing a poly A tail by using the 39Race adaptor (RLM-RACE kit, Ambion). The cDNA was subjected to PCR with an adaptor-specific primer and a virus-specific primer, and the amplicons were subjected to sequence analysis.
VeroE6 cells, mouse 3T3 cells, mouse macrophage-derived J774.1 cells and MRC-5 cells were infected with viruses at an moi of 0.02 at 37uC for 1 h, and washed 3 times with PBS. Then medium was added. Culture supernatants were harvested at various times p.i., and the virus titer was measured by plaque assay.
Virus titers were determined by plaque assay in Vero cell monolayers grown in 24-well plates. For the viral plaque assay, serial tenfold dilutions of each specimen were prepared in HBSS with 2% fetal bovine serum added (HBSS-FBS), and 50 ml of each dilution was adsorbed on duplicate cell monolayers for 1 hour at 37uC. After 1 hour, the monolayers were overlaid with 0.6 ml of a mixture of 1 part 1% agarose and 1 part 2X Eagle's basal medium with Earle's salts, 17 mm HEPES, 8% fetal bovine serum, 100 U of penicillin/ml and 100 mg of streptomycin sulfate/ml. After incubation for 72 h at 37uC in a 5% CO 2 atmosphere, each cell monolayer was stained by adding 0.6 ml of a second overlay, identical to the first, but containing 4% neutral red. After an additional 24 h incubation, plaques were enumerated and viral titers were calculated.
Five-week-old female outbred CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). The mice were housed, 5 mice per cage, in micro-isolator cages, in a BSL-4 biological containment laboratory with a 12-h day-night photoperiod. Virus inocula were prepared in HBSS, pH 7.4, with no supplements. Each mouse was inoculated i.p. with 0.1 ml of inoculum in a tuberculin syringe fitted with a 26-gauge, 3/8-inch needle. Moribund mice or mice pre-selected for euthanasia were anesthetized with isoflurane, whole blood was collected via cardiac puncture, and the mice were euthanatized by cervical dislocation. Liver, spleen, and brain tissues were aseptically collected immediately following euthanasia or as soon as dead mice were discovered. All experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of the University of Texas Medical Branch and the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996). The facilities used are fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Tissues were prepared as 10% (w/v) homogenates in HBSS supplemented with 10% heat-inactivated (56uC for 30 minutes) fetal bovine serum and antibiotics (200 U/ml penicillin and 50 mg/ml streptomycin). Serum for virus and neutralizing antibody assays was separated from whole blood by centrifugation at 1,500 x g for 15 minutes and stored at -80uC until assayed. Tissue homogenates were similarly clarified by centrifugation, and supernatants were harvested and stored at 280uC until assayed.
Serum neutralizing antibody was determined using an 80% plaque-reduction neutralization test. Serial fourfold dilutions of serum were prepared in HBSS-FBS. An equal volume of the ZH501 strain of RVFV suspension containing approximately 80 PFU/50 ml was added to each dilution. After incubation at 37uC for 1 hr, 50 ml of each dilution was adsorbed on duplicate Vero cell monolayers for 1 hr at 37uC and then overlaid with 0.6 ml of the agarose-medium mixture used in the viral plaque assay. After 72 hr incubation at 37uC in a 5% CO 2 atmosphere, each monolayer received 0.6 ml of a second agarose containing neutral red dye. Plaques were counted and an 80% reduction in the number of plaques inoculated was used as the endpoint for virusneutralization titers.
Anti-rZH501-M847-G serum demonstrating a PRNT 80 titer of 1:640 to ZH501 was a mixture of the sera, each collected at days 6, 7 and 8 p.i. from eight rZH501-M847-G-infected mice. Similarly, anti-rZH501-M847-A serum demonstrating a PRNT 80 titer of 1:80 to ZH501 was a mixture of the sera collected at 6 days p.i. from four rZH501-M847-A-infected mice. Diluent (HBSS-FBS) and normal mouse serum served as negative controls, while convalescent goat anti-ZH501 serum showing a PRNT 80 titer of 1:5,120 to ZH501 served as a positive control. We incubated at 4uC overnight vials containing 100 ml of approximately 5.0 log 10 PFU/ml of rZH501-M847-G, rZH501-M847-A or ZH501 combined with 100 ml of each serum sample or diluents. After incubation, virus titers were determined by using viral plaque assays.
One hundred microliters of 10% tissue homogenate were mixed with 900 ml of TRIzol reagent (Invitrogen, Carlsbad, CA). After addition of 200 ml of chloroform, tubes were shaken vigorously by hand and centrifuged at 15,000 rpm for 10 min at 4uC. Following centrifugation, the aqueous phase was transferred to a new tube and 500 ml of isopropanol was added to the tubes. Samples were centrifuged at 15,000 rpm for 25 min at 4uC. RNA pellets were washed with 75% ethanol and dried. Thirty microliters of RNasefree water was added to dissolve the RNA pellet. The samples were then treated with RQ1 RNase-Free DNase (Promega, Madison, WI), and the RNAs were purified by addition with phenol-chloroform.
The total RNA of infected VeroE6 cells or mouse liver, spleen, kidneys and brain were extracted with Trizol reagent (Invitrogen). First-stranded cDNA was synthesized with a random hexamer by RTG YouPrime RXN Beads (GE Healthcare, Bucks, UK) according to the manufacturer's instructions. PCR primers which anneal to nucleotide 411 to nucleotide 430 (M430F: 59-ATG GCA GGG ATT GCA ATG AC-39) or nt.1041 to 1060 (M1041R: 59-ACT GCA AAG GGC ACA ACC TC-39) of anti-viral-sense M were used for PCR reaction. PCR was performed for 30 cycles at 94uC for 40 sec, 55uC for 1 min, and 72uC for 1 min using the Expand High Fidelity PCR System (Roche, Mannheim, Germany). The PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Germantown, MD), digested with Sac I and then separated on a 1% agarose gel.
The PCR product consisting of a wild-type ZH501 M-segment by M430F and M1041R was directly sequenced, or cloned into pSTBlue-1 by AccepTor Vector Kits (Novagen, Darmstadt, Germany) according to the manufacturer's instruction. Thirtyfive clones were sequenced by T7 primer.
Specimens for histopathologic examination were collected in 10% neutral buffered formalin. The livers, spleens, kidneys, and brains obtained from infected mice and control animals were processed for histopathological and IHC examination as previously described [47] . Formalin-fixed and paraffin-embedded tissue sections were subjected to hematoxylin and eosin (H&E) by standard methods for evaluating histopathology and IHC staining for detecting RVFV antigens, respectively. For detecting RVFV antigens, the tissues were incubated with rabbit anti-N antibody [48] (1:500). Color was developed by using the fuchsin+ substratechromogen system (DAKO cytomation, Carpentaria, CA). Figure S1 Growth curve of rZH501-847-A and rZH501-847-A in MRC-5 cells. MRC-5 cells were inoculated with rZH501-847-A or rZH501-847-A at an moi of 0.02. Culture fluids were collected and virus titers were determined by a plaque assay that used VeroE6 cells. The results were obtained from three independent experiments.  Confronting Potential Influenza A (H5N1) Pandemic with Better Vaccines Influenza A (H5N1) viruses are strong candidates for causing the next influenza pandemic if they acquire the ability for efficient human-to-human transmission. A major public health goal is to make efficacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. Important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human H5 strains. This strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. The concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype H5N1–infected persons is underscored. Better understanding of host–virus interaction is essential to identify tools to produce effective vaccines against influenza (H5N1). Infl uenza A (H5N1) viruses are strong candidates for causing the next infl uenza pandemic if they acquire the ability for effi cient human-to-human transmission. A major public health goal is to make effi cacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. Important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human H5 strains. This strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. The concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype H5N1-infected persons is underscored. Better understanding of host-virus interaction is essential to identify tools to produce effective vaccines against infl uenza (H5N1).
has spread with alarming speed across Europe, Africa, and parts of Asia in which the infection was not reported earlier. Establishment of the highly pathogenic avian infl uenza (H5N1) as an endemic virus within duck and poultry populations and its capacity to cross species barriers increase the possibility of adaptation to humans and a pandemic. Human infl uenza infections with subtype H5N1 viruses are often fatal. As of June 4, 2007 , 309 laboratoryconfi rmed cases of human infection have been reported to the World Health Organization (WHO); 61% were fatal, mainly in persons 10-39 years of age (www.who.int/csr/ caculatordisease/avian_infl uenza/en). If a pandemic is triggered by transmissibility of infl uenza (H5N1) from person to person, millions of people could die, and economies would likely be crippled for 6-24 months.
In the event of a pandemic, vaccination against infl uenza (H5N1) could limit the impact of infection at a public health level. However, no evidence exists that available vaccines would be protective against the pandemic strain of the virus. We comment on some of the limitations of currently available vaccines and propose novel strategies to improve vaccine formulations against infl uenza (H5N1).
The host response to infl uenza (H5N1) infection has not been defi ned, which has proven a considerable challenge in epidemiology and public health research. To develop effi cient vaccines, understanding how the virus interacts with the host in natural infection is necessary. Having insights into the hosts' responses to infl uenza (H5N1) would help defi ne targets for therapeutic intervention. Whether humans can develop immunity during a primary infection that would control replication and spread of subtype H5N1 viruses has been questioned (1). However, marked infl ammatory responses develop after infection with infl uenza (H5N1) in humans and other animals (2) (3) (4) . This condition is associated with statistically signifi cant synthesis of various proinfl ammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, interferon (IFN)-γ, IL-1α, and chemokines, including IP-10, MIG, monocyte chemoattractant protein-1 (MCP-1, IL-8, and RANTES. i.e., regulated on activation, normal T-cell expressed and secreted). If this is the case, these observations are consistent with the possible induction of innate immune responsiveness in the persons infected with infl uenza (H5N1 (5) . The death rate of ≈25% was half that of previously known outbreaks, and 5 mild or completely asymptomatic cases have been reported. One theory holds that milder cases have been occurring elsewhere but are not being recorded. Recently, 3 persons among 120 apparently healthy volunteers from the People's Republic of China, showed detectable virus-neutralizing antibody response to subtype H5N1 before vaccination (6) . Moreover, pigs infected with subtype H5N1 have become asymptomatic in Indonesia. Are these signs of development of some degree of immunity to virus, containing its replication and thus causing milder infection in naturally infected mammals? Recently, clusters of bird fl u cases were reported in Western Java, Indonesia (7); fatal disease developed in 6 persons there from the same family. Two other family members became ill but survived. All the family members likely had similar levels of exposure because they all lived in the same household. Other cases of nonfatal infections have been seen in Thailand and Vietnam. Unfortunately, there is little information about the immune response to the virus in those who survived, which would be valuable for understanding the mechanisms of protection. Indeed, following up the persons (cohort) living in the same affected villages, presumably mostly not exposed to virus, should clarify whether the maintained response refl ects boosting through natural exposure. Persons with prior exposure, as measured by antibody or viral RNA at recruitment, would likely have substantially higher responses to the vaccine than those naïve at recruitment if the vaccinating antigen contains homologous or cross-reacting determinants. Conceivably, boosting the "natural" immunity is a desirable outcome to improve protective effi cacy of any vaccine approach. Additional studies are required to evaluate the merits of priming populations in advance of an infl uenza (H5N1) pandemic.
After initial hesitation about using a wide-scale program of poultry vaccination, some European and Asian countries have begun vaccination. Inactivated vaccines are widely used in poultry but lack of critical potency testing, standardization, and quality control has led to variable and suboptimal immune responses. Moreover, a legitimate concern remains that the fowl vaccinated by attenuated live viruses may survive the disease but still carry the virus; thus, they would continue to spread infl uenza (H5N1) silently at the fl ock level (8) or to humans who come into contact with them. Vaccination that resulted in low levels of seroconversion facilitated the emergence of the Fujian-like sublineage of infl uenza (H5N1) in poultry (9) .
The immune responses elicited by subpotent vaccines may exert selection pressure that favors antigenic drift and shift ( Figure) . Antigenic drift relies on the accumulation of mutations within the antibody-binding sites in the hemagglutinin (HA), neuraminidase (NA), or both that abrogate the binding of antibodies. This makes infl uenza A virus strains able to evade neutralizing antibody from prior infection or vaccination. Antigenic shift, which is seen only with infl uenza A viruses, is a more drastic change. It results from genetic shift by reassortment exchange of the HA, and sometimes the NA, with novel subtypes that have not been present in human viruses for a long time. Antigenic shift leads to replacement of circulating strains with new variants that are able to reinfect hosts immune to earlier types; the result is usually a pandemic. Antigenic shifts caused 2 of the major infl uenza A pandemics in the last century, including the 1957 subtype H2N2 and 1968 subtype H3N2 outbreaks (10) .
Most infl uenza vaccines used in the United States and Europe are produced in embryonated hens' eggs and are formaldehyde-inactivated preparations (11) . Because highly pathogenic infl uenza (H5N1) subtypes may kill embryonated eggs, use of viruses that are no longer pathogenic, such as H5 (which lacks the polybasic cleavage site), to reduce the virulence of infl uenza (H5N1) vaccine strains so that these can be effi ciently propagated in eggs for vaccine production is feasible (10) . Virus particles that lack the gene for the nuclear export protein or are defective for the matrix (M2) gene were used as live vaccines in animal models (12, 13) ; however, whether these replication-defective vaccines will work in humans is not known. Live attenuated (cold-adapted) infl uenza vaccines have long been used in Russia, and a similar product has been approved for use in the United States (14) . These vaccines will replicate in the host, and thus lower doses may be effective; however, the preexisting antibody to the virus is more likely to diminish the value of a live vaccine. Moreover, such live vaccines are reported to cause asthma-like reactivity in children (15) . Monitoring live infl uenza vaccines is important because the risk for reversion to pathogenicity remains.
With the use of a technique known as reverse genetics, a prototype of infl uenza virus (H5N1) has been produced for the development of an inactivated subvirion vaccine. The gene segments encoding HA and NA were derived from A/Vietnam/2004, and all other genes were derived from the backbone (A/PR/8/34) virus, commonly used as a platform for infl uenza vaccine production. The HA gene was further modifi ed to replace the stretch of 6 basic amino acids at the cleavage site, and the resulting virus was avirulent in chickens. In a recent trial, healthy adult volunteers were given 2 intramuscular doses of this inactivated infl uenza (H5N1) vaccine. This split vaccine induced an antibody response predictive of protection in 54% of healthy adults tested, but only when given intramuscularly at high doses (two 90-μg shots) (16) . The large amounts needed (2 doses of vaccine, each 6 times the dosage of that used in a standard infl uenza shot) means that hundreds of millions of doses are needed to tackle a pandemic. Dose-sparing approaches, including the use of an effi cient nontoxic adjuvant to boost persons' immune responses, may improve the vaccine. Another trial was performed with 300 healthy participants 18-40 years of age, in which aluminum hydroxide adjuvant was used with similar split-virus vaccine (17) . However, the alumadjuvanted vaccines did not improve the immunogenicity or percentage of seroconversion at lower vaccine doses and only slightly improved immunogenicity at the 30-μg dose. This diffi culty underscores the importance of vigorous fundamental research to address the question of how to increase the immunogenicity of such vaccines, whether by better antigen presentation or by choosing alternative routes of administration, so that lesser amount of antigen could be given to induce protective response. The present annual global production capacity is ≈300 million doses of trivalent vaccine containing 15 μg HA per strain. This is equivalent to 900 million doses of monovalent vaccine, a quantity markedly insuffi cient for the world's 6.5 billion people. Clearly, dose-sparing formulations are urgently needed.
To test the hypothesis that whole-virion would be more immunogenic than conventional split-virion or subunit vaccines and may be adaptable to the antigen-sparing strategy, an inactivated, monovalent infl uenza A (H5N1), whole-virion vaccine was prepared from a highly virulent strain A/Vietnam/1194/2004 strain by removing the polybasic amino acids at the cleavage site, making the virus no longer pathogenic. The seed virus was grown to a high titer in embryonated eggs, inactivated with formalin, and purifi ed. These viruses were then adjuvanted with aluminum hydroxide and used in a phase 1 trial (6) . The highest immune response of 78% seropositivity was observed in the group given 2× 10 μg HA, which is equivalent to that elicited by higher doses of nonadjuvanted (90 μg) or adjuvanted (30 μg) split-virion vaccines (16, 17) .
Not knowing which particular genetic variant will sustain human-to-human transmission makes our ability to formulate a vaccine in advance all the more diffi cult. An inactivated vaccine that induces not only high levels of neutralizing antibody to surface proteins but also CD8 Tcell response against well-conserved antigens derived from internal viral proteins might provide superior protection in an epidemic or pandemic. In cases of established intracellular infl uenza A infection, infected cells are mainly eliminated by effector CD8+ T cells (CTLs) (18) . Any vaccine that will induce and direct these CTLs to the site of infection and generate a long-lasting memory response will be more effective for mounting protection against a pandemic form of infl uenza (H5N1). Inactivated vaccines need to be presented to the host's immune system with an appropriate adjuvant, but inactivated vaccines that use an adjuvant currently approved for human use (alum or MF-59) usually have lower immunogenicity than live attenuated vaccines (10) . Therefore, the pursuit for other nontoxic adjuvants, including TLR ligands and agonists that could effectively activate dendritic cells for the presentation of viral antigens to CD4 and CD8 T cells, should vigorously be continued. Use of cytokines such as IL-12 or IL-18 may enhance the immunogenicity of antiviral vaccines. Recombinant fowlpox vaccines coexpressing HA of subtype H5N1 and chicken IL-18 have been shown to induce complete protection in vaccinated chickens (19) . Use of adjuvants may enhance broader cross-reactive immune responses among infl uenza viruses (20) .
The evolution of many sublineages of infl uenza (H5N1) with antigenic diversity in Southeast Asia and southern China favors the wisdom of developing broadly cross-reactive vaccines for protection against an epidemic or pandemic (21) . Genetically engineered viruses could be constructed; these would express several variant antigens or determinants, thereby generating a broader immune response. The goal would be to develop vaccines that would induce broad-spectrum immunity-conferring protection to infl uenza including subtype H5N1. Ferrets vaccinated with A/PR/8/34 single-gene reassortants that differed only in their H5s were protected against a lethal challenge with A/Vietnam/1203/04 virus, suggesting generation of crossprotection (22) . Vaccination of mice with a live attenuated infl uenza vaccine or an alum-adjuvanted inactivated infl u-enza vaccine based on a related H5 HA from a nonpathogenic avian infl uenza virus, A/Duck/Pottsdam/1042-6/86 (H5N2), limited the disease severity and reduced deaths following challenge with a current highly pathogenic infl uenza (H5N1) (23) . Such cross-protective vaccines may provide clinical protection and prevent deaths in the early stages of a pandemic.
Genes of highly conserved proteins such as the nucleoprotein or M2 proteins could be included in adenovirus vector-based vaccines because immune responses against these infl uenza viral antigens provide protection in animal models (24, 25) . Recently, human adenoviral vector-based HA subtype 5 infl uenza vaccine induced protection in mice against infl uenza (H5N1) viruses isolated from humans (26, 27) . However, pre-existing immune response to human adenoviruses could be a potential problem in the generation of immune response against a foreign gene of interest. Delivering the vaccine nasally could largely overcome this problem because there appears to be no pre-existing immunity in the upper airways. Moreover, a robust CD8 T-cell response would likely be fl exible and able to fi ght infl uenza (H5N1).
Ideally, we need an effective vaccine for persons of all ages. However, if the vaccine is in short supply, priming fi rst those persons at high risk (e.g., young children, persons >50 years of age, healthcare workers) may be justifiable. During the early stages of an emerging H5 pandemic, such persons at high risk might be given an adjuvanted vaccine produced from a currently circulating strain, even if it is antigenically distinct, until an optimally matched and approved vaccine is available. This strategy is to produce a vaccine from an antigenically distant infl uenza (H5N1) strain that could induce broad-spectrum immunity capable of neutralizing newly emerged human H5 strains.
Vaccine development based on a cell culture system has advantages over egg-based technology because H5 strains are highly pathogenic for chickens and supplying large numbers of embryonated eggs could be diffi cult in a pandemic. In addition, potential allergic reactions to egg components would be avoided by growing the vaccine virus in tissue culture cells. Recently, mammalian cell culture was used for propagating viruses to prepare killed infl uenza vaccine (28) . Inactivated infl uenza vaccines produced with Madin-Darby canine kidney (MDCK) and Vero cells, which served as vaccine substrates, have been licensed in the Netherlands. Of note, the human cell line PER.C6 may provide a useful cell-based system because, unlike MDCK and Vero cell systems, it does not require a solid matrix support for the growth of cells. Selecting background viruses that grow well in these cell cultures and monitoring them for antigenic changes and contaminating microbes during propagation of the virus in cell culture need to be considered.
For the development of a universal infl uenza vaccine, a possible target is the relatively conserved M2 homotetramer. The concept is based on identifying alternative infl uenza antigens that are not as susceptible to antigenic shift and drift. Some degree of protection was induced in mice by priming with an M2 ectodomain peptide in adjuvant (29) . Studies that used the M2eA peptide conjugated to keyhole limpet hemocyanin and Neisseria meningitidis outer membrane protein illustrated good immune responses not only in mice but also in ferrets and rhesus monkeys (30) . In a recent study, 3 M2eA sequences, representing a range of epidemic strains and the (H5N1) strain, were fused to a proprietary hydrophobic protein domain. The resulting fusion proteins, formulated in liposomes, stimulated a protective response in mice challenged with subtypes H1N1, H5N1, H6N2, or H9N2 (31) . Previous studies have shown that when M2e is linked to hepatitis B virus core (HBc) particles, it becomes highly immunogenic, eliciting protective antibody response in mice (25) . Recently, a series of M2e-HBc constructs were made by increasing the copy number of M2e inserted at the N terminus from 1 to 3 per monomer. The best protection was seen when mice were vaccinated intranasally with these constructs combined with CTA1-DD, a cholera toxin A1-derived mucosal adjuvant (32) .
M2 serves as a pH-induced proton channel on the surface of all infl uenza A viruses but is present in low quantities. Further studies are warranted for understanding the mechanism of immune response to M2eA and for defi ning the appropriate immunization conditions for humans.
The lack of established correlates of immunity in animals and humans poses challenges to developing consistent immunologic endpoints for clinical trials and appropriate criteria for vaccine effi cacy. Serum antibody titers, mainly those determined by hemagglutination inhibition (HI) or virus neutralization (VN) assays, or both, are considered surrogate measures of protection. However, the HI test is insensitive for the detection of antibody to avian HA; there also are no recognized clinical correlates of immune protection for neutralization antibody (33, 34) . Recently, HI or VN assay failed to detect antibodies in ferrets protected by vaccination with whole-virus vaccines containing internal protein from Dk/Sing virus against a heterotypic virus (34). Whether the cross-protection reported is mediated by Tcell response is not known.
In recent years, attempts were made to improve the sensitivity of the HI test. More sensitive detection of anti-body to avian HA was seen when horse erythrocytes were used in place of turkey erythrocytes in the HI test because infl uenza virus was better able to bind to a2,3Gal-specifi c receptor sites on these erythrocytes (35) . The presence of asparagines at aa223 (H5 numbering) in H5 HA leads to improved sensitivity of the HI test (22) .
Often the immunogenicity of H5 vaccine candidates is assessed by HI or VN assays, but the basis of protection remains unclear. Nevertheless, the tests that are used to evaluate effi cacy of candidate vaccines are based on the assumption that antibody would mediate the protection against infection induced by vaccination, although this has yet to be critically established.
On the basis of initial evidence, infl ammation has been proposed as a possible cause or driving force of avian infl uenza (H5N1). However, components of the infl ammatory response might even be benefi cial. To address these possibilities, we need to determine whether infl ammation in avian infl uenza is an early event and a manifestation of innate immune response. If it is, some of the mediators of innate immune response, such as cytokine/chemokine levels, can be included in the evaluation of the potency of candidate vaccines. Further humoral response as a correlate for protection can be fi ne-tuned by determining the titer and isotype of antibody after vaccination. Several issues concerning vaccine effi cacy are unresolved: What are the consequences of vaccination for existing infl uenza (H5N1) infection, the extent of serologic cross-reactivity between the most closely related types of the virus, and the role in clinical protection? Vaccine administration may provide some therapeutic effects for infected persons who have not yet made an immune response but provide none for those with persistent infection associated with measurable humoral immunity.
Clearly, more studies are warranted to establish a highly reproducible assay to measure immunogenicity of a candidate vaccine and to determine adequate correlates of immune protection. Safety and immunogenicity of adjuvanted vaccines or new formulations should be critically assessed, and any fast-track approval of marketing vaccines must not compromise safety.
The marked virulence of the 1997 outbreak suggests that infl uenza A (H5N1) infection may have novel pathogenic mechanisms not seen in human infl uenza strains. To attempt to understand pathogenicity of this virus, an infl uenza virus bearing all 8 gene segments of the 1918 pandemic virus, which claimed at least 20-40 million lives, was recently generated in cultured cells. The reconstructed 1918 infl uenza viruses displayed accelerated activation of host immune response in mice with high levels of chemo-kines and cytokines in the lungs, resulting in infi ltration of infl ammatory cells and extensive damage to the lungs with severe hemorrhaging (36) . The pathogenicity induced by the reconstructed virus showed marked similarity to that reported with infl uenza (H5N1).
Increasing evidence from mouse models and humans suggests that certain infl ammatory mediators are potent drivers of the disease. If this is true, this could have important implications for developing new therapeutics. Acute respiratory distress syndrome, hemophagocytosis, or both, develop in a substantial fraction of patients with infl uenza (H5N1) infection; both of these conditions are thought to be promoted by overproduction of proinfl ammatory cytokines (known as a "cytokine storm") (37) . Consistent with these observations, cytokine release was markedly enhanced in human macrophages after infection with infl uenza (H5N1) (38) . Further, marked enhancement of chemokine and cytokine levels was observed in infl uenza (H5N1)-infected persons, particularly in those who died, and these correlated with high and disseminated viral replication (4). Additionally, infl uenza (H5N1) viruses appear relatively resistant to the inhibitory effects of host antiviral cytokines, such as interferons (IFNs) (39) . Thus, the severity of human infl uenza (H5N1) infection may be related to the induction of excessive proinfl ammatory responses that can accompany a primary infection and high viral shedding. Increased infl ammation was associated with viral replication in the respiratory and extrarespiratory organs of cats experimentally infected with infl uenza (H5N1) (3) . Mice infected with the highly pathogenic infl uenza (H5N1) strain A/HK/156/97, originally obtained from diseased chickens and an ill child in Hong Kong, China (HK), showed reduced ability to activate transforming growth factor-β (TGF-β), a potent antiinfl ammatory cytokine, compared to mice infected with less virulent A/Env/HK437/99 viruses (2) . The reduced ability to activate TGF-β may produce greater infl ammation at the site of infection and thus cause more severe disease. Alternatively, the low levels of activated TGF-β in the sera of A/HK/156/97-infected mice may allow the viruses to replicate and spread unchecked in the respiratory tracts of the mice, causing more severe disease. Recently, the impact of the nonstructural (NS) gene variation of Hong Kong (H5N1)/97 on cytokine production was illustrated (40) . The NS gene reassortant induced elevated pulmonary concentrations of the infl ammatory cytokines IL-1α, IL-1β, IL-6, IFN-γ, and chemokine KC and decreased concentrations of the anti-infl ammatory cytokine IL-10. This cytokine imbalance is reminiscent of the clinical fi ndings in humans infected with infl uenza (H5N1)/97 virus and may explain the unusual severity of the disease.
The ability to site specifi c engineering changes in the virus genome allows us to consider a novel vaccine approach. By engineering a virus with site-specifi c changes in the genome (for example in NS gene), we may produce infl uenza virus vaccine that favors the production of benefi cial anti-infl ammatory cytokines but remains highly immunogenic. In another approach, a human replicationincompetent, adenoviral vector-based infl uenza vaccine could be developed, in which genes of anti-infl ammatory cytokines are coexpressed, which will inhibit overproduction of proinfl ammatory cytokines. Such vaccines would be considered therapeutic vaccines (e.g., antidisease vaccines), which would inhibit infl ammation at the site of infection and protect against severe disease (Figure) . Excessive production of anti-infl ammatory cytokines may result in an inappropriate suppression of the host immune response. Further studies will validate the benefi cial effect of the anti-infl ammatory response for temporizing the cytokine storm seen in infl uenza (H5N1). Development of an immunization protocol that uses an adjuvant that allows selective priming of an antigen-specifi c immunoregulatory cytokines (e.g., IL-10, TGF-β) would be a major advance in the development of a vaccine for bird fl u with a substantial infl ammatory component. The search for potential adjuvants, such as TLR ligands and agonists that will favor the synthesis of inhibitory cytokines including IL-10, should be pursued. By testing whether manipulation of infl ammatory pathways changes the pathologic course, we would identify new targets for disease intervention.
Vaccination is the best option by which to prevent the spread of a pandemic virus and reduce the severity of disease. Defi ning the host response to infl uenza (H5N1) in natural infection is urgently needed to better understand the basis of protection and subsequent development of effi cacious vaccines. Improved vaccine strategies, which will require less antigen and be more robust in inducing both antibody and cell-mediated immunity for neutralizing infl uenza (H5N1) viruses, should be considered. To create an effective vaccine, a combination of factors must be optimized-such as number of doses, formulation without or with better adjuvant, and dose range. We also need to develop a reproducible assay that measures immunogenicity of a vaccine and to establish adequate correlates of protection. The effi cacy of potential cross-reactive vaccine candidates to induce broad-spectrum immunity to infl uenza (H5N1) viruses should be assessed critically; stockpiling of such vaccines may be justifi ed in the absence of optimally matched and approved vaccine during early stages of an H5 pandemic. Search for therapeutic vaccines (antidisease vaccines) aimed at controlling innate immune responses should be pursued, given the clinical evidence that the H5N1 subtype elicits a cytokine storm that contributes to disease pathogenesis. Vaccine development and deploy-ment need to be undertaken by a partnership of academia, government, and industry. The risk for dissemination of pandemic virus will remain if the disease is controlled in 1 area but not in others. A global approach is vital for combating the next infl uenza pandemic, a monumental public health challenge. Global Public Health Security National public health institutes will play a key role in implementation of the revised International Health Regulations. from transnational health threats demands a global public health perspective and investment in global public health infrastructure. The theme of this year's World Health Day and the World Health Report 2007 is "Global public health security-the need to reduce the vulnerability of people around the world to new, acute, or rapidly spreading risks to health, particularly those that cross international borders" (5) . With a call to all nations to "invest in health, and build a safer future," the World Health Organization (WHO) emphasizes the need for collaboration among nations to increase our collective capacity and infrastructure to respond to potential international health emergencies and other public health risks. As recent events have shown, global public health security is a complex, costly, and information-intense undertaking that requires strong national public health leadership and infrastructure, cross-border collaboration, capacity to identify problems rapidly and design real-time evidence-based solutions, well-trained and well-equipped workforces, well-functioning laboratories and service-delivery systems, capacity to sustain interventions, and ability to respond to unexpected events (5, 6) . Investment in these elements will strengthen not only global public health security but also the infrastructure needed to help broaden access to healthcare services and improve individual health outcomes, which would help break the cycles of poverty and political instability and thus contribute to national economic development and achievement of the Millennium Development Goals (7) .
A key driver in the effort to strengthen global public health security is the framework of the newly revised International Health Regulations (IHR [2005] ) (8) , the legally binding global agreement designed to build and strengthen national alert and response systems. Unanimously agreed upon by the World Health Assembly on May 23, 2005, the regulations are the result of experience gained and lessons learned about global public health security over the past 30 years. This global legal framework constitutes a "major development in the use of international law for public health purposes" (9) . It includes a commitment from WHO and from each of its 193 member states to improve capacity for disease prevention, detection, and response and provides ground rules to address national public health threats that have the potential to become global emergencies. The adoption of the new regulations ended a 10-year process of revision, stimulated by the pneumonic plague outbreak in India in 1994 (10) and the Ebola hemorrhagic fever outbreak in the former Zaire in 1995 (11) . The revised regulations have now entered into force for all WHO member states.
The revised regulations refl ect a growing understanding that the best way to prevent the global spread of diseases is to detect and contain them while they are still local.
WHO member states have obligations to rapidly assess and alert the global community about potential disease threats as well as to prevent and control the spread of disease inside and beyond their borders. Compared with the previous regulations, adopted in 1969 (12) , IHR (2005) expands the scope of internationally reportable diseases and events, provides criteria for identifying novel epidemic events, and specifi es conditions for involvement of the international community in outbreak responses. The revision includes the following 5 substantive changes.
The previous regulations applied to only 3 infectious diseases: cholera, plague, and yellow fever. IHR (2005) refl ects shifting concepts about disease control, shaped by recent and impending disease threats and the experiences of the past 2 decades in detecting and responding to disease outbreaks. The emergence and reemergence of a cascade of infectious diseases fueled by globalization and international travel (13) , the threat of biological terrorism, and novel environmental threats (14) have spotlighted the need for heightened vigilance and increased capacity to recognize and manage public health risks and emergencies. The appearance and rapid international spread of SARS and the pandemic potential of circulating avian infl uenza (H5N1) strains-with their combined health and economic effects-confi rmed the inapplicability of the 1969 IHR to most emerging and reemerging infectious diseases.
The revised regulations replace the previous diseasespecifi c framework with one built on timely notifi cation of all events that might constitute a public health emergency of international concern, taking into account the context in which an event occurs (15) . The advantage of this approach is its applicability to existing threats as well as to those that are new and unforeseen. The regulations also recognize the existence of threats to public health outside the infectious disease context, such as those associated with natural disasters, industrial or chemical accidents, and other environmental changes, which might cross international borders.
Expanding the scope of the IHR beyond reporting of 3 diseases to reporting of any public health emergency of international concern required an algorithm to assist in identifi cation of such events. The resulting decision instrument (see [8] , Annex 2, p. 43) identifi es a limited set of criteria for use by member states for fulfi lling the obligation to determine whether an event occurring within their territory might constitute a public health emergency of international concern and therefore require formal notifi cation to WHO within 24 hours of assessment. Essentially, the events that must be reported are those that fulfi ll at least 2 of the following criteria:
• Is the public health impact of the event serious? • Is the event unusual or unexpected? • Is there a signifi cant risk of international spread? • Is there a signifi cant risk of international trade or travel restriction?
To facilitate the use of the decision instrument, which requires some judgment to answer each of the questions, Annex 2 of the Regulations provides specifi c examples of events that might constitute a public health emergency of international concern. In addition to this broad scope for notifi cation, IHR (2005) includes a list of diseases for which a single case must be reported to WHO immediately, regardless of the context in which the disease occurs. This list includes smallpox, poliomyelitis due to wild-type poliovirus, human infl uenza caused by a new subtype, or SARS. In addition, an event involving certain other diseases (e.g., cholera, pneumonic plague, yellow fever, viral hemorrhagic fevers) calls for a careful evaluation using the decision instrument to determine whether notifi cation is indicated. The need for recognition of specifi c diseases requires adequate diagnostic laboratory capacity.
After an event is reported, only the Director General of WHO can determine whether the event formally constitutes a public health emergency of international concern. However, the Director General shall fi rst consult with the affected state party and hear the view of the emergency committee. This committee, composed of experts from the newly established IHR roster of experts, is specifi cally set up to review a reported event and provide advice to the Director General on whether an event constitutes a public health emergency of international concern and whether a temporary recommendation must be issued. On request, WHO will be able to provide technical support to affected countries, including the mobilization of the Global Outbreak Alert and Response Network.
A third innovation under IHR (2005) is the requirement for member states to designate "national IHR focal points" as the operational link for notifi cation and reporting to WHO and for WHO to name corresponding "IHR contact points." Effective communication between these 2 organizational entities will be central to the rapid management of a possible public health emergency of international concern. IHR focal points, or their designees, are required by IHR (2005) to be accessible at all times.
Experiences during the past several years have shown that public health emergencies expose the weaknesses and vulnerabilities of national and subnational public health infrastructure. The fourth change calls for member states to develop, strengthen, and maintain core capacities to 1) detect, assess, notify, and report disease events, and 2) respond promptly and effectively to public health risks and public health emergencies of international concern. State parties are required to complete a capacity assessment within 2 years of the revised IHR entering into force and, after this assessment, to develop public health infrastructure and human resources that ensure full compliance within 5 years of the IHR entering into force. This assessment must lead to the development of national action plans to meet the core capacity requirements that Annex 1 of the Regulations specifi es for different levels (i.e., local community or primary, intermediate, and national public health response) as well as designated airports, ports, and ground crossings. For these national points of entry, IHR (2005) also introduces special provisions for travelers, including the obligation to treat them with respect for their dignity, human rights, and fundamental freedom.
WHO is required to assist all member states in fulfi lling the new obligations. On request, WHO will collaborate with countries to evaluate their public health capacities and facilitate technical cooperation, logistical support, and mobilization of fi nancial resources for strengthening capacity in surveillance and response. Countries will build on existing national or regional strategies such as the Asia Pacifi c Strategy for Emerging Diseases in WHO's Southeast Asia and Western Pacifi c Regions (16) and the Integrated Disease Surveillance and Response strategy in the African Region (17) . In many countries, national action plans can also build on the infl uenza pandemic preparedness plans developed with WHO's guidance. Specifi c WHO guidelines and initiatives, particularly in the areas of external quality assessment for laboratories, data gathering and analysis at the health district level, and the central and coordination functions of national public health institutes, are being developed. WHO's Lyon Offi ce for National Epidemic Preparedness and Response is specifi cally dedicated to supporting countries in meeting the core national capacity requirements of IHR (2005) .
Under IHR (2005), new powers for WHO include an information-gathering responsibility that is not limited solely to offi cial state notifi cations or consultations but covers all available scientifi c evidence and other relevant information. WHO can consult nonoffi cial reports and require countries to collaborate with a request for verification. WHO is also empowered to recommend and coordinate measures that will help contain the international spread of disease, including public health actions at ports, airports, and land borders, and on means of transportation that involve international travel.
Because weak national public health capabilities undermine efforts to strengthen global public health security, IHR (2005) imposes substantial responsibilities on countries to improve public health capacity and infrastructure. However, despite the broad new goals included in IHR (2005), improvements in global public health security will depend on what member states are actually able to do. Success will rely on the capacity and performance of national public health systems (15) , anchored by strong national public health institutes (NPHIs). Low-resource countries, which are particularly vulnerable to emerging threats, will be particularly challenged by the IHR (2005) requirements and the need to ensure an appropriate and coordinated public health response to health emergencies.
Many countries have been well served by centralizing their public health expertise and activities within 1 institution or network of institutions that provides leadership and coordination for public health (18; unpub. data). Examples include the US Centers for Disease Control and Prevention, the National Public Health Institute of Finland, and the Chinese Center for Disease Control and Prevention. These NPHIs are usually governmental or quasi-governmental agencies with a central focus and organizational structure that allow coordination of national public health service delivery and ensure a country's ability to detect, investigate, and respond to public health emergencies. The core functions of an NPHI have been defi ned (unpub. data) and include evaluation and analysis of health status; public health surveillance, problem investigation, and control of risks and threats to public health; and public health research.
Given the scope and range of their activities, NPHIs are a vital asset to health development and security and will have a critically important role in implementing IHR (2005), whether as national focal points or as operational partners in fulfi lling the requirements of the regulations. Unfortunately, however, many countries still either have no NPHIs or have institutes with severely limited capacities and capabilities relative to the need. Even in countries with strong NPHIs, unpredictable and rapidly evolving health threats can quickly overwhelm capacity and inhibit a timely and complete response.
A new organization, the International Association of National Public Health Institutes (IANPHI, www.ianphi.org), was created to address these gaps through the enhancement and proliferation of NPHIs throughout the world. Founded in 2006 by 39 NPHI directors who recognized the importance of strong national public health capacity and the mutual benefi ts of shared information, experience, and expertise, IANPHI aims to be a catalyst for sustained improvements in public health capacity and infrastructure globally. With the partnership of WHO and funding fi rst from the Rockefeller Foundation and now from the Bill and Melinda Gates Foun-dation, the organization focuses on strengthening public health capacity in low-resource countries by strengthening NPHIs and on providing tools and a context that will support all NPHIs. IANPHI is also a professional association for NPHI directors; it fosters leadership development and advocacy for public health and collaborates with WHO.
Since early 2006, the founding members have continued to expand the network and put their shared vision into practice. IANPHI is managed by an executive board and a secretariat located both in Finland and in the United States. With nearly 50 current members and an ambitious agenda for collaboration, service, and growth, IANPHI is committed to a vision of a robust and fully integrated global network of NPHIs equipped to address critical public health challenges. Its mission is to strengthen and reinvigorate existing NPHIs, create new NPHIs where none exist, and provide funded grants to support NPHI capacity development priorities.
IANPHI achieves its service mission through a 3part approach of advocacy, technical assistance, and linkages. IANPHI advocates for NPHI development and proliferation through partnerships with key global health organizations, such as WHO. Through these partnerships, IANPHI ensures that NPHIs are considered in major global public health initiatives and that public health and the work of NPHIs are included in efforts to strengthen health systems.
Assistance to NPHIs in low-resource countries is provided through 3 grant programs. A short-term technical assistance program helps countries quickly resolve priority gaps in NPHI capability and infrastructure. A medium-term capacity-building program helps NPHIs address high-priority needs for up to 3 years. IANPHI's long-term grant program, the most intensive of the assistance efforts, will help create NPHIs in low-resource countries that currently lack a central public health focus. With funding from a $20 million grant from the Bill and Melinda Gates Foundation, the organization is committed to implementing 60 NPHI development projects by 2011.
As of June 2007, IANPHI had awarded technical assistance grants to public health institutes in 5 nations. The new awards include 3 short-term grants to NPHIs in Thailand, Uganda, and Iran to support training and infrastructure development. A medium-term grant to the Nigerian Institute of Medical Research will support sustainable improvements in disease surveillance, outbreak investigation, and emergency preparedness and strengthen linkages with other groups working to advance public health in the country; special focus will be on public health laboratory capacity building and integration of surveillance, epidemiology, and laboratory programs. Colombia's Institutos Nacional de Salud was awarded a medium-term grant to establish a pilot chronic disease study site to generate, collect, and dissemi-nate chronic disease data by using multiple mechanisms. The activities are designed to yield a sustainable network of surveillance and research sites to guide national-level public health decision making.
The cornerstone of the IANPHI approach is a peer-assistance model for NPHI strengthening and enhancement, with an emphasis on countries without NPHIs or with NPHIs in their early stages. Experts from IANPHI member institutes provide technical assistance and project support targeted at critical NPHI needs. Teams are guided by the Framework for the Creation and Development of NPHIs (www.ianphi.org/?action=arkisto&RYHMA=47&ID=&valittu=8), a product of IANPHI in partnership with WHO. The Framework provides a working defi nition of an NPHI and suggests a process for creating or enhancing an institute. By defi ning the critical characteristics of an NPHI, IANPHI hopes to bring specifi city to the organization's vision, align efforts to assist low-resource countries in building NPHIs, and provide benchmarks and resources to help any country assess and improve the functioning of its NPHI. To that end, IANPHI has also developed an NPHI toolkit (www. sph.emory.edu/IANPHI), which provides access to a variety of Web-based information resources for countries, NPHIs, and IANPHI peer-assistance teams to use as they work to assess, develop, and improve NPHIs and build public health capacity around the world.
Through strategies to defi ne and develop core public health functions and to share expertise, IANPHI helps NPHIs sharpen their focus and raise standards of performance. IANPHI also links NPHIs through annual meetings, regional events, leadership development activities, research seed grants, and communication outlets including a website, newsletter, and listserv. By fostering an international community of public health leadership, IANPHI helps NPHIs gain the benefi ts of shared information, experience, and expertise to address public health threats and opportunities. Through its grant program and other activities, IANPHI aims to help national governments develop organizational infrastructures to devise and implement comprehensive public health priorities, meet global public health goals, develop workforce capacity, effectively absorb donor funds, address emerging threats, and improve the health of their populations (18) .
In today's global environment, every country confronts similar challenges in keeping its population healthy and preventing the cross-border spread of disease. SARS demonstrated this dramatically in 2003, and the ongoing challenges posed by avian infl uenza have focused attention on the need for global pandemic infl uenza preparedness. Polio has reemerged in countries that had virtually eradicated it, while HIV/AIDS and other diseases continue to threaten the stability of communities around the world. Recent examples of emerging and reemerging diseases of global signifi cance are the resurgence of dengue in tropical and subtropical areas of the world; the spread and establishment of Japanese encephalitis and West Nile viruses in new habitats and environments; and the reoccurrence and spread of chikungunya virus in India, East Africa, and several Indian Ocean islands (19, 20) . As life expectancy increases worldwide, issues related to noncommunicable conditions are also becoming increasingly common to all. By working within the collaborative framework provided by IHR (2005), countries can benefi t through improved national and international surveillance; improved systems for rapid detection of and response to public health emergencies; standardized rules for evaluation, control, and resolution of urgent events; and mechanisms to increase national and local public health security.
Nonetheless, the success of IHR (2005) and other global public health initiatives such as the Millennium Development Goals depends on strong national public health systems with competent, well-trained staff and well-equipped facilities. By targeting the core of public health systems, especially in low-resource countries that currently lag behind in public health capacity and infrastructure, IANPHI will play a key role in improving the capacity of countries to effectively detect, investigate, and respond to public health emergencies. The result will be better control of endemic diseases such as HIV/AIDS, acute lower respiratory tract infections, diarrheal diseases, measles, tuberculosis, malaria, and the neglected tropical diseases. These efforts will strengthen the practice of public health worldwide, yield global public health benefi ts of disease control and prevention, and ultimately accelerate social and economic development in the poorest countries of the world and progress toward achieving the Millenium Development Goals. Dr Rodier is director of International Health Regulations coordination at WHO in Geneva. Dynamics and Control of Diseases in Networks with Community Structure The dynamics of infectious diseases spread via direct person-to-person transmission (such as influenza, smallpox, HIV/AIDS, etc.) depends on the underlying host contact network. Human contact networks exhibit strong community structure. Understanding how such community structure affects epidemics may provide insights for preventing the spread of disease between communities by changing the structure of the contact network through pharmaceutical or non-pharmaceutical interventions. We use empirical and simulated networks to investigate the spread of disease in networks with community structure. We find that community structure has a major impact on disease dynamics, and we show that in networks with strong community structure, immunization interventions targeted at individuals bridging communities are more effective than those simply targeting highly connected individuals. Because the structure of relevant contact networks is generally not known, and vaccine supply is often limited, there is great need for efficient vaccination algorithms that do not require full knowledge of the network. We developed an algorithm that acts only on locally available network information and is able to quickly identify targets for successful immunization intervention. The algorithm generally outperforms existing algorithms when vaccine supply is limited, particularly in networks with strong community structure. Understanding the spread of infectious diseases and designing optimal control strategies is a major goal of public health. Social networks show marked patterns of community structure, and our results, based on empirical and simulated data, demonstrate that community structure strongly affects disease dynamics. These results have implications for the design of control strategies. Mitigating or preventing the spread of infectious diseases is the ultimate goal of infectious disease epidemiology, and understanding the dynamics of epidemics is an important tool to achieve this goal. A rich body of research [1, 2, 3] has provided major insights into the processes that drive epidemics, and has been instrumental in developing strategies for control and eradication. The structure of contact networks is crucial in explaining epidemiological patterns seen in the spread of directly transmissible diseases such as HIV/AIDS [1, 4, 5] , SARS [6, 7] , influenza [8, 9, 10, 11] etc. For example, the basic reproductive number R 0 , a quantity central to developing intervention measures or immunization programs, depends crucially on the variance of the distribution of contacts [1, 12, 13] , known as the network degree distribution. Contact networks with fat-tailed degree distributions, for example, where a few individuals have an extraordinarily large number of contacts, result in a higher R 0 than one would expect from contact networks with a uniform degree distribution, and the existence of highly connected individuals makes them an ideal target for control measures [7, 14] .
While degree distributions have been studied extensively to understand their effect on epidemic dynamics, the community structure of networks has generally been ignored. Despite the demonstration that social networks show significant community structure [15, 16, 17, 18] , and that social processes such as homophily and transitivity result in highly clustered and modular networks [19] , the effect of such microstructures on epidemic dynamics has only recently started to be investigated. Most initial work has focused on the effect of small cycles, predominantly in the context of clustering coefficients (i.e. the fraction of closed triplets in a contact network) [20, 21, 22, 23, 24] .
In this article, we aim to understand how community structure affects epidemic dynamics and control of infectious disease. Community structure exists when connections between members of a group of nodes are more dense than connections between members of different groups of nodes [15] . The terminology is relatively new in network analysis and recent algorithm development has greatly expanded our ability to detect sub-structuring in networks. While there has been a recent explosion in interest and methodological development, the concept is an old one in the study of social networks where it is typically referred to as a ''cohesive subgroups,'' groups of vertices in a graph that share connections with each other at a higher rate than with vertices outside the group [18] . Empirical data on social structure suggests that community structuring is extensive in epidemiological contacts [25, 26, 27] relevant for infectious diseases transmitted by the respiratory or close-contact route (e.g. influenza-like illnesses), and in social groups more generally [16, 17, 28, 29, 30] . Similarly, the results of epidemic models of directly transmitted infections such as influenza are most consistent with the existence of such structure [8, 9, 11, 31, 32, 33] .
Using both simulated and empirical social networks, we show how community structure affects the spread of diseases in networks, and specifically that these effects cannot be accounted for by the degree distribution alone. The main goal of this study is to demonstrate how community structure affects epidemic dynamics, and what strategies are best applied to control epidemics in networks with community structure.
We generate networks computationally with community structure by creating small subnetworks of locally dense communities, which are then randomly connected to one another. A particular feature of such networks is that the variance of their degree distribution is relatively low, and thus the spread of a disease is only marginally affected by it [34] . Running standard susceptible-infected-resistant (SIR) epidemic simulations (see Methods) on these networks, we find that the average epidemic size, epidemic duration and the peak prevalence of the epidemic are strongly affected by a change in community structure connectivity that is independent of the overall degree distribution of the full network ( Figure 1 ). Note that the value range of Q shown in Figure 1 is in agreement with the value range of Q found in the empirical networks used further below, and that lower values of Q do not affect the results qualitatively (see Suppl. Mat. Figure S1 ).
Epidemics in populations with community structure show a distinct dynamical pattern depending on the extent of community structure. In networks with strong community structure, an infected individual is more likely to infect members of the same community than members outside of the community. Thus, in a network with strong community structure, local outbreaks may die out before spreading to other communities, or they may spread through various communities in an almost serial fashion, and large epidemics in populations with strong community structure may therefore last for a long time. Correspondingly, the incidence rate can be very low, and the number of generations of infection transmission can be very high, compared to the explosive epidemics in populations with less community structure (Figures 2a and 2b ). On average, epidemics in networks with strong community structure exhibit greater variance in final size (Figures 2c and 2d) , a greater number of small, local outbreaks that do not develop into a full epidemic, and a higher variance in the duration of an epidemic.
In order to halt or mitigate an epidemic, targeted immunization interventions or social distancing interventions aim to change the structure of the network of susceptible individuals in such a way as to make it harder for a pathogen to spread [35] . In practice, the number of people to be removed from the susceptible class is often constrained for a number of reasons (e.g., due to limited vaccine supply or ethical concerns of social distancing measures). From a network perspective, targeted immunization methods translate into indentifying which nodes should be removed from a network, a problem that has caught considerable attention (see for example [36] and references therein). Targeting highly connected individuals for immunization has been shown to be an effective strategy for epidemic control [7, 14] . However, in networks with strong community structure, this strategy may not be the most effective: some individuals connect to multiple communities (so-called community bridges [37] ) and may thus be more important in spreading the disease than individuals with fewer inter-community connections, but this importance is not necessarily reflected in the degree. Identification of community bridges can be achieved using 
Understanding the spread of infectious diseases in populations is key to controlling them. Computational simulations of epidemics provide a valuable tool for the study of the dynamics of epidemics. In such simulations, populations are represented by networks, where hosts and their interactions among each other are represented by nodes and edges. In the past few years, it has become clear that many human social networks have a very remarkable property: they all exhibit strong community structure. A network with strong community structure consists of smaller sub-networks (the communities) that have many connections within them, but only few between them. Here we use both data from social networking websites and computer generated networks to study the effect of community structure on epidemic spread. We find that community structure not only affects the dynamics of epidemics in networks, but that it also has implications for how networks can be protected from large-scale epidemics.
the betweenness centrality measure [38] , defined as the fraction of shortest paths a node falls on. While degree and betweenness centrality are often strongly positively correlated, the correlation between degree and betweenness centrality becomes weaker as community structure becomes stronger ( Figure 3 ). Thus, in networks with community structure, focusing on the degree alone carries the risk of missing some of the community bridges that are not highly connected. Indeed, at a low vaccination coverage, an immunization strategy based on betweenness centrality results in fewer infected cases than an immunization strategy based on degree as the magnitude of community structure increases ( Figure 4a ). This observation is critical because the potential vaccination coverage for an emerging infection will typically be very low. A third measure, random walk centrality, identifies target nodes by a random walk, counting how often a node is traversed by a random walk between two other nodes [39] . The random walk centrality measure considers not only the shortest paths between pairs of nodes, but all paths between pairs of nodes, while still giving shorter paths more weight. While infections are most likely to spread along the shortest paths between any two nodes, the cumulative contribution of other paths can still be important [40] : immunization strategies based on random walk centrality result in the lowest number of infected cases at low vaccination coverage (Figure 4b and 4c ).
To test the efficiency of targeted immunization strategies on real networks, we used interaction data of individuals at five different universities in the US taken from a social network website [41] , and obtained the contact network relevant for directly transmissible diseases (see Methods). We find again that the overall most successful targeted immunization strategy is the one that identifies the targets based on random walk centrality. Limited immunization based on random walk centrality significantly outperforms immunization based on degree especially when vaccination coverage is low (Figure 5a ).
In practice, identifying immunization targets may be impossible using such algorithms, because the structure of the contact network relevant for the spread of a directly transmissible disease is generally not known. Thus, algorithms that are agnostic about the full network structure are necessary to identify target individuals. The only algorithm we are aware of that is completely agnostic about the network structure network structure identifies target nodes by picking a random contact of a randomly chosen individual [42] . Once such an acquaintance has been picked n times, it is immunized. The acquaintance method has been shown to be able to identify some of the highly connected individuals, and thus approximates an immunization strategy that targets highly connected individuals. We propose an alternative algorithm (the so-called community bridge finder (CBF) algorithm, described in detail in the Methods) that aims to identify community bridges connecting two groups of clustered nodes. Briefly, starting from a random node, the algorithm follows a random path on the contact network, until it arrives at a node that does not connect back to more than one of the previously visited nodes on the random walk. The basic goal of the CBF algorithm is to find nodes that connect to multiple communities -it does so based on the notion that the first node that does not connect back to previously visited nodes of the current random walk is likely to be part of a different community. On all empirical and computationally generated networks tested, this algorithm performed mostly better, often equally well, and rarely worse than the alternative algorithm. It is important to note a crucial difference between algorithms such as CBF (henceforth called stochastic algorithms) and algorithms such as those that calculate, for example, the betweenness centrality of nodes (henceforth called deterministic algorithms). A deterministic algorithm always needs the complete information about each node (i.e. either the number or the identity of all connected nodes for each node in the network). A comparison between algorithms is therefore of limited use if they are not of the same type as they have to work with different inputs. Clearly, a deterministic algorithm with information on the full network structure as input should outperform a stochastic algorithm that is agnostic about the full network structure. Thus, we will restrict our comparison of CBF to the acquaintance method since this is the only stochastic algorithm we are aware of the takes as input the same limited amount of local information.
In the computationally generated networks, CBF outperformed the acquaintance method in large areas of the parameter space ( Figure 4d ). It may seem unintuitive at first that the acquaintance method outperforms CBF at very high values of modularity, but one should keep in mind that epidemic sizes are very small in those extremely modular networks (see Figure 1a ) because local outbreaks only rarely jump the community borders. If outbreaks are mostly restricted to single communities, then CBF is not the optimal strategy because immunizing community bridges is useless; the acquaintance method may at least find some well connected nodes in each community and will thus perform slightly better in this extreme parameter space.
In empirical networks, CBF did particularly well on the network with the strongest community structure (Oklahoma), especially in comparison to the similarly effective acquaintance method with n = 2. (Figure 5c ). As immunization strategies should be deployed as fast as possible, the speed at which a certain fraction of the . Assessing the efficacy of targeted immunization strategies based on deterministic and stochastic algorithms in the computationally generated networks. Color code denotes the difference in the average final size S m of disease outbreaks in networks that were immunized before the outbreak using method m. The top panel (a) shows the difference between the degree method and the betweenness centrality method, i.e. S degree 2 S betweenness . A positive difference (colored red to light gray) indicates that the betweenness centrality method resulted in smaller final sizes than the degree method. A negative difference (colored blue to black) indicates that the betweenness centrality method resulted in bigger final sizes than the degree method. If the difference is not bigger than 0.1% of the total population size, then no color is shown (white). Panel (a) shows that the betweenness centrality method is more effective than the degree based method in networks with strong community structure (Q is high). (b) and (c): like (a), but showing S degree 2 S randomwalk (in (b)) and S betweenness 2 S randomwalk (in (c)). Panels (b) and (c) show that the random walk method is the most effective method overall. Panel (d) shows that the community bridge finder (CBF) method generally outperforms the acquaintance method (with n = 1) except when community structure is very strong (see main text). Final epidemic sizes were obtained by running 2000 SIR simulations per network, vaccination coverage and immunization method. doi:10.1371/journal.pcbi.1000736.g004 network can be immunized is an additional important aspect. We measured the speed of the algorithm as the number of nodes that the algorithm had to visit in order to achieve a certain vaccination coverage, and find that the CBF algorithm is faster than the similarly effective acquaintance method with n = 2 at vaccination coverages ,30% (see Figure 6 ).
A great number of infectious diseases of humans spread directly from one person to another person, and early work on the spread of such diseases has been based on the assumption that every infected individual is equally likely to transmit the disease to any susceptible individual in a population. One of the most important consequences of incorporating network structure into epidemic models was the demonstration that heterogeneity in the number of contacts (degree) can strongly affect how R 0 is calculated [12, 13, 34] . Thus, the same disease can exhibit markedly different epidemic patterns simply due to differences in the degree distribution. Our results extend this finding and show that even in networks with the same degree distribution, fundamentally different epidemic dynamics are expected to be observed due to different levels of community structure. This finding is important for various reasons: first, community structure has been shown to be a crucial feature of social networks [15, 16, 17, 19] , and its effect on disease spread is thus relevant to infectious disease dynamics. Furthermore, it corroborates earlier suggestions that community structure affects the spread of disease, and is the first to clearly isolate this effect from effects due to variance in the degree distribution [43] . Second, and consequently, data on the degree distribution of contact networks will not be sufficient to predict epidemic dynamics. Third, the design of control strategies benefits from taking community structure into account.
An important caveat to mention is that community structure in the sense used throughout this paper (i.e. measured as modularity Q ) does not take into account explicitly the extent to which communities overlap. Such overlap is likely to play an important role in infectious disease dynamics, because people are members of multiple, potentially overlapping communities (households, schools, workplaces etc.). A strong overlap would likely be reflected in lower overall values for Q; however, the exact effect of community overlap on infectious disease dynamics remains to be investigated.
Identifying important nodes to affect diffusion on networks is a key question in network theory that pertains to a wide range of fields and is not limited to infectious disease dynamics only. There are however two major issues associated with this problem: (i) the structure of networks is often not known, and (ii) many networks are too large to compute, for example, centrality measures efficiently. Stochastic algorithms like the proposed CBF algorithm or the acquaintance method address both problems at once. To what extent targeted immunization strategies can be implemented in a infectious diseases/public health setting based on practical and ethical considerations remains an open question. This is true not only for the strategy based on the CBF algorithm, but for most strategies that are based on network properties. As mentioned above, the contact networks relevant for the spread of infectious diseases are generally not known. Stochastic algorithms such as the CBF or the acquaintance method are at least in principle applicable when data on network structure is lacking.
Community structure in host networks is not limited to human networks: Animal populations are often divided into subpopulations, connected by limited migration only [44, 45] . Targeted immunization of individuals connecting subpopulations has been shown to be an effective low-coverage immunization strategy for the conservation of endangered species [46] . Under the assumption of homogenous mixing, the elimination of a disease requires an immunization coverage of at least 1-1/R 0 [1] but such coverage is often difficult or even impossible to achieve due to limited vaccine supply, logistical challenges or ethical concerns. In the case of wildlife animals, high vaccination coverage is also problematic as vaccination interventions can be associated with substantial risks. Little is known about the contact network structure in humans, let alone in wildlife, and progress should therefore be made on the development of immunization strategies that can deal with the absence of such data. Stochastic algorithms such as the acquaintance method and the CBF method are first important steps in addressing the problem, but the large difference in efficacy between stochastic and deterministic algorithms demonstrates that there is still a long way to go.
To investigate the spread of an infectious disease on a contact network, we use the following methodology: Individuals in a population are represented as nodes in a network, and the edges between the nodes represent the contacts along which an infection can spread. Contact networks are abstracted by undirected, unweighted graphs (i.e. all contacts are reciprocal, and all contacts transmit an infection with the same probability). Edges always link between two distinct nodes (i.e. no self loops), and there must be maximally one edge between any single pair of nodes (i.e no parallel edges). Each node can be in one of three possible states: (S)usceptible, (I)nfected, or (R)esistant/immune (as in standard SIR models). Initially, all nodes are susceptible.
Simulations with immunization strategies implement those strategies before the first infection occurs. Targeted nodes are chosen according to a given immunization algorithm (see below) until a desired immunization coverage of the population is achieved, and then their state is set to resistant.
After this initial set-up, a random susceptible node is chosen as patient zero, and its state is set to infected. Then, during a number of time steps, the initial infection can spread through the network, and the simulation is halted once there are no further infected nodes. At each time step (the unit of time we use is one day, i.e. a Figure 5 . Assessing the efficacy of targeted immunization strategies in empirical networks based on deterministic and stochastic algorithms. The bars show the difference in the average final size S m of disease outbreaks (n cases) in networks that were immunized before the outbreak using method m. The left panels show the difference between the degree method and the random walk centrality method, i.e. S degree 2 S randomwalk . If the difference is positive (red bars), then the random walk centrality method resulted in smaller final sizes than the degree method. A negative value (black bars) means that the opposite is true. Shaded bars show non-significant differences (assessed at the 5% level using the Mann-Whitney test). The middle and right panels are generated using the same methodology, but measuring the difference between the acquaintance method (with n = 1 in the middle column and n = 2 in the right column, see Methods) and the community bridge finder (CBF) method, i.e. S acquaintance1 2 S CBF and S acquaintance2 2 S CBF . Again, positive red bars mean that the CBF method results in smaller final sizes, i.e. prevents more cases, than the acquaintance methods, whereas negative black bars mean the opposite. Final epidemic sizes were obtained by running 2000 SIR simulations per network, vaccination coverage and immunization method. doi:10.1371/journal.pcbi.1000736.g005 time step is one day), an infected node can get infected with probability 12exp(2bi), where b is the transmission rate from an infected to a susceptible node, and i is the number of infected neighboring nodes. At each time step, infected nodes recover at rate c, i.e. the probability of recovery of an infected node per time step is c (unless noted otherwise, we use c = 0.2). If recovery occurs, the state of the recovered node is toggled from infected to resistant. Unless mentioned otherwise, the transmission rate b is chosen such that R 0 ,(b/c) * d<3 where d is the mean network degree, i.e the average number of contacts per node. For the networks used here, this approximation is in line with the result from static network theory [47] , R 0 ,T(,k 2 ./,k.21), where ,k. and ,k 2 . are the mean degree and mean square degree, respectively, and where T is the average probability of disease transmission from a node to a neighboring node, i.e. T<b/c. Note that the variation in the degree is too small to be of relevance here (see further below and Figure 1d ). The reason we chose c = 0.2 (i.e. an average length of infectious period of 5 days) and R 0 <3 in most simulations (unless mentioned otherwise) is that these parameter values reflect, very roughly, some of the most widespread infectious diseases to which our study is relevant (i.e. flu-like infectious diseases that are transmitted directly from person to person by the respiratory or close-contact route [8, 9, 48, 49, 50] ).
After a simulation, we record the total number of cases infected (the epidemic size), the maximum frequency of infection at any point during the simulation (the peak prevalence), and the number of days that have passed between the first infected case and the simulation stop (the duration of the epidemic).
In order to understand the effect of community structure, we generated networks with 2000 nodes from scratch with varying degrees of community structure. The strength of community structure is generally measured as network modularity Q, which is defined as
where e ij is the fraction of all edges in the network that link nodes in community i to nodes in community j, and a i~X j e ij [15] . Thus, a i represents the fraction of edges in the network that connect to nodes in community i. If edges were to fall between nodes without any regard for communities, we would have e ij = a i a j , and thus Q = 0. There are numerous methods to Figure 6 . Assessing the speed of stochastic immunization algorithms acquaintance2 and CBF. The speed of an algorithm is assessed by counting the nodes that have to be visited by the algorithm until the desired vaccination coverage is achieved. Each visit is counted, even if the same node has been visited before. The bars show the difference of node visits (n visits) between the acquaintance2 method and the CBF method. Red bars mean the CBF method has visited fewer nodes -the difference is given by the height of the bar. A black bar indicates that the acquaintance2 methods has visited fewer nodes. With the exception of vaccination coverage 30% in the North Carolina network, the CBF method is always faster. (Data for speed comparison between acquaintance1 and CBF is not shown -the acquaintance1 method is always faster, but significantly less effectivesee middle column in Figure 5 ). doi:10.1371/journal.pcbi.1000736.g006
calculate the value of Q for a given network, and the development of more accurate and efficient methods is still a very active research field. In particular, one has to be careful when comparing values of Q because some measures are normalized while others or not [51] . We have used the spin glass method introduced by Reichhardt and Bornholdt [52] to measure Q throughout this manuscript.
To generate networks with community structure, we initialize a network by creating 50 small-world communities (as found in various social networks, see e.g. ref. [53] ) of 40 nodes using the Watts-Strogatz algorithm [54] such that each node has exactly 8 edges connecting to nodes of the same community. We then add 2000 edges randomly between randomly chosen nodes, making sure that the edges fall between communities only. Thus, we create a graph with 2000 nodes and 10000 (i.e. (2000+50 * 40 * (8/2))) undirected edges where one out of five edges falls between communities. The average degree of the network is 10, which is in line with recent reports on social contact patterns [55] . Starting from this initial network where Q<0.76, we create networks with increasing community structure by rewiring between-community edges so that they become within-community edges. More precisely, at each rewiring step, we (i) randomly choose a betweencommunity edge, (ii) randomly choose one of the two communities that the edge connects, (iii) pick a random node of the chosen community, and (iv) rewire the edge by detaching it from the node of the community that was not chosen in step (ii), and attaching it to the new node in the community that was chosen in step (iii). At all times, edges must always fall between two distinct nodes, and there can only be one edge between any two pair of nodes. We've also tested if all networks thus created consist of only a single connected component (they do).
The quantity (CV) 2 is the square of the coefficient of variation in degree (i.e. the squre of the ratio of the standard deviation of degree to the mean degree, where degree is defined as the number of edges incident to a node). (CV) 2 is important for the spread of infectious diseases since it is known that
where r 0 is the value of R 0 under the assumption of a homogenous network (i.e. no variance in the degree distribution) [1, 56] .
We used the network data collected on the social network website Facebook (www.facebook.com) by Traud et al. [41] . The data contains the friendship network at five US universities, where nodes represent individuals (i.e. members of the university), and edges represent friendship links between two individuals. Additionally, the data includes covariate information (if available) about the individuals, such as the gender of the individual, the dormitory residence, major (field of specialization) etc. While such friendship network data are interesting for various reasons, they do not necessarily reflect the contact network relevant for the spread of infectious diseases. Clearly, a friendship connection between two individuals on a social network website does not necessarily mean that there is also a connection between the two individuals in the contact network relevant to the spread of infectious diseases.
Thus, in order to obtain contact network data that are relevant for the spread of infectious diseases transmitted directly from person to person by the respiratory or close-contact route, we make the following assumptions: Individuals who have a friendship relation in the network, and who either (a) have the same dormitory residence, or (b) who major in the same field and the same class year, are likely to be in close enough physical contact on a regular basis as to be able to transmit an infection to each other. Thus, using the raw friendship data and the available information on dormitory residence, major, and class year, we extract the subgraph which reflects our assumptions. Having extracted the subgraph, we remove all nodes who are not part of the largest connected component (i.e. small subgraphs that are not part of the larger network). The networks thus reduce to the following contact networks: We note that the modularity Q of these networks is within the range of modularities measured in the computationally generated networks (see for example Figure 1) , with the exception of one network (Georgetown). Clearly, these networks will contain contacts that are not relevant for the spread of diseases (false positives) -at the same time, they will also miss some relevant contacts (false negatives). However, given the accuracy and amount of data, these networks are well suited to study the spread of infectious diseases on human contact networks, in particular for diseases transmitted directly from person to person by the respiratory or close-contact route. Degree distributions of these networks are shown in Suppl. Mat. Figure S2 .
The algorithms used to identify nodes can be divided into two classes: deterministic and stochastic algorithms. Deterministic algorithms require the complete information about each node (i.e. either the number or the identity of all connected nodes for each node in the network), and they rank nodes by processing that information by a procedure specific to that algorithm. Target nodes are then chosen by their rank (from high to low). Thus, for a given network structure, deterministic algorithms always give the same result, i.e. they identify the same target nodes (except for random choices when two nodes have exactly the same rank). Stochastic algorithms, on the other hand, do not require such detailed structural information -they identify target nodes by collecting information locally from randomly chosen nodes in the network. These algorithms represent the type of investigationrelated information in actual epidemics. We will now describe a number of deterministic and stochastic algorithms as we have used them in the main text.
Deterministic algorithms. We identifiy target nodes by ranking nodes to one of the three following criteria: degree, betweenness centrality, and random-walk centrality.
The degree of a node simply denotes the number of edges incident to a node.
The betweenness centrality C B (i) of a node i is defined as
where s, t and i are distinct nodes of the graph, s st is the total number of shortest paths between nodes s and t, and s st (i) is the number of those shortest paths that go through node i [38] . The random-walk centrality of a node i is a measure based on random walks, counting how often the node i is traversed by a random walk between any pair of nodes s and t. Following Newman [39] , we rank nodes according to the random-walk measure
for i?s, t. Here, A ij is the element in the adjacency matrix of the graph (0 or 1 in our case), and T is is the element in the voltage matrix which is calculated as described in detail in Newman [39] . Nodes are ranked according to the measure chosen (i.e degree, betweenness centrality, or random-walk centrality). We then immunize nodes going from high to low rankings, until the desired immunization coverage is achieved.
Stochastic algorithms. We use two stochastic algorithms to identify target nodes without knowledge of the full network structure. In the algorithms described below, targets are identified and immunized if they have not been immunized before.
The first algorithm, acquaintance immunization, has been described by Cohen et al. [42] and it works as follows: pick a random node v 0 , and then pick a random acquaintance v 1 , i.e. a randomly picked neighboring node of v 0 . Immunize nodes that have been referred to as acquaintances at least n times until the desired immunization coverage is achieved. In the case n = 1, every acquaintance will be immunized immediately. The acquaintance strategy has been shown to identify highly connected individuals, particularly in fat-tailed networks (such as so-called scale-free networks).
We propose another strategy, the community-bridge-finder (CBF) strategy, which rests on the observation that some individuals act as bridges between communities. The goal of the CBF algorithm is to identify such individuals based on random walks, without knowledge of the network structure, and thus without knowledge of the communities in a network. The algorithm works as follows: pick a random node v i = 0 and follow a random path (one node at a time, with the only condition that a The algorithm then picks two random neighbors of v 3 to check for connections to previously visited nodes -and finds one (to v 0 ). (e) Hence, v 2 is dismissed as a potential target, and the random walk continues to v 4 . Again, v 4 does not back-connect to any previously visited node (except, of course, to v 3 ), and thus v 3 is identified as a potential target -(f) thus again, two random neighboring nodes are picked to check for connections to previously visited nodes. Since no back connections can be found, v 3 is identified as a target and immunized. doi:10.1371/journal.pcbi.1000736.g007 node has not been visited by the random walk before). At every node v i$2 , check if there is more than one connection from v i to any of the visited nodes (the requirement for more than one connection stems from the simple fact that every node v i will have at least one connection to v i21 ). If there is just one back connection (i.e. from v i to v i21 ), a potential target v i21 has been identified. As an additional check, pick two random neighboring nodes of v i (other than v i21 ) and check for connections back to the previously visited nodes v j,i . If such connections exist, v i21 is not a potential target -continue the random walk at v i21 . If no such connections exist, immunize the potential target. Discard all information about visits, and start again at a randomly picked node v 0 . A schematic sketch of the algorithm is outlined in Figure 7 .
An algorithmic search for community bridges as described above can potentially take a very long time, depending on the structural features of the network. For example, the frequency of nodes that can potentially meet the immunization requirement set by the algorithm might be smaller than the desired immunization coverage. To prevent endless searches for community bridges, two additional checks are implemented. First, the number of nodes in any running random path does not exceed 10 (this is implemented using a first-in-first-out list that keeps track of the visited nodes). Second, we keep track of all nodes visited, and if a node has been visited at least k times (on any random walk), it will be immunized. In all results presented in this manuscript, we use k = 2. Figure S1 Results from simulations with the same parameters and settings as Figure 1a in the main text, but based on networks with lower community structure. The initial creation of these networks was identical to those created for Figure 1 in the main text (see description in Methods in the main text), but rather than rewiring between-community edges and turn them into withincommunity edges, we randomly rewired within-community edges in the following way: at each rewiring step, we (i) randomly choose a within-community edge, (ii) randomly choose one of the two nodes, (iii) pick a random node in the network, and rewire the edge by detaching it from the node that was not chosen in step (ii), and attaching it to the new node that was chosen in step (iii). At all times, edges must always fall between two distinct nodes, and there can only be one edge between any two pair of nodes. Note that this algorithm is essentially the reverse of the algorithm used to create networks with increased community structure in the main text.  Generation of Human CEACAM1 Transgenic Mice and Binding of Neisseria Opa Protein to Their Neutrophils BACKGROUND: Human CEACAM1 is a cell-cell adhesion molecule with multiple functions including insulin clearance in the liver, vasculogenesis in endothelial cells, lumen formation in the mammary gland, and binding of certain human pathogens. PRINCIPAL FINDINGS: Three genomic BAC clones containing the human CEACAM1 gene were microinjected into pronuclei of fertilized FVB mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by PCR for the presence of the human CEACAM1 gene. Feces of the PCR positive offspring screened for expression of human CEACAM1. Using this assay, one out of five PCR positive lines was positive for human CEACAM1 expression and showed stable transmission to the F1 generation with the expected transmission frequency (0.5) for heterozygotes. Liver, lung, intestine, kidney, mammary gland, and prostate were strongly positive for the dual expression of both murine and human CEACAM1 and mimic that seen in human tissue. Peripheral blood and bone marrow granulocytes stained strongly for human CEACAM1 and bound Neisseria Opa proteins similar to that in human neutrophils. CONCLUSION: These transgenic animals may serve as a model for the binding of human pathogens to human CEACAM1. Introduction CEACAM1 (carcinoembryonic antigen related cell adhesion molecule-1) is a type 1 transmembrane protein expressed on most epithelial cells, phagocytes and activated lymphocytes [1, 2] . In the case of epithelial cells, its cell adhesion activity is associated with diverse functions including angiogenesis [3, 4] , lumen formation [5, 6, 7] and the binding of extracellular pathogens [8] . In the case of neutrophils, it may play a role in the binding of neutrophils to activated endothelium and extravasation into inflamed tissues [9, 10] , and similar to epithelial cells, may bind extracellular pathogens [8, 11, 12] . In the case of activated lymphocytes, it serves as an inhibitory molecule, controlling the strength and duration of the immune response [2] . CEACAM1 is a critical molecule well situated to play a communication role between epithelial cells, that represent the first barrier to infection, and immune cells that control infections that disrupt the epithelial barrier. Thus, pathogens that bind to CEACAM1 may interfere with its normal function, namely maintenance of communication between these two barriers to infection. CEACAM1 has multiple splice forms that are differentially expressed between epithelial and immune cells. The so-called ''long form'' has a cytoplasmic domain containing two ITIMs (immunoreceptor tyrosine-based activation motifs) that, when phosphorylated, may bind Src homology 2 domain-containing tyrosine phosphatases SHP1 and SHP2 [13] to convey inhibitory signals to the T-Cell receptor [14, 15, 16] , B-Cell receptor [17] , and epithelial growth factor receptor [18] . The long form also has a bcatenin binding site [19] and a filamin A binding site [20] . The socalled ''short form'' lacks the ITIMs, but has the ability, like the long form, to bind actin, calmodulin, and annexin II [21] . These activities indicate a strong association of CEACAM1 with actin cytoskeleton [7, 21, 22] .
Among the human extracellular pathogens known to bind to human CEACAM1, Neisseria gonorrhoeae and Neisseria meningitidis have been shown to bind via the expression of their Opa (colony opacity-associated) proteins leading to adherence and invasion into epithelial cells [11, 23] and engulfment by neutrophils [8, 23, 24] , as well as suppression of the T-lymphocyte response [25] and killing human B cells [26] effects that presumably inhibit antibody production. As mentioned above, the inhibitory activity is mediated by phosphorylation of the ITIMs on the long form of CEACAM1 and subsequent recruitment of SHP1/2 [13, 27] that can disrupt phosphorylation of important receptors, including the insulin receptor [28] , EGFR [29] , TCR [14, 15] , BCR [17] , and TLR2 [30] . In the case of Neisseria gonorrhoeae, neutrophils, due to their high levels of CEACAM1 expression, are a major target for spreading the infection in the urogenital tract. A major limitation to studying these interactions in vivo has been the lack of an animal model, since murine CEACAM1 is not a receptor for these human pathogens. In order to overcome this limitation, we have generated a human CEACAM1 transgenic mouse that faithfully expresses human CEACAM1 in the expected tissues, including neutrophils. We demonstrate here that the neutrophils from the transgenic mice as well as human neutrophils bind gonococcal Opa 52 protein expressed on E. coli. Thus, these mice should serve as an appropriate animal model at least for studying some aspects of the human pathogenesis of Neisseria gonorrhoeae infections.
Growth and isolation of Bacterial Artificial Chromosome (BAC) Three BAC clones were purchased from Invitrogen. The DNA was cloned into the EcoRI sites of pBACe3. 6 The DNA of the BAC clones was extracted with Nucleobond PC 2000 EF kit (Macherey-Nagel, Düren Germany) according to the manufacturer's protocol. DNA pellets were dissolved in endotoxin-free water overnight at room temperature, and then used for microinjection.
Murine bone marrow cells were collected from femurs (see later section in Methods) and treated with colcemid (6 ml/mL in 0.075 M KCl) for 2 hours. The cells were fixed using Carnoy's fixative (3:1; methanol:acetic acid), dropped onto non-silanized slides, and air-dried. The slides were baked at 60uC for 12 hours and aged at room temperature for 72 hours. Prior to banding cells were aged at 90uC for 15 minutes. Metaphases were GTG banded and photographed with the BandView imaging system from Applied Spectral Imaging (ASI). Upon completion of the GTG analysis, the slide was de-oiled in Americlear. Slides were de-stained in methanol for 5 minutes and rehydrated in an ethanol series: 100%, 80%, and 70% ethanol. Subsequently, the slides were post-fixed in 1% formaldehyde/1XPBS/50mM MgCl 2 for 2 minutes at room temperature, washed in 1x PBS for 5 minutes and dehydrated in 70%, 80%, and 100% ethanol series. Once the slides were airdried, they were denatured in 70% formamide/2x SSC at 72uC for 30 seconds and immediately placed in cold 70%, 80%, and 100% ethanol and air-dried. Denatured mouse SkyPaint probe (10 ml, Applied Spectral Imaging) was applied to each slide, covered with a glass coverslip and sealed with rubber cement. Slides were transferred to a humidified chamber and placed in a 37uC incubator for 48 hours. Post-washes and detection was done following ASI SKY protocol. SKY metaphases were captured and analyzed with the SkyVision spectral imaging system (Applied Spectral Imaging). CEACAM1 BAC2 DNA was used as a probe. Three nick reactions were prepared using digoxigenin-dUTP. Reactions were combined for precipitation and re-suspended in 10 ml of 10 mM Tris buffer pH 8.5 for hybridization. The probe was hybridized to a human male metaphase to check for probe intensity and correct integration site. The probe (100 ng) was combined with 9 mL hybridization buffer, co-denatured on an 80u hotplate for 5 minutes and incubated overnight at 37uC in a humidified chamber. Post-wash consisted of 50% formamide/2x SSC at 45uC for 15 minutes, 2x SSC at 37uC for 8 minutes and 1x PBD at room temperature for 2 minutes. Probe was detected with rhodamine anti-digoxigenin and following SKY imaging, the slide was placed in 1x PBD for coverslip removal and immersed in room temperature 2x SSC for 1 hour to remove the SKY probe. After SSC treatment, slides were dehydrated and co-denatured for 3 minutes. Hybridization and post-wash were run as described above. Bone marrow preparations from a wild type mouse were used as a negative control and normal human male preparations were used as a positive control. Total murine bone marrow cells were prepared as described in a later section in Methods. Male bone marrow cells were discard tissue from the City of Hope bone marrow transplantation unit.
Growth and FITC labeling of Opa 52 E. coli and vector control E.coli E. coli Top10 containing pTrc99A (Amp) vector only or pTrc99A vector with an IPTG-inducible opa 52 gene were a kind gift from Dr. Scott Gray-Owen (University of Toronto, Canada). Frozen stocks were streaked on a LB plate containing 100 mg Ampicillin, and incubated at 37uC overnight. After 24 h, a single colony was picked and streaked on a LB/Ampicillin plate containing 0.1 mM IPTG, and incubated at 37uC overnight.
FITC was prepared at a final concentration of 1 mg/ml in 50 mM carbonate, 150 mM NaCl buffer (pH 9.2). Bacteria were collected in 1 ml PBS/Mg/Ca and re-suspended by gently pipetting and pelleted at 3000 rpm for 3 min. After removal of the supernatant, the bacteria were re-suspended in 500 ml of FITC solution and incubated at room temperature with gentle shaking in the dark for 30 min. After washing (56) the bacteria were resuspended in PBS/Mg/Ca with 1% BSA to quench remaining FITC. The labeled bacteria were diluted to a working ratio of 30 bacteria to 1 neutrophil.
All experiments involving live animals were conducted with the approval of the City of Hope institutional animal care and use committee. Mice used in the derivation of transgenic mice were maintained on a 14 hr light/10 hr dark cycle. Female mice (FVB/ NJ, Jackson Laboratories) were superovulated with 5 IU of pregnant-mare serum-gonadotropin (National Hormone and Peptide Program, UCLA, Los Angeles, CA) followed 48 hours later by 5 IU of human-chorionic gonadotropin (Sigma-Aldrich, St. Louis MO) and mating to fertile FVB/NJ males. Fertilized oocytes were collected ,12 to 14 hours after the middle of the dark cycle, and treated briefly with bovine hyaluronidase in M2 medium to remove the cumulus cells. Immediately prior to microinjection the purified BAC DNA was diluted to a concentration of 3 ng/mL in 1 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 100 mM NaCl containing 30 mM spermine/70 mM spermidine [31] . The BAC DNA solution was microinjected into the pronuclei of fertilized oocytes using standard techniques, and the manipulated oocytes were transferred surgically into the oviducts of 0.5 dpc pseudo-pregnant recipient female mice and allowed to develop to full term.
The initial screening of founder mice for the human CEACAM1 transgene was carried out by PCR analysis on mouse tail DNA. Briefly, tail samples (1-3 mm) were digested in 0.25 mL 0.4 N NaOH at 95uC for 10 to 15 min until tails were fully digested. Followed by adding 0.375 mL neutralization buffer (0.5 M Tris pH 8.0+20 mM EDTA). Digests were then diluted 10-fold. PCR were then performed by using Bio-Rad iCycler PCR (Bio-Rad Laboratory, Hercules, CA) with 2X fideliTaq PCR master mix (USB, Cleveland, Ohio). Two mL of diluted tail DNA was added into a final 20 mL volume reaction. The sense strand primer 59-CACCATGCCCAGCTAATTTT-39 and antisense strand primer 59-GTGGTCTGTTGCTCCCAGAT-39 only target the human CEACAM1 gene. The amplified product is 260 bp. The amplification parameters were initiated at 95uC for 5 min, then 95u C for 30 s, 53uC for 30 s, and 72uC for 30 s for 35 cycles, followed by 7 min at 72uC for the final extension. The primer sets for the mouse CEACAM1 gene are: sense 59-ATGAACAGTGAGG-CAGTCCC-39 and antisense 59-CAGAGGAAGAGTGTC-CAGGC-39. The amplification parameters were initiated at 95uC for 5 min, then 95u C for 30 s, 55uC for 30 s, and 72uC for 30 s for 35 cycles, followed by 7 min at 72uC for the final extension. The final product length is 155 bp.
Eight weeks old F2 pups (one human CEACAM1 gene positive and one negative by PCR) were euthanized by CO 2 inhalation. Liver, heart, lung, brain, kidney, spleen, small intestine (ileum), and neutrophil (from peripheral blood) or fecal pellets were homogenized and lysed in 1% NP-40 lysis buffer. Cell lysates were incubated on ice for 30 min, followed by centrifugation for 30 min at 4uC at 14000 rpm. Total protein (50 mg) was separated by sodium dodecyl sulfate-gel polyacrylamide electrophoresis, transferred to nitrocellulose membranes and probed with either anti-murine CEACAM1 mAb CC1 (1 mg/mL, kind gift from Dr. Kathryn Holmes, University of Colorado health Sciences, Denver, CO) and anti-b-actin antibody (1 mg/mL, Abcam, Cambridge, UK) antibody or anti-human CEACAM1 mAb T84.1 (1 mg/mL) [32] . Signals were detected on the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Mice were euthanized by CO 2 inhalation. Tissues were collected and fixed in 4% paraformaldehyde overnight. Immunohistochemical staining was performed on 5 mm thick sections prepared from formalin fixed, paraffin-embedded blocks. Sections were deparaffinized in xylene and rehydrated in a graded alcohol series. Slides were quenched in 3% hydrogen peroxide, steamed for antigen retrieval in DIVA/citrate buffer (pH 6. media. Blood smears on positive charged glass slides (Surgipath, Richmond, IL), fixed in methanol for 15 sec and stained as above. Mouse anti-human IgG1 (eBioscience, San Diego, CA) was used as an isotype control.
Transgenic and wild type FVB mice, born within the same litter, were euthanized by CO 2 inhalation. The bone marrow cells were flushed from femurs and tibias by using a 23-gauge needle with 10 mL of cold PBS. Single cell suspensions were prepared by flushing the cell solution twice through a 22-gauge needle and passing the cell solution through a sterile 40-mm nylon mesh. Spleens were removed aseptically, injected with 2 mg/mL of collagenase D and 20 mg/mL of DNase I (Roche Diagnostics, Mannheim, Germany) in Hanks solution, minced, incubated for 30 min at 37uC under 5% CO 2 , and teased through a 40 mm nylon mesh. Red blood cells in bone marrow cells and spleen cells were lysed twice using Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich, Saint Louis, MO 63103). Cells were washed twice with RPMI-1640 medium and subsequently suspended in RPMI-1640 culture medium containing 10% FBS and antibiotics. Cell viability was determined by trypan blue dye exclusion.
Mouse Gr-1 + granulocytes from bone marrow were positively isolated using Anti-Ly-6G MicroBead Kit according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Human neutrophils were isolated from discarded blood. This study was approved by the Institutional Human Subject's Review Board (City of Hope National Medical Center). Neutrophils were isolated by centrifugation over Ficoll-Paque Plus (GE Healthcare Biosciences, Pittsburgh, PA, USA) density gradient. Cell purities were determined by forward light-scatter/ side light-scatter gating of cells stained with PerCP-Cy5.5conjugated anti-CD16 mAb using a flow cytometer (FACSCanto TM II, BD Biosciences) and neutrophil purity was more than 95% [33] .
Neutrophils from mice or human were suspended at 1610 6 cells/mL in RPMI-1640 medium. Degranulation was induced by adding fMLF (Sigma-Aldrich, Saint Louis, MO 63103) to a final concentration of 100 nM followed by incubation at 37uC for 30 min. FITC-labeled E. coli (30610 6 cells/mL) with either Opa 52 or vector control were added to neutrophils (neutrophil/bacteria; 1/30) and incubated at 37uC for 60 min. Infection was arrested by washing the neutrophils with PBS three times.
Neutrophils were fixed in 4% paraformaldehyde with 50 mM HEPES buffer for 20 min at room temperature, followed by 15 min of 0.1% triton X-100, and 15 min of 0.2% BSA in PBS. Anti-human CEACAM1 mAb T84.1 was then added to the fixed cells at a final concentration of 1 mg/mL for 1 hour. After 3 washes with PBS, goat anti-mouse Alexa 555-conjugated antibody (Invitrogen, Eugene, OR) was added at the concentration of 2 mg/ mL for another 30 min followed by 3 washes with PBS. Stained cells were plated on glass slides with Hard Set mounting medium and counterstained with DAPI from Vector Laboratories Inc (Burlingame, CA 94010, USA). Confocal microscopy was performed on a Zeiss Model 310 (Oberkochen, Germany) confocal microscope.
Single cell suspensions from bone marrow or spleen from transgenic or wild-type mice were blocked by anti-mouse CD16/ 32 antibody (Clone: 93, Biolegend, San Diego, CA 92121), and (4) (5) (6) in liver (1, 4) , kidney (2, 5) , and small intestine (3, 6) . B. RT-PCR analysis for Ceacam1 isoforms in transgenic (7) (8) (9) or wild type mice (10) (11) (12) in liver (7,10), kidney (8, 11) , and small intestine (9, 12 Single cell suspensions (1610 6 /mL) from spleen from transgenic or wild-type mice were cultured in a 24-well plate for 72 hours in the absence or presence of 1 mg/ml plate-bound anti-CD3 mAb (145-2C11) and 2 mg/ml soluble anti-CD28 (37.51; BD Pharmingen). Cells were harvested, stained and analyzed as above methods by FACS.
Total RNA was isolated from transgenic mouse liver, kidney and small intestine by using TrizolH method (GIBCO-BRL/ Invitrogen Corp., Carlsbad, CA). Briefly, samples were homogenized in 800 ìL TrizolH (GIBCO-BRL/Invitrogen Corp., Carlsbad, CA), followed by the addition of 160 ìL chloroform. After centrifugation at 4uC at 14,000 rpm for 15 min, the top layer was transferred to a clean microcentrifuge tube and 400 ìL of 2propanol was added, incubated at room temperature for 20 min and centrifuged at 14,000 rpm for 10 min at 4uC. Total RNA was washed in 75% ice-cold ethanol once and after 5 min was centrifuged at 14,000 rpm at 4uC. RNA pellets were air dried and dissolved in 50 ìL RNase-free water. Complementary DNA (cDNA) was prepared using the OmniscriptH reverse transcription system (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. The reverse transcription system contains oligo primers, RNase inhibitor, MMLV reverse transcriptase, dNTP mix, buffer and 3 ìg total RNA in a final volume of 20 ìL. The reaction mixture was incubated at 42uC for 1 h. CEACAM1 isoforms were detected by PCR on a Bio-Rad iCycler PCR (Bio-Rad Laboratory, Hercules, CA) with 2X fideliTaq PCR master mix (USB, Cleveland, Ohio). cDNA (5 ml) was added to a final 25 mL volume reaction. The sense strand primer 59-AACGT-CACCCAGAATGAC-39 and antisense strand primer 59-CTCAG-GACCACTCCAATGA-39 target human CEACAM1 gene. The amplified products are 1118 bp (4L), 1066 bp (4S), 831 bp (3L), 779 bp (3S). The amplification parameters were initiated at 95uC for 5 min, then 95u C for 30 s, 55uC for 90 s, and 72uC for 30 s for 38 cycles, followed by 7 min at 72uC for the final extension. For mouse CEACAM1, the sense strand primer 59-GA-CTCTGTCGACGACATGGAGCTGGCCTCAGCACATC-39 and antisense strand primer 59-GACTCTATCGATTCACTTC-TTTTTTACTTCTGAATAAAC-39 were used to PCR amplify the murine Ceacam1 cDNAs. The four RT-PCR amplified products for murine Ceacam1 correspond to the 4L, 4S, 2L and 2S mRNA splice isoforms. The amplification parameters were same as above.
Three BAC clones containing the human CEACAM1 gene were microinjected into the pronuclei of fertilized oocytes of FVB N/J mice and founder transgenics were generated with BAC2 and BAC5 clones. BAC2Clone (214 kb) covered a large region proximal to human CEACAM1 on chromosome 19, and two other BAC clones (BAC3 and BAC5; 138, and 211 kb, respectively) covered shorter regions flanking the human CEA-CAM1 gene. Although litters were obtained from mice fertilized with all three microinjections, only those from BAC2 and BAC5 transmitted the germline gene to their offspring (Fig. 1A and data not shown), and only those from BAC2 exhibited expression of the huCEACAM1 protein in their feces (Fig. 1B) . Since CEACAM1 is heavily expressed in the GI tract, from the liver throughout the small and large intestine, and is shed into the lumen of the gut like CEA [34] , we used its detection in the feces as an early diagnostic test for protein expression. As can be seen in Fig. 1B , both murine and human CEACAM1 were detected in the feces of the founders. Only the offspring from founder 12 (BAC2) expressed the transgene. As expected, roughly 50% of the pups were positive for human CEACAM1 by both PCR analysis of tail samples (Fig. 1C) and western blot analysis of fecal samples (Fig. 1D ) of the F1 generation from founder 12 (BAC2). Further breeding of the BAC2-12 transgenic mice demonstrated transmission of an expressed CEACAM1 transgene from the F1 to F2 generation at the expected (,0.5) frequency for heterozygotes. No attempt was made to breed the BAC2-12 line to homozygosity at this stage of the study. Expression of the human CEACAM1 transgene was not detected in any of the BAC5 founders or their F1 offspring. Given the small number of litters obtained from each microinjection, it was not feasible for us to evaluate the success rate in terms of stable integration and expression of the various BAC clones.
The chromosomal insertion point for the BAC clone is random in transgenic animals produced by pronuclear microinjection. Using human specific probes for CEACAM1, we were able to demonstrate that the human gene is on chromosome 19 of human cells ( Fig. 2A) , as expected, and the BAC2 clone containing the human CEACAM1 gene was on chromosome 11 of the transgenic mice (Fig. 2C-D) . Although we did not confirm the location of the endogenous murine Ceacam1 gene, it has been reported to be on chromosome 7 [35, 36] . Thus, the murine Ceacam1 and human CEACAM1 genes are on different chromosomes of the transgenic mice. Since mRNA from the human CEACAM1 can be alternatively spliced, it was also important to determine if the expected splice forms were present. For this analysis, RNA was isolated from BAC2-12 transgenic mouse liver, kidney and small intestine and analyzed by PCR using specific primers for the major splice forms expected in human CEACAM1. As seen in Fig. 3A , transgenic mouse liver expressed human CEACAM1-4S and -3S isoforms, while transgenic kidney and small intestine expressed the -4L and -3L isoforms. Analysis of the endogenous expression of murine CEACAM1 isoforms revealed the -4S and -2S isoforms in the liver and intestine, while the kidney expressed mainly the -2S isoform (Fig. 3B) . Since mice express a highly homologous second allele, namely CEACAM2, mainly expressed in the testes and kidney, we cannot be sure if the kidney PCR products are from murine CEACAM1 or CEACAM2. The differences in tissue specific CEACAM1 isoform expression between mouse and man has been previously described [37, 38] .
Having obtained successful germline transmission of the human CEACAM1 gene and CEACAM1 protein expression in the BAC2-12 line of mice, we analyzed tissues from the transgenic mice by immunohistochemical staining to determine if expression of the human and murine genes was similar. First we analyzed heart as a negative tissue to eliminate the possibility that inappropriate expression occurred. Neither murine nor human CEACAM1 was expressed in the hearts of BAC2-12 transgenic mice, and wild type FVB mouse heart is shown as an additional control (Fig. 4A-C) . Three tissues that exhibit high expression of CEACAM1 in both mouse and human are the intestine, kidney, and liver. As seen in Fig. 4D -F, the expression of human CEACAM1 is similar to murine CEACAM1 in the small intestine in terms of distribution and intensity. As expected, both the murine and human CEACAM1 are expressed on on the luminal surface of intestinal epithelial cells. In the case of the kidney, the expression of human CEACAM1 is noticeably higher than the expression of murine CEACAM1 (compare Fig. 4H to Fig. 4G , transgenic, and Fig. 4I, wild type FVB) . This difference may be explained by the fact that mice have two Ceacam genes (Ceacam1 and Ceacam2) that are differentially expressed in the kidney [35, 37] , while humans have a single CEACAM1 gene. Thus, the expression of the human gene in the kidney may reflect the combined expression of the two genes in mouse; however, the anti-murine CEACAM1 antibody used here does not detect murine CEACAM2. The reason for the differential expression of the two mouse genes in the kidney is unexplained, but may reflect a role for the kidney in insulin clearance (see below). In the case of the liver, the staining pattern for murine and human CEACAM1 in the transgenic animals is identical (Fig. 4J-K) . The pattern shows highest expression around the portal triads with decreased expression around the portal artery. This may reflect the known function of CEACAM1 in effecting insulin clearance [39] . The observed CEACAM1 expression pattern follows the entrance of blood from the portal circulation and exit via the portal artery. The staining pattern for the murine CEACAM1 of wild type mice liver (Fig. 4L) is similar but less intense. Further quantitative studies are required to determine if there is a real difference in degree of expression of the endogenous murine Ceacam1 gene between the wild type and transgenic animals.
The high expression of CEACAM1 in kidney and liver was examined at higher magnification. As seen in Fig. 5A -B, the expression pattern for human CEACAM1 is identical to murine CEACAM1, but more intense. The pattern is consistent with the highest levels of expression in proximal tubules (less open lumen) and the lowest in distal tubules (more open lumen). In all cases the staining is on the luminal surface of the renal tubules. CEACAM1 is also expressed on the luminal surface of epithelial cells of the Bowman's capsules surrounding the glomeruli in the kidney (arrow, Fig. 5B ). In the case of the liver, the expression pattern of both murine and human CEACAM1 in the transgenic animals is confined to the bile canaluculi (Fig. 5D-E) . This expression pattern has been described previously [40] and suggests the possibility that the mechanism of insulin clearance by CEACAM1 [39] may be related to the function of bile canaliculi.
The consistent luminal expression of CEACAM1 on epithelial cells supports the hypothesis that CEACAM1 plays a role in lumen formation in several tissues [5, 6, 7] . This role has been documented in mammary epithelial cells grown in either a 3D culture [5, 6, 7] or in humanized mammary fat pads in NOD/SCID mice [41, 42] . CEACAM1 is also expressed on the luminal surface of prostate glands [43] , and like its expression in the mammary gland, is down-regulated in cancer [44] . As a first step in using these transgenic animals as a model for mammary and prostate cancer we examined its expression in these tissues. In the case of the mammary gland, it was necessary to study pregnant females, since the mammary gland is minimally developed in non-pregnant females. As shown in Fig. 6A -C, there was strong luminal expression of both murine and human CEACAM1 in the mammary glands of pregnant transgenic mice. There was also evidence for cytoplasmic expression, perhaps indicating active transport of CEACAM1 from the site of synthesis to the lumen. Similar results were seen for the prostate glands of male human CEACAM1 transgenic mice (Fig. 6 D-F) . These results suggest that these mice may be an appropriate model for studying the role of human CEACAM1 in tumorigenesis within these organs.
Another potential use of human CEACAM1 transgenic mice is to study pathogenesis where CEACAM1 functions as the pathogen receptor. The situation is especially critical in the case of Neisseria gonorrhoeae infections where there is a pressing need for the development of a vaccine [2] . Since the high expression of CEACAM1 on neutrophils plays an important role in its pathogenesis, we determined the expression levels of huCEA-CAM1 on neutrophils isolated from the bone marrow and blood of the transgenic animals. As shown in Fig. 7 , the expression of murine and human CEACAM1 in the transgenic animals was strong. As shown in Fig. 8(A-C) , the staining of murine and human CEACAM1 was strong for neutrophils in blood smears taken from the human CEACAM1+/2 transgenic animals, while staining for human CEACAM1 was negative for a CEACAM12/ 2 littermate. For purposes of comparison, neutrophils from a human blood smear were stained (Fig. 8D) . Notably, human neutrophils stain more intensely than those from the transgenic animals, suggesting a possible effect of an expression difference between the species. In addition, we examined the expression of human CEACAM1 in the spleen of transgenic animals. Based on cell surface staining, there was no expression on T-cells and significant expression on B-cells (Fig. 9A) . Consistent with these results, CEACAM1 has been shown to be inducible on both murine [45] and human T-cells [14] . The expression of CEACAM1 on murine [46] and human [17, 47] B-cells has been previously reported. Activation of splenocytes from transgenic animals by anti-CD3/28 antibodies for three days revealed increased expression of murine, but not human CEACAM1 in T-cells (Fig. 9B) . B-cell expression of murine and human CEACAM1 is shown for comparison.
Having demonstrated expression of human CEACAM1 on the neutrophils of the transgenic animals, it was important to demonstrate that they could bind Opa proteins, the requisite Neisseria gonorrhoeae ligand for human CEACAM1 [8] . For this analysis, we utilized E. coli expressing recombinant Opa 52 protein.
As shown in Fig. 10 , primed neutrophils from transgenic animals were able to bind E.coli-Opa 52 much greater than E. coli-vector control. Controls included binding of the parental (Opa-) strain on wild type neutrophils and Opa52-expressing bacteria binding to human neutrophils. These results suggest that the neutrophils in the transgenic animals may mimic the behavior of human neutrophils towards binding of Neisseria pathogens.
The generation of human CEACAM1 transgenic mice is an important model to demonstrate equivalence in function of the murine and human gene orthologs. Since pathogens that utilize human CEACAM1 as their receptor do not recognize murine CEACAM1, such a model is essential to study their pathogenesis. Furthermore, in order to demonstrate that CEACAM1 specific anti-pathogen vaccines are effective, the model must mimic the human tissue specific pattern of CEACAM1 expression. Thus, our approach was to generate transgenic mice that expressed the entire human CEACAM1 gene rather than a cDNA for CEACAM1. Although we attempted to generate several lines using BAC clones that spanned different regions of chromosome 19 encoding the CEACAM1 gene, only a single line that actually expressed the protein was generated. Given the large number of variables that affect both integration, expression, and transmission of a gene, the reason for this result is unknown. Fortunately, the BAC2-12 line obtained transmitted the transgene from the founder mouse to F1 and F2 generations and demonstrated the expected frequency for heterozygotic expression in subsequent generations. Furthermore, human specific splice forms were found in three major tissues (liver, intestine and kidney) of known CEACAM1 expression and tissue staining for human CEACAM1 demonstrated the expected staining pattern, namely at the lumen of epithelial cells.
Both murine Ceacam1 and human CEACAM1 genes produce multiple mRNA splice isoforms. Murine Ceacam1 generates mRNAs having mainly 4 or 2 ectodomains with long or short cytoplasmic domains. Indeed, the expected major splice isoforms of murine Ceacam1 were found in the liver, intestine, and kidney of both control and transgenic mice with a predominance of the -2S isoform. The human CEACAM1 gene, in contrast, generates mRNAs with different splice isoforms, namely -4L/S and -3L/S. The expected human splice isoforms were observed in the BAC2-12 transgenic line, with a predominance of -4S in the liver and -4L in the intestine and kidney. Given the differences in murine and human splice isoforms observed in the transgenic animals, we Figure 9 . Expression levels of murine and human CEACAM1 on spleen B and T cells of BAC2-12 transgenic and wild type mice. Freshly isolated spleen cells (A) and Spleen cells treated with 1 mg/mL anti-CD3 and 2 mg/mL anti-CD28 mAb for three days (B) from TG or WT mice were stained with either the anti-murine CEACAM1-PE or anti-human CEACAM1-PE and co-stained with anti-CD3-FITC and anti-B220-APC. Results shown are representative of three independent experiments with similar results. doi:10.1371/journal.pone.0010067.g009
conclude that the information governing the production of differentially spliced mRNA isoforms must be encoded in their respective genes. A comparison of the gene sequences at putative splice recognition sites may reveal which motifs are responsible for the observed mRNA species and tissue specific splicing. Preliminary studies on the human gene have begun to identify motifs responsible for generation of the long and short cytoplasmic domain splice forms [48] . The ratio of long and short CEACAM1 protein isoforms could critically affect cell function, since the short form lacks the ITIM motif. While the significance of the number of ectodomains is less well understood, the relative strength of cellcell adhesion may be affected [2] . The expression of murine CEACAM1 in the developing embryo at both the message and protein level has been studied in detail [49] . Notably, murine CEACAM1 was found in the developing gut, lung, and kidney epithelium. However, the occurrence of two Ceacam genes in the mouse (and not in human) complicates the picture, with the Ceacam2 gene mainly expressed in the kidney and testis [37] . The high expression of CEACAM1 in murine liver is striking and has been shown to regulate insulin clearance [39] . The pattern of human CEACAM1 expression closely mimics the mouse, with perhaps a caveat about the differential expression of the two murine Ceacam genes in the mouse. In our study of the human CEACAM1 transgenic mouse, we were able to compare the staining of tissues for mouse and human CEACAM1. Except for the kidney, the tissue staining patterns and intensities were similar. In the case of the kidney, the patterns were identical but the intensity of staining for human CEACAM1 was greater than for murine CEACAM1. This result probably reflects the higher expression of murine Ceacam2 (not measured) over that of Ceacam1 in the kidney.
We were especially interested in the expression of the human gene in the immune system. Murine CEACAM1 is known to be expressed on spleen B-cells [50] , serving as a receptor for the murine hepatitis virus. Similarly, human CEACAM1 is expressed on germinal center B-cells [17] . In the case of murine and human T-cells, little or no surface expression is observed on resting Tcells, but is highly expressed on activated T-cells [14, 51] . As expected, we found cell surface expression of both murine and human CEACAM1 on splenic B-cells, but not on splenic T-cells. Similar expression of murine and human CEACAM1 is expected on the surface of neutrophils, whether from the bone marrow or blood. Indeed, we found high levels of murine and human CEACAM1 on neutrophils from either source. Based on this preliminary analysis, it is likely that the CEACAM1 expression in the immune system mimics that found in mouse and man.
The model system was further validated by examining the binding of one of the Opa proteins found in Neisseria and known to bind to human CEACAM1. In this example, we chose Opa 52 a Neisseria specific protein which had previously been shown to confer binding to human neutrophils or to Hela cells transfected with human CEACAM1 [8] . Since Neisseria may adhere to and be phagocytosed by neutrophils via a variety of receptors, we chose to demonstrate Opa 52 binding using E. coli expressing this protein. As expected, the Opa 52 positive bacteria were bound in greater numbers by the transgenic neutrophils than the Opa 52 negative bacteria. Thus, the model appears to mimic what is observed with human neutrophils using Opa 52 positive Neisseria. Furthermore, the model should allow investigation into insulin clearance, vasculogenesis, lumen formation, and other functions ascribed to human CEACAM1 in a physiological setting. TUBERCULOUS SARCOIDOSIS: DOES IT EXIST?   DotKnot: pseudoknot prediction using the probability dot plot under a refined energy model RNA pseudoknots are functional structure elements with key roles in viral and cellular processes. Prediction of a pseudoknotted minimum free energy structure is an NP-complete problem. Practical algorithms for RNA structure prediction including restricted classes of pseudoknots suffer from high runtime and poor accuracy for longer sequences. A heuristic approach is to search for promising pseudoknot candidates in a sequence and verify those. Afterwards, the detected pseudoknots can be further analysed using bioinformatics or laboratory techniques. We present a novel pseudoknot detection method called DotKnot that extracts stem regions from the secondary structure probability dot plot and assembles pseudoknot candidates in a constructive fashion. We evaluate pseudoknot free energies using novel parameters, which have recently become available. We show that the conventional probability dot plot makes a wide class of pseudoknots including those with bulged stems manageable in an explicit fashion. The energy parameters now become the limiting factor in pseudoknot prediction. DotKnot is an efficient method for long sequences, which finds pseudoknots with higher accuracy compared to other known prediction algorithms. DotKnot is accessible as a web server at http://dotknot.csse.uwa.edu.au. RNA is a versatile nucleic acid, which is no longer seen as the passive intermediate between DNA and proteins. Numerous functional RNAs with an astonishing variety have been uncovered in the past decade. For example, non-coding RNAs participate in a wide range of cellular processes, are able to regulate gene expression and can act as catalyst (1, 2) . Macromolecule function is closely connected to its 3D folding and structure prediction from the base sequence is thus of great importance. RNA structure formation is understood to be hierarchical and, therefore, secondary structure prediction is the foundation for determining the tertiary folding (3, 4) .
There are two streams in computational RNA secondary structure prediction: comparative approaches and single sequence methods. In general, comparative approaches give accurate results for a set of wellconserved sequences (5) . However, comparative methods rely on the quality of multiple alignments and are not always feasible for RNA structure prediction, due to a lack of reliable data sets (6) .
When only a single sequence is given, the most popular approach for RNA structure prediction is free energy minimization. In the minimum free energy (MFE) model, continuous base pairs contribute enthalpic terms and loop regions are purely entropic. RNA comprises various secondary structure elements, i.e. stems, hairpin loops, bulge loops, internal loops and multiloops. Much experimental work has been done to determine their free energy parameters (7, 8) . The key concept that allows for dynamic programming is that all of these motifs are non-crossing and self-contained in terms of their free energy. The MFE secondary structure based on the additive free energy model can be predicted in Oðn 3 Þ time and Oðn 2 Þ space using dynamic programming (9, 10) . MFE prediction has been extended in several ways (11) . Suboptimal structures with free energy close to the MFE can be calculated (12, 13) . Using the dynamic programming principle, the full equilibrium partition function for RNA secondary structure is computed in Oðn 3 Þ time and Oðn 2 Þ space (14) . From the partition function, probabilities for base pairs and structure elements are derived. The main advantage of the MFE algorithm is its guarantee to find an optimal structure with regards to the underlying energy model. However, the inability to predict crossing structure elements, so-called pseudoknots, is a major drawback.
Pseudoknots are functional structure elements, which have been reported in most classes of RNA (15) . A pseudoknot forms when unpaired bases in a loop pair with complementary bases in a single-stranded region outside the loop (Figure 1 ). Pseudoknots play key roles in viral genome replication and regulation of protein synthesis (16) . Pseudoknots are also found in the cell where they participate in processes such as splicing, ribosomal frameshifting, telomerase activity and ribosome function (17) (18) (19) . In many cases, they assist in the overall 3D folding (20, 21) and should not be excluded from computational structure prediction.
RNA secondary structure prediction with arbitrary pseudoknots under a basic energy model is NP-complete (22, 23) . Restricted classes of pseudoknots can be included in the dynamic programming algorithm for prediction of the MFE structure, resulting in high computational complexity. In dynamic programming, there is always a trade-off between the generality of pseudoknots, which can be predicted, and runtime. Rivas and Eddy (24) cover a broad class of pseudoknots including kissing hairpins in their algorithm, which requires Oðn 6 Þ time and Oðn 4 Þ space. More restricted pseudoknots are included in other dynamic programming algorithms, which have runtime of Oðn 5 Þ using Oðn 4 Þ or Oðn 3 Þ space (22, 23, 25) . All of these algorithms are only feasible for short RNA sequences. The most practical method is pknotsRG, which computes the MFE structure with canonical simple recursive pseudoknots in Oðn 4 Þ time and Oðn 2 Þ space (26) . Dynamic programming does guarantee to find a structure with minimum free energy with respect to the underlying energy model. However, the energy model for pseudoknots used in dynamic programming is only a simple parameterization adopted from the affine multiloop energy model (24) (25) (26) . It was first introduced by Rivas and Eddy (24) because of the lack of experimentally measured parameters for pseudoknot energies. Predictive accuracy of MFE folding is always limited by the underlying energy model, and hence pseudoknot prediction results are poor for longer sequences.
Due to the computational complexity of dynamic programming for pseudoknot prediction, heuristic approaches were developed as an alternative. Heuristic methods do not necessarily return the MFE structure; however, they can include a wide class of pseudoknots and more advanced energy models in reasonable runtime. RNA secondary structure prediction including pseudoknots has been approached using genetic algorithms (27, 28) , stochastic context-free grammars (29, 30) , kinetic folding simulations (31, 32) and maximum weighted matching in a folding graph (33) . Iterative stem adding procedures have also been developed (34) (35) (36) . In several heuristic algorithms, the underlying energy model for secondary structures is simple base pair maximization, neglecting loop entropies (33, 34) . This may lead to unreliable results, especially for longer sequences. Another drawback is that most heuristic methods reported in the literature employ the same affine pseudoknot energy model as dynamic programming.
A different algorithmic framework is used in heuristic pseudoknot detection programs (37) (38) (39) (40) . Here, one attempts to find promising pseudoknot candidates in a sequence as a first step. These potential pseudoknots are subsequently analysed and verified. After pseudoknot detection, the remaining sequence can be folded using free energy minimization in Oðn 3 Þ time and Oðn 2 Þ space. Pseudoknot detection has two main advantages over dynamic programming. First, it is computationally much more efficient and, therefore, practical for scanning long RNA sequences for pseudoknots. Second, the underlying framework is less restrictive and allows for easy incorporation of sophisticated energy rules for pseudoknots or even comparative information. Pseudoknot detection delivers accurate pseudoknot prediction results for longer sequences in many cases (37, 39) .
Pseudoknot energy models have been studied in more detail in the past years and reliable energy parameters are in high demand. It is widely accepted that pseudoknot energy cannot be estimated with an additive model as used in MFE folding. There is strong interference between opposite loops and stems (L 1 and S 2 , L 3 and S 1 ), which come in close contact in the 3D fold. In a simple hairpin type (H-type) pseudoknot, loops L 1 and L 3 span across the deep narrow (major) groove and the shallow wide (minor) groove, respectively. The corresponding loop entropies are, therefore, not equivalent and depend on the structure and length of the opposite stem (41, 42) .
The lack of loop entropy parameters is a critical issue in pseudoknot prediction (43) . For H-type pseudoknots with interhelix loop size 1 nt, loop entropy values were derived using several fitted parameters (41) . Gaussian chain approximation based on polymer physics for pseudoknot loop entropies was also proposed (44) . Lattice-based models were developed to take into account volume exclusion effects (45) (46) (47) . The most successful models are based on the atomic coordinates of the RNA backbone. Using polymer statistical mechanics, Cao and Chen (42) calculated loop entropy parameters for pseudoknots with interhelix loop size 1 nt. Recently, their so-called virtual bond model has been extended to pseudoknots with interhelix loop size 6 nt (48) . Inclusion of such a pseudoknot energy model is not straightforward in dynamic programming in reasonable runtime due to dependencies between opposite loops and stems. Calculation of the full partition function under the virtual bond model takes Oðn 6 Þ time and Oðn 2 Þ space, making the approach only feasible for sequences shorter than say 150 nt (42) . However, pseudoknot energy parameters derived from the virtual bond model are readily available in tabular form for many stem and loop length combinations (42, 48) . It is straightforward to incorporate such energy parameters in a pseudoknot detection approach, making prediction using much improved energy models feasible for long sequences.
We present DotKnot, a pseudoknot prediction method that incorporates stem-loop correlated pseudoknot energy parameters from the virtual bond model (42, 48) . The workflow is similar to the detection approach used in KnotSeeker (39) , with two main improvements. First, a secondary structure partition function calculation in Oðn 3 Þ time and Oðn 2 Þ space is the basis for finding a set of promising structure elements with high confidence (14) . This set includes heuristically derived bulge loops, internal loops and multiloops with low free energy. Second, pseudoknot candidates are assembled using the set of promising structure elements and, therefore, loop entropy parameters can readily be used for pseudoknot energy evaluations (42, 48) . There is no dynamic programming kernel for verification of pseudoknot candidates. This is a major step towards successful pseudoknot prediction as the vast majority of methods in the literature use simple approximations of pseudoknot energies. However, improving the folding model behind the algorithmic framework is clearly the key to accurate prediction (43) .
DotKnot predicts the class of recursive H-type pseudoknots where one of the pseudoknot stems can be interrupted by bulge or internal loops. All of the three pseudoknot loops are allowed to form internal secondary structures. Using the set of structural elements derived from the probability dot plot as a construction kit, we could predict a broad class of pseudoknots, including kissing hairpins, in an efficient manner. However, our knowledge about energy parameters and folding mechanisms become the limiting factor for a pseudoknot search tool such as DotKnot. Given an RNA sequence, DotKnot returns only the (possibly empty) set of detected pseudoknots. These detected pseudoknots can subsequently be verified using laboratory techniques or comparative information. The remaining non-crossing sequence can then be folded in Oðn 3 Þ time and Oðn 2 Þ space using state-of-the-art RNA secondary structure prediction algorithms to obtain a global folding.
The detailed workflow for DotKnot is shown in Figure 2 . Given an RNA sequence, DotKnot finds sequence fragments with pseudoknot folding potential. These candidates are analysed in terms of their free energy values and credibility in the folded sequence. The output is a (possibly empty) set of detected pseudoknots, which can then be examined closely.
RNA structure forms through complementary base pairing, resulting in stabilizing stems and destabilizing loop regions. The basic building blocks for a pseudoknot are two crossing stems. We use the probability dot plot derived from the partition function as a guide for finding such building blocks. Given an RNA sequence, the partition function Q is defined as the weighted sum over the set of all possible secondary structures S, i.e. QðTÞ ¼ P s2S e ÀÁG s =RT where R is the universal gas constant and T the temperature. Once the partition function is known, probabilities for base pairs and structure elements can be calculated. The software RNAfold returns the probability dot plot representing both base pair probabilities and stack probabilities (11) . The stack probability P ij for a base pair ði, jÞ is defined as the probability that pair ði, jÞ and the subsequent pair ði þ 1, j À 1Þ are formed simultaneously (49) .
It has been shown that choosing base pairs with high probability can improve secondary structure prediction (50) . There are also several approaches that discuss the exclusion of base pairs with low probability for improving runtime. Here, the common technique is to use a cut-off value for base pair probabilities in order to determine only significant base pairs (51) . However, for pseudoknot stems we cannot simply dismiss base pairs with low probability. The partition function calculation is based on the ensemble of secondary structure elements, which are non-crossing interactions. In general, the pseudoknot stems are visible in the probability dot plot as they are members of the folding space. One can expect that at least one of the pseudoknot stems will have low base pair and stack probabilities. By default, RNAfold only displays probabilities larger than 1 Â E À5 . For pseudoknot detection in the dot plot, we use a cutoff probability of 1 Â E À11 to make sure that all pseudoknot stems can be found for long sequences. Given the probability dot plot, stems are assembled according to certain criteria. First, a stem must have at least three base pairs. Second, one can expect that the stack probabilities in a stable stem do not rise or drop sharply (49) . Helix elongation is an energetically favourable process; however, wobble base pairs can be destabilizing (8, 52, 53) . Furthermore, there may be other stable secondary structure elements competing for base pairs. Therefore, we demand that the absolute percentage increase or decrease of stack probabilities for subsequent base pairs ði, jÞ, ði þ 1, j À 1Þ in a stem has to be smaller than a certain threshold .
One has to keep in mind that base pair probabilities are not independent. Therefore, stem probabilities can never be calculated by simply multiplying the base pair probabilities. However, the average probability of participating base pairs in a stem can be used as a confidence indicator (54) . In our approach, we calculate the confidence c for a stem as the average stack probabilities. For each stem, an absolute weight is introduced in addition. The confidence c is based on the energy model for secondary structures, excluding pseudoknots. Therefore, pseudoknot stems tend to have low average probabilities especially for longer sequences, which does not necessarily correspond to their dominance in native RNA structures. We assign two additional weights for a stem based on a local energy evaluation. The simple stacking model employs the favourable base pair stacking parameters in a stem; however, it excludes the destabilizing energy contributed by the hairpin loop. The more sophisticated free energy model includes all entropy and enthalpy parameters derived by the Turner group (8) . The tool RNAeval is used to evaluate the local free energies of the stem candidates according to the two energy models introduced above, taking into account dangling ends on both sides (11) . DotKnot stores two stem weights w stack ðs i Þ (simple stacking model) and wðs i Þ (free energy model) for a stem s i . Only stems s i satisfying the following conditions are kept: w stack ðs i Þ < 0:0 kcal/mol and wðs i Þ < 4:0 kcal/mol. The resulting stems are stored in a stem dictionary D s ¼ s 1 ,s 2 , . . . , s n f g . Each stem s i has a unique key a i ,b i ð Þ, where a i is the start position and b i the end position of the stem in sequence S. For each stem s i , we store its length and the following values in the stem dictionary: cðs i Þ,w stack ðs i Þ and wðs i Þ.
In RNA structures, stems are often interrupted by bulge loops or internal loops. In the following, a stem interrupted by bulge loops or internal loops is denoted as s L i ( Figure 3 ). Naive construction of interrupted stems s L i from the stem dictionary D s would be inefficient due to a large number of possible stem combinations. Therefore, we employ a heuristic strategy for finding interrupted stems with low free energy.
Given the stem dictionary D s , interrupted stems are constructed using maximum weight independent set (MWIS) calculations with the confidence indicators as stem weights (Supplementary Algorithm 1). Using confidence c instead of local free energy gives better results in the MWIS calculation. First, it implicitly penalizes the formation of long bulge or internal loops. Second, the confidence is a relative measurement based on the whole folding ensemble. Only stems s i with confidence cðs i Þ ! 1 Â E À3 are considered as base pairs below this threshold are unlikely to participate in non-crossing secondary structure formation (55) . This cutoff step also significantly reduces runtime for longer sequences. Each stem s i 2 D s has two (left and right) endpoints a i and b i and is represented as an interval on the line. First, the sorted endpoints list for all stems is constructed and scanned from left to right. If a right endpoint is discovered, a candidate list of stems nested in the interval
is returned for the corresponding stem s i . A MWIS calculation on the candidate list returns the set of internal stems with maximum weight in linear time (56) . The outer stem s i can, therefore, become an interrupted stem s L i with bulges and internal loops or the exterior stem of a multiloop. The sum of confidences serves as the updated weight for outer stem s i . After the whole endpoints list has been scanned, stems interrupted by bulge loops or internal loops are stored in a new dictionary D L s . Multiloops are stored in a separate dictionary D M s . As a last step, the free energies of the interrupted stems and multiloops are evaluated with the tool RNAeval (11) . Only structure elements with negative free energy are stored.
So far, DotKnot derived a set of regular stems, interrupted stems and multiloops in a heuristic fashion from the probability dot plot (Figure 2 ). There is no guarantee to find the structure element with local minimum free energy for a given sequence stretch. However, dictionaries D L s and D M s will contain structures with free energy value close or equal to the local minimum free energy. These elements will be of great benefit for the elimination of false positive pseudoknots.
One major advantage of the detection approach is that the pseudoknot prediction target class is predefined and transparent. In contrast, dynamic programming algorithms construct pseudoknots through their recursion scheme, which in some cases leads to unspecific pseudoknot target classes (24, 57) . In this work, recursive H-type pseudoknots will be considered similar to the class of pseudoknots predicted by pknotsRG (26) . A recursive H-type pseudoknot has two crossing stems S 1 and S 2 , resulting in three loops L 1 , L 2 and L 3 . All of the three loops are allowed to form internal secondary structures; however, loop-loop interactions are not allowed. In this work, we also include pseudoknots where one of the stems S 1 or S 2 is interrupted by bulges or internal loops. This leads to a more comprehensive prediction class as in pknotsRG, where only bulges of size 1 nt are considered (26) . In general, the three dictionaries D s , D L s and D M s allow for the construction of complicated pseudoknot folds. For example, it is straightforward to construct kissing hairpins from the stem dictionary. The pseudoknot energy parameters become the bottleneck in this approach, not necessarily the complexity of pseudoknots. The three main steps for constructing recursive H-type pseudoknots are shown in Figure 4 . First, so-called core H-type pseudoknots form through simple combination of two crossing stems. They become recursive H-type pseudoknots when additional secondary structure elements fold in each of the three loops. Note that recursive pseudoknots are not allowed in the loops as this may lead to sterically infeasible configurations. The overall recursive H-type candidate pseudoknot is assembled in a third step.
On the first level, two crossing stems are combined to form a core H-type pseudoknot (Figure 4) . Core H-type pseudoknots are the building blocks for more complex pseudoknots. The pseudoknot stems can either be regular stems taken from the dictionary D s or interrupted stems from the dictionary D L s ( Figure 5 ). Here, we only allow pseudoknots with at most one interrupted stem because for more complex and less rigid pseudoknots we would have to employ a highly assumptive energy model. Certain loop length restrictions are applied because more meaningful results can be expected from prediction of shorter and well-studied pseudoknots. Loop L 1 and L 3 are both required to have at least 1 and 2 nt, respectively. Interhelix loop L 2 can have a size of 0 nt. All three loops are restricted to a maximum length, as there are no reliable energy parameters for very long pseudoknots. Loops L 1 and L 3 can have a length of up to 100 nt, whereas interhelix loop L 2 is restricted to a maximum length of 50 nt. During construction of the pseudoknot candidates, we discovered that crossing stems may compete for a base pair. This leads to an overlap at loop L 2 . In such a case, one of the stems is truncated according to certain rules (Supplementary Figure S2 and Supplementary Algorithm 2).
There is little knowledge about the 3D folding of complex pseudoknots with interrupted stems, which may lead to bending or distortions of the RNA A-helix. Loops L 1 and L 3 need to cross the major and minor groove of stems S 2 and S 1 , respectively. To disallow sterically infeasible configurations for long interrupted stems, we make the following assumptions (41) . For interrupted stems with more than 10 bp (including bulges and internal loops), the loops bridging the stems must have a minimum length: loop L 1 must be ! 2 nt and loop L 3 must be ! 6 nt.
After the first level of pseudoknot construction, free energy values are evaluated for each core H-type pseudoknot, which allows to filter unlikely pseudoknots. Only pseudoknots with low free energy (and therefore likely to form) will remain for the next step, which involves recursive secondary structure formation in the three pseudoknot loops. For all core H-type pseudoknots p 1 , . . . , p n , free energy ÁG is calculated as
where TÁS L 1 ,L 2 ,L 3 is the purely entropic free energy for loops L 1 , L 2 and L 3 . Three different pseudoknot energy models are employed for the loop entropy calculation, each of that comprise pseudoknots with certain characteristics. The length of loop L 2 determines which energy I II III Figure 4 . Construction of a recursive H-type pseudoknot. On the first level, two stems form a core H-type pseudoknot. On the second level, recursive secondary structure elements may form in each of the three loops. On the third level, the recursive H-type pseudoknot is assembled. model is used for the respective pseudoknot candidate (Table 1) . For details on pseudoknot loop entropy calculation using the virtual bond models CC06 and CC09 see (42) and (48), respectively. For pseudoknots with long interhelix loop L 2 , there is no physical loop entropy model available and we have to employ heuristics (24) (25) (26) . This heuristic energy model (LongPK) is also used for pseudoknots with one interrupted stem regardless of the length of loop L 2 . Stems interrupted by long bulge or internal loops are likely to result in bending rather than rigid formations (48) . Therefore, the loop entropy calculation becomes intricate. We calculate the loop entropy as TÁS L 1 ,L 2 ,L 3 ¼ þ Â L, where L is the number of unpaired nucleotides in the three pseudoknot loops with ¼ 7:0 kcal/mol and ¼ 0:1 kcal/mol. For pseudoknots with regular stems and loop length L 2 of 0 or 1 nt, one can generally assume that the two pseudoknot stems are coaxially stacked. This leads to a stabilizing effect for the two base pairs at the interhelix junction. Here, coaxial stacking is calculated using the Turner energy model, multiplied by an estimated weighting parameter g < 1 and added to the free energy of a pseudoknot (7, 8, 24) . For pseudoknots with interrupted stems and absent loop L 2 , we also add the appropriate coaxial stacking energy multiplied by an estimated weighting parameter g < 1. After energy evaluation, only pseudoknots with negative free energy are stored in the pseudoknot dictionary D p . Additionally, we demand that the free energy of a core H-type pseudoknot needs to be lower than the free energies wðS 1 Þ and wðS 2 Þ of the pseudoknot stems S 1 and S 2 .
Second level: recursive structure formation Secondary structure elements often form in pseudoknot loops, resulting in a recursive pseudoknot. After finding stable core H-type pseudoknots, the three loops L 1 , L 2 and L 3 are examined for likely secondary structure elements. It is a valid assumption that the three loops form recursive elements independently and can be treated separately (Figure 4) . From an algorithmic point of view, it is efficient to find recursive structure elements using a MWIS calculation (Supplementary Algorithm 3) . Given a core H-type pseudoknot, three candidate lists hold all possible secondary structure elements from dictionaries D s , D L s and D M s contained in each of the loops L 1 , L 2 and L 3 . A standard MWIS calculation with free energy weights for each of the three lists returns the set of secondary structure elements with best local free energy for each loop. The results are combined to form a recursive H-type pseudoknot.
For each recursive H-type pseudoknot, we need to evaluate the free energy with respect to the recursive structure elements. As described in Table 1 , the set of pseudoknots is divided into the three different classes according to the length of loop L 2 . To account for recursive structure elements, the loop entropies need to be recalculated. Following the notation in Cao and Chen (48) , we first calculate the effective loop lengths. The effective loop length l eff i for a pseudoknot loop L i (i ¼ 1, 2, 3) with internal structure elements is the number of unpaired nucleotides outside those internal structure elements plus the number of internal structure elements. (c) 5 3 For all pseudoknots with recursive structure elements, the loop entropy is recalculated using the effective loop lengths. Internal secondary structure elements add free energy values as given by the Turner energy model (8) . We keep only pseudoknots p i in the pseudoknot dictionary D p , which satisfy the following two conditions. First, a recursive H-type pseudoknot must have free energy ÁGðp i Þ < À5:25 kcal/mol. Second, the normalized pseudoknot free energy must fulfill ÁGðp i Þ=l i " where l i denotes the length of pseudoknot p i . Setting the threshold to " ¼ À0:25 is not too restrictive; however, it helps us to eliminate pseudoknots with high free energy (58) . For example, for the 3 0 -UTR TMV sequence with length 214 nt, we have 445 candidate stems in dictionary D s and 2026 pseudoknot candidates before filtering. After the length-normalized filtering step, only 274 pseudoknot candidates remain.
The set of recursive H-type pseudoknots stored in dictionary D p will certainly contain false positive pseudoknots. Therefore, a MWIS calculation with local free energy weights is employed using the structure elements from all four dictionaries: D s , D L s , D M s and D p . Stems s i 2 D s need to have confidence cðs i Þ > 1 Â E À3 to participate (55) . Furthermore, stems are allowed to contain nested structure elements, including pseudoknots. The MWIS procedure with nesting is described in detail in KnotSeeker (39) . Note that we do not include the free energy gain of À1:5 kcal/mol for the outer loop as in KnotSeeker, because favourable nested structures are already included through dictionaries D L s and D M s .
We evaluated our algorithm on a set of pseudoknotted and pseudoknot-free sequences of different RNA types ( Table 2) . Given a sequence, DotKnot is a method that predicts only pseudoknots. Therefore, predictive accuracy is measured for base pairs belonging to a pseudoknot. Two measurements are used for comparison of DotKnot and other selected algorithms from the literature. For each published pseudoknot in a sequence we report sensitivity S ¼ 100 Â TP=TP þ FN ð Þ and the positive predictive value PPV ¼ 100 Â TP=TP þ FP ð Þ . True positive (TP) corresponds to the number of correctly predicted base pairs in the predicted pseudoknot, False negative (FN) to the number of base pairs in the published pseudoknot that were not predicted and False positive (FP) to the number of incorrectly predicted base pairs in the predicted pseudoknot. A pseudoknot is said to be predicted by an algorithm if it is a crossing structure element and at least one of the two pseudoknot stems is partially predicted. Furthermore, the ratio r ¼ (number of correctly predicted pseudoknots)/(number of predicted pseudoknots) is reported. We compare DotKnot to two dynamic programming methods, namely pknots (24) and pknotsRG (26) , the pseudoknot detection tool KnotSeeker (39) and the heuristic approach HotKnots (35) . HotKnots returns a number of sub-optimal scenarios; however, we only evaluate predictive accuracy for the best solution.
5S rRNA, tRNA and miRNA are pseudoknot-free types of RNA. For the 5S rRNA and miRNA sequences chosen in our test set, DotKnot does not introduce any spurious pseudoknots. For two of the tRNA sequences, DotKnot predicts false positive pseudoknots. KnotSeeker, pknotsRG and HotKnots also predict false positive pseudoknots for some of the tRNA sequences. Minimum free energy prediction is known to have low accuracy for tRNAs. This is due to modified bases as well as coaxially stacked helices, which determine the characteristic 3D cloverleaf fold of tRNAs (24, 26) . Coaxial energies are implemented by pknots, which might explain why it does not predict false positive pseudoknots for the tested tRNA sequences.
Several of the test sequences contain pseudoknots where one of the core pseudoknot stems is interrupted by bulges or internal loops: Escherichia coli tmRNA, Legionella pneumophila tmRNA, the SARS frameshifting pseudoknot, human telomerase and Tetrahymena telomerase. For all of these pseudoknots, DotKnot delivers the best results in terms of sensitivity and specificity. For example, the Tetrahymena telomerase RNA (TER) contains a pseudoknot with a conserved central GA bulge in one of its stems, which is vital for telomerase function (76) . DotKnot perfectly predicts this bulged pseudoknot, while all other methods do not predict a pseudoknot structure. The biological relevance of pseudoknots with bulged residues should not be underestimated. Therefore, a prediction algorithm that can handle pseudoknots with interrupted stems such as DotKnot is highly desirable.
For complex pseudoknot foldings such as the hepatitis delta virus (HDV) double pseudoknot configuration, DotKnot gives the best prediction results out of all tested algorithms. The CrPV IRES has a long pseudoknot, which contains another nested pseudoknot. For this pseudoknot, DotKnot also gives the best prediction in terms of sensitivity and PPV.
DotKnot has excellent accuracy for simple H-type pseudoknots as those reported in many viral 3 0 -UTRs and frameshifting regions. We found that the energy models CC06 and CC09 used for predicting such 
Note that 5S rRNA, tRNA and miRNA are pseudoknot-free.
pseudoknots give better results than the heuristic pseudoknot energy parameters employed by the other algorithms. For example, the NeRNV and TMV 3 0 -UTRs both have five pseudoknots where four are simple H-type pseudoknots with interhelix loop 1 nt. For these pseudoknots, DotKnot gives the most accurate predictions, which we claim is due to the improved energy parameters by Cao and Chen (42, 48) . In terms of computational performance, DotKnot is very efficient due to the sparseness of the probability dot plot, the resulting low number of pseudoknot candidates and the implementation using dictionaries in Python. DotKnot runs in the order of seconds for all of the test sequences except T2 and T4, which take several minutes. For T4 with 1340 nt, we have 6567 candidate stems in dictionary D s and 7534 pseudoknot candidates before filtering. After the length-normalized filtering step, only 100 pseudoknot candidates remain for verification. Overall, it takes DotKnot <5 min to predict the correct pseudoknot in this sequence on our reference machine (Intel QC 2.66 GHz, 4 GB RAM). This is significantly faster than HotKnots, which takes 29 min, and pknotsRG, which takes 31 min. KnotSeeker is even faster than DotKnot and takes <2 min for the T4 sequence, because it does not rely on a partition function calculation. However, DotKnot is a more powerful prediction algorithm than For each pseudoknot, the best results in terms of both sensitivity S and positive predictive value PPV are marked in bold. The * symbol indicates that we were not able to run the algorithm due to the high time and space requirements. PK corresponds to the number of pseudoknots in the sequence as reported in the literature. We use pknots 1.05 with coaxial energies, pknotsRG 1.3 and HotKnots 1.2 without suboptimal solutions.
KnotSeeker due to the inclusion of pseudoknots with one interrupted stem.
We presented DotKnot, a program that detects recursive H-type pseudoknots given an RNA sequence. Pseudoknot detection is a promising and efficient approach for determining the folding of an RNA. Using pseudoknot detection tools such as DotKnot, KnotSeeker or HPknotter, one can find likely pseudoknots in a sequence with high accuracy (37, 39) . The structure of the detected pseudoknots can subsequently be investigated using laboratory or bioinformatics techniques. The remaining non-crossing sequence can be folded using secondary structure prediction algorithms in Oðn 3 Þ time and Oðn 2 Þ space. DotKnot and other pseudoknot detection approaches are very time efficient, even allowing scanning of long regions in viral genomes. DotKnot assembles pseudoknot candidates from a set of structural building blocks. This set contains stems, bulge loops, internal loops and multiloops. In general, complex pseudoknots can be constructed. However, there is a trade-off between the generality of predictable pseudoknots and the biological relevance of the result. Therefore, we restrict DotKnot to the prediction of recursive H-type pseudoknots where one of the pseudoknot stems can contain bulges and internal loops. For these recursive H-type pseudoknots, we are confident that the pseudoknot energy parameters used give a good approximation. For more complex pseudoknot folds such as those with loop-loop interactions, we would have to employ a highly assumptive energy model, thus sacrificing predictive accuracy. In the future, DotKnot will be extended to the prediction of kissing hairpins and other biologically relevant classes of pseudoknots.
DotKnot uses stack probabilities from the probability dot plot as the basis for finding pseudoknot building blocks. At this stage, only a single sequence is accepted as an input. It is well-known that functional pseudoknots are highly conserved in nature, for example in virus families. The pseudoknot detection framework used in DotKnot allows for incorporation of comparative information using aligned probability dot plots. One can expect that reliable alignments will greatly improve confidence in the predicted pseudoknots. Especially for complex pseudoknot foldings such as those found in bacterial tmRNA or in the telomerase RNA component, inclusion of comparative information will be invaluable. Risk of Importing Zoonotic Diseases through Wildlife Trade, United States The United States is the world’s largest wildlife importer, and imported wild animals represent a potential source of zoonotic pathogens. Using data on mammals imported during 2000–2005, we assessed their potential to host 27 selected risk zoonoses and created a risk assessment that could inform policy making for wildlife importation and zoonotic disease surveillance. A total of 246,772 mammals in 190 genera (68 families) were imported. The most widespread agents of risk zoonoses were rabies virus (in 78 genera of mammals), Bacillus anthracis (57), Mycobacterium tuberculosis complex (48), Echinococcus spp. (41), and Leptospira spp. (35). Genera capable of harboring the greatest number of risk zoonoses were Canis and Felis (14 each), Rattus (13), Equus (11), and Macaca and Lepus (10 each). These findings demonstrate the myriad opportunities for zoonotic pathogens to be imported and suggest that, to ensure public safety, immediate proactive changes are needed at multiple levels. M ost emerging infectious diseases are caused by zoonotic pathogens (1, 2) . The number and proportion of these diseases that originate in wild animals in particular has increased substantially in the past few decades, even after accounting for increased reports of new emerging infectious diseases (1) . This trend and recent pandemics of wildlife-origin infectious diseases (e.g., HIV, severe acute respiratory syndrome) suggest that targeted surveillance efforts should focus on activities that bring humans and wildlife in close contact (1, 3) .
The United States is among the world's largest importers of live wild animals (4) and imported >1 billion indi-vidual animals during 2000-2004 (5) . Little disease surveillance is conducted for imported animals; quarantine is required for only wild birds, primates, and some ungulates arriving in the United States, and mandatory testing exists for only a few diseases (psittacosis, foot and mouth disease, Newcastle disease, avian infl uenza). Other animals are typically only screened for physical signs of disease, and pathogen testing is delegated to either the US Department of Agriculture (for livestock) or the importer (6) . The process of preimport housing and importation often involves keeping animals at high density and in unnatural groupings of species, providing opportunities for crossspecies transmission and amplifi cation of known and unknown pathogens. Thus, imported wildlife remain a major public health threat, as exemplifi ed by the importation of Ebola virus in primates from the Philippines (7), monkeypox from imported African rodents (8) , and possibly HIV from chimpanzees in central Africa (9) . Wildlife importation also poses a great threat to domestic wildlife and the US agriculture industry (5) .
To analyze the volume and diversity of live mammals that have been imported into the United States in recent years, we used data from the US Fish and Wildlife Service Law Enforcement Management Information System. We focused on mammals because of the frequency and severity of previously reported mammal-borne zoonoses and because of the frequent close association between humans and many mammalian species (e.g., as pets). We then assessed the zoonotic diseases that imported mammals are known to host. Our results may be used to inform policy decisions about wildlife importation and zoonotic disease surveillance and may alert clinicians to the broad range of possible zoonoses that may be encountered in patients who have been exposed to imported animals.
We Memphis, Tennessee; and Newark, New Jersey. For each importation, we acquired information on the taxonomy, quantity, source (e.g., wild-caught, farmed), country of origin, intermediate port of call, port of entry, and declared purpose of all live specimens. Descriptive analyses were performed to determine the volume of trade from various regions of the world and the types of mammals imported. Individual importation events were then grouped into genera to determine the diversity of taxa imported. The phylogenetic relationships and geographic ranges of host mammals were determined by using the Animal Diversity Web at the University of Michigan Museum of Zoology (http:// animaldiversity.ummz.umich.edu/site/index.html).
We searched the literature to identify the zoonotic pathogens known to occur in animals of each taxon in the database. Only data on live animal importations (as opposed to animal products) and importations for which the genus was known were retained for analysis. Statistical analyses were performed by using Intercooled Stata 9 (StataCorp, College Station, TX, USA). In our fi nal risk assessment, we did not account for the origin of each specifi c importation because of limitations in the database, likely caused by a complicated system of exportation and reimportation.
We created a list of relevant zoonotic diseases at risk for importation (hereafter referred to as risk zoonoses) by searching the Centers for Disease Control and Prevention website (www.cdc.gov) and the World Health Organization website (www.who.int), reviewing the list of Select Agents (agents with bioterrorism potential) of the US Department of Health and Human Services (10), and consulting experts in the fi eld. To be on the list, diseases had to meet the following 5 criteria: 1) the pathogen must be zoonotic (there must be a recorded instance of infection of a human from an animal source); 2) the pathogen must be capable of causing signifi cant illness or death (e.g., fungal skin infections would not be on the list because although they are extremely common zoonoses, their effects are rarely debilitating); 3) the pathogen must be present in animals in the wild (i.e., not only in experimental models); 4) the pathogen must not currently be widespread in the United States, or it must have the potential for new epidemiology with regard to transmission (e.g., Yersinia pestis is presently found in wild rodents in the western United States, but it is not expected to be found in animals sold as pets); and 5) if the pathogen uses an intermediate vector, competent vectors must exist in the United States. The resulting list comprised 30 risk zoonoses (20 viral diseases, 9 bacterial diseases, and 1 helminthic disease); no fungal, protozoal, or prion diseases were on the list, and thus they were not analyzed.
Determination of the host range of the risk zoonoses was accomplished through systematic genus-driven and pathogen-driven searches of PubMed databases (www. pubmed.gov), the Google search engine (www.google. com), and references within published works. Confi rmed presence was defi ned as either isolation of the pathogen from an animal or serologic evidence of past infection. For all animals identifi ed in the literature as carrying a risk zoonosis, genus and family were recorded. The host ranges of all of the risk zoonoses were then cross-referenced against the imported genera to generate tables showing diseases found in each imported genus (affected genera). If the disease was found in a different genus within the same family, this was also noted (potentially affected genera). The justifi cation for this expanded risk assessment is the host nonspecifi city of many infectious diseases; lack of evidence for the presence of a given disease in a given host should not be construed as evidence against its presence.
During 2000-2005, a total of 4,067 shipment fractions of mammals were imported (a shipment fraction is the sum of all animals of a single species in a given shipment; a single shipment may contain several shipment fractions), totaling 246,772 individual mammals and representing 190 genera and 68 families. The average number of animals per shipment fraction was 61 (range 1-8,000). The most common declared purpose for importation was commercial use (not classifi ed according to pet trade, food, traditional medicine, etc.), accounting for 66% (163,760 individuals) of the total. The second most common declared purpose was biomedical research, accounting for 28% (69,986 individuals) of the total. Only a small number of individuals were imported for breeding, educational, zoo, personal, and other uses. Numbers of the most commonly imported animals were 126,014 (>50% of all imported individuals) long-tailed macaques (Macaca fascicularis), 30,058 small desert hamsters (Phodopus sungorus), 19,724 rhesus macaques (Macaca mulatta), 19,537 raccoons (Procyon lotor), and 7,112 chinchillas (Chinchilla lanigera). Together, these 5 species accounted for 82% of all imported individuals. By number of shipment fractions, the most common animals were 1,343 M. fascicularis macaques, 332 Cal-lithrix jacchus marmosets, 229 M. mulatta macaques, 165 C. lanigera chinchillas, and 107 Potos fl avus kinkajous.
The most common countries of origin for animal shipment fractions were People's Republic of China (717 shipment fractions); Guyana (635), United Kingdom (359), Vietnam (314), and Indonesia (305). These values must be interpreted cautiously, however, because many animals are imported and then reexported; thus, their true origin may become obscured. For example, a "wild-caught" chinchilla with a "country of origin" of Czech Republic must have originated elsewhere because chinchillas are native to Chile. A comparison between the natural geographic range of all wild-caught animals and their stated countries of origin showed that >25% of the pairings were impossible (i.e., the animals could not have come from their stated country of origin). This limitation is inherent in the way US Fish and Wildlife Service Law Enforcement Management Information System data are collected, and we were unable to correct these data.
The source of the animals was largely uninterpretable because 49% of all individuals were declared as being sourced from "animal derivatives and parts," despite the fact that we had selected only live animals for our analysis, and despite the fact that "animal derivatives and parts" is not one of the permitted responses to this question. Another 29% were declared as "captive-bred" and 15% as "wild-caught."
For the fi nal list of risk zoonoses, 3 of the original 30 agents (Hendra virus, Menangle virus, and Rickettsia prowazekii) were removed because few, if any, genera were found to harbor these infections; the fi nal tables therefore include 27 diseases (Tables 1-3) . The risk zoonoses capable of infecting the greatest number of genera were: rabies viruses, in 78 genera; Bacillus anthracis, the causative agent of anthrax, in 57 genera; Mycobacterium tuberculosis complex, in 48 genera; Echinococcus spp., the agents of hydatid cyst disease, in 41 genera; Leptospira spp., in 35 genera; Brucella spp., the agents of undulant fever, in 32 genera; Francisella tularensis, the agent of tularemia, in 31 genera; Crimean-Congo hemorrhagic fever virus, in 27 genera; Y. pestis, the agent of plague, in 24 genera; and Coxiella burnetii, the agent of Q fever, in 20 genera ( Table  2 ; online Technical Appendix, available from www.cdc. gov/EID/content/15/11/1721-Techapp.pdf).
If each genus within affected families is counted as potentially capable of harboring a risk zoonosis (according to the principle that many diseases are not entirely host specifi c), the number of genera potentially capable of harboring rabies viruses rises to 155 (82% of all imported taxa); potential carriers of Leptospira spp. increase to 131; M. tuberculosis complex to 124; F. tularensis to 115; B. anthracis to 113; C. burnetii to 108; and Y. pestis to 101.
The genera capable of harboring the greatest number of risk zoonoses were Canis (dogs) and Felis (cats), 14 risk zoonoses each; Rattus (rats), 13; Equus (horses), 11; Macaca (macaques), 10; Lepus (rabbits and hares), 10; and Ovis (sheep) and Vulpes (foxes), 9 each (Table 3) . Of the individuals in these high-risk genera, 49% were intended for commercial purposes and 44% were intended for biomedical research.
The families found to harbor the most risk zoonoses (excluding Hominidae because, by defi nition, they are capable of harboring all zoonotic diseases) were Muridae (Old World mice and rats, gerbils, whistling rats, and relatives), 21 risk zoonoses; Cricetidae (New World rats and mice, voles, hamsters, and relatives), 20; Canidae (coyotes, dogs, foxes, jackals, and wolves), 16; and Bovidae (antelopes, cattle, gazelles, goats, sheep, and relatives) and Felidae (cats), 15 each.
Our data demonstrate that myriad opportunities exist for key zoonotic pathogens to be imported into the United States or, if already present, to be introduced in a new context (e.g., in an animal sold as a pet). Imported animals of a large number of taxa were found to be capable of carrying risk zoonoses; these diseases include such serious public health threats as rabies, the fi lovirus hemorrhagic fevers, tuberculosis, and highly pathogenic avian infl uenza.
This study likely underestimates the broad nature of risk associated with the importation of wild animals. We examined only families in the class Mammalia that have been shown to harbor risk zoonoses; however, many pathogens routinely cross boundaries at least as high as the class level (e.g., human psittacosis from birds), if not higher. Furthermore, we included only live animals in this analysis; recent outbreaks associated with animal products (e.g., cutaneous anthrax from an imported goat hide used for making drums) attest to the risks associated even with dead animals (13) . Finally, the study can neither estimate the risk for unknown pathogens, which may be imported but not yet identifi ed, nor assess the volume and zoonotic risk created by illegal wildlife trade. Animals may be smuggled specifi cally because they have been banned from trade as a result of perceived or recognized health threats. Some animals on our list of risk zoonoses have already been banned from importation (e.g., masked palm civets, birds from countries affected by highly pathogenic avian infl uenza [H5N1]) (14) . However, pathogens have been identifi ed in illegally imported wildlife; e.g., a pair of crested hawkeagles (Spizaetus nipalensis) smuggled from Thailand and recently confi scated in Belgium were infected with highly pathogenic avian infl uenza (H5N1) (15) .
We did not quantitatively assess the risk for transmission of each pathogen at each importation event. Rather, we attempted to demonstrate the breadth of risk associated with importations of wild animals in general. Quantitative prevalence of the various pathogens in each wildlife host is highly variable, and determining it is beyond the scope of our analysis. Some genera represent the primary reservoirs of certain pathogens (e.g., Peromyscus for certain hantaviruses), whereas proof of the permissiveness of other genera to certain pathogens is limited to isolated case reports (e.g., Ebola Zaire virus in the duiker Cephalophus). Perhaps the greatest unknown associated with quantifying risks for each of the zoonoses is a pathogen's infectivity in various hosts. Some pathogens may increase to a high enough load in their hosts to be infectious; others may cause nothing more than a measurable serologic response in what is otherwise a dead-end host (though explicitly known dead-end hosts have been excluded from these analyses).
Our analysis highlights several ways that the US Fish and Wildlife Service could improve data collection. To enhance public health offi cials' ability to trace back the sources of imported zoonotic diseases, record keeping of the point of origin of shipments could be expanded to include not just their most recent and previous point of origin (as is currently done with the "Country of Origin" and "Country of Importation/Exportation/Re-importation") but also their actual origin. Accurate recording of the source of the animals (e.g., wild-caught, captive-bred) is also needed. Our results showed that half of all individuals had a declared source that was not one of the allowed choices (e.g., wild-caught, captive-bred). The source of an animal affects not only the likely level of risk (i.e., one would expect captive-bred individuals to carry fewer zoonotic diseases than wild-caught individuals) but also mitigation strategies when zoonotic diseases are identifi ed (e.g., euthanizing a colony vs. improving quarantine after capture).
The potential for importation of zoonoses that would pose a major public health threat suggests that increased surveillance should be applied to imported wildlife in the United States. One opportunity to reduce this threat is restriction of importation of key high-risk species, as was done when the Centers for Disease Control and Prevention used emergency powers to restrict importation of Gambian pouched rats during the monkeypox outbreak (14) . Given the great diversity of animals identifi ed by our analysis as potentially hazardous, broad importation bans would likely be necessary if the goal were to substantially decrease the overall risk. Political or social support may be limited for such broad bans, both in the United States (as one of the world's largest purchasers of wildlife) and abroad (where wildlife trade can have profound economic benefi ts).
Furthermore, illegalizing trade may only increase underground (illicit) trade, thereby eliminating the possibility of screening shipments for potential hazards. A more effective and acceptable strategy would be enhancing surveillance for the specifi c pathogens noted for the key risk genera (those harboring the greatest number of risk zoonoses, i.e., Canis, Felis, Rattus, Equus, Macaca, Lepus, Ovis, and Vulpes). Notably, the numbers of shipments of mammals is low relative to other wildlife groups (e.g., fi sh and reptiles). Lawmakers' interests in protecting our borders from external bioterrorism threats intersect with the need to protect ourselves from zoonotic diseases; many Category A bioterrorism threats (e.g., anthrax, plague, tularemia, and the viral hemorrhagic fevers) (10) are represented in the risk zoonoses outlined above. Finally, to facilitate the standardization of surveillance and detection of infection events, the Council of State and Territorial Epidemiologists should include all of the risk zoonoses among their states' notifi able diseases (most of which are already included).
Perhaps one of the simplest practical interventions for minimizing zoonotic disease risk is reduction of opportunities for transmission from wildlife to humans. Although a large proportion of imported animals are destined for biomedical research (in which potential occupational risks are largely understood and quarantine procedures likely mitigate risk), a greater proportion (even among the high-risk genera) are destined for commercial use and therefore could expose a wider group of persons to zoonotic diseases. Education of professionals likely to come in close contact with imported animals (e.g., veterinarians, importers, pet store employees), as well as the general public, should emphasize the risks for contracting zoonotic diseases from wildlife and pets (16) and the need for proper hygiene, safety procedures, and personal protective equipment (17) .
The recommendations above mirror others that exist in policy documents by the Defenders of Wildlife (5), in the 2003 joint position statement by the National Association of Public Health Veterinarians and the Council of State and Territorial Epidemiologists (18) , and in a recent Policy Forum article (19) . These reports describe clear steps for mitigating the risks presented by imported wildlife, yet their recommendations have so far gone largely unheeded. To ensure public safety, immediate proactive changes are needed at multiple levels. Such measures would be most effective if organized in consultation with groups involved in the wildlife trade. The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1 Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19(th) century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins. Live attenuated virus vaccines have been successfully employed in veterinary medicine to prevent the economic impact of many diseases in poultry and livestock. However, the role of vaccination with attenuated viruses on the evolution of wild type strains is not often considered. Antigenic escape because of strong selection by vaccines, emergence of new strains through recombination, and increased virulence to expedite transmission of new genotypes in vaccinated populations are of potential concern. In this paper, we explored the consequences of vaccination on the evolution of class II aPMV-1, which is the etiological agent of ND.
NDV, a single-stranded, non-segmented, negative-sense RNA virus of the genus Avulavirus, family Paramyxoviridae, infects a wide range of domestic and wild bird species worldwide, and causes a significant economic burden to the poultry industry [1] . The first outbreaks of NDV were reported during the mid 1920s in Java, Indonesia and Newcastle-upon-Tyne, England [2] , and within a few years were occurring throughout the world [3] . The name ND is reserved exclusively for the disease that results from infection with strains of aPMV-1 that are pathogenic for domestic chickens [4] . aPMV-1 has been grouped by virulence phenotype, with lentogenic, mesogenic, and velogenic strains representing increasing levels of virulence, ranging from subclinical infections with moderate respiratory involvement to extensive hemorrhagic lesions and neurological signs [5] . Inactivated vaccines were first made commercially available to the poultry industry in 1946, but because they provided incomplete protection against ND [6] , they were replaced with live lentogenic NDV vaccines. Although these vaccines reduce disease, they do not always prevent infection and birds can shed both vaccine and challenge strains of the virus [7, 8, 9] .
aPMV-1 genome size is approximately 15 kb and encodes six genes, which produce nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutininneuraminidase (HN), and polymerase protein (L) [10, 11] . RNAediting of P gene creates two additional proteins, V and W [12] . There are nine serotypes of aPMV-1; viruses associated with ND are in serogroup 1. Within serogroup 1 there are 2 major subdivisions, class I and II, based on phylogenetic grouping of the F gene [13] . Class I aPMV-1 are primarily recovered from waterfowl or samples from U.S. live bird markets, while the isolates from class II are commonly derived from poultry and other avian species [5, 14] . Eight genotypes of class II aPMV-1 can be identified [13] . Viruses belonging to genotypes I-IV have circulated since the 1930's. Genotype I and II consist of both lentogenic and velogenic viruses and have been associated with ND outbreaks in Australia and North America, respectively [5, 15, 16] . These viruses have been attenuated in culture and are used as modified live vaccines [16] . Genotypes V-VIII were first recognized in the mid-1960s [13] and contained only virulent viruses [16] . Genotype V was responsible for the second panzootic of ND in Europe from 1970-1974 and has been detected sporadically thereafter [17] . Genotype VI was described mainly from the Middle East and Asia during the 1980's-1990's [18] and genotype VII and VIII were reported in the 1990's from several countries [19, 20, 21, 22, 23, 24] . All genotypes, except IV [16] , are still in circulation.
RNA viruses typically have a high mutation rate due to low fidelity and processivity of their polymerase [25] , which coupled with a high replication rate and short generation time [26] lead to high evolutionary rates. In addition, evidence is accumulating that recombination is an important process driving genotype diversity for many RNA viruses [27, 28, 29] . Although recombination was not thought to contribute to aPMV-1 evolution [30, 31] , evidence of recombination in NDV has recently been reported [32, 33, 34] . This debate may be due, in part, to the reliance on a single gene for determining virus diversity and phylogeny. Because recombination can lead to the emergence of novel virus strains of unknown virulence [35, 36, 37, 38] , a better understanding of the role of recombination in circulating aPMV-1 is warranted.
In this study, we explored how vaccination strategies in poultry farming have shaped the evolution of this important avian virus using complete genome sequences available in GenBank. Specifically our objectives were to 1) determine if recombination was evident among full length class II aPMV-1; 2) estimate evolutionary rates of each genotype; 3) estimate the effective population size of each genotype; and 4) determine the selective forces on vaccine-related and nonvaccine-related wild type genotypes. Our results confirm that recombination is an important process in this negative sense RNA virus and that vaccine-related strains have an evolutionary history that is unique from nonvaccine-related strains, which includes distinct evolutionary rates, temporal changes in population size, and selection profiles.
The current phylogenetic classification of aPMV-1 strains is based on either full or partial nucleotide sequence of the M, F, or L genes. To determine if all genes in the viral genome provided consistent phylogenetic profiles, we obtained 54 full length class II aPMV-1 sequences from Genbank and generated nucleotide data sets for each of the six genes and a concatenated sequence of all protein coding regions. Maximum likelihood (ML) phylogenies were reconstructed for all sequence data sets under the appropriate nucleotide substitution model selected for each data set ( Figure 1 ). Each gene tree and the concatenated tree revealed seven distinct genotypes within the class II aPMV-1. However, genotype affiliations were not congruent among different genes ( Figure 1A ). While genotype III, IV, V, VI, and VII were monophyletic, genotype I and II showed inconsistent phylogenetic relationships ( Figure 1A and B). Three distinct patterns of affiliations of genotype I and II were observed among different gene trees. The NP, HN, L, and concatenated gene trees consistently placed genotypes I and II in a sister clade to other genotypes. In the P gene tree, genotype II formed a basal clade while genotype I clustered with the remaining genotypes and in the M and F gene trees, genotype I was the basal clade and genotype II clustered with the remaining genotypes.
Several taxa changed genotype affiliations in different gene trees and all discordant taxa were affiliated with vaccine-related genotype II in some gene trees ( Figure 1C ). For example, isolate AY562985 (Cockatoo/14698/Indonesia/1990) was affiliated with genotype II in the NP gene tree but with genotype VII in all other gene trees. This isolate occupied a long branch in the genotype VII sequences in the P gene tree. Isolate AY562989 (Dove/2736/ Italy/2000) was affiliated with genotype II in the M gene tree but with genotype VI in all remaining trees except the P gene, in which it was an outlier to all other genotypes. Isolate AY225110 (HB92 isolate V4 vaccine/China) affiliated with genotype I in M and L gene trees but with genotype II in other gene trees. Isolate EU167540 (Layer/SRZ03/China/2003) affiliated with genotype VII in all gene trees but it occupied a long branch in genotype II in F gene tree. A Shimodaira-Hasegawa test (SH-test) provided statistical support of taxon incongruence (p,0.005) among the gene trees (data not shown). Phylogenetic incongruence among genes suggests that recombination might play a role in class II aPMV-1 diversity.
To further investigate the possibility of recombination among the full length aPMV-1 sequences, we used seven different algorithms implemented in the RDP3 program [39, 40] . Chimeric NDV vaccine strain EU140955, which has the genotype II La Sota vaccine strain backbone and the F and HN genes from a contemporary genotype VII virus ( Figure 1C) , was included as a control to evaluate the prediction capability of the program. The predicted recombination breakpoint (detected by five methods with p-value,10 25 ) at position 7119 of the concatenated EU140955 matched correctly with the end of a SpeI restriction site of this chimeric strain where HN sequences were inserted from the KBNP-4152 strain. The MluI restriction site used to generate the chimera is within the intergenic region between M and F genes
Modified live virus (MLV) vaccines have been effective in reducing disease burden and economic loss caused by Newcastle Disease (ND) in domestic poultry. Because the vaccine is a live virus, it is transmissible among birds. Thus, vaccination strategies have the potential to impact the evolutionary genetics of wild type strains of aPMV-1 including those that cause ND. In this report, we provided evidence that viruses isolated from wild and domestic birds have recombined with vaccine strains, because vaccinated birds are protected from disease but not infection with other strains of aPMV-1. Despite the use of vaccines since the 1950s, the population size of the strain from which the most widely used vaccine was derived has steadily increased. In contrast, other contemporary genotypes, which emerged in the 1960s, experienced a decline in population size in 1998, which may reflect a change in poultry farming practices or disease. Vaccination imposed a unique selection profile on the genotypes derived from the vaccine-related strains when compared with nonvaccine-related strains. Although modified live viruses are important for controlling Newcastle Disease, the potential of vaccination strategies to change viral diversity and population dynamics should be considered. and is not present in our sequences. However, the RDP3 program reasonably identified the 59 breakpoint at the 8th nucleotide of the F-gene. Two additional recombination breakpoints within this insert were also detected by the GENECONV and Bootscan methods (p-value 2.29610 23 and 3.35610 22 ). These corresponded to the positions in the F gene segment of KBNP-4152 strain that were mutagenized to attenuate recombinant strain EU140955. Thus, we conclude that the RDP3 program accurately identifies recombination if five or more methods have statistical support of p#10 25 for the breakpoints and we propose that any breakpoints statistically supported with only one or two methods should be carefully interpreted.
Using the stringent criteria defined above, a total of five putative recombinant isolates were detected ( Figure 2 ). Four of these isolates, AY562985, EU167540, AY562989, AY225110, were those that showed discordant phylogenies described above ( Figure 1C ), and in each case, some recombinant regions were derived from genotype II sequences. Isolate AY562985 is predominantly genotype VII and had evidence of two recombination events based on RDP3 predictions. We confirmed that regions 508 to 926 (in NP) and 927 to 1511 (NP and P) are related to genotypes V and II, respectively by partitioning the data sets at the predicted break points and reconstructing a ML phylogeny ( Figure 3 ). In the region 927 to 1511, AY562985 had seven unique synonymous substitution sites compared to the other sixteen genotype II sequences in our dataset. Isolate AY562989, which is predominantly of genotype VI origin, also contained a putative recombinant region spanning positions 2039 to 3225, which was derived from genotype II and had three unique non-synonymous substitution sites compared to other genotype II sequences. Isolate AY225110 is a chimera of genotype I and II sequences. Compared to genotype II sequences, there were seven unique sites, four of which were non-synonymous substitutions, from the 59 end of NP to position 2702; two synonymous and two non-synonymous substitution sites in the 3757-7149 fragment; and two synonymous and seven non-synonymous substitution sites within the region from 13758 to the 39 end of L. For isolate EU167540, a putative recombination region between position 3753 and 4345 was affiliated with genotype II and had one non-synonymous substitution site compared to other strains. Thus, genotype II, which is used in vaccines globally, has recombined with at least three other class II aPMV-1 strains and the recombinant viruses have been isolated from both domestic and wild birds.
The fifth virus identified by all RDP3 methods, DQ485230, was a genotype VII isolate that contained a small region within the HN gene contributed by genotype III. In addition, a region spanning 1479 to 3751 in P and M appeared to be derived from a different genotype VII virus. Inter-genotype recombination was also detected by fewer than five of the RDP3 methods in DQ486859(GM/China), DQ485231(Guangxi11/China/2003) and AF309418(Fowl/B1/USA/1947). Genotype VII isolate DQ659677(NA-1/China) contained a 640 bp region within L contributed from genotype VI. The origin of the putative recombinant fragment spanning the M and F genes of EF065682(rAnhinga/USA) could not be determined. These data provide compelling evidence that genotypes II and VII are most commonly associated with recombinant viruses and that both intra-and intergenic recombination events can be detected using full genome sequence analysis.
Phylogenies of individual and concatenated genes were reconstructed after the removal of the five putative recombinant isolates and chimeric vaccine strain EU140955 (Figure 4 ). Consistent with Figure 1A and 1B, genotypes III -VII clustered together as a monophyletic group in all trees. All taxa were consistently affiliated with a single genotype and there were no long branches associated with any genotype. Two of the three original patterns of phylogenetic affiliation were retained following removal of recombinant sequences. The placement of genotypes based on HN and L was the same with or without recombinants; genotype I and II were sister groups to genotype III, IV, V, VI, and VII ( Figure 1A; Figure 4 ). All of the remaining trees presented a topology similar to that of the P gene-tree before recombinant removal, which placed genotype I with III-VII. It is noteworthy that in the absence of recombinant sequences, genotype II is never clustered with genotypes III-VII, as was seen with trees based on M and F in the presence of recombinant sequences ( Figure 1A ).
We inferred the evolutionary rates and past population dynamics of class II aPMV-1 using a Bayesian coalescent approach [41] . This analysis was based on all full length genome sequences in the data set which had a date of isolation and excluded the six recombinant sequences. Bayesian estimates of the evolutionary rates of each gene and concatenated coding genome of class II aPMV-1 were between 0.98610 23 -1.56610 23 substitutions/site/year (Table 1 ). Evolutionary rate estimates under a relaxed clock with HKY+G 4 (Table S4 ) and GTR+G 6 ( Table 1) substitution models were consistent. The time to the most recent common ancestor (TMRCA) of class II aPMV-1 was estimated to be between 114 and 137 years before 2005, or between year 1868 and 1891. Bayesian skyline plots (BSP) were used to infer how effective population size has changed with time [41, 42] . All six protein-coding genes and the concatenated genome maintained constant effective population size until the late 1990's ( Figure 5A ). In 1997-8 there was an abrupt decline in the population with recovery from this event in the early 2000.
To determine if all genotypes exhibited the same population history observed for the composite genotype data set, we repeated the analysis based on 97 dated full length F genes, which provided a larger data set for this analysis. Isolates from genotype III, IV, and V were not included because limited numbers of dated sequences were available. The TMRCA for genotype II was estimated to be 1899620 years, and the estimated evolutionary rates were between 0.3-1.1610 24 substitutions/site/year, making this the slowest evolving aPMV-1 genotype ( Table 2 ). Genotype I and VI emerged in the early 1960's and had higher evolutionary rates than genotype II. The most recently emerged strain was genotype VII, which dates to the late 1970's and had the highest evolutionary rate. BSP analyses based on the F gene demonstrated that each genotype had a unique population history. Prior to the emergence of genotype VII in the 1970s (phase i), genotype II showed an increase in population size ( Figure 5B ). After the emergence of genotype VII (phase ii) the population size of genotype VI began to increase, while that of genotypes I and VII were relatively constant. Phase iii depicts the time of the population bottleneck observed in Figure 5A , which was based on all genes in the composite genotype data set. Only genotypes I and VI show a trend for decreasing population size during this time. The last decade has been the most dynamical for the four genotypes of aPMV-1 (phase iv; Figure 5B ). Genotypes I and VII showed a marginal increase in effective population size followed by a decline; genotype I has continued to decline, whereas genotype VII appears to have stabilized. Genotype VI showed continuous decline in population size in phase iv ( Figure 5B ). Although estimates of population sizes for genotypes I, VI, and VII, have some degree of overlaps at the 95% posterior limit, genotype II shows no sign of reduction in effective population size since its origin ( Figure 5B ).
We compared the selection profiles on protein coding genes of genotypes I and II, which include strains that are circulating in the face of homotypic vaccination pressure (designated as the vaccinerelated group), and genotypes III-VII (designated as the nonvaccine-related strains) (Table 3) . Overall, the global rate of nonsynonymous to synonymous substitutions (dN/dS) for all protein coding genes were less than 1, indicating purifying selection has been the major driving force in the evolution of class II aPMV-1 viruses. There were no codons identified to be under positive selection in M and L genes in either group (Table 3) . However, there was a clear difference in the codon based selection profiles of N, P, F and HN genes between the vaccine-and nonvaccinerelated groups. In the vaccine-related group, only the surface protein encoding genes, F and HN, had positively selected codons. Both groups had a single site identified in F; these were at codon 115 within the F 0 cleavage site for the vaccine-related group and codon 28 in the signal peptide for the nonvaccine-related group. There were 3 positively selected sites identified in the HN gene in the vaccine-related group and one in the non vaccine-related group. In contrast, the P gene of nonvaccine-related genotypes III-VII had three sites predicted to be under positive selection but no sites were identified in the vaccine-related group. Thus, selection is focused on HN in vaccine-related groups and on P in nonvaccine- 
Our study explored the forces shaping the evolutionary history of class II aPMV-1 using available full genome sequences. We demonstrated that genotype affiliations based on individual genes and concatenated full length genomes of class II aPMV-1 were not consistent. This may account for discrepancies reported for genotype groupings that are based on partial or complete sequences of a single gene [13, 18, 43] . Topological incongruence among the gene trees reflects different evolutionary histories of each gene [44] ; recombination is the most plausible explanation for this. The role of recombination in the evolution of aPMV-1, and negative sense RNA viruses in general, has been debated. For example, Sakaguchi et al. [30] and Toyoda et al, [31] reported consistent topological placement of different NDV strains in both F and HN gene trees. Seal et al. [45] also reported that there was no evidence of recombination among NDV M gene sequences. Although recombination is more common in positive-sense RNA viruses and can be explained by several genetic mechanisms [46] , there is increasing evidence of homologous recombination in several non-segmented negative-sense RNA viruses [32, 47, 48, 49, 50] . Our approach differs from those used in previous studies of aPMV-1 evolution because we evaluated full genome sequences and tested individual recombinant regions with phylogeny-based incongruence tests. Thus, we show that all class II aPMV-1 genes have evidence of recombination breakpoints, that multiple recombination events are discernable in some isolates, and that both intragenic and intergenic recombination events are evident. We considered the possibility that the recombinants detected in our analysis were the result of laboratory artifacts, as has been previously suggested [32, 51] . Laboratory contamination is of concern because vaccine derived strains contributed the majority of the recombinant regions and these strains might have been present in laboratories sequencing field aPMV-1 isolates. The presence of unique nucleotide substitutions in the recombinant regions compared to the comparable region of the predicted parental genotypes suggests that these regions did not arise due to contamination with vaccine strains deposited in the sequence databases.
Our identification of recombinants derived from vaccine strains indicates that birds can be simultaneously infected with the live virus vaccine and other circulating aPMV-1 genotypes. Indeed vaccination is reported to protect poultry from disease but not always from infection with other strains [7, 8, 9] . Suboptimal vaccination strategies could also lead to birds becoming infected with both a vaccine strain and circulating genotype, which can alter viral virulence [16] . This is an important issue for poultry management. Lack of vaccine efficacy has not frequently been reported in the United States but other countries such as Nigeria [52] , Korea [53] , Taiwan [54] , and China [34, 55] have experienced vaccine failures. Recombination between wild type virus and vaccine strains is not unique to aPMV-1; vaccine recombinants of bovine viral diarrhea virus (associated with fatal mucosal disease) [35] , poliovirus (associated with paralytic poliomyelitis) [36, 37] ; and infectious bursal disease virus [38] have all been reported. This raises concerns that modified live virus vaccines, although efficacious, may facilitate emergences of new strains with unpredictable phenotypes through recombination with circulating viruses.
The evolutionary rates presented here for class II aPMV-1 are compatible with the rates estimated for other RNA viruses (e.g. [56, 57, 58] ), suggesting that class II aPMV-1 is also a rapidly evolving RNA virus. The relatively lower evolutionary rate for genotype II is consistent with the rate that was previously reported for avirulent NDV [33] . The larger effective population size, which counters the impact of genetic drift, is a possible explanation for lower evolutionary rates of genotype II. Based on these rate estimates, the TMRCA of this virus is estimated to be between 1868-1891, which is earlier than the first recorded outbreak of ND in Indonesia and England in the 1920's [2] . However, our data are in line with observations by Macpherson [59] , who suggested that an outbreak of disease in domestic birds that occurred in Northwest Scotland from 1897-1898 was due to NDV.
The demographic history of class II aPMV-1 determined by Bayesian skyline plots indicated that there was an abrupt decline in population size during 1997-98. Although the factors responsible for such an abrupt decline in class II aPMV-1 are not known, the impact of a severe El-Nino event during that time frame [60] or the slaughter of millions of domestic fowl during the first outbreak of H5N1 avian influenza virus in 1997 [61, 62] could be possible explanations.
In contrast to the other genotypes, genotype II was not impacted by factors causing population decline in the late 1990s. We expected that vaccination, which started worldwide in the Table 3 . Site-specific selection analysis for each coding gene of vaccine-and nonvaccine-related groups. Strains used for selection analyses in both groups are mentioned in Table S1 . 1950s, should have limited the number of susceptible avian hosts, thus causing a bottleneck for this genotype. However, the impact of NDV vaccination is not seen in the BSP. It is possible that the data available in GenBank is insufficient to capture the population history of this genotype. However, a plausible explanation for the absence of a population bottleneck could be that genotype II NDV is maintained as an asymptomatic infection because it is continually introduced to susceptible populations as a modified live vaccine. Vaccination effectively prevents birds from developing disease when exposed to a virulent strain, but does not prevent shedding [7] . Thus, the number of available susceptible hosts may not dictate genotype II population demographics.
Although overall all genes of class II aPMV-1 are evolving under purifying selection consistent with other paramyxoviruses [33, 56, 63] , distinctive profiles of positively selected codons were shown in both vaccine-and nonvaccine-related groups. Notably, P gene had the highest dN/dS ratio of any gene and had three sites predicted to be under selection in the nonvaccine-related group; there were no sites under selection in P gene in the vaccine related group. In contrast, HN had three sites predicted to be under selection in the vaccine related group. Of interest, codon 115 in the F cleavage domain is positively selected only in vaccine-related groups. Previous studies have reported that a single amino acid substitution at codon-115, which falls within the F0 cleavage site, resulted in a dramatic change from an avirulent infection to highly virulent NDV [64, 65, 66] . In contrast to the results reported in the present study, Miller et al [33] did not identify codon115 in F gene under positive selection. This is likely due to differences in the data sets because factors such as sequence length, sequence divergence, and the number of sequences can determine the ability to detect positively selected sites [67] . Thus, the vaccine-related genotypes I and II maintain a phenotypic mixture of strains with different infection and pathogenic potential and selection profiles.
A total of 54 complete genome sequences of class II aPMV-1 representing different avian hosts, geographic regions, year of isolation, and genotypes (based on previous published phylogenetic grouping) were retrieved from GenBank. The coding genome sequences were aligned using MEGA version 4 [68] . Six separate coding gene sequences datasets (for NP, P, M, F, HN and L genes) were generated (see Table S1 ) and a concatenated genome sequence from these six coding gene sequences was generated using Mesquite version 1.12 (http://mesquiteproject.org). Appropriate model of nucleotide substitution for each dataset was selected by the hierarchical likelihood ratio test implemented in Modeltest version 3.7 [69] . Maximum likelihood (ML) trees were reconstructed for all data sets using the heuristic search option, implementing stepwise addition with 100 random addition replicates and tree bisection-reconnection branch swapping in PAUP* version 4beta10 [70] and PHYML 3.412 [71] with 100 non-parametric bootstrapping replicates analyses. The inferred trees were visualized with FigTree version 1.12 (http://tree.bio.ed. ac.uk/software/figtree/) and the congruency of topology placement of class II aPMV-1 genotypes based on each gene and concatenated genome was tested using the Shimadoira, Hasegawa (SH) test [72] implemented in PAUP. The concatenated tree was constrained and tested versus other gene trees.
The recombination predictions of the concatenated genome sequences were conducted with a suite of programs within the RDP3 package [39, 40] . The individual programs RDP [39] , GENECONV [73] , Bootscan [40] , Maximum Chi [74] , Chimaera [75] , SiScan [76] and 3Seq [77] , were implemented for the analysis. Since no single program provided optimal performance under all conditions, any event supported by five or more methods with p-values #10 25 was the criteria used for positive recombination breakpoints identification. The breakpoint position and the putative parental sequences were also determined.
Twenty six full length genome sequences for which year of isolation was available were used to infer evolutionary rate and dates using BEAST version 1.4.8 [41] . Demographic history of a population/species using multi-locus data, even from a small number of individuals, can precisely recover past bottlenecks in population size that cannot be characterized by analysis of a single locus [78] . Given this fact, estimates based on the whole genome sequence data are expected to be more reliable. To determine the population history of individual genotypes, 149 complete F gene sequences, which had dates of collection, were retrieved from GenBank. Phylogenetic analyses were done as described previously (Table S2 ). From the ML tree, a total of 97 isolates from genotype I, II, VI, and VII were selected to infer evolutionary rates and population dynamics. These included all available dated full length sequences from Genotypes I (24 sequences), II (28 sequences), and VI (23 sequences). The majority (95%) of genotype VII sequences were of Chinese origin. Thus, we included the seven non-Chinese sequences and picked an additional 15 sequences based on phylogenetic diversity to represent genotype VII in our analyses. The evolutionary rate (nucleotide substitutions per site per year) of each gene and concatenated genome was estimated using the Bayesian Markov chain Monte Carlo analyses (independent assumption of codivergence). Substitution models of both HKY + G 4 and GTR + G 6 with estimated base frequencies, gamma and invariable site portion were used, with uncertainty in the data reflected in the 95% high-probability density (HPD) intervals. Strict clock and uncorrelated exponential (UCED) relaxed clock models were attempted independently, and the best-fit clock model was determined to be UCED based on the Bayes Factor calculated from their posterior distributions (Table S3 ). The Coalescent Bayesian skyline plot (BSP) was used to infer the past population dynamics. The BSP was constructed using the growth rate and demographic parameters from the selected best-fit models. Bayesian Markov Chain Monte Carlo (BMCMC) analyses were run for 5-10610 8 generations depending on each dataset. Convergence of trees was checked using Tracer v1.4.1 (http://beast.bio.ed.ac.uk/Tracer).
Selection analyses were done based on datasets without putative recombinant sequences because recombination can result in falsely identifying positive selection [79] . The datasets were split into a vaccine-related group, which included strains from genotypes I and II, and nonvaccine-related group, which included strains from genotypes III, IV, V, VI, VII (see Table S2 ). Positively selected codons were detected using Fixed-Effect Likelihood (FEL) via the Datamonkey website (http://www.datamonkey.org/) and ML approach implemented in CODEML (PAML package version 3.15) [80] . For FEL analysis, p-values less than 0.05 were used to support positive selection. For PAML analysis, the likelihood ratio test was used to compare M1a, M7 and M8a models that assume no positive selection (v,1) with those M2a and M8 models that assume positive selection (v.1) [81] .  China's Engagement with Global Health Diplomacy: Was SARS a Watershed? As part of the PLoS Medicine series on Global Health Diplomacy, Lai-Han Chan and colleagues provide a case study of China's growing engagement in global health diplomacy following the SARS epidemic. This article is part of the PLoS Medicine Global Health Diplomacy series.
Severe acute respiratory syndrome (SARS) was the first global epidemic of the 21st century. It not only caused mass panic but also generated a discourse on health insecurity around the world. Table 1 shows a chronological account of the disease outbreaks. Owing to China's belated response, particularly its obstruction in early 2003 of the entry of World Health Organization (WHO) assessment teams into the country for investigation of the virus, the subsequent mapping of the disease during the outbreak period kept global attention on China. In retrospect, there appear to be valuable lessons China can draw from its experience with SARS and several implications of SARS on China's engagement in global health diplomacy. This case study examines China's policy changes in the area of public health since the SARS outbreak. Using literature reviews, personal experience, and informal interviews with Chinese health officials, we provide insight into the extent of China's increased engagement in public health, at both the domestic and the international levels.
We spoke with three high-ranking health officials in China's Ministry of Health in August 2009 who admitted that the SARS outbreak had alerted Chinese citizens as well as the government to the danger that public health, particularly infectious diseases, could become a dire threat if not properly controlled. This perceived threat extended beyond their country to the world. In the face of criticism from abroad about China's handling of the SARS epidemic, the new Hu Jintao±Wen Jiabao leadership, taking office in early 2003, swiftly adopted a more open and proactive attitude to the WHO member countries and southeast Asian nations containing the disease.
Indeed, SARS appears to have prompted a national discourse on the interrelationship between infectious diseases and non-traditional security inside China. This is evidenced by the vast amount of literature on the subject of non-traditional security issues generated by Chinese scholars since the SARS outbreak. Using`^ ßh(fei chuantong anquan, non-traditional security)'' to search for articles contained in a database known as``China Academic Journals Full-text Database: Economics, Politics and Law (Zhongguo qikan quanwen shujuku: jingji, zhengzhi yu falu È zhuandang, -ýhpn: ÏN ?»Õc),'' there are barely anỳ`n on traditional security'' articles published before the SARS outbreak. However, subsequent to the outbreak it became a flourishing subject in China's scholarly world. Among the articles that includè`n on-traditional security'' in their titles since the start of economic reforms in 1979, more than 95% of them were published after 2003 (see Figure 1) .
At the domestic level, the SARS outbreak exposed a fundamental shortcoming of China's health care system. As such, China required a national health reform in order to improve its surveillance system and reorient its single-minded pursuit of economic growth since the late 1970s to a more balanced development between economic growth and social infrastructure building [1±3] . The health officials in Beijing were also of the view that SARS could be seen as a turning point for China's health reform because it provided a political rationale for the government to accelerate the reform. According to the Asian Development Bank, SARS cost China US$6.1 billion, or 0.5% of its GDP, in 2003 [4] . This economic loss may seem insignificant, but for a regime that prioritizes economic growth and stability, the political repercussions of an economic decline caused by a health crisis cannot be underestimated. Indeed, SARS alerted the Chinese leadership to the pitfalls of a public health care system in disarray [2] . In order to maintain a sustained economic growth, the central government has increased its public health funding significantly since the SARS outbreak. For example, in 2003, the central and local governments altogether allocated 111.69 billion yuan for public health, an increase of 23% over the previous year. Between 2002 and 2006, the government's public health spending grew by almost 100%. There was a further increase of 29.1% in 2007 to 229.71 billion yuan. The share of public health spending in the country's GDP was 0.89%
The Policy Forum allows health policy makers around the world to discuss challenges and opportunities for improving health care in their societies.
in 2007, compared to merely 0.75% five years ago [5, 6] .
External pressure has also impacted on the development of China's public health. During the SARS outbreak, the WHO directly told the Chinese government in its mission report in April 2003 that`` [t] here was an urgent need to improve surveillance and infection control'' in the country [7] . Two years later, the Chinese government officially admitted its health care system was ailing in a joint report issued by State Council's Development Research Centre and the WHO [8] . The recent decision on a new rural cooperative medical system is one of its efforts to provide its rural residents by 2010 with more equitable and accessible health care [9±11] and improve its diseases surveillance system at the local level. In addition, both the``loss of face'' in the Summary Points N SARS not only exposed a fundamental shortcoming of China's public health surveillance system as well as its single-minded pursuit of economic growth since the late 1970s, but also forced China to realize that, in the era of globalization, public health is no longer a domestic, social issue that can be isolated from foreign-policy concern. N Its ailing health care system, its aspiration to be seen as a``responsible state,'' and international demands for health cooperation have compelled China to be more proactive in the global health domain.
N There are signs that China is now using public health as a means to strengthen its diplomatic relations with the developing world, in particular the African continent.
N While China has embraced multilateral cooperation in a wide array of global health issues, its engagement remains``state centric'' and therefore leaders attach primary significance to intergovernmental organizations, particularly the UN agencies. SARS outbreak and its aspiration to be seen and respected as``a responsible state'' have pushed China to enhance its cooperation with international institutions in dealing with other pressing health issues [12] . as well as non-governmental organizations inside the country to combat the disease. However, while Beijing calls for and welcomes involvement of multiple actors in combating the disease inside its territory, it maintains little tolerance of anyone or any activity that would attenuate its absolute control over the country or threaten the supreme authority of the government. A major feature of China's multilateral public health engagement, then, is that of``state-led health governance.''
Nevertheless, compared to its initial handling of SARS, China now reacts in more timely fashion in releasing information on contagious diseases, despite implementation problems that include sluggish responses to disease outbreaks on the part of local officials and technical incapacity to detect sudden outbreaks at the local level. In addition, China has shown increased willingness to engage with international organizations on a wide array of global health issues. For example, in order to align with international interests in tobacco control, Beijing signed the WHO Framework Convention on Tobacco Control (FCTC) treaty in December 2003. It was ratified in 2005 by the Chinese National People's Congress and its legislature and took effect in 2006 [13] . Being the largest producer as well as the largest consumer of tobacco in the world, Chinese tobacco policy has long been influenced more by economic concerns than by public health [14] . Its ratification of the FCTC came as a surprise for many.
China's recent responses to the 2009 outbreak of swine flu (influenza A/H1N1) give an impression that the dreadful effect of SARS six years ago has taught China a lesson. As soon as the WHO raised its pandemic alert phase from 3 to 4 on 28 April 2009, Premier Wen Jiabao convened a cabinet meeting to discuss a set of response measures designed to deal with the disease, although there was neither any reported case of swine flu in China at that time nor a similar virus found in the pigs in the country. Two days later, the Communist Party of China (CPC) General Secretary Hu Jintao convened a meeting of the Standing Committee of the Politburo. That the holding of the highest level meeting was announced immediately after its adjournment was regarded as unusual by many China watchers [15] . China's aggressive and visible approach towards swine flu appears to demonstrate the government's determination in tackling the disease. However, this aggressive or even excessively stringent measure against swine flu, as some observers have said, aroused international debate. The WHO asked China to justify its decision to keep travelers from Mexico in quarantine. The Mexican government criticized China's response as``unjustified,'' threatened to take retaliatory action, and sent an airplane to Shanghai on 5 May 2009 to repatriate its quarantined citizens [16±18]. In contrast, others including health experts reportedly praised China for exercising extra vigilance against the virus [19, 20] .
At the international level, there have been signs since the SARS outbreak that public health is high on China's foreign policy agenda. First, Beijing has become more proactive in participating in global health governance. China had for a long time played a passive role in the WHO since gaining its membership in the organization more than three decades ago. The SARS outbreak let China experience the power of the WHO, which has become increasingly more influential while other international organizations, such as the United Nations Security Council, International Monetary Fund, World Bank, G8, and the World Trade Organization, are facing legitimacy, accountability, and representativeness challenges. WHO's authority in dealing with disease outbreaks is still widely recognized [21] . Without China's prior consent, the WHO issued a travel advisory against unnecessary travel to Guangdong province, putting China under the global spotlight for spreading infectious disease to other countries. Perhaps this lesson has prompted the Chinese government to realize the political importance of the WHO and to increase its participation in global health governance.
In the WHO Director-General election in 2006, China, for the first time since it gained its membership in UN agencies in 1971, nominated and supported a Chinese national, Margaret Chan, as a candidate for the top post. It is widely believed that Chan's success was a diplomatic triumph both for her and for China. Wang Yizhou, then with the Chinese Academy of Social Sciences, Beijing, told one of the authors (LHC) in March 2008 that Margaret Chan's nomination as the Director-General of the WHO was not a fortuitous incident. The health officials we spoke with in Beijing concurred with Wang's view and explained that China has recently realized and valued the increasing importance of the WHO at the world stage. It is also a source of national pride to have a Chinese national at the top post of the global health organization [22] . Chan was the Director of Health of Hong Kong during the SARS outbreak in 2003. Her nomination could be seen as a case of China's smart play and rising clout at the global stage, displaying its confidence in her managing of Hong Kong affairs and the successful implementation of China's`O ne Country, Two Systems'' policy [23] . Furthermore, China's WHO role politically could be regarded as a pre-emptive measure to block Taiwan's attempts to seek WHO membership [23] . On the other hand, with improved relations with the Ma Ying-jeou administration in Taiwan 
The second sign that China has put public health high on their foreign policy agenda since SARS is their provision of development assistance and global public goods for health. As such, China is now using public health as a means to strengthen its diplomatic relations with the developing world, including African countries. China began in the 1960s to send``angels in white'' and``barefoot doctors'' to the sub-Saharan region to provide some of the poorest African countries with medical services. However, as argued by Huang Yanzhong of Seton Hall University, China's health diplomacy was``flimsy, passive, and asymmetric,'' at least until the 1980s [3] . After the SARS outbreak, in spite of its own failing health system, the Chinese government reiterated in its China's African Policy, published in early 2006, the nation's commitment to improving Africa's public health service.
To balance the criticisms that its energy and resource extraction in Africa grab the scarce resources there and that it shields disreputable regimes in such countries as Sudan and Zimbabwe from international opprobrium, China has stressed``win-win'' relations in its deepening engagement with African countries. In response to the claims of exploitation in the natural resource sectors [26, 27] , China emphasizes a nostrings-attached policy in offering financial aid and technical support to less developed countries, including those in the African continent. In contrast, donor countries in the West and international financial institutions often attach conditionalities to their foreign aid programs, which are linked to market and political liberalization and good governance [28] . China has expanded its public health initiatives, such as in infrastructural building and health practitioner training, in Africa in recent years [29] , as well as commitment to cooperation with many African countries to help prevent and treat infectious diseases, particularly HIV/ AIDS and malaria [30±34]. In his African visit in June 2006, Chinese Premier Wen Jiabao asserted that China would promote sustainable development and help African countries tackle their burning social problems, of which public health was one of the top priorities [35] . Again in November 2009, during the fourth ministerial meeting of the Forum on China-Africa Cooperation in Egypt, Wen announced eight new measures to strengthen China-Africa cooperation in the following three years, including a 500 million yuan (US$73.2 million) assistance package that allows China to build 30 hospitals and 30 malaria prevention and treatment centers and to train 3,000 practitioners in the continent [36] .
Undoubtedly China has been learning from itself as well as from other developed countries the importance of providing sustainable development and global public goods for improving one's reputation on the world stage. At the``First International Roundtable on China-African Health Collaboration ± New Health Initiative'' in December 2009 in Beijing, one of us (LC) observed representatives from the WHO, World Bank, and the Bill & Melinda Gates Foundation praising China's development packages for their positive contributions to African development.
In addition, China's State Council has established in recent years a coordinating mechanism to facilitate cross-ministry dialogues and cooperation in global health and foreign aid initiatives. Chinese scholars have noted that a State Council`G lobal Health Diplomatic Coordination Office'' (quanqiu weisheng waijiao xietiao bangongshi, hÃk¤OÃl¤), led by a senior official at vice-Premier level, is crucial to effectively coordinating and developing policies of health diplomacy [37] . In order to increase the capacity of China's health diplomats to deal with global health challenges, a training course, the first in a series, for Chinese officials, including officials from the Ministries of Foreign Affairs and Health, was held in August 2009 in the Institute for Global Health at Peking University [38] .
Following the Chinese government's acknowledgement of a SARS outbreak in the country, it began to acknowledge the importance of public health to national development and to accordingly strengthen its multilateral cooperation in combating contagious diseases inside and beyond its borders. For example, in the midst of the recent global economic downturn, the Chinese government announced in 2009 an injection of 850 billion yuan (US$125 billion) into its health care system to improve its operation. Since the SARS outbreak, it has not only deepened its engagement with other nations and international organizations, and cooperated with a variety of actors in dealing with its own fledgling health care system including the problem of HIV/AIDS, but China has also developed a vision for global health diplomacy. A ground-breaking implication of the SARS outbreak for China is that it was struck to realize that public health is not simply a domestic, social issue that can be isolated from foreign-policy and security concerns. In a globalizing world, the Chinese government appears to have learned that its health policy will be scrutinized by the world, and hence, it has become more open to and actively participates in global health governance. The government is now learning from such European countries as the UK, France, and Switzerland in the provision of the global public goods for health. Its substantial health assistance to sub-Saharan Africa in building hospitals and training health practitioners forms part of its health diplomacy and contribution to global health governance. It has also been proactively engaging with both regional and global health institutions since 2003 and set up different health surveillance networks with its ASEAN partners as well as other intergovernmental organizations, such as the Asia-Pacific Economic Cooperation (APEC) forum [12] .
Despite its increasing engagement with global health governance since the SARS outbreak, China's approach remains, however, fundamentally state-centric, contrary to the essence of global health diplomacy and governance. With grave concern about the loss of national sovereignty to external or nongovernmental actors, Chinese senior leaders have therefore attached primary significance to intergovernmental organizations, particularly the UN agencies. In evaluating the impact of SARS, Andrew Price-Smith has put the same point succinctly:``while the SARS epidemic may have generated moderate institutional change at the domestic level ¼, it resulted in only ephemeral change at the level of global governance'' [2] . In other words, national sovereignty is still of paramount importance for the Chinese leadership. Because of its sensitivity to foreign interference into its internal affairs, the Chinese government has not yet formally or officially endorsed the notion of``human security.'' Under the umbrella concept of national security,``human safety,'' instead of``human security,'' is discussed throughout all of China's five white papers on national defense since 2000 (i.e., 2000, 2002, 2004, 2006, and 2008 ). Taiwan's participation in the World Health Assembly is predicated on the condition that it is considered part of China, not an independent entity. Having no tolerance in ceding its supreme authority, the central government has adopted a multi-faceted attitude towards its civil society organizations. While Beijing shows its willingness to cooperate with a wide array of actors inside China, it refuses to let its domestic NGOs and activists establish direct links with their counterparts overseas.
It is still uncertain whether this sovereign concern will trump the provision of global public good for health. Nevertheless, in a highly globalizing world, infectious diseases know no border. While China is seeking to adhere as much as possible to the underlying norms and rules of global health governance (and sometimes even applies them to their extremes), as evidenced by its handling of the recent swine flu outbreak, the major step forward is perhaps to reframe health as a global public good that is available to each and every individual of the world, rather than merely as an issue of concern to nation-states. Electron Tomography Reveals the Steps in Filovirus Budding The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses. Marburg virus (MARV) and Ebola virus, the two genera in the family Filoviridae, cause fulminant hemorrhagic disease in humans and nonhuman primates, resulting in high mortality rates [1, 2, 3] . Outbreaks of MARV disease in sub-Saharan Africa underline the emerging potential of this virus, which is classified as a highest-priority bioterrorism agent by the Centre for Disease Control [4, 5, 6, 7, 8] .
The filoviruses are members of the order Mononegavirales and contain a single-stranded negative-sense RNA genome, which is encapsidated by the nucleoprotein (NP). The MARV genome encodes seven structural proteins [9, 10] : the polymerase (L), VP35 and VP30 associate with NP to generate the helical nucleocapsid (NC) [11, 12, 13] . The viral glycoprotein (GP), which is inserted in the viral envelope, mediates cell entry [14, 15] . The major matrix protein VP40 plays a key role in virus assembly, and VP24, the second matrix protein, is suggested to support the template function of the NC [12, 16, 17, 18] .
MARV infected cells develop viral inclusions in the perinuclear region [19, 20, 21] . These contain NC proteins and are most likely centres of NC assembly [22] . MARV particles bud from the plasma membrane (PM) of long filamentous cellular protrusions that contain parallel actin bundles and other markers of filopodia [23] . The released virus particle has a membrane envelope and contains an NC that is surrounded by the viral matrix protein VP40. It is unclear how NCs are transported from viral inclusions to the PM, whether they adopt their virion conformation before, during, or after, transport, or where NCs associate with VP40 that is not co-transported with NCs but is necessary for budding. Released MARV particles appear filamentous, hooked, six-shaped or round by electron microscopy (EM) [24] but their three-dimensional (3D) morphology is unclear. It is also unknown whether production of differently shaped viruses depends on different budding mechanisms and whether they differ in infectivity. Experiments to address these issues are complicated by the need to perform all infection experiments under BSL-4 containment conditions.
The processes of assembly, budding and release of spherical viruses have been extensively studied [25, 26, 27] and it is well established that spherical enveloped viruses are produced by budding away from the cytoplasm in a process that is related and topologically equivalent to the formation of small vesicles in multivesicular bodies [28, 29] . In contrast, the basic steps of assembly and release for large, filamentous, enveloped particles such as the filoviruses are poorly understood. EM studies have shown MARV and Ebola virus particles protruding perpendicularly from the cell [23, 30] . These observations, together with similar findings of protruding intermediates of other important filamentous or rod-shaped viruses such as rabies virus [31] , influenza virus [32, 33, 34] , or vesicular stomatitis virus (VSV) [35] have led to the suggestion of a vertical ''rocket-like'' mode of budding. Ebola virus NCs have been seen associated parallel to the PM [36] , leading to the suggestion that a second, horizontal, ''submarine-like'' mechanism is the major mode of budding. This unusual mechanism has not been described for any other virus.
In this study we use electron tomography (ET) to describe and analyse in 3D the structure of MARV budding intermediates and released viruses at different stages of infection. In contrast to conventional EM of ultrathin sections, ET allows complete MARV virions and budding structures to be studied in 3D. This permits unambiguous determination of virus morphology, dimensions, stage of budding and position relative to the infected cell. The samples are prepared by high-pressure freezing followed by embedding in resin and staining with heavy metals. Unlike cryo-ET of vitreous samples, this method is not appropriate for the study of high-resolution protein structure. Nevertheless, it gives excellent preservation of the features studied here such as membranes and protein assemblies including viral NCs [37, 38] . It also has, for this particular purpose, substantial advantages over cryo-ET. The features of interest are imaged at much higher contrast, including at high tilt angles. The samples are also easier to handle and stable under the electron beam, allowing efficient screening, which facilitates the collection of larger datasets.
Our observations suggest that interplay between the virus and the infection state of the cell determines virus morphology. Furthermore, the 3D data allow us to describe the sequence of steps that take place in infected cells during assembly and budding of filamentous virions, and to reconcile the horizontal and vertical models for filovirus budding as representing different snapshots of a single budding process.
To determine the time course of production of infectious MARV during a prolonged infection period, supernatants of HUH-7 cells, infected with MARV under BSL-4 conditions, were collected from day one to four post infection (p.i.) and tested for viral protein content, specific infectivity and virion morphology. The amounts of viral NP and VP40 released from the cells were measured by quantitative immunoblotting. This showed that viral protein release peaked between day one and two p.i. ( Figure 1A ). The TCID 50 of each supernatant was determined and normalized to the amount of released NP to estimate the specific infectivity per virus ( Figure 1B , grey areas). Specific infectivity was also at a maximum between day one and two p.i.. Virus morphology in the supernatants was monitored by EM and revealed that at the peak of viral protein production and specific infectivity, 80% of the virus particles displayed the characteristic filamentous morphology of the filoviruses, with the remainder appearing bent or round ( Figure 1B) . Later in infection, the specific infectivity was lower and correlated with fewer filamentous particles and more round or bent particles ( Figure 1B) , suggesting that infectivity can predominantly be attributed to filamentous virus. There was also an increase in the release of cellular vesicular material over time (Table 1) .
Quantitative EM analysis of infected adherent cells that were fixed and embedded in situ revealed that cell morphology also changed over time. At day 1 p.i. 95% of the observed cells appeared intact and displayed filopodia-like PM protrusions ( Figure 1C , D). The cytoplasm often contained densely stained viral inclusions ( Figure 1D ) and virions were readily seen in the immediate periphery of cell profiles; they were easily identified by means of their electron-dense stain and a rod-shaped NC and were frequently found 'trapped' between or underneath adherent cells ( Figure 1E ). Over 90% of all observed virions around the cells were filamentous or hooked ( Figure 1F ). In contrast, when the monolayer was fixed at 4 days p.i., only 17% of the cell profiles were intact and 83% appeared vesiculated and resembled apoptotic cells ( Figure 1C , G). Most of the virus particles around such vesiculated cells were bent or round ( Figure 1F , H).
The above characterisation indicates that production of fully infectious, filamentous virus is highest at 1-2 days p.i., when the cells still have intact membrane profiles. This was therefore selected as an appropriate time point for studying assembly and budding of filamentous particles.
To study the assembly and budding of MARV we performed ET on MARV-infected cells. ET allows the different steps of assembly and budding to be visualized in 3D and the dimensions of viral structures to be measured and analysed. Infected cell monolayers were fixed at 1 day p.i., removed from the BSL-4 facility, processed for EM and cut into 300 nm thick sections in their in situ orientation. Dual-axis tilt series of the sections were acquired in the electron microscope, as described in Materials and Methods, and 3D reconstructions were computationally generated. Viral NCs could be readily identified in 3D reconstructions. They were found around viral inclusions, within the cytoplasm, associated with the PM, incorporated into budding viruses, and in released virus particles ( Figure 2 ). We therefore used the NC itself as a convenient marker for identifying virus assembly intermediates.
The 3D data allowed us to measure the length of complete viral NCs even when they were bent or tilted with respect to the sectioning plane. In the periphery of viral inclusions in the cytoplasm individual NCs could be found. They appeared as rodshaped, striated structures that were more densely stained than the cytoplasm and were on average 711 nm long ( Figure 2A , Table 2 ). NCs were frequently seen at the PM or in filopodia-like membrane protrusions, where they were associated with the membrane along
The filoviruses, Marburg and Ebola, cause lethal hemorrhagic fever and are highest-priority bioterrorism agents. Filovirus particles contain a rod-like nucleocapsid and are normally filamentous, though other shapes are seen. It is poorly understood how such large filamentous particles are assembled and released from infected cells. Here we have studied Marburg virus production in infected cells using electron tomography. This technique allows virus particles to be visualized in three dimensions at different stages during assembly. We find that in early stages of virus production, highly infectious filamentous viruses are produced, whereas after prolonged infection poorly infectious spherical viruses are released. We also define the sequence of steps in filamentous virus release. The intracellular nucleocapsid first travels to the plasma membrane of the cell, where it binds laterally along its whole length. One end is then wrapped by the plasma membrane and wrapping proceeds rapidly until the virus protrudes vertically from the cell surface. The rear end of the virus particle then pinches off from the cell. We propose that other important filamentous and rod-shaped viruses also follow this series of steps of assembly and budding.
their whole length. The length of PM-associated NCs was 707 nm ( Figure 2B , Table 2 ), the same as that of cytoplasmic NCs. Viral budding structures localized predominantly to filopodia-like protrusions of infected cells, in agreement with previous data [23] . They appeared as filamentous finger-like extensions emerging either from the tip or the sides of filopodia-like protrusions ( Figure 2C -E). Each bud accommodated a rod-shaped NC that was surrounded by densely stained material and had the same length as intracellular and PM-associated NCs ( Table 2) . Of the budding structures reconstructed in 3D, 13% appeared as extrusion intermediates (Table 2) . These structures contained a full length NC (729 nm) that was only partially extruded ( Figure 2C ): one end of the NC was tightly wrapped on all sides by the PM, whereas the other was attached to the PM along one side ( Figure 2C ). The majority (87%) of budding structures had the NC completely inserted into the finger-like membrane extension ( Figure 2D , E). Scission at the base of these fully extruded buds would lead to the release of filamentous virions ( Figure 2F and Video S1).
Most released viruses in the periphery of infected cells appeared as straight filaments in 3D reconstructions ( Figure 2F ). Some filamentous viruses displayed one bent or buckled end giving rise to hooked or six-shaped particles ( Figure 2G and Video S1). Filamentous viruses were on average 789 nm long and had a diameter of 88 nm (Table 2 ), in agreement with previous studies [20, 24] . All virions had a membrane envelope and contained a rod-shaped NC that displayed the regular striated pattern previously described [12, 20] . The volume between membrane envelope and NC was filled with a densely stained material, most likely the VP40 matrix protein, which appeared to link the envelope to the NC on all sides and along the whole length ( Figure 2F ). 3D analysis revealed that NCs in all released virions had the same length, on average 735 nm, and appeared bent or kinked when particles were hooked or six-shaped ( Figure 2G , Table 2 ). These length averages exclude a single outlier, which was a double-length filamentous virus with a double-length NC ( Table 2 ). The presence of NCs of the same length in the cytoplasm, at the PM, in filopodia, in all budding structures and in filamentous viruses suggests that the NC assembles into a helix of a defined and final length prior to being transported to the PM.
The 3D data also allowed us to closely examine both ends of reconstructed filamentous virions. This revealed morphological differences between the two virus ends: 19 out of 21 3Dreconstructed filamentous virions displayed a membrane bulge or hook at one end ( Figure 3A ), while the membrane formed a round, intact hemisphere at the opposite tip ( Figure 3B and Table 2 ). Only 2 out of 21 filamentous viruses had two round, intact tips. Virions with membrane distortions at both ends were never observed. Although we cannot exclude that such membrane distortions are due to chemical fixation, they suggest that a local instability of the viral membrane is specifically induced on one virus end. Thus, we made use of the fact that cells and extracellular viruses were fixed and examined in their in situ orientation. Measurement of the average distance of intact and distorted virus ends from the nearest cellular membrane revealed that intact virus ends were predominantly found at distances .250 nm away from a cellular membrane, whereas distorted virus ends were more frequently localized within 50 nm distance ( Figure 3C ). This suggests that the membrane distortions are found at the rear end of filamentous viruses where scission took place (also shown in Figure 2G and Video S1).
We also carried out ET of infected cells fixed at 4 days p.i.. At this time point the majority of cells exhibited heavily vesiculated membranes and produced rounded viruses of lower infectivity. The 3D analysis revealed that most viruses were roughly spherical in shape. They were generally found in close proximity to convoluted, vesiculating areas of the PM (figure 4A) and often surrounded by large numbers of cell-derived vesicles ( Figure 4A and Video S2). This suggests that release of spherical viruses occurs rapidly and simultaneously with the shedding of other cell-derived vesicles. NCs within spherical virions were bent or kinked but never broken or segmented, as might be suggested by 2D EM of thin sections (see, for example the 2D and 3D views of the spherical particle shown in Figure 4B ). None of the virus particles were toroidal, as has previously been suggested [39] . Interestingly, kinked NCs in spherical viruses had a total length of 734 nm, the same length as NCs in filamentous virions ( Figure 4B , Table 2 ) and they were associated along one side with the viral membrane ( Figure 4B ). These findings demonstrate that preassembled full-length NCs are packaged into each virion, irrespective of virus shape.
The analysis of MARV-infected cells by ET allowed us to take 3D measurements of NCs and revealed that NCs have uniform length at all stages in the virus assembly and budding process described here, from intracellular NCs to released virus particles. This suggests that the length of the complete viral genome and the number of nucleoproteins required to encapsidate the genome dictate the length of the intracellular NC prior to its transport to the PM.
Once the NC has reached the PM, two contrasting mechanisms have been proposed for filovirus budding. In the ''submarine-like'' model, budding proceeds via lateral association of the NC with the membrane, followed by horizontal budding. In the ''rocket-like'' model, the NC is extruded vertically from the PM. The analysis of budding MARV particles presented here allows the sequence of steps resulting in filamentous virus budding and release to be described in 3D. Viral NCs are assembled in the cytoplasm ( Figure 5A ) and delivered in their full-length form to the PM, with which they associate laterally, along one side, for their entire length ( Figure 5B ). Envelopment of the PM-associated NC is initiated at one end, and proceeds along the length of the NC ( Figure 5C ) until the nascent virion protrudes from the membrane, remaining attached at only one end ( Figure 5D ). Only very few particles are seen which appear to be partly extruded, whereas larger numbers of NCs are either associated with the PM prior to extrusion or are fully extruded and protruding from the PM. This strongly suggests that extrusion is a rapid process in comparison with its initiation or the subsequent membrane scission event.
Scission of the filamentous virus particle from the PM then takes place with a bud-neck shape which can have a circular cross-section. This is the same shape as the bud-neck which would be present in the budding of spherical enveloped virions, or in the budding of vesicles into a multi-vesicular body [40] . Release of a horizontally budding particle would require scission of a membrane neck with a noncircular cross-section. Our observations indicate that no such unusual scission mechanism needs to be proposed.
Scission of the protruding bud leaves local membrane instability at the rear end of the virus particle ( Figure 5E ), which may be exaggerated during sample preparation. In contrast, the front end of the filamentous particle is a well-defined hemisphere. The difference between the two ends could reflect a destabilization at the rear end of the particle induced by scission. Alternatively, the hemispherical front end of the particle may be stabilized by a structure involved in initiating envelopment, similar to the front end of bullet-shaped rhabdovirus particles [41, 42] .
The budding of filamentous MARV therefore proceeds via an NC that is laterally associated with the PM, and a vertically protruding bud. Any changes in the rates of individual steps in the budding process could dramatically alter the appearance of the budding structures in EM. For example, if the rate of scission were significantly increased, or the rate at which extrusion is initiated were dramatically decreased, then large numbers of NCs might be expected to collect horizontally under the PM, giving the appearance of a predominant ''submarine'' mode of budding [36] . If the rate of initiation of extrusion were significantly increased, or the rate of scission decreased (for example by inhibiting recruitment or function of the cellular endosomal protein sorting system), then large numbers of protruding buds would accumulate, giving the appearance of a predominant ''rocket-like'' mode of budding. We suggest the contrasting appearance of Ebola virus and MARV infected cells in EM does not reflect different budding mechanisms, but rather different rates for the individual steps of the process presented here. These rates are likely to be both virus and cell type dependent.
The sequence of steps in budding described here results in a very different mechanistic understanding of filovirus budding. Firstly, the proposed two budding modes, one vertical and one horizontal, can be reconciled as representing different snapshots of a single budding mechanism. Secondly, rather than filoviruses adopting a unique horizontal mode of budding not seen in other systems, the budding process can now be placed within the established framework of other cellular budding events. In the new model, budding is initiated by wrapping of one end of the NC with a hemispherical membrane, and completed by scission at a bud-neck with a classic round cross-section. These are the membrane shapes present during budding and scission events of cellular vesicles and spherical viruses. Thirdly, comparison of our data with published electron micrographs of filamentous virus budding structures suggests that other viruses may also follow the sequence of steps described here, and that rather than being unique, filovirus budding belongs within a more general budding mechanism also adopted by other rod-shaped viruses. For example, budding VSV, a rhabdovirus, can be found protruding perpendicular to cell membranes in infected cells, but the NC can also be seen underneath the cell membrane, with which it associates laterally along one side [35] . The rounded end of the virus, easily distinguished in VSV, seems to associate more tightly with the membrane [43] , and buds first. After budding, under certain preparation conditions, the rear of the virus is seen to show small membrane blebs or instabilities [41] similar in appearance to those described here for MARV. These striking observations are consistent with VSV following the same sequence of budding steps as MARV. A general assembly and budding mechanism for filoviruses and rhabdoviruses might represent a target for future antiviral drugs.
After a prolonged period of infection, the released MARV particles have lower specific infectivity and the majority is roughly spherical in form. Like the filamentous virions, the spherical particles contain a single full-length NC, which is kinked in a number of places, but not broken, suggesting that these particles still contain a full-length viral genome. The NC is not tightly wrapped on all sides as it is in filamentous virions, but is associated with the membrane along one side for its entire length. This lateral association is also seen for intracellular NCs prior to extrusion from the PM. Since formation of spherical particles is paralleled by convolution of the PM and shedding of cellular vesicular material into the supernatant, we propose that spherical viruses are released by large-scale membrane instability at sites of budding, leading to vesiculation of viruses prior to extrusion. In this model, kinking of the NC might be induced by the forces which lead to invagination and vesiculation of the PM, though other factors could contribute, such as lack of a viral or cellular factor which rigidifies the NC. The interplay between the budding process and the dynamics or composition of the cellular membrane, as well as possible changes in the relative rates of the different steps of the budding process in different virus strains [44] , mutants [45] , cell types [46] or stages of infection, may also contribute to the variable morphology observed in other viruses.
In summary, our observations reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of MARV virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses. Furthermore, we demonstrate that virus shape is determined both by viral and cellular factors. The model for filamentous virus budding presented here raises a number of new questions. Do elements of the cytoskeleton or other cellular components play a specific role in mediating envelopment or budding of filamentous or spherical virus particles? Is one end of the PM-associated NC specifically able to initiate the extrusion of the filament, as in rhabdoviruses, or is the directionality of the extrusion process random? What is the organisation of the NC and VP40 matrix layers in the released Table 2 . 3D analysis of virus shape and NC length at different steps of filamentous MARV budding. 
The HUH-7 human hepatoma cell line and Vero cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, l-glutamine and penicillin-streptomycin at 37uC under 5% CO 2 . All work with infectious MARV was performed under BSL-4 conditions at the Institute of Virology in Marburg. The MARV Leiden strain, isolated in 2008 in Leiden, the Netherlands [47] , was propagated in Vero E6 cells and purified as described previously [48] . HUH-7 cells were infected with MARV with a multiplicity of infection of approximately 1 plaque-forming unit per cell for one to four days. At the indicated time points, cell culture supernatants were collected and used for viral infectivity assays (see below), or viruses in the supernatants were purified by centrifugation over a sucrose cushion and fixed for 48 hours (h) with 4% paraformaldehyde in PBS for analysis of particle morphology (see below). For EM and ET of infected cells, HUH-7 cells were grown on carbon-coated sapphire disks, infected as above and cell monolayers were fixed at the indicated time points in the cell culture dish with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1M PHEM buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl 2 , 10 mM EGTA), pH 6.9 for 30 min, after which the fixative was replaced with 4% paraformaldehyde in 0.1M PHEM. Fixed cells were removed from the BSL-4 lab after 48 h of inactivation.
Infectivity of MARV particles released into the supernatant of cells 1 to 4 days p.i. was assayed by a 50% tissue culture infective dose (TCID 50 ) assay: Vero cells were grown in 96-well plates to 30 to 40% confluence. Cells were inoculated in quadruplicate with 10-fold serial dilutions of supernatants of HUH-7 cells infected with the MARV Leiden strain for one to four days as described above. The assays were evaluated at 10 days p.i.. TCID 50 values were calculated using the Spearman-Karber method [49] . Equal volumes of each supernatant were separated by SDS-PAGE followed by quantitative immunoblotting on a LiCor Odyssey system using a mouse monoclonal anti-NP antibody, and secondary antibodies, protocols and software (Odyssey version 2.0) provided by the manufacturer. TCID 50 values were normalized to NP levels detected in each supernatant, and the TCID 50 value in the supernatant collected at day 2 p.i. was set to 100%.
For quantification of virus morphology at different time points p.i., fixed cell culture supernatants were purified by centrifugation over a sucrose cushion, pelleted for 30 min at 40,000 g, 4uC in a Beckman ultracentrifuge using a TLA-55 rotor. Pellets were embedded in 12% gelatin and prepared for EM as described previously [50] . 70 nm cryosections were obtained with a Leica EM UC6 microtome, FC6 cryochamber (Leica Microsystems, Wetzlar, Germany) and a diamond knife (Diatome, DiS-Galetzka Weinheim, Germany). Thawed cryosections were counterstained with uranyl acetate as described elsewhere [51] . For EM and ET of infected cells, HUH-7 cells were grown on carbon-coated sapphire disks, infected and fixed as described above. Samples were high-pressure frozen with a BalTec HPM-010 and freeze substituted with 0.1% (w/v) uranyl acetate, 1% osmium tetroxide (w/v) and 5% (v/v) water in glass distilled acetone in a temperature-controlling device (Leica EM AFS I). Cells were kept for 40 h at 290uC and warmed up to 0uC (slope 5uC/h) with an additional 3 h infiltration period at 230uC. Samples were washed three times with glass distilled acetone, infiltrated at room temperature with increasing concentrations of epoxy resin (Glycidether 100, Roth, Karlsruhe, Germany) in acetone over 12 h, and polymerized at 60uC for 48 h. 150 nm and 300 nm sections were obtained with a Leica Ultracut UCT microtome and a diamond knife.
Thin sections of virus and infected cells were examined on a FEI Morgagni 268 TEM equipped with a 1K side mounted CCD camera (SIS, Muenster, Germany). Quantification of cell morphology and cell-associated virus morphology was carried out on 150 nm resinembedded sections in a systematic random sampling manner [52] . Starting points for sampling were chosen randomly on each grid, and all grids were examined in the same systematic manner. At least three grid squares per EM grid and three EM grids per time point were sampled. Morphology of viruses from cell culture supernatants was quantified by EM in the same systematic random sampling manner on thawed 70 nm cryosections of virus pellets, purified and embedded as described above. At least 200 particles from three different grids were evaluated per sample. ET was carried out essentially as described elsewhere [53] . Dual axis tilt series from 300 nm sections were recorded on a FEI TECNAI TF30 microscope operated at 300kV (4K FEI Eagle camera; binned pixel size 0.77 nm or 1 nm on the specimen level) over a 260u to 60u tilt range (increment 1u) and at a defocus of 20.2 mm. Tomograms were reconstructed using the IMOD software package (version 3.12.20) [54] . 3D measurements of virus end distance, analysis of virus end morphology and 3D surface renderings were carried out using the AMIRA Visualisation Package (version 5.2.0, Visage Imaging, Berlin, Germany). In total, 68 3D reconstructions from three independent infection experiments were analysed, of which 48 of high image quality were used for detailed analysis and measurements. NCs in different samples displayed variable contrast, which resulted from differential access of electron-dense stain to individual cells and different subcellular regions during sample preparation. NC representations in Figures 2 and 4 and Video S1 and Video S2 were generated and displayed using the AMIRA EM package [55] and MatLab (version 7.4.287).
Video S1 Release of filamentous Marburg virus particles at one day post infection. Animation through a z-series of 2 nm digital slices of a double axis tomogram, reconstructed from a tilt series taken of a ,300 nm thick section of a MARV-infected HUH-7 cell at one day post infection. Coloured overlay shows a 3D surface model of membranes and cytoplasm (yellow) and representations of the viral NCs (blue). A filamentous viral budding structure with the NC completely inserted into the budding site as well as several released filamentous viruses are seen.  Peptide-Mediated Liposomal Drug Delivery System Targeting Tumor Blood Vessels in Anticancer Therapy Solid tumors are known to recruit new blood vessels to support their growth. Therefore, unique molecules expressed on tumor endothelial cells can function as targets for the antiangiogenic therapy of cancer. Current efforts are focusing on developing therapeutic agents capable of specifically targeting cancer cells and tumor-associated microenvironments including tumor blood vessels. These therapies hold the promise of high efficacy and low toxicity. One recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics is to encapsulate anticancer drugs into targeting liposomes that bind to the cell surface receptors expressed on tumor-associated endothelial cells. These anti-angiogenic drug delivery systems could be used to target both tumor blood vessels as well as the tumor cells, themselves. This article reviews the mechanisms and advantages of various present and potential methods using peptide-conjugated liposomes to specifically destroy tumor blood vessels in anticancer therapy. One of the primary goals of a successful cancer treatment regimen is to deliver sufficient amounts of drug to tumors while minimizing damage to normal tissues. Most chemotherapeutic agents enter normal tissues in the body with indiscriminate cytotoxicity and do not preferentially accumulate at tumor sites. At times the dose reaching the tumor may be as little as 5% to 10% of the doses accumulating in normal organs [1, 2] . One reason for the inability for drugs to accumulate at target sites is that the interstitial fluid pressure (IFP) in solid tumors is higher than in normal tissues, that blocking transcapillary transport of chemotherapeutic drugs or antibodies [3] [4] [5] . In this way, the anticancer effect is decreased and toxic effect to normal cells is increased. Fear of severely harming the patients often limits the dose of anticancer drugs that can be given to a patient. These lower than optimal doses elicit incomplete tumor responses which leads to disease relapse and drug resistance. Therefore, most cancer drugs fail in clinical studies not because they are ineffective in killing cancer cells but because they cannot be administered in doses high enough to eradicate the tumor without severely harming the patient.
Several approaches have been developed to improve the ability of anticancer drug to more specifically target tumors and avoid normal organs. One of the most effective strategies is to encapsulate drugs in particles that deliver them preferentially to tumor sites. For example, liposome particles have been found able to deliver radionuclides, genes, and chemotherapeutic agents to tumor sites. [6] [7] [8] [9] [10] . Another promising strategy is to encapsulate anticancer drugs in liposomes conjugated with moieties, such as antibodies and peptides, that target particular types of target tumor cells or tumor vasculatures [11] [12] [13] . Use of internalizing ligands for targeting liposomes conjugated with such moieties makes it possible to deliver the chemotherapeutic drugs encapsulated within them to the cytosol through the receptor-mediated endocytosis [14] [15] [16] [17] . This article reviews the current research in developing liposomal drug delivery systems that use peptide ligands to target blood vessels in solid tumors. We discuss the identification of peptides that can target tumor blood vessels and the use of targeting and nontargeting 12 GGT GGA GGT TCG-3 Figure 1 : Selection of peptides that target tumor blood vessels using in vivo phage display. Peptide or antibody libraries are expressed as fusion proteins with a coat protein (pIII) of a bacteriophage, and the fused proteins are displayed on the surface of the virion. A phagedisplayed peptide library was injected through the tail vein of tumor-bearing mice. Eight minutes after injection, the mice were perfused through the heart. Phage recovered from the tumor was amplified and reinjected in mice for another four rounds. Tumor-targeting phages were further identified by in vivo tumor-homing assay, synthetic peptide binding and competition assay, and immunohistochemical staining. The identified peptides can be used as ligands to recognize cell surface markers or tumor antigens to develop targeted therapy. SCID mice bearing human cancer xenografts were successfully treated with ligand-conjugated antiangiogenic targeting liposomes.
liposomes to encapsulate and deliver chemotherapeutic drugs to tumor sites.
Virtually every conventional cytotoxic drug has been found to be antiangiogenic in in vitro and in vivo models [18] . One treatment approach known as metronomic therapy uses frequent administrations of low-dose antiangiogenic agents to destroy vessels in tumors while decreasing the toxicity to normal tissues [19] [20] [21] . For example, it has been found in mice that frequent administration of relatively low, noncytotoxic doses of liposome-encapsulated doxorubicin can shrink various solid tumor xenografts [13, 16] . The antiangiogenic agent bevacizumab (Avastin), a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has been used with some success to treat advanced colon cancer. One study compared the effect using three chemotherapeutic agents alone to treat advanced colon cancer with using the three agents combined with bevacizumab [22] . They found that the combined use of chemotherapeutic agents and bevacizumab extended overall survival by approximately 4.7 months compared to the use of chemotherapeutic agents alone [22] . Other angiogenesis inhibitors, including sunitinib and sorafenib, have also been found to improve clinical outcomes when used to treat various cancer types [23, 24] . The targeting of proliferating endothelial cells in the blood vessels of tumors has several advantages. First, endothelial cells in malignant tumors are genetically stable, nonmalignant, and rarely drug resistant, compared to the cancer cells [19, 21] . However, some recent studies show that tumor-associated endothelial cells can acquire cytogenetic abnormalities while they are in the tumor microenvironment [25, 26] . Second, the destruction of endothelial cells using this method amplifies the drugs antitumor effect. It has been reported that the elimination of one endothelial cell can inhibit the growth of as many as a hundred tumor cells [27, 28] . Third, antiangiogenic therapy decreases IFP within the tumor allowing better penetration by chemotherapeutic agents [29] [30] [31] [32] . For example, Jain found that bevacizumab could decrease IFP by normalizing tumor vasculature and decreasing vascular leakage [29, 33] . Fourth, antiangiogenic therapy is known to inhibit the growth of both primary and metastatic solid tumors. Finally, intravenously injected angiogenesis inhibitors can directly reach endothelial cells.
In addition, we can take advantage of the differences between endothelial cell plasma membrane proteins (i.e., vascular zip codes) to develop drug delivery systems capable of guiding therapeutic or imaging agents to a particular [13, 14, 50] . The reaction was completed and confirmed by quantifying the remaining amino groups using TNBS (Trinitrobenzenesulfonate) reagent [51] . Peptidyl-PEG-DSPE was transferred to preformed liposomes after coincubation at a temperature above the transition temperature of the lipid bilayer [52] . There were 500 peptide molecules per liposome [53] . The mean diameter of the targeting liposome is approximately 75 nm [2, 13] .
organ or tumor [34, 35] . Endothelial cells of blood vessels within solid tumors express certain molecular structures that are absent or minimally detectable in normal blood vessels [13, 36, 37] . These structures can be used as molecular targets for antitumor treatment.
The key to delivering drugs specifically to these targets is to identify and use ligands that specifically bind to and that can be internalized by endothelial cells in tumors. Combinatorial peptide libraries displayed on microorganisms have become a research tool for identifying cell surface-binding peptides that can become targets for antitumor treatment. Of the many molecular display techniques, phage display has been the most popular approach. Phage display is a selection technique in which a peptide or protein is fused with a coat protein of bacteriophage and displayed on the surface of the virion. Phage-displayed random peptide libraries have helped researchers map B-cell epitopes [38] [39] [40] , discover protein-protein contacts [41, 42] , and identify bioactive peptides bound to receptors [43, 44] or proteins [45, 46] . Peptide libraries can be used to find disease-specific antigens [47, 48] and cell- [2, 49] and organ-specific peptides [16, 35, 36] .
Recently, using affinity selection (biopanning) of phagedisplayed peptide libraries, researchers have discovered molecules that are expressed on tumor blood vessels exclusively [16, [34] [35] [36] . The strategy for identifying tumortargeting ligands and developing ligand-mediated targeted therapy is shown in Figure 1 . Researchers have used in vivo affinity selection of phage libraries to identify peptides that interact with the molecules found on endothelia in tumors [34, 36] . The NGR peptide motif targets angiogenic blood vessels [36] and the tumor-homing property of NGR motif relies on recognition of a CD13 isoform selectively expressed within tumor blood vessels [54] . Compared with the nontargeting liposomal doxorubicin (Caelyx), NGR peptide-conjugated Caelyx significant improvements in survival was seen in clinically relevant animal models of neuroblastoma, ovarian, and lung cancers [17] . Another peptide, SP5-52, has been found to recognize blood vessels created in tumors but not normal blood vessels in severe combined immunodeficiency (SCID) mice bearing solid tumors. Several selected phage clones display Pro-Ser-Pro, a motif crucial to peptide binding to tumor neovasculature [13] . Several tumor homing peptides have been found to bind to blood vessels in surgical specimens of human cancer and they have also been found to home to tumor tissues of different human tumor xenografts as confirmed by in vivo homing assays [16] . These studies found a greater correlation between increased tumoral accumulation of the targeting liposomes and antitumor efficacy than the accumulation of free drugs or drugs formulated in the nontargeting liposomes [2, 13, 16 ].
Most of the drug delivery systems approved for marketing are liposomal-or lipid-based formulations or therapeutic molecules linked to polyethylene glycol (PEG) [6, 10, 55, 56] . One such product is PEGylated liposomal doxorubicin, Figure 3 : Diagram of the molecular mechanism of peptide-conjugated liposomes on cancer therapy. These liposomes prolong circulation time in blood and improve pharmacokinetic and biodistribution of their encapsulated drugs. After intravenous administration, liposomes are large enough to be excluded from normal endothelium. In solid tumors, the angiogenic tumor vasculature becomes leakiness that particulate liposomes can extravasate and localize in the tissue interstitial space making it possible for more drug delivering liposomes to accumulate within the tumor by EPR effect. Coupling liposomes with peptides targeted to tumor cells or tumor vasculature further enhances the specificity and accumulation of liposomes in the tumor. On arrival in the tumor tissues, the liposomes are bound and internalized by tumor cells or tumor-associated endothelial cells through receptor-mediated endocytosis, fused with the low pH compartments of the endosomes, and subsequently broken down the liposomes and to release encapsulated drugs into the intracellular space of the cells.
which is known as Doxil in the US and Caelyx in Europe [57] . It is currently approved for the treatment of AIDSrelated Kaposi's sarcoma and recurrent ovarian cancer in North America, Europe, and other countries, and for metastatic breast cancer in Europe. Liposome-encapsulated doxorubicin has been found to significantly improve the therapeutic index of doxorubicin both in preclinical [58] [59] [60] and in clinical studies [61] [62] [63] [64] . An important advantage of PEGylated liposomal doxorubicin is that the heart muscle uptakes much less of it than free doxorubicin [58, 65] .
One study found no cardiotoxicity in 40 patients receiving cumulative doses of 500-1500 mg/m 2 of doxorubicin [62] . Free doxorubicin, on the other hand, is limited to a maximum recommended cumulative dose of 450-550 mg/m 2 . Colbern et al. found that the activity of PEGylated liposomal doxorubicin 1-2 mg/kg was almost equivalent to that of free doxorubicin 9 mg/kg in mouse Lewis lung carcinoma [59] .
One clinical study reported that most (>98%) of the drug circulating in the blood stream remains in encapsulated in liposomes [61] , suggesting that little of the liposomal drugs will be leaked to the circulation system during its journey to the tumor tissues. The hyperpermeability of tumor vasculature is a key factor for the success of liposome-delivered chemotherapy agents. The "leakiness" of the angiogenic tumor vasculature is estimated to have an average pore size of 100-600 nm [66] . These pores are significantly larger than the gap junction found in normal endothelium, which are typically <6 nm wide [67] . Liposomes with diameters of approximately 65-75 nm [13, 14, 50] are small enough to passively infiltrate tumor endothelium but large enough to be excluded from normal endothelium. Hence, they selectively extravasate into the tumor interstitial space. In the tissue of solid tumors, vasculature becomes so permeable that particulate liposomes can extravasate and localize in the tissue interstitial space [6, 10] . In addition, tumor tissues frequently lack effective lymphatic drainage [3] , which means that the liposomes can be retained longer. Together, these factors increase the Journal of Oncology 5 accumulation of the drug within the tumor, which has been referred to as the "enhanced permeability and retention (EPR) effect" by Maeda et al. [68, 69] . EPR-mediated passive tumor targeting by liposomes can increase the concentration of drugs in solid tumors by as much as ten times, compared to free drugs [70] .
Passively targeted liposomal drug delivery systems have some disadvantages. Normal organ uptake of liposomes leads to accumulation of the encapsulated drug in mononuclear phagocytic system cells in the liver, spleen, and bone marrow [63] , which may present hazards to these tissues. For example, with increased circulation time of these drugs may come increased toxicity inducing such problems as handfoot syndrome, mucositis, and hematological toxicities such as neutropenia, thrombocytopenia, and leucopenia [71] [72] [73] [74] . Therefore, ongoing research aims at enhancing the tumor site-specific action of the liposomes by attaching ligands to surface molecules of tumor cells and tumor vasculature, a process called active or ligand-mediated targeting liposomes [5, 6, 13, 75] .
The disadvantage of the passive PEGylated liposomes can be overcome by creating ligand-mediated targeting liposomes with more selective anticancer activity. The activity of anticancer drugs can be enhanced by coupling targeting moieties to the surface of liposomes to promote selective binding to tumor-associated antigens and facilitate the delivery of drug-containing liposomes to the intended cellular sites. This drug delivery system has a higher drug-to-carrier ratio than immunoconjugates and multivalent presentation of ligands, which increases their binding avidity [11] .
Antibodies that bind to tumor-specific antigens have so far yielded little success as a drug delivery system for solid tumors, which make up more than 90% of all cancers in humans. Although monoclonal antibodies have shown clinical potential as tumor targeting agents, they are limited by their large molecular size and poor tumor penetration [76] , by the immunogenicity associated with immunoliposomes, and by their toxicity to liver and bone marrow from nonspecific antibody uptake. These limitations can be overcome by using peptide ligands, which are smaller, less immunogenic molecules, and easier to produce and manipulate. Furthermore, peptide ligands have moderate affinity to antigens, which is beneficial because extremely high affinity of antibody-binding can impair tumor penetration [77] . Compared with antibody ligands, peptide ligands can improve tumor penetration and decrease MPS clearance of conjugated liposomes [50, 78] . The increasing use of peptides as targeting ligands has been aided by the use of phage display to identify novel ligands (Figure 1 ). Researchers have already produced liposomes conjugated with ligands that specifically target tumor cells or tumor vasculature [5, 16, 17] .
Peptide-conjugated liposomes have three main components: anticancer drug, a liposome carrier, and targeting peptide ( Figure 2 ). Remote loading methods such as the ammonium sulfate method [13, 79] and the pH gradient method [80] can encapsulate weak bases such as doxorubicin or vinorelbine into the liposomes with more than 95% efficiency. Schedule-dependent drugs such as vinca alkaloids, topotecan, and 5-fluorouracil are also potential candidates for liposomal delivery because they can extend the time when cancer cells are exposed to therapeutic levels of the drug.
The bioavailability and pharmacodynamics of liposomeencapsulated chemotherapeutic drugs must be considered in developing these delivery systems. To take advantage of the EPR effect, liposomes need to have long half-lives so that the drug stays within the carrier as long as possible in blood circulation until it accumulates in diseased tissues [81] . Once liposomes are localized to a solid tumor, the drug they contain must be released and become bioavailable at a rate remains therapeutically effective for a period of time. The rate of active drug's release into tumor cells, not the total drug concentration in the tumor tissues, is critical for measuring the actual bioavailability of the liposomal drug [16] . Some targeting liposomes have not been found to have greater therapeutic efficacy than passive liposomal drugs, possibly because the lack of internalizing ligands does not give the drug greater access inside tumor cells [82, 83] . Drug delivery can be further enhanced if the liposome-attached ligands bind selectively to internalizing antigens which would help increase the concentration of drugs inside tumor or tumor-associated endothelial cells resulting in higher drug concentration inside the cells [13, 15, 84, 85] . This binding to internalizing antigens by ligands can induce receptor-mediated endocytosis of liposomes into endosome compartments with low pH, where the liposomes break down and release the encapsulated drug into the intracellular space ( Figure 3 ). These steps lead to higher intracellular drug concentration and greater destruction or inhibition of tumor cells. Studies have confirmed greater cytotoxic effects produced by liposomes with peptides that target internalizing antigens through enhanced specificity and improved drug bioavailability [2, 16] .
The use of drug-encapsulated liposomes with ligands to target tumor blood vessels allows us to destroy both tumor blood vessels and tumor cells. In mice bearing human cancer xenograft, low dose of peptide-conjugated liposomal doxorubicin has been found to markedly inhibit vascularization and reduce total volume and weight of tumors [13, 16, 17] . The immunofluorescent analysis of the tumors in several studies has revealed an association between significant decreases in microvessel density and increases in the apoptosis of tumor cells and tumor-associated endothelial cells. The severe damage to tumor vasculature caused by peptide-conjugated liposomal doxorubicin throughout the tumors suggested an improvement in chemotherapeutic efficacy over nontargeting liposomes and conventional drugs [13, 16, 17] . This dual action may produce a greater, more durable anticancer effect than is found with the use of simple antiangiogenic therapy.
One peptide-conjugated liposome can deliver over ten thousand anticancer drug molecules directly into target tumor cells efficiently and effectively. The targeted and sustained release of the drug molecules can increase the maximum tolerated dose (MTD) of the cytotoxic drugs 6 Journal of Oncology and dramatically lower dose-limiting toxicities, and in turn prevent treatment delay or discontinuation. The affinity of targeting ligands may allow the liposomes to move past the high IFP barrier within tumors [4, 5, 13, 16] .
Advances in nanotechnology and molecular biology are moving us closer to developing an ideal "multifunctional smart nanodrug delivery system" using various types of ligands and drugs based on the kinds of diagnosis, imaging, or therapy needed. Such smart nanodrug delivery systems will allow accurate, specific, and noninvasive disease treatment, early diagnosis, and monitoring. In the future, combining ligands that specifically bind to cancer cells (including cancer stem cells) and tumor blood vessels with multifunctional liposomal drug delivery systems may help improve the effectiveness of cancer treatment and minimize the side effects traditionally associated with chemotherapy.
The development of highly selective anticancer drugs that can discriminate between tumor cells and normal cells is the most important goal of current oncology research. The potential use of ligand-conjugated liposome-encapsulated drugs to target tumor cells and vasculature is very promising. Peptides that specifically bind to tumor targets can be coupled to the PEG terminus of sterically stabilized liposomes and subsequently precisely deliver chemotherapeutic agents to tumor cells or blood vessels. Peptide-mediated liposomes that target vasculature are a new generation of chemotherapy delivery systems with superior pharmacokinetics, controlled biodistribution, efficacy, and safety profiles. Elevation of Intact and Proteolytic Fragments of Acute Phase Proteins Constitutes the Earliest Systemic Antiviral Response in HIV-1 Infection The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. Although human immunodeficiency virus type 1 (HIV-1) induces a chronic infection ultimately culminating in the development of an acquired immunodeficiency syndrome, it is now recognized that critical damage to the host immune system is mediated during the acute phase of infection, when an exponential burst of viral replication takes place, associated with massive depletion of the central memory CD4 + T cell pool [1, 2] . Prophylactic strategies to combat HIV-1 infection thus need to modulate events in the earliest stages of infection leading up to and impacting on this acute viral burst -which prompts an urgent need to understand the virus-host interactions occurring during this ''window of opportunity''.
Adaptive responses are known to play an important role in containment of the acute burst of viral replication in AHI [3] , but events in the earliest stages of infection are also likely to be heavily influenced by components of the innate immune system [3] . These include cellular determinants of the efficiency of viral entry into and replication within host cells such as the CCR5-delta32 allele, CCR2, CCL5 (RANTES), CX(3)CR1, CXCL12, or TRIM5, all of which can influence host resistance or susceptibility to HIV infection [3] . Interaction of virions with dendritic cells (DCs) early after virus transmission can have outcomes including virion destruction (following binding to langerin on resting Langerhans cells), efficient viral transmission to CD4+ T cells (following binding to DC-SIGN on sub-epithelial DCs), or the triggering of DCs (via interaction with TLRs) to produce cytokines/chemokines that may mediate antiviral activity, but may also drive immunopathological immune activation including cellular apoptosis [4, 5] . Genetic studies have helped to cast light on the in vivo importance of certain components of the innate immune system in acute/early HIV infection. These include associations between expression of certain KIRs and their cognate HLA alleles and resistance to, and/or control of HIV replication, implicating NK cells in control of HIV replication [6, 7, 8] . Furthermore, b-defensins, secreted from oral and mucosal epithelial cells appear to inhibit HIV-1 infection [9] . More recently, a peptide fragment derived from alpha-1 antitrypsin (AAT), a serine protease inhibitor and acute phase protein present in blood plasma, was shown to inhibit HIV host cell infection by blocking gp41 mediated cell entry [10] . Other natural factors exist that modulate HIV infection, such as a proteolytic product of the prostate phosphatase that is present in semen, which has the ability to dramatically enhance HIV infection [11] .
Much of our current picture of events in the eclipse and earliest viremic phases of acute HIV-1 infection is derived from in vitro studies and work carried out in non-human primate simian immunodeficiency virus (SIV) infection models, as the critical initial stages of infection are very difficult to study in humans. The availability of plasma sample series collected over a time-frame spanning the eclipse and viral expansion phase of HIV infection provide a unique opportunity to gain insight into the systemic activation of immune responses during this time. Previous reports have quantified an array of cytokines and markers of apoptosis in plasma panels and described a massive systemic ''cytokine storm'' occurring during the viral ramp-up phase, associated with an increase in plasma levels of apoptotic microparticles [12, 13, 14] . Importantly however, no systemic elevation in apoptosis markers or cytokine levels was detected during the eclipse phase when virus is being amplified at local infection sites prior to systemic dissemination. In this study, we used a proteomics-based approach combined with biochemical and cell biological assays to characterize factors that are elevated in plasma during the earliest stages of acute HIV-1 infection in humans. We describe increases in plasma levels of acute-phase reactants and proteolytically processed fragments that have anti-HIV activity during the eclipse phase prior to detection of HIV viremia or the first increases in systemic cytokine levels, which may represent the earliest systemic host antiviral response activated following infection.
Elevated plasma levels of acute phase markers in AHI prior to detection of viremia Samples collected at sequential time points spanning the eclipse and viral ramp-up phases from 19 US plasma donors who acquired HIV-1 infection were studied to gain insight into the kinetics of the earliest systemic anti-viral defenses activated in the acute phase of infection. Plasma was typically obtained from each donor at intervals of 2-5 d. Plasma panels were tested for HIV-1 by RT-PCR analysis of viral RNA titers, and time courses from different donors were aligned relative to the time point (T 0 ) when viremia first reached levels detectable by conventional assays (.100 RNA copies ml 21 ; Fig. 1A ). Most panels covered a timeframe from around d 220 to d +20 relative to T 0. It is currently thought that the eclipse phase in HIV-1 infection is in the range of 7-10 d [12, 15] hence most panels likely included samples collected from time points prior to the acquisition of infection onwards.
In order to determine whether there are detectable changes in plasma proteins or peptides accompanying the emergence of viremia, an initial mass spectrometry-based screen was performed on three plasma donor panels (Fig. 1B) . Analysis of the longitudinal MALDI-TOF data revealed mass peaks that were elevated at viremic time points ( Fig. 2A) . One mass peak with a molecular mass of 2178 Dalton [M+H] + was found to be considerably elevated in HIV-1-positive plasma ( Fig. 2A) . Sequencing by MALDI-TOF/TOF and LC-MS/MS identified this mass as peptide 86-105 derived from A-SAA ( Fig. 2B and Fig.  S1A) . A semi-quantitative analysis of mass peak intensities of the 2178Da [M+H] + peptide mass revealed that this peptide was elevated coincident with the increase in viremia, and in 2 of the 3 subjects, immediately prior to the detection of viremia (Fig. 2C) , suggesting that A-SAA protein levels are elevated at these times. A second mass peak with a molecular mass of 2213 Dalton [M+H] + was identified as peptide 960-979 of complement C3 (Fig. S1B) . This peak was also elevated prior to as well as during viremia (Fig. 2C ).
Plasma levels of A-SAA are increased prior to and during detection of viremia in AHI A-SAA was shown previously to be elevated in patients with AIDS [16] , and is commonly used as a general marker for inflammation [17, 18] . A recent study demonstrated that A-SAA has anti-viral activity in vitro [19] . We therefore examined a larger set of plasma donor panels (19) by ELISA to test whether elevation of A-SAA levels may be a general feature associated with acute HIV-1 infection, and how its induction is related to the increase in plasma viral RNA titers. As a control and to establish a baseline for use in statistical analysis of the data, we also measured A-SAA levels in plasma panels from five control plasma donors who did not become infected with HIV (Fig. S2A) . Baseline levels of A-SAA (calculated as described in the methods section) varied between individuals and were generally between 600-3800ng/ml. Analysis of A-SAA levels in the plasma panels from HIV-infected donors confirmed that A-SAA was elevated relative to baseline prior to and/or concurrent within the earliest detection of viremia. Importantly, significant A-SAA elevations (i.e. falling above a 90% prediction interval) were observed prior to T 0 (viral RNA .100 copies ml 21 ) in 15 out of 19 subjects (Fig. 3A) . In the subject group as a whole, A-SAA levels were thus elevated significantly prior to T 0 , the time of first detection of plasma viremia (p = 0.02, as determined using a Binomial test).
To monitor alterations of the acute form (A-SAA) as well as the constitutively expressed form (C-SAA) of serum amyloid A, we
Acquired immune deficiency syndrome (AIDS) remains a major health problem worldwide, affecting predominantly the adult population in the western world and in developing countries in particular. Despite a tremendous effort to develop a cure or a vaccine that confers protection against human immunodeficiency virus (HIV-1) infection, this has not been achieved in a satisfactory manner to date. Recent research efforts have suggested that the earliest immune responses activated after exposure to the virus have an influence on virus spread, containment and disease progression. In this study, a panel of donors who provided plasma samples collected over a time-frame spanning the period before and immediately after detection of HIV-1 infection permitted an insight into the activation of the earliest systemic immune responses. We describe increases in plasma levels of acute-phase reactants and proteolytically processed fragments that have anti-viral activity in vitro. These inductions occur prior to detection of HIV-1 virus in the blood and before the first increases in systemic cytokine levels, which may represent the earliest systemic host antiviral response activated following infection.
performed immunoblot assays with specific antibodies (Fig. 3B ). In 4 out of 10 plasma donor panels tested by immunoblotting (9011, 9013, 9016 and 9018), we confirmed the initial increase in A-SAA levels prior to viral ramp-up. In addition to this first wave of A-SAA induction that occurred prior to detection of viremia, immunoblotting also confirmed a second, more intense phase of elevation, observed in six of the ten donors tested (9011, 9012, 9016, 9018, 64012, 64122) that coincided with the increase in viral load. Other individuals either had very low or consistently high levels of A-SAA over the time-frame analyzed. By contrast to A-SAA, a major acute phase reactant that is inducible during the infection process, C-SAA, which was observed in an unmodified and glycosylated form, was detected with only minor inductions in all panels examined. To further explore whether elevation of A-SAA was specifically attributable to HIV-1 infection, we examined 6 plasma panels from donors who became positive either for hepatitis C virus (HCV) or hepatitis B virus (HBV) during the period of sample collection [14] . We detected increased levels of A-SAA over the time course of infection in 1/3 panels from subjects with acute HBV infection and 3/3 panels from subjects with acute HCV infection ( Fig. 3C and Fig. S2 B and C), indicating that A-SAA induction may represent a common host response to microbial infection.
A more comprehensive analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed a number of other plasma components including complement factors, apolipoproteins and alpha-1-antitrypsin (AAT) that are present in plasma during AHI ( Table S1 ). The factors found to be elevated in plasma during AHI included a C-terminal peptide derived from AAT, residues 377-396, referred to as VIRIP (Table S1 and Fig. 4A ), which was shown to inhibit HIV-1 entry into host cells by targeting the gp41 fusion peptide [10] . Seven plasma panels from HIV-infected individuals and five panels from uninfected controls were evaluated for the presence of VIRIP by tandem mass spectrometry in a semi-quantitative fashion. Ion counts detected for the precursor ion representing the expected molecular mass of VIRIP were correlated to viremia, and revealed an elevation of VIRIP coincident with and after the initial increase in viremia in two of the seven plasma donor panels from infected individuals, but none in the five controls (Fig. 4B) . A semi-quantitative titration of VIRIP peptide by mass spectrometry indicated that the amount detected corresponds to an estimated value of 0.1-0.3mM of VIRIP in plasma at peak concentrations (Fig. 4C ). Considering the sample loss during the isolation of VIRIP peptide from plasma, the effective VIRIP concentration will likely be in the range of low mg/ml, which is approximating the IC 50 value at which VIRIP interferes with HIV-1 entry [10] (see also below).
The appearance of VIRIP in plasma samples from HIV-1infected subjects raised the question of how proteolytic processing of AAT may be mediated under these conditions. Previous studies indicated that MMPs (collagenases and elastases) interact with and cleave AAT [20] . Inspection of the regions of AAT flanking the VIRIP sequence predicted cleavage sites for MMP-1, -6, -7, -8, -9, -12, and MMP-26 at the N-terminal, and MMP-7 at the Cterminal end of VIRIP (Fig. 5A) . In vitro digestion of purified AAT with recombinant MMP-7 revealed a degradation product at 5 kDa detectable by immunoblotting consistent with the Cterminal AAT fragment 377-418 containing VIRIP (Fig. 5B) . Subsequent analysis was carried out using LC-MS/MS, which identified peptides containing both cleavage sites required for the formation of VIRIP at Phe 376 -Leu 377 and Phe 396 -Leu 397 (Fig. 5C) . A third cleavage within the VIRIP sequence at Pro 381 -Met 382 was also observed. Importantly, formation of VIRIP itself was detected at the 2 h time point, confirming MMP-7 as a candidate protease involved in generation of the peptide in vivo. Antiviral activity of factors elevated in the plasma during AHI In order to test for potential interference with the infection process we evaluated the ability of acute phase proteins AAT, A-SAA and C-reactive protein (CRP) and the C-terminal AAT fragments 397-418 and 377-396 (VIRIP) to inhibit HIV-1 replication using in vitro peripheral blood mononuclear cell (PBMC) and dendritic cell (DC) infection models. PHA-activated PBMCs or monocyte-derived dendritic cells (MDDCs) were incubated with the test analytes both prior to and during infection with either an R5-or an X4-tropic virus and subsequent HIV-1 replication was monitored by the analysis of supernatant p24 levels or reverse transcriptase activity. VIRIP, derived from near the Cterminus of AAT, markedly inhibited the replication of both the R5 and the X4 virus, consistent with previous findings [10] (Fig. 6A) . In contrast, no effect was observed with either fulllength AAT or the C-terminal 22mer 398-418 (Fig. 6A ). In addition, CRP did not inhibit the replication of either the R5 or the X4 virus in PBMCs (Fig. 6C) . A-SAA did not exhibit any inhibitory activity in the PBMC infection system, but did inhibit the replication of the R5 virus in MDDCs as early as 12-24 h after infection (Fig. S3) and to a greater extent after 7 d (Fig. 6B) . Importantly, inhibition of MDDC infection was still greater than 50% at a 1mgml 21 A-SAA concentration, which is well in the range of the levels detected in infected individuals (Fig. 3A) . A-SAA was recently reported to inhibit MDDC infection by an X4/ R5 dual tropic virus via down-regulation of CCR5 expression [19] . We conclude that components of the acute phase response indeed have the capacity to interfere with HIV-1 infection, thereby potentially helping to control viral dissemination in the eclipse phase.
The availability of sequential samples from plasma donors who became infected with HIV-1 provides a unique opportunity to study changes in plasma in the eclipse and viral expansion phases of acute infection. Recent studies described the induction of a ''cytokine storm'' and massive cellular apoptosis during the phase of exponential viral replication [12, 13, 14] . Here a proteomicsdriven approach was used to demonstrate for the first time that acute phase proteins, some of which exhibit antiviral activity, are induced systemically even prior to the first detection of viremia and also before any detectable increase in plasma cytokine levels. The factors elevated included A-SAA, a protein primarily synthesized by cells in the liver, high-level production of which is known to be induced during the acute phase response to infection, trauma or stress by pro-inflammatory cytokines including TNF-alpha, IL-6 and IL-22 [21] . Notably, A-SAA was frequently elevated with biphasic kinetics in the plasma of subjects acquiring HIV-1 infection, an initial elevation occurring during the eclipse phase and a second elevation during the viral ramp-up phase. The latter was temporally coincident with elevations in circulating levels of multiple pro-inflammatory cytokines [14] and likely reflected a hepatic response to this systemic stimulus. The initial elevation in plasma A-SAA levels was found to occur significantly prior to detection of viral RNA in the plasma, well before any systemic elevations are detected in plasma cytokine/ chemokine levels [14] . Following sexual transmission of HIV-1, virus replicates in the local genital or rectal mucosa, then spreads to the draining lymph nodes and subsequently to the gutassociated lymphoid tissue (GALT) [22] . The mechanism by which acute phase protein production is triggered during this process is currently unknown, but it may involve transfer of inductive factors to the liver (including pro-inflammatory cytokines produced at local sites of viral replication), triggering production of acute phase reactants prior to widespread virus dissemination and systemic increases in cytokine levels. Alternatively, A-SAA can be produced at extra hepatic sites: A-SAA expression has been reported in macrophages, adrenal glands, kidney and intestine, albeit at lower levels as compared to hepatocytes [17] . The initial burst of A-SAA levels may also contribute to the subsequent ''cytokine storm'' observed later at the systemic level, since A-SAA was shown to induce an array of immunomodulatory cytokines including of the Th1-type in monocytes, macrophages and lymphocytes [23, 24] .
Activation of acute phase reactants may represent a very early line of anti-viral defense in HIV-1 infection, since A-SAA, AAT and a C-terminal peptide derived from AAT (referred to as VIRIP) were each shown to exert anti-viral activity in vitro [10, 19, 25, 26] (Fig. 6A,B) . However, not all acute phase proteins that are induced in response to inflammation have anti-viral properties as was observed with CRP ( Fig. 6C) , which was also elevated systemically in plasma of some donors prior to viremia (data not shown). We found selective inhibition of HIV-1 infection of MDDCs but not PBMCs by A-SAA. Its ability to inhibit HIV-1 replication was previously proposed to be mediated by downregulation of CCR5 expression [19] . The capacity to inhibit R5 virus replication in MDDCs but not PBMCs may thus be due to the fact that MDDCs express only low levels of CCR5, whereas CCR5 expression on CD4 + T cells is much higher. It is unlikely that the inhibitory effects of A-SAA are limited to specific pathogens only, as this acute phase reactant was described to be up-regulated in a number of pathological processes [17] including inflammation and diverse bacterial and viral infections [27, 28, 29] . Consistent with this, we observed marked elevation of A-SAA in plasma panels from donors acutely-infected with HCV. Notably, A-SAA has been shown to mediate antiviral activity against this viral pathogen too, an effect proposed to be mediated by mechanisms distinct from its inhibitory effect on HIV-1 infection [30, 31] . In acute HCV infection, A-SAA induction may be stimulated as a consequence of viral replication in the liver, and the associated cytokine response. In acute HBV infection little A-SAA elevation was observed. This may relate to the different Fig. S1 ) before and during viremia. Viral load and T 0 are as described previously [14] . Top panels: viral load; second and third panels: peak intensities of mass peaks 2178 Da and 2213 Da normalized to the corresponding peak intensities observed for the first time point of 64012; bottom panels: relative peak intensities of mass peaks 1555 and 2552 that were used as standards, illustrating use of comparable MS acquisition conditions for all time points. The arrows in the middle and bottom panels indicate viremic time points. doi:10.1371/journal.ppat.1000893.g002 [14] . The solid circles show A-SAA protein levels. Subject-specific background levels of A-SAA (dotted grey lines) and the 90% prediction interval (threshold for defining a significant elevation, black lines) were calculated as described in the methods section. 15 out of 19 subjects show a significantly elevated A-SAA level before the first detectable viral load. (B). A-SAA and C-SAA protein levels in sequential plasma samples from 10 donors acquiring HIV-1 infection as assessed using anti-A-SAA and anti-C-SAA immunoblotting. The two sets of blots are positioned replication kinetics of HBV as compared to HIV-1 and HCV, and/or the relatively muted cytokine response activated in acute HBV infection [14] .
Genetic studies suggest that polymorphisms observed in AAT, another component of the acute phase response, are linked to susceptibility to HIV-1 infection [32, 33] . AAT was initially reported to block the activity of HIV-1 protease [26, 34, 35] . Subsequently, an AAT-derived 26mer C-terminal peptide was shown to inhibit HIV-1 LTR gene expression in vitro [36, 37] . However, the strongest effect reported so far is exerted by VIRIP generated as a proteolytic product from AAT, which inhibits viral entry by binding to the gp41 fusion peptide and is active in the low micromolar range [10] . For both A-SAA and VIRIP, we were able to detect endogenous levels in plasma that are in the range capable of mediating viral inhibition, which raises the possibility that such factors play a role in combating viral replication, particularly in the earliest stages of infection.
No strong correlation was found between the initial timing of A-SAA elevation and either R 0 (the viral reproductive rate), the slope of viral ramp-up or the highest recorded viral load in the 19 subjects studied here. Nevertheless, the relationship between the magnitude and dynamics of early acute-phase protein production and the acute viral burst and subsequent efficiency of control of viremia should be addressed in a future study on a larger cohort from whom samples were collected over a longer time-frame extending into early infection. Interestingly, we also noted that levels of AAT proteolytic fragments in plasma from subjects chronically-infected with HIV-1 were elevated as compared to those in healthy controls (data not shown), suggesting that acute phase proteins may also play a role in chronic viral infection. It is possible that components of the acute phase response may not only contribute to the control of viral replication in infected individuals, but may also be involved in mediating resistance to infection. For instance, HIV-1-exposed uninfected individuals have been shown to have elevated levels of cleaved forms of A-SAA [19] , which suggests that their anti-viral activity contributes to resistance against infection.
Evidence from the literature and our own data suggest that MMPs are responsible for AAT proteolysis, in particular MMP-7, MMP-9 and MMP-26 [38, 39] . Altered levels of MMP-9 have been reported to correlate with HIV-1 infection, and breakdown of extracellular matrix has been suggested to aid dissemination of the virus [40, 41] . Our in vitro experiments confirmed the known MMP-7 cleavage sites on the C-terminal part of AAT and also demonstrated that VIRIP can be generated despite presence of an additional cleavage site within the sequence of the peptide. This cleavage site between Pro 381 -Met 382 , directly neighbouring the active site Ser 383 , has been shown previously to be sensitive to the oxidation state of the Met 382 residue [42] , thereby protecting VIRIP from further degradation. In addition, our results support the notion that the biological function of cleavage of AAT by MMP-7 may not solely be inactivation of its serine protease function, but also to generate new proteolytic peptides that have additional activities in themselves.
The abundant acute phase proteins that we were able to detect elevations in during AHI may be only selected examples of constitutively-produced or inducible analytes that play a role in combating infections. There is an increasing amount of evidence suggesting that ''endogenous factors'' exist that have inherent inhibitory activity towards infectious pathogens [43] . These include antimicrobial polypeptides [44] and antiproteases such as cystatins that have also been detected in cervical mucosa [45] . Insights gained into the mechanism of action of innate factors and acute phase reactants against HIV-1 and how they can be induced should be considered for novel vaccine strategies and therapeutics.
Plasmapheresis samples from the US plasma donor cohort used in this study were purchased from Zeptometrix Corporation and SeraCare Life Sciences. Donors were recruited via the SeptaCare Special Donor Program and enrolled for plasma donation after a medical examination and an interview with medical staff, in which it was explained that the donated plasma will be used by dedicated drug and vaccine researchers to perform research to help others. This study was approved by the Oxford Tropical Research Ethics committee (OXTREC, University of Oxford) and the NIH Office of Extramural Research (Nr. 201029 to B.M.K).
Panels of sequential samples obtained by plasmapheresis from US plasma donors who became infected with HIV-1, HBV or HCV and control subjects were purchased from Zeptometrix Corporation and SeraCare Life Sciences and were stored at 280uC before use. Details of the panels and methods used for analysis of HIV-1, HBV and HCV viral loads are as described [14] . Each plasma panel included samples collected prior to detection of plasma viremia through to seroconversion. The plasma panels from HIV-infected individuals were temporally aligned relative to a common time origin (T 0 ), defined as the time point when the viral load first reached detectable levels (.100 viral RNA copies ml 21 ), as described previously [14] . The median and range duration of observation prior to the first detectable HIV RNA level (T 0 ) for the 19 panels used in this study was 21 days with a range of 7 to 58 days.
Plasma donor samples were fractionated using weak anion exchange (WAX) magnetic beads (Bruker Daltonics, Bremen, Germany) according to the manufacturer's recommendation (Text S1).
For analysis by MALDI-TOF/TOF, a solution of a-cyano-4hydroxycinnamic acid (matrix, 3 mg) in ethanol:acetone (10 ml, 2:1) was prepared freshly. Samples processed as described above and matrix solution were mixed in a ratio of 1:4, and 1 mL aliquots were spotted in triplicates on an Anchor Chip MALDI plate (Bruker Daltonics, Bremen, Germany). Data was acquired on an UltraFlex MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) at 60% laser power until a total intensity of 2610 5 ion counts was reached. Spectra were analyzed using Bruker Daltonics FlexAnalysis software (version 2.4). For LC-MS/ MS tandem mass spectrometry, analysis was carried out by so that the time point when plasma viral RNA was first detected is aligned (vertical line). Post-viremic time points are indicated by the horizontal arrow. (C) A-SAA and C-SAA protein levels in sequential plasma samples from 3 donors acquiring HCV infection (top panel) and 3 donors acquiring HBV infection (bottom panel). As above, the two sets of blots are positioned so that the time point when plasma viral nucleic acids were first detected is aligned (vertical line); and post-viremic time points are indicated by the horizontal arrow. doi:10.1371/journal.ppat.1000893.g003 injection of 3 ml of sample prepared as described above on a nanoAcquity UPLC system coupled to a Waters Q-TOF Premier tandem mass spectrometer in MS E (high/low collision switching) mode as described previously [46] . Processing of raw data was performed using the ProteinLynx Global Server 2.2.5 software and the data were interrogated on an in-house MASCOT server (version 2.2). Alternatively, samples were analyzed on a Bruker HCTplus Ion Trap tandem mass spectrometer (Bruker Daltonics, Bremen, Germany) as described [47] . Semi-quantitative analysis of MS data was based on measuring peak heights observed for the peptide masses that were assigned to A-SAA 86 
Recombinant active MMP-7 (EMD Biosciences, Gibbstown, NJ) (7.2 mg, 4 ml) was added to a solution of AAT (Sigma Aldrich, St. Louis, MO) (100 ml, 2.9 mg ml 21 ) in 100 mM ammonium bicarbonate buffer and the mixture was incubated at 37uC. Aliquots (5 ml) were removed for immunoblotting at 0, 5, 20, 60 and 120 min, mixed with RSB (5 ml) and retained for analysis. Immunoblotting was carried out following gel electrophoresis on 4-12% Bis-Tris or 16% Tris-Tricine gels (Invitrogen, Carlsbad, CA) for increased resolution of low molecular weight species. LC-MS/MS analysis was carried out with the 5 min and 120 min time point samples following desalting and concentration by methanol chloroform precipitation [48] .
Analysis of plasma samples using an anti-SAA ELISA assay was performed using a commercial SAA ELISA kit (Abazyme, Needham, MA) and conducted according to the manufacturer's instructions. Plasma samples were diluted with assay buffer to obtain final concentrations within the linear range of the assay of 1-80 ng ml 21 . Inclusion of recombinant protein standards demonstrated that A-SAA was sensitively detected by the assay while C-SAA was non-reactive up to final concentrations of 750 ng ml 21 .
HIV-1 infection assays used viruses derived from the infectious molecular clones pNL4.3-BaL.ecto (R5-tropic) and pNL4.3 (X4tropic), supplied by John Kappes and Christina Jambor (University of Alabama at Birmingham, USA). Cryopreserved healthy donor PBMCs were stimulated with 2.5ug ml 21 PHA (Sigma Aldrich, St. Louis, MO) for 2 d in R10 medium (Invitrogen, Carlsbad, CA) and 1 d with 50U ml2 1 IL-2 (Roche, Nutley, NJ) in R10 medium adjusted to contain 20% serum. MDDCs were generated by culturing CD14 + cells isolated from buffy coats (National Blood Service, London, UK) using a Human Monocyte Isolation Kit II medium was never more than 0.2%. Control cultures were treated with a similar volume of 2% DMSO OptiMEM or plain OptiMEM (vehicle only) to match the addition of inhibitor. PBMCs were preincubated with inhibitors for 2 h prior to infection, then were infected with R5 or X4 virus at a MOI of 0.01 for 16 h at 37uC in the presence of inhibitor. Excess virus and inhibitor were washed out and cells were cultured for 7 d in R10 medium plus 50U ml 21 IL-2. Supernatants were harvested and p24 levels were determined by ELISA (Advanced BioSciences Laboratories, Inc., Kensington, MD). MDDCs were preincubated with inhibitors for 1 h prior to infection at a MOI of 0.1 for 2 h at 37uC in the presence of inhibitor; and after washout of inhibitor and excess virus, cells were cultured for 4 before reading out supernatant p24 levels as described above. Statistical analysis of the data from the viral inhibition assays was performed using a one-way ANOVA.
Statistical analysis of ELISA data on plasma A-SAA levels Two statistical tests were performed on transformed data from the sample time-courses from the five control non-HIV-infected plasma donors and the time points prior to D-15 in the timecourses from HIV-infected plasma donors: a Wilcoxon test for a two-group comparison (control vs. HIV-infected, p = 0.28) and an ANOVA test in a linear mixed model for the group difference (p = 0.4). As no significant differences were found between the controls and pre-D-15 data from the HIV-infected subjects, both were used to determine baseline A-SAA levels in the HIV-infected subjects. Linear mixed-effects models were fit to these data to estimate the subject-specific baseline level of A-SAA in each HIVinfected individual. A-SAA values above the 90% upper prediction bound was considered significantly elevated (see Fig. 3A and S2A). A two-sided Binomial test was conducted to examine whether the first elevation of A-SAA occurred significantly before T 0 . Additional tests of associations between the timing of first A-SAA elevation and three different parameters of viral replication (viral reproductive rate R 0 , slope of viral ramp-up and the highest recorded viral load) were also performed using linear models. Figure S2 A-SAA levels as measured by ELISA in plasma panels from 5 control plasma donors, 3 plasma donors acquiring HBV infection and 3 donors acquiring HCV infection. (A). A-SAA levels in plasma panels from five control non-HIV-infected plasma donors. The solid circles show A-SAA protein levels. Subjectspecific background levels of A-SAA (dotted grey lines) and the 90% prediction interval (threshold for defining a significant elevation, black lines) were calculated as described in the methods section. (B). A-SAA levels in plasma panels from three HBVinfected donors. In each graph, time is plotted relative to T200 (first time point where DNA levels are above 200 copies ml 21 ). A-SAA protein levels are indicated by black squares and solid black lines and HBV DNA levels by black triangles and dotted black lines. (C). A-SAA levels in plasma panels from three HCV-infected donors. In each graph, time is plotted relative to T600 (first time point where RNA levels are above 600 copies ml 21 ). A-SAA protein levels are indicated by black squares and solid black lines and HCV RNA levels by black triangles and dotted black lines.  Synthesis and Pharmacological Evaluation of Schiff Bases of 4-(2-Aminophenyl)-Morpholines In the present study, a novel series of 4-(2-aminophenyl)morpholines were synthesized and characterized by IR, (1)H-NMR, (13)C NMR and mass spectral analysis. The synthesized compounds were screened for analgesic (100 and 200 mg/kg), antiinflammatory (200 and 400 mg/kg), antibacterial (Bacillus subtilis, Bacillus cereus, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli) and antifungal (Candida albicans and Aspergillus niger) activities. The minimum inhibitory concentrations of the compounds were also ascertained by agar streak dilution method. N-benzylidine-2-morpholoino benzenamine (1) and N-(3-nitro benzylidine)-2-morpholino benzenamine (3) exhibited significant analgesic, antiinflammatory and antimicrobial activities.  Proper Distance Metrics for Phylogenetic Analysis Using Complete Genomes without Sequence Alignment A shortcoming of most correlation distance methods based on the composition vectors without alignment developed for phylogenetic analysis using complete genomes is that the “distances” are not proper distance metrics in the strict mathematical sense. In this paper we propose two new correlation-related distance metrics to replace the old one in our dynamical language approach. Four genome datasets are employed to evaluate the effects of this replacement from a biological point of view. We find that the two proper distance metrics yield trees with the same or similar topologies as/to those using the old “distance” and agree with the tree of life based on 16S rRNA in a majority of the basic branches. Hence the two proper correlation-related distance metrics proposed here improve our dynamical language approach for phylogenetic analysis. Whole genome sequences are generally accepted as excellent tools for studying evolutionary relationships [1] . Traditional distance methods with multiple alignment or various sequence evolutionary models for phylogenetic analysis are not directly applicable to the analysis of complete genomes.
A number of methods without sequence alignment for deriving species phylogeny based on overall similarities of complete genomes have been developed. These include fractal analysis [2] [3] [4] , dynamical language model [5] , information-based analysis [6] [7] [8] , log-correlation distance and Fourier transformation with Kullback-Leibler divergence distance [9] , Markov model [10] [11] [12] [13] [14] [15] , principal component analysis [16] and singular value decomposition (SVD) [17] [18] [19] . The analyses based on the Markov model and dynamical language model without sequence alignment using 103 prokaryotes and 6 eukaryotes have yielded trees separating the three domains of life, Archaea, Eubacteria and Eukarya, with the relationships among the taxa consistent with those based on traditional analyses [5, 11] . These two methods were also used to analyze the complete chloroplast genomes [5, 12] . The SVD method was used to analyze mitochondrial genomes of 64 selected vertebrates [19] . A correlation-distance method without removing the random background (similar to [7] ) was used to analyze rRNA gene sequences as DNA barcodes [20] .
In the above approaches of SVD, Markov model and dynamical language model, there is a step to calculate the correlation-related distance between two genomes after removing the randomness or noise from the composition vectors. A drawback is that these correlation-related distances are not proper distance metrics in the strict mathematical sense (Professor Bailin Hao, personal communication, 2009; see also [21] ). There are some ways to overcome this problem. One way is to change the concept of distance to that of dissimilarity proposed by Xu and Hao [15] in the Markov model approach. Another way is to replace a pseudo-distance by a proper distance metric, which requires that the results are not worsened from the biological point of view. In the first way, there is no widely accepted mathematical definition for the concept of dissimilarity or similarity. Chen et al. [22] defined a similarity metric, but unfortunately the sample correlation between two vectors in a vector space does not yield a proper similarity under their definition.
In this paper, we follow the second way and propose two proper correlation-related distance metrics to replace the pseudo-distance in the dynamical language approach used by Yu et al. [5] . We then evaluate the effects of this replacement on the analysis of a wide range of complete genomes from the biological point of view.
Three kinds of data from the complete genomes can be analysed using the dynamical language approach proposed by Yu et al. [5] . They are the whole DNA sequences (including protein-coding and non-coding regions), all protein-coding DNA sequences and the amino acid sequences of all proteincoding genes. We outline this approach here.
There are a total of N = 4 K (for DNA sequences) or 20 K (for protein sequences) possible types of K-strings, that is, the strings with fixed length K. We denote the length of a DNA or protein sequence as L. Then a window of length K is used to slide through the sequences by shifting one position at a time to determine the frequencies of each of the N kinds of K-strings in this sequence. We define = ) ... (
as the observed frequency of a K -string K α α α ... 2 as components and arrange according to a fixed alphabetical order all the K -strings to form a composition vector ) ,..., , (
Then we view the N components in the vectors X and Y as samples of two random variables respectively. The sample correlation ) , ( Y X C between any two genomes X and Y is defined in the usual way in probability theory as:
between the two genomes is then defined by 2 / )) ,
A distance matrix for all the genomes under study is then generated for the construction of phylogenetic trees. This distance method to construct phylogenetic tree is referred to as the dynamical language model method [5] . Finally, we construct all trees using the neighbour-joining (NJ) method [24] in the software SplitsTree4 V4.10 [25] or in the Molecular Evolutionary Genetics Analysis software (MEGA 4) [26] based on the distance matrices.
To determine a best length of strings (K) in our model, we plot the mean value of X over all K-strings from a genome (whole DNA sequences or protein sequences) as a function of K (see Figure 1 for examples from our data). The mean value of X starts to approach zero at K = 6 or 7 if we use protein sequences from genome and at K = 11 or 12 if we use whole DNA sequence. The mean value of X being close to zero means that the value of p (from the sequence) is almost equal to value of q (from the model). Hence these K values are suitable for phylogeny reconstruction using our approach. This result is also confirmed later in this paper from a biological point of view. 
Each genome can be considered as a point in N = 4 K (for DNA sequences) or 20 K (for protein sequences) dimensional space represented by its composition vector ) ,..., , (
between two vectors X and Y is said to be a distance metric if it satisfies the following properties:
for any X and Y.
If we denote:
| | X and | | Y are the lengths of the vectors X and Y respectively, then u X and u Y are unit vectors (i.e., have length 1). Let θ be the angle between two vectors of X and Y. It is well known that
The distance defined by 2 / )) ,
is not a proper distance metric because it does not satisfy condition (i) (except for unit vectors) and the triangle inequality (iii) [21] . In the following we describe two proper distance metrics related to the sample correlation.
The chord distance is defined on the set of unit vectors in a vector space as the length of the chord constructed from two unit vectors. Mathematically, let X u = (X u1 , X u2 ,…,X uN ) and Y u = (Y u1 , Y u2 ,…, Y uN ) be two unit vectors; then the chord distance D chord (X u ,Y u ) is defined as:
It is seen that 0 ) , ( = 
. This distance is also called Cavalli-Sforza chord distance [27] or described on pp. 163-166 of [28] . This distance performed well in simulations of tree-building algorithms by Takezaki and Nei [29] . It has also been used to analyze microarray gene expression data [30] .
This distance metric is also defined on the set of unit vectors in a vector space. For any two unit vectors u X and u Y , we define:
where ρ is any positive real number which is not smaller than 3. We call
, which means that the two vectors u X and u Y are identical as shown above. It is also obvious that ) , (
Using the facts
for any two unit vectors and 
. Usually we may take 3 = ρ .
We propose to replace the pseudo-distance in the dynamical language approach [5] by the chord distance or piecewise distance. We need to examine the effects of this replacement from the biological point of view. In order to do this, we evaluate the new distance metrics on four datasets, namely Dataset 1 of 109 complete genomes of prokaryotes and eukaryotes used in [11] , Dataset 2 of 34 prokaryote and chloroplast genomes used in [12] , Dataset 3 of mitochondrial genomes of 64 selected vertebrates used in [19] , and Dataset 4 of 62 complete genomes of alpha-proteobacteria used in [31] . (Note: Chan et al. [21] recently tested the chord distance with different denoising formulas on Dataset 2).
We used the dynamical language approach for Datasets 1 and 2 in [5] and Dataset 3 in [32] . Some biological comparisons of this approach with the Markov model approach on Datasets 1 and 2 were given in [5] . Recently we found that wrong data of the Archaea Crenarchaeota bacterium Pyrobaculum aerophilum (Pyrae) from Dataset 1 was used in [5] . Using the right genome data, Pyrobaculum aerophilum (Pyrae) groups with the other Archaea Crenarchaeota bacteria correctly (when we use the amino acid sequences of all protein-coding genes from genomes and K = 6). After this correction, the resulting tree is better than the one in [11] from the biological point of view, with all firmicutes group together and the other branches are similar. For Dataset 2, we obtained two trees with the same topology to those using the dynamical language approach in [5] and the Markov model approach in [12] (also using the amino acid sequences of all protein-coding genes from genomes and K = 6). For Dataset 3, we reported in [32] a good tree in agreement with the current understanding of the phylogeny of vertebrates revealed by the traditional approaches using the dynamical language approach (based on the whole DNA sequences of genomes and K = 11). This tree is better than the one in [19] and the one obtained by the Markov model approach. Hence we just need to compare the best trees obtained by the dynamical language approach using the two proper distance metrics with the best trees obtained from the pseudo-distance in [5] based on the first three datasets. In 2009, Guyon et al. [31] compared four alignment free string distances for complete genome phylogeny using Dataset 4. We will compare our method in this paper with the results in [31] based on Dataset 4.
The whole DNA sequences (including protein-coding and non-coding regions), all protein-coding DNA sequences and the amino acid sequences of all protein-coding genes from genome data are used for phylogenetic analysis. For Dataset 1, we have seen that amino acid sequences of all protein-coding genes from genomes give better results than those given by the whole DNA sequences and all proteincoding DNA sequences. We evaluated the dynamical language approach with chord distance and piecewise distance on the amino acid sequences of all protein-coding genes from genomes for K = 3, 4, 5 and 6. We find the trees using the new distance metrics have the same topology as the trees using the old "distance" for the same value of K, and the trees for K = 6 are the best. Here we present the tree for K = 6 using dynamical language approach with chord distance in Figure 2 . The phylogeny shown in Figure 2 supports the broad division into three domains and agrees with the tree of life based on 16S rRNA in a majority of basic branches. For further biological discussions, one can refer to [5] with the correction for the position of Pyrobaculum aerophilum (Pyrae). For Dataset 2, we have seen that the amino acid sequences of all protein-coding genes from genomes give better results than those given by the whole DNA sequences and all protein-coding DNA sequences. We evaluated the dynamical language approach with chord distance and piecewise distance on the amino acid sequences of all protein-coding genes from genomes for K = 3, 4, 5 and 6. We find the tree using the piecewise distance has the same topology as the tree using the old "distance" for the same value of K, the tree using the chord distance has similar topology (a little bit worse because Pinus thunbergii is separated from its correct position) to the tree using the old "distance" for the same value of K. And the trees of K = 6 are the best. Hence we present the tree for K = 6 using the dynamical language approach with piecewise distance ( 3 = ρ ) in Figure 3 . We also note that the topology of the tree in Figure 3 is the same as that of the tree obtained by the Markov model in [12] ). The phylogeny of Figure 3 shows that the chloroplast genomes are separated to two major clades corresponding to chlorophytes s.l. and rhodophytes s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution. For further biological discussions, one can refer to [12] . Genomes" on http://www.genome.jp/kegg/genes.html)), we find that the whole DNA sequences give better results than those given by the amino acid sequences of all protein-coding genes from genomes and all protein-coding DNA sequences. We evaluated the dynamical language approach with the proposed distance metrics on the sequences of whole genomes for K = 6 to 13. We find the tree using the piecewise distance has the same topology as the tree using the old "distance" for the same value of K, the tree using the chord distance has similar topology (a little bit better because Dasypus novemcinctus.(Dnov) is close to but does not remain in a branch of primates) to the tree using the old "distance" for the same value of K. And the trees for K = 11 are the best. Hence we present the tree for K = 11 using the dynamical language approach with chord distance in Figure 4 . The tree ( Figure 4 ) generated is similar in topology to the tree obtained using the SVD method in the case 4 = K [19] , and is also similar to a recently generated tree of 69 species [33] , placing a vast majority of species into well-accepted groupings. As shown in Figure 4 , our distance-based analysis shows that the mitochondrial genomes are separated into three major clusters. One group corresponds to mammals; one group corresponds to the fish; and the third one represents Archosauria (including birds and reptiles). The interrelationships among the mitochondrial genomes are roughly in agreement with the current understanding of the phylogeny of vertebrates revealed by the traditional approaches. For further biological discussion, one can refer to [32] .
For Dataset 4, Guyon et al. [31] first reconstructed a reference tree using Maximum Likelihood (ML) method based on the large (LSU) and the small (SSU) ribosomal subunits sequences (i.e., the traditional alignment method). Then they compared the results using four alignment free string distances for complete genome phylogeny. The four distances are Maximum Significant Matches (MSM) distance, k-word (KW) distance (i.e., the Markov model in [11] ), Average Common Substring (ACS) distance and Compression (ZL) distance. Guyon et al. [31] found the MSM distance out performs the other three distances and the KW cannot give good phylogenetic topology for the 62 alpha-proteobacteria (see Figure 3 in [31] ). We tested our dynamical language approach with pseudo-distance in [5] and the two proper distances in this paper on Dataset 4. We found that amino acid sequences of all protein-coding genes from genomes give better results than those given by the whole DNA sequences and all protein-coding DNA sequences. We evaluated the dynamical language approach with pseudo-distance in [5] and the two proper distances in this paper on the amino acid sequences of all protein-coding genes from genomes for K = 3, 4, 5 and 6. We found the trees using the new distance metrics have the same topology as the trees using the old "distance" for the same value of K, and the topology of trees for K = 5 and 6 are the same and the best. Here we present the tree for K = 6 using dynamical language approach with chord distance in Figure 5 . As shown in Figure 5 , all Rhizobiales (Bartonellaceae, Brucellaceae, Rhizobiaceae and Phyllobacteriaceae) (A), Rhizobiales (Bradyrhizobiaceae) (B), Rickettsiales (Rickettsiaceae and Anaplasmataceae) (C), Rhodospirillales (D), Sphingomonadales (E); Rhodobacterales (Rhodobacteraceae) (F) group into correct branches respectively. Even inside each lineage (groups A to F), our phylogentic topology is more similar to that of ML reference tree (the right side tree in Figure 1 of [31] ) than that obtained by the MSM distance (the best result in [31] ). After comparing our Figure 5 with the tree obtained using KW distance (i.e., the Markov model in [11] ) (the tree in Figure 3 of [31] ), our dynamical language model performs much better than the KW distance.
There is no significant effect by the normalization of the distances and different values of 3 ≥ ρ .
Using the proposed distance metrics, we compared the trees before and after normalization and found that the topology of the trees is the same. Then we set , 4 = ρ 6, 8, 10 and found that we could get the trees with the same topology as the tree for 3 = ρ . As a result, there seems to be no noticeable effect by normalization of the distances and different values of 3 ≥ ρ . Figure 4 . The NJ tree of mitochondrial genomes based on the whole DNA sequences using the dynamical language approach with chord distance in the case K = 11. In this tree the birds and reptiles group together as Archosauria. Figure 5 . Phylogeny of 62 alpha-proteobacteria using the dynamical language approach with chord distance in the cases K = 5 and 6 based on all protein sequences. The topology of trees obtained by the dynamical language approach with pseudo-distance in [5] and piecewise distance in the cases K = 5 and 6 based on all protein sequences are the same as that in this figure.
We proposed two new mathematically proper distance metrics based on the lengths of the chords constructed from unit vectors and on proportions of the sample correlation function of unit vectors to replace the pseudo-distance in the dynamical language approach [5] . The results showed improvements with this replacement from a biological perspective. These results confirm their usefulness in phylogenetic analysis. A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1 BACKGROUND: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses. Influenza A viruses are enveloped, negative-strand RNA viruses with a segmented genome. They infect a large variety of animal species, although birds are considered to constitute the reservoir from which all influenza A viruses in other species originate [1, 2] . On the basis of the antigenic properties of their two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are classified into 16 HA (H1-16) and 9 NA (N1-9) subtypes. The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. HPAI H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. It is widely accepted that continued human exposure to influenza viruses circulating in wild and domestic avian species poses a permanent pandemic threat [3, 4] . Effective vaccination against HPAI H5N1 would protect com-mercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird as well as birdto-human transmission. Therefore, the development of efficacious influenza vaccines is of high veterinary and public health importance.
Conventional vaccine preparations against HPAI H5N1 are produced by propagating virus in embryonated chicken eggs or mammalian cells. The vaccine viruses used are either reassortants or low pathogenic wild-type avian viruses. Either way, the viruses need to be selected for their ability to multiply in eggs or cultured cells, which may preclude the best genetic match to the circulating HPAI H5N1 strains. In addition, these vaccines have several other limitations as reviewed elsewhere [5, 6] . Hence, other vaccine strategies against HPAI H5N1 have been explored including the use of, among others, live attenuated influenza vaccines [7, 8] , live vaccines based on heterologous viral vectors such as pox virus [9] , adenovirus [10] , baculovirus [11] and Newcastle disease virus [12] , and DNA vaccination [13] . While these different strategies often showed promising results, their applicability ultimately will depend on various important issues including safety, efficacy, production and costs [5] .
Protective immunity against influenza virus infection and disease is primarily conferred through HA via the induction of anti-HA antibodies. As protection from influenza virus infection correlates with anti-HA titers, nearly all vaccine approaches aim to induce high levels thereof. Therefore, the HA protein is the antigen of choice for the development of recombinant subunit vaccines to protect against HPAI H5N1. An influenza vaccine based on recombinant purified HA could offer the following advantages: I) The HA antigen can be produced using safe, quality-controlled and scalable conditions. II) There will be no need for virus cultivation, thus avoiding the necessity a) to obtain viruses that replicate efficiently in eggs or cell culture, b) to use biocontainment facilities and c) to inactivate the virus using procedures that may affect antigenicity and raise safety concerns. III) The recombinant HA protein can be highly purified thereby limiting adverse reactions caused e.g. by the presence of egg contaminants. IV) Immunization with recombinant HA will allow the serological differentiation of naturally infected from vaccinated animals/flocks (the so-called DIVA principle; [14] ). IV) Recombinant HA vaccines are manufactured with a relatively short lead time, allowing an accelerated response to emerging influenza strains.
Recombinant HA can be produced using different expression systems. When expressed in E. coli the resulting HA protein gave rise to the induction of hemagglutination inhibition (HI) titers upon immunization of mice [15] . However, as proper folding and trimerization of the HA protein requires multiple posttranslational modifications including glycosylation and disulfide bond formation, expression of the HA protein in higher eukaryotic systems is likely to result in superior antigenicity. Thus, mammalian cellderived HA trimers were found to induce much higher levels of neutralizing antibodies than similarly produced monomeric HA protein [16] . The baculovirus expression system has been used to produce strain specific HA antigens in insect cells, which were shown to protect against a HPAI H5N1 challenge [17] . Such fullsize HA proteins may, however, be limited in their efficacy because the membrane proteins may not retain their native membrane-bound structure upon purification [18] . Hence, the production of recombinant, soluble, stable HA trimers that are secreted from the cells seems like an attractive alternative approach. Such HA trimers, expressed either in insect or mammalian cells, were indeed shown to elicit neutralizing antibodies [16] and to partially protect mice against HPAI H5N1 challenge infection [19] .
In view of their promising potential we have evaluated recombinant soluble HA trimers in chickens and mice for their ability to induce protective immunity against infection with HPAI H5N1. To this end, recombinant soluble H5 proteins provided with a GCN4 trimerization motif and a STREP-tag II, the latter for purification purposes, were expressed in mammalian cells. The recombinant soluble H5 trimers (sH5 3 ) were purified from the culture supernatants using a simple one-step purification protocol and characterized with respect to their oligomeric state and bioactivity. Subsequently, vaccination with the sH5 3 preparation was shown to provide complete protection against challenge with HPAI H5N1 both in mice and in chickens, in the latter already after a single immunization.
A cDNA clone corresponding to residues 18 to 523 (H3 numbering) of the HA from A/Viet Nam/1203/2004 (H5N1) (Genbank accession no. ABW90137.1) was synthesized using human-preferred codons by GenScript USA Inc. In this clone the predicted HA ectodomain protein lacks a multibasic cleavage site. The cDNA was cloned into the pCD5 expression vector for efficient expression in mammalian cells [20] . The pCD5 vector had been modified such that the HA-encoding cDNA was cloned in frame with DNA sequences coding for a signal sequence, an artificial GCN4 isoleucine zipper trimerization motif (KRMKQIEDKIEEIESKQKKIENEIARIKK) [21] and the Strep-tag II (WSHPQFEK; IBA, Germany). The resulting vector encodes the soluble trimeric H5 protein designated as sH5 3 .
pCD5 expression vectors containing the HA ectodomainencoding sequences were transfected into HEK293S GnTI(2)cells [22] using polyethyleneimine I (PEI) in a 1:5 ratio (mg DNA: mg PEI). At 6 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), Streptomycin (100 mg/ml), glutaMAX (Gibco), and 1,5% dimethyl sulfoxide. Tissue culture supernatants were harvested 5-6 days post transfection. HA protein expression and secretion was confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using a mouse anti-Strep-tag antibody (IBA, Germany). HA proteins were purified using Strep-tactin Sepharose beads according to the manufacturer's instructions (IBA, Germany). The concentration of purified protein was determined by using a Nanodrop 1000 spectrophotometer (Isogen Life Sciences) according to the manufacturer's instructions.
The oligomerization status of the sH5 3 proteins was determined by analyzing the elution profile using a Superdex200GL 10-300 column (GE Healthcare). Sialic acid-binding activity of sH5 3 was assessed using a fetuin solid phase assay. 1 mg/ml fetuin per well was used to coat 96-well Nunc MaxiSorp plates. When indicated in the figure legend, fetuin was treated with Vibrio Cholera derived neuraminidase (VCNA; Roche) followed by three washing steps. sH5 3 was pre-complexed with horseradish peroxidase (HRP)linked anti-Strep-tag antibody (2:1 molar ratio) for 30 min at 0uC prior to incubation of limiting dilutions on the fetuin-coated plates (60 min, room temperature [RT]). HA-binding was subsequently detected using tetramethylbenzidine substrate (BioFX) in an ELISA reader (EL-808 [BioTEK]), reading the OD at 450 nm.
Animal studies were conducted at the Central Veterinary Institute (CVI), Lelystad, under BSL3 conditions and after approval by the Animal Ethic Committee. In the first chicken experiment, 6 weeks old SPF white Leghorn chickens (Lohmann, Cuxhaven, Germany) were used (10 chickens per group). One group of animals was immunized twice (on day 0 and 21) by intramuscular (i.m.) injection (0.5 ml) of 10 mg For the next experiment, 70 one-day-old layer hens (white Leghorn) were purchased from a local breeder. The chickens had been vaccinated against Newcastle disease virus and infectious bronchitis virus at the age of one day according to the farm's routine and were raised in CVI's animal facility. At the age of 6 weeks, the birds were transported to the BSL-3 facility and allocated to 7 experimental groups of 10 birds each. The animals of six groups were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection of 10, 2 or 0.4 mg sH5 3 antigen adjuvanted with Stimune. When immunized twice, the same doses were given on day 0 and 21. As a challenge control, one group was mock-vaccinated twice (on day 0 and 21) with PBS in Stimune. Four weeks after vaccination (day 49), blood samples were taken and the chickens were challenged as above and observed daily for clinical signs during 14 days. Trachea and cloaca swabs were taken from each chicken on day 2, 4 and 7 p.i.
Female, specified pathogen-free (SPF) 9-week-old BALB/c mice (Charles River Laboratories; 10 animals per group) were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection (0.2 ml) of 2 mg sH5 3 adjuvanted with Stimune. As a challenge control, one group of mice was mock vaccinated. Three weeks after the vaccination, mice were anaesthetized with ketamin/xylazin by intraperitoneal injection and inoculated intranasally with 50 ml of H5N1A/Viet nam/1194/04 containing ,10 median lethal dose (LD 50 ; 3.7 10 log TCID 50 ; provided by Dr. Alan Hay from the WHO Influenza Centre at the National Institute for Medical Research, London). The mice were weighed daily and examined for signs of illness during the next 14 days. Clinical signs were recorded using a scoring system (0 = no clinical signs; 1 = rough coat; 2 = rough coat, less reactive, passive during handling; 3 = rough coat, rolled up, laboured breathing, passive during handling; 4 = rough coat, rolled up, laboured breathing, unresponsive). Animals reaching a score of 4 were euthanized. Surviving animals were bled and sacrificed on day 14 p.i.
Trachea and cloaca swabs were stored in cold tryptose phosphate broth supplemented with antibiotics. The medium was clarified by low-speed centrifugation and the supernatant was harvested, aliquoted and stored at 270uC. Upon thawing, trachea and cloaca swabs sampled from the same bird on the same day were pooled and the viral RNA was extracted from 200 ml using a MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche). Subsequently, cDNA was synthesized using reverse primer 59-CACTGGGCACGGTGAGC-39 and part of the M1 gene was amplified by running 45 cycles of Light Cycler PCR using primer 59-CTTCTAACCGAGGTCGAAACGTA-39 as the reverse primer in the presence of the TaqMan fluorescent probe 59-6FAM-TCAGGCCCCCTCAAAGCCGA-X-ph. Negative and positive control samples were tested in parallel. The lower limit of detection was determined to be approximately 500 TCID 50 . Some samples gave inconclusive results, meaning that they gave only a very weak signal (fluorescence,0.07) after .31 cycles.
Heat-inactivated immune sera from chicken blood samples were tested for hemagglutination inhibition (HI) activity with 1% chicken red blood cells and 4 hemagglutinating units (HAU) of H5N1 (A/Viet Nam/1194/04 NIBRG-14). In addition, the chicken sera were tested for HI activity using 8 HAU (67 ng) of recombinant soluble trimeric HA protein. To this end the recombinant proteins were precomplexed with the anti-Strep-tag antibody as described above, mixed with limiting dilutions of the chicken sera and incubated with 0.5% chicken red blood cells. Red button formation was scored as evidence of hemagglutination. Antibody titers were expressed as the reciprocal of the highest serum dilution showing HI. Immune sera prepared from mouse blood samples were treated with VCNA, heat-inactivated at 56uC for 30 min and tested by HI assay using 8 HAU of sH5 3 as described above.
Total antibody titers against sH5 3 were determined by using a sH5 3 -specific ELISA. To this end, 96-well Nunc MaxiSorp plates coated with 0.5 mg sH5 3 per well were incubated with limiting dilutions of chicken or mouse sera. After extensive washing, the plates were incubated with goat-anti-chicken or goat-anti-mouse antibodies conjugated with HRP. Peroxidase activity was visualized using tetramethylbenzidine substrate (BioFX) and an ELISA reader (EL-808 [BioTEK]), reading the OD at 450 nm.
The OD values of the 250-fold diluted samples, which were in the logarithmic phase of the curve, were plotted.
In order to express soluble trimeric subtype 5 HA (sH5 3 ) in mammalian cells, the H5 ectodomain-coding sequence was first cloned into an appropriate expression vector. In the pCD5 vector used, the H5-sequence was preceded by a signal peptide-encoding sequence, to allow efficient secretion of the recombinant protein, and followed by sequences coding for the GCN4 isoleucine-zipper trimerizaton motif [21] and the Strep-tag II, the latter for purification purposes (Fig. 1A) . Expression of the H5 ectodomain was achieved by transient transfection of the expression plasmid into HEK cells. Expression and secretion of the H5 protein was verified by subjecting cell culture supernatant to gel electrophoresis followed by western blotting using an antibody directed against Strep-tag II (Fig. 1B) . The results show that the recombinant H5 protein could be readily detected in the cell culture supernatant after transfection of the cells with the expression plasmid, but not after mock transfection. The secreted H5 protein was purified in a single step protocol by using Streptactin sepharose beads. Protein yields varied between 0.4-1 mg of recombinant protein per 100 ml cell culture medium. After purification of the H5 protein from the cell culture supernatant, the oligomeric state of the H5 protein was analyzed by gel filtration column chromatography (Fig. 1C) . The bulk of the H5 protein eluted with the velocity of an oligomer, while only a minor fraction was found as aggregates in the void volume. The trimeric nature of the H5 oligomer was confirmed using blue-native gel electrophoresis followed by western blotting (Fig. 1D) . When the H5 preparation was heat-denatured for increasing time periods prior to electrophoresis, the initially trimeric HA species dissociated into dimers and monomers. The biological activity of the purified sH5 3 was studied using a solid phase-binding assay with the sialylated blood glycoprotein fetuin. Binding of sH5 3 was measured by means of the HRP conjugated to the anti-Strep-tag II antibody as detailed in the Material and Methods section. The H5 preparation exhibited a concentration dependent binding to the fetuin. This binding was sialic acid-dependent, as no binding was observed when the fetuin had been treated with neuraminidase (VCNA; Fig. 1E ). In conclusion, biologically active sH5 3 was efficiently produced using a mammalian expression system and readily purified.
To examine the immunogenicity of sH5 3 and its potential as a vaccine a first experiment in chickens was performed, in which 10 chickens were vaccinated twice (on day 0 and 21) i.m. with 10 mg of Stimune-adjuvanted sH5 3 and challenged 3 weeks later by i.n./i.t. inoculation of a lethal dose of A/Viet Nam/1194/04 virus. Another 10 birds were mock-vaccinated to serve as challenge controls. As shown in Fig. 2 , the boost vaccination with 10 mg sH5 3 conferred complete protection. None of the vaccinated chickens died or showed symptoms indicative of influenza-related disease, whereas all mockvaccinated chickens succumbed within 2 days. Serological analysis showed that none of the mock-vaccinated animals contained antibodies against sH5 3 as determined by a sH5 3 -specific ELISA (Fig. 2B) . In contrast, all immunized animals demonstrated appreciable levels of HA antibodies already after a single immunization and these levels increased further after the boost. These total antibody levels against sH5 3 correlated nicely with the HI titers against H5N1 (Fig. 2C) . All mock-vaccinated chickens had a HI titer below the detection limit. HI antibody titers observed after the sH5 3 boost reached with a maximum of 1024 and a minimum of 64, which was apparently still sufficient to protect the animal against the lethal challenge. Interestingly, 50% of the birds developed HI antibody titers equal to 64 or higher already three weeks after the first vaccination. In addition, HI titers were also determined by using sH5 3 rather than H5N1 virus as the hemagglutinating agent, which gave essentially similar results (compare Fig. 2D and 2C) , demonstrating the reliability of the assay. In summary, the results show that chickens vaccinated twice with sH5 3 are protected against a lethal challenge with H5N1. The HI titers observed suggested that one vaccination might already be sufficient to confer protection against HPAI H5N1.
These results prompted us to determine the minimal sH5 3 dose required to confer protection and to examine whether a single dose could already be sufficient. In the second vaccination-challenge experiment, chickens were thus vaccinated with 10, 2 or 0.4 mg of sH5 3 either once or twice and challenged four weeks later by infection with a lethal dose of A/Viet Nam/1194/04 as described above. The results are shown in Fig. 3 . Again, all mock-vaccinated birds succumbed to the infection within 2 days. Vaccinating twice with a dose of 0.4 mg of sH5 3 was sufficient to protect 90% of the chickens against mortality, while all chickens survived when a dose of 2 or 10 mg was similarly administered (Fig. 3A) . Interestingly, also single vaccination with sH5 3 could induce sufficient immunity to protect chickens against lethal infection (Fig. 3B) ; when a dose of 2 mg was given only one chicken died (90% protection), whereas a dose of 10 mg was protective to all birds. Even after a single dose of 0.4 mg, 60% of the chickens were protected against death.
Serological analysis showed that protection against the lethal H5N1 challenge correlated well with the observed antibody levels against sH5 3 as determined by ELISA (Fig. 3C-D) and by HI assay (Fig. 3E-F) . Both assays revealed a dose-dependent antibody response, which was substantially enhanced after the booster immunization. Relatively high HA antibody levels were observed after two immunizations, even with the lower dose, except in two animals, one of which did not survive the challenge (Fig. 3C) . Also after a single immunization, significant antibody levels were measured, except again in the low dose group. Here, 5 animals had hardly measurable ELISA titers. Consistently, 4 of these animals succumbed to the challenge. Also the one animal that died after a single immunization with 2 mg of sH5 3 did not have detectable sH5 3 antibodies. Essentially the same results as with the ELISA were obtained with the HI assay using sH5 3 as the hemagglutinating agent ( Fig. 3E-F) . Thus, the animals that succumbed to the lethal challenge after a single immunization also exhibited the lowest HI titers.
We analyzed whether vaccination with sH5 3 decreased or prevented chickens from shedding challenge virus. For practical reasons, virus shedding was analyzed by a quantitative RT PCR rather than by measuring infectious virus titers. To this end, trachea and cloaca swabs were taken from the chickens of the vaccine dose titration experiment of Fig. 3 at 2, 4 and 7 days after the challenge inoculation. The tracheal and the cloacal swab sampled from each chicken at each particular day were pooled and the presence of viral RNA in the pooled swabs was analyzed using a quantitative RT PCR assay detecting the M1 gene. The results are shown in Table 1 . Of the chickens that received a booster vaccination, only 2 birds, both of which had received the lowest amount of antigen, tested positive. Notably, these were the two animals that developed the lowest sH5 3 -specific antibody titers (Fig. 3C) . One of these animals did not survive the challenge. Virus shedding could not be detected in any of the other birds, although 3 swabs gave inconclusive results. Of the chickens vaccinated only once, all animals that died tested positive. None of the birds vaccinated with 10 mg sH5 3 tested positive. Of the chickens vaccinated with a lower dose and surviving, two tested positive, but only at day 2 p.i. In conclusion, the vaccinated birds that could control the lethal HPAI H5N1 challenge infection exhibited minimum or no virus shedding.
Finally we examined whether sH5 3 would also confer protection in mice. Therefore, 2 groups of 10 mice were vaccinated either once (on day 21) or twice (on day 0 and 21) with 2 mg of sH5 3 adjuvanted with Stimune and challenged three weeks later by intranasal inoculation with ,10 LD 50 of H5N1 A/Viet Nam/ 1194/04. The percentage of mice surviving the infection, median clinical scores and body weights per group observed after the challenge inoculation are shown in Fig. 4 . All mock-vaccinated mice succumbed to infection or had to be euthanized by day 9 p.i.. These mice showed severe clinical signs, including respiratory distress and significant weight loss, which continued until the animals died. A booster vaccination with sH5 3 provided 100% protection against the lethal dose of A/Viet Nam/1194/04; none of the mice showed significant signs of disease and their body weights remained constant. A single vaccination with sH5 3 did not protect mice against disease and concurrent weight loss; yet 40% of the mice survived. These mice started to recover from day 9 p.i. onwards as demonstrated by their regain of body weight. (Fig. 4C ). Fig. 4D shows the pre-challenge anti-sH5 3 antibody levels in individual serum samples,as determined by ELISA. Such antibodies could be detected in most vaccinated animals. However, after a single immunization, these levels remained very low compared to those in animals that received a booster vaccination. These results were essentially confirmed by determining the HI titers against sH5 3 in the same serum samples. With the exception of one animal, the serum of which demonstrated high levels of auto-agglutination, all mice displayed high HI titers when vaccinated twice and low HI titers when vaccinated once. Thus, mice vaccinated twice with sH5 3 were protected against a lethal challenge with HPAI H5N1, while a single vaccination provided partial protection. The differences in protection correlated with the observed serum HI titers.
Despite all the-understandable-attention drawn in the past year by media and scientific community to the new pandemic influenza A virus H1N1, a large influenza threat continues to be posed by HPAI H5N1. In 2009, HPAI H5N1 was found in wild birds in Germany, China, Mongolia and the Russion Federation, several outbreaks of the virus in poultry were reported in Viet Nam, Hong Kong, Nepal, India, Bangladesh, Egypt, Lao DPR, and Cambodia, while H5N1 remained endemic in large areas of Indonesia. That same year also dozens of confirmed human cases were reported (WHO timeline of major events; http://www.who.int/csr/disease/avian_influenza/ ai_timeline/en/index.html). Even though the virus has so far remained restricted in its ability to infect humans and initiate efficient human-to-human transmission, its persistence and spread among wild birds and poultry holds a continual risk of the emergence of a pandemic strain. This threat can be reduced by vaccination of poultry against H5N1 as this would limit the propagation of the virus and minimize the risk of bird-to-human transmission. In addition, in case a human pandemic H5N1strain would emerge, there would be an immediate need for effective and reliable vaccines matching the pandemic strain. In the present study we evaluated the vaccine potential of a recombinant soluble H5 protein (sH5 3 ) to protect chickens and mice against a lethal infection with HPAI H5N1. The recombinant HA vaccine, which has a short development cycle, proved to be protective after a single immunization in its natural host, while a booster vaccination was needed to confer complete protection in a mammalian model. In addition, the sH5 3 induced immunity prevented viral shedding from chickens. These promising results warrant further research into the development of recombinant soluble HA as a fast, safe and effective alternative vaccine not only against H5N1, but against other influenza A viruses as well.
The recombinant HA expression cassette was constructed such that the HA protein was produced and secreted by cells in high yields as a bioactive trimer. The importance of the oligomeric state of the HA protein for the induction of neutralizing antibodies has recently been demonstrated [16] . High-molecular-weight oligomers and trimers, but not monomers, were found to efficiently induce neutralizing antibodies in mice. This difference was attributed to the preferential induction of antibodies against epitopes present in the monomeric, but not in the trimeric form [16] . While the soluble recombinant HA trimers were purified using metal affinity chromatography followed by ion-exchange chromatography in previous studies [16, 19] , we used a protocol based on the Strep-tag system [23] . Proteins with a Strep-tag exhibit high affinity towards Strep-tactin, an engineered form of streptavidin. By exploiting this highly specific interaction, Streptagged proteins can be isolated in one step. Furthermore, because the Strep-tag elutes under gentle, physiological conditions it is especially suited for the generation of native proteins [24] , a characteristic that in the case of HA may contribute to the ability of the recombinant protein to induce neutralizing antibodies.
The efficacy of the sH5 3 vaccine was first studied in chickens. Adjuvanted with Stimune, a water-in-oil adjuvant also known as Specol, the sH5 3 protein formulation induced an immunity that was completely protective against a lethal H5N1 challenge after administration of two doses ($2 mg sH5 3 /dose). Importantly, our vaccine preparation also protected chickens after a single immunization. While 100% of the chickens were protected after vaccination with 10 mg sH5 3 , 90% were protected when using 2 mg. Similar HA doses were previously needed to protect chickens with a vaccine preparation consisting of full length HA proteins, which had been purified from insect cell cultures infected with recombinant baculovirus expressing the H5 gene, emulsified in a water-in-oil adjuvant [17] . These results are consistent with the observation that mammalian cell-produced HA trimers elicited similar levels of neutralizing antibodies as trimeric HA produced in insect cells [16] . The efficacy of our sH5 3 vaccine preparation was furthermore demonstrated by the absence of viral RNA in the protected birds. This would imply that a vaccinated flock can pose a barrier against further spread of circulating virus.
Most conventional influenza vaccines require two vaccination rounds to produce antibody titers sufficiently high to confer full protection to chickens. In this regard, vaccination with sHA 3 provides potential advantages over other vaccine approaches. However, the production costs per dose of sHA 3 compared to egg-cultured inactivated whole-virus vaccines might be higher, even though recombinant protein expression in mammalian cell culture systems has been shown to be highly scalable and productive, with expression levels up to the order of grams of protein per liter [25, 26] . sHA 3 vaccination could however be economically feasible in epidemic situations when millions of chickens have to be vaccinated individually, provided that a single preventive vaccination would suffice. Moreover, vaccinated flocks housed in endangered regions rapidly achieve a state of protective immunity. This may be a particularly valuable feature in the event of a pandemic, when the virus transmission cycle needs to be interrupted as soon as possible and the risk of exposure of farmers, veterinarians and people in monitoring teams to potentially zoonotic HPAI should be limited to the upmost extent. The efficacy of the sH5 3 vaccine was also studied in mice. The vaccine preparation was completely protective against a lethal H5N1 challenge after 2 doses. Immunization with a single dose resulted in 40% survival. These differences in protection levels correlated with the observed anti-sH5 3 titers in the animals' sera. As the dose (2 mg) received by the mice is at least comparable, relative to their body weights, to the dose that conferred complete protection in chickens after a single immunization (10 mg), these results appear to suggest that the sH5 3 is more effective in conferring a protective immune response in chickens than in mice. The reason for this apparent discrepancy is unclear and warrants further investigations. In the only other study so far that used soluble HA trimers as a vaccine preparation in mice, much less protection against challenge with H5N1 was observed after two immunizations [19] . Although the H5 trimer produced in this latter study differed from the one that we used, with respect to the trimerization motif (t4 foldon vs GCN4 trimerization motif) and the purification tag (His tag vs Strep tag II), the difference is more likely to be explained by the different adjuvant used (Alum vs Stimune). Alum is known to induce low antibody titers when used with subunit vaccines [27] , while Stimune has been reported to generate long-lasting, functional antibody responses [28, 29, 30] . Stimune is however not licensed for human use.
In conclusion, we have shown that the sH5 3 protein produced in mammalian cells elicited protective immune responses in mice and chickens when adjuvanted with Stimune. Chickens protected against the lethal H5N1 challenge also did not shed the virus at day 7 post infection. As these recombinant HA vaccines can be manufactured with high yields and a relatively short lead time, they offer an attractive alternative vaccination strategy, which will allow a rapid response to circulating and potentially pandemic influenza viruses. Hospital Triage System for Adult Patients Using an Influenza-Like Illness Scoring System during the 2009 Pandemic—Mexico BACKGROUND: Pandemic influenza A (H1N1) virus emerged during 2009. To help clinicians triage adults with acute respiratory illness, a scoring system for influenza-like illness (ILI) was implemented at Hospital Civil de Guadalajara, Mexico. METHODS: A medical history, laboratory and radiology results were collected on emergency room (ER) patients with acute respiratory illness to calculate an ILI-score. Patients were evaluated for admission by their ILI-score and clinicians' assessment of risk for developing complications. Nasal and throat swabs were collected from intermediate and high-risk patients for influenza testing by RT-PCR. The disposition and ILI-score of those oseltamivir-treated versus untreated, clinical characteristics of 2009 pandemic influenza A (H1N1) patients versus test-negative patients were compared by Pearson's Χ(2), Fisher's Exact, and Wilcoxon rank-sum tests. RESULTS: Of 1840 ER patients, 230 were initially hospitalized (mean ILI-score = 15), and the rest were discharged, including 286 ambulatory patients given oseltamivir (median ILI-score = 11), and 1324 untreated (median ILI-score = 5). Fourteen (1%) untreated patients returned, and 3 were hospitalized on oseltamivir (median ILI-score = 19). Of 371 patients tested by RT-PCR, 104 (28%) had pandemic influenza and 42 (11%) had seasonal influenza A detected. Twenty (91%) of 22 imaged hospitalized pandemic influenza patients had bilateral infiltrates compared to 23 (38%) of 61 imaged hospital test-negative patients (p<0.001). One patient with confirmed pandemic influenza presented 6 days after symptom onset, required mechanical ventilation, and died. CONCLUSIONS: The triaging system that used an ILI-score complimented clinicians' judgment of who needed oseltamivir and inpatient care and helped hospital staff manage a surge in demand for services. The severity of seasonal influenza epidemics is unpredictable and influenced by the predominant circulating virus strains and level of immunity in the population [1] . During peak community influenza activity, hospitals and emergency rooms may be overwhelmed by patients presenting with influenza-like illness (ILI) and more severe disease [2, 3] . Illness attack rates may be higher among most age groups during pandemics than observed for seasonal influenza due to limited immunity among exposed populations [4] . The re-emergence of highly pathogenic avian influenza A (H5N1) virus among poultry with sporadic transmission to exposed persons and the resulting high mortality has stimulated global influenza pandemic preparedness [5] .
Key features of pandemic influenza planning are developing strategies to meet expected increased demand for patient care, and how to allocate limited resources, including ventilators and critical care [6] [7] [8] [9] . Guidance has been developed for clinical triage of patients with ILI, including special populations (e.g. children, pregnant women), during a pandemic [10] [11] [12] . A key clinical decision is determining which ill persons can be managed as outpatients and which require hospitalization. Scoring systems, with varying predictive power, have been developed to determine who will require hospitalization, need ICU care, require a ventilator, or is at high risk of death (e.g. CURB-65) [13] [14] [15] . The emergence of 2009 pandemic influenza A (H1N1) virus has presented a great challenge for clinicians throughout the world [16] . Overwhelming demand for medical care by patients with ILI and limited availability of oseltamivir necessitated that clinicians rapidly triage patients for outpatient care or hospital admission. These challenges are compounded by the need for early oseltamivir treatment of influenza patients for optimal efficacy [17] . At the Hospital Civil de Guadalajara, Fray Antonio Alcalde (HCGFAA), Mexico, clinicians from the Adult Infectious Diseases Unit used a modified ILI scoring system to systematically triage adult patients with respiratory complaints and determine who would be prioritized for hospitalization and antivirals. We describe this triaging system during the peak 2009 pandemic in Guadalajara (April-August, 2009).
HCGFAA is a 1000-bed tertiary care facility with a 30-bed infectious diseases unit. In response to high demand for emergency medical services among adult patients with acute respiratory complaints, infectious disease specialists implemented an ILI scoring system on April 25, 2009. This scoring system was adapted from a system developed by Hak et al in the United States for hospitalization decision-making among elderly patients with pneumonia or influenza during influenza epidemics [18] . In the emergency room (ER), a questionnaire was used to record patients' demographics, signs and symptoms, history of health care utilization, chronic medical conditions, laboratory, and radiology findings to calculate patients' ILI-scores ( Figure 1 ). Clinicians used an ILI-score $16 (high-risk), their judgment of patients' severity of illness and proximity to the hospital to decided whether to admit the patient and treat them with oseltamivir. Patients with intermediate ILI-scores (7) (8) (9) (10) (11) (12) (13) (14) (15) were discharged from the ER, treated with oseltamivir and followed daily by phone for 10 days. Those with low ILI-scores (#6) were discharged without antiviral treatment, and instructed to return if their symptoms worsened.
Nasal and throat swab specimens were collected from all highrisk and intermediate-risk patients. Swabs were combined in phosphate-buffered saline viral transport media and split into aliquots for influenza testing. One aliquot was tested by rapid diagnostic test (QuickVue Influenza Test, Quidel, San Diego, CA) and immunofluorescence at the hospital. A second aliquot was sent frozen at 270uC to the National Public Health (InDRE) laboratory in Mexico City. InDRE tested the samples with realtime RT-PCR (rRT-PCR) using a multiplex assay and 4 sets of primers (i.e. influenza A, universal swine, 2009 pandemic influenza A (H1N1), and a control for human genetic material) [19] . Each hospitalized patient had a chest x-ray and a chest CT scan performed at admission.
Clinicians prescribed standard doses of oseltamivir 75 mg BID for five days [17] . Hospitalized patients assessed to have severe illness received 150 mg of oseltamivir PO BID 65 days, amantadine 300 mg PO BID 610 days, broad spectrum antibiotics (e.g. linezolid), and paracetamol. Patients were discharged when afebrile and without dyspnea.
Patients' demographics, clinical presentation, treatments, and outcome data were entered into an SPSS database. The ILI-score, treatment, disposition, and virology results of triaged patients were compared by Pearson's X 2 , Fisher's Exact, Student t-tests, and Wilcoxon rank-sum tests.
The study was approved by the research ethics committee of the Hospital Civil de Guadalajara, Fray Antonio Alcalde and the final draft for publication was also approved by the research ethics committee of the Hospital Civil de Guadalajara, Fray Antonio Alcalde. Investigators kept the datasets in password protected systems and presented data without identifiers to protect the anonymity of case-patients.
During April 25-August 9, hospital staff triaged 1840 persons with acute respiratory infections ( Figure 2 (Table 1) . Two-hundred and thirty (12.5%) were admitted to hospital (median ILI-score = 15 [IQR = 11-19]) ( Figure 3 ). Of 286 ambulatory patients who were prescribed oseltamivir (median ILI-score = 11, IQR = 7-15), none required subsequent medical evaluation. Of 1324 ambulatory patients who were not treated with oseltamivir (median ILI-score = 5, IQR = 1-8), 14 (0.8%) returned a median of 8 days after their initial visit. Three (21%) of the 14 returning patients (i.e. one pregnant and two with a history of tobacco abuse), were hospitalized and treated with oseltamivir (with a median ILI-score = 19). Two of these 3 returning patients who were subsequently hospitalized tested positive for 2009 pandemic influenza (H1N1). One (7%) of the 14 returning patients was prescribed oseltamivir and discharged from the ED, and 10 (71%) were discharged home without oseltamivir. One patient visited triage three times, but was not treated with oseltamivir. Three deaths occurred in hospitalized patients (aged 18, 37, and 54 years). Decedents presented to the ER a mean of 4 days after symptom onset with a mean ILI score of 16. One decedent was confirmed with pandemic H1N1, one had seasonal influenza A, and one was not tested. All other hospitalized patients improved and were discharged home.
Hospitalized patients presented within a median of 2 days after symptom onset with dyspnea and abnormal findings on chest imaging. Sixty-seven (30%) of the 230 hospitalized patients smoked tobacco (for a mean duration of 8 years), 45 (20%) had a 
Of the 1840 persons triaged, 379 (21%) were tested for influenza (i.e. 371 (20%) by rRT-PCR, 112 (6%) by rapid diagnostic test, and 89 (5%) by immunofluorescence). Of the 371 patients tested by rRT-PCR, 104 (28%) had pandemic (H1N1) and 42 (11%) had seasonal influenza A detected. There was a 0.51 correlation between rRT-PCR and rapid diagnostic test results among the 85 patients who were tested by both methods (p,0.001). In contrast, there was a 0.15 correlation between rRT-PCR and immunofluorescence results among the 57 who were tested by both methods. In comparison to patients with seasonal influenza, patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) were younger (Figure 4 ). The median age of patients who tested positive for 2009 pandemic influenza A (H1N1) was 23 years versus 31 years for patients who tested positive for seasonal influenza A (p = 0.0007)( Table 1) . Patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) were not more likely to be pregnant, report substance abuse, have other medical conditions (e.g. obesity), or require hospitalization within 2 days of developing symptoms than other patients (Table 2) . At ER presentation, 69 (66%) of the 104 patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) reported a dry cough (mean duration = 3 days) versus 145 (55%) of 264 test negative patients (p = 0.03). Thirty-two (31%) the 104 patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) had a productive cough compared to 53 (20%) of 262 test negative patients (p = 0.03). Patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) presented with a median temperature of 38.5uC which, on average, started 2 days before admission [IQR1-3]. There were no differences in WBC at ER presentation between patients whose rRT-PCR tested positive for 2009 pandemic influenza A (H1N1) and patients who tested negative for 2009 pandemic influenza A (H1N1).
Eighteen (60%) of 30 hospitalized patients infected with 2009 pandemic influenza A (H1N1) with chest X-rays had abnormal findings while all 22 with chest CT scans had abnormal findings (Table 4) . Similarly, 5 (25%) of 20 hospitalized patients infected with seasonal influenza A who had chest X-rays had abnormal findings while all 10 who had chest CT had abnormal findings. Hospitalized patients infected with 2009 pandemic influenza A (H1N1) were more likely to have abnormal chest X-rays than patients infected with seasonal influenza A (p = 0.02) ( Table 4) . 
Testing positive for 2009 pandemic influenza A (H1N1) was not associated with prolonged stay. On average, 2009 pandemic influenza A (H1N1) patients were hospitalized for a median of 2 days [IQR 1-3days]. 2009 Pandemic influenza A (H1N1) infected patients with dyspnea on admission had a mean hospital stay of 2.1 days while those without dyspnea had a mean hospital stay of 1.3 days. The one decedent infected with pandemic influenza A (H1N1) presented 6 days after symptom onset with dyspnea and a 10 year history smoking history. There were no reported adverse events among patients associated with the use of oseltamivir.
During 6 weeks when there was co-circulation of pandemic and seasonal influenza A viruses in the community, hospital staff triaged more than eighteen-hundred patients with respiratory complaints and identified 12% for inpatient care. The triage system was based on assumptions about who is at risk for developing complications from seasonal influenza (e.g. patients aged over 65 years). Our analyses, however, suggested that patients infected with 2009 pandemic influenza A (H1N1) tended to be younger than seasonal influenza A patients. Nevertheless, our data suggest that clinicians used the ILI-score to help them determine, with minimal misclassification, which patients needed hospitalization versus who could be managed as outpatients [18] . The ILI-score helped guide clinicians to decide who needed hospital care and antiviral treatment when timely laboratory confirmation of influenza was not available. Only 1% of patients triaged needed re-evaluation. Such a system could be readily used to efficiently triage patients during outbreaks and epidemics by adapting the system's scores to match the anticipated characteristics of patients who are at highest risk of developing complications. While the triaging system led clinicians to hospitalize traditional groups at risk for complications from seasonal influenza (i.e. those with chronic medical illnesses), patients infected with 2009 pandemic influenza A (H1N1) were often young and had few pre-existing conditions [20] . These data are comparable with Mexican Directorate General of Epidemiology data that suggest 56% of pandemic (H1N1) confirmed deaths occurred among those aged 30-59 years, many of whom were previously healthy [21] . The age shift in 2009 pandemic influenza A (H1N1) cases may be caused by cross-reactive immunity from prior influenza infections in 33% of those aged more than 60 years [22, 23] . Health officials should adjust pandemic triaging tools to account for the younger age distribution of cases [24] . Pregnancy should also be included as a risk factor in triaging tools. Although there were too few pregnant women in our case series for subgroup analyses, other data suggest pregnant women are at high risk of developing severe complications from 2009 pandemic influenza A (H1N1) [25] .
In this case series, hospitalized patients who tested positive for 2009 pandemic influenza A (H1N1) received oseltamivir within 2 days of symptom onset and appeared to recover quickly with a median hospital stay of two days. Similarly, no ambulatory patients treated with oseltamivir required further medical care. In contrast, 3 patients initially discharged from the ED without oseltamivir returned to triage and required hospital admission. Two of these 3 later tested positive for 2009 pandemic influenza A (H1N1). One additional patient who required mechanical ventilation and subsequently died had presented 6 days after symptom onset. Another 5 hospital decedent whose care was transferred to the infectious disease service and therefore not part of our triaged case-series presented a median of 15 days after symptom onset. These cases suggest the importance of early oseltamivir treatment. Only one (1%) of 104 patients who tested positive for 2009 pandemic influenza A (H1N1) case-patients died. These findings contrast those of the National Institute of Respiratory Diseases in Mexico City where 12 (67%) of 18 patients required mechanical ventilation and 7 (39%) patients died [24, 26] . The discrepancy between these two case-series may be explained by when the populations served by these hospitals were affected by the pandemic. National Institute of Respiratory Diseases data were collected during March 24-April 24, 2009, when it was still unclear that a proportion of cases with severe acute respiratory infections had 2009 pandemic influenza A (H1N1). In Guadalajara, the outbreak started later. Hospitalized patients we described received earlier oseltamivir. Our patients were hospitalized during April 25-August 9. Seventy-five percent of our case-patients received oseltamivir within 72 hours of symptom onset. Patients in the Mexico City case-series presented with severe disease an average of 8 days after illness onset and received late oseltamivir.
Our findings have important limitations. A minority of all patients had respiratory specimens tested by RT-PCR, a large number of patients who were triaged were not confirmed with seasonal influenza or 2009 pandemic influenza A (H1N1) virus infection. No testing for other etiologies of acute respiratory illness was performed. Oseltamivir treatment among hospitalized patients was not randomized among cases and control. No comparison group was available to assess oseltamivir effectiveness for the treatment of 2009 pandemic influenza A (H1N1).
The triaging system with its ILI-score needs further validation. Nevertheless, such a triaging system can help guide the clinical management of patients presenting to the ED with acute respiratory illness in settings that lack timely diagnostic testing and have limited antivirals supplies. With some adaptation, the system may be especially useful in resource-poor countries, during the peak of pandemic influenza, or during other respiratory virus activity. Although no scoring system will replace clinical judgment, our experience suggests that the triaging system may have helped clinicians effectively triage patients and determine who needed hospital care and who could be managed as outpatients. The triaging system and the ILI-score should be modified to the local 2009 pandemic influenza A (H1N1) situation based upon hospital surge capacity, antiviral susceptibilities and supply, and the evolving epidemiology of this virus. Acute Encephalopathy Associated with Influenza A Infection in Adults We report acute encephalopathy associated with influenza A infection in 3 adults. We detected high cerebrospinal fluid (CSF) and plasma concentrations of CXCL8/IL-8 and CCL2/MCP-1 (CSF/plasma ratios >3), and interleukin-6, CXCL10/IP-10, but no evidence of viral neuroinvasion. Patients recovered without sequelae. Hyperactivated cytokine response may play a role in pathogenesis. Enterovirus RNA was detected by one-step real-time RT-PCR (3, 4) . Amplification is specific for the 5 untranslated region. Enterovirus RNA extracted from cell-culture fluid of clinical isolate confirmed by immunofluorescent was used as the source of positive control. RNA from the plasmid pAW109 was used as internal control for nucleic acid extraction as well as for the presence of inhibitors in the clinical samples. Primers and probes used were as follows: primer for enterovirus (EV), 5-ACATGGTGTGAAGAGTCTATTGAGCT-3, respectively. The performance of the assay was demonstrated by analysis of 3 replicates of quality control samples at 3 levels. The coefficients of variation (CV%) during the analysis of OP were 4.1%,1.9% and 2.1% at 3 ng/mL, 20 ng/mL, and 150 ng/mL, respectively (limit of detection of OP, 0.25 ng/mL). The coefficients of variation (CV%) during the analysis of OC were 2.3%, 0.5%, and 3.1% at 30 ng/mL, 400 ng/mL, and 4,000 ng/mL, respectively. Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool. groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient largescale evolutionary biosurveillance tool.
While the current influenza A H1N1 2009 virus is known to be sensitive to neuraminidase-inhibitor chemoprophylaxis, it has limited diversity at neutralizing antibody binding sites and overall mortality rates comparable with seasonal influenza (1) ; variations at numerous sites within the influenza A genome are predicted to alter these characteristics, with potentially important consequences for healthcare provision.
In order to enable large-scale identification of variations of H1N1(2009) viruses from multiple patient samples, it is necessary to develop a low-cost method for rapidly wholegenome sequencing the H1N1 samples. Historically, sequencing of viral genomes is performed using standard dye termination technologies. These conventional sequencing technologies produce accurate data but are too slow, costly and labour-intensive to be practical for large-scale epidemiologic or evolutionary investigations in viral outbreaks. Oligonucleotide resequencing microarrays that are capable of identifying nucleotide sequence variants may offer an alternative solution (2, 3) and in recent years, have been used for detecting and subtyping influenza viruses (4, 5) . By analysing sequences generated from tiling probes across targeted regions of various strains of the influenza virus [e.g. partial fragments of the haemagglutinin (HA) and neuraminidase (NA) genes], important information such as viral subtypes, lineages and sequence variants can be determined. Apart from influenza, resequencing microarrays have also been used to obtain whole-genome primary sequences for orthopoxviruses (6) , biothreat viruses (7) and SARS (8) . The reported studies mainly use platform accompanying software that employs probabilistic base-calling algorithms such as ABACUS (3) and Nimblescan PBC (8) . Although statistically sound, these methods are susceptible to hybridization noise caused by factors such as poor probe quality, poor amplification or mutations. This results in numerous ambiguous and false positive base calls that may affect the accuracy of downstream evolutionary analysis. Efforts have been made to improve the call rates and accuracies of existing probabilistic base-calling algorithms. For example, Model-P uses probe and sequence features to build intensity-prediction models that compute maximum likelihood scores for base-calling (9) . Another approach filters low-confidence base calls from problematic regions (e.g. regions with high mutation rates or repeats), thereby reducing the number of false-positive base calls (10) . Depending on the stringencies of the filters used, call rates may suffer as a result.
To address if these arrays can be used as a practical, large-scale re-sequencing tool, we have developed a system comprising customized sequence amplification primers, a 12-plex DNA resequencing array and an automated base-calling and variant analysis software (EvolSTAR). We demonstrate that the sequences obtained from the array are highly reproducible with !99.99% accuracy and 99.02 ± 0.82% genome coverage. The short turnaround time from sample to sequence and analysis results ($30 h for 24 samples) makes this kit an efficient large-scale evolutionary surveillance tool.
This article describes the development of the various genetic analysis components, and their validation using clinical samples. Accession numbers for 84 complete H1N1(2009) genomes generated are listed in Supplementary Data File 1.
Viral RNA from the diagnostic swabs or RNA extracted from MDCK cell cultures was extracted using the DNA minikit (Qiagen, Inc, Valencia, CA, USA) according to manufacturer's instructions. RNA was reverse-transcribed to cDNA using customized random primers designed using LOMA (11) and then amplified by PCR using proprietary H1N1(2009) specific primers. The presence of H1N1(2009) in the samples was confirmed using a separate real-time PCR assay based on the published primer sequences from the Centre for Disease Control and Prevention (CDC), USA.
We found 36 mutation hotspots in the alignments where mutations occurred near one another (within 20 bp). A perfect match (PM) probe residing in a mutation hotspot may contain mismatches that will have a detrimental effect on its hybridization intensity. To avoid this problem, we designed additional PM probes that contain all possible combinations of mutations found in each mutation hotspot. Thus, if two mutations are found within 20 bp of each other in the alignments, then we need in total four (2 2 ) PM probes to encode them. In general, 2 x PM probes are needed to completely encode a cluster of x mutations that occur within 20 bp of one another in the alignments.
We have identified five distinct types of neighbourhood hybridization intensity profile belonging to true nonmutations (wild-type), true mutations, isolated errors/ 'N's, long consecutive errors/'N's, and unknown errors/ 'N's, respectively. For each non-high-confidence query base, we determine the type of its neighbourhood hybridization signal intensity profile (NHIP) by the following criteria:
(i) True-non-mutation-The PM probe (of both strands) of the query base must be a high-confidence call [i.e. it has hybridization intensity ! 1.4-fold that of its mismatch (MM) probes]. Neighbourhood PM probes are also high-confidence calls. Let the mean hybridization intensity of the three nearest PM probes to the immediate left of the mutation base (at position À1, À2 and À3), denoted as {À1,À2,À3} , the mean hybridization intensity of the three PM probes to the far left of the mutation base (at position À4, À5 and À6), denoted as {À4,À5,À6} , the mean hybridization intensity of the three nearest PM probes to the immediate right of the mutation base (at position 1, 2 and 3), denoted as {1,2,3} , and the mean hybridization intensity of the three PM probes to the far right of the mutation base (at position 4, 5 and 6), denoted as {4,5,6} . We impose that {À1,À2,À3} % {À4,À5,À6} and {1,2,3} % {4,5,6} (ii) True-mutation-The PM probe (of both strands) of the query base must have hybridization intensity ! 1.4-fold that of its MM probes. To detect the characteristic dip, we check four mean hybridization intensities: the mean hybridization intensity of the three nearest PM probes to the immediate left of the mutation base (at position À1, À2 and À3), denoted as {À1,À2,À3} , the mean hybridization intensity of the three PM probes to the far left of the mutation base (at position À4, À5 and À6), denoted as {À4,À5,À6} , the mean hybridization intensity of the three nearest PM probes to the immediate right of the mutation base (at position 1, 2 and 3), denoted as {1,2,3} , and the mean hybridization intensity of the three PM probes to the far right of the mutation base (at position 4, 5 and 6), denoted as {4,5,6} . If {À1,À2,À3} < {À4,À5,À6} and {1,2,3} < {4,5,6} , we say this is a dip pattern and the query base is likely to be mutated. (iii) Isolated error/'N'-The PM probe (of both strands) of the query base has hybridization intensity < 1.4fold that of its MM probes. Neighbourhood PM probes are high-confidence calls. (iv) Long consecutive errors/'N's-The PM probe (of both strands) of the query base has hybridization intensity < 1.4-fold that of its MM probes. A majority of neighbourhood PM probes are nonhigh-confidence calls. (v) Unknown error/'N'-All other neighbourhood hybridization profile patterns that do not fall under the previous categories.
We define that a probe encodes the base b if b is located in the centre-most position of the probe and is the base to be To validate if the observed PM probe encoding b 1 is indeed the true PM probe of the sample sequence, we compute the likelihood ratio of f obs and f rand , where f obs is probability of observing the hybridization intensity reduction order b 1 b 2 b 3 b 4 given that the PM probe encodes b 1 and f rand is the probability of observing the hybridization intensity reduction order b 1 b 2 b 3 b 4 by chance. Precisely,
where #(wxyz) is the number of observed hybridization intensity reduction orders from high-confidence base-calls and t is the total number of hybridization intensity reduction orders excluding b 1 b 2 b 3 b 4 obtained from high-confidence base-calls. If the likelihood ratio >2, we expect that the observed PM probe encoding b 1 is indeed the true PM probe of the sample sequence.
EvolSTAR employs a two-step process for base-calling ( Figure 1 ). In the first step, each base query is scrutinized for signs of hybridization intensity abnormalities. If the gain-of-signal of the query base is strong and has no mutation, the base is called. In the second step, EvolSTAR then tries to recover base queries that have any hybridization intensity abnormalities with two analysis methods, namely neighbourhood hybridization intensity profile analysis and nucleotide substitution bias analysis.
Step 1: Identification of base queries with ambiguity. On our array platform, the hybridization intensity of each probe is given by the mean and standard deviation of the fluorescence intensities of nine individually scanned pixels. Hence, we define the signal-to-noise ratio (SNR) of a probe as the ratio of the mean to the standard deviation of the intensities of the nine pixels associated with the probe. In our experiments, we found that >95% of all probes had SNR less than T SNR (T SNR = m SNR + 2s SNR , where m SNR and s SNR are the mean and standard deviation of SNR of all probes on the array). The remaining 5% of probes with SNR ! T SNR are unreliable. Hence, base queries with one or more probes with SNR ! T SNR are analysed further in step 2. Furthermore, all base queries whose PM probe in the forward strand and PM probe in the reverse strand are non-complementary, or have weak PM/MM hybridization intensity differentiation (<1.4-fold) are also passed to step 2. Lastly, we also pass all putative mutation calls to step 2 for confirmation.
Step 2: Mutation confirmation and base query recovery. A high-confidence mutation call may be a result of coincidental non-specific hybridization of the same MM probe in both strands. As such, it may be inadequate to discern true mutations based solely on differences in the hybridization intensities of PM and MM probes. From our analysis of true-mutation calls made by PBC, we have found that true mutations have a signature NHIP type as per described in Figure 2b . Thus, query bases that result in a mutation call must have this signature NHIP. Finally, to confirm the mutation, we perform nucleotide substitution bias analysis on these query bases. For each of the query bases with NHIP of type described in Figure 2b , we compute the likelihood ' that the observed PM probe (representing the mutation) is indeed the true PM probe of the sample sequence given the hybridization intensity-based ordering of its MM probes (see 'Materials and methods' section). If ' > 2, the query base results in a strong mutation call (represented by upper case base calls 'A', 'C', 'G' or 'T'). If ' > 1, the query base results in a mutation call with weak support (represented by lower case base calls 'a', 'c', 'g' or 't'). Otherwise, they are re-assigned an unknown 'N' call.
For query bases that results in a mutation call but have NHIP of type described in Figure 2c , they are most likely isolated errors caused by poor PM probe quality. Hence, we correct the base-calls of these query bases to their respective reference bases (but represented by lower case Figure 2 . Summary of characteristics of neighbourhood hybridization intensity profiles for different type of calls. Summary of the characteristics of the NHIP for five types of call: (a) true-non-mutation, (b) true-mutation, (c) isolated error or 'N', (d) long chains of consecutive errors or 'N', (e) unknown error or 'N') based on their respective observed neighbourhood hybridization intensity profiles. The PM probe (red circle) of query base is at position 0 while neighbourhood PM probes (black circles) are numbered according to their distance away from the query base. A PM probe is significantly differentiated from its MM probes if its hybridization intensity is at least t-fold that of all its MM probes. base calls 'a', 'c', 'g' or 't') in the reference sequences. We also perform the same correction to non-high-confidence query bases with NHIP of type described in Figure 2c .
We try to recover the remaining query bases that have NHIP of type described in Figure 2d or 2e by analysing the substitution bias from their PM and MM probes in the forward and reverse strands separately. Similar to how a mutation is confirmed, we compute the likelihood ' f that the observed PM probe (representing the unsure base call) is indeed the true PM probe of the sample sequence given the hybridization intensity-based ordering of its MM probes in the forward strand. We also compute a similar likelihood ' r for the PM probe in the reverse strand. If the PM probes in both strands are complementary and ' f , ' r > 2, the query base results in a strong base call (represented by upper case base calls 'A', 'C', 'G' or 'T'). However, in many cases, the PM probes in both strands are not complementary due to non-specific hybridization of MM probes in one or both strands. For such query bases, we make base calls based on ' f and ' r : if ' f > ' r and ' f > 2, a base call with weak support (represented by lower case base calls 'a', 'c', 'g' or 't') is made from the PM probe in the forward strand. Else, if ' r > ' f and ' r > 2, a base call with weak support is made from the PM probe in the reverse strand. Otherwise, they are assigned an unknown 'N' call.
Note that since nucleotide substitution biases may vary depending on the experimental conditions, experimental reagents or input samples, for each experiment, we obtain a set of high-confidence base-calls and use them to infer the hybridization intensity reduction orders for each PM probe encoding. This is then used to compute likelihood scores for base-calling non-high-confidence query bases and mutation confirmation.
We generated a consensus sequence for each segment of the H1N1(2009) virus by aligning all 1715 complete and partial sequences available from the NCBI H1N1 flu resources database (http://www.ncbi.nlm.nih .gov/genomes/FLU/SwineFlu.html) as of 11 June 2009 using MAFFT (12) with high-accuracy option.
Tiling probes spanning the entire genome segments on both the forward and reverse strands were created at one base resolution (8) . Analysis of the sequence alignments revealed that there were no deletions, insertions or recombination. However, we found 36 mutation hotspots in the alignments where mutations occurred near one another (within 20 bp). Thus, we added additional probes that represent all possible combinations of mutations in these mutation hotspots onto the array. To ensure that we have accurate sequence of the drug binding pocket targeted by NA inhibitors (13) 
Due to the small amount of virus present in samples relative to human or cell-line total RNA, it was necessary to amplify the viral RNA through PCR. We employed a combination of sequence-specific and random PCR approaches using LOMA-optimized primers as previously described (11) . The addition of random primers ensured complete genome amplification, even if mutations were present at the specific-primer binding sites. PCR conditions were optimized by conducting five duplicate hybridizations of the same virus sample cultured from a patient sample under different PCR conditions. The optimized method was then tested on RNA isolated directly from nasal swabs obtained from the same patient and from virus grown in cell culture. Microarray sequences generated from these replicate experiments were compared with capillary sequencing to estimate sequencing accuracy.
Following PCR product labelling, hybridization and scanning, signal intensities for each probe was generated using Genepix 4.0 software, and annotated using Nimblescan 2.5 software. Initially, the standard Nimblescan software which employs a gain-of-signal approach [PBC algorithm (8) ], was used to determine the viral sequence. The PBC algorithm assumes that the signal intensity of the PM probe (which matches exactly to the sequence in the sample) will be significantly higher than that of the MM probes. While this approach sufficed for $90% of base queries, we observed that the discrimination between the PM and MM signals was not clear for the remaining probes.
These ambiguous signals were caused by the presence of multiple mutations in the probe sequence, homopolymers and hybridization artefacts. We developed a novel algorithm, Evolution Surveillance and Tracking Algorithm for Resequencing arrays (EvolSTAR), to resolve this problem. EvolSTAR improves upon PBC by adding an analysis of the NHIP and nucleotide substitution bias (described below).
Due to the use of tiling probes in resequencing arrays, a single nucleotide mutation at a particular query base could cause a dramatic reduction in the hybridization intensities of neighbouring PM probes up to six bases away (14) . This effect can be measured by studying the NHIP of each query base. We defined the NHIP of each query base as the observed pattern of hybridization intensities of its PM and MM probes and neighbouring (±6 bases from query base) PM and MM probes. To study the effects of sequence variation (mutation) and noise on the NHIP of a query base, we sequenced RNA from H1N1(2009) patient 380 by capillary sequencing and on duplicate microarrays. We compared sequence calls generated using by Nimblescan or by capillary sequencing and compiled a list of true (correct) calls, error calls and 'N' (unknown) calls. In total, of the expected 13 588 bases of the H1N1 virus (based on genome described at http:// www.ncbi.nlm.nih.gov/genomes/taxg.cgi?tax=211044) the microarray called 13 449 bases while capillary sequence was able to call 12 832 bases. Figure 3 shows the NHIPs of a representative set of 40 randomly selected query bases that result in true-nonmutation calls (wild-type calls). We observed that in these NHIPs, the PM probe of the query base together with neighbouring PM probes, have hybridization intensities significantly higher (>1.4-fold) than that of their MM probes in general. We also identified 10 mutations using capillary sequencing in the patient sample. The NHIPs of these 10 true-mutation calls (Figure 4 ) are very different from NHIPs of wild-type calls. The presence of a mutation at the query base created a MM in neighbouring PM probes and caused a drop in their hybridization intensities. The closer this mutation is to the centre of a neighbouring PM probe, the bigger the drop in hybridization intensity. This results in a distinctive dip to the immediate left and right of the centre of the NHIP where the mutation is.
Unlike the NHIPs of wildtype and true-mutation calls, the NHIPs of most errors and 'N' calls appear haphazard ( Figure 5 ). However, when we traced the locations of these errors and 'N' calls on the genome, we found that some are isolated among good calls while others are conjugated in a small locality of the genome. We investigated the NHIPs of isolated errors and 'N' calls that occurred among good calls and found that in these NHIPs, only the PM probe of the query base that is an error or 'N' call has poor hybridization differentiation with its MM probes while other PM probes have hybridization intensities significantly higher than that of their MM probes in general ( Figure 6 ). This suggests that for such calls, only the PM and MM probes of the query base are noisy while neighbouring PM and MM probes are unaffected. In addition, we also found that long chains of consecutive error and 'N' calls (especially at the 5 0 -and 3 0 -end of the sample sequences) often have NHIPs where the PM probe of the query base together with neighbouring PM probes, have poor hybridization differentiation with their MM probes (Figure 7) . These error and 'N' calls usually occur at the ends of the genome segments. In summary, NHIP analysis showed that all true mutation calls had a characteristic profile (Figure 2b ) that differed from wild-type sequence calls (Figure 2a ). Ambiguous calls arising from different causes, such as homopolymers, isolated errors and hybridization artifacts also have profiles that are distinct from true mutation profiles ( Figure 2 ). See 'Materials and Methods' section for details.
The presence of nucleotide substitution bias in Nimblegen resequencing arrays has been previously described (15) . However, this knowledge has so far been used only to improve probe design. In this article, we propose a novel method that makes use of nucleotide substitution bias in the array to improve base-calling accuracy and call rate. The key idea is to build a likelihood model of the substitution bias among the probes of non-ambiguous calls on the array; then use this to call bases with ambiguous signals.
To build the likelihood model, we first determined the substitution bias on our platform by comparing the PM and MM probes (of both strands) of 25 028 true calls made by PBC from the two replicate microarray experiments of patient sample 380 mentioned in the previous section. For each true call, we generated a hybridization intensity reduction order by ranking the PM and MM probes of a particular strand in decreasing order of hybridization intensity and recording their respective frequencies (Table 1 ). Table 1 shows that for each PM probe encoding, certain hybridization intensity reduction orders occur much more frequently than others. For example, if the PM probe encoding is 'A' (regardless of strand), then it is most likely that the hybridization intensity reduction order is 'TGC' or 'GTC'. Thus, by matching the hybridization intensity reduction orders of its PM/ MM probes with that in Table 1 , we can compute the likelihood that the putative base call for a query base with ambiguous signals is correct (see 'Materials and Methods' section). In this way, we can recover base calls of ambiguous query bases exceeding a reasonably high likelihood threshold and achieve better accuracy and call rate than PBC.
Grading the quality of the sequence calls EvolSTAR employs a two-step process for base-calling (details in 'Materials and methods' section). First, it determines if the gain-of-signal from the PM probe is strong. If strong, then the base sequence is called and annotated as 'high quality'. If the gain-of-signal is ambiguous, or if the base called is different from the expected sequence, then NHIP and nucleotide substitution bias is deployed to verify the sequence call. From empirical experiments and comparison with capillary sequence data, we observed that high quality sequence calls are made when the signal from the PM probe for both forward and reverse strands are at least 40% higher than that of the MM probes.
The second-step processes the ambiguous bases. For bases confirmed to be mutations through NHIP analysis and satisfy the substitution bias rule, they are graded as high quality sequence calls and denoted in the FASTA sequence as UPPER CASE characters. For bases confirmed to be an isolated error through NHIP analysis and also satisfy the substitution bias rule, they are graded as low confidence sequence calls and denoted in the FASTA sequence in lower case characters. The rest of the bases are called 'N' in the FASTA sequence. The flowchart of EvolSTAR is shown in Figure 1 .
To validate the software, we hybridized 14 patient samples in duplicate onto the microarray. The microarrays were analysed in parallel using Nimblescan (PBC algorithm) and EvolSTAR, and the sequences obtained were compared to Sanger capillary sequencing. We counted the number of true-non-mutation calls, true-mutation calls, error calls and ambiguous ('N') calls for both methods ( Table 2 ; Supplementary Data File 3). We also confirmed that the substitution bias in all 14 duplicate hybridization experiments (Table 3) were consistent with that found in Table 1 . Compared with the available capillary sequences for the 14 samples, EvolSTAR had an average error rate of 0.0029% and 12 ambiguous calls per sample (346 in total). This is far superior than Nimblescan PBC, where we obtained an average error rate of 0.083% and 158 ambiguous calls per sample (4434 in total). Furthermore, EvolSTAR called all true mutations correctly. The genome coverage attained by EvolSTAR (99.02 ± 0.82%) is also much higher than that of Nimblegen PBC (94.3 ± 6.06%).
We wondered if, and by how much, incorporating NHIP and substitution biases analysis to the PBC results would improve the performance of the PBC algorithm. We observed that more than 70% of the 65 error calls (false mutation calls) made by PBC did not have the characteristic NHIP of a true-mutation shown in Figure 2b . The remaining 30% of the error calls had a NHIP reminiscent of a true-mutation NHIP but did not satisfy the substitution bias rule. Using NHIP and substitution biases analysis together, we were able to reduce the number of false mutation calls to only two. Most of the 4434 'N' calls made by PBC were due to conflicting base calls from the forward and reverse strand. By analysing the NHIP and hybridization intensity reduction order of the query base in the forward and reverse strand individually, we were able to identify the noisy strand and hence, make the base call only from the non-noisy strand. We were able to recover 92% of the 'N' calls made by PBC using this approach.
In addition, we evaluate the robustness and reproducibility of EvolSTAR by employing six pairs of replicate experiments consisting of one pair nasal swab and five pairs of cell culture isolates, belonging to the same patient sample 305 (Supplementary Data File 4) .
Of the experiments, two pairs of replicates (305_nasal and 305_cell_cond1) were amplified under the same optimal experimental conditions while each of the other pairs (305_cell_cond2, 305_cell_cond3, 305_cell_cond4, 305_cell_cond5) were amplified under different suboptimal experimental conditions (simulating experimental volatility). Compared with the available capillary sequences for sample 305, EvolSTAR had an average error rate of 0.0012% and 28 ambiguous calls per sample (338 in total). On the other hand, Nimblescan A  CGT  547  246  CTG  558  237  GCT  957  367  GTC  2215  1407  TCG  1049  611  TGC  3015  2873   C  AGT  2035  2712  ATG  1752  2400  GAT  382  341  GTA  159  134  TAG  360  377  TGA  165  129   G  ACT  1474  1043  ATC  976  624  CAT  1639  1534  CTA  868  788  TAC  594  410  TCA  542  454   T  ACG  432  529  AGC  562  636  CAG  623  841  CGA  1066  1616  GAC  1421  1878  GCA  1637  2841 Hybridization intensity reduction orders found in 25 028 true calls from two replicated hybridization experiments of patient sample 380. For each true call, for each strand, we rank the PM probe and its MM probes based on their hybridization intensities in decreasing order. We count the frequency of each hybridization intensity reduction order. 1  6002  6  5888  6  0  0  EvolSTAR  2  6002  6  5993  6  0  0  PBC  2  6002  6  5895  6  0  1   507  EvolSTAR  1  3921  8  3913  8  0  0  PBC  1  3921  8  3736  8  0  3  EvolSTAR  2  3921  8  3916  8  0  0  PBC  2  3921  8  3758  8  0  2   581  EvolSTAR  1  8574  10  8567  10  0  0  PBC  1  8574  10  8458  10  0  2  EvolSTAR  2  8574  10  8566  10  0  0  PBC  2  8574  10  8461  10  0  5   582  EvolSTAR  1  3057  4  3051  4  0  0  PBC  1  3057  4  2986  4  0  0  EvolSTAR  2  3057  4  3053  4  0  0  PBC  2  3057  4  3001  4  0  0  593  EvolSTAR  1  3054  3  3053  3  0  0  PBC  1  3054  3  3007  2  1  0  EvolSTAR  2  3054  3  3053  3  0  0  PBC  2  3054  3  2992  2  1  0   9 061 364  EvolSTAR  1  5129  5  5123  5  0  0  PBC  1  5129  5  5064  5  0  0  EvolSTAR  2  5129  5  5122  5  0  0  PBC  2  5129  5  5042  5  0  0   9 061 365  EvolSTAR  1  3000  3  2993  3  0  0  PBC  1  3000  3  2956  3  0  1  EvolSTAR  2  3000  3  2991  3  0  0  PBC  2  3000  3  2941  3  0  0   9 061 366  EvolSTAR  1  1683  3  1683  3  0  0  PBC  1  1683  3  1649  3  0  1  EvolSTAR  2  1683  3  1682  3  0  1  PBC  2  1683  3  1636  3  0  1   923  EvolSTAR  1  4373  5  4365  5  0  0  PBC  1  4373  5  4187  5  0  1  EvolSTAR  2  4373  5  4330  5  0  1  PBC  2  4373  5  3738  5  0  6 Types of calls and their frequencies generated by EvolSTAR and PBC in replicated microarray hybridizations of 14 patient samples. Partial or complete capillary sequences were generated for each sample and used to verify the calls made by EvolSTAR and PBC on each replicate. We then count the frequency of true-non-mutation, true-mutation, error and 'N' calls in each replicate.
PBC obtained a relatively higher average error rate of 0.169% and 237 ambiguous calls per sample (2855 in total). Our results showed that EvolSTAR is robust and performs well when samples are prepared under sub-optimal conditions. Even for nasal swab samples that tend to have much less concentration of virus RNA than cell cultures, EvolSTAR suffered only a slight drop in performance compared to Nimblescan PBC. In conclusion, we have shown that EvolSTAR is robust and generates sequence calls of high accuracy and reproducibility in this pilot study consisting of 40 microarray experiments. Meanwhile, efforts will be put in to continually evaluate EvolSTAR with more samples and update it on a regular basis as the H1N1(2009) influenza virus evolves.
Besides a FASTA output of the virus sequence, EvolSTAR generates a visualization map of the sequence calls using a heat map based on the percentage identity of the called sequence to the reference sequence measured at 50 bp windows (Figure 8 ). The map template consists of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the NA gene. Locations of all mutation calls are denoted by red triangles beneath the heat map bar. Sequences that are of low coverage (<90%) are automatically flagged, and the overall PM/ MM discrimination ratio for each segment is displayed. The heat map bar allows the technician to rapidly assess the quality of the sequence data obtained from the microarray and identify regions where PCR did not work well, or presence of potential recombination/ reassortment events. Mutations, especially those in close proximity to drug binding sites, can be quickly visualized. Other details such as coverage, number of base calls successfully made, number of mutations and number of 'N' calls for each sequence call are also shown on the visualization map.
Traditional statistical and probabilistic sequence-calling techniques ascertain that a base call is of high confidence if they exceed pre-defined significance or probability thresholds. This approach works well for high-confidence base-calls but is inadequate to extract sufficient information from noisy base-calls. It is also difficult to determine the validity of a mutation call purely based on the distribution of hybridization intensities of its PM and MM probes. In this work, we have described two new hybridization intensity analysis methods that enable us to confidently identify true mutations and recover some noisy base calls. Compared to PBC, EvolSTAR has achieved superior call rates and accuracies, especially in low-concentration samples with high CT values. The robustness of the base calls enables our approach to be a practical large-scale evolutionary surveillance tool.
Although we are confident that our resequencing array can successfully generate complete sequences for the H1N1(2009) virus and its variants at the current stage, we cannot rule out the possibility of reassortments between the H1N1(2009) virus and other influenza viruses. Clearly, our resequencing array cannot fully sequence such events and will generate sequences with poor quality and coverage of the reassorted segments. To investigate the effects of a reassortment event on our array, we independently amplified segments 1, 2, 3, 5, 6 and 7 of the 2009 influenza A(H1N1) virus and segment 4 of a H3N2 influenza A virus, and hybridized them onto our array. The visualization map of this experiment is shown in Figure 9 . As expected, the sequence call for segment 4 [based on PM/MM probes from the segment 4 consensus of the 2009 influenza A(H1N1) virus] is poor in quality and coverage. However, we observed that we were able to get good base calls from region 1150-1547. This region turns out to be the only significantly similar (70% matched) region between the segment 4 consensus of the 2009 influenza A(H1N1) virus and segment 4 of a H3N2 virus (CY039087). This shows that identifying regions of high similarity between the 2009 influenza A(H1N1) virus with other influenza viruses and checking if these regions have good sequence calls may be a plausible way of detecting reassortments. The drawback of this approach is that it will fail to detect reassortment of Table 3 . Hybridization intensity reduction orders found in 14 hybridization experiments certain segments where there are no regions of high similarity between the H1N1(2009) virus and the parental influenza virus. It is also difficult to annotate and differentiate every region that the H1N1(2009) virus and all other influenza viruses share similarity with. We propose an alternative approach to detect reassortments. By analysing the PM/MM hybridization intensity foldchange of high confidence calls of all eight segments, we found that the average PM/MM hybridization intensity fold-change of high confidence calls in segments 1, 2, 3, 5, 6 and 7 belonging to the 2009 influenza A(H1N1) virus is $4.5 while the average PM/MM hybridization intensity fold-change of high confidence calls in segment 4 belonging to the H3N2 influenza A virus is only 1.9. The most likely reason for this huge drop in the average PM/MM hybridization intensity fold-change of high confidence calls is that the signal gained by most of the segment 4 PM probes on our array are through cross-hybridization to the segment 4 sequence of the H3N2 influenza A virus, and thus much lower than signal gained from true specific binding. Thus, by computing and comparing the average PM/MM hybridization intensity fold-change of high confidence calls in each segment, we can identify potential reassortments in a given H1N1(2009) virus sample. Virus samples with possible reassortments can then be sequenced using . A heat map bar is used to represent the quality and coverage of its sequence calls. The locations of all mutation calls made by EvolSTAR are represented by red triangles beneath the heat map bar. Sequences with coverage <90% are automatically flagged as 'low coverage'. Other details such as coverage: percentage of base calls successfully made, match: number of base calls that match the reference sequence i.e. non-mutation base calls, strong mismatch: number of high confidence base calls that do not match the reference sequence i.e. mutation base calls, weak mismatch: number of low-confidence base-calls that do not match the reference sequence i.e. mutation base calls and Ns: number of 'N' calls, for each sequence call are also shown on the visualization map. capillary sequencing or customized reassortment resequencing arrays.
So far, the sequence diversity of H1N1 2009 influenza virus has been rather limited. From our analysis, it would be possible to resequence all the published isolates using this resequencing approach. However, as antigenic drift is expected to occur, it is likely that the resequencing array would need to be updated at least annually. Updating the array requires only bioinformatics input, and does not require any other additional manufacturing costs. Thus, this combination of sample amplification primers, low-cost multiplex array and robust interpretation software allows sustainable, rapid, large-scale biosurveillance of the influenza H1N1(2009) virus.
From a broader perspective, this study has highlighted the feasibility of using resequencing microarrays for high-throughput full genome sequencing of viruses. In our application, resequencing microarrays are relatively low-cost, costing only a 10th that of a 454 run, and equivalent to that of a traditional capillary sequencing run. However, through multiplexing, our system can generate full genomes of 24 different H1N1(2009) samples in 30 h. In comparison, capillary sequencing and next-generation technologies such as 454 may obtain full genomes of only one or two different samples in the same time-frame. In practice, capillary sequencing is labour-intensive and thus impractical for large-scale full genome sequencing of viruses in an outbreak. In our experience with the 454 system, much of the amplified material is still human (as the bulk of the patient sample material is human RNA with very little influenza RNA), requiring very deep sequencing to obtain a complete flu genome sequence, with one compartment of a run not yielding sufficient viral information. Furthermore, assembly of the sequence fragments is required before any analysis can be done. Any abnormalities or gaps in the assembly would then require additional runs of 454, incurring more cost and time. Hence, our approach based on resequencing microarrays presents a cost-effective and efficient solution for high-throughput full genome sequencing of viruses.
Supplementary Data are available at NAR Online. Journals, Academics, and Pandemics In the wake of the SARS epidemic and the H1N1 pandemic, the PLoS Medicine editors ask whether journal publishing is an efficient enough mechanism for information sharing. The PLoS Medicine Editors* Two articles published recently in PLoS Medicine highlight the problem of how to effectively share information in the wake of a rapidly spreading disease, and prompted us to ask the question ''How well are journals doing?'' with regard to this important goal. The answer, sadly, seems to be ''not well enough.'' Although the potential of the Internet for improving the dissemination of information is now taken for granted, it would seem that the attitudes of those involved in sharing this information have not kept pace with the technology. Accordingly, it is fair to ask whether the flow of information in the face of a crisis is truly enabled by publication in medical journals (even online journals) or whether we need new avenues for rapid data sharing.
An article appearing this month in PLoS Medicine by Weijia Xing and colleagues [1] dissected the publication of a subset of epidemiological papers during the severe acute respiratory syndrome (SARS) epidemic of 2003. Based on their findings, it would be hard to conclude that journal publication was a successful mechanism for rapidly sharing information. As the authors note, ''Only 22% of the studies were submitted, 8% accepted, and 7% published during the epidemic.'' What were the reasons behind these findings? The authors argue that the lack of standard methods for data collection and manuscript preparation may have played a part. In addition, despite journals' best efforts to speed up times to publication (reflected in shorter publication times compared with control articles on unrelated topics, submitted at the same time), the time to publication was over 200 days for many articles. It's not possible to know whether these delays were compounded by articles being sequentially submitted to a number of different journals before being published. But it is notable that while the 311 SARS articles were published in 137 different journals, the first ten published studies appeared in The Lancet (n = 7) and The New England Journal of Medicine (n = 3). However, the impact factors of journals publishing articles on SARS decreased significantly as time went on. Put another way, it seems that at the beginning of the epidemic, highprofile journals were willing to publish papers on SARS, but their interest waned rapidly. In addition, it is likely that, as with publishing other types of article, authors will try high-impact journals first.
Fast forward to the H1N1 pandemic of 2009-10. It's too early to carry out the same type of analysis that was done for SARS by Xing et al., but a paper we published in early April indicates that many of the same problems remain. The paper, ''Association between the 2008-09 Seasonal Influenza Vaccine and Pandemic H1N1 Illness during Spring-Summer 2009: Four Observational Studies from Canada'' [2] reports potentially worrying findings about the impact of seasonal flu vaccination on illness from H1N1. We're the first to admit we were not quick-we received the paper on 2 November 2009, accepted it on 1 March 2010, and published it on 6 April 2010. It's quite possible that this delay could have exacerbated the problem of making decisions about vaccination for the public health physicians and policymakers who had heard informally about some of the results and wondered if they should be changing course. At the same time, the need for careful review of these controversial data meant that we could not rush the review process. In addition, as the lead author, Danuta Skowronski, herself indicated, prior rejection from another journal added further to the delay in publication [3] .
Taken together these two papers highlight an inherent limitation in the journal publication system with regard to rapid dissemination of results in a time of crisis: the processes that ensure careful evaluation come at the expense of immediate dissemination.
Journals are, of course, only one source of information for health and scientific research, and may be over-relied upon, especially in emergencies. International bodies such as WHO provided efficient regular [4] updates during both emergencies, as did local health bodies such as the Health Protection Agency in the UK [5] and the Centers for Disease Control in the US [6] . Face-to-face meetings and teleconferences provide further mechanisms for sharing of information, and when linked to sharing of resources were powerful catalysts for accelerating influenza research. PLoS itself launched an experimental site, PLoS Currents: Influenza (http://knol.google.com/k/plos-currents-influenza#) for early sharing of information after only the lightest of moderation.
Despite other mechanisms of dissemination (which need not preclude later publication in more formal peer reviewed journals) even in the face of a public health emergency, authors seem tied to publishing information first in peer-reviewed journals, possibly because they may perceive readers as reluctant to take results seriously until they have successfully emerged from review by a respected journal. It seems therefore timely to ask whether journals, and others involved in publishing, are confusing the valid role of a journal as a place of scientific record with an equally valid role, that of a news outlet? In a Canadian press report [3] on the Skowronski paper, Ross Upshur, head of the University of Toronto's Joint Centre for Bioethics, was quoted as saying ''Making public health policy during a pandemic based on data most people hadn't seen was far from ideal,'' adding that he believes the question of when public health priorities trump rules of scientific publication remains to be resolved: ''Putting the imprimatur of a high impact peer review journal first is I think what we need to have the discussion about.'' Of course, the authors of this paper, and of all the articles published from study of the SARS epidemic and the H1N1 pandemic, would say they were not seeking such an ''imprimatur'' for the sake of it, but because as yet there is no widely accepted alternative to the ''quality'' stamp that peer review imparts. Similarly, journal editors would maintain that highprofile papers generated by such emergencies need intense scrutiny lest the public health suffer from the premature publication of unreliable results.
But in the case of a public health emergency, are these concerns enough to override the need for information-any information-however preliminary and unconfirmed? Should the whole paradigm of publishing be rethought in such instances? In the age of blogs, Twitter, and the 24-hour news cycle, are journals a realistic avenue for rapid publication at all? Instead, is there a role for datasharing-i.e., the news outlet functionrather than traditional publication in an emergency situation? If so, who would set up data repositories and oversee them, how would authors get academic credit for their work, and how would readers and reporters learn to interpret data presented through such a system? Whatever the answers may be, it seems clear that before the next public health emergency strikes, the scientific publishing establishment needs to ask itself how it can respond in the way the world needs. Age groups and spread of influenza: implications for vaccination strategy BACKGROUND: The unpredictable nature of the potentially devastating impact of 2009 pH1N1 influenza pandemic highlights the need for pandemic preparedness planning, where modeling studies could be most useful for simulations of possible future scenarios. METHODS: A compartmental model with pre-symptomatic and asymptomatic influenza infections is proposed which incorporates age groups as well as intervention measures such as age-specific vaccination, in order to study spread of influenza in a community. RESULTS: We derive the basic reproduction number and other effective reproduction numbers under various intervention measures. For illustration, we make use of the Pneumonia and Influenza (P&I) mortality data and vaccination data of the very young (age 0-2) and the very old (age >64) during 2004-2005 Taiwan winter influenza season to fit our model and to compute the relevant reproduction numbers. The reproduction number for this winter flu season is estimated to be slightly above one (~1.0001). CONCLUSIONS: Comparatively large errors in fitting the P&I mortality data of the elderly (>64) were observed shortly after winter school closings in January, which may indicate the impact of younger, more active age groups transmitting influenza to other age groups outside of the school settings; in particular, to the elderly in the households. Pre-symptomatic infections seemed to have little effect on the model fit, while asymptomatic infection by asymptomatic infectives has a more pronounced impact on the model fit for the elderly mortality, perhaps indicating a larger role in disease transmission by asymptomatic infection. Simulations indicate that the impact of vaccination on the disease incidence might not be fully revealed in the change (or the lack thereof) in the effective reproduction number with interventions, but could still be substantial. The estimated per contact transmission probability for susceptible elderly is significantly higher than that of any other age group, perhaps highlighting the vulnerability of the elderly due to close contacts with their caretakers from other age groups. The relative impact of targeting the very young and the very old for vaccination was weakened by their relative inactivity, thus giving evidence of the lack of impact of vaccinating these two groups on the overall transmissibility of the disease in the community. This further underscores the need for morbidity-based strategy to prevent elderly mortality. In the spring of 2009, the novel H1N1 influenza virus first emerged in Mexico and later spread widely throughout the world within just a few months. The World Health Organization (WHO) announced on June 11 the start of 2009 influenza pandemic [1] , and further issued an advisory on August 28 for countries in the northern hemisphere to prepare for a second wave of pandemic spread in the coming fall/winter [2] . As of November 8, more than 206 countries and overseas territories or communities worldwide have reported laboratory confirmed cases of the pandemic pH1N1 virus, including over 6250 deaths [3] .
To lessen the severity of this pandemic, developing an effective flu vaccine and a global vaccination strategy is considered to be among the most important medical interventions [4] . However, to have the greatest impact, pandemic vaccines need to be available quickly and in large quantities, and to be delivered to the population optimally. Moreover, vaccines against a novel pandemic strain might take up to six months to manufacture and deliver, even in developed countries [5] . Given the potential threat of drug-resistance resulting from widespread use of antiviral treatment against pandemic flu, vaccine appears to be our primary weapon to prevent and to mitigate a pandemic. However, in addition to the need to consider the logistics of implementing largescale vaccination, distinctly different age-specific mortalities had also been observed during some past flu pandemics (e.g., in 1918 [6] ), which require different priorities when large-scale vaccination is to be implemented.
Moreover, vaccine for influenza is known to have different efficacy (i.e., reduction in the number of infectives) and effectiveness (i.e., reduction in symptomatic case number) for different age groups, see e.g., [7] [8] [9] . Setting priority for vaccination by targeting age groups most vulnerable (the elderly, infants, etc.) to prevent mortalities is commonly employed in most countries. However, when vaccinating those at greatest risk of mortality becomes impractical (if, e.g., medical care is relatively inaccessible) or inefficient (if, e.g., immune response is deficient), targeting those most likely to expose them to infection might be more preferable [10] . In this way, the very young and the old might be better protected by vaccinating those who are most likely to be in contact with them (thereby reducing their risk of exposure), rather than by being vaccinated. Comparison of influenza mortality among elderly Japanese during time periods when schoolchildren were and were not vaccinated suggests that the infected children pose a risk to others [11] , including the elderly. Moreover, several past US experiences (as summarized in [12] ) also are consistent with this conclusion. Nonetheless, influenza policymakers have typically advocated protecting those individuals of ages 6-24 months and >65 years directly.
Bansal et al. [13] recently carried out a comparative analysis of two classes of suggested vaccination strategies, namely, the mortality-based strategies that target the high-risk populations and the morbidity-based strategies that target the high-prevalence populations, by applying the methods of contact network epidemiology to a model of disease transmission in a large urban population. Using a range of mortality rates reported previously for past influenza epidemics and pandemics, they concluded that the optimal strategy depends critically on the viral transmission level (or reproduction number) of the virus. That is, the morbidity-based strategies outperform the mortality-based strategies for moderately transmissible strains, while the reverse is true for highly transmissible strains. However, they also cautioned that when information pertaining to viral transmission rate of a particular disease and the frequency of new introductions into the community prior to an outbreak is unreliable or not available, a mortality-based vaccination priority is recommended. This further demonstrates the importance of targeting and, moreover, the uncertainty surrounding this issue.
To further the uncertainties regarding influenza pandemic preparedness planning, it is widely believed that asymptomatic cases (i.e., individuals who had been infected but showed little or no symptoms) and asymptomatic infection of influenza (i.e., infection caused by an asymptomatic case) do indeed occur regularly (e.g., [14] [15] [16] [17] ).
Model with only asymptomatic infections, by either asymptomatic or subclinical infectives, during their infectivity period had been recently studied in [14] . In this current study, we will consider a traditional compartmental model which incorporates both pre-symptomatic and asymptomatic infections, in order to explore the role which they could play in the overall spread of disease, if any. Moreover, the age-group structure of the model, by dividing the population into seven groups of the very young, preschool children, younger and older schoolchildren, young adults, adults, and the elderly, allows us to study targeted public health policies (e.g., immunization) aimed at different age groups. Our model also allows for inclusion of immunity and other age-dependent intervention measures such as quarantine and voluntary home withdrawal (see e.g., [15, 16] ). A full model will be proposed to take into account of the above-mentioned factors that may be important in determining the best vaccine strategy.
Our model is an age-dependent compartmental model. The model flowchart is given in Fig. 1 , where the subscript i denotes the i th age group. The model variables are described as follows, with the time unit t in days:
S i (t): number of susceptible individuals of the ith age group at time t; V i (t): number of vaccinated individuals of the ith age group at time t; E i (t): number of exposed (infected) individuals of the ith age group at time t; π i : age-specific vaccine efficacy for age group i. l i (t) and  i V t ( ) : disease incidence rates for the susceptible and vaccinated individuals of age group i. See [Additional file 1] for detailed formulae.
The rest of the model parameters are listed in Tables 1-2, with the age-specific parameters given in Table 2 . The detailed description of the model is given in [Additional file 1]. Our main model assumptions are as follows:
(1) Exposed individuals are infective during the incubation period. It is commonly known (e.g., [18] ) that the pre-symptomatic (exposed) individuals cannot transmit Hsieh BMC Infectious Diseases 2010, 10:106 http://www.biomedcentral.com/1471-2334/10/106 the disease in the noninfectious latent period, during which the viral titres gradually increase to detectable and transmissible levels when they became infective for only a short period (0.25 days in [19] ) before the onset of symptoms. To avoid adding an extra compartment to account for the (short) period of infectivity after the end of the latent period and before the end of incubation period (see e.g., [20] ), we assume that the individuals are infective during the incubation period with the infectivity averaged out over the whole incubation period. This simplification is reasonable since the incubation period for influenza is typically very short, e.g., 1.48 days in [21] .
(2) Influenza vaccine is efficacious in preventing influenza infection and effective against influenza-like illness, albeit at different levels of efficacy and effectiveness for different age groups [8, 9] . Moreover, the vaccinated individuals are less infectious, once they become infected, when compared to those who had not been vaccinated.
(3) The quarantined individuals will be hospitalized directly following the onset of symptoms (see [22, 23] for modeling of quarantine for 2003 SARS outbreak).
(4) A fraction of the infectives has no symptoms or only subclinical symptoms, and is classified as asymptomatic infectives with reduced infectivity [14] . migration rate of symptomatic infectives 0 θ immigration rate of the population 0 θ 1 immigration rate of symptomatic infectives 0 q quarantine rate of unvaccinated exposed individuals 0 q V quarantine rate of vaccinated exposed individuals 0 1-home withdrawal rate of untreated symptomatic infectives 0 1-1 home withdrawal rate of all "well" individuals 0 τ reduction in infectivity of unvaccinated pre-symptomatic infectives 0.4 τ 1 reduction in infectivity of asymptomatic infectives 0.5 [15] τ 2 reduction factor in contact due to hospital isolation 0 reduction in infectivity of vaccinated infectives 0.5 [15] Table 2 Age-specific model parameters with the following sources: the age-specific values of vaccine efficacy and effectiveness [8, 9] , fraction of the symptomatic infectives [15] and [28] , and all other values from 2003-2006 Taiwan flu monitor surveillance data. 
The last four rows (in bold) were obtained by least-squared curve-fitting with data using MATLAB software.
(5) A hospitalized person is removed from isolation either by death or discharged due to recovery from illness.
(6) Homogeneous mixing within subpopulations is assumed.
(7) Negligible births and deaths (excluding disease deaths) during the course of the disease outbreak are assumed.
The basic reproduction number R 0 , the average number of infections by an infective in an immunologically naive population (see, e.g., Diekmann et al. [24] or van den Driessche and Watmough [25] ), is an important epidemiological quantity which gives indication to the potential severity of an epidemic. More precisely, the epidemic cannot be eradicated without interventions if R 0 exceeds unity. 
where R 11 , the average number of infections in group j caused by an infective from group i in an immunologically naive population, is given by
For illustration, the case n = 2 is described graphically in Fig. 2 
The two blue arrows denotes within-group infections (R 11 and R 22 ) and the black arrows denote inter-group infection cycle (R 12 R 21 ). The term R 11 R 22 subtracted in (3) accounts for the redundancy that resulted when adding R 11 and R 22 . Similar results for the basic reproduction number of a multi-group model were also obtained in [26, 27] .
Moreover, we have the following effective reproduction numbers due to interventions:
(i) The effective reproduction number with interven-
i, j = 1, 2, ... , n.
(ii) The effective reproduction number with vaccination only over a time period of immunization [0, T], R V , is: 
where
and    
Detailed derivations of the reproduction numbers are also given in [Additional file 1]. Model fit using 2005-2006 Taiwan winter seasonal influenza data and agespecific vaccination data as it was implemented during that flu season, as well as simulation studies of hypothetical scenarios, will be carried out.
With the issue of morbidity-based vs. mortality-based vaccine strategy for pandemic influenza still open to debate, the Taiwan Centers for Disease Control (TCDC) launched a new program of free flu vaccination for 1st and 2nd grades elementary school students (age 6-7) prior to the 2007-2008 winter flu season which was expanded further to include grades 1-4 in the fall of 2008. The aim of this vaccine program is hopefully to lower the seasonal influenza incidence across all age groups of the population. In anticipation of future investigation on public health impact of this program, we carry out model simulations by dividing the Taiwan population into 7 age groups (see Table 2 ), and by making use of the weekly Taiwan influenza vaccination data for young children of age 2 or less (age group 1, with free There were no other nonpharmaceutical intervention measures during this winter flu season. That is, all parameters pertaining to quarantine and home withdrawal in the model are set to be 0 in Table 1 . Moreover, for the sake of simplicity, we assume no noticeable level of migration and no waning of immunity during the flu season. The rest of the parameter values used are given in Tables 1-2 . We also assume a conservative 20% preepidemic immunity in our simulation based on a recent sero-epidemiological survey conducted during 2005-2006 winter flu season in Taiwan [28] .
For the contact rates between different age groups, we make use of the age-specific contact matrix obtained by Wallinga et al. [29] for Utrecht, the Netherlands, 1986. We adjust for the discrepancy in the population age distributions of Taiwan in 2005 and the Netherlands in 1986, by considering the ratios of demographic age structures of Netherlands in 1987 (Appendix Table 1 in [29] ) and of Taiwan in 2005 [Additional file 1: Table  A1 ]. The resulting contact matrix, of the average daily number of contacts for each individual in a certain age group with individuals in another age group, is given in [Additional file 1: Table A2 ].
The age-dependent hospitalization rates and per contact transmission probability in the last four rows (in bold) in Table 2 were obtained by least-squared curve-fitting with the 2004-2005 Taiwan winter P&I (Pneumonia and Influenza) mortality data from October 9, 2004 to March 5, 2005 for age groups 6 (age 22-64) and 7 (>64) using MATLAB software. ν 7 (τ) and ν 1 (τ) are piecewise linear (by week) approximations of the respective weekly vaccination data for elderly (>64) and young children in Taiwan during this time period. To simply our data fitting, we first fitted the data by assuming the per contact transmission probability of the infectees in i th group are averaged, i.e., b ij ≡ b i (see next to the last row in bold in Table 2 ).
The result of data fit is given in Fig. 3 . The reason for using only groups 6 and 7 for data fit is that the P&I mortality data during that winter season consists mostly of older people. More precisely, 5038 (88.5%) of the 5694 individuals who died of P&I are of age 65 or older (age group 7), 607 (10.7%) are between age 22-64 (age group 6), followed by 17 between age 0-2 (age group 1) and 14 between age 15-21 (age group 5). Each of other age groups has only a handful of cases. Therefore, to avoid large errors due to fitting data with small data size, we only use the data from the two largest groups for our model fit.
We also fitted the data by assuming the per contact transmission probability of the infectors in i th group are averaged, i.e., b ij ≡ b j (see the last row in bold in Table  2 and Fig. 4 ). We will compare these results to ascertain the difference in age groups on per contact transmission probabilities to and from particular age groups. To give more insight to the data used and the goodness of fit, we also provide the daily observed and predicated mortality corresponding to the case b ij ≡ b j in [Additional file 1: Figs. S1-S2].
By fitting only the hospitalization rates and the per contact transmission probabilities to the 2004-2005 Taiwan winter season P&I mortality for age groups 6 (age 22-64) and 7 (>64), we are able to obtain satisfactory model fit (see Fig. 3 ), although we note that fitting P&I deaths might conceivably lead to an overestimate of hospitalizations rates. However, simulation studies showed that the model fit is less sensitive to the hospitalization rates than to the transmission probabilities, and intuitively, most sensitive to changes in these rates for the elderly group.
It is interesting to note that comparatively significant errors in both theoretical curves occur in late January roughly after day 110. We note that day 105 was January 22, 2005 , when all schools in Taiwan closed for the winter vacation which lasted until after the traditional lunar New Year holiday, in mid-February of that year. The school closure, and subsequent shutdown of all non-essential venues during the week-long New Year holiday, surely had a significant impact on the contact rates which was not reflected in our constant contact matrix that implicitly assumes that inter-age group contact patterns remain the same during the whole season. More precisely, the P&I deaths for the elderly exhibits a slight increase for about 3 weeks after the closing of schools (shortly after the New Year) when compared to the model predicted values, while the P&I deaths for the adults of age 22-64 dropped substantially below the theoretical curve. These results indicate that the school closure and the subsequent New Year holiday led to more frequent contacts by the elderly in the households with family members who spent more time at home during the holidays At the same time, there were less contacts for the adults at the workplace (especially for those working in the educational facilities who had longer holidays) during this time period. Since most individuals in the elder group (and the very young) are not directly affected by the school closings and the holidays, one may speculate that the difference in the actual mortality and the theoretical mortality of the elderly, as averaged over the whole time period, is due at least in parts to the impact of interaction between the elderly and younger children with the school-age children and adults, when the activity levels (in terms of frequency as well as whom to make contact with) of the latter groups were significantly changed by the holidays. Furthermore, the elderly had a higher per contact transmission probability (Table 2 ) but lower contact frequency [Additional file 1: Table A2 ]. This study showed that, with the combination of these two factors, morbidity-based vaccination strategy still could be effective for the prevention of elderly mortality. Although a recent study to quantify the effect of school closures during the 2008 winter influenza season in Hong Kong did not find the school closures as having a substantial effect on the community transmission [30] , our results indicate the need for further studies on this topic. We also note that, normally, one would like to use averaged excess winter season P&I mortality data over other seasons for the data fitting of seasonal influenza. However, Taiwan often experiences summer influenza epidemics which would offset any attempt to obtain a reasonable "excess" P&I mortality for the winter season.
Using Equations (2-3) we obtain R 0 = 1.0001, just above unity. However, we note that it is more appropriately the reproduction number R of the winter flu epidemic, given the effect of pre-epidemic immunity that must exist. Chowell et al. [31] used several weekly seasonal flu mortality data, derived from P&I excess deaths and influenza-specific deaths from US, France, and Australia during 1972-1997 (1972-2002 for US), to estimate the (mean) reproduction number R p over 3 decades of seasonal flu. They found that the mean of R p to be around 1.3, with year-to-year variability of 0.9-2.1. Our estimate is lower, but within their range.
It is also interesting to note that for our set of parameter values used in Figs. 4, 5, the effective reproduction number with vaccine only (see Equations 5-6) has almost the same value as R 0 , to the fourth decimal digit. One explanation for this apparent lack of impact of vaccination during the 2004-2005 flu season, as indicated by the value of R V , is that the vaccination data we used is only for groups 1 (age 0-2) and 7 (age >64). In the formula for R V , or more precisely for R ij V 0 in Equation (7), c ij 2 is the daily contact frequency between groups i and j. The contact frequencies of groups 1 and 7 [Additional file 1: Table A2 ] are substantially smaller than the contact frequencies of the other groups, with the exception of within-group contacts and contact between groups 1 and 2 (age 3-5) in daycare facilities. Therefore, the relative impact of targeting these two groups for vaccination was weakened by their relative inactivity, thus giving further evidence of the lack of impact of vaccinating the very young and the very old on the overall transmissibility of the disease in the community. However, simulation with the same parameter values as in Fig. 3 except assuming no vaccination in the young children and elderly age groups, i.e., υ 1 (t) = υ 7 (t) = 0, and everything else the same produces Fig. 5 , where the result indicates that the impact of no vaccination (red curves) would still be significant, resulting in nearly 4-fold increase in mortality of elderly and 2-fold increase in mortality of adults. Therefore, the impact of vaccination on the disease incidence sometimes might not be fully revealed in the change (or the lack thereof) in the effective reproduction number with intervention but could still be substantial, since it is a simple mathematical property that distinctly different (next generation) matrices may have similar eigenvalues.
Furthermore, in our simulations we had assumed vaccine efficacy (proportion of infection prevented) of 40% for elderly and 70% for young children, to be in line with current literature [8, 9] . However, efficacy depends very much on matching of the circulating strains with vaccine strains each year, where mismatch often causes low efficacy and might have affected our resulting data fit. We also assumed vaccine effectiveness (proportion of reduction in symptomatic cases) to be 60% for elderly and 40% for young children (also see [8, 9] ).
The per contact transmission probability for the susceptible elderly (>64) infectees (see next to the last row in Table 2 ) is estimated to be 0.155, more than threefold of any other age group (with the young children of 0-2 being the next highest), which may be due to the common need for very close contact while the elderly (or younger children of age <3) are being cared for, typically by individuals from adults of ages 22-64 in age group 6, even though the frequency of contact might be less than that of with other age groups. This further highlights the high vulnerability of the elderly (or the younger children) to exposure from other age groups, and demonstrates the need for morbidity-based strategy to prevent the elderly (or younger children) influenza Figure 5 Simulation with the same model parameters as in Fig. 3 , except υ 1 (t) = υ 7 (t) = 0. The black dots are real data, the red curves are model fits in Fig. 3 , and the blue curves denote simulation without vaccination. mortality. On the other hand, the estimated per contact transmission probabilities for the younger (<3) and the elderly (>64) infectors are also both higher than those of the other groups (see the last row in Table 2 ), but not significantly so except when compared with those between ages 8-14. The higher transmission probabilities of young children and elderly infectives could reflect, again, the fact that contacts with these individuals are usually of a more intimate nature, although these probabilities are not as drastically different as their vulnerability to be infected. The less likelihood of schoolchildren of 8-14 to infect others might also be attributable to signs of their less intimate contacts with others as they grow into adolescence.
To further explore this situation, we note that from Equation (6), the partial derivatives of R ij v with respect to ν i π i are: (2) and (7). We also know that [0, 1] and ν i π i for all i, j. Subsequently,
effective reproduction number with vaccination only, R V , does not necessarily decrease as the effective vaccination rate ν i π i increases. In other words, vaccination is not always beneficial in reducing incidence and the design of an effective vaccination program in multigroup model is highly nontrivial, unlike in simple epidemic models where there is a simple formula for the critical vaccination coverage level necessary for eradication (p c ) [20] . In fact, it has been shown mathematically by Hadeler and Castillo-Chavez [32] , using a model with a core group, that partially effective vaccination program may actually increase the total number of cases. Explicit quantification of an optimal vaccination policy ( [33] ) in a multi-group population scenario remains to be a challenge for mathematical modelers and is beyond the scope of this work. Moreover, the model fit was carried out with vaccination (of the elderly and small children of age 2 or less) as the only intervention since vaccination was the only intervention that was implemented during the flu seasonal for which the fitted P&I data was collected. Further simulations with quarantine and home withdrawal also can be easily carried out.
A related issue is that of pre-epidemic immunity. Further sensitivity analysis using pre-immunity in the range of 10%-30% has shown that the results of curve-fittings are not sensitive to small changes in the pre-epidemic immunity. One would expect that pre-epidemic immunity does impact the outbreak, but nonetheless it is not reflected, at least not in the reproduction numbers. Pre-symptomatic infection and asymptomatic infection by asymptomatic infectives were incorporated into the proposed model. To gauge the roles these two features of influenza might play in a seasonal influenza epidemic, we performed the following simulations. Again using Fig. 3 as a benchmark, we first assume no pre-symptomatic infection (τ = 0) with all other parameter values unchanged (Fig. 6 ). Next we assume no asymptomatic infection by asymptomatic infectives (a i = a i V = 0) (Fig. 7) . The results indicate that pre-symptomatic infection seemed to have little effect on the model fits and the fitted parameters; while asymptomatic infection by asymptomatic infectives has a more pronounced impact on the model fit for the elderly, perhaps indicating the comparatively larger role asymptomatic infection plays in disease transmission.
Finally, as the WHO Strategic Advisory Group of Experts (SAGE) recently made its recommendations for priorities in vaccination for the H1N1 pandemic in terms of the social groups (e.g., healthcare workers those with chronic medical conditions), health groups (pregnant women), and age groups [34] , the model also can be used to divide population into social/health groups. For example, to study vaccine policy for the elderly, we could divide the elderly into those living in households and those living in old age homes, since they mix differently in these two distinct settings. The model is also useful for simulations of the cost-effectiveness of vaccine and other intervention measures, such as prophylaxis treatment, perhaps in future work.  Extracorporeal life support for management of refractory cardiac or respiratory failure: initial experience in a tertiary centre INTRODUCTION: Extracorporeal Life Support (ECLS) and extracorporeal membrane oxygenation (ECMO) have been indicated as treatment for acute respiratory and/or cardiac failure. Here we describe our first year experience of in-hospital ECLS activity, the operative algorithm and the protocol for centralization of adult patients from district hospitals. METHODS: At a tertiary referral trauma center (Careggi Teaching Hospital, Florence, Italy), an ECLS program was developed from 2008 by the Emergency Department and Heart and Vessel Department ICUs. The ECLS team consists of an intensivist, a cardiac surgeon, a cardiologist and a perfusionist, all trained in ECLS technique. ECMO support was applied in case of severe acute respiratory distress syndrome (ARDS) not responsive to conventional treatments. The use of veno-arterial (V-A) ECLS for cardiac support was reserved for cases of cardiac shock refractory to standard treatment and cardiac arrests not responding to conventional resuscitation. RESULTS: A total of 21 patients were treated with ECLS during the first year of activity. Among them, 13 received ECMO for ARDS (5 H1N1-virus related), with a 62% survival. In one case of post-traumatic ARDS, V-A ECLS support permitted multiple organ donation after cerebral death was confirmed. Patients treated with V-A ECLS due to cardiogenic shock (N = 4) had a survival rate of 50%. No patients on V-A ECLS support after cardiac arrest survived (N = 4). CONCLUSIONS: In our centre, an ECLS Service was instituted over a relatively limited period of time. A strict collaboration between different specialists can be regarded as a key feature to efficiently implement the process. Extracorporeal circulation support techniques have been proposed either for treatment of cardiac and/or pulmonary failure refractory to conventional treatments in adult patients. The first device, which assured blood extracorporeal oxygenation and perfusion of isolated organs, was developed by von Frey and Gruber in 1885 [1] . The first heart-lung machine was projected by Gibbon in 1937 in order to allow open-heart surgical operations [2] . Over the years, extracorporeal circulation circuit has been improved and the technique optimized, and it is now available for clinical practice. From a general point of view, two methods of support are outlined: venovenous extracorporeal oxygenation, commonly known as ECMO, Extracorporeal Membrane Oxygenation, for respiratory function substitution and extracorporeal life support technique (ECLS) with a veno-arterial circulation for both oxygenation and hemodynamic assistance. The major indications for ECMO, in adult patients, are severe acute respiratory distress syndrome (ARDS) refractory to conventional treatments [3, 4] , and, in selected cases, post-traumatic respiratory failure, severe asthma [5, 6] , and chronic lung disease waiting for lung transplantation [7, 8] . The indications for ECLS and cardiac support are cardiac failure due to any cause, and cardiac arrest not responsive to Advanced Life Support manoeuvres. In the first case, the in-hospital mortality rate is still high (between 33% and 38%) and ECLS represents a rescue-therapy useful for refractory patients [9] . In case of in-hospital cardiac arrest, when ECLS was used after ten minutes of unsuccessful cardiopulmonary resuscitation, an increase in survival rate at ICU discharge, at 30-day and at 1-year survival was reported [10] . At a tertiary referral trauma center (Careggi Teaching Hospital, Florence, Italy) an ECLS program was developed beginning April 2008 by the Intensive Care Unit of Emergency Department in association with the Intensive Cardiac Coronary Unit of Heart and Vessel Department.
Here we describe our experience in implementing a multidisciplinary ECLS team for cardiac and respiratory failure. In addition to reporting our clinical experience, we present the algorithm for ECLS activation for in-hospital cardiac arrest and the experience of a national referral center for treatment of H1N1 influenza related respiratory failure.
The ECLS team consists of an intensivist, a cardiac surgeon, a cardiologist and a perfusionist, all trained on ECLS technique and management. According to our activation protocol, ECLS team can be summoned within one hour, with 24 hour coverage. In most cases, the intensivist primes the process on the basis of clinical and radiological findings and activates the full ECLS team. The cardiologist's main task is to evaluate cardiac function in the pre-ECLS phase and guides the correct positioning of ECLS cannulas by transesophageal ultrasonography. Furthermore, the cardiologist is directly involved in selecting patients with cardiac failure suitable for ECLS treatment. The cardiac surgeon, in addition to actively participating to the clinical decision making process, is responsible for selecting and inserting the cannulas and starting the extracorporeal circulation, with the assistance of the perfusionist. In case of an ECLS run, irrespectively of the unit where the patient was admitted (General or Cardiac ICU), all the professionals of the team were available for consultation and performed at least one daily evaluation. This study, supported by institutional funds only, followed the principles of the Helsinki declaration and was approved by the Internal Review Board. Informed consent for data publication was obtained.
Veno-venous ECLS treatment (ECMO) was applied in case of severe ARDS not responsive to conventional treatments, but potentially reversible. Conditions of severe hypoxia or hypercapnia, where the limits of a pro-tective ventilation strategy could not be maintained (tidal volume less than 6 mL/Kg of predicted body weight and plateau pressure less than 30 cmH 2 O), were the indications for starting extracorporeal circulation [11] . The Careggi Teaching Hospital had started a collaboration with the ICUs of 12 district hospitals in Tuscany in a pilot project for centralization of acute lung injury/ARDS patients who require (or may require) ECLS treatment. In 2008 and spring 2009, preliminary meetings were organized to inform the peripheral hospitals' ICU staff and Administrations about the availability of the new ECLS program. During the H1N1 influenza A pandemic, the knowledge of ECMO treatment rapidly spread among the medical community and the Regional Ministry of Health issued indications to transfer all patients affected by severe respiratory failure related to influenza to Careggi Hospital. In Appendix 1 is reported the set of parameters that were adopted to quickly detect patients suitable for extracorporeal treatment in the peripheral hospitals. Patients deemed suitable for ECMO treatment were evaluated on site by the ECMO team. Depending on clinical condition, the transfer was performed on conventional ventilation or, alternatively, ECLS treatment was initiated in the peripheral hospital and maintained during transportation [12] . We preferentially adopted a high flow technique (5-6 litres per minute of blood flow), to maximize the opportunity of providing protective ventilation, aiming to achieve a plateau pressure below 28 cm H 2 O and PEEP 2 cmH 2 O above the lower inflection point of the quasistatic pressure volume curve, regardless the delivered tidal volume (in any case less than 6 ml/kg). Controlled respiratory frequency was reduced to 4-10/min to maintain normocapnia. Inspired oxygen fraction was reduced to 0.5 or lower, whenever possible. A recruitment manoeuvre was performed at least once a day, and ventilation with an intermittent high pressure breath ("sigh") was adopted to improve lung aeration [13] . During ECMO, nitric oxide administration [14] , vasoactive support, and prone positioning were maintained or initiated according to clinical conditions.
The use of ECLS for cardiac support was reserved for cases of cardiac shock refractory to standard treatments and cardiac arrests not responding to conventional resuscitation. According to our internal protocol, ECLS was adopted also as a bridge to implantation of Left Ventricular Assist Device or to heart transplantation [15] . ECLS was employed in cases of in-hospital cardiac arrest when the patient was considered to have a good chance of recovery both for clinical conditions and for the timing of resuscitation. An age limit of seventy years, severe irreversible brain damage, terminal malignancy, pre-signed "do not attempt resuscitation" orders and contraindications to prolonged systemic heparin infusion were the only strict exclusion criteria taken into account. In case of cardiac arrest, hypothermia was rapidly initiated and was maintained for 24 hours at a temperature between 32-34°C [16] . Veno-arterial (V-A) ECLS treatment was considered contraindicated, when a severe aortic incompetence, aortic dissection or ventricular thrombosis was detected by echocardiography.
The ECLS circuit consisted of a Rotaflow Maquet Centrifugal Pump (Maquet, Rastatt, Germany) and a hollow fiber membrane oxygenator (Quadrox-D Oxygenator, Maquet, Rastatt, Germany), connected with biocoated tubes. In the V-A circuit, blood was drained through femoral vein and reinfused into aorta through femoral artery. For V-V ECLS two types of cannulas were used. At the beginning, Raumedic cannulas ranging from 21 to 28 french (Raumedic AG, Germany) were employed with femoral and jugular vein cannulation. Since July 2009, Avalon Elite™ Bi-Caval Dual Lumen Catheters have become available. These specially designed dual lumen cannulas, inserted in the right internal jugular vein, permit both drainage and reinfusion of blood. In V-A ECLS, the distal perfusion of the limb could be jeopardized by the relatively large bore inflow cannula, inserted in the femoral artery at the groin: to prevent leg ischemia, we usually inserted a small shunt cannula (14 french) in the femoral artery, distally to the ECLS cannula. Heparin therapy was titrated by bedside measurement of activated partial thromboplastin time (aPTT) with Hemochron (Hemochron Jr. Sign. plus, ITC Europe, Milan, IT) every two hours. Numerical data were summarised as median and interquartile range.
A total of 21 patients were treated with ECLS during the first one year of activity (April 2008 -December 2009). Among them, 13 were treated with ECMO for respiratory failure (Table 1) , and 8 were treated with V-A ECLS due to cardiac arrest ( Table 2 ) and cardiogenic shock ( Table  3 ). The most frequent complication observed was local bleeding from the insertion points of the cannulae, central line access site and tracheostomy (36%). In one case, oxygenator failure occurred due to clots formation; in this occasion a rapid increase of D-Dimers was observed, followed by a worsening of oxygenation and decarboxylation performance of the artificial lung. Circuit change was promptly carried out with no further complications. In one case of V-A ECLS, major bleeding occurred at site of cannulae insertion several days after successful wean-ing, requiring multiple transfusions. At surgical inspection a femoral artery wall lesion was found and required prosthetic repair. Five patients received renal replacement therapy (continuous veno-venous hemofiltration, CVVH). The CVVH was connected in-line to the extracorporeal circuit with the withdrawal line before oxygenator and return line after the oxygenator. Renal function recovered in all cases, and both ECLS and CVVH run was uneventful on this configuration. During extracorporeal support, invasive procedures were carried out without any immediate complications. Among these, four bedside percutaneous tracheotomies (Ciaglia technique) were performed, and two narrow bore pleural catheters were inserted under ultrasound guidance for massive pleural effusions. Autopsy was performed in all non surviving patients and no lesion of vessels due to the presence of cannulae was observed.
A total of 13 patients were treated with ECMO for ARDS: six patients were affected by bacterial pneumonia, five patients had H1N1-related ARDS (two with Legionella Pneumophila superinfection), and 2 patients presented trauma-related respiratory failure. Data of each patient are represented in Table 1 . Median age was 59 years (IQR 44-65), with a prevalence of male sex (85%). Median ICU length of stay was 17 days (IQR [13] [14] [15] [16] [17] [18] [19] [20] . Eight out of 13 patients were successfully weaned from ECMO and discharged from ICU (overall survival rate of 62%). All H1N1 patients were discharged from ICU and from hospital. The median duration of ECMO was 235 hours (IQR 151-269), with a difference between survivors (221 hours) and non survivors (257 hours). We considered time from verification of ECMO criteria to extracorporeal support start as an efficiency parameter ("time to ECMO"), and it was 6 hours (IQR 4-9). From October 2009, when our ECMO Service became the referral centre of Central Italy for H1N1-induced ARDS, extracorporeal support was initiated in the peripheral hospital in 3 cases. Inter-hospital transport was safely performed on extracorporeal support and all patients were discharged alive from ICU. One young patient (19 years) died due to severe traumatic brain injury. In this patient, ECMO was maintained in the first 12 hours without systemic heparin infusion and no complications occurred during extracorporeal treatment. After cerebral death confirmation, multiple organ donation was accomplished. One patient (a 64 year-old woman) died due to subarachnoidal hemorrhage, although coagulation parameters were normal. 
Four victims of intra-hospital cardiac arrest received V-A ECLS for cardiac support (Table 2) . Patients were 41 years old (median, IQR 21-61; male sex 75%). The median duration of advanced cardiac life support manoeuvres before ECLS start was 58 minutes (IQR 53-68), and the median duration of ECLS was 16 hours (5-30). In two patients, the ECLS support started in the Emergency Room. All four patients died during their ICU stay (one patient after ECLS withdrawal). Data of the four patients treated for cardiogenic shock are represented in Table 3 . Median age was 49 years (IQR 38-58, male sex 50%). Among them, 2 patients survived and were discharged from ICU. In these patients, median duration of ECLS was 96 hours (IQR 60-137). Intraaortic balloon pump was necessary in all four patients. Survival rate was 50%.
From the experience here reported, we can state that, with a close cooperation between different specialists (intensivist, cardiologist, cardiac surgeon, nurse, perfusionist), an ECLS Service can be started over a relatively limited period of time, achieving a high level of efficiency. Our model of ECLS team has allowed us to start extracorporeal support in different hospital scenarios, such as ICU and Emergency Room. This feature of flexibility and adaptability of our ECMO system has made it particularly beneficial during the Influenza A pandemic, making this resource available also in peripheral hospitals.
The management of a patient on ECLS is still challenging in terms of utilization of resources and commitment of health personnel. Beyond the insertion procedure, a multidisciplinary team can better accomplish the tasks of daily management of the patient, as an intensivist, a cardiac surgeon and a perfusionist should repeatedly evaluate the circuit and the patient to guarantee a safe and uneventful treatment. Furthermore, every ECLS patient needs a dedicated nurse. With the assistance of these dedicated professionals, also in-hospital transportation can be safely carried out (i.e. to radiological suite). In our population 63% of patients received a CT scan during ECLS treatment, and no transport-related complications occurred. The most common complication was local bleeding, usually simple to manage. In this regard, the use of Bioline surface-heparinized circuits allowed a limited dose of heparin, and may have reduced the incidence of complications such as coagulation, complement activation, thrombus formation and the need for transfusions [17] [18] [19] .
The survival rate of 62% of our patients treated with ECMO for respiratory failure is comparable to other published studies. In 2004, Hemmila and co-workers retrospectively reviewed 255 patients with ARDS treated with ECMO between 1989 and 2004, showing a 67% of patients successfully weaned from ECMO and a hospital discharge of 52% [20] . More recently, the CESAR (Conventional ventilation or ECMO for Severe Adult Respiratory Failure) trial has shown an increase of survival rate, without severe disability, 6 months after randomization in patients treated with ECMO in comparison to conventional ventilation (63% vs 47%) [4] . From the first phase of implementation, our service was conceived to provide extracorporeal support even in peripheral institutions, therefore a dedicated ambulance was specifically prepared and all equipment arranged for transportation. In our opinion, this is a key feature for an effective ECMO service as inter-hospital transportation of patients with severe respiratory failure can be challenging due to the fact that limited possibilities of intervention are available and clinical deterioration may occur [21] . Therefore, several centres recommend the start of extracorporeal assistance before transfer [22, 12] . In our out-of-hospital ECMO experience, one patient was safety transferred by ambulance from a distance of 400 Km. We report quite a short time to establish V-A ECLS in case of in-hospital cardiac arrest (58 min). Furthermore, there is a trend towards a progressive reduction of this interval over time. Despite this remarkable performance of our ECLS system in terms of speed of response, no patient receiving extracorporeal support for cardiac arrest survived. In a large series of patients on extracorporeal support for in hospital cardiac arrest, Jaski and coworkers reported a long term survival rate of 23% in witnessed events and no survival in non-witnessed arrest. At multivariate analysis cardiac arrest in the critical care unit was found to be the only independent variable predictive of outcome [23] . In another series of 40 in-hospital cardiac arrest victims, time before ECLS was 105 minutes, and 20% survival rate was reported [24] . In our experience, the number of cardiac arrest patients with ECLS is so limited that comparison to published data is not feasible. Nevertheless, the reason for not responding to V-A ECLS treatment in our cases might be possibly related to the severity of previous clinical condition (2 traumas, 1 septic shock) and to the underling organ dysfunction.
ECMO and V-A ECLS might be considered a therapeutic option in patients with severe ARDS and/or with cardiac failure or cardiac arrest. In our experience, a well-timed start of ECMO in case of ARDS, prevents the progression of ventilator-induced lung injury and increases the chances of lung recovery. Also in case of cardiogenic shock, an extracorporeal technique seems a viable option and increases the possibility of early cardiac recovery avoiding neurological damages and multi-organ failure.
To guarantee a safe treatment, the involvement of several properly trained physicians and nurses seems advisable.
• An ECLS Service can be effectively organized in a Center were the needed competencies are available (intensivist, cardiologist, cardiac surgeon).
• When physicians and nurses are skilled in the technique, the Service can provide a safe transfer of critically ill patients from remote hospitals.
• ECMO should be considered in the initial phase of ARDS, when failure to ventilation strategy occurs.
• The resource of ECMO has resulted to be particularly important in the event of cases of severe respiratory failure, as in the last pandemic of Influenza A. How necessary is a fast testkit for mitigation of pandemic flu? It is widely feared that a novel, highly pathogenic, human transmissible influenza virus may evolve that could cause the next global pandemic. Mitigating the spread of such an influenza pandemic would require not only the timely administration of antiviral drugs to those infected, but also the implementation of suitable intervention policies for stunting the spread of the virus. Towards this end, mathematical modelling and simulation studies are crucial as they allow us to evaluate the predicted effectiveness of the various intervention policies before enforcing them. Diagnosis plays a vital role in the overall pandemic management framework by detecting and distinguishing the pathogenic strain from the less threatening seasonal strains and other influenza-like illnesses. This allows treatment and intervention to be deployed effectively, given limited antiviral supplies and other resources. However, the time required to design a fast and accurate testkit for novel strains may limit the role of diagnosis. Herein, we aim to investigate the cost and effectiveness of different diagnostic methods using a stochastic agent-based city-scale model, and then address the issue of whether conventional testing approaches, when used with appropriate intervention policies, can be as effective as fast testkits in containing a pandemic outbreak. We found that for mitigation purposes, fast and accurate testkits are not necessary as long as sufficient medication is given, and are generally recommended only when used with extensive contact tracing and prophylaxis. Additionally, in the event of insufficient medication and fast testkits, the use of slower, conventional testkits together with proper isolation policies while waiting for the diagnostic results can be an equally effective substitute. A global influenza pandemic has the potential to cause extensive morbidity and mortality, as well as severe social and economic disruptions. Past and present pandemics such as the Asian flu (1957, H2N2) , the Hong Kong flu (1968, H3N2) and the recent swine flu (2009, H1N1) outbreaks have demonstrated the extent to which the virus can be transmitted swiftly on a global scale (Cox & Subbarao 2000; Fraser et al. 2009 ). In the case of the 2009 H1N1 flu pandemic, although the H1N1 strain is not as lethal as originally thought, there are fears that a deadlier strain may emerge, sparking off the next wave of health crisis. This is in addition to the existing threat of the highly lethal H5N1 avian influenza virus (Hatta et al. 2001) mutating into a human-transmissible variant (Hatta & Kawaoka 2002) .
It is recognized that a combination of early use of antiviral medicine and social distancing measures can help contain a pandemic, or at least slow its spread until proper vaccines can be developed (Nuñ o et al. 2007 ). However, due to limited medicine stockpiles and resources, policies must be made such that they optimize the drug usage while minimizing the cost and economic impact of the intervention strategies. Since it is not possible to experimentally assess the overall effectiveness of the various pandemic control strategies, mathematical models play a major role as they allow us to address such issues through simulations. Notable past works include examining how to contain a novel influenza strain at its source (Ferguson et al. 2005; Longini et al. 2005) , and failing that, how we can limit its impact within the population (Ferguson et al. 2006; Germann et al. 2006; Wu et al. 2006; Nuñ o et al. 2007) .
Diagnosis is an important component in the overall pandemic management framework. With proper diagnosis, potential flu carriers can be identified before they become symptomatic. Non-pharmaceutical measures such as quarantine and contact tracing can then be activated, thus maximizing the probability of containment (Ferguson et al. 2005; Longini et al. 2005) .
Even in the event of an ongoing outbreak, it was found that early detection and intervention can help mitigate the spread of influenza. For instance, studies have revealed that during the 1918 flu pandemic, cities in the United States that enforced early interventions had significantly reduced mortality rate (Hatchett et al. 2007) . Furthermore, during a pandemic, various influenza-like illnesses (ILIs), as well as non-pandemic seasonal influenza cases will continue to present themselves within the population. The ability to differentiate between the pathogenic strains and the less harmful ones can help channel antiviral drugs to where they are needed. Such targeted treatment is invaluable in the face of limited drug supplies, especially in developing countries.
Current influenza diagnostic tests, especially when the strains are evolving constantly, vary differently in terms of efficiency, specificity and sensitivity (for a list of diagnostic tests, see Centers for Disease Control and Prevention 2009). These factors will impact the intervention policies that should be made during an outbreak. It has been argued that a slow, low throughput laboratorybased diagnostic test, such as immunofluorescence DFA antibody staining and RT-PCR (real-time polymerase chain reaction), may not be able to effectively assist pandemic mitigation (Wu et al. 2006) . Ideally, a rapid PCRbased diagnostic testkit should be able to detect the relevant virus in less than 30 minutes, requiring nothing more than a patient sample, and a portable device to perform extraction and drive the PCR cycle. This permits testing to be done quickly, outside a laboratory setting, and allows diagnosis to be performed more easily in rural areas, where novel influenza is likely to emerge. However, such testkits require time to develop for novel strains, and may not be available during the initial phases of a pandemic, during which one may have to fall back on conventional laboratory-based approaches.
The constraints imposed by the availability of fast and accurate diagnostic methods are factors that should not be overlooked in flu intervention policy making. However, previous works on pandemic modelling either treat ILIs as false positives that consume resources, or assume perfect diagnosis without accounting for the costs associated with the use of the different diagnostic techniques. In this work, we aim to investigate the cost and effectiveness of various intervention policies when implemented with different diagnostic methods. Specifically, we want to address the following questions-Is a fast and accurate testkit necessary in mitigating the spread of an ongoing pandemic? In addition, are there any other dominating factors that should be given due consideration in forming pandemic mitigation policies? To do so, we developed a stochastic agent-based epidemic framework and implemented it on a small city-scale model, typified by developed countries such as Singapore and the United States. The impact of testkits when used in the context of different intervention policies is then assessed by comparing the severity of the outbreak with an aggregate cost function which encompasses the various costs and resources involved.
For our work, we developed a stochastic agent-based pandemic model on a small-scale city. We extended the typical transmission model (Wu et al. 2006 ) by adding a pre-symptomatic phase, thus resulting in the SEPIR (susceptible-exposed -pre-symptomaticinfectious -recovered) model. In addition, we further refined the infectious phase into one of the three possible categories-asymptomatic, symptomatic and critical (for details, refer to figure 1a). To capture the constraints imposed by societal norms, such as the different population response patterns during different times of the day, we set the granularity of the time scale to be in terms of hours, rather than days.
We populated the city with 10 000 people, and simulation proceeds in discrete time steps of 1 h. Within the city, we identified the following location types-(i) household, (ii) workplace, (iii) school, (iv) mall, (v) hospital, and (vi) public transport. Whenever the context is clear, both workplaces and schools are simply referred to as workplaces, while malls, hospitals and public transport are termed communities. For workplaces, schools and malls, they are further divided The state transition diagram depicting the SEPIR (susceptibleexposed-pre-symptomatic-infectious -recovered) transmission model for pandemic influenza. The infectious phase is further sub-divided into asymptomatic (I A ), mild (I M ) and critical (I C ) cases. (b) The mean relative infectiousness of a person throughout the various phases.
into sub-locations. Workplaces have smaller work groups, schools are divided into classes, and malls are made up of several shops. At any point in time, each individual in the city will be in any one of the locations or sub-locations. Note that we classified hospital as a separate location entity, as it will be the main facility where diagnosis and treatment can be issued. The number of people in each location was obtained by fitting the model against demographic data for Singapore (Statistics Singapore 2009).
Each location in the model, with the exception of public transport, has an x -y coordinate assigned to it on a 10 Â 10 square grid. Inhabitants of the city move from one location to the next according to their individual schedules. Commuting between grids is done by either private (i.e. cars) or public transport. However, between locations on the same grid coordinates, it is assumed that movement does not require any forms of transport. Public transport is considered as a location.
Instead of x-y coordinates, it is assigned to a route it serves. The other locations in the city are grouped into four districts (each occupying a quadrant in the city grid). Travelling within and between the districts is done via a specific transport route, giving a total of 16 routes. All individuals travelling along the same route are considered to be in the same location, thus allowing the spread of pandemic flu.
In the city, we assumed that there are two hospitals, with one being designated as a flu treatment and isolation centre during a pandemic outbreak. Patients with flu-like symptoms will be directed to that hospital for diagnosis and treatment. The other hospital will handle other non-influenza-like illnesses (ONILIs) which include cases such as bacterial infections and accidental injuries. Normally, without a designated flu hospital, these ONILI cases will present themselves as susceptible targets for infection within the hospital. However, this scenario is not considered in depth within our model.
As mentioned in the previous section, ILI is one of the sources of drug wastage. It represents other types of illnesses as well as non-pandemic seasonal influenza, and is symptomatically indistinguishable from the pandemic strain. For these patients, they will be directed to the designated flu centre for diagnosis and treatment. Without proper diagnoses, these patients will consume resources unnecessarily. Since ILIs are non-specific illnesses, we assumed the number of new ILI cases to surface at a constant rate, independent from the spread of pandemic influenza. Variations due to seasonal effects are currently beyond the scope of this work. Based on published data from the Ministry of Health, Singapore (Ministry of Health Singapore 2006b), we estimated the rate to be three new infections per day, each lasting for 72 h. In the model, these individuals are randomly selected from the ILI-susceptible population; those suffering from pandemic flu and ONILIs can still be inflicted by ILIs. For ONILIs, we estimated the daily new occurrences to be 45 people (Ministry of Health Singapore 2006a), and they are randomly selected from the ONILI-susceptible population. Again, each ONILI case is assumed to last for 72 h. For details on how the values are derived, refer to the electronic supplementary material.
To model the spread of novel pandemic virus, parameters governing its infection dynamics are required. Since such parameters are not readily available for novel variants, we referred to the natural history of typical influenza viruses, adapting the values from previous works whenever possible. The basic function describing the dynamics of transmission is the instantaneous force of infection experienced by the ith individual, and it is summarized by the following equation:
ð2:1Þ
where X and K are the sets of people and locations in the city, respectively; b k is the transmission coefficient associated with a particular location type. The transmission coefficients are estimated such that without any forms of intervention, the proportions of transmissions within the household (P H ), workplace (P W ) and community (P C ) are approximately 30, 37 and 33 per cent, respectively (Ferguson et al. 2006) . This proportion is location-dependent, and the values we followed are reported to be estimated based on data from the United States. Using our transmission coefficients (b h ¼ 1.89 Â 10 21 h 21 , b w ¼ 3.2 Â 10 21 h 21 and b c ¼ 6.51 Â 10 23 h 21 ) the transmission proportions in the model are computed to be P H ¼ 30.7 per cent, P W ¼ 35.6 per cent and P C ¼ 33.7 per cent. G ijk is a proximity function for two individuals x i , x j and location k at a particular time instance. In the model, the function will give 3 if x i and x j are in the same sub-location and 1 if they are in the same location, but different sub-locations. Otherwise it returns 0. I j and S i are parameters that describe the infectiousness of x j and the susceptibility of x i , respectively. The probability of a person getting infected is then given by p(Infected i ) ¼ 1 2 exp(2l i Dt). To simulate external reseeding, we randomly select one person each day and infect him/her with pandemic influenza if he/she is susceptible. For the length of each phase in the transmission model, we did not define a fixed duration. Rather, guided by the values reported in Germann et al. (2006) , we divided the phases into a number of smaller substages, each lasting approximately 5 -6 h. This increases the granularity of the transmission model, and allows us to better scale the infectiousness profile upon consumption of medicine. The smaller substages do not include the susceptible and recovered phases. Hence, given the reported mean lengths of the exposed (1.2 days), pre-symptomatic (0.7 days) and infectious phase (4.1 days), in the model we divided them into 5, 3 and 17 substages, respectively. Simulating the model 20 000 times without any forms of intervention policies or diagnostic approaches gave us an average of 29 h for the exposed phase, 17 h for the presymptomatic phase, and 98.5 h for the infectious phase. Assuming that the total duration of infection follows a normal distribution, we computed the mean length to be 144.5 h and a standard deviation of 6.13 h.
How necessary is a fast testkit? J. Chin et al. 1035
Following previous works, presumably 33 per cent of the infected people are asymptomatic (i.e. do not show any symptoms) while the remaining 67 per cent show symptoms of infection (Longini et al. 2005; Germann et al. 2006; Wu et al. 2006) . Of those who are symptomatic, 6 per cent are assumed critical and require hospitalization (Wu et al. 2006) . The other 94 per cent will show only mild symptoms. For the critically ill, they are 50 per cent more infectious as compared with those showing mild symptoms, while the asymptomatic cases are 50 per cent less infectious. In the current model, we ignored the occurrence of deaths. However, it can be assumed that the death rate is directly proportional to the total attack rate. For each individual, the susceptibility is assumed to be constant (unless the person is on medication, or immunized against the disease). However, the infectiousness (for a mildly symptomatic person) follows a baseline profile as shown in figure 1b. Both the susceptibility and infectiousness can be modified by the consumption of antiviral drugs. While an infected person is on medication, his/her infectiousness is reduced by 60 per cent. On the other hand, the susceptibility of an uninfected person will be reduced by 30 per cent. In addition, the probability of symptoms appearing will be reduced to a minimum of 23.45 per cent depending on the length of medication. Being on medication also shortens the duration of the disease by a maximum of 25 h if the consumption of drugs is sustained throughout the course of infection. Finally, once a person has recovered from the disease, he/she is assumed to be immune to further infection, and we set the susceptibility to 0 (see the electronic supplementary material for more details).
In our model, each index case is handled by a decision analytical model. The overview of the decision flowchart is described in figure 2. For simplicity, it is also assumed that each decision is made independently of other index cases. For instance, in the event that two or more people in the same household display flu-like symptoms, the decision for each of them visiting a doctor is made independently. The probability of each decision is also independent of any previous decisions made. To simulate scenarios where some intervention policies are not put in place, the associated probabilities are simply adjusted to reflect them accordingly.
The decision process is triggered when a person starts to show symptoms. Depending on the severity, if the person is critically ill, he/she is admitted to the flu hospital for diagnosis and given mandatory treatment. Regardless of diagnosis outcome, the person will be hospitalized and kept isolated. For one showing mild symptoms, there is a 50 per cent chance that he/ she will visit the hospital to seek medical attention, i.e. diagnosis compliance rate. Note that the diagnosis compliance rate is 100 per cent for a person who is currently following quarantine orders. As our model is simulated in steps of 1 h, it will not be the case whereby the person will visit the hospital (if he/she decides to go) the moment symptoms appear. Instead, the trip will only be made after a certain delay, or when the hospital is open, whichever comes later. Currently, we set the delay to 6 h. At the hospital, a patient is diagnosed using one of the four possible approaches, depending on the diagnostic test we wished to assess. The time required varies with the type of diagnostic tests being used. If the patient is tested positive for pandemic influenza, treatment will be offered. For the treatment, we assumed that the drug used is the commonly stockpiled Oseltamivir (US National Library of Medicine and National Institutes of Health 2009). A patient being tested positive (index case) will be given two doses a day for 5 days, consuming a total of 10 doses. A negatively tested patient will be sent home where he/she will decide whether or not to take absenteeism from work or school. Unlike patients who are admitted due to critical illnesses, the class of positively tested patients may not comply with the given treatment (i.e. refuse to take medication even when it is prescribed to them). In the current model, the treatment compliance rate is set at 95 per cent. However, regardless of compliance, drugs given are considered expended and will be added to the total medication used. After the required drugs have been prescribed, patients with mild symptoms will be issued quarantine orders, where they are to stay at home for a pre-determined amount of time. Again, the patient may or may not comply with the quarantine order. Currently, we set the quarantine compliance rate at 50 per cent.
A person with mild symptoms may refuse to go to the hospital, or he/she could be tested negative for pandemic flu, or simply does not comply with quarantine orders. Nonetheless, he/she is still unwell and may decide to take absenteeism from work (for adults) or school (for children). For our simulation, adults have a 50 per cent chance of absenting themselves from work, while children will always stay away from school, until they recover.
Contact tracing is a non-pharmaceutical social distancing measure for limiting the spread of influenza by identifying people who may have come into contact with an infected person, and giving them prophylaxis or issuing them with quarantine orders. It has been shown theoretically that through contact tracing, major outbreaks can be reduced significantly at a small additional cost (Huerta & Tsimring 2002) . We divided the contacts of each patient into one of the following three categories: household, workplace and friends. When contact tracing for a class has been activated, i.e. the index case is being tested positive for pandemic influenza, all the contacts in that class will be traced successfully. To emulate logistical delays in tracing them, we set the time for successful traces to be uniformly distributed between 5 and 7 h after positive diagnosis of the index case.
Similar to the index cases, contacts are handled according to a decision flowchart as shown in figure 3. Upon being traced, the person will be offered prophylaxis-giving a person who is not obviously sick a course of antiviral drugs, so as to prevent him/her from being infected, or to reduce the severity of the illness when he/she does get infected. Again, there is a 95 per cent chance of the person consuming the medication. The prescription to be given is 1 dosage per day, for 10 days.
An insufficient drug stockpile is a real world issue faced by all countries. For instance, the United States has only enough antiviral medicine for 25 per cent of its population (US Department of Health & Human Services 2005). In order to conserve limited drug supplies, different countries have implemented policies that prioritize intended drug recipients according to their risk groups (US Department of Health & Human Services 2005). In addition, prophylaxis is only administered if deemed necessary. However, Longini et al. (2004) demonstrated that targeted antiviral prophylaxis could be an effective measure for containing influenza until the proper vaccines are developed. Here, some of the intervention policies include prophylaxis being given to contacts. In addition, to prevent drug wastage, we tracked the actual amount of drugs being consumed by the contact. If the contact becomes symptomatic later on and is subsequently diagnosed with pandemic flu, he/she will only be issued enough drugs to top-up his/her current supply to the required dosage levels as per index cases. Taking into account that not all patients will be upfront about the amount of drugs they already have, we set the average declared amount to be 50 per cent of the actual dosages remaining.
The various intervention policies implemented in our model consist of different combinations of handling How necessary is a fast testkit? J. Chin et al. 1037
index cases and their contacts. Specifically, we have the following intervention policies:
-P1: base case (no controls), -P2a: treatment of index cases only, -P2b: treatment and quarantine index cases, -P3a: as per P2b, trace household contacts only, with 10 days prophylaxis, -P3b: as per P2b, trace household contacts only, with 2 days quarantine, -P3c: as per P2b, trace household contacts only, with both prophylaxis and quarantine, -P4a: as per P2b, trace all contacts, with 10 days prophylaxis, -P4b: as per P2b, trace all contacts, with 2 days quarantine, and -P4c: as per P2b, trace all contacts, with both prophylaxis and quarantine.
Note that the duration of quarantine is set at 2 days as it is the length of time after exposure for symptoms to appear. For strains where the incubation period is longer, the quarantine duration may need to be adjusted accordingly. With the exception of P1, each intervention policy will be used with a particular testing method for the diagnosis of individuals who show influenza-like symptoms. The diagnostic approaches are:
-D1: assume all symptomatic cases are pandemic influenza positive, -D2: fast testkit, -D3: slow testkit, but the patient will resume his/her schedule during the testing period, and -D4: slow testkit, with the patient staying in the hospital while waiting.
For the base diagnosis approach (D1), we assumed that all the people who display influenza-like symptoms are pandemic positive (regardless of whether they are really infected with pandemic virus) and are handled according to the prevailing intervention policies. Through this approach, there is no need for any testkits.
If fast testkits are to be used (D2), a patient will only have to spend 1 h in the hospital for diagnosis and the result (of whether he/she has contracted pandemic influenza) to be released. Alternatively, slow testkits can be used. However, with slow testkits, there is a waiting time of 12 h. Depending on the diagnostic approach, the patient can either resume his/her daily routine (D3), or wait in the hospital (D4). Under D3, if the test turns out positive the person will be recalled back to the hospital where the appropriate treatment (for policies P2 -P4 only) is carried out. We assumed that the recall success rate is 100 per cent. By default, all tests (regardless of actual techniques) are set to be 100 per cent specific and 70 per cent sensitive.
For each intervention policy and diagnostic approach, we ran the model 50 times, with each iteration simulating the spread of the influenza virus for 60 days. We assumed that a global pandemic is underway, and the city is already in the mitigation phase of its pandemic response strategy, whereby the aim is to reduce the total number of people affected and maximize care for those infected. We examined the efficiency of the various testkits in mitigating the outbreak within the city when positive cases have already appeared.
We measured the severity of the pandemic (and hence the efficacy of the diagnostic method) using two key indicators-total attack rate (AR Total ) and peak attack rate (AR Peak ). The total attack rate indicates the total number of people who display influenza-like symptoms due to the novel strain. It is not the same as the total number of people infected as there is a fraction of people who recover without showing any symptoms. The peak attack rate measures the highest number of people (at any one time point) showing symptoms due to the pandemic virus infection. It is an indication of the maximum burden on the healthcare system, as well as a partial reflection of the economic impact due to absenteeism among working adults.
Other auxiliary indicators measured are the costs incurred due to the amount of testkits being used (C t ) and the antiviral drugs being issued (C d ). This is in view of the limited supply of drugs and additionally, in our case, diagnostic testkits. Another resource that is often overlooked is the holding capacity of the influenza hospital. While patients are waiting for diagnosis or their test results, they are required to stay at the hospital (with the exception of diagnostic approach D3). Hence the peak waiting room occupancy (C w )-the maximum number of people waiting at any one time-is another indicator that is measured. To gauge the economic impact of the various intervention policies, we measure the number of work hours lost (C h ) by all individuals who underwent diagnosis, treatment or were traced.
We formulated a function to compute the aggregate cost (C Aggr ) incurred by the various intervention policies and diagnostic methods. This function is described by the following equation:
C Aggr is a value that ranges from 0 to 1. The term a is the weight associated with the type of resource and it reflects the importance of each component to the aggregate cost. Note that there are no universal values for the weights, and they vary across cities with different resource priority.
For instance, a model depicting a developed nation may be assigned a higher value for a h due to its requirement to remain economically competitive whereas for a country without a proper healthcare system, one may assign higher values for a d and a t instead. Currently, we set a t ¼ 0.15, a d ¼ 0.45, a h ¼ 0.35 and a w ¼ 0.05 as we are investigating the use of testkits in a small city of a developed nation, but at the same time, we wish to minimize drug wastage.
We first simulated the spread of the pandemic influenza under various intervention policies and diagnostic methods using the default parameters (see the electronic supplementary material). For the testkits, we assumed a specificity of 100 per cent and sensitivity of 70 per cent. From our simulations, the basic reproduction number (R 0 ) is computed to be approximately 1.9, representing a moderately infectious pandemic outbreak. The results are shown in figures 4 and 5.
There is generally a similar trend in the comparison of the total attack rates, the peak attack rates and the amount of testkits being used across the different intervention policies, regardless of the diagnostic approach. An increase in the total attack rate corresponds to an increase in the peak attack rate and amount of diagnostic testkits being used. At the same time, to mitigate pandemic influenza, i.e. reduce the total attack rate, How necessary is a fast testkit? J. Chin et al. 1039 more antiviral drugs need to be consumed, a policy that may not be always feasible due to limited supplies. When treating only the index cases (figure 4a, policies P2a and P2b), the total attack rate shows a moderate decrease, from an average of 4463.9 cases (base line) to the lowest rate of 4016.2 cases (figure 4a, policy P2b, diagnosis approach D1). We start to observe a more significant decrease only when contact tracing comes into effect. Even so, this decrease is only significant when prophylaxis of the contacts is included in the policies. The work hours lost from the various diagnostic approaches and intervention policies, interestingly, does not follow the same general patterns observed in the other indicators. In particular, increasing the coverage of contact tracing and assuming all cases to be pandemic does not necessarily equate to more work hours being lost (figure 5d, policies P4a and P4c, diagnosis D1). This is due to the decrease in total attack rate, which offsets the work hours lost by the contacts.
Within each implemented intervention policy, the impact of having a fast testkit on the total and peak attack rates is only matched when a slower diagnosis method is used and the patient stays at the hospital. In some cases, requiring the patients to stay while waiting for the test results may even be more effective in containing the virus, as exemplified by policies P3b and P4b shown in figure 4a. However, the peak waiting time associated with such an approach may prohibit its effective implementation. Of course, one can simply assume that all patients who exhibit influenza-like symptoms have contracted pandemic influenza and issue them with the antiviral drugs. The downside of this approach is then the increased drug usage, and the proportion being wasted due to consumption by those inflicted with ILIs (figure 5a). Nonetheless, it may still be a viable option if the testkits constitute another limited resource that has to be managed properly. Figure 6 shows the plot comparing the total attack rate with the aggregate cost. From the plot, we see that in the context of most intervention policies, diagnostic approach D1 is distributed closer to the desired lower-left quadrant-which corresponds to an optimal ratio of number of people infected compared with resources consumed-especially when contact tracing has been implemented. Even with the use of diagnostic testkits, a slower one that requires the patient to stay, coupled with an appropriate intervention policy, is comparable to using a fast testkit. The only consideration for such an approach would then be the peak hospital waiting room occupancy. However, given that the availabilities of antiviral drugs and testkits are usually the main limiting factors, a slow testkit with the patient waiting in the hospital may still be preferred when a fast and accurate kit is not yet ready.
In addition, using the current weights for the aggregate cost function, data points derived from the same intervention policies can be clustered and grouped together (shown in figure 6 as dashed circles). This suggests that, based on the current priorities given to the cost components, intervention policies are the dominating factors in mitigating an influenza pandemic. It should be noted that, in particular, prophylaxis policies guided by contact tracing have stronger effects on reducing the total attack rate (lower right quadrant in figure 6 ). Within each intervention policy, the different diagnostic methods then fine-tune the allocation of limited resources.
3.2.1. Basic reproduction number. The basic reproduction number R 0 is an indicator that is often used to measure the transmissibility of a pandemic outbreak. Simply put, it is the average number of other individuals each infected person will infect in a completely susceptible population. Analyses of previous influenza pandemics estimated R 0 for large communities to lie between 1.2 and 3 (Mills et al. 2004; White & Pagano 2008) . For the recent H1N1 pandemic, some early studies have suggested R 0 to range between 1.4 and 1.6 (Fraser et al. 2009 )-indicating relatively low transmissibility-while others estimated it to be as high as 3.1 in certain countries such as Mexico (Boglle et al. 2009 ).
We obtained R 0 by first turning off all intervention policies prior to simulation. We then randomly infect one susceptible person and simulate the model, noting down the number of people he/she transmits the virus to. This process is repeated 1000 times, and the average number of transmissions is reported as the R 0 value. With the default parameters, we computed R 0 in the model to be approximately 1.9. To see the effectiveness of the various diagnostic approaches and intervention policies under different viral transmissibility, we vary R 0 from 1.6 (low transmissibility) to 2.3 (high transmissibility) by manually adjusting the transmission coefficients b h , b w and b c (equation (2.1)), while ensuring that the transmission proportion between the different location types remains relatively constant. Figure 7 shows the changes of various indicators (AR Total , AR Peak , C d , C t , C w and C h ) for different diagnostic methods and intervention policies with varying transmissibility. In general, there is a monotonic increase across all indicators as R 0 increases. Again, within each graph, it is observed that values from the same intervention policy tend to cluster together, regardless of viral transmissibility (with the exception of testkit usage for diagnostic approach D1, figure 7d ). Hence during the onset of an outbreak, where the transmissibility is still unknown-which is often the case-it may be more prudent to first implement proper social distancing intervention policies, followed by the use of testkits to ensure that drug wastage is minimized.
In the model, the current compliance rates for diagnosis, quarantine and absenteeism are set at 50 per cent (figure 2). However, in the event of a pandemic outbreak, there may be heightened awareness among the population, such that individuals displaying flu-like symptoms are now more likely to seek medical attention and follow quarantine orders. To investigate the effects of varying compliance, we simulate the model with varying levels of compliance rates (from 30 to 70% for a medium transmissible pandemic outbreak, i.e. R 0 ¼ 1.9).
The graphs in figure 8 show the results of varying the compliance rates. The general trend shows total attack rate decreasing as more symptomatic individuals seek some forms of diagnosis or antiviral medication. One would expect that with more people visiting the hospital, the amount of drug and testkit usage will increase as well. However, the contrary is observed for some cases, particularly for policies P4a and P4c. Under these policies, the total attack rate can drop by as much as 28.4 per cent as the compliance rates increase from 50 to 70 per cent ( policy P4c, diagnosis approach D2), and instead of seeing an increase in the medicine consumption, we get a decrease by 2.3 per cent (figure 8). Similar patterns are observed when other diagnostic approaches are implemented in tandem with P4a and P4c. We noted that the reversal of the correlation between total attack rate and cost occurs only in the presence of two conditions: (i) full contact tracing and (ii) prophylaxis of the contacts. Additionally, a high diagnosis compliance rate can be loosely associated with a lower false negative rate for viral detection. Hence, we expect measures that reduce the false negative rate to have a more significant impact on pandemic mitigation when implemented with the abovementioned conditions. The critical issue that follows is then how limited resources such as antiviral drugs and testkits can be used effectively with those conditions.
3.2.3. Antiviral drug efficacy. The effectiveness of antiviral drugs may be a potential factor influencing the impact of intervention policies and testkits on pandemic outbreak mitigation. Drug efficacy is represented in the model by the levels of reduction to infectiousness and susceptibility, as well as the probability of symptoms How necessary is a fast testkit? J. Chin et al. 1041
appearing for an infected person. We simulated the scenario for lower drug efficacy by lowering the amount of infectiousness reduction from 60 to 30 per cent, and susceptibility reduction from 30 to 15 per cent for infected and uninfected individuals, respectively. In addition, there is also a reduction of symptomatic probability to 45 per cent.
To examine the effects of lower drug efficacy, we look at the scatter plot of total attack rate against aggregate cost for both sets of simulations-base and reduced drug efficacy (figure 9). From the plot, there is a general increase in both total attack rates and costs with decreased drug effectiveness. Again, the results can be clustered by the intervention policies. However, it is interesting to note that, similar to varying the diagnosis compliance rate, points from policies P4a and P4c collectively experience a larger amount shift along both axes. Conversely, for a more effective antiviral drug, the impact on reducing both attack rates and costs through the implementation of policies involving full contact tracing and prophylaxis to the contacts will most probably be more significant.
To test the robustness of the observations for diagnostic approaches D2 to D4 within each intervention policy, the simulations are repeated but with varying levels of testkit sensitivity (50 and 100% sensitive). In general, increasing the sensitivity of the testkits has the effect of decreasing the total attack rate ( figure 10) . Surprisingly in (c) Figure 8 . Effects of varying compliance rates. The graphs show the changes of the (a) total attack rate, (b) drug usage and (c) testkit usage with increasing compliance rates for a particular diagnostic approach (D2). In particular, for policies P4a and P4c (highlighted in dashed circles), higher compliance rate (from 50 to 70%) will result in lower total attack rate, but without a corresponding increase in the resources required (light grey bar, 30% compliance; striped bar, 50% compliance; checked bar, 70% compliance). . Reduced drug efficacy. The black dots are the various data points obtained by simulating the model using base drug efficacy (base data points), while the white ones are derived using reduced drug efficacy (reduced data points). The aggregate cost for the base data points differs slightly from the original plot (figure 6) due to the increased set of data points used in the computation. Most of the reduced data points are shifted by a small amount, except for those obtained using policies P4a and P4c, as indicated by dashed circles. some cases, a highly sensitive, but slow testkit, coupled with proper diagnostic approaches, can bring about a lower total attack rate as compared with simply assuming that all patients are infected with pandemic influenza (figure 10, diagnosis D4 with policies P2a, P2b, P3b and P4b). One possible explanation is that confining the pandemic-positive patients in the hospital tends to remove them from infectious circulation while they are at their peak infectiousness.
Despite the decrease in total attack rate, the amount of drug usage increases with better sensitivity, mainly due to more people being correctly diagnosed and administered with antiviral drugs. This increase is not observed for the amount of testkits being used (figures 11 and 12). One interesting observation is that as the sensitivity of the testkits improves, the amount of drug usage becomes almost as much as if we were to assume that all patients are positively infected with pandemic influenza (figure 11). In some cases, the amount of drug dosage even exceeds the base approach D1 (e.g. figure 11 , policy P4c, diagnosis D2), possibly due to the higher total attack rate. Regardless of the testkit sensitivity, variations to the amounts of drug and testkit usage between diagnostic approaches D2 to D4 are not as significant as compared with the variations due to the different intervention policies being implemented.
Hospitals provide another dimension in shaping the spread of an influenza pandemic and affecting the efficacy of the various policies and diagnostic approaches. Unlike other location types, individuals who show influenza-like symptoms will deliberately go to the hospital for diagnosis, providing an opportunity for potential spread of the virus (Nuñ o et al. 2007 ). In our current model setting, hospital transmission does not play a major role as we have implemented a simple hospital segregation policy through the designation of a flu hospital. However, without such policies, some diagnostic methods may not work as well as shown previously.
We briefly study the importance of implementing proper hospital segregation policies by simulating our model without a designated flu hospital. Each symptomatic person will randomly select a hospital to visit for diagnosis and treatment. In addition, we increase the number of ONILI cases by threefold to 135 individuals daily in order to assess the results under a more severe setting. These people will also go to any of the two hospitals for treatment with a 50 per cent compliance probability.
The total attack rate, drug usage, testkit usage and amount of work hours lost are shown in figure 13 . From the results, we see an overall increase in the total number of people infected and the consumption of the different resources. However, an interesting observation is that the effectiveness for diagnostic approach D4-where the patient stays in the hospital while waiting for the test results-starts to degrade regardless of intervention policy. One reason for this phenomenon is that the force of infection experienced by an ONILI patient is increased dramatically, not only because they visit the same hospital as infected individuals, but also due to the higher concentration of infectious people in the hospitals as compared with other location types. In fact, in the absence of a fast testkit, simulations showed that it is better to collect the samples from the patient and allow him/her to go off while diagnosis is being performed using conventional laboratory-based methods. Nonetheless, the better policy will still be to prevent the mixing of genuine influenza cases and those with ONILIs. How necessary is a fast testkit? J. Chin et al. 1043
From the simulation results presented in the previous section, the impact of a fast testkit on mitigating pandemic influenza may not be as significant as the various social distancing policies themselves. In particular, for most cases within our model set up, assuming that the patient is positive for pandemic flu will result in better allocation of limited resources (drug dosage and amount of testkits) while minimizing the total number of people being infected. Then why is a fast testkit necessary? In the current model, the implementations of diagnostic methods and intervention policies are homogeneous, i.e. assume pandemic positive, slow and fast testkits are not being used in a way such that their intended effects are maximized according to whether the individual is an index case or a contact. In general, we find that three of the most important factors that guide the use of diagnostic testkits are (i) the resource consumption by false positives, (ii) the frequency of false positives, and (iii) the propagation potential of false negatives. In this work, an individual suspected to be infected may consume the following resources-antiviral drugs, testkits, work hours lost (which represents an intrinsic economic opportunity cost), and less importantly, the peak waiting room occupancy. Allocating an individual these resources when it is not really required (whether it is due to assuming that he/she is pandemic positive or the inadequate specificity of the testkits) is wastage which any policy maker would try to minimize. This is especially true for antiviral drugs, which often represents a hard constraint. Depending on the frequency and proportion of such false positives, proper diagnostic approaches can then be implemented. For instance, when the proportion of false positives is high, such as the case of a high ILI rate, assuming that all patients are pandemic positive would put a strain on the limited drug supplies. Of course, such rates may not be possible to obtain for a novel pandemic strain with no outbreak history. Nonetheless, by not advocating a blanket approach and instead, identifying areas where false positives are most likely to occur, the proper use of fast testkits can possibly achieve better mitigation outcomes and minimizing wastage.
To further illustrate our point, when a local pandemic is already underway, as depicted by our model, most patients who turn up at the hospitals for diagnosis are likely to have been inflicted with pandemic influenza. As such, it may be more prudent to assume positive and administer antiviral drugs without diagnosis. However, a different approach may be adopted for their contacts. From figure 5a, there is a significant increase in the amount of drugs being used when the tracing of all contacts is in place (except for policy P4b). Policy P4a uses almost 5 times more drugs than policy P2a despite reducing the total attack rate by only slightly more than half. This suggests that there is a huge amount of wastage due to the contacts. Hence, instead of applying prophylaxis to the contacts, using diagnostic testkits to identify infected contacts and treating them with antiviral drugs might then possibly yield a better attack rateto-drug usage ratio. The question then remains as to whether a fast or slow testkit makes a difference to the attack rate and drug usage. Assuming that the contacts need not go to the hospital and instead a respiratory specimen is collected and sent for analysis while the contact is ordered to stay at home (a diagnostic approach similar to D4), then a fast testkit may not be required. However, if a trip to the hospital is needed for diagnosis, the availability of a fast testkit may minimize the time the contact spends not under quarantine, preventing further spread of the influenza virus.
Another factor influencing the necessity of testkits, which is closely related to their sensitivity, is the repercussions of not treating a pandemic-positive individual. In figure 4, the total attack and peak attack rates for non-treatment are shown as base lines. For a moderately infectious variant as simulated in our model, an untreated case may not spread the virus in an exponential manner. As such, a testkit may be used to reduce drug wastage, but a highly sensitive one may not be required since the infectiousness of the individual is limited. However, for the more transmissible cases, such as the ongoing H1N1 swine flu outbreak (at the time of writing; Neumann et al. 2009 ; Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team 2009), it may be unwise to leave a potentially infectious person untreated. If a sensitive testkit is not yet available, then assuming that a symptomatic individual has (d) Figure 13 . Simulation results without hospital segregation. The diagram shows the (a) total attack rate, (b) drug usage, (c) testkit usage and (d) total work hours lost for the various intervention policies and diagnostic approaches without designating one hospital as a flu hospital. Note that for all cases, D4 has a higher total attack rate and it consumes more resources as compared with D2 and D3 (black bar, D1; light grey bar, D2; dark grey bar, D3; white bar, D4; solid line, base P1).
How necessary is a fast testkit? J. Chin et al. 1045
contracted the pandemic virus is probably more efficient in bringing down the attack rates. Aside from the transmissibility of the virus, geographical features may also affect the propagation potential of a false negative. In urban areas such as cities, where close contact due to work and public transport is inevitable, a single untreated case can rapidly lead to the formation and spread of influenza clusters. One other aspect which can be further investigated is the role of public transport in transmitting the pandemic virus. Public transport such as buses and subways not only facilitates the displacement of carriers between locations within the city, it also brings several individuals into close proximity, increasing the likelihood of the virus infecting several other people. A limitation of current diagnostic measures in most countries is that a symptomatic person has to travel to a local hospital for testing, which requires commuting, mostly on public transport. As mentioned previously, it is possible for a symptomatic individual to either send a respiratory specimen to the hospital, or request for a fast testkit for diagnosis at home, hence alleviating the need for public transport. Both measures can potentially reduce the risk of exposure to others significantly.
There are several other aspects to pandemic influenza that determine the efficacy and necessity of a fast testkit in minimizing drug wastage while mitigating an outbreak. Parameters and policies such as compliance rate of the general public, as well as activating prophylaxis to priority groups (US Department of Health & Human Services 2005) will influence how a testkit can be effectively used. However, considering these parameters and policies is beyond the current scope of this work.
For future works, we may add more flexibility to the policies being implemented, such as differential handling of index cases and contacts as briefly discussed. One mitigation means that can also be further explored is the role of vaccines and how they would shape the spread of the pandemic outbreak. Additionally, more complex social interaction patterns (Barrett et al. 2008) can be implemented to represent the spread of the pandemic in a more realistic manner. Data mining techniques (Baily-Kellogg et al. 2006) can then be used on the simulation results to better assist policy makers in assessing the implications and effectiveness of their policies both spatially and temporally. Finally, the aggregate cost function (equation (3.1)) may also be modified to present a more systematic cost -utility assessment of the various diagnostic approaches and intervention policies, taking into account factors such as quality-adjusted life-years (Sander et al. 2009 ).
Managing limited resources such as testkits and antiviral drugs while trying to contain the spread of pandemic influenza is a major challenge faced by several countries. In the event where a fast and accurate testkit is not yet available, one has to rely on slower testkits, coupling their use with effective social distancing measures to minimize the wastage of antiviral drugs. In this work, we have developed a stochastic agent-based pandemic model to assess the necessity of a fast testkit. From our simulation results, we showed that intervention policies, and not testkits, are the key means to successfully contain an outbreak, and that casting a wider net for contact tracing is crucial for minimizing the total attack rate. However, although not yet included in our model, we can infer from the results that most of the drug wastage is due to prophylaxis being administered to the contacts. Considering the use of testkits on the contacts may be a better resource allocation strategy. Within each intervention policy, the use of slower testkits while holding the individual at the hospital can be an equally effective means of mitigating a pandemic outbreak as compared with a fast testkit. However, this is provided that proper infection controls, such as a hospital segregation policy, are in place. Otherwise, hospital transmission may limit the usefulness of other efforts such as contact tracing and quarantine orders. Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction. Dendritic cells (DCs), a highly specialized subset of professional antigen-presenting cells, play a central role in the initiation of innate and adaptive immunity. There are two known major subsets of primary human and murine circulating DCs: myeloid DCs, which function principally in antigen presentation, and plasmacytoid DCs (pDCs), which secrete the type I interferons (IFN), IFNa and IFNb [1] , as well as a variety of other cytokines and chemokines. Viral induction of type I IFN expression has been well studied in recent years and has been shown to be mediated by multiple pattern recognition receptors (PRRs), including retinoic acid-inducible gene (RIG)-I, melanoma differentiation-associated gene (MDA)5, toll-like receptor (TLR)3, TLR7 and TLR9. PRR expression is restricted in pDCs, with only TLR7 and TLR9 implicated in viral-induced IFNa production through the recognition of single-stranded (ss)RNA or DNA, respectively [2, 3, 4, 5, 6, 7, 8, 9] . IFNa production by pDCs is also observed following TLR7/8 stimulus with synthetic resiquimods and imiquimods, or TLR9 antagonism by CpG oligodeoxynucleotides (ODN). pDCs are not generally thought to be able to respond to double-stranded (ds)RNA, as stimulation with poly I:C or long (500 base pairs) dsRNA molecules fail to elicit IFNa or upregulate activation or maturation markers, such as CD86 and CD83 [10, 11, 12] . However, low levels of IFNa production have been reported following pDC stimulation with poly A:U [13] or in vitro transcribed viral dsRNA [11] , and a recent report indicates a role for RIG-I-like helicases in recognizing replicating virus in murine pDCs lacking the IFN receptor [14] . Short interfering dsRNAs have also been demonstrated to elicit an IFNa response through TLR7, although this appears dependent on a specific sequence, and not on the dsRNA structure [12] .
It is well understood that pDCs activate natural killer cells [15] , macrophages [15, 16] and T and B cells [17, 18] , presumably, in part, through IFNa stimulus. Recent evidence suggests that pDCs may have additional antiviral effects, including the direct inhibition of viral replication in target cells secondary to the secretion of type I IFN [19, 20, 21] . pDCs are also implicated in restricting viral replication in vivo, as there is an inverse correlation between circulating pDC numbers and human immunodeficiency virus (HIV) or hepatitis C virus (HCV) viral load [22, 23, 24, 25] .
Rotavirus, a dsRNA icosahedral virus in the Reoviridae family, is the leading cause of severe dehydrating diarrhea in young children worldwide, with 500,000 to 600,000 annual deaths attributed to rotavirus infections [26, 27, 28, 29] . Significant morbidity and economic impact are the main effects in the United States, accounting for approximately 50,000 to 60,000 hospitalizations a year and loss of time from work for caregivers [30] .
Rotaviruses are characterized by a triple-layered protein capsid composed of four major structural proteins. Viral protein (VP)2 comprises the innermost layer, in which the dsRNA genome is contained, while the middle layer consists of VP6. The outer layer of the virion is composed of the VP7 glycoprotein and proteasesensitive VP4 spikes [31] . Although both triple-and doublelayered particles (TLPs and DLPs, respectively) are generated during rotavirus replication, only TLPs are infectious, due to the requirements of VP4 and VP7 for cell binding, and trypsin cleavage of VP4 for viral entry and infectivity [32, 33, 34, 35, 36] . Formation of noninfectious empty TLPs and DLPs, which lack the viral genome, is also observed during infection. The nonstructural proteins (NSP1 through 5) are involved in viral replication, morphogenesis and assembly, but they are not expressed by nonreplicating virus and are not part of the infectious virion [31] .
Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms required to control rotavirus infection. Rotavirus structural and nonstructural proteins have been detected in primary DCs from murine mesenteric lymph nodes and spleens [37, 38] , and murine bone marrowderived DCs have been shown to secrete type I IFN and tumor necrosis factor (TNF)a following rhesus (RRV) or bovine rotavirus (RF-81) challenge, respectively [39, 40] . Mature human monocytederived myeloid DCs (moDCs) have been demonstrated to be more susceptible to rotavirus infection than immature moDCs, although infection did not result in substantial cell death [41] . Additionally, infection did not induce moDC maturation, but instead promoted priming of Th1 cells [41] . A recent study of total human peripheral blood mononuclear cells (PBMCs) exposed to RRV or human rotavirus indicated that both myeloid DCs and pDCs are susceptible to infection, and that infection results in the secretion of IFNa, presumably from pDCs [42] . While increased levels of IFNa have also been correlated with a positive clinical outcome in infected children [43, 44] , several rotaviruses, including RRV, have recently been demonstrated to antagonize the production of type I IFN through the degradation of interferon regulatory factors (IRF)3, IRF5, and IRF7, and the inhibition of NFkB activation, by a viral nonstructural protein, NSP1 [45, 46, 47, 48] .
Here we report the effects of rotavirus infection on highly purified primary human pDCs. We demonstrate that although rotavirus initiates detectable transcription and translation in only a small percentage of pDCs, a significant percentage of pDCs activate and mature following exposure to virus. Importantly, we demonstrate that stimulation of pDCs with live or inactivated RRV effects secretion of IFNa and multiple proinflammatory cytokines and chemokines. This response is dependent on the presence of the viral dsRNA genome. As IFNa production by pDCs is classically triggered in response to ssRNA or DNA, the induction of this response by a replication-incompetent dsRNA virus indicates a potentially novel mechanism of viral sensing by pDCs.
To characterize the interaction between pDCs (phenotypically defined as viable lineage -HLA-DR + CD11c -CD123 + cells, Figure  S1 ) and rotavirus, pDCs were purified from human blood by negative selection (mean purity 6 standard error mean [SEM]: 86.48%60.92) and exposed to RRV at a multiplicity of infection (moi) of 5 or 10, or to equivalent quantities of inactivated RRV (iRRV). Expression of NSP2, a viral nonstructural protein expressed only in cells in which rotavirus replicates, was detected by intracellular flow cytometry at 6 and 12 hours post infection (hpi). As illustrated in Figure 1 , NSP2 was not detected in pDCs receiving MA104 supernatant (mock-treated; Figure 1A ) or iRRV (data not shown), but was expressed in a small but significant percentage (mean 6 SEM, moi 5: 1.18%60.23 6hpi, 1.43%60.28 12hpi; moi 10: 1.45%60.25 6hpi, 3.02%60.99 12hpi) of pDCs inoculated with live RRV (Figure 1B and 1D) . Additionally, NSP2 was detected only after pDCs were permeabilized (data not shown). Two populations of NSP2 + cells, NSP2 dim and NSP2 bright , were frequently apparent in pDCs exposed to live RRV ( Figure 1B and 1C) ; approximately 20% of donors had only a single NSP2 dim population. While the total percent of pDCs expressing NSP2 following RRV challenge increased from 6 to 12hpi, this increase was not significant (moi 5: p = 0.77; moi 10: p = 0.30; Mann-Whitney) ( Figure 1D ). The percent of NSP2 + pDCs also increased with moi (6hpi: p = 0.26; 12hpi: p = 0.063; Mann-Whitney), but remained a minor proportion of the total pDC population (median: 2.01%, moi
Rotaviruses cause severe dehydrating diarrhea and are a leading cause of death in children worldwide. A potent antiviral, interferon-a (IFNa), is rapidly secreted by plasmacytoid dendritic cells (pDCs) in response to viral single-stranded RNA or DNA genomes. Here, we examined the effects of rotavirus on pDCs purified from human blood. We found that very few pDCs supported rotavirus replication, and that pDCs retained similar functionality in response to live or inactivated rotaviruses. While pDCs produced large quantities of IFNa shortly after rotavirus exposure, this was impaired in cells supporting viral replication. We also found that two viral proteins and the rotavirus double-stranded RNA genome were required for the initiation of the pDC IFNa response to rotavirus. Additionally, we found that cleavage of one of these viral proteins, a traditional prerequisite for rotavirus infection in other cell types, was not required for the infection of pDCs or production of IFNa. This may enable the host to rapidly initiate an immune response to rotavirus that subsequently restricts infection to the intestine and contributes to the resolution of disease. Our study provides novel insight into the interaction between rotavirus and the host innate immune response, and also identifies a unique mechanism for the production of IFNa by pDCs.
10, 12hpi) ( Figure 1D ). Further increasing the moi to 25 or 100 modestly increased the NSP2 + population, but it remained relatively small (mean: 4.99%, median: 2.07%, moi 100, n = 4, data not shown). pDCs from multiple donors (n = 5, mean purity: 91.51%) were titered after overnight culture to determine whether rotavirus infection was productive. A significant increase in viral titer (approximately 20-fold, p#0.03; Wilcoxon signed rank test) (data not shown) was observed in two of these donors, while the other three showed no significant change over time (p$0.17; Wilcoxon signed rank test) (data not shown). While pDC viability was significantly decreased from 6 to 12hpi in cultures receiving mock, RRV (moi 5) or iRRV stimulus (p#0.004; Mann-Whitney), viral exposure did not result in significant cell death compared to mock stimulus at a given time point (p.0.063; Wilcoxon signed rank test) ( Figure 1E ). The low frequency of NSP2 + pDCs shows that the majority of human pDCs are resistant to RRV replication, as defined by evidence of expression of a non-structural rotavirus protein.
To establish whether pDCs activate or mature following inoculation with RRV, the frequency of pDCs expressing markers for activation (CD86) or maturation (CD83) was determined by flow cytometry. pDC activation (Figure 2A and 2C) and maturation ( Figure 2B and 2D) occurred at similar levels 12h after stimulation with either live or inactivated rotavirus (p$0.125; Wilcoxon signed rank test), and were significantly increased compared to mock-stimulated cells (p#0.03; Wilcoxon signed rank test). Stimulation with CpG ODN 2395 resulted in activation of a significantly greater percentage of the pDC population than that observed in pDCs exposed to RRV (p = 0.03; Wilcoxon signed rank test). The percent of pDCs matured by CpG ODN 2395 stimulus was also increased, but not significantly compared to those exposed to RRV (p = 0.094; Wilcoxon signed rank test). Viral replication did not inhibit maturation or activation, as pDCs positive for NSP2 expressed CD86 and CD83 at a significantly higher frequency (p#0.008; Wilcoxon signed rank test) than bystander pDCs ( Figure 2E -H). Expression of both molecules was significantly increased in both populations compared to pDCs receiving mock stimulus. Together, these data demonstrate that pDCs retain the ability to activate and mature after viral exposure, likely due to secondary effects, as the presence of replicative rotavirus is dispensable for induction of these phenotypes.
Production of IFNa is an important component of the pDCderived antiviral response. To assess this component of the pDC anti-rotaviral response, the frequency of cells expressing IFNa was determined by flow cytometry. Intracellular IFNa was detected within 4hpi of RRV stimulus, peaked at 6hpi and was sustained until at least 12hpi ( Figure 3A and 3B). IFNa was detected intracellularly at similar frequencies in pDCs exposed to iRRV (which was both infectivity and transcriptionally negative) (p = 0.815; Mann-Whitney) ( Figure 3A and data not shown), suggesting that replication or transcription of rotavirus is largely dispensable for IFNa induction in human pDCs. Intracellular IFNa was abrogated by the addition of RRV neutralizing VP7 or VP4 monoclonal antibodies (either mAb 159 or 2G4), indicating that the observed induction was virus-specific and likely dependent on an event occurring after virus binding and entry ( Figure 3C ) [49] . To identify and quantify IFNa secretion, and to detect other cytokines and chemokines secreted in response to RRV exposure, Luminex assays or ELISAs were performed on supernatants collected from inoculated pDCs at various times post infection. Only supernatants from pDC cultures $85% pure (mean: 90.84%60.75; n = 3-18) were analyzed to minimize the contribution of contaminating cells to the cytokine milieu. A significant IFNa response, approaching 10,000 pg IFNa/ml, was detectable 6h following exposure to live or inactivated RRV ( Figure 4A ). In addition, significant quantities of IFNb, TNFa, interleukin (IL)6, IL8, CXCL10 (IP10), CCL3 (MIP1a), CCL4 (MIP1b), CCL5 (RANTES) and TNFb were detected at various times post infection ( Figure 4B -I and data not shown). Together, these data suggest that live or inactivated RRV rapidly induce production of significant levels of IFNa and other proinflammatory cytokines and chemokines by pDCs.
In contrast to what was observed with pDC activation and maturation, IFNa production was significantly reduced in NSP2 + pDCs ( Figure 5 ). NSP2bystander pDCs produced IFNa at a significantly increased frequency (p,0.0001; Wilcoxon signed rank test) compared to pDCs dim or bright for NSP2 ( Figure 5A and 5B). The percent of IFNa + pDCs was significantly increased in the NSP2 dim population compared to that in the NSP2 bright (p = 0.0006; Wilcoxon signed rank test), supporting the hypothesis that increased levels of RRV infection are inversely correlated with IFNa production. More significant was the sustained production of IFNa in uninfected (NSP2 -) pDCs following RRV stimulus. A similar correlation between NSP2 expression and IFNb production was also observed ( Figure 5C ). These data indicate that infection in a minor subset of pDCs results in the local suppression of the IFNa response in these specific cells.
To further investigate the impairment of the IFNa response in pDCs undergoing rotavirus replication, phosphorylation of IRF7, the key transcription factor for the IFNa response in pDCs [50] , was assessed by phosflow ( Figure 5D and 5E). In accordance with the decreased production of IFNa by NSP2 bright cells compared to that of NSP2 dim pDCs, as well as previous findings of IRF7 degradation in rotavirus-infected cells [45, 46] , phosphorylated IRF7 (pIRF7) was substantially decreased in pDCs bright for NSP2 (p = 0.03 versus NSP2 dim pDCs; Wilcoxon signed rank test) ( Figure 5D and 5E), and is similar to that of IFNa -NSP2 -pDCs (p = 0.56; Wilcoxon signed rank test) ( Figure 5E ). To determine whether this decrease in pIRF7 was due to decreased levels of total IRF7 in pDCs, the MFI of total IRF7 was assessed ( Figure 5F ). While the total IRF7 MFI was slightly depressed in NSP2 bright compared to NSP2 dim pDCs, this decrease was not significant (p = 0.44; Wilcoxon signed rank test) and thus does not account for the observed differences in IRF7 phosphorylation. The increased levels of pIRF7 in NSP2 dim pDCs may represent continual, direct stimulus of the IRF7 pathway in virus-positive pDCs, as well as a mechanism by which the IFNa response is directly initiated. Alternately, pIRF7 may be sequestered in a nontraditional cellular compartment prior to degradation, leading to increased protein accumulation. Interestingly, levels of IRF7 phosphorylation in IFNa + NSP2 -pDCs were intermediate to that of NSP2 dim (p = 0.31; Wilcoxon signed rank test) and NSP2 bright or NSP2 -IFNa -pDCs (p = 0.0313; Wilcoxon signed rank test) ( Figure 5E ). The slight depression of pIRF7 in the IFNa + population compared to that observed in NSP2 dim pDCs may be attributable to the enhanced IFNa production in this subpopulation. As pIRF7 is degraded following the initiation of IFNa transcription, cells in which IFNa has been transcribed would be expected to have lower levels of pIRF7 than those negative for IFNa. Notably, inhibition of IFNa secretion, achieved by simultaneous addition of brefeldin A with viral inoculation, decreased the percent of IFNa + pDCs by an average of 63.5% (n = 6, data not shown). This supports the notion that recognition of secreted IFNa is an essential component of the IFNa response following rotavirus exposure, as has been reported with Newcastle disease virus [14] . Importantly, this is the first demonstration of impaired IRF phosphorylation in primary human cells infected with rotavirus.
Trypsin-mediated cleavage of VP4 is canonically required for productive entry of rotavirus into permissive cells [33, 34, 35, 36] . However, it has previously been reported that murine bone marrow-derived DCs activate following exposure to uncleaved bovine rotavirus [40] , indicating that VP4 cleavage may be dispensable for the induction of the DC response. To investigate the role of trypsin-mediated cleavage of VP4 in both rotavirus infection of, and IFNa production by, human pDCs, we exposed pDCs to RRV grown in the absence of trypsin (non-trypsinized; NT-RRV). Although subsequent exposure to trypsin (thus cleaving VP4) increased infectivity of the uncleaved NT-RRV preparation 63-fold by plaque titration and intracellular NSP2 staining by 20fold in MA104s (data not shown), examination of NSP2 staining showed that similar proportions of pDCs were infected with either the cleaved or uncleaved NT-RRV preparations (p = 0.625; Wilcoxon signed rank test) ( Figure 6A-C) . Likewise, uncleaved NT-RRV induced IFNa production by a similar (p = 0.375; Wilcoxon signed rank test) percentage of pDCs, compared to cleaved NT-RRV ( Figure 6A , 6B and 6D). Thus, cleavage of VP4 by trypsin is dispensable for both pDC infection and IFNa induction by RRV.
Although the ability of pDCs to mount an IFNa response to viral infection is well described, the only documented viral TLR ligands for pDCs are ssRNA and DNA [2, 3, 7, 8, 9] . As rotavirus is a dsRNA virus and since psoralen-treated, transcriptionallyinactive rotavirus efficiently induced IFNa (Figures 3 and 4A) , we investigated the viral requirements for IFNa induction. pDCs stimulated with equal quantities (see Methods describing particle quantification) of purified TLPs, DLPs, empty particles, or genome-free recombinant virus-like particles (VLPs) were stained for intracellular IFNa. As expected, density gradient purified RRV TLPs stimulated similar levels (p = 0.695; Wilcoxon signed rank test; n = 10) of IFNa production compared to pDCs treated with unpurified live or inactivated RRV expressing equivalent amounts of VP4, as measured by hemagglutination titer (Figure 7 ). pDCs exposed to 2/6 genome-containing RRV DLPs expressed neither IFNa nor NSP2, indicating a likely dependence on VP4 or VP7 for viral entry, as previously reported [32] . Alternately, one or both of these viral proteins could be serving as the pathogenassociated molecular pattern (PAMP) responsible for inducing the pDC IFNa response. To address this, pDCs were stimulated with 2/4/6/7 VLPs or 2/6/7 green fluorescent protein (GFP)-VLPs. Both 2/6/7 GFP-VLPs and 2/4/6/7 VLPs were capable of entering pDCs, but failed to induce IFNa (Figure 7 and data not shown). 2/6 VLPs were comparatively defective in the ability to enter pDCs (data not shown), thus supporting the hypothesis that DLPs fail to induce IFNa due to inefficient viral entry. The ability of infectious SA11, the parental virus from which the 2/4/6/7 VLPs were derived, to induce IFNa production ( Figure 7) suggests that the lack of IFNa following inoculation with 2/4/6/7 VLPs was due to the absence of a viral genome, and not to strain variation in surface proteins. Consistent with this hypothesis, IFNa was detected in very few pDCs exposed to empty TLPs, as compared to pDCs exposed to equal quantities of genomecontaining TLPs. Instead, IFNa production by pDCs exposed to empty particles was consistent with that observed in pDCs inoculated with an equal infectious dose of TLPs, suggesting that residual contaminating viral genome was responsible for IFNa production by the empty TLPs. Together, these data indicate that viral entry, mediated by VP4 and/or VP7, and the viral dsRNA genome are likely required for rotavirus-induced IFNa production by pDCs.
Endosomal acidification is required for initiation of the pDC IFNa response to rotavirus To begin to characterize the mechanism by which pDC recognition and subsequent induction of the IFNa response occurs following rotavirus exposure, pDCs were treated with concanamycin A, a potent inhibitor of endosomal acidification, and thus TLR7/9 function [9, 51, 52] , prior to inoculation with either live or inactivated RRV at an moi of 10 ( Figure 8A-8C) . Subsequent examination of intracellular IFNa production revealed significant inhibition in concanamycin-treated pDCs compared to untreated cells (mean 6 SEM, RRV: 83.3%64.6; iRRV: 83.2%64.5; p = 0.01; Mann-Whitney). To determine whether the observed lack of IFNa production was due to a defect in viral entry, concanamycin-treated pDCs were examined for NSP2 expression. Interestingly, NSP2 expression was significantly increased in concanamycin-treated pDCs compared to untreated cells ( Figure 8D and 8E) (mean fold increase: 5.9; p = 0.03; Wilcoxon signed rank test; n = 6). These data indicate that concanamycininduced inhibition of endosomal acidification does not inhibit viral infection of pDCs and hence, presumably, does not restrict viral entry. These data support the conclusion that concanamycin A treatment prevents the pDC IFNa response at the level of ligand recognition, implicating TLR7 or TLR9. Additionally, the data suggest that the observed low levels of viral replication in pDCs ( Figure 1D ) are due, at least in part, to suppression by the pDC IFN response, as NSP2 expression significantly increases following IFNa inhibition ( Figure 8C and 8D) . Notably, these experiments demonstrate that endosomal acidification is required for the pDC IFNa response to rotavirus, and provide indirect evidence for the involvement of an endosomal receptor, such as TLR7 or TLR9, in the induction of this response.
pDCs play a vital role in the generation of innate and adaptive immunity following viral infection, primarily through the production of large quantities of IFNa in response to stimulus of TLR7 or TLR9 PRRs by ssRNA or DNA PAMPs, respectively. Traditional receptors for dsRNA, such as RIG-I, MDA5 and TLR3, are largely thought to not play a role in the initiation of the IFN response in human pDCs [2, 3, 4, 5, 6, 7, 8, 9] . Although Newcastle disease virus normally elicits an IFNa response in murine pDCs through triggering TLR7, RIG-I-like helicases have been recently implicated in IFNa induction by replicating virus in murine pDCs lacking the IFN receptor [14] . Hence, the mechanisms governing efficient IFNa induction in these cells by transcriptionally inactivated, replication-incompetent dsRNA rotavirus are not yet understood. As rotaviruses represent a large burden of human disease, it will be important to better understand the immune mechanisms underlying their recognition and subsequent generation of antiviral immunity.
In the present study, we demonstrate that primary human pDCs are largely resistant to rotavirus replication, as intracellular Representative histograms showing pDC activation (C) or maturation (D) following exposure to MA104 supernatant alone (dotted line), CpG 2395 (4 mg/ml; shaded) or RRV (moi 5; bold line). (E-H) To determine whether viral replication affected the pDC response, cells exposed to live RRV were gated based on NSP2 expression. The expression of CD86 (E, G) and CD83 (F, H) by NSP2 + and bystander (NSP2 -) pDCs was subsequently assessed. (E, F) Summary of percent CD86 + (E) or CD83 + (F) pDCs in NSP2 + or bystander pDC populations from multiple donors 12hpi (n = 8-9); p#0.0078; Wilcoxon signed rank test. (G, H) Representative histograms showing pDC activation (G) or maturation (H) of NSP2 + (bold) or NSP2bystander (thin line) pDCs in cultures exposed to RRV (moi 5) or mock stimulus (dotted line). doi:10.1371/journal.ppat.1000931.g002 Figure 3 . Intracellular production of IFNa by pDCs after exposure to live or inactivated RRV. (A) Representative FACS plots of IFNa vs. CD123 expression by pDCs exposed to mock, iRRV or RRV stimulus 2-6hpi (moi 5). (B) Time course of IFNa induction in pDCs after mock or RRV (moi staining for NSP2 was observed in only a small percentage (#5%) of cells following RRV exposure, even at a non-physiologically relevant moi of 100. While productive infection was observed in a subset of donors, this was significantly less than that observed in cells highly permissive for rotavirus infection [53] . As rotavirus is generally a lytic virus [31, 54, 55] , the observed lack of significant death in pDC preparations exposed to live RRV, compared to those receiving mock stimulus or inactivated virus, further indicates that few pDCs are permissive to viral replication. These findings are in agreement with previous reports of low frequencies of pDC infection in virus models such as herpes simplex virus 2 [56] , Dengue [57] , human cytomegalovirus [17] , influenza [58] , HCV [24] and HIV [59] . The inability of the primary human peripheral pDC population to be uniformly infected by rotaviruses may be attributable to multiple factors, including age of the individual pDC, stage of the cell cycle, or cell-surface marker expression. Additionally, functionally-distinct pDC subsets have been recently identified on the basis of CD2 expression [60] ; studies are currently underway to elucidate the role of these subsets in the pDC response to rotavirus.
Consistent with the general resistance to rotavirus infection, pDCs retained several important functional abilities following RRV exposure, as evidenced by their activation, maturation and cytokine production. Evidence of this functionality was observed primarily in the major NSP2bystander population, although activation and maturation were significantly enhanced in the few NSP2 + pDCs, as well. The induction of this phenotype by inactivated RRV further indicates that the pDCs are responding to the presence of input virus or to secreted factors, and not to viral replication. The upregulation of DC costimulatory and maturation markers suggests antigen presentation to T cells is preserved, in line with previous reports that pDCs are necessary for stimulation of IFNc-secreting memory T cells [42] . The antigen presentation hypothesis is supported by increased expression of CD86 and CD83 on NSP2 + pDCs compared to bystander pDCs, indicating that the presence of virus enhances this phenotype. Conversely, IFNa/b production is impaired in NSP2 + pDCs. Combined with decreased IRF7 phosphorylation in NSP2 bright pDCs, this suggests the direct inhibition of the type I IFN response by rotavirus replication, as has been previously observed in fibroblasts and epithelial cells [45, 46, 47, 48] .
This study is the first to demonstrate that rotavirus, a segmented dsRNA virus, directly induces IFNa and IFNb production by primary human pDCs. While it has previously been reported that exposure to rotavirus induces IFNa secretion by murine FLT3 ligand-driven pDCs [39] and total human PBMCs [42] , and that this response required pDCs [42] , it was unclear until now whether pDCs produced IFNa directly in response to virus, or if pDCs amplified IFNa production in response to type I IFN or some other signal from a non-pDC. In a limited number of experiments, Mesa et al [42] observed not only pDCs singly positive for IFNa or rotavirus, but also cells double-positive for rotavirus and IFNa at a greater frequency than that reported here. It is important to note that Mesa et al used NSP4 as a marker for rotavirus infection. Extracellular NSP4 has been identified on uninfected cells [61] . Conversely, NSP2, employed in this report, to date has only been detected intracellularly. As such, the percentage of cells identified as positive for rotavirus on the basis of NSP4 staining could be inflated versus those expressing NSP2, as NSP4 staining may include pDCs not truly supporting viral replication; in turn, the percent of NSP4 + IFNa + pDCs would be artificially increased. This, combined with differences in sample size, may explain the discrepancies in the incidence of NSP4 + IFNa + pDCs (n = 3) observed by Mesa et al and the frequency of NSP2 + IFNa + pDCs (n = 27) reported here.
pDCs possess a seemingly unique phenotype in the context of rotavirus infection, as the ability of NSP1 to degrade multiple IRFs appears to subvert the type I IFN response in many other cells, including epithelial and fibroblastic cells [45, 46, 62] . Presumably, the constitutive expression of IRF7 in pDCs [63] allows for rapid induction of IFNa following rotavirus exposure, thus effectively suppressing viral replication-and the production of NSPs-in the majority of pDCs through the establishment of a largely paracrineinduced antiviral state. This conclusion is supported by the observed lack of significant increase in the NSP2 + or IFNa + pDC populations when the moi is increased to 25 or 100 (data not shown). Importantly, the decrease in IFNa and pIRF7 in pDCs expressing high levels of NSP2 illustrates that the local suppression of the interferon response by actively replicating virus [45, 46, 47, 48] is conserved in human pDCs at an individual cell level. However, inhibition in this minor population is unlikely to substantially modulate the amount of cytokine produced by the total pDC population. The present study provides the first formal demonstration of type I IFN inhibition in primary human cells in which rotavirus replicates.
The uniqueness of pDCs in the context of rotavirus infection is further demonstrated by the ability of trypsinized and nontrypsinized rotavirus to induce IFNa and NSP2 expression at similar frequencies. The long-standing understanding of the requirement for VP4 proteolytic cleavage by luminal trypsin to activate viral entry and infection in the gut led to the assumption that efficient rotavirus infection could not occur systemically due to absence of appropriate extracellular proteases. Rotavirus infection was thought to be largely constrained to the intestine because this was the only anatomical location with substantial amounts of active extracellular trypsin available. The apparent dispensability of this proteolytic requirement for pDC infection may represent an alternate mechanism of rotavirus entry. This ''non-trypsin dependent'' mechanism of infectious entry might also account for the low levels of systemic organ infection and spread that normally occurs during rotavirus infection, and the elevated levels seen during rotavirus infection of highly immunocompromised people or animals, where the systemic availability of trypsin to proteolytically activate rotavirus is unlikely. Additionally, this provides a mechanism for the establishment of an antiviral state systemically, where circulating virus would not be expected to be able to infect cells in the traditional, trypsin cleavage-dependent, manner.
We have demonstrated that both live and inactivated rhesus rotavirus efficiently stimulate secretion of type I IFN, in addition to many other cytokines, suggesting that transcription of viral nucleic acid is not required for either viral recognition or subsequent cytokine production by primary human pDCs. Conversely, viral replication is required for activation of the IFN response in fibroblasts [62] . Indeed, further analysis of the viral requirements for IFNa induction suggests that pDCs recognize and respond to input rotavirus dsRNA or some degradation product of the rotavirus genome. This is supported by the inability of genome-free TLPs to effect IFNa secretion, indicating that the lack of IFNa induction following VLP-5) stimulus (n = 3-30). *: p = 0.0313; ***: p#0.0002; Wilcoxon signed rank test. (C) IFNa induction and NSP2 production in pDCs exposed to RRV in the presence or absence of neutralizing VP4 (2G4) or VP7 (159) mAb. doi:10.1371/journal.ppat.1000931.g003 stimulus is due to the absence of a nucleic acid ligand and not to the lack of a minor structural protein, such as VP1 or VP3. To our knowledge, this is the first direct demonstration of the requirement for native viral dsRNA in the initiation of an IFNa response from primary human pDCs.
Importantly, this finding may represent an alternative mechanism of IFNa induction in primary human pDCs. The ability of a pDC that takes up rotavirus to mount a vigorous type I IFN response independently of rotavirus mRNA or protein expression provides a potential mechanism by which the host could effectively circumvent the anti-IFN effects of NSP1-mediated IRF degradation [62] . At least three possible mechanisms exist for this phenotype: first, input or self nucleic acid released from necrotic or apoptotic cells may serve as PAMPs in neighboring cells, as has been previously described [64] . As RRV exposure, either to replicationcompetent or inactivated virus, does not induce substantial pDC death (Figure 1) , and as the cytokine response occurs rapidly following infection, this scenario seems unlikely. Secondly, input viral dsRNA may be exposed in the cytosol, the traditional cellular location for rotavirus replication, through the process of viral replication and interact with a cytosolic receptor such as RIG-I or protein kinase R (PKR). Rotavirus and reovirus, both members of the Reoviridae family, have been demonstrated to induce IFNb production in epithelial and human embryonic kidney 293T cells, respectively, via RIG-I, but not PKR or TLR3 [65, 66] . The mechanism by which input viral genomic RNA recognition might occur is unclear, however, as viral dsRNA is thought to remain encapsidated in the DLP and not be free in the cytoplasm during viral replication. Finally, rotavirus particles may be taken up by and degraded within the endosome, where dsRNA or its degradation products, including ssRNA fragments or a single strand of the dsRNA [12] , would be able to interact with TLR3, TLR7 or TLR9. As a biological role for known dsRNA receptors, such as RIG-I, PKR or TLR3, has not been demonstrated in human pDCs [2, 3, 4, 5, 6] , it is unlikely that these receptors are responsible for the observed IFNa induction in this system, although RIG-I like helicases have been recently implicated in the generation of a type I IFN response in pDCs from IFN receptor knockout mice [14] .
Signaling by a cytosolic receptor should be abrogated if inhibition of endosomal acidification in turn prevented rotavirus entry into the cytosol. However, the persistence and even increase of NSP2 expression following treatment with concanamycin A indicates that pDC entry by rotavirus is conserved under conditions that alter endosomal pH and decrease the pDC IFN response (Figure 8 ). (B, C) pDCs from multiple donors were exposed to RRV and the percent of IFNa + (B) (n = 26) or IFNb + (C) (n = 4) pDCs negative, dim or bright for NSP2 were determined. *:p = 0.0286, ***: p#0.0006; Wilcoxon signed rank test. Donors with fewer than 0.5% of pDCs staining positive for NSP2 were excluded from this analysis. (D) Representative histograms of IRF7 phosphorylation in NSP2 bright (red), NSP2 dim (blue), and NSP2 -(green) pDCs 6 hours post RRV or mock (gray) stimulus. (E, F) Percent increase in pIRF7 (E) or total IRF7 (F) MFI by indicated pDC populations relative to mock treated pDCs from multiple donors (n = 6), 6 hours post RRV inoculation. *: p = 0.0313; Wilcoxon signed rank test. doi:10.1371/journal.ppat.1000931.g005
Thus, the substantial reduction of the IFNa response following concanamycin A treatment supports the notion that an endosomal receptor, such as TLR7 or TLR9, is involved in the initiation of the pDC response to rotavirus. Hence we predict that dsRNA derived from input virus is exposed following viral degradation, and not replication, as inactivated RRV, but neither genome-free TLPs nor VLPs, is sufficient for pDC activation/maturation and IFNa production. While it is possible that recognition of some combination of VP4, VP7 and dsRNA are required for IFNa induction, we instead believe that viral entry-mediated by VP7 and/or VP4-and subsequent recognition of genomic dsRNA or its degradation products are the critical components of this pathway.
The induction of both IFNa and TNFa by rotavirus may create a synergistic effect in the suppression of viral replication, as has been observed following RIG-I stimulus by myxoma virus [67] . Although the precise kinetics of induction are unclear, both TNFa and IFNa are also observed in pDC supernatants following influenza infection [24, 68, 69] . Interestingly, production of IFNa is delayed until 14-20 h following infection with a laboratory strain of H1N1 influenza, at which point ,4 ng/mL per million pDCs is detectable; this production was dependent on viral replication [69] . Conversely, we report that pDCs exposed to either live or inactivated rotavirus secrete approximately 10 ng/mL of IFNa by 6hpi. In agreement with previous findings [14] , the recognition of secreted IFNa is an important component of the amplification of the pDC IFNa response to both live and inactivated rotavirus. As such, the recognition of input viral genome and subsequent induction of an initial IFNa response prior to possible IRF7 inhibition allows for the establishment of an antiviral state by the global pDC population. While inactivated herpes simplex virus also induces IFNa, replication of vesicular stomatitis virus, Sendai virus and human respiratory syncytial virus has been demonstrated to be required for IFNa induction in pDCs [70, 71] . Together, this demonstration of rapid, potent, replication-independent induction of cytokine by a dsRNA virus, largely dependent on endosomal acidification, points to a novel mechanism leading to production of IFNa by human pDCs.
Peripheral pDCs likely represent a significant source of the systemic IFNa observed following in vivo rotavirus infection [43, 72, 73, 74] ; the dispensability of proteolytic activation of VP4 for pDC infection and IFNa stimulation by rotavirus provides a mechanism for the establishment of the antiviral state extraintestinally by a small amount of virus. Increased levels of serum IFNa have been reported in human pediatric patients infected with rotavirus [43, 44] ; levels peaked within two days of symptom onset and disease resolved more quickly in patients with the highest levels of IFNa [43] . Likewise, type I IFN responses were observed in newborn calves infected with bovine rotavirus: animals receiving both high and low doses of viral inoculums generated multiple waves of IFN production, with higher doses correlating with both more rapid IFN responses and a lack of clinical signs [74] . As the ability of type I IFN to activate multiple arms of the innate and adaptive immune response is well documented (reviewed in [75, 76, 77] ), and recent data have shown that type I IFN plays a critical role in restricting systemic rotavirus infection [78] , we predict that pDC-derived type I IFN plays a significant role in confining rotavirus replication to the intestine while initiating the systemic immune response in the periphery.
In summary, these studies show that primary human pDCs are largely resistant to rotavirus replication, but that they remain responsive and functional following viral exposure, as indicated by cellular activation, maturation and production of multiple cytokines, including TNFa and large amounts of IFNa. The pDC response is demonstrated to require both the viral dsRNA genome and acidification of the pDC endosome, implying the likely initiation of the type I IFN response by TLR7 or TLR9. To our knowledge, these data are the first to show that inactivated rotavirus can efficiently induce a type I IFN response, which differs from what has been seen in fibroblasts and epithelial cells exposed to rotavirus [62] . The restricted ability of rotavirus to replicate in pDCs, combined with the ability of the pDC to effect a type I IFN response in the absence of viral replication, likely serves the purposes of enabling the host to mount an innate antiviral response despite the efficient degradation of the IRFs by rotavirus NSP1 in many host cells. Combined with the novel dispensability of trypsin cleavage of VP4 for the initiation of the pDC response, this creates a mechanism for the establishment of an antiviral state in the systemic extraintestinal environment and likely contributes to the substantial restriction of systemic replication compared to that observed in the intestine [38] . These experiments also provide the first indication that rotavirus infection is capable of locally suppressing IFNa production in primary human cells. Importantly, these findings demonstrate that rotavirus infection of primary human pDCs provides a valuable experimental system for the study of cellular processes, receptors and signaling pathways underlying pDC-mediated antiviral immunity while revealing a potentially novel dsRNA-mediated pathway of IFNa induction.
Simian (RRV and SA11) tissue-culture adapted rotaviruses were grown in fetal monkey kidney (MA104) cells in the presence of trypsin, except where noted, as previously described [79, 80] . Where indicated, RRV empty and genome-containing TLPs were purified from MA104 cell lysates by cesium chloride density gradient centrifugation following genetron extraction and pelleting through a sucrose cushion, as previously described [81, 82] . DLPs were created by treating TLPs with 20 nM EDTA, thus removing the outer viral proteins VP4 and VP7, as previously described [32] . Purified viral preparations were dialyzed to remove residual cesium chloride prior to pDC stimulus. A portion of the RRV lysate preparation was inactivated by treatment with 40 mg/ml psoralen, followed by exposure to ultraviolet light for 40 minutes, as described [83] . The inactivation of infectivity and transcriptional activity of the RRV in the psoralen preparations was confirmed by cell culture assay and the elimination of endogenous RNA polymerase activity by in vitro assay. Except where noted, viral preparations were trypsin activated (5 mg/ml) at 37uC for 20-30 minutes prior to pDC infection. Preparations were endotoxinfree, as determined by Limulus amebocyte lysate test (Charles River, Wilmington, MA).
Rotavirus-like particles (VLPs) expressing VP2 and VP6 (2/6 VLPs), or VP2, VP6 and VP7 (2/6/7 VLPs), were generated by coinfecting Spodoptera frugiperda 9 cells with two or three recombinant baculoviruses, respectively, at a moi $5 plaque forming units per cell, as previously described [84] . Briefly, the baculoviruses expressed RF (bovine rotavirus) VP6, RRV VP7, or a fusion protein consisting of GFP fused to the N terminus of RF VP2 deleted in the first 92 amino acids. Infected cultures were collected 5-7 days post infection and purified by cesium chloride density gradient centrifugation. VLPs composed of RF VP2, SA11 Cl3 VP6, SA11 Cl3 VP7 and SA11 4F VP4 (2/4/6/7 VLPs) were similarly prepared, as previously described [85, 86, 87] . The protein concentration of the purified VLP preparations was estimated by the Bradford method.
All viral preparations were titrated by plaque assay on MA104 cells and expressed as plaque forming units per ml, as described [80] . Hemagglutination assays were performed to determine VP4 content of purified genome-containing and empty TLPs, unpurified RRV and 2/4/6/7 VLPs, as described [88] , and used to ensure that equal quantities of TLPs were added to pDC preparations. Additionally, DLPs were prepared by treating purified genome-containing TLPs with 20 nM of EDTA, thus removing the outer capsid from the virion and resulting in equal particle counts between the two preparations.
PBMCs were isolated from leukoreduction chambers obtained from the Stanford Blood Center (Palo Alto, CA) by centrifugation over ficoll-hypaque (GE Healthcare, Uppsala, Sweden). Plasmacytoid DCs were negatively selected using the pDC Isolation Kit (Miltenyi Biotec, Auburn, CA), according to manufacturer's instructions. To increase the purity of the pDC preparations, consecutive purifications were performed using an AutoMACS (Miltenyi Biotec, Auburn, CA). An average of 2 million pDCs, defined as viable, lineage -HLA-DR + CD11c -CD123 + cells, were isolated per donor; preparations were routinely .85% pure.
Approximately 2610 5 pDCs were exposed to rotavirus particles or 4 mg/mL CpG ODN 2395 (Coley Pharmaceuticals, Ottawa, Canada), as indicated, for 1 hour in serum-free RPMI-1640 (CellGro, Manassas, VA) supplemented with penicillin/streptomycin and L-glutamine (Gibco, Carlsbad CA). pDCs were washed and suspended at a concentration of 1 million pDCs/mL in RPMI-1640 with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin, L-glutamine and 10 ng/mL IL3 (Peprotech, Rocky Hill, NJ) until harvest. Where indicated, rotavirus infection was blocked using neutralizing monoclonal antibodies to VP5 (mAb 2G4) or VP7 (mAb 159), or as a non-neutralizing control, a VP6 monoclonal antibody (mAb 1E11). In studies determining whether pDC infection was productive, highly-purified pDCs (mean purity: 91.51%) were exposed to RRV at an moi of 10 for one hour; neutralizing mAb 159 was then added for 30 minutes. pDCs were washed 3 times, and were frozen immediately or after overnight culture. The resulting pDC pellets and supernatants were freeze/thawed three times, and virus titer determined by FFU assay, as previously described [89] .
Purified pDCs were incubated with 20 nM concanamycin A (Sigma-Aldrich, St Louis, MO) at 37 degrees for one hour prior to infection with live or inactivated RRV (moi 10).
Supernatants were harvested by centrifugation of cultured pDCs, which were then washed once with PBS (CellGro, Manassas, VA). The LIVE/DEAD Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) was utilized to assess cellular viability via amine exclusion. Surface staining was performed using antibodies against human CD3, CD14, CD16, HLA-DR, CD83, CD86, CD123 (BD Biosciences, San Jose, CA), CD11c, CD19, CD20 (eBioscience, San Diego, CA), and BDCA2 (Miltenyi Biotec, Auburn, CA); pDCs were defined as being lineage -HLA-DR + CD11c -CD123 + . Cellular fixation and permeabilization were performed using Cytofix/Cytoperm (BD Biosciences, San Jose, CA), per the manufacturer's instructions. Following blocking of Fc receptors (Miltenyi Biotec, Auburn, CA), intracellular staining was performed with antibodies to IFNa, total IRF7, phosphorylated IRF7 (pS477/pS479) (BD Biosciences, San Jose, CA), IFNb (Antigenix America, Huntington Station, NY) or NSP2 (mAb 191). The presence of NSP2 staining of exposed pDCs was considered to be indicative of rotaviral replication in those cells. Direct conjugation of mAb 191 to APC was performed by Chromaprobe Inc (Maryland Heights, MO). Data were acquired using a LSRII and DIVA software (BD Biosciences, San Jose, CA); analysis was performed using FlowJo (Treestar Inc, Ashland, OR).
Supernatants of cultures .85% (mean 6 SEM: 90.84%60.7499) pure for pDCs were analyzed by Luminex using MILLIPLEX MAP (Millipore, Billerica, MA) per the manufacturer's instructions for the presence of IFNa, IFNc, TNFa, TNFb, IL1b, IL1RA, IL2, IL4, IL6, IL8, IL10, IL12p40, IL12p70, CXCL10 (IP10), CCL3 (MIP1a), CCL4 (MIP1b), CCL5 (RANTES) and sCD40L. Secreted IFNb was detected by ELISA, per the manufacturer's instructions (PBL Interferon Source, Piscataway, NJ).
Mann-Whitney and Wilcoxon signed rank tests were performed using GraphPad Prism (GraphPad Software Inc, La Jolla, CA). A p-value of #0.05 was considered significant. Figure S1 pDC gating strategy. (A-D) pDCs were defined in these studies as amine -(viable) lineage -HLA-DR + CD11c -CD123 + cells. (E) Representative FACS plot demonstrating BDCA2 vs. NSP2 staining of pDCs (red) vs. contaminating HLA-DR-(blue) cells exposed to RRV (n = 12). Found at: doi:10.1371/journal.ppat.1000931.s001 (0.39 MB TIF) Role of CD14 in lung inflammation and infection This article is one of ten reviews selected from the Yearbook of Intensive Care and Emergency Medicine 2010 (Springer Verlag) and co-published as a series in Critical Care. Other articles in the series can be found online at http://ccforum.com/series/yearbook. Further information about the Yearbook of Intensive Care and Emergency Medicine is available from http://www.springer.com/series/2855. Toll-like receptors (TLR) on the surface of cells of the respiratory tract play an essential role in sensing the presence of microorganisms in the airways and lungs. Th ese receptors trigger infl ammatory responses, activate innate immune responses, and prime adaptive immune responses to eradicate invading microbes [1] . TLR are members of a family of pattern-recognition receptors, which recognize molecular structures of bacteria, viruses, fungi and protozoa (pathogen-associated molecular patterns or PAMPs), as well as endogenous structures and proteins released during infl ammation (damage/ danger-associated molecular patterns or DAMPs). To date, ten diff erent TLR have been identifi ed in humans and twelve in mice. TLR are expressed on all cells of the immune system, but also on parenchymal cells of many organs and tissues. Th e binding of a PAMP to a TLR results in cellular activation and initiates a variety of eff ector functions, including cytokine secretion, prolifera tion, co-stimulation or phagocyte maturation. To facilitate microbial recognition and to amplify cellular responses, certain TLR require additional proteins, such as lipopolysaccharide (LPS) binding protein (LBP), CD14, CD36 and high mobility group box-1 protein (HMGB-1). In this chapter, the role of CD14 as an accessory receptor for TLR in lung infl ammation and infection is discussed. Th e central role of CD14 in the recognition of various PAMPs and amplifi cation of immune and infl ammatory responses in the lung is depicted in Figure 1 .
CD14 was characterized as a receptor for bacterial endotoxin (LPS) in 1990, almost a decade before the discovery and characterization of TLR, and can be regarded as the fi rst described pattern-recognition receptor [2] .
Th e protein was fi rst identifi ed as a diff erentiation marker on the surface of monocytes and macrophages and was designated CD14 at the fi rst leukocyte typing workshop in Paris in 1982. Th e genomic DNA of human CD14 was cloned in 1988 and the gene was later mapped to chromo some 5q23-31. Several polymorphisms have been found in the CD14 gene, of which nucleotide polymorphisms at position -159 and -1619 correlated with decreased lung function in endotoxin-exposed farmers [3] .
Th e CD14 gene consists of two exons which code for a single mRNA that is translated into a protein of 375 amino acids. Th e CD14 protein is composed of eleven leucin-rich repeats, which are also found in TLR and which are important in PAMP binding. Moreover, the crystal structure of CD14 revealed that the protein has a `horseshoe' shape, similar to TLR4, and that LPS is bound within the pocket [4] . In contrast to TLR, however, CD14 lacks a transmembrane domain, and thus cannot initiate intracellular signal transduction by itself. Th e CD14 protein is processed in the endoplasmatic reticu lum and expressed as a 55 kDa glycoprotein on the cell surface via a glycosylphosphatidyl (GPI) anchor [5] . Like other GPIanchored proteins, CD14 accumulates on the cell surface in microdomains known as lipid rafts, which are fairly rich in cholesterol and accumulate several kinases at the intracellular site. CD14 is expressed pre dominantly on the surface of `myeloid' cells, such as mono cytes, macrophages and neutrophils, but at lower levels also on epithelial cells, endothelial cells and fi broblasts.
In addition to being expressed as a GPI-anchored membrane protein, CD14 is also expressed in a soluble form (sCD14) [2] . sCD14 may result from secretion of the protein before coupling to the GPI anchor or from shedding or cleavage from the surface of monocytes. sCD14 is present in the circulation and other body fl uids and levels of sCD14 in plasma increase during infl ammation and infection. Since interleukin (IL)-6 induces sCD14 expression in liver cells it is regarded as an acute phase protein. In bronchoalveolar lavage (BAL) fl uid from patients with acute respiratory distress syndrome (ARDS), sCD14 levels were strongly increased and correlated with total protein levels and neutrophil numbers in the BAL fl uid [6] , suggesting that sCD14 contributes to the infl ammatory process in the lung.
CD14 is a molecule with a wide range of functions. In addition to functioning as a pattern recognition receptor for a variety of microbial ligands, CD14 also acts as a receptor for endogenous molecules like intercellular adhesion molecule (ICAM)-3 on the surface of apoptotic cells, amyloid peptid, ceramide, and urate crystals. Ligation of CD14 by these ligands, except for apoptotic cells, mediates activation of infl ammatory responses.
LPS is the major constituent of the outer membrane of Gram-negative bacteria and is one of the most potent TLR ligands. CD14 together with LBP plays an essential role in binding of LPS to the TLR4/MD-2 complex [7] . LBP, which, among others, is present in the bloodstream and BAL fl uid [8] , binds to LPS aggregates and transfers LPS monomers to CD14. CD14 associates with TLR4/ MD-2 and transfers the LPS monomer to this complex [7] . Likewise, sCD14 is able to mediate LPS-activation of cells with low membrane CD14 expression, such as epithelial and endothelial cells [9] . However, at high con cen trations, LBP and sCD14 are also able to downregulate LPS-induced responses by transfer of LPS to lipoproteins for subsequent removal [10] . Recent data indicate that LPS is bound by MD-2 within the TLR4/ MD-2 complex [11] and that subsequent conformational changes in TLR4 lead to reorganization of its cytoplasmic domain, enabling the recruitment of the adaptor proteins, myeloid diff erentiation primary-response protein 88 (MyD88) and TIR-domain-containing-adaptorprotein-inducing-inter feron (IFN)-β (TRIF) [12] . Th ese adaptors initiate signal transduction to the nucleus by activation of nuclear factor (NF)-κB and IFN regulatory transcription factor (IRF)-3, leading to the production of cytokines that regulate infl ammatory cells [12] . In macrophages, TRIF-dependent signaling is essential for the expression of the majority of LPS-induced genes, including IFN-α/β. CD14, which lacks an intracellular domain for signal transduction, is expressed on the surface of alveolar macrophages, infi ltrating monocytes and neutrophils, and at lower levels also on epithelial and endothelial cells in the lung. CD14 recognizes and binds various structures from invading microbes, such as lipopolysaccharide (LPS) from Gram-negative bacteria, lipoteichoic acid (LTA) from Gram-positive bacteria, lipoarabinomannan (LAM) from mycobacteria, viral double stranded (ds) RNA and F glycoprotein (F-gp) from respiratory syncytial virus (RSV). CD14 subsequently transfers these bound components to Toll-like receptors (TLR) which than trigger cell activation. Binding of LPS to CD14 is regulated by additional accessory receptors in the lung, including LPS-binding protein (LBP) and a number of surfactant proteins (SP). Furthermore, soluble CD14 (sCD14) enhances LPS-induced activation of cells with low CD14 expression. Depending on the microbe and the PAMPs it expresses, CD14-amplifi ed responses can either be benefi cial to the host by induction of an adequate infl ammatory and immune response to eradicate the invading microbe, or detrimental to the host by excessive infl ammation and/or dissemination of the pathogen. Recently, it was reported that, in the absence of CD14, the TLR4/MD-2 complex can distinguish between diff erent chemotypes of LPS [13] . Smooth LPS is synthesized by most Gram-negative bacteria and consists of three modules: Th e lipid A moiety, a core poly saccharide, and an O-polysaccharide of variable length (made up of 1 to over 50 monosaccharide units) [7] . Gram-negative bacteria that fail to add the core polysaccharide or the O-polysaccharide chain to the lipid A moiety produce `rough' LPS, named after the rough morphology of the colonies these bacteria form. Lipid A, the bioactive part of both smooth and rough LPS, is responsible for most of the pathogenic eff ects in Gram-negative bacterial infections [7, 12] . Murine macrophages lacking CD14 secreted equal amounts of tumor necrosis factor-α (TNF) to macrophages expressing CD14 upon stimulation with rough LPS, but failed to secrete TNF in response to smooth LPS, an eff ect which was reversed by addition of sCD14 [13] . Moreover, macrophages lacking CD14 failed to secrete IFN-α/β in response to either rough or smooth LPS. Th ese fi ndings indicate that CD14 is required for activation of the TLR4/TRIF pathway by either smooth or rough LPS, and required for the activation of TLR4/ MyD88 pathway by smooth but not by rough LPS [13] . In addition to LPS, CD14 also facilitates TLR4 activation by other PAMPs including certain viral components [13, 14] .
In the lung, binding of LPS to TLR4 is infl uenced by a number of surfactant proteins (SP), including SP-A, SP-C and SP-D [15] . Th ese surfactants are able to infl uence the interaction between TLR4 and LPS by direct binding to LPS; i.e., SP-A binds to rough LPS and lipid A, but not to smooth LPS, SP-C also binds to rough LPS, and SP-D binds to both rough and smooth LPS. SP-A and SP-C binding to LPS inhibits TNF secretion by alveolar macrophages, whereas SP-D binding to LPS moderately enhances TNF secretion by alveolar macrophages. In addition, SP-A, SP-C and SP-D also bind to CD14 at the site which recognizes LPS. Strikingly, binding of SP-A to CD14 enhanced the binding of rough LPS and binding of SP-C to CD14 augmented binding of smooth LPS [15] , whereas binding of SP-A to CD14 reduced binding of smooth LPS and binding of SP-D to CD14 decreased binding of both smooth and rough LPS. Furthermore, SP-D infl uences LPS-induced TNF secretion by alveolar macrophages by regulating matrix metalloproteinasemediated cleavage of CD14 from the surface of these cells [16] .
Together, these fi ndings suggest that LPS recognition in the lung and subsequent induction of infl ammatory immune response is a complexly regulated process.
In addition to LPS-induced activation of TLR4, CD14 also amplifi es a number of TLR-dependent responses triggered by other bacterial PAMPs, including peptidoglycan, lipoteichoic acid (LTA) and lipoarabinomannan (LAM) [17] [18] [19] .
Peptidoglycan is an essential cell wall component of virtually all bacteria. Peptidoglycan is a polymer of Nacetylglucosamine and N-acetylmuramic acid, crosslinked by short peptides. Breakdown products of peptido glycan are recognized by diff erent classes of pattern-recognition receptors [19] . Polymeric soluble peptidoglycan is recognized by TLR2 on the surface of cells, and the interaction of peptidoglycan with TLR2 triggers MyD88-dependent activation and nuclear translocation of NF-κB, and subsequently the transcription and secretion of cytokines. Muramyl dipeptide and γ-Dglutamyl-meso-diaminopimelic acid, which are lowmolecular weight breakdown fragments of peptidoglycan, are recognized by intracellular pathogen recognition receptors, nucleotide-binding oligomerization domain containing (Nod)2 and Nod1, respectively [19] . Ligand binding to these receptors triggers interaction with the receptor-interacting protein kinase, RIP2, which activates NF-κB. Of these peptidoglycan breakdown products, only polymeric peptidoglycan binds to CD14, and CD14 enhances polymeric peptidoglycan-induced TLR2 activation. Th e low molecular weight fragments of peptidoglycan, like muramyl dipeptide, do not bind to CD14, do not induce cell activation through CD14 and also do not interfere with the binding of polymeric peptidoglycan to CD14 [19] . Furthermore, unlike LPS, peptidoglycan bound to sCD14 is not able to activate epithelial and endothelial cells with low membrane CD14 expression.
LTA is a constituent of the cell wall of Gram-positive bacteria, anchored on the outer face of the cytoplasmic membrane and commonly released during growth and antibiotic therapy. Like polymeric peptidoglycan, LTA induces NF-κB activation and cytokine secretion in a TLR2-dependent manner. LTA is recognized by LBP and CD14, and these accessory receptors both enhance LTAinduced cell activation [18] . Presumably in a similar manner, CD14 also enhances TLR2-dependent cellular activation by LAM derived from the cell-wall of mycobacteria. LAM derived from slowly growing virulent mycobacteria like Mycobacterium tuberculosis and M. leprae is capped with mannose (ManLAM), whereas LAM from avirulent and fast growing mycobacterial species is uncapped (AraLAM). Strikingly, AraLAM from avirulent mycobacteria is much more potent in inducing TNF secretion by macrophages than ManLAM from virulent mycobacterial strains [12] . AraLAM-, but not ManLAM-induced TNF secretion by monocytes and macrophages was largely CD14-, TLR2-and MyD88dependent [17] .
Recently CD14 was also found to enhance the innate immune response triggered by the TLR3 ligand poly(I:C), a synthetic mimic of double stranded RNA [20] . TLR3 together with TLR7 and TLR8 are regarded as sensors for viral infection, since these receptors recognize viral nucleic acids, like single and double stranded RNA. Th e potentiating eff ect of CD14 on TLR3 activation resulted from increased uptake of poly(I:C) and intracellular delivery to the compartment where TLR3 resides [20] . Taken together, these fi ndings suggest that CD14 plays an important role in the induction and amplifi cation of infl ammatory responses evoked by a wide variety of pathogens.
Th e contribution of CD14 to TLR ligand-induced lung infl ammation has been investigated in several animal studies (Table 1) . Intratracheal administration of LPS did not signifi cantly induce TNF release and neutrophil accumulation in the lungs of rabbits, unless LPS was complexed with LBP [21] or the animals were subjected to mechanical ventilation [22] . Intratracheal instillation of anti-CD14 antibodies together with LPS/LBP or intravenous pretreatment with anti-CD14 or anti-TLR4 antibodies before mechanical ventilation markedly reduced these infl ammatory responses [21, 22] . Despite a reduction in lung neutrophil number, intravenous anti-CD14 treatment of rabbits exposed to LPS and subjected to ventilation did not cause a decrease in lung chemokines, including CXCL8 (IL-8), growth related oncogene (GRO) and monocyte chemoattractant protein (MCP)-1, whereas anti-TLR4 treatment did lower the level of GRO moderately and of CXCL8 signifi cantly [22] . Th ese fi ndings reveal that LPS alone does not cause signifi cant lung infl ammation in rabbits and suggest that additional accessory signals are required. Whether mechanical ventilation induces increased release of LBP or release of (endogenous) DAMPs which potentiate the LPS-induced response remains to be determined.
In contrast to rabbits, administration of LPS alone to lungs of naive mice induced severe pneumonitis, irrespective of the manner of LPS delivery (inhalation or intra tracheal or intranasal instillation) or the source of LPS (Escherichia coli or Acinetobacter baumannii). Using antibody-treated and gene-defi cient mice, CD14 was found to be critically involved in the development of LPS-induced lung infl ammation [23] [24] [25] [26] . A study with CD14-defi cient mice and TLR4 mutant mice (lacking a functional TLR4) showed that LPS-induced vascular leakage, neutrophil infi ltration, nuclear translocation of NF-κB. Th e release of cytokines (TNF and IL-6) and chemo kines (CXCL1 and CXCL2) in the lung was completely dependent on these pattern recognition receptors [24] . Similar observations were made by others using mice treated intravenously with anti-CD14 Infl uenza A mouse CD14 -/-/~clearance, ~lymphocyte recruitment and activation, ~neutrophil infl ux, 50 ~cytokines antibodies [23] and by our group using CD14-defi cient and TLR4-defi cient mice [25] . Furthermore, intratracheal treatment of CD14-defi cient mice with sCD14 restored the infl ammatory response to the level present in wildtype mice, whereas treatment with wild-type alveolar macrophages restored the neutrophil infi ltration of the lung but not pulmonary TNF release [26] . Moreover, treatment with wild-type alveolar macrophages also restored neutrophil infi ltration in the lung of LPSexposed TLR4-defi cient mice [27] . Th ese fi ndings indicate that sCD14, and CD14 and TLR4 on the surface of alveolar macrophages contribute to the development of LPS-induced lung infl ammation. However, when a high dose of LPS was administered to the lungs of mice, acute lung infl ammation was absent in mice lacking functional TLR4, but only partially reduced in CD14 defi cient mice [24] . Th us, LPS-induced lung infl am mation is entirely dependent on TLR4 and, depending on the dose of LPS, also on the presence of CD14 in the lung.
Our group determined whether CD14 also contributes to the development of lung infl ammation induced by LTA, a TLR2 ligand from the cell wall of Gram-positive bacteria [28, 29] . Lung infl ammation induced by Staphylo coccus aureus LTA was completely dependent on TLR2, but independent of LBP and only moderately dependent on CD14 expression. As compared to wildtype mice, S. aureus LTA-induced neutrophil infl ux was unchanged in CD14-defi cient mice, whereas TNF and CXCL2 release in the lung were partially reduced [28] . Strikingly, however, pulmonary infl ammation was also greatly diminished in TLR4-defi cient mice, as well as in mice defi cient for platelet activating factor receptor (PAFR), a known receptor for LTA on epithelial cells. Similarly, lung infl ammation induced by Streptococcus pneumoniae LTA, which is less potent compared S. aureus LTA, was also completely dependent on TLR2 expression. However, in contrast to S. aureus LTA, neutrophil infi ltration of the lung was moderately reduced in CD14-defi cent mice treated with pneumococcal LTA, whereas TNF and CXCL2 release in the lung was unchanged [29] . Moreover, pneumococcal LTAinduced lung infl ammation was moderately diminished in TLR4-defi cient mice. Th us, despite the amplifying eff ect on LTA-induced TLR2-mediated responses in vitro, CD14 contributes minimally to lung infl ammation induced by LTA. Th e unexpected contribution of TLR4 to LTA-induced lung infl ammation may result from DAMPs generated during the infl ammatory process in the respiratory tract.
In line with the fi ndings that CD14 contributes to LPSinduced lung infl ammation in mice, a number of studies have shown that CD14 is essential for the host defense response in the lung against Gram-negative bacteria, such as nontypeable Haemophilus infl uenzae, a possible cause of community acquired pneumonia, and A. baumannii and E. coli, which are frequent inducers of nosocomial pneumonia (Table 1) . Nontypeable H. infl uenzae expresses the TLR4 ligands LPS and lipooligosaccharide on its cell wall, as well as several TLR2 ligands, including lipoproteins and porins. Previously, we found that activa tion of alveolar macrophages by nontypeable H. infl uenzae depended on expression of TLR4, TLR2, and CD14 [30] . Moreover, bacterial clearance after intranasal infection with nontypeable H. infl uenzae was markedly reduced in CD14-defi cient and TLR4-defi cient mice, as well as in TLR2-defi cient mice at later stages of the disease [30] . Interestingly, despite impaired bacterial clearance in CD14-defi cient and TLR4-defi cient mice, the infl ammatory response in the lung was strongly reduced in TLR4 defi cient mice, but elevated in CD14 defi cient mice. Similar observations were made with encapsulated H. infl uenzae in TLR4-mutant mice [31] . Furthermore, clearance of nontypeable H. infl uenzae was also significantly impaired in MyD88-defi cient mice, but not in mice lacking functional TRIF [30] . In a similar manner, CD14 was involved in the host defense response against A. baumanii [25] . CD14-defi cient mice, like TLR4defi cient mice, suff ered from impaired bacterial clearance in the lungs and enhanced bacterial dissemination after intranasal infection with A. baumannii. However, unlike TLR4-defi cient mice, CD14-defi cient mice developed similar infl ammatory responses compared to wild-type mice. Th ese fi ndings suggest a role for CD14 in antibacterial responses against nontypeable H. infl uenzae and A. baumannii. Although the role of TLR4 (and TLR2) in phagocytic killing is controversial, it is unknown whether CD14 is involved in such processes. Th e role of CD14 in E. coli-induced pneumonia was determined in anti-CD14 antibody treated rabbits. Intravenous anti-CD14 antibody treatment of rabbits inoculated with E. coli by bronchial instillation, resulted in decreased bacterial clearance from the lungs, but had no eff ect on neutrophil infi ltration or cytokine release in the lungs [32] . However, anti-CD14 treatment protected against sustained hypotension and reduced the levels of nitrate and nitrite in the blood. Th e contribution of CD14 to E. coli-induced pneumonia has not been investigated in mice, whereas the role of the other components of the LPS receptor complex (TLR4, MD-2, MyD88, TRIF) has been determined using gene-defi cient or mutant mice. Although analysis of bacterial clearance after intranasal infection of TLR4-mutant mice with E. coli produced inconsistent results [33] , lack of MD-2 or TRIF resulted in impaired bacterial clearance after E. coli instillation in the lungs [34, 35] . Moreover, E. coli-induced neutrophil accumulation and cytokine release was signifi cantly reduced in mice devoid of functional TLR4, MD-2, MyD88 or TRIF [33] [34] [35] . Th ese fi ndings indicate that signaling through the TLR4 receptor complex is essential in the host defense response against E. coli, and suggests that CD14 may contribute to these E. coli-induced responses.
To our knowledge, it is unclear whether CD14 contributes to host defense against Pseudomonas aeruginosa, a frequent cause of nosocomial pneumonia, and Burkholderia cepacia, a prevalent Gram-negative bacterium, together with P. aeruginosa, in patients with cystic fi brosis. Recently, it was found that both TLR4 and TLR5 are critical in the host response to P. aeruginosa and that TLR4-defi cient mice were not susceptible to intratracheal P. aeruginosa infection unless a bacterial mutant devoid of fl agellin production was used [36] . A similar approach is required to determine a role for CD14 in Pseudomonas-induced pneumonia. It is plausible that CD14 also contributes to the host response against B. cepacia, since LPS from this bacterium signals through TLR4 and anti-CD14 antibodies dramatically inhibited B. cepacia-induced chemokine secretion by lung epithelial cells [37] . Whether CD14 contributes to host defense response against Klebsiella pneumoniae, a known cause of nosocomial pneumonia, also remains to be determined, but data from our study with TLR4-mutant mice indicate that signaling through TLR4 is essential for successful clearance of this bacterium [38] .
In contrast to the essential role of pulmonary TLR4 and CD14 in the host defense response against most Gramnegative bacteria, we found that TLR4 was not involved and CD14 played a remarkable detrimental role in the host response to B. pseudomallei, the causative organism of melioidosis (the most common cause of communityacquired sepsis in Southeast Asia) [39, 40] . CD14defi cient mice infected intranasally with B. pseudomallei were protected from mortality, accompanied by enhanced bacterial clearance in the lung, blood and liver, and reduced cellular infi ltration in the lung [39] , whereas the course of disease in TLR4-defi cient mice was indistinguishable from wild-type mice [40] . Moreover, intranasal administration of sCD14 to CD14-defi cient mice partially reversed the phenotype into that of wild-type mice [40] . Interestingly, these fi ndings in B. pseudo mallei-infected CD14-defi cient mice strongly resemble our previous results found with TLR2-defi cient mice, and are in line with the observation that B. pseudomallei expresses an atypical LPS which signals through TLR2 [39] . Whether CD14 interacts with TLR2 in B. pseudo mallei-induced responses, and by which mechanism these receptors facilitate the growth and dissemination of B. pseudomallei after intranasal infection remains to be determined.
In the model for S. pneumoniae-induced pneumonia, we observed an unexpected detrimental role for CD14 in the innate host defense response. S. pneumoniae, a Gram-positive bacterium and the single most frequent pathogen causing community-acquired pneumonia, induces severe lung infl ammation and sepsis in wild-type mice after intranasal instillation. Strikingly, CD14defi cient mice were protected against pneumococcal pneumonia, presumably as a result of reduced bacterial spread to the circulation and reduced lung infl ammation [41] . In contrast, TLR2-defi cient and TLR4-mutant mice were not protected against pneumococcal pneumonia [38, 42] , but in fact TLR2 seemed redundant for effi cient bacterial clearance and TLR4-mutant mice were more susceptible to pneumonia, accompanied by impaired bacterial clearance. However, as in CD14-defi cient mice, lung infl ammation was also reduced in pneumococciinfected TLR2-defi cient mice [42] . Since intrapulmonary treatment with sCD14 rendered CD14-defi cient mice equally susceptible to S. pneumoniae as wild-type mice [41] , these results suggest that S. pneumoniae abuses (s) CD14 in the lung to cause invasive respiratory tract infection. Interestingly, the phenotype of CD14 defi cient mice strongly resembled the phenotype of mice defi cient for PAFR [43] , a receptor for phosphoryl choline from the pneumococcal cell wall which facilitates pneumococcal invasion of cells. Further studies are required to determine whether CD14 serves as a chaperone in the presentation of S. pneumoniae to the PAFR so that the phosphoryl-PAFR-mediated invasion is facilitated.
Since M. tuberculosis expresses a number of molecules, such as lipoproteins, which activate immune cells in a CD14-dependent manner, we and others investigated whether CD14 also contributed to the host immune response in mice with lung tuberculosis [44] . Although initially after intranasal infection of wild-type and CD14defi cient mice no diff erences in bacterial loads, cell infi ltration and release of most cytokines in the lung were found [44, 45] , at later time points (> 20 weeks after infection) CD14-defi cient mice were protected from mortality presumably as a result of a reduced infl ammatory response in the lungs [44] . Th ese fi ndings are completely opposite to the results from M. tuberculosisinfected TLR2-defi cient and TLR4-mutant mice, which suff ered from reduced bacterial clearance, chronic infl ammation, increased cellular infi ltration of the lungs and reduced survival [46] [47] [48] . Th e mechanism underlying the detrimental eff ect of CD14 in the host response against M. tuberculosis remains to be established.
In addition to its role in (myco)bacterial infections, CD14 may also play a role in the pulmonary host response against respiratory syncytial virus (RSV), the most common cause of lower respiratory tract disease in infants and young children worldwide, and infl uenza A virus, a cause of pneumonia in very young children, the elderly and immunocompromised patients. Th e envelop F glycoprotein from RSV and certain infl uenza A virus components activate macrophages in a CD14-dependent manner [14, 20] . Experiments with wild-type and TLR4mutant mice infected intranasally with RSV showed that viral clearance was reduced in the absence of functional TLR4 [14] , due to impaired natural killer (NK) cell migration and function and impaired cytokine secretion. Recently, it was found that TLR2 and TLR6 are also involved in recognition of RSV [49] . Whether CD14 contributes to these TLR-mediated immune responses against RSV remains to be determined. Using CD14defi cient mice, we demonstrated that CD14 played a minimal role in infl uenza A virus-induced pneumonia [50] . During the entire course of disease, viral loads were slightly reduced in CD14-defi cient mice, but this did not result from improved lymphocyte recruitment or lympho cyte activation, or consistent changes in pulmonary cytokines [50] . Th us, despite the fact that infl uenza A expresses ligands that require CD14 for immune cell activation [20] , CD14 seems redundant in the host defense response against infl uenza A virus.
CD14 plays a central role in the lung in the recognition and binding of a variety of (myco)bacterial and viral components, and in the amplifi cation of subsequent host responses. Th e studies discussed in this chapter indicate that the contribution of CD14 to the pulmonary host defense responses may range from benefi cial to detrimental, depending on the microbe and the PAMPs it expresses. Interfering with CD14-LPS or CD14-LTA inter actions reduced lung infl ammation. Interference with CD14-pathogen interactions, however, did not have a signifi cant eff ect on M. tuberculosis or infl uenza A virus infection, resulted in reduced clearance of nontypeable H. infl uenzae, E. coli or A. baumannii in the lung, but enhanced clearance (and reduced dissemination) of B. pseudomallei or S. pneumoniae. Th e latter observation indicates that certain pathogens may abuse CD14 in the lung to cause invasive disease. Whether CD14 is a suitable target for intervention in these latter infectious diseases and/or in aberrant infl ammatory responses during pneumonia requires further study.
Abbreviations ARDS = acute respiratory distress syndrome, BAL -broncoalveolar lavage, DAMP = damage/danger-associated molecular pattern, F-gp = F glycoprotein, GPI = glycosylphosphatidyl, GRO = growth related oncogene, HMGB-1 = high mobility group box-1 protein, ICAM = intracellular adhesion molecule, IFN = interferon, IL = interleukin, IRF = IFN regulatory transcription factor, LAM = lipoarabinomannan, LBP = lipopolysaccharide binding protein, LPS = lipopolysaccharide, LTA = lipoteichoic acid, MCP = monocyte chemoattractant protein, MyD88 = myeloid diff erentiation primary-response protein 88, NF = nuclear factor, NK = natural killer, Nod = nucleotide-binding oligomerization domain containing, PAFR = platelet activating factor resceptor, PAMP = pathogen-associated molecular pattern, RIP = receptorinteracting protein kinase, RSV = respiratory syncytial virus, SP = surfactant protein, TLR = Toll-like receptors, TNF = tumour necrosis factor, TRIF = TIR-domain-containing-adaptor-protein-inducing-interferon-β Non-invasive ventilation for critically ill patients with pandemic H1N1 2009 influenza A virus infection  We read with interest the study reported by Rello and colleagues [1] . Th e authors described the fi rst 32 documented patients with pandemic infl uenza A H1N1 (PIAH1N1) virus infection hospitalized in an intensive care unit (ICU) in Spain. Twenty-four patients (75.0%) had refractory hypo xemia and required advanced mechanical ventilation. Eight patients (33.3%) received noninvasive mechanical ventilation at ICU admission. Six of these patients (75%) required further orotracheal intubation and invasive mechanical ventilation and two (33%) died.
Non-invasive ventilation (NIV) is not recommended for patients with PIAH1N1 virus infection complicated by pneumonia, acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) because although NIV temporarily improves oxygenation and reduces the work of breathing in these patients, it does not necessarily change the natural disease course. On the other hand, NIV may increase the risk of respiratory pathogen transmission [2] and there is not enough evidence to support the treatment of ALI/ARDS with NIV. To date, three studies have suggested that NIV has not been successful in critically ill patients with hypoxemic respiratory failure attributable to PIAH1N1 virus infection [1, 3, 4] . In these studies a total of 76 patients received NIV, but 64 (84.2%) of these patients required subsequent intubation and invasive ventilation.
Considering the high failure rate of NIV therapy in patients with PIAH1N1 virus infection and ALI/ARDS, the treatment of ARDS associated with the PIAH1N1 virus infection should be based upon published, evidence-based guidelines for sepsis-associated ARDS. Standard lung-protective ventilation strategies are appropriate initially [2, 5] . We appreciate the interest of Dr Ñamendys-Silva and colleagues in our article [1] and their insightful observations regarding ventilator management of severe PIAH1N1 virus infection. We agree that NIV is not recommended for patients with respiratory failure due to PIAH1N1 virus infection. However, several points should be clarifi ed. Use of NIV in ARDS remains controversial and the etiology of hypoxemia seems to be an important determinant of successful outcome. Our results describe our national clinical practice in the current pandemic and it is consistent with other reports [3, 6] . Other authors have recently reported that, in centers with expertise on NIV, 30% of patients with ARDS were treated with NIV as a fi rst-line intervention and 30 to 50% of these avoided orotracheal intubation [1, 3, 6] . Th us, only a small number of patients with ARDS benefi ted from NIV in expert centers, always needing close monitoring in the ICU setting. In selected patients with milder presentation a conservative ventilator approach should be considered until additional data from 2009 PIAH1N1 is obtained.
Abbreviations ALI, acute lung injury; ARDS, acute respiratory distress syndrome; ICU, intensive care unit; NIV, non-invasive ventilation; PIAH1N1, pandemic infl uenza A H1N1.  Immune Response to Lactobacillus plantarum Expressing Borrelia burgdorferi OspA Is Modulated by the Lipid Modification of the Antigen BACKGROUND: Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively. CONCLUSIONS/SIGNIFICANCE: Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics [1] . A number of studies of oral vaccines generated from genetically engineered pathogenic or commensal bacteria have been reported [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] . In humans, Lactobacillus plantarum belongs to the natural flora of the vagina and the gastrointestinal tract. We have recently developed an oral vaccine based in L. plantarum expressing the outer surface protein A (OspA), a lipoprotein from Borrelia burgdorferi [12] .
Evidence suggests that the peptidoglycan layer of some LAB promote natural immuno-adjuvanticity [13] , [14] , [15] , [16] and antigen localization on the cell wall makes it more accessible to the immune system as compared to intracellular or secreted proteins [17] , [18] . Leader peptides mark proteins for translocation across the cytoplasmic membrane and lipid modification is of major importance both for anchoring exported proteins to the membrane and for protein function [19] . It has been shown that lipidation at the first aminoacid of the mature OspA protein is essential to induce an immune response [20] , [21] . In this study, we examined the influence of post-translational processing in the localization of OspA lipoprotein on the cell envelope of L. plantarum.
The normal immune response to harmless gut antigens and commensal bacteria is the induction of a local and systemic immunological tolerance, known as oral tolerance [22] , [23] . Immunological tolerance can be exploited to develop immunotherapies for autoimmune and inflammatory diseases but it is also the obstacle to the development of oral vaccines [23] . As a result of our observation that an oral vaccine based in L. plantarum expressing the OspA lipoprotein induced a protective, IgG-based, immune response in mice [12] we set out to further understand the processes that determine the immunological consequences of oral administration of antigen. We investigated the role of L. plantarum expressing OspA lipoprotein in breaking the oral tolerance of the gut and if this immune response was dependent on the lipid modification of OspA.
The procedures involving human blood were approved by the Institutional Review Board (IRB) of the University of Tennessee Health Science Center, under the provisions of the Department of Health and Human Services Regulations for Protection of Human Subjects (45 CFR 46) and similar FDA Regulations (21 CFR 50 and 56). All participants provided written informed consent. The procedures involving mice were in accordance with the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center.
Bacterial strains, cell lines and culture conditions L. plantarum was grown at 30uC in LM medium [1% proteose peptone (w/v), 1% beef extract (w/v), 0.5% yeast extract (w/v), 0.5% lactose (w/v), 9 mM ammonium citrate, 61 mM sodium acetate anhydrous, 0.4 mM magnesium sulfate, 0.3 mM manganese sulfate, 11.2 mM dipotassium phosphate, 0.5% Tween 20 (v/ v)], supplemented with 10 mg/ml of chloramphenicol (Cm). E. coli was grown at 37uC in TBY medium (BD, Franklin Lakes, NJ). T84 human colonic carcinoma epithelial cells were obtained from the American Type Culture Collection (ATCC, CCL-248, Manassas, VA). T84 cells were maintained at 37uC, 5% CO 2 in DMEM-F12K medium modified by ATCC, containing 10% FCS, 100 U/ ml penicillin and 100 mg/ml streptomycin.
A vector that expresses the full-length ospA gene from B. burgdogferi in L. plantarum was previously described [12] . To generate a vector expressing the OspA mutant, we designed primers in which we replaced the codon corresponding to Cys 17 with Asp 17 . Expression vectors were then transformed into L. plantarum strain 256 to obtain the clones LpA and LpA D17 , respectively. Protein expression was checked by immunoblot as follows. Recombinant L. plantarum cells were disrupted with a FrenchH press (Thermo Electron Corporation, Milford, MA), supernatants were analyzed on a 12% denaturing polyacrilamide gels and electrotransferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Billerica, MA) for analysis with an OspAspecific monoclonal antibody (mAb 184.1). To evaluate export and lipidation of OspA and its mutant expressed by recombinant Lactobacillus, we performed Triton X-114 phase partitioning [24] . L. plantarum cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in PBS. Bacteria were disrupted with a FrenchH press and the insoluble material (membrane and cell wall) was separated from the cytosol fraction by centrifugation. This cell envelope fraction was suspended in 1 ml of ice-cold 2% Triton-X114 (v/v) in PBS. The fractions were rotated end over end at 4uC for 1 h and were phase-separated by warming the solution for 30 min in a water bath at 37uC followed by centrifugation for 15 min at 25uC. The separated detergent and aqueous phases were each washed three times. The solutions were then rewarmed and recentrifuged as described and the detergent and aqueous phases were collected. Ten (10) ml of each phase was analyzed on 15% denaturing polyacrylamide gels, electrotransferred to PVDF filters, and used for immunoblot analysis. OspAspecific monoclonal antibody LA2.2 (1:100) was used as primary antibody, goat anti-mouse IgG (H+L) conjugated to alkaline phosphatase (1:1000; Pierce Rockford, IL) was used as secondary antibody and the immunoblot was developed by BCIP/NBT TM (KPL, Washington, DC). The protein bands corresponding to each OspA antigen were quantified by densitometry using a Multi Image TM Light Cabinet and the AlphaEase TM software (Alpha Innotech Corporation, San Leandro, CA). The results were plotted as a percentage of the total OspA content for each recombinant Lactobacillus.
A vector that expresses the full-length ospA gene from B. burgdogferi in Escherichia coli (EcA) was previously described [25] . To generate the vector pET9cmut OspA that expresses the OspA mutant ( mut OspA), we designed primers to amplify the B. burgdorferi ospA gene lacking the signal sequence and we cloned the PCR fragment into pET9c. The expression vectors were transformed into E. coli BL21 (DE3) pLysS, the recombinant proteins were purified and the endotoxines were removed using the Detoxi-Gel TM Endotoxin Removing Gel (Pierce Biotechnology, Rockford, IL).
Lactobacillus cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in TGE buffer [25 mM Tris-HCl pH 8, 50 mM glucose, 10 mM EDTA pH 8]. For cell wall removal, aliquots of 1 ml were treated with 250 KU/ml of lysozyme (Lyz; Novagen, Gibbstown, NJ) for 45 min at 37uC. Protoplasts and cell wall fractions were separated by centrifugation and the supernatants containing the cell walls collected. Protoplasts were washed, resuspended in TED buffer [25 mM Tris-HCl pH 8, 1 mM EDTA, 1 mM DTT] and disrupted with a FrenchH press. Supernatants were ultracentrifugated for 1 h to separate membranes from cytosol fractions. Three (3) mg of each fraction was analyzed on 15% denaturing polyacrylamide gels, electrotransferred to PVDF filters and used for immunoblot analysis with mAb LA2.2 as described above.
Recombinant Lactobacillus were treated with and without 250 kU/ml of Lyz in TGF buffer [100 mM Tris-HCl pH.8, 50 mM glucose, 1% FBS (v/v) (Hyclone, South Logan, UT)] for 30 min. Cells were washed and resuspended in TGF buffer with mAb LA2.2 (1:100) for 1 h at room temperature, washed three times with 500 ml TGF buffer and resuspended on 100 ml of the same buffer. Aliquots of 10 ml were placed on slides and air-dried at 37uC for 1 h. Slides were incubated with Alexa Fluor 488labeled goat anti-mouse IgG antibody (1:250) (Molecular Probes, Invitrogen, Carlsbad, CA) in 100 ml TGF buffer at 23uC for 1 h with intermittent gentle mixing. After incubation, slides were washed three times with TGF buffer and fixed with 4% PBSbuffered formaldehyde (methanol free; Ted Pella Inc., Redding, CA) for an additional 15 min at room temperature. Labeled cells were mounted in VectaShield medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and visualized using a Zeiss inverted Axiovert 200 motorized microscope and analyzed using the Axiovision 4.3 software.
To further investigate the localization of antigens on the Lactobacillus cell envelope, we developed an indirect live-cell enzyme-linked immunosorbent assay (lcELISA). Lactobacillus cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in TG buffer [100 mM Tris-HCl pH.8, 50 mM glucose]. For cell wall digestion, 1 ml aliquots were resuspended in TG buffer with or without Lyz (250 kU/ml) for 5 or 45 min at 37uC. Cells were washed twice with TG buffer, resuspended in the same buffer supplemented with 3% BSA (Bovine Serum Albumin, Sigma), and incubated with mAb LA2.2 (1:500). Samples were washed twice and incubated for 30 min with goat anti-mouse IgG (H+L) antibodies conjugated to alkaline phosphatase (1:1000). After an extensive wash, labeled cells were incubated with pNPP Microwell Substrate System (KPL). Microtiter plates were loaded with 100 ml of each cellular suspension, and optical densities were measured at 405 nm by a Spectra MAX plus ELISA reader (Molecular Devices, Sunnyvale, CA).
T84 cells (human colon carcinoma epithelial cell line) were seeded in 24-well tissue culture plates (BD Biosciences, San Jose, CA) at a density of 1610 6 cells/well and grown until they reached 90 to 95% confluence. L. plantarum cells were killed by exposure to UV light for 1 h and the lack of cell viability was confirmed by culture in MRS agar. T84 cells were co-cultured with UV-killed recombinant L. plantarum at a MOI 10:1 bacteria per cell, for 48 h. L. plantarum control and 0.5 mg/ml TNFa were used as negative and positive controls, respectively. Supernatants were collected and the human IL-8 production was measured by ELISA (Quantikine, R&D Systems, Minneapolis, MN).
Human peripheral blood was collected into heparin vacutainer tubes (BD Bioscience, Franklin Lakes, NJ) from healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). A final suspension was made in complete RPMI 1640 (Hyclone), supplemented with 10% [v/v] FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml and fungizone. Cell viability (greater than 95%) was determined by trypan blue exclusion. Human monocytes were purified from PBMC using the Monocyte Isolation Kit II (Miltenyi Biotec, Auburn, CA), according to the manufacturer's recommendations. These monocytes were then used to produce Monocyte Derived Dendric Cells (MDDC) as described below.
To derive dendritic cells from PBMCs and monocytes we cultured 1610 6 or 2610 5 cells/well, respectively, in 24-well tissue culture plates for 5 days in 2 ml of complete RPMI 1640 supplemented with 10 ng/ml IL-4, and 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D system, Minneapolis, MN). Cultures were placed at 37uC in a 5% CO 2 humidified incubator. Every two days the medium was removed and 2 ml of fresh complete medium was added. At day 5 the cells were co-cultured with 2.5 mg/ml of purified recombinant OspA or UV-killed recombinant Lactobacillus at MOI 10:1 colony-forming units per cell for 48 h at 37uC. 100 ng/ml of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (LIST Biological Laboratories, Campbell, CA) and L. plantarum were used as positive and negative control, respectively. Supernatants were collected and TNFa, IL-12, IFNc, IL-6, and IL-10 were quantified by ELISA (Quantikine, R&D Systems).
L. plantarum expressing the target antigen was cultured in LM medium supplemented with 10 mg/ml Cm, and grown at 30uC to an OD 600 of 1.0. That is the equivalent of 1610 9 cells/ml corresponding to approximately 125 mg of total protein. The cells were harvested by centrifugation at 3000 g for 10 min at 4uC and resuspended in 20% glycerol/phosphate buffered salt solution (Gibco, Grand Island, NY) in 1% of the initial volume. Cell suspensions in aliquots of 2 ml were frozen quickly in a dry ice bath and stored at 280uC. Aliquots were thawed at 4uC and 400 ml (4610 10 cells) were placed in a ball-tipped syringe for oral gavage inoculation. Groups of four female C3H-HeJ mice (6-8 weeks old, Charles River, Boston, MA) were immunized by intragastric inoculation of 4610 10 Lactobacillus expressing OspA recombinant antigens. L. plantarum (Lp) was used as control. Mice received the first immunization, twice daily, for 8 days (days 1-4 and 8-11). After resting for two weeks the mice were bled (day 28). On days 29-32 they received the 1 st oral boost and rested for an additional 2 weeks. On day 49, the mice were bled. On days 50-53 they received the 2 nd oral boost and rested for an additional 2 weeks. On day 68 mice were terminated, and blood, stool and bronchoalveolar lavage fluids (BAL) were collected. The animals were housed in a pathogen free colony on hardwood chip bedding in microisolator cages at the University of Tennessee Health Science Center, Memphis, TN. They were maintained on a 12 h light-dark cycle, in a room kept al 23uC with 50-60% relative humidity; they were given tap water and sterile irradiated rodent chow ad libitum.
Serum, stool and BAL from orally inoculated mice were tested by indirect ELISA for the presence of total IgG, IgG1, IgG2a or IgA to OspA. Purified recombinant OspA was coated at 0.5 mg/ ml on Nunc MaxiSorp TM flat-bottom ELISA plates (eBioscience, San Diego, CA) and indirect ELISA was performed using serum (1:500), stool (1:10) or bronchoalveolar fluid. Anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgA horseradish peroxidase-conjugated antibodies (1:2,000) (Jackson Immu-noResearch, West Grove, PA) was used as secondary antibody.
All data is represented as mean 6 standard deviation. Statistical analyses were performed using Student's t-test. p,0.05 are considered statistically significant.
Previously, we reported the cloning and expression of full length OspA from B. burgdorferi in a lactobacilli expression vector and designed an oral vaccine candidate for Lyme disease (LpA) [12] . For this study, we mutated the conserved lipidation motif of ospA by replacing the cystein at position 17 with aspartic acid to generate a protein that lacks the post-translational lipid attachment (Fig.1A) . Total extracts of L. plantarum carrying the empty vector (Lp), and expressing OspA (LpA) or OspA D17 (LpA D17 ) were analyzed by SDS-PAGE and protein expression was confirmed using anti-OspA monoclonal antibody, mAb 184.1 (Fig.1B) . The two bands seen for LpA may represent the lipidated unprocessed OspA protein (the leader peptide has not been cleaved, 31 kDa) and the lipidated mature protein (the leader peptide has been cleaved, 30 kDa). The single band seen for LpA D17 (30.4 kDa) may represent the unlipidated OspA (containing the leader peptide).
We further evaluated the export and lipidation of OspA and its mutant by Triton X-114 phase partitioning of the cell envelope ( Fig. 2 ). In the cell envelope of Lactobacillus, OspA partitions to the detergent phase, suggesting that OspA is exported through the membrane and that the amount of OspA that is hydrophobic (,50%) is higher than the hydrophilic form (,40%), although not significantly so. In contrast, the mutant OspA D17 partitions mostly to the aqueous phase, suggesting that, in addition to being exported, the mutant generated more hydrophilic OspA (,70%) than hydrophobic (,25%) ( Fig. 2A and 2B ). If about 25% of OspA is associated with the cell envelope due to hydrophobicity of the leader sequence (i. e. OspA D17 in the detergent phase of LpA D17 ), then the difference (i. e. ,30% additional wildtype OspA in the detergent phase of LpA) should be protein that is associated with the cell envelope via the lipid anchor, or the lipidated form of OspA. Differences for the mutant OspA D17 partitions are statistically significant (Fig. 2B , *p,0.05).
Next, we performed serial fractionation experiments in order to determine where the recombinant proteins are localized within the cell envelope of L. plantarum (Fig. 3A) . We treated recombinant L. plantarum with Lysozyme (Lyz) for 45 min and separated the cell wall fraction from the protoplast by centrifugation. We then disrupted the cells and separated the membrane from the cytosol fractions by ultracentifugation. The three fractions were then analyzed on denaturing polyacrylamide gels and OspA was identified by Immunoblot with anti-OspA monoclonal antibody (mAb LA2.2). Lipidated OspA expressed in L. plantarum is exported through the membrane and accumulates in the cell wall, while unlipidated OspA D17 remains mostly attached to the membrane (Fig. 3A) . Next, we wanted to analyze the localization of the recombinant proteins on the surface of L. plantarum. We incubated live recombinant L. plantarum with and without Lyz and we performed both live-cell ELISA (lcELISA) and immunofluorescence (IFA) assays. For lcELISA, we incubated the recombinant L. plantarum with Lyz for 5 and 45 min, the cells were washed and incubated with mAb LA2.2 (Fig. 3B) . For immunofluorescence, we performed a 30 min incubation with Lyz after which the cells were washed, incubated with mAb LA2.2 followed by Fluor 488-labeled goat anti-mouse IgG (1:250). Staining was visualized using a Zeiss inverted Axiovert 200 microscope (Fig. 3C) . In both assays, IFA and lcELISA, reactions without lyz (No Lyz) detect protein that is exposed on the surface of the cell. The epitope used to detect OspA and OspA D17 is located in the carboxyl terminal of the protein and is not surface exposed ( Fig. 3B and 3C) . Reactions with Lyz digest the peptidoglycan that releases the OspA that is trapped within the cell wall and expose OspA that is attached to the membrane (Lyz 5 min, 45 min or 30 min, Fig. 3B and 3C ). Our results indicate that lipidated OspA (LpA) is trapped within the cell wall. That is, a 5 min digestion with Lyz exposes a high amount of OspA that is associated with the peptidoglycan layer (Fig. 3B ) and a 45 min or 30 min digestion exposes a lower amount of OspA that is associated with the peptidoglycan layer ( Fig. 3B and 3C) , suggesting that OspA might not be attached to the membrane. In contrast, unlipidated OspA D17 (LpA D17 ) is trapped within the peptidoglycan layer of the cell wall, maybe attached to the membrane. That is, a 5 min digestion with Lyz exposes a low amount of OspA that is associated with the peptidoglycan layer (Fig. 3B ) and a 45 min or 30 min digestion exposes a high amount of OspA that is associated with the peptidoglycan layer and the membrane (Fig. 3B and 3C) . In order to analyze the potential inflammatory response to the oral administration of L. plantarum expressing OspA lipoprotein, we performed an assay using monolayer cultures of intestinal epithelial cells (T84), a human colon carcinoma cell line, stimulated with UV-killed LpA and LpA D17 and determined the production of IL-8 (Fig. 4) . The co-culture of T84 cells with UVkilled LpA or LpA D17 did not induce significant production of the pro-inflammatory chemokine IL-8 in comparison to the positive control (TNFa).
Dendritic cells (DCs) play a decisive role in the innate and adaptive immune response, as they produce cytokines in response to antigen stimulation and activated T cells. Monocytes were isolated from human Peripheral Blood Mononuclear Cells (PBMCs) and were treated with GM-CSF and IL-4 to derive to MDDCs. Because OspA is known to trigger TLR2-based receptors we stimulated MDDCs with purified proteins in order to access the effect of the naked protein (wildtype OspA ( wt OspA) versus a OspA mutant lacking the leader peptide ( mut OspA)), on cytokine induction. We co-cultured MDDCs with 2.5 mg/ml of purified wt OspA or mut OspA during 48 h and the production of pro-inflammatory cytokines TNFa, IL-12, IFNc, and antiinflammatory cytokine IL-10 was quantified by ELISA (Fig. 5A , 5B, 5C and 5D, respectively). wt OspA induced TNFa and IL-10 production (Fig 5A and 5D ) but it did not induce neither IL-12 nor IFNc ( Fig. 5B and 5C ). Compared to wt OspA, the OspA mutant lacking the leader peptide, mut OspA, showed a 10 fold-decrease in the TNFa production (Fig. 5A ) and a 2 fold-decrease in the IL-10 production (Fig. 5D ). Similarly to wt OspA, mut OspA did not induce neither IL-12 nor IFNc cytokine production ( Fig. 5B and 5C ). Differences between wt OspA and mut OspA are statistically significant (*p,0.001 and **p,0.05).
Next, we treated PBMCs with GM-CSF and IL-4 to derive the monocyte population into immature dentritic cells (PBMC/ DCs), and we investigated the cytokine production of human PBMC/DCs co-cultured with UV-killed recombinant L. plantarum expressing either lipidated OspA (LpA), the mutant unlipidated OspA (LpA D17 ) or the control (Lp). The amount of pro-inflammatory cytokines TNFa, IL-12, IFNc and IL-6, and anti-inflammatory cytokine IL-10 was quantified by ELISA (Fig. 6) . As compared to the control, LpA induced TNFa by 9 fold, IL-12 by 3 fold, IFNc by 4 fold and IL-6 by 10 fold (Fig. 6A, 6B, 6C and 6D) . Additionally, anti-inflammatory cytokine IL-10 was 7 fold upregulated by stimulation with recombinant L. plantarum expressing lipidated (LpA) as compared to the control (Lp) (Fig. 6E) . Compared to LpA, the mutant L. plantarum expressing the unlipidated OspA (LpA D17 ) induced significantly less TNFa, IL-12, IFNc, IL-6 and IL-10 ( Fig. 6A, 6B, 6C, 6D and 6E ). Differences between LpA and LpA D17 are statistically significant (*p,0.001 and **p,0.02). Further, compared to the control (Lp), LpA D17 induced TNFa, IL-6 and IL-10 (p,0.001). LPS was used as a positive control and upregulated secretion of all cytokines tested (data not shown). In order to dissect these results, we further purified monocytes by MACS to derive MDDC and analyzed cytokine production after stimulation (Fig. 7) . As expected, we determined that LpA induced higher production of proinflammatory cytokines TNFa (17 fold upregulated), IL-12 (,4 fold upregulated) and IL-6 (1-2 fold upregulated) as compared to the control (Lp) (Fig. 7A, 7B and 7D, respectively). There was no production of IFNc by monocyte derived dendritic cells (Fig. 7C) . Compared to LpA, the mutant L. plantarum expressing the unlipidated OspA (LpA D17 ) abrogated this response as seen by the decreased production of TNFa, IL-12 and IL-6 ( Fig. 7A, 7B and 7D) . Additionally, IL-10 was ,2-3 fold upregulated after stimulation with recombinant L. plantarum expressing lipidated (LpA) and the mutant appeared to impair this response (Fig. 7E) . Differences between LpA and LpA D17 are statistically significant (*p,0.05 and **p,0.001).
Antibody response to oral administration of recombinant L. plantarum Lastly, to assess the systemic and mucosal antibody immune response induced by the oral administration of recombinant L. plantarum, we immunized mice and tested serum levels of OspAspecific total IgG, IgG1 and IgG2a antibodies (Fig. 8) , and the levels of mucosal OspA-specifc IgA in bronchoalveolar lavage (BAL) and stool suspensions (Stool) (Fig. 9) , by indirect ELISA. In contrast to the controls, mice orally administered with L. plantarum expressing lipidated OspA (LpA) or the unlipidated mutant developed high titers of IgG specific antibody 68 days after the first inoculation (Fig. 8A) . However, mice inoculated with LpA produced higher levels of OspA-specific IgG1 compared to mice inoculated with LpA D17 , as shown on day 68 after the first inoculation (**p,0.01) (Fig. 8B ). In contrast, mice inoculated with either LpA or LpA D17 developed equivalent levels of IgG2a (Fig. 8C) . Furthermore, the same mice inoculated with LpA and LpA D17 developed a high OspA-specific IgA antibody titer in BAL (Fig. 9A) and stool (Fig. 9B) , 68 days after the first inoculation. Differences between LpA and LpA D17 were not statistically significant.
It is now generally accepted that mucosal vaccines that can elicit both secretory IgA and effective systemic immune responses could have advantages over many existing vaccines [1] . A large body of research has accumulated recently, whereby mucosal vaccines have been developed using LAB against a vast number of pathogens to various degrees of efficacy success [26] , [27] , [28] , [29] , [9] , [30] , [31] , [32] , [33] , [11] , [34] . We have recently found that L. plantarum expressing B. burgdorferi OspA lipoprotein induced the production of OspA-specific IgA and IgG antibodies as a result of oral administration [12] . In the present study, we investigated the effect of the lipid modification of OspA in the localization of the antigen in our mucosal delivery vehicle, L. plantarum. Furthermore, we investigated how recombinant L. plantarum expressing OspA lipoprotein breaks the oral tolerance of the gut. We show that the leader peptide of OspA targets the protein to the cell envelope of the delivery vehicle L. plantarum, and that the Cys 17 is the substract for OspA lipidation. Further, we show that our recombinant L. plantarum based mucosal delivery system does not induce secretion of IL-8 by intestinal epithelial cells and that it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity.
Expression of the full-length B. burgdorferi OspA gene in E. coli, a Gram-negative bacteria, produces a protein that is processed posttranslationally by signal peptidase II and contains an attached lipid moiety. In E. coli this lipoprotein partitions mostly into the detergent phase following extraction with Triton X-114 [35] . As seen in E. coli, we show that in Gram-positive L. plantarum, the leader peptide also marks OspA for translocation or export across the cytoplasmic membrane given that wildtype OspA partitions to the detergent phase following extraction with Triton X-114, suggesting that it is lipidated. In contrast, the mutant OspA D17 , which lacks the cystein that is needed for the lipid moiety attachment, partitions mostly to the aqueous phase. This shift on the solubility of the mutant OspA D17 appears to be due to the lack of the lipid attachment, that makes the antigen more hydrophilic than the native lipidated OspA, and therefore the mutant appears to be unlipidated. It appears that OspA is indeed lipidated and localizes mostly at the membrane-cell wall interface. This observation is consistent with a previous study of Gram-positive Lactococcus spp. lipoprotein processing that found that lipid modification is predominantly required to retain proteins at the membrane cell-wall interface [36] . The intestinal immune system is the largest and the most complex part of the immune system [23] . Crosstalk occurs between the luminal microorganisms and the mucosal tissue, with intestinal epithelial cells (IECs) mediating this dialogue. Microbial invasion of the gut epithelium stimulates IECs to produce CXCchemokine ligand 8 (IL-8) and activate other pro-inflammatory pathways that in turn coordinate the innate immune response [37] . The absence of IL-8 secretion by IECs in the presence of recombinant L. plantarum is consistent with previous findings [38] , [39] , [40] and suggests that an oral vaccine based in L. plantarum expressing the OspA lipoprotein would not induce the local adverse inflammatory effects attributed to pathogenic bacteria.
In the lamina propria of the intestine, the adaptive immune response is oriented towards tolerance and it involves suppression of Th1 cytokines such as IL-12 and activation of regulatory T cells that inhibit the proliferation of other T cells through production of IL-10 and TGFb1 [22] , [23] , [41] , [37] . Together with IECs, dendritic cells (DCs) interact with luminal bacteria [37] either through receiving M-cell delivered bacterial cargo or through extension of their own pseudopods across the IEC monolayer [42] , [43] , [43] . When we stimulated human PBMC-derived-dendriticcells (PBMC/DCs) with recombinant L. plantarum expressing the OspA lipoprotein, but not the unlipidated mutant, we observed that pro-inflammatory cytokines TNFa, IFNc and IL-6 were markedly upregulated. In addition, we observed an increase of anti-inflammatory cytokine IL-10. When we stimulated monocyte derived dendritic cells (MDDC) with L. plantarum expressing lipidated OspA, but not unlipidated OspA, we observed that proinflammatory cytokines TNFa and IL-12 were markedly upregulated. Here too, we observed an increase of anti-inflammatory cytokine IL-10. Differences in detection of cytokines in both assays can be explained by the fact that in the former (PBMC/DC) assay we expect to have a mixed population of monocyte derived dendritic cells, T cells, B cells and NK cells, and in the later (MDDC), we expect to have a relatively pure population of dendritic cells. These data indicate that recombinant OspA- expressing-L. plantarum stimulates the immune response via Th1/ Th2 cell mediated immunity and that, lipidation of the antigen in the delivery agent plays an active role in this response.
The lipid attachment in OspA seems to be a critical determinant of the mature protein immunogenicity [35] . Furthermore, the OspA leader peptide has been fused to different proteins to generate potent lipo-antigens that induce strong B (IgG) and T-cell (helper CD4 + and cytotoxic CD8+) responses [44] , [45] . Because lipoproteins and OspA are known to trigger TLR2-based receptors [46] , [47] , we stimulated monocyte derived dendritic cells with purified proteins in order to access the effect of the naked protein on cytokine induction. We observed that wildtype OspA ( wt OspA) induced TNFa and IL-10 but it did not induce IL-12 or IFNc. In the absence of the leader peptide, as in mut OspA, the TNFa response was abrogated and IL-10 was reduced in half. In contrast, if OspA is delivered in Lactobacillus we also observe an effect on the production of IL-12. This effect appears to be related to the delivery vehicle in which OspA is presented. Furthermore, our results show that mutation of the Cys 17 on the leader peptide of OspA decreases significantly the production of pro-and antiinflammatory cytokines, and that this effect is related to the lipidation of OspA. In addition, we observed that lipidation of protein is not essential to induce a specific humoral immune response to OspA (systemic IgG and mucosal IgA) when the antigen is orally delivered by Lactobacillus. The adjuvant effect of Lactobacillus as oral carrier, exerts a synergized effect along with the intrinsic antigenicity of unlipidated OspA. This result differs with others, where the lipid modification of OspA seems to be essential to induce a humoral immune response, when the soluble unlipidated antigen is delivered subcutaneously without a cellular carrier [35] . In addition, we have found that the mutation of OspA Cys 17 skewed the systemic IgG production towards IgG2a, probably as result of a shift from a Th2 to a Th1 response. Over all, our data suggest that the lipidation of OspA has a clear role on the mixed Th1/Th2 cellular and humoral immune response to orally administered Lactobacillus expressing OspA.
Given that we inoculate our animals with very high doses of vaccine for long periods of time our results appear to contradict the dogma that high and repeated doses of stimulating antigen induces oral tolerance [48] . This might be due to the fact that our antigen is delivered via a bacterial system rather than purified protein and therefore the amount that is actually exposed to the mucosal immune system might be much less than the naked antigen.
In summary, we investigated the mechanism by which a mucosal vaccine based in recombinant LAB breaks the immunological tolerance of the gut in order to elicit a protective immune response. It is possible that mucosal tolerance may have been broken by two different mechanisms. In the first scenario, M cells might transport whole recombinant L. plantarum or OspA lipoprotein as it is released from lysed recombinant L. plantarum across the epithelium where they can induce primary immune responses [1] . In a second scenario, as dendritic cells uptake recombinant LAB and recognized it as the pathogen, the bacteria are no longer retained at the mesenteric lymphnode, as it happens when DCs are loaded with commensal bacteria, but instead migrate to secondary lymphoid tissue and present antigen to T-cells in a systemic environment that is not tolerigenic [49] . Neutralization of X4- and R5-tropic HIV-1 NL4-3 variants by HOCl-modified serum albumins BACKGROUND: Myeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H(2)O(2 )and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected in vivo. FINDINGS: This report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV) glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner. CONCLUSIONS: Our data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide. An important event in HIV-1 infection is the step-by-step binding of the external envelope complex, the gp120/ gp41 trimer, to (i) CD4 [1] and to (ii) a family of seventransmembrane chemokine receptors including CXCR4 and CCR5 which are the two major coreceptors [2] on the cellular membrane as well as to (iii) heparan sulfate structures [3] . HIV-1 entry can therefore be inhibited by heparan sulfate [4] , its analogs [5] or other synthetic polyanions [6] , ligands for CD4 [7] , CXCR4 [8] or CCR5 [9] , and by factors that bind to gp120 like neutralizing antibodies [10] . Since HIV-1 variability is prodigious, viral escape to all these antiviral substances has been documented and therefore there is a pressing need to find new strategies to efficiently block HIV-1 infection.
In 1992, Klebanoff and coworkers [11] showed that stimulated polymorphonuclear (PMN) cells released an unknown factor which neutralized HIV-1. PMN from patients with hereditary myeloperoxidase (MPO) deficiency indicated that the antiviral activity was correlated with the presence and the release of the enzyme into cell culture medium. Adding MPO to PMN-(MPO-deficient) cell cultures restored the ability to block HIV-1 infection [11, 12] . In addition to MPO, the presence of two other factors, H 2 O 2 and chloride, was absolutely necessary to observe the antiviral activity in cell culture supernatants of stimulated PMN cells. Since the enzyme MPO catalyzes the reaction between H 2 O 2 and chloride to generate HOCl (bleach), Klebanoff and coworkers [12] suggested that this product of the MPO/H 2 O 2 /halide system was directly responsible for HIV inactivation. A clue as to the substrate of the MPO/H 2 O 2 /halide system was provided by the detection of HOCl-modified proteins in human tissue by a specific monoclonal antibody (clone 2D10G9) [13] . This antibody recognized an HOCl-modified protein in glomerular and tubulointerstitial inflammatory and fibrotic lesions and its binding was inhibited by HOCl-modified human serum albumin (mHSA) [14, 15] .
Recently we demonstrated that mHSA was active against West Nile virus (WNV) [16] . Low doses of HOClmodified human serum albumin (mHSA) inactivated WNV entry into VeroB4 cells in vitro and we observed an interaction between mHSA and the domain III of the WNV external envelope. Similar to WNV, HIV-1 also enters its target cell following membrane fusion of the viral envelope and the plasma membrane and it is conceivable that enveloped viruses share common functions to promote membrane-membrane contact to allow binding of cell membrane-anchored receptors.
Here we report, that mHSA, mBSA (bovine) and mMSA (mouse) bind to recombinant HIV-1 gp120 of the laboratory strain NL4-3. Binding of HOCl-modified serum albumins to gp120 also inhibited binding of recombinant CD4 (rCD4) and V3 loop-specific antibodies. All three HOCl-modified serum albumins inhibited viral infection and syncytium formation in a dose dependent manner.
HIV-1 strain NL4-3, an X4-monotropic laboratory isolate, was used for virus inhibition tests. HeLa-P4 cells expressing human CD4, CXCR4 were kindly provided by Matthias T. Dittmar, University Heidelberg. GHOST-CXCR4 cells, antibody sera, monoclonal antibody, and gp41 (ARP671, spanning the entire extracellular domain of HIV-1 gp41 MN ) were obtained from the European Vaccine Against AIDS Programme (EVA, Potters Bar, UK). Recombinant HIV-1 gp120 (LAV, CXCR4 monotropic, identical to NL4-3 gp120, glycosylated) and human rCD4 (soluble form, transmembrane region removed, glycosylated) was obtained from Protein Sciences Corporation, Meriden, CT, USA. These commercially proteins were expressed in insect cells by a baculovirus vector system, secreted in the serum-free cell culture medium and purified under non-denaturing conditions. The env expression plasmid pSVATGrev carries a subgenomic HIV NL4-3 fragment coding for rev, vpu and env under the transcriptional control of the SV40 early promotor. The plasmid sequence can be obtained upon request. Construction of NL4-3 mutants with R5X4 and R5 tropism and mutants lacking N-glycosylation site g15 was carried out as described earlier [17] .
The HOCl reaction was carried out as described in the literature [18] with modifications. In brief, freshly pre-pared HOCl was added to an aqueous solution of serum albumin (1 gr/liter phosphate buffered saline, pH 7.5). The concentration of HOCl was monitored by UV spectroscopy (ε = 375 mol/cm liter). Serum albumin was treated with HOCl at different molar ratios (serum albumin:HOCl, 1:10, 1:20, 1:100, 1:200, 1:500, 1:1000, 1:10000). After incubation for 30 min at RT, remaining HOCl was inactivated by adding the antioxidant 2 aminoethanesulfonic acid (taurine, final concentration 5 gr/l). The mixture was incubated again for 30 min at RT. Purification of the HOCl-modified serum albumin and the elimination of remaining 2 aminoethanesulfonic acid was achieved by concentrating the reaction mixture to a final volume of 50 ml by ultrafiltration (GE Healthcare, hollow fibre column UFP-10-C 3X2MA, membrane nominalmolecular-weight cutoff 10 kD) and then further purified by a continuous ultrafiltration process using the Quix-Stand ® apparatus (GE Healthcare). A total volume of 20 litres of water was slowly added over a time period of 48 h to purify the mHSA product which was continuously pumped over the hollow fibre column at a maximum pressure of 15 PSI. After this ultrafiltration process the solution (400 ml) was again concentrated to a final volume of 50 ml. The final yield of HOCl-modified serum albumin was between 250-500 mg/50 ml (i.e. 25-50%).
ELISA plates (Nunc, MaxiSorb) were coated with gp120 (3 μg/ml in carbonate buffer 0.1 M NaHCO 3 , 0.1 M Na 2 CO 3 , pH 9.6). To each well of a 96-well plate 100 μl of the gp120 solution was added and the plates were sealed with adhesive foil (Sarstedt) and incubated at 8°C overnight. Subsequently 250 μl of a BSA solution (20 μg/ml PBS) was added to each well and the plates incubated for 1 h at room temperature. After blocking, the plates were washed 5-times with PBS containing 0.05% Tween 20 (PBST). Modified serum albumin was added to the wells in a total volume of 50 μl PBS. After incubation for 30 min. rCD4 or antibody was added to each well in a total volume of 50 μl PBS. The plates were incubated for 1 h at room temperature and then washed 5-times with PBST. To detect gp120-bound rCD4 we used the anti-CD4 mouse monoclonal antibody NCL-CD4-1F6 (Novocastra). Bound anti-CD4 antibody was detected by a secondary anti-mouse HRP-antibody conjugate diluted 1:500 in PBS (BioRad). Bound gp120-specific monoclonal antibody was monitored directly using the anti-mouse-HRPantibody conjugate (BioRad). After binding of the secondary antibody, the plates were washed 5 times with PBST and 5 times with PBS. To each well 100 μl of a commercially available one-component ready to use solution of 3,3',5,5'-tetramethylbenzidine (TMB, Sigma Aldrich) was added, the reaction stopped with 1 M H 2 SO 4 , and the plate read at 450 nm.
Purified recombinant LAV gp120 (1 μg/100 μl 10 mM NaOAc pH 4.0) was immobilized by NHS/EDC coupling on a dextran coated, CH-activated C5 sensorchip (Biacore, Sweden) at densities yielding 3500-5000 response units (RU). Surface plasmon resonance was carried out using a Biacore 1000. Binding to immobilized proteins diluted in HBS-EP buffer (Biacore, Sweden) was tested at various concentrations at a flow rate of 5 μl/min for a period of 6 min.
To each well of a 96-well plate, containing 10.000 CD4 + HeLa-P4 cells inhibitor and control proteins were added at various concentrations and the cells were incubated for 48 h. Thereafter the medium was changed and 10 μl MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) solution (5 mg/ml MTT dissolved in PBS pH 7.4) was added. The plates were incubated at 37°C for 4 h after which the medium was removed and 100 μl of lysis buffer (9.94 ml DMSO, 60 μl HCl, 1 g SDS) added to each well. The plates were incubated for 10 min at 37°C. 
(a) GHOST-CXCR4 cells were infected with 1000 TCID (Tissue Culture Infective Dose) of the X4-tropic laboratory strain NL4-3. To study inhibition, virus was given to the cells after preincubation of cells with modified serum albumin for 30 min. Cell culture supernatants were tested on day 5 for p24-antigen by a standard p24-antigen ELISA. GHOST cells were cultured in DMEM (Gibco) including 10% FCS (Biochrom KG). (b) TZM-bl cells were infected with virus supernatant representing an infectious dose of 500 foci/96-well. Viral entry into TZM bl cells causes the expression of β-galactosidase, which can be monitored 2 days after infection. To detect βgalactosidase-expressing cells, the cell culture supernatant was removed and the cell layer was treated with 0.25% glutaraldehyde in phosphate-buffered saline (PBS, 5 min, RT) and washed three times with PBS. To stain infected cells, 100 μl of the X-gal solution (0.5 mg X-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside]/ ml, 1 mM MgCl 2 , 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide in PBS) was added to each well and the plates were incubated at 37°C. The staining reac-tion was monitored by microscopy. Finally, the cell layer was washed again with PBS and blue foci were counted.
GHOST CXCR4 cells (10 4 cells per 96-well) cultured in DMEM/10% FCS were infected with 1000 TCID of the HIV laboratory isolates NL4-3 and were also infected in the presence of 50 μg mHSA per ml cell culture medium (DMEM/10% FCS). Virus and mHSA were added to the cells simultaneously. The cultures were assessed microscopically 5 days after infection by visualization of induced syncytia and cell layer destruction. To visualize syncytia, cell layers were treated with 3% formalin for 10 min to inactivate virus and then fixed with methanol (Solution 1 of the Azur B and Eosin G based Hemacolor ® rapid staining set, Merck, Germany) for 5 min. After fixation, cells were stained with color reagent red (solution 2 of the Hemacolor ® staining set) for 30 sec followed by a staining with color reagent blue (solution 3 of the Hemacolor ® staining set) for 30 sec. Alternatively, HeLa-P4 cells, cultured in DMEM/10% FCS, expressing human CD4, CXCR4 were transfected with 1 μg pSVATGrev plasmid. This plasmid expresses the gp160 protein precursor, which is processed and expressed on the outside of the cellular membrane as the functional gp120/gp41 complex. Cells were transfected for induction of syncytia and mHSA was given to the cells 30 min after transfection. Syncytium formation was assessed after 28 h by standard phase contrast microscopy.
In a first experiment we tested different samples of the HOCl-modified HSA, which were generated by using different HOCl concentrations, in a syncytium inhibition assay. In HIV-1 cell cultures, infected cells fuse with other, non-infected CD4 + target cells. Cell-cell fusion is known as syncytium formation. Syncytium formation is the result of the binding of gp120, which is present on membranes of the infected cells, to the receptors on the target cells and subsequent insertion of the gp41 N-terminus into the host membrane. This gp120-receptor binding event promotes recurring cell-cell fusion which leads to the generation of large syncytia. The mHSA samples generated with low HOCl concentrations (molar ratios HSA:HOCl 1:10 and 1:50) showed no inhibitory effect on syncytium formation (Figure 1 ). At higher concentrations (molar ratio 1:100) an inhibitory effect of mHSA was detected with 90% inhibition at a concentration of approximately 50 μg/ml. The mHSA samples produced at molar ratios 1:1000 and 1:10.000 showed strong inhibition with 90% at 10 μg/ml and complete inhibition of gp120-induced syncytium induction at 20 μg/ml. Thus, HSA treated with higher concentrations of HOCl is transformed into an antiviral form in contrast to HSA samples treated with low HOCl doses.
Since a 1000-fold molar HOCl excess was sufficient to transform HSA into a viral entry inhibitor we generated HOCl-modified samples of three serum albumins from human, mouse and bovine by using a 1000-fold excess of HOCl. Using these three serum albumin preparations, we studied binding of mHSA, mBSA and mMSA to LAV (NL4-3) gp120 by surface plasmon resonance spectroscopy (SPR) (Figure 2a , c, e). The experiment demonstrated that serum albumin modified by a 1000-fold molar excess of HOCl bind to the immobilized gp120. No binding of mHSA, mBSA and mMSA to gp41 immobilized on a biosensor (binding < 5 RU, data not shown) was observed. Thus, binding was specific for gp120 envelope. Bound protein was removed from the gp120-biosensor by treatment with 100 mM HCl and 100 mM NaOH. This regeneration procedure retained the ability of immobilized gp120 to bind rCD4 and V2-and V3-loop specific antibodies. Also no binding to gp120 was measured using normal, non-modified HSA, BSA and MSA (Figure 2b , d, f). In these control experiments response units (RU) in SPR were also below 5 RU. Binding of HOCl-modified proteins was compared to gp120-binding of rCD4 and gp120-specific antibodies (Figure 2g ). At 50 μg/ml the modified proteins showed between 50-70% of the gp120 binding activity observed with rCD4 at 10 μg/ml. Binding of antibodies generated by immunization with CHOderived SF2-gp120 (Serum ADP429) or by a V3 loop peptide (IRIQRGPGRAFTIGC) (mab EVA 3047) showed binding responses at 1:80 dilution (Serum ADP429) or 25 μg/ml (mab EVA 3047) about 30 40 times as high as the HOCl-modifed serum albumins at 50 μg/ml.
Next we investigated whether binding of rCD4 or V2and V3-loop specific antibodies to gp120 was influenced by HOCl-modified serum albumin ( Figure 3 ). These binding studies showed that mHSA, mBSA and mMSA completely inhibited binding of rCD4 to LAV gp120 at concentrations above 10 μg/ml (Figure 3a) . In control experiments, neither binding of the anti-CD4-1F6 monoclonal antibody to CD4 nor binding of the secondary HRP-conjugated antibody was inhibited by HOCl-modified serum albumins (data not shown). In addition to rCD4, binding of V3 loop-specific monoclonal antibodies was reduced but not completely inhibited at 10 μg/ml (Figure 3b ). In contrast to binding of V3 loop antibody, binding of V2 loop antibody was not inhibited by mHSA, mBSA or mMSA.
For evaluation of virus neutralizing activity, the three HOCl-modified serum albumins were first tested in an MTT-assay (Figure 4a ). Using up to 200 μg/ml of HOClmodified protein, which is at least 10-times of the mHSA concentration necessary to neutralize NL4-3 >95%, we observed no anti-cellular activity in comparison to normal serum albumin. As shown in Figure 4b , virus infection was inhibited by mHSA, and mBSA in a dosedependent manner to >95% and >90% at a concentration of each 20 μg/ml. Virus inhibition by mMSA was slightly lower and inhibition >90% was observed using 50 μg/ml of mMSA. In addition to viral infection we also tested the inhibition of syncytium formation. As shown in Figure 5 , syncytium formation induced by virus strain NL4-3 (Figure 5b) was completely inhibited by mHSA at a concentration 50 μg/ml ( Figure 5c ). As demonstrated before (Figure 4a ) mHSA did not inhibit cell proliferation and, in agreement with this observation, cell morphology and staining of the nucleus proved vitality of the GHOST cells grown in the presence of 50 μg mHSA/ml (Figure 5a, c) .
Since syncytium formation is based on the presence of viral envelope and viral receptors on the cellular surface, syncytia can be simply induced by gp160 expression. Therefore, HeLa P4 cells were transfected with gp160 expression vector pSVATGrev. Transfected HeLa P4 cells expose the gp120/gp41 complex on their outer membrane surface and syncytia could be detected after 28 h (Figure 5e ). In contrast to non-transfected cells, incubation with mHSA did not show any syncytia (Figure 5d ). 1000) to LAV-gp120 was tested by surface plasmon resonance spectroscopy (Biacore 1000, GE Healthcare). Purified LAV gp120 (10 μg/ml, Protein Sciences Corporation) was covalently linked using EDC/NHC to a dextran coated, CH-activated sensor chip (CM5, GE Healthcare). After blocking with ethanolamine the biosensor was washed three times with 30 μl 100 mM NaOH and 30 μl 100 mM HCl (flow rate 5 μl/min). Proteins were tested for binding at 1, 5, 10, 25 and 50 μg/ml at a flow rate of 5 μl/min. (g). Binding of mHSA, mBSA and mMSA (values marked by the black arrow in Figure 1a, 1c and 1e ) was compared to the binding of CD4, and gp120-specific antibodies to a LAV gp120 coated CM5 biosensor. ADP429: anti-serum, derived from HIV-1 SF2 gp120-immunized sheep (AIDS reagent project, NIBSC, UK). EVA3047: monoclonal antibody, derived from IRIQRGPGRAFTIGC-peptide immunized mice (AIDS reagent project, NIBSC, UK). Figure 5f , incubation of pSVATGrev transfected cells with mHSA (50 μg/ml) again totally inhibited syncytium formation.
Next we investigated the effect of mHSA against infection of V3 loop recombinant NL4-3 viruses of X4, R5X4 and R5 tropism. The tropism was switched due to the exchange of the V3 loop as described earlier [17] . In addition we also tested mHSA against NL4-3 mutants which lack the N glycosylation site g15 within the V3 loop. As Figure 3 Inhibition of CD4 and antibody binding to gp120. Binding of CD4 or antibody, in the presence of mHSA, mBSA and mMSA, to LAV gp120 was tested by ELISA. ELISA plates were coated with LAV gp120 (1 μg/ml, Protein Sciences Corporation) over night. After gp120 binding, the wells were coated using BSA (20 mg/ml). Modified serum albumin was added to the wells and (a) CD4 (1 μg/ml, Protein Sciences Corporation) or (b) antibody diluted in phosphate buffered saline (PBS) pH 7.5, was added 30 min later. After 1 hour, the plates were washed 5-times with PBS. Binding of CD4 to gp120 was carried out at various mSA concentrations. Gp120-bound CD4 was detected by a CD4 specific mouse monoclonal antibody (NCL-CD4-1F6, Novocastra) followed by a staining with an anti-mouse HRP-antibody conjugate (BioRad). Shown are the averages of triplicate wells. Binding of V2-and V3-loop-specific monoclonal antibodies (mab) was carried out in the presence of 10 μg/ml of mHSA. Gp120-bound antibody was detected using an anti-mouse-HRP-antibody conjugate (BioRad). Antibodies were designated according to the repository reference of the Centralized Facility for AIDS Reagents. In brackets: originators antibody designation. All measurements were carried out in triplicates. White symbols: V2 loop specific mab. Black symbols: V3 loop-specific mab. shown in Figure 6 , all NL4-3 mutants were neutralized efficiently using 100-150 μg mHSA/ml. A significantly higher sensitivity against mHSA was observed for X4 viruses and the viruses lacking N glycan g15.
In the present study, we demonstrated that HOCl-modified serum albumins can bind to LAV gp120, and that HOCl-modified serum albumins possess the ability to efficiently neutralize entry of the NL4-3 laboratory strain. Since the 5'-half of NL4-3 was constructed from NY5 and the 3'-half from LAV the NL4-3 external envelope is iden-tical to the commercial LAV gp120 used in our SPR binding studies.
Binding between the LAV gp120 and our inhibitor was observed in a SPR assay with gp120 covalently linked to a biosensor chip. Binding to gp120 was only monitored with HOCl-modified serum albumin and non-modified serum albumin showed no binding activity. As shown before [16] , mHSA binding is also specific to the West Nile virus domain III and does bind to similar domains of Yellow Fever and Dengue-2 virus, respectively. Since mHSA also binds to WNV envelope, we suggest that the binding of HOCl-modified serum albumin to HIV-1 or WNV envelope is neither a highly epitope specific event nor does it mirror a fundamental, yet undiscovered, molecular step of the WNV or HIV-1 entry process. Such non-specific binding events are common in many other pathogen-host interactions. For example malaria parasites use negatively charged chondroitin-sulfate-proteoglycan (CSPG) structures to attach to the membrane surface of their host cells [19] . For HIV-1, heparan-sulfate-proteoglycan (HSPG) is such an attachment factor [20] and heparan sulphates as well as other polyanions are efficient inhibitors of HIV-1 infection [21, 22] . Our current hypothesis is that pathogens which coat their core particle with the membrane of the host cell need a simple mechanism to attach to cells possessing an identical membrane structure. This membrane-membrane contact must be mediated by the external envelope glycoprotein complex present in the viral membrane and might be a first non-specific binding event to initiate the next step, binding to specific receptors.
Masking the envelope complex to prevent membrane attachment seems to be a simple mechanism to prevent viral entry. The blockade of CD4 and V3-loop antibody binding by mHSA supports this hypothesis. The masking by mHSA completely inhibited rCD4 binding and partially inhibited the V3-loop antibody binding, indicating that a large and important proportion of the gp120 molecule is masked by mHSA. The surface of gp120 is per se covered by complex carbohydrate structures, which represent more than 55% of the gp120 molecular weight. Especially the elimination of the carbohydrate structure g15 within the V3 loop plays an important role in viral entry and these mutants showed higher infectivity. Due to higher infectivity, these viruses can escape from neutralizing antibodies present in human sera [23] . An interesting advantage of mHSA is the ability to neutralize these antibody resistant viruses. A more efficient neutralization of such viruses by mHSA might be the result of the lack of N-glycan g15 that renders the V3 loop more exposed. Such an unmasked V3 loop seems to be a better binding target for HOCl-modified albumins since the binding of V3-loop-specific neutralizing antibodies was blocked by mHSA. Masking the CD4 and V3 loop area by mHSA was also efficient for the blockade of CCR5-specific infection, but 3 times higher levels were necessary compared to the neutralization of CXCR4-viruses. Thus, V3-driven selection for higher infectivity of CXCR4 viruses or the switch from CCR5-to CXCR4-usage is not beneficial for promoting escape from mHSA neutralization. Blocking both coreceptor pathways is an important step to block HIV-1 transmission. In an evolving epidemic, CXCR4-tropism is present in high frequency among all HIV-1 subtypes except subtype C [24, 25] . The high frequency of CXCR4-tropism, 86% that was found in subtype A and CRF02_AG in patient samples from West Africa, demonstrates that a topical microbicide has to inhibit CXCR4-viruses frequently present in HIV-1 infected individuals. On the other hand, cervico-vaginal tissue preferentially supports the productive infection by CCR5-tropic viruses [26] . Thus, a microbicide has to block both coreceptor pathways to prevent HIV-1 sexual transmission. The HOCl-modified serum albumins, when applied as a topical microbicide, can be applied in an adequate concentration (>200 μg/ml) that is high enough to block both viruses completely.
In our experiments we added HOCl-modified serum albumin directly into cell culture media. Viral infection assays in cell culture have the advantage that cell culture ingredients such as (i) antioxidants present in fetal calf serum or (ii) free amino acids like methionine or cysteine present in cell culture medium at 0.2 M are present during the test period. In addition, the mHSA samples produced with low amounts of HOCl showed no inhibitory effect indicating that potential traces of taurine or HOCl are not contributing to the inhibitory effect. We also tested our inhibitor in three different neutralization assays using three different cell types (HeLa, GHOST and TZM-bl) which are well established as host cells, widely used in the HIV literature. Since virus is neutralized under all these different cell culture conditions, all the cell culture ingredients will not suppress the antiviral activity of mHSA. Preincubation of cells with mHSA also had no suppressive effect, showing that mHSA is not non-specifically adsorbed by the cells or their different membrane components. Moreover, the normal growth of other viruses like yellow fever and dengue-2 virus in cell cultures, over a period of up to 9 days, containing mHSA [16] indicates that mHSA has no unspecific overall neutralizing activity or toxic activity that inhibits cellular growth and more especially viral production. We have also tested mHSA in other sensitive cell culture systems (Plasmodium falciparum growth in erythrocytes, Lassa-, Marburg-, SARS virus growth) over a time period of 14 days (data not shown). In all these cell culture experiments we observed no inhibitory effect on pathogen growth indicating that mHSA in not a cellular toxin per se.
The data implicate that mHSA, or a well defined structure thereof, might be applicable as a drug against viral infection, and that the inoculation of mHSA into infected individuals might overcome adaptive immune tolerance. As was shown in animals, the active immunization of rats with Freund's adjuvant together with a high dose of chlorinated autoantigen induced an immune response against the HOCl-modified protein [27] . Induction of autoantibody might be a risk factor in the direct application of HOCl-modified proteins into body fluids but seems to be no major problem when applied as a topical microbicide. On the other hand, serum albumin is an important carrier for drugs and enzymes. In particular the transport of the enzyme MPO across the endothelial barrier depends on a specific interaction between HSA and MPO [28] . The HSA-binding epitope on MPO was identified as a short linear region (aa 425-454), causing high-affinity binding to HSA. The very close proximity of HSA and MPO implicates that HSA might be highly susceptible for MPO-dependent modifications. This also suggests that MPO-modified serum albumin is a common self antigen and therefore might be tolerated by the immune system.
It is known that HOCl is a powerful oxidant and modifications of protein structure and function are well documented.
In published studies, HOCl-induced modifications were analyzed after treatment of peptides or proteins with a 0.5-25-fold molar excess of HOCl [18, [29] [30] [31] . As shown by Vossmann et al. [16] as well as in the present study, an antiviral activity of serum albumins was observed only after treatment of serum albumin with HOCl concentrations above the molar ratio of 1:100. Using lower HOCl concentration, e.g. 1:10 or 1:50 we were unable to detect any antiviral activity in our HIV-1 infection assays. This observation is important and demonstrates that HOCl modifications, which occur under low HOCl conditions [18, [29] [30] [31] might be totally different to those induced with higher HOCl concentrations as shown here. Another minor aspect is that we have modified albumin at concentrations ≤ 1 mg protein/ml. When serum albumin was treated with HOCl at higher concentrations, as described in the literature (10 mg/ml) [29] , we observed no inhibitory activity in our viral infection and syncytium assays (data not shown). Thus, based on our current knowledge, the antiviral activity of serum albumins was induced in diluted aqueous solution and only with a >100-fold molar excess of HOCl.
The modification of proteins by HOCl seems to be a highly complex reaction. Data describing this process are available mainly based on the structure analysis of modified amino acids or peptides at low HOCl concentration. In a report by Salavej et al. [31] , HSA was treated with HOCl at a 25-fold molar excess, similar to the procedure by Pattison et al. [18] . In their analysis, oxygenation was detected for Trp238, Met147, Met353, and Met572, but no chlorination of any of the HSA residues was detected. Using HOBr, Salavej and coworkers detected the incorporation of one or two bromines at Tyr425, but no chlorination of any HSA residues was detected in HOCl treated samples [31] . However, the treatment of amino acid residues with HOCl leads to the documented side chain modifications, but HOCl-modification seems not to be limited to these side chain products. In the context of a linear peptide or protein HOCl-modified side chains undergo intra-or inter-molecular cross linking. Thus, in the context of a complex protein and probably under conditions with excessive concentrations of HOCl, the process is pushed towards the development of complex protein conformations the nature of which is still unknown. One final product of HOCl-treatment seems to be the generation of stable antigenic epitopes on human serum albumin.
Monoclonal antibodies against HOCl-induced epitopes, have been produced. These monoclonal antibodies were mainly raised against oxLDL. One of these monoclonal antibodies was able to bind to the HOClinduced epitopes on HSA and based on this antibody (clone 2D10G9) HOCl-modified proteins can be detected in human tissue [13] [14] [15] 29] . It was suggested that the epitope recognized by this monoclonal antibody is of a complex conformational type, but almost nothing is known about the nature of the 2D10G9 epitope. The fact that antibodies can be induced by immunization and HOCl-induced epitopes can easily be detected in human tissue [32] indicates that these HOCl-induced protein structures are chemically and structurally related and seem to be very stable.
Since a 100-fold molar excess of HOCl was necessary to induce the antiviral activity, all the reactions and modifications documented at low HOCl-concentrations might not explain the mechanism by which our HOCl-modified serum albumins inhibit viral entry of HIV-1 as well as West Nile virus. However, our study supports the hypothesis that HOCl might be part of the host defense against pathogens and apart from its direct toxic activity it may have an additional indirect but important activity, the transformation of a protein into an antiviral weapon [16] . From the cell culture experiments of Chase et al. and Klebanoff et al. [11, 12] , in which activated PMN cells release MPO and HOCl was generated, HOCl was suggested as the HIV-1-killing agent. Based on our results it is conceivable that the generated HOCl reacts with bovine serum albumin, which is usually present in high concentrations in cell culture media, and therefore mBSA would be present during viral infection. As we have now demonstrated, mBSA has a strong antiviral activity and might be also responsible for the antiviral effects observed in PMN cell cultures. The biological relevance of HOCl-modification of serum albumin or other proteins might be ques-tionable since virus replication in vivo is not limited to inflammatory loci, where MPO is expressed in vivo leading to the production of HOCl. To our understanding, the discovery of HOCl-modified serum albumin as an antiviral agent opens a new field for the development of nontoxic agents which can be added to blood products or can be used as a local microbicide to prevent viral transmission. Since the neutralizing activity of HOCl-modified serum albumins was documented against West Nile virus and the HIV-1 NL4-3 mutants, irrespective to their coreceptor type, future work will show how efficient HOClmodified serum albumins can neutralize other HIV-1 and HIV-2 variants as well as other viral species.
Taken together, the generation of HOCl-modified serum albumins is very simple, cost effective and needs no highly specialized laboratory equipment. We suggest that HIV-1 neutralization by HOCl-modified serum albumins is the result of masking the gp120 molecule. We have shown that the CD4 binding site and other regions, like the V3 loop, are partially masked by HOCl-modified serum albumins. Masking the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when applied, for example, as a topical microbicide. Human Pregnancy Specific Beta-1-Glycoprotein 1 (PSG1) Has a Potential Role in Placental Vascular Morphogenesis Previous studies suggest that human pregnancy specific beta-1-glycoproteins (PSGs) play immunomodulatory roles during pregnancy; however, other possible functions of PSGs have yet to be explored. We have observed that PSGs induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties. The present study investigates a potential role for PSG1, the most abundant PSG in maternal serum, as a possible inducer of proangiogenic growth factors known to play an important role in establishment of the vasculature at the maternal-fetal interface. To this end, we measured TGFB1, vascular endothelial growth factors (VEGFs) A and C, and placental growth factor (PGF) protein levels in several cell types after PSG1 treatment. In addition, tube formation and wound healing assays were performed to investigate a possible direct interaction between PSG1 and endothelial cells. PSG1 induced up-regulation of both TGFB1 and VEGFA in human monocytes, macrophages, and two human extravillous trophoblast cell lines. We did not observe induction of VEGFC or PGF by PSG1 in any of the cells tested. PSG1 treatment resulted in endothelial tube formation in the presence and absence of VEGFA. Site-directed mutagenesis was performed to map the essential regions within the N-domain of PSG1 required for functional activity. We found that the aspartic acid at position 95, previously believed to be required for binding of PSGs to cells, is not required for PSG1 activity but that the amino acids implicated in the formation of a salt bridge within the N-domain are essential for PSG1 function. Pregnancy specific beta-1-glycoproteins (PSGs)-previously known as SP1-are members of the carcinoembryonic antigen (CEA) family and are highly glycosylated proteins secreted by the placenta [1] . Human PSGs have been detected in the maternal serum as early as 3 days postfertilization, coinciding with the attachment of the blastocyst to the uterine wall [2] . Previous studies have reported abnormal levels of PSGs in complicated pregnancies and have shown their importance for the maintenance of a successful pregnancy [3] .
Eleven human PSG genes are clustered on chromosome 19q13.1-13.3 [4] . The human PSGs are comprised of a leader peptide followed by one N-terminal immunoglobulin (Ig) variable region-like domain (N-domain) and two or three Ig constant region-like domains [5] . PSG homologues have been identified in species with hemochorial placentation, including nonhuman primates, rats, and mice, all of which are species having several PSG genes that result in expression of several PSG proteins because of alternative splicing [6] . The function of human and murine PSGs is believed to be that of immunomodulators, as one of many reported mechanisms that prevent rejection of the allogeneic fetus by the maternal immune system. This proposed role of PSGs has been suggested by several investigators and is supported by different experimental findings, including the ability of PSGs to induce transforming growth factor beta 1 (TGFB1) [2, [7] [8] [9] [10] .
One of the best-known functions of TGFB1 is that of immunoregulation, but during pregnancy, TGFB1 has been suggested to play a role in several other processes, including implantation, trophoblast differentiation, and in the activation and resolution phases of angiogenesis [11, 12] . The recognized importance of TGFB1 during the angiogenic process has been strengthened by its ability to regulate the vascular endothelial growth factor (VEGF) family, which includes VEGFs A, B, C, and D as well as placental growth factor (PGF) [13] [14] [15] [16] . Therefore, we hypothesized that PSGs may induce TGFB1 to promote immune tolerance, affect trophoblast function, and promote angiogenesis in the pregnant uterus. We explored this possibility by examining the effect of PSG1 in cell types involved with development of the placental blood supply, including primary monocyte/macrophages, low-passage primary endothelial cells, and two extravillous trophoblast cell lines.
Previous studies from our laboratory showed that the Ndomain of PSGs is sufficient for their activity in target cells [9, 17] . The N-terminal domain of human PSG1 contains a Gly-Asp-Asp (GDD) motif on a solvent-exposed loop [18] in a region believed to be important for receptor binding and activity [19, 20] . The studies reported here indicate that amino acids G93 and D95, corresponding to the first and third amino acids in this potential receptor binding region, respectively, are not essential for the PSG1 functions we have described, whereas mutations in the salt bridge of the N-domain and elimination of two potential N-linked glycosylation sites render a protein without activity.
A better understanding of the molecules that control the process of angiogenesis and trophoblast-mediated vascular remodeling during pregnancy is important, because disturbanc-es in placental blood flow and vascular development impair fetal growth [21] . Angiogenesis occurs at all stages during pregnancy to ensure that the embryo will be supplied with sufficient nutrients and oxygen [22, 23] . The results presented here suggest that PSGs could contribute to the process of establishing the maternal-fetal vasculature in two ways. First, PSG1 has the ability to induce TGFB1 and VEGFA by different cell types, including monocytes/macrophages and extravillous trophoblasts. Second, PSG1 has the capacity to interact with endothelial cells, inducing tube formation, as well as to enhance vascular morphogenetic processes induced by VEGFA.
For immunoblotting, anti-FLAG M2 antibody (Sigma), the anti-PSG monoclonal antibody BAP1 (Genovac), and the anti-human Fc antibody (KPL) were employed. Neutralization of TGFB1 was performed by adding 10 lg/ml of the anti-human TGFB1 neutralizing antibody (R&D Systems) to the cells 10 min before PSG1 addition. Normal chicken Ig antibody, also obtained from R&D Systems, was used as the control. Recombinant human VEGFA was obtained from Immunotools.
The cDNA encoding the PSG1 leader peptide (L), N, A2, and B2 domains followed by a FLAG tag (Sigma) without the stop codon was synthesized by GenScript Corporation. The cDNA was subcloned into the BglII and NotI sites of the pEF1/V5-His vector (Invitrogen), resulting in the in-frame addition of the nucleotide coding for the V5 and histidine (His) tags at the C-terminus. The final construct, designated here as PSG1-FLAG, codes for a secreted PSG1 protein composed of the N, A2, and B2 domains followed by three tags-FLAG, V5, and His-at the C-terminus. To generate PSG1-Fc, the PSG1 cDNA encoding the L, N, A2, and B2 domains of PSG1 was subcloned inframe into the EcoRI-BglII sites of pFuse-IgG1 e3-Fc1 (InvivoGen), resulting in the addition of the hinge region, CH2 and CH3 domains, of the IgG heavy chain at the C-terminus.
We transfected each of the PSG1-encoding plasmids described above into dihydrofolate reductase-negative (DHFR À ) CHO cells along with pDCHIP, which encodes the Dhfr minigene, in a 1:10 molar ratio using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Positive transformants were obtained by methotrexate selection until maximum levels of protein were obtained from the cells, as determined by ELISA of the collected supernatants. Stably transfected cells were seeded into 5-kDa molecular weight cutoff hollow-fiber cartridges (FiberCell Systems, Inc.) and grown in Dulbecco modified Eagle medium supplemented with 2% fetal bovine serum (FBS; low IgG for PSG1-Fc production), 10 mM Hepes, 50 IU/ml of penicillin, and 50 lg/ ml of streptomycin. The supernatants from the cartridges were harvested daily, centrifuged at 5000 3 g for 10 min to remove cell debris, and then kept frozen until processed as described below. The control protein, FLAG-Fc, was harvested from the supernatant of DHFR À CHO cells that were a kind gift from Dr. Gerardo Kaplan (Center of Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD).
Recombinant proteins were purified by affinity chromatography using the Ä KTAprime Plus system (GE Healthcare). PSG1-FLAG was dialyzed into 20 mM sodium phosphate buffer (pH 7.4) containing 20 mM imidazole (EMD Chemicals, Inc.) and purified using a HisTrap column (GE Healthcare). The obtained fractions were pooled, buffer-exchanged into PBS, applied to a column packed with anti-FLAG M2 agarose (Sigma), and eluted with 33 FLAG peptide (Sigma). PSG1-Fc and the FLAG-Fc were dialyzed into 20 mM sodium phosphate buffer (pH 7.4) and purified using a HiTrap protein A column (GE Healthcare). The proteins were eluted with 0.1 M glycine (pH 2.7) and collected into tubes containing 100 ll of 1 M Tris-HCl (pH 8.0). Fractions containing the purified PSG from the anti-FLAG agarose or protein A columns were identified by Western blot analysis with the anti-PSG1 MAb BAP1 and were pooled. The pooled fractions were concentrated and buffer-exchanged with PBS using Amicon Ultra-15 10-kDa MWCO centrifugal filter units (Millipore Corp.). The purified proteins were run on a SDS-PAGE gel, stained with GelCode Blue Stain Reagent (Pierce), and quantitated against bovine serum albumin standards. In addition, the recombinant PSG1 proteins were immunoblotted with anti-FLAG (for PSG1-FLAG and FLAG-Fc) and antihuman Fc (for PSG-1Fc, the PSG1 mutants, and FLAG-Fc) after separation using SDS-PAGE. In all cases, 500 ng of protein were loaded per lane, and the antibodies were used at a concentration of 1 lg/ml overnight in 5% milk in Tris-buffered saline Tween-20 and were followed by a 1:10 000 dilution of the horseradish peroxidase-labeled secondary antibody (Bio-Rad).
To mutate selected amino acids in the N-domain of PSG1, we designed a PSG1 cDNA containing an Acc65I and an XhoI restriction site recognition sequence as silent mutations and cloned the cDNA into pFuse-IgG1 e3-Fc1 vector (InvivoGen). A 406-bp cDNA fragment was synthesized by Genscript Corporation in which the nucleotides coding for amino acids G and D-in positions 93 and 95, respectively, of the N-domain of the mature PSG1-were replaced for nucleotides coding for amino acids S and L, respectively. The fragment containing the mutations had an EcoRI site at the 5 0 end and an Acc65I site at the 3 0 end. To generate the mutant, we replaced the EcoRI-Acc65I fragment in PSG1 cloned into pFuse-IgG1 e3-Fc1 for the fragment containing the mutations. The successful exchange, resulting in PSG1 G93D95-S93L95, was confirmed by sequencing both strands of the cDNA.
The same strategy as described above was utilized to generate the R64, N79, and N77 mutant, in which all three amino acids were replaced for alanines. Briefly, a fragment with the mutated nucleotides with a 5 0 EcoRI site and a 3 0 XhoI site was synthesized by Genscript Corporation and replaced in the wild-type PSG1cDNA cloned into pFuse-IgG1 e3-Fc, previously digested with the same enzymes. The plasmids encoding the mutated PSG1s were transfected into CHO-K1 cells with Lipofectamine LTX (Invitrogen) according to the manufacturer's recommendations. Five hours posttransfection, the medium was aspirated, and the cells were kept in OPTI-MEM I (Invitrogen) for 72 h. The recombinant PSG1 G93D95-SL mutant, designated PSG1GDD!SDL, and the PSG1 R64N70N77-AAA mutant, designated PSG1RNN!AAA, were purified from the media using a protein A column as described above for wild-type PSG1-Fc. All proteins were determined to be endotoxin-free by their inability to induce tumor necrosis factor (TNF) in macrophages and with the HEK-Blue lipoplysaccharide (LPS) detection system (InvivoGen). The endotoxin levels in the proteins employed during these studies were tested, because LPS has been shown to induce VEGFA in some cell types.
All cells-both primary and established cell lines-employed for these studies were cultured in 5% CO 2 /95% air in a 378C humidified incubator. The RAW 264.7, CHO-K1, CHO DHFR À , and BeWo cells were obtained from American Type Culture Collection and cultured in the media and cell densities recommended. Because FBS contains high levels of TGFB1, we used 5% or 10% fetal clone III serum alternative (Hyclone) for culturing cells in the experiments in which TGFB1 was to be measured. The human invasive trophoblast HTR-8/SVneo cell line was provided to us by Dr. Charles Graham (Queen's University, Kingston, ON, Canada) and was cultured in HyQ RPMI 1640-RS (Hyclone) or RPMI 1640 (Gibco) with 5% fetal clone III or FBS, 13 normocin (InvivoGen), and 100 U/ml of penicillin/streptomycin (Gibco) [24] . The trophoblast cell line SGHPL-4 was a kind gift or Dr. G.S. Whitley (Division of Basic Medical Sciences, St. George's University of London, U.K.) and was cultured in Ham F-12 nutrient mix supplemented with either 10% fetal clone III or 10% FBS, 2 mM L-glutamine, and 103 penicillin/streptomycin. The human endometrial endothelial cell line (HEEC) was a gift from Dr. Gil Mor (Yale University School of Medicine, New Haven, CT). HEEC was cultured as described by Aldo et al. [25] and Krikun et al. [26] .
Human monocytes were isolated from the peripheral blood of healthy adult donors and purified by centrifugal elutriation as previously described [9] . The experiments employing monocytes were repeated six times, each time with cells from a different donor. Monocytes were maintained in RPMI 1640 (Invitrogen) supplemented with 2 mM glutamine and 50 lg/ml of gentamicin. Samples were obtained in accordance with National Institutes of Health Institutional Review Board approved protocols. Macrophages were derived from blood monocytes by culturing the adherent cells for 7 days in RPMI 1640 supplemented with 2 mM glutamine, 50 lg/ml of gentamicin, and 2% human type AB serum (Sigma). Human monocyte-derived dendritic cells were generated by culturing blood monocytes in RPMI 1640 supplemented with 2 mM glutamine, 10 ng/ml of CSF2 (granulocyte macrophage colony-stimulating factor; R&D Systems), and 20 ng/ml of interleukin 4 (R&D Systems) for 7 days; the day before PSG1 treatment, 100 ng/ml of LPS (Sigma) were added to the cells.
Uterine microvascular endothelial cells (UtMVECs) and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and utilized before 28 HA ET AL. passage 7. These cells were cultured in the basal media supplied by the company, supplemented with the recommended kit of media additives (EGM2 SingleQuots-hydrocortisone, human epidermal growth factor [hEGF], FBS, VEGF, human fibroblast growth factor beta [hFGFb], E3R-insulin-like growth factor 1 [R3-IGF], ascorbic acid, heparin, and gentamicin/amphotericin-B). Human dermal microvascular endothelial cells (HDMECs; Promocell) were cultured on gelatin-coated dishes in endothelial cell growth medium MV (Promocell) supplemented with 5% fetal calf serum. HDMECs were used at passage 3 or 4.
For ELISAs, cells were plated in triplicate wells the day before treatment in tissue culture, treated in 24-, 48-, or 96-well plate formats (BD Biosciences) and incubated in a 378C humidified incubator with 5% CO 2 . Cells treated in 24well plates were seeded as follows: RAW 264.7 cells at a density of 1 3 10 6 cells/well, HTR-8/SVneo cells at 0.4 3 10 6 cells/well, human monocytes at 1.2 3 10 6 cells/well, and endothelial cells (UtMVECs, HEEC, and HUVECs) at 0.4 3 10 6 cells/well. When the experiments were performed in 48-well plates, the cells indicated above were seeded at half the density as that used in the 24-well plates. BeWo cells and, in some experiments, human monocytes and HEEC were seeded in 96-well plates at a density of 1 3 10 5 , 4 3 10 5 , or 2 3 10 5 cells/ well, respectively. Human macrophages and human monocyte-derived dendritic cells were seeded in 96-well plates at a density of 0.3 3 10 6 cells/ well. SGHPL-4 cells were seeded at a density of 5 3 10 5 cells/well in a 96-well plate.
Cells seeded in 24-or 48-well plates were treated with recombinant proteins in 300 ll of media for treatment times of less than 6 h or in 1 ml of media for treatment times of greater than 6 h. Cells treated in 96-well plates were treated in 200 ll of media for 24 or 48 h. When primary endothelial cells were used in experiments designed to measure VEGFA, the cells were incubated in supplement-free EGM medium (Lonza) 2 h before treatment because of the presence of VEGFA in the complete media. Treatment of HEEC was performed in OPTI-MEM I (Invitrogen).
After treatment, the supernatants were collected and centrifuged at 5000 rpm for 5 min and used immediately for measurement of the specific factor by ELISA or stored at À808C. For measuring TGFB1 secretion by ELISA, supernatant or medium alone was activated according to the manufacturer's instructions (R&D Systems). VEGFA, VEGFC, and PGF ELISA kits were purchased from R&D Systems. TNF in the supernatants was determined using the OptEIA TNF (Mono/Mono) ELISA Set (BD Biosciences). At least three different protein preparations were tested, and experiments were repeated a minimum of three times.
The endothelial tube formation assay was carried out using Purecol, a threedimensional type I collagen gel (Nutacon) prepared in 48-well cluster tissue culture dishes as previously described [27] . After polymerization of the gel at 378C, wild-type HDMECs were seeded onto solidified gels at a concentration of 6 3 10 4 cells/well in 300 ll of endothelial growth medium MV containing 5% fetal calf serum and SupplementMix (containing heparin, epidermal growth factor, and hydrocortisone; Promocell). At confluence, the medium was replaced by basal medium containing 5% heat-inactivated fetal calf serum without further supplements. After 24 h, cells were treated with PSG1-Fc or FLAG-Fc (100 and 500 ng/ml) alone or in combination with VEGFA (25 ng/ ml). This treatment was repeated every 3 days, and photographs were taken with a phase-contrast microscope (Carl Zeiss, Inc.) equipped with a digital camera (ProgRes C10 plus ; Jenoptik AG).
For wound healing assays, HDMECs were seeded in a 48-well tissue culture plate until they reached confluence. In the middle of each well, a wound line was induced using a pipette as previously described [28] . Each well was treated with VEGF alone (25 ng/ml), PSG1-Fc alone (100 and 500 ng/ml), FLAG-Fc alone (100 and 500 ng/ml), PSG1-Fc (100 and 500 ng/ml) plus VEGF (25 ng/ml), and FLAG-Fc (100 and 500 ng/ml) plus VEGF (25 ng/ml). Basal medium alone was used as a negative control. The wounding area of each well was observed by phase-contrast microscopy (Leica). These areas were observed daily and photographed using a digital camera mounted on the phasecontrast microscope. The wounding distance was measured using the morphometric program Optimas (Optimast).
SPSS (SPSS, Inc.) and Microsoft Excel were used for statistical analysis of data. The two-tailed Student t-test was used to determine statistical significance in experiments comparing protein-treated cells versus untreated cells, with a Pvalue of less than 0.05 as a cutoff. One-way ANOVA was used to determine statistical significance of dose-response assays, with a P-value of less than 0.01 as a cutoff. All data are representative of at least three independent experiments.
The human PSG family has 11 members, and PSG1 is the most abundant family member [29] . We generated recombinant PSG1-FLAG, which consists of the leader peptide, followed by the N, A2, and B2 domains of PSG1 and then a FLAG, V5, and six His tags. The recombinant protein has an approximate molecular mass of 55 kDa and was harvested from the supernatant of stably transfected CHO cells, as described in Materials and Methods. Recombinant PSG1-FLAG protein was sequentially purified on a HisTrap column followed by an anti-FLAG affinity column. These two purification steps rendered a glycoprotein that was more than 90% pure based on Coomassie blue staining (Fig. 1, lane 1) .
We generated recombinant PSG1-Fc, which contains the same PSG1 domains as PSG1-FLAG but has a different tag at the C-terminus. The FLAG, V5, and His tags of PSG1-FLAG were replaced by the Fc tag, which consists of the mutated human IgG hinge domains, CH2 and CH3, from the pFuse-hIgG1e3-Fc1 vector. The recombinant protein has an approximate molecular mass of 70 kDa (Fig. 1, lane 2) . The FLAG-Fc protein (Fig. 1, lane 3) secreted from CHO was used as the control protein, as described in Materials and Methods.
Two different PSG1 mutants were generated. We mutated two residues at positions 93 and 95 within the N-domain of PSG1 to generate PSG1GDD!SDL-FC and three amino acids in positions 64, 70, and 77 to generate PSG1RNN!AAA-FC (see Fig. 6A, lanes 2 and 3, respectively) . The level of purity achieved after protein A purification of all proteins was more than 90%.
We have previously reported that recombinant GST-PSG1 purified from insect cells induces TGFB1 in human monocytes and in murine macrophages [9] . Human monocytes secreted PSG1 AND ANGIOGENESIS 29 TGFB1 in response to PSG1-Fc treatment, starting at a concentration of 10 lg/ml and up to the highest concentration tested (50 lg/ml) compared to FLAG-Fc-treated cells ( Fig.  2A) . We also observed a significant induction of TGFB1 in human monocytes (between two-and threefold the amount of TGFB1 secreted over the control protein-treated cells, depending on the donor) at a PSG1-FLAG concentration of 10 lg/ml.
In macrophages, TGFB1 has been shown to induce the proangiogenic factor VEGFA [14] . Therefore, we investigated the possibility that in addition to inducing TGFB1, PSG1 might regulate VEGFA expression in monocytes and macrophages. Human monocytes were treated with PSG1-Fc or control protein starting at a concentration of 10 lg/ml. Induction of VEGFA was seen in human monocytes at 50 lg/ml of PSG1-Fc over FLAG-Fc control (Fig. 2B) . In some experiments, significance was observed at PSG1 concentrations of 30 lg/ml (data not shown). When PSG1-FLAG was used to treat a mouse macrophage cell line (RAW 264.7), concentrations of 30 lg/ml were sufficient to observe induction of VEGFA over controls (data not shown).
Treatment of human macrophages derived from blood monocytes required concentrations of PSG1-Fc of 50 lg/ml to observe a significant induction of VEGF-A over FLAG-Fc (data not shown). While we found that monocyte-derived dendritic cells secreted TGFB1 in response to PSG1 treatment, VEGFA secretion was not induced by PSG1-Fc even at the highest concentration tested (80 lg/ml; data not shown).
Based on our recent observations that murine PSG23 induces TGFB1 and VEGFA in mouse and human trophoblast cell lines [30] , we explored the possibility that human trophoblasts could also respond to PSG1 treatment. Because we cannot obtain material to perform these experiments with primary first-trimester human trophoblast cells, we relied on the well-characterized, first-trimester extravillous trophoblast (EVT) cell lines HTR-8/SVneo [24] and SGHPL-4 [31] . These cell lines were derived from first-trimester chorionic villous tissue and, after immortalization, have been shown to retain many features of normal EVT and to behave in the same manner as primary cells [32, 33] . HTR-8/SVneo and SGHPL-4 cells were treated with increasing concentrations of PSG1, starting at 1 lg/ml and increasing up to 50 lg/ml. Upregulation of TGFB1 was observed at 10 lg/ml for both cell lines (Fig. 3, A and D) . Significant up-regulation of VEGFA was observed at 30 lg/ml in HTR-8/SVneo (Fig. 3B ) and 10 lg/ml in SGHPL-4 cells (Fig. 3E) .
Chung et al. [13] showed that culturing HTR-8/SVneo cells in the presence of TGFB1 resulted in an increase in the expression of VEGFA. Figure 3C shows that neutralization of TGFB1 affected the increase in VEGFA expression by HTR-8/ SVneo cells after treatment with PSG1. Therefore, the induction of VEGFA was TGFB1 dependent.
To investigate the possible interaction of PSG1 with endothelial cells, we treated primary human myometrial UtMVECs, which have been previously used in coculture experiments to mimic the uterine endothelial lining [34] , with 1, 10, 25, and 50 lg/ml of PSG1 or control protein. In addition, PSG1 and FLAG-Fc were added at the same concentrations to HUVECs and HEEC, immortalized by telomerase-mediated transformation. Significant PSG1-induced secretion of TGFB1 in UtMVECs (Fig. 4A) , HEEC (Fig. 4B ), and HUVEC (not shown) was observed starting at concentrations of 25 lg/ml. When testing the ability of PSG1 to induce VEGFA in these cells, we did not observe a significant induction of VEGFA with concentrations of PSG1 from 1 to 80 lg/ml (data not shown).
The experiments described above strongly suggest that at least some endothelial cells express receptors for PSG1. Therefore, we hypothesized that PSG1 could play a role in uterine angiogenesis and capillary morphogenesis. To investigate this further, we performed in vitro endothelial tube formation assays. HDMECs were cultured and seeded onto a collagen gel at passage 3 or 4 and then cultured until confluence. Afterward, they were stimulated with PSG1-Fc or the control protein (FLAG-Fc) alone or in combination with VEGFA. HDMECs exposed only to VEGFA were used as a positive control and formed tubes as expected (Fig. 5A) . Cells cultured in basal medium, the negative control, showed no tube formation (Fig. 5B ). PSG1 alone, applied at concentrations of 100 and 500 ng/ml, induced tube formation as shown in Figure  5C (100 ng/ml of PSG1). Simultaneous application of PSG1 at 25 and 100 ng/ml of VEGFA increased the network of VEGFA-induced endothelial tubes (Fig. 5D) as assessed semiquantitatively under microscopic evaluation. When the control protein FLAG-Fc was used instead of PSG1, we could not observe the formation of tubes or the increase in VEGFAmediated tube formation (data not shown). Neither PSG1-Fc nor FLAG-Fc applied at concentrations 100 and 500 ng/ml had an effect on wound distance in comparison to the negative control, in which the cells were exposed to basal medium only (data not shown). In contrast, VEGFA treatment of the cells, which was used as positive control, reduced the wounding distance within 12 h after wound induction (not shown).
Two other members of the VEGF family, VEGFC and PGF, have been shown to be important for normal placental development [35] [36] [37] [38] . We tested whether PSG1 induced VEGFC and PGF in different cell types. Expression of PGF has previously been reported in trophoblasts and endothelial cells [39] . Our results showed that PSG1 did not induce PGF in HEEC (Fig. 6A) , HUVECs (Fig. 6B) , and UtMVECs (data not shown). The choriocarcinoma cell line BeWo secreted high basal levels of PGF, and no significant difference in control protein and PSG1-treated wells was found (Fig. 6C) . On the other hand, the basal level of PGF secretion by HTR-8 SVneo cells was low-at the level of the lowest ELISA standard-but as for BeWo cells, no induction by PSG1 could be detected (data not shown).
The major source of VEGFC during pregnancy has been postulated to be the uterine natural killer (uNK) cells, but some positive staining for VEGFC in the extravillous trophoblast has also been observed [40] . We saw significant induction of VEGFC by PSG1 in HTR-8/SVneo cells, which express high basal levels of this growth factor, but this was not reproducible in four other independent experiments (Fig. 6D ).
The availability of the crystal structure of the murine CEArelated cell adhesion molecule CEACAM1 has made it possible to model the structure of other members of the CEA family [41] . The N-terminal domain of most human PSGs, with the exception of PSG1, PSG4, and PSG8, contain an Arg-Gly-Asp (RGD) motif [18] . This motif is on the FG loop, which is solvent exposed and considered to be a biologically important element in CEACAM1. PSG1 has the sequence GDD rather than RGD, but the importance of this region for receptor binding and activity for all PSGs has been frequently cited [19, 20] . To investigate the possible importance of this region of the PSG1 protein, we mutated the G at position 93 and the D at position 95 to S and L, respectively. We selected the amino acids S and L based on their presence in the corresponding position in human CEACAM3. These changes are not expected to affect the folding of the protein while being substantially different from the amino acids present in wild-type PSG1. Like wild-type PSG1-Fc, the resulting protein has an approximate molecular mass of 70 kDa (Fig. 7A) . The PSG1GDD!SDL mutant was compared to the wild-type protein for its ability to induce TGFB1 in HTR-8/SVneo, monocytes, and HEEC. Our In C, HTR-8/SVneo cells were pre-treated with 10 lg/ml of anti-TGFB1 antibody or isotype-matched control antibody (IM) 1 h before addition of 50 lg/ml of PSG1-Fc or FLAG-Fc. After 24 h, VEGFA in the supernatants was analyzed by ELISA. All cells were treated in triplicate, and data are representative of at least three independent experiments. For A, B, D, and E, statistical significance was determined by one-way ANOVA, and the Pvalue between doses was less than 0.01. For C, statistical significance between the PSG1-Fc-treated and FLAG-Fc-treated samples was determined by two-tailed Student t-test (*P , 0.05). Error bars represent the SEM. results show that the PSG1GDD!SDL mutant induced TGFB1 over control protein (Fig. 7B) .
Comparison of the PSG family members in human, baboon, mouse, and rat show some conserved elements. For example, an arginine at position 64 and an aspartic acid at position 82 are believed to form a salt bridge [18] . Between the amino acids forming the putative salt bridge are two (out of a total of three) potential N-linked glycosylation sites in the N-domain of all human PSGs. These potential N-linked glycosylation sites are also conserved in all mouse PSGs and in the majority of baboon and rat PSGs. Because the importance of N-linked glycosylation and the putative salt bridge has never been explored, we introduced three mutations that render a protein without the conserved N-linked glycosylation sites and the ability to form the salt bridge. As expected, this protein has a lower molecular weight than wild-type PSG1 (Fig. 7A, lane 3) . The PSG1RNN!AAA mutant did not induce TGFB1 in any of the cells tested, as shown in Figure 7B for HTR-8/SVneo cells.
Angiogenesis is required for embryonic development and growth for successful hemochorial placentation (for recent reviews, see [23, 42] ). Distinct vascular processes occur during pregnancy, starting with adaptations of the endometrial vasculature to support blastocyst implantation, followed by expansion and de novo formation of blood vessels to support embryo growth and development. Even at later stages of pregnancy, the placental vasculature continues to be remodeled to enable blood flow to the increasing metabolic demands of the fetus. Sufficient uteroplacental blood flow requires remodeling of the spiral arteries by the extravillous trophoblasts [43] . Failure of spiral artery remodeling has been associated with complications of pregnancy, such as preeclampsia and fetal growth restriction [44] [45] [46] . Therefore, a better understanding of the stimuli needed for cell production of angiogenic growth factors during the processes of uteroplacental vascular remodeling and the generation of the placental vascular network is of major clinical interest.
Trophoblasts play an important role in the physiological changes of the spiral arteries as well in other processes during establishment of the placental vasculature, but together with trophoblasts, other cell types clearly are involved in the regulation of angiogenesis during pregnancy. Among them are decidual and placental macrophages as well as dendritic, endothelial, uNK, and mesenchymal cells. Many of these cells secrete or respond to angiogenic factors, such as members of the VEGF family [40, [47] [48] [49] [50] .
Besides its well known role in angiogenesis, VEGFA has been suggested to regulate trophoblast behavior in an autocrine manner [51] . Other members of the VEGF family, such as PGF and VEGFC, promote endothelial survival and vascular remodeling [38] . In addition, PGF was shown to play an important role in successful uNK cell proliferation and/or differentiation [37] and VEGFC to facilitate immune tolerance and endovascular activity of uNK cells [35] . Therefore, we decided to investigate whether PSG1 induces the expression of different members of the VEGF family. When exploring the possible induction by PSG1 of VEGFC in HTR-8/SVneo cells and of PGF in BeWo cells, we found that these cells express very high basal levels of these growth factors and that whereas we saw significant induction in some experiments, this induction was not reproducible in others. Therefore, at this time, we are unable to reach a definite conclusion.
Expression of VEGFA by placental macrophages has been documented [52] , and whereas macrophages have been shown to be involved in angiogenesis, they require stimulation by activating factors [53] . We observed that PSG1 induced significant secretion of VEGFA and TGFB1 in primary monocyte/macrophages, with a small variation in the concentration of PSG1 required depending on the donor. The cells used in our in vitro studies most likely do not exactly replicate the phenotype of placental macrophages, but we propose that PSGs could be one of the placental products, which increases FIG. 5. In vitro endothelial tube formation assay on three-dimensional collagen gel in the presence or absence of PSG1 and VEGFA. Human dermal microvascular endothelial cells were seeded on the top of polymerized collagen gel and cultured until they achieved subconfluence. The cells were then stimulated with PSG1 alone or in combination with VEGF. VEGF induces endothelial tubes (A, arrows) as expected, whereas no tubes were seen in the control when the cells were exposed to basal medium only (B). Addition of PSG1-Fc to basal media induces endothelial tubes (C, arrows) and also supports VEGF-induced tube formation (D, arrows). Original magnification 3450.
HA ET AL. the secretion of VEGFA by these cells. Whereas we found that monocyte-derived dendritic cells secreted TGFB1 in response to PSG1 treatment, VEGFA secretion was not induced. Recent reports indicate that uterine dendritic cells fine-tune decidual angiogenesis by producing TGFB1 and secreted FLT1 and that they have an important role in vascular development [49, 54] . Interestingly, only alternative activation-not TGFB1 treatment-was reported to lead to VEGFA production by dendritic cells [55] . Therefore, it remains to be investigated whether, in the decidual microenvironment, PSG1 may induce dendritic cells to secrete VEGFA.
When exploring the potential interaction of PSG1 with nonimmune cells, we found that PSG1 induced the proangiogenic factor TGFB1 in different primary endothelial cells, HEEC, the choriocarcinoma cell line BeWo, and two firsttrimester extravillous trophoblast cell lines, HTR-8/SVneo and SGHPL-4. Of the cells listed above, VEGFA induction was only observed in the HTR-8/SVneo and SGHPL-4. Therefore, PSG1 could potentially play a role in trophoblast invasion, migration, and differentiation through its ability to regulate VEGFA secretion in EVT.
Our data show that PSG1 is involved in capillary morphogenesis and that it also influences vascular morphogenetic processes induced by VEGFA. PSG1 induced endothelial cell tube formation in the presence and absence of VEGFA. When VEGFA was added to the cells together with PSG1, we observed that the network was enhanced over the cells in which PSG1 or VEGFA was added alone. Further experiments are required to determine the molecular and signaling mechanisms by which PSG1 induces endothelial tube formation. Tube formation likely is not the result of induction of VEGFA by PSG1, because we did not observe significant PSG1-mediated VEGFA secretion in endothelial cells at PSG1 concentrations employed in the tube assays. Additionally, we did not observe an increase in endothelial cell migration upon PSG1 treatment, which would be expected as a result of up-regulation of VEGFA. Therefore, while PSG1 may increase the availability of VEGFA in the placenta, the effect on endothelial cells, because of its ability to induce VEGFA secretion from other cell types, may be of a paracrine nature or could be mediated by the observed up-regulation of TGFB1. PSGs belong to the CEA family, and it is interesting to note that other members of this family, which are membrane bound, have been implicated in the processes of immunoregulation, human trophoblast invasion, and angiogenesis [27, [56] [57] [58] [59] [60] [61] .
The PSG concentration increases progressively to reach a plateau in the last 4 wk of pregnancy. At 200 lg/ml, PSGs are the most abundant fetal proteins in the maternal bloodstream during late pregnancy [62] . At this time, and to our knowledge, no specific antibodies can distinguish the different members of the human PSG family, but splice variants for most of them have been reported to be expressed, with most of these including the N-domain [19] . Recently, transcripts for PSG1 were found in sperm, and PSG1 protein was detected during zygotic development, suggesting a possible role for this protein in the early stages of embryogenesis and/or implantation [63] . Within the N-domain, most PSGs have an RGD or an RGDlike sequence. The RGD motif forms the minimal functional binding unit in some integrin ligands [64] , and its presence in one of the solvent-exposed loops of most PSGs was assumed to be required for PSG function [20] . Within this sequence, the conservation of the aspartic acid (D) at position 95 has been cited as essential for function [18, 19] . Our results indicate that at least for some functions of PSGs, the G in position 93 and the D at position 95 are not essential. Binding assays showed that although the PSG1GDD!SDL mutant binds to target cells, such as HTR-8/SVneo, significantly over the control protein, the binding (measured as mean fluorescence intensity) was approximately half that of the wild-type protein. Whether other, as-yet-unknown functions of this protein could be mediated by these amino acids, or how the mutation affects the affinity of the interaction with the receptor, remains to be determined.
Mutations of the two conserved potential N-linked glycosylation sites and of one of the amino acids (R64) involved in the formation of a salt bridge rendered a protein with no activity. The introduced mutations very likely resulted in major conformational changes in the protein. Preliminary data from our laboratory indicates that enzymatic removal of N-linked carbohydrates with peptide-N-glycosidase F results in a significant reduction of the activity of the protein. In addition, we have previously established that N-linked glycosylation is required for interaction of murine PSG17 with CD9 [65] . It remains to be determined whether the removal of the carbohydrates just in the N-domain is enough to destroy the activity of the protein.
At present, the receptor for human PSGs remains unknown. Because human PSGs share 80% identity at the amino acid level, it is worth exploring whether all 11 members of the family bind to the same receptor to perform these functions. We performed our studies with two recombinant PSG1 proteins, which differ in the nature of the tag at the Cterminus. Both proteins had the same activity in target cells with small variations in the concentration of protein required to observe the effects reported.
In the present study, we show that different cell types can respond to PSG1, secreting growth factors known to regulate vascular development and trophoblast behavior. In addition, the in vitro studies described here indicate that the interaction of PSG1 with endothelial cells can have functional consequences. Therefore, we propose that besides their ability to regulate the immune response, PSGs may have the previously unrecognized ability to contribute to the establishment of the vasculature during pregnancy. The precise processes during angiogenesis in the maternal-fetal unit that could be modulated by this family of proteins require further investigation. Structural Optimization and De Novo Design of Dengue Virus Entry Inhibitory Peptides Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding “pseudoenergies”, we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 µM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery. Fusogenic viral envelope glycoproteins are multimeric proteins that facilitate the fusion of viral and target cell lipid membranes during the initiation of infection. The membrane fusion process is energetically favorable and essentially irreversible, but has a considerable kinetic energy barrier [1] . These proteins allow rapid membrane fusion by drawing the opposing membranes together and either stabilizing or providing the activation energy to surmount the transition state [1, 2] . In this way, they behave in many aspects like a fusion catalyst. Because they effect a macromolecular process that involves large scale conformational changes in the substrate membranes and the proteins themselves, these proteins possess multiple interacting surfaces that could be targeted by inhibitors [3] .
There are several distinct types of viral fusion proteins, including the class I, primarily alpha helical proteins (such as HIV TM and influenza HA), the class II, primarily beta sheet proteins (such as the flavivirus E and alphavirus E1), and mixed helix/sheet proteins (including herpes virus gB and rhabdovirus G) [3, 4] . To date, most progress with viral fusion protein inhibitors has focused on class I alpha helical proteins. The HIV TM protein provides an excellent example of targeting distinct, interacting surfaces for inhibition. The HIV TM functions as a homotrimer with each monomer contributing two alpha helical regions that interact to form a post-fusion six-helix bundle. Inhibition of the formation of this six-helix bundle can be accomplished by exogenous peptides mimicking either of the two reciprocally interacting helices [5] [6] [7] .
Only a few examples of viral entry inhibitors with activity against the primarily beta sheet envelope proteins (E) from flaviviruses have been described [8] [9] [10] . However, few of these have taken advantage of the available crystal structures of flavivirus E proteins, including both pre-fusion and post-fusion forms [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . The authors of some of these structures have predicted that several regions of these proteins might be targets for inhibition [11, 14, 15] . Here we report the use of structural data from the pre-fusion dengue virus-2 (DENV-2) E protein as a model for a computational approach to the design of new peptide inhibitors of DENV-2 entry. This approach makes use of a residue-specific all-atom probability discriminatory function (RAPDF) score to identify in situ amino acid sequences that are likely to have high structural and binding stability [23, 24] . Out of seven computationally designed peptides that were synthesized and tested, two were identified as possessing fifty percent in vitro inhibitory activity (IC 50 ) below 10 mM and another with IC 50 activity below 40 mM. Two of the inhibitors (DN57opt and DN81opt) are binding optimized variants of peptides originally designed from DENV inhibitory peptide sequences located in domain II near the domain I/domain II hinge region [9] . The other (1OAN1) is an entirely novel peptide designed from an extended beta sheet region comprising the first connection between domains I and II. We show that the two peptides with the highest inhibitory activity interfere with virus:cell binding, cause structural changes to the surface of DENV-2 virions, and bind specifically to purified DENV-2 E protein.
The causative agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, DENV has emerged in the past several decades as the most important mosquito borne viral disease with an estimated 2.5 billion people living in areas at risk for epidemic transmission and 50-100 million people infected annually [25, 26] . Complicating this situation, the four distinct serotypes of DENV generate only low level immunological cross protection, allowing for repeated epidemic outbreaks in the same populations [27, 28] . The phenomenon of antibody dependent enhancement has been shown to result in more severe disease in individuals who have been previously infected with a different serotype [29] [30] [31] [32] [33] . With no specific treatment or prevention available other than vector control, DENV is an important target for the development of antivirals and vaccines. The results presented here indicate that the DENV E glycoprotein has multiple accessible surfaces that can be targeted by distinct inhibitors and is an amenable target for rational inhibitor design.
Peptide inhibitors were designed to have improved in situ binding compared to naturally occurring sequences using the residuespecific all-atom probability discriminatory function (RAPDF) [24] . The x-ray diffraction structure of DENV-2 envelope protein (Protein Data Bank identifier 1OAN) was used as a template for creating mutant structures from which the peptides were derived [14] . For each peptide, we randomly selected a residue side chain and substituted it with a new side chain. The substitution was performed using a backbone-dependent side chain rotamer library and a linear repulsive steric energy term provided by SCWRL version 3.0 [34] . The resulting all-atom models were energy minimized for 200 steps using the Energy Calculation and Dynamics (ENCAD) program [35] [36] [37] . RAPDF scores were then calculated to estimate the structural stability of a given E protein structure derivative. For a selected residue, side chain substitution was carried out ten times. The amino acid that produced the best RAPDF score was selected and used as a template for further mutation. The entire mutation process was repeated 100,000 times to enable a rigorous search for peptides that produced the best RAPDF score (i.e., highest predicted stability).
A 20 residue acid sliding window that moved from the N to the C terminus of the E protein in 10 residue acid increments was evaluated by a structural stability (pseudoenergy) optimization protocol using the RAPDF. A Metropolis Monte Carlo search algorithm [38] was used to change each amino acid in the 20 residue window to one of the other 19 naturally occurring amino acids, and the stability of corresponding peptide in the context of the entire E protein structure was evaluated. This process was iterated 100,000 times using RAPDF as the target scoring function. The Metropolis criterion was used to select a particular change in the simulation: if a particular change resulted in a better RAPDF score (lower pseudoenergy), then it was retained. If a particular change resulted in a worse RAPDF score (higher pseudoenergy), then a random choice, based on the score difference between the previous change and the current one, was made to retain the corresponding change. This procedure enables not only enables design of peptides that will result in high structural and binding stability (i.e., the best RAPDF scores/ pseudoenergies), but also enables surmounting local minima encountered during the search. Computational optimization was performed on the four regions corresponding to the best RAPDF score, and therefore the highest binding potential, within the E protein as described above to generate variant peptides sequences.
DENV-2 strain NG-C was obtained from R. Tesh at the University of Texas at Galveston. Virus was propagated in the Macaca mulatta kidney epithelial cell line, LLC-MK2 (ATCC catalog number CCL-7). Cells were grown in Dulbecco's modified eagle medium (DMEM) with 10% (v/v) fetal bovine serum (FBS), 2 mM Glutamax, 100 U/ml penicillin G, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B, at 37uC with 5% (v/v) CO 2 .
Peptides were synthesized by solid-phase N-a-9-flurenylmethyloxycarbonyl chemistry, purified by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray mass spectrometry (Genemed Synthesis,
Virus surface proteins mediate interactions with target cells during the initial events in the process of infection. Inhibiting these proteins is therefore a major target for the development of antiviral drugs. However, there are a very large number of different viruses, each with their own distinct surface proteins and, with just a few exceptions, it is not clear how to build novel molecules to inhibit them. Here we applied a computational binding optimization strategy to an atomic resolution structure of dengue virus serotype 2 envelope protein to generate peptide sequences that should interact strongly with this protein. We picked dengue virus as a target because it is the causative agent for the most important mosquito transmitted viral disease. Out of a small number of candidates designed and tested, we identified two different highly inhibitory peptides. To verify our results, we showed that these peptides block virus:cell binding, interfere with a step during viral entry, alter the surface structure of dengue viral particles, and that they interact directly with dengue virus envelope protein. We expect that our approach may be generally applicable to other viral surface proteins where a high resolution structure is available. Focus forming unit assay LLC-MK2 target cells were seeded at a density of 1610 5 cells in each well of a 6-well plate 24 h prior to infection. Approximately 200 focus forming units (FFU) of virus were incubated with or without peptide in serum-free DMEM for 1 h at rt. Virus/peptide or virus/ control mixtures were allowed to infect confluent target cell monolayers for 1 h at 37uC, with rocking every 15 m, after which time the medium was aspirated and overlaid with fresh DMEM/10% (v/v) FBS containing 0.85% (w/v) Sea-Plaque Agarose (Cambrex Bio Science, Rockland, ME) without rinsing. Cells with agar overlays were incubated at 4uC for 20 m to set the agar. Infected cells were then incubated at 37uC with 5% CO 2 for 5 days. Infected cultures were fixed with 10% formalin overnight at 4uC, permeablized with 70% (v/v) ethanol for 20 m, and rinsed with phosphate buffered saline, pH 7.4 (PBS) prior to immunostaining. Virus foci were detected using a specific mouse mAb from hybridoma E60 (obtained from M. Diamond at Washington University), followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Pierce, Rockford, IL), and developed using AEC chromogen substrate (Dako, Carpinteria, CA). Results are expressed as the average of at least two independent trials with three replicates each. IC 50 values were determined using variable slope sigmoidal doseresponse curve fits with GraphPad Prism 4.0 software (LaJolla, CA), except for DN81opt, which was determined graphically due to a lack of data points to produce a reasonable curve fit.
Cytotoxicity of peptides was measured by monitoring mitochondrial reductase activity using the TACS TM MTT cell proliferation assay (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. Dilutions of peptides in serum-free DMEM were added to confluent monolayers of LLC-MK2 cells in 96-well plates for 1 h at 37uC, similar to the focus forming inhibition assays, and incubated at 37uC with 5% (v/v) CO 2 for 24 h. Absorbance at 560 nm was measured using a Tecan GeniosPro plate reader (Tecan US, Durham, NC).
DENV-2 NGC strain used for the cryoEM reconstructions was propagated in mosquito C6/36 cells. Virus was purified by precipitation with 40% PEG 8000 and then ultracentrifugation onto a 25% sucrose cushion. Virus was further purified by banding on a 10%-30% potassium tartrate gradient. The virus band was removed and dialyzed against 12 mM Tris pH 8.0, 120 mM NaCl, 1 mM EDTA, and concentrated using a Millipore Centricon filter. Purified virus was mixed with 1OAN or DN57opt at a concentration of 1 molecule of peptide for every E protein on the surface of the virus. The complex was incubated for half an hour at 37uC followed by half an hour at 4uC and then flash frozen on holey carbon grids in liquid ethane. Images of the frozen complex were taken with a Philips CM200 FEG transmission electron microscope (Philips, Eindhoven, The Netherlands) at a magnification 51,040 using an electron dose of approximately, 25e-/Å 2 using a Charge-Couple device.
Real time binding assays between peptides and purified DENV-2 S1 E protein were performed using biolayer interferometry on an Octet QK system (Fortebio, Menlo Park, CA). This system monitors interference of light reflected from the surface of a fiber optic sensor to measure the thickness of molecules bound to the sensor surface. Purified, recombinant, 80% truncated DENV-2 S1 E protein was obtained from Hawaii Biotechnology (Honolulu, HI). Peptides were N-terminally biotinylated with a 5:1 molar ratio of NHS-LC-LC-Biotin (Pierce/ThermoFisher, Rockford, IL) in PBS pH 6.5 at 4uC. Excess biotinylation reagent was removed using Pepclean C-18 spin columns (Pierce/ThermoFisher, Rockford, IL). Biotinylated peptides were coupled to kinetics grade streptavidin high binding biosensors (Fortebio, Menlo Park, CA) at several different concentrations. Sensors coated with peptides were allowed to bind to E protein in PBS with 0.02% (v/v) Tween-20 and 1 mg/ml BSA at several different E protein concentrations. Binding kinetics were calculated using the Octet QK software package, which fit the observed binding curves to a 1:1 binding model to calculate the association rate constants. E protein was allowed to dissociate by incubation of the sensors in PBS. Dissociation curves were fit to a 1:1 model to calculate the dissociation rate constants. Binding affinities were calculated as the kinetic dissociation rate constant divided by the kinetic association rate constant.
Approximately 200 FFU of DENV-2 without peptide was allowed to bind and enter target cells for 1 h at 37uC as described for the focus forming unit assay. Unbound virus was then removed by rinsing with PBS and peptide was added to the cells for 1 h at 37uC. Cultures were washed again in PBS and agarose overlays, incubation, and immunological detection was conducted as described for the focus forming unit assay.
Approximately 200 FFU of DENV-2 were allowed to attach to cells for 45 min at 4uC, and then rinsed with cold PBS before peptide was incubated with the target cells for 45 min at 4uC. The cells were rinsed again with cold PBS, and agarose overlays, incubation, and immunological detection were conducted as described for the focus forming unit assay.
Hemagglutination inhibition (HI) was performed according to [39] adapted to microtiter plates.
Binding inhibition assays were modified from Thaisomboonsuk, et al [40] .}. Briefly, LLC-MK2 monolayers were rinsed in 4uC DMEM containing 0.8% BSA and 25 mM HEPES, pH 7.5. Virus was incubated at 4uC with peptides, control anti-dengue serum, or heparan sulfate in DMEM/BSA/HEPES for one hour before adding to the monolayers for 2 hours at 4uC. Monolayers were rinsed 3 times with cold DMEM/BSA/HEPES media prior to RNA extraction using the Qiagen RNeasy mini kit (Valencia, CA) per manufacturers instructions. Quantitative, real time, reverse transcriptase polymerase chain reaction (qRT-PCR) was conducted utilizing the Roche Lightcycler RNA Master SYBR Green 1 qRT-PCR kit (Basel, Switzerland), using primers Den_F (TTA-GAGGAGACCCCTCCC) and Den_R (TCTCCTCTAACCT-CTAGTCC) from Chutinimitkul et al [41] .}. and the following cycling conditions: 1 h at 61uC, 30 s at 95uC, followed by 45 cycles of: 5 s at 95uC, 20 s at 61uC, and 30 s at 72uC. Cp values were used to estimate infectious units according to a standard curve. Independent assays were repeated three times, in duplicate or triplicate.
Graphs were generated using KaleidaGraph v.3.6 graphing software (Synergy Software, Reading, PA). Statistical analyses were performed using the GraphPad Prism 4.0 software package (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant.
We had previously identified several E protein regions where peptides mimicking the E protein sequence might function as inhibitors. Several of these mimic peptides did not show substantial DENV inhibitory activity [9] . These included a peptide derived from the domain II fusion sequence (DN80, corresponding to amino acids 96-114 in the DENV-2 E protein) and two overlapping peptides derived from the domain II hinge region (DN57 and DN81, corresponding to amino acids 205-223 and 205-232, respectively). Predictions from crystal structures [11, 14, 15] , as well as the previously confirmed inhibitory activity of an analogous WNV domain II hinge region peptide [9] lent support to the idea that the domain II hinge region was an attractive target for inhibition. Energy minimized peptides with sequences computationally optimized for structural stability and binding to the target regions, as evaluated by our residue-specific all-atom probability discriminatory function (RAPDF), were selected for further characterization and evaluation. These sequences generally had the best RAPDF scores (or ''pseudoenergies'') for structural stability and binding, much better (lower) than the original wild type sequences (see Table 1 for original and optimized sequences.). These sequences, DN57opt, DN80opt and DN81opt, were selected for synthesis and evaluated for antiviral activity.
To identify additional novel peptide inhibitors and their corresponding targets, a 20 residue sliding window that moved from the N to the C terminus of the DENV-2 strain S1 E protein (PDB ID 1OAN) in 10 residue acid increments was evaluated by a structural stability (pseudoenergy) optimization protocol using the RAPDF. A Metropolis Monte Carlo search algorithm [38] was used to change each amino acid in the 20 residue window to one of the 19 other naturally occurring amino acids, and the stability of each corresponding peptide in the context of the entire E protein structure was evaluated. Our approach identified four E protein regions with the potential for the highest in situ binding affinities. These correspond to DENV-2 strain S1 E protein amino acids 41-60, 131-150, 251-270, and 351-370 (see Figure 1 ) that were selected for synthesis and antiviral testing (1OAN1, 1OAN2, 1OAN3, and 1OAN4).
In order to verify the effectiveness of the binding optimization process and peptide design, synthesized peptides were tested for antiviral activity against DENV-2 strain NG-C in a focus forming unit (FFU) reduction assay. DENV-2 strains S1 (GenBank accession number M19197.1) and NG-C (GenBank accession number AF038403.1) share 98% amino acid sequence identity in the E protein and the majority of differences are conservative. Dose response curves generated for the optimized peptides DN57opt, DN80opt, and DN81opt are shown in Figure 2A . The domain II region peptides, DN57opt and DN81opt displayed IC 50 values of 861 mM and 3666 mM (mean 6 sem) respectively, while no inhibition of infection was observed with the fusion region peptide, DN80opt. Correspondingly, maximum inhibition of 97% and 57% was achieved at 20 mM and 50 mM for DN57opt and Names and sequences for previously tested wild type peptides are denoted with an asterisk [9] , computationally designed peptides, wild type sequence peptides, and scrambled control peptides are shown. 50% inhibitory concentrations (IC 50 values 6 sem) determined from sigmoidal curve fits to the dose response curves in Figure 2 are given for DN57opt, DN81opt, and 1OAN1. Tested peptides that did not achieve 50% inhibition are noted with a dashed line. Peptides that were not tested for antiviral activity are noted ND (no data DN81opt. Both DN57opt and DN81opt showed improved inhibition of DENV-2 compared to their non-optimized counterparts, with DN57opt and DN81opt showing a nearly 14 fold and a 2 fold increase, respectively, in inhibition of DENV-2 at equivalent concentrations [9] . The most active inhibitor, DN57opt was chosen for further study. A scrambled version of DN57opt (DN57optscr) did not display inhibition at any concentration tested ( Figure 2B ). Four de novo designed peptides, 1OAN1, 1OAN2, 1OAN3, and 1OAN4 were also tested for inhibitory activity using the same FFU reduction assay ( Figure 2C ). 1OAN1 was found to be an effective inhibitor of DENV-2 infection with an IC 50 of 764 mM and a maximum inhibition of 99% at 50 mM. A scrambled version of 1OAN1 (1OAN1scr) did not inhibit infection by DENV-2 at any concentration tested ( Figure 2D ). In addition to these full dose response inhibition experiments using approximately 100 infectious units of virus, both the DN57opt and 1OAN1 peptides were also capable of inhibiting 4,000 infectious units of virus (data not shown).
Because toxicity could result in a decrease in focus formation and be interpreted as evidence of antiviral activity, the inhibitory peptides and their scrambled versions were assessed for cellular toxicity. Confluent monolayers of LLC-MK2 cells used in FFU reduction assays were exposed to increasing concentrations of peptide before measuring mitochondrial reductase activity using an MTT mitochondrial reductase activity assay (Figure 3 ). When we initially performed these assays to exactly mimic the focus forming unit assay by waiting five days after peptide exposure, we saw no evidence of toxicity at any concentration of any peptide (data not shown). However, we found that a shorter postexposure incubation time revealed a subtle toxicity on the part of one of the peptides. Apparently, waiting more than 24 h postexposure gives the cells a chance to recover and conceals this effect. At 24 h post-exposure, DN57opt was found to be mildly toxic to cells at 40 mM (one-way ANOVA with Dunnet's post hoc test, P = 0.0004, N = 18), so only inhibitory data using lower, nontoxic concentrations was considered. Peptides DN57optscr, 
Cryoelectron microscopy (cryoEM) was used to visualize the effect of the DN57opt and 1OAN1 peptides on DENV-2 viral particles. Control dengue virions exhibited the normal, nearly smooth outer surface typical of mature flaviviruses [42] . The surfaces of the virus particles werebecome followingrough after treatment with peptides, implying a possible rearrangement of the envelope glycoproteins (Figure 4) . The treated virions no longer showed icosohedral symmetry, Attempts to reconstruct the structure of virus complexed with DN57opt and 1OAN1 structures by imposing icosahedral symmetry failed, indicating the viruses are no longer icosahedral. Control treatments with equivalent DMSO alone did not produce this morphological alteration.
Biolayer interferometry was performed to examine binding of the peptides to purified, truncated DENV-2 E protein. Amino terminally biotinylated peptides were immobilized onto streptavidin biosensors and then the association and dissociation of truncated E protein with the immobilized peptides was monitored. The interactions of three different concentrations of truncated E protein to peptides DN57opt and 1OAN1 are shown ( Figure 5) . A buffer blank containing no E protein was run for each peptide. The affinities of the peptides for the truncated E protein were calculated with a 1:1 binding model: The dissociation rate constants were: DN57opt k d = 7.7610 24 61.7610 24 s 21 , 1OAN1 k d = 1.6610 23 60.26 10 23 s 21 . We have previously used this system to characterize the binding affinities of several human monoclonal antibodies for DENV E proteins [43] .
In order to determine if the peptides were exerting their effects on post-entry steps in the virus replication cycle, DENV-2 was allowed to infect LLC-MK2 cells before peptide was added to the cells ( Figure 6 ). No inhibition of viral replication was observed at any concentration of DN57opt ( Figure 6A ) or 1OAN1 in these assays ( Figure 6B) , indicating that the peptides are not acting at a post-infection step.
Since we had determined that inhibition with both peptides occurs at a viral entry step, we asked if infection could still be inhibited after virus had bound to the surface of target cells. We bound virus to cells at 4uC, then treated with increasing concentrations of DN57opt or 1OAN1 before warming the cells back to 37uC and allowing the infections to progress ( Figure 6C and D). Inhibition of viral entry was observed for both peptides when added to the virus after it was bound to target cells.
To determine if the peptides interefere with virus:cell interactions, we conducted two different experiments. We first performed Design of Entry Inhibitor Peptides www.plosntds.org hemagglutination inhibition assays, but were unable to detect any inhibition of the ability of viral antigen to agglutinate red blood cells (data not shown). To further investigate virus:cell binding in a more relevant system, we treated virus with DN57opt or 1OAN1, bound the virus to cells, and washed the cells repeatedly at 4uC before measuring the amount of virus remaining on the cells by quantitative rt-PCR. Both peptides showed evidence of ability to block virus:cell binding compared to control virus without peptide (Figure 7) . Treatment of virus with pooled human anti-dengue serum or heparan sulfate similarly showed reduced cell binding.
We have used computational methods to design multiple peptide inhibitors of the DENV E glycoprotein. Importantly, out of seven peptides synthesized and tested, two peptides with high activity and one peptide with intermediate activity were identified. A high resolution crystal structure of the pre-fusion conformation of the DENV-2 E [14] was used as the starting point to generate in situ energy minimized peptides. Two distinct approaches were used for the design of these peptides. First, we built upon previous work targeting DENV fusion peptide and domain II hinge regions with naturally occurring E protein sequences from these regions [9] . No inhibitory activity was found for the optimized fusion peptide region sequence (DN80opt), indicating that this region may not be a promising target mechanistically for DENV peptide inhibitors. Since an analogous, naturally occurring WNV domain II hinge region peptide was shown to be inhibitory against WNV [9] , we reasoned that a more tightly binding analog of this region in the DENV E protein could be designed and might have improved inhibitory activity. This turned out to be correct, and we identified two distinct binding-optimized peptide sequences to this region with antiviral activity, DN57opt and DN81opt. This supports previous predictions of hinge region inhibitors and the proposed mechanism of fusion based on hinge region movements [11, 14, 15] . The second approach to designing peptide inhibitors was to identify peptides with non-native sequences derived from E protein regions that are highly stable in terms of structure and binding as evaluated by an all-atom scoring function (RAPDF). This identified four regions that were used to derive additional optimized peptides (Figure 1 ). Of the four resulting peptides tested, one, 1OAN1, was identified as having antiviral activity. This confirms the use of the sliding window RAPDF minimization approach for finding tightly binding protein ligands [23, 24] . It is perhaps not surprising that computational binding optimization increased the activity of previously inactive peptides that were based on naturally occurring E protein sequences. Naturally occurring sequences have multiple balancing selection pressures that may limit their binding stability in vivo. The combined use of primary sequence prediction tools [9] and structural optimization tools [23, 24] should be a valuable approach for finding binding partners and inhibitors for other protein targets.
Neither peptide showed inhibitory activity when added directly to cells after infection had already occurred, indicating that the peptides were acting during an entry step in the virus life cycle, and sequence scrambled versions of the two most active peptides were inactive, confirming sequence specific activity. Both peptides also block virus:cell binding, but are still capable of inhibiting infection even when added after virions have already bound to the surface of target cells.
CryoEM was used to visualize the effect of the peptides on DENV-2 virions. The surface of virions appeared to change from smooth to rough after incubation with the antiviral peptides. This suggests that there may be an alteration of the arrangement of the surface envelope protein (Figure 4) . Biolayer interferometry was used to measure the kinetics of binding between the peptides and Design of Entry Inhibitor Peptides www.plosntds.org soluble, truncated E protein ( Figure 5 ). These binding studies showed a direct interaction between the peptides and DENV-2 E protein with affinities in the 1 mM range and relatively fast on/off rates. The cryoEM images demonstrate that these inhibitory peptides probably cause structural deformations in intact viral particles, but do not provide information about the kinetics of these changes. It is possible that the peptides trap the viral E proteins in some conformation that is part of the normal breathing of the viral particles, and that this interferes with cell binding and entry.
The DN57opt and 1OAN1 peptides were designed for optimized binding to the pre-fusion E structure and we show direct evidence for this interaction, both with the purified, monomeric E protein, and with virion particles. These peptides likely function by displacing portions of the E protein and interfering with normal cell binding or the structural changes during entry. Although separate in the primary protein sequence, the regions targeted in the design the DN57 and 1OAN1 peptides are partially adjacent to each other in the crystal structure, with the C terminus of the 1OAN1 region occupying a pocket surrounded by the DN57 region (See Figure 1) . We stress that we do not know the structures of the bound and inhibited peptide/ E protein complexes, but these structures may shed light on the mechanistic details of cell binding and fusion. Taken together, our results support the hypothesis that both of these peptides interact directly with DENV-2 E proteins and are entry inhibitors.
Despite difficulties with oral administration and degradation in the digestive tract, peptides may make useful antiviral agents when targeted against viral envelope proteins. Directing inhibitors to viral surface proteins avoids the major difficulty of crossing cellular membranes in order to reach the target. For example, peptide inhibitors of intercellular viral targets, such as proteases or polymerases, would need to cross the cell plasma membrane, and in the case of flaviviruses, possibly internal membrane bound Figure 7 . Quantitative reverse transcriptase PCR virus:cell binding. Virus pre-incubated with either DN57opt or 1OAN1 shows reduced binding to cells compared to control virus without peptide. Pre-incubation of virus with pooled human anti-dengue serum or heparan sulfate similarly shows reduced cell binding. * Indicates a significant difference (p,0.05) from all others by 1-way ANOVA followed by Tukey's posthoc test. doi:10.1371/journal.pntd.0000721.g007
Design of Entry Inhibitor Peptides www.plosntds.org replication and assembly compartments. The HIV entry inhibitor T-20 (Fuzeon) is a peptide, and in the context of a chronic infection, repeated life-long injections are problematic. DENV is an acute infection and most severe DENV infections require intravenous fluid support, facilitating delivery of anti-DENV peptides by this route.
We have established the existence of multiple, distinct inhibitory peptides targeting the DENV E glycoprotein and confirmed the utility of rational design using structural data for developing DENV E protein inhibitors. Applications of this strategy should also be possible for the generation and refinement of lead compounds for other viral envelope fusion proteins. It would be optimistic to propose that any single antiviral would provide an effective treatment for DENV given the enormous genetic variability of the four serotypes and multiple substrains. Different classes of inhibitors targeting the E protein and other DENV targets [44, 45] , could form the basis for the development of a combination treatment plan to combat this disease.
Alternative Language Abstract S1 Translation of the abstract into Thai by Ekachai Jenwitheesuk. Author Contributions Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture BACKGROUND: The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. RESULTS: Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. CONCLUSION: As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents. In the last few years the interaction of viral matrix proteins or precursors with cellular proteins has attracted much attention in the field of medical virology due to the increase in the understanding of their interplay in late viral processes like protein transport, virus assembly and budding. Viral matrix proteins establish the link between outer shell and capsid core of enveloped viruses and bring together these parts in the virus assembly step. Moreover, matrix proteins frequently determine the place where the assembly step occurs. In influenza A viruses two M proteins are located on RNA7 of the negative-stranded, segmented RNA virus. The M1 protein functions as a typical matrix protein, while M2 exerts multiple tasks in the early and late phase of virus infection. M2 tetramers form an ion channel and in the early phase of virus infection M2 serves for the release of viral nucleocapsid by acidification of endosomes. In the late phases, M2 prevents premature activation of newly synthesized HA [1] and -in concert with M1-contributes to virus budding and morphology. The involvement in virus exit has been assigned to the cytoplasmic tail of the protein [2] [3] [4] . Influenza viruses bud from lipid rafts and for this event the components of the viral envelope (haemagglutin HA, neuraminidase NA, M2) and the RNA containing protein complex (vRNP) must come together to form infectious virus [5] [6] [7] . Interestingly, the endosomal sorting machinery (ESCRT), which has been involved in late steps of other viruses, does not contribute to influenza virus budding [6, 8] . Accordingly, other routes and gates have been suggested for the transport of influenza proteins and virus assembly/budding [5] .
In several previous investigations caveolin-1 (Cav-1), a multifunctional, raft-resident membrane protein has been linked to the virus replication of retroviruses HIV-1 and amphotropic mouse leukemia virus, rotavirus and respiratory syncytial virus [9] [10] [11] [12] [13] . Interestingly, a contribution of Cav-1 to HA transport has been reported for influenza virus infected MDCK cells [14] . In a recent investigation of the enveloped γ-retroviruses budding from lipid rafts we showed that caveolin-1 (Cav-1) interacts specifically with the MLV retroviral matrix protein in the Gag precursor, suggesting that Cav-1 serves in positioning the Gag precursor at lipid rafts [13] . Not surprisingly, Cav-1 is incorporated into MLV virions released from mouse NIH3T3 [13, 15] . Subsequently, competition and inhibition experiments provided evidence that Cav-1 modulates MLV retrovirus production [13] . Taken together, these findings pointed to a general contribution of Cav-1 in virus replication strategy and opened the possibility that other virus families budding from lipid rafts may co-opt the functions of Cav-1. In our search for cellular/viral targets a database screen for Cav-1 binding sites notably revealed that structural proteins like matrix proteins of other viral families, e.g. Orthomyxoviridae with influenza A virus as a representative, exhibit regions of homology with a consensus motif for Cav-1 binding (Cav-1 binding domain, CBD) (Wirth, M, unpublished) .
To address the biological relevance of the interplay of Cav-1 with influenza proteins we performed inhibition experiments with a dominant-negative Cav-1 mutant, knock-down by Cav-1 RNAi as well as competition experiments with M2 fusion proteins. We found, that the yield of human influenza virus progeny is affected by the presence/absence of Cav-1. The data suggest that Cav-1 can support the human influenza virus A life cycle. Pulldown and co-immunoprecipitation experiments were performed which showed binding of M2 and Cav-1.
We used MDCK (ATCC CCL-34), a canine kidney cell line commonly used in basic influenza virus research and vaccine production [16] [17] [18] [19] . To elucidate the biological importance of Cav-1 in the influenza life cycle, MDCK cells were infected with a selectable retroviral Cav-1 RNAi vector carrying a puromycin-resistance gene (RVH1-Puro-Cav-1) as well as control RVH1-Puro alone [20] . We found that the Cav-1 content decreased gradually to 25% of the value in wild-type MDCK at 14 1, 1, 10) . Notably, the experiments revealed similar results with an average decrease of influenza titres to 57.3% of wild-type levels ( Fig. 1) . The statistical analysis of nine independent experiments revealed that the 1.5-3 fold reduction in titres observed is highly significant (paired t-test, >99% confidence, p > 0,01) When cells stably transduced with control virus vector devoid of Cav-1 interfering sequences (RVH1puro) were infected with influenza A virus (m.o.i. = 10) titres of released virus was affected only marginally. Thus, we conclude that Cav-1 reduction in MDCK is correlated with a decrease in influenza virus progeny. This suggests, that Cav-1 directly or indirectly affects the human influenza virus life cycle in MDCK cells.
A dominant-negative Cav-1 mutant has been described which functionally inactivates caveolin-1 upon binding [21] . The mutant carries a F92A/V94A double mutation in the scaffolding domain (SD) of canine caveolin-1. Expression in rat adipose and COS-1 cells has been shown to interfere with the interaction of Cav-1 with the insulin receptor and impairs receptor function.
To confirm our results from knock-down experiments, we investigated the effect of expression of the dominantnegative SD mutant and over-expression of wild-type caveolin-1 on virus production. Expression efficiency Analysis using a paired t-test (n = 9) revealed that the 1,5 to 3 fold reduction in titres compared to MDCK control cells is statistically highly significant (p > 0.001). could be monitored easily, as endogenous and transfected, recombinant caveolins differ in their mobility in SDS-PAGE due to a C-terminal myc-tag (Fig. 2 bottom) . Cav-1 appeared in two isoforms, with molecular weights of 21 and 24 kD, respectively [22, 23] . Expression efficiencies ranged from 7-29% (SD) and 20-50% (wt-Cav-1) with respect to endogenous Cav-1 level. Provided that the myc-tag does not interfere with Cav-1 antibody detection and assuming a 1:1 interaction of SD mutant and endogenous Cav-1, sufficient competitor amounts should be available in successfully transfected cells. Next, transiently transfected MDCK and mock-transfected MDCK cells were infected with influenza A/PR/8/34 virus one day after transfection. 24 h later supernatants were used for titre determination (Fig. 2 top) . To account for between-session-variations in cell culture, values were normalized to virus production from infected wt MDCK (100%). To exclude sensitivity of influenza infection to the actual transfection process, pEGFP-N1 transfected control cells were infected with PR8 virus in a control experiment. Strikingly, SD mutant expression in MDCK cells interfered with human influenza A virus replication and decreased the viral titres on average 1.6 fold to 62% of titres from wild-type MDCK (average of three independent experiments, standard deviation = ± 15,95). Compared to processed EGFP control cells, virus yield from Cav-1 wt-or SD-transfected MDCK cells was reduced considerably, which excludes that effects observed on virus production are derived from the transfection process (data not shown). This strongly suggests that interference with Cav-1 function in MDCK cells interferes with human influenza A virus replication. Interestingly, over-expression of wild-type Cav-1 also diminished influenza virus production, since viral titres reached only 56% ± 10.53 compared to non-treated MDCK (n = 3). Thus, surplus exogenous Cav-1 interferes with endogenous Cav-1 function, too. However, compared to the SD mutant twice the amount of Cav-1 wt is necessary to account for a comparable level of inhibition, as judged by Western Blot analysis.
In order to elucidate the molecular basis of the interaction we scanned influenza A virus proteins for putative binding motifs. Cav-1 binds to various cellular proteins like membrane receptors, soluble or membrane-associated molecules [24] as well as several viral proteins and exerts functions in localisation, transport and cellular signalling (Table 1 ). Signalling is preceded by phosphorylation of Cav-1, which initiates events leading either to activation of specific signalling pathways [21] or maintenance of signalling-competent, yet inactive complexes [24] . A specific, lumenal domain termed caveolin scaffolding domain (CSD, aa 82-101) which resides adjacent to the region of membrane insertion, is responsible for specific protein binding in the vast majority of cases [24, 25] . Two consensus sequences have been identified in phage-display experiments and in the primary structure of Cav-1 binding partners which were termed caveolin binding domains (CBD) [26] . CBDs have been recognized in cellular [24] and viral proteins ( Table 1 ). The consensus sequence comprises a run of 3 aromatic residues (W, F, Y) separated by a characteristic spacing (ΦxxxxΦxxΦ; ΦxΦxxxxΦ; where x stands for any amino acid and Φ for W, F, Y). Our screening for CBDs identified putative binding regions in HA, PB2 and M2 of influenza A virus. Especially, a region in the M2 channel protein turned out to be highly conserved among human influenza A viruses (Fig. 3B ). The putative CBD overlaps with a loop/helical domain immediately following the M2 transmembrane region at the lumenal site of M2 (Fig. 3A) . The CBD surrounds Cys 50, which is palmitoylated and faces the membrane allowing for insertion of the palmitoyl residue into the lipid bilayer. Thus, the CBD would be located favourably for interaction with the Cav-1 scaffolding domain [27, 28] . Strikingly, compared to M2 of A/PR8/34 as a reference the CDB core motif (positions F47, Y52, F55) and immediately adjacent amino acid residues are completely conserved in 8 M2 sequences available for the pandemic influenza virus of swine origin A/2009 (H1N1) (Fig. 3C) . Furthermore, motif conservation is observed in a former H1N1 strain appearing in 1977, but homology is restricted to the aromatic core and to a lesser extent to adjacent residues. Surprisingly, in M2 of influenza A/ 1918 the CBD motif is not conserved, as its third aromatic residue phenylalanine is changed to leucine, a residue commonly found in the M2 of avian influenza A viruses at that position (G.-V. Hemgård and M. Wirth, unpublished observation).
These hints prompted us to investigate the effect of M2 over-expression on the influenza A virus life cycle in MDCK cells. We hypothesized, that surplus M2 fusion protein may reduce the concentration of available, functional Cav-1 by complexing. To monitor M2 protein levels and localization we generated mammalian expression vectors containing cDNAs for fusion proteins of M2 (A/ PR/8/34) with desRedExpress, a red fluorescent, tetrameric protein (pM2PR8DsRed) or EGFP (enhanced green fluorescent protein) (pM2PR8_EGFP) and transfected purified DNA into MDCK cells. Expression levels and localization of the fluorescent proteins were followed 1 and 2d after transfection. The transfection efficiency (ratio of fluorescent/nonfluorescent cells) ranged between 10 and 15%. M2 fluorescent fusion proteins initially were found in the cytoplasma and started to localize at the plasma membrane at day 1 post transfection. As expected M2DsRed and M2EGFP localization did not differ from localization of M2 after infection with A/PR8/ (Fig. 4) . Interestingly, M2 expression in infected MDCK decreased viral titres to 40% (M2DsRed) and 85% of (M2EGFP) of the level of non-transfected cells. Thus, M2 over-expression interferes with human influenza virus propagation, presumably by competing with endogeneous M2 for Cav-1 interaction.
To verify the predicted M2/Cav-1 binding, pull-down as well as co-immunoprecipitation experiments were carried out. For pull-down experiments, biotinylated peptides carrying the putative CBD of M2 or a mutant CBD with alanines instead of the motif's core aromatic residues were incubated with cell lysates and complexes were processed as specified in Material and Methods (Fig. 5A) .
Results from two independent experiments show that the M2 CBD-peptide indeed pulls down caveolin-1, while the alanine-CBD mutant exhibits a strongly reduced tendency to interact with Cav-1. These results indicate that M2 of influenza A/PR/8/34 indeed exhibits at least one caveolin-binding domain.
To confirm data of the pull-down experiments, coimmunoprecipitation experiments were performed using NIH3T3 or MDCK cells after transfection of pEP24c, an expression vector containing M2 PR/8 [29] or a vector harbouring fusion protein of M2 with fluorescent marker EGFP. 24 h later cell lysates were prepared in the presence of octylglucoside, a detergent that disrupt lipid rafts, as described previously [13] . In the first series of experiments, polyclonal anti-Cav-1 antibodies were used to pull-down Cav-1 complexes from lysates. Precipitated complexes were probed for the presence of M2 after Western Blot and immunostaining. In these experiments, the Cav-1 antibodies clearly pulled down a complex that contained a M2 from pEP24c transfected MDCK cells (Fig. 5B, a) or the M2 fusion protein from pM2PR8-EGFP transfected MDCK or NIH3T3 cells (Fig. 5B, b) as well as infected MDCK cells (Fig. 5B, c left panel) . In the second experimental setting, vice versa, monoclonal anti-EGFP antibodies were used to precipitate M2 binding partners and a rabbit anti-Cav-1 antibody was used to probe for the presence of caveolin (Fig 5B, c right panel) . These types of experimental settings identified M2 complexed with Cav-1 and vice versa in both cell lines, NIH3T3 and MDCK. Thus, the results suggest that M2 has the capability to interact directly or indirectly with caveolin-1 in different cell lines. With respect to the type of interaction, it is notable, that caveolin-1 as well as M2 have been reported to bind cholesterol via cholesterol specific recognition domains [30, 31] . This prompted us to investigate, whether cholesterol is involved in the M2/caveolin-1 interaction. For that purpose methyl-β-cyclodextrin (MβCD) was used to deplete cell lysates from cholesterol before co-immunoprecipatation (Fig. 5B b and 5c) . Interestingly, in pM2PR8-EGFP transfected NIH3T3 cells as well as in PR8 virus-infected MDCK cells, signals from co-immunoprecipated proteins decreased to a certain extent, if cholesterol was removed from the lysate before pull-down. These findings imply, that cholesterol seems to support the interaction of M2 with caveolin-1.
Viruses recruit the cellular machinery to support their own multiplication and elicit an early host response to overcome the unwanted viral invaders. In our contribution we investigated the ability of caveolin-1, a multifunctional protein, to interact with components in the influenza A virus life cycle and to interfere with influenza A virus production. Cav-1 represents an organizing element at the plasma membrane and serves on localization and accumulation of proteins in lipid rafts and transmission of signalling events [24] . Furthermore, the protein contributes to intracellular cholesterol transport and has been identified as the main determinant of caveolae, invaginations of the plasma membrane used for entry of molecules and particles into the cell.
Based on previous findings of Cav-1 involvement in the late retroviral life cycle [13] we investigated the influence [20] . This question may be answered in a Cav-1 (-/-) MDCK cell line, which yet has to be established. Data from knock-down experiments in MDCK were supplemented by transfection of a dominant-negative Cav-1 mutant as well as Cav-1 over-expression, which decreased viral yields by 38-44%. The results are reminiscent of experiments of Nystrom et al. who observed impairment of the insulin signalling pathway upon expression of both, the dominant-negative Cav-1 mutant and the over-expressed Cav-1 wt cDNA as well [21] . Finally, competition with M2 fusion proteins impaired virus replication, too.
Taken together Cav-1 supports virus multiplication in MDCK, but the cellular pathway directing this Cav-1 property is not known. It is conceivable, that the cellular protein level of Cav-1 is important for the outcome, as it has been suggested for Cav-1 involvement in the insulin pathway [21] .
Hints for the molecular basis of influenza virus/Cav-1 interaction may come from other viruses which co-opt Cav-1. It is evident that individual stages in the various viral life times are affected and different roles are allocated to Cav-1 as well (Table 1) . For example, the CBD region in the HIV-1 gp41 transmembrane protein can permeate membranes and is supposed to augment the fusion step upon virus entry. Remarkably, respiratory syncytial virus (RSV), induces Cav-1 phosphorylation, which results in intracellular relocation of proteins during the paramyxovirus life-cycle. In several cases, Cav-1 functions in positioning of viral proteins to intracellular membranes (Rotavirus, SARS) or specialised regions of the plasma membrane like lipid rafts (retrovirus MLV).
To understand the molecular basis of the Cav-1 contribution to influenza A virus propagation we focussed on Cav-1 interactions mediated by the caveolin-scaffolding domain (CSD, aa 81-102) [25] . Database searches and subsequent peptide pull-down assays in combination with co-immunoprecipitation experiments suggested binding of caveolin-1 to M2 presumably to a motif in the M2 protein fitting the CBD consensus [26] . Strikingly, the motif is shared in M2 of nearly all human influenza A viruses. M2 functions within the viral life cycle as a viroporin with proton channel activity that is crucial in the entry phase [1] and as a maturation cofactor in virus budding. The cytoplasmic tail is implicated in M1 binding and facilitates virus assembly and production [2] [3] [4] 35] . Furthermore, Schroeder et al. showed that avian M2 is a cholesterol-binding protein [31] . Most avian influenza A viruses contain two cholesterol recognition motifs (CRAC I, CRAC II) in close vicinity to the transmembrane domain in the cytoplasmic region of M2 [31, 36] . Thus, cholesterol-binding and palmitoylation in combination with a short transmembrane region may direct M2 to the raft periphery in membranes and may promote clustering and merging of rafts which is then followed by the pinching-off of avian viruses [31] . [30] . Notably, to some degree Cav-1 binding to M2 is sensitive to the cholesterol depletion (this investigation). Preliminary results of mutagenesis as well as localization experiments indicate a certain role of the M2-CBD in M2 transport and localization (unpublished observations). Taken together our results demonstrate, that Cav-1 exerts an influence on influenza A virus replication and data imply that the binding of Cav-1 to the matrix protein M2 is involved. However, which function or pathway in MDCK cells actually is triggered via Cav-1 interaction with M2, remains to be determined.
The appearance of the aggressive bird influenza (H5N1), the 2009 outbreak of a pandemic influenza (H1N1) of swine influenza origin, and the recent occurrence and rapid dissemination of oseltamivir-resistant human influenza strains are motors that have accelerated the search for new antiviral targets and agents within the last time [37] [38] [39] . The investigation of cellular mechanisms involved in 'early' and 'late' viral processes and the identification of cellular actors provides a means to interfere with viral strategies. With this respect, the observed Cav-1/M2 interplay may represent a new, conserved target for e.g. therapeutic intervention with circulating and newly emerging strains of human influenza A virus. Thus, appli-cation of high-throughput screening of compound libraries will follow target identification and may result in a new antiviral agent, as exemplified for a cellular target involved in the late retroviral life cycle [40] . When this manuscript was in preparation Zhou et al. reported binding of a cytoplasmic fragment of M2 from human influenza to Cav-1 in an in vitro assay based on a Cav-1 protein fragment expressed in E. coli and CBDdependent perinuclear co-localization upon expression in CHO cells [41] . However, no experiments on the functional importance of M2/Cav-1 were performed in this investigation.
MDCK Madin-Darby canine kidney (ATCC CCL-34) and NIH 3T3 (ATCC CRL-1685) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 2 mM L-Glutamine at 37°C in 5% CO 2 . Influenza A/Puerto RicoR/8/34 (H1N1, Mount Sinai strain) virus was generously provided by Stephan Ludwig (Virology, ZMBE, Muenster, Germany).
BCA protein assay kit (Pierce) Methyl-β-cyclodextrin (MβCD, Sigma), octyl glucoside (Applichem) and other chemicals were of the highest grade commercially available.
pCav-1 wt (myc-tagged canine Cav-1 cDNA in pCIS2) and pCav-1 SD (point mutations F92A V94A in scaffolding domain) are described elsewhere [21] . pM2PR8-EGFP and pM2PR8-dsRED were constructed by PCRcloning of M2 (A/PR/8/34/(H1/N1) into BamHI/AgeI linearized pEGFP-N1 and pDsRed-Express-N1 (Clontech), respectively. M2 identity was verified by DNA sequencing. pEP24c (M2 cDNA) [29] . pRVH1-Puro-Cav-1 and pRVH1-Puro [20] are described elsewhere.
Rabbit anti-caveolin 1 polyclonal antibody (pAb), mouse anti-caveolin 1 monoclonal antibody (mAb) mouse anti-EGFP mAb (JL-8) (all BD Transduction Laboratories) mouse anti-Influenza A virus M2 monoclonal antibody (14C2, ABR) were used according to the suggestions of the supplier. 
Plasmids were transfected into cells via Lipofectamine 2000 (Invitrogen or by calcium phosphate transfection [42] .
Lysates were prepared as described previously [13] .
Influenza A/PR/8/34 titre was determined by plaque assay on MDCK cells. PBS-washed MDCK were inoculated with 500 μl of virus dilution for 1-2h at 37°C. Cells were covered with 2 ml of MEM medium containing 1% purified agar (Oxoid, England) and 1-2 μg/ml trypsin (Sigma). After three days incubation at 37°C, plates were stained with 0.03% neutral red staining to facilitate plaque counting.
20 μM biotinylated peptide encompassing either the conserved CBD within human influenza M2 (Bio-β-Ala-LDRLFFKCIYRFFKHGL-amid) or a mutant where the CBD core motif is exchanged by alanine residues (Bio-β-Ala-LDRLAFKCIYRFAKHGL-amid) were inoculated with 50 μl NIH3T3 cell lysate (2 ml, T75 flask) for 90 min. Complexes were immobilized using 10 μl streptavidin coated paramagnetic microbeads and μ column (Miltenyi). Washed samples were eluted with 1× sample buffer preheated at 95°C for 2 min and 15 μl out of 70 μl eluate were separated by SDS PAGE, blotted to PVDF membrane and probed with rabbit anti-caveolin-1 antibody.
Cell lysates were incubated with rabbit anti-caveolin-1 antibody (1:2000) or mouse anti-EGFP antibody (1:100) at 4°C for 1 h, treated with 20-50 μl protein A-or G microbeads (Miltenyi) at 4°C for 1 h, and processed as described previously [13] . To deplete cholesterol, cell lysates were treated with 10-20 mM MβCD at room temperature for 1 h before co-immunoprecipitation.
Protein concentrations were determined using the BCA kit (Pierce). 5 μg total protein was separated on a vertical 12% separating gel. Subsequently, proteins were transferred to PVDF membranes using a Transblot™ Semi-dry transfer cell (Bio-Rad). After blocking for 1 h (0.2% CA blocking reagent, Applichem) immunostaining was performed with primary antibody followed by 4 washing steps (TBS 0,02% Tween 20) and addition of the secondary antibody at appropriate dilution. The blots were developed with chemoluminescent substrate (Supersignal Femto West, Supersignal Pico West, Pierce). The band intensities were quantified using QuantityOne software (Bio-Rad) and ImageJ.
The recombinant retroviral vectors were produced from 293T triple transfection of pCMV1MLVGP1, encoding MLV gagpol, pVSV-G, pRVH1-Puro-Cav-1 encoding a shRNA for Cav-1 inhibition and a puromycin resistance gene, as described [20] . For knock-down MDCK (60%-80% confluency) were infected with the respective shRNA retroviral vectors in the presence of 4 mg/ml polybrene for 48 hours. Puromycin-resistant clones were pooled and further analysed 10-27 days after infection.
Plasmids pCav-1 wt or pCav-1 SD (Scaffolding domain mutant) were transiently introduced into MDCK, NIH3T3 or MEF 3T3 KO cells using lipofectamine 2000.
Plasmids pM2PR8_EGFP or pM2PR8DsRed were transiently transfected into MDCK cells by lipofection. The cells were infected with influenza A/PR/8 virus 1 day after transfection. Virus titres were determined from supernatants after additional 24 h of incubation at 37°C. Class II Transactivator (CIITA) Enhances Cytoplasmic Processing of HIV-1 Pr55Gag BACKGROUND: The Pr55(gag) (Gag) polyprotein of HIV serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. Gag localizes to the plasma membrane (PM) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. The host factors involved in Gag trafficking to these sites are largely unknown. Upon activation, CD4+ T cells, the primary target of HIV infection, express the class II transcriptional activator (CIITA) and therefore the MHC class II isotype, HLA-DR. Similar to Gag, HLA-DR localizes to the PM and at the membranes of endosomes and specialized vesicular MHC class II compartments (MIICs). In HIV producer cells, transient HLA-DR expression induces intracellular Gag accumulation and impairs virus release. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that both stable and transient expression of CIITA in HIV producer cells does not induce HLA-DR-associated intracellular retention of Gag, but does increase the infectivity of virions. However, neither of these phenomena is due to recapitulation of the class II antigen presentation pathway or CIITA-mediated transcriptional activation of virus genes. Interestingly, we demonstrate that CIITA, apart from its transcriptional effects, acts cytoplasmically to enhance Pr160(gag-pol) (Gag-Pol) levels and thereby the viral protease and Gag processing, accounting for the increased infectivity of virions from CIITA-expressing cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that CIITA enhances HIV Gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator. HIV polyprotein Gag serves as a scaffold to promote assembly of progeny virions at cellular membranes [1] and recruits components of the vesicular protein sorting pathway to facilitate virus budding [2, 3, 4] . Concomitantly, the virally encoded protease begins to cleave Gag, which is required for complete virion maturation and infectivity [5, 6, 7] . Gag proteins can be detected at both the PM and the membranes of endosomes among different cell types, suggesting that budding is not limited to one cell-type specific locale [8, 9, 10, 11, 12, 13, 14, 15, 16] . Further, host factors which participate in targeting Gag trafficking to particular membranes are largely unknown. As Gag and infectious virus can originate from two cellular locations, two models for Gag trafficking have emerged. The first model proposes that following synthesis, Gag traffics to endosomal membranes, and upon exocytosis is deposited on the PM, where it serves as the site for productive virus assembly [14, 17] . The second model proposes that Gag is first trafficked to the PM, where virus assembly occurs, and then excess Gag is internalized to intracellular compartments [14, 18, 19, 20] , that serve as sites of productive virus assembly [15, 21] .
MHC class II heterodimers follow a similar trafficking route, appearing at both the PM and specialized multivesicular bodies (MVBs) called MHC class II containing compartments (MIICs) [22] . MHC class II is utilized by antigen presenting cells (APCs) to present exogenous processed antigen to CD4+ T cells [22, 23, 24] . MHC Class II genes, including: HLA-DR, -DP and -DQ and the accessory molecules, invariant chain (Ii) and HLA-DM, are transcriptionally activated by the class II transactivator (CIITA), the global regulator of coordinate class II MHC gene expression [25, 26] . As CIITA is induced in CD4+T cells upon activation, these cells express MHC class II [27, 28] . Upon synthesis, HLA-DR heterodimers are assembled in the ER and the immature complex (HLA-DR+ Ii) travels through the secretory pathway to MIICs, where the specialized HLA-DM chaperone loads the HLA-DR heterodimer with peptide [22, 29, 30] . Interestingly, both immature and mature forms of HLA-DR can be found at the PM and can be subsequently internalized to MIICs due to a di-leucine motif in the cytoplasmic tail of Ii (immature HLA-DR) and a dileucine motif and/or ubiquitination of conserved lysine residues within the HLA-DR b chain (mature HLA-DR), respectively [22, 29, 31, 32, 33, 34, 35, 36] . Therefore, a connection between HLA-DR and Gag trafficking would not be surprising as both have an alternative route to intracellular compartments by way of the PM. Indeed, expression of HIV-1 Nef, Vpu and Gag have been shown to alter HLA-DR trafficking [37, 38, 39, 40] . In addition, HLA-DR is preferentially acquired on the viral envelope of budding virions, which enhances virion infectivity and may play a role in bystander apoptosis of T lymphocytes [41, 42, 43] . Therefore, HLA-DR localization at virus assembly sites is not unexpected. Finzi et al. (2006) addressed the contribution of MHC class IIinduced MIIC formation to Pr55Gag trafficking by monitoring virus budding in the presence of transiently expressed HLA-DR heterodimers [8] . In HEK-293T cells expressing HLA-DR, there was a marked redistribution of Gag to intracellular compartments, and reduced virus release at the PM; mature virus budded into HLA-DR containing multivesicular bodies and was retained [8] . We hypothesized that recapitulating endogenous expression of the entire class II antigen presentation pathway in producer cells via expression of CIITA would restore infectious virus release and provide a more physiologically relevant model for HIV-1 assembly studies. As expected, stable or transient expression of CIITA did not induce intracellular retention of Gag. However, HLA-DR induced Gag retention could not be alleviated by transient co-expression with HLA-DM and/or Ii, or CIITA-mediated upregulation of the recycling endosome GTPase, Rab4B [44] . Further, mutating ubiquitinatible lysine residues or complete truncation of the cytoplasmic tails of the HLA-DR a and b chains did not restore virus release. Rather, limiting the amount of HLA-DR DNA transfected into cells restored virus release and alleviated Gag retention. Curiously, CIITA expressing cells produced virus that was significantly more infectious than CIITA deficient cells, and this was independent of the class II antigen presentation pathway. CIITA enhances infectivity of virions from producer cells through a novel function, by improving maturation through an increase in viral protease-dependent Gag processing. Using a panel of CIITA mutants, we demonstrate that cytoplasmic CIITA increases Gag-Pol polyprotein levels. Overall, our work reveals a novel cytoplasmic, post-transcriptional function of CIITA, which is expressed upon T cell activation and is constitutively expressed in macrophages and dendritic cells, all known targets of HIV infection.
Transient expression of the class II heterodimer, HLA-DR, induces intracellular accumulation of Gag and a marked reduction in extracellular virus release from producer cells [8] . This previous study focused on HLA-DR in the absence of Ii and HLA-DM, critical components of the antigen processing and presentation pathway which influence MHC class II trafficking. To determine if expression of the complete MHC class II pathway could overcome Gag and virus retention we used CIITA to coordinately express these genes. Cells were either transiently (pcCIITA) or stably (A293T-CIITA) transfected with CIITA ( Figure S1 ) and HIV-1 NL4-3 DNA, and Gag localization was monitored by immunofluorescence and compared to cells transiently expressing the HLA-DR a and b heterodimers (pcDRab1b5), or vector-only (pcDNA3.1). As expected, a weak but uniform Gag signal was present in vector-only transfected cells (Figure 1Aa ) and transient HLA-DR expression induced a marked redistribution of Gag as indicated by a dense, punctuate Gag staining pattern (Figure 1Ad ). Conversely, Gag accumulation was not observed when CIITA was either transiently or stably expressed (Figure 1Ab or c, respectively). Therefore, transient expression of HLA-DR in producer cells does indeed lead to Gag retention, which is not observed in the presence of CIITA. However, recapitulation of the MHC class II pathway in trans (pcDRab1b5+Ii+HLA-DMab), also resulted in dense accumulation of Gag signal (Figure 1Ae ), suggesting that CIITA-mediated coordinate activation of HLA-DR, -DM and Ii expression is insufficient to overcome Gag retention. Flow cytometric analysis confirmed these findings, as cells transfected with HLA-DR heterodimers and/or co-transfected with some or all of the components of the class II antigen presentation pathway stained as Gag hi , indicating Gag accumulation ( Figure 1B and C). However, this population was absent in cells transiently or stably expressing CIITA ( Figure 1B and C) . Therefore, absence of Gag accumulation in CIITA expressing cells is likely not due to its transactivation of the MHC class II antigen presentation pathway.
To assess whether CIITA-mediated alleviation of Gag retention in class II expressing cells correlated with restored virus production, virus particle and infectious virus release were measured by p24 ELISA and GHOST cell infectivity assays, respectively. Virus release, both infectious and particle titers) were reduced when cells were transfected with either HLA-DR or other components of the MHC class II antigen presentation pathway ( Figure  S2 ), confirming a correlation between Gag retention and reduced virus titers in the presence of HLA-DR, as previously demonstrated [8] . These assays also demonstrate a dramatic increase in infectious virus release from CIITA-expressing cells as compared to cells expressing vector only (Figure 2A & S2), despite significantly fewer virus particles being released from a CIITAexpressing cell (as measured by pg/mL of p24 in the culture supernatant, Figure 2B & S2). Therefore, while fewer virus particles are released from a CIITA-expressing cell, those particles are significantly more infectious ( Figure 2C ).
Previously, HLA-DR incorporation into the envelope of budding virions was demonstrated to enhance virion infectivity [45, 46] . To determine if CIITA-enhancement of virion infectivity was due to HLA-DR or other components of the class II antigen presentation pathway, we measured virion infectivity from cells expressing these proteins. However, virions from these cells were just as infectious as virions released from vector only controls, yet not as infectious as virions from cells either stably or transientlyexpressing CIITA ( Figure 2D ). Therefore, CIITA enhancement of virion infectivity is independent of the MHC class II antigen presentation pathway. Together, these data suggest CIITA has two effects on the HIV replicative cycle in producer cells, both of which are independent of the MHC II antigen processing pathway; i) it does not induce HLA-DR, mediated intracellular retention of Gag and ii) it increases the infectivity of HIV virions.
CIITA has been shown to upregulate expression of Rab4B, a small GTPase involved in endocytic recycling [44, 47] . Therefore, we hypothesized that CIITA via the action of Rab4B, could increase Gag recycling back to the PM, thereby alleviating HLA-DR-mediated Gag retention. Commercially available Rab4 antibodies cannot distinguish between Rab4A and 4B. However, Krawczyk et al. (2007) demonstrated by chromatin immunoprecipitation that CIITA specifically associates with the Rab4B and not the Rab4A promoter [44] . Immunoblotting demonstrated that there is an increase in Rab4 in CIITA-expressing cells ( Figure 3A ), therefore it is likely that this is in the form of Rab4B. However, when Rab4B and HLA-DR were co-expressed in producer cells with the HIV-1 NL4-3 plasmid, it did not rescue intracellular retention of Gag ( Figure 3B ). In fact, infectious virus release from cells transiently co-expressing Rab4B and HLA-DR was further reduced, suggesting that Rab4B does not alleviate HLA-DRmediated retention of Gag ( Figure 3C ).
Up until this point, we had not co-expressed CIITA with HLA-DR in producer cells to determine if CIITA could alleviate retention of Gag and/or restore infectious virus release as would be expected. Interestingly, when CIITA was transiently expressed with the HLA-DR heterodimers in producer cells, Gag still accumulated intracellularly and infectious virus production remained reduced ( Figure 3B and C, respectively), suggesting that expression of CIITA is not sufficient to compensate for HLA-DRmediated Gag retention. However, when HLA-DR is endogenously expressed under the control of CIITA, retention is alleviated. [8] . Ubiquitination of a conserved lysine residue on the HLA-DR b chain induces intracellular accumulation of class II heterodimers in MHC class II compartments (MIICs) [35, 36] . Thus, the ubiquitination state of HLA-DR, might influence Gag retention. To address this idea, Lys225Arg (pcDRabK225R) and Lys222/ 225Arg (pcDRabK222/225R) point mutations were made in the HLA-DR beta chain. Despite these mutations, HLA-DR expression on the surface of A293T cells was not significantly altered (flow cytometric data not shown), nor was intracellular retention of Gag alleviated or infectious virus release restored ( Figure 4A and B, respectively). Therefore, we attempted to alleviate Gag retention and restore infectious virus release, as demonstrated by Finzi et al. (2006) [8] , by complete truncation of the cytoplasmic tails of both HLA-DR a and b (pcDRaDcyto and pcDRbDcyto, respectively). However, loss of either cytoplasmic tail (pcDRaDcytob and pcDRabDcyto), or both cytoplasmic tails (pcDRaDcy-tobDcyto), from the heterodimer did not alleviate Gag retention and even further reduced infectious virus release from these cells ( Figure 4A and B, respectively). Our results may differ from Finzi's because of the differences in cell type, or HLA-DR gene alleles, nevertheless they strongly suggest that transient HLA-DR expression in producer cells induces Gag retention. Further, flow cytometry to monitor surface HLA-DR expression demonstrates that transient transfection of HLA-DR induces an approximate half-log to log-fold increase in HLA-DR as compared to cells stably or transiently expressing CIITA, respectively ( Figure S1 ). Therefore, it is possible that HLA-DR induced Gag retention is a consequence of HLA-DR overexpression in this transient system To test this possibility, virus producer cells were transfected with increasing amounts of HLA-DR in the presence of HIV-1 NL4-3 DNA, and infectious virus release was monitored. As HLA-DR expression increased, the level of infectious virus release decreased ( Figure 4C ). Further, while not statistically significant, there was a positive correlation (r = 0.9954) between Gag accumulation (Gag hi ) and increasing HLA-DR expression ( Figure 4D ), suggesting Gag retention is likely related to HLA-DR overexpression. Similarly, when HLA-DM, which is structurally similar to HLA-DR, was overexpressed in producer cells, Gag retention was also induced ( Figure 4D ) and infectious virus release reduced (data not shown). As endogenous expression of the class II antigen presentation pathway by CIITA does not induce Gag retention and limiting expression of HLA-DR or -DM likewise has a limited effect on Gag retention in producer cells, these data collectively suggest that overexpression of the class II pathway alters Gag trafficking in producer cells leading to reduced infectious virus release. However, these data do not provide an explanation for the increased infectivity of virions released from CIITA-expressing cells.
Viral protease cleavage of the Gag and Gag-Pol polyproteins is required for virion maturation and infectivity [5, 6] . Figure 5A ) [13, 48, 49, 50, 51] . As there is an increase in virion infectivity from CIITA-expressing cells, we hypothesized that the processing of Gag polyproteins may be enhanced in these cells. As suspected, analysis of cell lysates from equal numbers of HIV-1 transfected A293T-CIITA and A293T cells demonstrated a higher ratio of mature CAp24 to Pr55Gag in CIITA-expressing cells ( Figure 5 B&C), demonstrating that Gag processing in the presence of CIITA is enhanced. Additionally, increased levels of processing intermediates are present in A293T cell lysates. Specifically, the p41 (MA-CA-SP1) and p25 (CA+p2) products were increased (Figure 5 B&C) in these cells, indicating that Gag processing in the presence of CIITA is more efficient. Gag processing is also enhanced in cell-free virions from cells either transiently or stably expressing CIITA, as demonstrated by the increased levels of CAp24, and the loss of the p41 and p25 intermediate products relative to Pr55Gag ( Figure 5D&E ). Collectively, these results suggest that CIITA increases Gag polyprotein processing, leading to enhanced production of infectious virions.
HLA-DR is incorporated into the HIV virion envelope [52] and is transcriptionally activated by CIITA, therefore its contribution to Gag processing was assessed. Virions from HLA-DR-expressing cells exhibited less Gag processing than virions from CIITAexpressing cells ( Figure 5F , bottom panel). When virions from both cell lines were affinity-purified on HLA-DR binding columns, Gag processing was reduced in those produced in the HLA-DRexpressing cells as compared to virions from cells which express CIITA ( Figure 5F , upper panel and 5G), suggesting that HLA-DR alone does not contribute to increased Gag processing. Similar to our previous results, there are fewer processing intermediates present in virions from CIITA-expressing cells, suggesting that CIITA expression enhances virion infectivity by increasing maturation through more complete Gag processing.
Cleavage of the Gag and Gag-Pol polyprotein is mediated specifically by the virally-encoded protease [53, 54, 55] . We considered that CIITA might increase viral protease processing of Gag; thus, we examined Gag processing and infectious virus release from CIITA-expressing HIV producer cells in the presence of a protease inhibitor. If CIITA is increasing protease processing, such an enhancement should be overcome by lower doses of the HIV protease inhibitor, Lopinavir. As expected, between concentrations of 0.6 and 1.25 nM of Lopinavir infectious virus release from CIITA-expressing cells was reduced to that of A293T cells treated with vehicle only ( Figure 6A ). To determine if the reduced Gag processing correlated with decreased infectious virus release, we monitored Gag cleavage by western blotting and as expected, between 0.6 and 1.25 nM of Liponavir, Gag processing in virions from CIITA-expressing cells was returned to that of vehicle treated, vector-only control cells ( Figure S3 ). These results directly demonstrate that CIITA-mediated enhancement of Gag processing and infectious virus release is through the HIV protease.
The only known function of CIITA is as a transcriptional coactivator; therefore, we thought it likely that it drives the expression of a gene which enhances viral protease-mediated Gag processing. Therefore, we reasoned that expression of CIITA mutants which fail to localize to the nucleus should not mediate increased Gag processing or enhanced infectious virus release. To test this idea, a panel of CIITA mutants (GTP2, a magnesium ion coordination site mutant and GTP3, a guanine ring-binding domain mutant and L1035P, a point mutant in the C-terminal leucine rich repeat domain, which are all defective for nuclear localization and fail to activate HLA-DRA transcription [56, 57, 58] ), were individually co-expressed with HIV-1 NL4-3 DNA, and infectious virus release and Gag processing were monitored. Interestingly, producer cells expressing these mutants also produced more infectious virus as compared to vector-only control ( Figure 7A ) and had increased Gag processing in both cell-bound virions and cell-free virions ( Figure 7B and Figure S4 , respectively). Loss of CIITA nuclear localization (and therefore coactivation potential), did not hinder increased Gag processing and infectious virus release, strongly suggesting that CIITA acts cytoplasmically to increase HIV virus maturation independent of its transactivation function. We further evaluated CIITA transactivation potential as a mechanism of enhanced virus release utilizing a different model system. NIH 3T3 BALB/c cells (expressing human p32 to allow for virus like particle production [59] ) expressing CIITA and transfected with an LTR-deficient HIV genome construct (pcHIV PAL),under control of a CMV promoter had a dramatic increase in virus-like particle production as compared to NIH 3T3 cells in the absence of CIITA ( Figure S5 ). This result provides further evidence that CIITA enhancement of virus production is independent of its transactivation potential on the HIV LTR.
To determine the mechanism by which CIITA mediates protease activity in these cells, we analyzed the expression of the Gag and Gag-Pol in CIITA-expressing cells. Expression of the HIV protease is extremely toxic to cells, and is thus very tightly regulated during virus replication [60, 61, 62] . The viral protease arises from the alternative Gag translation product ( Figure 5A ), Gag-Pol which, accounts for only 2-10% of Gag polyproteins and results from ribosomal frameshifting at a conserved heptamer ''slippery sequence'' and a secondary stem-loop structure on gag-pol transcripts [51, 63] . Therefore, increased viral protease-mediated processing of the Gag polyproteins from CIITA-expressing cells may be due to an increase in overall viral protease levels, due to increased Gag-Pol synthesis. To establish the ability of CIITA to mediate the enhanced production of HIV protease, cells were transiently co-transfected with either CIITA or pcDNA and HIV-1 NL4-3 DNA, and were treated with 80 nM of Lopinavir to inhibit the majority of Gag polyprotein processing. Gag-Pol levels relative to Gag were then determined by western blotting. While 1-2% of all Gag polyprotein in cell lysates of vector-only cells was in the form of Gag-Pol, expression of CIITA increased this level to almost 5% ( Figure 7C and D) , indicating that CIITA increases Gag-Pol levels in virus producer cells. We also measured the potential of the cytoplasmic CIITA mutants to increase Gag-Pol levels relative to Gag and, interestingly, Gag-Pol made up approximately 13, 20 and 15% of all Gag polyproteins in GTP2, GTP3 and L1035P -expressing cells, respectively. This result further demonstrates a novel transcription-independent role for cytoplasmic CIITA during HIV infection, where CIITA enhances Gag-Pol protease expression, which subsequently enhances virus maturation and infectivity. [16] . We speculated that complete expression of the antigen presentation pathway, through expression of CIITA, would alleviate this phenotype in virus producer cells. Interestingly, we noticed two phenomena: i) CIITA did not induce intracellular retention of Gag or impair virus release in producer cells despite expression of HLA-DR and ii) the virus released from cells expressing CIITA was significantly more infectious. Upon investigation of CIITA-mediated alleviation of HLA-DR-induced Gag retention, we found that this effect was not due to the lack of Ii or HLA-DM (key components of antigen presentation), as had been expected, nor was it a consequence of Rab4B expression in these cells. Further, when lysine residues in the HLA-DR chain were mutated or both of the HLA-DR chain cytoplasmic tails were truncated, Gag was still retained and virus release inhibited. In addition, when CIITA was expressed in conjunction with HLA-DR this effect could not be alleviated, suggesting that Gag retention is a consequence of HLA-DR overexpression. Indeed, retention of Gag is directly correlated to levels of HLA-DR expression. Further, we did not observe Gag retention when class II antigen presentation pathway genes were expressed at endogenous levels via CIITA expression. Overexpression of HLA-DM, which is structurally similar to HLA-DR also induced Gag retention, suggesting that retention of Gag is a likely consequence of altered trafficking of overexpressed class II antigen presentation pathway components rather than a physiologically relevant phenomenon.
Despite observing similar HLA-DR and Gag retention/reduced virus release effects as Finzi et al.(2006) , our results differed in that we did not observe the requirement for intact HLA-DR a and b-chain cytoplasmic tails for the induction of Gag retention. In our hands, when the ubiquitinatible lysine residues in the HLA-DR b-chain were mutated or the cytoplasmic tails of the HLA-DR dimer were completely removed, there was no alleviation of Gag retention or virus release. Beyond the obvious differences between the previous work [8] and our own (i.e. provirus construct and cell type), our mutant data may differ from theirs because arginine residues were substituted for lysine 215 and tyrosine 220 residues in the HLA-DR a and b chain, respectively, in order to ensure stabilization of the truncated HLA-DR molecule at cellular membranes. These differences in mutant constructs may potentially explain discrepancies in our results.
We also observed that expression of HLA-DM was sufficient to induce Gag retention and impede virus release from cells. However, Finzi et al.(2006) did not observe retention in the presence of HLA-DM or HLA-DO [8] . This difference may be explained by their use of a bicistronic construct for expression of the HLA-DM heterodimer; however, this strategy was also used to express HLA-DR, which still induced retention [8] . Irrespective of these differences, Gag retention and loss of virus release correlates with increasing HLA-DR expression. These results do not exclude the possibility that HLA-DR and Gag trafficking may be linked. Indeed, we have demonstrated that Gag co-immunoprecipitates with HLA-DR from CIITA expressing cells and this interaction is independent of the cytoplasmic tails of HLA-DR (data not shown). Further, other HIV proteins may be linked to class II trafficking. Stumptner-Cuvelette et al. (2003) and Chaudhry et al.(2009) , have independently demonstrated that HIV Nef induces internalization of surface class II in epithelial and monocytic cell lines, respectively [37, 64] . However, we did not observe downregulation of HLA-DR from the cell surface, following transfection of the HIV genome into CIITA-expressing epithelial cells (data not shown) or HIV infection of CIITA-expressing CD4+ T cells (unpublished data). Further, Gluschankof and Suzan demonstrated that expression of Gag-Pol restored HLA-DR presence at the cell surface in the H78-C10.0 line, a T cell clone deficient for surface HLA-DR expression [40] . Collectively, our work and that of others suggests a link between HLA-DR and Gag trafficking, where localization may be cell-type dependent.
One of the more interesting findings of this study is that while overall viral titers from CIITA-expressing cells decrease the infectivity of these particles is significantly enhanced. Increased infectivity was due to improved virion maturation in a viral protease-dependent manner. Not only was processing to capsid p24 more complete in CIITA expressing cells, but vector-only control cells and those only expressing HLA-DR contained increased levels of processing intermediates. The presence of higher levels of processing intermediates in virus produced in these cells may help explain the reduced infectivity, as studies in both MLV [65] and HIV [7] demonstrate that partially cleaved Gag products act transdominantly to reduce virion infectivity through reduced reverse transcription of viral RNA, despite the presence of functional Reverse Transcription polymerase [7] .
Next we demonstrated that CIITA increased Gag processing through the viral protease and evaluated whether this enhancement was a consequence of CIITA-mediated transcriptional activation. The CIITA L1035P mutant, which fails to translocate to the nucleus [58] , demonstrated increased Gag processing and virus release compared to vector-only control, suggesting a cytoplasmic role for CIITA in the later stages of the HIV infection cycle. This does not preclude the possibility that an undetectable level of CIITA L1035P might translocate to the nucleus. However, no L1035P was detected in the nucleus after a 24 hour treatment with the nuclear export inhibitor, leptomycin B [58] . Further, the predominantly cytoplasmic GTP-binding CIITA mutants, GTP2 and GTP3, had a similar increase in infectious virus release versus vector-only expressing cells.
Previously, it was demonstrated that increased Gag-Pol levels severely impair virion infectivity through disruption of RNA genome dimerization [62] . Further, HIV protease is known to be toxic to cells as it leads to the production of the novel Procaspase 8 cleavage product, Casp8p41, which induces apoptosis through loss of mitochondrial membrane potential [66] . Therefore, we would expect that virions from CIITA-expressing cells would be reduced in titer and infectivity, as there is increased Gag-Pol and protease. However, while overall viral particle numbers (as measured by p24 ELISA) from CIITA expressing cells were decreased, the infectivity of these virions was significantly increased when calculated by infectious units per pg p24. Interestingly, at 0.6 nM Lopinavir, the infectivity of virions from CIITA-expressing cells increased over vehicle-treated controls, despite reduced Gag processing. Further, the mutants of CIITA, which produced the highest level of Gag-Pol, did not demonstrate a linear increase in Gag processing or virion infectivity, which may be explained by previous work demonstrateing that increased Gag-Pol levels impairs viral infectivity [61, 62] . Therefore it is likely that CIITA is capable of increasing Gag-Pol levels to a point which can impede virus maturation, albeit not enough to reduce it to levels observed from vector-only expressing cells. Therefore, overall infectivity of virions is increased from cells expressing CIITA despite an altered Gag to Gag-Pol ratio. Future studies should focus on how CIITA can increase this ratio without severely impairing virus release as well the novel mechanism by which CIITA increases Gag-Pol levels. Preliminary studies in this laboratory suggest that CIITA enhancement Gag-Pol may be due to increased ribosomal frameshifting (data not shown).
Finally, it is tempting to speculate that CIITA, which is expressed upon CD4+ T cell activation and increases viral protease levels, may also contribute to Casp8p41-mediated apoptosis, which may link CIITA to the decimation of T cells in the GALT during primary viremia [67] . Thymic epithelial cells [68] , cervical epithelial cells [69] , human colonic epithelial cells [70] and oral keratinocytes (normal human oral epithelial cells) [71] have all demonstrated in vitro the capability of being productively infected with HIV. Infection of epithelial cells provides potential in vivo significance to this study, especially considering that thymic epithelial cells constitutively express MHC class II (and thus CIITA) [28] . In addition, most other cells can be stimulated to express CIITA in the presence of IFN-c (i.e. a pro-inflammatory environment), which is induced during HIV infection of the GALT [72] . Overall, this study demonstrates that the function of CIITA may be more broad than previously thought. Given this previously undescribed role in enhancement of virion maturation, the precise consequences of CIITA expression during the HIV replicative cycle may provide rationale for the development of novel antiviral therapeutics.
A293T cells [73] were maintained as previously described [74] and CIITA-stable A293T cell lines were generated by cotransfecting PVU I linearized pDNA3.FLAG.CIITA8 [75] and pCMV4His, a mammalian selection vector which encodes the histidinol dehydrogenase gene under control of the CMV promoter into the cells. Stable clones were selected in DMEM medium containing 5 mM L-histidinol (Sigma-Aldrich., St. Louis, MO) and cloned by limiting dilution assay. GHOST cell medium was supplemented with 0.2 mg/ml G418, 0.1 mg/ml hygromycin B and 1 mg/ml puromycin (Sigma) as previously described [76] .
Using cDNA reverse transcribed from A293T-CIITA RNA, we amplified HLA-DRa, HLA-DRb1 and HLA-DRb5 sequences (GenBank accession nos. NM019111, NM002121, and NM002125, respectively), using primers containing forward restriction site XbaI and the reverse restriction site HindIII (Table S1 ). The haplotyping of HLA-DR isotypes expressed by A293T cells was determined by the Transplantation Immunology Lab, Albany Medical College. Primers were designed to include an intact Kozak sequence upstream of the translation start site. HLA-DRa and b5 plasmids were then used for site-directed mutagenesis using primers indicated (Table S1 ). Transient transfections of all plasmids, including: pDNA3.FLAG.CIITA8 [75] , CIITA-GTP2 and -GTP3 [56] and CIITA -L1035P [58] , p33-143 (coding for the p33 and p35 isoforms of invariant chain-a kind gift provided by Dr. Eric O. Long, NIAID), HLA-DMa and b (pMCFR-PAC and pDMb/MCFRkindly provided by Dr. Lisa K. Denzin, Sloan-Kettering Cancer Center), eGFP-Rab4B (kindly provided by Dr. Marci Scidmore, Cornell University), and HIV Gag-iGFP [77] (kindly provided by Dr. Benjamin Chen, Mount Siani School of Medicine) were performed using a 3:1 ratio Fugene HD Transfection Reagent to DNA in serum-free media as suggested by the manufacturer (Roche, Indianapolis, IN). Virus plasmid DNA provided half of total DNA used in transfection reactions.
NIH 3T3 Balb/c cells are transfected with 1.5 mg CIITA and 4.5 mL FuGene HD (7:2 ratio of FuGene to DNA for optimal cells growth) and selected for with L-histidionol for two weeks and analyzed for MHC II expressing using fluorescence-activated cell sorter (FACS) with PE-conjugated mouse IgG2a (Cedar Lane) against I-A d and clones were selected by limiting dilution. These cells were co-transfected with p32cDNA and pcHIV PAL (which contains all HIV genes except the LTR sequences) at a 2:1 ratio. The p32cDNA was donated by the Peterlin laboratory [59] . Both MHC II expressing VLPs and MHC II-negative VLPs were produced and concentrated 10-fold in a 100 K molecular weight cut-off filter tube (Millipore) for 15 minutes at 4000 rpm prior to titering via HIV-1 p24 CA Antigen Capture Assay Kit were performed following the manufacturer's instructions (NIH AIDS Research and Reference Reagent Program).
Cytoplasmic RNA from 48 hr cultures of each cell type was collected using the RNeasy Mini Kit according to manufacturer's instructions (Qiagen, Inc., Valencia, CA).
1ug of DNA-free RNA was run out on a non-denaturing gel to ensure equal concentrations of each sample followed by reverse transcription of 1 mg of each RNA sample using the iScript cDNA synthesis kit, following manufacturer's instructions (BioRad, Hercules, CA). 50 ng of cDNA was used to amplify HLA-DM, CIITA, gapdh, and Ii from each cell type to confirm expression. Forward and reverse primers sequences used in RT-PCR experiments indicated in Table S1 .
Cells were analyzed for HLA-DR expression as previously described [56] . Briefly, cells were incubated with murine clone L243 [78] . Densitometric analysis was performed as previously described [80] and the percentage of total HIV-1 a CA p24 (183-H12-5C) [79] reactive bands was used as a measure of Gag processing [81] . Intracellular Gag staining 10 6 cells were permeabilized with IntraPrep Permeabilization Reagent (Beckman-Coulter, Fullerton, CA), following manufacturer's instructions. Gag was stained with FITC-conjugated FH190-1-1 (Beckman-Coulter) for approximately 15 m prior to analysis and data interpretation as performed above, with the exception of the percentage of Gag+ cells was determined after gating on HLA-DR+ cells.
Virions were purified using the mMACS Streptavidin Kit (Miltenyi Biotec Inc., Auburn, CA.), according to the manufacturer's instructions using biotinylated-L243 (Biolegend). GHOST assay [82] to determine infectious virus production and p24 ELISAs to determine virus particle release using the HIV-1 p24 CA Antigen Capture Assay Kit were performed following the manufacturer's instructions (NIH AIDS Research and Reference Reagent Program).
Cells were transfected with either pcCIITA or empty pcDNA vector 4.5 hours prior to Lopinavir (NIH AIDS Research and Reference Reagent Program) or DMSO (vehicle-only) treatment at indicated concentrations. Virus and cell supernatants were collected approximately 17 hours post-treatment. For determina-tion of p160Gag-Pol to p55Gag ratios, producer cells were transfected with CIITA constructs (pcCITIA, GTP2, GTP3, L1035P) or vector only control (pcDNA) and then transfected with pNL4-3 16 hours later, prior to being treated with 80 nM Lopinavir. Cell lysates were used to monitor the levels of Pr55Gag to Pr160Pol via western blotting with HIV-1 a CA p24 (183-H12-5C) after 17 hours.
Table S1 Primers used in this study.  On computational approaches for size-and-shape distributions from sedimentation velocity analytical ultracentrifugation Sedimentation velocity analytical ultracentrifugation has become a very popular technique to study size distributions and interactions of macromolecules. Recently, a method termed two-dimensional spectrum analysis (2DSA) for the determination of size-and-shape distributions was described by Demeler and colleagues (Eur Biophys J 2009). It is based on novel ideas conceived for fitting the integral equations of the size-and-shape distribution to experimental data, illustrated with an example but provided without proof of the principle of the algorithm. In the present work, we examine the 2DSA algorithm by comparison with the mathematical reference frame and simple well-known numerical concepts for solving Fredholm integral equations, and test the key assumptions underlying the 2DSA method in an example application. While the 2DSA appears computationally excessively wasteful, key elements also appear to be in conflict with mathematical results. This raises doubts about the correctness of the results from 2DSA analysis. The use of sedimentation velocity analytical ultracentrifugation (SV) has significantly expanded in the last decade (Howlett et al. 2006; Scott and Schuck 2006; Cole et al. 2008) , and new computational methods for SV analysis are being actively developed by several groups (Balbo et al. 2005; Philo 2006; Brown et al. 2007 Brown et al. , 2009 Behlke and Ristau 2009; Brookes et al. 2009; Correia and Stafford 2009) . In particular, diffusion-deconvoluted sedimentation coefficient distributions calculated from direct boundary modeling of experimental data (Schuck 2000; Schuck et al. 2002) have proven to be very useful tools in many biophysical applications (for a list of references see Schuck 2007) . They can achieve relatively high hydrodynamic resolution of pauci-and polydisperse macromolecular mixtures, exhibit exquisite sensitivity for trace components (Berkowitz 2006; Liu et al. 2006; Brown et al. 2008a, b; Gabrielson et al. 2009 ), and can be related to sedimentation coefficient isotherms and Gilbert-Jenkins theory for the analysis of slowly or rapidly interacting systems (Dam and Schuck 2005; . The extension of sedimentation coefficient distributions to two-dimensional sizeand-shape distributions was introduced (Schuck 2002 ; Brown and Schuck 2006) and applied in numerous studies (Markossian et al. 2006; Chang et al. 2007; Deng et al. 2007; Race et al. 2007; Broomell et al. 2008; Brown et al. 2008; Chebotareva et al. 2008; Iseli et al. 2008; Moncrieffe et al. 2008; Paz et al. 2008; Sivakolundu et al. 2008; Wang et al. 2008; Eronina et al. 2009; Mortuza et al. 2009 ). More recently, the Demeler laboratory has described the concept of a novel algorithm (''2DSA'') for determining size-and-shape distributions, as implemented in the software ULTRASCAN (Brookes et al. 2006 , 2009 Demeler et al. 2009 ). In the present work, we critically compare the background of the different algorithms and assess their performance.
The SV experiment was carried out with a Beckman-Coulter XL-I analytical ultracentrifuge, following standard protocols as described by Brown et al. (2008a, b) . A monoclonal immunoglobulin G (IgG) preparation in phosphate-buffered saline (PBS) buffer was inserted in 12mm Epon centerpieces, temperature equilibrated at 18°C, and then accelerated to 45,000 rpm and scanned with absorbance optics at 280 nm. Data analysis was performed with SEDFIT 11.8 using c(s) models as described by Schuck et al. (2002) , the two-dimensional size-and-shape model c(s, f r ) as described by Brown and Schuck (2006) , and applying Bayesian prior knowledge as described in detail by Brown et al. (2007) . The computer used for these analyses was a Dell Precision T5400 workstation, with dual 32-bit quadcore 3.16-MHz processors and Windows operating system. 1
For clarity of the analysis of the algorithms, we first provide a mathematical outline of the problem. This is followed by a more detailed discussion of appropriate discretization parameters, and from this we derive the demands on the computational platforms. Then we discuss algorithmic aspects for calculating Lamm equation solutions and for computing a size-and-shape distribution from the experimental data, and finally comment on methods for estimating their true information content.
The size-and-shape distribution problem is a Fredholm integral equation of the form aðr; tÞ ¼ 
where the data a(r, t) are the measured evolution of the radial signal profiles, and c(s, f r ) is a differential size-andshape distribution, expressed most conveniently for the modeling of SV data in coordinates of sedimentation coefficient s and frictional ratio f r (Brown and Schuck 2006) . v(s, f r , r, t) are normalized solutions of the Lamm equation (Lamm 1929 )
which predicts the evolution of the concentration profiles of an ideally sedimenting species with sedimentation coefficient s and diffusion coefficient D(s, f r ) that is initially uniformly distributed between the meniscus and bottom of the solution column at loading concentration of 1.
Equation (1) can be discretized on a rectangular grid with (S 9 F) size-and-shape values (s i , f r,j ) comprising all combinations of S equidistant sedimentation coefficient values from s 1 = s min to s S = s max (with constant mesh size Ds ¼ ðs S À s 1 Þ=ðS À 1Þ ¼ s iþ1 À s i ), and F frictional ratio values from f r,1 = f r,min to f r,F = f r,max (with constant mesh size Df r ¼ ðf r;F À f r;1 Þ=ðF À 1Þ ¼ f r;jþ1 À f r;j ). With the data being (N 9 M) discrete signal values at radius r n and time t m , abbreviated as a nm , (1) leads to the linear leastsquares problem Min c i;j !0 X n;m a nm À X S i¼1 X F j¼1 c i;j vðs i ; f r;j ; r n ; t m ÞÀbðr n ÞÀbðt m Þ
The c i,j provide an estimate of the size-and-shape distribution with cðs; f r Þ % c i;j ðDsDf r Þ. Signal offsets from systematic time-invariant [b(r n )] and radial-invariant [b(t m )] noise contributions are indicated in Eq. (3), but their simultaneous optimization with the method of separation of linear and nonlinear parameters (Ruhe and Wedin 1980) poses no significant further complications (Schuck and Demeler 1999) and therefore they will be dropped from further consideration in order to make the notation more transparent in the following. 2 We can introduce a new index l that lexicographically orders all data points (a total of L = N 9 M), and a single index k that enumerates all size-and-shape grid points (s i , f r,j ) from 1 to K = S 9 F, which allows us to write (3) as a simple sum 1 We also analyzed the data with ULTRASCAN II version 9.9 to confirm our results as far as possible. Unfortunately, the current lack of a manual section for the use of the 2DSA analysis and the excessive computational times involved prevented us from a direct comparative analysis of the same data with the full 2DSA model as described by Brookes et al. (2009) . Further, a detailed comparison does not seem possible due to seemingly unavoidable data truncation steps when loading data in ULTRASCAN II, and due to our inability to write the entire calculated distribution into a text file.
This highlights the nature of the problem being a standard nonnegative linear least-squares problem. The unconstrained problem can be solved with the method of normal equations (Lawson and Hanson 1974; Golub and VanLoan 1989) 
with the K 9 K matrix P (sometimes referred to as the Gram matrix) with elements P jk ¼ P l v jl v kl , the K 9 1 vector d with elements d k ¼ P l a l v kl , and the K 9 1 vector c representing the unknown distribution. The unique best-fit solution with the nonnegativity constraint c k C 0 can be found unambiguously with the algebraic algorithm NNLS, which was introduced and proven by Lawson and Hanson (1974) . We first used the NNLS algorithm in the context of SV distribution analysis, in a form where we expressed all requisite quantities with elements of the normal equations (Schuck 2000) . NNLS is an active set algorithm that divides the unknowns into sets with active (c k = 0) and inactive (c k [ 0) inequalities, and iteratively establishes the active set producing the best-fit solution. For the inactive set, the problem takes the same form as (5), but with all matrix and vector elements from components with active constraints deleted (Gill et al. 1986) .
Frequently the problem of fitting distributions of the form (1) is ill posed, meaning that many different solutions will fit the data statistically indistinguishably well (Louis 1989; Hansen 1998; Engl et al. 2000) . For example, Provencher (1982) has illustrated this point via the Lemma of Riemann-Lebesgue, showing that one should expect a large set of very different solutions to fit the data equally well within the experimental error. In practice, noise of the data can amplify to determine even the overall features of the best-fit solution c; and often the strictly best-fit solution consists of a series of spikes whose number, location, and height may not reflect the presence of such species in the physical experiment, but are governed by the details of the noise and other imperfections in the data.
It is therefore desirable to suppress, among all possible solutions, those that contain a potentially misleading amount of detail arising from noise amplification. Towards this goal, regularization is a standard approach that determines the most parsimonious solution of all that fit the data statistically indistinguishably well. It minimizes a measure of the information content of the solution while optimizing the quality of fit. A well-known and widely applied strategy to suppress artificial spikes is Tikhonov-Phillips regularization (Phillips 1962; Provencher 1982; Louis 1989; Hansen 1992; Press et al. 1992) , which uses, for example, the square of the second-derivative matrix (H kj ) to stabilize the solution of (4):
or, formulated with normal equations,
where a is a parameter that scales the regularization constraint (Louis 1989; Press et al. 1992) . Again, (7) has an unambiguous best-fit solution that can be determined algebraically with NNLS for any value of a, and the latter can be adjusted in a simple one-dimensional search such that the least-squares fit remains at a statistically indistinguishable quality compared with the initial best fit in the absence of regularization (Bevington and Robinson 1992). A Bayesian variation of this approach is possible that modulates the regularization matrix to enhance the information content of the solution in view of existing (or hypothesized) prior knowledge (Sivia 1996; Brown et al. 2007; Patel et al. 2008 ).
We will refer to this approach as the ''standard algorithm,'' because it is firmly rooted in textbook linear algebra and basic linear least-squares optimization, and utilized in many applications throughout the biophysical literature and physical sciences. We have introduced this approach previously into the SV analysis, and it underlies all size-distribution analyses in SEDFIT and SEDPHAT. If used without regularization, it provides exact solutions (within numerical precision) to the least-squares problem (3), and when used with regularization, the algorithms ensure that fits with statistically indistinguishable quality are obtained.
The 2DSA method by Demeler and colleagues aims to solve the same Eqs. (1), (3), and (4), respectively. This is described by Brookes et al. (2009) , and with less mathematical detail by Demeler et al. (2009) . The Demeler approach deviates in key aspects from the strategies described above. Apparently in order to circumvent perceived computational limitations, a novel multigrid scheme is conceived that would allow a sequence of fits with lowresolution 10 9 10 (S 9 F) grids to approximate the solution of (1) and (3) with high-resolution S ) 10 and F ) 10. For achieving parsimonious results Monte Carlo iterations are applied (Brookes et al. 2009 ). Some of the key ideas will be discussed in the following.
Appropriate mesh sizes for the two-dimensional problem First, in order to assess the size of the problem and computational requirements, we need to clarify how fine the grid of s-values and f r -values needs to be in order to fully extract all information from a typical set of sedimentation velocity data. Let us consider as an example the experimental data from a preparation of IgG molecules sedimenting at 45,000 rpm, as shown in Fig. 1a . It is useful to start the analysis with a one-dimensional sedimentation coefficient distribution analysis c(s), since the sedimentation coefficients are the experimentally best determined quantities. c(s) eliminates the shape dimension by using hydrodynamic scaling laws such as the traditional s * M 2/3 law for globular particles (Schuck 2000) , theoretical models for wormlike chains (Yamakawa and Fujii 1973) or any user-defined exponential scaling laws for polymers (Pavlov et al. 2009 ). For the given data we can determine from the c(s) analysis (not shown) that s-values from 0.1 to 15 S will be sufficient to describe all sedimenting species. Equidistant discretizations with S = 100 or S = 200 lead to statistically indistinguishable quality of fit, as measured by F-statistics (Bevington and Robinson 1992; Straume and Johnson 1992) , and therefore we preliminarily conclude that S = 100 will be a reasonable choice.
Typically, the resolution in the frictional ratio dimension cannot be expected to be very high, even in combination with data from SV experiments at a range of rotor speeds (Schuck 2002) . Therefore, a discretization providing F = 10 values between 1.0 and 2.5 (ranging from extremely compact to very extended protein structures) should be a sufficiently flexible basis to describe the actual frictional ratio for each species (knowing that we have inserted folded proteins into the sample solution, and keeping in mind the average frictional ratio of 1.68 estimated from the c(s) analysis). The resulting 10 9 100 grid with a total of K = 1,000 species was fitted with the standard algorithm to the data in Fig. 1a , leading to virtually random distribution of residuals (1b), with a root-mean-square deviation (rmsd) of 0.00672, consistent with the noise in the data acquisition. The resulting distribution is shown with and without regularization in Fig. 1d and c, respectively. As expected, while the s-values of the species are well defined, the shape dimension is highly underdetermined, resulting in the Fig. 1 Illustration of the standard algorithm for size-and-shape distributions applied to the experimental data of an immunoglobulin G sample, sedimenting at 45,000 rpm. a Experimental data acquired with the absorbance optical system (solid lines). The color temperature indicates the temporal order of the scans, with blue for the early and red for the late scans. The dotted lines, virtually overlapping the experimental data, are the best-fit distribution from modeling with Eq. (3) for a grid of K = 10 9 100 (f r , s)-values ranging from f r -values of 1.0 to 2.5 in 10 equidistant steps, and from s-values of 0.1 to 15.0 S in 100 equidistant steps. b Residuals of the fit, presented as a bitmap (Dam and Schuck 2004) and as an overlay plot for all traces. The root-mean-square deviation is 0.00672 OD. c Raw size-and-shape distribution without regularization. As in (Brown and Schuck 2006) , the 2D grid of (f r , s)-values is indicated by solid lines, combined with a color temperature contour map in the plane below. The solution is a series of spikes in f r -dimension, with a comparatively well-defined s-value of *5.8 S for the main species. An observation familiar in the study of IgG (and many other protein) samples is the low-level population of dimeric species at *9 S, as well as trimeric traces at *11-12 S. d Tikhonov-Phillips regularization applied to produce the most parsimonious size-and-shape distribution of all that fit the data statistically indistinguishably well at a P = 0.95 confidence level (i.e., that produce a rmsd value of 0.00677 OD or better) c typical series of peaks in 1c, and in a smooth relatively uniform distribution after regularization in 1d. (This justifies, in retrospect, the choice of F = 10 values as a sufficiently detailed discretization of the frictional ratio dimension.)
We can compare the rmsd achieved with this 10 9 100 grid (0.00672) with a fit under otherwise identical conditions but different grids: a coarser 10 9 50 grid leads to an rmsd of 0.00678, which is barely statistically worse (on a one standard deviation confidence level), and a finer grid with 20 9 200 grid leads to an rmsd of 0.00670, which is statistically indistinguishable. Thus, a 10 9 100 grid is of sufficiently high resolution to extract the entire information content of the experimental data.
After outlining the structure of the problem and the discretization parameters typically required for a size-andshape analysis of SV data, it is possible to discuss the computational requirements. Demeler's 2DSA method was implemented with the goal of accessing a high-performance computing environment (TeraGrid) in order to avoid prohibitive memory limitations that Demeler and colleagues perceive to occur when using ubiquitous ordinary laboratory workstations. Specifically, the authors (Brookes et al. 2009 ) estimate the memory needs for modeling a set of M = 50-100 sedimentation velocity scans with typically N = 500-800 data points each by only a low-resolution 10 9 10 (S 9 F) grid. They conclude that ''Performing just a 10 9 10 grid search on such an array would require close to half a gigabyte of memory just for data storage of a single experiment.' ' (Brookes et al. 2009 ). We will examine this estimate in more detail.
In practice, when using the absorbance optics with the recommended and widely applied setting of 0.003 cm (Brown et al. 2008a, b) for the radial intervals, in order to diminish errors from sample migration during the scan (Brown et al. 2009 ), we obtain only on the order of *200-250 points per scan in a long-column SV experiment. In typical high-speed SV experiments with eight-hole rotors, we can acquire usually only 50 scans or fewer before depletion occurs and/or migration and backdiffusion approach steady state, even with small solutes. This is sufficient for a highly detailed analysis of multicomponent systems, as discussed by Balbo et al. (2005) . Predicted values v(s i , f r, j , r n , t m ) need to be calculated for each species (s i , f r, j ) with arrays of the same size as the data a(r n , t m ). Since the experimental data have a precision not better than four decimal places, their representation as a standard 32-bit floating-point data type with eight significant figures is already wasteful. Nevertheless, calculating conservatively with 32-bit floats we arrive at a memory requirement of only *4.8 MB for storage of model data, rather than 0.5 GB [250 9 50 9 10 9 10 9 4 bytes 9 (1,048,576 bytes/MB) -1 = 4.76 MB]. With interference optical (IF) data, the native radial density of points is higher (*1,500 per scan). Since the radial density of points of interference scans is not exploited experimentally, it could be safely reduced to the level of absorbance data by preaveraging, which reduces the statistical noise approximately by a factor of 2. However, again calculating conservatively and using the native resolution of IF data, this would lead to *28 MB storage space, or *57 MB if 100 scans were used to represent the evolution in a SV experiment.
We find that the *5-50 MB actually required for calculating size-and-shape distributions with 10 9 10 grids is compatible with the available memory on many different platforms, ranging from [200,000 MB available on Tera-Grid systems, to *2,000-3,000 MB typically available on 32-bit Windows, and even the *50-90 MB available on current smartphones.
Consistent with this result, we and others (Markossian et al. 2006; Chang et al. 2007; Deng et al. 2007; Race et al. 2007; Broomell et al. 2008; Brown et al. 2008; Chebotareva et al. 2008; Iseli et al. 2008; Moncrieffe et al. 2008; Paz et al. 2008; Sivakolundu et al. 2008; Wang et al. 2008; Eronina et al. 2009; Mortuza et al. 2009 ) have regularly used full high-resolution grids (such as 10 9 50, 10 9 100, or higher) in SEDFIT on ordinary personal computers or laptops, an exercise that is a regular part of the Workshop on Hydrodynamic and Thermodynamic Analysis of Macromolecules with SEDFIT and SEDPHAT at the National Institutes of Health (Schuck 2009 ). This is possible due to the fact that the memory requirement for the high-resolution grid would be 48-286 MB to store the model data (assuming 50 scans for data absorbance or native interference data modeled with a 10 9 100 grid). It is readily verified that, even for the complete high-resolution grid and when globally analyzing many experimental data sets, this is well below the memory limit of currently common 32-bit Windows operating systems.
Further, all computations can be condensed to the normal Eq. (5), requiring essentially only a matrix P of 1,000 9 1,000 numbers to be operated on, which even as double-precision data type requires less than 8 MB, trivial by current standards on any platform. Once condensed to the form of Eq. (5), our SV problem is far smaller (often several orders of magnitude) than common problems of analogous mathematical structure, for example, in astronomical image analysis (Narayan and Nityananda 1986) . For the data shown in Fig. 1 , in the implementation in SEDFIT (which does not optimize memory allocation), *20 MB of RAM are used.
The necessary computational power will strongly depend on the implementation of the algorithms, of course. Parallelization can be readily achieved in the standard algorithm, which can in many places decrease the computation time by a factor virtually proportional to the number of threads. This is true, for example, for solving the Lamm equations, and for the most time-consuming step of building the normal equations matrix. The time for a complete calculation with a full high-resolution grid (10 9 100) for the data shown in Fig. 1 , on a current dual-processor quadcore 3.16-MHz PC (Dell Precision T5400), is only 42 s. 3 The time required for a 10 9 50 grid, which we have already seen leads to an adequate fit within the noise of data acquisition, is 10 s. Finally, it is *15 min for a 20 9 200 grid. In the standard algorithm a Monte Carlo statistical analysis may be desired, for example, in order to examine the statistical accuracy of a particular species population as determined from the integration of the distribution in a certain range. In the standard algorithm, each iteration requires only updating the vector d of the normal Eq. (5) and solving these equations. For the data shown in Fig. 1 , one iteration takes *3 s on a single thread on our PC.
We conclude that ordinary current workstations do not pose a limitation for rigorously determining the size-andshape distributions, neither with regard to available memory, nor with regard to processor capabilities.
Modeling a distribution of species with different size and shape to the data depends critically on the accuracy of the Lamm equation solutions (2) that predict the sedimentation profiles for all species. For calculating Lamm equation solutions, Demeler and colleagues apply the ASTFEM algorithm that was recently introduced by Cao and Demeler (2005) . In that work, the authors report two criteria for the performance of their ASTFEM algorithm in comparison with the reference (true) solution: (1) the overall rmsd (referred to by Cao and Demeler as ''L 2 error,'' in a nonstandard definition), and (2) the maximum error in the evolution of concentration profiles.
That the rmsd is small (compared with the noise of data acquisition) is a necessary but not sufficient condition for the algorithm to be useful in modeling experimental data. In fact, the majority of points of the predicted concentration profiles typically fall into the plateau regions, which are trivial to predict (those in the solvent plateau are constant zero) but have limited or no information about the sedimentation process. These plateau points can keep the overall rmsd error of the solution below the statistical errors of the data acquisition, even though the maximum errors in the sedimentation boundaries may be much larger.
The accuracy of the description of the shape of the sedimentation boundary (rather than the plateaus) is critical for modeling the size-and-shape distributions. Therefore, a sufficient condition is that the maximum error is smaller than the noise of the data acquisition. For example, in order to model experimental data with signal-to-noise ratio of up to *1000:1, the maximum errors of the Lamm equation solutions at unit concentration should be less than 0.001.
For numerically solving the Lamm equation, an overriding question is the discretization of the radial coordinate. Solutions with fine radial mesh are generally more accurate but computationally more expensive, and conversely, coarsely discretized Lamm equation solutions are quicker to calculate. Even though it has not been explicitly mentioned in the SV literature until recently (Brown and Schuck 2008) , it is easy to see that a fundamental limitation for any finiteelement algorithm with linear elements is the obligate error that occurs when approximating a smooth, curved function with piecewise linear segments. This is illustrated in Fig. 2 for a system chosen by Cao and Demeler (2005) as a benchmark in the introduction of their ASTFEM algorithm. Figure 2 shows the deviations of the curved, accurate solution from a series of linear segments with a total of only 100 (red) or 200 (blue) radius values from meniscus to bottom.
For the determination of suitable radial mesh sizes for calculating the Lamm equation solution, Cao and Demeler applied the L 2 error criterion. This led to the recommendation of very coarse grids with *100 points, and indeed the main benefit of the ASTFEM algorithm perceived by Cao and Demeler (2005) is numerical stability even for such very coarse radial grids.
Unfortunately, large maximum errors in the approximation of the sedimentation boundaries are an unavoidable consequence of coarse radial discretization. In fact, the errors in the sedimentation boundaries shown in Fig. 2 are similar in magnitude to those of Figs. 8b and 9b in Cao and Demeler (2005) . Remarkably, none of the examples provided by Cao and Demeler (2005) led to maximum errors below 0.001, and in most cases it was a factor of 10 or more above this mark. Such errors can be expected to significantly impact the result of the size-and-shape distribution analysis.
We have recently derived a new finite-element algorithm (Brown and Schuck 2008 ) based on the recognition that the approximation of the concentration profiles as linear segments does not only generate an obligate error (independent of the algorithm), but that this also represents the dominant source of error in the finite-element approach as described by Claverie et al. (1975) . Accordingly, we generate a set of nonequidistant radial grid points with optimal spacing to achieve Lamm equation solutions with constant, predetermined accuracy (as measured by the maximum error for the radial data range to be analyzed).
To optimize the efficiency, all points in the solvent and solution plateaus are calculated with the trivial analytical expressions (Brown and Schuck 2008) . We note that, for the 10 9 100 grid shown in Fig. 1 , the calculation of the Lamm equation solutions for all 1,000 species with an accuracy of better than 0.001 (maximum error) requires a total of less than 2 s on our PC. Thus, computational expense for achieving high-accuracy Lamm equations should not be limiting the size-and-shape distribution analysis of SV data.
The 2DSA ''divide-and-conquer'' algorithm by Brookes et al.
The 2DSA algorithm applied by the Demeler laboratory consists of a large number of repeated applications of Eq. (4) with K & 10 9 10 and similarly low-resolution grids. Figure 3 shows the results of fitting the same data as shown in Fig. 1 with a 10 9 10 grid under otherwise identical conditions. The deviations are ±10% of the maximum signal, and clearly this model does not even qualitatively describe the data well. As a consequence, we cannot assume that the distribution obtained from this model reflects in any way the species present in the experiment. (It is grossly different, for example, from the distribution shown in Fig. 1c, d. ) Brookes et al. (2009) recognize that such a fit is insufficient and consistently attribute the idea of using 10 9 10 grids to Brown and Schuck. For example, the authors state ''…a 10 9 10 grid as proposed by Brown and Schuck a Experimental data, with the color temperature blue to red indicating the temporal evolution of the sedimentation, as in Fig. 1 . Shown as black lines are the best-fit distributions with the 10 9 10 grid distribution model, ranging from f r -values of 1.0 to 2.5 in ten equidistant steps, and from s-values of 0.1 to 15.0 S in ten equidistant steps. b Residuals bitmap and overlay. The rmsd of the fit is 0.03088 OD. c Best-fit size-and-shape distribution with the 10 9 10 model, in the same presentation as the 10 9 100 model in Fig. 1c Fig . 2 Accuracy of the solution of the Lamm equation. Whenever using linear elements for the finite-element solution, an obligatory error is the approximation of the true boundary shape by piecewise linear segments. This is illustrated here for a system chosen as model system by Cao and Demeler (2005, compare Fig. 8b) , with s = 10 S and D = 2 9 10 -7 cm 2 /s, for which very accurate Lamm equation solutions were calculated with a very fine discretization (black thin line). If the radial range from meniscus to bottom is divided evenly in a set of only 100 radial points and the boundary shape is approximated by piecewise linear segments (red line, residuals shown in enhanced scale in the graph below), very large deviations occur, even if at these points the correct Lamm equation solutions were calculated. For an even division with 200 radial points (blue) the obligatory errors are smaller but still approximately ten times the experimental noise. Grids with 100 radial points were proposed by Cao and Demeler (2005) , leading for samples at unit concentration to maximum errors far exceeding the experimental noise. As shown by Brown and Schuck (2008) , the minimum number of radial points that for this system allow for this obligate error to be \0.001 is *300, based on an optimized nonequidistant spacing of radial points (using high density where boundaries are steep) (2006) is insufficient to reliably describe the experimental parameter space. If the actual solute is not aligned with a grid point, false positives are produced,'' and even declare the second major point in their results as ''A 10 9 10 grid suggested by Brown and Schuck (2006) is clearly insuffi-cient…,'' and state in the summary ''We have shown that low resolution grids as proposed by Brown and Schuck (2006) are insufficient to obtain reliable information.'' This attribution is not based on reality. Unmistakably, we have used in the referenced work (Brown and Schuck 2006) exclusively high-resolution grids (11 9 100 in Fig. 1 and 2, 12 9 60 in Fig. 3, 15 9 50 in Fig. 4, 13 9 100 in Fig. 5, 11 9 100 in Fig. 6 , and finally 13 9 50 in Fig. 7) , all of which are shown to describe the data well to within the noise of data acquisition (Brown and Schuck 2006) , and similar is true for other published applications of the method by other laboratories and by us. Thus, the idea of using 10 9 10 grids is entirely a product of the Demeler laboratory, and, to our knowledge, first described in the Brookes et al. (2009) paper.
Despite the failure of overly coarse grids, remarkably, Demeler's 2DSA algorithm consists exclusively of repeat applications of such coarse grids: They are considered ''subgrids'' of a hypothetical grid with much higher resolution, which is never actually completely fitted to the data, but nevertheless suggested to reflect the final resolution of the distribution. The details are not entirely clear, but there are two key ideas: (I) The coarse grids are translated relative to each other multiple times by increments D 2 s and D 2 f r , and their results are joined. (II) The joined set of grid points with inactive nonnegativity constraints from (I) is used to form a new, second-stage irregular grid of similarly low number of grid points as the initial grid. 4 The Demeler scheme of repeat application of different coarse subgrids, storage, and combination of their results, is termed a ''divide-and-conquer'' strategy. Divide-and-conquer algorithms are well-known tools in numerical mathematics that facilitate the use of parallel computation to solve problems, such as singular value decomposition (Arbenz and Golub 1988; Gu and Eisenstat 1995; Xu and Qiao 2008) . Generally, such algorithms are established by proof of their correctness. This criterion has not been attempted for the 2DSA algorithm. Concerns arise from the following arguments:
The premise underlying (I) is that the results from independent application of different grids can be meaningfully combined. Following the idea of the Demeler laboratory that low-resolution subgrids can be ''refined into a grid of any desired resolution'' through their combination scheme, let us consider that putative final regular high-resolution grid, which would have mesh size Ds = D 2 s and Df r = D 2 f r . As shown above, one can actually solve the size-and-shape distribution problem directly using the standard algorithm with this full-sized high-resolution grid with even mesh size, via the normal equation (5) with the K 9 K matrix P and K 9 1 vector d, where K is the total number of species of the two-dimensional grid. In our example of Fig. 1 , K = 1,000 for the 10 9 100 grid that is of sufficient resolution to describe all aspects of the experiment. Now going backwards, one may consider our high-resolution grid to be represented by a total of C different equal-sized subgrids, each referenced with index c (e.g., ten grids of 10 9 10 resolution), such that merging all grid points of the subgrids produces the high-resolution grid. For each subgrid, one can solve the distribution with the normal matrix method, and it is easy to show that the relevant matrix equations are P ðcÞ c ðcÞ ¼ d ðcÞ ; where P ðcÞ are square submatrices of P of size (K/C) 9 (K/C) and d ðcÞ are subvectors of d of size 1 9 (K/C). One can use a nomenclature for the elements of the high-resolution grid such that the points are ordered in sequence of the low-resolution subgrids.
In general, it is not true that the individual results c ðcÞ from the individual problems P ðcÞ c ðcÞ ¼ d
can be combined to a concatenated vector c ð1Þ ; . . .; c ðCÞ that would represent the result c of the full solution (with or without nonnegativity). This would require the cross-correlation between points from the different grids to vanish, and the high-resolution K 9 K matrix P to have a structure P ¼
This is not the case, as illustrated in Fig. 4 for our example data. As can be discerned clearly, the structure of P when sorted along subgrids (Fig. 4b) is different from merging the submatrices P (c) (Fig. 4c) , which neglects very substantial features of the model. If we ignore this problem and calculate the distribution with the matrix of Fig. 4c (or, equivalently, if we simply merge all solutions from consecutively fitting the distribution data with all ten 10 9 10 grids and plot them at their appropriate points in the high-resolution grid), we arrive at a size-and-shape distribution as shown in Fig. 5b . This is very different from the known exact solution shown in Fig. 1 , which is reproduced for convenience in Fig. 5a .
Apparently to address this problem, the 2DSA method takes from the concatenated solution of the subgrids only the pattern of active/inactive nonnegativity constraints. Demeler and colleagues construct from the points with nonzero concentration values in the concatenated partial solutions (or a subset thereof) a new grid, conceived to be equal in size to the original low-resolution grids, but now with uneven spacing of the grid points. Again, we can analyze this approach best by comparison with the full, high-resolution grid with the full matrix P, where the unambiguous best-fit nonnegative solution is found exactly with the proven NNLS algorithm (Lawson and Hanson 1974) . The ad hoc exclusion of grid points that did not produce positive concentration values in any of the subgrids is in direct conflict with NNLS. Nothing guarantees that the (s, f r )-values populated in the exact solution will be correctly recognized as populated species (be assigned nonzero values) in the fit with the low-resolution grid of which they are a part. The points populated in the exact solution may therefore simply not be part of the second-stage grid.
Illustrating this problem, the crosses in Fig. 5d represent all the grid points that made positive contributions in any of the preliminary sequence of low-resolution fits (which covers all grid points of the high-resolution grid, as described above). All the dots (red and blue) are the positive solution components of the exact high-resolution solution. They are colored blue if they coincide with a cross, i.e., have been correctly identified in the first stage as being part of the solution, and they are colored red if they were never part of any low-resolution fit and were therefore excluded from the second-stage grid. If the analysis proceeds with the second-stage grid (i.e., preconstraining the analysis to the values indicated by crosses in Fig. 5d) , we arrive at the solution shown in Fig. 5c . This is very different from the true high-resolution solution shown in Fig. 5a . Thus, the second stage cannot correct for the errors that occur from a naïve subdivision of grids in (I). Fig. 4 Magnitude of the elements of the normal matrix P calculated for the 10 9 100 model shown in Fig. 1 . P is symmetrical and has 1,000 9 1,000 values, plotted here by row and column numbers as indicated in the abscissa and ordinate of the picture, and the values P kl j j are plotted using the color scale. In principle, the nomenclature indexing the 10 9 100 grid points for the f r 9 s grid to form the vector of 1,000 parameters is arbitrary. a Here, all grid points are sorted by increasing s-value, i.e., (s 1 , f r,1 ), (s 1 , f r,2 ),…(s 1 , f r,10 ), (s 2 , f r,1 ),…(s 2 , f r,10 ),…(s 100 , f r,10 ). As can be discerned from the smooth appearance, the matrix elements are not strongly dependent on the f r -value. b The same matrix can be reordered to reflect subdivision along ten regular subgrids c, each of the form (s 10(c-1)?1 , f r,1 ), (s 10(c-1)?1 , f r,2 ),…(s 10(c-1)?1 , f r,10 ), (s 10(c-1)?2 , f r,1 ), (s 10(c-1)?2 , f r,2 ),…(s 10(c-1)?2 , f r,10 ),…(s 10(c-1)?10 , f r,1 ), (s 10(c-1)?10 , f r,2 ),… (s 10(c-1)?10 , f r,10 ) with c = 1…10. Each of the subgrids represents an evenly spaced 10 9 10 grid with origin shifted by D 2 s = 0.1505 S. c The idea that one could determine a high-resolution size-and-shape distribution from merging the results obtained separately in fits with subgrids corresponds to the assumption that there be no correlation between points from the different grids, i.e., that P can be subdivided into the ten submatrices P (c) . For the present data, this corresponds to the solution of the problem with a normal matrix as shown in c. Clearly, this is very different from the true matrix shown in b b Eur Biophys J (2010) 39: 1261-1275 1269 Brookes mentions multiple stages of the sequence (I) and (II) with different mesh sizes D 2 s and D 2 f r , and an ''iterative refinement'' of the procedure that utilizes in stage (I) the coarse starting grids that have been extended with populated points from the results of stage (II) of the previous iteration (Brookes et al. 2009 ). The same fundamental concerns apply to this iteration. To the extent that the results from (II) may not contain the grid points of the exact solution, it is unclear how the inclusion of these additional grid points would aid in the recognition of the correct solution. Even if the added grid points in (I) do represent part of the correct solution, it is not certain that they would be correctly maintained as part of the solution after (II). Empirically, the Demeler laboratory reports convergence of this iteration series in the absence, but not in the presence, of systematic noise corrections to the data (Brookes et al. 2006) . Even if the iteration does converge, it is unclear whether it is convergent to the correct solution.
Parsimony: suppressing artificial detail Since the 2DSA algorithm never actually applies a model with a full regularly spaced high-resolution grid, the traditional regularization methods, such as Tikhonov or maximum-entropy regularization described above, do not seem to be easily applicable. In fact, Brookes et al. (2009) express the view that the fit of c(s, f r ) with a high-resolution grid in conjunction with regularization suffers from ''lack of resolution,'' and ''produces unnecessarily broad molecular weight distributions.'' We believe that, if prior Fig. 5 Contour plots of the size-and-shape distributions calculated with different models for the IgG data shown in Fig. 1 . The color temperature (blue to red) indicates increasing height of the peaks. (a) For comparison, this panel shows the same distribution as shown in Fig. 1c , calculated with the high-resolution grid of 10 9 100 (f r , s)values. b Distribution obtained in stage (I) by merging the distributions calculated sequentially and independently with different lowresolution grid of 10 9 10 (f r , s)-values, each translated by D 2 s = 0.1505 S. One example for the low-resolution grid analysis is shown in Fig. 3 . All low-resolution grids are chosen such that they are evenly spaced subgrids of the high-resolution grid, and such that, by joining the grid points of (f r , s)-values of all the low-resolution grids, the high-resolution grid of a is obtained. c A new grid is defined in stage (II) by joining all grid points from the entire sequence of lowresolution grids that yielded positive contributions to the fit. This is the set of grid points for which b displays nonzero populations of the distribution. In a secondary analysis, a fit to this irregular grid is performed, and the results are shown as a contour plot. Although the smallest differences Ds and Df r in this secondary grid are the same as those of the high-resolution grid, it considers only a small subset of the points from the high-resolution grid. This causes the deviations from the exact results in a and those in c. d Illustration of the grid points used in c, showing as black crosses all points that yielded positive contributions in any of the first-stage low-resolution fits. For comparison, solid circles are the grid points that make positive contributions in the exact direct high-resolution analysis of a. Blue circles indicate those that coincide with grid points in the Demeler scheme, and red circles indicate those that are populated in the exact solution but not found in the grid of the Demeler scheme knowledge about the sharpness of the expected c(s, f r ) peaks is available, this can be inserted with a Bayesian refinement of the Tikhonov regularization as we have reported for SV analysis (Brown et al. 2007 ) and implemented in SEDFIT.
In the absence of such prior knowledge, however, the resolution of the regularized solution is limited not by the analysis (assuming reasonable discretization), but rather by the information content of the experiment. It is important to recognize the nature of this limit, in order not to overinterpret the data. Of course, it also would be trivial, although usually misguided, to perform a distribution analysis simply not applying this regularization step at all, and to rely on the exact solution of the fit with the high-resolution model, which usually produces artifactual detail that is the result of noise amplification due to the ill-conditioned nature of the basic Eq. (3).
In our example, these aspects can be discerned when comparing the most parsimonious solution in Fig. 1d from Tikhonov regularization with the spiky exact solution in Fig. 1c , or with the incorrect solution in Fig. 5c from one iteration adapted from the Demeler scheme. Even though the spiky solutions suggest very few and discrete species to be in solution, the smooth Tikhonov solution fits the data indistinguishably well from the exact best-fit solution. Its nearly featureless appearance in the f r -dimension highlights simply the lack of sufficient information in the raw data in order to determine the f r -values well.
In order to address the impact of noise and error amplification on the results of the 2DSA algorithm, it was combined by Brookes et al. (2009) with a Monte Carlo analysis. Fifty iterations were performed by the Demeler laboratory in order to determine 95% confidence intervals. This seems to be an unusually low number of iterations, in particular since the high confidence limits require estimating the quantiles of rare events, in this case the 2.5 and 97.5 percentiles. With 50 iterations, they are determined by the extreme 1.25 occurrences of parameter values, which makes these estimates of the confidence intervals quite variable statistical quantities themselves. As is well known, usually the number of Monte Carlo iterations required to produce meaningful results is typically on the order of 1,000-10,000. However, it seems this would lead to excessive computational effort, several orders of magnitude more costly than the Tikhonov regularization in the standard approach with the full high-resolution grid, which requires for our standard example only a few seconds on our PC.
The authors report confidence intervals for molecular parameters of the identified solutes, but it is not clear whether these were determined (1) by statistical analysis of the results obtained in each Monte Carlo iteration after the integration of putative solute peaks, or (2) if these confidence intervals reflect the uncertainties of the putative solute peaks in a distribution that, as a whole, has gained error bars at each grid point from the statistics of the Monte Carlo iterations. For method (1), the problem arises of how to identify the group peaks representing a putative solute species. For method (2), the question arises of whether the Monte Carlo approach is effective in providing parsimonious ''average'' distributions.
Generally, Monte Carlo simulations are not part of the diverse set of regularization methods explored in the standard literature (Louis 1989; Hansen 1992 Hansen , 1998 Kress 1999; Engl et al. 2000) , although Monte Carlo methods have been used for estimating the regularization parameters of standard regularization functionals (Ramani et al. 2008) . The concept of a statistical distribution of parameter values should be confused neither with the real population distribution of coexisting species in the sample mixture nor the estimate of the latter in the form of a calculated sizeand-shape distribution. Nevertheless, one could ask to what extent one can rely on the statistical nature of the noise in the data, in combination with noise amplification, to produce a parsimonious two-dimensional histogram of (s, f r )species populations. As an example, we compare in Fig. 6 the standard analysis of our model data with the 10 9 100 grid and Tikhonov regularization (6a) with the histogram of all distributions from 50 Monte Carlo iterations (each based on an exact standard fit with the 10 9 100 grid; 6b). After 50 iterations the histogram clearly shows multimodal and spiky behavior suggesting the presence of multiple species, in contrast to the single broad peak representing the smoothest solution of all that fit the originally measured data. Thus, the 50 Monte Carlo iterations do not provide an effective means to correctly identify the information content of the data. If, on the other hand, we are independently knowledgeable about the monodisperse nature of the sample, we can use the Bayesian approach (Brown et al. 2007 ) to calculate the size-and-shape distribution that is closest to a single peak, and these results are shown, for comparison, in Fig. 6c .
In the present letter, we have examined the different algorithmic elements that were conceived and applied in the recently suggested ''2DSA'' size-and-shape distribution by Brookes et al. (2009) . We have compared this with the standard approach that is well established for solving illposed integral equations problems in many fields, which rests on well-established linear algebra and related numerical tools of linear least-squares analysis. Contrary to the assertion of Brookes, Cao, and Demeler, the application of the standard approach to the size-and-shape distribution problem is quite feasible on ordinary laboratory computers within only a few minutes of computation time, even when using high-resolution grids suitable to fully extract the experimental information content. As implemented in SEDFIT, this approach is being applied in many laboratories (Markossian et al. 2006; Chang et al. 2007; Deng et al. 2007; Race et al. 2007; Broomell et al. 2008; Brown et al. 2008; Chebotareva et al. 2008; Iseli et al. 2008; Moncrieffe et al. 2008; Paz et al. 2008; Sivakolundu et al. 2008; Wang et al. 2008; Eronina et al. 2009; Mortuza et al. 2009 ). Since this can supply exact (up to numerical precision) best-fit solutions, we have applied it to a data analysis example to serve as a reference solution in a study of the performance of different computational strategies on which 2DSA relies. This illustrates the consequences of the deviations from the established mathematical reference frame that should be expected to arise in Demeler's 2DSA approach.
First, there are concerns about the accuracy of the evaluated Lamm equation solutions serving as kernel to the size-and-shape distribution integral. This could likely be addressed by deviating from the discretization parameters advocated by Cao and Demeler (2005) .
Second, a more fundamental problem is the use of grids with extremely small number of points, far below the resolution required to describe the data. If, as illustrated in Fig. 3 , the predicted concentration profiles from these coarse models do not even qualitatively follow the experimental data, we question whether there are any meaningful conclusions that can be drawn from these results. Brookes et al. (2009) distract from this problem by incorrectly stating that such grids were the basis of the implementation of c(s, f r ) models in SEDFIT, which is well described in the literature to achieve excellent fits of the data to within their statistical noise. To the best of our knowledge the attempt to utilize coarse grids is without precedent prior to the Brookes et al. paper.
Despite the inability of these grids to describe the data, Demeler and colleagues suggest that the combination of results from the application of a large number of different, but similarly coarse, grids (all with 10 9 10 or lower resolution; Demeler et al. 2009 ) can be used in some way to achieve an analysis equivalent to that of a high-resolution grid. In the simplest form, this argument would be incompatible with basic matrix algebra, because it neglects cross-correlation between points from different grids. Discarding the magnitude of species' populations in this concatenated distribution, and using only the pattern of b Fig. 6 Comparison of strategies to compute parsimonious distributions that display the information content of the IgG data shown in Fig. 1 . a Contour plot of the size-and-shape distribution obtained with the high-resolution grid of 10 9 100 (f r , s)-values, after application of Tikhonov regularization, as shown in Fig. 1d . b The sum of 50 sizeand-shape distributions calculated with the exact standard method using the same high-resolution grid, but each based on synthetic data sets generated from the best-fit distribution of Fig. 1 with added normally distributed noise at the same magnitude as exhibited by the experimental data. c Integration of the main 6 S peak of the size-andshape distribution as calculated in Fig. 1 allows to determine the weighted-average s-value and f r -value, which can be used in the Bayesian framework to calculate the size-and-shape distribution c d (s, f r ) (Brown et al. 2007 ) that is closest to that of a single species, within the limits imposed by the requirement to produce a fit of statistically indistinguishable quality to that shown in a. As can be discerned from the secondary peak at *6 S with low frictional ratio, a strictly monodisperse interpretation of the main peak is contradicted by the experimental data. (Note the different scales on the color bar) nonnegativity constraints from such an analysis, is in conflict with the established Lawson and Hanson algorithm NNLS. The effect of the empirical extension to multiple stages is uncertain, and may not converge. Although one could construe cases where it will certainly work (such as distributions consisting of a single species), the Demeler scheme for generating nonequidistant small grids in multiple stages appears fundamentally flawed for the general case. The strategy of sequentially applying different, equally coarse, grids is in contrast to established multigrid methods for integral equations, which provide successfully finer parameter discretization (Kress 1999) . The division of the full problem into separate subproblems to be solved in parallel, followed by merging their partial solutions, has been used successfully in some image restoration problems (Bevilacqua and Piccolomini 2000) where the image regions are known to be uncorrelated with each other due to a localized point-spread function. However, this condition is not fulfilled in the present case. In SV analysis, the cross-correlation of signals from different species can be very large. This is reflected by the fact that (1) is ill posed, and illustrated by the fact that the matrices in Fig. 4b and c are different. For a correct solution of the SV problem, the regular high-resolution grid should be considered fully and unbiased by any scheme of preselection of excluded parameter regions. The latter is quite feasible with standard algorithms and commonly available computational resources, and we note that the problem is fairly small compared with many image analysis problems of similar structure.
Finally, the application of the Monte Carlo approach to achieve greater parsimony of the results (i.e., simplicity of the distribution in the sense of suppressing artificial detail) is equally novel, but not very successful when we applied this idea to our example data analysis. An example of the lack of regularization in the 2DSA method resulting in artificial detail can be found in the data shown by Planken et al. (2008) , where a standard c(s) analysis of SV data with maximum-entropy regularization exhibits only a single broad skewed distribution [ Fig. 3c in Planken et al. (2008) ], consistent with the expected continuous size distribution of the material studied, yet the 2DSA analysis of the same data suggests the presence of more than 14 discrete peaks (at different s-values and all at similar frictional ratio) [ Fig. 4 in Planken et al. (2008) ]. The Monte Carlo approach is certainly an extremely computationally costly step, in particular if one would carry it out with statistically meaningful iteration numbers. In contrast, application of the standard Tikhonov regularization to the full highresolution problem, with or without Bayesian modulation, takes a small fraction of the computational effort of the original problem, i.e., on the order of seconds on a PC.
In conclusion, we would regard the computational effort to be a secondary problem, and the choice of computational platform rather inconsequential, relative to the main concern arising from simple mathematical arguments that Demeler's algorithm may not give correct results. The authors do qualify their algorithm to be empirical, and that ''the results are not generally in exact correspondence with the original problem'' (Brookes et al. 2006) . They argue that ''[the results] can be made sufficiently close through careful use of the given heuristics'' (Brookes et al. 2006) . We are uncertain of the process referred to here, and how closeness to the exact solution would possibly be assessed without explicitly calculating the exact best-fit solution. So far Demeler and colleagues have not brought forward any proof that the distributions returned by the 2DSA method are at least close in the major attributes to the correct solution. We believe that the question of correctness of the algorithm is critical, especially since the authors invite the general application of this method, as implemented in the ULTRASCAN software, to address data analysis problems in novel biophysical and biochemical studies, rather than simple model problems with known solutions. Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1α (HIF-1α), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses. The Spanish influenza pandemic of 1918-1919 was the most devastating outbreak of infectious disease in recorded history, with an estimated 20-50 million human deaths following an unusually severe and rapid clinical course [1, 2] . The molecular pathogenesis of this pandemic is still poorly understood, but a prevailing hypothesis is that the severe tissue necrosis, influx of inflammatory cells and profound vascular leakage in the lower respiratory tract precipitated an acute respiratory distress syndrome (ARDS) leading to multiple organ dysfunction syndrome (MODS). Similar mechanisms have been proposed for the clinical symptomatology and the pathology induced by the currently circulating avian H5N1 influenza A virus strains [3] .
Reports of extrapulmonary distribution of highly pathogenic influenza viruses (HPIV) in humans are limited to a few recent reports of detection of viral antigen in or isolation of H5N1 virus from brain, CSF, intestine and fecal material from individuals who died with influenza virus-induced coma and diarrhea [1, [4] [5] [6] . Infection with influenza viruses is generally restricted to the respiratory tract in humans, although encephalopathy has been reported for seasonal influenza, particularly in children [7] .
There appears to be wide disparity in results concerning extrapulmonary spread of avian H5N1 viruses and pathology in animal models. In mice, virus has been recovered from most extrapulmonary tissues examined, including brain, but viral titers were often low and inconsistent [8] [9] [10] . In cynomolgus macaques infected with the H5N1 virus-strain A/Hong Kong/156/97, virus replication was mainly confined to the respiratory tract [11, 12] . In contrast, in cats infected with H5N1-strain A/Vietnam/1194/2004, high titers of virus were re-isolated from most extrapulmonary tissues and organs regardless of infection route [13] . Similar results were obtained in ferrets with the virus strain A/Hong Kong/486/97, despite the latter strains having relatively low pathogenicity in mice [8, 10, 14] . These species differences may reflect variations in virus receptor distribution [15] , variations in inflammatory and immune responses [16] [17] [18] [19] , or differences inherent to the H5N1 strains used, such as replication efficiency in host cells, interference with interferon responses [8, 10, 20, 21] , or any combination of these variables.
Using a macaque model of influenza [22, 23] and employing a recent human isolate of H5N1 influenza A virus as well as reassortant H1N1 influenza A viruses containing genes encoding two (HA, NA) or four (HA, NA, NS1 + NEP) proteins of the 1918 Spanish influenza virus strain, we previously reported on pulmonary pathology [23] . We subsequently have sought to further address questions regarding extrapulmonary virus replication and inflammationassociated extrapulmonary effects caused directly or indirectly by these virulent viruses. In particular, we hypothesized that the transcription factor hypoxia inducible factor-1 (HIF-1) would be up-regulated in animals with severe pneumonia, either as a result of hypoxia or the inflammatory response. HIF-1 has emerged as a key regulatory molecule due to its responsiveness to both microenvironmental tissue conditions, such as hypoxia, and inflammatory mediators [24] [25] [26] [27] [28] . We found that the expression of HIF-1α appeared to reflect the extent and kinetics of lung inflammation following infection with these influenza viruses and was an indicator of extrapulmonary tissue reactions in the absence of frank pathology or virus replication.
A detailed description of the experimental protocol, including animal sources, virus sources and the infection protocol has been published [23] . Briefly, 34 Chinese-origin M. fascicularis were assigned to five experimental groups matched for age, weight, and gender to the extent possible. Four groups of eight animals were inoculated through the combined intratracheal, intranasal, tonsillar, and conjunctival routes with a total of 10 7 Two animals per group were euthanatized at days 1, 2, 4, and 7 postinfection (pi). Two animals were used as mock-infected control animals and terminated at day 7 post-challenge. At necropsy, all tissues were examined grossly and harvested. Samples for histology and immunohistochemistry were fixed in 10% neutral-buffered formaldehyde for 48 h, followed by transfer into 70% methanol-free ethanol, and stored until processing. The tissues were routine embedded in paraffin and sectioned (4-5 μm). Hematoxylene-and eosin (HE)-stained sections were examined on an Olympus BX41 microscope equipped with a Q-Color3 camera (Olympus) and corresponding computer software. Immunohistochemistry (IHC) detection of influenza virus antigen in formaldehyde-fixed, paraffin-embedded tissues was carried out as described previously [22, 23] , except that proteinase K was used for antigen retrieval. The H1N1 viruses were detected using the influenza virus nucleoprotein-specific monoclonal antibody M322211 (Fitzgerald Industries, Concord, MA, USA). Because this antibody appeared to cross-react poorly with the H5N1 nucleoprotein, a polyclonal rabbit antibody specific for H5N1 viruses (source: CDC) was used on tissues of the H5N1 group. Tissues from our previous studies were used as positive controls [22, 29] . Other controls included substitution of the influenza-virus-specific antibodies with monoclonal antibodies specific for various flaviviruses (West Nile virus, St. Louis encephalitis virus, bovine pestivirus) and omission of the primary or secondary antibodies.
To characterize the inflammatory reactions in pulmonary and extrapulmonary tissues, sections from lungs [23] , tracheobronchial and retropharyngeal lymph nodes, tonsils, spleen, heart, liver, right kidney, colon and ileum as well as several areas of the brain were immunolabeled for Mac387 (myelomonocytic lineage marker), CD83 (mature dendritic cells), HIF-1α, hemeoxygenase-1 (HO-1), cyclooxygenase-2 (COX-2), inducible nitric oxide syntase (NOS2), vascular endothelial growth factor-A (VEGF-A), phospho-p38α mitogen-activated protein kinase (phospho-p38α MAPK), and the apoptosis marker activated caspase-3. Brain sections were also labeled for glial fibrillar acidic protein (GFAP) to test for gliosis or loss of astrocytes, for microglial cells using the Iba-1 marker and for oligodendrocytes, using the transcription factor OLIG2 as a cell marker [30] . Sources of antibodies and immunolabeling protocols have been described in detail elsewhere [30, 31] . Positive control tissues included samples from inflammatory processes in lungs, intestine and brain of macaques, cattle, mice, guinea pigs, and cats [30] [31] [32] . The sections were counterstained with Meyer's hematoxylin (Scytek Laboratories; Logan, Utah), mounted with coverslips, and examined on an Olympus BX41 light microscope. The immunolabeling was scored semi-quantitatively on a scale of 0-6, based on the number of positive cells and the overall intensity of labeling, with 0 indicating no apparent labeling, 1 through 6 indicating increasing expression above normal levels, and a score of 6 denoting diffuse occurrence of positive cells with high-intensity labeling. For HIF-1α, the scores relate to nuclear expression (nuclear translocation) rather than cytoplasmic expression. In the instances of a marker for normal cellular constituents, such as microglia in the brain or dendritic cells in the lymphoid tissues including bronchiole-associated lymphoid tissue (BALT), normal levels were given a score of 0. The scale therefore differs for each marker but is consistent for a particular phenotype, depending on the normal tissue levels of expression in naïve animals. Photomicrographs were acquired with an Olympus Q-Color 3 camera and associated computer software and are reproduced without manipulation.
Cynomolgus macaques (Macaca fascicularis) infected with a human HPAI H5N1 virus isolate (A/Vietnam/1203/2004), or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection, with inflammation peaking on days 2-4 pi in the 1918 reassortant groups, followed by resolution. In contrast, no cessation of inflammation was apparent in the H5N1 group [23] . Grossly, the most notable extrapulmonary changes occurred in the lymph nodes. The retropharyngeal and tracheobronchial lymph nodes appeared mildly to moderately enlarged after all of the influenza virus infections, most notably on days 4 and 7 pi. Early in the infections, there was variable edema of the lymph nodes, and by day 4 and 7 pi, the nodes in the HANA, HANANS and A/Texas groups were characterized microscopically by marked follicular and paracortical hypertrophy. In contrast, the follicles in the lymph nodes of H5N1-infected animals were involuting and appeared with variable degrees of hyalinization of the germinal centers, except at 7 days pi, where there were signs of follicular hypertrophy in the one surviving animal. Only in the H5N1-infected animals was there histological evidence of tonsil involvement, with frank neutrophilic and necrotizing tonsillitis on days 1 and 2 pi (Fig. 1a) . By day 7, the inflammation was resolving. Notably, the histopathological changes in the lymph nodes and tonsils of the H5N1-infected animals correlated with the extent of virus infection as detected by IHC (exemplified by the tonsil in Fig. 1b) . A few virus-infected cells were found in the tonsil and retropharyngeal lymph nodes of HANA-infected animals on day 1 pi (not shown). No virus-infected cells were detected in these tissues in any animal of the other groups. The number of CD83-positive cells in lymph nodes and tonsil either remained within normal levels or increased over time in the HANA, HANANS and A/Texas groups, while these cells decreased in frequency and intensity of labeling in the tissues of the H5N1 animals (Fig. 1d) . The disappearance of CD83-positive cells from the lymphoid tissues of the H5N1-infected animals over time correlated temporally with a pronounced occurrence of apoptotic cells, as reflected by expression of activated caspase 3 (Fig. 1f) .
The spleen of the H5N1-infected animals presented with very pronounced accumulation of Mac387-positive cells, mainly neutrophils, in the red pulp, most notably on days 1 and 2 pi (Fig. 2) and then decreasing, but not completely disappearing, over the following 5 days. Small numbers of scattered virus-antigen-positive cells were detected in the spleen of H5N1-infected animals on days 1 and 2 pi, but not in any of the other groups, where the spleen morphology tended to follow that of the lymph nodes, reflecting immune stimulation. The occurrence of CD83-positive cells also followed the pattern of the lymph nodes in these latter groups.
Virus-infected cells were detected in the mesothelial cells of the meninges of one H5N1infected animal on day 4 pi, most pronounced over the occipital lobes (Fig. 3a) , while neurons and glia cells were negative for virus antigen in all animals at all time points examined. Apart from the meninges, the only other extrapulmonary and non-lymphoid tissues with virusantigen-positive signals were the colon (macrophage or dendritic-like cells in lamina propria; Fig. 3b ) of one H5N1-infected animal on day 2 pi, and the abdominal mesothelium (mesothelial cells, most notably the lining associated with the kidney capsule) of one animal on day 4 pi. No extrapulmonary virus antigen was detected in any animal of any other group at any time point.
While HIF-1α, HO-1, NOS-2, phosphor-p38-MAPK and VEGF-A were markedly upregulated in the lungs within 24 h pi in animals infected with either of the reassortants or with H5N1, and remained highly expressed in the latter group throughout the course of the study (Fig. 4 , and data not shown), no consistent pattern was discernable in the expression of these inflammatory markers in the lymphoid tissues. Nevertheless, all five markers were markedly upregulated above control levels at one or more time points postinfection in all lymphoid tissues of all infection groups (data not shown).
Histologically, the only tissue outside the respiratory tract and lymphoid tissues with discernable changes on HE sections was the liver of animals in the H5N1 group, where the Kupffer cells appeared mildly hypertrophic. This change was more apparent in sections stained for Mac387 and HIF-1α (Fig. 5a , c, and data not shown). Using these same phenotypic markers, it became evident that intravascular blood monocytes in many tissues and resident, interstitial macrophages in kidneys and heart of these animals were likely also activated, or alternatively, these tissues were infiltrated with a small but significant population of activated macrophages (Fig. 5e) . A further indication of extrapulmonary activation of the macrophage lineage was seen in the brains of H5N1 infected animals, where a mild, but very notable hypertrophy, and possible increase in numbers, of microglial cells was apparent in sections labeled for the Iba-1 molecule (Fig. 6) . Moreover, there were signs of astrogliosis in the H5N1-infected animals on days 4 and 7, as reflected by increased GFAP expression (data not shown). VEGF-A expression was more pronounced in the brain in this group from day 2 pi through day 7, although on days 1 and 2, the expression in the other groups was also notable (data not shown).
We previously reported that H5N1 virus as well as reassortant H1N1 viruses containing the HA and NA genes from the 1918 pandemic influenza virus induce a severe pneumonia within 24 h of challenge, but that only the former virus gives rise to a sustained inflammatory response and massive parenchymal necrosis in the respiratory tract [23] . In this study, we show that this also applies to the tonsils, and it is accompanied by reactions in extrapulmonary tissues consistent with a systemic cytokinemia. The 1918, HA and NA genes significantly increased the virulence of a seasonal human H1N1 virus of low pathogenicity but did not confer a virulence phenotype comparable to that of the H5N1 virus or the complete 1918 pandemic influenza virus [33] , suggesting that other viral genes, such as PB1 [34] , probably also play significant roles in the pathogenesis. Furthermore, only the H5N1 virus appeared to spread beyond the respiratory tract to lymphoid tissues, intestines and meninges. Previous studies with other H5N1 isolates and the fully reconstructed 1918 virus in macaques similarly found evidence of only very limited virus spread outside the respiratory tract [12, 33] , whereas in cats and mice, H5N1 virus spread more widely [9, 13, 35] . Virus has also been isolated from or detected at extrapulmonary sites in human patients succumbing to H5N1 virus infection, including brain and intestine [4] . Thus, it would appear that the virus strain, timing postinfection, species, age and infection dose play important roles in determining virus spread beyond the respiratory tract, or at least the possibility of detecting it.
While overt pathology ascribable to the virus infection was not seen in any tissues outside the respiratory tract and associated lymphoid tissues by conventional histology, immunohistochemistry for inflammatory reactants suggested that the effect of at least the H5N1 virus infection was not limited to those tissues. Notably, severe hypoxia, as a result of the pronounced lung pathology [23] , cytokinemia, or both may have given rise to extrapulmonary reactions. Both pathways may be regulated by HIF-1 [28] . HIF-1 was originally discovered as a biological O 2 sensor that enables the organism to adapt to hypoxia. Hypoxia is a reduction in the normal level of oxygen tension and occurs during acute and chronic pulmonary disease, vascular disease and cancer. Under hypoxic conditions, the rate of oxygen supply limits the rate of oxygen consumption, and aerobic metabolism is reduced. In cases of severe O 2 deficiency, the respiratory chain succumbs, and as a consequence, cellular death by necrosis or apoptosis may result. HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits. Whereas HIF-1β is constitutively expressed, HIF-1α is targeted to ubiquitinylation by the von Hippel-Lindau tumor suppressor protein and is rapidly degraded in the proteasome. Under hypoxic conditions, hydroxylation of HIF-1α is blocked, which promotes protein stability and transactivation of HIF-1 [26] . Upon translocation to the nucleus, HIF-1 promotes the expression of genes encoding proteins that increase the cellular supply with oxygen and with energy-providing substrates [27] . HIF-1 initiates the defense against hypoxia at different levels, but in virtually all tissues, hypoxia induces the synthesis of proteins controlling local blood flow, notably VEGF and HO-1 [26, 27, 36] .
Several of the aforementioned agents downstream of HIF-1 are also potent inflammatory mediators, and recent studies have shown that HIF-1 plays a central role in stress responses beyond hypoxia (reviewed in [25, 28] ). Many pro-inflammatory cytokines and reactive oxygen species (ROS) can activate HIF-1 even under normoxic conditions [25, 37] , with subsequent HIF-1 induction of proteins that promote inflammation in a seemingly positive feedback loop of inflammation regulation (reviewed in [28, 38] ). HIF-1 has also been shown to be essential for the regulation of the glycolytic capacity of myeloid cells. In the absence of HIF-1, the cellular ATP pool is drastically reduced, and this metabolic defect causes profound impairment of macrophage and neutrophil cell aggregation, motility, invasiveness, and antimicrobial activity [24] .
In the context of severe pneumonia caused by infection with highly pathogenic influenza virus, HIF-1 induction by both tissue hypoxia, due to compromised respiratory capacity, and the cytokine cascade initiated by innate immune responses, notably IFN-α/β, would be possible [28, 39, 40] . Pulsoximetry was not carried out in the present study due to logistic constraints, and thus it is not possible to determine with certainty whether the marked up-regulation of HIF-1 expression was due to systemic hypoxia, the inflammatory cascade, or both. Regardless of the proximal cause, up-regulation of HIF-1 expression, notably nuclear expression, could then promote the amplification of the cytokine cascade, contributing to the systemic inflammation response syndrome (SIRS). We have demonstrated that H5N1-infection in macaques induces profound and sustained up-regulation of mRNA for IFNs and their downstream signaling molecules, as well as for IL-1, IL-6 and TNF-α, accompanied by very significant levels of circulating IL-6 and TNF-α proteins [23] . Furthermore, pronounced nuclear and cytoplasmic expression of HIF-1α was demonstrated by IHC in peripheral blood leukocytes and tissue macrophages (described in this paper), and phospho-p38α-MAPK expression was up-regulated in both the respiratory tract and lymphoid tissues, lending support to the contention that increased cytokinemia is indeed induced during H5N1 infection, with systemic effects as a consequence. This interpretation is corroborated by findings by Lee et al. [3, 41] , who also demonstrated that high cytokine levels in peripheral blood correlated with increased phospho-p38α-MAPK expression in H5N1 infection in human patients.
During SIRS and MODS, the brain appears to be a main target organ, with activation of glial cells, upregulation of NOS2, apoptosis and loss of neurons [7, 42] . The proximal inducers of this encephalopathy include TNF-α, type I IFNs, IL-1β and IL-6 [17, [43] [44] [45] , all of which were highly up-regulated in animals with H5N1 virus infection throughout the 7-day observation period [23] . Neurological symptoms, other than 'depression', are difficult to monitor in macaques, and this, for logistic reasons, could not be done in this study. However, the finding of microglial activation, as evidenced by increased cell size and Iba-1 expression [30] and upregulation of VEGF-A expression in the neuropil and of HIF-1α expression in intravascular leukocytes and leukocytes in the interstitium of the choroid plexus of H5N1 infected animals, suggests that these animals might have experienced an encephalopathy, albeit relatively mild, that could have contributed to their constitutional symptoms and consequent high clinical scores [23] .
Collectively, the data presented here suggest that extrapulmonary tissue responses are part of the pathophysiology of infections with highly pathogenic influenza viruses. This is in accord with recent clinical observations in human patients [46] , where treatment for hypoxia improved survival rates. This would also appear to link in with the observed expression of HIF-1α, which in our study appeared to reflect the extent and kinetics of lung inflammation following infection of macaques with these various influenza viruses and, notably, was an indicator of extrapulmonary tissue reactions in the absence of frank pathology or virus replication. Thus, measurement of HIF-1 expression, e.g. in peripheral blood mononuclear cells, might be used as a prognostic biomarker in severe respiratory infections.  Using Complementary and Alternative Medicines to Target the Host Response during Severe Influenza It is now accepted that an overwhelming inflammatory response is the cause of human deaths from avian H5N1 influenza infection. With this in mind we sought to examine the literature for examples of complementary and alternative medicines that reduce inflammation, and to place the results of this search in the context of our own work in a mouse model of influenza disease, using a pharmaceutical agent with anti-inflammatory properties. Two Chinese herbs, Angelica sinensis (Dang Gui) and Salvia miltiorrhiza (Danshen), have been recently shown to protect mice during lethal experimental sepsis via inhibition of the novel inflammatory cytokine High Mobility Group Box 1 protein (HMGB1). Biochanin A, a ligand of the peroxisome proliferator activated receptors (PPAR) alpha and gamma and the active isoflavone in Trifolium pratense (red clover), has anti-inflammatory properties, and thus could be used as an influenza treatment. This is of great interest since we have recently shown that gemfibrozil, a drug used to treat hyperlipidemia in humans and a synthetic ligand of PPAR alpha, significantly reduces the mortality associated with influenza infections in mice. The inflammation-modulating abilities of these natural agents should be considered in light of what is now known about the mechanisms of fatal influenza, and tested as potential candidates for influenza treatments in their own right, or as adjunct treatments to antivirals. It is now generally accepted that the infectious agent in isolation does not cause the illness and fatal outcome seen in acute systemic infectious diseases. Instead, the pathogen induces host cells to generate excessive amounts of pro-inflammatory cytokines, the prototypic example being tumor necrosis factor (TNF), that alter organ function and host metabolism, thus generating the disease we observe (1, 2) . This general concept, originally proposed to describe the severe disease caused by malaria infection (3) , then sepsis (4) and influenza (5) , has taken root in the mainstream, and is now often referred to as the 'cytokine storm' (6) .
Influenza researchers have embraced the cytokine storm mechanism as an explanation for the fatal human disease caused by avian H5N1 influenza (7) (8) (9) . Likewise, the pandemic influenza outbreak of 1918 is likely to have induced an overwhelming and fatal cytokine response in humans, as mice and monkeys infected with a reconstructed 1918 influenza virus exhibited a dysregulated immune response and hypercytokinemia, which leads to death (10) (11) (12) . The inevitable 3-6-month delay in vaccine availability in the event of a pandemic (13) means that treatments to prevent death (but not necessarily infection) are a necessity. Existing problems with antiviral drugs (stockpiles not big enough to treat populations, drugs need to be taken prophylactically or very soon after infection, resistant virus strains have emerged) further necessitate investigation into other treatments (14) . Targeting the detrimental host response generated by particularly pathogenic strains of influenza virus would allow disease severity to be reduced to the point where people will be sick, but will not die as a result of infection. We see agents with an ability to damp inflammatory cytokine release [be they natural or pharmaceutical (15) ] as ideal candidates for influenza disease treatments, particularly during the period while a new vaccine is being generated, and as useful adjunct treatments to antivirals. As has been suggested for statins (16) , using agents that are in use in the human population for the treatment of other conditions means that the safety of such agents is already established, as is the optimal human dose range.
A New Pharmaceutical Treatment for Severe Influenza?
We have found that the pharmaceutical agent, gemfibrozil, already used in the human population to treat hyperlipidemia, protects a significant proportion of mice infected with influenza virus (H2N2) from developing a fatal disease (17) . Gemfibrozil belongs to the fibrate class of drugs, which, like statins, are used to lower blood lipid levels, although fibrates differ in their mechanism of action. Gemfibrozil is a synthetic ligand for the peroxisome proliferator activated receptor (PPAR) alpha. This nuclear receptor controls lipid homeostasis, with fatty acids and eicosanoids as endogenous ligands (18, 19) . We chose gemfibrozil for its known ability to reduce release of inflammatory cytokines, including TNF, interleukin (IL)-6 and interferon-g (IFN-g), as well its ability to inhibit the nuclear translocation of the transcription factor nuclear factor k-B (NF-kB) (20) (21) (22) , and hypothesized that these drug side-effects might be useful in ameliorating influenza disease, as has already been suggested (23) . We have found that the protective ability of gemfibrozil during influenza infections is likely to be a fibrate class effect, since fenofibrate also reduces the mortality of influenza virus-infected mice (unpublished data). Gemfibrozil lowers serum IL-6 levels in mice infected with influenza virus (unpublished data) but its effects on other aspects of the host immune response are not yet known, and remain the subject of our further study.
It is important to note that the use of fibrates and statins in the human population for lipid-lowering has not, to our knowledge, enhanced susceptibility to viral disease. This is probably due to the fact that the innate antiviral response to infection, for example expression of the antiviral protein, Mx, is regulated by type I interferons and not by TNF or IL-1 (24) . While TNF has been reported to exert direct antiviral effects against influenza virus in vitro (25) its antiviral effects in vivo are less clear. Administration of neutralizing anti-TNF antibodies during the course of influenza in mice did not increase viral titers, and the infection still resolved (26) . Furthermore, the course of infection of H5N1 influenza virus was not worsened in mice genetically unable to produce TNF (27) . It is therefore unlikely that agents that interfere with expression of pro-inflammatory cytokines such as TNF will enhance susceptibility to influenza, but this remains to be experimentally tested.
The strategy of selecting agents with anti-inflammatory properties need not only apply to pharmaceutical agents. There are already precedents in the literature for the use of natural agents to treat severe infections such as influenza. Ginseng and Salviae (28) have been trialed for their abilities to improve influenza vaccination outcomes with good results, and may also modulate cytokines to reduce immunopathology during influenza infections. Researchers working during the SARS outbreak used complementary medicines to treat a small number of SARS patients with good results, and suggested that these treatments might be useful in treating cases of avian influenza (29, 30) . A recent review of the antiviral and immunomodulatory effects of compounds isolated from plants and traditional Chinese medicines describes a Chinese study in which FM1 influenza virus-infected mice were given the traditional Chinese medicine Yiqi Qingwen Jiedu Heji (31) . Researchers found that this treatment reduced expression of the cytokines TNF, IL-6 and IFN-g in the lungs of FM1 influenza-infected mice, all of which are implicated in inflammation, and increased levels of the anti-inflammatory cytokine IL-10, the combined effect being a shortened course of influenza disease (32) . Glycyrrhizin, an active component of liquorice roots, when given to mice from one day before infection with influenza virus (H2N2), protected all treated mice from fatality while all control mice died (33) . What is most interesting about this result is that glycyrrhizin binds to, and inactivates, the novel pro-inflammatory mediator High Mobility Group Box 1 protein (HMGB1) (34) , which is elevated in the serum of sepsis patients who succumbed to infection (35) .
With these non-pharmaceutical precedents in mind, we searched the literature for examples of complementary and alternative medicines used to treat conditions other than influenza that are characterized by increased pro-inflammatory cytokine levels, for their potential to mitigate the host response during severe influenza.
Sepsis Experimental models for sepsis provide an excellent avenue for understanding pathophysiology and mechanisms of disease during severe inflammatory states. Because sepsis is characterized by a prolonged inflammatory state that, if untreated, results in death (36) , it is comparable to the cytokine storm that ensues in H5N1 influenza infections in people. Any agents that have proven to be of benefit in sepsis models should be considered as candidates for influenza treatments, and we have found several examples in the literature of complementary medicines that do this.
The low molecular weight fraction of an aqueous extract of the Chinese herb Angelica sinensis (Dang Gui) is dose-dependently protective during lethal experimental sepsis and endotoxemia (37) . Ninety percent of mice were saved during lethal endotoxemia (when only 30% of controls survived) when the extract was given daily, and 70% of mice were saved during lethal sepsis (when only 25% of controls survived) when the extract was given daily. What is most impressive is that in the sepsis model, the repeated administration of the extract was not begun until 24 h after the onset of sepsis, when some animals had already died. The authors also showed that this late administration significantly reduced serum levels of HMGB1, and attributed the rescue of mice in part to the attenuation of systemic HMGB1 accumulation by the herbal extract. This effect on HMGB1 is important as HMGB1 is the only high mobility group protein with an additional extracellular function, a cytokine-like activity (38, 39) . It is this function that many researchers have become interested in since the first demonstration in 1999 of the presence of high levels of HMGB1 in the serum of mice and human patients with sepsis (35) . What is most unusual about HMGB1 is that its secretion continues for an uncommonly long time, with peak levels detected in cell cultures 18 h after stimulation (35) . This is in contrast to TNF secretion, for example, which peaks 90 min after the initial stimulus. In turn, once released into the circulation HMGB1 generates a further wave of inflammatory cytokine production (40) through its receptors, the receptor for advanced glycation endproducts [RAGE; (41, 42) ], and toll-like receptors 2 and 4 (43) . This ability to amplify and prolong inflammation is consistent with observations in sepsis where both HMGB1 and other inflammatory cytokines are persistently elevated (44, 45) .
Recently, the same group of researchers showed that the aqueous extract of another Chinese herb, Salvia miltiorrhiza (Danshen), traditionally used to treat cardiovascular disorders, was also protective against lethal endotoxemia and sepsis by decreasing HMGB1 levels in vivo (46) . As for the Dang Gui extract, the Danshen extract was administered 24 h after the onset of sepsis yet still rescued mice. As a potential anti-influenza treatment Danshen may have a double-pronged attack, as it also possesses anti-viral activity (47, 48) .
Artemisinin, a Chinese herb traditionally used as an anti-malarial drug, has been shown to possess potent anti-inflammatory effects via inhibition of NF-kB (49) . It, too, protects against lethal endotoxemia (50) thus warranting its consideration as a potential influenza treatment. Chloroquine possesses antiviral (51) and anti-inflammatory activities (52) and has already been suggested as a potential treatment for pandemic influenza (23) .
Ginseng is another herb with potent anti-inflammatory effects. An extract from Korean red ginseng (Panax ginseng, C.A. Meyer) significantly protected mice in experimental sepsis by decreasing TNF, IL-1, IL-6 and IFN-g production via inhibition of NF-kB activation (53) (54) (55) . It is likely that Korean ginseng will also reduce HMGB1 levels, because cytokines under the control of NF-kB, such as TNF and IFN-g, induce HMGB1 (56). Green tea (Camellia sinensis) reduces endotoxin-induced release of HMGB1 and is also proposed to possess the ability to decrease mortality from sepsis if taken regularly (57) .
Chronic pancreatitis is characterized by increased serum levels of pro-inflammatory cytokines (58) . Acute pancreatitis attacks are often followed by multiple organ failure and death, due to the overproduction of inflammatory cytokines (59) and like H5N1 influenza, the 'cytokine storm' mechanism has been used to describe this syndrome (60) . Serum levels of HMGB1 are known to be elevated during acute pancreatitis (61) . Danshen (S. miltiorrhiza), described above as being protective during experimental sepsis, also protects against acute pancreatitis by reducing levels of reactive oxygen species (62) , downstream by-products of inflammation. Its use in two syndromes that are characterized by an altered host response, leading to multiple organ failure and death, should make it a priority for influenza researchers interested in examining the effect of immunomodulatory treatments on the course of influenza disease.
Pro-inflammatory cytokines are increased during menopause with ensuing pathologies including osteoporosis and atherosclerosis (63) . Red clover (Trifolium pratense) isoflavone extracts are widely used for relieving postmenopausal symptoms in women. Biochanin A is the major isoflavone present in red clover, and activates both PPAR alpha and gamma receptors in vitro (64) . Gemfibrozil, the synthetic PPAR alpha ligand that increases survival of mice during severe murine influenza (17) , possesses anti-inflammatory side effects including inhibition of NF-kB activity; this transcription factor is essential for the transcription of pro-inflammatory genes (21) . Similarly, biochanin A has been recently shown to decrease production of the pro-inflammatory cytokine eCAM 2010;7(4) TNF, as well as its downstream mediators nitric oxide and superoxide (65) . The fact that biochanin A activates both PPAR alpha and gamma would be an advantage in treating severe influenza, as the PPAR gamma agonist pioglitazone was recently shown to reduce the mortality associated with PR8 influenza in mice (66) . It is possible that combining PPAR alpha and gamma activation in one treatment, using biochanin A, may prove more effective in curbing influenza-related mortality than activation of either PPAR receptor alone.
The above examples of complementary medicines, used in seemingly disparate syndromes to treat essentially the same thing-pathological cytokine excess-should provide researchers working in the influenza field with a solid starting point for in vivo studies in animal models of the disease.
Prescribed mixtures of Chinese herbs are given to patients with influenza. A common formula contains Jin Yin Hua (honeysuckle flower), Lian Qiao (forsythia fruit), Bo He (field mint), Jing Jie (schizonepeta), Jie Geng (root of balloon flower), Gan Cao (licorice root), Dang Gui (A. sinensis root), Dang Shen (Codonopsis root), Chai Hu (thorowax), Qianghuo (Notopterygium root) and Sheng Jiang (fresh ginger rhizome). Intriguingly, nine of these 11 herbs have been shown to possess antiinflammatory properties. A. sinensis is dose-dependently protective during lethal experimental sepsis and endotoxemia (37) via its ability to limit release of HMGB1, as has already been discussed. There are many examples of Jie Geng (root of balloon flower), reducing both production of pro-inflammatory cytokines and proinflammatory mediators (such as reactive oxygen species and nitric oxide), with these effects likely to be mediated through suppression of activation of NF-kB [e.g. see ref. (67) ]. Sheng Jiang (fresh ginger rhizome), Gan Cao (licorice root) and Lian Qiao (forsythia fruit) also reduce levels of inflammatory cytokines via suppression of NF-kB induction (e.g. see refs (68) (69) (70) . Thus it seems that a mixture of herbs, as traditionally prescribed, is likely to have profound effects on the inflammatory cytokine balance, warranting further investigation of the mixture itself in vitro and in vivo.
HMGB1 has a growing literature on its involvement in the pathogenesis of severe inflammatory states. Large amounts of HMGB1 have been detected in the serum of sepsis patients who succumbed to infection (35) . Similarly, during falciparum malaria levels of HMGB1 are higher in patients who do not recover (71) .
However recent observations paint a more complex picture of the significance of elevated serum HMGB1 measurements. Sepsis patients with severe organ dysfunction had the lowest serum HMGB1 levels of all sepsis patients tested (72) , which probably points to a well-known sideeffect of acute infection, immunosuppression. We have shown that despite plasma levels of HMGB1 being low at the time of peak mortality in mice infected with A/Japan/305/57 influenza virus (H2N2), HMGB1 may be elevated via its passive release in the lung at this time (73) . Therefore it would be important to first ascertain, using animal models of influenza, how levels of HMGB1, in the serum and/or lung, were associated with pathology and/or fatality. Our own work in this area continues, as does our focus on optimizing the way in which HMGB1 is measured for study. Figure 1 shows the likely sources and role of HMGB1 during infection with influenza virus.
Therapeutic interventions in sepsis models, such as administration of ethyl pyruvate (74) , an agent that limits HMGB1 release in vitro, or administration of an anti-HMGB1 antibody (75) , save a significant proportion of mice, implicating the inhibition of HMGB1 release or activity as a therapeutic target during immune pathology. As previously mentioned, glycyrrhizin protects influenza virus (H2N2)-infected mice from fatality (33) potentially via its ability to bind to, and inactivate, HMGB1 (34) . Therefore it would be important to establish whether or not therapeutic intervention with any of the agents discussed here reverse the fatality of severe influenza in animal models via effects on HMGB1 and/or other aspects of the host response.
However, endogenously produced molecules that dampen the effects of HMGB1 in the circulation are also elevated during severe disease states. Thrombomodulin, which binds to HMGB1 and prevents it binding to RAGE (76) is elevated during severe malaria (77), sepsis (78, 79) and influenza (80) . Paradoxically, it is this binding of HMGB1 to thrombomodulin that also prevents activated protein C generation (81) and thus its ensuing anti-inflammatory activities (82, 83) . Thus the balance between HMGB1 and thrombomodulin in vivo is likely to be important (81) and should also be determined as part of these studies.
A second, broader strategy could be to examine the traditional Chinese medicine formula discussed here, indeed, any other traditional formulas in their entirety, for anti-inflammatory activity in vitro. Cultured mouse macrophages could be stimulated with either commercially available recombinant influenza virus H5N1 haemagglutinin (HA) or LPS to induce production of pro-inflammatory cytokines, in the presence or absence of the formula, prepared as traditionally described (simmered in boiling water for 45-60 min). Cytokine assays to profile pro-and anti-inflammatory cytokines secreted over time into the culture supernatant could then be conducted, using a system such as multiplexing to examine many different cytokines in each sample (such as anti-inflammatory cytokines IL-4 and IL-10, and pro-inflammatory cytokines IL-1, IL-2, IL-6, IL-8, IL-17, IFN-g, TNF and HMGB1). Given results which point towards levels of pro-inflammatory cytokines being reduced by treatment of the cells with the formula, the next step would be to conduct studies in mice infected with influenza virus (such as mouse-adapted PR8) and administered the formula orally, daily, adjusted according to body weight. Parameters measured in the in vivo experiments could initially include analysis of serum and lung cytokine levels, body weight and temperature, and even survival, providing researchers could interact with their institutional ethics committee to arrive at a mutually appropriate end-point for euthanasia for experiments of this nature.
Systematic reviews of the literature on clinical trials using traditional Chinese medicine in influenza patients have demonstrated the need for proper design, using randomized controlled trials and sufficiently large study groups to gain meaningful results (84, 85) as well as a more systematic approach to disease diagnosis (86) . Thus it would be informative to take the insights gained from results in mice into the clinic. Patients hospitalized with influenza could be recruited [see ref. (87) for parameters relating to recruitment] to a randomized controlled trial to determine if treatment with a traditional Chinese medicine formula (for example, the formula previously discussed here) affected the course of disease. It would be prudent to begin treatment immediately, before laboratory confirmation of influenza virus infection. This is because symptoms are apparent at the time of increased serum cytokine levels (87) . Obviously patients testing negative to influenza virus would subsequently be excluded from analysis. The decoction would be prepared in the traditional manner (simmering in boiling water for 45-60 min) and administered to patients by hospital staff. The control treatment could simply be a flavored water solution that is stored in the same way as the decoction (at 4 C for not more than 1 week), and consumed daily in the same quantity as the decoction. Both solutions should be colored identically (with a food-grade dye, for example). It would be ideal to ensure the trial was conducted in a double-blind fashion so that neither the patient nor the staff knew which treatment was active. Previous studies have compared the incidence of influenza-like illness in healthcare workers (93), secrete pro-inflammatory cytokines and may also secrete HMGB1 both actively and passively. MIP is macrophage inflammatory protein.
Cytokines (excluding IL-10) commonly reported to be involved in pathogenesis were compiled from (94) (95) (96) . IL-10 from effector T-cells was recently shown to limit inflammation during acute influenza (97) . HMGB1 up-regulates pro-inflammatory cytokine expression via its cellular receptors, the receptor for advanced glycation end products [RAGE; (41, 42, 98) ] and to a lesser extent, toll-like receptor (TLR) 2 and 4 (43, 99) . RAGE expression was recently shown to be associated with influenza A virus pneumonia, accompanied by increased levels of HMGB1 in bronchoalveolar lavage fluid (100).
using an herbal formula against those using no agent (88) but results were questioned because workers given the herbal formula knew that they were taking the 'active' treatment. While these trials recruited thousands of patients, we suggest that patient numbers could be kept relatively small in the first instance (20 patients per group) if, in addition to patients being unaware of the activity of the treatment they are given, daily blood samples are taken to measure serum levels of cytokines, as for the in vitro assays described above. One milliliter of blood would provide enough serum to analyze cytokine levels, and daily measurements on the same individual would allow for paired statistical analysis. The appropriate end-point would be convalescence, but obviously if symptoms worsened with treatment (or control) then standard interventions should immediately take place. These basic but necessary experiments could pave the way towards viable, low cost alternatives to planned influenza pandemic treatments-approximately $8 for a fortnight's treatment (88) . Figure 2 summarizes the possible inflammation-reducing effects of traditional Chinese medicines (e.g. A. sinensis, S. miltiorrhiza and glycyrrhizin), a natural agent (biochanin A), and pharmaceutical interventions (e.g. statins, fibrates), during influenza.
Direct stimulation of the vagus nerve has been shown to exert potent inflammation-reducing activities in the body (89) . Transcutaneous stimulation of the vagus nerve improves survival in mice with sepsis by reducing serum levels of HMGB1 (90) . Thus there is a distinct possibility that vagus nerve stimulation by acupuncture could control immune-mediated pathology, by limiting release of pro-inflammatory cytokines. The potential for using acupuncture in this way was recently reviewed (91) with the authors noting that 'the use of acupuncture as an adjunct therapy to conventional medical treatment for a number of chronic inflammatory and autoimmune diseases seems plausible and should be validated by confirming its cholinergicity'. Thus stimulation of the vagus nerve in patients with influenza could also represent an adjunct treatment in the management of severe influenza cases, provided it can be further investigated. Figure 2 . Potential mitigation of the action of HMGB1 and other pro-inflammatory cytokines by traditional Chinese medicines, a natural agent, and pharmaceutical drugs. The Chinese medicine glycyrrhizin, from liquorice root, binds to HMGB1 and inactivates its biological activities (34) , while the Chinese herbs A. sinensis and S. miltiorrhiza reduce the release of HMGB1 (37, 46) . Release of pro-inflammatory cytokines is reduced via inhibition of NF-kB activation, by glycyrrhizin (101-103), A. sinensis (104, 105) , and S. miltiorrhiza (106, 107) ; by the natural agent biochanin A (65, 108, 109) ; and the pharmaceutical drugs statins (110, 111) and fibrates (21, 112, 113) . Pro-inflammatory cytokine release is reduced by activation of PPARs (21) and conversely, anti-inflammatory cytokine release is increased by their activation (114, 115) . PPAR alpha is activated by fibrates (21); PPAR alpha and gamma are activated by biochanin A (64); and statins upregulate PPAR alpha and gamma and show molecular signaling properties similar to fibrates (116).
The example given here of an ancient Chinese medicine formula used to treat influenza containing nine (out of 11) herbs with anti-inflammatory properties provides compelling evidence that the way forward for the treatment of influenza in a pandemic should be immunomodulation. The host response is targeted, rather than the virus itself. Our observation that treatment of influenza virus-infected mice with a pharmaceutical used in humans, the lipid-lowering and immunomodulatory PPAR alpha agonist gemfibrozil, resulted in significantly decreased influenza-induced mortality, further shows that targeting the host response is a valid possibility. The natural immunomodulatory agent biochanin A from Red Clover, which is both a PPAR alpha and PPAR gamma agonist, could have similar effects to gemfibrozil on the course of influenza disease in vivo. Chinese herbs such as A. sinensis and S. miltiorrhiza which independently reduce secretion of the novel inflammatory cytokine HMGB1, and glycyrrhizin from liquorice root, which binds to HMGB1 and inactivates its activity, provide another avenue for investigation, as does the establishment of the role of HMGB1 in severe influenza. The wide availability and low economic price of these agents could make such agents an inexpensive alternative treatment, particularly in countries with large populations who will have no access to pandemic vaccines or antivirals. Pandemic (H1N1) 2009 Influenza Community Transmission Was Established in One Australian State When the Virus Was First Identified in North America BACKGROUND: In mid-June 2009 the State of Victoria in Australia appeared to have the highest notification rate of pandemic (H1N1) 2009 influenza in the world. We hypothesise that this was because community transmission of pandemic influenza was already well established in Victoria at the time testing for the novel virus commenced. In contrast, this was not true for the pandemic in other parts of Australia, including Western Australia (WA). METHODS: We used data from detailed case follow-up of patients with confirmed infection in Victoria and WA to demonstrate the difference in the pandemic curve in two Australian states on opposite sides of the continent. We modelled the pandemic in both states, using a susceptible-infected-removed model with Bayesian inference accounting for imported cases. RESULTS: Epidemic transmission occurred earlier in Victoria and later in WA. Only 5% of the first 100 Victorian cases were not locally acquired and three of these were brothers in one family. By contrast, 53% of the first 102 cases in WA were associated with importation from Victoria. Using plausible model input data, estimation of the effective reproductive number for the Victorian epidemic required us to invoke an earlier date for commencement of transmission to explain the observed data. This was not required in modelling the epidemic in WA. CONCLUSION: Strong circumstantial evidence, supported by modelling, suggests community transmission of pandemic influenza was well established in Victoria, but not in WA, at the time testing for the novel virus commenced in Australia. The virus is likely to have entered Victoria and already become established around the time it was first identified in the US and Mexico. The first confirmed case in Australia of pandemic (H1N1) 2009 influenza (pH1N1) was recorded in Queensland in a returned traveller from the United States (US) on 9 May 2009 [1] , almost four weeks after the first cases were confirmed in the US on 15 and 17 April 2009 [2] . Victoria confirmed its first case in a traveller from the US on 20 May [3] and the first case in Western Australia (WA) was notified on 24 May in a traveller returned from Canada via the US [4] .
The response to the identification of cases of pandemic influenza initially followed the guidelines of the Australian Health Management Plan for Pandemic Influenza (AHMPPI). [3, 5, 6] , Management of the pandemic moved through three phases, described as Delay, Contain, and Protect. An additional phase, Modified Sustain was applied only in Victoria. The phases were designed to delay the entry of pandemic virus into Australia, to contain the virus once it had entered the country, to sustain a response once community transmission had been established and to protect the vulnerable once infection was deemed to be widespread [5, 7] .
Modified Sustain was announced in Victoria on 3 June 2009 -just more than two weeks after the first confirmed case in Victoriaand was followed by a decrease in active case finding. Two weeks later, on 17 June, the Australian Government announced the Protect phase, one that had not been included in Australia's pandemic plan, but was added to the original AHMPPI [5, 7] once the less severe nature of the pandemic had been accepted. WA pre-empted formal adoption of the Protect phase on 13 June, when all doctors and hospitals were asked to cease active case-finding, and prioritise influenza testing only to persons with severe influenza-like illness or established medical risk conditions. Victoria formally moved to the Protect phase on 23 June.
Although the first laboratory-confirmed cases were identified in Victoria and WA within four days of each other, reported case numbers immediately escalated in Victoria but not in WA [8] . We suggest this observation is explained by the unrecognised establishment of community transmission of pH1N1 in Victoria, but not in WA, around or before 26 April, when public health agencies and laboratories in all Australian states and territories (''jurisdictions'') commenced investigating and testing incoming travellers with influenza-like illnesses from North America for pandemic influenza. We support our argument with a detailed review of case follow-up data for both states, a review of other surveillance data relevant to Victoria and modelling of the epidemic in both states.
We compared Victoria and WA because they do not share a border and a large distance separates these two states, allowing for importation between states to be more readily recognised. The state capitals, Melbourne and Perth, are approximately 3,400 km apart by road, on the south-east and west coasts respectively. Victoria has a population of approximately 5.4 million of whom 70% live in Melbourne, while the WA population is estimated as 2.2 million, with 74% living in Perth.
As part of the Delay and Contain phases of the Australian response to the pandemic, active case-finding involved identification, isolation, testing and antiviral treatment of incoming travellers with influenza-like illnesses; and prophylactic treatment and home quarantine of the close contacts of suspect/confirmed cases. Influenza is a notifiable disease in all Australian jurisdictions. Public health reference laboratories in Victoria and WA developed pH1N1-specific nucleic acid amplification tests in the first week of May.
Early spread of the pandemic virus in both states was concentrated in the capital cities [3] . High quality case ascertainment and contact follow-up data were available from both states. Of all Australian jurisdictions, community transmission of pandemic influenza was established earliest in Victoria and latest in WA.
Case ascertainment and follow up has been described in detail for Victoria [3] . Until 3 June, when the Modified Sustain phase was implemented, an attempt was made to identify and confirm every case and to follow-up every contact of suspected or confirmed cases. Until this date, 977 cases were identified and 5,807 contacts were followed-up. In WA prior to the formal implementation of the Protect phase on 13 June, all suspected or confirmed cases were actively followed up and travel histories were recorded. By this date, 102 cases had been confirmed and 232 household contacts of these cases followed-up, plus a large number of other contacts, including those on aeroplanes and at schools.
Other relevant data were gathered from international outbreak reports, postings on the electronic noticeboard ProMED-mail (http://www.promedmail.org) and a range of other electronic media reports.
The basic reproduction number (R 0 ), indicates the average number of people each infected person infects in a totally susceptible population. By contrast, the time dependent effective reproduction number (R), indicates the average number of people each infected person infects, given the current interventions in place, and any prior immunity that reduces the susceptible pool. The effective reproduction number is always less than or equal to the basic reproduction number and typically declines gradually as a disease spreads through the population and collective immunity increases. The effective reproduction number was calculated using an adaptation of the method of Bettencourt and co-workers [9] [10] [11] to allow for imported cases and a distributed serial interval. The adaptation consists of cases being partitioned into local (L) and imported (M) cases and these are tracked in the data so that the new imported cases are not considered to be locally acquired infections and hence are not attributed to infection from previous local cases.
This method uses a stochastic version of the standard SIR (susceptible, infective, recovered) model and Bayesian inference to determine a probability distribution for R that best matches the case report data.Let t denote the time interval between case reports (taken to be daily here) and in the time interval (t2t, t) the number of locally acquired cases is L(t) and the number of imported cases is M(t). Implicit in the adaptation used here is the assumption that the imported cases spend their infectious period in the jurisdiction of interest which is reasonable given those cases where reported in that jurisdiction. The model uses discrete Euler time step approximations to the derivatives and so the new imported cases can be added at each discrete time step without affecting the model. The usual SIR infective equation can be written to the same degree of accuracy as in [9] [10] [11] as
According to the SIR model, detailed in [9] [10] [11] , the number of newly acquired local cases at time t+t due to the L+M cases at time t is then given by:
where c is the mean infectious period. At each time step the new imported cases are added to obtain the total number of cases that give rise to locally acquired infections in the following time period.
Since daily case numbers are highly variable a probabilistic model is needed to allow for this variation. For a given R the probability of L local cases at time t+t depends on the number of local and imported cases at time t and is given by:
where P[l] is a suitable probability distribution with mean l. The difference between this and that presented in [9] [10] [11] is that the number of locally acquired cases is used as the data at time t+t rather than all cases. The standard SIR model only deals with the average number of cases so a suitable probability distribution for P[l] is a Poisson distribution which is the most general form (highest entropy) if only averages are known.
We are interested in estimating R and how it evolves over time as new cases are reported. That is, we want to know the probability distribution of R that best fits the available data. From Bayes theorem:
The denominator of the right hand side is simply a scaling factor that can be calculated from the sum of the probabilities being 1 and does not need to be explicitly determined. The first term in the numerator is calculated using the Poisson distribution, as discussed above, using the case numbers at time t+t. P[R] is the prior probability distribution of R, which reflects earlier values of R, either from calculated values or initially from knowledge of the disease. Here an initial unbiased estimate of P[R] is chosen to be a uniform distribution on [0 4], that is any value of R in [0 4] is equally likely. The above equation is iterated to obtain progressively better estimates for the probability distribution of R as time progresses and more data become available. As a probability distribution for R is obtained, compared to other methods that produce a single value for R, a 95% credible interval for the R value is easily obtained.
We have verified this method using many thousand numerically simulated outbreaks with known values of R and different imported cases distributions. Over all different importation scenarios the method gave a reliable estimate of the underlying reproduction number. Previously, imported cases have either been removed from the calculations [12] or treated as local cases [13] . Both of these approaches overestimate the true effective reproduction number as either too much transmission is assigned to local cases or imported cases are assigned as being locally acquired, respectively.
For daily case report data considered here t = 1, which is shorter than the serial interval of influenza. Using only the previous day's data (t = 1), as outlined above, results in slow convergence of the method since changes in the case numbers are due mostly to the case numbers more than t days earlier. Faster convergence and tighter bounds on R are obtained if R is calculated using a weighted sum of L and M stretching back in time beyond the serial interval. The weighting used is the temporal distribution of the serial interval, taken to be gamma distributed here, which weights earlier days relative to how likely the serial interval was to be that long. See below for further discussion on the serial intervals used in the analysis.
We used mathematical models to calculate the number of days required for an outbreak initiated by a single imported case to reach the cases observed on 29 May in Victoria. The epidemiological parameters that most affect the growth rate of an outbreak are the reproduction number (defined above) and the serial interval. The serial interval measures the number of days between the time of infection of a secondary case and the time of infection of its infector. As discussed elsewhere [14, 15] , both the distribution of the serial interval and its mean influence the growth rate of outbreaks. Following earlier work [12, [16] [17] [18] we have assumed a gamma distribution for the serial interval, and have considered a range of values for both the reproduction number and the mean serial interval.
Numerical simulations were performed in MATLAB using a stochastic version of a SEIR (susceptible, exposed, infective, recovered) type model. The code is available from the author (GM). A stochastic model was used rather than a deterministic SEIR model as it better reflects the variability inherent in the early stages of an outbreak. Inputs to the model were a fixed reproduction number (R) and a serial interval distribution (f(t), t = 1,…M) as described above. New cases at time t were sampled from a Poisson distribution with mean RS(t)gf(t)I(t2t), where S(t) is the fraction of the population susceptible and I(t) are the number of infected individuals at time t. Initially, there were one million susceptible individuals and one infective case was introduced at time zero. We performed 1,000 simulations for each pair of values, and recorded the mean and standard deviation over these simulations. The simulations were not run beyond a total of 5,000 cases, so the results are insensitive to the initial population size, which was chosen large enough so that susceptible depletion was not an issue. Due to the stochastic nature of the method, not all simulations result in an established outbreak, with some resulting in what is known as stochastic die out [19] . Only those simulations that resulted in at least 20 cases were retained.
In order to obtain a robust estimate of the time taken for case numbers to reach those observed on 29 May, we considered values of the reproduction number ranging from 1.2 to 1.8 and mean serial interval from 2 to 4 days, consistent with other estimates of the mean serial interval of pandemic H1N1 of 1.9 days [20], 2.8 days [12, 16, 21] , 3.2 days [22] and longer [23] . We performed 1000 simulations for each pair of values, and recorded the mean and standard deviation over these simulations.
While the serial interval and the reproduction number are the key factors that determine the speed at which an outbreak takes off, heterogeneities in contact patterns may also have some impact. One of the most likely sources of heterogeneity for this data arises from age structure [17] . The stochastic SEIR model described above is homogeneous with respect to age structure. This may be a limitation of the model. We therefore tested the impact of age heterogeneity using an alternative model with different reproduction numbers for adults and children (but with the overall reproduction number equal to that of our basic model). In order to test the impact of very high levels of heterogeneity, we assumed that the reproduction number for children was twice that of adults, due to heightened mixing between children and lack of prior immunity. The structured model estimated a reduction in 20-25% in the days required to reach the case numbers. In particular, for the intermediate case of a reproduction number of 1.4 and mean serial interval of 2.8 days, the delay was reduced from 33 to 26 days. The Victorian data could not be reproduced with this agestructured heterogeneity without using unrealistically large values of the reproduction number. The same was true of the stochastic SEIR model described above and other common simulation models such as deterministic SIR and SEIR type models [24] . We concluded that, although model structure influenced the estimate of the delay, even very high levels of heterogeneity had a relatively minor impact relative to the effect of the reproduction number or the serial interval, which are the dominant factors in determining the speed of the spread of the outbreak.
This research was exempted from ethical review under the Australian Government National Health & Medical Research Council's 'National Statement on Ethical Conduct in Human Research' because it was defined as negligible risk and involved the use of existing collections of data and records that contain only non-identifiable data about human beings. This study used aggregated notifiable diseases data that were collected under the relevant public health legislation in Victoria and WA.
The first laboratory confirmed case in Victoria was notified on 20 May. Figure 1 shows notified cases by date of onset and location of acquisition until the commencement of the Modified Sustain phase; pandemic phase changes and case identification milestones are also indicated. Only 5% of the first 100 cases in Victoria were imported, and only eight of the 977 (0.8%) cases diagnosed prior to the introduction of the Modified Sustain phase reported a travel history. The first five diagnosed cases reported travel to the Americas: three brothers from one family returned from the US, a visitor from Mexico and another traveller returned from the US. All five cases were reported on 20-21 May. Two cases diagnosed on 1 June (numbers 368 and 374) were reported to have travelled to an affected country in the seven days prior to illness onset although the country was not specified for either case. One other case (number 398) diagnosed on 2 June, was reported to have acquired her infection in Japan.
An outbreak of influenza due to both pH1N1 and seasonal H3N2 influenza occurred on the cruise ship, the Pacific Dawn at a time prior to there being recognised transmission of pH1N1 in Australia. Of almost 3,000 passengers on the cruise, nine passengers with a Victorian residential address were subsequently confirmed to have pH1N1 infection. The earliest onset date of symptoms amongst the Victorian passengers was 18 May. Symptom onset for this patient occurred two days after boarding, consistent with a prodromal infection at the time of embarkation. Despite recognition of numerous passengers with influenza-like illness when the ship berthed in Sydney on 25 May, public health authorities allowed passengers to disperse into the community because the ship had not visited any port where there were confirmed cases of pandemic influenza. Retrospectively, it appears plausible that the Melbourne passenger who joined the cruise on 16 May 2009 with prodromal infection may have been the source of the shipboard pH1N1 outbreak.
Two other observations, which may also reflect a high point incidence of disease in Melbourne prior to recognition that local transmission was occurring. The eighth case to be diagnosed with pandemic influenza in Victoria was an eight-year-old male ascertained from routine general practice sentinel surveillance for influenza (Figure 1 ). This boy had no travel history or contact with travellers and symptom onset was on 18 May, two days prior to notification of the first confirmed Victorian case, the traveller from the US, who also had symptom onset on 18 May. Around that time in May, pandemic influenza was also exported from Melbourne to China. Amongst the first 12 cases in China, diagnosed between 11 and 25 May, one was a traveller from Melbourne who had arrived in Beijing on 21 May [25] , only one day after Victoria's first case was confirmed.
Calculation of the reproductive number from the early Victorian data for all notified cases was performed as described in the methods section. Values ranged from R = 2.7 around 20 May, when the first Victorian case was reported, and fell steadily and dramatically to 1.5 by 29 May (Figure 2) .
To test the hypothesis of early community transmission of pH1N1 in Victoria, numerical simulations were performed using plausible reproductive numbers ranging from 1.2 to 1.8 and with mean serial intervals (MSI) ranging from 2 to 4 days, consistent with previous estimates [12, 16, [20] [21] [22] [23] . The length of time needed to obtain similar case notifications to the number observed around 29 May was determined. For statistical robustness 1,000 simulations for each R and MSI pair were performed and the average length and the standard deviation calculated (Table 1) 
The first confirmed case of pandemic influenza was notified on 24 May, four days after notification of the first Victorian case, in a traveller returning from Canada via the US. Only 23 additional cases had been notified more than two weeks later, with five linked to travel from North America and the remainder in travellers from Victoria or linked directly to Victorian-origin cases. Of the first 102 cases notified in WA, 53% were imported from Victoria or linked directly to Victorian-origin cases.
By 30 June, 247 cases had been notified in WA (Figure 3 ). Of these 16 (6%) were travellers from overseas countries with documented transmission, 94 (38%) were travellers from Victoria or locally acquired cases linked to Victorian-origin cases, 29 (12%) were associated with travel from other Australian jurisdictions, 106 (43%) were locally acquired with no travel history and no identifiable links to imported cases and two (0.8%) were lost to follow up. Amongst the 94 clearly documented Victorianassociated cases, there were 72 individual importations over this period, demonstrating repeated seeding of WA by persons infected in Victoria.
Utilising the same method as used for estimating R in Victoria, and allowing for imported cases, R in WA was estimated to be well 
We have shown that epidemic curves and estimates for the effective reproduction number confirm the very different nature of the pH1N1 outbreaks in Victoria and WA. There appeared to be no pandemic virus in WA prior to multiple importations from Victoria, other Australian jurisdictions and overseas. Estimates of R in WA were consistently around 1.2 and never above 1.4. By contrast, initial estimates of R in Victoria approached 3, followed by a rapid -and implausible -decrease over a very short period of time. This precipitous fall cannot be explained satisfactorily by potential reduced transmission associated with early control interventions (such as partial school closure, t isolation and treatment of cases, and chemoprophylaxis and home quarantine of contacts), or depletion of susceptible people within the population. For instance, it took 20 days for the estimated value of R for the 1918-19 pandemic in San Francisco to fall from ,2.4 to ,1.2 [9] . We suggest the high initial value of R = 2.7 in the Victorian pandemic and the dramatic decline in 9 days is more likely explained by unrecognised cases due to an earlier commencement of the epidemic in Victoria.
The reproduction number for pH1N1 has been calculated for a number of different countries and ranges from 1.2-1.7 in Mexico, the US and Peru [20] [21] [22] [23] . Higher values of R of ,2 have been reported for Japan [16] and New Zealand [12] but these are acknowledged to be influenced by social clustering and increased diagnostic testing, and are hence most likely over-estimates of the true value of R. The estimates of R in Victoria and WA reported here have accounted for imported cases.
When the Victorian pandemic was simulated to reflect a plausible value of R = 1.4 and a serial interval of 2.8 days, earlier undetected cases needed to be invoked to reflect the observed epidemic pattern. In this scenario, modelling suggests that community transmission of the pandemic virus was most likely established by 25 April, around the time the virus was first recognised in the US and Mexico, two weeks before the first reported case in a traveller to Australia, and almost six weeks before community transmission was recognised in Victoria. Had simulations used higher values for R and lower values for the serial interval, the Victorian pandemic would have been modelled to commence even earlier. Conversely, there was no need to evoke undetected cases in WA in order to estimate a plausible range of values for R.
There was a marked difference in the proportion of imported cases in Victoria and WA. In WA 50 (49%) of the first 102 cases had travelled (eight overseas, 38 to Victoria and four to other Australian states) and a further 20 were directly linked to those cases that had travelled interstate. This is similar to the experiences of countries in the northern hemisphere. For example, in Spain 78% of the first 98 cases had acquired their infection abroad [26] ; in the United Kingdom 44% of the first 65 cases reported travel to the United States or Mexico [27] ; in Germany 47% of the first 198 cases were described as imported [28] and in Turkey 77% of the first 111 confirmed cases in the Turkish community were imported [29] . In Ireland 84% of the first 156 cases were imported, 14 (9%) were infected in Ireland by an imported case and two (1%) were infected in Ireland without any identifiable travel association [30] .
We have previously highlighted three observations to support our hypothesis of early community transmission of pH1N1 in Victoria [3] . We have now elaborated on the first of these observations, that a low proportion of Victorian cases had any travel history or link to travellers. Travel history and exposure were collected for all 977 cases reported before commencement of the Modified Sustain phase, so that no cases with a travel history or exposure to travellers should have been missed.
Secondly, there was a rapid rise in the number of notifications of locally acquired cases. In Peru a period of almost five weeks elapsed from identification of the first imported case before a dramatic increase in cases was recorded [31] . This rapid rise in Victoria occurred almost immediately and could not have been a consequence of exposure to the five documented imported cases, given that all these cases were isolated and their household contacts quarantined. Either transmission was already well established in Victoria by this time, or there were continuing undetected imported cases that fuelled the epidemic. The latter is unlikely, given widespread media attention and active case-finding at that time that targeted travellers reporting influenza-like illness.
Thirdly we noted the difference in the median age of 15 years in the first 977 cases to the median age of 21 years in patients notified through the general practice surveillance scheme [32] , and suggested this implied an amplification of an established epidemic in school-aged children. We have now supported these three arguments with modelling of the Victorian and WA notification data and two further circumstantial observations related to Victoria, namely, export of a case to China and an outbreak on a cruise ship in which the index case was a Melbourne resident.
Other observations also support the hypothesis of early community transmission in Victoria. We have previously established thresholds for the surveillance of influenza-like illness (ILI) in the state [33] . Normally ILI levels are below the baseline threshold when surveillance commences but, in 2009, ILI levels were above this threshold when surveillance commenced at the end of April [34] . None of the first 112 patients admitted with pH1N1 to seven hospitals in Melbourne had acquired their infection overseas [35] . Finally, it was possible to identify a presumed infectious source for only 3.7% of the first 1000 cases in Victoria (James Fielding, unpublished data).
Sub-typing of influenza A specimens archived at the Victorian Infectious Diseases Reference Laboratory between January and April 2009 did not identify any pandemic influenza viruses. We assume that mild disease would generally not have resulted in presentation for medical care and that retrospective identification of cases will be difficult.
The first alarm about infection with pH1N1 was related to increased rates of hospitalisation and death due to severe pneumonia in young adults in Mexico [36] . Identification of pH1N1 virus from Mexican patients was in response to this concern. However, the identification of the virus in the US at around the same time was serendipitous, following the identification of two influenza A viruses, one from a study on a point-of-care test and the other from a routine surveillance system, that could not be sub-typed [2] . Concern rose in the US when it was realised that the Mexican and US viruses were essentially identical [2] .
The most likely explanation for the discrepancy between the way the novel virus was detected in Mexico and the US is that the virus had been circulating far longer in Mexico than the US. One phylogenetic analysis suggests that the pandemic virus may have entered the human population between November 2008 and March 2009 [20] while a second study suggests the virus may have been causing human infections as early as September 2008 [37] . Widespread unrecognised community transmission causing mild infections may have been occurring in Mexico for some weeks or months, eventually leading to recognition of a cluster of severe pneumonia in a sub-group of susceptible young adults in April 2009. This cluster would have represented the apex of the infectious pyramid.
Indeed, identification of the pandemic virus as the cause of respiratory illness in a 6-month old Mexican infant with disease onset on 24 February has been informally reported, confirming at least two months of virus circulation in Mexico prior to recognition of the outbreak [38] . It is similarly conceivable that pH1N1 was circulating unrecognised in Victoria for several weeks before it was first detected. In those weeks specific testing was targeted at incoming travellers from North America, with no hint that the virus was already circulating in the Victorian community.
A clinical attack rate below 1.4% due to pH1N1 has been estimated for the spring of 2009 in the US [39] , a clinical attack rate of 7.5% has been estimated in New Zealand for the entire influenza season between April and August 2009 [40] and in England the estimated clinical attack rate was 10 times lower than the cumulative incidence of infection of 20% suggested from serosurveys of 15-24 year olds [41] . We suggest that a relatively low clinical attack rate -but a much higher infection rate -by a virus causing generally mild disease would allow community transmission of the virus to go unrecognised for many weeks. We further suggest that this occurred in Mexico and Victoria and may indeed have occurred in other countries [42] . Radiological and Clinical Characteristics of a Military Outbreak of Pandemic H1N1 2009 Influenza Virus Infection OBJECTIVE: To describe detailed clinical and radiological features of the pandemic H1N1 2009 influenza viral infection among healthy young males in a semi-closed institutionalized setting. MATERIALS AND METHODS: A total of 18 patients confirmed with the pandemic H1N1 2009 influenza virus infection from July 18 to July 30, 2009 were enrolled in this study. Each patient underwent an evaluation to determine detailed clinical and radiological features. RESULTS: All patients presented with high fever (> 38.0℃), with accompanying symptoms of cough, rhinorrhea, sore throat, myalgia and diarrhea, and increased C-reactive protein (CRP) values with no leukocytosis nor elevated erythrocyte sedimentation rate (ESR). All patients, including one patient who progressed into acute respiratory distress syndrome, were treated with oseltamivir phosphate and quickly recovered from their symptoms. Chest radiographs showed abnormalities of small nodules and lobar consolidation in only two out of 18 patients. However, six of 12 patients who underwent thin-section CT examinations showed abnormal findings for small ground-glass opacities (GGOs) in addition to poorly-defined nodules with upper lobe predominance. CONCLUSION: In a population of healthy young adults, elevated CRP with normal ESR and white blood cell levels combined with GGOs and nodules on thin-section CT scans may indicate early signs of infection by the pandemic H1N1 2009 influenza virus. I individuals live in close contact and have higher reported rates of viral infections (14) . Furthermore, there have been a few reports providing detailed descriptions of the radiologic features of the pandemic H1N1 2009 influenza virus infection such as in thin-section CT (11) (12) (13) .
In this article, we aim to describe detailed clinical characteristics and radiologic features of chest radiographs and thin-section CT findings of patients confirmed with the pandemic H1N1 2009 influenza virus infection occurring in a semi-closed setting.
This study was approved by the ethics committee of the Armed Forces Medical Command, which waived the requirement of informed patients consent for this retrospective study.
From July 18 to July 30, 2009, 18 out of 57 patients who presented with acute febrile respiratory illness (fever of 38� C or higher, and/or a cough or shortness of breath) (8) , were confirmed with the pandemic H1N1 2009 influenza virus infection in a tertiary military hospital in South Korea. We retrospectively reviewed the medical charts, as well as the laboratory and radiologic findings of these patients.
All patients were young male soldiers with a mean age of 20 years (age range: 19 to 22 years). Fifteen of the 18 patients confirmed with the pandemic H1N1 2009 influenza virus infection were from the same barrack, and they visited the emergency department due to acute febrile respiratory illness from July 18 to July 20, 2009. The other three patients were from different barracks and were admitted with suspected cases of rhabdomyolysis after a long distance march (n = 1), acute gastroenterocolitis (n = 1), and acute febrile respiratory illness with leukocytopenia and thrombocytopenia (n = 1). All three patients came into contact with one or more of the previously-mentioned patients lying in nearby beds in our hospital.
Among the 15 patients who presented to the emergency department with acute febrile respiratory illness, two patients had returned from a brief visit to their hometowns one week prior to the outbreak of acute febrile respiratory illness and were suspected as the source of the illness.
Specimens from a nasopharyngeal swab (n = 17), and tracheal aspirate (n = 1) were collected at the emergency department or wards when the 15 patients of the same barrack visited the emergency department, or when symptoms developed in the remaining three patients who came into contact with them. Reverse-transcriptasepolymerase-chain-reaction (RT-PCR) tests and additional viral cultures were performed in accordance with published guidelines from the WHO in all patients (8) .
A malaria smear and triple antibody tests of Hantann, leptospirosis, and Tsutsugamushi virus were also performed to rule out other potential causes of fever, and revealed no remarkable abnormalities.
Two authors retrospectively reviewed the clinical and laboratory findings. For the review of the clinical features, initial symptoms such as fever, cough, sputum, rhinorrhea, sore throat, myalgia, headache, dizziness, vomiting, and diarrhea were recorded. In turn, patient symptom progression while under treatment was also reviewed. For review of the laboratory data, the hematological analysis of the white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin level, hematocrit level, platelet count, neutrophil count and the biochemical analysis of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were recorded.
Chest radiographs and thin-section CT examinations were obtained from 18 and 12 patients, respectively, prior to the administration of antiviral agents. Two out of the 12 patients undergoing thin-section CTs received follow-up thin-section CT examinations a week after starting antiviral therapy.
All radiographic examinations were performed using digital radiographic equipment (TDR4600-F80; Gold Mountain Medical System, Seoul, Korea) and a standardized technique (110 kV, 2 mAs, and a 180-cm film-focus distance for the posteroanterior views). Thin-section CTs of the thorax were performed using a 16 channel CT scanner (BrightSpeed; GE Medical Systems, WI), with the following parameters: 1.25 mm slice thickness with a 2.5 mm gap, supine position, scanning during inspiration, 6 seconds scan time, 120kV, auto mA.
All chest radiographs and thin-section CT images were reviewed by three experienced chest radiologists (8, 10, and 16 years of experience in chest radiology, respectively) using a picture archiving and communication systems (PACS) viewer. Decisions were reached by consensus.
To review chest radiographs, the presence of nodular opacity, consolidation (focal, multifocal, or bilateral) or interstitial patterns, distribution of findings including central (inner two-thirds of lung) or peripheral (outer onethird of lung) and upper, middle or lower lung zones, atelectasis, mediastinal abnormalities, and the presence or absence of a pleural effusion were analyzed (15) .
Thin-section CT findings were interpreted using the descriptors proposed by the Fleischer Society Nomenclature Committee (16) . Ground-glass opacity (GGO) was defined as an increase in the lung parenchymal attenuation that did not obscure the underlying vascular architecture. A nodule was defined as round opacity, at least moderately well-defined, and no more than 3 cm in diameter. Consolidation was defined as increased lung attenuation that obscured the underlying vasculature (16) . The lesion size was described as small (diameter, less than 1 cm), medium (diameter, 1 to 3 cm), large (diameter, 3 cm to 50% of the segment), or segmental (50% to 100% of the segment). The sites were described by the name and number of involved segments of the lung. Location of the lesion was defined as peripheral if it was in the outer onethird of the lung; otherwise, it was defined as central (17) . Attention was also paid to the presence of other abnormalities such as pleural effusion, lymphadenopathy, cavitation, calcification, or septal thickening.
The clinical course and outcome of all patients are summarized in Figure 1 . In the analysis of 15 patients who visited the emergency department, the mean interval between symptom onset and emergency room visit was 1.7 days ± 1.5 days (range, 0 to 5 days). In the analysis of all 18 patients, the mean interval between symptom onset and the start of oseltamivir phosphate (Tamiflu � , Roche, Basel, Switzerland) administration was 3.9 ± 1.6 days (range, 1 to 7 days). In addition, the mean interval between symptom onset and symptom improvement was 4.7 ± 1.7 days (range, 2 to 8 days).
The clinical characteristics of the 18 patients in our study are summarized in Tables 1 and 2. All patients presented with high fever (> 38.0� C, median value and range; 38.5� C Among our study population, one patient presented with high fever, severe nocturnal cough, and blood tinged sputum, and the respiratory symptoms eventually progressed into acute respiratory distress syndrome (ARDS), which further led him to receive mechanical ventilation (case No. 12). A retrospective medical chart review revealed that this patient was a solider serving in a different unit from the majority of the other patients and had been in contact with a patient with pandemic H1N1 2009 influenza virus infection lying in a nearby bed in our hospital prior to symptom aggravation. The laboratory data of this patient showed leukocytopenia, thrombocytopenia, and an increased CK level. Other laboratory tests were within normal limits. He was initially treated with antibiotics such as macrolide and a 3rd generation cephalosporin under suspicion of bacterial pneumonia, which was never confirmed. However, his symptoms did not improve. phosphate and antibiotics, the patient's symptoms improved and was taken off mechanical ventilation five days after antiviral agent administration.
Sixteen out of 18 patients in our study population showed no abnormalities on chest radiographs. Moreover, only two patients in our study showed abnormal findings of a small central nodular opacity in the right upper lung zone in one ( Fig. 2A) , and consolidations in the left middle lung and both lower lungs in the other (Fig. 3A) on chest radiographs.
On thin-section CT images, six of 12 patients (50%) showed abnormal findings. Of these six patients, five (83%) had small GGOs; four patients had them in the upper lobe of the lung and one had a small GGO discovered in the lower lobe of the lung. Also, in four out of the five patients with small GGOs (80%), the lesions were localized in the periphery of the lung, and two of the five patients also showed bilateral involvement. For segmental involvement in terms of lesion size, three patients showed one segment, one patient showed two segments, one patient showed three segments, and one patient showed eight segments. Nodules were observed in all patients (100%), which were small in size with showing poorly defined margins (Fig. 2B) . In five patients (83%), the nodules were combined with GGOs in the same area. In addition, the nodule was a solitary finding in one patient (17%), which was located in the peripheral area of the right upper lobe. The patient who progressed into ARDS had bilateral lobar consolidations combined with GGOs, nodules, and bilateral pleural effusion (Fig. 3B) . One other patient showed right paratracheal lymphadenopathy. In our study, there was no evidence of calcification, mass, cavitation, or septal thickenings in any of the patients. The thin-section CT findings of the 12 patients are summarized in Table 3 .
In two patients, follow-up thin section CT images acquired one week after the start of antiviral therapy showed complete resolution or a remarkable decrease in size and opacity of the previously observed GGOs and nodules (Fig. 2C) .
With increasing concern over the spread of the pandemic H1N1 2009 influenza virus infection worldwide, detailed knowledge of both its clinical and radiologic features can assist the clinical practice and decision-making of physicians facing this emerging pandemic infectious disease. In addition, preparation for this pandemic infectious disease among institutionalized populations in semi-closed settings such as schools, dormitories, military barracks, or prisons should be made since this infection can spread very rapidly and may quickly become unmanageable without prompt and proper management in the early stages of disease spread. In this study, we attempt to describe one incidence of a military outbreak of the pandemic H1N1 2009 influenza virus with a focus on the clinical and radiological features of these patients, as well as the outcome of treatment.
We found common clinical features for all patients with the pandemic H1N1 2009 influenza virus infection in the present study, which included a high fever with accompanying symptoms of cough, rhinorrhea, sore throat, myalgia, or gastrointestinal symptoms such as vomiting or diarrhea. These findings are similar to the symptoms of other influenzas (8) and those found in previous studies that dealt with unspecified populations (3, 5, (9) (10) (11) (12) (13) .
Generally, laboratory tests, with the exception of serology and culture examinations, are not useful for the specific diagnosis of influenza. Leukocyte counts are variable according to stage; frequently low during the early stages of illness, and later become normal or slightly elevated (15) . In the present series, there were no patients with elevated WBC counts and 67% of patients showed lymphocytopenia. These findings are consistent with those found in previous studies that dealt with unspecified populations (3, 5) . One additional diagnostic laboratory feature we were able to find was an elevated CRP level while both the ESR and WBC counts remained within a normal range in all patients in our study with pandemic Korean J Radiol 11(4), Jul/Aug 2010 - (14) . Further detailed comparative studies with a larger number of patients would be required to fully explore this issue. Most patients in our study did not show a severe hospital course, although one patient's symptoms aggravated into ARDS and needed mechanical ventilation before laboratory tests revealed that he had the pandemic H1N1 2009 influenza virus infection. We considered the possibility that co-infection of the pandemic H1N1 2009 influenza virus with other unknown respiratory pathogens under the leukocytopenia status could have attributed to this symptom aggravation (9, 10), particularly as he was a soldier serving in a different unit from the majority of other patients. In addition, the symptom aggravation had occurred after he had been in contact with a patient infected with the pandemic H1N1 2009 influenza virus and there were no pandemic H1N1 2009 influenza virus cases in the unit the patient had been enrolled in. However, since his respiratory symptoms and high fever did not respond to other previous antibiotic treatment, there is still the possibility that the disease course in this case could have been solely due to a severe manifestation of the pandemic H1N1 2009 influenza virus infection.
In the present study, chest radiographs were abnormal in two of 18 patients with H1N1, which was less frequent than that of a previous report, which showed a 42% rate of chest radiograph abnormality (12) . This suggests that negative findings on chest radiographs cannot rule out the possibility of the pandemic H1N1 2009 influenza virus infection, particularly in its early stage or for an uncomplicated influenza infection (14) . Thin-section CT examination results revealed that abnormal findings were found in six out of 12 patients (50%). The most common CT abnormalities included small GGOs in five out of six patients and nodules in all six patients. However, in the previous studies (12, 13) , multifocal consolidations (two of seven patients and 50%) and GGOs (two of seven patients and 25%) were the most common CT findings of the pandemic H1N1 2009 influenza virus infection. The differences between the present study and previous studies could be attributed to the fact that radiological examinations in the present study were performed at the initial presentation, and probably the early stages of the disease; whereas in previous studies (12, 13) , radiological examinations were performed at various times after symptom onset.
In our study, there was one patient with bilateral lobar consolidations, which progressed into ARDS. Other investigators (3) also reported similar radiographic features in patients who progressed into ARDS. Although extensive consolidations in the lung might be severe manifestations of primary viral pneumonia from the pandemic H1N1 2009 influenza virus infection, there still remains the possibility of combined pneumonia caused by other pathogens or pulmonary manifestations of ARDS (14) .
The clinical courses and outcomes of patients in our study with the pandemic H1N1 2009 influenza virus infection were much better than those of previously reported cases (3, 4) . We attribute this to the patient population of our study. All of our patients were healthy young soldiers without any risk factors for complications. Furthermore, according to military rule, any problems concerning health must be notified to the medical department immediately, and thus, our patients may have been detected at an earlier stage of the disease course than would be found for the general population. In comparison with a previous report (3) that showed a high mortality rate in patients with the pandemic H1N1 2009 influenza virus infection, the interval between symptom onset and the emergency department visit (mean 1.7 days in 15 among 18 patients) and symptom onset and the start of antiviral agent therapy (mean 3.9 days) were shorter. In addition, detection and treatment were made more promptly. Therefore, we conclude that an early diagnosis and early antiviral management can improve the clinical course and outcome of the pandemic H1N1 2009 influenza virus infection. However, to understand the detailed relationship between symptom onset to admission or antiviral management and clinical courses or outcomes, further study, including a comparative analysis with a large case series, is warranted.
In our series, most patients (15 out of 18) were from the same barrack, a semi-closed setting; whereas, the three remaining patients from different barracks were thought to be infected from these 15 patients. All patients were healthy young males without any combined chronic disease. Clinical and laboratory results from this homogeneous study population can be helpful in understanding the clinical characteristics of the pandemic H1N1 2009 influenza virus infection in semi-closed settings and in institutionalized populations composed of a young healthy population, such as in schools, dormitories, prisons, or military units. Furthermore, this report demonstrated that a prompt diagnosis and treatment resulted in excellent outcomes and could provide as an example for the detection and management strategy for cluster outbreaks of this virus. However, there may be limitations in applying these results to the larger general population.
In conclusion, in a population of healthy young adults, elevated CRP with normal ESR and WBC levels combined with GGOs and nodules on thin-section CT scans may indicate early signs of infection by the pandemic H1N1 2009 influenza virus. A Clathrin Independent Macropinocytosis-Like Entry Mechanism Used by Bluetongue Virus-1 during Infection of BHK Cells Acid dependent infection of Hela and Vero cells by BTV-10 occurs from within early-endosomes following virus uptake by clathrin-mediated endocytosis (Forzan et al., 2007: J Virol 81: 4819–4827). Here we report that BTV-1 infection of BHK cells is also dependent on a low endosomal pH; however, virus entry and infection were not inhibited by dominant-negative mutants of Eps15, AP180 or the ‘aa’ splice variant of dynamin-2, which were shown to inhibit clathrin-mediated endocytosis. In addition, infection was not inhibited by depletion of cellular cholesterol, which suggests that virus entry is not mediated by a lipid-raft dependent process such as caveolae-mediated endocytosis. Although virus entry and infection were not inhibited by the dominant-negative dynamin-2 mutant, entry was inhibited by the general dynamin inhibitor, dynasore, indicating that virus entry is dynamin dependent. During entry, BTV-1 co-localised with LAMP-1 but not with transferrin, suggesting that virus is delivered to late-endosomal compartments without first passing through early-endosomes. BTV-1 entry and infection were inhibited by EIPA and cytochalasin-D, known macropinocytosis inhibitors, and during entry virus co-localised with dextran, a known marker for macropinocytosis/fluid-phase uptake. Our results extend earlier observations with BTV-10, and show that BTV-1 can infect BHK cells via an entry mechanism that is clathrin and cholesterol-independent, but requires dynamin, and shares certain characteristics in common with macropinocytosis. Virus internalisation is initiated by binding to specific receptors at the cell-surface and culminates in host-cell membrane penetration and delivery of the viral genome to the site of replication. For many viruses, membrane penetration follows virus endocytosis. Several different endocytosis pathways operate in mammalian cells which deliver cargoes to distinct intracellular locations [1, 2] . Clathrin-mediated endocytosis (CME) is the major pathway for receptor-dependent endocytosis in many cell types and is dependent on adaptor proteins, such as the AP-2 complex, the GTPase dynamin [2, 3] , and several other important accessory molecules including AP180 and Eps-15 [4, 5, 6] . CME delivers cargoes to acidic endosomes; initially to early-endosomes where they are sorted for transport to either late-endosomes/lysosomes for degradation or to recycling-endosomes for recycling back to the cell surface. Several acid-activated viruses are known to exploit the low pH within vesicles derived from the clathrin pathway, to trigger capsid un-coating and/or membrane penetration [7] .
Clathrin-independent endocytosis includes the caveolae pathway which is dependent on caveolin, lipid-rafts and cholesterol [1, 2] . Cargoes internalised by caveolae are delivered to neutral pH caveosomes [8, 9] . From caveosomes, cargoes can traffic to the ER or Golgi [10, 11, 12] . Like CME, caveolae-mediated uptake requires dynamin and can be inhibited by expression of dominant-negative (DN) mutants of dynamin-2 [10, 12] . A number of viruses use caveolae for infection [10, 13, 14, 15, 16] and much of our knowledge of caveolae comes from studies with simian virus-40 [10, 17, 18] . A number of caveolin-independent pathways also originate from lipid-rafts, and novel clathrin-and raft-independent processes have also been identified. These pathways have different requirements for cellular proteins such as dynamin, flotillin, small GTPases, and for cholesterol and other specific lipids [1, 2, 19, 20, 21, 22, 23, 24, 25] , and can also be exploited by viruses for infection [21, 26, 27, 28, 29, 30, 31] .
Macropinocytosis is the major form of endocytosis responsible for the majority of fluid-phase uptake for a number of cell types [32] . Macropinocytosis is characterised by actin-dependent reorganisations of the plasma membrane to form macropinosomes, morphologically heterogenic vesicles that lack coat structures. Macropinocytosis is constitutive in many cell types but in others requires activation by growth factors [32, 33, 34] and contributes significantly to antigen presentation by specialised antigen presenting cells [32, 34, 35] . Macropinocytosis is also exploited by a wide range of important animal pathogens for cell invasion and possibly the avoidance of immune surveillance [36] .
Here we have investigated the entry route used by bluetongue virus serotype 1 (BTV-1) to infect BHK cells. Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae which includes many important pathogens for man and animals [37] . BTV exists as at least 25 serotypes [38, 39] and is the etiological agent of bluetongue, a severe and economically important haemorrhagic disease of ruminants. Bluetongue is arthropod-borne and primarily transmitted between susceptible animal hosts by certain species of Culicoides biting midges [40] .
BTV is non-enveloped and consists of a 3 layered protein capsid (outer-capsid, core and sub-core) surrounding a genome of ten linear segments of double-stranded RNA. The outer capsid is formed from two viral proteins, VP2 and VP5, while the inner core-particle consists of a 'core-surface' layer composed of VP7, which surrounds a sub-core shell composed of VP3. VP2 interacts with host cellsurface receptors serving as a virus-attachment protein while VP5 is involved in host-cell membrane penetration [41, 42, 43] . The outer capsid proteins are removed during cell entry, and transcriptionally active core particles are released into the cytosol where virus replication occurs [44, 45] . However, the core-particle is also infectious in its own right demonstrating that VP7 can also mediate cell attachment and membrane penetration, possibly by a distinct mechanism [46] . Other viral encoded proteins (NS1, NS2 and NS3) are also produced in infected cells where they are involved in virus replication and release of progeny virus particles [47] .
Infection by BTV is acid-activated and requires the low pH within endosomes for disassembly of the outer viral capsid and membrane penetration, and BTV particles have been described inside endosomes which have the appearance of clathrin-coated vesicles [48] . Recent studies have concluded that entry and infection of Vero and Hela cells by BTV-10 occurs via CME, with capsid disassembly and membrane penetration within earlyendosomes [45] . Here we describe the use of pharmacological and DN inhibitors of endocytosis to investigate entry and infection of BHK cells by BTV-1. We found that the clathrin pathway is not the major entry route used by BTV-1 to infect BHK cells. Instead we found that the entry mechanism shares certain characteristics in common with macropinocytosis and appears to deliver virus directly to late endosomal compartments. These studies extend earlier observations and show that BTV joins an increasing number of viruses that can exploit multiple endocytosis pathways for infectious entry.
Baby Hamster Kidney (BHK)-21 cells (clone 13) were obtained from the European Cell Culture Collection and maintained at 37uC, 5% CO 2 , in Glasgow Minimum Essential Medium (GMEM) (Sigma) containing 10% foetal bovine serum (FBS) (Autogen Bioclear), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 5% tryptose phosphate broth solution (Sigma).
The South African reference strain of BTV-1 (IAH reference number RSArrrr/01, ICTVdb isolate accession number 41010B4F) was grown on BHK cells and gradient purified according to previously published methods [49] . Purified virus was stored at 4uC in the presence of sodium-N-lauroylsarcosine (0.1%) to prevent virus aggregation and was used for all experiments [46, 49] . Viruses were diluted immediately before use thereby reducing the concentration of sodium-N-lauroylsarcosine to ,0.01% which showed no cytotoxic effects.
Rabbit anti-BTV/NS2 (Orab 1) and the Guinea-pig anti-BTV/ VP5 (PM10) antibodies were produced at the Institute for Animal Health using recombinant NS2 and BTV-1 as immunogens respectively. The specificity of these antibodies was verified by western blotting against purified BTV-1 and a BTV-1 infected BHK cell lysate using uninfected cells as a negative control, and by showing a lack of cross-reactivity with uninfected BHK cells by confocal microscopy. Mab 9E10 (anti-c-myc) was from the Developmental Studies Hybridoma Bank (University of Iowa). The mouse monoclonal antibody (Mab 4A1) to Lysosomal Antigen -1 (LAMP-1) was from Jean Gruenberg (University of Geneva). Species specific, Alexa-Fluor conjugated secondary antibodies were from Invitrogen.
Methyl-b-cyclodextrin, filipin, cytochalasin-D, dynasore monohydrate, and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) were from Sigma. Ammonium chloride and concanamycin-A were from Fluka. Latrunculin-A, and Alexa-Fluor labelled human transferrin, dextran and phalloidin were from Invitrogen. Stock solutions of Methyl-b-cyclodextrin, transferrin and dextran were made in GMEM, and ammonium chloride in sterile water. Stock solutions of phalloidin were made in methanol. Stock solutions of other inhibitors were made in dimethyl sulfoxide (DMSO). Where appropriate, an equivalent dilution of DMSO (or methanol) was included in the mock treatment. Cells were seeded on glass coverslips (BDH) in antibiotic-free cell-culture medium and transfected when ,60% confluent. Cells were transfected in Optimem (Invitrogen) using a ratio of 1 mg plasmid DNA to 1 ml Lipofectamine 2000 (Invitrogen) according to the manufacturers guidelines. Cells were incubated at 37uC, 5% CO 2 for 4 h when the transfection medium was replaced with antibiotic-free cell-culture medium. Cells were used for experiments at 12 h post-transfection. Cells expressing a transgene were identified by confocal microscopy. Transfection efficiencies (determined by GFP or c-myc expression) ranged from 30-43%.
For entry experiments with transfected cells, BTV-1 (13 mg/ml in GMEM) was bound to the cells at 4uC for 40 minutes. Unbound virus was removed by washing and the cells placed at 37uC (in GMEM) to initiate entry. Where specified, inhibitors were added to the cells for 0.5 h before the virus binding step and remained present throughout the assay. All entry experiments were ended at the times indicated by the addition of 4% paraformaldehyde (PFM), with the exception of those involving transferrin which were ended using 4% PFM with 0.25% glutaraldehyde. Input virus was detected using the anti-VP5 antibody, PM10. For quantification of virus uptake, cells with cytoplasmic labelling for BTV were scored as positive. In all experiments, ,94% of the control cells showed virus uptake.
For infections with transfected cells, the cells were inoculated with BTV-1 at a multiplicity of infection (m.o.i.) of 1 infectious virus particle per cell, in serum-free medium for 1 h at 37uC. Unbound virus was removed by washing with GMEM and the cells returned to 37uC in cell culture medium with reduced (2%) FBS.
Inhibitors (Methyl-b-cyclodextrin, cytochalasin-D or latrunculin-A) were added to cells for 0.5 h before infection. The cells were then inoculated with BTV-1 (as described above) also in the presence of an inhibitor. Mock-treated cells were infected in the absence of an inhibitor. After the 1 h incubation with virus, the cells were washed with GMEM to remove unbound virus and returned to 37uC in cell culture medium with reduced (2%) FBS, and supplemented further with 25 mM ammonium chloride. To control for any effects on intracellular virus replication the inhibitors were also added to other cells immediately after the virus inoculums were removed and coincident with the addition of ammonium chloride.
In the experiment shown in figure 2 , BTV-1 (m.o.i. = 1) was bound to the cells at 4uC for 40 minutes. The cells were then washed to remove unbound virus and placed at 37uC (in GMEM) to initiate infection. This allowed the addition of inhibitor to be synchronized with the start of infection. Concanamycin-A was added to the cells at the noted times. When used as a pretreatment, concanamycin-A was added for 0.5 h at 37uC before the addition of virus and remained present only during the virus binding step. Concanamycin-A was also added to other cells, either coincident with the start of infection (i.e. on shifting the cells to 37uC) or at various times after infection was initiated.
All infection experiments were ended after 12 h by the addition of cold 4% PFM for 40 minutes. Infected cells were identified using confocal microscopy and the anti-NS2 antibody, Orab1. For quantification of infection, cells with cytoplasmic labelling for BTV NS2 were scored as positive.
The ability of cells to internalize through the clathrin pathway was determined by observing transferrin uptake by confocal microscopy. Cells were serum starved for 0.5 h prior to the addition of Alexa-568 labelled transferrin (25 mg/ml) in GMEM at 37uC for the times indicated in the results. For quantification of transferrin uptake, cells with cytoplasmic labelling for Alexa-568 transferrin were scored as positive. Alexa-568 labelled dextran (5 mg/ml) was added to the cells as described for transferrin. In all experiments, .98% of the control cells showed transferrin or dextran uptake.
After cell fixation with PFM, the cells were washed with PBS pH 7.5 (Sigma) and permeablized with 0.1% Triton X-100 (Sigma) in Phosphate Buffered Saline (PBS) for 20 minutes. When using Mab 4A1 to identify LAMP-1 positive compartments, the cells were permeablized with 0.5% Saponin (in place of Triton X-100) for 15 minutes and 0.1% Saponin was included in all subsequent steps. Non-specific binding sites were blocked with block buffer (100 ml Tris-buffered saline supplemented with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10% Normal Goat Serum (Harlan Lab Sera) and 1% Teleostean Gelatin [Sigma]) for 0.5 h. The cells were washed with PBS and incubated with primary antibodies in block buffer for 1 h, washed again with PBS, and incubated with the appropriate species specific, Alexa-Fluor conjugated secondary antibody (also in block buffer) for 1 h. The cells were washed and the nuclei labelled with 49, 69-Diamidino-2-Phenylindole (DAPI) (Sigma) in H 2 O for 10 minutes and then washed with H 2 O. The coverslips were mounted in Vectashield mounting medium for fluorescence (Vector Laboratories) and sealed with clear nail varnish.
Transfected cells expressing dynamin or Eps15 constructs were identified by the GFP fusion tag. Transfected cells expressing AP180C were identified using the anti-c-myc antibody (9E10) and a goat, anti-mouse Alexa-488 conjugated secondary antibody. LAMP-1 positive compartments were labelled using Mab 4A1.
All data were collected sequentially to eliminate cross talk between fluorescent dyes on a Leica SP2 Confocal Scanning Laser Microscope. For experiments that involved dual labelling of virus and cellular compartments, the specificity of the secondary antibodies for the target primary antibody was confirmed by showing a lack of cross-reactivity with non-target primary antibodies. The data are shown as single optical sections through the middle of the cell.
Cellular cholesterol was visualised at 430 nm immediately after labelling with 125 mg/ml filipin diluted in PBS for 15 minutes at room temperature [50] . To label for actin filaments, cells were fixed and permeabilized (as above) and incubated with Alexa-488 labelled phalloidin diluted in block buffer for 10 minutes at room temperature.
For experiments with DN-dynamin-2 or DN-Eps15, uptake of transferrin and virus was normalised to the results obtained for cells expressing the control (wt dynamin-2 or the Eps15 control respectively). Over-expression of wt AP180 inhibits CME and cannot be used as a control for AP180C [6] . Therefore, for experiments with AP180C, transferrin or virus uptake was normalised to the results obtained for cells of the non-expressing population (i.e. the non-green cells). Similarly, for DN-dynamin-2 or AP180C, infection was normalised to the level of infection of cells expressing wt dynamin-2 or cells of the non-expressing population, respectively. The level of infection of inhibitor-treated cells was normalised to the levels of infection of the mock-treated controls. Student's t test was used to determine statistical significance.
It has been reported that BTV-10 infects Hela and Vero cells using CME for entry [45] . Here we have investigated the involvement of the clathrin pathway in BTV-1 entry and infection of BHK cells using deletion mutants of AP180 (AP180C) and Eps15 (Eps15-Ed95/295; from here known as DN-Esp15) which act as DN inhibitors of CME and block uptake of transferrin [5, 6, 51] . BHK cells were transiently transfected to express AP180C-c-myc, GFP-DN-Eps15, or a control GFP-Eps15 (D3D2; from here known as 'Esp15 control') that does not interfere with the clathrin pathway [5, 51] . At 12 h posttransfection, the cells were incubated with Alexa-568 labelled transferrin for 15 minutes or with BTV-1 for 0.5 h. The cells were then fixed and processed for confocal microscopy (Fig. 1) . Virus was detected using PM10 (anti-VP5) and an Alexa-568 conjugated secondary antibody. Cells expressing Eps15 proteins or AP180C were identified by the GFP or c-myc fusion tags respectively (see methods) and are shown in green on figure 1. Cells were scored for transferrin or BTV uptake as described in the methods. For each DN protein, uptake of transferrin or BTV was normalised to the results obtained for the controls (see methods). In cells expressing GFP-DN-Eps15 the frequency of transferrin uptake was reduced by 53% when compared to cells expressing the Eps15 control (Fig. 1A , B, E, F and O) indicating a block to clathrin-mediated endocytosis. Similarly, the frequency of transferrin uptake was reduced by 82% in cells expressing AP180C when compared to cells of the non-expressing population (Fig. 1I, J and O) . In contrast, expression of DN-Eps15 (Fig. 1C , D, G, H and O) or AP180C (Fig. 1K , L and O) did not significantly inhibit virus uptake. In virtually all cells expressing the DN proteins the pattern of virus labelling was indistinguishable from cells expressing the Esp15 control or cells that were not expressing a transgene. These results show that under conditions where clathrin-mediated uptake of transferrin is inhibited BTV-1 is taken up normally by BHK cells.
The above experiments required using a relatively high BTV-1 concentration (13 mg/ml), to allow virus detection during entry. This raises the possibility that the virus may have been forced to enter the cells via an endocytic pathway that is not normally used, or is non-productive for infection. Therefore we also investigated the effect of AP180C expression on BTV-1 infection at a low m.o.i., to encourage virus uptake by the most efficient route. At 12 h post-transfection, the cells were incubated with BTV-1 (m.o.i. = 1) for 1 h at 37uC. The cells were then washed to remove excess virus and infection continued for a further 11 h when the cells were fixed and processed for confocal microscopy. Cells expressing AP180C were identified using expression of the c-myc tag as described above. Infected cells were identified using a rabbit antibody (Orab1) that binds the viral NS2 protein (a marker of virus replication) and an Alexa-568 conjugated secondary antibody. Intense areas of NS2 labelling were detected within infected cells (Fig. 1M and N) . Cells expressing AP180C were scored for infection (indicated by labelling for NS2) and the results normalised to the results obtained for the non-expressing cell population. In the cells expressing AP180C the frequency of infection was similar to that of the non-expressing cell population, indicating that AP180C does not inhibit infection (Fig. 1O) . These results show that virus uptake by cells expressing AP180C leads to infection and support the conclusion that CME is not the major pathway used by BTV-1 for entry into BHK cells.
BTV infection is known to be dependent on the low pH within endosomes [45] . Concanamycin-A is a potent and specific inhibitor of the vacuolar proton ATPase and is commonly used to raise endosomal pH [52] . Pre-treatment of BHK cells with concanamycin-A inhibited BTV-1 infection ( Fig. 2A and 2B ) confirming the requirement for active endosomal acidification. Similarly, when concanamycin-A was added coincident with the start of infection (i.e. at time 0 on figure 2C) a strong inhibitory effect was also seen. When added after the start of infection, concanamycin-A also had an inhibitory effect on infection. When added 2 h after infection was initiated, the frequency of infection was reduced by ,50% when compared to mock treated cells (Fig. 2C) . Near identical results were obtained using ammonium chloride in place of concanamycin-A (data not shown). These results suggest that BTV-1 is delivered to acidic endosomes much slower than expected for uptake by CME [53] .
Raising endosomal pH may prevent receptor recycling, which could deplete the cell surfaces of attachment receptors and thereby reduce infection. However, the pattern of virus uptake by concanamycin-A treated cells (detected at 0.5 h post-uptake by confocal microscopy using the PM10 antibody) was indistinguishable from mock-treated cells (data not shown), indicating that the inhibitory effect of concanamycin-A on infection was not due to a failure of the virus to be internalised.
It has recently been shown that cargo internalised via caveolae can be delivered to acidic early-endosomes [17] . Caveolaemediated endocytosis is dependent on the integrity of lipid-rafts, and like other raft mediated endocytic pathways can be inhibited by cholesterol depletion [8, 20] . The effect of cholesterol depletion on BTV-1 infection was investigated using methyl-b-cyclodextrin (MbCD) which binds and extracts cholesterol from the plasma membrane. BHK cells were mock-treated or treated with MbCD for 0.5 h at 37uC. The cells were then incubated with filipin which binds cholesterol and can be detected by fluorescence microscopy. Filipin fluorescence was detected at the plasma membrane and within the cytosol of mock-treated cells (Fig. 3A) . In contrast, although fluorescence was detected within the cytosol, the intense plasma membrane fluorescence generated by filipin binding was greatly reduced by MbCD, indicating extensive and selective cholesterol depletion from the cell surfaces (Fig. 3B) .
MbCD treated cells were also infected with BTV-1 (m.o.i. = 1) for 1 h in the presence of the drug (see methods). Mock-treated cells were infected in parallel. After washing the cells to remove excess virus and drug, infection was continued for a further 11 h. At the end of infection, the cells were processed for confocal microscopy using NS2 to detect infected cells. Figure 3C shows that pre-treatment of cells with MbCD did not inhibit infection when compared to mock-treated cells. These results show that the cell entry mechanism used by BTV-1 to infect BHK cells does not require cholesterol and is, therefore unlikely to be mediated by caveolae or other lipid-raft mediated endocytic pathways.
The involvement of dynamin in BTV-1 entry and infection was investigated using a DN mutant of the 'aa' splice variant of dynamin-2 [3, 54, 55] . BHK cells were transiently transfected to express GFP-wt 'aa' dynamin-2, or GFP-DN 'aa' dynamin-2. At 12 h post-transfection, the cells were incubated with Alexa-568 labelled transferrin for 15 minutes or with BTV-1 for 0.5 h. The cells were then fixed and processed for confocal microscopy (Fig. 4) . Virus was detected using PM10 (anti-VP5) and an Alexa-568 conjugated secondary antibody. Cells expressing wt or DNdynamin-2 were identified by the GFP fusion tag and are shown in green. The cells expressing GFP-DN-dynamin-2 showed transferrin uptake at a lower frequency than cells expressing the wt construct ( Fig. 4A-D and 4I) indicating a block to clathrinmediated endocytosis. In contrast, BTV-1 was taken up normally by cells expressing DN-dynamin-2 ( Fig. 4E-H and 4I ).
At 12 h post-transfection, cells were also infected with BTV-1 (m.o.i. = 1) for 1 h at 37uC. The cells were then washed to remove excess virus and infection continued for a further 11 h when the cells were fixed and processed for confocal microscopy. Infected cells were identified using a rabbit antibody (Orab1) as described above. The frequency of infection of the cells expressing DNdynamin-2 was normalised to the level of infection of the cells expressing the wt protein. Figure 4I shows that expression of DNdynamin-2 does not inhibit BTV-1 infection. These results are consistent with those obtained using DN-Esp15 and AP180C and support our conclusion that BTV-1 can enter BHK cells using a clathrin-independent pathway. Furthermore, as the 'aa' splice variant of DN-dynamin-2 also inhibits caveolae-mediated endocytosis, the above results also support our conclusion that caveolae are unlikely to be required for virus entry and infection.
To further investigate the role of dynamin in BTV-1 infection, BHK cells were treated with the general dynamin inhibitor, dynasore [56] . Mock-treated and dynasore-treated cells were incubated with BTV-1, Alexa-568 labelled transferrin or Alexa-568 labelled dextran. Figure 5A -F shows representative cells and that dynasore effectively inhibited uptake of all three ligands. Virtually all of the drug treated cells (n.300 cells per ligand) showed no uptake of virus, transferrin or dextran.
We also investigated the effects of dynasore on BTV-1 infection. However, these experiments were inconclusive as ,6 h after the drug was removed, the cells detached from the coverslips indicating a delayed cytotoxic effect. Nevertheless, the observation that dynasore inhibits entry of BTV-1 into BHK cells shows that the entry mechanism is dynamin dependent.
A number of endocytic processes are dependent on functional actin dynamics [2] . Therefore we determined if BTV-1 entry into BHK cells requires actin using cytochalasin-D which disrupts actin filaments. Cells were mock-treated, or treated with the drug and successful disruption of actin filaments confirmed by confocal microscopy after labelling the cells with Alexa-488 labelled phalloidin (data not shown). Mock or drug-treated cells were allowed to internalise BTV-1 (for 0.5 h) in the presence of the drug. The cells were then fixed and processed for confocal microscopy using PM10 to detect input virus as described above. Figure 6A and 6B show representative cells and that cytochalasin-D effectively blocked entry of BTV. Virtually all of the drugtreated cells (n.200 cells) showed a pattern of virus labelling as shown on the figure. In contrast, figure 6C and 6D show that cytochalasin-D did not inhibit uptake of 568-Aexa labelled transferrin, or 568-Alexa labelled dextran. These results show that BTV-1 uptake by BHK cells is actin-dependent and confirms that BTV-1 and transferrin enter cells via different mechanisms.
Cytochalasin-D treated cells were also infected with BTV-1 for 1 h in the presence of the drug (see methods). Mock-treated cells were infected in parallel. After the 1 h incubation with virus the cells were washed to remove excess unbound virus and inhibitor, and infection continued for a further 11 h. At the end of infection the cells were processed for confocal microscopy using NS2 to detect infected cells. The effects of cytochalasin-D on actin filaments are reversible and it is possible that following drug washout, virus that remained bound to the outsides of the cells (as a result of actin disruption) may cause an infection. Therefore ammonium chloride was added to the culture medium immediately after the virus inoculums and cytochalasin-D was removed. Ammonium chloride is a membrane permeable weak base that rapidly raises endosomal pH [57] and was added to prevent further infection events by virus that have not yet entered the cell and undergone acid-induced membrane penetration. Under these conditions infection can only occur during the period when cytochalasin-D was present. To control for possible effects on intracellular virus replication, cytochalasin-D was also added to other cells immediately after the virus inoculums were removed and co-incident with the addition of ammonium chloride. Under these conditions, cytochalasin-D can only have an inhibitory effect on infection by interfering with post-entry virus replication. Figure 6E shows that pre-treatment of BHK cells with cytochalasin-D inhibited BTV-1 infection. In contrast, when added after the virus internalisation step no inhibitory effect was seen (Fig. 6E) . Similar results were obtained using latrunculin-A in place of cytochalasin-D (Fig. 6E) . These results confirm that BTV-1 infectious entry into BHK cells is actin dependent.
Macropinocytosis is actin dependent and is increasingly recognised as an important cell entry route for a number of viruses [36] . Macropinocytosis is also dependent on the amiloridesensitive Na + /H + exchangers [36] and is inhibited by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). EIPA has previously been shown to inhibit macropinocytosis without affecting other endocytic pathways such as CME [58] . Figure 5 shows representative cells and that EIPA effectively inhibited uptake of dextran (Fig. 5I) and BTV-1 (Fig. 5G) . Virtually all of the drug treated cells showed no uptake of dextran or virus (n.300 cells counted for each ligand). In contrast, EIPA did not inhibit uptake of transferrin (Fig. 5H ) as virtually all cells showed transferrin uptake; (n.300 cells counted). These results also suggest that BTV-1 entry may be mediated by macropinocytosis or a macropinocytosis-like mechanism. In addition, as EIPA blocked uptake of BTV-1 but not transferrin, these observations strengthen our conclusion that BTV-1 entry is not mediated by the clathrin pathway.
Although commonly used as a marker to indicate uptake by macropinocytosis, dextran is likely to enter cells by other pathways that participate in fluid-phase uptake. Certainly, our results with cytochalasin-D show that dextran can enter BHK cells using an actin-independent pathway, as although actin filaments were disrupted by cytochalasin-D, dextran uptake appeared to be unaffected (see Fig. 6D ). Nevertheless, figure 7 shows that at 0.5 h and 2 h post uptake, BTV-1 was co-localised with co-internalised Alexa-568 labelled dextran, which also suggest that BTV-1 may use macropinocytosis or a macropinocytosis-like mechanism to enter BHK cells.
The results described above show that BTV-1 infection of BHK cells occurs via a clathrin-independent entry mechanism that shares certain characteristics with macropinocytosis. Macropinosomes can fuse with lysosomes or recycle their contents back to the extracellular space, most likely via early-and recycling-endosomes [32, 59, 60] . Therefore, we investigated whether BTV-1 was delivered to these compartments during entry. BTV-1 was internalised for 15, 30, 60, 90 and 120 minutes before the cells were fixed and processed for confocal microscopy using PM10 (anti-VP5) to detect internalised virus. Early-and recyclingendosomes were labelled by incubating the cells with Alexa-568 labelled transferrin during the final 15 minutes of virus uptake (i.e. for 15 minutes before the cells were fixed). Alternatively, other cells were double labelled for virus (as above) and LAMP-1 (a marker for late-endosomes and lysosomes) using Mab 4A1. Figure 8 , shows representative cells from these experiments. After 15 or 30 minutes of virus uptake, little or no virus colocalisation with transferrin was observed (Fig. 8A-F) . Similarly, at the other times investigated, virus co-localisation with transferrin was not observed (data not shown). These results show that during the first 2 h of entry, BTV-1 does not appear to enter early-or recycling-endosomes, supporting the conclusion that CME is not required for BTV-1 infection. Similarly, at 15 minutes postuptake, virus was not co-localised with LAMP-1 (data not shown). In contrast, at 0.5 h and 2 h post-entry BTV-1 was observed co-localised with LAMP -1 ( Fig. 8G-L) . At 0.5 and 2 h post uptake, ,34% and ,54% of the virus was judged to be colocalised with LAMP-1. These results are consistent with an uptake mechanism that delivers BTV-1 to LAMP-1 positive compartments (late-endosomes/lysosomes) without the need to first pass through early-endosomes.
Infection by Bluetongue virus is acid activated and can be inhibited by raising endosomal pH. Consistent with these observations, Forzan et al [45] concluded that BTV-10 infection of Vero and Hela cells is mediated by CME, with capsid disassembly and membrane penetration occurring from within early-endosomes. In this report, we have confirmed that active endosomal acidification is required for BTV-1 infection of BHK cells. However, in contrast to previous studies we found that the clathrin pathway is not the major entry route used by BTV-1 to infect BHK cells. This conclusion is based on a number of observations, including; (1) although a low endosomal pH was shown to be essential for infection, the kinetics of BTV-1 delivery to acidic compartments was slower than expected for uptake by the clathrin pathway [53] ; (2) during entry, BTV-1 did not colocalise with transferrin, a commonly used marker for CME; and (3) cell-entry and infection was not inhibited by expression of three different DN inhibitors of CME (AP180C, DN-Eps15 and DNdynamin-2). Furthermore, both EIPA and cytochalasin-D were shown to inhibit uptake of BTV-1 but not uptake of transferrin indicating that BTV-1 and transferrin are internalised by different endocytic pathways.
Our observations suggest that during entry into BHK cells, BTV-1 is delivered to acidic compartments via a clathrin independent route. One possible route is uptake via caveolae as they can undergo fusion with early-endosomes [17] . However, the failure of DN-dynamin to inhibit BTV-1 entry and infection of BHK cells suggests that virus uptake is independent of caveolae. This conclusion was further supported by showing that BTV-1 infection was not inhibited by cholesterol depletion, which disrupts several lipid-raft dependent processes including caveolae-mediated endocytosis. Another clathrin-independent uptake route that leads to acidic endosomes is the CLIC/GEEC (CLathrin Independent Carriers/GPI-AP Enriched Endocytic Compartments) pathway [61, 62] . However, in contrast to the entry mechanism used by BTV-1 to infect BHK cells, the CLIC/GEEC pathway is highly sensitive to cholesterol depletion [62] and is, therefore, unlikely to mediate BTV-1 uptake. Recent studies have shown that vesicles derived from a number of other clathrin-independent pathways can fuse with early-or late-endosomes [22, 24, 63, 64] , and that viruses can use such mechanisms for infectious entry [15, 16, 24] . For example, Lymphocytic Choriomeningitis Virus (LCMV) and Kaposi's Sarcoma associated Herpesvirus (KSHV) are internalised by clathrin-independent mechanisms that result in virus delivery to late-endosomes without first passing through early-endosomes [65, 66, 67] . However, the uptake pathway used by LCMV appears distinct from that described here for BTV-1 entry into BHK cells in several ways including that LCMV uptake and infection are not blocked by inhibitors of macropinocytosis. The entry route used by KSHV appears more similar to that described here for BTV-1 since it also shares characteristics of macropinocytosis; however, unlike the BTV-1 BHK cell entry mechanism, entry of KSHV is not inhibited by dynasore and is therefore dynamin independent.
On the basis of virus co-localisation with dextran, and the sensitivity to EIPA or actin disruption, the BTV-1 BHK cell entry route appears to be most closely related to macropinocytosis [36] . A number of viruses including vaccinia virus [68, 69, 70] , coxsackievirus B3 [71] , HIV [72] , KSHV [66] , echovirus 11 [73, 74] , human rhinovirus-14 [75] and Adenovirus type 3 [76] have been shown to exploit macropinocytosis, or macropinocytosis-like endocytic pathways for cell-entry and infection. Macropinosomes can deliver their contents to late endosomal compartments (late-endosomes or lysosomes) without the need for early-endosomes [32, 59] . Similarly, the route described here for BTV-1 entry into BHK cells also appears to by-pass early-endosomes and delivers virus to late endosomal compartments. These observations suggest that infection (i.e. acid induced membrane penetration) might occur from within late-endosomes or lysosomes. However, we cannot yet be certain that these compartments are the site of infection as the intermediate steps of virus trafficking between the plasma membrane and late-endosomal compartments are not known, and it is possible that virus could be exposed to a low pH on transit.
The involvement of dynamin in macropinocytosis is not clear and studies investigating its role have given conflicting results [55, 58, 69, 70, 77, 78] . Similarly, viruses that use macropinocytosis for infection display different sensitivities to dynamin inhibition [69, 70] . Our studies showed that a DN mutant of the 'aa' splice variant of dynamin-2 does not inhibit BTV-1 uptake or infection of BHK cells. This DN mutant was shown to inhibit uptake of transferrin (see Fig. 4 ) but not dextran (data not shown), indicating that macropinocytosis/fluid-phase uptake, but not CME is active in BHK cells expressing this mutant. These observations are consistent with recent studies by Cao et al., (2007) which show that different splice variants of dynamin-2 regulate different endocytosis pathways, and that expression of a DN mutant of the 'aa' splice variant of dynamin-2 resulted in attenuation of transferrin, but not dextran uptake [55] . In contrast, BTV-1 uptake was inhibited by the dynamin inhibitor, dynasore. Since it is likely that dynasore inhibits all forms of dynamin, this observation suggests that the mechanism used by BTV-1 to enter BHK cells is dynamin dependent but relies on a form of dynamin that cannot be inhibited by DN mutants of the 'aa' splice variant of dynamin-2.
In conclusion, we have described here a pathway for BTV-1 uptake and infection of BHK cells that is independent of clathrin and cholesterol requires dynamin, appears to deliver virus directly to late-endosomes/lysosomes without first passing through earlyendosomes, and shares characteristics with macropinocytosis. Recently, Forzan et al., [45] reported that BTV-10 infection of Vero and Hela cells is mediated by CME with acid-induced infection occurring within early-endosomes. Hence, BTV joins an increasing number of viruses that appears able to use more than one endocytic pathway to initiate infection [21, 66, 79, 80, 81, 82] . BTV infects a wide variety of cell types in its mammalian hosts including endothelial cells [37] , mononuclear phagocytes [83] , cd T cells [84] , dendritic cells [85] , and a variety of leukocytes [37, 86] . It is likely that this broad tropism, and the ability of BTV to replicate in such evolutionary distant hosts results, in part from an ability to use multiple entry routes to initiate infection. Moreover, BTV can exist in at least three different forms that are all considered to be infectious. These include intact virus particles, infectious sub-viral particles, and virus-cores [46] . These different particle types have different surface components, and may therefore also use different entry mechanisms for infection. However, the entry route used by BTV to infect its natural target cells and the relevance of these pathways to pathogenesis in the animal and insect hosts will require further study. Assessing the human immune system through blood transcriptomics Blood is the pipeline of the immune system. Assessing changes in transcript abundance in blood on a genome-wide scale affords a comprehensive view of the status of the immune system in health and disease. This review summarizes the work that has used this approach to identify therapeutic targets and biomarker signatures in the field of autoimmunity and infectious disease. Recent technological and methodological advances that will carry the blood transcriptome research field forward are also discussed. outgoing lymphatic vessels, the cells again reach the bloodstream to be transported to tissues throughout the body. Upon patrolling these tissues, they gradually drift back into the lymphatic system to re-enter the blood and begin the cycle all over again. Th e complex patterns of recirculation depend on the state of cell activation, the adhesion molecules expressed by immune and endothelial cells, and the presence of chemotactic molecules that selectively attract particular populations of blood cells. Circulating immune cells are, in addition, exposed to factors that are released systemically.
A wide range of molecular and cellular profi ling assays is currently available for the study of the human immune system ( Figure 2) . Th e level of sophistication of instruments such as polychromatic fl ow cytometers, one of the immunologist's favorite tools, has increased over the past few years. Major technological breakthroughs have also occurred in the fi elds of genomics and proteomics, thus creating today a unique opportunity for the study of human beings in health and disease where inherent heterogeneity dictates that large collections of samples be analyzed. Among the high-throughput molecular profi ling technologies available today, genomic approaches are the most scalable, have the most breadth and robustness, and therefore are best suited for the study of human populations.
Th e human genome can be investigated from two diff erent angles that consist of either determining its make up or measuring its output. Sequence variation can be detected using, for instance, single nucleotide polymorphism (SNP) chips, which permit the identifi cation of common polymorphisms or rare mutations associated with diseases. Hundreds of thousands of SNPs can be typed using these platforms, yielding a genome-wide, hypothesis-free scan of genetic associations for a given phenotype of interest. Many such genome-wide association studies (often referred to as GWAS) have been published in recent years, a number of them investigating the genetic underpinning of immune-related diseases [1] . Notably, such studies have been useful to pinpoint genes and pathways that may be involved in the pathogenesis of Figure 1 . Blood is the pipeline of the immune system. Transcriptional profi ling in the blood consists of measuring RNA abundance in circulating nucleated cells. Changes in transcript abundance can result from exposure to host or pathogen-derived immunogenic factors (for example, pathogen-derived molecular patterns activating specialized pattern recognition receptors expressed at the surface of leukocytes) and/or changes in relative cellular composition (for example, infl ux of immature neutrophils occurring in response to bacterial infection). The main blood leukocyte populations circulating in the blood are represented in this fi gure. Each cell type has a specialized function. Eosinophils, basophils and neutrophils are innate immune eff ectors playing a key role in defense against pathogens. T lymphocytes are the mediators of the adaptive cellular immune response. Antibody producing B lymphocytes (plasma cells) are key eff ectors of the humoral immune response. Monocytes, dendritic cells and B lymphocytes present antigens to T lymphocytes and play a central role in the development of the adaptive immune response. Blood leukocytes can be exposed in the circulation to factors released systemically from tissues where pathogenic processes take place. In addition, leukocytes will cross the endothelial barrier to reach local sites of infl ammation. Dendritic cells exposed to infl ammatory factors in tissues will be transported via the lymphatic system and reach lymph nodes via the aff erent lymphatic vessels. These dendritic cells will encounter naïve T cells that are transported to the lymph node via high endothelial venules. 'Educated' T cells will then exit the lymph node via eff erent lymph vessels that collect in the thoracic lymph duct, which in turn connects to the subclavian vein, at which point these T cells rejoin the blood circulation. [2] . Associations between common genetic variants and resistance to infection have also been reported [3, 4] . However, parameters measured by this approach are determined by heredity and will not change throughout the life of an individual. Th is is in contrast to transcript abundance, which is the parameter measured by the second genome-wide profi ling approach. Transcriptional activity is largely dependent on environmental factors and, as a result, RNA abundance will change dynamically over time. For instance, sets of transcripts may be induced in response to an infectious challenge and return to baseline levels following pathogen clearance. Dynamic changes in the cellular make up of a tissue will also eff ect changes in transcript abundance that will be measured on a genome-wide scale.
Transcriptional profi les have been obtained from many human tissues -including, for instance, the skin [5, 6] , muscle [7] , liver [8, 9] , kidney [10, 11] or brain [12] -but the status of the immune system can be best monitored by profi ling transcript abundance in blood. Indeed, profi ling transcript abundance in blood provides a 'snap shot' of the complex immune networks that operate throughout the entire body. However, while this has proven to be a valid approach to fi nding clues about The number of high-throughput molecular and cellular profi ling tools that can be used to profi le the human immune system is increasing rapidly. Proteomic assays are used to determine antibody specifi city or measure changes in serum levels of cytokines or chemokines using multiplex assays. Cellular profi ling assays are used to phenotype immune cells based on intracellular or extracellular markers using polychromatic fl ow cytometry. In vitro cellular assays can measure innate or antigen-specifi c responsiveness in cells exposed to immunogenic factors. Genomic approaches consist of measuring abundance of cellular RNA and also microRNAs that are present in cells or in the serum. Other genomic approaches consist of determining gene sequence and function (for example, genome-wide association studies, RNA interference screens, exome sequencing). patho genesis as well as to identifying potential biomarkers [13] [14] [15] [16] , a number of challenges and limitations exist. Data interpretation is one of them. Firstly, the volume of data generated from such studies can be overwhelming, and it is necessary to integrate information from a multitude of sources (study design, quality control data, sample information, and importantly clinical information) in order for the results to be interpretable. Secondly, the changes in transcript abundance observed in complex tissues such as blood can be caused not only by regulation of gene transcriptional activity but also by relative changes in abundance of cell populations expressing transcripts at constant levels. Th irdly, in addition to pathogenic processes, a number of factors may aff ect blood transcript abundance and confound the analysis. Medications and co-morbidities are two such factors that often restrict patient selection and complicate data interpretation. Th is review will discuss some of the strategies recently developed that will address some of these limitations.
Real-time PCR technology is currently considered the gold standard for the analysis of gene expression. However, it can be used to measure abundance of only a limited number of transcripts. Introduced over 10 years ago, DNA microarrays are now in routine use and can measure transcript abundance on a genome-wide scale. Th is technology relies on dense arrays of oligonucleotide probes that will capture complementary sequences present in biological samples at various concentrations. Th e probes can be deposited on a solid surface (printed microarrays), synthesized in situ (Aff ymetrix GeneChips), or bound to glass beads lodged into wells etched in the surface of a glass slide (Illumina BeadArrays). Th e labeled material captured by the microarray is imaged and relative abundance determined based on the strength of the signal produced by each oligonucleotide feature. It should be noted that, while they provide a means to survey transcript abundance on a genome-wide scale, the sensitivity of microarray assays is low compared to other approaches such as real-time PCR. A microarray is not a fully quantitative assay and changes in transcript abundance must be measured in reference to control samples that need to be included in each study. However, some of these limitations may be lifted by methods relying on high-throughput sequencing for the genome-wide measurement of RNA abundance [17] . Building on the legacy of the SAGE (serial analysis of gene expression) technology introduced in the 1990s, RNA sequencing (RNA-seq) [18] uses either total or fractionated RNA, for example poly(A)+, as a starting point. Th is material is converted to a library of cDNA fragments. High throughput sequencing of such fragments yields short sequences or reads that are typically 30 to 400 bp in length, depending on the technology platform used. For a given sample, tens of millions of such sequences will then be uniquely mapped against a reference genome. Th e higher the level of expression of a given gene, the higher the number of reads that will be aligned against it ( Figure 3 ). Th us, this approach does not rely on probe design and provides several types of information, including not only transcript abundance but also transcriptome structure (splice variants), profi les of non-coding RNA species, and genetic polymorphisms. RNA-seq is expected to become sufficiently cost-eff ective and practical that it will eventually supersede microarray technologies.
Other technologies should be considered for the profi ling of focused sets of genes. Nanostring technology can, for instance, detect the abundance of up to 500 transcripts with high sensitivity [19] . Th e approach is 'digital' since it counts individual RNA molecules using strings of fl uorochromes as reporters to identify the diff erent RNA species. Other technology platforms developed by, among others, Luminex, High Th roughput Genomics or Fluidigm round up the off ering for 'subgenome' transcript profi ling.
Th e fi eld of autoimmunity has proven a fertile ground for blood transcriptional studies. Alterations in transcript abundance in the blood of patients refl ect the sustained response against self-antigens and, more generally, uncon trolled infl ammatory processes. Such diseases often present with recurring-remitting patterns of activity, with episodes of fl aring that may be refl ected by fl uctuations in transcript abundance. Th e work has initially focused on diseases with clear systemic involvement such as systemic lupus erythematosus (SLE) [20, 21] . Multiple cell types and soluble mediators, including IL10 [22, 23] and IFNγ [24] [25] [26] , have been proposed to be at the center of lupus pathogenesis. While some scattered evidence indicated the potential role of type I interferon in lupus, several observations did not support the hypothesis: fi rst, not every SLE patient has detectable serum type I IFN levels [27] ; second, dysregulation of type-I IFN production is not found in most murine SLE-models [28] ; and third, genetic linkage and association studies had not identifi ed candidate lupus susceptibility genes within the IFN pathway [29] . However, in one of our earliest microarray studies we demonstrated that all but one of the pediatric patients exhibited upregulation of IFNinducible genes, and the only patient lacking this signature had been in remission for over 2 years [20] . In addition, it was found that treating SLE patients with high dose IV steroids, which are used to control disease fl ares, results in the silencing of the IFN signature. A surprise from these initial studies was the absence of type I IFN gene transcripts in the face of an abundance of IFN-inducible ones in the blood cells of SLE patients. A likely explanation is that the cells producing type I IFN, and therefore transcribing these genes, migrate to sites of injury. Altogether, results from microarray studies played a key role in convincing the community of the potential importance of type I IFN in SLE pathogenesis [15, [30] [31] [32] [33] [34] . A phase Ia trial to evaluate the safety, pharmacokinetics, and immunogenicity of anti-IFNα monoclonal antibody (mAb) therapy in adult SLE patients was recently conducted [35] . Th e antibody elicited a specifi c and dosedependent inhibition of overexpression of type I IFNinducible genes in both whole blood and skin lesions from SLE patients, at both the transcript and protein levels. As expected, overexpression of BLyS/BAFF, a type I IFN-inducible gene, also decreased with treatment. Th us, this fi rst trial supports the proposed central role of type I IFN in human SLE.
Systemic onset juvenile arthritis (SoJIA) is another disease with systemic involvement that greatly benefi ted from the study of blood transcriptional profi les with the development of both therapeutic and diagnostic modalities [14, 16, 36, 37] . Diseases with specifi c organ involvement have also been the subject of signifi cant, yet not always extensive, blood profi ling eff orts. Blood signatures have, for instance, been obtained from patients with Following extraction, RNA is used as a template and amplifi ed in a labeling reaction. The labeled material captured by the microarray is imaged and relative abundance determined based on the strength of the signal produced by the fl uorochromes that serve as reporters in this assay. The Nanostring technology measures RNA abundance at the single molecule level. RNA serves as starting material for this assay, which does not involve the use of enzymes for amplifi cation or labeling. Capture and reporter probes form complexes in solution with RNA molecules. These complexes are captured on a solid surface and imaged. Molecule counts are generated based on the number of reporter probes detected on the image. The reporter consists of a string of seven fl uorochromes, with four diff erent colors available to fi ll each position. Up to 500 diff erent transcripts can be detected in a single reaction on this platform. For RNA sequencing (RNA-seq) the starting RNA population must fi rst be converted into a library of cDNA fragments. High throughput sequencing of such fragments yields short sequences or reads that are typically 30 to 400 bp in length. For a given sample tens of millions of such sequences will then be uniquely mapped against a reference genome. The density of coverage for a given gene determines its relative level of expression. Similarities and diff erences between these technology platforms should be noted. For instance, microarrays and Nanostring technologies rely on oligonucleotide probes to capture complementary target sequences. Nanostring and RNA-seq technologies measure abundance at the single molecule level, with results expressed as molecule counts and sequence coverage, respectively. Microarray and RNA-seq technologies require extensive sample processing, which include amplifi cation steps. dsDNA, double-stranded DNA. Nanostring RNA-seq multiple sclerosis [38, 39] . Given the inaccessibility of the brain, blood constitutes a particularly attractive source of surrogate molecular markers for this disease. Th ese eff orts have yielded a systemic signature and identifi ed potential predictive markers of clinical relapse and response to treatment [40] [41] [42] . Transcriptional signatures have also been generated in the context of dermatologic diseases. In this case, the target organ being readily accessible, eff orts have been focusing on profi ling transcript abundance in skin tissues [43, 44] . However, systemic involvement has been recognized in recent years to be an important component of autoimmune skin diseases and unique blood transcriptional profi les have also been identifi ed in patients with, for example, psoriasis [45] [46] [47] .
Blood transcriptional profi les have been generated in the context of many other autoimmune diseases. Indeed, the range of autoimmune/autoinfl ammatory diseases that have been investigated encompasses SLE [20, 21, 48, 49] , juvenile idiopathic arthritis [16, [50] [51] [52] [53] , multiple sclerosis [54, 55] , rheumatoid arthritis [56] [57] [58] [59] , Sjogren's syndrome [60] , diabetes [61, 62] , infl ammatory bowel disease [63] , psoriasis and psoriatic arthritis [45, 47] , infl ammatory myopathies [64, 65] , scleroderma [66, 67] , vasculitis [68] and anti-phospholipid syndrome [69] . Th e body of work produced that focuses on blood transcript profi ling in the context of autoimmune diseases has been covered at length in a recent review [70] .
Global changes in transcript abundance have also been measured in the blood of patients with infectious diseases. In this context, alterations of blood transcriptional profi les are a refl ection of the immunological response mounted by the host against pathogens. Th is response is initiated by specialized receptors expressed at the surface of host cells recognizing pathogen-associated molecular patterns [71] . Diff erent classes of pathogens signal through diff erent combinations of receptors, eliciting in turn diff erent types of immune responses [72] . Th is translates experimentally into distinct transcriptional programs being induced upon exposure of immune cells in vitro to distinct classes of infectious agents [73] [74] [75] . Similarly, patterns of transcript abundance measured in the blood of patients with infections caused by diff erent etiological agents were found to be distinct [13] .
Predictably, dramatic changes were observed in the blood of patients with systemic infections (for example, sepsis) [76, 77] . However, profound alterations in patterns of transcript abundance were also found in patients with localized infections (for example, upper respiratory tract infection, urinary tract infections, pulmonary tubercu lo sis, skin abscesses) [13, 16, 78] . Measuring changes in host transcriptional profi les may therefore prove of diagnostic value even in situations where the causative pathogenic agent is not present in the test sample. Importantly, it may also help ascertain the severity of the infection and monitor its course.
Infections often present as acute clinical events; thus, it is important to capture dynamic changes in transcript abundance that occur during the course of the infection from the time of initial exposure. Blood signatures have been described in the context of acute infections caused by a wide range of pathogenic parasites, viruses and bacteria, including Plasmodium [79, 80] , respiratory viruses (infl uenza, rhinovirus, respiratory syncytial virus) [13, [81] [82] [83] [84] , dengue virus [85, 86] , and adenovirus [82] , as well as Salmonella [87] , Mycobacterium tuberculosis [78] , Staphylococcus aureus [88] , Burkholderia pseudomallei [76] and the general context of bacterial sepsis [77, [89] [90] [91] . Some of those pathogens will persist and establish chronic infections (for example, human immuno deficiency virus and Plasmodium) that may lead to a state of latency (for example, tuberculosis), and transcript profi ling may be used in those situations as a surveillance tool for monitoring disease progression or reactivation.
Blood profi ling of infectious diseases remains limited in scale. In particular, additional studies will be necessary to ascertain dynamic changes occurring over time.
In addition to autoimmune and infectious diseases, blood transcript profi ling studies have been carried out in the cancer research fi eld. While hematological malignancies have led the way (reviewed in [92] ), blood profi les have also been obtained more recently from patients with solid organ tumors [93] . Notably, these signatures can refl ect not only the immunological or physiological changes eff ected by cancers but also the presence of rare tumor cells in the circulation [94] [95] [96] .
Blood signatures have also been obtained from solid organ transplant recipients in the context of both tolerance [97] [98] [99] and graft rejection [10, 100, 101] . While such signatures can also be detected in biopsy material [102] [103] [104] , blood off ers the distinct advantage of being accessible for safely monitoring molecular changes on a routine basis. Some work has also been done in the context of cardiovascular diseases where infl ammation is known to play an important role. Hence, profi les have been identifi ed in a wide range of conditions, including stroke, chronic heart failure or acute coronary syndrome [105] [106] [107] [108] .
Th e body of published work is too large to be cited in this review -and it is likely to be only the tip of the iceberg, with a lot more unpublished data scattered through out public and private repositories. Other eff orts have yielded, for instance, blood transcriptional signatures in patients with neurodegenerative diseases [109] [110] [111] , and those associated with disease exacerbation or responsiveness to glucocorticoids in patients with asthma [112, 113] , and with responses to environmental exposure [114] [115] [116] , exercise [117, 118] or even laughter [119] . Unfortunately, too many published studies are underpowered and sometimes lack even the most rudimentary validation steps. All too often primary data are not available for reanalysis either, refl ecting a lack of enforcement of editorial policies, or the absence thereof in some journals. Hence, one of the main challenges for this fi eld is to move beyond the proof of principle stage and consolidate the wealth of data being generated.
Collectively, studies published thus far demonstrate that alterations in transcript abundance can be detected on a genome-wide scale in the blood of patients with a wide range of diseases. Th is statement is far from trivial given the skepticism that initially met studies investigating the blood transcriptome of patients. We have also learned that: 1) multiple diseases can share components of the blood transcriptional profi le -for instance, the case for infl ammation or interferon signatures; 2) while no single element of the profi le may be specifi c to any given disease it is the combination of those elements that makes a signature unique; and fi nally, 3) the work accomplished to date highlights the importance of carrying out analyses aiming at directly comparing transcriptional profi les across diseases. Indeed, much can be learned, for instance, about autoimmunity from studying responses to infection, and vice versa. Furthermore, such eff orts may eventually lead us closer to a molecular classifi cation of diseases. First, however, technological and methodological advances are necessary for the blood transcriptome research fi eld to move beyond the proof of principle stage.
Recent progress in blood transcriptome research has been possible thanks to the development of robust sample collection techniques and the introduction of high throughput gene expression microarray platforms. Such advances have been necessary but the margin for progression in the fi eld is still very signifi cant. We describe here some of the current hurdles and discuss potential solutions for overcoming them.
For years the scale of blood transcriptional studies has been constrained by the cost of the technology. With the price tag on a commercial whole genome microarray below the $100 US mark, this is not the case anymore. Th us, data management has now become the fi rst essential step to making large scale molecular profi ling a viable proposition. Beyond storing the output of microarray instruments, data management must capture and organize information that is essential for the interpretation of the results (Figure 4) . Th is includes sample information, data quality metrics, clinical information collected at the time of sampling, details about the experimental design, and materials and methods. Capturing such information ensures that the large volumes of data generated, which are often not published immediately, will remain exploitable for years to come. Th is point has become critical given the fact that results from genome-wide profi ling studies can never be exploited to their fullest extent and possess considerable cumulative value when re-analyzed collectively. Notably, the results generated by other cellular and molecular profi ling platforms will also need to be integrated in order to complete the picture. Th erefore, implementing eff ective data management solu tions and practices is essential to sustain the necessary increase in the scale of blood transcriptional studies (Figure 3 ) [120] . Unfortunately, implementing data management solutions in the laboratory is often an expensive proposition, requiring customization of off -the-shelf products or development of custom software adapted to handle specifi c workfl ows. Managing data also takes time and requires dedicated personnel. Th us, while the need is widely perceived, the commitment and steps necessary to implement eff ective data management solutions and practices are rarely adopted.
A myriad of approaches have been developed for the analysis of genome-wide transcriptional profi ling data [121] [122] [123] [124] . However, there is no silver bullet when it comes to microarray data analysis. Th e challenges encountered are several fold: 1) dimensionality, or how to cope with the fact that the number of parameters measured exceeds by several orders of magnitude the number of conditions included in most experiments; 2) noise -a direct consequence of the fi rst point is that results from microarray analyses are particularly permissive to noise (false discovery); 3) 'seeing' the data -data visualization is critical as it helps promote insight and supports data interpretation; 4) biological context -it is important to keep the biology in sight at all times. Indeed, while it is easy to become absorbed by the data, it is essential to use biological knowledge when designing analysis strategies. Finally, there is hardly a one-size-fi ts-all approach to micro array data analysis and what works in one situation may not be universally applicable. Indeed, the most common response from experts when questioned on the best way to analyze a given dataset is that 'it depends…': it depends, for instance, on the extent of the diff erences being observed or on the variability inherent to a given disease or study population; it depends on what questions are being asked; or it can depend on whether follow-up confi rmatory experiments are planned. In Table 1 we provide a data mining primer that explains the basic steps involved in microarray data analysis and the considerations that arise [125] [126] [127] [128] [129] . Ad hoc data mining approaches can be developed to meet specifi c needs. For instance, we have developed a data mining strategy for the specifi c purpose of analyzing blood transcriptional profi les [15] . Th is approach simply consists of a priori grouping of sets of genes with similar transcriptional patterns. Th is is repeated for several diff erent datasets and subsequently, when comparing the cluster membership of all the genes across those datasets, the genes with similar membership Figure 4 . Data management is key to progress. Extensive cellular and molecular profi ling of human subjects generates vast amounts of disparate data. Eff ective data management and integration solutions are essential to the preservation of this information in an interpretable form. Thus, data management eff orts occurring 'behind the scenes' have an essential role to play in realizing the full potential of high throughput profi ling approaches in human subjects. are grouped together to form what we have termed a transcriptional module. Structuring the data permits focusing downstream statistical testing on these sets of transcripts that form coherent transcriptional and functional modular units. Th is is in contrast with more traditional approaches that rely on iterative statistical testing for thousands of individual transcripts that are treated as independent variables. Th e modular transcriptional framework that we have developed reduces the number of variables by collapsing sets of coordinately expressed genes into a new entity, the module. Reducing data dimensionality as such can: 1) facilitate functional inter pretation; 2) enable comparative analyses across multiple datasets and diseases; 3) minimize noise and improve robustness of biomarker signatures; and 4) yield multivariate metrics that can be used at the bedside [15] . Data visualization is also of critical importance for the interpretation of large-scale datasets. We have devised a straightforward visualization scheme for mapping global transcriptional changes for individual diseases on a modular basis ( Figure 5 ).Briefl y, diff erences in expression levels between study groups are displayed for each module on a grid. Each position on the grid is assigned to a given module; a red spot indicates an increase and a blue spot a decrease in transcript abundance. Th e spot intensity is determined by the proportion of transcripts reaching signifi cance for a given module. A posteriori, biological interpretation has linked several modules to immune cells or pathways (see legend of Figure 5 ). Hence, in the example provided in Figure 5 
Here we provide basic analysis steps and important considerations for microarray data analysis:
-Per-chip normalization: This step controls for array-wide variations in intensity across multiple samples that form a given dataset. Arrays, as with all fl uorescence based assays, are subject to signal variation for a variety of reasons, including the effi ciency of the labeling and hybridization reactions and possibly other, less well defi ned variables, such as reagent quality and sample handling. To control for this, samples are normalized by fi rst subtracting background and then employing a normalization algorithm to rescale the diff erence in overall intensity to a fi xed intensity level for all samples across multiple arrays.
-Data fi ltering: Typically more than half of the oligonucleotide probes present on a microarray do not detect a signal for any of the samples in a given analysis. Thus, a detection fi lter is applied to exclude these transcripts from the original dataset. This step avoids the introduction of unnecessary noise in downstream analyses.
-Unsupervised analysis: The aim of this analysis is to group samples on the basis of their molecular profi les without a priori knowledge of their phenotypic classifi cation. The fi rst step, which functions as a second detection fi lter, consists of selecting transcripts that are expressed in the dataset and display some degree of variability, which will facilitate sample clustering. For instance, this fi lter could select transcripts with expression levels that deviate by at least two-fold from the median intensity calculated across all samples. Importantly, this additional fi lter is applied independently of any knowledge of sample grouping or phenotype, which makes this type of analysis 'unsupervised' . Next, pattern discovery algorithms are often applied to identify 'molecular phenotypes' or trends in the data.
-Clustering: Clustering is commonly used for the discovery of expression patterns in large datasets. Hierarchical clustering is an iterative agglomerative clustering method that can be used to produce gene trees and condition trees. Condition tree clustering groups samples based on the similarity of their expression profi les across a specifi ed gene list. Other commonly employed clustering algorithms include k-means clustering and self-organizing maps.
-Class comparison: Such analyses identify genes that are diff erentially expressed among study groups ('classes') and/or time points. The methods for analysis are chosen based on the study design. For studies with independent observations and two or more groups, t-tests, ANOVA, Mann-Whitney U tests, or Kruskal-Wallis tests are used. Linear mixed model analyses are chosen for longitudinal studies.
-Multiple testing correction: Multiple testing correction (MTC) methods provide a means to mitigate the level of noise in sets of transcripts identifi ed by class comparison (in order to lower permissiveness of false positives). While it reduces noise, MTC promotes a higher false negative rate as a result of dampening the signal. The methods available are characterized by varying degrees of stringency, and therefore they produce gene lists with diff erent levels of robustness.
• Bonferroni correction is the most stringent method used to control the familywise error rate (probability of making one or more type I errors) and can drastically reduce false positive rates. Conversely, it increases the probability of having false negatives.
• Benjamini and Hochberg false discovery rate [125] is a less stringent MTC method and provides a good balance between discovery of statistically signifi cant genes while limiting false positives. By using this procedure with a value of 0.01, 1% of the statistically signifi cant transcripts might be identifi ed as signifi cant by chance alone (false positives).
-Class prediction: Class prediction analyses assess the ability of gene expression data to correctly classify a study subject or sample. K-nearest neighbors is a commonly used technique for this task. Other available class prediction procedures include, but are not limited to, discriminant analysis, general linear model selection, logistic regression, distance scoring, partial least squares, partition trees, and radial basis machine.
-Sample size: The number of samples necessary for the identifi cation of a robust signature is variable. Indeed, sample size requirements will depend on the amplitude of the diff erence between, and the variability within, study groups.
A number of approaches have been devised for the calculation of sample size for microarray experiments, but to date little consensus exists [126] [127] [128] [129] . Hence, best practices in the fi eld consist of the utilization of independent sets of samples for the purpose of validating candidate signatures. Thus, the robustness of the signature identifi ed will rely on a statistically signifi cant association between the predicted and true phenotypic class in the fi rst and the second test sets.
modules. It should also be noted that no changes were observed for other modules, such as module M3.1, which includes interferon-inducible genes, abundance of which would be increased in the context of a viral infection.
MicroRNA (miRNA) control has emerged as a critical regu latory circuit of the immune system. Measuring changes in miRNA abundance in the blood of human subjects in health and disease is therefore a promising new fi eld of investigation. Th ese short non-coding singlestranded RNAs about 22 nucleotides in length have been found to play essential regulatory roles [130] [131] [132] . Th ese molecules exhibit highly specifi c, regulated patterns of expression and control protein expression by trans lational repression, mRNA cleavage, or promotion of mRNA decay. Interestingly, thanks to their small size, miRNA molecules are stable and can be measured not only in blood cells but also in circulation in the serum [133] . Th ey are thus not only potentially important contributors to immune function, but also potential sources of biomarkers.
Blood transcriptome research will also benefi t from concep tual advances that may help address shortcomings inherent to whole blood profi ling. First, blood is a complex tissue and changes in transcript abundance can be attributed to either transcriptional regulation or relative changes in composition of leukocyte populations. Two approaches exist for 'deconvoluting' these two phenomena. First, one can isolate and individually profi le diff erent cell populations present in the blood. Th is approach may also permit the identifi cation of transcripts expressed at low levels or the detection of diff erences in expression that would otherwise be drowned in whole blood [134, 135] . However, isolation methods may introduce technical bias, and require extensive sample processing. A second approach consists of deconvoluting whole blood transcriptional profi les 'in silico' . Th is type of analysis attempts to deduce cellular composition or cell-specifi c levels of gene expression using statistical methodologies [136] [137] [138] [139] [140] [141] .
Finally, we must also keep in mind that the immune status of a human subject is not entirely refl ected by its blood profi le obtained at the steady state. Indeed, an individual's capacity to respond to innate as well as antigen-specifi c immune signals may also provide useful and complementary information.
In conclusion, blood transcript profi ling has earned its place in the molecular and cellular profi ling armamentarium used to study the human immune system. Changes in transcript abundance recapitulate the infl uence of genetic, epigenetic, cellular and environ mental factors. Initially considered to belong to the 'cutting edge' , this approach has become both robust and practical. As discussed in this review, it has become a mainstay for the study of immune function in patients with a wide range of diseases. Furthermore, recent studies have demonstrated the utility of blood transcriptome profi ling for monitoring immune responses to drugs or vaccines [35, 142, 143] . Th us, blood transcript profi ling is developing Relative changes in transcript abundance in the blood of patients with S. aureus infection compared to that of healthy controls are recorded for a set of 28 transcriptional modules. Colored spots represent relative increase (red) or decrease (blue) in transcript abundance (P < 0.05, Mann Whitney) within a module. The legend shows functional interpretation for this set of modules. Fingerprints have been generated for two independent cohorts of subjects (divided into a training set used in the discovery phase, n = 30, and an independent test set used in the validation phase, n = 32).
into a mainstream tool for the assessment of the status of the human immune system. Bid Regulates the Pathogenesis of Neurotropic Reovirus Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence. Tissue injury in response to infections by many viruses occurs as a consequence of apoptosis. Multiple studies using animal models of viral disease demonstrate a correlation between apoptotic potential and disease severity [1, 2, 3, 4] . These observations highlight proapoptotic signaling following virus infection as an attractive target for antiviral therapy. However, despite its central importance in viral pathogenesis, gaps in knowledge about the identity of death signaling pathways that modulate virus-induced apoptosis in vivo, along with an incomplete understanding of how these signaling cascades are activated during virus infection, have hampered the deployment of this strategy for treatment of viral disease.
Mammalian reoviruses injure infected cells via apoptosis both in culture and in tissues of infected animals. As such, studies of these viruses have contributed to an understanding of how virus infection culminates in apoptotic cell death. Unlike other viruses in which virulence correlates with cell-death capacity, the identity of viral and cellular factors that regulate reovirus-induced apoptosis in cell culture are for the most part known [4, 5, 6, 7, 8, 9, 10] . Moreover, many of these intermediaries also modulate reovirus-induced apoptosis in vivo [4, 7, 11, 12] . Studies using reassortant reoviruses [5, 6] , ectopically expressed proteins [13] , and genetically engineered reovirus mutants [4, 7] highlight a critical role for reovirus outer-capsid protein m1 in apoptosis induction. Collectively, these studies indicate that prodeath signaling evoked by m1 occurs subsequent to membrane penetration but prior to synthesis of viral RNA or protein [4, 7, 14, 15] .
Classical death-receptor-mediated extrinsic apoptotic pathways stimulated by reovirus infection execute the death response [16] . Treatment of cells with soluble TRAIL receptors or expression of a dominant-negative form of Fas-associated death domain (FADD) protein blocks apoptosis, demonstrating that signaling via death receptors is required for execution of the apoptotic program [17] . In keeping with the function of extrinsic apoptotic signaling in reovirus infection, caspase-8 activation [18] and Bid cleavage [19] are observed in cells infected with reovirus [16] . Reovirus infection also stimulates intrinsic apoptotic pathways, as evidenced by release of cytochrome c and Smac/DIABLO from the mitochondria and activation of caspase-9 [16, 20, 21, 22, 23] . Concordantly, reovirus-induced apoptosis is dampened by over-expression of Bcl-2 [24] , which inhibits mitochondrial apoptotic pathway activation [25] .
Bid is a proapoptotic BH3-only member of the Bcl-2 family that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. Following death-receptor signaling, cytoplasmically resident Bid is cleaved by activated caspase-8 to generate a truncated form of Bid known as tBid [26] . tBid translocates to the mitochondria and triggers the release of cytochrome c and activation of the core mitochondrial apoptotic machinery [19, 27] . It is not known whether Bid plays a functional role in apoptosis induction by reovirus. Moreover, the relationship between apoptosis effector pathways and early events in viral replication are not understood.
In addition to these classical apoptotic pathways, the innate immune response transcription factor, NF-kB, is activated following reovirus infection [8] . NF-kB activation by reovirus depends on the viral m1 protein and can be accomplished by genome-deficient reovirus particles [4, 7, 8] . Blockade of NF-kB signaling using chemical inhibitors or cell lines genetically deficient in NF-kB p50, NF-kB p65/RelA, IkB kinase (IKK)-a, or IKK adaptor IKKc/NEMO significantly diminishes reovirus-induced apoptosis [8, 28] . Consistent with these findings, activation of NF-kB occurs within the first few hours of reovirus infection and precedes the biochemical and morphological hallmarks of apoptotic cell death [8, 28] . These observations suggest that NF-kB couples m1-mediated events to the cellular apoptotic machinery. Although regulation and function of NF-kB has been extensively studied, the precise relationship between NF-kB and the cell-death machinery remains undefined.
In this study, we examined the function of cellular apoptosis regulator Bid using genetically deficient murine embryo fibroblasts (MEFs) and mice. We found that while Bid is dispensable for reovirus replication in cell culture, its function is required for reovirus-induced apoptosis. Blockade of NF-kB signaling, which diminishes apoptosis induction by reovirus [8, 28] , prevents cleavage of Bid. In comparison to wild-type mice, Bid-deficient mice display diminished susceptibility to reovirus-induced CNS disease following either peroral (PO) or intracranial (IC) inoculation. Attenuated reovirus virulence in the absence of Bid is associated with decreased reovirus replication in the murine CNS. These results define an important role for Bid in virusinduced apoptosis and disease and illuminate Bid-dependent prodeath signaling as a viable target for antiviral therapy.
Reovirus infection of HEK293 epithelial cells leads to a biphasic loss of full-length (FL) Bid [16] . Since a mitochondrial amplification loop through Bid is required for apoptosis only in some cell types, such as hepatocytes [29, 30] , it is not known if Bid is cleaved in all cell types infected by reovirus. In addition, although calpains [31, 32] , caspases [26, 33, 34, 35] , and cathepsins [36, 37, 38, 39] can mediate Bid cleavage and have been implicated in apoptosis induction by reovirus [15, 16, 40] , the precise identity of the protease that generates tBid following reovirus infection is not known. To determine whether tBid is generated following reovirus infection of fibroblasts, and to define the mechanism of Bid cleavage following reovirus infection, we infected murine L929 fibroblasts with reovirus strain type 3 Dearing (T3D) and monitored levels of FL Bid and tBid over 48 h ( Figure 1A ). While levels of FL Bid remained unchanged in mock-infected cells, we observed loss of FL Bid between 24 and 48 h post-infection. Decreased levels of FL Bid correlated with a corresponding increase in levels of tBid. To determine whether the generation of tBid results in activation of the mitochondrial loop of the intrinsic apoptotic pathway, we assessed
Viruses injure host tissues by activating signaling pathways that trigger cell death by a process called apoptosis. Hence, blockade of apoptosis may serve as a useful strategy to dampen the severity of viral disease. However, deployment of such a strategy requires identification of host signaling networks that control cell death and a detailed molecular blueprint of how these pathways are activated by a virus. In this study, we used mammalian reovirus, an important experimental model for studies of viral encephalitis, to elucidate how cell death pathways are activated following viral infection and whether these signaling cascades influence the capacity of a virus to produce lethal CNS disease. We found that Bid, a host regulator of cell death, influences apoptosis induction by reovirus. Moreover, Bid is required for efficient reovirus replication in the CNS and modulates reovirus neurological disease. These findings highlight Bid as a critical regulator of viral pathogenesis and illuminate a potential new target for development of antiviral therapeutics. (A) L929 cells were adsorbed with PBS (mock) or reovirus T3D at an MOI of 100 PFU/cell. Following incubation at 37uC for the indicated intervals, whole cell extracts were prepared, resolved by SDS-PAGE, and immunoblotted using antisera specific for Bid, actin, or procasapse-9. (B) L929 cells were adsorbed with reovirus T3D at an MOI of 100 PFU/cell. Following incubation at 37uC for the indicated intervals in the presence of 0 or 10 mM of Z-IETD-FMK, whole cell extracts were prepared, resolved by SDS-PAGE, and immunoblotted using antisera specific for Bid, actin, or procasapse-9. Protein bands are indicated on the right. A non-specific band is indicated by an asterisk (*). doi:10.1371/journal.ppat.1000980.g001 levels of procaspase-9 as a surrogate for the formation of the caspase-9-containing apoptosome ( Figure 1A ). In a time frame consistent with cleavage-induced generation of tBid, we observed a decrease in procaspase-9 levels in reovirus-infected cells. These findings suggest that following reovirus infection of murine fibroblasts, Bid serves to activate the mitochondrial apoptotic pathway.
The adaptor molecule FADD is required for cleavage of Bid following reovirus infection of HEK293 cells [16] . Based on these data, we hypothesized that caspase-8 activity as a consequence of extrinsic prodeath signaling, is required for cleavage and activation of Bid. To test this hypothesis, we assessed the capacity of reovirus to mediate Bid cleavage in L929 cells treated with caspase-8 inhibitor Z-IETD-FMK ( Figure 1B ). As anticipated, Bid cleavage was not observed in mock-infected cells or mock-infected cells treated with Z-IETD-FMK (data not shown). Although tBid was generated at 36-48 h following reovirus infection of vehicle-treated cells, reovirus failed to efficiently induce activation of Bid in Z-IETD-FMK-treated cells until 48 h post-infection, providing evidence that reovirus evokes cleavage of Bid via caspase-8. In response to a variety of death agonists, Bid amplifies death signaling by linking the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways [30] . Since our findings with reovirus parallel this pattern, our results suggest that Bid functions similarly following reovirus infection by linking the death-receptor and mitochondrial apoptotic pathways.
Signaling via the intrinsic pathway is essential for reovirusinduced apoptosis [16] . This observation, along with the dependence of mitochondrial apoptotic signaling on cleavage of Bid, suggests that Bid serves an essential function in reovirusinduced apoptosis. To directly test whether Bid is required for apoptosis induction following reovirus infection, we compared reovirus-induced apoptosis in wild-type and Bid-deficient MEFs. For these experiments, MEFs were infected with T3D, and apoptosis was assessed by chemiluminescent measurement of the activity of caspase-3 and caspase-7, which serve as effector caspases for both the extrinsic and intrinsic apoptotic pathways ( Figure 2A ). In comparison to mock-infected cells, infection of wild-type cells resulted in a significant increase in caspase-3/7 activity at 24 h post-infection. Since MEFs are poorly permissive for reovirus infection [41] , staining of infected cells by indirect immunofluorescence indicated that adsorption with 100 PFU/cell of T3D resulted in infection of only ,8% of cells at 20 h post infection (data not shown). Despite a low frequency of infection, this MOI resulted in an ,3-fold increase in caspase-3/7 activity. When infection was initiated at 1000 PFU/cell, ,20% cells were infected (data not shown), and caspase-3/7 activity increased ,5fold. In contrast, infection of Bid-deficient cells resulted in minimal caspase-3/7 activity following infection at either MOI. Increase in caspase-3/7 activity following treatment of each cell type with a broad-spectrum protein kinase inhibitor, staurosporine, was equivalent (,5-fold), demonstrating that although Bid-deficient cells possess functional death-signaling pathways, they resist apoptosis induction by reovirus.
As an alternative means to quantify apoptosis, we compared wild-type and Bid-deficient MEFs for the onset of morphological characteristics of apoptosis following reovirus infection using an acridine orange (AO) staining assay ( Figure 2B ). Infection of wildtype cells resulted in a significant increase in the fraction of apoptotic cells at 48 h post-infection with 40% and 100% of the cells exhibiting apoptotic features at MOIs of 100 and 1000 PFU per cell, respectively. In contrast, Bid-deficient cells infected with T3D at either MOI displayed levels of apoptosis equivalent to mock-infected cells, ,10%. Similar results were obtained following infection with another apoptosis-proficient reovirus strain, T3SA+ (data not shown). These data indicate that Bid is required for apoptosis induction following reovirus infection.
To determine whether decreased apoptosis in Bid-deficient cells is attributable to alterations in reovirus infection in the absence of Bid, we compared reovirus infectivity in wild-type and Biddeficient cells using an indirect immunofluorescence staining assay ( Figure 2C ). An equivalent proportion of reovirus antigen-positive cells was detected at 20 h post-adsorption of wild-type and Biddeficient cells. These data indicate that reovirus is capable of initiating infection in Bid-deficient cells. To determine whether reovirus completes a full infectious cycle in Bid-deficient cells, wild-type and Bid-deficient cells were adsorbed with T3D, and viral titers were determined by plaque assay at 0, 12, 24, and 48 h after infection ( Figure 2D ). Reovirus replicated with similar kinetics and produced equivalent yields in wild-type and Biddeficient cells. Thus, the failure of Bid-deficient cells to undergo apoptosis in response to reovirus is not a consequence of diminished reovirus infection of these cells. We conclude that Bid is a key regulator of reovirus-induced apoptotic cell death.
The identification of an essential role for Bid in apoptosis induction following reovirus infection allowed us to examine the relationship between NF-kB activation and Bid cleavage. To determine whether Bid is required for activation of NF-kB following reovirus infection, we compared reovirus-induced NF-kB activation in wild-type and Bid-deficient cells using a reporter assay. Wild-type and Bid-deficient MEFs were transfected with an NF-kB-luciferase reporter plasmid and infected with reovirus. Analogous to treatment with TNFa, a control NF-kB agonist, reovirus infection resulted in equivalent (,2-to 3-fold) activation of NF-kB-driven gene expression in wild-type and Bid-deficient cells ( Figure 3A ). These results indicate that Bid is dispensable for NF-kB activation following reovirus infection and suggest that either reovirus-induced NF-kB activation occurs prior to Bid cleavage or that NF-kB activation and Bid cleavage occur in parallel but independent pathways that both function in apoptosis induction by reovirus.
To determine whether cleavage-induced Bid activation is dependent on NF-kB, we examined Bid cleavage in cells lacking p65/RelA, an NF-kB subunit required for apoptosis induction following reovirus infection [8] . Infection of wild-type MEFs with reovirus results in generation of tBid at 36-48 h after infection ( Figure 3B ). In contrast, infection of p65/RelA-deficient MEFs with reovirus did not lead to tBid generation even though efficient viral replication is observed in these cells [8] . Treatment of both wild-type and p65/RelA-deficient MEFs with apoptotic agonists TNFa and cycloheximide resulted in efficient cleavage of Bid, indicating that cell-death pathways leading to Bid cleavage are intact in both cell types. These findings suggest that cleavage and activation of Bid following reovirus infection requires NF-kB and place Bid cleavage subsequent to NF-kB signaling in response to reovirus infection. Moreover, since Bid amplifies death responses from the extrinsic apoptosis pathway by activating the mitochondrial loop, these findings suggest that death-receptor signaling during reovirus infection occurs in an NF-kB-dependent manner.
Apoptosis-signaling pathways involving death receptors DR4 and DR5 and death ligand TRAIL, as well as Fas and FasL, have been implicated in apoptosis induction by reovirus [17, 42, 43] . However, it is not known which of these pathways mediates cleavage-induced activation of Bid. It is also not understood whether NF-kB regulates the activation of these pathways. Since upregulation of Fas following reovirus infection is dependent on prodeath signaling via c-Jun N terminal kinase (JNK) [43] , and because JNK is activated via a mechanism distinct from NF-kB following reovirus infection [44] , we focused our efforts on assessing the regulation and function of death-receptor signaling via TRAIL following reovirus infection. For these studies, we assessed the capacity of reovirus to induce apoptosis in MEFs lacking TRAIL-R, the only known receptor for TRAIL on murine cells [45, 46, 47] (Figure 4 ). In comparison to mock infection, T3D infection of wild-type cells resulted in an MOI-dependent ,5-to 20-fold increase in caspase-3/7 activity at 24 h post-infection ( Figure 4A ). Although T3D infection of TRAIL-R-deficient cells also resulted in an increase in caspase-3/7 activity in comparison to mock-infection, the magnitude of this increase was only ,2-to 6-fold. Assessment of apoptosis in wild-type and TRAIL-Rdeficient MEFs using AO staining also showed an increase in apoptosis both in wild-type and TRAIL-R-deficient cells in comparison to mock-infected cells ( Figure 4B ). However, a substantially greater fraction of wild-type cells showed morphologic features of apoptosis in comparison to TRAIL-R-deficient cells infected at equivalent MOI, suggesting that efficient induction of apoptosis by reovirus requires TRAIL-R. T3D displayed comparable replication kinetics and produced equivalent yields in wild-type and TRAIL-R-deficient cells ( Figure 4C ). Thus, Figure 2 . Bid is required for apoptosis induction following reovirus infection. (A) Wild-type or Bid-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 24 h, caspase-3/7 activity in cell lysates was determined. Results are expressed as the mean ratio of caspase-3/7 activity from infected cell lysates to that from mock-infected cells for triplicate samples. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. Cells were treated with 10 mM staurosporine (Sts) for 24 h as a control. (B) Wild-type or Bid-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 48 h, cells were stained with AO. Results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (C) Wild-type and Bid-deficient MEFs were adsorbed with 10 4 particles/ cell of T3D. After incubation at 37uC for 18 h, cells were visualized by immunostaining with polyclonal reovirus-specific antiserum, followed by incubation with Alexa546-labeled anti-rabbit IgG. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (D) Wild-type and Bid-deficient MEFs were adsorbed with T3D at an MOI of 2 PFU/cell. The inoculum was removed, and cells were incubated at 37uC for the times shown. Viral titers were determined after two cycles of freeze-thaw by plaque assay using L929 cells. Results are presented as mean titers from three independent experiments. Error bars indicate SD. doi:10.1371/journal.ppat.1000980.g002 differences in the apoptotic potential of reovirus in wild-type and TRAIL-R-deficient cells are not associated with differences in reovirus growth in these cells.
To determine whether reovirus-induced cleavage of Bid is dependent on signaling via TRAIL-R, we monitored Bid cleavage following infection of TRAIL-R-deficient cells ( Figure 4D ). At 48 h post-infection of wild-type cells with T3D, FL Bid was cleaved to generate tBid. In contrast, FL Bid was not cleaved in T3D-infected TRAIL-R-deficient cells. While the apparent difference in the levels of FL Bid in wild-type and TRAIL-Rdeficient cells was not reproducible, we consistently observed that levels of FL Bid remained unchanged in TRAIL-R-deficient cells following reovirus infection. These data indicate that TRAIL-R contributes to the induction of apoptosis by reovirus and suggest that cleavage of Bid following reovirus infection is dependent on TRAIL-R signaling.
Reovirus virulence correlates with its capacity to cause apoptosis [4, 7, 11, 12, 48, 49] . Given the central role of Bid in apoptosis induction by reovirus in cell culture, we hypothesized that reovirus apoptosis and virulence would be diminished in the absence of Bid. To test this hypothesis, we inoculated two-day-old wild-type and Bid-deficient mice perorally with a highly virulent, enteric, neurotropic reovirus strain, T3SA+ [50] , and monitored infected animals for signs of neurological disease and infection-induced morbidity over a period of 21 days ( Figure 5A ). Following inoculation with 10 4 PFU of T3SA+, most wild-type mice developed paralysis and respiratory distress. In contrast, the majority of Bid-deficient mice were asymptomatic. Consistent with this observation, ,91% of wild-type mice succumbed to reovirus infection with a median survival time of 11 days, whereas only ,30% of Bid-deficient mice died. Due to the relative resistance of Bid-deficient mice to reovirus-induced encephalitis, a median survival time could not be determined. Thus, the cellular apoptotic regulator Bid modulates reovirus-induced encephalitis.
To determine whether the enhanced survival of Bid-deficient mice in comparison to wild-type mice following T3SA+ infection results from reduced reovirus replication, we compared titers of reovirus at sites of primary and secondary replication at 4, 8, and 12 d post-inoculation ( Figure 5B -E). Peak titers of reovirus were comparable or slightly higher (,5-to 10-fold) in the intestine, liver, and heart of wild-type mice in comparison to Bid-deficient animals. In contrast, substantially greater differences in peak reovirus titers were observed in the brain, with wild-type animals showing ,25-to 100-fold higher titers in comparison to those in Bid-deficient mice at 8 d post-inoculation. However, by 12 d postinoculation, titers of reovirus in wild-type and Bid-deficient mouse brains were equivalent. These findings suggest that reovirus infection is inefficient in the absence of Bid, especially in the CNS.
Although titers of reovirus in the CNS were decreased in Bidnull mice following PO inoculation, it was not clear whether reduced reovirus titer in the CNS was a consequence of diminished reovirus dissemination to the CNS or diminished reovirus replication at that site. To distinguish between these possibilities, we inoculated wild-type and Bid-deficient mice intracranially with 100 PFU of T3SA+ and monitored infected animals for signs of CNS disease and mortality for 21 days (Figure 6A ). At this dose of T3SA+, most wild-type and Biddeficient mice displayed symptoms of neurological disease. Concordantly, both strains of mice succumbed to reovirus-induced disease with equivalent frequency and a median survival time of 13 days. Reovirus titers in the brains of wild-type and Bid-deficient mice also were comparable at 4, 8, and 10 d post-inoculation ( Figure 6B ). These results indicate that following a high-dose Whole cell lysates were prepared at the indicated times after infection or 12 h following TNFa/CHX treatment and resolved in 18% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with polyclonal antisera specific for Bid or actin and appropriate HRP-conjugated secondary antibodies and visualized using chemiluminescence. doi:10.1371/journal.ppat.1000980.g003 inoculation, Bid is dispensable for reovirus growth in the murine CNS and attendant encephalitis.
Peak titers of reovirus in the brains of intracranially-inoculated wild-type mice were ,1000-fold higher than those in perorallyinoculated wild-type animals (compare Figures 5E and 6B) . We thought it possible that this difference in viral load might contribute to the dramatic difference in the requirement for Bid in the pathogenesis of reovirus-induced CNS disease following PO and IC inoculation. To test this hypothesis, we inoculated wild-type and Bid-deficient animals intracranially with a considerably lower but still lethal dose of T3SA+, 5 PFU, and monitored infected animals for signs of reovirus encephalitis ( Figure 6C ). In comparison to wild-type mice in which ,95% succumbed to disease, ,70% of Bid-deficient mice developed lethal encephalitis. Moreover, the median survival time of wildtype mice infected with T3SA+ was significantly less (13 days) than that of Bid-deficient mice (15 days). To determine whether this difference in survival correlates with the efficiency of reovirus replication in the CNS, we compared titers of reovirus in brains resected from infected mice at 4, 8, and 12 d post-inoculation ( Figure 6D ). Titers of reovirus in brains of wild-type mice were substantially higher (,10-to 100-fold) at each interval in Results are expressed as the mean ratio of caspase-3/7 activity from infected cell lysates to that from mock-infected cell lysates for triplicate samples. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (B) Wild-type or TRAIL-R-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 48 h, cells were stained with AO. Results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (C) Wild-type and TRAIL-R-deficient MEFs were adsorbed with T3D at an MOI of 2 PFU/cell. The inoculum was removed, and cells were incubated at 37uC for 0, 12, 24 and 48 h. Viral titers were determined after two cycles of freeze-thaw by plaque assay using L929 cells. Results are presented as mean titers from three independent experiments. Error bars indicate SD. (D) Wild-type and TRAIL-R-deficient MEFs were mock-infected or infected with T3D at an MOI of 100 PFU/cell. Whole cell lysates were prepared at 48 h after infection and resolved in 18% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with polyclonal antisera specific for Bid or actin and appropriate HRP-conjugated secondary antibodies and visualized using chemiluminescence. doi:10.1371/journal.ppat.1000980.g004 Figure 5 . Bid modulates reovirus replication and pathogenesis following PO inoculation. (A) Two-day-old wild-type and Bid-deficient mice were inoculated perorally with 10 4 PFU of T3SA+. Mice (n = 23 to 27) were monitored for survival for 21 days. ***, P,0.005 as determined by logrank test in comparison to wild-type mice. (B-E) Two-day-old wild-type and Bid-deficient mice were inoculated perorally with 10 4 PFU of T3SA+ and euthanized at the times shown. Intestines (B), liver (C), heart (D), and brain (E) were resected and homogenized by freeze-thaw and sonication. Viral titers in organ homogenates were determined by plaque assay. Results are expressed as viral titer in organs of single infected animals as indicated by comparison to those in Bid-deficient mice, with those at 4 and 12 d post-inoculation reaching statistical significance. These findings indicate that at a lower viral inoculum, Bid promotes efficient replication of reovirus in the CNS. Collectively, these data suggest that Bid influences reovirus virulence by regulating the growth of reovirus in the brain.
To assess the capacity of T3SA+ to produce neurological injury in the presence and absence of Bid, we examined hematoxylin and eosin (H&E)-stained coronal brain sections prepared from wildtype and Bid-deficient mice euthanized 10 d following IC inoculation with 5 PFU of T3SA+ (Figure 7) . This time point was chosen to coincide with the presence of maximal viral titers following inoculation by this route. Since the inoculum used for these experiments was at least ,10-fold lower than that used for most other studies of reovirus CNS pathogenesis [4, 7, 11, 12, 43, 48, 51] , the extent of injury following infection of wild-type mice was not as extensive. Nonetheless, inoculation of wild-type mice with T3SA+ resulted in neuronal death in the cerebral cortex, hippocampus, thalamus, and hypothalamus, consistent with previous reports [4, 7, 11, 12, 43, 48, 51] . While the majority of infected wild-type mouse brains showed signs of injury, tissue damage was minimal in all of the brains examined from similarly infected Bid-deficient animals. Examination of the hippocampal region of a representative wild-type mouse brain at higher magnification showed damage to the CA3 region, with the pyramidal cells showing condensed nuclei characteristic of apoptosis ( Figure 7B ). In contrast, little damage was detected in an equivalent region of a Bid-deficient mouse brain ( Figure 7B ). These findings indicate that Bid is required for neurological injury produced by reovirus in mice.
To determine whether these differences in neurological injury are attributable to alterations in tropism of reovirus in the absence of Bid, sections of mouse brain were stained for reovirus antigen. Reovirus displayed similar tissue distribution in wild-type and Biddeficient mouse brains, indicating that Bid expression does not influence reovirus tropism (data not shown). The CA3 region of a wild-type mouse brain showed reovirus antigen in areas coincident with extensive neuronal damage ( Figure 7B ). Regions positive for reovirus antigen also stained with an antibody for activated caspase-3. Although similar regions of Bid-deficient mouse brains contained reovirus antigen, staining was of diminished intensity and frequency ( Figure 7B) , consistent with the decreased efficiency of reovirus replication in the CNS ( Figure 6B) . Accordingly, few cells showing intense caspase-3 staining were observed in regions that contained reovirus. These data suggest that neuronal apoptosis following reovirus infection is diminished in the absence of Bid. Thus, Bid links reovirus replication and apoptosis induction in the production of fatal encephalitis.
Early steps in reovirus replication elicit apoptosis via a signaling pathway dependent on NF-kB [4, 7, 8, 14, 15] . It is not understood how virus-induced NF-kB activation leads to cell death. In this study, we evaluated the function and regulation of Bid in apoptosis caused by reovirus. We found that although Bid is dispensable for reovirus replication in cell culture, it is required for the induction of apoptotic cell death following reovirus infection. In this context, Bid is converted to its active, proapoptotic form, tBid, in an NF-kB-dependent manner. Generation of tBid in reovirus-infected cells requires signaling via TRAIL-R and caspase-8. These findings indicate that NF-kB signaling following reovirus infection results in activation of the extrinsic apoptotic pathway. In turn, the extrinsic apoptotic pathway evokes the mitochondrial apoptotic cascade via cleavage-induced activation of Bid. Together, these events culminate in the induction of apoptotic cell death.
Many viruses induce apoptosis via activation of host-encoded apoptosis-regulating factors. For example, the VSV M protein induces apoptosis by inhibiting the transcription of antiapoptotic factors such as Bcl-xl [52] . In other cases, virus-encoded polypeptides insert into mitochondrial membranes and trigger cytochrome c release, leading to activation of the mitochondrial apoptotic pathway. For example, influenza A virus PB1-F2 is thought to directly activate proapoptotic signaling by interaction with the mitochondrial membrane-associated factors ANT3 and VDAC1 [53] . Although one model for apoptosis induction by reovirus suggests that the w fragment of reovirus m1 protein induces apoptosis by directly targeting mitochondria analogous to PB1-F2 [13] , our studies using apoptosis-defective reovirus mutants [4, 7] , coupled with data presented here, support the idea that w-mediated NF-kB signaling activates the mitochondrial apoptotic pathway indirectly via death-receptor signaling and Bid cleavage. This indirect mechanism of mitochondrial pathway activation by a proximal signal transducer also explains the timing of prodeath signaling in the reovirus replication cycle. We think that events associated with viral entry into cells, which are mediated by the m1 w fragment subsequent to membrane penetration, activate NF-kB within 1 h of infection [7, 28] . Unlike other NF-kB agonists such as TNFa which rapidly and transiently activate NF-kB, activation of NF-kB following reovirus infection is gradual and sustained and occurs maximally at 6-8 h post infection [28] . Activated NF-kB complexes lead to expression of genes that promote cleavage-induced activation of Bid at 24-36 h post infection and elicit characteristic features of apoptosis, including effector caspase activation and DNA fragmentation. These changes occur subsequent to completion of viral replication, and, therefore, apoptosis appears to have little detectable effect on viral growth in cell culture [4, 7, 8, 28] . Although unusual, NF-kBdependent apoptotic pathways are also utilized by other viruses such as Dengue virus [54] , HIV [55] , infectious bursal disease virus [56] , and Sindbis virus [57] . Thus, our studies may have uncovered a potentially conserved signaling pathway utilized by viruses to induce apoptosis via NF-kB.
It is not known how activation of NF-kB by reovirus culminates in cell death. Three previous studies have attempted to identify proapoptotic host genes that serve as effectors of the death response following reovirus infection. In the first, gene-expression profiles following infection with reovirus strains type 1 Lang (T1L) and type 3 Abney (T3A), which differ in the capacity to induce apoptosis [58] , were compared by microarray analysis [59] . These experiments did not demonstrate differences in expression of death ligands or their respective receptors following infection by either strain. Thus, it was concluded that expression of these death mediators by reovirus is unlikely to contribute to apoptosis induction by reovirus. However, some differences were observed in expression of regulators of death-receptor signaling [59] . But since T1L and T3A display significant genetic diversity and vary in the modulation of multiple signaling pathways [9, 60] , the contribution of NF-kB to the expression of prodeath genes could not be established. In the second study, gene-expression profiles following T3D infection in the presence and absence of functional NF-kB were compared [61] . Although this study identified several NF-kB-dependent genes that coordinate the cellular antiviral immune response, including numerous interferon-stimulated genes (ISGs), no classical components of death receptor-mediated signaling pathways or proapoptotic Bcl-2 family members were significantly upregulated in response to reovirus infection. In the third study, gene-expression profiles of reovirus strains that differ in the capacity to elicit translational shutoff were compared [62] . This study also demonstrated an increase in ISG expression but did not identify obvious NF-kB-dependent candidates that could serve to activate death receptor signaling.
We think there are three possibilities to explain why apoptosisregulating, transcriptional targets of NF-kB, such as death ligands (e.g., FasL and TRAIL) [63, 64] , death receptors (e.g., Fas and DR) [65, 66] , and death-signaling regulators (e.g., Bax and Bcl-X s ,) [67] , were not identified in these studies. First, changes in the expression of prodeath genes activated by reovirus infection may be too transient to have been detected in the intervals selected for analysis. Second, the transformed nature of the cell lines used in these studies may not have been amenable to detection of alterations in gene expression induced by reovirus infection. Third, NF-kB activation following reovirus infection may regulate death signaling at a post-transcriptional level by an as yet unknown mechanism. In support of this idea, levels of DR5 protein increase following reovirus infection [17] but not its mRNA [59] . Additional studies using primary, non-transformed cell lines and genetically engineered viruses that differ only in the capacity to activate NF-kB are required to define how reovirus activates extrinsic apoptotic pathways to evoke cell death.
In addition to enhancing an understanding of mechanisms by which virus-induced signaling leads to activation of Bid, our studies highlight a critical role for Bid in controlling the pathogenesis of a viral disease. We found that Bid-deficient mice are less susceptible to lethal encephalitis produced by a neurotropic reovirus strain following either PO or IC inoculation.
Reovirus replicates with slower kinetics in the absence of Bid, and virus-induced apoptosis and CNS injury are diminished in Biddeficient animals. Although Bid contributes significantly to reovirus pathogenesis, our data do not allow us to determine whether diminished reovirus virulence in Bid-deficient animals is attributable to reduced capacity of reovirus to replicate in the CNS, diminished capacity of reovirus to injure neurons by apoptosis, or both effects. It is also not clear whether the decreased capacity of reovirus to evoke apoptosis in the CNS is a cause or effect of the lower viral titers at that site.
Because Bid serves to amplify the death response, it is not universally required for apoptosis induction. In some cell types, known as type I cells, caspase-8 activation results in direct, Bidindependent activation of the apoptosis effectors, caspase-3 and caspase-7 [29] . In others, known as type II cells, apoptosis requires amplification of death signals through stimulation of the mitochondrial pathway. In these cases, Bid serves to link the extrinsic and intrinsic apoptotic pathways [30] . Since the requirement for Bid in reovirus virulence is dependent on viral dose, we think that the role of Bid as an apoptosis regulator contributes to viral replication and consequent neurovirulence. Thus, we hypothesize that neurons function like type I cells when infected at a higher dose of virus and do not require the amplification of the mitochondrial apoptotic pathway via Bid to undergo apoptotic cell death. However, at lower infectious doses, neurons function like type II cells and require Bid-driven activation of the intrinsic mitochondrial apoptotic cascade to elicit cell death. This model also may explain why primary neuronal cultures infected with reovirus at a high MOI do not appear to require cytochrome c release and caspase-9 activation for apoptosis induction [42] .
It is not known how Bid controls the efficiency of reovirus replication in the CNS. One possibility is that Bid-regulated apoptosis is required for efficient release of virus from neurons. Therefore, cell-to-cell spread of reovirus within the CNS may be inefficient in the absence of Bid. As an extension of this idea, blockade of apoptosis by other means also should cause a delay in reovirus replication. However, although symptoms of encephalitis are alleviated in NF-kB p50-deficient mice or in wild-type mice treated with a JNK inhibitor due to a reduction in virus-induced neuronal apoptosis, reovirus replication kinetics are not substantially diminished [11, 12] . Consistent with these findings, diminished virulence of apoptosis-defective reovirus mutants is not accompanied by significant decreases in reovirus replication efficiency in the CNS [4, 7] . Apoptosis following reovirus infection can occur in absence of p50, albeit at low efficiency [8] . Similarly, apoptosisdefective reovirus mutants retain some capacity to induce apoptotic cell death [4, 7] . Therefore, it is possible that in comparison to reovirus infection of Bid-deficient mice, CNS apoptosis was incompletely blocked in these other studies. Such a difference in the efficiency of apoptosis inhibition could explain the observed discrepancy in the requirement for Bid and other host or viral modulators of apoptosis for efficient replication of reovirus. A second possibility is that a Bid function not related to its capacity to regulate apoptosis contributes to reovirus replication in the CNS.
Analogous to its role in reovirus-induced cell death, Bid is implicated in apoptosis caused by many viruses [68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79] . However, prior to our study, it was not known whether Bid modulates the pathogenesis of viral disease. The function of Bid in viral pathogenesis has been examined in a previous study, which found that the BH3-only protein, Puma, but not Bid, contributes to apoptosis-mediated elimination of antigen-specific T cells following acute infection with herpes simplex virus-1 [80] . Here, we demonstrate a pathogenic function for Bid in viral infection. Should Bid similarly modulate disease outcomes following infection by other virulent viruses, antiapoptotic compounds targeting Bid [81, 82, 83] may serve as useful antiviral therapeutics.
Murine L929 cells were maintained in Joklik's minimal essential medium supplemented to contain 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B (Invitrogen). Wild-type and Biddeficient MEFs were maintained in Dulbecco's minimal essential medium (DMEM) supplemented to contain 10% FBS, 2 mM Lglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B. TRAIL-R-deficient MEFs, prepared from D13 embryos, were maintained in DMEM supplemented to contain 10% FBS, 2 mM L-glutamine, 16 MEM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 20 mM HEPES, 100 U/ ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B. Reovirus strain T3D is a laboratory stock. T3SA+ was generated by reassortment of reovirus strains T1L and type 3 clone 44-MA as described [84] . Purified reovirus virions were generated from second-or third-passage L-cell lysate stocks of twice-plaque-purified reovirus [85] . Viral particles were Freon-extracted from infected cell lysates, layered onto 1. 
Rabbit antisera raised against T1L and T3D have been described [87] . Rabbit antiserum specific for procaspase-9 was purchased from Cell Signaling. Goat antiserum specific for Bid was purchased from R & D systems, and goat antiserum specific for actin was purchased from Santa Cruz Biotechnology. HRP-conjugated antirabbit and anti-goat secondary antibodies were purchased from Amersham GE Biosciences. Alexa Fluor-conjugated anti-mouse immunoglobulin (Ig) G, anti-rabbit IgG, and anti-goat IgG secondary antibodies were purchased from Invitrogen.
Plasmids pRenilla-Luc and pNF-kB-Luc [88] were obtained from Dr. Dean Ballard (Vanderbilt University).
L929 cells or wild-type, Bid-deficient, or TRAIL-R-deficient MEFs were either adsorbed with reovirus at an MOI of 100 PFU/cell or mock-infected in serum-free medium at 4uC for 1 h, followed by incubation in serum-containing medium at 37uC for various intervals. Whole cell lysates were prepared by washing cells in phosphate-buffered saline (PBS) followed by lysis using 16 RIPA buffer (50 mM Tris [pH 7.5], 50 mM NaCl, 1% TX-100, 1% DOC, 0.1% SDS, and 1 mM EDTA) containing a protease inhibitor cocktail (Roche). Following centrifugation at 15,0006g to remove debris, the lysates were resolved by electrophoresis in polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked for at least 1 h in blocking buffer (PBS containing 5% milk or 2.5% BSA) and incubated with antisera against Bid (1:1000), actin (1:2000), or procaspase-9 (1:1000) either at room temperature for 1 h or 4uC overnight. Membranes were washed three times for 10 min each with washing buffer (PBS containing 0.1% Tween-20) and incubated with1:2000 dilution of horseradish peroxidase (HRP)-conjugated or Alexa Fluor-conjugated goat anti-rabbit Ig (for procaspase-9) or donkey anti-goat Ig (for Bid and actin) in blocking buffer. Following three washes, membranes were incubated for 1 min with chemiluminescent peroxidase substrate (Amersham Biosciences) and either exposed to film (for HRP-conjugated secondary antibodies) or scanned using an Odyssey Infrared Imager (LiCor).
Wild-type, Bid-deficient, or TRAIL-R-deficient MEFs (10 4 ) were seeded into black clear-bottom 96-well plates (Costar) and adsorbed with reovirus in serum-free medium at room temperature for 1 h. Following incubation of cells at 37uC for 24 h, caspase-3/7 activity was quantified using the Caspase-Glo-3/7 assay (Promega).
Wild-type, Bid-deficient, or TRAIL-R-deficient MEFs (5610 4 ) were grown in 24-well plates (Costar) and adsorbed with reovirus at room temperature for 1 h. The percentage of apoptotic cells after 48 h incubation was determined using AO staining as described [6] . For each experiment, .200 cells were counted, and the percentage of cells exhibiting condensed chromatin was determined by epi-illumination fluorescence microscopy using a fluorescein filter set (Zeiss Photomicroscope III; Thornwood, NY).
Wild-type or Bid-deficient cells (2610 5 ) were grown in 24-well plates and adsorbed with reovirus at room temperature for 1 h. Following removal of the inoculum, cells were washed with PBS and incubated in complete medium at 37uC for 18 h. Monolayers were fixed with methanol, washed twice with PBS, blocked with 2.5% Ig-free bovine serum albumin (Sigma-Aldrich) in PBS, and incubated successively for 1 h with polyclonal rabbit anti-reovirus serum at a 1:1000 dilution and for 1 h with Alexa Fluor 546labeled anti-rabbit IgG at a 1:1000 dilution. Monolayers were washed with PBS, and infected cells were visualized by indirect immunofluorescence using a Zeiss Axiovert 200 fluorescence microscope. Reovirus antigen-positive cells were quantified by counting fluorescent cells in at least two random fields of view in triplicate wells at a magnification of 206.
Wild-type, Bid-deficient, or TRAIL-R-deficient MEFs (2610 5 ) in 24-well plates were adsorbed with reovirus at room temperature for 1 h in serum-free medium, washed once with PBS, and incubated in serum-containing medium for various intervals. Cells were frozen and thawed twice prior to determination of viral titer by plaque assay using L929 cells [89] .
Wild-type and Bid-deficient cells in 24-well plates were transfected with 0.72 mg/well of an NF-kB reporter plasmid, which expresses firefly luciferase under NF-kB control (pNF-kB-Luc), and 0.08 mg/well of control plasmid pRenilla-Luc, which expresses Renilla luciferase constitutively, using Fugene6 (Roche). After incubation for 24 h, transfected cells were adsorbed with reovirus in serum-free medium at room temperature for 1 h and incubated at 37uC in serum-containing medium for 24 h. Luciferase activity in the cultures was quantified using the Dual-Luciferase Assay Kit (Promega) according to the manufacturer's instructions.
Wild-type C57BL/6J mice were obtained from Jackson Laboratory. Bid-deficient mice backcrossed on to a C57BL/6J background for at least 8 generations have been previously described [30] . Two-day-old mice were inoculated either perorally or intracranially with purified virus diluted in PBS. PO inoculations were delivered in a volume of 50 ml by passage of a polyethylene catheter 0.61 mm in diameter (BD) through the esophagus and into the stomach [90] . The inoculum contained 0.3% (vol/vol) green food coloring to allow the accuracy of delivery to be judged. IC inoculations were delivered to the left cerebral hemisphere in a volume of 5 ml using a Hamilton syringe and a 30-gauge needle (BD Biosciences) [91] . For analysis of viral virulence, mice were monitored for weight loss and symptoms of disease for 21 days. For survival experiments, mice were euthanized when found to be moribund (defined by rapid or shallow breathing, lethargy, or paralysis). For determination of viral titer and immunohistochemical staining, mice were euthanized at various intervals following inoculation and organs were resected. For analysis of virus growth, organs were collected into 1 ml of PBS and homogenized by freezing, thawing, and sonication. Viral titers in organ homogenates were determined by plaque assay using L929 cells [89] . For immunohistochemical staining, organs were fixed overnight in 10% formalin, followed by incubation in 70% ethanol. Fixed organs were embedded in paraffin, and 5-mm histological sections were prepared. Consecutive sections were stained with H&E for evaluation of histopathologic changes or processed for immunohistochemical detection of reovirus antigens or activated caspase-3 [11] . Animal husbandry and experimental procedures were performed in accordance with Public Health Service policy and the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care and approved by the Vanderbilt University School of Medicine Institutional Animal Care and Use Committee.  Effects of Pulsed Electromagnetic Fields on Human Osteoblastlike Cells (MG-63): A Pilot Study BACKGROUND: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes. QUESTIONS/PURPOSES: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures. METHODS: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome. RESULTS: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). CONCLUSIONS AND CLINICAL RELEVANCE: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine. PEMFs have been used for many years [44] . They reportedly are effective for treating nonunions [1, 7, 10] , delayed unions [1, 42, 44] , osteotomies [32] , avascular necrosis of the femoral head [5, 34] , bone grafts [11] , and spinal fusion [36] . Although the therapeutic properties of PEMFs are well known, the sequence of events by which electromagnetic stimulation can bring about its desirable effects on bone healing is not completely understood. PEMFs modify some important physiologic parameters of cells, such as proliferation, transduction, transcription, synthesis, and secretion of growth factors [24] . PEMFs induce cell proliferation in mitogen-stimulated lymphocytes [10] and improve IL-2 receptor expression and IL-2 use in lymphocytes from aged donors, which are characterized by defective production and use of this growth factor [10] . PEMF exposure induces cell proliferation in human osteoblasts and chondrocytes cultured in vitro [18, 20, 38, 44, 45] . PEMFs determine signal transduction by means of intracellular release of Ca 2+ leading to an increase in cytosolic Ca 2+ and an increase in activated cytoskeletal calmodulin [9] . PEMFs induce a dose-dependent increase in bone [2] and cartilage differentiation [2] [3] [4] 33] , and upregulation of mRNA expression of extracellular matrix molecules, proteoglycan, and Type II collagen [3] . The acceleration of chondrogenic differentiation is associated with increased expression of TGF-b1 mRNA and protein [4] , suggesting the stimulation of TGF-b1 may be a mechanism through which PEMFs affect complex tissue behavior such as cell differentiation and through which the effects of PEMFs may be amplified [4] . PEMFs also are postulated to act at a membrane level influencing signal transduction of several hormones or growth factors such as parathyroid hormone, IGF 2, and adenosine A2a, producing the amplification of their transmembrane receptors [1, 19, 21, 23, 31, 46] . Studies of single genes using RT-PCR suggest activation of osteocalcin, osteopontin, and TGF-b transcription during osteogenesis [22] and inhibition of cyclooxygenase 2 in synovial fibroblasts stimulated with TNFa or lipopolysaccharide [21] . A wide analysis of gene expression in cells exposed to PEMFs has not been performed: most studies focus on a few aspects of cell activities or they have been performed using different types of signals in different experimental conditions.
We therefore asked (1) whether PEMFs affected a wide array of genes in human osteoblastlike cells (MG63), and (2) whether and to what extent PEMFs induce proliferation and differentiation of osteoblasts.
We treated osteoblastlike cell cultures (MG-63) with PEMFs for 18 hours, and maintained similar nontreated controls. Gene expression of both groups therefore was evaluated with cDNA microarray analysis, containing 19 ,000 genes spanning a substantial fraction of the human genome. All experiments were performed in triplicate in the same culture conditions for control and treated cells.
Osteoblastlike cells (MG63) were grown in sterile Falcon wells (Becton & Dickinson, Franklin Lakes, NJ) containing Eagle's minimum essential medium supplemented with 10% fetal calf serum (Sigma-Aldrich, St Louis, MO) and antibiotics (penicillin 100 U/mL and streptomycin 100 lg/mL; Sigma-Aldrich). Cultures were maintained in a 5% CO 2 humidified atmosphere at 37°C. For the assay, cells were collected and seeded at a density of 1 9 10 5 cells/mL in two multiwells (one for the control and one for the treated). Each multiwell was comprised of six wells, 9-cm 2 , in which 3-mL of complete medium was added.
After 24 hours, cells were exposed to PEMFs for 18 hours using a PEMF generator system (Igea, Carpi, Italy). The PEMF used in this study is used clinically to treat nonunions or delayed unions and avascular necrosis of the femoral head [32] [33] [34] . The solenoids were powered using a Biostim pulse generator (Igea), a PEMF generator. The electromagnetic bioreactor applied to the cells has the following characteristics: intensity of the magnetic field, 2 ± 0.2 mT; amplitude of the induced electric tension, 5 ± 1 mV; signal frequency, 75 ± 2 Hz; and pulse duration, 1.3 ms. The stimulated multiwell was placed parallel between the two solenoids of the PEMF generator. The solenoids were placed at a distance of 10 cm and the multiwell was located on an acrylic support exactly at the center of the two solenoids. Control cultures were placed in the same incubator; nevertheless, the presence of the electromagnetic field was checked and its value was less than 0.05 mT. This value was ineffective in previous studies [38] [39] [40] [41] [42] [43] [44] [45] [46] . After 18 hours, when cultures were subconfluent, cells were processed for RNA extraction.
For DNA microarray screening and analysis, we used the same protocol as described previously [12] [13] [14] [15] [16] . Briefly, RNA was extracted from cells by using RNAzol. Ten micrograms of total RNA was used for each sample. cDNA was synthesized by using Superscript II (Life Technologies, Invitrogen, Milano, Italy) and amino-allyl dUTP (Sigma-Aldrich). Monoreactive Cy3 and Cy5 esters (Amersham Pharmacia, Little Chalfont, UK) were used for indirect cDNA labeling. RNA extracted from untreated cells was labeled with Cy3 and used as control against the Cy5-labeled treated (PG) cDNA in the first experiment and then switched. For 20 K human DNA microarrays slides (MWG Biotech AG, Ebersberg, Germany), 100 lL of the sample and control cDNAs in DIG Easy hybridization solution (Roche, Basel, Switzerland) were used in a sandwich hybridization of the two slides, constituting the 20 K set at 37°C overnight. Washing was performed three times for 10 minutes with 19 saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate at 42°C and three times for 5 minutes with 0.19 SSC at room temperature. Slides were dried by centrifugation for 2 minutes at 2000 rpm. Hybridized arrays were scanned with a GenePix 4000 scanner (Axon Instruments) at variable photomultiplier tube (PMT) voltage to obtain maximal signal intensities with less than 1% probe saturation.
The Foreground Median intensity for Cy3 and Cy5, Background Median intensity for Cy3 and Cy5, spot size data were imported into BRB-ArrayTools software [43] using the Import wizard function. Global normalization was used to median center the log-ratios on each array r to adjust for differences in labeling intensities of the Cy3 and Cy5 dyes.
The normalized Log ratios also were imported to Significance Analysis of Microarray (SAM) [48] software to identify differentially expressed genes. SAM assigns a score to each gene on the basis of a change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance-the false discovery rate (FDR). Analysis parameters (Delta) were set to result in zero FDR.
PEMF affected gene expression in MG-63 osteoblastlike cells (Fig. 1) . The genes differentially expressed in cells treated with PEMFs were either upregulated (268 genes) ( Table 1) or downregulated (277 genes) ( Table 2) . PEMF induced osteoblast proliferation and differentiation and regulated genes involved in bone formation in the direction of an enhancement of osteogenesis (Tables 3, 4 ).
In particular, PEMFs induced upregulation of several genes at the transcriptional level like STAT3, homeobox A10 (HOXA10), and V-akt murine thymoma viral oncogene homolog 1 (AKT1). Some genes acting at the transductional level also are upregulated including calmodulin (CALM1), activator protein 1 (AP-1), Nuclear factor kappaB (NF-KB), cAMP response element binding (CREB), and P2RX7 (Table 3) . Several interesting overexpressed genes are components of cytoskeleton and involved in cell adhesion (Table 3) . Examples are fibronectin (FN1) and vinculin (VCL). PEMF also increased the expression of genes encoding for collagenous and noncollagenous extracellular matrix proteins including collagen Type 1a2 (COL1A2), osteonectin (SPARC), and metallopeptidase inhibitor 1 (TIMP1) ( Table 3) . Some genes downregulated by PEMFs are related to degradation of extracellular matrix (ECM) (Table 4) , specifically, matrix metallopeptidase 11 (MMP11), or stromelysin 3 and dual specificity phosphatase 4 (DUSP4).
The improvement of osteogenesis is important because of the wide clinical applications it may have. PEMFs reportedly restart osteogenesis in disorders in which it has stopped [34] and in disorders in which osteogenesis needs to be enhanced [32] . Although considerable basic and clinical research on PEMFs has been reported, their mechanism of action is not completely clear. Moreover, studies in the existing literature have so far focused only on a few aspects of cell activities [9, 10, 46] , or they have been performed by using different types of signals in different experimental conditions [1, 9, 22, 23] . To address these limitations in the literature, we asked (1) whether PEMFs affected a wide array of genes in human osteoblastlike cells (MG63), and (2) whether and to what extent PEMFs induce proliferation and differentiation of osteoblasts.
We acknowledge several limitations. First, the experiment was performed using a human osteosarcoma cell line (MG63), whereas the use of a primary human osteoblast cell culture might better replicate what happens in humans in vivo. We chose the MG63 cell line because these cells show a phenotype similar to that of normal human osteoblasts, while also providing a reproducible experimental model suitable for the microarray analysis. Second, as it is still difficult to explain the roles of all genes, whose expression was modified, we focused on the role of genes with well-known functions related to osteogenesis. Third, although microarray technology is widely accepted as a valid approach to describe changes induced by a factor on cell environment, additional research using, for example RT-PCR, might be useful to provide supplementary support for the results obtained. Fourth, we studied responses at only one time. We chose 18 hours exposure time on the basis of a previous time experiment, in which a peak in DNA synthesis was seen after 18 hours of stimulation in MG63 cultures maintained in the presence of 10% FCS [45] . In contrast, Lohmann et al. reported PEMFs enhanced cell differentiation in MG63 cultures and reduced cell proliferation [30] . The differences existing between the two sets of data regarding cell proliferation could be related to the different experimental conditions used. Lohmann et al. exposed MG63 cultures when they reached confluence. When cultures are confluent they stop to proliferate. We exposed cells to PEMF when cultures were subconfluent, therefore, they responded with an enhancement of proliferation. We cannot extrapolate our findings to shorter or longer exposures to PEMFs. PEMFs appear to act on bone formation by inducing upregulation of several genes related to osteoblast proliferation and differentiation. Among those genes, HOXA10, a transcriptional factor that acts positively on RUNX2, is the main transcriptional regulator of osteoblast differentiation [25] . HOXA10 controls osteoblastogenesis via RUNX2-promoted osteoprogenitor cell differentiation in immature osteoblasts [25] . This protein also is believed to be involved in activation of alkaline phosphatase, osteocalcin, and sialoprotein genes [25] . We also observed * SAM assigns a score to each gene on the basis of a change in gene expression relative to the standard deviation of repeated measurements. STAT3, P2RX7, and AKT1 upregulation. It has been suggested that osteoblast-specific disruption of STAT3 results in an osteopenic phenotype [27, 41] . STAT3, involved in bone turnover [27] , regulates the transcription of various genes that modulate cell proliferation and differentiation in a cell-specific manner [27] . P2RX7 is a purinergic receptor, which is correlated with calcium channels and interacts with the calmodulin-dependent protein [37] . Activation of P2RX7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells promoting osteogenesis [37] . V-akt murine thymoma viral oncogene homolog 1 (AKT1), is a phosphoinositidedependent serine-threonine protein kinase, and one of the key players in the signaling of potent bone anabolic factors [29] . The disruption of AKT1 in mice led to low-turnover osteopenia through dysfunction [29] . AKT1 deficiency causes decreased bone mass and formation [29] , impairs RUNX2-dependent differentiation and function of osteoblasts [29] , and impairs bone resorption via dysfunction of osteoblasts and osteoclasts [29] . AKT1 suppresses osteoblasts apoptosis through inhibition of FOXO3a and Bim [29] , and may mediate the osteoblastic bone formation by IGF-1 [29] . The IGF-1/AKT1 pathway might be a common pathway for bone anabolic action of parathyroid, thyroid, and growth hormone [29] . We also observed upregulation of genes involved in connective and bone tissue formation (COL1A2) and noncollagenous extracellular matrix (ECM) synthesis (SPARC, FN1, VCL). COL1A2 encodes for collagen Type 1a2. Collagen Type 1 is the most represented collagen in the human organism and is important for ECM stability [6] . Osteonectin (SPARC), the most abundant noncollagenous protein in bone tissue, modulates cell-matrix interaction and is involved in the tissue-remodeling process [47] . FN1 is important for ECM stability and involved in adhesion and migration cellular processes such as tissue healing [39] . VCL is a cytoskeletal protein associated with the intercellular junctions between the cells and the matrix [49] .
The effect of TIMP1 upregulation and of MMP-11 and DUSP4 downregulation can be interpreted as a decrease in the degradation process. TIMP1 promotes apposition of ECM by inhibiting collagen and other components of ECM degradation operated by the metalloproteinase [26] . DUSP4 inactivates the superfamily of MAP kinase, which is involved with proliferation and differentiation. DUSP4 downregulation, then, stimulates proliferation [17] . MMPs potentially can degrade almost all components of the periprosthetic ECM and contribute to prosthetic loosening and osteolysis through pathologic ECM degradation and bone remodeling around prostheses [28, 35] . The stromelysins especially have broad substrate specificity, including proteoglycans, laminin, and fibronectin [35] . Stromelysin-1 determines the release and activation ECMbound latent TGF-ß1 and is involved with ECM turnover [8] . Upregulation of CALM1 promotes enhancement of calmodulin1, a protein involved in proliferative cell activation [40] . Calmodulin also is involved in the transduction mechanism of PEMFs [9] .
Our data suggest many effects of PEMFs on human osteoblastlike cells in vitro. PEMFs seem to exert an 
Inactivates the superfamily of MAP kinase Inhibition of proliferation and differentiation Caunt et al. [17] anabolic effect on cells. In particular, they are consistent with abundant preclinical and clinical findings showing a positive effect of PEMFs on osteogenesis. Stimulation by PEMFs induces bone healing in patients, shortens the time of healing processes, and stimulates healing of nonunions. Exposure to PEMFs acts on cell behavior in different ways. More specifically, PEMFs stimulate cell proliferation and induce osteoblastogenesis and differentiation of osteoblasts. Moreover, PEMFs promote ECM apposition and mineralization, and decrease degradation and absorption processes of ECM. These data suggest a more comprehensive explanation of the observed clinical effect of PEMFs on the induction of osteogenesis. Given their broad effects, PEMFs might be useful in other fields such as regenerative medicine. Virology Experts in the Boundary Zone Between Science, Policy and the Public: A Biographical Analysis This article aims to open up the biographical black box of three experts working in the boundary zone between science, policy and public debate. A biographical-narrative approach is used to analyse the roles played by the virologists Albert Osterhaus, Roel Coutinho and Jaap Goudsmit in policy and public debate. These figures were among the few leading virologists visibly active in the Netherlands during the revival of infectious diseases in the 1980s. Osterhaus and Coutinho in particular are still the key figures today, as demonstrated during the outbreak of novel influenza A (H1N1). This article studies the various political and communicative challenges and dilemmas encountered by these three virologists, and discusses the way in which, strategically or not, they handled those challenges and dilemmas during the various stages of the field’s recent history. Important in this respect is their pursuit of a public role that is both effective and credible. We will conclude with a reflection on the H1N1 pandemic, and the historical and biographical ties between emerging governance arrangements and the experts involved in the development of such arrangements. With the recent outbreak of novel influenza A (H1N1), better known as swine flu, familiar faces became even more familiar. In the Netherlands, the experts who not only frequently informed the public and investigated the virus, but also predicted the pandemic's course and handled public health policy problems, were figures that had assumed this role ever since the 'revival' of infectious diseases in the late 1970s and early 1980s. The general public, politicians, journalists and sceptics alike were already familiar with their names and faces, reputations and views, and even with their tactics and styles of handling such affairs.
This article is about these actors. I have studied the way in which three Dutch virologists, Albert Osterhaus, Roel Coutinho and Jaap Goudsmit, became both prominent and visible experts, and how they dealt with the various challenges entailed by such a role. This article uses a biographical-narrative approach to analyse their advancement into public and policy experts on infectious diseases, against the backdrop of the broader developments in Dutch policy and society.
In this article, the genesis of the visible expert (visible for policymakers, the media and the general public) is studied on an individual or micro-level by examining a number of stages between the late 1970s and the present day. Although textual sources (media coverage, policy reports and so on) are used as a backdrop and frame of reference, the basic source of input for my study is a series of narrative interviews with the experts themselves. Before turning to the case study as such, I will outline the theoretical and methodological framework of my research. I will also place biographical research in the broader field of science studies and discuss the methodological opportunities and risks involved in such an approach.
In science studies the science-policy nexus has been extensively studied by looking at boundary organisations (viz. Jasanoff 1990; Guston 1999; Bijker et al. 2009; Huitema and Turnhout 2009) , at the role of experts in policy development (Rutgers and Mentzel 1999; Nowotny 2000; Hoppe 2005 Hoppe , 2009 Turnhout et al. 2008 ) and at policy cultures, or their absence (Hellström 2000; Halffman and Hoppe 2005; Halffman 2005) . In this context, a biographical approach has two major advantages. First of all, a biographical approach allows us to study the individual views of the experts themselves and the assessments of their own roles. Secondly, a biographical approach allows us to flesh out the relationship between experts, policymaking and public communication.
The views of experts working in close interaction with policymaking and the public sphere have been extensively studied. Hoppe, for instance, described the views of scientific experts as boundary workers, and discerned several ways of looking at the science-policy boundary (Hoppe 2009 ). Huitema and Turnhout (2009) focus on the institutional level and describe the 'political' roles of experts working in a Dutch boundary organisation. Claire Waterton (2005) also focused on the science-politics boundary, in particular in the field of research policy and funding. Together with Sarah Davies (2008) , who describes the views held by scientists regarding the science-public boundary, these authors analyse the outlooks of individual scientists working across the boundaries of their organisations and disciplines. One of the challenges of discussing the views of scientists in the science-policy and science-public zones is to point out how each of the experts acted in these boundary zones and how they negotiated tensions within these zones, creating an identity for themselves in the process.
In light of the 'Third Wave' debate on expertise (see Collins and Evans 2002; Collins and Evans 2007; Wynne 1992 Wynne , 2003 , a distinction can be made between a 'relational' versus a 'realistic' view on expertise. In this paper we will look at the experts and their expertise from a relational or social role perspective (Collins and Evans 2007, p. 2) . We treat the role of experts not as a given solid state, but as something in flux. This flux is the net result of interactions between the actions of individuals, and the attributing of trust and credibility by others (viz. Goffman 1959, pp. 15-16; Wynne 1992, p. 282; Huitema and Turnhout 2009, p. 579) , turning the experts into 'hybrid characters' (Daston and Sibum 2003) . A role in this understanding cannot be attributed solely to what the individual expert wants. Roles are contingent outcomes of these interactions, in which trust and credibility play a crucial part.
This article studies these interactions under specific circumstances, i.e. during moments of crisis. Crises as focusing events challenge experts to inform and comment on the crisis, to assist in the development of appropriate policies, and more generally to sketch the prospects of where the crisis is heading (Birkland 1997) . Understood as such, we will see how crises function as springboards for experts from relative invisibility to visibility, and how experts are thus invited or forced to develop their own style of crisis management. Some authors point to the strategic use of crises in inducing policy change (Kingdon 1995; Birkland 1997; Weingart et al. 2000) . These sources demonstrate that a crisis is not something that 'just happens' to experts, but that experts, as policy entrepreneurs, also shape and give voice to crises. They tend to use moments of crises to 'capture attention for their causes', at times even by 'overselling to the media' (Weingart 1999, p. 159 ; see also Edwards 1999, p. 169) .
In the expert narratives we will see how perceptions of science, policy and public boundaries, and strategic ways of acting on the boundaries, are viewed from a biographical perspective. This shows the developments, the challenges and crises that experts engage with, and the learning experiences of 'being an expert'. It opens up the biographical 'black box' of experts in policymaking and public debate.
The biographical-narrative approach has been gaining ground in the areas of science studies and social sciences (Shortland and Yeo 1996; Huisman et al. 2000; Plummer 2001) . We find examples of this approach in anthropological laboratory studies (Traweek 1988) , feminist studies of science (Abir-Am and Outram 1987), the sociology of science (Zuckerman 1977; Goodell 1977; Kissmann 2007) , the history of science (Porter 2006; Nye 2006; Terrall 2006) and the epistemology of science (Zwart 2008). The biographical-narrative method used here combines socialhistorical studies and narrative analysis, and provides an idiographic view on expertise in the form of 'expert narratives'. To produce these narratives, data from various types and sources were ordered around a specific 'plot' or thematic thread Virology Experts in the Boundary Zone 147 (Polkinghorne 2005, p. 69) : the advancement of virologists from laboratory experts into experts on policy and the public domain. The inductive development of narratives involved four iterative stages. In the first stage, data were collected on the academic and public track records of the three virologists, and on the socio-historical context of infectious diseases in which these virologists were situated. This was used to prepare biographical-narrative interviews, 1 the second step. These interviews focused on their experiences in policymaking and public debate. The experts also commented on each other in these interviews. The third step was to organise the data temporally, hereby focusing on the diachronic data (data on events, their relations and the effects of events). This step included the collection of additional data, for example by conducting a second round of interviews. In the final stage, the expert careers were written down as coherent and empirically-grounded narratives, thereby 'explicating an intrinsically meaningful form' (Polkinghorne 2005, p. 93) .
Rather than being the only true reconstruction of past experiences and events, the narratives constitute one possible approach to ordering them coherently. The labels used to structure these narratives must likewise be seen as provisional headings. As to the methodological validity of the narratives that emerge in this manner, it is important to see that the production process of narratives takes place on various levels. The first level is the level of the actual events as experienced by the experts involved, the so-called lived story (Rosenthal 2005, p. 27; Wengraf 2000) . Subsequently the experts themselves will reflect on these events retrospectively and from a certain distance. This level can be referred to as the told story. Finally, as a result of the interactions, post hoc reflections and reframings between interviewee and researcher, 2 the written story as a third level of narrative emerges 3 (Littig 2008, par. 17) . Thus, the written story builds on the two previous levels, and care is taken to secure the internal validity through iterative data collecting and interview sessions, as is also the case here.
Moreover, the written story also feeds back into the previous levels and shapes the lived story. Narrating is a way of constructing ones identity (Brockmeier and Carbaugh 2001, p. 15; Bruner 2001, pp. 34-35) . In the course of constructing a 'front region' narrative, the identities the virologists assume as experts shape their actions and interventions, allowing them to present themselves in a way they find desirable (Goffman 1959) . Narratives not only constrain, they also enable action (Deuten and Rip 2000, p. 72) . By being involved in narrative processes in biographical-narrative interviews, and by intervening in the identity constructions, one actually comes closer to the lived story. 1 Interviews with experts or elites differ remarkably from other kinds of interviews; for a brief discussion see Beate Littig (2008). 2 In this paper I unfortunately cannot illustrate this dimension with a structural analysis of the narratives. For reasons of space I therefore limit myself to a thematic analysis (see Riessman 2008, chapters 3-4) . 3 The virologists themselves also produce written narratives on their roles, creating fascinating comparative research material on narrativity and identity (see Goudsmit 2009; Coutinho 2 February 2010) .
Before turning to the actual expert narratives, I will give a brief overview of the main Dutch institutes and their functions, the institutional landscape as it were. The Ministries of Health and of Agriculture are the two major policymaking bodies in the domain of infectious diseases, while local (Gemeentelijke Gezondheidsdiensten, GGD) and national (Rijksinstituut voor Volksgezondheid en Milieu, RIVM) public health institutes have an executive task. University Medical Centres (UMCs) play a more independent role. A central advisory body is the Dutch Health Council (Gezondheidsraad, GR) . The Health Council is an advisory body that presents the 'state of affairs' of science, and explicitly advises policy, it does not make it (Interview Knottnerus & De Visser. 30 January 2006; Bijker et al. 2009 ). In 2005, the Centre for Infectious Disease Control was established as a specialised body for scientific advice and outbreak control, emerging as a new boundary organisation in the Dutch institutional landscape (CIDC, see Ministry of Health 7 December 2004). The Dutch advisory sector for infectious diseases can thus be characterised as a 'corporatist policy arrangement', with a restricted and formally accredited set of experts that act on personal title (Halffman and Hoppe 2005, p. 137; Bijker et al. 2009, pp. 85-86) . On an international level we find the European Scientific Working Group on Influenza (ESWI), the European Centre for Disease Prevention and Control (ECDC) and of course the World Health Organization (WHO).
This landscape constitutes the backdrop for the three biographical narratives. Osterhaus started his career as a veterinarian at the RIVM (1978) and went to Rotterdam UMC in 1994. Coutinho worked at the Amsterdam GGD from 1977 until 2005, after which he moved to the CIDC. He has always been affiliated with the Amsterdam Medical Centre (AMC). Goudsmit also spent much of his career at the AMC; in 2002 he joined Crucell, a Dutch biotech vaccine company.
In discussing the expert narratives, we divide the field of virology into a number of stages. Roughly, we identify five stages, based on the alternation of times of crisis and of control. Then, all of a sudden in 1982, HIV/Aids emerged as an unknown enemy in the Dutch gay scene. It constituted a challenge and problem for human virology, accompanied by a sudden influx of research funding, public concern and policy development. 5 Furthermore, there was an aggressive viral outbreak in seals in the North and Baltic Seas around 1988, which killed tens of thousands of seals. BSE was seen just before this, putting veterinary virology firmly on the map and introducing virologists into the midst of political and public debate. As it turned out, these crises were a springboard to visibility for the three virologists studied here.
At the start of the 1990s, these crises had more or less been brought under control. The causal agents were discovered and the era saw the development and implementation of policies in routine veterinary and human healthcare practices. The HIV/Aids epidemic stabilised in the Netherlands and government interest in infectious diseases decreased. This stability would last until 1997, when new infectious threats suddenly emerged. For the virologists this was a moment to once again assume visibility in public debate and involvement in policy development. The first H5N1 deaths marked the advent of a second crisis. Besides fears of an avian influenza pandemic, scientists and politicians were concerned about the growing threat of bioterrorism and outbreaks of foot and mouth disease. Finally, the outbreak of SARS in 2003 came as a complete surprise. It was SARS that highlighted the real danger of pandemic outbreaks, but that also triggered the awareness of preparedness and prevention plans as new instruments in preventing a pandemic from developing to its full capacity (Osterhaus 6 March 2009).
Towards the end of this period of declining public and political need for virologists, all three virology experts completed their studies. Their careers were to become heavily intertwined with the events that followed; they had positioned themselves in such a way that, unaware of future challenges, their involvement with policy and the media during the first crisis was evident.
Although Osterhaus had been in the media since 1978 (on the death of dogs at the world dog show), his story really begins in 1985 with Bovine Spongiform Encephalitis (BSE). In the early years, no one believed BSE could become a human pathogen; 'people said it was just scrapie' (Osterhaus 6 March 2008). 6 However, Osterhaus suspected it could indeed become a human pathogen, and in 1988 he pursued it to the EU policymaking arena with backing from his director. As chairman of the EEC Scientific Veterinary Committee he acted cautiously. Nevertheless, he met with scepticism, since pointing out the possible public health dangers went against the grain of policymakers. He did not refrain from setting policy machinery in motion, and a proposal for legislation on the various dangers of BSE was issued almost monthly. In his opinion, this had its effect on policy processes (Osterhaus 29 July 2008).
In 1988, he became more visible to the general public with the dramatic death of seals in the North Sea. 7 Osterhaus was called in to investigate these deaths, and discovered that the seals had died of a previously unknown virus. However, Greenpeace opposed his conclusion: they were convinced that pollution or poisonous algae had caused the fatalities, since this fitted their own objectives. This disagreement with the environmentalists challenged Osterhaus to prove he was right, and he conducted extensive experiments with seals. To his surprise these studies did identify pollution as a contributing factor in the seals' deaths, but as an indirect causal factor: it made them less resistant. Osterhaus considered this a success: he had discredited his own hypothesis and discovered a causal chain more nuanced than either he himself or Greenpeace had expected (Osterhaus 6 March 2008). The research on seal death, that started in 1985 and has continued ever since, has had an enormous impact on several aspects of Osterhaus' career. It delivered him his first four Nature publications in 1988 and 1989, and many other papers followed. His reputation grew, and the conflict with Greenpeace pulled him into political and public debates. Due to the seal death research he also became scientific advisor to a national seal shelter.
The controversies surrounding BSE and the seal deaths show two aspects of Osterhaus' role. He was self-confident in his estimations about risks, but also acknowledged criticism from his sceptics. The tricky question was when to stick to his original position, and when to adopt the opposing view, and, consequently, the appropriate strategy. This, he says, created a tension: on the one hand he had his own gut feelings, and at the same time he tried to think through competing viewpoints. There was not only an epistemological choice to be made, but also a balancing act to carry out: considering various interests and health threats. We will characterise his role as that of the strategic scientist: someone who carefully balances different viewpoints and forms of evidence, but who also has the strategic skills and will to further his own case.
Of the three virologists studied here, Coutinho's role was the one shaped the most by the nature of his research and the positions he held. As a public health director in Amsterdam with expertise in viral epidemiology and professional relations with homosexuals and intravenous drug users, it was obvious he would be involved in dealing with HIV/Aids, and he was very motivated to do so.
In 1984, Coutinho, Goudsmit and their superior, Jan van der Noordaa, initiated the Amsterdam Cohort Studies (ACS) 8 on HIV/AIDS, and began testing a cohort of homosexual men. The results were alarming: 30% of the participating 750 homosexuals were infected with HIV, revealing an urgent public health issue. However, as homosexuals were in a process of emancipation, they feared discrimination and blame for bringing HIV/Aids into the world. Disease prevention did not outweigh this concern. Since Coutinho saw that both homosexuals and drug users tended not to take the danger of infection seriously (as they had previously done with venereal diseases), he used the statistics to make them aware of the scale and nature of the epidemic. His rationale was that without being open about a problem, one cannot start solving it. Also, covering up the truth implies a disdain for society and is likely to result in a loss of credibility (Coutinho 13 May 2008; Mooij 2004; Andere tijden 26 November 2002) .
Although a sound approach for Coutinho, it met with substantial opposition on various fronts. Indeed, the slightest hint towards the identification of homosexuals as a risk group made them revolt; and to Coutinho's dismay the national government gave equal weight to the control of HIV and the prevention of discrimination. A chasm emerged between scientific and socio-political realities, 9 but one that could be handled within the National Committee on AIDS Control (NCAB) to which he belonged. 10 If the scientists were taken by surprise by the HIV/Aids epidemic, then the media were even more so. They desperately needed information, and Coutinho and other members of the NCAB were in a position to virtually control the coverage of HIV/ Aids (Mooij 2004, p. 21) . By doing so they also influenced policy (Coutinho 13 May 2008) , since politics, especially in those days, was very susceptible to public opinion.
Despite this chasm, in the beginning he perceived and experienced the collaboration with homosexuals as 'extremely good, very constructive and also very useful' (Coutinho 25 July 2008) . But tensions increased as time progressed: HIV/Aids spread, the number of parties involved increased and the decision-making structure grew more complex. From 1986 onwards, when a new information campaign was launched, Coutinho's critical, factual, and open attitude was too problematic for those working with him; he openly stated that the campaign masked the fact that homosexuals were a distinct risk population (Coutinho 25 July 2008) . Following this criticism, the productive working relationship with the gay community started to crumble.
On two occasions in 1989 the tensions between Coutinho, homosexuals and politics culminated in conflict. A clash with politics came when the Secretary of State for Health rejected a report on anonymous HIV testing. Coutinho, one of its authors, openly mocked the Secretary and he was brought to account. Although this was an unpleasant experience, Coutinho said he would do the same again (Coutinho 13 May 2008) . And so he did. Later that year he was appointed professor, and in his inaugural lecture he surprised friend and foe by again criticising the HIV/Aids communication policy of the NCAB. Members of the NCAB and of the gay community were outraged: how could someone like Coutinho threaten the societal position of homosexual men? Coutinho was forced to step down from the NCAB.
But how dramatic was his departure? The information campaigns were up and running (if not without some hiccups), a policy framework was in place and 'personal relations were fine' (Coutinho 25 July 2008) ; he actually felt relieved to be out of the NCAB.
Coutinho's role during this stage started out as critical collaborator, and it shifted to controversial collaborator after 1986: he was very outspoken, stubborn, did not negotiate with others on the contributions he made to the media, and he did not let himself be tamed by the sensitivities of gay emancipation. He described this period as being jammed between science and policy. 11 The collaboration in the control of HIV/Aids became gradually more problematic, which resulted in this shift in Coutinho's role.
Goudsmit's role contrasts those of Coutinho and Osterhaus. He was not heavily involved in the development of health policy frameworks or legislation, as his sphere of activity was more limited to public debates. These debates included the debate on gay blood donors and on the origins of AIDS. When invited to reflect on the 1980s during interviews, he did not recount specific moments, but rather gave a general impression of his contacts with the media. What dominated was the sense of losing control over his utterances in the media, and this had a lasting influence on Goudsmit's attitude towards the media (Goudsmit 2 April 2008) .
One event in the expert development of Jaap Goudsmit, which gradually takes us to the end of the second stage, is an affair that shocked many: the Buck-Goudsmit affair. 12 Early in 1990 Henk Buck, a professor of chemistry, wanted to check whether a DNA drug he had discovered could work on HIV. Goudsmit therefore ran tests with the substance, and mistakenly confirmed that it worked. A letter was sent to Science and Buck and Goudsmit held an official press conference. This was, after all, a discovery of great importance.
The positive atmosphere turned bleak when Buck started making claims that went beyond the original significance of the discovery, promising a cure within a few years. People were outraged, since HIV/Aids patients were given false hope. While Buck himself was scapegoated, Goudsmit got away, but not entirely untouched. The drama affected him, and he became more reluctant when called on as an expert and more cautious when in contact with the media (Goudsmit 2 April 11 Every time he spoke about this, he made a wrenching gesture with his hands. 12 See (Hagendijk and Meeus 1993; Eijgenraam 1991; Andere tijden 29 November 2005) .
Virology Experts in the Boundary Zone 153 2008). Despite criticism of his role in the controversy, Goudsmit kept his position at the AMC; and when three years later Science announced him as Europe's most important HIV/Aids researcher (Cohen 1993 ) the Buck-Goudsmit affair was not mentioned at all. During the second interview Goudsmit described his 1980s self as a hubristic expert (Goudsmit 19 May 2008) , and other sources support that view: someone who is very confident about his knowledge base. This stage of his career involves frequent loss of control over statements and quotes: being tempted by journalists to utter sweeping statements and daring analyses. Analysing the interviews with Goudsmit, we find two interrelated problems for the hubristic expert. The first is a lack of experience in controlling the media; the second is a lack of knowledge of the limits to his expertise. As we will see, a gradual shift in both these aspects later in his career would change his role.
Stage 3: 1993-1997: Reaching Relative Stability: Controlling the Current Crisis
In 1993, some 10 years after the first Dutch cases of HIV/Aids, the epidemic stabilised, a partial indication that the expert community had gained relative control over the situation. The general implications were that HIV/Aids became less newsworthy, which of course had implications for the roles of the scientific experts, in particular Roel Coutinho. Transformation is a recurring theme in this third stage: positions, whether in a political, institutional, public or epistemological sense, changed, and the role of our experts changed accordingly.
Osterhaus experienced a number of changes that were to make him even more visible than before. An important change in this respect is that of occupational context. In 1994, he left the RIVM, 'the institute that muzzled him' (Osterhaus 29 July 2008), when he refused to end his seal research (NRC Handelsblad 14 April 2003) . He and his staff moved to the Erasmus Medical Centre in Rotterdam, where he still holds a position.
This change from a muzzling institute to a university that was not familiar with 'ministerial responsibility' liberated him in his role as public expert; and with this change a second came along: he became director of the WHO National Influenza Centre. Influenza appeared on his radar screen and Osterhaus became interested in past influenza pandemics. He then became chairman of the European Scientific Working group on Influenza, the ESWI. On a more personal note, being struck by influenza himself made him experience the severity of flu. These personal, professional and scientific experiences with flu would from now on become a dominant factor in his contact with policy and the media (Osterhaus 29 July 2008). In 1995, he made some initial, cautious remarks that hinted at the lurking threat of influenza (Trouw 17 February 1995) . But in 1997 the cautiousness gave way to a much more alarming message when the first human bird flu casualty was reported (Osterhaus 6 March 2008). Osterhaus and others wrote a letter to Nature entitled A Pandemic Warning (De Jong et al. 1997) , in which they referred to H5N1 as a virus with 'unknown pandemic potential'. A few months later he would be more straightforward in a Dutch newspaper, saying that 'a future pandemic flu on the scale of the 1918 Spanish Flu was not unimaginable' (Algemeen Dagblad 30 December 1997) .
The flu deaths were to trigger the second crisis in the recent history of virology. In contrast to the first crisis, when the virologists were taken by surprise, Osterhaus became the advocate of awareness of and preparation for influenza. From now on, a discursive strategy announcing the coming pandemic was aired in public media and in policymaking, using the pandemic warning as an instrument to create awareness and a policy forum (Parool 26 April 2001) . This led to the charge from politicians and colleagues of being a prophet of doom (Osterhaus 6 March 2008), which marks the role he had during this stage.
Although not of his own bidding, Coutinho's preoccupation with policymaking and the media decreased significantly after 1992. A quite visible expert began to hibernate. He felt content with these developments since he had become worn out with endless debates in committees and with telling the same stories to the media over and over again. Expert hibernation in this sense does not mean he was entirely absent in the media or in policymaking, but compared to the preceding and following periods there was a significant decrease of exposure. There is one event, however, which demonstrates his view as an expert on health problems. After a decline in HIV/Aids cases in 1993, he later reported the epidemic to be far from over, and stated how he thought the problem should be prevented: 'It makes a huge difference when you get the problem out in the open' (Trouw 5 December 1998) . This strategy was put to the test during the 1980s, and it passed. He says that it has been the key to his expert policy and still is today.
Goudsmit's role also changed during this stage. Although at the end of the second stage he was recovering from the Buck-Goudsmit affair, in retrospect he claims the whole affair did not fundamentally change his ideas on how to be an expert.
The two processes that we identified earlier-learning to control the media and becoming more aware of his knowledge base-are drawn to a close at the mid-1990s by the realities of vaccine research. In 1990, Goudsmit and Osterhaus received funding to develop a candidate HIV vaccine. In 1992, the former was very confident about the prospects for such a vaccine (Trouw 24 March 1992) ; but just two years later we see a mixed attitude: Goudsmit is still optimistic regarding the possibilities of a vaccine, although he thinks that it will require international coordination and prolonged collaboration (Parool 26 March 1994) . That is what he Virology Experts in the Boundary Zone 155 therefore endeavours: in 1994, Goudsmit becomes a co-founder of the IAVI, the International Aids Vaccine Initiative. In the meantime, the joint project with Osterhaus fails, in part because they 'entirely underestimated the difficulties in developing a vaccine' (Goudsmit in Mooij 2004, p. 164 ). Goudsmit is currently still active in the development of vaccines, but his prospects for an HIV vaccine are sombre (NRC Handelsblad 1 December 2007; Goudsmit 19 May 2008) . In Goudsmit's own words, the hubristic expert discovered that he was not that knowledgeable at all, that despite all his knowledge about HIV he could not produce a vaccine. This is what in effect changed the way in which he performs as an expert in public. As he became painfully aware of the limits of his expertise, Goudsmit changed into an expert who wants to be in strict control of what to say, when, where and to whom (Goudsmit 2 April 2008).
It was during this stage that Osterhaus incited awareness of a potential pandemic. He became a key figure in the Dutch, European and WHO pandemic preparedness plans, and he aired his pandemic warning ever more frequently and urgently, acting as a catalyst for policymakers. But other threats, such as the threat of bioterrorism (Gezondheidsraad 2001 (Gezondheidsraad , 2002 , foot and mouth, and later SARS, were also posing challenges for virologists. The greatest fears were, and still are, zoonoses: animal viruses that evolve into human pathogens (Gezondheidsraad 2005) . Collectively, this amounted to the second crisis.
Following the H5N1 deaths in 1997, Osterhaus unfolded a scenario that matched his earlier response to BSE. Virologists, including Osterhaus, urged the WHO to develop pandemic preparedness plans, which was done instantly. In an individual capacity, he then initiated a meeting to discuss the appropriate policy responses for Europe. Besides him initiating the meeting, it is interesting to see how he organised this meeting and steered its outcomes. Osterhaus was a well-known and respected figure in the European health policy scene, belonging to some of the key scientific or health care committees. A first move he made when organising the 1998 meeting was to hand it over to an EU civil servant, now the meeting's captain. The civil servant would take credit for this meeting, and it would also be political. The second move was to select the participants, including the responsible EU commissioner and a range of influential virologists. By selecting the participants himself, Osterhaus brought together a specific body of political power and scientific expertise; and he managed to orchestrate the political process in a direction he deemed optimal without drawing attention to himself. Osterhaus strategically translated his pandemic warning into an official and visible EU committee.
From then on, a discursive strategy in the media invoked a coming crisis: 'We have had three pandemic outbreaks of influenza, in 1918, 1957 and 1968 , killing some 100 million civilians. We can be sure a new pandemic will arrive, and unless we take appropriate measures, hundreds of thousands will die'. Although the pandemic warning was a policy lubricant (Parool 26 April 2001) , Osterhaus believed in the reality of this message and he says it was a plain technical assessment (Osterhaus 6 March 2008). His colleagues, however, believed the message to be an overstatement, bordering on lobbying for research funds (Coutinho 13 May 2008; Goudsmit 2 April 2008 , 19 May 2008 Van der Noordaa 7 May 2008) . They also say that Osterhaus' tone has tempered over the years, perhaps because the discursive strategy has had its desired effects, perhaps because the scientific reality has changed. 13 This tension between his own scientific gut feeling and the scepticism of his critics is a dilemma he dubbed the 'cry wolf dilemma'. This 'visionary virologist' had to perform a balancing act between two extremes: on the one hand the credible but reticent academic, on the other hand the 'incredible fool' (Osterhaus 6 March 2008); and the longer it took for a pandemic to strike, the more his credibility would be at stake.
His public appearances became numerous as the concerns over bioterrorism, influenza, bird flu and SARS increased, and as the Dutch government neglected public communication. The media's solution to this latter problem was straightforward: call Osterhaus. He is always willing to spare a moment since he believes science communication is part of a scientist's professional ethos (Osterhaus 6 March 2008). These appearances amounted to a clear media policy with a handful of tricks to safeguard his public credibility.
It was not only the media who sought his assistance. In the midst of the BSE outbreak of 2000, Osterhaus was also asked to personally advise the Minister of Agriculture, who appeared to be ill-informed by his own staff. That same afternoon, the minister informed Parliament of his BSE policy. Osterhaus heard the same sentences he had spoken that morning, and in the interview says he felt relieved: 'Democracy was still able to function as it should' (Osterhaus 6 March 2008). Civil servants lack the expertise to make informed decisions and they need experts to give advice, even if that means importing their political agenda.
2003 was a busy year for virology, but especially for Osterhaus. In addition to his professorship and his extensive policy work, both avian flu and SARS struck in the spring of that year. He became involved in both of them. One moment that Osterhaus especially recalls regarding avian flu is a meeting of the Outbreak Management Team (OMT) to determine the necessary policy. The majority opposed Osterhaus' policy view, and rather than conforming, he tried to enforce his view by threatening them with a minority point of view, sketching what might happen should the OMT abstain from following his advice. It proved effective, but also demonstrated the fragility of his position: had he not been there, policy would have been very different (Osterhaus 6 March 2008).
When SARS struck, the pandemic preparedness plans came into effect. Under the direction of the WHO a small selection of laboratories, including Osterhaus' own lab in Rotterdam, were on the hunt for the cause; and on 16 April 2003 the Osterhaus lab managed to find the final and most rewarding piece of the puzzle. A press meeting was held at the WHO Geneva headquarters that same afternoon; a letter in Nature (Fouchier et al. 2003 ) and a royal decoration followed. Osterhaus reaped the rewards for the discovery and his status rose (Enserink 2003) . But what is more: the WHO prevented a pandemic. This marks the SARS crisis as a moment when virologists could control a crisis. Osterhaus himself noticed that his credibility, authority and power in science, politics and society rocketed.
However, the various cases he narrated illustrate the strategic position Osterhaus held, a position that slowly became problematic. The many 'hats' he wore and responsibilities he carried, both nationally and internationally, confounded his expert role. He says it was manageable to know when to speak from which position and responsibility, but outsiders saw this same person in quite different settings, taking up both scientific and political roles in one same interview (see for example Netwerk 21 February 2003) . Other virologists noted that he was doing the government's dirty work (Osterhaus 29 July 2008; Goudsmit in NRC Handelsblad 5 July 2003), and even a ministerial letter to parliament had to justify the way Osterhaus' view was accommodated in policy (Veerman 23-04-2003) . In the second crisis, Osterhaus had scientific credibility and political responsibilities, he made scientific and strategic statements, and he spoke in both a personal and collective capacity, making him an entangled expert.
The hibernation of Coutinho lasted until 2000, when after more than 20 years at the public health division of the Amsterdam GGD, Coutinho became scientific director of the entire institute. In his new position his role as expert changed. HIV/Aids was still on his agenda, as were venereal diseases in general, but as the second crisis called for experts to master a wide array of human and veterinary infectious agents, he also had to inform the public on a broad portfolio of diseases. The number of public appearances increased dramatically, and he once again became a familiar figure in the newspapers. 14 This increasing diversity of topics was welcomed, but it also implied challenges, as he now had to keep track of all kinds of developments and topics that went beyond his original expertise (Coutinho 25 July 2008) .
How did he cope with this? In really complex cases, Coutinho says, he referred the journalist to a knowledgeable colleague. But in most other cases, Coutinho did it himself. He read up on the subject matter, called a few people, and then appeared in the media to comment or inform. The major challenge was to decide when he felt knowledgeable enough not to consult others. We note that this back region (Goffman 1959) , the preparation, remains invisible to the public view; that which is visible in the front region is the knowledgeable expert, who then takes the credit.
The role he takes up is that of spokesperson and translator: speaking on behalf of the institute, and translating the specialist expertise residing there to public or political contexts (Coutinho 13 May 2008) . There are a number of reasons to do so. First of all, he believes that it is part of his responsibility to carry the risks involved here. Secondly, he is the experienced expert, aware of the intricacies of communication. Thirdly, he enjoys communicating with the media, and this is stimulated by the many successes he has experienced (Coutinho 13 May 2008) . Consequently, he is again a familiar face in the media. As the topics have become far less controversial and political than they were during the 1980s, the contributions to policy and to the public sphere are also more technical (determining risks, developing preparedness plans). This makes him a technical spokesman.
In the fourth stage we see several changes affecting Goudsmit's expert role. During the mid 1990s, he held a full professorship at the Amsterdam Medical Centre, and the IAVI was a side project. After 2002 the balance started to shift. He moved to biotech firm Crucell in 2002, where he is now director of research and member of the board. He ended his board membership of the IAVI in 2004, and he swapped his full professorship for an honorary one.
In addition to the professional changes, as a virologist he became more cautious and reserved regarding what he knew. From studying his narrative, we see that 'taking control', that lesson from the 1980s and 1990s, is accomplished at two levels: on the communicative level by knowing when to stop talking, and on the strategic level by choosing the right battlefields. It is his habit of selecting the more regulated battlefields that is a very distinct feature of his expert career after the mid-1990s (Goudsmit 2 April 2008) . He chooses spaces he feels comfortable and secure in, spaces that respect the strict boundaries of his expertise. Although these factors are important in understanding why Goudsmit takes up a certain role, in practice the different constituents are difficult to discern. Two loci where he prefers to act are the courtroom and the study, writing popular science books. In a long laudation on his experiences as a courtroom expert, 15 he describes it as the site where expertise is used in the 'purest way possible' outside of science, where all agendas lie open and only the expert's knowledge is questioned. The courtroom enhances the separation of facts and their interpretation; the juridical boundaries create a regulated environment, which is appreciated by Goudsmit (Goudsmit 2 April 2008) .
Another preferred site for showing his expertise is his writing desk, where he is in charge of the content and form. Since the turning point in his expert biography in the mid-1990s, a steady stream of popular science books has been published. He tries to communicate technical knowledge to the public in the purest possible way, although this has not been well received by either book reviewers or editors, who say his books are too technical, too hard to read, or promise more than they actually contain (Goudsmit 19 May 2008; Trouw 17 June 2000 , 1 March 2003 .
Next to these 'battlefields' that are cherished for their safety, Goudsmit avoids policymaking, for he loathes the conflicts of interest and the consensus finding. He has developed a love of purity in expertise that excludes policy rooms. Because of his love of purity, the preference for a clear expert role and the resulting choices, we refer to him as the puritan. Following years of a pandemic warning, the rapid spread of SARS, and numerous actual or possible outbreaks, politicians in the Netherlands were well aware that the lack of political responsibility, coordination, and strategic planning for outbreaks needed to be addressed. Furthermore, in 2003 a veterinarian died after being infected with bird flu. 16 This focusing event (Birkland 1997) triggered the shift to the fifth stage, during which there were endeavours to increase control over the viral threats. What is remarkable about this shift is that although the virologists occupied different positions and held different views on the science-policy-public nexus, they pushed it through collectively.
Goudsmit chose to write an open letter to a Dutch newspaper with the provocative title 'The Netherlands are unprepared for a viral attack'. The message was that the government was failing to take responsibility for the control of epidemics and public communication; people like Osterhaus were doing what the government should have been doing (NRC Handelsblad 5 July 2003). The Director-General of the Ministry of Public Health replied, in which he acknowledged part of the critique (NRC Handelsblad 17 July 2003). Osterhaus and Coutinho also campaigned for a new infectious disease infrastructure, referring to SARS and avian flu as the wake-up calls. Osterhaus continued his alarming predictions of a pandemic, and even Coutinho made dramatic statements to create awareness (Volkskrant 5 June 2003) , which is contrary to his factitious style of communicating. It was, in his words, steering between the Scylla of a failing outbreak management and the Charybdis of creating a panic (Coutinho 25 July 2008) .
The collective efforts proved effective: in the course of 2004, a decision was taken to install the Centre for Infectious Disease Control (CIDC) at the RIVM, a centre that, under political accountability, would coordinate outbreaks of infectious diseases and handle public communication (Ministry of Health 7 December 2004). This new instrument for control embodies the fifth stage.
When the government planned the CIDC in 2004, Osterhaus was officially consulted regarding these plans and asked to stand as candidate director. Alarmed by the fact that the government would set up the centre without proper consultation with the Health Council, Osterhaus informed them of this and suggested a consulting committee. The next step was the selection of a director. Osterhaus turned the offer down, as he had had his share of the RIVM in the past. In his view, the only other person capable of the job was Coutinho, and Osterhaus personally phoned him (Osterhaus 29 July 2008); Coutinho accepted. Again, Osterhaus was tying a policy process and its outcome together by using formal and informal relations and his credibility in policy; this, again, took place in the back region.
The implication of the CIDC was that the political complexity surrounding Osterhaus was temporarily resolved, and that there was a government voice for public communication. Meanwhile, Osterhaus' campaign for more pandemic vigilance continued. Starting in 1997, major steps were taken in this direction, such as the national storage of antiviral drugs (Gezondheidsraad 2005) .
Osterhaus' role in avian flu and in the successes regarding SARS resulted in a dramatic increase in public, political and scientific credibility. Through his countless media appearances he gained the status of a public figure, and some even call him 'the David Beckham of virology' (Volkskrant 10 February 2009) . He seems to be an invincible expert, not just because of this increase in credibility, but also because the risks of entanglement had been resolved. But as the H1N1 pandemic demonstrated, 'David Beckhams' are particularly prone to loss of credibility; there was great upheaval with regard to Osterhaus' commercial, scientific and political interests (Enserink 2009 ).
Coutinho has been director of the CIDC since January 2005 and his main responsibility has been to set up a new control structure for infectious diseases. Zoonoses are by far the greatest threat, and there is considerable conflict between the interests of agriculture (a melting pot for infectious agents) and of public health. Notorious cases are MRSA and Q fever bacteria (Coutinho 25 July 2008) . Coutinho is caught in the middle of both ministries, and acts as a kind of buffer. Whenever there is an outbreak, Coutinho and his centre are on top of it, with regard both to outbreak management and to public communication. The CIDC has the data, the expertise and the responsibility to do so. He is still a spokesman and translator, but now under ministerial responsibility.
Coutinho's role has led to a dispute between Coutinho and Osterhaus. Coutinho describes himself as an expert who is independent of the institutional setting and always does what he believes in. Osterhaus, however, depicts Coutinho as a governmental expert, since he falls under direct responsibility of the health minister. Osterhaus recounts how he has seen Coutinho change over the years, becoming less outspoken, and says that his political accountability has contributed to this (Osterhaus 29 July 2008). Although Coutinho clearly experiences the political pressures, he says it has not changed him; he is not the governmental representative Osterhaus claims he is. He is less outspoken, since there is no need for him to act otherwise (Coutinho 13 May 2008) .
The debate of his role is illustrative for the embedding of the CIDC as a new 'boundary object' in the Dutch institutional landscape, combining science, policy and communication in a novel way.
Following his interventions in infectious disease control in 2003, Goudsmit repeated the same strategy in 2004. In the same form and in the same newspaper, he castigated the Dutch education policy and pleaded for a dual university system (NRC Handelsblad 10 June 2004). This was not based on his expertise as a virologist, but on his experience in academia and in industry. Again, this letter evoked much response, some of it critical, and a documentary (Tegenlicht 5 February 2006) and book essay followed (Goudsmit 2006) .
In addition to this interest in education, he is still writing popular science books on virology. This is probably the only topic in which he still publicly discusses his expertise in virology. He is constantly experimenting with form, and in his present book he is again trying to communicate pure knowledge, which remains difficult. The resulting book is Dromen van vaccines ('Dreaming of vaccines', Goudsmit 2009 ).
The puritan expert keeps the hubris on a leash, and Goudsmit is very selective in his expert dealings. He nevertheless displays what he calls his 'hobbies' (Goudsmit 2 April 2008) : writing novels and sharing his views on issues he feels knowledgeable about. This combination of a strict expert acting within virology and a hobbyist outside of virology leads to the role of hobby expert.
This article has studied from a biographical-narrative perspective how Albert Osterhaus, Roel Coutinho and Jaap Goudsmit positioned themselves over the years as policy and public experts in the field of infectious diseases. Their narratives covered five stages, from the late 1970s until April 2009, when the swine flu pandemic struck. Right from the start, they were all simultaneously conducting research, drafting public health or veterinary policy based on that research, and communicating to various publics and patient groups on the diseases. In some cases, communication was used strategically to induce policy change (Birkland 1997; Kingdon 1995) , but often enough communication was aimed at solely informing the public on the status quo of outbreaks.
Of the various elements of the experts' biographies, two aspects stand out from the rest: the personal reflections of these experts on their roles; and the tandem development of governance arrangements and the role of these virologists therein.
To start with the first: the biographical approach shows us how the early, trial and error methods of 'being an expert' steadfastly developed into more crystallised views on their roles. The experts based their methods on the early-career understandings of the relationship between science, politics and the public domain. This article opens up the biographical-narrative 'black box' of these experts, and the enactment of that biography in practice (Goffman 1959; Deuten and Rip 2000; Brockmeier and Carbaugh 2001) . Learning experiences, personal or communal reflections on specific events, the continuous process of re-narrating and reframing past experiences, all added up to an individual conviction on 'how to perform as a credible expert'. In Coutinho's case, we might say that his self-perception as an expert has not changed dramatically, but solidified in a repertoire of communicating the facts. Goudsmit, on the other hand, following a redefinition of his expertise, radically changed the way in which perceived his role in policy and in public debate. Then we have Osterhaus, who still finds himself in a continuous balancing act between a credible scientist, a policy entrepreneur, a public communicator and an industry advisor. The biographical-narrative approach has elucidated how each of them have accommodated themselves in the boundary zone between science, policy and public debate, and thus juxtaposes with a more macro-sociological understanding of expertise, such as Nowotny's 'narrative of expertise' (Nowotny 2000) .
The second aspect that stands out is the development of governance arrangements. The studies of Coutinho and Osterhaus demonstrate that since they were pioneers in the development of new governance arrangements, they remained central figures in these arrangements for infectious disease control. The emerging governance structures and the pioneering virologists remained tied together. It is no accident that when the CIDC was being set up, Osterhaus and Coutinho were the prime, if not only, candidate directors. Up until the H1N1 pandemic, these two virologists were the key figures in policymaking and public debate. In cases where new governance arrangements emerge, we can thus expect the leading figures in that process to remain tied to those arrangements. It is certainly relevant to scrutinise the degree in which the experts themselves strive to remain tied, whether the arrangements as such keep them tied, and why. This article demonstrates how the involvement of certain figures is understandable from a historical and biographical perspective.
However, with shifting challenges and changing social, economic and political contexts, it may be desirable to renew the composition of expertise in governance arrangements. Without wishing to pass direct judgment on either Coutinho or Osterhaus, I suggest that biographical ties should not grant access by default. Probing these dimensions, as this study does, assists in the assessment of the nature and structure of contemporary governance arrangements, the persons involved and their relationships to science, politics and the public sphere. Although this does not provide a prescriptive approach to expertise, such as that suggested by Collins and Evans (Collins and Evans 2007) , it may help to critically question the almost automatic policy involvement of some experts.
The recent H1N1 pandemic demonstrates that this problematisation is not merely a scholarly exercise. From the first announcements of the new virus, Osterhaus and Coutinho dominated politics and the media. Their performances raised a plethora of comments and questions and, to put it mildly, were not undisputed (Van Rijswoud 2009) . This led to the organisation of a public debate with Osterhaus and Coutinho, inviting them to publicly reflect on their roles (see Dortmans and Van Rijswoud 2009) .
A last and important conclusion of this case study is that during the recent pandemic, the historically grown roles of Osterhaus and Coutinho did not resonate with the changing ideas of various public domains on the roles of experts. As norms and expectations for credibility and authority change over time, these experts cannot rely on the crystallised understandings of their roles and their settled positions in governance arrangements. During the course of the pandemic, for various reasons, Osterhaus' and Coutinho's credibilities were severely damaged and their authorities disputed. Osterhaus' balancing act could not withstand the growing wave of criticism (Enserink 2009) , and Coutinho's notion of medical authority was challenged down to the bone (Coutinho 2 February 2010). These controversies raise a call for a more thorough reflection on their roles, both within governance and public communication. Answering that call, this article tells us, is a continuous struggle and challenge. Human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent enteric infections A new species of parvovirus tentatively named human bocavirus 4 (HBoV4) was genetically characterized. Among 641 feces samples from children and adults the most commonly detected bocaviruses species were HBoV2>HBoV3>HBoV4>HBoV1 with HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children. HBoV3 and HBoV4 species combined were found in 12/192 cases of non-polio acute flaccid paralysis (AFP) from Tunisia and Nigeria and 0/96 healthy Tunisian contacts (p=0.01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within species was found. The multiple species and high degree of genetic diversity seen among the human bocaviruses found in feces relative to the highly homogeneous HBoV1 suggest that this world-wide distributed respiratory pathogen may have recently evolved from an enteric bocavirus, perhaps after acquiring an expanded tropism favoring the respiratory track. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases including AFP and diarrhea will require further epidemiological studies. B19 in 1976, several other human parvoviruses have recently been genetically characterized. PARV4 was found in the blood of a febrile adult intravenous drug user [5] , HBoV1 in the nasopharyngeal secretion of a child with respiratory problems [6] , HBoV2 in the stool of children with non-polio acute flaccid paralysis (AFP) [7] , and HBoV3 in the stool of Australian children with diarrhea [8] . In contrast to PARV4 or B19, bocaviruses contain a third open reading frame of unknown function [9, 10] .
The first bocavirus identified was in cows [10] , and the name of the genus is derived from its first known hosts (bovine-canine). Animal bocaviruses can cause both respiratory and gastrointestinal diseases, as well as embryonic and fetal death [11] .
HBoV1 infection has been linked with mild-to-severe, primarily lower respiratory tract infections in children, frequently in association with other viral infections [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . HBoV1 has also been detected in stool samples, although association with diarrhea appears weaker than with respiratory symptoms [26] [27] [28] [29] [30] [31] . HBoV1 strains show a very low degree of genetic variability worldwide [32, 33] .
In this study, pan-bocavirus polymerase chain reaction (PCR) primers were designed and used on human stool samples from several countries. All 3 recently identified bocaviruses plus a fourth species, HBoV4, were identified. A high degree of genetic diversity, relative to that seen for HBoV1, was seen among human bocaviruses in feces.
Stool samples were collected as part of previous clinical studies and were anonymized. This study was approved by the University of California San Francisco Committee on Human Research. Samples from Nigeria and Tunisia were collected as part of the World Health Organization's poliovirus eradication program from children with non-polio AFP between the ages of 4 months and 15 years. Stool samples from healthy contacts of case patients from Tunisia were matched for age. Stool samples from Nepal were obtained from adult travelers and resident expatriates with diarrhea with no known pathogens detected by standard microbiologic testing for enteric bacteria; enzyme immunoassay for rotavirus, adenovirus, astrovirus, Giardia and Cryptosporidium; and reverse transcription PCR for norovirus. Stool samples from healthy, asymptomatic control persons were collected from the same population. Stool samples from the Minnesota Department of Health are from individuals with diarrhea and healthy individuals, matched for age and residential area code.
PCR amplification of bocaviruses. We used nested PCR targeting the VP1/2 region of both HBoV1 and HBoV2 (nucleotide positions 3233-3808, numbered here and subsequently with use of the HBoV2 prototype sequence; GenBank accession number FJ170278). Nucleic acids (both DNA and RNA) were extracted (QIAamp Viral RNA mini kit) from 140 mL of clarified stool supernatant and were eluted into 60 mL of water. First-round PCR primers were AK-VP-F1 (5 -CGCCGTGGCT-CCTGCTCT-3 ) and AK-VP-R1 (5 -TGTTCGCCATCACAAA-AGATGTG-3 ) and second-round primers were AK-VP-F2 (5 -GGCTCCTGCTCTAGGAAATAAAGAG-3 ) and AK-VP-R2 (5 -CCTGCTGTTAGGTCGTTGTTGTATGT-3 ). PCR reactions contained 2.5 U of Taq DNA polymerase (NEB) in 1.1ϫ Thermopol reaction buffer with MgCl (2.0 mmol/L), 20 pmol/ L (each) of forward and reverse primers, and 2.5 mL of nucleic acids (first round) or 1 mL of the first-round PCR product (second round) as template in a 50 mL total volume. Firstround conditions were 10 cycles at 95ЊC for 35 s, 58ЊC for 1 min, and 72ЊC for 1 min, with a decrease of 0.5ЊC in annealing temperature each cycle; 30 cycles at 95ЊC for 30 s, 54ЊC for 45 s, and 72ЊC for 45 s; and a final extension at 72ЊC for 10 min. Similar conditions were used for the second PCR round, except that the initial annealing temperatures were 60ЊC and 58ЊC in the first and second group of PCR cycles, respectively. Amplicons of the appropriate size, as detected by agarose gel electrophoresis, were directly sequenced. The PCR products whose sequences are reported here produced unambiguous dideoxysequencing electrophoregram peaks indicating the predominance (190%) of a single bocavirus variant. The sensitivity of the pan-bocavirus nested PCR was determined using dilution of plasmids containing the target sequences from each of the 4 bocavirus species and was estimated at 10-100 plasmid copies for each species.
Complete genome sequencing. Nearly complete genomes were amplified using PCR primers designed from alignments of HBoV1 and HBoV2 genomes and were then directly sequenced by primer walking [7] . The terminal sequences were acquired by a modified protocol for rapid amplification of complementary DNA ends [5] . The terminal sequences are incomplete because of extensive hairpin structures that prevented extensions to the viral 5 and 3 extremities.
Distance measurements and phylogenetic analyses. Phylogenetic relationships were evaluated with use of Mega 4.1 (http:// www.megasoftware.net/mega41.html) [34] . Neighbor-joining trees were inferred using a matrix of pair-wise maximum-likelihood distances computed from a nucleotide alignment including the genomes obtained in this study and in GenBank, plus a matrix of PAM distances computed from the inferred amino acid alignment.
Recombination analyses. Similarity values based on Jukes-Cantor corrected nucleotide distances between full-length sequences were calculated using the program SequenceDist in the Simmonic2005 v1.6 Sequence Editor Package [35] . To assess similarity across the genomes, sequence scans were performed using a fragment length of 300 bases and an increment of 9 bases between fragments. For sequence comparisons with HBoV1 and HBoV2, a mean pair-wise distance was computed using a set of 14 HBoV1 and 7 HBoV2A sequences.
Nucleotide sequence accession numbers. The near-full genomes and partial VP1 gene sequences have been deposited in GenBank under accession numbers FJ973558-FJ973563 and GQ506558-GQ506661.
Pan-bocavirus PCR primers were designed that could anneal to both HBoV1 and HBoV2, amplifying a ∼576-nucleotide fragment of the VP1 capsid gene. Nucleic acids from human stools collected from Nigeria, Tunisia, Nepal, and the United States were then analyzed using this nested PCR (Table 1) . Of the 641 samples tested, 101 (16%) had confirmed positive results by PCR sequencing, with the highest prevalence in Tunisian children with AFP (33%). To determine the phylogenetic relationship of these strains, the sequences were aligned with available sequences of HBoV1, HBoV2, and HBoV3. Only 4 of 101 strains (from Nigeria and Tunisia) grouped with HBoV1. The remaining strains shared a more recent common ancestry with HBoV2, although the sequences clustered into 4 distinct genetic lineages, labeled HBoV2A, HBoV2B, HBoV3, HBoV4 ( Figure 1 ). The HBoV2A clade included all published HBoV2 genotypes [7] , plus 4 new strains from Nigeria and Tunisia and the recently reported W153 strain from Australia [8] . Because of the high degree of genetic diversity observed and to reduce splitting of HBoV2 into a multitude of genotypes, all strains in that cluster were reclassified into a new, now more diverse genotype A (HBoV2A). The nearly full genome of a new HBoV2A variant was sequenced (TU-C-114-06) and showed very high protein identity (198%) to the Pakistani HBoV2 prototype PK5510 (FJ170278).
The HBoV2B cluster included 76 of the 101 bocavirus strains reported here ( Figure 1 ). Two nearly full HBoV2B genome sequences demonstrated a pair-wise amino acid divergence in VP1 of 0.45% (Table 2 ) (NI-213 and NI-327; GenBank accession numbers FJ973560 and FJ973559). Greater divergence was observed when these strains were compared with the VP1 of HBoV2A strains (average divergence, 3.9% [range, 3.3%-4.5%] ( Table 2 ). The nucleotide and amino acid distances at the other loci are shown in Table 2 .
The HBoV3 cluster included 11 strains, and the genomes of 2 representative strains, NI-374 and TU-A-210-07, were sequenced (GenBank accession numbers FJ973563 and FJ973562). When compared with HBoV2, the NS1 region of both strains showed an average amino acid divergence of 26% in the NS1 region and 9% in the VP1 region. Compared with HBoV1, the HBoV3 strains showed an average amino acid divergence of 12% in the NS1 region and 20% in the VP1 region ( Table 2 ). All 11 strains were classified as members of the HBoV3 species, recently described by Arthur et al [8] . A distantly related variant of HBoV3 was also recently identified in a sewage sample in the United States (HBoV3B-CA-1-C1; Figure 1 ) [36] . To confirm whether this variant represents a second genotype of HBoV3 will require full genome sequencing.
The HBoV4 cluster included 6 strains. The complete genome of 1 representative strain (NI-385) was obtained (GenBank accession number FJ973561). The NS1 protein of HBoV4 (NI-385) showed an average of 11% divergence with HBoV2 and 25%-27% divergence with HBoV1 or HBoV3 (Table 2) . For the VP1 protein, the relative divergences were reversed, with an average divergence of 8.5% with HBoV3, 9.5% with HBoV2, and 19% with HBoV1. NI-385, therefore, also appears to be a recombinant, with the 5 genes NS1 and NP1 closely related to HBoV2 (particularly genotype A) and the 3 VP1 gene slightly more similar to HBoV3 than HBoV2 (Table 2 ). According to the International Committee on Taxonomy of Viruses species demarcation criteria in the genus Bocavirus, members of different species must show 15% divergence in their nonstructural gene nucleotide sequences [37] . The genetic distance of NI-385 to its closest relatives in the NS1 gene (HBoV2A) was 10.8% (range, 6.8%-12.6%), indicating that NI-385 qualifies, pending International Committee on Taxonomy of Viruses review, as the prototype of a fourth HBoV species (HBoV4). A distant VP1 (partial) variant of HBoV4 was also detected in the United States (US-MN-964-05; Figure 1 ). To determine whether the latter variant represents a second HBoV4 genotype will require full genome sequencing.
Nearly complete genomes and phylogenetic analysis of new bocavirus species. In a manner similar to HBoV1 and 2, all the new genomes of HBoV2, 3, and 4 encoded 3 large open reading frames (ORF). The left ORF encodes the nonstructural protein (NS), the middle ORF encodes NP1, and the right ORF encodes overlapping VP1/VP2 capsid proteins. Conserved motifs associated with rolling circle replication, helicase, and ATPase were identified within the NS protein. NP1 is a highly phosphorylated protein of currently undetermined function [38] ; NP1 differed in length between species, ranging from 214 to 219 amino acids. Situated within the VP1-unique (VP1u) region, the phospholipase A2 motifs required for parvovirus infectivity were found in all 6 genomes, together with the calcium-binding loop and catalytic residues.
Further evidence of recombination in human bocaviruses. To further determine the relationship between members of the Bocavirus genus, phylogenetic analyses of NS1, NP1, and VP1/ VP2 were performed, with use of both nucleotide sequences and deduced protein sequences (Figure 2 ). The NS1 and NP1 genes of HBoV3 clustered with HBoV1, whereas their VP1/2 gene clustered with HBoV2. The incongruence in phylogenetic association between loci provided further evidence that HBoV3 originated from a recombination event bringing together the NS1/NP1 gene of HBoV1 and the VP1/2 gene of HBoV2 [8] .
The likely recombinant origin of HBoV4, clustering with HBoV2 in the NS1/NP1 but with HBoV3 in the VP1, is also shown (Figure 2) .
A scan of sequence divergence between complete genome sequences further supported the hypothesis of past recombi-nation between HBoV1 and 2 in the generation of HBoV3 and recombination of HBoV2 and HBoV3 in the generation of HBoV4, with both recombination points near the NP1 and VP1 junction (Figure 3 ). When different HBoV2 variants were similarly analyzed for recombination, intraspecies HBoV2 recombinants were also detected (data not shown) [7] .
Diversity among respiratory HBoV1 and enteric HBoV2-4. We compared the intraspecies diversity of HBoV1 with that of HBoV2 by use of the partial VP1 sequence data generated with the pan-bocavirus PCR primers available for the greatest number of HBoV2, 3, 4 variants ( Figure 4A and 4B) . A very low average pair-wise difference was seen for HBoV1 collected worldwide ( Figure 4A ). HBoV2, including both genotypes, was more diversified than HBoV1, although HBoV2B alone showed a low level of diversity comparable to that of HBoV1 (HBoV2B generated the large low divergence peak in Figure 4A ). The homogeneity of HBoV1 and HBoV2B can also be visualized in the small branch lengths in Figure 1 . When the pair-wise dis- tances of all the enteric (non-HBoV1) sequences were plotted ( Figure 4C ), the distribution was much larger than that for HBoV1. The low level of intraspecies diversity of HBoV1 relative to the other species is also reflected in Table 2 .
The genomic organization of HBoV species are remarkably similar to those of animal bocaviruses, except that all HBoV NS ORFs encode a shorter NS2 protein (630-650 amino acids), compared with the longer NS1 of animal bocaviruses (716-726 amino acids) ( Figure 5A ). We noticed in all 4 HBoV species the presence of a stretch of encoded amino acids similar to the C-terminus of the longer NS1 of animal bocaviruses overlapping the NP ORF, but in a different frame ( Figure 5A) . Genomes of all HBoV species were aligned to determine the presence of conserved potential RNA splicing signals near the end of the smaller NS2 ORF and the putative second exon encoding the C-terminal region of NS1 (Figure 5B). The putative NS1 resulting from such a spliced transcript encoded a 750-780 amino acid protein with a carboxy terminus that showed significant similarity to that of the canine and bovine bocaviruses NS1 ( Figure 5C ) [39, 40] . A study using RT-PCR for the detection of HBoV1 viral transcript in human lung epithelial cells did not detect the NS splicing proposed here [41] . On the other hand, the proposed NS1 RNA splicing and NS1 protein expression itself were detected using Northern blots and NS1 C-termini specific sera, respectively, in 293 and human epithelial cells transfected with plasmids expressing HBoV1 transcripts (Jianming Qiu, personal communication).
Most PCR-positive stool samples contained HBoV2B (76 of 101), making this genotype the most commonly detected enteric human bocavirus (Table  1) . HBoV3 was identified in 11 stool samples and HBoV4 in 6, making them the second and third most common enteric human bocaviruses, respectively, in the regions analyzed here. HBoV1 was detected in only 4 of the 101 positive samples. Stool samples from patients with AFP in Nigeria and Tunisia both showed a high prevalence of HBoV2A+B (21%-26%). Because both patients with AFP and healthy contacts from Tunisia showed a comparable prevalence of HBoV2, no asso- ciation was observed between AFP and HBoV2 excretion. The prevalence of HBoV2 in adults with diarrhea from the United States and Nepal were also compared with those in healthy matched subjects. No associations were observed between HBoV2 shedding and diarrhea. Although the numbers of HBoV3 and HBoV4 detected were relatively small, it was noticed that the 8 HBoV3-and 4 HBoV4-positive samples were found among 192 samples from patients with AFP, whereas these 2 species were not detected among 96 healthy Tunisian matched contacts. Comparing the combined Nigerian and Tunisian patients with AFP infected with HBoV3 and HBoV4 to Tunisian controls yielded a 2-tailed Fisher's exact P value of .01, whereas comparing only the Tunisian patients with AFP with Tunisian controls gave a P value of .059. HBoV3 was also found in 1 patient with diarrhea from the United States and from 1 healthy person each from the United States and Nepal. HBoV4 was also found in a patient with diarrhea and 1 healthy adult from the United States. Further testing will be needed to confirm this trend of an association of HBoV3/4 with AFP in children or of the association of HBoV2 with diarrhea [8] .
We report on a previously uncharacterized species of human bocavirus which we tentatively named HBoV4. HBoV1, HBoV2, and HBoV3 were also detected using a pan-PCR approach. HBoV2 has been detected in the stool of Pakistani children [7] and HBoV3 in stool samples of Australian children [8] , and both were recognized as recombinant viruses. The newly reported Australian HBoV3 (EU918736) is closely related to a Tunisian strain (TA-210-07) (Figure 2) . A highly prevalent genotype of HBoV2 (HBoV2B), together with partial genomic support for second genotypes of HBoV3 and HBoV4, were also identified. The availability of novel bocavirus genomes will allow the design of species-specific PCR or microarray oligonucleotides for their detection and for the disease association studies that are now required for the 3 recently characterized enteric human bocavirus species (HBoV2, HBoV3, and HBoV4). Based on the phylogenetic clustering observed for a large number of partial VP1 sequences ( Figure 1 ) and the distances among full genomes (Table 2) , we propose for future classification that HBoV strains showing 18% protein and 110% nucleotide difference in the complete VP1 gene should be considered different species, whereas those showing 11.5% protein and 15% nucleotide difference should be considered different genotypes. This VP1-based classification retains the 4 proposed human bocavirus species. The VP1 locus was selected because it is likely to strongly influence tissue tropism and potentially pathogenesis [42] .
HBoV1 is primarily, although not exclusively [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] , a respiratory virus. We show here a higher prevalence of HBoV2 (particularly HBoV2B) than HBoV1 in stool samples from children and adults of different countries. A recent study testing for HBoV1 and HBoV2 DNA with use of species-specific nested PCRs failed to detect any HBoV2 in 16500 respiratory secretion samples from Edinburgh and Bangkok, whereas HBoV1 was found in 3% and 14% of these respiratory samples, respectively [43] . Another study found 5 (2%) of 212 nasopharyngeal samples from Korean children with acute lower respiratory track infections to be positive for HBoV2 DNA, but unexpectedly, no HBoV1 DNA was found [44] . Analyzing for HBoV1 and HBoV2 in both respiratory secretion and stool samples collected from the same individuals will be required to confirm whether the tropism of HBoV2 favors the digestive track and is distinct from that of the largely respiratory HBoV1.
Extensive evidence for recombination was observed through full genome analyses, including the likely recombinant origin of HBoV3 and HBoV4 and the high level of intraspecies recombination between HBoV2 variants [7, 8] . The high prev-alence of bocavirus infection does provide the opportunity for coinfections, the first step in generating recombinant viruses. Indeed, a HBoV3 and HBoV4 coinfection was detected based on the pattern of mixed bases in 1 directly sequenced PCR product (data not shown).
The frequent detection of HBoVs in stool samples from both healthy children and adults supports the likelihood of long periods of viral shedding and/or frequent reinfections. Determination of whether symptoms such as diarrhea are associated with acute infection in subsequently healthy viral shedders will require quantitative viral load measurements, analysis of longitudinally collected samples, and serological assays. Whether prior infection provides any protection against reinfection with the same or different genotype or species is also unknown.
In both diarrhea sample sets analyzed here, consisting of samples from adults, no association with HBoV2 shedding was detected. If HBoV2 causes diarrhea, it may do so in only a small subset of infected children, possibly those without passively transferred maternal antibodies or without protective immune responses from prior infections. Coinfections with other enteric viruses may also exacerbate symptoms. Given the very large number of childhood infections (viral shedding prevalence of 120% in some developing countries), even low virulence would translate into a large disease burden.
A trend of an association of HBoV3/HBoV4 detection with AFP was detected. The small numbers of cases will require independent confirmation. The neurological damages caused by bovine and canine bocaviruses in their animal hosts provide a precedent for infant nervous system pathogenicity [11] .
The totality of HBoV1 sequences collected worldwide by multiple groups show very low protein and nucleotide sequence diversity (Table 2 and Figure 4A ) [35, 36] . In contrast, this single study found substantial diversity among HBoV2 and HBoV3, a fourth species (HBoV4), and extensive viral recombination (Figures 3, 4B, and 4C ). Assuming comparable rates of evolution, the genetically homogeneous and largely respiratory HBoV1, therefore, appears to be the more recently evolved species relative to the more diverse HBoV2, 3, and 4 found predominantly in feces. We propose that HBoV1 evolved from an enteric bocavirus ancestor that acquired, through mutation and/or recombination, enhanced respiratory tract tropism. Single-stranded DNA parvoviruses have been shown to have a mutation rate approaching that of RNA viruses, and recombination among animal parvoviruses has also been reported [45] [46] [47] . Parvoviruses have also demonstrated the capacity to rapidly expand their host species tropism, resulting in a recent pandemic in dogs [42, 48, 49] . A recent study showed that HBoV1 could replicate in differentiated human airway epithelial cells [41] . Whether HBoV2, 3, and 4 show an in vitro tropism and in vivo distribution that is more biased towards digestive track cells will require further study. The Battle between Virus and Host: Modulation of Toll-Like Receptor Signaling Pathways by Virus Infection In order to establish an infection, viruses need to either suppress or escape from host immune defense systems. Recent immunological research has focused on innate immunity as the first line of host defense, especially pattern recognition molecules such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs). Various microbial components are recognized by their vague and common molecular shapes so-called, pathogen-associated molecular patterns (PAMPs). PAMPs induce inflammatory reactions mediated by the activation of the transcription factor, NF-κB, and by interferons, which lead to an antiviral immune response. Viruses have the capacity to suppress or escape from this pattern recognition molecule-mediated antimicrobial response in various ways. In this paper, we review the various strategies used by viruses to modulate the pattern recognition molecule-mediated innate immune response. The host immune system recognizes and eliminates invading pathogenic microorganisms such as viruses, bacteria, and fungi. The first line of defense in mammals is the innate immune system. Recently, the mechanisms by which the innate immune system recognizes pathogen have been extensively studied. Pattern recognition molecules/pathogen recognition receptors (PRRs) are classified into three families: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and nucleotide binding-oligomerization domain (NOD)-like receptors (NLRs) [1, 2] . Ten TLRs (TLR1 to 10) have been identified in humans. The RLR family contains retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) [3] . The NLR receptor family contains NOD1, NOD2, NLRP3, NLRPC5, NLRP1, NAIP, and CIITA [4] . In addition, DNA-dependent activator of interferon regulatory factors (DAI) has been identified as a DNA sensor [5] . Various microbial components are recognized as their vague and common molecular shapes by PRRs. Early responses against virus infection are initiated on recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition molecules, triggering two responses. One is the production of interferons (IFNs) resulting in an antiviral state as part of the innate immune response, and the second is maturation of dendritic cells (DCs) to establish acquired immunity. In order to establish an infection within a host, viruses must escape from and/or suppress the immune system by various strategies. An important strategy used by viruses is modulation of PAMPinduced immune responses.
TLR signaling proceeds via two pathways: the myeloid differentiation factor 88 (MyD88)-mediated pathway, and the Toll-interleukin-1 receptor (TIR)-domain-containing adaptor inducing IFN-β (TRIF)-mediated pathway [1, 2] . The former causes activation of the transcription factor NF-κB, which activates various genes contributing inflammatory reactions. The latter causes induction of IFNs, whose stimulation leads cells to antiviral state. TLR3 only activates the TRIF-mediated pathway. TLR3 signaling activates IRF-3, an important transcription factor for IFN-β, and IFN production is induced. TLR2 only activates the MyD88mediated pathway. However, TLR4 activates both pathways, so TLR4 agonists activate NF-κB and induce IFN production. Cytosolic PRRs, such as RIG-I, MDA5, and DAI, commonly activate IRF-3. The expression of PRRs differs depending on the cell type. Importantly, it is different between cells derived from myeloid stem cells (myeloid dendritic cells (mDCs), monocytes, macrophages, Langerhans cells, and neutrophils) and cells derived from lymphatic stem cells (plasmacytoid dendritic cells (pDCs), T cells, and B cells). For example, TLR7 and TLR9 are rarely expressed on mDCs, whereas TLR3 and TLR8 are rarely expressed on pDCs. TLR4, on the other hand, is expressed at very low levels on both pDCs and mDCs.
Initially, viruses invade the host epithelial tissues found in the oral cavity, respiratory tract, intestinal tract, and the urogenital apparatus ( Figure 1 ). Lamina propria DCs, Langerhans cells, and stromal cells are resident in these tissues. In the connective tissues, fibroblasts resident, and capillary vessel and lymphatic vessel are expanded. Monocytes, macrophages, T cells, B cells, pDC, and mDC circulate within the blood vessels and lymphatic vessels, and patrol the interstitial spaces. All these cells are potential targets for virus infection. Virus-infected epithelial cells and fibroblasts produce IFNs, mainly IFN-β and IFN-λ, which provide surrounding uninfected cells with antiviral state. Furthermore, chemokines and cytokines, such as interleukin-1β (IL-1β), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) are also produced. These molecules promote chemotaxis of the resident DCs (lamina propria DCs and Langerhans cells) toward virus-infected and dead cells. Neutrophils, monocytes, macrophages, plasma cells, mDCs, and pDCs also migrate from the blood vessels to the site of infection. IFN-γinducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), MIP-3α, and MIP-3β largely contribute to the transmigration of blood DCs. However, these blood-borne immune cells can also be infected by viruses, which can then modulate the production of various cytokines and chemokines.
To establish an infection, viruses need to suppress a number of host immune responses, the antiviral activity induced by IFNs, the chemotaxis of immune cells induced by chemokines/cytokines, the maturation and activation of DCs, activation of NK and NKT cells, transmigration of mature DCs to the lymph nodes, and the differentiation and activation of T cells and B cells in the lymph nodes, for example. When viruses infect immune cells, such as DCs, the infected cells frequently show suppression of maturation and differentiation, suppression of cytokine receptor and costimulatory molecule expression, secretion of molecules that mimic cytokines and cytokine receptors, and so on. Furthermore, infected cells often alter their cytokine profiles. These strategies are used by the virus to inhibit the acquired immune response. In addition, it has been suggested that virus infection induces regulatory T cells. Type 1 (HTLV-1) . HTLV-1, which is a retrovirus, infects CD4 + T lymphocytes, CD8 + T lymphocytes, DCs, B cells, macrophages, and astrocytes, and preferentially replicates in CD4 + T lymphocytes. HTLV-1 causes latent infection as a provirus, whose genome is integrated into the host DNA, and does not replicate in cells in G0 phase. When the infected T lymphocytes are stimulated with antigen presentation from DCs, they proliferate triggering HTLV-1 replication. During the replication stage, a viral protein, Tax, activates NF-κB and promotes the growth of infected cells via upregulation of IL-2 and IL-2 receptors [6, 7] . NF-κB also activates the long terminal repeat (LTR) of HTLV-1 genome, which further enhances viral replication [8] . The replicated virus induces a host immune response, and cells infected with virus are eliminated by the induction of cytotoxic T cells specific for HTLV-1 Tax. In patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), the high proviral load induces a strong HTLV-1-specific immune response [9] . This leads to the rapid elimination of infected cells through the induction of proinflammatory cytokines and cytotoxic T lymphocytes. So, in order to escape from acquired immune responses, HTLV-1 needs minimum replication and latent infection.
Some virus proteins are known as negative regulators of replication. The HTLV-1 basic leucine-zipper factor (HBZ) protein suppresses Tax-mediated transcription activation of the viral LTR [10] . The p30 protein suppresses transcription of mRNAs encoding Tax and Rev [11] . p30 also contributes to the expression of TLR2, TLR4, and TLR9, and activation of IRF4. In monocytes, the TLR2 gene promoter is regulated by the transcription factors Sp1, Sp3, and PU.1 [12] , and the TLR4 gene by ISRE and PU.1 [13] . p30 downregulates the expression of both TLR2 and TLR4, because it binds to PU.1 and prevents it from binding to DNA [14] . This leads to the suppression of DC maturation, and of their subsequent migration to the lymph nodes. Furthermore, p30 suppresses the enzymatic activity of glycogen synthetase kinase 3β (GSK3β) through promotion of the phosphorylation of nine serine residues. This leads to the induction of IL-10, which suppresses the function of macrophages, and also the maturation and activation of DCs. In fact, serum IL-10 levels are elevated in patients with adult T lymphocyte leukemia. These immunosuppressive properties of IL-10 indirectly contribute to the inhibition of virus replication and to the suppression of virus-induced immune responses. Activation of infected T lymphocytes by DCs in the lymph nodes and cell-to-cell transmission of virus are considered to be important for virus proliferation in human. The viral p12 protein suppresses cell surface expression of both MHC class I and the IL-2 receptor, and also suppresses linker for activation of T cells (LAT), which is an adaptor protein required for T cell activation [15] . This results in suppression of T cells and dystunction of the stimulation/activation by DCs via the T cell receptor. HTLV-1 causes proliferation of infected cells rather than virus and suppression of host immune responses, which helps it to maintain a latent infection in order to survive. can infect CD4 + T lymphocytes, monocytes, macrophages, and DCs, and preferentially replicates in activated T lymphocytes and activated macrophages. It replicates generally much more slowly in DCs regardless of maturation stage.
HIV establishes a latent infection in resting T lymphocyte, and T lymphocyte activation by DCs or stimulation with IL-2 is thought to trigger active replication of the virus. Resting T lymphocytes show resistance to infection and proliferation of HIV. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) is important for this resistance [16, 17] . The antiviral mechanisms of APBEC3G are considered to act via inhibition of viral reverse transcriptase (RT). It also produces a transition (G to A) in the DNA strand transcribed by RT due to its cytidine deaminase activity. However, a mutant lacking cytidine deaminase retains antiviral activity. Low molecular weight complexes of APOBEC3G (LMM: 70-100 kDa) have antiviral activity, but high molecular weight complexes (HMM: 700 kDa) do not. The Vif protein of HIV converts LMM to HMM, and promotes proteasome-dependent degradation of the complex, so HIV can counteract its antiviral activity [18] . T cell activation leads to conversion of LMM to HMM (proviral environment). On the other hand, IFNs induce LMM type (antiviral environment) [19, 20] .
During the early stage of HIV infection, clinical symptoms show sings of immune system activation such as flu-like symptoms, rather than immunosuppression. Immunological activation is caused by RNA40, an oligonucleotide derived from HIV, which activates pDCs, mDCs, T lymphocytes, and monocytes via TLR7 and TLR8 [21] . The virus proteins, Tat and Vpr, induce proinflammatory cytokines via activation of NF-κB [22, 23] , which then enhances the transcription of the virus genome via the LTR. IL-8 also contributes to the propagation of the virus via the accumulation of T lymphocytes.
Enhanced proliferation of HIV causes immune responses that attempt to eliminate the virus. So, HIV maintains a latent infection in order to survive within the host. The Vpu protein suppresses NF-κB activation by inhibition of proteasome-dependent degradation of IκB [24] . The Nef protein suppresses phosphorylation of ERK through induction of a phosphatase MKP-1 [25] . This causes suppression of TNF-α production by macrophages via TLR4 signaling. Because TNF-α is necessary for maturation and translocation of DCs (including Langerhans cells) to the lymph nodes, downregulation of TNF-α leads to suppression of acquired immune responses, and so prevents inhibition of virus proliferation by activated T lymphoctes. Furthermore, Nef inhibits T cell receptor induced lymphocyte activation [26] . These strategies contribute to the suppression of HIV proliferation in the lymph nodes, and also inhibit the propagation of infection. Other immunosuppressive mechanisms used by HIV have also been reported. Cosuppressive molecules, such as B7-H1 on DCs and PD1 on T lymphocytes, are upregulated in patients with HIV, and induce apoptosis through their interaction with DCs and T lymphocytes. In addition, HIV is thought to induce regulatory T cells [27] .
. HCV does not only infect hepatocytes. It can also infect DCs, macrophages, monocytes, and T lymphocytes. However, the virus does not replicate efficiently in these cell types. Although, virus particles and virus proteins are found in the blood of HCV patients. HCV causes no cytopathic effects. Immune responses against HCV may be weak, but infected cells are attacked and eliminated. In order to survive in the host, HCV maintains a chronic infection by suppressing the host immune responses. Both HCV core and NS3 proteins activate NF-κB and AP-1 via stimulation of TLR2, which requires TLR1 and TLR6 as costimulators, in monocytes and Kupffer cells [28] . This activation leads to the production of IL-10 and TNF-α, both found in HCV patients at a high titer. IL-10 suppresses the maturation of pDCs and the activation of T lymphocytes, and induces apoptosis in pDCs. The NS3-NS4A protein complex, which is a serine protease, degrades TRIF and IPS-1/Cardif/MAVS/VISA, which are essential for cellular signaling via TLR3, TLR4, and RIG-I [29] . This shutting off of TRIF-and IPS-1-dependent signaling results in the suppression of IFN-α and IFN-β production, and in dysfunction of mDCs. The NS5A protein suppresses activation of IRAK-1 through its interaction with MyD88. This leads to the shutting off of TLR7 and TLR8 signaling, and to the suppression of maturation and differentiation of pDCs [30] . On the other hand, NF5A suppresses TRAF2 dependent NF-κB activation via interaction with NF5A and TRAF2, but suppress neither MEK1 activation nor IKKβ-dependent NF-κB activation [31] . TNF-α-dependent activation of JNK is enhanced. Infected DCs are thought to affect the production of TNF-α, TNF-α signal transduction, and chemotaxsis and maturation. Dysfunction of DCs, suppression of T lymphocyte activation, and a decrease in DC number due to apoptosis allow HCV to establish a chronic infection [32] . It has also been reported that HCV-specific cytotoxic T lymphocytes share upregulated expression of the coinhibitory molecule PD-1, and that signaling from PD-1L (PD-1 ligand) on DCs results in suppression of HCV-specific cytotoxic T lymphocytes [33] .
Measles virus infection causes strong immunosuppression. Measles virus wild strains (clinical isolates) recognize CD150/SLAM (signaling lymphocyte activation molecule) as a receptor. However, laboratory (vaccine) strains only recognize CD46. SLAM is strongly expresseed on memory T cells and B cells, but is also expressed on monocytes, T cells, B cells, and matured DCs. SLAM is not expressed on immature DCs, epithelial cells, and endothelial cells. On the other hand, CD46 is expressed on variety of cell types. Infection of SLAM-negative mucosal epithelial cells with measles virus wild strains is thought to be mediated by an as yet unknown "third" receptor [34, 35] . The HA protein of wild virus strain, but not the laboratory strain, induces cytokines such as IL-1α, IL-1β, IL-6, IL-8, and IL-12 via the TLR2 signaling pathway [36] . These cytokines activate and recruit immune cells to the site of inflammation, where the activated immune cells are infected with measles virus via SLAM. The infection and cytokine production are thus propagated.
Interaction of the HA protein with SLAM suppresses TLR4-mediated IL-12 production, but not IL-6 and TNF-α production. However, IL-12 production mediated by other TLR signaling pathways (i.e., not via TLR4) is unaffected by the HA protein [37] . These observations suggest that SLAM is a coupling factor for TLR4, and that the HA protein inhibits this function. The HA protein also interacts with a C-type lectin, dendritic cell-specific ICAM-3grabbing nonintegrin (DC-SIGN) on DCs, and activates the serine/threonine protein kinase Raf-1 via the Ras signaling pathway. DC-SIGN-mediated Raf-1 activation induces phosphorylation of NF-κBp65 on Ser-276, and its subsequent acetylation. This leads to the enhanced transcription of IL-10 [38] . During infection stage that virus proteins are synthesized de novo, the host cells, such as monocytes and DCs, infected with measles virus show suppressed IL-12 production and TLR signaling, via TLR2, TLR4, TLR7, and TLR9. pDCs infected with the measles virus showed suppressed IFN production and dysfunction of maturation [39, 40] . Both monocytic cell lines U937 and THP-1 and human peripheral blood mononuclear cells infected with measles virus show markedly suppressed TLR2-and TLR4-mediated proinflammatory cytokine induction via NF-κB and AP-1 [41] . However, epithelial cells infected with measles virus show constitutive activation of NF-κB and proinflammatory cytokine production, and these are further enhanced by treatment with TLR agonists such as lipopolysaccharide (LPS). Monocytic cell lines infected with the mumps virus, which also belongs to the Orthomyxoviridae family, show constitutive activation of NF-κB and constitutively high levels of IL-8 production. In monocytic cells infected with the measles virus, LPS-induced ubiquitination (probably K63linked type) of TNF receptor-associated factor 6 (TRAF6) is suppressed and does not form active complexes of TAK1, TAB2, and TRAF6. An ubiquitin-modifying enzyme A20, 8 Mediators of Inflammation which is a host NF-κB negative regulator, is upregulated in measles virus-infected monocytic cells, but not in infected epithelial cells. The promoter region of the A20 gene shares two NF-κB binding sites and a negative regulatory motif, ELIE, which is located upstream of, and adjacent to the two NF-κB binding motifs. Measles virus P protein (phosphoprotein) interacts with the ELIE motif, and activates transcription of A20. P protein is thought to release the suppressed A20 transcription machinery independently of activated NF-κB [42] . The reason for cell-type specific A20 expression is unclear. However, the measles virus V protein, which is formed by RNA editing of the P genome and has an N terminal amino acid sequence identical to that of P protein, does activate NF-κB. The balance of the expression levels and time courses of the P and V proteins may also contribute to the cell-type specific suppression of TLR signaling pathways.
Virus. The NS1 protein of type A influenza viruses suppresses innate immune system signaling activated by the PAMPs, via TLR3, TLR4, RIG-I, and MDA5 system. The suppression should contribute to efficient replication of infected virus in respiratory epithelial. The mechanism of suppression is mainly via inhibition of IRF-3 phosphorylation [43] , leading to suppressed induction of IFN-α/β, and IFN-λ1, 2, and 3. In addition, inhibition of NF-κB and AP-1 activation in influenza virus infected cells leads to suppressed proinflammatory cytokine production, for example, IL-8 and TNF-α. Influenza virus NS1 protein is known to be a multifunctional protein able to inhibit the type I IFN induction, the IFN-induced antiviral activity, the binding and sequestration of dsRNA, the interference with the host mRNA processing, the facilitation of preferential viral mRNA translation, and the inhibition of DC activation [44, 45] .
. RSV F protein causes TLR4-mediated NF-κB activation during the early infection stages of infection that is dependent upon virus replication. IL-1β, IL-6, and IL-8 are induced via NF-κB activated by the stimulation of TLR4. During the late stages, the viral G protein is produced, and secreted, which then suppresses TLR4-mediated signal transduction [46] . The cysteine-rich (GCRR) region of the G protein is also important for NF-κB suppression. Following the interaction of virus proteins with host cell surface proteins, the viral M2-1 protein inhibits the translocation of RelA, a component of NF-κB, to the cell nucleus [47] . Expression of the viral nonstructural proteins, NS1 and NS2 inhibit activation of IRF3 induced by TLR3, TLR4, and RIG-I signaling, and also suppress IFN production [48] . However, NS1 and NS2 proteins have little effect on NF-κB or AP-1 activation, so the production of proinflammatory cytokines may be effectively induced in the RSV-infected cells.
Orthopox viruses, including the vaccinia virus, produce proteins that mimic cytokine receptors, such as those for IFN-α, IFN-β, IFN-γ, IL-1β, IL-18, and TNFα, and disturb the cytokine signal transduction system. Other viral proteins also disturb the intracellular signal transduction systems. The viral A46R protein has a TIR domain, and interacts with MyD88 and TRIF, suppressing both IL-1 and TLR signal transduction, but not TNF-α signal transduction [49] . The viral A52R protein binds to IRAK2, suppresses TRAF6-dependent IKK and NF-κB activation, and then inhibits production of IL-8 and RANTES. However, A52R also binds to TRAF6 and promotes polyubiquitination of TRAF6, TAK1 activation, MAPKK6 phosphorylation, and activation of the JNK-p38 MAP kinase pathway. The later leads to the induction of IL-10 production [50] . The viral N1L protein interacts with the IKK complex (IKKα-IKKβ-IKKγ), TBK1, and IKKε, and then suppresses the activation of NF-κB and IRF-3 [51] . Vaccinia virus not only suppresses proinflammatory cytokines, but also induces production of an immunosuppressive cytokine IL-10, which shifts the Th1 response and suppresses cellular immunity. Human monocytes infected with vaccinia virus produce IL-10, and this IL-10 is then further upregulated by stimulation with LPS [52] .
During the early stages of infection, adenovirus particles induce the MyD88-dependent production of RANTES, IP-10, and MIP-1 [53] . The cytokines produced enhance both sensitivity to LPS and the production of TNF-α. TNF-α suppresses the formation and maturation of virus particles, and induces apoptosis of infected cells. TNF-α also promotes the chemotaxis and maturation of dendritic cells. The induction of TNF-α is considered to be a host defense response. On the other hand, the adenovirus E1A and E3 proteins inhibit TNF-α-induced apoptosis. The receptor internalization and degradation (RID) complex, which consists two E3 products, E3(10.4 k)/RIDα and E3(14.5 k)/RIDβ, suppresses cell surface expression of Fas, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1, TRAIL receptor 2, and TNF receptor 1 [54, 55] . This results in the shutting down of the TNF-α-mediated signaling pathways in the infected cells. RID also suppresses the TLR4 signaling pathway. LPS-induced MCP-1 and IL-8 production are suppressed through the inhibition of NF-κB and AP-1 activation [56] . RID does not alter the expression levels of TLR4, and so is thought to affect other components of the TLR signal transduction pathway.
. HCMV modulates NF-κB activity during the various stages of infection. During the early stages, the membrane glycoproteins gB and gH interact with TLR2 and activate NF-κB [57] . NF-κB then contributes to the induction of proinflammatory cytokines, to the expression of virus immediate early genes, and to the replication of the viral genome. The viral US28 protein, which is a HCMV-encoded chemokine receptor, constitutively activates both NF-κB and phospholipase C signaling pathways [58] . The activated NF-κB mediates the upregulation of the host serine/threonine protein kinase, receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK).
RICK, which has a caspase-recruitment (CARD) domain, functions downstream of the pattern recognition receptors (which include the TLR, RLR, and NLR family members) and mediates NF-κB and MAP kinase activation. RICK suppresses the replication of HCMV in cooperation with active NF-κB and IFN-β [59] . Not to be outdone, the late gene products of HCMV also suppress NF-κB activation [60, 61] . TNF-α signaling is suppressed through downregulation of TNF receptor 1. IL-6, IL-8, and MCP are not induced in HCMV-infected cells and chemotaxsis, activation, and maturation are all suppressed.
although it can also infect T cells, NK cells, and epithelial cells. Both the attachment of EBV to receptor CD21 and the interaction of the virus glycoprotein gp350-gp250 with TLR2 activate NF-κB [62] . The virus gB and gH proteins are also candidates for TLR2 ligands. Immortalization of B cells by EBV infection is due to the activation of NF-κB by the latent membrane protein 1 (LMP1), and antigen stimulation-like signaling of the B cell receptor by LMP-2. TLR2 signaling suppresses the transcription of the TLR9 gene via activation of NF-κB containing p65 protein. So downregulation of TLR9 and upregulation of TLR7 and MyD88 are observed in EBV-infected cells [63] . Cell proliferation is thought to be driven by TLR7 signaling activated by virus RNA, because the TLR7 antagonist IRS661 suppresses cell division. Small RNA encoded by the EBV genome (EBV-EBERs) activates TLR7 signaling, and induces IL-10 production. TLR7 signaling also upregulates and activates IRF-5, and induces proinflammatory cytokines. However, activated IRF-5 is negatively regulated by EBV-induced IRF-4 and a splice variant of IRF-5 (V12IRF-5) [63] . This suggests that the TLR7 signaling system play a role in the cell division of EBVinfected cells, and in the establishment of persistent infection. Lytic infection and production of virus particles are observed during the late stage of EBV infection. Late proteins suppress NF-κB activation. This leads to the downregulation of proinflammatory cytokines, the upregulation of TLR9, and the suppression of TLR7 function through interaction of TLR7 and TLR9. So the TLR9 system should be important during late stage of EBV infection.
Viruses are in a constant battle with the host immune system. Viruses modulate both the innate and acquired immune systems using a number of clever strategies. The goal appears to be to survive within the host for a long time, rather than efficient replication. Excessive replication would lead to detection and elimination by the host innate and acquired immune systems, thus bringing about the death of the virus. The virus needs to strike a balance between activation and suppression of host immune response. It is likely that each virus has developed various strategies to modulate the host immune response individually, and viruses that have succeeded in creating a good balance between host and parasite have survived.
Modification of TLR signaling is a promising strategy for treatment of cancer, allergy, and infectious diseases [64, 65] . Especially, the immunomodulators should not generate resistant virus to drugs compared to antiviral drugs targeting viral proteins. As an existing example of virus infection, imiquimod, which is a TLR7 agonist, have been applied to an infectious disease caused by human papilloma virus, namely, condylomata acuminata [66] . TLR4 antagonists are trying to be applied to treatment of sepsis. Several issues must be considered for the clinical application of TLR signaling. Among the greatest is assessment of what are keys of host defense or virus survival in view of a series of viral infection process. For example, human herpes simplex virus 1 (HSV-1) requires activated NF-κB for its efficient replication [67] . On the other hand, NF-κB has a key role in inflammatory reactions via transcription activation of proinflammatory cytokines, cell adhesion molecules, and MHC. Thus, activation of NF-κB is a double-edged sword for HSV-1. In the host side, TLR signaling varies according to organs and tissues. For example, intestinal epithelial cells express low levels of TLRs and high levels of negative regulators of TLR signaling, such as Tollip [68, 69] . Also, in intestinal immunity, NF-κB activation in a subset TLR signaling in DCs and macrophages is suppressed by a negative regulation IκBNS [70] . The dysregulation of TLR signaling in the lumen of intestinal epithelial causes limit chronic inflammatory activation induced by commensal bacteria. Various TLR polymorphisms have been found, and some of them are shown to contribute to dysregulation of TLR signaling. The dysregulation has been suggested to be linked with a number of disease sensitivity and condition depending on individual differences [71, 72] .
In conclusion, the molecular mechanisms involved in modulation of host immune systems, including TLR signaling, give us important hints on how to overcome infectious diseases caused by viruses. Pneumonia Incidence and Mortality in Mainland China: Systematic Review of Chinese and English Literature, 1985–2008 BACKGROUND: Pneumonia is a leading infectious disease killer worldwide, yet the burden in China is not well understood as much of the data is published in the non-English literature. METHODOLOGY/PRINCIPAL FINDINGS: We systematically reviewed the Chinese- and English-language literature for studies with primary data on pneumonia incidence and mortality in mainland China. Between 1985 and 2008, 37 studies met the inclusion criteria. The quality of the studies was highly variable. For children <5 years, incidence ranged from 0.06–0.27 episodes per person-year and mortality ranged from 184–1,223 deaths per 100,000 population. Overall incidence and mortality were stable or decreased over the study period and were higher in rural compared to urban areas. CONCLUSIONS/SIGNIFICANCE: Pneumonia continues to be a major public health challenge in young children in China, and estimates of pneumonia incidence and mortality vary widely. Reliable surveillance data and new prevention efforts may be needed to achieve and document additional declines, especially in areas with higher incidence and mortality such as rural settings. Despite the availability of safe and effective antibiotics and vaccines for treatment and prevention, pneumonia is a leading cause of death worldwide and the leading infectious disease killer [1, 2] . Pneumonia is the single leading cause of death globally among children under 5 years of age accounting for approximately 2 million deaths annually [2, 3] . Children in developing countries have an estimated 0.29 episodes of pneumonia per person-year, compared with 0.05 episodes per person-year in developed countries [3] .
Pneumonia is one of the leading causes of death in adults and children in China [4] . In urban areas, pneumonia is the fourth leading cause of death, and in rural areas pneumonia is the leading cause of death [5, 6] . A recent article in the Chinese literature estimated that each year in China there are 2.5 million patients with pneumonia and that 125,000 (5%) of these patients die of pneumonia-related illness [5] . A 2008 global review by Rudan and colleagues estimated that there were 21.1 million new cases of clinical pneumonia annually in China in children under 5 years of age (0.22 episodes/person-year), which is second only to India in burden (43.0 million new cases, 0.37 episodes/person-year) [3] .
Available estimates of the burden of childhood pneumonia in China vary widely, and pneumonia accounts for an estimated 17% of all child deaths in China and 67% of all childhood pneumonia deaths in the Western Pacific region [3, 6, 7] .
Although the global community recognizes that pneumonia is an important public health issue in China, the disease burden is not well studied nor necessarily reported in the English language and these data have not been systematically reviewed for English-or Chinese-language readers [8] . Complicating an assessment of the pneumonia incidence and mortality in China is the lack of ongoing and systematic surveillance. With the exception of human avian influenza and severe acute respiratory syndrome (SARS), pneumonia and other respiratory diseases are not included in the 39 nationally notifiable infectious diseases in China [9] . However, in the wake of the 2003 SARS outbreak, enhanced surveillance using pneumonia of unknown etiology for early detection of suspected SARS was initiated in 2004 (case definition given in Table S1 ). We conducted a systematic review of the Chinese-and Englishlanguage literature in order to describe pneumonia incidence and mortality in China, evaluate the quality of published studies, and identify gaps in the literature that can be addressed through surveillance and epidemiologic research projects in the future.
No ethical review was required since all results are from the published literature.
Using PubMed, we searched the National Library of Medicine database for manuscripts published before October 31, 2008. The reference terms``pneumonia,''``acute respiratory infection,'' and`l ower respiratory tract infection'' were each combined with`C hina'' and``Mainland China.'' Using equivalent terms, we performed additional searches for publications in the Chinese medical literature using the Wanfang (http://www.wanfangdata. com/) and Chongqingvip (www.cqvip.com) databases [10, 11] . In these databases, publications were available since 1982 and 1989, respectively. The English search terms were translated into Chinese (by author X.G.) for use in the Chinese-language searches [º, %'|8SÓ, |8SÓ (P-ý-ý' F)]. In addition, journal articles cited in the identified manuscripts were collected and added to the review.
Clinical and community-based studies with primary data collection in humans were identified through a literature search conducted in November 2008 ( Figure 1 ). Studies conducted exclusively in Hong Kong special administrative region (SAR), Macao SAR or Taiwan, China, were excluded. We also excluded studies focusing exclusively on SARS and avian influenza, outbreak reports, diagnostic studies of pneumonia etiologies, and studies that did not include population-based estimates of incidence or mortality.
Two coauthors who read Chinese as a first language (X.G., W. L.) abstracted from these references the year of publication; province, prefecture, and city; study population (e.g., age group); study design; site of case detection (i.e., inpatient, outpatient, or both); pneumonia case definition; and estimates of pneumoniarelated incidence and mortality. English data abstraction was each checked by a native English and Mandarin speaker. Data abstractions were validated during direct discussions with English-speaking epidemiologists (B.J.S., A.T.F., S.J.O., A.L.C.). Incidence is reported as the annual number of pneumonia episodes per year (i.e., person-years). For manuscripts presenting incidence per 100,000 population, data were converted to personyears to be comparable. For example, if there were 200 cases in a year in a population of 10,000, the incidence would be converted from 2,000 per 100,000 per year to 0.02 person-years. When incidence was reported on an annual basis during a multi-year study period within a single study, a simple mean was calculated to report overall incidence during the study period. For mortality measures, when possible, we calculated case fatality rate (i.e., the percentage of pneumonia cases that died), mortality per 100,000 population, and mortality per 1,000 live births annually. We evaluated trends in incidence and mortality and made comparisons across studies by using studies with similar study methods and case definitions when possible.
There were three pneumonia case definitions used in the studies: I. The World Health Organization (WHO) case definition of pneumonia for Integrated Management of Childhood Illness [12] , II. Chinese medical association guidelines (IIa: communityacquired pneumonia (CAP) [13] or IIb: hospital-acquired pneumonia (HAP) [14] ), and III. physician assessment (IIIa: acute lower respiratory infection; IIIb: newborn pneumonia; or IIIc: pneumonia as a cause of death in children under 5 years of age). The specific signs and symptoms for each case definition are detailed in Table S1 .
For each study that we identified through the systematic review, we critically reviewed the quality of each manuscript. Based on published recommendations for measuring quality of epidemiologic studies of pneumonia [15] , we assessed quality using the following six criteria: (1) geographic location was reported, (2) study was conducted for a period of at least one year or multiples of one year to account for seasonal factors, (3) site of case detection or surveillance location was reported, (4) age and population size of cohort of at least 50 cases were reported, (5) quality assurance and monitoring methods were employed to assure that data was complete and high quality, and (6) a clearly defined case definition (e.g., not based solely on clinical diagnosis) was used and reported. These six criteria were selected to represent basic and essential indicators of epidemiologic study quality. Each criteria was dichotomous (1 = reported and 0 = not reported); the sum of all reported criteria yielded the manuscript's overall quality criteria score (0 to 6; Tables S2 and S3). At least one coauthor who read Chinese as a first language (X.G., W.L.) reviewed each of the studies to evaluate these six criteria; evaluations were then validated during direct discussions with an English-speaking epidemiologists (B.J.S., A.T.F., S.J.O., A.L.C.). We included all studies in this review to report the differences in study quality.
Thirty-seven published studies met the inclusion criteria ( Figure 1 ); 14 publications included data on pneumonia incidence and 28 publications reported pneumonia mortality estimates. At least three studies were conducted in Beijing, Guangxi AR, Hubei, Jiangsu, Shanghai, Shanxi, and Sichuan each ( Figure 2 ). Five (36%) of the 14 incidence articles and 3 (11%) of the 28 mortality articles were identified using Pubmed [16, 17, 18, 19, 20, 21, 22, 23] .
The studies were each evaluated on six quality indicator criteria (Tables S2 and S3) . First, all studies reported the geographic location of the study, but few, if any, reported more specific information on setting such as altitude and annual rainfall, prevalence of malnutrition and AIDS, or immunization coverage against pneumonia vaccines and access to health care [15] . Second, all but 2 studies (35 [95%] of 37) reported at least one year of data. Third, all studies reported the site of case detection and the age of the cases. Fourth, the population size was not given for 15 (54%) of the 28 mortality studies; when population was reported, at least 50 cases were reported. Fifth, four (22%) of 18 studies of incidence and 27 (97%) of 28 studies of mortality reported quality assurance and monitoring.
The sixth quality criteria evaluated the case definition used. Of the 14 studies that reported incidence, 5 (36%) used the WHO case definition for pneumonia and 4 (26%) used the Chinese Medical Association guidelines case definition; 5 (36%) used physician diagnosis, which was considered less reliable than the other case definitions. The incidence estimates using the WHO case definition were generally higher than those using a physician diagnosis, so we report incidence estimates separately by case definition; there was no clear trend in mortality estimates based on case definition, so we did not separate these estimates by case definition. Five (33%) of 15 incidence studies and only 1 (4%) mortality study included chest x-ray in their case definition. Most of the mortality data relied on physician diagnosis (23 [82%] of 28 studies). One quarter (7 of 28) of the mortality studies were reports of deaths for children under 5 years of age collected through the national death surveillance program.
In summary, all of the studies met at least four of the six quality criteria. Only 5 (18%) of the 28 mortality studies and none of the incidence studies met all six quality criteria.
Age. Based on 6 studies, the age-specific incidence of pneumonia in children ,1 year of age ranged from 0.01±0.68 episodes per person-year using either clinical or WHO case definitions [16, 17, 18, 19, 24, 25] . Based on 9 studies of children ,5 years, the incidence ranged from 0.06±0.27 episodes per personyear using clinical case definition and from 0.14±0.66 using the WHO case definition (Table S2) [16, 18, 19, 21, 24, 25, 26, 27, 28] . Incidence was lower in older children [16, 25] , and one study presented a pneumonia incidence for adults: 0.037 episodes per person-year for people $65 years of age [20] .
Time trends. A study conducted in two provinces in the West found that pneumonia incidence in children ,5 years of age decreased from 1997 to 2000 by 64% in Yunnan province (0.083 in 1997 versus 0.030 episodes per person-year in 2000) and by 47% in Qinghai province (0.017 in 1997 versus 0.009 in 2000) [21] .
Rural vs. urban areas. Seven incidence studies were performed in urban areas (50%), five in rural areas (36%), and two (14%) in both urban and rural areas. Pneumonia incidence rates were generally higher in rural areas compared with urban areas (Table S2) . For example, a study in Guangdong that compared pneumonia rates in both rural and urban settings found that rates in rural areas were more than four times higher than urban rates in children ,1 year of age (0.91 vs. 0.19 episodes per person-year, respectively) and more than six times higher in children ,5 years of age (0.79 vs. 0.12 episodes per person-year, respectively) [24] .
Regions. Most (83.3%) studies on pneumonia incidence were conducted in Northeast and Southeast China. The incidence of pneumonia in children ,5 years of age in Northeastern China (range: 0.06±0.27 episodes per person-year) [16, 18, 19, 26] , was lower than southern China (range: 0.32±0.66 episodes per personyear), which includes the Southeast, South Central, and Southwest regions (Table S2) [24, 27, 28] . There were no studies exclusively in the North Central or Northwest regions. One multi-site study showed that pneumonia incidence among children ,5 years of age was higher in Southern than in Northern provinces (0.057 versus 0.013 episodes per person-year in southern Yunnan and northern Qinghai, respectively) [21] . A multi-site study in 1986 reported that pneumonia incidence in children ,14 years of age in Eastern China (range 0.041±0.057 episodes per person-year) was higher than in Western and Central China (range 0.032±0.035 episodes per person-year) [25] .
Age. Twelve (43%) of 28 studies that evaluated pneumoniarelated mortality presented estimates for children ,1 year of age; 23 (82%) presented estimates for children aged ,5 years; most (20 studies, 71%) presented mortality as deaths per 1,000 live births per year (Table S3) . Mortality rates ranged from 485 to 890 deaths from pneumonia per 100,000 population for children ,1 year of age [25, 29, 30] , and from 184 to 1223 deaths from pneumonia per 100,000 population for children ,5 years of age [25, 26, 27, 28, 29] . When mortality was measured per 1,000 live births, the estimates ranged from 0.66 to 12.8 deaths for children ,1 year of age [23, 31, 32, 33, 34, 35, 36, 37, 38] and from 0.71 to 16 for children ,5 years of age [21, 23, 26, 31, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] (Table S3 ). Case fatality rates were higher in children ,1 year of age (4.67±4.88%) than in children ,5 years of age (0.52± 1.94%). One study in adults, which included older adults up to age 94 years, found a case fatality rate of 9.98% [22] .
Time trends. Nine (50%) of 18 studies that examined multiple years of data reported decreasing mortality trends during their study periods [23, 27, 29, 30, 35, 36, 37, 38, 47] . For example, a seven-year study in Shandong province found that the case fatality rate for pneumonia decreased from 1.94% to 0.50% from 1995 to 2001, mortality decreased from 421 to 119 deaths from pneumonia per 100,000 population, and mortality per 1000 live births decreased from 2.50 to 1.07 [26] . However, another nine studies did not show a clear declining trend [21, 33, 40, 41, 42, 43, 44, 45, 46] . For example, a separate study in Shandong province found that while the mortality rate was lowest in the most recent year of the 11-year study (0.14 deaths per 1000 live births in children ,5 years in 2006), there was no clear decrease in mortality over the previous decade [31] . No studies showed increasing mortality.
Rural vs. urban areas. Nineteen (68%) of 28 mortality studies were conducted in both urban and rural areas, while 6 (21%) were conducted in rural areas and 2 (7%) were conducted in urban areas only. As with incidence, pneumonia mortality in rural areas was generally higher than in urban areas. In one study conducted in Zhejiang from 1991 to 1993, mortality for infants ,1 year old ranged from 10.63 to 15.06 cases per 1,000 live births in rural areas compared with 2.29 to 2.54 cases per 1,000 live births in urban areas; for children ,5 years, pneumonia mortality ranged from 10.75 to 15.13 in rural areas compared with 2.60 to 2.79 in urban areas [46] . A study in Jiangsu province found 4.18 and 1.12 deaths per 1000 live births in rural and urban areas, respectively [35] .
Regions. Studies on pneumonia mortality were more geographically representative than studies of pneumonia incidence, and there was at least one study of mortality from each of the six regions (Table S3 ). Estimates of mortality from pneumonia in infants ,1 year old were consistent in four of the six studies conducted in southern China (3.04±3.73 deaths from pneumonia per 1,000 live births) [32, 33, 35, 36] ; only one study in northern China reported mortality rates from pneumonia in infants (0.66 deaths from pneumonia per 1,000 live births) [31] . In children aged ,5 years, the highest mortality rates were reported by four studies that were each conducted in multiple regions throughout mainland China (9.55±14.40 deaths from pneumonia per 1,000 live births; Table S3 ) [21, 23, 38, 48] . Relatively high mortality rates in this age group were also reported in the Northwest [42, 43, 44] and the Southwest [37, 47] (3.91±7.51 and 7.7±12.08 deaths from pneumonia per 1,000 live births, respectively).
Three studies evaluated hospital-acquired pneumonia [49, 50, 51] . In Shanghai where the studies were performed, the hospital-acquired pneumonia rate ranged from 1.6% to 2.4% of hospitalized patients.
Given the population size of China (1.3 billion persons) [6] , the varied health utilization and economic development, the diverse climate (tropical to subarctic), and the range of population densities, the burden of pneumonia in China would be expected to be large and highly variable across regions. Despite overall trends from these studies suggesting that pneumonia incidence and mortality are stable or decreasing, pneumonia continues to be a major public health concern in China [7] . The studies in this review found incidence of pneumonia in children ,5 years of age that were as low as what has been estimated for the developed world globally (0.05 episodes per person-year) and that were as high as what has been estimated for the developing world (0.29 episodes per person-year) [52] . The studies included in this review reported pneumonia incidence for children ,5 years of age (0.06± 0.27 episodes per person-year from 1985 to 2008) that was similar or less than what has been estimated for China (0.22 episodes per person-year in 2008) [3] . Although the studies reported a wide range of pneumonia mortality estimates (184±1,223 deaths per 100,000 population), these are consistent with pneumonia remaining the leading cause of childhood mortality in China [7] . While the data summarized here provide insights into pneumonia in China, they also serve as a reminder that reliable and high quality national and regional data on pneumonia incidence and etiology are needed to adequately direct prevention and control efforts.
Perhaps the largest limitation to this study is that study comparisons of morbidity and mortality rates were constrained by the wide variation and quality of the study designs. This is particularly evident in the wide range of mortality estimates among the different studies. In general, the incidence estimates were higher when the more standardized WHO case definition was used compared with estimates obtained from physician diagnosis; however, there was significant variability even among studies that used similar case definitions. The use of a standard pneumonia case definition that is designed for surveillance and epidemiologic research would improve generalizability and could allow for direct comparisons of incidence and mortality estimates in China and elsewhere [15, 53] . Most studies spanned multiple years, which would account for differences in seasonality of pneumonia, but a few were conducted for one year or less. Although a few of the studies reported large surveillance populations, many calculated incidence based on relatively small populations or did not report the population under surveillance. Most of these studies were conducted in large, urban centers primarily serving residents of densely-populated areas; few included adults or populations from rural western China. Although many of the studies were conducted prospectively, none calculated incidence using active, population-based surveillance and population denominators were not reliably measured in each study. Until standardized case definitions and appropriate surveillance methodology are applied, pneumo-nia incidence and mortality estimates should be interpreted cautiously.
In regions for which we identified published data, pneumonia incidence in China appears to be declining and mortality is stable or declining from the 1980s to the 2000s. There are several factors that could have contributed to these changes over time. China experienced substantial economic growth during these years, a trend that was more pronounced in the coastal (Eastern) areas. From the beginning of economic reforms in 1978 to 2006, China's gross domestic product (GDP) increased 5,719% from 362.4 billion RMB to 21,087.1 billion RMB [54] . The income of the average Chinese person also improved during this time period; GDP per capita rose by 4,144% from 379 RMB to 16,084 RMB [54] . Economic development may have led to improvements in healthcare quality and access to health services.
In addition to economic development, China is undergoing dramatic healthcare reform, including government-sponsored healthcare in rural areas [55, 56] . Declines in the incidence of pneumonia are likely attributable to the implementation of pneumonia intervention measures, such as technical training for village doctors, health education to parents, improved pneumonia surveillance and case management, and the use of vaccines against pneumonia in the routine immunization program (namely measles and pertussis). The improved detection and recognition of pneumonias following the SARS, avian influenza and 2009 influenza H1N1 epidemics could lead to more cases of pneumonia being promptly identified and treated. Large scale programs to introduce less polluting cookstoves in China have led to decreases in lung cancer and chronic obstructive pulmonary disease [57, 58] ; studies from other countries suggest that reductions in exposure to indoor air pollution from solid fuels used for cooking can also lead to fewer cases of pneumonia [59] . Other strategies, including better access to care, improved hygiene, and better nutrition may need strengthening to effectively reduce the incidence of pneumonia in China [60] .
For many Chinese, adequate healthcare remains difficult to access; this review revealed a disparate incidence and mortality of pneumonia across different regions of China [55, 61] , some of which is likely due to inequalities in health care. For example, respirators and ventilators for children are not currently available in many county hospitals. Eastern China is more developed, whereas Western China is more rural. According to a report on national maternal and child health endorsed by the China Ministry of Health, UNICEF, and WHO, pneumonia is the leading cause of death in children under 5 years of age in some rural areas, and comprises a larger proportion of deaths in children under 5 years of age as the area becomes more rural [62] . This report also demonstrated the decreasing trends in pneumonia mortality across China, especially in areas where few clinical studies have been completed.
Better access to proven public health interventions, including vaccines, is needed in the public sector in China. Vaccines against Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, and influenza are not part of routine childhood vaccination programs in many countries worldwide [63, 64] ; none of these vaccines are included in the routine childhood immunization schedule in mainland China. However, Hib and influenza vaccines are commonly available in many parts of China through vaccination clinics, and Hong Kong SAR is the first region in China, as well as Asia, where pneumococcal conjugate vaccine will be included in their routine childhood immunization program starting September 2009 [65] . In addition, Hong Kong SAR recommends seasonal influenza vaccine use in high risk groups. Vaccine clinical trials in other countries have estimated that 21% of radiologically confirmed pneumonia is caused by Hib [66] and 36% by pneumococcus [66, 67] ; over 10% of hospitalized pneumonia in children in nearby Thailand are due to influenza [68] . Studies within China have suggested that Hib and pneumococcus are common causes of pneumonia in children [69, 70, 71] , suggesting that widespread use of these two vaccines, as well as influenza vaccine, could reduce the incidence and mortality of pneumonia in China.
This paper has several strengths, particularly the inclusion of papers published in both the English and Chinese literature. A recent global review on childhood pneumonia incidence included only two of the 14 articles presented here, suggesting that articles not published in English are usually overlooked and difficult to obtain [3, 16, 18] . We did not search three of the five major Chinese-language literature databases (the China National Knowledge Infrastructure China Academic Journals Full-text Database, Chinese Biomedical Literature Database, and Chinese Medical Current Content), so we may not have captured all relevant manuscripts; however, the two databases that we did search include some of the greatest number of journals and articles of the five major databases [10, 11, 72] . In addition, this review includes information on all ages, although only two studies included adults. While global pneumonia prevention efforts often focus on children, the burden of pneumonia in adults and the elderly is also substantial. Importantly, interventions aimed at children may have underappreciated benefits on adults. For example, the introduction of universal childhood pneumococcal vaccination in the United States in 2000 resulted in significant declines in pneumococcal incidence in both children and adults. In 2003, the indirect effect of preventing invasive pneumococcal disease in adults was over twice the direct effect of preventing cases in children [73] .
Comprehensive data on pneumonia incidence and mortality are essential for monitoring disease trends, guiding policy decisions, and prioritizing disease prevention and control strategies. For China, accurate information on the incidence and mortality of pneumonia, as well as data on cost, will be central for planning the addition of new vaccines to routine childhood immunization programs. Also, seasonal and pandemic influenza remain ever present global threats. Continued surveillance and consideration of influenza vaccination and other control measures are needed. In collaboration with the U.S. Centers for Disease Control and Prevention (CDC), China's CDC is implementing active, population-based surveillance for pneumonia in certain areas using a standard approach and case definitions. The surveillance system will aim to better define pneumonia incidence, identify etiologies, and guide important clinical and public health decisions. Increased laboratory capacity needs to be built to ensure continued rigorous surveillance and quick response to emerging threats. Together these improved surveillance and laboratory data should help improve detection, prevention and control of pneumonia throughout China.  On the possible role of robustness in the evolution of infectious diseases Robustness describes the capacity for a biological system to remain canalized despite perturbation. Genetic robustness affords maintenance of phenotype despite mutational input, necessarily involving the role of epistasis. Environmental robustness is phenotypic constancy in the face of environmental variation, where epistasis may be uninvolved. Here we discuss genetic and environmental robustness, from the standpoint of infectious disease evolution, and suggest that robustness may be a unifying principle for understanding how different disease agents evolve. We focus especially on viruses with RNA genomes due to their importance in the evolution of emerging diseases and as model systems to test robustness theory. We present new data on adaptive constraints for a model RNA virus challenged to evolve in response to UV radiation. We also draw attention to other infectious disease systems where robustness theory may prove useful for bridging evolutionary biology and biomedicine, especially the evolution of antibiotic resistance in bacteria, immune evasion by influenza, and malaria parasite infections. In biology, robustness describes the relative capacity for a biological system to maintain constancy of phenotype ͑e.g., population growth, individual development͒, despite perturbation by mutation ͑genetic robustness͒ or by environmental change ͑environmental robustness͒. [1] [2] [3] [4] Epistasis is implicit in genetic robustness; a robust genome tends to retain phenotype when a mutation is introduced, whereas the identical mutation is expected to typically alter phenotype when placed in a brittle ͑nonrobust͒ genetic background. Both types of robustness are central to evolutionary biology because robustness dictates how organisms respond to environmental challenges, the very crux of natural selection.
Advancements in the understanding of robustness and its evolution have often arrived through theoretical studies, [5] [6] [7] [8] but empirical studies have made recent in-roads. Experiments using artificial life ͑"digital organisms," selfreplicating computer programs that can evolve͒ valuably demonstrated that elevated mutation rates can select for evolved increases in genetic robustness to tolerate mutation, even at the expense of reduced reproductive fitness. 9 The explanation was that high mutation rates could selectively favor genetic variants that were not necessarily productive and resided on flat regions of the "fitness landscape;" these robust genotypes formed an epistatic network that produced equally fit phenotypes despite mutation-induced movement across the landscape ͑Fig. 1͒. 6, 8, 10 Other landmark studies have successfully examined robustness by considering phenotypic effects of mutations underlying proteins using computational and in vitro approaches. 11, 12 Viruses with RNA genomes are natural systems that typically experience high mutation rates owing to their lack of error-repair during replication. Thus, RNA viruses have proved to be useful and tractable models for studying robustness evolution in biological populations. This work has focused on the success of robust versus nonrobust RNA virus variants when mutation rates are further elevated through exposure to ultraviolet ͑UV͒ light and other mutagens, 13 and on evolved changes in robustness under frequent virus coin-fection, which allows buffering of mutational effects via complementation. 14 Below we review some of the evidence from these studies, and present new findings from an increasingly popular model system for examining evolution of robustness: the segmented RNA bacteriophage 6. [14] [15] [16] In reviewing these results, we hope to highlight the importance of empirical work in RNA viruses for testing theory pertaining to robustness, as well as for better understanding the evolutionary biology and evolvability of infectious organisms in general.
Our second main goal is to review the evidence from biomedically important infectious diseases of humans, describing how the success of various disease agents may be commonly considered in light of their genetic and/or environmental robustness. The importance of robustness has been discussed in the context of noninfectious diseases such as cancers and metabolic syndromes. 17, 18 Here we focus particularly on infectious disease systems: the evolution of antibiotic resistance in bacteria, influenza virus host shifts, and malaria parasite invasion. A review of these studies seeks to further demonstrate the broad importance of robustness to theories of biological systems, and the evolution of infectious disease.
If a population is well-adapted to its environment, almost all mutations should lead to deviations from optimal performance in the selective habitat. Therefore, populations at equilibrium are expected to experience selection for genetic robustness. But study of this process in experimental populations is problematic for at least two reasons: equilibrium states are difficult to achieve ͑or definitively prove͒ in lab-evolved populations, and selection for mutational robustness is "second-order" because the benefit is not experienced until offspring carrying mutations arise. 1 However, theory and artificial-life data 9 support the idea that genetic robustness should be strongly favored when populations experience elevated mutation rates, suggesting that RNA viruses would be fruitful systems to explore how genetic robustness evolves. In general, success in these systems has come either from manipulating ecological conditions such that robustness becomes less important and evolves to decrease, or from manipulating competition environments to examine whether elevated mutation rates favor genetically robust populations.
In both strategies, the ultimate goal was to manipulate treatments and confirm that certain test populations better maintained a measurable phenotype in spite of mutational change, and thus were relatively robust.
Theory on adaptive genetic robustness under elevated mutation rate generally assumes that phenotype expression results solely from the underlying genotype. However, viruses can employ complementation, a mechanism whereby low-fitness genotypes can use, to their advantage, intracellular proteins made by coinfecting strains of high fitness. [19] [20] [21] Coinfection combined with complementation can therefore provide phenotypic buffering in the event of genomic mutations, similar to other buffering mechanisms such as gene duplication and diploidy that might be positively selected because they facilitate canalization in higher organisms. 22, 23 Complementation can buffer against negative fitness effects of deleterious alleles in coinfecting populations of viruses. 20 This buffering effect introduces an ecologically determined community-level robustness which renders their individuallevel robustness less necessary. This logic infers that the degree of coinfection-high multiplicity of infection ͑MOI; ratio of viruses to cells͒ versus low MOI-should influence evolution of robustness in virus populations. 14 We note that many other phenomena consequential for virus evolution can occur as a result of coinfection, especially genetic exchange ͑recombination͒ between viruses and selection for virus "cheaters" such as defective-interfering particles. 24 For brevity we limit our discussion to complementation that occurs during coinfection and its potential role in the evolution of virus robustness.
The segmented RNA phage 6 is typically grown in the laboratory on the plant pathogenic bacteria Pseudomonas syringae pathovar phaseolicola. To examine whether high-MOI populations of phage 6 will evolve decreased genetic robustness against mutations, virus populations were first propagated for 300 generations under high versus low MOI. 25, 14 The expectation was that high-MOI evolved viruses would become more brittle, in comparison to their low-MOI evolved counterparts. To reveal this assumed outcome, clones were randomly chosen from the treatment populations and used to initiate lineages subjected to mutation accumulation via passage through severe bottlenecks ͑population size= 1 individual͒; this method allows genetic drift to overwhelm selection, such that virus lineages amass mutations that are on average deleterious. 26 The mean magnitude and FIG. 1. Genotype and phenotype spaces are represented schematically in two dimensions. A brittle organism produces a phenotype that is a reflection of the underlying genotype, whereas a robust organism produces a constant phenotype regardless of the underlying genotype. ͓Reprinted with permission from M. Félix and A. Wagner, Heredity 100, 132 ͑2008͒. Copyright ©2008 Nature Publishing Group.͔ variance in fitness changes that occurred because of bottlenecking were statistically compared between lineages founded by presumed robust ͑low-MOI͒ and brittle ͑high-MOI͒ viruses. Results from the mutation accumulation analysis supported the hypothesis that viruses historically evolved under frequent coinfection became relatively less genetically robust than those evolved under rare coinfection. 14 One alternative explanation for the observations would be a higher mutation rate in the viruses evolved at high MOI, causing them to experience a greater number of fixed mutations ͑and hence, greater fitness loss͒ during the mutation accumulation; however, no evidence suggests that these viruses mutate at rates higher than their counterparts evolved at low MOI. 14, 15 Thus, the study provided the first evidence that genetic robustness could evolve to change in biological populations.
Other experimental studies have used RNA viruses or viroids ͑RNA viruslike plant pathogens that lack proteincoding genes͒ as models to study whether elevated mutation rates favor robust genotypes. One study examined two species of viroids that differed in robustness. 13 The robust viroid species had a low reproductive capacity in plants but a large neutral neighborhood of mutations ͑fraction of one-error mutations that do not change minimum free energy RNAsecondary structure͒, whereas the brittle viroid had the opposite combination of traits. It was hypothesized that brittle viroids should outgrow robust viroids when coinfecting plants under ordinary conditions, but the robust viroids should be favored in the presence of a mutagen because they better maintained phenotype ͑proper structure͒ despite mutational input. As predicted, under standard growth conditions in host plants, results showed that the brittle viroid was selectively favored. In contrast, when infected plants were subjected to irradiation by ultraviolet C ͑UVC; short wave UV light of wavelength 280-100 nm͒, this known viroid mutagen caused the robust viroid to be favored in competition, because it better withstood growth despite mutation. 13 Thus, this empirical outcome in biological populations was consistent with the earlier findings seen in artificial-life studies. 9 A second study used an RNA virus of eukaryotes and two different mutagens, but found very similar results to those described above. 27 Vesicular stomatitis virus ͑VSV͒ is an ssRNA virus that is vector-transmitted by some insects and causes disease in certain mammal species. 28, 29 Two diverged populations of VSV were characterized for reproductive growth on tissue culture cells, and for within-population variance among clones for this phenotype; this effort revealed that one population was slow-growing but relatively less variable in clone phenotypes ͑robust͒ and the other had the opposite characteristics ͑brittle͒. 27 Whereas standard growth conditions allowed the faster replicating brittle virus population to outcompete the slower replicating robust population, exposure to either of two mutagens favored the robust variant. These results echoed the earlier studies where competition under elevated mutation rates was observed to favor robust populations, even though robustness was associated with reduced reproduction. 9, 13 While several of these studies have provided evidence for extant, evolvable differences in robustness, the literature is generally lacking in examples of direct selection for robustness in biological systems. In an effort to examine direct selection for RNA virus resistance to UVC damage, here we present new data from a short-term experimental evolution study with phage 6. These results underscore some of the biological, methodological, and conceptual barriers to observing direct selection for robustness in RNA virus systems.
UVC is well-known to impact RNA viruses negatively 30, 31 and previous studies have directly examined the mutagenic effects of UV radiation on RNA genomes. 13, 32 Furthermore, evidence from the biochemical and biophysical literature suggests that UV radiation can interfere with RNA replication fidelity. [33] [34] [35] Regardless, preliminary experiments showed that UVC exposure for periods up to 30 min greatly increased mortality in wild type phage 6 ͑Fig. 2͒, indicating that this environmental effect should produce strong selection for UVC resistance in populations of the virus. We note that identical UVC exposure was even more damaging in another well-studied virus, the ssDNA phage X174 ͑Fig. 2͒; this difference suggested that RNA phage 6 may be relatively more robust to effects of UVC than other viruses, echoing our earlier suggestion that the virus was fairly robust to mutation accumulation. 14 For this reason, experimental evolution of phage 6 under UVC exposure may provide a conservative test for whether virus populations can adapt to resist the debilitating effects of UV.
A single clone of wild type phage 6 was used to found three replicate populations in each of three experimental treatments ͑nine populations total͒, where UVC exposure was manipulated using our published methods. 16 A 200 ml aliquot containing ϳ10 6 plaque-forming units ͑pfu; i.e., viable virus particles͒ of each population was suspended in Luria broth culture medium in the absence of cells. Aliquots were then placed in wells of a flat-bottomed polystyrene 96well plate. A UV illuminator ͑Spectroline Long Life Filter͒ was placed over the 96-well plate to expose the virus samples to UVC emission. "High dosage" treatment exposed FIG. 2. Death curves for virus strains in response to differing dosages of UVC exposure. Wild type RNA phage 6 ͑filled circles͒ better withstands UVC degradation than does DNA phage X174 ͑filled squares͒. A phage 6 population experimentally evolved for 20 generations under 2.5 min UVC exposure ͑open circles͒ survives better than its ancestor, except under prolonged dosage. In contrast, a representative phage 6 control population ͑gray circles͒ evolved in absence of UVC exposure presents a death curve nearly identical to the ancestor. See text for details. populations to UVC for 15 min, "low dosage" treatment exposed them for 2.5 min, and the "control" treatment populations were never exposed to UVC. Following this manipulation, each population was mixed ͑1:1 volumetric͒ with P. phaseolicola bacteria ͑i.e., ϳ4 ϫ 10 9 cells͒ and incubated at 25°C for an additional 120 min, sufficient time for at least one virus generation consisting of cell attachment/entry, intracellular replication of virus progeny, and cell lysis ͑death͒ that bursts the cell to liberate offspring. A typical burst in phage 6 produces ϳ200 viral progeny per infected cell 36 ͑we note that the host bacteria were never exposed to UVC, eliminating the possibility that UVC negatively impacted cell physiology or caused host mutagenesis͒. After 120 min, the mixture was centrifuged to pellet cells, and the supernatant was filtered to harvest a cell-free virus lysate containing up to ϳ2 ϫ 10 8 pfu of virus progeny. The lysate was then stored at 4°C for 22 h. The lysate was diluted 100-fold and used to initiate the next passage where the phage population was again subjected to treatment or control conditions, and allowed to infect naive bacteria freshly cultured from frozen stock ͑i.e., to eliminate possibility of phage-bacteria coevo-lution͒. Thus, experimental populations were challenged to produce sufficient progeny to sustain themselves in the face of the daily 100-fold dilution; UVC was an environmental stressor that could reduce the likelihood of this sustainability unless viruses responded through adaptation. A total of 20 passages ͑i.e., 20 generations͒ occurred in the short-term selection experiment, and samples at each passage were stored at −80°C for future analysis.
At the end of the study, replicated ͑n =4͒ assays were performed to gauge whether the survival of treatment populations had changed during the experiment. Evolved populations were measured for frequency of surviving virions, in assays where samples containing ϳ10 6 pfu of virus were exposed to UVC for six different time periods: 1, 2.5, 5, 10, 15, and 30 min. Results showed that after 20 passages the death curves for the populations in the control treatment were highly similar to that presented by the wild type phage 6 ancestor; Fig. 2 shows a representative outcome for one of the control populations. In contrast, we observed that all three lineages in the high dosage ͑15 min UVC exposure͒ treatment went extinct by passage 10 of the experiment ͑data not shown͒. These data were informative because they indicated that the strong selection created through high dosage of UVC exposure was too overwhelming for viruses to be ecologically sustained, and/or to experience spontaneous genetic variation useful for meeting the environmental challenge. The latter outcome clearly demonstrated an evolutionary constraint for the virus. Last, all of the evolved populations in the low dosage ͑2.5 min UVC exposure͒ treatment were extant by end of the study. However, only one of these three populations exhibited a death curve that differed from the ancestor, as shown in Fig. 2 . This result proved that a response to UVC selection was possible in phage 6 under our low dosage conditions. Furthermore, the data interestingly showed that the adaptation caused ϳ17% gain in survival ͑on average͒ relative to the ancestor at dosages away from the selection challenge of 2.5 min ͑i.e., 1, 5, 10, and 15 min͒. However, this response on the part of the one lineage was not overly impressive, because the same 50% mortality as the ancestor was evident under 30 min UVC exposure, demonstrating an additional constraint in the virus even though an adaptive response occurred at low dosage ͑Fig. 2͒.
Given the overall modest response in phage 6 populations subjected to strong selection for changes in the UVC survival phenotype, the available evidence strongly suggests that pre-existing constraints prevent the virus from easily adapting. Thus, we did not observe adaptive increases in virus resistance to UV degradation, presumably involving mutagenesis. However, the experimental evolution was of relatively short duration, and additional selection might produce adaptive resistance to UVC damage. Nevertheless, a lack of adaptive response may be unsurprising in phage 6, because elsewhere we showed that genetically robust and brittle strains of the virus generated in the aforementioned MOI experiment did not statistically differ when assayed for UVC survival. 16 Thus, neither direct selection ͑current findings͒ nor indirect selection for robustness against UVC damage was evident in this study system. These observations may be explained by a relatively high level of robustness to UV damage in phage 6, which has already run the gamut of selection, creating a constraint for the virus to improve any further. One ecological explanation may be that the virus naturally infects plant pathogenic bacteria that colonize leaf surfaces, 37 suggesting that prior adaptation to resist UV damage might have precluded observations of further adaptive response in the laboratory.
This study reveals the potential pitfalls to overinterpreting empirical results in the attempt to study robustness. First, claiming that an organism has directly evolved robustness through natural selection requires a nuanced understanding of how a given environmental stressor impacts an evolvable entity. Such knowledge can be dubious in the RNA virus context, as stressors such as UVC might impact RNA viruses in multiple ways. [30] [31] [32] Even further, if a test population evolves an adaptive response to an environmental challenge, one has to be cautious in distinguishing resistance from robustness. While both involve the ability to withstand the influence of perturbations, robustness describes a more generalized response that affects performance in multiple environments 1,2 and evolvability. 15, 16 
The existing empirical evidence for evolved changes and selective advantages in robustness reinforces the importance of microbial systems in the study of evolutionary biology. But later work examining the relationship between robustness and evolvability is perhaps even more relevant for elucidating how infectious diseases evolve. On the one hand, the ability for robustness to allow phenotypic constancy in the face of environmental and mutational perturbation provides obvious benefits, such as reliable cellular function, individual development, and offspring reproduction. However, rigidity in the face of change may pose problems; because natural selection acts on phenotypic variation, robustness that buffers this variation could impede evolution by natural selection. These conflicting necessities force organisms to strike a balance between robustness and evolvability, the capacity to adapt. By examining this balancing act, we may learn whether evolvability can itself evolve; i.e., whether natural selection can exert a second-degree effect on evolution. Below we briefly describe two recent experiments on the relationship between robustness and evolvability.
One experiment showed that genetic robustness improved evolvability of phage 6 populations when heat shock was used as an adaptive challenge. 15 Phage 6 is typically cultured at 25°C, and exposure to 45°C heat shock for as little as 5 min leads to ϳ20% survival ͑%S͒ in populations of the virus. Robust and brittle clones of the virus from the aforementioned MOI experiment 14 were used to found lineages passaged for 50 generations of experimental evolution with periodic ͑every fifth generation͒ exposure to 45°C heat shock. At the end of the study, we measured mean %S at 45°C measured for each founding clone and its derived end point population to estimate ⌬%S, the change in percent survival after heat-shock selection. The results showed that the lineages founded by robust genotypes were more evolvable ͑greater ⌬%S͒, indicating that robustness promoted evolvability in phage 6, under the test conditions. The explanation was that robust genotypes of phage 6 may have proteins that better tolerate mutations while maintaining proper folding especially at moderate temperatures, 16 similar to suggestions from in vitro studies where less-sensitive ͑relatively robust͒ proteins seem more likely to maintain their function in a new environment where innovation is needed. 11 That is, despite equivalent sensitivity to 45°C heat shock in the robust and brittle founding strains, the robust viruses may have proteins that tend to be capable of undergoing mutations while maintaining their proper folding when 45°C constitutes a selective environment. 15 Whether this exact relationship extends to other novel environments has yet to be explored. But the fact that a positive relationship exists at all is essential for tests of existing theory, 8 and begs the question of whether the observed relationship between genetic robustness and evolvability extends to evolution of human pathogens, as implicated in recent claims of evolved increases in genetic robustness in HIV. 38 Antiviral therapy consisting of chemical mutagenesis that induces an insurmountable mutational load seems promising for treatment of RNA virus infections. [39] [40] [41] But such measures may select for RNA viruses to evolve altered polymerases with improved replication fidelity that reduces genomic mutation rates. 42 Alternatively, viruses may be selected to evolve mechanisms of genetic robustness where high mutation rates are preserved but phenotypic effects of the mutagen are buffered. This outcome is perhaps unlikely given the empirical data thus far. 43 However, further data involving a variety of different viruses are warranted, and the results from experimental evolution studies caution that robustness may be positively related to evolvability and to competitive superiority in RNA viruses. 15 A second experiment centered closely on the potential link between robustness and pathogen evolvability on novel hosts. Environmental robustness of a virus may be defined in terms of its host generalization ͑host range, the number of host types it can productively infect͒. 44 Theory predicts that evolved generalization may be particularly useful if organ-isms tend to encounter environments that change unexpectedly. 45 That is, generalists may be favored by selection because they are better predisposed to survive and give rise to successful progeny in the face of variable environments, relative to specialists that possess a narrow niche. This idea may explain why pathogens with a pre-existing broad host range seem better able to emerge on novel hosts, relative to specialized pathogens. [46] [47] [48] To test this idea directly, we used populations of VSV that evolved differences in host use because of prior selection in constant versus variable host environments. 49, 50 The formal prediction was that VSV populations experiencing direct selection for host range would have higher mean growth and less variance in mean growth on a collection of challenge hosts, compared to VSV populations that were either relatively specialized or indirectly selected for host range. 51 When challenged to grow on four novel hosts in vitro, the viruses that had been selected for generalism exhibited higher or equivalent host growth, lower among-population variance in host growth, and lower variance in population growth across hosts. Thus, these three predictions relating to the hypothesis were generally supported because direct selection for host breadth more often allowed successful emergence. The results suggested that determination of current niche breadth should be further investigated as a potentially useful indicator in predicting pathogen emergence. 51, 52 Because these many recent empirical breakthroughs in the study of robustness have involved microbes, one might predict that infectious disease is the primary biomedical realm where these robustness results might have an immediate, practical impact. Below we describe three examples of biomedically important human disease systems where robustness theory appears highly relevant.
The effects of evolution are sometimes difficult to perceive in daily life, making public skepticism of the existence of evolution all the more challenging to address. Perhaps the foremost example used to counter these notions is the widespread problem of resurgent diseases that had been previously treated through antibiotics, especially evolved resistance in bacterial pathogens. Although naturally produced antibiotics evolved in microbial communities long before humans appeared as a species, the medical realm began earnestly administering antibiotic drugs only since their discovery in the 1950s. Widespread production and utilization of antibiotics in medicine and agriculture, however, has created a massive uncontrolled experiment in which bacteria have been selected to harbor antibiotic resistance genes, making existing therapies largely ineffective in many clinical circumstances.
Bacterial resistance to antibiotics and other harmful substances ͑e.g., heavy metals͒ often occurs through genes carried on plasmids: autonomously replicating DNA elements that are nearly ubiquitous in bacterial populations. Individual plasmids can sometimes include multiple genes conferring resistance to a variety of antibiotics. Also, plasmids can effectively spread within and among bacteria populations and species through conjugation ͑cell-to-cell horizontal transfer͒. Together, these features create the capacity for rapid spread of multiple drug resistance in bacteria. Furthermore, plasmid-borne mechanisms ͑e.g., postsegregational killing͒ often exist to ensure that vertical transfer ͑plasmid inherit-ance͒ is reliably maintained across host-cell generations. For these reasons, plasmid biology has undoubtedly contributed to the current crisis of ineffective antibiotic therapy.
Plasmid-bearing bacteria that are resistant to a wide variety of antibiotics can be defined as relatively environmentally robust, compared to bacteria strains unable to grow in the presence of antibiotic challenges. Whether this difference arose largely through selection acting at the level of bacteria, or purely at the level of plasmids, is unclear. In the former case, acquisition and maintenance of a large number of resistance genes could be evolved "bet-hedging" in bacteria, where selected retention of these alleles occurs despite only rare circumstances when they are actually needed to protect against antibiotics. Alternatively, selection may be acting purely at the level of plasmids, favoring infectious genetic elements that collect resistance genes which may be beneficial for "paying the rent" to their bacterial hosts, as plasmids cannot easily control the host backgrounds and environments in which they reside. Last, selection may be acting on the symbiotic microbe created through plasmid/cell association, where the combined interests of plasmids and their hosts may sometimes coincide and other times conflict. Regardless, environmentally robust bacterial pathogens of humans must have been selectively favored during the past halfcentury, or the ineffectiveness of antibiotic therapy would not be a prominent issue today.
The evolutionary consequences of environmental robustness in resistant bacteria have not been widely addressed. Bacteria able to robustly grow in a variety of antibiotic environments are generalists according to the ecological definition of niche breadth. This niche breadth may have been directly molded through selection if the bacteria descended from a lineage exposed to the various antibiotics. Alternatively, the niche breadth may be fortuitous if the bacteria obtained a plasmid harboring multiple-resistance genes that ran the gauntlet of selection in other contexts. No matter the circumstance, we noted above that some theory predicts evolved generalization may be particularly useful if organisms tend to encounter highly variable environments. 41 The phenomenon of drug cross-resistance and possible evolutionary modification of a resistance allele allowing survival in altered circumstances ͑i.e., evolutionary innovation͒ lend plausibility to the concept that multiple-resistance strains by an evolvability advantage would better thrive in the face of novel drugs. We are unaware of any studies addressing the relationship between number or diversity of resistance genes in bacteria and the evolvability of bacterial pathogens in new environments. Such research would be highly useful for examining the evolutionary processes underlying the widespread success of resistant bacteria.
We note that existing data already suggest that genetic robustness is relevant to the evolution of antibiotic resistance in bacteria. Research shows that compensatory mutation is an important underlying genetic mechanism for maintenance of resistance in the absence of antibiotic selection. 53 This epistasis mechanism explains how a resistance gene that is costly for bacterial growth in absence of an antibiotic may be compensated by one or more mutations in the plasmid or bacterial chromosome, effectively eliminating the cost of carrying the resistance gene ͑or the plasmid on which it re-sides͒ when no antibiotic is present. If a bacterial strain contains a compensatory mutation that interacts to reduce the cost of various different resistance genes, then this epistatic mutation would provide an example of a genetic-robustness allele responsible for environmental robustness across antibiotic environments. For example, the multiple antibiotic resistance locus in E. coli and several species of Salmonella is involved with production of an efflux pump that removes various antibiotics from the cell, affording low-level clinical resistance. 54 However, this mode of resistance can be rather costly if transport in and out of the cell is not tightly regulated. Evidence suggests that the low-level resistance may be a stepping-stone to more finely tuned evolved mechanisms allowing greater resistance, 55 and we can conceive that genetic changes simultaneously compensating to reduce the cost while maintaining the broad resistance would provide an example of a genetic-robustness allele.
Many of the above studies suggest ways that robustness might play a role in disease pathogenesis and treatment efficacy, and how robustness might be linked to emergence of future viral diseases in humans. However, contemporary examples already indicate that robustness theory is helpful in elucidating evolutionary dynamics of important RNA virus diseases. One example is the occurrence of flu pandemics in humans, a substantial public health concern in the last century.
Recent studies suggest that strains of influenza A virus can spread through human populations because they form a neutral network of virus genotypes that share the same phenotype. 55, 56 Essentially, this network allows the virus to drift through neutral space connected via mutation, until a single mutation shifts to a different serotype ͑Fig. 3͒. This shift then facilitates escape from host immunity, and fosters the ability for the virus to infect a new class of susceptible hosts. Although these studies make no explicit mention of the role of such genetic robustness in flu epidemics, 55,56 the available evidence strongly suggests that the phenomenon is relevant in this case of influenza strain H3N2. Genetic robustness in the hemagglutinin ͑HA͒ gene of strain H3N2 allows the virus to drift through neutral genotypic space without a change in phenotype until it reaches a rapid saltational phenotypic shift that permits the ability for strains derived from this virus to reemerge in humans. This model is compatible with other robustness-evolvability links outlined in the literature. 57, 7, 8 Judging from phylogenetic and molecular evolution analyses of clinical isolates, it appears that the HA gene of influenza A virus appears to be under strong selection by the human immune system, 58 and perhaps this has been true for much of the evolutionary history of flu disease in humans.
The genetic robustness of influenza viruses and the accompanying structure of neutral genetic networks probably arose to foster the epidemiological spread of the virus in humans and/or other animal hosts. The size of these neutral networks is of consequence in the evolution of influenza, as suggested by modern robustness theory. 8 A HA gene with relatively low robustness would feature a small neutral network; this characteristic might cause the virus to molecularly evolve rather quickly, perhaps outpacing the availability of susceptible individuals in a host population, slowing or halting spread of the virus. But a relatively larger neutral network would afford greater robustness of HA genes in influenza, affording long-term persistence of the virus as it drifts neutrally through sequence space before fortuitously achieving a genotype with a beneficial novel surface antigen that promotes reemergence.
Genetic robustness in the genetic networks of influenza A virus might have an environmental robustness correlate. Influenza A virus is notorious for its characteristically broad host range, including various bird species and mammals such as domesticated pigs whose proximity to humans may spur pandemics. [59] [60] [61] As noted earlier, relatively broad host range of a pathogen may be defined as environmental robustness. The ability for influenza A viruses to traverse the aforementioned neutral network of genetic robustness may have an important environmental robustness component. The virus may be able to easily create a robust neutral network of genotypes associated with attachment/entry of various host cells due to intrinsic environmental robustness that evolved via selection to infect a variety of host environments. In influenza A virus, whether environmental robustness preceded genetic robustness, or vice versa, is difficult to know. But environmental robustness of viruses such as influenza A virus undoubtedly fosters the ability to reside in various host species that may serve as reservoirs that allow occasional spillover into a species of interest such as humans. 52 Robustness may also be relevant in explaining aspects of the current AIDS pandemic. A global increase in robustness has been invoked to explain an apparent global decline in the virulence of HIV-1 strains. 38 Although it was not discussed extensively, the robustness concept in this example seems to be environmentally, not mutationally, determined. The argument is that a perceived decline in HIV-1 virulence might be due to generally evolved increase in environmental robustness of the virus, owing to its worldwide spread. The virus has thus encountered variable subpopulations of humans and has undergone selection for environmental robustness, which coincides with improved performance across environments at the expense of reduced reproduction ͑i.e., virulence͒ on average. This explanation assumes that the stated tradeoff in viral traits is a generalized phenomenon, and that results for increased genetic robustness at the expense of reduced reproduction 9,13 apply in the case of environmental robustness as well.
Regardless of the disease system, pharmacology involves the use of small molecule inhibitors of biomolecular interactions, and we believe that robustness may be a highly relevant concept in the identification of potential drug targets. This logic begins with identifying in pathogen systems the modules that may serve as good targets versus those that would be poor targets for pharmacological intervention. Presumably, poor targets would be modules that are robust to perturbations, pharmacological agents included.
When designing drugs to treat infections, the invasion step is where the pathogens might be most vulnerable to pharmacological intervention. This idea is supported by studies in which parasite exposure to chemical compounds neutralizes binding to host receptors, fortifying the importance of invasion to the establishment of a clinical infection. 62, 63 Thus, many therapeutic measures against a variety of pathogens rely on this vital receptor-ligand interaction. Virus examples include the seasonal influenza vaccine, and fusion inhibitor compounds, a class of anti-HIV therapies. 64, 65 In nonvirus parasites the situation is more complicated, because the process of invasion is a complex module involving several different effectors. One possible example of a robust invasion module is in Plasmodium falciparum, the causative agent of the most deadly form of human malaria. In P. falciparum and the apicomplexan parasites, an apical complex is central to the invasion process, which includes multiple steps involving protein interactions and temporally and spatially regulated expression of proteases and polymerization machinery. 63, 66, 67 Given the importance of erythrocytes as cells where parasite replication occurs, one might expect that P. falciparum interactions with erythrocytes should be particularly critical in a robustness module for the FIG. 3 . ͑Color online͒ Robustness in gene networks of influenza A HA genes. Phenotypes are genetically robust, allowing genotypes to drift through genotypic neutral space before instant shifts in phenotype. Such a scenario is a demonstration, from the actual epidemiological literature, of how genetic robustness can facilitate evolvability in infectious disease agents. ͓From E. van Nimwegen, Science 314, 1884, 2006. Reprinted with permission from AAAS.͔ pathogen. In fact, studies show that the erythrocyteattachment process involves redundancy in ligands used for binding of the pathogen to erythrocyte receptors. 68, 62 This evolved strategy might have been selected in P. falciparum because erythrocyte receptors are highly polymorphic, perhaps among the fastest evolving genes in the human genome. 69, 70 Research shows that P. falciparum utilizes a plethora of ligands to bind erythrocyte receptors. 62, 68, 70 Interestingly, a "molecular hierarchy" may be the ideal term to describe ligand usage in P. falciparum; the pathogen's invasion module may consist of a hierarchy whereby some ligands used in attachment are preferred over others, with the switch in usage only taking place when a ligand higher up in the hierarchy is ineffective ͑Fig. 4͒. 68 This observation may be usefully couched in terms of robustness, because redundancy is a possible mechanism for constructing a robust biological system. To ensure that an essential function is carried out, a biological system can be organized to contain several alternative pathways in the event that one or more become unavailable or dysfunctional. This evolved strategy decreases the probability that an environmental perturbation disrupts performance.
The concept of robustness has the continued potential to shift paradigms in biology by challenging assumed relationships between genotypes and phenotypes. Here we have highlighted some of the empirical evidence for evolution of robustness, and discussed its relevance for evolution of infectious diseases, particularly in humans. Despite the widereaching importance of robustness theory in biology and other disciplines, we emphasize that the origins and implications of robustness may be highly context specific. The underlying causes of diseases in humans and other organisms are manifold. To wield the robustness concept effectively in the evolution of infectious diseases, sufficient understanding of how ecological history might have selected for robustness is warranted. Robustness has often resided in the realm of mathematical theory, but future work would benefit from col-laborations between empiricists and theoreticians to test explicitly predictions using tractable models, or disease pathogens. These efforts would help to elucidate further the role of robustness in past evolution of parasites such as influenza A virus and P. falciparum, and to predict how it might play a role in their future evolutionary change.
Empirical evidence for unifying principles in biology is often sought, but is not easily achieved. One consequence of earth's biodiversity is that exceptions to general rules are often found if one searches hard enough. For example, a popular notion in biomedicine was that parasites should inevitably evolve to become mutualistic to their hosts, because this would afford long-term survival of a parasite species that might otherwise force its host into extinction. However, theory and experiments later overturned this conventional wisdom by demonstrating that infectious parasites may evolve greater or lesser virulence ͑damage to the host͒ depending on their unique ecologies. [71] [72] [73] [74] Similarly, it should be expected that evolution of robustness and its underlying mechanism might differ substantially across the biosphere. Therefore, caution is warranted when attempting to draw widespread conclusions from the handful of valuable empirical studies on evolution of robustness. Perhaps it is possible to construct a universal theory of robustness that applies everywhere, but this is unknown. Our hope is that the exciting studies and biological examples presented here will further motivate researchers in basic science and biomedicine to consider the potential importance of genetic and environmental robustness in a great variety of biological systems. With this impetus, we may amass enough empirical data to help bridge the intellectual gap separating abundant theory on robustness evolution and application of this thinking to applied problems in biomedicine and public health. Isolation and characterization of microparticles in sputum from cystic fibrosis patients BACKGROUND: Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients. METHODS: Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens. RESULTS: Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a(+)-and CD66b(+)-, but not CD11b(+)-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients. CONCLUSIONS: In summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF. In cystic fibrosis (CF), the lung disease is characterized by high concentrations of neutrophil chemokines, such as IL-8, and a sustained accumulation of neutrophils in the airways [1, 2] , in presence and absence of detectable infection [3] . In CF airways, neutrophils undergo conventional activation and functional reprogramming [4] [5] [6] [7] . For example, they show oxidative burst increase, enhanced production of leukotriens and elastase, increased IL-8 and decreased IL-1 receptor antagonist release (reviewed in [8, 9] ). However, the neutrophil response is not capable to clear bacteria from the CF airways ensuing in exaggerated apoptosis of neutrophils [10] [11] [12] [13] . Furthermore, neutrophils are targeted by Pseudomonas aeruginosa, the main pulmonary pathogen associated with the disease. Neutrophils killed by the bacteria release proteases that disable any neighbouring viable neutrophils [14] . Thereafter, bacterial persistence and the products of the damaged neutrophils spur further neutrophil recruitment, inducing inflammation, tissue damage and then generation of an environment that allows continued infection.
Sputum is recognized as a very useful sampling method in CF for both research and clinical use aiding both the diagnosis and monitoring of lung disease inflammatory status. A great advantage of the technique is that it enables sampling of the airways in a non-invasive manner, in contrast with other methods such as bronchial biopsy, bronchial brushing and broncho-alveolar lavage, all of which require bronchoscopy, discomfort and risk that it entails [15] . Furthermore, sputum may contain protein/peptide components that could act as biomarkers of disease or its severity [16] .
Microparticles (MPs) are small plasma membrane vesicles that are less than 1 μm released by several cell types (macrophages, platelets, endothelial cells, granulocytes, monocytes, lymphocytes) following chemical (cytokines, thrombin and endotoxin), physical (shear stress and hypoxia) [17] and apoptotic [18] stimuli. One of the first described roles for MPs was in the initiation and amplification of the coagulation cascade and furthermore they play a pivotal role in thrombosis, in the propagation of inflammation, modulation of vascular tone, angiogenesis, stem cell engraftment, and tumour metastasis. These MPs' effects depend on molecules harboured at their surface or within their cytoplasm due to their origin cell [18] . MPs are normally present in blood from healthy individuals but they increase in patients under pathological states associated with inflammation, such as sepsis [19] , preeclampsia [20] , metabolic syndrome [21] , pulmonary arterial hypertension [22] , and malaria [23] , strengthening the notion that MPs may play a role in these diseases. The phenotype of circulating MPs is also different in different pathological states, and detection of its cellular origin may serve as a predictor or marker of the diseases [24] .
Mutschler and colleagues, for the first time ever, showed the presence of MPs, derived from platelet, in pulmonary air-liquid interfaces in sedated pigs [25] . Recent investigation conducted in broncho-alveolar liquid fluid (BALF) has provided the characterization of intra-alveolar procoagulant MPs in patients with acute respiratory distress syndrome (ARDS) and hydrostatic pulmonary oedema. Intra-alveolar MPs from ARDS patients contain high levels of tissue factor, show an highly procoagulant activity, and are likely contribute to intra-alveolar fibrin formation, a critical pathogenic feature of acute lung injury [26] . To the best of our knowledge, no studies have been conducted to elucidate about the presence and role of MPs in other lung diseases. Since cellular activation and apoptosis, the main sources of MPs, are features of neutrophils in the CF airways, we have undertaken a study for the identification and characterization of MPs in the sputa of CF patients.
The study was approved by, and performed in accordance with, the ethical standards of our institutional review boards on human experimentation. Written informed consent was obtained from each subject.
We enrolled 10 CF patients who consecutively had been admitted at the CF Center of the Hospital of Cerignola "G. Tatarella" for parenteral (i.v.) antibiotic therapy during acute respiratory exacerbation, and 11 stable CF patients. Exacerbation was defined as a deterioration in symptoms perceived by the patient and included an increase in cough, sputum production, dyspnoea, decline in forced expiratory volume in 1 sec (FEV 1 ) compared with previous best, weight loss and fever [27] . Each patient was given a clinical score obtained from the sum of the individual parameters (0 = no symptom; 1 = moderate; 2 = severe). Serum C-reactive protein (CRP) was assessed as a marker of active inflammation [28] . CF patients were compared with 7 primary ciliary dyskinesia (PCD) patients.
Bacterial species in sputum specimens were identified accordingly to the North-American guidelines [29] . Sputum samples were directly spread-out in selective media, such as MacConkey agar for Pseudomonas aeruginosa and Alcaligenes xilosoxidans, manitol salt agar for Staphylococcus aureus, and BCSA for Burkholderia cepacia complex, and incubated at + 36 ± 1°C for a period of 18-72 h. Colonies were quantified and identified by classical (manual) phenotypical tests.
Spontaneous sputum was collected in sterile cup and immediately processed. The sputum was washed with NaCl 150 mM, mixed with an equal volume (1:1) of Sputasol ® (SR 0233A, Oxoid Ltd, Hampshire, UK), and then incubated in a water bath at + 37°C for 15 min until visible homogeneous.
Processed sputum was centrifuged at 37 × g for 3 min the supernatant was centrifuged at 253 × g for 10 min and then recentrifuged at 253 × g for 20 min to remove the cells and large debris, respectively. Two hundred μl of each MP-containing supernatant were frozen and stored at-80°C until characterization by flow cytometry and microbiological tests.
Remaining MP-containing supernatant was centrifuged at 14,000 × g for 45 min to pellet MPs. MP pellet was subjected at two series of centrifugations at 14,000 × g for 45 min. Finally, MP pellet was replaced in 500 μl of 0.9% saline salt solution and stored at + 4°C until total counting.
MPs population was characterized in sputum supernatant, according to the expression of membrane-specific antigens. Anti-human CD11a labelling was used to numerate leukocyte MPs, while numeration of granulocyte MPs and monocyte/macrophage MPs was performed using anti-human CD66b and anti-human CD11b, respectively. Human IgM was used as isotypematched negative control for CD66b staining, while IgG was used as isotype-matched negative control for CD11a and CD11b.
For these studies, 10 μl of supernatant MPs were incubated with 10 μl of specific antibody (1 μg/ml; FITC-conjugated; BioLegend, San Diego, CA). After 15 min of incubation at + 4°C, samples were diluted in 500 μl of 0.9% saline salt solution. Then, 10 μl of Flowcount beads were added to each sample and analyzed in a flow cytometer (Beckman Coulter coulter epics XL-MCL). Sample analysis was stopped after the count of 10,000 events.
To rule out whether sputum supernatant staining was due to bacterial cells, supernatants, used for phenotypic characterization, were plated onto agar plates and kept at + 37°C for 16 hours.
As a further control, we evaluated two bacterial strains for cross-reaction with antibodies. Pseudomonas aeruginosa PAO1 strain [30] and Staphylococcus aureus ATTC strain 29213 were thawed and bacteria were recovered on agar-blood plates. One colony of P. aeruginosa and S. aureus were allowed to grow in 1 ml of Trypticase Soy Broth (TSB) (Difco, Becton Dickinson, Sparks, MD) or BBL™ brain heart infusion (BD Diagnostic Systems, Sparks, MD) respectively, for 1 hour at + 37°C. Bacteria were then incubated with 400 μg/ml gentamicin for 2 hours at + 37°C, and subsequently with anti-granulocyte antibodies under the same conditions of MPs, then finally analyzed by flow cytometry with the same settings used for MPs.
MPs contained in the supernatant of a processed CF sputum were subjected to a single centrifugation at 14,000 × g for 45 min. MP pellet was fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 24 hours. The sample was then dehydrated in solutions of ethanol of increasing strength from 50%, 70%, 95% and 100% for 10 minutes in each solution. The sample was finally dehydrated in propylene oxide for 15 minutes. Finally, the sample was embedded in epoxy resin (Epon 12). After overnight polymerization, ultrathin sections (70 nm) were cut and examined in a JEOL (Tokyo, Japan) transmission electron microscope.
Data are shown as mean ± SEM (Table 1) or medians (quantification and phenotype of MPs present in CF and PCD sputum). Statistical significance of differences between acute and stable groups of CF patients was evaluated by a two-tailed unpaired Student's t-test. To compare the number of MPs and the amount of different antigens the non-parametrical Mann-Whitney test was used. All data were analyzed using Prism 4 (GraphPad Software, Inc., La Jolla, CA). p values of less than 0.05 were considered significant.
Characteristics of patient are summarized in Table 1 . CF patients had more expiratory airflow obstruction, as mea-sured by the FEV 1 % predicted, although not significantly different from PCD patients, and were more likely to be colonized with Pseudomonas aeruginosa and Staphylococcus aureus. In some CF patients, more than one bacterial strain colonized the same patient. The acute and stable groups of CF patients were well differentiated on the basis of decrease in FEV 1 as compared with the best one in the last year (ΔFEV 1 ), serum CRP, and clinical score ( Table 1) .
Sputa liquefied with Sputasol, a dithiothreitol formulation, were centrifuged at low speed to remove large debris, and studied by flow cytometry analysis. MPs were readily identified in dot plots ( Figure 1A ) and were positive for CD66b antigen ( Figure 1D ). To discriminate whether bacteria or bacterial bodies could give such image, supernatants were plated onto agar plates and no bacterial growth was observed in sputa obtained from CF patients. However, to evaluate if bacteria could be stained by anti-granulocyte antibodies, Pseudomonas aeruginosa PAO1 were grown for 1 hour and then killed by gentamicin treatment. Although detectable in the same region of MPs ( Figure 1B,) , killed bacteria, analyzed by flow cytometry after antibody binding did not show any positivity for the antibody directed against CD66b ( Figure 1E) .
Also, Staphylococcus aureus ATTC strain 29213 was incubated with anti-granulocyte CD66b antibody. S. aureus was partially detectable in the same region of MPs ( Figure 1C ) but like P. aeruginosa did not show any positivity for the antibody directed against CD66b ( Figure  1F ). Therefore, we conclude that the staining of sputum supernatant was given only by MPs. Figure 2 shows electron microscope picture of sputum-MPs from CF patients. Multiple spherical particles ranging in diameter from 100 to 500 nm were detected. Of note, no bacterial bodies were found associated with MPs.
We evaluated the level of MPs in the sputum of 21 CF patients compared with the sputum of 7 PCD patients. Although heterogeneous, comprising both stable and acute patients, the CF group showed a significantly higher number of MPs than the PCD group (Figure 3 ).
MP phenotype was analyzed by evaluating the presence of antigens representing different cell types: CD11a for leukocytes, CD66b for granulocytes, CD11b for monocyte/macrophages. In CF patients, amount of MPs expressing CD66b (median value of 53.8%) was higher than those expressing CD11a (median value of 16.1%) and CD11b (median value of 0%). Comparison of all CF patients versus PCD patients showed that amounts of MPs expressing CD66b and CD11a were significantly higher in CF than in PCD (CD66b: p = 0.0068; CD11a: p = 0.0226) (data not shown). No differences in the three populations of MPs were found between patients in acute and stable phase of CF. However, both acute and stable patients showed significantly higher levels of MPs expressing CD66b and CD11a in comparison to PCD patients (for CD66b: stable CF vs. PCD: p = 0.0373; acute CF vs. PCD: p = 0.0046. For CD11a: stable CF vs. PCD: p = 0.0464; acute CF vs. PCD: p = 0.0431; Figure 4 ).
Simple and non-invasive biomarkers of lung inflammation in CF are needed to monitor disease progression, Burkholderia cepacia complex 0 2 0
Alcaligenes xilosoxidans 0 1 0 FEV 1 , forced expiratory volume in 1 second as % predicted. In 1 stable CF patient analysis of CFTR mutations is missing. Statistically significant differences between stable and acute CF patients are shown. 100nm identify exacerbations, and evaluate the efficacy of novel therapies [31] . Sputum is a rich, non-invasive source of biomarkers of inflammation and infection, and has been used extensively to assess inflammation in the CF airways (reviewed in [15] ). There is compelling evidence from small single-centre studies supporting an association between sputum biomarkers and disease status in CF.
Recently, a multicenter cross-sectional study has found significant negative correlations between FEV 1 % predicted and spontaneously expectorated sputum inflammatory markers including free elastase, IL-8, neutrophil counts, and percent neutrophils [32] . In this study we provide evidence, for the first time, of the presence of MPs in sputa obtained from CF patients. The membrane composition of MPs reflects the plasma membrane of the original cell at the precise moment of MPs production and thus allows the characterization of the cellular source [33] using antibodies directed against these specific epitopes. Our data strongly support the notion that MPs are derived from granulocytes, while the presence of MPs derived from monocyte-macrophages is negligible. Although there are several potential sources of sputum MPs, including erythrocytes, platelets, and epithelial cells, our data suggest that granulocytes are the predominant source of MPs in CF. Our findings are consistent with massive influx of neutrophils into CF airways and their accumulation on the surface of the airway epithelium [34] . In this environment, neutrophils are activated by bacterial products, pro-inflammatory cytokines, and chemokines. Neutrophils undergo apoptosis, as normally happens in acute inflammation, but also post-apoptotic necrosis, releasing toxic enzymes and oxygen radicals. MPs in CF sputa likely reflect both activation and apoptosis of neutrophils. In order to evaluate the effect of bacterial infection on MPs production, it would be interesting to compare subgroups based on the presence or absence of infection with Pseudomonas aeruginosa or other bacterial strains, therefore further studies with larger number of patients are needed. PCD patients were selected as controls because healthy donors do not produce spontaneous sputum; moreover, PCD patients have similar respiratory infections to CF patients. In PCD, neutrophilic lung inflammation, incidence of lung infection, offending organisms, development of bronchiectasis and longitudinal declines in lung function are similar to CF but appear to be delayed, and serious lung disease tends to develop later in life [35] [36] [37] . This could be the reason for a significantly less presence of granulocytederived MPs in PCD when compared with CF patients.
The mechanisms of MP formation are complex and not completely elucidated. Following cell activation or apop- tosis, MPs formation is dependent on a sustained rise in the cytosolic Ca 2+ concentration with the consequent activation of different cytosolic enzyme relevant to MPs formation. Calpain is one of the most important enzyme and has several actions in MPs generation including cleaving of cytoskeletal filaments, facilitating microparticle shedding, and activating apoptosis through procaspase-3 [38] . These changes result in cytoskeletal reorganization, loss of the asymmetric distribution of aminophospholipid, membrane blebbing and MP formation [39] . Some release of shedding vesicles takes place from resting cells, however the rate of the process increases dramatically upon stimulation [40] . Ca 2+ is not the only second messenger involved. In various cell types, in fact, phorbol ester activation of protein kinase C (PKC) is also effective. In PC12 cells, shedding vesicles are released upon application of phorbol esters and not of Ca 2+ ionophores. The purinergic receptors of ATP, a ligand released by many cell types, have an important role. In dendritic cells, macrophages and microglia, activation of the purinergic receptor channel, P2X7, was found to induce intense release of MPs. In other cell types (such as PC12 and platelets), activation of the P2Y receptors coupled with the Gq protein was found to be effective.
Electron or confocal laser scan microscopy can be used for better characterization of morphological features or visualization of MPs. Indeed, here we show that sputum MPs display a range between 100 and 500 nm, larger than that previously shown for MPs obtained from edema fluid of a patient with ARDS [26] . However the most widely used method for studying MPs is flow cytometry due to its simplicity and the wealth of information that can be gleaned from the population under study [17] . The major advantage of flow cytometry is staining of MPs to determine the origin/cellular source of MPs. In addition, flow cytometry can also be used to enumerate blood MPs by adding a known number of fuorescent or non fuorescent latex particles to the sample prior to performing analysis [41] .
Microvesicles also originate from the endosomal membrane compartment after fusion of secretory granules with the plasma membrane, where they exist as intraluminal membrane-bound vesicles called exosomes. These exosomes are released from cells during exocytosis of secretory granules together with the proteins present inside these granules. MPs are released from the surface of membrane during membrane blebbing in a calcium flux and calpain-dependent manner and are relatively large (100 nm-1 μm). In contrast smaller exosomes that are more homogeneous in size (30-100 nm) are released from the endosomal compartment [42] . In the only paper reporting a direct comparison, the shedding vesicles of platelets 'could be detected by flow cytometry but not the exosomes, probably because of the smaller size of the latter' [43] .
MPs contain numerous proteins and lipids similar to those present in the cell membranes from which they originate. Furthermore, as MPs' membranes engulf some cytoplasm during membrane blebbing, they may also contain proteins derived from it, mRNA, and, as recently demonstrated, microRNA (miRNA) [44] . It is now emerging that miRNAs may play a key role in host defence and inflammation [45, 46] . Moreover, MPs may "hijack" infectious particles (e.g. human immuno deficiency virus (HIV) or prions) from the cytoplasm or possibly even whole intact organelles such as the mitochondria [42] .
A question remains unresolved: whether MPs present in CF sputa have functional consequences on the pathophysiology of CF lung disease. Recent data bring the evidence that MPs can transfer message from different type of cells. The mechanisms by which MPs may influence biology of target cell could be different; MPs may (i) stimulate other cells by surface-expressed ligands acting as a signalling complex, (ii) transfer surface receptors from one cell to another, (iii) deliver proteins, mRNA, miRNAs, and bioactive lipids into target cells or (iv) serve as a vehicle for the transfer of infectious particles (e.g. HIV, prion) [42] .
Sputum through its inflammatory cell, bacterial, volatiles, mucin and protein content represent an important tool for the diagnosis and monitoring of CF and other respiratory diseases, beside for the study of disease pathogenesis and its treatment. Measurement of these components is largely sophisticated and quantifiable, and the search for novel biomarkers of CF airways disease in this biofluid is under way [16, 47] . The finding of MPs in sputum raises some intriguing questions on the pathophysiological role of MPs in pulmonary epithelium. Our data strongly support the notion that MPs are derived from granulocytes of CF patients, and this correlates with massive influx of neutrophils into CF airways and their accumulation on the surface of the airway epithelium [34] . In this environment, neutrophil-derived MPs could contribute to selfperpetuating inflammatory cycle, and may account for the exaggerated proinflammatory response of cells in CF patients.
Independently of the potential role played by MPs in CF, taking in consideration the fact that CD66b + MPs are present in higher level in CF than in PCD sputum, they might be considered as biological markers of this pathology. Taken together, these data open a new opportunity for the study of lung pathology. HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response. Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases [1] [2] [3] [4] . Ad vectors have been utilized as vaccine vectors because of several attributes. This broad utility profile has derived from several key attributes: (a) the viral genome is readily manipulated allowing derivation of recombinant viruses; (b) replication-defective Ads can be derived and propagated easily in complementing cell lines making production of large scale vaccines feasible; (c) Ads infect a broad range of target cells [5, 6] ; (d) they possess a large gene delivery payload of up to 8kb; and (e) the vector can achieve unparalleled levels of in vivo gene transfer with high levels of induced transgene expression [6, 7] . Traditionally, Ad-based vaccines have been designed to express antigens through transgene expression of a given antigen [8] . However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to preexisting Ad serotype 5 (Ad5) immunity. 50-90% of the adult population has pre-existing immunity (PEI) to Ad5 and therefore, if an individual is vaccinated with an Ad vector for therapeutic purposes there maybe limited transgene/antigen expression in that individual [9] [10] [11] [12] [13] . In this regard, the ''antigen capsid-incorporation'' strategy has been developed to circumvent drawbacks associated with conventional transgene expression of antigen within Ad. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. This antigen capsid-incorporated strategy has been used for Ad-based vaccines in the context of many diseases [4, [14] [15] [16] [17] [18] . One of the first instances whereby the antigen capsid-incorporation strategy was used was in research performed by Crompton in 1994. Crompton and colleagues inserted an eight amino acid sequence of the VP1 capsid protein of poliovirus type 3 into two regions of the adenovirus serotype 2 hexon. One of the chimeric vectors produced from this methodology grew well in tissue culture and antiserum raised against the Ad with the polio insert specifically recognized the VP1 capsid of polio type 3.
Using this antigen capsid-incorporation strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. The major capsid protein hexon has been utilized for these antigen capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion [4, 14, 15, [17] [18] [19] [20] [21] . Of note, our previous work has shown that we can incorporate small heterologous peptides into Ad hexon hypervariable regions (HVRs) without perturbing viral viability and other biological characteristics [19] . Published studies have focused on antigen and/or epitope incorporations at HVR5 or single site antigen/epitope incorporation at fiber or protein IX (pIX) [22] . In this regard, antigenic epitopes including linker sequences ranging in size from nine to forty-five amino acids have been incorporated within the Ad5 hexon region or Ad2 hexon region. These epitope incorporations include epitopes derived from polio, Pseudomonas aeruginosa, B. anthracis, and HIV as well as model epitopes [4, 15, 16, 18] . Based on our ability to manipulate both HVR2 and HVR5 sites, recently we sought to explore the relative merits of antigen incorporation within these two hexon locales. In order to accomplish this we compared the flexibility and capacity of HVR2 and HVR5, respectively. We genetically incorporated identical model epitopes of increasing size within HVR2 or HVR5 of the Ad5 hexon. Our previous study demonstrated that hexonincorporated model antigens elicit a range of immune responses depending on antigen placement or antigen size at either the HVR2 or HVR5 locales. This study confirmed that HVR2 could be a potential incorporation site for antigens of considerable size and that vaccination with vectors that display incorporations at HVR2 is feasible for vaccine development [16] .
Our study herein focuses on the creation of multivalent vaccine vectors presenting HIV antigens in the context of the Ad capsid protein hexon, as well as expressing HIV antigens as a transgene. Specifically, these novel vectors utilize the hexon HVR2 as an incorporation site for a twenty-four amino acid region (EKNE-KELLELDKWASLWNWFDITN) of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). In addition, to presenting MPER within HVR2 these vectors express HIV-specific protein Gag from the Ad5 DE1 region. Our study herein illustrates that our multivalent anti-HIV vectors elicit a humoral and cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present model antigens at HVR2, elicit a HIV epitope-specific humoral immune response.
For these studies HIV-1 gp41 monoclonal antibody (2F5), cat# 1475 was used. The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 gp41 Monoclonal Antibody (2F5) was generated by Dr. Hermann Katinger. The human monoclonal antibody to HIV-1 gp41 is specific for ELDKWA epitope [23] [24] [25] . Goat anti-human horse radish peroxidase (HRP) antibody was purchased from Southern Biotech. (Birmingham, AL).
Human embryonic kidney cells (HEK293) were obtained from and cultured in the medium recommended by the American Type Culture Collection (Manassas, VA). All cell lines were incubated at 37uC and 5% CO 2 under humidified conditions.
In order to generate recombinant adenoviruses with the MPER insertions within the hexon regions, fragments of DNA corresponding to MPER, were generated by PCR from templates provided by Integrated DNA technologies. In our manuscript, the MPER sequence corresponds to EKNEKELLELDK-WASLWNWFDITN from HIV gp41. This fragment was subcloned into the BamHI site in the previously described HVR2-His 6 /pH5S plasmids [19] . To create Ad5 vectors containing HIV epitopes in the HVRs of hexon, these resulting plasmids were digested with EcoRI and PmeI. These resulting fragments containing the homologous recombination regions and the hexon genes were purified, then recombined with a SwaI-digested Ad5 backbone vector that lacks the hexon gene, pAd5/DH5 [26] . These recombination reactions were performed in Escherichia coli BJ5183 (Stratagene, La Jolla, CA). The resultant clone was designated as Ad5/HVR2-MPER-L15; in addition, there was another virus construct with this hexon modification, which contained the HIV Gag gene in the DE1 region: Ad5/HVR2-MPER-L15 (Gag). To create Ad5 vectors containing HIV Gag, the cytomegalovirus (CMV)-Gag construct was subcloned into an Ad DE1 shuttle plasmid. The resulting plasmid was digested with PmeI restriction enzyme. The resulting fragment containing the homologous recombination region at the Ad5 DE1 region was then recombined with the E1 deleted Ad5 backbone. AdCMVEnv was made as previously described [27] , This vector expresses the HIV 89.6 envelope (Env) gene under the control of the CMV promoter.
To rescue viruses the constructed plasmids were digested with PacI and two mg DNA were transfected (Lipofectamine 2000 Reagent, Invitrogen, Carlsbad, CA) into the Ad-E1-expressing HEK293 cells. Following plaque formation, they were processed for large-scale propagation in HEK293 cells. Viruses were purified by double cesium chloride ultracentrifugation and dialyzed against phosphate-buffered saline containing 10% glycerol. Viruses were stored at 280uC until use. Final aliquots of virus were analyzed for physical titer using absorbance at 260 nm. The infectious viral titer (IFU) per ml was determined by tissue culture infectious dose (TCID 50 ) assay. The TCID 50 titer was calculated by using KARBER statistical method: T TCID 50 titer = 10610 1 + d(S 2 0.5) /ml, in which d is the log 10 of the dilution and S is the sum of ratios from the first dilution. Modifications of the hexon gene was confirmed by PCR analysis with the primers 59HVR2 (sense), CTCACGTATTTGGG-CAGGCGCC and 3'HVR5 (antisense), GGCATGTAAGAAA-TATG AGTGTCTGGG, which anneal up and downstream of the site of the insertion within the hexon open reading frame.
To analyze Gag expression, in brief, 1610 6 HEK293 cells were infected with various vectors at 100 IFU per cell. Cell lysates were collected after 24 hours and subjected to four freeze-thaw cycles to obtain crude cell lysate. 10 mg of protein was boiled in Laemmli sample buffer for 10 minutes and resolved on 4 to 15% sodium dodecyl sulfate-polyacrylamide gel. The proteins were transferred to polyvinylidene fluoride membrane and staining was performed with Gag antibody (1:1,000) (Affi-anti-HIV matrix IgY, Genway Biotech, Inc.), followed by secondary staining with HRP-linked goat-chicken IgY (1:2,000) (Aves Lab, Inc). The proteins were detected on the polyvinylidene fluoride membrane by staining with 3939-diaminobenzidine tablets (Sigma-Aldrich, St. Louis, MO).
In brief, to analyze MPER presentation on selected vectors, 10 10 viral particle (VP) were boiled in Laemmli sample buffer for 10 minutes and resolved on 4 to 15% sodium dodecyl sulfatepolyacrylamide gel. The proteins were transferred to polyvinylidene fluoride membrane and staining was performed with HIV-1 gp41 monoclonal antibody (2F5) (1:1,000). Followed by secondary staining with HRP-conjugated goat anti-human antibody (1:2,000). The proteins were detected on the polyvinylidene fluoride membrane by staining with 3939-diaminobenzidine tablets (Sigma-Aldrich, St. Louis, MO).
The enzyme-linked immunosorbent assay (ELISA) was performed essentially as described previously [28] . In order to determine if the MPER peptide was surface exposed on the Ad virion whole virus ELISA was performed. Briefly, different amounts of viruses ranging from 4610 6 to 9610 9 VPs were immobilized on 96-well plate (Nunc Maxisorp, Rochester, NY) by overnight incubation in 100 ml of 100 mM carbonate buffer (pH 9.5) per well at 4uC. After washing with 0.05% Tween 20 in Phosphate-buffered saline (PBS) and blocking with blocking solution (2% bovine serum albumin and 0.05% Tween 20 in PBS), the immobilized viruses were incubated with anti-HIV 2F5 monoclonal antibody (cat# 1475) for 2 hr at room temperature (RT) followed by incubation with an AP-conjugated goat antihuman antibody. Colormetric reaction was performed with pnitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO) as recommended by the manufacturer, and optical density at 405 nm (OD 405nm ) was determined with a microplate reader (Molecular Devices).
For the anti-MPER response ELISA plates (Nunc Maxisorp, Rochester, NY) were coated with 10 mM of the MPER peptide (GenScript Co, Piscataway, NJ) in 100 ml of 50 mM carbonate (pH 9.6) per well, according to the method we described previously [29] . Plates were washed and then blocked with 3% BSA/PBS. After washing, 60 ml of 1:20 diluted sera was added. After incubation for at least 2 hr at RT, the plates were extensively washed and blocked with 3% BSA/PBS. The plates were then washed with HRP-conjugated goat anti-human antibody (Southern Biotech, Birmingham, AL). ELISAs were developed with TMB substrate. In order to determine MPER isotype-specific reactivity, ELISA plates were coated with 10 mM of the MPER peptide in 100 ml of 50 mM carbonate (pH 9.6) per well, according to the method we described previously [29] . Plates were washed and then blocked with 3% BSA/PBS. After washing, 60 ml of 1:20 diluted sera was added. After incubation for at least 2 hr at RT, the plates were extensively washed and blocked with 3% BSA/PBS. Isotype-specific mouse antibody (Sigma-Aldrich, St. Louis, MO) was then bound to ELISA plates. Plates were then washed and HRP-conjugated anti-mouse antibody (Dako Denmak, Denmark). ELISAs were developed with TMB substrate (Sigma-Aldrich, St. Louis, MO). OD 450 nm was measured on an Emax microplate reader.
Growth kinetics of Ad vectors were obtained essentially as described previously [26] . HEK293 cells were plated in 6-well plates at the density of 3610 5 cells per well 24 h before infection. The cells were infected with Ads at 5 VPs/cell in 500 ml growth medium containing 2% FBS. 1.5 ml more growth medium containing 10% FBS was then added into each well after 2 h incubation at 37uC in 5% CO 2 humidified incubator. The infected cells were monitored and harvested with medium at various time points post-infection until complete CPE was formed. The collected cells together with the medium were lysed by four freeze-thaw cycles, and subjected to centrifugation at 3000 6g for 30 min at 4uC for cell debris removal. The total viruses in each well were determined by multiplying TCID 50 titer with the total volume of the supernatant, and plotted as growth curves.
Heat inactivation assay was performed essentially as described previously [19, 26, 30, 31] . Briefly, viruses were incubated at 45uC for 0, 5, 10, 20 or 40 min in either PBS (without Ca 2+ and Mg 2+ ) or growth medium containing 2% FBS. Then their infectious titers were re-determined by standard TCID 50 method (AdEasy vector system, Qbiogene Inc., Carlsbad, CA). One day before analysis 10 4 293 cells plus 100 ml of growth medium with 2% Fetal Bovine Serum (FBS) were added in 96-well flat bottom plates. Eight serial dilutions of the virus ranging from 10 23 to 10 210 or 10 26 to 10 213 were made in medium containing 2% FBS depending on the virus, and 100 ml of each dilution was added into 96-well plates, one row for each dilution. After incubation for 10 days at 37uC in 5% CO 2 humidified incubator the plates were examined for cytopathic effect (CPE) under microscope. Observable CPE containing wells were counted for each row in order to determine the ratio of positive wells per row in the 96-well plates. Titer was calculated by using TCID 50 .
The following experiment was performed to determine antibody response after immunization with Ad vectors. Female BALB/c (H-2K d ) mice at 6-8 weeks of age were obtained from the Jackson Laboratory (Bar Harbor, ME). Groups of at least eight mice were analyzed in each experiment or at each time point. Ads were injected into each group of mice: Ad5, Ad5/HVR2-MPER-L15(Gag), Ad5/HVR2-MPER-L15DE1, AdCMVGag, Ad-CMVEnv at 1610 10 VP per mouse using intramuscular (i.m.) injection. These mice were boosted 14 days after prime with 1610 10 VP of the same vector. For CD8 T cell response analysis, blood was collected from mice vaccinated with Ad5/HVR2-MPER-L15(Gag) or AdCMVGag. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
The antigenic epitope of HIV MPER derived from gp41 was generated in an expression plasmid by (GenScript Co, Piscataway, NJ). This peptide was used as a positive control for the ELISA assays. Peptides were .98% pure as indicated by analytical highperformance liquid chromatography. Peptides were dissolved in 100% DMSO at a concentration of 10 mM and stored at 220uC until use. 
Mice were vaccinated and boosted, as described above, peripheral blood Gag-specific CD8 T cells were enumerated by Flow cytometric analyses. Whole blood (100 ml) was collected in heparinized PBS on days 26, 69, and 84 following initial vaccination. Leukocytes were collected using lymphocyte separation medium (Mediatech. Inc. Manassas, VA). Cells were collected and washed twice in 1%BSA, 2mM EDTA in PBS and counted. One to two million lymphocytes were stained with or without Gag Tetramer (APC) (created by Emory University) and a cocktail of CD4-Alexa488 and CD8-PE antibodies (BD Pharmingen, San Jose, California). Cells were washed with 1%BSA, 2mM EDTA in PBS. Cells were fixed with 1% paraformaldehyde and subsequently analyzed for flow cytometry on LSR II cytometer. Analysis of 3,000 CD8 T cells was collected per sample.
The data are presented as the mean 6 the standard error. Statistical analyses were performed with the nonpaired two-tailed Student t -test, assuming equal variance. Statistical significance was defined as P,0.03.
After establishing the technical feasibilities allowing us to place epitopes into HVR2 or HVR5 [16, 19] , we sought to explore whether we could present HIV antigens within the Ad protein hexon as well as express HIV antigens as a transgene within Ad5. The rationale for choosing a portion of the MPER (EKNEKEL-LELDKWASLWNWFDITN), derived from gp41 for incorporation into the Ad capsid is based on the fact that the gp41 envelope protein ectodomain is a target of three broadly neutralizing anti-HIV-1 antibodies [32] . Gp41 antigenic function is conformation dependent, therefore we planned to insert a linker sequence onto the MPER core region in order to have the MPER epitope presented on the Ad capsid as close to it's natural conformation as possible [33] . Our vaccine vector design also embodies the incorporation of the HIV Gag gene within the Ad DE1 region. The HIV Gag gene is a major structural protein of the HIV virus. The Gag protein has been frequently used for HIV vaccine schemes [34] . The DNA sequence corresponding to a 24 amino acid region of the MPER plus a 15 amino acid linker was amplified by PCR and then cloned into the hexon HVR2 shuttle vector as previously described [16] . In addition, the HIV Gag gene was subcloned into an DE1 shuttle vector under control of the CMV promoter and transferred into the E1-deleted Ad5 genome (Ad5/HVR2-MPER-L15(Gag)) by homologous recombination. In addition, a control vector was generated, which contained an E1-deleted Ad5 genome in combination with MPER within the hexon HVR2 region (Ad5/HVR2-MPER-L15DE1). A control Ad genome expressing the Gag gene under the CMV promoter with no hexon modification (AdCMVGag) was generated in this study as well. The resulting Ad genomes were partially sequenced to confirm that the correct genes were incorporated. Subsequent transfection of HEK293 cells with the resulting recombinant genomes resulted in rescue of the following vectors: AdCMVGag, Ad5/HVR2-MPER-L15DE1, and Ad5/ HVR2-MPER-L15(Gag). In order to further confirm vector identities, hexon and Gag-specific PCR analyses were performed using genomic DNA from the purified virions ( Figure 1A-B) . With regard to hexon-specific PCR, AdCMVGag was found to have a wild type hexon PCR profile producing ,450 base pairs (bp) PCR fragment using the hexon-specific primers designed to amplify a regions between HVR2 and HVR5 ( Figure 1A, lane 1) . A hexon specific PCR, on the Ad5/HVR2-MPER-L15(Gag) construct revealed a 500 bp fragment, suggesting the expected insertion ( Figure 1A, lane 2) . The addition of ,50 bp in Figure 1A , lane 2 indicates the incorporation of the MPER-L15 DNA within the hexon region. Both AdCMVGag and Ad5/ HVR2-MPER-L15(Gag) were found to be positive in a Gagspecific PCR assay, producing a ,1500 bp product with Gagspecific PCR primers ( Figure 1B, lanes 1-2, respectively) . A diagram depicting the vector construction of vectors used in this study is illustrated in Figure 1C . Expression and display of HIV antigens within adenoviral vectors After successful incorporation of HIV genes we next sought to verify expression of our transgene and capsid incorporation at the protein level by Western blot analysis. HEK293 cells were infected with unmodified Ad5 (as a negative control) or one of the HIV-Gag containing viruses (Ad5/HVR2-MPER-L15(Gag) or Ad-CMVGag, respectively. Cell lysates were prepared 24 hours post-infection and subjected to Western blot analysis with anti-Gag-specific antibody. Gag protein expression was evidenced by the presence of a ,55 kDa molecular weight protein band in the lysates from cells infected with either hexon-modified Gag expressing vector Ad5/HVR2-MPER-L15(Gag) (Figure 2 -A, lane 2) or the AdCMVGag (Figure 2-A, lane 3) . As expected no Gag protein expression was detected in the lysates from cells infected with the unmodified Ad5 virus (Figure 2-A, lane 1) .
In order to determine if the hexon-modified vectors were presenting the MPER epitope within the hexon region, purified unmodified Ad5, Ad5/HVR2-MPER-L15(Gag), or Ad5/HVR2-MPER-L15DE1 were subjected to Western blot analysis with anti-gp41 antibody. The MPER protein was detected as a 119 KDa protein band associated with Ad5/HVR2-MPER-L15(Gag) or Ad5/ HVR2-MPER-L15DE1 particles, Figure 2 -B, lanes 2 and 3, respectively. The size of the 119 KDa band corresponds to the expected size of the Ad5 hexon protein with MPER peptide genetically incorporated into the HVR2 region. There was no MPER protein detected on Ad5 wild type particles (Figure 2-B, lane 1) .
HIV antigens, incorporated within HVR2, are exposed on the virion surface
The foregoing studies validated our ability to derive stable vectors that incorporate MPER within hexon HVR2. Having established the technical feasibilities that allowed us to place large epitopes within hexon's HVR2, we next sought to explore the functional utilities of these modified vectors. To this end, we performed an ELISA assay to verify that the HIV MPER motif in the HVR2 was accessible on the virion surface ( Figure 3A ). In this assay, varying amounts of purified viruses were immobilized in the wells of an ELISA plate and incubated with anti-gp41 antibody. The results showed significant binding of the anti-gp41 antibody to the Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1, whereas no binding was seen in response to AdCMVGag control. These results indicate that the reactive MPER epitope was properly exposed on the virion surfaces when incorporated within HVR2.
In order to determine the capability of the gp41-specific antibody to bind capsid-incorporated antigen in a dose-dependent manner a dose-response ELISA assay was performed with anti-gp41 antibody. A single concentration of each of the following viruses: AdCMVGag, Ad5/HVR2-MPER-L15(Gag), or Ad5/ HVR2-MPER-L15DE1 was applied to ELISA plates, followed by the addition of serial dilutions of gp41 antibody. As predicted, the anti-gp41 antibody bound to Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 in a dose dependent manner ( Figure 3B ). Our data suggest that the MPER epitope is presented within the hexon in its native conformation as it can be recognized by a monoclonal HIV neutralizing antibody.
Previous studies have shown that capsid modifications, as well as some transgene incorporations, can compromise viral growth characteristics. In order to ensure that normal viral growth was not inhibited by epitope and/or transgene incorporation in the viral particle/genome we performed growth kinetic assays on vectors used in this study. To obtain a quantitative understanding of this effect all respective vectors were titered in HEK293 cells until full CPE was achieved in order to determine growth kinetics. The data illustrates that the unmodified Ad5 had the highest titer (,1610 14 IFU/ml) and produced full CPE at 96 hours postinfection. AdCMVGag also produced full CPE at 96-hours postinfection, however, AdCMVGag had a titer, several orders of magnitude lower than that of the Ad5. Ad5/HVR2-MPER-L15(Gag) showed comparable titers to that of AdCMVGag. The control vector expressing full-length envelope protein (AdCMVEnv) reached full CPE after 120 hours post-infection. Ad5/HVR2-MPER-L15DE1 yielded the slowest growth kinetics and relative titer, reaching full CPE at 192 hours post-infection (Figure 4) . These growth kinetic patterns were similar to other capsid and transgene modified vectors developed in our laboratory [31, 35] .
Previous work has shown that capsid modifications may result in the production of thermoliable vectors. Therefore, we performed thermostability assays with our respective vectors. These particular assays are important due to the complex nature of the incorporated antigens, i.e. MPER and/or Gag. Hexon-modified vectors, as well as controls vectors, were subjected to heating at 45uC for various time points (0,5,10, 20 and 45 minutes). These vectors were then used to infect HEK293 cells. The viral infectious titers were re-determined after heating using TCID 50 assays. According to the thermostability assay Ad5/HVR2-MPER-L15DE1 was the least infectious of all the vectors, showing a dramatic infectivity reduction relative to Ad5 following incubation at 45uC for 20 or 45 minutes ( Figure 5 ). These thermostability results are similar to what we have observed with other transgeneencoding or capsid-modified vectors in our laboratory [31, 35] .
To verify the immunizing potential of these vectors against HIV we next sought to determine if they were capable of eliciting an anti-HIV immune response in mice. Equal numbers (1610 10 VP) of the vectors were used to immunize BALB/c mice via an intramuscular route. The sera were collected from mice for ELISA assays at various days after priming and boosting. Purified MPER antigenic peptide was bound to ELISA plates. The plates were then incubated with the immunized mice sera. The binding was detected with HRP-conjugated secondary antibody. The amount of anti-MPER antibody in the sera was calculated based on a standard antibody curve dilution. The data demonstrates no binding of the sera from mice immunized with Ad5, AdCMVEnv, or AdCMVGag to the MPER on the plates at any time point. On the contrary, immunization with Ad that contains capsid-incorporated MPER antigen elicits an anti-MPER specific response and allows the formation of MPER-specific antibody in the serum ( Figure 6 ). This immune response is observed at the earliest time point that we observed, which was 14 days post-prime (d.p.p). The peak immune response was observed with the capsid incorporated MPER vectors at 54 d.p.p, which also corresponds to 14 days postboost (d.p.b.). At 54 d.p.p the determined concentration of gp41 antibody was ,7 mg/ml. A slight decline of the MPER antibody in the sera at 95 d.p.p was observed in the mice vaccinated with capsid-modified vectors, which corresponded to 55 d.p.b. At 55 d.p.b. MPER antibody levels in the sera was determined to be ,5 mg/ml. In summary, Ad5/HVR2-MPER-L15DE1 and Ad5/HVR2-MPER-L15(Gag) elicited an anti-gp41 response in vaccinated mice. This MPER-specific response was increased after boosting. It is important to note that the envelope expressed in AdCMVEnv was from the 89.6 HIV variant. The epitope inserted into the Ad5 hexon and the peptide were identical sequences with the 89.6 Env MPER region. The transgene expressed from the Ad5 vector was 89.6 Env (i.e. gp160), which is processed into gp120 and gp41. There were no point mutations within the transgene expressed Env.
We next performed experiments to determine the quantitative aspects of the isotype-specific humoral immune responses that were generated in response to the anti-HIV immunization vectors. The sera were collected from mice for ELISA assays at 54 d.p.p and 14 d.p.b., as described previously. Purified MPER antigenic peptide was bound to the plate. The plates were then incubated with immunized mouse sera and the binding was detected with HRP conjugated secondary antibody. Ad5/ HVR2-MPER-L15DE1 and Ad5/HVR2-MPER-L15(Gag) vectors produced an isotype-specific anti-MPER humoral response in vaccinated mice; whereas there was no isotype-specific response seen in mice vaccinated with Ad5, AdCMVGag or AdCMVEnv. The presence of isotypes IgG2b and IgG1 corresponds to Th1 and Th2 activation, respectively (Figure 7) . T cell activation is important for host protection against foreign antigens.
The incorporation of the HIV-1 epitope into the hexon protein of Ad5 may interfere with the viral infectivity and/or reduce recognition by anti-Ad5 neutralizing antibodies (NAb). We therefore compared infectivity of Ad5/HVR2-MPER-L15(Gag) to that of the original AdCMVGag virus in the presence or absence of anti-Ad5 NAb found in human ascites that have been described previously [36] . In the absence of ascites, the frequency of cells expressing Gag after infection with AdCMVGag or Ad5/HVR2-MPER-L15(Gag) was 81.8% and 80.6% respectively, indicating that the incorporation of HIV-1 epitope into the hexon protein of the Ad5 vector did not reduce virus infectivity ( Figure 8A ). Compared to the unmodified Ad5 vector, the presence of the HIV-1 epitope did not alter neutralization by anti-Ad5 NAb present in the human ascites ( Figure 8B ). Therefore the HIV-1 epitope did not facilitate escape from pre-existing anti-adenoviral humoral immune response, probably due to the polyclonal nature of the ascites NAbs. for ELISA isotype binding assays. 10 mM of purified MPER (EKNEKELLELDKWASLWNWFDITN) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera followed by isotype specific antibodies. The binding was detected with HRP conjugated secondary. OD at 405 nm represents of isotype-specific MPER antibody levels in sera. doi:10.1371/journal.pone.0011815.g007
The previous experiments rely on the immune recognition of capsid proteins and do not address the capacity of these modified vectors to transduce cells and direct expression of the delivered antigen-encoding transgenes in vivo. To test the capacity of our hexon-modified vectors to deliver and induce expression of the HIV Gag transgene, we measured the expansion of Gag-specific CD8 T-cells at various consecutive time points following vaccine priming and boosting, and later measured persistence of memory T cells. Equal amounts of viral particles were used to immunize mice. Blood was collected from mice on 21 d.p.p. and 26 d.p.b. Tcells were subjected to Gag tetramer staining. The results in Figure 9 illustrate that after mouse immunization with Ad5/ HVR2-MPER-L15(Gag) (5.63%) or AdCMVGag (4.05%) we observed comparable Gag responses between these immunization groups, this is an acceptable result in that the data shows that there was not a loss of Gag response due to the incorporation of MPER epitope within the hexon capsid. Mouse immunization with Ad5 vector resulted in background levels of T-cell activation (0.83%). In addition, Ad5/HVR2-MPER-L15(Gag) (9.51%) vector allowed boosting of the Gag transgene expression as compared to the AdCMVGag vector (2.25%) (Figure 9 ), these results were statistically significant at p,0.03. Intracellular IFNc staining in tetramer positive CD8 T cells was observed for all groups (data not shown), confirming the functional integrity of the expanded HIV Gag-specific CD8 T cells.
We have developed novel Ad vectors that have the potential to optimize Ad vaccine approaches. This strategy involves inserting antigenic epitopes into HVR2 or HVR5 regions of the Ad capsid protein, hexon, to stimulate epitope-specific antibody responses following vaccination. This method offers the ability to compare a range of identical epitopes incorporated within HVRs for antigenic optimization. Our current study is the first of its kind to genetically incorporate HIV antigen within the Ad5 hexon HVR2 alone or in combination with genomic incorporation of a transgene for HIV antigen expression. In this study we successfully incorporated the HIV MPER in the Ad5 HVR2 region as evidenced by PCR, Western blot analysis, and the purified, wholevirus ELISAs (Figures 1,2, and 3) .
We believe that further characterization of the gp41 binding antibodies is necessary; we performed additional ELISA experiments using purified gp41 protein and sera from our vaccinated mice. We were able to observe a slight binding of whole gp41 protein to the sera of mice vaccinated with Ad vector presenting MPER within the hexon capsid (data not shown). We speculate that this binding would have been increased if we were able to obtain an identical purified gp41 protein as that presented with our capsid-incorporated MPER sequence. This was not possible due to the divergency of HIV protein structures and reagents available. We also performed an ELISA assay with HIV pseudo particles and sera from our vaccinated mice (data not shown). The results were similar to that seen with purified gp41 protein. We speculate that it is difficult to detect binding of gp41 to the HIV particle due to the need of further optimization of this assay and/ or the limited amount of gp41 molecules available on a single HIV virion (,10-30 Env (gp120 and gp41) molecules per virion).
It is more than likely that the MPER epitope inserted into the Ad hexon is not presenting in the same conformation as it would be presented within the context of an HIV virion, therefore there are advantages and disadvantages to the antigen capsid-incorporation approach. The advantages of this approach include the fact that it is obviously much easier to insert a small peptide from HIV into hexon as compared to inserting a correctly folded version of the full HIV protein into hexon (which would likely be impossible). However, despite the fact that the MPER presentation might be different with respect to Ad capsid incorporation compared to that of MPER expressed on the HIV virion, we were able to detect the presence of this epitope by the means of human HIV monoclonal antibody 2F5, which recognizes 14 amino acids within MPER ( Figures 2B and 3) . Whereas, we were not able to detect the presence of MPER with the use of human HIV monoclonal antibody 4E10, which binds a 6 amino acid linear epitope immediately C-terminal to the 2F5 epitope and contained with MPER (data not shown). These observations suggest that the MPER conformation is similar but not identical to that presented by the HIV virion. One advantage of the antigen capsidincorporation approach is that the non-glycosylated MPER may be more immunogenic because of the absence of glycan-shielding. With the hexon/peptide presentation approach we should be able to address additional interesting questions, e.g. are there any glycosylation sites within or around the MPER region that affect immunogenicity. We plan to rigorously examine the ability of the peptide presentation approach to produce neutralizing antibodies, perhaps in a guinea pig or rabbit system.
When the MPER was incorporated into HVR2 in combination with transgene expression, we observed growth kinetics ( Figure 4) and thermostability ( Figure 5 ) changes similar to that of other capsid-modified vectors generated in other studies [31, 35] .
Although, there appears to be substantial growth kinetic and thermostability differences between Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (Figures 4 and 5 ); there appears to be no significant difference between cellular Gag immune response generated from vaccination with Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (Figures 6 and 7) . Most importantly, vaccination with hexon-modified vectors in this study resulted in a humoral anti-HIV response. This is noteworthy, because HVR2 has not been fully explored for 'antigen capsid-incorporation' strategies. Anti-MPER humoral responses could be boosted after homologous Ad vector administration. At 40 d.p.p the MPER response in mice, vaccinated with hexon-modified vectors was approximately from 6 to 7 times higher than when using AdCMVGag, AdCMVEnv, or Ad5 vector. This is a critical finding of our study. It appears that vaccination with AdCMVEnv does not yield an antibody response. This finding could be explained in various ways: (1) since the HIV Env protein is a precursor protein that is catalyzed by protease to give the final product gp41 and gp120, it is possible that the production of gp120 is masking the antibody recognition of gp41 [37] ; (2) this finding might indicate that the antigen capsidincorporation strategy is superior to antigen transgene expression with regards to generation an in vivo immune response to MPER and/or other antigens [14] . In order to confirm the validity of the results obtained from all vaccination groups, a separate set of ELISA experiments were performed whereby sera from all vaccinated mice were bound to unmodified Ad vectors. All of the sera from the vaccinated groups bound unmodified wild type Ad5 equally well, indicating equal vaccination by Ad5, Ad-CMVGag, AdCMVEnv, Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (data not shown). In addition, anti-MPER IgG1 and IgG2b isotype-specific responses were observed in mice vaccinated with the MPER capsid-modified vectors ( Figure 7) . Similar results were seen with IgG2a (data not shown), however; no MPER IgA-specific response was observed (data not shown). We did not expect to find much of the MPER IgA-specific immune response after vaccination with these vectors due to the route of vector administration. However, we plan to further investigate IgA-specific response in the context of targeted hexonmodified vectors in combination with alternative routes of administration. From a humoral immunity standpoint, IgA2 subclasses are reduced in saliva upon HIV infection, and total secretory IgA levels are reduced at later disease stages. Salivary IgA can be neutralizing to HIV-1 and HIV-2, and can block epithelial transmigration. In this instance, salivary IgA can be functional against HIV [38] [39] [40] . Figure 8 , compares the infectivity of Ad5/HVR2-MPER-L15(Gag) to that of the AdCMVGag vector in the presence or absence anti-Ad5 NAb found in human ascites. Both vectors were equally neutralized in the presence of human ascites fluid. We expected that Ad5/HVR2-MPER-L15(Gag) would have been neutralized to a lesser degree than AdCMVGag in the presence of human ascites. However, since this was not the case, we speculate that the polyclonal anti-Ad5 antibodies found in human ascites fluid is not masked by the addition of a single epitope incorporated within the hexon region. We also speculate that we might have observed different results with this assay if we had used monoclonal antibody, targeted to the hexon region where the insert was incorporated. If a monoclonal antibody was used in the presence of ascites fluid, Ad5/HVR2-MPER-L15(Gag) might escape neutralization largely as compared to AdCMVGag. However, this possibility was not explored further at this time because it does not diminish the clinical benefit of these vectors. Importantly, the Ad5/HVR2-MPER-L15(Gag) vector allowed boosting of Gag transgene production, as compared to the wild type Gag expressing vector (Figure 9 ), this result was statistically significant at p,0.03. This is an important finding for two reasons:
(1) it indicates the potential for a second administration of the same vector without the diminished production of transgene and (2) it demonstrates that the incorporation of HIV epitopes within HVR2 does not have a detrimental effect on in vivo transgene production ( Figure 9 ). We speculate that the MPER-modified vector allows boosting compared to AdCMVGag, possibly because the Ad5/HVR2-MPER-L15(Gag) Ad elicits less anti-Ad5 immune response. It is possible that the MPER epitope reduced the immunogenicity of the Ad5 vector. It is also possible that the anti-MPER antibody fraction was not neutralizing with regards to the hexon-modified Ad5 boost. Similar results were seen with experiments performed by Abe and colleagues, their studies support the concept that modified hexon thwarts Ad5 neutralizing antibodies and promotes cellular immune responses [20] .
There have been a range of studies that have now investigated the viral antigen capid-incorporation strategy as a viable means to improve vaccination in many disease or infection contexts [4, 14, 15, 17, 18, 21] . Due to the controversial HIV STEP trial and the continuing HIV epidemic, some groups have employed the antigen capsid-incorporation strategy to make strides against HIV/AIDS by attempting to make a safe and effective HIV vaccine [41, 42] . The main vector systems that have been utilized to derive a HIV vaccine include human rhinovirus (HRV) and Adbased vector systems [43] . With respect to the HRV system, researchers have constructed HRV:HIV chimeras in an effort to stimulate immunity against HIV-1 [44] . Furthermore or along these lines, in an effort to develop HIV vaccines researchers within this same group have generated combinatorial libraries of HRV capid-incorporated HIV-1 gp41 epitope. Their results indicate that they have been successful in eliciting antibodies whose activity can mimic the 2F5 effect [45] .
Commercial and clinical Ad development for HIV vaccines have progressed preferentially more than vector systems such as HRV because, the plasticity of Ad generally exceeds current rhinovirus systems. For example, because it is a relatively small RNA virus, the rhinovirus platform can display an array limited to 60 copies of a single HIV-1 epitope [44] . In comparison, the Ad hexon-incorporation display platform could present an array of 720 HIV-1 epitope copies and the pIX incorporation display platform could potentially present an array of 240 HIV-1 epitope copies. Another significant difference between the two platforms is in the number of locales that have been successfully utilized for heterologous epitopes insertion. In this regard, to our knowledge, the human rhinovirus 14-based platform utilizes a single-epitope insertion site in the major immunogenic portion of the viral VP2 loop 2 [46, 47] . In contrast, the Ad vector platform, could potentially allow incorporation of the HIV-1 MPER epitope into four structurally distinct locales, including: hexon, (HVR2 and HVR5) [20] , protein IX, penton base and/or fiber. Lastly, in contrast to the rhinovirus that lacks this capacity, the Ad platform has sufficient coding capacity allowing for HIV-1 transgene expression in combination with presenting the same or a different antigen on the viral capsid surface. Despite some of the noted limitations with the rhinovirus platform, one of its attractive features has been the previously mentioned development of mutagenic libraries of human rhinovirus 14 chimeras that each display randomized residues representing HIV-1 V3 or MPER epitopes [48, 49] . In this regard, it is worth mentioning that our lab has previously developed a similar approach for screening and identifying heterologous amino acid sequence insertions which have been incorporated into the adenovirus fiber knob region using a modified phage display approach [50] [51] [52] . Overall, through extensive and ongoing vector development by both our group and others, the Ad platform offers a dynamic level of plasticity that translates into the advantage of multiple vaccine development [20] .
Taken together, our current study demonstrates that utilization of the HVR2 for HIV MPER epitope incorporation, in combination with HIV Gag transgene incorporation within the Ad genome. Our previous work describes the precise optimal antigen size and configuration for HVR5 capsid incorporation.
Based on our ability to manipulate Ad5 capsid proteins as well as internal gene loci, we will be able to establish novel vectors that allow for the production of multivalent vectors that may act as a vaccine 'cocktail'. These vectors with capsid-incorporated antigens will have the potential to vaccinate patients against HIV by producing a robust anti-HIV humoral and T cell response. Our data illustrates that there is a diverse immunological response/ profile of vaccination with capsid-modified vectors compared to that of vector containing wild type hexon. This fact provides the rationale that a heterologous prime boost strategy with our hexonmodified vector followed by the wild type hexon vector could potentially achieve an even greater anti-HIV immune response. In addition, these vectors may have great potential to circumvent the drawbacks seen in the general population related to Ad5 PEI. Our future plans include transitioning these vectors to a 'gutless' [53] [54] [55] [56] [57] system in order to determine the duration and quality of HIV antibody production. In combination with the 'gutless system' we plan to decrease Ad5 PEI through the use of chemical conjugates such as Polyethylene Glycol [58, 59] and/or Ad vector chimeras. Model of end stage liver disease (MELD) score greater than 23 predicts length of stay in the ICU but not mortality in liver transplant recipients INTRODUCTION: The impact of model of end stage liver disease (MELD) score on postoperative morbidity and mortality is still elusive, especially for high MELD. There are reports of poorer patient outcome in transplant candidates with high MELD score, others though report no influence of MELD score on outcome and survival. METHODS: We retrospectively analyzed data of 144 consecutive liver transplant recipients over a 72-month period in our transplant unit, from January 2003 until December 2008 and performed uni- and multivariate analysis for morbidity and mortality, in particular to define the influence of MELD to these parameters. RESULTS: This study identified MELD score greater than 23 as an independent risk factor of morbidity represented by intensive care unit (ICU) stay longer than 10 days (odds ratio 7.0) but in contrast had no negative impact on mortality. Furthermore, we identified transfusion of more than 7 units of red blood cells as independent risk factor for mortality (hazard ratio 7.6) and for prolonged ICU stay (odds ratio [OR] 7.8) together with transfusion of more than 10 units of fresh frozen plasma (OR 11.6). Postoperative renal failure is a strong predictor of morbidity (OR 7.9) and postoperative renal replacement therapy was highly associated with increased mortality (hazard ratio 6.8), as was hepato renal syndrome prior to transplantation (hazard ratio 13.2). CONCLUSIONS: This study identified MELD score greater than 23 as an independent risk factor of morbidity represented by ICU stay longer than 10 days but in contrast had no negative impact on mortality. This finding supports the transplantation of patients with high MELD score but only with knowledge of increased morbidity. Liver transplantation is still a complex and cost-intensive procedure [1] and the results are influenced by many interrelated factors. As liver transplantation has become a universally accepted treatment for end-stage liver disease, the number of patients accumulating on the waiting list has gradually outweighed the scarce resources of available organs. Fair allocation of donor livers to patients with end-stage liver disease is a difficult task. The USA and Europe used prioritization systems based on waiting time and on the parameters of the Child-Turcotte-Pugh score [2] . Since February 2002, the United Network for Organ Sharing introduced a new allocation policy for cadaveric liver transplants, based on the model for endstage liver disease (MELD) score [3] . This new policy stratifies the patients based on their risk of death while on the waiting list [4] . The impact of MELD score on postoperative mortality remains elusive. There are reports of reduced survival in groups with high MELD scores [5, 6] , but also reports of no influence of MELD score on survival [7, 8] .
Furthermore, the unique pathophysiology of end-stage liver disease (ESLD) has important implications on critical care treatment after transplantation [9] . Although liver transplantation has been the sole treatment of patients with ESLD for over 20 years, only limited data are available addressing the intensive care management and complications of this patient population [10, 11] .
The current challenge is to optimize outcome with limited resources, because liver transplantation remains financially expensive with incremental costs when postoperative complications occur. Therefore, it is essential to identify and modify risk factors to improve postoperative ICU management.
In this study we addressed the question of whether MELD score affects postoperative morbidity, represented by an increased length of stay in the ICU and mortality in patients after liver transplantation. Furthermore, the study was undertaken to determine the major ICU problems in such patients and to outline and predict major clinical risk factors regarding length of stay in the ICU and mortality.
Therefore, data from all consecutive liver transplants performed in our institution over six years, from 1 January 2003 to 31 December 2008, were analyzed.
We included in the study a total of 144 consecutive patients who underwent liver transplantation between 1 January, 2003 and 31 December, 2008 in our transplant center. Five of these patients underwent seven retransplantations. Two of them underwent retransplantation twice and three patients only once, and two cases out of this seven were electively listed and five patients were high urgent listed. Thus, we included data of 151 liver transplantations in 144 patients over six years with a median follow up of 27.0 months into our study.
Patients were transplanted according to the MELD score, which is based on recipient kidney function, coagulation time and serum bilirubin, and ranges from 7 to 40. This score is a reliable parameter to predict mortality of liver transplant candidates on the waiting list [12] . In order to prevent discrimination of patients on the waiting list with a hepatic tumor or a metabolic and cholestatic disease, those patients received exceptional points, resulting in higher (corrected) MELD scores than the calculated laboratory (uncorrected) MELD would be [13] . Following approval by the local ethics committee, all patients gave written informed consent before transplantation for postoperative data analysis.
We included all adult (> 16 years of age) liver transplant recipients from January 2003 until December 2008 who were electively or high urgently listed. The only exclusion criteria were living related liver transplant recipients. One patient, who was retransplanted twice (electively listed) during this period was excluded from analysis, because the initial transplantation was before the study period.
We defined extended donor criteria (marginal grafts) as either age 65 years or older or cold ischemia time of 720 minutes or longer or biopsy-proven steatosis (micro-or macrovascular in ≥60% of hepatocytes or ≥30% macrovascular steatosis) [14, 15] .
As baseline characteristics we analyzed age, gender, height, weight, body mass index, creatinine, hematocrit and platelet count. Creatinine values of the patients with renal replacement therapy (RRT) prior to transplantation were excluded from the calculation. For analysis the last available values directly before transplantation were included. Furthermore, the following clinical data were collected: underlying liver disease, Child-Turcotte-Pugh classification, MELD score uncorrected and corrected for hepatocellular carcinoma according to the regulation of the government [13] , incidence of hepatorenal syndrome directly before transplantation (according to the definition described by Arroyo and colleagues [16] and Salerno and colleagues [17] ), and diabetes mellitus, electively or high urgent listing, pretransplant location (home, normal hospital ward or ICU) and finally the need for pretransplant RRT.
All patients were transplanted without veno-venous bypass, as described by McCormack and colleagues [18] . Management of coagulation and transfusion practice was performed according to the internal guidelines. Patient data were collected in respect to operating time, estimated intraoperative blood loss, transfusion of red blood cells (RBC), fresh frozen plasma (FFP) or platelets and intraoperative application of fibrinogen.
The following data were collected: length of stay in the ICU, incidence of readmission to the ICU, readmission cause, serum creatinine peak level, incidence of renal failure assessed by the RIFLE (risk, injury, failure, loss, endstage of kidney disease) criteria, incidence of RRT, incidence of sepsis, incidence of pulmonary failure (acute respiratory distress syndrome (ARDS), pneumonia with consecutive reintubations), ventilation days, serum peak values of bilirubin, alkaline phosphatase, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); incidence of primary graft nonfunction and retransplantation, incidence of rejection on the ICU and reoperations during the ICU stay, and the incidence of acute coronary syndrome. In the case of four primary graft nonfunctions in the ICU with a following four consecutive emergency retransplantations, we considered those four retransplantations as ICU complications and analyzed these patients as four ICU cases. Furthermore, we considered three electively listed retransplantations as three additional cases and therefore calculated the ICU parameters from 147 transplantation cases out of 144 patients. The graft specific parameters, that is peaks of bilirubin, alkaline phosphatase, ALT and AST, were analyzed from all 151 transplanted grafts.
The influence of patients MELD score on postoperative mortality and length of stay in the ICU longer than 10 days (morbidity) was univariately and multivariately analyzed in 128 electively listed and transplanted patients. High urgent listed patients were not included in these analysis because of another allocation system according to the Clichy criteria [19] .
We analyzed data in respect to graft survival after one year, three years and five years and patient's survival was calculated for one year, three years and five years, respectively. Furthermore, the ICU and hospital mortalities (mortality during the hospital period of the transplantation in our center without transfers to other hospitals) were analyzed. For graft survival we analysed the data of all 151 transplantations and all the 144 patients were included in the survival analysis.
We performed a Cox proportional hazard model to identify risk factors for mortality of liver transplant recipients. Through multiple logistic regression analysis we identified predictive factors for ICU length of stay of more than 10 days.
MELD influence on mortality and length of stay in the ICU of more than 10 days was univariately performed with an unpaired t-test. For multivariate analysis we used the method of multiple logistic regression to identify risk factors for length of stay in the ICU and a Cox proportional hazard model to identify independent risk factors for mortality. Calculation of mortality and graft survival was performed by Kaplan Meier analysis. We calculated the baseline characteristics, operative parameters, incidence of ICU complications, rejections and reoperation incidence as the relative and absolute numbers. Data are expressed as mean ± standard deviation; different data expression is stated in the text. All calculations were performed with Statview 4.5 (abacus concepts, Berkeley, CA, USA). Statistical significance was accepted with P < 0.05 (two-sided tests).
The baseline characteristics of the recipients are shown in Table 1 . The underlying liver diseases of the 144 patients are presented in Table 2 . The incidence of hepa-torenal syndrome and diabetes mellitus was 29 patients (20.1%) and 26 patients (18.1%), respectively. The mean MELD score of these 128 patients was corrected 19.5 ± 7.1 (median 19, range 8 to 40) and uncorrected 15.8 ± 8.6 (median 15, range 6 to 40), respectively. Sixteen out of 144 patients (11.1%) or 21 out of 151 transplantations (13.9%) (inclusive of four retransplantations) were high urgent listed and transplanted because of acute liver failure or primary graft nonfunction, respectively. The location of the patients directly before transplantation was 106 (70.2%) at home, 18 (11.9%) on a normal ward and 27 (17.9%) on the ICU. The incidence of pretransplant RRT was 7 out of 144 patients (4.8%).
The mean age of donors was 48.6 ± 17.1 years and the cold ischemia time was 539 ± 166 minutes. According to the chosen criteria for extended donor grafts 57 out of 151 (37.7%) marginal donor grafts used in our study population showed at least one of the defining criteria.
The mean operation time for the 151 transplantations was 391 ± 90 minutes (median 370, range 280 to 705). The estimated blood loss during the operating procedure was 2,559 ± 2,860 ml (median 1,300, range 200 to 15,000). Transfusion requirements during transplantation were 6.2 ± 8.1 units of RBC (median 4, range 0 to 47), 14.2 ± 12.9 units of FFP (median 12, range 0 to 77), 1.7 ± 2.9 units of platelets (median 1, range 0 to 18) and fibrinogen 3.2 ± 5.1 g (median 0, range 0 to 22).
In a total of 117 (81.8%) transplantations RBC were transfused, in 133 (86.9%) FFP and in 71 (50.7%) platelets were given. No transfusion of RBC, FFP or platelets was achieved only in seven (4.6%) transplantations. Fibrinogen was administered in 76 (49.6%) transplantations.
The analysis of the 147 ICU cases showed a mean initial ICU length of stay of 8.8 ± 13.6 days (median 4, range 2 to 94), a readmission rate of 34 (22.8%), whereas 7 patients were readmitted twice and one patient 4 times. The mean readmission length of stay was 2.0 ± 6.5 days (median 0, range 0 to 50) and in turn the overall length of stay in the ICU was 11.3 ± 16. (Figure 1 ). After transplantation, the serum peak levels of bilirubin was 136 ± 116 μmol/l, alkaline phosphatase was 170 ± 136 U/l, ALT was 1401 ± 1436 U/l and AST was 2199 ± 2734 U/l. The causes for readmission are shown in Table 3 .
The ICU mortality was 3.5% (5 of 144 patients) and the hospital mortality was 5.6% (8 of 144 patients). Cumulative graft survival was 86.5% after one year, 79.3% after three years and 67.9% after five years and the cumulative patients survival was 89.5% after one year, 84.1% after three years and 74.1% after five years, respectively ( Figure  2 ).
MELD score corrected was significantly increased in the patients, which stayed longer than 10 days in the ICU (22.3 ± 7.6 vs. 18.8 ± 7.2, P = 0.015), but had no influence on mortality (Figure 3 ). The odds ratio for longer (> 10 days) ICU stay was 7.0 (confidence interval: 1.7 to 28.4, P = 0.007).
The Cox proportional hazard model for mortality identified sepsis (P = 0.011), postoperative RRT on ICU (P = 0.002), transfusion of more than 7 units of RBC (P = 0.045) and hepatorenal syndrome before transplantation (P = 0.016) as independent risk factors for mortality. Transfusion of more than 10 units of FFP, gender, use of marginal grafts, age, pretransplant diabetes mellitus, or postoperative bilirubin peak level, did not affect mortality (Table 4) .
The multiple logistic regression analysis of predictive factors for ICU length of stay of more than 10 days identified use of marginal grafts (P = 0.022), development or renal failure of more than RIFLE class 2 (P = 0.006), transfusion of more than 10 units of FFP (P = 0.034), respiratory failure (P = 0.009), MELD score corrected above 23 (P = 0.007), transfusion of more than 7 units of RBC (P = 0.032) and sepsis (P = 0.046) as independent risk factors. Age, gender, preoperative incidence of diabetes mellitus, directly pretransplantation ICU admission (transplanta-tion from the ICU), postoperative bilirubin serum peak level were no predictors of length of stay in the ICU (Table 5 ).
Currently allocation of liver organs through the MELD system and the impact on patient outcome is a hot debate. Data on the impact of preoperatively assessed MELD score on the morbidity and mortality of postoperative recipients are only few. This study correlated morbidity, but not mortality with the MELD score in patients after liver transplantation in uni-and multivariate analyses and demonstrated a MELD score above 23 to be an independent risk factor for an ICU stay longer than 10 days (odds ratio 7.0). Siniscalchi and colleagues reported a correlation of MELD score and postoperative complications in 242 liver transplants [20] . Interestingly, the MELD scores in that study were similar to our findings (22.8 vs. 22 .3 in our study in the high morbidity group and 17.6 vs. 18.8 in the low morbidity group). Another study associated increased length of stay in the ICU in association with high MELD score above 30 [7] , but failed to find a difference in mortality. Only in patients exceeding a MELD score of 36, mortality seems to be predicted by MELD as reported from Saab and colleagues [21] . In our population, four patients showed MELD score above 35, three of them died in the postoperative course. In contrast, a study of 340 transplanted patients showed no difference in early death in respect to the MELD score [8] . Several other publications from the USA have also document that MELD score cannot predict survival after liver transplantation [22] [23] [24] . Nevertheless, the question of whether very high MELD scores affect mortality remains elusive. Taken together, despite no clear correlation of MELD score and postoperative mortality, there is strong [25] . Another finding of this study was a high incidence of postoperative renal failure and subsequently need for RRT. Cox proportional hazard model revealed RRT in the ICU as an independent risk factor for mortality. RRT in our population was necessary in 21.8% during ICU stay. Other studies reported an incidence ranging from 3% to 20% [26] [27] [28] depending on the severity of preexisting renal conditions. Our study population included seven cases of pretransplant RRT already in need of RRT. This fact probably contributed to a higher incidence of renal failure when compared with those studies.
Apart from higher postoperative costs, renal failure and subsequent need for RRT is associated with increased mortality in ICU patients in general [29] and in particular in liver transplant recipients, varying from 27% to 67% depending on the comorbidities [30] [31] [32] [33] . There is strong evidence that even mild renal failure after transplantation might lead to longer hospital stay, more infections and increased overall mortality [33] [34] [35] .
In our study population, 95 (67.9%) patients presented with renal failure at different stages according to the RIFLE criteria. Planinsic and Lebowitz observed renal failure in more than 80% of cases during the first 48 hours after surgery for liver transplantation. Mortality was extremely high in up to 50% of liver transplant recipients with renal dysfunction at 30 days following surgery and, if hemodialysis was required, it could reach 60% [35] . The etiology of renal failure after liver transplantation is certainly multifactorial. Most reported risk factors are pretransplant renal dysfunction, low serum albumin, dysfunction of the liver graft, bacterial infections and reoperations [36] .
Furthermore, the contribution of intraoperative stressors is not to be neglected: hypotension with or without hypovolemia, operation without veno-venous bypass [37, 38] and use of nephrotoxic agents as antibiotics or immunosuppressants may further contribute to progressive renal failure.
Interestingly in our study population among the patients in need of postoperative RRT, even patients with preoperative normal kidney function could be found, which underlines the impact of intra-and postoperative stressors on renal failure. The focus of postoperative management should lie on provisions to avoid renal failure and logically lower morbidity and mortality.
Looking at preoperative kidney function in our study population we found an incidence of hepatorenal syndrome of 20% with a hazard ratio of 13, which corresponds to other studies [39] [40] [41] . Although liver transplantation can correct hepatorenal syndrome [42] , the time frame for recovery of renal function seems to be too long for RRT-free management. Often RRT is needed as a bridging therapy until the kidneys recover. In our study population, if duration of dialysis pretransplant was less than 30 days only 8% of patients still require hemodialysis 8 weeks after transplantation, which is somewhat in contrast to the study by Lo and colleagues, where 25% of the patients with hepatorenal syndrome require longterm RRT after transplantation [43] . Thus, hepatorenal syndrome is not always reversed, in particular when pretransplant RRT is necessary [44] and additional kidney transplantation becomes an option [40] . Hepatorenal syndrome prior to transplantation and RRT postoperatively are strong predictors for mortality in liver transplant recipients and the postoperative renal impairment leads to a prolonged ICU stay for these patients. Allogeneic FFP and RBC transfusions are associated with well-known adverse effects, reflected by increased incidence in viral and bacterial infections, activation of inflammatory and coagulation pathways, and immunologic reactions [45] [46] [47] . In patients after liver transplantation, intraoperative transfusion of packed RBCs are associated with more complications [48, 49] and infections [50] . Our multivariate analysis revealed transfusion of more than 7 units of RBCs and transfusion of more than 10 units of FFP as independent risk factors for mortality and prolonged ICU stay. Other reports identified intraoperative transfusion as a risk factor for morbidity and mortality in liver transplant recipients [39, 50, 51] and Massicotte and colleagues could demonstrate that a restrictive transfusion regime was associated with better outcome in liver transplantation recipients with an average MELD of 18 [49] . Thus, avoiding transfusion of RBC seems to be crucial to reduce postoperative morbidity and mortality.
In our group ICU mortality was 3.5% and the hospital mortality was 5.6%. The hospital mortality is closely related to the hospital length of stay [52] . Our survival data are similar to other transplant programs [6] [7] [8] with a cumulative patient survival of 89.5% after one year, 84.1% after three years and 74.1% after five years, even though in our study population 38.4% of marginal donor grafts were transplanted. In our study population, the use of marginal liver grafts was associated with higher ICU length of stay, but did not lead to an increased overall mortality [53, 54] and is able to decrease wait list mortality.
Sepsis was also highly associated with prolonged ICU stay and increased mortality confirming the results of other studies [55, 56] . It is still a leading cause of death (20 to 50%) in non-cardiac ICUs [57] . In our study population sepsis occurred in 10.8%, that is, it was ranked fourth in the complication list after renal failure, readmissions and reoperations.
The gastrointestinal system might play a key role in the pathogenesis owing to a breakdown of intestinal barrier function. Gurusamy and colleagues concluded from their database review that the use of prebiotics and probiotics might be effectful in the prevention of sepsis [58] . However, our patients received no prebiotics or probiotics, but this might be a beneficial therapeutical option in the future. Most importantly these patients should be management according to the guidelines of the Survival Sepsis Campaign [59, 60] .
This study identified MELD score above 23 as an independent risk factor of morbidity represented by ICU stay longer than 10 days but it did not clearly affect mortality. This finding supports the transplantation of patients with high MELD score at the cost of increased postoperative morbidity, in particular when it is seen in the light of reduced waiting list mortality. Furthermore, we identified transfusion of more than seven units of RBCs as an independent risk factor for mortality and for prolonged ICU stay. Postoperative renal failure and transfusion of more than 10 units of FFP are strong predictors of morbidity and postoperative RRT was highly associated with increased mortality, as was hepatorenal syndrome prior to transplantation.
• High MELD scores greater than 23 did not affect mortality in liver transplant recipients.
• Sepsis, postoperative RRT on ICU, transfusion of more than seven units of RBC and hepatorenal syndrome before transplantation were strong predictors for mortality in liver transplant recipients.
• Transplantation of marginal grafts, development or renal failure greater than RIFLE class 2, transfusion of more than 10 units of FFP, respiratory failure, MELD score greater than 23, transfusion of more than seven units of RBC and sepsis are predictors for increased length of stay in the ICU. Angiotensin-converting enzyme 2 autoantibodies: further evidence for a role of the renin-angiotensin system in inflammation Traditionally viewed as important in the regulation of blood pressure, the renin-angiotensin system - and specifically the angiotensin-converting enzyme (ACE)-angiotensin (Ang) II-AT(1 )receptor axis - may play a prominent role to promote inflammation and fibrosis. ACE2, a new component of the renin-angiotensin system, has emerged as a key enzyme that selectively degrades Ang II and generates Ang-(1-7), a bioactive peptide with anti-inflammatory and anti-fibrotic actions. Takahashi and colleagues demonstrate circulating titers of inhibitory autoantibodies against ACE2 in patients with systemic sclerosis. The current study reveals a potentially novel mechanism to attenuate the catalytic activity of ACE2, thereby promoting the actions of Ang II. In the present issue of Arthritis Research & Th erapy, Takahashi and colleagues assessed circulating levels of ACE2 in patients with connective tissue pathologies including pulmonary hypertension and persistent digital ischemia [1] . In comparison with normal controls, patients with overt vasculopathy expressed signifi cantly higher amounts of ACE2 protein in the circulation. Th ese patients, however, exhibited reduced ACE2 activity in serum and circulating autoantibodies against the enzyme. Th ere are few reports on the circulating levels of ACE2 in humans or experimental models, possibly refl ecting the diffi culty of obtaining a consistent measure ment of the enzymatic activity. Th e current study reveals a potentially novel mechanism to attenuate the catalytic activity of ACE2, thereby promoting the infl ammatory actions of Ang II.
ACE and ACE2 are both chloride-activated metallopeptidases that are predominantly associated with the cell membrane and are widely distributed in various tissues and vascular beds. In contrast to ACE, which cleaves two amino acid residues from the carboxyl terminus of Ang I to form Ang II, ACE2 hydrolyzes a single amino acid from the carboxyl end of Ang II to form Ang-(1-7) [2] . ACE is considered the primary enzymatic pathway that catalyzes the generation of Ang II in the circulation and tissues. ACE inhibitors, which have become standard therapies in the treatment of hypertension and other cardiovascular disease, have little or no inhibitory activity against ACE2, but they reduce the metabolism of Ang-(1-7) [2] . Circulating levels of ACE activity are readily measurable in humans and other species using synthetic substrates or assessing the direct conversion of Ang I to Ang II.
In comparison with serum ACE, Rice and colleagues reported that the circulating levels of ACE2 were 100-fold lower and that <10% (40 out of 494) of their patients expressed measurable ACE2 activity [3] . Nevertheless, families with detectable circulating ACE2 exhibited a greater incidence of cardiovascular pathologies although the overall sample population was low. More recent studies by Epelman and colleagues fi nd that circulating levels of ACE2 are highly associated with increasing severity of progressive heart failure [4] . However, patients
Traditionally viewed as important in the regulation of blood pressure, the renin-angiotensin system -and specifi cally the angiotensin-converting enzyme (ACE)angiotensin (Ang) II-AT 1 receptor axis -may play a prominent role to promote infl ammation and fi brosis. ACE2, a new component of the renin-angiotensin system, has emerged as a key enzyme that selectively degrades Ang II and generates Ang-(1-7), a bioactive peptide with anti-infl ammatory and anti-fi brotic actions. Takahashi and colleagues demonstrate circulating titers of inhibitory autoantibodies against ACE2 in patients with systemic sclerosis. The current study reveals a potentially novel mechanism to attenuate the catalytic activity of ACE2, thereby promoting the actions of Ang II. were chronically treated with inhibitors of the reninangiotensin system including aldosterone antagonists which may increase basal ACE2 expression potentially contributing to the protective mechanisms of these therapies.
Th ere is increasing evidence for the interplay of the renin-angiotensin system and infl ammatory events [5] . Pre-eclampsia is associated with circulating autoantibodies against the AT 1 protein that act as functional receptor agonists to promote vasoconstriction and infl ammation [5] . Studies by Harrison and colleagues suggest that T-cell expression of the AT 1 receptor contributes to infl ammatory events and the development of hypertension. Moreover, activated T cells may themselves generate Ang II locally to infl uence cell function in an autocrine manner [6] . In experimental encephalo myelitis, AT 1 expression was increased and subsequent AT 1 receptor blockade or ACE inhibition ameliorated the autoimmune infl ammation [7] .
Th e present fi ndings by Takahashi and colleagues reveal increased expression of circulating ACE2 in patients with vasculopathy utilizing a novel protein capture assay [1] . Despite the increased levels of the enzyme, ACE2 activity was markedly lower in comparison with the control group. Indeed, the authors report the presence of circulating levels of ACE2 antibodies that exhibit inhibitory activity in vitro. Previous studies showed that commercial sources of antibodies against ACE2 also inhibit enzyme activity, suggesting the epitope may encompass the catalytic site [4] ; however, the present study is the fi rst to identify autoantibodies that attenuate enzyme activity in a patient population.
Th e current fi ndings are of potential importance in our understanding of the role of circulating and tissue sources of ACE2, particularly in various disease states. Increased circulating levels of ACE2 may refl ect a compensatory mechanism to alter the balance of the renin-angiotensin system to favor the ACE2-Ang-(1-7)-AT 7 receptor axis and promote the antifi brotic and anti-infl ammatory actions of the heptapeptide, as well as attenuate the Ang II-AT 1 receptor pathway. Clearly, generation of endogenous antibodies with inhibitory activity against ACE2 may undermine this compensatory response. Indeed, identifi cation of endo genous ACE2 inhibitors is important in lieu of optimizing the therapeutic benefi ts following administration of recombinant soluble ACE2, as recently demonstrated in models of diabetic nephropathy [8] and liver fi brosis [9] or in the genetic expression of ACE2 in pulmonary hypertension [10] .
Although the ongoing study of the renin-angiotensin system has now surpassed the century mark, the characterization of this system and identifi cation of the factors that regulate the expression or activity of its components continues to yield novel therapeutic targets in cardiovascular disease and other pathologies.
Abbreviations ACE, angiotensin-converting enzyme; Ang, angiotensin. The Role of Chemokines during Viral Infection of the CNS  Viral infection of the central nervous system (CNS) poses unique challenges to the immune system with regards to controlling and eliminating the invading pathogen. These obstacles include the presence of a blood-brain barrier (BBB) that provides a physical and physiological barrier that is difficult for cells and molecules to cross, the absence of classic lymphatic drainage that may impair the generation of an adaptive immune response, and limited MHC class I or II expression on resident cells of the CNS, even during periods of neuroinflammation. In addition, the CNS is composed of a variety of highly specialized cells, many of which have limited renewal capacity, that represent potential targets of infection by numerous different viruses. Nonetheless, antigen-specific lymphocytes are ultimately able to accumulate within the CNS and contribute to defense by reducing or eliminating the invading viral pathogen. Alternatively, infiltration of activated cells of the immune system may be detrimental, as these cells can contribute to neuropathology that may result in long-term cellular damage or death. Understanding the mechanisms that govern leukocyte trafficking from the microvasculature into the CNS parenchyma is therefore critical for comprehending the molecular and cellular events that control neuroinflammation following infection by neurotropic viruses. Chemokines, small (8-10 kDa) proteins expressed by almost all nucleated cell types, are divided into four subfamilies based upon the number and spacing of conserved cysteine residues present within the amino terminus of the protein.
Chemokine function is controlled through often promiscuous signaling via seven transmembrane G-protein-coupled receptors. While initially characterized as important in inflammation by targeting distinct leukocyte populations, chemokines are now considered critical mediators of a variety of biological processes, including development, tissue homeostasis, and coordinated immune responses during viral infection.
Chemokines are now recognized as critical regulators of leukocyte trafficking into the CNS. This leads to the inevitable questions, which cells are producing chemokines and how is this controlled? Numerous studies have revealed that resident cell populations of the CNS are able to synthesize and secrete a variety of chemokines. Astrocytes and microglia are the primary source of chemokines following infection with a wide range of neurotropic viruses, including the JHM strain of mouse hepatitis virus (JHMV), lymphocytic choriomeningitis virus (LCMV), Theiler's murine encephalitis virus (TMEV), herpes simplex virus 1 (HSV1), and human immunodeficiency virus (HIV) [1] [2] [3] [4] . Neurons are also capable of secreting chemokines during HIV and West Nile virus (WNV) infection [5, 6] , while endothelial cells express chemokines during simian immunodeficiency virus-induced encephalitis [7] . Both in vitro and in vivo studies have highlighted that CNS viral infection often results in distinct chemokine signature patterns. For example, Prehaud and colleagues have demonstrated that in vitro infection of neurons with rabies virus (RABV) results in robust production of chemokines, whereas HSV-1-infected neurons do not [8] . However, specific chemokines, e.g., CXCL10 and CCL5, are often expressed independently of either cellular tropism or viral genetics, suggesting that factor(s) either secreted in response to infection (such as type I interferon [IFN]) or utilized for viral recognition are shared between many neurotropic viruses. Tolllike receptors (TLRs) recognize both DNA and RNA and they are able to rapidly respond to viral infection, in part, by promoting chemokine gene expression. During TMEV infection, TLR2 and TLR3 cooperation leads to the expression of the macrophage chemoattractants CCL2 and CCL5 [3] , while TLR2 and TLR9 mediate chemokine expression during HSV-1 infection [4, 9] . Type I IFNs regulate glial-derived chemokine expression in response to CNS infection with LCMV (Traub strain) and HSV-1 [2, 9] ; however, this pathway is dispensable for expression of other chemokines, e.g., CCL2 following infection with JMHV [10] . Rather, JHMV viral proteins influence chemokine secretion through as yet undefined mechanisms [11] , while the HIV-1 protein Nef influences neuronal chemokine secretion [5] . Moreover, WNV-infected cerebellar granule cell neurons readily secrete CXCL10 in vitro, while CXCL10 expression by WNV-infected cortical neurons is muted [12] . The consequence of this differential expression of CXCL10 is reflected in altered migration of defined inflammatory cells into the cerebellum at the expense of other WNV-infected CNS regions [12] . Collectively, these data illustrate that viral infection of the CNS by a wide variety of neurotropic viruses induces highly orchestrated and individual patterns of chemokine secretion by resident cells of the CNS, evoked by disparate pathways that converge into often overlapping profiles of inflammatory cell infiltration.
Signaling events that occur early following viral infection are often critical in dictating outcome. Recent studies have highlighted the importance of innate immune cells in contributing to a ). Monocytes are also attracted into the CNS via the chemokine CCL5 and its receptor CCR5. Neutrophils and monocytes participate in the degradation of the bloodbrain barrier (BBB), in part through the release of the matrix metalloproteinase MMP-9, and therefore ensure successive infiltration of virus-specific lymphocytes into the CNS. (B) During the acute stage of disease, astrocytes, microglia, neurons, and endothelial cells continue to secrete chemokines, serving to attract activated T lymphocytes, NK cells, and monocytes into the CNS. CD8+ and CD4+ T lymphocytes bearing the receptor CXCR3 and/or protective response, and we are just now learning how chemokines are involved in attracting these cells to the CNS. Infection of mice with neurotropic virus such as HSV-1 and JHMV results in the rapid accumulation of neutrophils to the CNS [13, 14] . Studies using the JHMV model system have provided insight into the functional relevance of neutrophil migration to the CNS, as these cells are required to contribute to the permeabilization of the BBB [14] . During JHMV infection, astrocyte-and endothelial-derived expression of ELR+ (glutamic acid-leucine-arginine) CXC chemokines, including CXCL1, attracts CXCR2-reactive neutrophils to the CNS [14] . Neutralization of this signaling axis specifically abrogates neutrophil infiltration, thereby preventing BBB degradation and the ensuing entry of protective JHMV-specific T lymphocytes [14] . In the absence of CXCR2 signaling, JHMVinfected mice experience higher viral loads and quickly succumb to infection, indicating that neutrophil targeting of the CNS is critical in host defense [14] . Conversely, McGavern and colleagues have suggested that during acute LCMV infection (Armstrong strain), cytotoxic T lymphocyte (CTL)-mediated chemokine gene expression contributes to fatal meningoencephalitis, in part, by attracting neutrophils and monocytes into the CNS, and this is associated with fatal vascular permeability and seizures, thus highlighting a detrimental role for neutrophils in response to viral infection [15] . In addition to enhancing the permeabilization of the BBB by recruiting neutrophils and monocytes, chemokines can also function as gatekeepers regulating leukocyte penetration into the parenchyma. Following WNV infection of the CNS, CXCL12 retains antigen-sensitized lymphocytes within the perivascular space. Antagonism of CXCR4, the receptor for CXCL12, enhances T lymphocyte entry into the CNS parenchyma, and this correlates with reduced WNV burden, enhanced survival, and limited neuropathology [16] . Thus, expression of chemokines early in response to infection with neurotropic viruses aids in effective host defense by promoting vascular permeability and regulating parenchymal lymphocyte infiltration ( Figure 1A ). However, it should be emphasized that the consequences of BBB degradation can vary from efficient viral clearance to fatal encephalitis and seizures, depending upon the virus and the route of infection.
Although early signaling events are clearly important for host defense during viral infection, the infiltration and anti-viral activity of T lymphocytes are requisite for viral clearance and survival. CXCL10, which is prominently expressed within the CNS during many viral infections [1, 17] , functions to attract activated T lymphocytes bearing the receptor CXCR3. Neutralization or genetic silencing of CXCL10 following infection with HSV, JHMV, and WNV dramatically reduces T cell trafficking into the CNS, thus preventing efficient viral control and often resulting in poor resolution [6, 18, 19] . In addition to attracting T lymphocytes, the CXCR3 ligands CXCL10 and CXCL9 also attract natural killer (NK) cells during JHMV infection [20, 21] ; however, their role in viral clearance remains unclear. The macrophage and T lymphocyte chemokine CCL5, or one of its receptors, CCR5, also promotes leukocyte trafficking into the CNS and subsequent viral control during JHMV infection and WNV-induced encephalitis [22, 23] . The clinical relevance of this observation was revealed when homozygosity for the defective human CCR5 allele (CCR5D32) was associated with an increased risk for symptomatic WNV infection [24] . Collectively, these data demonstrated that chemokine expression during viral infection promotes the generation and infiltration of immune effector cells necessary for quelling viral replication ( Figure 1B ).
A potential consequence of chemokine secretion and the subsequent accumulation of leukocytes within the CNS, while important for viral control in many instances, is the development of neuropathology. For example, the fatal meningoencephalitis induced by LCMV infection is mediated by infiltration of virusspecific CTLs that promote subsequent myeloid cell and leukocyte entry [15, 25] . During infection with LCMV (Traub), genetic silencing of CXCL10 or its receptor CXCR3 reduces the infiltration of CD8+ T cells, conferring either partial or near complete protection from immunopathology and death [26, 27] . However, CXCL10 remains dispensable for T cell infiltration or the development of fatal inflammation during infection with LCMV (Armstrong) [28] , further highlighting underlying differences in viral strains and chemokine utilization with regards to disease outcome. During JHMV infection, sustained CXCL10 and CCL5 expression leads to continuing immune cell infiltration that manifests an immune-mediated demyelinating disease. Neutralization of either chemokine during persistent JHMV infection abrogates the immune infiltration and greatly reduces both disease severity and demyelination [29, 30] . In addition to attracting inflammatory cells that contribute to neuropathology, CXCL10, which is chronically expressed within the brains of patients suffering from HIV-associated neurological disorders, can directly induce neuronal cell death [5] . In addition, proteolytically cleaved CXCL12, which is also detectable within the brains of HIV-1infected patients, is capable of inducing neurotoxicity and apoptosis [31] . Although beyond the scope of this review, extensive work has focused upon the direct and indirect roles of the chemokine receptors CXCR4 and CCR5 (and their associated ligands) in contributing to HIV-associated dementia (reviewed in [32, 33] ). Therefore, chemokines are critical mediators of neuropathology during viral infections of the CNS, either by attracting pathogenic inflammatory cells or directly mediating neurotoxicity and cell death.
From this brief review, it is evident that the biological roles of chemokines in host defense and/or disease in response to viral infection of the CNS are constantly evolving. An emerging picture has developed that indicates that chemokines and their receptors are intimately involved in generation of effective host responses to viral infections within the CNS. Paradoxically, chemokine expression has also been associated with neuropathology. Thus, chemokines and/or chemokine receptors are potentially relevant targets for treating various viral-induced neuropathies by dampening specific biological functions associated with disease. Recent evidence has emerged implicating chemokines, specifically CCR5 are attracted by the chemokines CXCL10 and CCL5, respectively, and mediate viral control through direct cytolytic activity and/or cytokine secretion. CXCL12, which signals through CXCR4, may, however, sequester T lymphocytes within the perivascular space and regulate penetration of the parenchyma, thus inhibiting efficient viral clearance. doi:10.1371/journal.ppat.1000937.g001 CXCR4 and CXCL12, as important mediators of neurogenesis [34] ; thus, chemokines produced during viral infections may influence neural precursor cell function and therefore influence recovery and repair. We can only look forward to future research that will undoubtedly uncover new and exciting roles for the chemokines in host defense, disease, and recovery within the context of the virally infected CNS. A cross-sectional survey to evaluate knowledge, attitudes and practices (KAP) regarding seasonal influenza vaccination among European travellers to resource-limited destinations BACKGROUND: Influenza is one of the most common vaccine-preventable diseases in travellers. By performing two cross-sectional questionnaire surveys during winter 2009 and winter 2010 among European travellers to resource-limited destinations, we aimed to investigate knowledge, attitudes and practices (KAP) regarding seasonal influenza vaccination. METHODS: Questionnaires were distributed in the waiting room to the visitors of the University of Zurich Centre for Travel' Health (CTH) in January and February 2009 and January 2010 prior to travel health counselling (CTH09 and CTH10). Questions included demographic data, travel-related characteristics and KAP regarding influenza vaccination. Data were analysed by using SPSS(® )version 14.0 for Windows. Differences in proportions were compared using the Chi-square test and the significance level was set at p ≤ 0.05. Predictors for seasonal and pandemic influenza vaccination were determined by multiple logistic regression analyses. RESULTS: With a response rate of 96.6%, 906 individuals were enrolled and 868 (92.5%) provided complete data. Seasonal influenza vaccination coverage was 13.7% (n = 119). Only 43 (14.2%) participants were vaccinated against pandemic influenza A/H1N1, mostly having received both vaccines simultaneously, the seasonal and pandemic one. Job-related purposes (44, 37%), age > 64 yrs (25, 21%) and recommendations of the family physician (27, 22.7%) were the most often reported reasons for being vaccinated. In the multiple logistic regression analyses of the pooled data increasing age (OR = 1.03, 95% CI 1.01 - 1.04), a business trip (OR = 0.39, 95% CI 0.17 - 0.92) and seasonal influenza vaccination in the previous winter seasons (OR = 12.91, 95% CI 8.09 - 20.58) were independent predictors for seasonal influenza vaccination in 2009 or 2010. Influenza vaccination recommended by the family doctor (327, 37.7%), travel to regions with known high risk of influenza (305, 35.1%), and influenza vaccination required for job purposes (233, 26.8%) were most frequently mentioned to consider influenza vaccination. CONCLUSIONS: Risk perception and vaccination coverage concerning seasonal and pandemic influenza was very poor among travellers to resource-limited destinations when compared to traditional at-risk groups. Previous access to influenza vaccination substantially facilitated vaccinations in the subsequent year. Information strategies about influenza should be intensified and include health professionals, e.g. family physicians, travel medicine practitioners and business enterprises. Pandemic and seasonal influenza are still a challenging field of the public health system. Influenza -a mild to severe respiratory infection caused by RNA viruses of the family Orthomyxoviridae -is one of the most common vaccine-preventable disease in travellers. Worldwide, between 250'000 and 500'000 deaths are estimated to be due to seasonal influenza infection each year [1] . Influenza is also responsible for tremendous economic costs both from admissions to hospital and loss of productivity [2] . Influenza affects all age groups and is usually self-limited. Common symptoms include acute fever, muscles pain, headache, cough and chills [3] . Special risk groups, such as very young children, the elderly and those suffering from chronic lung or heart diseases are at risk for serious influenza complications, e.g. bacterial pneumonia [4, 5] . Influenza reaches peak prevalence in winter in the Northern hemisphere (Nov-Apr) -as well as in the Southern hemisphere (Apr-Oct) and circulates yearround in the tropics [6, 7] . Seasonal influenza vaccination is an effective prevention strategy and is therefore routinely recommended for special risk groups [8, 9] . Of note, the seasonal influenza vaccine recommendations of the U.S. Centres for Disease Control were recently expanded and include now about 80% of the population [10] .
Influenza is known to be a quite frequent infection among travellers to tropical and subtropical destinations compared to other infections, e.g. vector-borne ones. About one of hundred travellers abroad gets infected [7] . The risk of infection depends on the travel destination and the season. Travellers crossing hemispheres may be confronted with different antigenic variants of the influenza virus. By returning home, the new variant may be transmitted to contact persons [11] . The first pandemic of the 21st century has highlighted the need for international influenza prevention strategies [12] .
The objective of this study was to investigate the vaccination coverage as well as knowledge, attitudes and practices (KAP) regarding influenza vaccination among travellers to resource-limited countries to improve or adapt current preventive strategies.
Two cross-sectional surveys were conducted at the University of Zurich Centre for Travel' Health during January and February 2009 and January 2010, respectively. Selfadministered, anonymous questionnaires including 16 items were distributed to travellers waiting for pre-travel health advice. Participation was voluntary. Individuals above 17 years, understanding German or English, residing in Switzerland and planning to travel to a resourcelimited destination were included. Questions included demographic data (gender, age, nationality, education, profession), travel-related characteristics (destination country, duration of stay, influenza risk perception, previous travel health advice, travel purpose, travel costs) and general attitudes and practices towards influenza vaccination (vaccination coverage, reasons to be vaccinated, reasons to refuse vaccination, motivations to consider vaccination with options for multiple answers except for the vaccination coverage). In 2010, an additional question targeting the pandemic influenza A/H1N1 vaccination coverage was included. The questionnaires were checked for completeness. A written letter of exempt was received by the Ethical Commission of the Canton of Zurich.
Statistical analyses were conducted by using SPSS ® version 14.0 for Windows. Differences in proportions of demographics, travel-related data and attitudes and practices were compared using the Chi-square test. The significance level was set at p ≤ 0.05. For the multiple logistic regression analysis the surveys were analysed as well as pooled dataset and each survey, CTH-2009 and CTH-2010, separately. The seasonal influenza vaccination was used as outcome and all demographic, travel-related and attitude-and practices-related factors were evaluated as independent predictors. Odds Ratios (OR) were determined by stepwise backward elimination of variables with p > 0.150. For sensitivity analyses, each dataset of the CTH studies, 2009 and 2010, was analysed separately and additionally, predictors for pandemic influenza vaccination were determined by multiple logistic regression analyses.
From a total of 938 eligible individuals, 868 (92.5%) were included in the analysis ( Figure 1 ). Overall, 479 (55.2%) were females and 389 (44.8%) males. The great majority of participants (503, 57.9%) were between 18 and 35 years old with a median age of 32 years (range 18 -84 yrs). Only 46 (5.3%) responders were above 64 years of age. In general, participants were highly educated with 480 (55.3%) being university graduates. Overall, the characteristics of participants planning to travel to resource-limited destinations are presented in Table 1 . 
Of all vaccinated participants, 44 (37%) declared to be vaccinated for business reasons and 25 (21%) due to age ( 
Travel as risk factor for an influenza infection is poorly established among international travellers when regarding the low vaccination coverage as well as the low selfperceived travel-associated risk estimates. Of note, previous influenza vaccinations facilitated receiving an influenza vaccination in the following year by about 13 times. Therefore, easy access to the influenza vaccine is important. High media coverage was not considered sufficient to increase the vaccination rate substantially as is indicated by the low increase of the vaccination coverage between the two surveys in 2009 and 2010 and also by the low pandemic influenza vaccination coverage of only 14.2%. Therefore, multiple efforts need to complement one another including information strategies provided by family physicians and travel medicine practitioners, but also job-and age-related activities need to be considered. Our sample of travellers is comparable to other studies performed at our Centre for Travel' Health [7] with respect to the age distribution, educational level and travel duration. Inherent limitations include a selection bias: Frequently visited destinations such as the Middle East, North Africa and the Caribbean are underrepresented as travellers to those destinations generally do not consider a pre-travel health consultation as indicated [11] but destinations with higher risk for faecal-orally transmitted infectious diseases, such as TD or bacterial meningitis, are well represented, such as e.g. India and Sub-Saharan countries. Therefore, our sample may represent a best practice sample. The fact, that the high proportion of university graduates indicates a health literate population may result in an even overestimated risk perception as well as influenza vaccination coverage. All data collections relied on self-reported information. Hence, the results of the studies might be limited by a potential bias such as disclosure bias, although self-report of influenza vaccination status has been found to be reliable when checked against medical record documentation [13] . Most seasonal influenza activity occurs during November to April on the Northern hemisphere and vaccination is usually administered between October and November. Therefore, travellers visiting the opposite hemisphere have to be counselled accordingly and the seasonal influ-enza vaccine also for the Southern hemisphere has to be available as there is year-round influenza activity in tropical and subtropical areas.
Risk perception and vaccination coverage regarding seasonal and pandemic influenza was very poor among European travellers to resource-limited destinations  Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens. For serodiagnosis of human helminthic infections, many currently used tests rely on native antigens, either extracted from whole worms (somatic antigens) maintained in laboratory animals, or cultivated in vitro to obtain excretory/secretory products (metabolic antigens). These natural antigens are limited in availability and suffer from batch-to-batch variation. A number of Echinococcus proteins have been recombinantly produced and tested for use in serodiagnosis [1] [2] [3] [4] [5] [6] but to our knowledge, only recombinant EmII/3-10 [7, 8] and its related sequence Em18 [9] are successfully applied in commercial test kits. Recombinantly expressed antigens used in diagnostic tests require a high degree of purification to avoid cross-reactivity due to contaminants from the expression system. Unspecific binding and cross-reactivity are major problems with both, extracts of whole worms [10, 11] and recombinant proteins [12] . For improving diagnostic test performance, it is desirable to identify highly specific and highly reactive epitopes from the proteome of the pathogen in question and synthetically produce the corresponding peptide antigens. Synthetic peptides are advantageous for diagnostic applications since they are well defined, easily produced in large amounts, highly pure and often cost-saving if compared to the production of natural antigen in animal models or in vitro culture. Applications of peptides in immunodiagnosis of different parasitic diseases were given by Noya et al. [13] .
The availability of an increasing number of pathogen genomes is boosting basic research as well as applied science. In the field of parasitological diagnostics, sequencing of parasites genomes also creates new opportunities. Annotated genomes and proteomes are available for some of the medically important protozoan pathogens, such as Plasmodium species and some Kinetoplastida species. Following the genome of the parasitic nematode Brugia malayi [14] , the genome of Schistosoma mansoni, a blood vessel dwelling trematode, has recently been released [15] as well as a draft genomic sequence for Schistosoma japonicum [16] and more genome data from various helminth species are to be expected in the near future (see for example http://www.sanger.ac.uk/ Projects/Helminths/ or http://www.nematode.net/). Currently, proteins predicted from ESTs, whole genomes or contigs are available for data mining already from a considerable number of helminth species. Using the sequence data available in the public domain, we have designed and tested a pathway for identifying novel antigens for serodiagnostic test development.
Our selection procedure for peptide antigens relied on the application of bioinformatic filters to collections of protein sequences, including conceptually translated nucleotide sequence data. The selection criteria aimed at proteins located to the hostparasite interface. Such proteins are potentially seen by the host immune system and may elicit an immune response and thus represent good candidates of diagnostic antigens. Our in silico analysis therefore prioritized proteins containing a sorting signal directing the protein to the extracellular space (PSORT II [17] ), transmembrane domains (TMHMM II [18] ), or a C-terminal signature sequence for addition of glycosyl phosphatidylinositol (GPI) anchor (GPI-som [19] ). These prediction algorithms require full length amino acid sequences with complete N-and C-termini, thus excluding the use of most EST libraries. The majority of proteins generally display well defined three dimensional structures, which is mostly globular. Our selection procedure preferred sequences in which the chemically synthesized peptide also adopts a natural conformation in vitro. There are two structural motifs meeting this claim. Firstly, the intrinsically unstructured regions (IUR) that lack a well defined three dimensional structure displaying an extended conformation that can be identified for example by IUPred [20] . Secondly the alpha-helical coiled-coil motif predicted for example by Paircoil2 [21] . Alpha-helical coiled-coils are believed to readily fold into a stable structure in aqueous solution [22] . A microarray platform served for testing the diagnostic potential of the peptides selected in silico. Using a highthroughput microarray format brings together the advantages of low reagent consumption and rapid multiplexed analysis. Particularly in diagnostics, the possibility of testing the reactivity of a given serum sample with multiple antigens simultaneously harbors great benefits. Promising candidates identified on microarray were further explored and validated for use in ELISA.
We have carried out the proof-of-principle for bioinformatic selection of suitable long synthetic peptides (LSPs) for the tapeworms Echinococcus multilocularis and Echinococcus granulosus, both of major medical importance causing alveolar (AE) and cystic echinococcosis (CE), respectively. CE and particularly AE, due to its infiltrative and/or space-occupying growth, are severe diseases with high fatality rate and poor prognosis if managed incorrectly [23] . The importance of alveolar echinococcosis is not represented by the number of reported cases but rather by the severity of the disease in the individual patient [24] . Standard diagnostic tests for human echinococcosis, AE as well as CE, imply imaging techniques such as ultrasonography, x-ray and computer tomography, as well as serological tests based on enzyme-linked immunosorbent assay (ELISA) and immunoblots [25] . Antigens used in these serodiagnostic tests were developed from crude extracts to purified fractions to recombinant antigens and for E. multilocularis to vesicular fluid originating from in vitro cultivated metacestodes [26] . Despite substantial improvements have been achieved, the main screening test still relies on the availability and quality of native antigens, i.e. hydatid fluid of Echinococcus granulosus cysts collected from naturally infected intermediate hosts at the slaughterhouse. One study reported that source and quality or fertility of cysts are critical for test outcome. This called for standardization of antigens and test methods [10] . To produce a robust and reproducible test for the routine diagnostic laboratory, we have evaluated the performance of chemically synthesized peptides in comparison to natural (EgHF, EM2) [26, 27] and recombinant antigen (EmII/3-10) [7] .
Ethical clearance for retrospective use of anonymized patient sera for test development and quality control was obtained from the ethical committee (Ethikkommission beider Basel).
Sera of healthy blood donors living in Switzerland were used to define a cut-off for distinguishing between positive and negative test results. In ELISA, 50 blood donor sera were used. In microarray, a single serum and 2 pools made of 5 sera each were used. For testing diagnostic peptide reactivity in ELISA, 44 sera from E. multilocularisand 35 sera from E. granulosus-infected patients from Central Europe were used. All echinococcosis patients had active hepatic lesions of either CE1 or CE2 type (WHO-IWGE standardized classification) and all sera were sampled prior to any therapeutic intervention, i.e. before surgery and/or chemotherapy. Diagnoses were confirmed serologically as described by Müller et al. [26] , in complementation to the clinical diagnosis based on imaging procedures and, if available, retrospective histopathological investigations. For testing cross-reactivity of the peptides, sera from patients with following infections were used (concomitant echinococcosis was ruled out by clinical and serological criteria): 2 trichinellosis, 2 trichuriasis, 10 toxocariasis, 8 ascariasis, 1 anisakiasis, 2 hookworm infection, 8 strongyloidiasis, 10 filariasis (Loa loa, Mansonella perstans, Onchocerca volvulus), 10 fascioliasis, 1 paragonimiasis, 11 schistosomiasis (Schistosoma mansoni, S. haematobium, S. mekongi), 12 neurocysticercosis
Crude or purified, somatic or metabolic extracts of native antigens are routinely used for the serodiagnosis of human helminthic infections. These antigens are often crossreactive, i.e., recognized by sera from patients infected with heterologous helminth species. To overcome limitations in antigen production, test sensitivity and specificity, chemically synthesized peptides offer a pure and standardized alternative, provided they yield acceptable operative characteristics. Ongoing genome and proteome work create new resources for the identification of antigens. Making use of the growing amount of genomic and proteomic data available in public databases, we tested a bioinformatic procedure for the selection of potentially antigenic peptides from a collection of protein sequences including conceptually translated nucleotide sequence data of Echinococcus multilocularis and E. granulosus (Plathyhelminthes, Cestoda). The in silico selection was combined with high-throughput screening of peptides on microarray and systematic validation of reactive candidates in enzyme-linked immunosorbent assay. Our study proved the applicability of this approach for selection of peptide antigens with good diagnostic characteristics. Our results suggested the pooling of several peptides to reach a high level of sensitivity required for reliable immunodiagnosis. 
Protein sequences of Echinococcus multilocularis and E. granulosus were retrieved from NCBI Entrez Protein Database on October 16, 2007 . NCBI Entrez Protein Database are compiled from a variety of sources with daily updates, including SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq. To identify putative membrane or extracellular proteins, the protein sequences were analyzed using TMHMM II [18] for prediction of transmembrane domains, GPI-SOM [19] for prediction of glycosylphosphatidylinisotol (GPI) anchor signals, and PSORT II [17] for prediction of protein subcellular localization. All protein sequences were further screened for stretches of alpha-helical coiled-coils using Paircoil2 [21] and intrinsically unstructured regions using IUPred [20] . Both of these structures are believed to adopt native conformation in aqueous solution and therefore constitute major selection criteria. Due to the limited number of Echinococcus sequences in NCBI Entrez Protein Database and few predicted surface proteins containing stretches of alpha-helical CC and/or IURs, the analysis was extended to all proteins with alpha-helical coiled-coil and IUR predictions, irrespective of their predicted locations. This included known proteins previously tested in immunodiagnosis. To narrow down protein regions for selection of 30mer peptides, protein sequences were subjected to prediction of coiled-coil stability by STABLECOIL (http://biomol.uchsc.edu/cores/biophysics/ stablecoil). In case of IURs antigenicity predictions were performed using BepiPred [28] . Thus detected regions of high predicted CC stability or antigenicity were favored. To increase solubility, peptides were selected to start and end with a hydrophilic or neutral amino acid and to harbour on average one charged amino acid, either positive or negative, per five residues. In addition, the guidelines suggested by the manufacturer were followed (http://www.altabioscience.bham.ac.uk/ pdfs/Intro_to_series_SYNTHETIC_PEPTIDES.pdf). From 11 sequences listed in table S1, 45 peptides were selected according to the criteria mentioned above.
Peptides were produced by Fmoc solid phase synthesis (Alta Bioscience, University of Birmingham, UK). The length of the peptides was limited by the EpiScan synthesis procedure (Alta Bioscience, University of Birmingham, UK) to a maximum of 30 amino acids. Thus the length of the peptides used for spotting onto microarrays ranged between 24 and 30 amino acids with an additional aminohexanoid acid (AHX) spacer and a biotin at the N-terminus. Biotin was required to bind peptides to a streptavidincoated solid phase, i.e. microscope glass slide for microarrays (Alta Bioscience, University of Birmingham, UK) or to 96 well plates for ELISA. In order to remove electric charges from the free Cterminus, the synthetic peptides were modified by carboxyterminal amidation. Thus, both N-terminal and C-terminal peptide ends were uncharged, mimicking natural segments of internal protein sequences.
For defining optimal peptide length, we designed extended variants of the 8 most reactive candidates from the microarray. The length of extended peptides ranged between 40 and 47 residues (table 1). Modifications at the N-and C-terminus were identical to those of the microarray peptides. The extension of peptides was chosen to increase alpha-helical coiled-coil stability or to combine epitopes from two single peptides. To target these improved peptides to E. multilocularis diagnosis, we chose the E. multilocularis sequences for designing longer peptides. Sequences derived from antigen B8/1 (accession number BAC77657) and antigen B8/2 (accession number BAD89809). To our knowledge, these were the first synthetic peptides from any E. multilocularis antigen B sequence that were serologically evaluated. Alignments of homologous antigen B8/1-and antigen B8/2-sequences of the two Echinococcus species are shown in figures S1 and S2, respectively. Antigen B8/1sequences of E. granulosus and E. multilocularis showed 86% identity and antigen B8/2-sequences 93% identity. Peptide longD8-9 coincided with the region of a previously published E. granulosus epitope EVKYFFER [29] , but differed from another published synthetic peptide p176 [30] : p176 spans the N-terminal region of antigen B8/1 (amino acids 17-54), while longD8-9 covered the central part of the protein (amino acids . Within the overlap of 26 amino acids, three amino acids differed.
A microarray platform was used for screening in silico selected peptides for reactivity with patients' sera. The low-density peptide microarray (Alta Bioscience, University of Birmingham, UK) featured two blocks, each containing 45 Echinococcus peptide spots, a spotting control allowing positioning of grid for analysis (unrelated, TAMRA-labeled peptide), a control for serological detection (human IgG) and a blank (spotting solution). All microarray work was done at room temperature. Microarray slides were blocked for one hour in assay buffer (16PBS pH 7.4, 0.05% Tween20, 3% milk powder). Individual human sera or serum pools were diluted 1:50 in assay buffer and incubated for two hours in a moist chamber. Slides were washed for 365 minutes in assay buffer and incubated for one hour with a 1:100 dilution of Cy5-labeled goat anti-human IgG (H+L) secondary antibody (Jackson ImmunoResearch Laboratories product number 109-175-088). Slides were washed for 365 min- ELISA 96-well plates (NUNC Immobilizer Streptavidin) were prewashed three times using an ELISA plate washer (deionized H 2 O, 0.05% Tween20) and coated overnight at 4uC with 100 ml/well of synthetic peptide diluted to 2 mg ml 21 in PBS pH 7.4. All following steps were carried out at room temperature. After washing, the plates were blocked for one hour with 150 ml/well of assay buffer (3% milk in PBS pH 7.4, 0.05% Tween20, 0.5 mM biotin). The plates were washed and incubated for one hour with human sera diluted 1:200 in assay buffer (100 ml/well). Alkalinephosphatase-conjugated goat anti-human IgG antibodies (Sigma product number A 3187) 1:1000 in assay buffer were used as secondary antibodies. After washing, 100 ml of conjugate dilution was added to each well and incubated for one hour, followed by a final wash. Wells were then incubated for 15 minutes with 100 ml of p-nitrophenyl phosphate (Sigma product number N 4645) at a concentration of 1 mg ml 21 in substrate buffer (13.2 mM Na 2 CO 3 , 35 mM NaHCO 3 , 1mM MgCl 2 66H 2 O, pH 9.6). Absorbance values (A 405nm ) were measured at 405 nm in a Tecan Sunrise microplate absorbance reader. All serum samples were tested in duplicates. The two values were averaged and blankcorrected. For distinguishing between positive and negative ELISA test results, 50 sera from healthy blood donors living in Switzerland were tested. The cut-off value was determined by the mean of the blood donor samples plus two standard deviations. Sensitivity and specificity of the single peptide candidates were calculated using test results from confirmed echinococcosis patients as true positives (TP). Blood donors and individuals infected by other helminths were taken as true negatives (TN). The working characteristics of the peptide antigens were explored by receiver-operating characteristics (ROC) plot analysis (Analyse-It, version 2.21).
To test the impact of pooling peptide candidates, LSP longD12, longD1 and longD8-9 were mixed in equal parts (mixW) and applied at the same conditions as described above. The cut-off was determined by the mean A 405nm of fifty blood donor samples plus 2 standard deviations. Sensitivity and cross-reactive behavior was tested with 30 echinococcosis sera and 15 sera from various helminth and amoeba infections that had previously shown crossreactivity with the single peptides.
240 entries of E. multilocularis and 940 entries of E. granulosus proteins were retrieved from NCBI Entrez Protein Database including redundancies. Combining the outputs of PSORT II, TMHMM II, GPI-som, IUPred and Paircoil2, led to the selection of 11 protein candidates (table S1). According to the information available in GenBank, these proteins were isolated from metacestode or protoscolex tissue, with the possible exception of HSP70, for which no information on developmental stage was provided.
Six proteins had been reported previously to react with antibodies from patients' sera: EmII/3 [7] , EM13 [6] , P-29 [31] , GRP [32] , EG19 [33] , AgB8/1 and AgB8/2 [1] . These earlier findings supported the inclusion of these antigens in our analysis. In total, 45 peptide sequences from 11 proteins were chosen to be chemically synthesized and spotted onto microarray. 31 peptides derived from E. multilocularis and 14 from E. granulosus (table S1).
In silico selected peptides were screened for diagnostic potential on a microarray platform (Alta Bioscience, University of Birmingham, UK). From a total of 45 peptides contained in the microarray, 17 showed reactivity with pooled or single sera from echinococcosis patients (8/31 peptides from E. multilocularis, 9/14 peptides from E. granulosus). The peptides that reacted above the cut-off determined from blood donor sera (FI.1009) are listed in table 2. These 17 peptides were considered to have diagnostic potential and were therefore subjected to further testing of specificity and sensitivity. The predicted structure of the reactive peptide candidates was mostly alpha-helical coiled-coil (7 peptides) and alpha-helical (7 peptides). 3 peptides were predicted to be intrinsically unstructured.
The 17 peptides reactive on microarray were carried forward for evaluating their application in ELISA-based serodiagnosis, the current routine technique. Test sera from AE and CE patients and sera from patients with infections other than echinococcosis were used. Sensitivity, cross-reactivity and impact of peptide length were evaluated (data not shown). With respect to sensitivity, long peptides (more than 30 residues) were superior to shorter ones. 3 out of 17 peptides were selected for extensive validation: longD12 (deriving from E. multilocularis EmII/3-10), longD1 (EmII/3-10) and longD8-9 (E. multilocularis antigen B8/1). All 3 peptides did not clearly differentiate between AE and CE infections as can be seen by overlapping standard deviations of A 405nm values obtained from AE and CE sera (figure 1). Peptides longD1 and longD12 derived from EmII/3-10, a well established antigen specific for the diagnosis of E. multilocularis infections [8] . In our hands, longD12 reacted with both, AE and CE sera ( The cut-off values determined from 50 blood donors were similar for longD8-9 and longD1 with 0.165 and 0.200, respectively. The cut-off for longD12 was higher with an OD of 0.357. Compared to 90% sensitivity of a commercial E. granulosus hydatid fluid ELISA used in routine testing [34] , the sensitivity of each single peptide alone was rather low, candidate longD8-9 being the most sensitive with 57%. LongD1 and longD12 both reached 16%. The specificities of longD8-9, longD1 and longD12 were 94%, 91% and 94% respectively, thus performing in the range required for a routine diagnostic assay. There were minor cross-reactivities observed with sera from helminth infections other than echinococcosis (table 3) . Peptide longD8-9 was positive with 1/12 cysticercosis, 1/8 strongyloidiasis and 1/10 schistosomiasis Selection of Diagnostic Antigens by Microarray www.plosntds.org sera. Peptide longD1 was seropositive with 2/8 strongyloidiasis, 1/ 10 toxocariasis, 1/10 filariasis, 1/10 fascioliasis and 3/10 schistosomiasis sera. Peptide longD12 was positive with 1/8 strongyloidiasis, 1/10 toxocariasis, 2/10 schistosomiasis sera. Cross-reactions with Taenia, Strongyloides and filaria are also seen in conventional diagnostic assays. We further tested the specificity with sera from patients suffering from liver abscess due to Entamoeba histolytica infection. LongD12 was positive with 3/26, longD1 with 4/26 and longD8-9 with 3/26 amebiasis sera (table 3) . To compare the diagnostic performance of each peptide, a receiver-operating characteristics (ROC) plot was generated (figure 2). Candidate longD8-9 emerged as clear favorite, although on its own not meeting the sensitivity required for antigens applied in routine diagnostics.
The use of 30-50mer peptides for serology impairs sensitivity compared to the full length antigen, because peptides likely harbor less epitopes. We calculated the cumulative sensitivity of the 3 single peptide ELISAs and measured the experimental combination of the 3 peptides in one antigen mix (mixW). The cut-off of mixW was 0.352, which is in the same range as the cut-off of one of its components, namely longD12. By cumulating the individual results of the 3 assays, the theoretical sensitivity indeed increased from 57% to 70%, but specificity decreased to 82% (table 4) . Compared to cumulative positivity, pooling the 3 peptides in one well (mixW) led to a loss of positive test results. In return, the specificity increased due to a loss of cross-reactions. Positive test results obtained by single peptide ELISAs compared to pooled peptide ELISA are summarized in table 5.
The use of synthetic peptides as substitutes of native antigen in immunoassays has been highly recommended for the diagnosis of infectious diseases by different authors [13, 30, 35] . Synthetic peptides are applied successfully in diagnosis of viral and bacterial infections [36] [37] [38] [39] . Compared to a metazoan parasite, viruses and bacteria in general have smaller genomes and accomplish less complex post translational protein modifications, which might imply more rapid success in identification of immunodominant peptides. It even allows for analysis on whole proteome level [40] . Here, we investigated the use of synthetic peptides of 30-50 amino acids in length for the use in immunodiagnosis of human hydatid disease. We provided proof of principle for the selection of synthetic peptides by bioinformatic means and for screening on a microarray platform for diagnostic reactivity. 17 out of 45 peptides on the microarray proofed reactive with sera from echinococcosis patients.
The peptide performing best in our investigation was longD8-9, a 42mer peptide deriving from E. multilocularis antigen B8/1. It reached a sensitivity of 57% and a specificity of 94%. The reactivity of LSP longD8-9 with sera from AE as well as CE patients suggests its use for both, patient screening as well as for follow-up examinations of treated patients. For the latter purpose, it will be necessary to determine the proportion of patients seroconverting during treatment. The applicability of our peptide antigen for monitoring treatment follow-up samples is ongoing work in our laboratory. Table 3 . Numbers of sera tested positive with peptides longD12, longD1 and longD8-9. Our results confirm previous reports [38, 41] that a test based on a single peptide needs to be complemented by additional peptides to reach a sensitivity comparable to the antigen extract. To compensate for limited sensitivity of individual peptides, we performed ELISAs with combinations of synthetic peptides applied as mixtures for coating. We compared these results to the ''cumulative sensitivity'', a theoretical value obtained by summing up the positive results from individual peptide ELISAs for each serum tested. The cumulative result amounted to a theoretical sensitivity of 70%. We did not detect this high sensitivity. In contrast, pooling of 3 peptides in one well led to a loss of positive signals otherwise obtained with individually tested peptides. 8/29 echinococcosis samples were lost through pooling. One reason was suggested by an increased cut-off for pooled peptides. In some sera similar ODs were observed in individual test and in peptides mixtures, but due to increased cut-off for pooled peptides, these sera turned negative. The loss of sensitivity obtained with pooled peptides in mixW could also be due to partial occupation of streptavidin binding sites with less reactive antigen. The same reasons may account for increased specificity. Combination of peptides in one well led to a loss of unwanted cross-reactivity with 5/11 previously false positive signals disappearing. Thus in our study, pooling peptides increased specificity, but lowered sensitivity. In future work peptide mixes might be optimized by combining peptides with equally good diagnostic sensitivity and specificity. In particular, the sensitivity of mixes is likely increased by continuous search for new peptides with improved diagnostic operating characteristics as substitutes for the least reactive candidates. The behaviour of peptide mixtures applied as antigens in ELISA needs further investigation.
To select peptides with broad recognition by human echinococcosis patients, our bioinformatic selection aimed at exported or parasite surface exposed antigens. Such proteins are likely to elicit an immune response. A major drawback was the scarce number of full-length Echinococcus spp. protein sequences deposited in public databases. Thus it might be worthwhile for further studies to adapt the bioinformatic selection criteria to the use of partial EST sequences. Our data points out the need for screening more peptide candidates. Further investigations on whole genome level are likely to provide additional candidates with sufficient sensitivity and specificity, which can eventually be combined to a synthetic antigen pool. Single or pooled LSPs are compatible with high throughput platform technologies such as luminex or biacore technology that could replace ELISA in the future. Additionally to surface or extracellular localization, one criterion during bioinformatic selection was prediction of secondary structures. Alpha-helical coiled-coils and intrinsically unstructured proteins are believed to adopt native conformation in aqueous solution and might therefore be well suited for immunoassays. It is widely accepted that the majority of epitopes are of the conformational type [42] . Our results showed that for diagnostic purposes the predicted alpha-helical and alpha-helical coiled-coil peptides were superior to peptides deriving from intrinsically unstructured regions. Among the serum-reactive peptides identified by microarray screening, 14 peptides were of predicted alpha-helical organization, while only 3 peptides derived from intrinsically unstructured regions. Furthermore, during our extensive testing of candidates, all IUR peptides were dismissed due to lack of sensitivity.
In addition to the secondary and tertiary structure of a peptide, a further determinant for recognition is the immobilization of peptides to the solid phase used in an immunodiagnostic test. Antibody capture by peptides crucially depends on the accessibility of key residues and therefore the introduction of a spacer molecule to spatially separate the peptide from its carrier protein is vitally important [43] . The peptides in our study were synthesized with an N-terminal AHX-spacer coupled to biotin. The biotin was used to immobilize the peptide antigen to a streptavidin-coated solid phase, i.e. microscope glass slides and 96-well ELISA plates. Coating the biotinylated peptides to non-streptavidin surfaces such as Immulon 2HB (Thermo Scientific) or Poly Sorp (NUNC) ELISA plates, led in those samples tested to discrepancies of duplicate values and mostly reduced A 405nm values, in a non-linear manner (data not shown). Immobilization of peptides by direct adsorbance to the polystyrene surface might lead to sterical blockage of key residues necessary for interaction with antibodies and thus decrease the A 405nm values. Similarly, HIV-1 peptides showed in ELISA increased test sensitivity and specificity when immobilized via biotin-streptavidin [44] . Surfaces functionalized with streptavidin guarantee a directional, highly dense and reproducible coating of biotinylated peptide antigens. It has been shown that the direction of peptide immobilization is of secondary importance as long as peptides deriving from internal protein sequences are investigated [43] . Interactions of internal sequences do not depend on free N-or C-terminus and therefore attachment of spacer and carrier molecules can be achieved successfully at both termini of the peptide.
Our approach represents an alternative to peptide selection by phage display [45, 46] . The bioinformatic selection avoids construction and handling of phage display libraries and panning procedures. The most important limitation to successful panning consists in the lack of selective antibodies. Antibody purification from human sera requires pure and specific antigen, which generally is not yet available. Our bioinformatic approach to peptide selection reduces complex lab work and is compatible with screening on peptide microarray. In our hands, this platform proved highly suitable for investigation of antigen-antibody interaction. Figure S1 Alignment of E. granulosus and E. multilocularis antigenB8/1 sequences with location of peptide D8, D9 and longD8-9.  Protective Efficacy of Cross-Reactive CD8(+) T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8(+) T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP(336–374) peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8(+) T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D(b)NPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D(b), including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8(+) responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8(+) T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A. Virus-specific CD8 + T cells play a critical role in host defence via the production of antiviral cytokines, the direct killing of virusinfected cells and the establishment of immunological memory [1] . The selection of CD8 + T cells into an immune response requires specific interaction between the T cell receptor (TCR) and virus peptide bound to Major Histocompatibility Complex class I (pMHC-I) molecules on the surface of infected host cells. The processing of virus proteins into short fragments generates thousands of peptides that might potentially form pMHC-I epitopes, but only a few elicit CTL responses [2] .
Virus escape mutants are well documented for persistent infections and constitute a major problem for CD8 + T cellmediated control and vaccine design [3, 4, 5, 6, 7, 8, 9] . With regard to the influenza A viruses, mutational changes driven by CD8 + cytotoxic T lymphocytes (CTLs) are unlikely to result in long-term persistence within the individual, as other mechanisms (particularly antibody) can ultimately mediate virus clearance [10] . Even so, the fact that such mutants can be found in nature suggests that influenza virus-specific CTLs are of protective value. Perhaps this reflects that the infection of new subjects favours the selection of mutant viruses that are more slowly controlled (and thus shed for longer), particularly in the face of a seasonal ''bottleneck'' where much of the population is already immune [11] . In humans, influenza escape variants have been observed for CD8 + T cell epitopes presented in context of several HLAs, including HLA-B8, HLA-B27 and HLA-B35 [12, 13, 14, 15, 16, 17, 18, 19] . The immunogenic peptides can be modified at an MHC anchor residue, resulting in defective binding to the MHC-I glycoprotein, or at a TCR contact site. Mutations at TCR contact residues lead to partial (cross-reactive) or total (non-cross-reactive) loss of recognition by wt CD8 + T cells [13] , with some variants eliciting epitopespecific CD8 + T cell responses that are both novel and of substantial magnitude [12] .
Using influenza A virus infection of B6 mice [20] , we showed previously that virus variants with mutations at critical solventexposed residues that are important for TCR binding can generate effective but non-cross-reactive CD8 + CTL responses to what are essentially new epitopes [21, 22] . This raises the possibility that it might be worthwhile to think in terms of vaccinating against likely virus escape mutants. The present analysis focuses on the crossreactive (to wt D b NP 366 ) CD8 + T cell response to the mutant D b NPN3A epitope [22] formed by substitution of the partially solvent exposed, and non-prominent for TCR binding, residue at position (P) 3 of the influenza NP 366-374 peptide [21, 23] . The findings have implications for vaccines to combat virus mutants and tumour escape variants, and suggest that immunisation against likely cross-reactive variants would have to be carefully evaluated to see if the strategy is worthwhile.
Earlier analysis [21, 22] using sequential alanine substitutions in the immunogenic NP 366-374 peptide established that there is some cross-reactive, though diminished, stimulation following the incubation of wt D b NP 366 -specific CD8 + T cells with the NP mutant peptide containing a single asparagine to alanine substitution at position (P) 3 (NPN3A mutant). Such cross-reactive CD8 + T cell responses after NPN3A stimulation are not surprising as the partially solvent exposed P3N residue is unlikely to play any prominent role in contacting the TCR [23] . The wt NP 366-374 peptide binds to H-2D b in an extended conformation where the P2-Ser, P5-Asn and P9-Met are the anchor residues, P3-Asp is a semi-anchor (partially buried/partially exposed), and P4-Glu, P6-Met, P7-Glu and P8-Thr are solvent exposed and available for contact by the TCR (Fig. 1) . This provides the basis for a defined experimental system that can be used to determine what happens when a TCR repertoire is selected to what would seem (from in vitro analysis) to be a suboptimal, cross-reactive mutant epitope. The interpretation of in vitro analysis alone should, however, be tempered with caution, as another mutant (M6A) that did not cross-react at all with the wt epitope elicited a completely novel, in vivo endogenous CTL response of equivalent magnitude when expressed in an infectious influenza A virus [22] . What would be the case for TCR responses elicited by influenza A viruses expressing the mutant NPN3A peptide in the native viral protein?
The NPN3A mutation was engineered into PR8 (H1N1) and HKx31 (H3N2) influenza viruses (PR8NPN3A, HKNPN3A) to allow cross-challenge experiments in the absence of antibody neutralisation. The B6 mice were immunised i.p. with the virulent PR8 mutant and wt viruses, while the HK viruses were used for primary (1 o ) i.n. infection of naïve mice or secondary (2 o ) i.n. challenge of PR8-immune (.30d previously) mice. Naïve (primary) or PR8-immune (PR8, or PR8NPN3A) mice were challenged i.n. with the homologous virus (HK, or HKNPN3A) and CD8 + T cell responses were measured using the D b NP 366 and D b NPN3A tetramers (Fig 2) . It was immediately apparent that the splenic D b NPN3A + CD8 + set elicited by the HKNPN3A challenge was significantly smaller on d10 (p,0.05) than the D b NP 366 + CD8 + T cell response induced by the wt virus (Fig 2B) , though there was no significant difference between D b NPN3A + CD8 + and D b NP 366 + CD8 + T cell numbers at the site of infection (BAL, Fig 2A) . This has been seen before [24] and suggests that the need to clear virus from the lung results in preferential CTL localization to the site of infection when immune T cell numbers are limited. The profile of a diminished D b NPN3A + CD8 + T cell response was maintained into memory (d28, Fig 1C; p,0.02), supporting the view that the relative size of persistent T cell pools reflects the extent of antigen driven proliferation during the acute anti-viral response.
A high proportion of the wt D b NP 366 + CD8 + T cells in BAL (94.0% (1 0 , d10); 93.2 (2 0 , d8); Fig 2A) and spleen (69.8% (1 0 , d10); 93.7% (2 0 , d8); Fig 2B) bound the D b NPN3A tetramer and produced IFN-c when stimulated by the NPN3A peptide (data not Figure 1 . A stick conformation illustrating how the influenza NP 366-374 interacts with H-2D b . NP 366-374 peptide binds to the H-2D b in an extended conformation. The P3-Asp, P5-Asn and P9-Met are the anchor residues, whereas the P4-Glu, P6-Met, P7-Glu and P8-Thr are solvent exposed and available for contact by the TCR. doi:10.1371/journal.ppat.1001039.g001
Introduction of a new influenza strain into human circulation leads to a rapid global spread of the virus due to minimal antibody immunity. Established T-cell immunity towards conserved viral regions provides some protection against influenza and promotes rapid recovery. However, influenza viruses mutate to escape the protective immunity. We found that established T cell immunity can recognise influenza mutants with variations at positions that are partially involved in T cell recognition. However, an initial priming with the mutated variant decreases recognition of the original parental virus. This finding results from a markedly lower functional quality and limited structural interactions of the mutant. In terms of possible vaccination strategies for rapidly changing viruses or tumours, it appears that priming with crossreactive mutants that display such characteristics would be of no benefit as the same level of T cell immunity against such mutants can be elicited by exposure to the original virus.
shown). Similarly, the majority ($90%) of CD8 + T cells elicited by HKNPN3A infection bound the D b NP 366 tetramer and produced IFN-c (data not shown) at levels equivalent to those seen for the response to NPN3A, indicating a high level of cross-reactivity between the D b NP 366 + CD8 + and NPN3A + CD8 + T cell responses. Staining with the D b PA 224 tetramer was included to establish that the NPN3A mutation neither diminished nor enhanced other influenza-specific CD8 + T cell responses (Fig 2) .
Despite the decreased D b NPN3A + CD8 + T cell numbers generated following primary infection (Fig 2A-C) , the recall response was substantial following HKNPN3A challenge of PRNPN3A-immune mice ( Fig 2DE) and the NP.PA immunodominance hierarchy that has long been recognized for secondary responses to wt influenza A viruses in H2 b mice [25] was maintained ( Fig 2DE) . Similarly, the total cell numbers for memory D b PA 224 -specific T cells on d28 were comparable for those primed and boosted within the wt and mutant virus combinations (Fig 2F) , while the D b NPN3A + CD8 + set was 8-fold smaller than the wt D b NP 366 + CD8 + population (p,0.005). Again, the results following secondary challenge support the view that, at least in the earlier (d28) stages of memory, T cell numbers reflect the extent of clonal expansion during the acute phase [26] . In our experiments, we detected D b NP 366 + CD8 + and NPN3A + CD8 + T cells by two techniques, tetramer staining and IFN-c ICS. Both techniques gave us comparable antigen-specific CD8 + T cell numbers, indicating that both tetramers accurately detected epitope-specific populations (Fig. 2GH) .
Given that the NPN3A mutation was associated with a numerically diminished response following infection with either the wt or NPN3A influenza A viruses (Fig 2) , we also asked if there was any effect on CD8 + T cell function or phenotype, particularly for markers (CD62L and CD127) that discriminate between memory T cell subsets [27, 28] . More of the D b NPN3A + CD8 + T cells remained CD62L hi when sampled at the peak of the response (d10) following primary challenge (Fig S1A) , confirming the impression from the quantitative analysis (Fig 2 A-C) that there may be less clonal expansion. On the other hand, IL-7R expression was comparable for the D b NPN3A + CD8 + and D b NP 366 + CD8 + T cell populations generated in virus-infected naïve mice (Fig S1 CD) with both being (p,0.01) lower than the values for the D b PA 224 -specific set ( Fig S1C) . This suggests that IL-7R levels may be antigen dose-rather than magnitude-dependent. Neither of these differential effects was apparent for d28 memory T cells specific for the mutant or wt epitopes (Fig S1BD) . Functional analysis of cytokine production based on short term (5h) stimulation with cognate peptide in the ICS assay showed no obvious differences at any stage for the D b NP 366 and D b NPN3Aspecifc T cells, though the usual divergence [29] from the D b PA 224 + CD8 + T cell response was observed (p,0.01) (Fig S1 E-H) . These data suggest that NPN3A mutation leads to crossreactive, but diminished, CD8 + T cell responses with comparable cytokine production profiles.
Crystal structure and thermostability of the H2Db-NP-N3A complex
Can the smaller response to D b NPN3A be correlated with structural constraints or any decrease in stability for the pMHC-I complex? The D b NPN3A crystal structure containing the heavy chain of H-2D b (residues 1-275), the b2 microglobulin (residues 1-99) and the 9 residues of the NPN3A peptide was determined to a 2.6 Å resolution ( Fig. 3 and Table 1 ), with a final R factor of 22.1% and an R free of 30.4%. The structure of the H-2D b -NPN3A complex was compared (Fig. 3B , Table 1 ) to the wt D b NP 366 [23] . As observed for the wt D b NP 366 , the mutant NPN3A peptide bound H-2D b in an extended conformation, the P2-Ser, P5-Asn and P9-Met represent the anchor residues, P3-Ala semi-anchor residue, whereas the P4-Glu, P6-Met, P7-Glu and P8-Thr are solvent exposed and available for contact by the TCR (Fig. 3AB) . However, the mutation of P3-Asn to Ala leads to a loss of 35 contacts between the peptide and the MHC molecule. In comparison to the wt NP 366 that makes 636 contacts with the H-2D b molecule, the NPN3A peptide achieves only 601 contacts. Interestingly, the Asn3 Ala mutation abolishes contacts between the P3 residue of the peptide with His155 and the Tyr156 of the MHC and eliminates one hydrogen bond between the P3-Asn Nd2 and the P4-Glu O (Fig. 3CD ). The loss of inter-residue bonding between 3N and 4E within the NPN3A peptide may also be important for TCR recognition as it changes the characteristics of the wt D b NP 366 epitope (defined as type III constraint) [30] into an unconstrained D b NPN3A epitope. This change of constraint within the epitope could affect the dynamics of the peptide upon TCR ligation, and subsequently alter TCR reactivity toward the mutated epitope. In addition, the loss of contacts with the His155 of the MHC is of interest, as position 155, termed the ''gate-keeper residue'' [31] is involved in contacting the TCRs in all TCRpMHC-I complexes solved to date [32] . Thus, the lack of interactions between the NPN3A peptide and the MHC through His155 may also affect recognition of the complex by D b NP 366specific TCRs.
The loss of contacts between the peptide and the MHC molecule could lead to decreased stability of the peptide and subsequent changes in NPN3A presentation. To test this hypothesis, we performed a thermostability assay on both NP 366 and NPN3A bound to the H-2D b molecule. The NP 366 and NPN3A peptides are equally effective at stabilising H-2D b . The pMHC-I complex with the NP 366 wt peptide had a Tm of 51.860.7uC and D b NPN3A showed a comparable level of thermostability (Tm = 51.461uC), irrespective of the concentrations of the complex used for the assay. This suggests that the NPN3A mutation does not modify the stability of the pMHC-I complex when compared to the cognate epitope.
Naïve precursor frequency and TCR repertoire for D b NPN3A
Is the smaller D b NPN3A + CD8 + T cell response a consequence of diminished naïve CTL [33] ? We found (Fig 4A) similar naive CTLp counts for D b NPN3A (34.6613.08) and D b NP 366 (28.5611.0), indicating that the smaller response to D b NPN3A + CD8 + is not due to reduced number of naïve precursors. Furthermore, assessing the extent of Vb8.3 bias (the dominant Vb for the D b NP 366 + CD8 + set) within the naïve D b NPN3A + CD8 + population ( Fig 4B) showed that the extent of Vb8.3 usage (mean 13.2%64.3) (Fig. 4B ) was much the same as that determined previously [33] for naïve D b NP 366 + CD8 + CTLps (mean 17.1%67.4) However, sequencing the naïve D b NPN3A + Vb8.3 + CD8 + TCR CDR3b regions showed a clear difference from the comparable wt-specific set. The ''public TCR'' dominance characteristic of D b NP 366 + Vb8.3 + CD8 + T cells in both pre-immune [33] and immune [34] TCRb repertoires [34] was not a prominent feature of the D b NPN3A-specific TCR repertoire. These public TCRs were found in only one (SGGANTGQL and SGGGNTGQL) or two (SGGSNTGQL) of the 10 mice tested (Fig. 4C) . Thus, although the naïve CTLp frequencies are comparable for D b NP 366 + Vb8.3 + CD8 + and D b NPN3A + Vb8.3 + CD8 + T cells and there is some overlap of some cross-reactive TCRs, the two repertoires are far from identical. The roughly equivalent numbers of precursors specific for the wt D b NP 366 and mutant NPN3A peptides were unexpected considering the lower response after infection with the mutated virus. The lower magnitude of the NPN3A + CD8 + T cell response and narrower TCRb repertoire suggest that only a proportion of naïve NPN3A + CD8 + precursors are being recruited into the immune response or that, once recruited, these cells do not expand efficiently. Inefficient recruitment and/or expansion early after influenza infection, despite large naïve CTL precursor numbers, have been recently documented by our group as key determinants of subdominance for D b PB1-F2 62 and K b NS2 114 -specific responses [33] .
The next step was to dissect the immune D b NPN3A + CD8 + CTL repertoire to determine how TCR diversity relates to the size of the immune D b NPN3A + CD8 + T cell response. The D b NPN3Aspecific CD8 + T cells were first analysed for Vb usage by staining with a panel of anti-TCRVb mAbs and the D b NPN3A tetramer. After infection with the NPN3A viruses, the strong Vb8.3 bias characteristic of responding D b NP 366 + CD8 + T cells [34, 35] was prominent for the D b NPN3A + CD8 + sets in only 50% of the immune mice (n = 10). The mutant D b NPN3A + CD8 + T cells utilized a variable spectrum of TCRVb elements, with overrepresentation of Vb 7, 8.1/8.2, 9, 11, and 12 after primary ( Fig  S2A) or Vb6, 7 and 9 following secondary ( Fig S2B) NPN3A virus challenge.
We analysed TCRb clonotypes within the Vb8.3 as (i) it is still a preferred region (29.8%620.2) of NPN3A + CD8 + T cell response) and (ii) clonotypes within this region could be highly relevant for cross-reactive CD8 + T cell responses between NP 366 and NPN3A as they are prominent in both populations. Overall, the mutant and the wt immune Vb8.3 repertoires appear different. Single-cell RT-PCR and sequencing of the CDR3b region of tetramer + Vb8.3 + CD8 + T cells following either HK or HK-NPN3A infections showed that (Table S1) 2), and showed evidence of more variable CDR3b loop lengths (8-9 aa). The reduction (relative to the wt D b NP 366 ) in Vb8.3 usage by the D b NPN3A + CD8 + T cells reproduces the relative loss of ''public'' wt TCRs found for the naïve repertoire ( Fig 4B) . This likely reflects that the N3A mutation has disrupted an ''optimal'' TCR/ pMHC fit that maximizes antigen-driven clonal expansion. Indeed, the public TCRs were not a prominent feature of the D b NPN3A + CD8 + set, being found in only 4 of the 7 NPN3Ainfected mice at a very low frequency, namely 0% in the 1 0 response and 22.9% in the 2 0 response ( Table 2 ). This is in contrast to the D b NP 366 + CD8 + Vb8.3 + TCR repertoire [34] that is largely (90%) comprised of high-frequency, public clonotypes found in all infected B6 mice [34] .
These results also establish that the naïve D b NPN3A + CD8 + TCR repertoire (Fig. 4C) is predictive of the immune D b NPN3A + CD8 + TCRb response ( Table 2 ). The relative lack of a ''public'' response translated to a profile of reduced ''sharing'' between individual mice, and a total increase in the number of D b NPN3A + CD8 + clonotypes due to the 'private' nature of TCRb repertoire for each mouse (Table 2 ). Even so, the clonotypic diversity within individual NPN3A virus-infected mice was reduced compared to the spectrum found following wt virus-infection. This was true whether the cells were sorted using the wt D b NP 366 or the mutant D b NPN3A tetramer, reflecting the significant cross-reactivity between the D b NP 366 and D b NPN3A-specific populations recovered from mice infected with the NPN3A viruses. By the measure of tetramer binding, it thus seems that the NPN3A virus is selecting a less diverse repertoire than the wt virus, with the repertoire being almost completely cross-reactive with that elicited by the wt D b NP 366 epitope.
Similarly, when the D b NP 366 + Vb8.3 + CD8 + T cells induced by wt HK infection were sequenced, the majority of the TCRb clonotypes were detected with both the D b NP 366 and D b NPN3A tetramers (Table 3) . However, a switch in frequency was seen for some CDR3b-defined clonotypes, indicating selective binding of particular TCRs by the D b NPN3A tetramer. For example in M9 and M10 (Table 3) the 'public' SGGANTGQL CDR3b-sequence dominated the D b NP 366 + Vb8.3 + CD8 + T cell population, whereas this sequence was less commonly detected in the same mice by the D b NPN3A tetramer. The difference could, of course, reflect diversity in TCRa usage. Following clonotype selection with the D b NP 366 and D b NPN3A tetramers, sequencing of the secondary TCRb repertoire (M11-M13) induced by challenge with the homologous (PR8, then HK) viruses showed less divergence within the wt D b NP 366 + CD8 + T cell specific population. Interestingly, clonotypes like KGGS-NTGQL were enriched by the D b NPN3A tetramer in some but not other mice (Table 3 ; present in M9 but not M12) suggesting, again, the likely importance of Vb-Va chain pairing for recognition of the native D b NP 366 + CD8 + T cells with the mutant D b NPN3A tetramer. Thus, the analysis suggests that only a subset of the repertoire generated by wt infection is able to recognize the D b NPN3A epitope, though this population is more diverse than that generated in response to the mutant NPN3A virus. All the statistical differences ( Table 2, Table 3 ) were confirmed when the data were standardized to a number of sequences (data not shown).
We further analyzed the D b NPN3A + Vb9 + CD8 + set that was prominent in 2 of the HK-NPN3A secondarily challenged mice. An average of 1.561 TCRb clonotypes was found within this population (Table S2) . Again, the D b NPN3A + Vb9 + CD8 + TCRb repertoire emerged as essentially restricted and private. However, TCRb analysis within other Vbs for NPN3A + CD8 + T cells would need to be performed to compare the whole TCR repertoires.
Analysis of Va chain usage for the mutant D b NPN3A + CD8 + and wt D b NP 366 + CD8 + T cells by PCR with a panel of Va specific primers established that those two T cell responses indeed tend to utilise different Va chains. The wt D b NP 366 + CD8 + T cell population [36] (Day EB, unpublished) tended to use Va8 and Va17.3 (n = 3). Conversely, the D b NPN3A + CD8 + TCRs preferentially expressed Va4, Va5 and Va11 (Table S1 ). While the sample size is small and there are at least 72 different Va chains, these results provide a snapshot of the mutant D b NPN3A + and wt D b NP 366 + populations and suggest that there are differences in both Vb and Va TCR chain usage.
Response characteristics following heterologous challenge and TCR/pMHC-I avidity As there was substantial cross-reactivity in vitro (Fig. 2) for the D b NP 366 and D b NPN3A-specific responses, it was important to determine whether memory T cells that cross-react for the D b NP 366 and D b NPN3A epitopes are preferentially recalled by secondary infection with the heterologous virus. Mice that were primed with the PR8NPN3A and then challenged with either the wt HK or mutant HKNPN3A viruses showed equivalent recall of D b NPN3A + CD8 + T cells during the acute phase of the secondary response. This was detected by IFN-c production ( Fig 5A) and tetramer staining (data not shown) and is consistent with the TCR CDR3b analysis (Table 2) . Conversely, when mice were firstly primed with wt PR8, then later infected i.n. with either the wt HK or mutant HK-NPN3A, the D b NP 366 + CD8 + T cells were differentially recalled indicating that (in the absence of primary selection from the naïve repertoire by the mutant epitope) only some of the D b NP 366 + CD8 + memory T cells that were expanded by heterologous challenge bind D b NPN3A (Fig. 5A) . Interestingly, wt priming and challenge (1 0 PR8-.2 0 X31) resulted in significantly higher CD8 + T cells numbers (Fig. 5B ) than were found for any secondary CD8 + T cell responses after NPN3A priming (1 0 PR8-NPN3A-.2 0 X31-NPN3A and 1 0 PR8-NPN3A-.2 0 X31). These results lead to two main conclusions: (i) priming with the wt virus elicits CD8 + T cells that respond relatively well to a subsequent infection with cross-reactive variant (ie 1 0 PR8-.2 0 X31-NPN3A = 1 0 PR8-NPN3A-.2 0 X31-NPN3A); (ii) priming with the cross-reactive variant can be detrimental as the diminished primary response may limit the full expansion of CD8 + T cells that are able to respond to the subsequent wt infection (ie 1 0 PR8-NPN3A-.2 0 X31 is lower than 1 0 PR8-.2 0 X31) and skew the TCR usage. Thus, using NPN3A for either priming or the challenge gives an equally poor response.
To determine whether such decreased CD8 + responses elicited after heterologous challenge would affect influenza virus clearance, we performed experiments to examine the protective efficacy of cross-reactive CD8 + T cell repertoires. We performed prime-andchallenge studies in mMT mice lacking B cells to ensure that antibody responses did not mask any possible inhibitory effects of ''suboptimal'' TCRs on viral clearance. As suggested by CD8 + T cell data, assessment of lung viral titres showed delayed viral clearance on d6 after the secondary infection in case of heterologous prime-and-boost (PR8-.X31-NPN3A and PR8-NPN3A-.X31) compared to homologous infections (PR8-.X31 or PR8-NPN3A-.X31-NPN3A) (Fig. 5B) . These results indicate that recall of ''suboptimal'' TCRs for a single T cell specificity can lead to delayed viral clearance, despite the presence of other influenza CD8 + T cell responses (D b PA 224 These patterns of complete, or partial, cross-stimulation following in vivo virus challenge were reflected in the results found for in vitro measurements of ''functional avidity'' (Fig 6) . Pulsing immune spleen cells recovered directly ex vivo with graded doses of wt or mutant peptide in the ICS assay showed comparable levels of IFN-c induction (Fig 6AC) in every situation but one, the exposure of wt-primed T cells to the NPN3A peptide ( Fig 6B) . Thus, while the immune repertoire selected by D b NP3A shows evidence of equivalent avidity following stimulation with the NPN3A or NP 366 peptides, D b NP 366 induces a response that is of higher avidity for the wt than the mutant epitope. The same effect was seen even more clearly when three (all expressing the SGGGNTGQL CDR3b) T cell hybridoma lines [37] specific for D b NP 366 were stimulated with the two peptides ( Fig 6DE) . This result is in accord with findings from both the TCR repertoire analysis of crossreactive clonotypes, assessed by tetramer binding (Table 2) , and the response magnitudes determined following homologous and heterologous virus challenge (Fig 5) . Thus, priming and recall of cross-reactive CD8 + T cells recognising mutant viral epitopes reflects functional (defined as responsiveness to a peptide) pMHC-TCR avidity.
To determine the pMHC-I avidity of the responding D b NP 366 + CD8 + and NPN3A + CD8 + T cells, we additionally performed tetramer dilution (Fig. 7A -C) and tetramer dissociation (Fig. 7D-E) assays. While tetramer dissociation assay measured the ''off-rate'' component of pMHC-I avidity, tetramer dilution technique assessed the overall pMHC-I avidity (both the ''on'' and ''off'' rates). Furthermore, we also assessed CD8b-dependence for functional activation (a measure of low avidity CD8 + T cells) of both wt D b NP 366 + CD8 + and the mutant NPN3A + CD8 + T cells by anti-CD8b mAb blocking, followed by IFN-c ICS (Fig. 7G-I) . Our data obtained from those three additional measures of pMHC-I avidity confirmed the results obtained by the peptide titration combined with ICS (functional avidity, Fig. 6 ) and further suggested significantly lower pMHC-I avidity of the wt D b NP 366 + CD8 + (generated by the wt HK infection) for NPN3A variant.
The P3N within the immunodominant influenza virus-specific D b NP 366 epitope is a partially solvent exposed, and non-prominent for TCR binding, residue that is predominantly buried within the MHC cleft [21, 23] . The NPN3A mutation leads to both decreased recruitment of CD8 + T cells and a narrowed clonotype selection profile within Vb8.3 and Vb9 regions. Structurally, the mutation leads to a loss of a number of contacts between the NPN3A peptide and the MHC-I molecule, including a contact with the gate-keeper residue at position 155, and unaltered stability of the H-2D b -NPN3A complex. The fact that the NPN3A mutation affects contacts with the MHC-I at His155, known to play an important role in TCR-pMHC structures in general, is likely to indirectly compromise the TCR recognition. By loosing the bond with the Asn3, His155 may gain more flexibility and thus be inappropriately placed for the subsequent TCR ligation onto the NPN3A peptide. Alternatively, it is also possible that a small part of the solvent-exposed head group of the Asn3 residue in the wt NP 366 peptide might, to some extent, be directly interacting with the TCR following ligation. Surprisingly, despite the loss of several contacts between NPN3A peptide and H-2D b , the stability of the peptide-MHC-I complex remains constant for both NP 366 and NPN3A. This suggests that the Asn3 as a secondary anchor does not play an important role in stabilizing the peptide within the MHC-I. The structural basis for the diminished recruitment of D b NPN3Aspecific CD8 + T cells is thus likely to rest in the way that the partially-solvent exposed residue contacts MHC-I and modifies TCR ligation.
The emerging D b NPN3A + CD8 + T cell population was characterised by different Va and Vb preference, distinct CDR3b sequences and a lower overall TCR diversity in comparison to wt D b NP 366 + CD8 + T cells. These findings suggest that a very limited spectrum of CD8 + T cells can recognise the D b NPN3A mutant structure. Interestingly, a similar P3 substitution in the influenza virus D b PA 224 epitope had no affect on D b PA 224 + CD8 + T cell recognition and recruited a diverse array of TCR clones comparable to the spectrum found for the wt response [21] . This suggests that the partially-exposed P3 residue plays a greater structural and/or TCR recognition role for the ''featureless'' D b NP 366 than for the ''featured'' D b PA 224 complex reflecting, in turn, the more limited spectrum of TCRs that bind D b NP 366 [21] . Taken together, it appears that partially-exposed residues within viral peptides can provide important contacts with the MHC-I, which can in turn cause remote effects that modify antigenicity for the TCR-pMHC-I complex and impact on both TCR repertoire selection and the magnitude of CD8 + T cell responses.
Interestingly, there were no differences in function or phenotype characteristics for the D b NP 366 + CD8 + and NPN3A + CD8 + T cells, although those two CTL sets had a high proportion of different TCR clones. This is in accordance with previous studies showing that the simultaneous production of antiviral cytokines [29] and IL-7R [20] expression is antigen dose-rather than magnitude-related. Conversely, levels of the CD62L [38, 39, 40] activation marker differed for the D b NP 366 + CD8 + and NPN3A + CD8 + populations, indicating that response magnitude has some relationship to the activation status of CD8 + T cells [40, 41, 42] which may, perhaps, reflect the extent of CTL proliferation.
The TCR repertoires specific for D b NP 366 and D b NPN3A appear to be quite distinct. The response overall for wt D b NP 366 + CD8 + T cells is characterised by conserved, ''public'' clonotypes that constitute the majority (83.5% in 1 0 response and 92.3% in 2 0 response) of the selected TCR repertoire [34] . These public clonotypes are not a prominent feature of the D b NPN3A + CD8 + set, being found only in 4/7 NPN3A-infected mice at very low frequency. Since we know that these TCR clonotypes are present in all B6 mice [34] , the difference presumably reflects the lower TCR avidity for D b NPN3A, as indicated by the T cell hybridoma analysis where they were shown to require 1000 times more NPN3A than NP 366 peptide for optimal stimulation. Since the public clonotypes cannot be efficiently recruited into the immune response by the mutated N3A virus, this could have created a ''hole'' in TCRs capable of recognising the mutated epitope, which subsequently can lead to a reduction in T cell immunogenicity [43] . However, though both the D b NP 366 + CD8 + and D b NPN3A + CD8 + T cell responses are characterised by quite distinct TCR repertoires, the majority are bound by both the D b NP 366 and D b NPN3A tetramers and can be detected by stimulation with either the NP 366 or the NPN3A peptides, suggesting that a clonal dissection of TCR clonotypes is needed to make a valid interpretation about the truly crossreactive CD8 + T cell responses. These findings also raise questions concerning the true correlation between pHMC-I tetramer binding in vitro and the in vivo selection of a responding TCR repertoire.
Overall, the results indicate that a loss of a number of contacts between the NPN3A peptide and the MHC-I molecule and lower functional and structural pMHC-I avidity (for wt D b NP 366 ) D b NPN3A selects a narrowed TCR repertoire of ''best fit'' TCRs from a spectrum of naïve clonotypes that, once activated, clonally expanded and engaged in an immune response, have sufficient avidity to be recalled by exposure to the wt D b NP 366 epitope. Conversely, the ''better'' fit D b NP 366 finds a sufficient spectrum of high avidity TCRs within that available naive repertoire and does not (likely because of clonal competition) select most of the TCR ab pairs that interact optimally with D b NPN3A. Priming with the wt virus thus establishes memory for only a very limited secondary response to the mutant. Similar to our results, subtle variations within the anchor residue of H b peptide/I-E k also decreased peptide-MHC class II affinity and the activation of responding T cells [44] .
Thinking about this in terms of possible vaccination strategies for use against rapidly changing viruses or tumor epitopes, it appears that priming with cross-reactive mutants that have characteristics comparable to NPN3A would be of no benefit (or even could be detrimental as evidenced by delayed viral clearance) as the same level of T cell immunity against such mutants can be elicited by exposure to the wt epitope. On the other hand, changes like the non-cross-reactive NP-M6A mutation [22] that induce a completely novel, high quality response might merit incorporation in an experimental vaccine or immunotherapy strategy. Overall, working out the topographical constraints that determine these differential response profiles would seem eminently worthwhile.
A further reason for defining the structural rules governing TCR cross-recognition is that similar effects have been found for different epitopes derived from unrelated viruses. Published studies provide evidence for cross-reactive CD8 + T cell responses between influenza A virus and Epstein-Barr virus (EBV) [45] , influenza A virus and Hepatitis C virus (HCV) [46, 47] or lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV) [48] . Such heterologous cross-reactive immunity can unintentionally skew TCR recruitment, result in a narrow TCR repertoire and subsequent viral escape [48] as well as influenza disease severity [22] . doi:10.1371/journal.ppat.1001039.g006 [45] . The topic of TCR cross-reactivity in CD8 + T cell responses clearly merits more attention.
All animal experimentation was conducted following the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the University of Melbourne Animal Ethics Experimentation Committee in Melbourne. -NPN3A) influenza A viruses, in 500ml of PBS. Viruses share the same internal components for NP and PA from which CD8 epitopes are derived [49] . Virus stocks were grown in the allantoic cavity of 10 day old embryonated chicken eggs, from which the viral titre was determined by plaque assay on monolayers of Madin derby canine kidney (MDCK) cells.
Recombinant influenza viruses with the single amino acid substitution (N3A) within the NP 366 peptide, ASNENMETM, were generated using the eight-plasmid reverse genetics system [50] . The substitution was first incorporated by site directed mutagenesis using PCR primers encoding N3A 366 peptide, ASAENMETM, and the opposite primer encoding NP. Recombinant PCR products encoding N3A 366 were digested with BsmB1 and ligated into the alkaline phosphatase treated pHW2000 vector. Recombinant viruses (HK-NPN3A and PR8-NPN3A) were rescued following transfection of MDCK-293T cell coculture with the eight plasmids encoding influenza segments. Viruses were then amplified in the allantoic cavity of 10-day old embryonated chicken eggs, and the viral titre determined by plaque assay of allantoic fluid infecting monolayers of MDCK cells. There was any evidence of altered fitness in vivo for the HKNPN3A virus, as the kinetics of virus growth and clearance following i.n. challenge of naïve B6 mice were found to be equivalent for the wt HK and mutant viruses (Fig S3A) . Similarly, the levels of CTL activity found using target cells infected with HKNPN3A or the HK wt viruses were comparable, and the same was seen for peptide pulsed cells, suggesting that antigen presentation of NPN3A peptide remains constant (Fig S3 BC) .
Lungs taken from mice after primary viral infection (Fig. S3A) or prime-and-boost approach using homologous (PR8-.X31 and PR8-NPN3A-.X31-NPN3A) or heterologous (PR8-.X31-NPN3A and PR8-NPN3A-.X31) strategy (Fig. 5B) were homogenised and the virus-containing supernatant above the cell debris was harvested and stored at 270uC. Titres of infectious virus in the lung supernatants were determined by plaque assay on monolayers of MDCK cells.
Spleen and bronchoalveolar lavage (BAL) samples were recovered from mice at acute phases of the primary and secondary infections (d10 and d8 respectively), and the BAL samples were incubated on plastic petri-dishes for 1hr at 37 0 C to remove macrophages. The spleens were disrupted and enriched for CD8 + T cells using goat anti-mouse IgG and IgM antibodies (Jackson ImmunoResearch Labs, West Grove, PA, USA). For assessment of naïve precursor frequency of N3A 366 + CD8 + T cells, spleens and lymph nodes (inguinal, brachial, axillary, superficial cervical, and mesenteric) were collected from naïve mice and processed to single-cell suspensions.
Tetramer and phenotypic staining of CD8 + T cells Lymphocytes from the BAL and spleen were stained with tetramers conjugated to Strepavidin-APC or PE (Molecular Probes, Eugene, OR, USA) at optimal staining concentrations (10 mg/ml D b NP 366 , 40 mg/ml D b N3A 366 , and 10 mg/ml D b PA 224 tetramers) for 1hr at room temperature. Cells were washed twice in FACS buffer, and stained with 1 mg/ml CD8-PerCP Cy5.5, 5 mg/ml CD62L-FITC and 5 mg/ml CD127-APC mAbs (BD Biosciences) for 30 mins on ice, washed twice and analysed by flow cytometry on the FACS Calibur (BD Immunocytometry) and analysed by CellQuest Pro software (BD Immunocytometry). We titrated all the batches of all the tetramers used in this study. We used tetramers at optimal concentrations (10-40mg/ml) based on both the percentage of epitope-specific CD8 + T cells and the mean fluorescence intensity (MFI) of tetramer staining. A Scatchard analysis [51] based on the tetramer dilution assay (Fig. 7 A-C) was also plotted (Fig. S4 ) and confirmed our observations from routine tetramer titrations that the D b NP 366 tetramer displays slightly superior pMHC binding capacities over the NPN3A tetramer at a concentration ,5mg/ml.
CD8 + T cells from spleen were stained with the D b NP 366 and D b -NPN3A tetramers conjugated to Streptavidin-PE (Molecular Probes, Eugene, OR) for 60mins at room temperature. For a tetramer dilution assay, 2-fold dilutions of PE-conjugated tetramers were used at a range of concentrations (0.15-40mg/ ml). For a tetramer dissociation assay, lymphocytes were stained at the optimal concentration of PE-conjugated tetramers as assessed by tetramer titration as determined by both the percentage of tetramer + CD8 + T cells and mean fluorescence intensity (MFI). Cells were washed twice in FACS buffer (10%BSA/0.02% NaAz in PBS), stained with a FITC-conjugated mAb to CD8a (BD Biosciences Pharmingen) for 30mins on ice, washed and analysed by flow cytometry. As a measure of pMHC avidity, splenic T cells were used in tetramer dissociation assay [29] . After staining with tetramer, T cells were washed and incubated in the presence of anti-H2D b antibody at 5mg/ml at 37 0 C to prevent tetramer rebinding. Cells were removed at intervals, stained with the FITCconjugated mAb to CD8a and analysed by flow cytometry. Loss of tetramer + CD8 + T cells at particular time-points was calculated in comparison to tetramer staining at t = 0mins.
Enriched T cell populations from spleen and BAL were stimulated with one of the NP 366 , N3A 366 , PA 224 or PB1 703 peptides (AusPep) for 5 hrs at 37uC, 5% CO 2 in the presence of 1mg/ml Golgi-Plug (BD Biosciences Pharmingen) and 10U/ml recombinant human IL-2 (Roche, Germany) (BD Biosciences). Cells were washed twice with FACS buffer, stained with CD8-PerCP Cy5.5 for 30 mins on ice, fixed, permeabilised and stained with anti-IFN-c-FITC (5mg/ml), TNF-a-APC (2mg/ml), and IL-2-PE (2mg/ml) mAbs (Biolegend). Samples were acquired using flow cytometry, and the total cytokine production calculated by subtracting background fluorescence using no peptide controls. In selected experiments, lymphocytes were stimulated with varying concentrations of peptides, three-fold dilutions ranging from 300nM to 0.0008nM to determine the sensitivity specific peptides, defined as 'functional avidity' [52] .
Splenocytes were obtained from mice sampled on d6 after secondary infection. Lymphocytes were pre-cultured in the presence or absence of anti-CD8b antibody (53.5-8) (10 mg/ml). Cells were then stimulated for 5 h with peptide, IL-2 and GolgiStop also in the presence or absence of anti-CD8b antibody (5 mg/ml). Following stimulation, cells were analysed for CD8 and IFNc expression. Shown is the percentage of CD8 + cells producing IFN-c after stimulation in the presence of anti-CD8b blocking mAb.
Determination of N3A 366 + CD8 + T cell precursor frequency Naïve N3A 366 -specific CD8 + T cells were identified as described [33, 53] . Briefly, processed lymph nodes and spleen samples were resuspended in 100 ml of Sorter buffer, 100 ml FcR block (24G2 and CD16 culture supernatant, 1% mouse and 1% rat serum) was added, and clumps of dead cells were discarded. Tetramers at optimal staining concentrations (D b N3A 366 -PE at 40mg/ml and D b NP 366 -PE at 10mg/ml) were added to the cell mix and incubated for 1 hour at room temperature in the dark. Cells were washed and resuspended in 400 ml buffer with 100 ml anti-PE microbeads (Miltenyi Biotec), and incubated at 4uC for 25 mins. Following two washes, cells were resuspended in 3 ml of buffer and cells that had bound the microbeads were purified on a magnetic LS column according to manufacturers instructions (Miltenyi Biotec). Cells eluted from the column were centrifuged (5156g, 6 min, 4uC), supernatant carefully aspirated to leave 90 ml buffer remaining, and 10 ml antibody cocktail was added for 30 min at 4uC. The antibody cocktail contained anti-CD8-APC Cy7 (Pharmingen, BD), anti-CD4-PE Cy7 (eBiosciences), anti-B220-FITC (Pharmingen, BD), anti-CD11b-FITC (eBiosciences), anti-CD11c-FITC (eBiosciences), anti-F4/80-FITC (eBiosciences), anti-CD62L-APC (Pharmingen, BD), and anti-CD3-PerCP Cy5.5 (Pharmingen, BD) mAbs. Cells were washed in 2 ml buffer, centrifuged (5156g, 6 min, 4uC), and supernatant aspirated leaving 100 ml. Resuspended cells were passed through 45 mm sieve, and data acquired by flow cytometry on the LSR II (Becton Dickinson) and analysed with FlowJo (Treestar Inc.) software. In selected experiments, naïve NPN3A + Vb8.3 + CD8 + T cells were single-cell sorted for TCRb analysis. Experimental details of the single cell RT-PCR designed to amplify naive NPN3A + Vb8.3 + CD8 + T cells using 2 different sorting strategies are listed in Table S3 .
LacZ-inducible T cell hybridomas specific for NP 366 peptide [37, 54] were resuspended at 1610 6 cell/mL and aliquots (100ml) and dispensed into 96-well flat-bottom plates together with 5610 5 naïve splenocytes (APCs). Cells were cultured in the presence of NP 366 or NPN3A peptides at concentrations ranging from 10 215 M to 10 24 M for 18h at 37 0 C. The cells were then washed with PBS, fixed with 100ml of 2% formaldehyde/0.2% glutaraldehyde in PBS for 5min on ice, washed in PBS and incubated with 2.5mg of 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-gal) for 16h at 37 0 C. The LacZ + hybridomas were then counted using a light microscope, and the number of NP 366 -specific hybridomas was calculated by subtracting the ''background'' LacZ expression for cells cultured in the absence of the peptide.
Splenocytes were stained with 10 mg/ml D b NP 366 or 40 mg/ml D b N3A 366 -PE tetramer in sort buffer (PBS with 0.1% BSA) for 60 mins at room temperature, washed and stained with 1 mg/ml anti-CD8-Allophycocyanin and 10 mg/ml of either anti-Vb 8.3 or anti-Vb9 for 30 mins on ice, washed twice with sort buffer. Single lymphocytes were isolated by sorting with a FACS Aria (BD Immunocytometry), into 80 wells of an empty 96 well twintec plate (Eppendorf). mRNA transcripts were reversed transcribed to cDNA, using a Sensiscript kit (Qiagen) according to manufacturers instructions, and the CDR3b region amplified by a nested hot start PCR using Vb primers [34] . Positive PCR products were purified using Qiagen PCR purification kit and sequenced.
Protein expression, purification, crystallisation and structure determination H2-D b and b2-microglobulin molecules were expressed in Escherichia Coli as inclusion bodies, refolded with the NP-N3A (ASAENMETM) peptide and purified as previously described [55, 56] . The H2D b NP-N3A complex crystals were obtained at 3mg/ml by the hanging-drop vapour diffusion technique at 20uC. Crystals were grown with a reservoir containing 0.1 M sodium citrate at pH 5.7, 28% PEG 3350 (w/v), 0.2 M lithium sulphate. The crystals belong to space group I222 and the unit cell dimensions were consistent with one molecule per asymmetric units (Table S1 ).
The crystals were flash frozen to a temperature of 100K before data collection in-house with a Rigaku RU-200 rotating-anode Xray generator. The data were processed and scaled with the XDS [57] . The crystal structure was solved using the molecular replacement method using the program Phaser [58] from the CCP4 suite of programs [59] . The search probe used to solve the structure was the structure of mouse MHC class I H2-D b minus the peptide (Protein Data Bank accession number 3CPL) [22] . The progress of refinement was monitored by the R free value with neither a sigma nor a low-resolution cutoff being applied to the data. The refinement protocol used includes several cycles of refinement with REFMAC [59] followed by manual model rebuilding with O program [60] . 'Translation, liberation and screw-rotation' displacement refinement was used to model anisotropic displacements of defined domains was used during the refinement process. The electron density around the NPN3A peptide was unambiguous, and all the side chains were built at full occupancy. Some mobile loops in the heavy chain of the H-2D b molecule (residues 191-201; 220-228 and 247-254) have been removed from the final model due to missing electronic density. Final refinement statistics are summarized in Table I , the coordinates of the H2Db-NP-N3A complex have been deposited with the Protein Data Bank under accession numbers 3FTG.
Thermostability measurements of recombinant class I complexes using circular dichroism (CD) Circular Dichroism Spectra were measured on a Jasco 815 spectropolarimeter using a thermostatically controlled cuvette. A far-UV spectra was collected from 190nm to 250nm. The UV minimum was determined as 219 nm for H2Db-NP-N3A. The measurements for the thermal melting experiments was made at the minimum for H2Db-NP-N3A, at intervals of 0.1uC at a rate of 1uC/min from 20uC to 90uC. The Jasco Spectra Manager software was used to view and smooth the traces and then the GraphPad Prism software was used to plot Temperature versus % unfolded. The midpoint of thermal denaturation (Tm) for each protein was determined as the point at which 50% unfolding was achieved. The measurements were done in duplicate at two concentrations (4mM and 2.2mM) in a solution of 10mM Tris pH 8, 150mM NaCl.
The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3FTG).  Development of a Symptom Score for Clinical Studies to Identify Children With a Documented Viral Upper Respiratory Tract Infection The objective of this study was to develop a symptom scoring system for use in clinical studies that differentiates children with cold symptoms who have an identifiable viral etiology for their upper respiratory tract infection (URI) from those in whom no virus is detected. Nasal swabs for PCR testing for identification of respiratory viruses were obtained on children aged 2–11 y at baseline and when parents thought their child was developing a cold. Parental-recorded severity of specific symptoms in children with and without a documented viral URI were compared. Nasal swabs were obtained on 108 children whose parents reported their child was developing a cold. A viral etiology was identified in 62 of 108 (57.4%) samples. Symptom measures that best differentiated children with a viral etiology from those without were significant runny nose and significant cough on days 1–4 of the illness. A URI symptom score was developed based on these symptoms, with a sensitivity of 81.4%, specificity of 61.9%, and accuracy of 73.3%. Parental impression is only a moderately accurate predictor of viral URI in children. Our URI symptom score provided a more accurate method for identifying children with viral URIs for clinical studies. T he common cold is a ubiquitous feature of childhood. The average child develops up to 6 -8 colds per year, each lasting 7-9 d (1). Cold symptoms are an extraordinarily common reason for health care utilization in pediatric patients (2, 3) . Beginning in the 1950s, investigators discovered rhinovirus and other viruses that cause the common cold (4, 5) . Subsequently, the term "viral upper respiratory tract infection" (viral URI) has become virtually synonymous with "cold" for describing the illness.
In many settings, differentiating a viral URI from other respiratory illnesses may be of trivial importance. However, for epidemiologic studies of URI or randomized controlled trials of various cold remedies, it may be critically important to more accurately differentiate patients with an actual viral infection from those with other respiratory conditions. The "gold standard" for diagnosing a viral URI, PCR (6, 7) , is expensive and requires access to sophisticated laboratory facilities, which limit its applicability for large clinical or epi-demiologic studies. Thus, other methodologies for identifying patients with viral URI may be more practical.
Over a half century ago, Jackson et al. (8) developed a symptom scoring system to differentiate adults with viral URIs from other conditions. For that study, adult volunteers were randomized to have either saline solution or saline solution containing filtered nasal secretions from individuals with "typical colds" instilled in their noses. For the next 14 d, the study participants rated the severity of 12 symptoms of URI on a 0 -3 scale. Those who received the filtered nasal secretions had significantly more severe symptoms than those receiving saline alone; a total symptom severity score of Ն14 was found to be the best cut-off value for defining a URI. This symptom scoring system, commonly referred to as the "Jackson score," continues to be used in studies on URIs in adults (9) . Because the presence of some of the symptoms in the score, such as chilliness and myalgia, may be difficult to detect in preverbal children, the use of the Jackson score in pediatric studies may be limited. Other investigators have characterized symptom patterns in children and adults with known viral URIs (6, 10, 11) . There has been little effort to differentiate symptoms among pediatric patients with a documented viral URI from those of children who have some features of a URI but in whom no virus is detected. Thus, there is a need for a validated symptom checklist, or prediction rule, that accurately predicts the presence of a community-acquired viral URI in children enrolled in clinical studies.
As a precursor to a planned randomized controlled trial of the efficacy of Echinacea purpurea in preventing viral URIs in pediatric patients, we collected respiratory samples using nasal swabs on children with a clinical cold for PCR testing against respiratory viruses. Parents of these children completed comprehensive symptom diaries during the episode. Our goal was to identify a set of symptoms that could be used to develop a prediction rule that differentiated children with a viral URI from those with negative viral testing.
A prospective observational study was conducted by the Puget Sound Pediatric Research Network (PSPRN) and Bastyr University. PSPRN is a regional practice-based research network of community pediatric practices in the Puget Sound area; for this project, seven offices participated. Bastyr University is an academic alternative medical school located in Kenmore, WA. At PSPRN practices, participating providers discussed the study with parents of potentially eligible children; if the parent was interested, contact information was sent to the research assistants who explained the study in detail and completed the enrollment procedures. Bastyr University used a generalized recruitment strategy to enroll patients by advertisements.
Study participants. Eligible children were aged 2-11 y, in good health, and with no history of chronic lung disease. Eligibility was based on the inclusion/ exclusion criteria planned for a subsequent randomized trial of Echinacea. Thus, patients with a history of asthma, allergic rhinitis, autoimmune disease, or allergy to Echinacea or related species were not enrolled. Each participant was in the study until she/he developed a cold or completed a 120-d observation period without developing cold symptoms. Study data were collected from November 2007 to May 2008. The study was approved by the Seattle Children's Hospital Institutional Review Board and the Bastyr University Institutional Review Board. Written informed consent was obtained from the parents of study children; written assent was obtained from children aged 7 y and older.
Study design. At enrollment, parents completed a study form that included demographic information, use of daycare Ͼ 20 h/wk, school attendance, and exposure to cigarette smokers. In addition, a baseline nasal swab for PCR testing was collected. For patients with URI symptoms at the baseline visit, the baseline swab collection was rescheduled at least 14 d later. At enrollment, parents were given a cold symptom diary and asked to notify the research team as soon as they thought that their child was developing a cold.
Research assistants telephoned parents of study patients every 14 -17 d to remind them of the study procedures and ask about any cold symptoms in the child. If the parent indicated that the child had developed a cold, but that he/she had not contacted the study team, the patient was considered to have a "missed" cold and excluded from further analysis.
Parents notified a research assistant when they believed their child was developing a cold. The research assistant specifically inquired about the presence of four respiratory symptoms: runny nose, nasal congestion, cough, and sneezing. If the research assistant confirmed the child had one or more symptoms, he/she was considered to have a clinical cold, and arrangements were made to collect an "acute" nasal swab for PCR testing within 48 h.
Once the research assistant determined that a study child had met criteria for a clinical cold, the parent was asked to begin completing the daily symptom diary. The scoring system used in the diary was based on the Jackson score and other cold severity rating systems and included measures for 12 separate symptoms for each day of the cold, for up to 14 d (8,10,11). Symptoms included sneezing, runny nose, nasal congestion, cough, fever, headache, malaise, chilliness, scratchy throat, sore throat, hoarseness, and myalgias. Each morning, for as long as symptoms persisted, parents were asked to rate the severity of each of these symptoms using the scale: 0 ϭ none, 1 ϭ mild, 2 ϭ moderate, and 3 ϭ severe. An additional possible response was "can't tell," which was added because it was felt that it would be difficult for parents of young children to rate the presence and severity of some of these symptoms. In an attempt to standardize the reporting, a guide for rating severity of symptoms including examples of symptoms warranting a particular severity was provided to all parents of study children. The guide for rating symptoms is shown in Table 1 .
Initially, we planned to include data from only the first 3 d of recorded symptoms to develop a prediction rule for identifying a viral URI in a child. Ultimately, data from the first 4 d of symptoms were used to develop the prediction rule because inclusion of information from day 4 on symptoms improved the precision of the rule. Inclusion of data after day 4 did not increase the accuracy of the prediction rule.
Sample collection and testing. A deep nasal swab, obtained by a small flexible nylon flocked swab (Copan Diagnostics, Corona, CA) inserted approximately one half the distance between the nares and bridge of the nose, was used for all sample collections. After collecting the specimen, the swab was rinsed vigorously in 0.5 mL of lysis buffer. Samples were stored at room temperature until testing (12) . Total nucleic acid was extracted from 200 L of swab specimen in lysis buffer as described previously (13) . One thousand copies/reaction of an external control was added to identify false-negative PCR results (14) . Nasal swab samples were tested by quantitative PCR for respiratory syncytial virus A and B, human metapneumovirus (hMPV), influenza A and B, parainfluenza types 1, 2, 3, and 4, adenovirus, rhinovirus, and coronavirus (subtypes 229E, HKU1, NL63, and OC43) (7, (13) (14) (15) (16) .
Nasal swabs were also tested for bocavirus. During the course of the study, evidence emerged that bocavirus does not seem to be an etiologic agent of viral URIs in children older than 2 y (17, 18) . Therefore, we did not include bocavirus in our analysis.
A study patient was classified as having a viral URI if one or more viruses were detected that had not been found in the child at the baseline swab. If the same virus was detected at the baseline and acute swab, the child was classified as having a viral URI only if at least a 2 log increase in viral load was present in the acute swab.
Data analysis. An iterative process was used to develop the prediction rule for accurately classifying whether a study child had a viral URI (19, 20) . Ratings for each measured symptom for the first 4 d of the illness were coded into two variables. The first measure of each symptom was whether it was present, corresponding to scores of 0 versus scores of 1-3. The second measure was whether the symptom was present and of "significant severity," corresponding to scores of 0 -1 versus 2-3. With this procedure, 96 different variables defining the presence or absence of symptoms were created (12 symptoms ϫ 2 measures/symptom ϫ 4 d of assessment). The rate of presence of each symptom measure in children with a PCR confirmed viral URI and those with a negative PCR ("no URI") was then compared using 2 tests. Those symptom measures for which rates in the two groups (viral URI or no URI) were different (p Ͻ 0.20) were classified as candidate symptom measures and included in the next step. For symptoms in which both severity measures (e.g., any runny nose and significant runny nose) were different in patients with and without viral URI, only the measure with the more significant difference was included. In addition, candidate symptom measures in which greater than 10% of parents indicated "can't tell" regarding the presence of the symptom were excluded.
Stepwise forward logistic regression was used to identify combinations of symptom measures that differentiated children with and without viral URIs. The final regression model included all candidate symptom measures associated with the outcome of viral URI (p Ͻ 0.30). To develop a "URI symptom score," 1 point was given for the presence of each of the identified symptom measures; the total URI symptom score in a given patient was the sum of these points. Receiver-operating-characteristic (ROC) analysis was done to choose the cut-off score that maximized the accuracy of the cold score in predicting the presence of a viral URI. "Accuracy" was defined as the proportion of children with a viral URI with a "positive" URI symptom score plus the proportion with no URI with a negative score.
Before collecting study data, a decision was made to develop a URI symptom score measure that had "sensibility." This term is used to describe a decision rule that has face validity to clinicians caring for children (19, 20) . For example, if a symptom measure on days 2 and 4 of illness was associated with the outcome of a viral URI in the regression model, it would be sensible to also include scores for that symptom on days 1 and 3. Thus, additional models for the URI symptom score were developed by including symptom measures that we judged to increase the sensibility of the prediction rule. Alternative models of the score were developed in which one or more symptom measures were removed until the final model for the URI symptom score with the highest accuracy in differentiating children with and without a viral URI was derived. A final model, using the Jackson score, (8) was also constructed. For the Jackson score, the sum of the severity of eight symptom measures for 6 d is calculated; a score Ն14 is considered as indicative of a viral URI.
After determining the symptom measures to be used in calculating each model of the URI symptom score, ROC analysis was done to determine the optimal cut-off value. Based on this cut-off value, the sensitivity, specificity, positive predictive value (PPV), negative predictive value, and accuracy of the model in predicting a viral URI were calculated; 95% confidence interval (CI) around the point estimates were also calculated. The sensitivity of the final URI symptom score in predicting the presence of viral URIs caused by rhinoviruses (versus no URI) was determined. A similar analysis was conducted for viral URIs caused by viruses other than rhinovirus.
A total of 150 children were enrolled in the study including 126 by PSPRN pediatricians and 24 by the research team at Bastyr University. Disposition of study patients is summarized in Figure 1 . Acute nasal swabs for PCR analysis were obtained in 108 patients whose parents thought that their child was developing a cold and had at least one cold symptom. The rest of the analysis is focused on the 108 children in whom an acute nasal swab was obtained. Demographic characteristics for these 108 children are summarized in Table 2 . No differences for any characteristics between children who had an acute nasal swab and those who did not were noted. There were also no statistically significant differences in demographic characteristics between the 108 children in whom acute swabs were obtained and the eight participants who had a "missed" cold.
No virus was detected at baseline in 87 of the 108 children (80.6%) who had a nasal swab collected for an acute illness. Viruses detected at baseline in the remaining 21 children are shown in Table 3 . Five patients had rhinovirus detected at both baseline and with the acute nasal swab. In one child, there was Ͼ2 log increase in viral load from baseline to the acute swab; this patient was classified as having a viral URI. The other four patients had no substantial increase in rhinovirus load and were categorized as having no URI. In all other "positive" acute swabs, the virus detected had not been present at baseline. Overall, a respiratory viral etiology was identified in 62 of the 108 children (57.4%, 95% CI: 47.5-66.9%) considered to be developing a cold with at least one respiratory symptom (Table 3) . Rhinovirus was the most common etiologic agent, accounting for 49.2% of all viruses detected.
Completed symptom logs were returned by parents of 101 study children (93.5%). In addition, information from symptom logs was also collected by research assistants when acute swabs were obtained. Data on symptom severity were obtained on all 108 study children on day 1 of their cold The presence of 96 separate symptom measures in study children were compared. Twenty-one symptom measures met criteria for inclusion as candidate symptom measures (Table 4). Differences in the rates of significant runny nose and significant cough were present during the first 4 d of the illness. The most significant difference between the two groups was in the rate of significant runny nose on day 4 (50.9% in those with viral URI and 9.5% in those with no URI, p Ͻ 0.001). Although children with a viral URI were less likely to have any headache or significant headache on day 1 than those with no URI (p ϭ 0.035 and 0.048, respectively), parents of 14 children (age, 2.2-5.2 y) indicated they could not tell whether their child had this symptom. Two parents did not respond to this item on the symptom log, leading to missing headache data in 14.8% of study children. Similarly, 13 parents (12.3%) indicated they could not tell whether their child had a scratchy throat (or sore throat) on day 2. Because of the amount of missing data, neither headache nor scratchy throat was considered as candidate symptom measures.
All candidate variables were entered into stepwise forward logistic analysis; the following symptom measures were included in the final model: significant runny nose, day 1; significant cough, day 4; significant runny nose, day 4; and any fever, day 3. Based on these symptoms, three models for a URI symptom score were developed. For each model, a score was derived by assigning one point for the presence of each symptom included in the measure. ROC analysis was then used to determine the cut-off score that maximized the accuracy of the model. The properties of each model as a URI symptom score are summarized in Table 5 . Model 1 included only the symptom measures included in the final forward regression analysis. For model 2, in which the "sensibility" of the prediction rule was improved, symptom measures for significant cough on days 1-3, significant runny nose on days 2 and 3, and any fever on days 1-2 and day 4 were included. Using systematic removal of different measures, the inclusion * Number of children with data for the symptom measure and excluding those for whom no data were available for that day, those whose parents did not rate the severity of the symptom, or whose parents indicated "can't tell" for the severity of the symptom. * One point was given for the presence of each symptom measure present and points summed for each child. The cut-off value was the score that best differentiated children with and without a viral URI.
† Model 1 included the following symptom measures: significant runny nose day 1, significant runny nose day 4, significant cough day 4, and any fever day 3. ‡ Model 2 included the following symptom measures: significant runny nose days 1-4, significant cough days 1-4, and any fever days 1-4. § Model 3 included the following symptom measures: significant runny nose days 1-4 and significant cough days 1-4. Model 4 is based on the sum of the severity (rated 0 -3) for the following symptoms for 6 days: sneezing, headache, chilliness, malaise, runny nose, nasal congestion, cough, and sore throat (Jackson score) 8. of any or all the fever symptom measures tended to decrease the precision of the prediction rule. Model 3, which included the symptom measures of significant runny nose on days 1-4 and significant cough on days 1-4, was the most accurate model for differentiating children with a viral URI from those with no URI and was chosen as the final URI symptom score. Model 4 was based on the Jackson score. However, because many parents responded "can't tell" for severity of certain symptoms, the score was calculated on only 65 children.
The overall sensitivity of the final URI symptom score was 81.4%, the specificity was 61.9%, and the accuracy was 73.3%. The sensitivity of the URI symptom score for viral URIs caused by rhinovirus was 76.7% (95% CI: 57.7-90.0%) and 86.2% (95% CI: 68.3-96.1%) for those caused by other viruses (p ϭ 0.35).
In this study, nasal samples from children with clinical colds were collected to detect common respiratory viruses associated with viral URIs in pediatric patients. PCR techniques were used to identify viruses, a technique more sensitive than other detection or culture methods (6, 7) . Because children may have prolonged shedding with some respiratory viruses, particularly rhinovirus, in the absence of symptoms (21, 22) , we collected specimens on study participants both when they were well and when they were symptomatic. A child was classified as having a viral URI when either a new virus was isolated or a significant increase in viral load of a previously identified virus was detected in the presence of acute symptoms. With these techniques, we identified a virus in 57.4% of children whose parents thought their child was developing a cold and had at least one respiratory symptom.
Although it may seem surprising that a viral etiology was not identified in Ͼ40% of children with cold symptoms using the most sensitive molecular techniques currently available, the definition of a "cold" is actually somewhat nebulous (9) . In one study of 215 adults who reported they were developing a cold, only 54% had at least one respiratory symptom and only 17% met Jackson criteria for a cold (23) . Thus, the use of a "clinical cold" as the entry criterion for clinical trials on the efficacy of a potential viral URI treatment includes participants with heterogeneous clinical conditions and may bias findings toward the null if the therapy being tested is specifically acting by an antiviral mechanism. If a symptom scoring system could more reliably predict a viral URI, analysis of data from clinical studies could be conducted to assess efficacy in the subset of patients with a probable viral etiology. This approach would have the benefit of decreasing heterogeneity of the sample and eliminating the need for expensive laboratory testing to confirm viral etiology in clinical studies.
Viral URIs may be more difficult to identify in young children, who cannot articulate the presence of many symptoms. In our study, greater than 10% of parents indicated they could not tell whether their child had a headache or sore throat, both components of the Jackson score for defining a cold (8) . Furthermore, the diagnostic properties in patients in whom the Jackson score could be calculated were not as good as our URI symptom score. This confirms our assertion that the Jackson score is not appropriate for clinical trials in children, and a new symptom scoring system is needed to identify viral URIs in pediatric patients.
With the use of our prediction rule, the PPV for identifying a child with a documented viral URI (75%) was similar to that found in other studies in both pediatric patients and adults using a variety of detection methods (6, 24, 25) . In these previous studies, the estimated PPV may actually have been somewhat inflated because there was no baseline viral screening. Thus, in prior studies, a proportion of the viruses detected could potentially be related to prolonged shedding rather than as the cause of symptoms (26) . In our study, 8.7% of viruses detected during an acute episode had been present at baseline without a significant increase in viral load.
Some study children may have been infected by a virus that is yet to be discovered leading to a false-negative test. It is also possible that some false-negative test results were caused by the technique used to obtain nasal secretions, although every effort was made to collect a proper sample. We may have detected more viruses if both culture and PCR testing had been used, although the sensitivity of PCR is known to exceed that of culture in most studies. For example, in one study, PCR tests consistently and substantially increased detection of multiple respiratory viruses compared with culture, and, in our laboratory, the use of PCR detected at least one respiratory virus in 53.4% of subjects compared with 38.3% who had virus detected by the use of direct immunofluorescence (7, 27) .
One limitation of this study is that the diagnostic utility of our URI symptom score may be different in another population of children (28) . It is also possible that the utility of the symptom score would be different at another time when the distribution of circulating respiratory viruses is different. Subsequent investigation is needed to validate the URI symptom score that we developed. However, we purposefully incorporated a number of features into the development of the score that were designed to maximize its generalizability. First, rather than including symptom measures based solely on a statistical analysis, we modified the URI symptom score to have clinical "sensibility." The precision of the URI symptom score could have been increased in this particular population by weighting some symptoms (29) . We chose the more conservative approach of giving equal weight to relevant symptoms to minimize the effects of statistical quirks of the data set. Finally, we only included symptom measures for which Ͼ90% of parents could adequately rate severity. Because measures included in the URI symptom score (significant runny nose and cough) are intuitively important, it is more likely that the score will perform similarly in other children than if a more complicated prediction rule had been developed.
The results of this study indicate that parental perception of a child developing a cold and the presence of respiratory symptoms is a moderately accurate predictor of a viral URI; a respiratory virus was detected in 57.4% of children who met these criteria. Predicting which child has a viral URI was substantially increased by using our URI symptom score. The use of this symptom score as an analytic criterion for children enrolled in trials of remedies to treat and/or prevent viral URIs should increase the chance of detecting the efficacy of study interventions in the future. Cancer Biomarker Discovery: The Entropic Hallmark BACKGROUND: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. CONCLUSIONS/SIGNIFICANCE: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-througput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases. In a seminal review paper published nine years ago, Hanahan and Weinberg [1] introduced the ''hallmarks of cancer''. They are six essential alterations of cell physiology that generally occur in cancer cells independently of the originating tissue type. They listed: ''self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of the normal programmed-cell mechanisms (apoptosis), limitless replicative potential, sustained angiogenesis, and finally, tissue invasion and metastasis''. More recently, several researchers have advocated including ''stemness'' as the seventh hallmark of cancer cells. This conclusion has been reached from the outcomes of the analysis of high-throughput gene expression datasets [2, 3] . The new role of stemness as a hallmark change of cancer cells is also supported by the observation that histologically poorly differentiated tumors show transcriptional profiles on which there is an overexpression of genes normally enriched in embryonic stem cells. For example, in breast cancer the activation targets of the pluripotency markers like NANOG, OCT4, SOX2 and c-MYC have been shown to be overexpressed in poorly differentiated tumors in marked contrast with their expression in welldifferentiated tumors [4] .
Other authors suggest different hallmarks, with many papers pointing alternative processes as their primary focus of their research. The difference may stem from the fact that these authors prefer to cite as ''key hallmarks'' physiological changes which occur at a ''lower level'' scale closer to the molecular events. These authors cite, for example, ''mitochondrial dysfunction'' [5, 6] (including, but not limited to ''glucose avidity'' [7] and ''a shift in glucosemetabolism from oxidative phosphorylation to glycolysis'' [6, 8] , ''altered glycolysis'' [9] , ''altered bioenergetic function of mitochondria'' [10] ), ''dysregulation of cell cycle and defective genome-integrity checkpoints'' [11] , ''aberrant DNA methylation'' [12] (''promoter hypermethylation of hallmark cancer genes'' [13] and ''CpG island hypermethylation and global genomic hypomethylation'' [14] ), ''shift in cellular metabolism'' [15, 16, 17] , ''regional hypoxia'' [18] , ''microenviroment acidosis'' [19] , ''abnormal microRNA regulation'' [20, 21] , ''aneuploidy'' and ''chromosome aberrations'' [22, 23, 24, 25, 26] , ''disruption of cellular junctions'' [27] , ''avoidance of the immune response'' [28] , ''pre-existing chronic inflammatory conditions'' [29, 30] , ''cancerrelated inflammation'' [29] , ''disabled autophagy'' [28] , ''impaired cellular senescence'' [31] , ''altered NF-kappaB signalling'' [32] , ''altered growth patterns, not altered growth per se'' [33] , ''disregulated DNA methylation and histone modifications'' [34] , ''tissue dedifferentiation'' [35, 36] , and ''somatically heritable molecular alterations'' [37] . This research enriches the list of the most important cancer hallmarks. However, these physiological changes occur at a ''lower'' molecular level they are likely related sub events of the orginial seven instead of newly discovered ''key hallmarks''. More recently, Luo et al attempted a ''stress-based'' description of some of the hallmarks in terms of ''stresses'' (''DNA damage/replication stress, proteotoxic stress, mitotic stress, metabolic stress, and oxidative stress'') [38] . While this is an interesting descriptive grouping, it is still a phenotypical characterization. What is needed is a higher level unifying genotypical characterization, from which individual disregulated processes can be identified in a quantitative way using the existing high-throughput data capture methodologies. It is clear that a unifying hallmark is needed if we aim at quantifying the cell's progression. It is then evident for us that a unifying mathematical formalism is necessary to uncover the cell transcriptome's progression from a normal to a more malignant phenotype.
We start our quest assuming an implicit working hypothesis common to many research groups around the world: the macroscopic physiological changes (i.e. Hanahan and Weinberg's ''hallmarks'') must also correlate with global alterations of the molecular profiles of gene transcription. It is also assumed that the ''hallmark changes'' occur along a certain timeline, but that some of the sub-processes discussed before are concurrent. These processes may start in a slow incremental way with some of the major changes being early events while others (e.g. tissue invasion and metastasis) are likely later processes triggered by new events during cancer progression. The timeline is not explicit and it is also likely that cancer subtypes progress to similar timelines. In some cases the sequence of events are better understood (e.g. some leukaemia subtypes [39] ). The elicitation and regulation of molecular events is likely to be an ongoing quest during this century for many types of cancer.
It is not to be assumed that some of the transitions of the transcriptome are gradual. That is a hypothesis that is unnecessary in this study. We envision that the progression of cancer may have ''switches'', with a number of concurrent converging events leading to macroscopic observable changes in the gene expression profile resulting in dramatic variations of expression patterns. For instance, these molecular switches could not be characterized by an ''oncogene'' but by a large number of the genes that have changed its transcriptional state. These abrupt changes may be triggered by the confluence of several non-linear interactions, and are likely to be related to the physiological hallmarks we refer to above.
The presence of macroscopic observable changes that are computable from a large number of relatively smaller changes mean that it may be possible to find an objective mathematical formalism to infer the turning point at which these radical changes occur.
It is then evident that computing the Jensen-Shannon divergences, the Normalized Shannon Entropy, and the Statistical Complexity of samples reveal different global transcriptional changes. It is, however, not easy to infer if these changes would correlate with a gradual progression or sudden changes. However, one valid mathematical possibility is that the most important ''hallmark of cancer'', a unifying principle above all, is the existence of a measurable gradual ''progression'' from a well-differentiated gene expression profile (corresponding to a healthy tissue). This would reveal the timeline of a higher level process that is observable and measurable via a change of Normalized Shannon Entropy and an increment of Jensen-Shannon divergences from the originating tissue type. If this is the case, by correlating the changes in Information Theory quantifiers with the expression of the genes we would be able to not only uncover useful biomarkers to track this progression but to explain the ''hallmarks'' in an ordered timeline. The timeline also yields clinical and translational important outcomes. Such analytical methodology will naturally produce ''a continuous staging'' of the cancer samples, based on a solid foundations of Information Theory, based on the knowledge of transcriptional profile of healthy cells as reference to measure divergences. In addition, as a mathematical methodology, it can be applied to other high-throughput technologies for which a probability distribution function of observed abundances has been computed.
With these ideas in mind, we provide a ''transcriptomic-driven'' method revealing important biomarkers for cancer progression a direction of time for which they are presented. The method, however, is generalizable to other type of high-throughtput techonologies (e.g. proteomic studies). We have chosen two types of cancers to study which are almost at the antipodes in terms of progression rates: prostate cancer and melanoma.
Prostate cancer progresses very slowly. Pathological samples are common in autopsies of men as young as 20 years old. By the age of 70 more than 80% of men have these alterations, a fact that already shows a relationship of this cancer type with increasing age. The clinical management of prostate cancer requires the identification of the so-called Gleason patterns in the biopsies [40] , which after almost fifty years is still ''the sole prostatic carcinoma grading system recommended by the World Health Organization''. However, undergrading, underdiagnosis, interobserver reproducibility and variable trends in grading have been observed as major problems [41, 42] . Melanoma, on the other hand, differs from prostate cancer in its rapid progression [43] and it is considered one of the most aggressive types of cancer. One of melanoma's usual markers of progression and concern (i.e thickness) is measured in millimetres, which gives a rough idea of how devastatingly fast the disease can spread.
We will present our results starting with one prostate cancer dataset, followed by another in melanoma, to come back to the prostate cancer discussion using another highly relevant dataset. This is a departure from the alternative approach in which each disease is discussed in separate sections. However, after considering several possibilities, we are convinced that our approach is the most appropriate to showcase the technique and its power. Details on the datasets and methods used are given in the 'Materials and Methods' section of this paper. We also refer to the original studies and manuscripts associated to the three datasets we analysed. and available at the web address given above). After imputation of missing values, we first calculated the Normalized Shannon Entropy and the MPR-Statistical Complexity for the each sample.
The flowing section explains the context in which our results were generated (refer to the 'Materials and Methods' section for detail on how our quantities are computed). The Normalized Shannon Entropy measure is widely used in ecosystem modelling to quantify species diversity, where it is acknowledge as having great sensitivity to relative abundances of species in an ecosystem [45] . We utilise the same sensitivity to differentiate a samples in cancer datasets. Figure 1 shows that the Normalized Shannon Entropy of prostate cancer tumor samples do not differ much from normal samples. This is in contrast to lymph node metastasis samples that appear to have smaller values of Normalized Shannon Entropy.
A mathematical interpretation of this result is that the samples from lymph node metastases have cells that not only varied their transcriptomic profile, they have also ''peaked'' the distribution of expression values with significant fold increases on a smaller number of probes. This explains the reduction in Normalized Shannon Entropy. We note that there are several mechanisms that can explain a macroscopically observable global reduction of transcription. For instance, this may indicate that a relatively large number of genes have reduced their expression levels by genome damage, changes in gene regulation, or other silencing processes. It is reassuring to observe that the changes of the most prototypical quantitative measure we can draw from Information Theory, the Normalized Shannon Entropy correlate well with the transition between normal samples with to ones with metastases. However, it is also evident from that normal samples do not differentiate much from the tumor group (the Normalized Shannon Entropy values do not differ much). It is then not the number of genes with high expression values, but the change in the distribution of expression levels on the molecular profile, that can provide the other measure that could distinguish these other samples. This must be handled by the other statistical complexity measures to be discussed next.
Several statistical complexity measures can be defined which aim to clarify our argument. We will first discuss the results of computing the MPR-Statistical Complexity measure (in the previous figure the y-coordinates correspond to the MPR-Statistical Complexity values of each sample). The MPR-Statistical Complexity is proportional to both the Normalized Shannon Entropy associated to the transcription profile and the Jensen-Shannon's divergence between that probability density function and the uniform probability distribution. Again, we refer the reader to the 'Materials and Methods' section for an explanation of how these magnitudes are computed.
Although the results of using the MPR-Statistical Complexity might not seem particularly impressive, there are a few reasons why we introduce them at this stage. We want to illustrate a fact that can already be observed when we employ this measure on this dataset. In this dataset, for a given entropy value interval, normal tissue samples tend to have relatively lower MPR-Statistical Complexity values than tumor and lymph node metastasis. This means that both prostate cancer and metastases samples diverge from a ''more uniform'' distribution indicating that the distribution ''peaks'' in fewer active genes. It also means that, in terms of Jensen-Shannon's divergence, the transcriptional profile of a normal prostate cell sample is ''closer'' to a uniform distribution than to the one that is observed in a prostate cancer cell sample.
The reader will readily argue, and with reason, that the transcriptional profile of a normal cell is tissue-specific and that it hardly resembles that of a uniform distribution of expression values. That is correct and this observation motivates the introduction of two new statistical complexity measures. We generically call these two variants as 'M-complexities' (with 'M' standing for ''modified''). They have the same functional form as the MPR-Statistical Complexity, but instead of computing the Jensen-Shannon's divergence from a uniform probability distribution we compute it against an ad hoc probability distribution functions derived from the data. In this sense, these measures are more supervised then the MPR-Statistical Complexity is. Another perspective is that the MPR-Statistical Complexity is a special case of this measure in which the ad hoc probability distribution function of reference is the equiprobability distribution. The relevance of this measure derives from being a general definition that allows [44] . Metastatic samples have typically lower values of Normalized Shannon Entropy than normal samples and prostate cancer primary tumors. The reduction in Normalized Shannon Entropy indicates that there exists a significant reduction on the expression of a large number of genes, or that the gene profile of metastatic samples has a more ''peaked'' distribution (due to the upregulation of a selected subset of genes). Both possibilities just cited are not mutually exclusive. We also note that neither the Normalized Shannon Entropy, nor the MPR-Statistical Complexity (as a single unsupervised quantifier), can help differentiate between tumor and normal samples, indicating that other Information Theory quantifiers are required for this discrimination. doi:10.1371/journal.pone.0012262.g001 accommodating several different reference states. We will use it to measure divergences to the ''initial'' and ''final'' transcriptomic states (two states of reference). Taken as computed averages over normal samples, and respectively metastatic ones, these measures will allow tracking the processes of differentiation of a cancer cell from a particular tissue type.
For example, using Lapointe et al.'s dataset, the M-Normal statistical complexity quantifier first requires the computation of the probability distribution function of the average gene expression profile of all normal prostate samples. Afterwards, the Normalized Shannon Entropy and the Jensen-Shannon's divergence of any sample profile will be computed using the divergence to that averaged normal distribution. Analogously, we compute the M-Metastases statistical complexity quantifier by first calculating the average profile of the metastases samples, and then generating the corresponding probability distribution function, finally computing the Jensen-Shannon's divergence with that profile. We refer to the 'Materials and Methods' section for details of the calculations.
The results can be observed in Figure 2 . On the x-axis, the lymph node metastases have the largest values of M-Normal indicating a divergence from the normal profile. In addition, the M-metastases values of normal samples tend to be higher than most of the metastasis samples (with the exception of only one). Figure 2 shows a gradual progression of the samples positions on this plane from a well-differentiated tissue type specific profile, first to a more heterogeneous primary tumor cluster, and finally to an even less differentiated metastatic profile.
The result presented in Figure 2 shows that the prostate cancer samples, which are not metastases and therefore could have been scattered anywhere on the plane, are clustered on a particular confined area between the two other groups. We understand that there are reasons to be sceptical about this result being not just a simple consequence of the gene selection process used by Lapointe et al. For example, if we assume that the 5,153 probes singled out by Lapointe et al. in their figure one of Ref. [44] (and that constitute our original data) have been selected with a supervised method that try to distinguish between normal and metastases, then the relative position of normal and metastases samples is perhaps something to be expected. However, even under that assumption, what is not expected is the position of all primary tumor prostate cancer samples, linking the normal cluster of samples with the metastases one. Note that the definition of both the M-Normal and M-Metastases measures do not use any information from the primary tumor prostate cancer samples, so the location of these samples between the normal cluster and the metastases, bridging them naturally is something to highlight. Together with Figure 1 , it gives evidence that supports the working hypothesis that a gradual ''progression'' occurs, from the normal tissue specific profile to the metastasis one.
Indeed, following our line of argument, Figure 2 has even more relevance when we highlight the fact that the 5,153 probes have not been selected with a supervised method. The authors say that the only selection criteria was to single out the 5,153 cDNAs whose expression varied most across samples. In the supplementary notes of their paper the authors say: ''We included for subsequent analysis only well measured genes whose expression varied, as determined by (1) signal intensity over background .1.5-fold in both test and reference channels in at least 75% of samples, and (2) 3-fold ratio variation from the mean in at least two samples; 5,153 genes met these criteria.'' As a consequence, Figure 2 has been generated without class selection bias only using the genes that have the most varied expression pattern.
We now turn to another aspect of the statistical complexity and entropy analysis. We note that Figure 2 shows that the metastases samples have a clear reduction on Normalized Shannon Entropy in comparison with the values observed for the normal samples. At the same time, metastases samples, as expected, have higher Mnormal complexity than the normal samples ( Figure 2 ). It is then interesting to evaluate the value of the Jensen-Shannon divergence of these samples and to identify the genes that most correlate with the variations of Jensen-Shannon divergence to quantify one of the factors that is related to the statistical complexity changes.
We have computed the correlation of the gene expression profile corresponding to each of the 5,123 probes. For each of the 5,123 probes, we computed both the Pearson correlation (x-axis of Figure 3 ) and the Spearman correlation (y-axis of Figure 3 ) of each probe profile with the Jensen-Shannon divergence having as probability distribution of reference that of a metastasis profile (these values are called JSM2-Pearson and JSM2-Spearman in the accompanying Excel file provided). With this data, we have produced Figure 3 , a scatter plot of the values associated to each probe. In this figure, there are two probes that are immediately recognizable by any cancer researcher, and in particular for those in prostate cancer: KLK3/PSA (Prostate Specific Antigen) and FOS.
The interpretation of these scatter plots is not immediate and needs an introductory explanation. Each dot corresponds to one probe of the array. For example, a dot that is very close to the origin of coordinates (0,0) indicates a probe such that its pattern of gene expression (across all samples) is not correlated with the Jensen-Shannon divergence to the average profile of a metastasis pattern. It is, in essence, a probe which is highly uninteresting in this regard. Probes that have a high correlation, across all samples, either positive or negative with the Jensen-Shannon divergence to the average profile of a metastasis pattern are highly informative. They ''co-express'' with this measure.
Although we provide in the supplementary material the information corresponding to all probes, we will discuss just a few of them. This will allow the reader to understand these plots and will put our results in the perspective with current research in prostate cancer. We particularly highlight the position of KLK3/ PSA, FOS and CCL2. To our surprise, we have found which is perhaps the most famous biomarker in prostate cancer KLK3/ PSA (Kallikrein-related peptidase 3), probe G_914588 (correlations of 20.9312 and 20.9000 respectively). FOS and KLK3/ PSA are the second and the fourth most negatively correlated probes in this ranking of all the genes in the microarray. With opposite signs for correlations are CDKN2D, FOXM1, and BRCA2. The following is a discussion of a selection of probes (highlighted in Figure 3 ) in the context of prostate cancer.
CDKN2D (Cyclin-dependent kinase inhibitor 2D, p19, inhibits CDK4). One of the genes that has strong positive correlations is CDKN2D, (Cyclin-dependent kinase inhibitor 2D, p19, inhibits CDK4) (Pearson correlation of 0.7543, Spearman correlation 0.6833), probe G_145503. A gene that shows a positive correlation with the divergence of a metastasis profile indicates a gene that has a putative reduced expression on these samples. CDKN2D is a known regulator of cell growth regulator and controls cell cycle G1 progression [46, 47] . Loss of CDKN2D in cancer cells is one event which is generally associated to a more malignant phenotype.
FOXM1. Another probe that presents positive correlations is FOXM1 (Forkhead box M1), with Pearson correlation of 0.7039 and Spearman correlation 0.7500), probe G_564803. It has been recently shown that the depletion of FOXM1 still allows cells to enter mitosis but they are unable to complete cell division. As a consequence this leads to mitotic catastrophe or endoreduplication [48] . FOXM1 is considered a key regulator of a transcriptional cluster which is that is essential for proper execution of the mitotic program and the control of chromosomal stability [49] .
BRCA2 -(Breast cancer 2, early onset). Another gene with positive correlations is BRCA2 (Breast cancer 2, early onset), probe G_193736, with Pearson correlation of 0.8161 and Spearman correlation 0.7333). While the loss of BRCA2 function and its consequences in prostate cancer is being reconsidered [50, 51, 52, 53] , BRCA2 is generally regarded as a ''tumor suppressor'', with an established role in maintaining genomic stability via its function in the homologous recombination pathway for double-strand DNA repair. This result is supporting its proposed function. Loss of BRCA2 function is thus a warning sign of the existence of error prone cell processes. In prostate cancer BRCA2 has been associated to promotion of invasion through upregulation of MMP9 [54] . BRCA2 loss of function due to mutations is linked to poor survival in prostate cancer [55] and rare germline mutations have been associated with early-onset of prostate cancer [56] .
CCL2/MCP-1 (chemokine (C-C motif) ligand 2). Bone is one of the most common sites of prostate cancer metastasis; close to 85% of men who die of prostate cancer have bone metastasis [57] . The successful metastatic process to bone follows from the activation of osteoclasts with bone resorption, which in turns leads to the release of different growth factors from the bone matrix [58] . CCL2 has been previously reported as expressed in human bone marrow endothelial cells; the CCL2 stimulation promotes prostate cancer cell migration and proliferation [57, 59] and it has been proposed as a paracrine and autocrine factor for invasion and growth of prostate cancer [60] . As a consequence of this central role in the tumor microenvironment, CCL2 is being the object of several studies and is included in the list of potential targets for novel therapies [60, 61, 62, 63, 64, 65, 66, 67, 68, 69] .
FOS (V-fos FBJ murine osteosarcoma viral oncogene homolog). A probe for FOS (G_811015; correlations of 20.9380 and 20.9500 computed with Pearson and Spearman) has a similar correlation than KLK3/PSA. The high rank of FOS was unexpected, but perhaps it is less of a surprise for some experienced researchers in prostate cancer as its role has been highlighted in the past [70, 71, 72] . Amplification of members of the MAPK pathway was associated with androgen independent prostate cancer, and co-expression of RAF1, ERBB2/HER2 and c-FOS would lead to this phenotype [73] .
We will not discuss in depth the known relationships between FOS, Lamin A/C and prostate cancer. We leave this discussion for later, as Lamin A/C will also appear in our study of the other prostate cancer dataset studied in this paper. Lamin A/C appears as a member of a set of genes with reduced expression for higher grade primary prostate cancer samples (note that the current analysis that gave FOS as a biomarker is on lymph node metastatic samples like here). However, we would like to point out a connection that is currently hypothesized between Lamin A/C and FOS, the gene we have just discussed. Ivorra et al. have recently proposed that ''lamin A overexpression causes growth arrest, and ectopic c-Fos partially overcomes lamin A/C-induced cell cycle alterations. We propose lamin A/C-mediated c-Fos sequestration at the nuclear envelope as a novel mechanism of transcriptional and cell cycle control'' [74] . In addition: ''c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and lossof ERK1/2 activity, respectively.'' [75] . These novel interactions between LMNA and FOS, their putative role in prostate cancer metastasis and their seemingly different behaviours in prostate cancer lymph node metastases warrant further investigation.
SOX9 (SRY (sex determining region Y)-box 9). This transcription factor has been recently identified as having an importat role during embryogenesis and in the early stages of prostate development [76, 77] and in testis determination [78] , processes that link SOX9 upregulation to cancer development [79] . Basal epithelial cells do express SOX9 in a normal prostate. While there exists no detectable expression in lumina epithelial cells, SOX9 has already been reported as ''expressed in primary prostate cancer in vivo, at a higher frequency in recurrent prostate cancer and in prostate cancer cell lines (LNCaP, CWR22, PC3, and DU145)'' [80] . Wang et al., also in [80] add that: ''Significantly, down-regulation of SOX9 by siRNA in prostate cancer cells reduced endogenous AR protein levels, and cell growth indicating that SOX9 contributes to AR regulation and decreased cellular proliferation. These results indicate that SOX9 in prostate basal cells supports the development and maintenance of the luminal epithelium and that a subset of prostate cancer cells may escape basal cell requirements through SOX9 expression.'' An increased value of SOX9 expression in advanced prostate cancer has been associated to tumor progression and the epithelial-mesenchymal transition [81] . SOX9 expression has been associated with a putative subgroup of prostate cancer [82] , associated to lymph-node metastasis (as seems to be the case in this dataset) and has a know role in chondrogenic differentiation processes [83] .
KLK3/PSA -(Kallikrein-related peptidase 3)/Prostate Specific Antigen. To finalize our initial discussion on this dataset, we address KLK3. The high ranking of KLK3/PSA in our list is perhaps one of the most remarkable retrodictive outcomes of our approach. KLK3/PSA (also known as Prostate Specific Antigen) is a conspiquous member of our top rank list. It is perhaps the best blood biomarker for prostate cancer screening. Its relevance and popularity as a target of studies is so wide that it makes unfeasible any serious attempt to uncover its relevance in the prostate cancer literature. A search using PubMed using the keyword 'KLK3' (and the other alias names of this gene) reveals a total of 11,429 published papers. Of course, many of these publications relate to its role for early screening, but in this study we are uncovering its role as a tissue biomarker. Our results echoes a recent contribution by S. Miyano's and his collaborators [84] on a massive meta-analysis of microarray datasets. It is also in line with results from clinical studies that indicate that a 5-year PSA value is useful for predicting prostate cancer recurrence. [85] . Certainly the dynamics of PSA, now perhaps with FOS and SOX9 added to the set of biomarkers of interest, warrant further investigation for patient population stratification after initial treatment.
The biomarkers discussed in this section warrant further investigation in prediction of lymph-node metastasis and clinical management of prostate cancer [86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109] . We refer the reader to the Supplementary Material to have a complete list of probes and their correlations with the Information Theory quantifiers.
The following sections present the results that we obtained with a melanoma dataset. Our aim is to observe if variations of the Normalized Shannon Entropy and the statistical complexity measures, MPR-complexity and the modified forms M-normal and M-metastases, provide interesting results in a different disease and experimental setting.
In this case we have selected a gene expression dataset from Haqq et al. [110] containing information of 14,772 cDNAs in 37 samples (Figure two from the [110] ). The 37 samples include 3 normal skin, 9 nevi, 6 primary melanoma and 19 melanoma metastases. This datasets has more phenotypical characteristics for the group of samples.
After an initial process of data cleaning, we removed 35 probes which had an unsually high expression value on only a few samples, in some cases on a single one. The dataset we work with from original contributed by Haqq et al.consists of 14,737 probes. First, we computed the Normalized Shannon Entropy and the MPR-Statistical Complexity for each sample (refer to the 'Materials and Methods' section for a detailed presentation of these calculations). Figure 4 shows the values of these quantifiers for each sample.
We first observe an important difference between Figure 1 and Figure 4 . In this melanoma dataset, neither the use of the Normalized Shannon Entropy nor the MPR-complexity helps to discriminate between normal skin, nevi, primary and metastastic melanomas. Nevertheless, we decided to present this figure for methodological reasons. We envision that some researchers will calculate the Normalized Shannon Entropy and MPR-complexity using all the probes. We note that in Figure one of Haqq et al's original paper, the whole probe set was previously filtered by selecting those which vary across samples, thus indicating that they may have information about disease subtypes (although the phenotypic types were not biasing the selection). In this case we want to illustrate both the Normalized Shannon Entropy and MPR-complexity calculated using all the probes does not give the expected benefits. We will now see the benefits of using the M-complexities.
As we did for prostate cancer (see Figure 2 ), we aim at identifying if the use of the modified forms of the statistical complexity (the M-complexities) could give some insight where the Normalized Shannon Entropy and MPR-complexity measures fail. To compute the M-normal measure, we need to define the average gene expression profile for a normal cell (which we call P ave ). We thus resort to the three normal skin profiles and we produce the average based on these profiles (details for computing the average profiles are given in the 'Materials and Methods' section). We call M-skin the resulting measure that relies on this profile. Analogously, we need to compute a pattern for M-metastasis, and we proceed to calculate the P ave profile averaging over the 19 metastases samples. The result is encouraging, as samples plotted in the (M-skin, M-metastasis)-plane cluster in groups, showing an important M-skin complexity transition between normal skin cells and nevi. Most importantly, this method naturally shows that some of the metastatic samples have a large value of M-skin complexity, so we present the results of another experiment, aimed at clarifying this fact.
In their original publication, Haqq et al. classified the melanoma metastases in two groups due to their molecular profiles: five samples were classified as 'Type I' and fourteen as 'Type 2' based on a hierarchical clustering approach. Our result reinforced the view that the Type II melanomas metastasis is a pretty homogeneous group, we will present the results on the (Mskin, M-metastasis I)-plane. This means that now the P ave profile will not be obtained by averaging over the 19 metastases samples, but instead using only the 14 samples which have been labelled as 'Type II'. As such, we aim at revealing if Type I samples are indeed different in this plane, and if other clusters are also present. Figure 5 presents the results. The first fact worth commenting is the pronounced gap between normal skin samples and the nevi, primary, and metastatic melanoma samples as revealed by the Mskin measure. Note also that the M-skin is based on the average profile that of the normal samples, which indicates that no information about the profiles of metastasis are used, yet M-skin reveals that increasing values of this measure may be linked with a 'progression' from nevi to primary and metastasis melanoma profiles.
We now introduce another useful technique to identify genes which correlate with the transitions. The challenge is to find genes which are related with the progression towards metastases profiles, even when we recognize that there the group of metastasis samples is heterogeneous (containing at least two groups). Since the final outcome of Figure 4 and Figure 5 is that the Normalized Shannon Entropy does not help much in this experimental scenario, we will concentrate only on one of the multiplicative factors of the Mcomplexities, the Jensen-Shannon divergence. We compute two P ave profiles, one with the normal skin samples only, and the other with all the metastasis samples (regardless their type). We will call the two divergences JSM0 and JSM5 respectively. We then compute the Spearman correlation of the profile of all gene probes in the array across the 37 samples to both JSM0 and JSM5. We have listed all probes according to the absolute value of the difference of these correlations, i.e. Abs. Diff. (probe) = |JSM0(probe)2JSM5
(probe)| in decreasing order. The results are provided as Haqq-PLoSONE-SupFile.xls, in the sheet labelled 'Results-correlation'.
The rationale is to identify those probes which are highly correlated (both positively or negatively) with the Jensen-Shannon divergence of the normal tissue profile and that ''reverse signs''. For instance, a probe for the TP63 gene (Tumor protein p63, keratinocyte transcription factor KET), AA455929, is ranked in the third position. Its correlation with the Jensen-Shannon divergence of the normal skin type is relatively high and negative (JSM0 = 20.63632) while at the same time is has a positive correlation with the Jensen-Shannon divergence of the metastasis profile (JSM5 = 0.62138). In the ranking, the first probe that presents the opposite behaviour is one for ADA (Adenosine deaminase), AA683578. Figure 6 helps to understand the relationship of these correlations with expression. Not only are these genes well correlated with the divergences, they also seem to be good markers of the progression from one tissue type profile to the metastasis profile.
We will now discuss three of these genes in the context of current biological knowledge on melanoma drivers and metastatic progression. We provide many references for one of them, SPP1 (Secreted phosphoprotein 1 or Osteopontin). The discussion on this gene will be left for later, when we will discuss specifc oncosystems related to cell proliferation, chemotaxis and responses to external simulus. Figure 7 shows the expression of ADA (Adenosine deaminase, AA683578) as a function of TP63 (keratinocyte transcription factor KET, AA455929). All normal skin samples, as well as nevi and a couple of primary melanomas have relatively low values of ADA but they express TP63. There is a change of roles in metastatic and some primary melanomas, which have reduced TP63 expression but increased values of expression of ADA. As we will later see, these events correlate with other major transcriptional modifications which involve dozens of genes and that we have been able to map thanks to functional genomics bioinformatics tools. The role of SPP1 will be discussed in that context after some references to TP63, ADA, and PLK1 which follow.
TP63. The product of this gene [111, 112] belongs to the same protein family of its more famous relative, TP53, a gene that is often mutated in human cancers [113] and highly regarded as a key ''tumor suppressor''. TP63's product, p63, is a homologous protein to p53, which is considered to be phylogenetically newer [114] and also regarded as an important apoptotic and cell-cycle arrest protein. Mice that lack TP53 are born alive with a propensity for developing tumours; mice that lack TP63 do not appear to be tumour prone, although, new results are partially contradicting earlier findings [115] . It appears that the diverse roles of the isoforms of the p63 family reveal that there exists a crosstalk with the different isoforms of the p53 family that needs to be systematically investigated [116] . It has recently been shown that p63 is a key regulator of the development of stratified epithelial tissues [113] and that its deletion results in loss of stratified epithelial and of all keratinocytes [117] . Melanocytes also express two isoforms of p63 [118] , but p63 expression is not reported in 57 out of 59 tumors in a tissue microarray study performed by Brinck et al. [119] . It is clear that the the role of loss of expression of TP63 in melanoma warrants further investigation.
ADA -(Adenosine deaminase) and DPP4/CD26 (Dipeptidylpeptidase 4, CD26, adenosine deaminase complexing protein 2). A link between TP63 and ADA has already been reported in the literature. ADA is a gene involved in cell division and proliferatation [120] and it has been suggested to have a regulatory role in dendritic cell innate immune responses [121] .Translational modification is also a function of p63. Sbisa et al. have proved that ADA is a direct target of isoforms of p63, which is an important discovery as ADA has two TP53 binding sites, leading to a complex metabolic balance due to the different relationships between this trio and p21 yet to be completely elicitated [120, 122] . Several studies indicate elevation of adenosine deaminase levels in sera of breast [123] , head and neck [124] , colorectal [125] , acute lymphoblastic leukaemia [126] and laryngeal cancers [127] .
We observe a marked increase of expression of a probe for ADA with melanoma progression while at the same time we observe a loss of expression of a probe corresponding to DPP4/CD26 (Dipeptidylpeptidase 4, CD26, adenosine deaminase complexing protein 2), a membrane-bound, proline-specific serine protease [128] that has been attributed tumor suppressor functions [129] . It has been previously reported that loss of DPP4 immunostaining helps to discriminate malignant melanomas from deep penetrating nevi, a variant of benign melanocytic nevus [130] and early reports of their absence in metastatic melanomas exist [131, 132] . As deep penetrating nevi can mimic the vertical growth phase of nodular malignant melanoma, and ADA could potentially be downregulat-ing DPP4 [133, 134] we believe that the elicitation of the complementary role of these two biomarkers to distinguish these two entities is necessary and also warrants further clinical studies.
PLK1 (Polo-like kinase 1 (Drosophila)). Another probe for gene that ranks high as a positive marker of metastasis is PLK1, Polo-like kinase 1, Serine/Threonine protein kinase 13 (AA629262). PLK1 is a centrosomal kinase [135] which is Figure 7 . Scatter plot showing the expression of the probe corresponding to ADA (Adenosine deaminase), AA683578 (y-axis) and TP63 (Tumor protein p63), AA455929 (x-axis). All the samples that have TP63 expression are normal or nevi, with two primary melanomas still preserving TP63 expression but with higher ADA. The trend reverses for the rest of the primary melanoma samples and the metastatic ones, which all express ADA but not TP63. doi:10.1371/journal.pone.0012262.g007 Analogously, we compute the Jensen-Shannon divergence of each sample with the average metastastic profile and we also compute the correlation of each probe with this measure (y-axis). The position of one probe corresponding to the TP63 gene (Tumor protein p63, keratinocyte transcription factor KET), AA455929, is highlighted. The expression of this probe has a relatively high negative correlation with the Jensen-Shannon divergence of the normal skin type (JSM0-Spearman = 20.63632) while at the same time is has a positive correlation with the Jensen-Shannon divergence of the metastasis profile (JSM5 = 0.62138). The first probe that presents an opposite behaviour is one for ADA (Adenosine deaminase), AA683578. Probes for SPP1 (Secreted phosphoprotein 1 or Osteopontin) and PLK1 (Polo-like kinase 1 or Drosophila) are also highlighted. While PLK1 is currently less recognized as a biomarker in melanoma research, the importance of SPP1 in cutaneous pathology [315, 318, 320, 321] and in particular in melanoma [208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219, 222, 226, 264, 314, 315, 316, 317, 319, 322, 323, 324, 325, 326, 327, 328, 804, 805, 806, 807, 808, 809] is increasing. Using a 5-biomarker panel that included SPP1, Kashani-Sabet et al. used tissue microarrays on 693 melanocytic neoplasms to show that SPP1 expression collaborates significantly improving the detection of high percentage of melanomas arising in a nevus, Spitz nevi, dysplastic nevi and misdiagnosed lesions [253] . Like in the case of prostate cancer ( Figure 3 , in which KLK3/PSA -Prostate Specific Antigen was highlighted), our method allows the detection of important biomarkers with a high degree of concordance with current biological understanding of metastatic processes. doi:10.1371/journal.pone.0012262.g006 regarded as being linked to centrosome maturation and spindle assembly [135] . PLK1 expression has also been singled out as a biomarker of a ''death-from-cancer'' signature, sharing with others the function of being an activator of mitotic spindle check point proteins. With other proteins it would has a stem cell-like expression profile phenotypically characterized by enabling metastasis with anoikis resistance and disregulated cell-cycle control [136] . PLK1 inhibition could be a common target for gastric adenocarcinoma [137] , bladder cancer [138] , colon cancer [139, 140] , hepatocellular carcinoma [141] , medullary thyroid carcinoma [142] , esophageal cancer [143] , pancreatic cancer [144] and in some types of non-Hodgkin lymphomas [145] and breast cancer [146] .
PLK1's Spearman correlation with the values of the Jensen-Shannon divergence of samples with the normal skin profile is relatively high (0.5863). PLK1 also has a high value of (negative) Spearman correlation with the values of the Jensen-Shannon divergence of samples with the average metastatic profile (20.44571 In the comparison, it was found that metastatic malignant melanomas with expressed PLK1 at markedly elevated levels (median, 60.00% vs. 37.98%; p-value,0.000053), concluding that PLK1 is a reliable biomarker for patients at high risk of metastases, even when the most important prognostic clinical factor (Breslow's maximum thickness of the primary malignant melanoma) indicates the contrary [147] . We consider this an important finding as PLK1 silencing is already part of an integrated oncolytic adenovirus approach currently being studied in mice models of orthotopic gastric carcinoma [148] and has promise due to the lack of a reported measurable immune response of siRNA-based therapeutics [149] . Another positive note is the less sensitivity to PLK1 depletion of cells with a functional p53 [150, 151] , and can help to sensitize cells to chemotherapy (as observed in lung cancer [152] ). This constraint of aneuploid cancer cells to PLK1 expression, particularly in cells with inactivated p53 [153] , could be exploited by lentivirus-based RNA interference [154] .
Correlation analysis with Jensen-Shannon divergences reveals biomarkers for loss of cell adhesion, cell-cell communication, impairment of tight junction mechanisms and dysregulation of epithelial cell polarity.
As discussed before, the probe for ADA (Adenosine deaminase) is the first that has a different trend. Since we put all metastasis samples together in the same group when we calculated the average probability profile (and we have a heterogeneous group) we have on our ranking 58 probes that appear before ADA (we refer to the Supplementary File Haqq-PLoSONE-SupFile.xls). An analysis using GATHER (http://gather.genome.duke.edu/) [155] to interpret the collective influence of the lack of expression of all these genes in the metastasis samples reveals an interesting new perspective. Using Gene Ontology, we found that six of the 44 genes identified by GATHER are related to epidermis development (CDSN, DSP, EVPL, GJB5, KRT13, KRT5), p-value ,0.0001, Bayes Factor 16, and eight genes are related to cell adhesion (CDSN, CLDN1, DSG1, DST, LGALS7, LRIG3, PCDH21, PKP1), p-value,0.0001, Bayes Factor 7. ANK1 (Ankyrin 1, erythrocytic), AA464755 was also singled out as by our Gene Ontology analysis as related to the maintenance of epithelial cell polarity (p-value = 0.002, Bayes Factor 3). The use of another profiler of genome signatures (g:Profiler, [156] ) also reinforces the view that many genes that have lost expression are related to 'Epidermis Development' (COL17A1, DSP, EVPL, GJB5, KRT13, KRT5, LCE1C, MAFG, TGM3) with p-value = 7.78E-11. Thirteen are associated with Gene Ontology function of cell communication (ANK1, CDSN, CLDN1, DSG1, DST, GCHFR, GJB5, GPR115, LGALS7, LRIG3, PCDH21, PKP1, PTGER3), albeit with a pvalue of only 0.02. GCHFR is also involved in nitric oxide metabolism.
If we add to the list of 44 genes already recognized by GATHER the other 77 probes that after ADA in this ranking have also loss of expression (until we found PDXP (Pyridoxal (pyridoxine, vitamin B6) phosphatase), the evidence is stronger, now COL7A1, GJB5, KLK4, and KRT1 also is in this group (the Bayes factor of this association returned by Gather is now 21 for the GO term 'Epidermis development'). 'Cell adhesion' has now 13 genes, CDSN, CLDN1, COL7A1, DSC2, DSG1, DST, JUP, LGALS7, LRIG3, PCDH21, PKP1, SLIT3 THBS3 (p-value,0.001, Bayes factor 10). These results are considered statistically very relevant as identifiers of a particular process which seems to be undermined by this collective loss of expression.
If we put all this information together, we clearly observe a pattern of downregulation of gene expression that is associated with an impairment of epidermis development and the maintainance of its structure ( Figure 8 and Table 1 ). This is, perhaps, an instantiation of one of the ''extended hallmarks of cancer'' (that of ''tissue dedifferentiation''). This process includes the loss of function of genes that are essential for the maitainance of tight junction and epithelial cell-cell communication. While loss of epithelial structure is related to these genes, we observe that those that increase expression are associated to other developmental processes, not necessarily concerted in this panel. Instead they show a pattern of increasing cell motility, chemotaxis and positive regulation of cell proliferation. We will first discuss the processes related to the loss of adhesion, which could be linked to an increased probability of metastatic potential of these cells.
The loss of expression of Plakophilin 1, Junction plakoglobin, Desmoplakin and Desmoglein 1 indicate deficiencies in desmosome processes.
In general, this panel is composed of a number of genes that are losing expression during progression and that have Gene Ontology annotations related to tight junctions, gap junctions, adherens junctions and desmosomes, and an impaired set of processes that link, via intercellular channels and bridges, the cells of the epidermis. Mutations in these genes are linked to a number of skin genetic diseases [157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170] The desmosome are cell-cell adhesive junctions which provide a mechanical coupling between cells. These junctions are found in several epithelial tissues and the decreased assembly of the desmosome has been shown to be a common feature of many epithelial cancers [171, 172] . Plakoglobin helps to connect transmembrane elements to the cytoskeleton [173] . Plakophilin 1 [174] (PKP1, one of the genes in our panel above) is a desmosomal plaque component [175] that stabilizes desmosomal proteins at the plasma membrane [176, 177] and, with desmoplakin [178] , recruits filaments to sites of cell-cell contacts [179] . As a consequence, it has been proposed that the lack of PKP1 increases keratinocyte migration [180] and loss of PKP1 expression in head and neck squamous cell carcinoma and in esophageal squamous cell carcinoma may contribute to an invasive phenotypic behaviour [171] , perhaps as a consequence of the impaired recruitment of desmoplakin.
The desmoglein-specific cytoplasmic region (DSCR) is the site of caspase cleavage during apopotosis and is a conserved region of yet undefined function and unknown structure, but it specifies the function of the desmoglein family of cell adhesion molecules (of which DSG 1 is a member). It has been recently shown that the DSCR has a weak interaction with PKP1, Plakophilin 1 (ectodermal dysplasia/skin fragility syndrome) and the cytoplasmic domain of Desmocollin 1 [181] . Plakoglobin is cleaved by Caspase 3 during apoptosis [182] . In addition, Kami et al. in Ref [181] also report and conclude that: ''desmoglein 1 membrane proximal region also interacts with all four DSCR ligands, strongly with plakoglobin and plakophilin and more weakly with desmoplakin and desmocollin 1. Thus, the DSCR is an intrinsically disordered functional domain with an inducible structure that, along with the membrane proximal region, forms a flexible scaffold for cytoplasmic assembly at the desmosome''.
As previously discussed, all these genes progress towards a loss of expression, and they are highly correlated. Figure 9 shows the average expression of PKP1/Plakophilin 1 (ectodermal dysplasia/ skin fragility syndrome), (NM_000299) and JUP, Junction plakoglobin, (BX648177) on the x-axis against that of DSP, Desmoplakin (NM_004415 Hs.519873) on the y-axis. Again, we see a clear pattern of progressive reduction of expression from normal skin and nevi (green and yellow, respectively), primary melanomas (in orange) and melanoma metastases (red).
Joint loss of expression of Claudin 1 and members of the Aquaporin family are also linked to a transition to a more malignant phenotype
We note however, the Gene Ontology annotation is not the only way that we can make sense of this information. A detailed analysis of that list of 58 genes reveals other proteins involved in tight junction, like Aquaporin 3 (AQP3). Probes for AQP3 and Claudin 1 (CLDN1) have reduced expression with the progression of the disease as shown in Figure 10 .
AQP3 (Gill blood group) is a member of the aquaporin family of proteins, and currently is recognized as an 'aquaglyceroporin' [183] of great importance to maintain skin hydration of mammals epidermis [184] . Three proteins of this family (AQP1, AQP3, and AQP9) have probes that seem correlated with melanoma progression, all losing their expression in the process of going from normal skin to metastatic melanoma. AQP3 water channels have been pointed out as an essential pathway for volume-regulatory water transport in human epithelial cells [185] . AQP3 is also selective for the passage of glycerol and urea and it has been suggested that osmotic stress up-regulates AQP3 gene expression in cultured keratinocytes [186] . AQP3 was found to be the predominant aquaporin in human skin which increased expression and altered cellular distribution of AQP3 in eczema thus contributing to water loss [187] . The putative involvement of aquaporins in the progression of melanoma, uncovered by our method in our results, warrants further investigation as it has been recently shown that another member of this family (AQP8) also facilitates hydrogen peroxide diffusion across membranes [188] . It is suspected that AQP3 has other functions with a suggestion that it is involved in ultraviolet radiation induced skin dehydration [189] . There is no probe for AQP8 in Haqq et al.'s dataset that we could scrutinize from its trend with progression but we note that a novel strategy for drug development for melanoma (i.e. Elesclomol) works by inducing apoptosis via a mechanism of elevation of reactive oxygen species (of course, including hydrogen peroxide in cancer cells) thus exploiting the ''Achilles hell of cancer metabolism'' [190] .
Claudin 1, CLDN1 [191] , a gene which is reported to be ''normally expressed in all the living layers of the epidermis'' [192] , in concert with AQP3, is a key component of the tight junction complexes of the epidermis. Low CLDN1 gene expression was correlated with shorter overall survival in lung adenocarcinoma. Overexpression of CLDN1 was correlated with suppression of cancer cell migration, invasion and metastasis [193] . Hoevel et al. report that re-expression of CLDN1, in breast tumor spheroids, induces apoptosis and they conclude: ''These findings support a potential role of the tight junction protein CLDN1 in restricting nutrient and growth factor supplies in breast cancer cells, and they indicate that the loss of the cell membrane localization of the tight junction protein CLDN1 in carcinomas may be a crucial step during tumor progression'' [194] . Tokes et al.also report that malignant invasive breast tumors are negative Table 1 . Gene names and probe accession number of the 27 probes with genes annotated with functions on cell adhesion, cell-cell communication, tight junction mechanisms and epithelial cell polarity shown in the heat map in Figure 8 . for CLDN1 [195] . As in breast cancer [196] , in which reduced expression correlated with recurrence status, the low expression of CLDN1 and other tight junction proteins seems to contribute to cellular detachment.
The complementary set of correlations with the Jensen-Shannon divergences unveils biomarkers for cell proliferation, chemotaxis, and responses to external simulus.
If the use of Gene Ontology has produced very peculiar results, helping us to link the loss of expression of 44 genes with a significant change in epithelial structure and development. A natural question arises: ''Which is the significance of another set, now arbitrarily chosen to be also of the same cardinality (i.e 44 genes) with the complementary behavioural pattern?'' We have now listed all the probes according to Diff. (probe) = JSM0(probe)2JSM5(probe) in decreasing order. The results are provided as Haqq-PLoSONE-SupFile.xls ('Results-correlation' sheet). This now gives ADA as the first ranked gene. Again using GATHER [155] on the first 44 genes recognized by the software, and again using Gene Ontology, we observe as most important common function that of cell motility (CCL3, CXCL10, FPRL1, SEMA6A, SPP1), p-value = 0.0002, Bayes Factor 5, and chemotaxis (CCL3, CKLFSF7, CXCL10, FPRL1, SPP1), p-value,0.0001, Bayes Factor 7. The genes CXCL10, SPP1, and WARS, together with another gene that has been annotated as related to positive regulation of mitosis (SCH1), have also been annotated as regulators of cell proliferation (pvalue = 0.007, Bayes Factor 2). Using the g:Profiler software [156] , we obtain a complementary information. Sixteen genes (including SPP1, SEMA6A, LEF1 [197] , CD230, ALS2CR2, DKK1, CYFIP2, SHC1, ANKRD7, IFI6, CITED1, and MID1) have been associated to the Gene Ontology term of 'developmental process'.
SPP1 -Secreted phosphoprotein 1 (osteopontin). SPP1 is one of the most conspicuous melanoma biomarkers [198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222] (see also the references cited in Figure 6 and note its eminent position in this scatter plot). In 1990, Craig et al. reported that SPP1 may work as an autocrine adhesion factor for tumor cells (see also [204, 223, 224] ). They observed that ''SPP1 mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen'' [225] . The evidence is being constantly expanded on the role of SPP1 as a molecular prognostic biomarker in melanoma [226] . Activation of SPP1 may be an important event that allows the transformed melanocytes to invade the dermis as proposed by Geissinger et al. in 2002 [208] . This causes SPP1 to avoid the apoptotic stimulus, one of the ''hallmarks of cancer'', which invasive cells will be receiving from this new tissue.
If we extend the literature-based search so that we now include the first 200 gene probes recognized by GATHER then we have 27 gene probes associated with the Gene Ontology in terms of ''cell proliferation'' (p-value = 0.0002, Bayes Factor 5), and 'regulation of cell proliferation', p-value = 0.003, Bayes factor 3). However, other partners of PLK1 appear and their function in 'mitotic cell cycle' (pvalue = 0.0003, Bayes Factor 5) is increasingly present (in particular, the M phase of the mitotic cell cycle). The details of the Gene Ontology terms which are significant and the genes associated to them are listed in Table 2 .
The analysis using g:Profiler largely coincides with the analysis using GATHER, however, it retrieves 12 genes associated with the M phase of mitotic cell cycle, namely: AURKA and AURKB [227, 228, 229] , BUB1 [230, 231] , CDCA5A/Sororin/p35 [232] , CDC7 [233, 234] , CHEK1 [235] , KIF23/MKLP-1 [227, 236, 237] , MAP9/ASAP [238, 239] , NCAPD3, NCAPG2 [240] , NEK6 [241, 242, 243, 244] , PLK1 [147, 245, 246] , PTTG1/Securin [247] , SHC1/p66 [248, 249, 250] (discussed in the context of SHC4 signalling), and TFDP1/DP-1 [251] . These are a significant finding by g:Profiler (p-value = 4.03E-07).
We have listed above some of the genes gene associated to the M phase of mitotic cell cycle and associated references which are either to current research in melanoma and/or its biological function. We now list other genes which have been associated with the term 'cell proliferation' by GATHER. These genes are: ARPC1B [252] , ARPC2 (which, together with SPP1, is also in the novel 5-biomarker panel of Kashani-Sabet et al. [253] ), BCCIP (BRCA2 and CDKN1A-interacting protein)/P21-and CDK- Figure 10 . Expression of a probe for CLDN1 (Claudin 1) (y-axis) as a function of a probe for Aquaporin 3 (x-axis). Other members of the aquaporin family of proteins have a similar behaviour. AQP3, together with CLDN1 are key components of the tight junction complexes of the epidermis and their joint loss of expression seem to be related to a transition to a more malignant phenotype. We use the same color coding as Figure 9 . doi:10.1371/journal.pone.0012262.g010 associated protein 1) [254] , BST2/Bone marrow stromal antigen 2/Tetherin [255] , CCL3/MIP-1alpha [256, 257, 258] , CCT4, CDCA5/Sororin [259, 260, 261, 262, 263] , CENPF/Mitosin [264] , CXCL1/chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) [265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285] (in uveal melanoma see [286] ), CXCL10 [256] , FLT1/VEGFR1 [287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299] , FTH1/Ferritin Heavy Chain [300, 301, 302] (which may indicate a necessary condition for the mainainance of iron sequestration and suppression of reactive oxygen species accumulation [303] ), FPRL1, LIG3/DNA Ligase 3 [304] (which, together with XPA and ERCC5 is associated to DNA repair in ionizing radition studies [305] ), MCMDC1, PSEN2, NRP2/Neuropilin 2/Vascular endothelial cell growth factor 165 receptor 2 [306, 307, 308] , SEMA6A (a member of the Semaphorin family, of increasing importance in cancer research [309, 310, 311] and in particular due to its observed upregulation in undifferentiated embryonic stem cells [312] ), SLAMF1/CD150 (a marker associated with hematopoietic stem cells [313] ), SPP1/Osteopontin (which, together with ARPC2, is also in the novel 5-biomarker panel of Kashani-Sabet et al. [253] ) [206, 207, 208, 209, 210, 211, 212] [206,207,208,209,210, 211,212,214,215,216,217,218,219,220,221,222,226,314,315,316,-317,318,319,320,321,322,323,324,325,326,327,328,329] , STK6 [230, 330] , and WARS/Tryptophanyl-tRNA synthetise [331] . Figure 11 shows a heat map of discussed gene probes annotated with functions on cell proliferation.
The references provided next to each gene help to related these upregulated genes in the context of current research in melanoma or with the M phase of mitotic cell cycle, showing a high degree of correlation between our results and with published literature.
Another microarray dataset we have selected to evaluate for the relevance of transitions of Normalized Shannon Entropy and Statistical Complexity was contributed by True et al. [332] in 2006.
The original goal of True et al. was to identify a molecular correlate for Gleason patterns 3 and, if possible, the clinically most worrisome patterns 4 and 5. They partially succeeded by linking the expression of only 86 genes with Gleason pattern 3 [332] using a standard statistical analysis. In this study, we eliminated sample 02-209C since data was acquired using a different platform and would not be useful for our analysis. The remaining thirty one (31) samples were assayed with the GPL3834 (FHCRC Human Prostate PEDB cDNA Array v4) platform using 15,488 probes.
We also eliminated all the probes with missing values, remaining 13,188 probes.
We have first plotted the samples on the (Normalized Shannon Entropy, MPR-Statistical Complexity) plane ( Figure 12 ). It was interesting to observe that there exists a high correlation between the two measures. Samples that are entirely composed of Gleason pattern 3 tend to have a greater value of Normalized Shannon Entropy than 0.985. We can also identify a cluster of samples that present Gleason patterns which are either 4 or 5. Note that there seems to be two outliers (02_003E and 03_063) to the generic trend of the other 29 samples. The two outliers are samples that correspond to samples labelled as having Gleason 3 patterns and both have unusually low values of Normalized Shannon Entropy that are well below the values of the rest of the group.
This raised a suspicion about the true nature of this phenomenon. If the labelling is correct, this may indicate a subsampled group of prostate cancer that has Gleason 3 pattern characteristics but very low entropy. Alternatively, it may indicate an experimental bias for reasons we can not explain with the available clinical information. In order to clarify the situation, and see if we can declare these two samples as outliers of the other group, we performed another experiment. We have now computed two modified complexities, which we will call M- Gleason 3 and M-Gleason 5 ( Figure 13 ). The names are probably selfexplanatory, but a brief reminder follows. To calculate the MPR-Complexity, by definition, we have used the equiprobable distribution as our probability distribution of reference (for the computation of the Jensen-Shannon Divergence of the gene expression profile to this distribution). In the case of the M-Gleason 3, the probability distribution of the reference is obtained averaging all the probability distributions of the samples that have been labelled as Gleason 3 (analogously, we calculated M- Gleason 5) . Samples that have Gleason pattern 3 and 5 appear as separate clusters in the (M-Gleason 3, M-Gleason 5) plane with the two putative outliers of the general trend far apart (even if they have been used to calculate the average probability distribution function of the Gleason 3 pattern). Even samples with Gleason 4 pattern are located closer to samples of Gleason patterns 3, and 5, indicating that, perhaps, there exists a subsampled subtype of prostate cancer or there might be another experimental bias or factor that at present we can not resolve with the information we have for these samples. Consequently, we have decided to eliminate both samples (02-003E and PNA_03-063A) from further calculations. With these considerations, we now have a dataset with 13,188 probes and 29 samples as our dataset for further analysis. Figure 14 shows the distribution of the samples using the Normalized Shannon Entropy and the MPR-complexity. By definition, the positions of the 29 samples in the plane do not change (this figure is basically ''zooming in'' one region of Figure 12 that contains these samples). We note again, however, that the 29 samples seem to be separating in three different clusters. Whether we can argue about the existence or not of these gaps in Normalized Shannon Entropy, it is clear that there seems to be a progression as we have seen with Lapointe et al's dataset. There is a group of three samples with Gleason pattern 3 that seem to have the the largest Normalized Shannon Entropy values. There is also a cluster that only contains samples of either Gleason pattern 4 and 5, all with Normalized Shannon Entropy values smaller than 0.985.
There is also very little variation (see Figure 15 ) of the positions of the 29 samples on the (M-Gleason 3, M-Gleason 5)-plane, indicating a degree of robustness that the computation of these modified complexities have, even in the presence of some outliers.
After observing that Figure 14 shows a correlation of Gleason pattern score with Normalized Shannon Entropy, we asked ourselves: 'which are the genes that most positively and negatively correlate with the transitions of Normalized Shannon Entropy?' We have plotted Spearman versus Pearson correlation values of probe expressions to attempt to find those that best correlate, either positively or negatively, with the Normalized Shannon Entropy values of the samples. The results have revealed some of the most relevant biomarkers of progression, and some unexpected newcomers. Figure 16 shows the Pearson and Spearman correlations of all the 13,188 probes in the dataset with the Normalized Shannon Entropy values of the samples. We have highlighted some particular genes that are discussed below.
CDKN2C (cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4). When we compute the correlations of the probes expressions with the Normalized Shannon Entropy values of the samples, the gene that has the most negative correlations is CDKN2C (cyclin-dependent kinase inhibitor 2C -p18, inhibits CDK4 -NM_078626), which has been previously associated with the transition from prostatic intraepithelial neoplasia (PIN) to prostate cancer [68] Figure 12 . We plot the values of two modified statistical complexities, which we will call M-Gleason 3 and M- Gleason 5. Instead of using the equiprobable distribution as our probability distribution of reference (for the computation of the Jensen-Shannon Divergence of the gene expression profile to this distribution), as required for the MPR-Statistical Complexity calculation, we used a different one. For the M-Gleason 3, the probability distribution of the reference is obtained averaging all the probability distributions of the samples that have been labelled as Gleason 3 (analogously, we calculated M- Gleason 5) . This is analogous to our approach in melanoma ( Figure 5 ) in which we used normal and metastatic samples as reference sets for a modified statistical complexity. We observe that, even in this case, 02_003E and 03_063 continue to appear as outliers. In addition to the evidence, we have observed that the deletion of these two samples did not significantly alter the identification of biomarkers. doi:10.1371/journal.pone.0012262.g013 Samples 02_003E and 03_063 seem to be outliers to this trend, and in the case of 03_063 the sample is not even close to a hypothetical linear fit which seems to be the norm for all the samples. Figure 13 will provide further evidence that may indicate that these two samples are outliers or not to the overall trend. doi:10.1371/journal.pone.0012262.g012 with the dedifferentiation process, with preoperative PSA levels and the percent of Gleason 4 and 5 cancers [338] .
AMACR, Cyclin G2, CDK4 and CDK7. Other probes that also have high negative correlations with the Shannon Normalized Entropy correspond to CCNG2 (Cyclin G2) CR598707, CDK4 (Cyclin-dependent kinase 4), CDK7 (Cyclin-dependent kinase 7, TFIIH basal transcription factor complex kinase subunit) [339] , and AMACR (Alpha-methylacyl-CoA racemase), an ''obscure metabolic enzyme (that has taken) centre stage'' [340] as judged by the extraordinary convergence to this biomarker in prostate. We believe that our result is an important finding. AMACR was not judged of importance according to the methodology used in [332] and it was barely cited in that manuscript. Here we present results, from an unifying biological and informational principle, which allows (using Ref. [332] 's own data) the identification of the most central current biomarker with a truly compelling body of support in independent studies [ [490, 491] . Knockdown of BRCA1 results in the accumulation of multinucleated cells, indicating that BRCA1 regulates gene expression of an orderly progression during mitosis [492] , preserving chromosomal stability [490] . BRCA1 showed decreased expression in a study involving immortalized prostate epithelial cells before and after their conversion to tumorigenicity [493] . Lack of BRCA1 function may impair activation of STAT3 [494] . Inactivation of TP53 by somatic mutations is also associated to the panel of disruptions which are common for this ''tumor suppressor'' [113] . One possible mechanism for gene silencing is CpG island methylation. Rabiau et al.show in [495] that BRCA1, RASSF1, GSTP1 and EPHB2 promoter methylation is common in prostate biopsy samples. Mannicia et al. suggest that the mitochondrial localization of BRCA1 proteins may be a significant factor in regulating the mitochondrial DNA damage [5] .
SFPQ -(Polypyrimidine tract-binding protein-associated splicing factor). The most positively correlated gene with the loss of Normalized Shannon Entropy is SFPQ/PSF (Polypyrimidine tract-binding protein-associated splicing factor) (Spearman correlation of 0.7902), a multifaceted nuclear factor [496, 497] which is also a putative regulator of growth factor-stimulated gene expression [498] . This is extremely interesting as it has been recently shown that the AR/PSF complex interacts with human PSA gene and that PSF inhibits AR transcriptional activity [499] . The loss of expression of SFPQ and other proteins that together regulate androgen receptor-mediated gene transcription [500] (see also [501, 502] ) may indicate they have a role not only as a biomarker of the progression and well as transitions of the disease to androgen independence. In a study of human labor, Dong et al., also showed that SFPQ acts as a Progesterone Receptor corepressor, thus putatively contributing to the functional withdrawal of progesterone [503] . We will return to this particular gene later on the 'Discussion' section as new evidence of its role in nuclear organization has been documented.
CD40 -(TNFRSF5, B-cell surface antigen CD40). The loss of Normalized Shannon Entropy gives us several markers that indicate a de-differentiation from a epithelial basal phenotype and an increasing loss of control of cell cycle regulation (due to uncoordinated upregulation of CDK4, CDK7, CCNG2 with their functional partners). This poses the question: What can we observe while looking at the genes that most positively correlate with the loss of Normalized Shannon Entropy? We observe, second on the ranking of all samples, a probe for CD40 (TNFRSF5, B-cell surface antigen CD40), BX381481 with a Spearman correlation of 0.7616. Loss of CD40 expression has been previously reported in prostate cancer and it is the object of a study that attempts to establish dendritic cell gene therapies [504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522] . We will continue discussing CD40 in the following subsection in concert with other genes.
Another natural question can be asked: Which is the extra information that we can obtain the by analysing the correlations with the MPR-Statistical Complexity in this case? As we have discussed before, and can be appreciated from Figure 14 , there is a strong correlation between the MPR-Statistical Complexity and the value of the Normalized Shannon Entropy. It appears in prostate cancer, as in this gene expression dataset, the reduction of Entropy is not the major factor responsible for the increase in MPR-Statistical Complexity. Again, it is perhaps better to now look at one of the multiplicative factors of the statistical complexity measure, the Jensen-Shannon divergence to the equiprobability distribution, as this is increasing the MPR-complexity.
CD40. We present more evidence of the case of CD40 as a biomarker, since a probe for CD40 (BX381481) ranks 6 th (the Spearman correlation of the probe expression with the Jensen-Shannon divergence from the equiprobability distribution is 20.5764). CD40 is a member of the TNF receptor superfamily. Notably, in 56 out of 57 archival prostate cancer samples Palmer et al. have reported no CD40 expression [518] . However, CD40 expression was present in normal prostatic acini, so they proposed that ''invasive prostate cancer is a CD40-negative tumour'' (see the previous results of Moghaddami et. al. [514] ). Matching our observations, they proposed that CD40 provides ''insight into progression of cancer from normal epithelium''; our proposed methodology is revealing this fact as well. Depletion of CD40 in the tumour microenvironment may be central in avoiding the action of the immune system [506] , as prostate cancer induces a progressive suppression of the dendritic cell system [520] . It is perhaps a central piece which should be put together in the context of other pieces of information coming from immunotherapy [508, 512, 513, 516] and pharmacological studies [507] that warrant serious investigation towards the design of new and improved clinical studies [508, 517] .
CD59 molecule, complement regulatory protein. Four probes for protectin [335, 523, 524] , CD59, with Spearman correlations with the Jensen-Shannon divergence from the equiprobable distribution, ranging from 20.61823 to 20.5089, rank between the 1 st and 39 th position (when we rank genes according to this correlation in ascending order). CD59 is an interesting gene as ''a comprehensive investigation of CD59 expression in prostate cancer has not been conducted yet'' [524] . Like LMNA (which is ranked third and will be discussed later) the rank of CD59/ protectin means that these genes progressively loose expression of these probes. CD59 is expressed in the prostatic epithelium [525] and in prostasomes [526] ; secretory granules which are produced, stored and released by the glandular epithelial cells of the prostate [527] . Babiker et al. concluded in [335] that prostasomes (via expression CD59) contribute to the protection of malignant cells from complement attack. We now investigate if the ratio of deltacatenin to CD59 can is a more robust biomarker for non-invasive prostate cancer detection, particularly after the results presented in [528] . We also note that CD59 may be also relevant to reveal the heterogeneous nature of prostate cancer. Its correlation was good, but is not lower than 20.62, which in our experience, indicates that we may be dealing with at least two types tumors in this dataset. Indeed, Xu et al. obtained CD59 mRNA levels were determined by real-time PCR in matched (tumor/normal) microdissected tissues from 26 cases and they found that: ''High rates of CD59 expression were noted in 36% of prostate cancer cases and were significantly associated with tumor pT stage (P = 0.043), Gleason grade (P = 0.013) and earlier biochemical (PSA) relapse in Kaplan-Meier analysis (P = 0.0013). On RNA level, we found an upregulation in 19.2% (five cases), although the general rate of CD59 transcript was significantly lower in tumor tissue (P = 0.03)'' [524] . They concluded that: ''CD59 protein is strongly expressed in 36% of adenocarcinomas of the prostate and and is associated with disease progression and adverse patient prognosis'' [524] . Jarvis et al. have previously hypothesized that CD59 expression, in some cancer cells, may help to regulate the immunological response, protecting them from the cytolytic activity of complement [523] (see also [529, 530] ).
LMNA (Lamin A/C). The third probe in the ranking corresponds to a LMNA (Lamin A/C), AY528714. Mutations on LMNA have been linked at 10 different human diseases [531, 532] . LMNA, due to its functions, could be involved in important cell fate decisions as lamins are involved in the organization of the functional state (and position) of interphase chromosome [531] . Lamins are ''scaffolders'' for the function of nuclear processes such as chromatin organization, DNA replication, cellular integrity and transcription [532] . As a consequence Lamins are involved in several clinical syndromes [533, 534, 535] . Among the recent functions attributed to LMNA is as an intrinsic modulator of ageing within adult stem cells via a mechanism where LMNA act as signalling receptors in the nucleus. These observations correspond to Pekovic and Hutchinson who observed that dysfunction of LMNA leads to inappropriate activation of self-renewal pathways and initiation of stress-induced senescense [536] . In lmna-deficient mouse embryonic fibroblasts (lmna(2/2) MEFs), the loss of lmna''dramatically affects the micromechanical properties of the cytoplasm'', since ''Both the elasticity (stretchiness) and the viscosity (propensity of a material to flow) of the cytoplasm in Lmna(2/2) MEFs are significantly reduced'' [537] . Using ballistic intracellular nanorheology to evaluate the micromechanical properties of the cytoplasm of these cells, Lee et al. conclude: ''Together these results show that both the mechanical properties of the cytoskeleton and cytoskeleton-based processes, including cell motility, coupled MTOC and nucleus dynamics, and cell polarization, depend critically on the integrity of the nuclear lamina, which suggest the existence of a functional mechanical connection between the nucleus and the cytoskeleton. These results also suggest that cell polarization during cell migration requires tight mechanical coupling between MTOC and nucleus, which is mediated by lamin A/C'' [537] (see also [538, 539] ). In addition to these very interesting findings, a functional association of LMNA and the retinoblastoma protein (pRB) exists. Nitta et al. have shown that pRB needs to be stabilized by LMNA for INK4A-mediated cell cycle arrest and that somatic mutations in LMNA may also have a role in tumor progression [540] . In mammalian cells, LMNA a) colocalizes with c-FOS at the nuclear envelope, b) suppresses AP-1 through a direct interaction with c-FOS and, in LMNA-null cells perinuclear localization of c-FOS is absent (but it is restored when it is overexpressed, c) LMNA-null cells have enhanced proliferation [74] . These results obtained by Ivorra et al. are giving the indication that of yet another mechanism of cell cycle and transcriptional control mediated by LMNA [74] (see also [541] ). LMNA has also been proposed as an inhibitor of adipocyte differentiation [542] . Hutchingson et al. have proposed the alias of ''guardian of the soma'' for lamins A and C as they seem to have ''essential functions in protecting cells from physical damage, as well as in maintaining the function of transcription factors required for the differentiation of adult stem cells'' [543] .
From our results, we can not completely establish if the downregulation of CD40 and CD59 are enough to pinpoint an impaired or abnormal immune response. If we continue the inspection of the list, the first 20 probes give us more supporting evidence. The 20 probes correspond to 13 different genes. Five of these 13 genes have Genome Ontology information annotated as ''defense response'', the above mentioned CD59 and CD40 as well as IL4R (interleukin 4 receptor, CR616481), XBP1 (X-box binding protein 1, AK093842) and HLA-A (major histocompatibility complex class I HLA-A29.1, BU075230). Takahashi et al. [544] report an inverse correlation between XBP1 expression and histological differentiation in a series of prostate cancers without hormonal therapy, the expression of XBP1 was localized in epithelial and adenocarcinoma cells of the prostate and the majority of refractory cancer cases exhibited weak XBP1 expression), MST1/STK4 (along with MST2/STK3) act as inhibitors of endogenous AKT1, a mediator of cell growth and survival [545] .
We can not yet know what reason is behind their joint downregulation, but another interesting common denominator is that 12 out of 13 genes share a regulatory motif for NF-kappaB (according to TRANSFAC, V$NFKB_Q6_01). A putative role for NF-kappaB in prostate cancer has been reported based on the observation of the centrality of NFKB on two up-and downregulated networks compairing prostate tumors and healthy tissue [546] and in a larger study by McDonnel et al. [547] (255 core prostate cancer tissue microarrays from 47 prostatectomy specimens). Several other researchers are currently investigating different roles of the NFKB family in prostate cancer [548, 549, 550, 551, 552, 553, 554, 555] and it could be a promising target for intervention [555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571 ]. If we include other genes following the ranking order, the first 38 genes in the ranking include 33 that have the regulatory motif V$NFKB_Q6_01 (GATHER reports for this list a p-value of 0.0006). Even when we double the list to the probes that correspond to the first 76 different genes recognized by GATHER, 58 of them have the regulatory motif V$NFKB_Q6_01, with p-value = 0.003 (ATP6AP2, BCAT1, BTG2 [572, 573, 574, 575, 576, 577, 578] , C14orf123, C18orf45, CCL2, CD302, CD40 (already discussed), CD59 (already discussed), CHI3L1, COL16A1, COMMD6, CRABP2, CSRP1, CTBP2, CTGF (Connective tissue growth factor, [579, 580, 581, 582] ), DES, DMN, DNAJB1, EGF, EMP1, FHL2 [583, 584, 585, 586, 587, 588] , GRIPAP1, GSTM1 [589, 590] , HBEGF, IL4R, ITGA3, ITGA7, JUNB [591, 592] , KIAA0152, KIAA1191, KIAA1324, KLF6, LAMB2, LMNA (already discussed), NFATC1, NFKB2, NUDC [593] , P4HB, PDK2, PIM1, PISD, PXN, RAP1B, RNF40, SARA1, SEC61A1, SGTA [594] , SLC12A2, SRD5A2, STAT6 [595, 596] , TACSTD2, TBX1, TMED3, VPS39, WDFY3, XBP1 [544] , ZAK). This result indicates that our results support the importance of NFkappa-B and the huge amount of research effort to understand the role of the NFkappa-B activity and its potential as a target for intervention in prostate cancer (File S4).
The group of 58 biomarkers contains one of particular interest, STAT6. This gene is considered a survival factor in prostate cancer and a key regulator of the genetic transcriptional program responsible for progression [595] . STAT6 has been recently linked to HPN as one of the most robust pair of biomarkers for prostate cancer using an integrative approach that linked several microarray datasets [596] .
Analysis using GATHER of this group reveals that six of these 58 genes are in KEGG pathway path:hsa04510, Focal adhesion (EGF, ITGA3, ITGA7, LAMB2, PXN, RAP1B, p-value,0.0007) and from these there are three in pathway:hsa045122, ECM-receptor interaction (ITGA3, ITGA7, LAMB2, p-value,0.005) while four of these six are also in path:hsa04810: Regulation of actin cytoskeleton, (EGF, ITGA3, ITGA7, PXN, p-value,0.01).
LAMB2. Alterations of the gene profile of LAMB2 and CDKN2C/p18(Ink4c), a CDK4 inhibitor, have been reported on the transition from prostatic intraepithelial neoplasia (PIN) to prostate cancer [597] (see also [333] ).
3). The contribution of the loss of these integrins and the subsequent derived impairment on cell adhesion has been reported in several tumours. Ren et al. in [598] report that ''Focal or no integrin alpha 7 eexpression in human prostate cancer and soft tissue leiomyosarcoma was associated with a reduction of metastasis-free survival (for example, for prostate cancer with focal or no expression, 5-year metastasis-free survival was 32%, 95% CI = 24.4% to 40.3%, and for prostate cancer with at least weak expression, it was 85%, 95% CI = 79% to 91%; p-value,.001)''.
''Any method involving the notion of entropy, the very existence of which depends on the second law of thermodynamics, will doubtless seem to many far-fetched, and may repel beginners as obscure and difficult of comprehension.'' Willard Gibbs, Graphical Methods in the Thermodynamics of Fluids, (1873)
The changes of the Normalized Shannon Entropy and Statistical Complexity of the gene expression profile of a cancer cell are associated with the gradual deterioration of genome transcriptional information content due to the modification of its structural and functional integrity during disease progression. Our results clearly suggest that we can track the cancer cell's progression by following observable changes in the Shannon Entropy and, in particular, by employing the Jensen-Shannon Divergence of the gene expression profile of a sample to the normal expression profile. We have also shown if an average expression profile of some state of interest can be properly defined (i.e. distant metastasis) then the Jensen-Shannon Divergence can help us to identify which probes best correlate with these measures resulting in useful biomarkers.
Before any thermodynamical consideration could be discussed, we note that there is a clear and objective informational perspective that our study delivers. In this study we have chosen to position ourselves as the 'receivers' of a 'transcriptional message'. In this experimental perspective the tumor tissue is the 'sender' (the source of information) and the high-throughput technology (gene expression microarrays in this case) can be regarded as the transmission medium (providing noise and distortion). As we explain in the 'Materials and Methods' section, the Shannon Entropy of a gene expression profile is the average expected surprisal of that profile understood as a message. The Normalized Shannon Entropy makes this surprisal an intensive measure and the correlation of the gene expression patterns across samples with this measure can deliver useful biomarkers to track the progression of transcriptional change. After normalization, we have a measure that does not depend of the number of probes of the high-throughput technology, although, it obviously does depend on the type of probes used.
We believe that the readers may have already noticed an apparent paradox. While some researchers understand cancer progression as a mechanism that increases entropy, we actually observe a reduction of Normalized Shannon Entropy in this work. This means that our normalized average expected surprisal, as receivers of the transcriptional message, is smaller. We must then discuss the physical meaning of thermodynamic entropy, its current use in systems biology and cancer research genetics and the informational measure we use in this paper to clarify these notions in this context.
In biomedical research there exists a certain consensus among cancer researchers that genetic instability or ''mutability'' is a major critical force of cancer progression, but it is not the only one to consider. It is clear that the mutational damage of key genes (like TP53, TERT, BRCA1, RB1, etc.), and the collective damage inflicted on key DNA repair mechanisms (like Nucleotide-excision repair and Base-excision repair) collaborate for an increasing acceleration of the number of genomic changes. Sub-microscopic alterations of the genome accumulate in cancer progression in an irreversible way and ''are compounded by the widespread scrambling of the chromosome structure, and thus the karyotype, found in cells from the great majority of solid tumours'' [599] . In Weinberg's own words [599] : ''we learned that this chromosomal chaos also contributes this progression forward''.
This ''chromosomal chaos'' [600] or ''cancer as a chromosomal disease'' perspective is viewed by some researchers not as just a side consequence of mutational damage, but as the main core theme to understand a number of unexplained issues in cancer progression. ''In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy'' [601] . Regarding the cancer types studied in this paper, one particular ''measure of disorder of a system'', aneuploidy, has been observed in poorly-differentiated prostate cancer cells and it is often associated with a more agreessive phenotype [602, 603] , increased PSA levels [604, 605] , and correlate with Gleason score [606, 607, 608] . Gene fusions and chromosomal rearrangements are other source of increase in the ''disorder'' of the genome organization and they are increasingly being recognized as a major player in prostate cancer progression [609] . The increase in ''karyotypic complexity'' and ''extended aneuploidy and heteroploidy'' may be already enough to develop a malignant melanoma phenotype, as the report of Gagos et al. indicate [610] . The observed finding of aneuploidy in melanoma (also including uveal melanoma) is also increasingly important due to a number of different independent observations [247, 611, 612, 613, 614, 615, 616, 617, 618] . It is in this context that the word 'entropy' has been used.
The magnitude of the ''chromosomal chaos'' is also evident from comparative genomic hybridization (CGH) studies which show significant variations in the copy number of individual chromosomal segments. 'Chaos' is really a very appropriate word to describe what we observe from CGH data. The genomic changes are not distributed uniformly at random. 'Chaos' has been described by some researchers as ''a kind of order without any periodicity''. Some common changes seem to consistently appear in several independently arising tumours of the same type, and sometimes the researchers suggest common links [619] . Our work has addressed, in part, this question: ''Can we quantify the chaos observed in the genome from the increasingly available transcriptional data and relate it to tumour progression?'' If no commonalities were observed, we would not have found interesting biomarkers that seem that strongly correlate with the divergences from normal tissue types. We know from our results that these commonalities do occur.
We need to go back to basics to explain these evolving concepts and resolve this apparent paradox. The phrase ''karyotypic entropy'' has been used in the past to define what is actually a divergence from the normal chromosome structure and it genomic organization. This denomination has also been employed by several authors, notably [601] , but it has also been used in at least two other publications [620, 621] . These works have in common the use of this term to refer to a ''disorder'', fuelled by the undergraduate textbooks indoctrination of associating increase of entropy in natural spontaneous processes with the increase of ''observed disorder'' in the system. We propose that the use of a natural measure of divergence, the Jensen-Shannon divergence, could not only be a more formal, but also more appropriate modelling approach. As such, we propose to introduce the term 'karyotypic divergence' or 'karyotypic Jensen-Shannon divergence' to replace this concept and to avoid a subjective approach.
Why is it the case that we observe the Normalized Shannon Entropy of the transcriptional profile decreasing with cancer progression when intuitively our average expected surprisal (Shannon Entropy) should increase with progression?
Arieh Ben-Naim in his recent book ''A farewell to Entropy: Statistical Thermodynamics based on Information'' [622] comments:''It is interesting to note that Landsberg (1978) not only contended that disorder is an ill-defined concept, but actually made the assertion that 'it is reasonable to expect 'disorder' to be an intensive variable'''. Ben-Naim also states: ''In my view, it does not make any difference if you refer to information or to disorder, as subjective or objective. What matters is that order and disorder are not well-defined scientific concepts. On the other hand, information is a well-defined scientific quantity, as much as a point or a line are scientific in geometry, or mass or charge of a particle are scientific in physics.'' However, in a manuscript entitled ''Can Entropy and 'order' increase together ?'' Landberg defines (in an attempt to decouple the notions of order and entropy), for a thermodynamical system that can be on N states the 'disorder' D(N) to be the Normalized Entropy (which is a function of N) divided by Boltzmann's constant [623] . 'Disorder' then is an intensive magnitude bounded by 0 and 1, and 'order' is defined as 1-D(N) .
While Landberg's decoupling argument between order and entropy [623] may still be controversial in Physics, the question is pertinent for our apparent paradox (the question that motivates this subsection). Borrowing from the title of his paper we could now state the central question as ''Can Shannon Entropy increase while the Normalized Shannon Entropy decrease?'' The solution of this apparent paradox is a trick of escapologism, perhaps also paralleled by what a cancer cell may be experiencing (or ''reacting'' in response to increased sources of stresses), and it is worth discussing in this context. Let H[X] be Shannon Entropy for an ensamble X with N different values. We will now assume, and here is the trick, that N is not a constant, but a function of time N(t). Let D(X (N(t) )) be the Normalized Shannon Entropy. By definition D(X(N(t))) = H(X(N(t)))/log 2 (N(t)). Then, just by taking the time derivatives it can be shown that the time variation of D(X(N(t))) can be negative, although the time rate of H[X] can be positive.
where k is a constant. The escape to our paradox is ''achieved'' via making explicit the time variability of N(t). Landberg explicitly mentions that biological systems are examples where growth processes increase N(t), and perhaps the increased diversity in the transcriptome of a cancer cell during progression is one of such examples. This discussion somehow resolves the apparent disassociations due to language barriers that may exist between the different disciplines (physics, information theory, molecular biology and oncology). A biologist may regard a cancer cell as an entity that, during progression, may ''spread'' its transcriptomic profile, including the generation of a large number of novel molecular species (due to adquired characteristics during its ''devolution'' from the normal type). In our informational perspective, this would be analogous to a situation in which the sender of a message, after some time, decides to increase the size of the alphabet of transmitted symbols. Clearly, it is intuitive to think that the receiver would be in a situation of increased Shannon Entropy. However, if the receiver is not aware of the new symbols (or is not able to detect them) and some of the symbols of the previous alphabet are no longer used, the receiver would now perceive a reduction of Normalized Shannon Entropy, observing an increasing order.
We now borrow an illustrative example from Landberg [623] , but we add a twist to this argument for the purpose of illustrating this discussion. Suppose we have a sender transmitting only two possible symbols (N = 2), and we will assume that we have the same probability, let's denote this as (1/2, 1/2). Then the average expected surprisal (Shannon Entropy), is H(X) = 1, and the Normalized Shannon Entropy is also equal to one. Assume now that now our sender starts to transmit using another symbol, so that we now have theoretical probabilities of (0.5, 0.25, 0.25). Then N = 3, and the average expected surprisal increases to H(X9) = 1.5 the Normalized Shannon Entropy is now 1.5/log 2 (3) = 0.946… (a reduction). This 'third symbol' could actually represent a new ''molecular species'' or a protein isoform that would not be normally expressed in that tissue type [624] , or even something entirely new, product of a mutational/deletional event. If our hypothetical high-throughput technology can only be detecting the first two symbols, and following the conventions we established in the 'Materials and Methods' section, we would be ''observing'' frequencies of (2/3,1/ 3) since the other events would not be detected with our equipment. As a consequence, the both the log 2 (2) = 1, Shannon Entropy and the Normalized Shannon Entropy are both reduced to 0.918293. Obviously, we can not count what we can not observe. As a consequence, a degenerating transcriptional profile that produces novel molecular species, and at the same time reduces those which we can not measure with a particular technology, would look increasingly more ordered.
We envision that physicists may find here a fertile ground to explore new ideas and attempt novel mathematical formalisms for cancer progression from the realm of finite-state thermodynamics [625] and in particular endorevesible processes [626] and endoreversible thermodynamics [627] . Some molecular alterations would then be part of the set of revesible processes that could occur in a cancer cell, while other processes like aneuploidy or gene fusions could be truly ''irreversible genetic switches'' associated with cancer progression [628] . If we assume that the process is slow (i.e. the times required for significant variations of the transcriptome's profile is large in comparison with the cell's processes time scales), and follwing the results of Spirkl and Reis [626] , it may be possible that we have a constant entropy production rate exists during cancer progression leading to Hauptmann's ''entropic devolution'' [629] . Hauptmann sees a malignant tumour as ''a dissipative structure arising within the thermodynamical open system of the human body'' that starts when ''a localized surplus of energy exists and there is no possibility to export entropy. An energetic overload in most malignant cells is indicated by their abnormally high phosphorylation state.'' His perspective, preceeded in part by Dimitrov [630] , Klimek [631, 632] and Marinescu and Viculetz [633] might then fit well an endoreversible thermodynamic formalism. Hauptmann says in [629] ''I believe that cancer is a special kind of adaptation to energetic overload, characterized by multiplication and mutation of genomic DNA (generation of new biomolecules which enhance the probability of survival under harmful conditions), and by chiral alterations (reduction of entropy by entrapping energy) leading to abnormal configurated biomolecules. In this regard the genetic alterations are probably secondary changes. Cancer serves to dissipate energy in a type of developmental process but one in which the results are harmful to the whole organism: an entropic devolution.'' This thermodynamical perspective is now worth exploring and we will discuss it in this context. Assuming that a cancer cell is in a state of ''energy overload'', without ''the possibility of exporting entropy'', could it lead to some type of ''genetic alterations''? Which key mechanisms might be impaired? What consequences is this ''system'' delivering? Could this be another hallmark for oncosystems indentification?
In 1871, in this book called ''Theory of Heat'', Maxwell speculated the idea of ''a being, who can see the individual molecules'' and who has enough reactive intelligence to open and close a unique small hole existing between two communicating vessels (called 'A' and 'B'). An ideal gas filled both vessels, so that starting at uniform temperature the intelligent being could observe the molecules and close and open the hole accordingly to a mission: ''to allow only the swifter molecules to pass from A to B, and only the slower ones pass from B to A.'' The being, ''without expenditure of work raise the temperature of B and lower that of A in contradiction to the second law of thermodynamics.'' The ability of the ''being'' to use observable information about the system to lower the thermodynamical entropy has motivated many articles in physics and fuelled the imagination of many since it was originally introduced by Mawell, and named as ''demon'' by Thomson three years later [622] . An excellent collection of articles until 1990 [634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644] was edited by Leff and Rex [645] . The Maxwell ''demon'', far from being ''exorcised'' from Physics, still inspires interesting new perspectives [634,635,636,637,638,639,640,641,642,643,644,646,647 ].
In a letter to Peter Guthrie Tait, Maxwell writes about the ''demons'': ''Is the production of an inequality of temperature their only occupation? No, for less intelligent demons can produce a difference in pressure as well as temperature by merely allowing all particles going in one direction while stopping all those going the other way. This reduces the demon to a valve. As such value him. Call him no more a demon but a valve like that of the hydraulic ram, suppose.'' (from [645] , p. 6). Maxwell gives again here a sign of his brilliant mind, ''degrading'' the demon to a valve, but also offering an inspiring perspective to oncosystems research. Which types of mechanisms exist in biological systems, and particularly in individual cells, to control these differential values in key parameters? Could changes of key physical parameters for metabolic processes of the cytoplasm and cell's organelles like temperature, volume, pH or electrochemical potentials be also implicated in cancer progression?
The influence of temperature may be giving an interesting working hypothesis for further research. What are the consequences if cancer cells are a different type of open system which also operates at a different temperature than a normal cell? Butler et al. have studied p53 and they argue that at temperatures above 37 degrees centigrades wild-type p53 spontaneously loses DNA binding activity. While folding kinetics do not show important changes in a range from 5 to 35 degrees C, the unfolding rates accelerate 10,000-fold. This leads to a somewhat unexpected mechanism of p53 inactivation. It could be the case that a fraction of p53 molecules become trapped in misfolded conformations with each folding-unfolding cycle due to the increased frequency of cycling. The occurrence of misfolded p53 proteins can lead to aggregation and subsequent ubiquitination in the cell, leading to p53 inactivation [648, 649] . If a key ''guardian of the genome integrity'' [650, 651] and its remarkable conformational flexibility [652] is challenged by an increase of temperature [653] , its role in genotoxic damage and adaptive response (like that of the skin to UVB damage [654] ) may be impaired. The same may occur for other members of the DNA damage response. An increment in temperature has already been linked to skin carcinogenesis. Boukamp et al. report in that [655] ''exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 40 degrees C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors.''
On the other hand, natural gradients on physical biochemical properties can also be challenged in a cancer cell. This in turn derives in metabolic processes running under abnormal parametric circumstances. It is well-known that compartimentalization, in biological systems, naturally require the existence of mechanisms that would keep some key state variables relatively constant, or within bounds, for normal operation of the metabolic processes. One example is very illustrative and a case in point. Instead of demons, holes, or valves, the cell requires pores in its membranes to allow osmotic regulatory processes, yet it should preclude the conduction of protons. This is a nanotechnological design problem not faced by Maxwell, but certainly solved by biological systems without the need of an ''intelligent being'' as Mawell cleverly pointed to Tait in his letter.
This discussion brings us to one of the gene families we have already discussed in this paper, the aquaporins [184, 656, 657, 658, 659, 660, 661] . They are considered the primary water channels of cell membranes [662, 663, 664, 665] . The specific functions of each member of this family are now being slowly mapped by several research labs around the world [666] . Their clinical role in cancer [667, 668, 669, 670, 671, 672, 673, 674, 675] ,obesity [676] , malaria [677, 678] and other diseases is emerging [657, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689] . In [690] , our group observed the dowregulation of AQP3 in all melanoma cell lines studied of the NCI-60 dataset of Ross et al.; this dowregulation was also observed for the CNS and Renal cell lines. AQP3 was relatively upregulated for Leukaemia and Colon cell-lines (we refer the reader to the Supplementary Material of [690] for details). Inhibition of AQP3 in prostate cancer cells was already proposed as a mechanism that increases the sensitivity to cryotherapy treatment [691] .
The aquaporins are not ''an intelligent being'' in any real sense, yet they are so formidable selective that they could easily parallel Maxwell demon's efficiency in creating the right conditions for the cell. Wu et al. give us some clues on the role of point mutations in the AQP1 and how their effective electrostatic proton barrier can be impaired [692] . The elicitation of the detailed mechanistic explanation of this extraordinary selectivity is under intense investigation with a number of techniques, including sophisticated molecular dyanamics simulations, for an overview of this field see [665, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708] . One less known feature of aquaporins is that they may not only channel water, but also carbon dioxide and ammonia [709, 710, 711] , glycerol [712] and urea and other small solutes [713] and, very relevant for cancer research, hydrogen peroxide [188] . At least two of members of this family have been observed in the inner mitochondrial membrane in different tissues. This in turn may indicate mitochondrial roles for aquapotins in osmotic swelling induced by apoptotic stimuli [714] .
Could it be possible that we can track cancer progression by looking at some of these ''Maxwell demons''? We have seen in Figure 10 , that AQP3 has a reduced expression with increased progression in our melanoma dataset. Cao et al., reported that ultraviolet radiation induced AQP3 down-regulation in human karatinocytes; thus AQP3 has become a strong and plausible link between UV radiation, skin dehydration [186, 715] and photoaging [189] . This may indicate an impared function on skin hydration [184, 185, 716, 717, 718, 719] . The expression of AQP3, as well as AQP1, AQP5, and AQP9 seem to be correlated with melanoma progression, indicating a common pattern of downregulation from the higher values in normal skin and benign nevi (see Figure 17 ).
Does a similar pattern of aquaporin downregulation exist in prostate cancer? Wang et al. have looked at the expression and localization of AQP3 in human prostate using cell lines as well as patient samples. They have observed AQP3 mRNA ''in both normal and cancerous epithelia of human prostate tissues, but not in the mesenchyme. In the normal epithelia of the prostate, localization was limited to cell membranes, particularly the basolateral membranes. However, the expression of AQP3 protein in the cancer epithelia was not observed on the cell membranes.'' This finding seems to implicate the subcellular localization of AQP3 as a possible indicator of a transition to a more malignant phenotype. Lapointe's dataset allows us to see the downregulation of AQP3 and AQP1. A large subgroup of primary prostate tumors has reduced levels of AQP3 and AQP1 as most of the lymph node metastasis samples [ Figure 18 ].
One critique that we are aware we could receive is that the current manuscript presents a novel methodology and an underlying unifying theory based on retrodictions or postdictions. Indeed we have shown that the use of the Normalized Shannon Entropy and the Information Theory quantifiers (the M-complexities and the Jensen-Shannon divergence) allow to monitor cancer progression and to identify the best biomarkers that correlate with the transcriptomic changes. Our approach works in a retrodiction way in that it looks at data already obtained by other studies, but gives a unifying framework to track cancer progression. For instance, on True et al's dataset, our unifying hallmark of cancer gives not only MAOA, which was already identified in the original publication, but also AMACR, CD40, CDK4, etc. are very important biomarkers for prostate cancer. Analogously, the identification of KLK3/PSA in Lapointe's dataset is another important retrodiction which shows the power of the method.
In some sense our approach also works in a postdiction way, as it helps to evaluate the speculation that cancer cells have ''an entropic devolution''. Our results show that the variations of Normalized Shannon Entropy and Jensen-Shannon divergences indeed give measurable changes, and that these changes are related to important biomarkers in the two types of cancer studied in this work.
In addition, we remark that we are literally making hundreds, or even thousands of predictions. The results in the 'Supplementary Material' provide this information for the detailed scrutiny of our peers. We believe that other probes with gene expression patterns in high correlation with the probes discussed in this paper, and perhaps less studied by immunohistochemistry and other methods in the two cancer types studied here, are worth exploring as a group of biomarkers. These predictions can be tested with further studies on staging and patient stratification.
A very recent study by Ballal et al. have linked BRCA1 to telomere length and maintenance and its loss from the telomere in response to DNA damage [720] (see also [721] ). We have previously mentioned that BRCA1 is a conspiquous biomarker arising from the analysis of True et al.'s dataset using our methods. We found this to correlate with a preivous study that showed that BRCA1 has a reduced expression in immortalized prostate epithelial cells before and after their conversion to tumorigenicity [493] . We also mentioned that the knockdown of BRCA1 leads to anaccumulation of multinucleated cells [492] , preserving chromosomal stability [490] . Ballal et al. telomeric ChIP assays to detect BRCA1 at the telomere and reported time-dependent loss of BRCA1 from the telomere following DNA damage. Due to the role of telomeres in maintaining chromosomal stability [722] and the inverse correlation of telomere length and divergent karyotypes in prostate cancer cell lines [723, 724] (as well as the recognized role of telomere dysfunction in the induction of apoptosis or senescence in vivo [725, 726, 727, 728, 729, 730] , increase of mutation rates [731] , DNA fragmentation [732] , and their relation with DNA damage signalling [733] ), we checked for other probes of genes involved in telomeric function.
From those which we were able to identify in True et al's dataset, we have found a strong high correlation of the expression of BRCA1 with TERF2/TRF2 (telomeric repeat binding factor 2) [734] and a negative correlation with the expression pattern of TERF2IP (telomeric repeat binding factor 2, interacting protein) [ Figure 19 ].
Finally, one particular type of probes has also caught our attention, and we would like to refer to them before concluding this section.
With the denomination of 'non-coding RNA' we identify those RNA molecules which are functional but that are not translated into proteins. Many microarray chips contain probes that are annotated as 'non-protein coding', indicating that there might be some valuable expression data that we can also mine for information. We note that our method, although employing transcriptomic data, does not limit its application to proteincoding information, and that the combined use of protein-coding and non-coding protein probe expression would allow a more comprehensive view of the transcriptional state of the cell.
Among non-protein coding, microRNAs [735] are gaining acceptance as key players in several cancers [736, 737, 738] (including prostate cancer [739, 740] ), but the so-called ''long noncoding RNAs'' [741] are also gaining a place in the scenario of cancer biomarkers (see [742] , and [743, 744, 745] ). We thus turned our attention to these probes that have been annotated as ''nonprotein coding'' and we highlight some of them that have very high correlation values with the Normalized Shannon Entropy in True et al's prostate cancer dataset. In particular, the probes for MALAT1/ MALAT-1 [742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758] have a very conspiquous position (See Figure 20) . They located very closely to other protein coding biomarkers that have also lost expression and have been discussed in this work like SFPQ, CD40, BRCA1, and TP53 (see Figure 16 ). MALAT1 has been recently pointed as a biomarker in primary human lobular breast cancer as a result of an analysis of over 132,000 Roche 454 highconfidence deep sequencing reads [749] . An international team, searching on thousands of novel non-coding transcripts of the breast cancer transcriptome, has been able to identify more than three hundred reads corresponding to MALAT1 [749] . This is a noncoding RNA which was identified in 2003 in non-small cell lung cancer, was shown to be highly expressed (relative to GAPDH) in lung, pancreas and prostate, but not in other tissues including muscle, skin, stomach, bone marrow, saliva, thyroid and adrenal glands, uterus and fetal liver [758] . MALAT-1, also known as NEAT2, is considered to be ''extraordinarily conseved for a noncoding RNA, more so than even XIST'' [754] . Our results indicate that the reduction of expression of some non-coding RNAs, in particular of MALAT-1, and SNORA60 with respect to their normal expression in prostate, as well as the upregulation of SNHG8 and SNHG1 should be monitored as useful biomarkers to track disease progression.
We will now address another non-coding RNA called NEAT1 which, like NEAT2, is also conserved in the mammalian lingeage. Before we move onto NEAT1, we will first recall a previous result. We have noted before the conspiquous position of SFPQ/PSF (Polypyrimidine tract-binding protein-associated splicing factor) in Figure 16 . The expression of a probe for SPQF has the highest correlation with the values of the Normalized Shannon Entropy. We highlighted before that SFPQ/PSF is a putative regulator of growth factor-stimulated gene expression [498] . The loss of SFPQ expression during the progression of prostate cancer may be an important key to understand this disease or one of its subtypes. We have also mentioned that the AR/PSF complex interacts with the PSA gene (perhaps the most well-established prostate cancer biomarker) and that SFPQ/PSF inhibits AR transcriptional activity [499] . Kuwahara et al. showed that SFPQ together with NONO (Non-POU-domain-containing, octamer binding protein) and PSPC1 (Paraspeckle protein 1 alpha isoform, formerly known as PSP1) are expressed in mouse Sertoli cells of the testis and form complexes that function as coregulators of androgen receptormediated transcription [500] . While new research results [759] link SFPQ and NONO/P54NRB with the RAD51 family of proteins (largely regarded as another key protector of chromosome integrity as being involved in homologous recombination DNA repair), it is perhaps SFPQ and NONO's co-localization in paraspeckles that make this group also remarkable [760] .
Paraspeckles [760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777] are a novel nuclear compartment, of approximately 0.2-1 mm in size, discovered in 2002, by Fox et al. in Dundee Scotland, following the identification of the protein PSPC1 (AF448795) in the nucleolar proteomics project at Lamond's lab which is described well by Fox et al. [777] . Three years later, Fox, Bond and Lamond showed that NONO and PSPC1 form a heterodimer that localizes to paraspeckles in an RNA-dependent manner [773] . Paraspeckles are dynamic structures, observed in numbers that vary between 10 and 20, that seem to control gene expression via retention of RNA in the nucleus [772] . A long noncoding RNA called NEAT1/MEN epsilon/beta [754, 760, 762, 764, 778] , that colocalizes with paraspeckles, seems to be integral to their structure. Depletion of NEAT1 erradicates paraspeckles and a biochemical analysis by Clemson et al indicates that the NEAT1 binds with paraspeckle proteins SFPQ/PSF, P54NRB/NONO and PSPC1. NEAT1 is also known as TncRNA (trophoblast-derived noncoding RNA) [754, 779, 780, 781, 782, 783, 784, 785, 786] and probes for TncRNA exist on this dataset, We have observed in True et al.'s dataset that there exists a high correlation between the Normalized Shannon Entropy with the expression of SFPQ/PSF, P54NRB/NONO, and TncRNA. Overall, this implies that the disruption of the function of the paraspeckles is correlated with the increasing signs of deterioration of normal transcriptomic state of the cells. While a causal relationship still needs to be proved, we admire the mathematical elegance of the Normalized Shannon Entropy of the samples, a global measure of the average expected surprisal of the transcriptome, which in turn has lead us to consider the dysfunction of the smallest nuclear body as a putative biomarker of disease progression. The role of SFPQ/PSF in the control of tumorigenesis is under investigation [787] and the information coming from these studies would need to be integrated with their role, together with P54NRB/NONO and TncRNA, in paraspeckles if we want to achieve a better understanding of these mechanisms.
In this contribution we have shown that for the melanoma and prostate cancer datasets studied, the quantitative changes of Information Theory measures, Normalized Shannon Entropy, Jensen- Figure 19 . The stacked average gene expression of probes corresponding to BRCA1 and TERF2 (telomeric repeat binding factor 2) in True et al's prostate cancer dataset. The first group of samples (1 to 9 in green) correspond to Gleason 3 pattern, indicating that most of the samples in this group have no significantly reduced expression of this pair of genes. The second group of columns (10 to 21 in yellow) correspond to Gleason 4 patterns and the last 8 columns (22 to 29 in red) correspond to Gleason 5 samples. A very recent study by Ballal et al. have linked BRCA1, to telomere length and maintenance and its loss from the telomere in response to DNA damage [720] (see also [721] ). There is an increasing trend of dowregulation, so it would be interesting to evaluate if indeed this pair of proteins could be an early marker of dowregulation useful to evaluate samples with Gleason pattern 2, or if may constitute a biomarker useful to distinguish a prostate cancer subtype. doi:10.1371/journal.pone.0012262.g019
Shannon divergence and the novel Statistical Complexity quantifiers defined here are in high correlation with gene expression changes of well-established biomarkers associated to cancer progression. In addition, variations of the basic technique (i.e. a modified form of statistical complexity) which allows us to better understand the phenotypic changes observed in these samples which are associated with the progression and the transitions of the gene expression profiles. For instance, in a properly defined Statistical Complexity vs. Entropy plane, on a melanoma dataset first studied in Ref. [110] , samples appear in well differentiated ''clusters''. These clusters correlate well with the phonotypic characteristics of normal skin, nevi, primary and metastatic melanoma. In this ''Complexity vs. Entropy'' plane, primary melanomas samples appear ''bridging'' benign nevi and metastatic melanoma samples. Our results may also suggest that the evolution of metastatic melanoma leads to at least two different subtypes.
The Normalized Shannon Entropy of a transcriptional sample profile is calculated associating the measured expression values of a gene with the relatively probability of being expressed. We have observed that, in general, the transcriptomes of tumour progressing cells tend to have lower values of Normalized Shannon Entropy than normal ones. Given a population of normal cells of a given tissue type it is then possible to compute useful measure of divergence of cancer cell profiles from the normal expression average profile, in terms of Information Theory quantifiers, the Shannon Eveness normalized entropy and generalized statistical complexity [788, 789, 790] .
In addition, our observation of the correlation of the statistical complexity of tumours with its natural progression allows an unprecedented way of finding biomarkers that links with the gradual deterioration of the genome integrity. The proposed methodology uncovered, for the first time, evidence of the putative role of impared centrosome cohesion in melanoma progression. Statistical complexity has then been able to pinpoint otherwise unrecognized biomarkers in concert with existing ones, reinforcing the view that ''chromosomal chaos'' and ''cancer as a chromosomal disease'' can be a useful guiding principle to understand the molecular biology of cancer and uncover the timeline of its progression. This is a powerful method to uncover ''oncosystems'' instead of ''oncogenes''. ''Oncosystems'' are a highly differentially disregulated set of genes that, if linked with the molecular ''hallmarks of cancer'' described in the introduction, and existing databases with putative common functional genomic annotations, can help to understand the biological progression pathways that drive the disease.
On one of the prostate cancer dataset studied (obtained from a previous published study, [44] ), we observe a gradual pattern of reduction of Normalized Shannon Entropy from three well characterized tissue types: normal prostate, primary prostate tumours and lymph node metastases. On a different dataset on prostate cancer (from Ref [332] ), we observe that a group of samples having Gleason Figure 16 is that we have now highlighted the position of s ome probes which have been annotated as corresponding to ''non-coding RNAs''. In particular, we highlight those of MALAT1 (Metastasis associated lung adenocarcinoma transcript 1, (non-protein coding)), SNORA60 (small nucleolar RNA, H/ACA box 60); both increasingly downregulated, SNHG1 (small nucleolar RNA host gene 1 (non-protein coding)) and SNHG8 (small nucleolar RNA host gene 8 (non-protein coding)). The probes for MALAT1/MALAT-1 [742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758] have a very conspiquous position, which we could judge a priori to be equivalent in relevance to those of the previously discussed roles of SFPQ, CD40, BRCA1, and TP53 (see Figure 16 ). MALAT1 has been recently pointed as a biomarker in primary human lobular breast cancer as a result of an analysis of over 132,000 Roche 454 high-confidence deep sequencing reads. Within the thousands of novel non-coding transcripts of the breast cancer transcriptome, Guffanti al., identified more than three hundred reads corresponding to MALAT1 [749] . This non-coding RNA, first identified in 2003 in non-small cell lung cancer, was shown to be highly expressed (relative to GAPDH) in lung, pancreas and prostate, but not in other tissues including muscle, skin, stomach, bone marrow, saliva, thyroid and adrenal glands, uterus and fetal liver (see figure four of Ref. [758] ). Our results indicate that the reduction of expression of some non-coding RNAs, in particular of MALAT-1, and SNORA60 with respect to their normal expression in prostate, as well as the upregulation of SNHG8 and SNHG1 should be monitored as useful biomarkers to track disease staging and progression to a more malignant phenotype. Interestingly enough, a study published in 2006 by Nadminty et al. has shown that KLK3/PSA modulates several genes, reporting a 16.5 fold downregulation of MALAT1 [810] . While these results have been obtained using the human osteosarcoma cell line SaOS-2, our results indicate that MALAT1 expression in the normal prostate and in cancer cells could also be considered as a relevant biomarkers to be tested in the future. doi:10.1371/journal.pone.0012262.g020 patterns 4 and 5 (two patterns which are typically associated to an aggressive phenotype) have lower Normalized Shannon Entropy values than a subset of Gleason pattern 3 (a pattern which is normally associated to a less aggressive phenotype but which nevertheless is still of clinical concern). However, a group of samples having Gleason patterns 3, 4, and 5 is revealed; this mixed cluster has a mid-range entropy. This is an interesting fact which correlates with the limitations observed in Ref. [332] . We note the authors' comment: ''We were unable to identify a cohort of genes that could distinguish between pattern 4 and 5 cancers with sufficiently high accuracy to be useful, suggesting a high degree of similarity between these cancer histologies or substantial molecular heterogeneity in one or both of these groups.'' Our results provide a conciliatory middle ground that explains the perceived clinical usefulness of Gleason pattern classification, widely used around the world, while at the same time reveals the reason for the difficulties of obtaining a good transcriptional signature for the other two patterns [791] .
We have seen, through a detailed discussion of several biomarkers in three different datasets, that the variation of the gene expression distributional profile can be characterized via Information Theory quantifiers. Our study also showed that current established biomarkers of the two diseases studied seem to correlate with those that best co-variate with these quantifiers. For instance, AMACR, in our second prostate cancer dataset studied, naturally appears as one of the most correlated genes (in both the Pearson and the Spearman sense) with the pattern of variation of Entropy of the samples. Together with MAOA, which is the highlighted gene in True et al.'s [332] original publication, AMACR is now being recognized as one of the best biomarkers in primary prostate cancer with approximately 180 publications dedicated to it in the past five years. We have also shown that many gene probes that best correlate with the divergence of the normal tissue profile have been identified as useful biomarkers (via other accepted validation methods). This said, the use of other sources of information, like pathway or gene ontology databases has lead as to the identification of other cell processes that may be altered.
We have presented a unifying hallmark of cancer, the cancer cell's transcriptome changes its Normalized Shannon Entropy (as measured by high-througput technologies), while it increments its physical Entropy (via creation of states we might not measure with our devices). This hallmark allows, via the use of the Jensen-Shannon divergence, to identify the arrow of time of the process, and helps to map the phenotypical and molecular hallmarks of cancer as major converging trends of the transcriptome. The methodology has produced remarkable postdictions and retrodictions that show that it can predictively guide biomarker discovery.
We refer the reader to the original publications for details of methods for data collection, but we highlight here some aspects that are important to understand the data generation process for the purpose of our analysis.
Samples were obtrained from radical prostatectomy surgical procedures. Samples are labelled as ''tumors'' if they contain at least 90% of cancerous epithelial cells, and they were considered as ''non-tumor'' if they contain no tumor epithelium and are from the noncancerous region of the prostate. The later samples were labelled ''normals'' although the authors alert that some may contain dysplasia. In this dataset, Lapointe et al. have performed a gene expression profiling by using cDNA microarrays containing 26,260 different human genes (UniGene clusters). Using 50 mg of total RNA from prostate samples Cy5-labeled cDNA was prepared and Cy3-labeled cDNA used 1.5 mg of mRNA common reference, pooled from 11 human cell lines (see Ref. [792] ). The fluorescence ratios were subsequently normalized by mean centering genes for each array, a relatively standard procedure. In addition, to minimize potential print run specific bias, Lapointe et al. report that ratios were then mean centered for each gene across all arrays according to Ref. [793] . We have only used the genes that the authors report in their first figure, 5,153 genes that have been well measured and have significan variation in some of the samples. For the other details of their matrials and methods we refer the readers to the Supporting Notes and the Materials and Methods section of their original publication [44] .
Samples were obtained from nevus volunteers and melanoma patients and only those samples that have more than 90% of tumor cells were profiled. The 20,862 cDNAs used (Research Genetics, Huntsville, AL) represent 19,740 independent loci. (Unigene build 166).median of ratio values from the experiment were subjected to linear normalization in nomad (which can be accessed at http://derisilab.ucsf.edu), log-transformed (base 2), and filtered for genes where data were present in 80% of experiments, and where the absolute value of at least one measurement was .1.
In this dataset, samples have information of 15,488 spots per array, with a total of 7,700 unique cDNAs represented. The samples were obtained from frozen tissue blocks from 29 radical prostatectomies accessioned and selected to represent Gleason grades 3, 4, and 5. The samples are ''treatment naïve'', meaning that they were also selected such that their gene expression profile is also and the absence of any bias that the treatment before prostatectomy. The frozen sections (8 mm) were cut from optimal cutting temperature medium blocks and immediately fixed in cold 95% ethanol. Around 5,000 epithelial cells from both histologically benign glands and cancer glands were separately laser-capture microdissected (LCM). The authors of the study have also been very careful to include only one Gleason pattern in each laser-captured cancer sample, following a process in which the patterns were assessed independently by two investigators.The matched benign epithelium was captured for each cancer sample for a total of 121 samples.
An important characteristic of this dataset is the normalization procedure. For each spot and in each channel (Cy3 and Cy5), True et al. substracted the median background intensity from the median foreground intensity, and subsequently the log ratios of cancer expression to benign expression were computed. These ratios were obtained by first dividing the background-subtracted intensities (Prostate Cancer/Benign) and then taking the logarithm base 2. In the case that the median background intensity was greater than the median foreground intensity, the spot was considered missing. We refer to the original publication for the other aspects of imputation, spot quality and filtering, but, like in Lapointe et al's study, they also filter to keep informative (expression ratios of benign versus cancer should at least be 1.5-fold or greater in at least half of one of the Gleason groups as one of the selection criteria).
Shannon Entropy. In many circumstances, experimental measurements are associated with the accumulation of individual results which, ultimately, qualitatively and quantitatively characterized our experimental observations. The presence (or absence) of a particular result of an individual experimental measure is called an event. An event which can take one of several possible values is called a random variable. Analogously, a random event is an event that can either fail to happen, or happens, as a result of an experiment. An event is certain if it can not fail to happen and it is said to be impossible if it can never happen.
Following Andreyev [794] , we will define the probability p(x) of an event x, as the theoretical frequency of the event x about which the actual frequency occurrence of the event shows a tendency to fluctuate as the experiment is repeated many times. The Shannon information content of an event x (or the surprisal of an event x, [795] ), is defined as h(x)~log 2 1 p(x)
Following McKay [796] , an ensamble X is a triple (x,A X ,P X ), where x is the value of a random variable, which takes on one of a set of possible values, A X~f a 1 ,a 2 ,:::,a i ,:::,a N g, having probabilities P X~f p 1 ,p 2 ,:::,p N g, with p(x~a i )~p i , p i §0 and X a i [A X p(x~a i )~1.
The Shannon Entropy of an ensemble X (also known as the uncertainty of X), denoted as H[X], is defined to be the average Shannon information content. It is the average expected surprisal for an infinitely long series of experiments. We use the theoretical frequencies to compute this average, and then we have
Suppose that we have a fair dice, the theoretical frequency of an event 'the dice shows a three' is 1/6, (if the dice is assumed fair, the theoretical frequency is the same for any number from 1 to 6). In that case a hypothetical experimentalist guessing will have an average expected surprise of H[X] = log 2 (6) . We note the two natural bounds that the entropy can have. The Shannon Entropy of an ensemble X is always greater or equal to zero. It can only be zero if p(x~a i )~1 for only one of the N elements of A X~f a 1 ,a 2 ,:::,a i ,:::,a N g. On the other hand, the Shannon Entropy is maximized in the case that p(x~a i )~1=N. This is the so-called ''equiprobable distribution'', a uniform probability distribution over the finite set. Transcriptional Shannon Entropy. Let f (j) i the expression value of probe i (i = 1,…, N) on sample j (j = 1, …, M). For each sample j we first normalize the expression values. We interpret them as the theoretical frequency of a single hybridization event. We then define a probability distribution function (PDF) over a finite set as: The uniform (equiprobably) distribution is defined as i Let H e~H ½P e ~log 2 N, then in this paper we always use the Normalized Shannon Entropy, defined as:
i H e , j~1, . . . ,M
The Jensen-Shannon divergence and the Statistical complexity measures
Given a probability distribution function over a discrete finite set, is then straightforward to calculate its Normalized Shannon Entropy if we have the theoretical frequencies. Several measures of ''complexity'' of a probability distribution function have been proposed. In this work we have used Statistical Complexity measures.
All the complexity measures used in this work are the product of a Normalized Shannon Entropy of the probability distribution function, and a divergence measure to a reference probability distribution function. We follow earlier proposals by López-Ruiz, Mancini and Calbet who first introduced a statistical complexity measure based on such a product in [797] . The LMC-Statistical Complexity is the product of the Normalized Shannon Entropy, H[P], times the disequilibrium, Q[P]; the latter given by the Euclidean distance from P to P e , the uniform probability distribution over the ensemble. In this paper we used a later modification which we refer as the MPR-Statistical Complexity [43] which replaces the Euclidean distance between P to P e by the Jensen-Shannon divergence [788, 798] . The Jensen-Shannon divergence is linked in physics to the thermodynamic length [799, 800, 801, 802] .
We define the MPR-Statistical complexity [790] as:
where Q P (j) ,P e  à Q 0 J s P (j) ,P e  à , Q 0 is a normalization factor, and J s P (1) ,P (2)  à is the Jensen-Shannon's divergence between two probability density functions P (1) and P (2) , which in turn is defined as J s P (1) ,P (2)  à H P (1) zP (2) 2
In this work, in many cases we compute the Jensen-Shannon divergences of a probability with a probability of reference which is not the uniform probability distribution over the ensemble. In general, it is the average over a subset of probability distribution functions which are consider to be either the ''initial'' of ''final'' states of interest. Let P ave be such an average, then the M-Statistical Complexity of a probability distribution function P (j) , given a P ave of reference, is given by C (M) P (j)  Ã~H P (j)  à : J s P (j) ,P ave  à An illutrative example. In order to discuss a relatively simple example that can intuitively provide a grasp of the basic mathematical principles of Information Theory we present a hypothetical ''gene expression'' dataset involving four samples each with the expression of five unique probes corresponding to five genes (not necessarily different) as follows in Table 3 .
One of the quantifiers that we use in this contribution describes a measure of order for a sample: the Normalized Shannon Entropy also known as Shannon Evenness Index [803] . This section focuses on this quantifiers use and importance (refer to the 'Materials and Methods' section to see how this measure is calculated). In Sample 4 all probes have the same expression therefore it has the highest achievable value of Normalized Shannon Entropy (H = 1). The Normalized Shannon Entropy values for samples 1 and 2 are the same (H = 0.82). Sample 3, which tends to be less peaked and has the two most significantly expressed genes with the same value, has a higher value of Normalized Shannon Entropy (H = 0.92) (see Figure 21 ).
This simple example shows that the Normalized Shannon Entropy variations of the gene expression profile convey information about global transcriptomic changes; however, this measure alone is not enough to characterize the deviations from normal tissue profiles. For example, assume that Sample 1 is the normal profile of a particular tissue type. Assume that Sample 3 is the profile of a cancer cell that originated from that tissue type, the variation of Normalized Shannon Entropy can be related to this malignant change. However, as Sample 2 illustrates, Normalized Shannon Entropy is not enough to let us to measure the variation from a profile and at least another Information Theory quantifier is needed. We resort to Statistical Complexity quantifiers, which in turn use the Jensen-Shannon divergence [798] to provide this complementary dimension [800] (refer to the 'Materials and Methods' section for a mathematical definition of the Jensen-Shannon divergence). Figure 21 shows how the Jensen-Shannon divergence helps us to evaluate the variation between profiles. Samples 1 and 2, as perhaps intuitively expected, have the largest divergence between them, their Jensen Shannon divergence is 0.286636 (JS(1,2) = Table 3 . An example dataset to illustrate the principles of Shannon Entropy and the Information Theory quantifiers used in this work. Table 3 . Sample 4 has the largest attainable value since the expression of all probes is the same. Samples 1 and 2, which have the same set of expression values, although in different probes, have the same value of Normalized Shannon Entropy. As a consequence, there is a need for another quantifier of gene expression to address the permutational indistinguishability of these two expression profiles. The Jensen-Shannon divergence provides a natural alternative (see Table 4 ). doi:10.1371/journal.pone.0012262.g021 Table 4 . Jensen-Shannon divergence values using the example introduced in Table 3 . Table 4 . Let H P (j) Â Ã be the Normalized Shannon Entropy of a transcriptional sample profile, then the MPR-Statistical Complexity C (MPR) P (j) Â Ã is defined as being proportional to the product of the Normalized Shannon Entropy times the Jensen-Shannon divergence of the profile with the equiprobable distribution (in the example above the equiprobable distribution is that of Sample 4). Then we have
Where Q 0 is a normalization factor. Once again, we refer to the 'Materials and Methods' sections for the accompanying formal mathematical presentation. As a consequence, we can plot the MPR-Statistical Complexity of the samples of our example as a function of the Normalized Shannon Entropy as can be seen in Figure 22 . Annotated genes. A full list of gene references in this paper along with their descriptions from iHOP (http://www.ihop-net. org/UniPub/iHOP/) can be found in supplementary material reference File S5.  SYSGENET: a meeting report from a new European network for systems genetics The first scientific meeting of the newly established European SYSGENET network took place at the Helmholtz Centre for Infection Research (HZI) in Braunschweig, April 7-9, 2010. About 50 researchers working in the field of systems genetics using mouse genetic reference populations (GRP) participated in the meeting and exchanged their results, phenotyping approaches, and data analysis tools for studying systems genetics. In addition, the future of GRP resources and phenotyping in Europe was discussed. SYSGENET is funded through the COST framework (http://www.cost.eu/about_cost). COST is an intergovernmental framework for European Cooperation in Science and Technology that promotes and coordinates nationally funded research in Europe, through funding of research networks. SYSGENET is coordinated by Klaus Schughart (Helmholtz Center for Infection Research, Braunschweig, Germany) . More detailed information about SYSGENET can be obtained from the website http://www.helmholtzhzi.de/sysgenet/.
Infectious diseases continue to represent a threat to human health. Due to global warming and travel, newly emerging diseases are spreading at an unprecedented rate around the world. Examples are the dissemination of antibioticsresistant mycobacteria, the new swine influenza virus variant, SARS, and West Nile virus (WNV). Several research groups are using mouse GRPs to identify complex genetic influences on the host susceptibility to infections. GRPs have been and will continue to be an important basis for understanding infectious diseases in humans. A very good example for translational research was presented by Pascal Rihet, who identified genomic susceptibility regions to malaria in human populations in Africa (Delahaye et al. 2007 ) and then continued to compare these results with studies in mouse GRPs. In this way, a region on human chromosome 5 and its homologous regions on mouse chromosomes 11 and 18 were identified. Subsequent expression studies in mice will now help to determine the molecular networks and genes involved. Paul Denny described the mapping of genetic susceptibility to Streptococcus pneumonia infections in mouse inbred strains to chromosomes 7 (Denny et al. 2003) and 4 (unpublished) . Infection susceptibility to influenza was described by Klaus Schughart, who also pointed out that high susceptibility includes a hyperreactive immune response in the host (Srivastava et al. 2009 ). Xavier Montagutelli generated a unique resource of Mus spretus 9 C57BL/6 J interspecific recombinant congenic strains that carry different genomic fragments of Mus spretus on a C57BL/6 J background (Burgio et al. 2007 ). This GRP was used to identify resistance and susceptibility regions to various pathogens, including Rift Valley fever, West Nile virus, Yersinia pestis, and influenza. The first lines of the Collaborative Cross strains have been screened by Fuad Iraqi for a number of susceptibility loci to various pathogens (unpublished). It was remarkable to see that several quantitative trait loci (QTL) showed high significance and that the genomic intervals for several loci were very narrow, which should make it possible to identify quickly the underlying quantitative trait genes.
Metabolic diseases in humans are dramatically on the rise; obesity and related diseases in particular represent a serious challenge to future health systems. Several groups addressed the complex genetics of metabolic functions and disorders using different mouse GRPs. Gudrun Brockmann reported on the mapping of QTLs for obesity in a specific mouse strain isolated in Berlin and the BXD congenic strain set (Neuschl et al. 2010) . The future goal is to relate these QTLs to genetic polymorphisms that influence the immune system. Joan Campbell-Tofte presented the use of herbal extracts for the treatment of type 2 diabetes in humans. She nicely illustrated the use of mouse models: from human to mouse to humans and back to the mouse. Pénélope Andreux reported on the setting up of a mouse clinic in Lausanne for a systematic analysis of mouse GRPs for a large number of metabolic phenotypes, including mitochondrial functions (Koutnikova et al. 2009 ). Juan M. Falcon-Perez described the genomic, proteomic, and metabolic phenotyping capabilities of their technological platform and introduced extracellular microvesicles and metabolomic profiles as two new biological sources for identifying biomarkers for the detection and monitoring of hepatic diseases (Hackenberg et al. 2009 ). Abnormalities and diseases of the liver are also the subject of studies presented by Karl Kashofer (Kashofer et al. 2009 ). Several loci for (nonalcoholic) steatohepatitis have been mapped in chromosome substitution strains, and a more detailed mapping in subcongenic strains is ongoing.
Although rats in general were the species of choice for use by experimental psychologists to study behavior, mice have been the preferred animal for behavior geneticists since at least the 1940 s. In addition, the adaptation of behavioral assays and the development of new methods have confirmed the mouse as one of the most preferred experimental systems to learn more about the genetic underpinnings of behavior and associated phenotypes. Martien Kas described the currently underlying scientific rationale. Precise measurement of a well-described behavioral trait across a GRP will lead to the identification of associated genes and genomic regions. In the next step these genes can be used to find homologous genes and pathways that contribute to the development of neuropsychiatric disorders in humans. The translational value of this interspecies genetic approach was nicely exemplified in a study in which a QTL for avoidance behavior in mice was related to bipolar disorders in humans (de Mooij-van Malsen et al. 2009 ). In a similar approach, Iris Hovatta used a cross-species neurogenomics comparison to correlate brain region-specific gene expression patterns and anxiety-like behavior in mouse GRPs to polymorphisms in the Finnish population for anxiety disorders (Hovatta et al. 2005) . The mouse genes allowed identification of potential candidate genes in humans who predispose to anxiety disorders. Paul Franken presented studies on the identification of genetic traits that influence homeostatic and circadian aspects of sleep, and the electroencephalogram in the BXD GRP and in inter-and backcross panels of mice (Shaw and Franken 2003) . Several genes that play a decisive role were identified, and further phenotyping of the extended BXD GRP is planned. Eero Vasar and Sulev Kõks described the role of the Wfs1 gene in knockout mice for anxiety behavior, an altered response to morphine and the release of striatal dopamine (Koks et al. 2009; Luuk et al. 2009 ). Ewelina Knapska reported the use of a highly sophisticated cage system, IntelliCage, to automatically record a number of different complex behavioral traits in mice (Jaholkowski et al. 2009 ). Mice can be housed in social groups but nevertheless tested individually. Ryszard Przewlocki reported on a systematic study on various inbred mouse strains to identify genetic determinants of alcohol and drug addiction (Piechota et al. 2010) . Combining these studies with comprehensive gene expression analyses revealed that glucocorticoid receptor-activated gene expression pathways play an important role. Wim Crusio studied behavioral traits in learning and related them to brain anatomy (Crusio and Schwegler 2005) . Thereby, the extent of neuron projections in the hippocampus could be correlated to more efficient learning capabilities and these two phenotypes are very strongly correlated genetically. Guus Smit gave an overview on a collaborative effort in the Netherlands in which several research groups have determined various behavioral phenotypic traits and QTLs in a commonly used BXD population (Loos et al. 2009 ). Also, they established a mouse facility in which they are using automated screening cages with sophisticated video recording and analysis.
Cancer is still one of the most frequent causes of death in Western countries, and understanding its molecular causes as well as establishing appropriate animal model systems for the development of new treatment strategies is very important. Fragiskos Kolisis reported on the setting up of an infrastructure for a systems biology approach to carcinogenesis and aging (Chatziioannou et al. 2009 ). Understanding proteasome function and dysfunction as well as studying the alterations of the genome and proteome that account for different cancer phenotypes in a mouse skin carcinogenesis model are among the research goals. Javier Santos used GRPs to identify genetic traits for the susceptibility to radiation-induced thymic lymphomas in interspecific recombinant congenic and consomic mouse strains (Santos et al. 2009 ). Frank Lammert developed assay systems to determine genetic causes of liver fibrosis and inflammatory liver carcinogens in the BXD mouse recombinant congenic GRP (Weber et al. 2008) .
Leonard Schalkwyk studied allele-specific methylation in humans (Schalkwyk et al. 2010) . He estimated that potentially more than 35,000 sites in the genome exhibit allele-specific modifications, and of these 10% are not in cis, a number that largely exceeds the number of known imprinted loci. These findings suggest that individual genetic heterogeneity may be much larger than estimated thus far and may contribute to individual phenotypic differences. Jiri Forejt used inter-subspecific consomic strains (Gregorova et al. 2008) to investigate male sterility and its consequences for interspecies hybrid sterility (Mihola et al. 2009 ). Furthermore, in the livers of inter-subspecific hybrid strains he discovered new patterns of gene expression that were absent from both parental strains.
The capture, storage, handling, and analysis of large data sets will present a specific challenge for future systems genetics projects. Ritsert Jansen and Pjotr Prins presented their approaches to integrate data from various phenotypic studies, encompassing gene expression, metabolome, and classical traits, and to develop new tools for advanced and improved mapping of QTLs (Jansen et al. 2009; Li et al. 2008; Swertz and Jansen 2007) . These tools will be provided to the scientific community. Andreas Beyer gave a report on how to integrate data obtained at the post-transcriptional level with RNA expression data. Several loci that influence the post-transcriptional regulation of gene products could be identified in yeast. Steffen Möller presented his suite for the analysis of expression QTL (http://eqtl.berlios.de), which is being applied to the analysis of experimental autoimmune encephalomyelitis in mouse and rat. Anastasios Bezerianos presented a platform and developments for the identification of gene regulatory networks integrating protein-protein interactions and microarray data (Bezerianos and Maraziotis 2008). They started with yeast data and will soon expand to mouse, concentrating on time-varying gene regulatory networks. Morris Swertz presented XGAP, a software platform developed for data management and integration of large data sets from phenotyping and genotyping studies (Swertz et al. 2010) . Grant Morahan described the development of an extended tool for WebQTL that allows a genome-centric analysis of QTL interactions (unpublished).
The Collaborative Cross (CC) is currently being generated as a community resource for more sensitive and refined mapping of QTLs. The goal is to breed a large population of recombinant inbred strains starting from eight founder strains. The eight founder strains were selected to capture a large portion of the genetic variation in the mouse genome. In fact, the genetic variation represented in the CC will be twice the genetic variation present in the human population (reviewed in Valdar et al. 2006) . The three sites where the resource is being generated reported the present status of their breeding colonies; the final goal is to generate a total of 700 lines (Chesler et al. 2008; Iraqi et al. 2008; Morahan et al. 2008a) . Grant Morahan gave an update on the ''Southern Cross'' being established in Perth, Australia. An inbreeding depression was observed at generations 7-9. At present, about 200 strains have been bred beyond generation 10. The first 40 strains are expected to be inbred by the end of the year. David Threadgill reported the status of the breeding colony at the University of North Carolina, Chapel Hill, NC, USA. About 300 lines are currently breeding at UNC. The first 50 recombinant inbred lines will be available by the end of this year and 200 lines by the end of 2011. To speed up the inbreeding process, markerassisted breeding will be used to create homozygous lines beyond generation 12. Genome analysis demonstrated that all parental genomes are well represented in the advanced generations. The first phenotyping analysis showed a large variation in body weight, exercise propensity, and susceptibility to pathogens. Richard Mott described the genome structure of a smaller CC colony, funded by the Wellcome Trust, which was developed by Fuad Iraqi and is presently housed in Tel Aviv, Israel. A first phenotyping analysis for the QTLs that affect recombination frequencies was performed. A full-genome sequencing project to complete the parental strains with high coverage is underway at the Sanger Institute.
The two-day meeting in Braunschweig has clearly demonstrated the great value of mouse GRPs in identifying genetic determinants of complex genetic traits for various phenotypic traits related to diseases in humans. The partners of the network collectively have great expertise in disease phenotyping and analysis of genetic reference populations. Several examples that illustrated the translation of the knowledge gained in the mouse experimental systems to humans were presented. Links to clinical researchers already exist at several places but will have to be further expanded in the future. Furthermore, mouse GRPs can be ideally combined with mouse mutant lines carrying a gene-knockout mutation to determine the effect of a strong genetic defect in combination with modifier genes. It also became clear that a strong and sustained financial investment in mouse breeding and phenotyping facilities as well as in bioinformatic infrastructure is urgently needed to further advance a systems genetics approach in Europe.
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Greece; Sulev Kõks, University of Tartu, Estonia; Frank Adaptive Contact Networks Change Effective Disease Infectiousness and Dynamics Human societies are organized in complex webs that are constantly reshaped by a social dynamic which is influenced by the information individuals have about others. Similarly, epidemic spreading may be affected by local information that makes individuals aware of the health status of their social contacts, allowing them to avoid contact with those infected and to remain in touch with the healthy. Here we study disease dynamics in finite populations in which infection occurs along the links of a dynamical contact network whose reshaping may be biased based on each individual's health status. We adopt some of the most widely used epidemiological models, investigating the impact of the reshaping of the contact network on the disease dynamics. We derive analytical results in the limit where network reshaping occurs much faster than disease spreading and demonstrate numerically that this limit extends to a much wider range of time scales than one might anticipate. Specifically, we show that from a population-level description, disease propagation in a quickly adapting network can be formulated equivalently as disease spreading on a well-mixed population but with a rescaled infectiousness. We find that for all models studied here – SI, SIS and SIR – the effective infectiousness of a disease depends on the population size, the number of infected in the population, and the capacity of healthy individuals to sever contacts with the infected. Importantly, we indicate how the use of available information hinders disease progression, either by reducing the average time required to eradicate a disease (in case recovery is possible), or by increasing the average time needed for a disease to spread to the entire population (in case recovery or immunity is impossible). During recent years it has become clear that disease spreading [1] [2] [3] transcends geography: the contact process is no longer restricted to the immediate geographical neighbors, but exhibits the stereotypical small-world phenomenon with inescapable impact [4] [5] [6] [7] . The SARS and, at the time of writing, the influenza A outbreaks provide arresting examples of this effect in a biological context, although the spread of computer viruses constitutes perhaps the most obvious manifestation of such a property. Recent advances in the science of networks [2] also brought the impact of disease interactions beyond geography to the spotlight, providing compelling evidence of the role that the networks of contacts between individuals or computers play in the dynamics of infectious diseases [3, 5] . In the majority of cases in which complex networks of disease spreading have been considered [7] , they were taken to be static entities. However, social networks are intrinsically dynamical entities. In fact, modern societies have developed rapid means of information dissemination, both at local and at centralized levels, which one naturally expects to alter individuals' response to vaccination policies, their behavior with respect to other individuals and their perception of likelihood and risk of infection [8] . In some cases one may even witness the adoption of centralized measures, such as travel restrictions [9, 10] or the imposition of quarantine spanning parts of the population [11] , which may induce abrupt dynamical features onto the structure of the contact network. Furthermore, individual knowledge (based on local information) about the health status of acquaintances, partners, relatives, etc., combined with individual preventive strategies [12] [13] [14] [15] [16] [17] [18] [19] [20] such as condoms, vaccination, the use of face masks or prophylactic drugs, avoidance of visiting specific web-pages, staying away from public places, etc., may also lead to bias in the organization of disease paths along dynamical networks.
As a result, quite a few studies have recently investigated the impact of dynamical networks on disease progression, as well as the influence of the way information (disease awareness) flows in parallel with disease progression and the role of noise in disease dynamics [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . At par with these models, our model studies disease dynamics in a finite and constant population of individuals. Contrary to current models though (see Discussion Section for more details), our dynamical contact structure allows for a variable number of overall links between individuals, which in turn depends on the overall incidence of disease in the population. This increased complexity of the model, however, will allow us to describe analytically disease dynamics in finite populations. Infection occurs along the links of a contact network whose structure may change based on each individual's health status and the availability of information regarding the health status of others. We assume the existence of some form of (unsupervised) local information about the health status of social contacts, depending on which individuals may be more or less effective in avoiding contact with those infected while remaining in touch with the healthy. We investigate some of the most widely used models of disease spreading, such as the popular Susceptible-Infected-Susceptible (SIS) model [1, 3] , the Susceptible-Infected (SI) model [1] used to study, e.g., AIDS [1, 34] , and the Susceptible-Infected-Recovered (SIR) model [1, 35] , more appropriate to model, for instance, single season flu outbreaks [1] or computer virus spreading [5] . We provide analytical results which show that, on finite adaptive networks, the effective infectiousness of a disease depends on i) the size of the population, ii) the number of infected and ultimately iii) on the efficiency of local information spreading. These results, obtained under the assumption that network reshaping occurs much faster than disease spreading, are shown (numerically) to remain valid for a much wider range of time scales. When combined with the analytical results already known for static networks [3] , which describe well the opposite limit in which the network dynamics is slow compared to disease dynamics, we demonstrate that one can describe analytically most of the time scale spectra. We further show how availability of information may either drastically reduce the time required to eradicate a disease (when recovery from the disease is possible), or drastically increase the time after which the entire population becomes infected (when the disease cannot be cured). Finally, we discuss the implications of assuming that individuals exhibit diverse response profiles to available information [36] [37] [38] .
Let us start by introducing the well established SIS model [1, 3] , whereby individuals can be in one of two epidemiological states: Infected (I) or Susceptible (S). Individuals occupy the nodes of a graph, whereas the links of the graph define who interacts with whom. As usual, we define a transmission probability l and a recovery probability d. We assume a population of finite and constant size N, but with variable number of links [39, 40] . Links are established and removed at rates which also depend on the health status of the individuals connected: Links of different types -SS, SI and II -will tend to last differently. The SIR model, on the other hand, requires the introduction of a new epidemiological state -Recovered (or Removed, R) -representing individuals who, once recovered from the infection, acquire immunity. In SIR, the same parameters l and d defined above remain finite, but now we have three new kinds of links -SR, RI and RR. Finally, the SI model can be viewed as the limit of the SIS model when d~0, representing diseases from which recovery is impossible.
Suppose all individuals seek to establish links at the same rate c. For simplicity, we assume that new links are established and removed randomly, a feature which may not always apply in real cases, where the limited social horizon of individuals or the nature of their social ties may constrain part of their neighborhood structure (see Discussion). Let us further assume that links may be broken off at different rates, based on the nature of the links and the information available about the neighbors: Let us denote these rates by b pq for links of type pq (p,q [ S,I,R f g). This allows us to write down a system of ordinary differential equations [39, 40] for the time evolution of the number of links of each type. These and all other technical details of the model are presented in the Methods section. In the steady state of the linking dynamics, the number of links of each type is given by L Ã pq~wpq L M pq , where w pq~c czb pq À Á are the fractions of active pq-links, compared to the maximum possible number of links L M pq , for a given number of S, I and R. In the absence of disease only SS links exist, and hence w SS determines the average connectivity of the network under disease free conditions, characterizing the type of the population under study. In the presence of I individuals, to the extent that S individuals manage to avoid contact with I, they succeed in escaping infection. Hence, the intuition is clear: Reshaping of the contact network based on available information of the health status of individuals will contribute to inhibit disease progression. In the extreme limit of perfect information and individual capacity to immediately break up contacts with infected, we are isolating all infected, thus containing disease progression. Our goal here, however, is to understand how and in which way partial individual information affects the overall disease dynamics.
Often individuals prevent infection by avoiding unprotected contact with infected once they know the state of their contacts or are aware of the potential risks of such infection [12] [13] [14] [15] [16] [17] [18] [19] [20] 28, 32] : such is the case of many sexually transmitted diseases [12, [41] [42] [43] , for example, and, more recently, the voluntary use of face masks and the associated campaigns adopted by local authorities in response to the SARS outbreak [11, [13] [14] [15] . In the present study, we assume that individuals are not centrally supervised or informed: Individual decision is based on available local information about the health state of one's contacts. To the extent that such information spreads quickly and contacts are not too frequent, one can study analytically the limit in which the network dynamics -resulting from adaptation to the flow of local information -is much faster than disease dynamics. In this case, one may separate the time scales between network adaptation and contact (disease) dynamics: The network has time to reach a steady state before the next contact takes place. Consequently, the probability of having an infected neighbor is modified by a neighborhood structure which will change in time depending on the impact of the disease in the population and the overall efficiency of local information flow. It will be shown that a quickly adapting community induces profound changes in the dynamics of disease spreading, irrespective of the underlying epidemic model. Furthermore, we will demonstrate numerically that the two limiting cases amenable to analytic treatment -static networks on the one hand, and quickly adapting networks on the other hand
During the past decade, we learned that the structure of contact networks plays a crucial role in the spread of diseases. Most theoretical studies addressing this issue assume that contact networks are static entities, whereas the actual disease paths continuously reshape based on local social dynamics. This work aims to achieve a better understanding of disease spreading in populations characterized by a dynamically structured contact network where contacts appear and disappear over time. The network dynamics are entangled with the disease dynamics, as individuals may have access to local information that makes them aware of both the existence of the disease and the health status of their contacts, allowing them to minimize exposure to infection. Here we show the equivalence between disease propagation in an adaptive contact network and that in a well-mixed population with a rescaled transmission probability, which depends also on the fraction of infected in the population. Thus, one can emulate the effect of an adaptive contact network with a simple correction of the transmission probability. This result is obtained in the limit where network adaptation proceeds much faster than disease spreading, but we demonstrate that it also holds for a much wider range of scenarios.
-remain valid for a wide range of intermediate time scales, strengthening the power of the analytical predictions derived here.
The amount of information available translates into differences mostly between the break-up rates of links that may involve a potential risk for further infection (b SI , b IR , b II ), and those that do not (b SS , b SR , b RR ). Therefore, we consider one particular rate b I for links involving infected individuals (b I :b SI~bIR~bII ), and another one, b H , for links connecting healthy individuals (b H :b SS~bSR~bRR ). In general, one expects b I to be maximal when each individual has perfect information about the state of her neighbors and to be (minimal and) equal to b H when no information is available, turning the ratio between these two rates into a quantitative measure of the amount of information available or the efficiency of information use. Note that we reduce the model to two break-up rates in order to facilitate the discussion of the results. The general principles and conclusions remain valid when all break-up rates are incorporated explicitly. It is worth noticing that three out of these six rates are of particular importance for the overall disease dynamics: b SS , b SR and b SI . These three rates, combined with the rate c of creating new links, define the fraction of active SS, SR and SI links, and subsequent correlations between individuals [44] , and therefore determine the probability for a susceptible to become infected (see Methods). This probability will increase when considering higher values of c (assuming b I .b H ). In other words, when individuals create new links more often, therefore increasing the likelihood of establishing connections to infected individuals (when present), they need to be better informed about the health state of their contacts in order to escape infection. In the fast linking limit, the other three break-up rates (b II , b IR and b RR ) influence disease progression since they contribute to altering the average degree of the network.
In the Methods, we show that disease spreading in a quickly adapting network can be studied as if it took place in a well-mixed population with same average degree SkT as the original network and transmission probability l A . Since the lifetime of a link depends on its type, the average degree SkT of the network depends on the number of infected in the population, and hence becomes frequency (and time) dependent. Similarly, fast linking dynamics leads to a rescaling of the transmission probability, l?l A~g{1 l, with g depending on the particular model of disease spreading. In the SIR model, g is given by
where i denotes the number of infected individuals in the population and r the number of recovered (immune). The rescaling parameter g for the SI and SIS models is the same, but with r~0. Note that g scales linearly with the frequency of infected in the population, decreasing the more individuals get infected (assuming w SS =w SI w1), and depends implicitly (via the ratio w SS =w SI ) on the amount of information available. We would like to stress the distinction between the description of the disease dynamics at the local level and that at the population level. Strictly speaking, a dynamical network does not change the disease dynamics at the local level, meaning that infected individuals pass the disease to their neighbors with probability intrinsic to the disease itself. At the population level, on the other hand, disease progression proceeds as if the infectiousness of the disease effectively changes, as a result of the network dynamics. Hence, analyzing an adaptive network scenario at a population level can be achieved via a correction on the transmission probability, keeping the mathematically more attractive wellmixed scenario. In this sense, from a well-mixed perspective, dynamical networks contribute to change the effective infectiousness of the disease, which becomes frequency and information dependent.
One can define a gradient of infection G, which measures the tendency of the disease to either expand or shrink in a population with given configuration (defined by the number of individuals in each of the states S, I and R). For the SIS model, eradication of the disease is favored (G(i),0), irrespective of the fraction of infected, whenever R A 0 : l d Nw SI v1 (see Eq. 35 in Text S1), indicating how the presence of information (b H ,b I ) changes the basic reproductive ratio. This is illustrated in the upper panel of Figure 1 , which depicts G for different values of b I (assuming b H ƒb I ) and a fixed transmission probability l. The corresponding quasi-stationary distributions, which characterize the relative time the population spends in each configuration (and defined in Methods), are shown in the lower panel and clearly reflect the sign of G. Whenever G(i) is positive (negative), the dynamics will act to increase (decrease), on average, the number of infected. Figure 1 indicates how the availability of local yet reliable information hinders disease progression: For b I~0 :75 the interior root of G(i) disappears, making disease expansion unlikely in any configuration of the population.
The analysis of the gradient of infection of the SIS model has the natural advantage of showing the effect of adaptive networks in a one-dimensional simplex (the fraction of infected). Yet, an analogous result holds for the SIR model. The gradient of infection now also depends on the number of recovered (r) individuals in the population and, once again, allows us to identify when disease expansion will be favored or not. Figure 2 gives a complete picture of the gradient of infection, using the appropriate simplex structure in which all points satisfy the relation i+r+s = N. The dashed red line indicates where G i,r ð Þ~0 in case individuals do not have any information about the health status of their contacts, i.e., links that involve infected individuals disappear at the same rate as those that do not (b I~bH ). Disease expansion is more likely than disease contraction (G i,r ð Þw0) when the population is in a configuration above the line, and less likely otherwise. Similarly, the solid blue line indicates where G i,r ð Þ~0 whenever individuals share information about their health status, and use it to avoid contact with infected. Once again, the availability of information modifies the disease dynamics, inhibiting disease progression for a broad range of configurations.
Up to now we have assumed that the network dynamics proceeds much faster than disease spreading. This may not always be the case, and hence it is important to assess the domain of validity of this limit. In the following, we discuss the particular case of the SIS model. An analogous study for both SIR and SI yields qualitatively similar results, as discussed in the Text S1. Figure 3 shows the average SIT of the quasi-stationary distributions (circles) obtained via computer simulations (see Methods for details) as a function of the relative time scale t of network update (taking as unit time scale that associated with disease dynamics). Whenever t??, we can characterize the disease dynamics analytically, assuming a well-mixed population (complete graph), whereas for t?0 we recover the analytical results obtained in the fast linking limit. At intermediate time scales, Figure 3 shows that as long as t is smaller than 10, network dynamics contributes to inhibit disease spreading by effectively increasing the critical spreading rate. Overall, the validity of the time scale separation extends well beyond the limits one might anticipate based solely on the time separation ansatz. As long as the time scale for network update is smaller than the one for disease spreading (tv1), the analytical prediction for the limit t?0, indicated by the lower dashed line in Figure 3 , remains valid. The analytical result in the extreme opposite limit (t??), indicated by the upper dashed line in Figure 3 , holds as long as tw10 5 . Moreover, it is noteworthy that the network dynamics influences the disease dynamics both by reducing the frequency of interactions between susceptible and infected, but also by reducing the average degree of the network. These complementary effects are disentangled for intermediate regimes, in which the network dynamics is too slow to warrant sustained protection of susceptible individuals from contacts with infected, despite managing to reduce the average degree (shown by crosses). In fact, for tw10 the disease dynamics is mostly controlled by the average degree, as shown by the solid lines in Figure 3 . Here, the average stationary distribution was determined by replacing, in the analytic expression for static networks, SkT by the timedependent average connectivity SkT Ã computed numerically.
Note that the specific behavior of SkT Ã shown in Figure 3 results from the frequency dependence of SkT. When b I wb H , the network will reshape into a configuration with smaller SkT as soon as the disease expansion occurs. For tv1, SkT Ã reflects the lifetime of SS links, as there are hardly any infected in the population. For 10 0 vtv10 3 , the network dynamics proceeds fast enough to reduce SkT, but too slowly to reach its full potential in hindering disease progression. Given the higher fraction of infected, and the fact that SI and II links have a shorter lifetime than SS links, the average degree drops when increasing t from 1 to 10 3 . Any further increase in t leads to a higher average degree, as the network approaches its static limit.
Contrary to the deterministic SIS model, the stochastic nature of disease spreading in finite populations renders certain the probability that the disease disappears after some time. However, this result is of little relevance given the associated times required to reach the absorbing state (except, possibly, in very small communities). Indeed, the characteristic time scale of the dynamics plays a determinant role in the overall epidemiological process and constitutes a central issue in disease spreading.
In the Text S1 we derive an analytical expression for the average recovery time (t i ) of a population with i infected individuals. Figure 4A shows the recovery time t 1 in adaptive networks for different levels of information, illustrating the spectacular effect brought about by the network dynamics on the recovery time. While on networks without information (b I = b H ) the recovery time rapidly increases with the rate of infection l, adding information moves the fraction of infected individuals rapidly to the absorbing state, and, therefore, to the disappearance of the disease. Moreover, as shown in the Text S1, the size of the population can have a profound effect on the recovery times. With increasing population size, the population spends most of the time in the vicinity of the state associated with the interior root of G(i). For large populations, this acts to reduce the intrinsic stochasticity of the dynamics, dictating a very slow extinction of the disease.
When recovery from the disease is impossible, a situation captured by the SI model, the population will never become disease-free again once it acquires at least one infected individual. The Markov chain that represents such diseases therefore has another absorbing state, corresponding to networks where everyone has been infected, besides the previous one in which no one is infected. The time to reach this state again depends on the presence of information. When information prevails, susceptible individuals manage to resist infection for a long time, thereby delaying the rapid progression of the disease, as shown in the inset of Figure 4B . Naturally, the average number of generations needed to reach a fully infected population increases with the availability of information, as illustrated in the main panel of Figure 4B .
Finally, in all models discussed here we also investigated the effect of allowing for different individual rates associated with the way each individual creates or destroys her social ties. Due to agestructure of most populations or intrinsic individual or cultural differences, some individuals will tend to react differently whenever they, or a contact, get infected [36, 38] . In the Text S1 we show that the disease spreading remains unaffected when individual rates (of seeking and removing links) are drawn from a normal distribution with variable variance, as long as the average value for the rate remains unchanged.
Making use of three standard models of epidemics involving a finite population in which infection takes place along the links of a dynamical graph, the nodes of which are occupied by individuals, we have shown analytically that the bias in graph dynamics resulting from the availability of information about the health status of others in the population induces fundamental changes in the overall dynamics of disease progression.
The network dynamics proposed here differs from those used in previous models of disease spreading on adaptive networks [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . Similar to most models, the population size remains fixed. Unlike most of these studies, however, we do not impose any local or global linking constraints, meaning that individuals can create (or remove) a link without the need for removing (or creating) another one -in other words, the number of links will change in time, also adapting to the disease configuration of the population. Consequently, the average degree of the network results from the self-organization of the network structure, and co-evolves with the disease dynamics (cf. networks evolving towards critical values of connectivity, as studied in [45] ). A population suffering from high disease prevalence where individuals avoid contact in order to escape infection will therefore exhibit a lower average degree than a population with hardly any infected individuals. Such a frequency-dependent average degree prevents also that containment of infected individuals would result automatically in the formation of a dense healthy cluster, which is extremely vulnerable to future infection, as reported before in [21, 25, 26] .
The description of disease spreading as a stochastic contact process embedded in a Markov chain constitutes a second important distinction between the present model and previous studies. This approach allows for a direct comparison between analytical predictions and individual-based computer simulations, and for a detailed analysis of finite-size effects and convergence times, whose exponential growth will signal possible bistable disease scenarios. In such a framework, we were able to show that adaptive networks in which individuals may be informed about the health status of others lead to a disease whose effective infectiousness depends on the overall number of infected in the population. In other words, disease propagation on adaptive networks can be seen as mathematically equivalent to disease spreading on a well-mixed population, but with a rescaled effective infectiousness. In accord with the intuition advanced in the introduction, as long as individuals react promptly and consistently to accurate available information on whether their acquaintances are infected or not, network dynamics effectively weakens the disease burden the population suffers from. Last but not least, if disease recovery is possible, the time for disease eradication drastically reduces whenever individuals have access to accurate information about the health state of their acquaintances and use it to avoid contact with those infected. If recovery or immunity is impossible, the average time needed for a disease to spread increases significantly when such information is being used. In both cases, our model clearly shows how availability of information hinders disease progression (by means of quick action on infected, e.g., their containment via link removal), which constitutes a crucial factor to control the development of global pandemics.
Finally, it is also worth mentioning that the knowledge about the health state of the others may not always be accurate or available in time. This is for instance the case for diseases where recently infected individuals remain asymptomatic for a substantial period. The longer the incubation period associated with the disease, the less successful individuals will be in escaping infection, which reduces in our model to a lower effective rate of breaking SI links, with the above mentioned consequences. Moreover, the (social) network through which awareness of the health status of others proceeds may lead to different rates of information spread. In such cases, one may model explicitly the spread of the health state of each individual, as done in Refs. [28, 32] , and study the interplay between disease expansion and individuals' awareness of the disease. Of course, depending on the particular disease at hand and the contact network along which it propagates, one may have to take other factors into account, besides network adaptability, in order to make accurate predictions of disease progression. Creation and destruction of links may for instance not always occur randomly, as we assumed here, but in a way that is biased by a variety of factors such as social and genetic distance, geographical proximity, family ties, etc. The resulting contact network may therefore become organized in a specific way, promoting the formation of particular structures, such as networks characterized by long-tailed degree distributions or with strong topological correlations among nodes [2, [46] [47] [48] which, in turn, may influence the disease dynamics. The impact of combining such effects, resulting from specific disease scenarios, with those reported here will depend on their prevalence. A small fraction of non-random links, or of ties which cannot be broken, will likely induce small modifications on the average connectivity of the contact network, which can be incorporated in our analytic expressions without compromising their validity regarding population wide dynamics. On the other hand, when the contact network is highly heterogeneous (e.g., exhibiting pervasive longtail degree distributions), non-random events may have very distinct effects, from being almost irrelevant (and hence can be ignored) to inducing hierarchical cascades of infection [49] , in which case our results will not apply.
We define disease dynamics in finite populations of size N as a stochastic process. Time evolves in discrete steps and two types of events may occur which change the composition of the population: infection events and recovery events. Let us assume the SIR model, as this is the most general case (see Text S1 for a detailed analysis specific for each disease model). Each state of the population is characterized by two indices i,r ð Þ, where i is the number of infected individuals in the population and r the number of recovered (and immune) individuals (izrƒN). The number of infected decreases with a rate given by
where t 0 is the recovery time scale, i N the probability that a randomly selected individual is infected and d the probability that this individual recovers. Adopting t 0 as a reference, we assume that the higher the average number of contacts SkT, the smaller the time scale t INF at which infection update events occur (t INF~t0 =SkT) [3] . Hence, the rate of increasing the number of infected is given by
The first factor stands for the typical time scale at which infection events occur. The second factor represents the probability of randomly picking a healthy individual. This individual interacts with a random neighbor, who is infected with probability given by the third factor (well-mixed assumption). This contact leads to an additional infection with probability l. When r~0, Equations (M1) and (M2) describe both SIS and SI (d~0) models. We obtain the finite population analogue of the well-known meanfield equations characteristic of these models by recognizing that, in the limit of large populations, t 0 G i,r 
Equations (M1) and (M2) define a Markov chain M with variable states depending on the epidemic model (see Text S1). The fraction of time the population spends in each state is given by the stationary distribution of M, which is defined as the eigenvector associated with eigenvalue 1 of the transition matrix of M [50, 51] . In the SIS model, the state without infected (i = 0) is, however, an absorbing state of the Markov Chain. The quantity of interest is therefore the stationary distribution of the Markov chain obtained from M by excluding the absorbing state i = 0. This distribution is also known as the quasi-stationary distribution of M [52] and corresponds to the fraction of time the population spends in each state, assuming the disease does not go extinct (see Figure 1 ).
Consider a network of constant size N with variable number of links. New links are established randomly at rate c. Existing links between individuals with health states p and q (p,q [ S,I,R f g) disappear at rate b pq . The time evolution of the number L pq of pqlinks can be written as a system of ordinary differential equations _ L L pq~c L M pq {L pq {b pq L pq [39, 40] . L M pq denotes the maximum number of pq-links, which depends on the number of individuals in states p and q (L M pp~p p{1 ð Þ=2 and L M pq~p q for p=q) and thereby couples the network dynamics to the disease dynamics. In the steady state of the linking dynamics ( _ L L pq~0 ), the number of links of links of each type is given by L Ã pq~wpq L M pq , with w pq~c czb pq À Á .
When the time scale for network update (t NET ) is much smaller than the one for disease spreading (t DIS ), the number of infected increases with a rate
where we made t 0~1 . The effect of the network dynamics becomes apparent in the third factor, which represents the probability that a randomly selected neighbor of a susceptible is infected. In addition, Equation ( which shows that disease spreading in an adaptive network is equivalent to that in a well-mixed population with frequency dependent average degree SkT and a transmission probability that is rescaled according to l A~g{1 l, where
which is valid for both SIR, SIS (r~0) and SI (d~0,r~0) models.
All individual-based simulations start from a complete network of size N = 100. Disease spreading and network evolution proceed together under asynchronous updating. Disease update events take place with probability 1zt ð Þ {1 , where t~t NET =t DIS and t DIS~1 , network update events occur otherwise. For network update events, we randomly draw two nodes from the population. If connected, then the link disappears with probability given by the respective b pq . Otherwise, a new link appears with probability c. When a disease update event occurs, a recovery event takes place with probability 1zSkT ð Þ {1 , an infection event otherwise. In both cases, an individual j is drawn randomly from the population. If j is infected and a recovery event has been selected then j will become susceptible (or recovered, model dependent) with probability d. If j is susceptible and an infection event occurs, then j will get infected with probability l if a randomly chosen neighbor of j is infected. The quasi-stationary distributions shown in Figure 1 are computed as the fraction of time the population spends in each configuration (i.e., number of infected individuals) during 10 9 disease event updates (10 7 generations). The average number of infected SIT and the mean average degree of the network SkT Ã observed during these 10 7 generations are shown in Figure 3 . The results reported are independent of the initial number of infected in the network. Finally, the disease progression in time, shown in Figure 4b , is calculated from 10 4 independent simulations, each simulation starting with 1 infected individual. The reported results correspond to the average amount of time after which the population reaches a state with i infected.
Text S1 Adaptive contact networks change effective disease infectiousness and dynamics. 1  The role of infections in the pathogenesis and course of multiple sclerosis Interplay between susceptibility genes and environmental factors is considered important player in the genesis of multiple sclerosis (MS). Among environmental factors, a role for an infectious pathogen has long been considered central to the disease process. This opinion has support both from epidemiological data and the findings of immunological abnormalities in spinal fluid that reflect an immune response to an as yet undetermined antigen, possibly a pathogen, in the cerebrospinal fluid. Our review will outline the current understanding of the role of infection in the causation and progression of MS. We will review the data that point to an infectious cause of MS and consider the specific agents Chlamydophila (Chlamydia) pneumoniae, Human Herpes Virus 6, and Epstein-Barr Virus, that are implicated in either the development or progression of MS.  Calciomics: prediction and analysis of EF-hand calcium binding proteins by protein engineering Ca(2+) plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms. Viruses also utilize the universal Ca(2+) signal to create a specific cellular environment to achieve coexistence with the host, and to propagate. In this paper we first describe our development of a grafting approach to understand site-specific Ca(2+) binding properties of EF-hand proteins with a helix-loop-helix Ca(2+) binding motif, then summarize our prediction and identification of EF-hand Ca(2+) binding sites on a genome-wide scale in bacteria and virus, and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes and the nonstructural protein 1 (nsP1) of Sindbis virus. When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca(2+)-binding sites that have been previously underrepresented due to the limitation of conventional methodology. Ca 2+ , a signal for "life and death", is involved in almost every aspect of cellular processes. Due to its abundant bioavailability, Ca 2+ was selected through evolution to perform multiple biochemical roles, acting as a second messenger inside mammalian cells to regulate a myriad of important cellular processes from triggering life during fertilization to facilitating apoptosis [1, 2] . As best exemplified by fast responses controlled by highly localized Ca 2+ spikes and slow responses regulated by repetitive global Ca 2+ transient oscillation or intracellular Ca 2+ waves, Ca 2+ signals exhibit diversified spatio-temporal patterns to meet varying demand of cellular processes [3] (Figure 1 ). Errors in any step of the calcium signal pathway can be critical, resulting in uncontrolled cell death or abnormal gene expression [4, 5] .
Ca 2+ is able to bind to hundreds of cellular proteins over a 10 6 -fold range of affinities (nM to mM) (Figure 2 (a)), depending on the nature of the Ca 2+ -modulated events. Ca 2+ binding has been shown to be essential for stabilizing proteins as well as maintaining proper cellular free Ca 2+ concentrations as seen in buffer proteins such as calbindin D 9k and parvalbumin ( Figure 2 ). Generally the Ca 2+ modulated activity is achieved through Ca 2+ -dependent conformational changes in Ca 2+ -binding proteins. For example, one of the ubiquitous intracellular trigger (modulating) proteins, calmodulin (CaM), has been shown to interact with over 300 proteins [6] ( Figure 2 ). Interestingly, this protein has been recently shown to regulate a large class of membrane proteins that are essential for cell signaling and cell-cell communication such as gap junctions [6] and voltage-dependent Ca 2+ channels. Although bacterial cells do not have complex subcompartments or organelles, there is strong evidence that Ca 2+ plays an essential role in bacterial signaling, communication and stability similar to that observed in eukaryotic cells (Figure 1 (a)) [7] [8] [9] [10] [11] . Bacterial cells also have a well-regulated cytosolic free Ca 2+ concentration (ap- Viruses avidly perturb the intracellular Ca 2+ signaling network to achieve their own demand. A number of viral proteins (oval shape) from different families of viruses disrupt Ca 2+ signaling by targeting various Ca 2+ signaling components. Adapted with permission from ref. [3, 6] .
proximately 0.1-2 μM) that is significantly lower than that observed in the extracellular medium (mM) due to Ca 2+ transporters and channels [8] [9] [10] [11] . Similar to the eukaryotic systems, P-type ATPase Ca 2+ efflux pumps have been characterized from Synechococcus and Flavobacterium. A Ca 2+ transporter of S. pneumoniae is involved in Ca 2+ -DNA uptake, lysis, and competence [12, 13] . Uptake of Ca 2+ and other divalent cations can also accompany uptake of phosphate by the phosphate transport system of E. coli. Furthermore, it has been reported that bacteria contain Ca 2+ binding proteins that are essential for cell adhesion and communication [14] [15] [16] [17] . Viruses, on the other hand, utilize the universal Ca 2+ signal to create a specific cellular environment to achieve their own purposes (Figure 1(b) ). Ca 2+ plays important roles in viral gene expression, post-translational processing of viral proteins, virion structure formation, virus entry, and virion maturation and release. As shown in Figure 1 (b), the interplay between viruses and Ca 2+ in the infected cell falls generally into three major categories: (1) viral proteins directly or indirectly disturb Ca 2+ homeostasis by altering membrane permeability and/or manipulating key components of the Ca 2+ -signaling apparatus; (2) viral proteins directly bind to Ca 2+ for structural integrity or functionality; and (3) critical virus-host interactions depend on cellular Ca 2+ -regulated proteins or pathways.
According to their structural features, Ca 2+ -binding sites in proteins are classified as either non-continuous or continuous. In non-continuous sites the Ca 2+ ligand residues are located remotely from one another in the protein sequence. Most of the Ca 2+ binding proteins, such as cadherins, C 2 domains, site I of thermitase, phospholipase A 2 , and D- galactose binding protein (GBP) belong to this family. Continuous Ca 2+ -binding sites have binding pockets formed by a stretch of contiguous amino acids in the primary sequence (e.g. EF-hand proteins) ( Figure 2 ). EF-hand proteins have a conserved Ca 2+ binding loop flanked by two helices [18, 19] . Based on the conserved features of the Ca 2+ -binding loop, EF-hand proteins have been divided into two major groups: the canonical EF-hands as seen in CaM and the pseudo EF-hands exclusively found in the N-termini of S100 and S100-like proteins [18] . Their major difference lies in the Ca 2+ binding loop: the 12-residue canonical EF-hand loop binds Ca 2+ mainly via sidechains (loop positions 1, 3, 5, 12), whereas the 14-residue pseudo EF-hand loop chelates Ca 2+ mostly via backbone carbonyls (positions 1, 4, 6, 9) ( Figure  2 ). Each type of EF-hand loop has a bidentate Ca 2+ ligand (Glu or Asp) that functions as an anchor at the C-terminal of the binding loop. Among all the structures reported to date, the majority of EF-hand sites have been found to be paired either within multiple canonical EF-hand motifs or through the interaction of one pseudo EF-hand motif with one canonical motif [18] (Figure 2 ). For proteins with odd numbers of EF-hands, such as the penta-EF-hand calpain, EF-hand motifs are coupled through homodimerization or heterodimerization [20] [21] [22] .
Due to the spectroscopically-silent nature of calcium and its physiological abundance, determination of the calcium binding capability of proteins is challenging. First, most experimental methods such as dialysis are only sensitive to the total calcium content. In addition, overcoming the persistent background contamination of calcium during the preparation of calcium-free sample for proteins with strong calcium binding affinities is a non-trivial task. Further, since most calcium binding proteins contain multiple calcium binding sites that cooperatively bind calcium resulting in induced conformational change (e.g., CaM) ( Figure 2 ), obtaining site-specific calcium binding affinity is limited by complication from contributions from cooperativity and conformational entropy [23] . Hence, understanding the molecular mechanism of biological function related to calcium is largely hampered by the lack of site specific information about the calcium-binding properties, especially for the ubiquitous EF-hand calcium-binding motif. Progress in understanding the molecular mechanism of calcium modulated biological process requires us to answer several important questions. First, what are the site-specific calcium binding affinities of calcium binding proteins, particularly those that utilize multiple coupled calcium binding sites to respond to sharp changes in cellular calcium concentration? Next, how can we predict or identify calcium binding sites in proteins using genomic and structural information? Finally, how can we verify calcium binding capabilities in the bacteria and virus genomes?
In this paper we first describe our effort in developing a grafting approach to understand site-specific calcium binding affinities using calmodulin as an example. Next we discuss our progress in predicting EF-hand calcium binding sites in various biological systems such as bacteria and virus systems. We then report our results following application of the grafting approach to probe calcium binding capabilities in streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes and the nonstructural protein of Sindbis virus.
To overcome the above-mentioned barriers and limitations associated with naturally-occurring Ca 2+ binding proteins, we have developed a grafting approach for engineering a single Ca 2+ -binding site in order to dissect the key structural factors that control Ca 2+ -binding affinity, conformational change and cooperativity. In principle, the key determinants for Ca 2+ affinity can be systematically introduced into a stable host protein frame and evaluated by eliminating or minimizing the contribution of conformational change. The key factors that are essential for Ca 2+ -dependent conformational change can be further revealed by analyzing the folding, stability, dynamics, and conformations of the host protein upon binding of a designed Ca 2+ -binding site without the complication of cooperativity. The cooperativity of two-coupled Ca 2+ -binding sites can then be estimated once the intrinsic Ca 2+ -binding affinities of both sites are obtained based on the energetics relationship. Figure 3 shows our grafting approach in obtaining site-specific calcium binding affinity using domain1 CD2 as a scaffold protein.
We have shown that CD2 is an excellent scaffold protein [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . It retains its native structure following insertion of the EF-hand motif both in the absence and presence of Ca 2+ ions. This provides the foundation for measuring the intrinsic Ca 2+ binding affinity with minimized contribution of protein conformational change. In addition, the aromatic Grafting approach to probe site-specific metal binding properties of Ca 2+ -binding proteins. (a) Schematic representation of the grafting approach. Any predicted linear Ca 2+ -binding sequence can be inserted into the host protein CD2 domain 1 (CD2.D1) between residues S52 and G53 without disrupting the integrity of the host protein. Metal binding to the engineered protein can be monitored by taking advantage of a potential LRET pair, the buried Trp (W32) within the two layers of beta-sheets and the terbium ion bound to the inserted sequence. (b) Flow chart showing the application of grafting approach to confirm metal binding of predicted Ca 2+ binding sites. Adapted with permission from ref. [23, 29] . residues in CD2 enable us to obtain Tb 3+ affinity of the grafted Ca 2+ binding loop using FRET. Ca 2+ and its analog La 3+ are able to compete with Tb 3+ for the grafted metal binding site. We have also optimized the length of two glycine linkers that connect the Ca 2+ binding loop and CD2 to provide sufficient freedom for the loop. The grafted EFloop III of CaM in different protein environments and scaffolds (such as CD2) has similar metal binding affinities for La 3+ and Tb 3+ , which implies that the grafted EF-hand loop is largely solvated and functions independently from the host protein or the protein environment. More importantly, using high resolution NMR and 15 N labeled protein, we have shown that both Ca 2+ and La 3+ specifically interact with the residues in the grafted EF-loop [25] , suggesting that the grafted loop retains its native Ca 2+ binding property. In addition, to dissect the contribution of the EF-loop and its flanking segments on Ca 2+ affinity, we have inserted the EF-loop, the loop with the exiting F-helix, and the loop with both EF-helices of Site III of CaM into CD2. In contrast to the largely unfolded structure of the isolated peptide fragment, the inserted flanking helices are partially formed, as revealed by both CD and NMR. Ca 2+ affinity is enhanced about 3-10 fold when the flanking helices are attached. Further, we have first estimated the intrinsic Ca 2+ affinities of the four EF-hand loops of CaM (I-IV) by individually grafting them into CD2. EF-loop I exhibits the strongest while EF-loop IV has the weakest binding affinity for Ca 2+ , La 3+ , and Tb 3+ . EF-loops I-IV of CaM have dissociation constants for Ca 2+ of 34, 245, 185, and 814 μM, respectively. Based on the results, we proposed a charge-ligandbalanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are major determinants for the Ca 2+ binding affinities of EFloops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca 2+ binding properties and to estimate the cooperativity and conformational change contributions of coupled EF-hand motifs. We have shown that the contribution of the cooperativity and conformational change to the Ca 2+ affinity for the C-terminal is 40% greater than that for the N-terminal. The same approach will be used to probe the site-specific Ca 2+ affinity of bacterial proteins. Furthermore, we have applied high resolution pulsed-fieldgradient diffusion NMR (PFG NMR) and analytical ultracentrifugation to investigate the oligomeric state of the isolated EF-loop III of CaM in CD2 with and without the flanking helices. The loop without the helices (CaM-CD2-III-5G) remains unpaired in solution in the absence and presence of Ca 2+ . However, the loop with the flanking helices (CaM-CD2-III-5G-EF) is a dimer in the presence of Ca 2+ [34] . Our findings suggest that hydrophobic residues on flanking helices play an essential role in dimerization and coupling of two EF-hand motifs for stronger Ca 2+ affinity.
By taking advantage of the sequence homology of currently available EF-hand loops and the flanking structural contents, we generated a series of patterns for the prediction of EFhand proteins. We have modified the pattern PS00018 by allowing more choices (Glu, Gln, and Ser) at position 1 and adding constraints at the flanking helical regions for canonical EF-hand motifs. In addition, several patterns have been developed to identify EF-hand like sites with different structural elements flanking the loop. Further, to circumvent the problem of identifying the pseudo EF-hand loop, a pattern has been developed by moderately loosening the constraints at the paired C-terminal canonical EF-hand and incorporating reserved residues in the N-terminal pseudo EF-hand. Compared with the original pattern PS00303, the new pattern reflects conserved genomic information in both EF-motifs and significantly improved the predictive accuracy and sensitivity [41] .
To understand the role of Ca 2+ in bacteria, we have predicted and analyzed potential bacterial EF-hand and EFhand like Ca 2+ -binding motifs on a genome-wide scale using our developed bioinformatic tool (http://www.chemistry.gsu. edu/faculty/Yang/Calciomics.htm). A total of 390 putative Ca 2+ -binding proteins have been predicted. Of these, 40 proteins were identified with multiple EF-hands ranging from 2 to 6, and 16 of these 40 proteins have been reported previously [32] . The other 350 proteins contain mononuclear EF-hands. Several examples in three classes of these predictions with diversity in the Ca 2+ -binding loop and flanking structural regions together with one class of prediction from other methods are shown in Table 1 . These proteins are implicated in a variety of cellular activities, including Ca 2+ homeostasis [42] [43] [44] , chemotaxis [8, 45, 46] , binding to scaffold proteins [47] , resistance to acid stress [48, 49] etc. According to their sequence homology and based on the assumption that they evolved from a common ancestor, these proteins could be further classified into several major phylogenetic groups [41] .
A notable example is the streptococcal hemoprotein receptor (Shr), a surface protein with a role in iron uptake that has no significant homologues in other bacteria but shares partial homology with eukaryotic receptors such as Toll and G-protein dependent receptors (gi 15675635, GenBank). Additional sequence analysis identified a leucine-rich repeat domain, an EF-hand Ca 2+ domain, and two NEAT domains [50] . As shown in Figure 4 , the single EF-hand motif identified in Shr has a significant homology to that of CaM with all the conserved Ca 2+ binding ligand residues and two flanking helices (Figure 4 ).
Though EF-hands have been found abundantly in eukaryotes and bacteria, literature reporting EF-hand or EF-hand like Ca 2+ -binding motifs in virus proteins is scarce, possibly due to lack of accurate prediction methods and robust validating methodologies. A thorough search in PubMed with the key words "EF-hand and virus" only results in 4 examples (1, 3, 5, 7, 9, 12) and the hydrophobic residues (n, blue). The predicted EF-hand from Shr (Streptococcal hemoprotein receptor, S. pyrogenes) and PlcR (phospholipase accessory protein, P. aeruginosa) are aligned with some EF-hands known to form oligomers: CaM EF3, the third EF-hand from calmodulin; TnC EF3, the third EF-hand from troponin C; PV EF3, the third EF-hand from parvalbumin; D9K EF2, the canonical EF-hand from calbindin D9K, The search patterns used for the identification of the EF-hand loop and flanking helices (Helix E and Helix F) are also shown in the bottom. ). In addition, the functions of almost 20% of these matched proteins remain uncharacterized. We hope that our prediction will serve as a prelude to more extensive searching for additional viral Ca 2+ -binding proteins that are closely associated with virus-host interacting events ( Figure  1(b) ). Rubella virus (RUB), the only member of the genus Rubivirus, in the Togaviridae family, is the causative agent of a disease called rubella or German measles. Nonstructural protein (NS) open reading frame (ORF) of RUB encodes a polypeptide precursor which is able to cleave itself into two replicase components involved in viral RNA replication. A putative EF-hand Ca 2+ binding motif of the nonstructural protease that cleaves the precursor was successfully predicted across different genotypes of RUB and determined by established grafting approach [51] . The grafted EF-loop bound to Ca 2+ and its trivalent analogs Tb 3+ and La 3+ with dissociation constants of 214, 47, and 14 μM, respectively. The NS protease containing mutations of cal-cium binding sites elimination (D1210A and D1217A) was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca 2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. Interestingly, the same bioinformatics algorithm that successfully predicted the Ca 2+ -binding loop in the RUB NS protease also predicted an EF-hand Ca 2+ -binding motif in nsP1 of alphaviruses (Figure 4(b) ). NsP1 is one of the four nonstructural proteins produced by alphaviruses and is involved in membrane binding and has methyl/guanylyl transferase activity.
Next, we grafted two predicted 29-residue EF-hand motifs, one from the Shr of S. pyrogenes (CD2.Shr.EF) and the viral nsP1 of Sindbis virus (CD2.Sin.EF), into CD2.D1 to examine their Ca 2+ binding capability by using aromatic residue-sensitized Tb 3+ luminescence resonance energy transfer (Tb 3+ -LRET) ( Figure 5 ). Circular dichroism studies of both engineered proteins showed a notable trough at ~216 nm which is characteristic of β-sheet structure. More negative signals were observed below 240 nm due to the contribution from the insertion of the helix-loop-helix sequences. Both proteins were able to bind the Ca 2+ analog, Tb 3+ , with affinities of 25.1 μM for CD2.Shr.EF and 16.4 μM for CD2.Sin.EF ( Figure 5(c)-(d) ). The biological relevance of these EF-hand Ca 2+ -binding motifs will be further investigated.
Overall, based on sequence homology, we have developed a straightforward and fast method to detect linear Ca 2+ -binding motifs from genomic information. Genome-wide analysis of EF-hand Ca 2+ -binding motifs in bacteria and virus have been analyzed with this methodology. Experimentally, we have also developed a robust and reliable grafting approach to study Ca 2+ -binding properties of continuous Ca 2+ binding sites. This novel approach has been successfully used to dissect site-specific Ca 2+ binding affinity and cooperativity among the four canonical EF-hands in the prototypical Ca 2+ -binding protein, calmodulin. The combination of these two approaches is expected to enable us to explore more Ca 2+ binding sites that are underrepresented due to the limitation of available methodology. Proteomics Comparison of Cerebrospinal Fluid of Relapsing Remitting and Primary Progressive Multiple Sclerosis BACKGROUND: Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85–90%) and primary progressive (PP) MScl (10–15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins. METHODOLOGY/PRINCIPAL FINDINGS: CSF samples (n = 31) were handled according to the same protocol for quantitative mass spectrometry measurements we reported previously. In the comparison of PP MScl versus RR MScl we observed a number of differentially abundant proteins, such as protein jagged-1 and vitamin D-binding protein. Protein jagged-1 was over three times less abundant in PP MScl compared to RR MScl. Vitamin D-binding protein was only detected in the RR MScl samples. These two proteins were validated by independent techniques (western blot and ELISA) as differentially abundant in the comparison between both MScl types. CONCLUSIONS/SIGNIFICANCE: The main finding of this comparative study is the observation that the proteome profiles of CSF in PP and RR MScl patients overlap to a large extent. Still, a number of differences could be observed. Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling. It is suggested in literature that the Notch pathway is involved in the remyelination of MScl lesions. Aberration of normal homeostasis of Vitamin D, of which approximately 90% is bound to vitamin D-binding protein, has been widely implicated in MScl for some years now. Vitamin D directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes, and so it may be involved in neuroprotective elements in MScl. Multiple sclerosis (MScl) can be divided into two major subtypes based on clinical representation of disease symptoms in the patients [1] . Between 85-90% of patients can be classified as having the relapsing remitting (RR) MScl subtype, in which disease relapses are followed by periods of remission, and 10-15% of all MScl patients are diagnosed with the primary progressive (PP) subtype [2] . Even within a single large Dutch MScl pedigree of 26 patients with similar genetic background, the percentage of patients with a PP phenotype remained 15% [3] .
By definition, in PP patients disease progression is characterized by a progressive course without relapses or remissions from the onset of the disease [4] . PP patients tend to have lower inflammatory lesional activity, for which no immunological or genetic explanation has been identified yet. The scarce compar-ative neuropathological studies show a large overlap in lesional pathology, but indicate less inflammatory activity for PP, with still substantial axonal damage [5] . The general picture is that relapse onset and PP forms share substantial characteristics. In other words, it has remained a challenge to identify the biological parameters that determine a PP disease course.
Although proteomics analysis of active multiple sclerosis lesions may be a straightforward approach to study the processes involved in MScl disease pathways [6] , this is very difficult to perform in living individuals. In most cases the pathology of the disease can only be investigated in post-mortem material, which quite frequently represents the end-stage of the disease. The study of CSF taken during disease appears a good alternative. CSF is in close contact with the CNS parenchyma and collects the products of the inflammatory and neurodegenerative processes of MScl activity.
Proteomics analysis of CSF has detected a number of proteins that were elevated in MScl patients [7, 8] . Additionally, differentially abundant proteins identified by proteomics, such as apolipoprotein A1 [9] and chromogranin A [10] were validated by other techniques. Other studies added additional data on elevated immunoglobulin expression in MScl CSF, as well as increased levels of apolipoprotein E [11, 12] . Yet in all currently reported proteomics CSF studies of MScl patients either only a single subtype of MScl patients or a combined group of all subtypes of MScl was studied, whilst the differences between the subtypes of MScl remained unexplored. Because RR MScl and PP MScl are very different in terms of disease course and disease progression, this also has therapeutic consequences. Hence, there are probably also differences on a biological and pathological level, which could, if determined, be very useful for elucidation of the biology and pathology of both disease types.
In the current study we specifically aimed to differentiate between the MScl patients and healthy controls and between both subtypes of MScl by comparing CSF proteins and peptides. Subsequently, the identified biomarker proteins and peptides were discussed in relation to the different pathological processes observed in RR MScl and PP MScl. 
The CSF samples of MScl patients, divided into two groups, RR MScl [13] and PP MScl [14] , were collected from untreated patients undergoing routine diagnostic procedures by an experienced neurologist (RQH), and matched for presence or absence of oligoclonal bands. The healthy control CSF samples were taken from patients receiving spinal anesthesia prior to non-neurological minor surgical interventions, such as knee and hip replacements, groin rupture and Achilles tendon rupture. All samples were handled in exactly the same way after sampling, using a procedure that has been previously reported [12] . In brief, immediately after sampling, the CSF samples were centrifuged to discard cells and cellular elements and the total protein concentration and albumin concentrations were determined. The number of oligoclonal bands and the intrathecal cell count were also reported. The remaining volume of the samples was aliquoted and stored at 280uC, where they remained until sample preparation for this study. No extra freeze-thaw cycles were allowed.
The CSF samples were handled according to the same protocol for quantitative MALDI-FT-ICR MS measurements we reported previously [12] , which consists of a blinded experiment in which the samples were digested by trypsin and subsequently measured by MALDI-FT-ICR (APEX IV Qe 9.6 Tesla MALDI-FT-ICR mass spectrometer (Bruker Daltonics, USA)). After calibration by means of omnipresent albumin peaks, an analysis matrix is generated. A univariate analysis, in which a p-value was determined for every peak position, was used for statistical analysis, in which two groups were compared at a time, for a total of three individual comparisons between the groups. The differentially abundant peaks (p,0.01) in the comparisons were considered for identification purposes. The fold increase of every identified peptide with p,0.01 in the comparisons was determined to confirm differential abundance between groups. Assessment of the statistical background, by means of permutation of a series of 50 scramblings of the samples for each comparison, was used to define a cut-off number for the determination of statistically significant differences between groups. In this permutation procedure all samples are randomly assigned a new group number, scrambling the sample group compositions, prior to performing the univariate analysis to determine the p-values for each peak position. By this method the number of peaks that is assigned a p-value below 0.01 by chance is determined. Iterative repetition of this procedure allows for a statistically relevant mean with standard deviation that could be taken as a realistic background value for this not normally distributed data.
The differentially abundant peptides were identified by nano-LC-ESI-Orbitrap MS. These measurements were carried out on a Ultimate 3000 nano LC system (Dionex, Germering, Germany) online coupled to a hybrid linear ion trap/Orbitrap MS (LTQ Orbitrap XL; Thermo Fisher Scientific, Germany). Five mL digest were loaded on to a C18 trap column (C18 PepMap, 300 mm ID 65 mm, 5 mm particle size, 100 Å pore size; Dionex, The Netherlands) and desalted for 10 minutes using a flow rate of 20 mL/min 0.1% TFA. Then the trap column was switched online with the analytical column (PepMap C18, 75 mm ID 6150 mm, 3 mm particle and 100 Å pore size; Dionex, The Netherlands) and peptides were eluted with following binary gradient: 0%-25% solvent B in 120 min and 25%-50% solvent B in further 60 minutes, where solvent A consist of 2% acetonitrile and 0.1% formic in water and solvent B consists of 80% acetonitrile and 0.08% formic acid in water. Column flow rate was set to 300 nL/ min. For MS detection a data dependent acquisition method was used: high resolution survey scan from 400-1800 Th. was performed in the Orbitrap (value of target of automatic gain control AGC 10 6 , resolution 30,000 at 400 m/z; lock mass was set to 445.120025 u (protonated (Si(CH 3 ) 2 O) 6 ) [15] ). Based on this survey scan the 5 most intensive ions were consecutively isolated (AGC target set to 10 4 ions) and fragmented by collision-activated dissociation (CAD) applying 35% normalized collision energy in the linear ion trap. After precursors were selected for MS/MS, they were excluded for further MS/MS spectra for 3 minutes. The MS/MS identifications were obtained using in the Bioworks 3.2 (peak picking by Extract_msn, default settings) software package (Thermo Fisher Scientific, Germany), and its' SEQUEST feature, using minimum XC scores of 1.8, 2.2 and 3.75 for reliable identification of single, double and triple charged ions respectively in the UniProt-database (version 56.0, human taxonomy (20069 entries)). Carboxymethylation of Cysteine (+57.021 u) as fixed and oxidation of Methionine (+15.996 u) as variable modifications and tryptic cleavage were considered. The number of allowed missed cleavages was 2, the mass tolerance for precursor ions was 10 ppm and for fragment ions 0.5 Da. The cut-off for mass differences with the theoretical mass of the identified peptides was set at 2 ppm.
Contamination of CSF by serum of plasma is a possible issue in CSF peptide profiling, because if one or more of the samples is contaminated by serum or plasma, the comparison of CSF peptide profiles is inevitably skewed by the higher total protein concentrations in serum or plasma [16] . To prevent inclusion of contaminated CSF samples in this study, the CSF samples were checked for specific blood contamination. If a hemoglobin peptide could be identified by nanoLC-ESI-Orbitrap (C 18 column) with sufficiently high confidence score or if the mass peak 1274.7255 (part of hemoglobin gamma) has a signal to noise of 4 or higher in MALDI-FT-ICR measurements, the sample was discarded from further analysis due to plasma/serum contamination. Another blood specific protein, apolipoprotein B100 was checked in the same way as possible blood contamination.
For two proteins, for which we found differentially abundant peptides, we performed validation experiments. This was done by commercially available ELISA (for vitamin D-binding protein) and by western blot (for protein jagged-1), using a validation cohort of patients consisting of 10 RR MScl and 10 PP MScl samples. The samples of the validation cohort were demographically and clinically comparable to the original cohort of patients (average EDSS RR MScl group (standard deviation in brackets): 2.5 (0.8), average EDSS PP MScl group 2.8 (1.0), and the disease duration, presence/absence of oligoclonal IgG bands and male female ratio were all similar to the original cohort (no p-values below 0.05) using a t-test to compare the groups). Additionally we also measured the original samples using this ELISA and western blot. For the first protein, vitamin D-binding protein, we performed a commercially available ELISA (Immundiagnostik, Germany) according to the manufacturers specifications. For the second protein that was differentially abundant between the both MScl types, jagged-1, we performed a two-step western blot using goat anti-jagged1 antibodies (primary antibody) and anti-goat antibodies (secondary antibody) (Sigma Aldrich, United States). Protein transfer was checked by Ponceau staining. Quantitative assessment of the gel bands after photoluminescence was performed using Image J (freely available at www.rsb.info.nih.gov/ij).
In total 34 CSF samples were used for mass spectrometry analysis, while twenty of these samples and twenty additional samples were used for validation experiments. All samples analyzed by mass spectrometry were tested as being negative for serum/plasma contamination by MALDI-FT-ICR measurements. However, in three samples we were able to identify hemoglobin peptides with sufficiently high XC scores for confident identification using nanoLC-ESI-Orbitrap measurements. These three samples, 2 PP MScl and 1 RR MScl, were subsequently excluded from further analysis. The analysis matrix, which was used to profile the differences in peptide profile between the three groups, consisted therefore of 31 CSF samples (Table 1 and Supplementary information Samples S1 file).
After the MALDI-FT-ICR spectra were loaded into Peptrix TM software package, they were each tagged with a group number (1, 2 and 3 for PP MScl, RR MScl and controls respectively). Calibrating using five omnipresent albumin peaks was followed by generation of an analysis matrix with the intensity of all peaks of every sample recorded for all detected peaks (Supplementary Information Matrix S1 file). Using the Wilcoxon-Mann-Whitney test to compare the groups pair-wise, the comparison between both MScl types resulted in 15 peak masses with p-values below 0.01. By scrambling the sample groups the number of background peaks was determined at 17, so the number of differentially abundant peptide peaks in the comparison of the two MScl types is around the level of the number of background peaks, indicating that the difference between these two groups appears to be nonexistent or at least at background level. However, the proteins that were identified with low p-values in this comparison were of substantial interest in a MScl context.
A total of 43 peptide peaks with a p-value below 0.01 were observed for the comparison of PP MScl versus the controls. The comparison of RR MScl versus the controls had 41 peak masses with p-values lower than 0.01. Seventeen of the peak masses with p,0.01 were present in both comparisons.
Identification of the differentially abundant peptides was performed by measuring all samples using the nanoLC-ESI-Orbitrap. Due to the prefractionating by nanoLC far more peak masses and identifications are generated by ESI-Orbitrap than there are peak masses in the analysis matrix generated by quantitative MALDI-FT-ICR. Although many of the identified peptides do not correspond to peak masses in the analysis matrix, we were able to identify a number of differentially abundant peptides for all three comparisons (full list, including charge states and sequence coverage, can be found in the supplementary material (Supplementary Information Mass Spectrometry S1 file)). Of the 43 differentially abundant peptide masses that were observed using MALDI-FT-ICR mass spectrometry in the comparison of PP MScl versus the controls we were able to identify 29 peptides (Supplementary Information Table S1 ). These peptides included several peptides of Ig gamma-1 and Ig kappa. Another differentially abundant peptide was identified as a part apolipoprotein D, which has previously been shown to be elevated intrathecally in MScl patients [17] . In the comparison of RR MScl versus controls we were able to identify 24 of the 41 differentially abundant peptide masses, which, as was the case with the 
The comparison of PP MScl versus RR MScl showed a limited number of differentially abundant peptide peaks. Of these peaks 7 were identified, the most notable being protein jagged-1 ( Table 2 ). This particular protein was over three times less abundant in PP MScl compared to RR MScl. Another interesting differentially abundant protein is vitamin D-binding protein, which was not detected by mass spectrometry in the PP MScl samples but was detected in the RR MScl samples. Other proteins, such as serine/ threonine kinase NLK and sodium leak channel non-selective protein were more abundant in PP MScl than in RR MScl, although for the latter protein the difference was small (1.18 fold increase).
Because our main interest was focused on the differences between the two MScl types we selected two differentially abundant proteins from that comparison for validation purposes using 10 PP MScl and 10 RR MScl samples measured by mass spectrometry and an additional 10 PP MScl and 10 RR MScl samples from independent patients. Validation by ELISA showed that the concentration of vitamin D-binding protein was significantly lower (t-test, p,0.05) in the PP MScl group compared to the RR MScl group in the samples also measured by MALDI-FT-ICR MS (Table 3) . A similar result was observed in the new, clinically and demographically comparable validation samples (Table 4 ) After western blotting, quantitative assessment of the gel bands showed that protein jagged-1 was indeed less abundant in PP MScl than in RR MScl. A t-test on the photoluminescence readout values of the original sample set showed a p-value below 0.05 when comparing the RR and PP MScl samples ( Table 3) . The same comparison in the validation samples also showed a p-value below 0.05), indicating a significant differential abundance of jagged-1 in the two MScl disease types (Table 4) .
In order to place the identified proteins in a biological context, they were uploaded to the Ingenuity Pathways Analysis service (Ingenuity Systems) for to network analysis. Six of the seven differentially abundant proteins were placed in a network relating to neurological disease (Figure 1 ).
The main finding of this comparative study is the observation that the proteome profiles of CSF in PP vs RR MScl patients overlap to a large extent. This is in line with the lack of clear-cut differences between the two major clinical MScl sub-groups, at genetic, immunological and neuropathological levels. Interestingly, our approach using sensitive state-of the art mass spectrometry techniques, led to the identification of a few distinct CSF proteins, some of them with biological functions that appear of direct interest for MScl pathology. Two of these proteins were validated by other techniques as well as in a validation sample set. The lack of statistical power, due to the low number of biological samples available for this study, led to a less than ideal statistical analysis, meaning that the reported p-values may not be directly associated to disease pathology. However, the reported unvalidated proteins are of significant biological interest, and among these are several immunoglobulin proteins, which have been previously reported to be elevated in CSF of MScl patients [18, 19] .
The number of peaks with low p-values in the comparison of the two MScl types is lower than the number of background peaks, so this is a strong indication that, even though there may be peptides and proteins that are differentially abundant in the comparison, overall the difference between the two disease types appears to be undetectable by means of the univariate analysis. One of the peptides of protein jagged-1 is over three times less abundant in PP MScl and also is observed with a lower incidence in this group ( Table 2) . Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling, which influences neuronal function and development [20] . The Notch signaling pathway has long been known to influence cell fate in the developing nervous system. Jagged-1 has been found to be highly expressed in hypertrophic astrocytes within and around active MScl plaques lacking remyelination, while, in contrast, there was negligible jagged-1 expression in remyelinated lesions suggesting involvement of the Notch pathway in remyelination in MScl [21] . Later, linkage equilibrium screening implicated a number of genes, including the jagged-1 gene, as susceptibility genes for MScl in a large contingent of Europeans [22] . It has also been suggested that jagged-1 has therapeutic potential in the treatment of CD8 + T cell mediated diseases, due to it's ability to deliver indirect negative signals into CD8 + T cells in vivo [23] . Additionally, animal models have shown that elevated expression of Notch and jagged-1 expression does not appear to be a limiting factor in remyelination, but the animal model study reports that there were no quantitative differences in Notch1 expressing cells in slow and rapidly remyelinating lesions, indicating that Notch-Jagged signaling is not a rate-limiting determinant of remyelination in rodent models of demyelination [24] . Additionally immunohistochemistry experiments have shown that constituents of the Notch pathway are expressed in remyelination in an animal model of T-cell-and antibody-mediated CNS demyelination [25] . However, network studies based on the quantitative expression levels of 20 genes in over one hundred individuals identify jagged-1 as a new therapeutic target whose differential behavior in the MScl network was not modified by immunomodulatory therapy, illustrating how network analysis can predict therapeutic targets for immune intervention and identifying the immunomodulatory properties of jagged-1, making it a new therapeutic target for MScl and other autoimmune diseases [26] . The identified peptide of vitamin D-binding protein is not observed in any of the PP MScl samples, but small peaks of this peptide are detected in 6/11 RR MScl samples. Impaired vitamin D homeostasis has been widely implicated in MScl for some years now [27] [28] [29] . This vitamin directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes [30, 31] , and so it may be involved MScl. Considering that the geographic incidence of MScl indicates an increase in MScl with a decrease in sunlight exposure, that vitamin D is produced in the skin by solar or UV irradiation and that high serum levels of 25-hydroxyvitamin D have been reported to correlate with a reduced risk of MScl, a protective role of vitamin D has been suggested [32] . More than 99% of 25-hydroxyvitamin D, the principle circulating metabolite of vitamin D is bound to proteins, of which approximately 90% is bound to vitamin D-binding protein [33, 34] . Recently, a CSF proteomics study showed that serum levels of vitamin D-binding protein were decreased significantly in RR MScl patients compared to other neurological disorders [35] . While our results do not indicate a differential abundance difference between the MScl subtypes and the controls, the two disease types did vary significantly in CSF levels of vitamin D-binding protein, with RR MScl showing a higher abundance. Since a neuroprotective function has been suggested for vitamin D, it may be that in PP MScl this neuroprotective pathway is at least partially deficient, resulting in a significantly more disabling disease manifestation. However, it should not be forgotten that vitamin D-binding protein has pleiotropic functions, beyond vitamin D metabolism. It can significantly enhance the chemotactic response to complement fragment C5a [36] , and there are substantial stimulatory effects on macrophages [37] . In light of the increasingly recognized role of innate immunity in the progressive phase of MS pathogenesis [38] , vitamin D-binding protein appears an interesting candidate mediator.
The identification of a peptide of beta-2-microglobulin as differentially abundant in the comparison between both MScl disease types is somewhat misleading. Another seven peptides of this protein were identified among the peak masses in the analysis matrix that had high p-values in the comparison of PP and RR MScl, so it is very likely that the low p-value of the peptide of beta-2-microglobulin reflects high abundant protein variations, suggesting this low p-value is most likely a false positive. In contrast, the other proteins that are differentially abundant in this comparison were either identified by the single peptide listed in Table 2 or by multiple peptides that had low p-values.
The identification of peptides of albumin and serotransferrin stands out in the tables of the differentially abundant peptides and proteins. While the identifications are essentially correct it must be noted that only 2.4% of the identified albumin peptides and 4.8% of the identified serotransferrin peptides had p-values below 0.01, which means that the values for the peptides of these two particular proteins are most likely due to other reasons than large abundance differences in these two proteins. In comparison, most of the other differentially abundant proteins that we identified were found by a small number of peptides with a low p-value or a single peptide with a low p-value. For these proteins we did not observe any other peptides with non-significant p-values, with the exception of bromodomain adjacent to zinc finger domain protein 1A and Rho GTPase-activating protein 18-like. For both of these proteins another peptide was identified with high p-value, indicating these proteins were likely not differentially abundant. The peptides with low p-values of these proteins were also characterized by a low fold increase, making them less interesting for independent immunoassay follow-up. Because of the healthy state of the CSF control group, no intrathecal inflammatory response was to be expected in this group. Therefore the very clear difference in immunoglobulin abundance in the comparison with the both MScl disease types can be explained. In the two comparisons of both MScl types with the control group, several proteins are present in both comparisons, for example Ig gamma-1 chain C region and apolipoprotein D. Apolipoproteins have been previously implicated in MScl. Proteomics studies have shown apolipoprotein E abundances to be elevated in CSF of MScl patients compared to controls [9, 12] . In the central nervous system apolipoprotein D is a lipocalin that is mainly expressed in glia, but also in neurons. This protein has been repeatedly implicated in MScl, and it has been shown that it has a neuroprotective effect in a number of neurodegenerative diseases by controlling the level of peroxidated lipids, which coincides with glial activation in mouse models of encephalitis [39] . A previous proteomics study showed increased levels of apolipoprotein D in patients with a clinically isolated syndrome of demyelination, indicating that abundance levels of this protein are highest in MScl patients at the time of their first exacerbation [10, 17] . Another potentially interesting protein found to be differentially elevated in the comparisons of both MScl types with the controls is ryanodine receptor 1. This receptor is involved in the maintenance of the calcium-equilibrium in brain tissue. The release of toxic levels of positively charged calcium ions may, due to the deleterious effects of excitotoxicity, represent a key mechanism of axonal degeneration in disorders such as MScl [40] .
In conclusion, the CSF peptide profile of the control samples differed from both MScl types, with, not unexpectedly, proteins related to immune response showing the highest fold increase in abundance in the MScl types compared to the controls. Even though the CSF peptide profiles measured by MALDI-FT-ICR of PP MScl and RR MScl were quite similar, still a few differences could be observed, most notably regarding the molecules confirmed by immunoassay, protein jagged-1 and vitamin Dbinding protein. Author Contributions Interstitial lung diseases in children Interstitial lung disease (ILD) in infants and children comprises a large spectrum of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. These disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. Typical features of ILD include dyspnea, diffuse infiltrates on chest radiographs, and abnormal pulmonary function tests with restrictive ventilatory defect and/or impaired gas exchange. Many pathological situations can impair gas exchange and, therefore, may contribute to progressive lung damage and ILD. Consequently, diagnosis approach needs to be structured with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. Several classifications for ILD have been proposed but none is entirely satisfactory especially in children. The present article reviews current concepts of pathophysiological mechanisms, etiology and diagnostic approaches, as well as therapeutic strategies. The following diagnostic grouping is used to discuss the various causes of pediatric ILD: 1) exposure-related ILD; 2) systemic disease-associated ILD; 3) alveolar structure disorder-associated ILD; and 4) ILD specific to infancy. Therapeutic options include mainly anti-inflammatory, immunosuppressive, and/or anti-fibrotic drugs. The outcome is highly variable with a mortality rate around 15%. An overall favorable response to corticosteroid therapy is observed in around 50% of cases, often associated with sequelae such as limited exercise tolerance or the need for long-term oxygen therapy. Interstitial lung disease (ILD) in infants and children represents a heterogeneous group of respiratory disorders that are mostly chronic and associated with high morbidity and mortality (around 15%) [1, 2] . These disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. Typical features of ILD include the presence of diffuse infiltrates on chest radiograph, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange [3] .
There have been many different approaches to the classification of ILD, with major shifts based on clinical investigation, improvement in chest imaging, and collaboration with pathologists. In 1998, Katzenstein and Myers proposed four histopathologically distinct subgroups of idiopathic interstitial pneumonias: usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP) and a closely related pattern termed respiratory bronchiolitis-associated ILD, acute interstitial pneumonia (formerly Hamman-Rich syndrome), and non specific interstitial pneumonia (NSIP) [4] . In 2002, an international multidisciplinary consensus classification of idiopathic interstitial pneumonias was proposed by the American Thoracic Society (ATS)/European Respiratory Society (ERS) [5] . This classification defined a set of histologic pattern that provided the basis for clinico-radiologic-pathologic diagnosis, with the final pathologic diagnosis being made after careful correlation with clinical and radiologic features. However, as discussed in several reports, the classification schemes of adult ILD are not satisfactory for the pediatric cases which seem to comprise a broader spectrum of disorders with a more variable clinical course [6] . In addition, pediatric histologic patterns often do not resemble pathologic features of lung tissues from adults and some forms are only observed in children younger than 2 years.
Among the proposed classifications for pediatric ILD, one strategy frequently used is to separate the primary pulmonary disorders and the systemic disorders with pulmonary involvement. Recently, an additional group has been introduced which is based on the concept that some pediatric ILD are observed more frequently in infants, while others are more specific to older children. The last ERS monography on ILD provided a chapter on pediatric classification which is based on a clear distinction between children aged 0-2 years and children over 2 years-old [7] . Indeed the stage of lung development and maturation should be taken into consideration when approaching a diagnosis of pediatric ILD. In this view, a new term "diffuse lung disease" has recently been introduced that comprises a diverse spectrum of lung disorders with impaired gas exchange and diffuse infiltrates by imaging. These disorders, more prevalent in young children, include diffuse developmental disorders, lung growth abnormalities, neuroendocrine cell hyperplasia and pulmonary interstitial glycogenosis, surfactant dysfunction disorders, disorders related to systemic diseases, disorders of immunocompromised host, and disorders of normal host caused by various insults such as aspiration syndrome or infections [8] . Some diseases are mostly observed in older children such as systemic diseases, idiopathic disorders as described in adults (DIP, UIP, NSIP and lymphoid interstitial pneumonia (LIP)), unclassifiable ILD and also infectious disorders [9] .
It is important to point out that the pathologic processes underlying the so-called diffuse lung diseases involve not only the alveolar structure but also the distal part of the small airways and the conducting zone, i.e. the terminal bronchioles. Terminal bronchioles are lined with a simple cuboidal epithelium containing Clara cells, basal cells and a limited number of ciliated cells. Clara cells secrete nonsticky proteinaceous compounds to maintain the airway in the smallest bronchioles, which constitute the quiet zone between the conducting and the respiratory lung zones [10] . The terminal bronchioles are surrounded by a spiral of smooth muscle. Each of the terminal bronchioles divides to form respiratory bronchioles which contain a small number of alveoli. Consequently, the term of diffuse lung disease refers to disorders that can affect both the distal part of the conducting and the respiratory lung zones, and include ILD as well as pathological processes leading to obstruction/ obliteration of small airways [8] . Therefore, diffuse lung diseases encompass a broader group of diseases than ILD which refers to disorders that affect the respiratory function of the lung and consequently the pulmonary structure responsible of the diffusion of gases between blood and air (i.e. the alveolar epithelium, the interstitium, and the pulmonary capillary endothelium).
The present review focuses on ILD in immunocompetent children, and excludes pulmonary consequences of previous lung injury in situations of chronic aspiration syndromes, resolving acute respiratory distress syndrome, and bronchopulmonary dysplasia.
An estimated prevalence of 3.6 per million has been reported by Dinwiddie and coworkers through a national survey of chronic ILD in immunocompetent children in the United Kingdom and Ireland over a three year period (1995) (1996) (1997) (1998) [1] . This prevalence is certainly under-estimated due to the lack of standardized definitions and the absence of organized reporting systems. From the limited published data composed mainly of case reports and small series, it seems that pediatric ILD occurs more frequently in the younger age and in boys [11] . In addition, nearly 10% of cases appear to be familial [12] .
The understanding of the mechanisms underlying the development and progression of ILD remains elusive [13, 14] . Indeed, for a long time, chronic ILD and pulmonary fibrosis were believed to result mainly from chronic inflammation following an initial injury to the alveolar epithelial lining [15, 16] . In cases of limited injury, it was thought that the reparative attempt could reverse the trend toward fibrosis. By contrast, in situations of continuing injury, the repair process driven by inflammatory molecules produced by the local cells will result in scarring and structural changes. Therefore, by targeting the inflammatory response, the belief was that fibrosis could be prevented or controlled. This theory explains the large use of anti-inflammatory therapy with, however, limited clinical efficacy.
Based on clinical and experimental observations, a new paradigm has progressively emerged with the alveolar epithelium being viewed as a key actor in the development of ILD [17] [18] [19] . Following injury, alveolar epithelial cells (AEC) may actively participate in the restoration of a normal alveolar architecture through a coordinated process of re-epithelialization, or in the development of fibrosis through a process known as epithelial-mesenchymal transition (EMT) [20] . Complex networks orchestrate EMT leading to changes in cell architecture and behaviour, loss of epithelial characteristics and gain of mesenchymal properties. The reasons for epithelial cell loss and inappropriate re-epithelialisation are still debated, but ongoing apoptosis is believed to be a key component in the progression of the disorder [21] . Prolonged denudation of the basement membrane contributes to altered interactions and cross-talk between AECs and mesenchymal cells, resulting in profound modifications of cell functions with imbalanced production of oxidants, proteases, and polypeptide mediators including cytokines and growth factors such as Transforming Growth Factor (TGF)-β and Endothelin (ET)-1. A consequence is the perpetuation of a vicious cycle with TGF-β promoting epithelial cell apoptosis, which in turn increases the local production of TGF-β [22] . ET-1 is also considered to be an important actor, based on the current knowledge of its numerous functions including fibroblast and smooth muscle cell mitogen, and stimulant of collagen synthesis [23, 24] . Recent studies showed that ET-1 is produced by AEC, and could induce alveolar EMT via stimulation of endogenous TGF-β production.
ILD may be caused by myriad etiologies with differing prognoses and natural history. Indeed, multiple factors may injure the alveolar epithelium and initiate the development of ILD [25] . The initiating injury can be introduced through the airways and the circulation, or can occur as a result of sensitization. Consequently, the mechanisms underlying disease progression will be influenced by the causative event as well as by the host and the environment. These mechanisms are developed through interactions of multiple pathways, which include apoptotic pathways, developmental pathways, and endoplasmic reticulum (ER) associated pathways ( Figure 1 ).
Apotosis plays a central role in lung remodeling associated with ILD [26] . An important molecule in the events associated with epithelial cell apoptosis is TGF-β, which is overexpressed in ILD. Downstream events linked to upregulation of TGF-β include modifications in the expression of various components of the cell cycle machinery, mainly the cyclin-dependent kinases (CDK) system that plays an essential role in ensuring proper cell cycle progression. Recently, much work has been focused on the protein p21cip1, a member of the CDK inhibitor family. This protein promotes cell cycle arrest to apoptosis in cases of cellular DNA damage. Interestingly, upregulation of p21cip1 has been reported in the lung tissues of patients with pulmonary fibrosis, primarily in hyperplastic alveolar epithelial cells [27] The increased expression of p21cip1 can favour the process of epithelial cell apoptosis. Apoptotic cells can also produce TGF-β. A consequence would be the perpetuation of a vicious cycle with TGF-β promoting epithelial cell apoptosis, which in turn increases the local production of TGF-β.
Recently, it has been suggested that genes associated with lung development and embryonic pathways could be involved in aberrant epithelium-mesenchymal crosstalk and epithelial plasticity, and could therefore participate in the development of chronic ILD. Selman and coworkers reported that lung fibrosis is characterized by enrichment for genes associated with cell adhesion, extracellular matrix, smooth muscle differentiations, and genes associated with lung development [28] [29] [30] [31] . During EMT in the embryonic period, cells undergo a switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype [32] . Molecular processes governing EMT are induced by a cooperation of receptor tyrosine kinases or oncogenic Ras (RTK/Ras) pathway and TGF-β signaling [33] . Recently, additional pathways and effectors have been reported to play a role in the induction of EMT, such as Wnt//β-catenin, Notch and Sonic hedgehog signalling [34] .
Recent reports strongly suggest that the ER stress may represent an important mechanism of the altered repair process observed in the alveolar epithelium of fibrotic lung [35] . Situations associated with abnormal regulation of the cascade of events leading to the formation of mature protein result in either misfolding or mistargeting of the protein. These events trigger induction of intracellular aggregate formation and ER stress, which can lead to cell death through apoptosis and autophagic pathways [36, 37] . Several stimuli including oxidant-antioxidant imbalance, viral proteins, inflammatory molecules, nutrient deprivation may induce ER stress [38, 39] ( Figure 2 ). Among the cytoprotective mechanisms available are the ER chaperones such as binding immunoglobulin protein (BiP). Interestingly, mutant BiP mice have been reported to die within several hours of birth from respiratory failure due to impaired secretion of pulmonary surfactant by type 2 AEC. In these animals, expression of surfactant protein (SP)-C was reduced and the lamellar bodies were malformed, indicating that BiP Figure 2 Alveolar structure disorder-associated ILD and ER stress. The (Endoplasmic Reticulum) ER and its protein maturation machinery allow the synthesis of mature secretory and membrane proteins with specific folded conformation. In situations of stress induced by genetic mutations or environmental factors, unfolded or misfolded proteins are retained in the ER and induce a defence mechanism called the ER stress response. The induction of ER chaperones is critical to increase the ER folding capacity allowing the production of correctly folded protein.
When this defence mechanism is impaired, the misfolded proteins can either be degraded by the proteasome or form protein aggregates. Protein aggregates are toxic and can cause conformational diseases. Within the alveolar epithelium, misfolding of SP-C could trigger induction of intra-cellular aggregate formation and ER stress, with consequently development of alveolar structure disorder-associated ILD and conformational disease.
plays a critical role in the biosynthesis of surfactant [40] . Several recent reports suggest the possible implication of ER stress in ILD, with activation of stress response markers in fibrotic lung tissues.
It is now well established that surfactant dysfunction plays an important role in the development and progression of ILD. Pulmonary surfactant is a multimolecular complex constituted of phospholipids and proteins secreted by type 2 AEC into the alveolar space. It assures alveolar stability by reducing surface tension along the epithelial lining and this role involves mainly the lipids and the specific hydrophobic SP, SP-B and SP-C. Other important players in surfactant metabolism include the ATP-binding cassette, sub-family A, member 3 (ABCA3) and the thyroid transcription factor 1 (TTF-1).
Surfactant deficiency can be induced by a number of primitive and secondary mechanisms. Among them are genetic defects with mutations in SP-B gene (SFTPB) as well as genes coding for SP-C (SFTPC), ABCA3, and TTF-1 [41] [42] [43] . More than 30 SFTPB (located on chromosome 2) mutations have been identified among patients with a congenital deficiency in SP-B. For SFTPC located on chromosome 8, at least 35 mutations have been described, localized primarily in the COOHterminal Brichos domain [44, 45] . A proposed function of the Brichos domain is a chaperone-like activity, which could prevent misfolding and aggregation of the parent protein. Alterations in the Brichos domain could therefore lead to diseases through mechanisms related to abnormal protein processing and cell toxicity [46] . Recently, several studies have also documented the role of ABCA3 deficiency in ILD. ABCA3 functions in the transport of surfactant lipids into lamellar bodies and is required to maintain pulmonary surfactant phospholipid homeostasis. Another contributor of ILD is TTF-1 (NK2 homeobox 1) dysfunction. TTF-1 is a critical regulator of transcription for the surfactant protein SP-B and SP-C. It is encoded by a gene located on chromosome 14q13 and is composed of three exon and two introns [47] . It is expressed in the thyroid, brain and lung.
Stem cell dysfunction represents a new domain of investigation. Alveolar epithelium regeneration and repair requires activation and proliferation of tissue-resident (progenitor) cells and their differentiation to replace the damaged cells [48] . However, unlike cancer cells, stem cells are not immortal and display decreasing telomere length with aging [49] . Telomere shortening has been documented to be associated with reduced capacity for stem cell renewal, and decreased activity of telomerase, the polymerase responsible for telomere maintenance. The stem cells of the alveolar epithelium are the type 2 AEC, and expression of telomerase has been documented in these cells [48] . Experimental studies have also indicated that telomerase is expressed mainly during lung development with a peak expression before birth followed by a decrease to nearly undetectable levels in mature alveolar epithelium. Interestingly, telomerase expression and activity could be reinduced in normal quiescent type 2 AEC exposed to oxidative stress [50] . The current understanding is that a population of type 2 AEC may have the capacity to survive injury through telomerase activation, and consequently may be responsible for repopulation of the damaged alveolar epithelium. On the basis of reports of pulmonary disorders in dyskeratosis congenita (a rare hereditary disease of poor telomere maintenance), recent and exciting findings have documented mutations in the telomerase gene in familial idiopathic pulmonary fibrosis [51] . In addition, it is likely that environmental factors such as inflammation, oxidative stress, or virus infection may modify telomerase activity and account for the development of organ-specific disease associated with telomerase dysfunction. In this view, new data in chronic respiratory diseases support the concept that alveolar stem cell dysfunction may play an important role in the rate of progression or severity in ILD [52] . The question whether telomerase mutations or telomere dysfunction may be implicated in pediatric ILD needs to be addressed in prospective studies, one possible tool being determination of telomere length in circulating leukocytes.
The frequency of lung fibrotic disorders is much lower in children than in adults. Some clinical situations have features certainly unique to children, but many of these diseases overlap their adult counterparts with the primary event being injury and damage of the alveolar epithelium [11, 13] . Yet, the overall outcome and prognosis of the diseases in children are thought to be less severe than in adult patients. In addition, pediatric ILD is more responsive to therapeutic strategies than adult ILD [9] . These differences may be explained by the types of initial injury, which may not be similar due to changes in the host environment. Another explanation is the modifications of the process of wound healing with age. Comparison of the response to injury in foetal and adult skin shows clear differences [53] . Skin wound healing in the foetus is characterized by complete regeneration of the skin and the absence of scar formation. Progressively with age, the skin looses the capacity to regenerate the original tissue architecture with the result being scar formation that extends outside the wound bed. The process of healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors [54] . The slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. In a study on the role of aging in the development of cardiac fibrosis in the rabbit, differences in the cascade of events leading to myocardial remodeling were observed, with mainly the presence of more myofibroblasts synthesising collagen and expressing high levels of TGF-β in older animals [55] . A study of growth factors involved in skin wound healing in young and aged mice also showed age-dependent changes. Expression of all the fibroblast growth factors was diminished in aged mice, even in healthy skin. In addition, the post-wound regulation of expression of these factors and of TGF-β was less pronounced and slower than in young mice. These findings are in agreement with data observed in muscle that indicated significant alterations in the TGF-β production with age [56, 57] . Other potential mechanism is linked to the observation that injury in adult tissues does in certain circumstances stimulate tissue regeneration, depending on the presence of small subsets of primitive stem cells. Stem cells are the self-renewing, primitive, undifferentiated, multipotent source of multiple cell lineages [49] . While such cells are critical for development and growth through childhood, residual pools of adult stem cells are hypothesized to be the source of the frequently limited tissue regeneration and repair that occurs in adults [58] . Unlike embryonic stem cells, adult stem cells are not immortal, and show decreasing telomere length with increasing age. The naturally limited replacement capacity of such endogenous stem cell pools may occur via exhaustion of the stem cell pool or arise as a consequence of inherited or acquired mutations that alter proper stem cell function [59] . The limited life span of cells may result from replicative senescence in response to various stresses including DNA damage, oxidants, and telomere erosion [52] . All these forms of injury have been documented in the lung from adult patients with ILD.
The prevalence of children ILD is higher in the younger patients: more than 30% of patients are less than 2 years at diagnosis, as recorded by the recent ERS Task Force. 7% have parental consanguinity and nearly 10% of case siblings were affected by similar diseases. There is a male predominance with a sex ratio of 1.4. The presenting clinical manifestations are often subtle and nonspecific. The onset of symptoms is, in most cases, insidious and many children may have had symptoms for years before the diagnosis of ILD is confirmed. However, the majority of patients has symptoms for less than one year at the time of initial evaluation. The clinical manifestations vary from asymptomatic presentation with radiological features suggestive of ILD to more characteristic presence of respiratory symptoms and signs such as cough, tachypnea and exercise intolerance [9, 60] . These varying presentations are also reflected in the report published by Fan et al. who systematically evaluated the clinical symptoms and physical findings of 99 consecutive children with ILD [2] . Common symptoms at presentation included cough, dyspnea, tachypnea and chest wall retraction, exercise limitation and frequent respiratory infections. Cough is observed in almost 75% of the patients, is normally non-productive and does not disturb sleep. Tachypnea is observed in 80% of patients and is usually the earliest and most common respiratory symptom. Unexplained fever is also reported in almost one third of infants. Failure to thrive (37%), tiring during feeding and weight loss are also common symptoms, mainly in young patients. Although a history of wheezing may be elicited in almost 50% of the patients, wheezing is documented by physical examination in only 20% of the cases.
The frequent clinical findings are inspiratory crackles (44%), tachypnea and retraction. In a child with a normal birth history, these are strongly suggestive of ILD. Other findings associated with an advanced stage of lung disease include finger clubbing (13%) and cyanosis during exercise or at rest (28%) [9, 61] . During physical examination it is essential to check the presence of associated non-respiratory symptoms such as joint disease, cutaneous rashes, and recurrent fever suggestive of collagen-vascular disorders. Details should also be obtained on precipiting factors such as feeding history, infections, or exposure to dust and drugs. In addition, information on relatives or siblings with similar lung conditions should be gathered.
Plain radiographs are usually performed in a child suspected of ILD at first presentation, but the information provided is often limited and the key chest imaging tool for diagnosis is the High Resolution Computed Tomography (HRCT), which can visualize the parenchymal structure to the level of the secondary pulmonary lobule.
HRCT technique for ILD diagnosis has been extensively discussed [62] [63] [64] . To optimise spatial resolution, there is a general agreement to use thin sections, the smallest field of view and a sharp resolution algorithm. The most common HRCT feature of ILD is widespread ground-glass attenuation. Intralobular lines, irregular interlobular septal thickening and honeycombing are less common findings. Large subpleural air cysts in the upper lobes adjacent to areas of ground-glass opacities have been also reported in young children with ILD. These cysts are interpreted as paraseptal or irregular emphysema. HRCT is useful for ILD diagnosis and selection of lung area to be biopsied. It is proposed that it also may contribute to monitor disease activity and/or severity. However, evaluation is still needed to support a role of HRCT as a follow up tool in pediatric patients.
Pulmonary function testing (PFT) techniques are well established in children and adolescents. However, children aged 2-6 years represent a real challenge in pulmonary function assessment as they cannot be sedated and find it difficult to cooperate with all respiratory manoeuvres. In 2007, an ATS and ERS statement on PFT in preschool children summarized the current knowledge on the PFT techniques suitable for young children [65, 66] .
Although PFT does not provide specific information, it represents a useful investigation for both the diagnosis and the management of ILD [11] . Generally, in ILD, pulmonary function abnormalities reflect a restrictive ventilatory defect with reduced lung compliance and decreased lung volumes [67] [68] [69] . Vital capacity (VC) is variably diminished; the decrease in total lung capacity (TLC) in general is relatively less than in VC. Functional residual capacity (FRC) is also reduced but relatively less than VC and TLC, and residual volume (RV) is generally preserved; thus the ratios of FRC/TLC and RV/TLC are often increased. Airway involvement is observed only in a minority of patients. Lung diffusing capacity of carbon monoxide (DLCO) or transfer factor (TLCO) is often markedly reduced and may be abnormal before any radiological findings. However, DLCO corrected for lung volume may also be normal in many children. Hypoxemia as defined by a reduced resting arterial oxygen saturation (SaO 2 ) or a reduced resting arterial oxygen tension is often present. Hypercarbia occurs only late in the disease course. During exercise the above described dysfunctions become even more pronounced. Thus, gas exchange during exercise might be a more consistent and sensitive indicator of early disease [3] .
Bronchoalveolar lavage (BAL) usefully provides specimens for cytological examination, microbial cultures, and molecular analysis. Besides infections, BAL can be of diagnostic value in several situations. In the context of pulmonary alveolar proteinosis, BAL abnormalities are characterized by milky appearance fluid, abundant proteinaceous periodic acid schiff positive material, and presence of foamy alveolar macrophages (AM) [70] . BAL can also be diagnostic for pulmonary alveolar haemorrhage [11] . This diagnostic is easy when the BAL fluid has a bloody or pink color, but its gross appearance may be normal. Microscopic analysis may then be of value by documenting the presence of red blood cells in AM or haemosiderin laden AM [71] . Among other situations, the diagnosis of Langerhans cell histiocytosis can be performed with the use of the monoclonal antibodies revealing the presence of CD1a positive cells (in more than 5% of the BAL cells) [72] . Lipid disorders with lung involvement represent another indication of BAL. This includes congenital lipid-storage diseases (Gaucher's disease and Niemann-Pick disease) or chronic lipid pneumonia due to chronic aspiration [73, 74] . However, in cases of aspiration syndromes, the presence of lipid laden AM is sensitive but not specific [75] .
In other pathological situations, BAL can usefully serve to direct further investigations. Accumulation of BAL T-lymphocytes with prevalence of CD4+ cells is suggestive of sarcoidosis, whilst prevalence of CD8+ cells is suggestive of hypersensitivity pneumonitis [76] . Also, an increase in BAL eosinophils suggests pulmonary infiltrates associated with eosinophilia syndromes [77] . Depending of the underlying diseases, a number of cellular and molecular investigations can be proposed including the studies of various surfactant components, phospholipids and apoproteins [78] .
With increasing recognition of the different patterns of ILD and their clinical significance, histological investigation has become increasingly important. Depending on disorder presentation, biopsy may concern more accessible organs than the lung such as the skin or the liver in sarcoidosis. Histological evaluation of lung tissue usually represents the final step in a series of diagnostic approaches.
Different methods may be used to obtain lung tissue. The major difference between individual methods lies mainly in balancing invasiveness against the potential for obtaining adequate and sufficient tissue for diagnosis. The techniques of choice are open lung biopsy and video assisted thoracoscopy biopsy. In children, open lung biopsy usually provides sufficient tissue with few complications related directly to the biopsy procedure [79] . Video assisted thoracoscopy biopsy is an alternative to open lung biopsy, and it has been shown that the procedure can be safely performed, even in small children [80] . The place of other methods like transbronchial lung biopsy and percutaneous needle lung biopsy in appropriate diagnosis of pediatric patients with ILD has to be established [81] [82] [83] .
The lung histological patterns that can be observed in ILD have been reviewed by the ATS/ERS [5] . In children, they include mainly: DIP, NSIP, and LIP. DIP is characterized by airspaces filled with AM, thickened alveolar septa, scattered mixed inflammatory cells and minimal fibrosis. Many alveolar spaces are lined by hyperplastic type 2 AEC. Recently, association with surfactant disorders has been reported [41, [84] [85] [86] . NSIP encompasses a broad spectrum of abnormalities with varying degrees of alveolar wall inflammation or fibrosis. The cellular pattern of NSIP is characterized by mild to moderate interstitial chronic inflammation and type 2 AEC hyperplasia in inflammation areas. It has been reported in a variety of underlying conditions including connective tissues diseases and surfactant disorders. LIP features include a marked diffuse infiltrate of mature lymphocytes, plasma cells and histiocytes within the pulmonary interstitium, particularly the alveolar walls. They are often associated with either connective tissues disorders or immunodeficiency states, both congenital and acquired [9] . Another pattern described mainly in adults is diffuse alveolar damage (DAD), which includes diffuse homogeneous thickening of alveolar interstitial walls with myofibroblast accumulation, prominent type 2 AEC hyperplasia and atypia, and hyaline membranes containing surfactant proteins and cellular debris [87] . Usual interstitial pneumonia (UIP) is rare in children [88] . It is characterized by severe remodeling of the alveolar structure with heterogeneous appearance consisting of contiguous areas of normal lung, dense scarring, and bronchiolar abnormal proliferation. Interstitial inflammation is usually mild to moderate. Histologic patterns of ILD unique to infancy are described below.
Laboratory tests are used to exclude a number of respiratory diseases in childhood that does not typically present with ILD such as chronic aspiration syndromes, resolving acute respiratory distress syndrome, tuberculosis, cystic fibrosis, bronchopulmonary dysplasia and diffuse pulmonary disease such as cystic fibrosis. Laboratory tests also verify the absence of immunodeficiencies [3] .
When these conditions have been eliminated, the spectrum of investigations that should be performed for the diagnostic approach will be guided by the history and clinical presentation in each individual child. These investigations are discussed below for the various disorders. In addition, an increasing number of blood and BAL biomarkers for evaluation of disease severity and progression is currently investigated. The studied molecules include various cytokines and chemokines, surfactant protein D, Krebs von den Lungen-6 antigen (KL-6), matrix metalloproteinases MMP1 and MMP7 and defensins [89] [90] [91] [92] .
A large number of pathological situations can impair gas exchange and contribute to progressive lung damage and ILD. Consequently, diagnosis approaches need to be organized by cause, with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. Indeed, in a number of pathological situations, no final diagnosis is proposed and the conclusion reported by the physician in charge of the patient is ILD of unknown cause. However, information from recent studies highlights the concept that lung insults caused by substances from the environment or in the context of systemic diseases are largely under-estimated and should be more often discussed considered in the diagnostic process. Based on this consideration, the following diagnostic grouping for pediatric ILD can be considered 1) exposure-related ILD; 2) systemic disease-associated ILD; 3) alveolar structure disorder-associated ILD; and 4) ILD specific to infancy.
Accordingly, a step-by-step etiological diagnostic approach is required and is summarized in Figure 3 . Once the diagnosis of ILD is established on clinical, radiological, and functional findings, a careful history should be obtained for potential exposure-related diseases leading to discuss the need for specific serum antibodies against offending antigens. The following step focuses on the search for systemic disease associated ILD, oriented by the presence of clinical and functional extra-pulmonary manifestations. In such situations, additional investigations should include specific serum antibodies and possibly tissue biopsies in organs other than the lung. Finally, elimination of these 2 groups of causes with a lung restricted expression of the disease allows discussing the potential interest of a lung biopsy.
Exposure-related disease refers to diseases caused by a sufficient level of exposure (dose) to components with target organ contact, and subsequent biologic changes and clinical expression. Many agents have been associated with pulmonary complications of various types including ILD. The adult literature has provided extensive lists of candidate molecules [93] . In children, the potential involvement of these molecules is not similar as the environmental conditions and the use of therapeutic drugs differ. It is important to point out that exposure-related diseases are certainly under-estimated in the pediatric age. One reason is linked to the fact that the diagnosis is less often discussed than in adults as pediatricians and other child health care providers do not usually have the expertise necessary to take an environmental history. In this review, the most frequent causes of exposure-related ILD are discussed.
Hypersensitivity pneumonitis (HP) is a cell-mediated immune reaction to inhaled antigens in susceptible persons [94, 95] . In children, HP is often associated with exposure to antigens in the home environment as well as with certain hobbies. The most frequent types of HP include bird fancier's diseases, humidifier lung diseases, and chemical lung diseases. Bird fancier's diseases are induced by exposure to birds with the antigens being glycoproteins in avian droppings, and on feathers. Importantly, respiratory symptoms in exposed patients who have only one pet bird at home should raise the suspicion of HP [96] . Humidifier lung diseases (air conditioner lung, misting fountain lung, basement lung diseases) are caused mainly by free-living amoeba and nematodes, as well as bacteria and fungi. Chemical lung diseases can be induced by various inorganic antigens such as those from vaporized paints and plastics. Low-molecular-weight chemicals may react with proteins in the airways, thus forming complete antigens. Once exposure history is obtained, additional information is required and includes biologic tests allowing measurements of environmental contaminants and interpretation of the results by environmental medicine experts.
As HP is believed to be an adult disease, children are often diagnosed at the chronic stage of the disease resulting of a long-term exposure to low levels of inhaled antigens. Children can develop subtle interstitial inflammatory reactions in the lung without noticeable symptoms for months [97] . Clinical features in the classic form include non productive cough, dyspnea, malaise, asthenia and occasional cyanosis [95] . Lung function abnormalities are not specific and appear similar to changes observed in other ILD. HRCT abnormalities vary from ground glass attenuation predominantly in the mid-upper zone to nodular opacities with signs of Figure 3 Search for ILD etiology in children. ILD is defined by the presence of diffuse infiltrates on chest radiographs or chest high resolution computed tomography, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange. The search for etiology requires a systematic step-by-step diagnostic strategy for identifying: exposure-related ILD; systemic disease-associated ILD; alveolar structure disorder-associated ILD; and ILD specific to infancy.
air-trapping [62, 63, 98] . Laboratory tests focus mainly on the search for serum-precipitating IgG antibodies against the offending antigen [95] . However, the presence of these antibodies is considered to be of questionable clinical relevance for diagnosis, as it is observed in up to 50% of serum samples of exposed but asymptomatic individuals. BAL cell profile study typically shows an increase in total cell count with a remarkable elevation in the percentage of lymphocytes often over 50% with a decreased CD4/CD8 ratio [95, 97] . However, in contrast to studies in adults, the CD4/CD8 ratio could be within the normal range for children [76] . Histopathologic evaluation of lung tissue is usually not necessary for the diagnosis of HP.
At the present time, there is no diagnostic test that is pathognomonic for HP, and only significant predictors of HP are identified. The most significant diagnostic tool is a detailed environmental exposure history. Other diagnostic features include: positive precipitating antibodies to the offending antigen; recurrent episodes of symptoms; symptoms occurring 4-8 h after exposure; occurrence of diffuse parenchymal lung disease by lung function and HRCT; BAL abnormalities with lymphocytic alveolitis and increased CD8+ T cells.
Medication, drug, radiation and tobacco exposure Drugs used in inflammatory or cancer pediatric diseases can cause ILD. They include anti-inflammatory agents (e.g. aspirin, etanercept), immunosuppressive and chemotherapeutic agents (e.g. azathioprine, methotrexate, cyclophosphamide), antibiotics, cardiovascular agents, and, for teenagers, illicit drugs [99, 100] . There are no distinct clinical, radiographic or pathologic patterns, and the diagnosis is usually made when a patient is exposed to medication known to result in lung disease, with a timing of exposure appropriate for disease development and elimination of other causes of ILD. Treatment relies on avoidance of further exposure and corticosteroids in markedly impaired patients.
Exposure to therapeutic radiation in the management of pediatric cancer may also results in ILD. Patients presenting within 6 months of therapy generally have radiographic abnormalities with ground glass patterns in both radiation-exposed and unexposed tissue [101] .
The association between tobacco use and ILD is less well appreciated than the relation with chronic obstructive pulmonary disease (COPD). In addition, pediatric patients do not usually have a significant smoking history to develop respiratory disorders [102] .
Connective tissues disorders (CTD) are a heterogeneous group of immunologically mediated inflammatory diseases. Their origins are multifactorial with genetic, constitutional and environmental elements contributing to their development. CTD refers to any disease that has the connective tissues of the body as a primary target of pathology. The connectives tissues are composed of two major structural proteins, elastin and collagen, with different types of collagen proteins in each tissue [103] . Many CTD feature abnormal immune system activity associated with inflammation. Pulmonary manifestations of CTD may include both vascular and interstitial components. From recent reports, the incidence of ILD in the context of CTD appears to be higher than previously appreciated [104, 105] . Importantly, ILD may precede the development of clinically obvious CTD, sometimes by months or years. Table 1 provides information on suggestive clinical and serological features in selected conditions. The main disorders to be considered in childhood are rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. The other include Sjögren syndrome, dermatomyositis and polymyositis, ankylosing spondylitis, and mixed connective tissue disease. Rheumatoid arthritis Rheumatoid arthritis (RA) is an inflammatory disorder defined by its characteristic diarthroidal joint involvement. It is the most common CTD in children, but pulmonary involvement is less frequent than in adults. Genetic and environmental factors seem to be important contributors of disease progression, with influence of sex (more frequent in male), presence of two copies of the HLA-DRB1 "shared epitope" (HLA-DR SE) and anticyclic citrullinated peptide antibody (anti-CCP), and possibly tobacco exposure [106, 107] .Almost 50% of patients with RA have specific serologic abnormalities several years before the onset of joint symptoms, and the findings of elevated serum levels of IgM rheumatoid factor or anti-CCP is associated with a high risk for the development of RA [107] . Systemic sclerosis Systemic sclerosis (SSc) is characterized by a progressive dermatologic abnormality [108] . Its etiology remains unknown; it is believed to be a complex disease in which interactions between environmental, auto-immune, and genetic factors result in various disease phenotypes [109] . Although it is a rare disease in childhood, the diagnosis is based on skin disease. Cardiopulmonary complications are common and have been associated with death in young patients. Almost all patients with SSc have serum antinuclear antibodies. The other autoantibody markers are listed in table 1. Recently, the presence of anti-DNA topoisomerase II autoantibody has been reported to be a key factor in the development of ILD, in association with class II MHC status (HLA-DR3, HLA-DPBI) [110] . Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is an auto-immune disorder characterized by the involvement and dysfunction of multiple organ systems. The mechanisms of tissue injury involve autoantibody production and immunocomplex formation leading to an inflammatory process. Diverse clinical phenotypes are observed, including a variety of mucocutaneous lesions, non erosive arthropathy, renal disease (glomerulonephritis and interstitial nephritis), lung disease, pericarditis, and a spectrum of neurologic disorders. Laboratory abnormalities are characterized by the presence of antibodies reactive to nuclear (ANA) and cytoplasmic antigens.
Pulmonary vasculitis are observed in vasculitic syndromes that preferentially affect small vessels (arterioles, venules, and capillaries). They include the anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (Wegener's granulomatosis, Churg-Strauss syndrome, and microscopic polyangitis) that share histologic similarities without immune deposits; anti-glomerular basement membrane (GBM) disease; Henoch-Schönlein purpura and cryoglobulinemia vasculitis. Vasculitic syndromes that affect large/medium vessels (such as Kawasaki's disease, polyarteritis nodosa) only occasionally affect the lung [111] . Wegener's granulomatosis Wegener's granulomatosis (WG) is a rare disease of uncertain cause. It seems to affect children as much as adults with an increasing reported incidence around 2.75 cases/million/year, mostly in teenagers with a reported median age of 14.2 years (4-17 years) [112, 113] . It is characterized by inflammation in a variety of tissues including blood vessels (vasculitis). WG primarily affects the upper respiratory tract, lung, and kidneys. The diagnosis is based on the combination of symptoms and a biopsy of affected tissue with necrotising granulomatous vasculitis in the absence of an infectious etiology. The diagnosis is further supported by positive blood tests for cytoplasmic-staining (c)-ANCA PR3 type [114] .
Churg-Strauss syndrome Churg-Strauss syndrome (CSS) is a granulomatous small-vessel vasculitis. The cause of this allergic angiitis and granulomatosis is not known, but autoimmunity is evident with the presence of hypergammaglobulinemia, increased levels of immunoglobulin E (IgE), and perinuclear-staining (p)-ANCA. The diagnosis relies on biopsy evidence for vasculitis and at least 4 criteria among the following: moderate to severe asthma, blood eosinophilia (at least 10%), and nonfixed pulmonary infiltrates with extravascular eosinophils on biopsy [115] . Twenty-nine pediatric cases have been reported so far in the literature, with lung involvement in 72% of [116] . Anti-glomerular basement membrane disease Goodpasture syndrome is a rare disease that involves rapidly progressive kidney failure along with lung disease and is characterized by the deposition of anti-GBM antibodies. Several cases have been reported in the pediatric literature. The autoantibodies mediate tissue injury by binding to their reactive epitopes in the basement membranes. This binding can be visualized as the linear deposition of immunoglobulin along the glomerular basement membrane. The principle component of the basement membrane is type IV collagen which can be expressed as 6 different chains, from alpha1 to alpha6. The Goodpasture antigen has been localized to the carboxyl terminus of the noncollagenous domain of the alpha3 chain of type IV collagen. The anti-GBM antibody can usually be found in serum [117] . Strong evidence exists that genetics play an important role. Patients with Goodpasture disease have an increased incidence of HLA-DRB1 compared to control populations [118] .
Granulomatous disorders are characterized by the presence of granulomas defined as a focal, compact collection of inflammatory cells in which mononuclear cells predominate. Granulomas form as a result of tissue injury by a wide variety of agents including microorganisms, antigens, chemical, drugs and other irritants. In other situations including sarcoidosis, the etiologic factors remain to be determined. Sarcoidosis Sarcoidosis is a chronic inflammatory disease in which granulomatous lesions can develop in many organs, mainly the lung. Its cause remains obscure, and most likely involves environmental and host factors [119] . The current concept is that a still unknown stimulus activates quiescent T cells and macrophages leading to recruitment and activation of mononuclear cells, with, as a consequence, granuloma formation, alveolitis, and in some cases interstitial lung fibrosis [120] . Sarcoidosis is relatively uncommon among children. Its diagnosis is based on a combination of suggestive clinical features, with histologically-documented noncaseating granuloma, in the absence of other known causes of granuloma formation [121] . The incidence and prevalence of sarcoidosis are reported to be influenced by age, race and geographic localization [122] . Although the youngest patients reported were infants 2 and 3-months old, most of the cases in children occur in preadolescents and adolescents. From the national patient registry on patients with sarcoidosis in Denmark during the period 1979-1994, 81 patients with a confirmed diagnosis were ≤16 years of age [123] . The calculated incidence was 0.29 per 100.000 person-years. In children ≤4 years of age, the incidence was 0.06; it increased gradually to 1.02 in children aged 14-15 years. Marked racial differences in the incidence and prevalence of sarcoidosis have been reported by many authors [122] . Various reports in the literature also indicate that race and ethnicity affect both the patterns of organ involvement and disease severity. In a follow-up study we have conducted in 21 children with pulmonary sarcoidosis, 12 children were Black [124] . Also the number of organs involved was higher in the Black than in the Caucasian children.
Clinical manifestations in sarcoidosis are the consequences of local tissue infiltration with sarcoid granuloma. Therefore, disease expression depends on the organ or system involved and a variety of symptoms and physical findings can be observed [125] . The modes of presentation include non-specific constitutional symptoms, alone or associated with symptoms related to specific organ involvement. In the report of children with sarcoidosis in Denmark, the most common non specific symptoms were asthenia, weight loss, and fever [123] . Clinical findings mainly include respiratory manifestations, lymphadenopathy, skin lesions, ocular and central nervous system abnormalities. The most common radiographic findings are hilar lymph node enlargements, with or without lung changes. Lung function abnormalities are frequently observed in children with restrictive pulmonary pattern and abnormal diffusing capacity [126] . Other investigations such as BAL documenting a lymphocytic alveolitis with increased CD4/CD8 ratio, and elevated serum angiotensin-converting enzyme may provide additional evidence of sarcoidosis [127] . Other granulomatous disorders in children A number of pathological situations are associated with granulomatous disorders defined by the presence of non-caseating granuloma in biopsied tissues. Infections are the main causes of other granulomatous diseases, and are in some cases related to disorders of neutrophil function such as chronic granulomatous disease (CGD) [128] . Most children with CGD present with recurrent bacterial and fungal infections. The most frequently encountered pathogens are Staphylococcus aureus, Aspergillus, Burkholderia cepacia, and enteric gram negative bacteria [129] . The most prominent pulmonary lesions include an extensive infiltration of the lung parenchyma and hilar adenopathy. In some situations, a homogeneous distribution of small granulomatous lesions can occur, with a radiological appearance of miliary tuberculosis.
The other granulomatous diseases can be seen in other described diseases, such as immune disorders (including Crohn's disease and histiocytosis X), HS pneumonitis, vasculitis disorders or neoplasms.
Lysosomal diseases Gaucher's disease is an autosomal recessive disease and the most common of the lysosomal storage diseases. It is caused by a genetic deficency of the enzyme lysosomal gluco-cerebrosidase that catalyses the breakdown of glucocerebroside, a cell membrane constituent of red and white blood cells. The consequence is an accumulation of glucocerebroside in reticuloendothelial cells, leading to excessive deposition of fatty material in the spleen, liver, kidneys, lung, brain and bone marrow. Pulmonary expression is mainly characterized by physiologic involvement (reduction in lung the diffusion capacity and the functional residual volume). Lung imaging may show interstitial changes [130] .
Niemann-Pick diseases are genetic diseases primarily due to deficiency of sphingomyelinase resulting in the accumulation of sphingomyelin within lysosomes in the macrophage-monocyte phagocyte system, mainly the brain, spleen, liver, lung, and bone marrow. Histology demonstrates lipid laden macrophages in the marrow, as well as "sea-blue histiocytes" on pathology. The infantile form with a dominant neurologic expression is rapidly fatal. In older patients, cases of ILD have been reported [131] .
Hermansky-Pudlak syndrome is a heterogeneous group of autosomal recessive disorders associated with accumulation of a ceroid-like substance in lysosomes of a variety of tissues. It is characterized by albinism, bleeding tendency associated to poor platelet aggregation and systemic complications associated to lysosomal dysfunction. A chronic inflammatory process may explain the progressive development of ILD and fibrosis [132] . Familial hypercalcemia with hypocalciuria Familial hypercalcemia with hypocalciuria is caused by autosomal dominant loss-of-function mutations in the gene encoding the calcium-sensing receptor (CASR), a G-protein coupled membrane receptor expressed in many tissues [133] . Loss-of-function mutations in CASR impair the feedback inhibition of parathyroid hormone secretion in response to a rise in the blood calcium concentration. The result is hypercalcemia associated with inappropriately normal or mildly elevated levels of parathyroid hormone. In the kidneys, mutations in CASR prevent the feedback inhibition of calcium reabsorption in situation of hypercalcemia, leading to relative hypocalciuria. Respiratory symptoms are usually mild and associated with reduction in the lung diffusion capacity. Lung histology indicates the presence of foreign body giant cells and mononuclear cells infiltrating the alveolar interstitium, without circumscribed granulomas.
Langerhans'-cell histiocytosis is part of the histiocytosis syndromes, which are characterized by an abnormal proliferation of Langerhans' cells [134] . The Langerhans cells are differentiated cells of monocyte-macrophage lineage that function as antigen-presenting cells. The origin of the expanded population of Langerhans' cells is unknown; in adults, the only consistent epidemiologic association is with cigarette smoking. These cells may form tumors, which may affect various parts of the body. Most cases of pediatric Langerhans'-cell histiocytosis are observed in children between ages 1 and 15 years, with usually bone involvement (80%) including the skull. The tumors produce a punched-out appearance on bone X-ray, and can cause fracture without apparent traumatism. Langerhans'-cell histiocytosis can also affects various organs including the lung [135] .
Children with pulmonary Langerhans'-cell histiocytosis present in a variety of ways. They can be asymptomatic or present common symptoms such as nonproductive cough and dyspnea. HRCT of the chest is a useful and sensitive tool for the diagnosis. Indeed, the combination of diffuse, irregularly shaped cystic spaces with small peribronchiolar nodular opacities, predominantly in the middle and upper lobe, is highly suggestive of pulmonary Langerhans'-cell histiocytosis [63] . Other abnormalities include ground-glass attenuation. The presence of increased numbers of Langerhans' cells in BAL fluid (identified by staining with antibodies against CD1a) with a proportion greater than 5 percent is also strongly suggestive of pulmonary Langerhans'-cell histiocytosis.
Histologically, the cellular lesions forms nodules containing a mixed population of cells with variable numbers of Langerhans' cells, eosinophils, lymphocytes, plasma cells, fibroblasts, and pigmented alveolar macrophages.
Several forms of ILD have been reported to occur with inflammatory bowel diseases (Crohn's disease) and celiac disease [136] . Primary biliary cirrhosis and chronic hepatitis have also been reported to be associated with parenchymal lung dysfunction [137, 138] . In addition, there are reports on ILD in association with neurocutaneous disorders (tuberous sclerosis, neurofibromatosis, ataxia-telangiectasia) and amyloidosis [139] .
Depending on the causes, the components of the alveolar structure (the epithelium and the alveolar space, the interstitium, and the pulmonary capillary endothelium) can be involved differently and can serve as primary targets of the underlying pathological processes. Based on history, clinical presentation, BAL data, and, most important, on information from lung tissue studies, the disorders can be gathered in groups according to predominant structural targets (Figure 4 ).
The disorders affecting primarily the alveolar epithelium and the alveolar space share common histopathological description, with preserved pulmonary architecture, hyperplasia of type 2 AEC, interstitial infiltrates composed of immuno/inflammatory cells and scattered myofibroblasts, and the alveolar space filled with either immuno/inflammatory cells, desquamated materials, or components derived from surfactant lipid and protein complex. In the coming years, it is likely that the list of disorders will expand rapidly with the availability of specific tissue markers. Currently, the following grouping can be proposed: infections, surfactant disorders, and eosinophilic lung diseases. Infections The role of infection, mainly viral, in the development and progression of ILD is sustained by a number of human and experimental reports. From recent knowledge, it is strongly suggested that latent viral infections may be involved in the pathogenesis of ILD, through targeting of the alveolar epithelium. The main virus implicated include adenovirus, members of human herpes virus family (Epstein-Barrr virus and cytomegalovirus), and respiratory syncitial virus [140] . Number of other viruses can also be involved such as Influenza A, hepatitis C, or even Human Immunodeficiency Virus (HIV) in immunocompetent children [141] [142] [143] [144] .
Human adenovirus being predominantly respiratory pathogens, adenovirus infections can cause a variety of pulmonary symptoms and can persist for long periods of time. Several studies in adult patients have indicated that the adenovirus gene product E1A could be detected Figure 4 Alveolar structure disorder-associated ILD. Depending on the causes, the alveolar structure components can be involved differently and serve as primary targets of the underlying pathological processes. Based on history, clinical presentation, BAL and lung tissue information, the disorders can be gathered in groups according to the predominant alveolar targets: epithelium, vascular or interstitial components. in lung tissues by in situ hybridization in up to 16% of cases of idiopathic pulmonary fibrosis. The causative role of the virus in the initiation of the disease remains uncertain, but it may be an important factor in its progression as treatment with corticosteroids may make patients more susceptible to adenovirus infection or reactivation from latency. E1A has been shown to increase the production of TGF-β and to induce lung epithelial cells to express mesenchymal markers, thereby contributing to remodeling of the alveolar structure [145] . Isolation of the virus from the throat and serologic studies are diagnostic supportive, but the diagnosis is confirmed by the detection of the virus in lung tissues.
Epstein-Barrr virus (EBV) and cytomegalovirus (CMV) are widespread pathogens that share the characteristic ability of herpesviruses to remain latent within the body over long periods. In mice, the control of herpesviruses replication have also been reported to be associated with the arrest of lung fibrosis [146] . EBV is present in all populations, infecting more than 95% of individuals within the first decades of life. Infection by CMV is reported in 60% of individuals aged 6 and older and more than 90% of aged individuals have antibodies against CMV. In addition, CMV is also the virus most frequently transmitted to a developing fetus. Most healthy people who are infected by EBV and CMV after birth have no symptoms, but infection is important to certain high-risk groups of infants and immunocompromised individuals. Several studies in the adult literature have reported an increased incidence of EBV and CMV infection in patients with pulmonary fibrosis, associated with virus DNA-positive lung tissue biopsies in several cases [147] . However, so far, no evidence of causal relationship between viruses and pulmonary fibrosis has been provided.
Respiratory syncytial virus (RSV) is the most common cause of viral lower respiratory tract infection. It affects people of all ages, and can cause severe disease in infants, in older immunodeficient children and the elderly. An intriguing feature of RSV infection is the susceptibility of previously infected individuals to reinfection with antigenically closely related viruses or the identical virus strain. Recently, increased interest has been focused on the contribution of persistent RSV in several chronic lung diseases including chronic obstructive pulmonary disease [148] . The role of RSV in the physiopathology of theses disorders as well as and the mechanisms of its persistence remain to be elucidated [149] . Interestingly, in a recent work on the histopathology of untreated human RSV infection, the presence of the virus in AEC has been documented [150] . From these various data, a role of RSV in the development of ILD needs to be investigated. Immunostaining with RSV-specific antibodies of tissues from lung biopsy should be proposed.
Among the other pathogens, Chlamydophila pneumoniae and Mycoplasma pneumoniae are currently drawing increasing consideration. They are frequent causes of community acquired pneumonia in children. Before the age of 10 years, almost 70% of children have had Chlamydophila pneumoniae infection based on serological studies [151] . These pathogens are intracellular organisms that primarily infect respiratory epithelial cells and alveolar macrophages and have the propensity to persist within several cell types such as macrophages. They are well known to cause a wide variety of respiratory manifestations, with possible progression towards diffuse parenchymal diseases associated with interstitial infiltrates on chest imaging and reduction in the lung diffusion capacity [152] . Regarding Legionella pneumophilia infection, progression towards ILD has been infrequently reported in adult patients.
Results from recent studies provided evidence that viruses can infect the alveolar epithelium and may be documented in lung tissues from patients using virus DNA detection and immunohistochemistry. A number of specific antibodies are currently available and should prompt to investigate the presence of the above cited viruses in the lung tissues from children with ILD. Surfactant disorders Surfactant disorders include mainly genetic surfactant protein disorders and pulmonary alveolar proteinosis
The deficiency in SP-B is a rare autosomal recessive condition known to be responsible for lethal neonatal respiratory distress. Rare survivals have been described in partial deficiencies [153, 154] . The SFTPC mutation I73T (c.218 T > C) is the more prevalent mutation. Others are described in only one family. The phenotype associated with SFTPC mutations is extremely heterogeneous leading from neonatal fatal respiratory failure to children and adults chronic respiratory disease with ILD [45] . Recessive mutations in the ABCA3 gene were first attributed to fatal respiratory failure in term neonates but are increasingly being recognized as a cause of ILD in older children and young adults. Over 100 ABCA3 mutations have been identified in neonates with respiratory failure and in older children with ILD [86, [155] [156] [157] [158] [159] [160] [161] . Mutations in the TTF-1 gene are associated with "brainlung-thyroid syndrome" which combines congenital hypothyroidism, neurological symptoms (hypotonia, chorea), and ILD of variable intensity [162] [163] [164] [165] [166] [167] [168] . So far, few mutations have been reported, mostly in exon 3 [169, 170] .
Pulmonary alveolar proteinosis (PAP) is a rare lung disorder characterized by alveolar filling with floccular material derived from surfactant phospholipids and protein components. PAP is described as primary or secondary to lung infections, hematologic malignancies, and inhalation of mineral dusts. Recently, the importance of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of PAP has been documented in experimental models and in humans. GM-CSF signaling is required for pulmonary alveolar macrophage catabolism of surfactant. In PAP, disruption of GM-CSF signaling has been shown, and is usually caused by neutralizing autoantibodies to GM-CSF. Therefore, the emerging concept is that PAP is an autoimmune disorder resulting in macrophage and neutrophil dysfunction. In a recent report, it has been reported that GM-CSF autoantibodies are normally present in healthy individuals, but at lower levels than in PAP patients [171] . In addition, in vitro experiments indicated that these autoantibodies reduce GM-CSP signaling similarly in healthy individuals and in PAP patients. At levels above a critical threshold, GM-CSF autoantibodies are associated with multiple impaired GM-CSF dependent myeloid function [172] . Several cases of genetic defects in the common beta chain for the GM-CSF receptor have been documented [173] . Eosinophilic lung diseases Eosinophilic lung diseases constitute a diverse group of disorders of various origins. The diagnosis is suggested by the presence of pulmonary infiltrates on chest imaging and peripheral eosinophilia. It is confirmed by the presence of increased amounts of eosinophils in BAL and/or lung tissue eosinophilia. In this section, eosinophilic vasculitis will not be discussed (see chapter 6.2.2). The search for an etiology includes a combination of clinical and laboratory investigations. Eosinophilic lung diseases of known cause in children include mainly allergic bronchopulmonary aspergillosis, parasitic infections and drug reactions. Eosinophilic lung diseases of unknown cause comprise Loeffler syndrome (characterized by migrating pulmonary opacities), acute eosinophilic pneumonia, and chronic eosinophilic pneumonia [174, 175] . The idiopathic hyper-eosinophilic syndrome is a rare disorder observed mainly in adults; it is characterized by prolonged eosinophilia and a multiorgan system dysfunction due to eosinophil infiltration with pulmonary involvement documented in almost half of the patients [176, 177] .
Alveolar capillary dysplasia and pulmonary capillary hemangiomatosis The pulmonary capillaries form a dense sheet-like meshwork composed of short interconnected capillary segments. The capillary meshes are wrapped over the alveoli, with only a single sheet of capillaries between adjacent alveoli on the same alveolar duct. Impaired development of this vascular network can be caused by genetic defects, prematurity or injury. Aberrant angiogenesis documented in pediatric patients include mainly alveolar capillary dysplasia, and pulmonary capillary hemangiomatosis [178] . Alveolar capillary dysplasia is a rare disorder, presenting with persistent pulmonary hypertension of the newborn [179] . The strongest diagnostic features are poor capillary apposition and density, allied with medial arterial hypertrophy and misalignment of pulmonary vessels [180] . Pulmonary capillary hemangiomatosis is also a rare disease that is characterized by proliferation of capillary-sized vessels within the alveolar walls of the lung [181] . Intimal thickening and medial hypertrophy of the small muscular pulmonary arteries are present resulting in elevated pulmonary vascular resistance. Most cases appear sporadic. Chest imaging shows nodular pulmonary infiltrates and septal lines. A definitive diagnosis can be made only by histologic examination. Interestingly, capillary proliferation in the alveolar wall has been reported in hereditary haemorrhagic telangiectasia [182] . Lymphatic disorders Alveolar structure formation is characterized by refinement of the gas exchange unit and functional adaptation of endothelial cells into vessels including pulmonary lymphatics. The pulmonary lymphatic network promotes efficient gas exchange through maintaining interstitial fluid balance. Lymphatic disorders can be classified as primary or secondary.
Congenital errors of lymphatic development can lead to primary pulmonary lymphatic disorders that include lymphangiomas and lymphangiomatosis, lymphangiectasis, and lymphatic dysplasia syndrome [183, 184] . Lymphangiomas are focal proliferations of well differentiated lymphatic tissue, and lymphangiomatosis describes the presence of multiple lymphangiomas. Most of these disorders are discovered in fetuses or during the early postnatal period. Lymphangiectasis is characterized by pathologic dilation of lymphatics. The term "lymphatic dysplasia syndrome" includes congenital chylothorax, and the yellow nail syndrome (a triad of idiopathic pleural effusions, lymphedema, and dystrophic nails) [185] . Secondary forms of lymphatic disorders result from a variety of processes such as chronic airway inflammation that impair lymph drainage and increase lymph production [186] . Diffuse alveolar hemorrhage syndromes Diffuse alveolar hemorrhage (DAH) syndromes are caused by the disruption of alveolar-capillary basement membrane as a consequence of injury to the alveolar septal capillaries, and less commonly to the arterioles and veinules. The hallmarks are intra-alveolar accumulation of red blood cells, fibrin, and hemosiderin-laden macrophages. It is important to point out that approximately one third of patients with DAH do not manifest hemoptysis, and BAL can be extremely helpful if this entity is suspected by showing the presence of siderophages or red blood cells within the alveoli. DAH can be observed in association with systemic findings or without evidence of associated diseases.
In children, situations of DAH in the context of other disorders are reported in several forms of vasculitis discussed above. Other disorders that can also be accompanied by DAH include pulmonary hypertension and congenital heart diseases, pulmonary veino-occlusive disease, arteriovenous malformations and hereditary haemorrhagic telangiectasia, coagulation disorders, and celiac disease [187] .
In the absence of systemic findings, isolated pulmonary capillaritis should be discussed with the search for positivity of the antiglomerular basement membrane antibody with linear deposits in the lung tissue biopsy as well as suggestive serologic features such as p-ANCA antibodies [188] .
Idiopathic pulmonary hemosiderosis is a diagnosis of exclusion based on patient presentation with acute, subacute, or recurrent DAH, on the results of lung biopsy showing evidence of 'bland' pulmonary hemorrhage (ie, without capillaritis or vasculitis), and after exclusion of the conditions listed above [189] . In this situation, red blood cells leak into the alveolar space without evidence of damage and/or inflammation of the alveolar capillaries. In addition, the diagnosis of idiopathic pulmonary hemosiderosis can only be considered after exclusion of diseases induced by environmental factors such as pesticide and cow's milk (Heiner's syndrome) [190] . This syndrome is a hypersensitivity disease that affects primarily infants, and is caused by antibodies to cow's milk proteins. The diagnosis is supported by positive milk precipitin test and rapid improvement of symptoms and pulmonary infiltrates on chest imaging after exclusion of milk proteins.
In the resolution phase of tissue injury, elimination of mesenchymal cells and recruited inflammatory cells is essential for restoration of normal cellular homeostasis. Dysregulated repair process in ILD is associated with accumulation and dysfunction of interstitial fibroblasts [191] . In the coming years, it is likely that progress in the understanding of the mechanisms involved in the impaired myofibroblast apoptosis as well as evasion of these cells from immune surveillance will open new areas of investigations and will provide support for the characterization of disorders that affect primarily the alveolar interstitial components in pediatric ILD. Indeed, recently, distinct intrinsic differences in gene expression pathways has been reported between control and lung fibrosis myofibroblasts which suggests that ILD myofibroblasts are pathological cells with fundamental changes [192] .
In the context of ILD, pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia, and chronic pneumonitis in infancy have been reported to be exclusively observed in very young children [8] .
Pulmonary interstitial glycogenosis (PIG) is a non lethal disease, reported in neonates with respiratory distress syndrome developed shortly after birth [193, 194] . Very few cases are described so far but it seems to have a male preponderance [195] . The histological hallmark of pulmonary interstitial glycogenosis is the accumulation of monoparticulate glycogen in the interstitial cells on lung biopsy. It is thought to represent a maturation defect of interstitial cells that leads them to accumulate glycogen within their cytoplasm [8, 196] . It is discussed that PIG could meet "chronic pneumonitis in infancy" as this remains a generalized term [87] . As well, PIG could be considered as a premature lung disease, but more than half of published cases were in term infants [195, 197, 198] . The long term consequences in these infants need to be ascertained.
Neuroendocrine cell hyperplasia of infancy (NCHI) is also a non lethal disease characterized by tachypnea without respiratory failure. The human airway epithelium contains highly specialized pulmonary neuroendocrine cells (PNEC) system. It's function remains unknown but is hypothysed to act in modulation of fetal lung growth and in post-natal stem cell condition [199] . The PNEC system permits synthesis and release of serotonin and neuropeptides such as bombesin [200] . As normal bombesin levels decrease after mid-gestation, its overexpression in NCHI could be attributed to a nonregression of neuroendocrine cells [201] . Clinical presentation is typically a respiratory distress in post-natal young infant (mean age 3.8 months in a large serie, but cases in older children have been reported [202] . HRCT shows patchy centrally ground-glass opacifications and air trapping [203] . On lung biopsy, the histological abnormality is hyperplasia of neuroendocrine cells within bronchioles documented by bombesin immunohistochemistry. The follow-up reveals in some cases the persistence of tachypnea and oxygen requirement for several months. Usually, there is a good prognosis [7, 8, 196, 202] .
Chronic pneumonitis in infancy was first described by Katzenstein et al. [4] . The clinical and radiologic features are similar to those observed in other forms of ILD. Specific histologic abnormalities include diffuse thickening alveolar septa, hyperplasia of type 2 AEC, and presence of primitive mesenchymal cells within the alveolar septa. In some cases, foci of pulmonary proteinosis-like material have been observed in air spaces. The prognosis has been reported to be poor with a high mortality rate.
Other disorders associated with pulmonary development and growth abnormalities encompass a broader spectrum of respiratory manifestations and are more adequately integrated in the classification of diffuse lung diseases [8] .
Management of children with ILD includes administration of oxygen for chronic hypoxaemia, and maintenance of nutrition with an adequate energy intake, Immunization with influenza vaccine on an annual basis is recommended along with other routine immunizations against major respiratory pathogens [11] . In addition, aggressive treatment of intercurrent infections and strict avoidance of tobacco smoke and other air pollutants are strongly recommended.
A very few children do not require any treatment and recover spontaneously. In the majority of cases, treatment with immunosuppressive, anti-inflammatory, or anti-fibrotic drugs is required for weeks, months or even years [1, 9, 61] . Various drugs discussed below can be used, but no guidelines for treatment of ILD in children have been proposed so far. The major reason is the very limited number of pediatric patients available for a prospective clinical trial. In addition, controlled studies with a placebo arm are unacceptable because of the poor prognosis of untreated cases and the reported efficacy of anti-inflammatory therapies in a number of pediatric ILD.
At the present time, the main therapeutic strategy is based on the concept that suppressing inflammation may most likely prevent progression to fibrosis. Among the anti-inflammatory agents used in pediatric ILD, steroids are the preferred choice, administered orally and/or intravenously. This has been well illustrated by the results of the ERS Task Force on pediatric ILD [9] . Oral prednisolone is most commonly administered at a dose of 1-2 mg/kg/day [1] . Children with significant disease are best treated with pulsed methylprednisolone at least initially [61, 204] . This is usually given at a dose of 10-30 mg/kg/day for 3 days consecutively at monthly intervals. The minimum number of cycles recommended is 3 but treatment may need to be continued for a longer period of 6 months or more depending on response. When the disease is under control, the dosage of methylprednisolone can be reduced or the time between cycles can be spaced out. The disease may then be controlled with oral prednisolone preferably given as an alternate day regime. In few cases oral prednisolone is used from the beginning simultaneously with intravenous methylprednisolone but this is only recommended in those with very severe disease. Methylprednisolone may be effective when other forms of steroids administration fail without significant side effects.
An alternative to steroids is hydroxychloroquine with a recommended dose of 6-10 mg/kg/day. Individual case reports have described a response to hydroxychloroquine even in the presence of steroid resistance [1, 205, 206] . Some groups have proposed to base the decision as to which agent to use on the lung biopsy findings, with a preference for steroids in case of large amount of desquamation and inflammation and for hydroxychloroquine if increased amounts of collagen representing pre-fibrotic change are found. However, as documented in the ERS Task Force on pediatric ILD, the preferred choice between steroids or hydroxychoroquine in children is highly dependent on the expertise of the center in charge of the patient, and does not seem to be oriented by the histopathological pattern [9] . In case of severe disease, steroids and hydroxychloroquine may be associated. In situations of inefficiency of steroids and hydroxychloroquine, other immunosuppressive or cytotoxic agents such as azathioprine, cyclophosphamide, cyclosporine, or methotrexate may be used. These treatments have been used mainly in situation of autoimmune disorders.
Promising therapeutic options include macrolides. Indeed, these antibiotics have been shown to display a number of anti-inflammatory and immunomodulatory actions. Although the mechanisms and cellular targets specific to macrolide activity remain to be elucidated, beneficial effects in several chronic lung diseases including chronic obstructive pulmonary diseases (COPD) and cystic fibrosis have been reported [207, 208] . Of interest is the ability of macrolides to accumulate in host cells including epithelial cells and phagocytes. In a recent report, a favorable response to treatment with clarithromycine has been described in an adult patient with DIP [209] . Other new therapeutic strategies currently proposed in adult patients target fibrogenic cytokines. The Th1 cytokine interferon-γ has an antifibrotic potential through suppression of Th2 fibrogenic functions. Antagonists to TGF-β include pirfenidone and decorin. The use of molecules directed against TNF-α such as the soluble TNF-α receptor agent etanercept is also under investigation. To date, there are no reports on the use of these novel therapies in pediatric ILD. Finally, in the coming years, it is likely that an expanding number of molecules aimed at favoring alveolar surface regeneration and repair through activation and proliferation of tissue-resident (progenitor) cells will come out.
Depending on the underlying diseases, several specific treatment strategies needs to be considered. These include whole lung lavage for pulmonary alveolar proteinosis, which has been reported to be effective by removing the material from the alveolar space [210] . GM-CSF has also been shown of interest in this disease [171] . Other strategies such as interferon-α for pulmonary haemangiomatosis are effective [211] .
In recent years, lung transplantation has emerged as a viable option in children of all ages, even in young infants, and lung or heart-lung transplantation may be offered as an ultimate therapy for end-stage ILD [11] . The outcome and survival do not seem to be different from those reported in conditions others than ILD, although comparisons are difficult to establish due to the limited number of reported cases.
Response to treatment and outcome can be evaluated in children based on several criteria such as decrease in cough and dyspnea, increase in oxygenation at rest and sleep, and changes in pulmonary function tests [1, 11] . Improvement on thoracic HRCT may also be seen, but tends to occur over a much longer period of time. Reports in pediatric ILD have not shown a good correlation between histological findings and outcome. Some children with relatively severe fibrosis on lung biopsy make good progress, whereas others with mild desquamation have a poor outcome. This is probably due to the variable severity of the disease in different parts of the lung especially in relation to the particular area biopsied, despite HRCT guidance. Overall a favorable response to corticosteroid therapy can be expected in 40-65% of cases, although significant sequelae such as limited exercise tolerance or the need for long-term oxygen therapy are often observed. Reported mortality rates are around 15%. The outcome for infants is more variable [1, 61] .
Pediatric ILD comprises a large spectrum of disorders, with compelling evidence that some of these disorders are observed more frequently in infants, while others are more specific to older children. Ongoing basic research will provide new insights into the molecular basis of ILD pathogenesis (including genetic factors causing familial disease) in children, and is expected to identify important preclinical markers of disease, pathways of disease regulation, and novel potential targets for therapeutic intervention. For the future, there is a strong need for international collaboration which will allow collecting sufficiently large cohorts of patients with specific entities in order to perform proper therapeutic trials. As a prerequisite, however, a clear and standardised classification of the histopathology of the underlying conditions is critical. Such multicenter trials will help to reduce the still considerable morbidity and mortality in children with ILD. Predictive Power of Air Travel and Socio-Economic Data for Early Pandemic Spread BACKGROUND: Controlling the pandemic spread of newly emerging diseases requires rapid, targeted allocation of limited resources among nations. Critical, early control steps would be greatly enhanced if the key risk factors can be identified that accurately predict early disease spread immediately after emergence. METHODOLOGY/PRINCIPAL FINDINGS: Here, we examine the role of travel, trade, and national healthcare resources in predicting the emergence and initial spread of 2009 A/H1N1 influenza. We find that incorporating national healthcare resource data into our analyses allowed a much greater capacity to predict the international spread of this virus. In countries with lower healthcare resources, the reporting of 2009 A/H1N1 cases was significantly delayed, likely reflecting a lower capacity for testing and reporting, as well as other socio-political issues. We also report substantial international trade in live swine and poultry in the decade preceding the pandemic which may have contributed to the emergence and mixed genotype of this pandemic strain. However, the lack of knowledge of recent evolution of each H1N1 viral gene segment precludes the use of this approach to determine viral origins. CONCLUSIONS/SIGNIFICANCE: We conclude that strategies to prevent pandemic influenza virus emergence and spread in the future should include: 1) enhanced surveillance for strains resulting from reassortment in traded livestock; 2) rapid deployment of control measures in the initial spreading phase to countries where travel data predict the pathogen will reach and to countries where lower healthcare resources will likely cause delays in reporting. Our results highlight the benefits, for all parties, when higher income countries provide additional healthcare resources for lower income countries, particularly those that have high air traffic volumes. In particular, international authorities should prioritize aid to those poorest countries where both the risk of emerging infectious diseases and air traffic volume is highest. This strategy will result in earlier detection of pathogens and a reduction in the impact of future pandemics. Predicting the origin and emergence of new diseases is critical to preventing and controlling them [1, 2] . In particular, if the early spread of a newly emerging pathogen can be predicted and curtailed before it becomes pandemic, its impact on public health and global economies may be much reduced [3, 4, 5, 6] . In March and April of 2009, a novel H1N1 influenza A virus (2009 A/ H1N1) with gene segments from humans, swine, and birds led to the first pandemic of influenza in forty years [7, 8, 9, 10] . Current evidence points to a Mexican origin for the initial human-tohuman transmission of this virus, although preliminary genetic analyses suggest the virus has an older and highly-mixed lineage [8] . The virus' lineage and rapid spread suggest that global trade and travel may have played an important role in its early emergence [7, 8] . Here, we attempt to elucidate how these factors may relate to the emergence and spread of this newly detected virus.
One unresolved question is to what degree does a country's development affects its ability to detect and respond to an emerging disease in a timely manner? Development may affect spending on healthcare infrastructure, and particularly, spending on the high cost, intensive public health surveillance needed during the early stages of a pandemic [11, 12, 13] . Socioeconomic factors will also likely affect individuals' abilities or desire to seek diagnosis or treatment, and a country's capacity to test and identify pathogens. Here, we analyze socio-economic and travel data to understand the initial spread of this virus. We focus on the early stages of the epidemic, when travel from Mexico was likely to be the dominant mode of viral spread. Finally, we examine poultry and swine trade data prior to the 2009 A/H1N1 pandemic to add to our understanding the processes that led to the emergence of this virus.
As of May 8 th 2009, only two weeks after it was first reported, the 2009 A/H1N1 influenza strain had spread to 24 countries, 40 U.S. states (plus the District of Columbia) in the US, and 9 provinces in Canada ( Figure 1 ). This rapid spread resulted, in part, from the tight connectivity of the globe through air travel ( Figure 2) .
A log-logistic survival analysis regression model was used to predict the time-to-reporting of the first confirmed 2009 A/H1N1 case to each country. Of all the models evaluated, a multivariate model with three predictors, (1) total country-level healthcare spending per capita, (2) estimated passenger volume arriving from Mexico via direct flights (direct flight capacity), and (3) passenger volume from Mexico via indirect, or two-leg, flights (indirect flight capacity), provided the best fit to the data using AIC, as detailed under Methods (Table 1 , DAIC = 0, overall x 2 = 54.33 on 5 degrees of freedom, p-value,0.0001). The correlation between total country-level healthcare spending and the flight data was low (r,0.4). Although the correlation between direct and indirect flight data was high for countries with direct flights (r.0.9), the indirect flight information provided critical additional information for areas without direct flights. The AIC scores demonstrated this, as the model that included only direct flight information and healthcare spending did not explain the data as well as the best fit model (DAIC = 9.044). Alternate socio-economic measures, even those directly related to healthcare, such as the number of physicians per capita, GDP, or population density were much less predictive than total healthcare spending per capita. Notably, out of univariate analyses, the model with healthcare spending per capita as the sole predictor fit better than models with flight information alone (Table 1) , demonstrating just how informative this data is in predicting the date of reporting. In the best fitting multivariate model, indirect flight capacity had the largest effect size, but including healthcare spending per capita substantially increased the fit to the data (Tables 1, 2 ). For Canadian provinces and American states, we conducted an analysis with just the flight data (Table 3 overall x 2 = 22.89 on 2 degrees of freedom, p-value ,0.001). While the direct flight information does not have a statistically significant effect, the indirect does, most likely because only a few key hubs had direct flights, and these hubs also have a large volume of indirect connections.
For the country-level analysis, we compared the predicted reporting dates with the actual reporting dates, for countries where the disease arrived by May 8 th , 2009 ( Figure 3 , Supplemental Online Figure S1 ). We validated the model by determining how well a model fit to data up until May 8th predicted reporting dates for fourteen countries where the disease was detected between May 9 th and May 19 th (Supplemental Online Figure S2 ). The correlation between forward predicted and observed dates was 0.62, and the observed reporting date fell within the 95% confidence interval for all countries. Many of the actual reporting dates are earlier than predicted, which is expected due to the nonlinear nature a of log-log survival analysis regression. In particular, countries that had not reported disease by the cut-off date were included in the analysis by designating these as locations that ''survived'' the entire study period without acquiring the disease (i.e, censoring). This appropriately extends the predicted reporting dates by including information on both countries that had reported disease by the cut-off date as well as countries that had not. Using this methodology, we also estimated the reporting date of the disease in the remaining 103 countries and the 95% confidence intervals ranged from April 17 th to May 29 th , 2009 (Supplemental Online Figure S3 ).
To elucidate the potential origins of this novel viral strain, and to shed light on targets for future surveillance and prevention programs, we analyzed global trade in live poultry and swine during the decade preceding the current pandemic [14] . We estimate the trade in live swine between Canada, the United States and Mexico to be over 1.75 million animals over the last decade, 
Previous studies suggest that data on air travel can be used to predict the spread of newly emerged human pathogens and better target public health measures [15, 16, 17, 18] . Our analyses support this, but demonstrate that the ability of a country to rapidly detect, diagnose, and report the new infection is a critical element that enhances our predictive power and control capacity. Other studies suggest that analysis of the underlying drivers of disease emergence (e.g. agricultural intensification, land-use change) can be used to predict the geographic origins of new emerging diseases [2] . The currently circulating pandemic influenza strain is a triple reassortment virus with closest known relatives from Europe, Asia, and North America, but there is uncertainty regarding its origin due to the large temporal separation between this pandemic 2009 A/H1N1 strain and the nearest ancestors (10-15 years) [7] . Our analyses of swine and poultry trade demonstrate an enormous potential for intercontinental mixing of potentially zoonotic pathogens, including influenza A viruses. Although artificial insemination is the predominant strategy for interbreeding of commercial swine, live swine are still routinely traded for breeding purposes [19] . Large numbers of poultry are also traded globally, and low pathogenicity influenza viruses are likely to spread unnoticed among poultry until they reassort or mutate to highly pathogenic forms, such as the A/H1N1v strain. This strain notably was the results of reassortment of several relatively low pathogenic influenza strains, as explained by Garten et al. [8] . In addition, as the recent cases of workers exposing a herd of pigs to the 2009 A/H1N1 virus makes clear [20, 21] , even dramatic reductions in the international live animal trade may not prevent the exposure of local livestock to novel viral types from distant locations [9, 10] . Although extensive trade of poultry and swine between continents and within the North American countries almost certainly contributed to the emergence of this virus, surveillance of influenza strains circulating among traded animals is poor [10] , so that it is impossible to designate any single country, trade connection or market as the key point at which the new strain evolved. Expanded surveillance for influenza in livestock populations may allow more of the markers of transmissibility and virulence to be identified, or factors driving higher virus transmission to be determined [9, 22] . In particular, we need to analyze all influenza strains, including the non-and low pathogenic influenzas, in addition to the highly pathogenic ones, with greater regularity. Only by this thorough surveillance can we begin to understand what differentiates the strains that cause pathogenesis in humans from those that do not. Such that eventually we may be able to predict viral emergence and develop vaccines against pandemic influenza viruses in advance of their spread. In order to develop such capability, we need to do more surveillance of livestock and wild influenza strains now.
The speed at which 2009 A/H1N1 spread during the early phases of this pandemic is striking. It was detected in four continents within three weeks after Mexican authorities first reported it. In contrast, the 1918 Spanish flu took 3 years to circle the globe [23] . Our analyses of air-travel data support the WHO's decision to recommend against closing all air travel from Mexico, since the virus most likely had already spread to several other countries by the time it was first reported to be widespread in Mexico on April 29 th . In particular, cases had already been detected in the United States, which is a major hub for connecting flights [24] .
Our current report is the first published analysis of H1N1 spread to include indirect flight data, and this significantly increased the predictive power of our model. Our analysis suggests that airports serving as major hubs could be targets for disease surveillance, and could become facilities that train people and stockpile medicines in preparation for pandemics. This approach differs from previous reports that focus on the role of travel restrictions at hubs [6, 17] .
Our results further suggest a critical role for health care spending in determining a country's probability of detecting, confirming and reporting influenza cases in the early phases of a pandemic. The negative relationship between healthcare spending and detection of 2009 A/H1N1 influenza may be due to a delay in testing or in the collecting of specimens from individuals in countries lower healthcare resources. These countries likely have lower rates of health insurance, less healthcare infrastructure, lower self-reporting, and lower numbers of doctors per capita.
One consequence of lower health care resources is that the threshold for detection (i.e., the number of cases that need to occur before a case is detected, tested and confirmed by medical authorities) is likely higher in lower-income countries that cannot afford to invest as much in public health and healthcare infrastructure. Similar socioeconomic factors have been shown to play an important role in determining spatiotemporal patterns of diseases such as tuberculosis, schistosomiasis, West Nile virus, and HIV/AIDS [12, 13, 25, 26] .
We found that incorporating data on healthcare spending per capita significantly increased our power to predict the time of reporting of 2009 A/H1N1. This suggests important strategies for future disease control. During the early stages of a pandemic, countries with moderate to high air travel from a pandemic origin, but relatively low healthcare spending, are likely to significantly under-report cases. It is therefore in the best global health interest for intergovernmental and other aid agencies to specifically target these nations for assistance to test and report cases early in a new pandemic. We propose that subsidies for outbreak response to these nations with high connectivity and low resources would be the most effective strategy to reduce the spread and impact of a pandemic.
Efforts to better target pandemics would be more effective in reducing disease spread if they were set up in advance of a pandemic [5, 6, 16] , as there is a very small window of opportunity in which to act once a new emerging disease is detected. Such efforts could be strategically positioned to target emerging disease 'hotspots' [2] that are also hubs of trade and travel for surveillance and prevention [16, 27] . For influenza viruses, any future identification of a spillover of a novel strain from poultry or swine to farm workers should be rapidly followed by analyses of the travel routes out of the country where the index case was discovered. At that point, intergovernmental agencies such as WHO could best target limited resources to the poorer countries that are most likely to receive high numbers of airline travelers from the pandemic origin. These are the countries where reporting is likely to be poorest, and where a significant, undetected caseload is likely to exist by the time resources are allocated. These at-risk countries are also the least capable of affording control measures.
On the whole, this H1N1 strain appears to be relatively mild, although it is still inflicting additional morbidity and mortality. However, if a strain with a higher mortality rate, such as that observed with the H5N1 avian influenza subtype, were to spread in a similar fashion, the outcome would be catastrophic both in terms of human suffering and economic damage. For example, the impact of an H5N1 avian influenza outbreak, should the virus become easily transmissible between humans, on the United States economy has been estimated to be $71.3-$166.5 billion [28] . The measures we have proposed are likely to have economic benefits that far outweigh their costs.
We compiled the data on international air travel from the IATA database, supplied by Diio, LLC through their APGdat service [29] . Similar to prior analyses [15, 16, 17, 18] , we used direct connection information with regards to aircraft type and passenger capacity to calculate the connectivity of Mexico with all airports included in the database, and summarized this information (as direct flight capacity) at the country level. Additionally, we estimated the number of connecting passengers (indirect flight capacity) by calculating the number of passengers (p i,j ) arriving at airport j from airport i, and then estimating the number of passengers (p j,k ) going from airport j to airport k, based on all flights reported in the database. We limited the potential connections (trip jRk) to flights that departed no sooner than one hour after the first trip (iRj), and no later than six hours after the arrival of the first trip. We also disallowed return of passengers to Mexico once they left the country, and the return of passengers to North America once they left that region. We thus obtained a quantity, x i,j,k , that estimates the total potential connections to airport k available to passengers from the first trip (iRj). Setting constant the fraction of all passengers that connect (x), we obtained an estimate of the number of passengers with two leg itineraries for each potential destination (iRk; Eq. 1):
We summarized these connections at the country scale, thereby estimating connectivity for nearly every country on the globe with Mexico through either direct or indirect flights; the only countries excluded would require an overnight stay in a hub airport, or three or more connecting flights. We validated our algorithm (eq. 1) for connections within the contintental U.S.A. (the only data on actual itineraries, including connecting flight information, to which we had access). We randomly chose 50 connecting itineraries within the U.S.A. and compared our predictions to the actual routes. Our predictions were statistically significant, using a simple proportional model with log-normal errors, and explained over 60% of the variance in actual routes (F = 83.71, p,0.001 on 1, 49 d.f, adjusted R 2 = 0.6232).
We determined the date a country reported its first WHOconfirmed 2009 A/H1N1 case through May 8 th , 2009. We chose this date in order to limit the analysis, as much as possible, to initial spread from Mexico, because it served as a natural breakpoint in the distributions of reporting dates, as well as being the date our initial analysis. We performed a survival analysis using R [30] , and used an accelerated life time model using a log-logistic distribution. We also examined using a scale-free exponential distribution, as opposed to a log-logistic distribution, which requires a scale parameter, but these models did not fit nearly as well, as measured by AIC. We followed Burnham and Anderson [31] , in using Akaike Information Criterion (AIC) to choose the model that best explains the data (i.e., the one with the lowest AIC, or equivalently DAIC, score). Additionally we provided the Akaike weights, which estimate the likelihood that a specific model is the true model, assuming that the true model is in the set of examined models [14] . Using this methodology, we choose to evaluate 22 models that made mechanistic sense including a null model for a reference. We did not include any models with only the indirect flight data, and without the direct flight data, because we feel that this does not make mechanistic sense. To reduce multicollinearity we included at most two socio-economic indicators.
We evaluated four independent predictors for the date of first confirmed 2009 A/H1N1 case: the volume of (1) direct and (2) indirect passengers on international flights, (3) the country-specific Gross Domestic Product and (4) healthcare spending per capita, by both private and public entities, from 2006 (the most recent year with all data available) from World Bank estimates [32] . We also examined alternate socio-economic metrics as compiled by the World Bank [32] , such as the number of physicians, and average population density. However models including these predictors did not perform as well (as measured by AIC) and often had many more missing values if limited to most recent information.
For all analyses, dates were transformed to Julian day since February 15 th , and all predictor variables were standardized (mean subtracted, then divided by standard deviation) in order make possible the direct comparison of coefficients. This standardization has the added advantage of canceling out the x factor in equation 1 for the statistical analysis; thus, our analyses do not require any assumptions about the number of passengers who make connecting flights.
These statistical models were used to predict the expected time of detection for all countries in our database that had GDP, population density, healthcare, and flight data. Confidence intervals were constructed from the best model fit based on the variance of the data, using the ''predict'' functions in R [30] .
We obtained United Nations Food and Agriculture Organization data on trade in Live Swine (commodity code HS96:S0103) and Live Poultry (S0105) from the U.N. Comtrade data portal [14] . We analyzed data from the last ten years (the approximate time since 2009 A/H1N1 diverged from the nearest sampled virus) [7] , and focused on trade to North America (Mexico, Canada and United States) from outside this region, as well as trade to Mexico within the North American region. Figure S1 Model predictions compared with actual case arrival dates. Dates of case arrivals (black diamonds) for cases that were reported before our cut off of May 8th. Grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. Found at: doi:10.1371/journal.pone.0012763.s001 (0.02 MB PDF) Figure S2 Forward prediction of future case arrival dates. Dates of case arrivals (black diamonds) for cases that were reported after our cut off of May 8th, but before May 19th. Grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. Found at: doi:10.1371/journal.pone.0012763.s002 (0.02 MB PDF) Figure S3 Forward prediction of future case arrival dates. Grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. Found at: doi:10.1371/journal.pone.0012763.s003 (0.03 MB PDF) Evolutionary Entropy Determines Invasion Success in Emergent Epidemics BACKGROUND: Standard epidemiological theory claims that in structured populations competition between multiple pathogen strains is a deterministic process which is mediated by the basic reproduction number ([Image: see text]) of the individual strains. A new theory based on analysis, simulation and empirical study challenges this predictor of success. PRINCIPAL FINDINGS: We show that the quantity [Image: see text] is a valid predictor in structured populations only when size is infinite. In this article we show that when population size is finite the dynamics of infection by multi-strain pathogens is a stochastic process whose outcome can be predicted by evolutionary entropy, S, an information theoretic measure which describes the uncertainty in the infectious age of an infected parent of a randomly chosen new infective. Evolutionary entropy characterises the demographic stability or robustness of the population of infectives. This statistical parameter determines the duration of infection and thus provides a quantitative index of the pathogenicity of a strain. Standard epidemiological theory based on [Image: see text] as a measure of selective advantage is the limit as the population size tends to infinity of the entropic selection theory. The standard model is an approximation to the entropic selection theory whose validity increases with population size. CONCLUSION: An epidemiological analysis based on entropy is shown to explain empirical observations regarding the emergence of less pathogenic strains of human influenza during the antigenic drift phase. Furthermore, we exploit the entropy perspective to discuss certain epidemiological patterns of the current H1N1 swine 'flu outbreak. Recent years have seen an apparent acceleration in the rate of emergence of new infectious disease pathogens in the human population [1] . Some of these have their origins in animal (wild or domesticated) reservoirs [2] [3] [4] , and the years since 2003 have witnessed the appearance of SARS [5, 6] and swine flu [7] . The 20 th century saw, for example, the emergence of pandemic 'flu in 1918, 1957 and 1968 (with a limited H1N1 re-emergent outbreak in 1977) [8] , avian 'flu [9] and the rise of HIV in the 1980's [10, 11] . Additionally, antibiotic-resistant pathogens have become increasingly widespread in the past decade, particularly in healthcare settings [12] . Antigenically-variable pathogens are responsible for much of the burden of communicable disease in the world today. Therefore, developing an understanding of the factors that lead to the emergence and spread of novel pathogenic agents and strains is a topic of great interest. In recent months the emergence of a swine 'flu (H1N1 2009) with human-to-human transmission capability has re-focussed attention on this issue [7, 13] . Likewise, studies, such of those of Creanza et al. [14] who used a computational analysis of viral nucleotide and amino acid sequence data collected during seasonal 'flu epidemics show how diversity declines over the course of an epidemic. These observations underscore the role that ecological constraint play in the evolution of pathogens.
For antigenically variable pathogens it is competition between strains that is the fundamental mechanism which determines the observed patterns of disease spread and prevalence. Diseases in this category include influenza A virus, meningococcal and pneumococcal bacterial infection, malaria and dengue fever, to name but a few. The principal epidemiological characteristics of such diseases are the absence of life-long protective immunity, cross-reactive immunity between strains and the potential for future re-infection. Each strain is in competition with the others for resources. In this case the resources are susceptible hosts, and dominance goes to those variants that are able to outpace their neighbours in their ability to infect susceptibles. From a Darwinian perspective it is the ''fittest'' strains that will dominate. Translating the qualitative notion of fitness into quantitative terms constitutes one of the fundamental problems in evolutionary epidemiology.
Standard epidemiological theory as largely developed by Dietz [15] and Anderson and May [10] revolves around the basic reproduction number, R 0 , the number of secondary infectives, as the key parameter [15, 16] for analysing disease emergence, spread and vaccination strategy. In the case of structured populations, R 0 is defined as
where the function V (x) is the infectious net-reproductive function. It is an ''infectious age''-dependent function that defines the rate at which an infected host generates secondary infections in the time interval following its initial infection (File S1 Section i). This theory has been extended to address competition between emergent pathogen strains using basic reproduction number as the metric for competitive dominance [16] . Selective advantage,s s, in the case of competing strains is now given bỹ
where DR 0 is the difference in the basic reproduction number between the incumbent and invading strain [16] . The measure of selective advantage given by equation 2 implicitly assumes that the population is infinite -a mathematical idealisation. The fact that conditions of finite size may have an effect on the outcome of selection has been recognised in a population genetics context [17] but has not been explored analytically in multi-strain epidemic models [16] . These studies, however, assumed that the populations were unstructured or demographically homogeneous. The effect of finite size in studies of selection between competing types in structured populations was first developed in Demetrius [18] . The analysis focused on demographic structure and rested on the observation that in view of the heterogeneity in structure and the finite population size,fluctuations in population numbers will occur. The ergodic theory of dynamical systems was then exploited to generate a new family of demographic variables to describe the population dynamics and its fluctuations. A diffusion process was then applied to show that the outcome of selection will now be determined by the robustness or demographic stability of the population, and regulated by the population size and certain demographic parameters which characterise the geometric properties of the infectious net-reproductive function. Robustness, the rate at which the population returns to the steady state condition after a perturbation in age-specific fecundity and mortality variables, can be formalised in terms of the statistical measure evolutionary entropy. This macroscopic variable describes the uncertainty in the age of the mother of a randomly chosen newborn. The change in basic reproduction number, the classical criterion for selective outcome, was shown to be the limit, as population size tends to infinity, of the entropic selection principle. Hence the classical models of selection are limiting cases of the entropic models. This study of competition in age-structured populations was extended by Demetrius, Gundlach and Ochs [19] to the analysis of the dynamics of selection where the heterogeneity derived from individual variations in size, metabolic condition or spatial location. The entropy parameter in this general context describes the uncertainty in the state (size, metabolic condition or spatial location) of the ancestor of a randomly chosen individual.
The results of this study formed the basis of a general model of the evolutionary process which is called directionality theory. We now apply this theory to analyse the effect of finite size in multi-strain epidemiological models where the heterogeneity derives from variability in infection age. The quantity V (x) in this class of model pertains to the product of the survivorship and infectivity of infectious individuals. We will apply the formalism described in [19] [20] [21] [22] to show that the invasion dynamics of competing strains in populations of finite size is predicted in terms of the macroscopic variable evolutionary entropy, S, which is given by
The quantity p i is the probability that the parent of a randomly chosen infective is in the age class i. The statistical measure, S, describes the uncertainty in the age of an infected parent of a randomly chosen infective.
The statistical parameter evolutionary entropy describes the rate at which the population returns to its steady state condition after a random perturbation in the age-specific fecundity and mortality variables. Entropy is analytically related to the generation time, T (the mean age of infection).We will use this analytical fact to show that entropy is also analytically related with the duration that the host organism is infected, and hence it can be regarded as a basic metric of pathogenicity.
Directionality theory shows that entropy S predicts the outcome of competition between strains. The selective advantages s in the case of competing strains involves S and two additional macroscopic variables (W and c, the first and third moments of a random variable defined in terms of the net-reproductive function and the probability distribution p i ). The selective advantage is now [20] given bỹ
Here N denotes the population size of infectives and DS is the relative evolutionary entropy of the incumbent and the invader. The quantities W (the reproductive potential) and c (the demographic index) are statistical parameters, and both are functions of the agespecific fecundity and survival functions which determine the infectious net-reproductive function V (x). The parameters W and c define different scenarios for the epidemiological population biology that prevail during the competitive invasion process. These quantities, in contrast to entropy, can assume positive or negative values contingent on the geometry of the infectious net-reproductive function. The demographic index, c, relates to the flatness or peakness of the infectious net-reproductive function V (x): generally speaking cv0 implies a peaked net-reproductive function, whereas cw0 implies it is flat. This term is less influential on the dynamics as it is scaled by 1=N, so when N (the number of infectives) is moderately largẽ s s~{WDS; so competitive advantage is now determined by the relative entropy and the reproductive potential.
An equivalent formulation of the selective advantage,s s, can be written in terms of the growth rate r and a quantity called the demographic variance, s 2 [19] , which is the second moment of a random variable defined in terms of the infectious net reproductive function and the distribution p i ,
where Dr is the difference in growth rate between the incumbent and variant strains. Its equivalence to equation 4 is demonstrated in File S1, Section ii. It is evident that as N?large we recover equation 2, since Dr and DR 0 are positively correlated. We exploit this new theory of entropic epidemiology to explain detailed empirical observations regarding the emergence of less pathogenic strains of human influenza A virus, an issue that has remained elusive when viewed through the framework of classical epidemiological theory. Moreover, we discuss the current on-going swine 'flu (H1N1 2009) outbreak from the perspective of directionality theory, [18] [19] [20] [21] [22] .
Much recent work on strain dynamics in multi-strain pathogens has focussed on the adaptations of basic epidemic models to deal with multiple strains with differing assumptions about host immune responses [23] [24] [25] [26] . This has led to progress in understanding issues such as strain clustering effects, for example, but at the price of intractability when large numbers of strains are considered. By contrast, the model presented here takes a different approach, as it focuses on the emergent properties of multiple competing strains without a detailed rendering of all biological features. The penalty for generating this alternative model is that the biological detail is presented more crudely than in the more detailed epidemiological approaches. Strain selection takes place at a number of levels ranging from within hosts all the way up to the population level. In such multi-scale systems a variety of modelling approaches are needed. However, we believe that the approach presented here complements existing formulations. To make the presentation concise we will summarise certain general results of the dynamics of competition in structured populations, as elaborated in directionality theory [18] [19] [20] [21] [22] , and apply them in an epidemiological context. Specifically, our model shows that in the context of emergence of new human 'flu strains in SE-Asia there will be a progressive shift to less pathogenic strains. This empirically observed pattern is consistent with our entropic perspective.
Classical epidemiological theory associates increased competitive advantage with increasing basic reproduction number, R 0 [27, 28] . The argument is that a larger R 0 results in a faster rate of infection of susceptibles (as determined by the growth rate, r) thereby driving competitor pathogen strains with lower R 0 to extinction. The basic reproduction number varies for different pathogens (and their strains) and varies according to the circumstances (geographic location, age structure, population density, previous exposure etc) of the host population [10] .
The claim that the outcome of competition between variant strains is a deterministic process mediated by R 0 is the analogue of the claim, which goes back to Fisher [29] , that the rate of increase of total population numbers -the Malthusian parameterdetermines the outcome of competition between an incumbent and a variant type. These studies, in both epidemiology and population genetics assume that populations have infinite size.
Evolutionary entropy, in the context of epidemiological models, describes the uncertainty in the age of a parent of a randomly chosen infectives. This quantity is a demographic parameter that is positively correlated with the demographic stability.
Directionality theory, a study of the dynamics of competitive invasion [18] [19] [20] [21] [22] 30] of structured populations when population size is finite predicts that the outcome of selection is a stochastic process determined by evolutionary entropy and contingent on population size and two other demographic variables which characterise the geometry of the infectious net-reproductive function. Evolutionary entropy in this more general context describes the uncertainty in the state of the ancestor of randomly chosen infective. (File S1, Section i).
In this paper we exploit this general tenet to show that in finite populations with heterogeneity in age of infection the outcome of strain competition is a stochastic event determined by evolutionary entropy and contingent on the demographic parameters W and c. Selective advantage in this case is given by equation 4.
One of the most significant parameters in epidemiology is the duration of host infection, D, which is taken to represent the period of time for which an infective is capable of transmission to susceptibles. This parameter is related to T, the generation time. D can be generally expressed in the form D~kT where k is a parameter dependent on the strain, but the characterisation of D will depend on the model system under consideration. In a basic S-I-R model, for example, the fecundity function is a constant independent of infectious age. Consequently the generation time will depend uniquely on the mortality rate (i.e. the rate of recovery from the pathogen). The generation time T will be inversely related to the rate at which individuals leave the infected class, denoted by n. Hence for this class of models D~kT~1=n. In the model described in this paper, survivorship and fecundity are functions of age, so the generation time involves both survivorship and fecundity components. The generation time T can be expressed in terms of the entropy function S. As shown by Demetrius et al. [31] , the evolutionary entropy can be shown to be analytically related to T by This equation asserts, therefore, that in systems with demographic heterogeneity the duration of infection will now be regulated by the pathogen entropy, S. This important fact underscores the fact that demographic heterogeneity dictated by V (x) will induce significant changes in the epidemiological dynamics.
These results have particular significance in understanding the epidemiology of influenza A virus.
Influenza A epidemics in humans are characterised by infrequent (typically on a decadal timescale), but nevertheless significant, genetic re-assortments that lead to pandemics of new sub-types (antigenic shift) as well as within-sub-type evolution (antigenic drift) on an annual timescale. In the antigenic shift scenario it is assumed that there is very little or no host cross-immunity with previous virus types, whereas within-sub-type drift can generate strains with varying degrees of host immune response. This difference is important in determining how variants invade host populations.
Antigenic shift and antigenic drift can be characterised in terms of the demographic parameter W. This variable is analytically related to the population growth rate, r, and the entropy rate S/T by the identity r~S=TzW (where T is the generation time -see File S1 section i for parameter definitions). From this equation it follows that when Wv0, rvS=T.
Wv0[rvS=T i.e. r small -corresponds to a relatively small/ negligible growth rate of incumbents.
But when Ww0[rwS=T i.e. r large -corresponds to a relatively large growth rate of incumbents.
We will consider these two notable epidemiological characteristics in turn:
Antigentic Drift: Ww0. Recent work on the genetic and antigenic evolution of Influenza A H3N2 in humans has demonstrated that seasonal 'flu epidemics emerge from seed strains originating in countries of south-east Asia which subsequently spread sequentially through the global population [32] [33] [34] [35] . This suggests that novel H3N2 variants compete with existing strains within the E-SE Asia circulation network with the dominant strain being responsible for generating the next global seasonal 'flu strain. Once out of the seeding region there appears to be little subsequent viral evolution [32] , though as pointed out by Rambaut et al. [36] subsequent changes tend to be deleterious and so die out. When a new H3N2 variant is generated within the seeding region it competes for susceptibles with existing variants. The epidemiological picture in this densely populated SE-Asia seeding region is one of strong fluctuations of multiple circulating strains [32] . Much of this fluctuation is driven by the cross-reactive immunity and heterogeneity of host population immune response to the different strains and finite size population effects. The dynamics are characterised by repeated boom-bust cycles in the strain populations, so competition is taking place between a new variant and a rapidly growing incumbent pathogen population that has a large positive growth rate r, implying that the reproductive potential Ww0. Information on the infectious netreproductive function from which we would infer the value of c is less readily available [33] . It is plausible to assume, however, that the rate at which secondary infections are produced is broadly correlated with pathogen burden. This results in a netreproductive function for influenza [33, Figure 1 ] that is slightly skewed towards the early stages of host infection, implying that c is small and negative. From Table S1 , the constraints Ww0 and cv0 suggest that new variants will enjoy a selective advantage if their evolutionary entropy is lower than other competing strains (DSv0) during the competitive growth phase. In this model the invasion success of the new strain will be dependent on the relative evolutionary entropy of the variant contingent on the demographic variables W and c. The information given in Table  S1 can be invoked to determine the selective outcome of competing strains. This information underscores the difference between the deterministic process which defines the classical models and the stochastic process which characterises entropic epidemiology. A variant strain has epidemic potential and will dominate existing H3N2 strains providing that DSv0. By contrast, in a deterministic model (equation 2) the outcome of competition between strains is a deterministic process predicted wholly by R 0 -the strain with the largest basic reproduction number will always dominate. In the entropic model there is a probability of a downward drift in the evolutionary entropy, S, of the dominant strain. Within the classical framework (infinite population size) the outcome is deterministically ordained, that is, we expect that those variants which produce more secondary infectives to dominate all others. By contrast, in a finite population (and contingent on the values of W, and to a lesser extent c) the outcome has an intrinsic stochastic component: it is the evolutionary entropy that determines success so there may be dominant variants which produce fewer secondary infectives (i.e. have a lower growth rate and R 0 ) than their co-circulating competitors.
The analytic relation between evolutionary entropy and the duration of infection described above entails that lower entropy strains have a shorter duration of infection. Consequently, we expect new annual 'flu strains to have variable R 0 and short infectious duration relative to strains circulating in the SE-Asia seed network because of their smaller evolutionary entropy, S. This pattern is also seen in Table 1 where successful emergent epidemics have variable R 0 but short infectious durations. Additionally, these lower entropy strains will have a greater resilience in maintaining the chain of infection as the pathogen spreads because they have a selective advantage with respect to other strains with which they have to compete for susceptibles. Basic epidemic models [10] correlate the basic reproduction number R 0 with the duration of infection, D which implies that short duration infections will have smaller basic reproduction numbers and would be more likely to die out. By contrast, the analysis here suggests that in the context of emergent epidemics it is the minimisation of the evolutionary entropy (which is proportional to D -since S~log Dz(b{ log k)) that is the determinant of emergent epidemic success, Figure 1 shows the change in entropy over time for a simulation of the invasion process by variants, and it is clear that there is a tendency to decreasing entropy over time for each run. Classical epidemic models define the proportion of the population that need to be vaccinated to eliminate a disease in terms of R 0 , but the theory presented here shows that this is only true in the limit of infinite population which indicates that in future new vaccination criteria will be required for emergent epidemics which take into account finite population size, variability in infection profile and stochasticity effects.
Antigenic Shift: Wv0. On longer timescales completely new influenza A virus sub-types occasionally emerge. These events are unpredictable and usually result in global pandemics with significantly elevated levels of 'flu-related mortality. These new sub-types are thought to arise from hybridisation of human 'flu viruses with those circulating in pigs and/or poultry [8] . Because the new virus is generated by a complete change in the HA antigenic subunit on the surface of the virus the entire global population is essentially susceptible to the disease thereby generating a pandemic. The last major antigenic shift event was in 1968 and it generated the H3N2 (Hong Kong 'flu) strain that has been in circulation since.
Following antigenic shift the new influenza variant is in competition with an incumbent strain that is already at equilibrium in the population which suggests that the growth rate r is small, so Wv0. From the perspective of directionality theory (Table S1 ) the favourable condition for establishment of the new type is higher entropy relative to the existing circulating sub-types (DSw0), i.e. a longer duration of infection. In the antigenic shift case we are addressing infection in the entire global population so, in effect the population of infectives, N is very large. Therefore the role of the demographic index term c (though again it will be small and ,0, for influenza) is minimal.
The larger entropy of the variant corresponds to longer infectious duration than the circulating incumbents. However, the mechanism of antigenic drift (as described above) then begins to operate on this newly established sub-type and so there will be a gradual decrease in infectious duration of the dominant circulating variant with time. Given that the new virus is likely to be a hybrid animal-human type, it is likely that it is less well adapted to human hosts so it might have a low R 0 . If, rather crudely, we associate infectious duration in the host with pathogenicity, directionality theory implies high pathogenicity immediately following establishment of a new sub-type followed by decreasing pathogenicity in subsequent years. That is, the new Influenza A sub-type (such as 1918, 1957 and 1968) appears to be highly pathogenic in the immediate interval following establishment, but there is a contribution to the decrease in 'flu mortality in the era following the antigenic shift by the action of competitive selection of lower entropy variants during the antigenic drift phase in the SE-Asia seeding region. When a new Influenza A virus sub-type is generated due to antigenic shift the cycle is repeated again. Clearly, there will be a decrease in the absolute influenza mortality figure (number of fatalities) during the drift phase because as time passes the overall population immunity level increases. However, the model suggests that the case fatality rate (CFR -number of fatalities per infected individuals) will decline during the drift phase due to declining pathogenicity. A decline in the CFR is observed empirically when comparing a pandemic attack year and the next subsequent epidemic [39] but there does not appear to be any detailed epidemiological analysis of long-term CFR trends during the drift phase. A recent detailed study of the epidemiology of Influenza A H1N1 in the era 1918-1951 [40] shows, Figure 2 , that during the H1N1 era there is evidence of a sustained decrease in mortality rate between 1918 (autumn wave) and 1924 and likewise between 1928 and 1944. Whilst some of this progressive weakening of the influenza epidemics is due to increasing host population immunity it is likely that there is also a contribution to declining pathogenicity resulting from the mechanisms proposed in the directionality theory. H1N1 (2009) Swine flu. In March 2009 the first reports of an epidemic of a novel influenza-like pathogen emerged in Mexico [13] . Analysis showed that the infectious agent in this on-going epidemic to be Influenza A H1N1 (swine flu) [7] . H1N1 has generated epidemics in humans in the past and was responsible for the 1918 influenza pandemic and a 1977 'flu epidemic. It is possible to explain the relentless spread of this current outbreak in directionality theory. This new variant has emerged from a 'flu type associated with pigs, but now has human-to-human transmission capability. This successful variant has a shorter infectious duration than other influenza A strains [13] which suggests that it may have emerged with a competitive advantage founded on its lower entropy DSv0 ð Þrelative to other currently circulating 'flu strains. The extent of pre-existing immunity to H1N1 (swine flu) is currently unknown, but our results suggest that in humans there may be some pre-existing protection from previous exposure to influenza virus. This possibility is also noted by Fraser et al. [13] . Alternatively it may simply be a novel pandemic strain with short infectious duration. SARS had a higher R 0 and longer infectious duration (Table 1 ) than swine flu yet did not have the same global impact, which reinforces the generic suggestion from entropic considerations that it is those emergent diseases with shorter infectious durations that appear to have the greater pandemic potential.
Existing endemic infections. We noted above that many antigenically stable infectious diseases (measles, chickenpox for example) have comparatively long infectious durations compared with emergent infections (Table 1 ). Directionality theory suggests that this might be the consequence of a long evolutionary adaptation to humans by occasional mutations resulting in ever higher evolutionary entropy S. In this picture, for a mutation to dominate an established equilibrium incumbent strain it is necessary for the variant to have DSw0, so on evolutionary time scales we see an upward drift in entropy and, consequently, ever increasing duration of infectiousness. Although such diseases do have a seasonal component to their incidence they nevertheless exist at some stable mean prevalence within the host population. The next dominant strain measles has to compete against a long-established incumbent strain that is at overall equilibrium in the population. Figure 3 shows the change (upward drift) in entropy of the most frequent variants in the population using a simulation of the invasion process. In this picture the dynamics of invasion is considered at a global scale (number of infectives N large) so the conventional Malthusian picture re-asserts itself. Consequently in this situation there will be proportionality between the duration of infection and R 0 , so the concept of a basic reproduction number retains its conventional role as a measure of selective advantage and, hence, its usefulness as a metric for the amount of vaccination required to eliminate a given pathogen strain. It is apparent that within the approach presented here the epidemiological context (emergent pathogen versus established equilibrium) in which the competition takes place does matter to the outcome and the properties of strains that will dominate.
In summary, directionality theory shows that during the fluctuating (opportunistic) competitive growth phase of multi-strain pathogen epidemic establishment it is short infectious duration (low entropy) strains that are favoured over longer infectious duration (higher entropy) strains. Moreover, they will be resilient to competition from other strains thereby giving them pandemic potential. By contrast, in established (equilibrium) populations it is longer infectious duration (high entropy) strains that have competitive advantage. This suggests that the epidemiological circumstances, opportunistic or equilibrium, that are prevalent in a host population during competitive emergence are critical in determining the properties of the dominant pathogen strain. To be clear, the stochasticity and fluctuations present in this model arise from consideration of an infection process in a finite population that has infection demographics defined by the function V (x). In this case the straightforward application of the concept of the basic reproductive number can be of limited usefulness as a key determinant of epidemic dynamics as there is no longer an automatic correlation between R 0 and competitive dominance.
The model presented above has some explanatory power beyond that of conventional theory in that it suggests that for Influenza A new pandemic variants generated by antigenic shift will be more pathogenic (assuming pathogenicity correlates with infectious duration) than the subsequent seasonal strains generated by the process of evolutionary antigenic drift. From a public health perspective these results suggests that monitoring of those emergent strains with a shorter infectious duration is a better indicator of pandemic risk than focussing on just R 0 , as they present an elevated threat of triggering pandemics and may need to be the target of timely vaccine development. Moreover, these results suggest the calculations of R 0 may not provide a reliable guide to the vaccination effort required to eliminate an emergent pandemic strain. The limitation of a singular focus on R 0 has been highlighted by Meyers [41] in the context of epidemics on networks. However, further work is needed to develop the application of directionality theory to empirical epidemiological questions such as determining the optimal vaccination coverage. Table 2 contrasts the classical and entropic models in order to emphasize their fundamental differences in explanatory and predictive power.
The reasons why some flu strains are more pathogenic than others is a complex issue involving specific details of host-virus interactions, but the model we propose has the attraction of capturing (on a simple criterion of pathogenicity, at least) evolution to generally less-pathogenic strains. The determinants that drive empirically observed patterns of emergence and spread of novel infectious pathogens are incompletely understood, turning as it does on the interplay of epidemiological, immunological and genetic considerations. No single model is able to capture the full complexity of this reality, but the work presented here is intended to shed some light on the criteria for invasion success and subsequent evolution of emergent strains.
Our results show that conditions of demographics of the infection process, finite population size and consideration of the prevailing epidemiological dynamics against which strain competition occurs together impose limitations on the explanatory and predictive power of any analysis based solely on the basic reproduction number. The concept of evolutionary entropy provides a framework that is stochastic in its foundation for resolving these limitations.
The population of infectives is divided into a number of discrete ''age'' classes. Each day every individual either moves up to the next age class or moves to the Recovered class with probability b i .
An infective in age-class i produces on average m i infectives. These new infectives each begin their journey through the infective stage in age-class 1. Consequently, there are two functions, defined by l i and m i , (and hence V i~li m i see File S1 section i) that characterise a pathogen strain and its behaviour in the host.
Transitions in the simulation are decided by a stochastic process. The simulation starts with N wild-type infectives. Each day each infective has its infectious age increased by 1. A random number in the interval (0,1) is then generated for each infective. If this number is ,current recovery probability b i then the individual recovers, otherwise it generates its quota of secondary infectives m i . New infectives begin with infectious age = 0. For each new infective a random number in the range (0,1) is generated. If it is ,mutation rate then this new infective will be a variant strain. Mutations are generated by a small perturbation to the function V i (see below ''Mutation and Competition''). Each day the number of each strain is calculated so that the dominant (highest frequency) strain can be identified. The entropy, S, of this strain is then calculated using the demographic parameters.
To simulate the Ww0 scenario in Figure 1 (which corresponds to antigenic drift in the SE Asia region) requires rapid strain growth rates (r large). This is done by initially allowing the total population (of all strains) to grow rapidly. Once the supply of susceptibles becomes depleted the population collapses abruptly (resource availability variable). The supply of susceptibles is then re-instated and the boom-bust cycle repeats itself. New strain variants are generated through the cycle. The purpose of this is to mimic the conditions for emergence of new variants when there is competition and growth. In this scenario variants that compete against each other are not at equilibrium in the population so we are addressing a localised competitive situation. The total population size used for this simulation was 10,000 individuals with d(x)~a with a in the range +0:1 with a mutation rate of 10 {5 day -1 .
To simulate the Wv0 scenario in Figure 3 the supply of susceptibles is controlled to maintain the total population of infectives at a broadly constant level (i.e. resource availability constant). This reflects low-to-minimal growth rate (equilibrium, r small) of the incumbent. The number of infectives fluctuates around an equilibrium level and new variants attempt to invade the system whilst it is in this configuration. In this scenario the incumbent is already established at equilibrium, so we are addressing a global competitive situation. These simulations were run for 200,000 days with d(x)~a with a in the range +0:1 and with a mutation rate of 10 {4 day -1 .
Each variant is characterised by the net fecundity function V i . We assume that mutants are defined by V Ã (x)~V (x) 1zd(x) where d(x) is monotonic in x. As a consequence, mutants arise from translation on the function V (x) (corresponding to a change in the age of infector) or from a re-scaling of V (x) (corresponding to an increase or decrease in the net fecundity function). Monotonicity is imposed to preclude net-fecundity profiles that are large at early and late stages and low at intermediate stages.
The genotypes of mutant and wild-types are constructed so as to have positive growth rates. Consecutive time-steps of evolution are simulated by generating random numbers to decide which individuals recover or continue as infectives. To simulate competition between strains some additional growth constraints have to be applied to each scenario. In the Ww0 case the initial population is allowed to grow rapidly from an initial starting number N init . Following exhaustion of the supply of susceptibles (i.e. resources are depleted) an extrinsic mortality 1{N init =N ð Þis applied to all individuals to reduce the population of infectives back down to its starting value. This process is repeated over many time-steps. In the Wv0 case if the total population size (N max ) of infectives is exceeded an extrinsic mortality 1{N max =N ð Þis applied probabilistically so that the population is maintained at a level that fluctuates around N max .
In both scenarios, at each time step, the dominant (most frequent) genotype is determined and its entropy calculated from equation S4.The value of this entropy is recorded for the duration of the simulation. It should be noted that these simulations are not based on an elaboration of S-I-R models of the usual type where susceptibles and infectives interact via conventional mass-action terms. Here, the population of infectives is directly manipulated to reflect the kind of epidemiological dynamics that are typically seen in the emergent and equilibrium phases [42] .
Table S1 Invasion criteria in the entropy model. *''a.s. = Almost surely'' refers to the fact the result is a stochastic process. The criteria for large and small population size are defined in more detail in Demetrius et al. [19] . The criteria noted in Table S1 have been tested against simulation where they have been shown to be replicated. Found at: doi:10.1371/journal.pone.0012951.s001 (0.07 MB DOC) Figure S1 Life cycle for an infective corresponding to the matrix in equation S1 with 4 infectious age classes.  The Nature of Protein Domain Evolution: Shaping the Interaction Network The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. The protein universe is the collection of proteins of all biological species that exist or have once existed on Earth [1] . Our sampling and understanding of it began over half a century ago, when the first peptide and protein sequences were determined by Sanger [2, 3] and, subsequently, the sequencing of RNA and DNA [4] [5] [6] . In the meantime, the genome projects of the last decade have uncovered an overwhelming amount of sequence data and researchers are now starting to address a series of fundamental questions that should shed light onto protein evolution processes [7] [8] [9] [10] . For instance, how many gene encoding sequences are present in one genome? How many sequences are repetitive and are these sequences similar in the various organisms on Earth? Which genes were involved in the large scale genome duplications that we see in animals?
A comparison of sequences for evolutionary insight is best achieved by looking at the structural and functional (sub)units of proteins, the protein domains. By convention, domains are defined as conserved, functionally independent protein sequences, which bind or process ligands using a core structural motif [11] [12] [13] . Examples of domain modes of actions in signaling cascades for instance, are to connect different components into a larger complex or to bind signaling-molecules [14, 15] . Protein domains can usually fold independently, likely due to their relatively limited size, and are well known to behave as independent genetic elements within genomes [16, 17] . The sum of these features makes protein domains readily identifiable from raw nucleotide and amino acid sequences and many protein family resources (e.g., Superfamily and SMART [see Table 1 ]) indeed fully rely on such sequence similarity and motif identifications [18, 19] .
The algorithms that are used for domain identification are built around a set of simple assumptions that describe the process of evolution. In general, evolution is believed to form and mold genomes largely via three mechanisms, namely i) chemical changes through the incorporation of base analogs, the effects of radiation or random enzymatic errors by polymerases, ii) cellular repair processes that counter mutations, and iii) selection pressures that manifest themselves as the positive or negative influence that determines whether the mutation will be present in subsequent generations [20, 21] . By definition, each of these phenomena styles, reproductive strategies, or the lack of apparent polymerase-dependent proofreading such as in positivestranded RNA viruses [22] [23] [24] [25] . Consequently, substitution rates need therefore be calculated to correctly compare two or more sequences and hunt uncharted genomes for comparable domains. Particularly this last strategy, using general rate matrices like BLOSOM and PAM, is an elegant example of how new protein functions can be discovered [26] [27] [28] [29] [30] . Fast algorithms for pair-wise alignments can be found in the Basic Local Alignment Search Tool (BLAST), whereas multiple sequence alignments (MSAs, Fig. 1A) in which multiple sequences are compared simultaneously are commonly created with for example ClustalX and MUSCLE (see Table  1 ) [31] [32] [33] [34] .
Close relatives, sharing an overall sequence identity above for example 50% and a set of functional properties, can also be grouped into families and subfamilies. In turn, these families share also evolutionary relationships with other domains and form together so-called domain superfamilies [18, 35] . Evolutionary distances between related domain sequences can easily be estimated from sequence alignments, provided that the correct rate assumptions are made. Subsequently, these can be used to compute the phylogenies of the domain that share an evolutionary history. These, often tree-like graphs (Fig. 1B) , depend heavily on rate variation models, such as molecular clocks or relaxed molecular clocks (e.g., Maximum Likelyhood and Bayesian estimation), which are calibrated with additional evidence Fig. (1A) . It was computed using Bayesian estimation and presents the best-supported topology for the alignment. Numbers indicate % support by the two methods used, while # indicates gene duplication events in the common ancestor and * marks a species-specific duplication event. For computational details, please see [42] . such as fossils and may therefore also provide valuable information on aspects like divergence times and ancestral sequences [36] [37] [38] . Commonly used phylogenetic analysis strategies are listed in Table 1 .
A limitation of all inferred phylogenetic data is that it is directly dependent on the alignment and less so on the programs used to build the phylogenetic tree [39] . One of the shortcomings of automated alignments may thus derive from the fact that they commonly employ a scoring and penalty procedure to find the best possible alignment, since these parameters vary from species to species [22, 23] , as mentioned above. Careful inspection of alignments is therefore advisable, even though software has been developed that combines the alignment procedure and phylogenetic analysis iteratively in one single program [40] .
Although sequence and phylogenetic analysis provide a relatively straightforward way for looking at domain divergence, comparison of solved protein structures has shown that protein tertiary organizations are much more conserved (>50%) than their primary sequence (>5%) [41] . For this reason, protein structures and their models provide significantly more insight into the relations of protein domains and how domain families diverged [16] . For example, the inactive guanylate kinase (GK) domain present in the MAGUK family was shown to originate from an active form of the GK domain residing in Ca2+ channel beta-subunits (CACNBs) through both sequence and structural comparison [42] . Furthermore, identification of functionally or structurally related amino acid sites in a fold sheds light on the complex, co-evolutionary dynamics that took place during selection [43] .
As described above, the evolution of a protein domain is generally the result of a combination of a series of random mutations and a selection constraint imposed on function, i.e., the interaction with a ligand. The interaction between protein and ligand can be imagined as disturbances of the protein's energy landscape, which in turn bring about specific, three-dimensional changes in the protein structure [44, 45] . Binding energies however, need not be smoothly distributed over the protein's binding pocket as a limited number of amino acids may account for most of the free-energy change that occurs upon binding [45] [46] [47] . In these cases, new binding specificities (including loss of binding) may therefore arise through mutations at these hot spots. An example is a recent study of the PDZ domain in which it was shown that only a selected set of residues, and in particular the first residue of -helix 2 ( B1), directly confers binding to a set of C-terminal peptides [48] .
The folding of a domain is essentially based on a complex network of sequential inter-molecular interactions in time [49] . This has of course significant implications for domain integrity, particularly if one assumes that the core of a protein domain is and has to be largely structurally conserved. Indeed, even single mutations that arise in this area may easily derail the folding process, either because their free energy contribution influences residues in the direct vicinity or disturbs connections higher up in the intermolecular network [49] . It is therefore hypothesized that protein evolution took place at the periphery of the protein domain core, and that gradual changes via point mutations, insertions and deletions in surface loops brought about the evolutionary distance we see among proteins to date [21, [50] [51] [52] .
However, distant sites also contribute to the thermodynamics of catalytic residues. This is achieved through a mechanism called energetic coupling, which is shaped by a continuous pathway of van der Waals interactions that ultimately influences residues at the binding site with similar efficiency as the thermodynamic hotspots [53, 54] . Indeed in such cases, evolutionary constraints are not placed on merely one amino acid in the binding pocket, but on two or more residues that can be shown to be statistically coupled in MSAs [54, 55] . In addition to contributions to binding, these principles also explain why the core of a domain structure will remain largely conserved, while at functionally related places residues can (rapidly) co-evolve with an overall neutral effect [56] . Of course, these aspects of co-evolution are also of practical consequence for structure prediction and rational drug design [43] .
Through selective mutation, protein domains have been the tools of evolution to create an enormous and diverse assembly of proteins from likely an initially relatively limited set of domains. The combined data in GenBank and other databases now covers over 200.000 species with at least 50 complete genomes and this greatly facilitates genome comparisons [57] [58] [59] . Following such extensive comparisons, currently > 1700 domain superfamilies are recognized in the recent release of the Structural Classification of Proteins (SCOP) [60] and it has become clear that many proteins consist of more than one domain [17, 61, 62] . Indeed, it has been estimated that at least 70% of the domains is duplicated in prokaryotes, whereas this number may even be higher in eukaryotes, likely reaching up to 90% [35] .
There are various mechanisms through which protein domain or whole proteins may have been duplicated. On the largest scale, whole genome duplication such as those seen in the vertebrate genomes duplicated whole gene families, including postsynaptic proteins, hormone receptors and muscle proteins, and thereby dramatically increased the domain content and expanded networks [42, 63, 64] . On the other end of the scale, domains and proteins have been duplicated through genetic mechanisms like exon-shuffling, retrotranspositions, recombination and horizontal gene transfer [65] [66] [67] . Since the genetic forces, like exon-shuffling and genome duplication vary among species, the total number of domains and the types of domains present fluctuate per genome. Interestingly, comparative analyses of genomes have shown that the number of unique domains encoded in organisms is generally proportional to its genome size [60, 68] . Within genomes, the number of domains per gene, the socalled modularity, is related to genome size via a power-law, which is essentially the relation between the frequency f and an occurrence x raised by a scaling constant k (i.e., f (x) x k ) [69, 70] . A similar correlation is found when the multi-domain architecture is compared to the number of cell types that is present in an organism, i.e., the organism complexity or when the number of domains in a abundant superfamily is plotted against genome size (Fig. 2) [71, 72] .
Given the amount of domain duplication and apparent selection for specific multi-domain encoding genes in, for example, vertebrates, it may come as little surprise that not all domains have had the same tendency to recombine and distribute themselves over the genomes [68, 73] . In fact, some are highly abundant and can be found in many different multi-domain architectures, whereas others are abundant yet confined to a small sample of architectures or not abundant at all [68, 70] . Is there any significant correlation between the propensity to distribute and the functional roles domains have in cellular pathways? Some of the most abundant domains can be found in association with cellular signaling cascades and have been shown to accumulate non-linearly in relation to the overall number of domains encoded or the genome size [70] . Additionally, the on-set of the exponential expansion of the number of abundant and highly recombining domains has been linked to the appearance of multicellularity [70] . A reoccurring theme among these abundant domains is the function of protein-protein interaction and it appears that particularly these, usually globular domains, have been particularly selected for in more complex organisms [70] . This positive relation is underlined by the association of these abundant domains with disease such as cancer and gene essentiality as the highly interacting proteins that they are part of have central places in cascades and need to orchestrate a high number of molecular connections [74, 75] . Their shape and coding regions, which usually lie within the boundaries of one or two exons, make them ideally suited for such a selection, since domains are most frequently gained through insertions at the N-or C-terminus and through exon shuffling [76] [77] [78] .
From a mutational point of view, protein-protein interaction domains are different from other domains as well and this appears to be particularly true for the group of small, relatively promiscuous domains like SH3 and PDZ. These domains are promiscuous in the sense that they both tend to physically interact with a large number of ligands [79, 80] and are prone to move through the genome to recombine with many other domains. It has been found that particularly these domains evolve more slowly than non-promiscuous domains [70] . This likely stems from the fact that they are required to participate in many different interactions, which makes selection pressures more stringent and the appearance of the branches on phylogenetic trees relatively short and more difficult to assess when co-evolutionary data in terms of other domains in the same gene family or expression patterns is limited [42, 63] . Non-promiscuous domains on the other hand can quite easily evade the selection pressure by obtaining compensatory mutations either within themselves or their specific binding partner [70] .
The overall phenomenon that the number of protein domains and their modularity increases as the genome expands has not been linked to a conclusive biological explanation yet. A rationale for the increase in interactions and functional subunits, however, may derive from the paradoxical absence of correlation between the number of genes encoded and organism complexity, the so-called G-value paradox [81] . There is indeed evidence that domains involved in the same functional pathway tend to converge in a single protein sequence, which would make pathways more controllable and reliable without the need for supplementary genes [73] . Additionally, the number of different arrangements found in higher eukaryotes is, given the vast scale of unique domains present, relatively limited. This in turn implies that evolutionary constraints have played an important role in selecting the right domain combinations and the right order from N-to C-terminus in multi-domain proteins [13, 82] . In fact, the ordering and co-occurrence of domains was demonstrated to hold enough evolutionary information to construct a tree of life similar to those based on canonical sequence data [70] . Furthermore, the increased use of alternative splicing and exon skipping in higher eukaryotes likely supplied a novel way of proteome diversification by restricting gene duplication and stimulating the formation of multi-domain proteins [83, 84] . In plants, however, the latter notion is not supported since both mono-and dicots show limited alternative splicing and a more extensive polyploidy [85] [86] [87] .
It is clear that some of the above characteristics are underappreciated in the phylogenetic analysis of linear amino acid sequences. Moreover, the effects of evolution extend even further than these aspects and entail transcriptional and translational regulation, intramolecular domain-domain interactions, gene modifications and post-translational protein modifications [88] [89] [90] [91] [92] [93] [94] [95] [96] . New methods are thus being developed to take into account that when sequences evolve, their close and distant functional relationships evolve in parallel. Correlations of mutations have already been found between residues of different proteins [97, 98] and compensating mutational changes at an interaction interface were shown to recover the instability of a complex [99] . These observations are evidence for the current evolutionary models for the protein-protein interaction (PPIs) networks that are being constructed through large-scale screens [100] [101] [102] . In these, a gene duplication or domain duplication (depending on the resolution of the network) implies the addition of a node, while the deletion of a gene or domain reduces the amount of links in the network (Fig. 3) . In the next step, extensive network rewiring may take place, driven by the effect of node addition or node loss in the network (i.e., the duplicability or essentiality of a domain/protein) and mutations in the domain-interaction interface [67, 74, [103] [104] [105] .
Beyond mutations at the domain and protein level, regulation of protein expression provides another vital mechanism through which protein networks can evolve. Microarray studies are now well under way to map genome-wide ex-pression levels of related and non-related genes under a variety of conditions [91, [94] [95] [96] . For example, transcriptional comparisons have investigated aging [106] and pathogenicity [107] . Unfortunately, given the highly variable nature of gene expression and the fact that different species may respond different to external stimuli, such comparisons can only be performed under strictly controlled research conditions. To date most studies have therefore focused on the embryogenesis, metamorphosis, sex-dependency and mutation rates of subspecies [94, [108] [109] [110] [111] . Other studies have revealed valuable information on promoter types and duplication events [91] [92] [93] [94] .
To overcome the limitations mentioned in the previous paragraph, the analysis of co-expression data has been developed to supplement the direct comparison of individual gene expression changes [95] . In this procedure, a coexpression analysis of gene pairs within each species precedes the cross-comparison of the different organisms in the study. This approach thus primarily focuses on the similarity and differences of the orthologous genes within network, and is therefore ideally suited for the study of protein domain evolution and has already revealed that species-specific parts Fig. (3) . Evolutionary models for protein-protein interactions. The evolution of protein networks is tightly coupled to the addition or deletion of nodes. Additionally, events that introduce mutations in binding interfaces of proteins may result in the addition or loss of links in the network. Node addition may take place through e.g., domain duplication or horizontal gene transfer, while rewiring of the network is mediated by point mutations, alternative splice variants and changes in gene expression patterns.
of an expression network resulted via a merge of conserved and newly evolved modules [95, 112, 113] .
Finding evolutionary relationships protein domains is mostly based on orthology and thus commonly performed on best sequence matches. Identifying these and categorizing them depends largely on multiple sequence alignments and this will in most cases give good indications for function, fold and ultimately evolution. However, this approach usually discards apparent ambiguities that arise from speciesspecific variations (e.g., due to population size, metabolism or species-specific domain duplications or losses) and may therefore introduce significant biases [114] . Biases may also derive from the method of alignment, the rate variation model used to infer the phylogeny, and the sample size used to build the alignment [39, 40, 115] . Care should therefore be taken to not regard orthology as a one-to-one relationship, but as a family of homologous relations [91] , to select for appropriate analysis methods [39, 115] and extend comparative data to protein interactions and expression profiles [91] . Indeed, as our wealth of biological information expands, our systems perspective will improve and provide us with an opportunity to reveal protein domain evolution at the level network organization and dynamics. Large-scale expression studies are beginning to show us evolutionary correlations between gene expression levels and timings [94, 106, 107, 112, 116] , while others demonstrate spatial differences between paralogs or (partial) overlap between interaction partners [117] [118] [119] [120] . Indeed, when we are able to map the spatiotemporal aspects of inter-and intra-molecular interactions we will begin to fully understand the versatile power of evolution that shaped the protein universe and life on Earth [118] .  Quantitative Phosphoproteomics of Proteasome Inhibition in Multiple Myeloma Cells BACKGROUND: The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC) in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib. CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells. The ubiquitin-proteasome pathway is responsible for proteolysis of eukaryotic cellular proteins related to cell cycle regulation, cell survival, and apoptosis [1] . Inhibition of proteasome activity is a novel therapeutic strategy against cancer cells. Bortezomib (formerly known as PS-341), a cell-permeable boronic acid dipeptide, is a specific inhibitor of the proteasome pathway [2] and received Food and Drug Administration (FDA) approval for the treatment of MM and mantle cell lymphoma [3] .
Bortezomib has been reported to trigger pleiotropic signaling pathways in MM cells, including: (a) stabilizing cytoplasmic IkB and blocking NFkB nuclear translocation [4] ; (b) activation of stress response proteins such as heat shock proteins Hsp27, Hsp70, and Hsp90 [5] ; (c) up-regulation of c-jun NH2-terminal kinase [6] ; (d) induction of intrinsic cell death pathway [7] ; (e) activation of extrinsic apoptotic signaling through Bid and caspase-8 cleavage [8] ; (f) impairment of DNA repair machinery via inactivation of DNA-dependent protein kinase [9] ; (g) down-regulation of mitogen-activated protein kinase and phosphatidylinositol 3kinase/Akt signaling pathways [10] ; and (h) down-regulation of the p44/42 MAPK signaling cascade [11] . All these signaling events may collectively contribute towards the overall anti-MM activity of bortezomib. However, the exact number and identity of cellular signaling events involved in proteasome inhibition and the mechanisms underlying the associated apoptotic response in MM cells remain to be elucidated.
Elucidation of cellular signaling networks requires methodologies for large-scale quantitative phosphoproteomic analysis that can reveal dynamic system-wide changes in protein phosphorylation. Recent technological advances in mass spectrometry-based proteomics have enabled us to make a large-scale identification of signaling molecules through the enrichment of phosphorylated proteins or peptides [12, 13] . One of the most widely used and well known strategies currently used in phosphoproteomic studies is stable-isotope labelling by amino acids in cell culture (SILAC). Although introduced relatively recently, SILAC has been used extensively in the proteomics community [14] . With SILAC, the entire proteome of a given cell population is metabolically labeled by heavy, non-radioactive isotopic variants of amino acids, thus making it distinguishable by MS analysis [15, 16] . Thereafter, two or more distinctly SILAC-labeled cell populations can be mixed and analyzed in one MS experiment, allowing accurate quantization of proteins from the different cellular states. By coupling with a phosphoprotein or phosphopeptide enrichment method, such as titanium dioxide (TiO 2 ) [17] , strong cation exchange (SCX) [18] , or the two in combination [19] , SILAC has been widely applied to profile dynamic phosphorylation changes in signal transduction [20, 21] .
In this study, we investigated the differential MM phosphoproteome upon proteasome inhibition by using SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. Many potential novel signaling proteins and associated signaling pathways were confidently identified. Our further functional results indicated that perturbations in stathmin phosphorylation play a significant functional role in mediating apoptosis in MM cells exposed to bortezomib and the bortezomib-induced changes in the MT stabilization can be attributed to the bortezomibinduced phosphorylation of stathmin. By correlating the phosphoproteomic data with functional studies, the current results provided novel insights into the mechanisms of bortezomib actions in MM cells.
To obtain a global view of the changes of protein phosphorylation in bortezomib-treated myeloma cells, we compared the phosphoproteome of U266 cells treated with or without bortezomib. The workflow is outlined in Figure 1 . Cells in normal medium (light culture) were treated with bortezomib, and cells grown in medium containing stable isotopes (heavy culture) were treated with vehicle. These two populations of cells were lysed, mixed at a 1:1 ratio, and subjected to TiO 2 purifications followed by LC-MS/MS analysis. After LC-MS/MS analysis on the enriched phosphopeptides, all MS/MS spectra were searched, respectively, against the forward and reversed human protein sequence databases to estimate rates of false-positive matches. Search results were filtered based on peptide score of MASCOT and PTM score. In total 1024 phosphopeptides (redundant) from the target database passed our criteria, allowing 14 decoy matches. The phosphopeptide false-positive rate was therefore estimated to be 1.4%. Multiple filtering criteria were established to validate search results. For each of the phosphorylated peptides identified in this work, peptide sequences were manually confirmed. After validation we identified 418 unique phosphorylation sites from 244 unique phosphopeptides corresponding to 233 protein groups. This entire dataset is provided as Table S1 in which hyperlinks are built up to view all the MS/MS spectra. Using the PTM score, we could localize the phosphor groups with high confidence (Class I phosphorylation sites) in 197 cases (Table S1), indicating that the phosphorylation sites detected by current strategy are of high confidence. Figure 2A shows a representative MS/MS spectrum for a phosphosite-containing peptide in the detection, and all other MS/MS spectra are available via the hyperlinks in the Table S1 .
To quantify the phosphorylation change for each phosphopeptide, we used the MSQuant software to calculate area ratio, defined as the ratio of the ''heavy'' peak area over the ''light'' peak area in the chromatogram (Fig. 2B ). All together, we have achieved quantification of 259 unique phosphorylation sites from 154 unique phosphopeptides corresponding to 132 protein groups. Based on a predefined threshold of 1.5-fold change, 131 phosphosites from 75 unique phosphopeptides corresponding to 72 proteins showed a 1.5-fold or greater change as listed in Table  S2 . In other words, 31% of the total phosphosites detected showed significant alteration after bortezomib treatment, suggesting that dysregulated phosphorylation may play an important role in bortezomib-induced apoptosis.
To further confirm the results from the quantitative phosphoproteome analysis, we chose stathmin for Western blotting verification using an anti-phospho-Ser38 stathmin antibody. As shown in Figure 2C , SILAC results were very much consistent with Western blotting analysis for this protein. Upon bortezomib treatment, phosphorylation of stathmin at Ser38 increased, whereas steady-state stathmin remained almost unchanged in Western blotting verification.
To better characterize bortezomib-regulated phosphoproteins, we classified all the differentially expressed phosphoproteins (DEPPs) into 23 functional categories according to the PANTHER system. These proteins are implicated in a broad range of cellular activities (Fig. 3A ). Next to the unclassified proteins (17%), proteins involved in nucleic acid binding account for the second largest portion (14%). There are also a significant number of proteins involved in receptor (10%), regulatory molecule (6%), and kinase (5%). Previous studies indicated that bortezomib exerts its anticancer function by inhibiting protein degradation in cancer cells [22] . In this connection, many bortezomib-regulated phosphoproteins found in current study were nucleic acid binding (14%) and transcription factors (4%), as shown in Figure 3A . These data suggest that the cancer-inhibitory effect of bortezomib may also rely on its regulatory role in mRNA transcription. Table S2 only shows a list of all borterzomib regulated phosphoproteins in MM cells. It lacks the biochemical context. To create significance out of otherwise static proteomic data, we constructed an interaction networking of the DEPPs (Fig. 3B) . The DEPPs were linked by various evidences based on neighborhood analysis, experimental results or text-mining. These relationships are color coded, and the legends are provided next to the map. This does not mean that all the interactions took place within a single spatial and temporal situation, but it enables the identification of central nodes in these DEPPs. Notably, stathmin was a hub in this network, suggesting that stathmin may play an important role in mediating bortezomib-induced apoptosis in MM cells. Some proteins remained orphans because there is insufficient information in the database to link them to other proteins in the network.
To further investigate the reliability of the results, PhosphoSite (http://www.Phosphosite.org) was used to distinguish known phosphorylation sites from novel phosphorylation sites. Among the identified 418 phosphorylated sites, 51% were also reported by others previously (for details, see Table S3 ). In other words, many of the phosphorylation sites were also determined by researchers with other cancer cells, further demonstrating that the phosphorylation sites detected by current strategy are reliable.
To predict the kinase substrate relationships from the dataset, the computer algorithm SCANSITE was used. SCANSITE makes use of peptide library phosphorylation data to predict substrates recognized by specific kinases. Tables S4 & S5 show the results of phosphopeptides that were identified in this study and were predicted to be associated with a kinase binding motif by SCANSITE at the different stringency levels. It was found that most of the phosphorylation sites determined in this study were phosphorylated by acidophilic serine/threonine kinase and proline-dependent serine/threonine kinase. Notably, we found fifteen sites that can be phosphorylated by the Casein Kinase 2 (CK2). CK2 is a constitutively active protein kinase implicated in cellular transformation and the development of tumorigenesis [23] . Aberrantly active in MM cells, CK2 controls the cell survival [24] . Our SCANSITE analysis suggests that CK2 may play an important role in bortezomib-induced apoptosis and may represent a potential target in MM therapy.
SILAC phosphoproteomic analyses and Western blotting revealed an increase of phosphorylation of stathmin at Ser38 and the unchanged steady-state stathmin in U266 cells upon proteasome inhibition (Fig. 2 ). It has been reported that the functional alteration of stathmin resulting from specific phosphorylation events may be involved in the process of apoptosis induced by proteasome inhibitors in proliferating cells [25] . To elucidate the differential phosphorylation of stathmin isoforms, Western blotting was performed to analyze the phosphorylation pattern of stathmin in bortezomib-treated U266 cells using stathmin antibody and specific phospho-antibodies against three known phospho-sites (Ser16, Ser25, Ser38) [26] . As shown in Figure 4A , in accordance with SILAC results, phosphorylation of stathmin at Ser38 was increased whereas steady-state stathmin remained unchanged. Furthermore, phosphorylation of stathmin at Ser16 was increased whereas Ser25 was decreased after bortezomib treatment (Fig. 4A ).
It has also been reported that Ser16 is a target for calmodulindependent protein kinases (CamKII), Ser25 is specifically phosphorylated by mitogen-activated protein (MAP) kinase, and Ser38 is a target for cycline-dependent kinase-2 (CDK2) [26] . To test the activation status of upstream kinases catalyzing the incorporation of phosphoryl groups to each of these residues, specific antibodies against active forms of CaMKII, MAPK and CDK2 (responsible for the stathmin phosphorylation on Ser16, Ser25 and Ser38, respectively) were used. In accordance with the increase in p-Ser16 and p-Ser38 of stathmin in bortezomib-treated cells, a parallel activation of CamKII and CDK2 was detected after bortezomib treatment ( Fig. 4B ). At the same time, the decrease in phosphorylation levels of Ser25 was correlated with the inactivation of MAPK, a critical kinase for cell survival.
Hence, these results suggest that the regulation of the phosphorylation profile of stathmin at the level of residues Ser16, Ser25, and Ser38 may participate in the response of myeloma cells to proteasome inhibitors, and that stathmin is a target for multiple protein kinases, which are regulated by multiple signal transduction cascades.
The functional significance of stathmin phosphorylation in the response of MM cells to bortezomib was then examined more rigorously. To this end, stable U266 cell clones overexpressing the stathmin wild-type (U266-WT) or mutants (U266-S16A, U266-S25A and U266-S38A) were generated using His-tagged constructs. Figure 5A shows the expression of His-tagged target proteins as well as stathmin expression in these cells. The complete methods and characterization in terms of proliferation, cell cycle, colony-forming efficiency (CFE) and apoptotic ratio of these cells are described in the Supplemental Data S1. We observed that all these cells had a comparable growth pattern and CFE, similar proportions of cells in G1, S, and G2 plus M phases and similar apoptotic ratio (Table S6) . However, there were significant differences between these cells with regard to their sensitivity to bortezomib treatment. As shown in Figure 5B , cells transfected with wild-type stathmin (U266-WT) exhibited significant increase in bortezomib-induced cell death compared with parental cells (U266). In contrast, cells transfected with mutant stathmin (U266-S16A, U266-S25A or U266-S38A) were significantly less sensitive to bortezomib lethality than U266 cells (P,0.05 for U266-S16A and U266-S38A or P,0.01 for U266-S25A, respectively) (Fig. 5B) .
It has been reported that phosphorylation turns off the microtubule destabilizing activity of stathmin [27, 28] and that proteasome inhibitors increase tubulin polymerization and stabilization in myeloma cells [29] . We thus tested whether bortezomib actually induced changes in the polymerization status of the MTs in these cells. Indeed, when these cells were treated with bortezomib for 24 hours, we observed an increase in the amount of tubulin in the polymerized 'P' fraction as compared with untreated cells (Fig. 5C ). The baseline proportion of a-tubulin in the polymerized fraction ranged from ,43% to 52%, while the polymerized proportion observed after bortezomib treatment was ,60-90% (Table 1) .
To investigate whether these bortezomib-induced changes in the tubulin polymerization were mediated by phosphorylation of stathmin, we examined the tubulin polymerization in stable U266 clones that overexpressing WT stathmin and the phosphorylation site-deficient stathmin mutants S16A, S25A and S38A. As shown in Figure 5C , by comparing with U266 cells, overexpression of WT stathmin and phosphorylation site-deficient mutants resulted in a significant decrease in the percent of polymerized tubulin following treatment with bortezomib ( Fig. 5C and Table 1) .
Thus, our findings support the notion that bortezomib induces tubulin polymerization and stabilization through the mediation by phosphorylation of stathmin and this may be a contribution factor to the mechanism of proteasome inhibition and toxicity in MM cells. 
Quantitative phosphoproteomic approaches offer great promise for rapid progress in the analysis of drug targets or mechanism of action, especially when combined with traditional biochemical approaches presently used for studying individual proteins [30] . Moreover, the ability to simultaneously measure changes in phosphorylation state of many proteins in a single experiment can provide unique information needed for quantitative modeling of signaling pathways.
In the present study, we used a combined strategy that comprised phosphoprotein enrichment, SILAC, and LC/MS analysis to profile the differential phosphoproteome in bortezomib-treated MM cells. A total of 72 phosphoproteins were found to have a 1.5-fold or greater change upon bortezomib treatment (Table S2 ). According to their change pattern, these DEPPs can be categorized into up-(,0.75) or down-(.1.5) regulated groups (Table S2) . Notably, in comparison with the number of downregulated DEPPs, many more proteins are up-regulated in this process (70 versus 5). This unbalanced pattern has also been revealed by previous in vitro biochemical studies [22] . Many studies have demonstrated that bortezomib can trigger pleiotropic signaling pathways and suppress the proteasomal degradation of multiple phosphorylated singnaling molecules [8, 10, 31] . Therefore, we speculate that the up-regulation of phosporylation of these DEPPs may constitute one of the mechanisms of bortezomibinduced apoptosis in MM cells. The PANTHER classification system also revealed that the DEPPs implicated in a variety of molecular functions, such as nucleic acid binding, receptor, regulatory molecule and so on (Fig. 3A) , clearly showing that in addition to targeting proteins involved in apoptotic pathways, bortezomib also altered multiple signaling pathways to induce its anti-cancer effects.
In particular, as suggested by protein network analysis (Fig. 3B) , stathmin may play a central role in mediating bortezomib-induced apoptosis in MM cells. Stathmin (also termed as p19, 19K, p18, prosolin, and Op18) is a ubiquitous 19 kDa cytosolic phosphoprotein that is highly expressed in a wide variety of cancers, including a subset of leukemias and breast carcinomas and is a key regulator in the control of proliferation and cell cycle [32, 33] . Stathmin is a phosphorylation responsive regulator of microtubule (MT) dynamics that increases the catastrophe rate of MTs (depolymerization or shrinkage phase of individual MTs) in a dose-dependent manner [34] . Four Ser residues, Ser16, Ser25, Ser38, and Ser63 in stathmin are subjects for phosphorylation in intact cells [26] .
The regulation of stathmin phosphorylation is complex and, in all likelihood, multifactorial. For example, phosphorylations at all four Ser residues fluctuate during the cell cycle and CDK2 has been identified as the kinase system involved in cell cycle-regulated phosphorylation of Ser38. Besides, three distinct protein kinases have been identified to phosphorylate stathmin in response to external signals. These kinases are members of the MAPK family that phosphorylates Ser25, cyclic AMP-dependent protein kinase (PKA) that phosphorylates Ser63 and CamKII that phosphorylates Ser16 [26] . Because the kinases acting at these residues are distinct and may be functioning through different pathways, it is reasonable to speculate that regulation of stathmin phosphorylation can be achieved by multiple pathways, thus providing the cell with a finely tunable mechanism for controlling microtubule assembly and dynamics in relation to its needs.
In the present study, the role of stathmin and its phosphorylation in bortezomib-induced cell death was further investigated by overexpression of the WT stathmin and phosphorylation site-deficient stathmin mutants S16A, S25A or S38A in myeloma cells. Overexpression of WT stathmin significantly increased bortezomib-induced cell death. On the contrary, overexpression of the phosphorylation site-deficient stathmin mutants S16A, S25A and S38A significantly decreased, but did not block, bortezomibinduced cell death. Importantly, increased levels of tubulin polymerization were observed upon bortezomib treatment in cells overexpressing WT or mutant stathmin, but to a lesser extent than parental U266 cells (Fig. 5C, Table 1 ). There are several possible explanations as to why bortezomib-induced cell death is not completely blocked by the stathmin mutants. First, endogenous WT stathmin is still present, and thus the mutant protein has to compete with the WT protein. Second, bortezomib can also modulate the activity of other MT regulatory proteins that may also contribute to the cell death [29] . Furthermore, the MT system is not the only event involved in bortezomib-induced cell death in myeloma cells [22, 31] . This suggests that bortezomibinduced cell death is the result of a concerted series of events.
Therefore, we conclude that bortezomib-induced phosphorylation of stathmin promotes cell death and that phosphorylation on Ser16, Ser25 and Ser38 is necessary for this process. Furthermore, the bortezomib-induced changes in the MT stabilization can be attributed to the bortezomib-induced phosphorylation of stathmin, and MT stabilization is in fact responsible for the bortezomibinduced cell death-promoting activity of phosphorylated stathmin.
Another protein of interest uncovered in this study is BCL2associated athanogene 3 (BAG3). In the current study, BAG3 was found to have increased phosphorylation at Ser377 upon bortezomib treatment. BAG3 belongs to the evolutionarily conserved BAG family of proteins that were originally isolated based on their ability to interact with the anti-apoptotic protein Bcl-2 [35, 36] . It is involved in a wide variety of cellular processes, including cell survival, cellular stress response, apoptosis and virus replication [16, 37, 38] . Recent evidence implicates an additional function of BAG3 in the regulation of the autophagy pathway. These findings indicate that autophagosome formation and turnover may depend on BAG3 and that BAG3 can stimulate autophagy processes [39, 40] . Autophagy is a major intracellular degradation system. Unlike the ubiquitin-proteasome system (UPS), autophagy is mainly responsible for the degradation of long-lived proteins and subcellular organelles [41, 42] . Autophagy plays important roles in development, cellular homeostasis and cell survival and is frequently activated in tumor cells exposed to chemotherapy or radiation and confers therapeutic resistance [43, 44] . The UPS and autophagy have been viewed as distinct degradation systems, but recent studies suggested that they are functionally coupled and that suppression of the proteasome promotes autophagy [45, 46] . However, the functional connection and the inter-regulation between the two systems are not well understood.
Importantly, Zhu et al. has shown that proteasome inhibition activates autophagy through a phosphorylation of eIF2a-dependent mechanism to eliminate protein aggregates and alleviate proteotoxic stress [46] . However, their results also demonstrated that complicated mechanisms are involved in proteasome inhibition-mediated autophagy activation and the phosphorylation of eIF2a only partially accounts for this activation [46] . Thus, it is possible that the complexity of the process may be much higher than presently envisaged, and that other proteins may be as important in controlling autophagy activation as eIF2a.
Based on the critical role of BAG3 in the stimulation of the autophagy pathway, it is tempting to suggest that bortezomibinduced phosphorylation of BAG3 might play an important role in autophagy activation. Therefore, the increase in BAG3 phosphor-ylation is likely part of the cell's response to bortezomib treatment and appears to represent a novel mechanism with a link between the two protein degradation systems. This speculative idea, however, is not yet supported by the current experimental data, and further investigations are undergoing to determine the functional implication of BAG3 in coordination between the proteasome and autophagy.
In summary, we have, for the first time, performed quantitative phosphoproteomics to study the effects of bortezomib on MM cells. The current results expand the list of bortezomib-targeted phosphoproteins and their phosphorylation sites. Especially, our functional studies indicated that bortezomib-induced phosphorylation of stathmin promotes cell death and that the bortezomibinduced changes in the MT stabilization can be attributed to the bortezomib-induced phosphorylation of stathmin. Our comprehensive study of phosphorylation regulation in proteasome inhibition in MM cells may serve as a valuable resource for future research in the field and thus advance the general mechanistic understanding of bortezomib in MM.
The human myeloma cell line U266 was purchased from American Type Culture Collections (Rockville, MD). Myeloma cells were routinely maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1 mmol/L L-glutamine, and 10% fetal bovine serum at 37uC, 5% CO 2 in air. Bortezomib was provided by Millennium Pharmaceuticals (Cambridge, MA). To differentially label bortezomib-treated and -untreated U266 cells, the SILAC Protein Quantitation Kit (Pierce Biotechnology, Rockford, USA) was used according to the manufacturer's instruction. In brief, cells were grown in SILAC RPMI 1640 Medium (Pierce Biotechnology, Rockford, USA) containing 10% v/v dialyzed FBS, and either 0.1 mg/mL heavy [ 13 C 6 ] or light [ 12 C 6 ] L-lysine (Pierce Biotechnology, Rockford, USA). To ensure full incorporation of the heavy and light labeled amino acids, cells were grown for at least six cell doublings prior to analysis. U266 cells were treated with 3 nM bortezomib for 24 h, according to the half-maximal inhibitory concentration (IC50) measured by Hideshima et al [11] . After treatment, cells were washed three times with ice-cold washing buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.0) and transferred to a clean 1.5 mL Eppendorf tube. Cells were lysed with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0). Cellular debris was removed by centrifugation for 30 min at 13, 200 g and at 4uC. Protein concentrations were measured in duplicate using RC DC protein assay (BioRad, Hercules, CA, USA) and confirmed by SDS-PAGE.
Phosphopeptide enrichment was performed as previously described [17] . Briefly, equal amounts of proteins from untreated ( 13 C 6 -lysine) and bortezomib-treated ( 12 C 6 -lysine) U266 cells were mixed (1 mg in total) and subjected to disulfide reduction with 10 mM DTT (37uC, 3 h) and alkylation with 20 mM iodoacetamide (room temperature, 1 h in dark). The protein mixtures were mixed with four volumes of ice-cold acetone to precipitate proteins. Precipitated proteins were collected by centrifugation and washed with ethanol two times. The pellet was re-dissolved in 50 mM ammonium bicarbonate and then digested with sequencing grade modified trypsin (1:25 w/w) (Promega, Madison, WI) at 37uC for 20 h and then quenched by addition of TFA to a final concentration of 0.5%. The digests were evaporated to about 20 mL in SpeedVac centrifuge. The phosphopeptides from digested peptides were enriched by using Phosphopeptide Enrichment TiO 2 Kit (Calbiochem, San Diego, CA) according to the manufacturer's instruction with slight modifications. Briefly, the tryptic digest was dried, re-dissolved in 200 mL TiO 2 Phosphobind buffer containing 50 g/L 2,5-dihydroxybenzoic acid and then mixed with 50 mL TiO 2 Phosphobind Resin. After 30 min incubation, the supernatant was discarded, and TiO 2 was washed three times with the wash buffer. After that, 30 mL elution buffer was added two times to elute the phosphopeptides. The elutions were combined and acidified with 5 mL of 10% formic acid for SCX-LC-MS/MS analysis. All the buffers and the phosphopeptides purification resin were provided in the kit by the manufacturer.
The enriched phosphopeptides were analyzed with a Finnigan Surveyor HPLC system coupled online with a LTQ-Oribitrap XL (Thermo Fisher Scientific, Waltham, MA) equipped with a nanospray source. The phosphopeptides were firstly loaded on a strong cation exchange (SCX) column using an autosampler, and the peptides were eluted by NH 4 Cl with different concentrations (1 mM, 10 mM, 100 mM, 200 mM, 1 M). Then, each fraction peptide was respectively loaded onto a C18 column (100 mm i.d., 10 cm long, 5 mm resin from Michrom Bioresources, Auburn, CA) using an autosampler. Peptides were eluted during a 0-35% gradient (Buffer A, 0.1% formic acid, and 5% ACN; Buffer B, 0.1% formic acid and 95% AcN) over 90 min and online detected in LTQ-Orbitrap using a data-dependent method [47] . The general mass spectrometric conditions were: spray voltage, 1.80 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 200uC. Ion selection thresholds were: 1000 counts for MS 2 and 500 counts for MS 3 . An activation q = 0.25 and activation time of 30 ms were applied in MS 2 acquisitions. The mass spectrometers were operated in positive ion mode, employing a data-dependent automatic switch between MS and MS 2 acquisition modes. For each cycle, one full MS scan in the Orbitrap at 1610 6 AGC target was followed by five MS 2 in the LTQ at 5000 AGC target on the five most intense ions. Selected ions were excluded from further selection for 90 s. Maximum ion accumulation time was 500 ms for full MS scans and 100 ms for MS 2 scans. All MS/MS spectra were collected using normalized collision energy (a setting of 35%), an isolation window of 3 m/z, and 1 micro-scan. The resolution used in the MS step in the Orbitrap is 60000. An extra DDNL (data-dependent neutral loss) MS 3 method was applied for phosphopeptide detection [17] . MS 3 was triggered if a neutral loss peak at 298.0, 249.0, 232.7 or 224.5 Da was observed in the MS 2 and that peak was one of the three most intense ions of the MS 2 spectra. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by XCalibur data system (Thermo Fisher Scientific, Waltham, MA).
Peak lists for the database search were produced in the Mascot generic format using BioWorks 3.3.1 (Thermo Finnigan, San Jose, CA) and DTASuperCharge V 1.31 (SourceForge), and the derived peak lists were searched using the Mascot 2.2.04 search engine (Matrix Science, London, UK) against a real and false IPI human database (V3.56, including 153, 078 protein entries). The following search criteria were employed: full tryptic specificity was required; two missed cleavages were allowed; Carbamido-methylation was set as fixed modification, whereas Oxidation (M), Phospho (ST), and Phospho (Y) were considered as variable modifications. Precursor ion mass tolerances were 10 ppm for all MS acquired in the Orbitrap mass analyzer, fragment ion mass tolerance was 0.5 Da for all MS 2 spectra acquired in the LTQ. Mass spectra of identified phosphopeptides with peptide score .10 were further processed and validated with the MSQuant 1.5 software for post-translational modification (PTM) score analysis [48] . Two filters criteria for phosphopeptide identification were applied: 1) Peptide score threshold was 17; 2) The total threshold of PTM score and peptide score was 36. All fragmentation spectra were manually verified using the criteria as described by Mann et al [48] . For phosphopeptides with multiple potential phosphorylation sites, the probabilities for phosphorylation at each site were calculated from the PTM scores as described [48] . Phosphorylation sites that were occupied with probability .0.75 were reported as class I phosphorylation sites. For class II sites, localization probability was between 0.75 and 0.25. Phosphorylation sites with localization probability ,0.25 were discarded. The identified phosphopeptides were further processed with MSQuant 1.5 for statistics evaluation as well as quantization.
Differentially expressed phosphoproteins (DEPPs) were classified based on the PANTHER (Protein ANalysis THrough Evolutionary Relationships) system (http://www.pantherdb.org), which is a unique resource that classifies genes and proteins by their functions [49] . The DEPP interaction network was build automatically by the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) system with default setting except that organism, confidence(score), and additional (white) nodes were set to ''human'', ''0.20'', and ''10'', respectively [50, 51] . The gene name list of these proteins was input to search against the database which contains known and predicted protein-protein interactions. The retrieve included a detailed network which highlights several hub proteins. The identified phosphoproteins were compared to the public database of PhosphoSite (http://www.phosphosite.org/) to find out the novel phosphoproteins and phosphosites. Each confirmed phosphoprotein was searched with SCANSITE (http:// scansite.mit.edu) [52] for potential kinase motifs with high, medium, and low stringency.
Protein extracts (30 mg) prepared with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0) were resolved by a 10% SDS-PAGE gel, and transferred onto Immobilon-P PVDF transfer mem-braneS (Millipore, Bedford, MA) by electroblotting. After blocking with 5% non-fat milk, the membranes were probed with rabbit anti-CaMKII polyclonal, rabbit anti-phospho-CaMKII (Thr286) polyclonal, goat anti-actin polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CDK2 polyclonal, rabbit anti-phospho-CDK2 (Thr160) polyclonal, rabbit antiphospho-stathmin (Ser25) polyclonal antibodies (Abcam Inc., Cambridge, MA), rabbit anti-His-tag polyclonal, rabbit antistathmin polyclonal, rabbit anti-phospho-stathmin (Ser38) polyclonal, rabbit anti-phospho-stathmin (Ser16) polyclonal, rabbit anti-p44/42 MAPK polyclonal, mouse anti-phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibodies (Cell Signaling, Danvers, MA), and mouse anti a-tubulin monoclonal antibodies (DM1A, Sigma, St. Louis, MO). Blots were then incubated with peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG (KPL, Gaithersburg, Maryland) for 1 h at room temperature at a 1:2000 dilution and then developed by using the SuperSignal West Pico Kit (Pierce Biotechnology, Rockford, IL).
Human stathmin was cloned into the NH 2 terminal His-tagged pReceiver-M01 expression vector (Genecopoeia, Rockville, MD). Construction of mutant stathmin cDNAs, where the codons for Ser-16, Ser-25 or Ser-38 are exchanged to Ala, was performed by site-directed mutagenesis using a QuikChange kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Primers used, with the introduced mutations underlined, were: (Ser16RAla) 59-AGCGTGCCGCAGGCCAG-39; (Ser25RAla) 59-GCTGATT-CTCGCCCCTCGGTC-39; (Ser38RAla) 59-CCCCCTTGCC-CCTCCAAAG-39. All mutant constructs were confirmed by DNA sequence analysis.
The plasmids were introduced into U266 cells using the X005 mode of Nucleofector (Amaxa, Cologne, Germany), according to the Optimized Protocol for the U266B1 cell line. Electroporated cells were cultured in medium with the presence of 0.5 mg/ml G418 (Mediatech, Manassas, VA) for 14 days, and then cultured in the 96-well plates for dilution cloning. Finally, the clone selected from pReceiver-M01 blank vector transfected U266 cells was designated as U266-NC. The clones selected from wild type or mutant stathmin plasmids transfected U266 cells were designated as U266-WT, U266-S16A, U266-S25A or U266-S38A, respectively.
The extent of apoptosis was evaluated by using Annexin V/PI staining and flow cytometry as described previously [53] . In brief, 1610 6 cells were washed once in 16PBS and were stained with Annexin V-FITC and PI (2 mg/mL) according to manufacturer's instructions. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed with the WinMDI 2.8 software program.
Tubulin polymerization assay was performed essentially as previously described [29, 54] . Briefly, cells grown to confluency in 24-well plates were washed twice with 1X PBS. To separate polymerized (P) from soluble (S) tubulin, the cells were all incubated at 37uC for 5 min in the dark in hypotonic lysis buffer containing 5 mM paclitaxel, 10 mM Trichostatin-A (Calbiochem, San Diego, CA), 1 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 200 units/ml aprotinin, 100 mg/ml soybean trypsin inhibitor, 5.0 mM e-amino caproic acid, 1 mM benzamidine, and 20 mM Tris-HCl, pH 6.8, vortexed vigorously and centrifuged at ,15,000 g at 22uC for 10 minutes. The supernatants containing soluble 'S' tubulin were transferred to another Eppendorf tube separating them from the pellets containing polymerized 'P' tubulin. Upon separation, tubes were placed on ice and pellets of polymerized 'P' tubulin were resuspended by sonication for 10-20 seconds in a volume of lysis buffer equal to the soluble 'S' fraction. Each had gel sample buffer added, equal aliquots were separated by 10% SDS-PAGE, and western blots using anti-a-tubulin antibody were obtained. The immunoblots were scanned, and densitometric analysis was performed using the public domain NIH Image program ImageJ (available on the Internet at http://rsb.info.nih.gov/nih-image/). The percentage of polymerized 'P' tubulin was determined by dividing the densitometry value of polymerized 'P' tubulin by the total tubulin content (the sum of the densitometry values of soluble 'S' and polymerized 'P' tubulin). An advantage of this assay is that the amount of total protein loaded for each sample is irrelevant since the 'P' and 'S' fractions are equalized for each pair, and it is the proportion of the polymerized to the soluble tubulin fraction that is measured.
All data are expressed as mean 6 standard deviation. Statistical significance was determined by Student's t-test (two-tailed), while the significance of the differences was determined using the twotailed Mann-Whitney test. Statistical significance was assigned if P,0.05.
Data S1
Found at: doi:10.1371/journal.pone.0013095.s001 (0.03 MB DOC) Author Contributions Insights into the Evolution and Emergence of a Novel Infectious Disease Many zoonotic, novel infectious diseases in humans appear as sporadic infections with spatially and temporally restricted outbreaks, as seen with influenza A(H5N1). Adaptation is often a key factor for successfully establishing sustained human-to-human transmission. Here we use simple mathematical models to describe different adaptation scenarios with particular reference to spatial heterogeneity within the human population. We present analytical expressions for the probability of emergence per introduction, as well as the waiting time to a successful emergence event. Furthermore, we derive general analytical results for the statistical properties of emergence events, including the probability distribution of outbreak sizes. We compare our analytical results with a stochastic model, which has previously been studied computationally. Our results suggest that, for typical connection strengths between communities, spatial heterogeneity has only a weak effect on outbreak size distributions, and on the risk of emergence per introduction. For example, if [Image: see text] or larger, any village connected to a large city by just ten commuters a day is, effectively, just a part of the city when considering the chances of emergence and the outbreak size distribution. We present empirical data on commuting patterns and show that the vast majority of communities for which such data are available are at least this well interconnected. For plausible parameter ranges, the effects of spatial heterogeneity are likely to be dominated by the evolutionary biology of host adaptation. We conclude by discussing implications for surveillance and control of emerging infections. Zoonotic emergence of novel human infections poses a significant risk to global public health. For example, the 'Spanish flu' pandemic of 1918 probably originated in birds and caused millions of deaths worldwide [1] . While much less virulent, the subsequent influenza pandemics of 1957, 1968 and 2009 [2, 3] are potent reminders of the capacity of the influenza virus to cross the species barrier into humans. Many other pathogens share this capacity: the SARS outbreak of 2003 [4] [5] [6] has been linked to bats and palm civets [7, 8] . In 2008, a novel arenavirus which killed four out of five patients in South Africa was linked to rodents [9] .
Previous work [10, 11] has studied models of within-host evolution and between-host transmission, in which an initially poorly transmitting pathogen acquires adaptations to human hosts, following repeated zoonotic introductions until it achieves pandemic potential. These make the natural, simplifying assumption that the host population is homogeneous, so that changes in infection parameters entirely reflect adaptations in the biology of host infection. In reality, however, factors such as human contact patterns [12] and other host heterogeneity [13, 14] may also shape the risk and speed of emergence events. We concentrate here on the heterogeneity in the spatial structure of the human host population, an area which has hitherto received little attention in the context of adapting pathogens. We model spatially separated communities with varying types and strengths of interconnections, for example between a village and a city. Our aim is to study under what regimes such 'ecological' structure could have a strong effect on emergence, in comparison with 'evolutionary' factors governing the biology of infection.
In the following section we give an overview of the modelling approach. We then present new analytical results for the simple models studied previously, which ignored spatial host population structure. We use these expressions to provide useful answers to important questions: if we knew how a zoonotic pathogen would adapt to human physiology, could we anticipate its emergence? How reliable would such predictions be? Furthermore, can we predict which zoonoses will cause outbreaks which do not turn into epidemics? Next, we ask: how large does a single, finite host population have to be, for population size to have a negligible effect? We then incorporate spatial heterogeneity by separating the human host population into communities. We present a model in which a small village is connected, by human travel, with a large city as an example of the general case of two interconnected communities. We use this model to ask: how strong do community interconnections have to be for us to safely ignore the separation of a population into spatially structured communities, such as cities and villages? We review available commuting data to ask how these thresholds compare with typical human mobility patterns? We close with a discussion of implications for public health.
We build on a model of evolution and emergence originally presented by [10] in which a zoonotic pathogen infects humans, and initially has very poor onward transmissability. Thus for people who are infected by animals the average reproductive number, R 0 , is well below one (R 0 %1). We call this the first reproductive number a 1 for the wildtype strain. Occasionally, during such zoonotic infections, the pathogen acquires genetic changes that increase its ability to pass to other humans. During any chain of transmission the pathogen might adapt sufficiently that it achieves such ease of human-to-human transmission that R 0 w1 and an epidemic becomes possible. Such a process can be characterised by a vector of reproductive numbers (a i with i~1, . . . , n), and a vector of mutation probabilities (m i with i~1, . . . , n) where n{1 denotes the number of adaptive steps necessary to reach the fully adapted strain n.
In what follows we restrict our attention to the case of n~3, m 1,2~m and m 3~0 , allowing us to model two routes of adaptation with opposite and distinct characteristics while minimising the overall complexity in number of required strains. For both routes of adaptation, the first, wildtype strain has very low transmissibility, the third has pandemic potential, and the second strain has intermediate transmissibility. This intermediate transmissibility is not enough to sustain the novel pathogen within the human host population, but secondary infections by humans are possible. Thus we have a 1 va 2 v1. Finally, the human adapted strain has a reproductive number a 3 w1. Further, we assume an identical mean infectious period for all strains.
Following [11] , we first distinguish two routes to adaptation: the 'punctuated' scenario has an evolutionary course a i~0 , 0:1, 2 ½ , while the 'gradual' scenario has a i~0 , 0:9, 2 ½ , the only difference being a 2 , the fitness of the intermediate stage.
This leads to the following SIR-model, normalized with respect to the mean infectious period,
where S is the number susceptible, I i is the number infected with strain i, and R the number of recovered or removed. We do not include births and deaths as we expect a zoonotic emergence, or extinction, to happen on a much shorter timescale than the human lifespan. We translate this model to a stochastic simulation of a multitype branching process, using the Gillespie algorithm [15] . The infection is seeded in a single random, susceptible host with the wildtype strain.
In general, every introduction has only two possible outcomes: emergence or extinction. Extinction happens if the novel pathogen dies out because it fails to adapt for human transmission or just by stochastic extinction. Hence, the introduction only leads to a limited number of infectious hosts, which we refer to as the 'outbreak size'.
Conversely, a novel pathogen of zoonotic origin emerges if it is sufficiently adapted for human transmission and begins to spread in a self-sustaining way. Formally, in an unlimited host population the cumulative number of infectious hosts is unbounded as time goes to infinity. Computationally we use a threshold of I 3~1 00 infectious hosts with the fully adapted strain to distinguish between emergence and extinction. This threshold ensures a probability of extinction less than (1=R 3 ) I3~7 :9 Ã 10 {31 [16] . Therefore, the number of falsely identified emergences, which would truly be extinctions, is negligibly small. Moreover, these arbitrarily small probabilities ensure that our simulation results are insensitive to the precise choice of threshold used. In situations where the host population is very small we relax our emergence threshold to smaller numbers of infectious hosts as some population sizes are below 100.
In the special case N?? and S=N?1 it is possible to calculate the probability of emergence per introduction into the human host population, given the evolutionary course of the pathogen, and the mutation rate with which the pathogen adapts. Our derivations start with assuming one homogeneous human host population of infinite size. This assumption can be easily relaxed as we show later. To calculate the probability of emergence, we define next event probabilities of infection p i , mutation m i , and recovery r i for each individual infected host, therefore the probabilities for what type of event will come next for each infectious host are
Note that in general, we can extend this adaptation process to arbitrarily many adaptive steps. The mutations are uni-directional towards the adapted strain. Using a branching-process approach similar to [10] , we derive the probability of emergence per introduction as follows (see Text S1, A.1.1, for more details)
Author Summary
Emerging infections are a continuing global public health issue, the most recent example being last year's 'Swine flu' influenza pandemic. However, for many zoonotic pathogens, some adaptation is required to cross the species barrier from an animal reservoir into humans and cause sustained transmission. Previous work has explored the relationship between the evolutionary biology of an adapting pathogen, and the epidemiology of cases that may arise before such a pathogen becomes pandemiccapable. Here, we extend this work to incorporate what is often an important host ecological feature, the spatial distribution of the host population. Many zoonoses occur away from large population centres. For example, HIV is thought to have entered the human population through bushmeat hunters in the sparsely populated jungles of Central Africa. We ask: when a pathogen is evolving to adapt for human transmission, under what circumstances does the spatial structure underlying the human population become important? We approach this question using mathematical models to explore regimes of connectedness between communities. Our results suggest that most communities are sufficiently interconnected to show no effect on the emergence process. We finish by discussing the implications of these findings for public health.
The probability of emergence can be expressed by the next event probabilities and the probability of extinction q i given an index infection of strain i. This expression can be solved analytically for all possible routes of adaptation.
Regardless of the underlying population structure and the pathogen's biology, we can make an estimate of the number of introductions needed before an emergence arises M, given the probability of emergence per introduction p em with 0ƒp em ƒ1 (see Text S1, A.1.3, for more details)
Note that this is the average number of introductions without an emergence. The average number of introductions needed for an emergence event is SMTz1.
In addition, the variance can be obtained in a similar way (see Text S1, A.
This variance leads to a standard deviation of the same order as the average number of introductions, vMw. This makes the number of introductions before an emergence inherently unpredictable if the probability of emergence per introduction is small (p em %1).
Again, in the special case S=N?1, the branching-process approach can be extended to derive the probabilities of outbreak sizes before emergence (see Text S1, A.1.2, for more details). In general, the probability of an outbreak of size x i is defined as g i (I i,0 , x i ) with i denoting the strain. The number of infected hosts to start with is denoted by I i,0 . Furthermore, the overall outbreak size probability can be derived using conditional probabilities p out (X )~g 1 (I 1,0 , x 1 ) X I 1,0 zx 1 ,...,I n{1,0 zx n{1
where X~I 1,0 z P n i~1
x i is the total outbreak size, and f i (I i,0 ) is the probability of getting I i,0 patient zeros to start with in strain i. The summation in the derivation of the overall outbreak size probability is over all possible subsets of infectious hosts to start with.
We use a metapopulation model to explore the effects of spatial host heterogeneity, effectively dividing the human population into interconnected communities. As an example of the general case of spatially structured communities, we focus on a simple village -city model to approximate the spatial host heterogeneity in rural areas connected, by human mobility, to bigger cities. There are many different types of human mobility between communities such as villages and cities, including short-term commuting and long-term labour migration. Particularly in developing countries, however, information on dominant patterns is sparse. Nonetheless, anecdotal evidence from Vietnam [17] , for example, suggests that short term commuting plays an important role: here, a subset of the village residents collects agricultural produce for trading in local markets in the city, and travels to the city on a daily to weekly basis. Accordingly we present a model in which the residence time of villagers in the city is typically less than the infectious period. However, in the supplementary information we also present a model incorporating migration on longer timescales (see Text S1, A.2). These two different models illustrate that our results appear qualitatively robust to different types of human movement.
As before, we have a wildtype pathogen capable of acquiring adaptations for human transmission. Assume a finite number of hosts in the village, and an effectively infinite number in the city. To allow for daily commuting, we label individuals in the city according to whether they are commuters from the village or not (neglecting commuters originating from the city and present in the village). The superscripts (v),(c) represent village and city inhabitants respectively, while (vm) denotes villagers in the city. Village members commute to the city at a per capita rate x 1 , and return at per capita rate x 2 . At any one time, a proportion w of villagers, the commuters, are in the city with w being set by x 1 and x 2 as described below. Further, we neglect susceptible village commuters acquiring infection in the city (this arises formally from the infinite number of hosts in the city). The number of village residents is fixed at N~N (v) zN (vm)~1 000, and we define the average number of commuters as Village, non-commuting : Village, commuting :
City :
Commuting equilibrium :
Note that equation (11) arises from the fact that w~N (vm) = (N (v) zN (vm) ). To represent daily commuting, with an infectious period of 5 days, we set x 2~5 , and choose w to give the required average number of commuters vcw. To seed a wildtype infection in the village we set I (v) 1~1 . In the village-city model, an emergence event is defined as having 100 infectious hosts with the fully adapted strain in the city.
We use the three strain model described before to study the impact of the mutation rate, m, and the average reproductive number of the intermediate strain, a 2 , on the probability of emergence per introduction in a single, infinite population. We assume 0vmƒ10 {1 as an illustrative spectrum of possible mutation rates. Figure 2 shows the probability of emergence for different mutation rates and average reproductive numbers of the intermediate strain. Not surprisingly, the probability of emergence grows non-linearly with a 2 and m. The probability of having no mutation in the second strain is (1zm) {x where x is the number of infected hosts with strain 2. While the intermediate reproductive number affects the exponent, the mutation rate has a direct influence on the base. We validate our analytical results by comparing them with the average probability of emergence of 10 3 simulated emergence processes, using one homogeneous population as described in (1). Figure 2 reveals an excellent agreement between the analytical results and simulations.
For small host communities, the depletion of susceptible hosts can play a significant role in limiting an ongoing outbreak. What is the effect of a finite population size on these analytical results which assume an infinite host population? Figure 3A compares the simulated outbreak size distribution of different sized populations with our analytical predictions. Note that, for populations greater than 500, there is close agreement between numerical and analytical results. When considering populations of size 1,000 or more, we do not expect population size dependence to have a substantial effect.
The village's number of residents in our commuting model is sufficient to avoid finite size effects on the outbreak size. Furthermore, it is independent of any spatial heterogeneity. The number of residents only has a limiting effect on the outbreak size distribution. Figure 3B shows the outbreak size distribution for our short-term commuting model. The average number of commuters ranges from 1 to 100. As we expect, no significant effect on the outbreak size distribution can be seen, even for vcw~1. It validates our assumption of the independence of infectious hosts, necessary for a branching-process formulation, as the simulations closely match the analytical predictions. It is noteworthy here that only the biology of the novel pathogen determines the emergence process, as outbreak sizes group according to the intermediate average reproductive number a 2 . The minimal deviation for vcw~100 commuters is based on the fact that the effective village size is only N (v)~9 00 due to the absent commuters.
In Figure 4 , we extract the probability of emergence per introduction given a certain number already infected. These data are easily calculated using the outbreak size distribution and the probability of emergence per introduction. Assume an introduction has caused x infectious hosts already. The probability of extinction is the cumulative probability of getting an outbreak size equal to or larger than x, renormalized by all possible outcomes (extinctions and emergences) once x hosts are infectious. This yields the probability of emergence per number of infected. In Figure 4 , the effect of spatial heterogeneity can be seen directly. For vcw §10, the village-city simulations agree very well with the analytical results assuming a single, infinite population. But for vcw~1, the probability of emergence converges to approximately 0:42.
While Figure 4 shows the probability of emergence as a function of the number infected, the actual outcome is highly unpredictable even if the probability of an event is known as the average waiting time to an emergence shows (see equations 4 and 5). It can be generalized for the probability of emergence given x infectious hosts. For example, the probability of emergence given five infectious hosts in the gradual route (II) is p em (x~5)~0:567. It follows on average every 1:736 times this happens an emergence will happen. The standard deviation is +1:161, which leads to the conclusion that even if the probability is known, it is inherently unpredictable when this will actually lead to an emergence.
This confirms that a pathogen needs a sufficient connection between communities to emerge, despite its ability to cause outbreaks, regardless of the spatial structure. Hence we expect the existence of a threshold where spatial heterogeneity effectively does not matter any more. Previous research has shown that the effect of heterogeneity in spatially structured population models depends on the interconnectivity with a threshold effectively allowing the pathogen to spread between communities [18] [19] [20] . Our approach allows new insights, as we do not need to specify the actual number of infectious hosts migrating to a new community. We measure connectivity between communities in terms of the average number of commuters vcw for which rich empirical datasets can be found. Figure 5 presents illustrative examples of empirical data of commuting patterns in different parts of the world. Most data has been collected by Offices of Statistics of five countries on three continents [21] [22] [23] [24] [25] . A further, two independent studies have been used to estimate commuting patterns of towns in Indonesia [26] and China [27] .
We next attempt to quantify the regimes in vcw for which spatial heterogeneity may be neglected. We approach this question using a simple analytical derivation for the effect of spatial heterogeneity, which considers only the adapted strain. Assume a connected community such as the village in our village-city model, with a fully adapted pathogen introduced into the village. Given an emergence and epidemic in the village, the probability that this causes an emergence and epidemic in the city is
Therefore, H spatial is a spatial homogeneity coefficient, measuring the impact of spatial heterogeneity on an emergence process. It ranges from 0, leading to two isolated communities, to 1, effectively removing any spatial heterogeneity and forming one homogeneous population. f is the fraction of the village residents becoming infectious. It can be derived using [28] f~1{e
The spatial homogeneity coefficient depends only on the connection strength expressed in commuters vcw and the average reproductive number R 0~a3 of the fully adapted strain. Though it only considers the fully adapted strain, we expect this coefficient to be a good approximation for a multi-strain model as the vast majority of infectious hosts will transmit the fully adapted strain in the case of an emergence. Figure 6 gives an overview of the influence of spatial heterogeneity as a function of vcw. Effectively, spatial heterogeneity is negligible once a critical number of ten commuters connect the two communities. This is a very low threshold, and empirical data shows that the probability of having a community with less than the critical number of ten commuters is approximately 1% for all our data combined.
As illustrated by the close fit between analytical and numerical results in figure 6 , there is only a small error in the analytical expression arising from neglecting infections with mal-adapted strains. This error is greatest for the gradually adapting pathogen, because an intermediate strain with a 2~0 :9 tends to cause larger outbreaks than one with a 2~0 :1. Nevertheless, the deviation remains small.
In light of this agreement, how does the critical average number of commuters vary with the adapted reproductive number? While for R 0~2 ten average commuters are sufficient to dissolve spatial heterogeneity, this changes dramatically for smaller average reproductive numbers (see Figure 7) . If a well-adapted strain is only just pandemic capable (i.e. R 0 just above 1) villages with only ten commuters are only 50% likely to seed an epidemic in their local city, and spatial structure becomes important again. For example, the critical number of commuters is close to 100 for R 0~1 :2. For R 0~2 the spatial homogeneity coefficient is approximately 0:42 for one commuter. This agrees with what we find in Figure 4 using simulated results.
In this article we first present analytical results to calculate epidemiological parameters of a novel disease, adapting to humans. We explore the influence of spatial host contact structure, and validate our result with stochastic simulations of simple village-city models as an example of interconnected communities within a spatially structured population. Our study reveals that for plausible parameter ranges, spatial heterogeneity only has very limited impact on the probability of emergence, as well as the outbreak size distribution. Neither a change in strength of spatial heterogeneity (e.g. number of commuters), nor in its quality (e.g. short term versus long term commuting) shows a significant influence. Our results suggest that only the most remote rural communities would be subject to epidemiological isolation. In particular, the available empirical data suggests that communities tend to be highly interconnected with relatively high connection strengths. Of course, it is the most remote communities of the world for whom we have the least relevant data. More empirical research on spatial heterogeneity is needed to form a better understanding of its effect, and this need is greatest in developing parts of the world.
In addition, population size becomes an important factor only when that population is relatively small { fewer than N&500 individuals. Only a small number of infectious hosts are actually involved in the emergence process, which relates to the small reservoir of susceptibles needed for a successful emergence. Moreover, biological processes such as the speed of evolution and the adaptive route show a strong influence on the overall emergence process. We show that epidemiological parameters such as the outbreak size group according to the evolutionary route. Previous research has shown the effect of the pathogen's route of evolutionary adaptation and mutation rate on the probability of emergence per introduction [10, 11] . Our theoretical derivation of the probability of emergence extends this and offers the benefit of being analytically solvable for any possible route of adaptation and any mutation probabilities. We note here that previous work has highlighted the role of other, significant types of heterogeneity in emergence of a novel infection. For example, [29] describe the effect of the pathogens life history, such as the length of infection, on the emergence of a novel pathogen. Further, heterogeneity in human-to-human transmission within a population may have an influence on the course and probability of emergence and outbreaks [13, 14] , usually lowering the probability of emergence. While we have concentrated here on simple types of spatial heterogeneity, a significant question for future research is the role of mixed heterogeneities, for example spatially structured populations with additional heterogeneity in the human-to-human transmission.
We also find that the waiting time for an outcome of a novel pathogen's introduction is highly unpredictable, even if the probability for such an event is known. Conversely, this means that an estimate of the underlying epidemiological parameters from observed data will be highly uncertain. Unfortunately, a large number of observations will be necessary to achieve confidence in the parameters, and even a large number of introductions gone extinct do not rule out the possibility of emergence for a pathogen. We came to a similar result [11] using a measurement on the upper bound of the probability of emergence. Moreover, the probability of emergence given a certain number of infectious hosts can be surprisingly low. Even a comparatively large number of infectious hosts can end in extinction, especially for low mutation rates and intermediate-stage average reproductive numbers just below one. Figure 5 . Data of commuting patterns in different parts of the world. Shown is the cumulative fraction of all communities with equal or less than the specified number of commuters. The data was mostly collected by the National Statistical Offices of the respective countries. The gold line represents commuting data from Brazil [21] , the red line data from the USA [22], the blue line data from the UK [23] , the brown line data from Japan [25], the cyan line data from Hong Kong [24] , and the green line data from two independent sources. The green line has orange [27] and pink data points [26] , corresponding to its data sources. Our data represents the commuting flows between administrative units. The definition of administrative units varies highly between countries. For example, the US data is on a granularity of 3,141 counties, while the data from Japan is based on its 47 prefectures. However, heterogeneity can also be found within countries datasets. The Brazilian data is on a level of 5,507 municipalities with resident sizes ranging from 1,166 to 10, 435, 548. doi:10.1371/journal.pcbi.1000947.g005 Figure 6 . Deviation between simulated and analytical predicted probability of emergence. The deviation is a function of the average number of commuters vcw. The deviation is defined as Dp (ana) em {p (sim) em D=p (ana) em . The analytical probability of emergence p (ana) em is for an infinite ulation without spatial structure. The simulated probability of emergence p (sim) em is for short-term commuting with the blue data points representing the gradual route (II) of adaptation, and the orange data points representing the punctuated route (I). The solid black line is 1{H spatial (1=a 3 ) vcwf , as defined in equation 12 in the main text. It is the analytical expected deviation for spatial heterogeneity as a function of the spatial homogeneity coefficient. The simulations agree very well with the analytical expected deviation. The gradual route (II) is slightly more off from the theoretical prediction as a result of the small but significant number of infected with the intermediate strain. Effectively, it lowers the number of commuters infected with the fully adapted strain and therefore the probability of transmission from the village to the city. Nevertheless, the analytical prediction as well as the simulations show no significant impact of spatial heterogeneity from a critical commuter threshold of vcw~10. doi:10.1371/journal.pcbi.1000947.g006 Figure 7 . Impact of spatial heterogeneity on disease transmission between communities (I). The impact is measured with the spatial homogeneity coefficient with 0ƒH spatial ƒ1. Given H spatial~1 every emergence in the village automatically leads to an emergence in the city, and H spatial~0 represents no chance of successfully transmitting the pathogen into the city. The figure reveals that spatial structure becomes especially important for small average reproductive numbers. In addition, the average number of commuters needed to show an effect of spatial heterogeneity is surprisingly small. doi:10.1371/journal.pcbi.1000947.g007
Our work has relevance for important public health issues: if a novel disease is detected in a rural setting, and it appears to be spreading, how feasible is it to contain infection by restricting movements to and from the village? Our results suggest that first, an infeasibly tight level of quarantine would be required for any chance of containment, corresponding to enforcing a low level of vcw in Figure 6 . To all intents and purposes isolation would have to be absolute to be effective. In most circumstances such extreme intervention would not be acceptable. Second, given typical mobility patterns, it is likely that once there is a detectable number of cases in the village, there may already be a significant number of cases outside of it. Therefore quarantining interventions are likely to come too late.
Our work raises important questions for future research: where should surveillance be focused to detect an emergence as early as possible, especially if resources are limited? Given emergence of a novel infection in a rural setting, how much time can we buy through limiting travel to and from major urban centres? These and other questions will undoubtedly benefit from more systematic studies of emergence in the context of population distributions. Nonetheless, theoretical models such as those presented here can offer useful, fundamental insights to guide such studies.
Text S1 Supplementary material with figures for 'Insights into the Evolution and Emergence of a Novel Infectious Disease'. Found at: doi:10.1371/journal.pcbi.1000947.s001 (0. 16 MB PDF) Author Contributions Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients BACKGROUND: An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. METHODOLOGY: The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. PRINCIPAL FINDINGS: 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. CONCLUSIONS: Disease severity was associated with pronounced impairment of host immune response. The first pandemic of this century was unexpectedly caused by a novel swine-origin H1N1 virus, the pandemic H1N1 (2009) virus (p-H1N1-09). Mexico was the first country to be affected in early March with reports of mild respiratory infection as well as severe pneumonia cases and considerable mortality [1, 2] . Several countries were subsequently affected reporting variable mortality, smoking, pregnancy and obesity being important risk factors for severe disease [3, 4, 5, 6] .
On 1 st August 2009, a 14 year-old girl without history of known risk factors succumbed to p-H1N1-09 infection in Pune, western India representing the first fatality from the country. As of 21 st April 2010, India has reported 1483 deaths during the pandemic (http://pib.nic.in/h1n1/h1n1.asp), Pune contributing to 173 cases (http://www.maha-arogya.gov.in/march-april%202010.htm).
During the initial phase of the pandemic, designated wards in the government hospitals admitted every mild case, treated with Oseltamivir and discharged after recovery. A special intensive care unit treated the critically ill patients. The present study was undertaken during this very early phase of the pandemic. To understand the basis of differential disease presentation/outcome, we investigated 26 mild cases and 15 critically ill patients during 1 st August -19 th September 2009. This report provides comparative data on viral load, cytokines, gene-profiling, Toll-like-receptor (TLR) levels, antibody titres and antibody isotypes.
Ethical clearance for the study was obtained from, 'Institutional Human Ethical Committee' as part of the pandemic influenza investigations. Written consent was obtained from all the participants involved in the study. For minors and critically ill patients it was obtained from parent/guardian. Patients confirmed to have p-H1N1-09 infection by a positive real time PCR test (http://www.who.int/csr/resources/publications/swineflu/CDC RealtimeRTPCRprotocol_SwineH1Ass-2009_20090428) were studied. These included 15 patients admitted to Intensive Care Unit and requiring mechanical ventilator support and 26 suffering from mild respiratory symptoms. All the mild cases were ambulatory patients admitted to a designated hospital. In the initial phases of the pandemic during which this study was performed, patients suggestive of Influenza-like illness were admitted to designated ward of a Corporation hospital. Throat swabs were collected and sent to the National Institute of Virology for diagnosis. Osletamivir treatment was initiated immediately after the confirmation of diagnosis. These patients were discharged after the completion of the treatment. On the contrary, majority of the severe patients were admitted only after the development of serious respiratory consequences. Same protocol was followed for diagnosis and antiviral treatment.
A single blood sample was collected from mild cases, 1-3 days after the development of respiratory symptoms. As controls, blood samples from 20 apparently healthy individuals were collected. Sequential blood/lung aspirate samples (standardized tracheal aspirates) were collected from the critically ill patients. The first sample was collected within 3 days for 13 patients; while one each was collected on 4 and 8 (pregnant woman) days after the appearance of symptoms (Table 1 ). Blood and lung aspirates were transported to the lab within half an hour of collection. Lung aspirates obtained from severe cases were immediately aliquoted and frozen at 280uC. An aliquot was mixed in 1:3 proportions with the RNAlater and stored at 280uC for gene analysis. Hundred microlitre of the blood sample from every patient was processed immediately for TLR staining as described below. In parallel, blood samples were immediately processed for the isolation of Peripheral Blood Mononuclear Cells (PBMCs) by density gradient centrifugation using Ficoll-Hypaque (Sigma). Plasma layer was removed and stored at 280uC in aliquots; cell pellets were stored in 500 ml RNALater solution (Ambion) at 280uC.
A highly sensitive and specific ELISA was carried out by coating the wells with purified recombinant HA protein (expressed in baculovirus system) of p-H1N1-09 virus, sera at 1:100 dilution and anti-human-IgG-HRP conjugate as the detector antibody [7] . Isotyping was done as described earlier [8] .
Anti-human antibodies for TLR 2, 3, 4 and 9 (eBioscience, USA) and TLR 7 and TLR 8 (Imgenex, USA) were used for the staining. For surface staining of TLR 2 and 4, 100 ml of whole blood was lysed with BD FACS lysis solution (BD Biosciences, USA), washed, fixed and processed for staining with anti-human TLR 2 (FITC conjugated) and TLR 4 (PE conjugated) antibodies respectively. For intracellular staining, 100 ml of the whole blood was fully lysed with BD FACS lysing solution (Becton Dickinson), washed twice with Perm-wash buffer (BD bioscience) and processed for staining with the following anti-human antibodies: PE-TLR 3, FITC-TLR 7, FITC-TLR8 and PE-TLR 9 respectively. Stained cells were resuspended in 500 ml 1% paraformaldehyde, analysed on FACScalibur flow cytometer (Becton Dickinson).On the basis of forward and side scatter plot lymphocytes and monocytes were gated and data analysis was done using BD FACSDivasoftware.TLR levels were expressed as median fluorescence intensity (MFI).
Concentrations in the plasma/lung aspirates were determined for seventeen cytokines/chemokines (IL1a, IL1b, IL6, TNFa, 
Frozen lung aspirates were thawed, centrifuged to pellet down the cells and pellets were used to isolate RNA. Total RNA was extracted from PBMCs and lung aspirate cells by using Ribopure Kit (Ambion) as per the manufacturer's instructions. RNA was eluted in 100 ml elution buffer, quantitated using Nanodrop (ND-1000) and processed for quality check in Agilent bioanalyzer (Agilent, U.S.A). Equal quantities of RNA (500 ng) with $7 RIN value were processed further for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.K.). All cDNAs were tested in real time PCR assay using TaqMan primers and probe for 18S rRNA (Applied Biosystems, U.K.) to ensure efficient cDNA synthesis. cDNAs were mixed with equal volumes of TaqMan 2X PCR master mix from one-step RT-PCR kit (Applied Biosystems) and 125 ng (RNA equivalent) cDNAs were loaded per port of the TaqMan Low Density Array card (TLDA) of the Human Immune panel (Applied Biosystems, U.K.) and run on 7900HT Fast Real-Time PCR system (Applied Biosystems, U.K.). Relative gene expression values were obtained employing comparative Ct method using Applied Biosystems' Relative Quantification (RQ) Manager Software v1.2. cDNAs from six healthy individuals processed similarly were considered as calibrators. 18s RNA was used as an endogenous control. Relative quantitation values of each study group were used to calculate mean RQ values. For cluster analysis, relative quantitation values were log2 transformed and hierarchically clustered with analysis software (Cluster 3.0) [9] .
Viral RNA was extracted from 140 ml plasma/lung aspirates using QIAamp Viral RNA Mini Kit (Qiagen, Germany) as per the manufacturers' instructions. For viral RNA quantitation, CDC primers and probe were used. The target HA gene from an Indian isolate was cloned to obtain in vitro transcripts, serial 10-fold dilutions of the RNA were used to generate a reference curve. A linear relationship was obtained from 10 10 -10 2 starting copies/ reaction (r 2 = 0.99), the detection limit being 100 copies.
Levels of cytokines and chemokines were analyzed after log transformation and a value of 0.2 pg/ml was used in the case of undetectable concentration of cytokine or chemokine in the tested samples. The Mann-Whitney U or Fisher exact tests were used for group comparisons of numerical and categorical data respectively. For all analyses, a P value of less than 0.05 derived from a two tailed test was considered significant. All statistical analyses were performed with 'SPSS11.0 for Windows' software (SPSS Inc.).
All the 26 mild cases (Male: Female ratio 12:14, age range 6-51 yrs) had fever/history of fever in last 3 days. The other symptoms included sore throat/cough (14/26), nasal discharge (6/ 24), headache/bodyache (5/26), diarrhea (2/26) and breathlessness (4/26). Radiological examination was not indicated and hence not performed. Nasal oxygen was not required.
Of the 15 critically ill patients, 2 survived (Table 1 ). The male:female ratio was 6:9, the age range being 13-53 years. Associated co-morbidities were present in 1/2 surviving and 7/13 fatal patients. Importantly, radiological findings showed that all the fatal cases had $3/6 lung zonal involvement and required ventilator assistance. Both the surviving patients initially needed nasal oxygen but were put on ventilator subsequently. The patients were either treated with Osletamivir alone (n = 7) or in combination with Zanamivir (n = 8). From the endotracheal tubes of 4 patients Acenotobacter was isolated. None of the 26 mild cases had recognizable risk factors. S21, a 36 year female suffering from severe seasonal influenza survived after 39 days of hospitalization.
Plasma samples from both patient categories were negative for pH1N1-09 influenza virus RNA. Sequential lung aspirate samples of 6 patients (2-5 samples) demonstrated gradual/continued lower or no viral load. Of the single lung aspirate from 3 patients with rapid death, two were negative for viral RNA while one exhibited 1.51610 5 copies/ml ( Figure 1 ).
Serum samples available from 4 critically ill patients exhibited HI titres of 40-320. At admission, the geometric mean titres (GMT) of anti-rpH1N1-09-HA-IgG antibodies (ELISA) were significantly lower in the mild infections (800, 95% CI values 571-1120) than the critically ill patients (4996, 95% CI values 3970-6288) (p,0.0001). The last blood sample collected 4-11 days later from 11 critically ill patients showed .4 fold rise in antibody titres. IgG1 was the exclusive isotype in both the categories. 
Comparison among healthy controls and disease categories showed no difference in the levels of IFNc, IL12p70 and IL2RA were significantly lower in mild cases and higher in critically ill patients ( Table 2 ). All other molecules showed significantly higher levels in both patient categories. The overall pattern reflects pandemic-H1N1-09 influenza infection. The disease severity correlated with significant increase in IL1RA, IL2, IL6, CCL3, CCL4 and IL10 levels in critically ill patients.
Analysis of gene expression profiles revealed significant findings; please see Table S1 :
Comparison of controls with disease categories. 
Tables 2 and S1 represent the status of cytokines and chemokines at protein and gene levels respectively in the lung aspirates/cells of lung aspirates of critically ill patients. In the absence of similar samples from mild cases/healthy controls for ethical reasons, no comparison was possible.
Comparison of plasma and lung aspirates of critically ill patients identified higher levels of CXCL8 and IL12p70 in the lung aspirates. No significant difference was noted for the other molecules. None of the plasma markers of severity exhibited higher levels in the lung aspirates. Expression patterns of genes were also analyzed by hierarchical clustering. Results are graphically represented by assigning a specific color to each cell on the basis of the expression levels.
Genes showing no changes in expression levels (as compared to controls) are shown as black. Upregulated genes are shown in red with increasing intensities in proportion to the expression levels and in different intensities of green indicating levels of downregulation. Figure 3a shows cluster image of samples from mild and critically ill cases taken on admission. Seasonal flu sample (S21) showed a distinct gene expression pattern and formed a separate cluster (cluster 6) from all other samples. Three lung samples from critically ill patients also formed a separate cluster as most of the proinflammatory genes were upregulated (cluster 2). Remaining samples formed mixed clusters (cluster 4 and 5) which included both mild and critically ill patients. Overall, no distinct clustering pattern for the patients was observed for the severe and mild cases. With respect to gene clustering, clusters A and B contained genes associated with inflammation.
Comparisons of genes of lungs and PBMCs of the same 3 patients or all the critically ill patients as a group yielded similar results.
Comparison of lung cytokines and chemokines at protein and gene levels showed comparable elevated levels of IL12B, CXCL8, IL1B, CCL3, IL1A and IL6 while IL17, TNF and IL10 were elevated only at the gene level. IFNG was at basal level both at gene and protein levels.
Variable numbers of sequential blood samples were available from critically ill patients. Comparisons of TLRs did not show any distinguishable pattern (data not shown). For evaluating gene expressions, a separate cluster analysis was carried out for the sequential PBMC samples of critically ill patients (Figure 3b ). Samples formed two distinct clusters. Samples from S1, S2, S3 and S4 formed a single cluster (cluster 1) showing significant Figure 2 . TLR levels in the blood lymphocytes and monocytes. TLR levels of peripheral blood from the patients and healthy controls were determined separately by either surface staining or intracellular staining of the cells using TLR specific antibodies followed by FACS analysis. Scatter dot plots and histograms: A representative sample each from different categories, a) healthy individual, b) mild case, c) critically ill case, d) TLR levels: Expressed as median fluorescence intensities (MFI) for different categories of patients. doi:10.1371/journal.pone.0013099.g002 downregulation of most of the immune function genes. Cluster 2 contained all sequential samples from the seasonal flu case (S21) and one pH1N1 case (S7). Though most of the gene expression levels were similar to survived case, S7 did not survive. Genes formed four clusters, A, B, C and D. Cluster C contained majority of the analyzed genes (73/87) and all were downregulated in the cluster 1.
The dynamics of cytokines/chemokines at protein levels (6 patients) are shown in Figure 4 . Data for the single critical patient suffering from seasonal influenza (S21) is also presented. Comparison of plasma cytokines and chemokines did not show any distinct pattern in the sequential samples of the fatal or survived cases. Patient S6 did exhibit different pattern when compared to others.
This first comprehensive study addresses important issues of the identification of markers for severity of p-H1N1-09 infection and dynamics of immune responses in severe disease by evaluating several parameters. The investigations were initiated during the early phase of the pandemic when isolation of all the Figure 3 . Gene expression analysis. Gene expression profiles of total PBMCs from blood samples of mild and critically ill cases and lung aspirate cells from critically ill cases were determined using TaqMan Low Density immune panel arrays. PBMCs from six healthy individuals were taken as controls, which were treated as replicate arrays to calculate the mean baseline expression level for each gene. The fold changes in the gene expression levels were calculated in relation with the controls. Values for eighty-seven genes were hierarchically clustered on log2 transformation. The corresponding gene of each cluster is listed by a gene symbol on the right-hand side of the images. a) Cluster image of gene expression analysis of PBMCs from mild and critically ill cases and lung aspirate cells from critically ill cases: PBMC samples from mild cases are denoted as M (M1, M2, M13). First sample taken after admission from each critically ill case was taken for the comparison and are denoted as S1-1, S2-1, S21-1. Single samples from critically ill patients are denoted as S8, S12 etc. Lung aspirate samples are denoted as L. b) Cluster image of gene expression analysis of sequential blood samples from critically ill cases: Total PBMCs from the sequential blood samples of severe cases obtained on different post admission days are denoted as S1-1, S1-2, …S21-8). doi:10.1371/journal.pone.0013099.g003 p-H1N1-09 mild infections in designated wards was obligatory. Therefore, we could collect samples before the initiation of the antiviral therapy and the data truly represents acute phase of mild disease. On the other hand, critically ill patients were given Oseltamivir immediately after the confirmation of the infection and the samples were collected subsequently. As underlying medical conditions were present in one of the 2 survivors and 7/ 13 critically ill patients, association of co-morbidities alone does not seem to be responsible for complications or aberrant immune response.
The absence of viremia in both patient categories and relatively low viral load in the lung aspirates of the critically ill patients suggest that enhanced replication of the virus may not be an important contributor to the pathogenesis (Figure 1) . The viral load in lung aspirates was independent of fatality. In contrast, among the Spanish patients [10] , 93% and 57% of the mild and critical cases respectively were positive for serum viral RNA, with no significant difference in the viral load. Both studies used CDC primers for real time PCR (http://www. who.int/csr/resources/publications/swineflu/CDCRealtimeR TPCRprotocol_SwineH1Ass-2009_20090428) and the critical cases were bled when already on Oseltamivir treatment, negating sensitivity of the PCR, effect of antiviral therapy or delay in collection of samples to be responsible for different results. The absence of uniform mutations in the fatal casesderived Indian p-H1N1-09 isolates suggests limited/no role of mutant virus in the pathogenesis [11] . Viremia was associated with the outcome of H5N1 infection, 9/11 fatal and 0/5 nonfatal cases being viremic [12] .
Serum HI-antibody positivity was noted in 1/15mild, 2/10 critically ill Spanish patients and 4/4 critically ill patients from India. ELISA could detect antibodies in every patient. The absence of viremia among the Indian patients may be due to an early antibody response.
The presence of IgG-anti-p-H1N1-09 antibodies in all the severe Indian patients (HI antibodies in all the 4 screened) does indicate switch from the innate to adaptive immunity. Poor outcome despite the switch may probably be attributed to the timing of the shift or role of antibodies in disease severity. Significantly higher titres of IgG-anti-p-H1N1-09 antibodies in the critically ill patients supports the role in pathogenesis. This observation is in sharp contrast with that for the SARS patients examined from Canada [13] documenting significantly lower anti-SARS CoV spike antibody titres in the critically ill patients. Though the presence of neutralizing antibodies protect against Influenza virus infection, clearance of the infection is mediated by cellular immunity. The exclusive presence of IgG1 antibodies in both patient categories document Th2 bias with significant enhancement with severity. This observation correlates with the elevated levels of TLR3, 4, and 7, Th2 cytokines (IL6 and IL10) at protein level and IL4, IL5, IL6, IL10 at gene level.
We identified higher plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10 as markers of severity. Of these, IL6 is a known marker of influenza disease severity with probable involvement in tissue damage [12, 14] . CCL3 and CCL4 are important mediators of virus induced inflammation. IL10 is recognized as a regulatory (anti-inflammatory) cytokine and can act on multiple cell types to regulate immune and inflammatory responses [15] . IL6 is known to be responsible for regulating plasma levels of IL1RA and IL10 [16] . Among Spanish patients, serum IL15, IL12p70 and IL6 were recognized as the hallmark of severity [10] . Significant increases in TLR levels without corresponding rise in cytokines suggest aberrant immune response in critically ill patients. The possibility of viral proteins diminishing cytokine production cannot be ruled out. Similar to in-vitro studies [17, 18] , cytokine storm associated with pathogenesis of H5N1 infection [12] was not the feature of p-H1N1-09 infection. The observations of no increase in the levels of plasma IFNG in both patient categories when compared to controls as well as basal gene expression in the lungs of critically ill patients point out lack of co-ordination in the modulation of innate and adaptive immune responses.
At PBMC gene level, down-regulation of a large number of genes was associated with disease severity (Table S1, Figure 3b ). This profile is indicative of massive infiltration of monocyterecruited neutrophils, DCs/macrophages to the target organ for mounting immune response/tissue repair and/or viral proteininduced shut down of the cellular genes, as these cells are known to efficiently replicate the virus [19, 20] . Additional studies are required to examine if the virus specifically down regulates host antiviral genes or dictates generalized shut down of host mRNA synthesis [19, 21] , especially in relation to the disease severity. Paradoxically, constant inflammatory signals provided by the significant increase in several chemokines (CCL2, CCL3, CCL19, CXCL8, CXCL10, CXCL11) and pro-inflammatory cytokines (IL6, IL17, IL1A, IL1B, TNF) in the lung cells may have resulted in massive infiltration of leucocytes and excessive tissue damage. This is to our knowledge the first report on p-H1N1-09-induced gene expression in human lung cells.
As against differential cytokine levels and viral load observed in a male and pregnant female fatal H5N1 case [21] , except for higher down-regulation of IL1B/IL2, the pregnant woman (S3) in our series was similar to others. It was intriguing to note that despite attempts of mounting immune response similar to mildrecovered patients, the patient S7 did succumb to the disease. It is important to recognize that the current study examines virus-host interactions throughout the course of severe disease, limitation of the study being absence of similar data from mild cases.
We could investigate a critical case suffering from seasonal influenza (S21). As against similar patterns recorded for pandemic influenza cases, distinct differences were recorded for this case (Figure 3a, 3b) .
In the ferret model with 27% mortality based on the .20% weight loss following infection with A/California/07/2009 pandemic influenza virus [22] , sequential lung sampling documented decreased gene expression of CCL2, CXCL10, TNFA and IL1B on day 7 when animals show highest weight loss. Comparison of the human data presented in our study (Table S1) shows that in the lungs of the critically ill patients all these and several other chemokines/inflammatory cytokines are overexpressed. Thus, the gene expression profiles at the time of overwhelming symptoms in the lungs of the infected ferrets (decreased expression) do not match the data on severe human cases from the present series (elevated expressions).
While finalizing the manuscript, we came across two studies from Hong Kong [23, 24] . The mild patients (n = 22) were non viremic while 13% of the 23 fatal cases were viremic. Data from Spain, Hong Kong and India suggests the role of host genetics in immune response to the pandemic-causing virus.
In conclusion, our data confirms earlier findings of dysregulated host response in severe infections with H5N1 [12] and 1918 influenza viruses [25, 26, 27] . However, the mechanisms leading to similar end results seem to vary with the type of viral infection. Indepth studies to understand the role of virus/viral proteins in modulating host response need to be undertaken on priority. Identification of specific pathways might provide clues to include immunomodulators for the treatment of severe cases, albeit in conjunction with potent antivirals.  Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34(+)CD38(−) human hematopoietic progenitor cells, BALB/c Rag2(−/−)IL-2Rγc(−/−) mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19(+)CD27(+) B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens. Hyper-immune sera containing polyclonal immunoglobulins (Igs) have been widely used in both therapeutic and prophylactic clinical settings [1] . However, the use of polyclonal sera was associated with several problems, such as the stimulation of allergic reactions, low reproducibility between clinical batches and high off-label use, which finally caused a decline in their use [2] . The advent of technologies to make monoclonal antibodies (mAbs) derived from animals, especially mice, has overcome many of the problems associated with the use of polyclonal sera. The technology to make monoclonal cell lines of antibody-producing cells by fusing antibody producing plasma cells with myeloma cells was described for the first time in 1975 by Milstein and Kohler [3] . The therapeutic potential of mAbs was immediately recognized and in 1980 the first mAb, OKT3, was approved for therapeutic applications. This antibody inactivates T cells, thereby preventing rejections of organ transplants [4] . However, because of the animal origin of the first generation of mAbs that were used in clinical trials, human subjects treated with these antibodies developed vigorous immune reactions against the animal proteins, which were thereby eliminated preventing their therapeutic actions [5] . To overcome these problems technologies were developed to diminish the immunogenicity of mouse antibodies by replacing part or the complete mouse antibody backbone by its human equivalent, first generating chimeric, and subsequently fully humanized antibodies [6] . In a parallel approach transgenic mice bearing the human Ig region were created to obtain fully human antibodies following immunization. The use of these mice obviates the elaborate molecular engineering of antibodies that is needed to humanize antibodies generated in wild-type mice, however, the maturation process of the mouse B cells expressing human Igs is different from that of fully human B cells [7] .
Immortalization of B cells from immune humans seems to be the logical strategy to avoid these problems. However, the methods to achieve this goal have showed low efficiencies, although some progress has recently been reported [8, 9] . Nevertheless, the major disadvantage of human B cells immortalization is the need for cells from either vaccinated individuals or patients who had recovered from an infection. Thus, to fully exploit the Ig repertoire of human B cells in an in vivo setting, we explored the possibility to raise mAbs following de novo induction of human B cell responses in mice carrying elements of the human immune system (HIS). HIS mice are generated by engrafting immunodeficient mice with human hematopoietic stem cells (HSC) with or without human lymphoid tissues from fetal origin [10, 11, 12] . In particular, mice deficient for the recombinase activating gene-2 (Rag2) and the common gamma chain of the IL-2 receptor (Il2rg) on a BALB/c or a non-obese diabetic (NOD) background are permissive for human HSC xenografts. Inoculation of newborn mice from these strains with human HSC of fetal or umbilical cord blood origin gives rise to robust engraftment of a number of immune cells, including T, B, NK and dendritic cells. In this work, we describe a convenient approach to generate fully human mAbs based on the immunization of BALB/c Rag2 2/2 IL-2Rcc 2/2 engrafted with human CD34 + CD38 2 HSC [13, 14] . To this end, HIS mice were immunized with commercial vaccines against hepatitis B virus (HBV) and tetanus. Following immunization, human CD19 + B cells were sorted based on surface CD27 expression, as a marker of memory phenotype, and the isotype of surface Igs. The sorted B cell populations were immortalized in vitro by retroviral transduction with human B cell lymphoma (BCL)-6 and BCL-XL genes and antigen-specific B cell clones were established and characterized.
The obtained results provided the proof-of-concept for the usefulness of this generic approach based on HIS mice combined with immortalization of human B cells for the rapid and inexpensive development of human mAbs against a wide range of antigens.
The use of fetal liver tissue obtained from elective abortions with gestational age ranging from 14 to 20 weeks was approved by the Medical Ethical Committee of the AMC-UvA and was contingent on informed written consent.
BALB/c Rag2 2/2 IL-2Rcc 2/2 mice were bred and maintained in individual ventilated cages, and fed with autoclaved food and water. HIS mice were generated as previously described [13, 14, 15, 16] , with the approval of the Animal Ethical Committee of the AMC-UvA (permit number DHL-100970). In brief, human fetal livers were obtained from elective abortions with gestational age ranging from 14 to 20 weeks. Magnetic enrichment of CD34 + cells (.98% pure) was performed by using the CD34 Progenitor Cell Isolation kit (Miltenyi Biotech), after preparation of single cell suspensions and isolation of mononuclear cells by density gradient centrifugation over Lymphoprep (Axis Shield). Finally, newborn (,5 days old) sub-lethally irradiated (3.5 Gy) BALB/c Rag2 2/2 IL-2Rcc 2/2 mice were injected via intra-hepatic route with 5-10610 4 sorted CD34 + CD38 2 human fetal liver hematopoietic stem cells in 30 ml. All manipulations of HIS mice were performed under laminar flow. Cell suspensions were prepared in RPMI medium supplemented with 2% fetal calf serum (FCS).
Twelve to sixteen weeks after CD34 + CD38 2 HSC engraftment, HIS mice were killed and single cell suspensions of splenocytes were prepared. Red cells lysis was performed in 1 ml of red cell lysis buffer (Sigma) for 10 min. Splenocytes were washed, resuspended in 600 ml of RLT lysis buffer (Qiagen) and homogenized by passing through a 21-gauge needle several times using RNase free syringes. RNA was prepared using RNeasy mini kits (Qiagen) according to manufactures instructions. BCR V H immunoscope was performed as previously described [17] . Briefly, cDNA was prepared and real-time PCR performed by combining primers for the different V H chains (V H 1-7) and specific fluorochrome-labeled probes against the different constant regions (C H m, C H a and C H c). An additional four PCR cycles 'run-off reactions' were then performed on the PCR products using fluorescent primers specific for the constant regions (Fcm, Fca and Fcc). Products were gel separated to determine CDR3 lengths. Analysis of six individual HIS-mice containing greater than 30% human chimerism in the spleen was performed. The number of human CD19 + B cells in chimeric spleens ranged from 5-12610 6 .
Eight weeks after HSC transplantation, blood was taken from HIS mice to verify the level of engraftment by flow cytometry, as described elsewhere [18] . HIS mice with a good level of human reconstitution (.20% hCD45 + cells) were immunized by intramuscular route (biceps femoris) using a 29G needle, three times on weeks 14, 16 and 18 with either 100 ml of the HBV vaccine (Engerix-B, GlaxoSmithKline) or 50 ml of tetanus toxoid (TT) containing vaccine (Tetanus vaccine, The Netherlands Vaccine Institute). These amounts correspond to 1/10 of the normal human dose. Negative controls received the same volume of PBS buffer. Two weeks after the last immunization, HIS mice were exsanguinated under isofluran/oxygen narcosis. Spleens and mLN were removed aseptically and cellular suspensions were prepared. The BM cells were isolated from the femur and tibia. from Beckman Coulter; CD3 (SK7), CD4 (SK3), CD8 (SK1), CD19 (HIB19), CD38 (HIT2), CD45 (2D1 and HI30), CD45RA (HI100), CD138 (MI15), IgM (G20-127), IgD (IA6-2), IgG (G18-145) and CCR7 (3D12) from BD Biosciences; CD27 (LT27) from AbD-Serotec; CD27 (LG.7F9) from eBioscience. TT-specific B cells were also occasionally stained with PE-coupled TT, kindly provided by Dr. Andreas Radbruch (German Rheumatism Research Center, Berlin, Germany). Dead cells were excluded based on DAPI incorporation. All washings and reagent dilutions were done with PBS containing 2% FCS and 0.02% NaN 3 . Stained cells were analyzed with an LSR-II interfaced to a FACS-Diva software system (BD Biosciences). Cell sorting of B cell subsets were performed on HIS mouse BM and spleens using a FACS-Aria cell sorter interfaced to a FACS-Diva software system (BD Biosciences). For these experiments, all washings and reagent dilutions were done with 2% FCS supplemented PBS without NaN 3 .
The human BCL6 [19, 20] and BCL-XL [21] encoding cDNAs were further cloned in a LZRS retroviral expression vector, around a T2A cleavage-promoting peptide sequence and upstream a cassette containing an internal ribosome entry site (IRES) and the gene encoding GFP. We therefore obtained a LZRS vector in the following configuration: BCL6-T2A-BCLXL-IRES-GFP [9] . Transfection of Phoenix-GALV packaging cells and virus production were performed as previously described [22] . Before retroviral transduction, memory B cells were activated on c-irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L-L cells) in the presence of 25-50 ng/ml recombinant mouse interleukin-21 (rmIL-21, R&D systems) for 36 h [19] . The B cells were washed, mixed with retroviral supernatants in Retronectin-coated plates (Takara), centrifuged at room temperature for 60 min at 360 g, and subsequently incubated with the retroviruses at 37uC, 5% CO 2 for 6-8 h. Transduced B cells were maintained in co-cultures using CD40L-L cells (10 5 cells/ml) and in standard IMDM (Gibco) culture medium supplemented with 8% fetal bovine serum (FBS; HyClone), penicillin/streptomycin (Roche) and 25 ng/ml rmIL-21.
The analysis of human Ig-V H sequences was performed as follows. Total RNA was isolated from approximately 5610 5 monoclonal B cells with Trizol (Invitrogen). The cDNA was generated and subjected to PCR with primers specific to the different V H family members. PCR products were sequenced to determine the CDR3 region of the different clones. Sequence analysis was performed using BigDye Terminator chemistry (Applied Biosystems Inc.) and CodonCode Aligner software.
The plasma harvested from HIS mice (7 days after the first and second immunization; 10 days after the third immunization) and B cell clone culture supernatants were screened by ELISA for the presence of total human IgM, total human IgG and antigenspecific antibodies. Measurement of total IgM and IgG was performed by coating 96-well plates either with AffiniPure F(ab') 2 fragment goat anti-human IgM (Fc5m-specific, Jackson Immu-noResearch) or AffiniPure goat anti-human IgG (Fcc fragmentspecific; Jackson ImmunoResearch). Control human serum protein calibrator (Dako) with known IgM (0.8 mg/ml) and IgG (10.4 mg/ml) concentrations was used as a standard to be compared to the samples. For the detection of antigen-specific antibodies, 96-well plates were coated either with tetanus vaccine (Nederlands Vaccin Instituut) or Engerix B (GlaxoSmithKline) (106 diluted in PBS) for 1 h at 37uC or overnight at 4uC. Alternatively, Ridascreen Tetanus IgG ELISA plates (Biopharm)
were also used to screen for TT-specific antibodies. After coating, the plates were washed in ELISA wash buffer (PBS, 0.5% Tween-20). A PBS solution containing 4% of milk was used as a blocking agent, before adding serial dilution of HIS mouse plasma (starting at a dilution of 1:5) or cell culture supernatants (starting at a dilution of 1:2). Enzyme-conjugated detection antibodies were added at a dilution of 1:2500 for HRP-conjugated anti-IgG and a dilution of 1:5000 for HRP-conjugated anti-IgM (both from Jackson ImmunoResearch). Then, TMB substrate/stop solution (Biosource) was used for the development of the ELISA assay.
Statistical analyses were performed using GraphPad Prism version 5.02 for Windows (GraphPad Software). Data were subjected to two-tailed unpaired Student t test analysis. The obtained p values were considered significant when p,0.05.
We have generated HIS mice by transplanting human HSC into alymphoid BALB/c Rag2 2/2 IL-2Rcc 2/2 newborn mice ( Figure 1A ). As reported previously, multilineage human hematopoietic reconstitution is observed in HIS mice, which demonstrate human thymopoiesis, B cell differentiation, NK cell and plasmacytoid dendritic cell development, and myelopoiesis [10, 13, 14, 15, 16] . Human immune cells accumulate in lymphoid tissues, and several B cell subsets are observed in HIS mice ( Figure 1B) . We analyzed the human B cell repertoire present in naive HIS mice by using B cell receptor (BCR) immunoscope analysis based on quantitative PCR of Ig variable (V H ) and constant (C H ) region gene segments [17] . Due to the lack of human spleen samples, the cells isolated from HIS mouse spleens, which contained sufficient numbers of human B cells to perform the immunoscope analysis, were compared to control human peripheral blood mononuclear cells (PBMC) samples, which were considered acceptable for the purpose of the performed comparison. We observed that IgM-expressing B cells as well as Ig isotype-switched B cells are found in naive HIS mice ( Figure 1C) . The vast majority of B cells of HIS mice expressed an IgM (97.961.0%), whereas IgG (1.861.0%) and IgA (0.0760.04%) expressing B cells represented minor populations. Only the frequency of IgA-expressing B cells was found significantly higher in control human PBMC samples (p,0.0001). At 10-14 weeks post-transplantation (i.e. in steady state conditions), the human Ig concentrations in the blood were 12268 mg/ml (IgM) and 143612 mg/ml (IgG) ( Figure 1D ), as previously reported [8, 16] . In comparison, the normal range for Ig concentration in healthy humans is 400-3100 mg IgM/ml and 7200-14700 mg IgG/ml. In brief, despite a low frequency of IgG-expressing cells, both human IgM and IgG accumulated in the plasma of ,3 month-old HIS mice to levels representing around 10% and 1% of adult human IgM and IgG concentrations, respectively.
We further examined the antigen receptor repertoire diversity in HIS mice, by determining the length of CDR3 hypervariable regions for each Ig-V H gene family. The analysis of CDR3 length distribution of individual HIS mouse splenocytes showed that IgM repertoires are undistinguishable from normal human PBMC IgM repertoires, as measured by the BCR immunoscope analysis ( Figure 1E ). This observation suggests that HIS mice contain a broad variety of naive IgM + B cell clones. The V H -family usage was large and similar to control human PBMC ( Table 1) . The BCR immunoscope analysis was also performed for IgG and IgA repertoires and we observed more restricted repertoires, as expected from B cells undergoing clonal selection and Ig class switch recombination ( Figure S1 ). Immunization of HIS mice with HBV and tetanus vaccines results in the generation of antigen-specific antibody responses
Since HIS mice contained broad naïve B cell repertoires, we analyzed the induction of human antigen-specific B cell responses after immunization with commercially available human vaccines. We designed a vaccination protocol based on repeated intramuscular immunizations (3 injections with 2-week intervals) of 10-14-week old HIS mice with vaccines containing hepatitis B surface antigen (HBsAg) or TT. Seven days after the last immunization mice were sacrificed, the blood and the lymphoid organs were harvested, and the phenotype and function of human cells was analyzed.
All HIS mice showed human reconstitution (.20% hCD45 + cells) in the blood before starting the immunization protocol, which correlated with human engraftment in lymphoid organs. Overall, 42% of HBsAg-vaccinated (8 out of 19 vaccinated animals) and 40% of TT-vaccinated (6 out of 15) HIS mice showed significant production of antigen-specific IgM antibodies, as detected by ELISA (Figure 2A) . We performed a kinetic monitoring of antigen-specific plasma Ig levels in individual HBsAg-vaccinated responder HIS mice and we observed that after the first immunization antigen-specific Igs were rarely detected. In contrast, after the second immunization antigen-specific IgM was detected, which steadily increased after the third immunization with approximately 25-40% of responder mice also showing an antigen-specific IgG response (Figure 2A) . This suggests that repeated vaccination leads to enhanced antigen-specific antibody production. The responder mice exhibited higher total IgM (173641 mg/ml) and total IgG (4596140 mg/ml) concentrations in their plasma, as compared to PBS-injected (IgM: 37612 mg/ml; IgG: 191658 mg/ml) and non-responder vaccinated (IgM: 44611 mg/ml; IgG: 192667 mg/ml) animals ( Figure 2B) .
At the end of the immunization protocol, vaccinated animals showed significantly higher numbers of hCD45 + cells in all organs (i.e. spleen, bone marrow (BM) and mesenteric lymph nodes (mLN)) in comparison to mock-injected control mice. Responder HIS mice exhibited higher numbers of human T and B cells in the spleen, as well as T cells in the BM ( Table 2; Table S1 ), suggesting that the vaccination protocol had a positive impact on the accumulation of human B and T cells. Moreover, the mLN isolated from vaccinated HIS mice contained 4 to 5-fold more hCD45 + cells than those of control animals ( Figure 2C ), suggesting that the mLN structure might play a role in eliciting an immune response in the HIS mice.
In humans, the CD27 + memory B cell population contains the majority of antigen-experienced B cells [23, 24] , and we reasoned that the same should be true in vaccinated HIS mice. We therefore cell sorted several different CD19 + CD27 + B cell subsets from individual HIS mice. We used two strategies to isolate the following human B cell (CD45 + CD19 + ) subsets from BM and spleens of vaccinated HIS mice: (i) CD27 hi CD38 hi , (ii) CD27 + CD38 lo/int IgD + , and (iii) CD27 + CD38 lo/int IgD 2 on the one hand ( Figure 3 - , although the number of cells was increased for each of these subpopulations in the vaccinated animals, as expected from the enhanced number of total B cells ( Table 2) . We only observed a significant increase in the frequency of IgG + B cells within the CD27 hi CD38 hi plasmablast population of vaccinated responder HIS mice, as compared to PBS-injected animals (13.262.7% and 4.361.8% of CD27 hi CD38 hi B cells, respectively; p = 0.0311). In order to identify, isolate and immortalize the antigen-specific antibody-producing B cells, the aforementioned B cell subsets were transduced immediately after cell sorting with a retroviral vector encoding both human BCL6 and BCL-XL [9, 19] . By ectopically expressing BCL6 and BCL-XL in splenic or peripheral blood memory B cells and culturing them with factors produced by follicular helper T cells (CD40L and IL-21), we generated highly proliferative, BCR positive B cell lines that secrete Igs. Since these cells express BCL6, the differentiation of memory B cells to terminal plasma cells is blocked [28, 29, 30] . Therefore, the resulting B cells can expand extensively in vitro for long periods of time in presence of CD40L and IL-21, and provide a tool to generate antigen-specific human BCR-positive, antibody-secreting B cell lines.
The number of isolated cells from spleen and antigen-specific B cell clones that were generated with the BCL6/BCL-XL transduction approach is provided in the Table S2 . Since the frequency of antigen-specific B cell clones was unknown, we started with microcell cultures ranging from 0.6 to 640 cells per well. The wells containing antigen-specific B cells -as determined by HBsAgspecific or TT-specific ELISA -were subsequently cultured by limiting dilution to obtain monoclonal B cell lines. Overall, we generated 15 anti-HBsAg IgM + B cell clones from 3 HIS mice vaccinated with HBsAg, and 18 anti-TT IgM + B cell clones from 3 HIS mice vaccinated with TT ( Table S2 ). The estimated frequency of HBsAg-specific B cells (clones) in the HIS mice after vaccination was 1/350. The IgM secretion level of the B cell clones were in the range of 1 mg per 10 5 cells over 3 days in culture, which was in a similar range of secretion (0.6-5 mg/10 5 cells/3 days) to what was previously reported for B cell clones generated from human blood [9] .
The IgM V H regions of the BCR of the antigen-specific IgM + B cell clones were sequenced. Overall, the BCR of HBsAg-specific and TT-specific B cell clones exhibited a V H sequence close to the germ-line sequence, although limited frequencies of somatic hyper-mutations were observed (Table S3 and Table S4 ). Somatic hyper-mutations were occasionally detected in all framework regions (FR) and complementary determining regions (CDR), and most of the BCR diversity was the result of Nadditions in the CDR3 region. Based on the BCR sequence, we observed that 12 out the 15 anti-HBsAg IgM + B cell clones were unique, as well as 5 out the 18 anti-TT IgM + B cell clones ( Table  S2, Table S3 and Table S4 ).
The supernatants of TT-specific B cell clones were further tested for their capacity to recognize different antigens by ELISA. We observed that IgM mAbs did not cross-react with unrelated antigens (i.e., HBsAg and respiratory syncytial virus (RSV) antigens) ( Figure 4A) . The TT-specific B cell clones were also screened by flow cytometry for direct binding of the TT antigen labeled with a fluorochrome ( Figure 4B) . Interestingly, three types of clones that produced antibodies that gave a similar signal in ELISA were detected, with high, intermediate and low binding of the fluorescent TT antigen.
In the present work we established a new approach to generate fully human mAbs. We immortalized B cells from vaccinated HIS mice by transduction with BCL6 and BCL-XL followed by expansion in presence of IL-21 and CD40L. Antigen-specific B cell clones were obtained that expressed the BCR on their cell surface and secreted antigen-specific antibodies. Similarly to methods based on the immortalization of human memory B cells from individuals that were either vaccinated or exposed to pathogens, our strategy exploits the antibody repertoire of human B cells which is likely to be different from that of B cells of mice expressing human Ig gene segments.
Naïve HIS mice display an extensive human IgM-expressing B cell repertoire. Based on the analysis of the length of the CDR3 regions, this IgM B cell repertoire is similar to the repertoire of healthy individuals. Thus, HIS mice have no obvious limitations for the generation of human IgM mAbs against any possible antigen. Upon intramuscular vaccinations with either TT or HBsAg, approximately 40% of the HIS mice were able to mount an antigen-specific antibody response. Human IgM-producing B cell lines against both antigens were obtained after isolation of memory B cells followed by ex vivo differentiation into plasmablastlike cells. It is important to highlight that the selection of the antigen-specific human B cell clones relied on relevant bioassays (e.g., ELISA or neutralization test). In contrast to EBV-based approaches, human B cell immortalization using transduction with BCL-6 and BCL-XL preserves the expression of the BCR at the surface and antigen-specific B cell clones can also be selected by binding of labeled antigen to the BCR of immortalized memory B cells (e.g. by using a labeled antigen).
Even when IgG was used as a selection criterion, we were unable to establish antigen-specific IgG + human B cell clones. The reason for this might be that T cell help in this system is suboptimal as indicated by the absence of antigen-specific T cell responses after vaccination (not shown). We also observed that the BCR of the B cell clones had a close to germ-line sequence, suggesting that also the induction of somatic hyper-mutation is sub-optimal in HIS mice. In our hands the great majority of the vaccinated HIS mice showed a defective formation of germinal centers [14, 16] , which further explains the absence of antigenspecific Ig-class switched B cells. So far, humanized mouse models based on the transplantation of human HSC only -i.e. without additional human tissues -share these limitations, and immunization strategies result in the limited generation of class-switched antigen-specific B cell responses [14, 31, 32] . Similar patterns are observed in human HSC-transplanted immunodeficient mice infected with lymphotropic pathogens, such as HIV [33] or EBV [34] , although Dengue virus infection in HIS mice was reported to induce an IgG response in a majority of the responder animals [35] . It is not clear why IgG antigen-specific responses are limited while serum IgG can accumulate efficiently, considering the low frequency of IgG + B cells in HIS mice. It remains to be determined whether this apparent discrepancy might be explained by the conjunction of particularly effective IgG production on a cell basis by IgG + B cells (which might occur in a T cell independent manner, such as in the case of the IgG3 subclass), long-term stability of human IgG in the HIS mouse serum as compared to human IgM, and/or defective survival of IgG + B cells under specific conditions (e.g. after antigen-specific triggering of the BCR).
Although IgM mAbs might already be useful for some specific applications or could be modified by Ig class swapping to obtain IgG mAbs [36] , optimized humanized mouse models with improved B cell function are highly desirable. One reason for the suboptimal interaction of T and B cells may be the poor survival resulting in a high turnover of human T cells (discussed in [10, 37] ), making it very likely that procedures leading to improved accumulation of human T cells may promote B cell responses and isotype switching. It was already shown that human B cells undergoing isotype switching can be obtained in humanized mice, provided that a human environment supporting this process is present, e.g. in SCID mice transplanted with human fetal bones, thymus and lymph nodes [38] . Consistent with this notion, enhanced human peripheral T cell accumulation was observed in NOD/SCID mice transplanted with human bone marrow HSC, fetal liver and fetal thymus tissues (referred to as BLT mice), as compared to conventional humanized mouse systems [39] . Interestingly, BLT mice consistently generated an antigen-specific IgG response after HIV-infection [40] . Although it is yet unknown whether the isotype switch observed in BLT mice is truly T cell dependent, those data might support the idea that improved T cell homeostasis has a positive impact on B cell responses. To obtain humanized mouse models with improved B and T cell homeostasis, alternative strategies not relying on the transplantation of human fetal tissues -which are not necessarily easy to access to, for ethical, legal or practical reasons -will likely be favored in the future. The replacement of mouse genes involved in the hematopoietic system by their human equivalent is a valuable strategy to improve development, maintenance and/or function of several hematopoietic cell subsets in humanized mouse models, as shown with cytokines, such as IL-7 and Il-15 [15, 21, 41, 42] , or MHC molecules (N.D.H and J.P.D., manuscript submitted) [43, 44] . The fact that the human CD47 was shown to be unable to properly interact with the mouse SIRPa indicates that reintroducing a functional phagocyte inhibition mechanism via the CD47/SIRPa signaling axis is another strategy of potential interest [45] .
In conclusion, our results show using two standard vaccine antigens the general applicability of an innovative B cell immortalization method in combination with the HIS mouse model to generate human mAbs. Similarly to methods based on the immortalization of human memory B cells from vaccinated or convalescent individuals [9] , our approach exploits the broad antibody repertoire of human B cells, overcoming the potential limitations of conventional humanized murine mAbs such as laboriousness or impaired biological properties, synthetic antibody libraries that require a known target antigen, and transgenic mice bearing the human Ig locus that have limited B cell repertoires. In addition, our method enables to exploit experimental infection models and immunization regimes that would be unethical or untenable in humans. Considering the upcoming advances in HIS mice models [37] , this new approach will provide a powerful tool to generate human mAbs for either diagnostic or therapeutic purposes. Figure S1 IgG/IgA B cell repertoire in naïve HIS mice. Similarly to Figure 1E , the naive IgG (A) and IgA (B) B cell repertoires of HIS (BALB-Rag/c) mice were evaluated on splenocytes by performing a BCR immunoscope for each V H family. The profiles obtained with control human PBMC are also shown. Found at: doi:10.1371/journal.pone.0013137.s001 (1.61 MB TIF) Figure 3 . Limited dilutions of B cells transduced with BCL-6 and BCL-XL were performed with 6.4 and 0.64 cells/well. After sub-cloning of the positive wells, we generated 15 IgM + anti-HBsAg mAbs, of which 13 are unique (as determined by Ig-V H sequence, see Table S3 ), and 18 IgM + anti-TT mAbs, of which 5 are unique (see Table S4 ). In the case of HBsAg vaccination, the number of screened B cells was ((192*6.4)+(96*0.64))*3 = 3870, which eventually suggests that the frequency of HBsAg-specific B cells is at least 1/350 B cells. Found at: doi:10.1371/journal.pone.0013137.s003 (0.13 MB DOC)
IgM V H amino-acid sequence of generated HBsAgspecific B cell clones. The germ-line sequence is given for each V H family, with indication of framework regions (FR) and complementary determining regions (CDR). Highlighted amino-acids correspond to N-additions (in the CDR3 region) and somatic hyper-mutation events, whether it results in a silent mutation (green) or not (red). Clones with identical BCR sequences are grouped together. Found at: doi:10.1371/journal.pone.0013137.s004 (0.02 MB PDF)  Reflections on Pandemic (H1N1) 2009 and the International Response Gabriel Leung and Angus Nicoll provide their reflections on the international response to the 2009 H1N1 influenza pandemic, including what went well and what changes need to be made in anticipation of future flu pandemics. There is general consensus that the only predictable characteristic of influenza viruses and pandemics is their unpredictability [1] . Given such uncertainty, reasonable application of the precautionary principle should prevail in the responses. Indeed many of the initial responses to the 2009 pandemic went well. Once isolated, the pandemic virus strain was shared immediately, specific diagnostic assays were produced and distributed worldwide, antivirals were available in many countries, vaccine development started promptly, and clinical trials demonstrating vaccine safety and immunogenicity were conducted rapidly.
There were many inherently favourable features of the pandemic itself, not all of which were immediately apparent (Table 1) . This was not 1918 Spanish flu. The impact has been mostly confined to the health sector. But that impact has been significant and heterogeneous, with pressure experienced by primary and hospital care (especially intensive care and paediatric services). Distilling descriptions of the impact of a complex public health threat like a pandemic into a single term like ''mild,'' ''moderate,'' or ''severe'' can potentially be misleading [2] . Certainly the experience of hospital clinicians indicated that this pandemic, sometimes described as ''mild to moderate,'' was not limited to only mild or moderate illness. Many patients were severely ill and died, and undoubtedly, high-quality clinical management of patients with severe complications in intensive care units saved many lives of the critically ill, who often required prolonged hospitalisation [3] .
The epidemiology of this pandemic is different than for seasonal influenza epidemics, but not unlike previous pandemics. Young people have been disproportionately affected in terms of hospitalisation and deaths compared to seasonal influenza in which complications and mortality are predominantly borne by the elderly [4] . Similarly, the risk to pregnant women has been higher than for seasonal influenza [5, 6] , which was also noted in previous pandemics. The attributable premature mortality may remain unclear for some time. Recent American analyses have estimated many more deaths than those officially reported with laboratory confirmation of infection and that years of life lost were equivalent to the 1968 pandemic. The lower bound of such estimates is equivalent to the annual burden caused by a typical H3N2 seasonal epidemic in temperate climates [7, 8] . The years-of-life-lost metric captures the impact of a different agespecific mortality pattern which death counts cannot. Deaths involving the young and healthy incur many more potential years of life lost compared to those of older adults and of chronically ill individuals.
There are also a number of ''firsts'' for the 2009 pandemic after an interpandemic period of more than four decades (Box 1). These brought both opportunities and challenges. Under the auspices of the World Health Organization (WHO), the process of a global review by public health specialists from around the world has recently begun. They were nominated by national authorities and are led by an elected chair who assessed the handling of the 1976 swine influenza event among US military personnel at Fort Dix [9] . Here we offer some initial reflections on the first 12 months of the present pandemic.
Considerable effort in recent years had been dedicated to preparing for surveillance during a pandemic and to incorporating modelling in planning in some countries. The pandemic virus was detected and isolated reasonably early, although too late for any attempt at containment. It remains unclear precisely when or where it first emerged, but the earliest human infections were detected in North America and the best estimates of the timing of emergence are variously mid-February from field epidemiology in southeast Mexico or mid-January from a molecular clock model [10] . Situational awareness during the early phase allowed quick assessment by countries, notably those affected first (Mexico, US, Canada, and Southern hemisphere temperate countries). The integration of clinical, laboratory, and epidemiologic data proved essential and gave important insights into disease severity, transmission dynamics, and anticipated impact of interventions. Focused local or national studies with analyses shared through WHO or regional bodies proved more valuable than relying on collection of primary data for analysis in some regions [2] . Although there were modelling efforts underway, only a few governments incorporated such data for policy decisions.
Data from seroepidemiological studies have been limited, primarily due to the lack of routine influenza serosurveys, and
The Essay section contains opinion pieces on topics of broad interest to a general medical audience.
technical challenges with the assays, interpretation, and validation of results. Available serological data on prevalence or seroincidence of humoral immunity yielded age-specific attack rates that indicated a substantial proportion of asymptomatic infections and mild illnesses, similar to or greater than past pandemics and seasonal outbreaks. This was confirmed by a recent Hong Kong study showing the proportion of asymptomatic infection, secondary attack rates, viral shedding, and course of illness among household members were largely similar between infections with seasonal and pandemic influenza virus strains circulating during 2009 [11] . The few published serosurveys revealed heterogeneities in infection rates among different groups and between different places [12] [13] [14] . In particular there appears to be serological evidence of substantial preexisting humoral immunity among older adults, ranging from 23% (1:32 titre by haemagglutination inhibition in those 65 years or over) [14] to 34% (1:80 titre by microneutralisation assay in those 60 years or over) [15] in different studies. Further data on population susceptibility by age or the availability of a rapid and accurate serological test could allow health services to further target vaccine efforts for subsequent waves, as has been done in a few countries [14] .
Early on, some airports installed thermal screening and others asked travellers to declare fever or respiratory symptoms at disembarkation. The utility of these interventions has been repeatedly challenged [16] , although if executed well could delay the start of community transmission by a few weeks [15, 17] (Table 2) . Similarly, during the early stages of global or local spread, quarantine, isolation, school closures, and other social distancing measures were variously implemented in some populations (e.g., Mexico [18] , western Japan [19] , UK), although most have not yet been formally evaluated and published [20] . Two exceptions are in Hong Kong and the UK. In the former, it was estimated that transmission fell by 25% when schools closed [21] . In settings like Hong Kong, with the infrastructure and resources to implement such measures and N Decisions regarding pandemic response during the exigencies of a public health emergency must be judged according to the best evidence available at the time.
N Revising pandemic plans-to be more flexible and more detailed-should wait for WHO leadership if national plans are not to diverge. Surveillance beyond influenza should be stepped up, and contingencies drawn up for the emergence or re-emergence of other novel and known pathogens. [22] but some countries attempted containment in Phases 5 and 6. Some countries even instituted a ''containment phase'' using case-finding and various measures such as isolation and antiviral treatment of ill suspected and confirmed cases, and quarantine of exposed persons with or without antiviral chemoprophylaxis, while others never attempted or quickly moved from resource-intensive containment to mitigation [22] . A preliminary evaluation of intensive containment undertaken in parts of the UK during its spring/summer wave of 2009 demonstrates how resource-and labour-intensive community containment could have been and also how even with a lot of resources the measures had to be abandoned [23] . It is now recognised that the phrase ''containment'' was unfortunate and potentially misleading since at best the actions were only mitigating impact [24] . This pandemic virus transmitted efficiently among children and at least one study has shown that school closures were associated with reduced population transmission when implemented early [21] . Closures appear to have stopped school outbreaks in western Japan and might have also mitigated impact initially on the local communities [25] . However, decisions on this intervention were contextually specific, dependent on feasibility and their potential downsides [26] . In Europe and the US the judgement was generally that proactive school closures would not be justified as a community mitigation intervention in the context of a perceived mild-to-moderate pandemic among the general population, and reserve plans for widespread closure have not been activated in most jurisdictions. However, local decisions were made to close schools in some areas as a response to prevent transmission and high attack rates among schoolchildren or simply where there was too much illness and absenteeism to sustain teaching [21, 27] .
Personal protective interventions such as face masks, hand hygiene, and early isolation may have been beneficial in reducing transmission at the individual level in the home [27, 28] , although household secondary attack rates during the pandemic were similar to those with seasonal influenza [13, 29] . Their population level impact remains to be assessed. There was much debate over whether to use conventional masks or respirators in health care settings. One well-conducted Canadian trial on seasonal influenza virus transmission published during the pandemic suggested no additional advantage from N95 respirators [30] .
Oseltamivir and zanamivir (and later peramivir in some countries) played a role in the mitigation effort, sometimes drawing on national stockpiles. Except for Japan, widespread use of antivirals had not been the norm previously. It became standard to recommend neuraminidase inhibitors for treatment of inpatients and high-risk outpatients, and in restricted circumstances for chemoprophylaxis. Innovative delivery schemes were sometimes developed. Those who fell sick in England could have a telephone assessment (taking pressure off primary care) and then if appropriate receive empiric oseltamivir treatment from a local pharmacist. In Norway oseltamivir was made available ''over the counter.'' However in many European settings, reluctance remained among primary care providers to prescribe a drug they were unused to. Another controversy was whether to offer oseltamivir to all those with symptoms or target those at higher risk for complications. The observational data so far suggest that early treatment with neuraminidase inhibitors have worked to reduce severe disease and have not been linked to significant adverse risks [31, 32] . Late clinical presentation and delayed initiation of antiviral treatment have been implicated with more severe complications worldwide, indicating gaps in identifying and treating patients before disease severity increases. While sporadic cases of oseltamivir resistance have been reported in association with a specific mutation (H275Y in neuraminidase), such oseltamivir-resistant viruses have rarely transmitted [3] . Indeed, the pandemic virus has remained genetically and antigenically stable so far.
The core pharmaceutical preventive intervention was vaccines and this has Box 1. A Series of ''Firsts'' about Pandemic (H1N1) 2009 N The first pandemic to emerge in the twenty-first century. It has been more widespread and remains ongoing, compared to SARS. N The first pandemic to occur after major global investments in pandemic preparedness had been initiated. N The first pandemic for which effective vaccines and antivirals were widely available in many countries, thus requiring public health authorities to earn and retain the confidence of health care providers through whom such are usually distributed.
N The first influenza pandemic to coincide with the ongoing HIV/AIDS pandemic and for which preliminary data do not suggest a substantial, disproportionate impact on HIV-infected patients.
N The first pandemic that took place within the context of a set of International Health Regulations and global governance, which had not been widely tested until the present. N The first pandemic with early diagnostic tests that led to rapid diagnosis but also an early obsession in the media and of policymakers with having reports of the numbers of those infected. N The first pandemic with antivirals available in many countries that led to a hopeful expectation that the pandemic might be containable, leading to the preparation for and implementation of a ''containment phase'' in some places. N The first pandemic in which intensive care was available in many countries to treat critically ill patients, fostering an expectation that everyone could be treated and cured. N The first pandemic with a ''blogosphere'' and other rapid social media messaging tools that challenged conventional public health communication.
been a particular focus for critics citing the uneven and suboptimal uptake across countries. Development of a pandemic vaccine was a scientific success, but limited availability until after the autumn/winter wave had nearly peaked in the Northern Hemisphere contributed to lower coverage than anticipated [33] . Vaccination coverage depended on many factors, including availability, preordering, licensing and bureaucratic hurdles, logistics, convenience, and, most crucially, public and professional perceptions. This pandemic presented a particular risk communication challenge, since while infection usually results in mild illness, occasionally it is lethal, even in the young and previously healthy despite optimal treatment [34] [35] [36] . In the absence of any excess risk of serious side effects compared to annual seasonal vaccines [37] (despite the intensive effort to look for such) the benefits of immunisation far outweighed any potential down-sides at the individual level, particularly for those at higher risk for complications. Notwithstanding such evidence, the cost of pandemic vaccines was considerable and a loss of public confidence has sometimes been triggered by unsubstantiated media reports of serious side effects with a ''new vaccine'' that utilised the same manufacturing technology as for years of seasonal vaccines. Uptake among health care providers as role models has been mixed, as has their expression of the need for vaccination at all. This sometimes cast doubt in the minds of the public. Conversely, pandemic deaths in young healthy people abruptly changed public perception (such as in Canada, Romania, and Finland); supply and organisational issues then became crucial.
Another more fundamental criticism challenges whether vaccines should have been procured at all given an eventual surplus in the developed North. The unexpected finding that a single dose was immunogenic among all persons except for younger children, which reduced the required number of doses by half from the projected number needed in most countries, but this was not known in advance of countries placing vaccine orders. Had there been ''overpreparation''? The prior worry had been the reverse -would there be sufficient production capacity to meet needs [38] ? Even in retrospect, and with the observed burden of the pandemic, a vaccine was clearly justified for countries where annual vaccines for seasonal influenza are routinely recommended.
Field and pharmacovigilance data so far have shown that these vaccines were immunogenic, effective, and very safe [39] . However, the frailty was timing and availability. Generally supplies came in later and in smaller amounts than forecasted, in part due to lower yield in growth of the vaccine virus strain than expected. Reduce and delay community spread somewhat at the earliest stage to allow better preparation for mitigation response [15] Completely prevent entry of infected individuals due to suboptimal sensitivity and asymptomatic (including infected and within incubation period) or subclinical presentation [16] Many countries did not attempt these measures because of logistics, stage of pandemic [22] or other cost-benefit considerations [16] China Hong Kong SAR Japan Personal protective measures (e.g., face masks, hand hygiene, cough etiquette, early self-isolation when ill) Reduce risk of infection to self and close contacts (if self is ill and infected) [27, 28] Have not been evaluated whether they can provide significant populationlevel protection
Virtually all countries implemented these measures to varying degrees in health care settings according to the risk of the situation. Almost all encouraged hand hygiene, cough etiquette, and early self-isolation
Most countries recommended adoption of hand hygiene, cough etiquette, and early self-isolation when ill, but use of face masks in the community was uncommon except in East Asia.
Antivirals for treatment and chemoprophylaxis [21, 22] Mitigation: reduce illness severity and complications if administered early; reduce transmission from those receiving treatment; sometimes also used as chemoprophylaxis in high-risk circumstances Provide significant population-level protection or allow containment Attempts at source containment were not possible, as the pandemic was effectively already in WHO Phase 5 when what became the pandemic virus was first identified [22] . Initial observational studies suggest antivirals were successful when early treatment was administered Canada Germany Hong Kong SAR Japan UK US (these populations attempted the intervention initially but effort was not sustained towards the later stages of the pandemic)
Vaccines Mitigation (a) at individual level by conferring immunity to infection in those at higher risk of severe disease or (b) at a population level by immunizing population groups especially those who are transmitting most (i.e., children)
In most countries vaccine was not available early enough and/or arrived in insufficiently large amounts to achieve mitigation at a population level. Greater population benefit may occur in the next season Most of the orders arrived after the peak of the autumn/winter wave in the geographic north (whose countries had received most vaccines). Therefore, judgement on their impact in averting serious morbidity and deaths may come only after the second winter. Perhaps then, differential use by countries will allow for comparisons where there is good surveillance for severe disease and deaths.
There have been claims of extraneous influence on the independent and objective judgment of expert advice that in turn influenced decision-making [40] . These claims have been robustly countered as they relate to WHO's advisory and decision-making process [41] . As Harvey Fineberg, chair of WHO's external review, pointed out, when assessing any allegations of impropriety or bias, or the perception of such, it would be important to distinguish between financial or other conflicts with potential pecuniary gains versus predispositions arising from an individual's background and experience. Rather than aiming for a complete purge of any and all experts who had worked with vaccine manufacturers and received sponsorship, as these are often the very same group who possess the most relevant and useful expertise precisely because they have been closely involved in the research and development process, the focus should be on making the declaration of such interest wholly transparent and comprehensive according to a set of robustly established procedures that can withstand the strictest scrutiny. It is reassuring that WHO has honoured its commitment to making public the names and declarations of interest of the pandemic Emergency Committee when the pandemic was declared over on 10 August 2010. Additionally, receiving advice should be differentiated from making decisions. The people entrusted with undertaking the latter task should then judge the validity of the advice rendered by experts, having taken into account their interest declarations. The decision makers should also be prepared to justify their actions.
It is important to learn from our experience through the first year and beyond as we move into the new seasonal influenza [42, 43] . It is theoretically possible, although unlikely, that the second winter of this pandemic will be worse than the first, as happened for the 1968 pandemic when transmissibility increased [44] . Equally, if the pandemic virus outcompeted the A(H3N2) virus strains responsible for more intense seasonal epidemics, there may even be a diminution of disease burden in older people. As of this writing, seasonal influenza A (H3N2) and B virus strains continue to cocirculate. Antigenic drift in the 2009 H1N1 virus is expected to occur in the future, especially under the pressure of so many people now being immune through infection or immunisation, although the timing is unpredictable. The pandemic virus is included in the trivalent seasonal influenza vaccine composition for both hemispheres. Clear communication of public health messages will remain a particular challenge and not confusing what could happen (and should be prepared for) with what is most likely to happen. In assessing the pandemic response, decisions made during the exigencies of a public health emergency must be judged according to the best evidence available at the time. Hindsight always gives perfect vision and using post-hoc information to evaluate prior decisions at best confuses and often produces unfair conclusions.
Preparedness plans will have to be revised in due time, after the many lessons learned have been gathered. This should be done quickly in case the worst is not yet over [45] . However, rewriting plans should best wait for WHO leadership if national plans are not to diverge. A strong argument exists for making future plans more flexible and having extra descriptions including the many aspects of severity when a pandemic is emerging that then determine the consequential public health actions [2] . Broadening surveillance for a range of influenza A viruses among a wide range of animals (e.g., swine), not just in avian species, as well as strengthening the monitoring of seasonal influenza virus infections in humans will facilitate identification of novel influenza A viruses of pandemic potential, and earlier detection of the emergence of a pandemic virus. More broadly we should look beyond influenza and draw up contingencies for the emergence or re-emergence of other novel and known pathogens [45] .
One challenge faced initially in this pandemic was for timely collection and sharing of clinical data to inform optimal management of critically ill patients worldwide. Establishing clinical research infrastructure prior to a pandemic and a central institutional review board will facilitate data collection and analyses [46] , whether for the next influenza pandemic, SARS outbreak, or next novel respiratory pathogen of global importance. Clinical management of severe influenza disease should not be limited to the current antiviral regimen, and include the development of other therapeutics (e.g., novel antivirals and immunotherapy).
Ongoing improvements in the routine and timely monitoring of hospital admissions and deaths attributable to influenza, as well as representative serological surveys at regular intervals can provide epidemiological data with which to reduce uncertainty around the true burden of influenza and thus inform policy choices [47] .
Assessment of the humoral and cellular immune response over time in a subset of vaccinated individuals could reveal how vaccine-induced immunity differs from natural infection, and whether cross-reactive responses to other influenza virus strains are modulated by the two types of immunological response [48] . The latter could become important as the pandemic strain has already been cocirculating with other interpandemic influenza A virus strains in some parts of the world.
Greater access to antivirals and influenza vaccines worldwide is an ongoing challenge. Although WHO secured pledges of 200 million vaccine doses and monies for operations, and more than 80 less-resourced countries have signed agreements with WHO for supply of vaccines, this gap remains. It is an indefensible fact that these vaccines started to flow to the poorer countries well after they began going to the countries with advance purchase arrangements. Delivering timely pandemic influenza vaccination in countries without existing seasonal vaccine programmes is proving difficult. The longterm solution has to be improved surveillance, expanded monitoring of disease burden, and better prevention and control of influenza, including the development of seasonal vaccine use and production in all regions of the world [49] . Increased coverage of available bacterial vaccines (Hib, pneumococcal) will help prevent secondary invasive bacterial coinfections with either seasonal or pandemic influenza.
Finally accusations of ''overreaction'' can be countered by the observation that investment in fire services or insurance is usually judged against their ability to respond to conflagrations. If the first test is a lesser fire, that experience should be used for improvements rather than as a reason to scrap the fire engines and cancel the insurance [40] . Development of a large-scale isolation chamber system for the safe and humane care of medium-sized laboratory animals harboring infectious diseases The close phylogenetic relationship between humans and non-human primates makes non-human primates an irreplaceable model for the study of human infectious diseases. In this study, we describe the development of a large-scale automatic multi-functional isolation chamber for use with medium-sized laboratory animals carrying infectious diseases. The isolation chamber, including the transfer chain, disinfection chain, negative air pressure isolation system, animal welfare system, and the automated system, is designed to meet all biological safety standards. To create an internal chamber environment that is completely isolated from the exterior, variable frequency drive blowers are used in the air-intake and air-exhaust system, precisely controlling the filtered air flow and providing an air-barrier protection. A double door transfer port is used to transfer material between the interior of the isolation chamber and the outside. A peracetic acid sterilizer and its associated pipeline allow for complete disinfection of the isolation chamber. All of the isolation chamber parameters can be automatically controlled by a programmable computerized menu, allowing for work with different animals in different-sized cages depending on the research project. The large-scale multi-functional isolation chamber provides a useful and safe system for working with infectious medium-sized laboratory animals in high-level bio-safety laboratories. In high-level bio-safety laboratories, animals infected with Risk Group 3 pathogens (as defined by the World Health Organization) must be housed in isolation chambers (World Health Organization, 2004) .
In an effort to minimize the risks for scientists working with these animals, a new isolation chamber was designed in a bio-safety lab. The chamber was designed mainly to separate the internal controlled environment from the external environment, and the operator from the experiment and the experimental products. The primary aim was to prevent crosscontamination from the internal to the external environment or vice versa (Kruse et al., 1991; Wathes and Johnson, 1991; Huang, 2005; Tattershall, 2006) . Isolation chambers were designed to improve experimental safety by preventing this cross-contamination, reducing the likelihood of operator error, and minimizing the contaminated area. In addition, the comfort level of scientists working with the isolation chambers was taken into account. The safety level of these chambers allows for the operator to avoid wearing too many protective suits, which improves both the comfort and flexibility of the operator (Sawyer et al., 2007) .
There are two main types of isolation chambers: positive pressure and negative pressure. In general, positive-pressure isolation chambers are used to ensure that specified-pathogen-free (SPF) laboratory animals housed in the chambers are protected against outside contaminants (Hurni, 1981; Clough et al., 1995) . Negative-pressure isolation chambers are used with infectious animals housed in the chambers to prevent migration of hazardous contaminants to the outside (Wathes and Johnson, 1991) .
Both rigid and soft barriers are commonly used for physical separation in the construction of isolation chambers (ISO 14644-7:2004) . Rigid barriers can be made of many different materials, and the more rigid the material, the more reliable the physical barrier. Construction of these rigid barriers usually makes use of plastic enclosures, metal profile enclosures, or hot-worked metal enclosures (ISO 10648-1:1997) . The isolation chamber is designed to house a living animal, and therefore continuous airflow inside the enclosure is needed to drive out heat and moisture generated by the animal's metabolism and to decrease the concentration of odor, dust, and infectious substances (Hillman et al., 1992) . The resulting exhaust gas is subject to a filtering system designed to prevent pathogen contamination of the external environment (Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council, 1996) . The aerodynamics is allowed for one-way flow or turbulence of the airflow inside the isolation chamber, under negative pressure relative to the environment. Supply and exhaust air can be passed through high-efficiency particulate air (HEPA) filters to prevent the formation of aerosols that could potentially escape into the environment (Runkle et al., 1969) .
A double port transfer exchange (DPTE) system is commonly used in isolation chambers to allow the transfer of experimental materials from one chamber to another without exposing the experimental material to the outside environment (Allen et al., 2009) .
The acronym DPTE was originally derived from the French phrase 'double porte de transfert etanche', meaning double door sealed transfer or double door transfer port. A newly validated rapid transfer port boasts bi-directional transfer as one of its features, a system also known as an α-β transfer port or rapid transfer port (RTP) (Michael et al., 2004; Tsai et al., 2009) .
Non-human primates with a close phylogenetic relationship to humans are often susceptible to a number of diseases and parasites found in humans (Tauraso et al., 1969) . To establish an unknown pathogen as the cause of a disease Koch's postulates should be followed (Fouchier et al., 2003; Pan and Sun, 2004; Conly and Johnston, 2008) . The similarity of genetic, physiological, and behavioral characteristics between humans and non-human primates, and the occurrence of similar pathological changes upon infection, have led to non-human primates becoming an irreplaceable animal model for the study of pathogen infection, the screening of anti-pathogen drugs, and vaccine evaluation (Conly and Johnston, 2008) . Because of the critical role that non-human primates play in the study of these pathogens, it is critical to find safe and reliable methods for their physical containment.
In the present study, a negative-pressure isolation chamber was designed and an initial attempt at building the chamber was completed. Two extraction blowers are used, one working and one on standby, to ensure the isolation chamber internal pressure is both stable and secure. The isolation chambers contain two DPTE systems for the transfer of experimental materials and the collection of excrement into a small container. The sterilization chain for the isolation chamber is composed of a peracetic acid sterilizer and its associated pipeline. A programmable logic controller (PLC) system and WINCC 6.0 system are used to automatically control and monitor the isolation chamber. Rapid pressure changes and the exhaust system are adjusted via a variable frequency drive inlet air blower and extraction blower. Isolation chamber parameters are menu-managed by the automatic control system allowing the researcher to set different isolation environmental parameters depending on experimental requirements.
2 Negative-pressure isolation chamber system design
The structure of the negative-pressure isolation chamber system includes a top ventilation unit, an isolation chamber working zone, a lower part of the control system, and an isolation chamber support frame (Fig. 1) .
The isolation chamber working zone is composed of a stainless-steel box and two front doors. The welded box was manufactured using dumb-gloss stainless-steel 316L with a thickness of 3 mm. The interior chamber dimensions are 400 cm (L)×120 cm (W)×120 cm (H). The isolation chamber front door includes two damping braces on each front side with a dual-piston mechanism for holding the front door securely open to let the animal cages in, two operation panels with stainless-steel 316L framework, and two door hinges connected to the stainless-steel box. The operation panel is made of transparent polymethylmethacrylate (PMMA). The transparent front door allows for visualization of the contents of the isolation chamber. Silicone seals around the PMMA panel ensure that the system is air tight. The front door is manually fastened onto the box framework with a hammer bolt. There are seven circular polyethylene (PE)-machined glove ports on the operation panels. The port inner diameter is 300 mm and the center-tocenter spacing of each port is 450 mm. The glove port assembly includes a glove port ring, glove port gasket, pressure ring, and glove port inner-securing ring. The glove port ring and glove port inner-securing ring are jointed with a thread connection. The glove port ring edges are fixed on the PMMA panel by compressing the glove port gasket and pressure ring on each side of the PMMA panel by tightening a fixing screw.
The changeable sleeve and glove combination is mounted on the glove port through a sleeve fixing ring that secures the elastic Hypalon sleeve onto the glove port inner-securing ring. A glove port bung connects the glove and sleeve. Neoprene glove shapes are ambidextrous.
The transfer system for the isolation chamber is composed of two DPTE systems. One round, 270-mm diameter DPTE system with an α transfer door is built into the left wall of the isolation chamber using a transfer port assembly kit. The transfer port assembly kit includes the DPTE transfer port external flange, DPTE transfer port external flange sealing ring, and DPTE transfer port internal flange. The DPTE transfer port external flange is fixed onto the inside isolation chamber wall by compressing the DPTE transfer port external flange sealing ring on the outside of the isolation chamber wall with a tightening fixing screw.
The animal transfer container is autoclavable and contains a β door that can be manually docked to the port. The transfer container is 50 cm deep and is capable of transferring a 6-kg monkey into the isolation chamber. The transfer container can be autoclaved without compromising its containment and can be opened with a specialized tool to remove the sterilized waste. This system works very well for rapidly and safely transferring experimental materials and animals.
Another 270-mm diameter DPTE system with an α transfer door is mounted on the bottom of the isolation chamber. The autoclavable canister using a β door can dock to the port and with a depth of 20 cm is more suitable for transferring smaller materials such as animal blood samples. This type of short bottom transfer canister can be opened with a specialized tool in the biological safety cabinet, and the samples can then be removed for further stages of analysis (e.g., centrifugation), while the transfer canister is closed and put into a plastic bag for autoclaving.
The animal excrement collection system is composed of a disinfection pool, animal excrement collection DPTE transfer container, and scrubbing tools. The disinfection pool is oblong, has a slightly tilted bottom and a perimeter wall, and is close to and higher than the bottom transfer port. A drain valve is installed in the disinfection pool outfall. One end of a flexible diversion silicone tube is connected to the drain valve, and the other end touches the autoclave plastic bag inside the animal excrement collection DPTE transfer container docked to the bottom transfer port. The transfer container depth is 50 cm. There are stainless-steel lockable wheels under the bottom of the animal excrement collection DPTE transfer container.
One gas-tight water service valve with a serrated hose is mounted on the left-side of the interior. A spray gun is connected to the serrated hose for cleaning the isolation chamber.
Four primate cages can be placed on the stainless-steel-cage slideways in the isolation chamber. The slideways are attached to the isolation chamber bottom in a manner that allows cage movement in a direction along the axis perpendicular to the axis of the isolation chamber front door. If the primate cages are removed, other animal cages such as rabbit cages and guinea pig cages, can also be placed on the detachable frame, but each time only one kind of animal species cage can be inside.
The isolation chamber is supported by two type 304 stainless-steel isolation chamber support stands. The control cabinet stainless-steel shelf and fasteners are mounted to the right-side tubular frame of the base stand and provide a work surface to support the control cabinet.
Stainless-steel slideways are also mounted on the top of the isolation chamber box. The pipes, air blower, valve, and adjustable illumination lamp are fastened to the reserved mounting holes or mounting plates of the slideways by a fixing screw.
Two extraction blowers share one exhaust port in which an anemometer is installed. Each of the extraction blowers is connected with its own coupling clamp to the outlet ventilation pipe, exhaust ventilation pipe, and exhaust electronic control ball valve to form an exhaust channel. The two exhaust channels have a parallel connection. Two sets of sterilizing agent bypass tubes have a series connection with an ipsilateral sterilizing agent bypass electronic control ball valve and a parallel connection with the same side of the exhaust ventilation pipe on two ends of the exhaust electronic control ball valve. All of the valves are automatically controlled by the PLC.
The airflow direction through the isolation chamber via the air inlet and outlet is shown in Fig. 1 . Room air is drawn into the interior of the isolation chamber through the inlet pre-filter, inlet air blower, inlet ventilation pipe, inlet air electronic control ball valve, and inlet HEPA filter in the animal breeding mode. The air from the isolation chamber is drawn out of the exhaust export through the exhaust pre-filter, exhaust HEPA filters A and B arranged in series, exhaust ventilation pipe, and exhaust electronic control ball valve via an extraction blower. The HEPA filters are arranged in series to ensure that if one fails, the other can still ensure exhaust security and prevent pathogens from being discharged into the atmosphere.
During the sterilization process, the inlet air blower, inlet air electronic control ball valve, exhaust electronic control ball valve, and extraction blower are all closed. Following this closure, the sterilizing agent by-pass electronic control ball valve is opened. The sterilizing agent in the peracetic acid sterilizer evaporation tank is heated and its vapors are pushed by compressed air into the volume to be sterilized by a sterile connecting hose, a sterilization reagent import, a coupling clamp for the inlet ventilation pipe and an inlet HEPA filter. The exhaust sterilizing vapors escape from the interior of the isolation chamber through an exhaust pre-filter, two-in-series HEPA filters, a sterilizing agent by-pass tube, a sterilizing agent by-pass electronic control ball valve, and an extraction blower to the exhaust export.
The isolation chamber interior pressure is controlled by automated instrumentation that is connected to the supply and exhaust ventilation system. The automatic pressure regulation system is capable of maintaining the relative pressure inside the isolation chamber via the exhaust ventilation system, which can account for transient volume changes such as glove entry or withdrawal.
A videotape system mounted on the control touch panel stainless-steel box on the side of the isolation chamber box includes a rotatable camera and camera-installation base. The camera installation base is fastened to the control touch panel stainless-steel box by fixing screws. The position of the rotation camera can be adjusted by using the telescopic locking nut and rotating locking nut. This system enables recording of both the scientist's experimental procedures and the status of animals living in the isolation chamber. The video is displayed on the personal computer (PC) screen and saved automatically in the central control room through the control interface connected to the videotape system.
To ensure the comfort and welfare of animals in the isolation chamber, chambers are equipped with an automatic light control system and a television entertainment system. The automatic light control system includes adjustable illumination lamps and a lampshade. The adjustable illumination lamps are composed of three cold light lamps and their conditioners. The illumination system can be used to meet the needs of the animal's physiology, as well as experimental requirements. The television entertainment system consists of a flat television and two transparent television installation boxes fastened to the front door by fixing screws. The animal is able to watch television programs that are controlled by the central control room. The purpose of this design is to reduce depression associated with the space constraints faced by the animals and to ensure the ethical treatment of the animals.
The composition of the isolation chamber control system is depicted in Fig. 1 . This system includes an alarm indicator light tower, liquid crystal display (LCD) touch screen, and control cabinet. The LCD touch screen is fastened on the outside of the isolation chamber by a fixing screw.
The PLC is built into the control cabinet. The control cabinet, which has a fan and a filter cover, is mounted onto the stainless-steel shelf of the isolation chamber support frame through fasteners and a fixing screw.
Step 7 software installed in the PLC central processing unit (CPU) and through the LCD touch screen enables users to automatically control a variety of options. The animal breeding mode program, containment test program, sterilization program, auto/manual control mode, maintenance mode, and custom programs can all be automatically controlled by the PLC CPU and associated touch screen.
The management system for the isolation chamber touch screen was developed by Simatic WINCC flexible software. The operation can be recorded and printed, as can any system failures. The data interchange between the PLC and touch screen is made possible through an industrial trunk Profibus decentralized periphery (DP).
The temperature, humidity, illumination, atmospheric pressure, and air flow velocity are measured by appropriate sensors, and the values are imported to the PLC through control lines. Normal value ranges for each parameter can be programmed into the PLC, and if the parameter values deviate from the set upper and lower limits, the PLC automatically adjusts the interior environment of the isolation chamber to match the programmed values. For example, the levels of humidity, illumination, and ventilation can all be controlled by the PLC to adjust values back to pre-determined normal levels. If the PLC is unable to bring the parameter back into a normal range, the digital output module in the PLC lights an alarm bulb and sounds a buzzer, as all alarms are indicated with both a warning light and sound.
The control system controls alarms for a variety of isolation chamber problems including major equipment error alarms (such as the air blower or HEPA), major parameter alarms when values are out of the desirable ranges (such as temperature or humidity), an alarm when switching to the uninterruptible power supply (UPS)/emergency power supply (EPS), and alarms for experimental failure or error (such as negative-pressure breeding mode procedures or pressure test procedures).
To control pressure, a micro-differential pressure sensor is mounted on the side of the first exhaust filter. The analog module in the PLC compares the values of program settings with the values collected from the micro-differential pressure sensor, and automatically conducts proportional-integral-differential (PID) regulation. The adjusted output values are used to control blower velocity through the output module of the PLC, regulating the isolation chamber internal pressure. If a plug or leak occurs, the microdifferential pressure sensor transmits the detected signal to the PLC. If the detected values are beyond the scope of the pre-loaded high and low pressure settings, the exhaust electronic control ball valve and inlet air electronic control ball valve automatically shut down to maintain the isolation chamber as a fully-contained environment and to prevent the escape of pathogens into the outside environment. At the same time an alarm indicator light tower would start to sound an alarm, and the touch-screen would display information on the alarm. The alarm information would then be transmitted to the lab server through the industrial Ethernet module in the PLC. The alarm message displayed is recorded onto the lab server for analysis at a later date.
The blower rotation rate and frequency are automatically controlled by the PLC system to ensure that the airflow velocity, air exchange rate, and atmospheric pressure match the set values. If one exhaust blower fails, the PLC system responds by switching to another backup exhaust blower to ensure ventilation safety and the internal negative-pressure state of the isolation chamber.
The cold light lamp regulator is controlled by the PLC digital output module to automatically adjust the illumination time (12 h/12 h or 10 h/14 h light/dark cycle) according to animal behavior. The illumination time and intensity (5-10, 15-20, and 100-200 lx) can be set from the touch screen by the operator and automatically executed. The lamps also can be switched on or off manually to meet different lighting requirements during an experimental procedure.
Temperature and humidity sensors are fitted within the isolation chamber. The isolation chamber internal temperature is maintained at 18-22 °C, and relative humidity is kept within 40%-70%. The isolation chamber internal temperature and humidity readings are collected by the PLC analog module, visualized as the project value (actual values of temperature and humidity), and automatically displayed and recorded on the touch screen. The values are also recorded on the lab server.
To operate the isolation chamber, the main power on the control cabinet is first turned on. The current operating parameters are displayed on the touch screen interface and can then be adjusted by the human operator by following the interface prompts. The isolation chamber should be monitored for 48 h to ensure that it is running normally. This includes supplying filtered air to the isolation chamber and ensuring that the exhaust air is cleaned by the double in-line HEPA filters and passed through the exhaust air system into the open air. Fresh air exchanges should be conducted at a rate of about 36 times per hour. Following 48 h of monitoring, the inside temperature is 22-23 °C, the relative humidity is 60%, the working negative pressure in the isolation chamber is adjusted to −100 Pa with respect to the laboratory. Appropriate anesthesia should be used on healthy medium-sized animals to pass them through the quarantine system and into the isolation chamber via the DPTE system. The animal experiment should be performed according to bio-safety operation standard procedures while the animal is still under the appropriate anesthesia. If the gloves are removed during the operation, a low pressure audible/visual alarm system is activated, and a minimum velocity value of 0.65 m/s in the center of the glove port is maintained. If a glove is damaged by a needle, the blowers are capable of maintaining the isolation chamber pressure at −100 Pa, but the alarm system would not be activated because the micro-differential pressure sensor is not sensitive enough for a leak of this size. Proper procedure dictates that the small hole be labeled by the operator and a new glove exchanged. All of the feeds, experimental material, waste, feces, and other materials can be transferred into or out of the isolation chamber by the DPTE system. There are several isolation chambers in one room, and the autoclave does not connect with any of them. Instead, DPTE β canisters are filled with items and are then sterilized in the autoclave. After sterilization, the sterile items are removed after opening the β door with specialized tools. The sterile items are then sent to a centralized disposal center for medical waste. Following the completion of studies with single animals, each animal is treated as dictated by bio-safety operation standards and general animal welfare. The cadavers are autoclaved in β canisters and sent to animal carcass disposal sites. The isolation chamber is connected to the peracetic acid sterilizer to sterilize its interior.
The newly designed multi-functional isolation chamber system reported here achieves multiple technical improvements: (1) By using variable frequency drive blowers as the inlet air and extraction blowers, adjustments for rapid pressure changes (e.g., insertion of gloves) can occur automatically without breaching the inert atmosphere. The extraction blowers contain an integrated backup system with one blower running at full strength and another on standby to act as a backup. Negative or positive pressure state is kept at a stable and safe level through the automatic control system. The pressure is adjusted depending on different requirements for different animals and/or experimental conditions. (2) A DPTE 270 α door mounted on one side wall of the isolation chamber allows for the transfer of experimental materials. Another DPTE 270 α door is mounted on the bottom of the isolation chamber near the disinfection pool with a slightly tilted bottom. Animal excrement can be discharged to the DPTE container via the flexible silicone diversion tube. The excrement collection package is then minified. An experimental sample can also be transferred to the bio-safety cabinet through a shorter DPTE container. (3) The control cabinet installation is comprised mainly of the PLC, electric element (which includes the voltage transformer, secure alternating current contactor, circuit breaker, electric cable, indicating lamp, and button), printer port (for data output), and industrial Ethernet interface (which allows for the remote data control of multiple isolation chambers by the WINCC 6.0 program system). Automatic control and monitoring of the isolation chamber and sterilization system are achieved by the exchange of data between the touch screen and control cabinet through the industrial bus Profibus DP to meet different laboratory animal research project parameter requirements such as pressure, humidity, temperature, illumination, and disinfection. A human operator can set the isolation chamber environment parameters according to the requirements of the infectious animals or for cleaning animals, allowing for the acquisition of adequate and authentic data. (4) Animal welfare is ensured by installing adjustable illumination lamps, a rotatable camera, a flat television, a micro-differential pressure sensor, and a temperature humidity sensor to maintain comfortable living conditions for the animal.
In the future we will report on the use of this isolation chamber in specific experiments to further demonstrate its versatility and usefulness. Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed. The term proteomics describes the study and characterization of complete set of proteins present in a cell, organ, or organism at a given time [1] . In general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. The human genome harbors 26000-31000 protein encoding genes [2] ; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (PTMs), has been estimated to be close to one million [3, 4] . It is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions [5] . Moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than 100000 copies of a single protein [6] . In spite of new technologies, analysis of complex biological mixtures, ability to quantify separated protein species, sufficient sensitivity for proteins of low abundance, quantification over a wide dynamic range, ability to analyze protein complexes, and high throughput applications is not yet fulfilled [7] . Biomarker discovery remains a very challenging task due to the complexity of the samples (e.g., serum, other bodily fluids, or tissues) and the wide dynamic range of protein concentrations [8] . Most of the serum biomarker studies performed to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire blood proteome [9] . Processing and analysis of proteomics data is indeed a very complex multistep process [10, 11] . The consistent and transparent analysis of LC/MS and LC-MS/MS data requires multiple stages [12] , and this process remains the main bottleneck for many larger proteomics studies. To overcome these issues, effective sample preparation (to reduce complexity and to enrich for lower abundance components while depleting the most abundant ones), state-of-the-art mass spectrometry instrumentation, and extensive data processing and data analysis are required. A wide range of proteomic approaches are available such as gel-based applications include one-dimensional and twodimensional polyacrylamide gel electrophoresis [13, 14] , and gel-free high throughput screening technologies are equally available, including multidimensional protein identification technology [15] , isotope-coded affinity tag ICAT [16] ; SILAC [17] ; isobaric tagging for relative and absolute quantitation (iTRAQ) [18] . Shotgun proteomics [19] and 2DE DIGE [20] as well as protein microarrays [21, 22] are applied to obtain overviews of protein expression in tissues, cells, and organelles. Large-scale western blot assays [23] , multiple reaction monitoring assay (MRM) [24] , and label-free quantification of high mass resolution LC-MS data [25] are being explored for high throughput analysis. Many different bioinformatics tools have been developed to aid research in this field such as optimizing the storage and accessibility of proteomic data or statistically ascertaining the significance of protein identifications made from a single peptide match [26] . In this review we attempt to provide a overview of the major developments in the field of proteomics, some success stories as well as challenges that are currently being faced.
About 20-30% of all genes in an organism encode integral membrane proteins, which are involved in numerous cellular processes [27] . Membrane proteins constitute 30% of the typical proteome, yet their propensity to aggregate and precipitate in solution confounds their analysis [28] . The target residues for tryptic cleavage (i.e., lysine and arginine) are mainly absent in transmembrane helices and preferentially found in the hydrophilic part of these lipid bilayer-incorporated proteins. Because of the protein aggregation step of IEF, 2DE is unsuitable for the separation of integral membrane proteins and is limited to detection of membraneassociated proteins and membrane proteins with a low hydrophobicity [29] . Membrane solubilization methods have been deployed to analyze enriched membrane fractions and address the solubility issue by using detergents [30] , organic solvents [31] , and organic acids [32] compatible with subsequent proteolytic digestion/chemical cleavage, separation and analysis by LC/MS. In this approach, (1) an enriched yeast membrane fraction is solubilized with 90% formic acid in the presence of cyanogens bromide. The concentrated organic acid provides the solubilization agent, and cyanogen bromide, functional under acidic conditions, allows many embedded membrane proteins to be cleaved, (2) a membrane-enriched microsomal fraction is solubilized by boiling in 0.5% SDS and, following isotope-coded affinity tag (ICAT) labeling, is diluted to reduce the concentration of SDS, and (3) by using an enriched membrane sample, the proteins are thermally denatured and sonicated in 60% organic solvent (methanol) in the presence of trypsin. The resultant peptide mixture is then analyzed by LC/MS. All three of these methods are effective and optimize the identifications of membrane proteins. Another method using high pH and protenase K is optimized specifically for the global analysis of both membrane and soluble proteins [33] . High pH favors the formation of membrane sheets, while proteinase K cleaves exposed hydrophilic domains of membrane proteins. Commercially available nonionic detergents, dodecyl maltoside, and decaethylene glycol mono hexadecyl are proved most efficient membrane protein solubilizers [34] . Another more successful approach to isolate membrane proteins relies on cell surface labeling in combination with high resolution two-dimensional (2D) LC-MS/MS [35] . In addition, improved analytical tools should be developed, that is, multidimensional liquid chromatography of peptide mixtures generated from membrane proteins, nanoflow chromatographic techniques for hydrophobic transmembrane peptides, and native electrophoresis of membrane protein complexes, which, in combination with mass spectrometry, should lead to the identification of the majority of proteins in the membrane proteome of simple microorganisms. It is important to quantify not only the identified membrane proteins but also to determine the levels of interacting partners. Subcellular fractionation techniques that employ a combination of centrifugation steps are a common choice for preparing plasma membrane-(PM-) enriched fractions including detergent-resistant membrane fractions, commonly known as lipid rafts. These methods can offer a significant improvement in specificity for PM proteins over approaches that do not perform any subcellular fractionation, but rather use whole-cell or tissue preparations [36] . Chemical-tagging methods [37] have been a more applied technique used to enrich for PM proteins and are often used in conjunction with physical separation strategies. This method allows for a specific class of protein or modification of interest to be physically separated from other nontagged proteins. Importantly, when chemical tags are attached to the extracellular domain of PM proteins on intact cells, they offer an unrivaled specificity for PM proteins, because they offer a manner to distinguish true PM proteins from intracellular contaminants. Cell-surface biotinylation, the covalent attachment of a biotin tag to the extracellular domain of PM proteins, is also a popular choice [38] [39] [40] .
Serum is a complex body fluid, containing a large diversity of proteins. More than 10000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [41] . Serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact all tissues. Consequently, serum proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [42] . However, serum has been termed as the most complex human proteome [43] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [44] . The analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [45] . Albumin is a protein of very high abundance in serum (35-50 mg/mL) that would be a prime candidate for complete selective removal prior to performing a proteomic analysis of lower abundance proteins. Thus, removal of albumin from serum may also result in the specific removal of low abundance cytokines, peptide hormones, and lipoproteins of interest. Immunoglobulins, and antibodies are also abundant proteins in serum that function by recognizing "foreign" antigens in blood and initiating their destruction [46] . The presence of higher abundance proteins interferes with the identification and quantification of lower abundance proteins (lower than ng/mL in serum). Complexity and dynamic range of protein concentrations can be addressed with a combination of prefractionation techniques that deplete highly abundant proteins and fractionate. Heparin chromatography coupled with protein G appears to be an efficient and economical strategy to pretreat serum for serum proteomics [47] . Protein prefractionation by immunodepletion and reversed-phase separation of the depleted plasma on mRP-C18 column provide methods compatible with LC-MS-based analysis. A polyclonal antibody-based system to rapidly deplete multiple high abundant proteins in serum, plasma, CSF, and other biological fluids. Individual antibody materials are mixed in selected percentages and packed into a column format. Albumin can be removed by immunoaffinity columns [48] , isoelectric trapping [49] , dye-ligand chromatography [50] , and peptide affinity chromatography [51] . Another approach involves the removal of IgG by affinity chromatography using immobilized protein A or protein G [52] . A recently developed depletion method that mixes 6 high-specificity polyclonal antibodies (MARS) to remove the top 6 proteins in a single purification step is commercially available [53] . Human-14 multiple affinity removal column depletes the top 14 abundant proteins from human serum, plasma, CSF, and other biological fluids. To address 2D limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements [54] . In contrast, 2D-PAGE analysis, SELDI-TOF MS is a rather new method which is especially valuable for the identification of serum-derived biomarkers [55] . This method is based on ProteinChip Arrays which carry various chromatographic properties, such as anion exchange, cation exchange, and hydrophilic or hydrophobic surfaces [56] . For the analysis of serum, only 5-10 μL of serum sample is applied to these surfaces; after washing off unbound material, the protein fingerprint can be determined and visualized by time-of-flight mass spectrometry. The advantages of this method are the low amount of sample necessary for analysis, its speed, and high throughput capability. Many different groups have used this method and related methods based on prefractionation of serum proteins by beads and subsequent MALDI analysis for the identification of biomarkers in serum, urine, pancreatic juice, and other biological fluids [57] . The necessity of this removal or separation is also illustrated that many proteins found useful as biomarkers [58] . Different fractionation steps (such as electrophoresis, SELDI, and liquid chromatography) have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins [59] . 2DE and MALDI MS had applied to identify candidate biomarkers at early and late stages of lung cancer disease. This method identified 46 proteins in tumor bearing mice this included disease regulated expression of orosomucoid-8, a-2-macroglobulin, apolipoprotein-A1, apolipoprotein-C3, glutathione peroxidase-3, plasma retinol-binding protein, and transthyretin [60] . Recently 1065 proteins were identified by stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation with tandem mass spectrometry of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer [61] . Specimen collection (Blood, serum, plasma samples) is an integral component of clinical research. Access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research [62] . Variables that may impact analytic outcomes include (1) the type of additive in the blood collection tubes; (2) sample processing times or temperatures; (3) hemolysis of the sample; (4) sample storage parameters; (5) the number of freeze-thaw cycles [63, 64] .
The key variable in any analysis is that the case and control samples are handled in the exact same manner throughout the entire analytical process from study design and collection of samples to data analysis [63, 65] . These types of differences between samples could have a significant impact on the stability of proteins or other molecules of interest in the specimens. Small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. A representative working group, standard operating procedures internal working group, comprised of members from across early detection research network should be formed to develop standard operating procedures (SOPs) for various types of specimens collected and managed for biomarker discovery and validation work.
Limitations of Two-Dimensional Electrophoresis. Figure 1 gives the general work flow in proteomics and Table 1 addresses their strengths and limitations. Two-dimensional electrophoresis (2DE) was developed two decades before the term proteomics was coined [66, 67] . The 2DE entails the separation of complex protein mixtures by molecular charge in the first dimension and by mass in the second dimension. 2DE analysis provides several types of information about the hundreds of proteins investigated simultaneously, including molecular weight, pI and quantity, as well as possible posttranslational modifications. 2DE is extensively used but mostly for qualitative experiments and this method falls short in its reproducibility, inability to detect low abundant and hydrophobic proteins, low sensitivity in identifying proteins with pH values too low (pH < 3) or too high (pH > 10) and molecular masses too small (Mr < 10 kD) or too large (Mr > 150 kD) [2] [3] [4] [5] . Poor separations of basic proteins due to "streaking" of spots and membrane proteins resolution [68] are limiting factors in 2DE. However, 2DE is the only technique that can be routinely applied for parallel quantitative expression profiling of complex protein mixtures such as whole cell and tissue lysates [69] and most widely used method for efficiently separating proteins, their variants and modifications (up to 15000 proteins). There are two ways to study posttranslational modifications by means of 2DE. First, posttranslational modifications that alter the molecular weight and or pI of a protein are reflected in a shift in location of the corresponding protein spot on the proteomic pattern. Second, in combination with Western blotting, antibodies specific for posttranslational modifications can reveal spots on 2DE patterns containing proteins with these modifications [70] . Protein extraction and solubilization are key steps for proteomic analysis using 2DE, highly hydrophobic proteins tend to precipitate during isoelectro focusing (IEF), low copy number and the insolubility of transmembrane proteins renders quantitative analysis of these peptides and polypeptides are very challenging [71] . In order to enhance protein extraction and solubilization, different treatments and conditions are necessary to efficiently solubilise different types of protein extracts [72, 73] . The major challenge for protein visualization in 2DE is the compatibility of sensitive protein staining methods with mass spectrometric analysis. Therefore, several fluorescent staining methods have been developed for the visualization of 2DE patterns, including sypro stainings and Cy-dyes [74] . Although sypro ruby [75] and silver staining [76, 77] have a comparable sensitivity, sypro ruby staining allows much higher reproducibility, a significantly wider dynamic range and less false-positive staining. In addition, sypro ruby allows for the detection of lipoproteins, glycoproteins, metalloproteins, calcium-binding proteins, fibrillar proteins, and low molecular weight proteins that are less "stainable" using other methods. Finally, a large number of protein spots on 2DE patterns contain several proteins with a similar pI. A pH gradient with a narrow range allows zooming into different proteins with the same molecular weight. Increased separation distance 40 × 40 cm gels using CA-IEF [78] could increase the proteome coverage up to 5000 proteins. Use of overlapping narrow range IPGs "Zoom" gels and increase in separation area could yield better membrane protein separation [79] . This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins.
Proteins can also be fluorescently labelled with Cy2, Cy3, or Cy5 prior to 2DE [80] . CyDyes are cyanine dyes carrying an N-hydroxysuccinimidyl ester reactive group that covalently binds the e-amino group of lysine residues in proteins. During DIGE [81] , proteins in each of up to 3 samples can be labelled with one of these fluorescent dyes, and the differentially labelled samples can be mixed and loaded together on one single gel, allowing the quantitative comparative analysis of three samples using a single gel ( Figure 2 ). The DIGE technique has exhibited higher sensitivity as well as linearity, eliminated postelectrophoretic processing (fixing and destaining) steps and enhanced reproducibility by directly comparing samples under similar electrophoretic conditions [81, 82] . The resulting images are then analyzed by software such as De-Cyder which are specifically designed for 2D-DIGE analysis [83] . The major advantages of 2D-DIGE are the high sensitivity and linearity of its dyes, its straightforward protocol, as well as its significant reduction of intergel variability, increasing the possibility to unambiguously identify biological variability, and reducing bias from experimental variation. Moreover, the use of a pooled internal standard, loaded together with the control and experimental samples, increases quantification accuracy and statistical confidence [84] . The DIGE technique has dramatically improved the reproducibility, sensitivity, and accuracy of quantitation; however, its labeling chemistry has some limitations; proteins without lysine cannot be labeled, and they require special equipment for visualization, and fluorophores are very expensive [83, 85] . Tag (ICAT) . Gel-free, or MS based, proteomics techniques are emerging as the methods of choice for quantitatively comparing proteins levels among biological proteomes, since they are more sensitive and reproducible than two-dimensional gel-based methods. ICAT is one of the most employed chemical isotope labeling methods and the first quantitative proteomic method to be based solely on using MS [86, 87] . Each ICAT reagent consists of three essential groups: a thiol-reactive group, an isotope-coded light or heavy linker, and a biotin segment to facilitate peptide enrichment. In an ICAT experiment, protein samples are first labeled with either light or heavy ICAT reagents on cysteine thiols. The mixtures of labeled proteins are then digested by trypsin and separated through a multistep chromatographic separation procedure. Peptides are identified with tandem MS, and the relative quantifications of peptides are inferred from the integrated LC peak areas of the heavy and light versions of the ICAT-labeled peptides [88] . The ICAT concept has been widely used after its introduction [89] [90] [91] . Different software programs were developed to analyze ICAT labeled MS data (e.g., proICAT from Applied Biosystems, spectrum Mill from Agilent Technologies, and Sashimi from the Institute of System Biology [92] ). ICAT is extremely helpful to detect peptides with low expression levels, which is one of the bottleneck issues in analytic protein techniques [93, 94] . However, major limitations of this technique include selective detection of proteins with high cysteine content and difficulties in the detection of acidic proteins [95, 96] . The methods for direct comparison of DIGE and ICAT for the identification and quantification of proteins in complex biological mixtures are also being considered [97] .
While the ICAT reagent only interacts with the free sulfhydryl of homocysteine and 8% protein is noncysteine, the SILAC has emerged as a valuable proteomic technique [98] which becomes more common for cell types and have been applied in many fields [99] [100] [101] . The SILAC technique can be effectively expanded to compare the differential expression levels of tissue proteome at different pathological states, which allows to identify new candidate biomarkers [102] . Compared with the ICAT, a popular in vitro labeling, SILAC as an example of in vivo coding requires no chemical manipulation, and there is very little chemical difference between the isotopically labeled amino acid and its naturally occurring counterpart [103] . In addition, the amount of labeled proteins requires for analysis using SILAC technique is far less than that with ICAT. Therefore, the SILAC-based method has broadly applied in many areas of cell biology and proteomics. Except that the SILAC-based quantitative method is powerful in comparative/differential proteomics, it has been widely used in analyzing protein posttranslational modification, such as protein phosphorylation, detection of protein-protein or peptide-protein interactions and investigating signal transduction pathways [104, 105] .Though there are numerous advantages for using SILAC-based methods compared to chemical labeling, a major drawback of SILAC is that it cannot be applied to tissue protein analysis directly. To overcome this shortcoming, SILAC has been successfully applied to tissue proteome based on 15 N isotope labeling [106] . Microorganisms such as malaria parasite can be labeled with isoleucine [107] . Latterly the culturederived isotope tags (CDITs) method was developed as an alternative quantitative approach for studying the proteome of mammalian tissues based on the application of SILAC [108] .
18O Stable Isotope Labeling. Differential 16O/18O coding relies on the 18O exchange that takes place at the Cterminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen exchange in the presence of H218O [109] . The resulting mass shift between differentially labeled peptide ions permits identification, characterization, and quantitation of proteins from which the peptides are proteolytically generated. In contrast to ICAT, 18O labeling does not favor peptides containing certain amino acids (e.g., cysteine), nor does it require an additional affinity step to enrich for these peptides [110] . Unlike iTRAQ, 16O/18O labeling does not require a specific MS platform nor does it depend on fragmentation spectra (MS2) for quantitative peptide measurements. It is amenable to the labeling of human specimens (e.g., plasma, serum, tissues), which represents a limitation of metabolic labeling approaches (e.g., SILAC). Taken together, recent advancements in the homogeneity of 18O incorporation, improvements made on algorithms employed for calculating 16O/18O ratios and the inherent simplicity of this technique should result in increased use of 18O labeling [111] . In general, 18O labeling suffers from two potential drawbacks, inhomogeneous 18O incorporation and inability to compare multiple samples within a single experiment. A dual 18O labeling using a non-gel-based platform has been developed to overcome the major problems of existing proteolytic 18O labeling methods [112] .
(iTRAQ). The iTRAQ reagent is well known for relative and absolute quantitation of proteins. The iTRAQ technology offers several advantages, which include the ability to multiplex several samples, quantification, simplified analysis and increased analytical precision and accuracy [113] [114] [115] .
The interest of this multiplexing reagent is that 4 or 8 analysis samples [116] can be quantified simultaneously.
In this technique, the introduction of stable isotopes using iTRAQ reagents occurs on the level of proteolytic peptides ( Figure 3 ). This technology uses an NHS ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on N-methylpiperazine) to proteolytic peptides via the formation of an amide bond [117] . Due to the isobaric mass design of the iTRAQ reagents, differentially labelled peptides appear as a single peak in MS scans, reducing the probability of peak overlapping. When iTRAQ-tagged peptides are subjected to MS/MS analysis, the mass balancing carbonyl moiety is released as a neutral fragment, liberating the isotope-encoded reporter ions which provides relative quantitative information on proteins. An inherent drawback of the reported iTRAQ technology is due to the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity and this approach needs a powerful multidimensional fractionation method of peptides before MS identification.
Prefractionation of proteins based on electrokinetic methodologies in free solution essentially relaying on the isoeletric focusing (IEF) has gained wide acceptance. Many commercial devices are now constructed to take the advantage of this principle ( Table 2 ). Reproducible fractionation steps will break down the sample complexicity while concentrating low abundant species, resulting in more confident protein identifications and quantification by 2D gels, mass spectrometry, and protein arrays. A good example of a innovation is liquid-phase isoelectric focusing (IEF) as a prefractionation tool before the first dimension of 2D gel electrophoresis [118, 119] . For more consistent pI separation, the Zoom IEF fractionator [120] and multicompartment electrolyser (MCE) [121] are being used to prefractionate the proteins. The fractionated samples can be directly applied on standard narrow range IPG strips for 2D electrophoresis. This allows at least 10000 to 15000 separate proteins to be analyzed, including proteins of very low abundance. IEF, a highresolution electrophoresis technique, has been widely used in shotgun proteomic experiments [122] . IEF runs in a buffer-free solution containing carrier ampholytes or in immobilized pH gradient (IPG) gels. The use of IPG-IEF for the separation of complex peptide mixtures has been applied to the analysis of plasma and amniotic fluid [123, 124] as well as to bacterial material [125] . The IPG gel strip is divided into small sections for extraction and cleaning up of the peptides. This technique recovers the sample from the liquid phase and was demonstrated to be of great interest in shotgun proteomics [126] . IEF is not only a high resolution and high capacity separation method for peptides, it also provides additional physicochemical information like their isoelectric point [127, 128] . The pI value provided is used as an independent validating and filtering tool during database search for MS/MS peptide sequence identification [129] . The recent introduction of commercially available OFFGEL fractionator system by Agilent Technologies provides an efficient and reproducible separation technique [130] . This separation is based on immobilized pH gradient (IPG) strips and permits to separate peptides and proteins according to their isoelectric point (pI) but is realized in solution [131] . Its micropreparative scale provides fraction volumes large enough to perform subsequent analyses as reverse phase (RP)-liquid chromatography (LC)-MALDI MS/MS. The combined use of iTRAQ labeling and OFFGEL fractionation methods for the proteomic study of complex sample is also being considered [132, 133] .
In this procedure, a large well is used to separate the sample by PAGE and lanes are created on the membrane containing immobilized protein with the use of a manifold [134] . Compatible combinations of primary antibodies are predetermined, with the criterion of being able to identify proteins that do not comigrate. Different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. The scalability of the system depends on defining suitable combinations of primary antibodies, with up to 1000 antibodies in 200 lanes being used in the largest screens. Detection software is used to identify proteins based on their expected and observed gel mobility. Unlike 2D PAGE and HPLC-MS/MS, large-scale western blotting only identifies proteins for which antibodies are already available. While this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. In addition, this approach is highly flexible, and can be focused to particular sets of proteins or protein function, such as cell signaling molecules. Importantly, the foundation of this approach is the large amount of data on individual antibodies, which are already available and characterized in the literature [135] .
Another approach to analyse proteomes without gels is "shotgun" analysis using MudPIT [136] . In the MudPIT approach, protein samples are subject to sequencespecific enzymatic digestion, usually with trypsin and endoproteinase lysC, and the resultant peptide mixtures are separated by strong cation exchange (SCX) and reversed phase (RP) high performance liquid chromatography (HPLC) [137, 138] . Peptides from the RP column enter the mass spectrometer and MS data is used to search the protein databases [138] . The MudPIT technique generates an exhaustive list of proteins present in a particular protein sample, it is fast and sensitive with good reproducibility however, it lacks the ability to provide quantitative information [139] [140] [141] . A combination of HPLC, liquid phase isoelectric focusing, and capillary electrophoresis provides other multimodular options for the separation of complex protein mixtures [142] .
High throughput production of human proteins using different methods is being developed to make protein array approach more practical. Recently simple and efficient production of human proteins using the versatile gateway vector system has been developed [143] . In this approach, protein expression system is applied to the in vitro expression of 13364 human proteins and assessed their biological activity in two functional categories and developed "human protein factory" infrastructure which includes the resources and expression technology for in vitro proteome research. In another approach, DNA array to protein array (DAPA) is utilized, which allows the "printing" of replicate protein arrays directly from a DNA array template using cellfree protein synthesis [144] . Based on the nucleic acid programmable protein array (NAPPA) concept, high-density self-assembling protein microarray is developed to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates [145] . This method will enable various experimental approaches to study protein function in high throughput. The adventage of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, proteinnucleic acid, and protein-small molecule interactions, as well as posttranslational modifications [146, 147] . The microarray format provides a robust and convenient platform for the simultaneous analysis of thousands of individual protein samples, facilitating the design of sophisticated and reproducible biochemical experiments under highly specific conditions [148] . The principal challenges in protein array development are 3-fold: (1) creation of a comprehensive expression clone library; (2) high-throughput protein production, including expression, isolation, and purification; (3) adaptation of DNA microarray technology to accommodate protein substrates [149] . Functional protein microarrays differ from analytical arrays in that functional protein arrays are composed of arrays containing fulllength functional proteins or protein domains (Figure 4) . These protein chips are used to study the biochemical activities of an entire proteome in a single experiment. They are used to study numerous protein interactions, such as protein-protein, protein-DNA, protein-RNA, proteinphospholipid, and protein-small molecule interactions [150, 151] . Companies have introduced protein arrays aimed not only at proteomic analysis but also functional analyses of proteins (e.g., Biacore AB, Ciphergen Biosystems Inc., Phylos Inc.). Affinity proteomics aim to produce antibodies to every protein expressed by the human genome and these will be characterized against purified antigens and tested on tissue arrays to collect information about their specificity for tissue antigens [152] . Companies are focused to produce various binding partners, for example, affibodies, monoclonal antibodies, and their fragments [153] . Protein chips will likely be the next major manifestation of the revolution in proteomics and offer another solution to analyze low abundant proteins and have the potential for high throughput applications to identify biomarkers [154] . Protein chips differ from previously described methods; whereas screening by 2DE or LC MS/MS can potentially detect any protein, and protein chips can only provide data on set of proteins selected by the investigator [155] .
The development and application of high throughput, multiplex immunoassays that measure hundreds of known proteins in complex biological matrices, is becoming a significant tool for quantitative proteomics studies, diagnostic discovery, and biomarker-assisted drug development. Two broad categories of antibody microarray experimental formats have been developed [156] , direct labelling, single antibody experiments [157] , dual antibody, sandwich immunoassays are described [158, 159] . In the direct labelling method, all proteins in a complex mixture are tagged, providing a means for detecting bound proteins following incubation on an antibody microarray. In the sandwich immunoassay format, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. In addition, a variety of microarray substrates have been described, including nylon membranes, plastic microwells, planar glass slides, gel-based arrays and beads in suspension arrays.
Much effort has been expended in optimizing antibody attachment to the microarray substrate. Finally, various signal generation and signal enhancement strategies have been employed in antibody arrays, including colorimetry, radioactivity, fluorescence, chemiluminescence, quantum dots and other nanoparticles, enzyme-linked assays, resonance light scattering, tyramide signal amplification, and rolling circle amplification. Each of these formats and procedures has distinct advantages and disadvantages, relating broadly to sensitivity, specificity, dynamic range, multiplexing capability, precision, throughput, and ease of use. In general, multiplexed microarray immunoassays are ambient analyte assays [160] . Given the heterogeneity of antibody array formats and procedures currently in use in proteomics studies, and the absence of a "gold standard," there exists an urgent need for development and adoption of standards that permit platform comparisons and benchmarking.
Regardless of the choice of a given proteomic separation technique, gel-based or gel-free, a mass spectrometer is always the primary tool for protein identification. During the last decade, significant improvements have been made in the application of MS for the determination of protein sequences [161] . Mass spectrometers consist of an ion source, the mass analyzer, and an ion detection system. Analysis of proteins by MS occurs in three major steps (a) protein ionization and generation of gas-phase ions, (b) separation of ions according to their mass to charge ratio, and (c) detection of ions [162] . In gel-free approaches such as ICAT and MudPIT, samples are directly analyzed by MS whereas, in gel-based proteomics (2DE and 2D-DIGE), the protein spots are first excised from the gel and then digested with trypsin. The resulting peptides are then separated by LC or directly analyzed by MS. The experimentally derived peptide masses are correlated with the peptide fingerprints of known proteins in the databases using search engines (e.g., Mascot, Sequest). There are two main ionization sources which include matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) and four major mass analyzers, which are time-of-flight (TOF), ion trap, quadrupole, and fourier transform ion cyclotron (FTIC) which are currently in use for protein identification and characterization [163] . A combination of different mass analyzers in tandem such as quadrupole-TOF and quadrupole-ion trap has combined the individual strengths of different types of mass analyzers and greatly improved their capabilities for proteome analysis [162] . Simple mass spectrometers such as MALDI-TOF are used for only measurement of mass, whereas tandem mass spectrometers are used for amino acid sequence determination [164] . In MALDI the sample of interest is crystallized with the matrix on a metal surface and a laser ion source causes excitation of matrix along with the analyte ions, which are then released into the gas phase. MALDI measures the mass of peptides derived from a trypsinized parent protein and generates a list of experimental peptide masses, often referred to as "mass fingerprints" [165, 166] . In ESI, the analyte is ionized from a solution and transferred into the gas phase by generating a fine spray from a high voltage needle which results in multiple charging of the analyte and generation of multiple consecutive ions. Tandem mass spectrometry or MS/MS is performed by combining two different MS separation principles. In tandem MS, individual trypsin-digested peptides are fragmented after a liquid phase separation. Tandem MS instruments such as triple quadrupole, quadrupole ion trap, fourier transform ion-cyclotron resonance, or quadrupole time-of-flight are used in LC-MS/MS or nanospray experiments with electrospray ionization (ESI) to generate peptide fragment ion spectra [167] . Ion mobility spectrometry (IMS) has been utilized as a rapid gas-phase separations strategy for biomolecular ions [168, 169] . The strategy provides high sensitivity because the gas-phase dispersion of peptide ions separates features corresponding to low abundance species from interfering chemical noise [170] . Reduced spectral congestion also allows for the use of shorter experimental run times (LC separations) without sacrificing throughput; short analysis time scales are key to measuring the large numbers of samples required to determine normal protein variability prior to realizing individual plasma profiling. Additionally, mobility-dispersed ions can be fragmented and mobility linked to fragment ions without ion loss from precursor mass selection [171] . These advantages have been demonstrated in head-to-head comparisons with conventional LC-MS/MS technology using rapid (21 minutes) LC gradients [169] . Accurate mass and time (AMT) tag approach [172] addresses an analogous situation in LC-MS-based proteomics studies. In this approach, initial LC-MS/MS analyses are performed on prefractionated peptide samples in order to provide peptide sequence identifications. These experiments are relatively low throughput because the peptide prefractionation can be quite extensive and require separate LC-MS/MS analyses for each fraction. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-negative bacterium Rhodobacter sphaeroides 2.4.1. There has been a recent trend in proteomics toward the development and application of technologies for the targeted analysis of proteins within complex mixtures [173] . Selected reaction monitoring (SRM) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest [174, 175] . The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. The use of tandem mass spectrometry data acquired on an LTQ ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an SRM assay using a triple quadrupole mass spectrometer [176] . One of the biggest benefits of a targeted assay on a triple quadrupole mass spectrometer is high throughput. Using the selectivity of multiple stages of mass selection of a tandem mass spectrometer, these targeted SRM assays are the mass spectrometry equivalent of a Western blot [173] . An advantage of using targeted mass spectrometry-based assay over a traditional Western blot is that it does not rely on the creation of any immunoaffinity reagent. While its application is novel in the proteomics community, SRM has been utilized for several decades in the toxicology and pharmacokinetics disciplines [177] . Peptidebased immunofractionation methods show potential for proteome wide screening approaches but are limited by the availability of antibodies [178, 179] . The stable isotope standards with capture by antipeptide antibodies (SISCAPA) approach is based on the addition of stable isotope labeled standard peptides to the digested clinical sample followed by immunoaffinity enrichment of standard and analyte peptide by highly specific antipeptide antibodies [180, 181] . This approach enables the absolute quantification of selected diagnostic peptides from digested clinical samples down to physiologically relevant analyte concentrations (ng/mL) at high precision (10% CV) and accuracy [178, 179] . Further improvement of MRM-based biomarker quantification should be possible if whole sets of analyte peptides can be enriched by immunofractionation. Since this method relies on one specific antibody per target protein/peptide the generation of more than 10000 antibodies is necessary for proteome wide screening approaches. Novel peptide affinity enrichment strategies enabling proteome wide analyses of signature peptides may provide an important addition to future proteome workflows. Undoubtedly, the accuracy, high throughput, and robustness of MS technologies have made the characterization of entire proteomes a realistic goal [180, 181] .
The major bottlenecks in proteomics research today are related to data analysis to create an environment where computer scientists and biologists and the people who collect data can work closely together, so they can develop the necessary analytical tools that will help interpret the data [182] [183] [184] . Processing and analysis of proteomics data is indeed a very complex multistep process ( Figure 5 ). The meaningful comparison, sharing, and exchange of data or analysis results obtained on different platforms or by different laboratories remain cumbersome mainly due to the lack of standards for data formats, data processing parameters, and data quality assessment. Accurate, consistent, and transparent data processing and analysis are integral and critical parts of proteomics workflows [185] . We can now generate huge amounts of data, and currently there is an enormous challenge to figure out how to actually analyze this data and generate real biological insights. The necessity of an integrated pipeline for processing and analysis of complex proteomics data sets has therefore become critical.
Validation. This step consists of the assignment of MS/MS spectra to a database search using one of several engines available (e.g., Sequest, Mascot, Comet, X!tandem, etc.). One of the difficulties related to the use of sequest for peptide identifications is the lack of methods to globally evaluate the quality of data and the lack of methods to access global changes created by filtering schemes and/or database changes [186] . Most approaches are matching and scoring large sets of experimental spectra with predicted masses of fragment ions of peptide sequences derived from a protein database. Results are scored according to a scheme specific to each search engine that also depends on the database used for the search. Usually tools are linked to one specific platform or were optimized for one instrument type. The various search engines do not yield identical results as they are based on different algorithms and scoring functions, making comparison and integration of results from different studies or experiments tedious [187, 188] . Peptide identification via database searches is very computationally intensive and time-demanding. High quality data allow more effective searches due to tighter constrains, that is, tolerance on precursor ion mass and charge state assignment, which will drastically reduce the search time in case of an indexed database. In addition, accurate mass measurements of fragment ions further simplify the database searches and add confidence to the results. The association of identified peptides with their precursor proteins is a very critical and difficult step in shotgun proteomics strategies as many peptides are common to several proteins, thus leading to ambiguous protein assignments. Therefore it becomes critical to have an appropriate tool that is able to assess the validity of the protein inference and associate a probability to it. Protein Prophet database tool combines probabilities assigned to peptides identified by MS/MS to compute accurate probabilities for the proteins present [189] . 5:1921-1926, 2006. impossible. The lack of common standards and protocols has led to this situation and often resulted in duplication of efforts. Results were usually reported as a set of identified proteins (i.e., list of peptides identified and associated proteins) with minimal supporting data. Obviously the large volume of such data sets has made publication of detailed results using classical mechanisms very challenging. Sharing and exchange of data and results requires the definition of standard formats for the data at all levels (including raw mass spectrometric data, processed data, and search results) as well as a better definition (and/or standardization) of the parameters used for the data processing or the database searches.
Organellar proteomics aims to describe the full complement of proteins of subcellular structures and organelles. Identification of the proteins contained in subcellular organelles has become a popular proteomics endeavor [190] . When compared with whole-cell or whole-tissue proteomes, the more focused results from subcellular proteomic studies have yielded relatively simpler datasets from which biologically relevant information can be more easily extracted [191] . Subcellular fractionation consists of two major steps, disruption of the cellular organization (homogenization) and fractionation of the homogenate to separate the different populations of organelles. Such a homogenate can then be resolved by differential centrifugation into several fractions containing mainly (1) nuclei, heavy mitochondria, cytoskeletal networks, and plasma membrane; (2) light mitochondria, lysosomes, and peroxisomes; (3) golgi apparatus, endosomes and microsomes, and endoplasmic reticulum; (4) cytosol.
Each population of organelles is characterized by size, density, charge, and other properties on which the separation relies [192] . Analyzing subcellular fractions and organelles allows tracking proteins that shuttle between different compartments, for example, between the cytoplasm and nucleus. A high dynamic range of proteins can be partially achieved by fractionation of the proteome into subproteomes by applying affinity purification may allow proteomic analysis of low copy number proteins [193] . The nuclear, chloroplast, amyloplast, plasma membrane, peroxisome, endoplasmic reticulum, cell wall, and mitochondrial proteomes were successfully characterized in Arabidopsis [194] . Several groups have taken advantage of this approach to recover a higher percentage of membrane proteins from subcellular extracts using various nonionic and zwitterionic detergents or phase-partitioning methods. These efforts resulted in the successful determination of the protein complement of the thylakoid and envelope membrane systems of the chloroplast [195] . By enriching for the protein class of interest based on a particular chemical/physical characteristic(s), offer the advantage of reducing sample complexity and access to lower abundance proteins in a discoverydriven experimental approach [196] . Free flow electrophoresis (FFE) utilizes differences in electrophoretic mobility rather than density to separate cells or subcellular organelles [197] . FFE has previously been used in separating endosomes from hamster ovary cells [198] , plasma membrane from human platelets [199] , and insulin transporting vesicles in liver cells. The separation is based on the electrophoretic motility of cells or cell organelles suspended in a vertical free flowing buffer film on which an electric field is applied at a right angle to the flow direction. FFE has been a most valuable tool in the investigation of the composition of secretory vesicles and in addition, it has clarified how the membrane of plasma membrane vesicles is oriented after nitrogen disruption of human neutrophils [200] . Importantly, subcellular fractionation is a flexible and adjustable approach that may be efficiently combined not only with 2D gel electrophoresis but also with gelindependent techniques. However, they do have limitations of considerable cross-contamination with other subcellular organelles.
PTMs of proteins are considered to be one of the major determinants regarding organisms complexity [201] . To date, at least more than 200 different types of PTMs have been identified of which only a few are reversible and important for the regulation of biological processes. Specific functions are usually mediated through PTMs, such as phosphorylations, acetylations, or glycosylations, which places additional demands on the sensitivity and precision of the method [202] . One of the most studied PTMs is protein phosphorylation, because it is vital for a large number of protein functions that are important to cellular processes spanning from signal transduction, cell differentiation, and development to cell cycle control and metabolism. Enzymes and receptors can be switched "on" and "off " by phosphorylation and dephosphorylation. It was estimated that 10-50% of proteins are phosphorylated. Phosphorylation often occurs on serine, threonine, and tyrosine residues in eukaryotic proteins [203] . Analysis of the entire cellular phosphoproteome has been an attractive study subject since the discovery of phosphorylation as a key regulatory mechanism of cell life. Unfortunately, phosphoproteins analysis is not straightforward for five main reasons. First, the stoichiometry of phosphorylation is generally relatively low, because only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. Second, the phosphorylatation sites on proteins might vary, implying that any given phosphoprotein is heterogeneous (i.e., it exists in several different phosphorylated forms). Third, many of the signaling molecules, which are major targets of phosphorylation events [204] , are present at low abundance within cells and, in these cases; enrichment is a prerequisite before analysis. Fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, and minor sites might be difficult to identify. Finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates. In addition, various methods for protein phosphorylation site determination have been developed, yet this task remains a technical challenge [205] . Western blot has been widely used to determine the presence of PTMs. However, this technique relies on the prior knowledge of the type and position of specific modifications and the availability of antibodies. It has low throughput and not ideal for studying highly complicated samples. Specific chemical or affinity enrichment steps are usually incorporated into the sample preparation or fractionation stages of the general scheme of proteomic studies [206, 207] . Well established methods involving the analysis of 32P-labeled phosphoproteins by Edman degradation and two-dimensional phosphopeptide mapping have proven to be powerful but not without limitations. Consequently, mass spectrometry (MS) has emerged as a reliable and sensitive method for the characterization of protein phosphorylation sites [208] and may therefore represent a method of choice for the analysis of protein phosphorylation [209] . Immobilized metal affinity chromatography (IMAC), Metal oxide affinity chromatography (MOAC), and covalent methods are all capable of selectively enriching phosphopeptides [210] . MOAC based on adsorption to TiO2 is especially attractive, but as with all techniques, loading, rinsing, and elution solutions must be carefully selected to minimize nonspecific adsorption and to maximize the detection of both monophosphorylated and multiphosphorylated species. IMAC might not provide the selectivity available with TiO2 enrichment, but with appropriate reagents, IMAC can be selective and sensitive for monophosphorylated and tetraphosphorylated peptides. However, some buffers and reagents such as EDTA are not compatible with IMAC, so HPLC purification may be needed prior to this technique [211] . When trying to isolate and identify as many phosphoproteins as possible in a cell lysate, chromatographic column-based methods are required. Multiple elutions from IMAC or MOAC columns or even gradient elutions can help to simplify fractions of proteins and reveal more peptides [212, 213] . A combination of techniques can reveal large numbers of phosphopeptides in complex samples, but comprehensive phosphoproteomics is still not possible. For the highest protein coverage, future phosphoproteomic techniques will likely employ multiple enrichment techniques along with two-dimensional separations, but such studies are time consuming. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies, and mass spectrometry are particularly attractive for systematic investigation of posttranslationally modified proteins in proteomics [214] .
Organisms. The application of proteomics and related technologies for the analysis of proteome is severely hampered by the lack of publicly available sequence information for most of the unsequenced organisms [215] . Despite the precision of the mass information yielded by the SELDI technique, a significant number of proteins were found to have no similarity to known peptides, an aforementioned weakness of proteomics studies in nonmodel organisms [216] . In order to circumvent this limitation, different strategies and tools were developed to make unsequenced organisms amenable to high-throughput proteomics [217] (Figure 6 ). However, an evaluation of their performance in an integrated proteomics strategy using high-throughput shotgun MS data is currently missing. In principle, two different approaches can lead to an increase in protein identifications from unsequenced organisms. In the first approach, MS/MS data are searched against a protein database of an evolutionarily closely related organism. However, as a matter of principle of database-dependent searches, only proteins can be identified that contain at least one peptide with exactly the same sequence as the peptide from a protein in the database. With increasing evolutionary distance this will be an increasingly severe restriction [218] . In the second approach, the amino acid sequence of a peptide is extracted from the MS/MS spectrum for de novo sequencing, that is, in a fully databaseindependent manner using exclusively the information contained in the MS/MS spectrum. Several software tools for peptide de novo sequencing are now available and some of them provide sufficiently good results when applied to high-quality spectra [219] . A basic limitation of MS de novo sequencing methods is the necessity for backbone cleavage between each pair of adjacent amino acids; a mass value representing a terminal fragment containing only one of the two residues is a first requirement for ordering of a specific pair [220, 221] and this limitation urged the need for bioinformatics approaches that can help interpret the proteomics data [219] .
In the past several years there have been very important extremely useful advances in proteomics methods based on bottom-up display and bottom-up identification using peptides [222] . These methods offer more sensitivity, greater rapidity and greater proteome coverage are often made with the explicit or implicit assertion that these methods are bound to replace more traditional methods based on topdown analysis, especially using 2D gels [223, 224] . The combination of bottom-up display and bottom-up identification has achieved very important successes in detecting the presence of large numbers of different proteins in cells or subcellular organelles [225, 226] . The use of specific fractionation schemes and prudent adoption of methods to increase the number of proteins able to be identified and quantified is enabling significant biological advances to be made. Further technological developments that enable a larger proportion of the proteome to be visualized will further enhance our ability to characterize biological systems. As such, these advances in proteomics will impact not only academic pursuits but also pharmaceutical, biotechnology and diagnostic research and development [227] .
In the future gel-free techniques MudPIT, iTRAQ and 18O stable isotope labeling could be expected to gain more importance as they become more established. Sample prefractionation system provides a highly valuable tool to fractionate proteins and peptides from complex eukaryotic samples like plasma. This approach has a positive influence on the number of proteins identified compared to SCX method [228] . iTRAQ is a very powerful tool, recognised form its ability to relatively quantify proteins. iTRAQ reagent improves MALDI ionisation, especially for peptides containing lysine. Although SILAC labelling is easy for any laboratory that uses cell culture, the MS technology that is required is still beyond the capabilities of most groups. One of the factors that contributed to the rapid acceptance of the SILAC technology was the availability of an open-source program, MSQuant, for interpreting results. Protein microarrays offer the ability to simultaneously survey multiple protein markers in an effort to develop expression profile changes across multiple protein analytes for potential use in diagnosis, prognosis, and measurement of therapeutic efficacy [229] . This technology is an excellent high-throughput method used to probe an entire collection of proteins for a specific function or biochemistry. It is an exceptional new way to discover previously unknown multifunctional proteins, and to discover new functionalities for well-studied proteins [230] . A systematic and efficient analysis of vast genomic and proteomic data sets is a major challenge for researchers today. To overcome limitations of current proteomics strategies in regard to the dynamic range of peptides detected and alternative mass spectrometrybased approaches are being explored. Targeted strategies exemplified by multiple reaction monitoring detect, quantify, and possibly collect a product ion spectrum to confirm the identity of a peptide with much greater sensitivity because the precursor ion is not detected in the full mass spectrum [231] . A systematic and efficient evaluation of large-scale experimental results requires (1) automatic retrieval of user defined information to construct a customized, queryable database; (2) an intuitive graphical and query platform to display and analyze experimental data in the context of the customized database; (3) efficient utilization of webbased bioinformatics software tools for data interpretation, prediction of function, and modeling; (4) scalability and reconstruction of the database in response to changing user needs and an ever-expanding base of knowledge and bioinformatics tools [232] . Creating a software tool to encompass the four crucial features outlined above is a challenging and ongoing task, particularly with respect to the ever-expanding publicly available base of knowledge and bioinformatics tools. The data processing and analysis bottleneck can be overcome through integration of the entire suite of tools into one linear pipeline. The good news is that all of the various proteomics strategies are in phases of very rapid technological development and that important advances in sensitivity, throughput, and proteome coverage can be expected in the near future for all of them.
Post translational modifications 2DE:
Two-dimensional gel electrophoresis DIGE: Fluorescence 2D difference gel electrophoresis ESI:
Electrospray ionization FTIC:
Fourier transform ion cyclotron HPLC: High performance liquid chromatography ICAT:
Isotope-coded affinity tag iTRAQ: Isobaric tags for relative and absolute quantitation IPG:
Immobilized pH gradient LC:
Liquid chromatography MALDI: Matrix-assisted laser desorption/ionization MS:
Mass spectrometry MudPIT: Multidimensional protein identification technology TOF:
Time of flight PAGE: Polyacrylamide gel electrophoresis SCX:
Strong cation exchange MRM: Multiple reaction monitoring assay SELDI: Surface-enhanced laser desorption/ionization IMAC: Immobilized metal affinity capture. A Systematic Molecular Pathology Study of a Laboratory Confirmed H5N1 Human Case Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo. Influenza pandemic are characterized by the worldwide spread of novel influenza strains for which most of the population lacks substantial immunity [1, 2] . Pandemic viruses typically cause heightened morbidity and mortality [1] . The continued circulation of highly pathogenic avian influenza (HPAI) viruses H5N1 has resulted in occasional coincident infections among humans. Since late 2003, when widespread H5N1 virus poultry outbreaks were reported in multiple countries in Asia, there have been 467 laboratory confirmed human cases in ten countries reported to the World Health Organization as of December 2009 with a mortality rate of about 60% [3, 4] . Global public health concerns surrounding H5N1 viruses include not only individual transmission events between infected poultry and individual humans, but also their pandemic potential, should these viruses acquire genetic changes that result in sustained human-to-human transmission. To date, several case clusters of H5N1 infections have been reported [5] and limited epidemiologic information has suggested person-to-person transmission of H5N1 in a few instances, usually involving family members. Of additional concern to both human and animal health, is the extensive geographic spread of HPAI H5N1 viruses in recent years and their isolation from multiple species of wild birds and mammals [6] [7] [8] [9] . Despite the recent emergence of the 2009 H1N1 pandemic [10] , the pandemic threat from HPAI H5N1 viruses has not diminished [11] .
Human H5N1 disease is clinically and pathologically distinct from that caused by seasonal human influenza A H3N2 or H1N1 viruses [12] . The majority of confirmed human HPAI H5N1 virus infections have been characterized by a severe clinical syndrome including a rapid progression of lower respiratory tract disease, often requiring mechanical ventilation within days of admission to a hospital [13] [14] [15] [16] [17] [18] . In addition to pulmonary complications, other clinical manifestations of H5N1 virus infections may include severe lymphopenia, gastrointestinal symptoms, and liver and renal dysfunction [14, 16, 17, 19, 20] . Reactive hemophagocytosis in multiple organs, and occasional detection of viral antigen or viral RNA in extrapulmonary organs suggest a broader tissue distribution of H5N1 viruses compared with seasonal viruses in fatal human cases [21, 22] .
Patients with severe H5N1 disease have unusually higher serum concentrations of proinflammatory cytokines and chemokines. Levels of plasma macrophage attractant chemokines CXCL10 (IP-10), CXCL9 (MIG), and CCL-2 (monocyte chemoattractant protein 1, MCP-1) and of neutrophil attractant interleukin-8 (IL- 8) were substantially higher in patients with H5N1 disease compared with those experiencing seasonal influenza virus and were significantly higher in H5N1 patients who died compared with those who recovered [23, 24] . The elevation of plasma cytokine levels was positively correlated with pharyngeal viral load [23] and may simply reflect more extensive viral replication, and consequently, direct viral pathology rather than being causative of the pathology observed in H5N1-infected patients. Compared with human H1N1 and H3N2 influenza viruses, infection of human primary macrophage cultures in vitro with H5N1 viruses also lead to the hyper-induction of proinflammatory cytokines [25] .
Most studies on H5N1 pathology describe pulmonary features of human disease. Although H5N1 virus infection of humans is primarily one of the lower respiratory tract, more recent reports suggested that influenza A H5N1 may in rare, severe cases, disseminate beyond the lungs and infect brain [26, 27] , intestines [20, 27] and lymphoid tissues [27] , and result in extra-pulmonary clinical manifestations including encephalopathy or encephalitis [15, 28] . This extrapulmonary dissemination of HPAI H5N1 virus contrasts with seasonal influenza virus infection of humans which, even in fatal cases, is restricted to the respiratory tract. However, there have been relatively few reports describing histopathology and virus distribution in H5N1 cases [5, 16, 21, 24, 26, 27] . To better understand the pathogenesis of human H5N1 virus infection, and investigate the route of virus dissemination in vivo, we report on the use of different techniques to detect virus distribution and infection of 5 organ systems in a laboratory confirmed fatal human H5N1 virus infection, and analyze the relationship between viral load in tissues and host response. Our results suggested that the virus can infect multi-organs besides pulmonary. High viral load is associated with increased host response though the viral load is significantly difference in various organs. Cells of immunologic system could not be excluded to play a role in dissemination of the virus.
Virus culture, real-time RT-PCR, IHC and ISH were used to identify virus distribution in different tissues. As shown in Table 1 , live virus was recovered from respiratory tissues including lung, trachea, bronchus and aortopulmonary vessel. In the digestive system, virus was isolated from tissues collected from the ileum, colon and rectum, but not the stomach, duodenum or liver. Of note, virus culture was also positive on tissues collected from brains, ureter and axillary lymph-node. Sequencing results showed that the sequences of isolates are identical. Real time RT-PCR results were consistent with the detection of virus by culture, except for the liver and spleen tissues which were positive by real time RT-PCR but negative for virus isolation. The tissue distribution of viral RNA or antigen detected by ISH and IHC stains respectively, was also generally consistent with virus isolation by culture or real time RT-PCR result. However, several exceptions were noted. There was a lack of detectable staining by either method in bronchus tissue, despite virus detection. On the other hand, both staining methods detected viral product in the kidney, although virus isolation and real time PCR were both negative.
The viral load is associated with host response figured out by proinflammatory factors. The relative H5N1 viral load in different tissues by determining the ratio of viral HA copy number relative to the copy number of the beta actin gene for a given tissue sample. Tissues of the respiratory system yielded higher copy number ratios than tissues from all to other organ systems with the lower left lung lobe yielding the highest viral load, overall. Interestingly, ureter tissue had the highest viral load of non respiratory system tissues. All other tissues had lower viral loads that were not. Among the digestive system tissues, the viral load in the liver was the highest (See Fig. S1 ). Additionally, we detected mRNA copies of macrophage attractant chemokine CXCL10 (IP-10), macrophage inflammatory protein 3b (MIP-3b), RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNFa in tissues of brain, respiratory organ, spleen and lymph-node. RANTES and TRAIL can be detected in all selected tissues. MIP-3b is positive in selected tissues except right upper lobe lung with positive b-actin. IP-10 can be determined copies in cerebral context, left upper lobe lung, left lower lobe lung, right middle lobe lung, right lower lobe lung, and aortopulmonary vessel. TNF-a can be showed copies in left upper lobe lung, left lower lobe lung, right middle lobe lung, right lower lobe lung, aortopulmonary vessel and spleen. The correlation among levels of viral load and were showed by Fig. 1 . The Pearson's cross correlation analysis (See Table S1 ) showed that high viral load is associated with high host response figured out by proinflammatory factors (p,0.05) except TNF-a (P.0.05), and the correlation is significant between proinflammatory factors except between TRAIL and TNF-a (p.0.05), and between TRAIL and IP-10(p.0.05) as well.
The histopathologic features in different organs are shown in Fig. 2 . Lung showed diffused alveolar damages including intraalveolar edema, focal intra-alveolar hemorrhage, necrosis of alveolar line cells, focal desquamation of pneumocytes in alveolar spaces, interstitial mononuclear inflammatory cell infiltrates, and extensive hyaline membranes. Trachea showed focal denudation of the epithelium with edema, and mononuclear inflammatory cell infiltrates. Spleen showed depletion of lymphocytes with congestion and organized infarcts. Axillary lymph-node was congested with depletion of lymphocytes. The central nervous system showed extensive edema with focal neuronal necrosis in hippocampus. Diastem between Purkinje cells layer and particle cells layer showed focal augmentation in cerebellum. Liver was congested with edema and focal fatty degeneration. Kidney was congested with edema. Other selected tissues showed no significant histological changes. (Fig. 3I ). As influenza virus is a negative-strand RNA virus, ISH stain with sense and antisense probes detected both virus RNA (with sense probe), mRNA and cRNA(with anti-sense probe). ISH stain showed that the two sets of probes (for hemagglutinin and nucleoprotein) generated similar staining results. In the selected 24 autopsy tissue from 5 organ systems, positive staining of ISH was detected in all samples except Bronchus, stomach and duodenum. In the lung tissue samples, sense probes extensively hybridized in the nuclei of pneumocytes (Fig. 4A) , whereas antisense probes hybridized in both the cytoplasm and nuclei. In all other organs with positive staining, both sense and antisense were present mainly in the cytoplasm of infected cells. In respiratory system, the positive staining was found in epithelial cell of lung ( 
We systematically studied the tissue tropism of H5N1 virus in a fatal human case, based on 24 autopsy tissue samples from 5 organ systems, and analyzed the relationship between viral load and host response level in selected tissues. We presented evidence suggesting that the H5N1 virus can infect selected tissue of selected 5 organ systems including respiratory, digestive, nervous, urinary and lymphoid system. High viral load can induce high proinflamatory factors level in tissues, and immune cells could be the target of the virus.
There is a substantial amount of evidence that HPAI H5N1 virus can infect extrapulmonary organ tissues [16] [17] [18] 27] and precede other clinical manifestation [20, 29] . Our results presented that the viral antigens or viral RNA can be found in trachea, lung, brain, intestines, liver, spleen, lymph-node and kidney which were reported as same before [20, 26, 27, 30] , as well as in aortopulmonary vessel and ureter which were not reported before. Notably, the virus can be found in tissues of lower gastrointestinal tract including small intestine and large intestine but in stomach and duodenum. The origin of infection in the extrapulmonary organs could be blood-borne, which is supported by previous studies showing live H5N1 virus can be isolated from the serum [20] and plasma [31] . Unfortunately, we haven't obtained blood samples to [25, 30, [33] [34] [35] [36] and T lymphocytes [37] . The viral load shows various levels in different organs although the virus can be found in multiple organs. Quantitative detection of viral gene showed that viral load is the highest in tissues of respiratory system, especially, in left lower lobe of lung. The liver had the highest viral load in tissues of digestive system. Interestingly, renal duct showed a high viral load although PCR detection of kidney was negative. And viral load was high in spleen and lymphonode. Despite of the viral cells tropism, possible reasons of different viral load could include activation of low PH [38] , viral receptor distribution [39] , cell N-linked glycoprotein distribution [40] or other unknown mechanisms. On other hand, non-permissive or abortive infection is extra possible in some of the tissues where the virus cannot replicate effectively. Additionally, it should be possible that phagocytosis of viral antigen from pathologically significant lytic/replicative infection caused positively viral detection in these tissues.
High viral load is associated with increased host responses. To be different with seasonal influenza virus, the H5N1 virus caused intense transcription induction and secretion of inflammatory cytokines/chemokines, as documented in human cases [15, 22] , and increased induction of cytokines and chemokines has been demonstrated in H5N1-infected mice [41] . Moreover, High virulence of H5N1 virus is associated with increase with increased host responses [42] . However, the relationship between viral load and host immuno-response has never been reported. Our study suggested that the high viral load is correlated with proinflamatory factors including IP-10, RANTES, MIP-3b and TRAIL. This finding is particularly relevant to the mechanism of H5N1 pathogenesis which is associated not only with the virus but also with the host responses. Inflammatory protein can be produced by almost any infected cells and by immune cells, including alveolar macrophages [43] . However, type II pneumocytes have a marked ability to secrete large amounts of cytokines, such as TNF-a, GM-CSF, MCP, and IL-8, in response to various insults [44, 45] and can be induced to secrete IL-1a, IL-6, RANTES, and MCP-1, in response to TNF-a [45, 46] , the last being produced by alveolar macrophages during H5N1 infection. Therefore, it is possible that the much greater cytokine induction by the H5N1 virus was as much a consequence of the response of individual, infected cells as it was a consequence of the numbers of infected cells.
Immune cells could be a target of the virus infection. Previous findings showed that viral sequences and antigens have been detected in lymphocytes in lymph node tissue, as well as in Hofbauer cells (macrophages of the placenta), Kupffer cells (macrophages of the liver), and mononuclear cells in the intestinal mucosa [27] . This is consistent with our find. Our presented evidence shows that virus can be detected in macrophage. Moreover, our finding showed that viral RNA can be detected in CD3+ T lymphocytes, progenitor cells and follicular dendritic cells of spleen. The spleen is a complex organ with several functions, including the removal of senescent or aberrant red blood cells from circulation, as well as the removal of circulating pathogenic organisms [47] . So circulationg immune cell with H5N1 virus could play a role in dissemination of virus when those cells with virus can not remove the virus in cells completely. We unprecedentedly investigated 24 autopsy tissues of 5 organ systems from a dead patient of H5N1 infection. Our study results indicate that the H5N1 virus could be found in multiple organs. Viral load in infected tissues is correlated with host response. And circulating immune cell can not be excluded to play a role in dissemination of virus in vivo.
The patient was laboratory confirmed as an H5N1 infection by China CDC on February 20, 2008. He is a 42 years-old Chinese male living in Guangxi, China with a history of 6 days of fever, cough and dyspnea. Two weeks before hospital admission, he bought 3 hens at an open-air market, one of which died later at same day. The remaining birds exhibited symptom of chicken attack next day. The patient killed and cooked the birds, and ate them together with other family members who later did not experience any clinical symptom. On admission, the patient was febrile with a temperature of 40.3uC, had infiltration of lower left lung lobe based on chest radiography, bilateral lower lung moist rales, and substantially reduced oxygen saturation. He was placed on a ventilator and treated with antibiotics, spasmolysis, corticosteroids, and the dissipatation of phlegm and fluids. Despite treatment, the patient presented with function damage of multiple organ (lung, heart, liver and kidney) on the second day after admission, and died 59 h after admission, and 8 days after the onset of symptoms. Throat swabs were collected and performed RT-PCR and rRT-PCR detection at the day patient died. No antiviral drug treatment was given since the patient died before finally laboratory diagnosis.
After informed consent was obtained, the cadaver was stored at 4uC and underwent autopsy about 18 h after death. The autopsies were done following conventional protocols with strict adherence to biosafety procedures [48] . Twenty-four tissues were collected from respiratory, digestive, nervous, urinary and lymphatic organ systems. Duplicate tissue samples were collected; one sample was fixed in diethylpyrocarbonate (DEPC) treated 10% formalin for pathologic analyses, while a second sample was frozen at 280uC for virus isolation and molecular analyses.
Frozen tissues were thawed and homogenized before inoculation into embryonated eggs for viral culture. Briefly, 1.0 ml of phosphate-buffered saline (PBS) was added to 100-150 mg of tissue, which was homogenized, and, then centrifuged; 0.1 ml of the recovered supernatant was injected into the allantoic cavity of 11 days-old specific pathogen-free embryonated chicken eggs. The allantoic fluids were tested for haemagglutation activity with turkey red blood cells after 72 h incubation at 35uC. Samples containing virus were subjected to RT-PCR and sequencing. Three blind passages were performed on hemagglutinationnegative samples to confirm the absence of infectious virus. All steps were performed in BSL-3 containment laboratory.
Total RNA from the tissues samples was extracted using an Axygen total RNA extraction kit (Axygen, USA) as described in the manufacturer's instructions. Briefly, 400 ml of lysis buffer was added to 30-40 mg of ground tissue. The nucleic acids were eluted in 50 ml nuclease-free water and stored at 220uC. For every five samples, one negative control (water) was included to detect any possible contamination.
Control RNA products derive from in vitro transcription of the matrix (M) gene RNA of A/Anhui/1/2005(H5N1), human house keeping gene beta-actin and human cytokines/chemokines (including IP-10, RANTES, TRAIL, MIP-3b and TNF-a) genes were used as positive controls and to establish the detection limit of the assay. Recombinant plasmids with entire M gene, beta-actin gene segment and cytokines/chemokines gene segment were linearised by restriction enzyme, and then purified using a DNA clean-up kit. DNA concentration was measured as OD units at 260 nm. One mg of linearized plasmid DNA was transcribed using Riboprobe in vitro transcription system kit (Promega, USA) according to the manufacturer's instructions. The transcribed RNA was purified using phenyl/chloroform solution and was quantified by spectrophotometer. RNA copy number was then determined following the method of Fronhoffs [49] .
To analyze the H5N1 viral load and quantify proinflammatory factors in different tissues, real-time RT-PCR was performed with a Strategene detection system using a fluorescently labeled TaqMan probe to enable continuous monitoring of amplicon formation. The primer and probe of H5 HA gene is from the WHO-released primer sets [50] . The primers and probes of proinflammatory factors and beta-actin gene were obtained from the literature [51, 52] . The concentration of primer and probe used was 40 mM and 10 mM, respectively. The reaction was completed in a total volume of 25 ml performed by QuantiTect Probe PCR Kit (Qiagen, Germany). The reaction mixture was incubated with 5 ml DNase-treated total RNA (the template which was used to amplify b-actin gene was performed by 100-fold dilution) at the following temperature cycles. First, the reverse transcription reaction was completed by 1 cycle at 50uCfor 30 min. Next, H5N1 HA gene, cytokine/chemokine gene and house keeping (bactin) genes were amplified by 1 cycle at 94uC for 15 min and 45 cycles at 94uC for 15 s, 55uC for 30 s, and 72uC for 30 s each. As described in previous report [53] , the standard curve was generated using serial dilution of in vitro transcribed standard RNA (from ,10 to 10 7 copies). The viral load and cytokines/ chemokine levels are presented as the log 10 value of the ratio between copies of the target gene and b-actin gene.
Immunohistochemical stain was performed on 5 mm thick deparaffinized sections using monoclonal antibodies against the nucleoprotein of influenza A (Serotec, UK) by a two-step peroxidase method. For controls, we used unrelated antibodies in place of the primary antibody. Briefly, sections were deparafinized by 2 washes in xylene and were rehydrated through decreasing concentrations of ethanol. After washing in PBS at pH 7.6 for 5 min at room temperature (RT), sections were heattreated with antigen-retrieval solution (TRIS/EDTA buffer, pH 9, Dako, Denmark) for 10 min using microwave antigen retrieval method. After blocking with 10% normal horse serum for 10 min at RT, the sections were incubated with the specific antibody (1:100) for 30 min at RT. Unbound antibody was removed by 3 washes in PBS before adding HRP-labelled polymer for 30 min at RT (Dako CSA Detection System, Denmark). After washing unbound labeled polymer in PBS 3 times, peroxidase staining in tissue sections was revealed by DAB solution (CSA Detection Systems, DAKO, Denmark). After stopping the reaction in running water, sections were counter-stained with a quick rinse in Mayer's hematoxylin solution. After dehydrating with increasing concentrations of ethanol and xylene, the sections were mounted with DPX and examined by light microscopy (Olympus BX51, Japan).
The development of probes was based in analysis of the full haemagglutinin and nucleoprotein gene sequences of all Chinese human isolates of H5N1. Oligonucleotide DNA probes representing conserved gene regions were used. Probes were labeled by digoxigenin-UTP (Roche Diagnostics, Penzberg, Germany) and tested for specificty using human biopsy of gut tissue with in vitro infection of H5N1 virus. Since H5N1 is a negative-stranded RNA virus, sense probes were defined as the probes that detect the viral RNA (negative-stranded), whereas antisense probes detected mRNA and complementary RNA (cRNA), which are both positive-stranded. Briefly, before hybridization, all solutions were prepared with DEPC-treated water. After deparaffinization and rehydration, tissue sections of 5 mm thickness were treated with proteinase K digestion for 30 min. Tissue sections were then incubated with a hybridization cocktail containing 25 mg/mL of probes at 37uC for 16,18 h. All sense and antisense probes were applied separately on consecutive tissue sections. After blocking with normal horse serum (1:100), sections were incubated with alkaline phosphatase-labelled digoxigenin antibody (1:1000, Roche Diagnostics, Penzberg, Germany) for 1 h, and the reaction products were colorised with nitroblue tetrazolium/5-bromo-4choloro-3-indolyl phosphate (Roch Diagnostics, Penzberg, Germany) for 1-1.5 h, and counterstained in 2% nucleic fast red liquid for 1 min. As a positive control, we used human lung and gut biopsy tissues with in-vitro infection of H5N1 virus. Negative controls also included an unrelated antisense probe against the fragment of the heamaglutinin gene of the seasonal influenza virus H3N2 as well as H5N1 in-situ hybridization probes to tissues.
After completeing the colorization reaction of the ISH as outlined above, sections were incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. The sections were then blocked with 10% normal horse serum for 10 min at RT, before incubating with monoclonal antibodies (against CD3 + , CD68 + , CD34 + , D35 + ) for 1 h at RT. Unbound antibody was removed by 3 washes in PBS before the addition of HRP-labeled polymer for 30 min at RT (Dako CSA Detection System, Denmark). After washing 3 times with PBS to remove unbound labeled polymer, peroxidase staining in tissue sections was revealed by DAB solution (CSA Detection Systems, DAKO, Denmark). After stopping the reaction in running water, sections were counter-stained by 2% nuclear quick red solution for 30 s. For each tissue, double-labeled stain with IHC and ISH was repeated three times for data analysis. To strengthen further the results of colocalization studies, we performed ISH and IHC on consecutive sections. Tissue sections showing ISH-positive cells were carefully compared with consecutive tissue sections on which IHC with antibodies against specific cell markers was applied. Co-localization of a specific cellular marker and viral genome was clearly identified.
The correlation between viral load and quantitative proinflammatory factors profile was analyzed by Pearson's correlation test using Instat software (Vision 5.0, GraphPad prism). Differences were considered significant at p,0.05. Figure S1 The distribution of viral load in selected tissue samples. The viral HA gene and b-actin gene copies in tissues were determined by quantified real-time RT-PCR. The ratios between HA and b-actin gene copies which was showed by logarithm presented the viral-load level in different tissue. Found at: doi:10.1371/journal.pone.0013315.s001 (0.11 MB TIF)  Situational Awareness and Health Protective Responses to Pandemic Influenza A (H1N1) in Hong Kong: A Cross-Sectional Study BACKGROUND: Whether information sources influence health protective behaviours during influenza pandemics or other emerging infectious disease epidemics is uncertain. METHODOLOGY: Data from cross-sectional telephone interviews of 1,001 Hong Kong adults in June, 2009 were tested against theory and data-derived hypothesized associations between trust in (formal/informal) information, understanding, self-efficacy, perceived susceptibility and worry, and hand hygiene and social distancing using Structural Equation Modelling with multigroup comparisons. PRINCIPAL FINDINGS: Trust in formal (government/media) information about influenza was associated with greater reported understanding of A/H1N1 cause (β = 0.36) and A/H1N1 prevention self-efficacy (β = 0.25), which in turn were associated with more hand hygiene (β = 0.19 and β = 0.23, respectively). Trust in informal (interpersonal) information was negatively associated with perceived personal A/H1N1 susceptibility (β = −0.21), which was negatively associated with perceived self-efficacy (β = −0.42) but positively associated with influenza worry (β = 0.44). Trust in informal information was positively associated with influenza worry (β = 0.16) which was in turn associated with greater social distancing (β = 0.36). Multigroup comparisons showed gender differences regarding paths from trust in formal information to understanding of A/H1N1 cause, trust in informal information to understanding of A/H1N1 cause, and understanding of A/H1N1 cause to perceived self-efficacy. CONCLUSIONS/SIGNIFICANCE: Trust in government/media information was more strongly associated with greater self-efficacy and handwashing, whereas trust in informal information was strongly associated with perceived health threat and avoidance behaviour. Risk communication should consider the effect of gender differences. Pandemic influenza A/H1N1 has a clinical profile similar to seasonal influenza, despite initially appearing more severe [1] . Respiratory infectious diseases (RIDs) such as influenza are a major public health issue best dealt with by prevention, ideally vaccination. However, in the first six-months or so of a newlyemergent RID epidemic/pandemic vaccines are generally unavailable and non-pharmacological interventions can play a major role in minimizing RID spread [2] [3] [4] . Government health education messages are a major source of information for promoting self-protective practices against RIDs. These preventive messages generally emphasize improved hygiene, face-mask use by infected persons, and social distancing measures, including avoiding crowds during epidemics [5] [6] [7] .
Predictors of population uptake of health protective behaviours in RID epidemics have begun to be studied [8] [9] [10] [11] [12] [13] , yet related theory remains nascent and this is problematic: to effectively predict behaviour during future epidemics robust theory is critical. Effective models that enable comprehensive prediction of health protective behaviours remain limited mainly to two overlapping theoretical paradigms: the Theories of Reasoned Action/Planned Behaviour (TPB) [14] [15] [16] and Bandura's concept of self-efficacy [17] [18] [19] (the belief that one can successfully execute some behaviour), particularly regarding the core TPB concept of perceived behavioural control, which controversially is claimed by some to be largely synonymous with self-efficacy [19] [20] [21] and by others to be indistinguishable from intent [22] (the intention to execute a particular behaviour), the key predictive element of TPB [16] . When used to account for health-related behaviours TPBbased models typically account for ,35% of variance in outcomes [16] , while self-efficacy accounts for ,25% of variance in outcomes [23, 24] . However, neither TPB nor Self-efficacy allow for the social and affective influences that might be expected logically to be important in RID [25, 26] . We report on a theoretical model that incorporated elements of influenza causal knowledge, perceived self-efficacy and also social and affective influences ( Figure 1 ) because these latter variables have been less frequently studied in combination, but have theoretical and logical support for their potential importance in the context of RIDs. We tested this model against data collected in the early phase of the influenza A/H1N1 pandemic (Table S1 ) to examine how levels of trust in formal and informal sources of risk/prevention information associated with hand washing and social distancing.
Ethics approval was obtained from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. For this telephone interview, written informed consent was waived by the IRB but verbal consent was required from all the respondents and agreement to participate in the interview was taken as further consent. Before the interview began, a brief introduction about the study aims and interview contents was given and then respondents were asked whether the interview could start. If approval was received this was recorded and the interview performed. If not, respondents were thanked and the call was terminated.
More than 98% of Hong Kong households have landline telephones and all local calls are free. Random-digit dialled telephone numbers and within-household random-sampling grids (Kish grids) are a cost-effective way to survey highly representative random population samples. Kish grids are matrices containing random numbers for different sized households that facilitate random selection of individuals within households and help minimize sampling bias. The number of eligible household residents, ''n'', is determined by asking the person of first contact in the household. The Kish grid provides a randomly generated number ''k'' between 1 and ''n'' which is used by the interviewer. Ordering by age and starting from the oldest eligible member in the household, the k'th member is then invited to participate in the survey. Different grid values are used for each household. As part of a series of surveys to monitor A/H1N1 epidemic activity, a commercial polling organization administered the questionnaire using this telephone-survey methodology, targeting 1,000-1,500 participants on each occasion, a sample size calculated to give an estimate of A/H1N1 health protective behaviours with a precision of 63%. The survey with the largest sample was selected for this analysis. Sampling was performed during the evening to minimize exclusion of young working adults.
Data on attitudes, knowledge, situational awareness, risk perception and preventive behaviours (Table S1) were collected by household telephone interviews, based on random digit dialling. One Cantonese-speaking adult (age$18) who lives .4 nights per week in each household was selected using a Kish grid. All interviews were conducted between 8:30pm-10:45pm from 23 rd -25 th June, 2009, two weeks after the first community transmission had been identified in Hong Kong. 
Existing theoretical frameworks of behaviour change have been adapted to predict health-related behaviour-change for chronic, non-communicable diseases [15, 16] , but we lack a comprehensive evidence-based model of protective behaviour against RID threat [11] . A recent review of 26 papers on RID prevention behaviours concluded that 23 lacked a theoretical basis [13] . Existing applications of health behaviour change models in communicable disease are almost exclusively limited to HIV/AIDS research [24, 27] and to a lesser extent hepatitis B and C, which share the same transmission pathways as HIV. There are good reasons why sexually-transmitted diseases embody a different set of influences than do RIDs. For example people are highly motivated to seek sexual contact (or injection drug use) and have a high degree of potential control (e.g. condom use) over the nature of these encounters, even though they may be situationally constrained from executing that control, and are infected only by direct exchange of bodily fluids. In contrast, one can acquire an RID transmitted by air droplets, hand contact or fomites for up to 72 hours after the person who is the source departs [28] , or immediately by being sneezed on. Infection is much more casual. Clearly, the controllability of RIDs requires different behavioural imperatives to those in STDs and hence different psychological influences should be considered. Attempts by the TPB to accommodate social influences had relied on incorporating social norms [14] , the behavioural expectations within a group. However, norms, and hence theoretical models reliant on norms to account for social processes, cannot accommodate the fact that communicable respiratory diseases make other humans ambiguous sources of threat: one can usually control sexual encounters but not who shares public transport. In this respect social factors in communicable respiratory disease differ significantly from those in non-communicable diseases and warrant greater consideration than existing HBC models allow. Outbreaks of new infectious diseases constitute situations that are uncertain, dynamic, and embody highly personal threat, requiring rapid decisions on appropriate action [29] . Under such circumstances timely and relevant information on the best preventive actions become critical to such decision-making. Hence, health protective behaviour during the early stages of a novel epidemic would be more likely to resemble situational reactions using established or known default actions such as avoiding crowds (social distancing), rather than intention-based planning before any behavioural change, such as deciding to consult a doctor to administer a vaccination. Later in the epidemic as threat familiarity increases, different factors such as planned behaviour may become important.
Reporting delays, uncertainty and other biases affect publicly available information on the characteristics of newly-emergent communicable diseases, such as A/H1N1 lay knowledge of infection-related risks can be limited. The resulting uncertainty about disease severity and transmissibility at the epidemic onset extends to the utility and timing of adopting preventive measures. Information cues to individuals about initiating protective action must therefore be synthesized from various sources. Perceived information reliability or trustworthiness influences decisions to utilize any given information source [30] to inform awareness of the situation. More trustworthy sources are therefore likely to be more influential. Epidemic situational awareness is likely derived from formally-announced public information like news items, government press releases and health education messages, and also from informal, social sources [25, 29] ; observation of other peoples' behaviour and communications from family, peers and neighbours. Noting how others behave informs action decisions in the observer [19] . If those around you are wearing masks, this indicates others might have knowledge you do not possess, and that the threat level might be locally high and imminent, suggesting prudent precautionary or RID preventive behaviour. Observers are also subject to social conformity influences that can help adoption of group patterns of behaviour. Maintaining situational awareness, involving elements of perception, comprehension and prediction [31] , during epidemics probably relies on these two types of information. However, when uncertainty is high and widespread, or when there is low confidence in social and other information sources then individuals' HPBs might be expected to be more independent of formal and informal information sources.
Perceived risk is influenced by several stimulus characteristics, including unfamiliarity, invisibility, dreadfulness and inequity [32] , and by recipient characteristics, including demographics and trust in information source and content [33] . Perceived risk is an important determinant of protective behavioural responses [12, 34, 35, 36] , but is subject to optimistic bias, where for example people distort their risk of contracting influenza downwards relative to others [35, 37] . Nonetheless, susceptibility to risk remains an important measure in understanding variation in behavioural responses to threat and reflects the key element of perceived risk in an epidemic/pandemic situation. Worry is a cognitive process linked to anxiety [26, 38] and reflects negative affectivity, interacting with perception of susceptibility to risk [26, 39] and may also influence RID protective responses such as social distancing [13] .
Because data were collected using telephone interviews we had to adapt measures to suit a brief format in order to avoid people hanging up mid-way or providing invalid answers to hurry the interview, a problem encountered with this data collection method. We therefore used parsimonious measure to minimize assessment fatigue and low response rates which threaten representativeness.
Trust in government/media (formal) information: We asked about respondents' agreement with three statements (Table S1 ). Responses were made on categorical five-point scales ranging from ''strongly disagree'' to ''strongly agree''. Scalability of these three items was assessed using Cronbach's alpha, which at 0.61 indicated that the internal consistency between items was low, but acceptable. However, to minimize potential measurement error arising from the low internal consistency, this construct was treated as a latent variable in the subsequent analysis [40] . A latent variable is a concept opposed to an observed variable. A latent variable can not be measured directly but is inferred from one or more variables that are directly measured (observed variable) while an observed variable can be directly measured with a specific question or item or observed by the researchers. For example, an ''attitude'' is a concept that is difficult to measure directly with single items but can be inferred from various questions asking about different aspects of that attitude. Then within the analysis ''attitude'' is treated as the latent variable while the questions used to infer it are the observed variables.
Trust in interpersonal (informal) information: Respondents' agreement with two statements (Table S1 ). Responses were made on categorical five-point scales ranging from ''strongly disagree'' to ''strongly agree''. Scalability of these two items was assessed using Cronbach's alpha, which at 0.50 indicated that scalabilty was unsuitably low for two items. This suggests that these two items measure different aspects of social information. Again to minimize potential measurement error this construct was treated as a latent variable in the subsequent analysis.
Understanding cause of A/H1N1 (''I understand how Swine flu is caused'') and self-efficacy (confidence in one's ability to act in a way that achieves desired future outcomes) for A/H1N1 prevention (''I am confident that I can protect myself against Swine flu''): Each was assessed using responses on 5-point scales of agreement with these two single item statements (Table S1) .
Perceived personal susceptibility: Two items, one assessing absolute susceptibility (perceived absolute probability of developing A/H1N1) and another assessing relative susceptibility (perceived probability of developing A/H1N1 relative to peers) formed a latent variable for perceived personal susceptibility (Table S1 ). The Cronbach alpha of these two items was 0.66.
Worry about contracting H1N1. Respondents were asked to indicate their level of worry over the past one week about contracting influenza A/H1N1. Responses were 5-point scales of worry ranged from ''never thought about it'' to ''extremely worried'' (Table S1 ).
Hand hygiene. Respondents were asked to indicate frequencies of use of four hand hygiene practices over the three days prior to interview: hand washing after sneezing, coughing and touching nose; hand washing after returning home, use of liquid soap for hand washing, and hand washing after touching common objects. Responses were on a 4-point scale of frequency: 1 ''never'', 2 ''sometimes'', 3 ''usually'' and 4 ''always''. Cronbach's a was 0.62 (Table S1 ).
Social distancing behaviours: a. Social Avoidance. Respondents were asked to indicate if they had adopted any of four avoidance behaviours due to influenza A/H1N1 in the past 7 days: avoiding eating out, avoiding using public transport, avoiding going to crowded places, and rescheduling travel plans Responses were coded as 1 ''yes'' and 0 ''no''. Cronbach's a was 0.61 (Table S1 ).
We first compared the demographic structure of the sample against that of the general population derived from the Hong Kong government General Household Survey to identify any sample differences.
Our model proposes that trust in formal (government and media sources) and informal (from other people) information affects RID epidemic health protective behaviours, the former by informing about generic risk and response characteristics for dealing with a potential threat (causes and protective responses), the latter about threat imminence, severity and response effectiveness (seeing how others behave). We refer to the product of these combined processes as situational awareness, and propose that rather than driving behaviour directly information acts through altering the cognitive/affective domain of situational awareness. Thus the model is predicated on several premises: that understanding of the disease and perceived personal susceptibility influence self-efficacy [17, 18, 31] ; that the effect of perceived susceptibility to influenza on HPBs acts through increasing worry about the disease [26, 33, 38, 41, 42] ; and that more worry from perceived susceptibility prompts HPBs [39, 41, 42] . These cognitive/affective processes are represented in the hypothesized model ( Figure 1 ).
Structural Equation Modeling (SEM) is a method for simulating and testing multiple and interrelated causal relationships simultaneously in statistical data, making it suitable for theory development and testing [40] . SEM was applied to test the hypothesized model. SEM is usually performed when a model contains latent variables assessed with specified measurement models. Despite including estimations of a series of multiple regression equations, SEM differs from regression analysis in several ways, which make it advantageous for this kind of analysis.
First, SEM is usually theoretically based because it is performed after researchers specify the hypothesized model. Second, it can be used to refine the hypothesized model by estimating the measurement model and structural model simultaneously. Finally SEM analysis can accommodate measurement errors of the constructs in the model [40] . In our hypothesized model, trust in formal information, trust in informal information, perceived personal susceptibility, hand washing and social distancing behaviours were entered as latent (inferred) variables while other constructs were entered as observable (directly measured) variables because they were assessed with only one item. Two different health protective behaviors, hand washing and social distancing, were entered as the HPB outcomes because we hypothesized that different influences may act on each of these. We assumed that the ''disturbances'' of the two health behavior outcomes were correlated. Disturbance represents the unexplained variances of the latent variables predicted by the specified independent variables [40] . In making this assumption, we assumed that unexplained variance in the outcome variables could be correlated and the variables in question jointly influenced by other unknown factors, and so allowed for such constraints within the model by using more conservative criteria. Previous studies have shown that hand hygiene and social distancing behaviours during a pandemic could be influenced by some common causes which were not fully explored in our study such as current health, past experience of disease and cues to action [14] . In particular, in our study, the two kinds of health protective behaviours occurred in the same situation of the 2009 influenza pandemic, and so it is sensible and reasonable to assume that they could be influenced by some common causes which were not fully explored in our studies.
Adequacy of the measurement models was tested before testing the full structural model. To test the full structural model, all constructs ( Figure 1) were entered into the model and all factor loading, specified paths, covariance, measurement errors and disturbances were estimated simultaneously. Since the model contained categorical variables, Weighted Least Square with mean-and variance-adjusted estimation (WLSMV) was used to estimate the standardized parameter (b) for each path [43] . With this kind of estimation, chi-square difference testing is inappropriate. We therefore used the Comparative Fit Index (CFI), Tucker-Lewis Index (TLI) and Root Mean Squared Error of Association (RMSEA) to evaluate the model fit to the data. A CFI.0.95, TLI.0.95 and RMSEA,0.05 indicate a good fit of data to the model [43] . The analysis was conducted in Mplus 6.0 for Windows [43] . The proportion of missing values ranged from 0.1% for ''In the past one week, have you ever worried about catching influenza A/H1N1'' to 10.1% ''did you wash hands after sneezing, coughing or touching nose in the past 3 days''. Missing data were handled with multiple imputation to generate 10 datasets which were summarized into one for subsequent analysis. Multiple imputation was performed in AmeliaView [44] .
Responses are likely to differ by sociodemographic factors [13] . We therefore stratified the sample by gender and by age (,45 years old vs. .45 years old). Education is also likely to have a significant effect but there are difficulties in education stratification in Hong Kong. The age cut-off of 45 years was adopted to account for the introduction in Hong Kong of 6-year compulsory education in 1971 and 9-year compulsory education in 1978 [45] . This means that people aged 45 or above are much less likely to have a tertiary (college/university) level education and less secondary (high school) education than people aged ,45 years old [45] . Moreover, in traditional families in China, a son (who lived with his parents after marriage) was usually more educationally-favoured over daughters (who moved to their in-laws' home on marriage) to ensure support for the parents in their old age, so males usually obtained more education than females [45] . These distinctions were somewhat evidenced by our data which showed that 98% of the respondents aged ,45 compared to 71% of the respondents aged 45 or above (x 2 = 147.69, p,0.001), and 89% of male compared to 80% of female respondents, obtained at least secondary education (x 2 = 17.05, p,0.001). Since the numbers of tertiary educated respondents and primary (elementary) educated respondents were too small to produce stable models, we limited stratification to gender and age only and acknowledge that this also incorporates indefinable education and income effects.
Consequently, we used a multi-group SEM to assess the invariance of the model (Figure 1 ) across gender and age group (respondents aged 18-44 and aged 45 or above). We tried to test the model by stratifying the sample into four subgroups (female aged 18-44, female aged 45 or above, male aged 18-44 and male aged 45 or above). However, the sample size for males aged 18-44 was relative small (Table S2) . Moreover, all the model variables were treated as categorical variables and we used the WLSMV method to estimate the model. This method requires that each subsample covers all the categories of each variable. In the case of one category, younger males, not all variable values were present. To meet the assumptions for analysis we would need to recode all variables, intrinsically altering the model. In order to avoid this, we relinquished a combined four-group comparison and instead compared the model across gender and the two age groups separately. To perform multigroup comparison we first ran a model with all parameters unconstrained. We then identified factor loadings that were not significantly different (p$0.05) and set these as equal, while loadings that were significantly different were allowed to vary, and finally paths that did not differ significantly were constrained to be equal while those that differed significantly were allowed to vary and estimated separately by groups. The ''DIFFTEST'' option in MPlus 6.0 was used to obtain a correct chi-square difference test for the WLSMV estimators and was used to estimate the differences between the least constrained model (with all the paths freely estimated) and the most constrained model (with all the paths constrained to be equal) as well as the partially constrained model (with some of the paths freely estimated and others constrained to be equal) [43] . A p-value.0.05 for the ''DIFFTEST'' indicate a nonsignificant difference between the models.
Finally, to help interpret these multigroup SEM comparisons, we performed a post-hoc examination of the model variable means for different gender and age groups and tested differences using the Mann-Whitney test, which tests differences between two groups on ordinal scales of measurements.
A total of 1,001/1,449 (69.1% response rate) Hong Kong adults successfully completed the interview. The characteristics of the sample were compared against the Hong Kong 2006 by-census population data [46] , showing respondents to be better educated and more likely to have been born in Hong Kong compared to the general population (Table 1 ) but otherwise representative.
Both formal and informal information trust were correlated with all situational awareness variables except worry about contracting A/H1N1 (''Worry''), while formal information trust was also independent of perceived personal susceptibility (''Susceptibility''). In turn, understanding of H1N1 cause (''Understanding'') and Perceived self-efficacy (''Self-efficacy'') were significantly associated with hand washing while Worry and Susceptibility were significantly associated with social distancing (Table S3 ).
The SEM model fitted well to the data with CFI = 0.977, TLI = 0.969 and RMSEA = 0.026. Standardized coefficients indicated two primary features in the model; the first one linking Formal information and hand hygiene and a second linking Informal information and Social distancing ( Figure 2 ). Paths were seen via Formal information trust and Self-efficacy (b = 0.25) and Self-efficacy and hand hygiene (b = 0.23), and via Formal information trust and Understanding (b = 0.36), and Understanding and hand hygiene (b = 0.19) while Understanding and Selfefficacy were independent. These associations formed the first feature. Marginal associations between Worry and hand hygiene and between Self-efficacy and social distancing were seen, but the small standardized coefficients of b = 0.13 suggest that these paths are minor. Susceptibility and Worry were associated, but otherwise were functionally independent, both upstream from formal information trust, and downstream from hand hygiene.
The second feature of the model is reflected in a different set of paths associating informal information trust with social distancing. Trust in informal information sources was inversely associated with Susceptibility (b = 20.21), which was associated positively with Worry (b = 0.44), and inversely with Self-efficacy (b = 20.42). However, more confidence in informal information sources was associated with more Worry (b = 0.16) and finally, only Worry was associated with social distancing (b = 0.36). Trust in informal information was independent of Understanding and Self-efficacy.
The only remaining notable feature of the model was a strong inverse association (b = 20.42) between Susceptibility and Selfefficacy. This suggests some interaction between these two variables that could strongly influence both sets of paths mentioned so far. Overall, the model explained 11.3% of the variance in hand hygiene and 16.1% of the variance in social distancing behaviors.
Across gender, both the least constrained model and the most constrained models fit the data well with CFI.0.970, TLI$0.970, RMSEA = 0.025. The most constrained model did not differ significantly from the least constrained model (x 2 for ''DIFFT-EST'' = 29.30, d f = 19, p = 0.061). However, three sets of associations differed significantly between females and males: those between Formal information trust and Understanding, from Informal information trust to Understanding, and from Understanding to Self-efficacy. These paths were set free and estimated separately in female and male. The model with these paths freely estimated fit well to the data with CFI = 0.978, TLI = 0.976 and RMSEA = 0.023, and did not differ significantly from the least constrained model (x 2 for ''DIFFTEST'' = 15.07, d f = 16, p = 0.519). Figure 3 presents the results of multigroup comparison of the model applied to males and females with the three path parameters unconstrained. For a given path, if the path coefficients did not differ significantly between males and females, only the path coefficient for males is presented; if the path coefficients differed significantly between males and females, the path coefficients for both genders are presented with the coefficients for males presented on the left of the slashes and for females presented on the right of the slashes.
By comparison, the model shows that for both genders while the association between Formal information trust and Understanding was positive this association was stronger amongst females (b = 0.50) than males (b = 0.25); the association between Informal information trust and Understanding was weakly positive in males (b = 0.12) but weakly negative in females (b = 20.14), and; the association between Understanding and Self-efficacy was positive (b = 0.12) in males but non-significant in females (b = 20.01).
Across the two age groups, both the least constrained model and the most constrained model fit well to the data with CFI.0.960, TLI$0.950, RMSEA#0.030. The most constrained model did not differ significantly from the least constrained model (x 2 for ''DIFFTEST'' = 15.85, d f = 19, p = 0.667). No path was found to be significantly different between the two age groups.
Means and standard deviations for all model variables by gender and age group showed differences (Table S4 ). All the constructs did not differ by gender except for hand hygiene and social distancing with female being more likely to wash their hands and adopt social distancing behaviours. Trust in formal and informal information sources, Self-efficacy, and Hand hygiene significantly differed by age groups, with respondents of older age group being more likely to trust the information from both sources, perceive higher self-efficacy and wash their hands.
We tested a hypothesized model of associations between trust in (formal/informal) information, situational awareness variables (causal understanding, self-efficacy, susceptibility and worry) and different types of health protective behaviours (hand hygiene and social distancing) for influenza protection. The model suggested that two different sets of influences relate trust in information to hand hygiene, and to social distancing respectively. The strongest associations observed were between Susceptibility and Self-efficacy 
Neither age nor gender contributed significant variation to the association between Trust in Formal information and Self-efficacy, and Self-efficacy and hand hygiene. These findings are consistent with other studies showing self-efficacy is enhanced by procedural information [18, 19, 47, 48] and that attitudinally and actionoriented interventions are more successful in changing behaviour for communicable disease protection, such as in the case of HIV [24] . Similarly, exposure to relevant media stories during the 2009 A/H1N1influenza pandemic was associated with higher efficacy beliefs regarding hygiene, which in turn was associated with greater frequency of reported tissue access and sanitising gel purchase among British people [49] . However, there is evidence that coping style interacts with the ability of procedural information to enhance self-efficacy and under circumstances of high threat, such as during SARS-type epidemics where mortality is high, procedural information might be counter productive for some segments of the community who use an information avoidance (''blunting'') coping style [50] . Self-efficacy was only weakly associated with social distancing. People are limited in their ability to avoid crowds in Hong Kong, one of the most densely populated cities on earth, despite the Hong Kong government recommending this in order to limit the pandemic [51] . However, the relatively mild impact of A/H1N1 meant that people saw no reason to jeopardize their economic well-being and curtail other social activities, given such a low perceived threat [49, 52] . Hand washing was probably seen as sufficient protection.
The association between Trust in Formal information and Understanding of influenza cause differed by gender but not age, with females showing a stronger association. Men tend to have poorer health knowledge than women [53] . We found that females were more likely to wash their hands than were males. Older respondents reported significantly greater trust in formal information, marginally-significantly better understanding of influenza cause and were more likely to wash their hands. This is consistent with other studies reflecting that preventive practice is enabled by knowledge of causes [49, 54] . However, increasing knowledge is not itself sufficient to always ensure preventive behaviour [55] . In this context, Understanding has an independent contribution to hand washing practice only.
Trust in Informal information seems to be associated with less perceived susceptibility to health threat. This may reflect rational processes or cognitive bias. Trusting social cues involves comparison and conformity influences, and can enhance optimistic bias (the tendency to view oneself less likely to experience negative events but more likely to experience positive events) in personal risk estimates [56] , thereby reducing perceived Susceptibility. Conversely, others' behavioural cues about health threat proximity can arouse motivating worry and anxiety producing protective action [17, 29] . We found Trust in informal information was independent of both Understanding of influenza cause and Self-efficacy. However, when stratified by gender, the Trust in informal information-Understanding association was positive among males but negative among females. Education is probably an important influence in understanding and may have a bearing on these patterns which await clarification.
Susceptibility was strongly associated with both Self-efficacy (negatively) and Worry (positively). Neither Worry nor Susceptibility varied significantly by gender or age group. This is plausible and theoretically consistent [26, 34, 35, 39] . Worry was strongly associated with social distancing, again consistent with British data [49] . Although Worry was also significantly associated with hand hygiene, the association was weak. Elsewhere, using a generic measure of personal hygiene practices we have found a stronger association between disease worry and hygiene, suggesting a moderate effect of level of disease worry [57] .
The model tested explained only a modest proportion of the variance in adoption of HBPs, suggesting that there are significant theoretical gaps that remain to be filled. These await further research.
Social distancing is unassociated with formal HPB messages, suggesting potential susceptibility to a ''herd-like'' response in this Chinese community, particularly if confidence in formal (government or doctors) information is low. Voerten and colleagues describe such a pattern of response in the early stages of SARS [25] . These models support the hypothesis that social distancing is more likely to occur when perceived health threat is high [25] . Logically, when others seem to be behaving in a way that is informed and probably consistent then their actions provide clear information. If mixed social messages occur signalling uncertainty then the utility of social information will fall. This is likely to be associated with increase perceived susceptibility, and possibly greater worry and distancing behaviour. This pattern of responses would be most likely early in a novel RID epidemic where disease characteristics and behaviour are often uncertain. High threat uncertainty then drives social avoidance of potentially high-risk others. High levels of worry are associated with greater social distancing. Around 50% of 997/14,297 (response rate 7%) British respondents agreed that social avoidance would minimize risk of A/H1N1 infection, and respondents reporting more anxiety were more likely to engage in preventive actions; severity and likelihood of infection were the most important determinants of preventive action [12] . Further research on social influences on HPB during epidemic and pandemic RIDs is warranted. Providing more knowledge about disease causes can improve hand hygiene but is unlikely to influence social avoidance, which appears less amenable to formal health messages. However, as formal messages achieve acceptance across the population, and uptake of HPBs increases, then under circumstances where a critical mass of the population are practicing precautions trust in informal information should increase, reducing susceptibility and worry and leading to declines in social avoidance. Because others are likely adopting HPBs this makes them less of a contagion risk. Conversely maintaining a high level of hand washing practices may require sustained public education activities. Finally, different segments of the population probably communicate different types of information with their peers.
Self-efficacy in preventing A/H1N1 influences hand hygiene but has little influence on social distancing. Formal health education messages that focus on enhancing the public's sense of their ability to protect themselves by adopting hygiene practices would seem to be the most effective to improve hand hygiene, but where the practice is already established, high levels of trust in these messages are not likely to significantly increase hand hygiene.
This study is limited in being cross-sectional and relying on hypothesized modeling to infer causality. This is potentially errorprone and can only be confirmed by specific longitudinal tests of the hypotheses proposed above. There are potential limitations related to measurement imposed by the need to be parsimonious in questioning due to use of telephone interviews. Where this is not done refusal rates would have been unacceptably high [12] raising serious questions about representativeness. As a consequence, construct validity for some latent variables was weaker than expected, for example, only two items were scaled to measured trust in informal information giving a low internal consistency. We re-ran the SEM treating the two trust items as separate which gave almost identical associations with different situation awareness variables, so we entered their combined score as a latent variable in the final model. Only one item measured self-efficacy. This is generally not considered adequate but does have precedent indicating it is valid for predicting behavioral change [9] . Finally, this random sample, closely representative of the population of Hong Kong and collected early in the epidemic phase, nonetheless was slightly older and less-well educated than the general population. This was likely due to unavoidable sampling bias from surveying in the early evening to 10pm. Many young adults do not return home from work until after this time and were thereby not sampled. The results may in part reflect this bias. Otherwise the response rate was high at 69% and excellent compared to similar studies [12] . Some of these above limitations may also have contributed to the low explained variance of the model.
Many factors influence RID protective behaviour. This study has examined a very limited number of these. Confidence in formal information such as health education messages is associated with greater compliance to recommended preventive measures for influenza A/H1N1 [12] . However, the mechanisms for this were unclear. We have shown that this probably involves different mechanisms for hand washing and social distancing, and suggest how these might function. Formal messages may not reduce social distancing behaviours until such time that preventive behaviours are widely adopted in the community. Social distancing seems more likely to occur when there is high influenza-related worry and uncertainty, such as in the initial stages when epidemic circumstances are unknown, or if an epidemic is severe and appears poorly controlled, as during early SARS. This would seem to be largely worry/affect-driven. If so, then social distancing is likely to occur irrespective of government messages as population anxiety about an epidemic increases. Susceptibility may also increase and this may inhibit self-efficacy regarding hand washing. Finally, high levels of community uncertainty or rumour are likely to increase distancing by exacerbating perceived susceptibility and worry.
A simple version of our findings can be found it the supporting file (Text S1).
Table S1
Found at: doi:10.1371/journal.pone.0013350.s001 (0.05 MB DOC) Text S1 This is a simple version of our study findings for nonspecialists. Found at: doi:10.1371/journal.pone.0013350.s005 (0.03 MB DOC) Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans. Yersinia pestis (Y. pestis) is the causative agent of plague that has killed over an estimated 200 million people in previous pandemics [1] . The current incidence of plague is low but the animal reservoirs for Y. pestis exist worldwide. Sporadic cases have been reported recently with an average case number of 2,500 worldwide [2] . Y. pestis can be rendered airborne and its potential use as a bioweapon is recognized [3] as a category A agent on the NIAID list of biodefense-related pathogens. Current treatment for plague consists of antibiotics, while a live attenuated vaccine against plague is used in the former Soviet Union for prevention [4] . Nevertheless, these live attenuated whole-cell vaccines or killed whole-cell vaccines have adverse effects to varying degrees [4] . Though both types of treatment are efficacious, there is a need for an alternative treatment for plague [5] .
A multiple-antibiotic-resistant isolate of Y. pestis has been isolated, and drug resistance was shown to be mediated by a self-transferable plasmid [6, 7] . A subunit vaccine, which consists of two virulent factors, the F1 protein and V-antigen, is currently in human clinical trials [8] [9] [10] . Studies involving the vaccine antigens in various formats have provided the proof-of-concept data that humoral response can be efficient in protection against Y. pestis [11, 12] . There are multiple reports that mouse anti-plague monoclonal antibodies (mAbs) against a Y. pestis challenge can passively protect a mouse against plague [13] [14] [15] . Therefore, mAb therapy may be an attractive alternative to the existing treatments for plague. Despite the promising possibilities, there remains a major hurdle in the treatment against plague and that is the possible immune response of humans to the mouse mAbs that are currently available. One possibility to ameliorate the immune response against the mouse mAb is to humanize the mAb for use in humans, or another alternative is to develop new and fully human anti-plague monoclonal antibodies for clinical usage [16] .
We describe here the isolation of three mAbs from a large naive human phage-displayed Fab library. One, designated as m252, is against the F1-antigen and the other two (m253, m254) are against the V-antigen. When used alone, m252 displayed good protective effects, whereas m253 and m254 did not. However, a clear synergistic effect was found when they were used together. Maximum protection by m252 alone could be achieved by altering the antibody administration schedule. This is the first report describing the isolation of fully human anti-plague mAbs that show efficacy in a mouse model of plague. These antibodies represent a significant breakthrough toward possible adjunctive therapeutic treatment of Y. pestis infection in humans.
With the F1 antigen, only the plate format yielded positive Fab clones after four rounds of selection with the F1 antigen. Sequencing of the clones confirmed that they were identical and designated as m252. With the V-antigen, the plate and bead format each yielded two positive Fab clones after four rounds of selection with each format. One clone from each format, designated as m253 and m254, respectively, was selected for further analysis. Sequence analysis revealed that m252 has heavy and light chains originated from germlines IGHV1-2*02 and IGKV1-16*01 respectively. M253 originated from IGHV1-18*01 and IGKV1-9*01, while m254 was from IGHV3-43*01 and IGKV1-27*01. The mutational rate ranged from zero to less than 10%. This is typical for antibodies isolated from naïve human libraries by panning against viruses causing acute infection in contrast to neutralizing antibodies selected from immune human libraries by panning against HIV-1, which causes a chronic infection [17] . Each of the clones was then transformed into HB2152 cells and the respective Fab was expressed and purified ( Figure 1a ). After conversion to IgG1 expressing clones, the three antibody clones were transiently transfected into Freestyle HEK 293F cells, and the expressed IgG1s were purified ( Figure 1c ).
To determine both the specificity and affinity of the selected antibodies, ELISA with both Fab and IgG formats were conducted as described in the methods. All Fabs and IgGs bound to their respective antigens specifically without cross-reaction to other antigens tested (Figure 1b and d) . Anti-F1 Fab and IgG have apparent affinities in the low and sub-nM range, respectively. Both m253 and m254 Fabs have apparent affinities of approximately 100 nM (Figure 1b and d) . Their IgGs however have sub-nM apparent affinities (avidities). The avidity effect is very pronounced for all three antibodies.
Low level of competition between the human anti-F1 and anti-V Fabs and mouse anti-F1 and anti-V mAbs
The three human anti-plague Fabs were used in competition-ELISAs against a panel of mouse anti-plague mAbs. The mouse anti-plague mAbs included the anti-F1 mAb F1-04-A-G1, and five anti-V mAbs, which included the anti-V mAb 7.3 m that was highly protective. We found no apparent competition between the human anti-V m253 Fab and the mouse anti-V mAbs (Figure 2a ). However, we observed some weak competition between the human anti-V m254 Fab antibody and some of the mouse anti-V mAbs (7.3 m, 10-1 m, and 74-1 m) that we did not see with the human anti-V m253 Fab antibody (Figure 2b ). The competition between the human anti-F1 m252 Fab antibody and the mouse anti-F1 mAb was also minimal (Figure 2c ). We ran two controls in the competition studies with the human and mouse mAbs. For one control we did not add a primary antibody (labeled NC, Figure 2a -2c), and for a second control we used a nonspecific mouse isotype IgG1 mAb in the competition assay (labeled Bm, for a Burkholderia mallei IgG1 mAb). There was also a lack of competition between the human anti-V m253 and m254 Fab antibodies, suggesting that these two human anti-V Fabs recognize different epitopes (one which may be conformational) on the V-antigen (Figure 2d ). Of note, however, is that when the competition-ELISA was performed in a different fashion, namely when the human anti-F1 or anti-V mAbs were allowed to bind to the respective antigens before adding the mouse anti-F1 or anti-V mAbs, moderate competition was detected between the human anti-F1 m252 and the mouse anti-F1 mAbs, as well as between the human anti-V m253 and mouse anti-V 84-1 mAbs (data not shown).
To characterize the binding of the human anti-F1 and anti-V mAbs to the F1-and V-antigens, respectively, more closely, we examined the binding of the human mAbs to two separate panels of overlapping peptides. One panel covered the full-length of the F1 antigen (27 peptides) and the other -the V antigen (53 peptides). For the human anti-F1 m252 mAb, there was a weakmoderate binding signal with peptides 1 and 2, which are located at the N-terminus of the F1-antigen (Figure 3a ). This suggests that the m252 mAb may also recognize a conformational region that involves peptides 1 and 2. The binding by human anti-V m253 mAb resulted in a strong signal with peptide 2 and a weak signal with peptide 1 (Figure 3b ). The anti-V m254 mAb, did not bind to any of the peptides, suggesting that its epitope may be conformational ( Figure 3c ). In initial studies with m254 we saw some weak binding to peptides 36 and 42, but when we repeated the binding studies with the human anti-V mAbs we did not see binding to peptides 36 and 42. This might explain its weak competition with mouse antibodies that recognize diverse epitopes on the V-antigen ( Figure 2b ). The positive signals seen with the m253 and m254 mAbs with V-antigen peptides (numbers 19, 20, 27, and 28) are nonspecific signals that are generated by the secondary antibody (Amemiya et al. unpublished). The epitopes of the mouse antibodies have also been determined by the peptide binding assay (Amemiya, et al. unpublished). The data is consistent with the competition-ELISA presented in this study.
To test if the human anti-F1 and anti-V IgGs bind their respective targets on bacterial cells, we performed flow cytometry analysis. Both the mouse anti-F1 F1m mAb and human anti-F1 m252 mAb bound specifically to Y. pestis grown at 37uC and not to the same strain grown at 26uC. Neither did they bind to a control E. coli strain grown at 37uC (Figure 4 , bottom panel). This is consistent with previous reports that the expression of the F1-antigen is regulated by temperature (37uC), and it is not expressed at RT. The human anti-V m254 mAb showed minor binding to the Y. pestis grown at 37uC, although we do not normally see binding with the human anti-V m253 or mouse anti-V mAbs, which included 7.3 m, 74-1 m, and 141-1 m (Amemiya, unpublished). A mouse IgG1 isotype control mAb, which did not show any binding to the wholecells, was included to show binding by the mouse and human anti-F1 mAbs was antibody specific. To further confirm the binding data we used immunofluorescence technique. The results were consistent with the flow cytometry data, where only the mouse and human anti-F1 mAb bound to Y. pestis whole-cells ( Figure 5 ). Neither the human anti-V m254 mAb ( Figure 5D ) nor mouse anti-V mAbs, like 7.3 m (data not shown) nor control samples ( Figure 5A , nonspecific mouse IgG1 isotype; Figure 5E , no primary mAb) showed significant binding to Y. pestis.
Human anti-F1 and V mAbs protect synergistically against a Y. pestis challenge in a bubonic plague model
The ability of the human anti-F1 and anti-V mAbs to passively protect mice against a Y. pestis infection was evaluated in a bubonic plague model. The human anti-F1 and anti-V mAbs were used either separately or together in different combinations. When mAbs m252 and m253 and m254 were given to mice separately before challenge with Y. pestis CO92, only the human anti-F1(m252) mAb showed some efficacy. The mean-time-to-death (MTD) in the m252 mAb-treated mice was shifted to 13.0 days (1/ 6 survivors) when compared with mice given normal mouse serum (NMS), which had a MTD of 7.0 days (0/6 survivors) ( Figure 6A ). Unlike m252, however, the human anti-V mAbs, m253 and m254, did not show any significant protection [mean-time-to-death (MTD) of 6.7 days (0/6 survivors) and 7.3 days (0/6 survivors), respectively] when compared to the NMS -treated mice. The mouse anti-F1 (F1m) and anti-V (7.3 m) control mAbs both passively protected all (6/6) mice under the same challenge conditions (MTD of 21 days). When both human anti-V mAbs were given to mice passively ( Figure 6B ), no improvement in protection after challenge was observed (MTD of 6.8 days, 0/6 survivors), which was similar to that seen with the NMS-treated mice (MTD of 7.3 days, 0/6 survivors). However, when the human anti-F1 m252 mAb (MTD of 11.3 days, 2/6 survivors) was given together with the two human anti-V m253 and m254 mAbs, a greater number of mice were passively protected (MTD 14.0, 5/ 6 survivors) than when the antibodies were used separately, suggesting a synergistic effect. Although we saw some protection with the human m252 mAb, and a synergistic protective effect when the human m252 mAbs was given with the human m253 and m254 mAbs in the bubonic plague model, we wondered if there was any effect (positive or negative) with a nonspecific human IgG1 mAb by itself or when combined with the human anti-F1 m252 mAb in the number of survivors in the mouse model of plague. To answer this question, we injected five groups of mice with the following mAbs: one group of mice with only a nonspecific human IgG1 mAb (Hu-IgG1, 1500 mg); another group of mice with the mouse anti-F1 mAb (F1m, 500 mg); another group of mice with the human anti-F1 mAb (m252, 500 mg). Two other groups of mice were included, that were given either F1m (500 mg) or m252 (500 mg), with the nonspecific Hu-IgG1 mAb (1000 mg) one day before challenge with Y. pestis CO92 ( Figure 7 ). All mice in the group that received only the nonspecific Hu-IgG1 mAb died by day 8, which was similar to the control antibody mouse groups in Figure 6A and 6B. The same number of survivors was obtained (6/6) whether mice were given only mouse F1m mAb or F1m combined with the nonspecific Hu-IgG1. As we have seen previously ( Figure 6A ), only 1/6 mice survived in the group that received only human m252 mAb. When the human m252 mAb was combined with the nonspecific Hu-IgG1 mAb, we obtained one more survivior (2/6) than we obtained without the nonspecific Hu-IgG1 mAb. This variation in the number of survivors was not different than we saw previously ( Figure 6B ). In addition, the MTD was not affected by the presence of the nonspecific Hu-IgG1 (15.8 days) when given with the Hu-antiF1 mAb (15.8 days). These results suggest that the nonspecific Hu-IgG1 mAb had little effect on the survival of mice given the human m252 mAb or mouse F1m mAb.
Delaying time of delivery of human anti-plague mAbs provided better protection against a plague challenge One possible reason we observed less protection with the human anti-F1 and anti-V mAbs in the mouse plague model was that the level of the human IgGs may not have been sustained in the mouse over time compared to the mouse IgG mAbs, We tested this hypothesis in two separate studies. We first examined the concentration of the human antibody in mice directly, by measuring the level of m252 (anti-F1) and m253 (anti-V) in serum after they were given the human mAbs by i.p. injection. Mouse sera were collected at different time points after the initial dosing and human IgG levels were monitored by direct ELISA. As seen in Figure 8A , the anti-F1 m252 mAb appeared to have a half-life of approximately 8 days, and the half-life of m253 mAb was approximately 10 days. After 21 days, the levels of these two human antibodies were undetectable. In contrast, the level of both the mouse anti-F1 (F1m) and anti-V (7.3 m) mAbs may have decreased initially like the human anti-plague mAbs, but after 21 days, the levels were still approximately 40-50% of the initial concentration ( Figure 8B ). The half-live of human IgG mAbs in mice reported here is similar to what was found in another study where human mAbs were used against another biothreat agent [18] . In contrast, a human IgG molecule would have an average serum half -life of 21 days in a human [19] . Because of these findings we then administered the anti-F1 m252 mAb at different time points relative to the time of challenge. While the original regimen provided consistently modest protection, administration of the human m252 mAb 24 and 48 hours postchallenge provided increasing protection with the 48 hours schedule provided complete protection ( Figure 9 ). Antibody administration at even later time points was not performed since mice began to die 3-4 days after challenge without any treatment. However, we did evaluate the effect of a second dose of antibody at a later time point. In this group, mice first received an initial dose of human anti-F1 m252 mAb 24 hour before challenge as was done with the earlier protocols. These mice then received a second dose of the human anti-F1 m252 mAb 5 days after challenge. There was an increase in both the number of survivors (5/6) and MTD (20 . Epitope mapping of the human anti-F1 and anti-V antibodies by peptide binding assay. A. Each of twenty-seven peptides that covered the full length of the F1-antigen were used to coat an ELISA plate (0.05 ml of a 25 mg/ml solution of each peptide), and binding by the human anti-F1 m252 mAb (0.05 ml of a 10 mg/ml solution) was analyzed. The sample labeled F1 was the full-length antigen used to coat the plate as the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled Media was the negative control with no primary antibody added. B and C. Each of fifty-two peptides that covered the full length of the V-antigen was used to coat an ELISA plate (0.05 ml of a 25 mg/ml solution), and the human anti-V m253 (B) and m254 (C) mAbs were used (0.05 ml of a 10 mg/ml solution), respectively, to analyze for binding. The sample labeled V was the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled Media was the negative control without the primary antibody. doi:10.1371/journal.pone.0013047.g003 days) approaching the efficacy displayed by a single dose administered 48 hour after challenge. These data suggest that the optimum serum concentration of the human IgG1s was critically dependent on the time of administration, and that the optimum concentration of the human anti-plague IgG1s in turn determined the outcome of the treatment protocol.
Antibiotics have been at the forefront of combating bacterial infection for decades with great success. However, the develop-ment of new antibiotics is struggling to keep pace with the emergence of drug resistant bacterial strains, for example as in Y. pestis [6] . There has been an intense interest in developing antibody-based therapies as an alternative method of treatment [5] . Initially, antibody-based therapy was mostly limited to treating cancer or immune disorders. However, because of a better understanding of the pathogenesis of infectious agents, and the advancement in the development of protective or neutralizing antibodies, the use of antibody-based therapy against infectious agents has become more frequent [20] . In this report we described the first isolation of fully human mAbs against the Y. pestis Figure 6 . Human anti-F1and anti-V mAbs show synergistic protection when used together in the murine bubonic plague model. The human and mouse anti-F1 and anti-V mAbs were given i.p. to mice 24 hrs before parenteral challenge with Y. pestis CO92, and the number of surviving mice for each treatment group was monitored for 21 days after challenge. 6A. The following antibodies and amounts were used: normal mouse serum (NMS, 500 mg); mouse anti-F1 (F1m, 500 mg); mouse anti-V (7.3 m, 100 mg); human anti-F1 (m252, 500 mg); human anti-V (m253 and m254, 500 mg each). 6B. The following mAbs and amounts were used: NMS, 500 mg; F1m, 500 mg; m252, 500 mg; m253+ m254, 500 mg each; m252+ m253+ m254, 500 mg each. doi:10.1371/journal.pone.0013047.g006 virulence factors F1-and V-antigens. Previous studies have shown that mouse mAbs can be effective in protecting mice against Y. pestis ( [13] [14] [15] . However, because they are mouse mAbs they are not safe to use in their present form in humans [21] . Of particular concern is the immune reaction against mouse primary antibody sequences in human system. This may lead to severe adverse effects and at the same time reduce the potential benefits. It is highly desirable to have fully human antibodies for these reasons. The fully human anti-F1 (m252) reported here displayed moderate to good protection against a bubonic plague challenge with Y. pestis CO92. On the other hand, the two anti-V mAbs (m253, m254) when used separately did not show any efficacy, but when they were used together with m252, the combination of the human anti-plague mAbs resulted in better protection overall, suggesting a synergistic effect between the antibodies. A similar effect was reported in studies using mouse anti-F1 and anti-V mAbs in a mouse model of plague [15] . Further in our case with the human anti-F1 mAbs, when we gave mice the human anti-F1 mAb 1-2 days after challenge, we saw a greater protection against a plague challenge could be achieved. This suggested that the maintenance of serum concentration of the human mAbs in the mouse was possibly one critical factor for better protection. Kinetic studies revealed that indeed the serum concentration of the human antibodies dropped further than the mouse anti-plague mAbs over the course of the study. It is also plausible that the human antiplague mAbs might bind to other mouse antigens nonspecifically, thus decreasing the amount of free circulating human anti-plague antibody in the mouse. Figure 7 . A nonspecific human IgG1 antibody (Hu-IgG1) had little effect on the mean-time-to-death (MTD) and number of surviving mice that received the human anti-F1 mAb. The nonspecific human IgG1mAb, and the human and mouse anti-F1 and anti-V mAbs were given i.p. to mice 24 hrs before parenteral challenge with Y. pestis CO92, and the number of surviving mice for each treatment group was monitored for 21 days after challenge. Mouse or human mAbs and their amounts were given to the following groups of mice: nonspecific human IgG1 (Hu-IgG1, 1,500 mg); F1m (F1m, 500 mg); F1m (500 mg) + Hu-IgG1(1,000 mg); m252, 500 mg; m252 (500 mg) + Hu-IgG1 (1,000 mg). doi:10.1371/journal.pone.0013047.g007 Another important underlying factor for efficient protection by antibodies is the epitopes the antibodies recognize. Although the human anti-F1 (m252) mAb appeared to bind to the same region as the mouse antiF1, which was at the amino-terminal end of the F1antigen (Amemiya et al., unpublished), we could not demonstrate direct competition between these two antibody species. This observation may be the result of the nature of the antibody binding site or epitope, because several mouse mAbs isolated independently recognized the same region or epitope on the F1-antigen, behaved in the same manner (Amemiya et al., unpublished). It may be that once these mAbs bound to the amino-terminal end of the F1antigen, they may not readily come off the protein or may dissociate very slowly. Whether this is because the binding site involved both linear and conformational sites is not known, but both the mouse and human anti-F1 mAbs bound to the whole anti-F1 antigen very well, but only weakly -moderately to the 59-peptides. Nevertheless, the human m252 mAb was as protective as the mouse anti-F1 mAb when the human mAb was given after challenge. It has also been reported that a neutralizing epitope on the V-antigen was located in a region spanning amino acids 135 to 275, and a possible minor, secondary neutralizing epitope exists near the amino-terminal region of the V-antigen [14] . Neither of our human anti-V antibodies reported here competed with the mouse anti-V antibodies efficiently. The minor competition between the human anti-V Fab antibody and the mouse anti-V antibodies suggests that the recognition site of the human anti-V antibodies is slightly different or they may partially share conformational binding site. These differences might be one reason for their inability to protect as efficiently as the human anti-F1 m252 mAb.
Exactly how the human anti-F1 252 m mAb is able to protect mice may be directly related to the presence of F1 antigen on the surface of the plague organism. The F1-antigen has been reported to be anti-phagocytic [22, 23] . The ability of macrophages to take up the plague organism is directly related to the lack of the F1antigen, and resistance to phagocytosis is related to the presence of the F1-antigen. The binding of the human anti-F1 252 m mAb to the surface of the plague bacilli or opsonization may trigger phagocytosis of encapsulated bacilli into macrophages, thereby allowing phagocytic cells to clear the host of the pathogen.
The exact mechanism by how the V-antigen exerts it virulence is not completely known. There are reports showing that Vantigen is secreted into the growth medium and the secretion is important for virulence [24, 25] . The secretion of V-antigen in the medium has been described to be dependent on contact with the host cell, and could also be directed into the host cell by a Yersinia outer proteins (Yops) dependent secretion (Ysc) type III system (TTSS) [26] [27] [28] . It also has been suggested that free V-antigen may enter the cell by endocytosis besides being injected into the cell by the Ysc TTSS [29] . Once inside the host-cell, we do not know exactly what host proteins interact with the intracellular Vantigen [29] . Nevertheless, there is some evidence that anti-V antibodies enhance phagocytosis through possibly the Fc receptor, and thereby block Yop delivery into the host cell, and thus preventing Ysc dependent TTSS injection of V-antigen into the host cell [30, 31] .
In this study, however, we were not able to detect binding of the human anti-V mAbs on the surface of Y. pestis cells by flow cytometry or fluorescent microscopy, suggesting that the Vantigen was not on the bacterial cell surface under the conditions used in our studies or the expression level of V-antigen was below the level of detection. As has been discussed previously, however, the presence of V-antigen on the surface of the cell may be dependent on contact with the eukaryotic host cell. The highly specific region of the neutralizing epitope (s) on the V-antigen suggests a possible ligand-receptor interaction between the Vantigen and a cellular factor. This indicates that perhaps the Vantigen exerts its biological effect through mechanisms other than mediating the TTSS pathway.
In conclusion, the human anti-plague antibodies reported here represent perhaps the ones that are closest to practical clinical use. They may be safer and more efficient in the human system due to their fully human nature, and a likely longer half-life in humans. Also, intravenous application of the antibodies in humans may be a rapid delivery system that may augment antibiotics treatment in plague-exposed individuals. Finally, the affinity of all three antibodies can be further increased using readily available techniques, may reduce the dose required for efficient protection. The successful development of these three human anti-plague Figure 9 . Post-challenge administration of the human anti-F1 m252 mAb conferred better protection. The human anti-F1 m252 mAb was administered before or after Y. pestis challenge, and mice were monitored for 21 days after challenge. The mouse anti-V 7.3m mAb (100 mg) was used as a positive control mAb. Normal human serum (NHS, 500 mg), which was used as a negative control, had only 5 mice per group. The numbers behind each antibody represent the time in days in which the antibody was administered (500 mg) to mice relative to the day of challenge (day 0). The two numbers after the human anti-F1 m252 (21 and +5) represent two different days when the mAb (500 mg) was added to the same group of mice relative to the day of challenge. doi:10.1371/journal.pone.0013047.g009 antibodies in this model suggests that new and more potent anti-V antibodies can be potentially developed using the same approach but with restricted V-antigen subunit fragments containing the critical neutralizing epitopes, and perhaps other virulent factors.
The Y. pestis CO92 strain used in the challenge studies was originally obtained from T. Quan, Centers for Disease Control and Prevention, Fort Collins, Co. It was isolated from the sputum of a human case of pneumonic plague [32] . The Y. pestis CO92 was grown and inoculum prepared for challenges essentially as described previously [33] . A Y. pestis pgmstrain, which was originally isolated from Y. pestis CO92, and used in the antibody binding studies described below was obtained from Susan L. Welkos (USAMRIID, Frederick, MD).
Y. pestis purified F1,V, and F1-V [34] protein antigens were obtained from Brad Powell (USAMRIID, Fort Detrick, Frederick, MD). The 27-peptide array that covered the F1-antigen were 14to 17-mers with 11 amino acid overlaps; they were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI)(Manassas, VA). The 53-peptide array that covered the V-antigen were 15-to 17-mers with 11 or 12 amino acid overlaps and were obtained from BEI.
The anti-F1 mouse mAb F1-04-A-G1 (or mF1) was provided by George Anderson (USAMRIID) [13] , and anti-V mouse mAbs 10-1 m, 74-1 m, 84-1 m, and 141-1 m as well as the control IgG1 mouse anti-Burkholderia mallei (Bm) antibody were obtained from Sylvia Trevino (USAMRIID) and anti-V mouse mAb 7.3 (7.3 m) was obtained from Jim Hill (Porton Down, Wiltshire, UK) [14] and used in competition ELISAs and as positive controls in mouse passive protection experiments. All mice mAbs were IgG1 isotypes. The human IgG1 control mAb used in the passive protection study was an anti-human IGFII mAb.
Purified F1-and V-proteins were either coated directly to Maxisorp plates (Nunc, Denmark) in PBS buffer at 4uC, overnight for plate format panning or were biotin-labeled first with EZ-link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) for streptavidinconjugated magnetic bead format panning. The labeling was performed according to the manufacture's recommended protocol. For the plate format, approximately 10 12 Fabs displayed on the surface of phage amplified from a large naive library [35] were suspended in PBS with 2% dry milk and applied to wells coated with the F1-or V-proteins. After incubating for 2 hours at room temperature, each well was washed 5 times for the first round and 10 times for the subsequent four rounds before the phage were rescued with TG1 cells at the exponential growth phase. For the bead format, biotin-labeled F1-and V-antigens were first incubated with the same amount of phage as in the plate format in 1 ml of PBS+2% dry milk suspension at room temperature for 2 hour. Fifteen ml of Dynabeads MyOne Streptavidin T1(Invitrogen Dynal AS, Oslo, Norway) pre-blocked with PBS+2% dry milk was then added to the antigen/phage mixture for one hour at room temperature. The beads were then washed 5 times with PBS for the first round and 10 times for the subsequent four rounds of selection. Phage were then rescued with TG1 cells. A total of four rounds were performed for each antigen with each format.
Monoclonal ELISA was then performed to select for positive clones. One hundred clones were screened for each antigen from each format. Only clones displaying an OD405.2.0 were selected for plasmid preparation and sequencing.
Expression, purification, conversion to IgG1, and generation of stable clones Clones selected as described above were transformed into E.coli strain HB2151 for expression [36] . Briefly, a single clone was inoculated into 2YT supplemented with 100 units of ampicillin and 0.2% glucose and incubated at 37uC with shaking. When the OD600 reached 0.6-0.9, IPTG was added to achieve a final concentration of 1 mM and the culture was shifted to 30uC with shaking and incubated overnight. Cells were then collected, and lysed with polymyxin B (Sigma, St Louis) in PBS, and mixture subjected to Ni-NTA agarose bead (Qiagen, Hilden, Germany) purification. For IgG1 production, the heavy and light chains of the respective Fabs were cloned into the bi-cistronic expression vector pDR12 kindly provided by Dennis Burton (Scripps Research Institute, La Jolla, CA). For small scale IgG1 production, transient transfection and expression in Freestyle HEK 293F cells (Invitrogen, Carlsbad, CA) were used. For large scale production, stable clones were generated using CHO-K1 (ATCC, Manassas, VA) cells. Briefly, the heavy and light chains of the three human anti-plague IgG1s were cloned into pDR12 vectors and transfected into CHO-K1 cells. One day after transfection, the cells were replated and subjected to selection in GMEM medium supplemented with 25 mM MSX. Two weeks later, the MSX resistant clones were amplified further. The clones were tested for the expression of respective IgG1s and then adapted to growth in serum-free medium HyQSFM4CHO (HyClone, Logan, UT) supplemented with 30 mM MSX. The serum-free growth medium was then collected and passed through a protein A-sepharose resin column for IgG1 purification.
An ELISA assay was used to assess the binding ability of the Fabs and IgG1s. Briefly, F1-and V-antigens were coated to a Costar high binding 96-well plate (Corning, Corning, NY) and incubated overnight at 4uC. The next day, the plate was blocked with 2% dry milk in PBS before serial dilution of Fabs or IgGs were applied to the plate. After an incubation of the plate at 37uC for one hour, anti-His-horse radish peroxidase (HRP) for Fab detection or anti-human-Fc-HRP (for IgG detection) in PBS+2% dry milk was added to each plate and incubated for another hour at 37uC. The plates were then washed four times, and the ABTS substrate (Roche, Mannheim, Germany) was added. After approximately 10 min at room temperature, the OD405 was taken.
For competition studies between the mouse mAbs and human Fabs the antigen at 2 mg/ml (F1-protein or V-antigen) was used to coat 96-well plates (Immulon 2HB, Thermo Electron, Milford, MA), and the plates were incubated overnight at 4uC. After washing the plates, a blocking solution (1% bovine serum albumin with 0.05% Tween 20 in PBS) was added to the plates, and plates incubated for 1 hr at 37uC. The flag-labeled human Fabs, and biotinylated-mouse mAb were allowed to bind to the antigen simultaneously for 1 hr at 37uC. To detect the presence of the human Fab, an anti-flag-M2-peroxidase conjugate was used, and to detect the amount of biotinylated mouse mAb present, a streptavidin-conjugated HRP was added for 1 hr at 37uC, before adding a hydrogen peroxidase-3,39,5,59-tetramethylbenzidine solution. The color reaction was allowed to develop at room temperature for 15 min and read at 450 nm.
For analysis of IgG1 binding to F1-or V-antigen peptides, the peptides were added to the plates in 0.05 ml per well at 25 mg/ml. and the plates incubated overnight at 4uC in Immunlon 2HB 96well plates. After the washing and blocking steps as described above, binding of the human and mouse mAbs IgGs was detected as described above. The binding of all mAbs to the peptides was evaluated at 10 mg/ml.
Flow cytometry was used to analyze the binding of the human anti-F1 and anti-V antigen IgG1s to Y. pestis pgmwhole-cells. Y. pestis pgmwas first streaked onto a sheep-blood agar plate and grown at room temperature (RT) for 3 to 4 days until colonies were readily visible. A single colony was picked and inoculated into 10 ml of Heart Infusion Broth (Remel, Lenexa, KS) containing 0.2% xylose and 2.5 mM CaCl 2 , and cells grown overnight (o/n) at RT with rigorous shaking. The next day, an aliquot of the bacteria culture was shifted to 37uC, and the other remained at RT, and the cultures were allowed to continue for another 3 hours before the bacteria were collected and suspended in PBS. Ten mL of bacterial cells was mixed with 90 mL of Fc Block solution [PBS with 1% FCS and 10 mg/ml FcBlock (BD Bioscience)] to achieve a final density of 1610 7 cells/ml. Human or mouse IgGs were added to the bacterial cell suspension to a final concentration of 10 mg/ml. After incubating at 4uC for 30 min, the bacterial cells were collect by centrifugation and suspended in 1 ml of PBS, and cells washed twice. The bacterial cells were then suspended in the same Fc Block solution, and secondary antibodies, which included either goat anti-mouse IgG-FITC (Pierce, Rockford, IL) or goat anti-human IgG-FITC (Southern Biotech, Birmingham, AL) were added to the cells at a dilution of 1:100. After 30 min at 4uC, the cells were then washed three times with PBS and subjected immediately to FACS analysis using a FACSCalibur (BD Bioscience, San Diego, CA), after the cells were fixed with 4% paraformaldehyde. For detection of antibody binding to whole-cells by immunofluorescent microscopy, the growth of Y. pestis pgmand the sample preparation for binding by human or mouse anti-F1 or anti-V mAbs was identical as that for the FACS analysis, except a Nikon T-2000 fluorescent microscope (Nikon Instruments Inc., Melville, NY) was used for detection.
Passive protection by human or mouse anti-F1 or anti-V mAbs and Y. pestis challenge studies Antibodies to be evaluated for their efficacy against a plague challenge were given intraperitoneal (i.p.)(500 mg per mouse, except when stated differently in the figure legend) to 6-10 week old BALB/c mice 24 h before they were challenged or at time points post-challenge as indicated in the figure legend. The challenge dose was prepared from frozen stocks of Y. pestis CO92 that were streaked on tryptose blood agar slants and incubated at 28uC for 48 h. After the incubation period, the slants were rinsed with 10 mM potassium phosphate buffer, pH 7.0, and cell density adjusted to the required density with the same buffer. Mice were given the challenge dose of LD 50 ,25-40, subcutaneously in 0.2 ml, where 1 LD 50 is equal to 1.9 cfu [37] and observed for at least 21 days. Research was conducted in compliance with the Animal Welfare Act and other federal statues and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The studies involving mice were approved by the IACUC at the U.S. Army Medical Research Institute of Infectious Diseases, animal protocol number AP-07-040.
Half-life of human or mouse anti-F1 and anti-V mAbs given passively to mice An ELISA as described above was used to detect the amount of human or mouse anti-F1 and anti-V mAbs present in mice over time. Briefly, F1-V protein (2 mg/ml in 0.2 M carbonate buffer, pH 9.4)) was used to coat a 96-well plate (Immulon 2HB) overnight at 4uC before washing and blocking. Two-fold dilutions of mouse serum taken retro-orbitally after i.p. administration of the mAb were made in 1X PBS with 1% BSA and 0.05% tween-20, added to plates and incubated for 1 hr at 37uC before washing. The amount of human or mouse anti-F1 or anti-V mAb binding to the antigens was detected by the addition of goat-anti-human or goat-anti-mouse IgG conjugated to HRP (Southern Biotechnology). The results from 3 mice at each time point for each mAb was performed in triplicate and were reported as the mean of the reciprocal of the highest dilution giving a mean OD of at least 0.1, which is at least twice the standard deviation (SD). Diagnostic value of triggering receptor expressed on myeloid cells-1 and C-reactive protein for patients with lung infiltrates: an observational study BACKGROUND: Differential diagnosis of patients with lung infiltrates remains a challenge. Triggering receptor expressed on myeloid cells (TREM)-1 is a neutrophil and monocyte receptor up-regulated during infection. The aim of this study was to evaluate the diagnostic accuracy of TREM-1 and of C-reactive protein (CRP) from patients with lung infiltrates to discern community acquired lung infections. METHODS: 68 patients admitted to a medical ward with acute respiratory illness were enrolled in the study. Neutrophil and monocyte TREM-1 expression were measured by flow cytometry, sTREM-1 by an enzyme immunoassay and C-reactive protein by nephelometry. Clinical pulmonary infection score was recorded. RESULTS: 34 patients were diagnosed with bacterial community acquired pneumonia (group A) and 34 with non-bacterial pulmonary disease (group B). Median serum TREM-1 concentration was 102.09 pg/ml in group A and lower than 15.10 pg/ml (p < 0.0001) in group B. Mean±SE neutrophil TREM-1 expression was 4.67 ± 0.53 MFI in group A and 2.64 ± 0.25 MFI (p = 0.001) in group B. Monocyte TREM-1 expression was 4.2 ± 0.42 MFI in group A and 2.64 ± 0.35 MFI (p = 0.007) in group B and mean±SE CRP was 18.03 ± 2 mg/ml in group A and 7.1 ± 1.54 mg/ml (p < 0.001) in group B. A cut-off of 19.53 pg/ml of sTREM-1 with sensitivity 82.6% and specificity 63% to discriminate between infectious and non-infectious pulmonary infiltrates was found. sTREM-1 at admission greater than 180 pg/ml was accompanied with unfavourable outcome. CONCLUSION: TREM-1 myeloid expression and sTREM-1 are reliable markers of bacterial infection among patients with pulmonary infiltrates; sTREM-1 is a predictor of final outcome. Early diagnosis of lung infections remains a challenge. There is no gold standard for diagnosing microbial infection as clinical and laboratory signs are neither sensitive nor specific enough, and microbiological studies often remain negative. The presence of a new infiltrate on plain chest radiograph is considered indicative for diagnosing pneumonia, especially when is supported by clinical and laboratory findings. However it is difficult to differentiate a chest infiltrate of bacterial origin from a chest infiltrate of non-bacterial origin solely based on radiological criteria [1] . The diagnosis of infection is not always clear in the acute setting in patients with respiratory tract disease and a surrogate marker of infection would be a major benefit in the diagnostic armamentarium. Many inflammatory mediators and acute phase reactants, like C-reactive protein (CRP) and procalcitonin, have been described as reliable markers of infection; however none are specific enough, since they are also increased in non-infectious inflammatory conditions [2] .
Triggering receptor expressed on myeloid cells (TREM)-1 is a recently described receptor on neutrophils and monocytes. It behaves like a pattern recognition receptor (PRR) since its activation leads to the release of pro-inflammatory cytokines, namely of tumour necrosis factor-alpha (TNFα) and of interleukin (IL)-8. Although its ligand is still unknown, activation is mediated by bacteria and fungi [3, 4] . A soluble form of TREM-1, namely sTREM-1, is increased in the bronchoalveolar lavage (BAL) of patients with ventilator associated pneumonia (VAP) [5, 6] , and in the serum of patients with sepsis, with bacterial meningitis and with acute pancreatitis [7] [8] [9] [10] [11] [12] . This same soluble form of TREM-1 seems to be increased in patients bearing noninfectious processes like peptic ulcer, inflammatory bowel disease, viral infections, malignant pleural effusions and chronic obstructive pulmonary disease (COPD) but also among patients after cardiac surgery or cardiac arrest. Increase of sTREM-1 seems particular prominent when the latter non-infectious states are complicated with systemic inflammatory response syndrome (SIRS) without infection [13] [14] [15] [16] [17] [18] [19] .
Several published studies yielded contradictory results for the diagnostic and prognostic usefulness of TREM-1 and of sTREM-1 for infections [5, 7, [20] [21] [22] . The created impression is that more data are necessary to yield definitive results for its usefulness as a diagnostic and prognostic marker of community acquired pneumonia (CAP).
The aim of the present study was to define whether expression of TREM-1 on cell membranes of neutrophils (nTREM-1), of monocytes (mTREM-1) and serum sTREM-1 may help in the diagnosis of acute bacterial infections for patients admitted with a new pulmonary infiltrate or pleural effusion.
In this observation trial, all consecutive admissions to the Department of Critical Care and Pulmonary Services on predetermined and randomly selected emergency duty days were eligible. Inclusion criteria were: i) age above 16 yrs, ii) written informed consent; iii) acute respiratory illness and iii) presence of new pulmonary infiltrates or pleural effusion on chest x-ray or lung computed tomography. Exclusion criteria were: i) Human immunodeficiency virus (HIV) infection, ii) documented extrapulmonary infection, iii) neutropenia; and iv) oral intake of corticosteroids defined as any more than 1 mg/kg of prednisone for more than 1 month. The study protocol was approved by the Ethics Committee of the hospital and written informed consent was obtained from all patients within the first 12 hrs after admission.
Clinical, laboratory, and imaging data were recorded for each patient including: i) clinical presentation; ii) body temperature, iii) arterial blood gas, iv) peripheral blood cell counts, v) gram stains and cultures of all biological fluids obtained (blood, sputum, bronchial secretions, BAL, and pleural fluid); vi) imaging findings, vii) antigen serology (Legionella spp and Streptococcus pneumonia urinary antigen, serological testing for Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae) and viii) in-hospital mortality. The severity of illness was assessed by calculating Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA) and Clinical Pulmonary Infection (CPIS) scores at admission [23] .
A diagnosis of community-acquired pneumonia (CAP) was established in any patient presenting with a combination of fever, cough and purulent sputum, shortness of breath, chest pain, and new consolidation on chest X-ray or computed tomography. The severity of pneumonia was assessed the first 24 hours of admission according to Confusion, Urea nitrogen, Respiratory rate, Blood pressure (CURB) index. Patients having two or more criteria were identified to have severe pneumonia [24] . Sepsis, severe sepsis and septic shock were defined according to current recommendations [25] . Pneumonia was considered to be absent when: i) an alternative cause for pulmonary infiltrate was established (e.g. pulmonary embolus) and ii) full recovery was achieved without antimicrobial therapy.
Pulmonary embolism was diagnosed according to current recommendations [26] . Lung cancer was ruled out based on histology and/or cytology specimens. Congestive heart failure was diagnosed according to American Heart Association [27] , and interstitial lung disease according to American Thoracic Society guidelines [28] . All cases were evaluated by two clinicians blinded to TREM-1 and sTREM-1 results. Agreement about the diagnosis was achieved in all cases.
Patients with CAP were classified as having bacterial respiratory infection (group A). All other patients were classified as having non-bacterial respiratory disorders (group B). All patients assigned to group B were subject to chest computed tomography.
For the measurement of sTREM-1, mTREM-1, nTREM-1 and CRP 10 ml of peripheral venous blood were sampled after venipuncture of the antecubitul vein under sterile conditions on the day of admission and on days 3 and 7 of hospitalization. Seven ml were centrifuged and serum was stored in -80°C until assayed for sTREM-1. Three ml were collected into EDTA-coated tubes (Vacutainer, BD) for estimation of nTREM-1 and mTREM-1 expression. Briefly, red blood cells were lysed by ammonium chloride. White blood cells were labelled by phycoerythrin-conjugated anti-TREM-1 monoclonal antibodies (R&D InC, Minneapolis, USA) for 30 minutes in the dark. nTREM-1 and mTREM-1 expression were assessed after passage of labelled cells through a flow cytometer (Epics XL/MSL, Beckman-Coulter Co, Miami Florida) and expressed as the mean fluorescence intensity (MFI) with gating for neutrophils and for monocytes by their characteristic FS/SS scattering.
Determination of sTREM-1 was performed in duplicate by a developmental enzyme-linked immunoabsorbent assay according to the instructions of the manufacturer (R&D Inc, Minneapolis, USA). The lower detection limit and inter-day variation of the assay were 15.1 pg/ml and 5.23% respectively.
Measurement of serum CRP was performed by an immunoturbidimetric assay on Roche automated clinical chemistry analyzers and was expressed in mg/ml. CRP was used as a comparator due to its universal application in all studies of evaluation of biomarkers.
Asumming that measured parameters between groups A and B differed by 50%, it was calculated that 30 to 40 patients should be assigned into each group to yield a difference at the 5% level with 80% power.
Values for nTREM-1, mTREM-1 and CRP are presented as mean ±SE; those of sTREM-1 are presented as medians and 95% confidence intervals (CI) or interquartile range (IQR). Comparisons between groups for nTREM-1, mTREM-1 expression and for CRP were done by ANOVA, followed by the Tukey's test for multiple comparisons. Comparisons of sTREM-1 between groups were done by Mann-Whitney U test after Bonferroni corrections for multiple comparisons. Comparisons of sTREM-1 between consecutive days within one group were done by Wilcoxon's signed rank test. Receiver Operator Curves (ROC) were designed to asses sensitivity, specificity, positive and negative predictive values for the estimated parameters to disclose infectious from non-infectious infiltrates. Patients were divided into two categories according to serum levels of sTREM-1 upon admission: those with sTREM-1 below or equal to 180 pg/ml; and those with serum sTREM-1 greater than 180 pg/ml. This concentration has been proposed as a threshold defining final prognosis in septic populations [22, 29] . Since CAP is a common cause of sepsis, this threshold was considered of merit. Survival was assessed by Kaplan-Meier and comparisons were done by log-rank test. Correlations between severity scores and measured parameters were done according to Spearman. Probability values less than 0.05 were considered statistically significant. All statistics and graphs were done using the Statistical Package for the Social Sciences software version 17.0.0 (SPSS Inc, Chicago, IL).
The study flow-chart is shown in Figure 1 . Demographic and clinical data of the patients are summarized in Table  1 . Patients suffering from tuberculosis and enrolled in group B were presented with pleuritis.
Group A (n = 34) consisted of patients with community acquired pneumonia (CAP) likely to be caused by extracellural bacteria. Seventeen had microbiological evidence of pulmonary infection, with isolation of the offending pathogens from sputum, blood or BAL samples (when bronchoscopy was performed). Seventeen patients were diagnosed with CAP on the basis of typical clinical and radiological presentation and good response to antibiotic therapy. Main radiological Group B (n = 34) consisted of patients with non-bacterial respiratory disorders. Diagnoses were: lung cancer (13 patients); pulmonary embolism (six patients); interstitial lung disease (six patients); heart failure (n = 5); pulmonary tuberculosis (two patients); rheumatoid pleuritis (one patient); and Q-fever (one patient). Main radiological findings were: right pulmonary infiltrate (six patients); left pulmonary infiltrate (three patients); bilateral pulmonary infiltrates (11 patients); right pleural effusion (four patients); left pleural effusion (one patient); both right lung infiltrate and right pleural effusion (four patients); both left lung infiltrates and left pleural effusion (two patients); bilateral pulmonary infiltrates and left pleural effusion (one patient); and left pulmonary infiltrate and bilateral pleural effusions (one patient).
Among patients from group A with CAP nine (n = 9) died; six patients were admitted to the ICU and three were not admitted to the ICU due to relatives' denial. Mean age of patients not admitted to ICU was 80 years; the first two patients had a case-history of stroke and chronic heart failure; the third patient had a case-history of lung cancer. All three died from severe sepsis and multiorgan dysfunction syndrome (MODS). Mean age of patients admitted to ICU was 70 years; two patients had a case-history of aortic valve stenosis; two patients were under chronic intake of receiving corticosteroids; the fifth patient suffered from end-stage renal disease; and the sixth patient was suffering from hepatic failure due to alcohol intake. All six patients died from severe sepsis and multiorgan dysfunction syndrome (MODS). All patients in the ICU accomplished the clinical and radiological criteria for acute respiratory distress syndrome (ARDS) and were ventilated with the strategy of low tidal volume ventilation, according to current guidelines [30] , with volume limited mode ventilation, low tidal volumes (about 6 ml/kg ideal body weight), a maximum of 25-30 breaths per minute, high positive end-expiratory pressure (PEEP 10 cmH 2 O) and a goal plateau airway pressure < 30 cmH 2 O. Among patients admitted in the ICU, two died on the second day post-admission; one died on the third day post-admission; one on the seventh day post-admission; one the eighth day postadmission; and one on the twentieth day post admission.
Concentrations of sTREM-1 and of CRP in sera of both groups and expression of nTREM-1 and mTREM-1 are given in Table 2 . All four parameters were significantly greater in group A than group B.
ROC of sTREM-1, nTREM-1, m-TREM-1 and CRP to differentiate whether a chest X-ray infiltrate is due to CAP or to a non-infectious process is shown in Figure  2 . Area under curve (AUC) of sTREM-1 was 0.771 ± 0.068 (95%CI: 0.63 -0.9, p = 0.001). Sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were 82.6% and 63% respectively at concentrations above 19.53 pg/ml. AUC of nTREM-1 and mTREM-1 were 0.778 ± 0.063 (95%CI: 0.65 -0.9, p = 0.001) and 0.712 ± 0.07 (95%CI: 0.56 -0.86, p = 0.009) respectively. Sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were 78.3% and 58.6% for nTREM-1 above 2.55 MFI. Sensitivity and • Lung cancer (13) • Staphulococcus aureus (3) • Pulmonary embolism (6) • Haemophilus influenzae (2) • Congestive heart failure (5)
• Pseudomonas aeruginosa (2) • Interstitial lung disease (6) • Other (4 specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were 82.6% and 75.9% respectively for mTREM-1 above 3.05 MFI. AUC of CRP was 0.789 ± 0.065(95%CI: 0.66 -0.9, p < 0.001). Sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were 78% and 76% respectively at concentrations above 8.7 mg/ml. Positive correlations were found between APACHE II scores and expression of TREM-1 on monocytes on day 1 (r s : +0.363, p: 0.010); and between APACHE II scores and sTREM-1 on day 1 (r s : +0.262, p: 0.043). No significant correlations were found between APACHE II scores and expression of TREM-1 on neutrophils on day 1 as well as between SOFA scores and any of the measured parameters on day 1.
Correlations between serum levels of sTREM-1 and CRP and expression of TREM-1 on monocytes and neutrophils in relation to the identified causative pathogen of CAP are shown in Figure 3 . Serum levels of sTREM-1 were greater among patients with CAP caused by Gram (+) cocci and Haemophilus influenzae than among patients with CAP caused by other pathogens.
Death occurred in three out of 17 patients were no pathogen was defined (17.6%); in nil out of three patients infected by atypical pathogens (0%); in three out of seven patients (42.9%) infected by Gram-negative bacteria; and in three out of nine patients (33.3%) infected by Gram-positive cocci or H. influenzae (p: 0.034 between grouping according to pathogen). Survival of patients with sTREM-1 on day 1 below or equal to 180 pg/ml was prolonged compared with patients with sTREM-1 on day 1 above 180 pg/ml ( Figure 4) . 
The results of the present study indicate that TREM-1 can be used as marker of bacterial infection in patients with lung infiltrates. sTREM-1, nTREM-1, mTREM-1 and CRP were comparable to their discriminating ability between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin. sTREM-1 levels were decreased within the first 48 hours in patients with CAP with favourable outcome probably after the initiation of appropriate therapy followed by improvement of clinical symptoms. Finally, sTREM-1 levels above 180 pg/ml were an accurate independent predictor of in-hospital mortality from CAP.
Discrimination of the infectious or non-infectious origin of a pulmonary infiltrate remains an everyday clinical problem. CPIS was introduced for that purpose helping considerable in cases of ventilator-associated pneumonia (VAP) [23] . TREM-1 is a surface receptor on cells of the myeloid lineage. Activation of TREM-1 leads to the production of pro-inflammatory cytokines [9, 31, 32] . Binding of its ligand is possibly linked to the activation of several transcription complexes that synergize with NF-B in order to elicit transcription of genes of pro-inflammatory cytokines [8] . sTREM-1 is the soluble counterpart of TREM-1 and it is probably shed in the systemic circulation from cell membranes of neutrophils and monocytes [7, 33, 34] . The physiologic role of sTREM-1 remains under question despite data support a probable anti-inflammatory role [31, 35] .
TREM-1 has been studied in patients with pneumonia, especially VAP [5, 21, [36] [37] [38] . Few data are available on the diagnostic role of TREM-1 and of sTREM-1 in patients with lung infiltrates. Our data are in agreement with observations from the study by Phua [36] . Their proposed sTREM-1 cut-off point was 163 ng/ml, which is different than the one we found. This may be result from the different method of assaying sTREM-1 the used being Western blotting.
The results of our study are in contrast to those of another study [37] that did not disclose any difference in nTREM-1 expression between patients with and without a bacterial lung infection probably due to the small number of patients included in that former study. El Sohl et al [38] reported elevated alveolar levels of sTREM-1 in pulmonary aspiration syndromes, but not in serum. However, serial plasma sTREM-1 levels were not obtained and the possibility that plasma levels might rise on subsequent days cannot be excluded.
Two recent studies [39, 40] evaluated the diagnostic role of CPIS and of sTREM-1 in BAL fluid from patients with bilateral lung infiltrates in the intensive care unit (ICU). These studies reported controversial results. However authors did not measure sTREM-1 in serum on consecutive days.
The reported results of the present study are the first to our knowledge that evaluate the diagnostic value of TREM-1 among patients with lung infiltrates to discriminate CAP. They also disclose a relationship between levels of circulating sTREM-1 and causative pathogens. More precisely, infections caused by Streptococcus pneumoniae, Sthaphylococcus aureus and Haemophilus influenzae were accompanied by greater levels of sTREM-1 and by greater expression of TREM-1 on neutrophils than infections caused by other pathogens. Although it may be hypothesized that Gram-positive cocci and H. influenzae are strong inducers of TREM-1 expression, it should be emphasized that TREM-1 is one PRR, the exact agonist of which remains to be found [29, 31] .
A former study of our group [29] and another by Gibot et al [22] in heterogeneous populations of patients with severe sepsis of diverse aetiology investigated the role of early assessment of sTREM-1 as a determinant of final outcome. Results revealed that concentrations greater than 180 pg/ml are accompanied by survival benefit. The exactly opposing finding is reported here. This discrepancy may be explained by the enrolment of more homogeneous populations of patients, compared to these former studies [22, 29] , all suffering with CAP.
Our study presents two main limitations: a) no documented cases of CAP by Legionella pneumophila, Mycoplasma pneumoniae, protozoa or parasites were enrolled in group A; b) mortality in the CAP patient group was high probably due to the existence of severe co-morbid conditions.
In conclusion, the presented results indicate that serum sTREM-1 and expression of TREM-1 on neutrophils and monocytes may serve as markers of CAP in patients with pulmonary infiltrates. Concentrations of sTREM-1 in serum are particularly increased in CAP caused by Gram-positive cocci and Haemophilus species. The real clinical value of sTREM-1 assay comes when TREM-1 levels are low, allowing the clinician to withhold empiric antibiotics until culture results are available, and thus eliminating unnecessary antibiotic exposure to the patient. And finally, early serum levels of sTREM-1 greater than 180 pg/ml in CAP are associated with unfavourable prognosis. and Evangelos J. Giamarellos-Bourboulis Ass. Prof. MD have no conflicts of interest to disclose related to this study. Evangelos J. Giamarellos-Bourboulis Prof. MD has received reimbursement for attending the 29 th International Symposium on Intensive Care and Emergency Medicine where participated as a speaker and unrestricted educational grants from ABBOTT Hellas SA; Wyeth Hellas SA; Sanofi-Aventis Hellas SA. Nodeomics: Pathogen Detection in Vertebrate Lymph Nodes Using Meta-Transcriptomics The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-coding transcripts, we identified two new proteobacterial species; a Helicobacter closely related to Helicobacter cetorum in the Helicobacter pylori/Helicobacter acinonychis complex and an Acinetobacter related to Acinetobacter schindleri. Among viruses, a novel gamma retrovirus and other members of the Poxviridae and Retroviridae were identified. We additionally evaluated bacterial diversity by amplicon sequencing the hypervariable V6 region of 16S rRNA and demonstrate that overall taxonomic diversity is higher with the meta-transcriptomic approach. These data provide the most complete picture to date of the microbial diversity within a wildlife host. Our research advances the use of meta-transcriptomics to study microbiota in wildlife tissues, which will facilitate detection of novel organisms with pathogenic potential to human and animals. Information about the commensal and pathogenic microbial communities associated with host species, including humans, is limited. The endemic microbial community of a healthy host is important to characterize because its perturbation can be a cause of disease [1, 2] . Pathogenic microbes often escape detection if the clinical consequences of infection are similar to known pathogens or if they infect non-domestic species [3] . The maintenance of unknown pathogens in wildlife species is particularly problematic because many emerging human and livestock infections arise from contact with wild animals [4] [5] [6] [7] .
With the advent of meta-genomics methods, the entire community of microorganisms that exist in a given environment can potentially be identified. Pyrosequencing and other high throughput sequencing approaches have been applied to determine the microbial population in environmental samples such as soil and seawater [8] [9] [10] [11] and more recently to investigate the community of microbes on human mucosal surfaces [12] [13] [14] [15] , both of which are rich in microorganisms. Next generation sequencing methods have also been successfully applied to identify the microbial agents of several new diseases [16] [17] [18] [19] [20] . Recently, RNA based meta-transcriptomic studies [21] [22] [23] , which profile both protein-coding transcripts and ribosomal RNA (rRNA), have been used to study both functional and structural features of environmental microbial communities.
The key question behind this study was whether viable microorganisms could be detected within healthy mammalian lymphoid organs by employing massively parallel sequencing coupled with computational techniques able to detect transcripts of microorganisms among the abundant transcripts of the mule deer host. Lymph nodes are the specific replication sites for certain pathogenic viruses and bacteria [24] [25] [26] [27] [28] [29] . Moreover, although the blood and the lymph systems are considered to be essentially free of viable microorganisms in healthy individuals, the transient and often asymptomatic presence of both bacteria and viruses have been detected in the circulation [30, 31] . Phagocytic cells engulf these microbes and migrate to lymph nodes. Thus, lymph nodes should concentrate the commensal, endemic, and potential pathogenic microbial communities of a host species.
We evaluated the microbial community in retropharyngeal lymph nodes of mule deer to assess microbial exposure via the oral or respiratory route. Because ungulates browse and receive small punctures from sharp forage, we reasoned that healthy animals would potentially be exposed to microorganisms from their environment or to resident oral and rumen microorganisms that would be cleared in draining nodes. We used mule deer to highlight the utility of this approach in a wildlife host, but the method is broadly applicable to any host species.
Our studies document for the first time that there is a community of viable microorganisms in retropharyngeal lymph nodes of healthy wild ungulates. Furthermore, our findings demonstrate the applicability of meta-transcriptomic techniques for the detection of novel bacteria and viruses in internal organs.
The microbial community of mule deer lymph nodes Detection of protein-coding and ribosomal RNA transcripts provides strong support for the presence of viable and replicating microorganisms. Therefore, we enriched the total RNA obtained from lymph nodes for poly(A) + RNA to prepare cDNA libraries and subjected them to pyrosequencing on a Roche GS FLX sequencer (Roche-454 Life Sciences). Properties of sequencing runs are given in Table S1 . All reads were compared against the nonredundant NCBI protein database. The composite metatranscriptomic species profile for five individual and two pools of 4 or 8 mule deer samples, determined using the software MEGAN [32] , is depicted in Fig. 1A .
On average, 51% of total transcript-tags could be assigned to known taxa with a bit score cutoff of 50 (see Table S2 ). Of the assigned tags, 99.3% were of eukaryotic origin, predominantly matching to Bos taurus and other close relatives of mule deer that are represented in the protein database. Approximately 0.3% of the assigned tags were to bacteria. Proteobacteria represented 60% of all bacterial hits; Enterobacteriaceae in the Gammaproteobacteria were the most commonly identified within this group. Firmicutes and Actinobacteria represented 22% and 5% of the identified bacterial taxa. Table S3 lists all bacterial genera detected in the seven data sets. Transcripts assigned to Archaea, family Halobacteriaceae, were identified in both pooled samples but none of the individual libraries. Only 37 transcripts were assigned to viruses. Twenty-nine of these matched to the Retroviridae and Poxviridae while the remaining were to phages, insect viruses, and a single assignment to herpesvirus. These results suggest that representatives of many bacterial phyla, archaea, and two major virus families are transcriptionally active in mule deer retropharyngeal lymph nodes.
Meta-genomics studies evaluating microbial rich communities were pioneered based on genomic DNA sequences [8] [9] [10] 13] . Thus, we compared genomic libraries prepared from retropharyngeal lymph node tissue of MD 72360, MD 80228, MD 84709, and MD 84730 with our data from transcript libraries derived from those animals (Fig. 1 ). Many sequences from the genomic DNA libraries were to non-coding regions and could not be used for taxonomic profiling (Fig 1B, Table S2 ). Based on proteincoding sequences, only four bacterial genera were identified in the comprehensive MEGAN analysis of the four genomic data sets. Xylella and Burkholderia were identified in MD 72360, Acidovorax was found in MD 84709, and Bartonella was found in both MD 84709 and MD 84730. Bartonella and Xylella, as well as a member of the beta retroviruses (found in MD 80228 and MD 84709), were identified only in the genomic DNA data, suggesting that they might not represent actively replicating organisms. These findings indicate that meta-transcriptomics may be the preferred method for detecting the viable endemic microbial community in the tissues of healthy animals.
The most commonly detected microorganisms in the transcriptome libraries comprised intestinal and skin-dwelling bacteria and soil and freshwater bacteria. Ruminococcus, which is part of the commensal intestinal microbial community of ungulates, was detected in all seven libraries ( Fig. 1A and Table S3 ). Other bacteria found in at least three of the seven data sets were Propionibacterium, a commensal bacterium of skin and the gastrointestinal tract, and the environmental soil or water inhabitants Magnetospirillum, Streptomyces and Pseudomonas. Members of the latter genus are able to colonize a wide range of niches and are also potential pathogens. Other animal and human pathogenic genera detected in at least three different libraries were Burkholderia, Streptococcus, Flavobacteria, and members of the Enterobacteriaceae (Escherichia, Providencia).
The overall bacterial diversity and the number of unique transcripts assigned to each bacterial taxon varied among the samples. Notably, Helicobacter was only detected in the library constructed from MD 257 but there were 12 unique transcript-tags assigned to this genus. More commonly, bacterial taxon identification was based on a single tag. Many of the single transcript-tags came from MD 80228, which had the highest bacterial diversity profile of all libraries analyzed, and from MD 84730. Bacterial genera detected solely in either one or both of these two samples include Acinetobacter, Legionella, Enterobacter, Salmonella, Yersinia, Vibrio, Listeria, Mannheimia, and members of the Corynebacterineae, all of which contain known pathogens. In addition, both specimens depicted by far the highest numbers of reads taxonomically assigned to the family Enterobacteriaceae. The lowest diversity of bacterial genera was found in the MD OCT-pool, which was derived from eight different mule deer. Pooling RNA from several animals potentially increases the representation of transcripts common to all animals but might decrease the ability to detect transcripts that are unique to one animal. Consistent with this, the MD Bonner-pool, which was derived from four animals, provided a broader spectrum of bacterial genera than the MD OCT-pool. Thus, pooling samples did not improve our ability to detect microbial diversity in lymph node samples.
In contrast, viruses were detected in both pooled samples, although the total number of transcript-tags was low. Of the individual libraries, only MD 257 had evidence of viral transcripts (Fig. 1A) . The majority of viral transcripts were from a cervid poxvirus [33] , and a novel gamma retrovirus.
The computational analysis described above identified putative microorganisms in mule deer tissue based on detection of proteinencoding transcriptional activity. Although the cDNA used in our analyses was derived from total RNA enriched for polyadenylated RNA, it retained a considerable amount of the abundant ribosomal RNA (rRNA). These sequences contribute to the 'no hits' category in Figure 1 and Table S2 . Bacterial rRNA derived from the same dataset can, therefore, be used to provide additional support for species identification. By classifying the rRNA-tags from each library using the RDP rRNA classifier tool [34, 35] (http://rdp.cme.msu.edu/) we increased the number of bacterial genera identified (see Fig. 2 for MD 257, Fig. S1 for MD 80228 and MD OCT-pool, and Table S4 ). Abiotrophia, which is a component of the normal oral and intestinal microbial commu-nity, was detected in six of the seven libraries; environmental bacteria such as Thermoanaerobacter, which is frequently found in hot springs, were detected in four of the seven samples. Other genera that were identified based on rRNA in at least two of the libraries were Actinomyces, Campylobacter and Mycoplasma. Of particular importance, rRNA-tags supported the presence of Helicobacter in the MD 257 library (Fig. 2) , of Acinetobacter, Escherichia, Pseudomonas, Salmonella, Shigella and Variovorax in the MD 80228 library, and of Shigella in the MD Bonner-pool library ( Fig. 1 and Fig. S1 , Tables S3 and S4).
The support for Helicobacter in the MD 257 library was particularly compelling because there were 12 unique transcripttags and one rRNA-tag to this genus. We evaluated the phylogenetic relatedness of the mule deer Helicobacter with other Helicobacter based on four of the protein-coding transcripts and on the single 16S rRNA sequence. All analyses demonstrated that the Helicobacter detected in the mule deer lymph node is a unique organism that affiliates with the H. pylori cluster ( Fig. 3A and 3B, and Fig. S2 ). Because 16S rRNA sequence data is available for more species, we were able to further demonstrate that the closest Figure 1 . MEGAN comparison of the taxonomic profiles of (A) cDNA transcript-tags from 454 sequencing five individual lymph node samples and two lymph node sample pools and (B) genomic DNA-tags from four individual lymph node samples. Depicted are assignments with bit score cutoffs $50. Circle sizes are scaled logarithmically. Not assigned: sequencing-tags matching to sequences in the NCBI database that are not assigned to taxa; no hits: sequencing-tags not matching to any sequences in the NCBI database. doi:10.1371/journal.pone.0013432.g001 Figure 2 . MEGAN comparison of taxonomic profiles of MD 257 cDNA transcript-tags analyzed against the protein database (red) and the ribosomal database (blue), and of V6 amplicon 16S rRNA-tags analyzed against the ribosomal database (green). Bit score cutoff for the protein database comparison was set at 50, and confidence cutoffs for the ribosomal database comparisons were set at 80%. doi:10.1371/journal.pone.0013432.g002 relative to mule deer Helicobacter is a newly described H. cetorum isolated from different dolphin species (Lagenorhynchus acutus, Lagenorhynchus obliquidens, and Tursiops truncatus) and a beluga whale (Delphinapterus leucas) [36] (Fig. 3A) .
We also evaluated the phylogenetic placement of Acinetobacter detected in MD 80228 based on both 16S rRNA and rpo-b sequences. The number of rpo-b sequences for Acinetobacter in the database is limited. However, we demonstrated that the MD 80228 transcript-tag clustered with those of Acinetobacter (Fig. 3D ) [37] . Moreover, based on 16S rRNA, we determined that the Acinetobacter species identified in the MD 80228 cDNA library was distinct from all known Acinetobacter and was most closely affiliated with Acinetobacter schindleri (Fig. 3C) .
The low representation of viral sequences was not unexpected because viruses causing acute infections should be difficult to detect in healthy animals. Retroviruses integrate into the host genome as part of their replication cycle, thus transcription of viral genes can be persistent in infected animals. Overall four transcript-tags were assigned to gamma retroviruses of the family Retroviridae. Based on the transcript-tag from the MD Bonner-pool and an upstream region that is conserved in gamma retroviruses, PCR fragments were amplified and sequenced from MD 191 cDNA, which was used in the MD Bonner-pool, and from genomic DNA of MD 80228. These sequences were compared to other gamma retrovirus sequences using maximum likelihood methods (Fig. 4) . The mule deer gamma retrovirus forms a distinct clade within the gamma retroviruses, which has many well-described members of primate, murine, and feline origin. A newly described gamma retrovirus from killer whale (Orcinus orca) [38] is the closest relative of this mule deer retrovirus. The killer whale virus was described as an endogenous retrovirus based on its finding in various tissues and individuals. However, our detection of transcripts to this virus in only three of the libraries and the sequence variation in the PCR fragment between genomic (MD 80228) and transcript-derived (MD 191) mule deer samples suggest that both endogenous and exogenous gamma retroviruses might be present.
As an alternative approach to identifying bacterial microorganisms present in lymph node tissue, we utilized amplicon DNA library sequencing technology. The hypervariable region V6 of the 16S rRNA gene was used because it has been reported to differentiate between many bacterial species [39] . Amplicon libraries of V6 were generated from the 454 cDNA libraries of MD 257, MD 80228, and MD OCT-pool and subjected to multiplex pyrosequencing on a Roche GS FLX sequencer (for properties of amplicon sequencing runs, see Table S1 ). The V6 amplicon rRNA tags were evaluated using the RDP classifier tool (Table S5) .
The assigned bacterial genera cluster in the Gamma-and Betaproteobacteria, the Actinobacteria and in the order Bacilli. A comparison of the three methods used to detect bacteria in mule deer lymph node samples is shown for MD 257 in Figure 2 and for MD 80228 and MD OCT-pool in Figure S1 . Acinetobacter, Burkholderia, Corynebacterium, Escherichia, Providencia, Salmonella, Pseudomonas, Ralstonia, Staphylococcus, Streptococcus and Variovorax were identified by both amplicon and cDNA sequencing in MD 257, MD 80228, and/or MD OCT-pool (Tables S3 and S5) .
Although the overall taxonomic diversity in the V6 rRNA amplicon libraries was lower than that detected in the cDNA transcript libraries, the diversity within bacterial classes was higher. Newly identified genera comprised predominantly environmental soil, sediment and water inhabitants (e.g. Aeromicrobium and Bdellovibrio), and the potential pathogens Stenotrophomonas, Rhodococcus, Rothia, and Gardnerella [40] [41] [42] [43] . These findings indicate that the V6 rRNA amplicon sequencing technology is a valuable tool in complementing information about the bacterial community in host tissues.
Microbiome profiles of environmental samples and animals have mostly been based on the analysis of genomic DNA [8] [9] [10] 13, 44, 45] . Further, studies on the microbiomes of humans or animals have been restricted to habitats known to harbor large collections of microorganisms, in particular skin, oral cavity or gut [12] [13] [14] [15] [46] [47] [48] . In this study, we sought evidence for viable microorganisms in lymph nodes, an organ hitherto believed to be largely amicrobic in the absence of overt disease [26] [27] [28] [29] . Our data demonstrate that transcriptional activity of a variety of bacteria and a limited number of archaea and viruses, including novel organisms, can be confirmed in healthy animals using a meta-transcriptomic approach.
In our study, we faced the computational challenge of detecting a rare microbial community in a dominant pool of host genetic material. We utilized transcript-based libraries because there is an amplification of protein-coding sequences during transcription, which increased our detection ability and provided support that the identified microorganisms were viable. Further, the database for protein-coding regions is more extensive than that for noncoding regions for non-reference organisms. Thus, focusing on transcripts should facilitate classification of novel organisms and those without complete genome coverage. Indeed, our study demonstrates that at the moderate sequencing depth employed, there were more assignable sequencing tags to protein-coding regions utilizing cDNA compared to genomic DNA, which consequently increased our ability to detect microbial taxa. In addition, transcriptome sequencing yields bacterial ribosomal RNA, which is highly expressed in metabolically active microorganisms and is well documented as a taxonomic tool for bacteria. Because single protein-coding or rRNA transcript-tags from a putative microorganism were frequently encountered, our confidence in taxonomic assignment increased by employing bioinformatics methods to classify organisms based on both types of transcripts. Amplicon 16S rRNA sequencing increased the sensitivity to detect members of some bacterial classes. However, primer specific methods do not provide as comprehensive a perspective on the microbiota due to a possible amplification bias towards more abundant taxa or those exhibiting higher primer specificity. Therefore, neither our metatranscriptomic nor amplicon sequencing approaches should be considered quantitative. We note that in samples that are highly enriched for actively replicating microbial organisms, such as environmental samples or gastrointestinal tract specimens, cDNA-based approaches can yield an abundance of small RNA produced by complex microbial communities, which can facilitate studies on microbial ecology but be less useful for identification of individual microbes [49] . In addition established metagenomics or metatranscriptomic [11, 50, 51] approaches that utilize sample fractionation methods for microbial enrichment will likely provide a more comprehensive profile of the community structure. These methods were not applicable to our samples, which included phagocytized microorganisms and viable microbes that were not robustly proliferating. Nevertheless, as deeper sequencing of cDNA libraries using newer high-throughput sequencing methods becomes more accessible, it could complement the Roche-454 pyrosequencing data, potentially covering the entire viable microbial community.
Our study confirms that there are viable microorganisms in intact lymph nodes of apparently healthy mule deer. In the analyzed samples, we identified members of all bacterial phyla, as well as archaea, a DNA virus and a Retrovirus. The bacteria were representative of organisms that are commensal to mule deer and to their external environment. For example, we detected the common rumen and intestine dwellers, Ruminococcus and Abiotrophia, based on transcript-and rRNA-tags, respectively, in most libraries, indicating that commensal gut and mucosal microorganisms may routinely be sampled in secondary lymphoid tissue, presumably from transient bacteremia. Streptomyces was the most common soil dwelling bacteria identified. Of interest, Legionella, which is found near hot springs, was identified only in an individual mule deer from the Yellowstone region. The finding of a considerable number of archaeal transcripts in MD OCT-pool and MD Bonner-pool libraries implies that members of this domain of life are likely present in mule deer habitats or resident in mule deer gastrointestinal tracts, as has been recently documented in humans [52] . Correspondingly, environmental bacteria identified in healthy deer lymph nodes may reflect the animal's habitat.
Few viruses were identified with our analysis methods. This could represent the paucity of viruses in healthy animals. However, viral detection may be more difficult than bacterial identification using this technology in part due to extensive sequence diversity among viruses in the same family. For example, we were only able to detect the gamma retrovirus because a transcript was present which was homologous to a conserved region of the viral env gene, and the cervid poxvirus was detected because sequence data for this virus was present in the database. Other persistent viruses, such as herpes viruses (for which we detected a single transcript), would be expected to be present in some animals. However, detection of latent herpes virus infection may be difficult because protein-coding transcript levels are low and latent viruses express non-coding RNA [53] . In addition, viral detection can be compromised if viral sequence tags were misassigned to the host organism because of homology of viral and host genes. Thus, many virus tags might be found among the host transcripts or in the not-assigned or no-hits groups of the MEGAN analysis, which together comprise nearly half of the total sequenced transcript-tags of our data.
In addition to our finding of a novel gamma retrovirus, we also identified new species of Helicobacter and Acinetobacter. Phylogenetic evaluation of Helicobacter transcripts and 16S rRNA from the MD 257 cDNA library placed this new organism in the Helicobacter pylori/Helicobacter acinonychis/Helicobacter cetorum complex. All members of this complex have been associated with gastritis and peptic ulcer disease in humans and animals [36, [54] [55] [56] . Our detection of this bacterium in only one animal suggests that this Helicobacter is not a mule deer commensal. Of interest in this respect is the high incidence of H. pylori infections and gastric ulcers in American Indian populations from the same geographical area in central Montana [57] . Acinetobacter and Pseudomonas were identified in MD 80228 libraries based on all detection methods used (cDNA transcripts for protein-coding and rRNA, and amplicon rRNA). Phylogenetic evaluation of Acinetobacter transcripts and 16S rRNA from the MD 80228 cDNA library placed the respective reads in close relationship to Acinetobacter schindleri. Acinetobacter species are important environmental organisms, however they also are notable pathogens. In particular, Acinetobacter schindleri infections appear to be increasing in prevalence in hospitalized patients [37, 58] . Therefore, both of the newly identified bacteria are potential mule deer pathogens.
In conclusion, our study demonstrates that endemic microbiota can be detected in lymph nodes of healthy animals using metatranscriptomic approaches. These results suggest that metatranscriptomic analyses of secondary lymphoid organs could be valuable in monitoring endemic infections in wildlife or livestock as well as in detecting novel infectious organisms with the potential for causing emerging zoonotic or epizootic infectious diseases. Further, these studies have the potential to cast new light on the diversity of life within and among individuals.
Retropharyngeal lymph nodes were obtained from a total of seventeen individual Montana mule deer that were presented by hunters to check stations approximately 5 hr (range 2-11 hr) of being shot. Because our samples were obtained from legally killed animals, the study is exempt from Montana State University guidelines governing animal experimentation. Lymph nodes were dissected from animals with sterile scalpel and forceps, and rinsed in 70% ethanol. After dissection from the animal, the lymph nodes were either frozen directly or stored in RNAlater (Applied Biosystems, Ambion, CA) until further processing.
Lymph node tissue was taken from mule deer in several geographical regions. The Bonner pool consisted of tissue from four mule deer (167, 191, 196, 200) Preparation of genomic DNA, total RNA, poly(A) + RNA and cDNA Lymph node tissue cores were dissected into small pieces and further disrupted, lysed and homogenized using a TissueLyser with steel beads (Qiagen, Germany). Genomic DNA was isolated from lymph nodes of four individual Mule deer (MD 72360, MD 80228, MD 84709, and MD 84730) using either the Genomic DNA Buffer Set with 20/G Genomic-tips (Qiagen, Germany) or the AllPrep DNA/RNA Mini Kit (Qiagen, Germany). Total RNA was isolated using the RNAqueous-Midi Kit (Applied Biosystems, Ambion, CA). For the Bonner-and OCT-pools, equal quantities of total RNA from lymph nodes of four or eight individual Mule Deer, respectively, were combined. Poly(A) + RNA was enriched from total RNA using the MicroPoly(A) Purist Kit (Applied Biosystems, Ambion, CA). Poly(A) + RNA (0.9-5.0 mg each) was used for cDNA synthesis (Just cDNA Double-Stranded cDNA Synthesis Kit, Stratagene, CA) after elimination of residual contaminating genomic DNA using the Turbo DNA-free Kit (Applied Biosystems, Ambion, CA). In one case we explored an alternative empirical approach to enrich for rare microbial transcripts, using total RNA of the MD OCT-pool. Reverse transcription and amplification of cDNA was done as described by Cheung and coworkers [59] and included a normalization step, which effectively decreased overexpressed reads. The data resulting from this approach are included in the MD OCT-pool data.
Up to 5.0 mg of cDNA or genomic DNA was subjected directly to preparation of 454-DNA libraries and subsequently to pyrosequencing without any prior PCR or cloning steps. Library preparation and pyrosequencing were performed as described previously [60] on a Roche GS20 sequencer FLX (Roche Applied Sciences/454 Life Sciences, Branford, CT), producing sets of RNA-tags or DNA-tags, respectively. The runs were performed on either quarter or half plates, resulting in read numbers between 10,673 and 176,878 and base numbers in the range of 1,411,420 to 41,066,808. The MD OCT-pool cDNA library was run twice due to low read and base numbers of the first run, and the transcript-tags of these two runs and of the run following the normalization approach (see above) were combined for all subsequent data analysis. Sequences are deposited to the Sequence Read Archive (in progress).
The data of individual 454 runs (and the compilation of normal and normalized MD OCT-pool data) was compared against the NCBI non-redundant protein database (BLASTX-nr) with an evalue of 1e -4 to identify transcript RNA-derived tags. To filter repetitive elements, RepeatMasker (http://www.repeatmasker. org) was used to scan the mule deer sequences, with the latest version of Repbase 13.04 [61] . The output files were analyzed with the program MEGAN [32] version 3.7.2.
The 16S ribosomal RNA content of the cDNA pyrosequencing reads was analyzed by comparison to the ribosomal database of the Ribosomal Database Project (RDP) version 10 (http://rdp.cme. msu.edu/) [34] . The selected output reads were classified by the RDP Classifier tool (Naïve Bayesian rRNA Classifier Version 2.0) using the Taxonomic Outline of the Bacteria and Archaea, release 7.8, for the setup of the taxonomical hierarchy [35] . The output files were analyzed with MEGAN version 3.7.2 [32] . For the MD OCTpool, the combined data of three individual 454 runs was used.
The cDNA from MD 191, which was used in the MD Bonner pool, and genomic DNA from MD 80228 were subjected to PCR using forward primer 5-ATGTGGGGGAGTTGATTCTTTT-TA and reverse primer 5-CTGCGCCTGAGTGGTCTACATA. PCR conditions were 40 cycles of 95uC for 30 sec, 56uC for 30 sec and 72uC for 90 sec. Fragments were gel isolated, cloned using the Stratagene PCR cloning kit (Stratagene, La Jolla, CA) and Sanger sequenced.
Partial nucleotide sequences of 16S rRNA and rpo-b for Helicobacter and Acinetobacter and of flgK, GDP-D-mannose dehydratase and UDP-3-O- [3-hydroxymyristoyl] glucosamine Nacyltransferase for Helicobacter from cDNA sequencing, and of env gene for the retrovirus from a PCR product were aligned with the respective homologous sequences available in GenBank using the MEGA version 4 [56] software. The appropriate nucleotide substitution model for each data set was selected by the Akaike information criterion implemented in the Modeltest version 3.7 [62] , and maximum likelihood (ML) trees were reconstructed using PhyML version 2.4.4 [63] . Using the same program (PhyML) nodal supports were estimated with 100 bootstrap replicates. The trees were visualized in FigTree version 1.2.2 (http://tree.bio.ed.ac.uk/software/figtree/).
Fusion-primers were designed including the sequences of the 454-Amplicon DNA library specific primers A and B, respectively, (GS FLX Amplicon DNA Library Preparation Method Manual, www.roche-applied-science.com), 4-base barcode sequences for identifying amplicon products derived from mule deer specimen MD 257, MD OCT-pool, and MD 80228 (TGCA, ACGT, and CGAT, respectively), and the ''universal'' V6-specific PCR primer sequences V6F: 59 TCGATGCAACGCGAAGAA 39 and V6R: 59 ACATTTCACAACACGAGCTGACGA 39 (designed to conserved regions flanking V6 based on comparison of 110 bacterial DNA sequences [39] ).
The MD 257 template for amplicon generation was based on the total RNA fraction depleted of poly(A) + RNA (see ''Preparation of genomic DNA, total RNA, poly(A)+RNA and cDNA''). The supernatant was cleared of small RNA molecules using the MEGAclear Kit (Applied Biosystems, Ambion, CA) and depleted of host ribosomal RNA performing two cycles of the MICROBEnrich (Applied Biosystems, Ambion, CA) protocol. Subsequent depletion of bacterial ribosomal RNA yielded an RNA sample enriched for bacterial transcripts (MICROBExpress, Applied Biosystems, Ambion, CA), which was subjected to cDNA synthesis (Just cDNA Double-Stranded cDNA Synthesis Kit, Stratagene, CA) after elimination of residual contaminating genomic DNA using the Turbo DNA-free Kit (Applied Biosystems, Ambion, CA).
Either cDNA derived from RNA enriched for non-polyadenylated bacterial mRNA (MD 257) or cDNA sequencing library samples derived from reverse transcribed poly(A) + RNA (for MD OCT-pool and MD 80228) were used as templates for the generation of 16S rRNA V6 hypervariable region-specific amplicons using the FastStart High Fidelity PCR System (Roche, Switzerland). PCR conditions were 50 cycles of 94uC for 30 sec, 55uC for 45 sec and 72uC for 45 sec. The yielded amplicon products were purified using AMPure, and the resulting individual amplicon DNA libraries were clonally amplified by multiplex emulsion PCR followed by sequencing using the GS FLX pyrosequencing platform. The sequencing output data were computationally divided into subsets according to the barcodes (and the corresponding mule deer sample) and the primers A or B. Figure S1 Comparative MEGAN analysis of (A) MD 80228 and (B) MD OCT-pool transcript-tags analyzed by comparison to the protein database (red) and the ribosomal database (blue), and of amplicon 16S rRNA-tags compared to the ribosomal database (green). Bit score cutoff for the protein database comparison was set at 50, and confidence cutoffs for the ribosomal database comparisons were set at 80% and 80%, respectively.  In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors. The emergence and resurgence of human viral pathogens can be traced to a complex variety of causes including increased urbanization, human contact with animal reservoirs, a decrease in effective public health systems, and the spread of insect vectors that disseminate some viral infections [1, 2, 3] . Flaviviruses are a genus in the Flaviviridae family and include important emerging and resurgent human pathogens such as dengue virus (DENV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and yellow fever virus [2, 4] . Flaviviruses are transmitted by insects such as mosquitoes and ticks, and can cause severe human diseases characterized by encephalitis, meningitis, and hemorrhages [2, 3] . More than one third of the world's population lives in dengue fever endemic areas, and there are an estimated 50-100 million cases of dengue infection and 500,000 cases of the more lethal complication, dengue hemorrhagic fever, per year [5, 6, 7, 8] . There are currently no antiviral therapies for flaviviruses. DENV vaccine development is underway but is problematic due to the presence of four DENV serotypes and the potential for antibody-dependent enhancement of infection [2, 6, 9, 10] . Antiviral therapies could thus be an important alternative for DENV and for viruses such as WNV in which the cost and potential side effects of vaccination must be weighed against the relatively low number of human cases [2] .
Flaviviruses are small, highly organized enveloped viruses with a spherical shape [4, 11] . They contain a positive-sense RNA genome packaged by the viral capsid protein. The nucleocapsid is surrounded by a lipid bilayer containing the viral membrane protein E. Flaviviruses infect cells by receptor engagement at the plasma membrane, endocytic uptake, and a membrane fusion reaction triggered by the low pH of the endosome compartment [12, 13] . The viral E protein binds the receptor and drives the fusion of the viral and endosome membranes to initiate virus infection. The pre-fusion structure of the E protein ectodomain (here referred to as E9) shows that E contains three domains composed primarily of b-sheets: a central domain I (DI) connecting on one side to the elongated domain II (DII) with the hydrophobic fusion loop at its tip, and connecting via a flexible linker on the other side to the immunoglobulin-like domain III (DIII) [14, 15, 16, 17, 18, 19] (Figs. 1A, S1). Although these regions are not present in the truncated E9 ectodomain, DIII connects to a stem domain and C-terminal membrane anchor (TM). The E protein in mature infectious flavivirus is organized in homodimers that lie tangential to the virus membrane [20] . Within each dimer the E proteins interact in a head to tail fashion, with the fusion loop of each E protein hidden in a hydrophobic pocket formed by DI and DIII of the dimeric E partner.
The E protein mediates virus-membrane fusion by refolding to a hairpin-like E homotrimer with the fusion loops and TM domains at the same end [21, 22] . This reaction involves low pH-triggered dissociation of the homodimer, fusion loop insertion into the endosome membrane, formation of a core trimer composed of DI and DII, and the foldback of the DIII and stem regions towards the target membrane and their packing against the core trimer. The prefusion and postfusion conformations of the flavivirus E fusion protein are structurally and functionally similar to those of the E1 fusion protein from the alphavirus Semliki Forest virus (SFV) [23, 24, 25] , and these fusion proteins are often referred to as ''class II'' [26, 27, 28] . In addition to the ectodomains whose trimer structures are described above, truncated fusion proteins composed of domains I and II (DI/II) can reconstitute SFV and DENV core trimer formation on target membranes [29, 30] . Such core trimers act as specific targets for DIII binding, thus recapitulating the protein-protein interactions during class II trimerization and hairpin formation.
Flaviviruses bud into the endoplasmic reticulum (ER) and are transported as virus particles through the secretory pathway and released by exocytosis [4] . Given the low pH that is present in the Golgi complex and trans-Golgi network (TGN) [31] , how do flaviviruses avoid inactivation during their transport? The particles are assembled in the ER as immature non-infectious viruses containing heterodimers of the precursor membrane protein (prM) and E protein [4, 26, 32] . Subsequent exposure to low pH in the secretory pathway triggers a dramatic rearrangement to E homodimers and makes the prM protein accessible to furin cleavage [33, 34] . Processing of prM by cellular furin results in mature infectious virus in which E homodimers are poised to mediate fusion [33] . Important recent studies describe the structure of pr peptide in complex with E, and indicate that processed pr remains associated with the virus at low pH and can inhibit virus-membrane interaction [34, 35, 36] . Thus, pr on the virus could protect E protein from low pH in the secretory pathway.
The flavivirus prM/pr protein plays multiple roles in the virus life cycle (reviewed in [26] ). prM acts as a chaperone for E protein folding [37] and associates with the tip of E [34] . prM also appears to respond to low pH to permit E rearrangement on the virus surface and allow furin access for prM processing [34, 38] . Following cleavage, the pr peptide may prevent premature virus fusion through bridging interactions that stabilize the E homodimer and thereby prevent dissociation to E monomers, a key fusion intermediate [35, 36] . To better understand these multiple roles of prM/pr, separation of its chaperone and pH-protection functions and characterization of the pr-E interaction are needed.
Here we developed a system to produce DENV pr peptide and reconstitute the pr-E interaction in vitro. At low pH pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion and trimerization. Addition of exogenous pr to mature DENV particles inhibited virus fusion and infection. Mutation of a key histidine residue in the pr-E interface, E H244, reduced pr's binding and inhibitory activity, and reduced DENV secondary infection and particle production. The defect in particle Figure 1 . Expression and purification of DENV2 pr and truncated E proteins. A) Linear diagrams of the DENV2 prM-E proteins and the truncated DENV2 pr and E proteins used in this work (not to scale). Domain and construct boundaries are marked, with numbering based on the individual proteins in the DENV2 New Guinea C (NGC) strain. The sequences appended to the diagrams contain the Strep (ST) affinity tag(s) used for protein purification (underlined), joined in the case of two Strep tags by a flexible linker region (STST). Pr was expressed in 293T cells and contains prM residues 1-86 plus N-terminal GS residues from the vector and the STST tag. The DI/II and E9 proteins were expressed in S2 cells and contain E residues 1-291 and 1-395, respectively, plus ST or STST tags. DIII was expressed in E. coli and contains E residues 289-430, comprising the linker, DIII, helix 1 and conserved sequence (LDIIIH1CS). The names in parentheses are the detailed nomenclature from [30] . B) 4 mg samples of purified pr peptide were incubated with DTT, Endo H, or PNGase F as indicated, analyzed by SDS-PAGE and stained with Coomassie blue. The positions of marker proteins are shown on the right with their molecular masses listed in kilodaltons. Asterisks indicate the positions of the added glycosidases. C) 4 mg samples of purified truncated E proteins were reduced with DTT as indicated, analyzed by SDS-PAGE and stained with Coomassie blue. Marker proteins are shown on the right with their molecular masses listed in kilodaltons. doi:10.1371/journal.ppat.1001157.g001
Enveloped viruses infect cells by fusing their membrane with that of the host cell. Dengue virus (DENV) is an important human pathogen whose membrane fusion is triggered by low pH during virus entry into the cell. However, newly synthesized DENV must also transit through a low pH environment during virus exit. DENV is believed to escape premature fusion in the exit pathway via the small viral protein pr, which is processed and associates with virus after biosynthesis, and is released from the virus particle in the neutral pH extracellular environment. Here we have reconstituted the interaction of pr with the DENV fusion protein E using soluble protein components. The interaction has a low pH optimum and inhibits membrane insertion of the fusion protein. The recombinant pr peptide can ''add back'' to fully infectious mature DENV and block virus fusion and infection. We found that mutation of a critical conserved histidine on the fusion protein inhibits the interaction of E and pr, and makes the virus susceptible to low pH-induced inactivation during exit. This work characterizes the mechanism of pr protection, and suggests that the conserved multifunctional pr-E interaction may be an important target for antiviral strategies.
production could be partially rescued by neutralization of exocytic low pH, indicating the important role of pr in protecting DENV from premature fusion during transport to the plasma membrane.
A number of truncated E proteins have been successfully produced by co-expression with prM (e.g., references [30, 39] ), while the pr-E structural studies were based on a secreted hybrid protein containing truncated prM linked to truncated E [34] . Previous studies indicated that full-length TBEV prM could fold correctly when expressed in the absence of E protein [37] , suggesting that production of pr peptide alone might be possible. We generated a construct based on residues 1-86 of DENV2 prM, truncating pr just before the start of the furin cleavage recognition site at residue 87 (Fig. 1A) . This sequence was linked to a mammalian signal peptide at the N-terminus and to an affinity tag at the C-terminus, and expressed in 293T cells. The protein was isolated in a highly purified form by affinity chromatography and gel filtration (Fig. 1B) , and was recognized by mAb prM-6.1 against prM [40] (data not shown). The pr peptide migrated at a position of ,17 kDa in reducing SDS-PAGE, in keeping with its predicted size of 13 kDa plus the presence of carbohydrate due to the glycosylation site at position 69. This carbohydrate was removed by Peptide N-glycosidase F (PNGase F) to give a peptide of the predicted size. The protein was largely resistant to Endoglyosidase H (Endo H) digestion, indicating maturation of the carbohydrate chain as the protein transited through the Golgi complex. A mobility shift was observed upon reduction of pr, in keeping with the presence of 3 disulfide bonds in the structure of pr [34] .
We also produced and purified a dimeric ectodomain form of DENV2 E protein containing all three domains (E9), a monomeric form containing E domains I and II (DI/II), and E domain III (DIII) (Fig. 1A and 1C ), all as previously described in detail [30, 41] .
As a first test of in vitro pr-E binding, we coupled pr to sepharose beads and tested its ability to pull-down truncated E protein containing only domains I and II. This form of E protein is monomeric and the tip of DII is thus accessible even at neutral pH. Previous studies showed that this and other DENV DI/II proteins are active in membrane insertion and trimerization at both neutral and low pH [30] . We observed efficient pull-down of DI/II protein by pr-sepharose ( Fig. 2A) , but in spite of the accessibility of the pr binding site on DI/II at neutral pH, pulldown was low pH-dependent. The pull-down of DI/II protein by pr was specific, as it was blocked by inclusion of mAb 4G2 against the E fusion loop at the DII tip, and did not occur with BSAsepharose beads. These data suggested that the recombinant pr peptide could bind to the tip of DI/II in a low pH-dependent reaction.
For more detailed studies of pr-E binding, we performed surface plasmon resonance (SPR) assays using our various forms of recombinant E protein with immobilized pr peptide. Compared to the pull-down assay, SPR can detect low levels of protein-protein interactions as binding is detected in real time and does not require removal of unbound E. The E9 protein is a dimer at neutral pH and dissociates to monomers at low pH [30] . When SPR was performed with E9 protein buffered at pH 8.0 there was very low binding (low signal response) (Fig. 2B ). As the buffer pH was Figure 2 . Pr peptide binds DENV E proteins in a pH-dependent manner. A) Pull-down of DI/II protein by pr. DI/II was incubated with sepharose beads conjugated with pr peptide or BSA at the indicated pH for 1 h at room temperature. As indicated, reactions contained a 2:1 molar excess of mAb 4G2 to the E fusion loop or mAb to the ST tag (con.). Input lanes show an aliquot representing 20% of the reaction prior to pull-down. (Panels B-D) SPR analysis of pr-E binding. Pr peptide was immobilized on a CM5 sensor chip, and DENV2 E9 (B), DI/II (C) or SFV DI/II proteins (D) were flowed over the chip at concentrations of 1.2 mM in buffers of the indicated pH for 300 s, followed by injection of protein-free buffer at the same pH. Data are a representative example of two independent experiments. doi:10.1371/journal.ppat.1001157.g002 decreased, the signal gradually increased, with maximal response observed at ,pH 6.25 and no further increase at pH 6.0. A rapid decrease in signal was observed when the samples were shifted to protein-free buffer, indicating rapid dissociation of the pr-E interaction. Similar results were obtained using monomeric DI/ II, with the lowest binding at pH 8.0, highest binding at pH 6.25, and a slight decrease at pH 6.0 (Fig. 2C) . Thus, the dimeric E9 and monomeric E DI/II proteins bound pr peptide with similar pHdependence. Binding to pr was specific, as little interaction was observed using the structurally similar E1 DI/II protein of SFV (Fig. 2D ). In addition, binding of DENV E DI/II protein to pr was inhibited by preincubation with mAb 4G2 against the fusion loop (molar ratio 1:1) (data not shown). Determination of the affinity of pr-E binding was not performed as the data did not fit to a simple Langmuir model of 1:1 binding, presumably because of E protein aggregation at low pH.
Previous studies showed that retention of endogenous pr peptide on the furin-processed DENV particle inhibits virus interaction with liposomes at low pH [35] . Structural considerations suggested that this inhibition occurs primarily by blocking low pH-triggered dissociation of the E dimer, a required first step in the fusion reaction. To test this mechanism, we evaluated the effect of pr on the membrane interactions of dimeric and monomeric forms of E protein. The E9 dimer was preincubated with pr peptide or an unrelated protein with the same affinity tag for 5 min at pH 8.0, and then treated at pH 5.75 in the presence of target liposomes. Membrane-associated proteins were separated by liposome floatation on sucrose gradients. There was no liposome cofloatation when E9 protein was incubated with liposomes at neutral pH (Fig. 3A) . About 70% of the total E9 floated with liposomes in the top part of the sucrose gradient after treatment at pH 5.75 in the presence (Fig. 3A , top panel) or absence (data not shown) of a control protein. In contrast, when E9 was preincubated with pr peptide (pr:E9 molar ratio 12:1) and treated with low pH, only ,2% of E9-ST floated with the liposomes (Fig. 3A , middle panel). Inhibition by pr was not observed when it was added after E9 was treated at low pH in the presence of liposomes for 30 min (Fig. 3A , bottom panel), and thus pr needed to be present during the membrane insertion step. Inhibition was concentrationdependent, with 22% E9 co-floatation at a pr:E9 molar ratio of 3:1, 8% at 6:1, and 0.4% for 24:1 (data not shown; see also We then tested the effect of pr on the DENV E DI/II protein. This protein is monomeric and its stable membrane interaction requires DIII to ''clamp'' the core trimer [30] . As shown in Fig. 3B , ,25% of DI/II co-floated with liposomes at low pH in the present of DIII, while no co-floatation was detected when BSA was Figure 3 . Pr peptide inhibits E protein-membrane interaction. A) E9-liposome co-floatation assay. E9 protein was mixed with pr peptide or an ST-tagged control protein (Seap) at a final concentration of 50 mg E9 protein and 200 mg pr/Seap protein/ml (molar ratio 12 pr/1E). Liposomes were added at a final concentration of 1 mM, and the samples were incubated at the indicated pH for a total of 60 min at 28uC. Where indicated, E9 protein plus liposomes were incubated for 30 min, pr peptide added to a final concentration of 200 mg/ml, and the incubation continued for an additional 30 min. The liposome-bound proteins were then separated by floatation on sucrose gradients at the indicated pH. Aliquots of the top, middle and bottom of the gradients were analyzed by SDS-PAGE and western blotting for E protein. B) DI/II-liposome co-floatation assay. 40 mg/ml DI/II plus DIII or BSA (200 mg protein/ ml) were incubated with liposomes plus 200 mg pr peptide/ml as indicated and assayed for liposome co-floatation as in panel 3A. C) SFV DI/II-liposome co-floatation assay. SFV DI/II protein (40 mg/ml) was mixed with BSA or pr peptide (160 mg/ml). Liposomes were added at a final concentration of 1 mM, and the samples were incubated at the indicated pH for a total of 30 min at 28uC. Liposome co-flotation was assayed as in panel 3A. D-E) Loss of pr inhibition of E protein in the pH range of the late endocytic pathway. D) pH dependence of pr inhibition. E9 protein was mixed with liposomes in the presence or absence of pr peptide (molar ratio ,12 pr/1E), treated at the indicated pH as in The structurally related alphavirus protein SFV E1 DI/II is monomeric and efficiently interacts with membranes at low pH (80% cofloatation, Fig. 3C , middle panel). No inhibition occurred when pr peptide was added prior to liposome addition (Fig. 3C , bottom panel), in keeping with the lack of pr-SFV DI/II binding in the SPR experiments discussed above. Thus, pr peptide specifically inhibits target membrane interaction of both monomeric and dimeric forms of the DENV E protein.
E9 protein efficiently inserted into membranes over a wide range of pH values from 6.25-4.5 ( Fig. 3D-E) . However, pr's inhibition of E membrane insertion was less efficient in the pH range (pH 5.0) present in the late endocytic pathway (Fig. 3D-E) . This loss of pr inhibition at more acidic pH may be relevant to recent studies of infection by immature DENV [42] , as mentioned in the discussion section below.
All of the results above were obtained with soluble forms of the E protein. In order to test the ability of exogenous pr peptide to interact with and inhibit intact DENV, we took advantage of a previously described assay that monitors low pH-triggered fusion of DENV with cells [41] . In this fusion-infection assay, virus is prebound to target cells on ice, and then treated at 37uC for 1 min at low pH to trigger virus fusion with the plasma membrane. This fusion reaction is then quantitated by detecting the infected cells by immunofluorescence. We tested the effect of pr peptide during this 1 min low pH treatment using DENV1 WP and DENV2 NGC. The sequence of E DI/II is 68% identical between these two serotypes. Both serotypes showed efficient fusion and infection after treatment at pH 6.0, with about a 10-fold increase compared to samples treated at pH 7.9 (Fig. 4 ). The addition of pr peptide during the 1 min low pH treatment strongly inhibited DENV fusion and infection. Inhibition was dose-dependent, with 45-49% inhibition at 6 mM pr and 81-85-% inhibition at 30 mM pr. In contrast, pr did not inhibit low pH-triggered fusion by the alphavirus SIN (Fig. 4) . Thus, exogenous DENV2 pr peptide can specifically interact with mature DENV1 and DENV2 to block virus fusion and infection. We did not observe inhibition when DENV was preincubated with 30 mM pr at pH 7.0 and then added to target cells in a standard infection assay, suggesting that under these conditions an inhibitory concentration of pr was not present during low pH-triggered fusion reaction in the endosome. This result also indicates that the presence of pr did not affect virus-cell binding.
Although the interaction of pr with DENV can clearly prevent virus-membrane interaction and fusion (this study and [35] ), the importance of pr in protecting DENV during exocytic transport has not been defined. The binding interface between prM and E contains three complementary electrostatic patches containing 11 residues [34] (see also Fig. S1 ). Sequence analysis shows that these 11 residues (Fig. 5A , numbered residues) are highly conserved among the 4 DENV serotypes, and that D63 and D65 of pr, and the complementary H244 on E protein are conserved among all reported flavivirus sequences [34] . Optimal pr-E binding in vitro occurred at ,pH 6.25 (Fig. 2) , suggesting that protonation of H244 could be involved in this pH-dependence. To test this we substituted alanine for H244 in the DI/II protein. DI/II H244A was produced in highly purified form with electrophoretic mobility similar to that of the wild type (WT) protein in reducing and nonreducing SDS-PAGE (Fig. 1C) .
We first tested the effect of the H244A mutation on pr-E binding. In agreement with our earlier results, WT DI/II protein was efficiently pulled-down by pr-sepharose (Fig. 5B ). Pull-down was low pH-dependent and blocked by mAb 4G2 against the E fusion loop at the DII tip. In contrast, almost no H244A DI/II protein was pulled-down by pr-sepharose at either low pH or neural pH (Fig. 5B ). SPR analysis of WT DI/II protein showed most efficient binding at pH 6.0, and binding was blocked by preincubating the DI/II protein with mAb 4G2 (molar ratio 1:1) before dilution into SPR buffer (Fig. 5C, upper panel) . Equivalent concentrations of H244A DI/II protein showed greatly reduced binding to pr compared to that of WT protein (Fig. 5C , lower panel). Although H244A binding was decreased, the residual binding was still blocked by mAb 4G2 and had an acidic pH optimum. This suggests that binding also involves other residues in the pr-E interface, such as the complementary residues identified in the structural studies and shown in Fig. 5A .
We then asked if the H244A DI/II protein was still active in binding to target liposomes. WT or mutant DI/II proteins were mixed with liposomes at low pH in the presence of DIII protein to stabilize the core trimer. Both proteins efficiently bound liposomes in a DIII-dependent reaction (Fig. 6) , indicating that the mutant protein retains its ability to insert into target membranes and form a core trimer. In agreement with the results in Fig. 3C , floatation of the WT protein was blocked by inclusion of pr during the membrane insertion step (Fig. 6) . In contrast, the efficiency of floatation of the H244A mutant protein was 43% in the absence of pr and 47% in the presence of pr. Thus, the H244A mutation did not inhibit E-membrane interaction but made that interaction insensitive to the presence of pr.
Since the E H244A mutation disrupts E protein's interaction with pr, we used this mutation to address the importance of pr in protecting DENV during transport through the exocytic pathway. We introduced the E H244A mutation into the infectious clone of DENV1 WP. WT and mutant viral RNAs were prepared by in vitro transcription and were electroporated into BHK cells. After culture for 3 d at 37uC, both WT and mutant RNA-electroporated cells expressed abundant E protein as detected by immunofluorescence microscopy (Fig. 7) . Parallel cultures were incubated for 6 d and progeny virus in the culture media was detected by infectious center assays on indicator BHK cells. WTinfected cells produced infectious progeny virus with a titer of ,1.5610 5 IC/ml. However, two independent infectious clones of the H244A mutant produced no detectable progeny virus, even though the viral RNAs mediated efficient primary infection as shown in Fig. 7 . This agrees with previous studies indicating lethal effects of an H244A mutation on DENV2 [43] .
The absence of secondary infection by the H244A DENV1 mutant could be due to decreased virus particle production and/or production of particles that are non-infectious. Efficient DENV particle production is dependent on E protein folding, particle budding into the ER, and subsequent particle egress through the secretory pathway. To investigate these issues, we took advantage of the ability of the flavivirus prM and E proteins to assemble into virus-like particles (VLP) in the absence of other viral components or virus infection [44, 45, 46 ]. The VLP system avoids complications arising from selection of revertants of deleterious virus mutations such as H244A. Flavivirus VLP bud into the ER in the immature prM form, undergo furin maturation during transport through the secretory pathway, and display similar low pH- [34] , potential key residues in pr-E interaction are indicated by their numbers in the DENV2 NGC proteins. B) H244A mutation inhibits pr-E binding in pull-down assay. WT or H244A mutant forms of DI/II were assayed for binding to pr-sepharose beads as in Fig. 2A . C) H244A mutation inhibits pr-E binding in SPR assay. WT or H244A mutant forms of DI/II were assayed for binding to pr at various pH values using SPR as in Fig. 2C , shifting to buffer alone at 300 s. Where indicated, mAb 4G2 (molar ratio 1:1) was pre-incubated 15 min at room temperature with DI/II proteins at pH 6.0 prior to assay. Data are a representative example of two independent experiments. doi:10.1371/journal.ppat.1001157.g005 dependent fusion activity as infectious virions [44, 47] . The VLP system has been used extensively to follow the process of flavivirus particle production and the role of prM in this process [37, 44, 45, 48] .
We established stable HEK 293 cells that inducibly express the DENV1 WT or H244A prM-E proteins. After 36 h induction with tetracycline, both WT and H244A cells show abundant intracellular expression of the DENV1 E protein as detected by immunofluorescence, while the parent cell line is negative for E expression (Fig. 8A) . To evaluate whether WT and H244A E proteins were correctly folded, cells were induced for 36 h, lysed, and immunoprecipitated with a rabbit polyclonal antibody to E DIII, and with two conformation-specific mAbs. mAb 4E11 recognizes a discontinuous epitope on DENV E DIII and requires proper DIII disulfide bond formation for recognition [49, 50] . mAb 4G2 recognizes the fusion loop at the tip of flavivirus E DII and its epitope is sensitive to reduction [51] . Expression studies have shown that the 4G2 epitope is not formed if the E protein is expressed in the absence of prM [52] , indicating that this epitope is particularly useful for diagnostic tests of prM's chaperone interaction with E (see also reference [37] ). As shown in Fig. 8B , lysates from cells induced to express prM plus WT or H244A E proteins showed strong reactivity with all three antibodies. Quantitation of multiple experiments confirmed that WT and H244A E proteins were comparably recognized by the 4E11 and 4G2 mAbs. Thus, by these criteria H244A E protein interacts with prM protein and is correctly folded. This result also agrees with our finding that truncated H244A E protein expressed with prM in the S2 cell system was fully active in low pH-dependent membrane binding and trimerization, suggesting correct folding (Fig. 6) . . DENV E H244A mutation inhibits release of virus-like particles via a low pH-dependent mechanism. A) WT and H244A mutant E proteins are comparably expressed. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37uC. E protein expression was detected by immunofluorescence and the nuclei were stained with DAPI. Fluorescence images are shown at the same magnification and exposure time. Bar represents 30 mm. B) WT and H244A mutant E proteins are comparably immunoprecipitated by conformation-specific mAbs. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37uC. E proteins in the cell lysates were immunoprecipitated by Sango, a rabbit polyclonal antibody to DIII, and by the mouse mAbs 4G2 and 4E11, as indicated at the top of the panel. Samples were then analyzed by SDS-PAGE and western blot using mouse anti-DENV2 Ab for the Sango samples and Sango for the mAbs samples. Asterisks indicate the positions of the IgG and IgG heavy chain, which cross-react in the western blot. Equivalent sample input was evaluated by western blot for b-actin (lower panel). C) Effect of low pH on WT and H244A VLP production. WT and H244A mutant cells were incubated with tetracycline for 2 h and then in this medium plus 20mM NH 4 Cl where indicated for a total of 36h. VLP released in the culture media were pelleted by ultracentrifugation, and E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were analyzed by SDS-PAGE and western blot using Sango. 5-fold more culture media from the H244A cells than the WT cells were loaded. Data are representative examples of two or more independent experiments. doi:10.1371/journal.ppat.1001157.g008
We then used the inducible cells to examine VLP production. Expression was induced for 36 h. The cells were then lysed and the E proteins immunoprecipitated, and the VLP in the culture media were pelleted by ultracentrifugation. Analysis by western blotting showed strong E protein expression in both WT and H244A cells, and no expression in the parent cells (Fig. 8C) . The WT cells released E protein in VLP, but VLP release from cells expressing the H244A mutant E protein was greatly reduced (Fig. 8C, -media samples) . This result is in keeping with the hypothesis that the H244A cells assemble VLP in the neutral pH environment of the ER but that VLP release is inhibited by the lack of pr protection from the low pH of the secretory pathway. To test this idea, we induced WT and H244A prM-E expression and cultured the cells in the presence of 20 mM NH 4 Cl to neutralize the acidic pH in the Golgi and TGN compartments (Fig. 8C , +NH 4 Cl lanes). The cellular expression level of either E protein was not significantly affected by NH 4 Cl treatment, and WT VLP production was similar in NH 4 Cl-treated cells and untreated cells. However, production of VLP containing the H244A mutant E protein was increased 4-7 fold in NH 4 Cl-treated cells. While H244A VLP production was still significantly decreased compared to that of WT, it was selectively rescued by NH 4 Cl treatment.
During translation of the flavivirus polyprotein, prM is the first protein translocated into the ER lumen, where it acts as a chaperone during the folding of the subsequently translocated E protein [4, 37, 44] . In addition to this important role of prM during E protein synthesis, a variety of data suggest that the interaction of pr peptide with the viral E protein protects flaviviruses from low pH during their transport through the exocytic pathway [34, 35, 36] . Here we showed that a recombinant pr peptide was efficiently folded, glycosylated, and secreted from 293T cells in the absence of its normal prM context and furin processing. Recombinant pr bound to soluble E proteins at low pH, inhibited E-membrane insertion, and interacted with mature dengue virus to block fusion and infection. Alanine substitution of the conserved E H244 within the pr-E interface disrupted pr-E binding in vitro and blocked secondary virus infection. VLP production was inhibited by the H244A mutation and partially rescued by pH neutralization with NH 4 Cl. Together our data demonstrate the critical role of pr in protecting DENV from exocytic low pH.
The in vitro interaction of pr with various truncated forms of E protein was strongly pH-dependent, with a pH optimum of ,6.25. In situ measurements indicate that the pH of the TGN is ,6 [53] , while the pH optimum of DENV2 NGC fusion is ,6.2 [41] . The low pH of the TGN is critical for the rearrangement of immature DENV to allow furin cleavage, but once the virus is processed it becomes fusion-active in this same pH range. Thus the pH dependence of the pr-E interaction appears optimized to protect DENV during its continued transit through the secretory pathway. Pr's inhibition of E membrane insertion was less efficient at a pH value (pH 5.0) similar to that in the late endocytic pathway (Fig. 3D-E) . This loss of pr inhibition at more acidic pH could help to explain the recent finding that infection by immature DENV is enhanced by antibodies to prM [42] . The antibodybound immature virus is likely to be endocytosed and processed by cellular furin in the endocytic pathway [54] . The lower pH conditions of the late endocytic pathway could then cause the loss of pr inhibition and allow virus fusion.
The structure of furin-cleaved DENV at pH 6.0 shows that pr is bound to the virion through interactions with the DII tip of one E protein and DI on the neighboring E monomer [35, 36] . This suggested that pr might primarily block virus-membrane interaction by preventing dissociation of E dimers, a required first step in the fusion pathway [55] . Our results show efficient binding of pr to the dimeric form of the DENV E protein, but also to the monomeric DI/II form. We do not know if the E9 protein dimer is stabilized by pr interaction or if the dimer dissociates prior to interaction with pr, and experiments to address these points were inconclusive (data not shown). The similar pH dependence of pr binding to monomeric and dimeric E proteins suggests that pr may bind the same site in both cases. mAb 4G2 against the fusion loop inhibited pr interaction with E DI/II, confirming that pr was binding to the DII tip rather than to other sites on expressed E proteins. In keeping with its binding site in the vicinity of the fusion loop, pr peptide blocked the membrane insertion and liposome co-floatation of E9 and DI/II proteins. Prior studies showed that a monomeric DI/II protein with a single Strep affinity tag stably inserts into liposomes at either neutral or low pH [30] , and pr blocked this insertion even at pH 8.0 where its interaction with DI/II was suboptimal (data not shown). Thus, while the pr-E interaction is strongly low pH-dependent, its functional inhibition of membrane insertion can still be observed at neutral pH in the presence of excess pr.
Several other studies have addressed the role of E H244 in the flavivirus lifecycle. Experiments in TBEV evaluated particle production and membrane fusion activity using a VLP system [56] . Mutation of H248 (TBE numbering) to A or I blocks VLP secretion, in agreement with our results. However, an H248N mutant efficiently produces VLP, and these particles show WT levels of fusion activity. WNV E H246A or Q mutations inhibit release of infectious reporter virus particles from cells, as do a number of other substitutions at this position [57] . Replacement of H246 with aromatic residues such as phenylalanine allows both particle release and infectivity. An H244A mutation in DENV2 NGC inhibits infectious virus production [43] . E H244 and its interacting partners D63 and D65 on pr are conserved within the flaviviruses, and thus these data from several flaviviruses plus our DENV results support an important role for the E 244 position. However, a histidine residue at this position does not seem to be strictly required for particle production, suggesting that substitutions such as 244F and 244N can support the interaction of E with pr.
In contrast to the block in production of H244A VLP, the H244A DI/II protein was efficiently secreted from cells. Mutant protein secretion was somewhat reduced, with the final yield of DI/II H244A about half that of the WT protein in two separate preparations (data not shown), suggesting some effects of nonoptimal pr interaction. However, unlike the E protein in virus or VLP, the truncated DI/II protein lacks the TM region and does not mediate membrane fusion, and thus may be relatively independent of the pH-protection function of pr. The purified WT and mutant DI/II proteins were able to bind liposomes and form core trimers that were stabilized by DIII (Fig. 6) . Thus, the mutant protein is correctly folded and active in membrane insertion. Studies with conformation-specific mAbs also provided evidence for the correct folding of H244A E protein (Fig. 8B) . Together, these results suggest that the H244A E protein is still able to access the chaperone functions of prM, while its decreased pr binding indicates that it can no longer utilize the pH protection functions of pr.
These data are consistent with the idea that, similar to WT E, the mutant protein is assembled with prM into VLP in the ER. The membrane insertion and trimerization activity of H244A suggest that the full-length mutant protein would be fusion-active on such VLP once they are transported from the neutral pH of the ER to the low pH of the Golgi and TGN [31] . Thus, the decreased release of H244A VLP and its partial rescue by neutralization of the exocytic pathway support a critical role for pr in protecting DENV from exocytic low pH, and suggest that virus/VLP fuses in the TGN in the absence of pr-E interaction. Rescue of H244A VLP production by NH 4 Cl was clearly incomplete. This may be due to complex aspects of both virus and cell, such as direct effects of the H244A mutation on particle assembly in the ER, or difficulties in blocking fusion of a virus with the relatively high pH threshold of DENV.
Several strategies have been used to block flavivirus and alphavirus fusion reactions and thus inhibit virus infection. SFV and DENV fusion are specifically blocked by exogenous DIII, which binds to the core trimer and prevents the foldback of endogenous DIII and hairpin formation [41] . A later stage in DENV fusion is targeted by a stem-derived peptide, which binds to the ectodomain trimer in which DIII has folded back but stem packing has not yet occurred [58] . These virus protein-protein interactions can be reconstituted in vitro [29, 30, 58] , opening the possibility of using them as screens for small molecule inhibitors of virus fusion and infection.
The in vitro reconstitution of the pr-E interaction using soluble components could also act as a screen for small molecule inhibitors of this important flavivirus protein-protein interaction. Such inhibitors could act at multiple points in the virus lifecycle. During virus protein biosynthesis, an inhibitor could block the chaperone interaction of prM with E, leading to misfolding of E and its elimination by the ER quality control pathway. An inhibitor of pr interaction could make E protein susceptible to premature fusion in the TGN and could thus block virus production similar to the H244A mutation. It is also possible that small molecule inhibitors of pr-E binding could interact directly with the DII tip on mature virus particles, perhaps stabilizing the dimer and/or blocking membrane insertion of the fusion loop, thereby blocking virus fusion. Thus the in vitro system we describe here has the potential to identify molecules that could aid in the study of the flavivirus lifecycle and that could act to inhibit specific steps.
Previous studies showed that after cleavage endogenous pr is retained on the virus particle if the virus is maintained at acidic pH [35] . Under these conditions, the virus-pr complex does not bind target membranes, while virus from which pr is first released at neutral pH efficiently binds membranes upon shift to acid pH. Thus, the bound endogenous pr inhibits virus-membrane interaction and presumably blocks virus fusion [35] . Our results demonstrated that even after maturation to fully infectious DENV particles, exogenous pr could add back to the virus and inhibit low pH-triggered virus fusion and infection. The flavivirus membrane fusion reaction is very rapid, occurring within seconds of low pH treatment [47] . Recombinant DENV2 pr peptide inhibited fusion by both DENV1 and DENV2, suggestive of a fairly broad spectrum inhibition in agreement with the strong sequence conservation at the pr-E interface [34] .
The structure of the flavivirus E protein in its pre-fusion and post-fusion conformations defines the dramatic conformational changes between these two states. Many questions about the intermediates that connect the pre-and post-fusion conformations remain. In particular, it will be important to define the membrane protein rearrangements in the context of the highly organized flavivirus particle. For example, a neutralizing E mAb that blocks virus fusion was used to trap a West Nile virus fusion intermediate [59] . It will be interesting to evaluate if exogenous pr peptide could also be used as a novel probe to capture intermediates in the flavivirus fusion pathway.
BHK-21 cells and C6/36 mosquito cells were cultured as described previously [60] . 293T cells and T-REx TM -293 cells were cultured as previously described using tetracycline-deficient fetal calf serum for the latter cells [61] . The DENV2 New Guinea C (NGC) strain and the DENV1 Western Pacific (WP) strain were propagated in C6/36 cells in DMEM containing 2% heatinactivated fetal calf serum and 10 mM Hepes, pH 8.0, as previously described [41, 62] . Sindbis virus expressing green fluorescent protein was obtained as an infectious clone (a kind gift from Dr. Hans Heidner) and propagated in BHK cells [63] .
4G2 is a mouse monoclonal antibody (mAb) that recognizes the fusion loop of flavivirus E proteins [51, 64] . mAb prM-6.1 recognizes a linear epitope on prM, and was a kind gift of Drs. Chunya Puttikhunt and Nopporn Sittisombut [40] . 4E11 is a mouse mAb that recognizes DIII of DENV E protein and neutralizes all 4 serotypes of dengue virus [49, 50] , and was a kind gift of Dr. Fernando Arenzana-Seisdedos (Institute Pasteur, Paris). The anti-DIII polyclonal antibody Sango was raised by immunization of a rabbit with purified DENV2 DIII protein [30] . Western blot detection of truncated E proteins used 4G2 or Sango antibodies. A mAb to b-actin was obtained from Sigma and used to confirm equivalent loading of cell lysate samples. Immunofluorescence detection of DENV-infected cells used the antibody to DIII or mouse polyclonal anti-DENV2 hyperimmune ascitic fluid (obtained from Robert B. Tesh, University of Texas Medical Branch), with Alexa Fluor 488 or rhodamine-conjugated secondary antibodies (Molecular Probes).
The sequence encoding residues 1-86 of pr was amplified by PCR of an expression plasmid for DENV2 NGC prM-E DI/II [30] . The PCR product was ligated into the pPUR vector (Clontech), with the 21-residue TPA signal peptide [65] fused at the N-terminus and a tandem Strep tag at the C terminus (Fig. 1) . The plasmid, referred to as pPUR-TPA-pr-STST, was transfected into 293T cells using polyethylenimine (PEI, Polysciences). For optimal protein production, 3.5610 6 cells were plated per 10 cm dish and cultured for 24 h in 10 ml of complete medium. 7.5 mg plasmid in 1 ml DME was mixed with 30 mg PEI, incubated 10 min, then added drop wise to the cell culture medium. After 12 h, the medium was changed to 10 ml DME plus 2% serum. The culture medium was collected after 48h and again after 72h. Pr was purified by affinity chromatography on a Strep-Tactin column from IBA BioTAGnology and by gel filtration using a Sephadex G75 column [30] . Final yields were ,2 mg purified protein/1 liter culture supernatant.
Truncated DENV E proteins (Fig. 1) were obtained by inducible expression in Drosophila S2 cells and purified by affinity chromatography as previously described in detail [29, 30] . The H244A mutation was introduced into the DI/II protein by in vitro mutagenesis, and S2 cell expression and purification were performed as above. DENV2 NGC DIII (Fig. 1) was previously referred to as LDIIIH1CS [30] , and contains domain III, the linker between domain I and domain III, and the H1 and CS regions of the stem domain. DIII was expressed in E.coli and refolded as previously described [41] . SFV E1 DI/II protein was produced as previously described [29] . All purified proteins were stored in TAN buffer (20 mM Triethanolamine[TEA], pH 8.0; 130 mM NaCl) at 280uC.
SDS-PAGE analysis was performed using 10-12% acrylamide gels with a Bis-Tris buffer system (Invitrogen). Western blots were performed with Alexa Fluor 688-conjugated secondary antibodies (Molecular Probes), and were quantitated using an Odyssey Infrared Imaging system and Odyssey InCell Western software (LI-COR Biosciences) [30] . Standard curves with purified E proteins confirmed the linearity of this analysis (data not shown).
Pr or BSA was coupled to NHS-activated sepharose 4 fast flow (GE Healthcare) as described in the manual. In brief, sepharose was washed with 1mM HCl, and incubated with 660 mg pr or BSA/ml in 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3 at room temperature for 1.5 hr. The reaction was quenched with 0.1M Tris-HCl pH 8.5 for 30 min and free protein removed by washing with PBS. About 1mg of protein was coupled to 1ml beads. For the pull-down assay, 3 mg DI/II protein was pre-incubated where indicated with 24 mg 4G2 (molar ratio 1:2) or control mAb for 10 min at room temperature, and then incubated for 1 h on a rocker at room temperature with 10 ml of pr-or BSA-sepharose in a buffer containing 20 mM MES, 20 mM TEA, 130 mM NaCl, 0.2% Tween 20 at pH 8.0 or 6.25. The beads were then washed twice with the corresponding buffer and the bound DI/II was analyzed by SDS-PAGE and western blot.
SPR studies were performed on a BIAcore 2000 instrument (GE Healthcare). Purified recombinant pr was immobilized on a CM5 biosensor chip by primary amine coupling as described in the manual. In brief, pr peptide was diluted to 10 mg/ml in 10 mM sodium acetate pH 4.7 and pre-concentrated on the chip surface. The chip was then activated by a mixture of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, followed by quenching with 1M ethanolamine at pH 8.5. Under these conditions, pr was immobilized to a final density of 600 or 1000 response unit (RU). A control cell was mock-coupled with protein-free solutions. To test interaction, truncated E proteins were diluted to 1.2 mM in a MES/TEA buffer (20 mM MES, 20 mM TEA, 130 mM NaCl) at a pH range of 6.0 to 8.0, and flowed over the chip for 300 s at 0.3 ml/min, followed by buffer alone at the same flow rate. After each round, the chip was regenerated by washing with 50 mM NaOH in 1 M NaCl. The pr chip showed undiminished E binding activity for at least 50 rounds.
Liposomes were prepared by freeze-thaw and extrusion through 200 nm polycarbonate filters [66] , and were stored at 4uC in TAN buffer under N 2 and used within 2 weeks of preparation. Liposomes were composed of a 1:1:1:3 molar ratio of 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE), sphingomyelin (bovine brain) (Avanti Polar Lipids; Alabaster, AL), and cholesterol (Steraloids, Inc.; Wilton, NH), plus trace amounts of 3 H-cholesterol (Amersham; Arlington Heights, IL).
Protein-membrane interaction was monitored using a liposome co-floatation assay [29, 30] . E9 or DI/II proteins at a final concentration of 50 mg/ml were incubated in TAN buffer (pH 8.0) for 5 min at 28uC in the presence of 200 mg pr peptide/ml as indicated. Liposomes were then added to a final concentration of 1mM lipid and the samples were adjusted to pH 5.75 by the addition of 0.3 M MES or maintained at pH 8.0, and the incubation continued at 28uC for 30-60 min. The samples were then adjusted to 20% sucrose and loaded on top of a 300 ml cushion of 40% sucrose, then overlaid with 1.2ml 15% sucrose and 200 ml 5% sucrose. All sucrose solutions were at the same pH as the samples, and were wt/wt in TAN buffer at pH 8.0 or in MES buffer (50 mM MES, 100 mM NaCl) at pH 5.5. Gradients were centrifuged for 3 hr at 54,000 rpm at 4uC in a TLS55 rotor, and fractioned into the top 700 ml, middle 400 ml and bottom 1 ml. The 3 H-cholesterol marker was quantitated by scintillation counting. 200 ml of each fraction were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE and western blotting [29] . Purified human secreted placental alkaline phosphatase with a ST affinity tag (Seap) was used as a control protein [67] , and was a kind gift from Yves Durocher, Biotechnology Research Institute, Montreal.
The fusion-infection assay was performed essentially as described previously [41] . In brief, BHK cells grown on 96-well plates were washed twice with ice cold binding medium (RPMI without bicarbonate, 0.2% BSA, 10 mM Hepes, and 20 mM NH 4 Cl, pH 7.9). Virus stocks were diluted in binding medium and incubated with cells on ice for 3 h with gentle shaking. Cells were washed twice with binding medium to remove unbound virus and pulsed for 1 min at 37uC in 100 ml RPMI without bicarbonate, containing 0.2% BSA, 10 mM Hepes and 30 mM sodium succinate at pH 6.0 or 7.9, containing the indicated concentration of pr peptide. Infected cells were incubated in MEM plus 2% FCS and 50 mM NH 4 Cl for 4 h at 37uC, and then at 37uC for 2 d in the presence of 20 mM NH 4 Cl. The number of infected cells was quantitated by immunofluorescence using mouse polyclonal anti-DENV2 antibody. Infection observed at pH 7.9 represents virus that is endocytosed and fuses during 1 min at this pH.
The DENV1-WP infectious clone (reference [68] , a kind gift from Dr. Barry Falgout) was digested with KpnI and a 3.3kb fragment including the E sequence was sub-cloned into the pGEM3Z vector to generate pGDENV1 3.3. pGDENV1 3.3 was used as a template to generate the E H244A mutation, using circular mutagenesis as previously described [69] . A 2.6kb BstB1/ XhoI fragment containing the H244A mutation was sub-cloned into the DENV1-WP infectious clone to obtain DENV1-E H244A. The mutation was confirmed by restriction analysis and sequencing of the complete prM-E region. Two independent infectious clones were used to confirm the phenotype.
The WT and the mutant infectious clones were linearized by Sac II digestion and used as templates for in vitro transcription [70] . RNAs were electroporated into BHK cells and cells were cultured overnight at 37uC followed by 6 d at 28uC in MEM containing 2% FBS and 10 mM HEPES, pH 8.0. Progeny virus in the medium was quantitated by infectious center assay on indicator BHK cells, using mouse polyclonal anti-DENV2 antibody. To detect primary infection, aliquots of the electroporated cells were plated on coverslips, cultured 3 d at 37uC, and processed for immunofluorescence microscopy as above.
WT and E H244A mutant DENV1 prM-E sequences were PCR-amplified from the pGDENV1 3.3 subclones described above, and cloned into pcDNA4/TO (Invitrogen). These constructs were transfected into T-REx TM -293cells using Lipofectamine 2000 (Invitrogen) and selected in T-REx HEK medium containing 125 mg/ml Zeocin, all as previous described [61] .
To test E protein folding and expression, 1610 6 WT and mutant E expressing cells were seeded in 10 cm plates, cultured for 24h, and then E protein expression was induced by culture for 36 h in 1.5 mg/ml tetracycline in DME medium with 10% FCS at 37uC. Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 1mM PMSF, 16 Roche complete protease inhibitor cocktail) on ice for 1 hr. The cell lysates were cleared by centrifugation for 30 min at 10,0006g and protein concentrations were quantitated and normalized. E proteins were immunoprecipitated from cell lysate samples (500 mg total cellular protein) using 20 mg purified mAb 4G2 or mAb 4E11 and 20 ml protein-G sepharose, or 30 ml Sango antibody and 20 ml protein-A sepharose. 4E11 and 4G2 immunoprecipitated samples were blotted with Sango. Sango immunoprecipitated samples were blotted with mouse anti DENV2 serum.
For VLP secretion studies, 2-3610 6 cells were seeded in 10 cm plates, cultured for 24h, and then induced by culture for 36 h in 1.5 mg/ml tetracycline in DME medium with 10% FCS at 37uC. The culture media were centrifuged at 10,0006g for 30 min to remove cell debris. VLPs were then pelleted through a 0.5 ml sucrose cushion by centrifugation at 54,000 rpm for 2 h at 4uC using a TLS55 rotor. To test the effect of neutralizing the pH of acidic cellular compartments, cells were seeded and induced as above. After 2 h of induction the media were changed to DME medium containing 20 mM HEPES pH 8.0, 2% FCS, and 1.5 mg/ml tetracycline plus 20 mM NH 4 Cl as indicated, and the incubation continued for a total of 36 h. E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were then analyzed by SDS-PAGE and western blot using Sango. Figure S1 Open-book view of pr-E interface. Pr peptide is shown in cyan. DI, DII and DIII of E9 protein are colored red, yellow and blue, and the fusion loop at the DII tip is labeled. The important charged residues in the pr-E interface are numbered and shown as stick drawings in blue (positive) or red (negative). In this structure from DV2 16681, E9 residue 71 is a Glu, while the corresponding residue in NGC E9 protein is an Asp. Figure  prepared from Protein Data Bank accession number 3C5X [34] using PyMOL. Found at: doi:10.1371/journal.ppat.1001157.s001 (0.39 MB PDF) Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. CD200 is a type 1 membrane glycoprotein, delivering immunoregulatory signals through binding to its receptors (CD200Rs) (1) (2) (3) (4) . It is present on neurons, B cells, activated T cells, thymocytes, dendritic cells and endothelium in mice, rats and human (5, 6) . A large and growing body of studies demonstrates that expression level of CD200 regulates graft survival (7) (8) (9) , susceptibility to autoimmune diseases (10) (11) (12) , fetal loss (13) , inflammation/infection (14) and tumor immunity (15) (16) (17) (18) .
Alternative splicing is a major mechanism for regulating biological systems, producing multiple messenger RNA (mRNA) and protein isoforms. Some of these isoforms have distinct or even opposing functions (19) . Many genes in the immune system have been found to be alternatively spliced (20) (21) (22) and a growing number of human diseases are associated with aberrant splicing of the genes (23) (24) (25) . However, few studies to date have identified the mechanisms that regulate alternative splicing in the immune system. While CD200 exists as a single copy gene, data from Borriello et al. (26) , confirmed by our experiments (27) , have reported that a splice variant of CD200 exists. Although exon 2 deletion of CD200 caused by alternative splicing results in a frame shift and premature translational termination, we noted the existence of a downstream ATG start codon in a perfect Kozak context (27) . When the first start codon is followed shortly by a terminator codon and creates a small open reading frame (ORF; 5 0 -mini-cistron), the 40S ribosomal subunit remains bound to the mRNA, resumes scanning, and potentially reinitiates at the next ATG codon downstream (28) . It is known that the NH2-terminal region of CD200 is important for its biological interaction with CD200Rs (29, 30) , and translation from the second ATG start codon would produce a truncated form of CD200 (CD200 tr ) lacking the NH2-terminal 43 amino acids which includes regions important for the interaction with CD200Rs. Indeed, our previous studies have shown that expressed CD200 tr is a functional antagonist to CD200 (27) .
Exons often contain specific short oligonucleotide sequences that affect their ability to be spliced. Exonic splicing enhancers (ESEs) within exons promote splicing of the corresponding exons and subsequent exon inclusion mediated by splicing regulatory proteins. The best-studied family of splicing regulatory proteins are Serine/ Arginine-rich proteins (SR proteins), which include the proteins SF2/ASF, SC35, SRp20, SRp30c and many others (31, 32) . It has become clear that many exons *To whom correspondence should be addressed. Tel: 4163404800 (ext. 6759); Fax: 4169289466; Email: zhiqi.chen@utoronto.ca contain ESE elements that bind to specific members of the SR family (25) , leading to exon inclusion.
Since CD200 is involved in many diseases and its splice variant CD200 tr is an antagonist to CD200, identification of the mechanism controlling the relative expression levels of CD200 versus CD200 tr may provide insight into novel strategies for treatment of clinical disorders. In the present study, we have explored the mechanism controlling CD200 alternative splicing and show that SF2/ASF regulates CD200 alternative splicing through its direct binding to an ESE site in exon 2 of this gene. The level of SF2/ASF determines the alternative splicing patterns in different tissues or cells. Interestingly, in a mouse model of viral infection, we detected for the first time that the normal splicing pattern of CD200 was reversed in the lung tissue of A/J mice infected with mouse hepatitis virus strain I (MHV-1), following an increase in expression of SF2/ASF in this MHV-1 susceptible mouse strain.
All human cell lines were obtained from American Type Culture Collection. Human B cell lines Daudi, Raji and TEM were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS). The human neuronal cell lines SK-N and HCN-1A were cultured with 10% FBS in a-MEM media (Invitrogen).
Total RNAs from different human tissues were purchased from Clontech. A human BAC clone containing the whole human CD200 gene and a pcDNA3.2 expression vector containing SF2/ASF were obtained from The Center for Applied Genomics (Hospital for Sick Children, Toronto). Taq DNA polymerase, T4 DNA ligase and all restriction endonucleases were purchased from New England Biolabs. Random Primers, Superscript Reverse Transcriptase II, Elongase Enzyme, pcDNA3.0 expression vector and all competent cells were purchased from Invitrogen. EndoFree Plasmid purification Maxi Kit and QIAEX II Gel Extraction Kit were ordered from QIAGEN. A purified SF2/ASF recombinant protein was kindly provided by Dr. Blencowe (University of Toronto). Anti-human and mouse SF2/ ASF antibody was obtained from Santa Cruz Biotechnology. Anti-human and mouse b-actin antibody was purchased from BD Biosciences. Small-interfering RNA (siRNA) including SF2/ASF siRNA and a 'scrambled' siRNA were synthesized by Eurogentec. RNA oligonucleotides were synthesized by DNA and RNA Synthesis Center at Hospital for Sick Children (Toronto). All the primers used for polymerase chain reactions (PCRs), real-time PCRs and mutations were synthesized by Invitrogen.
Female A/J and C57BL/6J mice, 6-8 weeks of age were purchased from Jackson laboratories. The mice were maintained in microisolator cages, housed in the animal facility at The Toronto Hospital Research Institute, University of Toronto, and fed standard lab chow diet and water ad libitum. All protocols were approved by the animal Welfare Committee. Parental virus Mouse Hepatitis Virus strain 1 (MHV1) was ordered from the American Type Culture Collection. As previously described (33) , MHV1 infection was carried out in a viral isolation room. A/J and C57BL/6J mice were anesthetized by intraperitoneal injection with 0.2 ml 10% pentobarbital diluted in normal saline. Mice were left untreated or received 5000 plaque forming unit (PFU) of MHV1 intranasally. Mice were sacrificed 12, 36 and 96 h postinfection and lung tissue was collected.
Total RNA was isolated from human B cell lines (Daudi, Raji, TEM), human neuronal cell lines (SK-N, HCN-1A) and mouse lung tissue using TRIzol reagent. Five micrograms of total RNA from human tissues (brain, heart, skeletal muscle, colon, liver, thymus, kidney, intestine, lung, placenta and spleen), or human B cell lines (Daudi, Raji, TEM) and human neuronal cell lines (SK-N, HCN-1A), or mouse lung tissue was treated with DNase I and reverse transcribed in the presence of 250 ng of Random Primers, 1Â PCR Buffer, 10 mM dNTPs and 200 U of SuperScript II reverse transcriptase (RT; Invitrogen) in a final reaction volume of 20 ml. Reactions were carried out at 25 C for 10 min, 42 C for 50 min, followed by a 15-min step at 70 C to denature the enzyme.
For regular PCR, 2 ml of first strand complementary DNA (cDNA) was amplified in a 50-ml reactions in the presence of 1Â PCR buffer, 1.5 mM MgCl 2 , 2.5 mM of dNTPs, 5 U of Taq DNA Polymerase (New England Biolab). A first cycle of 5 min at 94 C was followed by 30 cycles of 30 s at 94 C, 30 s at a different annealing temperature (based on different primer pairs), and 1 min at 72 C. The final extension step was at 72 C for 15 min. For real-time PCR, first strain cDNA was diluted 1:20 and quantified using an ABI 7900HT Sequence Detection System (Applied Biosystems). The sequences of the primers used for regular and real-time PCR were indicated in Table 1 .
The endogenous human CD200 primer pairs for regular PCR were also used to construct an amplicon-containing plasmid (endogenous) for a standard curve. An exogenous amplicon-containing plasmid (exogenous) for a standard curve was constructed using the primers shown in Table 1 . Samples were tested in triplicate using 4 ml of first strand cDNA in a 20 ml total volume with 1Â universal master mix (Applied Biosystems). The results were normalized to that of the housekeeping gene GAPDH and HPRT. The copy number of transcripts was determined by comparison with a calibration curve of known amounts of amplicon-containing plasmid.
Control reactions were performed for the specificity of the real-time PCR primers. A DNA fragment, containing either exon 1, exon 2 and exon 3 or only exon 1 and exon 3, was gel purified and subcloned into pcDNA 3.0 between NotI and XhoI sites. The CD200-bearing plasmids were then linearized by XhoI. In vitro transcription was carried out using TranscriptAid T7 High Yield Transcription Kit (Fermantas Inc.) following the manufacturer's instruction. Transcribed RNA was treated with DNaseI to remove template DNA and purified by phenol:choloroform extraction and ethanol precipitation. First strand cDNA was then synthesized and real time PCR was performed. The primer pairs used for real-time PCR are shown in Figure 3A and Table 1 .
A human BAC clone containing the whole human CD200 gene was used as a template for long-distance PCR to obtain a region bearing exon 1, intron 1, exon 2, intron 2 and exon 3 of the human CD200. Two mixtures were prepared: mix 1 (20 ml) contained 0.1 mg of DNA template, 0.5 mM dNTP mix and 0.5 mm of sense and antisense primers; mix 2 (30 ml) included elongase enzyme mix and 1Â long-distance PCR buffer A and B provided by the manufacturer (the ratio of buffer A and B is 1:4). The sense primer started with the NotI cleavage site and the antisense primer with the SalI site. The sequences of the primers were shown in Table 1 . Mix 1 and mix 2 were combined on ice and subject to PCR under the following condition: 94 C for 1 min followed by three cycles at 94 C for 30 s, 59 C for 30 s, 69 C for 20 min, and then 29 cycles of 94 C for 30 s, 69 C for 20 min. The final extension was 69 C for 15 min. The 12-kb CD200 fragment was displayed on 0.7% TAE-agarose gel and purified using QIAEX II Agarose Gel Extraction Kit following the manufacturer's instruction. For more efficient elution of the large size DNA, the final incubation time was extended to 30 min at 60 C. The gel-purified DNA fragment was verified by restriction enzyme digestion with BamHI, BglII, EcoRI and HindIII, respectively, and DNA sequencing. For ligation to pcDNA 3.0 expression vector, the CD200 fragment was digested with NotI and SalI. Meanwhile, pcDNA 3.0 expression vector was digested with NotI and XhoI. Afterwards, pcDNA 3.0 vector was further dephosphorylated to remove the 5 0 phosphoryl group, preventing the vector from selfligation. The enzyme-treated CD200 fragment and pcDNA 3.0 were ligated, at a molar ratio of 3:1, using 
Primers for real-time PCR (the location of the numbered primers was shown in Figure 3A ) Endogenous human full-length CD200 (1) sense (exon 2) 5 0 -CAGCCTGGTTTGGGTCATG-3 0 (2) antisense (exon 3) 5 0 -GCAGAGAGCATTTTAAGGAAGCA-3 0 Endogenous human truncated CD200 
Ligation products containing the alternative splicing construct were transformed into DH 10b Escherichia coli cells by electroporation using a Cell-Porator Electroporation System (Life Technologies) at 401 V, 330 mF capacitance, low and 4 k (for Booster). The cells were plated onto LB/ampicillin plates and incubated at 37 C overnight. Twenty isolated clones were randomly picked. Only one clone showed a DNA supercoil band with much larger size than that of the vector clone on the gel. This clone was further characterized by the combination of restriction enzyme digestion and sequence analysis.
An ESE site was identified in exon 2 of the human CD200 using computational methods RESCUE-ESE (34) and ESEfinder (35) . To mutate the ESE site, site-directed mutagenesis was employed using QuickChange II XL site-directed mutagenesis kit from Stratagene. Two mutagenic primers were synthesized, in which the ESE site was replaced by a BsiWI site or deleted, and purified by polyacrylamide gel electrophoresis (PAGE). The sequences of the primers used are shown in Table 1 (the mutated region was underlined). The mutagenesis reaction was carried out in 50 ml total volume with 40 ng of template DNA, 125 ng of each primer and 2.5 U PfuUltra high-fidelity (HF) DNA polymerase and 3 ml of QuickSolution reagent provided by Stratagene. The cycling conditions included a 1-min initial denaturation at 95 C, 18 cycles with 50 s denaturation at 95 C, 50 s annealing at 58 C and 40 min extension at 68 C, and a final extension of 7 min at 68 C. The product was then subjected to digestion with 10 U of DpnI for 2 h at 37 C, selectively removing the parental, methylated, and nonmutated strands. Four microliters of DpnI-treated DNA was then transformed into XL10-Gold Ultracompetent cells. Cells were plated and incubated for selection of ampicillin-resistant clones. Ten isolated ampicillin-resistant clones were picked at random and their mutated or deleted regions were characterized by DNA sequencing.
The B cell line Daudi was washed and resuspended in 1Â Hanks Balanced Salt Solution (HBSS) to a cell density of 2 Â 10 7 cells/ml. The neuronal cell line SK-N was trypsinized and resuspended in 1Â phosphate-buffered saline (PBS) with 2% FBS at a density of 10 7 cells/ml. Thee-hundred microliters of the Daudi cells or 500 ml of the SK-N cells were transfected with 10 mg of the alternative splicing minigene construct, the minigene construct with the ESE site deleted or mutated, the minigene construct plus SF2/ASF expression vector, or the ESE deleted construct plus SF2/ASF expression vector. Electroporation was performed with square waves of 700 V, 99 ms pulse length for four pulses for Daudi and square waves of 200 V, 70 ms pulse length for one pulse for SK-N using T820 ElectroSquarePorator (BTX). Both Daudi and SK-N cells were cultured in 5 ml of pre-warmed complete medium for 48 h before harvesting.
The RNA oligonucleotides used for gel mobility shift assay were as follows:
CD200 exon 2 with the wild-type ESE, 5 0 -GUGAUCAG GAUGCCCUUCUC-3 0 ; CD200 exon 2 with the mutated ESE, 5 0 -GUGACGUAC GUGCCCUUCUC-3 0 ;
The RNA gel mobility shift assay was carried out as previously described (36) . The RNA oligonucleotides were 5 0 -end labeled with g-32 P-ATP (Perkin Elmer) using KinaseMax kit from Applied Biosystems following the manufacturer's instruction. Unincorporated nucleotides were removed by using G-25 Sephadex Columns. Fifteen femtomoles of radiolabeled RNA oligonucleotides were mixed with 4 pmol of SF2/ASF recombinant protein in a 20-ml binding reaction containing 2 mg yeast tRNA (Applied Biosystems). For competition, 100Â cold CD200 exon 2 oligonucleotide was added to the reaction containing the radiolabeled CD200 exon 2 oligonucleotide and SF2/ASF. After incubation for 20 min on ice, the RNA-protein complexes were separated from free RNA by electrophoresis on a 5% native polyacrylamide gel, run at 170 V for 2 h in 0.5% TBE buffer. The gel was then dried and autoradiographed at À80 C with intensifying screen.
SF2/ASF siRNA was designed based on the information described by Cartegni et al. (37) . A 'scrambled' siRNA, which has no match with any mRNA of the human database, was used as a control. The siRNAs were synthesized by Eurogentec with the following sequences:
7.5 Â 10 5 Daudi cells or 5 Â 10 5 SK-N cells were seeded into 12-well plates 24 h before transfection. Twoand-a-half micrograms of siRNA was transfected to Daudi or SK-N cells using Lipofectamine 2000 (Invitrogen) to examine endogenous expression pattern of CD200 following silencing SF2/ASF. Two-and-a-half micrograms of siRNA, together with 10 mg of the alternative splicing construct DNA, was transfected to Daudi or SK-N cells by electroporation to detect exogenous expression pattern of CD200 following silencing SF2/ASF. The cells were harvested 48 h posttransfection. Total RNA and protein were then extracted.
Nuclear extracts from Daudi and SK-N cells were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (38) from Pierce Biotechnology following the manufacturer's instruction. Western blotting was performed using 20 mg of nuclear extracts. After separation on a 10% SDS-PAGE gel, the proteins were transferred to a nitrocellulose membrane and probed with anti-human SF2/ASF antibody [1:200 dilution, goat polyclonal immunoglobulin G (IgG; Santa Cruz Biotechnology] followed by washing in 2% milk-PBS Tween. The membrane was then incubated with Donkey anti-goat IgG (1:5000 dilution; horseradish peroxidase-conjugated (BD Biosciences) and followed by washing again. Substrates, luminal and enhancer were added onto the membrane and incubated for 1 min. The membrane was exposed to Kodak XAR-5 film with intensifying screens for 5 min. Anti-human b-actin antibody (1:6000 dilution, goat monoclonal IgG; BD Biosciences) was used as loading controls. The exposure time for b-actin was 20 s.
Statistical significance was calculated with one-way analysis of variance (ANOVA) followed by Tukey tests. P-values 0.05 were considered significant and shown in the figures.
The existence of discrete CD200 splice variants is cell and tissue specific Human CD200 splice variants were examined in human tissues, B cells and neuronal cells. Total RNAs from different human tissues or human B cell and neuronal cell lines were used for RT-PCR using a sense primer located in exon 1 of human CD200 and an antisense primer in exon 3. As shown in Figure 1A and B, two transcripts were detected in all the human tissues, B cell lines (Daudi, Raji and TEM) and neuronal cell lines (SK-N and HCN-1A). The larger transcript was by far the dominant one seen in the brain and neuronal cell lines. Accordingly, for subsequent experiments, the B cell line Daudi and neuronal cell line SK-N were used as representatives of the two different splicing pattern of CD200. The only tissue not expressing CD200 was human skeletal muscle. The two transcripts were purified from the agarose gel and sequenced. It was confirmed that the larger one represented an exon 2 inclusion, whereas the smaller one represented an exon 2 exclusion (CD200 tr ).
Since alternatively spliced exons often contain ESEs for binding of splicing regulators that determine the fate of the exon (exon inclusion or exclusion), we wondered whether ESEs for binding of splicing regulatory proteins existed in exon 2 of CD200. For this purpose, both RESCUE-ESE (34) and ESEfinder (35) were used to search for ESEs in the exon 2 of CD200. Only one ESE was identified in exon 2 by both RESCUE-ESE and ESEfinder. The ESE existed in exon 2 of CD200 in human, mouse and rat, with the sequence TCAGGA ( Figure 2A ). The identified ESE represents a known binding site for a splicing regulatory protein SF2/ASF, a member of the SR protein family (35) .
Exogenous expression of CD200/CD200 tr shared the similar pattern with the corresponding endogenous one
To gain insight into the role of the ESE in exon 2 of CD200, we generated an alternative splicing minigene construct containing the genomic region from exon 1 to exon 3 of the human CD200 ( Figure 2B) . A 12-kb fragment bearing this genomic region was characterized by sequencing and restriction enzyme digestion, and ligated to a pcDNA 3.0 expression vector. The construct was transfected independently to human B cell line Daudi and neuronal cell line SK-N. After 48 h, RNA was extracted from each cell population for detection of the exogenous expression of splicing pattern of CD200. RNA was also isolated from nontransfected Daudi and SK-N cells for detection of the endogenously expressed splicing pattern. To measure quantitatively the expression levels of the two splice variants, real-time RT-PCR was performed using the primer pairs located in different regions ( Figure 3A ). The specificity of the primers for amplification of full-length and truncated CD200 was examined. As shown in Figure 3B , the primer pair used for full-length CD200 did not amplify the template from CD200 RNA lacking exon 2 (truncated form), whereas the primer pairs for truncated CD200 were not able to amplify the template from CD200 RNA containing exon 2 (full-length form). Each primer pair generated only a single product (Supplementary Figure 1A) and the standard curves generated from each primer pair are parallel with slopes between À3.1 and À3.6 (Supplementary Figure 1B) . The exogenous expression of CD200/ CD200 tr had a similar pattern to the corresponding endogenous one in Daudi cells or SK-N cells ( Figure 3D and E).
To examine further whether the ESE in exon 2 of CD200 determined the fate of the exon (inclusion or exclusion), site-directed mutagenesis was performed to mutate the ESE element in the alternative splicing construct, replacing the ESE (TCCTGA) with a restriction enzyme BsiWI site (CGTACG) ( Figure 3C ) or to delete the ESE. After characterizing the mutation or deletion construct by sequencing, the splicing construct was transfected to Daudi and SK-N cells. Total RNA was extracted from cells 48 h after transfection and real-time RT-PCR was carried out. As shown in Figures 3C and D, and 4A and B, expression of the full-length transcript (exon 2 inclusion) was reduced in both Daudi and SK-N cells after mutation or deletion of the ESE in exon 2. These data suggest that the ESE in exon 2 of CD200 promotes exon 2 inclusion.
A splicing regulatory protein SF2/ASF directly binds to the ESE and determines the fate of exon 2 of CD200
Since the ESE described above is known to contain a putative binding site for SF2/ASF, we investigated whether SF2/ASF binds to the ESE. An RNA-EMSA was performed. As shown in Figure 5 , an RNA-protein complex was detected after the SF2/ASF recombinant protein with ÁRS domain was mixed with a radiolabeled RNA oligonucleotide containing the ESE site. This protein/RNA interaction is specific since SF2/ASF did not bind to a radiolabeled RNA oligonucleotide containing mutated ESE site and the above binding was eliminated by competing 100Â unlabelled oligonucleotide containing the same ESE ( Figure 5 ). Moreover, this binding was not competed by the same level of cold oligonucleotide with the ESE site mutated (data not shown). As previously described, the full-length CD200 was expressed predominantly in brain and neuronal cells. One explanation of this observation is that the expression of SF2/ASF is higher in neuronal cells and brain. To test this hypothesis, we assessed SF2/ASF levels in Daudi and SK-N cells by Western blotting. As shown in Figure 6A , the natural level of SF2/ASF was clearly higher in SK-N cells than in Daudi cells.
To gain further insight into the role of SF2/ASF in controlling alternative splicing of CD200, an siRNA against SF2/ASF was employed to knock down SF2/ ASF in Daudi and SK-N cells. A scramble siRNA was used as a negative control. After Figure 3 . The pattern of expression of exogenous full-length CD200 or truncated CD200 in different cells parallels that of the endogenous molecules and mutation of the ESE in exon 2 abolishes exon 2 inclusion. (A) The location of the primers used for real-time RT-PCR. Primers 1 and 2 were used for endogenous expression of full-length CD200; primers 3 and 4 were used for endogenous expression of truncated CD200; primers 5 and 6 were used for exogenous expression of full-length CD200; primers 7 and 8 were used for exogenous expression of truncated CD200; primers 9 and 10 were used for the constitutive expression of V region of CD200. (B) The specificity of the primers used for full-length or truncated CD200. CD200 RNA containing exon 2 or lacking exon 2 from in vitro transcription was reverse transcribed and used for real-time PCR using the primer pairs labeled in the figure. (C) Mutation of the ESE in exon 2 was confirmed by DNA sequencing. (D) Endogenous and exogenous expression of the full-length and truncated CD200 in Daudi cells, and exogenous expression of two isoforms after mutation of the ESE. (E) Endogenous and exogenous expression of the full-length and truncated CD200 in SK-N cells, and exogenous expression of two isoforms after mutation of the ESE. The data represent the mean ± SE (three independent experiments, triplicate determinations). Broken lines reflect exogenous expression of the full-length CD200 decreased after mutation of the ESE relative to that of wild type (P < 0.01 in Daudi; P < 0.05 in SK-N). Continuous lines reflect exogenous expression of the truncated CD200 increased after mutation of the ESE relative to that of wild type in SK-N cells (P < 0.05). and total RNAs extracted for real-time RT-PCR, along with nuclear proteins for western blot. As shown in Figure 6A , SF2/ASF expression was eliminated after treatment with 2.5 mg of siRNA. b-Actin was used as a loading control. Real-time RT-PCR was then performed using RNA samples treated with siRNA. As shown in Figure 6B and C, the endogenous expression of full-length CD200 (exon 2 inclusion) was reduced in both Daudi and SK-N cells, compared with Mock (no siRNA) or scramble siRNA-treated cells. The same pattern was observed for the exogenous expression of CD200 in Daudi ( Figure 6D or SK-N cells ( Figure 6E ) following silencing SF2/ASF. Consistent with the observation resulting from the ESE mutation or deletion, the expression pattern of full-length versus truncated CD200 was reversed in SK-N cells after knockdown of SF2/ASF.
To investigate further the function of SF2/ASF in exon 2 inclusion or exclusion, we performed overexpression analysis by transfection of SF2/ASF expression vector to Daudi or SK-N cells and examined the fate of exon 2. As shown in Figure 4A -C, overexpression of SF2/ ASF induced exon 2 inclusion but this function was abolished in the absence of the ESE in exon 2, indicating that SF2/ASF regulates CD200 isoforms only via the ESE.
These results support the hypothesis that the splicing regulatory protein SF2/ASF, acting through binding to the ESE in exon 2 of CD200, plays an important role in controlling alternative splicing of CD200, and regulates Figure 4 . ESE deletion or overexpression of SF2/ASF affects exogenous expression patterns of CD200 isoforms. Ten micrograms of the wild-type minigene construct, the minigene construct with the ESE site deleted, the minigene construct plus SF2/ASF expression vector, or the minigene construct with the ESE site deleted plus SF2/ASF expression vector was transfected into Daudi or SK-N cells by electroporation. After 48 h, cells were collected and total RNA was isolated for real-time RT-PCR.The expression levels of the full-length and truncated CD200 as well as total CD200 in Daudi (A) and SK-N (B) were normalized to the housekeeping genes GAPDH and HPRT. The data shown are expression levels of full-length or truncated CD200 relative to total CD200. The data represent the mean ± SE (three independent experiments, triplicate determinations). the relative ratio of expression of full length to truncated CD200.
The alternative splicing pattern is altered in vivo in A/J mice infected with MHV-1
Previous studies have shown that several viruses express a viral protein which mimics human CD200 and down-regulates host immunity to the virus following interaction with a human CD200 receptor on host cells (39) (40) (41) . Whether viral infection itself affects the expression of CD200 in host is an issue which remains to be explored. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) has been described to induce pulmonary pathology with features reminiscent of severe acute respiratory syndrome (SARS) (33) . To examine the correlation between the viral (MHV-1) infection and expression of CD200 in host we collected lung tissues from MHV-1 susceptible A/J mice and MHV-1-resistant C57BL/6J mice after infection. RT-PCR was performed using a sense primer located in exon 1 and an antisense primer present in exon 3. Interestingly, we observed a reversal of the normal CD200 splicing pattern in lung tissues of A/J mice postinfection ( Figure 7A ). Real-time RT-PCR provided a more accurate result of this phenomenon. We documented that the full-length CD200 was increased after viral infection and was 2-fold higher at 36 h postinfection compared with that before infection ( Figure 7B ). All the susceptible A/J mice were dead at 96 h postinfection. In contrast, the relative ratio of full-length to truncated CD200 did not change in infected C57BL/6J mice ( Figure 7C and D) . Thus, the pattern of alternative splicing of CD200 was correlated with susceptibility of these strains to viral infection.
Since the above studies have shown that SF2/ASF regulates alternative splicing of CD200, we wondered whether expression of SF2/ASF increased in A/J mice post infection. We performed western blotting using anti-SF2/ASF antibody. As shown in Figure 7E , no obvious difference of SF2/ASF level was seen between A/J and C57BL/6J mice before viral infection. Increased expression of SF2/ASF was detected in lungs of A/J mice 12 h postinfection, whereas no increase of SF2/ASF in C57BL/6J mice even 36 h postinfection, suggesting that the role of virus on host CD200 expression is mediated by SF2/ASF.
The studies reported here show that the relative expression of two isoforms (CD200 and CD200 tr ) is tissue and cell specific and the alternative slicing patterns are different between the pattern in the lymphoid tissues and that of neuronal tissues. The relative expression of the two isoforms of CD200 is of interest, given our recent evidence that the truncated form (CD200 tr ) can antagonize the functional suppression induced by full-length CD200 (27) . Although Borriello et al. (26) reported no change in the alternative splicing pattern of murine CD200 in lymphoid tissue after stimulation by Con A or LPS in vivo, in our in vivo studies of mouse lung tissues before/after infection of MHV-1 virus we observed that, unlike in the natural condition, following viral infection the expression of total CD200 increased in lung of both MHV-1 susceptible A/J mice and MHV-1-resistant C57BL/6 mice. However, the splicing pattern of CD200 is reversed only in A/J mice, with the full-length transcript, capable of inducing immunosuppresion, becoming the predominant one. In contrast, for C57BL/6J, an MHV-1-resistant mouse strain, no change in the splicing pattern of CD200 was seen in the lung. This result importantly demonstrates that only the splicing pattern, but not the total transcription level, of CD200 determines the murine immune response to MHV-1 and is consistent with the hypothesis that the shift in the balance of expression of CD200/CD200 tr to decrease expression of the truncated product allowing CD200 to function in its immunosuppressive role, possibly contributing to the increased susceptibility to MHV-1 in the A/J mice. Further studies showed an increased expression of SF2/ ASF in A/J mice postinfection and the increase in SF2/ ASF occurred prior to increased full-length CD200, strongly suggesting that the regulation of alternative splicing of CD200 is mediated by SF2/ASF. It remains to be determined what viral proteins of MHV-1 have this effect and how the proteins regulate expression of SF2/ASF. Our studies suggest that viruses escape elimination by the host's immune system not only through producing viral proteins which mimic CD200 but also by inducing host CD200 expression and reducing expression of the antagonist CD200 tr . Posttranscriptional regulation, including mRNA stability, plays an important role for gene expression (42) . Whether the increase of full-length CD200 in A/J mice is also due to differential mRNA stability cannot be ruled out.
In this report, we searched ESEs in the human and murine exon 2 sequence using two ESE-detecting algorithms RESCUE-ESE and ESE finder (35, 43) . Only one ESE, which is a putative binding site for SF2/ASF, was detected by both RESCUE-ESE and ESEfinder 2.0. No ESE was identified in the whole exon 2 when using higher stringent ESEfinder 3.0. Thus, we focused on this ESE for the rest of the experiments.
Since an ESE can promote exon inclusion, mutation or deletion of the ESE would lead to less full-length but more truncated CD200. Our results showed that after mutating the ESE in exon 2, expression of full-length CD200 was reduced in both Daudi and SK-N cells. This expression pattern is the reverse of that seen for endogenous CD200 expression in SK-N cells, in which the predominant expression is of full-length CD200. To exclude the possibility that the mutation created a new exonic splicing silencer (ESS) which led to decreased full-length, and increased truncated CD200, we deleted the ESE and examined the changes in CD200:CD200 tr . Our result showed that deletion of the ESE promoted exon 2 exclusion, the same result as we obtained from mutation analysis, indicating that mutation of the ESE does not create an ESS.
Identification of a putative ESE for SF2/ASF binding does not provide direct evidence that SF2/ASF recognizes and binds to the ESE. To examine whether the identified ESE in exon 2 is bound by SF2/ASF, we performed RNA-EMSA using RNA radiolabeled oligonucleotides bearing the ESE in exon 2 and a recombinant SF2/ASF with ÁRS domain to reduce nonspecific binding. The result showed a binding of SF2/ASF to the ESE and the binding is specific because either mutated ESE or 100Â cold oligonucleotides abolished the binding.
Knockdown of SF2/ASF decreased expression of full-length CD200 in both Daudi and SK-N cells. Consistent with data seen following mutation or deletion of the ESE, the expression pattern of CD200 was again reversed in SK-N cells. The western blot performed confirmed the efficiency of knockdown of SF2/ASF. In contrast, overexpression of SF2/ASF increased expression of full-length CD200 but only in the presence of the ESE in exon 2, highlighting the critical role of the ESE in the mechanism of alternative splicing of CD200. Ubiquitously expressed splicing factors, among them is SF2/ASF, are thought to control tissue specific alternative splicing through their different expression levels in different tissues (44) . Our result showed that the natural level of SF2/ASF was higher in the neuronal cell line SK-N than in B cell line Daudi. This may help explain why endogenous full-length CD200 (exon 2 inclusion) is expressed at much higher level than that of truncated CD200 (exon 2 exclusion) in SK-N.
A recent report has described a higher expression level of SF2/ASF in many tumors, including lung, thyroid, kidney, colon, small intestine and melanoma, relative to their respective normal controls. One mechanism to explain this observation is that SF2/ASF abolished the tumor suppressor activity of BIN1, a tumor suppressor gene, by inclusion of exon 12A which interferes with MYC binding (45) . In contrast to its roles in transplantation, autoimmune diseases and inflammation, CD200 enhances the growth of malignant tumors and it has been suggested that a novel approach to anticancer therapy might include blockade of CD200 (15, 16, (46) (47) (48) (49) . Since CD200 tr is an antagonist to CD200 (27) , our data are consistent with the hypothesis that increased CD200 tr expression and decreased expression of full-length CD200 by blockade of SF2/ASF may also be of potential benefit for cancer treatment.
In conclusion, we have identified an alternative splicing pattern for expressed human CD200 in different cells and tissues, and compared this with the pattern observed in vivo following viral infection. Our data suggest that regulation of expression of alternative splicing transcripts may be important in controlling susceptibility to viral infection. An ESE in exon 2 of CD200 is a binding site for a splicing regulatory protein, SF2/ASF, which we have shown to control the alternative splicing pattern of CD200. A drug-mediated manipulation of alternative splicing has recently been reported which includes modulation of SF2/ASF (25) . It would be of interest to know if this drug treatment alters the expression ratio of CD200 to CD200 tr and thereby produces change in immune function.
Supplementary Data are available at NAR Online.
Funding for open access charge: The Heart and Stroke Foundation (NA6164 to R.M.G.). Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial BACKGROUND: Neuraminidase inhibitors are thought to be efficacious in reducing the time to alleviation of symptoms in outpatients with seasonal influenza. The objective of this study was to compare the short-term virological efficacy of oseltamivir-zanamivir combination versus each monotherapy plus placebo. METHODS AND FINDINGS: We conducted a randomized placebo-controlled trial with 145 general practitioners throughout France during the 2008–2009 seasonal influenza epidemic. Patients, general practitioners, and outcome assessors were all blinded to treatment assignment. Adult outpatients presenting influenza-like illness for less than 36 hours and a positive influenza A rapid test diagnosis were randomized to oseltamivir 75 mg orally twice daily plus zanamivir 10 mg by inhalation twice daily (OZ), oseltamivir plus inhaled placebo (O), or zanamivir plus oral placebo (Z). Treatment efficacy was assessed virologically according to the proportion of patients with nasal influenza reverse transcription (RT)-PCR below 200 copies genome equivalent (cgeq)/µl at day 2 (primary outcome), and clinically to the time to alleviation of symptoms until day 14. Overall 541 patients (of the 900 planned) were included (OZ, n = 192; O, n = 176; Z, n = 173), 49% male, mean age 39 years. In the intention-to-treat analysis conducted in the 447 patients with RT-PCR-confirmed influenza A, 46%, 59%, and 34% in OZ (n = 157), O (n = 141), and Z (n = 149) arms had RT-PCR<200 cgeq/µl (−13.0%, 95% confidence interval [CI] −23.1 to −2.9, p = 0.025; +12.3%, 95% CI 2.39–22.2, p = 0.028 for OZ/O and OZ/Z comparisons). Mean day 0 to day 2 viral load decrease was 2.14, 2.49, and 1.68 log(10) cgeq/µl (p = 0.060, p = 0.016 for OZ/O and OZ/Z). Median time to alleviation of symptoms was 4.0, 3.0, and 4.0 days (+1.0, 95% CI 0.0–4.0, p = 0.018; +0.0, 95% CI −3.0 to 3.0, p = 0.960 for OZ/O and OZ/Z). Four severe adverse events were observed. Nausea and/or vomiting tended to be more frequent in the combination arm (OZ, n = 13; O, n = 4; and Z, n = 5 patients, respectively). CONCLUSIONS: In adults with seasonal influenza A mainly H3N2 virus infection, the oseltamivir-zanamivir combination appeared less effective than oseltamivir monotherapy, and not significantly more effective than zanamivir monotherapy. Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower effectiveness of this combination calls for caution in its use in clinical practice. TRIAL REGISTRATION: www.ClinicalTrials.gov NCT00799760 Please see later in the article for the Editors' Summary Neuraminidase inhibitors (oseltamivir [O] , zanamivir [Z] ) are thought to be efficacious as compared to placebo in outpatients with uncomplicated seasonal influenza [1] [2] [3] [4] [5] [6] , both clinically in terms of reduction in duration of symptoms, as well as in terms of a reduction in viral shedding. In 2008, they were considered an important strategy to limit the impact of an influenza pandemic both individually, by reducing morbidity and mortality, and collectively, by slowing spread of the virus to allow time for vaccine production, the cornerstone of influenza control [2] [3] [4] 7] . It was hypothesized that the widespread use of a single antiviral might result in the emergence of resistant strains whose subsequent spread could dramatically reduce the effectiveness of antiviral therapy. The combination of two antiviral agents, if well tolerated, and if producing at least additive antiviral activity, theoretically offers several advantages: reducing disease severity, viral shedding, and viral excretion period, thereby also lowering the attack rate and risk of selection of resistant viruses, specifically in individuals with prolonged viral shedding, such as immunocompromised patients [8, 9] . Indeed, mathematical modelling showed a reduction in risk of emergence of resistant strains during early phases of a pandemic, associated with use of two antivirals as compared to single antiviral therapy [9] . Finally, another theoretical advantage of combining two drugs would be to ensure optimal treatment of all circulating influenza virus types, subtypes, or variants, as susceptibility of influenza viruses has been shown to vary, and seasonal H1N1 viruses naturally resistant to oseltamivir, which remain susceptible to zanamivir, emerged in 2008 [10] . Among antivirals active against influenza virus, the combination of neuraminidase inhibitors is attractive, because both compounds are licensed for seasonal influenza, they are delivered to the respiratory tract by distinct means (directly through a diskhaler for zanamivir, after gastrointestinal absorption and hepatic metabolism for oseltamivir), and key mutations associated with resistance are different for each drug. However, negative interactions cannot not be ruled out owing to the possible competition between these two drugs, which target the same binding pocket in the neuraminidase.
In 2006, in the context of pandemic planning, we designed a double-placebo randomized controlled trial in patients presenting with seasonal influenza-like illness to compare the oseltamivirzanamivir combination to each of the monotherapies plus placebo. The trial was conducted in France, during the winter of [2008] [2009] . Because of the emergence of the pandemic 2009 (H1N1) virus in humans, in April 2009 in North America, and its subsequent worldwide spread, the independent data-monitoring committee requested that we terminate the trial early and analyze the results earlier than planned, given the possible impact of the results on antiviral treatment management during the pandemic [11] .
From January 7th to March 15th 2009 (period of the winter 2008-2009 influenza epidemic in France), we enrolled throughout France adults 18 y old and older who consulted their general practitioner within 36 h of influenza symptoms onset (following the first influenza symptoms reported by the patient), with a temperature greater than or equal to 38uC (reported or observed by the practitioner), one or more respiratory symptoms (cough, sore throat), one or more general symptoms (headache, dizziness, myalgia, sweats and or chills, fatigue), and a positive nasal rapid test for influenza A (Clearview Exact Influenza A & B) performed by the practitioner. Enrolment of women required a negative urine pregnancy test. Exclusion criteria were vaccination against influenza during the 2008-2009 season, recent exacerbations of chronic obstructive pulmonary disease (COPD), asthma or severe chronic disease, previous history of depression, and prior inclusion in this trial. Prior to inclusion, patients gave informed written consent. The protocol was approved by the Ethics Committee of Ile de France 1 (Texts S1 and S2).
At enrolment (day 0), a nasal swab for virological analysis was performed using a standard operating procedure (sample kit plus instructional video). Patients were allocated to treatment by a randomization list, with an arm ratio of 1:1:1, balanced by practitioner. A computer random number generator was used to select random permuted blocks of size 3. This randomisation code was given to the central hospital pharmacy that prepared blinded treatment units in conformity with good manufacturing practices (GMP). Each general practitioner received six treatment units and was told to distribute them by order of inclusion of his patients in the trial. Allocation was concealed through the similarity of all the containers and the impossibility for the GP to identify the treatment arm when opening the container. The three treatments were (1) oseltamivir capsule for oral use plus inhaled zanamivir, (2) oseltamivir plus inhaled placebo, (3) zanamivir plus oral placebo. Oseltamivir dosage was 75 mg orally twice daily; zanamivir dosage was 10 mg by oral inhalation using the commercialized GlaxoSmithKline Diskhaler, twice daily. Active drugs and placebo were kindly provided by Roche and Glaxo-SmithKline laboratories. A visiting nurse performed a nasal swab for virological analysis on day 2. Patients returned to their general practitioner at day 7 for a follow-up examination, and were contacted by phone on day 14. Patients, general practitioners assigning the patients, and outcome assessors (practitioners, virologists, patients), were blinded to treatment assignment throughout the study and statisticians until the end of the analysis.
Nasal swabs placed into a transport medium (Virocult, Elitech) were transported at 4uC by special courier to the nearest National Influenza Centre (NIC) (Hospices Civils de Lyon, Lyon, or Pasteur Institute, Paris, France). Upon arrival, the swab samples were eluted into 2 ml of transport medium, processed for real-time reverse transcription (RT)-PCR analyses and inoculated onto MDCK cells for virus isolation and subsequent subtyping using a standard hemagglutination inhibition assay. For RT-PCR analyses, RNA extraction from 200 ml of specimen was performed using the QIAmp virus RNA mini kit (Qiagen) with RNA elution into a final volume of 60 ml. All real-time RT-PCR assays were performed in a final volume of 15 mL with 5 mL RNA, 0. mM of each primer, 0.2 mM probe, and 0.8 ml enzyme mix (Super-ScriptIII platinum one-step quantitative RT-PCR system, Invitrogen). Type A influenza virus RNA was detected by a real-time RT-PCR targeting the conserved matrix gene using GRAM/7Fw (59-CTTCTAACCGAGGTCGAAACGTA-39) and GRAM/ 161Rv (59-GGTGACAGGATTG GTCTTGTCTTTA-39) primers and GRAM probe/52/+ (59[Fam]-TCAGGCC CCTCAA-AGCCGAG-[BHQ-1]39) probe. The quality of the specimens was assessed by real-time RT-PCR targeting the GAPDH cellular gene [12] . Amplification was performed on a LightCycler 480 (Roche Diagnostics) (NIC, Pasteur Institute, Paris) or an ABI 7500 (Applied Biosystems) (NIC, Lyon). Cycling conditions are available upon request. Quantified synthetic RNA transcripts corresponding to the M and GAPDH genes were used as controls in parallel [13] .
To take into account the variability in the quantity of cells collected by nasal swab, we calculated a normalized influenza viral load for each specimen; this normalized viral load was defined as the ratio of the M RT-PCR and GAPDH RT-PCR multiplied by the average GAPDH RT-PCR at day 0 to express results in copies of genome equivalent/ml (cgeq/ml). The virological response was defined as a normalized viral load below 200 cgeq/ml at day 2. This threshold was determined according to results on specimens from patients with a positive influenza A rapid test from winter 2008-2009 included in the French influenza surveillance network (GROG), and analysed by the 2NICs, and because it resulted in 5% false positive and 5% false negative results with respect to virus isolation (Table S1 ). It was validated by the independent datamonitoring committee prior to any analysis. Sensitivity of the qRT-PCR was assessed using serial dilutions of quantified synthetic transcripts corresponding to the target genes (Text S3). To assess comparability of the data between the two NICs, specimens were exchanged showing excellent concordance. The threshold of the qRT-PCR used was set well above the limit of detection.
Oral temperature was recorded and severity of seven symptoms (nasal stuffiness, sore throat, cough, muscle aches, tiredness or fatigue, headache, and feverishness) was rated by the patient twice daily (morning and evening) up to day 5 and then once daily on a four-point scale (0, none; 1, mild; 2, moderate; 3, severe) [2] [3] [4] 14] . The time to resolution of illness was defined as time from study drug initiation to time of symptom alleviation. Symptom alleviation was defined as the first 24-h period during which the above seven symptoms were absent or only mild as previously described [3, 4] . Influenza-related clinical events were defined as incidence of a secondary complication (such as pneumonitis or otitis) independently of any antibiotic initiation, and/or occurrence of exacerbation of a preexisting chronic disease. Patients reported treatment compliance using a self-administered questionnaire; full compliance during the day 0 to day 2 period was considered when 100% of planned drug intakes had been completed.
The primary efficacy endpoint was the proportion of patients with RT-PCR,200 cgeq/ml on day 2 of treatment. Given the viral shedding kinetics in patients with seasonal influenza receiving neuraminidase inhibitors, the day 2 virological endpoint was considered to be best suited to measurement of virological effects [2, 4] . Other endpoints were (1) the decrease of log 10 viral load between days 0 and 2 in the patients with confirmed influenza A on day 0 and available samples both at days 0 and 2; (2) the time to resolution of illness; (3) the number of patients with alleviation of symptoms at the end of treatment (day 5); (4) the symptoms score at the end of treatment; (5) the incidence of secondary complications of influenza such as otitis, bronchitis, sinusitis, pneumonia, and the use of antibiotics; (6) the occurrence of adverse events in all participants having received at least one dose.
According to the protocol, the intention-to-treat (ITT) analysis was performed on two populations: (1) all enrolled patients (primary objective), and (2) enrolled patients with an influenza A virus infection confirmed by RT-PCR on day 0 (influenza Ainfected population).
Sample size evaluation assumed that virological response was obtained in 70% of patients in the oseltamivir-zanamivir arm, compared to 55% in each of monotherapy arms on the basis of the extrapolation of the results of previous trials [2, 4] . Samples of 300 subjects per arm had 90% power to detect this difference, with a two-sided test and a type I error of 0.025 because of the two tests (factoring 20% lack of follow-up). Proportions of response at day 2 were compared between the combination therapy arm (OZ) and each monotherapy arm (O or Z) separately using two tests with a type I error of 0.025 because of the two tests. Patients without a day 2 sample were considered treatment failures. Mean decreases of log 10 viral load were compared using a t-test in patients who had both day 0 and day 2 samples assuming a value of 0.5 cgeq/ml when RT-PCR was negative. For clinical endpoints nonparametric tests were used. Times to resolution of illness and symptoms score at the end of treatment were compared using Wilcoxon tests. If time to symptom alleviation was missing it was imputed to be 14 d, i.e., the end of the trial; 95% confidence intervals (95% CIs) of median differences were estimated by bootstrap. Probability of symptoms alleviation versus day of treatment was estimated using the Kaplan Meier method and was compared between groups with the log-rank test. Proportions of clinical events and of patients with alleviation of symptoms were compared using Fisher's exact tests. As an exploratory analysis, 95% CIs for differences of response between the two monotherapies were also estimated. All analyses were performed using SAS software, version 9.1 (SAS Institute). 
Out of the 900 patients initially planned, a total of 541 patients were enrolled by 145 general practitioners. They were randomly assigned to oseltamivir plus zanamivir (OZ, n = 192), oseltamivir plus inhaled placebo (O, n = 176), zanamivir plus oral placebo (Z, n = 173) ( Figure 1 ). Mean age was 39 y (standard deviation [SD] = 13), 49% were male, 14% had preexisting chronic diseases, mean fever was 38.2uC (SD = 0.8). The mean duration of illness before enrolment was 25 h (SD = 10). Other characteristics of patients appeared well balanced in the three arms ( Table 1 ). The rate of fully compliant patients was not significantly different among the three arms (84% for the OZ arm, 88% for the O arm, and 85% for the Z arm).
Out of the 541 enrolled patients, 447 (83%) had a RT-PCR laboratory confirmation of influenza A virus infection on the day 0 specimen, with a mean viral load of 4.38 log 10 cgeq/ml (interquartile range [IQR] 3.75-5.30). All the day 0 specimens were GAPDH RT-PCR positive with a mean value of 3.88 log 10 copies/ml.
Primary endpoint. In the ITT analysis, considering the 541 enrolled patients with positive influenza A rapid test, the proportion of patients with a RT-PCR,200 cgeq/ml on day 2 of treatment was 52.6% in the oseltamivir-zanamivir arm, 62.5% in the oseltamivir monotherapy arm (p = 0. Table 2 ). The same trends were observed in the 382 fully compliant influenza A-infected patients (Table S3) , and in the 395 patients with H3N2 infection with proportions of 42.4% in the oseltamivir-zanamivir arm, 58.6% in the oseltamivir monotherapy arm, and 30.3% in the zanamivir monotherapy arm.
Other virological endpoints. In the 414 influenza RT-PCR confirmed patients with both day 0 and day 2 available specimens, the day 2 to day 0 decrease was 2.14 log 10 cgeq/ml in the oseltamivir-zanamivir arm, 2.49 log 10 
The median time to resolution of illness in the 541 enrolled patients was 3. Sum of the severity of the seven day 0 influenza symptoms (feverishness, nasal stuffiness, sore throat, cough, muscle aches, tiredness-fatigue, and headache) using a four-point scale [2, 14] . b The score is expressed as a percentage of the maximal score of 21. (Tables 2  and 3 ). Other clinical outcomes showed similar trends (Tables 2, 3 , and S3).
Four serious adverse events occurred during the study, one of which was considered unrelated to study drugs (acute bacterial pneumonia at day 3 in a patient receiving oseltamivir-zanamivir combination). Two adverse events also occurred in patients receiving the oseltamivir-zanamivir combination: severe headaches leading to interruption of therapy and facial oedema following the first administration, disappearing within 24 h postdrug interruption. The remaining patient experienced repeated vomiting after oseltamivir monotherapy drug administration. All four patients completely recovered.
Other nonserious adverse events reported in more than 1% of the total population were in the OZ, O, and Z arms, respectively, nausea and/or vomiting (in 13, 4, and 5 patients), diarrhoea (in 2, 1, and 5 patients), and rash (in 1, 2, and 2 patients).
This large publicly funded clinical trial examined the effect of combination neuraminidase inhibitor antiviral therapy in influenza, as compared to each monotherapy plus placebo. It showed that, during the prepandemic winter of 2009 with a predominance of H3N2 viruses (more than 85%) in France, the oseltamivir-zanamivir combination seemed less effective than oseltamivir monotherapy, and not significantly more effective than zanamivir monotherapy in adults with seasonal influenza A virus infection.
Analysis of the different antiviral regimens' efficacy was based on a primary virological endpoint, which we hypothesized could be a sensitive and a more specific indicator than a primary clinical endpoint. Clinical endpoints, used as primary endpoints in previous studies, were used as secondary endpoints in the present study [1, 5] . Clinical endpoints, which are based on a global assessment of both general (mainly immunologically linked) and respiratory (mainly virologically linked) symptoms, are probably not the best way to monitor the virological effect of treatment, because clinical symptoms are not exclusive to influenza. We thus considered that a difference in viral shedding rate would be the best indicator of the virological effects of combined therapy, and consequently a valuable surrogate. Our initial hypothesis was that the combination of two antivirals may reduce the rate of resistant virus emergence (for a naturally susceptible pandemic virus and a nonimmune population). In addition, we hypothesized that for cases of infection with susceptible seasonal influenza viruses, this could not be easily shown, owing first to the rarity of this phenomenon in adults, and second, to the necessity of monitoring virus excretion for several days, whereas for cases of influenza due to H1N1 viruses, which are naturally resistant to oseltamivir, the question was not relevant. Given the viral shedding kinetics in patients with seasonal influenza receiving neuraminidase inhibitors, the day 2 virological endpoint was considered to be best suited to quantify virological effects. The 200 cgeq/l threshold was chosen, as it was the best compromise in terms of specificity and sensitivity as compared to standard culture. Of note, the same trends were observed when a 100 cgeq/l or a 1,000 cgeq/ml cut-off was used to define virological success (Table S2 ). Furthermore, the study was designed to be statistically two-sided to take into account the possibility that the combination would perform worse than either drug alone because of the theoretical concern of antagonism at the receptor level. The oseltamivir-zanamivir combination seemed, both virologically and clinically, significantly less effective than the oseltamivir monotherapy. This result seems robust because (1) it was found using a double-blind placebo methodology, (2) there was overall concordance both among virological endpoints, and between virological and clinical endpoints, (3) it was confirmed over the three different subgroups of subjects included in the global population (541 enrolled patients, 447 influenza A-infected patients, 382 influenza A-infected and fully compliant patients). This lower clinical and virological response to the combination may suggest a negative effect of zanamivir on oseltamivir, as in the absence of interactions the effect of the combination should at least be additive [15] . A negative interaction at the level of binding at the catalytic pocket of the neuraminidase is an explanation that should be further investigated in vitro for both seasonal H3N2 and H1N1 viruses. Recent in vitro data showing the lack of synergy between oseltamivir and zanamivir, and some antagonism at higher concentrations of zanamivir on pandemic H1N1 2009 virus, are in agreement with this hypothesis [16] . Furthermore, contrary to oseltamivir, which upon digestive absorption needs to be metabolized, thus delaying arrival of the active drug at the infection site (t max = 4 h), inhaled zanamivir is delivered directly to the primary site of influenza virus replication. The hypothesis that zanamivir is more likely to occupy the catalytic pocket first, thus preventing the action of oseltamivir, must be tested. According to this hypothesis, the combination would be largely reduced to a zanamivir monotherapy.
Whereas the results of the primary virological endpoints indicated a superiority of the oseltamivir-zanamivir combination to zanamivir monotherapy, clinical results were not significantly different, suggesting that oseltamivir adds essentially nothing to zanamivir monotherapy. This view is concordant with the above hypothesis of the predominant catalytic site occupation by zanamivir when the combination is administered.
As an exploratory analysis, oseltamivir showed a significantly higher clinical and virological efficacy as compared to zanamivir. This finding could be the consequence of a suboptimal treatment regimen in the zanamivir arm, since the IC50 values for the A(H3N2) viruses of the 2008-2009 season were 2-to 3-fold higher for zanamivir as compared to oseltamivir, but remained within the range for susceptible strains (GROG surveillance; NICs, unpublished data). The virological result is confirmed by the longer time Table 3 . Virological and clinical response according to treatment arms in the 447 influenza A-infected patients between day 0 and day 2 (ITT analysis). to alleviation of the influenza symptoms in patients receiving zanamivir.
As the present study was conducted before the 2009 H1N1 pandemic during an influenza season where A(H3N2) viruses predominated, the impact of prepandemic seasonal A(H1N1) oseltamivir-resistant viruses on the results is expected to be negligible. We observed the same trends after excluding patients infected with seasonal H1N1 from our population. Of note, A(H3N2) viruses are to date sensitive to both drugs. It remains to be determined to what extent the present results can be extrapolated to susceptible viruses of other subtypes, e.g., H1N1, and in particular to the pandemic H1N1 2009 virus, which displays significant differences in the catalytic pocket of the neuraminidase [17] , or to a mixed viral season with H3N2 and H1N1 cocirculating viruses.
We must acknowledge several limitations to our study. First, this preliminary analysis was conducted on a partial set of data after enrolment of 541 patients instead of the 900 initially planned. However, it is highly unlikely that the lower response of the combination as compared to oseltamivir would have been reversed if all originally planned 900 patients had been enrolled. Second, as previous randomized clinical trials had shown the superiority of each monotherapy as compared to placebo in terms of time to symptom alleviation and viral shedding, it was decided, on the basis of ethical reasons, that the study would not comprise a double placebo arm. Third, the proportion of patients with unavailable viral swab on day 2 was higher in the combination arm. As the missing value equals failure, this may have biased the results in the combination arm towards reduced performance. Indeed, in the analysis of the 414 patients with available day 0 and day 2 nasal swabs, the same trends were observed. Fourth, the virological response was assessed only in one site (nose) and at one time (day 2), which prevents extrapolation of the results to the entire virological response over time and throughout the respiratory tract. However, clinical endpoints completed the picture, giving information on the overall response. Fifth, as mentioned above, day 2 sampling was chosen to show the virological effect. However, this is probably not the best moment to look for resistance emergence induced by drug selective pressure, as it has been shown to occur later in the course of treatment [18] [19] [20] . Nevertheless, we looked for neuraminidase inhibitor resistance using a standard fluorimetric test in the 65 patients with day 2 positive viral culture; none of them carried a resistant virus, except for one patient infected with an H1N1 virus resistant to oseltamivir but susceptible to zanamivir, as were all H1N1 viruses circulating during the study period. However, the absence of resistance at day 2 does not rule out any further (postday 2) resistance selection. Finally, this trial was conducted in adult outpatients, which prevents any extrapolation of the results to adults with severe presentation necessitating hospitalisation, and to children, who usually have more prolonged viral shedding. We chose the outpatient adult population because it seemed to be the most homogenous and the easiest in which to test our hypothesis.
Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower efficiency of the oseltamivir-zanamivir combination found in this study calls for caution in its use in clinical practice. Thus, also considering the superiority of oseltamivir monotherapy over zanamivir monotherapy observed in this trial, oseltamivir should be the recommended primary anti-influenza treatment during influenza seasons with predominant H3N2 viruses naturally susceptible to oseltamivir. These results would need to be confirmed for the 2009 H1N1 pandemic virus and in the coming years, for future circulating influenza viruses.
Table S1 Proportion of false positive and false negative results for various viral load thresholds compared to viral isolation in the sample of GROG patients.  A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs. Abstract Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.
Noroviruses (NoVs) cause sporadic acute nonbacterial gastroenteritis in all age groups [12, 13] . NoVs are divided into five genogroups, GI to GV, of which GI and GII strains mostly affect humans [21] . Recently, NoV genotype GII-4 has been responsible for the majority of sporadic gastroenteritis cases and outbreaks [13] . The NoV genome consists of a single-stranded RNA of about 7.6 kb, organized into open reading frames (ORF 1-3). ORF-1 codes for the RNA-dependent RNA polymerase, and ORF-2 and -3 encode the two structural proteins VP1 and VP2 [10] .
Expression of the capsid VP1 gene by recombinant baculoviruses leads to self-assembly into empty virus-like particles (VLPs) that are morphologically and antigenically similar to native NoV [9] . NoV VLPs are widely used as antigens in diagnostic serological assays and as candidate vaccines against NoVs [2, 7] . NoV VLPs are highly stable and resistant to variable conditions, particularly to low pH [1, 9] .
There are limitations in NoV VLP production in terms of inadequate yield and quality of the VLPs [1, 3, 9, 20] . Both sucrose and CsCl gradients ultracentrifugation have been used for purification of NoV VLPs [1, 7, 9, 17] , even though studies on rotavirus-like particles demonstrated a low yield and impurities resulting from CsCl gradient purification [16] .
In the present study, we compared commonly used methods for NoV GII-4 VLP purification [1, 14, 17] and concentration [6, 19] , considering the purity, yield, morphological integrity, antigenicity and functionality of the purified VLPs.
The steps for cloning the NoV GII-4 (GenBank sequence database accession number AF080551) full-length capsid gene are described elsewhere [11] .
VLPs were produced in Sf9 insect cells infected with the recombinant baculovirus according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Baculovirus titers expressed as the multiplicity of infection (MOI) of the P2 stocks were determined using a BacPak Rapid Titer Kit (Clontech Laboratories, Mountain View, CA).
At day 6, infected cell culture (200 ml) was clarified by centrifugation at 30009g for 30 min at 4°C. VLPs in the supernatant were concentrated by ultracentrifugation (L8-60M ultracentrifuge, Beckman SW-32.1 Ti rotor) at 100,0009g for 2 h at 4°C, and pellets were resuspended in 0.2 M Tris-HCl, pH 7.3. VLPs were loaded onto a 10-60% discontinuous sucrose gradient and ultracentrifugated at 100,0009g for 1 h at 4°C as described before [17] . Fractions were collected by bottom puncture. The fractions containing VLPs were pooled. An additional discontinuous sucrose gradient (35-60%) ultracentrifugation was performed. Sucrose was removed by overnight dialysis against 1 liter of PBS. VLPs were concentrated by dialysis against polyethylene glycol (PEG; 50%) [6] or by ultrafiltration [19] . VLPs were concentrated using an Amicon Ultra 30 kDa centrifuge filter device (Millipore Corporation, Billerica, Germany). VLPs were stored at 4°C in PBS.
Alternatively, a less time-consuming sucrose density gradient purification method was employed [14] . Clarified supernatants were pelleted twice by ultracentrifugation. Pellets were resuspended in 0.2 M Tris-HCl, pH 7.3, and placed on a discontinuous sucrose density gradient (10-60%) for ultracentrifugation at 100,0009g for 16 h at 4°C. The VLP band, which was visible at the 35% sucrose layer, was collected. Sucrose was removed by dialysis against 1 liter of PBS, and VLPs were concentrated by ultracentrifugation at 100,0009g for 2 h at 4°C.
In addition, clarified supernatants were concentrated, and the pellets were resuspended in sterile water. VLPs were sedimented by ultracentrifugation through cesium chloride (CsCl) (0.4 g/ml) at 116,0009g for 18 h at 4°C as described earlier by others [1] . CsCl was removed by dialysis against PBS, and VLPs were concentrated using an Amicon Ultrafilter.
The total protein content of the purified VLP preparation was determined using the Pierce BCA Protein Assay (Thermo Science, Rockford, USA). Endotoxin levels in the VLP preparations were quantified using the Limulus amebocyte lysate (LAL) assay (Lonza, Walkersville, MD, USA). The level of endotoxin was\0.1 EU/10 lg of protein, which is below the international standard of B30 EU/20 lg of protein [15] . All samples were analyzed for protein expression by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The presence of NoV VLPs was verified by electron microscopy (EM). VLP preparations were negatively stained with 3% uranyl acetate (UA), pH 4.6. The VLPs were examined using an FEI Tecnai F12 electron microscope operating at 120 kV.
Binding of GII-4 VLPs to carbohydrate receptors was examined by the histo-blood group antigen (HBGA) binding assay as described by others [18] , with slight modifications. Briefly, VLPs were coated at 50 ng/well, and synthetic biotinylated H type 3 and Lewis b histo-blood group carbohydrates (Lectinity Holdings Inc. Moscow, Russia) were used in serial threefold dilutions, starting from 6 lg/ml. Wells lacking the synthetic carbohydrates were used as a negative control.
The conditions for Sf9 cell infections were optimized in order to obtain a high yield of the VLPs. For recombinant baculovirus P2 stock, an MOI of 1 was found to be optimal, and VLPs were harvested from the supernatant after 6 days of infection. VLPs were further purified by several different procedures, as described in Fig. 1a .
The best yield (2-3 mg/200 ml) was obtained after the purification procedure described in Fig. 1a, panel B. In comparison, the method described in Fig. 1a , panel C, yielded a ten times lower amount of NoV capsid VLPs. The purity of the VLPs obtained by each method was determined by 12% SDS-PAGE and staining of the proteins with Page-Blue (Fig. 1b) . 1 lg of the total protein was loaded into each lane. Each method resulted in equally pure protein bands corresponding to the size of the NoV capsid. Residual PEG present in the VLPs, concentrated by PEG dialysis, may have interfered with the protein concentration determination, and Fig. 1 Purification, concentration and characterization of NoV VLPs. a Several purification procedures (A-D) were used, and the purity, morphology, antigenicity and yield of the VLPs were compared. b NoV capsid protein analysis by 12% SDS-PAGE. c Western blot analysis using a human convalescent serum against norovirus GII-4. d EM of purified NoV VLPs observed at a magnification of 930,000. Bar 100 nm. Samples from purification procedures A, B, C, and D correspond to lanes and panels A, B, C, and D, respectively. M protein weight marker this could explain the lower capsid protein concentration observed (Fig. 1b, lane A) . NoV capsid identity and antigenicity of the VLPs were assayed by western blot using human NoV-specific convalescent sera (Fig. 1c) . The results show pure NoV capsid proteins without degradation and with similar antigenicity for each of the purification methods. Next, we determined the morphological integrity and homogeneity of the VLPs by EM (Fig. 1d) . The VLPs obtained by purification with sucrose density gradients followed by dialysis and either ultrafiltration (Fig. 1d, panel B) or ultracentrifugation (Fig. 1d, panel C) were approximately 38 nm in size, with the classical appearance of NoV capsid VLPs. By contrast, CsCl purification (Fig. 1d, panel D) and concentration of the VLPs by dialysis against PEG (Fig. 1d , panel A) resulted in VLPs of heterogeneous size, which appeared broken and aggregated. The VLPs purified and concentrated by the method schematically presented in Fig. 1a, panel B , were of the best quality and were subjected to further analysis.
To determine the optimal storage conditions and stability of the NoV capsid VLPs, VLPs were treated under different conditions, and the samples were analyzed by SDS-PAGE to test their protein integrity (Fig. 2a) and by EM to examine their morphology (Fig. 2b) . VLPs were stable for at least 12 months at 4°C in PBS, pH 7.4 ( Fig. 2a and b, panel 1) , and at room temperature (23°C) up to 7 days ( Fig. 2a and b,  panel 3) . The VLPs withstood the sterile filtration conditions when 0.22-lm filters (Durapore, Millipore, Ireland) were used. Next, we intentionally disrupted the VLP morphology by heat treatment at 60°C for 1 h (Fig. 2b, panel 4) [1] without degrading the capsid protein (Fig. 2a, lane 4) .
An HBGA binding assay [8, 18] was used to test the functionality of the purified VLPs as well as to determine the significance of the conformational binding sites on the VLP, which would presumable require intact VLPs. A comparison of the binding of VLPs purified by sucrose gradient centrifugation and by CsCl sedimentation, as well as heat-treated VLPs (60°C, 1 h), to synthetic biotinylated H type 3 carbohydrate is shown in Fig. 3 . The binding was clearly dependent on the morphology and preserved structure of the VLPs, with the lowest level of binding observed with the heat-treated VLPs with disrupted conformational binding sites. Lewis b antigen was used as a control in the assay, and this did not bind to any of the VLPs (Fig. 3) .
NoV VLPs have been used extensively to study protein interactions [8] , and virus assembly [17] , and have been used as a tool in diagnostic serological assays [7] . Clinical trials have been performed with the NoV VLPs used as a vaccine [2] . For all these applications, high-quality VLPs would be preferable. In this study, different methods of VLP purification and concentration were used, and the purity, integrity, morphology, antigenicity and functionality of the GII-4 NoV VLP preparations were examined.
VLPs purified by each method (Fig. 1a , methods A-D) had a similar appearance and migration pattern on the SDS-PAGE gel. A western blot with a human convalescent serum from an individual infected with GII-4 confirmed the identity of the capsids and the lack of degradation products.
The reason for the lower protein yield after purification procedure C might have been that a narrow visible band of VLPs was collected from 35% sucrose, causing some of the protein to be omitted, thus affecting the yield. However, a yield of up to 2-3 mg of VLPs, which was obtained by the best purification method described in the present study, is remarkably high when compared to other reports [3, 4] .
EM analysis of the morphological integrity and homogeneity of the VLPs showed that VLPs obtained by purification with sucrose density gradients followed by ultrafiltration or ultracentrifugation (procedures B and C, Carbohydrate concentration (µg/ml)
Sucrose VLPs+H type 3
Sucrose VLPs+Lewis b Heat treated VLPs+H type 3
Heat treated VLPs+Lewis b CsCl VLPs+H type 3
CsCl VLPs+Lewis b Fig. 3 Binding of NoV VLPs to synthetic ABH histo-blood group antigens. Sucrose-purified VLPs (Sucrose VLPs, purified according to procedure B in Fig. 1a) , heat-denatured NoV VLPs (60°C, 1 h) (Heattreated VLPs) and CsCl-purified VLPs (CsCl VLPs) were tested for binding to H type 3 and Lewis b carbohydrates at the indicated concentrations. The pH value in the binding assay was 7.4 respectively) were homogenous and intact, and approximately 38 nm in size. However, CsCl-purified VLPs appeared heterogeneous in size, with a few broken particles, although comparable to the morphology seen by others [7] . CsCl purification is known to introduce several impurities at the end of the process and cause aggregation of the VLPs during storage [5] . The poorest morphology was seen after concentration of the VLPs by dialysis against PEG, which resulted in aggregation. In addition, residual PEG, which leaks through the dialysis membrane, might interfere with further applications of the VLPs [19] .
Our data clearly demonstrate that the purification process affects the integrity of the native quaternary structure of NoV VLPs and, subsequently, the receptor-binding functionality of the VLPs. Although the majority of the VLPs purified by the CsCl method seemed intact in the EM image, the difference from sucrose-density-gradient-purified VLPs in HBGA binding is striking. This result is supported by the recent finding that CsCl has a negative impact on the functionality of VLPs [5] . We also demonstrated that even heat-disrupted VLPs have binding capability, but intact homogenous VLPs have a significantly greater binding intensity. Standardization of the purification method for NoV VLPs used in diagnostic serological assays and blocking assays [8] would greatly strengthen the results obtained.
To the best of our knowledge, this is the first time that VLPs purified by conventional purification and concentration methods were compared in terms of yield, purity, morphological integrity, antigenicity and functionality. The results show that NoV GII-4 VLPs purified twice by sucrose density gradient centrifugation followed by ultrafiltration maintain their icosahedral capsid structure and their capacity to bind HBGAs. Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis PURPOSE: Systemic juvenile idiopathic arthritis is a chronic pediatric disease. The initial clinical presentation can mimic other pediatric inflammatory conditions, which often leads to significant delays in diagnosis and appropriate therapy. SJIA biomarker development is an unmet diagnostic/prognostic need to prevent disease complications. EXPERIMENTAL DESIGN: We profiled the urine peptidome to analyze a set of 102 urine samples, from patients with SJIA, Kawasaki disease (KD), febrile illnesses (FI), and healthy controls. A set of 91 plasma samples, from SJIA flare and quiescent patients, were profiled using a customized antibody array against 43 proteins known to be involved in inflammatory and protein catabolic processes. RESULTS: We identified a 17-urine-peptide biomarker panel that could effectively discriminate SJIA patients at active, quiescent, and remission disease states, and patients with active SJIA from confounding conditions including KD and FI. Targeted sequencing of these peptides revealed that they fall into several tight clusters from seven different proteins, suggesting disease-specific proteolytic activities. The antibody array plasma profiling identified an SJIA plasma flare signature consisting of tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin (IL)-18, regulated upon activation, normal T cell expressed and secreted (RANTES), P-Selectin, MMP9, and L-Selectin. CONCLUSIONS AND CLINICAL RELEVANCE: The urine peptidomic and plasma protein analyses have the potential to improve SJIA care and suggest that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9058-8) contains supplementary material, which is available to authorized users. Clinical Relevance Sensitive and specific diagnostic biomarkers for systemic onset juvenile idiopathic arthritis (SJIA) would allow its differentiation from other febrile illnesses, such as Kawasaki disease (KD) or acute infections (febrile illness (FI)) and facilitate prompt initiation of appropriate treatment at disease onset. Early treatment may reduce the risk of long-term complications and subsequent disabilities. In addition, biomarkers that distinguish intercurrent SJIA flare from infection in patients with known SJIA would be clinically useful, as would markers that predict impending disease flare or responder status to particular therapies, or provide an early indication of a treatment response. Finally, biomarkers may provide clues to unanswered questions concerning SJIA pathogenesis. Our comparative analysis of SJIA, KD, and FI urine peptidomes identified a small subset of the urine peptidome that effectively discriminates SJIA patients in the active, quiescent, and remission disease states, and also discriminates patients with active SJIA from confounding conditions including KD and FI. Urine peptide diagnostic and prognostic biomarkers could be of clinical use, especially for serial sampling of pediatric SJIA patients. Targeted sequencing revealed that these peptide markers fall into several tight clusters indicating SJIA-specific proteolytic events. Plasma cataloging analysis of the normal plasma peptidome shows that at least some of these nested peptide markers originate in the circulation. A customized antibody array was used to compare the plasma abundance of proteins known to be involved in inflammatory and protein catabolic processes, revealing a SJIA flare signature. Taken altogether, the urine peptidomic and plasma protein and peptide analyses suggest a testable model that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites.
Electronic supplementary material The online version of this article (doi:10.1007/s12014-010-9058-8) contains supplementary material, which is available to authorized users. biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites. 
Systemic onset juvenile idiopathic arthritis is a chronic inflammatory disease of childhood characterized by a combination of systemic features [fever, rash, serositis (e.g., pericarditis, pleuritis)] and arthritis. Current diagnosis of SJIA is based solely on clinical findings [1] and requires arthritis, daily fever for at least 2 weeks, and at least one of the following: evanescent erythematous rash, generalized lymph node enlargement, hepatomegaly and/or splenomegaly, or serositis. This makes early diagnosis of SJIA challenging, as its clinical manifestations are similar to other diseases, including malignancy, infection, Kawasaki disease (KD), and other autoimmune or inflammatory disorders. Long-term disease outcome in SJIA is variable. About 50% of patients experience a single episode that resolves. However, the other half experience either polycyclic or non-remitting disease. Sensitive and specific diagnostic biomarkers for SJIA would allow its differentiation from other febrile illnesses, such as KD or acute infections (febrile illness (FI)), and facilitate prompt initiation of appropriate treatment at disease onset. Early treatment may reduce the risk of long-term complications and subsequent disabilities. In addition, biomarkers that distinguish intercurrent SJIA flare from infection in patients with known SJIA would be clinically useful, as would markers that predict impending disease flare or responder status to particular therapies, or provide an early indication of a treatment response. Finally, biomarkers may provide clues to unanswered questions concerning SJIA pathogenesis.
There have been several previous biomarker discovery efforts in SJIA. Initial studies, including ours [2] , attempted to identify early clinical variables that predict long-term outcomes, such as joint damage or functional disability at ≥2 years after disease onset [3] [4] [5] [6] . Studies of serum found elevated cytokines, chemokines, and acute-phase reactants in active SJIA [7] [8] [9] [10] . More recently, transcriptional profiling of peripheral blood mononuclear cells from SJIA subjects with active disease revealed a signature of active SJIA that normalized in association with clinical response to treatment [11, 12] . A single SELDI-based analysis of plasma identified serum amyloid A as a plasma biomarker of disease activity [13] . However, all these efforts fall short of robust diagnostic and prognostic biomarkers with practical clinical utility.
We sought to explore urine as a source of biomarkers. Such markers would permit frequent tests, which would be of use, especially in children, for a chronic pediatric disease with a polycyclic course. A normal adult human excretes 30-130 mg of protein and 22 mg of peptides per day in urine [14, 15] . Urine proteomic analysis has identified more than 1,500 proteins including a large proportion of membrane proteins [16] . Urine peptidomic analysis revealed over 100,000 different peptides [17] . Our own in-depth 2D mass spectra (MS)/MSMS analysis led to the identification of 11,988 different urine peptide sequences from 8,519 unique protein precursors in normal human urine [18] . Recent reviews have indicated that analysis of the urinary proteome/peptidome can be highly informative for both urogenital and systemic diseases and used for disease classification [18, 19] . Naturally processed urine peptides have certain advantages over proteins as biomarkers. The roughly equal mass of protein and peptide in urine translates into at least a tenfold greater molar abundance of peptides. While the urine proteome contains a number of abundant proteins that obscure the lower abundance proteins more likely to be biomarkers, this problem does not complicate analysis of peptides in urine. A one-dimensional HPLC separation is sufficient for the analysis of greater than 25,000 urine distinct peptides.
Among the emerging quantitative proteomics technologies, isobaric tags for relative and absolute quantification (iTRAQ) allows concurrent protein sequence identification and relative quantification of those peptides with known protein sequences in up to eight different biological samples in a single experiment [20] . However, due to its limited throughput and current cost, iTRAQ is not feasible for simultaneous comparison of the large number of disease subjects needed to achieve the discovery of differential features with sufficient statistical power. As an alternative, a label-free liquid chromatography-mass spectrometry (LC-MS)-based approach has been applied as a quantitative biomarker discovery method. The label-free LC-MS approach can compare and quantify peptides with precision and accuracy comparable to those based on isotope labeling [21] . Although LC/ESI mass spectrometry is typically used in label-free quantitative proteomics, matrix-assisted laser desorption/ionization-timeof-flight (MALDI-TOF) mass spectrometry is increasingly being used and demonstrates low average coefficients of variation for all peptide signals across the entire intensity range in all technical replicates [22, 23] . Using the label-free LC/MALDI-TOF profiling approach, we previously discovered candidate urine peptide biomarkers of renal transplant rejection [19, 24] . Subsequent urine peptide biomarker validation [19] by multiple reaction monitoring (MRM) [25, 26] showed significant correlation between the urine peptide measurements obtained from label-free MALDI-TOF and from MRM using stable isotope-labeled synthetic marker analogues to derive absolute quantification.
The label-free LC-MALDI-TOF approach involves the comparison of urine peptidomes of different samples, and thus, multiple LC-MS spectra. However, comparing multiple LC-MS spectra in a label-free analysis is computationally intensive, demanding robust detection of LC-MS peaks, alignment of all LC-MS peaks, and determination of the common peak indices across all assayed samples. The output of data processing is essentially a P X N table in which each of the indexed P peptides has been quantified across the N studied samples. This table, reduced from LC-MS spectra of all samples, can be subjected to downstream statistical learning including transformation, normalization, and unsupervised/supervised analyses suited to the experimental design to mine for a differential subset of the P peptides, which will then be subjected to MSMS protein sequence identification and future quantitative prospective MRM [25, 26] or antibodybased validation.
We identified naturally occurring urine peptides with specificity for active systemic SJIA compared with other sources of fever. We hypothesized that SJIA flare is associated with increased levels of circulating mediators of inflammation that activate catabolic pathways leading to the generation of novel peptide biomarkers that are found in urine. We tested this hypothesis through global LC-MS analysis of urine and plasma peptides as well as targeted analysis of plasma proteins using antibody arrays.
The following reagents were used for the proteomics sample analysis: nanopure or Milli-Q quality water (~18 megohm cm or better); Amicon Ultra centrifugal filtration tubes were obtained from Millipore (Bedford, MA, USA) ammonium bicarbonate, ammonium formate, and formic acid were obtained from Fluka (St. Louis, MO, USA); Tris-HCl, urea, thiourea, DTT, iodoacetamide, calcium chloride, and TFA were obtained from Sigma-Aldrich (St. Louis, MO, USA); HPLC-grade methanol (MeOH) and HPLC-grade ACN were purchased from Fisher Scientific (Fair Lawn, NJ, USA); 2,2,2-trifluoroethanol was obtained from Aldrich Chemical (Milwaukee, WI, USA); and sequencing grade-modified trypsin was purchased from Promega (Madison, WI, USA). Sodium tetraborate, glycine, and picrylsuofonic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Informed consent was obtained from the parents of all patients and assent from all patients >6 years of age. This study was approved by the human subject protection programs at UCSD, UCSF, and Stanford University. Urine samples were obtained from two new onset SJIA disease (ND), 18 active systemic disease plus arthritis (SAF), nine SJIAwith active arthritis (AF), 18 quiescent SJIA on medication (QOM), nine SJIA in remission off medication (RD), and ten healthy control (HC) from Stanford University Medical Center and UCSF. In addition, urine samples were obtained from 23 KD and 23 age-similar FI control patients evaluated for fever at Rady Children's Hospital San Diego. All KD patients had fever and ≥4 of the five principal clinical criteria for KD (rash, conjunctival injection, cervical lymphadenopathy, changes in the oral mucosa, and changes in the extremities) or three criteria plus coronary artery abnormalities documented by echocardiography [27] All FI control, patients had naso-or oro-pharyngeal and stool viral cultures. Urine sample patient demographics are described in Tables 1 (SJIA) and 2 (KD and FI). Plasma samples included 25 SJIA flare (F), 14 SJIA (Q) for the training analysis, and 41 SJIA F and 11 Q for the "bootstrapping" testing analysis. Instead of in silico bootstrapping simulation, samples belonging to different visits of the same patient and even the same samples were assayed, i.e., "bootstrapped" experimentally, for testing. For the bootstrapping testing, a total of 52 SJIA samples were analyzed by the antibody array, where 41 samples were from 19 patients at the time of SJIA flare, and 11 samples were from eight patients at the time of SJIA quiescence. Plasma sample patient demographics are described in Table 4 .
Urine samples (5-10 mL) were collected in sterile tubes and held at 4°C for up to 48 h before centrifugation (2,000×g× 20 min at room temperature) and freezing of the supernatant at −70°C. Urine processing, preparation of peptides, extraction, and fractionation are as previously described [18, 19, 24] . Urinary samples were processed by centrifugal filtration at 3,000×g for 20 min at 10°C through Amicon Ultra centrifugal filtration devices (10 kDa cutoff; Millipore, Bedford, MA) preequilibrated with 10 ml Milli-Q water. The filtrate (urine peptidome) containing the low MW naturally occurring peptides was processed with Waters Oasis HLB Extraction Cartridges (Waters Corporation, Milford, MA) and extracted with ethyl acetate. The resulting urine peptide samples were quantified by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, as previously described [28] . Three nanomoles peptides were injected on a 100 μm×15 cm C18 reverse-phase column (Michrom) and eluted with a gradient of 5% to 55% acetonitrile over 50 min using a Michrom MS4 HPLC. Twenty-second fractions were collected onto MALDI targets with a Probot fraction collector (LC Packings). A total of 100 fractions were collected and analyzed on 4700 MALDI-TOF/TOF (Applied Biosystems) in MS mode. One microliter of matrix solution containing 4.8 mg/ml α-cyano-4-hydroxycinnamic acid (Agilent Technologies, Palo Alto, CA, USA) and 30 fmol/μl glu-fibrinopeptide (Sigma-Aldrich, St. Louis, MO, USA) was automatically deposited by the Probot on each spot.
The plasma peptidome preparation protocol was adapted from that of the urine peptidome analysis. The plasma samples were centrifuged at 3,000×g for 20 min at 10°C through Amicon Ultra centrifugal filtration devices (10 kDa cutoff; Millipore, Bedford, MA) pre-equilibrated with 10 ml Milli-Q water. The retentate (plasma proteome) was washed twice, brought to the final volume of 400 μl with 20 mM Tris-HCl (pH 7.5), and quantitated by the BCA protein assay (Pierce, Rockford, IL). The filtrate (plasma peptidome) containing the low MW naturally occurring peptides was processed with Waters Oasis HLB Extraction Cartridges (Waters Corporation, Milford, MA), and extracted with ethyl acetate. The resulting plasma peptide samples were quantified by the TNBS assay, as previously described [28] . Three nanomoles of peptides were fractionated by two-dimensional chromatography-a SCX column as the first and a RP column as the second dimension, and then subjected to extensive MSMS sequence identification involving a Thermo Finnigan LTQ-FTICR spectrometer.
The ABI 4700 oracle database MS spectra were exported as raw data points via ABI 4700 Explorer software version 2.0 for subsequent data analyses. The m/z ranges were from 800 to 4,000 with peak density of maximum 30 peaks per 200 Da, minimal S/N ratio of 5, minimal area of 10, minimal intensity of 150, and 200 maximum peaks per spot. We previously had developed an informatics platform [18] which contains an integrated set of algorithms, statistical methods, and computer applications, to allow for MS data processing and statistical analysis of LC-MS-based urine peptide profiling. The MS peaks were located in the raw spectra of the MALDI data by an algorithm that identifies sites (mass-to-charge ratio, m/z values) whose intensities are higher than the estimated average background and the~100 surrounding sites, with peak widths~0.5% of the corresponding m/z value. To align peaks from the set of spectra of the assayed samples, we applied linkage hierarchical clustering to the collection of all peaks from the individual spectra. The clustering, computed on a 24-node LINUX cluster, is two dimensional, using both the distance along the m/z axis and the HPLC fractionation time, with the concept that tight clusters represent the same biological peak that have been slightly shifted in different spectra. We then extracted the centroid (mean position) of each cluster, to represent the "consensus" position as the peak index (bin) across all spectra. The normalization of the MALDI-TOF signal intensity for each peptide feature was performed at two steps: (1) within each LC fraction (MALDI plate spot), all peptide peak signal intensities were normalized to the externally spiked reference peptide (30 fmol/μl glu-fibrinopeptide) at each MALDI plate spot; (2) each clustered peptide, with unique m/ z and LC fraction time, was normalized to the total signal intensity of all the clustered peptides within the same sample.
For urine peptidome analysis, we used the approach of ion mapping [29, 30] , whereby biomarker candidate MS peaks were selected on the basis of discriminant analysis, and then targeted for MS/MS sequencing analysis to obtain protein identification.
Extensive MALDI-TOF/TOF and LTQ Orbitrap MS/MS analyses coupled with database searches [29, 30] were performed to sequence and identify these peptide biomarkers. The identity of a subset of peptides detected was determined by searching MS/MS spectra against the Swiss-Prot database (June 10, 2008) restricted to human entries (15,720 sequences) using the Mascot (version 1.9.05) search engine. Searches were restricted to 50 and 100 ppm for parent and fragment ions, respectively. No enzyme restriction was selected. Since we were focusing on the naturally occurring peptides, hits were considered significant when they were above the statistical significant threshold (as returned by Mascot). Selected MS/MS spectra were also searched by SEQUEST (BioWorks™ rev.3.3.1 SP1) against the International Protein Index human database version 3.5.7 restricted to human entries (76,541 sequences). mMASS, an open source mass spectrometry tool (http://mmass.biographics.cz/), was used for manual review of the protein identification and MS/MS ion pattern analysis for additional validation.
Customized antibody arrays, consisting of pairs of capture and detection antibodies against 43 proteins, were utilized (SDF-1), IGF1, IFNG, IGFBP3,  IGFBP4, IGFBP6, IL-1A, IL-1B, IL-1R1, IL-2, IL-4, IL-5 . Antibody array fabrication, processing, data extraction and analysis were performed as previous described [31] .
Patient demographic data were analyzed using "Epidemiological calculator" (R epicalc package, version 2.10.0.0). The binned LC-MALDI MS peak data obtained for all urine peptidome samples were analyzed for discovery of discriminant biomarkers using algorithms [32] of nearest shrunken centroid (NSC) for biomarker feature selection, tenfold cross-validation analyses, and Gaussian linear discriminant analysis (LDA) for classification analyses. To control the number of false significant features found during NSC mining, we permuted the data set 500 times to calculate GFDR [33] . To quantify the difference between classes for the identified peptide biomarkers, Student's t test and Mann-Whitney U tests were used for hypothesis testing, and local false discovery rate (FDR) [34] tool was used to correct multiple hypothesis testing. In order to test whether the selected discriminated features could serve as a diagnostic biomarker panel, a logistic regression model was used to find a linear combination of the biomarkers that minimizes the total classification error. In order to avoid bias in data sets, we utilized a bootstrapping technique to bootstrap 500 times to evaluate the impact of the data construction on overall classification performance of the biomarker panel. For each of the bootstrapping sets, we used the LDA-derived prediction scores for each sample to construct receiver operating characteristic (ROC) curves [35, 36] . To summarize the results, the vertical average of the 500 ROC curves was plotted, and the boxes and whiskers were used to describe the vertical spread around the average.
We collected 56 intraday urine samples from pediatric SJIA patients at two sites, Stanford and UCSF (Tables 1, 2, and 3). These included patients with ND (n=2), SAF (n=18), AF (n=9), SJIA quiescence (inactive disease on medication; QOM, n=18), SJIA remission (inactive disease off all medications) (RD, n=9). For comparison, samples from subjects with KD (n=23), and acute FI (n=23) ( Table 2; collected at UCSD), and healthy, age-matched controls (HC, n=10, collected at Stanford) were collected.
We also collected 66 plasma samples from pediatric SJIA patients (Tables 4 and 5 ). These included patients with SAF (n=25) and (n=14). If available, plasma samples from multiple visits, considered as experimentally "bootstrapped" samples, of the same SJIA patient at different disease states were also collected for confirmatory analyses using bootstrapping. Thirteen patients provided both urine and plasma samples.
As expected, based on known differences in demographics [37] , there were differences in the age and gender distribution of our SJIA and the KD and FI urine subjects. Except for ND patients (median age, 3 years; range, 1-5 years), the SJIA patients (SAF: median age, 12.5 years; range, 3-17 years; AF: median age, 13 years; range, 11-16 years; QOM: median age, 13 years; range, 5-17 years; RD: median age, 14 years; range, 6-21 years) are older than KD (median age, 3 years; range, 1-10 years) and FI (median age, 2 years; range, 1-10 years) patients. Except for ND patients (100% male), there are fewer male SJIA patients (SAF, 33% male; AF, 33% male; QOM, 39% male; RD, 22% male) than KD (82% male) and FI (61% male) patients. As expected, active Table 5) .
Mass spectrometry-based urine peptidomics analyses suffer from two major sources of variance [18] : analytical issues including mass spectrometric ion suppression; and biological issues including dilution of urine by different hydration states of the urine donors. To standardize amount of urine peptides for comparative analysis, we have quantified each extracted urine peptidome by the TNBS assay [28] and 3 nmol of peptides were subjected to the downstream LC-MALDI-TOF profiling analysis. The initial step in our biomarker discovery effort was to collect urine peptide spectra by LC-MALDI-TOF profiling from the 56 urine samples. The MS spectrum of each HPLC fraction was analyzed by "MASS-Conductor" software (Ling, unpublished) , which extracts peaks from raw MALDI spectra, enables common peak alignment, generates consensus representative peaks across all spectra via two-dimensional hierarchical clustering of both mass/ charge and the HPLC fractions, and normalizes peak signal measurements.
To discover an SJIA systemic flare signature, the urine spectra of the subjects with systemically active disease, SJIA ND (n=2), and SAF (n=18) patients, were compared simultaneously to the non-systemic group of SJIA AF (n=9) and the QOM (n=18) patients and the other systemic inflammation groups: KD (n=23) and FI (n=23). The data mining process included selection of the discriminative urine "Bootstrapping" differing from in silico bootstrapping (re-sampling) simulation, samples belonging to different visits of the same patient and even the same samples were assayed, i.e.
"bootstrapped" experimentally peptides, supervised classification, bootstrapping, and ROC analysis, as outlined in Fig. 1 .
Classifier discovery and feature selection by the NSC algorithm [32] were performed using all the features in the data set. NSC algorithm iteratively shrinks the standardized class mean of the abundance for each peptide. Eventually all urine peptides were ranked by the difference between the shrunken class means. Tenfold internal cross-validation analysis and LDA led to the discovery of a biomarker panel of 17-urine-peptide biomarkers effectively differentiating SJIA flare (ND and SAF) from contrasting group of AF, QOM, RD, KD, and FI samples. Extensive MALDI-TOF/ TOF and LTQ Orbitrap MS/MS analysis coupled with database searches [29, 30] were then performed to identify these peptide biomarkers.
As shown in Tables 6 and 7, the 17-peptide biomarkers were found to be degradation products of eight different proteins: alpha1 antitrypsin (A1AT, two peptides having overlapping sequences), collagen type I alpha 1 (COL1A1; five peptides and three of them having overlapping sequences), collagen type I alpha 2 (COL1A2; one peptide), collagen type III alpha 1 (COL3A1; one peptide), collagen type IX alpha 2 (COL9A2; one peptide), fibrinogen alpha (FGA; two peptides having overlapping sequences), fibrinogen beta (FGB; two peptides having overlapping sequences), and uromodulin (UMOD; three peptides having overlapping sequences). Sequence alignment of these peptide biomarkers revealed tight sequence clusters for A1AT-, COL1A1-, FGA-, FGB-, and UMODderived biomarkers. The Mann-Whitney U tests (Table 6) were performed to evaluate the significance of discriminations "Bootstrapping" differing from in silico bootstrapping (re-sampling) simulation, samples belonging to different visits of the same patient and even the same samples were assayed, i.e. "bootstrapped" experimentally (Table 7) Fig. 2 Evaluation of the 17-urine-peptide biomarker panel as a classifier of SJIA versus systemic inflammation from Kawasaki disease or acute febrile illness. a A logistic regression model was used to find a panelbased algorithm that minimizes the total classification error discriminating SJIA systemic disease from inflammation due to KD/FI. The maximum estimated probabilities for each of the wrongly classified samples, are labeled with arrows. b A modified 2×2 contingency table shows the percentage of classifications that agreed with clinical diagnosis. c The discriminant analysis-derived prediction scores for each sample were used to construct a receiver operating characteristic (ROC) curve; 500 testing data sets, generated by bootstrapping, from the SJIA systemic flare, KD, and FI data were used to derive estimates of standard errors and confidence intervals for our ROC analysis. The plotted ROC curve is the vertical average of the 500 bootstrapping runs, and the box and whisker plots show the vertical spread around the average. d Distribution of the standardized ROC AUC values of the 500 falsely discovered panels upon the 500 class-label permutated data set of the cohort of SJIA F and KD/FI urine peptidomes. Examining all the 500 falsely discovered biomarker panel ROC AUC values, the number of falsely discovered same-size panels that have ROC AUC values greater than that of the original urine biomarker panel (represented by the red vertical line) dividing the total number of the "falsely discovered" biomarker panels led to the estimation of false discovery rate FDR Seventeen-Urine-Peptide Biomarker Panel Effectively Discriminates SJIA Flare from KD and FI In order to test whether the 17-peptide biomarkers could collectively serve as a diagnostic biomarker panel, a logistic regression model was then used to find a linear combination of the 17-peptide biomarkers that minimizes the total classification error discriminating SJIA systemic ND and SAF patients from KD and FI patients. Figure 2a plots the linear discriminant probabilities of the peptide biomarker panel. Samples had good separation between the highest and next highest probability for the classification. Seventeen of the 20 SJIA flare and all 46 non-SJIA (KD and FI) patients were correctly classified. The maximum estimated probabilities for each of the wrongly classified samples, are labeled with arrows. A modified 2×2 contingency table (Fig. 2b) To evaluate the performance of our peptide panel for separating SJIA flare from KD and FI, we used the discriminant analysis-derived prediction scores for each sample and constructed ROC curves [35, 36] . In addition, we utilized bootstrapping, a re-sampling technique to construct multiple-testing data sets to further evaluate the classification performance of the 17-urine-peptide biomarker panel. Figure 2b summarizes the 500 bootstrapping runs of the SJIA systemic flare, KD, and FI samples to derive the estimates of standard errors and confidence intervals for our ROC analysis. The plotted ROC (Fig. 2c) curve is the vertical average of the 500 bootstrapping runs, and the boxes and whiskers plot the vertical spread around the average. The ROC analysis yielded an averaged area under the curve (AUC) value of 0.999, indicating high performance.
We next sought to determine whether the panel of the 17urine-peptide biomarkers could serve as a flare signature to discriminate SJIA flare samples from samples of patients at QOM and RD. A logistic regression model was used to find a linear combination of the 17-urine-peptide biomarkers to minimize the total classification error, classifying patients of SJIA systemic flare from QOM and RD. Figure 3a plots the linear discriminant probabilities of the peptide biomarker panel. Samples had good separation between the highest and next highest probability for the classification. Eighteen of 20 SJIA flare (90%) and all 27 SJIA quiescent or remission patients were correctly classified. The maximum estimated probabilities for each of the wrongly classified samples are labeled with arrows. A modified 2×2 contingency table (Fig. 3b) shows the percentage of classifications that agreed with clinical diagnosis. Overall, the 17-urinepeptide biomarker panel classified the SJIA flare samples with 90% positive agreement with the clinical diagnosis and quiescent or remission samples with 100% agreement with the clinical diagnosis (P=4.16×10 −11 ). Figure 3c summarizes the 500 bootstrapping runs of the SJIA systemic flare, quiescent and remission samples to derive the estimates of standard errors and confidence intervals for our ROC analysis. The plotted ROC (Fig. 3c) curve is the vertical average of the 500 bootstrapping runs, and the boxes and whiskers plot the vertical spread around the average. The ROC analysis yielded an AUC value of 0.998.
In the ROC analyses of the 17-urine-peptide biomarker panel for discriminating SJIA F versus QOM/RD or SJIA F versus KD/FI, bootstrapping (a re-sampling technique) was used to avoid bias due to the presence of outliers in our assayed samples. In both cases shown in the ROC plots, the ROC analyses (Figs. 2 and 3c) yielded a significant AUC, indicating the ROC curve was not affected significantly by the bootstrapping process and demonstrating the robustness of our 17-urine-peptide biomarker panel in discriminative analyses.
As observed in other high throughput analyses, e.g., microarray expression profiling, where the number of profiled features greatly exceeds that of the assayed samples, concurrent analysis of MALDI-TOF spectral peaks to evaluate null hypotheses for differential urine peptide biomarkers leads to the multiple-testing problem. To address the multiple-hypothesis testing problem, we estimated the FDR in concurrent statistical tests of peptide panels, of the same size as our biomarker panel; multiple permutated "random" training data sets were constructed. The class labels of our training samples in either cohorts of SJIA F and QOM/RD, or cohorts of SJIA F and KD/FI, were permutated 500 times such that each time every sample would be randomly assigned a new class label (SJIA F or QOM/RD in F and QOM/RD discrimination; SJIA F or KD/FI in SJIA F and KD/FI discrimination). For each of the 500 simulated "training" sets, NSC algorithm was applied to rank all the MALDI-TOF spectral peak features based upon their ability to discriminate the binary classes: SJIA F versus QOM/RD; and SJIA F versus KD/ FI, respectively. The NSC-selected top 17-peak features were then designated as the "panel" for LDA analysis. ROC analysis subsequently was used to calculate the AUC for this "falsely discovered panel". The AUC values of the 500 falsely discovered panels were standardized, and the density distribution was plotted in Figs. 2 and 3d. FDR was calculated as the number of AUC values greater than that of our 17-urine-peptide panel divided by the total number of AUC values of the "falsely" discovered panels. As shown in Figs. 2 and 3d , the FDRs of our urine peptide biomarker panel are estimated as 0.2% in SJIA F versus QOM/RD discrimination, and 0.2% in SJIA F versus KD/ FI discrimination respectively. These results support the notion that the discovery of our peptide biomarker panel is Fig. 3 Evaluation of the 17-peptide biomarker panel as a classifier of active SJIA versus inactive (quiescent or remitted) SJIA. a A logistic regression model was used to find a panel-based algorithm that minimizes the total classification error discriminating active systemic SJIA from inactive SJIA. The maximum estimated probabilities for each of the wrongly classified samples, are labeled with arrows. b A modified 2×2 contingency table shows the percentage of classifications that agreed with clinical diagnosis. c The discriminant analysis-derived prediction scores for each sample were used to construct a receiver operating characteristic (ROC) curve; 500 testing data sets, generated by bootstrapping, from the SJIA systemic flare, and inactive SJIA data were used to derive estimates of standard errors and confidence intervals for our ROC analysis. The plotted ROC curve is the vertical average of the 500 bootstrapping runs, and the box and whisker plots show the vertical spread around the average. d Distribution of the standardized ROC AUC values of the 500 falsely discovered panels upon the 500 classlabel permutated data set of the cohort of SJIA F and QOM/RD urine peptidomes. Examining all the 500 falsely discovered biomarker panel ROC AUC values, the number of falsely discovered same-size panels that have ROC AUC values greater than that of the original urine biomarker panel (represented by the red vertical line) dividing the total number of the "falsely discovered" biomarker panels led to the estimation of false discovery rate FDR unlikely to be the outcome of chance in multiple hypothesis testing.
Direct Sequencing and Cataloging of Naturally Occurring, Normal Plasma Peptides Revealed Nested COL1A1, FGA, and FGB Peptides That Are Related to SJIA Urine Peptide Biomarkers
We reasoned that, at least, some of the SJIA urine peptide biomarkers, such as FGA peptide biomarkers, are likely filtered from the circulation into urine. To explore this, we fractionated normal plasma by two-dimensional chroma-tography to extract the naturally occurring peptides for MSMS peptide sequencing. Similar to other plasma peptide direct sequencing efforts [38] , we observed FGA peptide clusters. We found seven FGA peptide clusters in plasma (Electronic Supplementary Table 1) , and our urine peptide biomarkers FGA (20-38) 1,536.69, FGA (605-628) 2,560.2, and FGA (605-629) 2,659.24 were observed in plasma FGA peptide clusters I and VII. We did not detect FGA (605-621) 1,826.80, although this peptide was found in a published plasma peptidome sequencing effort [38] . Urinary peptide FGA (607-622) Table 8 Identification of peptides found in normal plasma that are related to SJIA urine peptide biomarkers previously published one [38] . The urinary FGA peptides found in active SJIA samples (Table 8 ) lack one or two Cterminal residues compared with a related plasma peptide and thus appear to derive from exopeptidase activity. The urinary COL1A1 peptide that is most differentially expressed in active SJIA (Table 8) extends beyond the C terminus of a closely related peptide found in normal plasma, suggesting it may be generated by inhibition of normal protease activity during SJIA. A urinary FGB peptide found in SJIA (at similar levels in KD, but not comparison groups with less systemic inflammation) is identical to a peptide found in normal plasma, suggesting that the precursor protein is increased during inflammation.
Our data indicate that at least some SJIA urine peptide biomarkers likely originate in circulation and are filtered into the urine.
Identification of TIMP1, IL18, RANTES, P-Selectin, MMP9, and L-Selectin as SJIA Plasma Flare Biomarkers
Fibrinogen is degraded by MMP9 [39] [40] [41] , and the fibrinogen degradation fragments have been shown to be biologically important molecules with numerous proinflammatory actions [42] . We reasoned that the generation of the SJIA urine biomarker peptides, including those derived from fibrinogen, may be an outcome of the actions of inflammatory mediators on protease expression and regulation, generating a disease-specific degradation pattern of source proteins, such as fibrinogen.
To explore this hypothesis, we utilized an antibody array, consisting of pairs of capture and detection antibodies against 43 proteins of chemokines and cytokines, protein catabolism regulators, and cell surface molecules involved in leukocyte adhesion, to profile and compare the F and Q plasma samples (demographics shown in Tables 4 and 5 ). Our training data set derived from plasma samples from 25 patients at the time of SJIA systemic flare and 14 patients at the time of quiescence. Classifier discovery and feature selection by a nearest shrunken centroid (NSC) algorithm [32] was performed with all the 43 proteins. Ten fold internal cross-validation analysis led to the discovery of a candidate flare signature consisting of six proteins: TIMP1, IL-18, RANTES, P-Selectin, MMP9, and L-Selectin (Fig. 4a) .
We used the NSC algorithm to derive shrunken class means of biomarker protein abundance and gauged the relative quantity of each plasma protein in the SAF and QOM samples to assess the relative resolving power of each biomarker (Fig. 4a) . To validate the antibody array observations, TIMP1 and MMP9 concentrations in SJIA plasma were also determined using enzyme immunometric assay kits from RayBiotech, Inc (Norcross, GA; data not shown). All of the SJIA flare biomarker proteins were found at higher levels in plasma at SJIA flare state. The LDA classification results were used to calculate the percentage of classification that agreed with clinical diagnosis, as shown in a modified 2×2 contingency table (Fig. 4b, left panel) . The six-protein biomarker panel classified the SJIA flare samples with 92% positive agreement and the non-flare samples with 71.4% agreement (Fig. 4c, left panel) 
To assess the performance of the peptide biomarker panel in the classification of "unknown" samples, we carried out an experimental bootstrapping approach. Instead of in silico bootstrapping simulation, samples belonging to different visits of the same patient and even the same samples were assayed, i.e., "bootstrapped" experimentally, for testing. For the bootstrapping testing, a total of 52 SJIA samples (demographics shown in Tables 4 and 5) were analyzed by the antibody array where 41 samples were from 19 patients at the time of SJIA flare, and 11 samples were from eight patients at the time of SJIA quiescence. Figure 4b plots the linear discriminant probabilities of the peptide biomarker panel for the training (left) and bootstrapping data (right); in both cases, samples had good separation between the highest and next highest probability for the classification.
Our six-biomarker panel classified blindly the bootstrapping samples with 87.8% agreement with the clinical diagnosis for the flare samples and 81.8% agreement for the quiescent samples (Fig. 4c , right panel) (P=2.4×10 −5 for the bootstrapping test). Based upon the discriminant analysis-derived prediction scores for each sample, we constructed ROC curves [35, 36] to evaluate the performance of our plasma protein panel for distinguishing flare from quiescence samples. Figure 4d summarizes the 500 bootstrapping runs of the assayed SJIA flare and quiescent samples to derive the estimates of standard errors and confidence intervals for our ROC analysis. The ROC analysis yielded an AUC values of 0.922 for the training Fig. 4 Identification of six plasma proteins as a SJIA plasma flare panel. a All of the six plasma biomarker proteins are of higher abundance in SJIA flare. Relative abundance: the nearest shrunken centroid values [32] have been utilized to represented the relative abundance of biomarkers in either SJIA F or Q patient class. b A logistic regression model was used to find a panel-based algorithm that minimizes the total classification error discriminating SJIA F from Q. The maximum estimated probabilities for each of the wrongly classified samples, are labeled with arrows. c A modified 2×2 contingency table shows the percentage of classifications that agreed with clinical diagnosis. d The discriminant analysis-derived prediction scores for each sample were used to construct a receiver operating characteristic (ROC) curve; 500 testing data sets, generated by in silico bootstrapping, from the SJIA F and Q, both the training and the experimentally bootstrapped, data were used to derive estimates of standard errors and confidence intervals for our ROC analysis. The plotted ROC curve is the vertical average of the 500 bootstrapping runs, and the box and whisker plots show the vertical spread around the average Fig. 4d left panel) and 0.907 for the bootstrapping testing ( Fig. 4d right panel) , respectively.
Urine based proteomic profiling is a novel approach that may lead to the discovery of non-invasive biomarkers for diagnosing patients with different diseases, with the aim to ultimately improve clinical outcomes [18] . Given new and emerging analytical technologies and data mining algorithms, the urine peptidome has become a rich resource for the discovery of naturally occurring peptide biomarkers. For pediatric diseases, urine is expected to become one of the most useful body fluids in clinical proteomics for diagnosis and risk-stratification. Mass spectrometry-based urinary protein and peptide profiling has led to the discovery of highly informative biomarkers for both urogenital and non-urogenital diseases [43] . At the current time, urine proteomics have been applied primarily to diseases affecting the kidney and urinary tract. Our focus on changes in urine that reflect systemic inflammation is novel and potentially of broad use [18] . One of our long-term goals is to use urine biomarkers to develop clinical tests that are non-invasive and feasible for frequent sampling and determination. With this in mind, urine peptidomes from SJIA patients were profiled to identify naturally processed urine peptide biomarkers and 17-urine peptides emerged as a candidate SJIA flare panel. This panel was found to be robust using statistical analyses. Nonetheless, the panel requires validation using a new sample set of sufficient size, guided by power analysis.
The panel discovered in this study appears capable of discriminating patients with active SJIA from those with quiescent or remitted disease. Similar to other molecular changes, such as plasma protein profiles [44] , this urine peptide panel may detect incipient SJIA disease activity prior to clinical evidence of disease. In order to offer a significant clinical advantage to justify routine monitoring of urine biomarkers, a test would have to be more sensitive than the history and physical exam at predicting impending flare, and would need to predict those disease flares which do not self-resolve and therefore require escalation of medical therapy. Serial evaluation of urine samples from SJIA subjects using MRM analysis will be performed to test this hypothesis.
The urine peptide panel also identifies subjects with active SJIA when compared with those with KD and FI. Our ability to discriminate between SJIA patients and other acute systemic inflammatory conditions is promising for future development of diagnostic tools. However, the SJIA patients in this study, except for the two new onset patients, are older than KD and FI patients. Collagens, bone growth and other connective tissue production may differ substantially. Therefore, development of diagnostic markers discriminating new onset SJIA from confounding acute inflammatory diseases, e.g., KD and FI, requires agematched subjects. To continue the discovery efforts and to validate the current biomarker panel, we plan to assemble a larger cohort of new onset SJIA. Another potential utility of the SJIA urine biomarker panel is to distinguish SJIA flare from infection in a patient with known SJIA, as these scenarios require different therapeutic responses. Urine samples from cohorts of both SJIA flare and SJIA patients with known infection will be assembled to validate the urine peptide biomarker panel revealed from this study.
The parent proteins of the urine peptide biomarkers can be found in the circulation or kidney. For example, A1AT, FGA, and FGB are all acute-phase plasma proteins, which are synthesized by hepatocytes and megakaryocytes [45] and are found at high levels in the circulation in the setting of acute or chronic inflammation. Increased fibrinogen and fibrin deposition within joints are prominent indicators of active SJIA flare [46] and arthritis [47] . We hypothesize that the SJIA urine peptide biomarkers may result from changes in the concentrations of inflammatory mediators and protein catabolism regulators, altering the levels of peptides that are ultimately filtered into urine or generated in the urinary tract from local protease activity. The consequence is the generation of a disease-specific molecular phenotype in SJIA urine. In support of this model, we and others [38] have found urine FGA peptide biomarkers in plasma. This would suggest that the FGA urine peptide biomarkers are likely to be present in circulation. Future prospective studies are needed to determine whether the plasma A1AT, FGA, and FGB peptides have diagnostic or prognostic value in SJIA disease management. However, UMOD protein is not derived from blood, but is produced by the thick ascending limb of the loop of Henle in kidney. Our plasma analysis failed to find any UMOD peptides, suggesting that UMOD peptide biomarkers are coming from kidney. The differential abundance of UMOD urine peptide biomarkers in SJIA suggests that SJIA is likely to have an impact on normal kidney function. Renal disorders in SJIA patients are not well characterized. One 9-year-old SJIA patient was characterized by an aggressive disease course and developed renal amyloidosis just 2 years after the disease onset [48] . A variety of renal disorders can occur in patients with rheumatoid arthritis (RA), which may due to the underlying disease. The most common disorders associated with RA are membranous nephropathy, secondary amyloidosis, a focal, mesangial proliferative glomerulonephritis, rheumatoid vasculitis, and analgesic nephropathy [49, 50] . It is unclear whether SJIA directly affects renal function or indirectly causes renal inflammation.
It would have been of interest to assess changes in total protein or peptide excretion in our tested SJIA and contrasting KD/FI samples. Amounts of inflammatory proteins excreted in urine can change dramatically, and febrile states have often been associated with increased protein excretion [51, 52] . Future characterization of the SJIA and KD/FI urine proteomes can help determine whether disease-related changes in total of protein excretion explain the changes in urine peptide profiles we observe in SJIA. Disease-specific alterations of gene transcription in the affected tissue and change in the balance of proteolytic and anti-proteolytic activities in urine, as we have proposed previously [18] , may also contribute to the altered pattern of urine peptides in SJIA. To further explore the possible underlying mechanisms related to urine peptide generation, we used an antibody array with 43 proteins known to be involved in the inflammation of SJIA, including certain proteases and their regulators. Plasma profiling of SJIA flare and quiescent samples using this antibody array identified a biomarker panel of TIMP1, IL-18, RANTES, P-Selectin, MMP9, and L-Selectin, all of which are present at higher abundance in SJIA flare than in quiescence. Given that fibrinogen is a substrate of MMP9 [39] [40] [41] , it is possible that up regulation of MMP9 and TIMP1 in circulation may be directly associated to the generation of FGA peptide biomarkers that ultimately are enriched in urine during active SJIA. It has been shown that treatment of active rheumatoid arthritis with golimumab (human monoclonal antibody to TNF-α) plus methotrexate significantly decreases serum IL-18, E-selectin, TIMP1 and MMP9 levels [53] . IL-18 also has been reported to be a candidate for a key cytokine in the pathogenesis of SJIA [54] . Notably, IL-18 synthesis is increased in SJIA, but not in KD [55] , indicating that there are differences in the inflammatory milieu in these (sometimes clinically similar) diseases. Such differences may explain the differences in expression of the FGA peptide biomarkers in urine between SJIA flare and Kawasaki disease. The observation of P/L-Selectins as part of the plasma biomarker panel suggests that P/L-Selectin-mediated leukocyte migration might be important in SJIA pathogenesis, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into inflammatory foci. Previously analysis [56] of rheumatoid arthritis (RH)-specific collagen breakdown products indicates that RH-specific fragments are formed locally in synovial fluids during diseases process and then released into the circulation. It is likely that the SJIA urine peptide biomarkers, in a similar formation mechanism as RH-specific collagen degradation products, originate due to local inflammation, and then are released in the circulation, which are ultimately enriched and ended in urine.
Together, urine peptidomics and targeted plasma profiling revealed a urine biomarker panel of 17-urine peptides and a plasma biomarker panel of six plasma proteins as SJIA flare signatures. Shown in Fig. 5 , our integrated analyses suggest that the differential abundance of urine peptides in SJIA urine may be an outcome of both the pathophysiological changes initiated by IL-18 and RANTES and P/L-Selectinmediated inflammatory responses and the function of leukocyte-derived TIMP1/MMP9; the latter would influence protein catabolism in SJIA. The inflammatory cytokines may also directly affect levels of expression of substrate proteins and influence levels of expression of peptide derivatives of these proteins. Evaluation of urine peptide profiles in future prospective studies will test the robustness and diagnostic/prognostic values of these urine peptide biomarkers and may provide new insights into SJIA pathogenesis. Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely α-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.  Translation Elongation Factor 1A Facilitates the Assembly of the Tombusvirus Replicase and Stimulates Minus-Strand Synthesis Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication. Genome-wide screens for host factors affecting RNA virus infections have led to the identification of several hundreds host proteins in recent years [1, 2, 3, 4, 5, 6, 7] . These works demonstrated complex interactions between the host and plus-stranded (+)RNA viruses, the largest group among viruses. (+)RNA viruses contain relatively small genomes and greatly depend on the resources of the infected hosts in many steps during the infection process. These viruses recruit numerous host proteins to facilitate their replication and spread [8, 9, 10] . Many host RNA-binding proteins have been implicated in replication of (+)RNA viruses, including ribosomal proteins, translation factors and RNA-modifying enzymes [8, 9, 10, 11, 12, 13, 14] . In spite of the extensive effort, the actual function of host factors in (+)RNA virus replication is known only for a small number of host factors [8, 10, 15, 16, 17] .
Tomato bushy stunt virus (TBSV) and other tombusviruses are model plant RNA viruses with 4.8 kb genomic (g)RNA coding for two replication proteins, termed p33 and p92 pol , and three proteins involved in cell-to-cell movement, encapsidation, and suppression of gene silencing [18, 19] . Yeast (Saccharomyces cerevisiae) expressing p33 and p92 pol replication proteins can efficiently replicate a short TBSV-derived replicon (rep)RNA [20, 21] . The tombusviral repRNA plays several functions, including serving as a template for replication and as a platform for the assembly of the viral replicase complex [19, 22, 23] . The viral RNA also participates in RNA recombination [6, 18, 24] , which likely plays a major role in virus evolution.
One of the major advantages of studying TBSV replication is the availability of genomic and proteomic datasets on virus-host interactions [4, 5, 6, 7, 10, 15, 25, 26, 27] . For example, systematic genome-wide screens of yeast genes have revealed that TBSV repRNA replication is affected by over 100 different host genes [5, 7] . Additional genome-wide screens with TBSV also identified ,30 host genes affecting TBSV RNA recombination [4, 6, 28] . The identified host genes code for proteins involved in various cellular processes, such as translation, RNA metabolism, protein modifications and intracellular transport or membrane modifications [3, 5, 7] .
Additional global approaches based on the yeast proteome microarray (protein array) have led to the identification of over 100 host proteins that interact with viral RNA or the viral replication proteins [25, 26] . Also, proteomics approaches with the highly purified tombusvirus replicase has determined at least seven proteins in the complex, including the viral p33 and p92 pol , the heat shock protein 70 chaperones (Hsp70, Ssa1/2p in yeast), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, encoded by TDH2 and TDH3 in yeast), pyruvate decarboxylase (Pdc1p), Cdc34p ubiquitin conjugating enzyme [14, 26, 27] and eukaryotic translation elongation factor 1A (eEF1A) [25] . The functions of GAPDH and Hsp70 have been studied in some detail [14, 29, 30, 31] , but the roles of the other host proteins, such as eEF1A, in the replicase complex are currently undefined. eEF1A is a highly abundant cellular protein with a role in delivering aminoacyl-tRNA to the elongating ribosome in a GTPdependent manner. Many additional functions have been ascribed to eEF1A including quality control of newly produced proteins, ubiquitin-dependent protein degradation, and organization of the actin cytoskeleton [32, 33] . Although eEF1A has been shown to be part of replicase complexes of several RNA viruses [16, 34, 35, 36] , studies on determining its functions in virus replication are hindered by several major difficulties. These include (i) genetic redundancy: yeast has two eEF1A genes (TEF1 and TEF2), whereas animals and plants have 2-7 genes and several isoforms of eEF1A. (ii) eEF1A provides essential functions for cell viability and mutations could have pleiotropic effects on protein translation, actin bundling and apoptosis. (iii) eEF1A is a very abundant protein that constitutes 1-5% of total cellular proteins, making it difficult to completely remove eEF1A from biochemical assays using cell extracts. (iv) eEF1A is also required for the translation of viral proteins in infected cells, making it difficult to separate its effect on translation versus replication, processes that are interdependent.
The first evidence that translation elongation factors, such as EF-Tu and EF-Ts, play a role in (+)RNA virus replication was obtained with bacteriophage Qbeta [34] . The eukaryotic homolog of EF-Tu, eEF1A was found to bind to many viral RNAs, including the 39-UTR of Turnip yellow mosaic virus (TYMV) [37] , West Nile virus (WNV), Dengue virus, Tobacco mosaic virus (TMV) and Turnip mosaic virus (+)RNA [35, 38, 39, 40] . In addition, eEF1A has also been shown to interact with various viral replication proteins or the replicases, such as the NS5A replication protein of Bovine viral diarrhea virus (BVDV) [41] , NS4A of hepatitis C virus (HCV) [42] , the TMV replicase [43] , and the Gag polyprotein of HIV-1 [44] . It is also part of the replicase complex of vesicular stomatitis virus, a negative-stranded RNA virus [45] .
The actual biochemical functions provided by eEF1A for (+)RNA virus replication are currently poorly understood. In case of WNV, eEF1A is co-localized with the WNV replicase in the infected cells and mutations in the WNV (+)RNA within the mapped eEF1A binding site have led to decreased minus-strand synthesis [46] . On the contrary, eEF1A was shown to enhance translation but repressed minus-strand synthesis of TYMV in vitro [37, 47, 48] . Overall, eEF1A likely plays a role in the replication of many RNA viruses. The interactions of eEF1A with viral RNAs and viral replication proteins and its high abundance in cells might facilitate recruitment of eEF1A into virus replication. eEF1A has been shown to interact with the components of the tombusvirus replicase, including the 39-UTR of the repRNA, as well as the p33 and p92 pol replication proteins [25] . eEF1A is also known to interact with the yeast Tdh2p (GAPDH) [49] , which is also a component of the tombusvirus replicase. Overall, the multiple interactions of eEF1A with various components of the tombusvirus replicase could be important for eEF1A to regulate yet unknown functions of the viral replicase complex.
In this paper, we characterized the functions of eEF1A in TBSV replication based on identification of functional eEF1A mutants in yeast as well as using in vitro approaches. The obtained data support the model that eEF1A plays several roles during TBSV replication, including facilitating the assembly of the viral replicase complex. Moreover, using in vitro replication assays, we demonstrate that eEF1A enhances minus-strand synthesis via stimulating the initiation step of the viral RNA-dependent RNA polymerase. Since eEF1A is also associated with several other viral replication proteins or binds to viral RNAs, it is possible that the uncovered functions of eEF1A might be utilized by other RNA viruses during their replication as well.
To determine the functions of eEF1A during tombusvirus replication, we generated ,6,000 yeast strains expressing eEF1A with random mutations (see Fig. S1A ) and tested the level of TBSV repRNA accumulation in a high-throughput assay [50] . In this assay, we used yeast strains, in which the two wt eEF1A genes (TEF1/TEF2) were deleted from the chromosome, while the wt or mutated eEF1A was expressed from plasmids. Importantly, a given eEF1A mutant is the only source of eEF1A in the yeast cells used. Using the high-throughput assay, we identified one yeast strain (N21) expressing an eEF1A mutant that supported reduced TBSV repRNA replication, while the other three strains with eEF1A mutants (named C42, C53 and C62) showed increased level of repRNA accumulation ( Fig. 1A and S1B-G). Interestingly, the eEF1A mutants supporting increased steady-state level of repRNA accumulation did not increase the relative level of p33 and p92 pol replication proteins (Fig. 1A , bottom panel; S1D-E). Thus, these eEF1A mutants likely affect TBSV replication directly. Accordingly, affinity purification of the solubilized tombusvirus replicase complex from yeast cells, followed by in vitro replicase activity assay revealed that the replicase from C42, C53 and C62 mutant eEF1A-expressing yeast strains had ,2-fold increased activities when compared with wt eEF1A-expressing yeast strain (Fig. 1B , lanes 1-6 versus 7-8). The amounts of replication protein p33 and the co-purified eEF1A were comparable in the purified replicase samples (Fig. 1B, bottom panel) , indicating that the differences in replicase activities in the mutants are likely due to enhanced replicase functions, and not due to altered proteins levels in the replicase complexes. Testing the ability of C42, C53 and C62 mutant eEF1As to bind to the viral RNA or to the p33 and p92 pol replication proteins in vitro ( Fig. S2B -C) did not reveal significant differences between the mutants and the WT. This further supports that these eEF1A mutations likely increase the function of the viral replicase without altering the protein and RNA components in the replicase.
Placing the identified mutations in the three novel gain-offunction mutants of eEF1A (V 301 D, L 374 V/N 377 K, and F 413 L; Fig. 1C , indicated with yellow balls), which exhibited increased tombusvirus replication, over the known structure of eEF1A [51] revealed a cluster on one face of eEF1A (namely, the actin bundling domain III), away from the domains known to bind to tRNA and translation factor eEF1Ba. On the other hand, the new reduced function mutant (A 76 V, Fig. 1C , indicated with green balls) and the previously identified T 22 S [25] , which exhibited
Plus-stranded RNA viruses are important pathogens of plants, animals and humans. They replicate in the infected cells by assembling viral replicase complexes consisting of viral-and host-coded proteins. In this paper, we show that the eukaryotic translation elongation factor (eEF1A), which is one of the resident host proteins in the highly purified tombusvirus replicase complex, is important for Tomato bushy stunt virus (TBSV) replication in a yeast model host. Based on a random library of eEF1A mutants, we identified eEF1A mutants that either decreased or increased TBSV replication. In vitro studies revealed that eEF1A facilitated the recruitment of the viral RNA template for replication and the assembly of the viral replicase complex, as well as eEF1A enhanced viral RNA synthesis in vitro. Altogether, this study demonstrates that eEF1A has several functions during TBSV replication. eEF1A mutants enhance TBSV RNA replication in a cellfree extract
Since eEF1A is part of the tombusvirus replicase complex [25] , it is possible that C42, C53 and C62 eEF1A mutants might affect the assembly/activity of the tombusvirus replicase. To test this idea, we prepared cell-free extracts (CFE) from yeast strains expressing selected eEF1A mutants in the absence of the wt copy of eEF1A. These yeast extracts contained comparable amount of total proteins as well as the amounts of eEF1A, ALP, PGK and Hsp70 (Ssa) yeast proteins were comparable ( Fig. 2A) . The advantage of the CFE extracts is that they can then be programmed with the TBSV (+)repRNA in the presence of purified recombinant p33 and p92 pol obtained from E. coli that leads to the in vitro assembly of the viral replicase, followed by a single cycle of complete TBSV replication, resulting in both (2)stranded repRNA and (+)-stranded progeny [31, 52] . Therefore, this assay can uncouple the translation of the viral proteins from viral replication, which are interdependent during (+)RNA virus infections.
Using CFEs from yeast expressing one of the three mutant eEF1As resulted in ,3-fold increased TBSV repRNA accumulation when compared with the extract obtained from yeast expressing the wt copy of eEF1A ( Fig. 2A , lanes 2-4 versus 5). These data suggest that the viral replicase complex containing the mutant eEF1A can support in vitro TBSV repRNA replication more efficiently than the replicase with the wt eEF1A. In contrast, CFE from N21 yeast supported TBSV repRNA replication to similar extent as the CFE containing wt eEF1A ( Fig. 2A , lanes 1 versus 5), indicating that N21 eEF1A mutant can perform the same functions as the wt eEF1A in vitro, when the same amount of p33 and p92 pol was provided.
To test if the increased TBSV repRNA replication in vitro was due to enhanced (+) or (2)-strand synthesis, we analyzed the replication products under non-denaturing versus denaturing conditions (Fig. 2B) . These experiments showed that the amount of dsRNA [representing the 32 P-labeled (2)RNA product hybridized with the (+)RNA] increased ,3-fold in case of C42, C53 and C62 mutants (lanes 3-8, Fig. 2B ) in comparison with the wt (lanes 9-10). The dsRNA nature of these products was confirmed by the ssRNA-specific S1 nuclease digestion assay (Fig. 2C ). On the other hand, the ratio of dsRNA and ssRNA did not change in the various CFEs containing the eEF1A mutants or the wt (Fig. 2B) . These results are consistent with the model that the replicase complex carrying the eEF1A mutants increased mostly the level of (2)RNA production, which then led to proportionately higher level of (+)RNA progeny.
Cell-fractionation assay, followed by the cell-free TBSV replication assay demonstrated that the soluble fraction from the C42, C53 and C62 mutant yeasts stimulated the in vitro replication of TBSV repRNA by ,3-fold, while the membrane fraction when derived from C42, C53 and C62 mutant yeasts had a lesser effect (Fig. 2D , lanes 12-14 versus 7-9). These data are in agreement with the expected mostly cytosolic distribution of eEF1A, albeit eEF1A is also present in the membrane fraction in a smaller amount (Fig. 2D , bottom panel).
To test directly if eEF1A could stimulate RNA synthesis by the viral RdRp, we chose the E. coli-expressed recombinant p88 pol RdRp protein of Turnip crinkle virus (TCV), which is unlike the E. coli-expressed TBSV or CNV p92 pol RdRp, does not need the yeast cell-free extract to be functional in vitro [53, 54] . The template specificity of the recombinant TCV RdRp with TBSV RNAs is similar to the closely-related tombusvirus replicase obtained from yeast or infected plants [21, 54, 55, 56] . However, the recombinant TCV RdRp preparation lacks co-purified eEF1A, unlike the yeast or plant-derived tombusvirus replicase preparations, facilitating studies on the role of eEF1A on the template activity of a viral RdRp. When we added the highly purified wt eEF1A to the RdRp assay containing TCV RdRp protein and a TBSV derived (+)RNA template, which is used by the TCV RdRp in vitro to produce the complementary (2)RNA product (Fig. 3A , lanes 3-4) [55] , we observed a ,6-fold increase in (2)RNA synthesis by the TCV RdRp (lanes 11-12), while, as expected, we did not detect new (+)RNA progeny (not shown). This suggests that eEF1A can greatly stimulate TCV RdRp activity in vitro, confirming a direct role for eEF1A in (2)RNA synthesis by a viral RdRp.
Since it is known that eEF1A can bind to the 39-UTR of TBSV (+)RNA as well as to the tombusvirus replication proteins [25] and to the TCV RdRp ( Fig. S2A) , we wanted to test if the above stimulating activity of eEF1A in the in vitro RdRp assay was due to binding of eEF1A to the (+)RNA template and/or to the TCV RdRp protein.
Pre-incubation of the purified wt eEF1A with the TCV RdRp prior to the RdRp assay led to a ,5-fold increase in in vitro (2)RNA synthesis (Fig. 3A , lanes 9-10), while pre-incubation of the purified eEF1A with the TBSV (+)RNA template prior to the RdRp assay led only to a ,2-fold increase in (2)RNA products (lanes 7-8). Also, preincubation of the TCV RdRp with the (+)RNA template prior to the RdRp assay containing purified eEF1A led only to a ,2-fold increase in (2)RNA synthesis (lanes 5-6), suggesting that eEF1A can stimulate (2)RNA synthesis less efficiently after the formation of the (+)RNA-RdRp complex. Overall, data shown in Fig. 3 imply that eEF1A stimulates (2)RNA synthesis most efficiently when it forms a complex with the viral RdRp prior to binding of the template RNA to the eEF1A-RdRp complex.
To test if eEF1A stimulates the rate of initiation of (2)RNA synthesis, we analyzed the amount of abortive RNA products, which are generated during de novo initiation of RNA synthesis by the TCV RdRp [57] . We found that the amount of the 5-11 nt long abortive RNA products increased by 3.5-fold in the presence of purified eEF1A in the TCV RdRp assay (Fig. 3B , lanes 3-4 versus 1-2). We also tested the RdRp activity in the presence of Top panel: Replication of the TBSV repRNA was measured by Northern blotting 24 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the rRNA (middle panel, the 18S ribosomal RNA levels were estimated by Northern blotting). Bottom two panels: Accumulation of p33/p92 pol and eEF1A was estimated by Western blotting using anti-His and anti-eEF1A antibody, respectively. Note that * marks an SDS-resistant p33 homodimer band. (B) An in vitro replicase assay to test the relative activity of the tombusvirus replicase obtained from yeast expressing various mutants of eEF1A. Top panel: We tested the in vitro replicase activity using comparable amounts of affinity-purified replicase with added DI-72 RI(2) RNA template. Bottom panels: Western blot analysis showing p33 viral replication protein and the co-purified eEF1A in the above purified replicase preparations. (C) Critical eEF1A residues for tombusvirus replication. Three novel mutants of eEF1A were identified, which exhibited increased tombusvirus replication (V301D, L374V/N377K, and F413L; yellow balls) while the new A76V and the previously identified T22S exhibited decreased tombusvirus replication (green balls). The structure of eEF1A was generated using Jmol with PDB coordinates 1IJE. doi:10.1371/journal.ppat.1001175.g001 eEF1A using a (+)RNA template with a mutation opening the closed structure in the promoter region that leads to increased template activity [58] . The mutated template also showed 2-fold increased abortive RNA products in the RdRp assay with eEF1A ( Fig. 3B , lanes 5 versus 6). These data strongly support the model that eEF1A stimulates the de novo initiation step in the RdRp assay. The denaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. The full-length repRNA is pointed at by an arrow. Panels below show Western blot analysis of the whole cell extracts for the indicated yeast proteins based on specific antibodies. Bottom panel shows the coomassie-blue stained SDS-PAGE gel to visualize total protein levels in the whole cell extracts. (B) Detection of single-and double-stranded RNA products produced in the cell-free TBSV replicase assay. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (mostly ssRNA is present). The amount of dsRNA and the ratio of ssRNA/dsRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. (C) Denaturing PAGE analysis of the TBSV replicase products obtained in the cell-free replicase assay after S1 nuclease treatment, which cleaves the ssRNA, but not the dsRNA product. (D) The denaturing PAGE analysis of the 32 P-labeled repRNA products obtained in the in vitro reconstitution assay is shown. The membrane fraction of the whole cell extracts prepared from eEF1A mutant strains were mixed with the supernatant fraction of CFE prepared from WT eEF1A (lanes 6-10) or the supernatant fraction of CFE from the mutant strains were added to the membrane fraction from the wt strain (lanes [11] [12] [13] [14] [15] . The reconstituted extracts were programmed with purified recombinant TBSV p33/p92 pol and (+)repRNA. Bottom panel: Western blot analysis shows the amount of endogenous eEF1A in various fractions (see above) prepared from yeast expressing various mutants of eEF1A. doi:10.1371/journal.ppat.1001175.g002
To test if eEF1A stimulates the rate of RNA synthesis in the absence of de novo initiation, we analyzed the amount of 39terminal extension (39-TEX) RNA products, which are generated from an internal primer by the TCV RdRp (Fig. 3D ) [55] . Addition of purified eEF1A did not increase the amount of 39TEX products (lanes 2, 4, 6, Fig. 3D ), suggesting that the elongation step The TBSV (+)RNA template was the short 39 end region (SL1/SL2/SL3), which contain the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The second template was SL1m with a point mutation within the promoter sequence, which is being used more efficiently by the TCV RdRp in vitro. Note that eEF1A has been shown to bind to the replication silencer element. The RdRp assay had two steps: first, the shown components were incubated at room temperature to facilitate their interaction, followed 5 min latter the addition of the shown component and the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 3-4). (B) Detection of abortive RdRp products in the in vitro assay. 15% PAGE/UREA gel was used to resolve the 4-10 nt long products produced during initiation followed by rapid termination. Note that abortive RNA products are characteristic products for RNA polymerases that initiate de novo (in the absence of a traditional primer). (C) Lack of stimulation of 39-terminal extension by eEF1A in vitro. The template RNAs (shown schematically) contain a common artificial hairpin structure at the 39 end that facilitates 39-TEX by the TCV RdRp. The black bar represents 3 different sequences in the three constructs, derived from RIV(+)(includes SL1/SL2/SL3 sequences), RIII(2) and RIII(+) of DI-72 RNA, respectively. The gel image shows the results of 39-TEX in the presence of 0 or 1 mg eEF1A as shown in a TCV RdRp assay. doi:10.1371/journal.ppat.1001175.g003 during complementary RNA synthesis is not affected by eEF1A. Altogether, the obtained in vitro TCV RdRp data suggest that eEF1A can mostly stimulate the initiation step during de novo viral (2)RNA synthesis.
To further test the function of eEF1A in TBSV replication, we used chemical inhibitors of eEF1A, including Didemnin B (DB) and Gamendazole (GM). DB inhibits the activity of GTP-bound eEF1A during translation by binding to a pocket in eEF1A involved in the interaction with the aminoacylated tRNA and the nucleotide exchange factor eEF1Balpha [59, 60] . GM has been shown to inhibit the actin bundling function, while it does not inhibit protein translation or GTP binding functions of eEF1A [61] . We found that both DB and GM efficiently inhibited TBSV repRNA replication in the in vitro assay with CFE, which contains the endogenous eEF1A (Fig. 4A ). Time-course experiments revealed that the inhibition by DB was the most effective when the inhibitor was added at the beginning or during the first 10-15 min of the assay (Fig. 4B , lanes 2-5), while GM inhibited the cell-free replication of TBSV repRNA when added not only at the beginning, but up to 40 min after the start of the assay (lanes [12] [13] [14] [15] [16] [17] . It is known that the recruitment of the viral RNA and replication proteins as well as the assembly of the viral replicase complex take place during the first 40-60 min in the cell-free assay [31] . Since DB could inhibit translation, we also tested the effect of another translation inhibitor, namely cycloheximide, which did not affect TBSV repRNA replication in our assay (Fig. S3) . These data suggest that the inhibition by DB and GM is unlikely through decreased translation in the replication assay. Therefore, the above data are consistent with the model that DB and GM interfere with the assembly of the viral replicase complex in the CFE. Also, GM seems to be a more potent inhibitor of TBSV replication than DB.
To further test if DB and GM can interfere with the assembly of the tombusvirus replicase complex, we performed a two-step in vitro assembly/replication assay, also based on CFE containing endogenous eEF1A [31] . In this assay, first, we only provide ATP and GTP in addition to the replication proteins, the (+)repRNA and CFE, which can support the assembly of the replicase, but cannot perform RNA synthesis due to the lack of CTP and UTP [31] . After 1 hr incubation, once the replicase assembly had taken place, we collected the membrane fraction of the CFE by centrifugation and removed the supernatant containing the unbound p33, p92, repRNA as well as the cytosolic fraction of the CFE. Then we added all four rNTPs (including 32 P-labeled UTP) to the membrane fraction of the CFE to allow for RNA synthesis by the pre-assembled replicase complex (second step, Fig. 4C ) [31] . Interestingly, adding either DB or GM during the first step resulted in robust inhibition of TBSV repRNA synthesis during the second step of the assay (Fig. 4C, lanes 2-3 versus 1) , whereas providing the same amount of DB and GM at the beginning of the second step did not result in inhibition of repRNA replication (lanes 4-6) . These data support a model that DB and GM could inhibit the assembly of the tombusvirus replicase complex, but not the RNA synthesis by the already assembled replicase. Similarly, DB and GM failed to inhibit TBSV RNA synthesis in an in vitro assay with a highly purified RdRp from yeast (Fig. S4A) .
Since the assembly of the tombusvirus replicase also depends on events prior to the replicase assembly step, such as template RNA binding by the viral replication proteins/host proteins (such as eEF1A), and template recruitment to intracellular membranes [21, 52] , we also tested the effect of DB and GM on these processes as well based on purified recombinant eEF1A. We found that GM strongly interfered with the binding of eEF1A to the viral RNA in an EMSA assay (Fig. 4D, lanes 3-6 versus 2) , whereas DB did not affect the binding under the assay conditions (lanes 9-12). Since DB binds only weakly to eEF1A in solution, but it binds much more effectively to eEF1A in the presence of GTP and the ribosome [62] , we also performed in vitro co-purification experiments. First, 35 S-labeled eEF1A was produced in an in vitro translation system (containing ribosome and GTP) and, second, biotin-labeled viral (+)repRNA was added. After short incubation in the absence or presence of various amount of DB, we performed affinity-purification of the viral RNA. Phosphoimaging revealed that eEF1A was co-purified with the viral RNA and the amount of protein co-purified with the viral (+)repRNA was inhibited by increasing amount of DB in the assay (Fig. 4E , compare lane 1 with [2] [3] [4] [5] . This demonstrated that DB inhibits the binding of eEF1A to the viral repRNA. Moreover, both DB and GM interfered with the recruitment of the viral template RNA to the membrane of the CFE containing endogenous eEF1A (Fig. 4F,  lanes 5-8 versus 3-4) . On the other hand, DB and GM do not seem to affect the interaction between eEF1A and p33 or p92 replication proteins in vitro (Fig. S4B) . Altogether, these data suggest that inhibition of eEF1A function by DB and GM could block several steps during the assembly of the tombusvirus replicase complex, including template binding by eEF1A and viral RNA recruitment into replication.
(+)RNA virus replicases contain viral-and host-coded components, which likely provide many yet undefined functions to facilitate robust virus replication in infected cells. Translation factors, such as eEF1A, are among the most common host factors recruited for (+)RNA virus replication. eEF1A is an integral component of several viral replicases, including the highly purified tombusvirus replicase complex. Since eEF1A is an essential G protein involved in translation elongation, it is difficult to obtain evidence for its direct involvement in virus replication in living cells. Indeed, down-regulation of eEF1A in cells has led not only to decreased TBSV repRNA accumulation, but also reduced p33 levels [25] . However, using a small set of functional eEF1A mutants defective in various functions revealed that eEF1A is involved in stabilization of p33 replication protein in yeast [25] .
Based on the previous successful strategy of analyzing eEF1A mutants, here we generated ,6,000 random mutants covering the entire eEF1A sequence and found four mutants, which greatly affected TBSV repRNA accumulation in yeast (Fig. 1A) . Among these mutants, C42, C53 and C62 increased TBSV repRNA replication. Importantly, this effect by the eEF1A mutants was not due to changing the translation efficiency of p33/p92 pol , but likely via directly altering viral replication and affecting the activity of the viral replicase. On the other hand, N21 mutant of eEF1A resulted in decreased TBSV RNA accumulation and also led to reduction in the level of p33 replication protein. This is reminiscent of the previously characterized GDP-binding mutant T 22 S [25], which supported greatly reduced level of viral RNA replication and p33 accumulation due to shortened half-life of p33. Overall, N21 mutant further supports that one of the functions of eEF1A in TBSV replication is to stabilize the p33 replication protein in yeast.
In addition to this genetic evidence on the relevance of eEF1A in TBSV replication in yeast, we also obtained additional supporting data by showing that chemical inhibitors of eEF1A, such as DB and GM, strongly inhibited replication of TBSV eEF1A Role in the Assembly of Virus Replicase repRNA in the cell-free replication assay (Fig. 4A ). Since we used the same amount of purified recombinant p33/p92 pol in this in vitro assay (i.e., translation in the CFE is not needed for production of p33/p92 pol ), the role of eEF1A in TBSV replication must be separate from its role in protein translation. Altogether, these data strongly established that eEF1A is directly involved in TBSV replication, independent of the role of eEF1A in protein translation. Fig. 2 . DB (150 mM) and GM (100 mM) were added at various time points and the replicase assay was stopped after 3 hours for each treatment, followed by RNA analysis in a denaturing PAGE gel. The replicase activity in the samples containing DMSO added at the 0 time point was chosen as 100%. (C) A step-wise approach was used to separate the possible effect of DB and GM during either the assembly of the TBSV replicase or RNA synthesis steps. In step 1, the purified recombinant TBSV p33, p92 pol and (+)repRNA were added to the whole cell extract in the presence of ATP and GTP, which only supports the assembly of the TBSV replicase, but prevents RNA synthesis. This was followed by removal of the extra amount of p33, p92 pol and repRNA, which were not bound to the membranes of cell-free extract, and then by the standard replicase assay in a buffer containing 32 P-UTP and ATP, CTP and GTP (step ''RNA synthesis''). The denaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. Note that DB (150 mM) and GM (100 mM) were added to the assay either at the beginning (prior to replicase assembly) or after the replicase assembly. See further details in panel B. (D) The effect of DB and GM on binding between the purified eEF1A and 32 P-labeled template RNA (SL1/SL2/SL3) based on EMSA. The bound and unbound RNAs are pointed at by arrowheads. GM and DB were applied in the following amounts: 0, 5, 50, 250, and 1000 mM. Note that the amount of unbound RNA in the absence of eEF1A (lane 1) was chosen as 100%. (E) The inhibitory effect of DB on co-purification of eEF1A with the viral repRNA. WT 35 S-labeled eEF1A was produced in a translation assay using rabbit reticulocyte lysate, followed by incubation with biotin-labeled DI-72(+) repRNA in the presence of 0, 50, 150, 500 and 1000 mM DB. Then the repRNA was captured with streptavidin-coated magnetic beads, followed by elution of the co-purified proteins from the beads. SDS-PAGE analysis shows the amount of co-purified 35 S-labeled eEF1A. (F) The inhibitory effect of DB and GM on the template recruitment step in vitro. Purified recombinant p33/p92 and 32 P-labeled DI-72 (+)repRNA (indicated as W, lanes 1 and 3-8) or C 99 -G mutant (+)repRNA (indicated as M, lane 2) were added to a whole cell extract (CFE) in the presence of DMSO (control), 100 mM GM or 150 mM DB, followed by centrifugation/washing to remove the 32 P-labeled repRNA that is not bound to the membrane. Then the membrane-bound RNA was analyzed in a denaturing PAGE gel. Note that the recruitment deficient C 99 -G mutant repRNA bound to the membrane nonspecifically (,20% level). doi:10.1371/journal.ppat.1001175.g004 eEF1A selectively enhances minus-strand synthesis during TBSV replication
The identified eEF1A mutants were also useful to dissect the functions of eEF1A in TBSV replication. Based on a cell-free TBSV replication assay in CFE prepared from yeast expressing the C42, C53 or C62 mutants, we found that the minus-strand synthesis was enhanced by ,3-fold, while the rate of plus-strand synthesis was proportionate with (2)RNA synthesis, resulting in ,10-fold more (+) than (2)RNA products for wt and each mutant.
We confirmed a direct role for eEF1A in RNA synthesis in vitro by using a highly purified eEF1A and the recombinant TCV RdRp, which is closely homologous with the TBSV p92 pol . Interestingly, it seems that eEF1A stimulates the RdRp activity directly, since pre-incubation of eEF1A and the RdRp prior to the RdRp assay led to the highest level of stimulation of (2)RNA synthesis (Fig. 3A) . On the other hand, pre-incubation of eEF1A with the TBSV-derived template RNA led only to ,2-fold increase in RNA synthesis in vitro (Fig. 3A) . Analyzing the amount of short abortive RdRp products, which are produced through initiation followed quickly by abortive termination [57] , in the in vitro assays revealed that eEF1A strongly enhanced the initiation of minus-strand synthesis (Fig. 3B) . Although the actual mechanism of stimulation of RdRp activity by eEF1A is currently unknown, we propose that eEF1A might facilitate the proper and efficient binding of the RdRp to the 39 terminal sequence of the viral RNA prior to initiation of (2)-strand synthesis (Fig. 5) . Accordingly, eEF1A was shown to bind to the so-called replication silencer sequence (RSE) in the 39-UTR, which is required for the assembly of the viral replicase complex [22, 58] . The binding of eEF1A-RdRp complex to the RSE might assist in placing the RdRp over the 39-terminal promoter sequence, thus facilitating the initiation of (2)RNA synthesis starting from the 39-terminal cytosine. Similar function of eEF1A in stimulation of (2)RNA synthesis has been proposed for WNV, based on mutations in the viral RNA within the eEF1A binding sequence that reduced the binding affinity of RNA to eEF1A and inhibited (2)RNA synthesis in infected cells [46] . eEF1A stimulates the assembly of the viral replicase complex during TBSV replication Recent intensive work revealed that the assembly of the viral replicase complex is a regulated process involving viral-and host factors, cellular membranes and the viral (+)RNA [8, 10, 19, 63, 64, 65] . The assembly of the viral replicase also depends on steps occurring prior to the actual assembly process, such as selection of the viral template RNA and the recruitment of (+)RNA/protein factors to the sites of assembly. Although our current understanding is rather poor about the factors involved and their functions during replicase assembly, rapid progress is being made in this area due to the development of a new cell-free assay based on yeast CFE [30, 31] . The yeast CFE is capable of assembling the tombusvirus replicase complex in vitro in 40-60 min in the presence of recombinant p33/p92 pol and the viral (+)repRNA [31] , allowing for studies on direct roles of various factors. We find that inhibition of eEF1A activity by either DB or GM also inhibited the assembly of the tombusviral replicase complex based on time-course experiments (Fig. 4B ) as well as a direct replicase assembly assay (Fig. 4C) . On the contrary, the replicase activity was not inhibited by these compounds after the assembly took place (Fig. 4B-C) . It is possible that after the formation of the eEF1A-RdRp-repRNA complex DB or GM are not effective in inhibiting the stimulatory effect of eEF1A on the RNA synthesis by the viral RdRp. Additional in vitro experiments with purified tombusvirus replicase preparations confirmed the lack of inhibition of RNA synthesis by DB or GM (Fig. S4A ) on pre-assembled virus replicases.
The inhibition of the tombusvirus replicase complex by DB or GM might come from the ability of these compounds to inhibit the template RNA recruitment step (Fig. 4F ). If the recruitment of the viral (+)RNA is inhibited, then the assembly of the viral replicase cannot take place in yeast or in vitro [21, 22, 31] . A target for GM and DB could be the inhibition of binding between eEF1A and the viral (+)RNA (Fig. 4D, E) . Since the actual steps during the replicase assembly process are not yet known, it is possible that eEF1A might play additional roles in the assembly of the viral replicase complex.
The presented data are also in agreement with the function of eEF1A as a chaperone of the viral RdRp. Binding between the eEF1A and RdRp might alter the structure of the RdRp that favors de novo initiation for RNA synthesis. Indeed, the chaperone activity of eEF1A and its bacterial homolog EF-Tu has been shown before [66, 67] . Moreover, the EF-Tu-EF-Ts complex is thought to function in the Qbeta replicase complex as a chaperone for maintaining the active conformation of the RdRp protein [68] .
Overall, the current work demonstrates two major functions for eEF1A in TBSV replication (Fig. 5) : (i) stimulation of the assembly of the viral replicase complex, likely by facilitating the recruitment of the viral RNA template into the replicase; and (ii) enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation.
Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) was obtained from Open Biosystems (Huntsville, AL, USA). Plasmid-borne TEF1/2 TKY strains (MATa ura3-52 leu2-3, 112 trp1-D1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2D pTEF2 URA3) were published before [69, 70, 71, 72] . The plasmid pESCHIS4-ADH-His33/CUP1-DI-72 expressing Cucumber necrosis virus (CNV) p33 and the TBSV replicon RNA, called DI-72, was described earlier [25] . The LYS2-based plasmid pRS317-Tet-His92, expressing CNV p92 under the control of Tetracycline-regulatable (Tet) promoter was constructed as follows: the Tet promoter sequence was obtained from pCM189-His92/Tet [73] by digestion with EcoRI and BamHI, and CNV p92 coding sequences from pGAD-His92 [7] digested with BamHI and PstI, followed by ligation into pRS317 vector treated with EcoRI and PstI. To generate mutations within TEF1 coding sequence by random mutagenesis, we constructed the TRP1-based plasmid pRS314-pTEF1-TEF1, which expressed TEF1 under the control of its native promoter. The TEF1 promoter sequence, the TEF1 coding region and the Cyc1 terminator sequences were amplified by PCR with the following primer pairs, #2764 (CCGCGAGCTCATAGCTTCAAAAT-GTTTCTAC)/#2765 (CCGCGGATCCGTAATTAAAACT-TAGATTAGATTGC), #2768 (CCGCGGATCCAAAATGGG-TAAAGAGAAGTCTC)/#1877 (CCGCCTCGAGTTATTTC-TTAGCAGCCTTTTGAGCAGC), and #2769 CCGCCTCG-AGGAGGGCCGCATCATGTAA/#2770 (CCGCGGTACCA-GCTTGCAAATTAAAGCCTTC), respectively. This was followed by cloning the PCR products into pRS314 digested with SacI and KpnI.
The mutagenic PCR conditions were as follows: 50 mM KCl, 10 mM Tris (pH 8.3 at 25uC), 7 mM MgCl 2 , 0.3 mM MnCl 2 , 1 mM dCTP and dTTP each, 0.2 mM dGTP and dATP each, 0.2 mM of each primer, 20 pM of template DNA and 10 units of Taq polymerase in a 10 ml reaction volume in 10 aliquots. The PCR was performed for 30 cycles at 94uC for 1 min, 50uC for 1 min, and 72uC for 1 min in a conventional thermal cycler. Three overlapping ,300-500 bp N-, central-and C-terminal segments of the TEF1 gene were amplified separately by PCR using primer pairs: #2767 (GTT-TCAGTTTCATTTTTCTTGTTC)/#2788 (GAGTCCATCT-TGTTGACAG), #2787 (CATCAAGAACATGATTACTGGT-AC)/#2790 (GACGTTACCTCTTCTGATTTC) and #2789 (CGGTGTCATCAAGCCAGGT/#2771, (TTCGGTTAGAGC-GGATGTGG), respectively.
Yeast strain TKY102 was co-transformed with constructs pESCHIS4-ADH-His33/CUP1-DI-72 and pRS317-Tet-His92 to induce TBSV repRNA replication according to standard Lithium acetate-PEG protocol [74] . The transformed yeast cultures were grown in a Synthetic Complete (SC) media with 2% glucose lacking leucine, histidine, lysine and uracil (SC-ULHK 2 ) by shaking at 29uC overnight. To completely suppress TBSV replication before induction, 1 mg/ml Doxycycline was added to the media to inhibit the expression of p92. The plasmid pool carrying the randomly mutated TEF1 gene was introduced into the yeast cells already transformed with the two virus expression plasmids by in vivo gap repair mechanism via homologous recombination (Fig. S1A) [75] . Briefly, pRS314-pTEF1-TEF1 was digested with enzymes to truncate the TEF1 coding sequence, and then the digested plasmid was recovered. The gapped plasmid (5-10 mg) was transformed together with overlapping PCR (20 mg) products carrying the TEF1 mutations created by random mutagenic PCR (see above). The transformed yeast cells were selected on SC media lacking uracil, tryptophan, leucine, histidine and lysine. The colonies were further streaked onto SC media plate lacking tryptophan, leucine, histidine and lysine (SC-TLHK 2 ) with 0.1% (w/v final) 5-Fluoroorotic Acid (5-FOA) media to select against the URA3-based wild-type TEF1 plasmid (Fig. S1A) . This selection was repeated once and the loss of URA3 plasmid was confirmed by the inability of the yeast strains to grow on uracil-minus media. The yeast cells carrying the randomly mutated TEF1 were grown at 29uC for 24 h in SC-TLHK 2 media with 50 mM CuSO 4 to induce virus replication. Total RNA extraction from yeast cells and Northern blotting and Western blotting were done as previously described [7, 25] .
Whole cell yeast extract capable of supporting TBSV replication in vitro was prepared as described [31] . The in vitro TBSV replication assays were performed in 20-ml total volume containing 2 ml of whole cell extract, 0.5 mg DI-72 (+)repRNA transcript, 400 ng purified MBP-p33, 100 ng purified MBP-p92 pol (both recombinant proteins were purified from E. coli), 30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol, 0.4 ml actinomycin D (5 mg/ml), 2 ml of 150 mM creatine phosphate, 0.2 ml of 10 mg/ml creatine kinase, 0.2 ml of RNase inhibitor, 0.2 ml of 1 M dithiothreitol (DTT), 2 ml of 10 mM ATP, CTP, and GTP and 0.25 mM UTP and 0.1 ml of [ 32 P]UTP [31] . The reaction mixture was incubated at 25uC for 3 h. The reaction was terminated by adding 100 ml stop buffer (1% sodium dodecyl sulfate [SDS] and 0.05 M EDTA, pH 8.0), followed by phenol-chloroform extraction, isopropanol-ammonium acetate precipitation, and a washing step with 70% ethanol as described [52] . The newly synthesized 32 P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.56Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85uC for 5 min in the presence of 50% formamide [31] . S1 nuclease digestion to remove single-stranded 32 P-labeled RNA was performed at 37uC for 30 min in a buffer containing 5 mM sodium acetate (pH 4.5 at 25uC), 0.28 M NaCl, 4.5mM ZnSO4 and 40 U S1 nuclease (Boehringer).
Fractionation of the whole cell extract was done according to [52] . The total extract was centrifuged at 21,0006 g at 4uC for 10 min to separate the ''soluble'' (supernatant) and ''membrane'' (pellet) fraction. The pellet was re-suspended and washed with buffer A (30 mM HEPES-KOH pH 7.4, 150 mM potassium acetate, and 5 mM magnesium acetate) followed by centrifugation at 21,0006 g at 4uC for 10 min and re-suspension of the pellet in buffer A. In vitro TBSV replication in the fractions was performed as described [31] .
Expression and purification of the recombinant TBSV p33 and p92 and TCV p88C replication proteins from E. coli were carried out as described earlier with modifications [54] . Briefly, the expression plasmids were transformed separately into E. coli strain BL21 Rosetta (DE3). Protein expression was induced using isopropyl b-D-thiogalactopyranoside (IPTG) for 8 h at 16uC, then the cells were collected by centrifugation (5,000 rpm for 5 min). The recombinant TCV p88C protein was purified on an amylose resin column (NEB), as described [54] . The cells were suspended and sonicated in MBP column buffer containing 20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride (PMSF). The sonicated extract was then centrifuged at 27,000 g for 10 min, followed by incubation with amylose resin (NEB) for 1 h at 4uC. After washing the resin 3 times with the column buffer and once with a low salt column buffer (25 mM NaCl), the proteins were eluted with a low salt column buffer containing 0.18% (V/W) maltose and 6% (V/V) glycerol and stored at 280uC. MBP-p33 and MBP-p92 pol were purified as above, except 30 mM HEPES-KOH pH 7.4 was used instead of 20 mM Tris-Cl pH 8.0. eEF1A was purified from yeast as described [76] and stored in aliquots at the vapor temperature of liquid nitrogen. Protein fractions used for the replication assays were 95% pure, as determined by SDS-PAGE.
Yeast strains (WT, C42, C53, C62) were transformed with plasmids pESCHIS4-ADH-HF33/CUP1-DI-72 expressing 6XHis-and Flag-tagged CNV p33 and the TBSV DI-72 repRNA, and pRS317-Tet-His92, expressing CNV p92 under the control of Tet promoter [25] . Co-purification was done according to a previously described procedure with the following modification [25] . Gel mobility shift assay (EMSA) and co-purification of eEF1A-repRNA EMSA was performed in a 10 ml-reaction containing 20 mM HEPES [pH 7.6], 50 mM KCl, 2 mM MgCl 2 , 1 mM DTT, 0.1 mM EDTA, 10% [vol/vol] glycerol, 10 U of RNase inhibitor, 10 nM 32 P-labeled DI-72 (+) RNA probe and 0.5 mg purified eEF1A protein [76] . Reactions were incubated at room temperature for 20 min and then resolved by 4% nondenaturing polyacrylamide gel as described previously [25] .
For in vitro eEF1A-repRNA co-purification, DI-72(+) repRNA was biotin-labeled in standard T7 transcription reaction in the presence of 20 mM Biotin-16-UTP (Roche). After the T7 transcription, the unincorporated biotin-UTP was removed on a Bio-Rad mini gel filtration column. The biotinylated RNA was immobilized on a column containing Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs). Briefly, a 30-ml suspension of SA-PMPs (Promega) was washed three times with 1 ml of water and re-suspended in 16 Phosphate Buffered Saline (PBS). Biotinylated DI-72(+) RNA (5 mg) was then added to the suspension of SA-PMPs, followed by 30 min incubation at 4uC with gentle rotation. The SA-PMPs were collected on the side of the tube in a magnetic stand and washed 3 times with 16 PBS buffer. eEF1A was translated in vitro and labeled with 35 In vitro TCV p88C RdRp assay
The TCV RdRp reactions were carried out as previously described for 2 h at 25uC [54] . Briefly, the RdRp reactions were performed in a 20 ml reaction containing 50 mM Tris-HCl (pH 8.2), 10 mM MgCl 2 , 10 mM DTT, 1.0 mM each ATP, CTP, and GTP, 0.01 mM UTP plus 0.1 ml of [ 32 P]UTP, 7 pmol template RNA, 2 pmol affinity-purified MBP-p88C. 20 pmol eEF1A was added to the reaction at the beginning or as indicated in the text and Fig. 3 legend. The 32 P-labeled RNA products were analyzed by electrophoresis in a 5% or 15% PAGE/8 M urea gel [57] . The 86-nt 39 noncoding region of TBSV genomic RNA was used as the template in the RdRp assay [25, 54] .
The use of eEF1A inhibitors in the in vitro replicase assembly assay Purified Didemnin B (NSC 325319) was kindly provided by the Natural Products Branch, NCI (Bethesda, MD, USA), while Gamendazole was a generous gift from Dr. Tash (University of Kansas Medical Center). Both chemicals were dissolved in DMSO (the final concentration was 20 mM). The concentrations of chemical and time point of the addition of the chemicals to the in vitro reaction are indicated in the text. The cell-free TBSV replicase assay and the in vitro TBSV replicase assembly assay were performed according to [31] . Briefly, the purified recombinant TBSV p33, p92 pol and (+) repRNA were added to the cell-free reaction in the presence of 1.0 mM ATP and GTP in step 1. After incubation at 25uC for 1 h, the in vitro reactions were centrifuged 21,0006 g at 4uC for 10 min. The supernatant containing extra p33, p92 pol and repRNA, which were not bound to the membranes in the cell-free extract, was discarded, while the membrane pellet was re-suspended in a standard in vitro replicase assay buffer containing [ 32 P]-UTP and ATP, CTP, and GTP, and incubated at 25uC for 3 h [31] .
The TBSV viral RNA gets recruited to the membrane from the soluble fraction with the help of TBSV replication proteins and host factors present in the yeast CFE. The in vitro RNA recruitment reaction was performed according to [31] , except that 32 P-labeled DI-72 (+)repRNA were used and rCTP, rUTP, 32 P-labeled UTP, and Actinomycin D were omitted from the reaction. As a negative control, a recruitment-deficient repRNA, termed C 99 -G mutant, was used (Fig. 4F , lane 2) [23] . This mutant RNA is not recognized by p33/p92 replication proteins and it does not replicate in plants, in yeast or in the CFE in vitro [23, 31, 52, 77] . The RNA recruitment assay results in the assembly of the functional viral replicase, when wt repRNA is used, and nonfunctional replicase when the C 99 -G mutant is used in the assay (J. Pogany and P.D. Nagy, not shown) [31] . Inhibitors DB and GM were added at final concentration of 150 and 100 mM, respectively. After two hours of incubation at room temperature, 1 ml of reaction buffer was added to the in vitro assay, followed by incubation on ice for 10 min. Samples were centrifuged at 35,0006 g for 1h, and the pellet was washed with 1 ml reaction buffer, followed by centrifugation at 35,0006 g for 10 min. The membrane-bound repRNA was extracted from the pellet by adding 0.1 ml stop buffer and 0.1 ml phenol/chloroform and vortexing, followed by isopropanol/ammonium acetate precipitation [52] . The RNA samples were analyzed by denaturing PAGE and phophoimaging as described [52] . Figure S1 Schematic presentation of the random mutagenesis strategy used to obtain 6,000 eEF1A mutants. (A) The yeast strain (tef1Dtef2D carried a plasmid that expressed one of the random eEF1A (TEF1) mutants from the native promoter. Each yeast strain also carried pESCHIS4-ADH-His33/CUP1-DI-72 and pRS317-Tet-His92 to induce TBSV repRNA replication as described in the M&M section. (B) Additional experiments on the effect of eEF1A mutations on TBSV repRNA accumulation in yeast. The yeast strain expressed only one form of eEF1A, as indicated. Top panel: Replication of the TBSV repRNA was measured by Northern blotting 24 h after initiation of TBSV replication. (C) The accumulation level of repRNA was normalized based on the rRNA (the 18S ribosomal RNA levels were estimated by Northern blotting). Panels (D), (E) and (F) show the accumulation of p92 pol , p33 and eEF1A, respectively, estimated by Western blotting using anti-His and anti-eEF1A antibody. (G) SDS-PAGE analysis of total protein extract from the above yeast strains, after Coomassie blue-staining. Found at: doi:10.1371/journal.ppat.1001175.s001 (0.15 MB PDF) Figure S2 Binding of eEF1A to TBSV and TCV replication proteins in vitro. (A) MBP-tagged TCV p88C (lacking the p28overlapping domain from the N-terminus), MBP-TBSV p92, MBP-TBSV p92C (lacking the p33-overlapping domain from the N-terminus) and MBP-TBSV p33 or MBP (1 mg each) were separately immobilized on amylose beads, followed by incubation with a cytosolic extract prepared from yeast. The bound host proteins were eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TCV p88C, MBP-TBSV p92, MBP-TBSV p92C, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel). (B) The effect of eEF1A mutations on binding to the viral p33 and p92 proteins in vitro. MBP-tagged p92, p33 or MBP were separately immobilized on amylose beads, followed by incubation with a cytosolic extract prepared from yeast expressing wt or mutated eEF1A. The bound eEF1A was eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TBSV p92, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel). (C) The effect of eEF1A mutations on binding to the viral repRNA. CFE containing WT or mutated eEF1A was incubated with biotin-labeled DI-72(+) repRNA. Then the repRNA was captured with streptavidincoated magnetic beads, followed by elution of the co-purified proteins from the beads. Western blot analysis shows the amount of co-purified eEF1A using anti-eEF1A antibody. Found at: doi:10.1371/journal.ppat.1001175.s002 (0.12 MB PDF) Figure S3 Lack of inhibition of TBSV repRNA replication by Cyclohexamide in a cell-free TBSV replicase assay. The cell-free TBSV replicase assay was performed as described in Fig. 4 . Cyclohexamide was added in the following amounts: 0, 2, 10, 50, 100 mg/ml. The membrane-bound tombusvirus replicase in a yeast lysate was solubilized with Triton X-100/SB3-10 detergent, followed by purification on a FLAG-affinity column as described. The activity of the affinity-purified TBSV replicase was tested on the same amount of DI-72(2) RNA added to each sample. DB (panel on the left) and GM (panel on the right) were added in the following amounts: 0, 100, 150, 200, 250 mM for DB and 0, 25, 50, 100, 200 mM for GM. Denaturing PAGE analysis of the 32 P-labeled RNA products obtained with the purified tombusvirus replicase is shown. Note that this replicase preparation is only capable of complementary RNA synthesis on the added template RNA, but incapable of supporting a full cycle of replication. (B) The effect of GM and DB on binding to the viral p33 and p92 proteins in vitro. MBP-tagged p92 and p33 were separately immobilized on amylose beads, followed by incubation in the presence of 0 or 100 mM GM or 150 mM DB with a cytosolic extract prepared from yeast expressing wt eEF1A. The bound eEF1A was eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TBSV p92, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel). Found at: doi:10.1371/journal.ppat.1001175.s004 (0.09 MB PDF) The calculation of information and organismal complexity BACKGROUND: It is difficult to measure precisely the phenotypic complexity of living organisms. Here we propose a method to calculate the minimal amount of genomic information needed to construct organism (effective information) as a measure of organismal complexity, by using permutation and combination formulas and Shannon's information concept. RESULTS: The results demonstrate that the calculated information correlates quite well with the intuitive organismal phenotypic complexity defined by traditional taxonomy and evolutionary theory. From viruses to human beings, the effective information gradually increases, from thousands of bits to hundreds of millions of bits. The simpler the organism is, the less the information; the more complex the organism, the more the information. About 13% of human genome is estimated as effective information or functional sequence. CONCLUSIONS: The effective information can be used as a quantitative measure of phenotypic complexity of living organisms and also as an estimate of functional fraction of genome. REVIEWERS: This article was reviewed by Dr. Lavanya Kannan (nominated by Dr. Arcady Mushegian), Dr. Chao Chen, and Dr. ED Rietman (nominated by Dr. Marc Vidal). Organismal complexity is difficult to define and to measure, especially quantitatively. When DNA was discovered to be the material basis of inheritance in all organisms, it was thought that the DNA content of an organism should correlate with its phenotypic complexity, but soon thereafter the C-value paradox was found. C-value refers to the amount of DNA contained within a haploid nucleus, and usually equals to genome size. Salamanders and lungfishes have the largest genomes of 120pg, while the C-value of humans is only 3.5pg [1] . C-values vary enormously among species. In animals they range more than 3,300-fold. Variation in C-values bears no relationship to the complexity of the organism. The discovery of non-coding DNA in the early 1970 s resolved the C-value paradox. Although it is still unclear why some species have a remarkably higher amount of non-coding sequences than others of the same level of complexity, it was believed that the number of genes contained in the genome, rather than the genome size, correlated with the complexity of the organism. However, the human genome project and other model organism genome projects revealed that there are only about 25,000 genes in the human genome [2] , while simple organism nematode have 19,500 genes [3] and rice even has more genes than humans, 46,000~55,000 [4] . Obviously, the number of genes bears no direct relationship to phenotypic complexity. This is called Gvalue paradox.
As proteins are the ultimate bearers of organismal structure and function, it is now believed that the diversity of proteins as well as the interactions between the proteins correlates with the phenotypic complexity. The study of proteomics proceeds extensively after the genome projects. However, proteins are different from nucleic acids. Many proteins are subjected to a wide variety of chemical modifications after translation. A lot of these post-translational modifications, such as phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, nitrosylation and protein splicing, are critical to the protein functions. The modified proteins display different physical and chemical properties and biological functions from the unmodified. If a modified protein can be seen as a new protein, then the size of the proteome would be much larger than the number of genes. The number of proteins in a living organism may never be accurately counted as the number of genes, because every modified protein can only be individually studied. That is technically much more difficult because different proteins have different properties. A proteomic study can become quite complex even if the object of the study is very restricted. Therefore, it seems difficult to calculate the phenotypic complexity of an organism at least at the present time.
There are some studies about the problem of biological complexity. The traditional mathematical notion of complexity is Kolmogorov complexity, which can be thought of as the length of the shortest message in which the given sequence can be encoded [5] . The complexity is minimal for a homopolymer, and is maximal for a random sequence, in which case complexity is equal to the sequence length. The later case means the sequence cannot be simplified at all, so is the most complex. However, Kolmogorov complexity does not correspond to our intuitive notion of biological complexity. The other complexity is to calculate the Shannon's information entropy of a sequence [5] [6] [7] [8] . The complexity is the length of the sequence subtracting the Shannon's entropy, and is actually the information content of the sequence. This kind of complexity has biological meaning. The more complex means the sequence is more conserved and therefore carries more information. However, this complexity is only the complexity of sequences, which has nothing to do with the phenotypic complexity of organisms.
There are three parts of information contained in an organism's genome: first, the information to construct the organism; secondly, the information to constitute DNA structures, including replicons, centromeres, telomeres, etc; and finally, the information for the mechanisms of evolution, which we do not know at the present time. Here we take the amount of minimal information needed to construct an organism as a measure of organism phenotypic complexity because apparently more complex organisms should need more minimal information to construct and simple organisms are supposed to need less information to construct. The information to constitute DNA structure is relatively simple and less important, and we know very little about the mechanisms of evolution, so for the purpose of this article we will not be considering these areas of information. The information needed to construct organism, to put it simply, is the information needed to express proteins in time, in space, and in quantity. We actually still know very little about this information up to now. The conserved gene coding sequences are only part of the information. To calculate this information, we need to construct organism mathematically using permutation and combination formulas based on the numbers of proteins and cell types.
While some biologists may know how to calculate the information content of sequence, most are not familiar with how to calculate amount of information. According to Shannon's information concept, information is to decrease uncertainty. The more uncertainty information decreases, the more the information. Information is the difference between the entropy of known and unknown. How much uncertainty you need to decrease, how much information you need. If you know everything, you do not need information. The less you know, the more you need to know, the more information you need. The uncertainty can often be calculated as possibility. The certainty means only one possibility. The uncertainty means many possibilities. The more possibilities excluded, the more the information.
Shannon's information entropy can be calculated using formula:
where p are the probabilities of events.
When the probabilities of all events are equal, H gets the maximal value. Let N be the number of events or possibilities, then
The number of possibilities N can be calculated by using permutation and combination formulas. H is the entropy of unknown because you only know the probabilities or possibilities. When you know everything, H becomes 0 because there is only one possibility left with probability 1 and the probabilities of other possibilities are 0. The entropy of known is 0. In order to know, you need to reduce the entropy to 0, so you need information:
So information I has the same value as entropy H, but they are different concepts. Entropy is a quantity to describe disorder. Information is a quantity to reduce disorder. Information can be calculated based on the entropy you need to reduce. It is easy to calculate information if the number of possibilities can be calculated. For example, in order to guess a random 8-digits telephone number, you need information
) . The probability for each event is same here. Sometimes the probability for each event may not be same, but you do not know the probabilities, so any probability distribution is possible and equal probability distribution is also possible. In order to know, you have to need information to reduce the entropy from the maximum to 0. You have to assume equal probability because in this way you need minimal information. For example, we need to encode a protein sequence with 10 amino acids (all the amino acids are independent). Although the actual amino acids distribution across the sequence may not be equal, we do not know the distribution information. We have to assume equal probability to write the sequence. In this way, we need minimal information. The formula I=log(N) can still be used. We need information
to write a protein sequence with 10 amino acids. If we know the sequence is MMMMMMMMMM, we need information I=0 to write the sequence because it is already known. If we know the sequence is a DNA sequence, then the entropy of unknown is H = 10 log (4), so we need information I = ⋅ = 10 4 20 log( ) bits to write the sequence. If we know it is an English sequence but we do not know any English except 26 letters, then the entropy of unknown is H=10 log (26) . We need information
to write the sequence. If we know English quite well, we will need much less information to write the sequence. For example, for the last character of the sentence "I love yo_", you may need no information to guess the underscore is "u" if you know English, but I need information log (26) bits to guess the character if I do not know English except 26 letters. Even though the distribution of each English letter is actually not equal at all, but I do not know the probabilities of each letter, I have to assume equal probability. I still need information log(26) bits for each character. Different persons need different information to know because they know differently. Information is the difference between the entropy of known and unknown. How much information you need depends on how much you have known, therefore depends on the entropy of unknown. The more you know, the less the entropy of unknown, and the less the information you need. For a sequence, we can also say the information of the sequence is the information we need to write the sequence.
Another example is to calculate the regulatory information of viruses. If a virus has 10 protein coding genes and all the genes express one by one in sequence and only express once, how much should be the regulatory information? The probability for each gene expression is the same, so the formula I=log(N) can be used. For the first gene to express, you have 10 genes to choose. For the second gene, the number of genes to choose is 9. The information needed for the order of expression is:
. 10 21
This information is regulatory information, which is actually composed of regulatory sequences or Transcription Factor (TF) binding sites. Because every base pair of DNA contains information log(4) = 2 bits, the length of all TF binding sites should be at least 21.8/2 = 10.9 bp. A virus may use longer sequence for the binding sites, but the minimum should be 11 bp.
The concept of information content of sequence is a little different from the concept of information above. The calculation of information content also needs to use Shannon's information entropy. To calculate information content, you must know the probability distribution of each site of a sequence, which is based on the data of population genetics. The information content or complexity of the sequence can be calculated as [5, 7, 8] :
where L is the length of the sequence, H is the entropy of known. For example, for a sequence AXT, the first site A is very conserved with probability 1 for A, 0 for other base pairs. The information content of the first site is:
If the second site X is actually random, any bases are possible, the information content is: C 2 = 1-1/4·log(4)·4 = 1-1 = 0 or C 2 = log 2 (4)-1/4·log 2 (4)·4 = 2-2 = 0bit If the probabilities of the third site T are 1/2 for T, 1/4 for A, 1/4 for G, the information content of third site can be calculated as: The more conserved the sequence, the more the information content. The amount of information of the sequence is determined by how much you know to write the sequence. If you know it is a DNA sequence and the probability distribution of each site of the known sequence, you need information I = H unknown -H known = 6-3.5 = 2.5 bits to write the sequence AXT. The information of the sequence is 2.5 bits.
For each genetic codon, which is composed of 3 base pairs, the amount of information is I=H unknown -H known =3 log 2 (4)-0 = 6 bits. For each amino acid, because many amino acids have more than one codon, the information content is different. For example, proline has 4 codons and the third base pair of the codons is wobbled, the information content of proline C=H max -H known =6-2 = 4 bits. In the same way, the information content of Arg is 3 bits, of Asn is 5 bits, and of Trp is 6 bits. Organisms probably know the importance of each amino acid and how to regulate gene expressions with codon bias, and evolved this codon system to regulate translation efficiently and reduce the harm of mutation of important amino acids. The redundance of information is R=I-C. The less the information content is, the more the redundance of information, the more the space to silence or neutralize mutation. So Arg is more important, more protected from mutation, and more expression regulated than Trp. Although some amino acids may need less information to be encoded (for example, 2 base pairs for the amino acids with information content less than 4 bits), organisms actually use 3 base pairs for every amino acid, so it cost organisms 6 bits genomic information to encode every amino acid. In another word, the amount of information of every amino acid in genome is 6 bits although there is information redundance in the 6 bits.
Using fixed number of base pairs rather than different number to encode all amino acids manifests that organisms probably do not know the distribution of amino acids across all protein sequences and assume equal probability for all amino acids. In this way organisms can change protein sequences and amino acid distributions flexibly and efficiently under changing environments. Otherwise, organisms have to change the codon system when the distributions change. For evolution, it is impossible for a living organism to know the future protein sequences and the amino acid distributions across the sequences. In order to generate any unknown future sequences efficiently, living organisms need to use a mechanism that can encode any protein sequences efficiently. In fact, all organisms use a mechanism that can reduce the maximal entropy to 0, which means equal probability for all amino acids when encoding protein sequences. For the same reason, organisms may not know the distribution of bases across all nucleotide sequences, and may assume equal probability for all bases even though the actual probability distribution across some genomes may not be equal at all. For biological information, most cases are like this with maximum entropy needed to be reduced and equal probability need to be assumed by organisms, so formula I=log(N) can be used to calculate the information.
Fixed number of base pairs of genetic code also simplifies the reading of DNA protein coding sequence and reduces the total information needed to encode protein.
If the lengths of codons were different, "space" would have to be used to separate each codon in protein coding sequence, and the total mechanisms of translation would be more complex.
Although proteins and other molecules may contain information beside DNA genetic information, most information that can pass to next generation is in DNA, so DNA directly controls enough information to construct organism. All the processes not directly controlled by DNA, including post-translational protein modifications and interactions, are the functions of proteins and will proceed automatically when the proteins are synthesized. All the information necessary for these functions is already contained in DNA sequences. Therefore, we do not need to take the post-translational processes into consideration when calculating genomic information needed to construct an organism.
The term "effective information" can be used to describe the minimal amount of information needed to construct an organism. The effective information actually means the genome size except "junk DNA" or "functional fraction" of genome. There are different formulas to calculate the effective information of viruses, bacteria, and eukaryotes because they have different genomic information structures.
Every organism has proteins, which need to be expressed in order, in time, and in quantity. Viruses have several to hundreds of proteins. For a complex virus with hundreds of proteins, it may need to separate expression of early and late proteins and maintain precise ratios of different protein products expressed simultaneously. Because all virus proteins are expressed only once, we only need to consider the order of virus protein expressions, while the quantities of protein expressions are controlled by the feedback through the affinity of regulatory factors to the binding sites.
There are many possibilities for the order of virus protein expressions and the protein sequences. If each protein is expressed one by one, the number of possibilities N can be calculated by using permutation and combination formula:
where x is the number of proteins, n 1 , n 2 , ... n x are the length of protein sequences (the unit is the number of amino acid residues).
As the probabilities for all amino acids and proteins are equal as analyzed before, the effective information can be calculated:
where n are lengths of virus proteins. The protein expressions may not be one by one in sequence. Some proteins may be expressed simultaneously. In this way, organisms may use less information to express proteins, depending on the specific expression routes. However, this way will be inflexible to change the routes under changing environments. Like encoding protein sequence, organisms may possibly use a mechanism that can cope with any routes and reduce the maximum entropy to 0. The maximum entropy is the case that all proteins are expressed one by one in sequence. In this way, organisms can change the expression routes easily without changing the mechanism of how the information system works.
Genetic information is stored in the form of DNA. DNA genetic code has degeneracy. In order to better compare the value of I and the genome size, we will not calculate the actual minimal information but the DNA information. So the formula becomes:
where 3 is the number of base pairs that make up a codon. The information I 1 forΣn 6 is of the protein coding sequences, because it is just the length of protein coding genes in bits. The information I 2 for log(x!) is of regulatory sequences. The x should be the number of protein linkage groups or operons. If all virus proteins are in one operon or linkage group or expressed simultaneously, no regulatory information is necessary. Because the number of protein linkage groups is usually unknown, we still use the number of proteins instead. The real I 2 in viruses is very possibly equal to x log(x) because in this way viruses can use fixed length of regulatory sequence log(x) for every gene or operon. This may simplify the mechanism of regulation. Although the information I 2 is calculated by assuming one by one protein expression, the result is that each gene or operon has a regulatory sequence, which is very possibly true.
The amount of effective information of Avian infectious bronchitis virus is: I=76,512 bits. The genome size is: C=length of genome 2 = 55,216 bits. So the I value is larger than the C value. After checking the virus genome, we find there is an overlap between genes. It's the overlap that makes the I value too large. When calculating the information of the overlapping part of the genes for the second time, because these base pairs already cannot change any more and there are no possibilities to be excluded, the amount of information for the second time calculation is 0. For example, sequences AACCC and CCCGG have overlapping part CCC. The information of the first sequence is 5 2 = 10 bits. The information of the second sequence is 2 2 = 4 bits because the information of sequence CCC is already known and this part contains no new information. So the information of sequence AACCCGG is 14 bits rather than 20 bits. The actual value of I 1 should be exactly equal to the size of the protein coding area. For virus genomes with overlapping genes, we use the actual size of protein coding area instead of Σn 6 to calculate the effective information. The effective information of some viruses is as follows ( Table 1) .
The effective information I of viruses range from thousands of bits to hundreds of thousands of bits. In general, single strand DNA viruses have the minimum amount of effective information, down to 3 thousands bits, while double strand DNA viruses have the maximum amount of effective information, up to hundreds of thousands of bits. Single strand RNA viruses and retro-transcribing viruses are between the above two.
The amounts of calculated effective information of viruses are consistent to the complexity defined by the number of proteins or genome size. In fact, the effective information is roughly proportional to the number of proteins. For viruses, the advantage of using effective information is not obvious because viruses are simple and the number of protein coding genes or genome size can be used to determine the complexity of viruses. For higher multicellular organisms, due to G-value paradox, the effective information will be more useful.
The calculation of effective information of bacteria is more complex than viruses' because the genes may express more than once in the growth of bacteria. There are regulations on protein sequency and quantity, but no regulation on the spatial deployment. The proteins can be thought to reach their positions in a cell automatically after synthesized.
A grown bacterium has roughly fixed volume, mass, and the number of protein molecules. It needs to produce all the protein molecules in its growth. The production of proteins in the growth is like chain reactions controlled by complex regulatory cascades with feedbacks. Even though the production of proteins is complex, we can always think that a bacterium grows theoretically from producing the first protein molecule to the last molecule in sequence. In this way, we can calculate the effective information. The problem is that many molecules may be produced simultaneously in the process. This may need less information. However, like viruses, in order to change protein expressions flexibly, organisms may use an information mechanism that can produce protein molecules one by one in sequence to reduce the maximum entropy to 0, which means each protein or operon has at least one regulatory sequence. Even if the calculated information may be more than necessary, we can still use k value later to adjust the information to fit to the actual amount of effective information based on real regulatory information of organisms.
The number of possibilities for the first molecule is:
where x is the number of proteins, n 1 is the length of the first protein.
If all the molecules are independent each other, the number of possibilities for all molecules by using permutation and combination formula is:
where y is the number of all protein molecules in a grown bacterium, n 1 , n 2 , ... n x are the length of proteins.
The regulatory information is calculated as encoding the order of all protein molecules in a cell, which includes timing and volume of expression. If you know the first 10 molecules produced are protein A, the next 100 molecules are protein B, the next 50 molecules are protein A again, the last 200 molecules are protein Z, you know the timing and volume of expression.
Although the chance for each protein in an organism may not be equal, the distribution is actually unknown for the organism because the distribution always needs to change under changing environment. For similar reason for encoding protein sequences, in order to cope with changing environments, organisms have to use a mechanism that can generate any distribution, including equal distribution, to reduce the maximal entropy to 0. This kind of mechanism provides flexibility for organisms to change the quantities of certain proteins any time to adapt the environments.
The effective information can be calculated:
The effective information includes two parts: first, the information to encode the protein sequences (I 1 ), and secondly, the regulatory information (I 2 ). The formula to calculate I 1 is the same as virus's. Converting the information to DNA information, the calculation can be simplified as:
where n is the average length of bacteria proteins, which is 308 for all bacteria [9] . I 1 should be adjusted to the exact size of the proteins coding area if there is overlapping of genes.
For regulatory information I 2 , in fact, every protein molecule is not produced independently. A bacterium does not once synthesize only one protein molecule, but a batch of protein molecules. Only the first protein molecule in the batch is independent, the possibility is × kinds of protein. The other following protein molecules are not independent. The possibilities follow the previous protein, which can only be 1. If a bacterium synthesizes 100 protein molecules at a time, I 2 is:
It can be expected that the number of protein molecules a bacterium once synthesizes may be proportional to the average quantity of the proteins y/x, which means the bigger the bacterium of the same structural complexity, the more the protein molecules it synthesizes at once.
where k is the average number of times a protein is synthesized. The value of k can be estimated as 5 based on the genomic information structure of E. coli (about 3% of E. coli genome is estimated as regulatory information). This is equivalent to that E. coli synthesizes 67 same protein molecules on average at a time. When the bacteria are very small, y is very close to x. The quantity of a kind of protein molecules may be less than 5. In order to ensure at least one protein molecule synthesized at once, the formula needs to be calibrated as:
This formula means that each protein has at least one regulatory sequence. The average number of regulatory sequences is k. The quantity of a certain protein is controlled by the times the protein is synthesized. The more the quantity, the more the times the protein is synthesized, the more the regulatory sequences. The average number of times is k. The quantity of protein synthesized each time is not directly determined by DNA information, but determined by the feedback through the affinity of regulatory factors to the binding sites. It is the number of regulatory binding sites upstream the protein coding gene sequence that determines the quantity of the protein.
In fact, most bacterial proteins are synthesized within the unit of operons, i.e. all the proteins in an operon are linked. They are not independent and are synthesized together. So the x in the formula should be the number of the addition of all operons and independent proteins in the genome. For example, the number of operons in E. coli is 834 [10] , with the number of independent proteins, together amounts to about 2,584. The x should be 2,584 for E. coli. Because the number of operons in most bacteria genomes is still unavailable at the present time, we calculated I 2 based on the number of proteins.
The complete formula to calculate the amounts of information of bacteria is:
where y is the total number of protein molecules in a bacterium. The effective information of some bacteria is as follows ( Table 2) .
The amounts of effective information of bacteria range from millions of bits to tens of millions of bits, just one order of magnitude higher than viruses'. The regulatory information, except very small bacteria and mycoplasma, accounts for 2~3% of the genome. The I values correlate well with the bacterial complexity defined by number of protein-coding genes, genome size, and volume. Bacteria's I values are higher than virus's. This is also consistent with our knowledge about complexity. The only thing that looks like an anomaly is that the I value of Haemophilus influenzae and Streptococcus pneumoniae exceeds the C value. Checking the average length of the proteins, we find that the actual protein average length of Haemophilus influenzae is only 235 (< 308) and the actual size of protein coding area is only 3.26e6 bits, while the calculated I 1 value is 4.26e6 bits, which already exceeds the C value. The corrected actual I value of Haemophilus influenzae is 3.39e6 bits, accounting for 91.6% of C value. Similarly, the average length of proteins of Streptococcus pneumoniae is 250, which is also much lower than the average value of bacteria. The corrected actual I value of Streptococcus pneumoniae is 3.77 e 6 bits, accounting for 92% of C value. Therefore, if precise I 1 value is needed, it should be directly calculated from the actual size of protein coding area, which is:
While the calculation of effective information of unicellular eukaryotes is the same as bacteria's, the calculation of I of multi-cellular organisms is much more complex because multicellular organisms not only need to produce all proteins to build different cells, but also need to put all the cells in spatial structures to build the organisms.
The number of possibilities for all the cells put together and the effective information can be calculated by using permutation and combination formula similarly, but the equation is very long and the explanation can be quite complex. It is better to separate the effective information directly to three parts and calculate separately: first, the information to encode all the proteins (I 1 ); secondly, the information to produce all the differentiated cells (I 2 ); and finally, the information to construct the spatial structures (I 3 ).
The information to encode all the proteins is the size of the protein coding area in the genome, like bacteria's I 1 . There are also overlapping genes in eukaryotic genomes, and the overlapping genes account for considerable weight [11] . So these overlapping parts must be adjusted according to the actual size of protein coding area, otherwise these parts will mix with other parts of information and can cause confusing results.
If the genome is not yet sequenced, this information can be calculated:
where g is the number of protein coding genes, n is the average length of proteins of eukaryotes, which is 448 [9] . In this way, the calculated I 1 is usually larger than the actual size of protein coding area.
To produce all the differentiated cells, proteins need to be chosen from the complete proteome. For one type of cells, the algorithm is similar to bacteria's,
Let x be the size of complete proteome (number of functional proteins before post-translational modifications), let t be the average size of cellular proteome of differentiated cells, cn is the number of cell types, a 1 , a 2 , a 3 ,..., a cn are the diversities of differentiated cellular proteomes from t. Because many genes expressed in the differentiated cells are the same, t+a 1 +a 2 +a 3 +...+a cn =x, then the information to produce diverse differentiated cells is:
a k x t a a a a k 2 1 12 
where k 2 is a coefficient. The formula means choosing t+a 1 from × to construct the first type of cell, and choosing a 2 from x-t-a 1 to construct the second cell, and so on. The possibilities for every protein molecule in all cell types are t, and the numbers of free protein molecules are proportional to the diversities of cellular proteome. As the calculated value of Σlog(C) is actually quite small and can be negligible, the information to produce diverse differentiated cells is:
For unicellular eukaryotes, the calculation of I 2 of is the same as bacteria's:
where x is the number of proteins. k 1 is estimated as 30 based on the information structure of Saccharomyces cerevisiae (Baker's yeast) genome to make the amount of effective information account for about 80% of the genome. This is equivalent to that a yeast cell synthesizes average 1000 protein molecules at once.
Because there are regulations between the cells of multicellular organisms, k 2 can be larger than k 1 , estimated as 110, which is equivalent to a cell synthesizes average 820 protein molecules at a time.
The average number of genes expressed in specific tissues is about 5000. The proteome size of a specific tissue ranges from a few thousand to tens of thousands. Because a tissue contains diverse cells, the proteome size of one differentiated cell may be a few thousand. Because most proteome data are still unavailable and are confusing with the proteome containing post-translational modifications, transcriptome data from clustered EST can be used instead. The number of unique sequences of clustered ESTs from human breast tumors is 6501 [12] , so we estimated t=6500 as the average cellular proteome size of differentiated cells. With all the differentiated cells available, organism spatial structure can be constructed. Let z be the total number of cells in an organism.
In the process of organismal development, cell divisions are controlled by complex regulatory signals. We can always think that cells are produced from the first one to the last one in sequence. When the last cell is produced, the development ends and the organism reach its adult weight. In this way, information can be calculated. Although some cells may be produced simultaneously, organisms may use an information mechanism to reduce the maximum entropy to 0, which is the case that all cells are produced one by one in sequence, in order to keep the flexibility to change the cell development routes easily. Even if the calculated information may be more than necessary, it can still be adjusted by k 3 value later to fit to the actual amount of information.
Like cell division, there is only one appropriate position between two adjacent cells. In this way, the spatial structure can be easily constructed. Let us start from deploying the first cell. If all cells are independent, the number of possibilities to produce and deploy all cells can be calculated using permutation and combination formula:
The information is:
The chances for different cell types and different positions may not be equal, but they are unknown. Organisms have to use mechanisms that can generate all possible distributions of cells and positions, including equal distribution. This kind of mechanisms provides flexibility for organisms to change the quantities and positions of cells easily and efficiently to adapt the environments.
When z is larger than 10 5 , log(z!) is very close to zlog (z) (> 90%). In order to simplify the calculation, log(z!) is replaced by zlog(z). When z is small, log(z!) is still used. The actual situation is that all the same cells are generated and deployed by many times. 
where k 3 is the average number of times one type of cells is generated. cn should be the number of cell types or the number of linkage groups. In fact, in the organs or tissues of multicellular organisms, many cells are linked, causing repetitive pattern in the organ or tissue. So the numbers of cell types cn in the formula should be replaced by the number of linkage groups. There is no regulatory information needed for genes or cells inside a linkage group. However because the numbers of linkage groups in organisms are still unknown, we calculated I 3 based on the number of cell types.
The formula also means each type of cells has at least one regulatory sequence. The average number is k 3 . The quantities of each type of cells are controlled by the number of times this type of cells is generated. The more the quantity, the more the times this type of cells is generated. The quantity of cells generated each time is not directly determined by DNA information, but by the cellular feedback signals. It is the times of generating that determines the quantities of each type of cells.
k 3 can be estimated as 2.5e4, which is equivalent to that human body averagely generates and deploys 4.2e7 cells at once, that is about 0.012g. The k 3 value is determined by the information structure of Schistosoma mansoni and Caenorhabditis elegans. Because C. elegans is very small and the I 3 of which is almost negligible, and Schistosoma mansoni is bigger than C. elegans but the phenotypic complexity should be a little bit less than C. elegans, therefore we adjusted the k 3 value to make the I value of Schistosoma mansoni close to, but a little bit lower than the value of C. elegans. So we take k 3 value as 2.5e4.
When z is very small, z is close to cn. That may make organisms once generate less than one cell. To avoid this kind of error, the formula can be calibrated as:
The final formula to calculate the effective information of Eukaryotes is:
110 6500 2 5 10 1 2 5 10
The x in the formula is the size of complete transcriptome. Because the size of proteome before post-translational modifications is still unknown, we use the size of complete transcriptome instead.
The sizes of complete transcriptome come from clustered EST data. As the number of genes is quite different from the number of clustered EST, it is a problem how to use the data from EST databases. There are a few databases having EST fragments and mRNA assembled and clustered to reduce the redundancy for gene discovery, but different databases give different results. For example, the number of human UniGene clusters is about 120,000, while the number of unique sequences of human EST clusters in The Gene Index project (TGI) of Harvard University is 1,080,000 [13, 14] . The difference between the two databases is supposed to be that TGI separated alternative splicing sequences and tried to produce tentative consensus, while UniGene put all the overlapping sequences together in one cluster. However only knowing this does not help match the data from the two databases. We still did not know the actual size of human complete transcriptome.
Even in one database, the data are often conflicting each other. For example, the number of human Uni-Gene clusters is about 120,000 [15] . UniGene means unique gene, and is supposed to cluster the transcribed ESTs and mRNA into unique genes. So the number of UniGene clusters should be equal to the number of genes. However, there are only about 25,000 genes in human genome, while the number of UniGene clusters that contain only one sequence is more than 40,000. There must be errors inside. Zhang et al analyzed the results of UniGene clusters [16] and pointed out that most narrowly expressed transcripts (NETs), whose expression is confined to a few tissues, resemble intergenic sequences, and most NETs are singleton clusters containing only one EST or mRNA sequence. So those singleton clusters seem unreliable. The sequences in these clusters may come from non-coding RNA, contamination of pre-mRNA, genomic DNA, non-canonical introns or foreign sources, or simple sequencing errors.
Owing to the establishment of other specialized databases, we can resolve this problem, at least the size of the human transcriptome. The Alternative Splicing Prediction Data Base (ASPicDB) in Italy has predicted almost all human alternative-splicing transcripts and has them listed in detail [17] . They analyzed 18,193 human genes and found 319,745 transcripts, which on the whole can represent the size of complete transcriptome because their data correspond quite well with the data from TGI.
In TGI's data, 730,000 of 1,080,000 human unique consensuses are singleton. The total number of human genes is about 25,000, among which 35~60% contain alternative splicing, so the number of singleton should be about 10,000~15,000. Obviously, most sequences in the singleton are the result of errors. The real singleton sequences perhaps are already included in the tentative consensuses (TC). So we discarded all unique singletons and only count the number of TCs and obtain the result of 328,301. As ASPicDB only analyzed 18,193 genes, while human beings have 25,000 genes. If other genes do not have alternative splicing, then the size of total transcriptome should be 326,552, which is quite close to the result of TGI. It is a pity that only human data in ASPicDB is fairly comprehensive, the data of other species are quite sparse and cannot be used in the same manner.
Because TGI is not updating the data regularly and many data was released in 2006, which may be out of date, so we use UniGene data as supplement. In order to take advantages of both UniGene and TGI, we took the average value of the two databases as the complete transcriptome size x.
We discarded all the error prone clusters in UniGene that contain only one sequence. Having the number of the remaining UniGene clusters multiplied by possible average number of alternative splicing, we could match the two databases. For example, the number of human UniGene clusters after the treatment is 82,718. After multiplied by the possible number of alternative splicing 4, we got 330,872, which is close to the result of TGI. The UniGene result of mouse is 56,365 4 = 225,460, which is close to TGI result 210,249. We supposed the average number of alternative splicing for mammals is 4, for birds is 3, for fishes, amphibians and chordates is 2, for other animals is 1.5, for plants is 2~2.5, to make the results from the two databases as consistent as possible. We also referred to other databases.
The sizes of complete transcriptome of some eukaryotes are as follows ( Table 3 ). The species were chosen for two reasons: first, we chose the species with higher numbers of UniGene clusters among the close species because the data are still incomplete; secondly, the species should have TGI data or other data sources. Plant species were chosen only to illustrate that this method can apply to plant.
In general, the results from the two databases are consistent. Some results of mammals obtained from TGI are lower than UniGene's. It's probably because some of TGI's data are too old, or because there are real differences in the average number of alternative splicing among mammals, but if so, it will be difficult to understand the huge difference between mouse and rat. At the present time, although the transcriptome data (clustered EST data) of many species are available, most of them are incomplete.
The TGI result of Danio rerio is too high. We cannot explain why. Perhaps those clusters contain too much gene fragments. The data of UniGene and TGI are far from perfect because they cannot correspond to the number of genes. Only if every transcript corresponds to every gene, like ASPicDB, the data can be more reliable.
The data of the number of cell types of eukaryotes can be calculated. We know the number of cell types of adult human body is 210 [18] ; sponges have 12 kinds of cell types [19] ; the simplest multicellular organism Trichoplax adhaerens has 4 types of cell [20] ; C. elegans has 27 types of cell [21] . Because the tanscriptome size of specific cells are relatively fixed, it can be anticipated that the larger the size of complete transcriptome of an organism, the more the number of cell types. Based on the data of Valentine's [22] , a linear relationship between the number of cell types cn and the size of complete transcriptome x can be drawn (Fig 1) . The number of cell types can be roughly calculated as:
( ) Table 3 The size of transcriptome of eukaryotes There is not yet evidence if the formula apply to plants, but the cn of plants are calculated using the formula in this paper just to illustrate rough range of effective information of plants.
With all the data available, the effective information of eukaryotes can be calculated (Table 4 ). If the genomes of some organisms are not yet sequenced, the numbers of genes are estimated according to the close species (marked by * sign). Because eukaryotes also have the problem of gene overlapping, it is better to find the exact size of protein coding area to calculate I 1 . Sometimes the size of protein coding area is quite different from the result calculated from the number of genes. For unicellular organisms, if the transcriptome sizes x are not available, then x are the numbers of genes.
After attentive observation of the I values, one can clearly see that the results demonstrate a definitive correlation between the amounts of effective information and the organismal phenotypic complexity defined by biological taxonomy and evolutionary theory. C. pombe has the lowest I value, while human beings have the highest. Nematode, insects, amphioxus, fish, frog, bird falls in between. The I values of eukaryotes range from tens of thousands to hundreds of thousands of bits, which are just one order of magnitude higher than prokaryotes'. These results are also consistent to our intuition about organismal complexity, whilst the number of genes is a poor index here. P. tetraurelia has quite high number of genes 39,642, but is actually a simple single cellular organism with low I value. So the effective information is a much better index of organismal phenotypic complexity.
The I value of Danio rerio (Zebra fish) is too high because the x is too high (higher than chicken's). The effective information of Ciona intestinalis accounts for 69% of the genome (almost all the non-repetitive sequence), that means the genome is quite compact and there are less junk DNA, therefore it will be easier to study the genomic information structure of this organism.
The x should be the number of transcripts that produce functional protein sequences. One transcript should correspond to one protein sequence, and vice versa. Every UniGene cluster should correspond to one gene and every TC in TGI should correspond to one protein sequence, and vice versa. ASPicDB uses a better algorithm because it makes all transcripts correspond with genes and proteins very well. The value of x is very important because the effective information is roughly proportional to x. The diversity of proteins before posttranslational modifications can reflect the complexity of organisms.
The number of cell types was calculated with all neuron cells as one type. Some argued that neurons should be counted as different kinds of cells because they are functionally differentiated. It is difficult to count the number of neuron types at the present time. Perhaps in the future, a more objective and high throughput method can be found to count the number of neuron types. This may explain why the number of cell types reaches saturated at mammal level.
Among the alternative splicing isoforms in organism transcriptome, how many are functional is still disputable. Some alternative splicing may cause premature termination codon (PTC); and the alternative isoforms with PTC can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery. According to ASPicDB, the number of transcripts of the human genome is about 320,000, among which only 30,000 may generate PTC+ isoforms. If this part is discarded, there will be no important effect on the calculation. As PTC related data of most organisms are still unknown, this part is not taken into consideration at this time.
There is an implicit assumption in this paper: all prokaryotes have the same k value and so do eukaryotes. It needs to be verified for this assumption to hold. The exact values of k can be determined when the genomic information structures of model organisms are completely clear. For example, when the genomic information structures of C. elegans are completely known, the value of k 2 can be completely determined. When the genomic information structures of C. intestinalis are known, the value of k 3 can be more precisely determined.
The information contained in I 2 and I 3 cannot be included in the sequences of regulatory proteins. I 2 is composed of regulatory sequences or regulatory factor binding sites. I 3 may be composed of regulatory noncoding RNA sequences.
It is clear that the effective information increases along with the increase of organismal phenotypic complexity defined by taxonomy, evolution, and intuition. The simpler the organism, the lower the I value. The more complex the organism, the higher the I value. The effective information in viruses is between 10 3~1 0 5 bits, while in bacteria is between 10 6~1 0 7 bits, and in eukaryotes between 10 7~1 0 8 bits. For multicellular organisms, the effective information increases from 4.68e7 bits of placozoa to 8.38e8 bits of human beings. Worm, insects, amphioxus, fish, frog, bird falls in between. These results are consistent to other observations with the number of cell types [22] , and the number of miRNA families [23] . Aburomia et al calculated the morphological complexity of 21 extant higher-order chordate groups based on the presence or absence of 479 morphological characters [24] . Their results are consistent to ours. Therefore, the effective information can be used as a quantitative measure of organismal phenotypic complexity from the simplest viruses to the most complex human beings. While limited by the incomplete data presently available, some results may not be so accurate, but the approximate range will remain true. When data become more complete and accurate, more precise calculation can be conducted.
Studies have reported increasing morphological complexity in multiple parallel lineages of the Crustacea [25] . When the phenotypic complexity of more organisms can be precisely calculated as effective information, it will be easier to study the evolution of organismal complexity.
The results of effective information of mammals are also consistent to the recently published article regarding the amount of constrained sequence in genome shared between eutherian mammals [26] . The constrained sequence means the sequence under functional constraint. The total amount of constrained sequence in rodents is estimated as 260Mb (5.2e8 bits), which is close to 5.09e8~6.34e8 bits effective information of rat and mouse. 300Mb (6.0e8 bits) of human genome is estimated under functional constraint. This is also close to 8.38e8 bits effective information of human. For fruit fly, the amount of constrained sequence is estimated as 55.5~66.2Mb (1.1~1.32e8 bits), which is also close to 1.09e8 bits effective information. Therefore, the effective information can be used as an estimate of functional fraction of genome.
The genomic information of viruses, bacteria, and some eukaryotes can be found at GenBank. where 128 and 18 is the mean molecular weight of amino acids and water respectively, 6.02e23 is the number of molecules of one mole.
The data for x comes from the genome database of GenBank. The data for volumes of bacteria can be found at website: http://www.ionizers.org/Sizes-of-Bacteria.html. The volumes of bacteria can be calculated based on rod lengths and rod or coccus diameters.
Given the average cell volume v of multicellular eukaryotes as 300 μm 3 , the total cell number z of an organism is: z = w/v = (w/3) 10 10 , where w is the weight of the organism.
The data of UniGene can be found at website: http:// www.ncbi.nlm.nih.gov/sites/entrez?db=UniGene. On the homepage of UniGene, you can see the number of Uni-Gene clusters of diverse organisms. Click on the organism name and enter the page of statistic data, you can see "Final Number of Clusters" and "Histogram of cluster sizes". With the number of total sets subtracting the number of cluster sizes with only one sequence, you can obtain the number of UniGene clusters.
TGI's data can be found at website http://compbio. dfci.harvard.edu/tgi/tgipage.html. Click on the organism names, you can enter the database of that organism. There are statistical data for every organism. Only the number of TC sequences is what we need. The singleton data can be ignored.
Alternative splicing data can be found at http://t.caspur.it/ASPicDB/. There is statistic data of human alternative splicing on the homepage, including number of genes, transcripts, etc. Although there are also alternative splicing data of other species, they are not yet complete enough. You can also obtain the numbers of genes expressed in a specific tissue from this database. In the advanced search page, you can search genes with different specific tissue names in the search bar, and then you can get the number of genes. We had these numbers averaged and got about 5000. The database does not give the data of transcripts expressed in different tissues; otherwise the average size of transcriptome t can be obtained this way.
The data of average adult weights of eukaryotes are calculated based on the body sizes, which can be found or estimated from various sources.
All the calculation can be conducted by simple Perl scripts, which are available on request.
Reviewer #1: Dr Lavanya Kannan (nominated by Dr. Arcady Mushegian) and Dr. Arcady Mushegian
The paper presents a method to calculate the phenotypic complexity of organisms. The phenotypic complexity of an organism is a measure of uncertainty associated with the size of the genome sequence, and is mathematically defined as the information entropy of the system. The amount of effective genomic information needed to produce a gene/protein sequences from a random sequence is at least the information entropy. The approach uses permutation and combination formulas to model the information needed to encode proteins for simple organisms like viruses, bacteria and other single celled organisms; and also extends the method to compute the information needed to produce differentiated cells and to construct spatial structures formed by the cells in higher organisms. The approach is not without interest, but several questions need to be addressed.
1. The main question is whether the complexity estimates given by the computations in this manuscript are any better than simply the number of protein-coding genes. Examining the I values for various species, from viruses to higher eukaryotes, one gets an impression that I is roughly proportional to gene numbers. Is this the case or not? If yes, what is the advantage of using I, and if no, then the relationship between the two is worth discussing in some detail.
2. In the calculation of I =Σn 6 for viruses (which is also I 1 for all the higher organisms discussed), where Σn is the summation of the lengths of all protein sequences, the authors make the following note: I is the same as the size of the protein coding area in the DNA sequence. It would be helpful if this equality may be explained. This paragraph, as many others, suffers from simplistic explanation of biological phenomena. In p.4: \...there are no regulations of quantity of gene products. Viruses only need to produce their proteins one by one in order." -this is not true, most if not all viruses have elaborate mechanisms of, e.g., separating the expression of early and late proteins; of maintaining quite precise ratios of different protein products expressed simultaneously; etc. Many of these processes require action of virus-encoded signals and cellular proteins. But is this relevant for computing complexity? in p. 5:\In fact, mutations are not normally allowed for a real protein sequence" -not true, viruses are notorious for rapid evolution that is facilitated, in the case of RNA viruses, by a particularly high mutation rate (but again, is this information even needed for what authors are proposing?). Why does the quantity I hold for cases of overlapping genes? A simple example that exemplifies both the above facts would be beneficial for the readers.
3. In the calculation for eukaryotes: For genomes that are not sequenced, it is noted that I 1 =6n g. It is also mentioned that in the case of overlapping genes, this quantity should be adjusted to the size of the proteincoding genes. How can this be done for the genomes that are not sequenced? Can this be elaborated?
Authors' response The comments are insightful. We have revised the manuscript based on the review, especially the calculation of effective information of viruses. raised if viruses are therefore more primitive than rickettsiae, bacteria, fungi, algae, plants or animals. Indeed, a single cell alga is more complex than bacteria. Can gastrula be more advanced than a parasite in the gut of a termite? Can an organism like a viroid more primitive than free living algae? To prevent such issues, the domain of study and the scale of complexity must be clearly defined.
4. The manuscript needs a technical editing. Just to mention some example problems here: the sentence "Set × is the size of complete proteome.." is not clear. I believe what authors intend to say is "Let × be the size of complete proteome..". I also notice that the word "once" has been misplaced or misused in some sentences. Note that "once synthesized" means differently from "synthesized at once"
Authors' response The comments are constructive. We have revised the manuscript based on the comments. We focus on the methodology how to calculate effective information rather than evolutionary claims in this manuscript.
Reviewer #3: Dr. ED Rietman (nominated by Dr. Marc Vidal)
The authors are to be commended for taking on a challenging and important biological question. Their basic hypothesis is that one can use the standard information measures on DNA strings and induce similar information measures on numbers of proteins in all types of cells. The premise is that there is in increase in complexity over the course of biological evolution and that this increase in complexity comes about as a result of a reduction in entropy.
The soundness of the hypothesis will not be commented upon, because there is so much doubtful with the basic premise. Start with a simple self-replicating autocatalytic set of molecular species. If mutation-based evolution can operate on this set, there will be an increase in the complexity of the molecular species in the set. This increase in complexity is driven primarily by chemical potential and reduction in free energy. The increase in the number of new molecular species capable of participating in the reaction set results in more ways to dissipate the free energy, and thus an increase, not a decrease, in entropy [28] [29] [30] .
Similarly, in a microbiological ecosystem with competing microorganisms, mutation-based evolution will increase the microorganism-based species diversity and drive up the number of ways the free energy may be dispersed. Again this results in an increase in entropy.
Besides misunderstanding fundamental thermodynamic issues, there are cellular biology errors. To support the calculations it is assumed that all proteins are produced at once. This conjecture can again be argued away, because chemical reaction networks cannot produce all molecular species at once. The chemical potential imbalance, at various points in the network, is the driving factor to produce other chemical species (Le Chatelier's principle). This same logic carries over into molecular systems biology and thus into cellular biology.
There is an insufficient review of the pertinent literature. The paper is not a review, but still a few paragraphs of review of other approaches to addressing this important question would have put this new work in perspective.
Authors' response It is true that mutation-based evolution is an entropy increase process, but evolution may not only be mutation-based. There may be other evolutionary mechanisms. Anyway, we have deleted those parts regarding entropy in this manuscript. Environmental factors preceding illness onset differ in phenotypes of the juvenile idiopathic inflammatory myopathies Objective. To assess whether certain environmental factors temporally associated with the onset of juvenile idiopathic inflammatory myopathies (JIIMs) differ between phenotypes. Methods. Physicians completed questionnaires regarding documented infections, medications, immunizations and an open-ended question about other noted exposures within 6 months before illness onset for 285 patients with probable or definite JIIM. Medical records were reviewed for 81% of the patients. Phenotypes were defined by standard clinical and laboratory measures. Results. Sixty per cent of JIIM patients had a reported exposure within 6 months before illness onset. Most patients (62%) had one recorded exposure, 26% had two and 12% had three to five exposures. Patients older than the median age at diagnosis, those with a longer delay to diagnosis and those with anti-signal recognition particle autoantibodies had a higher frequency of documented exposures [odds ratios (ORs) 95% CI 3.4, 31]. Infections were the most common exposure and represented 44% of the total number of reported exposures. Non-infectious exposures included medications (18%), immunizations (11%), stressful life events (11%) and unusual sun exposure (7%). Exposures varied by age at diagnosis, race, disease course and the presence of certain myositis autoantibodies. Conclusion. The JIIMs may be related to multiple exposures and these appear to vary among phenotypes. The juvenile idiopathic inflammatory myopathies (JIIMs) are a heterogeneous group of acquired systemic autoimmune diseases characterized by symmetric proximal weakness, the presence of characteristic rashes and other systemic features. While the aetiology of these disorders remains unknown, many lines of evidence suggest that they result from the interaction of multiple genetic risk factors and environmental exposures [1] .
The JIIMs, like other autoimmune disorders, appear to be comprised of a number of clinical and serological phenotypes, each of which defines more homogeneous subsets of patients in terms of demographic features, the presence of certain myositis-associated autoantibodies, immunogenetics and outcomes [2, 3] . For example, patients with anti-p155 autoantibodies form a phenotype characterized by the frequent presence of cutaneous involvement and characteristic photosensitive rashes of JDM and the HLA-DQA1*0301 allele, whereas patients with anti-synthetase autoantibodies frequently have moderate to severe weakness, arthritis, RP, mechanic's hands, fevers, interstitial lung disease and HLA DRB1*0301 [3] [4] [5] . Clinical features of illness also appear to differ by age, gender, race and even disease course phenotypes [6] [7] [8] . Such homogeneous phenotypes might share unique combinations of environmental and genetic risk factors that result in a discrete disorder [9] .
Several genetic risk factors for the JIIM have been defined, including MHC Class II alleles [10, 11] , cytokine polymorphisms [12, 13] , the protein tyrosine phosphatase gene N22 [14] and Gm and KM allotypes [15] .
Environmental risk factors in JIIMs are not as well understood, and most efforts have focused on the potential role of infections in their aetiologies. Studies of cohorts of patients with JDM indicate that respiratory and gastrointestinal infections may be temporally associated with the onset of JIIM [16, 17] . Prior studies of other autoimmune diseases suggest differences in environmental risk factors in different phenotypes [9] , but the relationship between environmental risk factors and phenotypes has not been examined in the JIIMs [16, 17] .
We, therefore, undertook this study to examine whether environmental factors that are temporally associated with the clinical onset of JIIM differ in selected phenotypes, focusing on a large, well-characterized population with data on both infectious and non-infectious exposures.
Four hundred and twenty-three patients with probable or definite JDM or juvenile PM (JPM) [18] were enrolled into the NIH Clinical Center or Food and Drug Administration's investigational review board-approved natural history protocols from September 1994 until July 2008; subjects' written consent/assent was obtained according to the Declaration of Helsinki. The study was approved by the NIDDK/NIAMS Institutional Review Board. Enrolled patients provided a blood sample for autoantibody testing and the treating physician completed a questionnaire that included clinical, demographic and laboratory data. For 285 of these patients, questions about factors temporally associated with illness onset were also completed, which is the basis of the present study. Informed consent/parent assent was consistent with the Declaration of Helsinki. Phenotypes were defined by age of illness onset, clinical features, disease course, race or autoantibodies. Disease course was classified as monocyclic if the patient achieved remission without evidence of active disease, based on clinical examination and laboratory testing, within 2 years of diagnosis; as polycyclic if the patient had recurrence of active disease after a definite remission; as chronic continuous if disease activity persisted for >2 years; and as undefined if follow-up was <2 years from the time of diagnosis [8] . Clinical, demographic and autoantibody characteristics of the study population are described in Table 1 . Only the autoantibody phenotypes defined as anti-aminoacyl-tRNA synthetase, anti-signal recognition particle (anti-SRP), anti-Mi2, anti-p155, anti-MJ, anti-U1 RNP and autoantibody negative were included in the analyses of environmental factors.
The physician questionnaire contained three questions about environmental exposures that had been previously suggested to be possibly associated with the onset of JDM [16, 17, 19, 20] . These included whether the patient had any documented infections, received any immunizations or took any medications (including vitamins, minerals, herbal preparations and dietary supplements) within 6 months before illness onset. The questionnaire also included an additional open-ended question about other environmental exposures within 6 months before illness onset relating to other possible triggers of disease and to specify these and when they occurred. Stressful life events were categorized as major vs minor and as One hundred and twenty-one patients were tested by IP immunoblotting. a Other myositis autoantibodies, which were not examined in the environmental exposure analysis, included: anti-Ro (n = 15), anti-PM/Scl (n = 5), anti-Sm (n = 3), anti-La (n = 2), anti-U5 RNP (n = 1), anti-U3 RNP (n = 1), anti-Ku (n = 1) and anti-Th (n = 1). Some patients have more than one myositis autoantibody. One hundred and one patients were not tested for myositis autoantibodies.
network, family, academic or unknown type, based on the Adolescent Perceived Event Scale of Compas et al. [21] (personal communication: B. Compas, Vanderbilt University). Illness onset was defined as the month and year when the first symptom related to myositis developed. A paediatric rheumatologist (L.G.R. or G.M.) reviewed available medical records for 81% of the patients in order to confirm the reported exposures, as well as the diagnostic and clinical material contained in the questionnaire. Patient sera were tested for myositis autoantibodies by validated methods [22, 23] . For anti-p155/140 and anti-MJ autoantibodies, serum samples were screened by immunoprecipitation (IP), and this was confirmed by IP blotting [5, 24] . Sera were considered positive if they blotted the antigen in immunoprecipitates prepared using reference serum (direct) or if reference serum blotted the antigen in immunoprecipitates prepared using patient serum (reverse). Since some IP-positive sera do not react by immunoblotting, reverse IP blotting was used for most sera [5] .
Case-only analyses were conducted to describe the frequency of exposures overall and in relation to patient phenotype. Statistical analysis was performed using Sigma Stat Version 3.1 (Systat Software, Inc., Chicago, IL, USA), including 2 and Fisher's exact tests to determine differences in the proportion of patients with different environmental exposures. Odds ratios (ORs) and 95% CIs were calculated using GraphPad InStat version 3.06 (GraphPad Software, San Diego, CA, USA). P-values were adjusted for multiple testing using Holm's procedure [25] , using SAS System for Windows, version 9.1.3 and SAS Enterprise Guide Version 4.1 (SAS Institute, Cary, NC, USA).
Sixty per cent of JIIM patients had one or more reported exposures within 6 months before illness onset ( Table 2 ). The total number of reported exposures was more frequent in white patients than in other racial groups (P = 0.008; OR 2.1; 95% CI 1.2, 3.5; Table 2 ). Although most patients (62%) had only one reported exposure, 26% had two exposures, and 12% had three to five recorded exposures in the 6 months before illness onset ( Table 2) . Of the 64 patients with more than one exposure, 50% had a combination of infection and medication, and 27% had a combination of infection and immunization. The combination of infection and immunization was more frequent in patients from non-white racial groups (P < 0.0001; OR 22.0; 95% CI 5.9, 81.7).
Patients who were >7.5 years of age at diagnosis (the median age at diagnosis) more often had three to five reported exposures compared with younger patients (P = 0.027; OR 3.7; 95% CI 1.3,10.6; Table 2 ), and patients with anti-SRP autoantibody more often had three to five exposures compared with patients without a myositis autoantibody (P = 0.027; OR = 31.0; 95% CI 1.9, 507). Patients with a longer delay to diagnosis (>4 months, the median delay) were more likely to have three to five exposures than patients with a shorter delay (P = 0.034; OR 3.4; 95% CI 1. 2, 9.8) . There were no other significant differences in the frequency or number of noted exposures between clinical phenotypes [JDM vs juvenile PM (JPM)], nor by gender, disease course, delay to diagnosis or between other autoantibody phenotypes (data not shown).
Infections were the most common type of exposure identified within 6 months before diagnosis, consisting of 45% of the total number of reported exposures, followed by medications (18%) and immunizations (11%) ( Table 2 ). Patients 47.5 years of age at onset, those with 44 months delay to diagnosis, those with a polycyclic illness course and those who were myositis autoantibody negative were more likely to have an infection in the 6 months before diagnosis than older patients, those with greater delay to diagnosis, those with a monocyclic or chronic continuous illness course or those who had anti-p155 or anti-SRP autoantibodies (ORs 1.8-4.3, Table 2 and data not shown). There were no other differences between documented types of exposure between clinical or other autoantibody phenotypes, nor by gender, disease course, race or delay to diagnosis.
From the open-ended exposure question, stressful life events constituted 11% of the reported exposures. Patients >7.5 years of age reportedly experienced a stressful life event more frequently in the 6 months before diagnosis than younger patients (P = 0.003; OR 3.5; 95% CI 1.5, 7.9). Unusual sun exposures comprised 7% of the total exposures in the 6 months before diagnosis and occurred exclusively in patients with JDM, not JPM. Unusual sun exposures included those resulting in sunburn, as well as receiving more sun than usual or travel to a more sunny location. An unusual chemical exposure was recorded 3% of the time and included application of pesticides inside or around the home, painting the home, use of formaldehyde to clean the child's bed and application of a hair perming chemical. Seven (2%) reported exposures involving unusual animal contact within 6 months of illness onset, including a dog or cat scratch, exotic bird bite or multiple flea or mosquito bites. Four (2%) exposures involved weight-training exercise or physical trauma, and two (0.6%) exposures involved dietary supplement usage before illness onset, including creatine monokinase and Echinacea. These less-frequent exposures were present exclusively in patients with JDM, except that weight-training exercise was also noted in one JPM patient. Weight training, physical trauma and dietary supplements were seen exclusively in patients >7.5 years of age at diagnosis and in patients with a greater delay to diagnosis.
White patients and patients who did not have an identified myositis autoantibody were more likely to have a documented infection within 6 months before illness onset than those from other racial groups or those with the anti-p155 autoantibody (P = 0.0008; OR 2.7; 95% CI 1.5, 4.8 and www.rheumatology.oxfordjournals.org None 114 (40) 62 (41) 52 (38) 69 (35) 45 (52) 15 (32) 31 (48) 49 (42) 20 (46) 19 (36) No. of patients exposed (22) 8 (25) 10 (30) 15 (22) 8 (34) 8 (24) 3-5 20 (12) 5 (6) (12) 4 (10) 2 (7) 1 (3) 12 (18) 2 (8) 2 (6) (18) 27 (17) 35 (20) 48 (18) 14 (20) 11 (19) 13 (22) 26 (18) 15 (33) zz
Immunization 37 (11) 16 (10) 21 (12) 29 (11) 8 (12) 10 (18) 4 (7) 14 (10) 5 (11) 6 (8)
Other 85 (25) 28 (18) P = 0.007; OR 3.9; 95% CI 1.5, 9.9, respectively; Table 3 ). The majority (68%) of patients with an infectious exposure had one documented infection, but 28% had two and 4% had three to five infections documented within 6 months of illness onset. Patients >7.5 years of age were more likely than younger patients to have two infections (P = 0.038; OR 2.7; 95% CI 1.1, 6.3; Table 3 ). Respiratory infections were the most common type of infection reported, followed by mucocutaneous and gastrointestinal infections (Table 3) . Pharyngitis was the most frequent specific infection and was more prevalent in patients >7.5 years of age than in younger patients (P = 0.017; OR 2.7; 95% CI 1.2, 6.0; Table 3) . A flu or febrile illness and otitis media were each seen in 13% of patients, and an upper respiratory infection in 10% of patients within 6 months before illness onset. There were no other differences noted in the infection site or type of infection among clinical or autoantibody phenotypes, nor by gender, race, disease course or delay to diagnosis.
Patients 47.5 years of age were more likely to have one drug exposure (P = 0.004; OR 15.4; 95% CI 1.8, 135), whereas those >7.5 years of age were more likely to have two drug exposures (P = 0.008; OR 18.9; 95% CI 1.0, 358) in the 6 months before illness onset (Table 4) . Of interest, >25% of the medication usage documented included drugs that were potentially photosensitizing or myopathic [26] [27] [28] [29] [30] [31] . There were no other differences noted in medication usage between clinical or autoantibody phenotypes, nor were there any differences by gender, race, disease course or delay to diagnosis.
There was no difference in the proportion of patients who received an immunization in the 6 months before illness onset or in the number of immunizations received, between clinical or autoantibody phenotypes, nor by age, gender, race, disease course or delay to diagnosis. Patients with a polycyclic illness course were more likely than patients with a monocyclic illness course to have received an immunization or to have received a measles-mumps-rubella (MMR) vaccine in the 6 months before illness onset (21 vs 6%; P = 0.023; OR 4.1; 95% CI 1.2, 13.9 and 50 vs 6%; P = 0.035; OR = 17.0; 95% CI 1.3, 223, respectively). Given the time period under study, it was not surprising that patients >7.5 years of age at diagnosis were more likely to have received a hepatitis B vaccine than younger patients (47 vs 12%; P = 0.002; OR 6.2; 95% CI 2.0, 19.7), whereas patients 47.5 years of age were more likely to have received a diphtheria-(pertussis)-tetanus vaccine (22 vs 6%; P = 0.033; OR 8.2; 95% CI 0.98, 69.8).
Nine per cent (n = 26) of patients had at least one stressful life event in the 6 months before illness onset, with 72% of these being major stressors and the remainder being minor stressors. The majority of these patients (65%) had one stressor, but 31% had two recorded stressors and 4% had three stressors. The categorization of stressors included network (50%), family (25%), academic (19%) and unknown types (6%). Patients >7.5 years of age had a stressful life event more frequently than younger children (P = 0.003; OR 3.5; 95% CI 1.5, 7.9). There were no differences in the proportion of patients with a reported stressor or in the number or type of stressor in 6 months before illness onset between clinical or autoantibody phenotype, nor by gender, race, disease course or delay to diagnosis.
The availability of a large, well-characterized population enabled us to examine the relationship between environmental exposures before illness onset and phenotypes in JIIM. We confirmed a number of exposures that had also been seen in prior studies of JDM, particularly the temporal association of respiratory infections preceding illness onset [16, 17] . We identified for the first time that a number of other non-infectious exposures occurred within 6 months of the first signs of illness, including medications, many of which are potentially myopathic or photosensitizing, immunizations, stressful life events and sun exposure. The main novel findings of this study were differences in some exposures by age at diagnosis, delay to diagnosis, race, disease course and autoantibody phenotypes. For example, children younger than the median age at the time of diagnosis had a higher frequency of documented infections, whereas older children had a higher frequency of stressful life events in the months before illness onset. Patients without a myositis autoantibody had a higher frequency of infections in the 6 months before illness onset than was seen in patients with anti-p155 or anti-SRP autoantibodies, whereas patients with anti-SRP autoantibodies had a greater number of documented exposures than patients without a myositis autoantibody. These findings suggest that environmental exposures may differ by phenotype, and that they could be useful in understanding pathogeneses [1] . We found that an infectious illness, particularly a respiratory infection, frequently occurs within several months before juvenile myositis onset, supporting the findings of other studies of exposures temporally associated with the onset of JDM. In one study, a prospective registry of patients within 6 months of illness onset in which data were based on a parent environmental interview and medical record review, respiratory infections were identified within 3 months of illness onset in 57% of patients [16] . The other, a retrospective cohort with review of medical records by infectious disease specialists, identified infections within 3 months before Conventions as per Table 2 . Bold values represent P 4 0.05 after Holm's adjustment for multiple comparisons (using family-wise error rates of 5%). a Based on total number of drug exposures, because some patients had >1 documented drug exposure. Potentially myopathic drugs included penicillin (n = 2) and ranitidine (n = 1) [28] [29] [30] [31] . Potentially photosensitizing drugs included loratadine (n = 1), diphenhydramine (n = 1) and sertaline (n = 1) [26, 27] . Drugs classified as potentially both myopathic and photosensitizing included ibuprofen (n = 5), trimethroprim/sulphamethoxazole (n = 3), isoniazid (n = 2) and erythromycin-sulfisoxazole (n = 1). Drugs not known to be either myopathic or photosensitizing included amoxicillin (n = 4), cefaclor (n = 3), pseudoephedrine (n = 2), cetirizine (n = 1), albuterol inhaler (n = 1), flecainide (n = 1), bromopheniramine maleate (n = 1), nedocromil (n = 1), oxybutynin (n = 1), permethrin (n = 1), pyrethrine (n = 1), cantharidine (n = 1), cefadroxil (n = 1), acetaminophen (n = 1), streptomycin (n = 1), erythromycin (n = 1) and nystatin (n = 1). Drugs whose classification is unknown included unknown antibiotic (n = 10), birth control (n = 3) and anaesthetic (n = 1). *P = 0.004; OR 15.4; 95% CI 1.8, 135. y P = 0.008 ; OR 18.9; 95% CI 1.0, 358.
the first symptoms of JDM in 33-50% of patients, and respiratory infections accounted for 80% of the infections [17] . The lack of control comparator groups in all of these studies, however, does not enable one to conclude that these exposures differ from a healthy population, nor that they are associated with the onset of illness. While infections, particularly upper respiratory infections, are reported frequently in school age children [32] , a prospective matched cohort of new-onset JDM patients reported a higher frequency of antecedent illness in the JDM patients compared with friend controls from the same geographical region [19] . We identified for the first time that a number of other non-infectious exposures also occurred within 6 months of the first signs of illness, including medications, many of which are potentially myopathic or photosensitizing, immunizations, stressful life events and sun exposure. Pachman et al. [16] noted medication use in >60% of patients, including medications for symptoms of early illness or antibiotics to treat associated infections. A listing of medications taken by patients in the present study and in others includes similar medications (Table 4) , and we noted that many of the medications could be potentially myopathic or phototoxic [26, 27, 29, 30] . Drug-induced myositis has been well described with a number of different medications, including D-penicillamine, lipid-lowering agents, L-tryptophan and IFN-a [33, 34] . Myopathic or phototoxic drugs, however, could lead to the first symptoms of myositis. Other environmental factors reported here, including ultraviolet light exposure, emotional stress and heavy weight lifting, have been reported as possible risk factors for adult DM or PM in case-controlled studies [35] [36] [37] [38] .
Almost 40% of the patients in this study had two or more reported exposures within 6 months before illness onset, rather than a single documented exposure. This is consistent with the concept that, just as systemic autoimmune diseases are polygenic [39] , they might also be polyenvironmental, meaning that patients may have more than one exposure before developing the disease. These exposures may also be dependent on gene-gene, environment-environment and gene-environment interactions. In diseases such as cancer, multiple infectious and non-infectious environmental factors have been associated with specific malignancies, and these environmental exposures have been shown to affect the development of disease in different ways, including altering mutagenesis, promotion and direct carcinogenesis [40] . Synergistic interactions between some of these environmental factors, including viral and non-infectious exposures, have also been seen in certain malignancies [41, 42] . It is possible, though, that there was a confounding between exposures, such as an infectious illness and the use of antibiotics, as noted by Pachman et al. [16] . Our data suggest that further investigation of the interaction between environmental exposures may be useful.
It is important to emphasize that the temporal association of environmental exposures with illness onset does not imply causality. For example, certain exposures, such as trauma or weight training, could have occurred after the onset of illness as a consequence of the first unrecognized symptoms of disease, such as fatigue or muscle weakness. Rather, exposures with temporal relationships to disease onset, as were seen in this hypothesisgenerating study, constitute a first step for determining which factors may trigger the onset of illness and warrant further investigation. Additional support for a relationship between these exposures and disease pathogenesis could be provided by dechallenge data, which did not exist in this cohort-based study, from laboratory investigations and from case-controlled epidemiological studies [43] . A case-control study by Pachman et al. [19] did not find any significant differences in pesticide use, psychological stress or exposure to animals in 80 JDM patients within 6 months of illness onset compared with 63 age-matched geographically similar healthy controls with similar school or daycare experiences, nor was parvovirus found to be an aetiological factor in recent-onset JDM patients compared with age-, genderand race-matched controls [44] . However, both of those studies may not been adequately powered to detect differences between the cases and controls. Also, the extent of matching of controls may have obscured differences with JDM patients. For example, in the parvovirus study [44] , the controls were age, race and gender matched to patients, but they were not geographically matched, whereas in the study of Pachman et al. [19] , the healthy controls, frequently age-matched classmates and neighbours, may have been geographically overmatched, but they were not gender or race matched. An appropriately powered prospective case-controlled study is needed to confirm the observations from this and other previous reports.
There are a number of potential limitations in this study. A primary limitation is the absence of a control group. Thus, the frequencies of exposures observed in juvenile myositis patients overall may not differ from healthy control populations and these exposures may not be associated with the onset of illness. In addition, there could be under-or over-reporting of potential exposures, including a selection bias in the patients who had the environmental component of the questionnaire completed. We also found more exposures, including infectious illnesses, in white patients compared with patients in other racial groups. This could potentially be the result of differences in access to health care, resulting in better documentation of such exposures. The somewhat arbitrary period of 6 months before the onset of illness for identification of environmental factors might not be relevant to the initiation of myositis for all exposures. Certain exposures could require a longer period to induce their effects, as has been reported in malignancies, silicosis and other disorders, while for other exposures a shorter time frame might be more relevant [37, 38] . Also, exposures other than infections, drugs and vaccines were reported in an open-ended manner, and patients were not required to be directly interviewed to obtain information about environmental exposures. We attempted to www.rheumatology.oxfordjournals.org overcome these possible biases by conducting a formal review of most of the medical records of the study subjects. However, the medical records might also have selection bias by reporting only some of the significant environmental exposures. Certain exposures, such as exposures in the home and use of certain chemicals, are likely not captured uniformly in the medical record by the treating physician. Nonetheless, the fact that our data on infections before illness onset are similar to those of other large cohorts suggests that the quality of the data and reporting are reliable [16, 17] . Finally, while some of the ORs in our study are large, the CIs may be wide and estimates could be inflated due to relatively small numbers of patients in some groups.
In summary, we have identified a number of environmental exposures, including infectious and non-infectious agents that occurred within 6 months before illness onset, varied by phenotype and may be important in the pathogenesis of JIIM. These findings suggest that a search for a single environmental factor that causes or triggers a single disease as currently defined, such as JIIM, may be unproductive, as patients could have several environmental exposures and these could vary with the disease phenotype that develops. These exposures require confirmation in case-controlled studies to identify whether they are associated with illness onset and whether they play any role in aetiology, yet they suggest focused areas of further research to better understand the environmental factors associated with the onset of JIIM phenotypes and their possible interrelationships with genetic risk factors.
Rheumatology key messages . Environmental exposures before the onset of juvenile myositis include infections, medications, vaccinations, sun exposure and stressful life events. . Exposures vary by disease phenotype, defined by age of illness onset, race and autoantibody status. Network Analysis of Global Influenza Spread Although vaccines pose the best means of preventing influenza infection, strain selection and optimal implementation remain difficult due to antigenic drift and a lack of understanding global spread. Detecting viral movement by sequence analysis is complicated by skewed geographic and seasonal distributions in viral isolates. We propose a probabilistic method that accounts for sampling bias through spatiotemporal clustering and modeling regional and seasonal transmission as a binomial process. Analysis of H3N2 not only confirmed East-Southeast Asia as a source of new seasonal variants, but also increased the resolution of observed transmission to a country level. H1N1 data revealed similar viral spread from the tropics. Network analysis suggested China and Hong Kong as the origins of new seasonal H3N2 strains and the United States as a region where increased vaccination would maximally disrupt global spread of the virus. These techniques provide a promising methodology for the analysis of any seasonal virus, as well as for the continued surveillance of influenza. Influenza, a negative-sense RNA orthomyxovirus, is one of the few diseases that is truly global in scale. It is responsible for approximately three to five million cases of severe acute respiratory illness and 250,000 to 500,000 deaths each year throughout the world [1] . In 2009, the swift isolation of swineorigin H1N1 strain (S-OIV) from all continents within several weeks of onset reinforced the idea that influenza is a highly infectious agent circulating worldwide [2, 3] .
Although vaccination remains one of the most powerful ways of combating influenza, choosing a representative strain for vaccine composition poses a challenging problem. Due to the virus's high evolutionary rate, significant resources must be spent to update vaccines each year in order to match the dominant epitope of the season. Even with annual strain selection, major antigenic reassortment can obviate otherwise promising vaccine candidates, as occurred with the 'Fujian/411/2002'-like H3N2 strain in 2003 [4, 5] . To prevent such vaccine failures, a solid understanding of the global spread of influenza must inform the design process. If reservoirs for new viral strains can be identified, surveillance in these areas can better optimize prediction of seasonal variants in seeded regions.
Previous papers investigating the global circulation of H3N2, the major seasonal influenza subtype prior to pandemic H1N1, focused on transmission within and between climate zones. Important motivating factors for such analysis include increased aerosol transmission in cold and dry conditions, as well as increased indoor crowding and decreased host immunity in cold and wet conditions [6, 7] . In the temperate zones, influenza exhibits distinct seasonality with flu-related cases spiking in the winter. However, several papers have confirmed the presence of viral diversity even between these epidemic peaks [8, 9, 10] , suggesting two possible scenarios during the inter-epidemic period: either viral infections locally persist at a low level only to reemerge as the dominant strains of the epidemic season, or an outside source introduces new genetic diversity into temperate populations each year. Although a degree of local persistence may occur, phylogenetic analysis supports the latter scenario, with few direct links between strains of the same region but successive seasons [8, 9, 10] .
For a given temperate zone, these conclusions suggest the tropics or the opposite temperate zone as plausible external seeding regions. At first blush, northern-southern temperate oscillations seem credible. Each year, northern and southern temperate climates have alternating seasonal influenza epidemics, lasting from November to April, and May to September respectively [11] . A possible mechanism of viral spread could involve transmission from the seasonal peak of one temperate zone into the season ebb of the other. On the other hand, specific epidemiological characteristics suggest a tropical origin for influenza. For example, although both climates share a similar yearly burden of mortality from influenza, the tropics do not possess the same consistent seasonal peaks during the winter months [9, 12, 13] . With a constant, low-level circulation of viruses year-round, the tropics represent an ideal epicenter for the extended transmission of new viruses to the rest of the world [14, 15, 16] .
Several papers tracking H3N2 across continents have asserted that this tropical reservoir of influenza strains lies within East-Southeast Asia [12, 14, 17] . Russell, et al. analyzed H3N2 data to identify regions of the world that are antigenically and genetically leading or trailing. They found that newly emerging strains appeared in E-SE Asia roughly 6-9 months earlier than in other parts of the world, while South America experienced delayed transmission of roughly 6-9 months following other parts of the world [8] .
However, such studies have been limited by several drawbacks. Most papers focus on H3N2 as a single entity, when in reality, it co-circulates with several other subtypes, the most important of which is seasonal H1N1 [11] . Although they possess different surface antigens, H3N2 and H1N1 share enough genetic similarity to display cross-immunity. As a result, seasonal H1N1 may demonstrate transmission patterns distinct from H3N2's [18, 19] . Such codependence between different subtypes is exemplified by the pandemic years of 1957 and 1968, when H2N2 replaced preexisting H1N1 and H3N2 replaced preexisting H2N2, respectively [20, 21] . Similarly, the antigenically different pandemic H1N1 strain of 2009 has largely overtaken previously circulating H1N1 and H3N2 [22] . During the years our dataset took place, evidence that H3N2 and H1N1 rarely co-dominate in a season further supports the idea of codependent dynamics [7] .
A second shortcoming stems from biases in the number of sequences from different regions and different seasons [8] . Most isolates of H3N2 and H1N1 were sampled from North America, whereas Africa and South America have been largely neglected [23] . Many sequences were obtained within the last 15 years, making reliable tracking over long periods of time problematic. On the level of climate zones, the number of temperate isolates far outstrips the tropics. Although hemagglutinin (HA), the HA1 domain, and neuraminidase (NA) have the most globally representative distributions of sequences, even these remain skewed ( Figure S1 , Figure S2 ).
In this paper, we present a novel probabilistic model for tracking the spread of influenza that employs two strategies to eliminate regional and seasonal data bias. The first involves clustering isolates of high sequence similarity by region and season. Since we would expect highly similar sequences from the same time and location to be related, we considered seeding events between clusters to be of greater significance. Consideration of clusters rather than individual sequences nullifies the overrepresentation of a high number of isolates from a single region and season ( Figure 1 ). As a second strategy for eliminating bias, we determined statistical significance of inter-cluster seeding events by modeling transmission as a binomial distribution with prior probabilities based on the proportion of sequences isolated before a given time point. To illustrate our methodology, Figure 2 depicts the 2003-2004 flu season, which was marked by failure to predict the dominant, tropically-derived Fujian/411/2002-like H3N2 strain. We identified a strong seeding pattern from the tropics to all three climate zones, supporting the effectiveness of our methodology.
We applied this model to the H3N2 and H1N1 coding regions of HA and NA, the most antigenic proteins of the eight viral segments. Clustering H3N2 sequences confirmed previous findings that this strain originates in the tropics, specifically E-SE Asia, and seeds South America by way of North America last. Clustering H1N1 NA also revealed a similar pattern of circulation beginning in the tropics. However, similar H1N1 analysis by continent and country was not possible due to the absence of a larger number of countries in the dataset.
Applying the same methodology to the H3N2 HA1 domain increased the geographic diversity enough to enable reconstruction of the global influenza network prior to the 2009 pandemic strain at a country level. Our results suggest a possible flu seeding hierarchy beginning in China and spreading throughout a highly interconnected E-SE Asian subnetwork. From there, viruses transmit to an Oceanic subnetwork dominated by interchange between Australia and New Zealand. Both subnetworks seed into the USA, which in turn seeds many countries, particularly in South America.
Expanding upon the sink-source hypothesis of global influenza dynamics proposed by Rambaut, et al. [15] , we applied techniques of graph theory to identify important source and sink regions in the global flu network. These techniques better describe the dynamic nature of influenza movement across the globe, as well as suggest different vaccination strategies to disrupt maximally viral flow around the world.
Spatiotemporally clustering the complete H3N2 and H1N1 coding sequences for HA and NA allowed the determination of multiple statistically significant seeding seasons between 1988 and 2009. For our initial analysis, we clustered sequences into three climate zones-northern temperate, tropical, and southern temperate. To determine seasonal boundaries, we defined the northern temperate season to last from 1 st July to the 30 th June of the following year and the southern temperate season to last from 1 st January to the 31 st December of the same year [11] . Although the tropics do not have a well-defined seasonal pattern, we determined a consensus tropical flu season from 1 st October to 30 th September of the next year (Text S1, Table S1 ).
Results for H3N2 showed that the overwhelming majority of statistically significant seeding seasons came from the tropics, confirming previous findings ( Figure 3A , Figure S3A ). Clustering H3N2 by the six major continents rendered an even more detailed picture. For HA, Asia was the primary seeder of Asia, North America, and Oceania. Prominent transmission from North America to Europe and South America was also observed ( Figure  S3B ). Interestingly, this hierarchical seeding structure reflects the findings of Russell, et al., which identified Asia and South America as antigenically advanced and lagging continents respectively [8] . This network of hierarchical seeding can be visualized as a directed graph plotted against the world map ( Figure 4A ). Analysis
As evidenced by several historic vaccine failures, the design and implementation of the influenza vaccine remains an imperfect science. The virus's rapid rate of evolution makes the selection of representative strains for vaccine composition a difficult process. From a global health viewpoint, how to optimally implement a limited stockpile of vaccines is another fundamental question that remains unanswered. An understanding of how influenza spreads around the world would greatly aid the design and implementation process, but regional and seasonal bias in collected virus samples hampers epidemiologic analysis. Here, we show that it is possible to counter this data bias through probabilistic modeling and represent the global viral spread as a network of seeding events between different regions of the world. On a local scale, our technique can output the most likely origins of a virus circulating in a given location. On a global scale, we can pinpoint regions of the world that would maximally disrupt viral transmission with an increase in vaccine implementation. We demonstrate our method on seasonal H3N2 and H1N1 and foresee similar application to other seasonal viruses, including swine-origin H1N1, once more seasonal data is collected.
of NA produced similar findings with the exception of North America being its own primary seeder ( Figure 3B ). No complete HA and NA isolates existed in the NCBI Influenza Virus Resource database [24] for Africa.
The complete dataset of HA and NA represented only 17 and 21 countries respectively. Despite the sparse number of countries for analysis, both HA ( Figure S3C ) and NA ( Figure 3C ) consistently identified Hong Kong (considered a country by NCBI sequence annotation) as the primary external seeder of USA and New Zealand among others, and New Zealand as the primary external seeder of Australia.
Due to fewer available sequences, clustering H1N1 did not yield as many significant seeding events as H3N2; however, our tests suggest that H1N1 adopts a similar seeding pattern with the tropics as a source. Of the two segments, NA sequences display a broader geographical profile than HA. In particular, our HA dataset for H1N1 contained no sequences from Hong Kong and only 1 (0.091%) China sequence, while NA contained 9 (0.69%) Hong Kong and 3 (0.23%) China sequences. Consequently, we considered NA to be more suitable for comparison between H3N2 and H1N1 and HA to be a background signal to assess the effect of Hong Kong and China on global influenza transmission. Even so, the number of these H1N1 Hong Kong and China sequences remained vastly disproportionate to the 361 (7.42%) Hong Kong and 133 (2.73%) China sequences of H3N2.
Clustering H1N1 NA by climate zone supported the theory of global viral spread from the tropics ( Figure 5B ). Unlike H3N2, H1N1 analysis by continent and country was inconclusive due to low (typically fewer than 3 seeding events), homogeneous counts. Although inconclusive, the fact that a tropical signal could be detected at all from such few tropical countries, including Hong Kong and China, suggests that H1N1 adopts a similar seeding pattern out of the tropics. Due to insufficient sampling, however, a more detailed transmission pattern could not be discerned.
Although using the complete HA and NA coding genomes facilitated differentiation of isolates by Hamming distance, the absence of data from certain countries limited the information gained from clustering at this geographic detail, a problem that has plagued previous studies [8] . To increase the amount of data from different geographical regions, we clustered H3N2 sequences of the HA1 epitope, expanding the number of isolates in the dataset from 2,251 to 4,864, and the number of countries from 17 to 81. A necessary consequence of expanding geographic coverage was an increase in the number of non-unique solutions (Text S1). Importantly, clustering HA1 by climate and continent was corroborated by findings from the complete HA and NA sequences, lending credence to the validity of the dataset. Due to the inclusion of isolates from Africa, which was hitherto not present in our datasets, H3N2 HA1 analysis also revealed Europe and North America tied for being the primary seeders of Africa.
Country clustering of the HA1 data produced a highly detailed global network of influenza variants. USA, Hong Kong, Australia, and China were identified as the four most prominent seeding countries in that order ( Figure 3D , Table S2 ). From the data, an inferred seeding hierarchy would begin with China at the epicenter of an E-SE Asian influenza subnetwork. Our analysis supports China as the most predictive seeder of many Asian countries, including Hong Kong. Both China and Hong Kong then serve as a launching pad for the dispersal of new seasonal variants to the rest of the world [14, 17] , in particular USA and an Oceanic subnetwork dominated by interchange between Australia and New Zealand. Viruses from USA, the largest seeder of the entire world, then spread to a number of South American, European, and African countries. Interestingly, Australia and Hong Kong are equally probable seeders of the USA ( Figure 3D ). Detailed transmission events are enumerated in Table S2 . An inset of the Asian subnetwork is depicted in Figure 4C , a demonstration of this study's high geographic resolution. After clustering, there were a total of 10 observed seeding events into the northern temperate zone: 1 from the north, 4 from the tropics, and 5 from the south. Up until that year, the skewed regional distribution of HA sequences included 541 (48.3%) northern temperate, 240 (21.4%) tropical, and 339 (30.2%) southern temperate isolates. Multiplying these percentages with the 10 observed seeding events yielded expected counts of 4.8, 2.1, and 3.0. Therefore, the number of seeding events from the north was less than expected, and from the tropics and the south, more than expected. Corresponding binomial p-values-0.986, 0.043, and 0.049, respectively-indicated that there were two statistically significant events, the most significant of which was transmission from the tropics into the northern temperate zone. Similar analysis for transmission into the tropics and the southern temperate showed that only the tropical zone was a significant seeder. doi:10.1371/journal.pcbi.1001005.g002
As can be seen with the world map plots ( Figure 4A,B) , a natural representation of the global influenza network is a directed graph with each node representing a clustered region (climate, continent, and country) and each edge representing a seeding event with a weight equal to the number of significant seeding seasons. To quantify observed patterns, we employed principles of graph theory to measure the importance of nodes using four different metrics.
By counting the number of indegrees and outdegrees of each node for H3N2, we identified that the tropics and the northern temperate zone ( Figure S4A ), specifically Asia and North America ( Figure Figure  S5A ), transmit and receive the most seeding events to and from the rest of the world, respectively. In a similar manner, we identified USA, Hong Kong, Australia, and China as the greatest seeders, and USA, Japan, Australia, and Hong Kong as the most seeded ( Figure 6A) .
In this analysis, we differentiated between internal (self-seeding) and external (seeding between nodes) transmission events. Importantly, we can accurately detect internal events in temperate countries since their flu seasons are discrete. On the other hand, the specificity for internal events in the tropics is much lower due to unpronounced seasonal peaks. To minimize the number of local false positives, we demarcated seasons within the tropics on a per country basis. We found that for all climate zones except the tropics ( Figure S4A ) and all continents except Asia ( Figure S5A ), the number of internal seeding events paled in comparison to the proportion of external seeding events,. The more numerous internal events in the tropics and Asia indicate a high level of circulation between tropical countries and between Asian countries. This pattern is supported by the highly interconnected E-SE Asian subnetwork depicted in Figure 4C . The small proportion of internal events for countries supports the notion that local persistence often plays only a minor role in influenza transmission [8, 9, 10] (Figure 6A ).
Beyond the absolute number of seeding events, a region's influence on global viral spread is also dependent on the topological structure of the graph itself. As an analogy, consider the influenza network as a system of connected train stations each representing a single region seeding influenza. In such systems, trains begin and end their routes at terminal stations. Similarly, influenza commuters begin their journeys at terminal sources and end at terminal sinks in each season. These start and end terminals can represent regions where new influenza variants respectively originate and ultimately spread to. To quantify the terminal characteristic, we calculated the outdegree minus the indegree of each node, which we term ''degree flow.'' Positive degree flow indicates terminal sources, while negative indicates terminal sinks. Countries were also ranked by calculating the proportion of nodes in a 1,000 randomized networks with a greater, or lesser, degree flow (Text S1). For analysis by climate zone, the tropics was identified as the only terminal source, suggesting that flu spreads from the tropical belt outward to both temperate zones ( Figure S4B ). As for continental clustering, Asia was the only terminal source, indicating that global circulation begins in Asia and ends in terminal sink continents, of which North America was the most prominent ( Figure S5B) . On a country level, Hong Kong and China were the greatest terminal sources, corroborating our observations ( Figure 3D) . Australia was also a conspicuous terminal source, especially within the Oceanic subnetwork where it seeded the greatest terminal sink, New Zealand. Several South American countries, including Chile and Argentina, figure as terminal sinks too, correlating with such countries as antigenically delayed [8] (Figure 6B ).
Trains also stop at waypoint stations, which can be the junction of a large number of routes. Correspondingly, certain regions act as waypoint sources: important intermediate launch pads to other destinations. Others act as waypoint sinks: important points of convergence for multiple routes. Eigenvector centrality can gauge this property on the principle that connections to high-scoring nodes contribute more to the score of the node in question than equivalent connections to low-scoring nodes. We used a method akin to PageRank, Google's method of assigning importance to web pages [25] .
Using this method, the northern temperate zone was the most important waypoint source and sink ( Figure S4C) . Similarly, the predominantly northern temperate continents of North America and Europe were identified as prominent waypoint sources and sinks. Asia, however, was the greatest waypoint source but a poor waypoint sink, correlating with its role as a greater terminal source than North America or Europe ( Figure S5C ). Interestingly, USA was both the greatest waypoint source and sink ( Figure 6C ). H1N1 NA clustering by climate zone produced results similar to that of H3N2 NA. The tropics consistently scored highest by seeding outdegree, positive degree flow, and PageRank source. In addition, the tropics possessed a large amount of internal seeding events. These results emphasize that similar to H3N2, H1N1 circulates within the tropics across seasons only to spread eventually to the temperate zones.
Betweenness measures the number of shortest paths between any two vertices in a network that lie on a given node. In the context of influenza, increasing vaccinations in regions of high betweenness would hypothetically have the greatest effect on diminishing the spread of infection worldwide. This novel strategy contrasts with previous studies simulating containment only at the source of influenza [26, 27] . For H3N2, this criteria highlighted Europe and North America as promising candidates for vaccination programs ( Figure S5D ). Clustering by country revealed USA, Japan, and Australia as sites in the influenza network vulnerable to disruption ( Figure 6D ).
Using statistical and network theory analysis, we analyzed H3N2 and H1N1 sequence data to determine the global spread of influenza. Our novel method employs two main strategies to eliminate geographic and seasonal bias: 1) Spatiotemporal clustering of sequence data to count seeding events between clusters and 2) Use of binomial prior probabilities based on the regional proportion of viral isolates to screen for significant seeding events.
Applying these techniques to coding HA and NA segments of H3N2 by climate zone and continent revealed a seeding pattern stemming from the tropics, particularly Asia. HA1 analysis produced a more detailed picture: each year, a wave of seasonal flu originates in China to feed an E-SE Asian subnetwork. From there, China and Hong Kong seed two major subnetworks, each dominated by Australia and USA.
Similar clustering of H1N1 NA sequences by climate zone reproduced tropical transmission to the rest of the world. However, due to inadequate geographic coverage, clustering H1N1 by continent and country proved inconclusive with few significant seeding events detected. One explanation for these results is that important seeding countries, such as China and Hong Kong, were too underrepresented in the dataset. Alternatively, global patterns may be weaker for H1N1 due to crossreactivity between the two strains [18, 19] , a conclusion reflected by the smaller number of seeding events for the strain.
In our analysis, the total number of seeding seasons for each region did not necessarily correspond to the total number of isolates from each region, indicating that our methodology counters data bias. However, certain confounders may affect results. First, selection bias in sampling remarkable variants, such as patients suffering severe rather than mild or non-symptomatic influenza, would poorly represent flu in the general population. Moreover, many sequences had to be excluded from our dataset due to poor annotation and lack of date information. Finally, although our probabilistic methodology accepts regional and temporal variability, it has low sensitivity for detecting anything but particularly significant seeding events for regions with very few sequences. This issue becomes important in analyses with regions that have no sequences whatsoever, as with near-absent sequences from Hong Kong and China for H1N1 HA. The persistence of such bias highlights the continuing need to sequence viruses in underrepresented areas, especially the tropics.
Each year, the current influenza vaccine is formulated separately for the Northern and Southern Hemisphere; one can surmise that two viral strains may not be enough to represent the entire pool of influenza strains around the world. Although there are many other economic and political concerns to consider, our methodology suggests several ways of guiding vaccine strain selection based on biological and epidemiological principles. Graph theory metrics-terminal and waypoint sinks and sources, as well as degree and betweenness centralities-pinpoint potential regions in which increased vaccinations could stem the transmission of influenza globally as well as locally. Increased analytical resolution could optimize vaccine design by choosing the dominant antigenic strain of a country's most predictive seeder. Vaccines could be catered to each country, rather than each hemisphere. At the very least, our analysis advises strain selection from the tropics, from which seasonal strains are dispersed each year. On the other hand, local strain selection within a country should prove comparatively ineffective, as few viruses persist in the inter-epidemic period to seed the following flu season.
Our analysis of terminal sources resonates with an old hypothesis that in southern China, zoonotic infection from liveanimals markets [28] selling in particular duck-a natural host of influenza [29] -combined with a dense population for sustained viral circulation, could be the main ingredients for the creation of new seasonal influenza variants. In support, two major acute respiratory infections-SARS [30] and H5N1/97 [31, 32] -have been definitively traced back to southern China, with Hong Kong serving as an important sentinel post for the rest of the world. Other influenza pandemics, 1968 H3N2 (Hong Kong) [28] and even as early as 1889 pandemic influenza [33] , have suspected origins in southern China.
It would be interesting to dissect the factors that govern waypoint sources and sinks. For example, air travel and other transportation may play a major role in the dispersal of virus worldwide [8, 19, 34, 35] . Many important hubs of the global flu network, including USA, Australia, Hong Kong, and China, have several of the world's busiest airports [36] . Understanding the reasons for these seeding patterns may offer other strategies for arresting the movement of flu.
The advent of 2009 pandemic S-OIV has largely depleted the number of seasonal H3N2 and H1N1 infections, most likely via cross-reactivity between novel and seasonal strains [22] . Consequently, the conclusions of this paper may not necessarily apply to current dynamics of seasonal H3N2 and H1N1. However, the fact that H1N1 shares a tropic-centric movement pattern with H3N2 despite cross-reactivity suggests that these patterns may still persist even in the presence of the cross-reactive S-OIV. Moreover, this paper demonstrates that when more sequence data is deposited in NCBI, a similar methodology can be applied to predict global circulation of S-OIV as well.
All sequence data used in this study was publicly available from the National Center for Biotechnology Information database (NCBI) [37] . For each segment, only protein coding regions were considered. Furthermore, we only used sequences with full date (year, month and day) and location information to build hierarchies. Geographical coordinates of each isolate were obtained using geolocation information from Google Maps. Sequences were then aligned using the ClustalW v. 1.83 multiple sequence alignment package using default parameters for H3N2 and H1N1, respectively. For each segment, sequences were aligned and those that were poorly aligned compared to the rest of the dataset were removed until all sequences aligned with a Hamming distance no greater than 0.15. Given estimated mutation rates of 6.7610 23 nucleotide substitutions per site per year [12, 19] , Hamming distances over the 20-year span of our dataset are expected to be no more than 0.15 of the sequence length. Outlying sequences were most likely incorrectly sequenced and were discarded from analysis.
Our methodology aimed to minimize data bias from geospatial and temporal variability in sequences from NCBI. First, we determined the most parsimonious evolutionary paths traversed by the flu virus. To this end, we sorted sequences from earliest to most recent viral isolates. Working backwards from newest to oldest, we calculated the sequence similarity of each virus to all earlier isolates regardless of geography. We defined a virus's most likely ancestor to be the sequence with minimum Hamming distance. From this data we built evolutionary paths for each virus. Related sequences were clustered (grouped) together by common geography and season to simplify the paths. For example, a chain of related viruses in the same region and season would be collapsed into a single umbrella node representing all of them. Our analysis was then based on looking at the transitions between clusters rather than individual viruses. We counted these ''seeding events,'' where the closest ancestor of a given cluster of sequences is from a different region or season [8] (Figure 1 ). When tallying seeding events, non-unique solutions were not considered where a given viral isolate possessed multiple closest ancestors from different geographical zones or seasons (Text S1, Figure S6 ).
The observed frequencies of seeding events between clusters were compared to expected frequencies based on the prior probability of randomly choosing a sequence from a given geographical zone in the past. Using the binomial distribution with the proportion of prior NCBI sequences as a binomial probability, a p-value was calculated for observing more seeding events than expected. The best predictor of a seeding region for each season had the greatest ratio of observed to expected seeding events with a p-value smaller than 0.05 ( Figure 2 ). Figure S4 Rankings of significant seeding and seeded climate zones for H3N2 and H1N1 using different graph theory metrics. (A) The indegree and outdegree of a node represent the total number of seeding events into and out of a region, respectively. Local seeding events depicted in gray play little role in overall seeding except in the tropics. (B) Degree flow measures the difference between seeding events out of and into a node and determines whether it is a terminal sink or source. (C) PageRank uses an algorithm similar to that employed by Google to categorize nodes based on the number and quality of links pointing to that node. Found at: doi:10.1371/journal.pcbi.1001005.s004 (0.88 MB EPS) Figure S5 Rankings of significant seeding and seeded continents for H3N2 using different graph theory metrics. (A) The indegree and outdegree of a node represent the total number of seeding events into and out of a region, respectively. Local seeding events depicted in gray play little role in overall seeding except in Asia. (B) Degree flow measures the difference between seeding events out of and into a node and determines whether it is a terminal sink or source. (C) PageRank uses an algorithm similar to that employed by Google to categorize nodes based on the number and quality of links pointing to that node. (D) Betweenness measures the number of shortest paths in a network passing through a given node. Text S1 Detailed description of the methodology, including evaluation of clustering, determining flu seasons, timing of observed seeding events, and network randomization. Detailed description of the methodology, including evaluation of clustering, determining flu seasons, timing of observed seeding events, and network randomization. Found at: doi:10.1371/journal.pcbi.1001005.s009 (0.02 MB DOCX) Willingness to accept H1N1 pandemic influenza vaccine: A cross-sectional study of Hong Kong community nurses BACKGROUND: The 2009 pandemic of influenza A (H1N1) infection has alerted many governments to make preparedness plan to control the spread of influenza A (H1N1) infection. Vaccination for influenza is one of the most important primary preventative measures to reduce the disease burden. Our study aims to assess the willingness of nurses who work for the community nursing service (CNS) in Hong Kong on their acceptance of influenza A (H1N1) influenza vaccination. METHODS: 401 questionnaires were posted from June 24, 2009 to June 30, 2009 to community nurses with 67% response rate. Results of the 267 respondents on their willingness to accept influenza A (H1N1) vaccine were analyzed. RESULTS: Twenty-seven percent of respondents were willing to accept influenza vaccination if vaccines were available. Having been vaccinated for seasonable influenza in the previous 12 months were significantly independently associated with their willingness to accept influenza A (H1N1) vaccination (OR = 4.03; 95% CI: 2.03-7.98). CONCLUSIONS: Similar to previous findings conducted in hospital healthcare workers and nurses, we confirmed that the willingness of community nurses to accept influenza A (H1N1) vaccination is low. Future studies that evaluate interventions to address nurses' specific concerns or interventions that aim to raise the awareness among nurses on the importance of influenza A (H1N1) vaccination to protect vulnerable patient populations is needed. The 2009 pandemic of influenza A (H1N1) infection has alerted many governments to make preparedness plan to control the spread of influenza A (H1N1) infection. With evidence on the effectiveness of vaccination in the control and prevention of seasonal influenza [1, 2] , vaccination for pandemic influenza is one of the most important primary preventative measures to reduce the disease burden associated with influenza A (H1N1) infection [3] .
Several high risk groups have been identified as "the priority group" to receive the influenza A (H1N1) vaccination and among these, healthcare workers have been identified "as a first priority" to be vaccinated against influenza A (H1N1) by the World Health Organization [4, 5] . Although it is considered essential for all healthcare workers to be immunized against influenza A (H1N1) to prevent the spread of influenza A (H1N1) to patients as the pandemic evolves, previous studies that have examined the acceptability of seasonal influenza vaccination among healthcare workers have generally demonstrated a low acceptance rate of vaccination in this group [6, 7] . Among all healthcare workers, nurses constitute the largest group with the highest frequency of contacts with patients and staff [8] . Previous findings of the acceptability of seasonal influenza vaccination in nurses showed that their acceptance of vaccination was lowest among all healthcare workers [6, 7, 9, 10] . Acceptability of influenza A (H1N1) vaccination in healthcare workers has been shown to be low [11] [12] [13] . A survey conducted in Greece found that only 17% of hospital healthcare were willing to receive influenza A (H1N1) vaccination [11] . Of all healthcare workers, nurses were found to have the lowest rate of acceptability of influenza A (H1N1) vaccination [12, 13] . A study of Italian healthcare workers showed 31% of nurses willing to accept vaccination compared to 67% of physicians [12] . In a study conducted of Hong Kong healthcare workers in hospitals, it was found that only 25% of nurses were willing to accept influenza A (H1N1) vaccination, compared with 47% of doctors and 29% of allied professionals [13] . General practitioners working in the community in France also report a high rate of acceptability of influenza A (H1N1) vaccination at 62% [14] . It is therefore not surprising that a recent online poll conducted in the UK suggested that nurses may be unwilling to receive pandemic influenza vaccination [15] . In a cross-sectional survey that was conducted on experienced nurses who were members of the nursing professional organizations in Hong Kong, the vaccination rate for seasonal influenza vaccination was about 50% [16] . In a more recent survey that explored influenza A (H1N1) acceptance rate in the same group of nurses [17] , it was found that only 13% were willing to accept vaccination for influenza A (H1N1) compared to 38% who plan to receive the seasonal influenza vaccination. However, in the study, there was a low response rate of 28% of nurses with different clinical settings. There is a lack of studies in Hong Kong looking at influenza A (H1N1) vaccination acceptability particularly in the community setting. Nurses who work in the community may be the first group to be in contact with patients who are affected with the influenza A(H1N1) infection. A recent study [18] showed differences in the concerns in using new vaccines during a pandemic than using established vaccine in a non-crisis situation. Therefore, we undertook the current study to examine the willingness of frontline registered nurses who work in the community in Hong Kong to receive vaccination against influenza A (H1N1) at the time of a pandemic.
All participants in this study were specially trained nurses, who provided nursing care and treatment for patients in their own homes (also known as Community Nursing Service) in Hong Kong. The responsibility of these community nurses is to provide nursing care and health education to patients through home visits. CNS nurses are employed by Hospital Authority in Hong Kong and provide continuity of care for patients who have been discharged from hospitals such that patients can recover in their own homes. Community nurses were chosen because of their frequent contacts with patients in their homes which is likely to increase their risk for exposure to influenza. We have only included CNS nurses who provide medical services in the study. The rest of the CNS nurses (around 100 nurses) provide psychiatric services in the community.
Currently, there are a total of 401 nurses who provide medical related services for the Community Nursing Service (CNS) centres that are distributed among the 7 geographical clusters in Hong Kong (in Hong Kong, public hospital and primary care services are organized in 7 clusters that covers all of Hong Kong).
In this study, twelve major CNS centres were contacted first and all CNS nurses were invited to participate in the current study through these 12 major centres. All 12 centres responded to this study and 270 questionnaires were returned with 267 completed questionnaires [19] . The response rate for this study was 67% and all questionnaires were received within a 2week period at a time when there was widespread H1N1 in the community.
The survey was sent out from June 24 th to June 30 th , 2009 when the WHO influenza pandemic alert level assigned to H1N1 was phase 6. Phase 6 signifies a widespread human infection, indicating that the virus has caused sustained community level outbreaks in at least one other country in another WHO region (WHO pandemic phase description). The pandemic in Hong Kong started on 1 st May, when a Mexican traveller was confirmed with influenza A (H1N1). Till the end of our data collection, there were 1389 confirmed cases and no death were reported.
All general managers of the involved community nursing centres were contacted through telephone to obtain approval to send questionnaires to their nursing staff. In total, 401 self administered, anonymous questionnaires were posted to general managers of centres who then passed these questionnaires to the community nurses in their centres. The general managers of centres were then reminded via telephone during the period from 2 nd July and 8 th July one week after the questionnaires were sent out and advised to return the completed questionnaires within the week. Once completed, questionnaires were collected and returned by their supervisors, except for one of the (Sau Mau Ping) sub-offices, where nurses mailed back their questionnaires individually. All centres sent their questionnaires back after one telephone reminder. The last pile of completed questionnaires was received on 14 th July, 2009.
The questionnaire consisted of six parts with 44 questions and the full questionnaire can be accessed by contacting the authors. The first four parts were based on a conceptual framework developed by Patel et al [20] to guide systematic planning for community primary care service response to pandemic influenza with modifications to make it more relevant for nurses. We added a fifth part on psychological responses to pandemic influenza and a sixth part on demographics of respondents which were based on two studies previously published (one on general practitioners' response to SARS and one on general public response to swine flu) [21, 22] . In summary, these sections were 1) clinical services change as a response to pandemic influenza; 2) internal environment changes as a response to pandemic influenza e.g. wearing of mask; 3) macro-environmental changes as a response to pandemic influenza e.g. use of guideline etc; 4) professional and public health responsibilities with respect to pandemic influenza; 5) attitude and psychological responses to pandemic influenza; and 6) demographics and year of education of respondents. The willingness to accept influenza A(H1N1) vaccination was asked in the professional and public health responsibility sections and the question "will you receive the new influenza A (H1N1) vaccine when it is available" was asked with a dichotomous "yes" or "no" response. For those who answered no, they were further asked to give their reasons for refusing to receive the vaccine. Only results on willingness of accept influenza A (H1N1) vaccination and information related to the analysis on willingness to accept vaccine are reported in this paper. Other results from this survey will be presented in a separate report.
Descriptive results were cross-tabulated. χ 2 test was used to examine characteristics between nurses who were willing to accept influenza A (H1N1) vaccination against those who were not willing to accept vaccine. Univariate analysis was performed with demographic information (age, post year education and working district), personal protective behaviour (hand washing practice), experience of taking care of SARS patients, and influenza vaccination in the previous 12 months as independent variables. Dependent variables were the willingness to receive pandemic influenza vaccination. Multiple logistic regression analysis was conducted to examine the relationship between pre-defined factors that we think might be associated with the acceptance of the influenza A (H1N1) vaccine when constructing the questionnaire and the dependent variable. The level of statistical significance was set at a p-value of ≤ 0.05.
Among the respondents ( Table 1) , most of them were females who had worked an average of 8.8 years as a community nurse (ranging from 2 months to 32 years) and having been a registered nurse for 16.5 years (ranging from 1 year to 36 years). The mean age of respondents was 39.1 years and about a third (30%) had had the experience of dealing with SARS. One third of them had received vaccination for seasonal influenza in the past 12 months. Nurses from each geographical cluster in Hong Kong participated, with 11% of respondents working in Hong Kong Island, 47% working in Kowloon and 42% working in the New Territories (Hong Kong is geographically divided into Hong Kong Island, Kowloon peninsula and the New Territories).
Overall, 194 (73%) participants do not want to receive new influenza A (H1N1) vaccine when it is available. The reasons for their not intending to receive vaccination when it is available are summarised in Table 2 .
The characteristics of respondents who were willing to accept influenza A (H1N1) vaccination and with those who were not willing to accept influenza A (H1N1) influenza vaccination were compared by χ2 test and were presented Table 1 . Nurses who were willing to receive influenza A (H1N1) vaccine were different from nurses who were not willing to receive influenza A (H1N1) vaccine with respect to "being vaccinated against seasonal influenza vaccination in the previous 12 months". There were no statistical significant differences in other characteristics as analyzed by chi-square test.
The relationship between demographic and other characteristics of the nursing respondents and their willingness to accept vaccination were analyzed further using forced entry logistic regressions (Table 3) . Having seasonal vaccination in the past 12 months was significantly independently associated with the willingness to accept influenza A (H1N1) vaccination (OR = 4.03; 95% IC: 2.03-7.98). Washing hands before and between patient contact, however, was negatively independently associated with willingness to accept influenza A (H1N1) vaccination (OR = 0.49; 95% IC: 0.23-1.06). To confirm the results, we have also conducted backward logistic regression and the results also indicated that having seasonal vaccination in the past 12 months was significantly associated with the willingness to accept influenza A (H1N1) vaccination (OR = 3.56, 95% CI: 1.87-6.80, p < 0.001).
Consistent with findings from previous surveys conducted in hospital healthcare workers and nurses [13, 17] , we have shown that the majority of nurses from community nursing services in Hong Kong were not willing to be vaccinated against H1N1 influenza when the vaccine becomes available. Similar to findings from previous studies in healthcare workers [13, 17, 23, 24] , we showed that the major concerns for vaccination against pandemic influenza was fear of side effects and concern of efficacy of the new vaccine (Table 2) . Moreover, influenza vaccination in the previous 12 months was significantly associated with their willingness to accept the pandemic influenza vaccination.
We also showed that in addition to previous vaccination with seasonal influenza, preventive behaviours such as frequent hand washing practice were independently associated with nurses' willingness to accept influenza A (H1N1) vaccination. We showed that "have been washing hands between and before patient contact" was negatively associated with willingness to accept vaccination independently although the reason for this is unclear and be a result of our relatively small sample. We can only postulate that the barrier to pandemic influenza vaccination is probably not related to the willingness of nurses to protect themselves against infections or their personal hygiene in general. Researchers [17] have suggested one of the barriers to pandemic influenza vaccination in nurses was misconceptions about the purpose of vaccinations in which nurse might think that the aim of vaccination was for self protection rather than to protect at risk populations in contact with them [17, 23, 25] . Specific vaccination policy for health care workers may improve vaccination in this group as nurses have different concerns and priorities when compared to the general public's concerns [17, 25] .
Although some may suggest that more educational programs for healthcare workers may be a solution to the low vaccination uptake [13] , studies have reported low influenza vaccination rates among healthcare workers even when educational programs were implemented [10] . Other studies including randomized controlled trials also failed to show that better knowledge or educational programmes (27) Other concerns (i.e. pregnancy, poor health status, and the severity of the epidemic of H1N1) 10 (<0.1)
Note: The total percentage exceeds 100% because multiple responses were allowed.
were effective in increasing acceptability of vaccination in healthcare workers [26] . Indeed, some suggested that educational campaigns based on the Health Belief Model were unlikely to be enough to change healthcare workers' acceptability of vaccination as evidence showed that perceived seriousness of infection, acknowledgement of increased risk of infection and knowledge of vaccine being safe were unrelated to vaccine uptake in healthcare workers [26] . Others suggested that educational programmes may be counter-productive as many of these healthcare workers do not perceive themselves to be at risk for contracting the infection. Recently, Ofstead et al [25] suggested that an ecological model, which included engaging organizations, communities and policy makers to create environments that were more conducive to risk reduction, might be more effective in increasing vaccination rates in healthcare workers.
To our knowledge, this is the first study to explore the willingness of nurses who work in the community to be vaccinated for pandemic influenza and our results confirmed that their acceptability of influenza A (H1N1) vaccination is low. A strength of our study is our response rate of 67% which is higher than similar report conducted in Hong Kong with a response rate of 28% [18] . A limitation of our study is that we have only documented nurses' intentions of when a vaccine is available and not the actual uptake of vaccination. Furthermore, all data from this study were from self-reports and recall bias, such recalling influenza vaccination in the previous year, might have occurred. A possible contributory factor e.g. recent episode of influenza-like illness which may influence the willingness of vaccination was not enquired. Our analysis of results was limited by the relatively small sample size in nurses who are part of the Community Nursing Service in Hong Kong with no information available on non respondents. However, our results are similar to recent studies conducted in hospital healthcare workers [13] and members of professional nursing organizations [17] in Hong Kong.
Consistent with previous findings which were conducted in healthcare workers and nurses [13, 17] , we confirm that the acceptance rate of pandemic influenza vaccination is low amongst community nurses. Since community nurses are at high risk of contracting influenza infection, and play a significant role in caring for community cases, special attention should be paid to this group as successful vaccination strategy has been shown to be beneficial in disease transmission [27] . Future work, including interventional studies evaluating potential interventions based on the ecological model or interventions that aim to increase awareness among nurses on the importance of vaccination in healthcare workers to protect vulnerable populations [16] is needed. The need to address low influenza vaccination rates in this high-risk group is urgent in the context of pandemic response. Development of an optimized RNA-based murine norovirus reverse genetics system Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of in vitro transcribed capped RNA directly into cells. The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.  On epidemic modeling in real time: An application to the 2009 Novel A (H1N1) influenza outbreak in Canada BACKGROUND: Management of emerging infectious diseases such as the 2009 influenza pandemic A (H1N1) poses great challenges for real-time mathematical modeling of disease transmission due to limited information on disease natural history and epidemiology, stochastic variation in the course of epidemics, and changing case definitions and surveillance practices. FINDINGS: The Richards model and its variants are used to fit the cumulative epidemic curve for laboratory-confirmed pandemic H1N1 (pH1N1) infections in Canada, made available by the Public Health Agency of Canada (PHAC). The model is used to obtain estimates for turning points in the initial outbreak, the basic reproductive number (R(0)), and for expected final outbreak size in the absence of interventions. Confirmed case data were used to construct a best-fit 2-phase model with three turning points. R(0 )was estimated to be 1.30 (95% CI 1.12-1.47) for the first phase (April 1 to May 4) and 1.35 (95% CI 1.16-1.54) for the second phase (May 4 to June 19). Hospitalization data were also used to fit a 1-phase model with R(0 )= 1.35 (1.20-1.49) and a single turning point of June 11. CONCLUSIONS: Application of the Richards model to Canadian pH1N1 data shows that detection of turning points is affected by the quality of data available at the time of data usage. Using a Richards model, robust estimates of R(0 )were obtained approximately one month after the initial outbreak in the case of 2009 A (H1N1) in Canada. Epidemics and outbreaks caused by emerging infectious diseases continue to challenge medical and public health authorities. Outbreak and epidemic control requires swift action, but real-time identification and characterization of epidemics remains difficult [1] . Methods are needed to inform real-time decision making through rapid characterization of disease epidemiology, prediction of shortterm disease trends, and evaluation of the projected impacts of different intervention measures. Real-time mathematical modeling and epidemiological analysis are important tools for such endeavors, but the limited public availability of information on outbreak epidemiology (particularly when the outbreak creates a crisis environment), and on the characteristics of any novel pathogen, present obstacles to the creation of reliable and credible models during a public health emergency. One needs to look no further than the 2003 SARS outbreak, or ongoing concerns related to highly pathogenic avian influenza (H5N1) or bioterrorism to be reminded of the need for and difficulty of real-time modeling.
The emergence of a novel pandemic strain of influenza A (H1N1) (pH1N1) in spring 2009 highlighted these difficulties. Early models of 2009 pH1N1 transmission were subject to substantial uncertainties regarding all aspects of this outbreak, resulting in uncertainty in judging the pandemic potential of the virus and the implementation of reactive public health responses in individual countries (Fraser et al. [2] ). Multiple introductions of a novel virus into the community early in the outbreak could further distort disease epidemiology by creating fluctuations in incidence that are misattributed to the behavior of a single chain of transmission.
We sought to address three critical issues in real time disease modeling for newly emerged 2009 pH1N1: (i) to estimate the basic reproduction number; (ii) to identify the main turning points in the epidemic curve that distinguish different phases or waves of disease; and (iii) to predict the future course of events, including the final size of the outbreak in the absence of intervention. We make use of a simple mathematical model, namely the Richards model, to illustrate the usefulness of near realtime modeling in extracting valuable information regarding the outbreak directly from publicly available epidemic curves. We also provide caveats regarding inherent limitations to modeling with incomplete epidemiological data.
The accuracy of any modeling is highly dependent on the epidemiological characteristics of the outbreak considered, and most epidemic curves exhibit multiple turning points (peaks and valleys) during the early stage of an outbreak. While these may be due to stochastic ("random") variations in disease spread, and changes in either surveillance methods or case definitions, turning points may also represent time points where epidemics transition from exponential growth processes to processes that have declining rates of growth, and thus may identify effects of disease control programs, peaks of seasonal waves of infection, or natural slowing of growth due to infection of a critical fraction of susceptible individuals. For every epidemic, there is a suitable time point after which a given phase of an outbreak can be suitably modeled, and beyond which subsequent phases may be anticipated. Detection of such "turning points" and identification of different phases or waves of an outbreak is of critical importance in designing and evaluating different intervention strategies.
Richards [3] proposed the following model to study the growth of biological populations, where C(t) is the cumulative number of cases reported at time t (in weeks):
Here the prime "′" denotes the rate of change with respect to time. The model parameter K is the maximum case number (or final outbreak size) over a single phase of outbreak, r is the per capita growth rate of the infected population, and a is the exponent of deviation. The solution of the Richards model can be explicitly given in terms of model parameters as Using the Richard model, we are able to directly fit empirical data from a cumulative epidemic curve to obtain estimates of epidemiological meaningful parameters, including the growth rate r. In such a model formulation, the basic reproduction number R 0 is given by the formula R 0 = exp(rT) where T is the disease generation time defined as the average time interval from infection of an individual to infection of his or her contacts. It has been shown mathematically [4] that, given the growth rate r, the equation R 0 = exp(rT) provides the upper bound of the basic reproduction number regardless of the distribution of the generation interval used, assuming there is little pre-existing immunity to the pathogen under consideration. Additional technical details regarding the Richards model can be found in [5] [6] [7] .
Unlike the better-known deterministic compartmental models used to describe disease transmission dynamics, the Richards model considers only the cumulative infected population size. This population size is assumed to have saturation in growth as the outbreak progresses, and this saturation can be caused by immunity, by implementation of control measures or other factors such as environmental or social changes (e.g., children departing from schools for summer holiday). The basic premise of the Richards model is that the incidence curve of a single phase of a given epidemic consists of a single peak of high incidence, resulting in an S-shaped cumulative epidemic curve with a single turning point for the outbreak. The turning point or inflection point, defined as the time when the rate of case accumulation changes from increasing to decreasing (or vice versa) can be easily pinpointed as the point where the rate of change transitions from positive to negative; i.e., the moment at which the trajectory begins to decline. This time point has obvious epidemiologic importance, indicating either the beginning of a new epidemic phase or the peak of the current epidemic phase.
For epidemics with two or more phases, a variation of the S-shaped Richards model has been proposed [6] . This multi-staged Richards model distinguishes between two types of turning points: the initial S curve which signifies the first turning point that ends initial exponential growth; and a second type of turning point in the epidemic curve where the growth rate of the number of cumulative cases begins to increase again, signifying the beginning of the next epidemic phase. This variant of Richards model provides a systematic method of determining whether an outbreak is single-or multi-phase in nature, and can be used to distinguish true turning points from peaks and valleys resulting from random variability in case counts. More details on application of the multi-staged Richards model to SARS can be found in [6, 7] . Readers are also referred to [8, 9] for its applications to dengue.
We fit both the single-and multi-phase Richards models to Canadian cumulative 2009 pH1N1 cumulative case data, using publicly available disease onset dates obtained from the Public Health Agency of Canada (PHAC) website [10, 11] . PHAC data represent a central repository for influenza case reports provided by each of Canada's provinces and territories. Onset dates represent best local estimates, and may be obtained differently in different jurisdictions. For example, the province of Ontario, which comprises approximately 1/3 of the population of Canada, and where most spring influenza activity was concentrated, replaces onset dates using a hierarchical schema, whereby missing onset dates may be replaced with dates of specimen collection (if known) or date of specimen receipt by the provincial laboratory system, if both dates of onset and specimen collection are missing.
Data were accessed at different time points during the course of the "spring wave (or herald wave)" of the epidemic in May-July of 2009, whenever a new dataset is made available online by the PHAC. By sequentially considering successive S-shaped segments of the epidemic curve, we estimate the maximum case number (K) and locate turning points, thus generating estimates for cumulative case numbers during each phase of the outbreak. The PHAC cumulative case data is then fitted to the cumulative case function C(t) in the Richards model with the initial time t 0 = 0 being the date when the first laboratory confirmed case was reported and the initial case number C 0 = C(0) = 1, (the case number with onset of symptoms on that day). There were some differences between sequential epidemic curves in assigned case dates. For example, data posted by PHAC on May 20 indicated an initial case date of April 13, but in the June 3 data this had been changed to April 12, perhaps due to revision of the case date as a result of additional information.
Model parameter estimates based on the explicit solution given earlier can be obtained easily and efficiently using any standard software with a least-squares approximation tool, such as SAS or Matlab.
Daily incidence data by onset date were posted by PHAC until June 26, after which date only the daily number of laboratory-confirmed hospitalized cases in Canada was posted. For the purpose of comparison, we also fit the hospitalization data to the Richards model in order to evaluate temporal changes in the number of severe (hospitalized) cases, which are assumed to be approximately proportional to the total cases number. The case and hospitalization data used in this work are provided online as Additional file 1.
We fit the model to the daily datasets, acquired in real time, throughout the period under study. The leastsquared approximation of the model parameter estimation could converge for either the single-phased or the 2-phase Richards models. For the sake of brevity, only four of these model fits are presented in Table 1 to demonstrate the difference in modeling results over time. The resulting parameter estimates with 95% confidence intervals (CI) (for turning point (t i ), growth rate (r), and maximum case number (K)), time period included in the model, and time period when the data set in question were accessed, is presented in Table 1 . Note that all dates in the tables are given by month/day. We also note that the CI's for R 0 reflect the uncertainty in T as well as in the estimates for r, and does not reflect the error due to the model itself, which is always difficult to measure.
In order to compare the 1-phase and 2-phase models, we also calculate the Akaike information criterion (AIC) [12] for the first, third, and fourth sets of data in Table  1 , where there is a model fit for the 2-phase model. The results, given in Table 2 , indicates that whenever there is a model fit for the 2-phase model, its AIC value is always lower than that of the 1-phase model and hence compares favorably to the 1-phase model.
Parameter estimates fluctuate in early datasets, and the least-squared parameter estimations diverge within and between 1-phase and 2-phase models in a manner that seems likely to reflect artifact. In particular, for the earliest model fits, using data from April 13 to May 15, the estimated reproductive number for the second phase is far larger than that obtained in the first phase, and that obtained using a single-phase model, and illustrating the pitfalls of model estimation using the limited data available early in an epidemic. Estimates stabilize as the outbreak progresses, as can be seen with the final data sets (April 11 to June 5 and April 12 to June 19). For comparison, we plot the respective theoretical epidemic curves based on the Richards model with the estimated parameters described in the table above in Figure 1 .
As noted above, model can be used to estimate turning points (t i ) and basic reproductive numbers (R 0 .), if the generation time T is know. We used T = 1.91 days (95% CI: 1.30-2.71), as obtained in [2] by fitting an age stratified mathematical model to the first recognized 2009 influenza A (H1N1) outbreak in La Gloria, Mexico. Estimates are presented in Table 1 . We also conducted sensitivity analyses with R 0 # calculated based on longer generation times (T = 3.6 (2.9, 4.3)) for seasonal influenza in [13] (see last column in Table 1 ). Excluding implausibly high estimates of R 0 generated using initial outbreak data (April 13 to May 15), we obtain the estimates of R 0 for the 2-phase model that range between 1.31 and 1.96. Inasmuch as Richards model analyzes the general trends of an epidemic (e.g., turning point, reproductive number, etc.), it can be used to fit any epidemiological time series for a given disease process, as long as the rate of change in the recorded outcome is proportional to changes in the true number of cases. As such, for comparison, we fit our model using the time series for 2009 pH1N1 hospitalizations in Canada posted by PHAC on July 15 [11] (that last date these data were made available) ( Table 3) . This time series was easily fit to a one-phase model ( Figure 2) . Further examples of using hospitalization or mortality data to fit the Richards model can be found in [14] .
We used the Richards model, which permits estimation of key epidemiological parameters based on cumulative case counts, to study the initial wave of 2009 influenza A (H1N1) cases in Canada. In most model fits, April 28-29 and May 4-7 were identified as early turning points for the outbreak, with a third and final turning point around June 3-5 in models based on longer time series. Although this modeling approach was not able to detect turning points using some earlier data sets (e.g., those limited to the period from April 12 to May 27), in general the turning points identified were consistent across multiple models and time series. Perhaps the most important divergence between models occurred with the detection of an April 29 turning point in the case report time series, but not in the time series based on hospitalized cases. We believe this may be attributable to the small number of hospitalizations, relative to cases, that had occurred by that date, as well as the fact that hospitalization data only became available on April 18.
The turning point can correspond to the point at which disease control activities take effect (such that the rate of change in epidemic growth begins to decline) or can represent the point at which an epidemic begins to wane naturally (for example, due to seasonal shifts or due to the epidemic having "exhausted" the supply of susceptibles such that the reproductive number of the epidemic declines below 1). This quantity has direct policy relevance; for example, in the autumn 2009 pH1N1 wave in Canada, vaccination for pH1N1 was initiated at or after the turning point of the autumn wave due to the time taken to produce vaccine; as the epidemic was in natural decline at that point, the impact of vaccination has subsequently been called into question. Although the Richards model is able to capture the temporal changes in epidemic dynamics over the course of an outbreak, it does not define their biological or epidemiological basis. As such, determining the nature of these turning points requires knowledge of "events on the ground" for correlation. We suspect that the last Note that all dates in the tables are given by month/day. Dates of posting are listed in parentheses. Model duration indicates whether they fit a 1-phase or 2phase model. Note that the maximum case number is rounded off to the nearest integer. R 0 # is obtained using the generation interval of T = 3.6 (2.9, 4.3) for seasonal influenza [13] . Table 2 Comparison of Akaike information criterion (AIC) values between 1-phase and 2-phase models for time periods with 2-phase model fit in Table 1 Time Table 1 , last line) and a 1-phase model using hospitalization data (June 11), this lag in turning points would actually be expected, due to the time from initial onset of symptoms until hospitalization, which was reported to have an interquartile range of 2-7 days in a recent study from Canada [15] . Timelines for the 2-phase model for case data of 4/12-6/19 and the 1-phase model for hospitalization data are presented graphically in Figure 3 . In addition to identifying turning points, the Richards model is useful for estimation of the basic reproductive number (R 0 ) for an epidemic process, and our estimates derived using a Richards model were consistent with estimates derived using other methods. For example, our R 0 agrees almost perfectly with that of Tuite et al., derived using a Markov chain Monte Carlo simulation parameterized with individual-level data from Ontario's public health surveillance system [16] . Our estimates of R 0 is smaller than that derived by Fraser et al. [2] using Mexican data, but such differences could relate in part to the different age distributions of these two countries [17] , and may also reflect the fact that our estimate is obtained Canadian data at a national level, while empirical Mexican estimates were based on data from the town of La Gloria with only 1575 residents.
Most epidemic curves in the early stage of a novel disease outbreak have multiple phases or waves due to simple stochastic ("random") variation, mechanisms of disease importing, initial transmission networks and individual/community behavior changes, improvements in the performance of surveillance systems, or changes in case definitions as the outbreak response evolves. However, changes in phase (signified by the presence of turning points identified using the Richards model) may also pinpoint the timing of important changes in disease dynamics, such as effective control of the epidemic via vaccination or other control measures, depletion of disease-susceptible individuals (such that the effective reproductive number for the disease decreases to < 1), or the peak of a "seasonal" wave of infection, as occurs with [4, 18, 19] , some competing methods require more extensive and detailed data than are required to build a Richards model, which requires only cumulative case data from an epidemic curve. As we also demonstrate here, the Richards model produces fairly stable and credible estimates of reproductive numbers early in the outbreak, allowing these estimates to inform evolving disease Table 1 , derived using early case data accessed on May 20, closely approximate our final estimates (Table 1, last row) . Thus, while early estimation with the Richards model failed to correctly detect turning points or accurately estimate the final outbreak size, it was nonetheless useful for rapid estimation of R 0 within a month of first case occurrence in Canada.
As with any mathematical modeling technique, the approach presented here is subject to limitations, which include data quality associated with real-time modeling (as data are often subject to ongoing cleaning, correction, and reclassification of onset dates as further data become available), reporting delays, and problems related to missing data (which may be non-random). In our current study, the hierarchical approach used by Canada's most populous province (Ontario) for replacement of missing data could have had distorting effects on measured disease epidemiology: the replacement of missing onset dates with dates of specimen collection could have resulted in the artifactual appearance of early turning points identified by our model, due to limitations in weekend staffing early in the outbreak. If, as we believe to be the case, public health laboratories did not have sufficient emergency staffing to keep up with testing on weekends such that weekend specimen log-ins declined sharply, this would have created the appearance of epidemic "fade out" on weekends. Other factors that might distort the apparent epidemiology of disease include changes in guidelines for laboratory testing of suspected cases, improved surveillance and public health alerts at later stages of the outbreak leading to increased case ascertainment or over-reporting of cases [20] . However, the quality of the time series will tend to improve with the duration of the epidemic, both because stochastic variation is "smoothed out", and also because small variations become less important as the cumulative series becomes longer. We note that a further application of the Richards model in the context of influenza would relate to comparison of the epidemiology of the 2009 influenza A H1N1 epidemic to past Canadian epidemics, though such an endeavor is beyond the scope of the present study.
In summary, we believe that the Richards model provides an important tool for rapid epidemic modeling in the face of a public health crisis. However, predictions based on the Richards model (and all other mathematical models) should be interpreted with caution early in an epidemic, when one need to balance urgency with sound modeling. At their worst, hasty predictions are not only unhelpful, but can mislead public health officials, adversely influence public sentiments and responses, undermine the perceived credibility of future (more accurate) models, and become a hindrance to intervention and control efforts in general.
Additional file 1: Electronic Supplementary Material. 2009 Canada novel Influenza A(H1N1) daily laboratory-confirmed pandemic H1N1 case and hospitalization data. Antiviral and Neuroprotective Role of Octaguanidinium Dendrimer-Conjugated Morpholino Oligomers in Japanese Encephalitis BACKGROUND: Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment. CONCLUSIONS/SIGNIFICANCE: Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation. The genus Flavivirus is composed of more than 70 different closely related species [1] . Many flaviviruses are arthropod-borne and causes significant human diseases. Among these, the four serotypes of dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are categorized as emerging global pathogens [2] . JEV is a mosquitoborne, positive sense, single stranded RNA virus, responsible for frequent epidemics of encephalitis, predominantly in children, in most parts of Southeast Asia and adjoining regions. It is the causal factor for 30,000-50,000 cases of encephalitis occurring every year and accounts for about 10,000 deaths annually with serious neurological squeal in the survivors [3] . JEV has been expanding its 'geographical footprint' into previously non-endemic regions and with several billion people at risk, Japanese encephalitis (JE) represents an internationally emerging concern in tropical and sub-tropical countries. Currently three types of JE vaccine are in use-the inactivated mouse-brain derived, the inactivated cellculture derived and the live attenuated cell-culture derived. However, there are limitations for their usage in terms of availability, cost and safety [4] . At present, chemotherapy against JEV is largely supportive and not targeted towards the virus. A lot of avenues has been explored in the past and are also being currently tried, so as to develop a safe and effective molecule that would be able to prevent the virus from replicating within the host.
The JEV genome is approximately 11 kb in length that carries a single long open reading frame (ORF) flanked by a 95-neucleotide 59 untranslated region (59 UTR) and a 585-neucleotide 39 UTR. The ORF encodes a polyprotein which is processed by viral and cellular proteases into three structural and seven non structural proteins [5, 6] . The 59 and 39 UTRs of the JEV genome contain conserved sequence elements and can form conserved stem loop structure. 59 UTR contain secondary structures which are required for the formation of translation pre-initiation complex [7] . JEV requires long range RNA-RNA interaction between 59 and 39 regions of its genome for efficient replication; one such interaction occurs between a pair of 10 complementary nucleotides, located in coding sequence for the capsid protein at 136-146 nucleotides from 59 end of the genome, and 39 cyclization sequence, commonly denoted as 39CSI (39 conserved sequence I) located at 104-114 nucleotides from 39 end of the genome [8, 9] . The 39CSI is highly conserved across members of JEV serocomplex, indicating the possibility that RNA elements within the 59 and 39 UTRs in JEV genome are essential for its replication.
Anti-sense oligonucleotides have been shown to be effectively used as therapeutic agents against viral infection. In one such study siRNA generated against the cd loop-coding sequence in domain II of the viral Envelope protein (which is highly conserved among all flaviviruses because of its essential role in membrane fusion) has been found to protect against lethal encephalitis [10] . Similarly siRNAs has also been generated against various nonstructural proteins of JEV and were found to be effective in inhibiting viral replication [11, 12] . Anti-sense approach has also been employed to inhibit flaviviral replication by generating anti-sense molecules against RNA elements within the 59 and 39 UTRs in flaviviral genome. In one such approach, DNAzyme against 39 UTR of JEV genome has be found to be effective in controlling virus infection in a murine model [13] . Under the same approach but with different kind of anti-sense oligonuleuotide called Morpholino, flaviviral replication has been inhibited in cultured cells as well as in animal models [14, 15] .
Morpholino oligomers are single stranded DNA analogues containing same nitrogenous bases as DNA but joined by backbone consisting of morpholine rings and phosphorodiamidate linkages [16] . For efficient delivery into cells these Morpholino are often conjugated with arginine rich peptide [17] . However, in the current study we have used a different type of Morpholino oligomer called Vivo-Morpholino against 39CSI and one of the secondary structures present in 59 UTR of the JEV genome. Vivo-Morpholino are specialized type of non-peptide Morpholino oligomers, conjugated with a new transport structure that provides effective delivery into a wide variety of tissues in living animals, thereby raising the possibilities of their use as therapeutic agents. The transporter comprises of a dendritic structure assembled around a triazine core which serves to position eight guanidinium head groups in a conformation effective to penetrate cell membranes. Vivo-Morpholinos have also been shown to effectively enter and function within cultured cells [18] . Vivo-Morpholinos are also cost effective, non immunogenic, and stable under physiological conditions as compared to other types of Morpholinos. This study was designed to evaluate whether the use of Vivo-Morpholinos as therapeutic agents, is possible in an experimental model of JE. We intend to show that these specifically designed Vivo-Morpholinos are effective in countering the viral load in the body, thereby imparting significant protection to the animals that were infected with a lethal dose of JEV.
All animal experiments were approved by the institutional animal ethical review board named ''Institutional Animal and Ethics Committee of National Brain Research Centre''. The animal experiment protocol approval no. is NBRC/IAEC/2007/ 36. Animals were handled in strict accordance with good animal practice as defined by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forestry, Government of India.
Vero cells (a kind gift from Dr. Guruprasad Medigeshi, Translational Health Science and Technology Institute, Gurgaon, India) and Neuro2A (obtained from National Centre for Cell Science, Pune, India) cells were grown in DMEM (Dulbecco's modified Eagles medium, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The GP78 strain of JEV was propagated in suckling BALB/c mice and their brains were harvested when symptoms of sickness were observed. A 10% tissue suspension was made in MEM (minimum essential medium), followed by centrifugation at 10,0006g and finally filtered through a 0.22 m sterile filter [19] . JEV was titrated by plaque formation on Vero cell monolayer. Vero cells were seeded in six-well plates to form semi-confluent monolayer in about 18 h. Cell monolayer were inoculated with 10-fold serial dilutions of virus samples made in MEM containing 1% FBS and incubated for 1 h at 37uC with occasional shaking. The inoculum was removed by aspiration and the monolayers were overlaid with MEM containing 4% FBS, 1% low-melting-point agarose and a cocktail of antibiotic-antimycotic solution (Gibco, Invitrogen Corporation, Grassland, NY, USA) containing penicillin, streptomycin, and amphotericin B. Plates were incubated at 37uC for 72-96 h until plaques became visible. To allow counting of the plaques, the cell monolayer was stained with crystal violet after fixing the cells with 10% formaldehyde.
All Vivo-Morpholino (MO) oligos were commercially procured from Gene Tools LLC, (Philomath, OR, USA). MOs were
Japanese encephalitis (JE) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the Culex sp. The threat of JE looms over a vast geographical realm, encompassing approximately 10 billion people. The disease is feared because currently there are no specific antiviral drugs available. There have been reports where other investigators have shown that agents that block viral replication can be used as effective therapeutic countermeasures. Vivo-Morpholinos (MOs) are synthetically produced analogs of DNA or RNA that can be modified to bind with specific targeted regions in a genome. In this study the authors propose that in an animal model of JE, MOs specifically designed to bind with specific region of JE virus (JEV) genome, blocks virus production in cells of living organisms. This results in reduced mortality of infected animals. As the major target of JEV is the nerve cells, analysis of brain of experimental animals, post treatment with MOs, showed neuroprotection. Studies in cultured cells were also supportive of the antiviral role of the MOs. The potent anti-sense effect in animals and lack of obvious toxicity at the effective dosage make these MOs good research reagents with future therapeutic applications in JE.
Vivo-Morpholino in Japanese Encephalitis www.plosntds.org designed to be complementary to sequences in the JEV (GP78 strain) genome, as shown in Table 1 . These oligonucleotides targeted specific regions in the 39 and 59 UTRs of the JEV genomic RNA (Figure 1) . A 21 base scrambled MO of random sequence (SC-MO) was used as a negative control in all the experiments. All MO sequences were screened with BLAST (http://www.ncbi.nlm.nih.gov/BLAST) against primate and murine mRNA sequences and the SC-MO was additionally screened against all flaviviral sequences. All MOs were procured in 300 nanomole quantities as a liquid of 0.5 mM stock (approximately 4 mg/mL) in buffered saline. They were diluted with sterile 16 PBS to achieve desired concentrations, and stored at 4uC as aliquots.
Five to six weeks old BALB/c mice of either sex were randomly distributed into 5 groups-Sham, JEV-infected, JEV-infected and treated with scrambled Morpholino (JEV+SC-MO), JEV-infected and treated with Morpholino against viral 39 conserved region (JEV+39 MO) and JEV-infected and treated with Morpholino against secondary structure in the 59UTR of viral RNA (JEV+59 MO). Initially each group contained 8 animals. Animals belonging to all groups except Sham were infected with 3610 5 plaque forming units (PFU) of JEV (GP78 strain) and that day was considered as day zero [20] . Animals of Sham group received equal volume of filtered MEM. Starting from 3 h post infection on day zero, 100 mg of SC-MO, 39 MO and 59 MO, diluted in 0.1 mL of sterile 16PBS (corresponding to 5 mg/kg body weight), were administered to animals belonging to JEV+SC-MO, JEV+39 MO and JEV+59 MO groups respectively, once per day, for 5 consecutive days. Animals belonging to the Sham-treatment group received equal volumes of sterile 16 PBS only. Survivality of animals in each group following JEV infection and Morpholino treatment were monitored daily upto 15 days post JEV infection (or till their death, whichever was earlier). Toxicity of the Morpholinos in mice was evaluated by weight loss and abnormal behavioral & clinical observations (including tremors, ruffled fur, hunching, ataxia, gait abnormalities), in a masked manner to minimize bias [14, 20] .
Mouse cytokine bead array (CBA) kits were used to quantitatively measure cytokine levels in mouse whole-brain lysates. 50 mL of bead mix containing a population of beads with distinct fluorescence intensities that have been coated with capture antibodies for different cytokines, and 50 mL of whole-brain lysates were incubated together, along with equal volume of phycoerythrin (PE)-conjugated detection antibodies, for 2 h at room temperature, in dark. The beads were then washed and resuspended in 300 mL of supplied 16 wash buffer. The beads were acquired using Cell Quest Pro Software in FACS Calibur and analyzed using BD CBA software (Becton Dickinson, San Diego, CA). Standard curve was prepared by incubating 50 mL of supplied mouse inflammation standards with 50 mL of bead mix and PE-conjugated detection antibodies [21] .
Protein concentrations of whole brain lysates were estimated by Bradford method. Sample volumes containing 20 mg of protein were electrophoresed on polyacrylamide gel and transferred onto nitrocellulose membrane. After blocking with 7% skimmed milk, the blots were incubated overnight at 4uC with primary antibodies against JEV E-glycoprotein (Abcam, USA), and JEV NS5 (a kind gift from Dr. Chun-Jung Chen, Taichung Veterans General Hospital, Taichung, Taiwan), iNOS (Upstate-Chemicon, USA), HSP-70, SOD-1 (Santa Cruz Biotechnology, CA, USA), TRX (AB Frontiers, Korea; a kind gift from Dr. Ellora Sen, NBRC), phospho NFkB, phospho ERK1/2, total ERK1/2 and phos-phoP38 MAP kinase (Cell Signaling, USA) at 1:1000 dilutions. After extensive washes with PBS-Tween, blots were incubated with appropriate secondary antibodies conjugated with peroxidase (Vector Laboratories, CA, USA). The blots were again washed with PBS-Tween and processed for development using chemiluminescence reagent (Millipore, USA). The images were captured and analyzed using Chemigenius, Bioimaging System (Syngene, Cambridge, UK). The blots were stripped and reprobed with antib-tubulin (Santa Cruz Biotechnology, USA) to determine equivalent loading of samples [22] .
For immunohistochemical staining, brains from scarified animals were excised following repeated transcardial perfusion with ice-cold saline and fixed with 4% paraformaldehyde. Twenty micron thick cryosections were made with the help of Leica CM3050S cryostat and processed for immunohistochemical staining to detect presence of JEV antigen in the brain and to label activated microglia. Sections were incubated overnight at 4uC with mouse anti-JEV antigen (Nakayama, 1:250) (Chemicon, CA, USA) and rabbit anti-Iba-1 (1: 500; Wako, Osaka, Japan), respectively. After washes, slides were incubated with FITCconjugated anti-mouse or anti-rabbit secondary antibodies (Vector laboratories Inc. Burlingame, USA) and following final washes, sections were sections were cover slipped after mounting with 49-6diamidino-2-phenylindole (DAPI, Vector laboratories Inc.). The slides were observed under Zeiss Axioplan 2 fluorescence microscope and Zeiss Apotome microscope (Zeiss, Gottingen, Germany), respectively [21] .
Cryosections of brain from Sham-treated, JEV-infected and JEV-infected and MO treated animals were rinsed in de-ionized water followed by incubation with the thionin dye. The excess dye 
The level of ROS produced within brain tissue of each treatment groups were measured by the cell permeable, nonpolar, H 2 O 2 -sensitive probe 5(and 6)-chlromethyl-20,70-dichlorodihydrofluoresceindiacetate (CM-H2DCFDA; Sigma, USA). CM-H2DCFDA diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. Subsequent oxidations of CM-H2DCFDA yields a fluorescent adduct dichlorofluorescein that is trapped inside the cell. Brain homogenates were treated with 5 mM solution of CM-H2DCFDA followed by incubation in dark at room temperature for 45 min and then the relative fluorescence intensity were measured with the help of Varioskan Flash multimode reader (Thermo Electron, Finland) at excitation 500 nm and emission 530 nm. The fluorescence intensity of intracellular CM-H2DCFDA is a linear indicator of the amount of H 2 O 2 in the cells. The measured mean fluorescence intensity was then normalized to equal concentrations of protein in each sample [23] .
Nitric oxide released from brain homogenates following MO treatment was assessed using Griess reagent as described previously. Briefly, 100 mL of Griess reagent (Sigma, St. Louis, USA) was added to 100 mL of brain homogenate and incubated in dark for 15 min. The intensity of the color developed was estimated at 540 nm with the help of a Benchmark plus 96-well ELISA plate reader (Biorad, CA, USA). The amount of nitrite accumulated was calculated (in mM) from a standard curve constructed with different concentrations of sodium nitrite [21] .
Mouse neuroblastoma cells (N2a) were plated in five 60 mm plates at a density of 5610 5 cells/plate, and were cultured for 18 h. After 6 h in serum free DMEM, cells were either mock- Vivo-Morpholino in Japanese Encephalitis www.plosntds.org infected with sterile 16PBS or infected with JEV at multiplicity of infection (MOI) of 5. After 1K h, cells were washed twice with sterile 16PBS to remove non-internalized virus. Three of the four plates that were infected with JEV, were treated with SC-MO, 39 MO and 59 MO at 10 mM concentrations and all plates were incubated for 24 h in serum free media.
After two washes with 16 PBS, cells were first fixed with BD cytofix solution (BD Biosciences) for 15 min and permeabilized by resuspending in permeabilization buffer (BD Cytoperm plus; BD Biosciences) and incubated at 25uC for at least 10 min. Cells were then washed twice in wash buffer (PBS containing 1% bovine serum albumin) then resuspended in wash buffer at 1610 6 cells per 100 mL. Primary antibody (JEV Nakayama strain; Chemicon, USA) were added in 1:100 dilutions and incubated for 30 min at 25uC. The cells were washed with wash buffer and pelleted by centrifugation followed by incubation with FITC conjugated secondary antibody for 30 min. After final wash with wash buffer, cells were resuspended in 400 mL FACS buffer and analyzed on a FACS Calibur. The percentage of population of JEV-positive cells was calculated after gating the populations on a Dot plot using Cell Quest Pro Software (BD Biosciences).
Statistical analysis was performed using SIGMASTAT software (SPSS Inc., Chicago, IL, USA). Data were compared between groups using one-way analysis of variance followed by post hoc test. Differences upto p,0.05 were considered significant.
MOs confer protection to animal from Japanese encephalitis MO treatment conferred significant protection to mice following JEV infection. The survival of mice following JEV infection was dramatically increased with treatments of both 39 and 59 MO. Approximately 90% of all the animals that were treated with 39 MO survived as compared to 75% survival of those animals that were treated with 59MO, post infection with JEV ( Figure 2A ). Infection with JEV was accompanied with distinct symptoms and weight loss whereas treatments with both 39 and 59 MO post JEV infection, prevented animals from suffering. Not much considerable changes in the average body weights of JEVinfected animals treated with both 39 and 59 MO were observed when compared to animals belonging to JEV and JEV+ SC-MO groups showing significant reductions in their body weights 6 days post infection ( Figure 2B ). The symptoms associated with JE in murine model were observed on daily basis and scores were attributed accordingly. The animals that had most symptoms received the highest scores. It was observed that 39 and 59 MO treated animals scored lesser than those belonging to the JEVinfected or JEV+SC-MO groups ( Figure 2C ).
To assess whether the MOs has any effect on reduction of viral load in brain, homogenized brain samples from all the treatment groups were subjected to plaque assay as described in materials and methods section. Number of PFU/mL of the brain homogenate was found to be significantly higher in both JEV and JEV+SC-MO groups when compared to Sham (p,0.001). Viral PFUs were found to be significantly reduced following 39 MO and 59 MO treatment when compared to only JEV-infected or JEV+SC-MO group (p,0.001) ( Figure 3A) .
To further validate the results obtained from the plaque assay, immunoblot for some of the JEV-specific proteins were performed.
The expression of NS5, a non structural protein of JEV, was significantly increased in JEV and JEV+SC-MO groups when compared to Sham (p,0.01), but its level were found to be significantly reduced after both 39 and 59 MO treatments when compared to JEV-infected group (p,0.01). Similarly, E glycoprotein level showed significant increase in JEV-infected and JEV+SC-MO groups when compared to Sham (p,0.01) which were then drastically reduced following 39 and 59 MO treatments (p,0.01) (Figure 3B-D) . Immunostaining of brain sections showed greater presence of JEV antigen in JEV-infected and JEV+SC-MO groups, whereas 39 and 59 MO treatments resulted in lesser presence ( Figure 3E ).
To further characterize the inhibitory effects of MO on JEVinduced neuronal death, brain sections from all the treatment groups were subjected to thionin staining. Numerous healthy cells were seen in sections obtained from Sham, JEV+39 MO and JEV+59 MO groups when compared to sections belonging to only JEV-infected or JEV+SC-MO groups which contained numerous unhealthy/dying neurons with altered morphology ( Figure 4A ).
Microglial activation and increased proinflammatory cytokine production are the hallmarks of JEV infection [24] . To see whether MO treatment helps in vitiation of these effects, immunostaining for microglial specific marker Iba-1 was performed in brain sections of all treatment groups. In brain sections of JEV and JEV+SC-MO groups the number of activated microglia with characteristic morphology, appeared to be more frequent when compared to sections belonging to Sham, JEV+39 MO and JEV+59 MO groups ( Figure 4B ). CBA performed to check the proinflammatory cytokines levels in the brain homogenates obtained from different treatments showed that levels of MCP-1, IFN-c, TNF-a, and IL-6 were found to be significantly increased in both JEV and JEV+SC-MO groups when compared to Sham infected groups (p,0.01). The elevated levels of these proinflammatory cytokines were drastically reduced with 39 and 59 MO treatments (p,0.01) ( Figure 4C -F).
Increased oxidative stress in CNS is a major outcome of JEV infection [20] . To evaluate whether MO treatment of mice resulted in abrogation of oxidative stress following JEV infection, we measured ROS and NO levels in brain homogenate obtained from all treatment groups. Two fold increases were observed in the ROS levels in the brain samples of JEV and JEV+SC-MO groups when compared to Sham (p,0.01), significant reduction in the ROS levels were observed in JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected groups (p,0.01). Although ROS levels has decreased in JEV+39 MO group when compared to JEV group, it remained significantly higher than that of Sham (p,0.01) ( Figure 5A ). Superoxide dismutase 1 (SOD-1) and Thioredoxin (TRX-1) are the proteins associated with oxidative stress. SOD-1 levels were found to be elevated approximately 2-and 3-fold in JEV-infected and JEV+SC-MO groups respectively when compared to Sham (p,0.01). Its levels in JEV+39 MO and JEV+59 MO groups were reduced significantly when compared to JEV-infected group (p,0.01). TRX-1 levels were also found to be increased significantly in JEV-infected and JEV+SC-MO groups when compared to Sham (p,0.01) but it were significantly reduced in brain samples obtained from JEV+39 MO and JEV+59 MO groups when compared to only JEVinfected groups (p,0.01) (Figure 5B, D&E) . HSP-70 is a heat shock Vivo-Morpholino in Japanese Encephalitis www.plosntds.org protein that has been associated with intracellular stress. Significant twelve fold increases in the levels of HSP-70 were observed in JEV and JEV+SC-MO groups when compared to Sham (p,0.01), this drastic increases in the levels of HSP-70 in JEV and JEV+SC-MO groups were reduced in JEV+39 MO and JEV+59 MO groups (p,0.01) ( Figure 5B&C ).
JEV infection leads to increased nitric oxide (NO) production in CNS [25] . Significant two fold increases were seen in the NO levels in brain samples obtained from JEV-infected and JEV+SC-MO groups when compared to those obtained from Sham (p,0.01). NO levels subsequently got down to significantly lower levels following 39 and 59MO treatments (p,0.01) ( Figure 5F ).
Immunoblot analysis showed nearly 8-fold increases in levels of iNOS in JEV-infected and JEV+SC-MO groups when compared to Sham (p,0.01). iNOS levels showed significant decreases in JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected groups (p,0.01) ( Figure 5G&H ).
Western blot analysis demonstrated a significant inhibition in the expression of different stress related proteins whose levels were elevated following JEV infection. Upon MO treatments there were approximately 4-fold increases in the levels of pNFkB in JEV and Figure 2 . Mice are protected from JEV following MO treatment. The survival of mice following JEV infection was dramatically increased with treatments of both 39 and 59 MO, though the survival in 39 MO treated mice was greater (,90%) than those treated with 59MO (75%) (A). Considerable changes in the average body weights of JEV-infected animals treated with both 39 and 59 MO were not observed when compared to animals belonging to JEV and JEV+ SC-MO groups that showed significant reductions in their body weights from 6 th day post infection till their death. Black arrows points to the days by which all the animals died. (B). Infection with JEV was accompanied with distinct symptoms that were alleviated following treatments with both 39 and 59 MO. Animals were assigned scores according to the symptoms, in a blinded manner. The graph was plotted by taking the scores of one animal that was considered as the representative of that group (C). n = 8 for all experiments; data shown are representative of duplicate sets of experiments. doi:10.1371/journal.pntd.0000892.g002 Vivo-Morpholino in Japanese Encephalitis www.plosntds.org JEV+SC-MO groups when compared to Sham (p,0.01). The levels of pNFkB were found to be significantly reduced in JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected groups (p,0.01) ( Figure 6A&B ). Phospho p38 MAPK levels also showed significant 3-fold increases in JEV and JEV+SC-MO groups when compared to Sham (p,0.01), its levels were also found to be reduced significantly following treatment with 39 and 59 MO when compared to only JEV-infected groups (p,0.01) ( Figure 6A&C ). Both phospho ERK1 and ERK2 levels were found to be significantly increased in JEV and JEV+SC-MO groups when compared to Sham (p,0.01). The levels of phospho ERK1 and ERK2 showed considerable decreases in JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected groups (p,0.01) ( Figure 6A&D ).
To assess whether MO has any effect on viral load in vitro N2a cell lysates from all the treatment groups were subjected to plaque assay. PFU/mL of the cell lysates was found to be significantly higher in both JEV and JEV+SC-MO groups when compared to mock-infected cells (p,0.01). Viral loads were found to significantly reduced in both JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected group (p,0.01) ( Figure S1A ). To further ascertain the results obtained from plaque assay, intracellular staining of JEV antigen in N2a was performed and number of JEV-positive N2a cells was then counted by flow cytometry. Only 16% and 9% of the total gated cells were found to be positive for JEV antigen in JEV+39 MO and JEV+59 MO groups respectively as compared to 30% in JEV-infected group, and 34% in JEV+SC-MO group ( Figure S1B ).
Use of anti-sense molecules for targeted inhibition of viral replication has been under investigation for quite sometime. Though the application of these molecules has raised the possibilities of their future use as novel therapeutic agents, there Vivo-Morpholino in Japanese Encephalitis www.plosntds.org are many issues regarding their effectiveness in terms of their stability and delivery to targeted cells. Recent studies are involved in developing techniques to minimize or eliminate these issues so that anti-sense therapy can be employed to a wide variety of intractable diseases such as splice-modifying genetic defects and viral diseases. The role of various anti-sense molecules in the inhibition of replication of JEV has been reported with positive outcomes [10, 11, 12, 13, 26] .
Morpholino oligomers are single stranded anti-sense molecules that exert their action by steric blocking of complementary RNA. Unlike other types of anti-sense oligonucleotides, Morpholinos provide all the desired properties of stability, nuclease resistance, high efficacy, long-term activity, water solubility, low toxicity, and exquisite specificity. Morpholino oligomers has been used previously for the inhibition of flaviviral replication [14, 27] including JEV [15] though all of them has utilized peptide based Morpholinos. The peptide based Morpholinos contain delivery moiety evolved from natural peptides whose active components are 6-9 arginine residues in a bio-available 6-aminohexanoicspaced structure [28] . However, these arginine-based peptides are not commercially available for research purposes and their greatest efficacies have been in delivering Morpholinos to the cytosol of tissues like liver [29] or leaky muscle [30] , which would be considered as easily deliverable. As a result, the reach of peptide based Morpholinos into a wide spectrum of tissues remains questionable [18] . Also, owing to the peptidic nature, degradation of the peptide portion of the conjugates was found to be time and tissue dependent [31] . Furthermore, the applications of the arginine-rich peptide transporters are limited due to their high cost, scalability and stability. Added to that are the risks of immune responses against the peptides which limits repeated administrations for diseases requiring long-term treatment [32] . Increases were observed in the ROS levels in the brain samples of JEVinfected and JEV+SC-MO groups in comparison to Sham, that were reduced following 39 and 59 MO treatments. Although ROS levels were decreased in JEV+39 MO group when compared to JEV-infected group, it remained significantly higher than that in Sham (A). Approximately 13-fold increases were observed in the levels of HSP-70 in JEV-infected and JEV+SC-MO groups as compared to Sham. These drastic increases were significantly reduced in JEV+39 MO and JEV+59 MO groups, the levels remained significantly higher than Sham (B&C). SOD-1 levels were found to be elevated 2and nearly 3-fold in JEV-infected and JEV+SC-MO groups respectively compared to Sham. 39 and 59 MO treatment caused significant reduction of SOD-1 levels compared to JEV-infected group (B&D). Alterations in TRX-1 levels were similar to that observed in SOD-1 except that its level in JEV+ SC-MO was not significantly different than only JEV-infected group (B&E). Nearly 2-fold increases were observed in NO levels of JEV-infected and JEV+SC-MO groups when compared to those obtained from Sham. NO levels were subsequently reduced to significantly lower levels following 39 and 59MO treatments (F). iNOS expression was found to increase 8-fold in JEV-infected and JEV+SC-MO groups when compared to Sham. Following 39 and 59MO treatments iNOS levels decreased significantly as compared to JEV-infected group (G&H). ( * p,0.01 for JEV when compared to Sham; ** p,0.01 for JEV+SC-MO when compared to JEV-infected only; # p,0.01 for JEV+ 39MO To minimize the problems encountered by the peptideconjugated Morpholinos, octaguanidinium dendrimer-conjugated Morpholino oligomers have been developed that are commonly referred to as Vivo-Morpholino (MO). These custom-sequence anti-sense molecules have been reported to enable Morpholino applications in adult animals. MO was our choice of anti-sense molecule as this enabled us to test the specifically designed oligonucleotides in both animal as well as cell culture models. Though 'outstanding' results have been reported to be achieved by intravenous (i.v.) administration of the MO, we preferred the intraperitoneal route via which modest systemic delivery can be achieved. This was so done because brain has been reported to be an ineffective tissue when MOs are administered i.v. [33] , though there is no direct evidence showing that MOs can cross blood brain barrier, when administered via other routes. According to the manufacturer's (Gene Tools LLC) instructions the maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. Our aim was to determine the minimum dose at which our desired effects could be achieved. Initially we had chosen two doses of 5 mg and 10 mg per kg body weight (b.w.) of the animals. We found that there was no significant difference between the survival rate of JEV-infected and MO treated mice in either dose (data not shown for 10 mg/kg b.w.). The survival rate was approximately 90% in those JEV-infected animals that were treated with 39 MO and 75% in animals treated with 59 MO. Thus we decided to proceed with the 5 mg/kg b.w. dose for all subsequent experiments.
Plaque assay from the brain homogenates of animals of all groups revealed that the number of infective viral particle production was dramatically reduced following 39 and 59 MO treatment. The 39 MO was generated against the 39 CSI region of the JEV genome that interacts with 59 CS region located in coding sequence for capsid protein at 136-146 nucleotides from 59 terminal of the genome. This interaction results in cyclization of JEV genome that is necessary for its efficient replication. The 59 MO was targeted towards one of the secondary structures of the 59 UTR that are required for the formation of translation preinitiation complex. Blocking of these two sites in the JEV genome leads to the most likely effect, i.e. inhibition of replication and translation of viral genome. This was further corroborated by the decrease in the expressions of viral proteins (NS5, E glycoprotein Vivo-Morpholino in Japanese Encephalitis www.plosntds.org and general flaviviral envelop protein) in the brain. Flaviviral NS5 is known to possess guanylyltransferase activity that helps in the synthesis of methylated cap structure at the 59 end of the viral genome that plays a crucial role in the translation and stability of mRNAs [34] . The JEV E glycoprotein is believed to be involved in viral adhesion and entry into host cells, hemagglutination, cellular tropism, viral virulence, and the induction of protective immune responses [35] . Decreased expression of these proteins indicates that viral replication and production of new infective viral particles are inhibited due to the MOs. Immunohistochemical staining for viral antigen also provided visual confirmation of the fact that JEV antigen was detected at much lower amounts in the brain following MO treatment. However, these data does not prove that MOs directly inhibit infective viral particle production in the brain itself, as it cannot be conclusively stated whether the MOs can reach brain. These data merely suggests that the number of replication-competent infective JEV in the brain was significantly reduced, which subsequently leads to neuroprotection.
It is well known that JEV infection causes microglial activation. Activated microglia releases an array of chemical mediators that are detrimental for the neurons in brain [24] . Since there was reduction in the production of infective viral particles following 39 and 59 MO treatments, we studied the effect on microglial pathophysiology in mouse brain. Our results show that there was significantly reduced number of activated microglia in the brain sections of both 39 and 59 MO-treated animals as compared to only JEV-infected or JEV-infected and SC-MO treated animals. Since there were little or no activation of microglia, proinflammatory cytokine levels in the brain were found to be significantly downregulated. Histochemical staining also revealed that neuronal population and morphology remained largely unaffected in 39 and 59 MO-treated animals' brains as compared to only JEV-infected or JEV-infected and SC-MO treated animals.
Generation of ROS with the generation of oxidative damage has been implicated in neurodegenerative diseases and in the degradation of nervous system functions and are also reported to increase following JEV infection [22] . Increase in ROS levels initiates various responses within the cell, including damage to proteins, DNA and lipid [36] . In this study, ROS levels were found to be many-fold increased in JEV-infected or JEV-infected and SC-MO treated animals that were then found to be counteracted by the treatment of 39 and 59 MO. The levels of stress related proteins such as SOD-1, HSP-70 and TRX where also found to be positively modulated following 39 and 59 MO treatment. NO is a known antagonist of JEV. It has been shown that NO inhibits JEV infection by preventing viral replication [37] . In our study NO levels were increased in the brain in response to JEV infection possibly due to the upregulation of inducible nitric oxide synthase (iNOS). Treatments with 39 and 59 MO caused a decrease of NO to basal levels as observed in Sham-treated animals.
Activation of pNFkB regulates apoptotic genes, especially the TRAF1 and TRAF2, and thereby checks the activities of the caspases, which are central to most apoptotic processes. JEV is known to activate pNFkb via a PI3K-dependent pathway in the brain of infected animals, which is associated with apoptosis [38] . JEV infection has also been shown to activate stress kinases, which in turn results in activation of ERK1/2, and p38 MAPK pathway leading to apoptotic death of neurons [39] . In accordance with the established results, here also we found that there was similar activation pattern of these molecules in JEV-infected and JEVinfected and SC-MO treated animal brain samples. Treatment with the MOs resulted in abrogation of those changes that led to greater survivality of brain neurons as observed by histochemical staining. Activation of p38MAPK is also related to the transcriptional activation of proinflammatory genes in the brain [40] . Thus the decrease in phophoP38 MAPK levels correlates with the decreased levels of proinflammatory cytokine levels in obtained from the brain.
To confirm the anti-viral and neuroprotective property of the MOs observed in in vivo models, cultured neuroblastoma cells were infected with JEV, followed by MO treatment. Though the MOs are specifically developed for in vivo studies, they are also known to be taken up by cells in culture conditions [18] . There was a significant decrease in viral titer in samples obtained from the cells that were treated with 39 and 59 MOs as compared to either JEVinfected or JEV-infected and SC-MO treated cells, as revealed by plaque assay. This data was supported by FACS analysis following intracellular staining for JEV antigen.
This study was undertaken to determine the antiviral and neuroprotective efficacy of Vivo-Morpholinos in an experimental model of JE so that it can be considered as a therapeutic agent in the near future. There have been studies regarding the anti-JEV effects of other types of Morpholino oligomers though none of them are yet to be considered for therapeutic purposes. This is the first study that investigates the role of Morpholino oligomers specially designed for effective delivery into live animal models. Generally, the i.p route of administration of any drug is preferred in animal studies over any other routes. However, the efficacy of these antisense molecules needs to be checked by administering through other applicable routes, as i.p. administration in humans is uncommon, though not unheard of. The amounts of oligomers required and the route of administration in this study marks these molecules as practicable therapeutic agents in JE, though further studies are required before these can be recommended for clinical trials. Figure S1 MO treatments decrease viral load in vitro. Mouse neuroblastoma cell (N2a) lysates from all the treatment groups were subjected to plaque assay in order to determine viral loads. PFU/mL was found to be significantly higher in both JEV and JEV+SC-MO groups when compared to Sham. Viral loads were found to be significantly reduced in both JEV+39 MO and JEV+59 MO groups when compared to only JEV-infected group (* p , 0.01 for JEV and JEV+SC-MO when compared to Sham; # p , 0.01 for JEV+39 MO and JEV+59MO when compared to only JEV-infected group) (A). Intracellular staining for JEV antigen in N2a was performed and number of JEV-positive N2a cells was then sorted by flow cytometry. 30% of the total gated cells were found to be positive for JEV antigen in JEV-infected group as compared to 34% in JEV+SC-MO group. Only 16% and 9% of the total gated cells were found to be positive for JEV antigen in JEV+39 MO and JEV+59 MO groups respectively (B). Found at: doi:10.1371/journal.pntd.0000892.s001 (0.25 MB TIF) Severe novel influenza A (H1N1) infection in cancer patients Background: The natural history and consequences of severe H1N1 influenza infection among cancer patients are not yet fully characterized. We describe eight cases of H1N1 infection in cancer patients admitted to the intensive care unit of a referral cancer center. Patients and methods: Clinical data from all patients admitted with acute respiratory failure due to novel viral H1N1 infection were reviewed. Lung tissue was submitted for viral and bacteriological analyses by real-time RT-PCR, and autopsy was conducted on all patients who died. Results: Eight patients were admitted, with ages ranging from 55 to 65 years old. There were five patients with solid organ tumors (62.5%) and three with hematological malignancies (37.5%). Five patients required mechanical ventilation and all died. Four patients had bacterial bronchopneumonia. All deaths occurred due to multiple organ failure. A milder form of lung disease was present in the three cases who survived. Lung tissue analysis was performed in all patients and showed diffuse alveolar damage in most patients. Other lung findings were necrotizing bronchiolitis or extensive hemorrhage. Conclusions: H1N1 viral infection in patients with cancer can cause severe illness, resulting in acute respiratory distress syndrome and death. More data are needed to identify predictors of unfavorable evolution in these patients. Patients with malignancies are more susceptible for acquisition of infections than the general population and are thought to potentially develop more complications [1] . Due to many disruptions in both innate and acquired immunity, even organisms with low virulence potential are able to cause significant morbidity and mortality in cancer patients [2] .
During April 2009, a novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in California and in Mexico as the cause of human respiratory disease, originating a pandemic [3, 4] . As of February 2010, 700 000 laboratory-confirmed cases of novel H1N1 influenza virus and 14 000 deaths have been reported globally. Most affected patients present with influenza-like symptoms and have a benign course [5] . However, patients with comorbidities as cancer and chronic diseases may show a serious clinical presentation, characterized by respiratory failure with variable severity [6, 7] .
Although the presence of a malignancy is considered to have a negative impact on the H1N1 disease severity, there are few reports on clinical outcomes in cancer patients affected by the disease. Redelman-Sidi et al. [8] recently described a cohort of 45 patients with solid and hematological malignancies with H1N1 infection. From this population, one single patient required intensive care admission and there were no viral infection-related deaths. On the other hand, fatalities have been described in oncologic patients, particularly in the ones with hematological malignancies [9] .
So far, there are no reports describing clinical characteristics of cancer patients with a severe form of the disease. These patients represent a vulnerable population, which require rapid diagnostic work-up and intensive management in specialized units. Therefore, we believe that it would be important to report on the clinical characteristics of cancer patients with a severe form of the H1N1 infection.
During the 2009 Southern Hemisphere H1N1 pandemics, Sao Paulo was among the cities with the highest incidence of disease in Brazil. In this study, we describe the clinical and pathological findings in critically ill patients with cancer and respiratory failure related to novel H1N1 infection admitted to original article an oncologic intensive care unit (ICU) in a reference cancer center in Sao Paulo, Brazil.
This analysis and report was approved by the Institutional Medical Ethical Committee. Clinical and laboratorial data at admission and during ICU stay are described, including the management of respiratory support and nonventilatory strategies. Data for the Simplified Acute Physiology Score II (SAPS II) and for the Acute Physiology And Chronic Health Evaluation (APACHE II) were reported as the worst value within 24 h after ICU admission. A daily evaluation of organ function according to the Sequential Organ Failure Assessment (SOFA) score was performed Radiological findings were evaluated through X-ray in all patients and computerized tomography when appropriate.
The in vivo diagnosis of H1N1 infection was confirmed by real-time RT-PCR (rRT-PCR) test using nasopharyngeal swab specimens, in accordance with guidelines from the Centers for Disease Control and Prevention (CDC) [10] .
autopsies Five patients who died had their autopsies performed in the Department of Pathology of the Universidade de Sao Paulo. The pathological findings on part of this population have been previously described by our group [11] .
Tissue fragments were formalin fixed, paraffin embedded and hematoxylin-eosin stained. For lung sections, Grocott, Brown-Hopps and Ziehl-Neelsen stainings were performed for the identification of fungi, bacteria and acid-fast bacilli, respectively. Lung fragments were sent for microbiological investigation using rRT-PCR to the Instituto Adolfo Lutz in Sao Paulo. Seasonal influenza A and swine influenza A detection was performed using the CDC protocol [10] . The RT-PCR test used for bacteria identified DNA from Haemophilus influenzae and Streptococcus pneumoniae.
results baseline characteristics Table 1 shows the baseline characteristics of the eight patients. Patient ages ranged from 55 to 65 years (median, 58 years). Four patients (50%) were male. All patients (100%) had preexisting medical conditions other than the neoplasm. Most of the patients presented no functional impairment before the infection-seven patients (87.5%) had Karnofsky scale >70.
Five patients (62.5%) had solid neoplasms and three patients (37.5%) had hematological malignancies. Two patients had been submitted to chemotherapy in the last 4 weeks (Table 1) . However, most patients were considered as having active disease regarding cancer status (seven patients, 87.5%). Four patients (50%) had metastatic disease. The patient with myelofibrosis had been submitted to stem-cell transplantation 1 year ago. All patients presented cough and fever, most patients had myalgia and dyspnea (87.5%). Hemoptysis, rhinorrhea and wheezing were present in 25% and diarrhea was related by 12.5% of patients.
At admission, all patients presented with signs of systemic inflammatory response syndrome, defined as two or more of the signs and symptoms described in Table 2 . All patients had fever or hypothermia and tachypnea. Leukocytosis was present in 75% of cases. At admission, only two patients were hypotensive (25%). However, hypoxemia was present in 100% of cases, and four patients (50%) had oxygen saturation <90% at admission (Table 2) . Initially, lung disease was in most cases localized in one or two quadrants of lung (75%). However, most patients developed a more extensive and progressive In all the eight patients, the diagnosis of acute respiratory distress syndrome (ARDS) could be established based on the presence of bilateral pulmonary infiltrates, a ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PO 2 /FiO 2 ) £200 and no clinical evidence for an elevated left atrial pressure. Due to rapidly progressive hypoxemia (PO 2 /FiO 2 117) and the rapid worsening of lung infiltrates (Figure 1 ), five patients (62.5%) needed invasive mechanical ventilation. Four patients were intubated in the first 24 h of ICU and one patient 48 h after unsuccessful noninvasive ventilation (NIV). NIV was successfully used in three patients (37.5%), who had milder forms of disease as showed by a computerized tomography that revealed sparse bilateral infiltrate ( Figure 2 ).
Patients who needed mechanical ventilation were managed with pressure-cycled ventilation, with a low tidal volume (target 6 ml/kg) open-lung strategy of ventilation, and a positive endexpiratory pressure (PEEP) titrated based on FiO 2 for goal plateau pressure (Pplat) < 30 cm H 2 O and SpO 2 88%-90% according to ARDS Network protocol. In some cases, due to refractory hypoxemia, PEEP levels of 14 to 16 cm H 2 O were applied. In all patients, recruitment maneuvers were usedcontinuous positive airway pressure 35-40 cm H 2 O for 30 swith short-term improvements in oxygenation in three of five patients (initial mean oxygen saturation rate increased from 88% to 94%). Neuromuscular blockade was used in two patients ( Table 2) . Despite the aforementioned ventilatory strategy, this particular group of patients developed refractory persistent hypoxemia. Lung mechanics of patients showed very low static and dynamic compliance and high airway resistance.
Most patients presented risk scores that predicted high-risk mortality (APACHE II 24 and SAPS II 52). Respiratory failure was the most commonly encountered dysfunction, followed by cardiovascular, renal and hematological failures. Two patients needed dialysis. Most patients needed vasopressor and inotropic drugs. Patients presented with a serious, rapidly Oseltamivir was initiated early in all patients. Corticosteroids were administered in all patients admitted in the ICU, at a dose of methylprednisolone 2 mg/kg/day. Antibiotics were empirically administered in all patients due to clinical suspicion of bacterial coinfection. Despite early treatment, these patients had a high mortality rate, with five deaths (62.5%). The median period from admission to death was 3 days (1-8). Interestingly, three patients who died showed persistent positive rRT-PCR in nasopharyngeal swabs after 5 days of oseltamivir. Hematological evaluation showed leukocytosis (median 16 100/mm 3 ) and anemia (median hemoglobin 9.3 g/dl) in majority of cases. Also, signs of tissue hypoxia as acidosis (median pH 7.2), high levels of lactate (median 4.2 mmol/l) and low levels of base excess (median 4.5 mEq/l) were noted in patients since admission until death. Reactive protein-C ranged from 99 to 447, revealing inflammatory response associated or not with coinfection (Table 3) .
During evolution, bacterial coinfection was diagnosed in seven patients: Staphylococcus aureus in blood stream in one patient, urinary tract infection due to Enterococcus faecalis in one patient and pneumonia due to Pseudomonas aeruginosa in one case and due to S. pneumoniae in four other cases. Also, one patient presented urinary tract infection due to Candida. An autopsy was performed in the five patients who died, and a summary of the findings is presented in Table 4 .
All patients presented extensive pathological alterations in the lungs; lungs were heavy, diffusely edematous and with variable degrees of hemorrhage. Diffuse alveolar damage (DAD) was present in most of the patients, but as previously described, there were three distinct patterns of pulmonary pathological changes [11, 12] : (i) four patients had classic exudative DAD, with alveolar and interstitial edema, hyaline membranes and reactive pneumocytes; (ii) one patient (esophagus neoplasm) had DAD and severe necrotizing bronchiolitis (NB) characterized by extensive necrosis of the bronchiolar wall and dense neutrophilic infiltrate within the bronchiolar lumen and (iii) one patient (myelofibrosis) presented with exudative DAD with an intense hemorrhagic component (Figure 3 ). Only one of the five patients did not present acute interstitial changes. In this patient with esophageal cancer, death was secondary to pulmonary thromboembolism and bacterial pneumonia. Two patients had pulmonary thromboembolism (two patients with esophagus neoplasm). In four of five patients, bronchopneumonia coinfection due to S. pneumoniae was confirmed at autopsy ( Figure 3) .
Metastatic disease was present in all patients with solid neoplasms, and two of those had lung involvement by tumor. All patients had atrophic or nonreactive white pulp in the spleen (Figure 3 ). In the lymph nodes, nonreactive follicles and sinusoidal erythrophagocytosis were found. The liver showed erythrophagocytosis and a few mononuclear inflammatory cells in the sinusoids in all patients and variable degrees of shockrelated centrilobular necrosis. The bone marrow was hypocellular in four of five patients. All patients had mild/ moderate kidney acute tubular necrosis. No patient had histological signs of encephalitis, myocarditis or myositis.
We report the clinical and pathological findings from eight patients with cancer and severe H1N1 infection who were admitted to an oncologic ICU during the winter period of the 2009 pandemic in Sao Paulo, Brazil. These patients were characterized by having active malignant disease, comorbidities and difficult to manage rapidly progressive acute respiratory failure.
Viral infections may represent up to 26% of the infections identified in cancer patients with pulmonary infiltrates. In a large study examining viral infections in hematological cancer patients, pneumonia was observed in 31% of the cases, and the overall mortality was 15% [12] . In this study, influenza A was the most common isolated agent, and the only independent predictor of fatal outcome was an absolute lymphocyte count £200 cells/ml.
Clinical outcomes in cancer patients with H1N1 infection have not been fully characterized yet. Due to potential severity of H1N1 infection in this group of patients, Crawford et al. [13] recommended during the 2009 pandemic that patients with cancer who are receiving chemotherapy and develop fever should be admitted to a hospital and receive oseltamivir after swab collection. In addition, patients with febrile neutropenia should be treated according to the usual protocol with the addition of oseltamivir.
Kharfan-Dabaja et al. [14] first reported H1N1 infection in two allogeneic hematopoietic cell transplantation recipients. In one case, the patient presented with fever, myalgia, sore throat and diarrhea without evidence of hypoxemia or lung progressive infiltrates. After treatment with oseltamivir, ciprofloxacin and doxycicline, the symptoms resolved without sequelae. In the second case, pulmonary symptomatology continued to deteriorate despite aggressive polymicrobial treatment, requiring mechanical ventilation and ultimately the patient died from respiratory failure [14] . Redelman-Sidi et al. [8] recently described 45 patients with cancer and/or hematological conditions and H1N1 infection at the Memorial Sloan-Kettering Cancer Center, with no reports of mortality or serious morbidities. In this report, only one patient was admitted in the ICU and did not need mechanical ventilation. In these patients with cancer, mortality was very high. The differences in outcomes may be related to specific characteristics of our population, including high prevalence of metastatic disease, comorbidities and high incidence of bacterial coinfection. Differently from the MSKCC experience [8] , most patients in our series initially presented with signs and symptoms of lung disease-hypoxemia, dyspnea and tachypnea-instead of only influenza symptoms.
The serious clinical presentation of the novel Influenza A (H1N1) infection in some cancer patients should be expected. Patients with cancer now live longer and immunosupression from malignant disease or its treatment renders many susceptible to infections [15] . Indeed, most of the patients in this series developed bacterial coinfections. The altered immunological response of these patients may contribute to the original article Annals of Oncology development of more severe forms of disease. In our study, three cases still excreted virus after 5 days in the ICU, as already reported [16] . Prolonged periods of viral shedding could be associated to disease severity [17] and perhaps with an oseltamivir-resistant strain of the virus [18] . Respiratory failure occurred in all patients who required ICU care and was characterized by rapidly progressive bilateral lung infiltrates with refractory hypoxemia and low left atrial pressure-ARDS. Most patients needed mechanical ventilation (five of eight), and they were all treated with protective strategies according to the ARDS Network protocol [19] . In all these patients, recruitment maneuvers were applied, with just transient improvement in oxygenation (initial median oxygen saturation rate increased from 88% to 94%), reflecting the extensive lung involvement of disease. In some cases of severe H1N1 infection, extracorporeal membrane oxygenation [20] has been proposed as an alternative support therapy with promising results [21, 22] .
Based on previous cases of H1N1 infection in noncancer patients, Ramsey et al. [6] described a significant proportion of patients with hypoxemic respiratory failure, specially when associated with comorbidities. Early intubation and admission in the ICU is prudent, given the rapid progression of hypoxemia. Based on current evidence, authors recommend patients should be managed with a low tidal volume open-lung strategy of ventilation, with PEEP titrated based on FiO 2 to achieve adequate SpO 2 and low Pplat [21, 22] .
This strategy was performed in all studied patients of our series; however, despite that there was no adequate response and all patients who required mechanically ventilated died with refractory hypoxemia and multiple organ failure. Similar to our findings, in a Mexican series of S-OIV cases, of 12 patients who needed mechanical ventilation, 7 died [23] .
Three patients presented with a milder form of lung disease, responding well to NIV, and with progressive improvement of hypoxemia. There is some controversy around the clinical Annals of Oncology original article efficacy of NIV in novel influenza A (H1N1) infection. Whereas NIV may be considered as a mode of ventilation for hypoxemic respiratory failure, there are concerns about its usefulness in an infectious epidemic [24] . In the Canadian experience, 30% of patients were noninvasively ventilated on admission, but 85% of these patients required subsequent intubation and invasive ventilation [6] . In addition, NIV machines have no bacterial or viral filters and do generate aerosols. There are often leaks from the mask, and it has to be removed sometimes for nursing care [24] . During the pandemic, NIV was associated with transmission of disease to health care workers [25] .
In our study, we detected bacterial coinfection in seven of the eight patients. The CDC reported that 29% of fatal cases in the United States presented at least one bacterial coinfection [26] . Mauad et al. [11] found evidence of bacterial coinfection in 38% of fatal cases in Sao Paulo.
The presented incidence of 87% of bacterial coinfection might explain in part the high mortality rate of this group, despite early antimicrobial therapy. Interestingly, biochemical and hematological data could not discriminate H1N1 infection from sepsis of bacterial origin in this group of patients.
Corticosteroids were administered to all patients as Meduri et al. [27] recommend in ARDS cases with low PO 2 /FiO 2 . Steroid use has been reported in some cases of H1N1associated ARDS without any adverse outcome [28] . However, some authors discuss the potential adverse effects of steroids in H1N1 infection, including higher mortality possibly related to virus spreading [29] . Although there is no consensus about its efficacy in this disorder, and despite the belief that corticosteroid could reduce pulmonary inflammation and fibrosis in severe cases, our poor results suggest that this strategy will need to be carefully reevaluated in the future.
By performing autopsies in the fatal cases in this population we could determine that the cause of death in all patients was extensive involvement of the lungs and alterations secondary to multiple organ failure in major organs such as kidney and liver [11] . Patients had severe DAD associated with severe NB and alveolar hemorrhage. Further, autopsy results showed that patients had metastatic disease and signs of cellular immunosuppression as depletion of white pump view on spleen analysis. Certainly, autopsies contributed to a better characterization of these patients. This report has some limitations. The unicentric characteristics of the study and the small sample size do not allow for definite conclusions about severe H1N1 presentation in oncologic patients. We characterized H1N1 infection in a selected population of patients with neoplasm, with a high incidence of metastatic disease and who needed ICU care. Our findings certainly describe the most serious presentation of disease.
The importance of describing this serious form of disease in patients with cancer is to reinforce prevention strategies in this group, as to recommend vaccine, hygienic measures, prophylactic antiviral treatment in cases of contact and adequate isolation in cases of hospital admission due to H1N1 infection [30, 31] . Also, a better understanding of clinical and pathological findings in the group of cancer patients could guide ventilator management and nonventilatory strategies to obtain lower rates of mortality.
In summary, our report of cancer patients highlights the severity of the Influenza A (H1N1) pandemic in this vulnerable population and the urgent need to establish specific protocols of care and management strategies designed to face this health care challenge. funding Conselho Nacional de Desenvolvimento Científico e Tecnoló gico (CNPq). The Tennessee Children's Respiratory Initiative: Objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases Background and objective: The ‘attack rate’ of asthma following viral lower respiratory tract infections (LRTI) is about 3–4 fold higher than that of the general population; however, the majority of children who develop viral LRTI during infancy do not develop asthma, and asthma incidence has been observed to continuously decrease with age. Thus, we do not understand how viral LRTI either predispose or serve as a marker of children to develop asthma. The Tennessee Children's Respiratory Initiative has been established as a longitudinal prospective investigation of infants and their biological mothers. The primary goals are to investigate both the acute and the long‐term health consequences of varying severity and aetiology of clinically significant viral respiratory tract infections on early childhood outcomes. Methods: Over four respiratory viral seasons, 2004–2008, term, non‐low birth weight previously healthy infants and their biological mothers were enrolled during an infant's acute viral respiratory illness. Longitudinal follow up to age 6 years is ongoing. Results: This report describes the study objectives, design and recruitment results of the over 650 families enrolled in this longitudinal investigation. The Tennessee Children's Respiratory Initiative is additionally unique because it is designed in parallel with a large retrospective birth cohort of over 95 000 mother–infant dyads with similar objectives to investigate the role of respiratory viral infection severity and aetiology in the development of asthma. Conclusions: Future reports from this cohort will help to clarify the complex relationship between infant respiratory viral infection severity, aetiology, atopic predisposition and the subsequent development of early childhood asthma and atopic diseases. The Tennessee Children's Respiratory Initiative (TCRI) has been established as a longitudinal prospective investigation of term, non-low birth weight otherwise healthy infants and their biological mothers. The primary goals of the study are: (i) to investigate both the acute and the long-term health consequences of varying severity and aetiology of clinically significant viral respiratory tract infections on the outcomes of allergic rhinitis (AR) and early childhood asthma; and (ii) to identify the potentially modifiable factors that define children who are at greatest risk of developing asthma following infant respiratory viral infection. This study is unique, in that it was designed in parallel with our Tennessee Asthma Bronchiolitis Study (TABS), which is a retrospective birth cohort study of over 95 000 infants and their biological mothers similarly designed to elucidate the factors predisposing to childhood asthma and allergic 
This report describes the study objectives, design and recruitment results of the Tennessee Children's Respiratory Initiative, designed to investigate the role of respiratory viral infection severity and etiology in asthma development. Future reports will address the complex relationship between infant respiratory viral infection and asthma and atopic diseases.
diseases, but lacking biospecimens. Thus, we designed the prospective TCRI to establish a base for the evaluation of both the risks and benefits of documented significant infant viral respiratory infection of varying severity and aetiology and other environmental exposures on childhood atopy outcomes and to establish a biospecimen repository for analyses including biomarker testing and genotyping. The prospective cohort has the longitudinal design properties that may overcome potential limitations intrinsic to retrospective studies, such as our TABS cohort. [1] [2] [3] [4] [5] It is our eventual goal that the findings from these investigations, in conjunction with other investigations that have helped to elucidate genetic and environmental factors associated with asthma development, will help in the identification of primary and secondary prevention strategies for asthma. This report describes the study objectives, design and recruitment results of this study cohort.
The TCRI is a prospective cohort of mother-infant dyads enrolled in a longitudinal investigation of the relationship of infant viral respiratory infection severity and aetiology and the interaction of other risk factors on the development of childhood asthma and allergic diseases. The study was approved by the Vanderbilt Institutional Review Board, and parents provided written informed consent for both their and their child's study participation.
Term, non-low birth weight, otherwise healthy infants were enrolled along with their biological mothers, at a single academic institution, Vanderbilt Children's Hospital, at the time of an acute visit (hospitalization, emergency department or unscheduled outpatient visit) for presumed viral bronchiolitis or upper respiratory tract infection (URI) during respiratory viral seasons September through May 2004-2008. Inclusion and exclusion criteria are outlined in Table 1 . Because of the grant funding start date, the first study season did not begin until November 2004. Recruitment was solely hospital-and clinic-based, and was performed 7 days/week during the first 2 years of cohort accrual, and 5 days per week for the two subsequent years. Screening and recruitment were done by experienced Congenital or acquired chronic heart or lung disease, prior requirement for mechanical ventilation for cardiac or pulmonary disease, immunodeficiency, neurologic disease with possible aspiration, significant gastroesophageal reflux disease felt to contribute to pulmonary disease, tracheomalacia Ever received one or more doses of RSV-IVIG or palivizumab Prior study inclusion Fever and neutropenia Children whose parents or guardians were not able to understand the consent process, or a language barrier † research nurses using computerized medical charts to screen infants with presumed respiratory viral illness.
The components and time line of the subject visits are outlined in Table 2 Mothers underwent prick skin testing to eight common aeroallergens and a blood specimen was obtained by venepuncture for serum immunoglobulin E (IgE) and DNA. A structured abstraction form was used to obtain information from the medical record regarding the index enrolment visit: current infant weight, confirmation of birth weight, room air pulse oximetry, requirement for supplemental oxygen, medication administration, prior wheezing episodes and detailed information on the current illness and hospital course. Following discharge from the hospital or outpatient setting, the final discharge diagnosis and results of culture data were obtained through chart review.
There are three phases of cohort follow up. First, mothers and children undergo an in-person wellchild follow-up visit during the child's second year of life conducted in the Vanderbilt Clinical Research Center, or during a home visit. Second, families are re-contacted every 12-18 months by phone and/or mailings for purposes of cohort retention, and to provide reminders about the remaining study components. Third, mothers, or the current guardians, undergo a phone interview during the fourth and sixth years of life to identify children with asthma, transient wheezing, AR and eczema.
The 2-year in-person well-child visit is conducted in the Vanderbilt Clinical Research Center, or during a home visit offered to those unable to return to the study institution. During the visit, the ISAAC questionnaire is administered, blood samples are obtained from children and mothers (if not previously collected) and a buccal swab is collected for DNA if blood cannot be obtained.
A structured telephone questionnaire is administered to the mother/parent when their child is 4 and again at 6 years. Trained interviewers employed in the Vanderbilt Survey Research Shared Resource use a web-based computer system to conduct structured telephone interviews, which capture detailed information on asthma and atopy diagnoses and symptoms, extensive environmental exposure history, physical activity, and comorbidities. Asthma, AR and atopic dermatitis outcomes are determined using the ISAAC questionnaire. For children with report of asthma and/or asthma symptoms in the previous 12 months, the Asthma Therapy Assessment Questionnaire is administered, and information on asthma medications, and asthma-related health-care visits are sought. 12, 13 Biospecimen collection and laboratory analyses Table 3 outlines the details of cohort biospecimen collection, repository and testing, which includes infant nasopharyngeal, urine, blood and nasal epithelial cell sample collection, and maternal prick skin testing and blood samples.
The discharge diagnosis and supporting clinical parameters of the infant acute respiratory illness visit were reviewed to confirm whether each child had lower respiratory tract infections (LRTI) or URI (n = 628) or another diagnosis (n = 46). LRTI and URI were defined using both the physician discharge diagnosis, as well as post-discharge chart review, and those cases that were not clearly identified as either LRTI or URI were reviewed by a panel of paediatricians who determined whether the illness represented an LRTI, URI, croup or other, which included those that could not be categorized with the available clinical information. Acute respiratory illness severity was determined using the ordinal bronchiolitis score that incorporates admission information on respiratory rate, flaring or retractions, room air oxygen saturation and wheezing, into a score ranging from 0 to 12 (12 being most severe). 27, 28 highly sensitive and specific qRT-PCR assays for many common respiratory viruses, including hMPV, HCoV, RSV, influenza A and B, parainfluenzaviruses 1-3 and rhinovirus. Real-time RT-PCR is performed using the Cepheid Smart Cycler II. All specimens are first tested for GAPDH to confirm integrity of RNA and monitor for potential PCR inhibitors. Negative and positive controls are included with each run, including RNA runoff transcripts to generate a quantitation curve. [14] [15] [16] [17] [18] [19] [20] [21] If rapid antigen and/or culture for RSV or Influenza were performed at the discretion of the admitting physicians during the index visit, these results are also captured and entered in the database. The cells are collected using Mid Turbinate Peds Nylon Flocked Swabs (MicroRheologics, Brescia, Italy), and placed into collection and digestion media, followed by processing and plating onto collagen IV coated tissue culture and grown at 37°C in a 5% CO2 incubator. To develop techniques for isolation, short-term culture, and in vivo modelling of epithelial and stromal cells, a xenograph model has been developed. Following short-term growth in culture, cells are transferred to denuded rat tracheas and implanted subcutaneously in nude mice.
Follow-up well-child visit Mother and child DNA is collected from both the mother and child during the blood collection, or using a buccal swab if blood can not be obtained. DNA is extracted by the Vanderbilt Center for Human Genetics Research Core Laboratory and stored following extraction for future studies.
Enrolment Infant Urine is collected from hospitalized infants during the acute infant illness at study enrolment, and from a convenience sample of outpatient subjects. Urinary measurements including, leukotrienes C4/D4/E4 (LTC4/D4/E4), and urinary metabolite of the isoprostane, 15-F2t-IsoP (8-iso-PGF2a) will be measured by a gas chromatographic, and other biomarkers and the remainder of the urinary biospecimens will be maintained in the repository. 25, 26 IgE, immunoglobulin E; LRTI, lower respiratory tract infections; URI, upper respiratory tract infection.
The family history of atopy was obtained using a family tree. Maternal atopy will be categorized as evidence of atopy by skin testing or specific IgE, and/or clinical symptoms of an atopic disease as assessed by the ISAAC questionnaire. Atopic status of the child will be determined by laboratory evidence of specific IgE during the second year of life, and by clinical evidence based on the above definitions.
The diagnosis of asthma will be determined at age 6 years based on responses to the ISAAC questionnaire. [6] [7] [8] The following criteria will define asthma during the sixth year of life: (i) 12-month prevalence of symptoms of asthma (current wheeze) or the presence of exercise-induced wheeze or dry cough at night not due to a cold or chest infection; and (ii) physician diagnosis as determined by the ISAAC questionnaire using either parental reported physician diagnosis of asthma or chronic use/ prescription of asthma-specific medications. Probable asthma will be defined as physician diagnosis only and analysed separately. Transient early wheezing will be defined as wheezing episodes present in the first 4 years of life, but not meeting the definition for childhood asthma at age 4 and 6 years. 29 Allergic rhinitis will be determined through the ISAAC core questions on AR. 7, 8 Children will be considered to have definite AR if each of three conditions is present between age 5 and 6 years: (i) a history of nasal congestion, runny nose, itchy watery eyes, sinus pain or pressure or headaches, sneezing, blocked nose, loss of sense of smell; (ii) substantial variability in symptoms over time or seasonality; and (iii) diagnosed as having allergic rhinitis by a physician or on medications for AR. Probable allergic rhinitis will be defined as meeting two of the three criteria, or only criteria 3.
Atopic dermatitis will be determined through the ISAAC core questions on atopic dermatitis, which are based on a list of major and minor criteria widely applied in clinical studies. 8, 30, 31 As eczema is probably more readily confirmed by objective tests than either asthma or rhinitis, patients will be considered to have definite atopic dermatitis if between age 5 and 6 years they report ever having an itchy rash that comes and goes for at least 6 months, and being diagnosed with eczema by a physician. 30, 31 Probable atopic dermatitis will be defined as one of the two above criteria.
In order to standardize and monitor the quality of data collection and processing, all study personnel received training and were certified for all the study procedures. Information is recorded on paper case report forms, data are entered and then checked by a second reviewer. Logical data checks are programmed and additionally performed by our systems analyst, investigators and again by our biostatisticians. For laboratory analyses, blind qualitycontrol samples are included in each biospecimen run. Telephone interviewers complete classroom training, orientation to the study population, computer modules, role play interviewing and training on study-specific protocols, and are formally evaluated at the end of training. A verbatim-recording of the interviewer and participant responses, and 10% participant re-contact allows quality-control staff to verify responses.
The outcome variables of interest are the incidence of asthma and allergic rhinitis. The primary exposure variables of interest are the severity and aetiology of the infant viral illness and maternal and familial atopic status and other environmental exposures. Cumulative asthma incidence over time, taking into account loss to follow up, will be used for illustrating incidence data. Incidence of asthma and allergic diseases among the enrolled infant population with viral respiratory illness will be calculated by dividing the number of incident asthma cases by the person time of follow up. A Kaplan-Meier plot of cumulative incidence over time, taking into account loss to follow up, will be used for illustrating incidence data. Incidence rates will be calculated by dividing the patient population into quartiles/quintiles of bronchiolitis severity scores. The adjusted risk of asthma and allergic rhinitis with bronchiolitis severity will be evaluated using the Cox proportional hazard regression model.
To assess the relationship between biomarker concentrations and increased risk of infant bronchiolitis, and early childhood asthma outcomes, we will conduct a nested case-control study. Geometric means of urinary biomarker concentrations for those who develop and do not develop asthma will be calculated separately and compared using the paired t-test. The association between these biomarkers and the risk of asthma and allergic rhinitis will be assessed using odds ratios and their corresponding 95% confidence intervals from adjusted logistic regression models. The potential confounders that will be considered in multivariable analyses will include demographic and exposure characteristics.
Subject recruitment for the TCRI Study occurred over 4 years, and was completed in May 2008. Overall, 9329 visits were screened, representing 7632 unique infants, and 2986 of these infants met study eligibility requirements. From the 2986 eligible infants, 674 infants and their biological mothers were enrolled (Fig. 1) . Among the 2312 subjects who were available during the recruitment periods, the major reasons for non-response were refusal (22%), insufficient time/ unwilling to stay for the visit (outpatients) (39%), conflict with or already enrolled in another study (20%), and other (18%) (includes language barrier, mother/ guardian not present, previously enrolled and other miscellaneous). In 99.9% of the cohort, the nasal/ throat swab was obtained, and in 79% of the hospitalized infants one spot urine sample was obtained at hospital admission. Weekly enrolment into the cohort is depicted in Figure 2 .
The TCRI is a large and comprehensive prospective epidemiologic study of mothers and their biologic children enrolled during infancy with a clinically significant LRTI or URI who are being followed through early childhood. This study will provide important information about the role of infant respiratory viral infection severity, aetiology, biomarkers and predictors important in the development of early childhood asthma and allergic rhinitis. The TCRI is additionally unique because it is designed in parallel with a large retrospective birth cohort of over 95 000 mother-infant dyads with similar objectives to investigate the role of respiratory viral infection severity and aetiology in the development of asthma.
As evidence suggests that the development of asthma may result in part from respiratory viral infection during infancy, which has a predilection for infecting, destroying and/or in some way biologically altering lower airway epithelium, this study will help to delineate whether the severity of that infection and other early-life events impact the risk of asthma and allergic disease development in later childhood. 3 Despite a high attack rate of developing asthma following viral bronchiolitis, the majority of children who have infant bronchiolitis do not develop childhood asthma. Thus while viral respiratory infections may alter lung physiology and target the inflammatory response to the lower airway, this may only occur during a vulnerable time period during development of the immune system or lung, or in the presence of other risk factors. This developmental component may further reflect important gene-environment interactions that regulate both short-and long-term airway physiological alterations that manifest themselves clinically as childhood asthma. Efforts to determine and define the role of these factors, including disease severity, maternal atopy and other environmental exposures, such as second-hand smoke, to asthma pathogenesis are the focus and goal of the TCRI.
Several limitations of this study should be noted. First, the study sample was not randomly selected from the general population, but instead was recruited from a single hospital and clinic-based setting, the Vanderbilt Children's Hospital. While Vanderbilt hospitalizations represent greater than 90% of Davidson County/Nashville infant hospitalizations, it represents a smaller proportion of emergency department visits (51%), and likely even fewer paediatric acute care visits. 2 The relatively low participation rate among eligible subjects is multifactorial and the result of the long-term nature of the study, the lack of study personnel to enrol all eligible subjects, as well as lower willingness among outpatients to extend their visit in order to participate in the study. While this impacts the demographics and exposures of the study population, and thus generalizibility of our study results, it should not impact the findings of the role of infant viral infection on the outcomes of interest. Next, while airway hyperreactivity is not assessed in making the diagnosis of childhood asthma, the identification of incident asthma cases will take into consideration the positive response to a validated written questionnaire that has been compared with bronchial provocation testing. 6, 8 While such an ascertainment strategy might result in the underdiagnosis of asthma, it is unlikely to result in false positive diagnoses during the sixth year of life. Finally, as with many studies, where all eligible participants were approached for participation, difficulty was encountered in follow up of those currently age-eligible for follow up. Strategies to address this include study personnel doing a significant number of follow-up visits at the subject's home, and shipping follow-up materials and requests to paediatricians of subjects who have moved from the region.
Future reports from this cohort will help to clarify the complex relationship between infant respiratory viral infection severity, aetiology, atopic predisposition, and the development of early childhood asthma and other atopic diseases. Ultimately, this study, along with the companion TABS cohort, has the potential to provide new approaches to identify infants at high risk of developing early childhood asthma and allergic diseases, as well as provide important information that may contribute to the development of prevention strategies.  Eosinophilic infiltrate in a patient with severe Legionella pneumonia as a levofloxacin-related complication: a case report INTRODUCTION: Legionella pneumonia can appear with different levels of severity and it can often present with complications such as acute respiratory distress syndrome. CASE PRESENTATION: We report the case of a 44-year-old Caucasian man with Legionella pneumonia with successive development of severe acute respiratory distress syndrome. During his stay in intensive care the clinical and radiological situation of the previously observed acute respiratory distress syndrome unexpectedly worsened due to acute pulmonary eosinophilic infiltrate of iatrogenic origin. CONCLUSION: Levofloxacin treatment caused the occurrence of acute eosinophilic infiltrate. Diagnosis was possible following bronchoscopic examination using bronchoaspirate and transbronchial biopsy. Since the pneumonia epidemic that struck the delegates of the American Legion Convention in Philadelphia in 1976, Legionella spp. has become a relatively frequent cause of community acquired pneumonia [1] .
Legionella may appear in different forms, from subclinical presentations to Legionnaires' disease, which has a mortality rate as high as 30 to 50% in cases of hospital infections and in cases of complications such as acute respiratory distress syndrome (ARDS). The fatality rate is 5 to 25% even in patients who are immunocompetent [2] .
Other complications are rare, although a significant number of drugs used in the treatment of Legionella pneumonia can be associated with the appearance of pulmonary eosinophilic infiltrates, especially non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics [3] . The diagnosis is mainly based on the temporal correlation between the administration of drugs and the appearance of the clinical condition, but it is often not easy to determine the etiologic agent with certainty.
This report concerns the case of a man with Legionella pneumonia that evolved into ARDS and then became complicated with eosinophilic infiltration as an effect of treatment with levofloxacin. Usually this drug is safe, though in some cases can cause eosinophilic pneumonia [4] .
A 44-year-old Caucasian man presented to our hospital for hyperpyrexia (over 39°C) for about a week, with general weakness and strong headaches; he had been treated by his general practitioner with amoxicillin and clavulanate administrated orally with no improvement.
His case history revealed that he was a smoker (20 packs/year). No other pathologies or trips abroad had been registered in the last 6 months.
On admission, he had hyperpyrexia (38.9°C), headache, dry cough, diarrhea, general weakness and sinus tachycardia (100 beats/minute); his oxygen saturation was 95% (no oxygen supplement).
The results of a physical examination of his chest were reduced vesicular respiration and crackling in the median axillary line to the left and in front; a chest X-ray showed extensive inconsistent parenchymal consolidation at the fissure of the left upper lobe ( Figure 1A ).
The results of initial laboratory examinations revealed his white blood cell count was 2020 cells/mm 3 , total bilirubin level was (1.6 mg/dL), he had reduced albuminemia (2.7 g/dL), increased alkaline phosphatase (382 U/L), γ-glutamyl transferase (69 U/L) and creatine phosphokinase (422 U/L). His serology test results were negative for Hepatitis B virus, Hepatitis C virus and HIV. His initial blood culture test results were negative for aerobic and anaerobic germs and mycetes.
Our patient began treatment with intravenous piperacillin and tazobactam (13.5 g/day) and clarithromycin orally (1 g/day). On the third day the results of his urinary antigen test were found to be positive for Legionella serogroup 1, so clarithromycin was suspended and substituted with intravenous levofloxacin (750 mg/day). We maintained the piperacillin and tazobactam treatment to help prevent secondary infection from other Gram-positive and Gram-negative bacteria.
On the sixth day, his clinical condition worsened. After consultation with an infectious disease specialist, we added rifampicin (900 mg/day) to support the levofloxacin action against Legionella pneumonia.
On the ninth day he showed respiratory distress (40 breaths/minute). An Arterial Blood Gas analysis in room air gave the following results: partial O 2 pressure (pO 2 ) of 50 mmHg, partial CO 2 pressure (pCO 2 ) of 30 mmHg, pH 7.50 and oxygen saturation (SaO 2 ) of 86%.
A computed tomography (CT) scan of his chest revealed multiple areas of parenchymal consolidation in the entire upper left pulmonary lobe, mixed with ground-glass areas and abundant pleural effusion. In the right lung, in the dorsal and basal regions, there were ground-glass areas mixed with consolidation areas (Figure 1B) .
On the 10th day PaO 2 /fraction of inspired O 2 (FiO 2 ) ratio was 101 and he was moved to our intensive care There was an absence of pleural effusion and no cardiomegaly. B) A chest CT scan taken on the ninth day: consolidation areas can be seen on the whole superior left lobe, mixed with ground-glass areas and air bronchogram. There was an absence of pleural effusion. C, D) A CT scan taken on the 21st day: on the left there is parenchymal consolidation with air bronchogram and pneumothorax, and several areas of parenchymal consolidation on the right superior lobe. There was an absence of pleural effusion.
unit. Here he was placed on a ventilator on continuous positive airway pressure modality, with noticeable improvement of the respiratory parameters (PaO 2 /FiO 2 ratio of 254). On the 17th day, levofloxacin was suspended in order to allow wash-out and taking of further blood cultures. On the 19th day levofloxacin was resumed; after advice from an infectious diseases specialist intravenous levofloxacin 1500 mg per day together with intravenous fluconazole 800 mg per day were given On the 21st day, after an initial improvement, he showed respiratory distress. A CT scan showed increased parenchymal consolidation with left pneumothorax ( Figure 1C, D) .
On the 22nd day, because of the unexpected occurrence of muscular exhaustion, orotracheal intubation was performed and he was placed on a mechanical ventilator in synchronized intermittent mandatory ventilation mode associated with appropriate kinetic therapy on a reclining bed. A fibrobronchoscopy study, carried out with bronchoalveolar lavage (BAL) for bacteriological reasons and in order to define the cytological profile, revealed the presence of numerous macrophages (32%), lymphocytes (26%; CD4/CD8 ratio 0.8), neutrophilic granulocytes (40%) and some eosinophilic granulocytes (2%). Protozoa, fungus and neoplastic cells were absent.
On the 23rd day, methylprednisolone (120 mg/day intravenously) was added to the therapy.
On the 26th day, he underwent another bronchoscopy, with BAL and transbronchial biopsy in the basal segments of the lower right lobe, which revealed a histological condition compatible with acute eosinophilic pneumonia (Figures 2 and 3) . The BAL confirmed the presence of eosinophils 28%, macrophages 57%, lymphocytes 15%, neutrophilic granulocytes 2% and a CD4/CD8 ratio of 1. Incidental findings showed masses of finely pigmented macrophages (due to our patient's smoking habit). Serum levels of total IgE were within normal limits, and the specific IgE antibody results for allergens (food, pollen, fungal) were also negative. Fecal and serological test results were negative for parasites.
On the 27th day, his steroid therapy was increased (methylprednisolone 1 g/day) while levofloxacin was suspended. His response to steroid therapy was rapid, with a general improvement starting from the fifth day of treatment (the 32nd day overall), associated with accompanying improvement of respiratory exchange and subsequent return to spontaneous breathing on the 41st day (PaO 2 /FiO 2 ratio of 357).
On the 51st day, a chest X-ray showed that the pneumonia bilateral consolidation had completely resolved ( Figure 4 ).
ARDS is a common medical emergency and is usually a complication of a previous illness, which is the etiological cause [5] . In our patient, the unusual fact was the overlapping of acute eosinophilic infiltrate in legionellosis.
Eosinophilic pneumonias include a wide range of pulmonary pathologies, characterized by alveolar and peripheral blood eosinophilia. Peripheral eosinophilia may be absent, in particular in the early stages of acute idiopathic eosinophilia pneumonia or in patients taking systemic corticosteroids. It may occur with extremely variable forms of seriousness, from asymptomatic pulmonary infiltrates to acute respiratory distress syndrome associated with respiratory insufficiency. The possible causes, such as drugs or parasitic infections, have been widely studied, but are, in most cases, idiopathic [6] . In our opinion, in accordance with the findings of other [7] , early low-dose steroid therapy leads to a better outcome of pneumonia with severe respiratory distress; however it could determine a delayed onset of eosinophilic pneumonia.
In our patient, we are inclined to consider it as having an iatrogenic etiopathogenesis. Other causes were excluded by laboratory tests for differential diagnosis options (serum total and specific IgE, fecal and serologic examinations for parasite infections).
Eosinophilic pneumonia has been linked to more than 80 drugs, although only 20 of these (for the most part NSAIDs and antibiotics) can be considered as common causes of this pathology [6] . All the drugs administered in the weeks prior to the appearance of eosinophilic infiltrate should be suspected as a possible cause of the pathology. Iatrogenic eosinophilic infiltrates usually develop progressively, with dyspnea, cough and fever in subjects who have taken certain drugs for weeks or months.
The diagnosis of drug-induced eosinophilic pneumonia is mainly based on a detailed history of drug exposure, evidence of eosinophil accumulation in the lung and exclusion of other causes. Numerous methods have been studied in order to demonstrate sensitivity to one or more drugs. One of the most commonly applied methods is the lymphocyte stimulation test (LST), which measures the proliferation of T lymphocytes in response to a drug in vitro, in order to diagnose a previous reaction in vivo. This concept was confirmed by the finding of drug-specific T lymphocyte clones that can interact with cellular receptors without being metabolized and without bonding to protein carriers [8] .
We did not consider it necessary to carry out the LST with our patient because this method is not specific and sensitive, and it has the major drawback of being difficult to interpret [8] .
With regard to the challenge test in vivo, this was not performed because of the serious clinical condition of our patient, who in any case did not give his consent. However, voluntary challenge may cause life-threatening adverse reactions and it should be limited to rare situations [9] .
Among the possible causes we considered, the first was levofloxacin. There are some reports in the literature regarding the possibility of development of eosinophilic pneumonia during the course of levofloxacin therapy [4] ; moreover, it was the drug administered to our patient for the greatest number of days (21 in total). Other points can be taken into account: (1) the drug was suspended for four days in order to allow for washout and subsequent blood culture; afterwards, the same drug was resumed. At the same time, the clinical radiological findings became worse, with an unintentional challenge effect. (2) The BAL on the 22nd day, as some other authors have reported, still showed compatibility with ARDS Legionella, [10] while the following BAL showed eosinophilia (28%) compatible with an acute eosinophilic pneumonia [6] , which histological exams confirmed ( Figure 3) .
With regard to the other drugs administered, there are reports of isolated cases of eosinophilia associated with parenchymal infiltrates as a consequence of rifampicin therapy [11] . There is only one reported case where clarithromycin may have led to eosinophilic pneumonia [12] , but our patient was only treated with this drug for two days. Moreover, it is possible that eosinophilic pneumonia could be an adverse reaction to smoking in predisposed subjects: this sometimes happens to patients who have recently started smoking or who have modified their 'way' of smoking (for example, increasing or changing type of smoking). Our patient, however, did not report any changes, either in quantity or in quality, in his smoking habits, so this would seem to exclude any relation to smoking [13] .
However, it is plausible that smoking could have acted as a cofactor (together with the drugs) in triggering the clinical condition, because it is a known fact that acute eosinophilic infiltrates are often frequent in smokers [14] .
In conclusion, levofloxacin may be the most probable cause of the occurrence of acute eosinophilic infiltrate in this patient. It is important to emphasize that we decided to change the diagnostic and therapeutic approach only when the presence of eosinophilic infiltrate was proven by transbronchial biopsy. Published studies dealing with risks of invasive endoscopic procedures in a patient who was critically ill on mechanical ventilation showed a higher incidence of complications such as hemorrhage and pneumothorax. Correlating the endoscopic risk to the percentage of correctly carried out diagnoses, which varies from 33% to 76%, with consequent change in therapeutic strategy, it may be stated that the risk/benefit ratio of the endoscopic procedure in terms of therapeutic response is surely in its favor and it is, therefore, recommended [15] .
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Functional analysis of the SRV-1 RNA frameshifting pseudoknot Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting. Ribosomal frameshifting is a translational recoding mechanism that allows the synthesis of multiple proteins from a single mRNA. During this process a certain proportion of the ribosomes is forced to move one or two nucleotides backwards (-1 or À2 frameshift) or forwards (+1 or +2 frameshift) whereafter they continue translation in the new reading frame. As a result the stop codon of the first open-reading frame is bypassed and a fusion protein is synthesized [reviewed in (1, 2) ] Frameshifting is frequently used by RNA viruses, in particular by those with a single genome, and is thought to lead to precise ratios of viral proteins, which is crucial for successful infection (3, 4) . Frameshifting is occasionally used by the eukaryotic cell, e.g., to regulate expression of antizyme (5) or by the prokaryotic cell to regulate production of release factor RF2 (6) and synthesis of the gamma subunit of DNA polymerase (7) .
The signal that makes a ribosome shift comprises two elements: a slippery sequence, where the ribosome switches the reading frame, and an adjacent stimulatory signal, usually a specific RNA structure. For À1 frameshifting, the slippery sequence usually consists of a heptanucleotide motif X XXY YYZ, where X can be any three identical nucleotides, Y can be three A's or U's, and Z is not G (8) . The slippery sequence has been shown to be shifty on its own in vitro, up to 2%, but is strongly stimulated, up to 40-fold, by the presence of a hairpin, a pseudoknot, a three-way junction (9) or an antisense oligonucleotide (10, 11) , located 5 to 8 nt downstream of the slippery sequence.
Despite recent progress (12) (13) (14) , the exact mechanism by which a downstream RNA structure stimulates À1 frameshifting remains unclear. The current view is that the downstream RNA element forms a physical barrier that causes a fraction of ribosomes to stall in their translocation step and puts tension on the mRNA-tRNA interaction (15) . This tension is relieved by realignment of A-site and P-site tRNAs in the 5 0 -direction, whereafter the ribosome resumes translation in the À1 reading frame. Several data also point to a role for the E-site tRNA in stimulating frameshifting (16, 17) .
In general, a pseudoknot is more efficient in stimulating frameshifting than a hairpin of the same sequence (18, R.C.L. Olsthoorn unpublished data). This difference is likely related to a higher thermodynamic stability of the pseudoknot. Indeed, from thermodynamic analyses it appears that pseudoknots are more stable than their hairpin counterparts (19) (20) (21) . Recent studies employing mechanical 'pulling' of frameshifter pseudoknots have shown a correlation between the mechanical strength of a pseudoknot and its frameshifting capacity (13, 14) and influence of major groove and minor groove triplex structures (22) . The higher strength of a pseudoknot can be primarily attributed to the formation of base triples between the lower stem S1 and loop 2 (Figure 1 ), making it more resistant against unwinding by an elongating ribosome (15, 23) . Base triples in the pseudoknots of Beet western yellows virus (BWYV) (24) , Pea enation mosaic virus (PEMV-1) (21) and Sugarcane yellow leaf virus (ScYLV) (25) have been shown to play an essential role in frameshifting.
We previously solved the solution structure of the pseudoknot present in the overlapping region of gag and pol genes of Simian retrovirus type-1 (SRV-1) by NMR (26) . The structure has a classical H-type fold and is further characterized by interactions between the minor groove of stem 1 and loop 2, which forms a triple helix by various tertiary interactions and extensive stacking ( Figure 1A) .
Here we present a detailed mutational analysis of the SRV-1 pseudoknot addressing the role of the triple helix for À1 frameshifting in vitro and in vivo. The data not only emphasize the functional role of the triple helix in À1 frameshifting but also suggest a role for refolding kinetics of frameshifter pseudoknots.
Mutations in the SRV-1 frameshifting signal were made in plasmid SF2 that is derivative of pSFCASS5 (8), a frameshift reporter construct used in earlier phenotypic studies. SF105-113 [except SF110 (A28G) see SF210] were obtained by a two-step PCR mutagenesis procedure using degenerated primers on plasmid pSF103 that contains the sequence of the 'NMR pseudoknot' (26) . The appropriate mutants were selected by dideoxy sequencing.
SF202-206 and SF209-211 were constructed as follows. First a BglII-NcoI fragment from the SF2 vector was replaced by a short DNA fragment obtained by hybridization of oligonucleotides (5 0 -GATCTTAATACGACT CACTATAGGGCTCAGGGAAACTGATCA-CGTGG C-3 0 ) and 5 0 -CATGGCCACGTGATCAGTTTCCCTGA GCCCTATAGTGAGTC-GTATTAA-3 0 ) creating a unique BclI restriction site just downstream of the slippery sequence. The resulting plasmid pSF201 was opened at the BclI and NcoI sites and sets of complementary oligonucleotides corresponding to mutants SF202-206 were inserted. In mutant SF207, which codes only for the À1 reading frame product, the BglII-NcoI fragment was replaced by two short complementary oligonucleotides: SF218/SF219: 5 0 GATCTGGCCACTAGTAC/5 0 CATGG TACTAGTGGCCA). Note that this in-frame product is 21 aa shorter than the frameshifted À1 product. A C B Figure 1 . Structure of the 'NMR' SRV-1 frameshift pseudoknot and the effect of substitutions in L2 on its frameshift-inducing capacity. This pseudoknot differs from the viral pseudoknot by having G2C18-to-CG, C10G32-to-UA, G20-to-C and deletion of GCU between C24 and A25 substitutions (26) . (A) The 3D model of the NMR pseudoknot (PDB 1E95); L2 bases, magenta; S1 and S2 base pairs in cyan, L1 base in red, 3 0 single-stranded sequence AC in yellow. (B) Substitutions in the NMR pseudoknot sequence. Dashed lines illustrate base triples. (C) Rabbit reticulocyte lysate translation products of mRNAs derived from BamHI-digested templates were separated on a 17.5% SDS polyacrylamide gel and detected by fluorography. The migration of the 19 kDa 0-frame product (NFS) and the 22 kDa 'frameshift' product (FS) are indicated. SF103, wild-type; see Table 1 for the explanation of other constructs.
SF213-224 were constructed as follows. First the entire BglII-NcoI fragment of pSF2 was replaced by a synthetic dsDNA fragment (5 0 -GATCTTAATACGACTCAC TATA-GGGCTCATTTAAACTAGTTGAGGGGCCA TATTTCGC-3 0 and 5 0 -CATGGCGAAATATGGCCC CTCAACTAGTTTAAATGAGCCCTATAGTGAGTC GTATTAA-3 0 , sequences forming a SpeI restriction site are underlined). The resulting plasmid pSF208 was opened at the BglII and SpeI sites and oligonucleotides SF226 (5 0 -GATCTTAATACGACTCACT-ATAGGGCTCAGG GAAA-3 0 ) and SF227 (5 0 -CTAGTTTCCCTGAGCCCT ATA-GTGAGTCGTATTAA-3 0 ) were inserted. This yielded pSF212, which was subsequently digested with SpeI and NcoI to insert sets of complementary oligonucleotides corresponding to mutants SF213-224. The sequences of these oligonucleotides are available on request. All mutants were checked by dideoxy sequencing and are listed in Table 1 .
In vitro transcription DNA templates were linearized by BamHI digestion and purified by successive phenol/chloroform extraction and column filtration (Qiagen). SP6 polymerase directed transcription was carried out in a 50-ml reaction containing 1-3 mg linearized DNA, 1 mM NTPs, 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 10 mM DTT, 6 mM MgCl 2 , 2 mM spermidine, 6 units of Rnase inhibitor (RNAguard, Pharmacia) and 15 units of SP6 polymerase (Promega). After an incubation period of 2 h at 37 C, samples were taken and run on agarose gels to determine the quality and quantity of the transcripts. Appropriate dilutions of the reaction mix in desalted and sterilized water were directly used for in vitro translations. Alternatively, transcripts were purified by phenol/ chloroform extraction and isopropanol precipitation as described earlier (27) .
Reactions contained 4 ml of RNA solution, 4.5 ml of reticulocyte lysate (Promega), 1 ml of 35 S methionine (ICN, in vitro translation grade), 0.5 ml of 1 mM amino acids (ÀMet) and were incubated for 60 min at 28 C. Samples were boiled for 3 min in the Laemmli buffer and loaded onto 12% SDS polyacrylamide gels. Gels were dried and exposed to phosphoimager screens. Band intensity of 0 frame and À1 frame products was measured using Molecular Imager FX (Biorad) and Quantity One software. Frameshift percentages were calculated as the amount of À1 frame product divided by the sum of 0 and À1 frame products, corrected for the number of methionines, multiplied by 100.
Pseudoknot mutants were cloned in KpnI/BamHI digested pDUAL-HIV(0) (4). The GGGAAAC slippery sequence was changed to the more efficient UUUAAAC (28) to obtain a better read-out. In these constructs the stopcodon of the first open-reading frame, Renilla luciferase, is located downstream of the pseudoknot. A non-frameshifting control was constructed by changing the slippery sequence to UUUAAGC, while in the in-frame control a C-residue was added directly downstream of this heptamer. In both constructs the pseudoknot was the NMR-pseudoknot. A list of oligonucleotide sequences is available on request. All constructs were confirmed by DNA sequencing. HeLa cells (24-well plate) were transfected with 250 ng of plasmid using lipofectamine-2000 (Invitrogen). Cells were lysed 20 h after transfection and luciferase activities were measured in a Glomax-multidetector (Promega) according to manufacturer's protocol.
The NMR structure of the SRV-1 pseudoknot (26) revealed a number of interesting features that may be relevant to the process of frameshifting. These features include stacking of adenosines A21-A26 in loop L2 and flipping out of cytosine 24, base-base and/or base-sugar interactions of A21, A26, U27 and A28 from L2 with the minor groove of stem S1 ( Figure 1A ). To test the importance of these interactions in frameshifting, mutations were introduced in these regions of the pseudoknot and their frameshifting capacity was measured in rabbit reticulocyte lysates and compared to the NMR wild-type pseudoknot ( Table 1) .
C20. For NMR purposes the wild-type G-residue at this position was replaced by a C (26) . Back mutation to a G (SF105) resulted in a 1.2-fold increase in frameshifting ( Figure 1C ). An adenosine at this position (SF106) was even more beneficial for frameshifting showing a 1.5-fold increase relative to the control NMR pseudoknot ( Figure 1C , compare lanes 'SF103 0 and '106 0 ). A uracil at this position was not tested since this would lead to a premature stopcodon in the À1 frame. Insertion of UUU (SF114) between C19 and C20 led to a slight decrease in frameshifting: 88% relative to the NMR wild-type (SF103). Even though in the latter mutant two additional base pairs might be formed with the 5 0 single-stranded sequence (AU and GU) these are probably melted when the ribosome is stalled over the slippery sequence.
A21. This nucleotide forms a base triple with the C2-G18 base pair. Mutation of A21 to G did not affect frameshifting, but replacement with C or U decreased the relative frameshifting activity to 60 and 63%, respectively (Table 1) . Although a guanine cannot substitute for the triple interaction with the C2-G18 base pair, its stacking properties may compensate for this. The two pyrimidines may be too small to bridge the distance here to form an interaction with C2-G18.
C24. This nucleotide is extruded from loop 2 by the stacking interaction of the continuous run of adenines and is not involved in any interaction. Thus, from a structural point of view, the presence of C24 in loop L2 seems superfluous or even a nuisance by hindering the stacking of the A21-A26 adenosines. Changing C24 to U (SF111) did not affect frameshifting ( Figure 1C) , suggesting that also a uridine at this position is flipped out. Replacing C24 by G (SF112) or deleting it (SF202) had no significant effect on frameshifting while an adenosine at this position slightly enhanced frameshifting activity (SF113), possibly as a result of extended adenosine stacking. These results show, as expected, that C24 is irrelevant and dispensable for frameshifting.
A26. A26 forms three hydrogen bonds with stem 2. Two hydrogen bonds from the N3 and hydroxyl group of A23 to the C16 2 0 OH together form a single ribose-zipper motif. The third hydrogen bond is between the N1 and amino group of G4. A pyrimidine seems too small to fulfil these interactions and indeed replacing A26 by C or U showed an approximately 3-fold lower frameshifting activity (Table 1, SF217 and SF216 ). G would be capable of forming the ribose zipper motif though a considerable clash between its N1 and the G4 amino group can be expected. The relatively high frameshifting activity, 70% of the A26G pseudoknot, suggests that, at this position in L2, stacking properties assisted by the ribose interaction may be more important than formation of the zipper motif itself.
U27. U27 forms only a single hydrogen bond via O2 to the amino group of G15, putting less structural restraints on frameshifting. Still mutation of this residue reduced frameshifting 1.4-to 2.5-fold ( Table 1 ). The 2-fold reduction seen by substituting C for U27 is not easily explained as this pyrimidine has an identically positioned O 2 to hydrogen bond to the amino group of G15.
A28. A28 is a key nucleotide in stabilization of the pseudoknot fold by anchoring L2 to the S1/S2 junction through formation of two hydrogen bonds with the 2 0 -hydroxyl group of G14. Replacement by U (SF108) or G (SF210) resulted in almost 2-fold decrease whereas a C (SF109), though having a similarly positioned N1 and amino groups as an adenosine, resulted in $4-fold drop in frameshifting ( Figure 1C) . Apparently, the relatively small pyrimidine ring is too far away to form interactions with the 2 0 OH of G14. In addition, the lower frameshift activity of the C-mutant could also be caused by formation of a base pair with G14, which could change the overall structure around the S1/S2 junction. In summary, the effect of single-nucleotide mutations in L2 show the functional, critical importance of the A26:G4-C16 and A28:C5-G15 triples that were previously identified by NMR spectroscopy (26) . Base triples at these positions, stabilizing the S1/S2 junction have been recognized to be essential for frameshifting in other pseudoknots as well, be it with different hydrogen bond patterns (21, 24, 25, 29) . To provide additional evidence for the importance of the specific interactions between L2 and S1 in frameshifting, we accumulated a number of the adverse mutations into one construct. Therefore, a construct was designed with a pyrimidine-rich L2 sequence CUCUCGUG (SF229, 'L2-mut') that included the A21U, C24U, A26G and A28G mutations. SF229 showed an 8-fold lower activity in frameshifting (Table 1) , which is approximately the sum of the single-nucleotide changes.
G1-C19. A previous sequence comparison showed that most frameshifter pseudoknots start with a 5 0 G-C3 0 base pair (30). Changing this base pair, which is not involved in any loop interaction, to C-G (SF209) affected frameshifting efficiency somewhat (73%). As stacking energies of this base pair are also lower (À2.4 versus À3.3 kcal/mol) this confers to the general notion that helix stability is an important determinant in frameshifting. Changing G1-C19 into an A-U base pair (SF211) or a G-G mismatch (SF107) reduced the efficiency even further to about 40%, again emphasizing the role of S1 stability.
G3-C17. This pair is also not involved in any triple interaction, but replacing it with A-U (SF342) resulted in a 60% drop in frameshifting. Again this indicates that the stability of S1 plays an important role in the frameshifting capacity of the pseudoknot. Previously, changing G3-C17 in the wild-type pseudoknot to A-U resulted in an almost 5-fold drop in frameshifting (31) . Since we do not have high-resolution structural information of the wild-type pseudoknot (L2: GAAACAAGCUUA), we cannot exclude that the G3-C17 base pair forms a base triple in this context, which could further attribute to the enhanced effect of mutating this base pair.
G4-C16. This base pair is involved in a base triple with A26, mutation of which had quite some impact on frameshifting (see above). Changing G4-C16 into A-U (SF204) is predicted to result in the loss of one hydrogen bond (G24N2H-A26N1), similar to changing A26 to G (SF218), which was still 71% active in frameshifting. The observed dramatic low activity of the A-U base pair mutant (13%) cannot be solely attributed to the loss of one hydrogen bond and must therefore also be due to compromising the stability of the S1 stem.
C5-G15. This pair forms a triple interaction with U27, mutation of which had at most a one-third lower frameshifting activity. Changing the base pair to U-A (SF203) caused an almost 4-fold drop in frameshifting activity, again showing the importance of stem stability.
C6-G14. This pair forms a base triple with A28 via the hydroxyl group of G14. This interaction is not expected to change by mutating it to a U-A bp (SF205). The 2.5-fold decrease must therefore be a consequence of reduced stem stability and/or stacking properties with the S2 U-A pair at the junction. This large decrease again illustrates the critical importance of the A28:C6-G15 triple interaction.
As with the single-nucleotide substitutions, the effect of the base pair mutations also correlates with the NMR structure, thereby illustrating the functional importance of the S1-L2 interactions. Also the stability of S1 as a primary determinant of frameshifting is clearly confirmed.
Using the above knowledge we attempted to create a supershifting pseudoknot (Figure 2 ). To this end we substituted G for C20, removed C24 and restored the G2-C18 and C10-G32 base pairs, as present in the SRV-1 pseudoknot. To our surprise this mutant was 3-fold less active in frameshifting than the parent SF206 pseudoknot. Close inspection of the sequence indicated a possible alternative structure containing two hairpins competing with pseudoknot formation (Figure 2 ). To prevent this alternative structure, we mutated C10-G32 to U-A (SF223) or G-C (SF224). In these constructs the U-A pair is predicted to destabilize the 4-and 7-bp hairpins and the G-C base pair is predicted to completely disrupt the 4-bp hairpin and destabilize the 7-bp hairpin. Frameshift levels of SF224 increased approximately 2-fold compared to SF222, while that of SF223 increased more than 3-fold suggesting that the role of alternative structure formation should not be underestimated.
Although SF224 is a slightly better shifter than SF206 (1.11-fold) with an absolute frameshift frequency of 24% it still cannot be considered a supershifter. Previous experiments with SRV-1 pseudoknot mutants yielded better shifters (31) . One of these, SF67, which differed from . Figure 2 . Putative alternative structure of the 'supershifter' pseudoknots. The boxed G-C base pairs and the circled G are also present in the wild-type SRV-1 pseudoknot. The position of the C24 deletion is indicated by the dot and arrowhead.
SF222 by still having C24, was capable of shifting 30% of the translating ribosomes. Thus, it appears that depending on the context deleting C24 is not always beneficial for frameshifting.
At present it is difficult to interpret these results. One explanation could be that C24 or other L2 nucleotides also play a role in refolding of the pseudoknot after ribosome passage. It can be envisaged that a faster refolding pseudoknot might be a more efficient frameshifter, because in order to cause a frameshift, a stable pseudoknot needs to be regenerated before it encounters the next ribosome.
If refolding kinetics of the pseudoknot is a determinant in frameshift efficiency the length of L2 is also expected to have an effect on frameshifting. To test this possibility we changed the length of L2 to 3 and 27 nt in mutants SF348 and SF350, respectively. The absolute frameshifting activity of SF348 was only 1.3%, i.e. a 16-fold decrease compared to SF206. SF350 showed 8.4% frameshifting, a 2.5-fold drop. Of course, when introducing such dramatic changes, one should also consider their effects on the pseudoknot structure and stability. In SF348 most of the base triples are presumably lost and a 3-nt loop crossing the minor groove of stem S1 may not be sterically and thermodynamically favorable. In fact, the very large-16-fold-reduction in frameshifting suggests that the pseudoknot is not formed at all (the presence of a pseudoknot structure was not checked by e.g. structure probing).
With SF350, containing the 27-nt L2, the situation is different. In this mutant some of the triples may still form but the additional unstructured 18 nt are expected to introduce a substantial entropic penalty into the system, which increases DG and therefore reduces frameshifting. In addition, a large loop may lead to a longer refolding time of the pseudoknot once a ribosome has passed through this structure.
To investigate whether the mutations that affected frameshifting efficiency in vitro would also exert their effect in vivo, we introduced some of the above mutations into a dual luciferase reporter plasmid and assayed their activity in vivo ('Materials and Methods' section). The frameshifting efficiency of the wild-type SRV-1 pseudoknot and GGGAAAC slippery sequence (SF400) was only $8%, against a background of 2% of a non-frameshifting control (data not shown). This is a factor of 3 lower than that reported in vitro (28) . The NMR pseudoknot showed $11% of frameshifting (SF402, data not shown) that increased to $19% in combination with the UUUAAAC slippery sequence (SF404). This 1.7-fold increase is very similar to the previously reported increase from 23 to 40% in vitro for changing the slippery sequence (28) . Mutations were made in the pseudoknot of plasmid SF404. As can be seen in Figure 3 , the substitutions had almost the same effect on frameshifting in vivo as in vitro. The only exception seems to be the effect of enlarging L2, which is consistently more detrimental for frameshifting in vivo than in vitro. This could be due to refolding kinetics of the pseudoknot, what in combination with heavier ribosomal traffic in vivo would result in a lower fraction of properly folded pseudoknots. However, other possibilities, like enhanced RNase susceptibility or alternative folds involving L2, may also account for this effect.
The interactions between L2 and S1 in the SRV-1 pseudoknot as previously determined by NMR (26) turn out to be important for frameshifting. Although single alterations of the base triples found in the NMR study had at most a 3-fold deleterious effect on frameshifting, combining several of these changes into one mutant, L2-mut, led to an 8-fold decrease in vitro and a 6-fold decrease in vivo. This is quite remarkable as the mutated sequence should still be able to fold as a pseudoknot, but apparently critical stem-loop interactions are lacking. As found for other frameshifter pseudoknots, the composition and length of L2 can greatly influence frameshifting. Most frameshifter pseudoknots have adenine-rich L2 loops, presumably because of the need for stable triple helix formation, which is best performed by adenosine's N1 and C6-amino groups (2) .
Deletion of the bulging C24 from the loop L2 did not affect frameshifting significantly in vitro and in vivo. This adds to the notion that this nucleotide can be safely squeezed out to allow stacking of adenosines in L2. It is noteworthy that other frameshifter pseudoknots also have a cytosine flanked by adenines in L2: GAACAAA in Figure 3 . Comparison of in vivo and in vitro frameshifting activities relative to that of the NMR pseudoknot. White bars, in vivo; gray bars, in vitro. The efficiency of the NMR pseudoknot ('wild-type') is set at 100%. Experiments were done at least three times in triplicate.
BWYV (29) , UAAAAACAC in ScYLV (25) , ACUCAAA A in MMTV (32) and AAACAA in HIV-1 type O (33) . Interestingly, L2 of the human telomerase pseudoknot, not involved in frameshifting, consists of CAAACAAA. This pseudoknot has recently been shown to function in frameshifting in vitro (22) . Also here the adenosines formed triple interactions with base pairs in stem S1 and were essential for frameshifting (22, 34) . The effect of substituting the second C in L2 has not been investigated; its presence may be required to prevent slippage of the RNA polymerase.
The main role of the triple interactions seems to be in stabilizing the pseudoknot such that high frequencies of frameshifting are possible. Mutations that remove triple interactions in general lead to lower frameshifting. Conversely, it seems that frameshifting pseudoknots with weaker stems need to be stabilized by such triples. For instance in the BWYV and PEMV-1 pseudoknots, S2 is only three base pairs but this is compensated by an additional triple between L1 and S2. The high number of triples in luteovirus pseudoknots may explain why for instance the ScLYV pseudoknot with a relatively high number of A-U base pairs in the bottom of S1 is still capable of inducing $15% of frameshifting (25) . On the other hand, when S1 is increased to 11 base pairs, triple interactions do not seem to contribute anymore as frameshifting becomes independent of the sequence of L2 (C-H. Yu and R.C.L Olsthoorn, unpublished data).
If we assume that ribosomes first sense the bottom of stem S1 we would expect base triples to be present at the bottom of the stem or in the 5 0 end of L2. This would be in line with the observation that the first three base pairs of a frameshift pseudoknot are usually G-C (30). However, most triples are found near the 3 0 end of L2 close to the junction with S2. Possibly, triples at the junction render the pseudoknot more resistant to forced unwinding by the ribosomal helicase, which would otherwise serve as a pivot.
Because the mere presence of a pseudoknot structure with a certain minimal thermodynamic stability might not be the sole primary determinant of frameshifting a number of alternative models have been proposed in which mechanical resistance of the pseudoknot to forced unwinding is the key (12, 15, 23) . In these models a pseudoknot trapped in the mRNA entry tunnel resists mechanical unwinding, which causes the ribosome to pause. Indeed, recent optical tweezers experiments, in which rupture forces of RNAs are measured by pulling 5 0 and 3 0 ends apart, showed a correlation between frameshifting and mechanical strength of the pseudoknot and confirmed a positive effect of base triple interactions on frame shift efficiency (13, 14, 22) . Frameshifting by the trapped pseudoknot is thought to be either passive, by stalling the ribosome for a sufficient amount of time, or active, by building up tension at the mRNA-tRNA interface by a counteracting ribosome, which can be relieved by a À1 frameshift (15) . Observations that ribosomes can stall for seconds to minutes without frameshifting to occur (35) and pausing appears to be necessary, but not sufficient for frameshifting (36, 37) , rather point to an active role. Although it now seems mechanical resistance to RNA unfolding is the key to ribosomal frameshifting, how the ribosome exactly 'chokes' on such a structure is still far from being understood. Remaining questions are for instance if torsional restraint of the pseudoknot resisting ribosomal unwinding is essential (23) , whether the pseudoknot truly plays an active role in for instance overbending the tRNA in the P-site (12) and at which stage of translational elongation frameshifting actually occurs.
One should also keep in mind the possible formation of alternative structures that can affect the frameshifting efficiency by lowering the fraction of properly folded pseudoknots. This possibility has not been investigated in detail but our own data ( Figure 2 ) suggest that this may be a relevant issue. Especially during heavy ribosomal traffic, as may be the case in vivo, fast and correct refolding of pseudoknots could be an important parameter for frameshifting. Under such circumstances it is conceivable that a hairpin is more preferred as it probably refolds faster than a pseudoknot. A recent study on the HIV-1 frameshift signal has shown that a decrease in the translation initiation frequency can lead to an increase in the frequency of frameshifting (38) . Also a previous study in yeast has shown a correlation between frameshifting and translation initiation frequency (36) . Our data with the enlargement of L2 also hint at a possible role for folding kinetics.
Although the time scale of folding (milliseconds) may at first seem irrelevant compared to the time scale of translation elongation (seconds) it has recently been reported that the conversion rate between two comparatively stable hairpins ranges from a few seconds to several minutes (39) . Also, slow-folding pseudoknots, 5-350 s, limit the activity of the Hepatitis Delta virus ribozyme (40) . Thus the efficiency of a frameshifter pseudoknot may not be solely dictated by its stability but also by its kinetic properties. A molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal A-site Although the ribosome is mainly comprised of rRNA and many of its critical functions occur through RNA–RNA interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. Prior studies demonstrated that two extended domains of ribosomal protein L3 form an allosteric switch between the pre- and post-translocational states. Missing was an explanation for how the movements of these domains are communicated among the ribosome's functional centers. Here, a third domain of L3 called the basic thumb, that protrudes roughly perpendicular from the W-finger and is nestled in the center of a cagelike structure formed by elements from three separate domains of the large subunit rRNA is investigated. Mutagenesis of basically charged amino acids of the basic thumb to alanines followed by detailed analyses suggests that it acts as a molecular clamp, playing a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, facilitating coordination of their functions through the elongation cycle. The observation that these mutations affected translational fidelity, virus propagation and cell growth demonstrates how small structural changes at the atomic scale can propagate outward to broadly impact the biology of cell. Analyses of a growing number of high resolution ribosome structures coupled with kinetic studies are revealing that the ribosome is highly dynamic, capable of assuming >40 different conformational states through the translation elongation cycle [reviewed in (1) ]. Thus, a critical question is how the ribosome coordinates all of these states to ensure the directionality and fidelity of protein synthesis. The general answer lies in allostery: the formation and breaking of specific intermolecular contacts in response to different ligand binding states serves as a series of switches to ensure that the ribosome is optimally configured to proceed to the next functional state. The current challenge is to identify and functionally map the specific allosteric switching components.
Numerous researchers have employed elegant biochemical, biophysical, structural, genetic, and computational approaches to this problem, a few of which are highlighted here. For example, biochemical approaches have been employed to define the kinetic parameters governing every step of the elongation cycle, revealing that selection of the appropriate aminoacyl-tRNA (aa-tRNA) depends on a two step kinetic process [reviewed in (2) ]. Biophysical methods have revealed that conformational switching by rRNA and ribosomal protein L1 ensures exit of deacylated tRNA from the ribosome (1,3), while real time single molecule fluorescence and force measurements are revealing dynamic motions of the ribosome and tRNAs, and directly probing the forces stabilizing ribosomal complexes [reviewed in (4) ]. Biochemical, computational and high resolution structural methods have been employed to map changes in rRNA structures during the transit of tRNAs through the ribosome and during the peptidyltransfer reaction (5) (6) (7) (8) (9) (10) . Pertinent to this study, we have previously used a combination of molecular genetics, biochemical and biophysical approaches to identify the contributions of specific ribosomal proteins and rRNA bases involved in coordinating the stepwise process of accommodation of aa-tRNA into the ribosomal A-site, activation of the peptidyltransferase center, and recruitment of the trans-acting translocase (EF-G in bacteria, eEF2 in eukaryotes) using yeast ribosomes as a model (11) (12) (13) (14) (15) (16) .
Ribosomal protein L3 in particular appears to have an important function in this process. Figure 1A shows L3 within the context of the large ribosomal subunit (LSU), including the following structural and functional elements; the peptidyltransferase center (PTC), the aa-tRNA accommodation corridor which lies in between Helix 89 and the complex Helix 90-92 structure, Helix 95 (the Sarcin/Ricin Loop or SRL), and the GTPase-associated center (GAC). The latter two structures interact with the eEF1A-aa-tRNA-GTP ternary complex and eEF2 (the eukaryotic translation elongation factor homologs of bacterial EF-Tu-aa-tRNA-GTP ternary complex and EF-G, respectively). The 3D rendering in Figure 1B shows that L3 contains a globular domain that interfaces with the solvent side of the LSU, and two structures, the N-terminal extension and a central loop that extend deep into the central core of the LSU. The central loop can be subdivided into two domains, the 'tryptophan finger' (W-finger) positioned at the tip of the central extension, and a cluster of basic amino acids that protrudes from the center of the internal loop like a thumb roughly perpendicular to the W-finger that we call the L3 'basic thumb'. Figure 1C shows that this basic thumb is nestled in the core of a cagelike structure formed by elements of three different 25S rRNA helices: H61, H73 and H90. We previously proposed a 'rocker switch' model describing how structural rearrangements of the N-terminal extension and the W-finger of L3 function to coordinate the stepwise processes of translation elongation (17) . Missing from the prior analyses was an explanation of how the movements of these extensions of L3 are communicated to functional centers of the ribosome. In this current study, analysis of the basic thumb of L3 illuminates this question. Mutagenesis of the indicated amino acids to alanines followed by genetic, biochemical and structural analyses suggests that the basic thumb acts as a molecular clamp to play a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, thus facilitating coordination of their functions through the elongation cycle.
Escherichia coli DH5a was used to amplify plasmid DNA. Transformation of E. coli and yeast, and preparation of yeast growth media (YPAD, synthetic drop out medium, and 4.7 MB plates for testing the Killer phenotype) were as reported earlier (18) . Restriction enzymes were obtained from MBI Fermentas (Vilnius, Lithuania). The QuikChange XL II site-directed specific mutagenesis kit was obtained from Stratagene (La Jolla, CA, USA). Macrogen Inc. (Piscataway, NJ, USA) performed DNA sequence analysis. Oligonucleotide primers were purchased from IDT (Coralville, IA, USA). The yeast strains used in this study were all derived from the rpl3-gene disruption (rpl3D) strain JD1090 (MAT ura3-52 lys2-801 trp1 leu2 À his3 RPL3::HIS3 pRPL3-URA3-CEN6 [L-A HN M 1 ]) (19) . Mutants of rpl3 were generated using the wild-type RPL3 gene in pJD225 (19) , synthetic oligonucleotides, and the QuikChange XL II kit. The pYDL dual luciferase reporter series of plasmids for monitoring programmed ribosomal frameshifting (PRF) were used as described earlier (20) . (36) . L3 is indicated in green. The aa-tRNA accommodation corridor is framed by Helix 89, and the complex structure formed by Helices 90-92. Elongation factors bind to the GTPase Associated Center (GAC) and the Sarcin/Ricin Loop (SRL) at the tip of Helix 95. The peptidyltransferase center (PTC) is in the center of the large subunit. (B) The 3D view of isolated L3, heat map colored from the N-terminus (blue) to the C-terminus (red). The N-terminal extension and the basic thumb and tryptophan (W) finger of the central extension are indicated. (C) The 3D view of interactions between amino acid residues of the L3 basic thumb investigated in this study. It is surrounded by a cagelike structure formed by 25S rRNA Helices 61-64, H73 and H90. Amino acids mutated in this study are labeled.
The killer virus assay was carried out as described earlier (18) . Briefly, yeast colonies were replica plated to 4.7MB plates newly seeded at an optical density at 595 nm (OD 595 ) of 0.5 of the 5 Â 47 killer indicator strain per plate. After 2-3 days at 20 C, killer activity was scored as a zone of growth inhibition around the Killer + colonies. To monitor programmed À1 frameshifting using the dual luciferase reporter plasmids, glass beads were used to prepare lysates from cells expressing the 0-frame, À1 (L-A derived), or +1 (Ty1 derived) dual luciferase plasmids (20) . After clarification of the lysates by centrifugation, typically 5 ml was used in a total volume of 100 ml of dual luciferase assay reagents (Promega, Madison WI, USA), and Renilla and firefly luciferase activities were quantitated using a TD20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Frameshifting efficiencies were calculated by dividing the firefly/Renilla luminescence ratios from lysates of cells expressing the PRF test reporters by the same ratio obtained from lysates of cells expressing the zero-frame control reporter. All assays were replicated enough times to achieve >95% confidence levels, and statistical analyses were performed as described earlier (21) .
Aminoacyl-tRNA synthetases were purified as described earlier (22, 23) .Yeast tRNA Phe was aminoacylated with unlabeled phenylalanine or with [ 14 C]Phe to make Phe-tRNA Phe and [ 14 C]Phe-tRNA Phe , respectively. [ 14 C]Phe-tRNA Phe was used to monitor enzymatic binding to the A site of poly(U) primed ribosomes, and acetylated-[ 14 C]Phe-tRNA Phe (Ac-[ 14 C]Phe-tRNA Phe ) was generated to monitor nonenzymatic P-site binding using poly(U) primed salt washed ribosomes. Phe-tRNA Phe and Ac-Phe-tRNA Phe were used in SHAPE structure probing experiments (see below). Yeast tRNA Phe was aminoacylated as described earlier (24) Reaction mixtures were incubated for 30 min at 30 C, and proteins were removed by extraction with acid-phenol-chloroform. Charged tRNAs were ethanol precipitated and purified using G25 spin columns. [ 14 C]Phe-tRNA Phe was separated from uncharged tRNA by high-performance liquid chromatography (HPLC) as described earlier (25) with the following modifications. Samples were loaded onto a 4.6-by 250-mm JT Baker wide-pore butyl column equilibrated with buffer A (20 mM NH 4 Cl, 10 mM MgCl 2 , 400 mM NaCl; pH 5.0) at 1 ml/min. The column was washed with 10 ml of buffer A, conditions under which free phenylalanine and aminoacyladenylate are eluted from the column. Uncharged tRNAs and residual free [ 14 C]Phe and nucleotides were eluted by isocratic elution of 19 ml at 15% of buffer B (20 mM NH 4 Cl, 10 mM MgCl 2 , 400 mM NaCl, 60% methanol; pH 5.0). [ 14 C]Phe-tRNA Phe was eluted using a programmed binary gradient of buffers A and B. Elution of aa-tRNA was monitored by OD 260 readings, and [ 14 C]Phe-tRNA Phe concentrations and specific activities were determined. The presence of aa-tRNA in the eluted material was confirmed by TLC (24) . Ac-[ 14 C]Phe-tRNA Phe was obtained in a similar manner. Yeast tRNA Phe was charged with [ 14 C]Phe as above, extracted with phenol and purified using G25 columns. Reaction mix (4 ml) contained 200 mM NaOAc, pH 5.2 and 7 nmol of [ 14 C]Phe-tRNA Phe . Acetylation was carried out by addition of 64 ml of acetic anhydride at 1 h intervals for 2 h on ice. After incubation, NaOAc concentration was raised to 300 mM and Ac-[ 14 C]Phe-tRNA Phe was ethanol precipitated. Ac-[ 14 C]Phe-tRNA Phe was further purified by HPLC as described earlier.
Sulfolink resin was charged with cysteine as described earlier (26) . Yeast cells were grown in YPAD media to mid log phase, collected and washed with binding buffer (10 mM Tris-HCl, pH 7.5; 5 mM MgCl 2; 60 mM NH 4 Cl; 2 mM DTT). Cells were suspended in binding buffer and disrupted using a Mini Bead Beater. Lysates were centrifuged at 30 000g for 30 min in Beckman MLS 50 rotor. Supernatant (2 ml) was removed and added to 2 ml of cystein-charged Sulfolink slurry (50% resin equilibrated with binding buffer) and incubated on ice for 15 min with mixing as resin sediments. After incubation resin was spun down at 1500g for 0.5 min. Supernatants were removed and resin washed five times with 5 ml of binding buffer. After washing, resin was suspended in 1 ml of elution buffer (10 mM Tris-HCl, pH 7.5; 10 mM MgCl 2; 0.5 M KCl; 1 mg/ml heparin; 2 mM DTT) and incubated for 5 min on ice with occasional mixing. The suspensions were centrifuged, supernatant collected, and elution was repeated two more times. Supernatants were combined (3 ml total volume) and GTP and pH neutralized puromycin were added to 1 mM each. After incubation at 30 C for 30 min, reaction mixtures were loaded on top of a 1 ml glycerol cushion (10 mM Tris-HCl, pH 7.5; 10 mM MgCl 2; 0.5 M KCl; 2 mM DTT; 25% glycerol) and centrifuged at 100 000g for 16 h. Ribosome pellets were suspended in 2 ml of elution buffer without heparin, loaded on top of 2 ml glycerol cushions and centrifuged at 100 000g for 16 h. Ribosome pellets were resuspended in storage buffer [50 mM HEPES-KOH pH 7.6; 5 mM Mg(CH 3 COO) 2; 50 mM NH 4 Cl; 1 mM DTT; 25% glycerol] at 5-10 pmol/ ml (1 OD 260 = 2 pmol) and stored at À80 C.
Complex C [ribosome-poly(U)-AcPhe-tRNA] was formed in 400 ml of binding buffer (80 mM Tris-HCl, pH 7.4, 160 mM ammonium chloride, 11 mM magnesium acetate, 2 mM spermidine and 6 mM b-mercaptoethanol) containing 0.4 mM GTP, 500 pmol ribosomes, 0.4 mg/ml poly(U) and 700 pmol Ac-[ 14 C]Phe-tRNA. Mixtures were incubated for 20 min at 30 C and then placed on ice. Complexes were purified from free Ac-[ 14 C]Phe-tRNA Phe by centrifugation through a glycerol cushion (0.5 ml; 20% glycerol in binding buffer by centrifugation at 50 000 rpm for 2 h in MLS 50 rotor). Ribosome pellets were rinsed twice with 1 ml of binding buffer and suspended in 1.15 ml of binding buffer. For puromycin reactions, 1.15 ml of complex C extract was pre-incubated at 30 C for 5 min, and reactions were initiated by adding pH neutralized puromycin (100 mM stock) to final concentrations of 10 mM. Aliquots of 100 ml were removed, and reactions were terminated at the indicated time intervals by addition of 100 ml of 1.0 N NaOH. Reaction products were extracted with 0.4 ml of ethyl acetate, 0.2 ml of organic phase was transferred to scintillation vials, and radioactivity was determined by scintillation counting. A 50-ml aliquot of initial reaction mixture was also transferred to scintillation vials, and total radioactivity (N o ) was determined. Controls without puromycin were included in each experiment, and the values obtained were subtracted as background. The percent of the bound Ac-[ 14 C]Phe-tRNA Phe converted to Ac-[ 14 C]Phe-puromycin was corrected with the extent factor a (determined if complex C were allowed to react for 1 h; C o = aN o ), as described earlier (27, 28) . The reaction plots were fit to a first-order exponential equation, and values of K obs (the apparent rate constant of entire course of reaction at a given concentration of puromycin) were calculated by using Graphpad Prism software.
Soluble protein factors were prepared as described earlier (28, 29) . aa-tRNA binding to the A-site of the ribosome was carried out as described earlier (13) . Ribosome mixtures (50 ml) contained 80 mM Tris-HCl, pH 7.4, 160 mM NH 4 Cl, 11 mM Mg(CH 3 COO) 2 ]Phe-tRNA Phe were added to ribosome mixtures and incubated for 20 min at 30 C. Aliquots were then applied onto nitrocellulose membranes, filters were washed with 6 ml of binding buffer, and radioactivity was measured by scintillation counting. Background levels of radioactivity were determined using a blank sample (without ribosomes) and subtracted from test samples. K d values were determined assuming single binding sites using Graphpad Prism software. eEF2 binding 6xHis-tagged eEF2 was purified from TKY675 yeast cells (kindly provided by Dr T. Kinzy) as described earlier (30) with the following modifications. EDTA was added to 5 mM to eluted eEF2 just before dialysis to bind leached Ni 2+ ions and prevent precipitate formation during dialysis due to aggregation of His-tagged protein. eEF2 concentration was determined by [ 14 C]ADP-ribosylation with diphtheria toxin (see below). Each preparation of eEF2 showed linear concentration response curves in the range of eEF2 amounts used in binding experiments. For eEF2-binding experiments, reaction mixes (25 ml) containing 12.5 pmol of salt washed 80S ribosomes and various concentrations of 6xHis-tagged eEF2 in binding buffer (50 mM Tris-HCl, pH 7.5, 50 mM ammonium acetate, 10 mM magnesium acetate, 2 mM DTT, 100 mM GDPNP) were incubated for 20 min at room temperature. Estimation of bound eEF2 was carried out as follows by assuming that ribosome bound eEF2 is not susceptible to ADP-ribosylation by diphtheria toxin (31) (32) (33) . Free (unbound) eEF2 was estimated by ADP-ribosylation of eEF2: 100 pmol [ 14 C] NAD + and 0.2 mg of diphtheria toxin were added to each reaction mix and incubated for 30 min at 30 C. Total eEF2 in each reaction mix was determined by ADP-ribosylation reaction after bound eEF2 was released by adding EDTA to 10 mM. Reaction mixes were precipitated with TCA, and amounts of [ 14 C]ADP-ribosylated eEF2 were determined by liquid scintillation counting. Control values (lacking diphtheria toxin) were subtracted. Ribosome bound eEF2 was calculated by subtracting free values from total amount. K d values were determined assuming single binding sites using Graphpad Prism software.
To prime ribosomes with poly(U), reaction mix (100 ml) in binding buffer (80 mM Tris-HCl, pH 7.4, 100 mM NaCl, 15 mM Mg(CH 3 COOH) 2 , 6mM b-mercaptoethanol) containing 55 pmol ribosomes and 50 mg poly(U), was incubated for 20 min at 30 C. Next, for P-site complex, 200 pmol of Ac-Phe-tRNA Phe was added. For structure probing of ribosomes with occupied A sites, ribosomal P sites were blocked with 4Â excess of deacylated tRNA Phe and Phe-tRNA Phe (200 pmol), GTP (0.5 mM) and 5 ml of crude elongation factor mix were added. Reaction mixes were incubated for 20 min at 30 C. Reactions were divided into two parts of 50 ml each (control and modification tubes) and 75 ml of binding buffer was added to each tube. 25 ml of 1M7 (130 mM in DMSO) was added to modification tubes. Control samples contained 25 ml of DMSO. After incubating for 85 min at 30 C ribosomes were precipitated with 450 ml ethanol. Ribosomal RNA was extracted using RNAqueous kit (Ambion). Pellets were dissolved in 100 ml of RNAqueous Lysis Buffer and processed according manufactures instructions. RNA was eluted in 50 ml volume and concentration adjusted to 1 mg/ml with elution buffer. Reverse transcriptase (RT) primer extension analyses of modified RNAs were performed as described (34) . The site-specificity of charged tRNA binding was confirmed using the puromycin reaction (35) .
The cryo-electron microscopy (cryo-EM) reconstruction of Thermomyces lanuginosus modeled with Saccharomyces cerevisiae rRNA and ribosomal proteins (36) was visualized using PyMOL (DeLano Scientific LLC).
The L3 basic thumb mutants affect cell growth, programmed À1 ribosomal frameshifting and yeast Killer virus maintenance rpd3Dcells harboring wild-type pRPL3-Ura were transformed with mutant rpl3 alleles expressed from a low copy TRP1 vector, and the viability of mutants was assessed by their ability to grow in the presence of 5-FOA. All of the L3 basic thumb single mutants (R232A, K236A, K237A, K241A, R244A, R247A and R248A) were viable as the sole forms of L3 with the exception of R240, which was lethal (Figure 2A , summarized in Table 1 ). Among the viable single mutants, only R232A and R247A conferred noticeable growth defects. A series of double mutants were constructed based on the viable mutants and their physical locations relative to one another. This analysis revealed that the K236A/R247A and K241A/R244A mutants significantly affected cell growth, while the R247A/ R248A mutant was inviable. The K236A/K237A double mutant did not grossly affect cell growth.
Most strains of S. cerevisiae harbor a symbiotic virus called 'Killer', a bipartite dsRNA viral system composed of the L-A helper virus and the M 1 satellite [reviewed in (37) ]. The 4.6-kb dsRNA L-A viral genome encodes the viral coat protein (Gag), and the Gag-pol replicase that is encoded by a programmed À1 ribosomal frameshift (À1 PRF) (38) . The M 1 satellite dsRNA is encapsidated and replicated inside of L-A encoded viral particles. The M 1 encoded preprotoxin is processed by the Kex1p and Kex2p cellular proteases into the mature secreted toxin. Cells infected by L-A and M 1 can kill uninfected cells, but are themselves immune, hence the name 'Killer'. The presence of Killer can be easily assayed by replica plating test cells onto a lawn of diploid uninfected (indicator) cells: Killer + cells will kill the nearby indicator cells, resulting in a ring of growth inhibition around the test cells. One of the first yeast mutants cloned, mak8-1, was first identified by its inability to maintain the Killer phenotype (Mak À phenotype) and encodes an allele of rpl3 (39) . Analyses of the L3 basic thumb mutants revealed that three of the single mutants (K236A, K237A and R247A) were completely unable to maintain the Killer virus, while R232A and R244A had weak Killer phenotypes. None of the double mutants were able to maintain the virus ( Figure 2B , summarized in Table 1 ).
The efficiency of À1 PRF determines the relative ratio of structural Gag to enzymatic Gag-pol available for viral particle self-assembly, and changing À1 PRF strongly inhibits virus maintenance (18, 40) . Previous studies have identified numerous L3 mutants that promoted altered rates of L-A promoted programmed À1 ribosomal frameshifting (À1 PRF), but did not affect Ty1 mediated programmed +1 ribosomal frameshifting (+1 PRF) (13, 17, 19, 23) . However, while all previous L3 mutants analyzed to date enhanced À1 PRF efficiency, all of the viable L3 basic thumb mutants promoted decreased À1 PRF, ranging between $55% and $75% of wild-type rates ( Figure 2C , summarized in Table 1 ). The significance of these changes in À1 PRF is confirmed by loss of the Killer virus, maintenance of which is known to be sensitive to even small decreases in À1 PRF rates (18, 41) . Consistent with prior studies none of the basic thumb mutants affected +1 PRF (summarized in Table 1 ).
The simultaneous slippage model of À1 PRF requires that both the aa-tRNA in the ribosomal A-site, and the peptidyl-tRNA in the P-site must shift on the mRNA (42), while in Ty1 mediated +1 PRF, only the peptidyl-tRNA slips (43) . Consistent with the frameshifting data, all of the L3 basic thumb mutants promoted decreased affinity for aa-tRNA to the A-site ( Figure 3A and B, and summarized in Table 1 ), but did not affect binding of Ac-aa-tRNA to the P-site ( Figure 3C and D, summarized in Table 1 ). Specifically, R232A, which does not directly contact any rRNA bases, had the smallest effect on aa-tRNA binding (K d values $126 nM compared to $94 nM for wild-type, i.e. 1.4-fold increase), while the K236A and K237A mutants, which participate in only a few rRNA contacts ( Figure 4B ), had moderate effects ($165 nM each, 1.8-fold wild-type). In contrast, R247A, which contacts both H61 and H90 had a very strong effect on aa-tRNA binding ($300 nM, 3.3-fold wild-type). The double mutants, which also affected multiple rRNA contacts had comparable effects on aa-tRNA binding (from $245 nM to $340 nM).
Eukaryotic elongation factor 2 (eEF2) catalyzes translocation and binds to the same site as the aa-tRNA-eEF1A-GTP ternary complex. Given the effects of the mutants on aa-tRNA binding, the effects of seven mutants on eEF2 binding were assayed ( Figure 3E and F, summarized in Table 1 ). For wild-type ribosomes, the dissociation constant for eEF2 was $383 ± 93 nM. Similar values were observed for R232A, K236A and K237A mutant ribosomes, but the R247A mutant promoted an $8-fold increase in affinity for eEF2 (K d $ 47 nM). Among the double mutants assayed, the K237A/K236A mutant promoted the largest increase in affinity for eEF2 ($84 nM, $4.5-fold increase), followed by K236A/R247A ($110 nM, $3.5-fold increase), and K241A/R244A ($229 nM, $1.7-fold increase).
Single round assays of peptidyltransferase activity were performed on puromycin treated salt washed ribosomes pre-loaded with Ac-[ 14 C]Phe-tRNA Phe and purified through glycerol cushions (Complex C) as described in the 'Materials and Methods' section. The observed K obs = 0.34 min À1 in wild-type ribosomes ( Figure 3G , Table 1 ) is comparable to similar reactions using E. coli ribosomes and the Ac-Phe-tRNA substrate (44) , confirming that these relatively low rates are determined by Ac-Phe-tRNA as a poor substrate for the peptidyltransferase reaction, and thus represent true measurements of peptidyltransferase activity, as opposed to peptidyl-tRNA turnover or other artifacts. The R232A, K236A and R247A mutants all promoted decreased rates of peptidyltransfer to approximately two-thirds of wild-type levels, while the double mutants (K236A/K237A, K236A/R1247A, and R247A/K248A) had stronger effects, decreasing rates to $50% of wild-type ( Figure 3G and H, Table 1 ). Unexpectedly, the K237A mutant enhanced the rate of peptidyltransfer by almost 2-fold above wild-type levels.
The L3 basic thumb mutants promote changes in 25S rRNA structure both locally and in elements associated with aa-tRNA and eEF2 related functions Inspection of atomic resolution ribosome structures reveals that the L3 basic thumb participates in a highly conserved set of interactions with the PTC proximal bases of Helix 73, and Helix 90, with bases on both sides of Helix 61, and with bases in a complex loop structure connecting Helices 61-64 of the LSU rRNA ( Figures 1C, 4B and D) (6, 36, (45) (46) (47) . Strikingly, while 2D maps of the LSU suggest that these structural elements are in physically separate domains from one another in (Figure 4B A-minor motif in the A-site of the peptidyltransferase center (48) . For ease of comparison, yeast 25S rRNA bases are listed with their E. coli homologs in Table 2 .
To assess the effects of the viable mutants on rRNA structure, SHAPE [Selective 2 0 -Hydroxyl Acylation and Primer Extension, (34, 49) ] using 1M7 [1-methyl-7nitroisatoic anhydride, (50) ] was used to probe wild-type and selected mutant ribosomes containing either Ac-aa-tRNA Phe at the P-site alone, or both tRNA Phe and aa-tRNA Phe at the P-and A-sites respectively (Figure 4) . Inspection of the results revealed two general trends. First, that the mutants promoted significant changes in rRNA structure in the peptidyltransferase center (U2953, U2955, G2977), along the path taken by aa-tRNA as it accommodates into the LSU (accommodation corridor, G2912, U2924, C2929, A2934), in Helix 94 where it interacts with the globular domain of L3 (G3003, A3006, G3009), and in Helix 95 (U3019, U3023, A3033). Secondly, while the mutants only altered the Helix 73-Helix 95 region when only the P-site was occupied by Ac-aa-tRNA, the majority of changes in the Helix 91-93 region were observed when both A-and P-sites were occupied. Detailed analyses reveal that the R247A mutant conferred the largest number of changes in rRNA structure, promoting deprotection of C2985 in Helix 91, G3003, A3006, G3009 in Helix 94, and U3019, U3023 and A3033 in Helix 95 when only the P-site was occupied ( Figure 4A and B). In contrast, this mutant promoted increased deprotection of G2912 in Helix 92, U2924 in the A-loop, A2934 in the bulge between Helix 92 and Helix 90, and U2955 in the peptidyltransferase center when the A-site was occupied by aa-tRNA. This mutant also caused hyperprotection of A2987 in Helix 73 when the A-site was occupied. Some of the other mutants had similar effects on some but not all of the same bases, e.g. K237A caused deprotection of G2912 and U2955, and enhanced protection of A2987 when the A-site was occupied. Other mutant specific effects were observed. For example, when only the P-site was occupied K247A promoted enhanced protection of G2977, while both K247A and K236A promoted deprotection of A2995 and U2953 under these conditions ( Figure 4A and B). K237A also promoted increased protection from 1M7 at G2977 when both the A-and P-sites were occupied. R232A had significant effects in the 3 0 loop between H90 and H92, and in the peptidyltransferase center (U2955). The observation that the bulged A2971 in Helix 93 was generally deprotected when only the P-site was occupied by Ac-aa-tRNA Phe , but became protected from chemical attack upon loading of aa-tRNA Phe into the A-site (Figure 4 ) serves as an important control, as this site occupancy-specific conformational change has also been observed for bacterial ribosomes (51) . Interestingly, this pattern was also observed for A2926 in the A-loop, the possible significance of which is discussed below.
How is information flow coordinated through the ribosome to ensure the directionality of protein synthesis?
Although the bulk of the ribosome is comprised of rRNA, and indeed, many of its critical functions are mediated through RNA-RNA interactions, it is clear that protrusions of ribosomal proteins, which can be thought of as loops, hooks and fingers, function to help 'switch' the ribosome between different conformational/functional states. For example, the C-terminal extension of E. coli S13 is thought to help coordinate movement of the peptidyl-tRNA with structural rearrangements at the ribosome interface that are critical for translocation by sampling the tRNA occupancy status at the decoding center (52, 53) . The N-terminal 'hook' of yeast L10 (E. coli L16) is believed to play an active role to coordinate switching of the ribosome between the pre-and post-translocational states (16) . Ribosomal protein L2, which is intimately intertwined with multiple domains of the LSU, is thought to coordinate long range interactions between tRNAs and the ribosome (14) . Ribosomal protein L3 is of particular interest because of its function as a 'gatekeeper' to the ribosomal A-site (13) . A follow-up study suggested that two critical structures of L3, the W-finger and the N-terminal extension, function together as a 'rocker switch' to coordinate LSU associated functions (17) . The L3 basic thumb is of interest because it appears to provide the structural link in this rocker switch mechanism. Protruding roughly perpendicular from the L3 W-finger toward the intersubunit face of the LSU, it is surrounded by a cagelike structure formed by a large bulge framed between Helices H61-64, Helix 73, Helix 90 and Helix 94. It is in the center of a nexus connecting the W-finger with the L3 globular domain, the peptidyltransferase center, the aa-tRNA accommodation corridor, and the SRL. Furthermore, it is proximal to the B5 intersubunit bridge, which involves multiple contacts involving bases in Helices 62 and 64 (54) .
Unlike the small subunit (SSU), where the four rRNA domains are largely physically distinct, the six rRNA domains of the LSU are highly intertwined (54) . With regard to the current study, the loop bounded by Helices 61-64 lie in domain IV, the PTC and Helices 90-93 and Helix 73 are in domain V, and Helices 94 and 95 are in domain IV. Previously, we demonstrated that the conformationally dynamic nature of the W-finger enables the central extension of L3 to function like a lever, and as such contribute to allosteric repositioning of rRNA structural elements (13, 17) . However, it was not clear how a small radial movement of thin, essentially planer element, could have such large and long ranging effects on rRNA structure and ribosome function. The perpendicular orientation of the basic thumb may answer this: we propose that it amplifies the action of this lever by adding three-dimensionality to the central extension in the form of a platform upon which structural elements from three different domains of 25S rRNA are anchored. In support of this, comparison of the L3 structures between EF-Tu and EF-G bound Thermus thermophilus ribosomes (55) reveals displacement of the a-carbon backbone of the W-finger and basic thumb structures by $2-3 Å , and of some individual sidechains by as much as 5 Å (Supplementary Figure S1) . This model explains how The large number of basically charged amino acids in the basic thumb enables it to participate in numerous hydrogen bonding/electrostatic interactions with rRNA bases, phosphates and riboses, functioning as a 'molecular clamp' to bridge these three domains. The current study focusing on eight amino acids in the L3 basic thumb neutralized their positive charges with alanine substitutions both singly and in selected pairs. The lethality of the R240A mutant as the sole form of L3 indicates that it may function to 'glue' the PTC proximal stem of H90 with the H61-H64 loop, and may well aid in coordinating formation of the C2876-G2922-G2951 Aminor motif that is critical for the 'induced fit' function of the peptidyltransferase center (5, 6) . Similarly, R247, which bridges Helix 61 with Helix 90 had profound effects on rRNA structure, ribosome biochemistry, translational fidelity, and cell growth, suggesting that it too is a critical bridging component between these two domains. Interestingly, the R247A/R248A double mutant was lethal, while R248A had a wild-type phenotype. This confirms the role of R247 and also indicates that the molecular defect conferred by the R247A single mutant might be partially complemented by the positively charged R248 adjacent to it. R240A and R247A appear to be the exception rather than the rule however, as the other single mutants had significantly lesser effects on these parameters. This is consistent with mutagenesis studies on other residues of L3, and with other LSU proteins and rRNA bases (11) (12) (13) (14) (15) 17, 23, (56) (57) , suggesting that the ribosome is an elegantly evolved molecular machine containing multiple levels of functional redundancy. Furthermore, all of the double mutants tested had strong effects on binding of aa-tRNA and eEF2, and on peptidyltransfer (Figure 3 ), suggesting that it multiple defects are generally required to disrupt the functionally redundant interactions between the basic thumb and the LSU rRNA. As an aside, the observation that A2926 (base paired to U2920) was protected from 1M7 modification in wild-type ribosomes when both Aand P-sites were occupied by tRNAs but deprotected when only the P-site was occupied ( Figure 4A ) is unique to yeast. To our knowledge, this has not been observed in E. coli ribosomes (51), which contains a G-C base pair (C2551-G2557) at this position, a conformational difference that suggests a potentially novel antibiotic target. Interestingly, Haloarcula marismortui appears to split the difference with a G-U base pair (U2576-G2582).
Some specific changes in rRNA structure are particularly telling. The base stack of aa-tRNA C74 with H. marismortui U2590 (E. coli U2555, yeast U2924) is thought to promote the induced fit of peptidyltransfer (6) . The enhanced deprotection of this base in R247A mutant ribosomes when both A-and P-sites were occupied by tRNAs is consistent with its strong effect on peptidyltransferase activity by this mutation (Figure 3 H) . Interestingly, this base was also strongly deprotected under the same conditions in K237A mutant ribosomes, but in this case peptidyltransferase activity as monitored using the puromycin reaction was actually enhanced ( Figure 3H ). The rates of peptidyltransfer Figure 4 . Continued arrowheads, and those deprotected relative to wild-type are indicated by black arrowheads. Bases marked in gray (A2926 and A2971) were deprotected when the A-site is unoccupied relative to when it contains aa-tRNA. (B) rRNA protection patterns of the L3 basic thumb mutants mapped onto the 2D diagram of 25S rRNA. Arrowheads indicate relatively protected and deprotected bases as above. Colored boxes indicate bases that interact with specified L3 basic thumb amino acid side chains. A2926 and A2971 are circled in gray, and C2925, which is the first gate in the aa-tRNA accommodation corridor, is circled in purple. The three bases participating the Type II A-minor motif that stabilizes the PTC are boxed and indicated. (C) Data from panels A and B mapped onto the 3D structure of the yeast ribosome. Indicated bases colored black correspond to bases deprotected in the mutants, while those colored gray are hyperprotected. Bases participating in the Type II A-minor motif (A m ) are colored purple. Helical structures and the PTC are color coded as indicated. Note that the loop formed between H61-H64 was removed from this figure because it obscures the L3 basic thumb. Table 2 . Homologous yeast 25S rRNA and E. coli 23S rRNA bases pertinent to this study observed in the current study ($0.3 min À1 ) and in similar analyses using E. coli ribosomes are significantly lower than naturally occurring rates [estimated to be >300 s À1 , (58) ] because Ac-Phe-tRNA is a poor substrate for this reaction. However, this property actually enables us to tease out the effects of local structural changes on PTC activity. Recent molecular dynamics simulations of portions of the ribosome reveal that individual bases can undergo a conservable degree of structural mobility due to local Brownian movements [reviewed in (59) ], suggesting that specific bases in the PTC are relatively free to assume either the induced or uninduced conformations in the absence of tRNAs, and that the equilibrium between these two states is influenced by the presence or absence of aa-tRNA in the A-site. Thus, we suggest that R247A mutant drives this equilibrium toward the uninduced conformation, while the K237A mutant favors the induced arrangement. The observation of distinctly different patterns of rRNA protection/ deprotection (e.g. compare G2912, C2929, U2953, G2977, C2985, A2995, G3003, A3006, G3009, U3019, U3023 and A3033 between the K237A and R247A mutants in Figure 4A ) is consistent with the idea that they also have opposing effects on PTC conformation and functionality. In addition, early studies demonstrated that while empty ribosomes are heterogeneous in their affinity for eEF2, consisting of two sub-populations having K d 's for eEF2 ranging from subnanomolar to hundreds of nanomoles (60), the affinity for eEF2 strongly depends on the functional status of the ribosome as determined by the occupancy status of the A-and P-sites (61, 62) . This suggests that the R247A and the double mutants that increased eEF2 affinity shift this equilibrium as well, possibly stabilizing ribosomes in the pre-translocation state, and that the interactions between the 25S rRNA bases and L3 amino acid residues investigated here are involved in transitions between the pre-and post-translocational states.
As discussed above, the effects of the K237A mutant are locally confined to the PTC, while those conferred by R247A are more global. The latter is reflected in their different effects on ligand binding to the A-site: R247A promoted very strong effects on both aa-tRNA and eEF2 binding as compared to the much weaker effects conferred by K237A. Examination of the ligand binding data reveals a reciprocal relationship between affinities for aa-tRNA and eEF2, i.e. increased aa-tRNA K d correlates with decreased eEF2 K d (Figure 3 ). This is consistent with the model that L3 plays a central role as an allosteric switch to coordinate binding of elongation factors, opening and closing of the accommodation corridor, and PTC activity to ensure the unidirectionality of protein synthesis (13, 17) . Interestingly, all of the mutants affected peptidyltransfer, consistent with observations that this process is highly sensitive to even minor structural changes in the ribosome [reviewed in (63) ].
In the end, the most important parameter is life; i.e. how does the L3 basic thumb contribute to the fitness of the organism? While only some of the mutants had gross effects on cell growth, they all affected translational fidelity as monitored by decreased rates of À1 PRF (Figure 2 ). In fact, the effects on À1 PRF correlated well with the A-site aa-tRNA-binding data. For example, R247A and K241A/R244A, which had the most pronounced effects on À1 PRF, also promoted >3-fold decreases in affinity for aa-tRNA. In contrast, R232A and K236A, which had the smallest effects on aa-tRNA binding, also promoted the smallest decreases in À1 PRF. As noted above, the mutants investigated in this report are unique in that they are the first examples that promoted decreased À1 PRF. This trend had only previously been observed with anisomycin, a competitive inhibitor for aa-tRNA 3 0 binding to the PTC (41) . We have suggested that the majority of À1 PRF occurs after aa-tRNA accommodation into the A-site, and prior to peptidyltransfer, while a smaller fraction can occur during translocation [reviewed in (64) ], a view that is supported by a recent study coupling kinetic modeling of À1 PRF within the translation elongation cycle with mass spectroscopic analyses of frameshifted peptide products (P.-Y. Liao et al., submitted for publication). By this model, decreased rates of aa-tRNA accommodation into the A-site should decrease the steady state abundance of substrate for À1 PRF, thus inhibiting this reaction. Changes in À1 PRF in turn alter the relative amounts of viral protein products available for viral particle self assembly, a ratio that is critical for virus propagation (18, 40) . This illustrates how minute changes in ribosome structure at the atomic scale can propagate outward, affecting ribosome biochemistry, translational fidelity, and the ability of cells to replicate viruses. Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes. Microbial genomes can be highly variable because of high mutation rates. Because of this extreme variability, it is often difficult to identify regions within a specific virus genome that are sufficiently evolutionarily conserved to serve as targets for specific detection primers and probes. RNA viruses are especially variable. The influenza virus, a negative sense, single-stranded RNA (ssRNA) virus with a highly variable RNA genome, for example, has been known to cause the diagnostic problem that is at the basis of this article, because of a high rate of mutation and genetic drift. In such situations, optimal detection primers and probes would be broadly targeted yet specific, and remain functional even if the genome sequence changed because of genetic drift.
Diagnostic nucleic acid hybridization probes are constructed from the most conserved portions of genes from viruses commonly causing infection. Long probes have a large inherent tolerance to microbial variation. The introduction into the introduction into the probe design of a base that can hybridize with all four normal bases (a universal base), or of multiple nucleotides (degenerations; wobbles) in a single position, can induce tolerance to natural viral variation (mismatch).
The naturally occurring (1-4) nucleotide (nt) deoxyribose-Inosine (dInosine) is one of many more or less generally hybridizing nt (5,6) known as universal bases. All four normally occurring DNA bases can hybridize to dInosine. The general trend in decreasing hybridization stability is I:C > I:A > I:T & I:G > I:I when using 1 M NaCl, 10 mM sodium cacodylate and 0.5 mM EDTA pH 7 (7, 8) . However, dInosine is readily available and can be recognized as a G by polymerases (5, 9, 10) . Alternative universal bases, e.g. 5-nitroindole, also exist.
3 M tetramethylammonium chloride buffer (TMAC) is a hybridization buffer that selectively raises the stability of A:T base pairs to approximately that of G:C base pairs (11) (12) (13) (14) . It was used in these studies to reduce the effect of sequence composition when comparing different probes of the same length. The term nucleation site is used in this article to indicate a stretch of contiguous perfectly matching nt, capable of initiating hybridization (15) .
The aim of these studies was to improve understanding of the design of probes to be used in a TMAC buffer system, by investigating variability and the inclusion of dInosines, other universal bases and wobbles. Specifically, we examined (i) variation (i.e. mismatch) tolerance, (ii) sensitivity to different mismatch distributions, (iii) utilization of dInosine as an nt analog, and (iv) specificity; we also present a new algorithm for prediction of hybridization results. In additional experiments, the question of the use of degeneracy versus a universal base was addressed. Furthermore, we investigated the use of the derived design criteria for detection of rotavirus RNA in clinical samples.
A 70-mer nucleic acid hybridization probe, named the InflA probe, was constructed from the most conserved portion of the matrix gene in segment 7 of the Influenza A H3N2 virus. Properties important for the design of variation (mismatch)-tolerant yet specific probes were investigated by studying the interaction between a set of virus-derived probes and complementary targets with different degrees and distributions of mismatch.
The 70-mer DNA probes were coupled to color-coded microspheres, hybridized with biotinylated target nucleic acids, incubated with streptavidin-phycoerythrin and analyzed in a Luminex Õ 200 TM system. The hybridization reaction was performed at a standard non-saturating concentration (0.2 nM target) in 3 M TMAC buffer.
In further experiments, in an attempt to make a probe with an extended mismatch tolerance, a series of dInosinecontaining probes were synthesized and analyzed for hybridization in the 3 M TMAC buffer system. A limited number of experiments comparing dInosine with an alternative universal base (5-nitroindole) or with wobbles (degenerations) were also conducted.
Many viruses harbor synonymous mutations (sm), which means that the third base in a codon can wobble without changing the amino acid in the protein. Targets with regions where every third base was mutated, to resemble the common phenomenon of sm (i.e. the nt sequence is varied without affecting the coding for a specific amino-acid code), were of special interest, since this is often the cause of the variation in coding viral sequences.
Rotavirus is a dsRNA virus that causes gastroenteritis. Clinical fecal samples, previously confirmed to contain rotavirus, were used to test the design strategies of this report. An asymmetric PCR, with biotinylated rev-primer in excess, was set up for the VP6 segment. The primers were designed using knowledge gained from this report, i.e. both degeneration and dInosine were used to create moderately degenerated probes with uninterrupted matching stretches as long as possible, in the most conserved regions. The biotinylated reverse (rev-) primer was made 77-nt long to be as tolerant to variation as possible, while the shorter forward (fw-) primer (23 nt) contained four locked nucleic acids (LNA) to increase the hybridization strength (represented by melting temperature, T m ) to match that of the rev-primer.
Single stranded 70-mer oligonucleotides with a C12 aminolink at the 5 0 end, with or without dInosines/ 5 0 -nitroindole/N wobbles, were obtained from Biomers.net (Ulm, Germany). The design of the probes was based on the programs BLASTn (16) , ClustalX (17) and ConSort ß (J.Blomberg et al., unpublished results). Briefly, matching viral sequences were retrieved from the GenBank database at NCBI, NIH, using the viral sequence of interest as a query, and alignments were performed by a BLASTn search. BLASTn and ClustalX alignments were analyzed in ConSort ß to define the most suitable probe sequence. ConSort ß provides the frequency of variation and the variation of nt composition in each base position, the number of aligned sequences, and a majority consensus sequence. The proposed probe sequence was further analyzed for its predicted Tm and probable homodimer and hairpin interactions using Mfold at the IDT OligoAnalyser site (http://eu.idtdna .com/analyzer/Applications/OligoAnalyzer/)
[http:// mfold.bioinfo.rpi.edu/ (18) ] and Visual OMP TM 7.0 (DNA Software). Visual OMP TM 7.0, which uses the Nearest Neighbor (NN) algorithm [DNA Software (19) ], was used to estimate the change in Gibb's free energy associated with hybridization of the two strands (ÁG) for the interaction between probes and targets at 45 and 55 C in 3M TMAC. IDT OligoAnalyser was used to estimate ÁG for the interaction between probes and targets using 50-mM Na + and 2-mM Mg 2+ . Each combination was tested with the heterodimer-formation function (http://eu.idtdna.com/analyzer/Applications/ OligoAnalyzer/).
To create the 70-nt InflA probes, nt 725-794 of the matrix protein 2 gene segment 7 of Influenza A H3N2, accession nr CY023083, was used as query in BLASTn (GenBank database at NCBI, NIH). The Norovirus probe sequence comprises nt 646-715 of the capsid gene of the Norwalk-like virus, accession nr AY274264. The probes for detection of rotavirus were made using sequence EU372725 of the Human rotavirus A strain CMH171/01 inner capsid protein (VP6) gene as query. The probe region chosen, nt 112-178, was analyzed using the haplotype function of ConSort ß . Two differently degenerated probes containing dInosines were designed from each of the two major haplotypes, which potentially covered all rotavirus group A variations recorded in GenBank.
Synthetic targets, complementary to the consensus sequence of the InflA, Norovirus and Rotavirus probes, with various numbers of mismatches, were purchased as 70-mer oligonucleotides with biotin attached to a 2-aminoethoxy-ethoxyethanol linker at the 5 0 end (Biomers.net, Ulm, Germany).
Specific synthetic 5 0 amine-C12 modified 70-mer probes for influenza A were designed and solid-phase coupled to xMAP carboxylated color-coded microspheres (Luminex Corp., Austin TX, USA), according to the protocol of the Luminex corporation (Austin TX, USA). Briefly, 2.5 Â 10 6 stock microspheres were collected by centrifugation and resuspended in 25 ml of 0.1 M MES, pH 4.5 (2N-morpholino-ethanesulfonic acid, Sigma). Subsequently, 0.2 nmol of the probe and freshly made 2 ml 10 mg/ml EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (water-soluble carbodiimide; Pierce; sold by Nordic Biolabs AB, Sweden)] in H 2 O were added to the microspheres and the suspension was incubated in the dark for 30 min at room temperature. Care was taken to store the EDC in a dry condition, in aliquots. After addition of another 2 ml (10 mg/ml) EDC in H 2 O and repeated incubation, the microspheres were washed with 0.5 ml of 0.02% Tween-20. The coupled microspheres were pelleted by centrifugation at 8000 Â g for 2 min and resuspended in 0.5 ml of 0.1% SDS. After a second spin at 8000 Â g for 2 min, the final pellet was resuspended in 50 ml of TE, pH 8.0.
An amount of 5 ml of 2.0 nM synthetic biotin-labeled target was mixed with hybridization buffer consisting of 33 ml 3 M TMAC buffer (3 M tetramethylammonium chloride, 0.1% Sarkosyl, 50 mM Tris-HCl, pH 8.0, 4 mM EDTA, pH 8.0; Sigma) and 12 ml 1Â TE-buffer pH 8.0 and 0.05 ml ($2500 microspheres) for each probe-coupled Luminex microsphere. The mixture was heated at 95 C for 2 min to denature the DNA targets and probes, followed by hybridization at 45 or 55 C for 30 min while shaking on the Thermostar (BMG LabTech; Offenburg, Germany) microplate incubator. An amount of 2 ml (0.05 mg/ml) of streptavidin-R-phycoerythrin (QIAGEN, Hilden, Germany) was added to the mixture, which was further incubated at 45 or 55 C for 15 min before analysis for internal microsphere and R-phycoerythrin reporter fluorescence on the Luminex Õ 200 TM system (Luminex corporation, Austin, TX). The amount of biotinylated target that hybridizes to the microsphere-bound probes is directly proportional to the Median Fluorescence Intensity (MFI) reported by the instrument (in all experiments, fluorescence was measured from a minimum number of each type of microsphere: a set of 100 beads). The term total MFI describes the hybridization signal from a perfectly matching probetarget duplex, while the percentage (%) of the total MFI describes the ratio between the hybridization signal of a mismatching probe-target duplex and the total MFI of that particular probe. Titration experiments established that $60% of the maximum hybridization capacity of the microsphere-bound probes was reached using the conditions under which total MFI was measured (i.e. microsphere-bound probes were not saturated). An MFI of 100 was used as the lower limit of detection (LLOD).
Fecal samples were obtained from children with gastroenteritis, from the Children's Hospital ward at Uppsala Academic Hospital. Samples were handled anonymously according to the rules of the ethical committee at the Academic Hospital. The study used samples that were positive in a rotavirus antigen detection test; 100 ml of the sample was diluted in 900 ml 1Â TE buffer. After centrifugation, 400 ml was added to a lysis buffer and total nucleic acid was extracted as described by the manufacturer (easyMag Õ , bioMe´rieux). The samples were eluted in 110 ml buffer and stored at À70 C.
A reverse transcriptase (RT)-PCR was set up to amplify and biotin-label the nucleic acid of human rotavirus A from these clinical samples.
The 545 sequences obtained from the rotavirus query in BLASTn were analyzed in the ConSort ß program to construct fw-and rev-primers. The fw-primer (nt 1-23), 5 0 -G GCTTTW+AAA+CGAA+GTC+TTCR-3 0 (+A, +C, +G, +T are LNA residues) and the biotinylated rev-primer (nt 502-426), 5 0 -TATGGAAATATATTAGG TTTATGAAAAACAAATCCIGTACGTTGTCTTCT ITTITGIARRTTCCAITTITCIATRTA-3 0 , resulted in a PCR product of 502 nt. After nucleic acid extraction in the easyMag Õ , the extracts were heated at 97 C for 5 min, followed by snap cooling on ice for 2 min to obtain ssRNA from the rotaviral dsRNA. The PCR reaction contained 5 ml of nucleic acid extract, 25 ml 2Â RT-PCR iScript buffer, 200-nM Fw-primer, 600-nM biotinylated rev-primer, nuclease-free water and 1 ml iScript RT enzyme (total volume 50 ml). The samples were run at 50 C for 30 min, 94 C for 10 min, 50 cycles at 94 C for 30 s, 55 C for 30 s and 72 C for 30 s, ending with 72 C for 7 min. An amount of 5 ml of the PCR product was used in the hybridization experiment with the microsphere-coupled Rotavirus probes (sequences shown in Supplementary Table S4A and B. The PCR products were sequenced with a 3130 Genetic Analyzer (Applied Biosystem) by utilizing the same fw-and rev-primers described above.
The discovery of the importance of long uninterrupted perfect matches and the long-range effects of mismatch (see 'Results' section), which are not embodied in current NN hybridization theories (19) , led us to formulate a simple new descriptive theory. The new algorithm includes aspects of NN theory (8, (20) (21) (22) but extends this to longer hybridizing segments and includes the effects of dInosine in the long oligonucleotides. Visual Omp TM 7.0 predicted that some 70-mer combinations would not hybridize, yet they did hybridize. In order to better predict hybridization, we investigated predictive strategies that took into account the matching nt, its neighbors, the length of the matching region, and the cooperativity of neighboring matching regions. We finally settled for an algorithm which attempts to model the nucleation and zipping stages during the hybridization process. The new model was termed NucZip ( Figure 5B ). The results obtained using this model was then correlated with those obtained using Visual OMP TM 7.0 and both were compared with experimental data.
NucZip simulates the hybridization process, starting with potential nucleation sites and then proceeding to 'zip' in both directions. The NucZip algorithm (written by JB in Visual FoxPro) was implemented as a module (procedure) in the ConSort ß sequence analysis program. The procedure is relatively simplistic, and does not contain the thermodynamic and secondary structure analyses provided by more sophisticated programs such as Visual OMP TM 7.0.
The algorithm starts by searching for perfectly matching hexa-, hepta-, octa-and nonamers, as potential nucleation sites (the 'Nuc' part). Every matching oligomer is given the number of matching nt as its NucScore. If the oligomer contains dInosine, the number of dInosines is subtracted from the score but the segment is still counted as uninterrupted, regardless of the dInosines. The NucScore is then used to select the two highest scoring potential nucleation sites which will undergoing zipping, up and downstream.
The score for the 'Zip' portion of the model is obtained from the number of consecutive matching trimers, tetramers etc, up to pentadecamers, each counted with equal weight, within a contiguous matching segment. Thus, ZipScore modex = AE k = 1 k = kmax (AE n = 3 n = 15 S n ), where kmax is the number of uninterrupted matching segments, including the chosen nucleation site, and S n is the number of successive segments of length n (varying from 3-15), i.e. the number of full length trimers, full length tetramers etc. up to full length pentadecamers, which fit into the matching segment. The same Zip scoring system was performed in two modes, counting dInosines either as matching (mode 1) or as mismatching (mode 2). In the second mode, dInosines will shorten the length of matching segments, decreasing the score. The final ZipScore was calculated as a weighted mean of ZipScore mode1 and ZipScore mode2 , where the weighting factor was based on the empirical data presented in the current report (Figures 2-4) . Since dInosine hybridizes more strongly to C than to the other nts, the algorithm adds a contribution based on the number of dI:C pairs weighted by an InoCfactor. The upstream and downstream ZipScores were obtained as: ZipScore downstream =ZipScore mode1 -dInosinefactor (ZipScore mode1 -ZipScore mode2 )+(dInoCnr * dInoCfactor) and ZipScore upstream = ZipScore mode1 -dInosinefactor (ZipScore mode1 -ZipScore mode2 )+(dInoCnr * dInoCfactor).
The ZipScores from up-and downstream zipping were then added; the final NucZipScore = ZipScore downstream+ZipScore upstream.
In this way, the contribution from longer matching segments was factored in with the contribution from the nearest neighbors (approximated by the trimer part of the algorithm). Figure 8 summarizes the principle of this computational work.
The probability of a match in a degenerated nt position is approximately predicted from the ConSortß analyses of target sequences. The behavior of the relatively few oligonucleotides with degeneration that were tested is approximately in line with NucZip reasoning, which places a premium on long uninterrupted matching stretches. However, a larger number of degenerated probes need to be analyzed before the contribution of probabilities of contiguous stretches extending through degenerated positions can be estimated and included into NucZip. The programming code is included in Supplementary Data.
ConSortß was used to demonstrate the variation in the nt sequence of segment 7 from 7333 genomes of Influenza A (GeneBank database at NCBI, NIH). All H and N influenza A types, HxNy, are represented in the alignment ( Figure 1 ). In comparison with the InflA probe designed to match a 70-nt region of the Influenza A H3N2 virus, the H5N1 virus differed in 5-nt positions, and the H1N1 virus in three other nt positions (Table 1) . Thus, if a detection probe could tolerate mismatches in nine positions, including the variant nt positions of the H1N1 and the H5N1 viruses, it would fully cover 67 different H and N combinations of Influenza A, i.e. nearly all recorded variants in the chosen region, as demonstrated in the BLASTn search.
The InflA probe was tested against 70 nt target molecules with 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 and 21 point mutations (pm) and 21 grouped mutations (gm) (Figure 2A -D; nt sequences of probes and targets can be found in Supplementary Tables S1A and B). The positions of the pm were based on the variations found by comparing H3N2, H5N1 and H1N1 viruses. Two targets had the same number of mutations, but different distributions: the 21 pm target had 21 evenly distributed pm, and the 21-gm target had seven groups of three mutations interspersed by 5 to 7 conserved nt. The InflA probe, coupled to Luminex microspheres, was allowed to hybridize with one of the biotinylated ssDNA targets in 3 M TMAC at two different temperatures: 45 C ( Figure 2A ) and 55 C ( Figure 2B ). The hybridization, given as MFI, was analyzed in the Luminex flow meter.
Introducing an increasing number of evenly distributed pm in the target had a negative effect on hybridization, as reflected in decreasing MFI; see Figure 2A 12-nt, one 7-nt and four 5-nt matching regions) still hybridized at 68% (MFI 4496, 45 C, Figure 2A ) and 71% (MFI 4001, 55 C, Figure 2B ) of the total MFI (i.e. of the MFI of the perfectly matching InflA target/InflA probe: MFI 6604, 45 C, Figure 2A ; and MFI 5657, 55 C, Figure 2B ) while the 15 pm target (containing one 6 nt, four 5 nt, and two 4 nt matching regions) hybridized at 13% (MFI 854, 45 C) and 1% (MFI 54, 55 C) of the total MFI. The InflA probe failed to hybridize with the 16-pm target (containing one 6-nt, three 5-nt, and two 4-nt matching regions) at either temperature, providing MFI values that were 6% (MFI 403, 45 C) and 0.5% (MFI 29, 55 C) of the total MFI.
The longest perfectly matching sequence between the mismatches in the InflA probe/21-pm target combination was 3 nt in length. No hybridization was detected at either temperature. In contrast, for the InflA probe/21-gm target combination, where the distribution of the 21 mutations created seven stretches of 5-7 perfectly matching nt between the mismatches, hybridization with the InflA probe was restored (Figure 2A-D) . The MFI signal increased to 20 and 30% (Figure 2A Table 1 sequence of H3N2). The number of dInosines included in the probes is indicated in the names Ino3-Ino21. Similarly, the wobbN_21 probe contains 21 N wobbles in the same position as the dInosines in the Ino21 probe. The complementary target of the InflA probe is named the InflA target. These targets have 0-21 pm, as indicated by the names; the 21-gm target has seven groups of three mismatches ( Figure 2D ). All target molecules were biotinylated at their 5 0 end. Each sample contained 0.2 nM of one of the synthetic biotinylated Continued and 7 and 8% ( Figure 2B and C, 55 C) of the total MFI, when the InflA probe hybridized with the 21-gm target instead of the 21-pm target.
In conclusion, a target with one to nine evenly distributed mismatches, preserving multiple contiguous matching stretches of at least 5 nt, has little reducing effect on hybridization with a 70-mer probe, while a target with >14 evenly distributed pm hybridizes inefficiently or not at all. Hybridization can be improved by utilizing a less stringent (lower) hybridization temperature. Furthermore, grouped mismatches tend to strengthen the hybridization compared to evenly distributed mismatches. Consequently, for hybridization between 70-mer strands with 10-20 mismatches, the distribution of the mismatching nt affects the hybridization more than the number of mismatches, indicating that the length and number of perfectly matching stretches are of greatest importance.
A panel of five 70-mer probes, Ino3-21, containing 3, 5, 7, 9 or 21 dInosines, was designed based on the InflA probe sequence. The dInosines were placed to match the positions of the pm in the above pm targets ( Figure 2D ).
Introduction of 3-9 dInosines in the probe resulted in only a small reduction in MFI when binding to the InflA target; i.e. the MFI signal decreased in the order InflA probe & Ino3 > Ino5 > Ino7 > Ino9. The Ino9 probe hybridized with the InflA target by as much as 76% (MFI 4970, 45 C) and 59% (MFI 3340, 55 C) of the total MFI, comparable with the hybridization of the InflA probe to the 9-pm target. In fact, all the Ino3-9 probes hybridized as efficiently with targets that had up to the same number of pm as the number of dInosines, including two mismatches not covered by dInosines, as they did with the InflA target. When the Ino probes hybridized with targets containing more than 12 pm, the probes with many dInosines worked better than the InflA probe; e.g. the Ino7 and Ino9 probes resulted in a signal 1.2-1.9 times higher than that for the InflA probe for targets with 13-16 pm at 45 C.
Interestingly, even the Ino21 probe hybridized quite strongly with the InflA target, at 26 and 39% (Figure 2A and C, 45 C) and 11 and 13% ( Figure 2B and C, 55 C) of the total MFI. Importantly, the Ino21 probe was able to restore hybridization with the 21 pm target (11 and 37% of total MFI) to which the InflA probe had totally failed to bind (Figure 2A and C, 45 C). The Ino21 probe hybridized with all the matching 3-21 pm targets with almost the same efficiency (mean 29.5% of the total MFI, 1946 MFI, at 45 C, Figure 2A; and 8% of the total MFI, 435 MFI, at 55 C, Figure 2B ), which is in the same range as the hybridization of the InflA probe with the 21-gm target (30.4% of the total MFI, 2005 MFI, 45 C, Figure 2A ; and 7.8% of the total MFI, 444 MFI, 55 C, Figure 2B ). The Ino21 probe and 21 gm target combination had 13 mismatches outside the dInosine positions and failed to hybridize (Figure 2A , B and C). In conclusion, the presence of dInosine in the probe decreased hybridization with a perfectly matching target, but a dInosine-containing probe bound more strongly than a dInosine-free probe when the target had many mismatches juxtaposed to the dInosine residues.
A minimum length of perfectly matching nt sequences is required for hybridization
The importance of uninterrupted matching regions was analyzed by making targets with different lengths of perfectly matching sequences at the 5 0 end or at both the 5 0 and 3 0 ends, in combination with a long region of 26, 33 or 74% randomly distributed mutations ( Figure 3A , B and D, nt sequences in Supplementary Table 2A and B) .
As expected, hybridization of the InflA probe to targets with 26% random mutations in a region between two flanking regions of 5 (26%5F), 7 (26%7F), 9 (26%9F) or 15 (26%15F) perfectly matching nt showed that shorter perfectly matching flanking regions reduced the MFI. All the 26% targets hybridized with the InflA probe at 45 C but, at 55 C, the 26%5F target failed to hybridize (5.6% of the total MFI, 271 MFI). Like the 33%12F and the 16-pm target, the 26%5F target contained 16 mutations (Figures 2A, B, 3A and B). The 33%12F target (45% of the total MFI, Figure 3A ), with its two regions of 12 uninterrupted nt, hybridized much more strongly than the 26%5F (27% of the total MFI) or 16-pm (6.1% of the total MFI) targets, which both contained several shorter matching regions, of 6, 5, 4 and 3 nt ( Table 2) . The same effect, i.e. that a few long perfectly matching regions result in better hybridization than several shorter regions, was also seen with the three targets that had 14 mismatches: the 26%9F, the 33%15F and the 14-pm targets at both temperatures ( Table 2 ). The 74%9F target, with only 12 matching nt dispersed in the central region, did not hybridize to the InflA probe (1.4% of the total MFI, 86 MFI, 45 C; and 0.6% of the total MFI, 30 MFI, 55 C), while the 26%9F target, with 38 matching nt (three regions of 5 nt and one region of 6 nt), did hybridize (44% of the total MFI, 2583 MFI, 45 C; and 24% of the total MFI, 1125 MFI, 55 C). This shows that the two flanking regions of nine matching nt did not cause hybridization alone. Furthermore, the 74%12F, with 11 matching nt dispersed in the central These experiments, taken in conjunction with the InflA probe hybridizing to the 21-gm but not to the 21-pm target, confirm that both the number of mismatches and their distribution are important. It is reasonable to assume that perfectly matching sequences of a minimum length function as nucleation sites (15) which initiate hybridization between the probe and the target.
Hybridization of the InflA probe with targets containing 74% mismatching 70-mers with one perfectly matching end of various lengths (74%xnt) was compared with hybridization of the InflA probe with short, perfectly matching targets (12-22 nt_free; Figure 3A , B and D; nt sequences in Supplementary Table S2B ) to analyze the effect of long mismatching ends. Utilizing the InflA probe / InflA target as reference, the 12nt_free target did not hybridize at 45 C (1.8% of the total MFI, 106 MFI) but the 15-, 18-and 22-nt_free targets gave successively stronger MFI signals (62, 98 and 123% of the total MFI; 3669, 5770 and 7269 MFI, respectively) than the 74%15nt, 74%18nt and 74%22nt targets (17.4, 55 and 62% of the total MFI; 1029, 3222 and 3637 MFI, respectively). Thus, the MFI is higher when the short, perfectly matching targets (xnt_free) hybridize with the InflA probe with only one long end protruding from the hybridized portion of the probe. Previous reports have demonstrated that 1-5 nt single dangling ends tend to stabilize duplex formation (23) (24) (25) (26) . This study shows that two long mismatching ends destabilize the hybridization of the matching part of the duplex. The long free mismatching ends could form intramolecular secondary structures that could have an effect on the hybridizing duplex. 
% MFI (InflA probe against target/InflA probe against InflA target).
Alternatively, the Brownian movements of the two long non-hybridized sections could mechanically stress the remaining base pairs.
The tolerance of the probe against sm was tested using targets (70 nt) with every third nt harboring an sm (referring to the reading frame of the matrix 2 protein of the H3N2 Influenza A) in regions of different length. The 33%9F, 33%12F and 33%15F targets have a region containing 18, 16, or 14 sm between two flanking regions of 9, 12, or 15 perfectly matching nt, respectively. The 33%9nt, 33%12nt and 33%15nt targets have a region of 20, 19 or 18 sm in combination with one region of 9, 12 and 15 perfectly matching nt at the 5 0 end ( Figure 3D , nt sequences in Supplementary  Table 2A and B) . As demonstrated in Figure 3A and B (and confirmed in Figure 3C ), the targets 33%9nt and 33%9F failed to hybridize with the InflA probe, while the 33%15F target hybridized at both hybridization temperatures (60% of the total MFI, 3517 MFI, at 45 C; 39% of the total MFI, 1934 MFI, at 55 C). The 33%12F target with its two 12 nt flanking regions only hybridized at 45 C (45% of the total MFI, 2635 MFI). The 33%15nt, with one contiguous region of 15 perfectly matching nt also hybridized only at 45 C (23% of the total MFI, 1352 MFI; MFI result taken from Figure 3A and B). Thus, for a 70-mer probe to hybridize with a target containing a relatively long stretch of sm, it must have (i) one uninterrupted perfectly matching region of at least 15 nt at 45 C, and longer than 15 nt at 55 C, or (ii) two uninterrupted matching regions of at least 12 nt at 45 C or 15 nt at 55 C.
dInosine-containing probes restore hybridization to targets containing sm
The targets containing sm were further tested against a set of dInosine-containing probes: one probe (the Ino18 probe) contained 18 dInosines matching the sm in the 33%9F target, two probes (21Ino_9nt5 0 and 21Ino_9nt3 0 ) contained 21 dInosines positioned as sm leaving a region of matching 9 nt at either the 5 0 or 3 0 end; and one probe (Ino24) contained 24 dInosines in every third base throughout the whole 70-mer probe.
Importantly, when dInosines matching the positions of the sm in the targets were included in the probes, the Ino18 probe hybridized with all the sm-containing targets, although with slightly varying MFI ( Figure 3A , B and confirmed in C). The 33%9F target, which did not hybridize with the InflA probe, was able to hybridize with the Ino18 probe (66% of the total MFI, 3894 MFI at 45 C, Figure 3A ; 55% of the total MFI, 2808 MFI at 55 C, Figure 3B ). Even the 33%9-15nt targets, with two mismatching nt at the 3 0 end not covered by dInosines, hybridized well with the Ino18 probe (48-51% of the total MFI, 2818-2994 MFI at 45 C, Figure 3A ; and 16-32% of the total MFI, 778-1463 MFI at 55 C, Figure 3B) . Thus, as shown in the previous series of pm matched to dInosine, when the sequence is interrupted too frequently by mismatches, leaving no suitable regions of perfect match, a probe with dInosines at the positions of variation will restore the hybridization by effectively creating the required longer matching region.
Introducing a dInosine in every third position throughout the Ino24 probe decreased the hybridization dramatically for all the 33% targets (6-16% of the total MFI, 361-920 MFI at 45 C; 1% of the total MFI, 53-79 MFI at 55 C). When utilizing the 21Ino_9nt5 0 probe with the 33%xnt targets, the MFI decreased because of the mismatches created by the two sm at the 3 0 end of the target (4-10% of the total MFI, 232-415 MFI at 45 C). In comparison, the 21Ino_9nt5 0 probe and the 33%xF targets, with no mismatch, hybridized at 26-43% of the total MFI (1692-2898 MFI at 45 C). Furthermore, the 21Ino_9nt3 0 , which did cover the two sm at the 3 0 end of 33%xnt, hybridized more strongly (10-21% of total the MFI, 450-695 MFI at 45 C) than the 21Ino_9nt5 0 probe (4-10% of the total MFI, 232-415 MFI at 45 C). Both these results demonstrate that using the less stringent temperature (45 C) permits hybridization even when a large number of Inosines is present (21 dInosines in a 70-mer probe), as long as a matching region of at least 9 nt is formed in the hybrid.
The hybridization of a long probe containing dInosines is comparable with that of a long degenerated probe with the same number of N wobbles, under lower stringency conditions
The effects of probes with dInosine or wobbles in the same positions were also investigated in 3M TMAC. The presence of a dInosine in a specific position instead of a wobble would theoretically decrease the degeneration of the probe and subsequently increase the concentration of the particular probe variant. A probe with 21 N wobbles, wobbN_21, at the same positions as the dInosines in the Ino21 probe, was tested. The surprising result was that the probe containing N wobbles hybridized very well with the InflA target (29% of the total MFI, 2220 MFI, Figure 2C ) and the 21-pm target (29% of the total MFI, 2190 MFI, Figure 2C ). This is in the same range as hybridization of the Ino21 probe with the InflA (26% of the total MFI, 1976 MFI, Figure 2C ) and 21-pm (11% of the total MFI, 794 MFI, Figure 2C ) targets (39 and 37% of the total MFI versus the InflA and the 21-pm targets, see Figure 2A ). These results also demonstrate that the wobb_N21 probe is not affected to the same extent as the Ino21 probe by increasing the hybridization temperature from 45 to 55 C ( Figure 2C ). The test was repeated by comparing an Ino18 with a wobbN_18 probe ( Figure 3C and D) . At 45 C, the Ino18 probe hybridized at least as well as the wobbN_18 probe while, at 55 C, the wobbN_18 probe hybridized better than the Ino18 probe. Interestingly, a probe containing 24 wobbles still hybridized better with all 33%xnt and 33%xF targets (30-45% of the total MFI at 45 C; 13-22% of total MFI at 55 C; Figure 3C ) compared with Ino24 (6-16% of the total MFI at 45 C; 1% of the total MFI at 55 C; Figure 3A and B). Obviously, the 70-mer probes can accommodate multiple degenerate positions and still hybridize because the majority of probe molecules will contain several long perfectly matching stretches created by chance. This is further deliberated under Discussion section.
The hybridization of a long probe containing dInosines is stronger than that of a long probe containing the same amount of 5-Nitroindole, at either high or low temperatures 5-Nitroindole is a second-generation universal base nt analog that was chosen for comparison with the first-generation dInosine with respect to hybridization properties in 3M TMAC. According to Loakes and Brown (1994) , 5-nitroindole is less destabilizing than its 4-and 6-isomers (27) and than 3-nitropyrrole (9). A probe with 18 5-nitroindole residues (5-NitroInd_18) was designed; the nt analogs were distributed to match the pattern of the dInosines in the Ino18 probe ( Figure 3D ). The probes were allowed to hybridize with the InflA target and the set of targets with sm, 33%_xF and 33%_xnt ( Figure 3C ). At 45 C, hybridization of 5-NitroInd_18 with the InflA (44% of the total MFI), 33%_xF (23-37% of the total MFI), and 33%_xnt (4-7% of the total MFI) targets resulted in hybridization signals that were much lower than those seen with the Ino18/InflA (73% of the total MFI), Ino18/33%_xF (54-78% of the total MFI), and Ino18/33%_xnt (47-59% of the total MFI) probes ( Figure 3C, 45 C) . Increasing the temperature to 55 C destabilized the 5-NitroInd_18 probe even more, resulting in hybridization of only 1-8% of the total MFI. In conclusion, dInosine functions much better than 5-nitroindole as a universal nt analog, under 3 M TMAC buffer conditions.
Hybridization of a probe containing dInosine is sensitive to mismatches neighboring the dInosine position, aiding specificity It has been shown above that when the dInosine and the mismatch have the same distribution pattern, i.e. the dInosine masks the mismatch, hybridization can be restored (Figures 2A, B, 3A and B). We analyzed how many mismatches outside the rescuing position of dInosine (mismatch outside dInosine; mmoi) a probe can tolerate. Norovirus, Ino18 and InflA probes were tested against a set of targets whose sequences were designed to range successively from a Norovirus sequence to the InflA sequence, allowing different amounts of mismatch and mmoi to be analyzed. Norovirus is a highly variable, positive-sense RNA virus belonging to the Caliciviridae, which causes 'winter vomiting disease'. The Norovirus sequence chosen (the capsid gene of the Norwalk-like virus, accession nr AY274264), after Blastn with the InflA probe sequence, has a short region of 8 nt that perfectly matches the end region of the InflA probe and has 10 dispersed matching nt ( Figure 4A ). The Norovirus target (70) 0_36_52 (0.8) (this code is explained below, and in the legend to Figure 4 ) was gradually changed to resemble the InflA target (0.8) 51_0_0 (70) by altering the central nt sequence. A set of targets was also created where the nine nt at the 5 0 end were changed into an InflA sequence and the central region was gradually changed from the Norovirus to the Influenza sequence, starting with (0.61) 9_27_42 (9.8). The targets were named according to the number of matching and mismatching nt in comparison with the three probes: (nt matching those of the Norovirus probe at 5 0 and 3 0 ) mismatching nt versus the Norovirus probe_Ino18 probe_InflA probe (nt matching those of the InflA probe at 5 0 and 3 0 ) (see Figure 4A ; nt sequences in Supplementary Table S3A and B) .
Two targets had 26 mismatches, with different dispersion patterns, after hybridization with the Norovirus or InflA probes; the distributions are shown in the (10.10) 26_10_26 (0.8) and (0.11) 26_10_26 (9.8) targets in the upper and lower panels of Figure 4A . The InflA probe did not hybridize with either of them, while the Noro probe hybridized weakly with both: 13% of the total MFI for the (0.11) 26_10_26 (9.8) target and 20% of the total MFI for the (10.10) 26_10_26 (0.8) target. The% MFI for the Noro probe was calculated by comparing the MFI with that of the Noro probe/Noro target hybridization. The stronger hybridization to the Noro probe than to the InflA probe can be explained by the distribution and length of the matching sequences between the 26 mismatches; the perfectly matching regions of 8 and 9 nt were not long enough to induce hybridization to the InflA probe, while one region of 11 nt (together with a 5-nt and a 6-nt region) or two longer flanking regions of 10 nt in the two targets was enough to induce hybridization with the Noro probe.
When the Ino18 probe (middle panel of Figure 4A ) was used with a target with 1 mmoi [i.e. (0.10) 35_1_17 (9.8)] or even 10 mmoi [(10.10) 26_10_26 (0.8)] located at the 5 0 and 3 0 ends of the target, outside the central region containing dInosines, there was no or little inhibition of hybridization at 45 C (71% of the total MFI, 4741 MFI, and 41% of the total MFI, 2773 MFI, respectively). Interestingly, when the 10 mmoi were evenly distributed within the region of 18 dInosines, as in the (0.11) 26_10_26 (9.8) target, hybridization was lost (0.9% of the total MFI, 59 MFI). The sensitivity to mmoi adjacent to dInosine was therefore investigated further. Targets with increasing numbers (2, 4 or 5) of mmoi neighboring the positions of dInosines successively reduced the hybridization signals: 2319 MFI (35% of the total) for the (10.10)_24_12_28_(0.8) target, 927 MFI (13%) for the (10.10)_22_14_30_(0.8) target, and 375 MFI (5.4%) for the (10.10)_21_15_31_(0.8) target, all at 45 C. Thus, dInosine is sensitive to neighboring mismatches. A comparison of the sensitivity of a dInosine-free probe (InflA) and a probe containing dInosine (Ino18) to neighboring mismatches showed that the dInosine-free InflA probe can hybridize to a target with 17 evenly distributed mismatches between two perfectly matching flanking sections of 9 and 8 nt, respectively [the (0.10) 35_1_17 (9.8) target, 24% of the total MFI, 1614 MFI at 45 C]. However, 7 mmoi adjacent to the dInosines completely destroyed hybridization between the Ino18 probe and the (10.10) 19_17_33 (0.8) target: 0.5% of the total MFI, 34 MFI, at 45 C. Results for 55 C are shown in Supplementary Figure S4B and Table 3 . The Ino18 probe failed to hybridize when 2 mmoi were placed next to the dInosines PAGE 11 OF . Thus, the hybridization capacity of a dInosine-containing sequence is severely reduced when the mismatch is adjacent to the dInosine. Figure 4A also shows how introduction of many dInosines affects the specificity. There was no hybridization between the influenza probe Ino18 with its 18 dInosines and the Norovirus target (70) 0_36_52 (0.8). Furthermore, although the dInosines mask 16 mismatches in the (0.11) 26_10_26 (9.8) target, the 10 mmoi that are in close proximity to the dInosines abolish hybridization (0.9% of total MFI, 59 MFI). In contrast, the other target containing 26 mismatching nt and 10 distant mmoi, (10.10) 26_10_26 (0.8), did hybridize to the Ino18 probe (41% of total MFI, 2773 MFI). Thus, the cross hybridization of a foreign (unrelated) dInosine-containing probe is dependent to a certain extent on the amount of mismatch, but is even more dependent on the distribution of mmoi. Figure 4B and Supplementary Table S6 show the origin of the nt that are not covered by the dInosines when using the Ino18 probe (MFI values from Figure 4A ). They demonstrate the number of InflA-matching nt outside the dInosine position (moi) needed for hybridization and the number of Norovirus moi causing cross hybridization. At least 37-38 InflA moi were needed to induce hybridization with the Ino18 probe but, as mentioned above, the distribution is at least as important as the actual number of matching and mismatching nt. Of the fewer than 30-31 nt that were of Norovirus origin in a target that hybridized with the Ino18 probe, 16 nt were common to both Norovirus and Influenza virus. If more than 31 nt were of Norovirus origin, hybridization to the Influenza Ino18 probe failed.
The region chosen for the 77-nt rotavirus probe, positions 112-178 in the alignment, was analyzed using the haplotype function of ConSortß, which decomposes highly variable stretches into a small number of less variable stretches (haplotypes). This resulted in two major haplotypes and probes which potentially covered all rotavirus group A variations recorded in GenBank. The two haplotype probes contained 14 and 8 dInosines, respectively, in combination with four degenerations. They were called Ino14_w4 and Ino8_w4 ( Figure 5C , Inosine as yellow and wobbles as light grey boxes). Two additional probes with fewer dInosines and more degenerations were also created: Ino11_w7 and Ino5_w7 ( Figure 5C ). The consensus sequence and the pattern of variation of the region chosen for the probe are shown in Figure 5A . The sequences of the four probes are shown in Supplementary  Table S4A and B and (schematically) in Figure 5C .
The degenerated LNA-containing fw-primer and the long degenerated dInosine-containing biotinylated rev-primer generated a single band of the correct size, 502 nt, when analyzed by electrophoresis using EtBr-stained agarose gel in all five clinical samples (data not shown). All four microsphere-bound probes detected the consensus synthetic rotavirus target as well as the amplified rotavirus nucleic acid from all five clinical samples. Interestingly, the four probes hybridized almost equally well within each sample. It was found that an asymmetric PCR of the clinical samples was necessary in order to obtain an MFI of reasonable strength from the probes (data not shown). This was probably because the complementary strand of the PCR product outcompeted the probe due to an affinity between the two strands that was higher than that between a dInosine-containing degenerated probe and the target strand. The data in Figure 5B are from one of the experiments using samples run in duplicate. The PCR products were sequenced (Supplementary Table S4 ), revealing that the Ino14_w4 and Ino11_w7 probes, which belonged to the same haplotype, covered the variations in all positions in all samples. However, the other pair of probes (Ino8_w4 and Ino5_w7) had one mismatch against clinical samples 1 and 2 and two mismatches against clinical samples 3, 4 and 5, as well as the consensus synthetic rotavirus target (magenta colored boxes in targets in Figure 5C , Supplementary Table S4 ). In conclusion, the long dInosine-containing degenerated probes worked well as variation-tolerant probes, covering variations, accepting a few mismatches, and still remaining specific (neither of the Rotavirus probes hybridized with the InflA target).
Once we had these experimental data, we tried to develop a unifying view of them. The ÁG predicted by the Visual Omp TM 7.0 software was compared with the percentage of the total MFI for each probe and target combination, including dInosine-containing probes (Figure 2A , 3A Figure 4B shows the total number of nt in each target that originated from either Noro (white triangles) or InflA (grey squares) and that are outside the position of the 18 dInosines when hybridized with the Ino18 probe, compared with the MFI (from Supplementary Figure S1A) for each combination. and 4A). For the sake of simplicity, data from 5-nitroindole and N-wobble-containing probes were omitted. The results demonstrated that some probe/ target combinations that hybridized well in practice, had very low predicted ÁG values ( Figure 6B ), e.g. the InflA probe hybridizing to the 74%12F (43% of the total MFI, ÁG = -20.62), 74%15F (77% of the total MFI, ÁG = À26.7), 74%18 nt (55% of the total MFI, ÁG = -29.24), and 74%22 nt (61% of the total MFI, ÁG = -34.55) targets.
When the results of the new NucZip scoring system were scored against the % MFI in Figure 6A , which shows all the target and probe combinations plotted in Figure 6B , it was found that they were more highly correlated with the experimental data than the predicted ÁG. To investigate these differences, each outlier in Figure 6B was connected to its plot position in Figure 6A ; see Figure 7A and B. Figure 7 shows that probe-target combinations containing many mismatches and dInosines were the main causes of the lower correlation between predicted and observed hybridization in Figure 6B . However, hybridizations between a long probe and a short target were not included in Figures 6  and 7 . Nor were data from probes containing 5-nitroindole or N-wobbles, because a full investigation such as this would require many more observations and would be out of the scope of this article. The NucZip results are further discussed in the Discussion section.
Thus, when the actual degree of hybridization for the entire data set (265 probe-target combinations) was matched with the predicted ÁG in Visual Omp TM 7.0, an only moderately precise correlation was obtained. The hybridization of combinations involving many mismatches and many dInosines was poorly predicted. However, when the NucZip algorithm was used, a higher degree of correlation was observed. The adjusted determination coefficient (R a 2 ) was 0.8636, indicating that 87% of the variation was explained by the NucZip algorithm, while the best fit of MFI% to the Visual Omp TM 7.0 predictions gave a determination coefficient of 0.7505, indicating that 75% of the variation was explained by NN theory (as embodied in Visual Omp TM 7.0). NN theory was thus insufficient for predicting hybridization under the hybridization conditions of our study. A high number of mismatches and dInosines gave hybridization predictions in Visual Omp TM 7.0 that were too low ( Figure 6B ). The NucZip algorithm, which takes into account the length of matching segments and cooperativity effects within and between matching oligonucleotide segments, increased the accuracy of hybridization prediction. dInosines were scored intermediate between matches and mismatches.
Other hybridization prediction algorithms are available on the Internet. However, when we compared the delta G predictions obtained from IDT Oligo Analyzer (http:// eu.idtdna.com/analyzer/Applications/OligoAnalyzer/), which uses a proprietary algorithm, with our experimental data, the correlation was poor (Supplementary Figure 2) . Although the exact experimental conditions (3M TMAC and 45 C) were not represented, this is not likely to have caused the low correlation.
Nucleic acid hybridization is fundamental to many molecular biology applications, and is expected to grow in significance as nanomedicine joins molecular medicine at the cutting edge of research (28) . In particular, biomedical applications of hybridization such as detection of variable viral target sequences are highly dependent on a precise understanding of the process involved. A probe that has a broad detection spectrum should be as specific as current narrower probes while retaining the ability to cover the biologically or clinically relevant sequence variants of specific microbes. The design of long mismatch-tolerant probes demands knowledge about hybridization in the presence of mismatches, degeneracy and nt analogs.
In pursuit of this level of understanding, and in order to obtain reliable hybridization data, we chose to use the Luminex suspension array system in our studies. The inherent ability of the system to report the median of a high number of measurements (i.e. measuring hybridizations signals from 100 different beads) provides highly reliable data. Moreover, hybridization equilibrium is reached more rapidly using the suspension array system (taking around 15 min) than by solid phase hybridizations such as micro arrays (often overnight). A probe length of 70 nt was selected for our studies because of the elevated mismatch tolerance of this length compared with shorter probes (29) . However, the advantage of the extended length of the probe could possibly be countered by a loss of specificity. Hybridization studies using long (50 or more nt) probes in a 3 M TMAC buffer system have not been reported previously; in reports using other hybridization systems, however, it has been suggested that 50-mer or 70-mer probes should contain no more than 15-20 contiguous nt complementary to non-targets (29, 30) . Previously, the hybridization properties of long probes have been analyzed using microarrays, with overnight (16) and ConSortß, to find suitable regions for primers to be used in reverse-transcription PCR and a conserved region for a detection probe. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis). The figure shows the alignment and variation of 214 Rotavirus A sequences in the nt position of the probes displayed in Figure 5C . ConSortß was used to group the variations of the probe region into two haplotypes, which were then used to construct the probes shown in Figure 5C and Supplementary Table S4A Letowski et al. found that, under microarray conditions, mismatches grouped at the 5 0 or 3 0 end of a 50-mer probe affected the binding to a target less than if the mismatches were distributed throughout or centered in the probe. Furthermore, the 50-mer probes with mismatches distributed along the whole probe were more destabilized than the probes that had mismatches centered in the duplex (34) . When Deng et al. studied mismatches in 50-mer microarray probes with 1-7 pm in different distribution patterns, they concluded that the signal intensity was decreased more by evenly distributed than by randomly distributed pm (32) . When 60-mer Figure 2A , 3A and 4A were plotted against the predicted ÁG calculated in Visual OMP TM 7.0 (DNA Software). The predicted ÁG was obtained for the interaction between probes and targets in 3M TMAC buffer at a hybridization temperature of 45 C. The percentage MFI is the MFI signal of a probe hybridized with a study target divided by the MFI signal of the same probe hybridized with its perfectly matching target (e.g. InflA probe against InflA target) at the same temperature. Regression lines were calculated using the SigmaPlot dynamic curve fitting system. A five parameter sigmoidal function gave the highest correlation [f = y 0 +a/ (1+exp (-(xx 0 ) oligonucleotides were hybridized in a microarray, mismatches located near the middle of the probe resulted in a greater reduction of signal intensity than those located at the ends (33) . Additionally, microarray experiments with short oligonucleotides (16-40 nt) and one mismatch or a nt insertion in all positions show that the hybridization signal decreases when the mismatching portion is centered (35, 36) . The matching segments are shorter when mismatches are centered than when they are located peripherally. Our results, using microsphere-bound 70-mer probes and Luminex technology in a buffer containing 3 M TMAC, confirm that the distribution of the mismatches is of great importance and that hybridization is stronger when there are a few longer uninterrupted sequences than when there are many short sequences. These effects are formulated in the NucZip algorithm.
Furthermore, it has previously been shown that two different oligonucleotides of 18 nt, complementary to the inner and outer portions of a 25 nt probe, could hybridize in solution with equal efficiency but, when the probe was coupled to a solid phase via a C6 linker, the 18-nt target complementary to the outer part of the 25-nt probe bound more efficiently than the 18-nt target complementary to the inner part, close to the solid phase (37-39), cf (33) . On our probes, the 5 0 end was coupled to the microspheres via an amino C12 linker. We did not observe any significant differences between matching stretches close to the bead surface and far from it and conjecture that perhaps the long linker allowed for greater accessibility.
It is possible to create a probe against a target with high nt variation, such as an RNA virus, by using degenerated bases at variable positions but the degeneracy of the probe is dramatically increased by each wobbling base, thus decreasing the effective probe concentration. For instance, a sequence with two wobbling bases present in nine positions of variation would give a degeneracy of 512 unique sequence combinations, while a target with 14 variations including A, T, C or G would demand a set of 268 Â 10 6 unique probes (degeneracy 268 Â 10 6 ). Honoreé t al. successfully used 18-23 nt probes with a degeneracy of up to 512 in 3 M TMAC buffer (40) . In the more stringent PCR buffers, the usage of probes with a degeneracy greater than 10 is not often reported (41) . Degenerated primers have the property of being 'forgiving' (41) (42) (43) . This is because the amplimer from a previously successful primer is a target for the same pool of primers in the next round of amplification, leading to an accumulation of amplifiable targets. However, the situation for a probe is different. A degenerate probe will always face the same target variation. Therefore, universal bases like dInosine may be more useful than degenerated sites for probes, as long as the hybridization strength (represented by T m or -ÁG) is good enough. The introduction into a probe of a universal base like dInosine instead of a wobbling base reduces the complexity of the oligonucleotide mixture and increases the actual number of hybridizing oligonucleotides.
Previously, Honore´et al. introduced up to three dInosines in radioactively labelled short oligonucleotide probes, 18-23 nt, in dot-blot hybridization, using a buffer containing 3 M TMAC (40) . The aim was to reduce the degeneracy in probes used for screening cDNA libraries. They found that dInosines had a slightly destabilizing effect on hybridization, especially when hybridizing against A, G and T, but that this could be minimized by reducing the hybridization temperature. However, the behavior of dInosine in long probes in 3 M TMAC has, to our knowledge, not previously been systematically explored. The 3 M TMAC is known to increase the binding contribution of the A:T base pairs, resulting in a similar contribution to T m to that from the G:C base pairs. The high ionic strength makes this an environment of relatively low hybridization stringency. The general trend shown in our study (e.g. Figure 2A and B), that dInosine in the probe decreases hybridization in 3 M TMAC, indicates that TMAC did not enhance the binding strength of dInosine base pairs as much as that of A:T base pairs; cf (8) . In a segment with dInosines at every third base, such as in the Ino18 probe, every matching nt neighbors a dInosine, i.e. it will not bind as strongly as a probe containing neighboring matches. Clearly, dInosine matches cause less destabilization than mismatches, and allow hybridization of probe/ target combinations with many short matching segments, like the InflA probe / 21-pm target combination. It is reasonable to assume that when a probe fails to hybridize due to a high number of mismatches in the target, dInosines at these positions will restore hybridization, since dInosine appears to bridge adjacent matching stretches, increasing their ability to nucleate.
Our results confirm the findings of Honore´et al., despite differences in (i) methods of detection, (ii) hybridization time, and (iii) length of probes. The experience gathered in this work indicates that dInosine base pairing can be considered intermediate between a match and a mismatch, when carried out in 3 M TMAC. Furthermore, our results indicate that dInosine causes less destabilization when hybridized with a C and an A, than when hybridized with a G and a T (7, 8, 40) , in 3 M TMAC; e.g. the Ino18 probe hybridized more strongly with the 33%9nt and 33%15nt targets than with the 33%12nt target ( Figure 3C and D and Supplementary Table S5 ). To lessen this effect and to be able to use the same hybridization temperature for a panel of probes containing no or different amounts of dInosine, it is preferable for the probes to be long, like the 70-mer probes investigated here.
The effects of the universal base dInosine were also compared with those of N wobbles. Thus, at the lower temperature, a dInosine-containing probe hybridized more strongly and, at the higher temperature, the N wobble probe hybridized more strongly. To understand how the highly degenerated probes, wobbN_21, wobbN_18 and wobb_N24, hybridized so well, we calculated the probability of randomly achieving an extension of the matching regions at the 5 0 and 3 0 ends of the wobbN_18 probe (Table 4 ). The probability that the closest N wobble to either the 5 0 or 3 0 end would be a perfect match is 0.5. Thus, 50% of the pool of degenerated probes have a 3 nt longer perfect match (12+9 or 9+12 matching nt at the 5 0 and 3 0 flanking regions) which, according to our results with non-degenerated probe/target combinations, should lead to rather good hybridization of the wobbN_18. In fact, wobbN_18 hybridization was similar in strength to that of the InflA probe to the 33%12F or 33%15nt targets. Furthermore, the probability of having several additional 5-nt matching regions in the central region is also high, probably giving rise to many more combinations in the same pool that matched There is a high probability of several additional matching regions of 5 nt in the central region, which will contribute to hybridization.
x, matching nt; N, wobble of A, C, G, or T. even better. By restricting the wobbles to 3 (e.g. a D or a B) or 2 (e.g. a Y or a T) nt, the probability of a match becomes even greater. Thus, in a highly degenerated probe with at least one continuous region of perfectly matching nt, a large part of the pool will extend this region and contribute to nucleation, zipping and hybridization. The behavior of the highly degenerated probes is encouraging and in accordance with the NucZip model, which predicts that the high likelihood of several matching stretches of 5 nt or longer will result in significant hybridization. One of the aims of this study was to investigate the binding capacity of dInosine in 3M TMAC. 5-NitroIndole was chosen as a comparative universal base. The results shown in Figure 3C demonstrate that 5-NitroIndole in the 5-NitroInd_18 probe had a much greater destabilizing effect than dInosine in the Ino_18 probe, without the same capacity to rescue hybridization with a target containing many sm. Furthermore, the 5-NitroInd_18 probe was more affected than the Ino_18 probe when the hybridization temperature was raised from 45 to 55 C. It is concluded that, under 3 M TMAC buffer conditions, dInosine is a better choice than 5-NitroIndole when designing a variation-tolerant probe with as little degeneration as possible.
Recently, Majlessi et al. (15) studied the nucleation process during double helix formation of short probes, 18-28 nt, with RNA or DNA targets of varying lengths and number of mismatches, in a buffer containing lithium succinate and lithium lauryl sulfate at pH 5.1. Hybridization is initiated by random collisions, but occasionally the complex is stable enough to nucleate the hybridization process. After investigating their model, they suggested that one nucleation region of 9 nt is not enough for further zipping and formation of a double helix. Instead, the first nucleation site needs a second nucleation site so that they can then cooperatively induce the zipping mechanism. They reported that inactivation of one of the 9-nt sites reduced hybridization >2-fold. Interestingly, one complete turn of a dsDNA molecule consists of 10.4 nt (44) . It is conceivable that the first (often temporary) contact between two single nucleic acid strands (nucleation) should not exceed one turn of the dsDNA helix in length, to avoid torsional disturbance and faulty interlocking of the strands. From this point of view, the nucleation site should be long enough to minimize false contacts, and short enough to have minimal steric effects on the strands. Nucleation sites of 6-9 nt fulfil these criteria. The chance of two random single strands matching at a hexanucleotide is 1/16 394, and at a nonanucleotide is 1/ 1 048 576. A matching nonanucleotide will thus reduce the ratio of random successful to unsuccessful nucleations, i.e. those which do not lead to further hybridization in the subsequent zipping phase, a million-fold. The subject is far beyond the scope of this article; however, our data, using 70-mer probes in 3 M TMAC buffer, reveal that a target with two separate regions of 9 nt was enough for efficient hybridization when several shorter regions of 5-6 nt were available between mismatches during the hybridization process (InflA probe/26%9F, 2583 MFI). Increasing the number of mismatches, i.e. shortening the matching regions between the 9 nt flanking regions, caused failure of hybridization (InflA probe/33%9F, 133 MFI and InflA probe/74%9F target, 86 MFI). In contrast, the Ino18 probe, with dInosines covering the mismatches and nine matching nt at the 5 0 and 3 0 ends, hybridized strongly (Ino18/33%9F, 3894 MFI). Furthermore, the InflA probe/74%15nt or InflA probe/74%18nt combinations showed that one region of 15-18 nt was enough to induce and sustain hybridization reproducibly, even if the rest of the 70-mer probe contained 74% mismatches. Having two perfectly matching regions of 15 nt (15F) compared to one region of 15 nt (15 nt) gave 2.6-fold higher MFI for 33%15F compared to 33%15 nt and 4.5-fold higher MFI for 74%15F compared to 74%15 nt, at 45 C. The Ino24 probe, with no dInosine-free matching trimers, hybridized inefficiently with all targets (361-920 MFI at 45 C), showing that dInosine is relatively inefficient in creating nucleating regions. Thus, two nucleation sites of 9 nt are enough to cause hybridization in 3 M TMAC when they are placed next to each other to form a longer region or when there are enough shorter matching regions of 5-6 nt between them. Alternatively, dInosines could bridge the mismatches between the 9-nt regions.
Earlier work has indicated that a region of 15 nt in a 50-mer probe or 20 nt in a 70-mer probe could cause significant cross hybridization in microarray hybridization experiments (30, 31) and our data agree with this. A 70-mer probe is able to hybridize with a region of 15 nt in an otherwise highly mismatching target in 3 M TMAC (74%15 nt, Figure 3A ). To summarize, the current study shows that a dInosine-free probe of 70 nt needs (i) at least three regions of at least six perfectly matching nt, (ii) two stretches of 12-15 perfectly matching nt, or (iii) one stretch of 15-18 perfectly matching nt to result in measurable hybridization. Probes with a high number of dInosines positioned at sites of variation need shorter matching regions than dInosine-free probes. It is suggested that this is probably because dInosine participates in nucleation and zipping during the hybridization process. Thus, a probe with 18 dInosines which match mismatches in the target needs either (i) two regions of 9 nt if the hybridization temperature is 55 C, or (ii) one region of nine perfectly matching nt at 45 C.
As also shown in the study, the risk of cross hybridization when using an nt analog like dInosine is minimal, since dInosine is sensitive to a mismatch in the position next to it and >5 mmoi will reduce hybridization. On the other hand, one should be aware that if an unintended target has many mismatches covered by dInosine and only a limited number of mmoi (<5 mmoi), this could lead to cross hybridization and false positivity. Furthermore, the assumption that sm all differ at the third codon base is an oversimplification. Some synonymous codons also differ at the first and second bases. Thus, even if the Ino18 probe could hybridize when 11 of 17 trimers were intact, with perfect matches at bases 1 and 2, the tolerance to mmoi of a highly dInosine-substituted probe like Ino18 is limited. Around half of its six surplus trimers must be reserved for sm occurring at codon positions 1 and 2. This leaves three trimers available for non-synonymous mismatches. However, a long probe is more likely than a short probe to have matches not neighboring mismatches.
The NN theory was developed for hybridization of short oligonucleotides in solution (45) . Its application to surface-bound oligonucleotides has not been precisely studied. Hooybergs et al. studied hybridization of 30-mer surface-bound oligonucleotides with a 20-mer linker to 30-mer targets in solution, with no, one or two evenly spaced mismatches (46) . Although NN theory was approximately corroborated, NN factors had to be recalculated to give an approximate fit to experimental data. Moreover, the adsorptive (Langmuir) behavior deviated from expectation at high target concentrations. Thus, many unresolved questions regarding hybridization behavior remain.
The concept of NucZip, with both local and distal cooperativity contributions, is an attempt to predict the hybridization behavior of most 70-mer probe-target combinations under the given conditions ( Figures 6A and 8) . The NucZip algorithm is now under revision to include probes containing 1, 2, 3 or 4 nt wobbles, as well as taking into account the results with the universal base 5-nitroindole. The algorithms, schematically described in Figure 8 , (i) were based on our experimental data (described above) and (ii) included highly matching nucleation sites extending beyond the neighboring nt. Thus, unlike the NN theory, NucZip takes the effects of matches at longer distances into account. The unique property of 3 M TMAC to provide a roughly equal contribution to hybridization by A:T and G:C pairs justifies simple computational approaches. The well known additivity of binding contributions per nt inherent in ÁG calculations according to the NN theory (22, 45) indicates that hybridization can be treated in a relatively simplistic way. Our concepts were based on the finding that the longer the sequence of uninterrupted matching nt, the more stable is the hybridization. Our calculations were thus focused more on binding than on destabilization, i.e. the use of positive rather than negative contributions. One of the weaknesses of the NN theory is that it adds all contributions, positive or negative, to a grand sum. The negative contributions are subtracted for the whole molecule, instead of in the local context where they belong. In our approach, the binding is first assessed locally, mimicking nucleation, and then extended cooperatively to the whole molecule, mimicking the zipping process. It is binding that keeps the hybrid together, and it is thus logical to focus on binding.
NN theory does, to some degree, predict that the distribution of the mismatches is an important factor insofar as it affects the nearest neighbors. However, the concentration of this theory on the nearest neighbor disregards the importance of longer matching stretches. The importance of mismatch distribution and uninterrupted matches is exemplified by the stronger signal for hybridization of the InflA probe with the 21-gm target (30% of the total MFI, 2005 MFI at 45 C; 8% of the total MFI, 443 MFI at 55 C) compared to the abolished signal when using a 21-pm target. This is demonstrated in Table 2 by the InflA probe hybridizations with targets containing 14 or 16 mismatches in different distribution patterns, where long uninterrupted perfectly matching sequences favoured hybridization. The cooperativity in hybridization beyond the nearest neighbor that was noticed when two or more matching trimers neighbored each other strengthened hybridization more than when the same numbers of matching trimers were separated by mismatches. In Figure 3A (45 C), the InflA probe hybridized better with 74%12nt (10% MFI), 74%15nt (26% MFI), or 75%18nt (57% MFI) targets than with the 74%9F target, with its 9 nt+9 nt of perfect match (3% MFI). Furthermore, at the higher temperature ( Figure 3B , 55 C), the 74%18nt (37% MFI) and 74%22nt (66% MFI) targets resulted in good hybridization while the 74%12F target, with its 12 nt +12 nt perfectly matching regions, failed (3% MFI).
Three matching trimers account for approximately one turn [10.4 nt (44) ] of the helix of a dsDNA molecule. It is thus likely that two long DNA strands become entangled or conformationally committed when at least one turn of the helix has been completed. Nucleation has to proceed rapidly, without too much torsion and entanglement, to allow many encounters in a short time. We envision that contact between two strands extending to 9 nt allows rapid comparison between strands with only local torsional disturbance. If the brief contact does not achieve binding strength over a certain threshold (the nucleation threshold), the strands separate and new comparisons are made. If the nucleation threshold is exceeded at initial contact, Total ZipScore mode1 : 25+12+12=49 Total ZipScore mode2 : 25+12+5+1=43 ZipScore downstream : 49-dInosinefactor*(49-43)+(dInoCnr*dInoCfactor) contiguous tri-to pentadecamers Figure 8 . The zipping component of NucZip. When a 6-9-bp sequence fulfilling the nucleation criteria has been detected, hybridization up and downstream of the nucleating site is attempted (zipping). The figure shows the downstream zipping process, with successive accumulation of score within a matching segment arising from the trimers, tetramers, etc. up to pentadecamers (each scoring equally) which fit into it. In this way, a longer matching segment gets more than a linear increase in score relative to a shorter one. Zipping extends from the potential nucleation site, terminating with a mismatch or the end of one of the strands. dInosines are counted as intermediate between a match and a mismatch, as described in the 'Materials and Methods' section. If several consecutive matching segments are encountered, their scores are added. binding extends further up and downstream, i.e. zipping occurs. This proceeds as long as the binding strength remains sufficient. The strands are held together chemically by base-base interactions and topologically by the multiple turns of intertwinement. It was a challenge to model this process. In the NucZip model, the program first tests for possible nucleation sites and selects the two highest scoring sites for further evaluation. Zipping is then performed up and downstream from each suggested nucleation site. The zipping algorithm symbolizes the successive cooperativity of binding by adding the number of successive tri-to pentadecamers for each matching segment, each of which ends either in a mismatch or at the end of one or two of the oligonucleotides. The contribution of added dInosine molecules is counted less than those of proper matches. The highest scoring nucleation point is chosen as the result. The results using degenerated probes were in line with this NucZip theory. A long matching segment created by a wobble position increased hybridization strength beyond the contribution of the additional single match.
At present, the NucZip model is intended for equally long nt segments. Exceptions to the model found in the experimental section of this work can be illustrated by the remarkable difference in hybridization between the short perfectly matching targets of 15-22 nt (15 nt_free, 18 nt_free and 22 nt_free) and the longer 70-mer targets (74%_15 nt, 74%_18 nt and 74%_22 nt) ( Figure 3A , B and D). Several groups have analyzed the effect of short so-called dangling ends of 1-5 nt (23) (24) (25) (26) , and report that they appear to stabilize hybridization of the duplex. Doctycz et al. observed that the dangling nt closest to the duplex contributed most to the stabilizing effect (47) . The lengths of our long mismatching ends ranged from 55 to 48 nt with a 74%-nt mismatch and no matching trimers. We speculate that the destabilization seen in a duplex with two long mismatching ends, one from the probe and one from the target, compared with a duplex between a long and a short oligonucleotide, with only one long protruding oligonucleotide, is due to shearing stress on the matching nt, or to competition from intra-strand secondary structures at the separate ends.
Although it can predict many aspects of long oligonucleotide hybridization in 3 M TMAC at 45 C, the NucZip concept has to be amended to include other temperatures, oligonucleotide concentrations and buffers in order to be generally useful. The NN model, which was developed with great precision by SantaLucia et al. (8, (20) (21) (22) and is used in the Visual OMP TM 7.0 computer program, is much more sophisticated than our procedure. It was considered out of the scope of this article to evaluate the Visual OMP TM 7.0 program in detail; however, importantly, its hybridization conditions can be adjusted. When Visual OMP TM 7.0 was used to calculate ÁG (Figure 6 ), the buffer and temperature conditions were set at 3 M TMAC and 45 C. Comparison of the NucZip and Visual OMP TM 7.0 models showed that the distribution of the targets with the long dangling ends (74%_Xnt) was corrected to a certain extent by NucZip; however, neither NucZip nor Visual OMP 7.0 accurately predicted the hybridization behavior of the short perfectly matching probe-target combinations of the InflA probe with the 15nt_free, 18nt_free and 22nt_free targets.
In conclusion, we have demonstrated that the distribution of mismatches greatly affects probe hybridization. A minimum number of continuous, perfectly matching stretches (nucleation sites) is needed to initiate hybridization. Thus, if the target contains many variations and no long uninterrupted matching segments, use of a dInosine-containing probe, which partially overcomes the obstacles caused by mismatches, will be beneficial. With respect to hybridization prediction algorithms, a simple statement of percentage mismatch, as used in many such algorithms, does not adequately reflect the hybridization properties of a long duplex. The insertion of dInosines in the positions of variation could detect target nucleic acid that a consensus probe would fail to catch. Hence, while high dInosine content in a probe decreases the probe's binding capacity, it also covers mismatches, as required when creating mismatch-tolerant, broaddetection probes against highly variable target nucleic acid sequences such as those seen in RNA viruses. The dInosine probes can be made even more forgiving by using a lower hybridization temperature. Furthermore, the probability of cross hybridization is low because the mmoi neighboring the dInosines have a destabilizing effect on hybridization. While the aim of this study was to improve our understanding of variability and the effects of dInosines, other universal bases and wobbles in the design of probes to be used in a 3 M TMAC buffer system, with more exploratory work the results may also be relevant to other hybridization systems and could aid the development of hybridization-based diagnostic tools, including nanotechnological applications such as the volume-amplified magnetic nanobead detection assay (48, 49) .
Supplementary Data are available at NAR Online. Asymmetry in the Presence of Migration Stabilizes Multistrain Disease Outbreaks We study the effect of migration between coupled populations, or patches, on the stability properties of multistrain disease dynamics. The epidemic model used in this work displays a Hopf bifurcation to oscillations in a single, well-mixed population. It is shown numerically that migration between two non-identical patches stabilizes the endemic steady state, delaying the onset of large amplitude outbreaks and reducing the total number of infections. This result is motivated by analyzing generic Hopf bifurcations with different frequencies and with diffusive coupling between them. Stabilization of the steady state is again seen, indicating that our observation in the full multistrain model is based on qualitative characteristics of the dynamics rather than on details of the disease model. In this work we study the stability of a multistrain disease model in two coupled populations. Multistrain diseases are diseases with multiple coexisting strains, such as influenza (Andreasen et al. 1997) , HIV (Hu et al. 1996) , and dengue (Ferguson et al. 1999b) . We consider two kinds of strain interactions: cross immunity and antibody-dependent enhancement. When the disease infects an individual, his or her immune system creates serotype-specific antibodies, which will protect the individual against that serotype. 1 However, there is evidence that antibodies also give some cross-protection to the other serotypes (Halstead 2007) . This reduced susceptibility to the other serotypes is temporary. When the temporary cross immunity wanes, heterologous secondary infections are possible. Low level antibodies developed from primary infections are believed to form complexes with the virus so that more cells are infected, and viral load is increased (Vaughn et al. 2000) . This effect is called antibody-dependent enhancement (ADE), and it has been observed in vitro in diseases such as Ebola (Takada et al. 2003 ) and dengue (Halstead and O'Rourke 1977) . Throughout this work we make the hypothesis that ADE increases the infectiousness of secondary infective cases due to the higher viral load Schwartz et al. 2005) . Alternative views of ADE as an increase in mortality associated with secondary infectives can be considered (Kawaguchi et al. 2003) . We focus in this paper on the multistrain disease dengue, which is believed to exhibit both temporary cross immunity and antibody-dependent enhancement (Halstead 2007) .
Dengue is a subtropical mosquito-borne disease that exhibits up to four serotypes. It is widespread in tropical regions of southeast Asia, Africa, and the Americas, infecting an estimated 50 to 100 million people every year (World Health Organization Website 2006) . Primary infections are sometimes asymptomatic, while secondary infections are more severe, with about 5% of secondary infections leading to dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the potentially fatal forms of the disease (World Health Organization Website 2006) .
An effective vaccine against dengue is very difficult to achieve. Because of ADE, infection with an unvaccinated strain following a single-strain vaccination could lead to the more severe symptoms associated with secondary infections (Halstead and Deen 2002) . Therefore, an effective vaccine must protect against all four serotypes simultaneously. A recent theoretical study on a 2 strains system (Billings et al. 2008) has shown that eradication of dengue using only single-strain vaccines is unlikely. Because a tetravalent vaccine is not immediately forthcoming, deepening our understanding of the dynamics of dengue in more realistic models is of great importance.
The dynamics of dengue in a single, well-mixed population has been studied in several recent publications, including but not limited to Ferguson et al. (1999a) , Schwartz et al. (2005 , Wearing and Rohani (2006) , Bianco et al. (2009) . ADE and cross immunity have been shown to play a fundamental role in mathematical models for the spreading of dengue, causing instability, desynchronization of serotypes, and chaotic outbreaks Cummings et al. 2005; Bianco et al. 2009 ). However, real populations may be spatially heterogeneous. To gain further insight into the wide spectrum of possible dengue dynamics, we relax the assumption of a well-mixed population. The division of a population into spatially distinct patches simulates the potentially heterogeneous environment in which the disease spreads. Spatial heterogeneity has been invoked in the past to account for the persistent character of some infectious diseases (Lloyd and May 1996; Hagenaars et al. 2004) and to explain in-phase and out-of-phase dynamics of diseases (He and Stone 2003) . A multipatch model with age structure has been used to explain bi-and tri-annual oscillations in the spread of measles (Bolker and Grenfell 1995) .
Human mobility patterns have a significant influence on the spreading of infectious disease. We will assume here that the coupling between patches is via migration, movement of individuals from one patch into another (as in Liebovitch and Schwartz 2004; Ruan et al. 2006; Sattenspiel and Dietz 1995; Grais et al. 2003 ). An alternative coupling strategy is mass action coupling, in which susceptibles in one patch are assumed to interact with infectives in another patch, introducing nonlinear coupling terms (Bolker and Grenfell 1995; Lloyd and May 1996; Rohani et al. 1999) . Stochastic coupling can be modeled (see Keeling and Rohani 2002 and references therein, and Colizza and Vespignani 2008) .
The purpose of this work is to analyze the stability of a model for multistrain diseases with interacting strains, using dengue as an example, in a system of two coupled patches. Since chaotic outbreaks are likely to produce a higher number of infected individuals, understanding the stability properties may play an important role in public health. The case of non-identical parameters in the two patches will be of particular interest, as this serves as a model for spatial heterogeneity. The paper is divided as follows. In Sect. 2 we introduce the epidemic model. Section 3 summarizes the bifurcation structure for a single patch. In Sect. 4 we present numerical results for bifurcations in two coupled patches. Section 5 motivates these results via analysis of a simple, lower dimensional model, and Sect. 6 concludes.
We use a compartmental model for multistrain disease spread with cross immunity and antibody-dependent enhancement, previously studied in a single, well-mixed population (Bianco et al. 2009 ). In this model, individuals can develop a primary infection with any of the serotypes. Immediately after recovering, the individual experiences a period of temporary partial cross immunity to all other serotypes. When the cross immunity wears off, immunity to the primary infecting strain is retained, but the individual can develop a secondary infection with a different serotype. Infectiousness of the secondary infectives is increased due to antibody-dependent enhancement. After the secondary infection, complete immunity to all serotypes is assumed. A flow diagram for the single patch model with two serotypes is shown in Fig. 1 for simplicity, but we present results here for all four serotypes. We extend the model to two spatially distinct patches, which are coupled by linear migration terms. For two patches (indexed by q) and n strains (n arbitrary), the model is as follows:
where the variables are s q , the fraction of susceptibles in patch q; x q,i , the fraction of primary infectives with strain i in patch q; c q,i , the fraction of individuals in patch q with partial cross immunity to strain i; r q,i , the fraction of individuals in patch q that are recovered from a primary infection with strain i and no longer have cross immunity to the other strains; and x q,ij , the fraction of individuals in patch q recovered from strain i and currently infected with strain j . The parameters are the number of strains n, the contact rate in patch q β q , the recovery rate σ , the ADE factor φ, the strength of cross immunity , the rate θ for cross immunity to wear off, the birth and mortality rate in patch q μ q , and the migration rate between patches ν. A list of parameters appears in Table 1 .
For clarity, we describe in detail the terms appearing in (5) describing the time evolution of the secondary infectives x q,ij . These are individuals who were previously infected with strain i and are currently infected with strain j . Individuals recovered from an infection with i and no longer retaining cross immunity (r q,i ) can become infected by any infective with strain j . We use mass action contact between infectives and non-invectives, contributing a term β q r q,i (x q,j + φ k =j x q,kj ) (where the secondary infectives have enhanced contagion given by the ADE factor φ). Cross immune individuals c q,i become infected with a reduced probability (1 − ). Linear terms −σ x q,ij and −μ q x q,ij appear as secondary infectives recover with rate σ or are lost to natural death with rate μ q . Finally, individuals are exchanged between patches q and q with rate ν, yielding migration terms −νx q,ij + νx q ,ij .
For simplicity, the birth and death rates in a patch are set equal to each other so that the total population of each patch is constant. The model of (1)-(5) allows for one reinfection. Tertiary infections are not considered (Nisalak et al. 2003) . The parameter determines how susceptible the cross immune compartments c i are to (Bianco et al. 2009 ). Note the reduction of susceptibility to a secondary infection through the cross immunity factor (1 − ) and the enhancement of secondary infectiousness due to the ADE factor φ. Mortality terms for each compartment are not included in the diagram for ease of reading Ferguson et al. (1999b) β, transmission coefficient, years −1 ∼200 Ferguson et al. (1999b) σ , recovery rate, years −1 50 Rigau-Perez et al. (1998) θ, rate to leave the cross 2 Wearing and Rohani (2006) immune compartment, years −1 φ, ADE factor ≥1 S c h w a r t z e t a l . ( 2005) , strength of cross immunity 0-1 Bianco et al. (2009) ν, migration rate, years −1 0-0.05 n, number of strains 4 a secondary infection, where = 0 means no cross immunity (the infection rate is identical for compartments c i and r i ) and = 1 confers complete cross immunity (cross immunes are immune to a secondary infection for an average time θ −1 ). We allow to take any value between 0 and 1. The ADE factor φ is the enhancement in the infectiousness of secondary infectives. φ = 1 means that secondary infectives are as infectious as primary infectives, while φ = 2 means that secondary infectives are twice as infectious as primary, and so forth. In contrast to Aguiar and Stollenwerk (2007), we will not consider values of φ smaller than 1. The migration rate from patch q to q , and from patch q to q, is ν. For simplicity, we assume that all individuals migrate with equal probability, independent of their infection status. This assumption may be relaxed in a future study. The migration rate is assumed to be slow compared to the infection spread. For convenience, we put it on the same order as the birth/death rate. We assume that the social parameters, which are the contact rate β q and birth/death rate μ q , may vary between patches, while the epidemic parameters are the same in all regions. Because the social parameters depend on human factors (and in the case of the contact rate also include mosquito levels, which are weather-dependent), these parameters are the most likely to be different in adjacent regions. We use parameter values compatible with dengue fever, which are summarized in Table 1 . Our contact rate β corresponds to a reproductive rate of infection R 0 of 3.2 − 4.8, which is consistent with previous estimates (Ferguson et al. 1999b; Nagao and Koelle 2008) .
A similar model to the one of (1)-(5) has recently been used to analyze the dynamics of dengue fever in a single well-mixed population (Bianco et al. 2009 ). The ADE φ and cross immunity strength were varied as bifurcation parameters. In the absence of cross immunity ( = 0), ADE alone generates instability, desynchronization, and ultimately chaotic outbreaks Schwartz et al. 2005) . A Hopf bifurcation is observed for a critical value of the ADE factor φ, above which oscillatory solutions are obtained. Weak cross immunity stabilizes the system, while strong cross immunity triggers instability and chaos even in Table 1 the absence of ADE (Bianco et al. 2009 ). In the latter case, destabilization occurs via a Hopf bifurcation for a critical value of the cross immunity strength . At the bifurcation, three identical complex pairs of eigenvalues of the Jacobian simultaneously become unstable. Although Bianco et al. (2009) discusses the full two parameter bifurcation structure (in and φ), we will consider the cross immunity and ADE effects separately in the present work. Figure 2a shows the bifurcation behavior of the single patch model with no cross immunity as the contact rate β is varied. The bifurcation structure was computed using a continuation routine (Doedel et al. 1997 ). The ADE value at which the bifurcation occurs increases slightly as β increases, and the dependence is approximately linear. The period of the periodic orbit, shown in Fig. 2b for a fixed ADE value, also varies with β. (Note that multistrain models with ADE can display subcritical Hopf bifurcations, Billings et al. 2007 , so the periodic orbit shown in Fig. 2b exists throughout the range of β values shown.) Similarly, varying the contact rate β in the absence of ADE affects the location of the Hopf bifurcation in cross immunity and its characteristic frequency of oscillation. Likewise, varying the other social parameter, the birth rate μ, affects the location and frequency of the Hopf bifurcations in φ and (data not shown).
We next examine the effect of coupling between distinct regions on the dynamics previously observed in the single patch model. In particular, we consider how migration between non-identical patches affects the stability of the steady state. We investigate the dynamics of the coupled systems by numerically integrating (1)-(5) and by tracking the bifurcations using a continuation routine (Doedel et al. 1997) . As mentioned in the previous section, the model has a Hopf bifurcation at critical values of the bifurcation parameters φ, the ADE factor, and , the cross immunity strength. We Fig. 3 Critical values of the parameters (a) and (b) φ at which the Hopf bifurcation occurs for coupled patches, as a function of the contact rate in patch 1, for two values of the migration rate, ν = 0.02 (solid black) and ν = 0.05 (dashed gray). The contact rate in patch 2 is fixed at β 2 = 200. In (a), φ = 1 (no ADE). In (b), = 0 (no cross immunity). μ 1 = μ 2 = 0.02 and other parameters are as in Table 1 study the effect of coupling and asymmetry on the stability by observing their effect on the location of the Hopf bifurcations. We consider cross immunity and ADE separately; that is, we analyze the system either with ADE and no cross immunity or with cross immunity and no ADE. Including both cross immunity and ADE together leads to qualitatively similar behavior to that reported here, at least locally when the asymmetry is not too large.
We proceed by fixing the patch-specific parameters in patch 2 and varying a social parameter (β 1 or μ 1 ) in patch 1 to observe the effect of increasing asymmetry on the dynamics. As previously mentioned, changing the social parameters modifies the natural frequency of the system. For each value of asymmetry, we look for the critical points at which the coupled system loses stability. We also consider several migration rates ν.
The effect of asymmetry in the contact rate is shown in Figs. 3(a) and 3(b) for two migration rates, namely ν = 0.02 (black) and ν = 0.05 (gray). The value of the critical parameter ( Fig. 3(a) ) or φ (Fig. 3(b) ) at which the Hopf bifurcation occurs is plotted against the varying contact rate. The symmetric case is at the bottom of the curve. We see that two identical patches have the same bifurcation point as a single, well-mixed population even when migration is present (cf. Fig. 2a) . However, a striking difference in the dynamics appears when we make the two patches weakly asymmetric. The values of φ and at which the Hopf bifurcation occurs are dramatically different from the symmetric case. The steady state stability persists well into the parameter regime where a single patch would display chaotic oscillations (Billings et al. 2007; Bianco et al. 2009 ). The location of the single patch Hopf point does not depend strongly on β, and decreasing the contact rate is actually destabilizing (Fig. 2a) , so the stabilization observed here is clearly a result of the coupling between asymmetric systems. Slightly increasing the migration rate (from ν = 0.02 to ν = 0.05 in Fig. 3 ) further increases the stability of the system. Fig. 4 Critical values of the parameters (a) and (b) φ at which the Hopf bifurcation occurs for coupled patches, as a function of the birth/death rate in patch 1, for ν = 0.02. The birth/death rate in patch 2 is fixed at μ 2 = 0.02. In (a), φ = 1 (no ADE). In (b), = 0 (no cross immunity). β 1 = β 2 = 200 and other parameters are as in Table 1 Similar results are obtained if the asymmetry occurs in the other social parameter, the birth rate, as depicted in Fig. 4 . Again, for asymmetric patches either stronger cross immunity or stronger ADE is needed to destabilize the steady state through a Hopf bifurcation.
We now motivate the results of the preceding section using a simple lower dimensional model and show that the qualitative features depend only on the bifurcation structure and characteristic frequencies rather than on details of the epidemic model. In this section we study the general behavior of two coupled systems, each displaying a Hopf bifurcation, but with different characteristic frequencies.
The generic form for a Hopf bifurcation is the following, in polar coordinates (Strogatz 2001 
A Hopf bifurcation occurs at α = 0, where periodic oscillations with frequency ω are observed. We now couple two systems of the sort in (6), each with a potentially different frequency ω q , via linear migration terms with migration rate ν. To lowest order (not displaying cubic terms), the coupled system in cartesian coordinates iṡ x 1 = αx 1 − ω 1 y 1 − νx 1 + νx 2 y 1 = αy 1 + ω 1 x 1 − νy 1 + νy 2 x 2 = αx 2 − ω 2 y 2 − νx 2 + νx 1 y 2 = αy 2 + ω 2 x 2 − νy 2 + νy 1
Stability is determined by evaluating the Jacobian of (7) at the steady state (x 1 , y 1 , x 2 , y 2 ) = 0. At the Hopf bifurcation, the real part of the largest eigenvalue crosses zero, with nonzero imaginary part. The roots of the characteristic polynomial f (λ) of the Jacobian are the eigenvalues {λ}. The four eigenvalues are
but it is not easy to see from these expressions where the Hopf bifurcation occurs. Instead, we solve directly for the critical value of α at the Hopf point. At a Hopf bifurcation, a pair of eigenvalues has zero real part. Thus to obtain the Hopf point, we can set λ = ib, where b is real. If ib is an eigenvalue, we require
and
If α = ν, (11) has nonzero roots b = ± 1 2 4α 2 − 8αν + 2ω 2 1 + 2ω 2 2 . When this b is substituted into (10), we obtain four potential roots for α at the Hopf point. Two are complex, which is unphysical and can be ignored. The one corresponding to the Hopf bifurcation is α c = ν − 1 2 4ν 2 − (ω 1 − ω 2 ) 2 . (The fourth root is larger and corresponds to loss of stability of the second pair of eigenvalues.) This α c is real and thus gives the Hopf point location when |ω 1 − ω 2 | ≤ 2ν. When ω 1 − ω 2 = 0 and ν > 0, α c is positive, indicating stabilization due to the asymmetry and coupling.
When α = ν, (11) is always satisfied. In that case, we must turn to (10) to determine b. Equation (10) has four roots, b = ± 1 2 (ω 1 + ω 2 ) ± 1 2 (ω 1 − ω 2 ) 2 − 4ν 2 . By our assumption, b must be real, and this occurs when |ω 1 − ω 2 | ≥ 2ν, which is precisely the case not covered by the above result. Thus when |ω 1 − ω 2 | ≥ 2ν, the Hopf point is at α c = ν.
Summarizing, the Hopf bifurcation occurs at
In Fig. 5 we show the location of the Hopf bifurcation given by (12) as a function of the asymmetry between the two systems for ν = 0.05. Comparison with Figs. 3 and 4 shows the qualitative agreement of the theoretical results for the lower dimensional system with the full multistrain system. Increasing the migration rate ν increases the value of the bifurcation parameter to which the Hopf point saturates, as in Fig. 3 . It is also worth noticing that, in the case of identical frequencies ω 1 = ω 2 , the Hopf bifurcation occurs at α c = 0, the location in the absence of coupling. This is consistent with the numerical results for the multistrain system in the case of symmetric patches. 
We have studied the endemic steady state stability properties for a multistrain epidemic model on two migration-coupled patches. Interactions between strains in the model were governed by temporary partial cross immunity and antibody-dependent enhancement. In the absence of coupling, the system displayed Hopf bifurcations in two epidemic parameters. Coupling between patches with non-identical parameters, which gave them non-identical characteristic frequencies of oscillation, was shown to shift the Hopf bifurcations, stabilizing the steady state. This behavior was observed for the Hopf bifurcation obtained by sweeping the cross immunity in the absence of ADE and the bifurcation obtained by sweeping the ADE in the absence of cross immunity. It occurred for asymmetry in either of our two social parameters, the birth rate and the contact rate.
To motivate this result, we diffusively coupled two low dimensional Hopf bifurcations with different characteristic frequencies and analyzed the stability of the steady state. We again saw that coupling between asymmetric systems led to stabilization. This indicates that the stabilization in the epidemic model is a result of the underlying dynamics, rather than the details of the model. We suggest that the stabilization may occur as a result of the two different coupled frequencies generating oscillations that tend to cancel each other because of phase differences. This topic will be studied in more detail in a future work.
Bifurcations from steady state to oscillatory behavior can be associated with an increased number of infection cases, particularly if chaotic oscillations occur, as in previous dengue models (Billings et al. 2007; Bianco et al. 2009 ). Our results suggest that if control strategies in one region are able to generate enough asymmetry, this could lead to a stabilization of the outbreaks, which would have a positive effect on adjacent regions. Asymmetry could be generated in the effective contact rate by mosquito control, which could include reducing mosquito breeding sites (Slosek 1986) , or through new genetic controls which are under development (Barbazan et al. 2008) . Asymmetry in the birth rate could be generated by lowering the effective birth rate through vaccination of new susceptibles once a vaccine is available. However, because the bifurcation point saturates rather than increasing indefinitely as asymmetry increases, such a strategy would be successful only if the epidemic parameters in the real system are moderately close to the bifurcation. (Extreme asymmetry may even be destabilizing, so this strategy works best locally when the asymmetry is not too strong.) Real systems exhibiting oscillatory behavior may already be well into an unstable region of parameters. For such systems, our suggested control strategy may not apply since asymmetry between adjacent regions is already likely, thus reducing that impact that an increase in the asymmetry could have. On the other hand, in the case of dengue fever a recent study (Cummings et al. 2009 ) has highlighted a decreasing trend in both the force of infection and birth and death rates in the population of Thailand during a time span of about 30 years. This phenomenon could potentially drive the system closer to the bifurcation point and make it more amenable to control.
In addition, the role of seasonality in exciting oscillations should not be ignored. Seasonal variations in the contact rate have been included in previous dengue models . The interplay of seasonality and coupling is a topic for future study.
The results of the present work may also be useful in estimation of the parameter values for ADE and cross immunity. ADE especially is difficult to measure in vivo and must be estimated by other means. Since recent publications have suggested that epidemiological data from Thailand show chaotic outbreaks , and given the certainty of human migration and asymmetry between adjacent regions of the country, it is possible that the actual parameter values for ADE and cross immunity are higher than the ones estimated by studying a single, well-mixed population.
Finally, the work discussed here shows a potential effect of human movement between heterogeneous regions. As spatial effects are further studied in epidemic models, it remains to be seen how this phenomenon will extend to more complicated spatial geometries, including more patches and perhaps non-symmetric coupling terms. This work represents a first step towards understanding the role of migration and spatial heterogeneity in dynamical properties of dengue observed in epidemiological data, such as traveling waves of infection in Thailand (Cummings et al. 2004 ). Furthermore, because the migration-induced stabilization depends only on the existence of a Hopf bifurcation in the model, it is expected that the stabilization will be observed in other population models that also contain Hopf bifurcations (e.g., Fussmann et al. 2000; Greenhalgh et al. 2004) . Clinical factors associated with severity in hospitalized children infected with avian influenza (H5N1) OBJECTIVE: The World Health organization received reports of 478 laboratory-confirmed cases of influenza A (H5N1) from 15 countries between November 2003 and February 2010. More than 50% of these cases involved patients <20 years of age. Determining an association between the clinical factors at the time of hospital admission and prognosis may be useful for timely and adequate consultation and treatment. It has been difficult to obtain these clinical factors adjusted with other confounding factors, such as age and sex, as published studies of H5N1 virus infection usually reported only a few cases. So, we performed a pooled analysis of the reported cases. METHODS: Five case reports (36 patients <18 years of age) of H5N1 infection from four countries published between 2004 and 2009 were assessed based on available individual clinical data. Using the pooled data for all patients, we investigated the associations between patients’ prognosis and available laboratory findings, such as white blood cell (WBC) counts, platelet (PLT) counts, and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by adjusting for age and/or sex. RESULTS: The linear regression analysis revealed that mortality was negatively associated with WBC and PLT counts adjusted with age and sex. Increased log AST tended to be associated with a poor prognosis (p = 0.054), but there was no significant association between survival and log ALT level. CONCLUSIONS: Both decreased WBC and PLT counts can be considered to be common predictors of poor prognosis in H5N1 influenza patients <18 years of age. Further studies are needed for clarification. The 2009 H1N1 influenza pandemic spread throughout the world, but since the beginning of 2010, the number of cases of this influenza has been declining. Reports of avian influenza (H5N1) infection in humans have also been reported, raising the threat of a new pandemic through a novel combination of the H5N1 virus with the 2009 H1N1 strain.
Cases of the highly pathogenic avian influenza A virus (H5N1) causing influenza symptoms in humans were observed in the Hong Kong Special Administrative Region, People's Republic of China in 1997 [1] . A total of 478 laboratory-confirmed cases of influenza A (H5N1) infection were subsequently reported to the World Health Organization (WHO) from 15 countries between November 2003 and February 2010 [2] . More than 90% of these cases were reported in five countries, namely, Indonesia, Vietnam, Egypt, China, and Thailand. Patients \20 years of age accounted for 63% of the cluster cases and 49% of the sporadic cases [3] . The case-fatality rate (CFR) in all cases was 62.1.
Of the 383 cases reported between November 2003 and May 2008 from these 15 countries, 10 .4% involved patients aged\9 years, 24% involved patients between ages 10 and 19 years, and 25% involved patients aged 20-29 years [4] . The overall CFR of H5N1 infection was 65%, and the CFR for patients aged 10-19 years was 78%, which was highest among all age groups [4] . Patients for whom the disease was ultimately fatal showed respiratory failure and progressed to pediatric acute respiratory distress syndrome (ARDS), which in patients infected with H5N1 influenza virus consisted of severe viral pneumonia accompanied by diffuse alveolar damage that was induced by intense cytokine reactions and inflammation [5] . Wiwanitkit investigated hematologic findings among reported H5N1infected patients and observed lymphopenia and anemia, but he did mentioned that the effects of the infection on platelet count were controversial [6] . A knowledge of the clinical factors present at admission to the hospital that are associated with prognosis may be useful when the aim is timely and adequate consultation and treatment. As many of the studies on the H5N1 virus have each only reported a few cases of H5N1 influenza, it has been difficult to get identify these clinical factors adjusted with other confounding factors, such as age and sex. Here, we report our investigation of the early indicators of prognosis in patients with H5N1 influenza aged \18 years which we identified from a pooled analysis of case reports.
For the case-investigation of H5N1 human infection reports containing individual clinical data, we selected five case reports from four countries published between 2004 and 2009 for investigation. From each report, we selected patients aged \18 years; ultimately, our study population consisted of 36 patients who were included in the pooled analysis. Four patients were from Tran et al. [7] (Vietnam), 12 were from Kawachi et al. [5] (Vietnam), 7 were from Chotpitayasunondh et al. [8] (Thailand), 8 were from Oner et al. [9] (Turkey), and 5 were from Kandun et al. [10] (Indonesia). H5N1 case-patients were identified by reverse transcriptase (RT)-PCR. The patients reported in Kawachi et al. [5] who showed severe ARDS were identified in the National Hospital of Pediatrics (NHP), and those in other reports were from passive surveillance. We investigated factors related with patients' prognosis from available laboratory findings, such as white blood cell (WBC) counts, platelet (PLT) counts, and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The AST and ALT levels were not available for the cases of Kandun et al. [10] and five cases from Kawachi et al. [5] , and the leukocyte and platelet counts were not available for one case from Kawachi et al. [5] . Data were analyzed using Wilcoxon and Kruskal-Wallis tests for continuous variables and Fisher's exact test for categorical variables. Linear regression analysis was used for investigating associations between laboratory findings and patients' prognosis. All statistical analyses were done by SPSS ver. 14.0J (SPSS, Chicago, IL).
We summarized the characteristics of patients from each report in Table 1 . The mean age of the pooled patients from all studies was 8.0 ± 4.4 years, and mean ages of the patients were significantly different among the different reports (p = 0.032). For the pooled data, the proportion of patients who showed ARDS or respiratory failure during hospitalization was 75%; this proportion was also significantly different among the different reports (p = 0.014). Overall, the CFR was 69.4%, and no significant difference between reports was observed. For the pooled data, median number of days from onset to admission was 6.0 and the median number of days from onset to outcome was 14.0; there no significant differences among the reports for both data sets.
In terms of the laboratory data at hospital admission, for the pooled data the median WBC count was 2900 (/mm 3 ), and the median PLT count was 125 (910 3 /mm 3 ); a significant difference in PLT count was observed among reports ( Table 2) . Based on available data on the liver function tests (three reports), the median AST and ALT levels were 280 and 52 IU/L, respectively; the levels of both liver enzymes were significantly different among reports (Table 2) .
Differences between laboratory data at hospital admission between fatal and nonfatal cases are shown in Table 3 . Leukopenia and thrombocytopenia at hospital admission were present in the fatal cases, but there were no differences in the serum levels of AST and ALT between fatal and nonfatal cases. In the linear regression analysis performed to investigate the association between blood cell counts and prognosis, WBC and PLT were dependent variables, and age, sex, and prognosis were independent variables. In the linear regression analysis performed to investigate the association between log-transformed liver function data and prognosis, log AST and log ALT levels were dependent variables, and age and prognosis were independent variables as the sample size was small enough to enable one covariate to be added. Standardized regression coefficients for the results of these linear regression analyses are shown in Table 4 . Death (the value of prognosis = 1) was negatively associated with WBC and PLT counts adjusted with age and sex. In contrast, increased log AST tended to be associated with poor prognosis, but there was no significant association between survival and log [1] with the results from the pooled data of our five studies reveals that the CFR for patients\19 years of age was 56.9% in the WHO report and 69.4% in our pooled data. The median time from onset of symptoms to admission was 4 days (mean 4.6 days) among all of the patients reported in our five studies and 5 days in patients reported to have died in the WHO report. The median time from onset to admission was 6 days in the pooled data. Cases were mainly identified through passive surveillance in the WHO report, whereas we also included cases collected through hospital medical records in our study. Thus, the CFR in the pooled data was higher than that of the WHO report, and the median time from onset to admission in the pooled data was longer than that in the WHO report. Our pooled data was biased to include more severe cases. Significant variations in patterns that may reflect differences in exposure related to social behavior, cultural and religious practices, access to care, and treatment or virulence of the viruses among countries have been pointed out in previous reports [4] . With respect to the virulence of viruses, viruses capable of infecting humans have been divided into groups from clades and subclades 0, 1, 2.1, 2.2, 2.3, and 7 from the phylogenetic tree for the hemagglutinin gene of highly pathogenic avian influenza A (H5N1) viruses. Clade 1 viruses were found to predominate in Vietnam, Thailand, and Cambodia in the early phase of the outbreak (2004) (2005) , and clade 2.1 viruses are endemic in Indonesia (2005) (2006) . Clade 2.2 viruses were associated with the outbreak of avian infection in Central and South Asia, the Middle East, Europe, and Africa and with human infection in western Asia, the Middle East, and Africa (including the Turkish outbreak from 2005 to 2006). The median time from onset to hospitalization were found to be unchanged among clades [11] . Differences in the proportion of fatalities were observed, but it is difficult to determine whether this difference was due to the difference in viral virulence, as other variations existed among countries.
Our pooled data also included these significant variations. Although our study is characterized by a number of the limitations shown above, our results may help generate hypothetical candidates for early indicators of prognosis in patients \18 years of age with H5N1 influenza.
In most of the earlier reports on H5N1 cases, the number of cases was not large enough to adjust for potential confounders. However, several published studies did investigate factors that predicted poor outcome, adjusting the confounders for the whole-age spectrum of the subjects. Kandun et al. [12] analyzed 127 confirmed infections from June 2005 through February 2008 in Indonesia and found that an individual case was significantly associated with the mortality. Liem et al. [13] reviewed medical records and analyzed 67 of 93 cases of H5N1 infection in Vietnam from January 2004 through December 2006. Applying a backward step-wise variable selection strategy for logistic regression analysis, these researchers were able to show that the presence of both neutropenia and raised ALT level predicted a poor outcome. Yu et al. [14] collected 26 H5N1 cases from hospital medical records of H5N1 cases in China from October 2005 through April 2008 and found that decreased PLT count, elevated lactic dehydrogenase level, ARDS, and cardiac failure were associated with mortality. In our study, decreased WBC and PLT counts were significantly associated with the prognosis adjusted with sex and age, but these results coincided with the results of individual previous reports. Both decreased WBC and PLT counts are considered to be common predictors of poor prognosis in H5N1 influenza patients \18 years of age. Further studies that include larger numbers of patients are needed to clarify these results.  Human monoclonal IgG selection of Plasmodium falciparum for the expression of placental malaria-specific variant surface antigens Pregnancy-associatedPlasmodium falciparum malaria (PAM) is a major cause of morbidity and mortality in African women and their offspring. PAM is characterized by accumulation of infected erythrocytes (IEs) that adhere to chondroitin sulphate A (CSA) in the placental intervillous space. We show here that human monoclonal IgG antibodies with specificity for variant surface antigens (VSA) specifically expressed by CSA-adhering IEs (VSA(PAM)) can be used in vitro to select parasites from nonpregnant donors to express VSA(PAM) and that this selection for VSA(PAM) expression results in preferential transcription of var2csa. The results corroborate current efforts to develop PAM-specific vaccines based on VAR2CSA. Children living in areas of stable Plasmodium falciparum transmission acquire substantial protective immunity against malaria during the first decade of life. Protection is mediated to a large extent by variant surface antigen (VSA)specific IgG. Nevertheless, women in such areas remain highly susceptible to P. falciparum infection if they become pregnant, as the parasites can switch to expression of particular VSA (called VSA PAM ), which allow infected erythrocyte (IE) sequestration in the placenta, but which are not compatible with parasite survival in a nonpregnant host. Acquired immunity to PAM is mediated by VSA PAMspecific IgG that either opsonizes IEs for phagocytosis or interferes with chondroitin sulphate A (CSA)-specific adhesion of IEs (1) . VAR2CSA, which is a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of VSA, appears to be the dominant or only pregnancy-associated Plasmodium falciparum malaria (PAM) type VSA in the P. falciparum genome, and is therefore the main target of current efforts to develop a vaccine against PAM (2) . To assist this work, we have developed a panel of VSA PAM -specific human IgG1 monoclonal antibodies (3) , which can opsonize VSA PAMexpressing IEs for phagocytosis and interfere with IE adhesion to CSA (Barfod et al . unpublished data) . These antibodies can be used to enrich for VSA PAM expression in parasites not expressing VSA PAM but obtained from women with PAM (3) . In the present study we used two of the above-mentioned monoclonal antibodies to select for VSA PAM expression in parasites from nonpregnant donors. One antibody (PAM1·4) was chosen because it appears to react with most or all parasites expressing VSA PAM . The other antibody (PAM8·1) was chosen because it reacts with a well-defined, but inter-clonally variant, epitope in the DBL3X domain of VAR2CSA.
We used eight VSA PAM -specific monoclonal IgG1 antibodies generated as described elsewhere (3; 4) . VSA PAM is defined here as IE surface-expressed VSA, which are significantly better recognized by plasma IgG from P. falciparumexposed multigravidae than from sympatric men, and where the recognition by plasma IgG from these men is not significantly different from recognition by plasma IgG from nonexposed controls. In contrast, typical non-PAM VSAs are better recognized by plasma IgG from P. falciparumexposed adults than from nonexposed donors, without marked sex-dependent differences. Seven of the monoclonal antibodies used here are specific for inter-clonally variant epitopes in either the DBL3-X or the DBL5-ε domain of VAR2CSA (3) . The exact specificity of the last antibody (PAM1·4) remains undefined, but it appears to recognize a conformational, and possibly discontinuous, epitope in VAR2CSA that is difficult to reproduce in recombinant constructs (3) . PAM1·4 recognizes VSA PAM expressed by most or all P. falciparum genotypes, whereas the VAR2CSA DBL3-X epitope recognized by PAM8·1 is present in some, but not all P. falciparum clones (3). Both PAM1·4 and PAM8·1 can opsonize VSA PAM -expressing IEs for phagocytosis and interfere with their adhesion to CSA (Barfod et al. in preparation) .
We used the two long-term in vitro -adapted parasites 3D7 and HB3. The 3D7 clone was originally derived from NF54 parasites isolated from a Dutch girl near Amsterdam airport (5) . It was chosen here because it can be selected for expression of VSA PAM that react with PAM1·4 but not PAM8·1 (3). HB3 was cloned from the Honduras I/CDC parasite strain (6) , and was chosen because it can be selected for expression of VSA PAM reactive with PAM1·4 and PAM8·1. All parasites were grown in 0 + erythrocytes as described (3) .
Selection for VSA PAM expression was done essentially as described elsewhere (7) . In brief, monoclonal PAM1·4 or PAM8·1 antibodies were immobilized on Protein A-coated magnetic beads (Dynal) and mixed with 3D7-or HB3-IEs.
IEs adhering to the antibody-coated beads were isolated in a strong magnetic field and subsequently returned to in vitro culture. Selection protocol was repeated when multiplication of the antibody-selected parasites allowed it. VSA expression was assessed by flow cytometry analysis as described elsewhere (8) . We used plasma from 10 P. falciparum -exposed multigravidae, 10 sympatric men, and 10 nonexposed controls to assess the sex-specificity of VSA recognition. According to the original criteria (9), sex-specific recognition requires that (i) levels of IE-surface reactive IgG are significantly higher in P. falciparum -exposed multigravidae than in sympatric men, and that (ii) the difference in IgG levels in P. falciparumexposed men and nonexposed controls is not statistically significant. We used term plasma from 30 P. falciparum -exposed women (10 pregnant for the first time, 10 for the third time, and 10 for the fifth time) to assess parity-dependency, which requires a statistically significant relationship between IgG levels and number of pregnancies in P. falciparumexposed women (9) . Parasite isolates were only considered to express VSA PAM if plasma IgG recognition of IEs was both sex-specific and parity-dependent.
Late-stage IEs were isolated by magnetic separation as described (8) and returned to culture overnight to obtain ring-stage IEs. Genomic DNA was isolated with a QIAamp blood kit (Qiagen), and total RNA extracted (TRIzol, Invitrogen) and treated with DNase I (Invitrogen) for 30 min (10) . The absence of DNA in RNA samples was confirmed as described (11) . Reverse transcription was performed using Superscript II (Invitrogen) and random hexamer primers, followed by real-time PCR to quantify var transcript abundances as described (11) . We used primer pairs specific for the 59 var genes in the 3D7 genome (12) and for 45 var genes in the HB3 genome (Table S1 in Supporting Information). The latter were tested on genomic DNA dilutions to ascertain appropriate fragment size, melting temperature, and amplification efficiency compared to the internal control seryl-tRNA synthetase . All primer pairs varied less than two Ct values from that of the internal control and amplified single fragments of the expected sizes.
Differences in antibody levels among plasma from P. falciparumexposed multigravidae, P. falciparum -exposed men, and nonexposed control donors were analysed by one-way anova , or by one-way analysis of ranks for non-normal distributed data. If statistically significant ( P < 0·05) overall differences were detected, significant pair-wise 
The VSA expressed on the surface of unselected 3D7-and HB3-IEs were significantly better recognized by plasma IgG from P. falciparum -exposed donors of both sexes than by IgG from nonexposed donors, whereas levels in exposed men and multigravidae were not significantly different from each other. Furthermore, IE surface-reactive IgG levels did not depend on parity (Table 1 and Figure 1a ). Thus, unselected 3D7 and HB3 both showed typical non-PAM-type plasma antibody recognition patterns of IE surface-expressed VSA. This was confirmed by the nonreactivity of 3D7-and HB3-IEs with all eight VSA PAM -specific monoclonal antibodies (Figure 1d ). When VSA expression was re-assessed after three rounds of selection (about 6 weeks after the first round of selection), the recognition of 3D7-and HB3-IEs selected on either PAM1·4 or PAM8·1 all showed an indeterminate VSA phenotype, where one but not both criteria for sex-specific antibody recognition were met (Table 1 and Figure 1b ). PAM8·1-selected 3D7 remained nonreactive with the four monoclonal antibodies used for testing at this time (PAM1·4, PAM3·10, PAM4·7, and PAM8·1), whereas the other three parasite lines showed reactivity with at least one of them (Figure 1e ). These results suggested that further rounds of selection might lead to definite VSA PAM expression, at least for PAM1·4-selected 3D7, PAM1·4-and PAM8·1-selected HB3. Indeed, PAM1·4-selected 3D7, as well as PAM1·4-and PAM8·1-selected HB3 had all acquired a typical sex-specific and parity dependent VSA PAM expression pattern after four additional rounds of selection (Table 1 and Figure 1c ), and reacted with all the monoclonal antibodies except the VAR2CSA DBL5-ε -specific PAM4·7 (Figure 1f ). In contrast, 3D7 selected seven times for reactivity with PAM8·1 retained an indeterminate sex-specificity pattern also seen after three rounds of selection, did not acquire the parity-dependent pattern typical of VSA PAMexpressing lines (Table 1) , and did not react with any of the eight VSA PAM -specific monoclonal antibodies (Figure 1f ). Thus, PAM8·1 could not be used to select 3D7 for VSA PAM reactivity, consistent with the absence of the predicted epitope for this antibody in the VAR2CSA DBL3-X domain of this parasite (3).
Transcripts of var2csa (PFL0030c) constituted 7% of total measured var gene transcripts in unselected 3D7 parasites, increasing to 80% after seven rounds of PAM1·4 selection. In contrast, PAM8·1 selection did not affect the proportion of var2csa transcripts in 3D7 (Figure 1g ). The HB3 genome contains two var2csa paralogs, var2csa-A and var2csa-B (13) . In unselected HB3 parasites, var2csa-A transcripts constituted 1% of the measured var transcripts, increasing to 28% and 4% after seven rounds of selection by PAM1·4 and PAM8·1, respectively (Figure 1h ). Transcript levels of the var2csa-B gene increased from 23% to 57% and 92% after seven rounds of selection by PAM1·4 and PAM8·1, respectively (Figure 1h ). No var transcript other than a Human monoclonal VSA PAM -specific antibody used for selection. b Determination of sex-specificity involves a two-step procedure: first establishment that IE surface-reactive IgG levels in at least one of the three donor categories (exposed multigravidae, exposed men, and unexposed controls) differs from at least one other category. Only if this is the case (as it was here: P < 0·001 in all cases), can sex-specificity be confidently assessed by post hoc testing for significant (P < 0·05) pair-wise differences. The result of this post hoc testing is indicated here as No: none of the two post hoc criteria for sex-specificity were met, Indeterminate: only one of the criteria was met. Yes: both criteria were met. See Materials and Methods for further details.
var2csa showed marked changes, and var2csa was therefore the dominant transcript in all the parasites following antibody selection. The dominance of var2csa-B transcripts relative to var2csa-A in HB3 after PAM8·1 selection, suggested that the DBL3-X domain in the protein encoded by var2csa-B might be of the FCR3-type recognized by PAM8·1, whereas the corresponding domain encoded by var2csa-A might be of the 3D7-type (PFL0030 c) not recognized by PAM8·1 (2).
Recognition of IEs by plasma IgG from 10 P. falciparum-exposed multigravidae (Exp. MG), 10 sympatric men (Ex. men) and 10 nonexposed controls (Unexp. ctrls) before selection (a), and after three (b) or seven (c) rounds of selection by human monoclonal IgG antibody PAM1·4. Results in panels A-C are presented as medians (horizontal line), central 50% of data points (boxes), central 80% of data points (whiskers) and outliers (•). In addition, statistically significant (P < 0·05) pair-wise differences are indicated by heavy horizontal bars along the top of the panels. Recognition of IEs by human monoclonal VSA PAM -specific IgG antibodies before selection (d), and after three (e) or seven (f) rounds of selection. Results in panels D-F are presented as individual data points. Negative cut-off, defined as the upper level of recognition of unselected parasites, is indicated as a dashed horizontal line. The proportion of var2csa transcripts among all var transcripts in 3D7 (g) and HB3 (h) before selection and after seven rounds of selection. Panel (i) shows the amino acid sequence of the PAM8·1-specific region of VAR2CSA DBL3-X in the two VAR2CSA paralogs in HB3, FCR3, and 3D7 parasites. Amino acid differences between the two HB3 sequences and the PAM8·1-reactive FCR3 sequence are underlined. However, var2csa-A and var2csa-B encode an identical amino acid sequence in the region spanning the PAM8·1 epitope, and this sequence was of the FCR3-type (Figure 1i ). The var2csa-A : var2csa-B transcripts may therefore reflect a founder effect.
The VSA subset VSA PAM is expressed by P. falciparum involved in pregnancy-associated malaria (PAM). VSA PAMspecific IgG mediates acquired immunological protection against PAM, and the PfEMP1 variant VAR2CSA appears to be the main or only target of these antibodies. VAR2CSA is therefore the leading candidate for development of vaccines against PAM. We used monoclonal human IgG antibodies to select erythrocytes infected by two genotypically distinct laboratory P. falciparum clones derived from nonpregnant donors for expression of VSA PAM . Parasites acquiring expression of VSA PAM following selection showed increased levels of transcripts encoding the PfEMP1 variant VAR2CSA, which appears to be the only PAM-type VSA in the P. falciparum genome. The results obtained in the study are important for several reasons.
First they support the hypothesis that all P. falciparum parasites have the capacity to express VSA PAM . This hypothesis is supported by previously published data that all P. falciparum genomes appear to contain at least one paralog of the gene encoding the only known VSA PAM -type antigen, VAR2CSA (11, 14) . Furthermore, this gene is selectively transcribed by placental parasites (and following selection for adhesion to CSA in vitro) and VAR2CSA is expressed on the IE surface (15) (16) (17) .
Second, they indicate that P. falciparum parasites regularly and spontaneously switch to expression of VSA PAM in the absence of an external signal, for example pregnancyassociated hormonal changes. It has been speculated that switching to VSA PAM expression requires signals from the pregnant host, for example hormones, and that selection therefore might not be possible unless the parasite is derived from such a host. It has also been argued that selection of parasites by panning on CSA in vitro might result in expression of antigens of dubious relevance to the antigens expressed as a result of in vivo selection occurring in the pregnant woman. Our data show that switching to expression of genuine VSA PAM can occur in vitro in the absence of external signals, in line with other recent evidence (18) . By extension, these findings suggest that switching to VSA PAM in vivo also occurs spontaneously regardless of the pregnancy status of the host. In a pregnant host, such parasites will often be at a selective advantage (because of the frequent absence of VSA PAM -specific immunity in women of low parity) (19) , whereas they appear to be unable to survive in a nonpregnant host (20) .
Third, our results support the hypothesis that although interclonal variation in VAR2CSA is the result of antibodydriven positive selection, the diversity of functionally important antibody epitopes in the molecule is constrained (21) . Thus, some parasites can express VAR2CSA without being vulnerable to recognition by certain VAR2CSAreactive antibodies, because of variation in defined parts of VAR2CSA. The persistent nonrecognition by PAM8·1 of VAR2CSA-expressing 3D7 is a case in point. At the same time, the PAM8·1 antibody, originally identified by its reactivity with VSA PAM -expressing FCR3-IEs (3), was effectively selected for VSA PAM expression in the genetically unrelated HB3 clone. The significance of these findings is underscored by the fact that PAM8·1 efficiently opsonizes IEs for phagocytosis as well as interferes with IE adhesion to CSA (Barfod et al. in preparation) . Our study also corroborates existing evidence that PAM1·4 recognizes most if not all VSA PAM -expressing parasites by recognizing a functionally highly constrained and conformation-dependent discontinuous epitope in VAR2CSA (3). Like PAM8·1, PAM1·4 is an opsonin and capable of interfering with IE adhesion to CSA (Barfod et al. in preparation) . Therefore, the PAM1·4 epitope is a highly attractive candidate for development of a PAM-specific vaccine, and its characterization is a matter of the highest priority in our current research. Although the theoretical possibility of another, non-PfEMP1 target of PAM1·4 remains, available evidence point to VAR2CSA.
In conclusion, our evidence support current efforts to develop PAM-specific vaccines based on VAR2CSA and highlight the versatility of human monoclonal antibodies generated from clinically immune donors in these investigations. Stimulation of ribosomal frameshifting by antisense LNA Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element. Programmed ribosomal frameshifting is a translational recoding event that increases the versatility of gene expression. It is mainly utilized by eukaryotic RNA viruses (1) (2) (3) , though some prokaryotic (4) and mammalian genes (5) (6) (7) are also controlled by ribosomal frameshifting. The requirements for À1 ribosomal frameshifting are the presence of a slippery heptanucleotide sequence X XXY YYZ (where X can be A, U, G or C; Y can be A or U; and Z does not equal Y; the spaces indicate the original reading frame) (8) followed by a downstream structural element, such as a pseudoknot, a hairpin or an antisense oligonucleotide duplex [for reviews, see (9) ]. Although the mechanism of frameshifting is still elusive, a promising model has been proposed by Brierley and co-workers using cryo-electron microscopy to image mammalian 80S ribosomes (10) . In their model, the ribosome is paused by its inability to unwind a pseudoknot structure resulting in a blockage of the A-site by eEF-2. During translocation, the P-site tRNA is bent in the 3 0 -direction by opposing forces. To release the tension, the P-site tRNA may un-pair and subsequently re-pair in the À1 frame with a certain frequency, followed by A-site tRNA delivery into the new À1 reading frame. These and other recent data obtained by mechanical unfolding of frameshifter pseudoknots suggest that mRNA secondary structures with certain conformational features that resist ribosomal helicase-mediated unwinding and eEF-2 catalyzed translocation are key players in ribosomal frameshifting.
Small oligonucleotides have been used for several years to regulate gene expression by RNaseH-dependent RNA degradation (11) , blocking translation (12) , or re-directing splicing (13) . More recently, microRNAs (miRNAs) (14) and small interfering RNAs (siRNAs) have appeared on the scene of post-transcriptional gene regulation (15) . siRNAs may be effective in treatment of chronic hepatitis-B virus infection (16) , HIV infection (17) , cancer (18) and age-related macular degeneration (19) . Very few antisense oligonucleotides, for example against the bcl-2 oncogene have reached the stage of clinical trials (20) or have actually been approved by the FDA, for instance for the treatment of human cytomegalovirus retinitis (21) .
Enhancing the stability of small oligonucleotides to prolong circulation and meanwhile increasing target specificity are major concerns for therapeutic applications. Various kinds of modifications in backbones, sugars or even analogs have already been studied extensively [for reviews, see (22, 23) ] to meet these requirements. Locked nucleic acid (LNA) is a rather novel nucleic acid analog comprising a class of bicyclic high-affinity RNA analogs in which the furanose ring of LNA monomers is conformationally locked in an RNA-mimicking C3 0 -endo/ N-type conformation (24) . The LNA modification also resists degradation by cellular nucleases. Furthermore, introducing LNA into DNA or RNA oligonucleotides improves the affinity for complementary sequences and increases the melting temperature by several degrees (25) . A recent study showed that LNA/DNA mix-mers against miRNA-122 can be acutely administered at high dosage with long lasting effects without any evidence of LNA-associated toxicities or histopathological changes in the studied animals (26) . These data suggests that LNA is a promising candidate for small oligonucleotide applications.
We and others have demonstrated that small RNA oligonucleotides are able to mimic the function of frameshifter pseudoknots or hairpins by redirecting ribosomes into new reading frames (27, 28) . In this article, we have investigated the length and concentration of RNA oligonucleotides for optimal frameshifting, as well as the effects of introducing LNA-type sugars in DNA oligonucleotides.
The À1 ribosomal frameshifting events were monitored by the SF reporter construct described earlier (27) . Complementary oligonucleotides (Eurogentec, Liege, Belgium) SF462 (CTAGTTGACCTCAACCCTTGG AA) and SF463 (CATGTTCCAAGGGTTGAGGT CAA) and SF468 (CTAGTTGAGCGCGCTGGAGGC CATGG) and SF469 (CATGCCATGGCCTCCAGCGC GCTCA) were annealed and ligated into SpeI/NcoI digested SF reporter to construct the SF462 and SF468 templates, respectively. All constructs were verified by DNA sequencing on an ABI PRISM Õ 3730xl analyzer (LGTC, Leiden, The Netherlands). RNA oligonucleotides (except for RNA13 which was obtained from Invitrogen) were purchased from Dharmacon (Lafayette, USA). The RNAs from Dharmacon carried a 2 0 -O-ACE protection group, which was removed by incubation with 100 mM acetic acid pH 3.8 and TEMED at 60 C for 30 min. The sequences of RNA oligos were as follows:
RNA6: GCGCGC, RNA9: CCAGCGCGC, RNA12: C CUCCAGCGCGC, RNA15: UGGCCUCCAGCGCGC, RNA18: CCAUGGCCUCCAGCGCGC, 18RNA: GCG CGCUGGAGGCCAUGG, and RNA13: CCAAGGGG UUGAGG.
DNA and LNA/DNA mix-mers were synthesized by Eurogentec. Custom oligonucleotides were extracted by phenol/chloroform followed by ethanol precipitated before use. The sequences of DNA and LNA/DNA mix-mers were as follows (lower case represents the LNA modification and capital represents DNA): 
Plasmids were linearized by BamHI and purified by phenol/ chloroform extraction followed by ethanol precipitation. In vitro transcription was conducted by SP6 RNA polymerase and carried out in the 30 ml reaction mixture of: 1 mg of linearized template, 5 mM of rNTPs, 20 units of RNase inhibitor and 15 units of SP6 RNA polymerase with buffer (all from Promega, Benelux). After 2 h incubation at 37 C, the integrity and quantity of transcripts were checked by agarose gel and appropriate amount of the RNA were diluted in nuclease free water for in vitro translation.
In vitro translations were carried out in nuclease treated rabbit reticulocyte lysate (RRL) (Promega). The amount of mRNA was 0.025 pmol and different amounts of oligonucleotides (0.025-15.625 pmol) were mixed with template for 20 min at room temperature. After incubation, 4 ml of RRL, 0.01 mM amino acids mixture except methionine, 2 mCi of 35 S methionine (10 mCi/ml, MP Biomedicals, in vitro translational grade) were added in total volume of 10 ml and incubated at 28 C for 1 h. After translation, samples were mixed with 2Â Laemmli buffer, boiled at 90 C for 5 min and resolved by 13% SDS polyacrylamide gels. Gels were fixed in 10% acetic acid and 30% methanol for 20 min, dried under vacuum, and exposed to phosphoimager screens (Biorad). The screen was scanned and the 0 frame and À1 frameshift protein products were quantified by Quantity One software (Biorad). Frameshift percentages were calculated by dividing the amount of À1 frameshift product by the amount of 0-frame and À1 frameshift products after correction for the number of methionines in the protein sequence, multiplied by 100.
Determination of the melting temperature of oligonucleotide duplexes RNA oligonucleotide 18RNA (5 0 GCGCGCUGGAGGC CAUGG3 0 , Dharmacon, USA) was mixed in a 1:1 molar ratio with RNA18, DNA18 or one of the various DNA/ LNA mix-mers, in UV-melting buffer (100 mM NaCl, 10 mM Cacodylate acid, pH 6.8). The analysis was performed on a Varian Cary 300 spectrophotometer using temperature ramps of 0.25 C /min during heating and cooling. The absorbance at 260 nm was recorded and normalized to the blank control.
Although antisense oligonucleotides were found to induce ribosomal frameshifting (27, 28) , the optimal number of base pairs has not been addressed yet. To investigate this we designed antisense RNA oligonucleotides that are 6, 9, 12, 15 and 18 bases complementary to the region downstream of an UUUAAAC slippery sequence in our reporter plasmid SF468 ( Figure 1 ). First, titration with RNA6 and RNA9 oligonucleotides revealed that a 625-fold molar excess of oligonucleotides over mRNA resulted in the highest level of frameshifting ( Figure 2a) ; this ratio was used in the following experiments. The shortest oligonucleotide, RNA6, was not capable of inducing significant levels of frameshifting (Figure 2b) , whereas RNA9 induced $3.5% of frameshifting. Maximum levels were obtained with RNA12, RNA15 and RNA18; all three induced $12% of frameshifting. In the following experiments oligonucleotides between 12 and 18 nt in length were used.
Since we have absent knowledge about the efficacy of LNA-induced ribosomal frameshifting, LNA/DNA mix-mers of 18 nt in length were designed to investigate this ( Figure 3) . A DNA oligonucleotide, as expected, was less capable (3.5%) of inducing frameshift due to the lower thermodynamic stability of RNA-DNA duplexes, see also below. Surprisingly, substituting the 3 0 -cytosine and guanosine in this DNA oligonucleotide by their LNA analogs enhanced its frameshift inducing capacity to 8.7%, i.e. as high as an RNA oligonucleotide (8.8%). Increasing the LNA content of this oligonucleotide further did not lead to higher frameshifting. On the contrary, the efficiency of LNA4 was with 7.7% lower than that of LNA2 and that of LNA6 was a mere 1.1%. Since the overall translation efficiency seemed not affected by LNA6 we suspected an effect of the oligonucleotide itself (see below).
To demonstrate that the enhanced effect of LNA oligonucleotides is a general feature we designed another construct (SF462) in which the target sequence was replaced by an unrelated sequence ( Figure 4 ). LNA/DNA mix-mers were designed in which nucleotides starting from the 3 0 -end were gradually replaced by LNA ( Figure 4 ). Increasing the number of LNAs from one to two and four in these DNA oligonucleotides improved their frameshift inducing ability, reaching an apparent optimum of 7.0% with four LNA substitutions. Further increase of the LNA content to 6 nt (LD6) did not improve frameshift efficiency, but, on the other hand, LD6 also did not lead to the dramatic decrease as observed above for the LNA6 oligonucleotide applied in the SF468 construct. We suspected that (partial) self-complementarity may be limiting the effective concentration of free LNA/DNA oligonucleotides. To check this possibility, we ran all the oligonucleotides on a non-denaturing polyacrylamide gel. Figure 5 showes that the LNA6 oligonucleotide indeed migrated more slowly indicative of partial dimer formation, presumably by intermolecular base pairing of the palindromic GCGCGC sequences in each oligonucleotide (compare the migration to that of the full dimer formed by annealing of oligonucleotides DNA18 and 18DNA). The LD series, as predicted, migrated as monomers. We noted that LD2, though loaded in equal amount, based on its UV absorbance, showed a higher affinity to ethidium bromide than its counterparts. At present we have no explanation for this unexpected behavior of LD2, since its migration and therefore its conformation was identical to the other LNA/DNA mix-mers.
These results demonstrate that LNA modifications indeed enhance the antisense-induced frameshifting efficiency probably due to higher thermodynamic stability and RNA-like structural properties. This phenomenon appears to be general, at least in our experiments.
To investigate which positions in a DNA oligonucleotide would exert the largest effect when substituted by an LNA analog, we designed LNA/DNA mix-mer mutants based on LNA2, which is the most efficient LNA/DNA mix-mer in our experiments and would give a good read-out. When the two LNA substitutions were moved two positions more inward (L2-1), compared to LNA2, frameshift efficiency decreased to 6.7% ( Figure 6 ). However, when the LNA modifications were moved another two positions more inward (L2-2), activity dropped to 2.7% ( Figure 6 ) which is comparable to an unmodified DNA oligonucleotide. Similarly, when the LNA groups were introduced at the other end of the oligonucleotide, activity was as low as DNA18 ( Figure 6 ). Finally, L2-4, in which the first and fourth position were LNA, was only half as efficient as LNA2. These results indicate that the choice of the location of the LNA modifications is crucial for the frameshift-inducing efficiency of an oligonucleotide.
Theoretically the position effect of the LNA substitutions could simply be explained by differences in thermodynamic stability of the resulting mRNA/oligonucleotide duplexes. To investigate this possibility we carried out Table 1 . The T m of the 18RNA/RNA18 duplex was the highest with 82 C in agreement with its high frameshifting efficiency. The 18RNA/DNA18 duplex had a much lower T m of 72 C, which is expected for an RNA/DNA hybrid, and also agreed with the lower frameshifting efficiency. The LNA substituted oligonucleotides, all had higher T m s (+4 to +9 C) than DNA18. The T m of L2-2 was with 81 C almost as high as that of RNA18.
Remarkably there was no correlation between the T m of the LNA oligonucleotides and their frameshifting inducing capacity. For example, the T m of LNA2 was rather low with 76 C but it had the highest frameshifting activity, and L2-2, which had the highest T m , actually had the lowest frameshifting activity. T m s of L2-3 and L2-4 were identical but their frameshifting activities were 2.3 and 4.6%, respectively. We also noted that both L2-2 and L2-3 were comparable to DNA18 in frameshifting activity but formed far more stable duplexes. These data suggest that the position effect of the LNA substitutions is related to the mechanism of frameshifting and not per se to their thermodynamic stability.
Previously, we have demonstrated that antisense oligonucleotides can induce high levels of À1 frameshifting (27) . The optimal length of small antisense oligonucleotides, however, was not investigated. Understanding the optimal length of trans-acting oligonucleotides that can induce the most efficient frameshifting and, at the same time, escape RNAi interference will be an important issue for future in vivo applications. Here we found that maximum levels of frameshifting were obtained with oligonucleotides of 12 nt and more. This is comparable to the stem lengths (S1+S2) of known examples of highly frameshift inducing H-type pseudoknots, such as the 6+6 bp of the Simian retrovirus typeÀ1 pseudoknot (29) , the 11+6 bp of the minimal Infectious Bronchitis virus (IBV) pseudoknot (30) , and the 6+6 bp chimeric Mouse Mammary Tumor virus (MMTV)-IBV pseudoknot (31) . In addition, in known examples of hairpin-induced frameshifts, the stem length of hairpins is around 12 bp (32) (33) (34) . This may imply that a full helical turn of an RNA helix either in one single stem or in two stacking stems of a pseudoknot (S1+S2) is selected by viruses to induce efficient ribosomal frameshifting.
In addition to RNA oligonucleotides, we demonstrated that LNA/DNA mix-mers are also capable of stimulating efficient À1 ribosomal frameshifting in contrast to DNA oligonucleotides. Replacing 2 nt in a DNA oligonucleotide by LNA was already sufficient to reach the same level of frameshifting as with a comparable RNA oligonucleotide. However, the excellent affinity of LNA oligonucleotides could be a double-edged sword in certain cases. In our experimental system, the oligonucleotides are partly self-complementary and this resulted in the formation of dimers ( Figure 5 ), which were apparently unable to induce frameshifting (LNA6, Figure 3 ). Hence, LNA substitutions should be optimized in a sequence that is prone to form dimers. In our SF462 construct (Figure 4) , the optimal number of LNA substitutions to induce the most significant amount of frameshifting is four. LD6 with two additional LNA substitutions did not improve the efficiency. Thus, our results suggest that the first 4 bp are critical for antisense-induced frameshifting. A likely explanation is that when a ribosome that is translating the slippery sequence, the helicase active site is around position +11, with respect to the first nucleotide of the P-site, which is close to the first base pair of the mRNA/ oligonucleotide duplex (35) . Increasing the local thermodynamic stability in this region may prevent ribosomes to unwind RNA structures, causing ribosomal pausing at the slippery sequence, and finally results in a higher frequency of ribosomal frameshifting.
Our data also showed that a single LNA modification is not sufficient to turn a DNA oligonucleotide into an efficient frameshift inducer but that a second LNA is needed. The best position for the second modification appeared to be also close to the 3 0 -end of the oligonucleotide. Although one could expect that spacing of two LNA groups by two non-modified sugars as applied in probes for miRNAs, results in the optimal induction of the 3 0 -endo conformation in the neighboring sugars (36), this was not the case in our frameshift assays. Here such a spacing was less efficient (see data for L2-4, Figure 6 ). However, we have not investigated if possible differences of self-dimerization behavior of these oligonucleotides accounts for the different stimulating activities, since such effects were only observed when six LNA modifications were introduced in an oligonucleotide of this sequence.
The observation that different positions of LNA substitutions induced different levels of ribosomal frameshifting is interesting. Even though the overall thermodynamic stability of these oligonucleotides is roughly the same, they still create different degrees of barriers for ribosomes to unwind and these differences could be the reason for different level of induced frameshifting.
The finding that local stability at the 3 0 -end of the LNA/ DNA mix-mers is important for frameshifting is in agreement with the observation that in natural examples of frameshift stimulators, most of them have high GC content in the first few nucleotides (1) . Hence, our data support the notion that the stability of the 3 0 -end of the oligonucleotide, which may reside in the active site of the ribosomal helicase, is critical for frameshift-inducing structural elements. In pseudoknots this stability is probably attained by triple interactions, since nature has no other way to increase the stability of a GC-rich A-type helix. Triplex structures have been documented for a number of frameshifter pseudoknots, e.g. BWYV (37), SRVÀ1 (38) and in a telomerase pseudoknot (39) . Several models of ribosomal frameshifting have been proposed (1, 40, 41) . The consistency from these studies is that ribosomal pausing at shifty sites by downstream structural elements is important but that pausing caused by RNA secondary structure, does not always result in frameshifting. In addition, a lack of correlation between the extent of pausing and the efficiency of frameshifting by IBV pseudoknots has been observed (42) . A recent study also showed that pseudoknots with a similar global structure can still induce very different levels of frameshifting although their thermodynamic stabilities were different (43) . These data complicate the view on the role of the downstream structure. Experiments involving simple oligonucleotides such as shown here may be better alternatives to elucidate the role of the downstream element.
Several groups have correlated the mechanical force of unfolding of a pseudoknot with its frameshifting efficiency by using optical tweezers (39, (44) (45) (46) and suggest that frameshift efficiency is dependent on the unfolding force rather than on differences of thermodynamic stability between folded and unfolded states. Since we showed here that antisense oligonucleotides can induce frameshifting presumably by serving as a physical barrier for the elongating ribosome, it will be interesting to measure the strength of these linear oligonucleotides in complex with (a piece of) mRNA by optical tweezers and see if there is a correlation with their frameshifting efficiency.
Finally, several properties of LNA, including its good aqueous solubility, low toxicity, highly efficient binding to complementary nucleic acids, high biostability, and, improved mismatch discrimination relative to natural nucleic acid (47) make LNA a promising candidate for in vivo applications of antisense-induced frameshifting.
Funding for open access charge: Leiden Institute of Chemistry, Leiden university. A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site. Over the past decade, atomic resolution ribosome structures have revealed the locations of critical elements. However, these static images do not reveal the dynamic movements within this complex macromolecule. The ribosome must coordinate multiple activities between spatially and functionally different sites in two subunits. These include three transfer RNA (tRNA)-binding sites, the peptidyltransferase and decoding centers and the elongation factor interacting regions. Events occurring in these regions must be carefully coordinated to assure rapid and accurate decoding of messenger RNAs (mRNAs). Current efforts in the field are focusing on determining the mechanisms by which these functional centers synchronize their actions and communicate with each other.
The eukaryotic ribosome contains nearly 80 intrinsic proteins. The high degree of similarity across species, from the primary amino acid sequences to their tertiary structures, suggests conserved functional roles beyond serving as mere scaffolding for the rRNAs. Ribosomal protein L11 of Saccharomyces cerevisiae is an essential, highly conserved component of the 60S subunit (in bacteria and archaea, the homologous protein is named L5; the yeast nomenclature is used throughout this text to minimize confusion). At the primary amino acid sequence level, L11 is well conserved among eukaryotes ($67-97% identity), while bacterial and archaeal L5 proteins are less well conserved (27-55% identical) (Supplementary Figure S1A ). L11 is uniquely positioned at the interface between the large subunit central protuberance ( Figure 1A and 1B) and the head of the small subunit ( Figure 1A ) (1) (2) (3) (4) (5) . In the small subunit, the head region undergoes significant rotational movement relative to the central protuberance between the pre-and post-translocational states (6) , and the protein-protein interactions between L11 and S18 (S13 in bacteria and archaea) on the small subunit (the B1b and B1c intersubunit bridges) undergo the largest intersubunit structural rearrangements between these two states (7) (8) (9) . These observations suggest that L11 may play a central role as an informational conduit between the two subunits. Detailed analysis of X-ray crystallographic and cryo-EM structures ( Figure 1B ) reveals that the concave surface of the b-sheet portion of L11 interacts with specific nucleotides in the minor groove of 23S rRNA helix 84 (10) . L11 also makes contacts with the helix III and loop C regions of 5S rRNA; these connections have been hypothesized to help stabilize 5S rRNA interactions and may participate in an information signal transmission network linking functional centers within the ribosome (1, 11) . Importantly, the B1b and B1c intersubunit bridges with S18 are the only protein-protein interactions between the two subunits (2, 3, 5, 8) . Analyses of these structures indicate that contacts involving L11 and S18 through the B1b and B1c bridges break and rearrange after eEF-2 binding and ribosome ratcheting, controlled in part by differentially charged amino acid side chains between the two proteins (2, 4, 5, 7, 9, 12) . An internal loop of L11 that we denote the 'L11 P-site loop', which is roughly formed by amino acid residues 48-68, also directly contacts the T-loop of the peptidyl-tRNA in the P-site through tRNA nucleotide 56 (3) (4) (5) . At the level of primary amino acid sequence, the P-site loop is highly conserved among eukaryotes (85-100% identity), while it is less well conserved among bacteria and archaea (42-57% identity) (Supplementary Figure S1B) . At the biochemical level, however, the P-site loop is significantly more homogeneous, containing a large number of well-aligned charged and aromatic amino acids. In particular, A50, F57, R60 and I65 (yeast numbering) are universally conserved. An alignment of the P-site loop structures from yeast, Haloarcula marismortui, Thermus thermophilus and Escherichia coli reveals that the P-site loop is extremely well conserved at the structural level (Supplementary Figure S1C) .
In yeast, L11 is encoded by the paralogous genes RPL11A and RPL11B located on chromosomes 16 and 7, respectively (13) . The 19-kDa proteins are 174 amino acids long and are identical except for an alanine (L11A) to threonine (L11B) difference at the third amino acid position. Analysis of L11 in the late 1980s (a.k.a. L16) showed that expression of either isoform was sufficient for cell viability (14) . However, when expressed as the sole form of L11, RPL11A mRNA transcripts accumulated to only 33-40% of wild-type levels as compared to cells expressing both isogenes, while RPL11B mRNAs accumulated to 60-66%. Expression of either isogene alone also affected 60S subunit assembly: a strain expressing only L11B grew at wild-type rates but synthesized fewer 60S subunits than wild-type cells (although apparently not below a threshold necessary for wild-type growth rates), while strains expressing only L11A grew more slowly than wild-type, and synthesized only 33-40% of wild-type levels of total L11 and 60S subunits (14) . A random mutagenesis screen of RPL11B for cold-sensitive mutants identified alleles that promoted 25S pre-rRNA processing and initiation defects (15) . The specific mutants identified in that study were S34P, S41P, S97F, A98V, S119C and G135D. In Arabidopsis, divergent 5 0 untranslated regions (UTRs) between the two isogenes were found to result in differential expression among plant tissues (16) . In addition to its function as a ribosomal protein, L11 has been implicated in p53 activation through its interactions with HDM2 in the nucleus of human fibroblast cells (17) , and mutant forms of L11 have been linked to Daimond-Blackfan anemia in humans (18) .
Although the structural information suggests that L11 should play a significant role in translation, functional analyses of the protein in this role have not been performed. In this report, a series of mutants were generated using a reverse genetics approach to parse the role of the L11 P-site loop. Detailed biochemical and structural analyses focused on two multi-amino acid mutants with opposing effects on rRNA structure and tRNA binding. We propose that prior to peptidyltransfer, the presence of peptidyl-tRNA in the large subunit P-site positions the L11 P-site loop to interact with the Helix 84 of the large subunit rRNA. After peptidyltransfer, spontaneous translocation of the deacylated tRNA to the large subunit E-site allows the L11 P-site loop to extend into the P-site, breaking contact with Helix 84. By this model, we hypothesize that the L11 P-site loop functions locally as a sensor of the occupancy status of the ribosomal P-site.
Restriction enzymes were obtained from Promega (Madison, WI, USA), MBI Fermentas (Vilnius, Lithuania) and Roche Applied Science (Indianapolis, IN, USA). The QuikChange XL II site-directed specific mutagenesis kit was purchased from Stratagene (La Jolla, CA, USA). DNA sequencing was performed by Genewiz (Germantown, MD, USA). Escherichia coli DH5a was used to amplify plasmid DNA. Transformation of yeast and E. coli and were performed as previously described (19) . YPAD, SD and 4.7 MB plates for testing the killer phenotype were as previously reported (19) . Plasmids for expression of dual luciferase reporters were described previously (20) .
Saccharomyces cerevisiae strain PSY2088 (MAT rpl11a::HIS3 rpl11b::HIS3 ura3-52 leu2D1 trp1D63 his3D200 + YCpL11B URA3), an rpl11a/rpl11b gene deletion strain in which L11 is supplied by a URA3-CEN6 based RPL11B clone, was a generous gift from Dr. Pamela Silver (21) . The L-A and M 1 viruses were introduced into PSY2088 by cytoplasmic mixing (cytoduction) through nonproductive mating with JD758 [MATa kar1-1 arg1 (L-AHN M 1 )] to produce the Killer + strain JD1313 as previously described (19) . Wild-type RPL11B was isolated from yeast strain PSY2088 plasmid (pYCP50L11B URA3). Using flanking BamHI restriction sites, a 2.2-kb fragment of DNA containing both the 525-bp wild-type RPLL11B ORF plus the native 5 0 and 3 0 UTR regions (1228 bp and 485 bp, respectively) was purified by agarose gel electrophoresis. This 2238-bp fragment was ligated into BamHI digested pRS314, a low copy TRP1-selectable plasmid (purchased from ATCC, Manassas, VA, USA) (22) to create pRS314L11B-TRP1. This plasmid served as the template for generation of rpl11B mutants by site directed mutagenesis using the primers listed in Supplementary  Table S1 . Wild-type and mutant pRS314L11B-TRP1 clones were transformed into JD1313, selected for growth on -trp medium, and cells having lost the URA3-based plasmid were identified by their ability to grow in the presence of 5-fluoroorotic acid (5-FOA) (23) .
The effects of temperature and translational inhibitors were assessed by standard 10-fold dilution spot assays. Yeast were grown in H-tryptophan synthetic deletion (SD) media (-Trp) to mid log phase. OD 595 values were obtained, and cells were serially diluted 10-fold from 10 5 to 1 CFU per 2.2 ml and spotted on -Trp plates. Growth was monitored at 20 C, 30 C and 37 C, and pharmacogenetic assays utilized 2 mg/ml paromomycin, 40 mg/ml anisomycin or 30 mg/ml sparsomycin incubated at 30 C for 3-5 days. Killer virus assays were performed as previously described (19) .
The dual luciferase reporter plasmids pYDL-control, pYDL-LA, pYDL-Ty1, pYDL-UAA (20) and pYDL-AGC 218 (24) were employed to quantitatively monitor programmed À1 ribosomal frameshifting, programmed +1 ribosomal frameshifting, suppression of a UAA codon and suppression of an AGC serine codon in place of an AGA argine codon in the firefly luciferase catalytic site respectively. In this study, the reporters were housed in LEU2-based reporters: the 0 frame dual luciferase reporter was pJD419, the L-A dsRNA virus À1 PRF containing reporter was pJD420 and the Ty1 containing +1 PRF reporter was pJD421. Cells were grown overnight in 5-ml volumes of -leu synthetic depletion media to mid log phase (A 595 = 0.8-1.5). Cells were washed, resuspended in lysis buffer (1X PBS pH 7.4, 1 mM PMSF) and lysed using 0.5-mm glass beads with a vortex mixer for 3-5 min at 4 C. Lysates were clarified by centrifugation for 5 min. at 8000 r.p.m. at 4 C. Samples were maintained on ice, and 5 ml of clarified lysate was added to 50 ml of pre-aliquoted Promega LARII reagent, mixed by pipetting, and read in a TD20/20 luminometer. Immediately upon completion of this read, 50 ml of Promega Stop and Glo buffer was added to the tube, pipetted to mix and read again. This was repeated 6-12 times per strain per reporter depending on the consistency of the data. Frameshifting rates were determined by taking the ratio of firefly to Renilla luciferases for each sample, and then taking the ratio of the average ratios of the 0 frame samples to that of test reporter ratios to obtain the rates for both À1 and+1 PRF. These results were then analyzed by t-test to determine statistical significance compared to wild-type levels as previously described (25) . Prior to determining rates of UAA readthrough (nonsense suppression), strains were cured of the endogenous yeast prion [PSI + ] by daily serial passage of cells in -trp liquid media containing 5 mM guanidine hydrochloride for 10 days. Rates of nonsense suppression were determined as previously described (20) using the LEU2selectable 0-frame control pJD419 and in-frame UAA containing reporter pJD702. Missense reporters were based on URA3 plasmids previously described for the sense reporter (20) and for the firefly luciferase 218 arginine codon (AGA) to serine (AGC) missense reporter plasmid pYDL-AGC (24) . Methodologies were the same as those for other dual luciferase assays described above.
Cells were grown overnight in a 30 C shaker in 500 ml of YPAD media to mid-log phase (OD 595 0.8-1.5), cooled to 4 C for 1 h to allow ribosomes to run off of transcripts while remaining tightly coupled. Cells were harvested by centrifugation and washed three times with 40 ml 0.9% KCl solution. Cell pellets were stored at À80 C until needed, at which time they were thawed and resuspended in 1 ml binding buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 60 mM NH 4 Cl, 2 mM DTT, 1 mM PMSF) per gram of cells. Cells were lysed with a 1:1 vol of Zirconian beads (BioSpec, Bartlesville, OK, USA) and disrupted using two 2-min pulses of a minibead beater. Lysates were clarified by centrifugation at 20 000 r.p.m. (50 000 g) using an MSL-50 rotor at 4 C for 25 min. Ribosomes were chromatographically purified using Sulfolink beads (Pierce, Rockford, IL, USA) as previously described (26) , and eluted from the resin in 8 ml of elution buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 500 mM KCl, 2 mM DTT, 0.5 mg/ml heparin). Eluted ribosomes were treated with 2 mM puromycin and 1 mM GTP for 30 min at 30 C and were layered on top of a 22-ml glycerol cushion [50 mM HEPES-KOH pH 7.6, 10 mM Mg(CH 3 COO) 2 , 50mM NH 4 Cl, 1 mM DTT, 25% glycerol] and pelleted by centrifugation at 30 000 r.p.m. at 4 C for 18-20 h. Pellets were washed with 1 ml of storage buffer [50 mM HEPES-KOH pH 7.6, 10 mM Mg(CH 3 COO) 2 , 50mM NH 4 Cl, 1 mM DTT, 25% glycerol], and resuspended in 200-400 ml of storage buffer. Concentrations were determined spectrophotometrically (1 OD 260 = 20 pmol ribosomes). The salt-washed ribosomes were aliquoted and stored at À80 C for up to 3 months. Ribosomal rRNA quality was checked on 1.3% agarose gels and rRNA to protein ratios were monitored by determining OD 260 to OD 280 ratios. Polysome profiles were obtained by sucrose density gradient centrifugation as previously described (27) . Samples were split, and 10 ml of dimethyl sulfoxide (DMSO) was added to half of the samples, while 10 ml of 60 mM 1M7 was added to the other half. Samples were incubated at 30 C for 20 min. Ribosomes were precipitated by the addition of 275 ml of ice-cold 100% ethanol and stored at À20 C for 1-2 h. Ribosomes were pelleted by centrifugation at 10 000 r.p.m. for 2 min and resuspended in lysis buffer and rRNAs were isolated using an Ambion (Austin, TX, USA) RNAqueous Õ -Micro RNA isolation kit. Optical densities were taken at 260 nm and 280 nm to monitor the quantity and quality of RNA, and samples were resuspended at a concentration of 1 mg rRNA/7 ml in pure water. HPLC purified oligonucleotide primers purchased from IDT (Coralville, IA, USA) are listed in Supplementary Table 2. Oligonucleotides were resuspended to 25 pmol/ml, 3 0 end labeled with g[ 32 P]ATP with T4 polynucleotide kinase (Roche, Indianapolis, IN, USA), and purified from free radiolabeled nucleotide by passage through a MicroSpin G-25 column (GE Healthcare, Piscataway, NJ, USA). Annealing reactions utilized 1 mg of modified rRNAs and 3 ml of labeled oligonucleotide heated at 80 C for 1 min, followed by a 4-7-min incubation at 5-10 C below the T m of each oligonucleotide. Annealed rRNA/ primers (2 ml each) were added to 3 ml of cold enzyme mix [0.25 ml 10 mM dNTP, 0.25 ml 100 mM DTT, 1 ml 5X Superscript III Buffer, 0.25 ml Superscript III (Invitrogen Life Technologies, Carlsbad, CA, USA), 1.25 ml H 2 O]. For sequencing samples, an additional 1 ml of each ddNTP was added to each C, T, A, G, sample, respectively. Primer extension reactions were performed at 52 C for 25 min, with potential 2-min-long extensions preceding the 52 C at lower temperatures depending on the individual T m values of the primers. Denaturing RNA loading dye (2 ml) was added to each sample, heated to 94 C for 2.5 min, and samples were resolved through 6% urea-acrylamide denaturing gels. Gels were dried and radiolabeled samples were visualized by phosphorimagery.
The published structures for the 70S ribosome from E. coli [PDB accession numbers: 2AVY, 2AW4; (2)], as well as yeast 80S structures from yeast (1S1I, 1S1H, 3JYV, 3JYW, 3JYX; (4, 12) ] were used in the analysis of this work and the generation of figures. Published T. thermophilus 70S subunits containing A-site, P-site and E-site Phe-tRNA were also employed (1G1X, (5) . All structures were visualized and manipulated using MacPyMol software (30) .
The visualization of a single salient loop of L11 interacting with peptidyl-tRNA indicated that it might play a vital role in sensing peptidyl-tRNA occupancy status and transmitting this information to other functional centers of the ribosome. As cells expressing RPL11B alone were healthier than those solely expressing RPL11A, genetic manipulations began with the yeast rpl11AD rpl11BD double knockout strain JD1313 expressing wild-type (WT) RPL11B from a low-copy, URA3-selectable episomal plasmid (pRPL11B-URA3). Oligonucleotide site-directed mutagenesis was used to construct a series of mutants, each containing changes of 1, 4 or 10 sequential amino acids ( Figure 1C ). Stretches of amino acids from arginine 51 to arginine 61 in the L11 P-site loop were targeted for site-directed mutagenesis expressed from a low-copy, TRP1-selectable episomal plasmid under control of the endogenous RPL11B promoter (pRPL11B-TRP1). After transformation and selection on SD medium lacking tryptophan (-trp), cells expressing only mutant rpl11B alleles were identified by their ability to grow on SD-trp medium containing 5-flouroorotic acid (5-FOA). Three of the multiple substitution mutants were inviable as the sole forms of L11B. These were R 51 YTVRTFGIR 60 !alanine (i.e. 51-60A); deletion of residues 51-60 (51-60Á); and F 57 GIR 60 !alanine (57-60A). Viable mutants, R 51 YTV 54 !alanine (51-4A), V 54 RTF 57 !alanine (54-7A), R51A, Y52* (mutations including Á, A, R, E, S, I, Q, N, H and F), F57A and R61A were rescued from yeast into E. coli, and the mutations were confirmed by DNA sequencing.
The L11 P-site loop mutants confer temperature-and drug-specific growth phenotypes displayed roughly wild-type growth. Cold sensitivity was assessed at 20 C and both mutants grew at wild-type rates. 51-4A showed enhanced growth at 37 C relative to itself at 30 C, while mutant 54-7A was similar to wild type. R51A grew at wild-type rates at 30 C and 37 C but showed enhanced growth at 20 C. The Y52* mutants displayed mutant-specific effects on growth rates at 30 C, but did not confer significant phenotypes at either 20 C or 37 C. F57A had wild-type growth rates at all temperatures, while R61A showed depressed growth at 30 C, which was rescued at 37 C.
Small molecule inhibitors of protein translation are useful probes for identifying changes in ribosome function. This study utilized three such molecules: paromomycin, anisomycin and sparsomycin. The effects of all three drugs were monitored using dilution spot assays at 30 C on SD-trp media containing various drug concentrations. Paromomycin is an aminoglycoside antibiotic that increases translational error rates by artificially stabilizing codon:anticodon interactions at the decoding center in the small ribosomal subunit (31) . As compared to their intrinsic growth in the absence of drug, both the 51-4A and 54-7A mutants were slightly hypersensitive to 2 mg/ml paromomycin, as were Y52Á, Y52N and Y52H. In contrast, R51A, F57A and R61A were all paromomycin resistant ( Figure 2B ). Anisomycin competes with the 3 0 end of the aa-tRNA for binding to the A-site pocket of the ribosome (32, 33) . Both 51-4A and 54-7A showed anisomycin resistance at 40 mg/ml, as did several Y52* mutants, and R61A ( Figure 2B ). Sparsomycin binds to the P-site and interferes with peptidyl-tRNA binding and peptidyl transfer (33, 34) . 51-4A and 54-7A mutants were hypersensitive to 30 mg/ml sparsomycin, as were most of the Y52* mutants, with the exception of Y52F, which conferred slight resistance to this drug ( Figure 2B ).
The yeast 'killer' system is composed of the L-A helper and M 1 satellite dsRNA viruses (35) . The L-A dsRNA viral genome encodes a capsid protein (Gag), and an RNA-dependent RNA polymerase (Pol) that is synthesized as a Gag-pol fusion protein consequent to a À1 Programmed Ribosomal Frameshifting (PRF) event (36) . The M 1 satellite dsRNA is encapsidated and replicated in L-A encoded viral particles, and the M 1 (+) strand encodes a secreted toxin that kills uninfected yeast through its interactions with the GPI-anchored Kre1p cell wall assembly protein (37) . Changes in À1 PRF efficiency alter the ratio of Gag to Gag-pol, and inhibit the ability of cells to maintain M 1 (19) . To monitor the effects of the mutants on Killer virus maintenance, colonies of JD1313 cells expressing either wild-type or mutant rpl11B alleles were spotted onto a lawn of diploid, Killer À indicator cells. Cells expressing wild-type RPL11B were Killer + as demonstrated by their ability to inhibit growth of the indicator cells ( Figure 2C ). In contrast, isogenic cells expressing the 51-4A, 54-7A and F57A mutants were Killer À . A weak killer phenotype, defined by decreased zones of growth inhibition, was observed in mutants Y52E, Y52N, Y52H and F57A.
The rpL11B mutants affect translational fidelity 'Translational fidelity' is generically used to describe the accuracy of protein synthesis. A series of bicistronic reporter plasmids were used to quantitatively monitor the effects of the L11B mutants on four aspects of translational fidelity: À1 PRF, +1 PRF, suppression of a UAA nonsense codon and incorporation of a missense near-cognate amino acid. In JD1313 cells expressing wild-type RPL11B, À1 PRF directed by the L-A dsRNA viral signal was 6.07% ± 0.16%. This compares favorably with other 'wild-type' strains in our laboratory (normal range from 4% to 8% (20, 25) . The 51-4A mutant promoted increased À1 PRF (1.33 ± 0.06-fold relative to wild type), while 54-7A trended in the opposite direction (0.84 ± 0.03-fold relative to wild type) ( Figure 3 , and Table 1 ). Both these values were statistically significant and correlate well with the Killer À phenotypes. Y52Á, Y52N, Y52E and Y52H mutants also showed increased rates of -1 PRF, with statistically significant rates ranging from Y52H at 1.20-fold wild type to Y52E at 1.49-fold wild-type. Y52A, Y52S, Y52Q and Y52F all had wild-type rates of À1 PRF.
While both À1 and +1 PRF are kinetically driven events, the substrates for the slippage are distinct: À1 PRF requires that both the ribosomal A-and P-sites are occupied by tRNAs, while+1 PRF occurs while the A-site is empty (38) . Rates of +1 PRF were monitored using a cis-acting signal derived from the Ty1 retrotransposable element using pYDL-Ty1. Baseline +1 PRF efficiencies in cells expressing wild-type RPL11B were 10.98% ± 0.30%. 51-4A had no effects on +1 PRF, while 54-7A promoted a small but statistically significant increase (1.18 ± 0.06-fold of wild type; Figure 3 ). Significant changes in +1 PRF were also observed in the Y52A, Y52S, Y52N, Y52E, Y52H and Y52F mutants. mRNA decoding occurs in the small subunit decoding center, and changes in termination codon recognition (nonsense suppression) is another indicator of altered translational fidelity. pYDL-UAA (39) , which contains an in-frame termination codon immediately 5 0 of the firefly luciferase gene, was used to monitor this parameter. The baseline rate of nonsense suppression in cells expressing RPL11B was 0.137% ± 0.003%. The 51-4A mutant slightly improved this aspect of translational fidelity, with nonsense suppression levels decreasing to 0.88 ± 0.04-fold of wild-type levels. 54-7A did not affect UAA recognition (Figure 3) . Y52Á, Y52A, Y52S, Y52N, Y52E and Y52H all promoted increased rates of nonsense suppression ranging from 1.31-to 1.78-fold wild type. pYDL-AGC 218 tests missense suppression levels by monitoring rates of incorporation of an arginine (AGA) near-cognate amino acid instead of a cognate serine (AGC) at the catalytic codon 218 within the firefly luciferase gene as previously described (24) . Thus, in this assay, mis-utilization of near-cognate tRNA Arg at the Ser AGC codon restores firefly luciferase activity. Wild-type missense levels were measured at 0.074% ± 0.002, comparable to previous studies (24) . Mutant 51-4A had significantly higher levels of missense suppression (measured at 1.21 ± 0.03-fold wild-type), while 54-7A did not significantly affect this phenomenon (1.07 ± 0.04 fold wild type) ( Figure 3 ). Missense suppression was not assayed for the single amino acid mutants.
The mutant rpl11b alleles promote opposing effects on tRNA binding to the ribosomal A-and P-sites Sucrose gradient analyses were employed to fractionate cycloheximide arrested elongating ribosomes on mRNAs in lysates generated from JD1313 cells expressing wild-type L11B, 51-4A, and 54-7A. In all strains the 60S peak was smaller than that of the 40S fraction which can be attributed to the presence of only a single copy of RPL11B, which has previously been shown to effectively reduce the number of 60S subunits produced by the cell to 60-66% of true wild-type levels while having no visible phenotypic effect on growth (14) . No significant differences were observed among the samples (data not shown). Phenotypic variation in PRF and in the presence of anisomycin and sparsomycin are indicative of altered interactions between the ribosome and tRNAs. P-site tRNA K d values were determined in vitro by binding 2-fold serial dilutions of N-acetylated-[ 14 C]Phe-tRNA to ribosomes until saturation was achieved ( Figure 4A ), and the resulting data were used to determine steady-state single site binding K d values ( Figure 4B ). Wild-type ribosomes bound this P-site substrate with a K d of 72.3 ± 7.9 nM. The 51-4A mutants promoted a slight increase in affinity for P-site substrate (K d = 50.9 ± 11.2 nM), while 54-7A had the opposite effect (K d = 89.3 ± 10.4 nM). Given the physical interaction between the L11 P-site loop and peptidyl-tRNA, it was imperative to determine whether the observed small changes in P-site affinities promoted by the mutants were biochemically significant. To this end, multiple turnover puromycin reactions were performed. In these experiments, puromycin was added to ribosomes pre-incubated with excess P-site substrate, i.e. Ac-[ 14 C]Phe-tRNA Phe , and accumulation of the peptidylpuromycin product was monitored over time. In these reactions, the first round of peptidylpuromycin synthesis is very rapid. Next, in a slow step, the ribosome intrinsically translocates the deacylated tRNA Phe into the E-site (40), followed by the slow diffusion of Ac-[ 14 C]Phe-tRNA Phe into the P-site where it can react with puromycin. Repetition of this cycle results in slow multiple rounds of product synthesis ( Figure 4C ). Assuming that the L11 mutants do not affect either rates of intrinsic translocation or of Ac-[ 14 C]Phe-tRNA Phe diffusion into the P-site, changes in product accumulation, i.e. K obs , should be due to differences in binding affinities for the P-site substrate. Consistent with this model 51-4A promoted 1.46 ± 0.14-fold increased K obs relative to wild-type ribosomes, while 54-7A decreased K obs to Figure 3 . The L11B mutants promote defects in translational fidelity. Isogenic yeast cells expressing either wild-type or mutant forms of L11B were transformed with dual luciferase reporters and control plasmids and rates of translational recoding were determined. All results are graphed as fold wild type. À1 PRF was measured using the yeast L-A virus frameshift signal. +1 PRF was directed by the frameshift signal derived from the Ty1 retrotransposable element. Nonsense suppression denotes the percentage of ribosomes able to suppress an in-frame UAA termination codon positioned between the Renilla and firefly luciferase reporter genes. Missense suppression rates were evaluated by incorporation of an arginine (AGA) near-cognate amino acid instead of a cognate serine (AGC) at the catalytic codon 218 within the firefly luciferase gene. Error bars denote standard error. P-values are indicated above samples showing statistically significant changes. (Figure 4E and F).
The P-site loop is flexible depending on the occupancy status of the P-site
The highly basic nature of the P-site loop, its interaction with peptidyl-tRNA, and its proximity to 25S rRNA Helix 84 (H84) suggested that it might interact with either of these two RNA components depending on the occupancy status of the P-site. Changes in interactions between the P-site loop and local rRNA structures may in turn propagate outward to more distant regions of the ribosome. To test this, SHAPE (41) (42) (43) was employed to probe for structural alterations in selected regions of the 25S, 18S and 5S rRNAs due to either the L11B mutants or in wild-type ribosomes with occupied or unoccupied P-sites. Due to the large size and complex three-dimensional structure of the ribosome, the entire rRNA content was not examined. Rather, approximately one-third of the rRNA bases were interrogated, focusing on those bases closest to L11, the A-and P-sites, and the decoding center.
In the first series of experiments, salt-washed wild-type and 51-4A, 54-7A, Y52Q and Y52F mutant ribosomes (chosen for structural analyses because they had the most pronounced genetic phenotypes) were treated with 1M7, an electrophile that adds an adduct onto the 2 0 OH groups of solvent exposed base sugars. Modifications were performed on salt-washed ribosomes because they represent the thermodynamic 'ground state' of the ribosome. Thus, the structural changes observed are indicative of changes in the full 'dynamic potential' of the ribosome as opposed to conformations locked in by e.g. occupation of binding sites by tRNAs or ribosome-associated factors. rRNAs were extracted, hybridized with 5 0 [ 32 P]-labeled oligonucleotide primers and reverse transcriptase primer extension reactions were performed. The products were separated through urea-acylamide denaturing gels, and visualized using a phosphorimager. 2 0 -OH ribose modification results in a strong stop 1-nt 3 0 of modified bases, and the intensity of the stops are proportional to the solvent accessibility and flexibility of riboses. Comparison of the protection patterns between wild-type and mutant ribosomes enables identification of specific bases which became protected or deprotected relative to WT.
In all areas examined, rpl11b ribosomes Y52Q and Y52F matched the wild-type rRNA base modification profile (data not shown), while 51-4A and 54-7A ribosomes revealed consistently reproducible differences. The most significant changes in rRNA structure were observed in bases C2675-A2679 (E. coli numbering: C2306-2310) located in the terminal loop of 25S rRNA H84 ( Figure 5A and E). The two mutants promoted opposing patterns of base protection/deprotection in this structure. Specifically, as compared to wild-type ribosomes, 51-4A promoted enhanced protection of this loop, while the loop was deprotected in the 54-7A mutants. Analysis of the recent cryo-EM yeast ribosome structure (4) revealed that these H84 loop bases are located within 3 Å of the stretches of amino acids changed to alanines in both the 51-4A and 54-7A mutants ( Figure 5B ). These findings suggested that the two mutants had the effects of displacing the P-site loop into two opposing conformational states: extended toward the P-site (54-57A), or retracted into H84 (51-54A). To test whether these two states are naturally dependent on P-site occupancy, the experiments were repeated with wild-type and mutant ribosomes with or without tRNA Phe in their P-sites. Consistent with this model, addition of tRNA to the P-site of wild-type ribosomes resulted in slightly enhanced protection of the H84 terminal loop bases closest to the P-site loop (A2676-A2679). Interestingly, C2675 showed significant deprotection when the P-site was occupied by tRNA. This base is on the far side of the terminal end of H84 from the P-site loop, suggesting that H84 itself alters its conformation upon tRNA occupancy of the P-site ( Figure 5C ). 51-4A's H84 bases were unchanged between P-site bound and unbound ribosomes, consistent with the P-site loop positioned in the 'retracted' state in this mutant, although small differences in the protection patterns suggest that the P-site loop is in a slightly different orientation in this mutant. In contrast, while 54-7A ribosomes, i.e. the P-site loop 'extended' state, showed deprotection at all bases (C2675-A2679) for both P-site bound and salt washed ribosomes, bases A2676-A2679 were less deprotected when tRNA was in the P-site and C2675 was even more reactive, consistent with the notion that the P-site loop interacts with H84 when peptidyl-tRNA is in the P-site.
Although no other SHAPE-specific changes were observed, several other phosphodiester bonds 3 0 of specific 25S rRNA bases were reproducibly more, or less, intrinsically labile as compared to wild type ( Figure 5D ). In both mutants, G2531 and G2534 located in expansion segment 31 (ES31) were more stable than in wild-type ribosomes as evidenced by reduced intensity of strong reverse transcriptase stops 1-nt 5 0 of these bases. Additionally, bases A2779-A2780 (E. coli A2407-U2408) located in the terminal loop of Helix 88 were hyper-labile in 51-4A mutant ribosomes as compared to WT, as shown by the presence of strong stops with increased intensity 1-nt 5 0 of these bases. These are mapped onto the two-dimensional structure of yeast 25S rRNA ( Figure 5E ).
The L11 P-site loop is largely comprised of polar amino acids and carries a net positive charge, making it ideal for interactions with the phosphate backbones of nucleic acids, e.g. rRNA and tRNA. Positioned between H84 and the peptidyl-tRNA T-loop, several of its amino acids are within H-bonding distance of H84 ($3.3 Å ), while C56 of the peptidyl tRNA T-loop comes within 2.1 Å of G58 in the L11 P-site loop (4, 5) , suggesting that the L11 P-site loop can directly interact with both of the RNA-based structures. While currently available X-ray crystal structures are unavailable for ratchet-state ribosomes, a recently published examination of tRNA movement through the E. coli ribosome using large-scale analysis of cryo-EM images implicates the P-site loop as a dynamic arm interacting with and moving in relation to tRNAs passing across the P-site (44) . Although these studies were performed at resolutions of 9-20 Å , leaving considerable ambiguity regarding the precise residues involved, they clearly reveal highly dynamic interactions between the P-site loop and both P-site, and E-site tRNAs.
Although death is not a phenotype per se, the inviable mutants are informative nonetheless in so far as they demonstrate that the amino acids F 57 GIR 60 are absolutely required for viability. While F57 is universally conserved, it does not appear to be essential on its own for viability, as witnessed in the mild phenotypes of the F57A mutant. Similarly, all single amino acid changes explored here resulted in viable cells, suggesting a certain degree of biochemical/biophysical redundancy within this essential loop. In support of this notion, the strongest growth phenotypes observed across a range of temperatures and small molecule translational inhibitors were concentrated in the multiple alanine substitutions, i.e. 51-4A and 54-7A, thus directing the bulk of the biochemical and structural analyses to these two mutants.
Analysis of the results of the assays performed on the viable multiple alanine substitution mutants (summarized in Table 1 ) provoke the hypothesis that the L11 P-site loop may dynamically function to help the ribosome sense the occupancy status of the large ribosomal subunit P-site. This is modeled in Figure 6 . When the P-site is unoccupied, the P-site loop can extend into this space, moving away from the terminal loop of H84. Upon occupation of the P-site, the peptidyl-tRNA T-loop displaces the L11 P-site loop, causing its retraction into H84. By this model, the rRNA SHAPE analyses depicting increased protection of Helix 84 by the 51-4A mutant show that this mutant drives the L11 P-site loop equilibrium toward the 'retracted' state. Conversely, increased deprotection of Helix 84 in the 54-7A mutant suggests that this more mimics the P-site unoccupied state, i.e. the 'extended' P-site loop state. This analysis directly explains the P-site binding data. Retraction of the P-site loop from the P-site results in 51-4A ribosomes having higher intrinsic affinity for this substrate while extension of this structure into the P-site creates a steric clash with the peptidyl-tRNA T-loop, resulting in decreased affinity for this substrate. That neither mutant conferred optimal peptidyl-tRNA P-site occupancy may account for their hypersensitivity to sparsomycin, especially for 54-7A in which the P-site loop is already competing with the tRNA for the P-site. Mutants 57-60A, 51-60A and 51-60Á appear to disrupt the normal function of the P-site loop to a lethal level. In addition, the observation that tRNA binding to the P-site results in deprotection of C2675 implicates H84 itself as a structurally dynamic unit. The functional consequences of this are not clear, although it is tempting to speculate that this conformational change may play a role in the structural rearrangements of the B1b and B1c bridges between the pre-and post-translocational states.
The lack of rRNA structural changes in the A-site or in the decoding center suggest that the biochemical and phenotypic effects observed are indirectly due to the changes described above. The reciprocal effects between Ac-aa-tRNA binding with the P-site and aa-tRNA interactions with the A-site are intriguing. In the aa-tRNA binding reactions, the ribosomal P-sites were occupied with daeacylated tRNA. We suggest that in the 51-4A mutant, the P-site ligand is more 'locked' into a suboptimal conformation, which in turn feeds back to the A-site, resulting in decreased affinity for its ligand. Conversely, the lessened ability of 54-7A mutant ribosomes to lock P-site ligand in a suboptimal conformation may account for the increased affinity of these ribosomes for A-site ligand. Anisomycin resistance by both mutants also followed the reciprocal P-site/A-site pattern, i.e. both mutants were sparsomycin hypersensitive. Paromomycin interacts with the decoding center in the small subunit, where it promotes misreading of near-cognate codons in the A-site by stabilizing codon-anticodon interactions (45) . This sensitivity may be attributable to an observed increase in missense incorporation of a near cognate arginine (AGA) over that of the sense serine codon (AGC) in mutant 51-4A. Intriguingly, 54-7A had wild-type levels of missense incorporation suggesting that its sensitivity to paromomycin was indirect. The reciprocal anisomycin/paromomycin phenotypes of the L11 mutants demonstrate the effects of this protein on A-site ligand based ribosomal functions over very long distances. Similar phenotypic patterns were previously observed with mutants of other large subunit components (46, 47) .
The observed effects on -1 PRF are consistent with a recent kinetic analysis demonstrating that aa-tRNA slippage is the most highly weighted parameter in determining the rate at which this process occurs (Liao,P.Y. et al., submitted for publication). Here, increased affinity for aa-tRNA by the large subunit suggests that the 51-4A ribosomes stabilize the frameshifted (i.e. near-cognate) tRNAs, reducing their ability to be proofread, thus promoting increased rates of À1 PRF. This is consistent with the observed increased rates of missense decoding in this mutant. Conversely, post-slippage A-site tRNAs are even less stable in the 54-7A mutants, leading these to be more efficiently proofread, and thus promoting decreased À1 PRF efficiency. In both cases, altering À1 PRF from the optimum 'golden mean' precludes these cells from maintaining the yeast killer virus (19, 48) . Programmed +1 frameshifting is completely dependent on peptidyl-tRNA slippage. Increased +1 PRF in the 54-57A mutant is consistent with decreased affinity for this substrate. The failure to observe decreased +1 PRF in the 51-54A mutant, despite its increased affinity for peptidyl-tRNA, is not entirely clear, although this may be due to the inability of these ribosomes to achieve a threshold beyond which +1 PRF effects can be observed.
The changes in rRNA stability observed in the terminal loop of Helix 88 and in ES31 are intriguing. Chemical protection experiments revealed the terminal loop of Helix 88 is involved in a kissing loop interaction with the terminal loop of Helix 22, and this interaction is apparent in the X-ray crystal and cryo-EM structures (4, 49) . Increased lability at A2779 and A2780 was tRNA H84 L11 S18 P-Site H84 L11 S18 C C Figure 6 . Model: the P-site loop acts as a sensor of the occupancy status of the P-site. (Left) When the large subunit P-site is unoccupied by tRNA, the L11 P-site loop is able to extend into this space leaving the distal loop of H84 partially deprotected from chemical attack. This conformation is favored by the 54-7A mutant of L11B. (Right panel) Occupation of the P-site by peptidyl-tRNA displaces the L11 P-site loop, causing it to tightly retract from the P-site and interact with H84, resulting in increased protection of the H84 terminal loop from chemical attack. H84 likely moves toward the P-site loop slightly, increasing the exposure of C2675 to the surrounding solvent. This conformation is favored by the L11B 51-4A mutant.
previously observed in the Y11C mutant of ribosomal protein L10 (homolog of E. coli L16) located at the base of the aa-tRNA accommodation corridor, and in the É2922C (E. coli U2554) 25S rRNA mutant located in the peptidyltransferase center (50, 51) . The observation that mutations located in three very different and topologically distinct regions of the large subunit conferred similar structural effects suggest that this kissing loop interaction plays an important role in ribosome function. Its location on the cytoplasmic face of the ribosome where deacylated tRNA leaves the molecule implies that the interaction between the terminal loops of Helices 88 and 22 may be involved in gating this deacylated tRNA exit corridor open and closed. This is consistent with the model of allosteric coordination between the A-and E-sites (52, 53) , which would indicate that the defects conferred by all of these mutants on aa-tRNA binding might impair this E-site gating function. The decreased lability of C2531 and G2534 in ES31 is similarly intriguing, raising more questions than answers. No function is currently associated with this expansion segment, but recent cryo-EM analysis shows it to be located on a solvent accessible surface of the large subunit (4) . Perhaps this site is also involved in A-site/ E-site coordination. Alternatively, it may be a site for recognition of defective ribosomes by the nonfunctional ribosome decay apparatus. Involvement of microRNAs in physiological and pathological processes in the lung To date, at least 900 different microRNA (miRNA) genes have been discovered in the human genome. These short, single-stranded RNA molecules originate from larger precursor molecules that fold to produce hairpin structures, which are subsequently processed by ribonucleases Drosha/Pasha and Dicer to form mature miRNAs. MiRNAs play role in the posttranscriptional regulation of about one third of human genes, mainly via degradation of target mRNAs. Whereas the target mRNAs are often involved in the regulation of diverse physiological processes ranging from developmental timing to apoptosis, miRNAs have a strong potential to regulate fundamental biological processes also in the lung compartment. However, the knowledge of the role of miRNAs in physiological and pathological conditions in the lung is still limited. This review, therefore, summarizes current knowledge of the mechanism, function of miRNAs and their contribution to lung development and homeostasis. Besides the involvement of miRNAs in pulmonary physiological conditions, there is evidence that abnormal miRNA expression may lead to pathological processes and development of various pulmonary diseases. Next, the review describes current state-of-art on the miRNA expression profiles in smoking-related diseases including lung cancerogenesis, in immune system mediated pulmonary diseases and fibrotic processes in the lung. From the current research it is evident that miRNAs may play role in the posttranscriptional regulation of key genes in human pulmonary diseases. Further studies are, therefore, necessary to explore miRNA expression profiles and their association with target mRNAs in human pulmonary diseases. A. miRNA definition, biology and function Discovery of microRNA (miRNA) lin-4 was the first short non-coding RNA discovered in 1993 as a regulator of developmental timing in Caenorhabditis elegans [1] . The first non-coding RNA identified in humans was let-7, which has been found involved in the control of developmental timing in humans and animals [2, 3] . Soon it became evident that these short non-coding RNAs are a part of much larger class of non-coding RNAs and the term microRNA (miRNA) was introduced [4] . To date, more than 900 miRNAs in Homo sapiens have been identified (940 in miRBase v15).
MiRNAs are small non-coding RNAs~22 nucleotides (nt) long involved in the negative post-transcriptional gene regulation via RNA interference mechanism [5, 6] . The sequences of miRNAs are highly conserved among plants-microorganisms-animals, suggesting that miRNAs represent a relatively old and important regulatory pathway [7] . MiRNAs belong to the most abundant class of human gene regulators [8] : up to a third of the human genes are regulated by miRNAs [9] . MiRNAs are, therefore, key regulators of numerous genes in biological processes ranging from developmental timing to apoptosis [e.g. [10] [11] [12] [13] [14] ]. It has been speculated that miRNAs may be associated with the regulation of almost every aspect of cell physiology [8] .
MiRNA genes are localized in the non-coding regions or in the introns of protein-coding genes in the genomic DNA. The miRNA genes are much longer than biologically active, mature miRNAs which originate through a multistep process [15] (Figure 1 ). Briefly, transcription by the RNA polymerase II leads to hundred or thousand nucleotides long primary miRNA transcripts (pri-miRNAs) [16] .
A local stem-loop structure of pri-miRNAs is then cleaved in the nucleus by the dsRNA-specific ribonuclease Drosha/ Pasha to 70 nucleotides long precursor miRNA (pre-miRNA) [17] in a process known as "cropping" [18, 19] . Pre-miRNAs are then actively transported from the nucleus to the cytoplasm [20, 21] . In the cytoplasm, pre-miRNAs are subsequently cleaved by RNase III Dicer into~22-nt miRNA duplexes [17, 20] . One strand of the short-lived miRNA duplex is degraded ("passenger" strand, miR*), whereas the other ("guide", miR) strand is incorporated into the RNA-induced silencing complex (RISC) and serves as a functional, mature miRNA [8] . Selection of the "guide" strand is based on the base pairing stability of both dsRNA ends [22, 23] .
Depending on the complementarity between miRNA and 3' untranslated region (UTR) of target mRNA there are two known mechanisms of miRNAs action on mRNAs: 1) target mRNA degradation and 2) translational inhibition with little or no influence on mRNA levels [24] (Figure 2) . Firstly, the deadenylation and subsequent degradation of the target mRNA occurs when miRNA is near-perfectly complementary with target mRNA [25, 26] . A recent study proved that mRNA degradation represents the major mechanism of miRNA regulation [27] . The authors showed that about 84% of all protein-coding mRNA targets undergo degradation while recognized by their cognate miRNA [27] . Secondly, the translational inhibition MiRNAs are transcribed by RNA polymerase II from the genomic DNA as long (hundred or thousand nucleotides) primary miRNA transcripts (pri-miRNAs). A local stem-loop structure of pri-miRNAs is then cleaved in the nucleus by the dsRNA-specific ribonuclease Drosha/Pasha to produce a 70 nucleotides long precursor miRNA (pre-miRNA). Pre-miRNAs in form of hairpins are then actively transported from the nucleus to the cytoplasm. In the cytoplasm, pre-miRNAs are subsequently cleaved by RNase III Dicer into~22-nt miRNA duplexes, consisting of the "guide" (miR) strand and the "passenger" (miR*) strand. The "passenger" strand is degraded, the "guide" strand is incorporated into the RNA-induced silencing complex (RISC) and serves as a functional, mature miRNA, acting by two different mechanisms according to the complementarity with the target mRNA. Adopted from Kim [15] . occurs when miRNA is only partially complementary to its target mRNA [28] [29] [30] . In light of the recent study by Guo et al [27] , this mechanism does not represent a predominant reason for reduced protein output.
Besides the complementarity between miRNA and mRNA, several other factors may influence the miRNA action such as impaired processing, methylation, gene polymorphisms, gene amplification, deletion of Dicer, translocations and others [31] .
It is evident that single miRNAs may regulate translation of numerous downstream mRNAs and each mRNA is likely to be regulated by several miRNAs simultaneously [30, 32] . Thus, identification of miRNA target genes has been a great challenge [33] . Numerous computational algorithms [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] were established which combined 5' seed matches, thermodynamic stability and conservation analysis in order to maximize specificity when predicting mRNA targets [44] (Table 1) . Nevertheless, various algorithms differ in the selection of mRNA targets and simultaneous application of several algorithms is, therefore, highly recommended. Nowadays, many web-based applications [45] [46] [47] [48] [49] [50] [51] [52] have been developed by combining existing prediction programs with functional annotations associated to many miRNA, gene, protein or biological pathway resources such as miR-Base, Ensembl, Swiss-Prot, UCSC genome browser, KEGG pathway and other databases [44] (Table 2) .
However, because of high similarities in miRNA sequences, computational algorithms may predict a large number of putative miRNA binding sites on mRNA targets [33] . Thus, experimental validation in biological system is fundamental to complete the target prediction [44] ; the currently available methods [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] are listed in Table 3 . Of these, antagomir studies or immunoprecipitation of Ago-bound mRNAs have been specifically developed for miRNA-mRNA studies. Antagomirs represent a novel class of chemically engineered oligonucleotides used to silence endogenous microRNAs [64, 65] . Immunoprecipitation is then based on the observation that each member of the Argonaute (Ago) protein family (catalytic components of the RNA-induced silencing complex) can bind to miRNAs and to partially complementary sequences in the 3'-UTR of specific target mRNAs. Thus, using highly specific monoclonal antibodies against members of the Ago protein family, Ago-bound mRNAs can be co-immunoprecipitated [66, 67] .
The lung has a very specific miRNA expression profile, highly conserved across mammalian species [68, 69] .
However, the knowledge of the role of miRNAs in physiological and pathological conditions in the lung compartment is still limited and it is based mainly on the studies in animal models. MiRNAs have been shown to be involved in 1) the lung development and homeostasis, 2) in inflammation and viral infections and 3) miRNA deregulation may contribute to several pulmonary diseases ( Figure 3 ). Hereby, we summarize the knowledge of the involvement of miRNAs in the lung and current information on their posttranscriptional regulation ongoing in the lung compartment. Besides pathology we pay attention also to physiological lung because understanding miRNA function in normal condition is prerequisite to description of its involvement in disease.
Several miRNAs such as miR-155, miR-26a, let-7, miR-29, miR-15/miR-16, miR-223, miR-146a/b and the miR-17-92 cluster have been shown to be involved in homeostasis and in the lung development ( Table 4 ). The pulmonary role of miR-155 was studied in murine lung, where it has been shown that miR-155 is crucially involved in the differentiation of naive T-cells into Th1 and Th2 cells [70, 71] . Mice deficient in bic/miR-155 became immunodeficient and displayed increased lung remodelling, higher bronchoalveolar leukocytes and impaired T-and B-cell responses to inflammatory stimuli [70] . Another member of miRNA family, miR-26a, has been shown to be selectively expressed in the bronchial and alveolar epithelial cells in murine lung [72] . Target mRNA of miR-26a is the transcription factor SMAD1, which is involved in the regulation of bone morphogenic protein signalling during lung development and pulmonary vascular remodelling [73, 74] . Thus, miR-26a might be important in controlling essential developmental and physiological events in the lung [75] . Also the miR-17-92 cluster is believed to regulate the lung development because its expression is high in embryonic development and steadily declines through development into adulthood [76] . Mice deficient in the miR-17-92 cluster died shortly after birth and lung hypoplasia/ventricular septal defects were demonstrated; moreover the absence of the miR-17-92 cluster let to upregulation of the pro-apoptotic protein Bim and inhibition of B-cell development [77] . On the other side, the overexpression of the miR-17-92 cluster in murine models resulted in an abnormal phenotype manifested by absence of terminal air sacs, which were replaced by highly proliferative, undifferentiated pulmonary epithelium [76] . Other miRNAs found to be involved in the pulmonary homeostasis are members of let-7 family [78] , miR-29 [79] , miR-15 and miR-16 [80, 81] , which Northern blot analysis [54] Quantitative real-time PCR [55] Ribonuclease protection assay [56] in situ hybridization [57] , [58] miRNA mimics [59] Western blot [60] Immunocytochemistry [61] Bead-based flow cytometry method [62] Suppression of miRNA expression in cells by antisense locked-nucleic acid oligonucleotides [63] Antagomir assays [64] , [65] Immunoprecipitation of Ago-bound mRNAs [66] , [67] function as tumor suppressors in lung cells. In addition, another miRNA, miR-223, has been shown to be crucial for normal granulocyte development and function in the lung [82] . MiR-223 mutant mice spontaneously developed neutrophilic lung inflammation with tissue destruction after endotoxin challenge [82] .
Two miRNAs, miR-146a and miR-146b, have been shown to play central role in the negative feedback regulation of IL-1β-induced inflammation; the mechanism is down-regulation of two proteins IRAK1 and TRAF6 involved in Toll/interleukin-1 receptor (TIR) signalling [83, 84] . Also other miRNAs have been shown to regulate the inflammation in mouse lung exposed to aerosolized lipopolysaccharide (LPS): miR-21, -25, -27b, -100, -140, -142-3p, -181c, -187, -194, -214, -223 and -224 [72] . Increase in these miRNAs correlated with the downregulation of pro-inflammatory cytokine production such as TNFα [72] . The deregulation of miR-155, the miR-17-92 cluster and miR-223, miRNAs involved in lung development and homeostasis, resulted in the uncontrolled lung inflammation in murine models [70, 77, 82] . Based on the studies in murine models, there is evidence that miRNA expression may influence also the course of pulmonary viral infections [85, 86] . MiR-200a and miR-223 were detected in lethal influenza virus infection presumably contributing to the extreme miR-155 important for normal lung airway remodelling (A) [70] alteration of T-cell differentiation (A) [71] miR-26a highly expressed within bronchial and alveolar epithelial cells, important for lung development (H) [75] let-7 highly expressed in normal lung tissue, functions as a tumor suppressor in lung cells (H) [78] miR-29 functions as tumor suppressor in lung cells (H) [79] miR-15, miR-16 function as tumor suppressor genes (H) [80] , [81] miR-223 control of granulocyte development and function (A) [82] miR-146a/b central to the negative feedback regulation of IL-1β-induced inflammation (H) [83] , [84] miR-200a, miR-223 contribution to the extreme virulence of the r1918 influenza virus (A) [85] miR-17 family, miR-574-5p, miR-214 upregulated at the onset of SARS infection (A, H) [86] virulence of the r1918 influenza virus [85] . MiR-17 family, miR-574-5p and miR-214 were upregulated at the onset of SARS infection: these miRNAs may help the virus to evade the host immune system and are responsible for effective transmission at the initial stage of viral infection [86] .
There is evidence that upregulation or downregulation of miRNAs is critical for the lung development/homeostasis and thus may contribute to development of pathological pulmonary conditions, namely to smokingrelated diseases including lung cancerogenesis, fibrosis, and other immune-mediated disorders including allergy (Table 5) .
Recent studies have implicated the miRNAs in the pathogenesis of immune system mediated pulmonary diseases. Tan and colleagues [87] described that a single nucleotide polymorphism in the 3'UTR of HLA-G, a known asthma-susceptibility gene, disrupts the binding sites of three miRNAs (miR-148a, miR-148b, miR-152) targeting this gene. Thus, it is likely that the association of the HLA-G gene to asthma-susceptibility may be due to the allele-specific regulation of this gene by miRNAs [87] . MiR-21 is a further miRNA crucially involved in allergic lung inflammation. Its molecular target is IL-12p35, a cytokine contributing to polarization of Th cells toward Th2 cells [88] . MiR-126 is another miRNA found to be involved in the pathogenesis of allergic airways disease [89] . The blockade of miR-126 suppressed the asthmatic phenotype leading to diminished Th2 responses, suppression of inflammation, reduced airways hyperresponsiveness, inhibition of eosinophil recruitment, and lower mucus hypersecretion [89] . In bronchial epithelial cells stimulated with IL-4 and TNFα, let-7, miR-29a and miR-155 have been involved in the regulation of allergic inflammation [90] . Multiple members of let-7 family were also found upregulated in experimental asthma model and the pro-inflammatory role of let-7 miRNAs on the allergic cytokine expression was confirmed [91] . Another study showed that expression of RhoA in bronchial smooth muscle cells (BSMCs), a new target for asthma therapy, is negatively regulated by miR-133a [92] . The same group later revealed that IL-13 is capable of reducing the miR-133a expression in BSMCs and that the miR-133a downregulation causes an Table 5 MiRNAs involved in pathological processes in the lung miRNA Function (A animal studies, H human studies) References miR-155, miR-17-92 cluster deregulation results in uncontrolled inflammation (A) [70] , [71] , [77] miR-21, miR-27b, miR-100, miR-181c, miR-223, miR-224 increased following exposure to LPS (A) [72] miR-155 overexpressed in solid tumors, inhibition of tumor suppressor genes (A, H) [81] miR-223 impaired granulocyte function, regulator of granulocyte production and inflammatory response (A) [82] miR-148a/b, miR-152 allele-specific regulation of asthma susceptibility HLA-G gene (H) [87] miR-21 key role in asthma (A) [88] overexpressed in solid malignancies (A, H) [103] up-regulated in bleomycin-induced fibrosis and IPF (A, H) [110] miR-126 suppression of the asthmatic phenotype by blockade of miR-126 (A) [89] downregulated in cystic fibrosis airway epithelial cells (H) [111] let-7, miR-29a, miR-155 regulation of allergic inflammation in bronchial epithelial cells (A, H) [90] let-7 pro-inflammatory effect in experimental asthma (A) [91] role in lung cancer progression (H) [99] miR-133a regulator of expression of RhoA, target for asthma therapy (A, H) [92] , [93] miR-146a reduced expression in COPD fibroblasts (H) [95] miR-218, miR-15a, miR-199b, miR-125a/b, miR-294 deregulated due to smoking (A, H) [96, 97] miR-218 tumor suppressor in non-small cell lung cancer (H) [98] miR-17-92 cluster overexpressed in lung cancers (H) [102] miR-34 regulation of apoptosis in lung cancer cells (H) [105] [106] [107] miR-210 overexpressed in lung cancer (H) [108] let-7d pro-fibrotic effect in pulmonary fibrosis (A, H) [109] upregulation of RhoA, presumably resulting in an augmentation of the contraction [93] .
Lung cancer and chronic obstructive pulmonary disease (COPD) share a common environmental risk factor in cigarette smoke exposure [94] . Although extensive studies of the involvement of miRNAs in lung cancer have been performed, there are only few reports focused on the role of miRNAs in COPD. Recent study on fibroblasts from COPD subjects stimulated in vitro with pro-inflammatory cytokines released less miR-146a than smokers without COPD [95] . The reduced miR-146a expression resulted in prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects [95] . There is evidence that smoking has influence also on other miRNAs. Expression profiling study in the rats exposed to environmental cigarette smoke revealed 24 downregulated miRNAs (especially let-7 family, miR-10, [96] . MiR-294, a known inhibitor of transcriptional repressor genes, was the only miRNA upregulated in smoke-exposed rats [96] . In another study, bronchial airway epithelial cells from current and never smokers differed in the expression of 28 miRNAs (especially miR-218, miR-15a, miR-199b, miR-125a/b, miR-294) in comparison to smokers, whereas the majority of deregulated miRNAs were downregulated in smokers [97] . Similar observation was observed in lung squamous cell carcinoma, where downregulation of miR-218 was associated with a history of cigarette smoking [98] . However, the majority of miRNA studies in smokingrelated diseases are focused on the role of miRNAs in lung cancer. Altered expression of miR-155 and let-7 has been reported in lung adenocarcinoma and expression of let-7 related to patient survival [99] . Moreover, it has been shown that let-7 may also play a role in lung cancer progression [99] [100] [101] . Further, increased expression of the miR-17-92 cluster has also been detected in lung cancer [102] . Another miRNAs involved in lung cancerogenesis are miR-21 and miR-34 families. MiR-21 was shown to regulate multiple tumor/metastasis suppressor genes in lung solid tumors [103] . MiR-34a/b/c have been identified to be a component of the p53 tumor suppressor network: p53 upregulates in response to DNA damage the members of miR-34 family [104] , thus regulating genes involved in the cell cycle and apoptosis [105] [106] [107] . Furthermore, miR-210 has been overexpressed in late stages of lung cancer, thus mediated mitochondrial alterations associated with modulation of hypoxia-inducible factor-1 activity [108] . Next, miR-218 was identified as a putative tumor suppressor in non-small cell lung cancer [98] .
Recently, it was reported that miRNAs may play pivotal regulatory role also in the fibrotic processes ongoing in the lung: the downregulation of let-7 d in idiopathic pulmonary fibrosis (IPF) resulted in the pro-fibrotic effects [109] . Also, upregulation of miR-21 was reported in the lungs of IPF patients and in the murine lungs with bleomycin-induced fibrosis, whereas miR-21 expression was enhanced by pro-fibrotic TGF-β1 [110] . Another disease associated with miRNA change was cystic fibrosis. Downregulation of miR-126 was detected in cystic fibrosis bronchial epithelial cells and its expression correlated with upregulation of TOM1 mRNA both in vitro and in vivo [111] . TOM1, a miR-126 target, was reported to be involved in the regulation of innate immune responses through its involvement in the TLR2/4 and IL-1β and TNF-α-induced signalling pathways [111] .
Small non-coding RNAs (miRNAs) play pivotal role in the posttranscriptional regulation of numerous human genes, mainly via degradation of target mRNAs. There is evidence that the lung has a very specific miRNA expression profile undergoing changes during the lung development. Studies namely in animal models have provided evidence that miRNAs participate in lung homeostasis and play pivotal role also in the control of pulmonary inflammation and viral infections. Recent studies showed evidence that upregulated or downregulated expression of various miRNAs play an active role in the pathogenesis of pulmonary diseases. Specific miRNA expression profiles were characterized for smoking related-diseases including COPD and lung cancer, immune-mediated pulmonary diseases and pulmonary fibrosis. Moreover, several miRNAs crucial for lung development and homeostasis such as let-7, miR-155 or miR-19-72 cluster have been identified to be deregulated in pulmonary allergy, asthma or lung cancer. The knowledge of altered miRNA expression profiles in diseased lung may thus offer new insights in the biology of pulmonary diseases. Moreover, miRNAs may represent attractive novel diagnostic biomarkers mainly due to their higher stability when compared to mRNAs [112] and could potentially provide possibilities for therapeutic intervention [31, 113, 114] . Rapid detection of pandemic influenza in the presence of seasonal influenza BACKGROUND: Key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. In addition, they must minimise false alarms during a normal influenza season. We develop a method that uses historical syndromic influenza data from the existing surveillance system 'SERVIS' (Scottish Enhanced Respiratory Virus Infection Surveillance) for influenza-like illness (ILI) in Scotland. METHODS: We develop an algorithm based on the weekly case ratio (WCR) of reported ILI cases to generate an alarm for pandemic influenza. From the seasonal influenza data from 13 Scottish health boards, we estimate the joint probability distribution of the country-level WCR and the number of health boards showing synchronous increases in reported influenza cases over the previous week. Pandemic cases are sampled with various case reporting rates from simulated pandemic influenza infections and overlaid with seasonal SERVIS data from 2001 to 2007. Using this combined time series we test our method for speed of detection, sensitivity and specificity. Also, the 2008-09 SERVIS ILI cases are used for testing detection performances of the three methods with a real pandemic data. RESULTS: We compare our method, based on our simulation study, to the moving-average Cumulative Sums (Mov-Avg Cusum) and ILI rate threshold methods and find it to be more sensitive and rapid. For 1% case reporting and detection specificity of 95%, our method is 100% sensitive and has median detection time (MDT) of 4 weeks while the Mov-Avg Cusum and ILI rate threshold methods are, respectively, 97% and 100% sensitive with MDT of 5 weeks. At 99% specificity, our method remains 100% sensitive with MDT of 5 weeks. Although the threshold method maintains its sensitivity of 100% with MDT of 5 weeks, sensitivity of Mov-Avg Cusum declines to 92% with increased MDT of 6 weeks. For a two-fold decrease in the case reporting rate (0.5%) and 99% specificity, the WCR and threshold methods, respectively, have MDT of 5 and 6 weeks with both having sensitivity close to 100% while the Mov-Avg Cusum method can only manage sensitivity of 77% with MDT of 6 weeks. However, the WCR and Mov-Avg Cusum methods outperform the ILI threshold method by 1 week in retrospective detection of the 2009 pandemic in Scotland. CONCLUSIONS: While computationally and statistically simple to implement, the WCR algorithm is capable of raising alarms, rapidly and sensitively, for influenza pandemics against a background of seasonal influenza. Although the algorithm was developed using the SERVIS data, it has the capacity to be used at other geographic scales and for different disease systems where buying some early extra time is critical. Rapid detection of pandemic influenza at national or regional level is a public health issue of critical importance [1, 2] . Huge excess mortality and morbidity have been associated with the pandemics of influenza outbreaks in the past [3] . In the aftermath of the highly pathogenic H5N1 avian influenza outbreaks worldwide [4, 5] , the growing concern [3,4] of a virulent form of a possible human influenza pandemic has led to the setting up of influenza surveillance systems across the globe [6] . One of the main purposes of such worldwide expansion of influenza surveillance systems is the timely detection of influenza outbreaks of pandemic potential [7] . The importance of timely detection lies in buying some extra time for being prepared to deal with a pandemic [3, 8, 9] . This has also been corroborated by some recent mathematical modelling studies [10, 11] of pandemic influenza outbreaks: a key finding suggests that there would be a small window of opportunity for getting ahead of pandemic outbreak fronts and thus helping early pandemic mitigation efforts if it could be detected early on.
Most developed countries as well as many from the developing world have some form of influenza surveillance in place [6] . These surveillance systems are based on the reporting of disease syndromes (e.g., reports of Influenza-like illnesses (ILI)) and are generally designed to monitor levels of seasonal influenza [12, 13] . Although the signature of pandemic influenza could be different from that of seasonal ones [14] , the traditional approach (patients presenting with clinical signs of ILI, collection of throat/nasal swab samples from some of these patients and, finally, laboratory confirmation of influenza) followed in influenza surveillance systems, in the absence of any detection algorithm applied to syndromic data, may not be able to pick it up early on. This is the reason that public health surveillance systems are being supplemented by the new state-of-the-art statistical tools [1, 2] . The development of these new statistical tools has demonstrated the potential to automate syndromic surveillance systems, to be able to raise specific and sensitive early alerts of adverse disease outbreaks. Indeed this is a fast growing and a very active area of scientific research at the moment [6] .
At present, a number of methods [12, 13, 15] exist to establish the onset of peak activities in the epidemics of seasonal influenza. These methods are mostly based on regression [16, 17] or time-series [12, 13, 15] analysis of seasonal ILI data. One such method is the Moving-Average Cumulative sums (Mov-Avg Cusum) method [18] [19] [20] . Originally developed for the industrial quality control [21] , it is now frequently used for detecting the outbreaks of seasonal and pandemic influenza [12, 22] . Recently there has been a flurry of new detection methods based on sophisticated statistical approaches [1, 2] , including those aimed at real-time monitoring and projecting of influenza cases [23, 24] . However, challenges remain in terms of how to use the ILI surveillance data in a simple and efficient manner for timely detection of influenza pandemics.
The basic reproduction ratio R 0 (i.e., the average number of new infections produced by a single infection in a totally naïve host population) plays a central role in our understanding of infectious disease dynamics. It determines whether a new infection will successfully invade the susceptible population [25, 26] . In the case of an ongoing epidemic, the effective reproduction ratio R replaces R 0 [25] . In the presence of disease tracing data, R can be estimated and the in-or out-of-control status of an ongoing epidemic can be established [27] . Where there is no availability of disease tracing data, as in influenza syndromic data, the weekly case ratio (WCR), defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week, may function as an indirect measure of R and may be suitable for raising public health alarms in the early stages of an emerging infection. Although pandemic influenza infections may grow exponentially in early invading stages (evident in the mortality data from the past pandemics [28] or in the mathematical modelling [10, 11, 29] of influenza pandemic), the detection algorithms so far employed in influenza surveillance systems largely ignore this natural behaviour as the basis for generating early warning of influenza outbreaks of pandemic potential.
The aim of this paper is to develop a detection algorithm, based on the estimates of WCR for expected influenza pandemics, to facilitate sensitive, specific and rapid detection of a pandemic outbreak at a regional level based on existing surveillance systems. Using the influenza surveillance data from Scotland, we first sift through the spatiotemporal patterns in the historical data by calculating WCR and N HB , the number of health boards (HBs) that show increases in the weekly ILI cases. The joint probability distribution of WCR and N HB is then contrasted with expected and observed patterns in the presence of pandemic influenza. As described in the next section, the expected patterns for pandemic cases are obtained from a previously published mathematical model [29] . Observed patterns for pandemic cases are based on records from the 2008-09 season when influenza A(H1N1)v was circulating in Scotland.
We compare the performance of our detection algorithm, using simulated influenza pandemics as well as data from the 2009 influenza A(H1N1v) epidemic in Scotland, with that of the Mov-Avg Cusum method and with the ILI rate threshold method, a slightly modified form of the baseline ILI activity indicator used by the Health Protection Agency (HPA) in the monitoring of seasonal influenza in the UK.
In developing our pandemic detection algorithm, we used historical seasonal ILI data, collected and compiled under the Scottish Enhanced Respiratory Virus Infection Surveillance (SERVIS) system. The data set spans from the 2001-02 season through to the 2008-09 season. Seasonal ILI cases are normally reported weekly over a period of 33 weeks (from the first week of October to the third week of May) in different age-and sex-classes, by sentinel general practices (GPs) across Scotland. The SERVIS sentinel GPs are drawn from 13 Scottish health boards. (There are currently 14 HBs in Scotland; all HBs except the Western Isles HB have participated in the SERVIS network of the sentinel GPs.) The Scottish health boards widely vary in their population sizes from 20,000 to 1,360,000. The total number of the sentinel GPs varied from 20 to 44 across the influenza seasons considered, with a minimum of 1 GP in a HB to a maximum of 9 GPs in a HB. Altogether, the numbers of people registered with sentinel GPs represent 2.7% to 4.9% of the Scottish population of around 5.15 million. This system was designed as a national surveillance scheme with regional coverage. It was not designed to be used as a surveillance system in each health board separately.
The weekly reported ILI cases at the national level are shown in Figure 1a . In our analysis we used the weekly ILI cases, aggregated at the HB level. An example of weekly HB-level ILI cases is shown for the 2004-05 influenza season in Figure 1b . Sentinel GPs are often recorded as reporting zero cases in a week in the SER-VIS data. It is unknown whether this represents true zeroes or non-reporting. However, we believe that certainly, in the data for the 2008-09 season, a blank means that no report was made by the practice and a zero means a report was made but no cases occurred.
The historical influenza data from 6 influenza seasons from 2001-02 through 2006-07 were used in estimating the background pattern of the seasonal ILI cases. The ILI data for the season 2007-08 from 23 sentinel GPs recorded just 93 cases compared with over 300 cases for the other seasons. The whole of the UK reported influenza cases below the HPA baseline activity threshold in this season [30] . We therefore excluded the 2007-08 data from our analysis. (As we checked this and will be clear later, the inclusion of the historical ILI data of this season increases the detection efficiency of the WCR method. The exclusion of the data of this season, therefore, ensures conservative estimate for the WCR method in the performance testing.) The 2008-09 data-set contains the 2009 influenza A(H1N1)v pandemic cases, so it was used for performance testing of our detection algorithm with real pandemic data. The SERVIS ILI data used in the study is freely available from Health Protection Scotland on request (NSS.hpsflu@nhs.net) to anyone wishing to use them for any non-commercial research purposes.
For our main analysis, we use simulated pandemic influenza data for Scotland. In brief, the pandemic model [29] is a stochastic, spatially structured, individual-based discrete time simulation. For the analyses carried out in this paper, the model pandemic outbreaks were run with a basic reproduction rate R 0 of 1.7 and a generation time of 3 days. (The R 0 value used here is slightly higher than what has been reported from various analyses [31] of the 2008-09 influenza A(H1N1)v outbreak data. But we have used a range of pandemic case reporting rates as discussed below.) Full model details are given in the Supplementary Information of [10, 29] .
The pandemic model was simulated 10 times for the whole of Great Britain, starting on day 1 seeded with a single infection at a randomly chosen location. In our analysis, however, we define the pandemic start week as the week when the first influenza infections occur in Scotland, which may or may not be reported. From the simulated pandemic infections, we sample ILI cases at case reporting rate of α as if they would have been reported by sentinel GPs to SERVIS (see Additional file 1). In this work we have used three values of α: 0.5, 1 and 5%. The sampled sentinel pandemic cases were then converted to the HB-level daily reported influenza cases by summing across all participating sentinel GPs of the HBs, which in turn were converted into weekly reported ILI cases to match the temporal resolution of the SER-VIS data. The first wave of pandemic influenza cases could occur in the presence of seasonal cases at any time of the year. To take this into account, we add the simulated pandemic ILI cases to the seasonal ILI cases from each of the six seasons and use the resultant ILI time series for detecting pandemic, sliding the pandemic start week across the entire influenza season. We present our results using 300 (30 samples times 10 simulated pandemics) sampled time series of pandemic cases. Here we use 10 simulated pandemics each sampled 30 times. (Note that the results are invariant with other sampling schemes, e.g., 300 time series, each sampled from 300 different simulated pandemics. The sampling is carried out after the first Scottish cases are reported, during the exponential growth phase of the epidemic. All simulated pandemics are therefore observed to have very similar temporal dynamics.) Each of these 300 time series were, in turn, overlaid with seasonal ILI time series from each of the 6 seasons, making a total of 1800 time series to be analysed for 33 (from week 1 to week 33) pandemic start weeks in a typical influenza season.
Pandemic detection algorithm Joint probability distribution of (WCR, N HB )
Our pandemic detection algorithm uses two metrics obtained from weekly reported ILI cases: the weekly case ratio (WCR) for cases aggregated across the region and N HB , the number of health boards reporting increases in the cases over the previous week.
The weekly WCR is defined as W CR = total ILI cases reported to all SERV IS sentinel GPs in week w total ILI cases reported to all SERV IS sentinel GPs s in week w − 1 Note that WCR is not defined for week 1 and, also, not defined for any week where in the previous week all sentinel GPs did report zero ILI cases. (In the historical data we have one instance (out of the total 214 weeks from all the 6 seasons) that for a week, which was not a start week but well within the influenza season, there were no ILI cases reported. In this situation we simply replace zero in the denominator by 1.) Since WCR is a continuous variable, in order to create a joint distribution, it is binned with a bin size of 0.1. We then construct a joint probability distribution of the two metrics using the historical ILI data from all the 6 seasons (see Figure 2 ). 
The historical seasonal ILI data are temporally heterogeneous, with substantial week-to-week variability ( Figure 1 ). For making any useful and robust statistical inference the probability distribution requires smoothing. The smoothing was done assuming that the weekly reported ILI cases in a HB have a Poisson distribution with rate parameter equal to the reported total weekly ILI cases in the HB. The Poisson distribution is preferred to a binomial distribution as the numbers of weekly reported cases are very much smaller than the total population of a health board. We simulated the Poisson model to generate weekly ILI cases at the HB level (see Additional file 2). The model-generated ILI counts from individual runs for each season were then used, as described above, to calculate the joint distribution of WCR and N HB . A set of 10,000 model runs per season's data were used in the estimation of this joint distribution. A smaller (1,000) or larger (100,000) number of runs were also tested for the robustness of the results.
We tested our algorithm for its specificity (i.e., not detecting a pandemic when no pandemic is occurring). To do this, we first calculate WCR and N HB for each week from a given seasonal HB-level ILI time series. We obtain the probability values of weekly pairs of (WCR, N HB ) from the joint probability distribution (the plots of the weekly probability values are given in Figure 3 ). In the second step, for a chosen threshold probability value δ, we count the number of weeks in which the probability of (WCR, N HB ) falls below the threshold. The total counts represent the number of false alarms (Fa s (δ)) in season s. The theoretical maximum number of false alarms ( FA s max ) in any given season is the total weeks minus 1 because WCR cannot be calculated for the first week. Finally, this leads us to calculate the specificity of the detection algorithm for a given threshold probability as follows:
A detection threshold is defined as the threshold probability δ that gives rise to a pre-set specificity of our detection method. We adjust δ in order to achieve specificities of 95% and 99%. This allows us to compare our method to other algorithms which also have the same constant specificity.
Detecting a pandemic Figure 4 shows an example of how our WCR detection algorithm works when applied to a simulated pandemic starting on week 1 and week 15. In the case of a pandemic starting on week 1 (week 15), the probability value of WCR and N HB falls below the threshold values for the first time on week 7 (week 21) triggering an alarm that week. We note here that the probability value is the probability mass function, i.e. P(WCR = xx and N HB = y), and not the more usual statistical significance level of P(WCR > = xx and N HB > = y).
We compared the performance (in terms of median detection time and sensitivity) of our detection algorithm with that of the Mov-Avg Cusum method. This method was recently used by Cowling et al. [12] as a statistically robust automation tool for generating early alerts for the onset of peak activity of ILI cases. We also compare the performance of an ILI rate threshold method, similar to the HPA baseline influenza activity level. The detection thresholds used by these methods were adjusted so that all three methods had the same detection specificity described earlier.
The d-week upper Cusum at time t is defined as follows:
where Cusum t d ≤ + + = 7 0 . X t  and st  are the 7-week moving average and standard deviation of ILI cases in weeks t-d-1 to t-d-7 Note that d stands for a delay period and this method will only be informative from the (d+8) th week onwards. An alarm is triggered using this method when Cusum t + on a week t crosses a pre-set threshold [21] . Using the SERVIS ILI data for six seasons we preset the threshold values for this method for all 16 (Table  in Additional file 3) . Therefore, this combination is used to make comparisons with other methods.
The HPA set ILI consultation rates as proxies for influenza activity in the United Kingdom. The threshold rate for baseline-and epidemic-level ILI activities has recently been revised: the baseline threshold is lower from 50 to 30 consultations per 100,000 population while the epidemic threshold has been decreased from 400 to 200 consultations per 100,000 population [32] . These thresholds are derived from the time series analysis of historical seasonal data, and serve the purpose of establishing when and/or whether the community ILI activity warrants some intervention of the public health departments. In the SERVIS data set (2001 -2007) considered, the epidemic threshold rate was never crossed [30] . Here the ILI rate threshold is denoted by η cases per 100,000 population per week and an alarm is generated when the aggregated ILI cases in any week crosses this threshold. In order to compare this method with alternatives we adjust η to obtain the pre-set specificity of 95% and 99%. The two respective values of η are 24 and 34.
Performances of the three methods are summarised in terms of sensitivity and median detection time (MDT) in Table 1 . Our algorithm is almost 100% sensitive (the lowest being 98% for a very low case reporting rate of 0.5%). Its MDT ranges from 3 to 5 weeks compared to 4 to 6 weeks for the Mov-Avg Cusum and threshold methods. While the threshold method is 100% sensitive, Mov-Avg Cusum is the worst performing method with sensitivity of 77% to 97%. Note that time to pandemic detection is counted from the start week of the first infections in simulated pandemics. There is a lag of about 3 to 5 weeks between the first infections arising in simulated pandemics and the first cases which get reported by sentinel GPs to SERVIS. If, therefore, the reference point is changed to the week of the first reported cases, then MDT of the WCR method will not be more than 2 weeks.
The distribution of detection times ( Figure 5 ) is to show whether the temporal pattern in the background seasonal ILI cases will have any effect on pandemic detection. The detection specificity (Sp) and the pandemic case reporting rate (α) were set for all methods at the following values: Sp = 99% and α = 5%. Detection times are typically within 5-6 weeks for our method (Figure 5a ). This compares favourably with the ILI rate threshold method at η = 34 cases per 100000 population (Figure 5b ). The slightly longer detection times are present for the starting weeks falling in the period of late-November to mid-January (week 9 to week 15 in Figure  5a ). This is the time period when the seasonal ILI incidences show widespread and peak influenza activities ( Figure 1 ). In the case of simulated pandemics starting during this period, the above two factors together mask the probability of (WCR, N HB ) as seasonal one in the first few weeks of pandemic. This masking causes delay in generating an alarm and the delay is more pronounced at lower case reporting rates and a higher specificity and is, for example, responsible for reducing the method's sensitivity to 98% at α = 0.5% and Sp = 99%. Conversely, during the same time-period the threshold method produces its shortest detection times because the peak-level seasonal cases having been added to the pandemic cases help the weekly ILI rate quickly cross the rate threshold η. However, our method outperforms the ILI rate threshold method in the beginning and end of an influenza season. Mov-Avg Cusum performs poorly in terms of detection times in the first few weeks of the season (Figure 5c ). This is because it requires 9 weeks to calculate the first Mov-Avg Cusum statistic. This situation improves as we move well within the seasonal period. But even for the later starting weeks, the method takes comparatively longer time to detect a pandemic. However, this method also outperforms the ILI rate threshold method towards the end of a season.
Detection of pandemics in the early weeks of its starting depends on the case reporting rate and specificity ( Figure 6 ). Our method outperforms the other two by rapidly detecting pandemics in a large fraction of model runs at specificity of 99% and case reporting rate α of 0.5%. It detects pandemics in >50% of total runs within the first 6 weeks of a pandemic starting while the Mov-Avg Cusum and the threshold methods detect pandemics, respectively, in <25% and <35% of total runs (Figure 6a) . The time to detection decreases when the specificity was lowered from 99% to 95% (Figure 6b ). In this case, about 25% and a slightly lower than 50% detection levels were achieved by the WCR method within the first 4 and 5 weeks while the Mov-Avg Cusum and the threshold methods still trailed below the 25% detection level. The same trend was observed when, for the fixed specificity of 99%, the case reporting rate α was raised from 0.5% to 1% (Figure 6c) to 5% (Figure 6e ). At the elevated reporting rates, decrease in specificity further increases the detection level for all methods. But the increase in pandemic detection within the first few weeks of pandemic is more pronounced for our method than the other two (Figs 6d & 6f) .
As shown in Figure 7 , our algorithm, retrospectively, detects the 2008-09 pandemic outbreak 12 weeks (i.e., The performances of the three methods compared in terms of sensitivity (Sen) and median detection time (MDT) for different values of pandemic case reporting rates and detection specificities of 95% and 99%. Sen is given as percentage (%) of the model runs summed across all 33 weeks, i.e. calculated from a set of 1800 overlaid time series times 33 weeks. MDT is in weeks and calculated from those of (1800 × 33) runs in which a given method was able to generate a detection alarm.
on week 41) after the first cases were reported on week 29 in Scotland (Figure 7a ). Clearly, here it does not perform as well as it does with the simulated pandemic data. In the next section we discuss possible reasons for this poor performance. The Mov-Avg Cusum method, which was the worst performer among the three methods using simulated pandemic data, also detects the pandemic in week 41 (Figure 7b ). Both methods outperform the ILI rate threshold method by 1 week (Figure  7c ).
In this paper we compare three methods of detecting an influenza pandemic using an existing surveillance system in Scotland called SERVIS. The ILI rate threshold method uses current ILI case data to detect pandemics. This method is motivated by the current HPA's threshold levels [30, 32] to monitor influenza activity at the national scale. The HPA thresholds serve the purpose of establishing whether seasonal influenza activity warrants some intervention (e.g., the start of antiviral prescription) of the public health departments. Mov-Avg Cusum and other variants of Cusum are already being used in public health surveillance systems [1, 6, 12, 18] . The Mov-Avg Cusum method detects a pandemic when the cumulative number of current ILI cases is substantially higher than the expected cumulative number. The Mov-Avg Cusum statistic keeps accumulating the deviation between observed and expected values over time and when the accumulated value crosses a pre-set threshold, an alarm is triggered [21] . It has three adjustable parameters that require optimisation for specific surveillance systems. Finally, the WCR algorithm introduced in this paper is based upon a characteristic of epidemics, their exponential growth in the early stages before control measures and depletion of susceptibles have occurred. It also assumes that pandemic influenza would occur synchronously across spatial units of influenza surveillance system in a region (as is predicted by the mathematical models [29] for pandemic influenza in Scotland). It makes use of the joint probability distribution derived from the historical seasonal ILI data to detect a pandemic influenza. The other methods do not use any information from the data, other than to set thresholds to achieve required specificities.
The WCR algorithm appears to provide a slightly more rapid and sensitive tool for detecting of pandemic influenza -median detection time for this method ranges from 3 to 5 weeks in comparison to 4 to 6 weeks for the other two methods. Although the WCR algorithm seems to do the job more efficiently with the simulated pandemics, it performed poorly with the 2008-09 pandemic data from SERVIS. There could be several possible reasons for this poor performance. First, the 2009 influenza A(H1N1)v epidemic happened outside the normal influenza season and, second, it was mild in severity [33, 34] . In addition, the number of SER-VIS sentinel GPs in the season 2008-09 was at its sparsest level -only 20 practices, as the SERVIS system was in the process of being phased out to be replaced by a system which automatically collected data from GP systems on a daily basis. These three factors will have contributed to poor reporting of the early pandemic cases, notwithstanding the huge media coverage given to the pandemic. This is consistent with the patchiness in the reported ILI cases through SERVIS sentinel GPs between weeks 29 and 41 (Additional file 4). No method will detect a pandemic in its early weeks if the early syndromic influenza data are not reported to the surveillance system. Finally, the 2009 pandemic influenza cases were more spatially heterogeneous than those predicted by the pandemic model. (It is interesting to note that during the period April to July 2009, when there was a sentinel practice within an outbreak area, Greenock and Govanhill in the GGC HB, there was no increased reporting of ILI.) This might have contributed to the observed patchiness in the sentinel reporting.
An important aspect of our algorithm is that the detection threshold remains constant throughout. An implementation of a time-varying detection threshold could make this algorithm capable of using the seasonal ILI pattern more efficiently. In principle, this could be implemented by calculating the joint probability of (WCR, N HB ) either on a week-by-week basis, or on a slightly more coarse temporal scale of the time-windows of high/low ILI activities in the seasonal data. Clearly increasing the number of sentinel GPs and the frequency of ILI case reporting would improve the temporal resolution of the WCR algorithm. In future work we will explore how many sentinel GPs are required to achieve this aim.
Furthermore, in outbreaks of a novel influenza strain, generally children and young adults of the population, who will have little or no prior immunity to the disease [4] , are disproportionately affected [28] . Implementing our detection algorithm using these data attributes will further improve the timeliness and specificity of the detection of pandemic influenza. SERVIS data contain age attributes which could be incorporated into our algorithm, but this requires more sentinel GPs to be of use.
The WCR algorithm could be applied to any syndromic surveillance data structured by space and time. Syndromic data-sets include, but are not limited to, the triage nurse calls [35] (e.g., the NHS24/NHSdirect calls in the UK [36] [37] [38] ), the over-the-counter medicine sales data [39, 40] available in most of the developed countries, or online web search queries [41] . These data sets are highly useful in the early detection of unusual health events [1, 2] . Generally these data sets come with spatiotemporal attributes and, therefore, could potentially be integrated with the seasonal ILI data; this should enhance the detection process (in terms of timeliness, specificity and sensitivity) of pandemic influenza. Responses of Human Endothelial Cells to Pathogenic and Non-Pathogenic Leptospira Species Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis. Leptospirosis is a geographically widespread zoonosis that has emerged as a significant public health problem in urban slums, particularly in the tropics. The infection is caused by species of spirochetes belonging to the genus Leptospira. There are more than 200 serovars of Leptospira distributed among both pathogenic and non-pathogenic species [1] . The pathogenicity of different strains can vary considerably depending on the host species and age, and on the infecting serovar [2] . The spirochete's mode of entry is through mucous membranes and cuts or abrasions on the skin [1] . Upon entry, the organisms travel through the bloodstream to multiple sites, and may cause liver and kidney damage, meningitis, and a variety of other inflammatory conditions. If the host survives the acute infection, leptospires can persist in the proximal renal tubules for weeks to months, protected from antibodies and causing little to no inflammation. The bacteria are then shed in the urine, and animal urine contamination of water is the primary source of human exposure.
Although little is known about how Leptospira species establish infection in their hosts, adhesion to the host cell surface and extracellular matrix (ECM) by pathogens is often the first critical step in the initiation of infection. Several groups have investigated the adhesion of Leptospira interrogans to endothelial, fibroblast, kidney epithelial, and monocyte-macrophage cell lines cultured in vitro [3] [4] [5] [6] [7] [8] [9] . It is likely that pathogenic leptospires can attach to several different types of mammalian receptors to establish the infection. In fact, infectious strains of Leptospira have been shown to adhere to ECM components including collagen type IV, fibronectin and laminin, and also to the plasma protein fibrinogen [4, [10] [11] [12] . Adhesion to several ECM components is mediated at least in part by the LigA and LigB proteins [11] and a group of additional related proteins that were identified through homology to a laminin binding protein [10, 12] .
Several studies have shown that the adhesion of pathogens to mammalian cells will provoke multiple changes in the physiology and/or gene expression of the host. The host-pathogen interactions that define a disease are clearly complex. Microarrays are a powerful tool to explore those host-pathogen interactions by analyzing the transcriptional profiles of host cells or pathogens. Although it has been documented that temperature and osmolarity alter leptospiral gene expression [13, 14] , no previously published research has focused on the mammalian cell responses to the bacteria. To understand how human endothelial cells alter gene expression in response to incubation with different strains of Leptospira, human gene arrays were probed with cDNA derived from the RNA purified from infected cells and uninfected controls. In this study, we discuss how global analysis of gene expression allows us to gain insights into host specific responses to infection with pathogenic Leptospira.
The human microvascular endothelial cell line of dermal origin (HMEC-1) [15] was obtained from Dr. Ades (Centers for Disease Control and Prevention, Atlanta, Georgia) and cultured in endothelial basal medium (Clonetics, San Diego, CA) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 1 mg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO) and 10 ng/ml epidermal growth factor (Sigma-Aldrich). The immortalized human macrovascular endothelial cell line EA.hy926 [16] was kindly provided by Dr. C.-J. Edgell (University of North Carolina, Chapel Hill, NC) and grown in Dulbecco's modified Eagle medium with high glucose supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and HAT Media Supplement (Sigma-Aldrich). Both cell lines were cultured in the medium recommended by the supplier in a humidified atmosphere of 5% CO 2 and both cell media were supplemented with 1 U/mL penicillin, 1 mg/mL streptomycin, and 2 mM L-glutamine for routine propagation. Cells to be used for experimental infection with Leptospira strains were cultured without the antibiotics.
The roles of proteoglycans in the endothelial cell response to L. interrogans were tested based on previously published protocols [17] . Briefly, chondroitin sulfate B was shown to bind L. interrogans and to competitively inhibit L. interrogans to mammalian cells, so it was tested for the ability to inhibit the endothelial cell responses to the bacteria described below. In addition, inhibition of proteoglycan synthesis by b-xyloside, which also decreases L. interrogans attachment to mammalian cells, was tested for any effect. Controls included chondroitin sulfate A, to which L. interrogans does not bind, and the sugar analog a-galactoside, which does not affect proteoglycan synthesis.
The reference strain Leptospira biflexa serovar Patoc was obtained from the American Type Culture Collection (ATCC 23582, Manassas, VA), and is a non-pathogenic species. L. interrogans serovar Canicola (pathogenic, strain ATCC 23606 and strain 11203-32) were obtained from the ATCC and Dr. Richard Zuerner (USDA, Ames, IA), respectively. L. interrogans serovar Copenhageni (pathogenic, strain designated Fiocruz L1-130) was provided by Dr. David Haake (UCLA, Los Angeles, CA). Bacterial strains were maintained in ambient air at 30uC. Bacteria utilized for this study were at low passage from the suppliers (#passage 6) and cultured in EMJH medium [1] supplemented with 100 mg/ml of 5-fluorouracil and 1% rabbit serum (Sigma-Aldrich). For some experiments, the bacteria were radiolabeled by addition of 35 S cysteine plus methionine to the medium as described previously [17] . The bacteria were enumerated using a Petroff-Hausser counting chamber and dark field microscopy.
Mammalian cells were plated in T-225 tissue culture flasks (BD Falcon, Bedford, MA) and grown up to 90% or higher confluence. When cells reached desired confluence, the monolayer was washed with PBS and the cells were lifted off the plastic culture flask with 5mM EDTA in PBS. This was done to allow access of the bacteria to endothelial cell surface receptors that are normally involved in attachment to the substratum, i.e. receptors that the bacteria may encounter when penetrating the vasculature. In addition, this approach minimizes degradation of mRNA that occurs during harvesting of adherent cells. After lifting, cells were spun for 10 minutes at 1,000 rpm, resuspended in the cell culture medium without antibiotics, and enumerated using a hemocytometer counting chamber. 2610 7 cells per sample were incubated in suspension with either L. biflexa serovar Patoc or L. interrogans serovar Canicola, or without any bacteria, for 1 h and 3 h at room temperature in the cell medium without antibiotics. The MOI (multiplicity of infection) used was 10 bacteria per mammalian cell. After incubation, cells were washed with phosphate buffered saline (PBS) and harvested for RNA isolation. The RNA was purified using RNeasy kit (Qiagen, Valencia, CA) with DNase digestion according to manufacturer's manual. The quality of RNA was checked using a Bioanalyzer (Agilent, Santa Clara, CA).
Human HEEBO (Human Exonic Evidence Based Oligonucleotide) Arrays, consisting of 44,544 70mer probes representing 30,718 known genes, were purchased from Microarrays Inc. (Nashville, TN). 5 to 20 mg of total RNA from uninfected control and infected samples was used to generate cDNA labeled with aminoallyl (aa)-dUTP through a reverse transcription reaction using anchored oligo(dT) primers. The purified aa-dUTP-labeled cDNAs were coupled in 10 ml 0.1 M NaHCO 3 with either Cy3 or Cy5 NHS-ester dye. Cy-dye labeled cDNA was purified using a Cyscribe GFX column (Amersham Biosciences, Piscataway, NJ). The two differently labeled cDNAs were mixed and hybridized using Pronto Microarray Hybridization Kit in a hybridization chamber (Corning, Corning, NY), with the same array slide for 38 to 42 hr according to manufacturer's instruction. After a series of washes using the buffers provided in the kit, slides were spun dry and scanned under two laser channels in a Scanarray 4000 scanner (Packard Bioscience, Meriden, CT).
Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but is seen occasionally in temperate regions as well. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Our analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa caused changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily maintaining cell layer integrity, while L. interrogans disrupted cell layers. In fact, L. interrogans caused significant disruption of endothelial cell layers, but this damage could be abrogated by the endothelial protective drug lisinopril. Our results suggest that L. interrogans binds to endothelial cells and disrupts endothelial barrier function, which may promote dissemination of the bacteria and contribute to severe disease manifestations. This disruption may be slowed by endothelial-protective drugs to decrease damage in leptospirosis.
Endothelial Cell Responses to Leptospira www.plosntds.org Images were overlaid and analyzed using Imagene (BioDiscovery, El Segundo, CA). Raw gene expression was imported from Imagene to GeneSifter (GeneSifter.Net, VizX Labs, Seattle, WA) for analysis. Data from 3 biological replicate experiments were normalized using Lowess normalization and by the median of the raw intensities for all spots in each sample for each array. The ratio of two fluorescence intensities of each spot reflected the ratio of each gene expressed in the infected and uninfected samples. Genes were considered to be induced or repressed when the ratio of infected/uninfected was at least 1.5 fold (increased or decreased), and the P value was ,0.05 by the Student's twotailed t test. For analysis involving more than one time point and/ or condition, the one way ANOVA test was performed. Microarray data are deposited in GEO archive under the accession numbers GSE23172 and GSE23173.
EA.hy926 cells were seeded in tissue culture treated glass slides (BD Falcon) and grown at 37uC as described above. After cells reached 100% confluence, the monolayer was washed three times with PBS and medium without antibiotics was added. Four compartments of each slide were inoculated with 1610 7 bacteria (MOI = 10) of either L. biflexa serovar Patoc or L. interrogans serovar Canicola. The remaining four wells were left uninfected to serve as negative controls. In some cases, parallel experiments were performed using cells plated on coverslips in 24 well culture dishes, which allowed centrifugation to facilitate bacterialendothelial cell contact. At the end of the incubation (1 h, 3 h and 24 h) the slides were washed three times with PBS and fixed with 3% (wt/vol) paraformaldehyde in PBS at room temperature for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS, washed three more times with PBS, and blocked overnight at 4uC with HEPES buffered saline (HBS) and 1% bovine serum albumin (BSA). On the next day the slides were washed again with PBS and incubated with fresh blocking solution for 1h at room temperature. After blocking, the layers were probed with either rabbit anti-L. interrogans (a gift from Dr. Richard Zuerner, USDA, AMES, IA) diluted 1:5000 or anti-L. biflexa antiserum (Biogenesis, Inc., Brentwood, NH) diluted 1:1000, followed by anti-rabbit IgG-TRITC conjugate (1:1000) plus phalloidin-FITC (200 U/mL) to stain filamentous actin. After repeated washing in PBS, chambers were removed from the slides and Prolong Anti-Fade (Invitrogen, Carlsbad, CA) was used to mount coverslips. Two different microscopes at two different institutions were used throughout the course of this work. At institution one, images were captured using a Zeiss Axioplan microscope with a digital charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan) and co-localization of the fluorescent labels was done using Volocity software (Improvision Inc., Lexington, MA). At the second institution a Zeiss Axioimager Z1 with an Axiocam HrC camera and a Nuance Multi-Spectral Imaging System (software CRI Inc, Woburn, MA, v.2.6.0) was used.
The endothelial cell lines EA.hy926 and HMEC were plated in 3.0 mm (2610 6 pores/cm 2 ) polyester transwell inserts (Corning) and cultured as described above. After reaching 100% confluence, as assessed by lack of penetration of the fluorescent dye FITCdextran 40,000 (and loss of penetration of the L. biflexa serovar Patoc), the monolayer was washed with PBS and cell medium without antibiotics was added to the inserts and wells. Inserts without cells were used as controls for these experiments. Bacteria were added to an MOI of 50 to allow reliable enumeration of bacteria crossing the cell layers or membranes without cells at early time points, and 10 mL from the insert and from the well were taken after 1 h, 3 h, 6 h, 24 h, 27 h, 48 h and 72 h. In addition to the non-pathogenic strain Patoc and the pathogenic Canicola, Leptospira interrogans serovar Copenhageni was also used to analyze the migration of leptospires through the cell monolayer. Motile leptospires were counted by dark-field microscopy using a Petroff-Hausser chamber. Data are shown for the time points through which the bacteria were motile; after 72 hr there was a progressive decrease in L. biflexa motility.
To determine whether the bacteria were affecting the viability of the endothelial cells, four methods were used. First, adherent and EDTA-lifted endothelial cells infected at an MOI of 10 were washed, then incubated with the vital dye CellTracker Green (CT-CMFDA, 10 mM) plus DAPI (0.02 mg/ml) (Molecular Probes, now part of Invitrogen, Eugene, OR) for 1 hour at 37uC under 5% CO 2 . The samples were mounted and viewed using the Zeiss Axioplan microscope described above, and live cells (bright green cytoplasm) and dead cells (bright blue nuclei) were enumerated in at least three fields per sample in at least three independent experiments. Second, the cells were stained using the Vybrant Apoptosis Assay Kit 2 (Molecular Probes), which stains for annexin V and membrane permeability. Third, the APO-BrdU TUNEL kit, also from Molecular Probes, was used. A second TUNEL-based kit, Alert DNA Fragmentation kit (Clontech Laboratories, Inc., Mountain View, CA) was also used. For methods two and three, the cells were also assessed using fluorescence microscopy. Finally, cells were harvested, and DNA was purified and analyzed for fragmentation (an assessment of apoptosis) using conventional agarose gel electrophoresis.
We identified statistically significant and reproducible changes in endothelial cell gene expression after incubation with each bacterial strain as compared to the uninfected controls and to each other. The data were analyzed using Webgestalt [18] to identify mammalian cell genes whose products comprise functional pathways in which multiple components showed alterations in gene expression (Table 1 ). Four pathways that show internally consistent changes in gene expression are the KEGG focal adhesion, regulation of actin cytoskeleton, leukocyte transendothelial migration, and ECM-receptor interaction pathways. They are considered together because a number of genes encode proteins whose functions participate in aspects of cell biology common to these pathways.
Actin microfilaments are one of the three major components of the cellular cytoskeleton. The cytoskeleton participates in maintaining adhesion to and communicating with the extracellular matrix, cell migration, division, and signaling. b-Actin (ACTB) mRNA was decreased in response to L. interrogans but increased in response to L. biflexa, both as compared to the uninfected control cells (Table 2) . Guanine nucleotide-binding protein alpha-13 subunit (GNA13) mediates the activation of the small GTPase RhoA [19] which when activated controls the assembly of focal adhesions and actin in the formation of stress fibers [20] . Although RhoA was not differentially regulated in response to the bacteria, Rho GTPase activating protein 5 (RhoGAP5) was differentially expressed following the same pattern as GNA13, in which both genes were downregulated in response to the pathogenic leptospires in comparison to the uninfected controls, and upregulated in response to the non-pathogen. The effect of decreased GNA13 may be to decrease stimulation of Rho, while The changes in expression of several additional genes are consistent with changes in cellular architecture as a result of leptospiral infection of these endothelial cells. For example, decreases in the mRNAs for radixin (RDX, a protein that links the actin cytoskeleton to the plasma), caveolins 1 and 2 (CAV1 and CAV2, which couple integrins to the Ras-ERK pathway, titin, the ECM component laminin b1, and integrin subunits a v and b 3 ( Table 2) , were seen in cells infected with L. interrogans Canicola as compared to the uninfected controls. In contrast, the L. biflexa Patoc caused increases in mRNA levels for the same genes in infected cells vs. uninfected controls (Table 2) . Together, all of these gene expression patterns are consistent with the hypothesis that one effect of L. interrogans serovar Canicola is to promote actin remodeling and detachment of the cells from the ECM. A fundamental stage in the pathogenesis of Leptospira infections is the ability of the bacteria to cross mucous membranes and underlying epithelial barriers, as well as endothelial cell barriers, and disseminate to different organs. Although Leptospira species are Ea.hy926 endothelial cells were plated in tissue culture treated glass chamber slides and allowed to reach near confluence (assessed visually). The bacteria were added at MOI = 10 and incubated with the endothelial cells for 1 or 3 hours at 37uC, then were washed and fixed. The slides were stained with phalloidin-FITC, which illuminates F actin, plus anti-Leptospira antibodies followed by TRITC-conjugated secondary antibody. Retraction of the cell bodies in response to L. interrogans Canicola, but not L. biflexa Patoc, is evident, particularly at 3 hr infection. The brighter staining of rounded and retracted cells with FITC-phalloidin may be due to disorganization of cellular architecture without complete depolymerization of the actin, which in the increased depth and decreased area of the cytoplasm would appear more concentrated and therefore brighter. Changes in endothelial cell morphology were most evident, and at earlier time points, in cells with which the L. interrogans bacteria were associated. One higher magnification micrograph of L. interrogans Canicola infected cells is included because the bacteria are small when viewing fields of endothelial cells that provide information on integrity of the monolayer. Micrographs are representative of multiple (.12) independent experiments. L. interrogans Copenhageni caused essentially the same changes in endothelial cell morphology as L. interrogans Canicola (data not shown). doi:10.1371/journal.pntd.0000918.g001 Endothelial Cell Responses to Leptospira www.plosntds.org extracellular bacteria apparently devoid of actin modifying exotoxins [21] [22] [23] , and devoid of the specialized secretion systems utilized by many bacterial pathogens to deliver toxins that disrupt the host cell cytoskeleton (as reviewed in [24] [25] [26] [27] [28] ), pathogenic leptospires might be indirectly targeting the cytoskeleton via cell surface attachment mechanisms that co-opt the host cell signaling to achieve the same result.
Decreased cellular adhesion to the ECM and rearrangement of the cytoskeleton may facilitate the migration of Leptospira through endothelial barriers as it disseminates from the site of inoculation. To further explore the possibility that actin rearrangements are triggered by Leptospira infection at the functional level, endothelial cells plated in chamber slides were infected at an MOI of 10 for 1 hour and 3 hours. As shown in Figure 1 , the bacteria were clearly more adherent to the cells than to the extracellular space, and the pathogenic bacteria caused dramatically more significant alterations in cellular morphology and integrity of the cell layer than did the non-pathogenic bacteria. The earliest change noted was a reduction in cortical actin (so the cell edges are less defined) and appearance of gaps in confluent cell layers, followed by loss of stress fibers and rounding of the cells. The images shown in Figure 1 are from cell layers that were just below confluence prior to infection, to allow better visualization of changes in individual cells. For example, while the cortical actin has largely disappeared in cells infected with L. interrogans Canicola by 1 hour postinfection, and stress fibers have disappeared and cell rounding is evident by 3 hours, the cells are largely unaffected at the same time points after infection with L. biflexa Patoc (Figure 1 ). L. biflexa Endothelial Cell Responses to Leptospira www.plosntds.org does adhere to mammalian cells in culture less efficiently than does L. interrogans (as shown and reviewed in [17] ), but even when bacterial contact with the cells was facilitated by centrifugation, the L. biflexa caused little disruption to cellular morphology and cell layer integrity (data not shown).
Although these and subsequent experiments were performed using adherent cells, the morphologic changes are consistent with changes in mRNA levels seen using lifted cells in the microarray experiments. Despite the alterations in cellular architecture and monolayer integrity, no decrease in endothelial cell viability was found by any of several criteria (see Materials and Methods), even after infection times extended as long as 48 hours (Figure 2 ). The disruptions in the layers did, however, result in the ability of the pathogenic strain to cross the monolayers more efficiently than did the non-pathogenic bacteria (Figure 3) . After a brief period in which the endothelial layer did prevent significant transmigration of the bacteria, the layer rapidly became essentially irrelevant as a barrier to the penetration of the pathogenic bacteria, as the bacterial counts in the lower chamber were unaffected by whether or not cells had been plated on the membrane.
Because Leptospira interrogans has been shown to bind to proteoglycans on the mammalian cell surface [17] , we tested a proteoglycan synthesis inhibitor, b-xyloside, for the ability to decrease damage to endothelial cell layers caused by L. interrogans Canicola. b-xyloside inhibits transfer of glycosaminoglycan chains to protein cores; a control sugar analog, a-galactoside, was tested in parallel. As shown in Figure 4 , inhibition of proteoglycan synthesis did not fully prevent the damage to the endothelial cell layers caused by L. interrogans. The inhibition of glycosaminoglycan chain attachment does not significantly affect the formation of holes in the cell layer caused by L. interrogans Canicola as assessed visually and by measurement of L. interrogans penetration of the cell layers (data not shown). b-xyloside does cause a reduction of L. interrogans Canicola and Copenhageni attachment to these cells ( [17] and data not shown), but does not abolish bacterial attachment, consistent with the hypothesis that additional nonproteoglycan molecules serve as substrates for L. interrogans attachment to cells. Direct bacterial attachment to the cells does appear to be required for the damage to the endothelial cell layers, as supernatants harvested from infected cell layers (infection times of 1-24 hr) and sterilized by centrifugation and filtration through 0.1 mm filters did not affect endothelial cell layer integrity (data not shown). Therefore, non-proteoglycan cell surface receptors are likely to be those primarily involved in the responses of the endothelial cells to L. interrogans attachment, and efforts to identify both the host cell and the bacterial cell molecules involved in these interactions are underway. As noted in the publication reporting the sequence of two L. biflexa Patoc strains [29] , there are a number of proteins predicted in the published L. interrogans genomes that are not present in the L. biflexa Patoc genome, including some that are postulated to have potential adhesin activities. These include proteins containing leucine-rich repeats, which are involved in many protein-protein interactions [29] . As stated in the publication of the L. biflexa genome, it is intriguing Ea.hy926 cell layers were treated with the proteoglycan synthesis inhibitor b-xyloside, or the control a-galactoside, as described in [17] prior to infection with L. interrogans sv. Copenhageni. After 3 hr, the cell layers were washed and fixed, then stained with phalloidin-FITC. Neither reagent significantly reduced disruption of the cell layers by L. interrogans, as alterations in cell morphology and significant gaps between cells were seen when L. interrogans was present, and trans-endothelial cell layer migration was not significantly affected (data not shown). Consistent with this result, chondroitin sulfates B and A, which do and do not inhibit L. interrogans attachment to mammalian cells, respectively [17] , also had no effect (not shown). doi:10.1371/journal.pntd.0000918.g004
Endothelial Cell Responses to Leptospira www.plosntds.org that a Treponema denticola leucine-rich repeat protein, LrrA, has been identified as an adhesion/tissue penetration factor [29, 30] . It is also possible that additional components of the surfaces of L. interrogans and L. biflexa might have different effects on host cells [31] [32] [33] . At this point, however, the determinants critical to the effects of L. interrogans-host cell interaction reported here remain to be identified, and neither bacterial adhesins nor host substrates can necessarily be predicted solely on the basis of the primary amino acid sequences.
Several drugs currently in use in humans have been reported to have endothelial barrier protective function; all are in use as antihypertensive therapeutics, and some for other therapeutic purposes as well. We therefore tested four different drugs with different mechanisms of action for the ability to prevent the damage to endothelial layers in culture caused by L. interrogans. Lisinopril binds to and competitively inhibits angiotensin 1 binding to angiotensin converting enzyme (ACE), which is expressed by endothelial cells, while telmisartan competitively inhibits angio-tensin 2 binding to its receptor AT 1 . Dopamine is an antagonist of VEGF/VEGFR2-mediated cell layer permeability in treatment of human umbilical vein endothelial cells (HUVECs) in vitro at 10mM, as well as VEGF-mediated angiogenesis in vivo and proliferation of HUVECs at 1 mM in vitro [34, 35] . Furosemide is an anion transport blocker and is used as a diuretic but has antihypertensive activity as a consequence, and was used as a control not expected to preserve endothelial layer integrity. While telmisartan, furosemide, and dopamine did not protect the endothelial layers from the damage due to L. interrogans Copenhageni infection, lisinopril did at 100 nM, 1 mM and 10 mM ( Figure 5 , representing 3 independent experiments, and data not shown). There are several possible explanations for this, including: 1) lisinopril inhibits L. interrogans attachment to the cells, and 2) that attachment is unaffected but the interaction of the bacteria triggers activation of a signaling cascade or release of a mediator whose action or activation is inhibited by lisinopril. We therefore investigated the possibility that lisinopril might prevent Figure 5 . Effects of specific drugs that protect endothelial barrier function on damage caused by L. interrogans. Panels A and B: Ea.hy926 endothelial cell layers were infected with L. interrogans sv. Copenhageni or L. biflexa sv. Patoc as described in Materials and Methods, except that just prior to the addition of the bacteria the drugs lisinopril, telmisartan, dopamine, or furosemide were added to 1 mM. The micrographs shown in Panel A (representative of three experiments) were taken at the 6 hour time point; the graphs in Panel B show the transmigration of leptospires over the entire 72 hr. time course. Shown are the means and standard deviations of all data from three experiments. Statistical significance was determined using repeated measures ANOVA followed by Bonferroni's multiple comparison test. For wells with cells, L. interrogans vs. L. biflexa, p,0.001, L. interrogans with no additions vs. telmisartan p.0.05 (not significant), L. interrogans with no additions vs. lisinopril p,0.001, L. interrogans with telmisartan vs. lisinopril p,0.001. There were no significant differences in the absence of cells, and the drugs did not affect bacterial motility or attachment of 35 S-labeled leptospires to the cells (Panel C and data not shown). doi:10.1371/journal.pntd.0000918.g005 Figure 6 . Lisinopril concentrations effective in protection of endothelial cell layers from damage due to L. interrogans. Ea.hy926 endothelial cell layers were infected with L. interrogans sv. Copenhageni as described in Materials and Methods, except that just prior to the addition of the bacteria the drugs lisinopril or telmisartan were added to the concentrations indicated. The micrographs were taken at the 3 hour time point. Lisinopril at 10 mM, 1 mM and 100 nM blocked endothelial disruption by L. interrogans; lisinopril at 10 nM or below did not. doi:10.1371/journal.pntd.0000918.g006
Endothelial Cell Responses to Leptospira www.plosntds.org endothelial damage by blocking L. interrogans Copenhageni attachment to the cells, but no inhibition of adhesion of 35 Slabeled bacteria [17] was seen even at a concentration of lisinopril 10 fold over the concentration used for these experiments ( Figure 5 ).
Although it was tempting to speculate that cell-surface-localized ACE could serve as a receptor for L. interrogans, as the enzyme is expressed by endothelial cells and proximal tubule epithelial cells [36] , and is therefore open to possible competition by the lisinopril, this is not consistent with our results to date. However, ACE2 is not inhibitable by lisinopril, but is a receptor for the SARS virus [37] , so there is precedent for ACE proteins serving as receptors for pathogens. It is also possible that the effect of lisinopril in our system is not related to ACE inhibition, but is instead due to additional effects of lisinopril, such as inhibition of isoprenoid synthesis, which is required for the post-translational modification of Rho GTPases, which in turn regulate the actin cytoskeleton [38] . In turn, this may lead to increased NO synthesis, which is protective of endothelial function in the face of a variety of insults. Given that doxycycline also has endothelial protective effects [39] , and that doxycycline is effective in treating leptospirosis [40] , our results may also provide a starting point for investigation into possible combinatorial therapeutic approaches to reduction of endothelial damage and consequent organ damage in human populations during leptospirosis outbreaks. Should this combinatorial approach prove useful in animal models, consideration as a focused approach to the treatment of human leptospirosis is warranted. The 1 mM dose shown in Figure 5 is at the high end of the physiologically relevant dosing range for humans, but administration of an antihypertensive to a patient with clinical manifestations of leptospirosis would be contraindicated, as further depression of blood pressure levels would be potentially lethal. However, in outbreak situations, this agent could potentially help to reduce endothelial damage if administered to affected populations as soon as an outbreak situation is recognized, prior to exposure of the majority of the population to pathogenic Leptospira species. In addition, protective effects of lisinopril were maintained even at a dose of 100 nM, which is well within the range routinely used in humans ( Figure 6 ). It will also be interesting to investigate the possibility that, on a population basis, patients on lisinopril fare better than patients not on this therapy during leptospirosis outbreaks.
Reorganization of the actin cytoskeleton, as indicated by our microarray studies and by phalloidin staining of F actin, is essential to the pathogenesis of diverse bacterial infections, and pathogens use many different strategies to provoke changes in the cellular cytoskeleton in order to facilitate invasion of tissues, invasion of host cells, or evasion of phagocytosis (as reviewed in [24, 41, 42] ). A different spirochete, Treponema denticola, produces the protein Msp, which disrupts the actin cytoskeleton in neutrophils and fibro-blasts, preventing phagocytosis of the bacterium and inhibiting the cellular migration required to respond to and repair the damage caused by the pathogen and the host response at the site of infection [43, 44] . These activities are likely to facilitate invasion and colonization of periodontal tissues by T. denticola. Previous work by another laboratory demonstrated that L. interrogans Copenhageni crosses MDCK canine kidney epithelial cell layers in culture more rapidly than does L. biflexa Patoc [45] , but without significant disruption to the cell layers or the actin cytoskeleton. Consistent with these results, in experiments not shown here we also observed no significant damage to NRK (normal rat kidney) 293 (human kidney) or HEp-2 (human laryngeal) epithelial cell layers infected with L. interrogans Canicola or L. interrogans Copenhageni. The calculations of the proportions of bacteria crossing the cell layers differed between the two studies, but our protocol accounted for the replication of the L. interrogans Canicola and Copenhageni in the co-cultures, while the L. biflexa Patoc did not replicate (data not shown). Thus the endothelial cells tested here respond very differently to the bacteria than did the MDCK epithelial cells, and our results are the first to suggest a mechanism: disruption of actin dynamics by bacterial attachment to the cell surface. Thus, while L. interrogans has not been shown to secrete a toxin that modifies actin, the bacteria are able to manipulate the actin cytoskeleton indirectly. Even the pore forming toxin activity reported for Leptospira [46, 47] does not appear to have as large an effect, as the endothelial cells here were viable throughout the experiments. The leptospires may be able to establish disseminated infection in part due to the binding of the bacteria to one or more mammalian cell surface receptors that in turn, regulate the dynamics of the actin cytoskeleton in the mammalian cell. Deciphering the role of, and mechanisms behind, actin rearrangement in response to pathogenic Leptospira will provide insights into the mechanisms that leptospires uses to disseminate to different organs of the host to cause infection and disease, and provides a possible avenue for therapeutic intervention in conjunction with antimicrobial therapy. A Porcine Adenovirus with Low Human Seroprevalence Is a Promising Alternative Vaccine Vector to Human Adenovirus 5 in an H5N1 Virus Disease Model Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5. Experimental adenovirus-based vaccine vectors are promising alternatives to conventional vaccine platforms. In particular, human adenovirus serotype 5 (AdHu5) vectors are well-characterized and are being developed against several infectious disease models including influenza, hepatitis C, dengue and viral hemorrhagic fever viruses [1, 2, 3, 4] . Several candidates have demonstrated unique protective efficacy and can generate robust immune responses in both animal models and clinical trials [4, 5, 6, 7] . Pre-existing immunity against AdHu5 is, however, frequent in the human population and has been associated with undesirable clinical outcomes and the suspension of clinical trials [8, 9, 10] . One promising alternative is the development and evaluation of rare human, chimpanzee, or other mammalian adenovirus vectors with low seroprevalence in humans. A chimeric simian adenovirus 21 vector protected mice against lethal Ebolavirus challenge and generated robust T-cell responses against the glycoprotein in nonhuman primates [11] . A bovine adenovirus 3 (BAV3)-based vaccine previously demonstrated successful protection against avian influenza A virus H5N1 challenge in mice and was able to escape preexisting neutralizing antibodies against AdHu5 [12] . Similarly, a porcine adenovirus 3 (PAV3) vector was successful in several swine vaccination studies against classical swine fever and pseudorabies virus [13, 14, 15] . PAV3-based vaccines were able to evade pre-existing immunity and provide long-term protection in pigs [14] . The antigenic profile and reported in vivo efficacy as an animal vaccine makes the PAV3 vector a promising alternative adenovirus vector for human administration.
Due to their genetic diversity and the availability of several significantly different isolates, avian influenza H5N1 viruses provide a valuable and challenging disease model for evaluating broad immune responses generated by potential adenovirus vectors. The external hemagglutinin (HA) glycoprotein mediates receptor binding, fusion, and can generate both strong antibody and cell-mediated immune responses [16] which can be directly assayed and provide useful comparison of adenovirus platforms. Highly pathogenic avian influenza H5N1 viruses have spread throughout domestic and aquatic bird populations in South East Asia and the World Health Organization (WHO) has confirmed 500 clinical cases of H5N1 cross-transmission into humans. Despite the limited incidence of human-to-human-transmission, high mortality rates (.60%) and continuous evolution of the virus represent a concern for future influenza pandemics [17, 18] . The emergence of pandemic swine-like H1N1 influenza A virus isolates in early 2009 highlights the need to generate cross-protective and lasting immune responses against diverging human and zoonotic influenza viruses. In addition to evaluation of different adenovirus platforms, the development of improved influenza vaccines would also help in better preparation against emerging pandemic viruses and could reduce the impact of infection on public health.
This study evaluates the protective efficacy following lethal homologous challenge of a replication-incompetent porcine adenovirus 3 (PAV3) vector expressing the HA gene from the A/ Hanoi/30408/2005 H5N1 (H5N1-H05) influenza A isolate (PAV3-HA). The immunogenicity of HA and the success of previous AdHu5 H5N1-HA vaccines [1, 19] suggested that avian influenza H5N1 may be a good comparative model to evaluate the efficacy of a similar PAV3 vector.
Previous studies showed that PAV3 does not exhibit crossreactivity with AdHu5 or BAV3 neutralizing antibodies [20, 21] . Additionally, PAV3 was not neutralized by the lowest dilution of 1:4 from 50 randomly selected human sera [20] In order to further address neutralization of PAV3 by an extended number of human sera, human Ig made of pooled sera from 10,000-60,000 individuals was evaluated. AdHu5, used as a control, was neutralized at the highest dilution of 1:160 (6.25610 23 mg/ml human Ig). In contrast, neutralization of PAV3 was not detected at 1:20 (5.061022 mg/ml), the lowest dilution tested. Previous studies showed little cross-reactivity between cell-mediated immune responses against AdHu5 and PAV3 [22] .
An optimized expression cassette containing the codonoptimized H5N1-HA was inserted by homologous recombination into a replication-incompetent PAV3 vector containing deletions in the E1/E3 genes (described in [23] ). An AdHu5-HA vaccine was also developed in parallel as a control to compare levels of protection and immune responses. Expression of the PAV3-HA and AdHu5-HA vaccines was evaluated in VRIBL E1, HEK 293, and mouse AB12 cells (Figure 1a ). BALB/c mice were also vaccinated with 10 10 virus particles (vp) and in vivo expression of PAV3-HA or AdHu5-HA in muscle tissue was detected 4 days post-immunization ( Figure 1b) . A robust antibody response is important for the prevention of influenza virus infection. The ability of the PAV3-HA vaccine to generate humoral immune responses following immunization was assayed through detection of HA-specific antibodies by hemagglutination inhibition (HI) or neutralizing antibody (NAB) titres. BALB/c mice were vaccinated with 10 10 vp/mouse of PAV3-HA or AdHu5-HA and the development of HI and NAB antibody responses was monitored from serum collected at days 8, 10, 14, and 21 post-immunization. All samples were treated with receptor-destroying enzyme (RDE), followed by complement inactivation the next day. An HI assay was performed using serial dilutions of the treated serum combined with H5N1-H05 virus and horse red blood cells to detect inhibition of cell agglutination by serum antibody. The reciprocal of the highest dilution which did not agglutinate red blood cells was scored as the HI antibody titre. HI titres of 2060, 120669, 213692, 5336184 were detected for PAV3-HA and 27611, 67623, 213692, 4266184 for AdHu5-HA, respectively, with no statistical difference between the two vaccines ( Figure 2a ). The presence of neutralizing antibodies to inhibit active H5N1-H05 infection was also evaluated as a measure of the humoral response. Serial dilutions of treated serum were incubated with 100 pfu/well of H5N1-H05 virus and cells were monitored for the presence or absence of cytopathic effects (CPE) using a light microscope. The highest serum dilution which did not exhibit CPE was scored as positive for neutralizing antibody and titres were reported as the reciprocal of the dilution. NAB titres of 060, 7612, 7612 and 90650 or 060, 7612, 10610 and 4060 reciprocal dilutions were observed with the PAV3-HA or AdHu5-HA vaccines at 8, 10, 14, and 21 days, respectively (Figure 2b ).
The increased neutralizing antibody response following PAV3-HA vaccination was statistically significant at day 21 (p = 0.045) relative to AdHu5-HA.
In order to assess antibody titres immediately before challenge, mice were vaccinated with different doses of each vaccine and serum was obtained 25-days post-immunization. HI reciprocal titres of 9866326, 10936293, and 1280 for the PAV3-HA vaccine or 10666330, 10666330, and 11736261 with AdHu5-HA were detected for 10 8 , 10 9 or 10 10 vp/mouse doses respectively (Figure 2c ). Both vaccines had similar HI antibody titres at all doses, with no significant differences detected between the PAV3-HA and AdHu5-HA (p = 0.57). Based on clinical data, a reference HI antibody titre of 40 is recommended for influenza vaccine candidates as the minimum level to confer fifty percent protection in humans [24, 25] . Both vaccines met this requirement at all doses prior to challenge. Serum samples had an average NAB titre of 65620, 75614, or 106646 reciprocal dilution at 10 8 , 10 9 or 10 10 PAV3-HA vp/mouse respectively ( Figure 2d ). The AdHu5-HA vaccine generated NAB titres of 65620, 55620 or 133646 at 10 8 , 10 9 or 10 10 vp/mouse, respectively.
Although a strong antibody response is important for immediate and long-term protection against influenza viruses, the induction of early cellular immune responses following vaccination may enhance clearance of virus-infected cells following H5N1 influenza virus infection. T-cell responses were assayed using an enzymelinked immunosorbent spot (ELISPOT) assay to detect secretion of interferon gamma (IFNc) by activated lymphocytes. Splenocytes were obtained from mice vaccinated at days 8, 10, 14, and 21 with 10 10 vp/mouse of PAV3-HA or AdHu5-HA and the cells were restimulated with pools of overlapping peptides corresponding to the entire H05-HA protein. The peptide corresponding to the immunodominant H5N1-H05 HA (IYSTVASSL, conserved influenza A virus) epitope was evaluated alongside an unrelated control peptide (TYQRTRALV, A/PuertoRico/8/34 H1N1 nucleoprotein).
In mice vaccinated with PAV3-HA, 13,59862066, 14,4426 2541, 69546392, or 20566633 spot-forming cells (sfc) per million splenocytes were detected at days 8, 10, 14, or 21 respectively (Figure 3a) . Immunization with the AdHu5-HA vaccine generated 1,9656341, 12,85861749, 7332693, and 19026372 sfc/million, respectively. The number of sfc/million splenocytes detected at day 8 was statistically significant between PAV3-HA and AdHu5-HA vaccines (p,0.005). The H05-HA immunodominant epitope also stimulated the strongest T-cell responses on day 8 for the PAV3-HA vaccine compared to AdHu5-HA. PAV3-HA vaccinated mice generated an average of 8126138 sfc/million at day 8 whereas 2206133 sfc/million were detected from AdHu5-HA mice at the same time point (Figure 3b , p = 0.048). At day 10, average responses following stimulation with the H05-HA immunodominant epitope were 1,2166178 or 1,364636 sfc/ million from PAV3-HA or AdHu5-HA immunized mice respectively (p = 0.258). T-cell responses of 770674 and 453684 were detected from PAV3-HA or 791658 and 590684 from AdHu5-HA at days 14 and 21 post-immunization, respectively. Restimulation of splenocytes from PAV3-HA or AdHu5-HA immunized mice by the NP control peptide repeatedly generated less than 65 sfc/million. Cellular responses following vaccination with PAV3-HA were further characterized by detecting the relative frequency of peptide-specific CD8 + T cells expressing IFN-c by flow cytometry. Splenocytes were harvested ten days post-immunization and restimulated in vitro with the H05-HA immunodominant peptide and unrelated NP or ZGP (TELRTFSI, Zaire Ebolavirus glycoprotein) control peptides. Cells were stained with anti-mouse CD8-FITC (fluorescein isothiocyanate) and an anti-mouse IFNc-PE (phycoerythrin). The frequency of IFNc positive CD8 + T-cells was 3.460.3 and 2.860.3 percent for PAV3-HA or AdHu5-HA, respectively (Figure 3c ). Cells stimulated with control peptide NP or ZGP showed frequencies of CD8 + IFNc positive T-cells equal to 0.360.05 or 0.660.05 percent, respectively. These results suggest that the PAV3-based vaccine can stimulate a robust immune response in mice faster than AdHu5HA and comparable in strength.
Protective efficacy of PAV3-HA following lethal challenge
The early detection of the T-cell response at day 8 postvaccination with the PAV3-HA vaccine suggest that it may be a good candidate for immediate administration just prior or following a suspected exposure to H5N1 virus. The success of rapid vaccination regimen was evaluated in two parts: first, overall survival and second, total viral load in lung tissue, as determined by TCID50 assay. BALB/c mice were challenged with a lethal dose of H5N1-H05 virus at 5, 8 and 10 days post-vaccination and lungs were harvested at day 3 post-infection. Although mice challenged 5 days post-vaccination did not survive lethal challenge (Figure 4a Survival was also assessed in groups of 10 BALB/c mice vaccinated with 10 8 , 10 9 , or 10 10 virus particles of recombinant adenovirus PAV3-HA or AdHu5-HA by intramuscular (I.M.) administration, challenged 28 days later with lethal homologous H5N1-H05 virus. Protection against clinical signs of disease with minimal weight loss and full survival was observed at the 10 10 vp/ mouse dose for both Ad-based vaccines ( Figure 5 ). Complete survival was also observed for both vaccines at a dose of 10 9 vp/ mouse, with the PAV3-HA vaccinated mice either asymptomatic or having milder signs of disease (14% weight loss) compared to AdHu5-HA (21% weight loss). Another desirable characteristic that influenza vaccines should provide is long-term protection. Therefore, long term immunity was evaluated in mice challenged 12 months after vaccination in order to determine whether protective immune responses could be maintained. PAV3-HA afforded full protection in mice challenged with 100 LD50 of H5N1-H05 virus 12 months post-immunization whereas 50% of mice vaccinated with AdHu5-HA succumbed (Figure 6a ). Higher HI antibody titers for PAV3-HA compared to AdHu5-HA (186665 and 60623, respectively (p = 0.006)) may have translated directly to the improved survival observed with the PAV3-HA vaccine. NAB titers were 23615 and 10611 for AdHu5-HA (Figure 6b ). An ELISA assay was also performed to detect total IgG antibody titres against the H5N1-HA antigen. Serum was obtained from mice 25 days and 1 year post-vaccination, and unvaccinated control mice (Figure 6c ). Total antibody titers were significantly lower for both vaccines after 1 year. On average, higher levels of IgG antibodies were detected for the PAV3-HA vaccine, however the difference was not statistically significant compared to AdHu5-HA (p = 0.241).
Currently, replication-deficient human adenovirus serotype 5 (AdHu5) vaccines are being evaluated against several pathogens. Several candidates have been shown to induce protective immune responses against emerging or re-emerging infectious pathogens such as Ebola and avian influenza (H5N1) viruses [4, 26, 27] Complete protection and long-term memory responses have also been reported in different animal models [7, 26, 28] ; however, the final development of AdHu5-based vector to approved human vaccines has been hampered by the presence of natural preexisting immunity to AdHu5 which is present in a large fraction of the human population. Neutralizing antibody to porcine adenovirus 3 (PAV3) was not detected from pooled immune globulin representing 10 to 60 thousand human sera suggesting that preexisting immunity to PAV3-based vaccines is not likely to be of concern for human applications. In addition, the documented compatibility of porcine and human tissues could translate into PAV3-based vaccines being efficacious while of low toxicity in humans [23] . The further development of a new adenovirus serotype also increases the spectrum of possible applications such as sequential vaccination with other adenovirus vectors (e.g. prime/boost or a second Ad-based vaccination against a different agent) [20, 29] .
The present study compares in parallel the immune responses and protection generated by a PAV3-based vector and a similar AdHu5-based vector using an avian influenza H5N1 disease model of infection. PAV3-based vector expressing the H5N1 HA antigen was able to generate quick and robust immune responses against H5N1. This finding was supported by the rapid induction of improved immediate protection by the PAV3-HA vaccine compared to AdHu5-HA. Interestingly, both vaccines shared similar in vivo expression, suggesting that the increase in vaccine efficacy observed with PAV3-HA may be due to specific interactions with the immune system rather than enhanced expression of the antigen. A recent study suggests that PAV3-HA may activate different innate immune pathways to AdHu5-HA [30] . Additionally, the PAV3-HA offered improved long-term protection against lethal H5N1 virus challenge with higher levels of detectable antibodies by HI assay. Although full short-term (28 day) survival was observed following vaccination with PAV3-HA or AdHu5-HA at the 10 9 vp/mouse dose, greater weight loss was observed with the AdHu5-HA vaccine. Comparing HI and NAB antibody titres with observed clinical outcome, this suggests that there may have been incomplete neutralization of the virus following vaccination with AdHu5-HA, perhaps explaining why there were differences in the levels of protection observed following challenge 1-year post-vaccination. It has been shown that levels of neutralizing antibodies in serum decrease over time following H5N1 infection [31] . Following vaccination, the exact levels of neutralizing antibodies correlating with full protection from infection with homologous virus is still uncertain. In comparison with total IgG antibody levels, neutralizing antibody titres correlated better with early and long-term protection, similar to previously described reports [19, 32, 33] .
Together the data suggests that PAV3 vector is an additional option to AdHu5 vector and could have several important applications including rapid or post-exposure protection against emerging influenza viruses or other infectious agents. A similar study described a BAV3-HA vaccine sharing comparable protective efficacy to the AdHu5-HA against an H5N1 virus. Two doses of the BAV3 vector generated similar humoral and cellmediated immune responses and was able to evade pre-existing neutralizing antibodies against AdHu5 [12] . Overall protective efficacy offered by PAV3-HA was similar to AdHu5-HA one month post-immunization and the immune response kinetics was also generally comparable at later time points. Although not explored in the current study, previous studies have evaluated the impact of pre-existing immunity to PAV3 and the potential reuse of PAV3-based vectors against different pathogens. Groups of outbred pigs with high PAV3 neutralizing antibody titres were vaccinated with a PAV3-based vaccine and vector re-administration did not result in hepatotoxicity or reduced transgene expression [14] . Doses of 10 13 particles/kg AdHu5 vector can also bypass pre-existing immunity, however, several pathologies including liver damage indicated by elevated transaminase, low platelets count, and lymphocytopenia were observed in nonhuman primates administered similar doses [9, 10, 34, 35] .
Even though conventional vaccines have been relatively successful against influenza A infection, the ability of adenoviral vectors to rapidly generate a strong immune response may be useful to specific applications such as rapid immunization of health care workers in anticipation of a probable exposure to an emerging virulent pandemic virus such as H1N1-1918. In addition to strong antibody responses against the H5N1-H05 virus, both survival and cellular responses suggest that induction of an earlier T-cell response by the PAV3-may complement the developing antibody response to improve protection against H5N1 challenge and contribute directly towards lower viral load. Although the influenza glycoproteins have high antigenic variation, the generation of faster T-cell responses by a PAV3 vector against well-conserved influenza antigens may supplement a mismatched antibody response and may improve protection against a wider range of influenza viruses.
Previous studies have shown that PAV3 vectors can transduce several human cell lines resulting in full transgene expression and adenoviral coat proteins [20, 21] . An additional safety feature is that wild-type PAV3 does not replicate in human cells [21] , Groups of 6 BALB/c were vaccinated with 10 10 vp/mouse of PAV3-HA () or AdHu5-HA (), or no vaccine (Control, ()) and challenged with 100LD50 of H5N1-H05 on days 5, 8, and 10 post-vaccination. Lungs were harvested from the mice on day 3 post-challenge. Virus titre was determined by TCID50 assay using serial dilution of lung homogenates on MDCK cells and monitoring for the presence of CPE over 48 hours. The TCID50 titre was calculated by the Reed & Muench method [39] and normalized/gram of lung tissue. Data is presented as log 10 TCID50/g of lung tissue. The data represent average values and standard deviations from one experiment performed with one vector preparation of each vaccine (* and ** represents p,0.05). doi:10.1371/journal.pone.0015301.g004
suggesting that a replication-competent PAV3 vector which would be easy to produce at high titres could also be a promising vaccine candidate. Nevertheless, performance of this new vaccine vector will need to be addressed in other animal species, including nonhuman primates, before its real utility as a human vaccine can be predicted more accurately. Overall, this study supports a complementary role for cellular immunity during early H5N1 infection and the further development of porcine adenoviruses as human vaccine candidates.
All animal procedures and scoring sheets were first approved by the Animal Care Committee (Animal Use Document ID# H-08-010) at the Canadian Science Centre for Human and Animal Health, according to the guidelines set by the Canadian Council on Animal Care.
Human embryonic kidney (HEK) 293 cells, Madin-Darby canine kidney cells (MDCK), and mouse AB12 cells were maintained in Dulbecco's modified eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), L-glutamate, sodium pyruvate (NaPyr), and antibiotics. Fetal porcine retina cells (VR1BL E1), expressing human adenovirus 5 (AdHu5) E1a and porcine adenovirus 3 (PAV3) E1b large genes, were maintained in minimum essential medium (MEM) alpha, supplemented with 10% FBS, L-glutamate, NaPyr, non-essential amino acids, HEPES buffered saline, penicillin/streptomycin, and 50 mg/ml Hygro- mycin B (BD Biosciences). Avian influenza H5N1 strain A/ Hanoi/30408/2005 (H5N1) was generously provided by Q. Mai Le and T. Hien Nguyen, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Virus was propagated on MDCK cells cultured with virus diluent (MEM, 0.3% bovine serum albumin, and antibiotics) containing 3.0 mg/ml TPCK-treated trypsin (TPCK-trypsin) and titered by plaque assay. All infectious recombinant adenovirus constructs were propagated on HEK 293 or VR1BL E1 cells and purified by a cesium chloride density gradient.
The full complementary DNA (cDNA) sequence from the A/ Hanoi/30408/2005 H5N1 hemagglutinin (H05-HA) gene was obtained, codon optimized, and synthesized from overlapping oligonucleotide primers, as previously described [36] . The H05-HA gene was first cloned into the pCAGa plasmid, containing a chicken-b-actin (CAG) promoter, to generate the pCAGa-HA construct. The pCAGa-HA expression cassette was then excised and inserted into two shuttle vector systems: pShuttle2 (Clontech) and pPAV227 (VIDO, University of Saskatchewan). Insertion of the expression cassette replaced the existing pShuttle2 cytomegalovirus (CMV) promoter with the CAG promoter, resulting in the pShuttle2-HA construct. Transfer plasmid pPAV227 was also modified to include the SV40 polyadenylation signal from pShuttle2, generating pPAV227-HA-PolyA.
The transgene cassettes from pShuttle2-HA and pPAV227-HA-PolyA were cloned into replication-deficient DE1DE3 adenoviral vectors: pAdenoX (AdHu5, Clontech) or pFPAV227 (PAV3, VIDO, University of Saskatchewan). pAdenoX-HA was generated through digestion of both pShuttle2-HA and pAdenoX by homing endonucleases I-CeuI/PI-SceI and ligation of cohesive ends using T4 DNA ligase (Invitrogen). pFPAV227-HA was generated through homologous recombination in Escherichia coli strain BJ5183 (recBC, sbcBC) [37] of linearized pPAV227-HA-PolyA (Eco47III/TthIII1) and pFPAV227 (PacI).
To obtain AdHu5-HA vaccine, HEK 293 cells were transfected with 10 mg of linearized pAdenoX-HA DNA in calcium phosphate (BD Biosciences) solution and cells were cultured until the appearance of cytopathic effects (CPE). Similarly, VR1BL E1 cells were transfected with 10 ug of linearized pFPAV227-HA DNA combined with Lipofectin (Invitrogen) and cells were cultured until CPE were apparent. Amplified adenoviruses containing cell lysates were harvested, freeze-thawed three times, and purified by CsCl gradients. The integrity of the H05-HA transgene cassette was confirmed through EcoRI restriction digests and by sequencing (DNA Core, National Microbiology Laboratory) with multiple primer sets. Total virus particles (vp) was determined by OD260 and total infectious particles was determined using anti-hexon antibodies against AdHu5 (AdenoX Rapid Titer kit, Clontech) or against PAV3 (VIDO, University of Saskatchewan). Four independent vector preparations were used for each vaccine. Total infectious particles and total viral particles were determined for both adenovirus vaccines, with ratios of 1:285, 1:333, 1:250, and 1:300 for PAV3-HA and 1:150, 1:250, 1:220, and 1:250 for AdHu5-HA.
Protein expression of H05-HA by both vectors was confirmed in HEK 293, VRIBL E1, and AB12 cells using standard Western blotting techniques. To evaluate in vivo expression, groups of 6 BALB/c mice were vaccinated with 10 10 vp of PAV3-HA or AdHu5-HA and muscle tissue was harvested 4 days following immunization. Muscle tissues were homogenized and normalized per gram of muscle tissue in radioimmunoprecipitation (RIPA) buffer. Expression of each vaccine was detected from 75 mg of loaded muscle tissue.
Groups of 10 BALB/c (Charles River Canada) were vaccinated with 10 8 , 10 9 , or 10 10 vp of recombinant adenovirus PAV3-HA or AdHu5-HA by intramuscular (I.M.) administration. Each vaccine was diluted in 100 ml and 50 ml was administered in each of the right and left hind limbs. All mice were anesthetized and challenged after 28 days through intranasal inoculation with 100 times the dose of A/Hanoi/30408/2005 virus required to obtain 50% survival (100 Lethal Dose 50 or 100LD50) in 50 ml virus diluent. The LD50 for the H5N1-H05 virus was 1.05 plaque forming units (pfu), therefore 100LD50 was 105 pfu. Mice were monitored for 15 to 20 days following challenge and signs of disease including weight loss, labored breathing, ruffled fur, and death were observed according to an approved scoring chart. All animal procedures were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) at the Public Health Agency of Canada (PHAC), according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the high biocontainment laboratory at NML/PHAC.
Groups of 4 BALB/c mice were vaccinated with 10 10 vp of PAV3-HA or AdHu5-HA vaccines. The day before each experiment, ELISPOT-IFNc (BD Biosciences) plates were coated with purified mouse IFNc and incubated at 4uC, overnight. As well, overlapping 15mer peptides (Mimitopes, Australia) spanning the entire H05-HA protein were resuspended overnight in dimethyl sulfoxide (DMSO) and allocated in pools by matrix format. Spleens were harvested on days 8, 10, 14, and 21 postimmunization and splenocytes were plated at 5610 5 cells/well in RPMI 1640 (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 5610 23 M 2-b-mercaptoethanol, and antibiotics) and restimulated with each of the peptide pools (2.5 mg/ml per well). An individual 9mer peptide representing the immunodominant H5N1 H05-HA (IYSTVASSL, conserved influenza A viruses) epitope was also evaluated, along with negative controls PR8-NP (TYQRTRALV, A/PuertoRico/8/34 (H1N1) nucleoprotein) and ZGP (TELRTFSI, Zaire Ebolavirus glycoprotein). ELISPOT-IFNc plates were incubated at 37uC overnight (18-20 hours) and washed the following day according to the manufacturer's instructions. AEC Substrate Set (BD Biosciences) was used to develop spots formed by interferon gamma secreting cells. Spots were visualized and counted using an ELISPOT plate reader (AID ELISPOT reader, Cell Technology, Colombia, Maryland).
Splenocytes obtained on day 10 post-vaccination, plated at 2610 6 cells/well, were restimulated with 9mer H05-HA, PR8-NP, or 8mer ZGP individual peptides in DMEM (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 5610 23 M b-mercaptoethanol, and antibiotics), IL2, and GolgiStop (Brefeldin A, BD Biosciences). Cells were stimulated for 5 hours and stained with anti-mouse CD8-FITC (fluorescein isothiocyanate) at 4uC for 30 minutes. BD Cytofix and permwash protocol was used to fix and permeabilized cells according to the manufacturer instructions. The following day, cells were stained Liposome-Coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8(+) T Cells We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8(+) T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8(+) T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8(+) T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8(+) T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity. Vaccines have played an important role in disease prevention and have made a substantial contribution to public health. Upon natural infection, it is known that the host responds by inducing both humoral and cellular immunity against the pathogen. However, most of the currently approved vaccines work by inducing humoral immunity [1] [2] [3] . For protection against viruses that are highly mutable and frequently escape from antibodymediated immunity, such as influenza A viruses, HIV, and HCV, humoral immunity is insufficient [4] [5] [6] [7] . Consequently, the development of vaccines that induce cellular immunity is critical to novel vaccine strategies.
T lymphocytes respond to peptide fragments of protein antigens that are displayed by MHC molecules on antigen-presenting cells (APCs). In general, extracellular antigens are presented via MHC class II molecules to CD4 + T cells while intracellular antigens are presented via MHC class I molecules to CD8 + T cells [8, 9] . However, a number of reports have demonstrated that a significant level of crossover, so-called 'cross-presentation', occurs in APCs [10] [11] [12] [13] [14] . Using this phenomenon, novel vaccine preparation inducing antigen-specific CTLs that effectively eliminate virus-infected cells is expected. The mechanisms of cross-presentation have been studied intensively [15] [16] [17] while the details have been left unclear. Part of the antigens taken via phagocytosis by APCs are known to be translocated into the cytosol and degraded by local proteases [18, 19] . In another pathway, some antigens internalized into endocytic compartments are loaded onto MHC class I molecules [20] .
We previously reported that antigens chemically coupled to the surface of liposomes induced antigen-specific IgG but not IgE antibody production [21, 22] . In addition, antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were presented not only to CD4 + -but also to CD8 + T cells by APCs [23] . Since liposome-coupled antigens induce antiviral immunity [24, 25] , they are expected to be applicable for the development of viral vaccines without inducing antigen-specific IgEs, which cause allergic reactions. In the present study, we investigated the mechanism by which the liposome-coupled antigens were cross-presented by APCs to CD8 + T cells.
Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates MHC class I of macrophages were stained with red fluoresceinlabeled anti-mouse H-2D d mAb (Fig. 1A : left column), and MHC class II of macrophages were labeled with DM-DsRed ( Fig. 1A : right column) as described in Materials and Methods. DQ-OVA, which exhibits green fluorescein upon proteolytic degradation, was coupled to liposomes consisting of unsaturated (oleoyl) or saturated (stearoyl) fatty acid, and added to the culture of macrophages. After incubation for 2 hr, the recovered macrophages were analyzed using confocal laser scanning microscopy. The results shown in Fig. 1 demonstrate that DQ-OVA coupled to oleoyl liposomes was processed at both MHC class I and class II compartments, while most of the DQ-OVA coupled to stearoyl liposomes was processed at the MHC class II compartment.
Alexa 488 -labeled OVA were coupled to liposomes and were added to the cultures of macrophages. As shown in Fig. 2 , OVA coupled to oleoyl liposomes were internalized by APCs more efficiently than those coupled to stearoyl liposomes at 37uC. Interestingly, OVA coupled to oleoyl liposomes but not stearoyl liposomes were internalized significantly by APCs even in a 4uC environment.
One of two kinds of inhibitors, cytochalasin B and DMA, which inhibit APC phagocytosis and pinocytosis of antigens, respectively, was added to the culture of macrophages 1 hr prior to the addition of Alexa 488 -OVA-or DQ-OVA-coupled oleoyl liposomes. One hour later, flow cytometric analysis was performed. As shown in Fig. 3 , the effect of cytochalasin B on the antigen uptake and digestion of liposome-coupled OVA by APCs was limited. On the other hand, DMA significantly reduced both antigen uptake and digestion of antigens by macrophages.
DQ-OVA-coupled oleoyl liposomes were added to the culture of macrophages in which either EEA1 or LAMP-1 were costained. The co-localization of the liposome-coupled antigens and intracellular organelles in the APCs was analyzed using confocal laser scanning microscopy. As shown in Figure 4 , although most of the DQ-OVA coupled to oleoyl liposomes was processed beyond LAMP-1-expressing compartments (green spots), a portion of DQ-OVA was processed at compartments expressing LAMP-1 (yellow spots). Co-localization of EEA1-expressing compartments with liposome-coupled-DQ-OVA was significantly less than that of LAMP-1-expressing compartments with DQ-OVA (Fig. 4B ).
In agreement with the results shown in Fig. 3 , antigen presentation by APCs pulsed with liposomal antigen was significantly inhibited by DMA but not by cytochalasin B in both CD4 + -and CD8 + T cell responses (Fig. 5 ).
In general, extracellular antigens are presented via MHC class II molecules to CD4 + T cells, whereas intracellular antigens are presented via MHC class I molecules to CD8 + T cells. Consequently, most APCs do not present exogenous antigens via MHC class I since exogenous antigens do not gain access to the cytosolic compartment. Therefore, exogenous antigens usually do not prime CTL responses in vivo. This segregation of exogenous antigens from the class I pathway is important to prevent CTL from killing healthy cells that have been exposed to foreign antigens but are not infected [26] . However, there are several exceptions to this rule, reflecting the ability of the exogenous antigens to be delivered into the cytosolic compartments [13] [14] [15] [16] [17] .
We have previously reported that antigens coupled to the surface of liposomes comprised of unsaturated fatty acid are presented to both CD4 + -and CD8 + T cells [23] . Confocal laser scanning microscopic analysis demonstrated that a portion of the liposome-coupled antigens were taken up and processed beyond the MHC class II compartment. In the present study, we confirmed that OVA coupled to oleoyl liposomes was processed at both the MHC class I and class II compartments ( Fig. 1 ). Flow cytometric analysis demonstrated that OVA coupled to oleoyl liposomes was incorporated more efficiently by macrophages than OVA coupled to stearoyl liposomes (Fig. 2) . Furthermore, OVA coupled to oleoyl liposomes was taken up by macrophages even in a 4uC environment, in which antigen entry could only occur via plasma membrane translocation. In general, antigen processing pathways largely depend on the route of antigen uptake, and liposomes with a certain lipid component are known to fuse with the plasma membrane [27] . The uptake of OVA coupled to oleoyl liposomes in a 4uC environment observed in the present study suggested that oleoyl liposome might fuse with the plasma membrane and thereby allow the liposome-coupled antigen direct access to the cytosol. The role of endocytosis in the uptake of the liposomal antigen was further examined by using specific inhibitors for antigen uptake (Fig. 3) . Cytochalasin B treatment of APCs prior to the addition of liposomal antigen in the culture had little effect. However, treatment of APCs with DMA significantly reduced the uptake of liposome-coupled OVA. Consequently, it was suggested that antigens coupled to oleoyl liposomes might be taken up by APCs via at least two pathways, penetration and pinocytosis. The analysis of intracellular pathways of antigens coupled to oleoyl liposomes using confocal laser scanning microscopy demonstrated that a portion of liposomal antigens taken up by APC were translocated to the lysosomal compartments expressing LAMP-1 (Fig. 4) , suggesting that the liposomal antigens processed at lysosomal compartment and beyond lysosomal compartment might be presented to CD4/ CD8 + T cells via MHC class II and class I, respectively. In agreement with the results of antigen uptake shown in Fig. 3 , the treatment of splenic CD11c + cells with DMA significantly reduced antigen presentation of liposomal antigens to both CD4 + -and CD8 + T cells as evaluated by T-cell activation (Fig. 5) .
It was reported that pinocytosis and scavenger receptor-mediated endocytosis by APC facilitate antigen presentation to CD4 + T cells; by contrast, mannose receptor-mediated endocytosis by APC has been shown to facilitate antigen presentation to CD8 + T cells [28] . However, as described in Materials and Methods, the oleoyl liposomes used in the present study do not contain mannose.
Thus, the data in the present study demonstrated that antigens coupled to oleoyl liposomes were internalized by APCs through both penetration and pinocytosis. The antigens coupled to the surface of oleoyl liposomes were processed at both MHC class I and class II compartments and presented to CD4 + -and CD8 + T cells. Although the detailed pathway leading to presentation to both CD4 + -and CD8 + T cells remains unclear, the observed behavior of antigens coupled to oleoyl liposome in APCs seems quite unique. Taken together, coupling of antigens to oleoyl liposome might potentially serve as a novel method to induce both humoral and cellular immunity.
Mice CBF1 mice (8 weeks of age, female) were purchased from SLC (Shizuoka, Japan). All experiments were approved (No. 208021 and 209082) by an independent animal ethics committee at National Institute of Infectious Diseases, Tokyo, Japan.
All phospholipids were provided by NOF Co. (Tokyo, Japan). Reagent grades of cholesterol were purchased from Wako Pure Chemical (Osaka, Japan).
Ovalbumin (OVA, Grade VII) was purchased from Sigma-Aldrich. For the analysis of the processing of liposome-coupled OVA by macrophages, DQ-OVA, which exhibits green fluores- Figure 2 . Uptake of liposome-coupled OVA by macrophages. Alexa-labeled OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages as described in Materials and Methods. Thirty minutes after the onset of the culture, macrophages were recovered and analyzed using flow cytometry. doi:10.1371/journal.pone.0015225.g002 Figure 1 . Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates. A, DQ-OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages expressing DM-DsRed (class II) or labeled with red fluorescein (class I), as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage DM or class I); cell only, macrophages without co-culture with DQ-OVA-coupled liposomes. B, the green-and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values 6 SD of the images shown in Fig. 1A . Asterisk, significant (p,0.01) difference of samples. doi:10.1371/journal.pone.0015225.g001 cence upon proteolytic degradation, was purchased from Molecular Probes, Inc. Synthetic CpG ODN (5002: TCCAT-GACGTTCTTGATGTT) was purchased from Invitrogen and was phosphorothioate-protected to avoid nuclease-dependent degradation.
OVA was labeled with fluorescence using an AlexaFluor 488 protein labeling kit (Invitrogen) according to the manufacturer's protocol.
Liposomes consisting of two different kinds of lipid were used in this study. Liposomes consisting of saturated fatty acids were composed of distearoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine, distearoyl phosphatidyl glycerol acid, and cholesterol in a 4:3:2:7 molar ratio (stearoyl liposomes), and liposomes consisting of unsaturated fatty acids were composed of dioleoyl phosphatidylcholine, dioleoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl glycerol acid, and cholesterol in a 4:3:2:7 molar ratio (oleoyl liposomes). The crude liposome solution was passed through a membrane filter (nucleopore polycarbonate filter, Coster) with a pore size of 0.2 mm.
Liposomal conjugates with plain OVA, Alexa-labeled OVA, or DQ-OVA were prepared essentially in the same way as described previously [22] . Briefly, to a mixture of 90 mg of liposomes and 6 mg of OVA in 2.5 ml phosphate buffer (pH 7.2), 0.5 ml of 2.5% glutaraldehyde solution was added in dropwise fashion. The mixture was stirred gently for 1 h at 37uC, and then 0.5 ml of 3 M glycine-NaOH (pH 7.2) was added to block excess aldehyde groups. This was followed by incubation overnight at 4uC. The liposome-coupled OVA and uncoupled OVA in the resulting solution were separated using CL-4B column chromatography (Pharmacia). The amount of lipid in the liposomal fraction was measured using a phospholipid content assay kit (Wako Pure Chemical). The OVA-liposome solution was adjusted to 10 mg lipid/ml in PBS, sterile-filtered using a Millex-HA syringe filter unit (0.45 mm, Millipore), and kept at 4uC until use.
For the measurement of OVA coupled to liposome, radiolabeled OVA (methyl-14 C; purchased from New England Nuclear) was mixed with cold OVA and used for coupling with liposome and for determining the calibration curve. The 
Mice were immunized subcutaneously (s.c.) with the OVAliposome conjugate at a dose of 1 mg lipid/100 ml/mouse in the presence of 5 mg/mouse CpG.
Macrophage hybridoma clone 39, obtained from the fusion of splenic adherent cells from CKB mice and P388D1 [29] , was used.
The DNA fragment coding the full-length H2-DMb2 [30] was amplified by PCR with two primers (59-ATGGCTGCACT-CTGGCTGCTGCTGCTGGT-39 and 59-GATGCCGTCCT- TCTGGGTAGGTGGATCC-39). The PCR product was cloned into the CMV promoter-driven expression plasmid pDsRedN1 (BD Clontech). This construct omitted the stop codon of H2-DMb2 and encoded the H2-DMb2 fused with DsRed. The cloned plasmid DNA was transfected to macrophage hybridoma clone 39 with Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. During the transfection to clone 39, the medium containing cDNA and the transfection reagent was replaced with fresh medium after an 8-h transfection, and then clone 39 was cultured for 40 h. To obtain stable cell lines, clone 39 was passaged at 1:5 into RPMI 1640 containing 10% FCS with 50 mg/ml geneticin (G-418; Sigma-Aldrich). Cells showing the best fluorescence were selected using a FACS Vantage cell sorter (BD Bioscience). After cell sorting, clone 39 expressing DM-DsRed was cultured in RPMI 1640 containing 10% FCS with 200 mg/ml geneticin.
To investigate the capture of OVA-liposome conjugates by macrophages, macrophage clone 39 was incubated for 30 min at 4uC or 37uC in the presence of fluorescence-labeled OVAliposome conjugates that contained a final concentration of 4 mg/ ml OVA. After the incubation, cells were washed with ice-cold PBS. In the case of using Alexa-labeled OVA-liposome conjugates, cells were then incubated with 1.2 mg/ml trypan-blue for 5 min at 4uC to block the fluorescence of Alexa-OVA attached to the cell surface. After the cells were washed, they were analyzed on a FACS Caliber flow cytometer (BD Bioscience). The histograms of fluorescence distribution were plotted as the number of cells versus fluorescence intensity on a logarithmic scale.
To investigate the localization of OVA-liposome conjugates by macrophages, macrophage clone 39 or DM-DsRed-expressing cloned macrophage 39 was cultured for 18 h at 37uC on 8-hole heavy Teflon-coated slides (Bokusui Brown) and was then incubated with DQ-OVA-liposome conjugates, prepared using oleoyl or stearoyl liposomes, for 2 h at 37uC. The slides were then washed with MEM and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, they were incubated for 10 min in 0.1 M glycine-HCl (pH 7.0) to block the remaining aldehyde residue. They were then washed two times in PBS. After washing, the slides were sealed with PBS:glycerin (1:9) and analyzed under an LSM510 confocal laser scanning microscope system (Zeiss). For analysis of co-localization of OVA and MHC class I, early endosomal antigen 1 (EEA1) or lysosomalassociated membrane protein-1 (LAMP-1) after blocking of the remaining aldehyde residue, cloned macrophage 39 was subsequently permeabilized with 0.05% saponin-TBS for 10 min at room temperature. After being washed twice with PBS, they were reacted with biotin-conjugated mouse anti-mouse H-2D d mAb (34-2-12, 10 mg/ml; BD Biosciences), goat anti-mouse EEA1 Figure 5 . IFN-c production by splenic CD4/CD8 + T cells of mice immunized with OVA after co-culture with CD11c + cells pulsed with OVA coupled to oleoyl liposomes. Splenic CD4/CD8 + T cells were taken from mice immunized with OVA and were cultured with CD11c + cells pulsed with OVA coupled to oleoyl liposomes with or without inhibitors as described in Materials and Methods. IFN-c production of T cells in the supernatants in the absence of inhibitors was normalized to 100%. Data represent the mean values 6 SD of triplicate culture. Asterisk, significant (p,0.01) difference as compared with the 'no inhibitor' group. doi:10.1371/journal.pone.0015225.g005 polyclonal antibody (N19, 1 mg/ml; Santa Cruz Biotechnology) or rat anti-mouse LAMP-1 monoclonal antibody (1D4B, 1 mg/ml; Santa Cruz Biotechnology) for 18 h at 4uC. After being washed three times with TBS, they were reacted with Alexa 546conjugated streptavidin (1:200 diluted; Invitorogen) to detect MHC class I, Alexa Fluor 568-labeled Ab (rabbit anti-goat IgG, 10 mg/ml; Invitrogen) to detect EEA1 or Alexa Fluor 568-labeled Ab (goat anti-rat IgG, 10 mg/ml; Invitrogen) to detect LAMP-1 for 4 h at room temperature. They were then washed two times in TBS. After the washing, the slides were sealed with PBS:glycerin (1:9) and analyzed under an LSM510 confocal laser scanning microscope system (Zeiss).
Quantification of confocal image analysis was done by single cell identification using the image analysis software MetaMorph (Molecular Devices Co., Tokyo, Japan), and the relative fluorescence intensity of green, red, and yellow pixels was assessed. The relative fluorescence intensity of all individual colors was then expressed as percent of the total fluorescence intensity. p values were calculated by the Student's t test with two-tailed distribution and two-sample unequal variance parameters.
In the case of inhibition studies, cloned macrophage 39 or CD11c + cells were incubated with indicated inhibitors 60 min before and throughout the antigen pulse. Cytochalasin B [31] and DMA [28] were purchased from Sigma.
Preparation of CD11c + cells and CD4 + -and CD8 + T cells CD11c + spleen cells of naïve mice and CD4 + T and CD8 + T spleen cells of mice immunized with OVA-liposome conjugates were prepared with the magnetic cell sorter system MACS, according to the manufacturer's protocol using anti-CD11c, anti-CD4 and anti-CD8 antibody-coated microbeads (Miltenyi Biotec).
Culture of CD4 + -and CD8 + T cells with CD11c + cells pulsed with OVA CD11c + cells were incubated with or without the indicated inhibitors for 60 min in a 24-well plate prior to the addition of OVA-liposome conjugates made using oleoyl liposomes. The final concentration of OVA-liposome added to the macrophage culture was 500 mg lipid/ml, which included 24 mg OVA. After 60 minutes' incubation, CD11c + cells were washed 3 times in ice-cold medium and 2610 5 cells were co-cultured with 5610 5 CD4 + T cells or CD8 + T cells, in a 48-well plate. A preliminary experiment showed that the optimal culture period in the above culture condition was 2 days for IFN-c production by CD4 + T cells and 5 days for IFN-c production by CD8 + T cells. After incubation in a CO 2 incubator for 2 or 5 days, the culture supernatants were collected and assayed for IFN-c.
IFN-c in the culture supernatants was measured using the Biotrak mouse ELISA system (GE Healthcare). All test samples were assayed in duplicate, and the SD in each test was always ,5% of the mean value.  Therapeutic Vaccination in Chronic Hepatitis B: Preclinical Studies in the Woodchuck Recommended treatment of chronic hepatitis B with interferon-α and/or nucleos(t)ide analogues does not lead to a satisfactory result. Induction of HBV-specific T cells by therapeutic vaccination or immunotherapies may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines in patients did not result in effective control of HBV infection, suggesting that new formulations of therapeutic vaccines are needed. The woodchuck (Marmota monax) is a useful preclinical model for developing the new therapeutic approaches in chronic hepadnaviral infections. Several innovative approaches combining antiviral treatments with nucleos(t)ide analogues, DNA vaccines, and protein vaccines were tested in the woodchuck model. In this paper we summarize the available data concerning therapeutic immunization and gene therapy using recombinant viral vectors approaches in woodchucks, which show encouraging results. In addition, we present potential innovations in immunomodulatory strategies to be evaluated in this animal model. World Health Organization estimates that about 2 billion people worldwide have been infected with hepatitis B virus (HBV). Since the introduction of preventive vaccination programs against hepatitis B in over 170 countries, the number of new infections is continuously decreasing. Despite the success of prophylactic vaccines, chronic HBV infection is still a global health problem. Over 360 million people are persistently infected with HBV, of whom 1 million die each year from HBV-associated liver cirrhosis or hepatocellular carcinoma (HCC). The outcome of HBV infection varies greatly from person to person. In most of the cases the infection is cleared spontaneously, however, 5%-10% of adults develop chronic infection. By contrast, 40%-90% of children which are born to HBV-infected mothers will progress to develop a persistent liver disease [1] . In the recent, years a marked progress has been made in the treatment of chronic hepatitis B. Currently, the two types of antiviral therapies are approved: treatment with pegylated interferon alpha 2a (PEG-IFNα) or nucleos(t)ide analogues, such as adefovir, entecavir (ETV), lamivudine, telbivudine, and tenofovir [2] [3] [4] [5] . However, the efficacy of those therapies in preventing liver cirrhosis and HCC is still limited. Treatment with PEG-IFNα leads to a sustained antiviral response in only one third of patients, regardless of combining the therapy with polymerase inhibitors. On the other hand, the treatment with nucleos(t)ide analogues significantly suppresses HBV replication that leads to a decrease of necroinflammation in the liver. However, those antivirals cannot completely eradicate the virus. After withdrawal of the drug, the rebound of viremia is observed in the majority of patients. Furthermore, the long-term treatment is subsequently associated with the appearance of drugresistant HBV strains that is often the cause of the therapy failure [6, 7] . Therefore, the new approaches in treating chronic hepatitis B are urgently needed.
It is well documented that an appropriate adaptive immune response is required to efficiently control the HBV infection. T cell-mediated immune response directed against 2 Hepatitis Research and Treatment hepatitis B virus antigens is crucial for resolution of the infection [8] [9] [10] [11] [12] . HBV-specific CD8 + T cells are able to clear HBV-infected hepatocytes by secretion of Th1 antiviral cytokines, such as interferons (IFNs) and tumor necrosis factor alpha (TNFα), and direct cytotoxic mechanisms (perforin/granzyme, ligand-ligand induced cell death, e.g., Fas-Fas-L) [12] [13] [14] [15] [16] . An early, vigorous, polyclonal, and multispecific cellular immune response against the viral proteins is associated with the clearance of hepatitis B in acutely-infected patients. In contrast, chronic HBV carriers demonstrate weak, transient, or often undetectable CD8 + T cell response that correlates with HBV persistence [17] [18] [19] [20] [21] . Humoral immune response, especially neutralizing antienvelope antibodies, play a key role in preventing HBV spread to noninfected hepatocytes [20, 22] .
Recent studies indicate that several mechanisms may be involved in the loss of the function of HBV-specific T cells during chronic hepatitis B. It was shown that high-level viremia negatively influences the virus-specific immune responses. High viral replication in the liver with viral load higher than 10 7 copies/mL is correlating with hyporesponsiveness of virus-specific CD8 + T cells in patients with chronic hepatitis B [23] . Moreover, the prolonged exposure to viral antigens occurring during the chronic viral infections can trigger the T cells to become tolerant and prone to apoptosis. The interaction between programmed death 1 (PD-1) receptor and its ligand PD-L1 (also known as B7-H1) plays an important role to prevent an overreaction of the immune system [24] . Recent studies revealed that inhibitory molecules such as PD-1 and CTLA-4 are markedly upregulated on virus-specific T cells, resulting in exhaustion (e.g., lack of IFNγ production and proliferation) [25] . Simultaneously, this mechanism can contribute to the development of the chronic infection by impairment of the effective antiviral response. This hypothesis was previously proven for hepatitis C virus (HCV) [26, 27] and human immunodeficiency virus (HIV) infection in humans [28] [29] [30] , as well as lymphocytic choriomeningitis virus (LCMV) infection in mice [31, 32] , and more recently for HBV [33, 34] . Furthermore, several studies imply that functional defects of antigen presenting cells (APCs), mainly dendritic cells (DCs), may contribute to the impaired T cell response in chronic hepatitis B patients [35] [36] [37] [38] [39] [40] [41] . In vitro studies showed that DCs isolated from HBV chronic carriers produce lower amount of antiviral cytokines, such as type I interferons and TNFα, in comparison to healthy controls [35, 36] . In addition, those DCs are less efficient in T cell activation and stimulation of T cell proliferation [35, [39] [40] [41] . The novel report demonstrated that myeloid DCs from chronic HBV patients express increased level of inhibitory PD-L1 molecule and therefore may down regulate functions of HBV-specific T cells [39] . Several investigations underline the significance of CD4 + CD25 + regulatory T cells in pathogenesis of persistent viral infections [42] . In HCVand HIV-infected patients, it was shown that regulatory T cells may downregulate HCV-and HIV-specific CD8 + and therefore influence the disease progression [43] [44] [45] . The role of regulatory T cells in HBV infection is still not clear. Nevertheless, the increased numbers of CD4 + CD25 + regulatory T cells were detected in the blood and the liver of patients with chronic severe hepatitis B [46] . In addition, the liver itself is an organ with tolerogenic properties that might contribute to the immunological tolerance during chronic HBV infection [47, 48] . Finally, viruses developed the strategies to efficiently evade the host immune response resulting in persistent infections. Viral immune escape due to the mutation of CD4 + , CD8 + , and B cell epitopes in a given HLA background have been observed in patients infected with HIV, HCV, and HBV [49] [50] [51] [52] [53] [54] .
Several studies demonstrate that the treatment with lamivudine alone, or in combination with interleukin-12 (IL-12), result in the restoration of the HBV-specific CD4 + and CD8 + immune response in chronic HBV-infected individuals. However, the therapeutic effect was not sustained in those patients [55] [56] [57] .
Over 20 years, continuous efforts have been undertaken to develop a therapeutic vaccine for chronic hepatitis B to enhance the virus-specific immune responses and overcome persistent HBV infection [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] .
Numerous clinical trials of therapeutic immunization exploited the conventional prophylactic hepatitis B surface antigen-(HBsAg-) based protein vaccines. These studies demonstrated reductions in viremia, HBeAg/anti-HBe seroconversion, and HBV-specific T cell responses in some patients. However, the anti-viral effect was only transient and did not lead to an effective control of the HBV [58] [59] [60] [61] [62] [63] [64] [65] . Combination of the HBsAg protein vaccines with antiviral treatment with lamivudine did not lead to a satisfactory improvement of the therapies [66] [67] [68] .
The strategies designed to specifically stimulate HBVspecific T cell responses were also not successful [69] [70] [71] . The lipopeptide-based vaccine containing a single cytotoxic T lymphocyte (CTL) epitope derived from HBV nucleocapsid was able to induce a vigorous primary HBV-specific T cell response in naïve subjects [76] . However, in HBV chronic carriers, the vaccine initiated only poor CTL activity and had no effect on viremia or HBeAg/anti-HBe seroconversion [69] . The DNA vaccine expressing small and middle envelope proteins proved to elicit the HBV-specific cellular immune response in chronic HBV carriers, however, this effect was only transient [70] .
Yang et al. presented the novel DNA vaccine for treatment of chronic hepatitis and combined the immunizations with lamivudine treatment [71] . The multigene vaccine contains five different plasmids encoding most of HBV antigens and human IL-12 gene as a genetic adjuvant. The combination therapy led to sustained antiviral response in 6 out of 12 HBV chronically infected patients. The responders were able to clear HBeAg and had undetectable viral load at the end of a 52-week follow-up. Those effects were correlating with a detectable T cell response to at least one of the HBV antigens. [71] . Nevertheless, further studies are needed to evaluate this strategy on a larger cohort of HBV chronic carriers.
The therapeutic vaccine-based HBsAg complexed with human anti-HBs was proposed by the group of Wen et al.
3 [77] . Immunogenic complexes (ICs) stimulate robust T cell responses by increasing uptake of HBsAg through Fc receptors on APCs and, therefore, modulate HBsAg processing and presentation. It was demonstrated that this vaccine administered to HBeAg-positive patients led to decrease of HBV DNA in serum, HBeAg seroconversion, and development of anti-HBs in part of the subjects [78] . Currently, the IC-based vaccine is the only one that entered phase III of clinical trials in chronic hepatitis B patients [79] . Even though the IC-based vaccine led to antiviral effect, clearance of HBV was not observed in treated patients. It seems that the vaccine alone is not sufficient to achieve the full control over HBV. Therefore, some steps have been undertaken to combine the IC-based vaccine with nucleos(t)ide analogues treatment, (Wen et al., personal communication) . The ongoing clinical trial will show whether IC are effective as a therapeutic vaccine in chronic hepatitis B.
Over the years, various animal models, including chimpanzees, woodchucks, ducks, and HBV transgenic mice, were established for development and evaluation of novel therapeutic strategies. Considering the cost, ethical reasons, and available amount, HBV transgenic mice are the most widely used models. Studies using HBV transgenic mouse models demonstrated that DNA immunization with the expression plasmids encoding different HBV proteins could induce HBV-specific antibodies and stimulate CTL responses. However, the functionality of HBV-specific CTLs induced in transgenic mice may be not fully developed [80] [81] [82] . Improvement of DNA vaccination regimen [83] and blockade of PD-1/PD-L1 interaction [34, 84] could enhance functional T cell responses and lead to inhibition of viral replication in vivo without causing hepatitis. Apart from the DNA immunizations, the other therapeutic approaches including administration of Toll-like receptor (TLR) ligands, HBV-specific siRNA, and direct activation of APCs were evaluated in HBV transgenic mice [85] [86] [87] . Those strategies were able to effectively reduce the HBV replication, and are currently under investigation as combined therapies. Nevertheless, this model has a significant limitation. As the HBV genome is inserted into the mouse chromosome, full HBV life cycle does not take place in the transgenic mice and no liver inflammation can be observed [88] . Thus, the animal models with naturally occurring hepadnaviral infection are required for the longterm evaluation of the therapeutic effect. In comparison to chimpanzees, woodchucks are easily available and affordable.
In this paper we would like to introduce woodchucks as a useful preclinical model for designing of the new therapeutic vaccines in chronic hepadnaviral infections. We will summarize the available data concerning therapeutic immunization approaches in woodchucks and present potential innovations in immunomodulatory strategies that yet to be evaluated on this animal model.
The Eastern woodchuck (Marmota monax) is naturally infected by woodchuck hepatitis virus (WHV). WHV was discovered in 1978 as a virus closely related to HBV [89] and classified as a member of Hepadnaviridae family. WHV and HBV show a marked similarity in the virion structure, genomic organization, and the mechanism of replication, but differ in several aspects, for example, regulation of transcription (Table 1 ) [90] . WHV causes acute self-limiting and chronic infection similar to HBV infection in the pathogenesis and profiles of the virus-specific immune response [91] . This feature of the woodchuck model makes it so significant for investigation of the new therapeutic approaches in chronic hepatitis B. Experimental infection of neonates or adult woodchucks with WHV reflects the outcome of HBV infection in humans. In adult woodchucks infection with WHV usually leads to the resolution of infection and only 5%-10% of animals will develop the chronic hepatitis. The exposure of woodchuck, neonates to WHV results in development of chronic WHV infection in 60%-75% of the cases [92] . The continuous replication of WHV in the liver during the chronic infection is nearly always associated with development of HCC in the woodchucks [93, 94] . After diagnosis of HCC the survival prognosis of the animals is estimated on about 6 months, like in humans. The common features of HBV-and WHVinduced carcinogenesis give the opportunity to examine the new anti-HCC therapies in the woodchucks [95] .
For many years, the studies on immunopathogenesis of WHV infection in woodchucks were restricted to determination of humoral immune responses [96] . The lack of appropriate methods to evaluate antigen-specific T cell responses was the serious limitation of this model.
Proliferation assay for peripheral blood mononuclear cells (PBMCs) based on incorporation of [ 3 H]-thymidine by cellular DNA, routinely used for human and mouse system, has been ineffective in the woodchuck PBMCs [97, 98] . The failure of this approach is consistent with the fact that woodchuck lymphocytes do not express the thymidine kinase gene (Menne et al., unpublished results). This obstacle had been overcome by usage of the alternative radioactively labeled nucleotide 2[ 3 H]-adenine [72] . Development of 2[ 3 H]-adenine-based proliferation assay enabled to detect the T-helper lymphocyte responses after stimulation of woodchuck PBMCs with WHV core, surface and X antigens (WHcAg, WHsAg, and WHxAg, resp.) [72, 99] . In addition, using the 2[ 3 H]-adenine-based proliferation assay in PBMCs from acutely infected animals, several T-helper epitopes within WHcAg [72] and WHsAg were identified [Menne et al., unpublished results] .
Recently established, a novel CD107a degranulation assay for woodchuck PBMCs and splenocytes made a significant breakthrough in studying pathogenesis of hapadnaviral infections in the woodchuck model [73] . Several studies demonstrated that detection of CD107a, as a degranulation marker, is a suitable method for determination of antigen 4 Hepatitis Research and Treatment 
Surface glycoproteins (large-L, medium-M, small-S), core protein, "x" protein, "e" antigen, DNA polymerase with reverse-transcriptase activity [22, 105] The corresponding proteins [91] Replication strategy
Replication of HBV DNA occurs by reverse transcription of an RNA intermediate within cytoplasmic nucleocapsids [22] The same mechanism [97] Genetic diversity 8 major genotypes [105] 1 major genotype (minor sequence differences) [91] Integration into host chromosome Yes [22] Yes, often close to N-myc oncogene region [106] Clinical course of infection were characterized ( Figure 1) . In contrast to self-limiting infection, WHV chronic carriers demonstrate weak or no virus-specific T cell responses against the identified epitopes [72, 73, 99] . The establishment of the assays for monitoring of cellular immune response in woodchucks is of great importance for a reliable evaluation of therapeutic and immunomodulatory strategies for treatment of chronic hepatitis B in the woodchuck model [96, 102, 103 ].
Recently described advancements in the characterization and monitoring of the woodchuck immune system during the WHV infection, made this animal model particularly useful for development of the immunomodulatory approaches in chronic hepatitis B. The natural occurrence of chronic WHV infection in woodchucks, that is closely related to HBV infection in humans, allows to evaluate the potentially new therapeutic strategies directly in chronic WHV carriers. Up to date, several studies of diverse therapeutic vaccinations have been carried out in woodchucks ( Table 2) . The pioneer investigations based on therapeutic vaccines based on WHV core [96] or surface antigens in combination with a helper peptide FIS [120] , or with potent Th1 adjuvants like monophosphoryl lipid A [121] did not lead to satisfactory results. Those experiments proved that vaccinations could induce specific B-and/or T cell responses in chronic WHV carriers. However, this alone was not sufficient to achieve the control of virus replication. It is assumed that high level viremia, during the chronic hepatitis B, can inhibit the therapeutic effect of the vaccination. Treatment of chronic HBV patients with lamivudine could transiently restore HBV-specific T cell immune response [55, 56] . Therefore, reduction of viral load by the nucleos(t)ide analogues pretreatment might support the efficacy of immunization to enhance the virus-specific immune responses. This hypothesis was tested in three experimental trials of the combination therapies in chronic WHV carriers.
The first study performed by Hervás-Stubbs et al. was based on lamivudine therapy [108] . Five chronically WHVinfected woodchucks were treated orally with the drug for 23 weeks. At week 10, after decline of WHV DNA by 3-5 logs, three animals were vaccinated with 3 doses of serum-purified WHsAg combined with T-helper FIS peptide derived from sperm whale myoglobin. The vaccination induced T-helper responses against WHV antigens, shifting the cytokine profile from Th2 to Th0/Th1. However, no beneficial effect on WHV viral load and WHsAg levels was observed in comparison to nonimmunized animals. After withdrawal of the lamivudine treatment the values of viremia returned to the pre-treatment levels.
The second trial evaluated the therapy with a very potent antiviral drug: clevudine (previously called L-FMAU) combined with a WHsAg-based immunization [74, 109, 110] . A large cohort of thirty 1-2-year-old chronically WHVinfected woodchucks was enrolled in the study. Half of the animals were orally treated with clevudine (10 mg/kg/day) for 32 weeks; the other 15 woodchucks received placebo. After withdrawal of clevudine treatment, 8 animals from each group were vaccinated with the four doses of formalin inactivated alum-adsorbed WHsAg and 7 were injected with the saline as a control. Combination of the drug and vaccine therapy resulted in marked reductions WHV DNA (6-8 logs) and WHsAg in serum during the 60-week monitoring period, in contrast to the vaccine only and placebo groups, where both markers remained at high levels. Combination therapy did not enhanced anti-WHs responses beyond those measured for vaccine alone. However, treatment with clevudine and vaccine together led to more sustained and robust lymphoproliferative responses to WHsAg and additionally to WHcAg, WHeAg, and WHxAg. Moreover, combination therapy delayed the onset of the liver disease and prevented HCC development in up to 38% of treated chronic WHV carriers in the long-term follow-up study [111] .
Recently, a novel therapeutic approach for treatment of chronic hepatitis B in a woodchuck model was described. The therapy combined the antiviral treatment with immunization with plasmid DNA and antigen-antibody immunogenic complex vaccines together [112] . DNA vaccines are considered to stimulate both humoral and cellular immune response, polarizing T cells in the direction of Th1 response [122] . Immunization of the naïve woodchucks with the plasmids encoding WHV core and preS2/S genes (pWHcIm and pWHsIm, resp.) induced the lymphoproliferative responses against the antigens and provided a protection against WHV challenge [123] . In addition, the DNA vaccine expressing HBsAg proved to elicit the vigorous T cell responses in chronic HBV carriers, however, this effect was only transient [70] . The HBsAg/anti-HBs IC vaccine is currently under the investigation in chronic HBV patients [77] [78] [79] .
To evaluate the efficacy of previously mentioned immunotherapy in woodchucks, firstly 10 chronic WHV carriers were treated with 15 mg of lamivudine, daily for 21 weeks. At week 10, four animals were pretreated with cardiotoxin and then received three immunizations with DNA vaccine containing three plasmids expressing WHsAg, WHcAg, and woodchuck IFNγ (pWHsIm, pWHcIm and pWIFN, resp.). Simultaneously, the other four woodchucks received three doses of the combination of DNA vaccine and WHsAg/anti-WHs immunogenic complex. Two chronic WHV carriers served as lamivudine monotherapy control. Lamivudine treatment resulted in only a slight decrease of WHV DNA levels in the woodchucks serum (0,7 and 0,32 log, resp.). Surprisingly, the DNA vaccination did not lead to any additional therapeutic effect beyond that observed for lamivudine treatment alone. In contrast, the triple combination of antiviral treatment, plasmid DNA encoding WHcAg, WHsAg, and wIFNγ and IC vaccines was able to decrease WHV viral load up to 2,9 log and the serum WHsAg up to 92%. Moreover, three of the four treated animals developed anti-WHs antibodies. Nevertheless, these effects were not sustained and all parameters reached the baseline levels shortly after withdrawal of lamivudine treatment. In addition, the vaccination did not induce WHVspecific T cell responses in the majority of woodchucks, even in animals that exhibited virological responses. Significant Hepatitis Research and Treatment 7 lymphoproliferative responses against WHV antigens were detected only in one animal after three immunizations with DNA vaccine [112] . The study demonstrated the benefit of using the combinatory therapy in chronically WHVinfected woodchucks. However, the transient therapeutic effects, suggest that this strategy needs further optimization.
The results from the previous studies clearly confirm the poor efficacy of the lamivudine therapy in woodchucks [108, 112, 124] . A new strategy evaluated the potency of an entecavir treatment and increased number of immunizations [Lu et al., unpublished results] . Chronically WHV-infected woodchucks were pretreated with the entecavir for 21 weeks; 10 weeks in a daily and 11 weeks in a weekly manner. During the weekly administration of the drug, one group of animals received 6 immunizations with two-plasmid DNA vaccine (pWHsIm and pWHcIm),the second group received combination of DNA vaccine together with purified WHV core and surface antigens, and the third group remained untreated. The entecavir therapy resulted in rapid and significant decrease of the viral load and WHsAg levels in serum of the animals. The effect was especially pronounced in animals that additionally received vaccines. In woodchucks treated only with entecavir, the increase of viremia was observed already during the weekly administration or immediately after withdrawal of the drug. By contrast, in both groups of animals, that were immunized with DNA or DNA/proteins vaccines, the delay before the rebound of WHV replication was significantly prolonged. In addition, entecavir treatment was effective to suppress WHV replication and enhanced the induction of WHV-specific T cell responses. An increased CTL activity was detected in individual woodchucks after DNA or DNA/proteins vaccinations. Moreover, two animals completely eliminated the virus from the blood and were WHV DNA negative in the liver [Lu et al., unpublished results] .
Altogether, the results obtained in the woodchuck model concerning combination of nucleot(s)ide therapy and immunization proved the synergistic effect of both therapeutical approaches. The therapeutic effects observed during such therapies were significantly increased and prolonged in comparison to the monotherapy alone. In addition, those therapeutic approaches could stimulate the WHV-specific T cell responses, usually impaired in WHV chronic carriers [72, 73] . A combination of antiviral treatment and vaccination is required for the improvement of virus specific T cell responses. Designing of the future therapeutic approaches should include pretreatment with the potent antiviral drugs, such as entecavir or clevudine, that proved their efficacy in the woodchuck model.
Previous results from therapeutic immunization trials on woodchucks, chimpanzees, and humans indicate that the licensed vaccines are not able to boost a functional antiviral T cell response. There is a need to use more potent strategies.
Vaccines based on recombinant viruses have gained a great interest because of their ability to stimulate robust humoral and cellular immune responses. Viral vectors were investigated as prophylactic and therapeutic vaccines against many human pathogens such as measles virus, herpes simplex virus (HSV), human papillomavirus (HPV), HIV, and rabies [126] [127] [128] [129] [130] . However, the utility of those recombinant vaccines in the treatment of chronic hepatitis B was not yet evaluated. Preliminary results obtained from the study in chronically HBV-infected chimpanzees immunized with retroviral vector, based on Moloney murine leukemia virus, encoding HBcAg suggest that further investigation of viral-vector based vaccines should be taken into consideration [131] . In the experiment, one of the three therapeutically immunized chronic carrier chimpanzees cleared the virus and showed HBeAg seroconversion. Significant ALT elevations observed in this animal implicate restoration of HBV-specific cytotoxic and humoral responses without causing fulminant hepatitis. Moreover, the other two chimpanzees demonstrated high anti-HBe titers after the therapy and one of them HBcAgspecific CTLs [131] . This study demonstrates not only the benefit of using the recombinant viral-vectors for treatment of chronic HBV infection in primate model, but also the possible advantage of using core antigen-based therapeutic vaccines. Even though the retroviral vector vaccination was well tolerated in the chimpanzees, several clinical trials suggest that gene therapy with traditional retroviral vectors can lead to oncogenesis [132, 133] . Therefore, the usage of another recombinant virus as a carrier of the proteins could be beneficial.
Recombinant adenoviruses have been one of the intensively investigated viral vectors for therapeutic purposes. Development of the novel methods for manipulating of the viral genome resulted in the three generations of the recombinant adenoviruses and with increasing capacity [125] (Figure 2 ). Several trials imply the usefulness of those vectors in gene therapy of genetic diseases and cancer [134] [135] [136] [137] . For many years, the first generation replication-deficient E1 or E1/E3-deleted adenoviral vectors have been explored as the vaccine carriers in prevention of the infectious diseases [138] . Adenoviral vectors have several advantages that can be beneficial for potent therapeutic vaccines.
First of all, adenoviruses are relatively susceptible for genetic modifications and can be easily produced in high titers. After transduction of the cells, adenoviral genome is not integrated into the host DNA and stays in the episomal form. As a result, the risk of the possible activation of the cellular oncogenes is minimal. Adenovirusbased vaccines proved to elicit a vigorous and sustained humoral and T cell responses to the incorporated antigen that is considered to be crucial in clearance of persistent viral diseases [127, [139] [140] [141] . The benefit of adenoviral vectors as a vaccine carrier is not only limited to stable delivery of proteins of interest. Several findings on additional immunostimulatory effects, for example, induction of the innate immune response, that originate from the nature of adenoviruses itself, may enhance the vaccine efficacy. Capsid of adenoviruses demonstrates immunostimulatory properties, that is why the coadministration of the adjuvant is usually unnecessary. Those vectors can directly transduce DCs causing their maturation and upregulation of MHC and costimulatory molecules on their surface, thus lead to enhanced antigen presentation. Moreover, it was shown that AdV-transduced DCs are secreting antiviral cytokines, such as IFNα, TNFα, and IL-6 [142] . Interleukin-6 is one of the most important factors that suppress the function of the regulatory T cells [143, 144] . Nevertheless, modified adenoviruses apart from the abovementioned advantages have one serious limitation. Thus far, vectors that were comprehensively examined as the vaccines have been based on the human adenovirus serotype 5 (Ad5) [127] . This serotype is the most common in the human population. Anti-Ad5 neutralizing antibodies are detectable in 45%-90% of adults [145] . The preexisting immunity directed against Ad5 is considered as a main reason of failure in the phase I clinical trial of a protective HIV-1 vaccine. STEP study guided by Merck pharmaceutical concern, based on 3-dose regimen of a trivalent Ad5 vaccine, suggested that the immunization might increase the risk of HIV-1 infection in the subjects with high neutralizing anti-Ad5 titers [146] [147] [148] . Moreover, even single immunization may induce immunity to the vector in seronegative individuals.
The negative effect of the pre-existing or Ad5-induced immunity against the vaccine, mostly when the therapy requires multiple dosages, may be overcome by heterologous prime-boost regimen. The utility of the rare human serotypes (e.g., serotype 35) [149, 150] or recombinant adenoviruses of nonhuman origin has been recently tested [151] . In particular, subsequent priming immunizations with plasmid DNA vaccine followed by a booster vaccination with AdV seem to be a very promising strategy. DNA primeadenovirus boost regimen proved to induce more robust and potent immune response in comparison to plasmid DNA alone and provided protection against the pathogen challenge in several animal models of infectious diseases [149, [152] [153] [154] . Furthermore, a clinical trial of multiclade HIV-1 DNA plasmid-Ad5 boost vaccine, HIVuninfected individuals demonstrated high immunogenicity even in the presence of high anti-Ad5 antibody titer. In addition, the vaccine proved to be well tolerated in the participants of the study [155] .
Several studies indicate that the transgene expression level can be increased from adenoviral vectors by the presence or insertion of an intron sequences [156] [157] [158] . Therefore, we constructed the new recombinant adenoviruses serotype 5 and 35 encoding WHV core protein and containing an intron between promoter and WHcAg gene sequences. Preliminary experiments showed that vaccination with the AdVs containing the intron sequences led to induction of robust cellular and humoral immune responses in mice. Moreover, immunization of the mice in DNA prime-AdV boost manner, using improved vectors, resulted in more vigorous and multispecific T cell responses in comparison to immunization with plasmid DNA alone [Kosinska et al., unpublished results].
Immunization of chronically WHV-infected woodchucks with plasmid DNA vaccine in combination with entecavir treatment showed a marked therapeutic effect. Addition of the recombinant adenoviruses to this regimen could be a new, more potent approach in treatment of chronic hepatitis B. We will apply DNA prime-AdV boost approach in WHV chronically infected woodchucks in combination with nucleos(t)ide analogs and evaluate its therapeutic potential.
Over the last 20 years, modified adenoviruses have been extensively studied as a vehicle for gene delivery to the liver, because of their high transfection efficiency and their natural tropism for hepatocytes [159, 160] . Moreover, the development of the third generation of adenoviral vectors that lack all viral coding sequences (e.g., helper-dependent adenoviral vectors), resulted in their increased capacity and minimized immunogenicity of the vector allowing longterm transgene expression [161] . High cloning capacity of those vectors enables usage of inducible or tissue-specific promoters and coexpression of multiple therapeutic or immunomodulatory genes [162] .
So far, several trials of virus-mediated gene therapy for treatment of chronic hepatitis and HCC were performed in chronically WHV-infected woodchucks and in cell culture systems. Those strategies were mainly based on delivery of antiviral cytokines, such as IFNα, IFNγ, IL-12 by recombinant adenoviruses, to reduce viral replication or modulate the immune response ( Table 3) .
Transduction of primary woodchuck hepatocytes from chronic WHV carriers with helper-dependent AdV encoding woodchuck IFNα (wIFNα) resulted in the reduction of WHV proteins expression in vitro [169] . In vivo studies on chronically WHV-infected woodchucks, demonstrated that a single injection of 1 × 10 12 vp of this vector into the liver's portal vein could inhibit WHV replication by 1 log up to 11 weeks after the treatment [163] . The same approach with helper-dependent AdV expressing woodchuck IFNγ (wIFNγ) did not show any antiviral effect, even though the transduction led to the production of biologically active interferon [163] . Another study combined intravenous delivery wIFNγ by recombinant adenoviral vector with nucleos(t)ide analogues therapy. Chronic WHV carriers were treated with clevudine and emtricitabine (FTC), together, for 8 weeks and after the initial drop in viral load one group of animals received additionally two i.v. injections of 3 × 10 10 PFU of Ad-IFNγ. Delivery of wIFNγ induced inflammation, caused by T cell infiltration, and increased hepatocyte turnover. However, this effect did not induce additional antiviral outcome in comparison clevudine/emtricitabine biotherapy alone [164] . Similarly, poor therapeutic effect was observed for gene therapy based on both wIFNγ and wTNFα. Intravenous injection of those recombinant adenoviruses during clevudine treatment led to decrease of replicative intermediates of WHV DNA in the liver, beyond what could be achieved by clevudine alone. Nevertheless, 6 weeks after injection there was no significant difference between the groups of WHV carriers receiving AdV expressing the cytokines or beta-galactosidase as a control [165] . The benefits of using the immunomodulatory genes in this study are difficult to assess, since it was reported that adenovirus infection alone is sufficient to transiently suppress the WHV replication in chronically infected woodchucks [170] . The lack of therapeutic effect by direct delivery of IFNγ is consistent with in vitro data obtained from persistentlyinfected woodchuck primary hepatocytes. Treatment of the cells with wIFNγ, even in the presence of wTNFα, was not able to inhibit the WHV replication. Moreover, high concentration of those cytokines resulted in the loss of the cells during the culture [171] . This observation underlines the cytotoxic effect of Th1 cytokines on the woodchuck hepatocytes. Rapid downregulation of the IFNγ expression, after transduction of the liver cells with viral vector, could be one of the mechanisms to protect the organism from the potential toxicity of this cytokine in vivo [163] . In addition, several reports indicates that the level of wIFNγ and wTNFα is higher in the liver of chronic WHV carriers in comparison to naïve animals [172, 173] . Therefore, continuous presence of inflammatory cytokines in the liver during the chronic WHV infection could result in hyporesponsiveness of hepatocytes to such a therapy.
The novel strategy to treat chronic WHV hepatitis is based on adenovirus-mediated delivery of murine IL-12 (mIL-12) gene into hepatocytes [166] . Interleukin-12 is a proinflammatory cytokine produced naturally by antigen presenting cells. IL-12 stimulates production of IFNγ and TNFα by T and natural killer (NK) cells and enhances their cytotoxic activity [174] . In the study, mIL-12 gene expression could be regulated by inducible promoter that was responding to progesterone antagonist RU486. Eight chronic WHV carriers received single dose of 2 × 10 10 i.u. of AdV expressing mIL-12 (HC-Ad/RUmIL-12) by intrahepatic injection at laparotomy. Two weeks after, the expression of mIL-12 was induced by the administration of RU486. The IL-12 treatment resulted in intense and sustained suppression of WHV replication in the liver as well as decreased viral loads in the serum. This effect, however, was visible only in the animals with basal viremia lower than 10 10 WHV copies per milliliter of serum. Animals, which responded to the therapy, developed a vigorous T cell response to WHcAg, measured by woodchuck IL-2 production, and demonstrated WHeAg and WHsAg seroconversion. Moreover, the FoxP3 levels in the livers of those animals were decreased, while in nonresponder woodchucks FoxP3 values were significantly upregulated [166] . This finding suggests that the intrahepatic expression of IL-12 may inhibit the regulatory T cells in the liver during the chronic WHV infection. Indirect induction of inflammatory cytokines, such as IFNγ and TNFα by IL-12, seems to be a more efficient strategy in breaking the tolerance to virus antigens than direct delivery of those cytokines. It suggests that probably additional events occur in the liver after AdV-mediated IL-12 transfer that supports the antiviral effects of this therapy.
Adenoviral delivery of genes for cytokines and other immunomodulators is widely used in cancer therapy in the animal tumor models as well as in patients [137, [175] [176] [177] [178] . The T cells play an important role not only in defense against the pathogens, but also in antitumor immunity and inhibition of the tumor growth. Interleukin-12 inhibits the angiogenesis and induces a potent antitumoral immune response by stimulation of IFNγ secretion. Therefore, IL-12 is a promising candidate for cancer gene therapy [179] [180] [181] [182] [183] . Strategy based on recombinant adenoviruses expressing IL-12 demonstrated antitumor effect in the murine models with transplantable HCC [184, 185] and was also evaluated in woodchucks [168] .
In the study, large (2-5 cm) intrahepatic tumors of 5 woodchucks were injected with a single dose of 1 × 10 9 PFU AdV expressing IL-12 and B7.1 molecule (AdIL-12/B7.1). The B7.1 molecule (also known as a CD80) is naturally expressed on the professional APCs and provides the synergistic effect in the tumor regression [181, 186, 187] . In 4 out of 5 animals, AdIL-12/B7.1 was delivered by laparotomy into the three HCC nodules and three nodules were injected with a vector expressing GFP as a control. Animals were sacrificed 7-14 days later and the tumor volumes were assessed. On average, treated tumors showed an 80% reduction in the volume whereas the size of the AdGFP-injected nodules increased. Remission of the tumors was associated with CD4 + and CD8 + T cell infiltration into the tumor tissue and increased local IFNγ levels after AdIL-12/B7.1 injection. One of the treated woodchucks received the intratumoral injection by magnetic resonance imaging (MRI) guidance and was monitored for 7 weeks. During this period the tumor size decreased from 8,6 cm 3 to 0,5 cm 3 [168] . This observation shows that administration of AdIL-12/B7.1 during MRI guidance, with therapeutic effect similar to laparotomy, could prevent the animals from harmful consequences of the surgery.
The study proved that the gene therapy based on IL-12 leads may be a promising strategy to treat HCC. By contrast, treatment with AdV encoding herpes simplex thymidine kinase combined with gancclovir administration did not lead to reduction in the tumor size [167] . Nevertheless, the short time of monitoring during the study makes it difficult to evaluate the prolonged antitumoral effect of this approach.
A recent study presents gene therapy with semliki forest viral vector expressing high levels of murine IL-12 (SFV-enhIL-12) on remission of HCC in chronically WHV-infected woodchucks. In the research, the vector was delivered by surgery into multiple sites of HCC tumors in the liver [75] . A total of nine woodchucks were enrolled in the experiment. Six of the woodchucks, two animals each, received different doses of SFV-enhIL-12: 3 × 10 9 vp, 6 × 10 9 vp, and 1, 2 × 10 10 vp, and three animals served as a control and received saline injections. The tumor size was monitored by ultrasound examination for 23 to 24 weeks. In all woodchucks, reduction in tumor volume was observed, however, this effect was transient and dose dependent. Animals treated with the highest dose of SFV-enhIL-12 showed the most spectacular reduction of the tumor size 71% and 80%. Nevertheless, the tumors started to grow between 6 and 14 weeks after the treatment. The antitumoral effect was associated with the induction of the immune response towards the tumor antigens, demonstrated by T cell proliferation assay, upregulation of leukocyte markers expression, and cytokine production, such as IFNγ, TNFα, IL-6, and IL-12. In addition, the therapy resulted in transient induction of lymphoproliferative responses against WHcAg and WHsAg and led to short-term reduction in WHV viral load [75] .
The results presented here indicate that viral-mediated gene therapy in treatment of chronic hepatitis B and HCC needs further optimization. However, treatment of the woodchucks with viral vectors allowed to achieve a long-lasting expression of the cytokines and their higher concentration preferably in the liver. Therefore, this strategy is proven to be more effective than an approach based on using of the soluble cytokines. In addition, adenovirusmediated gene transfer is proven to be a safe and a welltolerated strategy in the woodchucks.
The current progress indicates the feasibility of therapeutic approaches for treatment of chronic HBV infection. There is a general agreement that a combination of antiviral treatment and immunomodulation is essential to achieve a sustained control of HBV infection. However, many scientific questions are still not answered. The question how HBV infection leads to defective immune responses to HBV proteins remains to be investigated. This issue is the key to a more rational design of new therapeutic approaches. Recently, HBV proteins were found to suppress host innate responses [188] . It has to be clarified whether an early blockage of innate immune responses may further negatively influence the priming of adaptive immune responses. In addition, different groups reported consistently that TLR2 and TLR4 signalling may be impaired in chronic HBV infection patients [189, 190] . Thus, it is worthy to test whether an enhancement of innate immune responses in chronic carriers is necessary for restoration of specific immune responses. With the increasing number of available vaccine formulation, a more crucial question raised recently: what is the optimal combination of these vaccines. Obviously, it is necessary to test the mutual influences of different types of vaccines to maximize their effects and avoid the negative interference between the vaccines. Finally, the future design of therapeutic vaccines needs to be considered in nonnaïve hosts since patients have undergone other infections. It is yet not possible to foresee how the pre-existing infections and immunological backgrounds will influence the effect of therapeutic vaccines. Understanding these issues will be helpful for the translation of recent progresses for clinical use of therapeutic vaccines. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8(+)T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii BACKGROUND: Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8(+ )T cells from seropositive but not seronegative persons. RESULTS: Herein, when these peptides were administered with the universal CD4(+)T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam(2)Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam(2)Cys is an effective way to elicit IFN-γ producing CD8(+ )splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1(224-232)); AMLTAFFLR (GRA6(164-172)); RSFKDLLKK (GRA7(134-142)); STFWPCLLR (SAG2C(13-21)); SSAYVFSVK((SPA250-258)); and AVVSLLRLLK(SPA(89-98)). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera. CONCLUSIONS: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8(+ )T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles. parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera. Conclusions: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8 + T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.
Toxoplasmosis is a disease of major medical importance. Toxoplasma gondii causes congenital infections responsible for stillbirths and spontaneous abortions [1] [2] [3] [4] [5] . In addition, it causes neurologic disorders, uveitis, and systemic infections in immune-compromised patients. Toxoplasmic encephalitis is a cause of morbidity and mortality in those with congenital disease and persons with AIDS [6] . T. gondii is acquired by consumption of lightly cooked meat, especially lamb and pork contaminated with bradyzoites [7, 8] or by ingestion of food or water contaminated with oocysts containing sporozoites, which are the product of a sexual cycle in the intestine of the cat [8, 9] .
Development of an effective vaccine would prevent this disease. Attenuated T. gondii tachyzoites (e.g., S48, TS-4, T-203, ΔRPS13)have been employed for live vaccinations of non-human animals [10] [11] [12] [13] [14] [15] . Parasites attenuated by knockout of gene transcription recently have been proposed as a new type of attenuated vaccine candidate [13] [14] [15] . However, safety considerations may limit vaccination of humans with live organisms. Development of peptide-based vaccines created with epitopes which elicit IFN-γ production by CD8 + T cells [16] is a promising strategy to mobilize the immune system against T. gondii in humans [16] [17] [18] [19] [20] . Thus, an effort to create an immunosense epitope-based vaccine was initiated.
Recently, three peptide epitopes, KSFKDILPK (SAG1 224-232 ), AMLTAFFLR (GRA6 164-172 ), and RSFKD LLKK (GRA7 134-142 ), were found by our group to elicit IFN-γ from peripheral blood mononuclear leukocytes (PBMCs) from T. gondii seropositive HLA-A03 supertype humans but not from PBMCs of T. gondii seronegative HLA-A03 supertype humans [18] . Herein, initially we designed 4 CD8 + T cell epitope containing vaccine formulations comprised of lipopeptides that incorporate PADRE as well as a novel oil-in-water emulsion that includes a specially formulated synthetic MLA derivative called GLA-SE [21] [22] [23] [24] . First, efficacy of various vaccine formulations consisting of the three previously identified CD8 + T cell peptides eliciting HLA-A*1101-restricted, CD8 + T cell-mediated IFN-γ production in vitro from HLA-A*1101 mice was examined.
Then, in order to identify additional peptides from T. gondii tachyzoites, bradyzoites and sporozoites of Type I and Type II strains that are effective in eliciting IFN-γ from HLA-A03 supertype restricted CD8 + T cells, the first step was to select proteins with biologic properties, such as being secreted, compatible with MHC Class I processing. Then, bioinformatic algorithms to identify HLA-A03 supertype bound peptides were utilized to find additional, novel T. gondii-derived, potential CD8 + T cell eliciting epitopes restricted by the HLA-A03 supertype. We screened peptides from tachyzoite, bradyzoite and sporozoite proteins (GRA10, GRA15, SAG2C, SAG2D, SAG2X, SAG3, SRS9, BSR4, SPA, MIC) of the type II T. gondii strain, ME49, searching for those with high binding scores in a bioinformatic analysis (IC 50 < 50 nM) and then in binding assays. In addition, peripheral blood mononuclear cells from seropositive and seronegative persons were tested for response to these peptides using an IFN-γ ELISpot assay. These latter studies were performed to attempt to identify other peptides that would be promising candidates for inclusion in a multi-epitope, next generation immunosense vaccine. Then, information obtained from testing the first three peptides studied was used to guide formulation and administration of a peptide pool. This pool included the first three peptides identified earlier and tested in the initial experiments combined with the newly identified peptides that elicited IFN-γ from human HLA-A03 supertype restricted CD8 + T cells. All the peptides in this pool were tested with a universal T helper epitope called PADRE and a new, promising adjuvant called GLA-SE with Pam 2 Cys in HLA-A*1101 transgenic mice. Capacity to induce IFN-γ production by spleen CD8 + T cells, and to protect against parasite burden following subsequent challenge were determined. For parasite challenge, a luciferase transfected Type II Prugneaud parasite was administered, followed by luciferin administration and imaging with a Xenogen camera system a week later. This allows detection and quantization of bioluminescent parasites as a biomarker to assess efficacy of immunizations in protection.
Three CD8 + T cell epitopes had been identified from T. gondii proteins, based on their significant recognition by T cells from T. gondii seropositive HLA-A03 individuals [18] . A universal CD4 + T cell epitope, PADRE (AKFVAAWTLKAAA), was linked in sequence with the N-terminal end of each of the three different T. gondii CD8 + T cell epitopes: KSFKDILPK (SAG1 224-232 ), AML-TAFFLR (GRA6 164-172 ), RSFKDLLKK (GRA7 134-142 ). Also, these three epitopes were linked together with three alanines as the linker. The N-terminal end of each resulting CD4-CD8 peptide or polypeptide was extended by a lysine covalently linked to two molecules of the palmitic acid moiety. The lipopeptides (Lp) were named as LpKS9, LpAM9, LpRS9 and LpKS9-AM9-RS9. They are shown in Figure 1 .
Immunogenicity of lipopeptides in HLA-A*1101 transgenic mice HLA-A*1101 transgenic mice were immunized twice at intervals of three weeks with lipopeptides which were administered in PBS. Two weeks after the last immunization, the spleens were removed from immunized mice and the ability of splenocytes to produce IFN-γ upon stimulation with peptides was analyzed. Transgenic mice immunized with the three single peptide lipopeptide vaccines had T cells that produced IFN-γ ( Figure 2 ). The lipopeptide vaccines LpKS9 and LpAM9 stimulated higher IFN-γ production than LpRS9 ( Figure 2 ). However, LpKS9-AM9-RS9 with three peptide epitopes linked together did not stimulate strong IFN-γ responses when splenocytes from these mice were exposed to each of the individual peptides in vitro. Only results with LpKS9(KS9) and LpAM9(AM9) achieved statistical significance ( Figure 2 ).
In order to determine which formulation was most immunogenic, vaccination with a single peptide or a mixture of the peptides was compared with linked lipopeptide vaccines. Results of a representative experiment are presented in Figure 3 . Mice immunized with a vaccine formulated with a single peptide SAG1 224-232 , KS9-PADRE with the adjuvancy of GLA-SE and Pam 2 Cys, elicited IFN-γ production (SFC:248 ± 65; mean ± SEM) four-fold higher (p < 0.01) than mice that received LpKS9 GLA-SE vaccination (63 ± 17) (Figure 3a ). Similar results, with substantial IFN-γ production, were found when splenocytes from mice immunized with a mixture of peptides and adjuvants. These results were compared with IFN-γ production by splenocytes from mice immunized with a lipopeptide vaccine constructed with the three peptides linked together with alanine spacers LpKS9-AM9-RS9. Significant IFN-γ production was only present with KS9 and AM9 stimulation of spleen cells, and was much less robust and not significant when the spleen cells were stimulated by RS9 peptide.
To determine whether, and if so how, adjuvants effected immunogenicity of these peptides, HLA-A*1101 transgenic mice were immunized with pools of peptides that included all of the three peptides (KS9, AM9, RS9) alone or with varying adjuvants. HLA-A*1101 transgenic mice were immunized with: (1) CD8 + epitope peptide pool, (2) peptide pool plus PADRE, (3) peptide pool Figure 1 Schematic representation of the synthetic lipopeptide immunogens used in this study. The C-terminal end of a promiscuous CD4 + T cell peptide epitope (PADRE) was joined in sequence with the N-terminal end of one of three different T. gondii CD8 T cell epitopes: SAG1 224-232 (A), GRA6 164-172 (B); GRA7 134-142 (C) or three epitopes linked together (D) with a three alanine linker. The N-terminal end of each resulting CD4-CD8 peptide was extended by a lysine covalently linked to one molecule of palmitic acid. This results in a four lipopeptides construct. The abbreviation Lp is used throughout the manuscript whenever the lipopeptide (Lp) has been studied. When there is a mixture of components or undivided components they are named individually. Sequence of PADRE is AKFVAAWTLKAAA. Structure of Pam 2 Cys is PAM 2 KSS. a = abbreviation for name of protein from which peptide is derived. Figure 1 . The mice were immunized twice at intervals of three weeks. Ten to fourteen days after the last immunization, spleen cells were separated from immunized mice and stimulated by appropriate peptides in an ex vivo IFN-γ ELISpot assay. Data presented are averages of three independent replicate experiments. *, P < 0.05; **, P < 0.01. (Figure 3b ) were used as immunogens. For the lipopeptide immunizations, the mice were vaccinated twice at intervals of three weeks. For the immunizations containing individual components including the same peptides, the mice were vaccinated three times at intervals of two weeks. Ten to fourteen days after the last immunization, spleen cells were separated from immunized mice and stimulated by the appropriate peptide in an ex vivo IFN-γ ELISpot assay. Data presented are a representative example from three independent experiments. *, P < 0.05; **, P < 0.01. plus PADRE emulsified with GLA-SE, and (4) peptide pool plus PADRE and Pam 2 Cys emulsified with GLA-SE. Mice were inoculated three times at intervals of two weeks. Eleven to fourteen days post immunization, spleen cells were isolated and exposed to each individual peptide. A peptide was considered immunogenic if it induced IFN-γ spot formation that was significantly higher in the immunization group compared with the group inoculated with PBS. After the immunizations, only KS9 and AM9 were found to be immunogenic in HLA-A*1101 transgenic mice, and only when the iniversal CD4 + helper T cell peptide epitope PADRE was included. Robust responses were observed when GLA-SE was added in the vaccine. Greater responses were elicited when Pam 2 Cys was used as an adjuvant for some peptides but did not enhance responses to all of them, and in fact reduced the effect of some peptides. Figure 4 shows the representative data of IFN-γ spot formation from the four immunization groups which were stimulated by individual peptides.
Vaccination with peptide pools and adjuvants protects mice against type II parasite challenge HLA-A*1101 transgenic mice were immunized with peptide pools plus PADRE and Pam 2 Cys in GLA-SE three times at intervals of two weeks. Mice were challenged 2 weeks after the last immunization. They were imaged 7 days after they had been challenged with 10,000 Pru (Fluc) using the Xenogen in vivo imaging system. As shown in Figure 5 , numbers of luciferase expressing parasites in immunized HLA-A*1101 transgenic mice were significantly less compared to numbers of parasites in unimmunized mice. Mean 8 ] for immunized mice. Differences were significant (e.g., p < 0.0064, for the pooled experiments, using natural log transformed data and two-sample t test).
We initially identified 3 epitopes that provided protection against parasite challenge. As a next generation vaccine that might be even more robust, elicit CD8 + T cells that would be effective against all three life cycle stages and Type I and II genetic types of the parasite, we sought to identify additional HLA-A11 epitopes to be used for vaccine development. In order to identify additional peptides from T. gondii that were present in tachyzoites, bradyzoites, and sporozoites of Type II strains for HLA-A03 supertype restricted CD8 + T cells, we screened candidate peptides from tachyzoite, bradyzoite and sporozoite proteins (GRA15, GRA10, SAG2C, SAG2X, SAG3, SRS9, SPA and MIC) of the Type II T. gondii strain (Tables 1 and 2 ). For parsimony in numbers of peptides utilized, we attempted to include peptides present in Type I as well as Type II parasite genetic types. Peripheral blood mononuclear cells from seropositive T. gondii donors were tested for response to these peptides by using the IFN-γ ELISpot assay. Pooled peptides were tested initially. Pools 2 and 5 were significantly different (p < 0.05) from the control (data not shown). Then the individual peptides were tested ( Figure 6 ). Three out of 34 epitopes elicited responses greater than 50 IFN-γ SFC from seropositive donors PBMC, but not from seronegative donors PBMC ( Figure 6 ). These peptides and the proteins from which they are derived are: STFWPCLLR (SAG2C 13-21 ); SSAYVFSVK (SPA 250-258 ); and AVVSLLRLLK (SPA 89-98 ) adding proteins expressed in sporozoites and bradyzoites to the peptides selected. All peptides identified herein then were found to show high binding affinity to three to five HLA-A03 supertype alleles in the MHC-peptide binding assay ( Table 3 ). The numbers in Table 3 which indicate high binding affinity are those less than 500 IC 50 nM. They are indicated by bolded font numbers in Table 3 .
Vaccination with peptide pools including newly identified peptides and adjuvants elicits IFN-g and provides more protection to mice against Type II parasite challenge Then, HLA-A*1101 transgenic mice were immunized with peptide pools which included these newly identified peptides: KSFKDILPK (SAG1 224-232 ); AMLTAFFLR (GRA6 164-172 ); RSFKDLLKK (GRA7 134-142 ); STFWPCLLR (SAG2C 13-21 ); SSAYVFSVK (SPA 250-258 ); AVVSLLRLLK (SPA 89-98 ) plus PADRE and Pam 2 Cys in GLA-SE, They were immunized three times at intervals of two weeks. Significant IFN-γ spot formation responses were observed in vitro by the cells from immunized mice exposed to all the peptides except GRA7 134-142 . Figure 7a shows the representative data of IFN-γ spot formation from the four immunization groups which were stimulated by individual peptides. The inclusion of the newly identified peptides thus had potential to enhance immunogenicity in transgenic HLA-A*1101 mice.
Mice were challenged 2 weeks after the last immunization. They were imaged five days after they had been challenged with 10,000 Pru (Fluc) using a Xenogen in vivo imaging system. As shown in the initial experiment in Figure 7b , the numbers of luciferase expressing parasites in immunized HLA-A*1101 mice were significantly reduced compared to the numbers of parasites in unimmunized mice. Results 
An algorithm was developed to calculate projected population coverage of a T cell epitope-based vaccine using MHC binding or T cell restriction data and HLA gene frequencies [17] . We used this web-based tool http://www.iedb.org/ to predict population coverage of these HLA-A03 supertype peptide epitopes-based vaccine. The population coverage calculation results in Table 4 indicate that such coverage is varied in different geographic regions. The HLA-A03 supertype molecules that present these peptides would be expected to be present in 28 
In this study, we first evaluated immunization of HLA-A*1101 transgenic mice with either mixtures of peptides or lipopeptides derived from three identified T. gondii specific HLA-A*1101 restricted CD8 + T cell epitopes emulsified in 3-deacylated monophosphoryl lipid A (GLA-SE) adjuvant. Immunizations of transgenic mice with a mixture of CD8 + epitope peptide pools plus PADRE and adjuvants were able to induce splenocyte to produce IFN-γ and to protect against challenge with high numbers of Type II parasites.
Conjugation of CD8 + T cell determinants to lipid groups is known to enhance specific cell-mediated responses to target antigens in experimental animals and humans [25] [26] [27] [28] [29] , although mechanisms whereby immunity is achieved remains poorly understood. Lipopeptides hold several advantages over other conventional vaccine formulations; for instance, they are self-adjuvanting and display none of the toxicity-associated side effects of other Th1-inducing adjuvant systems. In our work, transgenic mice that were immunized with three short lipopeptide vaccines had T cells that produced IFN-γ. Among them the lipopeptide vaccine formulated with KS9 or AM9 In vivo protection demonstrated with imaging using Xenogen camera. HLA-A*1101 transgenic mice immunized with peptide pool and adjuvants were protected compared to control mice inoculated with PBS when they were challenged with 10,000 Prugneaud strain (Fluc)-T. gondii luciferase expressing parasites. There were a total of 5-9(usually 4-5 per group) mice tested in each control or immunization group. Differences between control and immunization groups were significant(p < 0.0064 using natural log transformed data and two-sample t test). Peptides derived from these proteins and the position within the proteins 2 Binding affinity was performed by MHC binding assay. 3 PBMC from four T. gondii-seropositive HLA-A03 supertype persons and four seronegative persons were stimulated with peptides, the T cell that produce IFN-γ were tested by ELISpot assay. 4 Splenic T cell were isolated from HLA-A*03 supertype(which includes the HLA-A*1101 haplotype) mice 10 to 14 days after peptide immunization and tested for their ability to generate IFN-γ in response to peptide. stimulated higher IFN-γ production than the lipopeptide vaccine formulated with RS9. Unexplained and variable responses have been observed to high affinity binding peptides in other models, e.g., studies of Livingstone, Alexander, Sette et al with Lassa fever virus. It will be of interest to better understand possible mechanisms for such lack of response in future studies. However, the lipopeptides with three epitope peptides linked together with alanine spacers did not stimulate an IFN-γ response by splenocytes from immunized mice when the splenocytes subsequently were exposed to each of the peptides in vitro. The reason why the lipopeptides with the three linked peptides did not work well in the transgenic mice might be related to a frame shift caused by the linkers that altered the response to the original peptides rather than the alanines functioning for the intended purpose of introducing a cleavage motif. The three linker "AAA" between the peptides had previously been demonstrated in other systems to result in sensitization to each linked peptide. However, surprisingly, it did not appear to work well herein. Because the three linked peptides in the lipopeptide formulation were not effective and we had found that a mixture of the components with a single peptide was as or more robust than the lipopeptide, we tried this approach with the three peptides that had been included in the linked lipopeptide with the universal helper CD4 + T cell peptide, PADRE, and adjuvants as described below. The response was robust both in vitro and in vivo (Figures 4 and 5) .
Some studies have shown palmitoylated lipopeptide constructs to elicit long-lived, protective cellular responses against a variety of pathogens, including Hepatitis B virus (HBV), influenza virus, and Plasmodium falciparum [25] [26] [27] [28] [29] . Our work herein shows that mice immunized with mixture of CD8 + and CD4 + eliciting peptides and lipid Pam 2 Cys emulsified in GLA-SE elicited higher IFN-γ production than mice immunized with lipopeptides constructed with the same components of CD4 + and CD8 + eliciting peptides, and Pam 2 Cys. The approach using cocktails of non-covalently linked lipid mixed to helper T lymphocytes(HTL) and CD8 + T cell (cytolytic T lymphcyte[CTL] and IFN-γ eliciting) epitopes for simultaneous induction of multiple CD8 + T specificities would have significant advantages in terms of ease of vaccine development.
HTL responses are crucial for the development of CD8 + T responses, at least in the case of lipidated covalently or non-covalently linked HTL-CTL epitope constructs formulated in PBS. Several previous studies have illustrated a role for CD4 + responses for development of CD8 + CTL responses, both in humans and in experimental animals [30] [31] [32] [33] [34] [35] . The inclusion of PADRE, a synthetic peptide that binds promiscuously to variants of the Figure 6 New peptides tested with PBMCs from HLA-A03 seropositive and seronegative donors. Then Peripheral blood mononuclear cells from seropositive T. gondii donors were tested for response to these predicted HLA-A03 supertype restricted CD8 + T cell epitope individual peptides by using IFN-γ ELISpot assay. Peptides that induced significant IFN-γ spot formation compared to DMSO are denoted by an asterisk. *, P < 0.05. Figure 7 Addition of peptides to pool robustly protects HLA-A*1101 mice against Type II parasite challenge. HLA-A*1101 transgenic mice were immunized with PBS (controls) or peptide pool with PADRE and Pam 2 Cys in GLA-SE. Splenic T cells were isolated 10-14 days post immunization and exposed to each peptide in an ex vivo IFN-γ ELISpot assay (Figure 7a ). HLA-A*1101 transgenic mice immunized with peptide pool and adjuvants were protected compared with control mice inoculated with PBS when they were challenged with 10,000 Pru (Fluc)-T. gondii luciferase expressing parasites (Figure 7b ). Mice were immunized and in a subgroup immune function was studied at the same time as the challenge shown was performed. Differences between control and immunized mice were significant (p < 0.0064 using natural log transformed data and two-sample t test).
human MHC class II molecule DR and is effective in mice, also augmented CD8 + T cell effector functions by inducing CD4 + T helper cells [30] [31] [32] [33] [34] [35] . Both CD4 + and CD8 + epitopes were targeted in order to drive a protective immune response [34, 35] . Adjuvanting antigens contributes to the success of vaccination. An example herein is that 3-deacylated monophosphoryl lipid A(GLA-SE), a detoxified derivative of the lipopolysaccharide (LPS) from Salmonella minnesota R595 was a potent adjuvant. This GLA-SE is a novel adjuvant which was formulated in an emulsion [21] [22] [23] [24] . This is a Toll-like receptor 4 (TLR4) agonist that is a potent activator of Th1 responses [21] [22] [23] [24] . It has been used as an adjuvant in human vaccine trials for several infectious disease and malignancy indications. It has been very effective as an adjuvant providing CD4 + T cell help for immunizations against other protozoan infections such as leishmaniasis [21] [22] [23] [24] . In our study, a robust response was observed when GLA-SE was included in preparation for immunization of mice. Pam 2 Cys (S-[2,3-bis(palmitoyloxy)propyl] cysteine) is a lipid component of macrophage-activating lipopeptide. Pam 2 Cys binds to and activates dendritic cells by engagement of Toll-like receptor 2 (TLR-2) [24] . Tolllike receptors (TLRs) function as pattern-recognition receptors in mammals [36] . We have found that both TLR2 and TLR4 receptors participate in human host defense against T. gondii infection through their activation by GPIs and GIPLs(Melo, Hargrave, Miller, Blackwell, Gazinelli, McLeod et al, in preparation, 2010). TLR2 and TLR4 likely work together with other MyD88-dependent receptors, including other TLRs, to elicit an effective host response against T. gondii infection [36] . In our study, there was a slightly more robust response observed when Pam 2 Cys was co-administered for some peptides, but not all of them.
The goal of the present study was to identify HLArestricted epitopes from T. gondii and evaluate whether they could provide protection against parasite challenge measured as protection against a luciferase producing Type ll parasite using a Xenogen camera system. In the future, additional more detailed studies involving analyses over longer times, other strains of the parasite and challenge with life cycle stages, evaluation of multiple organs including eye and brain, studies of protection in congenital infections, comparisons of delivery of these peptides as DNA encoding them versus other formulations. This future work will follow up and extend these intitial studies of reduction of parasite burden seen in Figures 5 and 7 .
Various peptide-based approaches to induction of IFN-γ responses were evaluated as part of ongoing efforts to develop immunosense vaccines for use in humans with each of the supermotifs which would in total include more that 99% of the human population worldwide. Robust protection was achieved in the HLA-A*1101 transgenic mice challenged with Type II parasites following immunizations. In order to identify additional peptides from T. gondii that were present in tachyzoites or bradyzoites [37, 38] or sporozoites of Type I and II strains and elicited IFN-γ from HLA-A03 + supertype (which includes the HLA*1011 allele) restricted CD8 + T cells, bioinformatic algorithms were utilized to identify novel, T. gondii-derived, epitopes restricted by the HLA-A03 supertype. Then PBMC cells were tested to determine whether the peptides elicited IFN-γ from human CD8 + T cells from seropositive persons. This was intended to collectively provide broad coverage for the human population with HLA-A03 supertype worldwide. The additional peptides we identified as immunogenic for human peripheral blood cells were also robust in eliciting IFN-γ from splenocytes of HLA-A*1101 mice and protection when used to immunize these mice. These findings will facilitate development of an immunosense epitope-based vaccine for human use.
A human immunome-based and parasite genome based bioinformatices approach was used to define candidate HLA-A03 supertype restricted peptides. Immunogenicity of a group of T. gondii HLA-A03 supertype restricted peptides, and therefore the proteins from which they are derived, for immune humans and HLA-A*1101 transgenic mice was demonstrated. These peptides elicit interferon-γ production by human CD8 + T cells. They also elicit interferon γ production by mouse splenocytes when utilized to immunize HLA-A*1101 transgenic mice with a lipopeptide with a universal CD4 + T cell eliciting epitope, PADRE, or in peptide pools with PADRE, Pam 2 Cys and GLA-SE, a novel adjuvant. Immunization studies demonstrate the need for and the efficacy of adjuvants in immunization of these HLA transgenic mice. Immunogenic peptides included KSFKDILPK (SAG1 224-232 ); AML-TAFFLR (GRA6 164-172 ); and RSFKDLLKK (GRA7 134-142 ); STFWBCLLR (SAG2C [13] [14] [15] [16] [17] [18] [19] [20] [21] ; SSAYVFSVK (SPA 250-258 ); and AVVSLLRLLK (SPA 89-98 ). The studies herein provide a foundation for immunosense based vaccines to prevent toxoplasmosis in those with the HLA-A03 supertype and information about how they can be adjuvanted.
HLA-A03 supertype CD8 + T cell epitopes included: KSFKDILPK (SAG1 224-232 ), AMLTAFFLR (GRA6 164-172 ), and RSFKDLLKK (GRA7 134-142 ). PADRE (AKF-VAAWTLKAAA) was the universal CD4 helper peptide used in vaccine constructs. Pam 2 Cys (Pam 2 -KSS) also was included. Lipopeptide constructs used in this study are shown in Figure 1 . Peptides and lipopeptides were synthesized by Synthetic Biomolecules, San Diego at > 90% purity. Additional HLA-A03 supertype bound peptides and their initial grouping into pools for in vitro studies are shown in Tables 1 and 2 . A TLR4 agonist, a GLA-SE adjuvant, was synthesized by the Infectious Diseases Research Institute (Seattle, Washington) as a stable oil-in-water emulsion. AMLTAFFLR (GRA6 164-172 ) and additional new peptides were first dissolved in DMSO and then diluted in PBS.
HLA-A*1101/K b transgenic mice were produced at Pharmexa-Epimmune (San Diego, CA) and bred at the University of Chicago. These HLA-A*1101/K b transgenic mice express a chimeric gene consisting of the 1 and 2 domains of HLA-A*1101 and the 3 domain of H-2K b , and were created on a C57BL/6 background. For each test, we used 4-5 mice for each group. Each experiment was repeated 2 to 3 times. Experiments in Figure 7b were performed including a subgroup analyzed for immune response in parallel with a subgroup in the challenge shown. All studies were conducted with approval of the Institutional Animal Care and Use Committee at the University of Chicago.
Transgenic T. gondii used for in vivo challenges was derived from Type II Prugniaud (Pru) strain and expresses the firefly luciferase (FLUC) gene constitutively by tachyzoites and bradyzoites. It was created, and kindly provided by S. Kim, J. Boothroyd and J. Saeij (Stanford University) and was maintained and utilized as previously described [18, 37, 39] .
To evaluate peptide immunogenicity, HLA-A*1101 transgenic mice were inoculated subcutaneously (s.c.) at the base of the tail using a 30-gauge needle with single peptides or a mixture of CD8 + T cell peptides (50 μg of each peptide per mouse) and PADRE (AKFVAAWTLKAAA) emulsified in 20 μg of GLA-SE (TLR4 agonist) with or without Pam 2 Cys. Pam 2 Cys concentration was 5 mg/ml. For immunization with lipopeptides, HLA-A*1101 mice received 20 nmol lipopeptide dissolved in PBS or emulsified in GLA-SE. As controls, mice were injected with PBS or PBS/GLA-SE. For the lipopeptide immunizations, the mice were vaccinated twice at intervals of three weeks. For the peptide immunizations, mice were immunized three times at intervals of two weeks. For challenge studies, mice were immunized with peptide emulsions and challenged intraperitoneally (i.p.) 14 days post-immunization using 10,000 Type II parasites.
Mice infected with 10,000 Pru-FLUC tachyzoites were imaged 7 days post-challenge using the in vivo imaging system (IVIS; Xenogen, Alameda, CA). Mice were injected i.p. with 200 μl of D-luciferin, anesthetized in an O 2 -rich induction chamber with 2% isoflurane, and imaged after 12 minutes. Photonic emissions were assessed using Living image® 2.20.1 software (Xenogen). Data are presented as pseudocolor representations of light intensity and mean photons/region of interest (ROI). All mouse experiments were repeated at least twice. There were 4-5 mice for each group. In the experiment in Figure 7b a subgroup of mice was used for studying immune response in parallel with the subgroup in the challenge shown.
Mice were euthanized 7 to 14 days after immunization. Spleens were harvested, pressed through a 70 μm screen to form a single-cell suspension, and depleted of erythrocytes with AKC lysis buffer (160 mM NH 4 Cl, 10 mM KHCO 3 , 100 M EDTA). Splenocytes were washed twice with Hank's Balanced Salt Solution (HBSS) and resuspended in complete RPMI medium (RPMI-1640 supplemented with 2 mM L-GlutaMax
[Invitrogen], 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 50 M -mercaptoethanol, and 10% FCS) before they were used in subsequent in vitro assays. Peptide concentration was 20 mg/ml and 0.20 μl was used per well. ELISpot assays with murine splenocytes were performed using α-mouse IFN-γ mAb (AN18) and biotinylated α-mouse IFN-γ mAb (R4-6A2) as the cytokine-specific capture antibodies. Antibodies were monoclonal antibodies. 5 × 10 5 splenocytes were plated per well.
PBMC were obtained, HLA haplotype was determined, and they were processed and cryopreserved as described [18] . ELISpot assays with human PBMCs were similar to those with murine splenocytes but used α-human IFN-γ mAb (1-D1K) with biotinylated α-human IFN-γ mAb (7B6-1) with 2 × 10 5 PBMCs per well. All antibodies and reagents used for ELISpot assays were from Mabtech (Cincinnati, OH). Antibodies were monoclonal antibodies. Both murine and human cells were plated in at least 3 replicate wells for each condition. Results were expressed as number of spot forming cells (SFCs) per 10 6 PBMCs or per 10 6 murine splenocytes.
Protein sequences derived from GRA10, GRA15, SAG2C, SAG2D, SAG2X, SAG3, SRS9, BSR4, SPA, and MIC were analyzed for CD8 + T cell epitopes based on predicted binding affinity to HLA-A03 supertype molecules using ARB algorithms from immunoepitope database (IEDB) http://www.immuneepitope.org [40, 41] . A total of 34 unique peptides IC 50 < 50 nM of all ranked nonameric peptides were selected. All protein sequences were from ToxoDB 5.1. Quantitative assays to measure binding of peptides to HLA class I molecules are based on inhibition of binding of radiolabeled standard peptide. Assays were as described [42] . Concentration of peptide yielding 50% inhibition of binding of radiolabeled probe peptide (IC 50 [43, 44] .
Statistical analyses for all in vitro assays were performed using 2-tailed student's T test. Natural log transformed data and two-sample t test were used to analyze data shown in Figures 5 and 7b . Two-tailed P values < 0.05 were considered statistically significant. Peptides were considered immunogenic in mice if they induced IFN-γ spot formation from immunized mice that were significant (P < 0.05) compared with spot formation from control mice. All mouse experiments were repeated at least twice. There were 4-5 mice for each group. The experiment in Figure 7b determined immune response and imaged mice in parallel. Conserved epitopes of influenza A virus inducing protective immunity and their prospects for universal vaccine development Influenza A viruses belong to the best studied viruses, however no effective prevention against influenza infection has been developed. The emerging of still new escape variants of influenza A viruses causing epidemics and periodic worldwide pandemics represents a threat for human population. Therefore, current, hot task of influenza virus research is to look for a way how to get us closer to a universal vaccine. Combination of chosen conserved antigens inducing cross-protective antibody response with epitopes activating also cross-protective cytotoxic T-cells would offer an attractive strategy for improving protection against drift variants of seasonal influenza viruses and reduces the impact of future pandemic strains. Antigenically conserved fusion-active subunit of hemagglutinin (HA2 gp) and ectodomain of matrix protein 2 (eM2) are promising candidates for preparation of broadly protective HA2- or eM2-based vaccine that may aid in pandemic preparedness. Overall protective effect could be achieved by contribution of epitopes recognized by cytotoxic T-lymphocytes (CTL) that have been studied extensively to reach much broader control of influenza infection. In this review we present the state-of-art in this field. We describe known adaptive immune mechanisms mediated by influenza specific B- and T-cells involved in the anti-influenza immune defense together with the contribution of innate immunity. We discuss the mechanisms of neutralization of influenza infection mediated by antibodies, the role of CTL in viral elimination and new approaches to develop epitope based vaccine inducing cross-protective influenza virus-specific immune response. Influenza remains a serious respiratory disease in spite of the availability of antivirals and inactivated trivalent vaccines, which are effective for most recipients. Influenza viruses are RNA viruses with strongly immunogenic surface proteins, especially the hemagglutinin. Error-prone RNA-dependent RNA polymerase and segmented genome enable influenza viruses to undergo minor (antigenic drift) as well as major (antigenic shift) antigenic changes, which permit the virus to evade adaptive immune response in a variety of mammalian and avian species, including humans. The unpredictable variability of influenza A viruses, which cause yearly epidemics in human population, is the main reason why no effective prevention against influenza infection exists up to date. Currently available vaccines induce antibodies against seasonal and closely related antigenic viral strains, but do not protect against antibody-escape variants of seasonal or novel influenza A viruses. Therefore, there is a call for development of a vaccine, which would be protective against virus strains of different HA subtypes and would not need to be updated every year. New approach to prepare a universal vaccine lies in the selection of conserved epitopes or proteins of influenza A virus, which induce cross-protective immune response, particularly M2, HA2, M1, NP [1] [2] [3] .
Influenza infection induces specific humoral immunity represented by systemic and local antibody response, as well as cellular immunity, represented by specific T-cell response ( Figure 1 ). Both of them are important in the host defense against influenza infection, because of their close cooperation mediated by various immune mechanisms. Dendritic cells and macrophages (antigen presenting cells, APCs) play an important role in initiating and driving of adaptive immune response [4] . Exogenous viral antigens, including inactive viral particles, intact viruses or infected cells, are taken up by APCs through endocytosis or phagocytosis. Their further processing results in generation of peptides that are presented via MHC I or MHC II molecules to CD8+ precursor T-cell and CD4+ helper T-cell precursors (Th0), respectively. Th0 cells are subdivided to Th1and Th2-type helper cells, based on the cytokine profiles they produce. Following influenza infection, APCs secrete IL-12 that contributes to the differentiation of Th0 into Th1 cells, which secrete IFN-γ and help to produce IgG2a antibodies [5, 6] . Th1 cells also produce IL-2, required for the proliferation of the virus-specific CD8+ CTLs. In contrast, when IL-10 is present early in The cellular immune response (right) is initiated after recognition of viral antigens presented via MHCI and MHC II molecules by antigen presenting cells (APC), which then leads to activation, proliferation and differentiation of antigen-specific CD8+ T or CD4+ cells. These cells gain effector cell function and either they help directly (Th1 or Th2 cell) to produce antibodies or, CTL effector cells recognize antigen peptides presented by MHCI on APC and kill the virus infected cells by exocytosis of cytolytic granules. The humoral immune response (left)is mediated by specific antibodies (e.g IgG, IgA) produced by antibody secreting plasma cells (ASC) which are the final stage of B cell development. This process is aided by CD4+ T helper and T cell-derived cytokines essential for the activation and differentiation of both B-cell responses and CD8+ T cell responses.
the immune response , Th0 cells differentiate to Th2  cells, which secrete IL-4, IL-5, IL-6 and help preferentially drive IgG1, IgA and IgE Ab production by anti- body-secreting plasma cells (ASCs) [6] [7] [8] [9] . CD8+ precursor T-cells, which maturate into CTLs (cytotoxic T lymphocytes), release antiviral cytokines (IFN-γ) upon recognition of short viral peptides presented by MHC I molecules on virus-infected epithelial cells, and destroy the virus infected cells by exocytosis of cytolytic granules. The granules contain cytolytic protein perforin and granzymes. Perforin is a protein that creates pores in membranes of infected cells. Granzymes are members of serine protease family. In the presence of perforin, granzymes enter into the cytoplasm of infected cells and initiate proteolysis, which triggers destruction of the target cell [10, 11] . CTLs could mediate killing of infected cells also by perforin-independent mechanisms of cytotoxicity. This involves binding of Fas receptor in the infected target cell membranes with the Fas ligand (FasL) expressed on activated CTLs. Interaction of FasL with corresponding Fas receptor leads to the activation of caspases, which induce apoptosis in influenza infected cells [12] [13] [14] .
Unlike T-cells, which recognize linear epitopes presented by MHC molecules, B cells can recognize antigen in its native form. Antibody response against influenza infection is mediated by secretory IgA antibodies and serum IgG antibodies. IgA are transported across the mucosal epithelium of the upper respiratory tract, where they represent the first immunobarrier to influenza viruses. IgG transude from the serum to the mucus by diffusion and are primarily responsible for the protection of the lower respiratory tract [15] .
Specific antibodies induced by influenza virus infection can neutralize infection by several different mechanisms ( Figure 2 ). They can directly block virus attachment to the target cells by interfering with virus-receptor interaction and thus prevent influenza infection ( Figure 2A ). These antibodies are directed to the globular domain of the surface antigen, hemagglutinin [16] . However, because of high variability of influenza A viruses, neutralization activity of these Abs is limited to viral strains, which are antigenically similar to the inducers of Ab Figure 2 Mechanisms of antibody-mediated neutralization during influenza infection. A. Serum IgG or B. mucosal IgA antibodies specific to hemagglutinin prevent influenza infection by blocking attachment to host cell receptors. C. After binding, the virus is internalized by receptor mediated endocytosis. The low pH in the endosome triggers conformational changes in hemagglutinin that expose fusion peptide located in HA2 required for membrane fusion. In this step, antibodies bound to HA2 block the fusion of viral and endosomal membranes and prevent release of ribonucleoprotein complex into the cytoplasm of target cell. D. Intracellular neutralization of influenza virus through transcytotic pathway of IgA that complex with viral proteins and inhibit assembly of progeny virions. E. Antibodies specific to neuraminidase inhibit release of budding viral particles and further spread of influenza infection by inhibition of neuraminidase activity. production. By contrast, it was shown that mucosal immunity mediated by secreted form of IgA Abs in the upper respiratory tract is more cross-protective against heterologous virus infection than systemic immunity mediated by IgG Abs [17, 18] . The strong cross-protective potential of IgA Abs appears to be the consequence of their polymeric nature, resulting in higher avidity of Abs for the influenza virus compared to the monomeric serum IgG Abs [18] . After synthesis by ASC, dimeric IgA (dIgA) Abs bind to the polymeric immunoglobulin receptor expressed on the basolateral surface of the epithelial cells and are transcytosed to the apical surface, where the poly-Ig receptor is cleaved, secretory IgA are released and prevent infection by blocking attachment to the epithelial cells ( Figure 2B ). Moreover, dIgA Abs are able to bind to the newly synthesized viral proteins within infected cells, thus preventing virion assembly ( Figure 2D ) [19] .
After attachment to the receptor on the target cell, influenza virus is internalized by receptor-mediated endocytosis. Conformational changes of hemagglutinin triggered by the low pH in the endosome activate viral and endosomal membrane fusion. In this step, antibodies, which bind to the non-receptor binding region of HA, could interfere with the low-pH induced conformational change in the HA molecule required for the fusion. Inhibition of the fusion between viral and endosomal membrane proteins mediated by such antibodies prevents release of the ribonucleoprotein complex (RNP complex) into the cytoplasm of the target cell, resulting in the inhibition of viral replication ( Figure 2C The interaction of opsonic complement proteins with complement receptor on macrophages (CR) increases the rate of phagocytosis of macrophages, causing direct virolysis or improvement of antibody-mediated inhibition of virus attachment to host cells [26, 27] . However, contribution of complement to the protective capacity of antibodies is contradictory, since it was shown that passive transfer of murine polyclonal anti-eM2 serum into C3-negative mice had protective effect [24], while human monoclonal anti-M2 antibodies could not protect complement-depleted mice [25] . It should be noticed that though some antibodies directed to conserved antigens such as M2 do not prevent infection by direct binding to virus, they can contribute to an earlier recovery from the infection by indirect antibodymediated mechanisms after binding to Fc-receptors on macrophages or NK cells. It is possible that the same mechanism of protection is mediated by antibodies to HA2 glycopolypeptide (HA2 gp), a conserved part of HA. They also do not prevent infection, but their strong protective potential has been proved in vivo [28-31]. For this reason understanding the role of the Fc effector function of antibodies in the clearance of influenza infection is required.
Both, ectodomain of M2 and HA2 gp are conserved antigens inducing antibodies protecting against influenza infection. Therefore, various studies are focused on these two antigens as inductors of heterosubtypic antibody response.
M2 protein is a single-pass type III membrane protein forming homotetramers representing pH-gated proton channel incorporated into the viral lipid envelope. This proton channel is essential for efficient release of viral genome during viral entry [32]. M2 protein is abundantly expressed at the apical plasma membrane of infected epithelial cells, but only a small number (16-20 molecules/virion) of M2 molecules are incorporated into virions [33, 34] . Great attention is paid to the extracellular N-terminal domain of M2 protein (eM2), a 23 amino acid peptide, which is highly conserved in all human influenza A strains. It is therefore an attractive target for preparation of a universal influenza A vaccine. In contrast to hemagglutinin and neuraminidase, eM2 is a weak immunogen [35] . Therefore, various approaches to increase its immunogenicity were used. All of them are based on increasing the immunogenicity of small antigen molecules by insertion of their multiple copies into a suitable immunogen Neirynck et al [36] prepared a fusion protein composed of eM2 and hepatitis B virus core (HBc) protein. This fusion protein has the ability to aggregate into the highly immunogenic virus-like particles inducing a long-lasting protection against lethal influenza A infection. High in vivo protective effect of described virus-like particles was proven after intraperitoneal or intranasal immunization of mice and subsequent infection with lethal dose of influenza viruses of various HA subtypes [37] . Efficacy of these particles has been increased by application of new adjuvant CTA1-DD. Combination of the eM2-HBc construct with the new adjuvant led to the protection of mice against lethal infection and a remarkably lower morbidity [38] . Various constructs of eM2 peptide engineered by conjugation to carrier proteins were evaluated as a vaccine, which successfully protected animals against infection with homologous but also heterologous human strains [24,36,37,39-42].
A different approach to increase immunogenicity of eM2 was described by other groups. Constructs composed of four tandem copies of the eM2 peptide fused to flagellin, a ligand of TLR5 (Toll-like receptor 5) [41], or glutathione-S-transferase fusion protein bearing various numbers of eM2 epitope copies [42], were used as immunogens. These studies showed that high eM2 epitope densities in a single recombinant protein molecule resulted in enhanced eM2-specific humoral response and higher survival rates of infected animals.
Another way to stimulate the immune system by small peptide was described by Ernst et al. Immunization of mice with these eM2-HD liposomes was protective against influenza virus strains of various subtypes and stimulated the production of specific IgG1 antibodies in mouse sera. Moreover, mice passively immunized with these antibodies were protected against lethal infection.
M2 protein in its native state forms a homotetramer, comprising also conformational epitopes, which might play important role in eM2 immunogenicity. It was shown that oligomer-specific antibodies were induced by recombinant eM2 protein mimicking the natural quaternary structure of M2 ectodomain in viral particle [43] . For this purpose, a modified version of leucine zipper from yeast transcription factor GCN4 was bound to eM2. High titers of antibodies recognizing M2 protein in the native conformation were obtained after intraperitoneal or intranasal immunization with this recombinant protein, and immunized mice were fully protected against lethal dose of influenza A virus [43] . Such vaccine could improve quality of humoral immune response with antibodies elicited not only against linear epitopes but also against conformational epitopes.
Above described results indicate that eM2 is a valid and versatile vaccine candidate to induce protective immunity against any strain of human influenza A viruses, and give a promise for finding new "universal" vaccine against flu.
Hemagglutinin (HA) is the major influenza virus target antigen recognized by neutralizing antibodies. It is a surface glycoprotein, synthesized as a single polypeptide, which is trimerized. Each monomer of HA is synthesized as a precursor molecule HA0 post-translationally cleaved by host proteases into two subunits, HA1 and HA2 linked by a single disulfide bond [16] . Cleavage into HA1 and HA2 gp is essential for the infectivity of the virus particle and spread of the infection in the host organism [44] .
The HA1 of influenza A virus forms a membranedistal globular domain that contains the receptorbinding site and most antigenic sites recognized by virus-neutralizing antibodies preventing attachment of virus to the host cell. Escape variants with mutation in the antigenic site easily avoid neutralization by existing host antibodies, leading to seasonal influenza outbreaks [45] . In spite of continual antigenic changes of hemagglutinin, common epitopes shared by various strains were identified. Although the degree of sequence diversity between HA subtypes is great, particularly in the HA1 glycopolypeptides, HA2 is its rather conserved part. According to documented results, HA2 has the prerequisite to be one of the potential inductors of protective heterosubtypic immunity [1, 28, 29, [46] [47] [48] . HA2
represents the smaller C-terminal portion of hemagglutinin, which forms a stem-like structure that mediates the anchoring of the globular domain to the cellular or viral membrane. N-terminal part of HA2 gp, termed the fusion peptide, plays a substantial role in the fusion activity of influenza virus. It was demonstrated that the rearrangements of HA as well as the fusion process is temperature-and pH-dependent [49, 50] . At neutral pH, the N-terminus of the fusion peptide is inserted into the inter-space of HA trimer. At low pH, which triggers the fusion process, N-terminus of the fusion peptide is exposed and inserted into the target membrane, allowing the release of the ribonucleoprotein complex into the cytoplasm [51, 16] . Although the epitopes of the [62, 63] . Moreover, it was shown that passive immunization with monoclonal antibodies against HA2 gp, as well as active immunization with recombinant vaccinia virus expressing chimeric molecules of HA, improve the recovery from influenza infection and contribute to a milder course of infection [28, 29] . A recent study showed that increased immunogenicity of HA2 gp could be achieved by unmasking of HA2 gp after removing the highly immunogenic globular head domain of HA1 gp. Headless HA trimers form the conserved HA stalk domain, on which HA2 epitopes are more accessible for B cells than in the native HA. Vaccination of mice with this headless HA immunogen elicited antibodies cross-reactive with multiple subtypes of hemagglutinin and provide protection against lethal influenza virus infection [31] .
Hemagglutinin HA1-HA2 connecting region, as well as N-terminal fusion peptide of HA2, are the broadly conserved parts of HA, the latter conserved even among all 16 subtypes of influenza A viruses [1, 47, 61, 64] . Protective potential of the fusion peptide or HA1-HA2 cleavage site of influenza A viruses were investigated by several groups. They found that mice vaccinated with a peptide spanning the HA1-HA2 connecting region exhibited milder illness and fewer deaths upon virus challenge [64, 65] .
Generation of monoclonal antibodies against universally conserved fusion peptide has attracted interest in the recent past, as such antibodies are known to inhibit the HA fusion activity and to reduce virus replication in vitro and also in vivo [28, 30, 54, 62, 63] . Additionally, passive immunotherapy with Abs reactive with all strains of influenza A could be an alternative for some populations at high risk of infection, like infants, the elderly and the immunocompromised patients, who may not benefit from active vaccination. Several groups described the potential of human monoclonal antibodies against HA2 subunit and its fusion peptide with broadspectrum protection as a universal passive immunotherapeutic agent against seasonal and pandemic influenza viruses [66] [67] [68] [69] . Sui et al. [70] obtained a panel of highaffinity human antibodies that bind to the highly conserved pocket in the stem region of hemagglutinin, comprising part of the fusion peptide and several residues of the HA1 subunit. These antibodies showed a broad degree of cross-reactivity. Moreover, it was suggested that the conformational epitope on HA recognized by one of these neutralizing antibodies (F10) is recalcitrant to the generation of escape mutants [70] .
Thus, identification of antibodies against conserved epitopes of hemagglutinin shows the way for their use in passive immunotherapy, designing of antivirals and represents an important step towards development of cross-protective universal vaccine against influenza virus that potentially does not require annual adjustment.
Nucleoprotein (NP) and matrix protein (M1) of influenza virus are conserved structural influenza antigens, to which antibody response is induced after natural infection. These antibodies, however, do not display a considerable effect on protection against influenza infection [22] . On the other hand, NP, M1 and other inner influenza antigens play important role in the cellular immune response. It was demonstrated that NP-or M1-specific Th cells could augment protective antibody response, aiding the B cells to produce antibodies specific to hemagglutinin [71] .
CTL play an important role in the control of influenza virus infections. They eliminate virus-infected cells, on which surface they recognize foreign antigens derived from endogenously expressed viral antigens presented by MHC class I molecules. Thus, they contribute to the clearance of the virus from the infected tissue and prevent the spread of viral infection. Although CTL do not prevent influenza infection, their beneficial effect on the course of infection was observed after the adoptive transfer of virus-specific CTL clones to mice, resulting in direct lysis of infected cells [72] [73] [74] . In addition, depletion of CTL in infected mice led to higher titers of the virus in lungs, increased mortality and more severe disease [75] . Depending on their antigen specificity, CTLs may be subtype-specific or, in case they recognize the internal antigens, they are broadly cross-reactive with various influenza A viral strains. Early studies in mice showed that the majority of influenza-specific CTLs were reactive across subtypes [76, 77] , what underlines their important role in heterosubtypic immunity. This high crossreactivity is explained by the antigenically conserved targets of CTL represented mostly by inner influenza antigens (e.g. NP, M1 and PB1, PB2) [78] [79] [80] [81] . However, some conserved T-cell epitopes were identified also on variable surface influenza antigens [82] [83] [84] .
Recent data support the beneficial role of T-cell response in reducing the severity of infection also in humans [85] [86] [87] [88] . Additionally, cross-reactive CTLs recognized different subtypes of influenza A virus and their protective effect was shown also in individuals, who did not have specific antibodies against a given influenza virus they were exposed to [89] . Therefore, vaccination strategies focused on generating T-cellmediated immune responses directed towards conserved epitopes of influenza virus are also considered.
T-cell epitopes are intensively studied as an alternative to the current vaccine strategy based mainly on the induction of the strain specific virus-neutralizing antibodies. Identification of conserved CTL epitopes shared by many influenza strains could represent the basis of vaccination strategies. This approach would be beneficial in the case of annual influenza epidemic and a potential pandemic, when humoral immunity is poorly or not protective due to the absence of pre-existing antibodies against emerging strains in the population [90, 91] .
While CTL mediated immunity is considered to be weak, epidemiological data indicate induction of crossprotective immunity in humans, who overcame influenza infection in the past [85] . It was shown that memory T-cells against the conserved epitopes confer protection from the infection with the virus strains of different subtypes in humans [82, 85, 86, 88, 89, 92, 93] .
Studies in mice demonstrated that, similar to the live influenza vaccine, adenovirus-based vaccine and DNA immunization induced CTL cross-protective immune response against infection with multiple influenza A subtypes [94] [95] [96] [97] [98] . The variable rate of cross-reactive CTL response was achieved also by using adjuvants, or various formulations and delivery systems with experimental influenza vaccines in preclinical animal studies [reviewed in [99] ]. It was shown that application of virus-like particles or virosomal vaccines could be successfully used for efficient delivery of multiple CTL epitopes to the target cells resulting in induction of CTL response [100, 101] .
Heterosubtypic immunity mediated by CTLs was described in naturally infected humans [88, 89, 102] . It is developed mainly against conserved epitopes of NP, M1 and NS1 [82, [103] [104] [105] [106] . Kreijtz et al. showed that virusspecific CTL developed in humans as a response to previous exposition to seasonal influenza A viruses of the H3N2 and H1N1 subtypes displayed considerable cross-reactivity also with avian influenza viruses (e.g. A/H5N1) [86] . Thus, it could be supposed that obtained pre-existing T-cell immunity in humans may help to decrease the severity of infection during a pandemic outbreak in comparison to those individuals, who lack cross-reactive influenza specific CTL populations [86, 88, 107] . Therefore, vaccines based on conserved CTL epitopes represent a reasonable approach to generate effective broadly protective cellular immunity against influenza viruses of various subtypes.
To develop vaccines capable of stimulating effective T-cell response, it is necessary to understand the factors contributing to the immunodominance of CTL epitopes. During viral infection, a large number of peptides are generated by processing of viral proteins in the proteasomes of infected cells. Only a small fraction of these peptides are presented by MHC class I molecules and subsequently recognized by specific CTL. This hierarchy of CTL response proved in animals [108] and in humans [104] is called immunodominance. There are several factors, which contribute to this phenomenon: HLA haplotype and its binding affinity to individual epitopes, repertoire of T-cell receptors, processing and presentation of viral peptides and interaction of CTL with antigen-presenting cells [109, 110] . It was shown that efficiency of epitope processing is one of the dominant factors affecting immunogenicity of multi-epitope vaccine [111, 112] .
The most frequently used models for such immunological studies are inbred mice, like B57BL6 (H-2 b ) or BALB/c (H-2 d ) mice. Therefore, T-cell influenza specific epitopes in inbred mice were studied by many authors. Comprehensive analysis regarding existing influenza A epitopes in mice among avian and human influenza strains was done by Bui et al. [113] . However, not all Tcell epitopes are equally immunogenic. In inbred mice B57BL6 (H-2 b ), peptides from nucleoprotein D b NP 366-374 and from a subunit of viral RNA polymerase D b PA 224-233 are immunodominant, while nucleoprotein epitope K d NP 147-155 is immunodominant in BALB/ c (H-2 d ) mice [84, [114] [115] [116] .
In contrast to inbred mice, the search for CTL epitopes suitable for development of CTL epitope-based vaccine in humans is more complicated [113] . The main reason is that HLA genes in humans are extremely polymorphic. Therefore, the knowledge of HLA restriction in population, which will be vaccinated, is necessary. The complexity of HLA molecules could be reduced by clustering them into sets of molecules that bind largely overlapping peptides. Such clustering was introduced by Sette and Sidney in 1999. They defined HLA supertypes as a set of HLA molecules that have similar peptide binding motifs and overlapping peptide binding repertoires [117] . Nine different supertypes (A1, A2, A3, A24, B7, B27, B44, B58, B62) were defined on the basis of their specifity for the main anchor positions of presented peptides. Later, other three HLA I supertypes (A26, B8 and B39) were described by Lund et al. [118] . Recent analysis provided an update of HLA I alleles classification into supertypes and is expected to facilitate epitope identification and vaccine design studies [119] .
An example of most frequently recognized conserved epitopes of influenza antigens in humans represents M1 58-66 CTL epitop, which is restricted by the high prevalence allele HLA-A*0201 and could be a promising vaccine candidate [120] . Computer programs available today can predict binding epitopes of a given protein for the most common HLA allele [121, 122] . In silico analysis supports the proposition that the T-cell response to cross-reactive T-cell epitopes induced by vaccination or seasonal viral exposition may have the capacity to attenuate the course of influenza infection in the absence of cross-reactive antibody response [123, 124] . The ability to predict the CTL epitope immunogenicity and recognition patterns of variant epitopes enhances the probability of the optimal selection of potential targets of immune response and can be utilized for vaccine design [93, 113, 125] . In spite of the differences in various classification schemes, the concept of HLA supertypes has been effectively used to characterize and identify promiscuously recognized T-cell epitopes from a variety of different disease targets, as are those of hepatitis C virus [126, 127] , SARS [128] or HIV [129, 130] but also influenza virus [131] .
A critical requirement for CTL epitope-based strategy is to identify and select promiscuous CTL epitopes that bind to several alleles of HLA supertypes to reach maximal population coverage. The utilization of supertyperestricted epitopes, which bind with significant affinity to multiple related HLA alleles, provides solution to this problem [117] . As described before, 80-90% population coverage can be achieved in most prominent ethnicities by focusing on only three major HLA class I supertypes -A1, -A3 and -B7 [132, 133] . By including two additional supertypes (A1, A24), 100% population coverage in all major ethnicities could be reached [117, 132] . Recently, HLA class I -A2, -A3 or -B7 supertype-restricted epitopes conserved among different viral subtypes of influenza virus were identified, what could be of relevance for the development of a potential supertype-restricted, multiepitope CTL-based vaccine protective against any subtype of influenza virus [82, 103, 113, 134] .
One of the drawbacks of currently available inactivated vaccines is the lack of broad cross-protective humoral and cell-mediated immune response against any influenza virus. Their efficacy is limited due to the genetic variation of influenza viruses. Therefore, their annual reformulation is necessary in an attempt to antigenically match the currently circulating strain for each of the three vaccine strains or their subunits (HA and NA of H1N1 and H3N2 of influenza A virus as well as of influenza B virus) from which they are composed. Increasing amount of information about conserved epitopes of influenza viruses brings us closer to the development of the universal vaccine. Such vaccine should contain both, conserved B-cell epitopes that are important for induction of cross-protective antibodies and CTL epitopes for the involvement of the cellular arm of the immune response to the overall protective effect [90] . It was shown that the pre-existing memory T-cell immunity as defense against seasonal influenza strains may have the capacity to moderate the course of disease in the case of newly emerging flu viruses in the absence of cross-reactive antibody response [86, 93, 123, 124] . It was also shown that it would be possible to elicit the CTL response simultaneously directed against multiple supertype-restricted conserved CTL epitopes [135] [136] [137] [138] [139] . This could be relevant for the development of a potential supertype-restricted multiepitope CTL based vaccine, with the effort to reach wide population coverage. Even though recent reports support a beneficial role of T-cell response in reducing human infections [86] [87] [88] 124] , there are still many questions regarding the feasibility of designing an effective supertype-restricted CTL epitope based vaccine in humans. In addition to CTL epitopes, B-cell epitopes from conserved influenza antigens that can elicit cross-protective humoral response should also be considered as a component of novel vaccines. Recently, highly cross-reactive monoclonal antibodies directed against conserved epitopes of HA2 subunit, including fusion peptide, were identified [28, 30, 66, [68] [69] [70] . HA2 subunit region as well as M2 protein are promising candidates for design of vaccine constructs aimed at providing broad-spectrum immunity to influenza viruses [1, 28, 31, 37, 45] . Cross-protective potential of HA2 and eM2 could be increased by optimization of their delivery and immunogenicity using vaccine vectors that target multiple Toll-like receptors for efficient stimulation of innate immunity and subsequent enhancement of the adaptive immune response [41, 140] . Conserved B-and T-cell epitopes, thus, could represent the basis for preparation of universal vaccine and bring new hope for development of pandemic or universal influenza vaccine.  The intracellular dynamic of protein palmitoylation S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation. Cellular proteins undergo a vast array of dynamic and static modifications to their amino acid backbone, which can dramatically extend and regulate functional output. Some of these modifications, phosphorylation in particular, have been the subject of intensive research, and their regulation and intracellular functions are extensively characterized. In contrast, our understanding of S-palmitoylation (hereafter referred to as palmitoylation), the attachment of palmitate (C16:0) or other long chain fatty acids to cysteine residues, has lagged behind some of its more popular cousins. Indeed, enzymes that catalyze palmitoylation reactions have only recently been identified after landmark studies in yeast, settling a long-running debate as to whether this process was predominantly enzyme mediated or spontaneous (Lobo et al., 2002; Roth et al., 2002; Fukata et al., 2004; Keller et al., 2004) .
Palmitoylation is one of a group of lipid modifications (collectively termed lipidation) that appears on eukaryotic proteins and includes the common N-myristoyl and isoprenyl modifications. N-myristoylation describes the addition of myristic acid (C14:0) to a glycine residue with an exposed NH 2 group after cleavage of the immediately adjacent initiating methionine (Zha et al., 2000; Resh, 2006a) . This process is predominantly cotranslational, mediated by soluble enzymes, and has a strict consensus sequence (MGXXXS/T). N-myristoylation can also occur posttranslationally, notably after caspase-mediated protein cleavage during programmed cell death (Zha et al., 2000) . Prenylation is a posttranslational process also catalyzed by soluble enzymes, involving the attachment of farnesyl or geranylgeranyl isoprenoids to a C-terminal cysteine present within a defined consensus sequence (Wright and Philips, 2006) .
Unlike the catalysts of N-myristoylation and prenylation reactions, the enzymes that mediate palmitoylation are polytopic membrane proteins (Fukata et al., 2004; Mitchell et al., 2006) , implying that cellular palmitoylation reactions occur at the cytosol-membrane interface. There are 23 putative S-palmitoyl transferases in mammals, characterized by the presence of a DHHC (aspartate-histidine-histidine-cysteine) motif within an 50 amino acid cysteine-rich domain. The large number of these DHHC proteins coupled with their localization to distinct membrane compartments (Ohno et al., 2006) implies that the cellular palmitoylation machinery is a highly regulated and coordinated system.
There is no strict consensus sequence for palmitoylation, however, palmitoylated cysteines do share some common characteristics: (a) they are often adjacent to myristoylation and prenylation sites, (b) the surrounding amino acids tend to be basic or hydrophobic, and (c) they are frequently located in the cytoplasmic regions flanking transmembrane domains (or within transmembrane domains). An elegant combined proteomic and genetic analysis in yeast revealed that some DHHC proteins appear to exhibit a preference for a particular class of substrate, e.g., transmembrane proteins or myristoylated/ prenylated proteins (Roth et al., 2006) . Similarly, some palmitoylated yeast proteins displayed a marked dependence on a specific DHHC protein (of which seven are expressed in yeast). However, this study also revealed that DHHC proteins can have overlapping substrate specificities, which is consistent with previous studies in mammalian systems showing that specific substrates can be palmitoylated by more than one DHHC protein (Fukata et al., 2004; Fang et al., 2006; Fernández-Hernando et al., 2006; Greaves et al., 2008 Greaves et al., , 2010 Tsutsumi et al., 2009; Shmueli et al., 2010) .
S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation. hydrophobicity/membrane affinity and preference for ordered cholesterol-rich membrane domains or rafts (Melkonian et al., 1999; Brown, 2006) .
A central function of palmitoylation is the regulation of protein sorting (Greaves et al., 2009b) , and it might be predicted that this simple lipid modification would have a very limited repertoire of effects on this process. However, this is not the case, and palmitoylation has been shown to act as a highly versatile sorting signal, which regulates protein trafficking to many distinct intracellular compartments. Several studies have highlighted regulatory effects of palmitoylation on either retention or anterograde trafficking of proteins at the ER-Golgi or protein cycling within the endosomal/lysosomal system (Linder and Deschenes, 2007; Greaves et al., 2009b) . Recent studies have extended our knowledge of the functional effects of palmitoylation on protein sorting by highlighting novel roles for this modification in targeting to cilia and flagella (Emmer et al., 2009; Cevik et al., 2010; Follit et al., 2010) and to the cleavage furrow of dividing cells (Hannoush and Arenas-Ramirez, 2009) .
What is the underlying mechanistic basis for the effects of palmitoylation on protein sorting? For some soluble proteins, palmitoylation may be a passive sorting signal, acting only as an essential membrane anchor that allows other domains of the protein to regulate subsequent trafficking. However, palmitoylation also has active effects on protein sorting, achieved by partitioning of proteins into cholesterol-rich membrane subdomains or rafts (Levental et al., 2010) , changing protein orientation at the membrane and thus affecting protein-protein interactions (Hayashi et al., 2005; Lin et al., 2009) , regulating the ubiquitination status of proteins and thereby modulating ubiquitinationdependent protein sorting (Valdez-Taubas and Pelham, 2005; Linder and Deschenes, 2007; Abrami et al., 2008) , or affecting the transmembrane orientation of palmitoylated proteins in the membrane bilayer (Abrami et al., 2008) .
Palmitoylation often couples with either N-myristoylation or prenylation to regulate membrane interactions of soluble proteins. N-myristoylation or prenylation of proteins in the cell cytosol provides a degree of hydrophobicity, although single lipid modifications are only sufficient for transient membrane interaction (Shahinian and Silvius, 1995) . In contrast, two closely positioned lipid modifications promote stable membrane attachment (Shahinian and Silvius, 1995) . Thus, at the cellular level, single myristoyl or prenyl chains facilitate transient membrane association, which is sufficient to allow access to membranebound DHHC proteins; subsequent palmitoylation by DHHC proteins will then promote stable membrane binding by inhibiting membrane dissociation (kinetic trapping; Fig. 1 A) . In this way, palmitoylation is essential for stable membrane association of many proteins, including farnesylated Ras and myristoylated G subunits (Hancock et al., 1990; Linder et al., 1993; Parenti et al., 1993) . Several soluble lipidated proteins are modified exclusively by S-palmitoylation, and these proteins may use an intrinsic weak membrane affinity for transient membrane interaction before palmitoylation (Greaves et al., , 2009a .
Therefore, it is clear that a major function of palmitoylation is to mediate stable membrane attachment of soluble proteins. However, the effects of this posttranslational modification are more complex and diverse than that of a simple membrane anchor, and indeed, many transmembrane proteins are also palmitoylated. Analyses of different proteins and systems have revealed that palmitoylation has many distinct effects on modified proteins, regulating protein trafficking, protein stability, membrane microlocalization, and protein-protein interactions (Resh, 2006a,b; Greaves and Chamberlain, 2007; Linder and Deschenes, 2007; Greaves et al., 2009b; Noritake et al., 2009) . Although these effects of palmitoylation appear diverse, they are likely determined by two particular properties of palmitate: Figure 1 . Regulation of membrane binding and trafficking of peripheral proteins by palmitoylation. (A) Proteins modified with single lipid groups (prenylation or N-myristoylation; green circles) have a weak membrane affinity that allows transient membrane interaction. Palmitoylation by membrane-bound DHHC proteins promotes stable membrane association by kinetic trapping (Shahinian and Silvius, 1995) . Note that some peripheral proteins are exclusively palmitoylated, and these proteins were suggested to interact with membranes before palmitoylation by way of an intrinsic weak membrane affinity (Greaves et al., , 2009a . (B) Palmitoylation of Ras-farnesyl by Golgi-localized DHHC proteins leads to a dramatic increase in membrane affinity by kinetic trapping. This increased membrane residency facilitates entry of palmitoylated Ras (red circles) into transport vesicles that deliver it to the plasma membrane. It is possible that palmitoylation also serves to move Ras into cholesterol-rich domains from which Golgi exit vesicles are formed . Depalmitoylation of Ras can occur anywhere in the cell, perhaps modulated by Apt1, resulting in membrane release and cytosolic diffusion before repalmitoylation at the Golgi. For simplicity, the figure only depicts depalmitoylation occurring at the plasma membrane. This palmitoylation/depalmitoylation regulation of protein sorting is not specific for Ras proteins and may be a common mechanism underlying the sorting of many peripheral palmitoylated proteins (Kanaani et al., 2008; Tsutsumi et al., 2009; Rocks et al., 2010) .
this approach indicated a half-life for N-Ras palmitoylation of 20 min (Magee et al., 1987) and showed that palmitoylation of G subunits of heterotrimeric G proteins can be modulated by agonist stimulation (Degtyarev et al., 1993; Mumby et al., 1994; Wedegaertner and Bourne, 1994) .
Recently, the intracellular dynamics of palmitoylation and palmitoylation-dependent protein trafficking have been investigated using fluorescence imaging. The power of biochemical radiolabeling techniques comes from the ability to selectively follow the fate of a labeled pool of protein by pulse chase. In contrast, conventional confocal microscopy analysis of fluorescent proteins is not well suited to allow older proteins to be distinguished from newly synthesized proteins, static protein localizations to be differentiated from dynamic trafficking, and resolution of palmitoylated and unpalmitoylated proteins. However, these issues can be addressed by synchronizing the protein under investigation or by selective photoactivation/ photobleaching of a specific pool of the protein or by using visible membrane accumulation of soluble proteins as an indicator of palmitoylation. These techniques were successfully used in the landmark publications that detailed the palmitoylationdependent cycling pathway of Ras proteins (Goodwin et al., 2005; Rocks et al., 2005) .
Recent studies used microinjection of semisynthetic N-Ras as a means to study real-time spatiotemporal dynamics of palmitoylation and membrane targeting (Rocks et al., 2005 ; microinjection of fluorescent protein allows the analysis of a synchronized (i.e., chemically identical and with the same initial localization) pool of protein. This elegant approach involves coupling chemically synthesized farnesylated peptides encompassing the C-terminal membrane targeting domain of N-Ras to a Cy3-labeled protein consisting of the remainder of N-Ras (amino acids 1-181) via a maleimidocaproyl linker. By using these semisynthetic farnesylated proteins with intact or mutated palmitoylation sites, high-resolution temporal dynamics together with spatial information on cellular palmitoylation and depalmitoylation reactions were open to investigation.
Immediately after microinjection, farnesylated N-Ras displayed a dispersed localization, which is consistent with its presence in the cytosol and transient association with intracellular membranes . However, a rapid enrichment of the Cy3-labeled construct became apparent at the Golgi region (t/2 14 s) with plasma membrane staining visible at later time points. The simplest interpretation of these observations is that palmitoylation of the farnesylated N-Ras occurs at the Golgi, promoting early accumulation at this compartment, and is followed by anterograde transport to the plasma membrane. This notion agrees well with previous analyses of Ras protein cycling (Goodwin et al., 2005; Rocks et al., 2005) . Nevertheless, it should be noted that palmitoylation of the microinjected semisynthetic Ras protein was not directly assayed in this study. Although technically challenging, it will therefore be of major interest in follow-up studies to perform correlative measurements of palmitoylation and intracellular localization of microinjected Ras. In addition, a recently published study reported that palmitoylated Ras proteins traffic from Golgi to recycling endosomes en route to the plasma membrane (Misaki et al., 2010) .
In contrast to other static lipid modifications, the versatility of palmitoylation as a membrane interaction and protein sorting module is greatly enhanced by its reversibility. It has long been appreciated that the palmitoylation of many (but not all) proteins is dynamic (Magee et al., 1987) and can be modulated in response to cell stimulation (Degtyarev et al., 1993; Mumby et al., 1994; Wedegaertner and Bourne, 1994) . Although palmitoylation dynamics of transmembrane proteins can impact sorting to distinct membrane compartments, depalmitoylation of soluble proteins can also mediate membrane release and cytosolic diffusion. Thus, the rapid palmitoylation and depalmitoylation dynamics of many proteins add an extra level of complexity to the effects of this posttranslational modification on protein sorting.
The effects of palmitoylation-depalmitoylation dynamics have been extensively analyzed for palmitoylated Ras isoforms (Goodwin et al., 2005; Rocks et al., 2005; Roy et al., 2005) . Farnesylated H-and N-Ras exhibit a weak membrane affinity that mediates interaction with Golgi membranes. Palmitoylation of Ras at this compartment promotes stable membrane association and trafficking from the Golgi to the plasma membrane. Subsequent depalmitoylation releases Ras from the plasma membrane into the cytosol, and the process of palmitoylation at the Golgi and trafficking to the plasma membrane is repeated ( Fig. 1 B) . This palmitoylation cycle achieves a constant flux of Ras proteins from Golgi to plasma membrane and was suggested to prevent spillover of Ras onto other cellular membranes by constantly resetting the intracellular localization (Goodwin et al., 2005; Rocks et al., 2005) .
The Ras cycling pathway is an example of how constitutive palmitoylation dynamics coordinate protein sorting. For some proteins, palmitoylation turnover and corresponding changes in intracellular localization can also be regulated by cell activity. Protein palmitoylation dynamics that are subject to regulation have been particularly well characterized for postsynaptic proteins; palmitoylation of the molecular scaffold PSD95 and AMPA and NMDA glutamate receptors are all regulated by synaptic activity (El-Husseini et al., 2002; Hayashi et al., 2005 Hayashi et al., , 2009 ). PSD95 is a peripheral palmitoylated protein that coordinates protein clustering at postsynaptic sites. This protein was suggested to display a decreased palmitoylation in response to glutamate receptor activation, which corresponded with a reduced synaptic clustering of both PSD95 and AMPA receptors. Palmitoylation of GluR1/GluR2 subunits of AMPA receptors and NR2A/2B subunits of NMDA receptors is also modulated by synaptic activity. The palmitoylation status of GluR1 affects association with the 4.1N protein, regulating activity-dependent receptor internalization and plasma membrane insertion dynamics (Hayashi et al., 2005; Lin et al., 2009) . Dynamic palmitoylation of NR2 subunits also modulates cell surface expression of the NMDA receptor ). Thus, neurons use dynamic palmitoylation as a response mode to couple changes in synaptic activity to changes in protein localization.
Classically, palmitoylation dynamics have been studied using radiolabeled palmitate and pulse-chase protocols; for example, proteins or could instead reflect a different rate of palmitate turnover and subsequent Golgi recycling (Fig. 2) . Interestingly, proteins in this category, including Fyn, TC10, R-Ras, RhoB, and Rap2C all displayed colocalization with a Golgi marker when vesicular traffic through the secretory pathway was blocked by low temperature . This observation implies that after their synthesis, these proteins traffic via the Golgi and is consistent with the notion that the Golgi may be a specialized reaction center for the palmitoylation of all newly synthesized peripheral proteins and for a subset that undergo rapid depalmitoylation dynamics.
The suggestion that palmitoylation of newly synthesized peripheral proteins is a Golgi-specific event is largely consistent with previously published data. Although DHHC palmitoyl transferases associate with a range of organelles, including the ER, Golgi, and plasma membrane (Ohno et al., 2006; Greaves et al., 2010) , independent analyses of DHHC substrate pairs have returned a high percentage of Golgi DHHC proteins as positive hits (Fukata et al., 2004 Huang et al., 2004; Fernández-Hernando et al., 2006; Greaves et al., 2008 Greaves et al., , 2010 Tsutsumi et al., 2009) . Indeed, DHHC9 displays specificity toward H-and N-Ras (Swarthout et al., 2005) and is Golgi localized (Swarthout et al., 2005) . However, Rocks et al. (2010) argued against any rigid requirements for DHHC substrate specificity and reported that DHHC9 knockdown (at the mRNA level at least) did not affect the intracellular localization of H-Ras or recovery kinetics after photobleaching of Golgi-localized H-Ras. To examine more closely whether the palmitoylation domain of N-Ras contained a recognition motif for interaction with specific DHHC proteins, the amino acids around the Rocks et al. (2010) did not report association of microinjected Ras proteins with recycling endosomes, and it will be important to clarify this apparent discrepancy between these studies.
The progressive pattern of localization of microinjected Ras (i.e., dispersed → Golgi → plasma membrane) was also observed for semisynthetic constructs in which the N terminus of N-Ras (1-181) was ligated to either myristoylated peptides derived from G i1 or Fyn or to the N-terminal 20 amino acids from GAP43, all of which contain palmitoylation sites. This result suggests that palmitoylation of N-Ras at the Golgi is not dependent on farnesylation per se but may instead be a common feature of peripheral palmitoylated proteins with an underlying membrane affinity. Note that GAP43 lacks any other lipid modifications and had the slowest rate of Golgi accumulation (t/2 = 29 s); this protein may bind to membranes before palmitoylation via an intrinsic weak membrane affinity (Greaves et al., , 2009a . It will be particularly interesting in follow-up studies to examine whether microinjected full-length G i1 , Fyn, and GAP43 display identical intracellular localization dynamics to the peptide sequences that were fused to N-Ras. This issue is relevant because previous work has shown that regions distant from palmitoylation sites can have a marked influence on the specificity of interaction with DHHC proteins; for example, the yeast vacuolar protein Vac8 was palmitoylated specifically by Pfa3 in vitro, but the isolated palmitoylation domain from this protein was palmitoylated by all five yeast DHHC proteins that were examined (Nadolski and Linder, 2009) .
Many other palmitoylated peripheral proteins do not display obvious steady-state localization at the Golgi but instead associate with the plasma membrane and endosomal membranes (Adamson et al., 1992; Kasahara et al., 2007; Sandilands et al., 2007) . The absence of such proteins from the Golgi might indicate that they are not modified by Golgi-localized DHHC Figure 2 . Regulation of protein localization by palmitoylation dynamics. The illustration depicts three palmitoylated proteins that have different rates of depalmitoylation. In this context, the term depalmitoylation refers to the complete absence of palmitoyl groups on the protein. Rapid depalmitoylation is associated with an enriched steady-state localization on Golgi membranes. This is achieved by depalmitoylation promoting membrane release and subsequent palmitoylation by Golgi-specific DHHC proteins leading to an accumulation at this compartment. Rapid depalmitoylation prevents excessive accumulation on endosomes via vesicular trafficking from the plasma membrane. In contrast, proteins that have a slower rate of depalmitoylation are maintained on membranes for longer and reach endosomal membranes via the plasma membrane. Note that a slower depalmitoylation rate may be achieved by a relative resistance to thioesterases, and/or the presence of many palmitoylated cysteines, and/or palmitoylation by DHHC proteins beyond the Golgi. All of these situations would limit the amount of the protein in a completely depalmitoylated state. This slower rate of depalmitoylation and membrane release limits the steady-state distribution on Golgi membranes.
Ras palmitoyl transferase (Erf2) is localized to the ER (Bartels et al., 1999) . Thus, there currently appears to be marked differences in the palmitoylation pathways of mammalian and yeast Ras proteins, which might reflect reported differences in the intracellular trafficking itineraries of these proteins (Dong et al., 2003; Wang and Deschenes, 2006) .
A key aspect of membrane targeting and cycling of dually lipidated proteins such as H/N-Ras, and the compartment where palmitoylation occurs, is the membrane interaction dynamics of the monofarnesylated protein. It is often implied that peripheral proteins bind to all or any intracellular membrane before palmitoylation; but is this the case? Does farnesylated Ras have an equal affinity for all intracellular membranes and, therefore, an unbiased membrane sampling before palmitoylation? In vitro experiments suggest that this may not be the case, as membrane interactions of farnesylated peptides are affected by membrane lipid composition (Gohlke et al., 2010) . Binding of farnesylated N-Ras to liquidordered membranes containing saturated phospholipids and cholesterol was reduced compared with liquid-disordered membranes made from an unsaturated phospholipid. These findings are particularly relevant given the high concentration of cholesterol in the plasma and endosomal membranes compared with membranes such as the ER (Mondal et al., 2009) . Semisynthetic proteins containing a single farnesyl or myristoyl group were suggested to interact with the plasma membrane based on total internal fluorescence microscopy analysis ; however, there was no indication of whether this interaction occurred with the same efficiency as interaction with ER-Golgi membranes. Visual inspection of the localization of farnesylated Ras proteins and peptides expressed in cells via plasmid transfection reveals clear ER and Golgi staining, whereas interaction with other membrane compartments (including the plasma membrane) is less obvious (Choy et al., 1999; Rocks et al., 2005 Rocks et al., , 2010 . Therefore, peripheral proteins may display a preference for interaction with specific membrane compartments rather than binding randomly to any intracellular membrane. This would clearly provide palmitoylation-dependent protein cycling with more specificity and directionality. The precise membrane interaction dynamics of peripheral proteins before palmitoylation therefore merits further high-resolution analysis.
Although the Golgi appears to function as a hub for palmitoylation of newly synthesized and cycling peripheral proteins, certain proteins undergo dynamic palmitoylation remodeling without accessing the Golgi. This suggests that active DHHC proteins are localized in post-Golgi compartments. Indeed, DHHC2 and DHHC5 associate with the plasma membrane in neuroendocrine cells (Greaves et al., 2010) , and these proteins are present on post-Golgi membranes in neuronal dendrites and at the postsynaptic density Li et al., 2010) . SNAP25, a multiply palmitoylated peripheral protein, is modified by Golgi-localized DHHC proteins (Fukata et al., 2004 Huang et al., 2004; Greaves et al., 2009a Greaves et al., , 2010 , which palmitoylation site were changed to their stereoisomeric (i.e., D-amino acids) counterparts. The insertion of these D-amino acids, which was predicted to disrupt any specific protein-binding site, had no major effect on Golgi accumulation of the microinjected proteins . Further analysis suggested that there was also not an essential DHHC recognition domain in the remainder of the Ras protein. These observations suggest that the DHHC proteins that modify Ras do not require a specific signature of the palmitoylated domain or upstream region and implies that palmitoylation of peripheral proteins may not be restrained by tight enzyme substrate specificities; the major requirement for palmitoylation presumably being a suitable cysteine in close membrane proximity. However, this idea is not readily consistent with other studies that have highlighted features of both DHHC proteins and substrate proteins that contribute to specificity of interaction (Greaves et al., 2009a (Greaves et al., , 2010 Huang et al., 2009; Nadolski and Linder, 2009) or that identified a requirement for a specific DHHC protein for palmitoylation of a specific substrate (Roth et al., 2006; Ohyama et al., 2007; Stowers and Isacoff, 2007; Emmer et al., 2009; Huang et al., 2009; Noritake et al., 2009; Tian et al., 2010) . Indeed, depletion of a single DHHC protein, Erf2, had a marked impact on the palmitoylation of yeast Ras (Bartels et al., 1999; Roth et al., 2006) . Further work is clearly required to delineate the reasons for these apparent inconsistencies.
If the Golgi really is a super-reaction center for palmitoylation of peripheral proteins in mammalian cells, it will be of great interest to determine how this is achieved. Are Golgilocalized enzymes highly efficient? Are cofactors required for palmitoylation enriched at the Golgi? Do peripheral proteins interact in a slightly different way with Golgi membranes, making them more susceptible to palmitoylation? Most peripheral palmitoylated proteins associate with post-Golgi membranes (particularly the plasma membrane) after palmitoylation. Thus, specific palmitoylation at the Golgi might be an important prerequisite to ensure plasma membrane trafficking. A particular feature of palmitate that might facilitate Golgi to plasma membrane trafficking is its affinity for cholesterol-rich lipid raft domains (Melkonian et al., 1999; Levental et al., 2010) . It was recently proposed that raft domains might act as platforms for vesicle budding from the Golgi , and thus, palmitoylation-dependent association with these domains would be predicted to promote protein traffic to the plasma membrane. In support of the idea that palmitoylation at the Golgi is important for correct trafficking of peripheral proteins, we recently identified a connection between the palmitoylation of cysteine-string protein (CSP) at the Golgi and its subsequent sorting . A mutant form of CSP with an enhanced membrane affinity associates with ER membranes and is only palmitoylated when ER and Golgi membranes are mixed by applying brefeldin A . Interestingly, however, after washout of brefeldin A, the Golgi recovers but the CSP mutant remains trapped at the ER , perhaps reflecting a disconnect between forward transport of peripheral palmitoylated proteins and the lipid environment of ER membranes (cholesterol poor).
Although the Golgi has been highlighted as a possible hub for palmitoylation of peripheral mammalian proteins, the yeast
In contrast to the wealth of information available on palmitoylating enzymes, our understanding of the proteins that regulate protein depalmitoylation is poor. Two main candidate thioesterases have been identified. Protein palmitoyl thioesterase 1 (Ppt1) depalmitoylates H-Ras and different G subunits in vitro (Camp and Hofmann, 1993) . Although there are reports that a cytosolic pool of Ppt1 may be present in cells (Kim et al., 2008) , this protein is thought to be predominantly localized to the lysosomal lumen (Hellsten et al., 1996) , where it is believed to function in depalmitoylation reactions occurring during protein degradation. Acyl protein thioesterase 1 (Apt1) reportedly displays thioesterase activity toward G i  1 , H-Ras, eNOS, and certain viral proteins (Duncan and Gilman, 1998; Yeh et al., 1999; Veit and Schmidt, 2001) but is inactive against other proteins such as caveolin (Yeh et al., 1999; Veit and Schmidt, 2001) . Importantly, Apt1 has a cytosolic localization, suggesting that it can regulate cellular palmitoylation dynamics. In support of this idea, overexpression of Apt1 into HEK293 cells was reported to increase the rate of removal of radiolabeled palmitate from G s  in pulse-chase experiments (Duncan and Gilman, 1998) .
Despite Apt1 being identified many years ago, the physiological importance of this protein as a thioesterase is not clear. However, a recent study reported an important function for Apt1 in controlling dendritic spine volume, possibly by regulating palmitoylation and membrane localization of G 13 (Siegel et al., 2009 ). The recent description of a novel Apt1 inhibitor (palmostatin B) should provide an important tool to more finely dissect the function of this protein in cellular palmitoylation dynamics (Dekker et al., 2010) . Initial analysis with palmostatin B suggests that it promotes a moderate increase in Ras palmitoylation and disrupts the intracellular localization of this protein.
Although our understanding of the mechanisms of depalmitoylation is limited, the spatiotemporal dynamics of this process were investigated by microinjection of semisynthetic N-Ras containing both farnesyl and palmitoyl chains . Previous analysis of an N-Ras protein in which the palmitoyl group was attached by a noncleavable thioether linkage revealed a dispersed intracellular localization without Golgi enrichment (Rocks et al., 2005) . This localization was suggested to reflect a requirement for active palmitoylation/ depalmitoylation cycling to achieve the correct localization of Ras. The palmitoylated protein with a cleavable thioester bond was therefore not expected to display any initial membranetargeting specificity. Despite this, the construct rapidly accumulated at the Golgi (t/2 = 27 s). This Golgi accumulation was suggested to follow on from binding of the farnesylated/ palmitoylated protein to any membrane, depalmitoylation, cytosolic diffusion, and subsequent repalmitoylation at the Golgi. There were two main interpretations made from this behavior of farnesylated and palmitoylated N-Ras: (1) depalmitoylation must be very rapid to account for the speed of Golgi accumulation and (2) depalmitoylation must occur throughout the cell because if it was confined to a specific location, association of the farnesylated and palmitoylated protein with some membranes would be irreversible.
promote stable membrane attachment of SNAP25 (Greaves et al., 2009a) . Recent work also reported that SNAP25 can be palmitoylated by DHHC2 but that this enzyme is unable to promote stable membrane attachment (Greaves et al., 2010) . This observation is consistent with the idea that palmitoylation of newly synthesized SNAP25 (which promotes membrane association) is restricted to the Golgi and that modification by post-Golgi DHHCs is only relevant once SNAP25 has been trafficked from Golgi to plasma membrane. This may reflect a weaker association between SNAP25 and DHHC2, such that productive interaction can only occur when SNAP25 is stably membrane associated. However, it is also consistent with the idea that unpalmitoylated SNAP25 may have a higher affinity for Golgi membranes than the plasma membrane, again reinforcing the importance of membrane affinities and preferences of peripheral proteins before palmitoylation (see previous paragraph).
In hippocampal neurons, DHHC2 is associated with mobile dendritic vesicles of unknown origin, and total internal reflection microscopy suggested that inhibition of synaptic activity promotes an increase in DHHC2 levels either at or just beneath the plasma membrane . Interestingly, this movement of DHHC2 correlates with enhanced palmitoylation and synaptic clustering of PSD95 (El-Husseini et al., 2002; Noritake et al., 2009) , and depletion of DHHC2 was reported to inhibit the increase in synaptic clustering of PSD95 after synaptic blockade. This work illustrates that dynamic palmitoylation can be achieved without peripheral proteins (such as PSD95) visiting the Golgi. It is interesting to note that depletion of DHHC3, which is localized to the somatic Golgi, also inhibited synaptic accumulation of PSD95. However, in contrast with DHHC2 depletion, knockdown of DHHC3 had no effect on the activity-dependent increase in synaptic clustering of PSD95 . This suggests that Golgi-localized DHHC3 is involved in the initial palmitoylation of newly synthesized PSD95, before dendritic targeting.
CSP, an important neuroprotective DnaJ chaperone, is palmitoylated by Golgi-localized DHHC enzymes (DHHC3, DHHC7, DHHC15, and DHHC17; Greaves et al., 2008) . In this regard, CSP is similar to most other peripheral palmitoylated proteins. Consistent with the analyses of CSP palmitoylation in mammalian cells, disruption of DHHC17 in Drosophila melanogaster resulted in a loss of palmitoylation and mislocalization of CSP (Ohyama et al., 2007; Stowers and Isacoff, 2007) . Surprisingly, however, DHHC17 does not exhibit a Golgi localization in Drosophila neurons but, instead, has a presynaptic distribution on synaptic vesicles or at the presynaptic plasma membrane (Ohyama et al., 2007; Stowers and Isacoff, 2007) . Although palmitoylation cycles have not been reported for CSP, it is possible that DHHC17 is important for regulating local palmitoylation dynamics of CSP in Drosophila presynaptic terminals.
Finally, there is also strong evidence to show that peripheral membrane proteins can undergo palmitoylation beyond the confines of the Golgi in yeast cells. As discussed earlier, Ras is modified by ER-localized ERF2 (Bartels et al., 1999) , and palmitoylation and membrane association of the yeast vacuolar fusion protein Vac8 are also markedly reduced after depletion of the DHHC protein Pfa3, which is localized to the vacuole membrane (Hou et al., 2005; Smotrys et al., 2005) . modulated by distinct posttranslational modifications present on the target protein.
Interplay between palmitoylation and phosphorylation was recognized many years ago for the  2 -adrenergic G proteincoupled receptor (Moffet et al., 1993) . Mutation of the palmitoylated cysteine in the C-terminal tail of this receptor led to an increased level of basal phosphorylation and a loss of coupling to Gs (O'Dowd et al., 1989; Moffet et al., 1993) . The effects of mutating the palmitoylation site were not caused by a loss of palmitoylation per se but rather by the increased phosphorylation of the palmitoylation-deficient mutant (Moffett et al., 1996) . Palmitoylation-phosphorylation interplay has also been reported to regulate trafficking of AMPA and NMDA receptor subunits Lin et al., 2009) . For GluR1 subunits of AMPA receptors, depalmitoylation was suggested to enable the more-efficient phosphorylation of neighboring serine residues. This phosphorylation in turn increased the interaction of GluR1 with 4.1N protein, which modulated plasma membrane internalization and insertion dynamics of AMPA receptors (Lin et al., 2009) . How does palmitoylation regulate phosphorylation? One potential mechanism is membrane insertion of palmitoylated cysteines restricting access of protein kinases to the adjacent phosphorylation sites (Fig. 3 A) .
If palmitoylation can regulate phosphorylation, can phosphorylation regulate palmitoylation? The STREX variant of BK potassium channels contains a PKA phosphorylation site within the cytoplasmic C-terminal tail that mediates channel inhibition (Tian et al., 2001) . A recent study reported that the STREX variant also contains palmitoylated cysteines adjacent to the PKA If depalmitoylation is not restricted to a specific membrane compartment, this suggests (a) a common depalmitoylase with access to many membranes (consistent with the cytosolic localization of Apt1), (b) a group of depalmitoylating enzymes with wide membrane compartment coverage, or (c) nonenzymatic depalmitoylation.
The same spatiotemporal pattern of localization was observed for a farnesylated/palmitoylated protein containing D-amino acids at the palmitoylation site , suggesting that depalmitoylation does not require a specific recognition sequence around this region. How would a cytosolic thioesterase like Apt1 recognize membrane-embedded thioester linkages without the presence of a defined consensus sequence? It is clear that much more research on Apt1 is required and that the identity of novel inhibitors will greatly facilitate this. One interesting angle is to determine whether the turnover of palmitate on cellular proteins correlates with the ability of Apt1 to promote depalmitoylation in vitro. For example, caveolin is not a substrate of Apt1 in vitro and does not undergo rapid dynamic palmitoylation in cells (Parat and Fox, 2001) , and the reverse is obviously true for proteins such as eNOS and Ras.
Interplay between palmitoylation and other posttranslational modifications DHHC proteins are clearly master regulators of intracellular palmitoylation reactions, and thioesterases (such as Apt1) may be equally important. However, are these enzymes the only means of regulating palmitoylation? In fact, there is evidence that palmitoylation/depalmitoylation dynamics can also be (1) Negatively charged phosphate group prevents palmitoylation of an adjacent cysteine by blocking membrane interaction. (2) Palmitoylation-mediated membrane association prevents access of protein kinases to an adjacent phosphorylation site. (3) Phosphorylation could alter the depalmitoylation rate of a neighboring cysteine, e.g., by increasing access to a thioesterase enzyme. (B) Phosphorylation of a soluble protein prevents palmitoylation by inhibiting transient membrane interaction. (C, 1) Possible regulatory effects of nitrosylation on palmitoylation. Nitrosylation may prevent palmitoylation by direct competition for cysteine residues. (2) It is also possible that nitrosylation could directly displace palmitate. Note that the examples shown do not illustrate the full range of effects that phosphorylation might have on palmitoylation and vice versa.
will be more physiologically relevant for palmitoylated proteins that interact either directly or indirectly with NOS enzymes.
Landmark studies in yeast highlighted the DHHC protein family as the catalysts of intracellular membrane fusion reactions and provided an essential spark to the palmitoylation field. This, together with the continuing development of new methodologies and reagents, has served as a platform for rapid expansion in our understanding of the mechanisms, spatiotemporal dynamics, and outcomes of protein palmitoylation. Techniques such as acyl-biotin exchange and click chemistry have offered highly sensitive alternatives to radiolabeling for the study of cellular palmitoylation (Drisdel and Green, 2004; Martin and Cravatt, 2009; Yap et al., 2010) . These approaches also permit the palmitoylation status of proteins to be studied in situ without cell labeling (acyl-biotin exchange) or the analysis of spatial patterns of palmitoylated proteins by fluorescence imaging (Hannoush and Arenas-Ramirez, 2009 ). Furthermore, both techniques have facilitated analysis of the cellular palmitoylome in both yeast and mammalian cells (Roth et al., 2006; Kang et al., 2008; Martin and Cravatt, 2009 ). The recent development of Apt1 inhibitors is an important step toward further delineating the function of this protein and developing an enhanced understanding of cellular depalmitoylation dynamics (Dekker et al., 2010) . It is expected that further technological developments will maintain the rapid pace of palmitoylation research. In particular, the development of more specific and selective inhibitors against the DHHC protein family (Resh, 2006b) will be a key to delineating the individual functions of these proteins and their contribution to the spatiotemporal dynamics of cellular palmitoylation reactions. site that regulate plasma membrane binding of the cytosolic C terminus (Tian et al., 2008) . Introduction of a phosphomimetic mutation at the PKA phosphorylation site or activation of cellular PKA perturbed palmitoylation-dependent membrane association of the C-terminal tail of STREX. Importantly, the full-length channel lacking the palmitoylated cysteines in the STREX domain was no longer subject to PKA-mediated inhibition, implying that phospho-regulation of the STREX channel is achieved via changes in palmitoylation and membrane association of the cytoplasmic C terminus.
Phosphorylation also appears to regulate palmitoylation of the cyclic nucleotide phosphodiesterase (PDE) isoform PDE10A2 (Charych et al., 2010) . The N terminus of PDE10A2 contains a palmitoylated cysteine residue (Cys11) that is essential for membrane anchoring and efficient dendritic transport in striatal neurons. Palmitoylation was perturbed when phosphomimetic mutations were introduced into a downstream PKA phosphorylation site . Does phosphorylation at Thr-16 actively promote depalmitoylation of Cys-11, or does it block palmitoylation of this cysteine? In fact, PKA activation markedly enhanced PDE10A2 phosphorylation but had no acute effect on membrane expression levels. This observation suggests that phosphorylation does not promote depalmitoylation and membrane release of PDE10A2 but, instead, likely inhibits membrane binding by blocking palmitoylation. This phosphoregulation of palmitoylation might be relevant to many palmitoylated peripheral proteins and could represent a mechanism to promote a shift toward the depalmitoylated state of a protein in the absence of active (thioesterase driven) depalmitoylation. Negatively charged phosphate groups are likely to inhibit palmitoylation of neighboring cysteines by interfering with membrane interactions before palmitoylation (Fig. 3) .
Another posttranslational modification that may impact palmitoylation dynamics is nitrosylation. Nitric oxide (NO) is produced from l-arginine by NO synthase enzymes (NOS) and can directly modify cysteines by S-nitrosylation (Stamler et al., 1992) ; this modification might therefore regulate palmitoylation dynamics by direct competition. The NO donor SIN-1 inhibited the basal level and the isoproterenol-stimulated increase in palmitate incorporation into 2 adrenergic receptor (Adam et al., 1999) . Palmitate incorporation into H-Ras, caveolin, SNAP25, and certain viral proteins has also been reported to be modified by NO donors (Hess et al., 1993; Baker et al., 2000; Akerström et al., 2009) . Indeed, the NO donor S-nitrosocysteine accelerated removal of radiolabeled palmitate from H-Ras in pulsechase experiments (Baker et al., 2000) , raising the intriguing possibility that NO may directly displace palmitate from modified proteins (Fig. 3 C) .
Overall, there is sufficient published data to suggest that NO may be an important regulator of palmitoylation dynamics. The development of more sensitive techniques to directly study nitrosylation is required to more rigorously delineate interplay between this modification and palmitoylation. It will be particularly important in future studies to determine how palmitoylation dynamics are affected by endogenously produced NO. The intracellular diffusion range of NO from its site of production is limited, and thus, interplay between palmitoylation and nitrosylation The impact of sex, gender and pregnancy on 2009 H1N1 disease Children and young adults of reproductive age have emerged as groups that are highly vulnerable to the current 2009 H1N1 pandemic. The sex of an individual is a fundamental factor that can influence exposure, susceptibility and immune responses to influenza. Worldwide, the incidence, disease burden, morbidity and mortality rates following exposure to the 2009 H1N1 influenza virus differ between males and females and are often age-dependent. Pregnancy and differences in the presentation of various risk factors contribute to the worse outcome of infection in women. Vaccination and antiviral treatment efficacy also vary in a sex-dependent manner. Finally, sex-specific genetic and hormonal differences may contribute to the severity of influenza and the clearance of viral infection. The contribution of sex and gender to influenza can only be determined by a greater consideration of these factors in clinical and epidemiological studies and increased research into the biological basis underlying these differences. Sex and gender differences can affect exposure to pathogens, vulnerability to infectious diseases, health seeking behaviours and immune responses to pathogens, resulting in differences between males and females in the incidence, duration, severity and case fatality rates following an infection [1, 2] . Sex refers to the biological and physiological characteristics that define males and females, whereas gender refers to the roles, behaviours, activities and attributes that individual societies consider appropriate for men and women. The impact of sex and gender on infection is tied to the age of the individual, as both biological and cultural factors can change dramatically with age. Consideration of these factors can result in a more effective public health response to infectious diseases, including influenza, and yet they are often inadequately addressed in clinical and basic research studies. A systematic review of the literature regarding sex, gender, pregnancy and the 2009 H1N1 pandemic indicates these are important factors which alter the severity of the disease as well as the prevention and treatment measures. A greater awareness of how sex and gender impact upon the biology of 2009 H1N1 infection could provide important insights into the unique morbidity and mortality patterns associated with this pandemic.
Virus An influenza pandemic was declared by the World Health Organization (WHO) in June 2009 and the virus, 2009 H1N1, became the primary influenza virus strain isolated from humans by the end of the winter influenza season in the southern hemisphere [3] . It was the dominant influenza A virus strain circulating in the northern hemisphere for the entire influenza season, effectively outcompeting both seasonal influenza A virus strains [3] .
The pandemic has been termed mild due to the relatively low mortality. Confirmed influenza virus infections, however, have increased substantially compared to recent years and the US Center for Disease Control (CDC) estimates of the number of people infected with 2009 H1N1 are greater than what would be expected in a standard influenza season [3] .
Most cases of severe disease and mortality after infection with seasonal influenza A virus occur in the ≥65 years population. In contrast, 2009 H1N1 has not been associated with a large number of infections in this age group but has the highest attack and hospitalization rates in individuals between the ages of 0-40. The reduced number of cases in those aged ≥65 stems in part from the fact that antibodies generated to pre-1950 H1N1 viruses cross react with 2009 H1N1, resulting in limited protection from 2009 H1N1 infection [3] .
Presence of co-morbidities or risk factors for severe disease Several populations are at risk for severe disease from seasonal as well as 2009 H1N1 infection [4] , including individuals who have pre-existing illnesses or medical conditions, pregnant women, immunosuppressed individuals (either through treatment, HIV infection, or as a result of a pre-existing immunosuppressive disorder) and children aged 0-4 years. Medical conditions associated with an increased risk of severe disease include chronic respiratory disorders (for example, asthma, bronchitis, chronic obstructive pulmonary disease [COPD] and cystic fibrosis), neuromuscular disorders (for example, cerebral palsy, myasthenia gravis and muscular dystrophy), metabolic diseases (for example, diabetes) and chronic renal, heart or liver disorders [3] . Factors such as obesity and hypertension are not normally associated with severe disease from seasonal influenza but have been suggested as risk factors for severe disease from 2009 H1N1 in some studies [5] [6] [7] .
The protective immunity induced by influenza vaccinations is mediated primarily by antibodies that recognize the viral haemagglutinin protein and neutralize virus infectivity. After virus infection, host innate immune responses, including production of cytokines and chemokines, are activated which initiate a cascade of immunological events that lead to the development of specific immune responses to the virus. Controlling and clearing influenza virus infection requires neutralizing antibodies and cell-mediated immunity (for example, activation of T cells) [3] . The influx of immune cells into an influenza-infected lung can lead to the overproduction of various cytokines and chemokines -often called a 'cytokine storm' -which can enhance the virus-induced lung damage resulting in severe illness. A limited number of studies suggest that an altered cytokine and chemokine response is contributing to severe 2009 H1N1 disease [8, 9] . Therefore, immunity to influenza viruses represents a balance between immune responses inducing protection and clearance of virus versus causing pathology.
Utilizing published observational reports of patients with confirmed 2009 H1N1 infection and those admitted into intensive care units worldwide, the incidence, severity and case fatality rates following infection appear to differ between males and females, but often are age-dependent and vary between countries. The outcome of infection with 2009 H1N1 is generally worse for females, but the magnitude of this difference varies across geographical regions.
Assessments of male-female differences in reported incidences of infection is confounded by two factors: (1) many countries do not disaggregate data by both sex and age which may mask sex differences among the age groups that are most likely to be exposed -children and young adults; and (2) the profound differences in health seeking behaviours between males and females [10] .
Household transmission studies of children and adults reveal that being female (female relative risk [RR]: 1.87, 95% confidence interval [CI]: 1.17-2.73) is a significant factor associated with higher secondary attack rates of influenza-like illness, with attack rates being higher among children and young adults than older adults (>55 years of age) [11] . Reported male-female differences in the incidence of infection vary with age in several countries, with a higher incidence of infection with 2009 H1N1 in young women than young men of comparable age [12] [13] [14] [15] . While pregnancy has been clearly linked with increased disease severity, the vast majority of infected females of reproductive ages are not pregnant, suggesting that additional factors are contributing to the increased incidence of infection. In contrast, in Asia, the majority of reported H1N1 cases have been male (57.1%) [16, 17] . In China, males (male odds ratio [OR]: 1.94, 95% CI: 1.07-2.66) also shed the 2009 H1N1 virus in pharyngeal and nasopharyngeal samples for a longer duration than females [18] suggesting that the transmission potential may be higher in males. Other countries reported no male-female differences in the number of cases of 2009 H1N1, but did not analyse the data stratified by both age and sex [19] [20] [21] [22] [23] [24] [25] .
One trend that appears consistent across more than 60% of the datasets evaluated is that more females are hospitalized with critical illness than males ( Figure 1 ). The first cases in the USA were in California (April-May 2009), where the a majority of hospitalized cases (21/26) were women, five of whom were pregnant [26] . Initial analyses of data from critically ill patients in the USA during the first wave reported no male-female difference [27] , but subsequent state-specific reports from the first and second waves illustrated differences between the sexes [28] [29] [30] . In Canada, a significant majority of critically ill patients have been young women (female RR: 1.3, 95% CI: 1.0-1.6) [5, 31] . Other countries also report that rates of hospitalization have been higher among females than males, with a majority of the females being of reproductive age (15-49 years of age) [14, [32] [33] [34] [35] [36] . Analyses of cohorts of patients in Mexico and Australia/ New Zealand revealed a trend for more females than males being hospitalized [6, 37] . Evaluation of these differences in some countries is confounded by age, as many studies do not report male-female differences according to age group [6, 27, 37] .
An examination of sex differences disaggregated by age is needed in larger, more complete datasets. The reason for the greater proportion of hospitalized women is not known, but many cases involve co-morbid conditions, including chronic respiratory diseases (for example. asthma and COPD), which are often more severe in females [38] [39] [40] .
Mortality from 2009 H1N1 is not common but data from South Africa, where the incidence of co-infection with HIV and tuberculosis is high, reveal that 65% of fatal cases were females of reproductive age, of whom almost half were pregnant [41] . RR of death is higher for young adult women (female RR: 1.5, 95% CI: 0.9-2.3) than men in Canada [31] . In Australia, 58% of fatal cases were male [35] and in Brazil and Peru case fatality rates have been equal between males and females [14, 20] . No consistent pattern of male-female differences in mortality from the 2009 H1N1 has emerged.
Increased morbidity and mortality in pregnant women has been documented during influenza pandemics and influenza seasons where virus infection rates are particularly high [42] .
Pregnant women represent a disproportionately higher percentage of severe cases with the increased risk ranging from four-to 10-fold greater compared with the general population ( Figure 2 ). Increased morbidity and mortality in pregnant women has been reported in many datasets [5, 14, 27, 28, 36, 37, 41, [43] [44] [45] . The disease course and clinical presentation [46] [47] [48] has been studied and comparisons of disease in pregnant women to age-matched non-pregnant women [37, 49, 50] or to the general population [51] have been made. Disease severity is increased during the second and third trimester. However, no clear clinical parameter has been associated with pregnancy-associated increased morbidity and mortality. There are no significant differences in the general symptoms of disease, the progression to viral pneumonia, acute respiratory distress syndrome (ARDS) or secondary bacterial pneumonia in pregnant women compared to the control populations. Severe disease was also associated with a greater than sixfold increase in adverse neonatal outcomes when compared with pregnant women suffering mild disease [49] . An increased risk of severe disease may be present during the early postpartum period but the reported number of cases is limited and requires additional investigation [37, 50] . Female to male ratios of hospitalization with confirmed 2009 H1N1 were calculated using published datasets [5, 6, 14, 16, [27] [28] [29] [30] [32] [33] [34] [35] [36] [37] 43, [134] [135] [136] . Pink bars = higher rates of hospitalization in females; blue bars = higher rates of hospitalization in males; grey bars = similar hospitalization rates in males and females. Details about sample sizes, time of data collection, and criteria for hospitalization are contained within each individual reference.
Pregnancy itself is considered a risk factor for severe disease [3] but the biological basis for this has not been established. Pregnancy-associated changes in immune function, hormone levels, cardiopulmonary stress and difficulties in treatment for respiratory disease are often cited as important factors [52] . The presence of other risk factors may increase the risk of severe disease in a pregnant woman (Figure 3) . The presence of a known co-morbidity in pregnant women with severe 2009 H1N1 disease can vary greatly and has been documented as 16% to 56% [37, [46] [47] [48] [49] [50] 53] . There is no one or cluster of co-morbidities associated with increased disease severity in pregnant women. Data indicate that when no additional co-morbidities were present, pregnant women still had a seven-to tenfold higher rate of severe disease when compared to age-matched, nonpregnant women [49, 50] .
Certain risk factors predispose patients to increased morbidity and mortality following exposure to influenza viruses [54] and the severity and prevalence of these underlying conditions often differ between males and females ( Figure 4 ). The 2009 H1N1 virus causes disproportionate disease among young adults, a population that has a distinct repertoire of risk factors associated with exposure and worse outcome following infection compared with very young or old.
Healthcare workers, as well as those in frequent contact with young children, are at a higher risk of exposure to influenza viruses than the general public [55] . Women represent over 50% of the healthcare workforce in many countries and nurses, teachers of young children and day-care workers are predominantly female [10] which potentially leads to a gender-specific occupational risk for influenza acquisition.
Hand hygiene compliance, one of the most effective ways to prevent transmission of influenza, is significantly better among female than male (male OR: 0.6, 95% CI: 0.4-0.98) healthcare workers [56] . Among healthcare workers in the USA, self-reported rates of use and knowledge about appropriate personal protective equipment in response to influenza are similar between the sexes [57] .
Differences in health seeking behaviour or healthcare access may impact both the acquisition and manifestation of influenza. A WHO survey in 59 countries from 2002-2004 revealed that adult women are more likely to seek healthcare in both higher and lower income countries [10] . The quality of care for women in some parts of the developing world is not equal to that received by men [58] . In some developing countries, knowledge of the pandemic was higher among men than women, which might reflect the fact that there are greater educational opportunities and greater chances of socialization for men [59] .
Chronic medical conditions predispose patients to increased influenza-related morbidity [54, 60] and malefemale, as well as pregnancy-associated, differences in disease prevalence have been reported.
(1) Respiratory disease Asthma has been a significant underlying condition in children and adults hospitalized with critical illness [27, 61] . Data from the USA and Canada illustrate that, prior to puberty, boys have more asthma exacerbations than girls. However, this trend is reversed in adulthood [39] . Rates of asthma attacks, numbers of asthma-related emergency room visits, numbers of asthma-related hospitalizations and duration of hospitalization are higher in women than men in the USA [39, 62] . Rates of asthma, as well as incidence of asthma attacks, appear to be the same in pregnant and age-matched non-pregnant women [63] .
Cystic fibrosis and COPD have been identified as risk factors for severe illness with 2009 H1N1 [3] and the [27] , Chicago, IL, USA [28] , California, USA [43] , New York, USA [49] , Australia and New Zealand [37] , Canada [5] and Brazil [14] . Estimates of the general population and pregnant woman are based on data from the US Census Bureau or the World Health Organization.
progression of cystic fibrosis and long-term survival is significantly worse for females than males, especially among individuals diagnosed in childhood [64] . Females with COPD report worse symptoms, lower exercise capacity, more airway hyper-responsiveness and worse health-related quality of life than males [65, 66] . Although morbidity from these conditions may be worse in females, mortality -both from all causes and from respiratory-related disease alone -is still higher in males with COPD [65] , illustrating the complexities involved in assessing the significance of sex and gender for a particular co-morbidity.
(2) Hepatic disease Chronic hepatic disease is a risk factor for severe 2009 H1N1 disease [3] . The development of hepatocellular carcinoma occurs at a 2:1 to 4:1 ratio for males to females [67] . The prevalence of serum hepatitis B virus (HBV) is consistently higher in men than women [68] . Males are more than twice as likely to die from liver cancer, which suggests that men may be more sensitive to the effect of HBV infection on the development of liver cancer [69] . Men also are twice as likely to develop cirrhosis [70] .
(3) Cardiovascular disease The rates and severity of cardiovascular disease differ between the sexes and these differences have been evaluated in the elderly [71] . As they have not been identified as an at-risk population for severe disease from 2009 H1N1 influenza, sex differences in cardiovascular disease may not be a critical factor. (4) Metabolic disorders Diabetes and morbid obesity have emerged as novel risk factors for severe 2009 H1N1 disease [3] . The lifetime risk of diabetes is higher in women than men, at least in the USA where approximately 55% of all diabetic-related deaths are women which may be a reflection of the fact that women tend to live longer than men [72] . In the USA, gestational diabetes and rates of diabetes in obese adolescent girls have been increasing [73, 74] . Gestational diabetes occurs in up to 14% of all pregnancies [75] . Women, particularly those of lower socioeconomic status, also receive less adequate diabetes care than men of the same socioeconomic status [76] .
Females, particularly in developing countries, tend to have higher rates of obesity [77] . According to the WHO, in 138 of 195 countries, females are over 50% more likely to be obese than males [78] . In some countries, the body mass index for women is 5-8 points higher than for men [78] . The higher rates of obesity and diabetes in females may be significant factors contributing to higher 2009 H1N1-related morbidity in women. It has not yet been determined whether prepregnancy obesity or excess weight gain during pregnancy represent equivalent risks. Precise parameters for documenting obesity in pregnant women have not been established [79] .
Influenza in immunocompromised individuals is associated with an increased severity of disease [54] and HIV is recognized as a co-morbidity for 2009 H1N1 influenza [54, 80] . The rate of HIV in females are approaching that of males worldwide [81] . HIV RNA levels are consistently lower in women than men [82] . However, women have a 1.6-fold higher risk of progression to AIDS than men with equal viral loads [83, 84] . There also are gender disparities in access to care for women with HIV, with women traditionally having greater difficulty accessing treatment [85, 86] . Whether infection with HIV and progression to AIDS differentially affects the outcome of influenza virus infections in males and females has not been evaluated.
The precise impact of sex, gender and pregnancy on responses to the 2009 H1N1 vaccines is not known [87] [88] [89] [90] . Data from clinical trials of seasonal influenza vaccines reveal pronounced sex differences in the rates of vaccination, antibody responses to the vaccines and adverse reactions to the vaccines and illustrate that these differences must be considered in response to the 2009 H1N1 vaccine. Seasonal influenza vaccination data further reveal that pregnant and non-pregnant women generate comparable immune responses and experience similar adverse side effects [54] .
Available data on rates of 2009 H1N1 vaccination have not been analysed by sex [91] but rates of seasonal influenza vaccination vary significantly with respect to sex and age [92] [93] [94] . Rates of vaccination among women are lower than men in some European countries [92] and may reflect greater negative beliefs about the risks associated with vaccination [95] , differences in physician recommendations regarding vaccination or occupational differences. Among healthcare workers in China, 73% of women reported intentions to decline both the H5N1 and 2009 H1N1 vaccines compared to 64% of men [96] . In France, acceptance (either receipt or intention to receive) of the 2009 H1N1 vaccine was higher among men and was higher among pregnant women and other groups with co-morbid conditions [97] . In the USA and Canada, vaccination against seasonal and 2009 H1N1 influenza during pregnancy is recommended irrespective of trimester [54, 98, 99] . The vaccination rate of pregnant women against 2009 H1N1 virus has been estimated to be only 38%, which is still higher than that normally seen with seasonal influenza vaccine [91] .
Numerous studies reveal that haemagglutination inhibition (HAI) titres following seasonal influenza vaccination are consistently higher in women than men of comparable ages [100] [101] [102] [103] [104] , which suggests that women may be better protected against influenza disease following vaccination than are men. Women aged 18-64 years generate a more robust neutralizing antibody response following vaccination than men [102] . Pregnant women appear to have similar responses to seasonal influenza vaccines compared to non-pregnant women. The National Institutes of Health reports that 47 out of 50 (94%) pregnant women immunized with 2009 H1N1 vaccine achieved antibodies levels considered to be protective within 21 days of inoculation [105] .
Women report more severe local and systemic reactions to influenza virus vaccines [100, 102, 104, [106] [107] [108] . Women also experience worse reactions to vaccine adjuvants [109] , which should be considered for 2009 H1N1 vaccines that are administered with adjuvant [88, 89] . The extent to which adverse reactions to the 2009 H1N1 vaccine differ in either frequency or severity between males and females has not been reported [60] .
Seasonal, H5N1 and MF-59-adjuvanted influenza vaccines are reported to be safe for pregnant women [110] [111] [112] . A study of 50 pregnant women who received the 2009 H1N1 vaccine reported it was well-tolerated with no significant adverse side effects documented [105].
Antivirals are an effective treatment following infection with influenza viruses when administered early during the course of disease. The 2009 H1N1 viruses analysed, to date, are all resistant to the adamantadine class of antivirals but remain sensitive to neuraminidase inhibitors [3] . Available data indicate that the rate of prescribing antivirals to seasonal influenza virus-infected individuals, ranging in age from infants to adults, is similar between males and females in the USA [113] [114] [115] . In contrast, inappropriate prescription of antibiotics for seasonal influenza is greater for women [114] . A meta-analysis of data from randomized, doubleblind clinical trials illustrates that, following treatment with oseltamivir, men return to their baseline wellness faster than women, suggesting that antiviral treatment for seasonal influenza may be more effective in men [116] . Whether this observation reflects patient reporting biases, need for differential drug doses or other confounding factors is not clear. These data do, however, indicate that sex and gender should be considered when evaluating the efficacy of antiviral treatment for 2009 H1N1.
Prompt administration of neuraminidase inhibitors is recommended for any pregnant woman with influenzalike symptoms [3] . Administration of antivirals within 48 h of symptom onset correlates with a mild or uneventful disease course in pregnant women [47, 48, 51] . Pregnant women who do not take antivirals, or begin treatment >72 h after symptom onset, have significantly higher morbidity and mortality rates compared to those who have early antiviral treatment [48, 49] .
Sex differences in the immune responses to influenza viruses have not been systematically examined [117] . Using data from other virus-host systems, several immunological, hormonal and genetic mechanisms have been identified as being differentially expressed between the sexes and altered during the course of pregnancy, which may account for male-female differences and pregnancyassociated increases in the severity of 2009 H1N1. Generally, women mount higher immune responses to viral infections [118] . Heightened antiviral immunity in women is beneficial for virus clearance, but may be detrimental if it becomes excessively high or prolonged, leading to pathology and even death. Over the course of pregnancy, inflammatory and antiviral immune responses are suppressed which can alter responses to viruses, such as influenza.
Women are at a greater risk of progressing to AIDS than men, despite having significantly less HIV RNA in circulation and host-mediated pathology is hypothesized to contribute to this sex bias [82] . Plasmacytoid dentritic cells (pDCs) are significant producers of type I interferons (IFN-α), which signal the activation of cytotoxic T cells for the elimination of virally infected cells. pDCs from women react more strongly to HIV-1 encoded toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in higher levels of immune cell activation [83] . Women with higher progesterone (P4) concentrations have greater numbers of activated pDCs in response to the HIV TLR7 ligand than women with lower P4 concentrations [83] . Several genes (for example, the Tlr7 gene that encodes a receptor that recognizes RNA viruses, including influenza viruses) that encode for immunological proteins are on the X chromosome and may escape X inactivation, resulting in higher amounts of expression in women [117] . X chromosomal variation also alters the course of progression of AIDS differently in women than men [84] . Whether female-biased immunopathology contributes to the severity of 2009 H1N1 disease in women requires consideration.
The prevalence of HBV, titres of HBV DNA and development of hepatocellular carcinoma are higher in males than females and involve the effects of hormones on viral and host gene expression [67, 68, 119] . Among HBV positive males, elevated concentrations of testosterone and expression of certain androgen receptor gene alleles correlate with an increased risk of hepatocellular carcinoma [120, 121] . In HBV transgenic mice, castration of males reduces, whereas replacement of testosterone in castrated males increases, serum HBsAg concentrations [122] . Chemically-induced hepatocellular carcinoma is more severe in male than female mice, which is mediated by increased inflammatory cytokine production by liver cells in males and can be reversed with oestradiol (E2) treatment [123] . Sex steroids modulate sex differences in the prevalence of HBV and development of liver cancer through effects on immune responses to HBV. Whether sex steroids affect the pathogenesis of influenza virus infection should be examined.
The impact of sex steroids, including androgens, oestrogens and progesterone (P4), on the activity of immune cells may contribute to sex differences and the effects of pregnancy on responses to 2009 H1N1. Generally, androgens, including dihydrotestoesterone and testosterone, suppress the activity of immune cells [124] . The immunosuppressive effects of androgens may reflect the inhibitory effects of androgen receptor signalling mechanisms on transcriptional factors that mediate the production of pro-inflammatory and antiviral cytokines [125] .
Oestrogens affect both innate and adaptive immune function. Oestradiol can have bipotential effects with low doses enhancing and high doses reducing proinflammatory cytokine production [126] . Low E2 concentrations promote helper T cell type 1 (Th1) responses and cell-mediated immunity and high concentrations of E2 augment helper T cell type 2 (Th2) responses and humoral immunity which may be responsible for some female as well as pregnancy-associated changes in immune responses [126] .
Another oestrogen that affects the functioning of the immune system is oestriol (E3), which is produced during pregnancy by the placenta. When E3 levels are high, inflammatory responses and the symptoms of Th1mediated autoimmune diseases -including multiple sclerosis -are reduced [127, 128] . Whether the effects of pregnancy on responses to 2009 H1N1 reflect the effects of E3 on immune responses requires investigation.
Progesterone suppresses innate immune responses [125, 129] . Elevated concentrations of P4 during pregnancy inhibit the development of Th1 immune responses that can lead to fetal rejection and promote production of Th2 immune responses [130, 131] . Progesterone also suppresses antibody production [132] . Recent data illustrate that pregnant women with severe 2009 H1N1 have lower levels of total IgG2 than healthy pregnant women or women with only moderate H1N1 disease [133] . As IgG2 levels are enhanced in a Th1dependent manner, this reduction in total IgG2 may be related to pregnancy-associated modulation of the immune response.
As data from the pandemic continue to be analysed, a number of factors should be considered by clinicians, epidemiologists and scientists in order to better understand the role of sex, gender and pregnancy on 2009 H1N1 disease.
• Age-and sex-associated differences in exposure and severity of infection must be documented, as many biological and behavioural differences occur over the course of the lifespan.
• The outcome of infection is worse for females, but the magnitude of this difference varies across countries and the differential contribution of gender and sex in different regions of the world must be considered.
• Excessively high innate and cell-mediated immune responses, including the production of cytokines and chemokines, may contribute to increased severity of influenza in females.
• Higher antibody responses to influenza vaccines in females may lead to an increased protection from disease.
• Sex should be considered when effective vaccine and antiviral dosages are determined in order to maximize efficacy while limiting adverse side effects.
• As the outcome of influenza infection can be worse for females, efforts should be made to increase acceptance of vaccines in both pregnant and nonpregnant females.
• The 2009 H1N1 infection of pregnant women needs to be studied carefully in order to determine the factors that are driving the increased morbidity and mortality rates.
• Sex hormones have profound effects on the immune responses to vaccines and infection and should be examined in clinical samples and animal models.
• Animal models of infection can provide important insights into the role of sex, pregnancy, and hormones on the immune response to vaccination, infection, and antiviral treatment.
Abbreviations CI: confidence interval; COPD: chronic obstructive pulmonary disease; DC: dendritic cell; E2: 17β-oestradiol; E3: oestriol; HBV: hepatitis B virus; OR: odds ratio; P4: progesterone; pDC: plasmacytoid DC; RR: relative risk; Th1: helper T cell type 1; Th2: helper T cell type 2. PhEVER: a database for the global exploration of virus–host evolutionary relationships Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus–virus and virus–host lateral gene transfers. PhEVER (http://pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems. Viruses are responsible for a large number of infectious diseases and cancers. Recently, new viral diseases have emerged leading to severe consequences on human activities. The emergence of many of these new viruses can be attributed to recombining viruses as well as to host species jump (1) (2) (3) . Therefore, understanding how viruses interact with their hosts and more specifically how the complex interactions between viruses and their host organisms are acquired and maintained throughout evolution, remains a major challenge (4) (5) (6) (7) . In order to assess this question, it is of prime importance to be able to detect and quantify the occurrence of lateral gene transfer events, and the impact of these events on viralhost co-evolution. Indeed, the mechanisms behind fast viral adaptation are far from being elucidated. Thus, we developed a global approach aimed at providing accurate evolutionary and phylogenetic information to tackle these questions.
The major drawback of currently available databases of homologous families to the study of viral homologies and lateral gene transfer in viruses is their taxonomic compartimentalization. Indeed, current databases present families of homologies either restricted to viruses only [Protein Clusters (8) , GeneTree (9) ] or to viral taxonomic groups [Viral Orthologous Cluster (10) ], some also not presenting viral information [HomoloGene (11) ]. The few databases that do present viral and non-viral sequences, such as Pfam (12) or the Conserved Domain Database (13) do not provide complete phylogenetic trees. This translates into the fact that it is not currently possible to have a global view on viral-host lateral gene transfers due to the difficulty of obtaining global information on cross-taxa transfers at the viral level.
We present the first public release of PhEVER, a unique database of homologous gene families containing sequences (i) of all completely sequenced viruses and (ii) from fully sequenced cellular organisms. The protein sequences are clustered-without a priori and according to similarity criteria-into families containing either only viral sequences or both viral and cellular sequences. PhEVER integrates extensive data from up-to-date completely sequenced genomes spanning a wide taxonomic range (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates). To our knowledge, this is the most complete database of families of homologous viral sequences. Indeed, it not only spans all known viral groups but it also has the unique feature of presenting homologies with eukaryotic and prokaryotic sequences. The database offers a clustering of proteins into homologous families containing at least one viral sequence, as well as pre-computed alignments and phylogenies for each of these families. Alignments and phylogenies are built according to state-of-the-art phylogeny procedures and we provide tools to edit them and recompute them on the fly (14) . We also provide the possibility for users to assign their sequence of interest to a family and to re-build the phylogeny accordingly through the HoSeqI tool (15) . PhEVER thus constitutes a comprehensive working tool to detect sequence homologies and possible gene transfer events. Public access and documentation is available through the database webpage and through all dedicated ACNUC retrieval systems (16) .
We developed a genome-wide cross-taxa approach to build a database of families of homologous sequences and to provide accurate alignments and phylogenies for the constructed families. The layout of the implementation of the PhEVER database is represented in Figure 1 and the details concerning its sequence content are available in Table 1 . In order to avoid redundancy due to the availability of numerous genomes of similar bacterial and viral strains in public databases, we collected all completely sequenced viral and bacterial genomes from RefSeq Viral (17) and Genome Reviews (18) , two non-redundant and curated databases of completely sequenced genomes. To this high-quality curated data composed of Archaea, Bacteria and Eukarya, we added nine eukaryotic genomes from Ensembl (Aedes aegypti, Anopheles gambiae, Bos taurus, Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Gallus gallus, Homo sapiens, Mus musculus) (19) to allow for a large representation of species from the different domains of life. The PhEVER database was structured under the ACNUC system (20) allowing it to be queried using a web interface and a large number of tools specifically developed for this database management system (14) .
From the flat files containing the genomes and their annotations, two databases were built under the ACNUC database management system (20) , which is specifically aimed at building, storing and querying biological sequence data. One of them contains the nucleic sequences, the other contains the proteins generated by translating all CDS of the complete genomes-using the Figure 1 . Flow chart of the PhEVER building process. Complete genomes and their annotations were retrieved from three external public databases (Ensembl, Genome Reviews and RefSeq Viral) to provide high-quality non-redundant data for Eukarya, Archaea, Bacteria and Viruses. Two databases (nucleic acids, proteins) were constructed from this data to form PhEVER. All annotated CDS and mature peptides were translated and used for the clustering procedure. The homologous families thus produced were annotated in PhEVER, and alignments and phylogenies were built for each family and incorporated in the databases. appropriate genetic codes. For viral genomes presenting polypeptides which further maturate in vivo into mature peptides, the mature peptides were added to the set of translated proteins according to the annotations specified in the given genome ( Figure 1 ). Table 1 lists the global content of the databases as well as their original sources. Annotations were extracted from UniProtKB via the cross-references found in the CDS (21) . Subsequently, sequences in the nucleic and protein PhEVER database were clustered into families and were assigned a family accession number. Alignments and phylogenies were built for each of these families according to state-of-the-art procedures ( Figure 1 ). The classification of all organisms present in the database was retrieved from the taxonomy database at National Centre for Biotechnology Information (11) and is available on the PhEVER web interface.
The clustering of proteins into homologous families was constructed using an automated procedure similar to the one described in (14) and implemented within a parallel framework in a software package called SiLiX. Briefly, sequences were assigned to protein families by simple transitive link using the following criteria. A similarity search of all translated proteins and mature peptides against all was performed using BLASTP2 similarity search with the BLOSUM62 substitution matrix, a 10 À4 e-value threshold and the 'm S' filter option (22, 23) . For each pair of sequences, HSPs which were not compatible with a global alignment were removed. Two sequences were included in the same family if the sum of the remaining HSPs covered >80% of the proteins length (and at least 100 amino acids) and if their identity was !35%. These two criteria were previously shown to provide a good trade-off between the ability of clustering sequences from divergent organisms and the quality of resulting alignments for subsequent phylogenetic analyses (14) . Finally, only families containing at least one viral sequence were integrated in the database. For each of the families, a small description built from the gene annotations ordered by frequency is available on the web interface. Figure 2 presents the distribution of viral species, proteins and families according to each viral group (A) as well as a Venn diagram representing the families content (B). Figure 2A presents PhEVER's broad taxonomical distribution covering all Baltimore groups (24). This distribution is naturally biased towards dsDNA, ssDNA and positive-sense ssRNA viruses reflecting the bias in genomic sequencing efforts. Indeed these groups contain long-studied viral families-either for their medical interest or for their economical impacts-such as Caudovirales, Poxviridae, Herpesvirales, Flaviviridae, Picornavirales or Parvoviridae. Figure 2 (B) shows a large number of orphan families indicating that a significant proportion of viral proteins (32%) do not contain any homologs with proteins from known genomes. These proteins, among which some might possibly be caused by annotation errors, are unfit for comparative functional analysis and should be the focus of future experimental studies to validate them and to provide with crucial information on viral mechanisms. Figure 2B also shows the small number of families sharing sequences from both viruses and eukaryotes compared to the relatively high number of families sharing sequences from both viruses and bacteria. This observation may be due to different underlying biological mechanisms but might also be an indicator of a still low coverage of the Eukarya domain.
One of the applications of PhEVER is the detection of horizontal gene transfer events by comparing a gene family tree with the expected species tree. The discrepancies between the gene family history and the species phylogeny can then be an indication of possible events including a gene duplication, a gene loss or a horizontal gene transfer. The quality of the gene phylogenies is therefore essential and in this perspective, we implemented a procedure based on a rigorous methodology. First, the clustering into families was built with criteria leading to conservative families. Second, maximum likelihood phylogenetic trees were inferred for all families based on conserved aligned blocks. In our databases, for all families containing at least three sequences, pre-computed alignments and phylogenies are therefore already available. This allows for a simple and accurate overview of any family without the need of heavy computations and Number of proteins associated to a family, followed by the proportion of proteins associated to a family in the taxonomic group. can be a useful tool to search for lateral gene transfers in viral genes. For each family containing at least three sequences, alignments were estimated using MUSCLE with default parameters (25) . All alignments were treated with Gblocks (26) to select conserved blocks. Phylogenetic trees were inferred by maximum likelihood using PhyML (27) with a JTT evolutionary model (28) . Branch support was inferred using the Shimodaira-Hasegawa-like non-parametric procedure implemented in PhyML (27, 29) . To accommodate for weak phylogenetic signal, a thorough exploration of the tree space was made through topological rearrangements using the Nearest Neighbor Interchange topology search method. Finally, for visualization purposes, the trees were then rooted using midpoint rooting. This procedure allowed us to build accurate phylogenies sustained by branches of high support values. Indeed, Figure 3 shows that $80% of all branch supports have a value higher than 75 and one-third are above 95. Global statistics are biased by the presence of low branch supports. These are mostly due to few small families of less than 10 leaves, indicating that most families present robust phylogenies ( Figure 3B ). Finally, Figure 3C indicates that the low branch support values are attributable to very similar sequences with small branch lengths and might be linked to unresolved topologies. By contrast, all branch lengths longer than 0.15 subst/site display a high branch support which reveals the accuracy of our phylogenetic inference procedure.
PhEVER is structured under the ACNUC sequence database management system and a large number of tools have been developed around this database management system [see (16) for an overview]. PhEVER can therefore be queried using (i) web applications, (ii) standalone software (iii) or embedded within Python, R or C code. The PhEVER web interface is available at http://pbil.univ-lyon1.fr/databases/phever and allows to search for sequences or families by combining several criteria (including species, gene names, annotation terms) as well as by crossing taxa. The graphical user interface QUERY_WIN and the terminal-based interface Raa_query (16) implement more features than the web interface and allow for remote ACNUC access and query as well as for the automatization of querying processes through standalone software. Finally the C language API, Python language API (16) and the seqinR package for R (30) implement tools for integrating queries in user designed code. Note that the PhEVER database can also be installed on a local machine or server for fastest data access. All files necessary for the PhEVER For all figures, only trees with more than four leaves are presented here. Branches with length smaller than 10 À5 were considered unresolved multifurcations and were discarded.
installation are provided through our ftp website or by simple request.
The PhEVER web interface allows for two query forms represented in Figure 4A and B. On the one hand ( Figure 4A ), the HoSeqI tool allows to search in PhEVER families with a user provided query (15) . This query is used to BLAST the proteins present in PhEVER and to match the most related family. Alignments and phylogenies for this new family containing the user provided sequence can be recomputed on the fly, visualized and manipulated with Java applets ( Figure 4F ) (31, 32) . On the other hand ( Figure 4B ), the query tool allows to directly query the database with a very diverse range of terms including gene name, annotation term, species name, protein accession number, genome accession number and family accession number. The species and sequences represented in each family detected by the query can then be viewed ( Figure 4D ) as well as the alignments and phylogenies which can be edited online via Java applets ( Figure 4E ) (31, 32) . More details on how to query PhEVER are presented in the Supplementary Data.
PhEVER is the first open access database to provide information at the cross-taxa scale for the analysis of virus-virus and virus-host protein transfers. It compiles information from all kingdoms of life, and handles data from the genomes of all completely sequenced viruses and prokaryotes and of a large range of eukaryotes. It is the largest database of viral homologous families and offers highly accurate pre-computed alignments and phylogenies, making it a powerful tool for the analysis of horizontal gene transfer and more widely for the analysis of gene history. Our objective is to continue the development of PhEVER around the analysis of protein evolution in the context of virus-virus and virus-host interactions. More specifically, the next step we have under development is the detection of evolutionary conserved modules in the proteins present in PhEVER. Indeed, there is strong evidence that proteins evolve in a modular way, where modules are defined as parts of proteins sharing a common evolutionary history. These modules act as small interchangeable blocks of sequences that may be combined into proteins and form novel functions (33) (34) (35) . We are interested in providing a global tool allowing to analyse the weight of modular evolution in viral adaptation. We will therefore implement the detection of modules in PhEVER proteins to provide information concerning the exchanges of genetic information at the sub-protein level.
In conclusion, PhEVER aims at being a comprehensive tool for the analysis of virus-virus and virus-host relationships from an evolutionary point of view, namely through the analysis of genomic interchanges. It should become a valuable tool for anyone working on viral evolution, but also to understand the general mechanisms behind protein evolution and functional innovation.
Public access and documentation is freely available through the database webpage (http://pbil.univ-lyon1.fr/ databases/phever/) and through all dedicated ACNUC retrieval systems. More information on dedicated ACNUC retrieval systems, such as standalone query software (16) , the seqinR package for R (30) or the C and Python APIs, can be obtained in the Supplementary Data or on the PhEVER webpage. The PhEVER flat files for local installation are available through our ftp server (ftp://pbil.univ lyon1.fr/pub/phever) and instructions are available on the database webpage. The PhEVER database is updated every 6 months. This update frequency allows to follow the fast pace of viral and prokaryotic genome sequencing as well as to obtain updated genomic annotations for large eukaryotic genomes. Previous versions of the database remain available upon request. Reporting errors in infectious disease outbreaks, with an application to Pandemic Influenza A/H1N1 BACKGROUND: Effectively responding to infectious disease outbreaks requires a well-informed response. Quantitative methods for analyzing outbreak data and estimating key parameters to characterize the spread of the outbreak, including the reproductive number and the serial interval, often assume that the data collected is complete. In reality reporting delays, undetected cases or lack of sensitive and specific tests to diagnose disease lead to reporting errors in the case counts. Here we provide insight on the impact that such reporting errors might have on the estimation of these key parameters. RESULTS: We show that when the proportion of cases reported is changing through the study period, the estimates of key epidemiological parameters are biased. Using data from the Influenza A/H1N1 outbreak in La Gloria, Mexico, we provide estimates of these parameters, accounting for possible reporting errors, and show that they can be biased by as much as 33%, if reporting issues are not accounted for. CONCLUSIONS: Failure to account for missing data can lead to misleading and inaccurate estimates of epidemic parameters. The recent outbreak of pandemic strain Influenza A H1N1 (pH1N1), as well as other infectious disease outbreaks that have taken place recently illustrate the need for a rapid public health response and the ability to collect and analyze data efficiently. Unnecessary panic and disruption to society is more likely to be avoided and appropriately measured public health responses are more likely to take place when we have accurate information on the virulence and pathogenicity of an emerging disease. For these reasons, quantitative methods have been developed and continue to be developed to facilitate the assimilation of emerging data. Important quantities to estimate include the basic reproductive number, R 0 , defined as the average number of secondary infections created by a single infected individual in an entirely susceptible population. Numerous methods have been proposed for the estimation of this quantity, including deterministic and stochastic compartmental models [1] , branching processes [2] , networks [3, 4] , and, more recently, a likelihood based method [5, 6] .
Another parameter of interest is the serial interval, or the distribution of the interval in time between an infector and infectee presenting with symptoms [7, 8] . It has been recently shown that this quantity may be time dependent; for example it can contract during the course of an epidemic as prevalence of the disease increases [9] . Other quantities of interest include the case fatality rate [10] and the attack rate in subpopulations, such as age groups.
Among methods that can be implemented with relatively straightforward data, we typically assume complete observations. Clearly this assumption is more often than not violated in practice, especially when dealing with national or even regional data. For instance the scare surrounding the anthrax attacks in the fall of 2001 in the United States led to a large number of individuals reporting to medical care facilities with suspected anthrax. Should analysis have focused on suspected cases in that situation, the magnitude and threat of the event would have been greatly exaggerated. During the recent H1N1 outbreak, the number of suspected cases of disease likely is composed of several cases that will not be confirmed, however there are undoubtedly an even larger number of undetected cases, at least in the initial stages of the epidemic before the large public health response was launched and later on as the growth of the epidemic in many locations rendered it impossible to continue to track a large portion of the cases. Further, as the pandemic progressed sick individuals were cautioned to stay at home, rather than seek medical attention unless they were acutely ill [11] , driving up the number of unreported cases. Several of the unreported initial cases could arise from individuals who are asymptomatic, but still carrying and transmitting the virus or from others whose illness was not sufficiently acute to warrant seeking medical attention. To our knowledge, the issue of the impact of this misspecification of the number of cases on the estimation of epidemic parameters has not been well-studied.
In what follows we use the likelihood based methodology described in [5] to broach the subject and investigate the impact of underreporting on estimates of both R 0 and the serial interval. First we provide an overview of the methodology we employ and introduce notation to describe the occurrence of misspecification of cases. Second we provide some theoretical results describing the impact on the estimation of the reproductive number. We illustrate this through a simulation study. Finally we investigate the impact that various plausible underreporting schemes would have on estimates obtained from the recent H1N1 outbreak in La Gloria, Mexico and compare these to estimates of R 0 obtained using the method proposed by Wallinga and Teunis (WT method) [3] and a simple exponential growth model [12] .
In what follows, we assume that the outbreak is in its initial phase and that there is an unlimited supply of susceptible individuals. This implies that all contacts that an infected individual has are with susceptible individuals. Additionally, we follow standard methods and assume homogenous mixing among individuals in the system being studied. Following [5] , we assume that N t = {N 1 , ..., N T } are the number of new cases each day of the epidemic, with T being the total number of days of data analyzed, and that the serial interval is given by p = {p 1 , ..., p k } where k is the maximal length of the serial interval and p j is the probability of an infectee presenting with symptoms j days after the infector. In practice we can model the p j with a multinomial distribution or a truncated continuous distribution, such as the Gamma or Weibull, and estimate the parameters of that distribution so that the dimensionality of the estimation is independent of k. Then, we show in [5] that the likelihood of the case counts N t = {N 1 , ..., N T } is given by a thinned Poisson:
. Consider that on a given day M t = q t N t of the cases are observed, where q t ≥ 0. Therefore little changes in (1) and it can be shown that the likelihood becomes
. Given that q t is known, estimation proceeds as described in [5] . In reality we seldom known q t and our intent here is to quantify the effects of ignorance of q t on estimation of R 0 and the serial interval.
We first consider the case where the serial interval (the p j ) is well known and specified, perhaps from contact tracing data or historical information. Then the MLE for R 0 in the complete data case is given bŷ which is comparable to the branching process estimator described by [2] , as illustrated in [5] . If p j is incorrectly specified we know that the estimates of R 0 are impacted [5] . Our interest here is the study of the impact of missingness therefore we assume that p j is correctly specified so as to avoid confounding the effect of these two issues when estimating R 0 . In the case where data is incorrectly reported, the estimator for R 0 obtained from [2] is 
We consider two simple missingness patterns in this scenario. First let the missingness be constant, i.e. q t = q for all t. Second let q t = q 1 for t ≤ t c and q t = q 2 for t > t c , where t c might correspond to a public health announcement or certain number of cases occurring so as to raise alarm of an epidemic. In these cases it is likely that q 1 < q 2 . This is the likely scenario initially in the current H1N1 outbreak, where cases were accumulating for some time before public announcements were made and increased surveillance was implemented. Numbers of confirmed cases available early on in the investigation likely underestimate the true number of cases dramatically. One can also imagine cases where q 1 > q 2 . This might occur in instances of overreporting such as occur in times of panic, for instance following the scare after the anthrax attacks in 2001 in the US [13] when more than 20,000 individuals started antibiotics until it was determined that they did not have anthrax. Additionally if all suspected cases of H1N1 were considered early in the epidemic, this could possibly overestimate the true number of cases. Further, as time has progressed in the H1N1 pandemic, it has become virtually impossible to ascertain all cases. Therefore it is likely that reporting initially increased and then began to decrease again as case counts escalated.
We now provide results to illustrate the impact of these reporting schemes on the estimation of R 0 . In the following scenarios we use the branching process estimator to avoid the complication of the serial interval. In essence this implies that we assume that the serial interval is one day long in all scenarios or that the data is grouped into generations, rather than days or some other time unit. We now compare the two branching process estimators of the reproductive number,R 0 and  R 0 that make use of the observed numbers of cases denoted by M t = {M 1 , ....,
In other words,R 0 is the naïve estimator that does not account for reporting issues and  R 0 is the true estimator. We prove the following two propositions in the Appendix. 
In summary, if the reporting fraction does not change through time, then the estimate of R 0 is unaffected. However if the reporting fraction increases (decreases) then we will overestimate (underestimate) R 0 if we ignore misreporting (labeled naïve, above).
In order to quantify the impact of missing data on the estimation of R 0 and the serial interval, we consider a simulation study. We use multinomial and gamma distributed serial intervals. The multinomial represents a recent estimate for the current Influenza A/H1N1 outbreak in the USA and has a mean, μ of 2.21 days and variance, s 2 of 0.89 with k = 4 [14] . The Gamma distributed serial interval represents a disease with a mean of 8 days and variance of 16 days with k set to 20. We consider three values for R 0 : 1.5, 2, and 2.5, making six simulation scenarios.
We then apply four missingness schemes to each dataset. The first two scenarios assume that the reporting fraction is constant through time. The second two schemes have the reporting fraction increase once 30 cases are accumulated. Following are the schemes that we consider:
1. q t = 0.1, for all t, 2. q t = 0.5, for all t, 3. q 1 = 0.05, q 2 = 0.5, 4. q 1 = 0.4, q 2 = 0.9.
Thus we have six sets of complete data and 24 sets of incomplete data, where each set of data has 10,000 simulated epidemics. We show results for data simulated with two initial cases (i.e. N 0 = 2). Epidemics are simulated to stop when 500 cases are created or they die out. We only consider those that have at least ten cases for analysis, since fewer than ten cases would likely not be detected and considered an epidemic. Thus all simulated epidemics that die out before ten cases are accumulated, as well as those which have fewer than ten cases after the missingness pattern is applied, are not analyzed. Additionally those simulations with more than k zeroes in a row after the missingness operation is applied are not analyzed. This would be comparable to an undetectable epidemic since cases are so sparse in time that they are likely not connected to the same source.
The results of the simulations are given in Figure 1 and 2. Consistent with our theoretical results we observe that when the reporting fraction is constant, the estimates of R0 are unaffected by a failure to control for missingness. However if the reporting fraction increases, then the estimates are smaller when we adjust for the missingness. We also note that [15] has recently described a tendency of this method to overestimate the mean of the serial interval when the serial interval is short, such as in cases of influenza. Thus part of the effect seen could be attributed to this phenomena, but likely will be uniformly so.
In [16] the authors report initial findings on the current H1N1 Influenza pandemic, including results from data collected on a localized outbreak in La Gloria, Mexico. Data for this analysis was obtained by surveying 1575 Figure 1 Simulation results for the estimate of the reproductive number. The first boxplot (All) in each frame shows results when all the data is used. Each subsequent couplet of boxplots first shows the estimates when missingness is correctly accounted for and second, when missingness is ignored when estimating. The numbers on the × axis denote the missingness scheme applied to the data (see text of manuscript for a detailed description). For example the first couplet corresponds to constant missingness of 0.05 with the left boxplot giving the results when missingness is accounted for and the right one for estimates when missingness is ignored. The horizontal line indicates the true value of the parameter. villagers out of 2243 villagers recorded in 2005 [17] . Of those surveyed, 615 cases were reported. It was later discovered that some of these reported cases were from seasonal flu. Figure 3 shows the observed data.
In addition to employing our likelihood based method (hereafter MLE method) to obtain estimates for the reproductive number and serial interval from the observed data, we also consider various schemes of underreporting among both those surveyed (due to asymptomatic cases or misclassification) and those not surveyed. Additionally we consider the possibility of overreporting, given that it was later noted that some of the cases reported were actually seasonal flu strains [18] . These reporting patterns are informed using the following pieces of information.
Estimates of the attack rate for Influenza vary greatly. In [19] the authors report an attack rate of 68% among servicemen during an H1N1 outbreak in Finland during the winter of 1977-78. Among the 1575 surveyed in La Gloria an attack rate of 39% (615/1575) was observed. Finally, a recent report in Peru [20] indicates that 33% of cases were asymptomatic, meaning that the attack rate in La Gloria could actually be as high as 58% if we Figure 2 Simulation results for the estimate of the mean of the serial interval. The first boxplot in each frame shows results when all the data is used. Each couplet thereafter shows estimates when missingness is correctly accounted for on the left and on the right, when missingness is ignored in estimation. The numbers on the × axis denote the missingness scheme applied to the data(see text of manuscript for a detailed description). The horizontal line indicates the true value of the parameter. For several of the gamma distribution scenarios the actual estimates are extremely large and thus the value of the median is included on the plot where necessary.
can extrapolate from Peru. The number of missing cases was calculated as a Poisson random variable with mean given by AR*(2243*AR-615). We generate 1000 epidemic sizes for each attack rate.
We first assume that there is a constant reporting fraction. In other words we attempt to study the impact of missing information on the individuals that were not surveyed, assuming that they would have followed the same trends as those who were surveyed. Using the simulated epidemic sizes, we superimpose the missed cases on the observed cases using a multinomial distri- The data simulated from these assumptions are shown in Figure 3 .
We consider three additional scenarios where we allow the reporting fraction to vary through time. In this case we let the reporting fraction follow a logistic function given by:
where r describes the growth rate of the epidemic and is calculated at 0.19 in this scenario and q 1 and q 2 are set to 0.4 and 0.9, respectively and represent the reporting fractions at the start and end of the epidemic. Again the multinomial distribution with q t , as calculated from the logistic function, is used to assign the missing cases to days of the epidemic. Figure 4 illustrates the simulated outbreaks.
Finally we consider the possibility that a significant fraction of the cases were not actually pandemic influenza strain and, in fact, cases were overreported. We still assume that there was an overall underreporting of influenza like illness (ILI) according to the schemes described above. However, we additionally assume that only a fraction of those cases are indeed of the pandemic strain. This is done by randomly sampling from all of the ILI cases using a binomial distribution with probability 0.25, 0.50 or 0.75, indicating that 25%, 50% or 75% of cases, respectively, are pH1N1.
For the MLE analyses, we calculate the estimates of R 0 and the serial interval, assuming the serial interval follows a multinomial with a maximal length of four days [14] . We estimate the parameters under two scenarios. First we consider the first 16 days when the epidemic curve is in exponential growth and 326 cases had been observed. We perform estimation assuming an infinite number of susceptible individuals. Second, we consider the entire epidemic curve and estimate the effective reproductive number R t , by allowing R t to following a four parameter logistic distribution, as shown in [6] . We report the value for R 0 from this analysis. The median of the estimates over the 1000 simulated datasets is shown for each scenario along with the interquartile range of estimates obtained.
Additionally we provide estimates obtained on the same data where all cases are assumed to be pH1N1 using the method described by Wallinga and Teunis [3] for the entire dataset with either the serial interval estimate obtained from the MLE based method or that obtained by [16] . R 0 is reported as the average R t over the first 16 days of the epidemic. We further use a simple exponential growth model to estimate the exponential growth parameter and R 0 , using both serial interval estimates [12] . Table 1 shows the results from the analysis for the MLE based method assuming that all the cases are pH1N1. The results that assume overreporting of pH1N1 are given in the appendix (Tables 2 and 3 ). We first consider the results obtained by considering the exponential growth phase of the epidemic (the first 16 days). Without any adjustments for underreporting, we estimate the basic reproductive number to be 1.41 and the mean of the serial interval to be 2.09 days with a variance of 1.15 days. When we assume constant missingness, the estimates for the reproductive number and the mean of the serial interval vary slightly from these estimates with their IQR containing the original values. Allowing the reporting fraction to vary according to a logistic function yields estimates of the reproductive number and the serial interval that are consistently less than the estimates from the original data.
When all of the data are considered, the results are similar. Without adjustment for missingness R 0 is estimated to be 1.42 and the mean of the serial interval is 1.96 days with a variance of 1.14. Under the constant missingness scheme the reproductive number estimates increase with the attack rate (between 1.49 and 1.54), as well as the mean of the serial interval (2.15-2.25 days). When the reporting fraction increases through time, the reproductive number is smaller and decreases as the AR increases (1.29 to 1.05). The mean of the serial interval follows a similar pattern ranging between 2.01 for AR = 39% and 1.45 for AR = 68%. The results from the overreporting scenarios follow a similar pattern, assuming that the amount of overreporting is consistent throughout the epidemic. The results indicate that if reporting were in fact increasing throughout the epidemic, then the original results could be overstating the magnitude of both the reproductive number and the mean of the serial interval. Table 4 provides the results using the method of Wallinga and Teunis [3] and the exponential growth model to estimate R 0 . The estimates using the MLE estimator of the serial interval (i.e. with mean of 1.96 for WT or 2.09 for exponential) are comparable to those obtained with the MLE method and are 1.48 for the WT method and 1.40 for the exponential method compared to 1.41 for the MLE method. Those with the Fraser et al [16] estimate (mean SI = 1.91 days) tend to differ as a direct function of the mean SI (WT R 0 = 1.57, exponential R 0 = 1.36). Overall the impact of Key: The first block of results consider the entire outbreak and fit the reproductive number using a four parameter logistic function. The second block consider only the first 16 days where the epidemic is in exponential growth. For each attack rate shown (39%, 58% and 68%) estimates are shown when data missingness is assumed to be constant and when it is assumed to follow a logistic pattern with the initial reporting fraction at 0.4 increasing to 0.9.
missing data is the same, regardless of the estimation method used.
We have shown the impact of reporting issues on estimates of the reproductive number and the serial interval. Using a MLE based method, we show that the estimate of the reproductive number is unaffected if the reporting fraction is constant through time. However, if the amount of reporting increases, then we will overestimate the reproductive number if no adjustment is made. The converse is true should reporting tend to decrease over time. The simulation results and the work from La Gloria tend to support this theoretical result. We note that using other methods of estimation of the reproductive number (Wallinga and Tuenis [3] and exponential growth) yield the same trends. From our simulation work, we notice that the mean of the serial interval appears to follow the trend of the reproductive number. For instance, if the reproductive number is overestimated, then the mean of the serial interval tends to be too large, as well. Thus missing data not only impacts the estimate of the reproductive number, but also the estimation of the serial interval.
We additionally note several caveats to the work presented here. First, we have assumed homogenous mixing by not accounting for any variability in the disease parameters among subgroups. Clearly disease outbreaks are dynamic and impacted by multiple factors in the effected population. We follow the precedent commonly used and assume homogeneity in the population. It is not clear how much results might change if this assumption is relaxed.
Second, we take a frequentist approach and do not allow for variability in the parameters that are assumed known [15] recently published work allowing for a Bayesian approach to estimation where prior information on the serial interval can be incorporated into estimation. The values for the end results are likely to not vary significantly, if the prior information is not informative, there is sufficient data for estimation, or the serial interval is longer than one day. However there are cases where this prior information can improve the estimates and might impact the conclusions drawn here. In our setting we assume that there is no prior information, or if there is, such as the serial interval being known, that it is known with certainty. In this case, this assumption might lead to overestimating of the mean of the serial interval.
Failure to account for ascertainment of cases can have a substantial impact on the estimation of key epidemiological parameters. If the fraction of cases reported does not change dramatically throughout the course of the epidemic, then estimates may not be impacted substantially. When the reporting fraction varies through the course of an epidemic, as it likely will, estimates can be substantially impacted. It is important that epidemiological studies of infectious disease outbreak seek to account and better understand the nature of reporting and make appropriate adjustments in the methods used to obtain results. We have shown how this can be done for the MLE method and illustrated its use for recent Influenza A/H1N1 outbreak data from La Gloria, Mexico. In that case results were off by as much as 34% when underreporting was not considered. Data do exist, however, to obtain an idea of the level of underreporting [13] use information on hospital admissions in the current H1N1 pandemic to obtain an estimate of the degree of underreporting. Further [15] has recently shown that incorporating contact tracing data into the estimation of the serial interval in a Bayesian framework can improve estimates. A similar approach could be used to improve estimates to account for suspected levels of misreporting. Clearly the need exists to use innovative methods to ascertain reporting issues in data using existing data, or by the collection of additional data or well-planned studies that can be rapidly initiated in the event of an outbreak. For instance, one might consider carefully studying a smaller population to determine the rate of reporting there. This could inform the overall rate of reporting. At the least, estimates should be reported as ranges, rather than as a single point estimate to indicate plausible values for the estimate.
It is straightforward to observe from the definition of  R 0 that when q t = q, the correct estimator simplifies to is not impacted by the missingness in the data.
Without loss of generality, we consider the simple branching process estimator, where p 1 = 1 and p j = 0 if j > 1. Additionally let q 2 = rq 1 , where r > 1. Then the estimator with missingness taken into consideration becomes .
We now illustrate the impact of reporting issues on the standard error of the estimators,R 0 and  R 0 using the formula provided in [21] and given by Proposition A2. If q t = q 1 , t ≤ t c and q t = q 2 , t > t c then i. If q 1 < q 2 < 1 then SE R R SE ( ) ( ) 0 0 >  .
ii. If q 1 > q 2 > 1 then SE R R SE ( ) ( ) 0 0 <  .
iii. If q 2 > q 1 > 1 or q 2 <q 1 < 1 then the results for the SE are not clearly defined.
Following are the results when we consider that only a fraction of the total cases in La Gloria were actually of the pH1N1 strain. For this scenario, we simulate 1000 datasets, as before. These datasets are interpreted as providing the total number of ILI cases. Of these, we assume that only a proportion is actually of the pandemic strain. We allow this to be 0.25, 0.50, or 0.75. Cases are randomly selected as pH1N1 cases and included in the analysis. Results are given in Tables 2  and 3 . Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine‐origin influenza type A/H1N1 and acute respiratory failure BACKGROUND: Cases of H1N1 and other pulmonary infections evolve to acute respiratory failure and death when co‐infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment. OBJECTIVE: To perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ARDS with confirmed H1N1. METHODS: Lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. Immunophenotyping was used to characterize macrophages, natural killer, T and B cells, and expression of cytokines and iNOS. RESULTS: The pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. Ultrastructural analysis showed viral‐like particles in all cases. CONCLUSIONS: Viral‐like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by RT‐PCR on nasopharyngeal aspirates. Bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair. Recently, a novel swine-origin influenza A (H1N1) virus with molecular features of North American and Eurasian swine, avian, and human influenza viruses [1] [2] [3] [4] has been associated with an outbreak of respiratory disease. According to the World Health Organization (WHO), between 25 April and 11 October 2009, 399,232 confirmed cases of H1N1 influenza virus and 4,735 deaths occurred throughout the world. 5 Brazil reported 1,528 deaths up to 10 November 2009. 6 Swine-origin influenza A (H1N1) virus infection can cause severe acute respiratory failure (ARF), requiring admission to an intensive care unit (ICU) in 15-30% of previously healthy young to middle-aged people. 3, 4, 7, 8 Death may occur when co-infections or lung injury prevail over the immune response, resulting in a progressive worsening of lung function (low compliance and oxygenation). Early diagnosis and a complete understanding of the pathological features of the H1N1 virus are important to help to improve treatment and the prognosis of this lethal disease. Analysis of the lung tissue from an open lung biopsy (OLB) of these severe cases can help in understanding the pathogenesis of this severe and sometimes fatal development. Until now, no reports of OLB findings used to guide the treatment of patients with H1N1 pneumonitis have been published, although according to many authors OLB is safe and diagnostically useful in patients with ARF, enabling appropriate therapy. [9] [10] [11] [12] The pathogenesis of ARF associated with swine-origin influenza virus (S-OIV) infection in humans is unknown. The influenza virus triggers pulmonary inflammation owing to an infiltration of inflammatory cells and an immune response. Bronchial epithelial cells are the primary target and the principal host for the virus. 13, 14 Normally, influenza viruses are recognized and destroyed by innate immune mechanisms which involve macrophages, interferon (IFN) a, b and other cytokines, natural killer (NK) cells and complement. When influenza viruses escape from these early defense mechanisms, they are captured and eliminated by adaptive immune mechanisms, where T and B cells and their antigen-specific effectors (cytotoxic T lymphocytes, cytokines such as IFNc and antibodies) target the virus. Additionally, antigen-specific memory cells (T and B cells) are involved in the prevention of the subsequent viral infection. 14 Thus, pathological findings obtained by an OLB, coupled to ultrastructural and immunologic analysis, may have an impact on decisions about changes in treatment strategies employed for these critically ill patients, and also provide a greater understanding of the pathophysiology of S-OIV infection. The objective of this study was to analyze pathologically and ultrastructurally S-OIV lung infection and the pulmonary immune response in a series of five cases with OLB.
We studied pathologically and ultrastructurally five patients suspected of having a pandemic S-OIV virus who developed ARF requiring ventilatory support. Nasal swabs for RT-PCR for H1N1 were collected from all patients. The OLBs indicated by the clinicians were carried out after receiving consent from the families. These patients had a severe evolution of the virus and more information about the physiopathology of the disease was required in order to provide adequate treatment. If no improvement of the respiratory status was seen in the patients with ARF after $5 days (defined as no decrease of the Lung Injury Score) an OLB was indicated. 15 Lung tissue sections (4 mm thick), prepared from 10% formalin-fixed, routinely processed, paraffin-embedded blocks, were stained with hematoxylin-eosin. The following methods of histochemical staining were carried out: Grocott's methenamine silver stain, Brown-Brenn, and Ziehl-Neelsen. The following pathological changes were analyzed: a) necrotizing bronchiolitis, b) alveolar collapse, c) dilatation of the airspaces, d) hyaline membrane, e) fibroplasia, f) squamous metaplasia, g) multinucleated cells, h) alveolar hemorrhage, i) acute inflammatory exudates, j) atypical pneumocytes. Pathological changes were graded, using two sections, according to a five-point semiquantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1-25%, 2 = changes in 26-50%, 3 = changes in 51-75%, and 4 = changes in 76-100% of examined tissue. This semiquantitative analysis is currently routinely used in most studies of the department of pathology of the University of Sã o Paulo Medical School. 16, 17 For immunohistochemistry, the avidin-biotin-peroxidase complex and streptavidin-biotin enzyme complex immunostaining methods were used with antibodies against: lymphocytes CD4 (clone: MO834, dilution 1:1000), CD8 (clone: M7103, dilution 120), CD20 (clone: M755, dilution 140), macrophages-histiocytes CD68 (clone: M814, dilution 130), mouse monoclonal antibodies from DAKO, Carpinteria, CA, USA; S100 (clone: Z311, dilution 11000) rabbit polyclonal antibodies from DAKO; CD1a (clone: MCA1657, dilution 1: 200) mouse monoclonal antibodies from Serotec, Oxford, UK; natural killer, NK (clone: MS136P, dilution 11000) mouse monoclonal antibodies from Neomarkers, Fremont, CA, USA; interleukin 4 (IL-4) (dilution 140), IL-10 (dilution 140) goat polyclonal antibodies from R&D Systems, Minneapolis, MN, USA; IFNc (clone: MAB285, dilution 130), mouse monoclonal antibodies from R&D Systems; tumor necrosis factor alpha (TNFa) (clone: AF210NA, dilution 140) all mouse monoclonal antibodies from R&D Systems; inducible nitric oxide synthase (iNOS) (dilution 1500) polyclonal rabbit from Calbiochem, La Jolla, CA, USA.
Immunohistochemical reactions were carried out in accordance with the manufacturer's instruction. Diaminobenzidine was used as the color substrate, and Meyer's hematoxylin was used for counterstaining. Cell immunophenotypes and immune expression of cells using the different methods of immunohistochemical staining were identified and graded according to a five-point semiquantitative intensity-based scoring system as: 0 = negative, 1 = positive in 1-25%, 2 = positive in 26-50%, 3 = positive in 51-75%, and 4 = positive in 76-100% of examined tissue. 18 Small blocks (1 mm 3 ) of lungs were fixed in 2% glutaraldehyde/2% paraformaldehyde in cacodylate buffer overnight, then fixed in 1% osmium tetroxide, dehydrated, and embedded in araldite. Ultrathin sections obtained from selected areas were double-stained and examined in a Philips TECNAI 10 electron microscope at 80 kV. For each electron microscopy image (15/case), the following structural changes were analyzed: a) cytoplasmic swelling, b) degenerative changes, c) sloughing of necrotizing alveolar epithelial cell type I (AECI) and II (AECII), d) denudation of the epithelial basement membrane, e) hyaline membranes, f) alveolar septal collapse, g) viral particles such as tubuloreticular structures (TRS) and cylindrical confronting cisternae (CCC), h) multinucleated AECII. Ultrastructural findings were graded according to a five-point semiquantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1-25%, 2 = changes in 26-50%, 3 = changes in 51-75%, and 4 = changes in 76-100% of examined tissue. 16, 17 
Five patients (two male, three female) mean age 48 years (range 35-81) were studied; only patient No 4 had preexisting medical illnesses (Table 1) and chest x-ray abnormality at disease onset. All the patients presented with a 4-10 days' (median 5 days) history of shortness of breath and flu-like symptoms and rapid clinical deterioration. They were transferred to the ICU for tracheal intubation and ventilation (range 8-25 days; median 17) and diagnosed as having ARF. 15 All the patients received 75 mg twice a day by nasal enteral tube of olsetamivir (range 4-14 days; median 10) and intravenous steroids (range 9-20 days; median 12). After obtaining these results the dose was changed from 75 mg twice a day to 150 mg twice a day through a nasal enteral tube, in accordance with the Brazilian guidelines. The presence of the H1N1 virus was confirmed in all five patients (Table 1) by nasal swab or lung tissue positivity of RT-PCR according to guidelines from the Centers for Disease Control and Prevention. 19 Other microbiological investigations, including the isolation of other viruses, were negative. During the evolution of disease in the patients in the ICU, Staphylococcus aureus was isolated from a blood culture (patients 2 and 3) and Klebsiella spp were identified in tracheal aspirate specimens (patient 1). Patients 1, 2 and 4 are alive, but patients 3 and 5 died of respiratory failure, with concurrent congestive heart failure, hepatic encephalopathy, and acute renal failure. Table 2) . Pulmonary specimens from patients 3 and 5 presented more intense changes at optical microscopy. The membranous and respiratory bronchioles were extensively compromised by epithelial necrosis, squamous metaplasia, and obliteration by fibroplasia ( Figure 1A -F). The parenchyma was modified by extensive alveolar collapse, dilatation of the airspaces, alveolar hemorrhage, and sparse hyaline membrane formation ( Figure 1G -I). There was interstitial thickening, with mild to moderate fibroplasia ( Figure 1I ), but a disproportionately sparse infiltrate of inflammatory cells, mainly histiocytes, including multinucleated forms, lymphocytes and megakaryocytes ( Figure 1J-K) .
Atypical bronchiolar and alveolar epithelial cells (AECs) were seen in all five patients, although the distribution was focal ( Figure 1J ). These atypical forms included multinucleated giant cells with irregularly distributed nuclei ( Figure 1K , L) or bronchiolar and AECs with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm ( Figure 1M ). However, distinct viral inclusions were not apparent.
The ultrastructural features were represented by bronchial and alveolar epithelium necrosis, a destroyed alveolar epithelium/basement membrane unity and the presence of viral-like particles (Table 3) . Patients 3 and 5 presented more prominent changes at submicroscopic level. Cytoplasmic swelling, necrosis, and degenerative changes of the endoplasmic reticulum and other organelles were present in bronchial and AECs (Figure 2A-C) . A large number of bronchiolar and AECs were detached from the basement membrane and were showing apoptosis (Figure 2A, B) . Lymphocytes also exhibited apoptosis. Sloughing of apoptotic bronchiolar cells and AECs causing denudation of the epithelial basement membrane was followed by deposition of hyaline membranes ( Figure 2D ).
Ultrastructural evidence of alveolar collapse was also present by the apposition of the alveolar septa ( Figure 2E -G). The regenerating bronchiolar epithelium extended along the adjacent alveolar septa showing features of cells with prominent surface microvilli with decreased or absent lamellar bodies and considerable cytologic atypia ( Figure 2H -L). Increased myofibroblasts and collagen fibers were also present ( Figure 2I ). Multinucleated epithelial cells with prominent nucleoli were noted in most cases, although such cells were sparse ( Figure 2K ). The proliferating bronchiolar and AECs containing TRS and CCC, probably representing residual viral-like particles, were distinguished in all cases ( Figure 2M -R). TRS appeared as reticular aggregates of branching membranous tubules located within the cisternae of the endoplasmic reticulum ( Figure 2M -O) or were compact ( Figure 2Q , R). CCC were identified as elongated, slightly curved cylindrical structures ( Figure 2P , Q), ring shaped ( Figure 2R ) or fused membranous lamellae, representing cisternae of endoplasmic reticulum. (Table 4) . Patients 3 and 5 presented with immunologic impairment.
In all patients small aggregates of macrophages, CD4+ Thelper cells, CD8+ T-cytotoxic cells, CD20+ B-cells, CD1a+ dendritic cells, S100+ dendritic cells, natural killer lymphocytes were present around vessels and bronchioles. Dendritic cells and TNFa were expressed sparsely in macrophages, AECs and endothelial cells, whereas IFNc was expressed in small mononucleated cells in lungs from patients with S-OIV. There was a very strong expression of IL-4, IL-10 and iNOS in small mononucleated cells.
This case series documents for the first time the pathological and ultrastructural findings of lung tissue from five patients admitted to the ICU with ARF and S-OIV infection who were submitted to OLB.
S-OIV (H1N1) virus and the pulmonary syndrome is an acute respiratory illness, first identified in Mexico with at present, 399,232 cases registered, 4,735 deaths, affecting more than 179 countries. 2, 5 Our patients, most of them previously healthy, had an atypical influenza-like illness that progressed during a period of 5-7 days.
The two patients who died showed a higher degree of pathological commitment of the disease at the OLB. Most of our patients were young to middle-aged and had previously been healthy. Increased risk for severe S-OIV illness is found in young children, 10-19 age groups, patients older than 65 years, pregnant women, obese people and those with comorbidities. 1, 7, 20 Fifteen to thirty per cent of patients with H1N1 infection required ICU admission. Mortality among the patients who required mechanical ventilation was around 58%. 7 In our case series the OLB findings showed that the lung damage was most likely due to infection by the influenza virus. The main pathological finding revealed necrotizing bronchiolitis and DAD, respiratory epithelial cells probably being the primary target of the infection. The extensive destruction of the respiratory and AECs and dysfunction in the immune and adaptative immune response led to DAD. As previously reported, possible mechanisms of damage include direct injury to the respiratory and alveolar epithelium exposing the basement membrane and leading to alveolar collapse by loss of surfactant, 13,14,21 with a secondary cytokine storm. 22 This is followed by exudation of macromolecules from the circulation, which finally form hyaline membranes. Activation of the cytokines is part of the immune reaction aiming to eradicate the virus. In this study, the systemic IFNc and TNFa cytokine activation probably resulted in reactive hemophagocytic syndrome in the bronchiole-associated lymphoid tissue and possibly also mediated the epithelial necrosis. 23 A mild inflammatory infiltration is most often seen in viral pneumonias; this has been explained by a cytokine-mediated blockade of lymphocytopoiesis and also by blockade of release from the bone marrow. 24 In our cases, expression in the lung of IFNc by small mononucleated cells and TNFa by macrophages and AECs was low. This finding may be supported by Kim and colleagues, 25 Conversely, we found a very strong expression of IL-4, IL-10 and iNOS by macrophages. The sparse inflammatory and immune reaction found in our samples, which involves targetting of the virus by NK cells, lymphocytes T and B cells, CD8+ cytotoxic T-lymphocyte cells, as well as CD1a and S100 cells, may be due to a combination of lymphoid tissue necrosis and apoptosis and exhaustion of lymphoid proliferation in response to the cytokine overdrive. In addition, the high IL-10 expression associated with its anti-inflammatory action may explain the low degree of inflammation observed in our cases. Taken together, our results suggest that in S-OIV infection, altered innate and adaptative immune responses may lead to incomplete virus eradication in the primary target of the infection and, consequently, imbalance between inflammation and reparation, resulting in bronchiolar obliteration and DAD.
DAD is likely to be a consequence of bronchiolar obstruction and consequent hypoxia rather than direct invasion of the viruses. It is a severe pattern of lung injury and could be secondary to various pulmonary and extrapulmonary insults. 26 In this series of cases we found DAD in which alveolar collapse was prominent, differing from classic DAD found in ARF or secondary to other pulmonary and extrapulmonary insults. This finding may have important implications in the ventilation strategy of the patients. 27 In addition, the presence of intra-alveolar hemorrhage may suggest virus-associated hemophagocytic syndrome. 23 
The lung tissue score was obtained independently by two different investigators. The pathologic findings were graded according to a five-point semiquantitative severity-based scoring system: 0 = normal lung parenchyma; 1 = changes in 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 76-100% of the examined tissue. Table 3 -Semiquantitative analysis of electron microscopy. In our current series, pulmonary ultrastructural analysis was important to obtain an understanding of the pathophysiology of this new disease. First, we demonstrated apoptosis and necrosis in the bronchiolar epithelium together with viral-like particles, thus suggesting the bronchiolar epithelium as the primary target of the virus infection. Second, we documented the submicroscopic pattern of a clastogenic DAD in S-OIV infection. Third, we found indirect evidence of virus infection in alveolar and bronchiolar epithelial cells represented by the TRS and CCC. These submicroscopic structures were demonstrated ultrastructurally in the lung tissue of all the patients and their presence suggests an inactivation of the virus by oseltamivir treatment or an altered innate immune response of these patients. They appeared mainly in respiratory cells and AECs and have previously been described in a variety of cell types. 28, 29 Usually, they occur in endothelial cells and lymphocytes from patients with autoimmune diseases and viral infections. 30 Patients with acquired immunodeficiency syndrome present TRS and CCC in these same cells. 31 The mechanism of TRS and CCC production in vivo is not definitely established. Nevertheless, clinical and experimental studies have shown that the presence of both structures in these diseases is directly associated with the increase of IFNa and IFNb but not with IFNc. 29 One theory to explain the nature and pathogenesis of TRS and CCC suggests that these structures are incomplete viral particles. 30 In our study, these viral-like particles were noted mainly in the respiratory epithelial cells, but not in the other cell types within the lung. These observations reinforce the hypothesis that the primary target cells for S-IOV infection are probably the bronchiolar epithelium. The atypical morphology of the bronchiolar and alveolar epithelial cells was probably related to viral cytopathic effects or reactive changes. In fact, the presence of multinucleated epithelial cells is not exclusive to S-IOV, and is seen in pneumonia caused by the family of Paramyxoviridae, including parainfluenza viruses, measles, mumps, respiratory syncytial virus and, perhaps, metapneumovirus. 31 Although multinucleated cells were seen in our cases, these probably reflect non-specific secondary changes.
We describe a case series of five patients with influenzalike illness with pneumonia and ensuing ARF who underwent OLB with subsequently confirmed diagnosis by RT-PCR testing for S-IOV infections. This report has some limitations. First, this study may not validate the importance of OLB in this population; however, it did provide information about this new disease. Second, it is difficult to compare our findings with those of others because to our knowledge no studies reporting an OLB in patients with S- The lung tissue score was obtained independently by two different investigators. The pathologic findings were graded according to a five-point semiquantitative severity-based scoring system: 0 = normal lung parenchyma; 1 = changes in 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 76-100% of the examined tissue. IFN, interferon; TNFa, tumor necrosis factor a. IOV have been published. Although there are already many autopsy series with patients with H1N1 that can be used for comparison, the pathological findings at autopsy are modified mainly by the presence of associated co-infections and mechanical ventilation. [32] [33] [34] [35] [36] [37] [38] [39] In summary, we have presented the pulmonary pathology in a confirmed and well-defined series of cases of S-IOV infection associated with ARF. The pathological features, in addition to necrotizing bronchiolitis and DAD, included the presence of multinucleated cells and intra-alveolar fibrin exudates (organizing pneumonia-like lesions). Although each of these features is non-specific, their combined occurrence, together with positive serologic, microbiologic, and immunologic investigations and/or ultrastructural tissue examination enables the diagnosis of S-IOV infection to be confirmed, and is particularly useful in clinically suspicious cases that do not fulfill the WHO criteria or in clinically inapparent cases.
We have shown that viral-like particles can be successfully demonstrated in lung tissue by ultrastructural examination, highlighting the importance of OLB, particularly in those patients without confirmation of the virus. We also showed that bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and that DAD is the consequence of airways obliteration and dysfunction on innate immunity, suggesting that the treatment should be focused on epithelium repair. Ventilatory and ECMO treatment of H1N1-induced severe respiratory failure: results of an Italian referral ECMO center BACKGROUND: Since the first outbreak of a respiratory illness caused by H1N1 virus in Mexico, several reports have described the need of intensive care or extracorporeal membrane oxygenation (ECMO) assistance in young and often healthy patients. Here we describe our experience in H1N1-induced ARDS using both ventilation strategy and ECMO assistance. METHODS: Following Italian Ministry of Health instructions, an Emergency Service was established at the Careggi Teaching Hospital (Florence, Italy) for the novel pandemic influenza. From Sept 09 to Jan 10, all patients admitted to our Intensive Care Unit (ICU) of the Emergency Department with ARDS due to H1N1 infection were studied. All ECMO treatments were veno-venous. H1N1 infection was confirmed by PCR assayed on pharyngeal swab, subglottic aspiration and bronchoalveolar lavage. Lung pathology was evaluated daily by lung ultrasound (LUS) examination. RESULTS: A total of 12 patients were studied: 7 underwent ECMO treatment, and 5 responded to protective mechanical ventilation. Two patients had co-infection by Legionella Pneumophila. One woman was pregnant. In our series, PCR from bronchoalveolar lavage had a 100% sensitivity compared to 75% from pharyngeal swab samples. The routine use of LUS limited the number of chest X-ray examinations and decreased transportation to radiology for CT-scan, increasing patient safety and avoiding the transitory disconnection from ventilator. No major complications occurred during ECMO treatments. In three cases, bleeding from vascular access sites due to heparin infusion required blood transfusions. Overall mortality rate was 8.3%. CONCLUSIONS: In our experience, early ECMO assistance resulted safe and feasible, considering the life threatening condition, in H1N1-induced ARDS. Lung ultrasound is an effective mean for daily assessment of ARDS patients. Since the first outbreak of a respiratory illness caused by Influenza A (H1N1) virus in Mexico [1] , several reports have described the need of intensive care [2] [3] [4] or extracorporeal membrane oxygenation (ECMO) assistance [5] in young and often healthy patients.
Beginning August 2009, the Italian Ministry of Health and the Tuscany Ministry of Health issued instructions to identify and establish referral centers able to care for the more severely ill influenza patients. Therefore, several referral centers were identified throughout the national territory among the hospitals already experienced in extracorporeal respiratory support techniques. The referral ECMO centres, in addition to being capable of guaranteeing the most advanced treatment in influenza related respiratory failure, were also entrusted with providing support to the nearby hospitals and assuring safe transportation.
In the present investigation we report our experience, as an ECMO referral center, in H1N1-induced acute respiratory distress syndrome (ARDS) and we present the critical care service planning in response to the H1N1 pandemic.
Following the instructions of the Italian Ministry of Health and Tuscany Regional Ministry of Health, an Emergency Medical Service was established in the Careggi Hospital in Florence Italy for the novel pandemic influenza.
The Careggi Hospital ECMO Team is composed of: an intensivist, a cardiac surgeon, a cardiologist, a nurse, and a perfusionist. All of the members of the team are properly trained in ECMO treatment. An ambulance and a car are equipped with an ECMO circuit, a transport ventilator and all of the materials needed to initiate extracorporeal support in the peripheral hospitals, and permit safe transportation while on extracorporeal circulation to our referral hospital.
The requirement of ECMO was decided based on the Italian Ministry of Health criteria (Table 1) .
From September 2009 to January 2010, all patients admitted to our ICU with severe respiratory failure due to H1N1 infection were included in this study. Patient demographics and clinical characteristics were collected from institutional ICU database (FileMaker Pro, File-Maker, Inc, USA), from Italian Group for the Evaluation of Interventions in Intensive Care Medicine database (GiViTI Margherita Project, Istituto Mario Negri, Bergamo, Italy) and from ECMO national network database. Discrete variables are expressed as counts and percentages, whereas continuous variables are reported as medians with 25th to 75th interquartile range (IQR). The Internal Review Board approved this retrospective study and informed consent for data publication was obtained from the patients or relatives.
Pressure volume curves were calculated with ventilator's built in application (Draeger Evita XL, Draeger Medical AG, Lubeck Germany) starting from a PEEP level of 5 cm H 2 O, with a pressure limit of 40. Ventilation parameters were set on the basis of this calculation, with a PEEP of 2 cmH 2 O above the lower inflection point of the pressure-volume curve, and a peek pressure below the upper inflection point. In all cases, pressure plateau was limited to 30 cmH 2 O and the tidal volume was kept below 6 ml/Kg [6] . Recruitment manoeuvres (40 sec at 40 cmH 2 O) were performed twice a day, if needed, to improve pulmonary gas exchange. Cannulation was conducted percutaneously with Seldinger technique in all cases, and cannulas position was confirmed by transesophageal echocardiography.
Heparin infusion during extracorporeal lung assistance was monitored every two hours by bedside aPTT measurement (Hemochron Jr. Signature plus, ITC Europe, Milan, IT), which was maintained between 50 and 80 seconds. In case of renal replacement therapy requirement in ECMO patients, a continuous veno-venous hemodiafiltration circuit was assembled on the ECMO circuit (aspiration on pre-pump line, restitution on preoxygenation line).
ECMO patients were ventilated with protective parameters, and respiratory rate and ECMO flow were adjusted to achieve normocarbia and oxygen saturation above 92%. assayed on pharyngeal swab, subglottic aspiration and bronchoalveolar lavage in accordance with published guidelines [7] . Bronchoalveolar specimens were obtained with a mini-invasive system (Kimberly-Clark BAL Cath, Kimberly-Klark N.V. Zaventem -Belgium), or by bronchoscopy.
Patients were isolated in negative pressure atmosphere rooms, and staff wore full protective garments (including FFP3 respirators, 3 M Italia SpA, Segrate, Italy), until 2 consecutive tests were confirmed negative. During the study period only one case of suspected transmission of influenza to a nurse occurred. Antiviral therapy consisted in oral oseltamivir (75 mg twice daily) and inhaled zanamivir (10 mg twice daily).
Blood and urinary cultures, tracheal aspirate, and pharyngeal swab were obtained upon patient admission. Empiric antimicrobial regimen at ICU admission was initiated with levofloxacin and amoxicillin/clavulanate; eventually specific antimicrobial therapy was varied or ended on the basis of microbiological results.
Steroids were administered at low dosage (20 mg metilprednisolon twice per day) to prevent lung fibrosis. Diuretics were administered at different dosages, depending on clinical judgment and the patient's renal function.
Lung ultra sound (LUS) examinations were daily performed by the attending physician, with a multifrequency convex probe (3.5-5 MHz, Mylab TM 30CV, ESAOTE, Genova, IT). With the patient in semirecumbent position, lateral and anterior views were obtained from base to apex of the chest. Posterior axillary line was followed during lateral transversal examinations. Chest quadrants defined by the intercostal spaces and the parasternal, mid-clavicular, and anterior axillary lines were scanned on the anterior chest wall [8] . The occurrence and extension of parenchymal consolidations, alveolar interstitial syndrome (measured by the number of B-lines), and morphology of pleural line were evaluated [9] [10] [11] . Pleural effusions were estimated by using Balik's formula [12, 13] . In order to ensure a uniform record, and allow to follow the evolution of the findings over time, all exams were recorded in an electronic form, in which the description of the main LUS features was predetermined [14] .
During the study period, 12 patients requiring invasive ventilation treatment and/or ECMO were admitted or transferred to our ICU. Baseline and clinical characteristics of patients admitted for H1N1-induced severe respiratory failure are summarized in Table 2 .
The median time between initial, non specific, symptoms and respiratory failure was 7 days (IQR 6-8.25), and severe hypoxia, unresponsiveness to non-invasive ventilation, was the main clinical feature. Our patients were young, median age 44.5 years, none of them older than 58 years, and eight (80%) younger than 50. Two patients were severely obese (BMI > 40), one woman was pregnant (18 weeks), two patients had a history of chronic obstructive respiratory disease (COPD), and one had diabetes. Two patients had Legionella Pneumophila coinfection at admission, and one young patient (16 years old) with suspect viral myocarditis and heart failure. At admission the patients, with the exception of the two coinfected, presented low leukocyte and platelet count and low plasma procalcitonin levels, significant levels of lactate dehydrogenase (LDH), creatine kinase (CK), and C-reactive protein ( Table 2 ). Median duration of mechanical ventilation (days) was 11.5 (IQR 9.8-16.3) and median ICU length of stay (days) was 14 (IQR 12-16.5). The pregnant woman continued the pregnancy without significant complications. In ICU infection rate was low with two ventilator associated pneumonia and two asymptomatic positive blood cultures in two ECMO patients. One ECMO patient died due to a systemic secondary infection by Aspergillus: this patient was the only non-surviving patient (overall mortality rate 8.3%).
RT-PCRs from bronchoalveolar lavage samples were positive in all patients included in this study. On the contrary, RT-PCR dosed on pharyngeal swab resulted positive in less than 70% of patients at ICU admission, and in 90% of patients in the second day ( Figure 1 ). Also efficacy of antiviral therapy was reliably followed through RT-PCR from bronchoalveolar samples, since analysis on pharyngeal swabs became negative quite early. Finally, no RT-PCR significant for H1N1 infection from subglottic aspirate sample was found.
In one patient, intravenous administration of zanamivir was needed, since the patient remained positive to viral infection after two weeks of therapy. Intravenous formulation of zanamivir is still subjected to pre-phase 4 clinical trial investigation, even if some reports on its safety profile are already available in literature. Therefore, local Ethical Committee approval was requested and the manufacturer provided the drug for use. Zanamivir was administered intravenously for five days (600 mg twice daily), as indicated by the producer. The patient's respiratory function improved and RT-PCR became negative after the third day. No adverse reaction was noted.
A total of 156 LUS have been performed. During every LUS, the following parameters were considered: pleural line aspect and motility, presence of consolidations, occurrence and severity of Alveolar Interstitial Syndrome (based on the number of B-lines), presence of pleural effusion and occurrence of pneumothorax.
Pleural thickness was described in 100% of cases and mostly bilaterally. Lung base was always involved. Lung gliding was present in 70% of LUS, even if decreased (20%). Pathological Lung Pulse was found in 20% of LUS, often in proximity to large parenchyma consolidations. Pleural effusion occurred in 7 patients. Two spontaneous pneumothorax have been detected with LUS during ICU treatment.
Alveolar Interstitial Syndrome was present in all ultrasound examinations, with the presence of normal lung pattern (spared areas). In 90% of cases, B-Lines were described as moderate/many. At lung recovery, residual B-Lines patterns were found mostly at both bases. White lung feature occurred in about 15% of LUS performed, mostly in the anterior and lateral scans. White lung was never uniformly distributed, but it was alternated to spared areas, or areas with a limited number of B-Lines.
Consolidations were found in 100% of cases. Most of them were multiple (65%), and lung bases were always involved. Contiguous subpleural consolidations were also present, increasing the pleural thickness laterally, mostly at the base and the apical part. Aerial bronchograms were always found within the consolidation pattern.
The routine use of LUS limited the number of conventional radiology examinations (Table 3 ). In ECMO patients group, the higher number of chest X-ray examinations was needed to verify the correct cannulae positioning. In both groups, bedside LUS limited the transportation to the CT-scan room, increasing patient safety and avoiding the transitory disconnection of the patient from the ventilator.
ECMO was needed in 7 patients (Table 3 ). In 4 cases, the ECMO Team was alerted and extracorporeal oxygenation was implanted directly at peripheral ICUs. No major transportation related problems were faced, even in the case of a long distance journey (400 Km). Median duration of ECMO support was 8 days (IQR 6-16.5), with a median duration of mechanical ventilation (days) of 19 (IQR 12-36).
Main clinical features and ventilatory and ECMO parameters of patients treated with ECMO are presented in Table 4 . Bleeding was the most important complication. In three cases, bleeding from vascular access sites due to heparin infusion required blood transfusions. Three patients presented prolonged oropharyngeal bleeding and transfusions were required. Among them, one needed electrical coagulation of a palatine injury, probably related to nursing manoeuvres. Two patients presented severe intra-bronchial bleeding, and several flexible bronchoscopy examinations and clot suctions were required. In one of these patients, bleeding from the lower airways during the weaning phase from ECMO, and ECMO removal has been hastened. Table 3 summarizes the main differences between patients who underwent to ECMO treatment and patients only ventilated. Despite the small sample, ECMO patients clearly showed a higher critical illness score (SAPS II), and worst pulmonary gas exchange compared to patients who did not required extracorporeal lung assistance. Coinfection and comorbidities at admission were present only in ECMO patients.
Our study population is young, comprising mainly healthy subjects, as previously reported [1, 2, 15] . Risk factors are similar to other studies, such as obesity, diabetes and pregnancy.
In the present case series, bacterial infection rate at presentation was low. Previous reports showed incidence of secondary superinfection by Streptococcus Pneumoniae, Staphylococcus Aureus, Pseudomonas Aeruginosa, Acinetobacter Baumannii, Escherichia coli [1, 3, 2] . In our experience, we found two cases (16.7%) of co-infection with Legionella Pneumophila, which is, to the best of our knowledge, a new epidemiological data, since no other case has been reported in literature. It is questionable whether Legionella Pneumophila infection occurred before or after H1N1 pneumonia. However, it could be that H1N1 pneumonia was associated with a lower reactivity of the immune system, as suggested by the low leucocytes count reported in our sample and by other Authors [3, 2, 1] .
One young patient presented heart failure, and viral myocarditis was suspected. The association of influenza with myocarditis is debated [16] , and H1N1 related myocarditis, has rarely been reported [17] . Furthermore, in our patient prolonged pre-hospital hypoxia was present and myocardial hypoxemia damage might have been involved. The patient required inotrope/vasoactive support for several days and eventually recovered fully with normal heart function.
Our observations confirm the responsiveness of this infection to antiviral therapy. We adopted a two-modality administration, both oral and inhaled. Our choice was made in consideration of the decrease in gut motility and adsorption usually observed in critically ill patients.
The World Health Organization (WHO) has questioned the sensibility of RT-PCR analysis for H1N1 in pharyngeal swab sample, encouraging analysis on samples from the lower respiratory tract. We routinely monitor H1N1 infection on three compartments: pharyngeal swab, subglottic aspiration, and bronchoalveolar lavage. In our experience, bronchoalveolar lavage at admission was positive in all patients while pharyngeal swab resulted positive in only 75% of cases.
As shown in Figure 1 , RT-PCR from pharyngeal swab at ICU admission failed to demonstrate the viral infection in 3 patients. Similarly, the time course showed that RT-PCR from pharyngeal swab resulted negative in an average time of 3 days after therapy start. Conversely RT-PCRs from bronchoalveolar lavage remained positive for a longer period and resulted more reliable for infection monitoring and assessment of the efficacy of administered therapy.
Based on our experience, RT-PCR from bronchoalveolar lavage resulted to be the most reliable method to diagnose and monitor H1N1 infection, since pharyngeal swab does not offer enough sensibility, neither for antiviral therapy initiation nor for antiviral therapy management. As subglottic aspiration resulted persistently negative, we do not recommend this sampling for diagnosis and monitoring of H1N1 infection.
Despite the severe clinical pictures, we experienced a very low mortality rate: only one patient out of 12 died (8,3%). One of the surviving patients presented a lung cavern for a past pulmonary infection, and deceased for a secondary superinfection by Aspergillus, probably already colonizing lung parenchyma before the onset of viral infection.
Our mortality rate is surprisingly low in comparison to a larger series of H1N1 patients, even when extracorporeal support technique were employed [5, 18] . Our finding can be related to the small number of patients included the study and definitive comparison with larger studies could be misleading. However, despite the severity of symptoms and the rapid progression to ARDS, H1N1 respiratory failure presents a relatively benign course when adequately treated, if compared to non-H1N1 induced ARDS, reported to have a mortality rate from 37% to 43% [19] [20] [21] [22] . Several factors may account for the favourable outcome in our series. All patients received protective ventilation. In particular, ECMO support permitted the maintenance of patients under a protective tidal volume with a respiratory rate below 12 per min, and a FiO 2 below 60%, compared with non-ECMO patients who needed a higher respiratory rate and FiO 2 to maintain an acceptable pulmonary gas exchange.
The availability of easily accessible tools for pulmonary mechanics evaluations on modern ventilators allowed an individualized and appropriate setting of ventilation pressure within the thresholds of so called "protective ventilation" [23] . Furthermore, early access to ECMO resource allowed the maintenance of protective ventilation even in more severe patients (Table 4 ). In this regard, lactate dehydrogenase is commonly considered a marker of lung damage, and in H1N1 pneumonia is reported as high [1] . In our ECMO patients, lactate dehydrogenase values presented lower levels than in non-ECMO patients (445 U/L vs 627 IU/L, respectively), suggesting that in ECMO patients the reduced need of pulmonary ventilation could reduce lung ventilatory stress and enhance healing, regardless of the more impaired lung condition.
However, it is possible that, since the technique has gained popularity and experience gathered to demonstrate its feasibility, we used ECMO also in patients who might previously have been successfully treated conventionally, and this may have influenced mortality.
Moreover, more than half of our ECMO patients needed to be land-transported from other hospitals in an advanced stage of respiratory failure. This may have further encouraged an early treatment with ECMO to ensure the safest transport. Bleeding is commonly reported during ECMO treatment [24] , and either anticoagulation or platelet and coagulation cascade activation through oxygenator and pump is involved [25] .
In our population bleeding also occurred more frequently in ECMO patients, and they required more transfusions compared to non ECMO patients. Nevertheless, in our experience, bleeding from cannulas insertion site or from upper airways, despite requiring transfusion, were not life threatening, and could be managed. In only two cases did severe bleeding occur in the lower respiratory tract. Fortunately in one case it occurred during weaning from ECMO, and it ceased after extracorporeal support removal. The other patient died from pulmonary aspergillosis and the haemorrhage could be also related to parenchyma disruption caused by the fungus.
Monitoring heparin regimen is extremely important during extracorporeal circulation, and activated clotted time is commonly measured bedside. Some debate exists regarding the optimal range and the accuracy of pointof-care measuring devices [26] [27] [28] . In our protocol, we usually measured aPTT every two hours with Hemochron Jr. in order to closely monitor heparin administration in the low range of dosage.
In our clinical practice, lung recovery and response to treatment are daily assessed by LUS examination, following several recent reports which underline the reliability of LUS in the evaluation and management of chest disorders [10, 29] . Despite CT-scan is the reference technique for evaluating lung lesions, it requires a transitory disconnection of the patient from the ventilator to permit the transportation radiology suite with potential risk of alveolar de-recruitment and worsening of oxygenation. Moreover, severe complications have been reported in intra-hospital transportation of critically ill patients [30, 31] . As we recently reported [13] , the routine use of bedside LUS has significantly reduce of the number of CT-scan and chest X-ray examinations in critical patients. The potential clinical benefit of reducing in-hospital transport for diagnostic radiology, it can be particularly relevant in patients with ECMO. In these patients, in fact, transportation requires time and a significant commitment of resources, although it was proved feasible both for inhospital [32, 33] and for inter-hospital long distance transportations [34, 35] .
Another advantage of LUS is the ability to evaluate the effectiveness of alveolar recruitment manoeuvres with the possibility to visualize real-time imagines of lung parenchyma re-aeration [8, 10, 29] . Finally, pleural effusions can be accurately diagnosed and monitored with LUS and in case of need for treatment an ultra-sound guided technique is recommended [36, 13] . This option seems to be particularly appropriate ECMO patients, where bleeding for conventional chest tube placement can occur in consideration of the need of heparin infusion. 
The present case series comprises a small number of patients, and naturally, it cannot be considered a high grade of evidence trial. However, our experience might be helpful for intensivists challenging H1N1-induced ARDS. For H1N1 infection monitoring (or diagnosis, if patient was intubated before) bronchoalveolar lavage can be more reliable than pharyngeal swab in order of the higher sensitivity. In our clinical practice, ECMO therapy resulted safe and feasible in the context of a life threatening condition, and it might be taken into consideration as a therapeutic choice rather than a rescue solution in experienced centers.
• ECMO might be taken into consideration as a safe therapeutic choice rather than a rescue solution in ARDS.
• RT-PCR from bronchial lavage is more accurate than from pharyngeal swab, in H1N1 diagnosis.
• Lung ultrasonography is a safe and reliable method to follow the pathology evolution/recovery of lung.
• Lung ultrasonography can limit the need of CT-scan and chest X-ray examinations.
List of abbreviations ARDS: acute respiratory distress syndrome; BMI: body mass index; CVVH: continuous veno-venous hemofiltration; ECMO: extracorporeal membrane oxygenation; ICU: intensive care unit; LOS: length of stay; LUS: lung ultrasound; RT-PCR: real-time reverse transcriptase-polymerase-chain-reaction; SAPS: simplified acute physiology score. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates. The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates. most other H1N1 and H5N1 influenza strains, especially in high-risk populations such as immunosuppressed patients and the elderly.
Influenza-specific plasmablasts are persistently induced throughout infection, providing a rich source of antiviral mAbs B cell responses were examined in nine patients infected with the pandemic 2009 H1N1 influenza virus. These patients had varying courses and severity of disease. The cases ranged from mild disease with rapid viral clearance within a few days after onset of symptoms to severe cases that shed virus for several weeks and required hospitalization with ventilator support. A majority of the patients were treated with antiviral drugs. The diagnoses were confirmed by pandemic H1N1-specific RT-PCR and serology. All patients had neutralizing titers of serum antibodies at the time of blood collection. A summary of the clinical patient data are shown in Table I . The majority of samples were obtained around 10 d after the onset of symptoms, with the exception of a particularly severe case where sampling was done 31 d after symptom onset.
Antigen-specific plasmablasts appear transiently in peripheral blood after vaccination with influenza or other vaccines (Brokstad et al., 1995; Bernasconi et al., 2002; Sasaki et al., 2007; Wrammert et al., 2008) , but the kinetics of their appearance and persistence during an ongoing infection remain unclear. Here, we have analyzed the magnitude and specificity of the plasmablast response in blood samples taken within weeks after onset of clinical symptoms of pandemic H1N1 influenza virus infection. Using a virus-specific ELISPOT assay, it was possible to show a significant number of pandemic H1N1-reactive plasmablasts in the blood of the infected patients, whereas none were detectable in a cohort of healthy volunteers (Fig. 1, A and B ). These cells were also readily detectable several weeks after symptom onset in the more severe cases. Fig. 1 (A and C) illustrates that, of the total IgG-secreting cells, over half of the cells were producing antibodies that bound pandemic H1N1 influenza virus. Moreover, plasmablasts specific for HA occurred at 30-50% the frequency of virus-specific cells (Fig. 1, C and D) , the specificity most likely to be critical for protection. Most patients also had a relatively high frequency of plasmablasts, forming antibodies that bound to past, seasonal influenza strains ( Fig. 1 C) or recombinant HA from the previous annual H1N1 strain, A/Brisbane/59/2007. Based on the overall frequency of pandemic H1N1-specific cells, it is likely that the cells binding other strains were overlapping populations and cross-reactive. None of the induced plasmablast cells bound to recombinant HA from the H3N2 strain from the same vaccine (A/Brisbane/10/2007). These findings demonstrate that influenza-specific human plasmablasts are continuously generated throughout an ongoing infection and that a fairly high proportion of these cells make antibodies that also cross-react with previous annual H1N1 influenza strains.
To analyze the specificity, breadth, and neutralizing capacity of these plasmablasts, we used single-cell PCR to amplify the heavy and light chain variable region genes from individually Hancock et al., 2009) . The Centers for Disease Control reports that there were an estimated 60 million cases of the 2009 H1N1 pandemic strain, which caused 256,000 hospitalizations. An unusually high frequency of severe disease occurred in younger and otherwise healthy patients (Hancock et al., 2009 ). In addition, rare infections with avian H5N1 influenza strains in humans had close to a 50% mortality rate (Subbarao and Joseph, 2007) . Emergence of a zoonotic or antigenically distinct strain that combined even a fraction of the morbidity and mortality of the pandemic H1N1 and H5N1 viruses would have dire consequences. Antibodies play a key role in protection against influenza infection in vivo (Puck et al., 1980; Gerhard et al., 1997; Luke et al., 2006; Simmons et al., 2007) . The fact that there were little or no preexisting antibody titers present before the emergence of this pandemic virus, and that the virus atypically caused such severe disease in young adult, illustrates the importance of comprehensively understanding the B cell responses and antibody specificities induced by infection with this influenza virus.
Here, we have analyzed the plasmablast responses induced by pandemic H1N1 infection and generated a panel of monoclonal antibodies (mAbs) from these cells to analyze their characteristics in detail. In contrast to seasonal vaccination, we show that a majority of the neutralizing antibodies induced by infection were broadly cross-reactive with all recent annual H1N1 strains, as well as the highly pathogenic 1918 H1N1 and avian H5N1 strains. These neutralizing antibodies bound predominantly to conserved epitopes in the hemagglutinin (HA) stalk region (Ekiert et al., 2009; Sui et al., 2009) , with some binding to novel epitopes in the HA globular head. The high frequency of these HA-stalk binding antibodies is of particular interest, as this epitope is a promising target for a broadly protective influenza vaccine (Steel et al., 2010) . Furthermore, the cross-reactive antibodies carried highly mutated immunoglobulin genes, indicative of extensive affinity maturation. Together, these findings support a model in which infection predominantly activated broadly cross-reactive memory B cells that then underwent further affinity maturation. We propose that the expansion of these rare types of memory B cells may explain why most people did not become severely ill, even in the absence of preexisting protective antibody titers. Recent studies in mice strongly support the idea that consecutive immunizations with antigens from divergent influenza stains can indeed hone the antibody response to preferentially target broadly protective conserved epitopes Wei et al., 2010) . Our findings demonstrate that cross-reactive antibodies can be preferentially induced in humans given the right immunogen, providing further support for the feasibility of generating a pan-influenza vaccine. Finally, in vivo challenge experiments showed that the neutralizing antibodies isolated protected mice challenged with a lethal dose of pandemic H1N1 influenza virus, even when administered therapeutically 72 h after infection, and also provided protection against antigenically distinct H1N1 influenza strains. These antibodies are thus promising as therapeutics against pandemic H1N1, as well as activated by pandemic H1N1 infection. Consistent with the frequency of plasmablasts secreting antibodies binding annual influenza strains by ELISPOT analyses (Fig. 1 C) , a majority (29/46, or 63%) of the pandemic H1N1-specific antibodies also cross-reacted with seasonal influenza viruses (Fig. 2,  A and B ). In fact, by ELISA, one third of these antibodies bind to the prepandemic strains at lower concentrations than they did to the pandemic H1N1 strain, suggesting higher avidity binding. By comparison, only 22% (11/50) of plasmablasts induced by annual H1N1 strains before the pandemic could bind the pandemic H1N1 influenza (Fig. S1 B) . We propose that the cross-reactivity of pandemic H1N1-induced cells derives from the activation of memory cells originally specific for past influenza immunizations in an original antigenic sin fashion.
Based on the 10-15-fold induction of plasmablasts and expression of intracellular Ki67 during ongoing immune responses (Brokstad et al., 1995; Bernasconi et al., 2002; Sasaki et al., 2007; Smith et al., 2009; Wrammert et al., 2008) , we can assume that most plasmablasts result from the ongoing infection sorted cells (defined as CD19 + , CD20 lo/ , CD3  , CD38 high , CD27 high cells; Fig. 1 E; Wrammert et al., 2008; Smith et al., 2009) . These genes were cloned and expressed as mAbs in 293 cells, and the antibodies were screened for reactivity by ELISA. Thresholds for scoring antibodies as specific to the influenza antigens were empirically determined based on being two standard deviations greater than the background level of binding evident from 48 naive B cell antibodies (Fig. S1 A) . Of 86 antibodies generated in this fashion, 46 (53%) bound pandemic H1N1 (Fig. 1 F) and one third (15 antibodies) were reactive to HA ( Fig. 1 G and Fig. S2 A) , most of them at sub-nanomolar avidities (based on surface plasmon resonance analyses; Fig. S2 B) . On a per donor basis, 55% of the mAbs bound to purified pandemic H1N1 virions (range: 33 to 77%). Of the virus-specific antibodies, 31% bound to recombinant HA (range: 14 to 55%). We conclude that virus-specific plasmablasts are readily detected after pandemic H1N1 influenza virus infection and that virus-specific human mAbs can be efficiently generated from these cells.
Plasmablasts from patients infected with pandemic H1N1 influenza were highly cross-reactive to prepandemic influenza strains As the plasmablasts are specifically induced by the ongoing immune response, we can learn about the origin of the B cells The mAb column indicates whether mAbs were made from the plasmablasts of these patients. (Table I) , mutations had accumulated at a significantly lower frequency than the IgG controls ( Fig. S3 A; P < 0.0001), suggesting a unique circumstance such as a low-level or lacking primary response. Detailed sequence characteristics for pandemic H1N1-induced plasmablasts are provided in Tables S1-S3. Though based on a limited or vaccine response. The ready detection of clonal expansions at a mean frequency of 16.5% of the cells for the six patients supports this view (based on CDR3 sequence similarity; Fig. 2 C). Since the discovery of somatic mutation, it has been appreciated that mutations progressively accumulate on variable genes after repeated immunizations (McKean et al., 1984) . Thus, we can gain insight into the origin of the pandemic H1N1 response by comparing the somatic mutation frequency of the plasmablasts present during H1N1 infection to that of other plasmablast responses. The PCR strategy allowed isolation of either IgG or IgA transcripts and identified 68% IgG and 32% IgA plasmablasts from the patients. Similar to plasmablasts induced by annual vaccination (Wrammert et al., 2008) , or after a fourth booster vaccine to anthrax, the variable genes of novel H1N1-induced cells from five of the six patients harbored high numbers of somatic mutations
Antibodies were tested at 10 µg/ml and threefold serial dilutions until a nonbinding concentration was determined. Each antibody was tested in at least two (and typically more) replicates for specificity and affinity estimations. Note that only 14 of 15 HA-binding antibodies have curves in G because one of the HA-reactive antibodies only binds HA on whole virions, not on the recombinant protein.
H1N1 reactive mAbs isolated from infected patients (1000, EM, 70, 1009) were assayed for binding to annual H1N1 influenza strain whole virus. The minimum detectable concentration is defined as two standard deviations above the mean binding of 48 randomly chosen naive B cell antibodies ( Fig. S1 A) . Bars are color coded to approximate levels of crossreactivity to the annual vaccine (circulating) strains of recent years. Panels A and B use the same color scheme. Each value is representative of at least two replicate ELISAs repeated until a single consistent minimum concentration was established. The center numeral equals total antibodies. (C) Analysis of the variable gene sequences from plasmablasts of the four pandemic H1N1-infected patients indicated that 16.5% of the pandemic H1N1-induced plasmablasts were clonally related (shared identical VH and JH genes and CDR3 junctions). (D) The average number of somatic hypermutations in the pandemic H1N1 patient plasmablast variable region genes compared with primary IgG plasmablast responses to vaccinia (small pox) or the anthrax vaccine, or after at least 4 boosters with the anthrax vaccine. To account for the obvious outlier in the pandemic H1N1 group (patient-EM), median values are indicated by the bar. Student's t tests excluding the outlier indicated a p-value of <0.04 for the remaining five pandemic H1N1 samples compared with the IgG memory and germinal center (GC) cells or the primary IgG plasmablast responses (0.2 with EM included) and a p-value of <0.0001 against the IgM populations. Notably, besides patient EM, each individual set of VH genes averaged significantly more mutations than the IgG memory and GC or the primary responses ( Fig. S3 A) . Each point represents one individual donor and is averaged from 25-75 sequences, except for the primary response to anthrax from which only 10 VH genes could be cloned from single cells because of the highly limited response. Mutations accumulated per individual sequence are depicted in Fig. S3 . Detailed sequence characteristics are provided in Tables S1-S3. The naive, IgG and IgM GC and memory populations are derived from historical data (Zheng et al., 2004 (Zheng et al., , 2005 Koelsch et al., 2007; Wrammert et al., 2008) .
indicating that they bound to sites other than the HA active site. Interestingly, antibodies of the latter type were predominant in the response (Fig. 3 A) . This specificity is reminiscent of antibodies against the recently discovered broadly neutralizing epitopes found on the HA stalk, rather than those located on the HA globular head that is more typical for neutralizing antibodies (Ekiert et al., 2009; Sui et al., 2009) . Importantly, five of these antibodies are indeed of similar specificity (including antibodies 70-5B03, 70-1F02, 1000-3D04, and a clonal pair from donor 1009: 3B05 and 3E06). These five antibodies bind with high affinity to most H1 strains including all from the vaccines of the past 10 yr, the 1918 pandemic strain, and to the H5 of a highly pathogenic number of patients, the frequent cross-reactivity and high number of somatic mutations support a model in which many of the plasmablasts induced by pandemic H1N1 infection arose from cross-reacting memory B cells.
A majority of the neutralizing antibodies bound to highly conserved epitopes in both the HA stalk and head regions A high frequency of the HA-specific antibodies was able to neutralize the virus in vitro (totaling 73% or 11/15; Fig. 3 A) . These neutralizing antibodies could be further categorized into two distinct groups: (a) neutralizing antibodies that displayed hemagglutination inhibition (HAI) activity (HAI + ) and (b) neutralizing antibodies that had no HAI activity, Fig. S2 A) . The antibodies are grouped based on whether they show HAI and/or neutralizing (neut) function. Antibody 1009-3B06 was only tested for binding to whole virus, as this antibody did not bind to rHA due to binding of a quaternary or conformationally sensitive epitope that is not present in the recombinant protein. HAI and neutralization assays were performed in duplicate and repeated at least three times. ELISA curves are provided in Fig. S2 A. (B) ELISA binding as shown by minimum positive concentration (defined for Fig. 2 ) of neutralizing mAbs to rHA or whole virions from pandemic H1N1 or other influenza strains (ELISA binding curves are provided in Fig. S2 A) . Three binding patterns (epitopes 1 and 2, and 3) were observed that coincided with specificity comparisons by competitive ELISA, as illustrated in Fig. 4 A. (C) Three representative neutralizing antibodies (EM-4C04, 70-1F02, and 1009-3B06) were used for HAI and microneutralization (MN) activity against pandemic H1N1 and several other annual or laboratory H1N1 influenza strains. Experiments were performed in duplicates and repeated at least three times. Minimum effective concentration is shown for both assays.
FACS analysis showed that the five antibodies bound to all 13 H5 variants tested at levels quite similar to F10, for which a crystal structure had been generated to define this epitope. Thus, half of the neutralizing and a surprising 10% of all antibodies induced by pandemic H1N1 infection bound to a conserved, critical epitope on the HA stalk. By comparison, none of 50 H1N1 strain-specific antibodies that we had previously isolated after annual vaccination before the 2009 pandemic had this reactivity (unpublished data). The frequency of pandemic-induced, stem-reactive antibodies (5/46) versus those from annual vaccine (0/50) is significantly greater (Chi-square test, P = 0.02). Further, this specificity is only rarely seen in human memory B cells (Corti et al., 2010) or from phagedisplay libraries (Sui et al., 2009 ). These observations support avian influenza strain (Fig. 3 B and Fig. S2 A) . In addition, these five antibodies cross-compete for a similar epitope that was not over-lapping with the HAI + antibodies (Fig. 4 A, epitope-1). These antibodies are competitively inhibited by a commercial antibody referred to as C179 that binds this HA stalk region (Okuno et al., 1993) , and four of five of these antibodies are encoded by the hallmark VH1-69 gene (Ekiert et al., 2009; Sui et al., 2009) . To verify HA stalk reactivity, these five antibodies were tested for binding to H5 variants predicted to affect the stalk epitope by the crystal structure, and their binding patterns were compared with that of the prototypical stalk antibody (mAb F10; Sui et al., 2009; Fig. 4 B) . Each H5 variant has a single residue mutation in the stalk region and was transiently expressed on 293T cells. (A) Competition ELISA assays were used to determine the similarity in specificity between the various neutralizing antibodies. Shown is the percentage of competition of each antibody in an ELISA binding assay against all other neutralizing antibodies. A 10-fold molar excess of unlabeled antibody was used to inhibit a biotinylated antibody. Percent competition is calculated as the reduction in absorbance relative to the level of inhibition of any particular antibody against itself. Colors indicate degree of inhibition of antibody binding, as indicated. Antibody C179 is a commercial antibody that binds to the stalk region of the HA molecule identifying epitope-1. Epitope-2 and -3 are each on the HA-head active site. 1000-2G06 and the nonneutralizing, but HAbinding, antibodies had no competition with any of the other HA-reactive antibodies and are therefore not shown. VH gene usage of the individual antibodies is listed on the right. All assays were performed in duplicate. (B) Plasmids encoding full-length WT H5-TH04 (A/Thailand/2-SP-33/2004 [H5N1]) and its mutants were transiently transfected into 293T cells. 24 h after transfection, cells were harvested for FACS analysis, and binding of indicated antibodies was tested at 10 µg/ml. The cell surface HA expression of each of the mutants were verified with a ferret anti-H5N1 serum (not depicted). Antibody F10 was one of the antibodies used to characterize the HA stalk epitope by x-ray crystallography (Sui et al., 2009 ) and served as a positive control for the binding pattern expected of HA stalk-reactive antibodies to these HA mutants.
to all recent H1 vaccine strains and reacted strongly to the 1918 pandemic strain (antibodies 1009-3E04 and 1000-3E01; Fig. 3 B and Fig. 4 A, epitope-3) . These mAbs bind to past vaccine strains with higher avidity than to the pandemic H1N1. Further studies are underway to precisely identify the epitopes of all neutralizing antibodies in this study.
Only two of 11 neutralizing antibodies were highly specific for the pandemic H1N1 strain alone (Fig. 3 B and Fig. S2 A) , including a low avidity antibody, 1000-2G06, which only showed slight neutralization capacity in vitro, and EM-4C04, which was very effective at neutralizing the pandemic H1N1 influenza. We conclude from these experiments that a surprising 82% (9/11) of the neutralizing plasmablasts that we isolated during pandemic H1N1 influenza infections were broadly cross-reactive to multiple influenza strains.
There is a distinct interest in developing monoclonal antibodies for use in a therapeutic setting. We selected three representative antibodies of the set we have identified for detailed functional analysis both in vitro (Fig. 3 C) and in vivo ( Fig. 5 and Fig. 6) , including: EM-4C04, 1009-3B06, and 70-1F02. The antibodies EM-4C04 and 1009-3B06 are specific for the active site of the HA molecule, whereas 70-1F02 binds to the stalk region. Furthermore, EM-4C04 is highly specific for pandemic H1N1, whereas 1009-3B06 and 70-F02 display the idea that a vaccine might be developed that preferentially targets the HA stalk, thus providing broad protection against many influenza strains.
The remaining neutralizing antibodies were HAI + and therefore bound to the HA globular head. Based on crosscompetition analyses, these antibodies fell into two groups binding nonoverlapping regions of the HA head, including epitope-2 and epitope-3 (Fig. 3 B and Fig. 4 A) . Indeed, using spontaneous escape mutant selection, we found that the EM4C04 mAb binds to the Sa region of the HA globular head (unpublished data). Thus, by proximity based on the competition assay (Fig. 4 A) , we can predict that all of the epitope-2 antibodies bind near the Sa/Sb region (including EM-4C04, 1009-3B06, and 1009-3F01).
Broadly reactive antibodies binding both pandemic H1N1 strains and common annual H1N1 strains have been identified both in humans Xu et al., 2010) and in mice (Manicassamy et al., 2010) . It is notable that three of five of the HA globular-head-binding antibodies induced by pandemic H1N1 infection were also broadly reactive to various H1N1 strains (Fig. 3 B) . One such novel antibody was the SF1009-3B06 antibody that reacts strongly with the pandemic H1N1 strain, as well as all recent H1N1 vaccine strains (Fig. 3 B and Fig. S2 A) . The precise epitope to which the 1009-3B06 antibody binds appears to be quite unique; it is only accessible on whole virions, not on recombinant HA, suggesting that the epitope is quaternary in nature. Finally, two antibodies cross-reacted and inhibited hemagglutination Figure 5 . In vivo prophylactic and therapeutic efficacy of human mAbs against pandemic H1N1 influenza virus. 6-8-wkold BALB/c mice were infected with a 3xLD50 dose of highly pathogenic, mouse-adapted 2009 pandemic H1N1 influenza (A/California/ 04/09). 24, 48, and 60 h after infection, 200 µg (10 mg/kg of body weight) of EM-4C04, 70-F02, or 1009-3B06 human mAb were injected intraperitoneally. All mice were monitored daily for body weight changes and any signs of morbidity and mortality. Percentage of initial body weight is plotted, and the number of surviving mice is shown in the lower right of each plot. Infected, untreated mice showed clear signs of sickness around day 4-5 after infection and perished by day 8-9. Prophylactic treatment is shown on the left for comparison. Antibody treatment conferred significant protection as determined by comparison of weights in untreated versus prophylaxis and at the time of treatment versus 12 d after infection (unpaired, twotailed Student's t test, P < 0.05). The log-rank test indicated significant survival as well (P < 0.001). Figure shows one representative experiments of at least three independent repeat experiments.
for the pandemic H1N1, had no protective effect on infection with PR/8/34 or FM/1/47. In conclusion, the antibodies characterized herein show promise for development as broadly reactive therapeutic agents against the pandemic H1N1 influenza virus, as well as against the majority of H1N1 and H5N1 influenza strains.
Our findings provide insight into the human B cell responses to a pandemic influenza virus strain. The unique genetic composition of the pandemic H1N1 influenza virus meant that our relatively young cohort probably had little or no preexisting specific antibody-mediated immunity to this virus before infection (Brockwell-Staats et al., 2009; Dawood et al., 2009; Garten et al., 2009; Hancock et al., 2009) . Thus, two sources of B cells could have contributed to this response: newly recruited naive B cells and preexisting memory B cells that bound to epitopes conserved between past seasonal strains and the pandemic H1N1 strain. We theorize that predominant activation of the latter, preexisting memory cells can account for the observed high frequency of neutralizing antibodies (11/15 HA-binding antibodies), the majority (9/11) of which are cross-reactive with seasonal H1N1 strains (Fig. 3 C) and other group 1 influenza strains, including H5 HA. Several observations support this conjecture.
Most convincingly, there was a particularly high frequency of cross-reactive antibodies overall, with a high level of somatic mutations found particularly among the variable genes of cross-reacting cells (Fig. 2 and Fig. S3 ). In fact, by ELISA most antibodies were cross-reactive and one third of the broadly cross-reactive binding (Fig. 3 B) and have functional activity against multiple recent and older H1N1 strains (Fig. 3 C) . These antibodies were all highly effective at providing prophylactic protection against infection with a lethal dose of mouse-adapted pandemic H1N1 in 6-8-wk-old BALB/c mice (Fig. 5) . Moreover, all three antibodies were effective therapeutically, even when they were administered as late as 60 h after the lethal challenge infection, well after the mice were symptomatic. For EM-4C04, we have successfully treated mice as far out as 72 h post-infection (unpublished data). Infected mice were already showing measurable weight loss that was reversed by administration of the antibody, demonstrating therapeutic potential even after the onset of disease. Viral clearance was analyzed in mice treated at 48 h after infection with EM4C04 (Fig. S4) . As early as day 4, the antibody-treated mice exhibited more than a log reduction in viral titers; titers continued to decline, such that by day 6, virus was undetectable or present at very low levels. The untreated mice perished by day 7 or 8, whereas the treated mice cleared the infection with no detectable virus on day 12. Finally, 1009-3B06 and 70-1F02, which showed activity against several current and older H1N1 seasonal influenza strains in vitro (Fig. 3 C) , were also tested in vivo against antigenically distinct influenza strains. For these experiments, mice were treated with 200 µg of mAb intraperitoneally 12 h before infection with a lethal dose of either pandemic H1N1 influenza or either of the two common influenza laboratory strains PR/8/34 or FM/1/47. 1009-3B06 and 70-1F02 showed protection against these antigenically distinct H1N1 influenza strains, as illustrated in Fig. 5 . EM-4C04, which is highly specific Figure 6 . Breadth of in vivo prophylactic efficacy in mice. 6-8-wk-old BALB/c mice were treated with 200 µg (10 mg/kg of body weight) EM-4C04, 70-1F02, or 1009-3B06 human mAb intraperitoneally. Control mice were treated with PBS only, a control mAb or polyclonal human IgG. 12 h later, they were challenged with a 3xLD50 dose of mouse adapted pandemic H1N1, PR/8/34, or FM/1/47 influenza virus. All mice were monitored daily for body weight changes and any signs of morbidity and mortality. Percentage of initial body weight (left) and survival curves (right) are plotted. Infected, untreated mice showed clear signs of sickness 4-5 d after infection and perished by day 8-9. Figure shows one representative experiments of at least three independent repeat experiments. Antibody treatment conferred significant protection as determined by comparison of weights in untreated versus prophylaxis, and at the time of treatment versus 12 d after infection (unpaired, two-tailed Student's t test, P < 0.05). The log-rank test indicated significant survival as well (P < 0.003). seroconversion, or by the presence of highly potent antibodies, such as EM-4C04, whose activities were less likely to titer out. The highly specific nature of the response from this patient may have contributed to this advantage, ultimately better targeting the epitopes of the pandemic H1N1 strain. In contrast, patient 1009 had relatively low HAI and MN serum titers but the highest frequency of broadly neutralizing antibodies and a less severe disease course. One possibility is that our sampling from this patient was done before peak serological responses. Another possibility is that the high frequency of these potent antibodies in the memory B cell compartment may have resulted in rapid resolution of infection, precluding the development of a high serological response. A third possibility is that despite broader protection, the stalk-reactive antibodies are on the whole less potent and more rapidly titrated out than the highly specific antibodies to the HA globular head. These various possibilities will be of significant interest to study in the future.
Finally, we report the development of a large panel of human mAbs induced by pandemic H1N1 infection. Prophylactic therapy with polyclonal or mAbs has successfully been used for RSV, rabies, Hepatitis A and B, and varicella. In the case of influenza, mAbs have been shown to provide prophylactic or therapeutic protection in mice and other animal models (Reuman et al., 1983; Sweet et al., 1987; Palladino et al., 1995; Renegar et al., 2004) . Passive transfer of maternal antibodies in humans has also been shown to confer protection (Puck et al., 1980) . Several of the antibodies we isolated have broad neutralization capacity in vitro against divergent influenza strains and show potent prophylactic and therapeutic activity when used to treat mice that were lethally infected with influenza. These antibodies could provide much needed pandemic therapeutics to treat severe cases of influenza and to protect high-risk populations.
In conclusion, analyses of the 46 mAbs induced by pandemic H1N1 infection indicated frequent activation of broadly reactive B cells. We propose that these cells had a memory cell origin caused by cross-reactivity to conserved and functionally important epitopes. If true, it will be important to characterize the efficacy of the pandemic H1N1 vaccine to induce a similarly cross-protective response.
All studies were approved by the Emory University, University of Chicago, and Columbia University institutional review boards (Emory IRB#22371 and 555-2000, U of C IRB# 16851E, CU IRB#AAAE1819). Patient clinical information is detailed in Table I. PBMC and plasma isolation. All work with samples from infected patients was performed in a designated BSL2 + facility at Emory University. Peripheral blood mononuclear cells (PBMCs) were isolated using Vacutainer tubes (BD), washed, and resuspended in PBS with 2% FCS for immediate use or frozen for subsequent analysis. Plasma samples were saved at 80°C or frozen in medium with 10% dimethyl sulfoxide for subsequent analysis.
Viruses and antigens. The pandemic H1N1 influenza virus (A/California/ 04/2009) was provided by R.J. Webby (St. Jude Childrens Hospital, Memphis, TN). Influenza virus stocks used for the assays were freshly grown in eggs, antibodies bound to past annual viral antigens at lower concentrations, suggesting higher avidity to past influenza strains than to the current pandemic H1N1 virus. Further, crossreacting cells that bind with higher affinity to the pandemic H1N1 strain also have the highest frequency of variable-gene mutations (Fig. S3 B) . Antibodies that were broadly crossreactive were among the more highly mutated clones (Fig. S3 B) . We propose that many of these cells were specific for crossreactive epitopes present in annual influenza strains that then underwent further affinity maturation and adaptation to the infecting pandemic H1N1 virus. Supporting this conjecture, Corti et al. (2010) first demonstrated that naturally occurring HA stalk-reactive memory B cells could be isolated from the blood of people recently immunized with the annual vaccine, before the outbreak of pandemic H1N1. The nature of that study was to screen EBV-transformed memory cell lines, thus precluding the determination of precise frequencies of these stalk-reactive B cells. However, these antibodies were estimated to be quite rare; occurring at one in thousands to one in hundreds of influenza-binding B cells, varying by individual. In contrast, we show that plasmablasts activated by infection with the highly novel pandemic H1N1 influenza strain have substantially increased targeting to the HA stalk region epitopes, totaling 10% of all influenza-specific antibodies and half of the neutralizing antibodies (Fig. 4) . In fact, most specific antibodies isolated in this study were cross-reactive to past influenza strains. Collectively, the data described supports a model in which divergent viruses that are conserved only at the most critical regions for function will elicit a higher proportion of cross-reactive and neutralizing antibodies. Thus, although the activated plasmablasts of relatively few patients could be analyzed in detail at the monoclonal antibody level, we proffer that with the proper immunogen, the long-sought development of a pan-influenza vaccine might be possible.
Interestingly, the highly specific antibody EM-4C04 was derived from a patient that had a very severe disease course, with persistent viral shedding over several weeks. In addition, the variable genes from the plasmablasts of this patient had the lowest average number of somatic mutations (Fig. 2 B , outlier, and Fig. S3 B) . Collectively, the unique specificity against pandemic H1N1, the low levels of somatic mutation, and the unusually severe disease in the absence of predisposing conditions suggest that this patient may have mounted a primary immune response to the pandemic H1N1 influenza infection. The complete lack of preexisting immunity may have contributed to the more severe disease observed in this patient. In contrast, the activation of broadly cross-neutralizing memory B cells in those with immune experience to annual strains might have contributed to the less severe disease of most infected patients during the pandemic.
It is notable that there is a discrepancy between patients for serum MN titers, the severity of disease, and the frequency of plasmablasts expressing neutralizing antibodies (Table I and Fig. 3 ). For example, patient EM, despite having the worst disease course, had the greatest HAI and MN serum titers. This may be caused by the time from infection (day 31), allowing full conditions that were previously published (Wrammert et al., 2008; Smith et al., 2009) . Variable genes were determined using in-house analysis software compared with the Immunogentics V gene dataset and the IMGT search engine (Ehrenmann et al., 2010; Lefranc et al., 2009 ). Background mutation rates by this method is 1 base-exchange per 1,000 bases sequenced (based on sequences of constant region gene segments). Comparisons were made to historical data, some of which was previously published (Zheng et al., 2005; Wrammert et al., 2008; Duty et al., 2009) .
Plaque assay and PRNT 50 assay. MDCK cells were grown in 6-well plates at a density of 8 × 10 5 /well. On the next day, cells were washed with PBS. 10-fold dilutions of virus were added in 500 µl DME and incubated at 37°C for 1 h, with mixing every 10 min. Cells were washed with PBS and overlayed with 199 media containing 0.5% agarose (Seakem), 1x antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 0.2% BSA (Sigma-Aldrich), and 0.5 µg/ml TPCK-Trypsin (Sigma-Aldrich). Cells were incubated for 36-40 h and fixed with 2% PFA for 10 min. Agarose plugs were removed and cells were stained with 0.1% crystal violet in 25% EtOH for 1 min. After removal from the crystal violet solution, plates were dried and used to count plaques in each well. For PRNT 50 assay, MDCK cells were prepared as above. On the next day, mAbs were threefold-diluted (60-0.74 µg/ml). 100 PFU of virus in 250 µl DME were incubated with equal volume of diluted mAbs at 37°C for 1 h before the plaque assay. Plaques were counted and the final concentration of antibodies that reduced plaques to <50 PFU were scored as PRNT 50 .
To determine the TCID 50 , MDCK cells were grown in 96-well plate at a density of 1.5 × 10 4 /well. On the next day, cells were washed with PBS and 10-fold diluted viruses in 100 µl DME were added into each well and incubated at 37°C for 1 h. After the incubation, cells were washed with PBS and 100 µl of DME containing 1x antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 0.5% BSA (Sigma-Aldrich), and 0.5 µg/ml TPCK-Trypsin (Sigma-Aldrich) was added. Cells were further incubated for 60 h, and 50 µl of the supernatant was incubated with equal volume of 0.5% of PBS-washed Turkey red blood cells (Rockland Immunochemicals) for 30 min. Four replicates were performed for each dilution, and complete agglutination was scored as HA + . Virus titers were calculated by the Reed-Muench method. For MN assay, 100 TCID 50 of virus in 50 µl DME were incubated with 50 µl of threefold-diluted antibodies (60-0.082 µg/ml) at 37°C for 1 h. Cells were washed and incubated in the media as described for the HAI assay for 60 h. The MN titer was determined to be the final concentration of mAbs that completely inhibited infection.
HAI and ELISA assays. Whole virus, recombinant HA, or vaccine-specific ELISA was performed on starting concentrations of 10 µg/ml of virus or recombinant HA and on 1:20 dilution of the vaccine, as previously described (Wrammert et al., 2008) . In brief, microtiter plates were coated with live virus strains totaling 8 HAU of total virus per well or with 1 µg/ml of recombinant HA protein. To standardize the various ELISA assays, common highaffinity antibodies with similar affinities and binding characteristics against each virus strain were included on each plate, and the plate developed when the absorbance of these controls reached 3.0 ± 0.1 OD units. Goat anti-human IgG (goat anti-human I-peroxidase-conjugate; Jackson ImmunoResearch Laboratories) was used to detect binding of the recombinant antibodies, followed by development with horseradish peroxidase substrate (Bio-Rad laboratories). Absorbencies were measured at OD415 on a microplate reader (Invitrogen). Affinity estimates were calculated by nonlinear regression analysis of curves from eight dilutions of antibody (10 to 0.125 ug/ml) using GraphPad Prism. The HAI titers were determined as previously described (Wrammert et al., 2008) . In brief, the samples were then serially diluted with PBS in 96-well v-bottom plates and 8 HAU (as determined by incubation with 0.5% turkey RBCs in the absence of serum) of live, egg-grown virus was added to the well. After 30 min at room temperature, 50 µl of 0.5% turkey RBCs prepared, and purified as previously described (Wrammert et al., 2008) . The hemagglutination inhibition activity was determined using turkey red blood cells (Lampire Biological Laboratories) as previously described (Wrammert et al., 2008) ELISPOT assay. Direct ELISPOT to enumerate the number of either total IgG-secreting, pandemic H1N1 influenza-specific, or vaccine-specific plasmablasts present in the PBMC samples were essentially done as previously described (Crotty et al., 2003) . In brief, 96-well ELISPOT filter plates (Millipore) were coated overnight with either the optimized amounts of purified pandemic H1N1 virions, recombinant HA from the pandemic H1N1 (as above), the 08/09 influenza vaccine at a dilution of 1/20 in PBS, or goat anti-human Ig (Invitrogen). Plates were washed and blocked by incubation with RPMI containing 10% FCS at 37°C for 2 h. Purified and extensively washed PBMCs or sorted plasmablasts were added to the plates in dilution series and incubated for 6 h. Plates were washed with PBS, followed by PBS containing 0.05% Tween, and then incubated with a biotinylated anti-huIgG () antibody (Invitrogen) and incubated for 1.5 h at room temperature. After washing, the plates were incubated with an avidin-D-HRP conjugate (Vector Laboratories) and, finally, developed using AEC substrate (3 amino-9 ethylcarbazole; Sigma-Aldrich). Developed plates were scanned and analyzed using an automated ELISPOT counter (Cellular Technologies, Ltd.).
Flow cytometry analysis and cell sorting. Analytical flow cytometry analysis was performed on whole blood after lysis of erythrocytes and fixing in 2% PFA. All live cell sorting and single cell sorting was performed on purified PBMCs using either a FACSVantage or ARIAII cell sorter system. All of the following antibodies for both analytical and cell sorting cytometry were purchased from BD, except anti-CD27, which was purchased from eBioscience: anti-CD3-PECy7 or PerCP, anti-CD20-PECy7 or PerCP, anti-CD38-PE, anti-CD27-APC, and anti-CD19-FITC. ASCs were gated and isolated as CD19 + CD3  CD20 lo/ CD27 high CD38 high cells. Flow cytometry data were analyzed using FlowJo software.
Generation of mAbs. Identification of antibody variable region genes were done essentially as previously described (Smith et al., 2009; Wardemann et al., 2003; Wrammert et al., 2008) . In brief, single ASCs were sorted into 96-well PCR plates containing RNase inhibitor (Promega). VH and V genes from each cell were amplified by RT-PCR and nested PCR reactions using cocktails of primers specific for both IgG and IgA using primer sets detailed in (Smith et al., 2009 ) and then sequenced. To generate recombinant antibodies, restriction sites were incorporated by PCR with primers to the particular variable and junctional genes. VH or V genes amplified from each single cell were cloned into IgG1 or Ig expression vectors, as previously described (Wardemann et al., 2003; Wrammert et al., 2008; Smith et al., 2009) . Antibody sequences are deposited on GenBank (accession nos. HQ689701-HQ689792 available from GenBank/EMBL/DDBJ). Heavy/light chain plasmids were cotransfected into the 293A cell line for expression and antibodies purified with protein a sepharose. Antibody proteins generated in this study can be provided in limited quantities upon request.
Mutational analysis. Antibody anti-H1N1 induced plasmablast variable genes were amplified by single-cell RT-PCR using primer sets and PCR efficacy of the mAb, mice were treated intraperitoneally with 200 µg (10 mg/kg of body weight) of the specific mAbs. 12 h later, mice were challenged with 3xLD50 of one of the mouse adapted influenza viruses used in the study. All mice were monitored daily for any signs of morbidity and mortality. Body weight changes were registered daily for a period of 14 d. All mice that lost >25% of their initial body weight were sacrificed according to the institutional animal care and use committee guidelines. To determine the therapeutic efficacy of the mAbs, mice were challenged with 3xLD50 of the mouse-adapted pandemic H1N1 virus. At various times after infection (12, 24, 36, 48, 60 , and 72 h) mice were treated intraperitoneally with 200 µg (10 mg/kg of body weight) of the specific mAbs. All mice were monitored daily and the body weight changes were registered daily as described above.
Statistical analysis. Data were collected and graphed using MS Excel and GraphPad Prism software. Efficacy of the therapeutic and challenge experiments was evaluated by analysis of variance using GraphPad Prism software.
Online supplemental material. Fig. S1 shows the binding characteristics of control mAbs. Fig. S2 shows further binding characteristics of the neutralizing mAbs. Fig. S3 shows further analysis of pandemic H1N1-induced plasmablast somatic mutations. Fig. S4 shows experiments demonstrating the therapeutic control of pandemic H1N1 viral titers in lungs after mAb treatment. Tables S1-S3 provide detailed characteristics concerning the variable gene sequences cloned from pandemic H1N1 induced plasmablasts. Online supplemental material is available at http://www.jem.org/cgi/ content/full/jem.20101352/DC1. Annual Symposium of the Society for the Study of Inborn Errors of Metabolism  A family of eukaryotic proline racemase-like genes has recently been identified. Many members of this family, including a human protein called C14orf149 lack a cysteine residue at the 300 position which is criticial for the racemase activity. The function of these enzymes has remained unresolved until now. We demonstrate that human C14orf149 catalyzes the degradation of trans-3-hydroxy-L-proline to delta-1-pyrroline-2-carboxylate (P2C). This is the first enzyme of this subclass of proline racemase-like genes for which the enzymatic activity has been resolved. It is also the first human enzyme that acts on 3-hydroxyproline. A mutant enzyme in which the threonine-300 in the active site is mutated back into cysteine, regains 3-hydroxyproline epimerase activity. This result and the evolutionary relationship between enzymes of the proline racemase-like family, suggests that a single mutation gave rise to this subclass of enzymes.
Presumably, human C14orf149 serves to degrade 3-hydroxyproline that is released by the degradation of proteins that contain this amino acid such as collagen IV, an important structural component of basement membrane. Interestingly, the 14q23 locus containing the C14orf149 gene has been linked to familial aneurisms.
A classic-MSUD patient was admitted at the age of 10 yrs because of severe neurological deterioration (ataxia, hallucinations, drowsiness) during severe decompensation due to gastroenteritis. Despite previous decompensation episodes, his neurological status was unremarkable. On admission, plasma leucine level was 1400 μmol/l, and BCAA-free aminoacid mixture (2 g/kg/ day) was immediately started. After 12 hours a brain MRI was performed, showing a picture resembling Wernicke encephalopathy (WE), characterized by FLAIR and DWI symmetric hyperintensity at the level of midbrain, hypothalamus, mamillary bodies and thalami. As WE is attributed to thiamine deficiency, thiamine supplementation (1 g/day i.v.) was started, with complete clinical and neuroradiologic recovery. A second classic-MSUD patient previously admitted with the same clinical picture and treated by analogous dietary adjustments, was retrospectively recognized with WE-like MRI lesions. The patient recovered completely even without thiamine supplementation. As WE pathophysiology seems to be mainly due to α-ketoglutarate dehydrogenase (α-KGDH) inhibition, we hypothesized a possible common pathogenesis of neuronal damage between the two disorders. Interestingly, α-ketoisocaproic acid was shown to selectively inhibit α-KGDH in brain mitochondrial preparations (Amaral AU, 2010) . These findings might explain the neuroradiological picture of WE in MSUD and its reversibility with decompensation treatment, furthermore MSUD should be included as a cause of WE. Background: Tyrosinaemia type III (4-hydroxyphenylpyruvate dioxygenase deficiency) is extremely rare and the cognitive outcome is not well defined.
Objectives: (i) to document cognitive profiles in tyrosinaemia III; (ii) to explore the relationship between dietary treatment and cognitive outcome.
Methods: Cross-sectional study of 11 patients with tyrosinaemia III (4 males, 7 females), age range 2-17 years (median 8) using age-appropriate cognitive tests. A questionnaire was completed by school relating to behaviour and special educational needs. Age at which dietary treatment was commenced and the pre-diet and post-diet plasma tyrosine levels until age 5 years were recorded. Results: Pre-diet plasma tyrosine concentrations were between 500 and 1500 μmol/l. Median age at commencement of dietary treatment was 12 weeks (range 5-112 weeks). Post-diet tyrosine concentrations ranged from 60-300 μmol/l. 75% of IQ scores were borderline (70-80) or extremely low (<70) with no significant differentiation between verbal and nonverbal scores. 2 patients had special educational needs and 2 had concentration difficulties. There was a weak negative correlation between the age at which patients started dietary treatment and full scale IQ (r=−0.514; p=0.192 Objectives: to examine the longterm outcome of treated homocystinuria patients (HCU, CbS deficiency), and to examine ways to quantify patient exposure to free homocystine and total homocysteine. Methods: A retrospective case note review of 46 HCU patients (92% pyridoxine nonresponsive), born between 1971-2009. Patients were divided into 4 groups: 23 New Born Screening (NBS) good/moderate control (Group I); 7 NBS poor control (Group II); 10 not detected on NBS (Group III); 6 born pre-NBS (Group IV). Median age of 28.23 years (0.4 -54.6 ) included 950 treatment years. Free homocystine measured between 1971-2009, and total homocysteine from 1997. Alternative statistical models were used to determine control: area under HCy curve; and expected homocystine level (free homocystine exposure/ years of exposure); level of exposure to free homocystine to time of clinical event.
Results: complication rate for Group I 2%, Group II 17%, Group III 45% and Group IV 36%. 84% of NBS attained 3 rd level education, compared to 43% for those presenting clinically.
Conclusions: (based on analysis of free homocystine): children with expected free homocystine levels >12.3 μmol/L are likely to develop ectopia lentis, and children, whose cumulative exposure to homocystine <100 μmol/L up to 4 years, are less likely to develop learning disabilities.
Background: Acute decompensations of maple syrup urine disease (MSUD) are usually treated by enteral feeding including amino-acids mixture without leucine, valine, isoleucine. However, it is difficult in case of gastric intolerance. Thus, we developed a new parenteral amino acids mixture.
Methods: 17 decompensations in 4 adult patients with MSUD treated by parenteral mixture (group P) were compared to 18 previous decompensations treated by enteral feeding in the same patients (group E). Total amount of leucine in blood was estimated as 0.65xbody weight xleucine concentration and clearance of leucine at day 3 (D3) was calculated by initial minus D3 amount of leucine/3. Results: Mean leucine concentration at presentation was similar in the groups P and E (15.7 and 15.9 mg/dL). Mean clearance of leucine at D3 was significantly higher in the group P than in the group E (1759.4±1021.0 vs 982.2±958.5 mg/day, P=0.026), without side effect. Mean duration of hospitalisation was similar (4 vs 4.5 days, P=NS) . No patient in the group P worsened and needed to be dialysed whereas one patient was dialysed in the group E.
Conclusion: This new parenteral amino acids mixture is safe and more efficient for the treatment of acute MSUD decompensation than classic enteral feeding. Background: Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism, especially in tyrosinemia type II, which is caused by deficiency of tyrosine aminotransferase. Hypertyrosinemia is associated with neurologic and development difficulties in several patients and the mechanisms of brain damage in this disorder are poorly known. Objectives: We investigated the effect of L-tyrosine administration on acetylcholinesterase activity in hippocampus, striatum, cerebral cortex and serum of rats. Methods: For acute administration, 10-and 30-day-old Wistar rats were killed one hour after a single intraperitoneal injection of L-tyrosine (500 mg/kg) or saline. For chronic administration, two injections (at 12 h interval) of L-tyrosine (500 mg/kg) or saline were given starting at postnatal day 7 for 21 days; twelve hours after the last injection, the animals were killed. Brain was removed and serum was collected for acetylcholinesterase activity evaluation. Results: We observed that acute (10-and 30-day-old) and chronic administration of L-tyrosine increased acetylcholinesterase activity in hippocampus, striatum, cerebral cortex and serum, as compared to control group. Conclusions: These results suggest that L-tyrosine administration may alter cholinergic synapses, and this may be involved in the pathophysiology of brain damage found in patients affected with hypertyrosinemia. Background: Maple syrup urine disease (MSUD) is an inherited aminoacidopathy resulting from dysfunction of the branched-chain keto acid dehydrogenase complex, leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine. Affected patients usually present CNS disturbances. Thus, we investigated BDNF levels in brain of rats submitted to an animal model of MSUD. Methods: For acute administration, three injections (1 h interval) of BCAA pool or saline were given subcutaneously to rats on postnatal day (PD) 10 or 28. For chronic administration, two injections (12 h interval) of BCAA pool or saline were given subcutaneously starting at PD 7 for 21 days. One hour (acute) or twelve hours (chronic) after the last injection, the animals were killed by decapitation and the brain was removed; BDNF levels were measured using a sandwich enzyme-linked immunosorbent assay.
Results: We observed that acute administration of BCAA increased BDNF in striatum and hippocampus in 10-day-old rats. In contrast, in 30-day-old rats, BDNF were increased in striatum and cerebral cortex. Chronic administration increased BDNF in hippocampus and cerebral cortex. Conclusion: Considering that an increase in BDNF in the brain may impair memory, we speculate that the present findings may be related to brain dysfunction in MSUD.
Background: Maple syrup urine disease (MSUD) is an autosomal recessive inborn error of metabolism caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain ketoacid dehydrogenase, leading to accumulation of the leucine, isoleucine and valine, branched-chain amino acids (BCAA). Neurological sequelae are present in most patients, but the mechanisms underlying the neurotoxicity in this disorder are yet unclear. Objectives: We studied the activity of acetylcholinesterase in hippocampus, striatum, cerebral cortex and serum of rats submitted to an animal model of MSUD. Material and methods: For acute administration, three injections (1 h interval) of BCAA pool or saline were given subcutaneously to rats on postnatal day (PD) 10 or 28. For chronic administration, two injections (12 h interval) of BCAA pool or saline were given subcutaneously starting at PD 7 for 21 days. One hour (acute) or twelve hours (chronic) after the last injection, the animals were killed by decapitation, the brain was removed and serum was collected; acetylcholinesterase activity was measured.
Results: Acute and chronic administration of BCAA pool increased acetylcholinesterase activity in brain and serum of rats.
Conclusions: The administration of BCAA pool may induce cholinergic synapses dysfunction; this may be an important factor in the pathophysiology of MSUD.
Backgrounds: To confirm the diagnosis of cases showing elevated levels of leucine in blood, we developed a simple enzymatic diagnosis method for maple syrup urine disease which measures isovaleryl-CoA converted from 2-isoketocaproate by crude lysate of lymphocytes using HPLC (J Inherit Metab Dis 27, 2004) . Patients: We applied the enzymatic diagnosis method to subjects with 4 mg/dl or higher concentration of leucine; in dried blood spots of (A) 9 symptomatic and (B) 12 asymptomatic newborns, and in plasma of (C) 5 symptomatic children.
Results: All subjects of group A, 5 of group B, and 1 of group C, were diagnosed with MSUD, respectively. Among the 11 subjects with normal results, marked elevation of blood leucine levels was observed in 2 sibling cases repetitively, suggesting thiamin-responsive MSUD; genetic analysis is under way. Discussion: Our simple enzymatic assay is a quite useful tool for clinical practice of the disease. It can be a laborious task to detect gene mutations for MSUD because branched-chain α-ketoacid dehydrogenase complex consists of many subunits. Enzymatic diagnosis enables us to focus on cases where genetic analysis is strongly required. We present the first case of a MSUD patient with a mild variant phenotype and bearing a null mutation in the PPM1K gene, which encodes for the regulatory enzyme, protein phosphatase 2Cm, of the branched-chain alpha keto acid-dehydrogenase complex. After completing the direct sequencing of the BCKDHA-E1alpha, BCKDHB-E1beta, DBT-E2 and DLD-E3 genes and considering that genomic rearrangements are the second most common cause of monogenic disorders, patient's DNA was analyzed using the single-nucleotide polymorphism genotype array technology. Results showed a copy neutral homozygous pattern for chromosome 4 compatible with a uniparental isodisomy (UPD) involving the entire chromosome. In other autosomal recessive disorders, homozygosity mapping has provided a hint about the chromosomal location of the probable disease-causing gene. Therefore, as the PPM1K gene is located in region 4q22.1 of chromosome 4, we sequenced the complete coding region of PPM1K (NM_ 152542.3) identifying a homozygous nucleotide change c. 417_418delTA (p.His139fs) in cDNA and genomic DNA from patient. The mutation was only inherited from the father, confirming a paternal UPD. Inmunoblotting assay of the expressed mutant PPM1K showed absence of PP2Cm protein. Our data are consistent with the previously assumed relationship between the loss of PP2Cm activity and an intermediate presentation of MSUD.
Background: The neonatal form is the most severe and most frequent type of maple syrup urine disease (MSUD). In Serbia, MSUD is not included in newborn screening programme. Objectives: Our aim is to investigate outcomes in critically ill newborns with early recognition and treatment of MSUD. Case report: Three newborns were diagnosed with MSUD in Neonatal Intensive Care Unit of our Institute during 2005-2010 period. All patients have been admitted for somnolence and poor suck beginning in the second week after birth. Diagnosis of MSUD was established within first 48 hours after admission with values of leucine ranging from 2351-2756 mcmol/L. Initial treatment included cessation of oral feeding and continuous hemodiafiltration for 24-60 hours. During the first 24 hours of treatment, we observed a 4-6 fold decrease in leucine concentration in these patients. Cognitive and physical development of all 3 patients is in normal range, with follow-up of 1-5 years. Conclusion: Early clinical recognition and urgent treatment that includes hemodiafiltration could provide normal development for children with neonatal form of MSUD. Maple syrup urine disease(MSUD)is an IEM caused by the deficiency of the branched-chain α-keto acid dehydrogenase complex,leading to the buildup of leucine, isoleucine and valine and their toxic by-products, causing acute cerebral damage and chronic sequelae.There are no epidemiological data for MSUD in Brazil, a disease not included on the national newborn screening panel. To characterize a cohort of Brazilian MSUD cases, data were collected through interview with doctors these patients. 79% of the patients were retrieved, and complete data obtained on 48 cases. Results: 72% of the cases were from Southern Brazil, 70% presented symptoms in the first 10 days of life. Metabolic formula was promptly available in 22% of the cases, and 88% presented delayed neuropsychomotor development. 52% had seizures and 62% respiratory abnormalities. There was a family history in 19% of the cases. Some of the patients who had early diagnosis did not have a normal development due to treatment failure during metabolic crisis or incorrect monitoring and biochemical control. 21% of the cases died before the age of 10 months, confirming a largely severe disease presentation.These results show that a comprehensive management program is needed in order to provide a better outcome to Brazilian MSUD patients. Objectives: There are few reports today about pregnancy in MSUD. We present two new cases with a special focus on the protocol to avoid mother metabolic decompensation (MD) at delivery. Case reports: The two patienta presented with the severe form of neonatal MSUD and experienced multiple MD from neonatal period through adulthood. The first patient is mentally handicapped and had 4 MDs during the first trimester of pregnancy. Delivery occurred at 39 weeks of pregnancy and the baby presented with intra uterine growth retardation (weight and height <10th percentile, head circumference: 25th percentile). He showed moderate developmental delay and at 12 years old, he required special schooling. Brain MRI performed at 1 year showed periventricular leucomalacia. The second pregnancy was well controlled from the beginning but the birth occurred at 32 weeks by caesarian section because of aging of the placenta. The boy also presented IUGR but has a normal development until now. The first patient experienced five MD episodes in the post-partum period, while a specific per and post-partum management protocol was performed to the second patient who did not experienced any MD. Conclusion: MSUD pregnancies must be carefully managed, mainly during the first trimester and delivery.
SINGLE DOSE NTBC-TREATMENT OF HEREDITARY TYROSINEMIA TYPE 1 Schlune A 1 , Thimm E 1 , Herebian D 1 , Spiekerkoetter U 1 1 Dept General Pediatrics, Univ Child Hosp, Duesseldorf, Germany
Background: NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione) is the mainstay of treatment in tyrosinemia type 1 (HT 1). The recommended administration is to be carried out in two divided daily doses. Patients and Methods: We monitored plasma NTBC concentrations in a series of seven patients who were changed from a multiple dose to a single NTBC dose. Two additional patients were directly started on single dose NTBC after diagnosis. In three patients, studies on NTBC kinetics were performed over 6 and 24 hours, respectively. Results: Plasma NTBC concentrations did not significantly decrease in any of the patients after changing from multiple to single dose NTBC treatment. In fact, in one patient, a significantly lower NTBC dose was required after changing to a single dose indicating a better compliance.
Kinetic studies as well demonstrated that plasma NTBC concentrations were completely stable over a period of 24 hours with a single dose application as expected with a known NTBC plasma half life of 54 hours. Conclusion: NTBC administration in tyrosinemia type 1 can be carried out in one single dose improving patients' compliance with the drug treatment.
TREATING TYROSINEMIA TYPE 1: EXPERIENCE FROM KUWAIT Sadeq Sameera 1 , Bin Nakhi Hanan 1 , Al Naqeeb Niran 1 1 Al Adan Hospital, Kuwait, Saudi Arabia
Background: Hereditary tyrosinemia type1(HT1)is a potentially lethal disease if not diagnosed and treated properly.Combined treatment with nitisinone (NBTC) and low-tyrosine diet has resulted in greater than 90% survival rate.
Case report: This report describes clinical data of 6 Kuwaiti patients having HT1.The index case was the product of consanguineous marriage male presented at five months age with hepatic failure, died at age of 7 months. Based on this family history all following siblings were screened ,three children (two males and one female)were affected. The second index case (the cusion of the first index case) was a female died at age of 6 months with hepatic failure. Her sister was diagnosed on birth based on family history. Once diagnosis was confirmed, all patients were started on tyrosine free diet and NTBC 1 mg/kg/day twice daily. They were monitored regularly. Results: Patients are leading normal life, 2 males aged 15 and 9 and 2 females aged 13 and 8. Although they have poor dietary control, their tyrosine level ranged between 800-1000. None of the patients had any complication of the disease or the drug. Conclusion: Medical advances improved prognosis of patients with HTI. Screening of HTI is recommended in Kuwait. 3 1 National Children Hospital "OHMATDET", Kyiv, Ukraine 2 National Medical Academy of Postdiploma, Kyiv, Ukraine 3 Research centre for medical genetics, Moscow, Russian Federation
Introduction: Tyrosinemia type I (HT1) is a rare congenital metabolic disorder of tyrosine metabolism, resulting in the accumulation of toxic metabolites that damage the liver and kidneys. Case Report: A 16 month old girl presented failure to thrive, rickets, significant visceromegaly (hepatomegaly with hepatic nodules, splenomegaly and nephromegaly) and anaemia. Her psycho-motor development was delayed. The height, weight were less than 2nd percentile. Fanconi syndrome was suspected. TMS showed highly elevated levels of tyrosine (568 mcmol/L, normal< 200), and phenylalanine (205 mcmol/L, normal<125). Other biochemical investigations showed high concentration of urine succinylacetone (542 mmol/mol de creat, normal<20) and alphaphetoprotein 1750 ng/ml normal<13.4). The hepatic transaminases, alkaline phosphates were elevated too. Genotype is IVS12+5 g/aSer352Arg. She has been treated since the age of 18 months with Orfadin (dose 1 mg/ kg/day) and with the phenylalanine-and tyrosine-restricted diet with good response and tolerance and adequate metabolic status is maintained. Her mental and motor developments have normalized.
Conclusion: In our case, the clinical manifestation was typical HT1. The Orfadin treatment and protein restrict diet with free tyrosine formula have improved considerably the prognosis for our HT1 patient. Objectives: Implementation of NTBC into treatment of hypertyrosinemia type I (HT I) greatly improved survival by prevention of acute liver failure and hepatocellular carcinoma. However, there are first reports of cognitive impairment in patients with elevated plasma tyrosine concentrations. Methods: Neurocognitive development in 9 patients with HT I under longterm NTBC therapy was assessed using standardized psychometric test batteries.
Results: High plasma tyrosine concentrations were frequently documented in 7 out of 9 patients. Psychometric testing revealed a total IQ score below the average and an inhomogenous test profile with significant differences between the different testing scales in 5 out of 7 patients, respectively. Motor abilities were subnormal in 4 out of 7 patients. Logopedic evaluation in children at school age documented dysfunction or retardation in language development in all but one of the tested patients, however, all but one patient had a migration background.
Conclusions: A high number of patients performed below normal in the assessment of development, motor function and speech. We propose intellectual impairment as a long-term complication in HT type I with elevated plasma tyrosine under NTBC treatment as observed in other hypertyrosinemias. These findings remain to be reproduced in greater patient numbers.
Background: Main clinical features of tyrosinemia type 1 include acute liver failure in infancy or chronic liver dysfunction and renal Fanconi syndrome in late-presenting cases. Enlarged spleen is usually an indicator of lysosomal storage diseases and is not described in patients with tyrosinemia type 1.
Case report: A 17 months old girl was referred to the neurologist because of walking difficulties which were in fact due to abdominal distension. Hepatosplenomegaly was documented clinically and by abdominal ultrasound and CT. Diagnostic work-up revealed renal Fanconi syndrome, the clue symptom leading to the correct diagnosis by determination of urinary succinylacetone. Severe liver cirrhosis with oedemas and ascites, and acute respiratory distress syndrome necessitated resuscitation in the ICU until a favorable response to treatment by NTBC. After two months, regression of hepatomegaly and of the enlarged size of the spleen was observed, even if splenomegaly in this case was indicative of severe liver cirrhosis with consecutive portal hypertension. Conclusion: Splenomegaly as a marker of hepatic cirrhosis may be a revealing symptom of tyrosinemia type 1. Background: Treatment of Tyrosinemia type I consists of NTBC and dietary restriction of Phenylalanine (Phe) and Tyrosine (Tyr). Case Report: Our patient was born after an uneventful pregnancy/delivery. At day 7 she was admitted because of positive NBS results for TYR1 (succinylacetone 4.7 μmol/l; threshold 1.2 μmol/l). Treatment was initiated with NTBC and restriction of Phe and Tyr (breastfeeding combined with Phe and Tyr free formula), based on blood Tyr concentrations. Due to increased Tyr concentrations, natural protein intake was decreased to a minimum of 0.5 g/kg/day. During the subsequent months, she developed first eczema, while later decreased growth parameters followed, and thereafter cortical myoclonus appeared and decreased psychomotor development seemed to arise despite adjustments of the natural protein intake both ways, and Tyr concentrations varying from some 215 to 609 μmol/l . Because of hypophenylalaninemia (< 20 μmol/l),~30 mg/kg/ day Phe-supplementation was started at day 192. Subsequently, all parameters improved. Conclusion: The need of Tyr and Phe varies among patients. Extra Phe usually is not considered as Phe is largely converted into tyrosine. However, this case shows that hypophenylalaninemia is an important complication, necessitating Phe-supplementation, even when this results in lower natural protein intake. Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in patients with alkaptonuria. The cause of the disease is deficiency of homogentisic acid oxidase. Patients with alkaptonuria excrete HGA in the urine. Therefore diagnosis of alkaptonuria can be confirmed by urine HGA analysis. Capillary electrophoresis (CE),a highly effective analytical technique, has been employed for the clinical analysis. The advantage of CE method is high separation efficiency, excellent resolution, short analysis time, electrolyte and sample consumption. CE is particularly suitable in the analysis of complex natural matrices, owing to its higher resolving power. Therefore, biological fluids can be directly analyzed without any pretreatment step or with a simple process of sample pretreatment by CE. In this work CE method for the determination of HGA in urine samples has been developed and validated. Separation and determination were carried out with ultraviolet detector at 190 nm. The optimum conditions were achieved using phosphate buffer 47 mM at pH 7.0 and voltage of 22 kV. Under these conditions the presence of HGA is detected in less than 8 min. Good linearity (r2>0.99) and repeatability (%RSD<2%) was obtained. The developed method was simple, easy, rapid and inexpensive for quantitative determination of urinary HGA. Background: Large amounts of glycine (Gly) occur in brain of patients affected by nonketotic hyperglycinemia (NKH), which is an inherited disorder characterized by brain abnormalities whose pathomechanisms are still poorly established.
Objectives: The effects of intrastriatal administration of Gly to rats on relevant oxidative stress parameters were investigated. Methods: Thiobarbituric acid-reactive substances (TBA-RS), carbonyl formation, sulfhydryl content, reduced glutathione (GSH) levels, nitric oxide production and the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PD) were measured in striatum from 30-day-old rats.
Results: Gly injection significantly increased TBA-RS (lipid peroxidation) and protein carbonyl content (protein oxidation). Furthermore, the activities of GPx, GR, SOD and CAT were altered by Gly. In contrast, Gly administration did not change sulfhydryl, GSH and G6PD activity.
Conclusions: Gly induces lipid and protein oxidative damage and also modulates the activity of antioxidant enzymes in striatum. In case these findings also occur in human NKH, it is feasible that oxidative stress may be involved in the pathophysiology of the brain injury observed in patients with this disease. Pérez-Cerdá C 1 , Navarrete R 1 , Sanz P 1 , Garcia Muñoz F 1 , Rodríguez-Pombo P 1 , Ugarte M 1 1 CEDEM,CBM,CIBERER,Univ Autonoma Madrid, MADRID, Spain NKH is caused by deficiency of the glycine cleavage multi-enzyme system (GCS) with three specific components encoded by GLDC, AMT, and GCSH. It is characterized by accumulation of glycine in body fluids and various neurological symptoms. In this study, we analyzed the mutation spectrum of twenty-two NKH patients grouped by clinical and biochemical findings as three subgroups: sixteen patients with neonatal presentation of convulsions and displaying high levels of CSF-glycine (88-246 μM); two cases with infantile outcome (CSF-glycine 24-26 μM); four patients with infantile onset, CSF-glycine from 12-34 μM and progressive neurological disease leading to death. The genetic screening included the exonsequencing analysis of GCS genes and a multiplex ligation-dependent probe amplification to detect GLDC deletions. GLDC mutations were identified in 15 of the 16 neonatal-onset NKH. The spectrum included six different types of GLDC deletions mostly affecting regions comprising exons 1-15 and 17 potential-pathogenic point mutations. Three different mutations in AMT gene were found in two patients; one neonatal (p.
[K294fs+T87_Q113del]) and one infantile (p. [Arg22Cys+Arg222Cys] ). For the third subgroup of patients no mutations were detected either in the GLDC, AMT, or GCSH gene suggesting a different primary gene lesion as responsible for the disease. Background: Low birth weight (LBW) is associated with increased morbidity and mortality for the newborn and increased risk on chronic diseases in adulthood. Choline has an essential role in the integrity of cell membranes, methylation reactions and memory development.
Objective: We examined whether umbilical/maternal choline and related methylamines betaine and dimethylglycine (DMG) concentrations were associated with LBW in Dutch women.
Methods: Blood was sampled from umbilical cords at delivery (n=1126). Maternal blood was sampled at 30-34 weeks of gestational age (n=366). We calculated birth weights standardized for gestational age (SBW) and defined LBW as SBW <2500 grams.
Results: Maternal concentrations of all analytes were lower compared to umbilical cord concentrations. Plasma betaine and DMG between mothers and newborns were strongly correlated. Higher umbilical cord choline and betaine were associated with lower birth weight (β=−60 [−89;-31] and β=−65 [−94;-36] ). Odds ratio for LBW was 4.12 [1.15;14.78 ] and 5.68 [1.24;25.91] for the highest umbilical choline and betaine quartile respectively compared to the lowest quartiles. Conclusion: We observed an increased risk of lower birth weight with increased umbilical choline and betaine in venous umbilical cord blood. These results might reflect a change in choline consumption or metabolism or a disturbed placental function.
Background: Molybdenum cofactor deficiency (MoCD) is characterized by severe and rapidly progressive neurological damage caused by the loss of sulfite oxidase activity, one out of four molybdenum-dependent enzymes. Without effective therapy death in early infancy has been the usual outcome. Objectives: MoCD type A patients carry mutations in MOCS1 causing a loss of cyclic pyranopterin monophosphate (cPMP), the first intermediate in the Moco pathway. An experimental substitution therapy with cPMP has been initiated in six patients. Patients: Patients have been diagnosed in utero or at the age of 2-20 days and treatment was initiated on days 0-36 with daily i.v. administration of 80 μg/kg and stepwise increase to 240 μg/kg body weight. Results: Within days, all urinary markers of sulfite oxidase (sulfite, S-sulfocysteine, thiosulfate) and xanthine oxidase deficiency (xanthine, uric acid) returned to almost normal and stayed constant throughout treatment. Clinically, all infants became more alert, convulsions and twitching disappeared within the first two weeks and the EEG showed the return of rhythmic elements and markedly reduced epileptiform discharges. Conclusion: Substitution of cPMP represents the first causative therapy available for MoCD patients. We demonstrate efficient uptake of cPMP and restoration of molybdenum cofactor-dependent enzyme activities. Further neurodegeneration was stopped. Conflict of Interest declared. We report on the efficacy of long-term cPMP substitution in two unrelated children with MOCD group A who both share the same homozygous mutation in the MOCS1 gene. Diagnoses were made on day 1 of life in Baby 1 due to a previously affected sibling and on day 4 in Baby 2 who started having intractable seizures from day 1 of life. Daily intravenous cPMP infusions were started on day 7 and 5 of life and continued for a period of 18 and 15 months in Baby 1 and 2, respectively, without interruption and using an identical protocol. Treatment efficacy and safety were carefully monitored. Both infants have been tolerating cPMP without adverse effects, apart from intercurrent central venous line infections. We observed a rapid and sustained good clinical and biochemical response. Baby 1 has shown very satisfying developmental progress whereas Baby 2, despite earlier treatment, suffers from static, dystonic cerebral palsy and cystic encephalopathy due to early non-progressive necrotic changes, probably pre-dating cPMP substitution. Infants with MOCD type A can be safely and effectively treated with longterm cPMP substitution. The extent of encephalopathy does not only depend on genotype and time delay between birth and start of cPMP substitution. Alpha-aminoadipic semialdehyde (α-AASA) is the hallmark accumulating metabolite in pyridoxine-dependent epilepsy due to Antiquitin deficiency (PDE). Elevated levels may be measured in urine by LC-MS for diagnosis of the disorder, according to age-dependent reference ranges. The typical ranges seen in Antiquitin deficiency are 7-138 mmol/mol Cr under 12 months and 1.3-40 mmol/mol Cr over 12 months. Patients with molybdenum cofactor deficiency (MoCoF) and PDE may both present with a neonatal epileptic encephalopathy (NEE). Urine samples analysed by LC-MS in our laboratory to aid differential diagnosis of NEE have shown elevated α-AASA in all cases subsequently confirmed by molecular genetic analysis to have MoCoF deficiency. α-AASA ranged from 9.6-16.9 mmol/mol Cr in two patients under 6 months (reference range <5) and 2.9 mmol/mol Cr in a single patient over 12 months of age (reference range <2). α-AASA may form a sulphonate with sulphite when present in excess. This may be excreted in urine and liberated during the alkalinisation step of sample analysis, providing a possible mechanism for the observation. Detection of elevated urinary sulphite or sulphocysteine in MoCoF deficiency will differentiate the two conditions guiding appropriate management. Background: Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurologic damage mainly caused by resulting sulfite oxidase deficiency (SOD). Elevated urinary levels of sulfite, thiosulfate and S-sulfocysteine (SSC) are hallmarks in the diagnosis of MoCD and SOD.
Objectives: Recently, a first human exposure and successful treatment of MoCD type A based on a substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been reported. Knowing the rapid progression of the disease symptoms in untreated patients, an early diagnosis of MoCD will be crucial for treatment outcome.
Results: Here we describe a fast and sensitive method for the analysis of SSC in human urine samples using high-performance liquid chromatography (HPLC). The analysis is based on pre-column derivatization with ophthaldialdehyde (OPA) and separation on a C18 reverse phase column coupled to UV detection. Total analysis time of the described method is three minutes and quantification is linear in the range of 0.5 to 500 μM SSC. SSC values from 45 control pediatric and 43 adult urine as well as 60 plasma samples are reported.
Conclusion: The described SSC quantification method is cost effective as well as useful for routine diagnosis. Conflict of Interest declared.
Augoustides-Savvopoulou P 1 , Ioannou C 1 1 1st Pediatr Dept,Arist Univ Thessaloniki, Thessalonikii, Greece
Background: Lysinuric Protein Intolerance (LPI) is an AR kidney and intestinal transport disorder (OMIM222700) affecting lysine, ornithine and arginine (SLC7A gene). Symptoms include protein-induced hyperammonemia, growth retardation, hepatosplenomegaly, osteoporosis, alveolar proteinosis, hemaphagocytosis.
Objective: Six patients with hyperdibasic aminoaciduria are described to highlight LPI clinical heterogeneity and a diagnostic pitfall. Case reports: Patients 1, 2: A soldier, 23y, with short stature, hepatosplenomegaly, osteoporosis, eye lesions, manifested psychotic episodes and seizures after high protein meals. Two SLC7A7 mutations identified in the patient and his 27y sibling ("hyperammonemia of unknown etiology", 1.80 m, centripetal fat, thin extremities, osteoporosis). Patient 3: Albanian 6y with failure to thrive (bone-age 3y), extreme aversion to protein, hepatosplenomegaly, lung lesions (HRCT), osteoporosis. Aminoacid profile indicated LPI. By 12, death from alveolar proteinosis and hemaphagocytosis. Patients 4, 5: Eight-year old with hyperammonemic encephalopathy. LPI diagnosed (two SLC7A9 mutations), also present in his asymptomatic 11y sibling. Patient 6: Male 4y with bronchiolitis (Singulaire regimen) and liver dysfunction. Symptoms with hyperdibasic aminoaciduria indicated LPI. Extensive SLC7A7 analysis negative but SLC7A9, SLC3A1 mutations present.
Conclusions: Our experience highlights clinical heterogeneity of LPI, the need for clinical awareness and effective treatment and that liver and pulmonary symptoms in the presence of cystinuria can masquerade as LPI.
Background: Lysinuric protein intolerance (LPI) is an autosomal resessive aminoaciduria affecting the transport of cationic amino acids (CAA; arginine, lysine, ornithine) at the basolateral membrane of epithelial cells in the intestine and kidney. LPI manifests with vomiting, attacks of coma, mental retardation due to hyperammonia episodes, diarrhea, failure to thrive, hepatosplenomegaly, bone marrow abnormalities, osteoporosis, pulmonary alveolar proteinosis, altered immune response and chronic renal disease. Case Report: 36 year old female patient followed with vomiting, failure to thrive during childhood consulted for glycogen storage disease type 7 with signs of mild haemolysis and glycogen storage at liver biopsy. Lysinuric protein intolerance was suspected with findings of vomiting attacks and failure to thrive during childhood, protein aversion, short stature, osteoporosis, hyperferritinemia, hemophagocytosis, mild hemolysis,glycogen storage at liver biopsy, elevated lactate dehydrogenase, hypercholesterolemia and hypertrigliceridemia. The patient was diagnosed as LPI by low plasma levels and high excretion to urine of CAA.
Conclusions: Presentation with variable clinical and laboratory findings because of the multisystemic involvement may cause missed or delayed diagnosis in patients with LPI. The diagnosis of lysinuric protein intolerance should be considered in patients presenting with hyperferritinemia, elevated lactate dehydrogenase and hemophagocytic lymphohistiocytosis in addition to protein intolerance or aversion. In the present study we investigated whether physical exercise would prevent proline-induced memory deficits in Morris water maze tasks, as well as its effects on brain-derived neurotrophic factor (BDNF) immunocontent and brain acetylcholinesterase (AChE) activity in cerebral cortex and hippocampus of Wistar rats. Animals were divided into 4 groups: (1) control; (2) proline; (3) exercise and (4) proline plus exercise. Rats were submitted to proline administration from the 6th to 29th day of life, when the treatment was discontinued and the treadmill exercise was performed from day 30th to 60th day. Twenty-four hours after the last exercise session, the rats were subjected to behavioral testing in the water maze and then sacrificed for determination of BDNF and AChE activity. Proline impairs memory on spatial reference and working memory tasks and exercise prevented such effects. BDNF was reduced in both cerebral structures and AChE activity was increased only in hippocampus. Our results suggest that memory deficit caused by hyperprolinemia may be associated, at least in part, to decrease in BDNF immunocontent and increased AChE activity. Taken together, these findings reinforce the potential neuroprotective of physical exercise as an experimental therapeutic strategy to minimize cognitive deficits. Support by CNPQ, FAPERGS.
COMPARTMENTALIZATION OF LYSINE METABOLISM AND ITS ROLE FOR GLUTARYL-COA DEHYDROGENASE DEFICIENCY Opp S 1 , Ruppert T 1 , Okun JG 1 , Koelker S 1 , Sauer SW 1 1 University Children' s Hospital Heidelber, Heidelberg, Germany
Background: Glutaryl-CoA dehydrogenase deficiency is an inborn error of lysine metabolism. Patients are characterized by neurodegeneration affecting basal ganglia induced by glutaric and 3-OH-glutaric acid accumulation. The precise pathway of lysine degradation and its subcellular localization is unclear. It is thought that there is a main, mitochondrial and a minor, peroxisomal pathway. We studied the individual steps of both pathways in Gcdh-deficient mice and potential therapeutic aspects of their modification. Methods: We tested activities and localization of saccharopine dehydrogenase, aminoadipate semialdehyde dehydrogenase, aminoadipate transaminase, 2-oxoadipate dehydrogenase, and pipecolate oxidase in liver, brain, and kidney. Further, we induced peroxisomal biogenesis by clofibrate and examined its effect on production of glutaric and 3-OH-glutaric acid.
Results: Saccharopine dehydrogenase, entry of lysine into the mitochondrial degradation pathway, was present in liver and kidney mitochondria but not detectable in brain. Aminoadipate semialdehyde dehydrogenase and aminoadipate transaminase activity were found in cytosolic and mitochondrial fractions of all tested tissue. Pipecolate oxidase, representing the peroxisomal pathway, was detected in kidney and brain. Strikingly, clofibrate treatment decreased instead of increasing glutaric acid levels.
Conclusions: In liver lysine is mainly oxidized by mitochondria, whereas in brain lysine oxidation occurs in peroxisomes. Renal lysine degradation is possible in both organelles.
NOVEL DELETION IN HYPOTONIA-CYSTINURIA SYNDROME Aydin HI 1 , Okur I 1 , Creemers JWM 2 , Coskun T 3 1 Div Metab Dis, Dep Ped, Gulhane MMF, Ankara, Turkey 2 Center for Human Genetics, Leuven, Belgium 3 Div Metab Dis, Dep Ped, Hacettepe Univ., Ankara, Turkey
Background: Hypotonia-cystinuria syndrome (HCS) that is caused by microdeletions of SLC3A1 and PREPL on chromosome 2p21 presents with generalized hypotonia at birth, failure to thrive, growth retardation and cystinuria type I. We here report a novel deletion in a Turkish patient with hypotoniacystinuria syndrome.
Case report: A 8 year-old-male patient born of consanguineous marriage was diagnosed as lysinuric protein intolerance by hypotonia, feeding problems and urine amino acid analysis at the age of 2 months and protein restriction and L-citrulline therapy was started. The patient was reevaluated when 8 years old because of the absence of hyperammonemia attacks even on a high protein intake, normal blood ferritin, lactate dehydrogenase levels and hypotonia-cystinuria syndrome was suspected. There was no sign of nephrolithiasis or urolithiasis. The plasma and urine amino acid analysis revealed normal plasma and high urine levels of lysine, arginine, ornithine, cystine. The demonstration of a homozygous deletion in SLC3A1 and PREPL genes confirmed the the diagnosis of HCS.
Conclusions: This deletion has not been described before. The deletion extends from the intron between PPM1B and SLC3A1 to the intron between exon 2 and exon 1 of PREPL. All exons of SLC3A1 and all coding exons of PREPL are deleted.
Cystinuria is an autosomal recessive disorder of dibasic amino acid transport in in kidney and intestine leading to increased urinary cystine excretion and nephrolithiasis. Two genes, SLC3A1 and SLC7A9, coding for rbaT and bO,+AT, account for the genetic basis of cystinuria. This study reports the clinical characterization and SLC3A1 (type A) and SLC7A9 (type B) mutations identified in a French cystinuria cohort. A total of 106 patients from 97 families with cystinuria and 18 relatives were investigated. A questionnaire addressing, age at first symptoms, stone recurrence rates, metabolic evaluations, medical therapy and follow-up was completed.
Mutation screening using sequencing analysis was performed. Quantitative Multiplex PCR of Short fluorescent Fragments analysis was developed for the detection of genomic microdeletions and microduplications. Splicing reporter minigene assay was used to evaluate the splicing effect on unclassified variants. 63 and 27 mutations have been identified in SLC3A1 and SLC7A9 genes respectively, of which 58 were novel. These mutations included 56 missense/nonsense, 13 deletion/insertion, 8 splicing, 13 large-scale rearrangements (18% of mutated alleles). Thus, 163 and 56 mutated alleles have been reported in SLC3A1 and SLC7A9 genes respectively. This report expands the spectrum of SLC3A1 and SLC7A9 mutations and supports that digenic inheritance is unlikely. Cystinosis is an AR lysosomal disorder. Free cystine accumulates continuously in lysosomes. Specific treatment with oral cysteamine results in long-term reduction of lylosomal cystine. However, corneal cystine crystals do not respond with oral cysteamine. A 26 years old female patient with typical nefro-cystinosis, had severe photophobia. The diagnosis was made on the first year of live and needs kidney transplant at 7 years of age. She developed epilepsy when she was 12 years. She has long been treated with oral Cystagon, which is not available in eydrops formulation. The Pharmacy Department of our Hospital have prepared and tested stability of cysteamine eydrops with the following formulation: cysteamine cloridrate 0,1136 g, sodic EDTA 0,1 g, benzalconium chloride 0,01 g and isotonic soluction of NaCl for 100,0 ml. This formulation is stable for 2 months and doesn't need refrigeration. The patient has now been treated with cysteamine eyedrops for 6 years. It is well tolerated and adherence to therapy is good. The severe photophobia disappeared. Ophthalmologic evaluation with confocal microscopy shows reduction on density corneal cystine crystals density. This cysteamine eyedrops formulation, used in this patient no needs refrigeration, is stable for a long period and most importantly, it is well tolerated and efficient. Most of the inborn errors of metabolism (IEM) are autosomal recessively inherited. Rarely, more than one IEM are seen in the siblings of one family or in the same sibling. Here we report a patient with both argininosuccinic aciduria (ASA) and methylmalonic acidemia (MMA) and our therapeutic approach. Chronic management of both diseases consist of a low-protein diet. But the treatment of more than one disorders affecting aminoacid metabolism was challenging. A 2 -day-old male patient was admitted to our hospital since a previous sibling had been diagnosed with ASA. The first child of the family had died due to MMA but the family refused prenatal diagnosis in this pregnancy. Laboratory investigations of the patient revealed elevated C3 propionyl carnitine, citrulline and argininosuccinic acid levels on serum tandem MS, elevated methylmalonic acid on urinary organic acid analysis and increased argininosuccinic acid levels on urine tandem MS. Both ASA and MMAwere diagnosed. The dietary regimen of the patient was planned with MMA being the basis and all the replacement therapies were given both for ASA and MMA. When 4 months old, his metabolic status was well, plasma ammonia level and blood gases were normal and he had no ketosis. Male infant presented at 8 weeks with respiratory difficulties and encephalopathy, a respiratory alkalosis, hyperammonaemia (355 μmol/l). Investigations revealed raised plasma ornithine (632 μmol/l) but no orotic acid or homocitrulline in his urine. Treatment with arginine, benzoate and phenylbutyrate resulted in rapid recovery. OAT deficiency was confirmed by enzyme and molecular studies. On treatment his ornithine increased to over 1000 μmol/l. Arginine supplements were discontinued at 11 weeks of age. However after a further 3 weeks ornithine had fallen to 10 μmol/l, and glutamine increased to >1000 μmol/l with ammonia at 144 μmol/l. Arginine supplements were therefore restarted at 4 months. Within a short period there was again a marked increase in ornithine to 983 μmol/l and so supplements were stopped. By 7 months his glutamine had increased to >900 μmol/l and ornithine fell to 31 μmol/l so arginine was again restarted. At 12 months his ornithine showed a persistent increase to so that arginine was stopped. Since then there has been no fall in ornithine levels or increase in glutamine. This case demonstrated that flux through the ornithine synthetic/catabolic pathway can reverse repeatedly in the first year of life so infants with OAT deficiency remain at risk from hyperammonaemia. Background: The outcome of childhood acute lymphoblastic leukemia has improved substantially after introduction of Asparaginase. Pegylated asparaginase (PEG-Asparaginase) has a longer half life and has been introduced recently to optimize therapeutic efficacy. We investigated whether introduction of PEG-Asparaginase affected the risk of hyperammonemia, an infrequent side effect of asparaginase treatment. Patients and methods: We analyzed blood samples and clinical data of eight consecutive patients with acute lymphoblastic leukemia receiving PEG-asparaginase in our hospital. Results: Maximum ammonia levels ranged from 89 to 400 μmol/L. Symptoms varied from mild anorexia and nausea to headache, vomiting, dizziness and lethargy and led to interruption of PEG-asparaginase in three patients. Analysis of plasma amino acid concentrations during PEGasparaginase therapy revealed a depletion of asparagine (6/6) and a marked reduction in glutamine concentration (5/6), in conjunction with strongly increased concentrations of both aspartic acid and glutaminic acid. Urea cycle intermediates were normal. Conclusions: Peg-asparaginase was associated with symptomatic hyperammonemia in all patients, unlike previous reports. We propose that the ammonia produced through hydrolysis of aspargine and glutamine is largely responsible for the hyperammonemia in patients receiving Peg-asparaginase. Background: L-Asparaginase is commonly used in chemotherapy protocols for lymphoproliferative disorders. Asparagine is an essential amino acid for tumor cells and required for their rapid malignant growth. The mechanism of tumor lysis of l-Asparaginase is hydrolysis through of the amido group of asparagine to aspartate and ammonia. L-Asparaginase has been reported to cause hyperammonemia, which theoretically may be induced by increased ammonia production exceeding the capacity to detoxify and/or decreased detoxification capacity. Case reports: A retrospective chart study was performed in 4 female patients, who were evaluated for encephalopathy caused by hyperammonemia after l-Asparaginase, PEG-Asparaginase or Erwinase (all incorporating l-Asparaginase). Extensive metabolic (blood amino acid profile, urine organic acids, and urine orotic acid) and molecular studies failed to identify a specific inborn error of metabolism Background: Ornithine Transcarbamylase (OTC) deficiency is a heterogeneous X-linked disorder comprising severe neonatal-onset hyperammonaemic encephalopathy and variable later-onset disorders. Case presentation: A developmentally normal 18 month boy presented with seizures, acute encephalopathy and elevated ammonia 115 μmol/L (10-50). CSF glutamine was 1085 μmol/L (319-742) and arginine was 7 μmol/L (12-30) with increased urinary orotate. His mother was asymptomatic.
Method: DNAwas extracted from urine and two blood specimens from the proband as well as maternal blood. The coding region and the intron-exon boundaries of the OTC gene (NM_000531.5) were sequenced. Results: The proband had a normal (46XY) karyotype excluding Klinefelters' syndrome. Sequence analysis in 3 samples consistently revealed two mutations at the same nucleotide in the proband: c.542A>T (p.Glu181Val) and c.542A>G (p.Glu181Gly); the former being 2-3 times more prevalent with neither being in the mother. Discussion: Mutations at residue 181 are consistent with OTC deficiency. p.Glu181Gly has been reported in a neonate. p.Glu181Val is novel and is predicted by PolyPhen-2 to be benign though SIFT predicts it to affect protein function with low confidence. Possible causes include de-novo mutation in mother followed by somatic mutation in the fetus, or initial somatic mutation in the fetus and a subsequent second mutation. Ornitine transcarbamylase deficiency (OTCD) is the most frequent urea cycle error and results from mutations in the OTC gene-that encodes a 354-residue polypeptide. The clinical picture in females is highly variable even within a family, depending on the X-inactivation pattern in liver. Mutations that lead to late-onset presentations may lead to life-threatening disease and may be unrecognized. We present a 14 months-old girl who manifested with recurrent stroke episodes after a respiratory infection that triggered a hyperammonemic crisis; investigations revealed hyperammonemia (384 μmol/L), coagulopathy, elevated liver enzymes, lactic acidosis and massive orotic aciduria (orotate/ creatinine ratio 5000 mmol/mol creatinine, reference range <11), being highly suggestive of ornithine transcarbamylase deficiency. DNA analysis revealed a novel mutation in the 6th exon of the OTC gene [c.628A>T (p.Lys210X)], leading to a premature stop of translation. Searching for OTCD mutations is laborious and expensive; if all OTCD causing mutations were known, mutation screening might be simpler. OTCD should be considered in the differential diagnosis of unexplained acute neurological presentation, even without a suggestive family history. This report expands the clinical, biochemical and molecular spectrum of OTCD with a female case heterozygous for the novel c.628A>T OTC mutation. Background: Hyperammonemia is well known in the cat family on an arginine deficient diet. Several studies have been conducted to explain this phenomenon. Morris and Rogers described arginine as an essential amino acid for the cat family and Jones found that cats are unable to produce ornithine. Levillain found that hyperammonemia in cats can be prevented and treated with either arginine or citrulline supplementation but not with ornithine. One cheetah breeding population, on a well balanced diet, in South Africa experienced chronic problems with hyperammonemia. The urinary creatinine excretion in the cat family is extremely high. Since creatine is synthesized from arginine, the high creatinine values indicated an enormous flux through arginine. Therefore arginine cannot be essential and the low arginine and citrulline concentrations indicated the presence of a possible catabolic pathway for at least one of the intermediates of the urea cycle.
Results: All the cheetahs who were unaffected by hyperammonemia excreted high concentrations 3-aminopiperidine-2-one. This metabolite was almost absent in the urine of the cheetahs prone to hyperammonemia. Conclusions: These results may indicate that cheetahs may use 3aminopiperidine-2-one as a source for ornithine or that an increased catabolism of ornithine to glutamic acid may result in hyperammonemia. Background: Patients with organic acidurias (OADs) and urea cycle defects (UCDs) have an enormous need for improved medical awareness, optimization of the diagnostic process and therapy, and improved networking between healthcare professionals and patients. Methods: An initiative named "European registry and network for Intoxication type Metabolic Diseases (E-IMD)" funded by the European Commission through DG Sanco has been started in January 2011. E-IMD aims to promote health for patients with OADs and UCDs.
Results: E-IMD already has 40 partners from 17 countries linking healthcare professionals, patient's representatives, industry and government authorities within Europe, Canada and the US. E-IMD will continue to expand its network by inviting new members. The registry will be launched in July 2011 and is expected to collect data on 600 individuals with an OAD or UCD over the next 3 years.
The new network will improve access to rapid diagnosis and care for patients with OADs and UCDs. E-IMD will help make this happen by 1/ objectively evaluating management strategies and outcome of patients (patient registry, www.e-imd.ukhd.de), 2/ providing evidence-based diagnostic and management protocols, and 3/ empowering patients and patient organisations by providing up-to-date information in their own language (website, www.eimd.net). OTC deficiency, first described in 1962, was recognised initially because the child's urine stank of ammonia and characteristically vomited repeatedly failing to gain weight when her diet was changed from breast milk to an artificial formula with a higher protein intake in the first few weeks of life. This leads to dehydration and a low c.s.f. pressure. Over a period of time brain size is limited with premature closure of the fontanelles and the child is irritable and screams. The urine contains fine needles of orotic acid. Hypertonia leads to opisthotonus as illustrated.
Because of high levels of blood ammonia and until the hepatic enzyme deficiency was demonstrated the disorder was termed hyperammonaemia.
It is now the most frequent inborn error of the urea cycle. Some of these features are illustrated in the attached DVD of the index patient, then twenty months old, being examined by one of the authors of the two papers first describing the syndrome. OTC deficiency presents in different ways neurologically and with different degrees of severity. The index patient was severely affected and it is suggested that the very marked muscular tonus illustrated is caused by the effect of ammonia on spinal reflexes. Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder affecting both sexes. We describe a large pedigree with OTC deficiency. A 15 months old girl was hospitalised with vomiting, poor appetite and lethargy. Elevated liver enzymes, lactic acid and ammonia (210 μmol/l) were found in blood. Glutamine in plasma was slightly elevated, otherwise aminoacids in plasma and urine were normal, orotic acid in urine also was normal. In anamnesis one of patient's sisters died suddenly at the age of 5 years with Rey syndrome, coma, encephalopathy. Another sister was strongly vegetarian. The mother's two brothers died in the neonatal period. The patient's niece died at the age of 11 months after viral respiratory disease with rapid coma and brain death and three cousins died in second and third day of life with respiratory problems, seizures, coma. Ammonia levels were not examined. OTC deficiency was suspected in our patient and the finding of the R142Q/N mutation in the OTC gene confirmed the diagnosis. The same mutation was found in the patient's mother, sister and in postmortem material in one of the deceased cousins. Our report shows the importance of early checking of ammonia in all patients with acute encephalopathy, coma. Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and is reported to correlate with the genotype. Activity of in vitro expressed recombinant mutant PAH proteins may predict the patient's phenotype and pharmacological response to tetrahydrobiopterin (BH4). We developed a PAH assay using a LC-MS/MS method for quantitative determination of phenylalanine and tyrosine after derivatization with Phenomenex EZ:faast kit. Several frequent PAH mutations (R158Q, R261Q, R408W, I65T) were expressed in eukaryotic COS-1 cells and quantified for PAH activity. In addition, PAH activity was measured in several cell lines (Huh-7, Hep3B) as well as in liver, brain, and kidney tissue from wild-type and PKU mice. The PAH assay is linear for both amino acids, phenylalanine as well as tyrosine (r2 ≥0.99) and exhibits a low limit of detection of less than 200 nmol/L. In addition the method allows differentiation of endogenous and enzymatically produced tyrosine in cell extracts. Intra-day coefficients of variance (CV) were less than 2.7% and inter-day CV less than 5.7% in lower tyrosine range. The accuracy determined through recovery was 105%. In conclusion, quantification of phenylalanine and tyrosine by LC-MS/MS is highly specific, reproducible, and faster than previously used methods. High variability in blood phenylalanine (phe) levels have been shown to be related to a poorer cognitive outcome in individuals with phenylketonuria (PKU) suggesting that intermittently high (or low) levels may have a deleterious effect in addition to that associated with high average blood phe concentrations.
We have analysed the standard deviation (SD) of blood phe and the dietary phe tolerance in 84 patients with PKU on dietary treatment during their first 4 years. Phe SD ranged from 70 to 460 μmol/l and phe intake from 120 to 940 mg/ day. There was a strong inverse correlation between phe SD and dietary phe tolerance (p <0.0001) confirming that those with 'severe' PKU showed a much higher variability in phe blood levels. Some individuals with a low phe SD but who, on the basis of their absolute blood phe levels had been assessed to need a relatively strict diet, were able to markedly increase their natural protein intake while still maintaining satisfactory phe control. We conclude that phe SD may be useful to provide additional information as to the severity of PKU and may suggest which patients might have a higher phe tolerance than previously thought. Missense mutations in the phenylalanine hydroxylase (PAH) gene can induce protein misfolding with loss of function of the PAH protein, the molecular basis of phenylketonuria. Sapropterin dihydrochloride, the synthetic form of tetrahydrobiopterin (BH4), was recently approved as the first pharmacological chaperone drug and shown to correct the loss-offunction phenotype by rescue of PAH misfolding. The aim of this study was to investigate the structure-function relationship of the BH4-molecule with respect to stabilization of PAH conformation. We compared the efficacy of BH4 and BH4-derivatives (BH2, Sepiapterin and 6-MPH4) with substitutions at the pterin ring system and/or the dihydroxypropyl side-chain to correct misfolding of wild-type and mutant PAH, respectively. BH4 and two dihydropterins (BH2, Sepiapterin) induced a compacted PAH conformation leading to mutation-specific increased thermal and thermodynamic stability. 6-MPH4, a cofactor without inhibitory potency lacking the dihydroxypropyl side-chain, led to protein destabilization. Interestingly, the presence of BH4 and BH2 but not Sepiapterin shifted the PAH unfolding model (2-state to 1-state). In conclusion, the structure-function relationship of these compounds is the molecular basis for structure aided drug design to improve the efficacy of pharmacological chaperones taking the patient's genotype into consideration.
L- PHE-TREATMENT OF ENU1/2 MICE LEADS TO STIMULATION  OF TETRAHYDROBIOPTERIN SYNTHESIS, PROBABLY  THROUGH GTPCH-GFRP MEDIATED ACTION, WITH  CONCOMITANT REDUCTION IN UBIQUITINATION  OF MUTANT PAH, AND INCREASE IN PAH Phenylketonuria is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). This protein has been regarded as a highly unstable enzyme, fact postulated to be responsible for the lack of the full-length hPAH 3D structure. We aimed to obtain hPAH mutant chimerical forms presenting a higher activity and stability. Firstly, the hPAH mutants C29D, C29S, C284S, C445S, D145K, D151K, E181K and E360K, were produced in a prokaryotic expression system. Due to their higher expression level the C29S and E360K proteins were selected for further characterization regarding the oligomeric profile, enzyme kinetics, substrate activation and thermal inactivation. When compared to the hPAHwt, the E360K presented a higher degree of aggregates. The C29S showed an increase in catalytic activity (1.3-fold) Results: GLM analyses of variance showed that PKU patients with lifetime Phe<=240 μmol/l outperformed patients with Phe >240 μmol/l. When patients with Phe<=360 μmol/l were compared to those with Phe >360 μmol/l, fewer and less pronounced differences were observed. Compared to controls, patients with Phe<=240 μmol/l performed comparable on all tests, which was not true for patients with Phe<=360 μmol/l. Discussion: When inhibitory control and working memory were required simultaneously, average Phe concentrations of<=240 μmol/l were advantageous compared to<=360 μmol/l, urging consideration of lowering the upper target Phe to 240 μmol/l during the first 12 years of life.
Background: Few studies have looked at acceptable serum phenylalanine levels in later life in patients with PKU. We examined the oxidative stress status of adolescents and adults with PKU. Methods: Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, blood anti-oxidant levels and nitric oxide production were examined. Results: Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level. Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level. Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level. Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level. The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 micro mol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q10 were lower. Serum nitrite/nitrate levels were higher together with low serum asymmetric dimethylarginine. Conclusions: Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine in PKU should be maintained at below 700-800 micro mol/l even in adult patients. Adults with phenylketonuria can develop neuropsychological abnormalities as a result of hyperphenylalaninemia. These symptoms can be detected with computerized neuropsychological tests. Brain hyperphenylalaninemia can in turn be measured with use of magnetic resonance spectroscopy. The aim of the study was to assess the brain phenylalanine concentration in hyperphenylalaninemic adults without neuropsychological deficits. Assessment of sustained attention, working memory and inhibitive control was performed in a group of 30 non-compliant adults with phenylketonuria by means of computerized CANTAB system. Brain/blood phenylalanine ratio was analyzed in patients without neuropsychological abnormalities (brain phenylalanine concentration was measured with use of magnetic resonance spectroscopy). Worsening of neuropsychological efficiency correlated with high levels of plasma phenylalanine. Neuropsychological abnormalities were recorded in 28 patients. In the remaining two patients relatively low brain/blood phenylalanine ratios were observed. This finding supports the hypothesis on the presence of mechanisms limiting brain phenylalanine concentration in selected patients with phenylketonuria, which was postulated by other authors. These potential mechanisms could result in lower brain toxicity of hyperphenylalaninemia. It should be stressed, however, that such situation can probably be expected very rarely. The study was sponsored by government research grant NN402329233. Several studies suggested a compromised BMD in HPA affected patients on diet-therapy. Our aim was to assess bone mineralization and evaluate effects of calcium integration and physical activity in HPA patients.
Patients and Methods: To analyze BMD we divided 117 patients basing on diet-therapy and diet-adherence; to analyze 12-months calcium integration we considered 29 patients on diet; to analyze physical activity we divided 117 patients basing on diet-therapy, calcium integration and physical activity. DEXA measured L1-L4 mineralization; we reported body mass index, Zscore-BMI, serum Calcium, Phosphate, Magnesium, Alkaline Phosphatase and Phenylalanine. Results: Zscore-BMD was statistically different between patients on diet and on free-diet, higher in the group with good adherence to diet than in the group with poor adherence. Zscore-BMD and serum Phe did not correlate proportionally. Zscore-BMD and blood-parameters improved after calcium integration. There was no significant difference between patients on diet with both calcium integration and physical activity versus patients on free-diet and physical activity.
Conclusions: Current analysis verified bone density reduction in HPA patients on diet. Restricted diet seemed to be associated to worse BMD. Treating with both calcium integration and physical activity we could optimize bone formation in order to prevent osteopenia and/or osteoporosis risk. In a non-intervention dietetic study of 20 patients (1-15 years, 12 f., 8 m.) with clinically classic phenylketonuria (PKU), food intake and phenylalanine (phe) blood levels were documented daily during 2 periods of 1 week each in 15 patients, and during 1 week only in 5 patients. The study periods were selected according to the current phe blood levels, with phe being within target range during one period, and above-target during the other one. By using a kinetic single-compartment model, the zero-order net protein synthesis was found diminished at elevated phe levels in 12 of the 15 patients, indicating sub-clinical catabolic states as a frequent cause of 'diet failure'. In contrast, the first-order constant of metabolic (plus renal) phe disposal was found almost identical during both periods (R=0.9782).
Beside the interpretation of inverse diurnal variation of serum phe (Güttler et al, 1969) , the question of catabolic states has been considered only very rarely in reports and guidelines on PKU management. Distinguishing such states from non-compliance may motivate compliant patients and their families to even stronger adhere to their dietetic schedule. Objectives: We report the first case of Munchausen by proxy in phenylketonuria (PKU). Case report: We report the case of a PKU boy whose metabolic follow up was difficult in the first year of life. He was referred to our centre for the management of very high Phe level (3300 μmol/L) which were non responsive to classical treatment. Despite an emergency regimen (PKU aminoacids substitutes+high caloric intake and 0 Phe intake) and a weight gain was 1.7 kg, the Phe level remained increased for 3 weeks. As we were suspicious of Munchausen by proxy, we asked to the mother to leave the hospital for a week and Phe level normalized within 3 days. Accidentally, we found in the child's bag, a bottle of a high caloric, high protein formula which could be the source of the extra protein given to the child. After the child was retired from his family, the Phe level never rise up again. Conclusion: Munchausen by proxy must be evoked when there is a discrepancy between the administered diet and the metabolic status and after elimination of all the other causes of metabolic decompensation.
Background: The involvement of reactive species in pathophysiology of phenylketonuria (PKU) is well established. In previous studies it was verified that PKU patients (treated with a protein-restricted diet supplemented with a special formula not containing L-carnitine and selenium) presented high lipid and protein oxidative damage as well as reduction in antioxidants. Our goal in this study was to evaluate the effect of a supplementation with L-carnitine and selenium, two well-known antioxidant compounds, on oxidative stress in PKU patients. Methods: We investigated various oxidative stress parameters in blood of 18 treated PKU patients before and after 6 months of supplementation with a special formula containing L-carnitine and selenium.
Results: It was verified that the treatment with the antioxidants was capable to revert the lipid and protein oxidative damage. Additionally, the supplementation normalized the glutathione peroxidase activity. It was verified a negative correlation between lipid peroxidation and L-carnitine levels as well as a positive correlation between glutathione peroxidase activity and selenium concentration. Background: In recent years, evidence has emerged indicating that oxidative stress is possibly involved in the pathology of phenylketonuric patients (PKU). PKU enzymatic and non-enzymatic antioxidant defences are decreased in plasma and erythrocytes of PKU patients, which may be due to increased free radical generation or secondary to the deprivation of micronutrients which are essential for these defenses Objectives: To compare the levels of the antioxidant enzyme activity of glutathione pathway: Glutathione peroxidase (GPx), and glutathione reductase (GRx) in PKU in relation to the healthy control group. Material and methods: We studied 42 PKU pateints compared with 30 healthy controls. Activity measurement was performed by enzymatic spectrometry.
Results: The GPx activity is decreased significantly in PKU (p=0.01). The GRx activity is significantly higher in the PKU group (p=0.006).
Conclusion: GPx activity correlated with levels of selenium in plasma (cofactor of the enzyme GPx). Our patients take selenium-supplemented formulas, so we should consider whether supplementation may not be effective.
The increased activity of GRx can be explained as occurring to avoid the toxic effect caused by increased levels of GSSG (oxidized glutathione) in PKU patients. Background: Alterations in antioxidant systems (AOS) have been related to multiple inborn errors of metabolism. Since many of the antioxidants come from the diet, patients with phenylketonuria (PKU) are susceptible to different dietary deficiencies of antioxidant vitamins and trace elements. It is important to identify alterations of this system in order that they can be corrected with supplements.
Objectives: Comparing the levels of total antioxidant capacity (TAC) and activity of antioxidant enzymes superoxide dismutase (SOD), and catalase (CAT) in PKU compared to the controls. Material and methods: We studied 42 PKU under dietary treatment and compared with 30 controls. TAC levels and SOD activity were determined by enzymatic spectrometry and CAT activity by spectrophotometry. Results: The activity of CAT and SOD is decreased significantly in PKU (CAT: p=0.02, SOD: p=0.001). We found no significant differences in TAC levels between PKU and controls. Conclusion: The antioxidant enzymes CAT, SOD are considered the main enzymatic defences against free radical production, and are decreased significantly in PKU in our area, which reinforces the hypothesis that oxidative stress is increased in PKU, and that pathogenic processes also occur in patients under protein dietary. Background: Phenylketonuria (PKU) is caused by deficiency of the phenylalanine hydroxylase, leading to accumulation of phenylalanine. Clinical features of PKU include mental retardation, microcephaly, and seizures. Mechanisms of brain damage still not clear, but increased oxidative stress is associated.
Objective: Verify the effects of exercise on oxidative stress parameters in the brain of hyperphenylalaninemic rats. Methods: Sedentary (Sed) and exercise (Exe) rats groups were subdivided into saline (SAL) and PKU. PKU groups were induced hyperphenylalaninemia through administration of alpha-methylphenylalanine and phenylalanine for 17 days, while SAL groups received saline. Exe groups conducted 2-weeks of aerobic exercise lasting for 20 min per day. At the 18th day, the animals were killed and the brain was homogenized to determine tiobarbituric acid reactives substances (TBA-RS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Results: PKU model caused an increase in TBA-RS and SOD, and reduces CAT and GPx. Exercise was able to prevent all changes seen in the PKU group, except for the SOD activity. Background: Phenylketonuria (PKU) is a rare genetic disease caused by a deficiency in phenylalanine hydroxilase activity, leading to an accumulation of phenylalanine (Phe). Clinically, patients present severe intellectual disability, whose pathophysiology is still uncertain. Objective: In the present work we investigated the levels of different cytokines in cerebral cortex of rats submitted to an experimental model of PKU.
Methods: Male 30-day-old Wistar rats received a single subcutaneous Phe injection (5.2 μmol/g) and/or p-chlorophenylalanine (p-Cl-Phe; 0.9 μmol/g), an inhibitor of phenylalanine hydroxilase. Control group received saline solution at the same volume. One hour after Phe administration, cerebral cortex was isolated and the levels of the cytokines interleukine-1B (IL-1B), interleukine 10 (IL-10) and α-tumoral necrosis factor (TNFα) were evaluated. Results: IL-1B and IL-10 levels were increased by the simultaneous administration of Phe and p-Cl-Phe, but not by isolated administration. On the other hand, TNFα levels increased in animals receiving Phe, p-Cl-Phe or Phe plus p-Cl-Phe, as compared to control group. Conclusion: Taken together, these data suggest that Phe administration alters cytokine homeostasis in brain of young rats. Our results may help to explain, at least in part, the characteristic brain impairment observed in PKU patients. Financial Support: L'Oreal/ABC/UNESCO, PIBIC/UNESC and CNPq. Background: Glutathione (GSH) is an important antioxidant that eliminates reactive species and acts as cofactor for many antioxidant enzymes. Several studies have reported the involvement of oxidative stress in PKU patients and changes in GSH/GSSG ratio (an important index of oxidative stress) were found in tissues from a hyperphenylalaninemia (HPA) animal model. Objectives: The activity of some enzymes involved in GSH metabolism was evaluated in brain of rats subjected to a chemically-induced HPA. Material and methods: Six-day-old Wistar rats received daily injections of α-methyl-phenylalanine (1,6 μmol/g), phenylalanine hydroxylase inhibitor, and Phe (2,1 μmol/g) for 7 days. Controls received saline instead. Animals were killed and the brain removed and homogenated to measure the activity of glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and glutamylcysteine ligase (GCL).
Results: The activities of GPx, GR and G6PD were decreased in HPA group while GCL activity was increased. On the other hand, HPA did not alter GST activity.
Conclusion/discussion: We demonstrated that alterations in the activity of enzymes involved in brain GSH metabolism is probably underlying the changes in GSH/GSSG ratio in HPA while reactions of phase II detoxification, represented by GST, seems to be not involved. ( Deficiency of 6-pyrovoyl-tetrahydropterin synthase is the most common cause of tetrahydrobiopterin metabolism disorders. Typical features of the disease are neuromotor signs, which are due to monoamine depletion. Isolated psychiatric symptoms are not commonly described.
We report a 17-year-old female, detected by neonatal screening for hyperphenylalaninemia and diagnosed at the age of 18 months as affected by PTPS deficiency (genotype T67M/K129E). In cerebro-spinal fluid (CSF), homovanillic acid (HVA) and 5 hydroxy-indolacetic acid (5HIAA) were reduced (253 nmol/L and 64 nmol/L respectively). Despite scarce evidence of characteristic extrapyramidal symptoms, our patient presented a stable cognitive deficiency (IQ 52), childhood-onset of anxiety disorders as separation anxiety and social phobia and pubertal onset of obsessive thoughts, hallucinations, odd behaviour and compulsiveness. These psychiatric features followed a fluctuant trend. On two different occasions of psychiatric symptom recrudescence during adolescence, CSF examination showed a normal level of 5HIAA and HVA and very high concentrations of 3-O-methyl-dopa and 5-hydroxy-tryptophan. Sertraline was introduced in augmentation of traditional therapy, with significant regression of symptoms. These findings suggest that normal levels of biogenic amines in CSF may not correspond to symptomatology abatement: normal concentrations of amine metabolites may not reflect neurotransmitter availability and activity at the receptor level.
We report the effect of cellular antisense therapy using a new transporter structure to suppress pseudoexon activation in primary dermal fibroblasts from patients with mutations in the PTS gene encoding 6pyruvoyltetrahydropterin synthase (PTPS), and also a deeper functional analysis of the mutant change c.164-712A>T that activates the exonization of an AluSq sequence previously reported. The results obtained using ex vivo minigenes showed that the change c.164-712A>T at position +6, but also at position +3 of the pseudoexon (c.164-715 T>A), seems to strengthen the pseudoexon 5´splice site thus improving complementarity to U1 snRNA. We have demonstrated that even though two different 3´ss could be used to insert a 70-bp or 45-bp pseudoexons, these changes predominantly activate the most upstream one. Regarding the use of a new vehicle effective for in vivo delivery into a wide variety of tissues, we have used a conjugates of an octa-guanidine dendrimer covalently linked to the morpholino oligos to perform a parallel set of experiments. The RT-PCR and also the pterin profile showed that the AMO transporter-conjugates successfully corrected the mis-splicing recovering the metabolic pathway and confirm the therapeutical option for this type of mutations. Background: The main treatment for PKU is lifelong dietary phenylalanine restriction. Early dietary treatment is effective in hyperphenylalaninemia, but this Phe restricted diet has negative aspects. A subset of patients shows a clinically significant reduction in blood phenylalanine when treated with pharmacological doses of Sapropterin. Sapropterin has been approved for the treatment of hyperphenylalaninaemia in patients>or= 4 years of age.
Objective: Assessing the treatment with sapropterin in children less than four years. Patients: PAH deficiency patients younger than 4 years treated with sapropterin. Results: Six children less than 4 years have been treated with sapropterin. All of them but one were responsiveness. Two of them began the treatment from the newborn period and the other three began it above one year. Considering all together, the Phe (nmol/ml) mean and SD was 215,4+ 108,49; the Phe intake (mg) mean and SD was 1025+745. Children who began when they were more than one year old, the Phe intake (mg) mean and SD before sapropterin treatment was 570,14+324,83 and after treatment was 1340,96+978,86. There has been no side effects. Comments. Sapropterin treatment is a valid alternative to the treatment with a diet limited in phenylalanine in hyperphenylalaninemia in patients less than 4 years. Background: Low birth weight is associated with increased neonatal morbidity and mortality but also with life-long consequences such as cardiovascular diseases. Maternal homocysteine concentrations (tHcy) have been linked to a wide range of adverse pregnancy outcomes and could influence birth weight. We performed a systematic review and metaanalysis on the association of maternal tHcy and birth weight. Design: A literature search using Pubmed revealed 78 abstracts. Studies were eligible if information on maternal tHcy, birth weight and the association between maternal tHcy and birth weight was available. Effect estimates were converted to odds ratios with a cut-off level of birth weight <p10 for gestational age (SGA) and maternal tHcy>p90. Cystathionine beta-synthase (CBS) deficient homocystinuria (HCU) if untreated, typically results in cognitive impairment, connective tissue disturbances, and atherosclerosis and thromboembolic disease. We investigated the expression levels of 25 separate cytokines/chemokines in both a transgenic mouse model of HCU and human subjects with the disease. HCU mice exhibited highly significant inductions of the pro-inflammatory cytokines Il-1alpha, Il-1beta and TNF-alpha which were normalized or significantly lowered by treatment with either betaine or the antioxidant amino sulfonic acid taurine. In untreated/poorly compliant human subjects with HCU, we observed constitutive induction of multiple pro-inflammatory cytokines (IL-1alpha, IL-6, TNF-alpha, Il-17 and IL-12(p70)) and chemotactic chemokines (fractalkine, MIP-1alpha and MIP-1beta) compared to normal controls. The expression levels of anti-inflammatory cytokines were normal in both HCU mice and humans with the disease. In human subjects, conventional therapy lead to either normalization or significant reduction of all of the proinflammatory cytokines and chemokines investigated.We conclude that HCU is a disease of oxidative stress induced chronic inflammation and that aberrant cytokine expression has the potential to contribute to multiple aspects of pathogenesis. Our findings indicate that antioxidant and anti-inflammatory strategies could serve as useful adjuvant therapies for this disease. Misfolding of mutant proteins is a common pathogenic mechanism in many genetic diseases including phenylketonuria. Since misfolding of mutant phenylalanine hydroxylase enzymes is successfully targeted by sapropterin administration we explored whether misfolding plays a role in cystathionine beta-synthase (CBS) deficiency and whether it can be modulated by chaperones.
We have studied a series of 27 disease-causing mutations located in different domains of the CBS molecule representing~70% of patient-derived mutant alleles. Expression in prokaryotic and eukaryotic cells showed a propensity of mutants to misfold/misassemble, which was accompanied by their impaired catalytic activity. Homocystinuria is an inborn error of metabolism biochemically characterized by cystathionine beta-synthase deficiency, leading to homocysteine (Hcy) tissue accumulation. Clinically, patients present mental retardation, seizures, and vascular complications. Folic acid therapy has been used, since it is a methyl donor in Hcy metabolism, as well as its own antioxidant properties. In the present study we evaluated the effect of Hcy on the activities of creatine kinase and respiratory chain enzymes (complex II, succinate dehydrogenase and cytochrome c oxidase) in parietal cortex of rats. We also evaluated the neuroprotector effect of folic acid on biochemical alterations elicited by Hcy. Acute treatment: 22 day-old rats received folic acid daily administrations for one week, 12 hours later the animals were subjected to a single Hcy administration. Chronic treatment: 6 day-old rats received daily folic acid and/or Hcy injections up to 28th day-of-life. Results showed that Hcy acute administration reduced cytochrome c oxidase activity, while chronic Hcy administration increased it. Hcy did not alter other enzymes evaluated. Folic acid prevented Hcy effects, probably by acting as an antioxidant. In conclusion, folic acid prevented the effects of Hcy administration on respiratory chain activity, suggesting a relevant role of this vitamin preventing neurotoxic effects of hyperhomocysteinemia. In the present study we evaluate the effect of chronic homocysteine administration on some parameters of inflammation, such as cytokines (TNF-α, IL-1β and IL-6), chemokine CCL2 (MCP-1), nitrite levels, prostaglandin E2, as well as acetylcholinesterase activity in rat hippocampus. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th day-ofage. Rats were sacrificed 1 or 12 h after the last injection. Results showed that chronic hyperhomocysteinemia significantly increased proinflammatory cytokines (TNF-α, IL-1β and IL-6), chemokine CCL2 (MCP-1), prostaglandin E2 and nitrite levels in hippocampus.of rats. Acetylcholinesterase activity also was increased after homocysteine administration. Our findings show that chronic hyperhomocysteinemia increases inflammatory parameters, suggesting that this process might be associated, at least in part, to the brain dysfunctions characteristic of some homocystinuric patients. Supported by CNPq and FAPERGS. The purpose of this study was to develop a chronic chemically-induced model of mild hyperhomocysteinemia in adult rats. We produced levels of Hcy in the blood (30 μM), comparable to those considered a risk factor for the development of neurological and cardiovascular diseases, by injecting subcutaneously homocysteine (0.03 μmol/g of body weight) twice a day from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Using this model, we evaluate the effect of chronic administration of homocysteine on redox status in blood and cerebral cortex of adult rats. Reactive oxygen species and thiobarbituric acid reactive substances were significantly increased in plasma and cerebral cortex, while nitrite levels were reduced in cerebral cortex, but not in plasma of rats subjected to chronic hyperhomocysteinemia. We also observed that homocysteine disrupted enzymatic and non-enzymatic antioxidant defenses in the blood and cerebral cortex of rats. Considering that experimental animal models are useful to understand the pathophysiology of human diseases, the present model of mild hyperhomocysteinemia may be useful in the investigation of additional mechanisms involved in tissue alterations caused by homocysteine. Supported by CNPq and FAPERGS. Homocystinuria is an inborn error of metabolism caused by cystathionineβ-synthase deficiency, leading to tissue accumulation of homocysteine (Hcy). Affected patients may present with seizures and mental retardation. It has been demonstrated that homocysteine might promote glutamatergic excitotoxicity due to overstimulation of NMDA receptors. Studies show that guanosine (Guo) prevents seizures induced by changes in glutamatergic system in rats. In the present study we evaluated the influence of Guo on the effects elicited by severe hyperhomocysteinemia on Na+, K± ATPase activity and glutamate uptake. Wistar rats received daily twice subcutaneous injection of D,L-Hcy (0.3-0.6 μmol/g body weight), and/or Guo (7.5 mg/kg) once a day from 6th to their 21th day of life. Twelve hours after the last injection the rats were sacrificed and hippocampus was dissected. Results showed that hyperhomocysteinemia reduced activity of Na+, K±ATPase and glutamate uptake in rat hippocampus. Guo prevents only the decrease Na+, K±ATPase activity. Our findings suggest that Hcy might alter neuronal excitability and increase levels of glutamate in the synaptic cleft which could lead to excitotoxicity. The mechanisms of prevention of Guo on the activity of Na+, K±ATPase is still unknown and need further studies to be elucidated. Supported by CNPq, FAPERGS. An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders of cobalamin metabolism, particularly with the cblC type of methylmalonic aciduria combined with homocystinuria . In order to analyze the role of homocysteine in stress response, we have evaluated several parameters related to oxidative stress and apoptosis in fibroblasts from patients with defects of MTHFR, MTRR and MTR genes causing homocystinuria. All cell lines showed a significant increase in ROS content and in MnSOD expression level, and also a higher rate of apoptosis with similar levels to the ones found in cblC fibroblasts. The amount of phosphorylated forms of p38 and JNK stresskinases was also increased. ROS content and apoptosis rate were increased in control fibroblasts and glioblastome cell line by shRNA-mediated silencing of MTRR gene expression, indicating the possible function of homocysteine in these processes. The toxic built-up of homocysteine in patients with homocystinuria might be partially responsible for the deleterious effects in stress response, and support the potential of using antioxidants as a novel therapeutic strategy to improve the severe neurological outcome of these rare diseases. The key regulatory point of transsulfuration pathway is catalysed by cystathionine beta-synthase (CBS), a homotetrameric enzyme activated by S-adenosyl methionine (SAM), which binds to CBS C-terminal domain. However, the activation mechanism remains unclear.
To study this mechanism we developed an expression system to produce recombinant CBS mutant proteins identified in a group of 18 CBS deficient patients presenting a disturbed SAM regulation, and localized away from the C-terminal domain, namely in the N-terminal domain (P49L) and in the catalytic core (G151R, G153R, K269del, I278T, R336H). CBS wild-type cDNA was cloned into pET28b expression vector. Mutations were obtained by site-directed mutagenesis. Enzyme activity and SAM activation ratio were determined according to standard procedures. Expression levels and purity grades were evaluated by SDS-PAGE and oligomeric profile by native-gel electrophoresis. Functional CBS wild-type protein was obtained in high yield and purity grade. From the studied CBS mutants only P49L presented a high residual activity (81% ofWT) but a lower SAM activation (20.3% of WT Inhibition of cellular methylation reactions by S-adenosyl homocysteine (AdoHcy), which accumulates in the setting of hyperhomocysteinemia, has been suggested to contribute to endothelial dysfunction in homocysteine (Hcy)-related vascular disease. We aimed at determining whether intracellular AdoHcy accumulation affects NO production by human endothelial cells. Human vascular endothelial cells were incubated with adenosine-2,3dialdehyde (ADA) (0, 5, 10 and 20 μmol/L), an inhibitor of AdoHcy hydrolase, for 12 to 24 hours. Extracellular Hcy and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (eNOS), were measured by HPLC. Intracellular AdoHcy was determined by LC-MS/ MS. Nitrite was quantified by Griess reaction to evaluate NO production. Coincubations with L-NG-nitroarginine (L-NNA) (1 mmol/L), an eNOS inhibitor, were also performed. eNOS transcriptional and translational levels were studied by qRT-PCR and Western blotting, respectively. eNOS function was evaluated by assessing the effectiveness of L-arginine to L-citrulline conversion in cell lysates. ADA elicited intracellular accumulation AdoHcy, while extracellular Hcy, ADMA and nitrite levels decreased. Co-incubation with L-NNA suppressed this effect. For ADA at 20 μmol/L, eNOS expression and activity decreased by 40% and 20%, respectively. Intriguingly, transcription of NOS3 increased. Thus, ADMA-independent postranscriptional mechanisms modulated by cellular methylation capacity may lower NO production by vascular endothelial cells. Mutations in the gene encoding methionine synthase result in methylcobalamin deficiency G (CblG) disorder leading to hyperhomocysteinemia and low methionine. The most frequent clinical findings reported are megaloblastic anemia and neurological symptoms. Patient 1 was hypotonic at birth and anaemia was observed at one month. She was weak, had feeding difficulties and a gastrostomy was installed at 7 months. Delayed development led to diagnosis and treatment start at one year. Almost 6 years old she presented with kidney failure due to an atypical hemolytic uraemic syndrome (HUS) from which she recovered after 6 months. Now at age eleven she has short stature and attends a special school. Patient 2 presented neonatally with apnoeas, hypotonia, seizures and poor weight gain. After initiation of treatment he recovered but not to complete normality. At two years he also developed an atypical HUS and a tracheal stenosis was diagnosed. Now, 3 years old, kidney function has normalized but he still suffers from hypertension, needs both a gastrostomy and a tracheostomy tube and has a short stature. His cognitive function is almost normal.
In conclusion these patients raise questions about treatment strategies and the mechanism behind the development of atypical HUS in CblG deficiency. Inborn errors of cellular cobalamin (Cbl) metabolism are disorders of decreased production of adenosylcobalamin or methylcobalamin either both or alone, cofactors for methylmalonyl CoA-mutase and methionine synthase, respectively. cblC deficiency is due to a defect in Cbl(III) to Cbl(II) conversion resulting in accumulation of methylmalonic acid and homocysteine. Neonatal presentation is typical but milder forms may present later with neurological deterioration. Features uncommon in inborn errors of metabolism such as dysmorphisms, ocular abnormalities, and congenital malformations are seen in cblC. Hydrocephalus, a recognised complication has been previously noted postnatally only and not secondary to cerebral haemorrhage. We describe a case with prenatal presentation of right sided ventriculomegaly and grade three intraventricular hemorrhage (IVH) diagnosed at 28 weeks gestation by ultrasound and MRI. Newborn screening showed elevated C3 acylcarnitine and confirmatory investigations led to the diagnosis of cblC disorder. A congenital bleeding disorder was excluded. At present his development is normal on hydroxycobalamin and betaine therapy despite marked right ventriculomegaly. To our knowledge, this is the first description of prenatal IVH and hydrocephalus associated with the cblC disorder and only the second report of prenatal abnormality. cblC disorder remains one of the few inborn errors of metabolism associated with prenatal malformations. Conclusions: Brain folate is essential for more than 100 metabolic processes in the nervous system and influences neurotransmitter turnover. The presence of FR autoantibodies over time predisposes to episodes associated with CNS folate depletion. Our findings may explain the alternating negative and positive symptoms characteristic of schizophrenia and may be due to fluctuations in antibody titer observed. Evaluation for neuro-immunologic causes, including FR autoantibodies is recommended for patients with intractable schizophrenia, who can benefit from folinic acid supplementation. Juvenile Megaloblastic Anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin (vitamin B12) malabsorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN that binds to intrinsic factor-cobalamin complex, in the distal intestine and the proximal renal tubule. We report in two siblings a compound AMN heterozygosity with c.742 C>T, p.Gln248X and c.208-2A>G mutations, that led to premature termination codon in exon 7 and exon 6, respectively and produced a dramatic decrease of receptor activity in urines, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742 T and c.208-2 G had no pathological signs. These results indicate that amnionless is indispensable for the proper luminal expression of cubilin, in humans. Severe mutations in the gastric intrinsic factor (GIF) gene lead to congenital megaloblastic anemia. Little is known on the frequency and influence of GIF variants on the cobalamin (vitamin B12) absorption. We therefore aimed at identifying GIF variants in subjects with biological evidence of cobalamin deficit, in 2200 subjects prospectively recruited in our University hospital (North East of France) during 2 years. The selection criteria included decreased cobalamin, increased homocysteine and/or methylmalonic acid and/or macrocytosis. Among the 2200 subjects, 176 cases presented with hyperhomocysteinemia and/or increased methylmalonic acid, including 45 cases with combined criteria of cobalamin deficit. These 45 cases were compared to 70 controls. Five GIF variants were identified in 14 cases. We report a 13-year old female with Asperger syndrome, epilepsy, sleep disorder, episodic ataxia and tremor. Investigations showed a reduced CSF pyridoxal 5'-phosphate (PLP) (6 nmol/L, range 10-37 nmol/L) with normal CSF monoamine neurotransmitters and low-normal plasma PLP (22 nmol/L, range 15-73 nmol/L). She had reduced whole-blood serotonin (539 nmol/L, range 600-1600 nmol/L), low aromatic amino-acid decarboxylase activity (24.4pmol/min/ml, range 36-129pmol/min/ml) and isolated reduction in plasma tryptophan (14,17,27 μmol/L, range 30-87 μmol/L). Urinary alpha-AASA excretion was normal and sequencing of the pyridox(am)ine 5'-phosphate oxidase gene revealed no mutations. She has shown good improvement on PLP therapy with cessation of seizures, tremor, ataxia and improvement in social functioning. Tryptophan depletion is associated with autistic symptomatology and disturbance of circadian rhythm. PLP deficiency may result in seizures. Plasma tryptophan levels are regulated by the inducible enzyme tryptophan-2, 3-dioxygenase (TDO2) which diverts metabolism of tryptophan into the kynurenine pathway away from the methoxyindole pathway for serotonin and melatonin synthesis. Increased TDO2 activity (via secondary induction/gain-of-function mutation) may lead to the biochemical phenotype described. The mechanism of reduced PLP levels is difficult to explain but, if kynurenine levels are elevated, it may be due to the formation of a Schiff base between PLP and kynurenine. We present two children with early infantile seizures, hypoglycorrhachia and increased CSF lactate. The clinical picture and routine biochemical findings raised the possibility of GLUT1 deficiency (CSF:plasma glucose <0.4) or mitochondrial disease. However urine organic acid analysis was suggestive of biotinidase deficiency, subsequently confirmed by low plasma biotinidase activity and clinical response to biotin. Both infants presented at 2 months with seizures. The CSF:plasma glucose ratios were 0.35 and 0.33 (reference range >0.60) and CSF lactates 5.1 and 6.4 mmol/L (reference range 1.1-2.2 mmol/L). There is an extensive differential diagnosis for infants with early infantile seizures. In the absence of infection, increased CSF lactate concentrations may raise the suspicion of a mitochondrial disorder, while low CSF glucose may be attributed to GLUT1 deficiency. However a low CSF lactate is expected in GLUT1 and low CSF glucose is not a typical feature of mitochondrial disease. Although hypoglycorrhachia is not generally reported in biotinidase deficiency, we suggest this diagnosis should be borne in mind where it occurs with seizures and raised CSF lactate. We speculate that the pyruvate carboxylase deficiency that arises in biotinidase deficiency may be responsible for these biochemical findings, perhaps due to lack of oxaloacetate. Background: Vitamin C is the cofactor in biosynthesis of collagen, catecholamines and iron metabolism.
Objectives: The clinical features of scurvy may resemble metabolic disorders and hamper early diagnosis. Case report: A three-year old male child was admitted to hospital with complaints of growth retardation, irritability, joint deformities, limited mobility, wheezing and frequent illness. Severe malnutrition, coarse facial appearance, gingival hypertrophy, organomegaly, significant osteoporosis, and anemia that did not respond to iron treatment were detected and differential diagnosis for metabolic disorders were considered. Screening for inborn errors revealed generalized aminoaciduria and elevated blood free carnitine. Enzyme analyses excluded lysosomal storage disorders. Dietary history of the patient revealed very poor consumption of vitamin C which was about 13% of RDA and had continued for a long period. Vitamin C level was 0.05 mg/L (n: 4-21) in plasma, 2.85 mg/24 hour (n:10-30) in urine, and 1.03 mg/6 hours in urine after intravenous loading. The patient was given 200 mg/day vitamin C orally. Irritability and bone sensitivity decreased after the first two days and clinical improvement ensued. Conclusion: Clinical features in scurvy resemble symptoms observed in some metabolic disorders. Careful clinical evaluation, dietary history and vitamin C assay may exclude uneccessary metabolic work-up. Background: Menkes disease (MD) is an X-linked disorder of copper transport caused by ATP7A gene mutations. Treatment with parenteral copper-histidine can change prognosis when initiated in early stages of neurological disease. Case Reports: Patient 1 was diagnosed of MD (15 bp del.exon1) at the age of 2 months, showing hypotonia, joint hyperlaxity and femoral fracture. No seizures were reported. Parenteral copper-histidine was started at the age of 5 months and continued for 9 years. At 10 years of age attends normal school and has no seizures. Current MRI shows marked tortuosity of intracranial vessels without brain atrophy. Connective tissue abnormalities include joint hyperlaxity, bone deformities and asymptomatic urinary bladder diverticula. Mild renal tubulointerstitial damage is due to chronic copper supplementation. Patient 2 was diagnosed of MD (c1971t>A mutation) at the age of 3 months after admission for refractory epilepsy. Treatment started immediately but neurological status did not improve. He developed severe mental retardation and intractable epilepsy. He was under treatment until the age of 5 years when he died. Discussion: Early treatment in Menkes disease is critical for the prevention of neurological damage although diagnosis is difficult in presymptomatic stage. Long term effects of treatment include bone deformities and tubulointerstitial damage. Background: Wilson disease is an autosomal recessive disorder in which copper accumulates in tissues due to deficit of hepatic ATPase. Clinical picture is extremely variable. Hepatic manifestation is the most common feature in childhood. Neuropsychiatric disorder appears in adulthood. Case Report: We present a case of a family with variable clinical picture in 2 siblings. 14 years old boy was sent with 3 years history of unexplained hepatopathy first revealed during acute gastritis. Detailed examinations were performed including ceruloplasmin, which was normal during first year. Accidently 17 years old brother was present at a first visit. He presented with hypertonic-hypokinetic syndrome which developed during 3 months and was explained by psychiatric treatment for anxiety and depression with progressive cognitive deterioration and rigidity. Suspected Wilson disease in both was confirmed by patognomic laboratory findings (low ceruloplasmin, high urine copper excretion), MRI findings in older one and definitely by DNA analysis. Because of persistent hyperbilirubinemia and otherwise negative findings in their sister all the siblings were examined and Gilbert syndrome was confirmed.
Conclusion: Clinical and laboratory variability during course of the disease and even in the same genotype may cause confusion in diagnostics. Therefore repeated laboratory investigation is strongly recommended. Background: Wilson's disease (WD, MIM #277900) is an autosomal recessive disorder leading to systemic copper accumulation and multiorgan damage. For pediatric patients, hepatic manifestations predominate. Untreated WD causes progressive liver and neurological deterioration. Early treatment is the most effective way of preventing these serious outcome.
Objective & Methods: We presented our experience of managing 10 paediatric WD patients. Data on clinical symptoms, laboratory findings, ultrasound findings, liver biopsies, genetic studies, treatment and outcome were studied. Results: Our patients were between 2 to 18 years of age at diagnosis. 1 patient presented in liver failure. 7 had abnormal liver functions detected incidentally. 2 were diagnosed through sibling screening. The most consistent abnormal liver function was an elevated alanine transaminase ranging from 60 to 419 IU/l (<58). Ceruloplasmin levels were all <0.1 g/l (0.21-0.59). Mean urinary copper excretion was 4 μmol/day (<1.0). 1 patient was treated initially with penicillamine and another trientine. 7 patients were started on zinc therapy. All remained well on follow up. Conclusion: Our patients represented a group of asymptomatic WD patients diagnosed and treated very early. With good treatment compliance, favourable outcome can be anticipated. One way of diagnosing these presymptomatic patients is through following up abnormal liver functions. Cerebral Folate Deficiency Syndrome(CFD) has been adopted for a group of neuro-psychiatric disorders associated with low spinal fluid 5 Nmethyltetrahydrofolate (MTHF) concentrations in the presence of normal folate metabolism outside the CNS. One important mechanism underlying CFD is the presence of serum autoantibodies of the blocking type directed against the folate receptor (FR) attached to membranes at the plasma-side of choroid plexus epithelial cells, which normally mediates the MTHF transport to the CNS. Two clinically recognizable syndromes of FR-antibody mediated CFD have been detected in young children: the infantile-onset CFD syndrome presenting 4 to 6 months after birth and a spastic-ataxic syndrome manifesting after one year. In addition, serum FR autoantibodies and CFD have also been reported in Rett, Aicardi-Goutières syndrome and infantile autism. During late adolescence CFD associated with fluctuating autoantibodies has been identified in intractable schizophrenia. This clinical heterogeneity associated with FR-autoantibody mediated CFD might be explained by differences in the time of onset and period during which these FR autoantibodies are generated leading to folate deficiency during various critical stages of early brain development. More awareness of CFD in association with FR autoimmunity should lead to earlier detection and diagnosis of these potentially treatable neuro-psychiatric disorders.
The creatine transporter (CRTR) defect is a recently discovered cause of Xlinked intellectual disability which is associated with cerebral creatine deficiency. Treatment with creatine monohydrate has not proved effective. Supplementation with creatine precursors L-arginine and glycine might increase endogenous cerebral creatine synthesis. The effect of this treatment is still controversial. We followed nine boys aged between 8 months and 10 years with molecularly confirmed CRTR defect with repeated 1H-MRS and neuropsychological assessments during 4-6 years of combination treatment with creatine monohydrate, L-arginine and glycine. Treatment did not lead to a significant increase in cerebral creatine content as observed with H1-MRS. After an initial improvement of locomotor and personal-social IQ subscales, no lasting clinical improvement was recorded. Additionaly, we noticed an age-related decline of IQ subscales in boys affected with the CRTR defect. Background: It is not known whether mouse models of creatine synthesis disorders AGAT and GAMT deficiency express any anatomical phenotype in the brain. Methods: Applying high-resolution magnetic resonance imaging combined with advanced image analysis techniques and statistics, the study aimed to characterize anatomical changes between AGAT and GAMT mutant mouse and there corresponding heterozygote and wild type genotype. Results: When analyzing absolute volumes of brain structures for the AGAT group, we found that 15 different brain structures had significant size differences among genotypes, including the corpus callosum, fimbria and internal capsule. These structures also had significant size differences when normalized for total brain size. Significant peaks (local areas of difference in relative size) were found for a number of areas in the brain, including the corpus callosum, hypothalamus and cerebral cortex. For the GAMT group, we found that the corpus callosum and internal capsule are significantly different in size among genotypes. These differences were found for both absolute and normalized volumes. Significant peaks were found in several regions of the brain, including the cerebellar cortex and hippocampus.
Conclusion: AGAT and GAMT knockouts express a similar neuroanatomical phenotype that, interestingly and in contrast to patients, is more pronounced in AGAT deficiency. Phosphocreatine serves as an important reservoir of high-energy phosphate for ATP synthesis in tissues that have fluctuating demands for energy. Human disorders of creatine biosynthesis and transport exist, leading to intellectual disabilities, epilepsy, and poor growth. Creatine biosynthesis requires two enzymes, the first, L-arginine:glycine amidinotransferase, the product of the Gatm gene, catalyzes the rate-limiting transfer of the amidino group from arginine to glycine, yielding guanidinoacetate and ornithine. We generated a mouse strain lacking Gatm. Homozygous Gatm deficient mice have an almost complete absence of creatine and guanidinoacetate, and exhibit poor growth and muscle weakness. There is a reduced amount of glycogen in the liver in conjunction with reduced glycogen synthase activity, lipid accumulation in liver, skeletal and cardiac muscle, and a marked hypoplasia of skeletal muscle. A consequence of creatine deficiency is the global reduction in skeletal muscle mitochondrial respiratory chain activities, and a compensatory increase in mtDNA content and citrate synthase activity. Muscle mitochondria accumulate striking paracrystalline arrays of cristae. Cardiac function as assessed by stress echocardiography is normal. Treatment with creatine leads to muscle proliferation and improves strength. This animal model of creatine deficiency demonstrates the central role creatine metabolism plays in growth and muscle homeostasis. Background: The creatine/creatine-phosphate system plays an essential role in keeping energy homeostasis in human tissues with high energy demand, such as brain and muscle. Deficiencies of creatine can cause developmental delay, intellectual disability and behavioral disorders. Three genes are involved in inborn errors of creatine deficiency syndromes: autosomal recessive GAMT and AGAT, as well as an X-linked SLC6A8 (CT1) encoding a creatine transporter. Deficiency of these enzymes can be distinguished by the concentrations of guanidinoacetate (GAA), creatine, and creatine/creatinine ratio in plasma and urine. However, biochemical analyses do not provide definitive diagnosis.
Method: Coding regions of AGAT, GAMT, and SLC6A8 (CT1) genes were sequenced on 25, 55, and 105 individuals, respectively. Results: Mutations in 3, 10, and 13 patients, respectively, were identified. The majorities (>70%) were novel, null mutations with an overall mutation detection rate of 14.2%, suggesting that biochemical and clinical diagnosis requires molecular confirmation.
Conclusion: Molecular analysis should be pursued on patients with suspicion of creatine deficiency. Since these diseases are treatable, early diagnosis assures early treatment, proper genetic counseling and patient management. Furthermore, identification of mutations aids in carrier and prenatal diagnosis. Background: X-linked creatine transporter (SLC6A8) deficiency presents with developmental delay, seizures and behavioral disturbance in boys due to cerebral creatine deficiency. Oral creatine supplementation generally has not resulted in an improvement in neurological symptoms. Case reports: The first boy presented at age 12 years with autism, developmental delay and seizures. A hemizygous c.1222_1224del TTC (p.408del) mutation was detected. The second boy presented at age 3 with mild global developmental delay with significant speech impairment. A novel hemizygous c.238 T>C (p.Y80H) variant was found in the SLC6A8 gene. MRS for both boys revealed a significantly decreased but not absent creatine peak. In view of the small residual creatine peak on MRS, treatment with oral creatine monohydrate supplementation was initiated. Results: Both boys demonstrated an improvement in developmental abilities, particularly speech, on creatine supplementation. In addition, the first boy became seizure-free and had a resolution of abnormal involuntary movements. Despite a clinical response to creatine supplementation in both boys, the size of the MRS peak remained unchanged on serial MRS studies. Conclusion: Boys with X-linked creatine transporter deficiency with a residual creatine peak on MRS may benefit from supplementation with oral creatine monohydrate. Creatine deficiency syndromes, due to defects in AGAT and GAMT (creatine synthesis pathway) or in SLC6A8 (creatine transporter) are inborn errors of metabolism essentially affecting the brain. Autosomal recessive AGAT deficiency is the least common of the three creatine deficiencies, having been described in only 3 families worldwide. In these patients, AGAT deficiency may be due to nonsense-mediated decay of mRNA due to stop codon mutations. Symptoms include speech delay, mental retardation, epileptic seizures, autism and brain atrophy.
To better understand the consequences of creatine deficiency in the brain, short-hairpin RNAs (shRNAs) were used to silence the expression of AGAT in the rat oligodendroglia-glioma hybrid cell line (ROC). Our results show an average of 88% decrease in AGAT enzyme expression in normal conditions. We show that in ROC cells, cell survival was not affected by AGAT silencing, both in normal conditions and under creatine deficiency (serum deprivation). Adeno-Associated Viruses (AAV) transducing shRNAs targeting AGAT mRNA are currently produced to test the effect of creatine deficiency in 3D organotypic primary cultures of brain cells. As creatine (Cr) plays an essential role for the maintenance of ATP levels in tissues with high demand, such as brain, creatine deficiency syndromes could result in energy depletion. This depletion appears as a plausible mechanism for causing overproduction of reactive oxygen species (ROS) that can lead to loss of the cell redox control resulting in cell aberrant proliferation even apoptosis. We selected two CRTR fibroblast cell lines (genotypes: p.F360del and p. W154X) with low intracellular Cr levels, overproduction of ROS, increased apoptosis and aberrant proliferation. The aim of this study was to explore the effect of Cr supplementation (500 μM 24 h) on intracellular Cr levels, and stress parameters. Results showed that intracellular Cr levels, measured by MS-MS, increased in both cell lines (7.9 and 4.6 fold) without reaching control levels. In addition, significant reduction of ROS levels, decrease of the apoptotic population (G0 phase) and progression of cells to S-G2/M stages were also detected by flow cytometry.
In conclusion, Cr at high concentrations is able to penetrate cells probably through passive transport, and may act normalizing cell cycle progression and ROS levels, outstanding the contribution of energy depletion toward metabolic stress at least in selected CRTR cells. Background: Arginine:glycine amidinotransferase (OMIM 602360) deficiency (AGAT-d) is an autosomal recessive disease characterized by specific biochemical, brain spectroscopy and genetic findings. So far only six AGAT patients were described in the literature (Bianchi MC. et al.2000; Battini R. et al.2002; Edvardson S.2010 ) and supplementation of Cr has been shown to improve clinical symptoms in affected cases. Presymptomatic treatment has been reported in only one subject (Battini R. et al.2006) . Patients: We report the neuropsychological follow up of 4 AGAT-d cases, all members of the same family, two sisters (aged 20 and 18 yrs), one male cousin (15 yrs) and one presymptomatic child (7 yrs Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of creatine biosynthesis. The enzyme catalyzes the conversion of guanidinoacetate to creatine. The clinical phenotype includes developmental delay/ intellectual disability, speech delay, seizures behavioral difficulties, and neurologic symptoms. Treatment with diet (arginine restriction/ ornithine supplementation) and creatine supplementation has been tried to decrease guanidinoacetate in the body. Treatment, both pre-symtpomatic and symptomatic, has shown significant improvement in clinical outcome. We evaluate a patient with GAMT deficiency. The patient is now 8.5 years old. He presented at 3.5 years with hypotonia, global developmental delay, and difficult to treat seizures. Testing revealed GAMT deficiency. After starting treatment, the patient no longer has clinical seizures, taking antiepileptic medication and has an improved EEG. Developmentally, he has made great strides. He has progressed from being unable to engage independently in classroom routines and activities, to reading at an approximately kindergarten to early first grade level, writing his name, counting objects and doing single digit addition. He has progressed form needing a PECS system for communication at the age of two and a half years to now consistently using 4-6 word phrases and descriptive speech. Our patient demonstrates the significant clinical improvement with treatment of GAMT deficiency. Creatine deficiency syndromes (due to AGAT, GAMT or SLC6A8 deficiencies) are inborn errors of metabolism characterized by an absence or a severe decrease of Cr in central nervous system, which is the main tissue affected. To investigate the effects of creatine deficiency on developing CNS, we developed a new experimental model of GAMT deficiency by gene knock-down, using RNA interference in 3D organotypic rat brain cell cultures in aggregates. A specific shRNA for the rat GAMT gene was transduced in brain cell aggregates by an adeno-associated virus (AAV2) under the control of the CMV promoter. The AAV2-transduced shRNA was able to efficiently knock down GAMT expression, as shown by the strong decrease of GAMT protein by western blotting. We show that GAMT knock-down strongly affected axonal growth (neurons, immunohistochemistry for pNF-M) and astrocytes (immunohistochemistry for GFAP), as well as increased cell death and apoptotic pathways in developing brain cells (TUNEL experiments and immunohistochemistry for activated caspase 3).
Our results may contribute to understand some of the alterations of CNS development affecting GAMT-deficient patients. Guanidinoacetate methyltransferase (GAMT) deficiency is a neurometabolic disorder characterized by tissue accumulation of guanidinoacetate. Affected patients present with epilepsy and mental retardation whose etiopathogeny is unclear. Since reports have shown that guanidinoacetate alters brain energy metabolism and that creatine, which is depleted in patients with GAMT deficiency, can act as a neuroprotector, in the present study we investigated whether creatine could prevent the alterations of respiratory chain complex II, Na+,K± ATPase and creatine kinase caused by intrastriatal administration of guanidinoacetate in adult rats. Animals were pretreated during 7 days with daily intraperitonial administration of creatine. After, these animals were divided into two groups: Group 1 (sham), rats that suffered surgery and received saline; and group 2 (guanidinoacetatetreated) and they were sacrificed 30 min later. Results showed that the administration of creatine was able to reverse the altered activities of complex II, Na+,K ± ATPase and creatine kinase. These findings indicate that the energy metabolism deficit caused by guanidinoacetate can be prevented by creatine that probably acts as an antioxidant. These data may contribute, at least in part, to a better understanding of the mechanisms related to the energy deficit and oxidative stress found in the GAMT deficiency. Supported by CNPq and FAPERGS. Background: Clinical symptoms of CDS are non specific and measurement of GAA and creatine is an important step for the diagnosis of these diseases.
Objectives: To determine sex and age matched reference values for plasma and urine GAA and creatine over a large cohort of control subjects and describe all known patients affected with CDS in France.
Patients and Methods: The controls cohort included 6417 subjects without CDS recruited in 6 French public hospitals during 28 months. Creatine and GAA were measured in plasma and urine and data were exploited by different statistical tools. Clinical, biological and genetic data from patients affected with CDS since 2003 were collected. Results: Analyses of control data highlighted new age reference range for plasma and urine GAA and creatine and a sex distinction for urine values. 51 patients affected with CDS (14 with GAMT deficiency and 37 with CRTR deficiency) were described. Conclusion: These new references values as a function of sex and age should improve the diagnosis of CDS, in particular to reduce the false positive rate. To conclude, we propose a diagnostic algorithm including biochemical investigations, gene studies and brain 1H-MRS, to improve the screening and the follow-up of the CDS patients. Elevated urinary excretion of creatine (CRE) is an important marker of creatine transporter (SLC6A8) deficiency. In addition to the alimentary sources of creatine (e.g. meat) the endogenous production comes from methylation of guanidinoacetate (GUA). The level of a biological component may be dependent upon its nearest metabolic precursor or other closely related substance, as has been shown for many other analytes where two dimensional reference areas do not have a quadratic form. Lg(U-creatinine) is fairly proportional to lg(age) in the interval 1-18 years of age, but deviates from that outside this interval. To investigate the functional relationship between the variables we performed a linear regression analysis from our laboratory production during the last 3 years, excluding 4 patients with documented SLC6A8 deficiency, one with muscular dystrophy and one with creatine supplementation. The model lg (U-CRE/creatinine)i=a+b.sexi+c.lg(age)i+d.lg(creatinine)i+e.lg(GUA)i+ fi where fi represents a residual, gave the following significant estimates: a**=1.11; c**=−0.548 ; d*=− 0.220 ; e**=1.009. (n=1255; **P<10-30; *P=7.10-8). Thus there is a one-to-one relationship between CRE and GUA, which requires the use of multidimensional reference ranges. Objetive: To present a sensitive and robust method for routine measurement of urinary Cr and GAA, combining solid-phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) separation, and detection by tandem mass spectrometry. Methods: Sample preparation: addition of deuterated internal standards, phosphate buffer and water in 96-well plates. Extraction: in-house assembled 96-well SPE using a strong cation-exchange resin. Separation of analytes was obtained on a Luna Silic column. Detection was done by positive-mode electrospray ionization using selected reaction monitoring with a Waters Quattro Micro TMS. Clinical validation was performed with NCCLS approved guidelines. Results and conclusion: Combination of cation exchange SPE with HILIC provided an efficient and specific method for simultaneous detection of Cr and GAAwithout derivatization, in less than 4 minutes. Achieved linearity was up to 10.4 mmol/L for Cr and 13.0 mmol/L for GAA. Total assay imprecision was bellow 16%. Limit of quantification were 5.9 μmol/L for Cr and 12.6 μmol/L for GAA. No ion suppression or enhancement was observed. The phospholipase C beta 1 (PLCbeta1) enzyme is essential for neurotransmitter-mediated signal transduction. Following receptor occupancy, PLCbeta1 catalyzes the production of myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol 4,5-bisphosphate (PIP2). PLCbeta1 gene defects may disrupt CNS development and function. One male infant with early-onset epileptic encephalopathy and a homozygous loss-of-function 0.5 Mb deletion in the PLCbeta1 gene has been reported (Kurian et al, Brain 2010). We report a second male with malignant migrating epilepsy of infancy, and a homozygous deletion of the 5' non-coding region and exons 1-3 of PLCbeta1. Seizures began at 6 months, occurred 50-60/day, and were refractory to antiepileptic drugs and ketogenic diet. Baseline MRI showed diffuse parenchymal volume loss. Mega-dose of myo-inositol (Ins) was administered gradually in an attempt to raise Ins in neurons and PIP2 levels at receptor sites and elicit a recruitment of other PLC isoforms. After 10 days of 500 mg Ins/kg/day, electroencephalographic seizures decreased to 5/day, and meaningful movements and alertness increased. TE MRS revealed a 2-4-fold Ins increase in the left basal ganglia and parietooccipital white matter regions.
After 10-weeks of Ins treatment, there has been a significant decrease in seizure activity that may be related to increased brain Ins levels. Using candidate gene approach and complete exome sequencing, we identified in two unrelated patients with autism spectrum disorders (ASD) and isolated low serotonin in brain-as reflected by the serotonin endmetabolite 5-hydroxindolacetic acid in CSF-a combination of heterozygous non-synonymous mutations in serotonin-related and/or autismassociated genes, including SLC29A4, SLC6A4, ITGB3, and others. Besides the previously reported codon-alterations from association studies, we newly identified in the brain monoamine transporter gene SLC29A4, encoding a putative serotonin-reuptake transporter protein PMAT, the alterations p.A138T or p.D326E with reduced activity. DNA sequencing of SLC29A4/PMAT in 125 individuals with ASD and 300 unaffected (control) subjects revealed the non-synonymous heterozygous alteration p.M24L or p.D29G in 7 additional subjects with ASD (and not in 300 control subjects). According to a hypothetical "genetic accumulationmodel" where the affected subjects must differ from non-affected family members (parents and siblings) in the sum of all mutations, the two ASD patients showed additional alterations in mainly serotonin homeostasis, but also other candidate genes that have previously been linked to ASD and/or intellectual disability. Our findings link a combination of mutations in several serotonin-related genes to ASD and mental retardation concomitant to low serotonin in CNS. Metabolomics refers to the comprehensive analysis of small organic molecules from biological fluids or tissues. The two main techniques used in metabolomics are (i) proton NMR spectroscopy (H-NMRS) and (ii) mass spectrometry (MS) coupled to liquid chromatography (LC) or gas chromatography.
We have developed a LC-MS-based analysis of the cerebrospinal fluid (CSF) metabolome and applied this approach to a cohort of 100 patients with various neurological disorders including patients with suspected neurometabolic disorders and patients with known metabolic disorders. Among these patients, 60 were previously studied by in vitro H-NMRS spectroscopy (Mochel et al., 2009) . From the 36 metabolites previously identified by H-RMNS only 6 could not be found with LC-MS, whereas the latter method allowed the detection of 106 additional characterized metabolites. All patients with abnormal metabolite profiles on H-RMNS were also highlighted with LC-MS but this technique allowed further identification of abnormal profiles in some patients considered normal with regards to H-NMRS.
In conclusion, LC-MS arises as a complementary tool to H-NMRS for metabolic profiling of the CSF. This technique appears promising for the identification of novel IEM involving the nervous system. (n=4), dopa-responsive dystonia (n=3), serine biosynthesis defect (n=1), cerebral folate deficiency (n=1), glycine encephalopathy (n=2), and pyridoxal phosphate dependent seizures (n=1). There was consanguinity in all, except one. Positive yield of a diagnostic LP for the diagnosis of inherited neurotransmitter metabolism disorder was overall 25.8%. Oculogyric crisis (50%) and diurnal variation (81.8%) were the only statistically significant variables, in patients with and without a specific diagnosis.
Conclusions: It is challenging to diagnose neurotransmitter defects, since there is not an ideal set of clinical symptoms. Consanguinity, diurnal variation and abnormal ocular movements are the most significant findings associated with a diagnosis of a specific neurometabolic disorder by CSF examination in our cohort. Early diagnosis is of great importance not only for specific treatments, but also for genetic counseling and prenatal diagnosis.
Eyskens FJM 1 1 Antwerp Univ Hospital, Div, Metab Dis, Antwerp, Belgium
Background: AADC-deficiency is a rare autosomal recessive inborn error of metabolism characterized by severe developmental delay, prominent motor abnormalities, oculogyric crises and autonomic features. Prognosis is poor and available treatment options only have marginal therapeutic effect.
Objective and hypotheses: We describe a five year old boy with AADCdeficiency. Severe, unpredictable, episodes of hypglycaemia were documented when he was switched from bromocryptine to pramipexol, a more potent dopamine agonist, in order to try to improve his motoric disabilities. Episodes of hypoglycaemia are documented in other patients with this disease. The pathogenesis of hypoglycaemia in these patients however is unknown. I hypothezise that a potent dopamine agonist in these patients can give rise to hypoglycaemia based on inhibition of growth hormone secretion through activation of dopanine D2 receptors and/or by the autonomic dysfunction in these patients with virtually no sympathetic activity left.
Methods: During episodes of hypoglycaemia, serum growth hormone, serum insuline and serum cortisol and urinary free cortisol and catecholamines were measured. Results: No overt hormonal abnormalities were found. The episodes of hypoglycaemia disappeared when the patient was switched back to bromocryptine.
Conclusions: Dopamine agnonists can give rise to episodes of hypglycaemia in patients with AADC-deficiency. Background: AADC deficiency is a rare autosomal recessive metabolic disorder, characterized by the lack of decarboxylation of the aromatic amino acids, L-dopa and 5-hydroxytryptophan, causing a dopamine, epinephrine, norepinephrine, and serotonine (metabolites) deficiency. Less than 100 cases have been diagnosed throughout the world. Methods: We report the case of a 7-month-old white male who was diagnosed with Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency. This diagnosis was based on CSF monamine neurotransmitter analysis and corroborated by enzymatic and genetic testing. Results: The proband was breech with neonatal hypotonia and has not met any developmental milestones. He presented for evaluation paroxysmal events which had been occurring for three months. The events are characterized by repetitive dyskinetic chewing motions without loss of consciousness. In addition to these events, the patient also had intermittent dystonic opisthotonic posturing, diaphoresis, occulogyric crises and sleep difficulties. He was tried on multiple antiepileptic medications which exacerbated his symptoms. Treatment with COMT inhibitor and Bromocriptine were not tolerated. The dystonic movements and sleep difficulties improved greatly with Lioresal and pyridoxine. Other symptoms such as diaphoresis are not Conclusion: Paroxysmal dyskinesia with stereotypic chewing and sleep difficulties due to AADC improved with Lioresal. The diagnostics of neurotransmitter disorders is almost exclusively based on the quantitative determination of the neurotransmitters or their metabolites in cerebrospinal fluid (CSF). The implementation of neurotransmitter analysis in clinical praxis is necessary for early diagnosis and treatment. Material and Methods: Neurotransmitter metabolites in CSF were analyzed in 82 children (at the age 1 months to 17 years) with clinical suspicion for neurometabolic disorders using HPLC with electrochemical detection. Results: The CSF level of homovanillic acid (HVA) was markedly decreased in three children (64, 79 and 94 nmol/l) in comparison to age related controls (lower limit 218-450). Neurological finding including severe psychomotor retardation, quadruspasticity and microcephaly accompanied with marked dystonia, excessive sweating in the first patient was compatible with the diagnosis of tyrosine hydroxylase (TH) deficiency (type B phenotype) and subsequent molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124 G>C in the TH gene. The treatment with L-DOPA/carbidopa resulted in the improvement of dystonia. MRI studies in two other patients with microcephaly revealed postischemic brain damage, therefore secondary HVA deficit was considered in these children. Introduction: Tyrosine hydroxylase deficiency includes two clinical phenotypes: an infantile progressive hypokinetic-rigid syndrome with dystonia (Type A) and a neonatal complex encephalopathy (Type B). Case Report We have revised video recording, that were obtained in a 15 years follow-up, of a male patient with a THD-type B. In the first months of life he had a truncal hypotonia, a severe hypokinesia and a reduced facial mimicry. Oculogyric crises and episodes of stupor were reported. THD was diagnosed when he was 18 months old according to CSF biogenic amine alterations and pathogenic mutation on TH gene. In the following years he suffered from generalized choreoathetosis and daily on-off phenomena. He walked unsupported at 11 years old while mental and language functions remained severely impaired. Different dopamine-mimetic drugs (including L-dopa-carbidopa, pramipexole, selegiline, entacapone, tolcapone, pyridoxine and rotigotine) were used. At the beginning he was a slow responder to pharmacological treatments. After the school age his response improved, despite fluctuations of symptoms and drugs-induced side effects (i.e. severe dyskinesia under pramipexole). Conclusions: Our experience demonstrates that the course of motor disorders in Type B variant requires a very slow increase of dopaminergic therapy. Mental retardation remains the main problems in these subjects. Background: Tyrosine hydroxylase (TH) deficiency is an IEM of neurotransmission with variable clinical presentation and L-dopa response, in which mechanisms of synaptic communication have not been described.
Objectives: To study clue synaptic dopaminergic and gabaergic proteins in CSF of patients with TH deficiency and their possible relation with phenotype and L-dopa response.
Methods: The following proteins: DAT, D2-receptor, VMAT2 and GABAVT were studied in the CSF of 10 subjects with TH deficiency (A phenotype: 4 patients; B phenotype: 6 patients; diverse mutations) by Western blot analysis. In 3 patients, these studies were pre and posttreatment. Results were compared to a control population.
Results: D2R pre-treatment was higher in patients than in controls. No significant differences were found between pre-treatment A and B phenotypes. D2R was higher post-treatment (paradoxical response) in a B patient (L-dopa induced dyskinesias, poor outcome), whereas it rapidly decreased in two A patients with an excellent L-dopa response. Coordinated regulation of DAT and D2R, as well as VMAT and GABAVT after L-dopa doses increase was observed.
Conclusions: Post-treatment D2R appears to have a paradoxical behaviour in B phenotype. Post-treatment GABAVT increase suggests GABA-dopa co-release. Study of CSF synaptic proteins may help understanding pathophysiological mechanisms in these disorders. Background: Homovanillic acid (HVA) is the main catabolic product of dopamine. Decreased cerebrospinal fluid (CSF) HVA values may be related to dopamine deficiency, but also in secondary HVA alterations. We have analysed biogenic amines and other biomarkers in CSF samples collected from neuropediatric patients. We aimed to study the association between CSF HVA and other biogenic amines and different neurological diseases. Methods: Samples from 1,386 subjects were analysed by HPLCelectrochemical detection. Subjects were classified in three groups according to HVA values compared to age-related reference value: low, normal or high levels.
Results: 16% (n=225) presented low values of HVA. Among them we detected patients with different primary deficiencies: tyrosine hydroxylase (n=10), aromatic L-amino acid descaboxylase (n=2), guanosine triphosphate cyclohydrolase 1 (n=4), pyridox(am)ine-5'-phosphate oxidase (n=1) and sepiapterin reductase deficiencies (n=1). As secondary deficiencies, we found hypoxic ischemic encephalopathy and mitochondrial cytopathies, as disorders associated with low HVA values. Regarding high HVA values, this finding was consistently associated with mtDNA deletion syndromes (Kearns-Sayre) Conclusions: Analysis of HVA and others metabolites biomarkers in CSF is useful for the differential diagnosis of primary neurotransmitters diseases. However, concentrations of HVA may be altered by several diseases of the central nervous system as a secondary event. Background: Dopa-responsive dystonia (DRD) is caused by partial defects of guanosine 5'-triphosphate cyclohydrolase I (GTPCH). In this study, we measured neopterin and biopterin level in DRD patients to confirm biochemical diagnosis. Methods: Eleven Japanese patients from 8 families were diagnosed as DRD patients due to heterozygous mutations in GCH1 gene, which codes for GTPCH (DRD (+/−)). Eleven non-dystonic controls were also measured. Their neopterin and biopterin concentrations in plasma and CSF were measured by using HPLC apparatus.
Results: The DRD patients had two missense mutations (A190V, T106I) and two frameshift mutations (K107fs, M211fs), one nonsense mutation (K239X), and a deletion of exons 2 and 3. Student's t test was used to compare values. It indicated that plasma neopterin level of DRD (+/−) was lower than those of controls (p=0.0002). Plasma biopterin level of DRD (+/−) and controls showed no significant difference (p=0.2866). Significant differences between DRD (+/−) and controls were also shown in CSF neopterin level (p=0.0014) and CSF biopterin level (p<0.0001). Data were expressed as average±SD.
Conclusions: The decrease in plasma neopterin concentration was related to DRD with GTPCH deficiency. It may be a useful diagnostic classification of DRD. Background: Monoamine oxidase A (MAOA) deficiency, a defect in biogenic amine neurotransmitter catabolism, was first described in 1993 in a large Dutch family with many borderline mentally retarded males with prominent aggressive behaviour disturbance. So far no new patients with MAOA deficiency were found. Methods: SNP-array analysis for genetic diagnostics and HPLC analysis for urinary metabolite analyses. Results: SNP-array analysis was performed on a young Swedish boy with ADHD-like behaviour, borderline mental retardation and gross obesity. A 1 MB deletion on the X-chromosome was found, encompassing the first three exons of the MAOA gene and no other known genes.
In urine samples of the boy, his older brother with the same clinical phenotype and their mother neurotransmitter metabolites were analysed for determination of the metabolic phenotype. As expected the neurotransmitter metabolite profile in the index patient was characteristically abnormal and confirmed the functional deficiency. Aminoglycosides and other compounds can promote premature stop codon (PTC) readthrough constituting a potential therapy for patients with nonsense mutations. Using an in vitro transcription-translation system we have established the proof of principle that nonsense mutations in the PCCA and PCCB genes causing propionic acidemia can be partially suppressed by aminoglycosides, with different efficiencies depending on the sequence context. To correct the metabolic defect, the amino acid incorporated at the PTC (usually Gln or Trp), should support protein function and this has been evaluated for 5 PCCA and 7 PCCB nonsense mutations in silico and by in vitro expression analysis of the predicted missense changes. Most missense changes retain partial activity confirming the feasibility of the approach. In selected patients' fibroblasts cultured for 5 days with different amounts of G418 and gentamycin we observe a significant increase (4-5 fold) Background: Cobalamin C (Cbl-C) defect is the most common inborn error of cobalamin metabolism causing methylmalonic aciduria and homocystinuria. Despite pharmacological treatment with OH-cbl, betaine, folate, and carnitine, the long-term outcome is unsatisfactory with progression of neurological and visual impairment. The pathophysiological mechanism(s) causing brain and ocular damage still remains to be elucidated. Recently, the contribution of oxidative stress has been hypothesized based on in vitro studies showing in Cbl-C fibroblasts a significant ROS increase that can be corrected by adding OH-cbl to cell culture (Richard, Hum Mutat 2009) . Objective: Since reduced glutathione plays an important role in ROS detoxification, we assessed in vivo by reverse-phase HPLC the glutathione status in blood cells obtained from 13 treated Cbl-C patients.
The results showed a relevant impairment of the glutathione homeostasis, as indicated by decrease of total-(p<0.005), reduced-(p<0.0001), and free-(p<0.001) glutathione in Cbl-C patients when compared to controls. Accordingly, the oxidized/reduced glutathione ratio was also significantly increased (p<0.03).
Conclusions: Our findings for the first time demonstrate in vivo a significant redox imbalance in Cbl-C defect that may contribute to the disease progression and indicating that antioxidant drugs should be used in combination with standard therapy. Case report: Bilateral visual loss occurred within five days in a 23-year old women with methylmalonic acidemia (MMA). The disease was metabolically well controlled by strict diet and carnitine supplementation since early childhood and the visual loss was not associated with metabolic decompensation. Ophtalmologic exams showed the typical signs of optic neuropathy, and common etiologies of this pathology were ruled out. Moderate enhancement of both optic nerves was present on MRI. Treatment attempts with high dose corticosteroids, and coenzyme Q10 combined with vitamin E were ineffective. Subacute partial bilateral neurosensory hearing loss occurred three months later, further complicating patient care.
Discussion: Very few cases of late onset optic neuropathy and neurosensory hearing loss associated with MMA have been reported, and a morphological correlate in MRI scans has not been documented before. Impaired mitochondrial function is suspected to be the underlying cause of bilateral optic atrophy and neurosensorial hearing loss in MMA, although the pathophysiology is not well understood. Managing late-onset or long term complications of metabolic disease is a central issue in the care of adult patients with inborn errors of metabolism. Methyl malonic aciduria (MMA) is a disease caused by a deficiency in the enzyme, methyl malonyl CoA mutase, which is responsible for the metabolism of branched-chain amino acids. MMA is generally characterised by attacks of ketosis, acidosis, nausea, vomiting, dehydration, lethargy and increased concentrations of methyl malonic acid in blood and urine. Patients with MMA are initially treated with vitamin B12, with those who do not respond placed on diets containing limited amounts of protein and branched-chain amino acids. Patients with uncontrolled MMA or infections due to catabolic stress may have skin lesions, specifically generalized exfoliative eruptions, a condition called acrodermatitis dysmetabolitica. These cutaneous eruptions are characterized by a deficiency in isoleucine, one of the eseential amino acids. We encountered a patient on an amino acid restricted diet who experienced skin lesions caused by a lack of isoleucine (plasma concentration, 4.71 μmol/L). Within days of liberalizing his restricted diet and after supplementation with 100 mg/kg/day isoleucine, the eruption resolved completely. We present two patients aged four years and fourteen years with methylmalonic acidaemia who were admitted to intensive care with severe metabolic decompensation. Both patients have end stage renal failure and are on the renal transplant list. The children's renal function had significantly deteriorated with the decompensation and both went on to receive haemodialysis while on intensive care. We present data to demonstrate the effect of dialysis on the MMA levels and correlate the levels with the progression of renal recovery. This data is of interest because it is assumed that MMA has a direct nephrotoxic effect, as patients with propionic acidaemia do not have renal disease as a complication while sharing other similarities with MMA. These cases raise the questions: (i) Does haemodialysis aid renal recovery at times of decompensation by offloading the effect of MMA on the kidneys (ii) Should we utilise MMA levels more in the management of MMA patients both when well and decompensated? (iii) Are there other biochemical parameters that can be linked with MMA levels, e.g. acylcarnitine profiles? Background: Short stature and low GH levels have previously been reported in patients with methylmalonic aciduria. Diagnosis of GH deficiency is usually based on GH response to a provocative stimulus like glucagon. Case Report: We describe a boy with vitamin B12 non-responsive methylmalonic aciduria. He presented in the neonatal period with intrauterine growth retardation and metabolic acidosis and following diagnosis was treated with carnitine and a low protein diet. He had mildly delayed developmental milestones. His renal function was deceased but stable with a glomerular filtration rate of 60 ml/min/1.73 m2. Despite satisfactory progress with infrequent metabolic decompensation, growth remained poor in the face of adequate nutrition. Coeliac screen, thyroid function, prolactin were normal but plasma arginine and Insulin-like growth factor (IGF1) were low. His bone age was normal for his chronological age. Glucagon stimulation test showed a sub-optimal response of GH in contrast to the arginine stimulation test which produced a good response with a sufficient peak of GH (21.4mcg/L). Treatment with arginine was initiated. Early follow up showed an improved height velocity and normalisation of his IGF1. Conclusion: In patients on protein restricted diets with faltering growth arginine levels should be measured and supplementation considered if low. Methylmalonic aciduria cblB type is due to mutations in the MMAB gene which encodes the ATP:cobalamin adenosyltransferase enzyme. We describe two siblings with identical genotype and different biochemical phenotype one detected through expanded newborn screening and classified as cblB type by somatic cell complementation. He presented deficient propionate uptake rescued after cellular B12 treatment. He was compound heterozygous for two novel variant changes p.H183L (c.287 T>C) and p.R190dup (c.568-570dup). The subsequent familiar genetic analysis revealed that his clinically asymptomatic sibling, nine years old, presented also both variant changes. She exhibited only mild excretion of MMA and close to normal propionate uptake. Functional analysis revealed that p.H183L mutant protein had specific activity and affinity parameters similar to wild-type. It is noticeable that the efficiency of expression and purification was nearly 70-fold lower that wild-type ATR and also had a high reduced stability compared to wild-type indicating that it is likely a new destabilizing mutation in the MMAB gene. Mutant protein p.R190dup was highly unstable and the specific activity was undetectable. The structural and functional analyses suggest that both mutations are likely disease-causing, but there must be an additional genetic defect in MMAB or phenotypic modifiers genes to explain the different phenotype. Women with methylmalonic acidemia may be at risk of metabolic acidosis, hyperammonemia or renal failure in the postpartum period. We describe the course and outcome of a pregnancy in a patient with methylmalonic acidemia due to Cobalamin-B-deficiency and a chronic renal failure caused by the underlying disease. Methylmalonic acidemia was diagnosed at 5 months of age; Cobalamin-Bdeficiency was genetically confirmed at the age of 26 years. Pregnancy occurred at the age of 27 years. Her long-term treatment consisted of the protein restricted diet, supplementation of isoleucine-methioninethreonine-valine-free amino-acid mixture, L-Carnitine and intramuscular application of Hydroxycobalamin (10 mg/every other week). During pregnancy the treatment was continued and control visits at the centre intensified. Her renal function was stable and the urinary excretion of methylmalonic acid decreased from 1600 to 477 mmol/mol Creatinine in the third trimenon. At term a healthy male infant was born spontaneously.
Postpartal the urinary excretion of methylmalonic acid increased markedly accompanied by a decline of the renal function, but improved partially in the following months. This is the first report of pregnancy in a woman with Cobalamin-Bdeficiency. A close observation is necessary to ensure an optimal care during and after pregnancy in patients with inborn metabolic diseases. Newborn screening for propionic (PA) and methylmalonic acidemias (MMA) based on measuring propionylcarnitine (C3) is neither sensitive nor specific. Due to a significant overlap in C3 concentration between affected and unaffected individuals, ratios such as C3/C2 were introduced to improve the newborn screening algorithms. However, the false positive rate of C3 disorders remains significant. While methylcitrate (MCA) is a specific marker for C3 disorders, it is not detectable by the current MS/MS screening method. To overcome this, we developed a simple and specific LC-MS/MS method for MCA in DBS samples. The method is based on derivatization with DAABD-AE which was undertaken to improve the poor MS properties of MCA. No separate extraction step was required and derivatization was achieved by incubating a 3.2 mm disc with DAABD-AE at 60:C for 45 min. MCA peak eluted at 2 min and the injection to injection time was 10 min. The method was successfully applied for the analysis of DBS samples from established PA and MMA patients (n=10) as well as controls (n=310). We anticipate that the implementation of this method will improve the screening process for PA and MMA by reducing the false positive rate and increasing the positive predictive value. Methylmalonic acidurias (MMA) constitute an important group of inherited metabolic disorders involving neurological deficits. Recent studies have shown an increased reactive oxygen species (ROS) production and apoptotic cell rate in fibroblasts especially from cblB patients with isolated MMA and cblC with combined MMA and homocystinuria. To gain insight into the pathophysiology of these disorders, the goal of this study was to examine mitochondrial dysfunction as a putative disease mechanism. An altered mitochondrial morphology of a grain-like structure was presented mainly in those fibroblasts with an increased ROS content (cblB and cblC), suggesting that ROS might lead to fission and a grain-like structure of the mitochondrial reticulum. Decreased oxygen consumption rate (OCR) was found in patients´fibroblasts. When cells were respiring in glucose free-medium supplemented with galactose, basal and maximal OCR increased in patients and control fibroblasts. Permeabilized fibroblasts from cblB patients compared to patients from other groups and controls, revealed that in vitro mitochondria display an enhanced Ca2+ uptake velocity and premature opening of the permeability transition pore which has been described to provoke the release of mitochondrial proapoptotic factors. These studies using cell models establish that mitochondrial dysfunction is directly or indirectly involved in these cobalamin metabolism defects. Background: Patients with propionic (PAemia) and methylmalonic (MMAemia) acidemias present severe crises of metabolic decompensation in the neonatal period, in which the levels of propionic (PA) and Lmethylmalonic (MMA) acids, respectively, can be as high as 2.5-5 mM. Treatment is constituted of a low-protein diet supplemented with Lcarnitine. Recently, some works have suggested that lipid and protein oxidative damage may be involved in the pathophysiology of these diseases, but DNA damage has not been fully investigated.
Objectives: In this work, we aimed to investigate the in vitro effect of Lcarnitine on DNA damage induced in vitro by PA and MMA.
Material and Methods: The alkaline comet assay was used to evaluate the DNA damage index induced in human leukocytes after incubation for 6 hours at 37:C with PA (2-5 mM) and MMA (0.5-5 mM) Background: Chronic tubulointerstitial nephritis is a frequent finding in patients with isolated methylmalonic aciduria. Among them, patients with mut0 and CblB disease have the highest risk of developing kidney disease. The underlying renal pathology has yet not been elucidated. Therefore, we aimed to establish an in vitro model for the human disease. Methods: We prepared cells from urine of healthy donors and patients with mut0, mut-, CblA, and CblB disease and purified proximal tubule epithelial cells (hPTEC). For long-term cultivation we used a method to immortalize these primary cells with pRSVneo vector containing SV40 (pRNS1) using electroporation. Afterwards, hPTEC were characterized regarding morphology and marker protein expression.
Results: By RT-PCR we showed expression of organic anion transporters 1 and 3, P-glycoprotein, ATP-binding cassette transporters 4 and 6. Expression of proximal tubule marker aquaporin 1 was demonstrated by western blotting. In light microscopy, hPTEC displayed characteristic cobblestone shapes. Using immunostaining, we demonstrated expression of the tight junction protein ZO-1. We found a maximum increase in methylmalonic acid and propionyl-carnitine concentrations in hPTEC of mut0 patients, and less pronounced elevations in mut-patients.
Conclusions: In summary, we present a new in vitro model to study the renal pathology of methylmalonic aciduria. Introduction: Stroke like episodes are a feature of inherited metabolic disorders, including propionic acidemia (PA) (1) . So far, it was unclear, whether these episodes are caused by cytotoxic or vasogenic edema (2) . Patient: At one week, PA was diagnosed in parallel through appearing symptoms and positive newborn screening. The boy is treated with a protein restricted diet, l-carnitine, ß-blockers and diuretics. At 4 years he had a prolonged gastroenteritis. Two weeks later he showed noticeable behavioural changes with regression, especially in speech, confusion and abnormal reaction to people and objects well known to him. EEG showed bilateral diffuse slowing of background activity with right-sided accentuation, and with sharp wave foci bilaterally over the temporo-occipital regions. Evaluation of T2-weighted images, T2 relaxation maps, diffusionweighted imaging (ADC maps) and T2-shine-through indicated the presence of cytotoxic edema in putamen and the cerebellum. After general anesthesia for imaging, EEG changes resolved. He recovered within some days.
Conclusion: This case illustrates that pathogenesis in propionic acidemia includes cytotoxic damage to the CNS, probably caused both by trapping of organic acids (3) and lack of anaplerotic substances for the Krebs cycle (4). Reference: (1) Acute respiratory distress syndrome (ARDS) is an acute condition characterized by bilateral pulmonary infiltrates and severe hypoxemia in the absence of evidence for cardiogenic pulmonary edema. Major risk factors associated with the development of ARDS include bacteremia, sepsis, trauma, fractures, burns, massive transfusion, pneumonia, aspiration, drug overdose, near drowning, postperfusion injury after cardiopulmonary bypass, pancreatitis, and fat embolism. Organic acidemias have not been previously reported as a risk factor for ARDS. We present herein two patients with organic acidemia who were followed up with ARDS. One of them was mut0 methylmalonic acidemia (MMA) and the other was propionic acidemia (PA). They were admitted with vomiting and subsequently developed neutropenia, trombocytopenia and lactic acidosis. In within several days on emergency care, they exhibited hypoxia and respiratory insufficiency, the chest X-ray were suggestive of ARDS. Despite intensive care and ventilatory support, the patient with MMA died, the other survived.
The patients with organic acidemia involving propionate metabolism might have tendenceny to ARDS due to the possible role of toxic metabolites in the ARDS pathogenesis. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to hyperinflammation caused by aberrant proliferation of activated lymphocytes and macrophages secreting high levels of cytokines. Hemophagocytic lymphohistiocytosis (HLH) may develop secondary to infections, malignancies, immune deficiency syndromes, rheumatologic and metabolic disorders. Associations between hemophagocytosis and inborn errors of metabolism including lysinuric protein intolerance, multiple sulfatase deficieny, galactosemia, Gaucher Disease, Pearson Syndrome, galactosialidosis have previously been reported in the literature. We report three cases with disorders of propionate metabolism one with methylmalonic acidemia and two with propionic acidemia developing secondary hemophagocytosis during the course of their metabolic disease. This is the first report of such an association.
In the patients, no infectious agent was isolated from the cultures of various specimens. All the patients presented with metabolic acidosis and ketosis, but the fact that whether HLH was triggered by the toxic metabolites or vice versa is unclear. Whether the molecular basis of the diseases in the presented three cases serve as a determining factor is not known. Considering all these issues the mechanism playing a role in the development of hemophagocytosis in our three patients with organic acidemia is not clear yet. In patient 2 VH recurred once, with seizure, abnormal EEG, and brain MRI. Acute and chronic brain abnormalities are well-documented in PA. Acute or chronic focal cerebral metabolic or toxic insult as part of the PA might etiologically explain the VHs in our patients.
Background: Accumulation of 3-hydroxyglutaric acid (3HGA) in body fluids is pathognomonic for type 1 Glutaric Aciduria (GA1). To date, methods for quantification of 3HGA mainly involve complex analyses using stable isotope dilution gas chromatography mass spectrometry (GC-MS). Here, we describe a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify 3HGA in dried urine spots (DUS) using DAABD-AE, a benzoxadiazole-type reagent designed for improving chromatographic and mass spectrometric properties of carboxylic acids. Material and Methods: No extraction was required and derivatization was performed directly on a 3.2-mm disc of DUS. Sample pretreatment involved heating the reaction mixture at 60°C for 45 min and 5 μl portion was analyzed by LC-MS/MS. Results: 3HGA measured in control samples (n=100) ranged between 0-7.1 mmol/mol creatinine. DUS samples from established low-and highexcretor GA1 patients revealed satisfactory results compared to those obtained previously by GC-MS (n=14).
Conclusion: This approach may be a useful primary screening method for low-or high-excretor variants in populations with high risk of GA1. Furthermore, for babies with elevated dried blood spot C5DC, follow-up testing of DUS GA and 3HGA will be a useful diagnostic test that may reduce the number of cases requiring enzymatic and molecular analyses. Background: The pathogenesis of the brain damage in glutaric acidemia type I (GA I) is not well understood. Objectives: Relevant oxidative stress parameters were investigated in cerebral cortex, striatum, hippocampus, liver and heart of 30-day-old knockout mice with glutaryl-CoA dehydrogenase deficiency (Gcdh −/−) and in wild-type animals (Gcdh +/+). Methods: Tissues were dissected, homogenized and assayed biochemically. Some Gcdh −/− and Gcdh +/+ mice received 8 μmol/g L-lysine i.p. 24 hours before the assays were performed. Results: Significant changes were observed only in the brain of Gcdh −/− mice that had received lysine, which is converted to glutaric and 3hydroxyglutaric acids. Cerebral cortex in these animals showed significantly higher thiobarbituric reactive acid substances (TBA-RS) and superoxide dismutase (SOD) activity and a diminution of reduced glutathione (GSH). In the striatum, TBA-RS, SOD and glutathione reductase were increased, while GSH levels and glutathione peroxidase activities were decreased. SOD activity was increased in the hippocampus. No changes were observed in liver and heart. Conclusions: Brain, and especially striatum, in Gcdh −/− mice is more vulnerable to oxidative stress following lysine administration than liver and heart. Increased vulnerability to oxidative stress may contribute to the pathogenesis of brain damage in human GA I. Case Report: We report three Iranian patients with GA1. All of them were from first cousin Iranian parents; had prolonged physiological Icter and macrocephaly. All three had highly elevated C5DC, glutaric acid and 3hydroxy glutaric acid. Two sibling were found to be homozygous for the GCDH gene mutation in the exon10 position390 (P.Gly390Ala). Surprisingly, in spite of similar mutation the 4 years old girl showed normal development without treatment whereas the development of her 15 months brother was severely impaired. The third patient, a 3 years old girl, was found to be homozygous for the GCDH gene mutation in the exon6 position 179 (C179Leu>Pro). Conclusion: If not diagnosed early and treated preventively before recurrent encephalopathic crisis occur, GA type1 is usually associated with a severe irreversible neurologic syndrome. This report indicates examination of urinary organic acid and acyl carnitine profile in children with symptoms of developmental delay and macrocephaly. Background: GA II is a disorder of fatty and amino acids metabolism due to defects of the Electron Transfer Flavoprotein (ETF) or ETF-CoQ oxidoreductase (ETF-QO). The disease is widely variable in its symptoms, severity, age of onset and rate of progression. Brain MRI typically shows T2-weighted hyperintensity of basal ganglia.
Objectives: To present a case in which leukodystrophy was a distinct and prominent imaging finding. Case Report: The index case presented at 22 months of age for investigation of developmental delay, anemia, hypothyroidism and slightly dysmorphic features. MRI disclosed supratentorial leukoencephalopathy, sparing intense capsule, basal ganglia and thalami. Brainstem was also affected with signal intensity in T2 -weighted images. In the differential diagnosis mitochondrial and "vanishing white matter" disease were considered. On subsequent follow-up the patient manifested an episode of acute encephalopathy with hypoketotic hypoglycemia, metabolic acidosis and hyperammonemia. Urinary organic acids and acylcarnitine profile were strongly abnormal and suggestive of GAII. Conclusion/Discussion: The unusual phenotype of this patient, characterized by progressive encephalopathy, along with the MRI findings were misleading and suggesting a leukodystrophic process rather than an organic aciduria. The presence of leukodystrophy should always prompt the investigation for an organic aciduria, even in the absence of acute decompensation. Background: The role of glial cells in glutaric acidemia I (GA-I) pathogenesis is mostly unknown. Objectives: We evaluated the effects of pathophysiological concentrations of glutaric acid (GA) and lysine on glial cells from the glutaryl-CoA dehydrogenase (GCDH) knockout (KO) mouse model. Methods: Functional and morphological parameters were analyzed in cortical and striatal cultures of astrocytes and oligodendrocytes from newborn C57 GCDH KO and wild type mice submitted to 5 and 10 mM GA or 10 mM lysine during 24 hours. Results: Lysine caused mitochondrial dysfunction, as verified by decreased mitochondrial potential in KO cultured astrocytes, measured by ratiometric fluorescence of the JC1 probe and MTT activity. Cellular glutathione levels decreased 25%, whereas carboxy-H2DFFDA signals augmented 35%. Astrocytic proliferation was increased by 30% related to untreated cells. GA causes similar effects without significant differences among the concentrations employed. No changes in glutamate transporter GLT1 and glutamine synthase expressions were found in KO astrocytes even upon GA or lysine exposure. In addition, 80% less of oligodendrocyte progenitors were obtained from the KO cultures.
Conclusions: Mitochondrial dysfunction together with increased oxidative stress and exacerbated astrocytic proliferation in GCDH KO mice could actively contribute to the neuronal loss and myelin defects in GA-I. Tissue-specific expression studies of Glutaryl-CoA dehydrogenase (Gcdh) in adult rats revealed expression in the whole rat brain, almost exclusively in neurons, and surprisingly high expression in the juxtamedullar cortex of the kidney. The organic anion transporter 1 (OAT1) mediates basolateral uptake of glutarate derivatives from proximal tubule cells and contributes to their renal clearance. In brain, OAT1 is expressed at the choroid plexus, in neurons of cortex and hippocampus. We hypothesized that Gcdh and Oat1 are co-expressed in the same cells in kidney and brain and analyzed their mRNA expression by in situ hybridization on cryosections of adult rat brain, kidney and liver. In brain, Gcdh and Oat1 were found co-expressed in most neurons. Only the Purkinje neurons of the cerebellum were found to be Oat1 negative. In the kidney Gcdh and Oat1 are widely co-expressed with a specific high expression in proximal tubule cells. In conclusion there seems to be a functional coupling of Gcdh and Oat1 on a renal and neuronal level. Further studies are ongoing to confirm these findings in human tissues. Background: Ethylmalonic acid (EMA) accumulates in short chain acyl-CoA dehydrogenase (SCAD) deficiency and ethylmalonic encephalopathy, disorders in which the pathophysiological mechanisms of brain damage are poorly unknown.
Objectives: It was investigated the in vitro effects of EMA on respiratory parameters measured by oxygen consumption supported by succinate or glutamate/malate in mitochondria from rat brain. Material and methods: Mitochondrial fractions were prepared from brain of 30day-old Wistar rats and incubated in a medium containing succinate or glutamate/ malate. Oxygen consumption was then measured under various conditions. Results: EMA increased state 4 (25%), decreased state 3 respiration (45%) and respiratory control ratio (RCR; 55%) with succinate-but not with glutamate/malate-supported oxygen consumption. EMA-induced decrease of state 3 in succinate-supported respiring mitochondria was significantly minimized by nonselective permeabilization of mitochondrial membranes induced by alamethicin. Malonic acid (MA) also diminished state 3 (40%) and RCR (40%), but did not affect state 4 respiration with succinate as substrate. However, mitochondrial permeabilization did not affect the MAinduced state 3 inhibition. Furthermore, the respiratory chain activities were not altered by EMA under these experimental conditions. Conclusion/Discussion: It is concluded that EMA inhibits succinate uptake into brain mitochondria. Background: Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is a neurometabolic disorder biochemically characterized by tissue accumulation and high urinary excretion of 2-methylbutyric acid (MB) and 2-methylbutyrylglycine (MBG). Although affected patients present neurologic symptoms, the pathophysiology of the brain damage in SBCADD is not yet established.
Objectives: We studied the in vitro effects of MB and MBG on important parameters of oxidative stress in cerebral cortex of young rats. Material and methods: Brain from 30-day-old Wistar rats was dissected, the cerebral cortex dissected, homogenized and utilized in the biochemical determinations.
Results: MBG, but not MB, increased thiobarbituric acid-reactive species (TBA-RS) (lipid oxidation). MBG also induced sulfhydryl oxidation and decreased glutathione (GSH) levels, reflecting a reduction of antioxidant defenses. In contrast, MB and MBG did not alter carbonyl formation. We also found that MBG-induced increase of TBA-RS levels and decrease of GSH were prevented by free radical scavengers, implying that reactive species were involved in these effects.
We therefore presume that lipid and protein oxidative damage induced by MBG may be involved, at least in part, in the pathophysiology of the neurological dysfunction found in SBCADD-affected patients. Mutations in the HIBCH gene lead to 3-hydroxyisobutyryl-CoA hydrolase deficiency, an inborn error of valine catabolism that leads to the build up of a highly reactive intermediate, methacrylyl-CoA. Methacrylyl-CoA undergoes addition reactions with thiol compounds leading to the formation of S-2-carboxypropyl-cysteine (S2CP-cysteine) and S-2-carboxypropyl-cysteamine (S2CP-cysteamine).
We have developed a method to quantitate S2CP-cysteamine in urine using liquid chromatography with tandem mass spectrometry in positive ion mode using a synthesised deuterated analogue, d5-S2CP-cysteamine, as an internal standard. Chromatographic separation was achieved using a Discovery HS F5 column (5 cm x 2.1 mm, 5 μm) with a mobile phase consisting of 4 mM ammonium acetate, 4 mM heptafluorobutyric acid buffer (pH 3.25) and acetonitrile. For S2CP-cysteamine and d5-s2CP-cysteamine parent ions of mass 164 and 169 were detected respectively. Quantitation was carried out using fragmented daughter ions 119 for our analyte and 152 for the deuterated internal standard. The method was validated in spiked urine over the linearity range of 0.2-100 μmol/L. Elevated urinary levels of S2CP-cysteamine were demonstrated in two patients initially picked up because of increased hydroxy-C4-carnitine then diagnosed by enzymology and HIBCH gene analysis. The method may prove useful in monitoring treatment e.g. by valine restriction. Beta-ketothiolase deficiency is an inborn error of isoleucine and ketone body metabolism, caused by mutations in mitochondrial acetoacetyl-CoA thiolase gene (ACAT1). We have so far analyzed gene mutations in more than 80 ACAT1 deficient patients. We previously identified a homozygous deletion including exons 2-4 in one patient and a homozygous duplication including exons 8 and 9 in another patient. However, such gene alterations are harder to identify in the heterozygous state than in the homozygous state through cDNA analysis. Since there are patients in whom one mutation cannot be detected by our routine genomic and cDNA analyses, we hypothesized that they would have heterozygous intragenic deletion or insertion. Hence, we established an MLPA method to detect copy numbers in each exon in the ACAT1 gene and, mixing patients' and control cDNA samples, successfully detected a heterozygous deletion including exons 2- Meanwhile organic acid profile showed an increased peak of mevalonic acid (13616 μmol/mmol Creat) and the diagnosis of mevalonic aciduria was confirmed by enzymatic and molecular studies. The initial prednisolone course had favourable response, but not sustained in subsequent cycles. The evolution has been marked by failure to thrive, needing parenteral nutrition, bicytopenia, with regular transfusions and reactivation of CMV infection with interstitial pneumonia that impose non invasive ventilatory support until the death at the fourth month of life. Discussion: The manifestations of mevalonic aciduria are similar to congenital CMV infection and differential diagnosis is often an issue. In this patient, both disorders were confirmed with problematic therapeutic options and a poor prognosis. Background: Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an autosomal recessive disorder biochemically characterized by accumulation and predominant high urinary excretion of 3methylcrotonylglycine . Affected patients usually have neurologic dysfunction and brain abnormalities, whose pathogenesis is practically unknown.
Objectives: We investigated the in vitro effects of 3-MCG (0.1-5 mM) on important parameters of oxidative stress in cerebral cortex of 30-day-old rats.
Methods: Wistar rats were sacrificed by decapitation, the cerebral cortex dissected, homogenized and used for the biochemical assays.
Results: 3-MCG significantly increased thiobarbituric acid-reactive substances (TBA-RS) levels (30%) and carbonyl formation (40%), indicating that this compound causes lipid and protein oxidative damage in the brain. In contrast, glutathione (GSH) levels, sulfhydryl oxidation and total reactive antioxidant potential (TRAP), that reflect non-enzymatic antioxidant defenses, were not significantly altered by 3-MCG. Finally, 3-MCG did not alter nitric oxide production, implying that reactive oxygen species were involved in the oxidative damage induced by this compound.
Conclusions: It is presumed that brain is susceptible to oxidative damage provoked by 3-MCG and that this pathomechanism may contribute to the neurological dysfunction characteristic of the patients affected by 3-MCCD. The girl now 4.5-year-old, at age of 3 yrs was admitted to our Dept, because glycaemia 500 mg/dl. Diabetes mellitus type 1 was diagnosed.
The girl was typically treated with insulin in constant iv infusion for 37 hrs. After 8 hrs she started to eat carbohydrate exchanges and take sc insulin injections. At the 4th day of hospitalization unexplained recurrent vomiting appeared. Only then the patient's mother mentioned about 3MCC deficiency identified by NBS, with no further specific recommendations. Urinary organic acid analysis by GC/MS confirmed the diagnosis. So additionally to carbohydrate, fat/protein counting, low-protein diet based on special formula and L-carnitine were introduced. Until now three hospitalizations occurred: 1st for implementation of personal insulin pomp, 2nd due to ketotic acidosis caused by technical problem with pomp/ catheter, 3 rd metabolic decompensation in the course of rotaviral infection, which required 6-day-long 10% glucose and insulin iv infusions. Current therapeutic recommendations are: low-protein ( Background: 3-Methylcrotonyl-CoA carboxylase deficiency is an inherited metabolic disorder biochemically characterized by high tissue accumulation and urinary excretion of 3-hydroxyisovaleriate, 3methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleryl-carnitine. Clinically, patients present severe brain abnormalities and neurologic dysfunction, whose pathomechanisms are still unclear.
Objectives: We investigated the in vitro effects of 3-MCG (0.1-5 mM) on important parameters of bioenergetics and Na+, K±ATPase in cerebral cortex of 30-day-old Wistar rats.
Methods: Rats were sacrificed by decapitation, the cerebral cortex isolated, homogenized and used for the biochemical assays.
Results: 3MCG significantly reduced CO2 production from acetate (30%) and the activity of complex II-III (35%). 3MCG also inhibited the activities of mitochondrial creatine kinase (mCK) (65%) and Na+, K±ATPase (45%). Furthermore, antioxidants attenuated or fully prevented the effect of 3MCG on the activities of mCK and Na+, K±ATPase, suggesting the involvement of reactive species on these inhibitory effects.
Conclusions: 3-MCG impairs brain bioenergetics at the level of energy formation, transfer and utilization. It is presumed that these mechanisms may be involved in the pathophysiology of the neurological dysfunction that occurs in patients affected by 3-MCCD. An increased level of N-acetylaspartate found in the pattern of urinary organic acids is a diagnostic hallmark of neurodegenerative Canavan disease (CD). Established confirmatory tests are the determination of aspartoacylase (aminoacylase 2) activity in cultured fibroblasts and mutation analysis by sequencing the ASPA gene. The enzyme activity test is of special importance, if mutation analysis yields no sequence variation with a clear pathologic significance. So far, a skin biopsy has been a prerequisite for testing aspartoacylase activity.
We have now studied by real-time PCR whether the ASPA gene is also transcribed in EBV-transformed lymphocytes and have determined enzyme activities in cell homogenates.
Our investigations demonstrate that the ASPA gene is transcribed in the lymphocytes to a similar extent if compared with fibroblasts. Based on the protein content, aspartoacylase activity is lower in EBV-transformed lymphocytes than in fibroblasts (< 20%), but even transformed lymphocytes revealed a difference in activities between patient cells and negative controls. This opens a new diagnostic perspective, and may also help with the systematic study and characterization of patients with CD which is currently conducted by our metabolic center. More comprehensive information on CD is a prerequisite for better counseling on the clinical outcome. Background: Canavan Disease(CD) caused by deficiency of aspartoacylase and increased excretion of N acetyl aspartate (NAA)is common in Ashkenazi Jews and has also been reported in other populations.Mutations causing Y231X (exon5) and E285A (exon6) are prevalent amongst Jews while A305E (exon6) accounts for 60% of non Jewish patients. Indian data is scarce which may have been due to non availability of NAA analysis by GCMS till recent past.Study design:We report a study of 35 clinically suspected CD cases (mean age 27 months) referred from across the country for NAA analysis. Clinically they presented with macrocephaly, developmental delay, poor head control and hypotonia. Results: A 20-200 fold increase in NAAwas obtained in around 8 patients (23% of total) of which only 50% were born to consanguineous couples suggesting prevalence of CD amongst several communities in India. As an initial molecular work up we screened 6 CD patients for prevalent mutations Y231X, E285A and A305E by sequencing exons 5 and 6. These mutations were not detected in our patients, suggesting a varied set of mutations amongst them.
Conclusion: A complete ASPA gene workup needs to be done to elucidate the mutation profile in our population. We present a series of patients with organic acidaemia complicated by pancreatitis. Patients A and B had propionic and methylmalonic acidaemias respectively. They presented with vomiting and developed severe, persistent abdominal pain requiring opiates, ketamine and intensive care support. Patient A had a marginally raised amylase but radiological findings were consistent with pancreatitis. She died after a prolonged PICU admission. Patient B had a 48 hour rise in amylase. He had a prolonged PICU stay and was discharged home on opiates. Patient C had propionic acidaemia and presented with vomiting. This resolved but he had an unexpected cardiac arrest, which he did not survive. Post-mortem showed pneumonia and pancreatitis. Patient D had methylmalonic acidaemia and died after a short illness. Post-mortem revealed acute on chronic pancreatitis, despite a normal amylase. These cases highlight: (i) Pancreatitis may be more common than thought (ii) Amylase is an unreliable marker. None of the patients had a significantly raised amylase. Diagnosis was based on clinical and radiological grounds (iii) Severe pancreatitis poses management difficulties in terms of analgesia and nutrition. Current best practice is to continue with enteral feeds and aggressive pain control, if necessary with intubation and sedation for a prolonged period. Background: Due to its property to enhance CPS-1, NCG could be used to treat OA-associated hyperammonemia. Design: Retrospective study in OAs patients with hyperammonemia episodes receiving NCG between 1995 and 2009. End-points were plasma ammonia (NH3), symptoms (neurological, psychiatric, psychomotor, hepatic) and safety Results: Overall, 57 patients were included. 48 episodes of hyperammonemia (60.4% of neonatal onset) in 41 patients (4 Isovaleric, 21 Methylmalonic, 16 Propionic Acidemias) were analyzed for efficacy. Median (range) episode duration was 6 days . Median (range) starting dose and duration of NCG was 75.5 mg/kg (13.3-303.0) and 4 days (1-15), respectively. The maintenance dose was slowly reduced; 43.8% of hyperammonemia episodes were treated with concomitant scavengers. Median (range) NH3 decreased from baseline of 215 μmol/L (76-1633) to 52.0 μmol/L (15-158) (last value under NCG). Consistent NH3 decrease was observed in all OAs, irrespective of concomitant scavengers. Median time to achieve NH3<=60 μmol/L under NCG was 36.5 hours. The clinical symptoms improved following NCG initiation. Among 74 adverse events observed, 22 were serious, mostly not drugrelated, except for 1 death (neurological damage). Conclusions: Carglumic acid rapidly and effectively reduces NH3 in OAassociated hyperammonemia, leading to an overall clinical improvement, with an acceptable safety profile. Over a period of 3 years 2498 patients with suspected metabolic disorder were evaluated. The patients showed 2:1 male:female ratio. The study group included asymptomatic screen positive neonates(n=25), symptomatic neonates and young children with acidosis, hypoglycaemia, hyperammonemia etc. Inborn error of metabolism was detected in~19.2% patients, prevalent being MMA(11.4%) and GA1(9.1%). Disorders such as PA, IVA,MSUD, tyrosinemia type1, canavan disease, biotinidase deficiency and others ranged from 0.5-2.4% of the affected subjects. In addition a few cases of rare disorders such as SSADH, 2 hydroxy glutaric aciduria etc were also diagnosed. Lactic acidosis and/or ketosis have been a consistent finding in several samples. Around 3.3% showed dicarboxylic aciduria either due to a metabolic defect / MCT administration or sickness. Early diagnosis has helped to initiate management in 7 MMA, 4 GA1, 2 IVA and 3 PA patients. Prenatal diagnosis in subsequent pregnancies was facilitated in referral laboratories for 2 MMA, 1 GA1 and 1 SSADH family. Thus it is observed that offering OA analysis within the country has helped to reveal inherited metabolic disorder profile amongst Indians and to initiate management and offer prenatal diagnosis to reduce the burden of recurrence in affected families. Human exposure of the mycotoxin fumonisin B (FB) is greatest in regions where maize products are the dietary staple. The tricarballylic acid moieties of FB are structurally similar to sphingolipids which can cause neural tube defects due to altered sphingolipid metabolism by inhibition of ceramide synthase. The toxic effects of FB can also lead to acute mycotoxicosis, esophageal cancer in humans and cancer, pulmonary edema, leukoencephalomalacia in animals. Case. A 5 days old female patient was admitted with elevated propionylcarnitine (5.5 μmol/L; cut-off 4.9 μmol/L) in expanded newborn screening. During second-tier tests, urinary organic acid analysis by GC-MS revealed excretion of "tricarballylic acid" (58 μmol/mol creatinine). The mother had vitamin B12 deficiency. Breast-feeding was withheld as a potential source of the baby's fumonisin exposure because her family traditionally consumed high amounts of maize and maize products. Tricarballylic aciduria ceased within three weeks. Samples of ground corn taken from the family revealed a high content of FB (4164 ppb; N< 100 ppb). This is the youngest baby reported to be exposed to FB. Early diagnosis of this baby with urinary organic acid analysis led to initiation of the regional investigation of contaminated maize products by the Turkish Ministry of Health. 
Our case is a 16-year-old girl with methylmalonic acidaemia. She has progressive scoliosis which caused restrictive lung function impairment and therefore spinal surgery was contemplated. There was limited experience in operating these patients who are extremely vulnerabe in encountering stress. Anterior spinal fusion with instrumentation T11 to T14 was performed. Pre-operatively, sufficient glucose load was given during fasting. We closely monitored her blood gas, glucose, ammonia level, lactate, electrolytes and urine ketones. Intraoperatively, we avoided lactate containing intravenous fluid, N2O, NSAIDs and relaxants that are metabolized by ester hydrolysis. We used fresh blood for transfusions as old blood would give higher protein and acid load. TPN and carnitine infusions were started intraoperatively. Postoperatively, patient was transferred to PICU. Excellent pain control was given. Oral feeding was resumed as fast as patient could tolerate. We ensured that she has good bowel motion as gut bacteria can produce large amounts of propionic acid which is the direct precursor of MMA. Carbaglu and haemodialysis machine were arranged in case patient went into decompensation with hyperammonaemia. Conclusion: Patients with organic acidaemia can undergo major surgeries under general anaesthesia. However, detailed planning and cooperation among different disciplines are necessary for the success. A 37 years old female patient with classical MSUD was admitted to our hospital with a sore throat, anorexia, and decreased responsiveness. Her medical history contained psychomotor retardation, spastic tetraplegia and epilepsy as a result of dietary noncompliance in early childhood. A Candida stomatitis and dehydration were diagnosed. She was successfully rehydrated and treated with Fluconazole 200 mg orally for 10 days. After 4 days she was discharged in good condition. Metabolic laboratory results showed derangement of branched amino acids which was likely to be caused by an accidental switch of her amino acid mixtures to a urea cycle disorder preparation 21 days before admission. Although often thought of as a common complication, there is no peer reviewed publication regarding fungal infections during metabolic derangement in MSUD. Experiments in healthy rats show that an excess of dietary leucine can result in immune impairment as does a decreased BCKDH-activity in leucocytes and an increase of oxidative stress as seen in MSUD. Thus, immune impairment in MSUD derangement is comprehensible. Why fungal and not bacterial infections are prevalent in MSUD derangement, still remains to be elucidated.
ESTABLISHMENT OF METHYLMALONYL COA MUTASE ACTIVITY MEASUREMENT WITH LYMPHOCYTE BY HPLC Nasu T 1 , Maeda Y 1 , Ito T 2 , Nakajima Y 2 , Tajima G 3 , Kato S 2 , Kurono Y 1 , Kimura K 1 , Sugiyama N 4 1 Dept Hosp Pharm, Nagoya City Univ, Nagoya, Japan 2 Dept Ped, Nagoya City Univ, Nagoya, Japan 3 Dept Ped, Hiroshima Univ, Hiroshima, Japan 4 Dept Ped, Aichi-Gakuin Univ, Nagoya, Japan
Background: An assay of the enzyme activity is very important for diagnosis of metabolic disorder. The activity of methylmalonyl-CoA mutase has been measured using fibroblast. However, the collections of the fibroblast from the patient and cell culture are troublesome task. We examined the simple and easy method using lymphocyte. Method: A mixture of methylmalonyl-CoA, lymphocyte and cobalamin in Tris-sulfate buffer (pH 7.5) was incubated for 15 min at 37°C. After the suspension was centrifuged (12,000 rpm, 10 min), the supernatant was analyzed by HPLC. STR-ODS 2 (6×150 mm, Shinwakagaku, Japan) was used as the HPLC column and detection of methylmalonyl-CoA and succinyl-CoA was performed by UV absorption (260 nm). Results: Although succinyl-CoA was detected in lymphocytes from a healthy volunteer, none or a small amount of succinyl-CoA was generated in the lymphocytes of a patient with methylmalonic acidemia. The amount of generated succinyl-CoA was proportinal to methylmalonyl-CoA mutase activity. Discussion: The measurement of methylmalonyl-CoA mutase activity with lymphocyte was possible. However, as succinyl-CoA decomposed with time by heat, analysis has to be performed carefully.
A-012 CANAVAN DISEASE: CASE REPORT Hasanoğlu A 1 , Küçükçongar A 1 , Ezgü FS 1 , Tümer L 1 , Kasapkara CS 1 , Biberoğlu G 1 , Salomons GS 2 1 Div Metab Dis, Univ Gazi, Ankara, Turkey 2 VU Univ Medical Center, Amsterdam, Netherlands
Canavan disease (CD) is caused by elevated levels of N-acetylaspartic acid (NAA). The triad of hypotonia, macrocephaly and head lag in infant after the age of three to five months should raise the suspicion of Canavan disease. The diagnosis of CD relies upon measurement of the concentration of NAA in the urine using gas chromatography-mass spectrometry (GC-MS). For certain diagnosis should be performed aspartoacylase enzyme activity in cultured fibroblast or molecular analyse of ASPA gene. Here presented a six months old girl patient applied with seizures to our clinic. In her physical examination lack of head control without macrocephaly and optic atrophy were found. We suspected neurometabolic disorder and performed metabolic screening tests. Urine GC-MS showed high level of NAA and magnetic resonance spectroscopy showed a peak of aspartic acid. After these results molecular analyse for CD were performed and confirmed the diagnose by the identified homozygous mutation (c.244dupA) in the ASPA gene. The first presentation of our patient were nonspesific neurological findings like seizures and hypotonia. Additionally there were no sign for macrocephaly which is generally seemed in CD.
Here emphasized the importance of metabolic screening test to arrive the diagnosis especially in non specific clinical manifestations.
Background: Methylmalonic acidemia (MMA) and propionic acidemias (PA) are the most common organic acidemias affecting the propionate pathway. Anemia, leukopenia and thrombocytopenia due to bone marrow supression are common findings in both disorders, specially in acute metabolic decompensation. It is suggested neurological injury in each disorder and renal dysfunction in MMA is related to mitochondrial dysfunction due to toxicity of methylmalonic acid, propionyl-CoA and 2-methylcitrate, but the pathogenesis of bone marrow supression is unknown. Objectives: To evaluate whether there is any relation between blood hemoglobin, white blood cell (WBC), thrombocyte levels and urine organic acid excretions, blood free carnitin, acylcarnitine levels. Material and Methods: The study group consisted of 37 children (MMA=30, PA=7). Blood and urine samples were obtained at the same time.
Results: There was no correlation between thrombocyte, WBC levels and urine methylmalonic acid, methyl citrate, propionic acid excretion and blood free carnitine, methylmalonylcarnitine, propionylcarnitine levels. There was a significant negative correlation between hemoglobin and propionylcarnitine levels in both diseases (MMA:r=−0.827, p<0.001 and PA:r=−0.692, p<0.001) Conclusions: The presence of negative correlation only between blood hemoglobin levels and blood propionylcarnitine levels in both diseases suggests that propionylcarnitin may have a role at the pathophysiology of anemia in these disorders. Methylmalonic aciduria (MMA) is an inborn error of organic acid metabolism. Patients with severe disease develop many complications despite treatment; often, the disease progresses to tubulointerstitial nephritis with progressive renal failure or to severe damage of the central nervous system. Liver, kidney, or combined liver and kidney transplantation is advocated when medical treatment is ineffective. Liver or combined liver and kidney transplantation is effective to improve the metabolic decompensation; however neurological deterioration has occurred in some individuals after the transplantation. Some individuals have received only renal allografts; Lubrano et al reported a 27-year-old woman with MMA MUT0, who received a kidney transplant at age 17 years, and presented no episodes of metabolic decompensation and normal renal function after the transplantation. We report a 26-year-old Japanese male with MMA MUT0 (R93H, IVS2+5 G>A), presenting end stage renal failure. The clinical symptoms appeared at age 7 months. He progressed into renal dysfunction at age 6 years. Recently serum levels of creatinine have reached above 5 mg/dl. Kidney transplantation is scheduled in June 2011. Background: Before extended neonatal bloodspot screening (NBS), MCADD patients presented with life-threatening hypoketotic hypoglycemia. Nowadays, asymptomatic newborns are identified by NBS with novel genotypes, which have not been found in clinically ascertained patients (i.e. ACADM variants). These children are treated as classical MCADD patients, while the necessity hereof is unknown. Objective: To determine the effect of ACADM variants on enzyme activity and fasting tolerance. Methods: MCAD enzyme activities were measured in cultured skin fibroblasts from 8 subjects with ACADM variants, using different substrates. In 5 subjects, a fasting study and phenylpropionic acid loading test were performed. Results: Using phenylpropionyl-CoA, MCAD activity in fibroblasts was significantly higher in variants (mean ±SD 8.1% ±7.5) compared to classical patients (<1%). Phenylpropionic acid loading caused urinary hippuric acid excretion in subjects with ACADM variants, indicating in vivo residual MCAD enzyme activity. Upon fasting, blood glucose, free fatty acid/ketone body (KB), and KB*glucose remained normal for 15-20 hours. Conclusion: ACADM variants show residual MCAD enzyme activity in vitro and in vivo, as opposed to classical patients, correlating with normal fasting tolerance. The effect of fever on in vivo residual enzyme activity remains unknown, and follow-up studies are warranted to determine the clinical relevance of these variants. Asymptomatic very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient patients are often supplemented with medium-chain triglycerides (MCT) to prevent the development of cardiomyopathy and myopathy. MCT is considered a safe dietary intervention, however, long-term observations into later adulthood are still missing. In the present study, we investigated the consequences of a long term MCT supplementation in VLCAD deficient (VLCAD−/−) mice. In vivo studies based on 1H and 13 C magnetic resonance (MR) techniques were applied to analyze noninvasively the MCT effects on abdominal fat distribution and composition as well as on liver fat. These data were subsequently correlated with serum transaminases and liver histology. In addition, hepatic biochemical markers of oxidative stress were assessed. The replacement of LCT by MCT, results in a dramatic accumulation of visceral fat and serum free fatty acids in VLCAD−/− mice followed by a profound shift in body triglyceride composition when applied over one year. Liver histology 1H MRS and biochemical analysis revealed pronounced steatosis and marked oxidative stress. MCT diet has been effective in prevention of cardiomyopathy and skeletal myopathy in FAO disorders. However, our data demonstrate that long-term MCT supplementation in the mouse model results in a severe hepatic phenotype similar to non-alcoholic steatohepatitis and metabolic syndrome. Background: LPIN1 mutations were identified as a cause of severe earlyonset rhabdomyolysis. The lipin family includes two other related members, lipin-2 and 3, which share strong homology and activity. Objectives: To determine the involvement of lipins in patients presenting with various symptoms ranging from rhabdomyolysis to myalgia at any age. Patients and Methods: LPIN1, LPIN2, LPIN3 coding regions were sequenced for 200 patients presented with rhabdomyolysis (n=170) of variable onset ranging from severe to mild (CK>or<10 000 UI/L) or myalgia without myoglobinuria (n=30). Results: 35 patients presenting with severe rhabdomyolysis, occurring before age 10 years but one, had 2 LPIN1 mutations. 86% Caucasian families shared the same intragenic deletion. 40% heterozygous relatives presented mild muscular symptoms. Muscle biopsies showed excessive lipid droplets. Rhabdomyolysis were mostly precipitated by fever. Incubation of lipin-1deficient myoblasts with pro-inflammatory cytokines leaded to significant increase of lipid droplets. Nine variants identified in milder phenotypes in either gene were non-functional in a yeast complementation assay except one. Conclusion: LPIN1-related disease, a new subtype of lipid storage myopathy, constitute a major cause of severe rhabdomyolysis in childhood and occasionally in adults. Relatives can be symptomatic. No major LPIN2/LPIN3 defects are associated with muscular diseases. Multiple Acyl-CoA dehydrogenation deficiency (MADD) is a metabolic disorder affecting fatty acid β-oxidation, resulting in a decrease of electron transference to respiratory chain. Pathophysiological mechanisms leading to clinical phenotype are believed to rely mainly on the resulting energy deficiency, although its consequences on mitochondrial function are still poorly understood. In order to bring new insights, a proteome approach was adopted. We isolated mitochondria from cultured fibroblasts, from a patient with a severe MADD presentation due to ETF-QO deficiency, characterize its mitochondrial proteome and compare it with normal controls. The used approach (2-DE-MS/MS) allowed the positive identification of 287 proteins in both patient and controls, presenting 35 of then significant differences in their relative abundance. Among the differentially expressed are proteins associated to binding/folding functions, mitochondrial antioxidant enzymes as well as proteins associated to apoptotic events. The overexpression of chaperones like Hsp60 or mitochondrial Grp75, antioxidant enzymes and apoptotic proteins reflect the mitochondrial response to a complete absence of ETF-QO. Our study provides a global perspective of the mitochondrial proteome plasticity in a severe case of MADD and highlights the main molecular pathways involved in its pathogenesis. An integrated perspective of mitochondrial plasticity face to MADD is given. Background: The physiologic role of long chain acyl-CoA dehydrogenase (LCAD) has long remained elusive. LCAD is expressed in type II pneumocytes, leading us to hypothesize that a patient with LCAD deficiency would present with congenital surfactant deficiency. Design/Methods: Term infants with unexplained respiratory distress were evaluated for known causes of congenital surfactant deficiency and had a normal genotype. Lung tissue from patient and controls were evaluated with electron microscopy for lamellar body structure as well as LCAD antigen expression using immunostaining and confocal microscopy. Genetic studies were performed. Results: One patient was identified. EM of lung tissue demonstrated eccentric deposits within the lamellar bodies. LCAD antigen was absent in patient lung tissue. Genomic DNA sequencing of patient DNA revealed a homozygous base pair change involving intron 6, causing a splicing error. At six months of age, a liver biopsy demonstrated accumulation of cis-3,4methylene-heptanoylcarnitine, likely derivative from a LCAD substrate. Conclusions: This is the first report of LCAD deficiency, presenting with a unique phenotype of congenital surfactant deficiency. MADD is an inherited disorder of fatty acid, amino acid and choline metabolism. There is good correlation between MADD disease severity and mutations in ETFA, ETFB or ETFDH. A neonate was diagnosed with severe MADD based on increased C5-C18 acylcarnitines and low (2-5% of control mean) fatty acid oxidation flux in fibroblasts. Mutation analysis revealed homozygosity for a novel c.158A>G (p.Lys53Arg) mutation in ETFDH exon 2. Surprisingly, PolyPhen analysis predicts the mutation to be benign. However, in silico analyses suggested that the mutation creates an exonic splicing silencer (ESS) motif (TAGGGA) for the splicing inhibitory protein hnRNPA1. Consistent with this, patient ETFDH cDNA showed complete exon 2 skipping, which results in removal of most of the mitochondrial targeting signal. A mutation in the APC gene also creates a TAGGGA motif and causes missplicing. This illustrates that splicing regulatory elements are general. We are currently analyzing if the TAGGGA motif functions as an hnRNPA1 dependent splicing silencer using RNA affinity purification experiments and a heterologous splicing reporter minigene.
In conclusion, our findings are consistent with the severe MADD phenotype of the patient and underscore the importance of testing mRNA splicing consequences when predicting the effect of exonic sequence changes. Background: Carnitine-acylcarnitine translocase (CACT) deficiency is a life-threatening, rare autosomal recessive disease often presenting in early infancy with seizures, hypoketotic hypoglycemia, hyperammonemia, cardiomyopathy, liver failure, muscle weakness, and apnea, similar to other defects of long chain fatty acid beta-oxidation, with indistinguishable acylcarnitine profile form infantile CPTII deficiency. Previously, diagnosis of CACT deficiency relied on in vitro enzymatic assay that is tedious and not practical. Objective: To develop Molecular methods for definitive diagnosis of CACT deficiency. Method: Sanger sequencing and oligonucleotide aCGH were used for the analysis of the SLC25A20 gene. Literatures were reviewed. Results: Among 100 individuals suspected of having CACT deficiency, mutations were identified in 7 unrelated families. Eight novel mutations were found, including a large 25.9 kb deletion encompassing exons 5 to 9 of the gene detected by aCGH. All patients presented clinical features and acylcarnitine profiles at early life consistent with CACT or CPTII deficiency. However, sequence analysis of CPT2 gene did not find mutations. Conclusion: CACT deficiency is pan-ethnic. It may not be as rare as originally thought. Patients with infantile form of CPTII deficiency should be analyzed for mutations, including large deletions, in the SLC25A20 gene. We report our experience in managing three surviving children (7 years, 5 years and 22 months) with carnitine-acylcarnitine translocase deficiency, confirmed by mutation analysis of the SLC25A20 gene. Patient A, born prematurely at 32 weeks, developed ST elevation and ventricular tachycardia on cardiac monitor on day 17 when feeding was reintroduced after recovery from necrotizing enterocolitis. Patient B, who was a term infant already discharged from hospital, presented on day 3 with cardiorespiratory failure requiring cardiopulmonary resuscitation (CPR) in the Accident and Emergency Department. Patient C presented on day 1 with cardiorespiratory arrest successfully revived with CPR. All 3 children were put on frequent feeding with a high carbohydrate / low fat diet, mainly with medium-chain triglycerides, supplemented with walnut oil rich in essential fatty acids. Patients A & C had significant feeding problems with food refusal requiring gastrostomy feeding and central venous line to maintain adequate glucose infusion, whilst Patient B was on a less meticulous diet with infrequent decompensations and no feeding difficulty. Patient B (aged 5 years) suffered from hypoxic brain damage resulting in mild grade mental retardation. The other 2 children have normal development despite frequent episodes of decompensation with hyperammonaemia and rhabdomyolysis. Background: To evaluate the relationship between the blood acylcarnitine profiles and clinical picture in patients with late-onset muscular phenotype of carnitine palmitoyltransferase (CPT II) deficiency (MIM 255110). CPT II deficiency is an autosomal recessive disorder of carnitine dependent transport of long-chain fatty acids into mitochondrial matrix. Methods: We analysed clinical data and the blood acylcarnitine profiles [C16, C18:1, the (C16+C18:1)/C2 ratios] in 32 samples of two adult male patients with the muscular phenotype of CPT II deficiency. Results: Patients presented with myalgia/weakness in 12/32 examinations. The elevation of the (C16+C18:1)/C2 ratios was found in 10/12 symptomatic and 9/20 asymptomatic cases. The levels of C16 acylcarnitine were elevated in 3/12 symptomatic and 2/20 asymptomatic cases. The elevation of C18:1 acylcarnitine concentrations was observed in 5/12 symptomatic and 5/20 asymptomatic cases. Higher levels of C18:1 acylcarnitine concentrations were accompanied by the elevation of C16 acylcarnitine concentrations in 3/5 symptomatic and 2/5 asymptomatic cases. Conclusions: Our patients had no correlation between the absolute values of the (C16+C18:1)/C2 ratios, C16 and C18:1 acylcarnitine concentrations and clinical symptoms. The (C16+C18:1)/C2 ratio was the most sensitive marker for the diagnosis of CPT II deficiency. Supported by the grant MZ0VFN2005 of Ministry of Health of Czech Republic. Since valproic acid interferes with mitochondrial energy metabolism, carnitine imbalance may occur in liver, the major target of VPA-toxicity. Aim: To investigate the acylcarnitine profile in animal liver tissues. Methods: Wistar rats were treated with one single injection of sodium valproate (100, 500 mg/kg) or subjected to a subchronic regimen (100 mg/kg/day for two weeks). Carnitine and acylcarnitines were quantified in liver tissues using ESI-MS/MS. Results: There was a significant dose-related increase of free carnitine and total acylcarnitine levels in the liver of rats treated with a single injection of valproate as compared with controls. Acylcarnitine levels were normal in livers of rats submitted to subchronic treatment except for malonyl-, isovaleryl-, 3-hydroxyisovaleryl-, adipoyl-, nonanoyl-, cis-4-decenoyl-and decanoylcarnitine which remained significantly increased. Discussion: The results clearly show an accumulation of acylcarnitines in liver at the onset of valproate administration. However, the disturbances in carnitine homeostasis appeared to recover after a subchronic regimen, except for specific individual acylcarnitines involved in the metabolism of branched-chain amino acids and medium-chain fatty acids. Endogenous biosynthesis of carnitine or its redistribution from peripheral organs may play an important role in the rescue of free carnitine levels in liver after a drug induced stress on mitochondrial metabolism. Background: Primary systemic carnitine deficiency (PSCD) is caused by mutation in the SLC22A5 gene encoding carnitine transporter OCTN2. The disorder is characterized by cardiomyopathy, weakness, hypoglycemic hypoketotic encephalopathy and highly responsive to L-carnitine therapy. Objectives: The aim was to investigate identification of clinical phenotype and genotypes with emphasis to cardiac manifestation and response to L-carnitine therapy in 14 patients in order to provide genotype-phenotype correlations. Material and Methods: Mutations for SLC22A5 gene in 14 patients with PSCD followed at Istanbul Medical Faculty was confirmed by direct sequence analysis. Clinical phenotype; ECG, echocardiography, 24-hours holter-ECG; genotype; dosage, duration of L-carnitine therapy was evaluated. Results: Age at diagnosis was 5.7+10.7 years (range:0.2-41.0). Follow-up duration was 4.4+2.2 years (range:2-9). L-carnitine 178.8+63.7 mg/day (range:75-300) was recommended. Symptoms at referral were Reye-like syndrome(4), cardiac insufficiency(4), convulsions(1), psychomotor retardation(1) and family history ( Plasma acylcarnitines are important biomarkers in the diagnosis of mitochondrial fatty acid beta-oxidation disorders (mFAOD). Although regarded as a reflection of the acyl-CoAs accumulating intramitochondrially in mFAOD patients, the true etiology of plasma acylcarnitines still requires clarification. It is generally assumed that these intermediates are formed within mitochondria via the activity of carnitine palmitoyltransferase 2 (CPT2). However, their transport across the mitochondrial and plasma membrane into the extracellular space remains undefined. Herein we aimed to resolve the mechanism by which intramitochondrial acylcarnitines are synthesized and further transported from the mitochondrial matrix to the extracellular space. Using lentiviral shRNA, medium-chain acyl-CoA dehydrogenase (MCAD) gene was silenced in control, CPT2 and OCTN2 deficient human fibroblasts. Transfected cell lines were incubated with 120 μM of n-decanoic acid plus Lcarnitine. In shMCAD transfected cell lines beta-oxidation proceeds until the formation of C8-CoA which will accumulate intramitochondrially. Extracellular acylcarnitine profiles were analyzed by ESI-MS/MS. Preliminary results show the absence of C8-carnitine in the medium of CTP2 deficient cells, pointing towards the involvement of this enzyme in the production of medium-chain acylcarnitines. In OCTN2 deficient cell lines however, extracellular accumulation of C8-carnitine is observed suggesting that the cellular export of acylcarnitines does not depend on OCTN2. Background: Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCAD or 2-methylbutyryl dehydrogenase) is a very uncommon autosomic recessive disorder of the isoleucine catabolic pathway, with variable and not well defined phenotype. We describe a case with clinical features not reported previously. Case Report: A 14 months old boy was referred for evaluation on arrival to our country. He is the first son of healthy consanguineous parents from Pakistan, with a normal psychomotor development until the age of 8 months. During pneumonia he suffered an acute neurological regression with seizures and feeding difficulties. On admission, the patient showed visual contact, severe generalized hypotonia, hyperreflexia, oral and upper limb dyskinesias. Metabolic screening revealed hyperlactacidemia and increase in 2-methylbutyrylglycine, 2ethylhydracrylic, ethylmalonic and methylsuccinic acids, all consistent with a diagnosis of SBCAD deficiency. Genetic testing is ongoing. Brain MRI shows bilateral striatal lesions. The patient is on a low-protein (lowisoleucin) diet. Discussion: The clinical spectrum of SBCAD deficiency is not well defined because of the small number of reported cases and their different phenotype. While it may be asymptomatic in some patients, in others it manifests as a severe neurological disorder. To our knowledge our patient is the first presenting with bilateral striatal involvement. The patient presented with brief hypoglycaemic seizures at the age of 3 and 4 days. Further diagnostic work-up revealed medium chain acylCoA dehydrogenase (MCAD)-deficiency. In the following years, she developed normally, liver and muscle function tests were normal. At the age of 11 years, isolated AST elevation was first noticed. AST values remained elevated ever since at 600-800 U/l (normal <35 U/l), all other liver enzymes, coagulation, CK and carnitine were normal. An infection with hepatotropic viruses could be excluded. Impaired fatty acid oxidation in MCAD-deficiency can compromise skeletal muscle, heart and liver function, especially in adolescents and adults. AST-elevation was considered to be related to MCAD-deficiency, further tests like liver ultrasound, echocardiography and ECG were unrevealing. At the age of 15 years, the girl presented first in our metabolic clinic. As organ functions were normal and there was isolated elevation of AST we considered macro-AST. The presence of macro-AST was confirmed by polyethylene glycol precipitation. Macro-AST is usually a harmless finding due to complex formation with immunoglobulins which cannot be excreted via urine. Conclusion: AST-elevation in this patient was unrelated to MCADdeficiency but due to macro-AST. IgE-elevation associated with pollenallergy may play a role. It has been estimated that 15-20% of the children with MCAD deficiency (MCADD) died suddenly during a first metabolic decompensation. Data from analysis of clinical symptoms preceeding sudden death are still rare. Case report: A 9 months old gypsy boy was found dead at home in the morning. A postmortal acylcarnitine profile from a dry blood spot raised the suspicon of MCADD. Mutation analysis revealed homozygosity for the 985A>G mutation in the ACADM gene. Retrospective analysis of the clinical course revealed that on the day preceeding his death the patient had developed vomiting and diarrhea without fever and any signs of respiratory infection. A consulted primary care paediatrician did not recommend hospitalization and suggested only oral rehydratation after vomiting had reiterated. Autopsy revealed fatty liver as well as severe cerebral edema. Microscopic examination of lungs revealed signs of acute viral infection. Virologic tests did not detect virus of seasonal influenza or pandemic influenza type A (H1N1). Sudden death in four children with MCADD have been described by Yusupov et al. despite newborn screening and in all vomiting was the dominant symptom preceeding sudden death (Molecular Genetics and Metabolism, 101, 2010, p. 33-39) . Clinical analysis of our case confirmed this observation. Introduction: MCADD is the most prevalent deficiency of mitochondrial fatty acid Beta-oxidation defects (FAOD) and is included in newborn screening program (NSP) of various occidental countries. France would implement MCADD in its NSP in future. Methods: through a retrospective study, we analysed 66 paediatric cases affected with MCADD, confirmed by enzymatic or molecular analysis by 3 French laboratories. Results: since the 1980s, 66 French cases have been collecte. According to these results, French MCADD's prevalence would be 1.1/million illustrating a probably large number of undiagnosed cases. Diagnosis was performed during acute decompensation in 86%, as sibling of a newly diagnosed case in 14%. Median age at diagnosis was 14 months. Clinical presentation was mostly hypoketotic hypoglycemia, liver involvement, and Reye syndrome. After 165months mean follow-up, 72% present full recovery from attack, 10% have neurological impairment, 18% died. Death rate was 63% before 3 months, 6% between 3 months and 3 years and 0% after 3 years. Death was always observed during the first metabolic attack. All patients screened after a diagnosis in sibship remained asymptomatic after 118-months mean follow-up. Conclusion: Ay present in France, MCADD is a severe under diagnosed disease, with high mortality and neurological sequelae. More than 60% of clinically manifested MCADD patients are homozygous for a single mutation the p. K304E (c. 985A>G). We have recently genotyped a cohort of 60 MCADD patients. During this study we found in heterozygosity with the p.K304E mutation a previously unreported variant p.G377V (c.1205 G>T). In order to infer the diseasecausing effect of this novel mutant we developed an E.coli expression system to produce a recombinant form of the p.G377V protein. This novel mutant showed 27% residual activity when compared with the hMCADwt recombinant protein. These results are consistent with the close proximity of residue 377 to the enzyme's substrate (Glu376) and FAD binding sites, the latter essential for dimerisation, and thus prove the disease-causing effect of the novel mutation. The developed expression system constitutes a powerful tool for the characterization of novel hMCAD mutants and open the way to the study of potential activity modulators of hMCAD variants aiming at the development of effective therapeutic approaches for this inherited metabolic disease. Background: MCADD deficiency is the most common disorder of fatty acid oxidation (1 in 15,000 live births in Western Europe). A single gene change, K304E, accounts for 90% of MCADD cases. The frequency of LCHADD is 1 in 70,000 live births in Western Europe. 60-86% of reported patients with isolated LCHAD deficiency have a prevalent E474Q mutation. Objectives: Following the recent diagnosis of 25 patients with b-oxidation defects (17 among them with LCHADD and 4 with MCADD) within 2 years of selective screening in our laboratory, we investigated the molecular features of LCHADD and MCADD in Russian patients. Patients and Methods: DNA extracted from dried blood spots of 21 patients and 792 randomized derived from newborns from Moscow city were studied. Results: All patients with LCHADD except one were homozygous for the mutation E474Q and all patients with MCADD were homozygous for the K304E mutation. 5/792 blood spots from newborns were heterozygous for the E474Q mutation , 2/722 blood spot heterozygous for the K304E and gives estimated newborn incidence of 1/84000 for LCHADD and 1/ 640000 for MCADD. Conclusion: LCHAD deficiency is prevalent among other disorders of mitochondrial b-oxidation and MCADD has unexpectedly low incidence in the Russian population. Background: Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of long chain fatty acid beta-oxidation, that compromises energy homeostasis and leads to accumulation of long-chain fatty acids and derivatives. Patients may develop hypoglycemia, rhabdomyolysis, hepatomegaly and cardiomyopathy. The main goal of treatment is to avoid catabolism. In addition, many patients use a long chain fatty acid restricted diet. Because essential fatty acids (EFA) are long chain fatty acids, those patient may be at risk for developing EFA deficiency. Methods: We retrospectively studied EFA measurements performed in erythrocytes of Dutch VLCADD patients on long chain fatty acid restricted diet, via gas-chromatography. Results: 25 blood samples of 11 patients were available for analysis. Eight patients (18/25 samples) had decreased levels of linoleic acid (C18:2n-6). Alpha-linoleic acid (C18:3n-3) was not decreased in any of the patients (0/ 25). Elevated mead acid (C20:3n-9), a putative marker for EFA deficiency, was found in only one sample. Conclusion: VLCADD patients are more prone to become EFA deficient, especially for linoleic acid. Metabolic consultants should be aware of this possibility and supplement EFA's. Furthermore, although patients were EFA deficient, mead acid did not increase. Mead acid might therefore not be a good marker for EFA deficiency. In our centre we have been providing preimplantation genetic diagnosis (PGD) since 2007. Up to now we have accomplished 117 IVF cycles followed by PGD in 40 genetic diseases. The success rate of the procedure (calculated on the fetal heart beat pregnancy) is around 43%. The case report descibes PGD in a 39 year old woman. Her son from a consanguineous marriage (r=1/8) has LCHAD deficiency. Molecular analysis in the child showed homozygosity for the 1528 G-C mutation in the HADHA gene which maps to 2p23 (MIM ID 609016). Both parents are heterozygous for the 1528 G-C mutation and the risk of recurrence for their children is 25%. We used genetic haplotyping technique by multiplex PCR on products of MDA (multiple displacement amplification) from 1 blastomere biopsied from the embryo. 9 embryos were biopsied, 4 of them were wild homozygotes, 4 of them were heterozygotes and in 1 embryo monosomy X was confirmed. 8 of the embryos were recommended to transfer, only 1 embryo was actually transferred. The pregnancy was confirmed by ultrasound investigation. The karyotype of the foetus from the chorionic biopsy was 46,XX. Molecular analysis of HADHA gene did not confirm 1528 G-C mutation. Background: Bacillus cereus occasionally causes diarrheic and emetic food poisoning. It was reported that cereulide, emetic toxin of Bacillus cereus, is associated with encephalopathy and Reye-like syndrome that resemble fatty acid oxidation (FAO) disorders. However, the underlying mechanism for Reye-like syndrome associated with Bacillus cereus is not known. Herein, the effect of cereulide on FAO was determined. Materials and Methods: Control fibroblasts were cultured in the presence or absence of cereulide along with octanoic or palmitic acid as substrate. FAO capacity was determined by in vitro probe assay and tandem-massspectrometry. Results: Octanoic or palmitic acid led to an increase of acetylcarnitine (C2) without causing accumulation of medium (C4, C6, C8 and C10) and long (C12, C14 and C16) chain acylcarnitines in the cells cultured without cereulide. In contrast, cereulide decreased C2 and markedly increased C4, C6, C8 or C16 compared to cells treated with octanoic or palmitic acid alone. Conclusion: The acylcarnitine profile in the presence of cereulide was similar to neonatal multiple acyl-CoA dehydrogenase deficiency. The results indicate that cereulide severely inhibits mitochondrial FAO toward a broad range of acyl-CoAs. Malfunction of FAO resulting from intoxication of cereulide may be responsible for Reye-like syndrome associated with Bacillus cereus. Investigation of a fat oxidation disorder by acylcarnitine analysis is common practice in the differential diagnosis of sudden unexpected death in infancy (SUDI). Taking samples as soon as possible after death minimises post mortem artefactual changes. We describe three recent cases that illustrate the benefit of a clear and well implemented SUDI policy by contrasting findings with samples taken at autopsy: Case-1: A male infant collapsed and died on day 2. Bloodspots taken immediately after death showed increased long chain acylcarnitines; those at autopsy were far less suggestive of a fat oxidation defect. Carnitine acylcarnitine translocase deficiency was later confirmed by fibroblast studies. Case-2: A 16 month old girl, initially found to have an isolated increase of urinary suberylglycine, but with normal bloodspot acylcarnitines, died unexpectedly of respiratory failure. Bloodspots taken at the time of death show increases in short and medium chain acylcarnitines but samples collected at autopsy were difficult to interpret. Investigations into a possible riboflavin transporter defect are ongoing. Case-3: A one month old male infant died suddenly. Bloodspot acylcarnitine analysis showed a deficiency of long chain acylcarnitines (notably C16, C18:1 and C18) and normal free carnitine. Subsequent fibroblast studies confirmed carnitine palmitoyl transferase type 1 deficiency.
Shigematsu Y 1 , Hata I 2 , Tajima G 3 1 Div Health Sci, Univ Fukui, Fukui, Japan 2 Dept Pediat, Univ Fukui, Fukui, Japan 3 Dept Pediat, Hiroshima Univ, Hiroshima, Japan
Background: Acylcarnitine analysis using serum samples or dried blood spots does not always provide diagnostic results to evaluate fatty acid oxidation defect. Instead, we analyzed acylcarnitines in lymphocytes loaded with stable-isotope labeled fatty acid for the diagnosis of long-chain fatty acid oxidation defects. Methods: [2H31] palmitic acid was added to glucose-free PBS solution of lymphocytes, which were collected from 3 ml of heparinized blood. The mixture was incubated for 2 hours, and washed lymphocytes were homogenized and centrifuged. Acylcarnitines in an aliquot of the supernatant, spiked with stable-isotope labeled acylcarnitines as internal standards, were analyzed by tandem mass spectrometry. Results: In lymphocytes loaded with [2H31]palmitic acid, a series of labeled acylcarnitines, from 2H31C16-to 2H1C2-acylcarnitine (d1C2AC), were detected. The ratios of d1C2AC/d31C16AC in patients with long-chain fatty acid oxidation defects, except for CPT-1 deficiency, were decreased as compared with those in controls. Decreased ratios of d27C14AC/d31C16AC were observed in patients with CPT-2 deficiency, decreased ratios of d23C12AC/ d27C14AC in patients with VLCAD deficiency, and increased ratios of d29C16OHAC/d31C16AC in patients with TFP deficiency, respectively. Conclusion: This simple test gives us diagnostic information for longchain fatty acid oxidation defects before mutation analysis, and is useful in newborn screening by tandem mass spectrometry. Background: Fatty acid oxidation defects are part of newborn screening programs worldwide. Since then disease incidences have significantly increased. New genotypes are identified with unknown clinical relevance. Methods: We performed octanoyl-CoA and palmitoyl-CoA oxidation studies in lymphocytes of newborns with acylcarnitine profiles suggestive for medium-chain acyl-CoA dehydrogenase (MCAD) or very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, respectively. In all individuals with residual activities <50%, molecular analysis of the respective gene was performed. Results: For MCADD, two mutations were found in patients with residual activities of 0-49%. Patients with the prevalent c.985A>G mutation presented with residual activities of 0-8%, compound heterozygotes with c.199 T>C on one allele had activities of 28-49%. Heterozygotes carrying the c.199 T>C mutation presented with residual activities of 70-100 % as controls carrying no mutation. In VLCADD, we identified two mutations in patients with activities of 0-22%. The majority presented with residual activities <10%. The mild c.848 T>C mutation resulted in activities of 10-13%, the c.339 C>A mutation was associated with a residual activity >20%. Conclusion: Our functional studies identify patients with mutations that are clinically not relevant, such as the known c199T>C mutation in the ACADM gene and allow prognostic predictions in these disorders known for their molecular heterogeneity. Background: Fatty acid oxidation disorders (FAOD) are group of inborn errors of metabolism. The most common cardiac abnormality are cardiomyopathy, arrhythmias, conduction anomalies, pericardial effusion and sudden death. The aim of the study was to describe the cardiac involvement in children with FAOD from Wielkopolska Region of Poland. Material and Methods: Cardiac evaluation was done in 12 patients with FAOD (VLCAD-2, LCHAD-5, MCAD-2, SCAD-1, primary carnitine deficiency-2) compared to 30 healthy controls by ECG, Holter-ECG, standard TTE, TDI and real time 4D. Results: Standard TTE revealed abnormalities in 8 patients: hypertrophic cardiomyopathy in 5, mild TI in 1, pericardial effusion in 2. Arrhythmia was observed in 2 cases. Additionaly in 2 patients congenital heart defects were diagnosed. Compared to controls, indexes of LV systolic and diastolic function of FAOD patients differed significantly, with reduced EF, FS as well as MVand TV E/A ratio, also MAPSE and TAPSE of LVand RV were significantly reduced. FAOD patients showed a reduction in PW-TDI measurements of lateral mitral annulus. In 2 patients reverse of cardiomyopathy have been observed. Conclusions: Cardiac involved is common in children with FAOD. Asymptomatic children with FAOD have an increased prevalence of subclinical LV dysfunction Background: Most fatty acid oxidation disorders (FAOD) present in childhood with cardiac or liver involvement, but exercise intolerance, myalgia, muscle weakness, and attacks of rhabdomyolysis may characterise presentation in adulthood. Objectives: To report the clinical, biochemical and genetic findings in adults with FAOD (either diagnosed initially in adulthood (n=15), or surviving childhood (n=19)). Methods: Clinical, biochemical and genetic data for 34 unrelated adult patients, aged 16 to 60 years, followed at four UK metabolic centres, were reviewed. Patients with MCAD deficiency were excluded. Results: Patients with CPT2 (17/34), VLCAD (7/34), LCHAD (3/34), PCD (3/34), MAD (2/34) and CPT1 deficiencies (2/34) were reviewed. Attacks of rhabdomyolysis were the most frequent manifestation of CPT2 and VLCAD deficiencies. Although muscle symptoms were rare in other defects, fixed muscle weakness and multisystem involvement were seen. In this cohort, acylcarnitine profile was frequently though not always indicative of the FAOD, with cultured skin fibroblast assays and/or identification of a pathogenic mutation confirming the diagnosis. Conclusions: FAOD presenting in adulthood may result in delayed diagnosis. Diagnosis can be achieved without resorting to invasive muscle biopsy. Recognition is crucial to allow appropriate management. Longterm outcome of childhood survivors is generally good, but many remain symptomatic. Background: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent fatty acid oxidation disorder. Patients present tissue accumulation of the medium-chain fatty acids octanoic (OA) and decanoic acids. Clinically, progressive encephalopathy, drowsiness and lethargy that may develop into coma and death are found. Objective: Since the pathophysiology of the brain injury in MCADD is uncertain, in the present work we investigated the effect of intracerebroventricular OA administration on oxidative stress parameters in cerebral cortex, striatum, hippocampus and cerebral spinal fluid (CSF) of rats. Material and Methods: Animals received a single intracerebroventricular OA injection (1.66 μmol). Control animals received artificial cerebral spinal fluid in the same volume. Animals were killed 1 h after OA administration. Thiobarbituric acid-reactive species (TBA-RS) levels, carbonyl content and superoxide dismutase (SOD) and catalase activities were evaluated. Results: It was observed that OA increased TBA-RS levels and carbonyl content in all tissues tested. In addition, catalase activity was increased only in cerebral cortex and hippocampus. On the other hand, OA administration did not alter SOD activity. Conclusion: Taken together, these data suggest that OA induces oxidative stress in brain. Our results may help to explain, at least in part, the characteristic brain dysfunction observed in MCADD patients. Background: fatty acid oxidation disorders (FAOD) are rare autosomal recessive inherited metabolic conditions.This paper will highlight the clues that can be obtained from history, clinical examination, and simple bedside tests characteristic for FAOD. Method: records of 15 patients admitted between 2000 to 2003 and diagnosed as FAOD were reviewed. Results: The final diagnosis was very long chain acyl-CoA dehydrogenase deficiency in ten patients and long chain 3-hydroxyacyl-CoA dehydrogenase deficiency in five. Eighty percent had a positive family history of either a previous sudden unexplained infant death or a similar diagnosis in a first-degree relative. History of consanguinity was positive in eleven patients. Diagnosis of FAOD can be difficult because patients tend to develop symptoms only during prolonged fasting and intercurrent acute illnesses. In our series seven patients presented with acute illness and four patients presented with respiratory distress, four had heart failure due to cardiomyopathy and another two presented with convulsions due to severe metabolic acidosis. Physical findings are usually non-specific, eight had hepatomegaly and four had marked hypotonia. Conclusion: Our group of 15 cases accumulated within 3 years is inordinately large and suggest that Kuwait provides a promising venue in which to study the biochemical and molecular genetics of FAOD. Background: Selective screening for Inborn Error of Metabolism was conducted among patients of pediatric clinics. Children were observed intensive care units with acute intoxication, hypoglycemia, resistant to therapy epilepsy, coma, and liver damage Methods: Clinical observation and selective metabolic screening was performed in the Kharkiv Specialised Medical Genetic Centre: blood amino acids (HPLC, Waters), urine organic acids (GS/MS, Agilent), biochemical blood indexes, ammonia, lactate levels were measured. TMS investigation from Guthrie cards was performed in other countries. Results: An 8 month old boy was observed by the geneticist in the intensive care unit. Clinical presentation included severe gastroenteritis, diarrhea, functional liver and kidney disorder, severe lactic acidosis, respiratory violations (artificial ventilation), fatty hepatosis seen on ultrasound observation, hypertrophic cardiopathy, hypoglycemia during infectious diseases. We suspected an inborn error of metabolism in the child. TMS analyses from the DBS sample were performed. The levels of long chain hydroxylated acylcarnitines and tetradecadienoylcarnitine (C14:2) were increased. The organic acid profile showed increased levels of suberic, 3-OH-suberic and sebacic acids. The levels of lactic acid and liver enzymes in blood were increased. Long-chain 3hydroxyacyl-CoA dehydrogenase/Mitochondrial trifunctional protein deficiency was dignosed. Background: Until recently, ATP synthase deficiency was considered as a relatively rare cause of OXPHOS dysfunction, mainly caused by pathogenic mtDNA alterations. The last two years an emerging group of patients with complex V defects of nuclear origin, mostly TMEM70 mutations, have been reported. However, technical difficulties for evaluating complex Vactivity and the heterogeneous clinical picture of patients seriously hamper correct identification. Methods: A diagnostic strategy combining spectrophotometrical analysis, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by in-gel activity staining, western blotting and diverse microscopic techniques was developed. Results: We report on a selection of complex V deficient patients harbouring pathogenic mutations in either mtDNA or nuDNA. Patients with mtDNA alterations usually have normal complex V activities measured by spectrophotometric analysis and show complex V subcomplexes in the BN-PAGE gel. Complex V deficiency due to nuDNA mutations results in a significant decrease in complex V activity using spectrophotometric techniques. The amount of detectable holocomplex V with BN-PAGE or western blotting is very low. Microscopic analysis in these patients shows aberrant mitochondrial morphology and severely decreased complex V immunoreactivities. Conclusion: Deficiencies of complex V either from mtDNA or nuDNA origin can efficiently be identified by combining several diagnostic techniques. Background: MELAS syndrome is one of the most common mitochondrial disorders. Mitochondrial proliferation may result in endothelial dysfunction and decreased nitric oxide (NO) availability leading to stroke-like episodes. This study aimed to assess NO production in adults with MELAS and the effect of oral supplementation with the NO precursors arginine and citrulline. Methods: Using stable isotope infusion techniques, we measured NO synthesis, amongst other variables, in healthy adults and subjects with MELAS before and after arginine or citrulline supplementation. Results: Adults with MELAS had lower NO synthesis rate, plasma arginine and citrulline concentrations, citrulline flux, and de novo arginine synthesis. In subjects with MELAS, arginine and, to a greater extent, citrulline supplementation increased the de novo arginine synthesis rate, the plasma concentrations and flux of arginine and citrulline, and NO production. Conclusions: The lower NO production in subjects with MELAS is primarily due to decreased de novo arginine synthesis secondary to decreased citrulline availability. De novo arginine synthesis increased markedly with citrulline supplementation, explaining the superior efficacy of citrulline in increasing NO production. The improvement in NO production with arginine or citrulline supplementation supports their use in MELAS syndrome and suggests that citrulline may have a better therapeutic efficacy. There are to date very few treatments for mitochondrial Respiratory Chain (RC) disorders and the management of patients remains largely supportive. In many cases, the RC defect is not total and some residual enzyme activity level can be measured. Thus, a therapeutic strategy aimed at stimulating residual capacities could correct partial RC deficiencies. In this context, one possible approach is to activate key factors capable to up-regulate the RC, using specific pharmacological agents. In line with this, we investigated the potential of resveratrol (RSV), a natural polyphenol compound, known to activate PGC-1alpha (Peroxisome proliferatoractivated receptor Gamma Coactivator-1 alpha), to stimulate the RC. The effects of RSV were tested in fibroblasts of controls and of five Complex I (CI) deficient patients harboring mutations in 3 nuclear genes encoding CI subunits (NDUFV1, NDUFV2 and NDUFS3). Spectrophotometric experiments indicated that exposure to RSV (75 microM, 72 h) increased the CI residual enzyme activity in controls (+30%) and in 4 out of 5 CI-deficient cells (+20 to +78%). Western-blot analysis showed that RSV significantly increased (+45 to +100%) the expression levels of mutated proteins in 4 out of 5 deficient cell lines. Finally, preliminary experiments suggest that these RSV effects might involve a mitochondrial biogenesis. Background and objectives: We recently reported on 24 statistical significant urinary organic acids from children with RCDs, disclosed through a metabolomics investigation (Reinecke, et al, Metabolomics, 2011-doi: 10 .1007/s11306-011-0309-0). The objectives here are: (1) to describe the outcome of a metabolomics investigation of urinary amino acids and acyl-carnitines from RCD patients and (2) to propose a putative biosignature for RCDs. Material and Methods: The urinary samples came largely from a previous patient group (Smuts, et al, J. Inherit. Metab. Dis., 2010-doi:10 .1007/ s10545-009-9031-8). The methods were: GC-MS analysis for the amino acids and tandem-MS for the acyl-carnitines. Statistical analysis included standard univariate analyses as well as principal component (PCA), partial least-square discriminant (PLS-DA) and a new method of concurrent class (CONCA) analyses. Results and Conclusions: From the metabolomics analysis a putative biosignature could be formulated which comprised of 16 metabolites: lactic acid, two Krebs-cycle intermediates, four organic acids from fatty acid catabolism, seven amino acids and two acyl-carnitines. This biosignature should still be validated with more RCD cases, but is already implementable as a screening instrument to establish the need for biopsying patients for confirmative enzyme analyses on RCDs as well as for monitoring of treatment. Background: Mutations in the RRM2B gene encoding the ribonucleotide reductase (RNR) p53R2 subunit were initially reported to cause childhoodonset mitochondrial DNA (mtDNA) depletion syndrome. Objectives: To determine the frequency of RRM2B mutations in adults with multiple mtDNA deletions, and to report the association of RRM2B mutations with Kearns-Sayre syndrome (KSS). Patients and Methods: RRM2B gene sequence analysis was performed in 50 adult patients with multiple mtDNA deletions in skeletal muscle, in whom mutations in known mtDNA maintenance genes (POLG and C10orf2) had been excluded. Functional studies of RNR protein included Blue-Native gel electrophoresis (BN-PAGE) and Western blot analysis. Results: A patient with KSS had two novel missense mutations in RRM2B. BN-PAGE demonstrated reduced heterotetrameric R1/p53R2 RNR levels, despite normal p53R2 levels on Western blot, suggesting failed assembly of functional RNR as a potential disease mechanism. Another patient, who presented with late-onset progressive external ophthalmoplegia and fatigue, had a heterozygous 3 bp deletion in RRM2B. Conclusions: 4% of our cohort had RRM2B mutations. KSS has not previously been linked to nuclear gene defects; we now show that disease pathogenesis may be caused by defective RNR assembly. RRM2B mutations should be considered in the differential diagnosis of multiple mtDNA deletions presenting in adults. Background: Complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) defect. X-linked AIF-deficient Harlequin (Hq) mouse is a faithful model for OXPHOS defect, with tissues-specific CI defect and phenotype closely resembling that of CI deficient-patients. No therapeutic solutions are available except "neutraceuticals" among which high-fat diet (HFD) has been proposed but never evaluated. Objectives: To evaluate the impact of HFD on disease progression in CIdefective mice. Methods: Hemizygous (Hq/Y) males were obtained by mating Hq/X females with control males and the diets given from weaning. Control diet was 8 KCal% in fat and isocaloric HFD 30 Kcal%. From one to six months, animals were monthly evaluated using an extensive battery of phenotype characterization: rotarod (indirect cerebellar ataxia evaluation), grip-test (muscular strength) and hindlimb clasping (HL, neurodegeneration marker). Results: HFD-treated Hq mice exhibited a significantly less severe loss of rotarod scores from one to six months (45 points of loss on average, n=21) when compared to Hq mice fed the control diet (100 points of loss, n=20). There was also a tendency to better HL-scores in HFD-fed mice. Conclusion: Isocaloric HFD slows down clinical neurodegeneration in CIdefective mice. This strongly favors its use in CI-defective patients. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common disorder leading to lactic acidemia. Phosphorylation of specific serine residues of the E1-alpha; subunit of the PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We recently found that phenylbutyrate prevents phosphorylation of the E1-alpha subunit of the branched-chain ketoacid dehydrogenase complex (BCKDC) and reduces plasma concentrations of neurotoxic branched chain amino acids in patients with maple syrup urine disease (MSUD), due to the deficiency of BCKDC. We hypothesized that, similarly to BCKDC, phenylbutyrate enhances PDHC enzymatic activity by increasing the portion of unphosphorylated enzyme. To test this hypothesis, we treated wild-type human fibroblasts at different concentrations of phenylbutyrate and found that it reduces the levels of phosphorylated E1-alpha; as compared to untreated cells. To investigate the effect of phenylbutyrate in vivo, we administered phenylbutyrate to C57B6 wild-type mice and we detected a significant increase in Pdhc enzyme activity and a reduction of phosphorylated E1-alpha; subunit in brains and muscles as compared to saline treated mice. Being a drug already approved for human use, phenylbutyrate has great potential for increasing the residual enzymatic activity of PDHC and to improve the clinical phenotype of PDHC deficiency. This study aimed to show feasibility of using the phosphorescence oxygen analyzer to screen for disorders with impaired cellular bioenergetics.
[O2] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, oxygen consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics. The rate of respiration (mean ±SD, in μM O2 per min per 107 cells, set as the negative of the slope of [O2] vs. time) for adults was 2.1±0.8 (n=18), for children 2.0±0.9 (n=20), and for fetuses 0.8±0.4 (n=18), p <0.0001. For an 8-year-old patient with reduced muscle NADH dehydrogenase and pyruvate dehydrogenase activities, the rate was 0.7±0.2 (n=3) μM O2 per min per 107 cells. For a 3-month-old patient with hepatocerebral mitochondrial DNA depletion syndrome (confirmed mutation in the MPV17 gene), the rate was 0.6 μM O2 per min per 107 cells. For two siblings with reduced cytochrome oxidase on muscle neurochemistry, the rates were 5.5 and 3.0 μM O2 per min per 107 cells. This novel approach allows non-invasive (preliminary) assessment of cellular bioenergetics. Background: The process of oxidative phosphorylation (OXPHOS) establishes a mitochondrial membrane potential gradient (DY) which is utilized by ATP synthase to generate cellular energy. Methods: We developed a microscopic method to evaluate DY in cultured fibroblasts using the fluorescent compound JC-1. We tested our method on controls and OXPHOS deficient cell lines. Results: DY was significantly lower in fibroblasts from patients with isolated and combined OXPHOS complex I to IV defects, while DY values were maintained in patients with ATP synthase defects. DY reduction correlated with severity of the defect and with the percentages of heteroplasmic mtDNA mutations. Cell lines displayed differential vulnerability to rotenone. Also, this complex I inhibitor induced perinuclear mitochondrial relocalization accompanied by cytoskeletal changes. In our set-up, the complex IV inhibitor KCN had not such an effect but caused cell death when used in high concentrations. The method was found especially efficient to visualize the heterogeneity of the defect in cells from patients with heteroplasmic mtDNA mutations. Conclusions: We found that JC-1 fluorescent microscopy allows to detect and differentiate OXPHOS defects and thus may be employed to study fibroblasts from suspected patients. Background: Mitochondrial disorders are a group of complex diseases that can be caused by mutations in nuclear or mitochondrial genomes. Current molecular diagnosis requires multiple different and complementary methods, including sequencing, qPCR, Southern blot or array CGH, for the detection and quantification of mutations. These procedures are labor intensive, time consuming, and costly. Methods: We have developed and validated a "deep" coverage Next Generation Sequencing (NGS) technique in clinical settings. This approach allows simultaneous analysis of a set of nuclear genes targeted to mitochondria and the whole mitochondrial genome for point mutations and deletions. We instituted proper qualitative and quantitative controls to be analyzed along with each sample for quality assurance. Results: We demonstrated an average coverage of >500X for targeted nuclear genes and >5000X for each of the 16,569 bases of the mitochondrial genome. Nucleotide changes are correctly called with quantitative information. The limit of detection of a heteroplasmic change is calculated to be about 1.5%. Small and large insertion/deletions were correctly detected with clear breakpoints and percentage of heteroplasmy. Conclusion: Our "deep" sequencing approach provides a one-step comprehensive molecular analysis for patients with suspicion of mitochondrial diseases in a timely, accurate, and cost-effective manner suitable for clinical application. Both showed an elevated level of citrulline in serum or whole blood. The ACOMG recommends considering the next disorders, as for further investigation of elevated Citrulline levels in the NBS test: Citrullinemia Types I&II, Argininosuccinic Aciduria & Pyruvate Carboxylase Deficiency. Since introduction of Citrulline level measurement in blood, as part of the NBS tests in Israel, 5 samples were found abnormal (above 40 mmol/Ltr), of which 1 was found to be linked to E3 deficiency (thanks to Dr. Almashanu for the data). Data is being gathered from the files of thirty E3 deficienct patients. The citrulline level of each patient is then weighed in relation to time of diagnosis & metabolic crisis, if had been present. Possible Conclusions: We highly suspect that elevated Citrulline in the NBS tests or serum analysis test for amino acids may mark a defect in the Citric Acid Cycle, such as E3 deficiency. A reasonable mechanism may resemble that for elevated citrulline in Pyruvate Carboxylase Deficiency: a decline in Aspartate production, as a result of a blockage in the transformation of Pyruvate to Oxaloacetate. Objective: Fetal infection is associated with considerable perinatal morbidity. As the umbilical vein regulates fetal blood flow from the placenta dysfunction of human umbilical venous endothelial cells (HUVEC) may compromise fetal blood supply. We incubated HUVEC with one of the key inflammatory mediators, LPS (Lipopolysaccharides), and measured the activity of respiratory chain enzymes. Material and Methods: Based on gestational age, HUVEC were divided into 1 mature and 2 premature groups from otherwise uncomplicated pregnancies (4 patients each) and incubated for one hour, six or twelve hours with LPS (100 ng/ml), respectively, incubation without LPS served as control. We analyzed the activity of respiratory chain enzymes and citratesynthase as a mitochondrial marker spectrophotometrically. Results: No significant differences in respiratory chain activities between the LPS-incubated cells and internal controls could be observed. However, we could demonstrate significant differences depending on gestational age: respiratory chain complexes C I+III (p<0.05) and C IV (p<0.01) showed higher activity at lower gestational age. No such differences were observed for C II+III, C V and citratesynthase. Conclusion: LPS on its own had little influence on the activities of mitochondrial respiratory chain enzymes. However, complex I+III and C IV activities are increased at lower gestational age. The most recent technological developments allows novel tools for biomarkers identification in mitochondrial respiratory chain deficiencies (RCD) through metabolome analysis. Using this approach, it has recently been shown in RCD that an extracellular creatine excess is in relation to intracellular P-creatine depletion. Furthermore, authors found that plasma creatine, appears to be the most specific and sensitive metabolite in RCD patients, exceeding lactate and alanine in magnitude of elevation and statistical significance (Shaham et al, PNAS 2010) . In order to confirm this single report, we tested plasma creatine as potential biomarker of mitochondrial dysfunction in samples obtained from 30 patients aged between 0.2-19 yrs. with pathogenic mutation (10 patients) or abnormally low RC enzyme activity in muscle (20 patients). Data showed that plasma creatine concentrations was significantly higher in mitochondrial patients (mean±SEM=81.1±3.2 vs. 57.7±2.0; p<0001) when compared with age-matched controls. In 8/30 plasma creatine clearly exceeded the upper control limit (97th percentile), and 4 of them had normal levels of plasma lactate and /or alanine on the same blood sampling. Our study confirms that plasma creatine is elevated in RCD and can be used in combination with other biomarkers for the diagnosis of mitochondrial diseases. mtDNA depletion syndrome (MDS) includes a group of autosomal disorders, generally recessive, characterized by a severe reduction in cellular mtDNA content in affected tissues. MDS is tissue-specific, affecting mostly tissues with higher energy demanding. As the cardiac muscle is one of the most energy requiring tissues, we aimed to establish control values for mtDNA copy number in myocardium in patients with cardiac involvement. We have analyzed mtDNA copy number in 14 samples of myocardium from paediatric patients (age: 4 days -11 years old) with possible mitochondrial OXPHOS disease (multiple OXPHOS deficit in 13 cases) and/or heart involvement. Total DNA was extracted by standard methods. The common mtDNA mutations were excluded and mtDNA copy number was determined by real-time PCR, using SYBR green and specific primers for target genes. The intensity of fluorescent signals was analyzed by 7500 Software v2.0.4 and relative mtDNA content was calculated by deltaCt. We performed a normal distribution and estimate 21% of cases (median 594, min-max 40-625, compared to 1293, min-max 1039-6474 in all other samples) with mtDNA depletion. The present work is an important contribution for gathering reference values in myocardium samples, given the scarcity of data in literature, namely in children. In 322 samples out of 921 muscle biopsies measured in Klinikum Schwabing between 2005 and 2009 a defect of respiration chain was found (35.0%). Isolated complex I defect was detected most frequently (118=36.6%), followed by combined complex I and IV defect (66= 20.5%). In 139 out of the 701 genetically investigated samples a positive result was found. In 35 cases (25.2%) mtDNA deletions and in 29 cases (20.9%) a depletion of mtDNA was detected. Interestingly within the 20 deletion positive cases also measured for respiratory chain activity in 13 cases no defect was detected (65 %). In contrast, within the 19 depletion positive samples measured for respiratory chain in only 3 cases no defect was found (16 %). In 253 samples genetic testing was performed initially without investigation of the respiratory chain activity due to clinical symptoms or histological findings. In 72 (28.5%) of these cases a diagnosis could be established.
Our results show the high value of the muscle biopsy in the elucidation of mitochondrial disease. However a normal respiratory chain activity in skeletal muscle can not rule out mitochondrial disease. From our experience testing for mtDNA deletions or depletion should be the initial step. Background: Barth syndrome is an X-linked genetic disorder presenting with cardiac and skeletal myopathies and caused by inborn errors in the tafazzin gene, resulting in defects in cardiolipin (CL) metabolism. CL is a unique mitochondrial phospholipid that is essential for assembly of electron-transport chain (ETC) and multi-enzyme complexes. The objective of this study is to elucidate the involvement of CL in the interaction of fatty acid oxidation (FAO) enzymes with mitochondrial ETC complexes. Methods: We investigated the effects of CL-deficiency on mitochondrial trifunctional protein (mTFP) interaction with ETC complex I in cardiac muscle of tafazzin knockdown mice. Results: Cardiolipin deficiency resulted in cardiac dysfunction, mitochondrial abnormalities, excessive autophagy and impaired energetics in tafazzin-knockdown mice. We found that cardiolipin-deficiency destabilizes the interaction of mTFP with ETC complex I. Analysis of extracellular fluxes revealed impaired utilization of palmitate as an energy substrate for mitochondrial FAO in tafazzin-deficient cardiomyocytes. Conclusion: Cardiolipin is essential for the normal physical association of TFP with ETC Complex I in cardiac mitochondria. Dissociation of TFP from ETC Complex I adversely affects the efficiency of fatty acid oxidation and aerobic energy production in heart. Impaired FAO may be one of the many pathogenic factors in Barth syndrome patients. Background: Barth syndrome (BTHS)(OMIM302060)or 3-Methylglutaconic aciduria type II, is a rare x-linked recessive disorder. It characterized by dilated cardiomyopathy, skeletal myopathy, cyclic neutropenia and elevated 3-methylglutaconic and 3 methylglutric acide in the urine. Variable clinical phenotypes being reported. Methods: Total of 3 patients, presenting with the constellation of findings suggestive of BTHS. Two had early evaluation by fetal echocardiogram (Echo).The diagnosis in all cases confirmed at the biochemical (urine gas chromatography-mass spectrometry (GC/MS)) and molecular level (TAZ gene sequencing).Periodic complete blood count, lactic acid, and Echo were performed. Results: Two families with 3 affected children; two patients were picked up early by abnormal fetal echocardiogram. The third presented with recurrent infection in infancy and failure to thrive. Subsequently all were found to have cardiomyopathy, cyclic neutropenia and the characteristic urine organic acid changes. Molecular analysis revealed that affected patients and their mothers were having hemizygous and heterozygous changes respectively in Taz gene. Conclusions: This is the first report of Saudi patients with Barth syndrome and their genotype. Early detection of cardiomyopathy by fetal echocardiogram and high index of suspicion would prevent unnecessary investigations and aid to better management in patients with BTHS. We report a 12 year old boy, who presented at 3 months of age with intractable seizures, optic atrophy, hypertonia, hyperreflexia in the lower extremities with clonus, and areflexia in the upper extremities. Family history was normal. Progressive cystic changes of the white matter noted on sequential MRIs was classified as a neurodegenerative spongiform leukoencephalopathy of unknown etiology. MRS demonstrated a large lactate peak and low NAA. Over the next 5 years, development was static, limited to smiling responsively. Muscle biopsy was done despite the MRI changes not considered classical of mitochondrial disease. Complex I deficiency was demonstrated and sequencing for known complex 1 genes available at that time including NDUFS 3, 4, 6, 7 failed to delineate the molecular etiology. A healthy brother was born unaffected suggesting recessive rather than maternal inheritance. In 2010, molecular analysis of NDUFV1 was pursued and the patient was found to have two heterozygous mutations: one previously reported in homozygous state in two siblings with similar presentations and MRI changes. The second mutation is a novel splice site mutation. This case report illustrates the importance of considering Complex 1 disease and in particular NDUFV1 molecular analysis in children with spongiform leukoencephalopathy. We report on two siblings, born to consanguineous Pakistani parents, with Sengers syndrome. The proband presented at 15 months of age following a cardio-respiratory arrest and was found to have hypertrophic cardiomyopathy with severely impaired cardiac function requiring ECMO support. The history revealed bilateral cataract surgery at 3 months and gross motor delay noted at 12 months. After assessment for cardiac transplantation eligibility, cardio-respiratory support was withdrawn. The mother was 32 weeks pregnant at the time and delivered a newborn male, noted to have bilateral cataracts at 1 week of age, lactic acidosis and hypertrophic cardiomyopathy at 8 weeks of age. The histochemical studies in the proband showed scattered COX negative fibers (previously unreported in Sengers syndrome) and pleomorphic mitochondria with abnormal structure. Respiratory chain complex activities were atypically all severely reduced. Histochemical studies in the sibling showed normal COX staining, but an increased number of morphologically abnormal mitochondria. The sibling had reduced respiratory chain complex activities reminiscent of, but less severe than, the proband. The sibling has reached 15 months of age and has had few symptoms, although his cardiomyopathy is progressive. This case report illustrates intra-familial variability of histochemical findings for two siblings with the same clinical phenotype. year-old girl, born to nonconsanguine Chinese parents, presented with global developmental delay within the first 6 months of life. She was evaluated for sensorial-neural hearing loss, ataxia, ophthalmoplegia and muscle weakness at age 3 years. In her physical examination, her growth parameters were at 50th percentile. There was no organomegaly. Her muscle tone was decreased. Deep tendon reflexes were not elicitable. She had marked ataxia and marked restriction in all gaze directions of each eye. Peripheral nerve conduction study showed generalized sensory-motor neuropathy. Her cranial MRI and MRspectroscopy were normal. Her liver function tests were normal. She had novel compound heterozygous (c.1279 G>A; p.E427K and c.1838A>G; p.D613G) missense mutations in the TWINKLE Helicase (C10orf2) gene. Conclusion: This is a patient with early onset mitochondrial encephalomyopathy caused by mitochondrial depletion in the TWINKLE Helicase (C10orf2) gene. Background: Mitochondrial complex I assembly is an intricate process which involves incorporating 45 subunit proteins, 1 flavin mononucleotide and 8 iron-sulphur clusters into the final~980 kDa holoenzyme. Mutations in any of the 45 subunits or in an unknown number of assembly factors and chaperones may cause complex I deficiency.
Objectives: To facilitate genetic screening in complex I deficiency by implementing biochemical and genetic approaches. Patients and Methods: 30 complex I deficient paediatric patients with heterogeneous clinical phenotypes were studied. Complex I deficiency and assembly state were analysed using spectrophotometric assay and Blue Native gel electrophoresis. Candidate gene analysis and homozygosity mapping were used to search for the underlying gene defect. Results: Abnormal complex I subassemblies accumulated in 3 cases: defects in NDUFS4 and a known assembly factor were causative in two cases, whereas the third defect is still unknown. Homozygosity mapping and bioinformatics analysis helped to identify a mutation in the novel complex I chaperone FOXRED1 in a fourth case. Conclusion: By using a combined biochemical and genetic approach we have found the underlying molecular defect in 13/30 [43%] of this complex I deficient patient cohort; further studies are ongoing to identify the genetic defects in the remaining patients. Background: Complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) defect. No therapeutic solutions are available except "neutraceuticals" among which high-fat diet (HFD) has been proposed but never evaluated. Objectives: To evaluate the effect of the long-chain fatty acid palmitate on human CI-deficient fibroblasts viability. Methods: Fibroblasts were obtained from three CI-defective patients genetically identified: two with NDUFS1 mutations and one with NDUFS4 mutations. They were cultured either in glucose-and glutamine-enriched DMEM, or in glucose-free, glutamine-enriched DMEM or in glucose-free, glutamine-, palmitate-enriched DMEM. Cell viability was evaluated daily using an automated cell counter. Succinate oxidation was assessed by polarography. Complex II activity was measured by spectrophotometry. Results: In one of the NDUFS-1 mutated cell line, death occurred within 80 hours after glucose withdrawal with full protection afforded by palmitate. In the two other CI-defective cell lines, death occurred within 170 hours after glucose withdrawal with full palmitate protection. The full protection from death provided by palmitate was associated with a slight but consistent increase (20-30%)in respiratory chain complex II activity. Conclusion: These data show that palmitate protects CI-deficient fibroblasts from death and provide a strong rationale for performing HFD trials in CI-deficiency. Complex I (CI) deficiency is a defect of OXPHOS caused by mutations in the mitochondrial or nuclear genomes. To date disease-causing mutations have been reported in all mitochondrial-encoded subunits and 21 nuclear genes. In about 50% of the patients no mutations are found, suggesting that undiscovered factors are an important cause of disease. In this study we report a consanguineous family from Southern Portugal with three affected children where CI deficiency could not be clarified. The affected children presented similar clinical findings with early onset of the disease having two of the patients a fatal outcome. A reduced activity of CI was detected in two of the patients, which led us to investigate, at the molecular level, some CI associated genes. However, no mutations were detected. Interestingly, all patients presented 3-methylglutaconic acid in the urinary organic acids and it is known that POLG gene is involved in the etiology of these syndromes. In only one of the patients, that also showed mtDNA depletion, the p.G848S/p.Q1236H mutations were found in POLG. It remains unsolved if this family due to the high consanguinity could have two different disorders or if a yet unknown gene, leading to complex I deficiency, could be involved. The role of a secondary respiratory chain deficiency as an additional mechanism to intoxication, leading to development of long-term energydependent complications, has been recently suggested in patients with propionic acidemia (PA). We show for the first time a coenzyme Q10 (CoQ10) functional defect accompanied by a multiple organ oxidative phosphorylation (OXPHOS) deficiency in a child who succumbed to acute heart failure in the absence of metabolic stress. Quinone-dependent activities in the liver (complex I+III, complex II+III) were reduced, suggesting a decrease in electron transfer related to the quinone pool. The restoration of complex II +III activity after addition of exogenous ubiquinone to the assay system suggests CoQ10 deficiency. Nevertheless, we disposed of insufficient material to perform direct measurement of CoQ10 content in the patient's liver. Death occurred before biochemical diagnosis of OXPHOS deficiency could be made. However, this case highlights the usefulness of rapidly identifying CoQ10 defects secondary to PA since this OXPHOS disorder has a good treatment response which could improve heart complications or prevent their appearance. Nevertheless, further studies will be necessary to determine whether CoQ10 treatment can be useful in PA complications linked to CoQ10 deficiency.
Background: Coenzyme Q10 (CoQ10) deficiency is a rare but often treatable condition. In view of the encephalopathic presentation of this condition the aim of this project was to establish a neuronal cell model of CoQ10 deficiency in cultured neuroblastoma, SHSY-5Y cells using paraaminobenzoic acid (PABA) treatment. Methods: Treatment of SHSY-5Y cells with 1 mM PABA induced a maximal 54% (46% residual CoQ10; p<0.01) decrease in celluar CoQ10 status compared to control levels.
Results: A progressive decrease in complex I, II/III and IV activities was noted with a maximal inhibition observed at 46% residual CoQ10 of 50-59%. ATP production also decreased by 67.5% in comparison to control. Residual CoQ10 levels of 77% and 46% resulted in an approximately 4x increase in mitochondrial oxidative stress. A 25% decrease in mitochondrial membrane potential was observed at 77% residual CoQ10. Conversely a 40% increase was observed at 46% residual CoQ10 compared to the controls suggesting a possible reversal of ATP synthase (complex V).
Conclusions: This cellular model has provided insight into the effect of CoQ10 deficiency on neuronal ETC function and oxidative stress and will enable the efficacy of potential treatments to restore mitochondrial function. This project is funded by Ataxia UK (www.ataxia.org.uk). Objective: coenzyme Q10 (CoQ10) deficiency due to mutation in the COQ2 with a new clinical presentation. Case report: At the age of 3 weeks, patient developed myoclonic seizures. Both plasma and CSF lactate were increased. Brain MRI was normal and muscle biopsy performed at 2 months of age did not disclose morphological or OXPHOS abnormality. Despite supplementation with riboflavin, biotin, pyridoxine and CoQ10 and several anti-epileptic drugs he continued to have partial and myoclonic seizures, evolving into epilepsia partialis continua. At 5 months he developed a nephrotic syndrome and CoQ10 was increased to 30 mg/kg /day but he expired. Results: Renal biopsy showed focal segmental glomerulosclerosis and on ultrastructural examination enlarged podocytes with hyaline cytoplasmic vacuoles. Respiratory chain activities of renal cortex demonstrated decreased activities of complexes II+III. A homozygous mutation c.326 G>A (p. Ser109Asn) in exon 2 of CoQ2 gene was found. Both parents were heterozygous carriers. CoQ10 concentration in fibroblasts was 16nmolCoQ/gr prot (mean 67). Conclusion: In contrast to previous reports of COQ2 nephropathy, in this case, kidney was not the only target. Therefore, this report extends in COQ2 mutation causing primary CoQ10 deficiency, the spectrum of the phenotype of severe early myoclonic epilepsy, subsequently nephrotic syndrome. 
We performed an open-label prospective study: patients were evaluated clinically (ICARS scale, MRI and video-tape registration) at baseline and every 6 months during a period of 2 years after CoQ10 treatment (30 mg/Kg/day). Results: Patients with CoQ10 deficiency showed a statistically significant reduction of ICARS scores (Wilcoxon test: p=0.018) after 2 years of CoQ10 treatment as compared to baseline conditions. In patients without CoQ10 deficiency, no statistically significant differences were observed in total ICARS scores after therapy, although one patient from this group showed a remarkable clinical amelioration. Conclusions: Biochemical diagnosis of CoQ deficiency was a useful tool for the selection of patients who are good candidates for treatment, since all of them responded to therapy. However, the remarkable clinical response in one case without CoQ deficiency highlights the importance of treatment trials for identification of patients with CoQ-responsive ataxia. We report the case of a 32 years woman in whom an exercise test as been useful for the diagnosis of mitochondrial related myopathy and for the evaluation of the treatment efficiency. This patient presented abnormal fatigability who was later shown to be related with a multiple respiratory chain deficiency on muscle biopsy. An homoplasmic mutation (m.8322 T>C) in the tRNALysine gene has been found in this patient. Initial incremental exercise test demonstrated a pathological increase in peak serum lactate (9251 μmol.L-1), consistent with a predominant glycolytic anaerobic activity. Following the diagnosis confirmation (muscle biopsy, genetic) a treatment by L-Carnitine and Co-Q10 was initiated. A second exercise test was performed 4 months later (same protocol). It showed an increase in aerobic capacity, resulting in a greater maximal work rate exercise (96% vs 85% of predicted), higher oxygen uptake at peak (96% vs 89%) and at anaerobic threshold (71% vs 51%). In contrast, no change in peak serum lactate level was assessed. Larger studies are needed to confirm whether such treatment could lead to significant improvement of exercise performance. Mitochondrial diseases are a type of neurodegenerative disorders which are associated with mitochondrial respiratory chain deficiency in muscles and in a wide variety of tissues. They are caused by mutations in nuclear or mitochondrial genome, what leads to extremely heterogeneous clinical manifestation of these diseases. A dramatic example of such disorder is Leigh syndrome which mostly has onset at first years of life and frequently induced by mutations in SURF1 gene or T8993C/G substitution in mitochondrial DNA (mtDNA). However there's a wide range of patients with rare mutations in mtDNA. The etiology of a mutation and a proportion of mutant mtDNA in a cell define the severity of a disease. Thus, point mutations in mtDNA can lead to a clinical polymorphism, thereby complicating the finding of a causative mutation. We performed mtDNA sequence analysis of a group of patients (c. 50) with Leigh/Leighlike phenotype. A total 8 different substitutions were identified. Among these, 3 unknown mutations (C3945A, G8839C, T14441C) were found. Also 5 other mutations were identified: G3697A, T8362G, G8363A, T13094C, G13513A. On a base of our results we've developed a specific primers panel for MLPA analysis which allows us to hasten a search of mutations in mtDNA.
Gavrilova RH 1 , Gurrieri C 1 , Weingarten TN 1 , Sprung J 1 1 Mayo Clinic, Rochester, United States MELAS could progresses to multiorgan pathology and early death. Impaired integration of pyruvate into Krebs cycle predisposes to lacticademia and increased perioperative risk. Controversy exists regarding increased susceptibility to certain anesthetics and malignant hyperthermia. Avoidance of lactated intravenous fluids (LR) was advocated because of impaired lactate metabolism. We describe outcomes of MELAS patients who underwent surgery at Mayo Clinic, USA. Perioperative data of confirmed MELAS patients were retrospectively characterized. 9 patients underwent anesthesia for 20 surgeries: general anesthesia with volatile anesthetics in 14; propofol-3; NMDA-14; succinylcholine-4. Lack of need for excessive amounts NMDA was not suggestive for resistance. Recovery from NDMR blockade appeared uneventful. Most patients were receiving levetiracetam/topiramate which do not alter response to NMDA. One had severe myopathy, succinylcholine was not used. MH triggering agents were routinely used without evidence of MH. Intraoperative LR in 13 cases did not result in acid-base decompensation. MELAS patients did not show aberrant response to muscle relaxants and to anesthesia management in this study. Response to muscle relaxants may depend on interplay between medications/antiepileptics and myopathy. NMDA should be dosed with aid of neuromuscular monitoring. Alterations of acid-base balance not encountered despite administration of LR. Background: Kearns-Sayre syndrome (KSS) is a mitochondrial disorder presenting as main biochemical findings high CSF protein and low 5methyltetrahydrofolate (5-MTHF) values, which reflect impaired transport across choroid plexus. Other compounds may also be affected. Our aim was to assess different biochemical parameters that may detect choroid plexus dysfunction in KSS patients. Methods: We studied 7 patients genetically diagnosed with KSS. The measured parameters in CSF were total proteins, 5-MTHF, homovanillic acid (HVA) and Selenium (Se) concentrations. Results: Together with high CSF Se values, increased CSF HVA and total protein concentrations and decreased CSF 5-MTHF values were observed in all cases. This pattern was only detected in the 7 KSS patients of 1,850 CSF samples analyzed in our laboratory during the last 6 years. Conclusions: The application of these biochemical analyses may allow early identification of new cases with undiagnosed KSS. These impaired metabolites seem very specific and may represent a good biochemical model for evaluating choroid plexus dysfunction. The accumulated Se in CSF might have cause toxicity effects or increased dopamine turnover. This last feature would be reflected by increased CSF HVA (marker of dopamine turnover). The association between Se and HVA and its possible clinical implications deserve further investigation. Human cytochrome c oxidase (COX) contains two copper centers CuA and CuB that are essential for enzyme catalytic activity. Aim of our study was to analyze copper content and COX activity and amount in muscle, liver, heart and brain tissue obtained at autopsy from 17 children with mutations in SURF1 (7x), SCO2 (9x) and SCO1 (1x) genes. Copper content was analyzed by atomic absorption spectrometry. COX activity and amount were analyzed by spectrophotometric and immunoelectroforetic methods. The copper content was more than 4-fold reduced in SCO2 and SURF1 liver in comparison with age related controls and more than 3-fold in SURF1 skeletal muscle. The severe copper deficiency of SCO1 muscle (5fold) was accompanied by 6-fold decrease of COX activity. The SCO2 heart showed 3-fold reduction of copper content and 10-fold reduction of COX activity. The SCO2 and SURF1 frontal cortex specimens displayed either normal or borderline copper levels although all of the SCO2 and SURF1 patients suffered from severe CNS involvement. While the copper deficient phenotype of SCO1/SCO2 tissues appears consistent with the function of both gene products in regulation of cellular copper homeostasis, the severe copper deficiency of SURF1 deficient tissues remains elusive. Supported by MSM0021620806, IGA-MZ-NS 10581/3, GAUK28410 In a family three children presented with severe neonatal lactic acidosis, hypertrophic cardiomyopathy and generalized muscular hypotonia. One child died in infancy, two survived a clinically severe neonatal period. At an age of 9 and 17 years, respectively, they present with exercise intolerance, proximal muscle weakness, non progressive hypertrophic cardiomyopathy and normal mental development. In a muscle biopsy normal activity of respiratory chain enzymes were found, however the amount of the mitochondrial phosphate carrier was decreased. This protein is expressed in two tissue-specific isoforms generated by mutually exclusive alternative splicing of the SLC25A3 gene transcript. We identified a homozygous mutation c.158-9A>G located in the 5'-intron next to exon 3A specific for heart and skeletal muscle. This creates a novel splice site resulting in a more than 95% decrease of the wild type allele. Although early onset mitochondrial liver disease is often rapidly fatal, there is spontaneous reversal in a small number of cases and these have a good long term prognosis. Identification of causative genetic defects in these patients has an important bearing on management decisions. A female neonate presented on day 6 with poor feeding, weight loss, jaundice and lactic acidosis. Serum transaminases were elevated and there was a coagulopathy. Liver histology showed distorted architecture and necrosis. Cytochrome oxidase activity in liver and muscle was profoundly reduced. However, by 3 months, she was progressing well and liver function, blood lactate and muscle cytochrome oxidase activity had all normalised. She is now developing appropriately. Two pathogenic mutations, c.835 G>A (V279M) and IVS11-3, C>G, were identified in the TRMU gene. This gene encodes an enzyme involved in mitochondrial tRNA modification and was initially recognised as a modifier of deafness due to the mtDNA A1555G mutation. Recently a small number of infants have been reported with mutations in this gene and reversible liver disease. It is therefore important to screen the TRMU gene in patients with early onset liver disease and lactic acidosis to identify those who will be expected to have a benign course. Deficiency of the pyruvate dehydrogenase E3 binding protein (E3BP) was identified in two sisters, the offspring of consanguineous Portuguese parents. The sisters, currently aged 17 and 27 years, both have episodic weakness and painful dystonic posturing of the torso and limbs associated with intercurrent illnesses. In addition, the older sib is prone to selfmutilation and aggressive outbursts, whilst the younger girl has an anxiety disorder. Dichloroacetate and a ketogenic diet appeared to be of some benefit, but triheptanoin oil was not tolerated. Both sisters have nonprogressive moderate to severe learning disability and modest elevations of blood and CSF lactate. Fibroblast pyruvate dehydrogenase activity was significantly reduced in both girls and there was a homozygous base substitution in the splice acceptor site of intron 7 of the PDHX gene, resulting in skipping of exon 8 and generation of a premature stop codon. Patients with complete E3BP deficiency have some residual enzyme activity, which may allow prolonged survival, although not usually to the extent seen in these sisters. A dystonic movement disorder developing during childhood is increasingly being recognised as a major manifestation of less acute presentations of pyruvate dehydrogenase deficiency, particularly those involving the E2 and E3BP subunits of the complex. Pyruvate dehydrogenase complex (PDC) deficiency is one of the most common neurodegenerative disorders associated with abnormal mitochondrial metabolism. The key feature is gray matter degeneration with foci of necrosis and capillary proliferation in the brainstem. Lactic acidosis with normal/low Lactate/Pyruvate is the main biochemical marker. The most common cause is mutations in the X-linked E1 alpha gene (PDH1 gene). Two girls with mutation c.904 C>T in the 10th exon of PDH1 gene have been diagnosed in Estonia. Before enzymatic/mutation analysis, organic and amino acids analysis in serum and urine were performed to determine lactate, pyruvate and alanine. For both analysis in serum protein was precipitated using 10% sulfosalicylic acid (0.25 ml for 1 ml of serum). Lactate and pyruvate in serum were elevated: 3400/372 μmol/l and 4060/ 1152 μmol/l (normal 3300/160 μmol/l). L/P was relatively low (< 10). Alanine in serum was elevated only in pt. I-829 μmol/l (N<495). In urine lactate, pyruvate and alanine were elevated in pt. II-4209 (N<151), 5146 (N<130) and 358 (N<254) mmol/mol creatinine. Conclusion: In both cases lactate and pyruvate were elevated in serum (L> 3000 μmol/l, P>300 μmol/l) with low L/P<10, which is specific to PDH deficiency. Background: Defects of both the pyruvate dehydrogenase complex and the mitochondrial respiratory chain are well recognised. It has been observed that patients with disease affecting the mitochondrial respiratory chain may have secondary defects of the pyruvate dehydrogenase complex, and vice versa. The interaction between these two systems is not fully understood. Aims: To identify how common it is to find deficiencies in both assays, to determine if it is possible to establish which is the likely primary defect and to comment on possible pathological mechanisms. Methods: The pyruvate dehydrogenase complex and mitochondrial respiratory chain assay results of patients from the north of England from January 1994 to December 2009 were matched. Results: The final data set was from January 1999 to December 2009. In total two hundred and thirty-nine pyruvate dehydrogenase complex assays were performed. Eighty-five were abnormal of which seventeen also had an abnormal mitochondrial respiratory complex. The majority had a complex I deficiency and, in all groups, the majority presented with a Leigh like illness. Conclusions: Advances in laboratory techniques means that more patients now receive a genetic diagnosis; this allows determination of the primary pathology and aids counseling. We report on 6 female patients with pyruvate dehydrogenase complex (PDHC) deficiency and a heterogeneous clinical and biochemical presentation. In all patients functional investigations of the mitochondrial energy metabolism were performed. The oxidation rates of pyruvate substrates were clearly decreased in 3/6 and mildly decreased in another 2/6. One patient had normal activities of the substrate oxidation rates. The activity of PDHC, however, was reduced only in 2 of the patients. Remarkably western blot analysis showed a reduced amount of E1α in relation to the other PDHC subunit in all patients. Analysis of PDHA1 gene expression showed an expression of the mutated allele in the range of 45-75% in the muscle biopsies of our patients. Sequence analysis of the X chromosomal PDHA1 gene revealed 4 novel missense mutations at conserved positions, 1 novel insertion-deletion and 1 large deletion, which also affected several neighbouring genes. All mutations were heterozygous. Heterozygous X-chromosomal diseases are a diagnostic challenge since the severity of the diseases depends on X-inactivation, which can be heterogeneous in different tissues. Concerning biochemical investigations 83% of the patients were detected by functional investigations of intact mitochondria while PDHC enzyme activity was decreased only in 33%.
Investigation of the mitochondrial energy metabolism from intact mitochondria by radiochemical substrate oxidation reveals defects of the pyruvate oxidation (PO) which is seen in an abnormal ratio of the oxidation of pyruvate+malate versus acetylcarnitine+malate. In approximately 3/4 of these patients defects of either one of the subunits of pyruvate dehydrogenase or of the regulatory phosphatases could be identified. Evaluation of the cofactor status in the remaining patients revealed an at least 4-fold decreased amount of thiamine pyrophosphate in the muscle of patients from 3 unrelated families. The clinical course of the 5 patients was characterised by metabolic crises after the first year of life with mild lactic acidosis. MRI showed a Leigh-like picture and lactate elevation in spectroscopy. Two of the patients died in infancy. Investigation of the known disease causing genes of the thiamine metabolism did not reveal pathogenic mutations. In another patient with reduced PO a severe deficiency of the lipoic acid prosthetic group was found. This patient presented with neonatal seizures. Brain sonography showed the development of a severe generalised multicystic encephalopathy. Echocardiography revealed an enlargement of the ventricular septum. He died at the age of 4 years. Pyruvate carboxylase (PC) deficiency is an autosomal recessive disease with three subtypes. Patients homozygous for the c.1828 G>A mutation in the PC gene belong to Type A, which typically has infantile-onset of developmental delay, hypotonia, and moderate lactic acidemia. We report the early brain MRI abnormalities of three patients homozygous for the c.1828 G>A mutation at 3 days, 4 days and 2 months of life respectively. The patients had bilateral cystic abnormalities in differing locations of the brain. One patient also developed diffuse brain edema. Neuropathology is available from two additional patients who died at 11 and 32 months. One patient had mild ventriculomegaly, and focal cavitation of the right caudate and right cerebellar white matter. The other patient had scattered multifocal demyelination and gliosis of the cerebral white matter. Although our patients have identical genotype and phenotype, the areas of neurologic injury in PC deficiency are diverse and varied. Our patients experience repeated episodes of severe metabolic decompensation, leading to profound developmental delay, independent of time of treatment implementation, frequency of metabolic decompensation and presence or absence of seizures. Despite the later clinical disease onset of the c.1828 G>A mutation, neurological injury can be observed on neuroimaging in the neonatal period. Elevated urinary excretion of 3-methylglutaconic acid (3-MGA) is considered rare in patients suspected of a metabolic disorder. In 3-MGAuria I, 3-MGA stems from leucine degradation. In all other types mitochondrial dysfunction is thought to be the common denominator. We investigated the clinical, metabolic and genetic data of 386 patients with 3-MGA-uria. 3-MGA-uria was often found in correlation with disorders not reported earlier in association with 3-MGA-uria (organic acidurias, urea cycle disorders, haematological/neuromuscular disorders). Mitochondrial dysfunction was indeed the common denominator for most of the patients. But, also a disturbed cholesterol biosynthesis can lead to 3-MGA-uria (Smith-Lemli-Opitz syndrome, glycogen storage disorder). Furthermore, we investigated 597 patients with mutations in 48 mitochondria-associated genes, thus having genetically proven mitochondrial disorders. 8.3% of these patients presented 3-MGA-uria. We show, that it was frequently seen in Complex V related disorders, in patients with mitochondrial DNA depletion or Pearson syndrome. Deficiencies of complex I and mitochondrial translational defects were less frequently associated with 3-MGAuria, which was also never reported in association with deficiencies of the complexes II-IV. With these data we will improve the diagnostic approach to the patient with 3-MGA-uria in general and type IV in particular. The paper further reclassifies the 3-MGA-urias. Background: Nuclear genetic defects in TMEM70 gene have been described in a subgroup of patients with 3-methylglutaconic aciduria type IV showing ATP synthase deficiency. Clinical presentation includes: cardiomyopathy, nervous system involvement and lactic acidosis. We report the clinical findings of two Spanish patients with TMEM70 mutations. Case reports: Patient 1 is the first son of healthy consanguineous parents diagnosed of intrauterine growth retardation and hypertrophic cardiomyopathy in neonatal period. He showed lactic acidemia with increased L/P ratio and 3-methylglutaconic aciduria, normal mitochondrial respiratory chain activities and normal brain MRI. He has developed: special facial phenotype, mild growth and psychomotor retardation and multiple decompensations. He is homozygous for c.317-2A>G mutation. Patient 2 is the second daughter of healthy consanguineous parents born premature after intrauterine growth retardation. She presented acute deterioration with lactic acidosis, increased L/P ratio and 3methylglutaconic aciduria. Mitochondrial respiratory chain activities showed deficiency of complex II+III. Brain MRI showed corpus callosum dysgenesis and cerebellar atrophy. She has developed: special facial phenotype, mild growth and psychomotor retardation, hypertrophic cardiomyopathy and multiple decompensations. She is compound heterozygous for the common c.317-2A>G and a new c.211-450_317-568del mutations. Conclusion: Patients with 3-methylglutaconic aciduria and cardiomyopathy should be screened for TMEM70 mutations. Today new diagnostic approaches are being developing for such "young" forms of mito diseases as mitochondrial hepatoencephalomyopathies. The algorithm for AS includes test for 4 frequent mutations (W748S, G268A, A467T, G848S) and afterwards whole POLG gene sequencing. For patients with clinical features different from AS, but still with strongly suggestion of hepatoencephalomyopathy we performed test for frequent POLG mutations and then sequencing of DGUOK and MPV17 genes. We revealed molecular defect in 9 AS patients and 5 MDS patients. The main pitfalls were: genetic heterogeneity-although AS has one gene, patients with other forms of hepatoencephalomyopathy may have mutation either in DGUOK, MPV17, POLG genes. Clinical data couldn't help us to foresee what gene is mutated; tissue specific accumulation of mtDNA depletionwe revealed only 2 patients with significant depletion of mtDNA (with DGUOK and MPV17 mutations consequently); search for 2nd allele-In our group of patients we didn't detect the second mutant allele in 4 cases (3 patients with hepatoencephalopathy and 1 patient with AS). Large POLG gene rearrangements are known being analyzed with CGH-method. 4) clinical polymorphism-differential diagnosis could be provide among several other forms if IMDs.
Kawachi Emi 1 , Murayama Kei 1 , Fushimi Takuya 1 , Fujinami Ayako 1 , Ajima Masami 1 , Harashima Hiroko 2 , Mori Masato 3 , Okazaki Yasushi 4 , Takayanagi Masaki 1 , Ohtake Akira 2 1 Div Metab Dis, Chiba Child Hosp, Chiba city, Japan 2 Div Ped, Saitama med Univ, Saitama, Japan 3 Div Ped, Jichi med Univ, Tochigi, Japan 4 Translational Reser Cent,Saitama Med Uni, Saitama, Japan
Background: Many enzyme defects show tissue-specificity in mitochondrial respiratory deficiency (MRCD), which sometimes make difficult for diagnosis. . Subject/Method: 158 patients were diagnosed to have MRCD out of 429 candidate patients. We classified clinically the following groups, Leigh's disease (LD), mitochondrial cytopathy (MC), neurodegenerative diseases (ND), hepatic disease(HD), cardiomyopathy (CM) and infantile mitochondrial disorder (IMD). We investigated the tissue specific pattern of enzyme defect to contribute correct diagnosis for MRCD. Results: Clinical diagnosis of lethal infantile mitochondrial disease was the greatest in number. The number of clinical LD, MC, ND, HD, CM and non-lethal IMD was 29, 23, 9, 21, 11 and 10. In LD, 14/18, 12/15, 3/3 and 0/0 were diagnosed in fibroblasts, muscle, liver and heart, respectively. In MC, 4/11, 11/16, 4/5 and 2/4, in ND, 3/3, 5/5, 1/1 and 0/0, in HD, 1/7, 0/0, 20/20 and 1/1, in CM, 2/6, 3/5, 4/9 and 10/10, in IMD, 14/25, 17/22, 22/24 and 8/13 were diagnosed in each specimen. Discussion: Almost HD and CM can be diagnosed only with affected organs. In other MRCD, enzyme defects can be detected in wide variety of organs and tissues, which support from many specimens can lead to greater confidence in the diagnosis achieved. Mitochondrial disorders (MD) may manifest in neonates, but the early diagnosis is difficult. The aims of the study were to analyse clinical and laboratory characteristics of MD with neonatal onset and identify possible association between clinical findings and specific mitochondrial diseases. Retrospective clinical and laboratory data were evaluated in 461 patients (331 families) with confirmed MD from two metabolic centers Prague and Salzburg. The neonatal onset of MD was reported in 129 patients (28%). Prematurity, intrauterine growth retardation, hypotonia necessitating ventilatory support were present in one third, cardiomyopathy in 40%, Leigh syndrome in 15% and profound lactic acidosis in half of neonates. 19 patients died in neonatal period. PDH complex deficiency was identified in 6, complex I in 15, complex III in one, complex IV in 23, complex V in 31, combined deficiency of several complexes in 53 patients. Background: Mitochondrial disorders affect~1 in 5000 births, and frequently present with combined deficiency of multiple respiratory chain (RC) enzymes. 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) deficiency is a rare disorder of valine catabolism. Objective: To report the association of multiple RC deficiencies with HIBCH deficiency. Patients and Methods: Two brothers, born to healthy distantly-related parents, presented with neonatal-onset neurodegenerative disease including seizures, dystonia and developmental regression. Relentless progression led to early death in both siblings. Investigation for mitochondrial disease revealed multiple RC defects (affecting complexes I, II+III and IV) in muscle from the older brother but normal activities in his sibling. The observation of persistently elevated hydroxy-butyrylcarnitine levels in the younger sibling led to investigation of HIBCH activity. Results: HIBCH activity was markedly reduced in cultured skin fibroblasts from both brothers. Subsequently a homozygous missense mutation affecting a highly conserved amino acid residue in the HIBCH gene was identified in both brothers. This mutation segregated with disease in the family. Conclusion: Multiple RC deficiencies are usually thought to arise from impaired maintenance and/or expression of the mitochondrial genome. We now show that HIBCH deficiency may lead to multiple RC defects. Objective: to study association of folate cycle polymorphisms (FCP) with mitochondrial dysfunction. Materials and Methods: 54 patients with a primary mitochondriopathy (PM) (Kearns-Sayre, MELAS, MNGIE, MERRF, organic acidurias) and 22 patients with secondary mitochondriopathy (SM). In both groups study of polymorphisms of MTHFR C677T and A66G MTRR by method of allelespecific PCR. Results: In 47 patients (87%) with PM were identified, compounds of allele frequencies MTHFR/MTRR as follows: N/N-7(13%); hmzg/hmzg-1 (1,9%); htrzg/htrzg-9(16.7%); htrzg/hmzg-17(31.5% ); hmzg/htrzg 2 (3.7%); N/hmzg-6(11.1%); N/htrzg-6(11.1%); hmzg/N-1(1,9%); htrzg/ N-5(9.3 %). In 19 (86.4%) patients with SM identified FCP, distribution of allele frequencies was as follows: N/N-3(13.6%); hmzg/hmzg-0(0%) ; htrzg/htrzg-2(9.1%); htrzg/hmzg-10(45.5%); hmzg/htrzg-0(0%); N/ hmzg-3(13.6%); N/htrzg-3(13.6%) ; hmzg/N-0(0%); htrzg/N-1 (4.5%). Conclusions: 66 (86.8%) patients with mitochondrial dysfunction had FCP and more specific to them was a polymorphism A66G MTRR-in 59 patients (77.6%) than the C677T MTHFR-in 48 patients (63.2%). This should be considered when developing an individual tactical correction energy deficit and conduct of prevention of complications associated with the presence of these polymorphisms. Background: Pristanic acid (Prist) concentrations are increased in peroxisomal disorders characterized by neurologic dysfunction and brain abnormalities, whose pathogenesis is poorly known. Objectives: Prist effects on important parameters of energy homeostasis were investigated in rat brain mitochondria. Material and methods: Purified mitochondrial preparations were obtained from brain of 30-day-old rats. Various parameters of energy homeostasis were then determined in the presence of 20-100 μM Prist. Results: Prist markedly increased state 4 respiration and diminished the RCR using both glutamate plus malate or succinate as substrates. In addition, Prist decreased state 3 respiring mitochondria, the ADP/O ratio, the mitochondrial membrane potential and NAD(P)H levels. Prist also induced mitochondrial swelling, probably through oxidative attack to the permeability transition pore (PTP) since cyclosporine A (PTP inhibitor) and N-acetylcysteine were able to prevent this effect. Conclusion/Discussion: The data indicate that Prist acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor, besides causing mitochondrial swelling. It is presumed that impairment of mitochondrial homeostasis may contribute to the neurological abnormalities presented by patients affected by peroxisomal diseases in which brain Prist concentrations are increased. Background: Peroxisomal abnormalities are biochemically characterized by tissue accumulation of branched chain fatty acids, including pristanic acid (Prist). Affected patients usually present brain abnormalities, however the pathomechanisms of cerebral injury in these diseases are still unknown. Objectives: We investigated the in vitro effects of Prist on Na+,K±ATPase and respiratory chain complex activities. Material and methods: Brain of young rats was dissected and the biochemical test performed after tissue preparation. Results: Prist markedly decreased the activities of Na+,K±ATPase (80 %) and of complexes I (65%), II (40%) and II-III (95%), without affecting complex IV activity. Conclusion/discussion: Considering the importance of Na+,K±ATPase for the maintenance of the membrane potential necessary to normal neuronal excitability and cellular cell volume control and of oxidative phosphorylation for brain energy production, the present data indicate that Prist compromises neurotransmission and brain bioenergetics. It is presumed that these pathomechanisms may be involved in the brain damage occurring in disorders in which Prist accumulates. Background: Pristanic acid (Prist) is a branched-chain fatty acid that accumulates in a variety of peroxisomal disorders. Although these disorders are characterized by neurological symptoms, the mechanisms involved in the pathophysiology are poorly known. Objectives: We studied the in vitro effects of Prist on important parameters of oxidative stress in cerebral cortex from young rats. Methods: Thiobarbituric acid-reactive substances (TBA-RS), carbonyl formation, sulfhydryl content, reduced glutathione (GSH) levels and nitric oxide production were measured in cerebral cortex from 30-day-old rats. Results: Prist increased TBA-RS levels, reflecting an increase of lipid peroxidation. This effect was totally prevented by the free radical scavenger melatonin, suggesting the involvement of reactive species. Prist also provoked protein oxidative damage, as determined by increased carbonyl formation and sulfhydryl oxidation. Otherwise, it did not alter nitric oxide production. Furthermore, the concentration of GSH was significantly decreased by Prist and this decrease was prevented by melatonin and alfa-tocopherol. Conclusions: It is therefore presumed that Prist elicits oxidative stress in the brain and that this pathomechanism may possibly be involved in the brain damage found in patients affected by peroxisomal disorders where Prist accumulates. Financial support: CNPq, FAPERGS, PRONEX and the FINEP research grant Rede IBN-Net and INCT-EN.
Ersoy M 1 , Tatli B 2 , Aydin K 3 , Saydam R 1 , Aktuglu-Zeybek Ç 1 , Özmen M 2 , Baykal T 1 , Demirkol M 1 , Gökçay G 1 1 Div Nutr Metab, Child Hosp, Ist Univ, Istanbul, Turkey 2 Div Ped Neurol, Ist Univ, Istanbul, Turkey 3 Div Radiol, Ist Uni, Istanbul, Turkey
Background: X-linked adrenoleukodystophy (X-ALD)(MIM 300100) results in inflammatory demyelination of the brain and spinal cord. The severity of MRI abnormality assessed with Loes score has a high predictive value. Objective: In X-ALD localization of the initial cerebral lesion can be more predictive about the severity of neuropsychologic impairment when similar MRI scores are encountered. Material/Methods: 28 patients with childhood cerebral X-ALD followed between 1991-2010 were studied. MR images, Loes scores; neurologic examination, X-ALD neurologic severity scale; Denver II test from 0-6 years, Wechsler Intelligence Scale for Children-Revised (WISC-R) from 6-16 years were evaluated. Results: 28 patients were grouped according to the pattern of cerebral involvement. Groups with parietooccipital (15), frontal (4), diffuse (2), cerebellar (1), corpus callosum (3) and no involvement (3) Objective: To evaluate the tolerability and effectiveness of treatment of pre-symptomatic X-ALD with lovastatin/coenzyme Q10 for the prevention of acute cerebral disease. Methods: Open-label, treatment of pre-symptomatic XL-ALD males <18 yrs old, with lovastatin, 1 mg/kg (max 40 mg) and coenzyme Q10 50-60 mg per day. Results: Seven pre-symptomatic boys currently aged 20.3±4.0 yrs were treated with 10-40 mg lovastatin and 50-60 mg coenzyme Q10 daily from age 8.5±3.5 yrs (3.3-12.6). One developed early MRI evidence of progression at age 5 yrs and underwent hematopoietic stem cell transplantation (HSCT). A second boy developed early signs of adrenomyeloneuropathy (AMN) at age 20 yrs. Of 16 boys who were never treated with lovastatin/coenzyme Q10, 3 are deceased (mean age 9.7±1.4 yrs), 3 are asymptomatic (mean age 12 yrs), 1 has adrenal insufficiency, and 9 have severe neurological impairment (mean age 14±6.3 yrs). No effect was demonstrated on plasma very long-chain fatty acids. Conclusion: Pre-symptomatic treatment of XL-ALD may prevent the development of acute cerebral degeneration; however, it does not appear to prevent the development of AMN. Further clinical trials are indicated. Structural variations of human genome emerge as novel major contributors to genetic diversity and disease susceptibility. Copy number variations, CNVs, are known to be involved in Mendelian disorders. Adrenoleukodystrophy X linked (XALD) is a rare disorder with two main phenotypes, a childhood form with cerebral involvement (CCALD) and a mild, adrenomieloneuropathy (AMN),adult chronic, that affects axonal tracts of the spinal cord. Both forms can be triggered by the same genetic mutation, indicating a participation of a modifier gene and or environmental factors. Therefore, to gain insight into what might be the potential source contributing to the differential phenotypes evolution we studied the CNV of genes involved in fatty acid metabolism, the inflammatory response and cell reparation. By using Illumina SNP data, Sentrix HumanHap 1 M-SNPs, we analyzed 8 XALD patients either with CCALD and AMN pure phenotypes. For gene candidate study (34) we used the semi-quantitative screening multiplex ligament-dependent probe amplification (MLPA) applied to 41 AMN, 46 CCALD and 26 Controls from Spain and Brazil. We found variable levels of AGPTA2, ABCA2, SLC27A2, SLC25A22, PCDH17, PCDH20, PNPLA2 . The data obtained may contribute to elucidation of clinical heterogeneity in XALD patients and to design specific therapeutic approaches. Mutations in PEX19 cause peroxisomal biogenesis disorders. PEX19 encodes a cytosolic protein that interacts with peroxisomal membrane proteins (PMPs) and functions as a cycling PMP-receptor protein. Besides full-length PEX19, the splice variant PEX19DeltaE2 accounts for a significant level of total PEX19 mRNA. Even though production of a protein was suggested by in vitro translation, PEX19DeltaE2 was shown not to complement PEX19 deficient cells. In the present work, we quantified PEX19DeltaE2 by real-time PCR showing significant variations among tissues. Furthermore, we found an increase in PEX19DeltaE2 mRNA upon inhibition of translation suggesting a sensitization to NMD without complete degradation. Using BRET, we showed that PEX19DeltaE2 has the capacity to bind PMPs. Nevertheless, PEX19DeltaE2 shows a distinct interaction pattern to the full-length protein lacking the interaction with PEX14, PEX13, and PEX10. This finding may explain that PEX19DeltaE2 is not able to restore the import of peroxisomal matrix proteins in PEX19 deficient cells.
In conclusion, PEX19DeltaE2 mRNA varies among tissues, its translation is regulated by NMD, and the protein partially retains PEX19 function.
These results indicate that this PEX19 splice variant is linked to a novel regulatory mechanism.
We present an 18 year old female with variant Zellweger syndrome. She presented at age 5 months with hypotonia, and failure to thrive. She was diagnosed at age 8 months on biochemical findings. Molecular analysis at 13 yrs showed compound heterozygosity for c.2528 G>A (p. Gly843Asp) and IVS2+1 G>A mutations. The child participated in a number of clinical treatment trials including Cholic acid and Docosahexanoic acid supplementation. She eats a low phytanic acid diet and takes vitamin K. She had a cochlear implant placed at 15 years resulting in marked improvement in her behavior. Currently she is ambulatory with support. She is functionally blind and is dependent for feeding, toiletry, dressing and all aspects of daily life. She has asymptomatic nephrocalcinosis and liver fibrosis. She has biochemically adequate adrenal function but with hypotension requiring glucocorticoids during illness. She has significant osteopenia. There is periventricular leukodystrophy. Her course has been slowly progressive and although generally healthy she is very fragile during illness. Currently the family is transitioning her to adult sheltered care. There are few reports of clinical outcome in long term survivors with PBD. Most have either one or 2 copies of the Gly843Asp mutation as in this child. Background: In peroxisomal disorders, accumulation of branched-chain fatty acids or bile acids intermediates can lead to severe neurological disease. SCPx is the last enzyme of the peroxisomal beta-oxidation system and until now, only one patient with SCPx deficiency has been described. Clinical description: The female patient presented at the age of 58 years with weakness of limbs, symmetrical sensory neuropathy, ataxia and moderate neuropsychological dysfunctions. Brain MRI demonstrated leukoencephalopathy. Metabolic investigations revealed increased levels of pristanic acid, whereas phytanic acid and very long-chain fatty acids were normal. The bile acid intermediates DHCA and THCA were undetectable, but there was excretion of abnormal bile alcohol glucuronides in urine. Fibroblast studies and SCP2 gene mutation analysis confirmed the diagnosis of SCPx deficiency. There was no clinical improvement on a phytanic acid restricted diet during six months. Discussion and conclusion: Like the first SCPx-deficient patient, this patient presented with neurologic symptoms but the onset was during adulthood. Most likely, SCPx deficiency is underdiagnosed, even if a peroxisomal disorder is considered, because the biochemical abnormalities are limited, and only analyzed in specialized laboratories. The excretion of bile alcohol glucuronides was similar in both SCPx-deficient patients and can be considered typical for this disorder.
Sharrard M 1 , Koodiyedath B 1 , Bowen J 1 , Beauchamp N 1 , Johnson D 1 1 Sheffield Children's NHS Trust, Sheffield, United Kingdom GSD IX is historically considered to be a recessively inherited disorder of glycogenolysis caused by mutation in the PHKA2 (X-linked) or PHKB or PHKG2 (autosomal) genes, coding the alpha, beta and gamma subunits of hepatic PhK respectively. PhK is a decahexameric enzyme composed of four of each of the subunits: alpha, beta, gamma, delta. A 19 month old boy with recurrent ketotic hypoglycemia had deficient erythrocyte PhK-0.75micromol/ml/gmHb (10-90) and normal glycogen phosphroylase and debrancher. He was a simple heterozygote for the novel missense mutation of PHKB (p.[=]+[Tyr167Cys]) with no mutation in PHKA2 or PHKG2. Both PHKB allelles generated stable mRNA transcripts. The mother and the half-brother (different father) experienced symptomatic hypoglycemia and PhK deficiency (3.8micromol/ml/gmHb, and undetectable respectively). Both were simple heterozygotes for the PHKB (p.[=]+[Tyr167Cys]) mutation, and both generated two stable mRNA transcripts. In this case the stable mutated betasubunit disrupts enzyme activity. Each PhK enzyme complex contains four beta subunits. If any one of these four is mutated, the resultant enzyme complex has no activity and a total PHK activity is 1/16 of normal. The Tyr167Cys mutation causes autosomal dominant negative inheritance of GSD IX, not previously reported in GSDs, with significant genetic counselling implication. The diagnosis of GSDs has traditionally relied on clinical features combined with biochemical profiling and enzymatic analysis. Diagnosis may then be confirmed by gene sequencing. Disadvantages of this approach include overlapping clinical and biochemical features; cost and time of sequential investigations; the need in some subtypes for tissue biopsy; the scarcity of laboratories offering enzyme analysis and the inherent enzyme instability. Unsupported by sequencing this can easily lead to misdiagnosis with inaccurate prognostic and recurrence risks provided and incorrectly targeted clinical surveillance. We present a new screening process of in-solution DNA capture and NGS to simultaneously screen 18 genes known to cause GSD. 120 bp RNA probes used to create a GSD library covering over 1 Mb Validation studies detected all point mutations, sequence variations and copy number variants. The results from over 30 suspected patients, confirm this technique improves diagnostic accuracy compared to stand alone biochemistry. Improving the original GSD library has allowed multiplexing of samples to further reduce costs. We propose using this technique early in the diagnostic pathway offers a more cost effective, efficient and accurate service and extends accurate diagnosis to patients from around the world who do not have access to the current diagnostic techniques. Glycogen Storage Disease type IV (GSD IV) is caused by deficiency of the glycogen branching enzyme (GBE). The diagnostic feature of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan. The disease is clinically heterogeneous, with variable tissue involvement and age of disease onset. Complete loss of enzyme activity is lethal in utero or in infancy, affecting primarily muscle and liver. However, enzyme activity as low as 5 to 20% leads to juvenile or adult onset of the disease that affects the central and peripheral nervous system and muscles. By using homologous recombination, two mouse models of GSD IV that reflect this spectrum of enzyme activity and disease were generated. By completely eliminating GBE activity, a phenotype similar to early onset GSD IV with neonatal lethality was created. In contrast, adult animals with some residual GBE activity accumulate significant polyglucosan in virtually all tissues, including the central and peripheral nervous system, and exhibit progressive neuromuscular dysfunction. Unlike in muscle, polyglucosan in liver is a degradable source of glucose and is readily depleted by fasting, emphasizing that there are structural and regulatory differences in glycogen metabolism between these tissues.
Behulova D 1 , Bzduch V 2 , Fabriciova K 2 , Sykora P 3 , Kolnikova M 3 , Syrova D 1 1 Dept Lab Med, Univ Child Hosp, Bratislava, Slovakia 2 1st Dept Pediatr, Univ Child Hosp, Bratislava, Slovakia 3 Dept Child Neurol, Univ Child Hosp, Bratislava, Slovakia Background: Glucose transporter 1-deficiency syndrome (GLUT1-DS) is a cause of the reduced glucose entry into the brain. Our aim was to compare biochemical findings, clinical features and genotypes in patients detected in Slovakia. Methods: Serum (S) glucose, cerebrospinal fluid (CSF) glucose (reference range, RR 2.5-3.9 mmol/l) and lactate (RR 0.70-2.10 mmol/l) were investigated and ratio CSF:S glucose (RR>0.65) was calculated in patients with various neurological and psychiatric symptoms. Genomic DNA for mutation analysis was obtained from blood samples. Results: Now, patients are at the age 7(M), 7(F), 8(F), 9(M) years, their initial biochemical findings were: CSF glucose 1.1, 1.7, 1.8, 2.2 mmol/l; CSF:S glucose ratio 0.19, 0.37, 0.40, 0.48; CSF lactate 0.82, 0.93, 1.30, 1.20 mmol/l and the first clinical symptoms appeared at 2.5, 8, 9 and 48 months respectively. Patient (Pt)1 presented with infantile epilepsy, developmental delay, Pt 2 with apathy, ataxy, Pt 3 with seizures, ataxy, Pt 4 with concentration disorders. Pathogenic mutations c.798_799insC, c.505_507del, c.1119delG were identified in Pt 1, 2, 3 respectively, analysis in SLC2A1 gene is being performed in Pt 4.
Conclusions: Despite small series we suppose CSF:S glucose ratio and CSF glucose could correlate with severity of clinical course in GLUT1-DS.
HYPERINSULINEMIC HYPOGLYCEMIA,REPORT OF 22 CASES Zaman TZ 1 , Rahmanifar AR 2 1 Metab Unit ,Tehran Univ, Tehran, Iran, Islamic Republic of 2 Iranian National Research Society,Div Me, Tehran, Iran, Islamic Republic of Background: Hyperinsulinemic hypoglycemia is the most important cause of hypoglycemia in the neonatal period or early infancy. It is a heterogeneous disorder with two different types of histopathological lesions; focal(islet-cell hyperplasia),always sporadic, and diffuse,a heterogenous disorder, recessively inherited, or rarely dominantly HI, including glutamate dehydrogenase gene involvement, when hyperammonemia is associated. These are clinically indistinguishable. Objective: The aim was to review clinical presentations, laboratory findings and outcome of 22 patients with hyperinsulinemic hypoglycemia. Methods: A Retrospective study of all patients diagnosed to have (PHH) between (19902010). Results: Total 22 patients;Male 11(50%);age at onset, 13(61%)in the first week,16(76%)in first 3 months and 22(100%)in first 6 months; related parents, 70%; birth weight >4 kg,54%. The most common presentation was seizures,95% followed by cyanosis,57%; insulin level,(10-25 micro IU/ml,mean 17.5). Plasma ammonia level was 2-3 times increased in 5/11 cases. All cases after emergency treatment were treated with Diazoxide, 13/ 22 were diazoxide sensitive and 9 underwent surgical intervention (pancreatectomy). Pancreatic biopsy was performed in 9 cases; Island hyperplasia,7 cases; B cells hyperplasia,2. 20 year FU; well in 18. Conclusion: 75% became symptomatic in the first week and 100% up to 6 months. Early detection and treatment are very important to prevent severe brain damage. It has been reported that rapid genetic analysis of ABCC8 and KCNJ11 genes could improve the clinical management of congenital hyperinsulinism (CHI). We evaluated retrospectively 5 patients (pts) with diazoxide-unresponsive CHI treated at our Centre in the last 30 years with respect to genetic analysis. Genetic analysis was performed after CHI management, at Royal Devon & Exeter NHS, UK and in 2 pts at Baylor College, Texas. Between 1981-2001, 2/5 pts (pts 1,2) underwent total pancreasectomy (TP) after multiple partial resections. Pt 3 underwent TP after medical treatment awaiting spontaneous remission. These pts showed diffuse CHI at histological examination. Pts 4 and 5 did not show focal CHI at venous pancreatic sampling (pt 2, 1995) or 18 F-Dopa PET/CT (pt 5, 2008) . Case 4 showed early CHI remission, pt 5 is stable on low dose octreotide. Pts 1-3 are homozygous for nonsense mutations (E51X case 1; A187V cases 2-3, sisters) on KCNJ11 gene. Pt 4 is heterozygous for nonsense mutation Q444H and pt 5 compound heterozygous (E529K and H125Q), both on the ABCC8 gene. In our pts rapid genetic analysis could be helpful to indicate the correct management (near-total pancresectomy or medical treatment), in combination with 18 F-DOPA PET/CT. Specific treatment of the acute porphyria attack is based on oral or intravenous carbohydrate loading. The rationale for this treatment has been unveiled in experimental animals; fasting of mice induces aminolevulinic acid synthase-1 (ALAS1) through actions of the peroxisome proliferatoractivated receptor-gamma coactivator-1alpha (PGC-1alpha). The aim of this study was to investigate the direct effect of glucose and insulin on mRNA levels of ALAS-1 in human hepatocytes. The rationale for carbohydrate loading in treatment of the acute porphyria attack was confirmed in primary human hepatocytes including a direct downregulatory effect of insulin on ALAS1 and PGC-1alpha. There was a linear correlation between ALAS1 and PGC-1alpha mRNA levels confirming a coregulation of ALAS1 and PGC-1alpha. The fact that increased expression of PGC-1alpha by dexamethasone at normophysiological levels of glucose (5 mM) and insulin (0.3 nM) did not increase ALAS1 points to importance of the mechanism of insulin stimulated export of the ALAS1 transcriptional regulator, forkhead box protein O1, from the nucleus. On the other hand, the potentiation of the phorphyrogenic action of phenobarbital by dexamethasone-induced expression of PGC-1alpha suggests that the role of PGC-1alpha in ALAS1 regulation extends to induction via the nuclear receptors pregnane X receptor and/or the constitutive androstane receptor. Glucokinase (GCK) is the enzyme controlling insulin release. Mutations in GCK gene can result in various phenotypes with autosomal dominant inheritance. Both persistent hyperinsulinemic hypoglycemia of infancy and hyperglycemia (MODY-2, PNDM) are observed, depending on the type of DNA changes. Activating mutations cause oversecretion of insulin despite hypoglycemia, with good response to pharmacological treatment. The prevalence of GCK-HH is 1.2% out of all HH cases. We present a girl, born at time to unrelated young parents. Father's history revealed serious HH in childhood, treated initially with diazoxide but finally pancreatectomy had to be performed. He developed insulindependent diabetes in age of 26 years. Some family members are probably also affected but not diagnosed Hyperinsulinemic hypoglycemia was diagnosed in our patient from first days of her life but was milder that in her father. DNA analysis revealed activating mutation in GCK gene, with protein effect: Val455Leu in both patients. Diazoxide therapy was introduced with good clinical response. Conclusions: 1.The identification of a GCK mutation provides information on the prognosis of the disease and helps therapeutic decision. 2.It implies also the diagnostic procedures for hypoglycemia directed to other family members. 3.In each case of neonatal hyperinsulinism, genetic counseling should be recommended.
Glucose is a metabolic fuel in the body especially for the brain. Glucose homeostasis is based on storage of carbohydrates, proteins and fats. Interconversion is regulated by several hormones and enzymes. Hypoglycemia in IEM is mainly due to inadequate stores, or immature enzymes, abnormalities in insulin production, disorders of carbohydrate, protein and fat metabolism. Material: A total of 40 neonates, Infants and children presenting with hypoglycemia of <50 mg/dl were investigated for inborn errors of metabolism. Results: Two of the neonates were premature at 28 weeks gestation, Beta ketothiolase deficiency in two neonates, CPT1 in one infant, 2 of MCAD deficiency, 2 of Glycogen storage disease (GSD 1 one case, GSD3 one case). 17.5% of children with hypoglycemia had an inborn error.
Maschke H 1 , Bergmann J 1 , Tsiakas K 1 , Santer R 1 1 Dept Pediatr, Univ Med Center Eppendorf, Hamburg, Germany
Background: The classification of hypoglycemia into ketHG and non-ketHG is important for the diagnostic approach. Among ketHG are disorders of glycogen formation, clinically recognizable by supranormal postprandial rise of plasma glucose and lactate concentration. In addition to hepatic glycogen synthase (encoded by GYS2), other proteins play a role in glycogen formation. In liver, glycogenin-2 (GYG2), glycogenin-1 (GYG1), shown to interact with glycogenin-2, and a glycogen synthase kinase are important. Defects of GYS2, GYG2 and GYG1 might result in ketHG, although GYG1 mutations have recently been associated with myopathic symptoms (Moslemi et al, NEJM 2010). Methods/Results: Nineteen consecutive patients with ketHG and typical laboratory findings were investigated (the majority without liver biopsy and/or enzymatic studies). When GYS2, GYG1 und GYG2 were systematically sequenced, only one patient with GYS2 deficiency was diagnosed. Defects of glycogenins were not detected. Conclusion: Glycogen synthase deficiency is a rare cause of ketHG even if the presentation is typical. In contrast to muscle glycogenin deficiency, recently reported for the first time, a deficiency of the most important hepatic glycogenin, GYG2, has not yet been diagnosed. We continue to offer GYG2 sequencing in suspicious patients, particularly those with low hepatic glycogen content and normal liver glycogen synthase activity.
Gautschi M 1 , Christ E 2 , Salvisberg C 1 , Zürcher T 1 , Schütz B 1 , Nuoffer JM 3 1 Interdiscipl Metab Unit, Univ Child Hosp, Bern, Switzerland 2 Div Endocrinology, University Hospital, Bern, Switzerland 3 Institute Clin Chem, University Hospital, Bern, Switzerland Dyslipidemia, with increased triglycerides (TG) and cholesterol (total [TC], VLDL-more than LDL-C, but decreased HDL-C), is a typical biochemical finding in glycogen storage disease type 1 (GSD1a). Intriguingly, this dyslipidemia is not associated with increased cardiovascular morbidity and mortality. We describe a 15-year-old female patient with a GSD1a chronically difficult to manage, whose blood lipids increased massively at puberty, with TG up to 29 mmol/L and TC up to 13 mmol/L. For the last 18 months, she has been repeatedly hospitalized for episodes of acute abdominal pain with nausea and vomiting; but no biological signs of pancreatitis, the much feared complication of massive hypertriglyceridemia, nor of any other underlying cause were found. Various therapeutic trials, including optimizing continuous glucose uptake, supplementation with medium chain triglycerides, or omega fatty acids, improved the clinical picture and the lipid profile only transitorily. PPARγ agonists have been suggested to act positively on lipid metabolism in this context. We have used pioglitazone (Actos.) and studied its impact on both glucose and lipid homeostasis in our patient: TG were reduced by 40%, TC by 25%, compared to baseline. Changes were also observed in adiponectin and insulin levels (and HOMA), whereas glycemia remained stable. Considering the essential role of vitamin D in bone homeostasis and the prevalence of hypovitaminosis D in our GSD I patients, we suggest to routinary evaluate this parameter in all patients.
Melis D 1 , Carbone F 2 , Della Casa R 1 , Minopoli G 1 , Parenti G 1 , Andria G 1 , Matarese G 2 1 Dept Pediatrics, Federico II University, Naples, Italy 2 Lab. Immunologia IEOS-CNR, Naples, Italy Background: Glycogen storage disease type 1b (GSD1b) is caused by mutations in the glucose 6-phosphate translocase (G6PT) gene. GSD1b patients present autoimmune disorders including inflammatory bowel disease (IBD), thyroid disease and Myastenia Gravis. Although these manifestations impact significantly on patients' quality of life, their pathophysiology has remained obscure. Patients and methods: Seven GSD1b and 14 age and sex-matched controls were enrolled. The presence of autoimmune disorders, serum levels of different autoantibodies, classes and subclasses of circulating lymphocytes, T cells activity in vitro, activity and number of T-cellsregulating system (TRegs) were investigated. Results: The CD3, CD4, CD8, NK, CD4CD28, CD3CD45RA, CD3CD45RO, CD4CD45RO lymphocytes counts were lower in patients than in controls. Aweak proliferation activity was observed in both homologous and heterologous sera. The patients also showed reduced TReg cells counts in the periphery. The presence of autoimmune thyroid disease correlated with CD8DR cells prevalence. The diagnosis of IBD inversely correlated with NK and CD4CD8 cells prevalence. The serum levels of acetylcholine receptor antibodies inversely correlated with CD4CD8, CD4CD45RA cells prevalence. Conclusion: The obtained data suggest that the association of lymphopenia, impaired T cell proliferation and reduced TReg cells is responsible for the development of autoimmune disorders in GSD1b patients.
Riva E 1 , Gasparri M 1 , Giulini Neri I 1 , Paci S 1 1 Ped Dep, San Paolo Hosp, Univ of Milan, Milan, Italy Glycogen storage disease Ib (GSD Ib) is characterized, in addition to the signs and symptoms of GSD I, by recurrent infections and inflammatory bowel disease (IBD), associated with neutropenia/neutrophil dysfunction. MGUS (Monoclonal Gammopathy of Unknown Significance) is a biochemical condition without clinical expression often described in patient with chronic diseases, with higher risk of transformation in myeloma. There are no cases of MGUS in GSD Ib patients described in literature. We describe the case of a 31 years old boy with diagnosis of GSD Ib at 3 months of age and of IBD at the age of 19, when anti-inflammatory therapy was started. Since 2007 the protein electrophoresis showed a monoclonal not determinable IgAλ component. Since 2010 a policlonality, attributed to his chronic inflammatory condition, was evidenced. The plasma and urine determination of κ and λ light chains showed higher values than in the normal range, with a normal κ/λ rate and a monoclonal component lower than 1,5 g/dl. The patient doesn't show either kidney insufficient function or peripheral neuropathy or bone lesions. This report confirms that GSD Ib is a clinical condition that still needs further investigations in order to better understand its evolution in adult patients.
McSweeney M 1 , Wood M 1 , Grunewald S 1 , Cleary MA 1 , Abulhoul LH 1 1 Great Ormond St. Hospital for Children, London, United Kingdom Background: GSD III has both hepatic and muscle manifestations of varying degrees. Muscle disease affecting function is not always clinically apparent early in childhood and may go undetected. One aspect of the musculoskeletal system that lends itself to monitoring is gait. Importantly, objective assessment of gait has the potential to quantify clinical change. Method: Four children with a diagnosis of GSD III were assessed using the GaitRite system. Specific parameters of gait-velocity, cadence, step length and base of support were measured. Changes for each individual were analysed and compared with age-matched controls. Results: All patients had slower and less efficient gait patterns than their peers. The most notable differences were in velocity and asymmetry in step length and base of support indicating an unstable and inefficient gait. These differences were not apparent on routine clinical examination. Creatinine kinase levels were elevated in all patients. Conclusion: The GaitRite identified abnormalities in gait parameters in GSD III, highlighting alteration in musculoskeletal function prior to overt clinical signs. This reliable instrument can monitor the muscle disease in this rare disorder. It could have considerable potential in clinical management and as an outcome tool in evaluating potential therapies.
Introduction: Dietary treatment of GSD is variable according to type and severity. There are few reports examining growth in GSD. Aim: A cross-sectional audit reviewing growth in children with GSD. Methods/Subjects: 18 children (10 boys) were studied. GSD Ia n=5; GSDI b, n=4; GSD III, n=5; GSD VI, n=1; GSD IX, n=3). Median weight/height z scores were calculated. Results: In GSD Ia (median age 8.3y, 4 Asian, 1 Caucasian), on 1.5 g/kg/ dose uncooked cornstarch (UCCS), normal diet and overnight tube feeds (CNTF), median height/weight z score was −1.63/-0.24. In GSD Ib (median age 7.2y; 4 Asian) on 0.7 g/kg/dose UCCS (n=2), milk-free diets (n=2) and CNTF, median height/weight z score was −2.04/0.68. In GSD III (median age 9.1y; 1 Arabic, 4 Caucasian) on 1.9 g/kg/dose UCCS, normal diets and CNTF (n=3) median height/weight z score was −0.5/-0.25. In GSD VI (age 6.9y; Asian) on 1.2 g/kg/dose UCCS only, height/ weight z score was −3.31/-2.26. In GSD IX (median age 8.7y, 3 Caucasian) one dose of UCCS pre-bed only, median height/weight z score was −2.09/-0.52. Conclusion: Suboptimal growth occurred in all GSD irrespective of dietary treatment. The impact on growth of metabolic control, infections, impaired exercise tolerance need investigation by collaborative trials.
Background: Previous studies examining reproductive parameters in men with galactosemia have inconsistently demonstrated abnormalities. We hypothesized that men with galactosemia would demonstrate evidence of reproductive dysfunction. Methods: Pubertal history, physical examination, hormone levels and semen analyses were assessed in 26 galactosemic men and compared to those in 46 controls. Results: The prevalence of cryptorchidism was higher in men with galactosemia than in the general population [9.1% vs. 1.0% (95%CI: 0.75-1.26; p=0.007)]. Testosterone (461±125 vs. 532±133 ng%; p=0.04), inhibin B (144±66 vs. 183±52 pg/mL; p=0.002) and semen concentration (46±36 vs. 112±75 x10^6 spermatozoa/mL; p=0.01) were lower and SHBG was higher (40.7±21.5 vs 26.7±14.6; p=0.002) in galactosemic men compared to controls. Semen volume was lower than normal in 7 out of 12 patients. Conclusions: Despite the limited sample size, this is the second time that a higher than expected prevalence of cryptorchidism is reported in men with classic galactosemia. The subtle decrease in testosterone and inhibin B levels and sperm count may mark mild defects in Sertoli and Leydig cell function. Follow-up studies are needed to determine the clinical consequences of these abnormalities. The low semen volumes may be an indicator of pathophysiological abnormalities in this disease and deserve further study. Background: Most female classic galactosemia patients suffer from ovarian insufficiency from an early age on. We characterized the phenotype of female galactosemia patients at different ages by MRI and/or abdominal or transvaginal ultrasound Patients and methods: 23 patients were included. MRI data of 14 patients were compared to different control patients (prepubertal, age-matched and postmenopausal) who underwent MRI for other reasons. Data on 17 ultrasounds were obtained retrospectively. Results: When comparing the ovarian volumes on MRI, the ovaries of the galactosemic girls were significantly smaller than those of the age matched controls (p=0.001) and the prepubertal ovaries (p=0.008), but did not differ significantly from postmenopausal ovarian volumes (p=0.161). Also, evidence for follicle activity was regularly seen in both the prepubertal and age matched control groups, but only rarely in the galactosemia patients and postmenopausal controls. On ultrasound, the ovaries were detected only occasionally. Conclusions: Imaging results point to early onset of ovarian abnormalities in female galactosemia patients. Little evidence for maturation of follicles was seen. Combined with the significantly smaller volumes of the ovaries we found no support for a maturation arrest alone as the main pathophysiological mechanism. This study supports the mechanism of increased follicle loss in these patients. Objectives: To define the prevalence and characteristics of motor complications in adult patients with galactosemia. Background: Early restriction of galactose prevents the neonatal complications of galactosemia, but long term problems, such as premature ovarian failure and neuropsychiatric disorders, often involving motor function, continue to develop. Their exact prevalence and phenotype are not well defined. Methods: All patients with classical galactosemia attending the Charles Dent Adult Metabolic Unit (London) from September 2010 to April 2011 were examined by a neurologist and their medical records reviewed. Results: 42 patients were examined. 26 patients (62%) had motor complications. This group also had a higher prevalence of other neurologic problems (65% vs 12%). 13 patients had symptoms related to their movement disorders and 8 reported progressive worsening. The remaining 13 had convincing signs on examination. The most common motor manifestation was tremor (N=21; 81%), followed by dystonia (N=14; 54%) and cerebellar signs (N=7; 27%). Several patients responded to standard treatments for tremor and dystonia. Conclusions: A high proportion of patients have central nervous system damage despite adhering to a galactose-restricted diet. Many develop symptomatic motor complications, mainly tremor, dystonia and cerebellar ataxia, which may progress, leading to disability and suggesting ongoing neurodegeneration. Classic galactosemia is an inborn error of galactose metabolism. Language production problems are among the most burdensome complications. This study hypothesized that galactosemia patients have language difficulties at the level of syntax (i.e. where grammatical information is retrieved and a sentence is constructed). Electroencephalography (EEG) was recorded in a group of galactosemia patients (n=22,mean age=14.9) and healthy controls (n=21,mean age=14.3), during task performance. The participants were instructed to either passively watch an animated visual scene or to utter an overt response describing this scene, requiring either minimal (separate words) or maximal syntax (sentence). Event-related-potentials (ERPs) were extracted from the continuous EEG, reflecting the brain's preparatory response to this task. Results indicate that the galactosemia ERPs start to diverge as early as 120 ms after scene presentation (P1). Also, the patient data deviates in the P2 and P6 ERP time windows (i.e. around 200 ms and 400-600 ms post-stimulus, respectively). Behaviourally, the patients need more time to prepare the utterance and make more mistakes. We suggest that galactosemia patients have not (only) late articulatory problems, but also impairments of early conceptualisation (P1), lexical access of the words (P2) and syntactic processing (P6), resulting in delays and more troublesome language production. Classical galactosaemia is an autosomal recessive inborn error of carbohydrate metabolism caused by mutations in the galactose-1phosphate uridyl tranferase gene (GALT). The objective of the present study was to investigate galactosaemia in Cyprus at the epidemiological, biochemical and molecular level. We identified two mutations in Cypriot patients with classical galactosaemia: A novel large deletion of 8489 bp encompassing all exons of the GALT gene, and the p.Lys285Asn mutation. Microsattelite analysis revealed the presence of a common haplotype suggesting a founder effect for this deletion in Cyprus. The frequency of galactosaemia carriers in the general population was estimated at 1/105 by means of GALT activity measurement in red blood cells. Molecular analysis of all carriers identified biochemically revealed three additional mutations, on one allele each: the p.Pro185Ser and the c.
[820+13A>G], previously found in Portuguese patients, as well as a new transition, the c. [378-12 G>A] , not encountered in the general population. Thus, five mutations account for all galactosaemia alleles studied: a novel whole gene deletion (55% of alleles), p.Lys285Asn (30%), p.Pro185Ser (5%), c.
[820+13A>G] (5%) and c.[378-12 G>A] (5%). Furthermore, the allele frequencies of the p.Asn314Asp, the Los Angeles variant and the Duarte 2 variant were determined at about 8%, 5.5% and 2.5% respectively.
Coelho AI 1 , Silva MJ 1 , Tavares de Almeida I 1 , Leandro P 1 , Vicente JB 1 , Rivera I 1 1 Metabolism and Genetics Group, iMed, Lisbon, Portugal
Herein we report comparative functional studies on wild-type and mutant forms of recombinant human galactose-1-phosphate uridylyltransferase (GALT), seeking a molecular basis for the phenotypes assigned to classical galactosemia patients bearing the respective mutations. GALT plays a key role in galactose metabolism. Mutations in the GALT gene resulting in GALT deficiency set the basis for classical galactosemia, an autosomal recessive disorder. To date, more than 200 mutations have been described, the majority being missense. Novel mutations were identified in the Portuguese galactosemic population (e.g. G175D and P185S), which will be studied in parallel with other known mutations (R148Q, Q188R and S135L). To characterize the wild-type and mutant forms, GALT wild-type cDNA was cloned into pET24b(+), bearing an N-terminal 6xHis-tag, the mutations generated by site directed mutagenesis, and expressed in E. coli BL21(DE3) Rosetta cells, in LB medium supplemented with iron and zinc. After purification by affinity-chromatography, comparative functional studies were employed to evaluate the cofactor loading, oligomeric profiles, conformational stability and enzymatic activity of wild-type vs. mutant GALT. The results provided biochemical information confirming that the selected mutations are disease-causing, and contributed for the understanding of how the mutational spectrum in galactosemic patients modulates GALT functional properties. Background: Galactokinase deficiency (GALK) is a rare autosomalrecessive disease with an estimated incidence 1:150,000 to 1:1,000,000. Cataract formation due to accumulation of galactitol may already occur in early childhood. This can be prevented, reversed or ameliorated by early diagnosis and therapy with a galactose restricted diet. Methods: From 2000 to 2010 the Screening-Laboratory Hannover analyzed 2 million dried blood spot samples from neonates. Enzymatic activities of galactose-1-phosphate uridyltransferase (GALT) and galactose plus galactose-1-phosphate were quantified. Indirectly screening for GALK was accompanied; in cases with elevated galactose plus galactose-1-phosphate and normal GALT activity, GALK activity was measured in erythrocytes to confirm GALK deficiency. Results: In 9/11 cases with elevated galactose levels and normal GALT activity the diagnosis of GALK was confirmed. In two cases screening results were false positive. 3/9 children developed cataracts. Two children were treated with lentectomy. Under a galactose-restricted diet existing cataracts were stable or reversed; concentrations of galactose normalized. Galactokinase activities and cataract formation did not correlate. Conclusion: For galactokinase deficiency a therapy with galactose restricted diet is available and useful to ameliorate ophthalmological complications. Measurement of elevated galactose levels as well as normal GALT activity in newborn screening may be helpful to detect galactokinase deficiency early Background: Transaldolase (TALDO) deficiency is a rare inborn error of pentose phosphate pathway. So far 14 patients from 8 families were described. In the majority of these cases clinical course of the disease was severe with neonatal presentation and early death. In 2009 we described two transaldolase deficient brothers (6 and 4 years of age) with relatively mild phenotype. Patients: Three years of follow up revealed in the older brother (currently 8 years old) advanced liver (nodular) cirrhosis, hyperbilirubinemia, persisted moderate anaemia and thrombocytopenia, clotting disturbances, splenomegaly, low body mass. Since the age of 7 the boy developed tubulopathy, proteinuria and progressing renal damage, mild pulmonary fibrosis and cardiomegaly. His intellectual development and social functioning are adequate to the calendar age. He has persisted tendency to generalized edema, and ascites requiring diuretics administration. His younger brother (6 years old) presents less advanced liver cirrhosis, as well as anaemia, thrombocytopenia, clotting disturbances, tubulopathy, proteinuria and osteopenia. Conclusions: TALDO deficiency is mostly pronounced in liver but taking into account the severe renal, pulmonary and cardiac involvement, TALDO deficiency should be considered as severe multisystemic disease. Hereditary fructose intolerance (HFI) is an autosomal recessive disease caused by mutations in the ALDOB gene encoding aldolase B enzyme. If undiagnosed, the persistent ingestion of fructose can lead to severe liver and kidney damage and death. Molecular genetic analysis are essential and useful diagnostic tool for HFI because of less invasive methods. Recently, promoter mutations in ALDOB gene were identified in patients with HFI beside over 50 different type of known mutations. In this study, mutation screening of 2-9 exons and promoter region (untranslated exon 1) in ALDOB gene was carried out in 13 Turkish patients with HFI by using direct sequence analysis. Six different type of mutations in ALDOB gene (one splice site, three missense and two deletion) were detected. In this new cohort with HFI, ALDOB gene harbored for three novel mutations including IVS2-3deltagG, c.71delTTGCins11GAATCTCTGGG, IVS1+ 1 G>A and three known mutation, p.A175D and p.C135R in coding region and g. -132 G>A mutation in promoter region of the ALDOB gene. Background/Objectives: Lactose intolerance is the inability to metabolize lactose, because of the lack of enzyme lactase in the digestive system. There are known three major types of lactose intolerance. We report a patient with adult type of hypolactasia who was picked up by selective screening for galactosemia. Case report: 2-month-old boy with recurrent complaints of restlessness and meterorism. Urinary Benedict's test was positive and urinary monoand disaccharide analysis revealed the elevation of galactose. Repeated analysis showed even larger excretion of galactose and also galactitol. Lactose-free diet showed positive impact. All galactosemia subtypes were ruled out by enzymatic analysis, but hypolactasia was confirmed on molecular level. He is homozygous for c.-13910 C>T polymorphism in MCMG gene, which regulates lactase gene transcription. Methods: Urinary galactose/galactitol content was evaluated by HPLC with refractive index/ultraviolet-visible spectrophotometrical detectors. Results: concentration of glucose in urine was 0.08 mmol/l (ref<0.8), galactose 334 mmol/mol cr (ref<377); 1-month later urinary glucose 1 mmol/l (ref<0.8), galactose 782 mmol/mol cr, galactitol 597 mmol/mol cr (ref<30); sugar derivatives in organic acid GC/MS analysis. Lactose contents in urine were respectively 44.3 mmol/mol cr and 20.3 mmol/mol cr. Conclusion: Urinary galactose analysis might be a valuable tool for picking up infantile hypolactasia cases. Patients with Pompe disease (Glycogen storage disease (GSD) type II) have been shown to excrete increased amounts of glucose tetrasaccharide (Glc4) in urine, thought to be derived from the action of serum amylase on glycogen accumulated as a result of lysosomal acid alpha glucosidase deficiency. We have developed a simple, rapid and reproducible method for the analysis of Glc4 by HPLC-ECD, by adaptation of an established method for urine and faecal sugars analysis. Using this method, we were able to demonstrate elevated urinary Glc4 concentrations in all Pompe patients studied (n=11), with all control patients having concentrations below the limit of detection (5 μmol/mmol creatinine) (n= 21); and importantly, we were able to show a distinction in Glc4 excretion between Pompe patients and an individual harbouring a pseudo-Pompe mutation. A clear decrease in Glc4 excretion was observed following commencement of enzyme replacement therapy (ERT) in 3 Pompe patients studied.
Our results therefore support the use of urinary GLc4 measurement by HPLC-ECD as an adjunct to enzymatic analysis in the diagnosis of Pompe disease, and as a biomarker for monitoring ERT in these patients. Additionally, raised urinary Glc4 was observed in patients with GSD type Ia and III, suggesting possible applications in other GSDs.
Timal S 1 , Morava E 1 , Hoischen A 1 , Veltman J 1 , Wevers RA 1 , Lefeber DJ 1 1 Radboud Nijmegen Medical Centre, Nijmegen, Netherlands Identification of disease genes in patients with a Congenital Disorder of Glycosylation type I commonly follows a series of biochemical assays, several of those with limited availability. After extensive biochemical and genetic research, still 10% of CDG-I families in our cohort remain unsolved. Next-generation-sequencing techniques offer new alternatives to identify disease genes, thus far mostly applied to several families with identical clinical features. We hypothesized that the functional knowledge of a glycosylation abnormality in CDG-I patients would greatly facilitate gene identification in single cases. Whole-exome sequencing was applied on 6 single cases with CDG-I. After removal of known public and in-house polymorphisms,~600 private variants remained. A number of~60 possible CDG-I gene candidates was selected, based on their location in ER glycosylation, dolichol synthesis or cytoplasmic monosaccharide conversions. In 4 patients, the genetic cause of disease was identified: one as DPAGT1-CDG (a single case reported so far) and 2 novel gene defects. Mutation analysis and biochemical assays confirmed the pathogenicity of the identified variants. In the 2 remaining patients, no variants were found in the 60 candidate genes. This approach shows the huge potential to apply whole-exome-sequencing in metabolic disease, where functional knowledge of the disease is available. In a group of patients with dilated cardiomyopathy, a diagnosis of Congenital Disorders of Glycosylation (CDG) type I was found. After homozygosity mapping in the consanguineous families, pathogenic mutations were identified in DK1 in all individuals, while enzyme activity was deficient in fibroblasts. DK1 encodes the dolichol kinase responsible for formation of dolicholphosphate. In comparison with the severe multisystem presentation in CDG, the clinical presentation in our cohort was dominated by dilated cardiomyopathy, a rare feature in CDG. Expression analysis of DOLK in different tissues showed highest expression in fetal and adult brain, thereby not explaining the tissue specific phenotype. The availability of patient heart biopsy material allowed us to study other dolichol-phosphate dependent glycosylation pathways in affected tissue. N-glycosylation was affected as shown by western blotting of CD63. In addition, reduced O-mannosylation of alphadystroglycan was found with functional loss of its laminin-binding capacity. Loss of functional dystroglycan is known in a subgroup of the congenital muscular dystrophies, commonly presenting with dilated cardiomyopathy. Conclusions: Dolichol kinase deficiency results in decreased availability of dolichol-P-mannose thereby leading to abnormal O-mannosylation of alphadystroglycan. We thus describe a combined deficiency of protein N-glycosylation and O-mannosylation, presenting as late-onset dilated cardiomyopathy. Congenital disorders of glycosylation (CDG) are genetic diseases with an extremely broad spectrum of clinical presentations due to defective glycosylation of glycoproteins and glycolipids. Some 45 CDG types have been reported since the first clinical description in 1980. Protein glycosylation disorders are defects in protein N-and/or O-glycosylation. Dolichol phosphate is the carrier of the N-glycans during their assermbly first at the outside and subsequently at the inside of the endoplasmic reticulum (ER) membrane, and hence is a key molecule in protein glycosylation. Recently, defects have been identified in the last three steps of the dolichol phosphate biosynthesis: dolicholkinase deficiency (DK1-CDG), steroid 5alpha-reductase type 3 deficiency (SRD5A3-CDG), and dehydrodolichyl diphosphate synthase deficiency (DHDDS-CDG). We report on a patient with SRD5A3-CDG carrying a novel (homozygous) mutation. The diagnostic features of this novel inborn error of glycosylation are psychomotor retardation, nystagmus, visual impairment due to variable eye malformations, cerebellar abnormalities/ataxia, and often ichthyosiform skin lesions.
THREE SIBLINGS WITH EXT1-CDG Ezgü Fatih Suheyl 1 , Kasapkara Çiğdem Seher 1 , Okur İlyas 1 , Küçükçongar Aynur 1 , Tümer Leyla 1 , Okur Arzu 2 , Saraç Avni 2 , Wuyts Wim 3 , Hul Els Van 3 , Hasanoğlu Alev 1 1 Div Metab Dis, Gazi University Hospital, Ankara, Turkey 2 Div Ped oncology, Gazi Univ Hospital, Ankara, Turkey 3 Dep Med Genetics,Univ Hosp of Antwerp, Edegem, Belgium EXT1/EXT2-CDG (HME, hereditary multiple osteochondroma) are common defects of O-glycosylation. HME is a rare autosomal dominant disorder characterized by the occurence of multiple benign cartilage capped tumors, that are typically located at juxta-epiphyseal regions of long bones. The diagnostic criteria are at least two osteochondromas of the juxta-epiphyseal region of long bones within the majority of cases with a positive family history and/or mutation in one of the EXT genes. Herein we report three siblings with an age range of 9-14 years who respectively manifested the features of hereditary multiple exostoses. HME affected siblings were referred to outpatient clinic with a lower lumbar spine BMD (z scores of −1.66, -1,9 and −3.18 respectively) and painless progressively increasing bony swellings located at the juxta-epiphyseal regions of long bones as well as other sites. A mutation in EXT1, c.1659 delC (p. Tyr553X), which cosegregated with the disease phenotype, was detected in three siblings. The EXT-1 gene may have a possible additional role in bone metabolism and we should follow these patients not only for the malignant change of exostoses but also for the other complications such as reduction in skeletal growth, restricted joint motion, premature osteoarthrosis and compression of peripheral nerves. Background: DPAGT1-CDG (CDG-Ij) is caused by a deficiency of the first GlcNAc transferase in the ER N-glycosylation pathway. Only one patient has been reported with microcephaly, dysmorphia, exotropia, hypotonia and seizures. Case report: The boy born as the second dizygotic twin by Caesarean section with BW 1410 g and asphyxia. Respiratory distress with frequent apnoeas occurred since birth. Physical examination showed a hypotrophic child with bilateral cataract, cryptorchism, dysmorphia, extremities hypertonia and joint contractures. During the first 3 months: jaundice treated by phototherapy, persistent anaemia-frequent transfusions, feeding difficulties-parenteral and naso-gastric feeding. Then refractory tonic seizures appeared, apnoeas and respiratory insufficiency. Cataract surgery (pars plana lensectomy) at 6 months showed calcification of lenses nuclei. Chest X-ray revealed broncho-pulmonary dysplasia. Biochemical tests showed low serum total protein, mildly elevated aminotransferases and elongated APTT. Metabolic work-up revealed abnormal, type 1 profile of transferrin isoforms suggesting CDG. Activities of PMM, PMI and LLO profile in fibroblasts were normal and patient was classified as unsolved CDG-Ix. He died at age of 2.5 years due to respiratory insufficiency. Genetic defect has been recently identified by whole-exome-sequencing. Conclusions: Congenital cataract is an uncommon manifestation of CDG and a novel finding is calcified nuclear cataract.
RFT1 is an enzyme involved in glycosylation pathway. Its defect causes one of the CDG syndromes. So far there were described six patients all up to the age of six. We present clinical, biochemical and molecular findings of two siblings with RFT1-CDG. First is 18 years old boy with severe mental retardation, profound bilateral hearing loss, epilepsy, obesity, craniofacial dysmorphy, hypothyreosis and mild coagulopathy. His sister is 16 and shows similar symptoms; just the hearing problems are milder. Both patients showed abnormal a type 1 transferrin isofocusing pattern. LLO analysis showed accumulation of DolPP-GlcNAc2Man5 at the cytosolic side of the ER membrane. Molecular analysis revealed two novel heterozygous missense mutations (c.1222A>G/c.1325 G>A) in the 12th exon of RFT1 gene. Our patients show milder phenotype and longer survival compare to the previously published patients. Several glycoproteins are essential in the normal production and regulation of thyroid hormones, including TSH, TBG, thyreoglobuline and the thyroid receptor. We prospectively examined glycosylation, the endocrine and clinical aspects of thyroid function, energy expenditure, and evaluated growth and development in 32 patients with congenital disorders of glycosylation. Thyroid function was abnormal in 19 patients with consistently elevated TSH in ten children, mostly in PMM2-CDG. In four patients, TSH elevations resolved without intervention. Enzymatic desialylation of TBG and TSH had no effect on the laboratory results in these patients and controls. Low free thyroxine was noted in three patients, corresponding to decreased energy expenditure and weight-increase. Low TBG was noted in half of the patients. Thyroid function tests in CDG patients are often abnormal. Most patients appeared to be clinically euthyroid even in the presence of abnormal TBG and TSH values. Still, in certain cases of significantly elevated TSH or low FT4, manifesting in clinical hypothyreoidism, thyroxine supplementation was necessary. TBG deficiency did not appear to have any clinical consequences. Hypoglycosylation of thyroid-proteins may affect laboratory values but may not always be of clinical importance for patients. Measuring energy expenditure in patients helps to decide on the appropriate therapeutic approach. Isoelectric focusing of transferrin and apolipoprotein CIII are used to diagnose N-linked and / or core-1-and 2 O-linked glycosylation defects. Plasma 2D-PAGE studies revealed altered charge and mass forms of C1inhibitor in CDG I and II patients. C1-Inhibitor is a protease inhibitor that plays a crucial role in various physiological pathways. It is heavily glycosylated: 49% of its molecular weight (MW) is composed of six Nand seven core-1 O-glycans. Investigations of C1-inhibitor as a potential marker for both N-& O-linked glycosylation defects was performed on patients with CDG type I and II, including congenital muscular dystrophies, by Western blotting. CDG I cases revealed reduced intensity of the native 100 kDa MW band and a lower MW band. In the CDG II group, samples from patients with FKRP and POMGnT1 mutations showed a normal profile (as expected as these are defects in Omannosylation). However, reduced MW bands were observed in the plasma from a patient with a LARGE mutation (which have defects in both O-mannose and O-GalNAc glycosylation) and also from a muscular dystrophy patient of undefined genetic cause. This demonstrates C1inhibitor could be a combined potential marker for various N-and Oglycosylation disorders. SLC35A1-CDG has been described in a single patient with spontaneous massive bleedings in the posterior chamber of the right eye, cutaneous and pulmonary hemorrhages, thrombocytopenia, neutropenia and recurrent infections. This new type of congenital disorder of glycosylation affects the transport of CMP-sialic acid into the Golgi apparatus. Due to the lethal outcome in the initial patient the genetic background could not be fully confirmed. We evaluated a patient of consanguineous origin, presenting with multiple dysmorphic features, psychomotor retardation, thrombocytopenia, persistent proteinuria and aortic insufficiency. Isoelectric focusing of transferrin showed a type II pattern, while the apolipoprotein C-III isofocusing pattern was normal. Mass spectrometry of serum N-glycans demonstrated severely abnormal sialylation. Mutation analysis in SLC35A1 confirmed a homozygous missense mutation. Functional analysis of the mutations in a CMP-sialic acid transporter assay in yeast showed a 50% reduction in transport activity. Expression studies showed significant gene expression in the central nervous system, and the kidneys, comparable with mental retardation and chronic proteinuria in our patient. We report on the second SLC35A1-CDG, and first adult, patient, presenting with intellectual disability, thrombocytopenia, renal and cardiac involvement due to a CMP-sialic acid transporter defect. The bleeding disorder remained asymptomatic until adulthood.
Lefeber DJ 1 1 Radboud Nijmegen Medical Centre, Nijmegen, Netherlands In the framework of the European program Euroglycanet, a quality control scheme was set up for the screening of Congenital Disorders of Glycosylation by analysis of transferrin glycosylation. Reproducibility problems after 3 rounds among Euroglycanet members necessitated an elaborate stability experiment. Serum samples were stored at different temperatures and lyophilisation conditions, after which the stability was tested using transferrin isofocusing during a 1-year follow up. Samples lyophilized in the presence of cryoprotectant were stable at shipment conditions for at least 2 months. Since 2009, the scheme is supervised by ERNDIM and open to other laboratories world-wide. Currently, 51 centres participate. In view of the very limited amount of patient material, the scheme operates with 20 microliter serum per sample. Different techniques are used for analysis of transferrin glycosylation, including isofocusing, capillary electrophoresis, HPLC and mass spectrometry. The samples originate from patients with a known CDG subtype or a secondary cause of abnormal transferrin glycosylation. The results are interpreted against a clinical information, provided with the samples. Scoring is based on profile interpretation and suggestions for follow-up diagnostics, and showed clear improved over the years. The results of this novel CDG QC scheme in ERNDIM's regular QC program will be presented.
Haas D 1 , Hoffmann GF 1 , Burgard P 1 1 Div Inb Metab Dis, Univ Children's Hosp, Heidelberg, Germany Background: Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation syndrome due to a deficiency of 7-dehydrocholesterol reductase resulting in an accumulation of 7-and 8-dehydrocholesterol in serum and amniotic fluid (AF). In affected pregnancies free estriol is often reduced and in prenatal ultrasound intrauterine growth retardation (IUGR) and malformations of CNS, heart, kidney, genitals and limbs are often detected. Objectives: Evaluating the specificity of features leading to a suspicion of fetal SLOS. Material and Methods: From 2002 to 2011 we determined sterol concentrations in AF samples from 69 pregnancies suspicious for SLOS by gas chromatography mass spectrometry. Results: Sterol analysis in AF was done in 24 cases because of the family history (affected sibling: n=14, other affected family member: n=5, lethal malformations in a sibling: n=5). In 39 cases prenatal abnormalities were determined (reduced estriol: n=10, multiple fetal malformations: n=15, IUGR: n=11). In 6 cases there was no clinical information. SLOS was diagnosed in 6 fetuses (8.7 %). 3 of those had an affected sibling, 3 had multiple malformations, one additionally a reduced estriol concentration. Conclusion/Discussion: The highest rate of confirmed SLOS was in at risk families, whereas fetal malformations were in most cases not caused by SLOS.
Kilic M 1 , Tokatli A 1 , Alanay Y 2 , Kilic E 2 , Kalkanoglu-Sivri HS 1 , Dursun A 1 , Önol S 1 , Haliloglu G 3 , Utine GE 2 , Boduroglu K 2 , Coskun T 1 1 Pediatr Metab Dis, Hacettepe University, Ankara, Turkey 2 Pediatr Genet, Hacettepe University, Ankara, Turkey 3 Pediatr Neurol, Hacettepe University, Ankara, Turkey Background: Smith-Lemli-Opitz Syndrome is an autosomal recessive disorder characterized by cholesterol synthesis defect. Yet there is no current proven therapy, studies so far indicate cholesterol therapy and HMG-CoA reductase inhibitors like Simvastatin improve growth, behavior and general wellness. Methods: Our study included four patients between 31 and 71 months of ages. Results and Conclusions: Male to female ration is 1/3 and consanguinity is present in 25 percent of cases. All patients presented around 2-3 months of ages with failure to gain weight and received a diagnosis before age 1. A characteristic facial appearance, microcephaly, submucosal cleft palate, cutaneous syndactyly, mental retardation, behavioral and feeding problems, postnatal growth retardation were present in all cases; and prenatal growth retardation was seen in half of cases. Cholesterol therapy of 100 mg/kg/day was started. During follow up increase in cholesterol level was observed while 7-dehydrocholesterol level was not decreasing. Patient's parents didn't consent with Simvastatin therapy due to unknown efficiency and possible side effects. With therapy; growth parameters, head circumference increased in accordance with growth chart rates. Except for one case, none of the patients reached to the 3 rd percentile. With regard to behavior, improved skills in every area was detected albeit little.
Roullet J-B 1 , Impey S 1 , Yang Q 1 , Steiner RD 1 1 
Background: SLOS is caused by inactivating mutations of the DHCR7 gene leading to cholesterol deficiency. It is characterized by a broad phenotypic spectrum typically including congenital malformations and mental retardation. SLOS pathophysiology remains poorly understood and there is no effective treatment. Objectives: to characterize the molecular changes caused by DHCR7 deficiency. Specifically, to obtain baseline gene expression profiles in affected and non-affected cells. Methods: Genome wide expression profiling (RNA-Seq) was performed using skin fibroblasts isolated from a patient with severe SLOS and neural stem cells (NSC) isolated from Dhcr7−/− mice (E18). Results: The SLOS fibroblast gene expression profile showed down regulation of genes involved in mitochondrial function, calcium homeostasis, cytoskeleton, lysosomal function, signaling (caveolin1, Na+/K+ ATPase), folate metabolism and defense against oxidative stress. Genes related to apoptosis, ubiquitination and extracellular matrix synthesis were upregulated. Such expression profile was not observed in Dhcr7−/− NSCs. In these cells, stem-cell marker nestin and cell-replication genes were downregulated whereas genes implicated in synaptic transmission, signaling, and cell differentiation were upregulated, suggesting abnormal NSCto-neuron differentiation in vivo. Conclusion: Cell biology is profoundly altered in SLOS. Future studies with cell lines from other patients and from NSC-derived neurons are needed to confirm and expand these findings. Background: An increase of plasma 25-and 27-hydroxycholesterols (25-OHC and 27-OHC) have been reported in hereditary spastic paraplegia type 5 (SPG5). In addition, 24-S-hydroxycholesterol (24-OHC) may be a biomarker of disease progression in patients with Huntington disease (HD) and Niemann Pick C1 (NPC1). Therefore, we have developed a highly sensitive and specific method to measure simultaneously these 3 plasma oxysterols. Method: Plasma oxysterols 24, 25 and 27-OHC were analysed by a LC-MSMS method with isotopic dilution after sterols derivatization into picolinyl esters (Honda et al; 2008) . Derivatized sterols were analysed by an UPLC-TQD system, consisting of a triple quadrupole mass spectrometer equipped with an ESI probe and UPLC-system. Oxysterols were separated on a C-18 RP-BEH column and detected by using multiple reaction monitoring. Results: This method provides high precision and accuracy (intra-assay reproducibility 7% CV, inter-assay reproducibility 10%, accuracy 93-106%). We identified a 15 fold increase of 25-OHC and a 7 fold increase of 27-OHC ratios to cholesterol in 8 SPG5 patients compared to 10 controls. Conclusion: The present UPLC-MS method provides a reliable and reproducible tool to quantify oxysterols in order to identify new SPG5 patients and to monitor disease progression in HD and NPC1 patients.
Eyskens FJM 1 , Singh B 2 , Simons A 2 , Beckx K 2 1 Antwerp Univ Hospital, Div,Metab Dis, Antwerp, Belgium 2 ZNA-UKJA, Child psychiatry, Antwerp, Belgium Background: CTX is an autosomal recessive defect in bile acid biosynthesis with accumulation of cholestanol in most tissues and body fluids. The enzyme Sterol 27-hydroxilase is deficient and mutation analysis is possible of the CYP27A1gene The clinical picture of juvenile cataract, xanthomas and a progressive neurologic disorder is very suggestive. Patients: Two brothers were diagnosed at the age of 12 and 14 years with cataract, a cerebellar ataxia, a peripheral neuropathy, absence of xanthomas, and a chronic diarrhea from early childhood. They had a low IQ (range 60-78), speech disturbances and normal neuroimaging. Molecular studies established the diagnosis of CTX. Treatment objectives and results: They first received only chenodeoxycholic acid which resulted in an improvement of well being, strength, growth and speech and a spectacular disappearance of the steatorrhea. When a HMG-coA reductase inhibitor Atorvastatin was added to the treatment, the bile alcohols excretion in urine normalized which could not be achieved with Simvastatin. Follow-up under the combined therapy showed an improvement of several cognitive functions although the IQ remained in the same range. The peripheral neuropathy disappeared under treatment. Conclusion: Chenodeoxycholic acid and a HMG-coA reductase inhibitor, especially Atorvastatin, should be combined in the treatment of CTX patients. We present a case of a consanguineous child diagnosed on newborn screening with medium chain acyl-CoA dehydrogenase deficiency (MCADD) who went on to develop significant failure to thrive. His weight, height and head circumference were all below the 0.4th centile. Investigations revealed a low vitamin E but only marginally low apo-B, not consistent with either abetalipoproteinaemia or hypobetalipoproteinaemia. Attempts at nasogastric, lactose free and hydrolysed feeds failed to achieve weight gain. An endoscopy and small bowel biopsy demonstrated lipid laden enterocytes. Further investigation demonstrated he fulfilled the criteria for chylomicron retention disease (CRD). Mutation analysis showed him to be homozygous for the SAR1b gene (c.409 G>A) with a polymorphism in exon 7. This mutation has previously been seen in eight French-Canadian patients, but the polymorphism seen is novel. Management of CRD usually requires a medium chain triglyceride (MCT) based diet however this was impossible due to his MCADD. He has instead been treated with a non-MCT based, high carbohydrate diet and high dose vitamin E. He has made excellent progress in terms of growth. This case is of interest as it provides additional insight into intracellular trafficking and describes the unique challenges in its management due to co-morbidity with MCADD. Background: Primary disorders of exogenous lipoprotein metabolism can cause recurrent morbidity including pancreatitis. Currently, restriction of long chain fat intake is the only established management. Methods: A retrospective review of the clinical course and biochemistry of all children who presented with type 1 hyperlipoproteinaemia over a 15 year period. Results: Of the 14 patients, 8 were diagnosed before age 6 months (4 presented incidentally, 1 on family screening and 3 with rectal bleeding). Of those diagnosed after age 6 months, 2 presented with pancreatitis, 2 on family screening and 2 were fortuitous. No patient whose serial triglyceride levels remained <10 mmol/l (n=6) was symptomatic, but 7 of the 8 patients whose levels were recurrently >10 mmol/l had episodic abdominal pain and 5 had pancreatitis. There was no significant difference (p=0.524) in mean triglyceride levels between those with abdominal pain (17.93+/− 2.339) and those with pancreatitis (15.59 +/−2.766). No adverse effects on growth were seen. Conclusions: In over half of all patients adherence to a strict low long chain fat diet was not achievable and was associated with increased morbidity, especially pancreatitis. Background: Sjogren Larsson syndrome (SLS) that is a rare inherited disorder caused by mutations in the ALDH3A2 gene for fatty aldehyde dehydrogenase (FALDH) presents with mental retardation of variable severity, spastic or quadriplegia, and generalised ichthyosis at birth. FALDH catalyzes long-chain aldehydes derived from lipid metabolism. More than 80 mutations in ALDH3A2 have been reported worldwide. Here we report two novel mutations in Turkish patients with SLS. Case Report: Patient 1: 7 years old male patient born of consanguineous marriage had generalised ichthyosis at birth. Evaluation because of mental retardation, spastic quadriplegia and ichthyosis at the age of 6 years demonstrated profoundly low level of FALDH [0.02 nmol/(min.mg)] in skin fibroblasts. Molecular analysis confirmed the diagnosis of SLS and revealed a novel mutation (homozygous c.274_281del8ins1) in the ALDH3A2 gene. Patient 2: 10 years old male patient born of non-consanguineous marriage had generalised ichthyosis at birth. SLS was suspected with the findings of severe mental retardation, spastic quadriplegia and generalised ihthyosis at 9 years of age. Subsequent molecular analysis revealed homozygous c.153+5_385+ 361del3011ins19 mutation (full-length exon 2 deletion) in the ALDH3A2 gene. Conclusions: We predicts that described novel mutations in our patients with typical clinical features of SLS cause premature termination codon. 3 Tarbiat Modares Univ,Depart of Medi Gen, Tehran, Iran, Islamic Republic of Background: Cerebrotendinous xanthomatosis is a rare genetic disorder of cholesterol and bile acid metabolism that results in systemic and neurologic abnormalities. It was first described by Van Bogaert and has been characterized clinically, biochemically, and genetically (mutations in the gene CYP27A1). The disease begins in infancy with chronic diarrhea. Cataracts become evident in childhood or adolescence, and xanthomata develop in the second and third decades of life. Significant neurologic impairment includes seizures, dementia, and extra pyramidal dysfunction and begins in the third decade of life and progresses until death. The presentation and course widely varies, and treatment can dramatically alter, especially with early initiation. Case Report: We report an Iranian family with three affected child who are suffering from Cerebrotendinous Xanthomatosis. Cardinal features were: Motor dysfunction, ataxia, spastic paresis, Xanthomas of the Achilles tendon, Cataracts double. MRI of the brain show diffuse cerebral atrophy and increased signal intensity in the cerebellar white matter on T2weighted scans. In this report, we present 3 patients with Cerebrotendinous Xanthomatosis, who confirmed by Molecular Analysis. The patients are suffering from disease resulting from a homozygous splice-mutation in intron 2 of the CYP27A1 gene. Key word; Cerebrotendinous Xanthomatosis, Motor dysfunction, CYP27A1 gene. Background: Fabry disease (FD) is characterized by deficiency of alphagalactosidase A leading to accumulation of globotriaosylceramide (GL-3). Urine levels of GL-3 are elevated in males and females. Objectives: To develop a fully validated GLP assay to measure isoforms of GL-3 (C22:0 and C24:0) that are representative of GL-3 found in the kidney. This assay is designed to accurately quantify GL3 in urine from females with high residual enzyme activity resulting in lower levels of GL-3. Methods: Well characterized synthetic reference standards were used to validate for C22:0 and C24:0 isoforms of GL-3. The assay is validated as per the 2001 US FDA Bioanalytical Method Validation guidance. Results: The validated assay is able to accurately, specifically and reproducibly quantify C22:0 and C24:0 isoforms. Lower limit of quantitation for this assay is 1 ng/mL for both isoforms. Inter-assay precision and accuracy for six validation runs is as follows: C22:0 Precision (4.5 to 15.1%) Accuracy (−0.92 to −4.9%), C24:0 Precision (5.6 to 7.1 %) Accuracy (−3.5 to −11.6%). Normal GL-3 range was determined in 38 healthy males and females using the validated assay. Background: Retrospective studies report discordant rates of change of GFR (ΔeGFR) (−2.9-12.2 ml/min/year) in untreated males with FD. Most studies of ERT rely on historical controls, so prospective data not affected by ascertainment biases on ΔeGFR are needed. Objectives: Describe prospectively ΔeGFR in FD subjects Methods: CFDI subjects not meeting criteria for ERT (Cohort1c) and those who met treatment criteria and were started on ERT through the CFDI (Cohort1b) were included. Data on ΔeGFR were analyzed by gender and stratified by baseline proteinuria (using a threshold of 0.3 g/day). Results: 97 subjects (84 F, 13 M; median followup 35 months) did not meet criteria for ERT (Cohort 1c). 44 subjects (29 F, 15 M; median followup 38.5 months) were randomized to ERT through the CFDI (Cohort 1b). ΔeGFR rates were: Cohort 1b: M-3.96 ml/min/1.73 m2/year F +1.56 ml/min/1.73 m2/year Cohort 1c: M-2.16 ml/min/1.73 m2/year F 0.0 ml/min/1.73 m2/year. As expected, ΔeGFR tended to be higher in subjects above the proteinuric threshold then those below but these trends were not statistically significant due to limited numbers. Conclusions: ΔeGFR is lower than previously reported. ERT does not restore ΔeGFR to values seen in subjects with no clinically apparent renal disease. Conflict of Interest declared. Female carriers of Fabry disease have a high tendency of developing vital organ damage causing severe morbidity and mortality. According to our newborn screening study, we found most female carriers were not detected by enzyme assay. In this study, we developed a streamlined method for HRM analysis to screen the mutations of GLA gene using a single PCR programme and a single melting profile. A total of 299,007 (156,179 males) newborns were screened for Fabry disease at our newborn screening centers. From this screening, we identified 121 (106 males) newborns carrying Fabry mutations. A total of 20 different mutations were identified in these patients. Both male and female patients were enrolled in the HRM analysis study. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919 G>A of GLA gene. PCR and HRM analyses were performed using a Roche LightCycler. 480. Both the heterozygous and hemizyous patients of these 20 mutations could be easily identified by HRM analysis. We also successfully used this method to test the dry bloodspots of the newborns with Fabry mutations without the need of determining DNA concentration before PCR amplification. Background: Fabry disease is an X-linked lysosomal disorder resulting from mutations in alpha-Galactosidase A (GalA) gene. Recent reports described that E66Q mutation in GalA gene is not a disease-causing mutation based on enzymatic studies. We carried out enzymatic and pathological studies on a patient with E66Q mutation in GalA gene. Case: A 34 years old male patients with end-stage renal failure and cardiomegaly who referred to our hospital for mutation analysis of GalA gene. He was diagnosed as gout in 15 years old and hemodialysis was started for gouty nephropathy from 31 years old. We carried out enzymatic and genetic analysis for GalA and cardiac biopsy. Result: He had E66Q mutation in GalA gene. GalA activity in leukocytes was 46.2% of average of normal controls and serum globotriaosylceramide (GL3) level was not elevated. The pathological study on cardiac biopsy sample showed no characteristic findings of Fabry disease. The immunostaining for GL3 of cardiac biopsy sample showed no positive cells. Conclusion: Although E66Q mutation reduced enzyme activity, the characteristic pathological findings of Fabry disease and the abnormal accumulation of GL3 were not detected. E66Q mutation of GalA gene was thought to be a functional polymorphism based on enzymatic and pathological studies. Conflict of Interest declared. We present PGD of Fabry disease (FD). Female in her thirties is FD carrier with positive family history. Her previous pregnancy was aborted. The risk 50% for offspring resulted in her informed decision for PGD in conjunction with genetic counseling. Our aim was to design specific-family test based on linkage analyses with GLA adjacent polymorphic markers avoiding the need to detect the mutation itself. Methods: Molecular analysis identified the pathogenic familial mutation c.1025 G>A (p.Arg342Gln) located in exon 7 of the GLA gene (GLA; EC 3.2.1.22), locus Xq22. In the IVF cycle we used genetic haplotyping technique by specific FD multiplex PCR on products of multiple displacement amplification (MDA) from one blastomere biopsied from the cleavage-stage embryo. Results: There was at least one unaffected embryo available for transfer on day 3, resulting in ongoing pregnancy after first attempt. Conclusion: The karyotype was denoted 46,XX from amniocentesis. Direct DNA analysis of the GLA gene did not confirmed causal mutation. The pregnancy is followed till delivery. Fabry disease is an X-linked disorder of alpha-galactocidase A which causes the accumulation of glycolipids in lysosomes. The incidence of the classical type of the disease is approximately 1 in 40,000 males. Recent studies have revealed the late-onset type of the disease to have a higher frequency than previously known. To determine the disease incidence in Japan, we screened newborns to measure alpha-galactosidase A activity in dried blood spots from Japanese neonates. Enzyme-deficient infants were retested, and infants who were double-screening positive were diagnostically confirmed by enzymatic activity and mutation analyses. Thirty eight neonates had a deficiency in alpha-galactosidase A activities and specific mutations, including 5 neonates with classical mutations identified previously. Based on our newborn screening in Japan, the incidence of alpha-galactosidase A deficiency was 1 in 5,600 male. Based on enzymatic activities, the incidence was 1 in 6,000 male. These results suggest that the late-onset phenotype of Fabry disease is underdiagnosed among both males and females in Japan. The recognition of the existence of these patients suggests the need for both early diagnosis and therapeutic intervention. However, ethical issues need to be taken into consideration in terms of when and whom the screening should be performed. Introduction: Fabry disease(FD) is a lysosomal disorder caused by the deficiency of a-galactosidase A, which leads to storage of globotriaosylceramide(Gb3) and endothelial disease with involvement of kidney, heart and the nervous system. Presentation in female patients can be very late in life and usually is suspected from family history. We report a female patient(20yo) with an unusually presentation of FD. Case report: Patient was admitted to the ER with purpuric rash in buttocks and lower extremities. After seven days she started with joint and abdominal pain. Lab exams showed proteinuria and hematuria and the diagnosis of Henoch-Schonlein Purpura was made; metilprednisolone 1 mg/kg IVand anti-hypertensive were started. Skin biopsy was performed and showed neutrophilic vasculitis with storage of IgM and granular C3. A kidney biopsy showed vacuolated podocites, suggesting FD. The assay of α-galA showed normal enzyme activity in leukocytes. Molecular analysis has shown one heterozygous mutation in exon 5(c.644A>Gp.N215S). Discussion:The patient has no other signs or symptoms of FD, and has no family history of FD. The clue to the diagnosis was a kidney biopsy, performed as she did not improve of the proteinuria with the standard treatment. Although unusual, Fabry disease may be considered in the evaluation of Henoch-Schönlein purpura. Methods: A 28-item measure of symptoms, Fabry Outcome Survey (FOS) Paediatric Health and Pain Questionnaire was developed. FOS is a registry for all patients with Fabry disease who are treatment naïve or receiving enzyme replacement therapy (ERT) with agalsidase alfa. A battery of psychometric analyses was performed to assess the measurement properties of this new instrument. Results: 87 children (age 4-18 years) completed the questionnaire. 23 items in three subscales emerged: Pain associated with heat or exertion; pain associated with cold; abdominal pain and fatigue. Internal consistency reliability for all three subscales was good (α≥0.84) and was high for all age groups (4-7, 8-12, 13-18 years) . Test-retest reliability was high (intraclass correlation coefficient≥0.74). Construct validity showed that each subscale measured unique patient symptom experiences. Conclusions: Psychometric analyses indicate that the measurement properties of the three subscales are valid and reliable for measuring patient-reported symptoms of FD. The questionnaire could be a useful tool for clinicians to understand the progression of disease and monitor treatment effects. Conflict of Interest declared.
Background: Globotriaosylceramide (Gb3) was assessed as a surrogate marker to predict change from baseline after 12 months of agalsidase alfa (agalα) treatment (CFB-M12) in estimated glomerular filtration rate (eGFR) and left ventricular mass index (LVMI) using pooled data from three 24-week, randomized, placebo-controlled trials (RCTs; TKT003/ TKT005/TKT010) and their open-label extension studies (EXT; TKT006/ TKT007/TKT013/TKT015) of patients with Fabry disease (FD). Methods: Males (≥18-year-old) with confirmed FD received agalα (0.2 mg/kg every other week) for 12 months. A backward elimination approach evaluated potential predictors (baseline and CFB-M12 Gb3 [urine; plasma], urine protein; baseline eGFR; age at first dose; baseline LVMI). Subgroups included patients randomized to placebo during RCTs (pbo->agalα) or to agalα (agalα->agalα), or with stage 2/3 chronic kidney disease (CKD2/3). Results: In the analysis population (n=73), eGFR (baseline) and plasma Gb3 (baseline; CFB-M12) significantly predicted CFB-M12 eGFR (all P< 0.05). No predictors of CFB-M12 LVMI were significant (n=39). In the pbo->agalα subgroup (n=36), only plasma Gb3 (baseline; CFB-M12) significantly predicted CFB-M12 eGFR (P<0.05). No significant predictors were found in other subgroups. Conclusion: Urinary and plasma Gb3 concentrations were not useful biomarkers for FD progression in agalα-treated patients. Conflict of Interest declared. Bioavailability was ≥50%; food reduced bioavailability by 40%. Plasma levels increased proportionally up to 1250 mg; no further increase was observed at 2000 mg. Steady-state was achieved within 5 days; accumulation ratios were 1.2 to 1.8. Unchanged drug was eliminated by the kidney with a half-life of 4 hours. Dose-related increases in WBC α-Gal A activity were observed after 7 days of dosing. AT1001 was well tolerated, with dizziness, headache and skin irritation the most commonly reported adverse effects. Conclusion: Migalastat was rapidly absorbed and exhibited linear pharmacokinetics. Dose-related increases in α-Gal A activity in PBMCs were observed. Conflict of Interest declared. Background: Fabry disease is caused by mutations in the gene (GLA) that encodes alpha-galactosidase A (alpha-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is an orally-available pharmacological chaperone that selectively binds and stabilizes alpha-Gal A, increasing total cellular levels and activity for some mutant forms. AT1001 is currently under clinical development for the treatment of Fabry disease. Methods: A validated, transient transfection-based assay was developed in HEK-293 cells to test 460+ Fabry disease-causing mutant forms (predominantly missense) for response to AT1001. Responses were measured by enzyme assay and western blot. Transfection efficiencies across independent assays and mutations were assessed by quantitative real-time PCR. Results: Increases in both alpha-Gal A protein level and enzyme activity were seen for~60% of the mutant forms. Importantly, the HEK-293 cell responses of 19 alpha-Gal A mutant forms to a clinically-achievable concentration of AT1001 (10 μM) were generally consistent with observed increases in alpha-Gal A activity in peripheral blood mononuclear cells from male Fabry patients administered AT1001 during Phase 2 clinical studies. Conclusions: These results suggest that the alpha-Gal A increase in cultured cells may be used to identify Fabry patients that could benefit from AT1001 therapy. Conflict of Interest declared.
Haynes BA 1 , Bragat A 1 , Sitaraman-Das S 1 , Schiffmann R 2 1 Amicus Therapeutics, Cranbury, NJ, United States 2 Baylor Research Institute, Dallas, TX, United States Background: Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by deficiency of alpha-galactosidase A with accumulation of globotriaosylceramide (GL-3) in tissues and body fluids, leading to progressive, multi-organ disease. Objective: To demonstrate the utility of a validated GLP assay to quantify two isoforms of urinary . Methods: Two kidney-related isoforms were measured in total 24 h urine in 120 untreated Fabry subjects from the FACETS clinical study and 38 healthy controls. C22:0 and C24:0 isoforms were quantified after urine homogenization and sonication; uGL-3 concentration is expressed relative to creatinine (ng/mg Cr). Preliminary Results: Males with Fabry disease have elevated levels of uGL-3 (n=38; mean+SD=888+865; median=846; range 9.8-3504) when compared to FD females and controls. Levels of uGL-3 in females ranged from 3.8 to1079(n=82; mean=183+192; median=143), overlapping with the range for the affected male population. In females, the mean level of uGL-3 was higher than that of the control group (control group mean= 17.1+8.3; median 14.5; range 7. 3-50.3) . Conclusion: This new analytically validated uGL-3 assay was used as a study screening tool to assess baseline uGL-3 levels in males and females with Fabry disease. It is anticipated that this assay may be useful to monitor treatment effects in FD subjects. Conflict of Interest declared. Background: NP-C diagnosis can be delayed for years due to heterogeneous presentation. A Suspicion Index was developed, ranking specific symptoms within and across domains, and with family history, providing a predictive score to help identify suspected patients with NP-C. Objectives: To construct a Suspicion Index and validate its sensitivity and specificity using patient data. Methods: A retrospective chart review in seven centres in Europe and Australia (N=216). Three patient types were selected: classical or variant filipin staining NP-C-positive cases (n=71); suspected NP-C cases that were filipin staining negative (n=65); or non-cases (without NP-C suspicion) with ≥1 characteristic symptom of NP C (n=80). Signs and symptoms of NP-C were categorized into visceral, neurological or psychiatric domains, and scores assigned according to their relative prediction for NP-C. Results: The Suspicion Index has good discriminatory performance (area under the ROC curve >0.75) with cut-points for grading suspicion of NP-C. Neonatal jaundice/cholestasis, splenomegaly, vertical supranuculear gaze palsy, cataplexy, and cognitive decline/dementia were strong predictors of NP-C, as well as symptoms occurring in multiple domains in individual patients, and siblings/parents with NP-C. Conclusion: The Suspicion Index is a valid tool that can help identify patients who may warrant further investigation for NP-C. Conflict of Interest declared. Methods: Retrospective study and review of laboratory/neuroimaging data were carried out in NPC patients diagnosed in the last 6 years. Results: Twenty-six patients were confirmed to have NPC by filipin staining ( 14 patients required molecular analysis). Regarding clinical form, 7 were perinatal , 16 infantile, 11 juvenile and 8 adults. Prolonged neonatal jaundice was a common feature and five of them were diagnosed with "neonatal hepatitis". Hepatosplenomegaly was present in all perinatal/ infantile patients, but absent in 6 adults. Ocular abnormalities were seen in 38 patients ( mostly, vertical supranuclear gaze paralysis). Leukoencephalopathy and progressive cerebral/cerebellar atrophy were the main MRI features. Other systemic manifestations included dystonia, cataplexia, immunodeficiency, dysphagia. At the time of the study, 13 patients were deceased. Conclusions: Onset of neurological disease in NPC patients is extremely variable, even in the same sibship. Better understanging of the natural history of the disease is crucial for evaluation of potential therapeutic approaches in such devastating disorder. Niemann-Pick type C (NPC) is a rare autosomal-recessive disorder caused by accumulation of unesterified cholesterol and glycosphingolipids in the lysosomes. Clinically is characterized by progressive neurological deterioration and hepato-splenomegaly. The aim of this study was to analyze the activity of sphingomyelinase, beta-glucosidase, beta-galactosidase and chitotriosidase in blood samples from patients with NPC and correlate this activity with that of normal individuals. Our results showed that NPC patients have a decreased sphingomyelinase activity (63%) than normal subjects (NPC=1.52 ±0.52 nmol/h/mgprot; normal=2.39±1.14 nmol/h/mgprot) and this difference was significant (p<0.04). NPC patients have 11.40±2.82 nmol/h/mgprot of beta-glucosidase activity and 1882±2461 nmol/h/mL of chitotriosidase activity compared to 14.8±4.49 nmol/h/mgprot for beta-glucosidase and 58.5±34.6 nmol/h/mL of chitotriosidase from normal individuals. The differences between the two groups are significant for both enzymes (p< 0.02 and p<0.0002 for beta-glucosidase and chitotriosidase, respectively). Beta-galactosidase did not differ between the two groups (NPC=152.6± 52.8 nmol/h/mgprot; normal=125.0±38.6 nmol/h/mgprot). These results allow us to conclude that cholesterol accumulation may be interfering with the metabolism of other sphingolipids such as glucosylceramide and sphingomyelin, affecting the activity of their degrading enzymes. Niemann-Pick disease type C (NPC) is an autosomal recessive condition caused by defects on cholestherol trafikking, which leads to a progressive and usually severe visceral and/or neurologic syndrome. The signs and symptoms overlap with other conditions, and diagnosis is difficult as usually requires a staining test performed on cultured fibroblasts and/or comprehensive molecular studies of the two NPC genes (NPC1 and NPC2). The possibility of treating affected patients with substrate reduction therapy (miglustat) makes more important the correct and timely identification of affected patients. With this aim we set up a comprehensive diagnostic program for NPC in Brazil, which includes: 1) providing information on diagnostic procedures, including a collection and transportation kit (for skin biopsy and blood collection); 2) assay of plasma chitotriosidase; 3) culture of skin fibroblasts and Filipin staining; 4)DNA isolation and molecular analysis of NPC1 and NPC2 gene, depending on results of the Filipin testing. This protocol was performed in 140 patients with suspected NPC, referred from physicians from all Brazilian regions. Diagnosis of NPC was confirmed in 15 cases, being abnormal but not conclusive in further 12 patients. These 27 cases were referred to molecular analysis of both genes to complete investigation (molecular analyses in progress). Conflict of Interest declared. Niemann-Pick C (NPC) disease is an autosomal recessive condition related to cholesterol trafficking defects. We describe a male patient and her aunt with NPC, with different genotypes and phenotype heterogeneity. Case 1: male,born from non-consanguineous parents,presented with normal development until the age of 2 years when started generalized seizures. The brain MRI showed hyperintense periventricular signal. At 3 years of age showed hepatosplenomegaly,umbilical hernia and spastic quadriplegia. Plasma chitotriosidase was increased (1749 nmoles/mL)and Filipin test in fibroblasts was strongly positive. Molecular analysis showed different mutations on the NPC1 gene(G1140V and L1157P). The patient died at 5 years due to respiratory complications. His aunt(father's sister),daughter of non-consanguineous parents,began at the age of 4 years with seizures, jerky movements who progressed with cognitive impairment, motor disorders,hallucinations and aggressiveness. At the age of 26 years she presents ataxia, dysmetria, choreoathetosis and absence of convergence and vertical saccades. The Filippin test in fibroblasts was positive. Molecular analysis of the NPC1 gene showed only one mutation (L1157P),indicating that the patient would be a carrier or would present a complex genotype. Although genetic heterogeneity could explain the different phenotypes observed in this family,further molecular studies(in progress)should help to clarify this interesting situation. (2-13.5y )] were followed in our centre between 2007 and 2011. They fell into 4 subgroups: 5 patients presented only with visceral symptoms (mean age of onset: 1 month) followed by neurological symptoms in the early infantile period in 3 of them (M: 12 m). Four patients (4-12 y) presented with neurovisceral symptoms and 2 patients presented with pure neuropsychological symptoms (3-7y) . Six patients were treated by miglustat for a mean period of 14 months (6-22 m). All patients underwent clinical assessments including neuropsychological tests, Pineda disability scale and MRI with diffusion tensor imaging (DTI) and spectroscopy (MRS). The mean dose of miglustat was 443 mg (457 mg/ m2). In treated patients neurological deterioration was observed in 4 and stabilization in 2. MRI showed retarded myelination but normal MRS. Fractional anisotropy (FA) was low in most patients compared to controls and seemed to correlate to age and disease severity. FA increased in some treated patients. Miglustat was well tolerated except in one patient who interrupted treatment. Response to treatment was variable with a trend for stabilization in patients treated at an early stage of the disease. Morbus Niemann -Pick type C (NPC) is a neurovisceral lysosomal storage disorder caused by mutations of either NPC1 or NPC2 gene. It results in abnormal cellular cholesterol trafficking and secondary accumulation of glycosphingolipids. The precise mechanism leading to clinical symptoms is unknown. One potential mechanism may be altered composition of cellular membrane lipids with subsequent impairment of lipid raft function and protein trafficking. Miglustat (N-butyl-deoxynojirimycin, NB-DNJ), an N-alkylated imino-sugar, inhibits the ceramide-specific glucosyltransferase has been advocated in the treatment of NPC. Methods and results: We examined the effects of NB-DNJ on membrane trafficking in fibroblasts of NPC patients and controls. We found substantial reduction in glycosphingolipid levels in the patient's fibroblasts after treatment with NB-DNJ. Furthermore, NB-DNJ resulted in a partial restoration of cholesterol-and sphingolipid-enriched membrane microdomains (lipid rafts) as assessed by increased levels of flotillin 2 (a protein marker of lipid rafts) in the floating fractions of sucrose density gradients in Triton X-100 lysates of NB DNJ-treated NPC fibroblasts. Finally, the enzymatic activities of mitochondrial inner membrane complexes II and IV were partially restored by NB-DNJ. Conclusion: Our study demonstrates that NB-DNJ as a potential therapeutic agent for NPC patients reverses membrane lipid abnormalities in NPC fibroblasts. Conflict of Interest declared.
PERSISTENT EFFECT OF MIGLUSTAT ON CHILDREN WITH NIEMANN-PICK C DISEASE Chien YH 1 , Peng SF 1 , Hwu WL 1 , Yang CC 1 , Lee NC 1 , Tsai LK 1 , Huang AC 1 , Su SC 1 , Tseng CC 1 1 National Taiwan University Hospital, Taipei, Taiwan
Niemann-Pick C disease (NP-C) is characterized by the accumulation of unesterified cholesterol and glycolipids in the lysosomes of cells in the nervous system and visceral organs. Miglustat, an iminosugar reversibly inhibits glucosylceramide synthase, has been approved for the treatment of Niemann-Pick C disease. We treated five children with NP-C disease. The median age for the first neurological symptom onset was 7.5 years (range 5-9 years). They were treated by miglustat since the median age of 12.3 years (range 9-14). Before treatment, all cases had mental impairment, 4 cases had motor impairment, and 4 cases had difficulties in swallowing. After treatment, improvement in swallowing was the most prominent and immediate finding. Although the condition of swallowing quality deteriorated in some patients after 1 year of treatment, probably due to drug interruption, both penetration-aspiration scales and dysphagia severity scores did remain stable in 3 of 5 patients after 3.7-5.7 years of therapy. Other domains which improvement has been found include motor function, cognitive function, and borderline horizontal eye pursuit movement. This study suggests that miglustat can provide therapeutic benefits in neurological symptoms and stabilize the disease in children affected by NP-C.
Introduction: Niemann-Pick type C (NPC) is an autossomal recessive disorder of cholesterol intracellular trafficking. The age of presentation extend into adulthood and the late forms have dominant neurological and psychiatric affectations. Substract deprivation therapy with miglustat could changed the course of the disease Case Reports: We present two brothers with the diagnosis of NPC at 21 and 26 years old. The first patient presented at 21 years old. He had an excellent school performance until 19 years old, when he begun a slow neurological deterioration associated with dysartic speech, saccadic eye movements, limb hypotonia, dismetry and an ataxic broad based gait. The biochemical study of cholesterol intracellular trafficking in fibroblasts confirmed NPC.The mollecular study identified a mutation in NPC1 gene. His oldest brother was being followed in Psychiatric consultation for psycosis and the same NPC1 gene mutation was confirmed. Both started therapy with miglustat. Four years later, the neurological picture is stabilized with a significative improvement in brain metabolic pattern in positron emission tomography study (PET). Discussion: NPC late presentations present a diagnostic chalange for the physician. Substract deprivation therapy with miglustat determined a favorable evolution. PET scan give good information to monitor neurologic progression and treatment response. Backgroud: Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid α-glucosidase. Accumulation of autophagosomes is a key factor for poor response to Enzyme replacement therapy in Pompe disease. We and others previously found that autophagy is also activated in patient fibroblasts. However, induction mechanism of autophagy in patient fibroblasts is not fully elucidated. In this study, we analyzed the glucose metabolism-related signaling pathway in patient fibroblasts to clarify their involvement in regulation of autophagy. Methods: Skin fibroblasts derived from infantile-onset patients with Pompe disease and normal infant were cultivated in the presence or absence of insulin, and were lysed with 2% SDS. These samples were analyzed by Western blotting using the antibodies against akt, and LC3. Results: The levels of phosphorylated akt were about 30-fold lower in patient fibroblasts than in normal fibroblasts, whereas the levels of LC3-II were about 7-fold higher in patient fibroblasts than in normal fibroblasts. When patient fibroblasts were treated with insulin, increased levels of phosphorylated akt and decreased levels of LC3-II were observed in them. Conclusion: These results suggest that activation of autophagy in fibroblasts from infantile-onset patients with Pompe disease is caused by downregulation of akt signaling. Conflict of Interest declared.
Shimada Y 1 , Fukuda T 2 , Nishiyama Y 1 , Kobayashi H 1 , Eto Y 3 , Ida H 4 , Ohashi T 1 1 Dept Gene Ther, Inst DNA Med, Jikei Univ, Tokyo, Japan 2 Div Neuropathol, Jikei Univ, Tokyo, Japan 3 Dept Genet Dis & Genom Sci, Jikei Univ, Tokyo, Japan 4 Dept Ped, Jikei Univ, Tokyo, Japan
Pompe disease is an autosomal recessive myopathic disorder arising from the deficiency of acid a-glucosidase (GAA). Autophagic buildup is a key factor for poor response to Enzyme replacement therapy in Pompe disease. This finding highlights the need for blood biomarkers which reflect autophagic dysfunction in skeletal muscles. In this study, we characterized the ubiquitin-protein conjugates in GAA-KO mouse plasma and skeletal muscles to investigate the possibility of them as a biomarker for autophagic buiidup. Skeletal muscle and plasma of GAA-KO and wildtype mice were lysed with 2% SDS and analyzed by using the antibodies against ubiquitin, K63-linked polyubiquitin, and LC3. Smears of ubiquitin-protein conjugates were more abundant in skeletal muscles of GAA-KO mice than those of wildtype mice. Additionally, the levels of specific ubiquitin-protein conjugates (approximately 58 kDa) were markedly increased in skeletal muscles of GAA-KO mice. Increased levels of the 58 kDa proteins were also observed in plasma from GAA-KO mice, and were prominently detected by anti-K63-linked polyubiquitin antibody. By immunohistological analysis, both LC3 and K63-linked ubiquitin-protein conjugates was colocalized in the myofibers from GAA-KO mice. These results suggest that the 58 kDa ubiquitin-protein conjugates in plasma may reflect the autophagic buildup in skeletal muscles from GAA-KO mice. Conflict of Interest declared. Background: N-linked oligosaccharides of glycoproteins represent important molecules that show significant alterations in a great number of conditions. Objectives: Given the fact that Pompe disease is characterized by altered glycogen metabolism, we hypothesized that N-glycan fingerprinting might represent a useful biomarker for the diagnosis and follow-up of Pompe patients. Patients and Methods: N-glycans in the serum of 2 patients with Pompe disease and an equal number of controls were isolated and digested with sialidase. The N-glycan profiles were analyzed by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Peaks corresponding to glycans were quantified and normalized to the total signal intensity. Results: We have found that the levels of profiles for peaks 1, 2, 4, 6, 7 and 9 displayed an increase, while those for peaks 3, 5 and 8 exhibited a decrease as compared to healthy controls. Peak 8, corresponding to a trigalactosylated, tri-antennary N-glycan structure (NA3), exhibited the most prominent difference, showing a 1.7-fold decrease. Measurements in consecutive blood samples before the initiation and during enzyme replacement therapy, showed no significant alterations in the concentrations of sugars. Conclusions: These data suggest that the changes in the serum N-glycans, especially NA3, could provide a useful biomarker for the diagnosis of Pompe patients. Background: Pompe disease (PD) is an autosomal recessive muscle disorder of glycogen metabolism caused by a deficiency of lysosomal alpha-glucosidase A (GAA) that results in the accumulation of glycogen primarily in muscle tissue leading to a severe hypertrophic cardiomyopathy associated with myopathy leading to death in early life. Objetive: We report a case of 8 months old baby affected by PD misdiagnosed as recurrent bronchiolitis episodes. Case report: Born of consanguineous parents (first cousin) without specific family history. He was admitted to hospital at 3, 4 and 5 months old affected by bronchiolitis; the last episode was associated to marked hypotonia. An echocardiography was performed and documented hypertrophic non obstructive cardiomyopathy and lab examinations showed a 3 fold increased CK levels. He was transferred to the regional centre of pediatric cardiology. Muscle biopsy suggested glicogen storage disease and blood spot revealed undetectable GAA activity. At 6 months of age he started enzyme replacement treatment in association of cardiac therapies and after 2 months the cardiac situation doesn't present any evolution. Conclusion: Respiratory disorder and progressive hypotonia could hide hypertrophic cardiomyopathy due to infantile PD form. The prognosis is strictly related to the early start of enzyme replacement treatment.
Kawagoe S 1 , Higuchi T 1 , Meng X 2 , shimada Y 1 , Shimizu H 1 , Fukuda T 1 , Nakahata T 3 , Fukada S 4 , Ida H 1 , Kobayashi H 1 , Ohashi T 1 , Eto Y . 1 1 Jikei University School of Medicine, Tokyo, Japan 2 Baylor Research Institute, Dallas, United States 3 Kyoto University, Kyoto, Japan 4 Osaka University, Osaka, Japan Background: Pompe disease (GSD-II) accumulates glycogen due to a deficiency of acid-α-glucosidase(GAA).Induced pluripotent stem (iPS) cell technology facilitates the study of the pathogenesis of Pompe disease and might eventually enable the development of autologous cell transplantation therapy. Objectives: We generated iPS cells from somatic cells by introducing reprogramming factors (Oct3/4, Sox2, Klf4 and c-Myc). Pompe-iPS cells were differentiated into skeletal muscle(SM) cells in Matrigel.-coated plates. Results: Our study first demonstrates the ability to generate iPS cells from a mouse model of Pompe disease. Initially, mouse fibroblasts were harvested from GAA knockout mice, and three reprogramming factors (Oct3/4, Sox2 and Klf4) were transfected into the isolated donor cells using a retroviral vector. These iPS cells also showed decreased levels of GAA enzymatic activity and strong staining with PAS and ACP. Spindle-shaped SM cells were successfully generated from Pompe-iPS cells and showed spontaneous contraction and positive staining with the MHC antibody. EM picture of SM cells showed typical morphological features. Furthermore, Pompe SM cells accumulated massive glycogen in lysosomes.
Conclusions: This study indicates that the iPS and skeletal muscle cells generated in this study could also be a useful disease model for studies investigating the pathogenesis and treatment of SM in Pompe disease. The heterogeneous response of skeletal muscle to enzyme replacement therapy (ERT) causes variable disability, from hypotonia to respiratory failure, in patients with infantile onset Pompe disease (IOPD). Histomorphometrical analysis reflects the extent of glycogen clearance and muscle damage, and may have a prognostic value. Two patients with IOPD, who started ERT at 2 months of age, had quadriceps biopsy during gastrostomy for feeding problem. Patient 1 responded well to ERT until late infancy, with motor regression and dependency on non-invasive ventilation since 14 months of age. Muscle biopsy at 15 months showed muscle fibres with abundant glycogen and minimal residual myofibril. He succumbed at 22 months of age. Patient 2 was well in the first 3 years. She then developed recurrent aspiration pneumonia and had gastrostomy at 43 months of age. She had satisfactory cardiorespiratory status and could walk independently. Light microscopy revealed variable glycogen content in muscle fibres and patchy involvement. More extensive glycogen overload was observed in the ultrastructural study. She eventually required walking-aid and non-invasive ventilation by 4 years of age. Our findings support the correlation between histology and clinical state, but caution the risk of sampling errors. Further studies are needed to evaluate its prognostic value. Conflict of Interest declared. Background: Pompe disease (glycogenosis type II) is a lysosomal storage disorder due to deficiency of glucosidase enzyme activity which leads to accumulation of glycogen in the cardiac and skeletal muscle. In the newborn predominant clinical presentation is hypertrophic cardiomyopathy, hypotonus and macroglossia. Case report: Our patient presented on the II day of life on a routine clinical examination with splitting of the second heart sound without any other clinical signs or abnormal fetal heart ultrasound. Electrocardiogram (ECG) showed typical Pompe disease related abnormalities and the heart ultrasound severe cardiac hypertrophy. Reduced enzyme activity (0,027, range 2,31-27,38 mcmol/L/h) on the blood spot was assessed within 48 hours by Tandem MS, further investigations (enzyme activity in leucocytes and fibroblasts, mutation analysis) were subsequently performed. In the meantime ERT was started on the VIII day of life. Conclusions: In the newborn unspecific cardiac findings may be Pompe disease related. Earliest detection of the reduced glucosidase activity by Tandem MS is a reliable method which allows timely introduction of the only therapeutic approach, the enzyme replacement therapy and normalisation of cardiomyopathy. Mutation analysis in our patient revealed a condition of compound heterozygousity of two novel splicing mutations: c.955+1 G>A and c.1438-2A>G.
In 2006 Enzyme Replacement Therapy (ERT) with alglucosidase alfa was registered as a treatment for Pompe disease. We present a primiparous 40-year-old woman with adult-onset Pompe disease who continued receiving alglucosidase alfa in a dose of 20 mg/kg every other week during pregnancy and lactation. The patient had moderate limb-girdle weakness and used nocturnal ventilation. The mother's clinical condition remained stable until the 25th gestational week. Thereafter she experienced more mobility problems and increased respiratory effort. Fetal growth was normal as monitored by regular ultrasound investigations. At a gestational age of 38 weeks and 5 days, a healthy baby-boy was born. There were no maternal complications and the child developed normally. After delivery pharmacokinetic studies were performed in breast milk and plasma. In breast milk, alpha-glucosidase activity levels peaked 2 hours after the end of the infusion, which was 2 hours later than in plasma. The alpha-glucosidase activity in breast milk disappeared over a period of 24 hours after the infusion. This case report indicates that ERT with alglucosidase alfa can be administered safely during pregnancy. We recommend refraining from breastfeeding on the day of the infusion, since Alglucosidase alfa transfers in small amounts into breast milk. Conflict of Interest declared.
Karabul N 1 , Goekce S 1 , Beck M 1 , Kampmann C 1 , Mengel E 1 1 Villa metabolica, Univ Child Hosp, Mainz, Germany
Recent studies show that immune response to rhGAA is associated with infusion related reactions and limited efficacy of treatment. Our study focus on the immune response to ERT in 4 patients with classical infantile phenotype followed up 1-6 years with ERT. All 4 patients had infusion-rate dependend IREs, temporally for 6-9 months in 3 patients, ongoing in 1 patient. No Ig-E antibody formation. Patient 1-3 had low titer IgG antibody formation in the first year of ERT and immuntolerizied there after. Patient 4 had high titer antibody formation without trend for immuntolerization. Patient 1 had partial response to ERT. Patients 2 and 3 had favourable outcome, as they are able to walk. Patient 4 showed initially some improvements followed by rapid detoriation after onset of IREs. Patient 1 is tested CRIM positve. In patients 2-4 CRIM status is not tested, but the mutations are highly suggestive for CRIM negative. In patients 2 and 3 alternative infusion protocols and preventive premedication were used. Immune response to rhGAA was associated with infusion related events. Poor outcome was observed in a patient with high antibody titer. Patients with immuntolerization had favourable outcome. For future immunmodulation strategies are mandatory to prevent high antibody formation. Background: Chitotriosidase (ChT) activity is 100 to 1000-fold higher than normal in Gaucher disease (GD) patients and falls with enzyme replacement therapy. It is not a useful marker in 6% of the population that is homozygous to a 24 bp duplication in exon 10 of CHIT1. The Hospital das Clínicas da Universidade Federal de Minas Gerais is a reference center in Minas Gerais state-Brazil for the treatment of GD type I. Only recently this center has included ChT in the follow-up. Objective: To establish the ChT activity and genotype for CHIT1 in patients with GD type I in treatment (n=26) in the state of Minas Gerais, Brazil. Methods: ChT activities (Hollak et al, 1994) and genotyping of CHIT1 duplication (Hise et al., 2003) in 26 GD patients. Results: Among 26 patients with GD, 18 were homozygous wild-type (HH=69%), 6 heterozygous (Hh=23%) and 2 homozygotes (hh=8%) for CHIT1 duplication. The ChT activity in patients HH (115-32,577, mean= 6,798 nmol/h/ml), Hh (200-4,417, mean=2,106 nmol/h/ml) was, respectively, around 200 and 62-fold greater than in controls (3-68, mean= 34 nmol/h/ml). The hh produced 1-1,4 nmol/h/ml. Background: In the last years lysosomal labs use Chitotriosidase as diagnostic screening parameter for LSDs. We evaluated the median and interquartile range (IQR) of chitotriosidase in 8 lysosomal storage disorders. Methods: We investigated 101 patients with confirmed diagnosis of Gaucher disease, Sphingomyelinase deficiency, Niemann-Pick disease type C, Fabry disease, GM1-Gangliosidosis, A-Mannosidosis and CESD. In 5% of the patients we found lack of chitotriosidase activity due to a common chito1-gen mutation. Results: It is a retrospective cross-sectional study in children and adults. Median chitotriosidase activity in the control group was 56 nmol/ml/h (IQR 42 nmol/ml/h) reflecting normal values. In Gaucher disease (GD: N=26) 18563 (IQR 13020), in Sphingomyelinase deficiency (SMD: N=9) 1111 (IQR 2009), in Niemann-Pick disease type C (NPC: N=35) 827 (IQR 1204), in GM1-Gangliosidosis (N=6) 776 (IQR 1533), in Fabry disease (N=10) 130 (IQR 151), in A-Mannosidosis (N=13) 171 (IQR 121), in one patient with Farber disease 3279 and in CESD 249 nmol/ml/h. Conclusion: Excluding those patients with lack of chitotriosidase activity 100 percent sensitivity was observed for elevated chitotriosidase in GD, SMD, GM1-Gangliosidosis, Farber disease and CESD. Limited specificity was seen in NPC, Fabry disease and A-Mannosidosis. Extreme high activity>5000 nmol/ml/h was exclusively noted in GD. Background: Monitoring efficacy of ERT in Gaucher type 1 by assaying plasma chitotriosiase activity is complicated by prevalence of its benign deficiency in all populations. Alternately HDL cholesterol measurement is advocated and observed to be increased on ERT.Study Design: We evaluated 102 suspected Gaucher type 1 patients and 165 healthy controls to assess chitotriosidase activity amongst Indians. ERT was initiated in 4 patients and both HDL cholesterol and chitotriosidase were assayed in baseline and follow up samples. Results: Gaucher type 1 was diagnosed in 32 patients based on the β glucocerebrosidase activity. Benign chitotriosidase deficiency was obtained in 21% of Gaucher type 1 and 16% of healthy controls. Chitotriosidase activity in remaining patients was significantly elevated and ranged from 5504-28,340 nmoles/hr/ml in Gaucher type 1 as compared to 8-87 nmoles/hr/ml in healthy controls. Three out of the four patients of ERT showed a significant decrease in chitoriosidase ( 17-24%) and a parallel increase in HDL cholesterol (32-42%) as compared to the pre treatment values. Conclusion: Chitotriosidase deficiency is consistent in India. Chitotriosidase reduces remarkably on ERT. Larger studies are required to assess utility of HDL cholesterol as a marker for monitoring the therapy.
β-Glucocerebrosidase gene (GBA) mutations have been identified as a risk factor for the development of Parkinson's disease (PD) in different ethnic groups. In Northern Greece significant association was only found in early onset PD patients (EOPD). Eight different GBA mutations were investigated in two cohorts of ethnic Greek patients with sporadic Parkinson's disease from Thessaly (n=100, cohort A) and Athens (n=105, cohort B). Age-gender-ethnicity matched healthy individuals from the same areas were included as controls (n=206). Statistically significant overrepresentation of GBA mutation carriers in the PD patients compared to controls was observed (p=0.006; OR=3.24, 95% CI=1.35-7.81). The association was reinforced in EOPD patients (p= 0.000; OR=11.37, 97% CI 3.73-34.6). PD patients harboring GBA mutations had an earlier onset of symptoms than non-carriers (p=0.034, p=0.004). Differences regarding the type of mutations and/or their relative frequencies were observed between cohorts. In conclusion, GBA mutations were identified with increased frequency in both geographical cohorts of sporadic PD patients compared to controls, the difference being statistically significant only in cohort A. An impressive association with EOPD was found, one third of the EOPD patients harboring a GBA mutation. Genetic and/or environmental factors may account for the observed differences in the two geographical cohorts studied. Objective: To characterize the saccadic eye movement abnormalities in patients with chronic neuronopathic Gaucher disease (GD3) in relationship to neurological and neurophysiological abnormalities. Methods: N=15 patients with GD3, age 8-28 years, prospective follow up (up to 4 years), saccadic eye movements (search coil method), neuropsychological and neurophysiological testing. Results: Patients with GD3 had a significantly higher regression slope of duration vs amplitude and peak-amplitude vs amplitude compared to healthy controls for horizontal/vertical saccades. Saccadic latency was significantly increased for horizontal saccades. Downward saccades were more affected than upward saccades. Regression slopes of horizontal vs vertical saccades for duration vs amplitude and peak-amplitude vs amplitude were significantly correlated (r=0.31, p<0.005) as was horizontal vs vertical saccade latency (r= 0.68, p<0.005). These abnormalities became more pronounced over time in some patients. There was a significant correlation between verbal IQ (p=0.03), performance IQ (p=0.04), full-scale IQ (p=0.02) and the downward saccade slope and between vertical saccade slope and the performance on the Purdue Pegboard when patient uses both hands (p=0.01). There was poor correlation between BAER, SSEP and saccadic eye movement parameters. Conclusions: Saccadic function and oculo-manual dexterity abnormalities are likely useful for studying neurological function in patients with GD3. Diagnosis for Gaucher Disease(GD) is based on beta-glucosidase(GBA) assay on leukocytes and chitotriosidase(CT) on plasma. Lately dried blood spots(DBS) are been used as screening, but not as final diagnosis because it's not established a measurement of specific activity. The aim of this study is to establish the best conditions of DBS elution to measure the specific activity of GBA and CT using total protein assay. DBS from 15 healthy volunteers and 8 GD patients were used. One punch of each sample was eluted using 40μL of sodium-phosphate-buffer(pH7) and tested for four incubation periods(10, 20, 30 and 60minutes) and two temperatures(30°C and 37°C). A microplate 4-MU fluorimetric assay for CT and GBA and a protein assay using 10μL of each elution was performed after. It was possible to measure activity of GBA and CT on DBS using a previous elution with pH7 buffer. The enzymes had higher activity with the 37°C elution than 30°C, so was protein(p<0.05). Because the determination of specific activity takes in consideration the protein amount, there was no statistically significant difference between temperatures and elution time. It was able to differentiate the healthy volunteers from the GD patients with a p=0.002 for CT and p=0.0002 for GBA.
Goldim M 1 , Garcia CS 1 , Coelho JC 1 1 Department of Biochemistry of UFRGS, Porto Alegre, Brazil
Gaucher Disease(GD) is caused by β-glucosidase(GBA) deficiency and chitotriosidase(CT) is increased in these patients. The dosage of GBA (leukocytes) and CT(plasma) are considered as standard for diagnosis. This measure can also be held in dried blood spots(DBS) just as screening. This study aims to establish reference values of GBA and CT activity in leukocytes, plasma and DBS and its correlation. We used heparinized-blood of 18 healthy-volunteers(HV), 18 GD-patients and 4 carriers. The techniques used 4-Metilumbeferil substrates. GBAwere incubated for 60 min(leukocytes) or 5 h(DBS) and CT for 15 min(plasma) or 30 min(DBS), at 37°C and stopped with pH10.3 buffer. Reactions were read at 365 and 450 nm spectrofluorimeter. The reference values for GBA in leukocytes raged from 8.6 to 18.4(HV), 0.4 to 9.0(carriers) and 0.0 to 4.6 nmol/h/mg protein(GD); while in DBS were 2.7 to 7.1(HV), 2.6 to 6.2(carriers) and 0.2 to 2.6 nmol/h/mL(GD). CT in plasma ranged from 0 to 246.9(HV), 165.1 to 338.7(carriers) and 0 to 49.58 nmol/h/mL(GD) and in DBS from 4.6 to 44.5(HV), 4.5 to 21.4 (carriers) and 0.0 to 2249.5 nmol/h/mL(GD). For both enzymes, the correlation was significant with p<0.0001, r=0.78(GBA) and r=0.89(CT). The results showed that DBS enzyme assay are reliable and can be useful as screening methods for high-risk populations. Background: Gaucher disease is an autosomal recessive disorder of lysosomal degradation of glucosylceramide. It results in a progressive accumulation of glucosylceramide in the lysosomes, leading to hepatosplenomegaly, anemia, thrombocytopenia and various skeletal complications. The most common mutation is the substitution A to G at nucleotide position 1226, which produces aAsn-Ser change at amino acid 370. There is variable frequency of this mutation: in Ashkenazic Jews carrier frequency 1: 15; 1 in 100 people in the United States. Objectives: Detect carrier frequency of mutation N370S in Latvian population Material and Methods: We studied 150 volunteers, aged 19-25 years. DNA was extracted from whole blood and purified by standard phenol/ chloroform extraction protocol. The presence of mutation N370S in gene GBA was analyzed using PCR with subsequent restriction enzyme XhoI digestion and detected in polyacrylamide gel. Results: One patient out of 150 was heterozygous for mutation N370S. Estimated Gaucher disease frequency when patients are homozygous for mutation N370S is 1 : 90 000 Conclusions: 1. Carrier frequency of mutation N370S in Latvian population is low. 2. Gaucher disease in Latvian population may be caused by other mutations 3. To obtain more precise results about mutation frequency control population should be enlarged.
Siebert M 1 , Bock H 1 , Michelin-Tirelli K 1 , Coelho JC 1 , Schwartz IVD 1 , Giugliani R 1 , Saraiva-Pereira ML 1 1 Medical Genetics Service-HCPA, Porto Alegre, Brazil
Gaucher disease (GD), an autosomal recessive lysosomal storage disorder, characterized by glucocerebrosidase (GC) deficiency due to mutations in the glucocerebrosidase (GBA) gene. To date, more than 250 mutations have been identified. The aim of present study was to identify sequence alterations in the GBA coding region in GD patients from Brazilian. We have evaluated a group of 125 unrelated patients from a total of over 800 individuals tested that were shown to have GC deficiency through enzyme assay. DNA samples from these patients were analyzed through long-range PCR, followed by specific screening for common mutations and direct sequencing. We have identified common and rare mutations in the mutant alleles. Besides p.N370S and p. L444P, p.G377S mutation was also frequently found in this cohort. Among rare mutations, alterations include insertions, deletions, complex alleles as well as single base changes. In addition, we have also described novel sequence variations that are likely to be disease causing mutations. As these sequences alterations are located in conserved residues of the protein or at a splicing site, these changes are likely to be responsible for changes in the structure and/or function of GC. Full screening of GBA gene is relevant to identified additional rare mutations. Forty-two adults with GD-1 stable after ≥3 years of ERT were enrolled. Endpoints were: change from baseline in liver volume (primary) and in spleen volume, haemoglobin concentration and platelet count (secondary). Mean (SD) patients' age was 45.1 (12.7) years and previous ERT duration was 9.5 (4.0) years. Median exposure to miglustat (100 mg t.i.d.) was 658 (range 3-765) days. Thirteen patients discontinued treatment as a result of adverse events (principally gastrointestinal), six for protocolmandated safety criteria for suspected disease progression, one for noncompliance and one withdrew consent. The primary endpoint was met, as the upper 95% confidence limit of mean percent change in liver volume from baseline to End of Treatment was below the noninferiority margin of 10% [−1.1%; 95%CI −6.0, 3.9%; mean (SD) baseline volume 1775 (484) cm3, mean absolute change −47.5 cm3 (95%CI −151, 56)]. Baseline values [mean (SD)] for spleen volume, haemoglobin concentration and platelet count were 510 (372) cm3, 14.8 (1.6) g/dL, and 198.9 (95.7) W10(9)/L, respectively. Mean (95%CI) absolute changes in these three parameters, respectively, were 102 (24,180) cm3, 0.95 (−1.38,-0.53) g/dL, and 44.1 (57.6, 30.7) W10(9)/L. Miglustat is a useful option for maintenance treatment in patients with GD-1 previously stabilised by enzyme therapy. Conflict of Interest declared. Background: Skeletal involvement is a frequent complication of Gaucher disease (GD), and represents an important cause of morbidity and disability. Objectives: To evaluate retrospectively the long-term efficacy of enzyme replacement therapy (ERT) in correcting bone mineralization in a paediatric cohort of patients with GD type 1. Patients and methods: 18 patients (age 9.2±4.7), receiving ERT (20-60 U/ kg) biweekly, up to a maximum of 17 years, were included. Bone symptoms, growth, imaging and lumbar bone mineral density (BMD) were assessed. Results: At baseline BMD was pathological (Z-Score range: -3.80 to −2.21) in 7/9 patients who started ERT after pubertal spurt, 5 of them showed growth delay. After 2 years of ERT, BMD significantly improved in 13/18 patients: mean values from −1.3 to −1.0 (p=0.02). A significant difference was found between baseline and last reading data (p=0.01). At the end of the study BMD normalized in all but 2 splenectomized siblings; growth delay normalized in all patients. Conclusions: ERT started in paediatric age correct osteopenia, maintain normal BMD and growth in the vast majority of patients (16/18). The improvement of BMD is mainly achieved during the first 2 years of treatment. Early splenectomy may hamper a normal bone mineralization.
COMBINED ENZYME-REPLACEMENT AND SUBSTRATE-REDUCTION THERAPIES IN GAUCHER DISEASE TYPE II/III Gautschi M 1 , Nava E 2 , Goeggel Simonetti B 2 , Nuoffer JM 3 1 Interdiscipl Metab Unit, Univ Child Hosp, Bern, Switzerland 2 Neuropediatric Unit, Univ Child Hosp, Bern, Switzerland 3 Institute Clin Chem, University Hospital, Bern, Switzerland
The patient from non-consanguineous Albanian parents presented at four months with isolated splenomegaly. Oculomotor apraxia and hepatomegaly progressively appared during the second half of the first year. Hematological values showed intermittent bicytopenia (hemoglobin 103-113 g/L [reference >100], neutrophils 1.25-1.80 G/L [>1.50] and thrombocytes 71-116 G/L [>150]). Chitotriosidase was 27.5 mU/ml (reference <1.03). Decreased beta-glucosidase activity in lymphocytes confirmed the diagnosis of Morbus Gaucher (residual activity 10%). The double homozygous mutations c.882 T>G and c.1342 G>C detected in the GBA gene of our patient (AMC Amsterdam) have been described earlier in Gaucher type II patients. At one year, tip-toe-posture and slight extension reaction of the upper limbs were the only additional neurologic abnormalities in an otherwise well developing girl, suggesting an intermediary rather than type II Gaucher, and prompted us to introduce combined enzyme-replacing (ERT) and substrate-reduction (SRT) therapies. Since treatment initiation (follow-up 9 months), both ERT and SRT are well tolerated, hepatosplenomegaly and blood cell counts have normalized almost completely. Moreover, psychomotor development is near to normal and neurological signs stable. Notwithstanding the necessarily cautious interpretation of the short follow-up, these findings suggest that early initiation of combined ER-and SR-therapies might improve outcome in Gaucher disease. Background: In GD1, deficient acid β-glucosidase activity causes glucosylceramide accumulation. Eliglustat, a potent and specific glucosylceramide synthase inhibitor, is under development as an oral substrate reduction therapy for GD1. Objective: Report long-term efficacy and safety results. Methods: Efficacy outcomes included changes in hemoglobin and platelet levels, spleen and liver volumes, and bone mineral density (BMD). Achievement of therapeutic goals was measured. Results: Of 26 enrolled patients, 19 completed 3 years. After 3 years, mean hemoglobin level and platelet count increased by 2.6 g/dL and 91%, respectively; mean spleen and liver volumes decreased by 61% and 29%, respectively (all P<.0001). The majority of patients met long-term therapeutic goals for these parameters; all met ≥3 long-term therapeutic goals. Mean lumbar spine BMD increased by 0.6 Z-Score; femur dark marrow was reduced or stable. There were no bone crises; new lytic lesions or bone infarcts; or worsening of pre-existing lytic lesions or bone infarcts. Eliglustat was welltolerated. Most adverse events (AEs) were mild and unrelated to treatment. Eight drug-related AEs, all mild, occurred in 6 patients. Conclusions: Eliglustat has shown promising efficacy and safety, with clinically meaningful improvements in hematologic, visceral, and bone parameters. Three Phase 3 trials are enrolling patients. Conflict of Interest declared.
Olkhovych N.V. 1 , Pichkur N.O 1 , Ivanova T. P 1 , Grishchenko O.N. 1 , Nedoboy A.N. 1 , Gorovenko N.G. 2 , Trofimova N.S 1 1 National Children Hospital "OHMATDET", Kyiv, Ukraine 2 National Medical Academy of Postdiploma, Kyiv, Ukraine Introduction: Gaucher disease (GD) is an inherited glycolipid storage disorder resulting from the deficiency of glucocerebrosidase. The GD accounts for 71% of all lysosomal disorders diagnosed in Ukraine. Materials and methods: The 17 GD patients (7 children, 10 adults) have been treated with ERT and monitored since 2004. The diagnoses of all patients were established by clinical, laboratory and morphological ("Gaucher cells" in bone marrow) data. The mean activity of glucocerebrosidase deficiency in leukocytes was 2,7 ±1,4 nmol/h/mg protein (reference range-7,2±2,1 nmol/h/mg protein). All patients were screened for gene mutations in the GBA gene. Results: The regularity of ERT with the recombinant glucocerebrosidase has reduced manifestations of hepatosplenomegaly and pancytopenia, bone pain and bone crises in the most GD patients, and led to significant improvements. The chitotriosidase activity has decreased from 4552-29614 nmol/h/ml pl (before the ERT) to 870-4897 nmol/h/ml pl (after the ERT). Conclusions: The efficacy of treatment depends on the regularity, the dose and the severity of the disease at the beginning of therapy. Background: In GD1, certain disease-related biomarkers (GL-1, GM3) have been identified that reflect perturbation of the glycosphingolipid metabolic pathway caused by acid beta-glucosidase deficiency, whereas others (chitotriosidase, CCL18, ACE, TRAP) represent secondary effects on tissues. Various biomarkers in GD1 are responsive to imiglucerase enzyme replacement therapy and are used clinically to monitor patients. Eliglustat, a novel, small molecule inhibitor of glucosylceramide synthase (GCS), is under development as an oral substrate reduction therapy for GD1. Objectives: Evaluate biomarker responses to eliglustat in GD1 patients. Methods: A Phase 2 study enrolled 26 adult patients with GD1 not on treatment; 19 completed 3 years of treatment with eliglustat. Plasma levels of GL-1, GM3, chitotriosidase, CCL18, ACE, and TRAP were measured periodically throughout the study. Results: GD1 biomarkers were elevated in most patients pre-treatment. Statistically significant decreases (P<.0001) following eliglustat treatment were seen in median plasma GL-1 and GM3 (80% and 64%, respectively), which normalized; and median chitotriosidase, CCL-18, and ACE (80%, 73%, and 62%, respectively), which all remained above normal. Two-year median TRAP decreased by 51% (P<.0020), but remained above normal. Conclusions: Eliglustat treatment led to reductions in GD1-related biomarkers that reflect both primary and secondary effects of the underlying enzyme deficiency. Conflict of Interest declared.
A PATIENT WITH ADULT ONSET GM2-GANGLIOSIDOSIS: 24 YEARS OF FOLLOW-UP Scarpelli M 1 , Tomelleri G 1 , Bertolasi L 1 , Salviati A 1 1 Dep Neurol, Univ of Verona, verona, Italy Sandhoff disease is a lipid-storage disorder caused by a defect in ganglioside metabolism due to mutations in the HEXB gene that disrupt the function of the N-acetyl-beta-D-glucosaminidase isoforms Hex A and Hex B. A late-onset form of Sandhoff disease is rare and its symptoms are heterogeneous, including cerebellar syndrome, motor neuron disease and autonomic dysfunction. We report on a 25 year-old man who suffered from mild proximal lower limb muscle weakness (4 MRC) and intense fasciculations. EMG demonstrated signs of denervation in lower limbs, whereas brain MRI was normal. Biochemical studies documented a 13% residual Hex activity in leukocytes; HEXB genetic analysis revealed a compound: delta5' deletion and C1214T (Pro405Leu) missense mutation. Twenty years later appeared bilateral Babinski sign and neurophysiological study showed peripheral sensory neuropathy. Twenty-four years from the onset, he was independent in deambulation, his weakness in lower limbs is slightly worsened (3 MRC) and mild weakness appeared in both arms. He didn't develop neither cerebellar or autonomic signs and brain MRI remained normal. To our knowledge, this is the longest follow-up in a patient with Sandhoff disease. It underlines how a clinical history of pure motor neuron disorder, in GM2-gangliosidosis, may have a relative benign course.
Simmons LM 1 , Wassmer E 1 , Preece MA 1 , Chakrapani A 1 1 Dept of IMD, Birmingham Children's Hosp, Birmingham, United Kingdom Background: Late infantile GM1 Gangliosidosis presents after the 1st year with neuroregression, absence of dysmorphism, and typical MRI changes affecting the thalami and basal ganglia. We present a case where diagnosis was delayed because of MRI findings of primary leucodystrophy. Case Report: The patient presented at 14 months with delayed walking & hypotonia without dysmorphism. After 2, she lost the ability to cruise, crawl and sit. Brain MRI showed typical features of leucodystrophy without abnormalities of the thalamus, basal ganglia and distal long tracts. Investigations for MLD, peroxisomal disorders and other metabolic causes were negative. Between 2.5-4 years she had progressive intellectual decline and onset of seizures, dystonia and spasticity. Further MRI scans at 4 and 5.5 years showed progressive loss of white matter with cerebral atrophy; the basal ganglia and thalami remained relatively preserved. At 8 years, she was in a near-vegetative state with spasticity, dystonia and complex epilepsy with mild dysmorphism; spinal Xray showed anterior beaking of vertebrae. Persistent oligosacchariduria and low leucocyte betagalactosidase led to confirmation of diagnosis by demonstration of 2 mutations, c75+2_3insT/c.601 C>T in the GLB1 gene. Conclusion: GM1 Gangliosidosis should be considered in the differential diagnosis of primary leucodystrophy presenting in late infancy P-378
Lee YM 1 , Kang HC 1 , Lee JS 1 , Kim HD 1 1 Dept of Ped, Yonsei Univ Col Med, Seoul, Korea, Republic of Tay-Sachs disease (TSD) is a metabolic disease, inherited in an autosomal recessive way, that is caused by a defect of hexosaminidase A. The gene responsible for the disease, HEXA gene, is located on chromosome 15q23-q24. Though TSD is rare in the general population, the frequency is reported to be 100 times higher in the Ashkenazi Jewish and eastern Quebec French Canadian. The disease is characterized by progressive weakness, loss of motor skills, decreased attentiveness, and increased startle response with progressive evidence of neurological degeneration. Hexosaminidase A activity study was performed in a boy who had shown delayed development since 6 months of age, regression since 9 months and exaggerated startle response. He had no unusual history nor medical event at birth. His hexosaminidase A activity was decreased and progressive brain atrophy was noted in serial brain MRI. HEXA gene study was performed and direct sequencing of the amplified genomic DNA revealed 2 kinds of mutations being in compound heterozygosity (c.913_915delTTC and IVS5-1 G>T). Family members received HEXA gene study and all were diagnosed as carriers. We would like to report the very first case of the classic infantile form of TSD that was confirmed by HEXA gene study in Korea.
Okur I 1 , Aydin HI 1 , Akin R 2 1 Div Metab Dis, Dep Ped, Gulhane MMF, Ankara, Turkey 2 Div Ped Neu, Dep Ped, Gulhane MMF, Ankara, Turkey Background: Hexosaminidase A deficiency results in a group of inherited neurodegenerative disorders characterized by impaired lysosomal catabolism of ganglioside GM2. Tay Sachs disease corresponds to deficiency of the alpha-subunit of beta-hexosaminidase. The infantile form of Tay-Sachs disease presents with progressive weakness, loss of motor skills, decreased attentiveness, and increased startle response beginning between three and six months of age with progressive evidence of neurodegeneration. More than 100 mutations have been described in HEXA gene which encodes the alpha subunit of beta-hexosaminidase. We here report a novel mutation in HEXA in a patient with infantile Tay-Sachs disease. Case Report: 34 month-old male patient, appeared to be completely normal at birth, presented with progressive weakness, severe psychomotor retardation, myoclonic jerks, an exaggerated startle reaction, and retinal cherry red spots. Leukocyte beta-hexosaminidase A activity in patient was profoundly low (1.72 nmol/h/mg protein of patient). The diagnosis of hexosaminidase A deficiency confirmed by a novel mutation (homozygote c.A1306G;I436V) in HEXA gene. Conclusions: The great majority of specific mutations described in the alpha subunit of the HEXA gene are associated with the acute infantile form. We here report a novel mutation associated with infantile form of Tay-Sachs disease. Background: Inborn errors of sialic acid metabolism include three lysosomal storage disorders: sialic acid storage disease (SASD), sialidosis and galactosialidosis. The phenotypical spectrum of these three diseases ranges from severe forms, which may present as hydrops fetalis, to relatively mild forms. Screening can be performed by determination of free (FSA) and conjugated (CSA) sialic acid in urine or amniotic fluid supernatant (AFS). Diagnosis of SASD can be performed by quantification of FSA in cultured fibroblasts. Objective: To develop simple quantitative procedures to determine FSA as well as CSA in AFS, and FSA in cultured fibroblasts, using isotope dilution LC-MS/MS. Results: In 6 SASD AFS samples FSA was 2-9-fold elevated compared to normal controls. CSA levels in AFS of 3 sialidosis and 4 galactosialidosis cases were all higher than the reference samples. The method was also validated for determination of FSA in fibroblast homogenates. In SASD fibroblasts FSA was clearly elevated compared to normal controls. Conclusion: We report simple quantitative procedures to determine FSA and CSA in AFS as well as FSA in fibroblasts, improving both prenatal diagnostic efficacy for sialic acid disorders and confirmatory testing in cultured fibroblasts following initial screening in urine or AFS. Introduction: α-Mannosidosis is a rare lysosomal storage disorder caused by deficiency of α-mannosidase. Characteristic symptoms of a-Mannosidosis are hearing loss in early childhood, skeletal abnormalities, immunodeficiency and mental retardation. Patients and Results: We investigated 31 patients, aged 3-44 years, with a-Mannosidosis. Common mutations found in 28 non-related patients were c.2248 C>T (9/ 56) and c.1830+1 G>C (6/56) in homozygous or heterozygous form. Mean age of onset was 1.5 years. Diagnosis was confirmed on average with 6.4 years. Most common first symptom was hearing loss, followed by skeletal abnormalities and development delay. Patients suffer from recurrent infections of upper airways in childhood and purulent infections of teeth and skin. 30 patients developed ataxia. 10 patients need a wheelchair. 6 patients showed psychiatric symptoms. Inguinal or umbilical hernia, mental retardation, amblyopia, gingivahyperplasia, mild hepatomegaly or splenomegaly can also be found in patients with a-Mannosidosis. Conclusion: Combination of hearing loss, recurrent infections, skeletal abnormalities and development delay are characteristic manifestations of a-Mannosidosis. A phase II-trial with enzyme replacement therapy for a-Mannosidosis is currently in process. Experience with other enzyme replacement therapies revealed that early diagnosis is important for efficacy of this therapy.
Okuyama T 1 , Kosuga M 1 , Kakee N 1 , Hirakiyama A 1 , Fuji N 1 , Kida K 1 , Eto Y 2 1 Crin Lab Med, NCCHD, Tokyo, Japan 2 Jikei Univ School of Medicine, Tokyo, Japan Background: Wolman disease is an autosomal recessive disorder of breaking down cholesteryl esters and triglycerides, resulting in accumulation of fat in the several organs. Major clinical manifestations are hepatomegaly and liver dysfunction. Enzyme replacement therapy is now under development. Its effectiveness is controversial because it is unclear whether significant accumulation and organ dysfunction initiate in fetal period or not. We had opportunity to analyze fetal liver and kidney of Wolman disease. Case report: A 36 year-old woman had two babies diagnosed as Wolman disease based on clinical manifestations. Gene test showed that the proband is a homozygote of 890 insert G of acid lipase gene. Prenatal genetic diagnosis was performed and the fetus was diagnosed as affected and aborted at 16 gestational weeks. An autopsy was done. Results: Liver and kidney tissue were obtained from the fetus. Cholesterol, Cholesteryl esters and triglycerides were separated by thin-layer chromatography. Apparent cholesterol ester accumulation was detected in liver and kidney tissue. In the liver, accumulation of triglycerides and cholesterol was also observed, indicating the accumulation had already started in fetal period. Conclusion: These data suggest enzyme replacement therapy for Wolman disease may not be effective when it starts after birth. Background: Tartrate-resistant acid phosphatase (TRAP), a lysosomal enzyme expressed in osteoclasts and macrophages, is a marker for growth and bone turnover, macrophage activation, and hairy cell leukemia. Recently, biallelic mutations in APC5, the gene encoding TRAP, have been found in patients with spondyloenchondrodysplasia (SPENCD), cerebral calcifications, spasticity and autoimmunity1. Abolished TRAP activity in serum and loss of expressed protein in patients' cells was demonstrated. TRAP deficiency causes hyperphosphorylation of osteopontin, a bone matrix protein, which appears to be the key pathogenetic factor for autoimmune disease in these patients. Here we report clinical characteristics of one of these patients. Case Report: The 15-years-old female patient was born to healthy consanguineous Turkish parents. First clinical findings evolved during the second year of age when chronic thrombocytopenia, progressive gait disturbance, spasticity, leukoencephalopathy, SPENCD, growth retardation, proteinuria, and episodes of severe hemolytic anemia developed. Her karyotype was found to be 46, XXX. Recently, homozygosity for an APC5 mutation was detected1; both parents were heterozygous for this mutation. Conclusion: SPENCD with leukoencephalopathy and autoimmunologic symptoms such as lupus erythematosus, hemolytic anemia and thrombocytopenia is highly suggestive of TRAP deficiency and requires further investigations with enzymatic and molecular genetic analyses.
Mercimek-Mahmutoglu S.M-M 1 , Ting J.T. 2 , Hochwald O.H. 2 , Au N.A. 3 1 Div Biochem Dis, Dept Pead, Univ of BC, Vancouver, Canada 2 Div Neonat, Dept Ped, Univ BC, Vancouver, Canada 3 Dept Pathol, Univ of BC, Vancouver, Canada
Background: Mucolipidosis-type-II (ML-II) is rare progressive neurodegenerative lysosomal storage disease (LSD). The characteristic clinical features become evident within the first year of life. Case presentation and results: This 6-month-old girl was born at term by Caesarean-section. She had blueberry muffin rash with multiple nonblanching purplish lesions and 2 cm palpable liver. Laboratory investigations revealed neutropenia, marked thrombocytopenia, unconjugated and conjugated hyperbilirubinemia and moderately elevated lactate dehydrogenase at age 4 days. Skeletal radiographs showed diffuse periostitis of all long bones suggesting congenital infections. However all serological investigations were negative. There were large, very prominent cytoplasmic vacuoles within the lymphocytes on peripheral smear, which led us to investigate for LSD at age 5 days. Serum total hexosaminidase, hexosaminidase A and alpha-N-acetylglucosaminidase enzyme activities were markedly elevated suggesting ML-II at age 10 days. She had known homozygous disease causing mutation in the GNPTAB gene (c.3503_3504delTC) confirming the diagnosis of ML-II. Conclusion: This is the first patient with ML-II presented with blueberry muffin rash. Peripheral blood film examination is a simple, cheap and readily available screening test to diagnose patients with LSD in the neonatal period. As bone marrow transplantation is widely used treatment option for the most LSD, early diagnosis is crucial.
Yoshino M 1 , Watanabe Y 1 , Yabe H 2 , Kato S 2 , Otomo T 3 , Sakai N 3 , Gasa S 4 , Hayasaka-Sukegawa K 5 1 Dept Pediatr & Child Health, Kurume Univ, Kurume, Japan 2 Dept Cell Tlanspl & Regen Med,Tokai Univ, Isehara, Japan 3 Dept Pediatr, Osak Univ, Osaka, Japan 4 Div Chem, Sapporo Med Univ, Sapporo, Japan 5 Dept Pediatr, Gifu Univ, Gifu, Japan
Background: Therapeutic effect of allogenic bone marrow transplantation (BMT) on I-cell disease has not been determined. Objective: To evaluate effects of BMT in a patient with I-cell disease. Case report: This girl, diagnosed with I-cell disease at age 8 months, underwent BMT at 14 months of age. She had achieved psychomotor development, though delayed, until 11 years of age, but her psychomotor function began to deteriorate after that time and she died at the age 21 years. Her affected elder sister achieved no psychomotor development at all and died at the age 2 years and 11 months. They both carried p. Q104X/p.R1189X mutations of the GNPTAB gene. Results: Activity of GlcNAc-1-phosphotransferase, alfa-mannosidase and beta-hexosaminidase in peripheral lymphocytes increased to a maximum value of 2.28-, 4.8-and 2.8-folds, respectively, after BMT. Chimerism analysis revealed that 94.8% of genome in the peripheral blood was of donor origin, as assed at 15 years of age. Conclusion/Discussion: BMT apparently restored enzymatic activity in the observed periods. Achievement of psychomotor development until the age 11 years may be attributable to possible therapeutic effect of BMT, as compared with the outcome of the affected sister. The reason of the deterioration remains unclear, as rejection was excluded. Conflict of Interest declared.
Wijgerde MGJM 1 , Verheijen FW 1 , Schoonderwoerd GC 1 , Ruijter GJG 1 , Huijmans JGM 1 1 Dept. of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands Mucolipidosis (ML) II and III are inherited lysosomal storage disorders caused by a deficiency of the enzyme N-acetylglucosaminyl-1-phosphotransferase (GlcNac-1-phosphotransferase), whose activity is required for the post translational processing of many hydrolases to form the mannose 6-phosphate (M6P) recognition site that permits lysosomal targeting. As a result of this enzyme deficit, lysosomal enzymes are secreted by cells and abnormal amounts of carbohydrates and fatty materials (lipids) accumulate in lysosomes, resulting in damaged tissues causing symptoms that range from joint problems and mild learning disabilities to severe retardation and skeletal deformities. Biochemical analysis of ML II/III patients show increased hydrolase (eg. beta-D-hexosaminidase, beta-D-glucuronidase, beta-D-galactosidase, and alpha-L-fucosidase) activities in blood plasma and elevated excretion of oligosaccharides in urine. Quantitative analysis using LC-MS-MS of free and bound sialic acid concentrations in urine samples of ML II/III patients reveal excessive excretion of bound sialic acid in 80% of investigated patients. In contrast, mildly elevated excretion of glycosaminoglycans (GAGs) detected by spectrophotometry after dimethylmethyleneblue staining and abnormal oligosaccharides detected by thin layer chromatography were each present in 60% of our patients. Excessive urinary excretion of bound sialic acid in a metabolic screen indicates, apart from sialidosis (ML I), also ML II/III as a putative diagnosis.
Pervaiz MA 1 , Matern D 1 , Rinaldo P 1 , Gavrilov D 1 , Tortorelli S 1 , Oglesbee D 1 , Raymond K 1 1 BGL, Mayo Clinic, Rochester, United States I-Cell disease is caused by defects in the targeting of lysosomal enzymes to lysosomes due to deficiency in the enzyme UDP-N-acetylglucosamine:Nacetylglucosaminyl-1-phosphotransferase. Multiple sulfatase deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 (SUMF1) which encodes formylglycine-generating enzyme (FGE). Patient 1 is a 7-year old female with extremetly elevated serum total hexosaminidase and α-N-Acetylglucosaminidase activity. Patient 2 is a 5year old girl with deteriorating clinical symptoms whose Arylsulfatase-B activity was only slightly below the normal range which triggered us to measure serum hydrolases. The activity of several serum hydrolases (Hexosaminidase, α-galactosidase, and α-N-acetylglucosaminidase) was high. A third patient had low enzyme activity for α-N-Acetylglucosamindase,α-L-Iduronidase, α-glucoronidase and Arylsulfatase B. Total serum hexosaminidase and α-Galactosidase activities were high which was consistent with a diagnosis of I-Cell disease. Patient 4 had reduced Arylsulfatase A and B, and α-NAcetylgalactosamine enzyme activities, findings suggestive of MSD. Conclusion: These cases demonstrate the difficulty of diagnosing as well as differentiating between I-Cell disease and MSD because of the overlap in abnormal enzyme activity levels in both conditions. It is very important to include I-Cell disease and MSD in the differential diagnosis of any abnormal lysosomal enzyme activity, especially if two or more enzymes are abnormal. Cases of I-cell disease diagnosed on the island of Ireland over 13 years (1/1/199-31/12/2010) were identified in collaboration with the clinical diagnostics laboratory. A database documenting details of diagnosis and clinical course where available was compiled and correlated with published birth rates including that of Irish Travellers (available only for the Republic). Twenty infants from 14 families were diagnosed with I-cell disease during the study period. 18 were born to Irish Traveller parents, one to non-Traveller Irish parents and one to parents from Southern Europe. Mutation analysis was available for 7 cases, of whom 6 (all Travellers) were homozygous for the c3503_3504delTC mutation. Median age of death in patients of the Traveller community was 232 days (range 3-936). Incidence estimated using population data for the Republic (ROI) and Northern Ireland was 1.56 per 100,000 live births. The incidence amongst Travellers (based on ROI cases and population data) was 114 per 100,000 live births, suggesting a carrier frequency of the common mutation of 1 in 15. The carrier rate amongst Irish non-Travellers remains rare at 1 in 512. This high incidence and carrier rate found in the Irish Traveller population is relevant for genetic counselling of this consanguineous community.
MUCOLIPIDOSIS TYPE II AND III: ECHOCARDIOGRAPHIC STUDY OF BRAZILIAN PATIENTS Alegra T 1 , Netto CB 2 , Souza CFM 2 , John AB 2 , Fagondes SC 2 , Schwartz IVD 1 1 Univ Federal do Rio Grande do Sul, Porto Alegre, Brazil 2 Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Mucolipidosis (ML) types II and III are lysosomal diseases due to GlcNAc-phosphotransferase deficiency. Multisystem complications are present, including cardiopulmonary ones. Objective: To characterize echocardiographic abnormalities in Brazilian ML II/III patients. Methods: Cross-sectional study based on echocardiograms and medical records of ML II/III patients seen in a Brazilian Reference Center for LSD. Results: 9 patients were included; 5/9 ML II (aged 0.8 to 5 years) and 4/9 ML III (11-42 years). Clinical exam: systolic murmurs (n=6/8), cyanosis and dyspnea (n=1/8). Echocardiography: some alteration (n=9/9), systolic function preserved (n=9/9), patent foramen ovale (n=1/8, 10 months, MLII), mitral thickening (n=5/9, of whom one MLIII), mild mitral regurgitation (n=8/9), aortic valve thickening (n=3/9, and 2/3 MLIII). Two MLII patients presented LVH (aged 4 and 5 years). PH was present in 1/9 patient (MLIII, good evolution after treatment with Sildenafil and continuous positive airway pressure during sleep). Conclusion: Our study is unprecedented in Brazilian patients and confirmed that ML II/III patients may present HP, aortic and mitral thickening, and mitral insufficiency. Differently from the literature, LVH occurred in one child in our sample. Following these patients is important to detect early abnormalities, especially PH, which can be adequately treated. Support: CNPq, CAPES, Brazil MPS Network Mucolipidoses α/β (ML II or III) and γ (ML III) are lysosomal diseases (LSD) due to deficiency in GlcNAc-phosphotransferase, which is encoded by GNPTAB and GNPTG genes. Objective: To identify mutations in the GNPTAB and GNPTG genes in Brazilian patients with MLII/III. Methodology: We analyze both genes in gDNA of MLII/III patients diagnosed in a Brazilian Reference Center for diagnosis of LSD. Exons and flanking regions are being amplified by PCR and sequenced in automated sequencer ABI3100.. Results: Fifteen patients were included: 7/15 have ML II (4 female; mean age±SD=2.2±1.9 years) and 8/15, MLIII (1 female; mean age±SD=20.9 ±12.3); 2/15 were siblings. Both pathogenic mutations in GNPTAB were identified in 8/14 unrelated patients and only one in 4/14. The most frequent pathogenic mutation was c.3503_3504delTC (exon 19); two novel pathogenic mutations are being described: c.2269_2273delGAAAC; c.2808A>G. To date, 9/11 exons of GNPTG were sequenced in 14 patients, but only 3 different mutations were found in 4/28 alleles. Discussion/Conclusions: MLII/III α/β appears to be the most common type of ML in Brazil. Our findings suggests that, in our country, screening for GNPTAB gene mutations should be initiated in exon 19.
Yano S 1 , Moseley K 1 1 Genet, Pediatr, Univ S California, Los Angeles, United States Background: Progressive cardiovascular changes have been known as one of the major causes of death in patients with mucopolysaccharidosis (MPS). All types of MPS have been reported to present with cardiovascular manifestations. Evaluation of endothelial function (EF) by flowmediated dilation (FMD) of the brachial artery has been studied and endothelial dysfunction (ED) characterized by decreased percent mediated dilatation was demonstrated in individuals with risk factors for atherosclerosis. Close relation of EF in coronary and brachial artery has been demonstrated. Studies to evaluate peripheral vascular EF with finger arterial pulse wave amplitude (PWA) with a finger plethysmograph (PAT) have been performed and showed that PAT hyperemia and FMD were significantly correlated. Objectives: To evaluate coronary artery ED in MPS patients for early detection of coronary artery lesions. Material and Methods: Finger arterial PWA with a PAT device (Endo-PAT2000) was used to evaluate coronary EF. Total of 17 patients were studied: MPS-I (11), II (2), III (2) Twenty-seven MPSpts (6MPS I, 16 MPS II, 5 MPS VI) on ERT were evaluated regarding urinary glycosaminoglycans(ur.GAGs) and the passive articular range of motion. Pts' mean age: MPS I 30.5 years(y)(range 16.7-44.0); MPS II 17y (11.3-29.9), MPS VI 7.4y (4.5-9.4) . ERT mean duration: MPS I 7.5y (6.3-8.7); MPS II 4.8y (3.2-7.1); MPS VI 3.7y (2.4-5.2) . ERT resulted in a mean reduction in ur.GAGs of 89% for MPS I (78.9-96.7); 76.1% for MPS II (30.4-97.2); 65.4% for MPS VI (37. 5-83.4 ). All the pts increased shoulder abduction: mean gain of 27.9degrees in MPS I; 20.8 in MPS II; 8.5 in MPS VI. In MPS I there was a hip mobility improvement (mean 11.2degrees) and a moderate improvement of wrists (6.7degrees) and elbows (4.9degrees). In MPS II there was a stabilization for each joint (gain of 2-3degrees), except for the foot (−1.6). In MPS VI, there was a gain of 2 degrees in foot mobility, there was a 1-3degree loss for the other joints. ERT is effective in decreasing ur.GAGs and in improving shoulder abduction. The best improvement was in MPS I pts (the ones treated longer), whereas there was a joint mobility stabilization in MPS II and MPS VIpts MPSII is the most prevalent type in Brazil with 30% of the cases (Rede MPS Brasil, 2010) , but in MG the most common is MPSI with almost 35% of the MPS. Most patients have classic phenotypes, but rare symptoms as heart aneurysm of the tip of the left ventricle and liver cirrhosis were observed in MPSII. As a differential diagnosis, GM1 gangliosidosis, mucolipidosis II, Geleophysic dysplasia, Dyggve-Melchior-Clausen syndrome, congenital hypothyroidism and Costello syndrome patients were sent to HC-UFMG. Enzyme replacement therapy has been indicated to most patients with MPSI, II and VI. One MPSI patient received successful stem cell transplantation 15 months ago at age of 1 year. Background: Mucopolysaccharidosis I (MPS I) is caused by a deficiency of α-L-iduronidase (IDUA), mainly due to nonsense mutations. Stop codon read through (SCRT) is an alternative to increase enzyme activity by the suppression of a premature stop codon. Objective: To treat MPS I fibroblasts with geneticin and chloramphenicol, which were described to induce misreading. Methods: Fibroblasts from three MPS I patients (p.W402X/p.W402X, p. Q70X/c.1739 G>T, p.R89W/p.W402X) were treated with geneticin (200ug/mL), chloramphenicol (200ug/mL) or had no treatment (n=4/ group). IDUA activity was measured by fluorimetric assay and mRNA expression by quantitative PCR. cDNA sequencing of compound heterozygous was performed. Statistical analysis was done using Friedman with pairwise compairisons (differences: p<0.05). Results: Geneticin was not able to enhance IDUA activity or expression and chloramphenicol enhanced 100-fold IDUA activity on compound heterozygous fibroblasts. cDNA sequencing showed that only the missense allele was being amplified. Conclusion: Nonsense alleles are probably being target to nonsense-mediated mRNA decay (NMD). Thus, positive response to SCRT is dependent on the patient's genotype and on the efficiency of NMD. Chloramphenicol apparently acts through a mechanism other than SCRT. This drug may be used ancillary to enzyme replacement therapy, as it is able to cross the blood brain barrier.
ALFA-L -IDURONIDASE GENE MUTATIONAL ANALYSIS OF 10 EGYPTIAN PATIENTS Ibrahim M 1 , Gouda A 1 , Fateen E 1 , Amr K 2 , Cooper A 3 1 1Biochemical Genetics Departments, Natio, Giza, Egypt 2 2 Molecular Genetics Departments, Nation, Giza, Egypt 3 3Willink Biochemical Genetics Unit , Man, Manchestar, United Kingdom Introduction: The various disorders of mucopolysaccharidosis (MPS) share many clinical features. MPS-I is an autosomal recessive disorder caused by a deficiency of the lysosomal enzyme α-L-iduronidase. Aim of work: Diagnosis of MPS-I among suspected cases referred to Biochemical Genetics Department. Detection of the mutation in the αiduronidase gene among some of the diagnosed Egyptian patients. Subjects and Methods: 10 patients were included; each one was subjected to quantitative determination of urinary GAGs, two dimensional electrophoretic separation, assay of α-L-iduronidase enzyme activity and mutational analysis. Results: Mutational analysis proved that 1 patient was homozygous for the mutation E299, 1 patient homozygous for the mutation C 593 ins 4 del 8 and another one for P533R. MPS-I mutations were not detected in the 4 other patients. 3 novel mutations (c. 854delC in exon 6, T141S in exon 4, and IVS2+6c>t) and G51D in exon 1 were detected. In addition; 9 sequence variants including 5 previously unreported polymorphisms (N73H, N297N, R363S, IVS10 (3025) g>t, and IVS11 (3318) c>a) were identified. Conclusion: Sequencing of the whole gene of α-L-iduronidase is needed to diagnosed patients to detect the commonest mutations among our population and their correlation with the response to enzyme replacement therapy when available. We present a primiparous 37-year-old woman with Mucopolysaccharidose type I, Scheie disease (MPSI-S) who continued to receive enzyme replacement therapy (ERT) with Aldurazyme in a dose of 100 I/U every week during pregnancy. Fetal development was normal and amniocentesis showed a normal karyoptype and enzyme activity for alpha-L-Iduronidase (IDUA). The patient underwent regular cardiac evaluation by echocardiography, which showed moderate mitral and aortic valve regurgitation with normal LV function. At the end of pregnancy she developed signs of pre-eclampsia with hypertension and albuminuria, which was treated with intravenous magnesium sulfate. After a gestational period of 37 weeks and 5 days a Caesarean section was performed and a healthy boy was born. After delivery the patient was admitted to the ICU because of congestive heart failure. Echocardiography revealed significant aggravation of the preexisting mitral and aortic valve regurgitation. After rigorous treatment, the patient recovered without sequelae and was discharged eight days after delivery. This case report indicates that ERT with Aldurazyme can be administered safely during pregnancy, but demonstrates that pregnant MPS I patients should monitored closely especially during the last months of pregnancy because of hemodynamic changes that may occur. Methods: A nationwide survey was conducted in Japan to collect clinical data of the patients with MPS II who received HSCT. The ability of daily life (ADL), joint mobility, height, IQ, MRI lesions, regurgitations of heart valves, and urinary glycosaminoglycans (GAG) were analyzed at the baselines and at the most recent visit in each patient. Results: The records of 26 patients collected from 8 distinct hospitals. The follow-up period was from 5 years 5 months to 16 years 3 months (mean, 9 years 6 months). ADL was maintained almost around baseline levels, joint mobility was improved, growth of height was better than untreated patient cohort. Cribriform changes and ventricular dilatation in brain MRI were improved in 9 and 4 patients, respectively. No improvement was shown in brain atrophy. Valvular regurgitations were diminished in 20 valves out of 66 valves, while worsening was observed frequently in the patients transplanted at older ages. The values of urinary GAG were remarkably lower in HSCT-treated patients than age-matched untreated patients. Conclusion: HSCT is effective and worthwhile in MPS II patients with both of attenuated and severe forms. Background. Mucopolysaccharidosis type II is an X-linked, progressive lysosomal storage disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase. In recent years, clinical trials and overall experience with enzyme replacement therapy (ERT) have been shown to clinically benefit patients with considerable preexisting disease, but no data exists on the effect of beginning ERT before the onset of significant clinical signs of disease. Material and Methods. We present the 3-year follow-up of a boy with MPS II who had idursulfase therapy initiated at the age of 3 months and compare his clinical course to his healthy twin brother. Results. After 3 years of treatment, the patient has not developed any clinical manifestations of MPS II. The most impressive findings were the preservation of normal ranges of movement for most joints, cardiac valves and facial appearance for our patient. The only difference when compared with his healthy twin brother was lower IQ and mild deformity of one vertebrae. Conclusion. We suggest that early treatment of MPS II may significantly delay or prevent the onset of the major clinical signs, substantially modifying the natural history of the disease. Introduction: Mucopolyssacharidosis type III (MPS III), known as Sanfilippo syndrome results from the deficiency of several enzymes needed to break down the glycosaminoglycan (GAG) heparan sulphate, which is particularly important in the central nervous system. MPS IIIC results from deficiency of acetylCoA:alpha-D-glucosaminide acetyltransferase. No specific treatment is available and is therefore limited to the management of symptoms. Genistein an isoflavone that inhibits glycosaminoglycans synthesis has been tried in some patients with MPS IIIA. Case report: We report a seven year old girl presenting with dysmorphia, mild developmental delay mainly of speech, hyperactivity, hepatomegaly high liver enzymes and mild bone abnormalities. Biochemical and molecular analysis showed increased urinary heparin sulphate, no residual activity in PBTL and two causal splicing mutations confirming the diagnosis of type C Sanfilippo disease. Having started genistein supplementation (10 mg/kg/day, in two daily doses) a significant decrease of GAGs and heparan sulphate in the urine were noticed three months later. This decrease was not sustained at a 6 month control evaluation and did not seem to have any clear clinical benefit. Conclusions: Although genistein may decrease the excretion of GAGS in the urine in patients with MPSIII, this effect may be transient and have no effect on symptoms.
Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an inherited lysosomal storage disease caused by deficiency of Nacetylgalactosamine-6-sulfatase (GALNS), an enzyme required for stepwise degradation of keratan sulfate (KS). KS is an important biomarker for MPS IVA as it appears in the circulation and urine, and KS levels can be used to differentiate between MPS IVA patients and unaffected individuals. We have developed a selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay for the KS-derived disaccharides Galb1-4GlcNAc(6S) and Gal(6S)b1-4GlcNAc (6S) in human urine and plasma using keratanase II digestion. This method expands on the existing LC/MS/MS method by allowing quantitation in both urine and plasma and using improved internal standards (heavy isotope-labeled Galβ1-4GlcNAc(6S) and Gal(6S)b1-4GlcNAc(6S)). The lower limit of quantitation was 0.026 μg/mL (plasma) and 0.104 μg/mL (urine), with a quantitation range of 0.026-5 μg/mL (plasma) and 0.104-20 μg/mL (urine). Clinical sample analysis in 168 MPS IVA patients and 225 healthy controls demonstrates the clinical utility of this method in identifying reference ranges for KS in urine and plasma and discriminating between individuals with MPS IVA and unaffected individuals. It also has the potential for longitudinal disease progression assessment. Conflict of Interest declared.
Park HD 1 , Ko AR 2 , Ki CS 1 , Lee SY 1 , Cho SY 2 , Kim SH 2 , Sohn YB 2 , Park SW 2 , Jin DK 2 1 Dept of Lab Med, Sungkyunkwan Univ, Seoul, Korea, Republic of 2 Dept of Pediatrics, Sungkyunkwan Univ, Seoul, Korea, Republic of Background: Mucopolysaccharidosis IVA (MPS IVA; OMIM #253000) is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The GALNS gene encodes GALNS, a lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Herein, we examined biochemical and genetic aberrations from six Korean patients presenting with classic MPS IVA. Methods: GALNS activity was measured in peripheral blood leukocytes or skin fibroblasts. PCR-direct sequencing was performed to investigate the molecular defects of GALNS gene. We also investigated the mutational status for the family members of the patients as well as 50 healthy subjects. Results: The mean age of six female patients was 8.0±5.2 years (range: 2-17 years). Severe reduction of GALNS enzyme was found in all patients. Introduction: MPS IVA is characterized by deficient activity of Nacetyl-galactosamine6sulfatase (GALNS), causing excessive lysosomal storage of keratan sulfate (KS). Patients experience impaired endurance and respiratory function. Case Description: This 16 y/o male patient with genotype homozygous c.452>T (p.Pro151Leu) started ERT with weekly infusions of GALNS (BMN 110) on 10Jun2009. Medical history included aortic regurgitation, genu valgum, limited mobility and overall weakness. Baseline physical exam showed short stature, cardiac murmur, shortness of breath, and joint laxity. Before ERT, he could not sign the consent form, was completely non-ambulatory, and required full assistance for self-care and activities of daily living. During treatment, he became stronger, gaining ability to rise from wheelchair and use toilet without assistance. After 79 weeks, mobility, breathing, and strength improved. He could crawl on his knees, ascend 30 steps, and he was able to sign the consent form for the extension study. Screening urine KS of 24.5 ug/mg creatinine had decreased to 10.8 by Week 72. Conclusions: ERT is a potential new treatment option for MPS IVA, which is expected to reduce KS storage, leading to improvements in respiratory function and endurance. This patient's markedly improved strength and endurance during trial participation improved his daily function. Conflict of Interest declared. Background:Mucopolysaccharidosis IV A(MPS IVA) is a rare autosomal recessive disease caused by deficiency of the lysosomal enzyme Nacetylgalactosamine-6-sulfatase(GALNS),resulting in storage of keratansulfate in many tissues and organs.This accumulation causes a severe skeletal dysplasia with short stature,and affects the eye, heart and other organs. Clinical trials with ERT for this disease are in progress. We describe an innovative fluorometric method for the assay of GALNS in dried blood spots(DBS). Materials and Methods: We used DBS as the enzyme source and compared it with leukocytes,having studied 25 MPS IVA patients and 54 healthy controls.We optimized the assay conditions in DBS,including incubation time and stability on different storage temperatures and along time.Results in DBS were compared to the ones obtained in leucocytes using the standard technique. Results and conclusions: The described fluorescent methodology was validated in our laboratory and the assay was found sensitive and specific, allowing reliable detection of MPS IVA patients.The use of DBS simplifies the collection and transportation,and is especially useful for testing patients from more remote areas of large countries,and when samples need to cross country borders.We believe this assay could be easily incorporated by reference laboratories and play a role in the screening for MPS IVA, contributing to the earlier detection of affected patients. Mucopolysaccharidosis(MPS ) VI is caused by deficient arylsulfatase B. Albinism is characterized by the absence of pigment in the skin, hair, eyes due to production of melanin. Case: The patient was the first of consanguineous parents. She had sensorineural deafness, generalized albinism., coarse face at the age of eighteen months old. Dysostosis multiplex by skletal X-Ray, high excretion of mucopolysaccarides in the urine, reduced arylsulfatase B enzyme activity in leucocytes (73,75 nmol/hour/mgprotein (209±96)) confirmed the diagnosis of MPS VI. The other sulfatase enzyme levels were normal. ARSB gene sequencing revealed homozygous c.908 G>A change in exon 5. This variant, predicted to result in an amino acid change from glycine to glutamic acid (p.G303E), has neither been shown to be associated with MPS VI nor has it been listed in the Ensembl SNP database. Conclusion: MPS type VI is caused by mutations in the ARSB gene, located in chromosome 5 (5q13-5q14). Over 130 ARSB mutations have been reported, causing absent or reduced arylsulfatase B activity. On the other hand the gene accounting for oculocutaneous albinism type 4 is locatad in chromosome 5 (5p13.2). We speculated that, possible chromosomal rearrengements should be further investigated in this case because of this extremely rare co-existence. Mucopolysaccharidosis VI(MPS VI,or Maroteaux-Lamy syndrome)is caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine 4-sulfatase (ARSB).This deficiency causes the storage of dermatan sulphate in tissues, leading to a progressive and severe bone dysplasia and to problems in many organs and systems.MPS VI is a very rare condition,which was a relatively high incidence(13 cases identified so far)in the county of Monte Santo (50,000 inhabitants),in Bahia state,Northeast region of Brazil, where a common mutation(H178L)was found in all cases.As MPS VI could be treated with ERT and as there are indications that a better outcome may be expected in early treated cases,a newborn screening program for MPS VI was set up on this specific area.To the program already in place for PKU,hypothyroidism and hemoglobin disorders,a screening for MPS VI was added by ARSB activity assay and by the detection of the common mutation, both performed on DBS. The standardization of the techniques of enzyme assay,DNA extraction and mutation detection were already completed and the test on newborn samples has started on January 1st,2011.The possibility of detecting carriers will help to calculate the expected frequency of this disease in the area, and will also be a tool to target genetic counseling to the more susceptible families. Mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disorder resulting from impaired function of the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB). In this study, the mutation spectrum of ARSB gene in 19 Turkish patients from 15 different families diagnosed with MPS type VI was determined by using direct sequence analysis. In total, 6 different types of disease-causing mutations (c.1036delG, p.L321P, p.R160X, p.R191X, p. C192R and p.E390K) and 4 polymorphisms (p.V358M, p.V376M, IVS1-26 T>C, IVS5-28A>C) were identified in the patients with MPS type VI. One of the six mutations (p.E390K) is novel in Turkish patients. As an interesting result, the most frequent mutation in our screened cohort was p. L321P (c.961 T>C) with 53% frequency. Our findings are shown inconsistency with the knowledge of the literature, since p.L321P (c.961 T>C) is not one of the common mutation in Caucasians.The mutational analyses on ARSB gene will identify structure/function relationships of the enzyme, more accurate interpretation of genotype/ phenotype correlations and for choosing appropriate treatments. Background: Cardiac abnormalities are frequent in patients with MPS VI and are an important cause of morbidity and mortality. Case Report: A four months old girl was admitted due to failure to thrive, bronchilolitis and heart murmur. The diagnosis of congenital mitral anomaly with severe regurgitation and severe dilatation of left atrium and ventricle was defined. Symptomatic treatment was started with high doses of diuretics and ACE-inibithor, but patient maintained severe heart failure, several respiratory infections and poor weight gain. At the age of 18 months, heart surgery was proposed to valve repair but was refused by parents. Meanwhile it became apparent a phenotype suggestive of MPS VI; diagnosis was established by enzymatic assay and ERT-Galsulfase was started. Now she accomplished 18 months of treatment and clinically is greatly improved, but still maintains aggressive therapeutics of congestive heart failure. Conclusions: This case presents with an unusual early severe valvular involvement with few systemic signs of MPS VI misleading the etiologic diagnosis initially. We emphasize the value of identification of a systemic disease in the context of apparently isolate mitral anomaly in an infant to timely begin specific treatment. Adequate management of these patients is still a challenge to multidisciplinary teams. Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome) is an autosomal recessive disorder caused by a deficiency in the lysosomal enzyme N-acetylgalactosamine 4 sulfatase ( arylsulfatase B, or ASB). We reviewed 10 patients with MPS VI ( five males and five females; age range 1-15.5 years) treated with weekly intravenous infusions of rhASB (1.0 mg/ kg) for at least 6 months. Gestational and perinatal data were normal for all patients. During the course of the disease, all patients except one whom we diagnosed at 4 months because of his sibling with the diagnosis of MPS VI developed coarsened facial features, short stature, heart valve disease, eye problems, musculoskeletal problems, hepatosplenomegaly and neurological abnormalities. We assesed the biochemical and clinical response every 3 months. All patients received rhASB enzyme replacement therapy (ERT) and showed improvement or stabilisation in clinical manifestations after onset of therapy. The most frequently noted benefits were improvement of endurance, reduced hepatosplenomegaly and decrease in urinary glycosaminoglycan excretion. ERT was well tolerated by all patients. This treatment is thus beneficial and appears to be safe for treatment of MPS VI in Turkish patients. These results indicate that the earlier ERT is started, the greater the response. Two siblings with mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome) have had seven years treatment with enzyme replacement therapy (ERT) using galsulfase (Naglazyme) weekly at a dose of1mg/kg starting from 8 weeks and 3.5 years of age respectively. Treatment has been well tolerated by both siblings with no infusion associated reactions recorded. In the younger sibling ERT has preserved joint movement, cardiac valves, liver and spleen size, height (10th centile) and facial morphology. He has mild scoliosis (23 degrees), stable mild corneal clouding but has recently developed evidence of carpal tunnel syndrome and has significant skeletal disease with a waddling gait from avascular necrosis of the hips. The older sibling initially had a marked improvement in joint and scoliosis mobility which have been maintained and has softening of her facial features and stabilised cardiac valve pathology and corneal clouding. Her height is 6 cms below the 1st centile after correcting for scoliosis which has progressed. Rodding of her spine, which was considered necessary prior to initiating ERT, has been deferred until completion of growth. She has required surgery for severe pes cavus. This paper adds further evidence that early initiation of ERT will slow or prevent the natural pathological progression of MPS VI. Mucopolysaccharidoses VI is an inherited lysosomal storage disease, associated with deficiency of arylsulphatase B. The accumulation of dermatan sulphate leads the symptoms of coarse face, short stature, dysostosis multiplex, contractures, cardiac valve disease, reduced pulmonary function, hepatosplenomegaly, hearing loss, sleep apnoea, corneal clouding, hernias, and generally normal intelligence. Diagnosis based on clinical findings, deficient enzyme activity and demonstration of normal activities of other sulphatase enzymes. With enzyme replacement therapy the prognosis is getting better. Case report: This 33 month-old-monozygotic-twin sisters are from a first degree cousin's marriage. They admitted with complaints of frequent respiratory infections, and limited mobility. Their older sister who had a similar facial appearance died from respiratory insufficiency at the age of 13. They have coarse face, short stature, corneal clouding, pectus carinatum, mitral valve insufficiency, claw hands, dysostosis multiplex, and normal intelligence. Enzyme analysis revealed undetectable levels of arylsulphatase B, with other sulphatases within normal limits. They have been on enzyme replacement therapy for one year. Discussion: With enzyme replacement therapy their effort capacity, and mobility have improved and the frequency and the severity of respiratory infections have decreased. To the best of our knowledge these patients are the first reported twins with mucopolysaccharidoses. Results: Ten patients were included. The median follow-up was 2.4 years (range 1.1-3.8 years). Galsulfase had a significant positive effect on the flexion of the right shoulder (p<0.001), liver size (p<0.001) and spleen size (p=0.017). Flexion of the left shoulder showed a positive trend (p=0.07). Two patients showed a significant decrease of left ventricular mass index (LVMI) Zscore. The one patient who showed a decreased forced vital capacity (FVC) at baseline, showed better FVC results during ERT (p=0.023). For the total group a mean decline of 84% in urinary glycosaminoglycan values was observed. ERT did not have an effect on cardiac valve regurgitation and hearing function. Background: Lysosomal storage disorders (LSDs) affect patient quality of life (QoL) but little is known about the disease impact on family life. We assessed the impact of LSDs on daily living of patients and their families and evaluated parent perceptions on the disease and enzyme replacement therapy (ERT). Patients and Methods: Adaptive behaviour, mental development, and cognitive function were assessed in 12 patients with LSDs (MPS I, MPS VI, Pompe, and Gaucher) over a 4-year period. Fifteen parents were evaluated for emotional well-being, satisfaction with life, perception on the severity and prognosis of the disease, disease impact on family QoL, and expectations on and satisfaction with ERT. Results: Adaptive behaviour, mental development and/or cognitive function were impaired in most patients. Depression and emotional disturbance was common among the mothers. Most parents considered their QoL as satisfactory. The disease impact and stress on the family was high, but appeared to decrease after onset of ERT. The high expectations of ERT before the start of treatment decreased to a more realistic level thereafter. Conclusions: LSDs greatly affect the lives of patients and their families. Psychological counselling of parents and providing realistic information about the expectable benefits from ERT is very important. Conflict of Interest declared. Background: Lysosomal storage disorders (LSDs) are a heterogeneous group of inherited metabolic diseases. The definitive diagnosis of these diseases includes the determination of enzyme activity in plasma, leukocytes, or cultured fibroblasts. In recent years, dried blood samples collected on filter paper (DBS) have been utilized. This technique offers a series of advantages, like the low transportation requirements which explain the increasing preference it enjoys currently. Objectives: To analyze the effect of storage conditions on activity of αgalactosidase A, arylsulfatase B and α-glucosidase. Material and Methods: Blood was collected and directly spotting on filter paper and stored at different temperatures (−20, 4, 25 and 37:C) and storage times (3, 10, 17 and 180 days) . The influence of filter paper size was also assessed (3.0 and 1.2 mm). Results: α-glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. Conclusions: Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection. -Laboratory (1992 -Laboratory ( -2010 were reviewed to screen individuals for MPS and ML by using CTS and/or tarsal tunnel syndrome (CTS/TTS) as a screening indicator. All individuals underwent CTS release surgery were included. Results: 2254 individuals (age 0-18 years) underwent NCS. The reason for referral was CTS/TTS in 1.6%. 3% (68/2254) had CTS/TTS. There was history of trauma in 47% (32/68). 26% (18/68) had an underlying disease. In the latter group 38.8% (7/18) had diagnosis of MPS (3 with MPS I, 2 with MPS II, 1 with MPS IV and 1 with MPS VI). 17/68 individuals underwent carpal tunnel release surgery. 5/17 had diagnosis of MPS in the latter group. One additional patient with MPS IIIA had normal NCVat age 4.5 years who will be followed with periodic NCS. Background: Oligosaccharidoses are Lysosomal Storage Disorders (LSDs) caused by mutations in genes encoding exoglycosidases and lead to accumulation of N-linked oligosaccharides within lysosomes. Due to overlapping clinical symptoms, diagnosis of oligosaccharidoses is challenging, involving individual enzymatic assays or urinary oligosaccharide analysis. Here, we aim to determine a universal buffer for the development of an in vitro multiplex assay to measure exoglycosidase activities from human tissues using tandem mass spectrometry (MS/MS). Methods: Classical 4-methylumbelliferyl (4MU) fluorometric assays were compared to determine an optimal universal buffer used to develop a MS/ MS based multiplex assay involving commercial substrates targeting exoglycosidases from fibroblasts. Results: A 30 mM citrate-phosphate buffer with a pH at 4.3 was the universal condition. Additionally, a MS/MS multiplex assay was established for beta-galactosidase and beta-hexosaminidase using substrates composed of 4MU and paranitrophenol (PNP) linked to particular monosaccharides. Enzyme activities from fibroblasts were successfully assayed in one vial by simultaneous measurement of 4MU and PNP reaction products. Conclusions: A universal buffer allows investigation of several exoglycosidases in a single vial. This was demonstrated for beta-galactosidase and beta-hexosaminidase using distinctive starting substrates and MS/MS technology. Substrates giving mass differentiable products for other exoglycosidases will delineate a multiplex assay that can rapidly identify oligosaccharidoses. Background: MPS disorders are the result of primary defects in lysosomal enzymes. Depending on the specific gene defect, the catabolism of one or more of the MPS is blocked leading to accumulation of the corresponding substrate(s). Objectives: To develop a LC-MS/MS method for glycosaminoglycan quantification: dermatan sulfate (DS) and heparan sulfate (HS) using urine filter paper samples. Methods: A urine filter paper disc was eluted, evaporated under nitrogen. Methanolysis was performed using methanol-HCl•3 N with heating at 65°C for 75 min. Deuterated DS and HS internal standards were used. An Alliance 2795XE-LC coupled to a Quattro micro MS/MS (Waters) was used. A chromatographic gradient allowed good separation with an Atlantis T3 column (9 min run). Data were recorded in positive electrospray ionization. Results: Validation of the method provided good results: linearity (r2> 0.995), accuracy (Bias<20%) and precision (CV<15%). Levels of DS and HS in controls were below LOQs. We analyzed 28 MPS patients: 9 MPS I, 2 MPS IH; 6 MPS IH/S; 1 MPS IS, 5 MPS II and 7 MPS VI. In every case, we were able to differentiate MPS patients from controls. Conclusion: A robust methodology was devised to measure glycosaminoglycans using urine filter paper samples. Results: The frequency of hospitalization for respiratory infections decreased in the 1 year following start of home mechanical ventilation. We have encountered no severe ventilator-related issues.No patient died during this study Conclusion: Home mechanical ventilation therapy is very helpful ; this approach helps patients to spend their lives calmly with their family at home. However, gaining regional support is paramount as many local services, such as ambulance departments, schools and regional nursing centres need to provide support to these patients and their families for home mechanical ventilation to be possible. Families often also require financial and psychological support. Olkhovych N.V. 1 , Pichkur N.O 1 , Ivanova T.P 1 , Trofimova N.S. 1 , Nedoboy A.N. 1 , Kormoz S.N. 1 , Gorovenko N.G. 2 1 National Children Hospital "OHMATDET", Kyiv, Ukraine 2 National Medical Academy of Postdiploma, Kyiv, Ukraine Introduction: Chitinases are enzymes that hydrolyze chitin and have been found in a wide variety of non-vertebrate species; recently a human analog of chitinases, chitotriosidase (CT) has been identified. Extreme elevations of plasma CT activity are observed in patients with Gaucher's disease (GD). The 24 bp duplication in the CT gene, resulting in an inactive protein, has been reported. The carrier prevalence is as high as 30 to 40% and the CT activity is half that in individuals with the wild-type gene. However no systematic evaluation of plasma CT activity has been carried out in GD patients taking into account the status of the allele defective for CT and dose in patients on enzyme replacement therapy (ERT). Case report: A 63 year old patient presented mild clinical presentation of GD: splenomegaly, thrombocytopenia. "Gaucher-like cells were revealed by bone marrow biopsy. The glucocerebrosidase activity-3,7 nmol/h/mg protein ( normal 5,5-9,5 nmol/h/mg protein) and chitotriosidase activity-0 nmol/h/ml pl. Genotype was N370S/N370S. The duplication 24 bp in both alleles was detected in CT gene. Conclusion: For monitoring treatment efficacy GD in this patient should be used in other biochemical markers, such as tartrate-resistant acid phosphatase.
Background: Hurler syndrome is the most severe form of mucopolysaccharidosis type I. Treatment options include enzymatic replacement therapy ( ERT) and bone marrow /stem cells transplantation. Case report: Female child with dilatated cardiomiopathy diagnosed at one month of age. Coarse features and growth failure suggested MPS I, confirmed at biochemical, enzymatic and molecular level. ERT with laronidase was started at 5 months of age, improving general clinical condition; patient was proposed to transplant. Ductus arteriosus surgery was done for stabilization of pulmonary involvement. and a bacillus Calmette-Guérin lymphadenitis postponed the bone marrow transplantation, successfully performed at the age of 14 months. A cutaneous graft versus host disease was controlled with immunosuppression but at 24 months a severe lung disease brought the doubt of pulmonary graft versus host disease. Despite leukocyte analyses showed non pathological α-L-iduronidase activity after transplant, there was an urinary GAG accumulation when ERT was postponed and so the patient is going on ERT until now. At the age of 36 months heart function and psychomotor development is close to normal, pulmonary symptoms are controlled with inhaled therapy, and she is growing well. Discussion: questions are highlighted concerning biomarkers significance and recommendations to ERT after graft. Mucopolysaccharidosis type I (MPS I) is a rare autossomal recessive lysosomal disease. Here we report a case of a female infant who, at the age of 8 months, was noticed to have hepatomegaly, failure to thrive, development delay and coarse facies. She was referred to our genetic clinics for investigation. At that time, physical examination showed: weight 6425 g, height 64 cm, OFC: 43 cm, mild coarsening of facial features, enlarged tongue, gibbus, hepatomegaly and development delay. The laboratory exams showed evidence of MPS I, with a deficient activity of α-L-iduronidase (0,17 nmol/hr/mg) and two null mutations at the IDUA gene (Hurler (W402X/ W402X), a genotype compatible with Hurler type preditction. The enzyme replacement therapy (ERT) with laronidase was started when she was 16 months old, while she was under evaluation for HSCT (Hematopoietic Stem Cell Transplantation), and stabilized her clinical status and improved respiratory condition. The allogeneic HSCT was performed after she was on ERT for 11 months (the time needed to find a donor), at the age of 27 months . Five months after HSCT the patient shows a very good outcome with leucocyte enzyme activity on the normal range. We emphasize the benefits of ERT before HSCT to stabilize the respiratory symptoms. Two siblings of consanguinous parents were noted to have a neurologic syndrome marked by regression of psychomotor performance from the first year, marked spasticity and progressive central nervous system degeneration (clinical manifestation was like late infantile Metachromatic Leukodystrophy.) Markedly delayed nerve conduction times were evident. Brain MRI showed diffuse white matter lesions without involvement of subcortical U-fibers., activity of arylsulfatase A white blood cells and cultured skin fibroblasts were below 5%. Molecular investigation of ARSA gene showed a homozygote DNA alteration in A905G position in exon 5 which changes amino acide Lys302Arg. The parents were heterozygote for this alteration. Because of some problem with prenatal diagnosis with enzyme assay molecular diagnosis according to sequencing finding was done for this family. Prenatal Diagnosis was done for this family and fetus was heterozygot for this mutation. This child was affected according to enzyme assay after birth. Molecular investigation for multiple sulfatase deficiency and Saposin B deficiency were negative in this family. Thus, we believe these patients may represent a new form of ARSA deficiency. Methods: We report follow-up of 29 French adult patients with stabilized type 1 GD treated with taliglucerase during 6 months. Results: 29 patients (12 females; median age 43.9). 7 were splenectomized, 2 had bleeding and 10 had a history of bone episodes (1 pathological fracture, 5 avascular necrosis & 4 bone infarcts). Previous treatment was discontinued for a few months because of a shortage of imiglucerase. Median posology was 120 U/kg/month. Biological evaluation at inclusion and at 6 months of treatment, median (min-max) was: hemoglobin G/dl: 14.1 (10.8-16.7) /13.9 (11.9-16.4); platelets 103/dl (only in 22 patients without splenectomy): 124 (50-182) /122. 5 (58.0-207.0) ; chitotriosidase: 1519 (110-12500) /1000 (108-11050). No clinical event related to the disease evolution was notified. 6 patients experienced 21 adverse events (AE) among which 2 patients had serious drug-related AEs with treatment discontinuation: severe anaphylactic reaction and chest pain. Conclusion: Taliglucerase seems to be an effective enzyme replacement therapy in 1GD stabilized patients. Potential side effects should be monitored in all patients and, in particular, those who have received previous exogenous ERT treatment. Conflict of Interest declared. Enzyme Replacement Therapy (ERT) for Pompe disease was approved for clinical use in 2006 based on the efficacy of alglucosidase alfa in classic infantile patients. In this ongoing prospective study, we included 71 adults who were treated with 20 mg/kg alglucosidase alfa every other week. Effects of ERT were compared with natural course. The median treatment duration was 23 months (range 5-47 months). Muscle strength increased significantly after start of ERT: 1.4% points/year for manual muscle testing and 4.0% points/year for hand held dynamometry. The calculated difference between natural course and treatment course was 3.3% points/year for manual muscle testing (P<0.001) and 7.9% points/year for hand held dynamometry (P<0.001). During ERT, FVC remained stable in upright position and declined further in supine position but less than before. For FVC in upright position, the calculated difference between natural course and treatment course was 1.75% per year (P=0.08). Favorable prognostic factors for treatment response were female gender for muscle strength and younger age for pulmonary function. Our study shows that ERTsignificantly alters the natural course in adult patients with Pompe disease. Muscle strength increased and pulmonary function in upright position stabilized, while both parameters declined before start of ERT. Conflict of Interest declared. Background: HPP, heritable rickets due to TNSALP gene mutations, may present in infancy with failure to thrive, fractures and respiratory compromise. ENB-0040 (bone-targeted, recombinant TNSALP) treatment is associated with respiratory function and motor development improvements (Greenberg, ASHG 2010) . Primary efficacy, radiographic change in rickets is reported. Objective: Evaluate rickets severity after 6 and 12-mos of ENB-0040 therapy. Design/Methods: 6-mo Ph2 study (with extension phase on-going); patients received 1 iv infusion, then thrice weekly subcutaneous injections (1-3 mg/kg/dose). A Radiographic Global Impression of Change (RGI-C) was developed to assess rickets in HPP. This is a 7-point scale, where −3 represents severe worsening and +3 complete healing of rickets. Rickets Severity Scale (RSS)(Thacher, 2000) is a 10-point scale scoring metaphyseal changes from 0(no evidence of rickets) to 10(severe rickets). Results: 11 patients enrolled. One withdrew and another died of sepsis unrelated to ENB-0040. "Responders" had a mean RGI-C score of>+2. At 6 and 12-mos, 9/10 and 8/9 patients were responders. Mean(±SD) RSS score for 9 patients at baseline was 8.5 (± 2.4) LSD Unit, Royal Free Hospital, London, United Kingdom Introduction: Gaucher disease is an inherited metabolic disorder in which a deficiency of the lysosomal enzyme glucocerebrosidase leads to pathology, primarily within the spleen and bone. Due to a global shortage, from 2009 to mid 2010, of the widely used enzyme replacement therapy (ERT), patients were allowed to be treated with VPRIV. (velaglucerase alfa, Shire Human Genetic Therapies), which was then in the late trial phase. Discussion on patient group: The patients receiving velaglucerase alfa at this time were newly presenting patients requiring ERT. All six patients were naïve to ERT. The subjects ranged in age from 17 to 69 years at start of treatment. Results: Average treatment duration for this group is now 11.5 months. Infusions have been well tolerated. The average dose used for this patient group is 30 units per kilogram two-weekly. The only adverse event reported, fatigue (by 1 patient), was present before ERT. Five subjects have shown improvement in platelet count within the first 10 months. Anaemia, where present (in 2 patients), has resolved. Visceral scans of these subjects and measurement of plasma chitotriosidase revealed a corresponding response. Conclusions: Initial results of velaglucerase alfa treatment in patients, now treated beyond the clinical studies, are encouraging.
Ferreira AC 1 , Sequeira S 1 1 Metab Unit, Dona Estefânia Hosp, CHLC, Lisboa, Portugal Background: Children with Gaucher disease type I (GD1) are usually treated with enzyme replacement therapy (ERT) at a dose of 30-60U/Kg/ 2 W. Recently, due to an acute shortage supply of imiglucerase, a reduced dose or a reduced infusion frequency was recommended. Objective: To evaluate the effects of a reduced infusion frequency of imiglucerase over 15 months of follow-up. Patients and Methods: Three patients were treated with ERT since a median age of 7 years (range 5-12). Only one had bone crisis and Erlenmeyer deformations. Median duration of treatment before dose reduction was 3 years (range 1-8). ERT resulted in total regression of symptoms, normalization of hematological parameters and progressive improvement of chitotriosidase in all patients. In August 2009 infusion schedule was changed from a media 45U/Kg every two weeks to every four weeks. Results: All patients remained asymptomatic and with no major change on hematological parameters except for one patient who presented subnormal platelet count. All patients showed an upward trend in chitotriosidase values. Comments: Although a longer follow-up is needed, is probable that even children completely stabilized can probably not be kept on lower doses even though the reduction of frequency of the infusions represent a lower social burden. Background: Bone pathology is a major concern in type 1 Gaucher disease. Methods: We evaluated BMD in all 10 patients enrolled in the ongoing, interventional study TKT025EXT (4 men, 6 women; median age 35 years ). Through 69 months, the mean velaglucerase alfa dose was 40 U/kg; 4 patients received concomitant bisphosphonates. Results: At baseline, at the lumbar spine (LS) and femoral neck (FN) respectively, 1 and 4 patients had osteoporosis; 8 and 5 had osteopenia; and 1 and 1 were normal (per WHO T-score-based categorization). Status change occurred only in patients not receiving bisphosphonates: osteopenic-to-normal (2 LS and 1 FN) and osteoporotic-to-osteopenic (1 FN) by Month 69. BMD improved significantly at the LS by Month 24 and at the FN by Month 33. In linear mixed models, Z-scores were significantly lower than the reference population at baseline and improved with treatment (LS and FN both P<0.01); subgroup analysis of patients not receiving bisphosphonates showed similar results. Conclusions: Velaglucerase alfa was associated with clinically meaningful and statistically significant LS and FN BMD improvements as early as Month 24 (LS) and 33 (FN), despite dose reduction (60 to 30 U/kg during Year 2 of therapy) and significant baseline skeletal pathology. Conflict of Interest declared. ). Most AEs were mild or moderate: most common were nasopharyngitis in 15 patients, headache in 15, nausea in 12. 11 patients (5%) experienced a severe AE, considered possibly drug-related in 3 (1%); 7 (3%) a serious AE, considered possibly drug-related (migraine) in 1 (0.5%). Mean hemoglobin and platelets improved in naïve patients and remained stable in switch patients. Conclusion: A clinically heterogeneous group of 211 GD1 patients successfully transitioned to velaglucerase alfa, which was generally well tolerated, supporting this ERT as a GD1 treatment option. Conflict of Interest declared. Giraldo P 6 , Kisinovsky I 7 , Bavdekar A 8 , Gupta N 4 , Kishnani PS 9 , Sureshkumar EK 10 , Wang N 11 , Crombez E 11 , Bhirangi K 11 Background: Therapeutic goals for GD1, a heterogeneous, multisystem disease, facilitate an individualized approach to long-term management. Methods: In HGT-GCB-039, treatment-naïve GD1 patients aged>=2 years were randomized to imiglucerase or velaglucerase alfa (60U/kg every other week [EOW]; 9 months). HGT-GCB-039 completers could enroll in extension HGT-GCB-044, receiving velaglucerase alfa (60U/kg EOW; ongoing). Results: 34 patients received study drug (17 imiglucerase, 17 velaglucerase alfa). 16 patients from each group entered HGT-GCB-044 and are included in this analysis. Hemoglobin concentration, platelet count, liver and spleen volumes were evaluated against therapeutic goals (based on Pastores et al, 2004) . At HGT-GCB-039 baseline, 0/32 patients met all goals (3 goals for splenectomized, 4 goals for non-splenectomized patients). After 9 months, 13/16 patients achieved all goals in both the continuous velaglucerase alfa and imiglucerase-to-velaglucerase alfa groups. After additional 15 months' exposure (in HGT-GCB-044; 24 months total), 16/16 and 15/16 achieved all goals, respectively; 1 splenectomized patient failed to achieve the hemoglobin goal. Conclusions: Most velaglucerase alfa-treated GD1 patients achieved all hematologic, liver and spleen therapeutic goals by Month 9, with comparable achievement in imiglucerase-treated patients. After 2 years, all patients who had received continuous velaglucerase alfa, and the majority transitioned from imiglucerase, had achieved all goals. Conflict of Interest declared. Harmatz P 10 , Fernhoff P 11 , Rhead W 12 , Longo N 13 , Giraldo P 14 , Zahrieh D 15 , Crombez E Background: We report outcomes in type 1 Gaucher disease (GD1) patients transitioned from imiglucerase to velaglucerase alfa. Methods: In trial TKT034, GD1 patients aged>=2 years, on imiglucerase (>=22 consecutive months at 15-60U/kg every other week), with a stable dose and hematologic parameters for>=6 months, switched to velaglucerase alfa (same U/kg, 60-minute intravenous infusion). TKT034 completers could enroll in the extension HGT-GCB-044. Results: 38/40 patients treated in TKT034 continued into HGT-GCB-044 (prior imiglucerase exposure 22-192 months; ages 9-71 years; 24% <18 years; 47% male; 8% splenectomized). After 2 years, the mean changes (95% CI) in hemoglobin concentration, platelet count, and spleen and liver volumes normalized to body weight were 0 g/dL (−0.3, 0.3), 8.0% (1.1, 15.0) , -8.1% (−14.1, -2.2) , and −1.2% (−4.8, 2.4) , respectively-all within the pre-specified clinically significant limits of stability: ±1 g/dL, ±20%, and ±15%, respectively. The majority of patients maintained all therapeutic goals met at baseline for these 4 parameters. The mean change (95% CI) in CCL18 concentration was −50.9% (−55.5, -46.3) and in chitotriosidase activity, -34.5% (−42.2, -26.9 Harmatz P 10 , Fernhoff P 11 , Rhead W 12 , Longo N 13 , Giraldo P 14 , Zahrieh D 15 , Crombez E 15 Background: Adverse events (AEs) are important considerations for physicians treating type 1 Gaucher disease (GD1) with ERT. Methods: In TKT034, 40 GD1 patients receiving stable imiglucerase for > =22 months switched to velaglucerase alfa for 12 months (protocol-specified continuous 60-minute infusion; otherwise, same regimen and U/kg as prior imiglucerase treatment). 2 patients discontinued (1 for personal reasons, 1 due to a hypersensitivity reaction during the first infusion); all 38 patients completing the study elected to continue in the extension. AEs were elicited by non-leading questions or discovered through observation, laboratory reports, or patient complaint. IRAEs were defined as beginning<=12 hours after infusion start and possibly or probably related to study drug. Results: In the 38 patients receiving 24 months of velaglucerase alfa, median prior imiglucerase exposure was 65 months (range 22-192) . The most common AEs (>20% of patients) were headache, nasopharyngitis, pharyngolaryngeal pain, cough, arthralgia, myalgia, and fatigue. Most AEs were mild or moderate and none led to study discontinuation. 6 SAEs occurred in 5 patients (none study drug-related). 9/38 patients experienced>=1 IRAE. Conclusions: In GD1 patients transitioned from imiglucerase, velaglucerase alfa was generally well tolerated through 2 years; no drug-related SAEs occurred and most patients experienced no IRAEs. Conflict of Interest declared. Background: Standard of care for Gaucher disease(GD), the deficiency of lysosomal enzyme glucocerebrosidase, is enzyme replacement therapy (ERT). ERT is highly effective in reversing the visceral and hematologic manifestations, but skeletal and pulmonary complications, and inflammatory component respond variably. We report the first-year experience with velaglucerase-alpha(VPRIV®), a gene-human glucocerebrosidase by providing specific case examples. Methods: 15 (12 F: 3 M) GD1 patients were treated with VPRIV®, 30 to 60 IU/kg, for one year. At initiation of therapy, splenectomized patients (n=4), 2 naïve to ERT, had significant disease-load, presenting with transfusion dependency, increased pulmonary pressures, systemic inflammation and skeletal involvement. Chitotriosidase(CHITO) was followed as a GD marker, and antibodies were obtained in selected patients. Results: Among patients with mild-moderate GD, hematological parameters and CHITO remained stable. In severely afftecteds, the clinical response was observed usually within first 3 months of therapy. Changes in CHITO ranged between normal levels to a more than 50% decrease. There were three infusion-related events, but antibody development was not observed. Conclusions: The success in clinical response may concur with better in vitro internalization of velaglucerase-alpha, into macrophages. Nevertheless, having different enzymes will provide an opportunity to re-evaluate initiation of ERT, and management of non-responders in GD. Conflict of Interest declared. Background: Pompe disease is caused by a deficiency in the lysosomal enzyme acid alpha-glucosidase (GAA), which results in progressive accumulation of glycogen in lysosomes of various tissues, mainly muscle. Recombinant human GAA (rhGAA) given as bi-weekly infusions, known as enzyme replacement therapy (ERT), is the primary treatment for Pompe patients. While generally effective, the rhGAA has a short circulating halflife, low tissue uptake, and can elicit immune responses that adversely affect tolerability and efficacy. AT2220 (1-deoxynojirimycin) is a small molecule pharmacological chaperone that binds endogenous GAA in cells and tissues, resulting in increased GAA lysosomal levels and activity. Objective: To determine if AT2220 also interact with exogenous rhGAA (i.e ERT) to improve its pharmacological properties. Results: In human plasma, AT2220 increased the stability and minimized denaturation of rhGAA at neutral pH/37°C. In rats, oral co-administration of AT2220 increased the circulating half-life of rhGAA by~2-fold. In GAA KO mice, oral co-administration of AT2220 resulted in up to 2.5-fold greater tissue uptake and glycogen reduction compared to rhGAA alone. Conclusions: Collectively, these data indicate that AT2220 stabilizes rhGAA, and leads to improved uptake and glycogen turnover. Based on these findings, a Phase 2a study of AT2220 with rhGAA has been initiated. Conflict of Interest declared. Background: Enzyme replacement therapy (ERT) in glycogen storage disease type 2 (GSD2) has been demonstrated to be safe and effective in short term studies. Objectives: To evaluate effects of ERT in juvenile GSD2 patients after 48 months of treatment. Patients and methods: 8 patients (mean age 10.9±1.9 years) treated with 20 mg/Kg of ERT biweekly up to 48 months were included. Walton Scale (WS), 6 minutes walking test (6 MWT), vital capacity, forced expiratory volume, arterial pCO2, and muscle enzymes were analyzed retrospectively. Results: After 48 months ERT was well tolerated in all subjects. Significant improvement of 6 MWT (p<0.03), and a stabilization or reduction of WS were observed in all patients. Parameters of respiratory function remained stable, and shortened time of required ventilatory support was observed in the 2 patients already ventilatory dependent at the beginning of the study. A significant reduction in muscle enzymes (p<0.03) was evidenced. Conclusions: Although the relative small number of patients included, this study demonstrate that ERT in juvenile GSD2 is able to improve motor function and stabilize ventilatory function after 4 years of therapy. The best results were achieved when ERT was started before the onset of the clinical manifestations of the disease. Maltase acid deficiency (Pompe disease), leads without treatment to hypertrophic cardiomyopathy muscle weakness, and premature death. Enzyme replacement therapy (ERT: rhGGA) is poorly efficient in crossreactive immunological material (CRIM) negative patients. In these patients, immunologic reaction can neutralize rhGGA . We described a CRIM negative (proven on fibroblasts) 30-months-old boy treated by ERT since two months of age. The disease is due to a homozygous mutation in the GAA gene (p.R870x). The cardiomyopathy rapidly improved, while the muscle weakness improved partially. Intially, he presented pulmonary infections, asthma, swallowing difficulties which led to a feeding by gastrostomy. The second year of life was marked by a real improvement in muscle weakness without anymore pulmonary infections. At 2 years old, he presented an anaphylactic reaction to the treatment while there was a slight increase of rhGGA antibodies (IgG: 1/100). Under steroid therapy he supported the next ERT infusions. Despite moderate psychomotor retardation, he clearly continue to progress on the gross motor development and on respiratory function. This is the first case of successful ERT in a CRIM negative child. Immunologic studies are pending to assess the immunological functions of this child who did not produce high levels of rhGGA antibodies.
SURVIVAL OF ADULT POMPE PATIENTS WITH AND WITHOUT ENZYME REPLACEMENT THERAPY Güngör D 1 , van der Ploeg AT 1 , Hagemans MLC 1 1 Center for Lysosomal & Metabolic Dis., Erasmus MC, Rotterdam,
Background: There is paucity of information on the impact of Enzyme Replacement Therapy (ERT) on survival of adult patients with Pompe disease. We assessed whether there is differential mortality in adult patients treated with ERT compared to untreated patients. Methods: Data of 270 patients with a median age of 48 years (range 19-79 years) were collected in an international observational study between 2002 and 2011. Kaplan-Meier survival curves were plotted and multivariate logistic regression analyses were performed.. Results: The ERT-group included 195 patients with 15 deaths and the nontreatment group 75 patients with 26 deaths. Survival was higher for patients on ERT compared to patients never on ERT and for patients with lower compared to higher levels of disease severity. For each level of disease severity (based on wheelchair and ventilator dependency) greater survival on ERT was observed. In logistic regression analyses controlling for age, gender, and national treatment patterns, both treatment with ERT and disease severity were associated with survival. Conclusion: Our results suggest that ERT expands the life span of adult patients even when they are severely affected. Longer follow-up is needed to further elucidate the relationship between ERT, disease severity and survival of adults with Pompe disease. Conflict of Interest declared. Background: The role of infections in IAR during ERT with the exogen protein is still not clear. We report a case of a patient who had been under ERT with Agalsidase Beta for more than four years who had an IAR when presenting an intercurrent infection. Case report: Male, 19 years, on ERT for 4 year. During his 90th infusion, he presented high fever, tremor, skin rash, which was treated with corticosteroids e antihistamine. On that day, even before ERT, he presented cough and rhinorrhea without fever. Acute rhinosinusitis was diagnosed, antibiotics were prescribed and the patient was discharged home. On the next infusion, he had already overcome the rhinosinusitis, was asymptomatic, didn't take any pre-medication and received the ERT without any other IAR. Discussion: The pharmacologic interaction with immune receptors concept (P-I concept) is a recently proposed addition to drug hypersensitivity classification. Infections can incite T lymphocytes to change their link to some drugs and this process may cause adverse reactions. Conclusion: ERT is often complicated by immune responses to the enzymes and the P-I concept must be reminded as an explanation to IAR in patients with infections, because in these cases the use of pre-medication is not necessary. Conflict of Interest declared. 
Bachground: MPS1 is an autosomal recessive disorder caused by deficient activity of the lysosomal enzyme alpha-L iduronidase, which leads to accumulation of heparan sulfate and dermatan sulfate, resulting in progressive multisystem disease with respiratory, skeletal, and neurologic manifestations. Treatment for MPS1 consists of supportive care and enzyme-replacement therapy with Laronidase. Bone marrow and hematopoietic stem cell transplantation is the treatment of choice for patients suffering from MPS1 with no or minimal central nervous system manifestation. Case Report: A 50 month old Iranian boy with coarse facial features, prominent forehead, corneal cloudy, sleep disturbance, hepatosplenomegaly, inguinal hernia, joint stiffness, and dysostosis multiplex congenital. He was diagnosed with MPS1 on the basis of clinical findings, an elevated urinary glycosaminoglycan level and low alpha-L-iduronidase activity in leukocytes. Mutation analysis revealed a homozygous splice mutation in the IDUA gene. He has been started on injection of aldurozyme intravenously every week from age 26 months. To attempt to prevent neurological impairment before bone marrow transplantation, he receives intrathecal enzyme replacement of aldurozyme and samples of his CSF are obtained for testing monthly. He tolerates intrathecal ERT with no adverse events. At age 50 months he will receive a bone marrow transplantation. Mucopolysaccharidosis I(MPS I)is a lysosomal disease caused by αiduronidase deficiency, leading to glycosaminoglycans(GAGs)storage in several organs.This may affect the vertebrae and meninges and cause spinal cord compression (SCC).As intravenous enzyme replacement therapy (ERT)is not expected to cross the blood-brain barrier,other approaches of drug delivery are necessary to treat SCC.We report a case of a 23 yo MPS I patient(Hurler-Scheie phenotype)with SCC who choose to stop intravenous ERT after 5 years of treatment since his perception was that symptoms were not improving.Laminectomy and occipital craniotomy were then performed, again with no improvement of his condition.Intrathecal infusions were offered on a compassionate use basis,and he agreed to perform 4 infusions with one month intervals.At the baseline and after the 4th infusion,CSF biochemistry(with GAGs measurement),clinical neurology assessments and some additional studies were performed.The patient referred improvement of his lower limbs sensitivity every first week after infusions with regression to previous status thereafter.Somatic sensitive potentials in upper and lower limbs showed improvement Intrathecal infusions showed to be safe and may be offered as an option when the neurosurgical risk is high or as an alternative to improve patient's QOL. We think that adjustments in dosis and infusion frequency may improve outcome of this approach.
The MPSII is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), characterized by the accumulation of glycosaminoglycans (GAGs). The major clinical manifestations are joint contractures and often mental retardation. The MPSII has been treated by HSCT/ ERT, but this effectiveness in CNS is limited because of poor uptake of enzyme to cross the BBB. To increase the efficacy of ERT in the brain, we tested intraventricular administration (IVA) procedure of IDS in Ids knockout mice. 20ug of IDS was administered into the ventricle of MPSII mice repeatedly 4 times every 3 weeks. The IDS enzyme activities, GAGs assay and histopathology were measured in mice tissues. The IDS activities were significantly increased in MPSII mice cerebral regions with IVA. In cerebellum, the activities were also elevated to similar levels of normal mice. The accumulation of total GAGs was decreased in the brain treated with IDS. The over expression of Lamp2 was also observed in Purkinje cell of MPSII mice. In MPSII mice tissues with IDS multiple IVAs, the high level IDS activities were maintained and GAGs storage levels were also decreased. These results demonstrate the possibility and efficacy of novel ERT procedure with IVA for MPSII treatment. Conflict of Interest declared. Introduction: MPS II is a X-linked lysosomal storage disorder causing a progressive multisistemic accumulation of glycosaminoglicans (GAGs). Enzimatic replacement therapy (idursulfase) has been introduced in the last years. Case report: We report the case of a 19 years old boy who received diagnosis of MPS II at the age of 4 years by the presence of an occasional heart murmur (R443X mutation). At 14 years he began therapy with idursulfase (dose 0,5 mg/kg/die): urinary GAGs have been decreased significantly. Despite the severe aortic and mitral regurgitation, no heart failure has been detected. The severe bilateral mixed hearing loss and the restrictive respiratory impairment are both stable, while the mild form of obstructive sleep apnea syndrome has been resolved. Hepatomegaly is still present, splenomegaly has been reduced and so the pancytopenia from hypersplenism. A mild thrombocytopenia is still present. Lower limbs muscular trophysm has improved but neither joint stiffness nor bone dysmorphisms did. Mild mental retardation is present. Conclusion: In this patient Idursulfase therapy can be considered effective even if started in adolescence.
A SYSTEMATIC LITERATURE REVIEW: ENZYME REPLACEMENT THERAPY IN MUCOPOLYSACCHARIDOSIS TYPE II Alegra T 1 , Eizerik D 1 , Cerqueira CCS 1 , Pereira TV 2 , Schwartz IVD 1 1 Univ Federal do Rio Grande do Sul, Porto Alegre, Brazil 2 Univ Federal de São Paulo, São Paulo, Brazil Enzyme replacement therapy with idursulfase (IDS) (0.5 mg/kg/every other week) is a therapeutic option for mucopolysaccharidosis (MPS) type II. Methodology: In order to examine the efficacy and safety of ERT, a systematic literature review was conducted until February 28, 2011. The inclusion criterion was being a randomized controlled trial (RCT); in the absence of at least 5 RCTs, prospective case series with ≥5 patients that evaluated relevant endpoints, previously defined by our research team, were also included. Results: Two RCTs comparing IDS to placebo and 2 open label trials were included. One RCT was a phase I/II trial (n=12 patients; 72 weeks), and the other a phase II/III trial (n=94; 54 weeks). Both demonstrated the following: reductions in urine glycosaminoglycans and hepatosplenomegaly, increase in the 6-minute walk test distance and pulmonary function; however, disease severity was heterogeneous between the groups at baseline. Both open-label studies revealed analogous benefits as described by the RCTs. Harmatz P 1 , Cheng S 2 , Martin K 2 , Burton B 3 , Giugliani R 4 , Guelbert N 5 , Guffon N 6 , Hendriksz C 7 , Hollak C 8 , Jones S 9 , Lin S 10 , Martins A 11 , Mengel E 12 , Mitchell J 13 , Parini R 14 , Valayannopoulos V Background: Mucopolysaccharidosis (MPS) Type VI is a lysosomal storage disorder characterized by a chronic, progressive course with multiorgan involvement. Methods: In our study, clinical, biochemical and radiological findings and response to enzyme replecement therapy for at least six months were analyzed in 20 patients with MPS VI. Any changes on clinical findings such as liver and spleen sizes, cardiac and respiratory parameters, endurance tests (6-minute walk test), changes in urinary glycosaminoglycan excretions, visual and auditory changes, and joints' range of motions before and after ERT were analyzed. Results: There was significant improvement in physical endurance with ERT (p<0.05). With ERT, there was an increase in growth rate but this was not statistically significant (p>0.05). Cardiac, respiratory, visual, auditory parameters were not been changed and there were no improvement on joint mobility (p>0.05). All patients and parents pointed out an increased quality of life, which did not correlate with clinical results. Conclusions: ERT caused increased physical capacity and decreased urinary dermatan sulphate/keratan sulphate ratios. No significant changes were observed in growth parameters, cardiac, respiratory, visual, auditory findings and joint mobility. ERT was safe but an expensive method for the treatment of MPS VI with mild benefits on severely affected cases.
Mucopolysaccharidosis VI (MPS VI) results in accumulation of the glycosaminoglycan (GAG) dermatan sulfate. Storage of GAG may be relieved by Naglazyme® treatment. The Clinical Surveillance Program (CSP) is a voluntary observational program which collects data to characterize progression of MPS VI and collect long term efficacy and safety outcomes of Naglazyme treatment based on patient standard of care assessments. Of the 151 patients enrolled in the CSP through 22 March 2011, 12 entered enrollment in the prior year and 139 have received Naglazyme treatment. To date, 6 patients have had bone marrow transplant (BMT), 2 of which were reported to not remain grafted, and 5 of the 6 patients have received subsequent Naglazyme treatment. There was an approximately 50% decrease in median urinary GAGs from baseline after Naglazyme treatment for up to 9 years. Improved endurance measurements (12-minute walk and 3-minute stair climb tests) and increased height and weight from baseline were observed in Naglazyme treated patients. Elevated antibody levels were not correlated with changes in urinary GAGs. Occurrences of serious adverse events related to Naglazyme treatment, including infusion related events, remain low. Naglazyme treatment appears to slow progression of MPS VI when compared to untreated patients in the CSP Conflict of Interest declared.
Ketteridge D 1 , Moore L 2 , Oates S 1 , Bratkovic D 1 1 Metabolic Clinic, SA Pathology, North Adelaide, Australia 2 Histopathology, SA Pathology, North Adelaide, Australia
Background: MPS VI is a multisystemic lysosomal storage disorder characterised by the accumulation of glycosaminoglycans (GAG) in lysosomes. The disorder affects most tissues of the body including the viscera, bone, cartilage and fibrous tissue. Enzyme replacement therapy (ERT), has drastically changed the disease course in MPS VI, however much remains unknown about longterm therapy. Case Report: We describe a female patient with rapidly progressive MPS VI who commenced ERT at 9 years of age and died at 16. Electron microscopy (EM) was performed on the tissues of a toe removed 6 months after commencing enzyme replacement therapy and on a contralateral toe at autopsy after almost 7 years of ERT. Results: Intital EM demonstrated significant staorage of GAGs in the cells of the bone, cartilage and fibrous tissue. Autopsy microscopy showed minimal to no storage of GAGs in bone or cartilage, but persistent storage in fibrous tissue. Conclusions: In this case there appeared to be microscopic clearance of storage from bone and cartilage, but not fibrous tissue. The differences observed may reflected differential cell turnover in bone and cartilage compared to fibrous tissue. This finding provides human evidence that ERT may influence the disease process in bone and cartilage. Lysosomal storage diseases constitute about 50 different diseases, with a specific lysosomal enzyme deficiency. They have characteristic features, and diagnosis must be established with specific biochemical assay or by DNA analysis. There is no specific cure for these disorders, however, a new era has emerged in recent years in the management with Enzyme replacement therapy (ERT).Although it is not a cure but it can modify or attenuate the disease phenotype and its progression. The treatable disorders include the non-neuronopathic Gaucher Disease, MPSI, MPSII, MPSVI, Fabry disease, and Pompe Disease by ERT. Results: ERT for Gaucher, Pompe and MPS I, and Fabry is being given in India by Genzyme, USA as a charity. At rainbow children Hospital, Hyderabad, India, a total of six patients were under ERT, Response to therapy was excellent with Gaucher disease, and not very encouraging with Pompe disease and reasonably satisfactory with MPS I. Conclusions: Treatable LSD is more often diagnosed in India and option for treatment is limited in view of the cost. Hypophosphatasia (HPP) is the metabolic bone disease caused by loss-offunction mutation within the gene encoding the "tissue nonspecific" isoenzyme of alkaline phosphatase (TNSALP). Perinatal/early infantile-onset HPP is usually fatal due to respiratory insufficiency. There is no approved medical therapy. We reviewed our 80-year (1927-2007) retrospective cohort of 19 Canadian patients with perinatal/early infantile-onset HPP. 14/19 were of Mennonite descent and those available for study were homozygous for the TNSALP gly317asp mutation with profound skeletal hypomineralization, severe rickets, and respiratory insufficiency. All died by 9 months-of-age, most often soon after birth, from pulmonary failure. Clinical trials of enzyme-replacement therapy for HPP using human, recombinant, bone-targeted TNSALP (ENB-0040) began in 2008. To date, 20 infants and young children (10 perinatal; 10 infantile-onset HPP) who met eligibility criteria have been enrolled and treated with thrice weekly subcutaneous injections. 1 withdrew after the first injection and 1 died of sepsis unrelated to ENB-0040. In the remaining 18 patients, including 2 perinatal homozygous for the Canadian Mennonite mutation, marked improvement in their radiological findings and respiratory function is paralleled by dramatic clinical gains with minimal side effects. The liver is a potential target for transgene delivery and expression for gene therapy of hepatic and various metabolic diseases, including amino acid metabolism or urea cycle disorders. Currently, we are developing and evaluating highly efficient non-viral gene transfer methods by targeting the murine liver as a potential alternative gene-therapeutic approach. In our study, we report the use of the minicircle (MC) technology for the gene therapy of phenylketonuria (PKU) mouse model. Our MC-DNA vectors contain a liver-specific promoter, Pah, the luciferase reporter gene plus a downstream mammalian scaffold/matrix attachment region (S/MAR) element for episomal maintenance and/or extra-chromosomal stability. Delivery was mediated by hydrodynamic tail vein (HTV) injection as a liver-targeted approach. Luciferase expression was monitored by quantitative bioluminescence in a non-invasive imaging technology in living mice (IVIS screening). We compared longitudinal luciferase expression between MC-DNA vector and conventional plasmid DNA (pDNA). Our data showed that vectors were exclusively delivered to the liver and the luciferase expression in mice injected with MC was more than 20-fold higher than mice injected with pDNA for at least 9 months. MC gene delivery for maximizing safety and sustained gene expression is a potential new approach for the hepatic treatment. Background: Lipoprotein lipase deficiency (LPLD) is a rare, monogenic lipid disorder causing hyperchylomicronaemia, increased risk of acute pancreatitis (360 x) and cardiometabolic complications. Alipogene tiparvovec (Glybera®) contains a gain-of-function LPL-gene mutation in an AAVvector. We conducted two interventional trials and one case note review study for pancreatitis (CT-AMT-011-03). Methods: In the interventional trials involving 19 LPLD adults, alipogene tiparvovec was administered IM one-time. These patients and subjects participating in an earlier observational study were enrolled in CT-AMT-011-03. Historical data for all hospital presentations due to abdominal pain were collected. Pancreatitis definition was according to modified Atlanta Diagnostic Criteria for acute pancreatitis. Statistical analysis to model the A Cox regression gap-time-model estimating hazard of pancreatitis pre/ post therapy was used. Results: Alipogene tiparvovec was well tolerated. Adverse events were mainly injection site related, mild-moderate and transient. A statistically significant reduction in the risk of acute pancreatitis was seen comparing the period from the first pancreatitis event to administration with the post-therapy period (median=2.9 years). The hazard ratio indicated a 63% reduction in risk of acute pancreatitis (95% CI 0.142-0.971). Conclusion: Alipogene tiparvovec was well tolerated, safe and reduced the risk of pancreatitis during long term follow up post one-time administration. Conflict of Interest declared. We have previously demonstrated the successful treatment of Pahenu2 mice, a model of human PKU, following liver-directed administration of a phenylalanine hydroxylase (PAH)-expressing rAAV2/8 vector. However, the therapeutic effect of this treatment was only temporary with transgene loss and reemergence of elevated blood phenylalanine within 4-6 months after vector injection. rAAV vectors that harbor 28S ribosomal DNA (rDNA) sequences flanking the therapeutic transgene cassette have been shown in other mouse models to permanently integrate at least ten times more frequently than standard rAAV. Portal vein injection into Pahenu2 mice of a novel rAAV2/8 vector that contains a PAH expression cassette flanked by rDNA sequences yielded complete correction of blood phenylalanine within one week. The treatment effect lasted almost one year after injection. Site-specific integration of the novel vector genome into mouse 28S rDNA has been demonstrated using nested PCR, but the true integration frequency is currently being evaluated. The inclusion of rDNA sequences into rAAV2/8 vectors to direct increased frequency of site-specific integration events is a promising novel step in the development of safe, effective rAAV-mediated liver-directed gene therapy for PKU and other inborn errors of metabolism. Introduction: Current treatments for Methylmalonic Aciduria (MMA) remain unsatisfactory and research on novel therapies remains a high priority. A lentiviral (LV) vector was developed to treat an in vivo model of MMA. Aim: To examine the therapeutic effect of lentiviral vector-mediated transfer of methylmalonyl CoA mutase (MCM) gene into a MCM knockout mouse. Methods: A LV-MMA vector was developed and injected intravenously into 5 MMA mice. Untreated MMA (n=6) and normal mice (n=6) were used as controls. Mice weight was measured weekly after treatment. Plasma and urine MMA were measured using mass spectrometry and posttreatment MCM enzyme activity was determined by HPLC, according to published methods. Results: The LV-MMA treated mice achieved near-normal weight for sex. Median plasma MMA levels in the LV-MMA treated group were reduced from 909+/−365uM at baseline to 377+/−166uM at 6 months, compared to untreated MMA mice 1114+/−328.54uM. Hepatic MCM enzyme 6 months post LV therapy ranged from 1.8 to 166.1 nmol/min/ug of protein (median 130.56+/−70uM), compared to not detectable in the untreated mice (N=6). Conclusion: These results confirm that the use of LV-MMA may be a viable approach for the treatment of this form of MMA. Background: Canavan Disease (CD), caused by aspartoacylase (ASPA) deficiency is a severe leukodystrophy with no effective treatment. Earlier gene therapy attempts using rAAV2 failed. Objective: To develop novel rAAV-based therapeutics for efficacious and safe gene therapy of CD. Materials Methods and Experimental Design: ASPA−/− KO mice created by us mimic the neuropathology and clinical manifestation of CD patients, as early as the 2nd week of life and die early. Novel rAAVs developed by us efficiently cross the BBB and transduce the CNS after an i.v. injection. We used miRNAs to regulate i.v. delivered rAAV towards CNS-restricted gene expression. This novel gene therapy approach was evaluated in the CD mice. Results: Single i.v. injection of rAAVASPA postnatally corrected metabolic, psychomotor deficits and spongy degeneration of the CNS. New findings of severe retinopathogy and renal pathology that were observed in the CD mice, were corrected with Gene Therapy. This treatment prolonged the life of the mice and restored their vision. Conclusion/Discussion: Our data demonstrate the feasibility, safety and efficacy of this novel approach for gene therapy in CD. Development of clinical trials for CD patients should be the outcome of these on-going studies. Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is one of lysosomal storage diseases (LSDs) caused by deficiency of betaglucuronidase (GUSB), resulting in progressive accumulation of glycosaminoglycans (GAGs) in various tissues including the brain. In this study, we evaluated the potential to perform gene therapy for MPS VII. Methods: We constructed a lentiviral vector, SMPU-R-MND-HBG -IE, to transduce cells with the normal human HBG cDNA. We injected the neonatal mice of MPS VII, and sacrificed 7 weeks and 31 weeks after treatment and analyzed GUSB activity by 4-MU method, Lentiviral DNA bio-distribution by real-time PCR and evaluation of autophagy performed by Western-Blotting and Immune stain of LC3 protein in brain and liver. Results: GUSB enzyme activities of the treated mice were elevated in all organs and decrease GAGs in all organs and a significant deceased LC3 protein in the treated mice and indicated the organs failure in MPS VII due to autophagy. We detected lentiviral DNA expression in all organs of treated mice 7 weeks and 31 weeks. Conclusion: We have demonstrated that the neonatal gene therapy for MPS VII mice using lentiviral vector resulted in long-term and efficient GUSB expression, which corrects the biochemical defects, and decreased LC3 protein. Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH). BH4 is synthesized de novo from guanosine triphosphate (GTP) by three enzymes: GTP cyclohydrolase I (GTPCH), 6pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Conventional treatment of patients with BH4 cofactor deficiency due to autosomal recessive mutations in the PTS gene, encoding PTPS, requires control of elevated blood-phenylalanine (Phe) levels by BH4 supplementation and oral replacement therapy with L-dopa and 5-OH-tryptophan, precursors of dopamine (catecholamines) and serotonin, respectively. We have recently developed a viable mouse model for PTPS deficiency, the compound heterozygous Pts-ki/ko mouse, which exhibit low PTPS activity, hyperphenylalaninemia and low brain dopamine and serotonin levels. Currently we are testing a triple-gene transfer to target liver using AAV2 pseudotype 8 vectors (rAAV2/8) in combination with luciferase coexpressed for in vivo hepatic imaging. Coordinate expression of PTPS, TH and TPH is thought to lead to hepatic synthesis of BH4, L-dopa plus 5-OH-tryptophan, respectively, and eventually to restoration of systemic phenylalanine clearance and endogenous supply of the essential precursors for monoamine neurotransmitter biosynthesis in the CNS.
HELPER-DEPENDENT ADENOVIRAL VECTORS FOR LIVER-DIRECTED GENE THERAPY OF PRIMARY HYPEROXALURIA TYPE 1 Castello R. 1 , Piccolo P. 1 , Borzone R. 1 , D'Aria S. 1 , Brunetti-Pierri N. 1 
Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT), which catalyzes the conversion of glyoxylate to glycine. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only therapeutic strategy available to prevent kidney failure. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we are investigating helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We have injected PH1 mice with an HDAd encoding the AGT under the control of a liver-specific promoter and observed a sustained reduction of oxalate urinary excretion and reduction in kidney stone formation. Recently, we have developed a minimally invasive method to improve the therapeutic index of HDAd (Brunetti-Pierri et al., 2009 ). This approach based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver, results in higher efficiency of hepatocyte transduction using clinically relevant low vector doses. Therefore, this method may permit correction of PH1 using clinically relevant doses of HDAd and may thus pave the way to clinical application of HDAd for PH1 gene therapy. The use of antisense genetic therapy for RNA mis-splicing diseases has gained increased attention as the splicing changes account for up to 15% of all mutations and with massive parallel genomic sequencing of individual patients the number of splicing mutations will be increased. Although the number of patients who can be potentially treated is low, it represents an excellent therapeutical option representing a type of personalized molecular medicine which is especially relevant for diseases for which there is to date no efficient treatment. In this work we summarize the splicing modulations explored to date especially targeted to deep intronic changes and the potential use to reprogram the splicing process using antisense therapy against intronic and exonic new or cryptic splice sites. In addition, we present our recent data in the investigation of new transporter structures that are thought to provide effective in vivo delivery. We are working on an octa-guanidine dendrimer covalently linked to specific morpholinos and also a new approach using locked nucleic acids monomers (LNA) bound to carbosilane dendrimers. We have successfully recovered the splicing process in MUT, PTPS, PCCA and PCCB disease cellular models suggesting that we are closer to applying the antisense therapy in animal models. A recently discovered chemical entity, PTC124 (Ataluren) was shown to selectively induce ribosomal readthrough of premature but not normal termination codons. In vitro experiments and subsequent treatment of mdx mice showed a promising improvement in the form of functional recovery. This led us to investigate the effect of PTC124 on nonsense mediated premature termination in two of the three cerebral creatine deficiency syndrome genes, namely the creatine transporter gene (SLC6A8) and the guanidinoacetate methyl-transferase gene (GAMT). We obtained PTC124 from Selleck Chemicals (Texas, USA) and incubated patient's cell-lines containing nonsense mutations with 17 μM of PTC124 for 46 days (SLC6A8, n=2) and 14 days (GAMT, n=3) and collected cells every 7 days. We measured intra-cellular creatine content and formation of 2H3-13 C2-creatine using a two-step derivatization procedure, followed by quantification with GC-MS. With all cell-lines we found no increase of function, indicated by low intra-cellular creatine content (SLC6A8) and no formed 2H3-13 C2-creatine (GAMT) as compared to the wild-type celllines. This led us to conclude that PTC124 (Selleck Chemicals) cannot induce ribosomal readthrough of the five tested nonsense mediated termination codons in the SLC6A8 and GAMT gene. However, it should be taken into account that we were unable to validate the obtained compound. Background: Batten disease (Juvenile Neuronal Ceroid-Lipofuscinosis, JNCL), is a recessively inherited untreatable neurodegenerative disorder caused by mutations in the CLN3 gene. The mechanism by which the abnormal or missing protein leads to neuronal cell death remains unclear. It has been shown that a downstream effect is abnormal intracellular calcium accumulation which may lead to apoptosis. Previously, we demonstrated reversal of the calcium effect in a neuroblastoma cell line using amlodipine and other calcium channel antagonists. Objectives: The aim of this study is to investigate the neuroprotective role of amlodipine in a primary neuronal model of CLN3. We studied calcium changes and indicators of apoptosis in amlodipine-treated primary rat neurons with siRNA CLN3 knock down. Results: We demonstrated that intracellular calcium is elevated in CLN3 siRNA-silenced primary neurons. We also demonstrate that amlodipine; an L-type calcium channel antagonist can reverse the aberrant calcium elevations at pharmacological levels. Furthermore, we demonstrated that amlodipine can protect from etoposide-induced apoptosis in the siRNAsilenced neurons. Conclusion: This study indicates that amlodipine, in addition to its wellestablished role as a calcium channel antagonist, can also function as a neuroprotective drug. It suggests that currently available calcium channel antagonists may be potential therapeutic agents for Batten disease. Background: Fabry disease (FD) is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-galactosidase A. Defects in this enzyme lead to accumulation of ceramide trihexoside (CTH) in the endothelium and with patients becoming susceptible to early onset stroke, cardiovascular and kidney disease. Objectives: The aetiology of the kidney damage associated with FD and CTH accumulation is poorly understood. We describe the development of novel glycolipid arrays by immobilising glycolipids to a chip surface and using them to study proteins which interact with CTH in the kidney. Methods: CTH and the ganglioside GM1 (positive control) were immobilised on RS100 arrays and incubated with adolescent rat kidney homogenates. Those proteins interacting with each glycolipid were then identified using QTOF mass spectrometry. Results: A number of proteins were identified that bound specifically to CTH including hamartin, chloride channel protein 1 and tubulin alpha 3 chain; which have been implicated previously in kidney disease. Interestingly, several other proteins were found to bind specifically to GM1. Conclusions: Glycosphingolipid arrays show much potential for studying protein: lipid interactions. Using this technology we have identified several proteins that could play a role in the complex pathogenesis of kidney disease in FD. Clinical molecular testing in IMD is currently PCR-based precluding the identification of deletions which account for a variable fraction (1-25%) of mutant alleles depending on the gene involved. We have developed a highresolution comparative genomic hybridization array (Metabolarray®) for the detection of exonic copy number changes in 205 genes involved in IMD which are currently diagnosed in the laboratory. The array consists of 62,979 oligos spread genome wide, with 40,555 hybridizing to target genes with an average spacing of about 250 bp and 26,678 covering the rest of the genome. For validation, we have retrospectively analyzed a series of IMD patients carriers of exonic deletions previously genotyped by different methods (MLPA, SNP-arrays). All the heterozygous and homozygous deletions even of a single exon were detected using the Metabolarray®. In a series of prospectively evaluated patients, we have identified a novel 2 Kb deletion in the PCCB gene encompassing exons 4 and 5. Our results show that this new molecular tool which can be easily modified to analyze additional genes may be highly efficient in screening for exon copy number changes in IMD patients with incomplete genotype. Background: Degradation of cytoplasmic tRNAs and of mitochondrial mRNAs are part of a cellular response to environmental stress, such as oxidative stress and may regulate protein synthesis or lead to apoptosis. PTCD1 is a novel human protein that was recently shown to decrease the levels of mitochondrial Leucyl-tRNAs. Objectives: To investigate, whether targeted degradation of mitochondrial tRNAs is part of the cellular response to oxidative stress, and whether PTCD1 might be involved in this process. Methods: We compared the effect of oxidative stress (induced by 500 μM tert-butyl hydroperoxide for 1 h followed by 0-3.5 h recovery) on PTCD1 knockdown , on PTCD1 over-expressing and on the respective control HepG2 cells. RNA levels were quantified by quantitative real-time PCR. Results and Discussion: Oxidative stress treatment rapidly decreased the steady state levels of the mitochondrial Leucyl-tRNAs (tRNALeuUUR, tRNALeuCUN), but increased the mRNA steady state levels of the mitochondrial superoxide dismutase (SOD2) and of the transcription factor ATF4 in mock-transfected control cells. PTCD1 knockdown could not prevent the decrease of tRNALeuUUR and tRNALeuCUN in response to oxidative stress. These results suggest, that PTCD1 is not involved in a targeted degradation of mitochondrial Leucyl-tRNAs in response to oxidative stress. Objective: To investigate the globle expression profiling of microRNAs in brain tissues of fetuses with anencephaly and normal control by using microRNA microarray, and predict the target genes of some specific miRNAs with bioinformatic method. Methods: The profiling of miRNAs from brain tissues of 3 fetuses with anencephaly and normal brains was detected using an Affymetrix GeneChip® miRNA microarray. Then some of the significantly misexpressed miRNAs were selected to validate the microarray assay results by real-time RT-PCR analysis. Furthermore, the target genes of these miRNAs were predicted with some online databases. Results: 1. Compared to the normal group, anencephaly group has a specific miRNA expression profile. There were 73 up-regulated miRNAs and 14 down-regulated miRNAs. 2. The microarray findings were extended using Real-time RT-PCR for 10 miRNAs. Of these miRNAs validated, 8 miRNAs were up-regulated, whereas one was down-regulated. In addition, mir-125a was found to be higher in anencephaly group than in the normal group, which contradicted the result detected by the microarray. Conclusion: This study shows that brain tissues of anencephaly group have a significantly misexpressed miRNA expression profile compared to that of normal group. And these misexpressed miRNAs may involve in the pathogenesis of NTDs. Conflict of Interest declared. PDHA2 gene encodes the testis-specific isoform of E1α, the target subunit for most pyruvate dehydrogenase complex deficiencies, and it was postulated that its activation in somatic tissues would represent a conceptual therapy. Accordingly, we aimed to unveil the mechanisms regulating PDHA2 tissue-specific expression. Basal transcriptional activity of different PDHA2 promoter constructs was assayed in three different somatic cell lines, and in vitro methylation experiments were also performed. A cell line (SH-SY5Y) was treated with 5'-aza-2'-deoxycytidine(DAC). PDHA2 methylation status was evaluate by MS-PCR, and mRNA levels analyzed by qRT-PCR; ChIP assays were performed and the recovered DNA analyzed by qRT-PCR. We found that PDHA2 promoter-directed transcription occurred in cultured somatic cells, with no obvious differences between cell lines, and that in vitro methylation clearly reduced transcriptional activity of the constructs. We observed that DAC treatment induced a significant demethylation of an exonic CpG island, while the core promoter remained fully methylated. Moreover, this demethylation was concomitant with PDHA2 gene derepression. Finally, ChIP assays confirmed the involvement of DNA methylation in PDHA2 gene expression.
In conclusion, these results show that PDHA2 tissue-specific expression is strongly controlled by epigenetic modification and demonstrate the possibility of activating its expression in somatic cells. Hereditary Methylmalonic Acidemias, MMAs, are severe autosomal recessive inborn errors of metabolism caused by the deficiency of methylmalonyl-CoA mutase (MUT). MUTconverts L-methylmalonyl-CoA into succinyl-CoA. A block at this enzymatic step results in elevated plasma levels of methylmalonic acid as well the accumulation of other propionyl-CoA-derived metabolites. The disorders are caused by mutations in the MUT apoenzyme or defective metabolism of the enzymatic cofactor, 5'-deoxyadenosylcobalamin. Two enzymatic phenotypes of apoenzyme deficiency are recognized: mut0 patients have no detectable residual enzyme activity while mut-patients have reduced enzyme activity that may be cobalamin-responsive. Mut0 patients can experience life-threatening metabolic instability. Liver specimens from six healthy donors and six mut0 patients were employed to identify deregulated proteins by using DIGE (Differential Gel Electrophoresis. We identified 150 deregulated spots, 80 down-regulated and 70 up-regulated. The spots were excised and identified by mass spectrometry. Proteomic results revealed decreased levels of proteins involved in energetic metabolism and cellular detoxification. A metabolic profiling is underway. The protein data set will be analyzed in the frame of metabolic networks to identify the most differentially altered cellular pathways. Defining altered protein profiles could lead to the identification of new therapeutic targets for MMA. The enzyme glutaryl-Coenzyme A-Dehydrogenase (GCDH) is a homotetrameric mitochondrial matrix protein, for which so far more than 150 diseases-causing mutations have been described, which lead to amino acid substitutions in different regions of the protein. Crosslink experiments show that distinct GCDH mutations with high residual enzymatic activity and amino acid substitutions localized on the surface of the protein affect the homotetramerization as well as the heteromeric complex formation with so far unidentified proteins. The electron-transfer flavoprotein (ETF) composed of the two subunits alpha-ETF and beta-ETF appeared as a promising candidate as interaction partners of GCDH. Pull down experiments with alpha-or beta-ETF immobilized to affinity columns and subsequent incubations with GCDHoverexpressing cell extracts revealed the first experimental evidence for a direct interaction of wildtype GCDH with alpha-as well as beta-ETF. The binding of alpha-or beta-ETF to distinct mutant GCDH proteins was reduced. The affinity of interactions between ETF and wildtype or mutant GCDH was characterized by surface plasmon resonance spectroscopy. Characterization of the interaction between alpha-and beta-ETF and different mutant GCDH proteins provided data on the site of the GCDH protein which is responsible for binding to ETF subunits. Herein we describe the use of aminoacid supplementation in restoring the in vitro folding and activity of natural arginine mutant proteins related to inborn errors of metabolism (IEM). Missense arginine mutations are the most frequent mutations in IEMlinked conformational disorders. Our group has observed that aminoacid supplementation reversed the clinical phenotypes of pyruvate dehydrogenase deficiency patients carrying mutations in arginine residues. Following this, we selected a range of arginine mutations in different proteins related to IEM, namely: E1 alpha subunit of pyruvate dehydrogenase complex (PDH-E1α), cystathionine-beta-synthase (CBS) and galactose-1-P uridyltransferase (GALT). The respective cDNA sequences were cloned into pET-based vectors, and the selected mutants generated by site-directed mutagenesis. By expressing the target proteins in E. coli BL21 (DE3) Rosetta cells, we evaluated the effect of aminoacid supplementation in the protein levels, folding and activity of WT and mutant proteins. The results on the in vitro restoration of protein function upon supplementation are currently being probed in a small set of patient fibroblasts. Altogether these results should provide clues as whether to employ aminoacid supplementation as a general therapeutic for IEM-conformational disorder patients carrying arginine missense mutations.
The extracellular matrix (ECM) is a complex meshwork of non-cellular material that surrounds cells in all tissues and organs. Correct assembly of ECM proteins is known to involve posttranslational processing in the endoplasmic reticulum (ER), but the exact mechanisms are only partially known. Recently it has been shown that autosomal-recessive forms of osteogenesis imperfecta may be due to deficiencies of ER chaperones.
Here we present evidence that the principle of disturbed ER processing represents a more general concept in ECM disorders. We report on six patients with a novel congenital myopathy Ehlers-Danlos overlap syndrome caused by autosomal-recessive mutations in an ER enzyme. Key clinical features include severe muscular hypotonia at birth, delayed motor development, muscular weakness, severe progressive scoliosis, joint hypermobility, sensorineural deafness, and normal intelligence. The candidate gene was identified by linkage analysis; truncating mutations were detected in all patients. The protein localises to the ER and is predicted to function as a protein folding catalyst. Western blot analysis and immunocytochemistry of cultivated fibroblasts of patients showed reduced amounts and/or disturbed organization of fibronectin and other ECM proteins. Deficient protein processing in the ER may not only cause autosomal recessive osteogenesis imperfecta but also other ECM disorders. Mutations in hydroxymethylbilane synthase (HMBS), the third enzyme of the haem synthesis, may induce acute intermittent porphyria (AIP). The molecular basis of the disease remains unclear and valuable information about phenotype-genotype correlations can be obtained by functional studies on the wild-type (wt) and mutants. Five mutations selected for this study (R116W, K132N, R167W, R173Wand V215E) represent: i) mutations around the active site and/or interfering with binding of the dipyrromethane-cofactor, expected to have catalytic, misfolding and/or destabilization effects and ii) mutations far from the active site predicted to affect overall folding and flexibility. Characterization of K132N and V215E has not been reported previously. We have characterized the steady-state kinetic parameters of the mutants comparative to the wt, and investigated the conformational stability by fluorescence and circular dichroism. K132N and V215E possess 87% and 54% activity, respectively, compared to wt. R116W and R167W show activity <20%, while R173W shows no activity, as expected from previous reports. Wt-HMBS shows a high thermostability (Tm=79°C). K132N, R167W and V215E reveal a wt-like conformation and stability, whereas R116W and R173W show decreased thermostability (Tm=54°C). These results together with on-going investigations will provide insights in the pathogenic mechanisms in AIP and help selecting potential therapeutic strategies.
ANALYSIS OF THE GIANT AXONAL NEUROPATHY FIBROBLASTS PROTEOME Van Coster R 1 , Mussche S 1 , De Paepe B 1 , Joel S 1 , Lissens W 2 , Rasic VM 3 , Murnane M 4 , Devreese B 5 1 Div Ped Neur, Univ Hosp, Ghent, Belgium 2 Center Med Gen, Univ Hosp, Brussels, Belgium 3 Clinic Child Neur and Psych, Univ, Belgrade, Serbia and Montenegro 4 Albany Med Coll, New York, United States 5 L-ProBe, Univ, Ghent, Belgium Background: Giant axonal neuropathy (GAN) is a hereditary disease characterized by severe loss of mental and motor functions. This disease is morphologically characterized by aggregation of intermediate filaments, a part of the cytoskeleton. Underlying mutations are found in the GAN gene encoding the protein gigaxonin which is involved in clearance of misfolded or damaged proteins by means of the ubiquitin-proteasome system, but the underlying mechanism still remains elusive. Methods: We compared the proteome of four control fibroblasts with that of four GAN patients by use of the quantitative iTRAQ 8-plex labeling method and mass spectrometry to test the hypothesis of accumulation of gigaxonin binding microtubule associated proteins (MAP's) in cultured skin fibroblasts and provide additional proteomics based insight into the cellular impact of gigaxonin mutations. Results: Among the differentially expressed proteins in GAN fibroblasts no upregulation of known gigaxonin binding MAP proteins could be demonstrated and no up or downregulation of cytoskeletal proteins in general was detected. Differentially expressed proteins were mainly associated with transcription/ translation and splicing and extracellular/membrane signaling pathways. Conclusions: Differentially expressed proteins indicated that the cytoskeleton organization was disturbed via several ways. Galectin-1 might have a central role is reorganization of the cytoskeleton. The Structural Genomics Consortium (SGC) spearheads the concept of open-source medical research by delivering structural biology knowledge on human proteins into the public domain. This wealth of 3D structures includes human enzymes implicated in inborn errors of metabolism. Understanding the effects of disease-associated mutations at the protein level is key to establish a causal relationship among sequence, 3D structure and patient phenotypes. To disseminate this structure-mutation data in an intuitive and approachable manner to the metabolic disease community, we are working with the Society for the Study of Inborn Errors of Metabolism (SSIEM) and Journal of Inherited Metabolic Disease (JIMD) to publish a collection of structure-mutation reports. Each article provides a jargon-free description of a protein structure and a table summary of associated missense mutations. Importantly the article is available online in an interactive visualisation format, where the reader can access each mutation to display a pre-programmed 3D scene, explore at will the molecular landscape in atomic detail and interpret the structural environment around the residue of interest. We hope that the interactive articles, the first of which is published in the 2011 June issue on fumarate hydratase deficiency, will close the knowledge gap between research communities and promote collaborations.
NUTRITIONAL ASSESSMENT OF PATIENTS WITH METABOLIC DISORDERS Thompson SM 1 , Yip Q 2 , Dennison B 1 , Watson P 1 , Coakley J 1 , Alexander I 3 , Bhattacharya K 3 , Ellaway C 3 , Christodoulou J 3 1 The Children's Hospital at Westmead, Sydney, Australia 2 Univ of Newcastle, Newcastle, Australia 3 Children's Hosp Westmead and Univ of Syd, Sydney, Australia Background: Patients with metabolic disorders may have inadequate, excessive or unbalanced micronutrient intake. A protocol assessing biochemical markers for 15 micronutrients was implemented in 2009. Methods: A chart review of 67 patients, fully or partially tested per protocol, compared blood results to normal ranges. Forty-six (69%) were on protein-restricted diets, of which 35 (52% total) were supplemented with amino acid medical food. Ten (15%) were on unrestricted diets and 11 (16%) on fat modified diets. Results: The most common deficiency across diet groups was docosahexaenoic acid (DHA) (76% of 58 tested). Two patients on protein-restricted diets had low plasma vitamin B12. Low plasma levels of magnesium, zinc and vitamins D and E were more common when a medical food was not prescribed. Plasma folate was elevated in 93% patients on medical foods; elevated levels of vitamin A, E and riboflavin were also reported. Low levels of plasma manganese were found across all diet groups. Vitamin D status was of concern in unrestricted diets. Discussion: Both elevated and low levels of micronutrients and long chain fatty acids were found in metabolic patients, despite regular assessment of nutrient intake and advice on suitable supplements. Regular monitoring under a revised protocol is recommended. Background: We performed a study to test the hypothesis that psychoeducation of patients and parents has an influence on compliance for patients with a metabolic disease. Methods: Thirty-five patients with different metabolic diseases were enrolled in the study. During an interview the following information was recorded: background of the patient, perception and knowledge of the disease and diet. Knowledge about disease, diet and consequences of noncompliance were scored on a Likert scale (1 to 5). Compliance was obtained by summing the scores, 0 (insufficient), 1 (acceptable) or 2 (preferable) of four items: 'number of contacts with the dietician (CD)', 'frequency of control of biochemical parameters (FC)', 'results of controls (RC)' and 'quantity of ordering diet products (QP)'.
Results: General knowledge about the diet and the disease was very good. In contrast, only a small majority showed good compliance, especially only moderate results for 'RC', 'QP' was achieved. No significant correlations were found between the degree of knowledge, the four individual compliance-scores, the total compliance-score and follow-up analyses of metabolites. An individualized approach by co-consultations with dietitian/psychologist is organized for "problem-cases". Background: low protein diet, avoidance of fasting and emergency regimen is the primary treatment of MMA. CKD as a complication necessitates dietary adaptation. Objectives: review of dietary manipulations in children with decreasing glomerular filtration rates (GFR), routinely measured from age 2y. Case series: MMAVitamin B12 non-responsive n=14; age at presentation, neonatal (n=9) to 1.5y, current ages: 1mth to 16y (median 7.8y). MMA B12 responsive n=12; age at presentation, neonatal (n=2) to 4y, current ages: 2mths to 16y (median 6.3y). Results: Cross section of GFR from last 2y. Non B12 responsive: mild CKD, GFR 60-89 ml/min/1.73 m squared n=3; moderate or severe CKD, GFR <59 n=9, aged 2 to 12y. Four children with moderate CKD, aged 2 to 5y. Vomiting, reflux, repeated illness and poor fluid intake cause episodic dehydration. Fluid intake is increased above normal as CKD progresses/ during illness. If renal bone disease, phosphate restricted further and vitamin D analogues/phosphate binders given. Normal calcium intake unless hypercalcaemic. Potassium restriction rarely necessary. B12 responsive: mild CKD n=7, others normal. No further dietary manipulations. Conclusion: Early monitoring of kidney function is indicated. Episodes of dehydration contribute to deterioration in kidney function, regular dietary assessment of fluid, electrolytes and energy intake are therefore essential. Introduction: In PKU, it is established that optimal blood phenylalanine control is dependent on adequate dietary knowledge of caregivers/patients. In practice, dietitians spend substantial time on caregiver education, in order to improve their diet understanding and treatment adherence. Aim: an audit to assess caregiver's practical knowledge and understanding of a low phenylalanine diet. Methods/Subjects: 26 caregivers of 32 children (median age 7.4y: range 10 months-16y) were visited at home. They completed a multiple choice questionnaires and a series of practical tests to review their understanding of dietary management of PKU. Results: 40% were unable to calculate all 1 g protein (50 mg phenylalanine) exchanges from food labels; 30% failed to identify the amount of natural protein in one phenylalanine exchange; 50% were unable to correctly estimate by 'eye' the number of phenylalanine exchanges in common foods; 40% and 80% respectively, were unable to correctly define a low phenylalanine 'free' food or the protein content of 'free' vegetable sauces. 80% reported they weighed phenylalanine exchanges >50% of time.
Conclusion: This audit identified a number of gaps in caregiver knowledge and awareness about diet and PKU. It is essential that caregiver understanding is regularly tested and reviewed with education updates given. Objective: Oxidative stress (OS) has been associated with chronic diseases. The concomitance of phenylketonuria (PKU) and hiperphenylalaninemia (Hphe) could increase the OS risk and its consequences. The aim waw to describe and compare oxidant and antioxidant biomarkers in individuals with PKU in adequate metabolic control, Hphe in follow-up and healthy controls. Design: Forty-five individuals (15 PKU, 15 Hphe and 15 controls) between 6 and 12-years-old participated; family smoking habits; anthropometric measurements; selenium (Se) and tocopherol intake along with thiobarbituric acid reactive species (TBARS), total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px) activity and plasmatic tocopherol were assessed. Results: Tocopherol intake was significantly higher in PKU individuals (p<0.0001); however, plasmatic measurement did not differ between groups. Se intake had a tendency to be higher in PKU (p=0.0573) nonetheless the GSH-Px activity was significantly lower in comparison to Hphe and controls (p = 0.0056) as well as TAC (p < 0.0001). Nevertheless TBARS did not show any difference (p=0.5803). Conclusions: Even though TBARS is not affected, the antioxidant capacity is lower in PKU patients. The elevated Se intake in PKU did not correlate with low GSH-Px activity: thus, Se nutritional status must be studied. Hphe individuals should be monitored closely throughout life hence their nutritional risk. The availability of medications and new metabolic formulas for the management of PKU in some cases has permitted the liberalization of the PKU diet. During this transition we recognized a need for nutrition focused educational material. We analyzed 3 day diet records to assess macronutrients and evaluated BMI's at baseline and post diet modifications.The diet analysis of >40% of our patient population that fell within this group revealed poor macronutrient profiles. In response to these analyses, we developed a series of educational modules to assist patients in making healthy, nutrient dense choices while maintaining their restricted and liberalized diets. Initially, a Foundations module was created to provide a base of information on healthy eating and became the template for the following modules: Early Childhood, Teenager, Adult, Pregnancy and Keeping it Simple (Low Literacy) . These modules emphasized the importance of incorporating colorful fruits/ vegetables, healthy fats and fiber in the diet. Recipes, menus, and snack suggestions provided guidance for patients appropriate for any level of dietary restriction. 3 day diet records post introduction of these modules revealed an improvement in nutrient profiles and a decrease in percent of weight gain.
PROPOSAL OFA NEW EDUCATION SYSTEM FOR THE PKU DIET Mannhardt SM 1 , Fekete A 2 , Heddrich-Ellerbrok M 3 , Rocker S 3 , Rohde C 4 , Bollhalder-Wäckerlig S 5 , Gebauer C 6 , Kiener C 7 , Knafl B 8 , van Teeffelen A 9 1 Mannhardt Coaching, Schliengen, Germany 2 Univ Child Hosp, Vienna, Austria 3 Univ Child Hosp, Hamburg-Eppendorf, Germany 4 Univ Child Hosp, Leipzig, Germany 5 Univ Hosp, Zürich, Switzerland 6 Univ Child Hosp, Berlin, Germany 7 Nutricia GmbH, Heilbronn, Germany 8 Univ Child Hosp, Graz, Austria 9 Pediatr Dept, Univ Klinikum, Münster, Germany
Background: Joyful and healthy eating is not in the focus in dietary treatment of PKU but rather the controlled intake of natural protein or phenylalanine. This is a burden on the families and the social life of the patients. Even relaxed diets due to sapropterin therapy are still complex. A working group of German, Austrian and Swiss Dieticians for relaxed PKU diet developed a new patient education system for strict and relaxed PKU diet. Results: The system indicates graphically in pyramidal form food categories that should be consumed during a day, e.g. foods from plant sources (fruit, vegetables, cereal based foods), foods from animal sources replaced by the corresponding amount of protein supplement and a few phenylalanine-rich extras. Portion sizes using a hand model for each food group are specified. The pyramid system emphasizes analogies to recommendations for healthy children. Patients are enabled to control their diet proactively and prospectively with regard to portions still allowed for the day instead of recording yet consumed and metabolized foods. The system was hitherto successfully applied in two metabolic centres. Conflict of Interest declared. Background: Exclusion of diary products in the treatment of inborn errors of amino acid (AA) metabolism results in a risk of deficit of absorbable calcium (Ca), phosphate ( P) and vitamin D with consequences of decreased bone mineral density (BMD). Patients and Methods: The following data from 28 patients (9 MSUD, 8 HT1, 4 MMA, 3 IVA, 2 MCC and 2 HOGA), in the age range 4-23 yrs: biochemical parameters of Ca-P metabolism, anthropometry, whole skeleton and lumbar spine BMD by DXA and nutritional status (based on diet records) were analyzed. Results: Body weight and height were within ±2SD in 90% of patients. Laboratory Ca-P markers were normal except decreased 25OHD3 (below 20 ng/ml) in 35% of patients. DXA examination revealed physiological results in 48% of patients, Z-score for whole skeleton BMD was below normal value in 32%, at lumbar spine low-normal/below normal BMD in 20% of patients. Analysis of diet records showed decreased vitamin D intake (mean 41% of RDA) with unfavourable Ca/P ratio. Introduction: There is little information about dietary practices in nonpyridoxine responsive homocystinuria (NPR-HCU) across Europe. Methods: 29 NPR-HCU dietary management questionnaires were returned from the SSIEM D-G network: (14 UK, 5 Germany, 3 Netherlands, 2 Switzerland, 2 Portugal, 1 France, 1 Norway, 1 Belgium). Results: 182 patients with NPR-HCU (57% (n=103) on a methionine/ intact protein restriction) were identified. Age distribution was: <1y, n=1; 1-10y, n−20; 11-16y, n=29; >16y, n=132. The use of diet with betaine increased with age: <1y, <1%; 1-10y, 10%; 11-16y, 12%; and >16y, 34%. The median intact protein intake (g\d) on diet only was <1y, 8 g; 1-10y, 12 g; 11-16y, 12 g; and >16y, 27 g. With diet and betaine, median intact protein intake (g\d) was: 1-10y, 14 g; 11-16y, 18 g; and >16y, 38 g. 41% (n=12) of centres used food methionine analysis for intact protein allocation; methionine exchange systems (primarily10mg or 20 mg) were common. 94% of patients on diet were prescribed methionine-free protein substitute. 48% of centres recommended cystine supplements for low plasma concentrations. Target concentrations for homocystine/homocysteine (free/total) and frequency of monitoring was inconsistent. Conclusion: In NPR-HCU, the use of dietary restriction declined with age. There is a need for European consensus guidelines on the management of NPR-HCU. Conflict of Interest declared. Background: Patients submitted to low protein diet (LPD) are susceptible to develop nutritional deficits, namely of vitamins, minerals and essential fatty acids, like docosahexaenoic acid (DHA). DHA is found mostly in fish, which isn't available in these patients' diet. Objectives: evaluate plasma DHA concentration in individuals submitted to LPD due to an inherited disorder, comparing with a control group and assess evolution after DHA supplementation. Methods: 20 patients (2 organic aciduria, 2 UCD, 10 PKU, 2 homocystinuria, 3 MSUD and 1 tyrosinemia type I), aged less than 18 years were submitted to analytic determination of plasma DHA in three moments (T0, T1 and T2), with 60 days intervals. In this period, patients were submitted to DHA supplementation (15 mg/Kg/day). Control group was randomly constituted by 33 individuals, aged matched and under normal diet. Results: in T0, patients' group presented a significantly lower DHA value than controls (27,65±13,69 μg/ml vs 55,23±21,84 μg/ml). Between T0 and T2, there was a significant improvement of patients' plasma values to 59,71±22,61 μg/ml, similar to control group. Conclusion: Daily supplementation of DHA allows plasma levels normalization and should be included in the nutritional schedule of these patients, in order to avoid deficits and optimize their growth and neurodevelopment. Methods: An LCMSMS method for quantification of DHAwas developed and fully validated. The assay was applied to establish a reference range (n=40), and to assess plasma DHA in 22 PKU patients. Results: DHA reference range was 131 μM-589 μM. PKU patients who were following a protein restricted diet demonstrated significantly lower plasma DHA than both the reference group and those PKU patients who were on a restricted diet and receiving DHA supplements (p<0.05). Conclusion: The method developed here is clinically useful in identifying patients deficient in DHA, and will be useful to clinicians who wish to supplement and monitor the DHA status of their patients in the future. Introduction: Propionic acidemia (PA) is caused by a congenital deficiency of propionyl-CoA carboxylase. Therapy includes a protein modified diet (with or without substitution of a precursor free amino acid mixture). Aim: Aim of this retrospective study was to investigate a possible correlation between protein intake (total and natural) and isoleucine concentrations in patients with PA. Methods: Laboratory parameters (e.g. concentrations of amino acids, n= 280) and up to 4 nutritional records (period of time 01/01-07/10, 3 days record estimated or weighted by parents, 2 days telephone record by a dietitian) of 7 patients with PA (diagnosis molecularly and enzymatically confirmed) were evaluated. Results: Isoleucine concentrations were in low or below reference range in all patients (mean: 33 μmol/l; median: 31 μmol/l, (range 7-67) n=25). They did not depend on total protein intake (mean: Background: Gastrointestinal symptoms and pancreatitis are recognised complications of Methylmalonic Acidaemia (MMA). Objective: Investigate incidence and review management strategies of feeding problems, gastrointestinal symptoms and pancreatitis. Case series: Fourteen patients with vitamin B12 non-responsive MMA. Age at presentation: neonatal (n=9) to 1.5y. Twelve patients with vitamin B12 responsive MMA. Age at presentation: neonatal (n=2) to 4y. Results: B12 non-responsive: 12 have feeding problems. Eleven are long term tube fed (from presentation n=4, by 6 months from diagnosis n=3 and all by 3 years) due to inadequate oral intake. 50% of those with feeding problems are on anti-emetics/anti-reflux medications and need feed slowly administered via feeding pump. Four have chronic diarrhoea and receive hydrolysed/elemental feeds. Four patients have presented with pancreatitis; isolated episodes n=3, acute on chronic n=1. Latter is now on anti-emetics and elemental feed with MCT via gastrostomy. During acute illness regimen is changed to slow jejunal feeds with increased fluid volume. B12 responsive group: one is tube fed (elemental). Conclusion: Feeding problems are frequent, presenting from diagnosis or acquired. Tube feeding becomes essential to meet prescribed diet and fluid requirements. Modifying feed rate, mode and 'type' may reduce gastrointestinal symptoms and enable enteral feeding in acute pancreatitis. Treatment of long chain fatty acid oxidation disorders (LCFAOD) is primarily by diet, but defining the ideal nutritional composition of a generic formula to treat all conditions remains challenging. Aim: To investigate the safety of a new MCT-containing (MCTF), nutritionally complete formula (Lipistart: Vitaflo International) in children with LCFAOD. Methods/Subjects: 5 well-controlled children (VLCADD, n = 2; LCHADD, n=1; CACTD, n=2; median age 9y; range 7-14y; 3 boys), who take daily MCT oil, completed a 21 day, phase 2 trial, on the safety of MCTF. MCTF (per 100 ml) contained 3.3% fat (2.6 g MCT); it replaced their usual formula (median volume 720 ml/d; range 500 ml-1900 ml/d).
Their usual formula was given on day −7 to 0 and 8 to 21; MCTF was given day 0-7. Blood samples were taken day −7, 0, 2, 7 and 14 (analytes included liver function tests, creatine kinase, glucose, acyl carnitines, free fatty acids and 3-hydroxybutyrates). ECG was monitored day 0 and 7. Results: There was no significant difference in biochemical control or ECG, and no child developed symptoms of metabolic decompensation on MCTF. Tolerance was good, although one subject developed constipation. MCTF was well accepted. There was consensus among clinics to eliminate dairy-based foods and ingredients, but a variation in restriction of more minor sources of galactose. Thirty-three percent of clinics routinely eliminate fruits and vegetables, such as tomatoes and blueberries, with a free galactose content >20 mg/100 g of food. Twenty percent restrict these foods only during infancy and early childhood, but allow them for older individuals. Another 30% of clinics do not restrict any fruits and vegetables. Similar variation in clinic policies was found for other foods and ingredients. Policies also vary regarding use of liquid soy-based formulas containing carrageenan and use of elemental galactose-free formulas for infants with slowly decreasing red cell galactose-1-phosphate concentrations. For infants with the Duarte variant, 43% of clinics treat with a soy formula for the first year while 27% of clinics do not treat this form of galactosemia. Results from this survey demonstrate a need for evidence-based recommendations to better standardize treatment for this disorder. Background: Pyruvate dehydrogenase complex deficiency (PDHCD) comprises a range of phenotypes and may be treated with ketogenic diet. We present 4 patients with enzymatically confirmed PDHCD treated with a modified ketogenic diet similar to the modified Atkins diet. In all families there were barriers of language, geographical distance and/or social circumstances to hospital admission and/or diet calculations, required by the traditional ketogenic diet. Case Reports: Three patients (3-5 years) commenced the diet at home with some or all of their usual milk drinks substituted with Ketocal.. Patient 1 was admitted to ensure safety of enteral feeding and parental understanding; 2 families made gradual changes at home to their child's usual eating plan whilst testing urinary ketones twice daily. All were advised on management strategies should urinary ketones exceed 16 mmol/ l. All patients are maintaining urinary ketones between 1.5-8 mmol/l and report clinical improvement (less irritability, improved motor function, less hospital admissions). A 17 year old previously on a high fat diet, commenced carbohydrate restriction due to worsening lower limb pain. The diet is well tolerated with some reported clinical improvement. Conclusion: A simplified diet to induce ketosis, commenced at home, may be less of a burden in PDHCD. Ketogenic Diet (KD) is an effective treatment for children with intractable epilepsy. Despite energy and protein prescription fulfilling the Recommended Dietary Intake for ideal body weight and normal growth, some patients fail to grow while on the diet. We hypothesised that growth failure in patients on a KD is due to an inadequate Protein:Energy (P:E) ratio. Retrospective dietary details (protein and energy intake; P:E ratio), growth parameters and biochemical markers were collected on all patients who had been on KD for >6 m since October 2002, excluding those with a metabolic disorder (n=39; male:female: 21:18; age range 10 m-16.5 yrs. Information was incomplete for 2 patients. Twenty-four children failed to grow: 10 had a P:E intake <1.3gm/100 kcal (n=5; age1-3y and>10y) or <1.5gm/100 kcal (n=5; age3-10y). Eight had selenium deficiency (or negligible intake). Appropriate selenium intake/ blood level was confirmed in all 10 patients with low P:E ratio. Two had severe iron deficiency. Four had low energy and protein intake. Two did not grow for unknown reasons. Surprisingly, three of those who grew well (age3-8 years) had <1.3 g protein/100 kcal, were not selenium deficient and had adequate selenium intake. P:E ratio and selenium deficiency are pivotal in the growth of children on a KD. Introduction: Accurate and safe feed production is challenging for caregivers of children with IMD requiring specialist feeds with multiple ingredients. Aim: To examine the accuracy of modular feed preparation. Methods/subjects: 52 subjects (38 mothers, 13 fathers, 1 patient; 67% Asian, 29% Caucasian; 4% Afro-Caribbean) with IMD children requiring specialist feeds were studied. Two feeds, with the same nutrient composition, were made with either 2 or 6 ingredients. Following a demonstration, caregivers of children with IMD prepared both feeds under controlled conditions. Ingredients were measured by digital scales, syringe and measuring jugs. Prepared feeds were analysed for nutrient content to determine preparation accuracy. Results: The nutrient composition of both feeds was inaccurate. Nutrients within 20% of the calculated amount for the 6 ingredient feed were: carbohydrate 92%; fat 2%; sodium 33%; potassium 79%; and zinc 23%. For the 2 ingredient feed: carbohydrate was 83%; fat 65%; sodium 4%; potassium 25% and zinc 76%. Fat was calculated as a % of expected minimum value. 6 ingredients feeds were more likely to be under concentrated and 2 ingredients feeds over concentrated. Conclusion: Preparation of specialist feeds is inaccurate and there is a need to simplify feed production for caregivers of IMD children. Conflict of Interest declared. In the UK, in young children >6 months of age with PKU, the most common way of administering phenylalanine-free protein substitute (PS) is in the form of a gel/paste given from a spoon pre-meals. It is prepared by adding water to powder. Disadvantages are associated with preparation inconvenience and changeable consistency, which are prevented with a ready-to-use (RTF) preparation. Aim: To study the efficacy, acceptability and tolerance of a RTF PS gel (PKU Squeezy; Vitaflo International) in young children with PKU. Methods/subjects: In a 7 day, observational trial, 10 subjects (9 PKU; 1 DHPR deficiency; 8 boys), median age 3y (1-10y), took the RTF, nutritionally fortified, PS gel at least once daily. Caregivers completed a daily diary examining tolerance, acceptability and ease of administration. Results: Appearance and tolerance were very similar between usual PS and RTF gel. However, caregivers rated the RTF as easy to use/prepare (100% vs. 50% usual PS); easy to administer outside the home (75% vs. 40% usual PS); and easy for others to prepare and administer (100% vs. 50% usual PS). Blood phenylalanine control remained excellent. Conclusions: This RTF PS gel developed for younger PKU children was successfully adopted by patients and more convenient for caregivers. Conflict of Interest declared. Background: Previous studies have suggested vitamin D insufficiency is associated with increased obesity and metabolic risk factors. However data on the relation between vitamin D status, adiponectin, and insulin sensitivity among obese children and adolescents are lacking. The aim of this study is to investigate the relation between serum 25(OH)D, adiponectin, and insulin sensitivity in obese and non-obese children and adolescents. Patients and Methods: Data from 40 obese and 40 non-obese children and adolescents were obtained. In every participants, anthropometric variables were recorded, fasting blood was assayed for 25(OH)D, glucose, adiponectin, insulin levels and insulin resistance was estimated by homeostasis model assessment (HOMA index) Results: The mean (±SD) age of participants was 13.2±3,6. Insulin resistance was obtained in 15 of obese patients (37.5%). In the overall population 25(OH)D was significantly inversely correlated with body mass index (BMI), insulin levels and HOMA index (p<0.05), and positively correlated with adiponectin (p<0.05). Adiponectin levels were negatively correlated with HOMA index in obese group (p<0.05). Adiponectin levels were lower in obese group (p<0.05). Conclusions: We observed realtionships between adiponectin, vitamin D, and insulin sensitivity in obese children and adolescents. Dietary therapy is the major treatment of urea cycle disorders. The aim is to describe the practical aspects of dietary management of a toddler with citrullinemia where education and compliance with dietary therapy has been challenging due to language and parental education level. A variety of repeated educational approaches have been used to manage this child including: visual resources, quizzes, supermarket tours, and inpatient bedside signs to prevent feeding of inappropriate foods. Dietary analysis of protein and nutritional adequacy is limited by the parent's inability to recall food intake and count protein. All modifications to the diet must be made in person. There have been four admissions in 20 months with hyperammonemia, necessitating formal education to nursing staff. Total dietetic time spent directly with this family has been 62 hours, plus preparation time for the resources above. Inborn errors of metabolism present a challenging case for dietitians: to translate their dietary therapy into food-based therapy for families. This case demonstrates dietary education can be a lengthy, time consuming, and ongoing process due to the barriers created by language and parental education. Despite the time and resources invested, understanding of the disorder by the family is still very limited. Renal hypouricemia is a heterogeneous inherited disorder characterized by impaired tubular uric acid transport, reabsorption insufficiency and/or acceleration of secretion with severe complications, such as acute kidney injury, renal failure and nephrolithiasis. Diagnosis is based on biochemical markers: hypouricemia and increased fractional excretion of uric acid. Therapy is not available, however avoiding dehydration, vigorous exercise and alkalization of urine will minimise renal impairment. More than one hundred cases with a loss-of-function mutation in the SLC22A12 gene have been found, most of the described patients are Japanese (OMIM #220150, type 1). Four patients with renal hypouricemia caused by defects in the SLC2A9 gene have been described (OMIM #612076, type 2). We describe the findings of 12 and 14 year old nonconsanguinous boys with acute kidney injury from UK; the concentrations of serum uric acid were 0.03 and 0.04 mmol/l and expressed as an increase in the fractional excretion of uric acid (46 and 93%). A diagnosis of renal hypouricemia type 2 was made on the bases of finding two novel missence transitions resulting in amino acid substitutions p. G216R Familial juvenile hyperuricemic nephropathy (FJHN, OMIM #162000) is an autosomal dominant inherited disorder characterized by hyperuricemia with decreased renal excretion of uric acid, gout and progressive renal failure. FJHN is caused by mutation in the UMOD gene (16p12.3) coding uromodulin. In the family examined by us, the father of the patient was treated with gout, he died of chronic renal failure. The patient has been monitored since her 27 of age with hyperuricemia; gradually she developed a chronic renal insufficiency in the course of 20 years. Hyperuricemia and borderline creatinine values were also observed with the patient´s son also at the age of 27. The FJHN was diagnosed on the basis of the family medical history, examination of creatinine in serum, uric acid in serum and in urine, determination of the value of extraction fraction of uric acid and uromodulin in urine. Results of biochemical examinations were subsequently confirmed by molecular genetic analysis of UMOD gene (c.334 T>C, p.C112R) with the two abovementioned family members. Allopurinol treatment leads to normalization of serum levels of uric acid and prevents gout attacks. Regular monitoring of renal functions is necessary, in case of renal failure hemodialysis or peritoneal dialysis is recommended, and kidney transplantation. Background: Hypoxanthine phosphoribosyltranferase (HPRT) deficiency is an X-linked disorder of purine salvage. Clinical manifestations range from uric acid over production with hyperuricaemia and gout to severe neurological impairment, self-mutilation and dystonia in Lesch-Nyhan Syndrome. Molecular testing is necessary to identify female carriers within families as a prelude to prenatal diagnosis. During the period 1999-2010 the Purine Research Laboratory studied 106 patients from 68 different families. We report the spectrum of mutations identified to date. Methods: Sequencing of genomic DNA or cDNA was used to define mutations in exons and flanking intronic regions. Results: Genomic sequencing revealed mutations in 60/68 families (88%). Overall, 24 of these mutations have not been previously reported. In 8 patients, exon sequencing was not informative. Copy-DNA analysis in one patient revealed an insertion derived from a deep intronic sequence with a genomic mutation flanking this region created a false exon. Carrier testing was performed in 21 mothers of affected patients, of these 81% (17) were found to be carriers of the disease associated mutation. Conclusions: Our results confirm the extraordinary variety and complexity of mutations in HPRT deficiency. A combination of genomic and cDNA sequencing may be necessary to define mutations. Background: Purine nucleoside phosphorylase (PNP) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction and autoimmunity. Biochemically, PNP deficiency may be suspected in the presence of hypouricaemia. We present clinical, biochemical and genetic data on 8 patients from 7 families identified as PNP deficient. Methods: Urine metabolites were separated and quantified on a Waters UPLC. PNP activity was measured in erythrocyte lysates. Identification of the underlying mutations was determined by DNA sequencing. Results: In seven of eight patients purine nucleoside excretion was massively elevated in urine. However, in one patient multiple blood transfusions resulted in normal PNP activity and barely detectable urine metabolites. Seven different mutations were characterised of which two were novel, c.257A>G (p.H86R) and c.770A>G (p.H257R). Interestingly, four out of eight patients diagnosed with PNP deficiency had normal serum levels of uric acid. Conclusions: To date 66 patients with PNP deficiency have been reported and 24 disease causing mutations identified. PNP deficiency should not be ruled out due to the presence of normal serum uric acid. Background: Human saliva has been extensively described for its composition of major proteins, electrolytes, cortisol, melatonin and some metabolites such as amino acids. Little is known, however, about nucleotide precursors in human saliva. Nucleotides are essential for synthesis of DNA/RNA, supplying energy, regulating G-protein signalling, and biosynthesis. Methods: Saliva samples were collected from full-term neonates, aged 1-3 days, using cotton swabs. Unstimulated fasting (morning) saliva samples were collected directly from adults. The samples were extracted and ultrafiltered, then nucleotide precursors were analysed by reversed-phase HPLC with UV-detection and mass spectrometry (with stable-isotope internal standards Discussion: Salivary concentrations of purine nucleotide precursors such as hypoxanthine, xanthine, adenosine, inosine and guanosine are surprisingly high in neonates and much higher than in plasma. Transition to lower adult levels appears to occur during the first year. These precursors in saliva may be useful biomarkers for diagnosis of inborn errors of nucleotide metabolism and the investigation of some pharmacogenetic disorders. Purine and pyrimidine disorders (PPD) represent a heterogeneous group with variable clinical signs and symptoms. Testing for PPD should ideally be done in all urine specimens investigated for inborn errors of metabolism (IEM), but this is seldom done due to the low prevalence. Thus, metabolic laboratories face the challenge of selecting the samples for determination of purines and pyrimidines (PP). In our laboratory, which is responsible for selective screening for IEM in Denmark, urine samples are analysed for PP in case of 1) increased/ decreased uric acid excretion or 2) if the patient has symptoms of an immunological disease, intractable seizures, self-mutilation, hypertonicity, autism or any symptoms that may be related to kidney stone formation or nephrolitiasis.
Since 2005 approximately 20 % (in total 1601 samples) of all urine specimens received for metabolic screening has been evaluated for PPD. Using our selection approach we have diagnosed seven patients, not previously suspected to have a PPD. These include: Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency; Adenylosuccinate lyase (ADSL) deficiency; Adenosine deaminase (ADA) deficiency and Dihydropyrimidine dehydrogenase (DPD) deficiency. Our findings highlight the importance of a selective screening approach for PPD.
Klein J 1 , Weinhold N 1 , Hennermann JB 1 1 Charité Universitätsmedizin Berlin, Berlin, Germany New therapeutic options, e.g. cPMP substitution in molybdenum cofactor deficiency type A (MoCD), raised interest in early detection of affected infants with inherited defects of purine and pyrimidine metabolism. Established methods of selective screening, e.g. HPLC with UV-or MS/ MS detection, use urine as matrix, not a common material in newborn material. In addition the cycle times are not suitable for high throughput screening. Therefore, a flow injection-MS/MS method with negative electrospray ionization was developed for fast screening of purines and pyrimidines in dried blood spots (DBS). The identical sample preparation for analysing amino acids and acylcarnitines in DBS can be used for determination of purines and pyrimidines, too. Analysis of purines includes the measurement of xanthine, uric acid, and sulfocysteine for MoCD, analysis of pyrimidines includes the measurement of orotic acid, ureidopropionic and ureidoisobutyric acid for urea cycle disorders. Thus, a single extract can be used for simultaneous measurement of amino acids, acylcarnitines, purines and pyrimidines in DBS. This approach allows the inclusion of purine and pyrimidine disorders into the newborn screening panel without extensive additional efforts. cPMP: cyclic Pyranopterinmonophosphate Deficiency of ornithine-δ-aminotransferase (OAT) in humans results in gyrate atrophy of the choroid and retina. Early diagnosis may allow initiation of treatment before irreversible damage has occurred. However, diagnosis is commonly delayed well into adulthood. Here, we report findings in a neonate who was evaluated because of a positive family history of OAT deficiency. The reversed enzymatic flux in early infancy resulted in borderline low ornithine concentrations-evoking urea cycle disturbances-an increased proline and low plasma citrulline concentration. Consequently, the proline/citrulline ratio was increased compared to controls. To find out whether newbornscreening (NBS) is suitable to detect this disorder, we performed aminoacid analysis on the original dried blood spot (from NBS) and compared it with >450.000 NBS data documented in the Minnesota database. The proline concentration (777 μmol/L) was just above the 99%ile (776 μmol/L), and citrulline concentration (4.5 μmol/L) was just above the 1%ile (4.37 μmol). The proline/citrulline ratio (172.9) was far above the 99% ile (97.6). Applying this ratio to NBS will lead to early and specific detection of neonatal OAT deficiency, with no additional expense for NBS laboratories quantifying aminoacids. Additional NBS samples from proven OAT deficient patients are required to provide disease reference ranges. Individuals with hereditary tyrosinemia type I(HT-1) are homozygous for one of several fumarylacetoacetate hydrolase (FAH)-mutations and are gradually deficient for the enzyme activity. As a result, the precursors of FAH accumulate and are metabolised to succinylacetone (SUAC).These precursors and SUAC have toxic effects. Affected individuals show hepatic and renal diseases and also neurologic manifestations. Untreated, the disease might lead to premature death. HT-1 has a general incidence of 1:100,000 with a higher occurrence in particular ethnic groups. Using general elevated tyrosine concentration as a marker for HT-1 has limited value because it is also common for tyrosinemia type II and III and other liver diseases. However, an increase in SUAC is pathognomonic for HT-1. The objective of this study was to evaluate a validated commercial LC-MS/ MS-test assaying newborn blood spots for SUAC among other markers for inborn errors of metabolism. Dried blood spots of 5,000 newborns were screened using the new method based on a simple extraction and derivatisation step. We compared the results with those obtained in parallel using our own in-house LC-MS/MS-method established at the Department of Pediatrics and Adolescent Medicine, Medical University of Vienna. Our data showed that the new method reliably identified all patients with HT-1. Background: Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. Two of these defects cblC/D defects can cause combined methylmalonic-aciduria and homocystinuria.
Objectives: Re-evaluation of the newborn screening for cblC/D defects based on the analysis of false positive and negative rates. Methods: Extended newborn screening was implemented in 2004 and 544,675 newborns were screened by MS/MS. Sequence analysis of genomic DNAwas performed to identify disease-causing mutations in MMACHC and MMADHC. Results: We identified five true-positive cblC defect cases (1/108,935) , one false-negative cblD defect case (1/544,675) and 248 false-positives. All cblC patients showed the most common MMACHC mutation-c.271dupA. The molecular study of cblD patient, a girl identified at 8-month-old, revealed p.R250X mutation in MMADHC gene. At screening time, acylcarnitines presented C3=6.1 μM (cut-off >6.0); Met=15.2 μM (cut-off>12) and C3/Met=0.40; C3/C2=0.35; and C3/C16=2.0. Discussion/Conclusion: Our results revealed that cblC/D defects are important conditions to be screened in our population. At sampling day, normal Met value associated with a borderline C3 value, lead to the misclassification of the cblD-patient as a normal-case. Until now, it was not possible to implement the second-tier tests, but in the future we intent to use this strategy to achieve a better assessment in the screening of these defects. Results: From 1999 to 2007 our recall rate for elevated C3-carnitine or ratio was 217 of 154,000 neonates (0.13%). Retrospective MMA testing of residual samples from these infants detected 19 with elevated MMA, reducing the predicted recall rate to 0.06%. To date 1,200 further MMA tests have been performed, and 48 neonates recalled for a second sample if MMA was above the 99th centile (recall rate below 0.025). 12 had persistent elevated MMA and were referred for clinical assessment. Of these, 11 infants were diagnosed with B12 deficiency and 1 was confirmed to have Cobalamin A deficiency (Dr B Fowler). Conclusion: The incorporation of second tier MMA determination into our routine newborn screening program continues to significantly reduce the false positive rate associated with the measurement of C3-carnitine and ratios. Glutaric Aciduria type 1 (GA1) is a rare disease affecting lysine, hydroxylysine, and tryptophan metabolism. Classically reported presentations include acute childhood encephalopathy precipitated by metabolic stress. However, there is a subset of individuals with GA1 who may remain asymptomatic into adulthood.
A 33 year old woman had an uneventful pregnancy and delivery. Newborn screening in her daughter was positive for Carnitine Uptake Defect (CUD). Investigations in mother revealed low total blood carnitine of 2.5 μM and a free carnitine of <1 μM. Medical history and physical examination were unremarkable. Investigations for secondary causes of hypocarnitinemia included an acylcarnitine profile noting an elevation in Glutaryl-carnitine at 1.06 μM (<0.06) and urine organic acids revealing elevations in glutaric acid and 3-hydroxyglutaric acids, thereby securing a diagnosis of GA1. Carnitine supplementation was initiated at 70 mg/kg/day. Molecular analysis of the GCDH gene is underway with results pending. This is the first reported case of an asymptomatic mother with GA1 ascertained by newborn screening of CUD in her unaffected child. Newborn screening has allowed us to detect asymptomatic mothers affected by inborn errors of metabolism, thereby forcing us to shift our paradigms with regards to the pathophysiology and management of these conditions.
QUANTIFICATION OF GLYCOSAMINOGLYICANS USING TANDEMMASSSPECTROMETRY FOR NEWBORN SCREENING OF MUCOPOLYSACCARIDOSES Kida K 1 , Fuji N 1 , Hirakiyama A 1 , Furujo M 2 , Kosuga M 1 , Okuyama T 1 1 Crin Lab Med, NCCHD, Tokyo, Japan 2 Nat Okayama Med Cent, Okayama, Japan Background: Mucopolysaccharidoses (MPSs) are a deficiency of lysosomal enzymes to digest Glycosaminoglycans (GAGs), such as dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS). The accumulation of GAGs into lysosome induces various symptoms. As enzyme replacement therapy (ERT) and bone marrow transplantation bring successful result for patients in early stage of MPSs, we need a suitable method for newborn screening (NBS) for MPSs using. dried blood spot samples (DBS) on Guthrie cards. Material and Methods: We used a highly sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) to analyze the disaccharides digested from GAGs in DBS. We measured GAGs in Guthrie paper from normal control newborn and adult samples (n=200), newborn and several types of MPS patients (I: 2; II:5; III:2, IV:2, and VI:2).
The results showed about 2-5 fold elevation of GAGs levels compared with those in normal control, and these levels decreased in ERTtreated MPS patients, suggesting this method is available for biomarker for monitoring ERT as well as NBS. Unexpectedly, KS levels were elevated in all MPS types. Conclusion: Method for early detection of patients with MPSs has established. Pilot NBS study is now under planning. Background: Newborn screening prompts early detection of infantile-onset Pompe disease and allows initiation of enzyme replacement therapy (ERT) before irreversible muscle damage. The incidence of infantile-onset Pompe disease is around 1 in 50 000 in Taiwan. Starting from 2009, newborns could be screened for Pompe disease on a self-paid basis. Recently the government is considering including Pompe disease as one of the universal newborn screening items. Therefore the purpose of this study was to conduct an economic evaluation of different screening strategies from the societal perspective. Study design: We used the modeling technique to perform a costeffectiveness analysis comparing two screening strategies, namely universal screening and self-paid screening. Results: Since over 90% of parents joined the screening program now on voluntary basis, from the point view of the society, a universal screening program would save USD$133 for every quality-adjusted life-year saved.
Conclusions: Compared to current screening a self-paid basis, a universal screening for infantile-onset Pompe disease could be cost-effective in spite of the low incidence rate. However, owning to the huge amount of treatment cost and the detection of late-onset patients as well, society may need more discussion about Pompe newborn screening in addition to cost-effectiveness only. Conflict of Interest declared.
PURSUING OBJECTIVE PERFORMANCE METRICS OF NEWBORN SCREENING (NBS) TESTS FOR LYSOSOMAL STORAGE DISORDERS (LSD) Matern D 1 , Orsini J 2 , Bentz Pino G 1 , Tortorelli S 1 , Oglesbee D 1 , Gavrilov D 1 , Rinaldo P 1 , Raymond K 1 1 Mayo Clinic College of Medicine, Rochester, MN, United States 2 New York Department of Health, Albany, NY, United States NBS for one or more LSD has been implemented in New York, Illinois, and Taiwan by enzyme assay using either MS/MS or fluorometry. Other assays have also been proposed. To prevent a recurrence of the significant variability in NBS performance that characterized the application of MS/ MS in NBS, a web site has been created to provide reference and disease ranges for all assays considered for LSD screening (www.nbstrn.org). Similar to the worldwide MS/MS collaborative project (McHugh DMS et al. Genet Med 2011; 13:230-54) , the goal is to collect assay-specific data sets of at least 50 true positive cases per LSD to establish disease ranges that will allow provision of cutoffs resulting in optimal analytical performance (high sensitivity and positive predictive value, low false positive rate). To achieve this goal, participation by screening laboratories and LSD specialists is crucial. All participants will have access to pages unique to their regional NBS program for data submission and comparison tools and to common pages inclusive of project tools and reports. LSD specialists are encouraged to facilitate the retrieval of any residual NBS samples of their patients. Background: Early detection of inborn metabolic disorders (IMD) by tandem mass spectrometry (MS/MS) enables early diagnosis and treatment to minimize morbidity and mortality. Objectives: To evaluate expanded newborn screening (ENS) results retrospectively and to identify the incidence of IMD in Turkey with our laboratory's data. Material and Methods: 107463 neonates were screened for IMD by tandem MS/MS from 2002 to 2010. Blood spots were collected between 2nd-7th days after birth. Acylcarnitine and aminoacid concentrations were determined by using tandem MS/MS. Further diagnostic tests were performed on newborns with elevated screening results by metabolic diseases centers and our laboratory. Results: We detected a total of 170 patients with IMD; 87 patients with aminoacid metabolism defects, 26 patients with fatty acid oxidation defects and 57 patients with organic acidemia. The overall incidence of these diseases was 1:632. Defects in aminoacid metabolism were the most common disorders. Conclusion: Our study showed that the incidence of IMD detected by ENS is very high in Turkey. The distribution of patients according to years revealed an increased incidence at recent years. This finding may be explained with the more widespread use of the ENS by tandem MS/MS. Background: Many Steps, much more links and mass data make it difficult to be operated and to manage its data in neonatal screening.
[Objective] To study and set up a new newborn screening information system (NSIS) as one of the basic platforms for neonatal screening of inherited metabolic disorders. Methods: The new NSIS-part A: was used to collect the information all of the newborn which collected DBSS(dried blood spots samples), and to deal out the screened results after laboratory test. The new NSIS-part B: to service laboratory and treatment for neonatal screening of inherited metabolic disorders.
[Results] All the data and information of total 343757 newborn screened from April of 2009 to March of 2011 include the information of newborn and their DBSS data, the laboratory test and its control data, the screening results and positive, the diagnosis and treatment information of babies with IMD can be managed freely. Parents can inquire about the baby' screening result on the appointed website by parents themselves. Discussion: NSIS is absolutely necessary. Appropriate NSIS make newborn screening to be high efficiency, high accuracy and high safety. NSIS should be improved and be perfected continually. Background: Approximately 1:1000 neonates are affected by congenital metabolic diseases in central Europe and 1:500 in Turkey. Undetected and untreated these diseases can lead to irreversible organ failures invalidity or death. Fully automated NMR spectroscopy of body fluids are used an analytical approach for diagnosis known, but also as yet unknown inborn errors of metabolism. Objectives: Primary objective of the study was to explore the range of variation (concentration and chemical shifts) of specific metabolites without clinically relevant findings. Secondary objective was the integration of the results from a healthy population of neonates into in NMRknowledge base to perform routine and completely automatic screening for congenital metabolic diseases using targeted and untargeted approaches out of one measurement per sample. Patients and methods: Urine samples of 512 neonates from 8 centers in Turkey were investigated by using fully automatic 500/600 MHz NMR spectroscopies. Results and Conclusion: In the urine of 20 neonates we found different pathological metabolites in high concentration. We present and discuss the NMR and clinical data of these children. The statistical analysis and quantification of metabolites allow developing a normal model in specific population also allowing a general assessment of the health state of newborn children. The UPLC-MS/MS method provided improved confirmation of analyte identity by the addition of a chromatographic separation step before mass spectrometric analysis. It decreased ion suppression from co-eluting substances, resulting in improved sensitivity that made it easier to distinguish between pathological and normal values. The method was in all cases able to distinguish between true and false positives. However, the positives were only considered really true positives when two diseasecausing mutations had been identified by mutation analysis. Background: Tandem mass spectrometry has allowed newborn screening programs to increase the number of disorders studied although more infants receive false-positive results. The number of these depends on the number of disorders screened and on the scenario(highly selected laboratories to real-world settings). Objective: To compare the expected number of false-positive results using available population-based studies with the real rate of these in our laboratory. Method:33.941 newborns were screened in 2010 from which we obtained the number of true-positives, false-positives, false-negatives and truenegatives, and calculated the sensitivity, specificity, PPV, NPV, prevalence and false-positive rate. We used the following equation (1):no. of false-positives= (1-(1-(1-prevalence) (1-specificity))k)*(no. of births), where k represents the number of tests performed with tandem mass spectrometry (24 primary targets) and prevalence refers to the average prevalence for an individual disorder screened (1 case per 111.997) (2) . Results: The estimated number of false-positive results was 34 for an expected specificity for an individual test of 99.995%(best-case scenario), 305 for a specificity of 99.95%(intermediate-case scenario)and 807 for a specificity of 99.9%(worst-case scenario). The real number of falsepositive results was 653 and the average specificity for each disease was 99.92%. Discussion: This estimation of specificities compares to real-world settings but all laboratories should document both their true-positive and falsepositive results.
(1)Tarini.Pediatrics2006.(2)ZytkoviczClinChem2001.
Schenone AB 1 , Specola N 2 , Colandre ME 1 , Frabasil J 1 , Vilche Juarez A 1 , Szlago M 1 1 FESEN-Lab de Nueroquímica "Dr Chamoles", Buenos Aires, Argentina 2 Hospital de Niños de La Plata, La Plata, Argentina
Argentinean newborn screening panel does not include the analysis of acylcarnitines and aminoacids by tandem mass spectrometry. There are currently no legislation regarding the use and conservation of RNBSBS. The aim of this work is to show how useful could be the storage of blood spots for retrospective diagnosis. A 4-month pregnant woman came to metabolic evaluation. Two previous children had died from severe liver failure of unknown cause. Autopsy was not performed; the only biological sample available was the RNBSBS from the last child. Acylcarnitines and aminoacids were analyzed on that sample. High levels of tyrosin(TYR), tyrosine/valine(TYR/VAL) ratio and succynil acetone(SUA) were found suggested Tyrosimenia type I. After birth samples were obtained from cord blood, 12, 24 and 48 hs of life. All the samples presented elevation of SUA, the 12hs sample also had a high TYR/VAL ratio with normal levels of TYR, and in the 24hs sample, TYR was also high. The newborn was put on NTBC treatment from day 4. After 1 and half year of follow-up the patient has a good evolution and better prognosis according the early onset of treatment. We demonstrate how important and helpful can be keeping RNBSBS for retrospective diagnosis of IEM.
FROM NEWBORN SCREENING TO OUTCOME EVALUATION: EXPERIENCE, CHALLENGES, AND OPPORTUNITIES FOR LONG TERM FOLLOW-UP OF METABOLIC DISORDERS. Botto LD 1 , Nabukera S 2 , Palmer M 1 , Piper K 3 , Feldkamp ML 1 , Romitti PA 2 1 Div Med Genet, Dpt Ped, Univ Utah, Salt Lake City, United States 2 Univ of Iowa, Iowa City, United States 3 Iowa Dpt Public Health, Iowa City, United States Background: Systematic collection of outcome data and public health benefits after expanded newborn screening is urgently needed, but scarce. One option is to enhance infrastructures of existing population-based birth defect surveillance programs. Methods: We expanded two such programs in the US (Utah and Iowa) to conduct surveillance for 19 selected metabolic conditions diagnosed by MS-MS. We developed case definitions and data dictionaries to track diagnoses, treatment, morbidity, mortality, and services used. Metabolic physicians assisted in case review. Results: The two-state cohort included 52 affected infants (cases) from among 229,353 live births delivered from 2005-2007. MCAD, PKU, and 3MCC accounted for 46 cases. Most cases had follow-up through age 2 years. 3MCC accounted for one-fifth of cases but one-half of those lost to follow-up. By age 2, one-fourth of children had an emergency room visit and one-third a hospitalization; such visits and hospitalizations decreased after age 1 year. Overall mortality was low (n=1, VLCAD deficiency). In terms of workload, these metabolic conditions added about 1% of cases to the surveillance programs. Discussion: Enhancing birth defect surveillance programs can be an efficient strategy to develop and quickly deploy long-term follow-up of metabolic conditions, provided a systematic approach and best practices are followed. Background: Newborn screening using tandem mass spectrometry for some urea cycle disorders (UCD), like citrullinemia type I and argininosuccinate lyase deficiency (ASLD), was introduced in Austria in 2003. This brought new challenges for follow up and treatment in patients with mostly mild variants. Patients: 13 patients with persistent hypercitrullinemia (Cit >50 μmol/l) were detected. Initially measured mean citrulline for citrullinemia was 214 μmol/l; range 72-704; for ASLD 85 μmol/l; range 60-96. Results: ASLD was confirmed by enzyme activity measurement in erythrocytes in three children. One of these patients had already developed a severe hyperammonemic crisis prior to arrival of the screening result; the others were asymptomatic in the neonatal period. In the other children citrullinemia type I was confirmed by ASS gene mutation analysis. One patient was symptomatic with classical neonatal-onset citrullinemia type I prior to the screening result. Conclusion: Newborn screening for UCD may not prevent severe hyperammonemic crisis in early-onset forms as the results is available too late. Screening identifies many individuals with attenuated citrullinemia type I and ASLD of uncertain clinical relevance. Further studies of long-term outcome and phenotype/ genotype correlations are necessary to determine the value of newborn screening for these conditions. Background: With more than 350 different diseases identified to date, IEMs represent about one-third of genetic diseases, nevertheless they are overlooked as cause of neurologic disorders in adults. Methods: Our study involved 192 patients evaluated in the neurogenetics clinics by geneticists and neurologists in a two-year period. Clinical anamnesis, physical exam, neuroimaging studies , ophtalmological and auditory evaluations, neurophysiological studies, biochemical tests, muscle biopsy with respiratory chain mitochondrial enzymes, lysosomal and peroxisomal studies and, when indicated, nerve/skin biopsy for EM studies and karyotype were performed in the course of the investigation. Results: IEMs were identified in 62 patients. Adrenomyeloneuropathy, AVED, abetalipoproteinemia, Fabry disease, Niemann-Pick type C, OTC deficiency, intermitent acute porphyria and mitochondrial disorders were the commonest IEMs diagnosed. Lesch-Nyhan syndrome, AMACR deficiency, cerebrotendineous xanthomatosis and coenzyme Q10 deficiency were other rare IEMs identified. In 93 patients, it was possible to rule out an IEM as cause of the neurological syndrome; 37 patients are still under investigation. Conclusion: Hereditary metabolic disorders can be an important cause of neurological disorders in adults and should not be missed, specially because many IEMs are often treatable diseases and, even the absence of a specific treatment, genetic counselling can be offered to the families. Objectives: The aim of our study was to evaluate IMD patients' satisfaction with the transition process and detect how it could be ameliorated. Methods: A group of 93 adult patients with amino acids, carbohydrate, urea cycle defects, organic acidurias and lysosomal disorders were included. The adult outpatient clinic is located at the Division of Neurology of the St. Bassiano Hospital. The transition process considered the last visit in the pediatric setting and the first two visits in the adult one. Perceived satisfaction was evaluated with a questionnaire-interview through three main domains: patients'care, psychological aspects, logistics. Results: The majority of patients were satisfied (90%). Positive features reported were: perception of independence and greater sense of control; the presence of new adult specialists required for adult care (i.e. vulnologist, cardiologist, gynecologist); regular clinical contacts with the adult metabolic expert, adult specialists and metabolic pediatricians; the neuropsychologist serves as the transition process reference. Major complaints were: lack of dietitian in the adult center; distance between the centers (50 km). Conclusions: The transition process for IMD patients is a fundamental process that cannot be deferred. The major achievement for the patients is an improved self-consciousness of their disease and increase personal management of therapy. Clinical guidelines suggest that adolescents should be seen on their own for part of clinic consultation. However, little is known of parental attitudes on this issue. We explored the views of parents of adolescents in our Metabolic Clinic, in which this is standard practice. Of 40 eligible parents, 33 completed a questionnaire handed to them in clinic, seeking parental understanding of confidentiality, their views about when information should be shared with them, and their perception of benefits/concerns of consulting with young people alone. The main advantages parents identified in adolescents seeing clinicians alone were: practicing talking to the doctor alone; taking responsibility for their own health; help them become more mature. Concerns identified included the possibility of not being informed about important issues (including eating disorders, cigarette smoking, alcohol and illicit drug use, pregnancy, and problems with parents); not being informed of the treatment plan, and concerns about their child possibly not understanding the issues or not remembering the treatment plan. Parental understanding of a confidential consultation with young people alone was remarkably similar to that in a general adolescent clinic (N=86). Background: Ornithine transcarbamylase (OTC) deficiency is an inherited metabolic disorder, better known to pediatrician because it is usually diagnosed during the neonatal period. Descriptions of late-onset forms are scarce but they are often fatal due to hyperammonemia. We report five adults who presented with various OTC deficiency symptoms after the age of 25 years. Patients and Methods: We retrospectively studied five patients (three males, two females), and collected clinical, biochemical and molecular features. Results: A 65-year old man, a 57-year old and a 43-year old female presented with acute neurological symptoms after unusually high protein intake. Ammonemia was high (78 to 327 μmol/L), associated with high orotic aciduria excretion. They were successfully treated with protein restriction and high caloric diet, a supply of ammonia scavenging medication, and hemodialysis in two cases. Two other asymptomatic brothers were diagnosed at 36 and 26 years of age after systematic screening owing to two fatal cases in their family. OTC deficiency was confirmed by DNA sequencing in four cases. Conclusion: OTC deficiency can present with a large spectrum of clinical characteristics. We report five cases of late-onset presentation of OCT deficiency, including the oldest one to our knowledge in the literature.
HYPERAMMONEMIA IN STARVATION FOLLOWING BARIATRIC SURGERY Estrella J 1 , Tchan M 1 , Bhattarchaya K 1 , Carpenter K 1 , Wilcken B 1 1 West Syd Gen Prog, WMH and CHW, Westmead, Australia Hyperammonemia complicating bariatric surgery has been only rarely described1-3. After exclusion of a urea cycle defect, the mechanism underlying this remains unclear. We describe a 52 year old woman with a BMI of 31 who underwent billary pancreatic bypass (duodenal switch) Following operation she had several months of very poor oral intake and developed recurrent episodes of hyperammonemic encephalopathy. Empirical antibiotic therapy for bacterial overgrowth was not beneficial. Amino acid profile showed uniformly very low levels of amino acids including glutamine. Plasma acylcarnitines were normal. Urinary orotic acid profile was below detection limit on a number of occasions. Gene analysis for possible CPS and NAGS deficiency was undertaken with no pathogenic mutations identified. Because of the persistence of her symptoms she underwent reversal of her gastric surgery and had a dramatic improvement, with a normalized amino acid profile and an improved appetite. There have been no further hyperammonenic episodes. We hypothesize that this nutritionally deficient patient developed substrate deprivation of urea cycle intermediates and tissue depletion of glutamine, impairing its use as an ammonia shuttle to the liver, both leading to decreased urea cycle activity. Phenylketonuria (PKU) adults modify diet by phenylalanine restriction, protein restriction, or relaxation of diet, potentially affecting body composition. We measured body composition in PKU adults (33 F, 9 M) aged 32.2± 9.5 years (mean±SD), and body mass index (BMI) 27.9±6.4 kg/m2 using air-displacement plethysmography (ADP) for total body fat (TBF), prompt gamma neutron activation analysis (IVNAA) for total body protein (TBP), and total body dual-energy x-ray absorptiometry (DEXA) for both bone mineral density (BMD) and skeletal muscle mass (SMM). In females, %TBF was 37.1±9.1, TBP using age and sex adjusted nitrogen index (normal NI=1.00±0.10) was 0.98±0.12, and SMM measured as height-adjusted appendicular lean tissue mass (ALTM) was 5.77±2.35 kg/m2. BMD z-scores were −0.27±1.26 (lumbar spine), -0.01±1.01 (femoral neck) and +0.07±1.15 (total body). In males, %TBF was 20.3±8.2, NI was 1.13± 0.13, and ALTM was 8.24±0.45 kg/m2. BMD z-scores were −0.75±1.22 (lumbar spine), -0.23±1.47 (femoral neck) and −0.09±1.32 (total body). PKU adults treated longterm with protein-modified but often high-energy diets have normal TBP and BMD, but adiposity is increased in women.
Background: life expectancy of patients with glycogen storage disease (GSD) type I has improved considerably, opening new problems correlated with adult age. Fertility and pregnancy represent a big issue for women. Reduced quality of life has been described in children, but no data are available for adults. Patients and methods: a total of 31 female with GSD I who were≥ 16 years (mean age 27±7) were included. Data about fertility and pregnancies were obtained from clinical records and interviews. 23 patients completed the questionnaire SF36 for quality of life assessment. Results: 25.8% of patients had delayed menarca; 16.1% of patients had documented polycystic ovaries. 5 successful spontaneous pregnancies in 4 patients with GSD Ia and 2 in a woman with GSD Ib were reported. The latter had development and enlargement of hepatic adenomas during pregnancies. While standardized physical and mental component scales were near normality in GSD Ia patients, they were lower in GSD Ib and inversely correlated with the number of drugs taken. Conclusion: successful pregnancies are possible in women with GSD Ia and Ib, but monitoring for adenomas is mandatory. Impaired quality of life is evident especially in patients with GSD Ib. Background: Fabry disease is a X-linked lysosomal storage disorder leading to multi-systemic life-threatening complications including an increased risk of stroke in young adults. Methods: The FIND ("Fabry: Initiative Nationale de Dépistage") was conducted between 2007 and 2009. Cases were men, aged 28 days to 55 years, with a first or recurrent ischemic stroke (50 adult neurology centers and 8 neuropediatric departments). Enzymatic activity of αgalactosidase A was measured by dried blood spots (DBS) using a filterpaper test. When activity was below a threshold a second visit was scheduled to answer a questionnaire oriented to confirm FD diagnosis using the gold standard leucocyte enzyme activity assay. Results: The study sample consisted of 902 men with a mean age of 43 years old. Low plasma α-galactosidase A activity was detected in 3 patients but enzyme activity didn't confirm FD diagnosis. A 59-year-old man, who was wrongly included , also showed a reduced activity of α-Gal A. FD diagnosis was confirmed by a reduced enzyme activity. Conclusions: Specific populations may be screened systematically for FD with a simple method using DBS. Our results suggest that the yield for FD screening in young patients with ischemic stroke is low. Supplementation of medium-chain triglycerides prior to exercise has been shown to reduce the biochemical abnormalities of long-chain fatty acid oxidation in individuals with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. It has also been shown to increase levels of βhydroxybutyrate and lower heart rate during exercise. Titrating the patient's dose to their metabolic needs may optimize the therapeutic benefit. By optimizing the dose of pre-exercise MCT oil, we hope to avoid exerciseinduced rhabdomyolysis and reduce the risk for arrhythmia by eliminating muscle catabolism and break down of endogenous long-chain fatty acids. In order to test this in the clinic setting, we designed a real-time protocol that could be implemented by the patient, without supervision, using facilities readily available in the community. We focused on monitoring CK, β-hydroxybutyrate, lactate and acylcarnitines for potentially toxic long-chain species. Monitoring was by serum samples and blood dot cards performed at measured time points, pre-and post-MCT oil supplementation and prescribed exercise. Preliminary results suggest that real-time monitoring of patients with LCHAD is feasible and allows determination of clinically relevant parameters; enabling fine tailoring of management plans to suit the patient's individual and potentially changing needs. deficiency is considered to be a rare but treatable cause of epileptic encephalopathy in children. Two clinical forms of PGDH deficiency have been described until now, that differ mostly by their degree of severity. The infantile form associates congenital microcephaly, severe psychomotor retardation and intractable seizures, inconstantly associated with cataracts, nystagmus, and spastic tetraparesis. A milder form has been reported in two siblings who presented with absence seizures of juvenile onset and mild developmental delay. Here, we report the case of genetically confirmed 3-PGDH deficiency in an adult who presented with congenital cataract, mild psychomotor retardation, and chronic axonal sensorimotor polyneuropathy mimicking Charcot-Marie-Tooth (CMT) disease. Amino acid analysis showed low serine levels in plasma and Cerebrospinal fluid (CSF). Treatment with high dose of serine resulted in normalization of plasma serine values and subjective functional improvement.
MATERNAL PROPIONIC AND METHYLMALONIC ACIDEMIA. HOW SHOULD FOLLOW UP BE DURING PREGNANCY? Bueno Delgado MA 1 , Delgado Pecellín C 1 , Lage S 2 , García Valdecasas MS 1 , Pérez Pérez M 1 1 Div Metab Dis, V. Rocio,Univ Child Hosp, Sevilla, Spain 2 Div Metab, Cruces Hosp, Barakaldo, Spain Background: Present experience in diagnosis and treatment of the organic acidemias and all Inherited Metabolic Diseases, has allowed that women, with these pathologies, consider being mothers. Fear of possible descompensations during pregnancy and/or childbirth, and the ignorance of sanitary personnel involved in the care of the patient, generates great anxiety to the patient in consdiering having a child. We report 3 successful pregnancies in women with organic acidemias. Methods: During each pregnancy profiles of amino acids, acylcarnitines, carnitine in plasma and organic acids in urine were performed. Patients sent samples of dry blood weekly for determination of amino acids and acylcarnitines and monthly for obstetric and diet evaluation and determination of urine organic acids. Results: We observed a great decrease of propionylcarnitine and total canitine. Oral carnitine supplement was required in three pregnancies. We also observed a decrease in maternal essential amino acids, but restricted protein diet was necessary all through the pregnancy. Changes in organic acids were not observed. Conclusions: We have observed a distinctive but common metabolic profile in 3 pregnancies. An exhaustive control of amino acids, acylcarnitines, carnitine and organic acids and diet management is essential for a good outcome in maternal organic acidemias. Molybdenum cofactor is essential for sulphite oxidase and xanthine dehydrogenase activities; and its deficiency is usually associated with devastating neurological manifestations. Although milder cases with later onset have been identified, the classical presentations are often fatal in the early age. We present a follow-up case of 23-year-old Caucasian female who was diagnosed at 11 years of age following similar diagnosis made in her younger sibling [1] . Biochemical profiles of her blood and fibroblast were consistent with the diagnosis. This was subsequently confirmed with MOCS2 gene analysis which showed compound heterozygous mutations. Despite a strikingly abnormal initial brain imaging, she was otherwise asymptomatic apart for mild lenses dislocation. Up until recently, she led a normal healthy life with stable employment and fully independence. Following recent serial family bereavements, she presented to a local neurology department with dramatic deterioration. She has marked apathy, dysarthria, dysphagia, associated with generalised dystonia but preserved comprehension. Serial brain imaging reveals significant progression in basal ganglia abnormalities and white matter changes. This case illustrates the dilemma in managing asymptomatic individuals over a prolonged period in this extremely rare condition. Late-onset Tay-Sachs (LOTS) is an autosomal recessive, neurodegenerative, lysosomal storage disease, caused by deficiency of ß-hexosaminidase A (Hex A). In vitro and in vivo studies suggest that Hex A activity can be partially rescued by the drug pyrimethamine. Here, four adult patients with LOTS were treated with different regimen of pyrimethamine for a total of 24 months. In vitro tests in patient fibroblasts prior to treatment shown a good response with 79 to 189% increase in basal Hex A activity. In vivo treatment response was assessed by repeated measures of Hex A activity in leukocytes. Although Hex A increased in all patients during the first four months of treatment, mirrored by some clinical improvement, prolonged inhibitory effect was observed in all patients after this initial period of time. This inhibitory effect was not reversed by decreasing the dosage of pyrimethamine, but only when the drug was reintroduced after at least two weeks of wash out. Attempts to reintroduce the drug at lower dosage (25 mg/d) resulted in the same inhibitory effect after 2-3 months. Finally, a cycle with continuous treatment for two months (25 mg/d) followed by two weeks of wash out resulted in the most sustained response.
Porphyria variegata (PV, OMIM 176200), is caused by a partial deficiency of protoporphyrinogen oxidase (PPOX, E.C. 1.3.3.4.) and its clinical manifestations include acute neurovisceral attacks and/or cutaneous photosensitivity. Biochemical diagnosis of PV is based on abnormal fecal porphyrin profile, fluorometric plasma scan, and increased urinary levels of porphyrin precursors during an acute attack. In this study, the PV diagnosis was determined in index patients from 9 unrelated Jewish families on the basis of clinical symptoms and biochemical findings. DNA analysis revealed 5 mutations in the PPOX gene, four novel (p.Trp42X, p. Gly187Arg, p.His333Arg, p.Ala466Glu), mutation p.Val84Gly has been previously described. Mutation p.His333Arg was found in five unrelated families, the remaining mutations were specific only for the individual family. All families with mutation p.His333Arg are from Morocco, the mutation might be a founder one in Moroccan Jewish population. This mutation was not found in DNA samples of healthy unrelated controls of Moroccan Jewish origin (n=280) and Ashkenazi Jewish origin (n=330). The identification of the asymptomatic family members will reduce risk of PVonset by avoiding triggering factors. Erythropoietic protoporphyria (EPP) is characterized by excess accumulation of protoporphyrin, particularly in the erythroid cells. EPP inheritance is complex, almost always associated with two molecular defects. In majority of EPP patients, clinical expression requires coinheritance of a private FECH mutation trans to a hypomorphic FECH*IVS3-48 C allele. This leads to decrease of FECH activity below threshold of cca 35%. Clinical manifestations of the disease are characterized by cutaneous photosensitivity in early childhood (burning, itching, swelling, and redness) in sun-exposed areas. Hepatic failure occurs in some patients (about 1-10% of EPP patients) which may necessitate liver transplantation (www.porphyria-europe.com; Lancet, 375: 924-937, 2010) . We investigated mother and son of Czech origin with manifest EPP and 7 members of their family in 4 generations and found a novel mutation in the FECH gene in 4 individuals including probands (G→A transition at position 84 in exon 2; W28X, located in a mitochondrial targeting sequence). Both clinically manifest probands (in son, recently, a liver transplantation was performed) inherited a hypomorphic allele as well, while two clinically latent individuals with FECH mutation did not. (Supported by grants # 1 M0520 and MSM 0021 620806 from MSMT of Czech Republic) P-543 Cerebrotendinous xanthomatosis (CTX) is a progressive neurodegenerative disorder caused by mutations in CYP27A1, the gene encoding sterol 27 hydroxylase. The enzymatic block leads to abnormal production of cholestanol, a toxic cholesterol derivative that accumulates in peripheral tissues and brain. Although treatment with chenodeoxycholic acid (CDCA) has been used for more than 25 years to stabilize or improve clinical manifestations, evidence of efficacy comes solely from isolated case reports and small clinical studies. Here, by using magnetic resonance spectroscopy (MRS), we show unambiguously from the follow up of 7 patients only, that treatment with CDCA improves brain function, as reflected by a significant and gradual decrease in the white-matter choline/ creatine and choline/NAA ratios. This improvement in spectroscopic parameters was accompanied by a significant improvement in the Mini-Mental State Examination (MMSE) score and in some nerve conduction velocities. These findings indicate that MRS can be used as an objective quantitative method for monitoring treatment efficacy in small groups of patients with rare neurological disorders. Several mutations in mitochondrial transfer RNA genes cause mitochondrial myopathy. The mtDNA gene MT-TK encoding tRNA lysine is commonly associated with MERRF. Common mutation is A-to-G transition at nucleotide 8344. This mutation can also be associated with isolated "limb-girdle" myopathy. We describe a 35-year-old caucasian female with a history of progressive exercise intolerance and increased CK. Physical examination showed a limb girdle weakness, facial diparesis, limitation of abduction on eye movements without ptosis and arreflexia. Lung function studies showed a restrictive pulmonary syndrome. The muscle biopsy revealed numerous ragged red fibbers. An A-to-G transition at nucleotide position 8344 in tRNA lysine gene was detected in 71% of the patient´s lymphocytes. At the age of 36 she gave birth to a male child with high plasma lactate levels and normal development. After she developed symptoms of nocturnal hypoventilation and non-invasive ventilation was initiated. Six years after the initial diagnosis she needed help to get up, but walked alone and needed nocturnal non-invasive ventilation. In our patient symptoms were limited to a skeletal myopathy, "limb-girdle" distribution and respiratory involvement. Isolated mitochondrial myopathy is a rare form of presentation of the mutation tRNA Lys A8344G. Severe cases like this have been rarely described Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome is a rare multisystemic disorder with typical onset in childhood caused by mutations in mitochondrial DNA. Cases reports 1: 28-year-old man, presenting with 2 acute episodes of behavioural and speech disorder with focal deficits and parieto-temporal lesion on MRI. The diagnosis of MELAS was based on his phenotype and history of hypogonadism and hypothyroidsm. MELAS was confirmed with 3243A>G mut. 2: A low stature boy beginning with seizures at 13 years. The MRI revealed multiple "vascular" lesions (thalamic and occipital bilaterally). Over the time, seizures increased in frequency and developed other neurological signs in relation with news lesions. The diagnosis was suggested by muscle biopsy. The respiratory chain was deficient in complex I. He died at age of 21. 3: 47-year-old man, presented at 45Y with partial seizure. CT appeared an extensive pseudo-stroke. About 7 months later he developed right homonymous hemianopsia and aphasia secondary a new left occipitotemporal lesion. Myoclonic epilepsy and metabolic acidosis began. Muscular biopsies suggest MELAS. We present 3 MELAS, age at onset 13-45, with stroke-like episodes and associated seizures. The diagnosis was obtained by typical MRI, muscular biopsy and positive genetic analysis in one of them.
LEIGH4S SYNDROME AND IT'S PHENOTYPIC VARIABILITY IN THE ADULT METABOLIC POPULATION Massano A 1 , Teotónio R 1 , Silva F 1 , Isidoro L 1 , Domingues J 1 , Garcia P 2 , Diogo L 2 , Grazina M 3 , Macário MC 1 1 Adult Neurology Department-HUC, Coimbra, Portugal 2 Metabolic Disease Unit-Pediatric Hosp, Coimbra, Portugal 3 Centro Neurociências de Coimbra, Coimbra, Portugal Leigh's syndrome is a mitochondrial disease related to various enzymatic defects that affect the oxidative metabolism and may occur in atypical way. Cases: 1: 34 years old male begins ataxia, gait impairment and frequent falls at 3 years old. Later develops uncontrollable vomiting, ophthalmoplegia, hemiparesis and right hemidystonia. Laboratory tests show mutation of the gene SURF1. 2: Male, 22 years old, with depressed mood at the age of 10 and behavioral changes associated with auditory-verbal hallucinations at 19 years old. 3: 22 years old lady, with hypertrophic cardiomyopathy diagnosed at 3 years old. She started loss of coordination, at 8 years, progressing to a lower limb dystonia, involuntary choreo-dystonic movements and cognitive deterioration with increasing learning disability. 4: Female who started at the age of 13, after a flu syndrome, a "Guillain-Barré syndrome like" with generalized hypotonia, distal tetraparesis and a bilateral hypoesthesia in glove and half. She died at 20 years old after development of neurologic deteriorations. All this 4 patients have lactic acidosis and typical Brain MRI. We bring 4 patients with Leigh's syndrome, who have distinct age or mode of presentation and clinical course. We intended to enhance the great phenotypic variability of this neurometabolic disease. Mitochondrial respiratory chain disorders (MRC), defined as primary diseases of the oxidative phosphorylation system are difficult to diagnose and classify. The aim is to characterize primary findings on histopathology of muscle biopsies performed in patients with definitive MRC. The study included patients of our neurometabolic adult consultation that fulfilled Walker's criteria for definitive MRC and had had a muscle biopsy performed. A total of 48 patients were assigned. 37.5% of the biopsies were considered normal. In the group with clinically predominant muscle involvement (group 1), the biopsy showed changes consistent with mitochondrial disease in 82% of cases against 37,5% on the other patients (group 2). COX-negative fibres were found in 64% of group 1 and 37,5% of group 2. In the first group, "red-ragged fibres" were seen in 71,4%, but only in sufficient number to be considered pathologic in 46,4%, while in the second group these numbers decreased to 50 and 25% respectively. Some other minor changes were seen: atrophy, blue-ragged fibres and increase in the variability of fibres diameter. Muscle biopsy is an important study in MRC disorders, and there appears to be good correlation between changes in it and a diagnosis of MRC with predominant involvement of muscle.
MULTIPLE SYMMETRIC LIPOMATOSIS (MADELUNG DISEASE): THINK MERRF! Tran CT 1 , Truffert AT 2 , Lobrinus JA 3 , Philippe JP 1 , Bonafé LB 4 , Morris MAM 5 , Bottani AB 5 1 Div of Endocrinology, Geneva Univ Hosp, Geneva, Switzerland 2 Dep of Neurology, Geneva Univ Hosp, Geneva, Switzerland 3 Div of Clin Pathology, Geneva Univ Hosp, Geneva, Switzerland 4 Div of Molec Pediatrics, CHUV, Univ Hosp, Lausanne, Switzerland 5 Serv of Gen Med, Geneva Univ Hosp, Geneva, Switzerland
Multiple symmetric lipomatosis (MSL), also called Madelung disease, is a disorder characterized by non encapsulated lipomas and fat accumulation around the posterior cervical region and upper trunk. It has been described in patients with myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. We report a 73-year-old woman with MERRF syndrome presenting with MSL. First symptoms consisted of progressive muscular fatigability of lower limbs at the age of 69 years. There was no history of myoclonus, epilepsy or ataxia. Physical examination showed an abnormal fat distribution and giant lipomas of shoulders and neck. There was no amyotrophy and muscular strength of limbs was normal. Serum CK was 207 U/l, lactate 3.2 mmol/l and pyruvate 135 μmol/ l (lactate/pyruvate ratio 23.7). Urinary organic acids were normal. EMG disclosed myopathic changes in the upper limb-girdle muscles. Histology of quadriceps muscle revealed lipid accumulation and ragged-red fibers. Blood DNA analysis identified a heteroplasmic m.8344A>G mutation of the tRNA-lysine gene. Although a rare initial presentation, MERRF should be sought for in all patients with MSL given its potential severity and the implications for genetic counselling. As the mitochondrial content of adipocytes is high, it is thought that the 8344A>G mutation interferes with the maturation of fat cells.
Male,63 years(y) old, normal weight and height, cognitively adequate. From 30 to 53y he had: membranoproliferative glomerulonephritis, atrial fibrillation, tibial vein thrombosis, polyneuropathy, pulmonary thromboembolism and a diagnosis of homocystinuria (plasma homocysteine 268 μmol/L) and antiphospholipid syndrome. He started oral anticoagulant, pyridoxine, folic acid, vitamin B12 therapy without benefit. From 56 to 62y: he had: transient ischemic attacks with following dizziness, subarachnoid hemorrhage, partial renal artery thrombosis. The analysis of cystathionine beta synthase, methylenetetrahydrofolate and Transcobalamin II genes did not show mutations. At 63y, urine and plasma analysis showed increased urinary methylmalonic acid (ur.MMA) (2706 mmol/mol creatinine) associated to normal vitaminB12 (789 pg/ml) and hyperhomocysteinemia (357 μmol/L) and a diagnosis of methylmalonic aciduria (MMA) with homocystinuria was done. He started treatment with betaine (up to 21gr/day), hydroxocobalamin (OHCbl; 1 mg/day orally) and a low-protein diet (40 g/day) decreasing dizziness, plasmatic homocysteine (latest value 107 μmol/L) and ur.MMA (latest value 259 mmol/mol creat ur). MMACHC gene analysis showed only one mutation (p.Arg2106Gln), the [1-14 C] propionate uptake in fibroblasts was reduced and normalized in OHCbl-supplemented medium. MMADHC gene analysis is ongoing. Before undertaking expensive genetic testing, a dosage of ur.MMA should be performed when homocystinuria is not associated to a vitamin B12 reduction Since our publication (Tuschl et al, JIMD, 2008) describing two siblings with inherited hypermanganesaemia, dystonia and liver cirrhosis, we have collected data on additional fourteen patients from seven unrelated families. All of these patients have sequence changes in the same gene; expression studies are in progress to confirm they disrupt the function of the gene product. Following normal initial development, patients typically present at the age of two to fourteen years, with gait disturbance and increased muscle tone in the lower limbs. Some patients also have upper limb involvement causing difficulties with fine motor movements. MRI imaging is characteristic with hyperintensity of the basal ganglia on T1 weighted scans and no corresponding abnormality on T2-weighted scans. Laboratory investigations show polycythaemia (Hb 16-22 g/dL) and whole blood manganese level above 2000 nmol/L. The degree of abnormality of liver function varies quite considerably-we have identified two further patients who died of the complications of liver cirrhosis. Chelation therapy plus iron supplementation, as used in the patient under our care, seems to halt progression of liver cirrhosis as well as improve dystonia. This treatable disorder of Mn metabolism needs to be considered in any patient presenting with dystonia or Parkinsonian symptoms.
A NOVEL BIOTIN-SENSITIVE LEUKODYSTROPHY (BSL) Sedel F 1 , Challe G 1 , Vignal C 1 , Assouad R 1 , Bellanger A 1 , Galanaud D 1 1 Pitié-Salpêtrière Hospital, Paris, France Biotin (vitamin H) is a water soluble vitamin which serves as a cofactor for (1) pyruvate carboxylase (gluconeogenesis), (2) 3-methylcrotonyl CoA carboxylase, (3) propionyl CoA carboxylase and (4) acetyl CoA carboxylase. In addition, biotin regulates in vitro and in vivo the expression of numerous genes. In humans, two inherited metabolic diseases directly affect the metabolism of biotin: biotinidase and holocarboxylase synthetase deficiencies. A third disease, Biotin responsive basal ganglia disease (BBGD) was identified in patients from the Middle East presenting with encephalopathies responding to high doses of biotin. The gene responsible for the disease, SLC19A3, encodes a second thiamine transporter (ThTr-2). Here we report the identification of a novel biotin-responsive disease in four unrelated individuals, three of them originated from North Algeria. All patients display a characteristic leukodystrophy that involves the periventricular white matter, corticospinal tracts, cerebellar peduncles, and optic radiations. Clinical features encompass relapsing episodes of cerebellar ataxia and optic neuropathy. In between episodes, patients may present psychiatric problems, cerebellar ataxia and/or optic atrophy. Visual evoked potentials show absence of P100 or increased latency of the P100, consistent with involvement of optic nerves. Clinical, electrophysiological, radiological and spectroscopic parameters improve after treatment with very high doses of biotin. Background: CYP7B1 encodes the cytochrome P450 alpha-hydroxylase and plays a role in the alternate/acidic pathway for primary bile acid production. Mutations in CYP7B1 were identified in children with severe liver disease and, recently, in adult SPG5 patients presenting with hereditary spastic paraplegias (HSP) (Tsaousidou, 2008) . Increased levels of 27-hydroxycholesterol (27-OHC) were reported in the plasma of 4 SPG5 patients (Schüle, 2010). Besides its role in hepatobiliary metabolism, in vitro studies suggest that 27-OHC may decrease bone mineral density, inhibit the cardiovascular protective effects of estrogens and increase oxidative stress in retinal pigment epithelial cells. Methods: We investigated 9 SPG5 patients from 6 families in order (i) to determine whether plasma 27-OHC can be used as a biomarker to screen HSP patients, and (ii) to explore the non-neurological manifestations that may result from the altered oxysterols metabolism in SPG5 patients-i.e. hepatobiliary and cardiovascular functions, bone homeostasis and retina. Results: The marked elevation of plasma 27-OHC was associated with altered liver functions, reduced bone density and optic atrophy in SPG5 patients. Conclusion: Following these investigations, we are now designing a clinical trial to determine the best candidate drugs-and doses-to lower 27-OHC in SPG5 patients. Background: Availability of sophisticated techniques for selective screening for inborn errors of metabolism is not available in Guatemala. Since 2002 the authors have a close collaboration between Guatemala and Germany/Switzerland. Methods: Aminoacids and Acylcarnitines were extracted from dried blood spots (DBS), and measured by MS/MS. If necessary for confirmatory diagnostics, organic acids were determined after extraction of urine dried on filterpaper. Results: Between 2002 and 2011 we have analysed 444 DBS samples from Guatemalan children. So far we found 11 children with a confirmed inborn error of metabolism (1*PKU; 1*GA-I; 1*Cit-type 2; 1*MCADD; 2*Arginase-def.; 1*VLCADD; 1*PA; 2*MSUD; 1*CAH), and 5 further cases with a presumptive, not yet confirmed, diagnosis (1*MADD; 2*OCTN2; 1*LCHADD, 1*CPT-II). The calculated effectivity index (confirmed cases only) is 2.4% (95% C.I.:1.2-4.4%) or 1 case in about 40 requests. Conclusions: Although the total number of samples is still quite low, the high rate of positive cases indicates that: (1) The overall incidence of inborn errors of metabolism is (at least) as high as in other countries. (2) The preselection of patients to be investigated, is at least as good as in other countries. International cooperation is a simple and effective way to provide state-of-the-art diagnostics for developing countries. Delivery of the best possible genetic healthcare to those with genetic disease requires access to all data of all individuals with their mutations and phenotype. The reasons and the utility of such data has been published recently (Cotton et al Genetics in Medicine, 11:843-9, 2009 ). Collection of such data has been suggested to be best by two complementary but partially redundant routes: (a) collection of all instances of each disease in each country and (b) collection of this data into gene or disease specific databases. There are examples of each of these: country (Australia) at www.hvpaustralia.org and colon cancer www.insight-group.org (Kohonen Corish et al Hum Mutat 2010; 31: 1374-81) . The latter is curated by representatives of a society, InSiGHT (www.insight-group.org). A similar grouping is emerging in inherited neurological disease (Haworth A et al Neurogenetics, 2011; in press) and being initiated in Mitochondrial disease. Such groupings have advantages such as load sharing, more capacity to raise funds, etc. There is a case for a similar initiative in Inborn Errors of Metabolism supported by the Human Variome Project. Those interested could contact Nenad Blau at: nenad.blau@kispi.uzh.ch Background: Metabolomics becomes an important tool in clinical research and diagnosing human diseases. In this work we focused on diagnosing of inborn errors of metabolism (IEMs) in plasma samples using a targeted metabolomic approach. Methods: Plasma samples were analysed using the AbsoluteIDQ p 150 Kit (BIOCRATES Life Sciences AG, Austria). The standard flow injection method of the kit comprising two subsequent 10 ul injections (one for positive and one for negative detection mode) was applied for all measurements. All experiments were performed on an QTRAP 5500 tandem mass spectrometer (AB SCIEX, USA) with electrospray ionization. Multiple reaction monitoring detection was used for quantification. Results: We analyzed 50 control samples and 34 samples with amino acids defects (phenylketonuria, maple syrup urine disease, tyrosinemia I, argininemia, homocystinuria, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, non-ketotic hyperglycinemia) and with acylcarnitine defects (methylmalonic acidemia, propionic acidemia, glutaric aciduria I, 3-hydroxy-3-methylglutaric aciduria, isovaleric acidemia, medium-chain acyl-coenzyme A dehydrogenase deficiency and carnitine palmitoyltransferase II deficiency A screening method for 36 metabolites related to inborn errors of metabolism (IEMs) of purines, pyrimidines, creatine, organic acid and galactose metabolism is presented. The method utilises ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-TMS) for analysis of the compounds in urine. Selected compounds were separated on a Phenomenex Luna 3 μ NH2 column in hydrophilic interaction mode with gradient elution. Mobile phase consisted of 20 mM ammonium acetate, pH 9.45 (A) and acetonitrile (B). Total analysis time was 13 min. Detection was performed by multiple reaction monitoring (MRM) in positive and/or negative mode.Two MRM transitions were chosen for each compound (with the exception of four compounds) and retention times were used for peak confirmation. Detector response was linear in the range of expected concentrations, except for pyruvate and uric acid. Limits of detection (LOD) lie below or within the normal concentration range of the metabolites. The chosen metabolites were analysed in normal urine, elevated concentrations of typical metabolites were found in urines of patients with IEMs. The presented UHPLC-TMS method is fast, sensitive and requires only minimal sample preparation. This work was supported by grants CZ. Background: 1H NMR spectroscopy has successfully been applied to the field of inborn errors of metabolism (IEM). However, body fluid NMR spectra lead to a wealth of data and it can be a daunting task to find the relevant information. In this study, chemometric methods were used to analyze urine NMR data. Objectives: To demonstrate the use of chemometric methods in NMRbased urine analyses to diagnose patients with IEM. Alkaptonuria (ALK) and methylmalonic aciduria (MMA) will be used as examples. Material and Methods: 1H NMR spectroscopy was performed on urine samples from 58 healthy volunteers, 5 patients with ALK and 6 patients with MMA. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied in order to establish models for discrimination between diseased and normal urine samples. Results: Discrimination of ALK and MMA urine from healthy urine was achieved by PLS-DA with excellent results. Not only is the technique able to find perfect separation of the groups, also the resonances of the diagnostic metabolites for both diseases can easily be found. Conclusion/Discussion: Chemometrics analyses can assist the interpretation of complex body fluid NMR data and may be useful in the rapid screening of patients with IEM.
NMR SPECTROSCOPY FOR DIAGNOSIS AND MONITORING OF METABOLITES IN SOME INBORN ERRORS OF METABOLISM: OTC DEFICIENCY, GALACTOSEMIA AND ALKAPTONURIA Vulturar R 1 , Nicolescu A 2 , Avram P 3 , Deleanu C 4 1 Dept Cell Molec Biol, "I Hatieganu" UMP, Cluj-Napoca, Romania 2 "P.Poni" Inst Macromolec Chem, Iasi, Romania 3 "A.Rusescu" Inst Mother and Child Care, Bucharest, Romania 4 "C.D. Nenitescu" Inst Org Chem, Bucharest, Romania
The emerging field of metabolomics, in which a large number of smallmolecule metabolites are detected quantitatively in a single step, promises immense potential for early-diagnosis, monitoring, and understanding the pathogenesis of many diseases. The clinical/ biochemical findings in some inborn errors of metabolism (IEM) are often nonspecific; an early differential diagnosis made in a single urinary sample it gives an important advantage. We present the spectrum of uriny metabolites from a 1 year-old girl with stroke-like episode, elevated transaminases, coagulopathy, being first interpreted as encephalitis. Beside the clinical presentation, the fast results gave by urinary NMR-spectrum showing a high concentration of orotic acid indicates the OTCD diagnosis. Beside this, we present our results and the utility of this method for rapid diagnosis and monitoring steps for galactosemia and alkaptonuria. The level of excretion of the metabolites in these three IEM has been well within the range of NMR detection. In the critical care setting, IEM that were not diagnosed through the neonatal screening should be considered as cause of acute neurologic, hepatic/ renal decline, rapid diagnosis being essential. We demonstrate the effective use of NMR-spectroscopic-profiles of urine in differential diagnosis for UCD and the possibility of management in other IEM. Background: Autism or autism spectrum disorder is a quite common neurodevelopmental disorder presenting in childhood. Although genetic factors play an important role in the etiology of autism, environmental toxicities, such as metal or xenobiotic toxicity are also important factors in this disorder among some of these children. The aim of this study is to examine the relationship between urinary porphyrins, biomarkers of oxidative stress and heavy metals in Turkish autistic children. Material and Methods: Urinary porphyrins were measured by an HPLC method; malondialdehide, arylesterase were assessed by colorimetric methods. Also, Pb, As, Cd, Hg were measured by AAS methods in the first morning urine samples of 36 autistic children and 26 age-matched control subjects. Results: Compared to the control group while there was no difference in the other urine parameters, urine total coproporphyrin and coproporphyrin-III levels were increased statistically significant (p<0.05) in autistic children (Patient group; copro-III:109.2 ±62.7, total copro: 176.5 ±146.9; and control group; copro-III:41.5 ±11.9, total copro:72.7 ±17.0 nmol/L respectively). There was no significant correlation between urinary porphyrin levels in urine and other parameters. Conclusion: Increased levels of urinary coproporphyrin may reflect the effect of environmental toxicity in Turkish autistic children Recessive hereditary methemoglobinemia (RHM) type 2 is an autosomal recessive metabolic disorder due to generalized deficiency of cytochrome b5 reductase (cytb5r). In contrast to RHM type 1, an almost benign disorder where cytb5r-deficiency is restricted to erythrocytes, a severe clinical picture with microcephaly, psychomotor retardation and dystonia develops. Cyanosis may be mild with methemoglobinemia values ranging between 10-42%. We hereby present a female patient, cyanosis noted immediately after birth, showing elevated methemoglobin values (8-19%). Erythrocyte cytb5r-activity was severely reduced leading to diagnosis of congenital methemoglobinemia. Excessive crying was observed from the sixth week of life, followed by vomiting and dystrophy. In the twelfth week of life deceleration of head growth, neurological impairment with no fixation and smiling, poor head control and opisthotonous were noted. Fibroblast cytb5r-activity was markedly reduced proving RHM type 2. Two novel mutations in the CYB5R3 gene were detected (maternal point mutation and paternal deletion) resulting in a premature stop codon. At 2,5 years the patient shows severe psychomotor retardation and mean methemoglobin values of 12%. Unexplained lactate elevation occurred intermittently. Medication with ascorbic acid, riboflavin and toluidine blue had no clear clinical benefit. We hereby draw attention to this rare disorder which is probably underdiagnosed.
IS HUTCHISON-GILFORD PROGERIA SYNDROME, A FARNESYLATION DEFECT, A TREATABLE METABOLIC DEFECT? Ferreira AC 1 , Sequeira S 1 1 Metab Unit, Dona Estefânia Hosp, CHLC, Lisboa, Portugal Introduction: Hutchison-Gilford progeria syndrome (HGPS), an extremely rare disorder, is a premature ageing syndrome caused by a de novo mutation in the LMNA gene. The most frequent mutation is p.G608G, causing an aberrant splicing of exon 11. More recently it is known that in this disorder results in intracellular accumulation of an abnormal farnesylated form of lamin A (progerin) producing premature cellular senescence. Intelligence is normal. Treatment until recently was largely supportive and death usually occurred at an average age of 13 years. As the metabolic pathway of the formation of lamin A became known the possibilities of treatment also improved. Case Report: We describe an eight year old child with failure to thrive and short stature, the characteristic facies of HGPS, alopecia, joint deformities and severe osteoporosis. Our patient is medicated with calcium, vitamin D, low dose of aspirin and is also currently integrated in an international clinical trial with a combination of pravastatin, zoledronic acid and lonafarnib (a farnesyltransferase inhibitor) drugs that act at different points of the pathway. Comments: Although the efficacy of this treatment is still currently being assessed an evaluation after one year shows some improvement of weight and hair growth. We measured the concentrations of 26 [C12-C24] long-chain fatty acids [PUFAs] in the red cells of 10 male patients with autism and 48 normal controls using the same methods of separation of their fatty acids by GC analysis of their methyl esters on a free fatty acid phase column [Dacremont and Vincent, 1995 ] . We also used innovative non-parametric statistical methods appropriate to the nature of the data. We have thus demonstrated that in 16 of these 26 fatty acids that this group of ten autistic patients had highly statistically significant reduced concentrations of their [C12-C24] Long Chain Fatty Acids in their red cells by comparison with those of a group of 48 normals Also using the known normal metabolic pathways, it was possible to locate and delineate defects in elongase and desaturase systems that normally lead to the formation of these fatty acids, ultimately leading to defects in the formation and incorporation of phospholipids sphingomyelin and sphingolipids in cellular membranes of affected patients. We speculate that this might constitute a diagnostic test. Cutis laxa is a disorder in which patients have wrinkled, abundant skin with abnormal elasticity. Skin symptoms may be associated with systemic involvement. Several new genetic defects have been discovered to cause cutis laxa. Surprisingly, a number of these syndromes are inborn errors of metabolism. These include disorders of glycosylation: COG7-CDG, ATP6V0A2-CDG as well as deficiencies in mitochondrial enzymes (P5CS and PYCR1). Despite these different etiologies, discriminating between ATP6V0A2-CDG and PYCR1-deficiency remains challenging due to overlapping features: cutis laxa, dysmorphisms, joint hyperlaxity, cognitive deficits, growth delay and late closing of the fontanel. We report on the metabolic and clinical features of eight patients with cutis laxa, diagnosed with a novel genetic defect. The four PYCR1 patients had a progeroid appearance and arthrogryposis. Two patients showed athetoid movements. MRI detected hypoplastic corpus callosum in one patient. Serum lactate and alanine were increased in two patients. The four patients with ATP6V0A2-CDG had glycosylation abnormalities and typical facial features. MRI revealed neuronal migration defects in three patients. Awareness of differences between these patient groups should lead to a timely diagnosis. We therefore suggest to measure lactate and alanine and perform cerebral MRI additional to screening for glycosylation in patients with cutis laxa.
NEURONAL CEROID LIPOFUSCINOSES 6 PRESENTING AS EARLY ONSET PARKINSONISM Leuzzi V 1 , Garavaglia B 2 , Vitali V 1 , Nardocci N 2 , Manti F 1 1 Dep Dev Neur Psyc Univ Sapienza, Rome, Italy 2 IRRCCS C Besta Neur Inst Found, Milan, Italy
The Neuronal Ceroid Lipofuscinoses (NCL) are the most common autosomal recessive neurodegenerative disorders in childhood. Ten different subtypes are classified based on age at onset, clinical features, biochemical aspects and genetic background. NCL6 (vLINCL gene on chromosome 15 q21-q23) has been associated with the Late-Infantile variant form and presents with epileptic seizures, myoclonus, speech delay, progressive mental and motor deterioration, and visual failure. We report a case of a boy of 13 years who present at the age of 5 with speech delay and mild coordination disorder. In the following few years he developed parkinsonism, ataxia, neuromotor regression, dysarthria with severe palylalia and generalized epilepsy. The mental functioning was relatively preserved. Brain MRI was initially normal and the possibility of a Dopa-responsive disorder was considered. However, CSF examination showed normal neurotransmitters and pterins. Afterwards a progressive cerebral and cerebellar atrophy became evident associated with a severe decline of white and gray matter NAA on H-MRS. In contrast there was no evidence of retinopathy with loss of vision. Genetic analysis of the vLINCL 6 gene revealed that the patient was homozygous for the F234L mutation. This case suggests that NCL6 should be considered in the differential diagnosis of early onset parkinsonism. We describe a 4 year old female patient born healthy and naturally, after normal pregnancy course. Neurophysiological development and anthropometric parameters were reported as adequate until 2=years old, when she showed episodes of acute vomiting, with concomitant language and neurologic disturbance and macrocephaly. The girl was diagnosed with a phonological disorder with specific language impairment. The following investigations were done when she came to our department, at 4 years of age: aminoacids, screening for congenital disorders of glycosylation (CDG), galactocerebrosidase, arylsulfatase A, VLCFA, N-acetylaspartate, karyotype, telomeres, urinary aminoacids, oligosaccharides, mucopolysaccharides and organic acids, electroencephalogram, were all normal. MRI showed frontal lobe white matter abnormalities. Mutation analysis for the GFAP gene was positive. Novel DNA mutations in exon 4: c.715 C>T (p.R239C) of the glial fibrillary acidic protein gene (GFAP), underlie the infantile form of Alexander disease, a rare autosomal recessive disorder (1:100.000) of the central nervous system of unknown etiology, characterized by the accumulation of Rosenthal fibers. Some authors have reported the p. R239C mutation in 4 patients with Alexander disease, 3 of them died before 4 years old and 1 before 6. So far this mutation was reported in patient with hereditary spastic paraplegia. Takahashi, T P-193 Takakura, H P-398 Takayanagi Tedesco, L P-392  Teerlink, T P-114, P-110  Teertstra, TK P-042  Tehrani, FT P-033  Tel, F A-007  Teles, EL P-  Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach. The spread of the cane toad, Bufo (Chaunus) marinus, into the Australian environment following the initial introduction at Gordonvale near Cairns, Queensland in 1935 has been spectacularly successful. Cane toads are now present throughout most of tropical northern Australia and their range is continuing to expand into world heritage areas. Predicted warming due to climate change could extend the range of the cane toad south into what are currently temperate regions [1] . The cane toad is a highly toxic and hardy introduced species and presents wide ranging ecological and social impacts within the Australian landscape. Native species such as quolls, goannas and native frogs are particularly susceptible to the cane toad toxin and many populations have been severely impacted by the arrival of cane toads [2, 3, 4] .
Attempts to halt the spread cane toads have so far been unsuccessful mainly due to the extensive, remote and inaccessible areas inhabited by the toads. An infectious biological agent appears to be the only viable option for controlling cane toads at such continental scales but as yet no known naturally occurring microbes have been confirmed as Bufo specific. An alternative option is to explore whether an infectious agent can be genetically modified to carry a gene that will specifically disrupt the cane toad life cycle, requiring selection of cane toad specific target genes as well as an infectious agent for delivery.
The concept of using genetically modified infectious agents to deliver antigens to wildlife is not new. Recombinant vaccinia virus expressing rabies glycoprotein delivered in baits to wild foxes has proved to be a highly effective strategy to combat rabies [5] . Since then other vaccines developed against diseases of wildlife include a rabies virus based vector used to immunise wildlife against SARS [6] . Extension of this concept has seen recombinant viruses developed to control a host's biological processes. An example is recombinant viruses expressing zona pellucida antigen that successfully deliver immunocontraception to pest animal species in laboratory trials [7, 8] .
Bohle Iridovirus (BIV) is a ranavirus in the family Iridoviridae. BIV was originally isolated from the Bohle River region in northern Queensland [9] and is the only documented isolation of a virus from amphibians in Australia. It is capable of infecting cane toad tadpoles [10] and is therefore a candidate for testing the viral delivery of genes in this species. Furthermore, in recent studies we have shown that BIV can carry and express foreign genes in vitro [11] .
Selection of target genes for delivery to cane toads has focused on metamorphosis since it is a critical phase in the amphibian life cycle. Metamorphosis is characterised by rapid and extensive morphological changes [12, 13] , accompanied by strong shifts in the expression of genes and proteins at the molecular level [14, 15] . Cane toad genes that are expressed in metamorphs but not in tadpoles therefore represent ideal targets to block and thus manipulate aspects of development. One documented example of the transition from a larval to an adult form in amphibians is haemoglobin [16] and we have used microarray analysis to establish that adult haemoglobin is significantly upregulated during cane toad metamorphosis [17] . Injecting tadpoles with adult globin interfered with expression of this protein in Rana catesbeiana, and induced changes in gene expression profiles of metamorphs [18] . Thus we hypothesise that it may be possible to alter metamorphosis by immunologically sensitising larval stages (tadpoles) to proteins expressed only in later post-metamorphic stages. Adult globin is not a cane toad specific gene; we used it here to determine whether autoimmunity might affect the protein profile of metamorphs. If successful, the concept would be extended to cane toad specific genes that were upregulated at metamorphosis.
This study outlines an investigation into the feasibility of an immunologically based biocontrol for cane toads. We demonstrate the presence of a clear larval to adult switch in haemoglobin mRNA and protein levels and hypothesise that this switch may be affected by early exposure, by either injection or viral delivery, to adult B. marinus haemoglobin. Our results indicate that the altered adult globin protein profile seen in Rana catesbeiana metamorphs after exposure of tadpoles to adult globin does not occur in B.marinus. The short larval stage in B. marinus compared with R. catesbeiana may preclude this approach to cane toad biocontrol.
All animals used in these studies were sourced from a colony of B. marinus maintained at CSIRO according to the methods described in Hamilton et al. [19] . Briefly, when tadpoles were required, adults were injected subcutaneously with a 0.25 mg/mL solution of leuprorelin acetate to induce ovulation and stimulate amplexus. Eggs were hatched and tadpoles maintained in aged water without chlorine at a temperature of 23-27uC.
Authority for the use of animals was provided by CSIRO animal ethics committees in accordance with the Australian National Health and Medical Research Council's code of practice [20] . These permits were (i) CSIRO Sustainable Ecosystems Animal Ethics Committee, Approval No. 08-05, exposure of pre-and post-metamorphic cane toads to proteins, DNA and RNA and produced RNA/cDNA and (ii) CSIRO Australian Animal Health Laboratory Animal Ethics Committee, Approval number 1132, biological control of cane toads.
Production and purification of recombinant globin and antisera B. marinus adult and tadpole globins (GenBank Accession numbers EL342145 and EU877979, respectively) were amplified using the following full length primer sets: adult globin sense 59-ATGGTCCATTTGACAGATCAC -39, and antisense 59-TTA-GTGGTAACCCTTGCCAAG -39 (444 bp), or tadpole globin sense 59-ATGGTTCATTGGACCGCTGAAGA -39, and antisense 59-TTAGAAATAGCCATGGCTCAGG -39 (444 bp). The fragments were cloned into the bacterial expression vector pDEST17 and expressed as His 6 -tagged proteins in E. coli BL21-AI cells (Invitrogen). Cultures were grown overnight (37uC) in LB supplemented with antibiotics, then diluted 100-fold and grown to an OD of 0.6 (600 nm). L-arabinose (Sigma) was added (0.2% final conc.) to induce protein production and incubation continued for 3-5 h. Bacteria were harvested by centrifugation, rinsed and resuspended in Tris-buffered saline (TBS: 50 mM Tris, 500 mM NaCl; pH 7.5), disrupted by freeze/thaw cycles and centrifuged at 10,0006 g for 30 min. The pellet was solubilised in TBS containing 8 M Urea for 30 min and then centrifuged at 20,0006 g for 30 min to remove insoluble materials. His 6 -tagged proteins were purified in the denatured state using Ni 2+ NTA agarose (Qiagen), washed via imidazole-containing steps (TBS+20, 30 or 40 mM imidazol) and eluted in TBS+500 mM imidazole. Size-based secondary purification was then achieved by continuous-elution electrophoresis (Model 491 Prep Cell, Bio-Rad). Globin proteins were dialysed against amphibian Ringers solution [4.89 g NaCl, 0.298 g KCl, 0.265 g CaCl 2 .2H 2 0, 0.197 g MgSO 4 .7H 2 0, 1.495 g NaHCO 3 , 0.127 g NaH 2 PO 4 .H 2 O and 1.982 g glucose per litre dH 2 O] overnight at 4uC and concentrations determined using the Bio-Rad Protein Assay. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) (15% gels) using the Bio-Rad Protean II System and visualised with Coomassie brilliant blue. Purified recombinant globin proteins were used as immunogens for the generation of rabbit antiserum. Briefly, two rabbits per immunogen were injected 10 days apart with vaccine containing 50 mg of either adult (rAdglob) or tadpole (rTadglob) recombinant globin. Two weeks later a third dose was administered if a boost to the antibody response was required. Each dose was prepared in CSIRO triple adjuvant (60% v/v Montanide; 40%, v/v rHb [combined with Quil A, 3 mg/mL, and DEAE-dextran, 30 mg/mL]), in water.
The time course of adult and tadpole globin production in normal animals was determined. Animals were sampled at stages 27, 28, 34, 36, 40, 41, 42, 45 , 46 (metamorph) and 1 month post metamorph, $3 animals per stage. Developmental stages of B. marinus were determined according to the method of Limbaugh and Volpe [21] . Levels of mRNA and protein were measured individually except for tadpoles at stages 27 and 28 where .10 animals were pooled due to the small size of tadpoles in the early stages of B. marinus development. 
Recombinant BIV (rBIV) expressing the neomycin resistance gene and adult globin (rBIV/neo r /Adglob), and the control virus without adult globin (rBIV/neo r ) were constructed according to Pallister et al. [11] .
To assess the effect of viral delivery of adult globin to B. marinus tadpoles, seven groups of tadpoles were infected at day 6 (stage 20) post hatching. Group A, uninfected cell culture supernatant; Groups B, C and D -10 2 , 10 3 and 10 4 TCID 50 /mL of the negative control virus, rBIV/neo r respectively; Groups E, F and G -10 2 , 10 3 and 10 4 TCID 50 /mL of the test virus, rBIV/neo r / Adglob respectively. Each group of 116 tadpoles was infected for approximately 6 h in 2 L of water containing the appropriate concentration of virus, rinsed in clean water for 5 min then divided into 4 tubs each containing 29 tadpoles. These tubs were randomly dispersed around 3 different rooms to allow for statistical variation due to the position of the tub.
Sampling was carried out at 5 different stages, 20, 28, 33, 42 and 1-2 weeks post tail resorption, according to the schedule in Table 1 . Tadpoles were sampled at each of the 5 stages for the detection of virus by real time PCR, and at all stages except stage 20, where tadpoles are very small, for the analysis of native adult globin profiles. However, tadpoles were only sampled up to and including stage 33 (just before the onset of native adult globin production) for the detection of viral adult globin. Five tadpoles per group were sampled at all stages except post tail resorption, where, for statistical purposes, 30 animals per group were sampled.
Tadpoles were euthanased by bathing for $5 minutes in 0.2% MS-222 (see Preparation of immunogen and inoculation trials). Tadpoles for real time PCR and for detection of viral adult globin were then frozen at 280uC until further processing. Tadpoles for blood sampling were euthanased as described then decapitated and blood was collected using a micropipette containing 0.2 M EDTA to prevent clotting. Red blood cells (RBCs) were pelleted at 13,0006g for 5 min, washed with PBS then lysed by resuspending in an equal volume of distilled water. Following centrifugation at 13,0006 g for 5 min the supernatant containing globin was removed and stored at 280uC.
Total RNA (5 mg) was extracted from tadpoles and toadlets using Trizol reagent (Invitrogen) according to the manufacturer's instructions and reverse transcribed using the cDNA First Strand Synthesis Kit (Invitrogen). Real time PCR was used to detect larval and adult globin mRNA at selected stages of development. Oligonucleotide sets were larval globin; sense 59-GCTGAAGA-GAAAGCCGC -39, and antisense 59-ATGGCGGTGACATTG-GAC -39 (151 bp), or adult globin; sense 59-CAGATCAC-GAGCTCAAGAG -39, and antisense 59-ATGGCATCAG-CAGAGCCA -39 (151 bp). Results were standardised with cane toad actin (GenBank Accession no. EL595572) by amplifying a 117 bp fragment using sense 59-ATGACACAGATAATGTTT-GAGAC -39 and antisense 59-ATCACCAGAGTCCATCA-CAAT -39 primers. Reactions consisted of QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.5 mM oligonucleotides and 200 ng of first strand cDNA template, and run on a RotorGene 2000 (Corbett). The thermal profile was as follows: 50uC for 2 min, 95uC for 2 min, 40 cycles of 95uC for 30 sec; 58uC for 30 sec; and 72uC for 30 sec. PCR target products were used as reaction standards at concentrations ranging from 1610 2 to 1610 8 molecules per mL. Analysis of real time PCR reactions and melting point dissociation curve reactions were performed using the programme Rotorgene version 6.0 (Corbett).
For DNA extraction the animal was thawed, weighed and Prepman Ultra (Applied Biosystems) was added directly to the tube at the following rates: #0.20 g, 450-500 mL; 0.21-0.30 g, 500-750 ml; .0.31 g, 900 mL. This was followed by approximately 100 mL sterile 1.0 mm zirconia silicone beads (BioSpec Products). The tadpole was homogenised in a mini-beadbeater (BioSpec Products) for 30 sec, microfuged at 13,0006 g for 30 sec then homogenised in a mini-beadbeater for a further 30 sec. To extract DNA the sample was heated to 100uC for 20 min, left to stand for 4 min at room temperature (RT), microfuged at 13,0006 g for 6 min and the DNA (350-650 ml) transferred to a clean tube.
Each reaction in the TaqMan assay contained 12.5 mL of Platinum Quantitative PCR SuperMix-UDG (Invitrogen), 90 nM sense (59-CTCATCGTTCTGGCCATCAA -39), 90 nM antisense (59-TCCCATCGAGCCGTTCA -39) primers, 25 nM MGB TaqMan probe (59-CACAACATTATCCGCATC-39), 5 mL of a 1/10 dilution of template DNA in dH 2 0, 0.05 mL of Rox dye, and water to a final volume of 25 mL. The reaction was run on the ABI 7500 Fast Real-Time PCR machine with a thermal profile was as follows: 2 min at 50uC, 10 min at 95uC, 45 cycles of 15 sec at 95uC then 1 min at 60uC. Results were automatically plotted by the Sequence Detection System Software version 1.3.1 (Applied Biosystems).
Proteins for western blot were extracted either from Trizol samples according to the manufacturer's instructions, or from virally infected tadpoles homogenised in 10 mM Tris with 100 mL sterile 1.0 mm zirconia silicone beads (BioSpec Products). The tadpole was homogenised in a mini-beadbeater (BioSpec Products) for 30 sec, centrifuged at 13,0006 g for 30 sec then homogenised in a minibeadbeater for a further 30 sec. SDS was added to a final concentration of 5% and the homogenate heated to 100uC for 1 min. After 30 min at RT to allow SDS to penetrate the sample, the homogenate was centrifuged, the supernatant decanted, heated to 100uC for 1 min and stored at 280uC until used. To extract globin from RBCs, blood was processed as previously described (see Infection and sampling) and the protein content of each sample was determined 
Samples were collected from stage 46 metamorphs anaesthetised by bathing for 2 min in 0.22% MS-222 followed by decapitation. Samples were collected by aspiration and pelleting of RBCs prior to the collection of sera-like fluids. Adult blood samples were collected as per Zupanovic et al. [23] . Microtitre plates (Beckton-Dickonson) were coated with 50 mL/well of rAdglob (10 mg/mL) and positive control ovalbumin (4 mg/mL; Sigma) in Ringer's solution overnight at 4uC and washed (x3) in TBS with Tween-20 (0.05%; TBST). The plates were blocked for 2 h with 5% skim milk powder (Bio-Rad) in TBST at 37uC. Metamorph sera (treated and control) and adult toad aovalbumin control sera were serially diluted in 1% skim milk/TBS and applied to plates (50 mL/well). After incubation for 1 h at 37uC the plates were washed with TBST three times. Rabbit antisera against toad IgG (1:1000; Zupanovic et al. [23] ) was added in 1% skimmed milk/TBS, incubated for 1 h at 37uC and washed (x3) in TBST. Next, goat a-rabbit IgG (1:2000; KPL) was added to each well, incubated for 1 h at 37uC and again washed (x3) in TBST. HRP-conjugated streptavidin (50 mL; KPL) was incubated for 30 min at RT, washed three times and developed with peroxidase substrate solution (50 mL of TBM; KPL) and the absorbance measured (405 nm).
Fitness was assessed using burst swim speed for tadpoles and maximum jump distance for metamorphs. At 11 days post injection (,1 week prior to metamorphic climax/stage 46) average burst speed in tadpoles was measured according to the method described by Van Buskirk and McCollum [24] where specific speed is normalised to body length rather than measuring absolute speed. Temperature at the time of testing was constant at 23-24uC. Jump distances were calculated in stage 46 metamorphs approximately 2 days after removal from aquatic tanks according to Wilson and Franklin [25] . Jumps were recorded using a digital camera (Canon; 15 frames sec 21 ) and distances calculated with MouseZoom version 1.4 (Freeware 1998 1.4 (Freeware -2003 and Microsoft Windows Movie Maker version 5.1 (1998) (1999) (2000) (2001) . Absolute distances were determined from screen pixel units with conversion to mm via background graph paper. It was determined that optimal jumps were performed on plastic rather than paper.
The time course for the appearance of adult globin mRNA and the disappearance of tadpole globin mRNA during normal cane toad metamorphosis were first assessed by real time PCR (Fig. 1a) . Tadpole globin mRNA was detected at all early stages until the level declined rapidly at the metamorphic climax (Stages 42-46) and was undetectable one month after metamorphosis. Conversely, adult globin mRNA was first detectable at stage 40 and at all following stages until the experiment was terminated.
Analysis of total protein extracts at selected stages by Western blot showed that larval haemoglobin was expressed in stage 40 tadpoles but not in stage 46 toadlets (Fig. 1b) . Adult globin protein was first detected in stage 36 tadpoles (faint signal) and continued to increase in abundance after metamorphosis. The positive control used for the experiments was recombinant adult globin that migrates as a larger protein than the native globin on PAGE gels due to the addition of a 6XHis-tag and plasmid linker sequence. The detection system was specific; anti-larval globin antibody detected larval globin, but not recombinant and native adult haemoglobins, and vice versa for the anti-adult globin antibody. Thus we confirmed that the globin switch seen at the mRNA level was also seen at the protein level.
rAdglob within inoculated tadpoles was clearly detected by rabbit antibody to adult globin as an 18 kDa band for several days after injection (Fig. 2) . rAdglob levels appeared reduced by half every 2-3 days, until 14 days post injection where no protein was detected.
Gross morphology during metamorphosis determined by wet weight and length was similar for treated (rAdglob) and control groups (no injection, or Freund's adjuvant only) (Fig. 3a) . Treatment with adult haemoglobin did not significantly delay metamorphosis. There was no significant difference in tadpole fitness between treatment and control groups just before metamorphic climax measured by swimming performance (burst swim speed). A p-value of 0.086 was determined in Microsoft Excel using the student t-test, 2-tailed distribution, 2-samples of unequal variance. Likewise there was no significant difference between treatments in the fitness of metamorphs as measured by maximum jump length (Fig. 3b) . A p-value of 0.996 was determined using the same student t-test as for the swim speed.
Metamorphs from each of stages 36, 40, 42 and 46 were pooled and analysed for differences in adult globin mRNA between treated and untreated groups. The results indicated no significant differences in adult globin mRNA levels between treated and untreated groups at any of these 4 stages (Fig. 4a) . A p-value of 0.914 was determined using the same student t-test as for the swim speed and jump length. As pooling animals from each stage could have masked effects in individual animals, 6 animals were taken from the treated and untreated groups at stage 46 and analysed individually. Again, no significant difference in globin mRNA levels was observed between individuals from treated and control groups. A p-value of 0.095 was determined using the student t-test outlined previously. As seen for the mRNA studies, we detected no change in the protein profiles of adult globin immunised metamorphs (n = 10) compared to control (n = 9) animals by Western blot (Fig. 4b) . Similar results were recorded in preliminary trials conducted using native globin purified from the blood of adult toads rather than recombinant globin. These inoculations had no effect at the morphological or mRNA and protein levels (data not shown). We were unable to detect reactive antibodies (IgY) against recombinant globin protein in any of the animals by ELISA. However, we were also unable to detect antibody to a normally highly immunogenic test antigen (ovalbumin) in metamorphs, although reactive antibodies generated by immunising one adult with ovalbumin were readily detectable (Fig. 5 ).
The effect of recombinant virus infection on mRNA expression levels in tadpoles was assessed using real time PCR. Treated and control animal groups were sampled at stages 20, 28, 33, 42 and 1-2 weeks post tail resorption (Table 2) . At stage 20, after infection and rinsing, no virus was detected in any animals indicating that there was no background level of BIV detectable by real time PCR and that any virus detected at later stages was the result of virus replication.
BIV was detected in all of the infected groups, with more infected animals detected as the inoculum increased. The control 
Analysis of adult globin profiles was carried out on RBCs from tadpoles taken at stage 42 and metamorphs at 1-2 weeks post tail resorption, by which time the switch has been made from tadpole to adult globin production. Blood from tadpoles given all three doses of the test virus (rBIV/neo r /Adglob that does express adult globin) and the control virus (rBIV/neo r that does not express adult globin) were analysed by polyacrylamide gel electrophoresis (PAGE) and silver staining (Fig. 6a) . The protein profiles of blood from animals infected with the rBIV/neo r control (n = 51) and rBIV/neo r /Adglob (n = 89) viruses were all similar. A western blot using rabbit anti toad adult globin confirmed that the main 14 kDa band detected by silver stain was adult globin (Fig. 6b) . We were thus unable to detect any change in the adult globin profile in RBCs taken from the animals that had been exposed to adult globin as tadpoles.
In this report we outline the steps we have undertaken to determine whether interference with cane toad tadpole develop- Burst swim speed represents the absolute swim speed normalised to body length, and the full data range (vertical line), standard deviation (box) and mean (horizontal line) are indicated. c: Jumping performance of rAdglob treated and FCA control amimals at stage 46 is shown. Longest jump distance was normalised to body lengths. doi:10.1371/journal.pone.0014576.g003 ment can be achieved using an immunological approach. Our proof of concept approach was largely influenced by previous observations that in bullfrog tadpoles immunised with purified adult globin, the adult globin protein profile was altered in surviving metamorphs [18] . Here we extended the concept to test whether a similar effect could be induced in B. marinus by injecting tadpoles with purified native and recombinant globin as well as viral delivery of this antigen.
We first established that the larval to adult globin switch reported in other amphibian species also occurred in B. marinus. It is well documented that a tadpole form of globin in anurans is replaced by adult globin during the course of metamorphosis [18, 26, 27] and here we demonstrate the existence of this switch for the first time in a Bufo species. We have previously demonstrated strong upregulation of adult globin genes during cane toad metamorphosis and that this was more pronounced than for any of the other genes induced at metamorphosis [17] . We have also previously shown that the recombinant BIV can be genetically modified to express adult globin in vitro [11] and confirmed here that this virus (rBIV/neo r /Adglob) is capable of infecting cane toad tadpoles. We therefore used rBIV/neo r /Adglob to assess the effect of viral delivery of an adult specific gene or protein to tadpoles on subsequent metamorphosis, and compared this to the effects of immunisation with purified protein and adjuvants.
A number of parameters were used to assess the effect of adult globin delivery to tadpoles. These included behavioural (average burst speed in tadpoles and maximum jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin by PAGE) and genetic (mRNA levels for adult globin) measures. However, we were unable to detect any differences between treated and control animals following immunisation with purified globin or exposure to recombinant virus. This contrasts markedly with the effects of globin immunisation in R. catesbeiana reported previously by Maniatis et al. [18] , who speculated that some form of immune response was instrumental in the altered adult globin profile observed in injected tadpoles.
Possible explanations for the differences observed between these studies is that tadpoles of B. marinus are inherently less immunocompetent than those of R. catesbeiana and/or they had less time than R. catesbeiana to mount an effective immune response to the globin antigen. Firstly, the cane toad has a very short larval stage of approximately 50-60+days, depending on tadpole density and temperature [28] whereas the larval stage in the bullfrog lasts at least 120 days and up to 2 years depending on environmental conditions [29] . The long larval stage in the bullfrog enabled an initial immunisation using FCA, followed by a boost 1 month later [18] . By contrast, cane toad tadpoles in our study were only large enough to be first injected at stage 26 (approximately day 9), barely 3 weeks before the onset of adult globin synthesis at stages 36-40 (day 30-43) and so a similar boost was not given. Nevertheless we considered that there should be sufficient time for a primary immune response to develop before adult globin appeared, provided cane toad tadpoles recognise the adult globin as a foreign protein. Secondly, due to the large difference between the cane toad and bullfrog developmental time frames, and as age at inoculation was not specified, the bullfrog tadpoles may have been inoculated later in development and been more immunocompetent than in our study. In support of this, studies in Xenopus have demonstrated that immune responses improve with age. The affinity of specific IgY antibodies against dinitrophenol (DNP) in Xenopus larvae is reported to be less than in adults, and in turn much lower than the affinity of mammalian anti-DNP IgG antibodies [30, 31] . The range of antibodies produced to DNP were also less heterogeneous in larval than in adult Xenopus [32] .
Cell mediated immunity may also be impaired in tadpoles. In mammals the antiviral response relies on cytotoxic T lymphocytes and these are Major Histocompatibility Complex Class I (MHC class I) restricted. Larval Xenopus reportedly lack MHC classical class I expression [33] suggesting the antiviral response in larvae may be compromised. Studies of the adaptive immune response in Xenopus adults and larvae to frog virus 3 (FV-3), the type virus of the ranavirus genus, indicate this may be so. While adult Xenopus cleared an initial infection and showed an accelerated response to a second injection, tadpoles were much more susceptible, suffering a high mortality rate and a reduced ability to clear the infection compared with infected adults [34] . In spite of lacking MHC class I, tadpoles do have CD8 T cells [35] and so the role played by the lack of MHC class I in the poor antiviral response is unknown. While most of these studies have been carried out in Xenopus, limited studies indicate bullfrogs are capable of mounting a detectable antibody response to an antigen, but apparently not to influenza virus despite repeated inoculations and a substantial and anamnestic response to bacteriophage T7 [36] . Our own studies indicate that B. marinus tadpoles did not mount a detectable antibody response to a widely used and well characterised immunogen, ovalbumin, to which adult B. marinus did respond.
Precedents do exist for immune interference in development. Arif et al. [37] showed that when affinity purified antibody to a protein involved in insect metamorphosis was injected into late stage larvae, the development of the larvae into adult moths was defective. Other studies in rabbits [38] and mice [7] have shown Table 2 . Detection of viral DNA in treated and control animals. that the immune response to an antigen can be enhanced by viral delivery. However, all indications are that targeting autoimmune responses in larval amphibians may not be a useful strategy as the capacity of tadpoles to respond to immunological stimuli may be too weak to affect the chain of events at metamorphosis.
In conclusion, we have shown that the globin switch occurs in B. marinus and, while we have not been able to perturb this switch immunologically, it remains a viable target for other approaches such as RNA interference. Given the short larval phase in the B. marinus life cycle, antigens produced later than globin may be more effective immunogens and we are currently investigating this possibility. It is also possible that this approach would be more successful in an amphibian with a longer larval phase. Finally, we have designed, developed and delivered a recombinant viral system that will enable the development of future strategies to prevent the spread of the toad. Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil. Eosinophils were discovered in the blood of humans, frogs, dogs and rabbits in 1879 by Dr. Paul Ehrlich [1] . At that time, the German chemical industry was flourishing and Ehrlich took advantage of newly developed synthetic dyes to develop various histological staining techniques. The coal tar derived, acidic and bromide containing dye eosin identified blood cells containing bright red "alpha-granules" and the cells were named eosinophilic granulocytes. Due to the acidity of the staining solution Ehrlich could not at the time say with certainty that the eosinophilic granules contained protein, though he speculated that if present, protein might be denatured by the low pH of the dye [1] . Subsequently it was shown that eosin binds highly basic proteins which constitute the granules of these cells. These charged proteins are contained in on average twenty large granules dispersed throughout the cytoplasm of each cell, which the eosin stain awards the characteristic red spotted appearance that discriminates eosinophils from other leukocytes [2] . More than a century later the physiological roles of these granular proteins have yet to be fully identified.
In eosinophil granules pH is maintained at 5.1 by an ATPase [3] where the basic proteins are packed forming crystals [2] . The main content of these granules are four proteins, the major basic protein (MBP) present in their cores, surrounded by a matrix built up of eosinophil peroxidise (EPO), the eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) and ECP. Vesicotubular structures within the granules direct a differential release of these proteins [4] . The granule proteins were all discovered and characterised about one hundred years after the discovery of the eosinophils [5] [6] [7] [8] . ECP is the best know of the proteins, assessed and used extensively as a marker in asthma and other inflammatory diseases. ECP has been scrutinized in a number of functional studies. The aim of this article is to review some of the findings of ECP quantifications in various diseases and set those in context of the experiments that have functionally analysed the protein. The findings will be used as guidance in a speculation of the biological role of eosinophil.
ECP is mainly produced during the terminal expansion of the eosinophils in the bone marrow Eosinophil progenitors (EoP's) in the bone marrow are the first cell identified exclusively of the eosinophil lineages. These EoP's express the cell surface markers IL-5R + CD34 + CD38 + IL-3R + CD45RA -, haematopoietic lineage associated transcription factor GATA-1, ECP mRNA transcripts and have visual characteristics of early eosinophilic blast cell [9, 10] . Most of the granule protein production takes place as EoP's undergo the final stages of maturation [11, 12] . ECP is synthesised, transported and stored in the mature secondary granules at such a high rate as that when the eosinophils are ready to leave the bone marrow, they contain 13.5 μg ECP/10 6 cells [13] ( Figure 1B ). Eosinophils are the major ECP producing cell while monocytes and myelomonocytic cell lines produce minute amounts in comparison [14] . Activated [15] but not resting neutrophils also produce some ECP and have the ability to take up further ECP from the surrounding environment storing it in their azurophil granules [16, 17] . In the myelo-eosinophilic cell line HL-60 clone 15, ECP production is dependent on a nuclear factor of activated T-cells (NFAT)-1 binding site in the intron of the ECP gene (denoted RNASE3) [18] . The RNASE3 gene was formed by gene duplication of an ancestral gene about 50 million years ago, the other duplication gene product being the eosinophil granule protein EPX/EDN gene (RNASE2). ECP and EPX/EDN are two ribonucleases with such a high degree of homology that they are unique to humans and primates and not found in other species. After this gene duplication however, ECP lost part of its ribonuclease activity, but acquired cytotoxic activity, whereas EDN/EPX remained a potent ribonuclease [19] .
ECP has homology to pancreatic ribonuclease and has the ability to degrade RNA [20] . The amino acid sequence of ECP has eight cysteine residues spaced all throughout the peptide establishing the tertiary structure of the protein by the formation of four cysteine double bonds. Two catalytic residues, a lysine and a histidine, responsible for the RNA degradation have been identified, K38 and H128 [20, 21] (Figure 2 ) and these residues together with the cysteines are present in all members of the pancreatic ribonuclease family [20] . Analysis of the crystal structure of ECP verified this relationship to these other members of RNase family; namely a β-sheet backbone and three α-helices [22] . In a grove between two of the alpha helices the catalytic site for RNA degradation is located, with ECP showing a preference for cleaving poly-U RNA but not doublestranded RNA [23] . ECP consists of a single-chain peptide of 133 a.a. containing three sites for N-linked glycosylation, a.a.'s 57-59, 65-67 and 92-94 [24] (Figure 2 ). The glycosylation is composed of sialic acid, galactose A. Blood, negative control B. Blood positive, ECP Figure 2 The RNASE3 (ECP) gene and ECP protein sequence with numbers referring to the amino acid sequence. Below the protein sequence is a schematic diagram of the peptide sequence where the beta sheet domains and the alpha helix domains are shown as red arrow and green barrel structures, respectively. Amino acids involved in RNase activity are represented by scissors. Amino acids involved in membrane interference, heparin binding and bactericidal activity are represented by red arrows. Glycosylated amino acids are represented with a glycomoiety while the letter N highlights the nitrated amino acid. A blue box shows the site of the amino acid altering polymorphism rs2073342. and acetylglucosamine [25] explaining the variation in its detected size by Western blot of between 16 and 22 kDa [26] . Nineteen arginine residues facing the outside of the protein giving rise to the proteins basicity (pI > 11) [27] and possibly also its extraordinary stability compared to other ribonucleases [28] . In the presence of H 2 O 2 ECP can be nitrated on tyrosine Y33 by EPO. This inflammation-independent nitration occurs during granule maturation and was suggested to enhance interactions after secretion between several of the otherwise repulsive, positively charged granule proteins ( Figure 2 ) [29] . ECP has been shown to interact with artificial lipid membranes [30] and two tryptophan residues, W10 and W35 facing the outside, similar to the present arginine's, have been associated with this lipid membrane interaction [31] . ECP also has RNase independent cytostatic activity on tumour cells and the tryptophan residues contribute to this activity [32] . W35 was additionally found necessary for killing gram negative and gram positive bacteria [31] . The tryptophan's also facilitate ECP binding to heparin [33, 34] . Another study found that the residues R34, W35, R36 and K38, all part of loop 3 (a.a.'s 32-41) contributed to heparin binding and cytotoxicity [35] (Figure 2) . Surprisingly, when purified from granules of circulating cells, large quantities of the protein were found to lack cytotoxic activity [36] . ECP has not, like EPX/EDN, been found have alarmin activity, stimulating dendritic cells during Th2 immune responses [37] , but ECP has the ability to bind lipopolysaccharide (LPS) and other bacteria cell wall components [38] which might have a priming influence on the immune system. The binding of LPS was mainly attributed to a.a.'s 1 to 45 [39] . The 1 to 45 a.a. region was found to retain bactericidal activity as well as membrane destabilization activity. One commonly occurring polymorphism in the gene is leading to the replacement of an arginine residue with a threonine, R97T [40] ( Figure  2) . The a.a. alteration reduced ECP cytotoxicity to the cell line NCI-H69 assessed by using both recombinant protein [36] and pools of naive protein variants [41] . RNase activity was however not influenced by the R97T alteration. Deglycosylation of the recombinant T97 restored the proteins cytotoxicity suggesting that glycosylation are responsible for this inhibitory role.
The physiological function of the granule contained cytotoxic ribonuclease Eosinophils contain a large amount of ECP but the question is why? What is the function of this protein? There is a constitutive baseline level of the eosinophils in many tissues and certain stimuli cause elevated production and influx of eosinophils in different organs. Moreover levels of the ECP in tissue and peripheral blood robustly correlated with the number of eosinophils present, which might be indicative that the function of ECP is also key to the role of eosinophils (see table 1 ). Since the discovery of ECP in 1977 [8] it has been used and evaluated as a biomarker to assess activity in various inflammatory diseases. This analysis has given indirect information of the proteins role in disease. For a comprehensive review of advantages and pitfalls of the usage of ECP as a biomarker in allergic disease see ref [42] . Furthermore, a number of in vitro studies have addressed the direct functional activities of the protein. Detailed following is a comprehensive review of these studies with summaries in table 1 and 2. To simplify comparison the concentrations used have been recalculated to μg/mL using the mean M w of 19.000 for the native protein (average of 16-22 kDa).
At homeostasis the eosinophil contributes 1 -4 percent of the circulating leukocyte pool. ECP is readily detectable in blood with plasma levels on the average 3 ug/L (serum 7 μg/L) in healthy individuals which correlates with circulating eosinophil numbers [43] . ECP in blood shows a turnover time (t 1/2 ) of 45 min [44] , and the plasma protein α 2 -macroglobulin (α 2 M) is found to be associated to the protein, in vitro at a molar ratio of 1.6 (ECP/α 2 M). This interaction is facilitated by proteolytic activity of cathepsin G or methylamine [45] , and conceivably takes place to facilitate the clearance of ECP [46] .
When eosinophils encounter adhesion molecules expressed on the endothelial cells of post capillary venule wall, the cells adhere and emigrate through the cell layer [47] . Local signals do however drive a low level influx of eosinophils in specific tissues at homeostasis. Eosinophils are present in almost all mucosal associated tissues, nasal mucosa [48] (Figure 3B ), lungs [49] ( Figure 4B ), gastrointestinal mucosa [50] , the reproductive tract, the uterus [51] , breast mucosa of mice [52] and skin [53] . The chemokine eotaxin is responsible for homeostatic eosinophil influx in the gastrointestinal tract in mice [54] whereas the mechanism of influx in other organs remains unknown. In addition, lymphocyte-associated tissue: lymph nodes [50] , thymus [55] and spleen [50] will have some cells stained red by eosin (see Figure 5 ).
The majority of ECP is released after the cell has left the circulation [56] . Several types of inflammatory stimulation have been shown to cause eosinophil degranulation. Interaction with adhesion molecules [57, 58] , stimulation by leukotriene B 4 (LTB 4 ), platelet activating factor (PAF) [59] , interleukin (IL)-5 [60] immunoglobulins and complement factors C5a and C3a [61] all cause ECP release. Upon stimulation of eosinophils small variants of ECP with sizes 16.1 and 16.3 kDa are released [62] . One line of studies have suggested that during Plasma cell line 0.5 ng/mL inhibition of Ig production anti ECP ab [87] B lymphocyte cell line 1 ng/mL inhibition of Ig production [88] Rat Peritoneal Mast Cells 17 45 min 50 percent increased histamine release [92] Human heart Mast cells 4.7 60 sec 10-80 percent increased histamine release PGD 2 synthesis Ca 2+ , temperature [94] Guinea-pig tracheal epithelium 103 6 hr exfoliation of mucosal cells [79] Feline tracheal epithelium 2.5 1 hr release of respiratory conjugates [99] Human trachea 2.5 [99] Human primary epithelial cells 10 6 hr rECP, necrosis [80] Bovine mucus 100 3 fold altered structure [97] Nasal epithelial cells 2.1 ng/mL upregulation of ICAM-1 [100] Human corneal epithelial cells 100 decreased cell viability [98] Epithelial cell line NCI-H292 20 ng/mL 16 hr upregulation of IGF-1 [102] Human fetal lung fibroblast (HFL1) 10 48 hr release of TGF beta, collagen contraction [81] Human fetal lung fibroblast (HFL1) 10 5 hr rECP and naive, migration anti ECPab [107] Human fetal lung fibroblast (HFL1) 10 6 hr 6 fold increased proteoglycan accumulation [108] Potential effects due to high ECP levels in circulation and skin inflammation whole eosinophil granules are released from disrupted cells ( Figure 4B ) and that internal proteins are subsequently released differentially through the process of piece meal degranulation [4] . Several diseases are associated with eosinophils and ECP. Most common are diseases associated with atopy and the T helper lymphocyte type 2 (TH2) phenotype. Cytokines such as IL-5 [63] , or chemokines such as eotaxin are produced in elevated levels and attract elevated numbers of eosinophils to the lumen and bronchi of the lungs in asthma [49] (Figure 4B ), the nasal mucosa in allergic rhinitis [48] ( Figure 3B ) and to the skin in atopic dermatitis [64] . In addition, the gastrointestinal tract and esophagus are infiltrated during conditions such as ulcerative colitis [65] and eosinophil esophagitis [66] . ECP has been measured in disease and the increase in number of activated eosinophils is associated with elevation of serum ECP (sECP) and plasma ECP levels [67] . Anticoagulants such as EDTA attenuate ECP release from eosinophils giving a snapshot of the in situ ECP level in plasma. sECP level on the other hand is often higher than plasma ECP as it's an artificial measure obtained by detection of the protein released during the blood clotting process in the test tube. sECP is thought to reflect the activation state of eosinophils [68] . ECP has also been detected in several other biological fluids such as bronchoalveolar lavage fluid (BALF), sputum, nasal lavage and in mucosa of the intestine [69] . ECP levels in various biological fluids in various diseases are presented in table 1. ECP measurements in allergic asthma have been found useful in monitoring the disease as sputum ECP correlates with forced expiratory flow (FEV) [70] and the need for glucocorticosteroid (GC) therapy while sECP correlate with eosinophil numbers in blood [71] . sECP is also elevated in some but not all cases of TH2 cytokine associated atopic dermatitis [72] eosinophil esophagitis [73] , parasite infection [74] and childhood respiratory syncytial virus (RSV) infection [75] . Raised levels of ECP have also been found in some cases that are not TH2 associated; a group of patients with bacterial infections had elevated sECP [76] , very high levels were found in nasal secretions from patients with bacterial sinusitis [77] and in sputum of a patient with tuberculosis and drug-induced acute respiratory distress syndrome (ARDS) [78] . Malignancies with primary eosinophilia are associated with the highest measurable sECP levels (see HES and malignancy section). Polymorphisms have been shown both to alter expression level and the function of the protein which might complicate the usage of the protein as a biomarker (see polymorphism section). The pathology attributed to eosinophils and ECP has been of both acute character such as defoliation of airway epithelium or activation of other cells [79] [80] [81] and of a chronic type, such as fibrosis in lungs [49] (Figure 5 ). Below we discuss the studies that indicate how ECP release influence other cell types locally ( Figure 6 ).
Lymphocyte activation mutually with ECP level has been shown to correlate with acute exacerbations in asthma [82] . sECP is also reduced during immune therapy which is a regimen that suppresses lymphocyte activity [83] . Eosinophils have been shown to migrate to lymph nodes where they might interact with T-lymphocytes. Eosinophils up-regulate major histocompatibility complex class II [84] for antigen presentation, thereby possibly contributing to T-lymphocyte activation and the increased inflammatory response during allergic inflammation [85] . Eosinophils are also present in the lymphocyte rich organs, the thymus and spleen and lamina propria of the gastrointestinal (GI) tract [50] . Although no studies have shown any direct link between ECP release and lymphocyte function, ECP released during the inflammatory processes, co-localises with lymphocytes. In vitro ECP has been shown to influence the proliferation of T and B lymphocytes which indicate that the protein could regulate those cells in vivo ( Figure 6 ). This was shown when mononuclear cells (containing lymphocytes, 2 × 10 5 ) were incubated with or without phytohaemagglutinin (PHA) and low levels of ECP (1 nM -0.1 μM, 190 ng/mL-2 μg/mL) for 48 hr, resulting in 50-67 percent inhibition of proliferation of the lymphocyte fraction [86] . The cells were not killed by these low levels of ECP. B lymphocyte activity might also be influenced by ECP since low levels (0.5-1 ng/mL) inhibit immunoglobulin production by plasma cells [87] and by B lymphocyte cell lines [88] . This effect was inhibited by anti-ECP antibodies and ECP was not toxic to the cell lines as cell proliferation was not inhibited with these low concentrations. IL-6 could restore the immunoglobulin production by the plasma cells and IL-4 had the same influence on the B lymphocytes. Primary human A. Healthy Control B. Allergic rhinitis plasma cells and large activated B lymphocytes responded to ECP in a manner similar to that of the cell lines [87] . Thus, ECP might influence the immune system in that immature lymphocytes are inhibited in their proliferation by ECP while activated B lymphocytes respond by decreased immunoglobulin production (see Figure 6 ).
Mast cells are found in the skin and in all mucosal tissues at homeostasis, and numbers are elevated in asthmatics lungs [49] . Mast cell and eosinophil numbers in mucosa are correlated to bronchial hyperactivity (BHR) [89] and mast cell products and eosinophil MBP but not ECP induces BHR [90] . Several lines of evidence suggest that there is a cross talk between eosinophils and mast cells [91] which to some extent are related to ECP release. Mast cells produce and secrete IL-5, PAF and LTB 4 known to augment ECP release from eosinophils. Rat peritoneal mast cells on the other hand incubated with moderate levels of ECP (0. 9 μM/17 μg/mL) for 45 min released 50 percent of their histamine. Histamine is not released from peripheral basophils by ECP treatment (as by MBP) [92] . However, the release of histamine may be location specific as no release was observed from human skin mast cells treated with up to 200 μg/ mL ECP [93] . Histamine and of some tryptase was though released from human heart mast cells, purified from traffic victims or from individuals undergoing heart transplantation, when stimulated with moderate levels of ECP (2.5 μM; 4.7 μg/mL). Between 10 and 80 percent of preformed mediators were released from these cells and MBP had a similar effect whereas EPX/ EDN did not induce any release [94] . This ECP induced histamine release occurred within 60 sec of stimulation and was found to be Ca 2+ -, temperature-and energy dependent, and ECP was not toxic to the cells. Another mast cell product, prostaglandin D 2 (PGD 2 ) was synthesised de novo by the same amount of ECP added. PGD 2 is a chemoattractant for eosinophils and TH2 lymphocytes, through binding the CRTH2 receptor [95] . Therefore these findings suggest that in some tissue the interactions between mast cells and eosinophils can be attributed to the positive feedback of ECP release.
ECP is detected in nasal mucosa in association with damaged epithelium [48] , in damaged corneal epithelium [96] as well as in BALF (at 40 ng/mL, table 1) [97] . The function of ECP has been assessed using several assays in the view of the presence of the eosinophil in the airways. Both destructive and non-destructive consequences have been found when analyzing various concentrations of the protein in interaction with the epithelium. High levels of ECP (5.4 μM/103 μg/mL) caused exfoliation of guinea-pig mucosal cells after 6 hr incubation with tracheal epithelium [79] . Confluent primary human corneal epithelial cells incubated with 0-100 μg/mL ECP, displayed a concentration-dependent gradual increase in morphological change and with the highest concentration, 100 μg/mL, being cytotoxic [98] . Lower concentration of the ECP (2.5 μg/mL) caused release of respiratory glycoconjugates (marker of mucus secretion), with a peak after 1 hr, from feline tracheal B. Allergic asthma A. Healthy control Figure 4 Eosinophil granulocytes in the bronchial mucosa. Sections of bronchial biopsies from (A) a healthy control or (B) an individual with allergic asthma were stained with ECP antibody visualizing eosinophils in the mucosa. The figures show that only a few eosinophils are present in the tissue of the healthy control, but many eosinophils accumulate in areas of reduced epithelial integrity in a specimen from a patient with allergic asthma. Original magnification ×420; Mayer's haematoxylin.
explants [99] . The short incubation time and possibility to repeat the stimulation suggested a non-toxic mechanism. MBP, which is almost as basic as ECP, in the same assay, showed the opposite effect; therefore these effects on mucus secretion are unlikely to be due to electrostatic charge. ECP at these moderate levels (2.5 μg/mL) displayed the same effect on human trachea [99] . However human primary epithelial cells underwent necrosis at higher levels (10 μg/mL) in another study [80] . ECP has also been shown to acting directly on airway mucus in vitro. At high levels (100 μg/mL) ECP altered bovine mucus three fold, as measured by a capillary surfactometer 
Helminth defence (F) Bacterial defence (F) Skin Ulceration (P) Figure 5 Known anatomical locations of eosinophil granulocytes and suggested activities of released ECP at these sites. On the left side are eosinophil granulocytes locations at homeostasis shown. On the right side are areas speculated to be affected by increased numbers of eosinophils and elevated levels of released ECP, in disease (pathology, P) and in physiological defense (function, F). This is a speculation by the authors of the review.
[97]. At low levels ECP (0.1 nM; 2.1 ng/mL) was instead found to increase the expression of intracellular adhesion molecule (ICAM)-1 on nasal epithelial cells [100] . ECP has previously been shown to be released from eosinophils when the cells adhere with their β2 (CD18) integrins to ICAM-1. Therefore the ECP triggered up-regulation of ICAM-1 on epithelial cells might mediate a positive feedback mechanism [101] . ECP has also been proposed as a mediator of tissue remodelling, see the fibroblast section below. When low levels of the protein (20 ng/mL) were used to stimulate the bronchial epithelial cell line NCI-H292 for 16 hr, the insulin growth factor (IGF)-1 receptor was found to be up-regulated [102] . ECP was speculated therefore to be involved in IGF-1-dependent lung tissue repair processes perhaps present during homeostasis and abnormally amplified during inflammatory conditions.
The persistent high number of eosinophils and ECP in the lungs of allergic asthmatics has led to the suggestion of their participation in the development of chronic lung tissue remodelling. Remodelling has also been found in the esophagus of patients with eosinophil esophagitis [103] and sECP has been found elevated in one case [104] . The remodelling in asthmatic lungs is in part caused by collagen and proteoglycan secretion from interstitial fibroblasts. Eosinophils have been suggested to participate in this by secretion of transforming growth factor (TGF) beta [105, 106] but here is additionally described how ECP could influences fibrosis development. Stimulation of a human fetal lung fibroblast cell line (HFL1) with moderate/high levels of ECP (5-10 μg/mL) for 24-48 hr resulted in increased release of TGF-beta [81] . ECP also augmented fibroblast mediated contraction of collagen gel and stimulated migration of HFL1 fibroblasts which could be blocked with antibodies to ECP [107] . In addition, ECP incubated with the fibroblast cell line for 6 hr resulted in a 6-fold increase of intracellular proteoglycan accumulation [108] .
Bronchial smooth muscles cells are involved during the progression of asthma development by secretion of cytokines as well as remodelling due to proliferation. Eosinophils have been found located in close proximity with smooth muscle cells. ECP does not influence smooth muscle cells by causing BHR [90] but high levels of ECP, similar to used for epithelial cells, appears to be cytotoxic, inducing cell death by necrosis in 1 hr. TNF alpha in contrast causes apoptosis of the smooth muscle cells [109] .
Conditions where eosinophils are overproduced lead to detrimental effects for the host. One such condition, HES is defined by the presence of more than 1.5 × 10 6 eosinophils/mL blood during a time period of at least 6 months, organ involvement and with no other etiology identified. One form of HES, the myeloproliferative form, is caused by an 800 bp deletion on chromosome 4 during the haematopoiesis in the bone marrow, resulting in a fusion between the gene FIP1L1 and the PDGFRA gene [110] . A fusion protein is produced which constitutively phosphorylates tyrosine residues leading to malignant expansion of eosinophils. Another form of HES is a clonal lymphocytic variant (L-HES) where aberrant cytokine production by malignant lymphocytes causes HES. For other cases the cause of the overproduction of the eosinophils is unknown but HES is associated with high levels of ECP in plasma and serum, of up to 0.2 μg/mL [111, 112] . It is not know however whether theses high levels of the protein are pathological. A few in vitro studies might relate to the etiologies of HES. Eosinophil infiltration of the skin of HES patients is the most common clinical manifestation [113] . Some of these patients present with erosive and ulcerative lesions and ECP was found both deposited and taken up by cells in those lesions [114] . ECP's ability to cause ulcerations in the skin has been analysed by injecting the protein intradermally into guinea pig skin, where it was found that the protein can persist there for two weeks [64] which is possibly attributed to its high stability [28] . Injections of high levels of ECP (48 and 190 μg/mL/2.5 and 10 μM) caused ulcerations which were most severe after seven days [114] . Inflammatory cells were found infiltrating the inflamed area and ECP was found taken up by cells within 48 hr. Injection of poly-lysine, other basic granule proteins MBP, EPO and the basic ribonuclease onconase showed that the severity of the lesions was not directly correlated with level of basicity. ECP and EDN were found to be more potent in lesion formations than MBP and EPO. Addition of RNase inhibitor or obliteration of the RNase activity by carboxymethylation of the RNase site of ECP reduced the ulcerations by 60 percent suggesting RNase activity is important, but not wholly responsible for the activity [114] . Some studies have shown that patients with HES have an slightly elevated risk for thrombosis formation systemically [115] and in the cardiac ventricle [116] . ECP has been shown to shortened the coagulation time for plasma which was dependent on an interaction with coagulation factor XII [117] . Eosinophils also infiltrate the endomyocardium of some patients and this has been suggested to be the cause of development of scaring in the ventricle [116] . High levels of ECP (16.25 μg/mL) degrade the muscle protein component, the myosin heavy chain in vitro [118] but it is not known whether ECP directly interacts with muscle fibres of the heart. The final stage is endomyocardial fibrosis in which eosinophils and ECP have been postulated to participate [119] by their influence on fibroblast function. Although a rare finding, a few patients with the myeloid form of HES have been reported to have central nervous system (CNS) manifestation [113, 120] . It is not known whether ECP can reach the brain but ECPs effect on the CNS has been assayed by direct intracerebral injection. Guinea-pigs injected with ECP, showed with doses of 0.1 μg and up, cerebral symptoms up until the end of the experiment at day 16 [121] . Purkinje cells in the brain were decimated in this model, suggesting that the circulating ECP could affect the CNS of some HES patients if the protein reached the brain.
Eosinophils have occasionally been found to infiltrate developing tumours and have been suggested to have a role in fighting these malignancies [122] . The involvement of the eosinophils have been suggested by the finding of elevated sECP levels in patients with renal tumours (table 1) [123] . ECP assayed in urine from patients with urinary bladder tumours showed a twofold increase compared to normal's [124] . The elevated levels suggest presence of activated eosinophils in some patients with these malignancies. In the analysis of the possible involvement of ECP in tumour defence, ECP has been evaluated in respect of altering proliferation of various cell lines. The cell lines K562 and HL-60 were incubated with 1.1 uM (21 ug/mL) ECP and the cell line A431 with 4 μM (76 ug/mL) and this resulted in 50 percent inhibition of proliferation after four days. To analyse whether growth inhibition was related to positive charge or RNase activity, poly-lysine or RNase A was used with no effect [34] . ECP exists in two forms dependent on a polymorphism, R97 and T97. It was found that the T97 form had reduced capability to kill K562 and NCI-H69 cells [36] . These recombinant (r) ECPs were produced in a baculovirus system and deglycosylation restored the cytotoxic activity.
Furthermore, high levels of bacteria expressed rECP had 50 percent cytostatic effect on HL-60 and HeLa cells [31] , compared to non-affected controls. ECP was found binding the surface of HeLa cells and caused cell death after 24 hr, accompanied by increases in intracellular radical oxygen species (ROS) generation and caspase 3-like activity [125] . A mix of ECP and EDN purified from urine and incubated with the Kaposi's sarcoma cell line KS Y-1 for 16 hr caused complete cell death at 0.625 μg/mL while 1 μg recombinant ECP produced in yeast and incubated with the same time span decreased the viability of the KS cell line by 29 percent. Proteins expressed in yeast lack glycosylation and the possible implications of this were speculated [126] .
Levels of serum ECP are elevated in TH2 engaging parasitic and helminth infections and eosinophils have long been thought to be a major defence against these types of infection. Elevated ECP have also been reported in some cases of bacterial and viral respiratory infections. Given that ECP is a cytotoxic ribonuclease, the ability of the protein to exterminate parasites, bacteria and virus in vitro has been extensively investigated (see also Figure 6 ).
Parasitic and helminthic infections drive the immune system towards TH2 cytokine production and concurrent eosinophilia. Since eosinophil infiltration in infected organs and skin is a common finding, eosinophils are thought to have a specific role in parasite killing [127] . Although, a challenged theory; the deposition of the cytotoxic protein ECP could be a mechanism by which the immune system kills off the intruders. Indeed, the eosinophilia in parasitic diseases is associated with elevated ECP in circulation (table 1) [72, 128] . ECP is also found released from eosinophils in proximity to parasites in skin and lymph nodes [129, 130] . The ability for ECP to kill or paralyse parasites and helminths have been analysed in vitro and high quantities were needed to influence the organisms. Three-hr-old larvae of Schistosoma mansoni were incubated with 10 μM (190 μg/mL) ECP and 60 percent were killed. S. mansoni, 3 days of age, were paralysed by the protein [131] while 50 μM (950 μg/mL) ECP killed 40 percent of Trypanosoma cruzi by 6 hr and 90 percent of Brugia malayi by 48 hr. This cytotoxicity of ECP to parasites was inhibited by heparin [132] and dextran sulphate, probably by interfering with the tryptophan and arginine residues as discussed earlier.
In addition, heat obliterated the toxic effect of ECP to parasites, highlighting the importance of the conformation of the protein [133] . The RNase activity of ECP was clearly shown not to be important for parasite toxicity, similar to that observed for EPX/EDN.
Eosinophils are found lining and degranulating in both the respiratory and gastrointestinal mucosa [50] . Eosinophils are generally not thought of as defendants during bacterial inflammation. However sECP has been found elevated in septic patients [76] and very high levels of ECP in nasal secretions from patients with normal cold (13 μg/mL) or severe community acquired rhinosinusitis has been described in one case (11.7 μg/mL, table 1) [77] . Moreover, a recent study has shown that eosinophils expel mitochondrial DNA coated with ECP and other granule proteins which are bactericidal in mice in vivo [134] . Additionally, a few studies have described neutrophils producing ECP [15] . In view of these findings the anti -bacterial properties of ECP has been evaluated. Bacterial strains chosen for analysis were Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). High levels of ECP (50 μg/ mL) decreased the number of colony-forming units (cfu) by 72 percent and close to 100 percent, respectively, for the two strains after a very short 2 hr of incubation. ECP only killed E. coli growing in logarithmic phase and acted on both the inner and outer membranes of E. coli [135] . Recombinant ECP was also cytotoxicity to S. aureus. Overnight incubation of rECP with the bacteria (16 kDa, 16 ug/ mL/1 uM) left 35 percent of the cfu. rECP in which a.a.'s involved in RNase activity had been substituted (K38R and H128D), terminating the RNase activity, had no effect on the bacterial killing activity [21] . In conclusion therefore, eosinophils and ECP might have a role in bacterial defence. Due to its stability, it might be feasible to speculate that ECP over time accumulate in mucus fluids such as nasal secretions and act as a first line of defence against bacterial intrusion.
ECP has been found significantly elevated in sputum from atopic subjects subjected to experimental rhinovirus infection [136] and in nasal secretions from atopic infants with respiratory RSV infection (table 1) [75] . Eosinophils and ECP are associated with RSV infection in children's lungs [137] and RSV can infect, and replicate in eosinophils [138] . Recombinant ECP expressed in a baculovirus system was used to evaluate whether ECP can inactivate the B subtype of RSV. ECP (0.5 μM; 9.5 μg/mL) incubated with the virus showed a 6-fold reduction of the infectivity of the virus to a human pulmonary epithelial cell line [139] . This antiviral activity was lower than that found with EPX/EDN (54-fold reduction) [140] , but the infectivity was increased by addition of RNase inhibitor (RI) to both proteins during incubation. Mixing the two proteins did not mediate any synergistic effects on antiviral activity. RNase A, however [up to 4 mM (76 mg/mL)], did not exert antiviral activity, suggesting that the RNase site but not activity is important for inhibition of infectivity.
Polymorphisms in the RNASE3 gene and association to production and disease Table 3 summarizes data from the NCBI entrez nucleotide site regarding polymorphisms detected in the ECP gene. Two polymorphisms are found in the protein coding region, two in intronic regions and two in the 3' untranslated region (UTR). ECP polymorphisms are differentially distributed according to ethnicity [141] . Two studies have evaluated polymorphisms in intronic and UTR regions of the ECP gene, and linked them with ECP production. One polymorphism rs11575981 (-393T > C) located in the promoter, in an C/EBP binding site was associated with decreased ECP level in serum, and decreased binding of C/EBP alpha [142] . Another polymorphism, in the 3'UTR, rs2233860 (499G > C or 562G > C) was associated with content of ECP in the eosinophils [143] . Three studies have analysed whether any polymorphisms are linked to allergic asthma and allergic rhinitis. The presence of the C allele in the nonsynonymous rs2073342G/C (371G > C/434G > C) polymorphism in the ECP gene, causing a.a. alteration Y97T, was found to be associated with absence of asthma in one Swedish study [40] . A study of Norwegian and Dutch subjects instead found that the haplotype C-G-G for the three polymorphisms rs2233859/rs17792481 (-38C/A), rs2073342 (371G/C/434G/C) and rs2233860 (499G/C/ 562G/C) being protective [144] . In a third, Korean study, which was the largest, the genotype rs2233860CC (499/562CC) was associated with allergic rhinitis [145] . Eosinophils occasionally infiltrate oral squamous cell carcinoma tumours. A study found a tendency for association of the rs2073342G/C C/C (371/434GC/CC) genotypes with a poor clinical outcome in patients with eosinophil rich such tumours [146] . As discussed earlier, eosinophils are present during helminth infections. The rs8019343 polymorphism T (1088TT) in the 3'UTR was exclusively present in the genome of a patient with tropical pulmonary eosinophilia [147] . Furthermore a study has found the rs2073342 with C (371/434C) polymorphism overrepresented in helminth infected Ugandans rs2233858
protein coding, Y > G is associated with allergic asthma [40] a , poor outcome in oral squamous cell carcinoma tumours [146] , C over represented in helminth infected Ugandans [148] rs12147890 A/G protein coding rs2233860 G/C 3' UTR,G is correlated to higher intracellular ECP [143] , G is associated with allergic rhinitis [145] , a rs8019343 A/T 499G > C, 562G > C 3' UTR T is only present in one patient with helminth infection [147] Polymorphisms found in the ECP gene and surrounding chromosomal sequence. Listed are Polymorphism i.d.'s, altered bases, alternative names, and types of associations a) C-G-G haplotype associated with allergic rhinitis [144] [148]. Interestingly, from the -550 polymorphism over a stretch of 272 bases to the mRNA transcription start site, thirteen polymorphism sites are located (NCBI Reference Sequence: NC_000014.7, J. Bystrom unpublished observation). Similar to the protein coding region and the 3'UTR, this region is highly homologous to the RNASE2 gene region, with the only differences being the sites of the polymorphisms. The replacement base's for twelve of the thirteen polymorphisms is to the same base as in the RNASE2 promoter sequence. This is also the case for two of the 3'UTR polymorphisms. This further highlights the extremely close relationship between RNASE3 (ECP) and RNASE2 (EPX/EDN).
ECP was first discovered in 1977 and since then, evidence has been gathered to understand its roles in physiology and pathophysiology. ECP is a peptide of 133 a. a., with the first 40 a.a. necessary for membrane interfering, heparin binding and cytotoxic activity. The heparin binding ability of ECP might enable the protein to bind proteoglycans on other human cells for possible uptake [34] or heparan sulfate in extracellular matrix for later use such as is the case for CXCL10 [149] . In a similar manner ECP might bind microorganisms peptidoglycans for uptake and cytotoxicity [32] . The non-synonymous polymorphism rs2073342 reduces cytotoxicity suggesting an alteration of the three-dimensional structure influencing catalytic site elsewhere in the protein. ECP is glycosylated, and as recently discovered can be nitrated. The development of increasingly sophisticated assays will determine whether other modifications, perhaps function associated, are also important in ECP activity.
Since the discovery of ECP, assays have been developed to determine its levels in biological fluids in various diseases (table 1) . ECP in serum can reach 0.1 -0.2 μg/mL for HES patients [111] and parasitic diseases infected individuals [72] and this is a 30 fold elevation compared to ECP in serum of healthy individuals. In BALF and nasal lavage from atopic patients the ECP levels are lower, 0.050 μg/mL but the sample are diluted during the collection process. In undiluted tears, sputum and nasal secretions the highest ECP levels have been found: 0.5, 0.7 and 10 μg/mL, respectively. The ECP measurements correlate with eosinophilic disease but have been found elevated also in some diseases without known eosinophil involvement [76] [77] [78] . The biological activity of ECP has been studied by incubation of the protein with several different cell types in vitro. Both human cells and pathogens have been assayed analysing different parameters (see table 2 ). In general, 10 -20 μg/mL and above, result in growth inhibitory and destructive consequences to mammalian cells, parasites and bacteria. ECP released in situ in diseases engaging high levels of eosinophils might reach these destructive concentrations (e.g. ECP accumulated in air way mucus of asthmatics, in nasal secretions of some sinusitis patients or released in skin of atopic dermatitis/HES patients, table 1 and Figure 5 ). Although it remains to be proven, there is a possibility that destructive activity to multiple cell types as well as induction of fibrosis is part of the etiology of disease where ECP levels are elevated during prolonged periods, e.g. in HES and helminth infection. There is also evidence that neutrophils are carriers of significant amounts of ECP. Using the murine system, granule proteins have been found associated with expelled eosinophil mitochondrial DNA and this DNA/protein complex trapping and killing bacteria in the gut [134] . It is intriguing to speculate whether the high levels of ECP present in various human mucosal secretions would equally be associated with eosinophil mitochondrial DNA and whether such complexes had the ability to capture and kill microorganisms.
The role of eosinophils in asthma has been under scrutiny since clinical trials showing that anti-IL-5 therapy did not improve the disease symptoms for allergic asthmatics albeit eosinophil numbers were reduced [150] . However, two recent clinical trials have shown that anti-IL-5 antibodies actually could relieve symptoms in eosinophil rich, late onset asthma, suggesting that eosinophils can have a pathogenic role in this disease. In these trials inflammatory exacerbations were reduced when anti-IL-5 antibodies were administrated [151, 152] . Earlier studies using diagnostic ECP measurements seem to agree with these findings as ECP levels correlate with severity of asthma: FEV (sputum ECP) [70] , need for GC treatment (sputum ECP) [153] and blood eosinophilia (sECP) [71] . Results from in vitro studies presented in this review may well suggest several roles for ECP in this type of allergic asthma. The protein might act as an inflammatory amplifier by augmentation of release of, for eosinophils chemotactic, PGD 2 from mast cells in asthmatic patients. Moreover, protein released in the interstitium might influence fibrosis development ( Figure 3B , 4B and 5). One might speculate that blocking antibodies to ECP could be a symptom relieving addition to the already established GC and anti-IL5 therapies used in eosinophil rich asthma and other eosinophilic diseases [113, 151, 152] . Table 2 shows that the level of protein needed to influence proliferation of lymphocytes and their antibody production is 1000 times lower than the destructive levels described above, i.e. in the ng/mL range. In murine system eosinophils have been ascribed a novel role in inflammation; the cells enter and contribute to the well orchestrated process of inflammation resolution of by release of the pro-resolving lipid protectin D1 [154] (for a review see [155] ). Whether ECP is released during this resolution process for the dual role of sequestering subpopulations of inflammatory lymphocytes [86] and promoting tissue repair by TGF beta augmentation [81] is an intriguing speculation. Eosinophils are also present at homeostasis at low numbers in lymphocyte rich organs at various locations but degranulated only in the GI tract [50] . A single eosinophil contains 13 pg ECP. Do eosinophils have a role in maintaining homeostasis and do low levels of ECP also have a role here? EDN, the sister protein has been found to play an active role during inflammation development influencing the maturation of DC's [37] . If EDN is proinflammatory, perhaps the two proteins divergence could be because ECP might have acquired a novel role as yet unknown role.
Finally, analysis of the DNA sequence of the ECP gene and surrounding regions have unravelled a number of polymorphisms. These studies have linked different polymorphisms and haplotypes to TH2 diseases, asthma, and allergic rhinitis. The studies have in some cases come to different conclusions but used different patients and different ethnic groups which might explain the variations. Diseases such as allergic asthma are multifactoral and to determine the role of certain polymorphisms one might need to look at larger defined groups to get a clear association. Altered expression levels might also influence both destructive functions and possible homeostatic roles. A careful analysis using all polymorphisms and corresponding haplotypes and large groups of defined populations would more clearly determine the role of ECPs genetic make-up, and its potential functions in physiology and disease.
The eosinophil granulocyte was discovered 130 years ago but its roles are still being revealed. The most characteristic feature of the eosinophil is the large secondary granules filled with basic proteins. The purpose of these proteins is still not fully understood. One of the proteins, ECP is a highly basic, cytotoxic, heparin binding ribonuclease that seems to need its ribonuclease site but not activity for its activities. Sensitive assays have been developed for its measurement in biological fluids which have contributed to the understanding of the role of the eosinophils in disease. In vitro studies have shown that high levels of ECP are necessary for development its destructive actions. Diseases engaging high levels of eosinophils might reach these levels locally in the tissue. At those high levels polymorphisms altering expression level and protein sequence might play a role within certain populations. Whether ECP also has roles at lower concentrations, such as the growth inhibitory influences on lymphocytes found in vitro, remain to be shown with in vivo models or clinically. These additional roles for ECP when discovered, might provide critical answers to the functions of eosinophil granulocytes and is therefore well worth waiting for. Excess healthcare burden during 1918-1920 influenza pandemic in Taiwan: implications for post-pandemic preparedness BACKGROUND: It is speculated that the 2009 pandemic H1N1 influenza virus might fall into a seasonal pattern during the current post-pandemic period with more severe clinical presentation for high-risk groups identified during the 2009 pandemic. Hence the extent of likely excess healthcare needs during this period must be fully considered. We will make use of the historical healthcare record in Taiwan during and after the 1918 influenza pandemic to ascertain the scope of potential excess healthcare burden during the post-pandemic period. METHODS: To establish the healthcare needs after the initial wave in 1918, the yearly healthcare records (hospitalizations, outpatients, etc.) in Taiwan during 1918-1920 are compared with the corresponding data from the adjacent "baseline" years of 1916, 1917, 1921, and 1922 to estimate the excess healthcare burden during the initial outbreak in 1918 and in the years immediately after. RESULTS: In 1918 the number of public hospital outpatients exceeded the yearly average of the baseline years by 20.11% (95% CI: 16.43, 25.90), and the number of hospitalizations exceeded the corresponding yearly average of the baseline years by 12.20% (10.59, 14.38), while the excess number of patients treated by the public medics was statistically significant at 32.21% (28.48, 39.82) more than the yearly average of the baseline years. For 1920, only the excess number of hospitalizations was statistically significant at 19.83% (95% CI: 17.21, 23.38) more than the yearly average of the baseline years. CONCLUSIONS: Considerable extra burden with significant loss of lives was reported in 1918 by both the public medics system and the public hospitals. In comparison, only a substantial number of excess hospitalizations in the public hospitals was reported in 1920, indicating that the population was relatively unprepared for the first wave in 1918 and did not fully utilize the public hospitals. Moreover, comparatively low mortality was reported by the public hospitals and the public medics during the second wave in 1920 even though significantly more patients were hospitalized, suggesting that there had been substantially less fatal illnesses among the hospitalized patients during the second wave. Our results provide viable parameters for assessing healthcare needs for post-pandemic preparedness. The 2009 pandemic H1N1 (pH1N1) virus spread swiftly to all parts of the world in a matter of a few months after it was first identified in Mexico in March. In August 2010, World Health Organization (WHO) declared the world to be in the post-pandemic period, when the pH1N1 virus is expected to continue to circulate as a seasonal virus for some years to come [1] . Moreover, active transmission of pandemic influenza virus still persists in some local areas, and it is still unclear whether the pandemic influenza activity has already transitioned into a seasonal pattern [2] . It is further speculated that groups identified during the recent pandemic as at a higher risk of severe or fatal illness will probably remain at a heightened risk during the post-pandemic period, although the number of such cases may decrease. In addition, a small proportion of people infected during the 2009 pandemic developed a severe form of primary viral pneumonia that is not commonly seen during seasonal epidemics and is especially difficult to treat. Therefore, quantitative ascertainment of the likely healthcare burden is an important aspect of post-pandemic preparedness planning.
More than 90 years ago, the first pandemic of the last century was initially observed in the early spring of 1918. It was quickly followed by much more fatal second and third waves in the fall and winter of 1918-1920, causing an estimated 50 million deaths [3] . It still proves to be a major dilemma for the scientific community to understand what had happened precisely, how it had happened, and why it was in several ways unlike any other influenza pandemic in recorded human history [3] . Several studies have focused on quantifying the global impact of that pandemic, either by using records of cases and mortality of the affected countries (e.g., [4] [5] [6] ), or by using vital statistics data from the affected countries to estimate the excess mortality of these countries. For example, Murray et al. [6] estimated the excess mortality rate of each affected country and extrapolated to conclude that an estimated 62 million people would be killed in a similar pandemic in 2004. However, to the best of our knowledge historical healthcare records had not been used to directly assess the excess healthcare burden during a pandemic.
The 1918-1920 pandemic swept through Taiwan in two distinct waves, both occurred during winter influenza seasons -the first at the end of 1918 and the second in early 1920, and with devastating loss of human lives. Increased influenza cases were initially reported in mid-October 1918 in Keelung, the main seaport in north [7] . A report published in the Taiwan Medical Association Journal in February 1920 [8] on the devastation brought by this first wave of influenza outbreak reported that 20.8% of the population had been infected with a case fatality rate of 3.26%. A second wave appeared at the end of 1919 and affected Taiwan through the early months of 1920, also with a severe death toll. A recent study [9] made use of the monthly mortality data in Taiwan during that time period to estimate that the total number of excess deaths during the pandemic months of November-December 1918 and January-February 1920 was 51,048 (95% CI 41,998-61,853).
The 1918-1920 influenza epidemic in Taiwan was intriguing in several aspects. First, it was one of the few regions in the world that a wave had occurred as late as 1920 [5] . Moreover, the two waves of the epidemic were separated by almost a full year, in contrast to intervals of a few months in most countries in the world [3] , and both occurred during the months of yearly winter influenza season in Taiwan. The relatively late occurrence of the initial outbreak in November of 1918, as well as the second wave in early 1920, perhaps signifies the relative lack of international travel due to its status at that time as a fairly recent Japanese colony (since 1895), in contrast to nearby regions such as Singapore which is geographically similar but more globally connected [10] .
Moreover, Taiwan is located in the tropical-subtropical zone with similar excess influenza deaths to those observed in temperate zone during periods of previously recognized influenza epidemics in Taiwan [11, 12] , and has been known to be one of the evolutionarily leading regions for global circulation of influenza [13] . Therefore, the island population in Taiwan could serve as a good model for studying spatial and temporal spread of influenza outbreak in a confined region during distinct waves of a pandemic. In this study, we will make use of the healthcare records from 1918-1920 in Taiwan to examine the level of excess healthcare burden under which healthcare system was extended in the years immediately following the initial wave of the pandemic in 1918, and to ascertain the possible post-pandemic demands on a modern healthcare system, such as we might face in the coming influenza seasons.
Our main source of data is the 1895-1945 Statistical Abstract of Taiwan [14] which contains the complete and detailed vital statistics of Taiwan during all 50 years of the Japanese occupation including detailed yearly healthcare records. We will use this data to explore the public health events that had occurred during those years during and immediately after the initial epidemic in 1918. During 1918-1920, there were 12 large public hospitals, 18-19 smaller public hospitals, and 60-68 private hospitals in Taiwan. In addition, there was a large network of trained "public medics" which was responsible for, among other duties, providing basic and primary medical care in the local community for people with clinical symptoms and for reporting local incidence of illnesses (including epidemic intelligence) to the government [15] . However, only the numbers of outpatients, in-patient hospitalizations, and all-cause deaths for the large public hospital and the public medics system were given in the Statistics Abstract [14] .
A measure of the severity of an epidemic and its burden on the healthcare system is the excess number of hospital visits and hospitalized patients during the epidemic and during the post-epidemic years. Yearly excess numbers of outpatients, in-patient hospitalizations (abbreviated to "hospitalization" hereafter), and all-cause deaths reported by the large public hospital and the public medics system during 1918-1920 were computed by the method of Serfling et al. [16] . We first computed the yearly mean numbers of outpatients, hospitalizations, and all-cause deaths over the two adjacent baseline years before the epidemic (1916, 1917) and the two adjacent baseline years after (1921, 1922) . We then subtracted these means from the corresponding yearly numbers of outpatients, hospitalizations, and all-cause deaths for each year during 1918-1920 to obtain the yearly excess numbers during 1918-1920. A yearly excess number is considered to be statistically significant if the number (of outpatients, hospitalizations, or allcause deaths) for that pandemic year exceeds the corresponding mean of the adjacent baseline years of 1916, 1917, 1921 , and 1922 by 2 SDs or more [9] . In order to compare the yearly excess healthcare burden of the pandemic years of 1918-1920, we computed the percentages of these yearly excess numbers over the means of the adjacent baseline years, to ascertain the impact of the pandemic on the healthcare system during each of the years in 1918-1920.
The yearly excess number of patients and all-cause deaths reported by 12 public hospitals and public medics system during 1918-1920 compared with the yearly averages during the adjacent "baseline" years of 1916, 1917, 1921, and 1922 are shown in Figures 1, 2, 3 . The percentages of the excess number of medical treatments and hospitalization for each year during 1918-1920 over the averaged yearly numbers of the adjacent baseline years of 1916, 1917, 1921, and 1922 are given in Table 1 with the 95% confidence intervals (CI). In 1919, the numbers of hospitalizations and treatments by public medics are clearly excessive, exceeding even the corresponding numbers in the epidemic years in 1918 and 1920 in some instances (see Figures 1, 2, 3 Moreover, the percentages of excess yearly number of deaths reported by 12 large public hospitals and public medics for each year during 1918-1920 over the averaged yearly number of deaths of the adjacent years of 1916, 1917, 1921, and 1922 , are given in Table 2 . Only 
There is an underlying assumption of our method that there is no drastic change in the Taiwanese population during 1916-1922, when the population size increased steadily but only by less than 10%, from 3,596,109 to 3,904,692 [14] . Methods to detect significant changes over time can be found in, among others, [17] .
We also note that the decline following 1919 in all the three sets of numbers reflecting healthcare burden might be partly attributable to a regression to the mean, given that the second wave was still in full force in the first two months of 1920. The limitation in the data, where only yearly numbers (and not monthly numbers) are given, makes it impossible to determine the months in which the drop had occurred, and whether the decline is attributable to the decrease in healthcare demand after the pandemic was over or to the low level of healthcare demand even during the pandemic months early in the year.
The percentages of excess deaths reported by the public hospitals and the public medics in 1918 were both statistically significant, corroborating the results from another study [9] . Moreover, given that the excess hospitalizations in the public hospital were not statistically significant and yet the percentage of excess deaths reported by the public hospitals was statistically significant and exceeded even those reported by public medics, one could infer that a comparatively larger proportion of hospitalized patients had lost their lives in 1918. However, the corresponding percentages of excess deaths were not statistically significant in 1920, even though similar levels of excess numbers of deaths were found in both waves [9] . This gives indication that the second wave in early 1920, although with a significantly greater number of hospitalizations, had substantially fewer fatal illnesses among the hospitalized patients when compared with the initial wave in 1918. 
Our results indicate that there was a considerable extra burden on the public medic system during the initial wave of the epidemic in 1918, with a significant loss of lives reported by both the public medic system and the 12 large public hospitals. In comparison, only a substantial number of excess hospitalizations in the public hospitals was reported in 1920, indicating that the population was relatively unprepared for the first wave in 1918 and did not fully utilize the public hospital system.
The most surprising part of our findings is the significant increases in the numbers of hospitalizations and treatments by the public medics for 1919, the year between the two waves when only the beginning of the second wave in December of 1919 had contributed 9 .24 (7.90, 11.12) *Excess deaths in 1918 are statistically significant (more than 2 SD). some initial influenza deaths over the monthly means of neighboring "baseline" years [9] . One possible reason for this is the contribution to hospitalization/treatment due to other diseases that were prevalent in 1919 (e.g., a cholera outbreak which led to 2,693 deaths in 1919 and 1,675 deaths in 1920 [14] ). However, limited by the retrospective nature of the study design, we are unable to identify or rule out other non-relevant diseases or conditions solely from our hospitalization/treatment data due to the lack of more detailed historical data.
It is also possible that the severe first epidemic wave during the previous winter of 1918 had alarmed the population to being more readily willing to quickly seek medical assistance at the first sign of an ailment, even though many of these illnesses might be unrelated to influenza. That is, the populace was more readily alerted to seek treatment from local public medics with any initial symptom of illness (as compared to visiting large hospital), while patients with more severe illness (of any kind) are more likely to be hospitalized by physicians. This type of overreaction on the part of the healthcare system and the general public had also been observed during the 2003 SARS outbreak where many non-SARS patients were hospitalized unnecessarily as suspected SARS cases. Adding the fact that both the numbers of hospitalizations and treatments by the public medics dropped drastically next year in 1920, the last scenario seems plausible. Another possibility is that pathophysiological or social processes [18] may be at play where the end of World War I could have contributed to movement of people and affected the pandemic's spread, although the Taiwan data indicated no noticeable increase in migration,.
Our results suggest that the excess burden on the healthcare system was high in the post-pandemic period, which would be a major challenge to any well-managed healthcare system. But it could contribute to fewer fatal illnesses. It has been noted that any present-day projection based on the 1918-1920 pandemic merely presents a worst-case scenario which we can avoid with diligence [19, 20] . However, one should note that the situation today, 90 years later, is very different in many aspects. While modern communication systems may facilitate more rapid spread of infections, implementation of interventions (school closures, masks, hand washing, bans on spitting in public, etc.) may reduce the overall transmission of influenza. Moreover, population demographics, health status and prior exposure to influenza are also different. In 1918-1920 life expectancy was shorter, so the population would have been on the average younger with less prior exposure to influenza, and therefore less compounded by past circulation of influenza as mentioned previously. Our results provide a basis to learn from the past to obtain projections of pandemic scenario and the viable hypothetical parameters for assessing healthcare needs specifically for the current post-pandemic preparedness in every country, including antivirals and vaccines needs for speedy, adequate, and equitable distribution. Finally, while this study is retrospective in design, the study methods can be easily modified for a prospective design and incorporated into a part of syndromic surveillance during a future influenza pandemic to monitor and adjust resources accordingly. Multifunctional nanoparticles as simulants for a gravimetric immunoassay Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-010-4419-8) contains supplementary material, which is available to authorized users. Viruses are the smallest form of life on earth with the ability to replicate and spread within living cells [1] . As they pass from cell to cell, they adapt to evade host immunity and spread disease, creating some of the worst pandemics in history [2] . Improving diagnostics for viruses, such as influenza, would help slow the spread of infection in the event of an emerging virus. It is known that even with reassortment, a common viral defense mechanism, the majority of anti-hemagglutinin (anti-HA) antibodies recognize a specific nine amino acid sequence within the epitope, AYDPVDYPY, which has been the focus of many assays to improve the detection of influenza [3] . Using this immunodominant sequence for the HA epitope, the influenza epitope can be mimicked with a functionalized nanoparticle yielding a comparable affinity to the linear peptide [4] . Another virus, Ebola, has also been effectively mimicked with a monolayer-protected cluster (MPC) through functionalization of the MPC with the antigenic determinant of the Ebola glycoprotein [5] . Integrating biology and materials chemistry using biomimicry in this way has allowed materials chemistry the opportunity to improve current diagnostic treatments, techniques, and limits of detection, but further improvements are still yet to be made [6] .
The utilization of immuno-molecular recognition in the assembly of nanoscale sensors has applications in medical diagnosis, treatment, and the understanding of diseases [7] . Enzyme-linked immunosorbent assay (ELISA) is widely utilized in clinics and hospitals as an initial screening for several infectious diseases. While ELISA can be effectively employed in laboratory settings for common infectious agents, several obstacles inhibit the adaptation of this standard clinical assay to portable or select agent detection schemes. Unfortunately, pathogenic agent detection requires calibration with irradiated or otherwise attenuated samples of the organism. This requirement limits the widespread use of immunosensor diagnostics because of the scarcity of these agents and the logistic difficulty in safe transportation to remote locations. Recent cases like the development of meningitis and tularemia infections in researchers who were working with the causative agents of these diseases alert scientists to the hazards accompanying work with live calibrants [8] . The possibility of exposure and high cost are enough to warrant investigation into a safer positive control for these disease detection assays and devices. Without a positive control, the operational status of the sensor cannot be determined. Thus, the development of a nanoparticle mimic would be a safer alternative to current methodology and could be extended to address the needs of other assays that incorporate well-defined epitopes.
Many traditional clinical assays lack the ability for electronic adaptation and timely results. The need exists for rapid, sensitive, and inexpensive methods that could be utilized in clinical settings [9] . Therefore, the rapid realtime quantification of a QCM has been combined with the selectivity of monoclonal antibodies to create an immunosensor for evaluation of the multifunctional nanoparticles. The use of a QCM for immunological detection has been demonstrated previously, where it has been shown to detect Staphylococcus epidermidis in clinical samples using nanoparticle amplification [10] , SARS virus in sputum samples [11] , plant pathogens [12] , an antigenic mimic of Ebola [5] , and influenza A and B from nasal washes [13] . In QCM assays, nanoparticles have enabled simultaneous parallel detection and amplification for gravimetry, thus lowering the limit of detection [6, [14] [15] [16] [17] . A QCM-based sensor with a nanoparticle control would allow for the simultaneous rapid and accurate analysis of multiple viral or biological hazards, without posing a safety or health threat. The work reported here builds on this previous work and addresses the specificity of detection of antibody-antigen binding at the QCM using polyepitope-functionalized gold nanoparticles.
Gold nanoparticles have favorable characteristics for their use as the basis for multifunctional microorganism simulants [18] . Nanoparticle size, shape, and capacity for surface modifications, based on chemical characteristics and environments, make the use of nanoparticles advantageous for detection, discovery, and diagnosis [7, 19] . Previous studies have shown that nanoparticles are capable of accepting a wide array of functional molecules via the Au-thiol bonds at the interface of the ligand and particle [20] . Since the physical and chemical properties of nanoparticles are dependent upon size [6] , the gold nanoparticles can be customized to simulate the variability in pathogen size. MPCs in this work had an average diameter of 2.6± 0.6 nm. Influenza virions vary in size, normally around 100 nm in diameter, but smaller nanoparticles were chosen in this study since they have a larger surface area to volume ratio and therefore increase the ratio of possible antigen presentation to gold core [21] . These nanoparticles were polyfunctionalized to increase the presentation of the nanoparticle epitope to the antibody. Previous studies have found multivalent ligand attachment to gold nanoparticles enhances the affinity measured in binding studies [22, 23] . The selectivity of antibody sensors for functionalized MPCs has been examined using two orthogonal epitopes: FLAG and HA. The FLAG epitope is a biological peptide sequence commonly used for identification of proteins in biological samples [24] . HA-and FLAG-functionalized MPCs can be used as a synthetic simulant and negative control for the HA epitope of the influenza virus [25, 26] . The binding of these nanoparticles was also compared with binding of negative-control Au-tiopronin nanoparticles and an authentic sample of H5 HA proteins from influenza virus (H5N1). The simulants work as safe controls whose application could be extended to address various pathological threats, in which using attenuated controls is problematic.
Chemicals Gold shot was purchased from precious metal vendors (Canadian Maple Leaf, 99.99%) and was initially converted to HAuCl 4 ·3 H 2 O by boiling Au 0 in HCl/HNO 3 solution [27] . N-(2-Mercaptopropionyl)-glycine (tiopronin, reagent grade), bovine serum albumin (BSA, fraction V, 96%), and sodium phosphate (monobasic, reagent grade) were purchased from Sigma. Other chemicals were obtained as follows: protein G was purchased from Southern Biotech, Fmoc-dPEG-COOH from Quanta Biodesign, anti-FLAG mouse monoclonal antibody from Stratagene, and anti-HA mouse monoclonal antibody from the Vanderbilt Molecular Recognition Core facility. The following reagent was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: H5 HA protein from influenza virus, A/Hong Kong/156/97 (H5N1), recombinant from baculovirus, and NR-652 (56 kDa) [28] . Analytical grade solvents for nuclear magnetic resonance (NMR) were obtained from Cambridge Isotope Laboratories, and water was purified using a Modulab Water Systems unit (∼18 MΩ/cm). Buffers were prepared according to standard laboratory procedure. Other chemicals were reagent grade and used as received.
MPC synthesis and characterization Gold-tiopronin-protected MPCs were synthesized as previously described [29] [30] [31] . Briefly, tiopronin-protected gold nanoparticles were synthesized by dissolving tiopronin and HAuCl 4 ·3H 2 O (3:1) into a MeOH/acetic acid solution (6:1). The reaction was stirred for 30 min and then cooled in an ice bath. NaBH 4 was then added in 10× molar excess of gold. Reaction product was stirred for 1 h. The solvent was removed under vacuum, and the pH was lowered to 1 with concentrated HCl. Reaction product purification by dialysis with cellulose ester membranes (Spectra/Por CE, MWCO= 10,000) removed any excess tiopronin. Average particle diameter was determined by transmission electron microscopy (TEM). TEM images were taken on a Phillips CM20 instrument after applying aqueous MPC samples to Formvar-coated 200-mesh copper grids (Ted Pella). The microscope operated at 200 keV with magnification in the range ×150-750,000. Thermogravimetric analysis (TGA) was performed with a TGA 1000 (Instrument Specialists, Inc.) to calculate Au-tiopronin stoichiometry. From TEM and TGA data, the nanoparticle size and Au-tiopronin stoichiometry were obtained. MPCs used in the following experiments had an average diameter of 2.6±0.6 nm and a base composition of Au 544 Tiop 204 (140.4 kDa), calculated from TEM and TGA data (at 650°C), respectively (Electronic Supplementary Material Figs. S1, S2, and S4) [32] [33] [34] .
Peptide synthesis and characterization The FLAG and HA epitopes were synthesized with standard 9fluorenylmethoxycarbonyl (Fmoc protocols on a solid resin support) [35, 36] . Epitope sequences were modified with a linker region comprised of a discrete polyethylene glycol (PEG, purchased as Fmoc-dPEG™ from Quanta Biodesign) and a C-terminal cysteine. PEG was added to attenuate nonspecific binding, while the C-terminal cysteine provided a thiol linkage to the gold surface. PEG formed the link between the cysteine and the rest of the peptide, HA-PEG-C for example. After initial MPC, studies performed without the PEG linker were found to have no binding; all future studies incorporated this linkage. The notations HA-Au, HA-FLAG-Au, and FLAG-Au are assumed to include the incorporation of this linkage. Epitope antigens HA (AYDPVDYPY-(PEG) 6 -C, 1562.6 Da) and FLAG (KDDDDKYD-(PEG) 6 -C, 1451.5 Da) were synthesized on an Apex 396 (Advanced Chemtech) equipped with a 96-well reaction block capable of vortex mixing. 2-Chlorotrityl resin was swollen in dichloromethane (Fisher) prior to synthesis [37] . Fmoc amino acids (Synpep) and Fmoc-dPEG 6 acid (Quanta Biodesign) were coupled using O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU, 5 eq with respect to resin, Synpep), 1-hydroxybenzatriazole (Hobt, 5 eq, Synpep), and N,N-diisopropylethylamine (DIEA, 10 eq, Advanced Chemtech) in N,N,-dimethylformamide (DMF, Fisher). Peptides were cleaved with 90% (v/v) trifluoroacetic acid, 5% anisole (Sigma), 3% thioanisole (Sigma), and 2% ethanedithiol (Sigma). Final purification was performed on a Waters C18 semi-prep RP HPLC column using a water (0.05% trifluoroacetic acid and acetonitrile) gradient. Peptide identity was confirmed via matrix-assisted laser desorption/ionization-time of flight analysis.
Place exchange Place exchange reactions between tiopronin-MPCs and thiolate-containing peptides, HA and FLAG, followed a method similar to Hostetler et al. [18, 38, 39] and assumes that epitope-tiopronin S N 2 place exchange is one for one. While it has been shown by 1 H NMR that the interchange of ligands is 1:1, the final ratio of the original ligand to exchanged ligand depends on ligand length, concentration of entering and exiting ligands, as well as the placement of the ligand (vertice, edge, or terrace) on the surface of the cluster [18, 38] . It should also be noted that the reported ratio will be an average exchanged ratio calculated from 1 H NMR, as the dispersity of MPC size will cause variations in the exact number of exchanged ligands on each cluster [18] . With the X-ray diffraction identification [40] of staple motifs, which are composed of one Au(I) and two thiolates, as part of the capping structure on gold-thiolate nanoparticles, place exchange must insert the new thiolate into the existing staples at the same time as the previous thiolate is lost in the S N 2 mechanism. Tiopronin MPCs were co-dissolved in DI water with free thiolated epitope (1:10). If necessary to aid the solvation of the epitopes into water, the epitopes were first dissolved into ethanol. The place exchange reaction took only one night for the exchange of a single epitope, while requiring 3 days for the exchange of two epitopes simultaneously. Solutions were dialyzed at room temperature for approximately 3 days as previously described and then dried under air [29] . The epitope composition of the MPCs was determined by TGA and 1 H NMR analysis (Electronic Supplementary Material Figs. S2 and S3).
Nuclear magnetic resonance 1 H NMR experiments were run at 300 MHz on a Bruker DPX-300 instrument with 5 s relaxation times. Samples were dissolved in D 2 O. The extent of place substitution of tiopronin by epitope on gold nanoparticles was determined by 1 H NMR, using methods previously described [18, 31] .
Enzyme-linked immunosorbent assay Initial tests to determine whether the functionalized nanoparticles were suitable as both a viral simulant and a synthetic calibrant were performed using ELISAs. Positive activity was established by adhering gold nanoparticles (250-500 ng/per well) with a variety of functionalities (Au-Tiop, HA-Au, FLAG-Au, and HA-FLAG-Au) to a 96-well Immulon 2HB plate. Buffer and free peptides (25 μg/well) were plated and tested in a similar fashion. Blocking was achieved with BSA (1 mg/mL), followed by exposure to either anti-FLAG or anti-HA primary antibodies (at recommended dilutions, monoclonal mouse). Horseradish peroxidase linked antimouse antibodies (1:5000 dilution) were then added, followed by exposure to tetramethylbenzidine (TMB, Sigma) substrate solution. The reaction was halted with 2 M H 2 SO 4 , and the plates were read at 450 nm (BioTek Synergy HT plate reader).
Immunosensor assembly The immunosensor was assembled on a 5-MHz AT-cut quartz crystal. Before assembly, the gold electrode was triple cleaned in piranha, and then received a final ethanol or acetone rinse and dried in a stream of N 2 (grams). The quartz crystal was then mounted in a flow cell holder, rinsed with phosphate buffer (PB, 50 mM phosphate, pH 7.2), and brought to resonant frequency at room temperature. For the work described herein, a Stanford Research Systems quartz crystal microbalance model 200, which measures both frequency and resistance, was used. Solutions were passed through the cell at a flow rate of 28 μL/min controlled by a Masterflex peristaltic pump. The sequence for biosensor assembly was protein G (20 μg/mL, 10 min), BSA (1 mg/mL, 5 min), antibody (20 μg/mL, 16 min), and viral simulant (500 μg/mL, 11 min) all in PB. The nanoparticles were filtered prior to use with a 0.22-μm filter from Millipore. The crystal was washed for a minimum of 10 min with PB between each step. Deposition of protein G allowed for the immobilization of the Fc region of anti-HA and anti-FLAG antibodies. The binding of non-functionalized Autiop nanoparticles (500 μg/mL, 11 min) with anti-HA antibodies was used as a negative control and as a test for nonspecific binding. Positive control was established utilizing anti-HA's recognition of recombinant H5 HA protein (5 μg/mL, 10 min).
The adsorbance of each of these molecules resulted in changes in both frequency and resistance. The bound analyte mass is proportional to changes in the oscillation frequency of the quartz crystal as described by the Sauerbrey equation (Eq. 1), where Δf is the change in frequency, C f is the known sensitivity factor of a 5-MHz crystal (56.6 Hz cm 2 μg −1 ), and Δm is the change in mass [41] :
In addition to mass, the frequency of the crystal is also dependent on the density and viscosity of the contact medium, a consequence of the solution's resistance to crystal oscillation [42] . The crystal frequency and resistance were recorded during the QCM experiments to allow for corrections to be made for solution resistance, following the work of Kanazawa and Martin [43] [44] [45] , which modifies the standard Sauerbrey equation to:
Frequency change for solution resistance is nominally −2.464 Hz/Ω given an active crystal surface area equal to 0.40 cm 2 per sucrose calibrations similar to work reported previously [32] . The resulting change in mass is therefore a combination of changes in resistance and frequency, while taking into account solution resistance. The QCM results corroborated results from the ELISAs.
In the development of a synthetic positive control for this influenza immunoassay, functionalized gold MPCs were synthesized and characterized. TEM analysis of the tiopronin-gold nanoparticles (Au-tiop) resulted in an average cluster size of 2.6±0.6 nm, with a range from 1.5 to 4 nm (Electronic Supplementary Material Fig. S1 ). The TGA tiopronin MPC mass loss equaled 37.9% and yielded a 2.67 Au:Tiop stoichiometric ratio for the nanoparticles assuming the loss of the Au(I)-thiolate staples (Electronic Supplementary Material Figs. S2 and S4 ). The assumption of the loss of the Au(I)-thiolate staples is justified by the observation of gold-thiolate compounds upon thermal decomposition in a mass spectrometer [46] . The average number of gold atoms calculated per cluster equaled 544 atoms. Combining the TEM and TGA yields a calculated empirical formula Au 544 Tiop 204 for the 2.6-nm diameter particle. Following place exchange of epitope(s) for tiopronin, the epitope loading was determined through 1 H NMR (Electronic Supplementary Material Fig. S3 ). The epitopes HA and FLAG each contain an exclusive amino acid whose proton signal occurs at a unique location in the 1 H NMR spectrum. Specifically, valine (V) is unique to the FLAG epitope and lysine (K) to the HA epitope. Tyrosine (Y) is common to both epitopes, but occurs in different stoichiometric ratios. The integrated resonance for each functionalized nanoparticle can be compared with that for nanoparticles protected solely with tiopronin to determine the average epitope stoichiometry of the nanoparticles. Peaks used for quantification consisted of signals at 1.2 ppm (V, 2 CH 3 ), 1.45 ppm (tiopronin, CH 3 ), 2.90 ppm (K, 2 ε-CH 2 ), 6.75 ppm (Y, δ-CH), and 7.05 ppm (Y, ε-CH Material Fig. S4) .
The ELISA results confirmed specificity of the functionalized gold nanoparticles to the anti-HA and anti-FLAG antibodies (Fig. 1 ). Evidence of cross reactivity between the gold nanoparticles presenting both peptides and both antibodies corroborates with QCM data. HA-FLAG-Au and FLAG-Au were detected with the anti-FLAG antibodies, while HA peptides and HA-Au were not. Correspondingly, HA peptides, HA-Au, and HA-FLAG-Au were recognized by anti-HA, while FLAG-Au was not. The LOD was calculated to be 0.146 OD using the average absorbance of wells incorporating buffer only in the initial step, which served as a negative control. The gold particles displaying only tiopronin (Au-tiopronin) appeared to nonspecifically bind with anti-HA. Nonspecific binding could be reduced in ELISAs with the use of higher concentrations of BSA or with the use of more stringent rinsing protocols, such as the use of Tween 20, a nonionic surfactant, between steps, which might also remove bound particles and lower the sensitivity of these assays [47] . MPCs functionalized without the PEG linker between the epitope and the cysteine linkage showed no binding with their respective antibodies in either ELISA or QCM experiments, but once the PEG link was incorporated into the mimic's a b designs, the antibodies were able to bind with the epitopefunctionalized nanoparticles. Calculations using Spartan computational software show the spatial projection of tiopronin molecules to be about 7 Å from the gold surface, and the peptides, due to the PEG addition, extend 20 Å, from the gold surface [48] . The addition of the six-mer PEG link between the epitope and the terminal cysteine enhanced the probability for cluster-epitope interaction with antibody by distancing the epitope from the gold surface. This enhanced binding was demonstrated with both ELISA and QCM studies.
Several observations can be made by inspection of the QCM responses (Fig. 2 , recorded data and schematic of adsorption) during sensor formation. Both antibodies are found to bind well to protein G. Observed spikes during assembly occur when reagent flowing through the peristaltic pump is momentarily interrupted for reactant exchange. A time delay (1-2 min) between spike and sensor response was due to the requisite transport time for the analyte to move through the peristaltic pump to the QCM sensor. binding of the nanoparticles and the HA protein to the HAantibody (Fig. 3a) and to the FLAG-antibody (Fig. 3b) . Detection of the HA protein confirms the ability of this assay to detect HA specific to the H5 HA subtype, and thus the use of this HA-functionalized nanoparticle as a viral simulant. This change in mass is based on the relationship that both frequency and resistance changes have on mass load. Originally, the Sauerbrey equation was applied in air or in a vacuum, and the resulting equation was only valid for thin solid layers deposited on the resonator [49]. Since then, extensive work has been done to establish the use of the QCM to probe interactions in a liquid environment, involving suitable oscillator circuits, fluid modeling in viscous and lossy fluids, as well as determination of the relationship between motional resistance and mass load [12, 45, 50] . The sensing layers utilized in this study should yield at most a layer 30-nm thick, using liberal estimates, where the Sauerbrey equation can be applicable to thin films less than 250 nm [51] [52] [53] [54] . Previous work, which uses a sucrose calibration, modifies the Sauerbrey equation to account for the changes that do occur in part from motional resistance, and therefore allows the ideology behind the Sauerbrey equation to apply in environments where energy is dissipated in the non-rigid liquid environment [30] . The nanoparticles increase the resistance of the crystals, and thus the rigidity, as well as decreased the frequency, resulting in a detectable increase of mass adsorbed on the surface of the crystal. Binding to the HA-antibody (positive Δm) occurs if the gold nanoparticle is functionalized with either HA or both HA and FLAG epitopes, but does not occur if the cluster is only FLAG functionalized (Fig. 3a) .
The nonspecific binding observed with ELISA of Autiopronin to anti-HA was not observed with the QCM. QCM naturally prohibits nonspecific binding through the acceleration of adhered particles. This acceleration is generated by the oscillation of the quartz and can help remove weakly bound or nonspecifically bound molecules [55, 56] . Similar binding of anti-FLAG to FLAG-Au and HA-Au-FLAG but not HA-Au was measured with the QCM (Fig. 3b) . Thus, orthogonal and normalized binding at the QCM is observed consistent with immunological results obtained from ELISA, but is observable in a much shorter time interval (10 min compared to the 4 h it took to complete the ELISAs) when using the developed immunoassay and the QCM. The demonstrated binding between bi-functionalized nanoparticles and their respective antibodies makes evident the practicality of their use as a simulant for microorganisms, while lacking the difficulties associated with use of an attenuated or killed pathogen. When a change in Fig. 4 Calibration of HA protein binding to anti-HA. a The average change in mass of H5N1 HA protein binding was determined at varying concentrations. Briefly, (black line) 40 μg/mL (n=2), (red line) 20 μg/mL (n=5), (blue line) 10 μg/mL (n=3), (teal line) 5 μg/mL (n=3), and (pink line) 1 μg/mL (n=4). Also shown for comparison is the binding of HA-Au (green line) and HA-FLAG-Au (orange line). b A linear representation of the Langmuir isotherm produced by this average binding is shown with a linear relationship of y ¼ 0:035 AE 0:004 ð Þ x þ 4:90 AE 15:5 ð Þ Â 10 À10 and R 2 =0.96. c Calibration curve for an assay time of 1.5 min where the black squares are HA proteins, and the red and blue dots are HA-FLAG-Au and HA-FLAG, respectively b mass is observed upon introduction of the functionalized MPC to the immunosensor, the immunological response is shown to be above the limit of detection (∼3 ng, calculated by three times the average noise). Results show the average change in mass for protein G to be 128±48 ng, for antibodies to be 276±75 ng, and for the nanoparticle simulants to be 37±8 ng (Table 1 ). While the time was held constant for each step, the binding varied slightly, which could be due to slight variations in surface roughness and the surface coverage of prior adsorption steps. Even with a standard deviation of 75 ng for antibody adsorption, a deviation of only 8 ng was measured for the final detection step. The binding of the nanoparticles demonstrates saturation behavior (Fig. 3) . Based on the shape of the QCM curves and the rapid increase in mass with time, it can be assumed that the kinetics would occur quickly and with presumably large equilibrium association constants. To test this theory, a calibration of anti-HA to HA binding was determined, and the binding of the functionalized nanoparticles was compared. The HA protein from H5N1 was exposed to anti-HA antibodies at concentrations ranging from 1 to 40 μg/mL (Fig. 4a, b) . This binding can be compared at any time point to generate a calibration curve (Fig. 4c , example shown is at 1.5 min). Based upon the desired separation of the lower data points, the binding can continue for several more minutes. Also, the amount of nanoparticle exposed to the surface can be lowered to prevent overloading of the sensor. Measuring the maximum binding that occurs, as opposed to lower time points, can be used to determine the equilibrium association constant (K a ) and increase our understanding of the affinity of our sensor. This constant was determined by fitting to a linear rearrangement of the Langmuir adsorption isotherm, where C was plotted versus C/Δm (Eq. 3, Fig. 4b ) [30] :
This yielded an equilibrium association constant for the binding between the HA protein and anti-HA of 7.14± 0.26×10 7 . This K a is in the range expected for antibodyantigen interactions, from 10 6 to 10 10 M −1 [57] . In fitting the binding of the nanoparticles to this Langmuir isotherm calibration (at the experimentally used nanoparticle concentration of 500 μg/mL), the sensor response to HA-FLAG-Au would have the same binding as 0.92±0.01 μM HA protein, and HA-Au would generate the same sensor response as 1.50±0.02 μM HA protein. The large response seen is at the maximum of the Langmuir isotherm. This demonstrates that even at lower concentrations, these functionalized nanoparticles can be used as a positive control. The functionalized MPC-antibody binding is not inhibited by the presence of an additional non-interacting epitope (either FLAG or HA) on the polyfunctionalized nanoparticle; therefore, multiple binding interactions can be explored simultaneously.
The ability to create multi-epitope-presenting nanoparticles that can orthogonally bind to specific monoclonal antibodies has been demonstrated using both ELISA and immunological QCM. Determination of the extent of antibody-functionalized nanoparticle binding is rapid using the QCM compared to ELISA. Also, like ELISA, the immunological response is specific, with QCM incurring less nonspecific binding. Interaction of the epitope with its antibody was improved through the use of a PEG linkage for epitope attachment to the MPC. Binding studies at the QCM show that polyfunctionalized gold nanoparticles exhibit the expected affinity to both antibodies that a normal immunological response is achieved from matched antibody-antigen couples and that an orthogonal response results otherwise. The results demonstrate that binding of polyfunctionalized gold nanoparticles could be used to determine sensor functionality, without resorting to the use of attenuated or killed microorganisms, or extracted and purified whole proteins. Hepatitis G Virus associated aplastic anemia: A recent case from Pakistan BACKGROUND: Aplastic anemia (AA) is a serious and rare disorder characterized by a hypocellular bone marrow. Hepatitis associated aplastic anemia (HAAA) is a variant of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. Several reports have noted an association between HGV and hepatitis-associated aplastic anemia besides other hepatitis causing viruses. CASE PRESENTATION: A female girl of age 11 year with a history of loose motion for one month, vomiting for last 15 days and poor oral intake for last few days is reported here. The physical examination presents fever, pallor whereas bleeding, hepatomegaly, Splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, Splenomegaly and lymphodenopathy. The laboratory investigation parameters were: haemoglobin 6.2 g/L, total leucocytes count 1.51, neutrophils 0.47%, absolute reticulocyte count 0.5%, Monocytes 0.16%, red cell count 3.2 mil/uL, Picked cell volume (PCV) 30.13%, Mean Corpuscular Volume (MCV) 78 fL, Mean Corpuscular Hemoglobin (MCH) 26.3 pg. The liver enzymes were alanine aminotransferease (ALT) 98 IU/L, aspartate aminotransferase (AST) 114 IU/L. Serologic and molecular tests for hepatitis A, B, C, D, E, TTV, B19 were negative, whereas HGV RNA PCR test was found positive for hepatitis G virus. The bone marrow aspirate and trephine biopsy examination revealed hypo- cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis. CONCLUSION: HAAA is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. Our finding suggests the involvement of HGV in the development of aplastic anemia. In patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by RT-PCR and if possible by bone marrow biopsy. Hepatitis G virus was reported first time has a non-A-E hepatitis and placed as flavivirus [1] .The induction of this new agent in the family of Hepatitis has attracted significant attention because of its etiology [2] . Hepatitis G virus has been marked as a cause of non-A through E acute viral hepatitis and sharp liver failure. Aplastic anemia complicating hepatitis is an uncommon but well recognized phenomenon. Hepatitis associated aplastic anemia is a severe disorder with a high mortality (85%) [3] .Hepatitis associated aplastic anemia (HAAA) is a deviation of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. The marrow failure can be severe and is usually lethal if untreated. Lorenz and Quazier has documented first time HAAA in two case back in 1955 [4] , by 1975 more than 193 cases had been reported [5] . Adil et al has reported, severe aplastic anaemia (SAA) 51.4%, very severe (VSAA) in 16.7% of 144 patients of aplastic anemia cases [6] . A number of reports have mentioned alliance between HGV and HAAA [7] [8] [9] [10] . Number of HAAA cases with a history of multiple blood transfusions has been reported [11, 12] . Crespo et al has documented a case of 24 year old man have community acquired HGV that later progress into severe aplastic anemia, point out HGV for both hepatitis and aplastic anaemia. However greater number of serum samples are needed to prove the association of hepatitis G virus and aplastic anaemia [13] . Moatter et al. has reported 5/43 patients of haemodialysis with raised liver enzyme, reduced platelet count and 21/100 patients of polytransfused b-thalassemia major children infected with HGV RNA form Pakistan [14, 15] . 
In this study well characterized samples of 93 aplastic anaemia patients before blood tranfusion were included. These characteristics include history, physical examination, haematological investigation, bone marrow aspirate and trephine biopsy examination, liver function test (LFTs), renal parameters, viral profile and abdominal ultrasonography. The diagnosis of HA-aplastic anemia was made on the basis of hepatitis (elevated serum aminotransferase enzymes, jaundice, absolute neutrophils counts, platelet counts and reticulocytes. All the 93 samples were checked for serological marker of HAV, HBV, HCV, HDV, HEV, HGV,TTV and B19. One of 93 samples from patients with HA-aplastic anemia has hepatitis G associated aplastic anaemia with positive HGV RNA.
A female girl of age 11 year is reported here. The patient had a history of loose motion for one month, vomiting for last 15 days and poor oral intake for last few days. The physical examination presents fever, pallor whereas bleeding, hepatomegaly, Splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, Splenomegaly and lymphodenopathy. The laboratory investigation parameters were: haemoglobin 6.2 g/L, total leucocytes count 1.51, neutrophils 0.47%, absolute reticulocyte count 0.5%, Monocytes 0.16%, red cell count 3.2 mil/uL, Picked cell volume (PCV) 30.13%, Mean Corpuscular Volume (MCV) 78 fL, Mean Corpuscular Hemoglobin (MCH) 26.3 pg. The liver enzymes were alanine aminotransferease (ALT) 98 IU/L, aspartate aminotransferase (AST) 114 IU/L. Serologic and molecular tests for hepatitis A, B, C, D, E, TTV, B19 were negative, whereas HGV RNA PCR test was found positive for hepatitis G virus. The bone marrow aspirate and trephine biopsy examination revealed hypo-cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis.
Flaviviruses belong to enveloped viruses with a single positive sence RNA about 10 kb. Hepatitis G virus medium of transmission mostly through blood. The possible roel of hepatitis G virus infection in the pathogenesis of rare non-liver disease has been suggested but need to be recognized. By some unknow reseason aplastic anemia some time proceded by hepatitis. Few reports have analysed the role of HGV in the development of HAAA [8, 9, 11] . Crespo et al in print a case through negative serological markes for HAV, HBV, HCV, HEV, hypoplastic marrow low platelet and white cell counts but detected HGV-RNA before any blood transfusion [13] . Byrnes et al has described hepatit G Virus positive case of 26 year old man before the use of medication, blood tranfusion or intravenous drug abuse [9] . Zaidi et al, reported a 19 year male before blood transfusion with positive HGV by RT-PCR and suggested that in the absence of any other clincial manifestions the possible infectious agent may be HGV for hepatitis G virus associated aplast anaemia [8] . In the list of studies of Hepatitis G Virus associated aplastic anaemia before blood transfusion we report a case of 11 years female girls.
Whereas some reports confirms the presence of HGV viraemia in the patient aplastic anaemia after blood transufion [8, 9, 11, 12, [16] [17] [18] . But however concluded that HGV viraemia is frequent in patients with aplastic anaemia [19] . Kiem et al reported similar finding after logistic regression analysis, that HGV RNA in transfused patients was 5.9 times higher compare to untransfused patients (P = 0.001). This implicates transfusion as major source of HGV with aplastic anaemia [18] .
The published literature point out that studies must be performed on many more aplastic anaemia patients prior to blood transfusion [20] . However, to find such patients in large number are not normally avaible to study, so far individual cases are reported. The ideal case regarding Hepatitis G associated aplastic anaemia are pre blood transfusion.
In conclusion, HAAA is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. Our finding suggests the involvement of HGV in the development of aplastic anemia. In patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by RT-PCR and if possible by bone marrow biopsy.
Written informed consent was obtained from the patients a copy of which is available for review by Editor-in-Chief of this journal. Authors' contributions SARS aided in acquistion of data that was included in this case report and drafted the manuscript. MI aided in acwuision and interpretation of the data analysed. AH helped in statistical analysis of the data and in editing of this manuscript. All authors have read and approved the final version of this manuscript. Travel Patterns in China The spread of infectious disease epidemics is mediated by human travel. Yet human mobility patterns vary substantially between countries and regions. Quantifying the frequency of travel and length of journeys in well-defined population is therefore critical for predicting the likely speed and pattern of spread of emerging infectious diseases, such as a new influenza pandemic. Here we present the results of a large population survey undertaken in 2007 in two areas of China: Shenzhen city in Guangdong province, and Huangshan city in Anhui province. In each area, 10,000 randomly selected individuals were interviewed, and data on regular and occasional journeys collected. Travel behaviour was examined as a function of age, sex, economic status and home location. Women and children were generally found to travel shorter distances than men. Travel patterns in the economically developed Shenzhen region are shown to resemble those in developed and economically advanced middle income countries with a significant fraction of the population commuting over distances in excess of 50 km. Conversely, in the less developed rural region of Anhui, travel was much more local, with very few journeys over 30 km. Travel patterns in both populations were well-fitted by a gravity model with a lognormal kernel function. The results provide the first quantitative information on human travel patterns in modern China, and suggest that a pandemic emerging in a less developed area of rural China might spread geographically sufficiently slowly for containment to be feasible, while spatial spread in the more economically developed areas might be expected to be much more rapid, making containment more difficult. Good morning/afternoon/evening ! With the spread of H5N1, potential next pandemic will be a terrible threat to human beings. It is necessary for us to develop feasible and effective pandemic preparedness and response strategy. The resident traveling survey is implemented by Chinese Center for Disease Control and Prevention. The purpose of the survey is to learn about the residents' traveling habits through collecting individual traveling data (including the distance from home to work/school and other traveling within past 7 days) to provide scientific evidence for establishing pandemic modeling and developing the strategy of pandemic influenza preparedness and response in China.
We mainly collect the information about your everyday traveling such as going to work or school and other traveling within past 7 days. Simple demographic data such as gender and age will also be collected. There are not any sensitive or private questions in the questionnaire, so you will not feel any discomforts. Meanwhile we will not begin the face to face survey until obtain oral informed consent from you. We promise that all the information you provide will be kept secret strictly. You will be voluntary for the survey. If you are not willing to take part in our survey, it would not bring any bad impact for you. It will take you less than 10 minutes to finish the survey, we feel sorry for bothering you. If you have any question, please consult our investigators, we will try our best to explain for you.
If you consent to participate, let's begin the survey. Please answer all the questions truthfully. We really appreciate you for your kindly help. 2. Fill the ID No. of the interviewees who has journeys caused by non-work/non-school in the last 7 days. and the sequence should be consistent with section one.
3. If the destinations are more than one place, please fill all the destinations in the blank.
If the destinations are more than one place, please fill all the GPS distance in the blank.
. For national travel, if the journey includes flows between rural area and urban area, please mark "√" in the blank. For international travel, if the destination is HK SAR, please fill①in "HK&Macao " column, if the destination is Macao, please fill②in "HK&Macao " column, if the destinations include other countries besides or except HK&Macao, please fill the actual address in Other column.(Other: all other countries except HK SAR and mainland China).
6. ①Business ②Tour ③Visit relatives and friends ④Other (If choose ④, write down travel reason directly in the blank).
Don't need to fill.
Good morning/afternoon/evening ! With the spread of H5N1, potential next pandemic will be a terrible threat to human beings. It is necessary for us to develop feasible and effective pandemic preparedness and response strategy. The resident traveling survey is implemented by Chinese Center for Disease Control and Prevention. The purpose of the survey is to learn about the residents' traveling habits through collecting individual traveling data (including the distance from home to work/school and other traveling within past 7 days) to provide scientific evidence for establishing pandemic modeling and developing the strategy of pandemic influenza preparedness and response in China.
We mainly collect the information about your everyday traveling such as going to work or school and other traveling within past 7 days. Simple demographic data such as gender and age will also be collected. There are not any sensitive or private questions in the questionnaire, so you will not feel any discomforts. Meanwhile we will not begin the face to face survey until obtain oral informed consent from you. We promise that all the information you provide will be kept secret strictly. You will be voluntary for the survey. If you are not willing to take part in our survey, it would not bring any bad impact for you. It will take you less than 10 minutes to finish the survey, we feel sorry for bothering you. If you have any question, please consult our investigators, we will try our best to explain for you.  Spatial dynamics of the 1918 influenza pandemic in England, Wales and the United States There is still limited understanding of key determinants of spatial spread of influenza. The 1918 pandemic provides an opportunity to elucidate spatial determinants of spread on a large scale. To better characterize the spread of the 1918 major wave, we fitted a range of city-to-city transmission models to mortality data collected for 246 population centres in England and Wales and 47 cities in the US. Using a gravity model for city-to-city contacts, we explored the effect of population size and distance on the spread of disease and tested assumptions regarding density dependence in connectivity between cities. We employed Bayesian Markov Chain Monte Carlo methods to estimate parameters of the model for population, infectivity, distance and density dependence. We inferred the most likely transmission trees for both countries. For England and Wales, a model that estimated the degree of density dependence in connectivity between cities was preferable by deviance information criterion comparison. Early in the major wave, long distance infective interactions predominated, with local infection events more likely as the epidemic became widespread. For the US, with fewer more widely dispersed cities, statistical power was lacking to estimate population size dependence or the degree of density dependence, with the preferred model depending on distance only. We find that parameters estimated from the England and Wales dataset can be applied to the US data with no likelihood penalty. Spatially explicit models are critical to understanding the spread of infectious diseases through populations and to better inform policy aimed at controlling that spread. Indeed, recent outbreaks of communicable diseases in human populations have triggered a series of studies addressing the spread of directly transmissible infections at a country level [1 -5] . Identifying a possible backbone of high probability transmission paths through populations may underpin the development of effective interventions to curtail spread on the population network [6] . For example, in human diseases, spatial models and microsimulations can quantify the possible role of border control, quarantine or transport reductions in curtailing local and international spread [2 -4,6 -11] . Spatial microsimulation models like these are critical to making effective policy decisions. Spatial models also allow limited control resources to be used where they might be most effective. For instance, during the initial stages of an outbreak of a new virus, disease incidence tends to occur in spatial clusters with occasional long-range infection events [10] . As case numbers increase in the start location, the frequency of long-range infection events increases. This spatial pattern was seen during the early stages of the 2009 influenza pandemic in Mexico, leading to local foci seeded by long-range interactions to other countries [12, 13] .
However, those epidemic models rely on a set of structural assumptions that need to be validated from data. A basic assumption of many spatially explicit transmission models is that flows between urban centres are a function of the distance between them and their attributes, most notably residential or worker population sizes [14, 15] , resulting in a so-called gravity model. However, for human diseases, little work has been done to validate the underlying assumption that human travel patterns are predictive of the spatial spread of diseases.
Early models of spatial coupling in ecology assumed that connectivity between populations was inversely related to the distance between them [16] . For people (and most animals) distance-based coupling is too simplistic an assumption. Movement between large population centres is disproportionately more frequent than between smaller ones [17] . Xia et al. [18] found that a distance-only model for spread of measles in the UK was a poor fit to weekly measles data from England and Wales from 1944 to 1967. The gravity model used in that study measures connectivity between population centres as a function of distance and a function of the population sizes of the origin and destination cities. However, measles is a childhood disease, and so the spatial dependencies of the host are different than for infections that affect both adults and children, such as pandemic influenza. Gravity models and other spatial interaction models allow understanding of the movement of populations from one location to another in the absence of movement data. Models, once validated, can predict modifications in connectivity when populations grow or shrink, when workflows vary owing to economic changes or if restrictions are imposed on one city and not others. This is in contrast to movement surveys, which are context-specific and provide a snapshot of the movement habits of a population.
The strength of connection between cities may be density independent, that is, the sum of connectivity of a city to all its neighbours does not depend on the number of neighbours that city has. In contrast, density-dependent connectivity links two cities at a strength solely determined by the sizes of those cities and their distance apart, so that the total connectivity of any one city scales with the number of close neighbours. Density-independent transmission gives a total force of infection, which is independent of the remoteness of the population, whereas density-dependent transmission will cause populations with many neighbours to experience a higher force of infection than those cities that have few neighbours. Thus, a densitydependent model will predict that isolated populations are less likely to become infected than populations with many neighbours, or few very large neighbours. The concepts of density dependence/independence have not only been used to model interactions between cities; they have also been used extensively in individual based models of disease spread. Most past studies tend to assume either density dependence (e.g. for animal epidemics; [19, 20] ), or density independence (e.g. for most human diseases) [3, 4] . Here, we explore the extent to which city-to-city (rather than individualto-individual) contacts are density independent by constructing a model that can capture intermediate levels of density dependence.
In this paper, we analyse mortality datasets from England and Wales and the United States from 1918 to 1919 to examine the pattern of spatio-temporal spread and the extent to which gravity models can reproduce observed trends. The 1918 pandemic constitutes a rare example of a well-documented epidemic in a largely susceptible human population, where the high mortality gives a clear incidence signal, and is therefore a rare opportunity to validate models of epidemiologically relevant geographic coupling. We examine the effect of city-specific characteristics (e.g. location, distance from other cities, population size and the number of influenza-related deaths) on the pattern of spread seen. We also investigate the impact of the distribution of cities in each country. The analysis provides further insight on the spatial variation in the spread of the 1918 influenza pandemic at a country level, much of which remained unexplained in past studies [18, [21] [22] [23] .
The 1918 pandemic H1N1 virus appears to have entered the general population of the UK and US in the spring of 1918 causing a reportedly mild disease [24] . This early wave was associated with increased mortality, but was probably only noticed because influenza is rare in summer. This epidemic waned later in the summer, but infection reappeared in the autumn with much increased mortality. It is unclear whether the viruses causing the spring and summer waves were closely related, but there is increasing evidence that the spring wave gave immunological protection against the autumn wave at a population level [25, 26] . By September 1918, the pandemic was a prominent global phenomenon. The autumn wave was virtually universal, albeit with some variation between countries in precise timing. In both the UK and the US, a third wave of influenza occurred in early 1919 although with greater heterogeneity in mortality rates between cities [24, 27, 28] . US cities had more variation in the severity of the major wave than the UK, probably in part because some enacted more stringent non-pharmaceutical interventions to mitigate the epidemic [27, 28] . The third wave was less pronounced in US cities than in England and Wales, again perhaps partly because of the effect of interventions.
The England and Wales dataset shown in figure 1a was published in the Supplement to the 81st Registrar General Report [29] . It provides weekly death counts and annualized mortality rates per 1000 from 83 county boroughs, 84 municipal boroughs, 71 urban districts and three unclassified urban centres in England and Wales, for a 46 week interval, 29 June 1918 -10 May 1919. The first 10 weeks are designated as wave 1, the next 19 as wave 2 and the last 17 as wave 3. Our analysis focuses on the second wave because it occurred in all cities (unlike wave 3) and because recording of mortality had begun in all cities before its arrival (unlike wave 1). In addition, reporting of influenza and influenza-related mortality changed between the first and second waves. Point locations of all urban centres were determined from the current or historical records, with Euclidean distance used to quantify inter-centre separation. Further information is given in the electronic supplementary material.
We compiled a US city dataset (figure 1b) from five publications reporting the Weekly Health Index as collated by the Bureau of the Census [24, [30] [31] [32] [33] . It covers the period 14 September 1918 to 15 March 1919 and contains weekly pneumonia and influenza death counts for 47 cities in the US. The US data therefore covers a period of two waves, with not all cities experiencing the later wave. There is very good agreement between different sources where they overlap. We used the Euclidean distance between cities (accounting for curvature of the Earth) to measure separation. Further information is given in the electronic supplementary material.
The analysis requires an estimate of when each city became infected to allow potential sources of that infection to be identified. For each city infected in week t, the candidate infectors are those infected in any week before t. We define the infection week of city i, t i , to be the first that meets a set of conditions on mortality in weeks t i þ 1, t i þ 2 and t i þ 3. We use mortality values ahead of t i to include the time from infection to death. A week could be designated the infection week if either (or both) of two sets of criteria were met for mortality in the following weeks. The first set of criteria required the mortality rate in week t i þ 1 to be above a certain threshold, to have increased in t i þ 2 and to be above a higher threshold in t i þ 3. These criteria are intended to ensure that the epidemic in that city is patently increasing. The second set of criteria was designed to capture cities where there was a rapid onset of increased influenza-related mortality. They therefore used a higher threshold on mortality in week t i þ 1, but less strict conditions on rate of growth in the following two weeks. The week of infection determined was found to be relatively robust to the precise choice of thresholds used. For further details on the algorithm and a spatio-temporal display of the result, see the electronic supplementary material.
In formulating our inter-city transmission model, we take the city as our unit of study. Each of N cities, i, has an infection time t i , an invariant population size P i and a time-varying mortality rate, r i,t at time t. Infected city i is separated from susceptible city j by distance d ij . Each week, each city can be in one of the three disease states: Susceptible, Latent or Infectious. We assume that all cities are susceptible at the start of a wave, they are latently infected for one week on infection, and that a city becomes infectious the week after it becomes infected. We assume that all transmission is endogenous to England and Wales or US after external seeding to the first infected city in each territory. If a city becomes infected in week t i , the candidate infectors are only those cities that are infectious in week t i . We assume that the transmission parameters are constant through time.
The model formulation aims to capture the effect of distance and population size on the connectivity of cities. Three modes of spatial transmission are considered: density-independent connectivity, densitydependent connectivity and an intermediate form where the degree of density dependence is estimated. The model also examines the different assumptions regarding a city's infectivity over time.
The force of infection, l is the hazard of infection from one city to another. From infected city i on susceptible city j at time t, it is:
where source city population and distance are normalized together. n and m are estimated parameters on source and destination population sizes, respectively. d ij represents the distance between cities with power parameter g to be estimated. w is an estimated parameter relating the infectivity of a city to its mortality rate. When w ¼ 1, the infectiousness of a city at time t i is proportional to the death rate in that city at time t i þ 1. We use one week as a lag from infection to death [24, [34] [35] [36] . A value of w ¼ 0 gives a flat infectiousness profile, independent of the death rate in the source city. Intermediate values of w give variation in infectiousness, which scales sub-linearly with weekly mortality. Estimating w allows us to assess whether mortality rate is a good proxy for infectiousness in an infected city. b is a time-invariant estimated infectivity term.
Parameter 1 describes the strength of connection of a susceptible city to all possible infectors. 1 ¼ 0 gives the density-dependent model and 1 ¼ 1 gives the density independent model. By allowing 1 to vary, we allow the model to estimate the degree of density dependence in connectedness between the cities.
The total force of infection on city j at time t is given by:
where Since the force of infection is a hazard, the probability that a susceptible city j is infected in a week t j is given by:
t¼0 Àl j;t ! ð1 À expðÀl j;t j ÞÞ:
We used a Bayesian framework for statistical inference.
The log-likelihood is given by:
ln P t j À Á :
We explore the joint posterior distribution of parameters by Markov Chain Monte Carlo (MCMC) sampling [37, 38] . We sampled parameters on a logscale using a random walk update scheme. b and g were jointly updated, while 1, m and n were updated singly. Five MCMC chains were started from a variety of start points within a credible range to assess convergence. Convergence was achieved within 100 000 iterations for all models from all starting parameter values. For each model, the chain was run for 500 000 iterations including a burn in of 100 000. Parameter estimates and equal-tailed 95 per cent credible intervals were obtained from the posterior distribution of 80 000 values thinned from the last 400 000 samples of the MCMC chains.
To investigate which components are most important for describing the spread of influenza, we consider a set of simplified variants of the model presented above. In those variants, each parameter can be either fixed at 0, at 1 or be estimated by MCMC. For a full comparison of each component of the model, see the electronic supplementary material.
The Deviance Information Criterion (DIC) is used to compare models [39] . This is calculated using the median parameter values owing to non-normality in the likelihood [39, 40] . Lower values are preferable and a difference of around 5 units is considered important [41] .
The model is used to generate epidemic trees [42, 43] . We sample 1000 parameter sets from the joint posterior distribution and calculate the probability of infection for each potential infector city. The most likely tree for each parameter set is generated by calculating which 'infector' city has the highest probability of infecting each 'infectee' city. The distance to this infector, the probability of the infector-infectee pair and the number of infectees each infector creates are calculated for each parameter set. Mean values are weighted by the frequency of infector -infectee pairs from 1000 trees.
We examine the ability of the models to recreate the observed epidemic by simulation. We use 1000 parameter sets sampled from the joint posterior distribution and for each set, we simulate an epidemic using the first infected city as a source of infection. Once a city is infected, the observed mortality curve is used to model the infectiousness of that city through time.
We also calculate the probability distribution of the week of infection for each city conditional upon the observed epidemic up to that time point. We use 1000 parameter sets sampled from the joint posterior distribution and for each set, we calculate the probability of infection each week for each city given the epidemic observed up to that time point. We use Welch's two-tailed t-test to differentiate outlying groups.
Comparison of spatial and non-spatial population-independent models shows that inclusion of distance substantially improves model fit for both England and Wales and the US (D DIC ¼ 64.7 and D DIC ¼ 33.3, respectively). DIC and parameter estimates for the distance-only model are given in column 1 of tables 1 and 2.
Previous formulations of the gravity kernel in the literature have considered either density-dependent (1 ¼ 0) or density-independent transmission (1 ¼ 1). Figure 2 compares the fit (expressed by the posterior deviance) of these two formulations and with that from the model where the degree of density dependence, 1, is estimated. This comparison is made for models assuming no linear or a fitted power-dependence of spatial coupling on both source and destination city population size. In figure 2a , w is estimated, whereas in b it is fixed at 1. See model components in the electronic supplementary material for further comparisons.
For England and Wales (figure 2a), in each population context the variant that estimates the degree of density dependence (the lightest curve of each colour) gives a slightly better fit than models with no density dependence, with pure density dependence fitting substantially less well. The comparison also shows that the models which estimate the effect of origin and destination city population sizes on the connectivity of cities are much better than either the populationindependent or linear population size-dependent models.
The same set of comparisons is made for the US in figure 2b . The situation is more complex with the posterior distribution of many model variants lying in the same area. Comparisons by DIC value cannot distinguish these models. Unlike in England and Wales, there is no density-dependence variant which has lower deviance for all three of the population sizedependence variants examined. Inclusion of nonlinear population size-dependence does not penalize the fit of the US model, and so cannot be definitively excluded as being consistent with the data. The models presented in columns 5 and 6 in table 2 have different population relationships, but the same DIC score. The credible intervals on the population parameters of the densitydependent population with infectivity model (column 5) are very wide suggesting that little information is added by the inclusion of these parameters.
In England and Wales, the lowest DIC model is one where the degree of density dependence is estimated and the effect of population is also estimated. This is in contrast to the US where population-independent models either with density-dependent or estimated density dependence spatial interaction terms are indistinguishable.
We tested models with three types of infectiousness profile through time: constant infectivity, a linear relationship between infectivity and mortality in the week ahead, and an estimated power-law relationship between mortality and infectivity. Mixing was poor when estimating w with the US data so we only compare the first two models in that setting.
In England and Wales, the linear infectivity model has a DIC value of more than 25 above either the constant or estimated infectivity model. Parameter estimates for the constant-infectivity and estimated-infectivity model variants are shown in columns 4 and 5 of table 1 using the density-dependent population-dependent framework from the previous comparison in England and Wales. These two models are indistinguishable by DIC (D DIC ¼ 0.1). Estimates for all other parameters are very comparable between these two models.
The estimated relationship includes two inputs from the infected city: the mortality rate and the population of the city. It can be more difficult to estimate parameters regarding infectivity, so we tested a model which takes only one piece of information from the infected city. The final column in table 1 shows a model which takes the mortality rate from the infected city into account but does not include the population size of that city. There is an improvement in the DIC score for this model of 4.4 over the constant infectivity model. Table 2 shows parameter estimates for models in the US. The difference in DIC score between a constant infectivity model and one with a linear relationship between mortality and infectivity is negligible in either a distance-only model framework (columns 1 and 6) or a population-dependent framework (columns 2 and 5). Adding infectivity information does not improve the fit of the model.
In the England and Wales dataset, the lowest DIC model is the single infected city parameter model in column 6 of table 1. The model is dependent on the destination population size, has estimated dependence of infectivity on mortality and an estimated intermediate degree of density dependence. The distance power g was estimated as 1.18 (0.96, 1.39). A lower value was found for models in the US, where for the most parsimonious low DIC model (density dependent, population independent), g was estimated as 0.79 (0.54, 1.00). Figure 3 shows the distance kernels for the two datasets. The credible intervals for g overlap for the two datasets.
The power parameter on the destination city population, m, was estimated at 0.40 (0.25, 0.54) in England and Wales. The credible intervals exclude 1, demonstrating that as population size increases, the susceptibility of the city increased more slowly.
We used the posterior median parameter estimates fitted to the England and Wales dataset to calculate a likelihood value in the US dataset. By likelihood ratio test, this value was not different from the most parsimonious low DIC US model (255.23, 257.72, p . 0.97). We therefore cannot reject the assumption that spread had the same characteristics in the US and England and Wales, though clearly the smaller size of the US dataset reduces inferential power. the epidemic during which each city was infected. Inferred city-to-city infection events more frequent than 70 per cent (in 1000 trees) are shown in black, events of lower frequency are shown in grey. Interactions in weeks 0 -3 are longer range than those in weeks 4 -7 ( p , 0.01), which are in turn longer range than those in weeks 8 -10 ( p , 0.01) (figure 4d ). The probability that the most likely infector was responsible for each infection falls as the epidemic progresses because there are many more potential infectors available later (figure 4e). Cities infected early give rise to more infections than those infected late in the wave, as expected, but the range is large, with some early cities giving rise to no new infections (figure 4f ). Figure 5 shows results for two models using the US data. We compare the most likely infection trees for the distance-only constant infectivity model with parameters inferred from the US data (figure 5a) with a model where parameters used to generate the trees are taken from the England and Wales single-infected city parameter model (figure 5c). In the distance-only constant infectivity model, the nearest infected city is always the most likely infector. In contrast, with the England and Wales parameters, some links between cities are high frequency, while other cities have several potential infectors of intermediate frequency ( figure 5b) . As in England and Wales, infection events inferred early in the epidemic have a higher support than those later in the epidemic. There are some exceptions owing to the distribution of cities in the US dataset-Oakland and San Francisco are distant from all other cities but very close to each other. In the distance-only constant infectivity model, some cities may give rise to a large number of new infections (e.g. Pittsburgh gives rise to nearly a quarter of infections) (figure 5d ). The effect of a city acting as a hub of infection is reduced in the more complex model, as the risk of infection from one city to another is the combined effect of several factors including distance.
For England and Wales, there is a relatively good agreement between observed and simulated epidemic curves (figure 6b). The observed epidemic curve rises more steeply than the simulation curves in the early stages of the epidemic, and peaks one week earlier than the simulation mean. This suggests that the model may underestimate the external infection pressure early in the wave. We calculated the probability that a city was infected in each week given the observed behaviour of all other cities up to that time. In England and Wales, 245 of 246 cities lie within the 95 per cent interval of their expected distribution. Figure 6a shows the cities which the observed infection week lies outside the stricter inter quartile interval. For further information see the electronic supplementary material. There are no population size ( p ¼ 0.36) or density trends ( p ¼ 0.11) in these cities, which are typically infected later in the epidemic ( p , 0.01 for difference in infection week). In the US, all cities lie within the 95 per cent probability interval and all but three lie within the inter quartile interval. Those three outlier cities are smaller than other cities ( p ¼ 0.01) but equally distributed in space ( p ¼ 0.88) and time ( p ¼ 0.06).
We have tested the effect on parameter estimates in England and Wales of relaxing the single-introduction assumption inherent in the model. We re-estimated the parameters conditioning on infections that occurred from week 3 of the epidemic onward. There is a small increase in the kernel power parameter estimate, which causes the kernel to decay more rapidly with distance (electronic supplementary material, figure S9 ). This suggests that the very long-range interactions, which are forced to occur early in the epidemic impact the shape of the kernel. However, the credible intervals largely overlap which indicates this assumption does not affect the fit of the model to a large degree.
In the US the simulated curves for the distance-only constant infectivity model are shown in figure 5e and for the England and Wales parameters in figure 5f. In both cases, the mean simulated and observed curve are very comparable, with the distance-only constant infectivity model giving peak incidence in the same week as observed. Figure 5g shows the observed week of infection against the simulated week of infection for all 1000 simulated epidemics. There is good correlation between the observed and simulated weeks of infection for both parametrizations.
We have tested the effect of thinning the England and Wales dataset so that it more closely resembles the US dataset to determine if the differences in formulation between the best models for each dataset are owing to the smaller number of cities in the US dataset. We removed all cities with fewer than 90 000 inhabitants in England and Wales leaving 46 cities distributed quite evenly in England and Wales as shown in the electronic supplementary material, figure S10. There were identifiability problems in estimating the density-dependence parameter 1 using the thinned dataset. The best model by DIC comparison gave a distance-only interaction (no dependence on population size) with infectivity scaling linearly with mortality in a density-independent framework. As we found with the US data, it is difficult to disentangle the effects of population and infectivity parameters because these feature in different combinations in comparable DIC models.
We have presented a statistical analysis of the spatiotemporal spread of the 1918 influenza pandemic between cities in England and Wales and the US. The results demonstrate that for England and Wales, a model with intermediate levels of density dependence in the connectivity between cities gives the best fit to the observed pattern. For the US dataset, where there are few, large and widely spaced population centres, estimating the degree of density dependence does not improve the fit. In both contexts, city population size affects inter-city coupling sub-linearly. Parameter estimates and model formulation inferred from the data of England and Wales explain the US dataset well. Gravity model parameter estimates generated in this study are comparable with values found in studies describing the spread of seasonal influenza [5, 44] .
Our analysis demonstrates the degree of spatial locality in the large-scale geographical spread of influenza in both England and Wales and the US in 1918. However, it is difficult to directly compare the kernel power estimate from this study with those from other studies owing to differences in the functional forms used. For instance, Viboud et al. [5] estimate two power parameters above and below a given distance threshold when modelling the spread of seasonal influenza in the US. Gravity models used to describe the spread of measles in the UK by Xia et al. [18] assumed a kernel power of 1, rather than fitting this parameter. The distance power estimates we found for England and Wales and the US are quite different from each other. It is not surprising that there is a disparity in the distance kernel in England and Wales and the US, as the spatial scale in the US is much larger than in England and Wales. In comparing the US and UK, it should be noted that the mean distance between cities is of course much larger in the US (see electronic supplementary material, table S1). In theory, this gives better resolution for estimating the kernel shape, as the range of inter-city separations is an order of magnitude larger than for the UK. However, this is counterbalanced by the smaller size of the US dataset, which reduces inferential power. The low kernel power parameter estimates we have found in both England and Wales and the US suggests that long-distance interactions were important in spreading influenza between distant cities in both countries. At the start of the major autumn wave in 1918, the armistice was more than two months away and it is likely that travel relating to the war effort, including troop movements, might have enhanced the frequency of long-distance movements. Density-dependent gravity models are frequently used to explain the connectivity of urban centres for human diseases [5, 18] . There is good evidence from the estimates of 1 in this analysis for England and Wales that the density-dependent model underestimates the total force of infection on remote cities. The 95 per cent credible interval for 1 includes 1, which indicates that the density-independent model formulation cannot be definitively excluded as an explanation for the data. There was limited statistical power to estimate the degree of density dependence in the US context, but use of the England and Wales best-fit model to describe the US data gave a very similar DIC to the best-fit US model. Hence, it is not clear if the difference in the estimated density dependence found between the US and the England and Wales is because of the large differences in the degree of population coverage between the US and England and Wales datasets. Our results using a subset of the England and Wales dataset suggest that the degree of density dependence is a difficult parameter to estimate when coverage is low. The low power on destination city size found in fitting the gravity model to the England and Wales dataset shows that connectivity of a city increases sub-linearly with population increase. When modelling spread of influenza in the US, Viboud et al. [5] found very comparable low values of the population exponents with the infectious city lower than the susceptible. Differing results come from the analysis of measles data in Great Britain with a power coefficient on infectious populations estimated at approximately 1.5 [18] . We found the best-fit model in England and Wales does not include the population size of the infector (origin) city, a result which needs further examination in future work. Differences in our population parameter estimates and those from studies on contemporary populations are likely to differ owing to changes in human mobility patterns since 1918. Our estimate for England and Wales that the infectivity of a city is sub-linearly related to mortality, suggests that the rate of death in a city is not as important to infectivity as the presence or absence of disease. Other studies have used constant infectivity terms for the analysis of human seasonal influenza [21] . However, our estimates do support some level of mortality dependence, suggesting that cities with a very high influenza burden, usually later in the epidemic wave, are more infectious than newly infected cities. In the US dataset, the best-fit model gave constant infectiousness, but again this may be due to a lack of power to estimate such parameters from the US dataset. It may also be caused by non-uniform infection pressure from cities not in the dataset, which could mask an infectivity relationship for the cities that are given.
Future possible extensions of this work include relaxing the assumption that all cities were equally susceptible at the start of the autumn wave of the pandemic. The variation in the onset of infection in cities may, in part, be due to the differing susceptibility of each city owing to differing attack rates experienced in the spring -summer wave, or population-level immunity from the 1890 pandemic or seasonal strains. However, the low case fatality of the first wave and age-specificity of infection between waves need to be understood before spatial heterogeneity in susceptibility can be discerned. There are varying reports on the magnitude and mechanism of the effect of infection during the first wave on attack rates in subsequent waves [24] [25] [26] [45] [46] [47] . Further analysis of the datasets considered here may provide an opportunity to disentangle these effects.  Charge-Surrounded Pockets and Electrostatic Interactions with Small Ions Modulate the Activity of Retroviral Fusion Proteins Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry. Bovine Leukemia Virus (BLV) and Human T-Cell Leukemia Virus Type-1 (HTLV-1) are related deltaretroviruses that cause aggressive lymphoproliferative disorders in a small percentage of infected hosts [1, 2, 3, 4, 5, 6] . Like other enveloped viruses, retroviruses must catalyse fusion of the viral and target cell membranes to promote entry of the viral capsid into the target cell. The retroviral class I fusion protein consists of the transmembrane glycoprotein (TM) component of the envelope glycoprotein complex [7] . Envelope is displayed on the surface of the virus or infected cell as a trimer, with three surface glycoprotein (SU) subunits linked by disulphide bonds to a spike of three TM subunits [8] . Experimentally validated models suggest that SU-mediated receptor engagement induces isomerisation of the inter-subunit disulphide bonds and initiates a cascade of conformational changes that activate the fusogenic properties of TM [9, 10] . Membrane fusion is achieved by re-folding of the TM from a native non-fusogenic structure through a rod-like pre-hairpin intermediate, in which the C-and N-terminal segments are embedded in the viral and target cell membranes respectively [7, 8] . The pre-hairpin intermediate then resolves to a trimer-of-hairpins structure, which pulls the membranes together and facilitates lipid mixing and membrane fusion [7, 8, 11, 12] . For several viruses membrane fusion is sensitive to inhibition by peptides that mimic a C-terminal region of the trimer-of-hairpins [13, 14, 15, 16, 17, 18, 19] . The C-terminal fragment of the HTLV-1 trimer-of-hairpins exhibits a short a-helical motif embedded in an extended non-helical peptide structure referred to as the leash and a-helical region (LHR) [20, 21] . The LHR-based mimetics are structurally distinct from the prototypic extensively a-helical peptide inhibitors of human immunodeficiency virus but are reminiscent of the leash regions observed in influenza haemagglutinin [20, 21, 22, 23] .
Importantly, amino acid residues that are required for potent inhibitory activity of the HTLV-1 and BLV peptides are not fully resolved in the available HTLV-1 TM structure, yet this information is critical to the development of therapeutically relevant peptide or low-molecular-weight inhibitors of HTLV-1 entry [17, 22] . Moreover, other class I fusion proteins have extended non-helical elements in the C-terminal region of the trimer-of-hairpins and understanding how these elements contribute to the leash in a groove mechanism of fusion protein function will have broad relevance to anti-viral therapies [23, 24] . We therefore sought to examine the structure and function of the BLV TM and to compare this information with data derived from diverse class I fusion proteins and the related HTLV-1 TM structure.
Here we show the structure of the post-fusion conformation of the BLV TM ectodomain and demonstrate that coordinated ions and a network of hydrogen-bonded water molecules make critical contributions to the assembly and stability of the trimer-of-hairpins form of the BLV TM and are essential for TM-mediated fusion. Additionally, we resolve a region of the LHR that is critical to the activity of peptide inhibitors of HTLV-1 and BLV entry. We provide evidence that basic residues in a membrane proximal helical element of the LHR interact with charged residues that surround an extended hydrophobic pocket on the coiled coil. This charge-surrounded pocket represents an attractive target for antiviral drugs. Our data indicate that coordinated ions and charge-surrounded hydrophobic pockets are functionally significant leitmotifs of class I fusion proteins.
The structure of the BLV TM ectodomain To obtain crystals for structural studies, the N-and C-terminal limits of the coiled coil region of BLV TM were identified using LearnCoil VMF [25] . The TM ectodomain, including the predicted coiled coil and LHR from Ala326 to Trp418, were fused to the C-terminal end of maltose binding protein via a threealanine spacer following the methodology of Center et al [26] . The soluble purified protein was crystallised and the structure solved to a resolution of 2.0 Å (Fig. 1 ). As anticipated, the overall fold of the BLV TM ectodomain is that of a trimer-of-hairpins, with the Nterminal a-helices twisting around each other to produce the central triple-stranded coiled coil that is characteristic of class I fusion proteins. Buried leucine and isoleucine residues within the coiled coil predominantly mediate the interactions between monomers, but strikingly there are two polar layers within the coiled coil that establish interactions with ordered water molecules and a chloride ion respectively (see below). At the base of the coiled coil the peptide backbone undergoes a 180u loop forming the chain reversal region (Fig. 1A, B) , within which is a short helical segment containing the first cysteine of the conserved CX 6 CC motif [9, 10] . The predicted disulphide between Cys384 and Cys391 is reduced in the resolved structure but this does not appear to affect the overall protein fold. In support of this view, preliminary lower resolution structures obtained for crystals formed in the absence of TCEP-HCL reveal an intact disulphide (data not shown). These data indicate that the disulphide is not essential for constraining the chain reversal region in the folded protein, even though it might play a role in the folding process. Therefore, the known defects in membrane fusion associated with substitution of these cysteines are likely due to direct perturbation of the inter-subunit disulphide isomerisation step of the envelopemediated membrane fusion process [9, 10] . After Cys391, the LHR begins with an extended non-helical leash followed by an eightresidue a-helix. This helix is followed by a three-residue linker incorporating a single proline, allowing a sharp kink in the LHR prior to the start of a second a-helix that adopts an orientation almost 90u to the first helical segment of the LHR (Fig. 1A , B, C).
A panel of mutants in BLV envelope are processed and expressed identically in vivo Guided by the crystal structure, a panel of mutants in BLV envelope were designed to perturb key features of the trimer-ofhairpins structure. A common difficulty with mutagenesis of viral envelopes, particularly the TM, is that at particular positions even conservative substitutions can dramatically impair the proteolytic processing and cell surface expression of envelope [27, 28] . Therefore, we compared each of the mutants with the parental ''wild-type'' envelope and confirmed appropriate expression, processing and cell surface presentation of the TM mutants. No significant differences in either the expression level of gp51 or the cleavage of the precursor protein, gp72, were observed ( Fig. 2A) . Moreover, flow cytometry analysis confirmed that in each case the mutant envelope was displayed on the cell surface at levels equivalent to wild type (Fig. 2B) . Subsequently, the envelopeexpressing cells were used as effector cells in syncytium formation assays, and the efficiency of each mutant envelope relative to wildtype in catalysing membrane fusion was calculated as the relative fusogenic index. The effect of each mutation on fusogenicity (Fig. 2C) , and the mutant phenotypes are interpreted with reference to the crystal structure below. Solvent molecules are critical for stable assembly of the core coiled-coil The trimerisation of the N-helices appears to be facilitated by presentation of aliphatic residues to the interacting faces of the TM monomers, thereby forming a hydrophobic core down the axis of the central coiled coil. Notably, in the BLV trimer-of-hairpins there are two positions along this interface that harbour polar residues, Thr353 and Asn367 (Fig. 3A, B ). Thr353 is located approximately half way up the N-helix, and it is oriented such that the methyl group of the side chain points toward the centre of the coiled coil and the hydroxyl group faces toward the neighbouring N-helix. In this position, Thr353 participates in a complex network of hydrogen bonding via several buried ordered water molecules. In particular, Thr353 makes a contact through one water molecule with the main-chain of Glu397 and through a separate water molecule to the side-chain of His354, one of the residues which forms the wall of the groove into which the LHR
Human T-cell leukaemia virus types-1 (HTLV-1) and bovine leukaemia virus (BLV) are divergent blood borne viruses that cause hematological malignancies in humans and cattle respectively. In common with other enveloped viruses, infection of cells by HTLV-1 and BLV is dependent on the membrane fusion properties of the viral envelope glycoproteins. Here we have solved the crystal structure of the BLV transmembrane glycoprotein, and, through a functional and comparative analysis with HTLV-1, we have identified features that are critical to fusion protein function. In particular, we demonstrate that electrostatic interactions with small ions dramatically stabilize the assembly and fusion-associated forms of the BLV TM, but are not required for the cell surface display of native prefusogenic envelope. Moreover, we show that charged residues that border a deep hydrophobic pocket contribute directly to appropriate folding of fusion-active envelope and are critical to membrane fusion. Importantly, the charged residues that border the pocket are key features that determine the specificity and activity of peptide inhibitors of envelope function. Our study demonstrates that charge-surrounded pockets and electrostatic interactions with small ions are significant leitmotifs of diverse class-1 fusion proteins and that these elements represent ideal targets for novel small-molecule inhibitors of viral entry. binds (Fig. 3A ). In the context of envelope the T353V substitution, which replaces threonine with a non-polar residue of equivalent size, completely abrogates envelope-mediated membrane fusion (Fig. 2C) . Moreover, the introduction of the T353V substitution into the pMBP-BLVhairpin vector yields a recombinant protein that completely fails to trimerise (Fig. 3E) . Interestingly, the data demonstrate that substitutions at Thr353 do not affect expression, processing, or surface expression of native envelope but severely impair the fusogenic activity of envelope by preventing assembly of the trimer-of-hairpins. Our data therefore provides further evidence that the trimeric coiled coil likely forms during the fusion process.
A spherical density feature was observed on the central axis of the molecule; comparison of refined temperature factors with those of the surrounding protein atoms suggests an entity more electron-dense than water. Based on the chemical environment, the shape of the density and B factor comparisons we have modeled this feature as a chloride ion. This chloride ion is situated towards the chain reversal end of the coiled coil between a group of three asparagines, one from each monomer of TM (Asn367). These asparagine residues establish electrostatic interactions with the chloride ion by creating a slightly positively charged microenvironment between the N-helices. An alignment of envelope sequences from diverse viral groups (Fig. 3C ) reveals that this asparagine is conserved. Moreover, comparison of the crystal structures for the fusion protein ectodomains of HTLV-1 [20] , MoMLV [29] , Ebola [24, 30] , and Syncytin-1 [31] , a protein derived from a human endogenous retrovirus that plays a critical role in the implantation of the trophoblast into the wall of the uterus [32] , show that the ability to coordinate chloride is retained (Fig. 3D) . To test the importance of the chloride interacting asparagines, we introduced amino acid substitutions of similar size and geometry. Introducing a leucine residue at position 367 of BLV TM renders envelope entirely non-fusogenic. The N367L substitution does not completely prevent trimerisation of the BLV coiled coil in vitro but it significantly destabilises the trimer and a large broad peak corresponding to higher order oligomers and aggregated material is observed by gel filtration chromatography (Fig. 3E ). Interestingly, a substitution whereby Asn367 is replaced by an aspartic acid residue produces a decrease in fusogenicity of almost 90% (Fig. 2C ). The N367D substitution replaces an amide with a carboxylate group in the centre of the coiled coil, which inverts the surface charge of the binding pocket, and therefore would not support interaction with a chloride. In contrast to N367L, the N367D mutation should maintain the hydrogen bonding in and around the chain-reversal region mediated by the carbonyl group that is common to both asparagine and aspartic acid, and therefore the disruption of fusion should be due primarily to the absence of the chloride ion, albeit we cannot exclude the possibility that the close proximity of negatively charged aspartate side chains impairs trimerisation. However, our biological assays demonstrate that the N367D substitution, unlike N367L, retains some membrane fusion activity. In vitro, rather than producing a range of higher order species, the introduction of the N367D mutation into pMBP-BLVhairpin produces a recombinant protein with a major peak at an elution volume consistent with the molecular weight of a tetramer rather than a trimer (Fig. 3E ). Tetramerisation is unlikely to be the reason for the compromised fusogenic activity of the N367D-envelope; nonetheless, the results demonstrate the importance of the chloride-interacting asparagines to the stability of the trimeric coiled coil.
Along the length of the LHR multiple contacts are made with the coiled coil and such interactions are required for the activity of inhibitory LHR-mimetic peptides [14, 17, 21, 22] . Amino acid residues that are critical determinants of peptide potency map to the C-terminal region of the LHR [21, 22] , but key residues and the manner in which they interact with the coiled coil are not resolved in the published HTLV-1 TM structure [20] . A detailed view of the BLV LHR bound to the coiled coil and a sequence comparison of the HTLV-1 and BLV LHRs are shown in Fig. 1C and D. Towards the N-terminus of the BLV LHR, conserved residues Leu394 and Ile396 dock into a hydrophobic pocket on the coiled coil, and an array of polar side chains at one side of the pocket interact with the LHR peptide backbone (Fig. 1C) . We have previously demonstrated that interactions with the peptide backbone contribute to the activity of the BLV and HTLV-1 peptide inhibitors [17, 22] . By contrast, the side chain of Arg395 projects out into solvent suggesting that this residue is not essential for interaction of the LHR with the coiled coil or for trimer-ofhairpins formation (Fig. 1C ). In keeping with this view, the R395A substitution has no significant effect on envelope fusogenicity (Fig. 2C) and therefore serves as a useful control for our analysis of envelope structure and function. As the N-helices twist around one another the groove between them accommodates the first conserved a-helical segment of the LHR. Within this a-helix, BLV residues Ile401 and Leu404 extend down and pack into the groove (Fig. 1C) . Significantly, substitution of Leu404 with alanine results in an 80% loss of fusogenic activity relative to wild type (Fig. 2C) , indicating that this interaction contributes substantially to the stability of the LHR/coiled coil interaction.
The first a-helix of the BLV LHR ends at Asp406, and between the end of this helix and the beginning of the next is a threeresidue linker, comprised of Leu407, Gln408 and Pro409. Although Leu407 is positioned such that it faces the groove between N-helices, a ridge across this groove prevents Leu407 from docking particularly deeply (Fig. 1C ). This Leucine is not conserved among leukaemia viruses. Instead, there is an Arginine residue at this position of the HTLV-1 LHR that makes contact with the coiled coil. Such differences may account, in part, for the specificity and significant differences in potency that are observed for the BLV and HTLV-1 peptide inhibitors [17] . The conserved proline (BLV Pro409) of the extended non-helical linker induces a sharp kink in the LHR thereby allowing a second a-helix to bind almost directly across the groove, at right angles to the rest of the LHR. Crucially, this second helix is not resolved in the published HTLV-1 TM structure [20] . At the point at which this change of direction occurs, a conserved leucine, Leu410, docks into the start of a deep hydrophobic pocket. Adjacent to Leu410 in this pocket and within the second LHR helix is Val414, which is also conserved in HTLV-1 (Fig. 1C, D) . Substitution of either Leu410 or Val414 with alanine markedly impairs the fusogenic function of envelope (Fig. 2C) . The substitution of L410A is somewhat more detrimental than substitution of Val414, with an activity loss of 75% and 65% respectively.
Embedded within the second helical element of the LHR is an arginine (Arg413) that participates in electrostatic interactions with the coiled coil (Fig. 1C ). This residue is conserved in HTLV-1 and is critical to the inhibitory activity of the HTLV-1 LHR mimetic peptide P cr -400 [22] . Intriguingly, Arg413 projects back along the axis of the coiled coil and binds between Leu407 and Leu410, where it forms hydrogen bonds with Gln343 from the same Nhelix and Asp342 from an adjacent N-helix and also donates a hydrogen bond, through the e-nitrogen, to the main chain carbonyl of Glu408 of the LHR (Fig. 1C) . The substitution of Arg413 with alanine dramatically disrupts the fusogenic activity of envelope and reduces envelope function by more than 90% relative to wild type (Fig. 2C) . Moreover, Arg413 cannot be functionally replaced by lysine. The R413K mutant, while more fusogenic than the alanine substituted derivative, is still 85% less effective at catalysing membrane fusion than wild-type envelope (Fig. 2C) . These data indicate that the electrostatic and hydrogen bonding interactions made by Arg413 are essential to the envelope-mediated membrane fusion process.
Contrasting effects of two arginine residues on the stability of the trimer-of-hairpins Based on homology modelling, we previously suggested that Arg403 of the LHR projects out from one side of the a-helix and is repelled electrostatically by N-helix residue Arg345 [17] . This prediction is substantiated by the crystal structure presented here (Fig. 1C) . Moreover, the R403A substitution yields an envelope that produces extensive syncytia and is significantly more fusogenic than wild-type envelope (Fig. 2C and Fig. 4C) . Thus, the substitutions R403A and R413A have very different effects on the fusogenicity of BLV envelope, increasing activity by over two-fold and almost completely abolishing activity respectively (Fig. 2C  and 4C ). Using a modified thermal aggregation assay [33] , we assessed the relative effects of these two substitutions on the thermostability of the BLV trimer-of-hairpins. Control experiments revealed that after a five minute incubation at 40uC, on average 53% of the wild-type MBP-BLV-hairpin protein was present in high-order aggregates (Fig. 4A, B) . However, when the same heat treatment was applied to the protein bearing the R403A mutation, we found that only 37% of protein aggregated. The difference to wild-type hairpin was significant (P#0.005, t-test) (Fig. 4A, B) . By contrast, the R413A substitution resulted in aggregation of over 70% of total protein, again a significant difference to wild type (P#0.005, t-test) ( Fig. 4A and B) . Notably, even without heat treatment over 50% of the R413A protein was aggregated (Fig. 4A) . Furthermore, these relative differences in the propensity of the recombinant proteins to aggregate were maintained following heat treatment at 50 and 60uC (Fig. 4B) . Hence, the contrasting effects of the arginine substitutions on envelope fusogenicity are directly due to changes in stability of the post-fusion trimer-of-hairpins structure.
A prediction based on the increase in thermal stability of the R403A substituted trimer-of-hairpins is that such substitutions should confer reduced sensitivity to LHR-mimetic peptide inhibitors. To test this view we compared the activity of the P BLV -391 antagonist of envelope fusion [17] against wild type or R403Asubstituted envelope. This peptide mimics residues Cys391 to Gln419 of BLV Env. In syncytium interference assays, P BLV -391 inhibited fusion catalysed by wild-type envelope with an IC 50 of 3.1760.09 mM (Fig. 4D ). However, P BLV -391 only inhibited fusion catalysed by R403A envelope with an IC 50 estimated to be .9 mM, and even at peptide concentrations up to 15 mM inhibition of syncytium formation was markedly damped indicating that both the potency and efficacy of the peptide was reduced against R403A substituted envelope (Fig. 4D) . Moreover, though more active against native envelope, a reciprocally substituted peptide antagonist, P BLV -R403A, failed to exhibit full potency against the R403 envelope derivative; P BLV -R403A inhibited wild-type envelope catalysed membrane fusion with an IC 50 of 1.360.1 mM, but inhibited the R403A envelope with an IC 50 of .6 mM (Fig. 4E) . In both experiments, C34 (a peptide mimetic of HIV-1 gp41 residues Gly627 to Leu661) was used as a negative control. Thus, improving the thermal stability of the trimer-of-hairpins form of a class-1 fusion protein significantly reduces the sensitivity of envelope to peptide inhibitors targeted to the coiled coil.
The structure of the C-terminal segment of the BLV LHR and the accumulated data suggest a key role for the conserved arginine (Arg413) in the mechanism of envelope-mediated membrane fusion. Moreover, our recent data indicate that the equivalent arginine residue, Arg422, of the HTLV-1 LHR-mimetic peptide is critical to inhibitory activity [22] . Notably, for the HTLV-1 LHRbased inhibitor residues equivalent to Leu413, Arg416 and Leu419 are also of importance to the biological activity of the peptide [21, 22] . We therefore sought to establish whether or not the conserved arginine in the HTLV-1 LHR docks with the coiled coil in a similar manner to that of BLV and if this residue is important to membrane fusion mediated by the HTLV-1 TM. We generated a small panel of mutants in HTLV-1 envelope, substituting Arg416 of the LHR with alanine and lysine (R416A and R416K), and making similar substitutions for Arg422 (R422A and R422K). Using a monoclonal antibody recognising HTLV-1 SU, we confirmed by Western blotting and flow cytometry that all four mutant envelopes were expressed and post-translationally processed in a manner identical to wild type (Fig. 5A, B) . In syncytium formation assays, the individual alanine substitutions of Arg416 and Arg422 resulted in envelopes that are 91% and 97% less fusogenic than wild-type envelope respectively (Fig. 5C) . Significantly, the lysine substitution of Arg416 produced an envelope that was not significantly less fusogenic than wild type (P.0.05, t-test) (Fig. 5C ). However, Arg422 could not be functionally replaced with lysine, the R422K mutation resulted in an 88% reduction in fusogenic activity. Taken together with the data from the BLV R413K mutant, this suggests that HTLV-1 R422 adopts a near-identical conformation to BLV R413 at the Cterminal segment of the LHR.
The likely conservation of position and orientation of HTLV-1 R422 in the trimer-of-hairpins allowed us to construct a model for the binding of the C-terminal segment of the HTLV-1 LHR to the coiled coil (Fig. 5D) . The model suggests that R422 could bind to a negatively charged ridge that is orientated across the groove between N-helices and that lies between the binding pockets for Leu413 and Leu419 and that R422 docks adjacent to R416. As such, four critical interactions between LHR side chains and the coiled coil are contained within a binding hotspot of ,16 Å x ,8 Å and disruption of these interactions profoundly impairs envelope-mediated membrane fusion.
There is greater sequence divergence between BLV and HTLV-1 than, for example, between SIV and HIV-1 [34, 35] , nonetheless the crystal structures of the BLV and HTLV-1 trimerof-hairpins are similar. The incorporation of ordered water molecules and ions to facilitate trimerisation of the coiled coil and assembly of the trimer-of-hairpins is seen in several viral fusion proteins [20, 29, 30, 36] . The crystal structure of SIV gp41 implicates multiple water molecules in the folding of the trimerof-hairpins [37] . However, the number of water molecules concentrated in one region of the BLV coiled coil is unusual. The interaction of Thr353 with an array of water molecules appears to play a critical role in assembly of the coiled coil, as substitution of T353 eliminates both in vitro trimerisation and in vivo fusogenicity. Notably, for HTLV-1 the threonine residue is replaced by an asparagine, which by virtue of its larger side chain directly hydrogen bonds with the adjacent N-helix. By contrast, Thr353 of BLV makes this contact through water-mediated hydrogen bonds. However, the contribution of polar interactions to coiled coil assembly is maintained at this location in HTLV-1 by interaction of a chloride ion on the symmetry axis of the coiled coil. Moreover, for both viruses three conserved asparagine Fusogenicity of HTLV-1 C-terminal LHR arginine mutants. The fusogenic index was calculated as described (mean 6SD from triplicate assays). (D) A ''hotspot'' for binding of HTLV-1 fusion inhibitors based on the previously published HTLV-1 structure and modelled incorporating the data from this study. Four key residues (Leu413, Arg416, Leu419 and Arg422, shown as yellow sticks) interact with the HTLV-1 coiled coil (grey space-filling model, region interacting with LHR residues shown in red) within a compact binding site. The contact made by Arg422 is critical to the binding and functional activity of HTLV-1 peptide inhibitors. doi:10.1371/journal.ppat.1001268.g005 residues located towards the membrane distal end of the coiled coil interact with a chloride ion. The properties of the N367L substitution and the failure of N367D to rescue envelope function indicate that the observed chloride-mediated interactions are essential for stable assembly of the coiled coil and for TMmediated membrane fusion. Significantly, the asparagine (Asn367) and adjacent arginine (Arg368) residues of BLV are conserved between BLV, HTLV-1 and a number of disparate viral fusion proteins, and the ability of the asparagines to interact with a chloride ion is maintained. Critically, the chloride ion is present within the post-fusion conformation of Ebola GP2 (30), but is conspicuously absent in a pre-fusion structure resolved by Lee et al [38] wherein the conserved asparagines adopt a more open arrangement and the coiled coil is only partially trimerised. We suggest that Asn367, as a direct consequence of conformational changes in SU resultant from receptor binding, and in concert with the equivalent asparagines from neighbouring monomers, mediates electrostatic interactions with a chloride ion, which brings the N-helices together, thus driving the trimerisation of the coiled coil to yield the pre-hairpin intermediate in an event pivotal to successful fusion. In addition, our results indicate that trimerisation of the N-terminus of TM is not a requirement for Env maturation and surface display of trimeric pre-fusogenic envelope, offering a novel insight into the conformation of retroviral Env spikes prior to activation. Taken together, the data suggest that while the ''knobs-into-holes'' interactions of aliphatic residues at the centre of the coiled-coil regions of class 1 viral fusion proteins are important for trimerisation of the fusion-active coiled coil the polar layers among these hydrophobic interactions are critical. This information should be considered when designing trimeric immunogens based on the coiled coil of TM. Moreover, the structure in and around the chloride-interacting region has been conserved in diverse viral fusion proteins and may be susceptible to coiled coil destabilising drugs.
It is interesting to note that removal of a steric clash between R403 and the BLV coiled coil yields an envelope that is more fusogenic than wild type. For HTLV-1 an isoleucine, at a position equivalent to residue Arg403 in the BLV LHR, also appears to make a steric clash with the coiled coil and an alanine substitution of this residue in the context of an LHR-based peptide inhibitor yields a peptide with improved coiled coil-binding properties and increased inhibitory activity [21] . It is therefore clear that viral envelope has not evolved to optimise LHR binding to the coiled coil or to maximise fusogenicity. One plausible explanation is that membrane fusion needs to be kept in check. An overly fusogenic envelope may induce rampant membrane fusion and syncytium formation leading to cell death by apoptosis and thereby provide additional stimuli for a robust anti-viral immune response. Clearly, sub-optimal LHR/coiled-coil interactions have been maintained during viral evolution and suggest that the development of compounds that bind to the coiled coil with higher affinity than the LHR may be an achievable anti-viral strategy.
The most notable difference between the HTLV-1 and BLV TM structures is the resolution of a second helical motif within the LHR of the trimer-of-hairpins. Residues that form the start of the second a-helix are conserved in HTLV-1 and are critical to envelope-catalysed membrane fusion and to the inhibitory activity of LHR-mimetic peptides [21, 22] . Just three residues from the end of our structure is the first tryptophan of a conserved tryptophanrich membrane-proximal region (MPR). The MPR is believed to interact with the opposing membranes during the fusion process and substitution of the aromatic residues severely compromises, but does not abolish, membrane fusion [39] . The conformation of the MPR has received considerable attention because broadly neutralising anti-HIV-1 antibodies bind epitopes contained within the MPR [40, 41] . It is likely that the HIV MPR adopts a helical structure when interacting with membranes and the coiled coil during fusion [40, 42] . Examination of the BLV envelope sequence and TM structure suggests that the second helical segment of the LHR is likely to continue and that in the fusion-activated state the tryptophan-rich MPR is also helical and inserted into the membrane at an oblique angle. For the leukaemia viruses, the conserved arginine (Arg413 in BLV), assists in defining the orientation of the MPR, as the interactions of Arg413 with the coiled coil and the LHR backbone propagate a sharp kink in the LHR towards the membrane-proximal end of the coiled coil. A lysine substitution of Arg413 retains only ,10% of the fusogenic activity of wild-type envelope lending further support to this view.
The BLV crystal structure and our analysis of HTLV-1 and BLV envelope-mediated membrane fusion reveal an important role for electrostatic interactions in binding of the LHR to the grooves of the coiled coil. For both BLV and HTLV-1 a conserved basic residue in the second helical segment of the LHR interacts with the charged rim of a deep pocket lined by non-polar residues. For HTLV-1 the contribution of charge is enhanced by the interaction of an additional basic residue (Arg416) with the opposite rim of the pocket. Thus, while the insertion of non-polar residues into hydrophobic pockets likely drives the docking of the LHR with the coiled coil, we suggest that it is the electrostatic attraction and hydrogen bonding of charged side chains around these hydrophobic interactions that serves to ''cement'' the LHR in place and thereby establish the precise geometry and affinity of this interaction. In keeping with this view, substitution of these basic residues ablates envelope-mediated membrane fusion and dramatically impairs the inhibitory and coiled-coil-binding properties of LHR-based peptides [22] . Moreover, only one of these important basic residues is conserved in BLV compared to HTLV-1 and it is notable that the synthetic BLV LHR-mimetic is approximately 10-fold less potent than the corresponding inhibitor of HTLV-1 [17] . Examination of the electrostatic surfaces of BLV, HTLV, HIV and MoMLV coiled coils reveals, in each case, the presence of a pocket surrounded by charged residues, though the polarity of the charge and positioning along the groove is variable (Fig. 6 ). The importance of the pocket and surrounding charge to the assembly of the trimer-of-hairpins and the potency of C-helixbased mimetic peptides has been demonstrated for HIV [43, 44] , and taken with our findings for BLV and HTLV-1 suggest that such charged regions are critical to retroviral envelope-mediated membrane fusion in general. We suggest that structure-assisted design of small molecules to target these charge-surrounded pockets is a viable objective for anti-retroviral therapy.
We expect that the data produced in this study will be pivotal to the development of more drug-like inhibitors of HTLV-1 envelope catalysed membrane fusion, and thereby provide therapeutic antiviral agents for adult T-cell leukaemia and HTLV-associated myelopathy/tropical spastic paraparesis, diseases for which there are currently no effective treatments [45, 46] .
The plasmids pCMV-BLVenv-RRE and pRSV-Rev have been described [17, 47] . To construct pCMV-HTLVenv-RRE the BLV env sequences in pCMV-BLVenv-RRE were replaced by a HTLV-1 env coding region amplified by PCR from pHTE-1 [48] . To construct the plasmid pMAL-gp30hairpin (MBP-BLV-hairpin), a modified fragment of the MBP open reading frame from pMAL-c2 (New England Biolabs) with a 59 BglII site and a 39 PvuII site replacing a SacI site was PCR amplified. A fragment of BLV env encoding amino acid residues 326 to 418 was PCR amplified with a 59 PvuII site and a 39 PstI site. The PCR fragments were ligated into a pMAL-c2 backbone digested with BglII and PstI. The Quikchange mutagenesis kit (Stratagene) was used to introduce point mutations into pCMV-BLVenv-RRE and pMAL-gp30hairpin following the manufacturer's instructions.
Expression of the MBP fusion protein was carried out as previously described [14, 49] . Briefly, Escherichia coli BL21(DE3) cells transformed with the MBP-BLV-hairpin vector were grown at 37uC with vigorous shaking in LB supplemented with 10 mM glucose and 100 mgml 21 ampicillin until an optical density at 600 nm of ,0.6 was reached. Protein expression was induced with Isopropylthio-b-D-galactoside (IPTG) at a final concentration of 0.5 mM for 4 h at 37uC. Cells were harvested by centrifugation and the pellet was resuspended in column buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 mgml 21 aprotinin) and frozen O/N at 220uC. The cell suspension was thawed and the cells lysed by sonication. Cell debris was pelleted by centrifugation at 9,0006g for 30 min. The crude lysate was diluted 1:5 in column buffer and loaded onto an amylose column pre-equilibrated with column buffer. The column was then washed with 15 column volumes of column buffer, and the bound protein was eluted with column buffer supplemented with 10 mM maltose.
The trimeric recombinant MBP-BLV-hairpin protein was purified by Superdex 75 gel filtration, and the required fractions pooled and concentrated to 12 mg ml 21 . Tris [2-carboxyethyl]phosphine hydrochloride (TCEP-HCl) was then added to a final concentration of 5 mM. Protein concentration was estimated by absorbance measured at 280 nm. Crystallization conditions were identified using the sitting drop vapour diffusion technique, with crystal screens from Hampton Research and Emerald Biostructures. Optimal crystallization was achieved by streak seeding with a reservoir solution composed of 24.5% (v/v) isopropanol, 13.5% (v/v) PEG-4000 and 0.1 M sodium citrate pH 5.6, and optimal diffraction was achieved using a cryoprotectant composed of mother liquor supplemented with 20% (v/v) glycerol. Diffraction data was collected at beamline BM14 at the European Synchrotron Radiation Facility (ESRF) in Grenoble. The structure was solved by molecular replacement using a truncated model of MBP-HTLV Hairpin (PDB ID 1MG1). Refinement proceeded through cycles of model building using Coot and O [50, 51] and refinement using REFMAC5 [52] . Data and refinement statistics are given in Table 1 Peptides Peptides were synthesised using standard solid-phase Fmoc chemistry and unless stated otherwise have acetylated N-termini and amidated C-termini. The peptides were purified by reversephase high-pressure liquid chromatography and verified for purity by MALDI-TOF mass spectrometry. All peptides were dissolved in dimethyl sulfoxide (DMSO), the concentration of peptide stock solutions was confirmed by absorbance at 280 nm in 6M guanidine hydrochloride and peptides were used at the final concentrations indicated.
HeLa cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% foetal bovine serum (FBS). For syncytium formation assays, HeLa cells were transfected with equal quantities of pCMV-BLVenv-RRE (or empty pcDNA 3.1 for mock samples) and pRSV-Rev using the GeneJuice transfection reagent (Novagen). After 24 h, 3610 5 transfected cells were added to 7.0610 5 non-transfected HeLa target cells in 6-well dishes (Nunc), and cocultured for 16 hrs, in the presence of peptides where appropriate. The cells were washed with phosphatebuffered saline pH 7.4 (PBS), fixed using 3% paraformaldehyde in PBS, and stained with Giemsa. Assays were performed in triplicate. To calculate fusogenic indices, the fraction of nuclei contained within syncytia in a 20x light microscope field was expressed as a percentage of the total number of nuclei within the field.
Western blotting was carried out using standard methods; bmercaptoethanol was used in sample preparation. BLV and HTLV-1 envelope was detected using supernatant from murine hybridoma cell lines expressing monoclonal antibodies raised against recombinant antigen derived from BLV or HTLV-1 envelope, and anti-mouse horseradish peroxidase conjugated secondary antibody at a dilution of 1:10,000 in PBS containing 5% (w/v) marvel and 0.025% Triton X-100.
To detect surface-expressed protein, cells transfected with the appropriate envelope expression vector were detached from culture flasks using PBS +2 mM EDTA, and washed twice with PBS. 5.0610 5 cells were incubated with agitation for 1 hr at room temperature in 1 ml DMEM +10% FBS containing 15 mgml 21 immunoglobulin purified from the BLV anti-Env antibodyexpressing hybridoma supernatant, or 150 ml supernatant from the HTLV anti-Env antibody expressing hybridoma. Cells were washed twice with PBS and incubated in the dark at room temperature for 45 mins in DMEM containing anti-mouse FITC (Sigma) at 1/1,000 dilution. Cells were washed once with PBS and once with PBS +0.1% sodium azide before fixing with PBS +0.5% paraformaldehyde. Bound fluorescence was detected using a FACScan flow cytometer (Becton Dickinson).
The oligomerisation states of MBP-BLV-hairpin and mutant derivatives were examined by gel filtration using a Superdex 200 column equilibrated with MBP elution buffer. Fractions containing the trimeric species were retained. To assess the thermostability of the trimers, samples from these fractions were re-run over the column either without heat treatment or with heat treatment at the temperature specified for 5 min, followed by cooling for 2 min on ice. The areas under the peaks observed were calculated, and the area of the peak corresponding to the aggregate was expressed as a percentage of the total area under both this peak and the peak corresponding to the trimer.  Tracheostomy and mechanical ventilation weaning in children affected by respiratory virus according to a weaning protocol in a pediatric intensive care unit in Argentina: an observational restrospective trial We describe difficult weaning after prolonged mechanical ventilation in three tracheostomized children affected by respiratory virus infection. Although the spontaneous breathing trials were successful, the patients failed all extubations. Therefore a tracheostomy was performed and the weaning plan was begun. The strategy for weaning was the decrease of ventilation support combining pressure control ventilation (PCV) with increasing periods of continuous positive airway pressure + pressure support ventilation (CPAP + PSV) and then CPAP + PSV with increasing intervals of T-piece. They presented acute respiratory distress syndrome on admission with high requirements of mechanical ventilation (MV). Intervening factors in the capabilities and loads of the respiratory system were considered and optimized. The average MV time was 69 days and weaning time 31 days. We report satisfactory results within the context of a directed weaning protocol. During winter in 2009, Argentina as well as several other countries in the world, was affected by Influenza A H1N1 pandemic [1] .
Our Pediatric Intensive Care Unit (PICU) was the referral center for this pandemic in the province of Buenos Aires.
Between June 1 and July 31 2009 113 patients were admitted in our PICU, 79 of them with respiratory pathology. Twenty presented positive PCR (polymerase chain reaction) by Influenza A H1N1and 22 presented positive IFI (indirect immunofluorescence) by RSV (respiratory syncitial virus). Out of the 69 patients who survived 3 presented difficult weaning and needed tracheostomy.
This paper describes the clinical behavior and presentation of these 3 patients who were admitted into our PICU in that period and who had a prolonged stay due to respiratory complications after viral infection (Table 1) .
We report the results obtained according to a directed weaning protocol.
Below, we describe each patient's evolution before tracheostomy was performed and before the weaning plan was begun.
A six-month-old male patient was admitted in our PICU after intubation owing to respiratory insufficiency due to bilateral pneumonia. Through nasopharyngeal secretions a positive PCR by Influenza A H1N1 virus was found. He was a 28-week preterm infant with a birth weight of 900 grams. He had bronchopulmonary dysplasia (BPD) because of mechanical ventilation during 2 months in the perinatal age. He also presented some malformations compatible with a genetic syndrome which was still being studied before admission (retromicrognathia, abdominal angioma in the medial line, low implantation of the ears, among others).
On admission the patient presented severe acute respiratory distress syndrome (ARDS) for which he required high MV parameters ( Figure 1 ). He presented serious refractory hypoxemia and remained 36 days on MV. Using a low tidal volume ventilation protocol we were not able to achieve an adequate minimal oxygenation. So, as we do not count on high frequency oscillatory ventilation (HFOV) because of certain socioeconomic limitations within our hospital, we resorted to standard modalities, reaching mean airway pressure (Paw) of 15. 2 Extubation failed by respiratory insufficiency in 2 opportunities requiring reintubation in the first 24 hours. In both opportunities the patient had passed a spontaneous breathing trial (SBT).
A respiratory endoscopy was performed which showed an inflammatory pathology of the upper airway (UA).
These findings together with presenting prolonged MV (36 days) led to the decision of carrying out a tracheostomy and of beginning a weaning plan.
A six-month old male patient born in due term with adequate weight, previously healthy. He was referred to our PICU for presenting bronchiolitis by co-infection with RSV and A H1N1 virus.
He presented severe ARDS and hypoxemia and required high MV parameters remaining ventilated for 51 days (Figure 2 ). Three SBTs were performed during his evolution with 3 elective extubations, failing in all cases. He also presented failure during disconnection from positive pressure with non-invasive positive pressure ventilation (NPPV) due to respiratory insufficiency.
A respiratory endoscopy was performed which showed an important edema of the larynx crown and posterior synechia due to which tracheostomy was performed on the 44 th day of hospitalization. Then, the weaning plan was started.
A female patient of 1 month and 20 days of age, born in due term with adequate weight, with no perinatologic history. She was admitted with low acute respiratory failure following pneumonia in the right superior lobe. Co-infection with RSV and Influenza A H1N1 virus was found. During her prolonged stay in PICU (111 days) she required MV due to ARDS with severe hypoxemia ( Figure 3 ). In five opportunities she passed an SBT but in all cases re-intubation was required within 48 hs owing to low respiratory involvement. Also, repeated presence of condensation in the right superior lobe was observed. Due to everything mentioned above, a respiratory endoscopy was performed, reporting a right anomalous tracheal bronchus and mild subglottic edema. Given the fact that the patient had several respiratory infections which led to an obstruction of the segment corresponding to the anomalous bronchus, an exeresis of the right anomalous tracheal bronchus and a segmentectomy of the right superior lobe were performed.
After 51 days on MV a tracheostomy was performed and respiratory weaning was started. Disconnection of positive pressure was achieved after 98 days.
The 3 patients described coincided in positive PCR for Influenza A virus (2 of them with co-infection with RSV), prolonged MV, ARDS with severe hypoxemia and successful SBTs with failed extubations during the course of 48 hours.
Those patients presented upper airway pathology by pathologic endoscopy and lower airway pathology (post viral sequelae). These two factors affected their weaning from MV.
In our PICU upon meeting the following criteria we initiate SBT based on current consensus: resolution of the basal cause of respiratory failure, peak inspiratory pressure (PIP) < 25 cm H 2 O, positive end-expiratory pressure (PEEP) < 5 cm H 2 O, tidal volume 6-8 ml/kg, FIo 2 < 0.40, intermittent and decreasing sedation, patient alert, hemoglobin > 10 g/dl, and decreasing vasoactives [2] [3] [4] .
In our PICU SBTs are performed with a T-piece and supervised by respiratory therapists. The variables to be considered are registered on charts that we have developed for that purpose and which are filled in at the start of the test and every 15 minutes during the two hours the test lasts.
It is PICU's policy to perform respiratory endoscopies in those children who present two or more failed extubations.
In all three cases, because of presenting a pathologic endoscopy the decision of performing a tracheostomy was discussed and accepted by the patients' parents. All of them were trained in the handling, hygiene and comfort of the tracheostomy.
For the patients we presented, a strategy was designed with the objective of achieving disconnection from positive pressure by means of an orderly and progressive protocol.
In the first place we measured the maximum tolerated time on CPAP in each patient. We have defined maximum time as the longest period the patient is able to remain on CPAP without altering his vital signs in +/-20%. This determination was carried out comparing the The mean value of the maximum tolerated time on CPAP was 4 hours (3-6 hours).
Out of the maximum time reached, between 50 and 75% was used for the respiratory training of the patient.
The objective was to train the patient without exposing him to fatigue and muscular tiredness due to the decrease in his respiratory capacitance. This allowed to program rest periods which became less frequent as training progressed. This plan was carried out during day time while during the night, sleeping time was respected supplying MV in PCV [5] .
A gradual diminution of the ventilatory support, combining PCV with increasing periods on CPAP + PSV, was performed. Once the patient managed to remain 12 hours on CPAP + PSV, this modality was alternated with T-piece whose duration was progressively incremented [6] (Figure 4) .
The objectives of the programed tasks for the day were described in a chart in which the physiological parameters found during the training period were constantly recorded: respiratory rate, heart rate, blood pressure, oxygen saturation, maximal inspiratory pressure (PIMAX), maximal expiratory pressure (PEMAX), ventilatory mechanics, use of accessory muscles, comfort index and rapid shallow breathing index (respiratory rate/tidal volume per kg of weight) [2] .
For the measurement of PIMAX and PEMAX a Marshalltown ™ manual vacuum meter was connected to the endotracheal tube. We selected the most negative and positive value of 3 efforts during spontaneous breathing over a period of 20 seconds occluding the airway with a one-way valve [2, 3] .
In case the patient presented signs of muscular fatigue which kept us from accomplishing the programmed task we returned to the work plan of the previous day (when he tolerated the exercise adequately) and he was reassessed the following morning with the same parameters.
With this muscular training, nutritional support and the supply of vitamins and minerals, an improvement of the respiratory mechanics was observed, focusing on the measured parameters managing to effectively disconnect patients from positive pressure [7] . This objective was considered as accomplished when the patient remained on a T-piece over a period of 48 hs.
The average MV time was 69 days. The average weaning time was 31.
Finally, the 3 patients were referred to a general pediatric ward, two of them with supplementary oxygen.
Children with severe respiratory disease due respiratory virus presented a high percentage of ARDS on admittance to PICU with severe hypoxemia, requiring high MV parameters. Among the surviving patients, a torpid evolution with long periods on MV and difficulties for its disconnection were observed [8] .
Unfortunately, up to date there aren't available protocols based on evidence to guide a difficult weaning process in tracheostomized children. The bibliography on adults is ample, but not always applicable to our infant population due to anatomic, functional and cognitiveaffective reasons [9] .
Due to the functional anatomic differences between children's and adults' air way, the ideal time to indicate tracheostomy after a prolonged period on MV is still being discussed [10] . This is why, in the bibliography, more prolonged periods on MV through an endotracheal tube (ETT) are found in children than in adults.
Tracheostomy facilitates weaning with respect to ETT because it produces: dead space reduction, less airway resistance, decreased breathing work, better secretion removal with suctioning, less likelihood of tube obstruction, improved patient comfort, less need for sedation and better glottic function with less risk of aspiration [5, 6, 11] .
The term weaning refers to the transition from ventilatory support to completely spontaneous breathing, during which time the patient assumes the responsibility for effective gas exchange while positive pressure support is withdrawn [12] .
Weaning consumes between 40 and 60% of mechanical ventilation time. About 25% of ventilated patients present difficulties in the disconnection from the ventilator, being unable to be disconnected by means of SBT, requiring a progressive procedure which can take several days, weeks or even months [13] . These patients would benefit from a directed weaning protocol.
Ventilatory failure may be generated by a deterioration of the neuromuscular capacity or by increasing the loads of the respiratory system. The decrease of the capacity may be owed to a depression of the respiratory center (whether by sedation, alkalosis or encephalic lesion), neuropathies (polyneuropathy of the critical patient), muscular disorders (malnutrition, hyperinsuflation, electrolyte disorders and the use of muscle relaxants and corticoids) and chest wall abnormality (unstable chest, post-operative pain). The increase in the load may be due to the high ventilation requirements (sepsis, anxiety and pain), resistive loads (bronchospasm, secretions), elastic loads (auto PEEP, decrease in compliance) and related to the ventilator and the endotracheal tube (valves, small tubes) [6, 12] .
All these factors were considered and optimized in our patients before proceeding to their extubation.
Among the elements we count on, we opted for the use of the described combination of ventilatory modalities as part of an orderly and progressive protocol.
This permitted the favoring of the muscular training, the improvement of the patient-ventilator interaction and therefore the enhancement of the respiratory work.
The use of PCV enabled us to ensure total ventilation during night rest, eliminating energetic waste used in training which allowed the recovery.
PSV allowed the patient to control his own frequency and therefore the circulating volume and minute volume. In this manner the PSV generated an adequate respiratory work and oxygen consumption of the respiratory muscles, diminishing fatigue and favoring re-training [12] . The combination of this modality with CPAP allowed to increase the functional residual capacity thus achieving a better oxygenation. This is owing to the increase in the number of effective alveolar units in gas exchange (alveolar recruitment). CPAP is able to offset the abnormal closing of the airway and the effect of the auto PEEP, noticing the beneficial effects when the level of inspiratory efforts to be developed during spontaneous breathing diminishes.
The use of a T-piece allowed for greater independence from the ventilator with less resistance in the airway since the opening of an inspiratory valve is not necessary. In addition it permitted a clinical evaluation of the diaphragmatic function which was not possible if the patient received any kind of inspiratory support [14] .
The three described patients had pulmonary sequelae for infection caused by respiratory virus with a pathology of the airway evidenced by respiratory endoscopy, and in spite of all this, their effective disconnection from positive pressure was achieved. This was possible combining tracheostomy with an orderly, monitored and progressive muscular training plan (directed weaning protocol).
Even though more studies are necessary to develop weaning protocols based on evidence, we believe that this type of approach may be useful in pediatric patients with difficult weaning due to other etiologies.
Written informed consent was obtained from the parents of the patient for publication of this report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5′ untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle. coding sequences. This PCR introduced three stop codons to terminate the Rluc coding sequence. The rest of the 5'UTR was amplified directly from a proviral molecular clone of HIV-1 group M subtype B (pLAI) (4) . The initiation codon for Fluc expression is located within the 5'UTR IRES and the context of the AUG encompassing 30 nt from the Gag sequence was included. A peptide linker (GGGGSGGGGS) was inserted by PCR immediately upstream of the Fluc coding sequence. The first half of the linker was inserted using PCR amplification on pLAI with the 5'UTR-AflII(+) and 5'UTR-BamHI(-) primers and the second half of the linker with the 5'UTR(+) and Linker-BamHI(-) primers. The linker was cloned in the AflII and BamHI restriction sites of pDual-HIV(-1)ΔAflII-TAR to generate pDual-IRES-HIV. This last step removed the frameshift region originally present in pDual-HIV(-1). Finally, we cut the fragment containing the Rluc coding sequence, the 5'UTR region of HIV-1 RNA and the Fluc coding sequence, using PmeI and ApaI restriction sites. This fragment was inserted into pcDNA5FRT (Invitrogen) previously linearized with SciI and ApaI to produce pFRT-IRES-HIV. Prior to this last cloning step, the KpnI and BamHI restriction sites from pcDNA5FRT were eliminated to facilitate subsequent cloning of mutant IRESes. To this end, an oligonucleotide cassette formed by the K7ΔKpnIΔBamHI(+) and
K7ΔKpnIΔBamHI(-) primers was inserted in the HindIII and EcoRV restriction sites of pcDNA5FRT.
The different mutants of HIV-1 IRES were made by PCR amplification with four primers. The external primers were Ext-KpnI(+) et Ext-BamHI(-). The details for all the primers used can be found in Supplementary Table 1 . The resulting PCR products were cloned in the KpnI and BamHI restriction sites of pFRT-dual-IRES-HIV.
ΔAflII-SpeI(+) 5'GACCCGGGGTACCAAGCTTGAGTTTAAACGCTAGCCAGC'3  Further Characterisation of the Translational Termination-Reinitiation Signal of the Influenza B Virus Segment 7 RNA Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAA UG and ∼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAA UG, known as the ‘termination upstream ribosome binding site’ (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAA UG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning. Eukaryotic viruses have evolved a variety of translational control strategies to facilitate expression of downstream open reading frames (ORFs) on polycistronic mRNAs and examples have been described at all three steps of protein synthesisinitiation, elongation and termination [1] . These include leaky scanning of 40S ribosomal subunits past the most 59 AUG [2] , the de novo recruitment of ribosomes to intercistronic internal ribosomal entry sites (IRESs; [3] ); programmed ribosomal frameshifting [4, 5] and the circumvention of normal termination by programmed stop codon readthrough [6, 7] . Generally, these processes allow the expression of two (or more) proteins from a single mRNA and may also permit a level of control over their relative quantities. Another way of accessing a downstream ORF in viral mRNAs is by termination-dependent reinitiation of translation (termination-reinitiation), a phenomenon first described in the expression of the influenza B virus BM2 protein [8] . The dicistronic mRNA that is derived from genomic segment 7 of this virus has two ORFs encoding matrix protein 1 (M1) and BM2, with the termination codon of M1 in close proximity to the start codon of the BM2 ORF (UAAUG; stop codon of M1 in bold, start codon of BM2 underlined) [8] [9] [10] . Following translation of M1, some 10-20% of ribosomes terminating at the M1 stop codon go on to reinitiate translation at the immediately adjacent BM2 start codon [8, 11] . This capacity to reinitiate protein synthesis following translation of a long upstream ORF was unexpected. During the elongation phase, initiation factors are likely to be rapidly lost, thus reinitiation of translation following termination was believed to be restricted to cases where the upstream ORF (uORF) is very short [12] [13] [14] . Our knowledge of the mRNA signals that allow efficient reinitiation following translation of a long upstream ORF has largely been obtained from studies of caliciviruses, namely in the expression of the VP2 protein of feline calicivirus (FCV; [15] [16] [17] ) and the VP10 protein of rabbit haemorrhagic disease virus (RHDV; [18, 19] ). Here, expression of the downstream ORF by termination-reinitiation requires a stretch of mRNA (between 69 and 87 nt in length) upstream of the stop codon of the first ORF (termed the termination upstream ribosome binding site or TURBS) and the close proximity of the stop and start codons of the two ORFs [15, 17, 19] . Within the TURBS, two essential sequence motifs (motifs 1 and 2) have been described. Motif 1 is highly conserved amongst caliciviruses and is composed of a short stretch of 6-9 nt (with a conserved core of UGGGA) with complementarity to the apical loop of helix 26 of 18S rRNA. This motif, which lies towards the 59 boundary of the TURBS, likely acts to tether the 40S subunit to the mRNA posttermination, facilitating the recruitment of the initiation factors necessary for reinitiation [15, 19] . Motif 2, which lies some 15-20 nt upstream of the termination-reinitiation window (AUGA in FCV), is a second essential stretch of 4-8 nt but shows little sequence conservation amongst caliciviruses. Recent work has revealed that this motif in fact forms the 39 arm of a stem-loop structure whose formation is necessary for efficient terminationreinitiation [16] .
The features identified in caliciviral TURBS suggest a model for termination-reinitiation in which post-termination 40S subunits are tethered to the mRNA through interactions between the mRNA (through motif 1) and 18S rRNA, initiation factors are recruited and the AUG restart codon located, processes which may require precise RNA folding (involving motif 2) within the TURBS. The TURBS is not highly active as an IRES and 40S subunit recruitment probably requires high local 40S subunit concentrations [17, 18] . An important question, therefore, is how the interaction between TURBS motif 1 and 18S rRNA helix 26 is facilitated. Analysis of primary and secondary structural features of the BM2 signal has revealed that it contains a short TURBS (of 45 nt) which is largely single-stranded, with motif 1 likely to be located in the apical loop of a metastable stem-loop structure when the ribosome is positioned at the termination codon of the upstream ORF [11] . We have suggested that motif 1 may thus be ''presented'' to the solvent-accessible apical loop of helix 26, promoting the interaction and subsequent 40S tethering. However, this hypothesis remains contentious. The paucity of stable RNA secondary structure within the BM2 TURBS [11] , the TURBS of FCV [I. Brierley, unpublished observations] and the calicivirus murine norovirus (MNV; [20] ), limits the predictive power of RNA folding programs in the assessment of the likely RNA folds present before and after ribosomal transit through the TURBS. It is also known that eukaryotic initiation factor 3 (eIF3) plays a role in the termination-reinitiation process, perhaps interacting with the TURBS to provide another link between mRNA and the 40S subunit [17] .
In this paper, we describe further analysis of the BM2 TURBS. In contrast to the caliciviral TURBS, which contain stretches of non-essential bases between motifs 1 and 2, all of the BM2 TURBS appears to be required for function perhaps due to its shorter length, with sequence-specific and sequence-independent elements. Evidence is provided, from oligonucleotide targeting and from expression studies in yeast cells, to support the hypothesis that BM2 motif 1 interacts directly with helix 26 of 18S rRNA, consistent with a tethering role. We also provide evidence that a close spacing between the M1 stop and BM2 start codons is not critical; rather, there is dependence upon the distance between the terminating ribosome and the TURBS. Indeed, the ribosome is able to locate start codons placed some distance downstream of the wild-type position if termination occurs at the normal distance relative to the TURBS. The role of RNA secondary structure in TURBS function was also investigated by mutagenesis, but the experiments gave only limited support for the predicted secondary structures. However, the capacity of eIF3 to stimulate terminationreinitiation, particularly from a defective TURBS, was confirmed, including a TURBS rendered defective through a point mutation in motif 1. Together, these data support the view that efficient termination-reinitiation requires both mRNA-rRNA interactions and the participation of eIF3.
The p2luc-BM2 plasmid series was prepared by subcloning sequences encompassing the influenza B virus terminationreinitiation signal (prepared by RT-PCR) into the dual-luciferase reporter plasmid p2luc [21] . Most of these plasmids have been described previously [11] . The p2luc-BM218S-AG, 18S-AT and 18S-AC plasmids were generated by site directed mutagenesis of p2luc-BM2wt or p2luc-BM2-204 (see below).
The CrPV-p2luc-BM2 plasmid series was generated by digestion of p2luc-BM2wt with PstI and NheI, and insertion of a PCR fragment comprising the cricket paralysis virus (CrPV) IRES downstream of a bacteriophage T7 promoter. The T7-CrPV fragment was generated by PCR from plasmid CrPV/+8norm [14] . The resulting plasmid contains a T7 promoter located 25 nt upstream of the CrPV IRES. Translation starts on an alanine codon within the inserted CrPV fragment, resulting in an Nterminal extension to rluc of 10 amino acids relative to that of the p2luc-BM2wt. Derivative plasmids were generated by site-directed mutagenesis using primers described previously [11] .
Termination-reinitiation in yeast cells was studied using the yeast dual-reporter plasmid pAC99 [22, 23] . The wild-type termination-reinitiation signal of BM2 was prepared by PCR as an EcoRV fragment that was subsequently cloned into MscIdigested pAC99. pAC99-BM2 derivative plasmids were generated by site directed mutagenesis as described below.
Sequences were confirmed by commercial dideoxy sequencing (using the facility at the Department of Biochemistry, University of Cambridge).
Site-directed mutagenesis was performed using the Quikchange II site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. Mutagenesis to introduce insertions longer than 6 bp was performed in two steps [24] , by first subjecting the mutagenesis reactions (containing either the sense or antisense primer) to three cycles of PCR, then mixing the reactions and performing a further 18 cycles as previously described [11] .
In vitro transcription and translation p2luc-BM2 reporter plasmids were linearised with HpaI and capped run-off transcripts generated using T7 RNA polymerase as described previously [25] . Messenger RNAs were recovered by a single extraction with phenol/chloroform (1:1 v/v) followed by ethanol precipitation. Remaining unincorporated nucleotides were removed by gel filtration through a NucAway spin column (Ambion). The eluate was concentrated by ethanol precipitation, the mRNA resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry.
Unless otherwise stated, mRNAs were translated in FlexiH rabbit reticulocyte lysate (FlexiHRRL, Promega) programmed with template mRNA at 50 mg/ml. Typical reactions were of 10 ml and composed of 60% (v/v) FlexiHRRL, 20 mM amino acids (lacking methionine), 500 mM MgOAc, 2 mM DTT, 5U RNase inhibitor (RNAguard, GE Healthcare Life Sciences), 130 mM-160 mM KCl (optimised for each batch of FlexiHRRL) and 0.2 MBq [ 35 S]methionine. Reactions were incubated for 1 h at 30uC and stopped by the addition of an equal volume of 10 mM EDTA, 100 mg/ml RNase A followed by incubation at room temperature for 20 minutes. Samples were prepared for SDS-PAGE by the addition of 4 volumes of 4X Laemmli's sample buffer, boiled for 3 minutes and resolved on 12% SDS-PAGE gels. The relative abundance of products on the gels was determined by direct measurement of [ 35 S]-methionine incorporation using a Packard Instant Imager 2024. eIF4G-depleted RRL was prepared and used as described previously [26] .
Purified eIF3 was a kind gift of Dr. Chris Fraser (Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California). Dominant-negative eIF4A-R362Q was prepared as described [14] .
Reporter gene assay in yeast cells pAC99 reporter plasmids were transformed into yeast strain Y349 using the LiOAc/ssDNA/PEG method [27] . For each experiment three transformants cultivated under the same conditions were assayed. Cells were disrupted by vortexing with acid washed glass beads (Sigma) at 4uC for 30 minutes. Cell debris was removed by centrifugation and reporter enzyme assays carried out as described [23] . Termination-reinitiation efficiency was calculated as the ratio of firefly luciferase activity to b-galactosidase activity relative to that of constructs in which the open reading frames were fused in frame.
The BM2 TURBS is streamlined into 45nt of RNA essential for termination-reinitiation, containing sequence-specific and sequence-independent elements
In RHDV and FCV, TURBS motifs 1 and 2 are separated by some 30 nt, much of which appears to be non-essential [15] [16] [17] 19] . However, given that the minimal sequence requirement for termination-reinitiation of BM2 (,45 nt; [11] ) is shorter than that documented in the caliciviruses (69 and 87 nt; [15, 17, 19] ), we wished to test whether any of the residues were non-essential. To do this, we introduced 6 nt deletions throughout the length of the sequence upstream of the M1 termination codon in the context of the BM2 termination-reinitiation reporter plasmid, p2luc-BM2-204 ( Figure 1A ; [11] ). As expected, translation of the BM2-204 reporter mRNA generated products corresponding to the capdependent upstream product (rlucM1-204 ,36 kDa) and the termination-reinitiation product (BM2fluc ,62 kDa). The latter protein was not observed in translations of the negative control reporter mRNA, p2luc-BM2ps (rluc'M1 ,33 kDa, [11] ), which contains an in-frame stop codon immediately upstream of the TURBS. Significantly, deletion of any part of the minimal required region led to a strong inhibition of BM2fluc synthesis, suggesting that the entire TURBS is required for efficient termination-reinitiation on the segment 7 RNA ( Figure 1A ). Alternatively, it was possible that the defect in BM2 synthesis in this deletion series may be a consequence of the altered spacing between the M1 stop codon and upstream features required for termination-reinitiation. To test this, we selected three of the 6 nt deletion mutants surrounding motif 1 (mutants 6.1, 6.4 and 6.6) and replaced the deleted regions with two different random 6 nt sequences ( Figure 1B ). No restoration of BM2 synthesis was observed with either of the 6.1 nucleotide replacement mutants ( Figure 1B) . Replacement of nucleotides into the 6.4 deletion mutant had no effect with the first of the mutants (6.4r1), but some recovery was observed in the second of these mutants (6.4r2), albeit the frequency of termination-reinitiation was diminished compared to that of the wild-type mRNA ( Figure 1B ). In contrast, insertion of 6 nt back into the 6.6 deletion mutant fully restored BM2 synthesis in both cases ( Figure 1B) . These results suggest that the minimal 45 nt TURBS region is split into both sequencespecific and sequence-independent elements, where the sequence towards the 59 end of the minimal region is important in basespecific interactions, whereas the 39 end of the TURBS may act solely as a spacer, important in placing motif 1 relative to the terminating ribosome.
Targeting the 18S rRNA:mRNA interaction using antisense oligonucleotides Termination-reinitiation on the BM2 ORF is likely to be dependent on interactions between the influenza B segment 7 RNA and helix 26 of the 18S rRNA [11] . However, other work has revealed that motif 1 may also be important in eIF3 binding [17] . To investigate the mRNA-18S rRNA interaction further, antisense 2-O-methyl oligonucleotides (AONs) were synthesised that would target either the loop of helix 26 of 18S rRNA ( Figure 2A ) or motif 1 of the BM2 TURBS ( Figure 2B ). Control oligonucleotides were also prepared to target sequences both upstream and downstream of motif 1 to control for potential nonspecific effects on translation ( Figure 2B ). An AON designed to target helix 26 of the 18S rRNA had no effect on BM2 synthesis ( Figure 2A ), although high concentrations (320-fold to 2960-fold molar excess of the AON to the mRNA) inhibited global translation ( Figure 2A and data not shown). Conversely, an AON targeting motif 1 specifically inhibited BM2 synthesis, with little effect on overall translation ( Figure 2B ). Importantly, the upstream and downstream control AONs had little effect on BM2 synthesis or global translation within the range tested for the BM2 motif 1 complementary AON ( Figure 2B ). It should be noted that the 80S ribosome will strip annealed AONs from the mRNA as it translates through the TURBS. However, both the upstream and motif 1 complementary AONs will be able to re-anneal once the ribosome reaches the UAAUG. However, it is possible that any effect of the downstream oligo is masked due to it being unable to reanneal in the presence of the terminating ribosome. Nevertheless, these data indicate that the effect of the BM2 motif 1 complementary AON is specific and not an effect on ribosome processivity, and provide further evidence in support of an interaction between motif 1 and 18S rRNA. However, we cannot rule out that these effects may also be due to perturbations of RNA secondary structure within the TURBS or due to interactions of motif 1 with an unspecified molecule (see later).
The dependence of BM2 synthesis on interactions between the mRNA and 18S rRNA raises the possibility that increasing 18S rRNA complementarity within motif 1 would lead to increased synthesis of the BM2fluc polypeptide. To test this, the bases adjacent to motif 1 were substituted such that the complementarity to 18S rRNA was increased to the 59, to the 39 or in both directions ( Figure 3A ). In all cases, however, the substitution mutations reduced the frequency of termination-reinitiation events, albeit to a lesser extent with the 39 complementary extension mutant ( Figure 3A ).
We went on to examine the possibility that multiple copies of motif 1 would stimulate termination-reinitiation, through the provision of an increased number of tethering sites. As a precedent for this, the murine Gtx mRNA (also known as Nkx 6-2) contains a short 9 nt IRES element that is known to act more efficiently when present in multiple copies. Similarly to motif 1, ribosomes are recruited through interactions between the Gtx IRES and helix 26 of the 18S rRNA [28] [29] [30] , although with the arm of the helix [31] rather than the apical loop ( Figure 3C , left panel). Two sets of constructs were prepared. In the first, one or two additional copies of the 6 nt (AUGGGA) core of motif 1 were introduced alongside the original ( Figure 3B ). Control constructs were also generated in which one or two copies of the hexanucleotide AUGCGA were looped in as a negative control, since this motif 1 mutation was shown previously to be unable to support termination-reinitiation, presumably due to the abolition of mRNA:rRNA base pairing [11] . In the second set of constructs, the bases downstream of the wild-type AUGGGA sequence were substituted to generate one or two extra copies of the AUGGGA motif ( Figure 3B ). In vitro translations revealed, however, that none of the mutations had a stimulatory effect on reinitiation on the BM2 ORF. Looping in a single extra copy of the AUGGGA (or AUGCGA) hexanucleotide had very little effect on the process, whilst insertion of two copies led to inhibition in both contexts ( Figure 3B ). In constructs where motif 1 was duplicated by virtue of nucleotide substitutions, termination-reinitiation was inhibited to a similar extent in constructs containing either one or two copies of the 18S complementary motif ( Figure 3B ). These data indicate that there is no additive effect on termination-reinitiation of adding extra copies of the 18S complementary region. In fact, the inhibitory effects seen most likely reflect the importance of the precise spacing of the ribosome with respect to motif 1 (in the loop-in mutants) or, alternatively, some effect on TURBS RNA secondary structure.
As discussed above, the Gtx mRNA is thought to be able to recruit ribosomes by virtue of interactions between helix 26 of the 18S rRNA and the mRNA [28] [29] [30] ). It was therefore of interest to determine whether the minimal 7 nt Gtx motif could functionally replace motif 1. Two constructs were generated, one in which the inserted Gtx element was complementary to murine 18S rRNA (which would result in a mismatch with helix 26 of rabbit 18S rRNA) and one with full complementarity to the rabbit 18S rRNA, such that efficient tethering would be expected in rabbit reticulocyte lysates (RRL; Figure 3C , top-right panel). Termination-reinitiation was found to be inhibited in either mutant and to a similar extent as mutants in which a single nucleotide substitution was present to abolish 18S rRNA:mRNA base pairing ( Figure 3C ; the single nucleotide mutants have been described previously [11] ). These results suggest that motif 1 and the Gtx IRES element cannot functionally replace each other.
Termination-reinitiation in yeast cells: further evidence for mRNA:18S rRNA interactions in ribosome tethering It is known that nucleotide substitutions within motif 1 that are predicted to destabilise the motif 1:18S rRNA interaction are inhibitory to termination-reinitiation, but it is possible that these mutations may affect TURBS structure or interaction with other translational components, like eIF3 [17] . In an attempt to resolve this issue, we assayed termination-reinitiation in yeast cells, whose 18S rRNA helix 26 equivalent has a somewhat different primary sequence. This experiment was carried out in the context of the yeast dual reporter vector pAC99, into which we had cloned the relevant BM2 information appropriately framed between bgalactosidase and firefly luciferase reporter genes. In pAC99-BM2wt, b-galactosidase is synthesised by cap-dependent translation, whereas firefly luciferase should only be synthesised following reinitiation on the BM2fluc ORF ( Figure 4A ). To confirm that firefly luciferase was being synthesised as a result of a genuine termination-reinitiation event (as opposed to internal ribosome entry, for example), we generated a premature stop (ps) mutant (pAC99-BM2-ps) in which a stop codon was inserted into the M1 sequence 233 nt upstream of the start codon of BM2fluc. In pAC99-BM2-yeast, motif 1 was changed to give full complementarity to the yeast 18S rRNA partner ( Figure 4B ). The plasmids were transformed into yeast strain Y349, and b-galactosidase and luciferase assays were carried out. Termination-reinitiation frequency was determined in comparison to the reporter gene activities observed in a vector in which the reporter ORFs were fused in-frame (pAC99-BM2IFC). As expected, termination reinitiation was significantly (p,0.01) reduced in pAC99-yeastps, although there was still some background synthesis of fluc ( Figure 4C ). Importantly, mutation of the motif 1 homologue such that it was fully complementary to yeast 18S rRNA led to a significant (p,0.01) increase in BM2fluc synthesis relative to the wild-type BM2 reporter ( Figure 4C ), supporting the view that mRNA:rRNA base pairing is a key determinant in BM2 ORF expression.
It has been suggested previously that termination-reinitiation is dependent on the stop codon of the uORF and the start codon of the downstream ORF lying in close proximity [8, 11, 15, [17] [18] [19] 32, 33] . Indeed, movement of the M1 stop codon more than 24 nt downstream of its original context in the terminationreinitiation 'window' of the BM2 signal results in inhibition of reinitiation on the BM2 ORF [11] . However, in all previous investigations into proximity effects of the start and stop codons, the termination site was moved downstream of the start codon of the upstream ORF [8, 11, 15, [17] [18] [19] 32] ). Given that terminationreinitiation almost certainly depends on interactions between the terminating ribosome and the TURBS, it is conceivable that these experiments have overestimated the importance of the relative proximity of the stop and start codons and that the distance between the terminating ribosome and the TURBS is the crucial issue. To investigate this, a series of mRNAs were prepared in which the UAAUG overlap was moved downstream en masse such that the distance between the stop and start codons was conserved but the ribosome would terminate at varying distances relative to the TURBS ( Figure 5B) . A series of control mRNAs were also examined in which the stop codon alone was shifted downstream of the BM2 start codon ( Figure 5A ; details in [11] ). In all constructs, nucleotides at -3 and +4 relative to the BM2fluc AUG were maintained to control for context effects of the start codon. As observed previously in the control series [11] , reinitiation occurred efficiently when the stop codon was moved up to 24 nt downstream of its original placement but was abolished when the stop was moved further ( Figure 5A ). Importantly, BM2 translation The 44 kDa product produced from this mRNA corresponds to the rlucM1 product, the 62 kDa product is the BM2fluc reinitiation product. The molar excess of AON (oligo) in relation to the mRNA is shown above the autoradiograph. (B) The sequence upstream of the termination-reinitiation site (UAAUG) is shown, with the A residue at the start of the TURBS underlined. Also shown is the core sequence of motif 1 in bold. The regions targeted followed the same pattern in mRNAs where the UAAUG motif was moved as a unit, with ablation of synthesis observed when this sequence was moved any further than 24 nt downstream of its original site ( Figure 5B) . These experiments reveal that the placement of the 40S subunit relative to the TURBS, posttermination, is quite critical.
As reinitiation is not absolutely dependent on the close proximity of stop and start codons ( Figure 5A) , it seemed conceivable that termination-reinitiation could still occur efficiently if the start codon of the BM2fluc ORF was moved downstream of the stop codon of rlucM1. A series of mRNAs were prepared in which the original BM2fluc start codon was mutated to AGC and new AUG restart codons inserted independently at +12, +24, +36, +48 and +63 nt, again preserving the wild-type Kozak consensus. As can be seen in Figure 6A , movement of the start codon to +12 resulted in 50% inhibition of fluc synthesis, but there was no further reduction in product yield when the restart codon was displaced further downstream. However, the electrophoretic mobility of this reinitiation product did not show the clear increase that would be expected given that the restart product would become progressively smaller with increased displacement of the restart codon. Rather, the reinitiation product band seemed less sharp and more diffuse with the +48 and +63 mRNAs. This suggested the possibility that reinitiation might be occurring at two sites: one in the region of the wild-type reinitiation site (and so necessarily at a non-AUG codon), and the other at the displaced AUG restart codon.
The most obvious potential non-AUG codon for the first type of reinitiation event is the AUA codon located immediately upstream (-1 position) of the wild-type reinitiation site at +1 ( Figure 6A ). We therefore tested whether reinitiation can indeed occur at this -1 position, by generating an AUG in this position and removing the wild-type +1 AUG. The results showed that the efficiency of reinitiation at an AUG in the -1 position was similar to when it was in the normal (+1) position ( Figure 6B , compare lanes 8 and 10), in agreement with the finding that reinitiation efficiency in the RHDV system was only slightly compromised if the restart codon was moved one codon upstream [19] . Given that quite efficient reinitiation still occurs in FCV and BM2 RNAs when the wild-type AUG is mutated to non-AUG codons [11, 15, 17] , this observation suggests that when the wild-type +1 AUG has been mutated, there is a strong possibility of some reinitiation occurring at the -1 AUA codon.
As for reinitiation at the displaced AUG codon, the data of Figure 6B (lanes 1-6) demonstrate that reinitiation certainly occurs at these downstream sites, because on this higher resolution gel the product size clearly decreases as the displacement is increased, although the efficiency is only 20-25% of the reinitiation frequency in the wild-type mRNA (lane 1). In fact, in this particular experiment there is much more downstream cistron product from the displaced AUG than product from the putative -1 AUA reinitiation site, which was only just visible on the autoradiogram (highlighted by the asterisk). In reviewing the results of many experiments of this type we conclude that the relative use of the two potential reinitiation sites shows quite a large degree of variability, which seems related to the use of different batches of reticulocyte lysate and so may possibly be due to small batch to batch differences in the endogenous ionic conditions. The variability seems to mainly affect the yield of putative -1 AUA reinitiation product, whereas the yield of product from the displaced AUG codon is relatively invariant at ,20% of the wild-type mRNA yield. The two extremes of the variability range are well illustrated by Figure 6B (very minimal use of the -1 AUA site) and Figure 6C , where there is actually more putative reinitiation at the -1 AUA than at the displaced AUG codon.
The fact that reinitiation, albeit at reduced efficiency, can occur when the first AUG is moved 63 nt downstream raises the question of whether the reinitiating 40S subunits access this site by linear scanning from the TURBS. We examined this by exploiting the dependence of ribosomal scanning on eIF4G [34] . The BM2wt and BM2start +63 mRNAs were modified such that translation of the upstream M1 ORF was driven by the cricket paralysis virus (CrPV) IRES, which does not require eIF4G for function. These mRNAs (CrPV-BM2wt, CrPV-BM2start+63) were then translated alongside capped brome mosaic virus RNAs (BMV, Promega) and BM2wt RNAs (as a control for the efficacy of eIF4G depletion) in mock-and eIF4G-depleted rabbit reticulocyte lysates ( Figure 6C ). Translations were also performed in the absence or presence of dominant-negative eIF4A (eIF4A R362Q [35] ) to inhibit the activity of any residual eIF4G that had escaped depletion. As expected, the translation of the capped wildtype RNA (BM2wt) and BMV RNAs were inhibited in the eIF4Gdepleted RRL, but the CrPV IRES-containing mRNAs were translated efficiently in both control and depleted RRL ( Figure 6C ). Importantly, depletion of eIF4G had no effect on the reinitiation efficiency in either the CrPV-BM2wt or CrPV-BM2start +63 mRNAs, suggesting that the reinitiation site can be located in a scanning-independent manner.
When two AUG codons were present, one at +63 and the other at either the +1 (wild-type) or -1 position, the upstream site took complete precedence over the +63 site ( Figure 6B, lanes 8 and 10) , and this precedence was maintained even when the stop codon was moved 12 codons downstream ( Figure 6B, lanes 11-13) . Thus the strongly preferred site for reinitiation is an AUG codon just downstream of the TURBS. If there is no AUG in this region, the reinitiation mechanism apparently seeks alternatives: either an acceptable non-AUG codon in this same region or an AUG codon further downstream. Reinitiation in the later case does not involve eIF4G/4A-dependent linear scanning, and so it is more likely to involve a direct transfer of the 40S subunit from the TURBS to the AUG, a transfer which might be facilitated by looping out the mRNA between the tethered 40S ribosomal subunit and the AUG.
Studies of the FCV signal have indicated a role for eIF3 in termination-reinitiation as, for example, addition of supplementary eIF3 to RRL is able to specifically stimulate synthesis of the ORF3 reinitiation product [17] . Furthermore, greater stimulation is observed with mRNAs that show a partial defect in reinitiation in the absence of eIF3, suggesting that the increase in eIF3 concentration may rescue the negative phenotype, perhaps by allowing increased binding of eIF3 to the mRNA [17] . To analyse whether the same is true of reinitiation on the BM2 ORF, we examined the effect of exogenous eIF3 on reinitiation on mRNAs containing a fully functional signal (BM2wt, BM2-204), or mutants by AONs are shown above the sequence, colour coded to match the relevant AON sequence. The BM2wt RNA was translated as above in the presence of increasing AON concentrations as indicated above each autoradiograph. doi:10.1371/journal.pone.0016822.g002 defective by virtue of a deletion (BM2-207, BM2-210, described in [11] ) or a substitution in motif 1 (BM2-AG, BM2-AC). To focus specifically on the effect of eIF3 on termination-reinitiation rather than 59-dependent initiation, the BM2 sequences were sub-cloned such that the rlucM1 ORF would initiate on an alanine codon provided as part of the CrPV IRES. As can be seen in Figure 7 , exogenous eIF3 specifically stimulated synthesis of the BM2fluc reinitiation product with all of the mRNAs tested, including those mutants that were essentially inactive (BM2-210 and BM2-AC; most evident in long exposures, see right panel, Figure 7) . Importantly, the addition of an unrelated, similarly purified protein had no effect on reinitiation in any of the RNAs described above (Figure S1 ), further highlighting the specificity of eIF39s effect on reinitiation.
Given the likely importance of the interaction between motif 1 and 18S rRNA, we previously carried out RNA structure mapping and minimal free energy predictions to assess whether motif 1 was in basepaired or single stranded conformation in the native mRNA. This work implicated two potential structures, mfold 1 and mfold 2 ( Figure 8A ; [11] ). In mfold 1 the 18S complementary region is sequestered in a base-paired region of stem 2. We previously hypothesised that translation through the segment 7 mRNA and termination of ribosomes at the M1 stop codon would prevent these interactions, creating a structure similar to mfold 2 ( Figure 8B ). In this structure, the 18S complementary region would then be presented to helix 26 of the 18S rRNA on the apical loop of a metastable stem-loop structure.
Given that translation through the TURBS up to the termination-reinitiation window is a prerequisite for efficient reinitiation [11] , one can speculate that mfold 2 is the biologically relevant fold, and that destabilisation of the central stem of mfold 1 (stem 2) would have little effect on the reinitiation process. To test this hypothesis we created destabilising mutations within either arm of stem 2 (chosen to avoid disruption of the 18S rRNA complementary region) in the context of the p2luc-BM2-204 parental plasmid (which encodes the minimal required region for termination-reinitiation [11] ). The mutations created were of either 2 or 3 nt to control for context effects on the BM2 AUG, creating the arm 1 mutants p2luc-BM2A1-xnt, and the arm 2 mutants p2luc-BM2A2-xnt (where x = the number of nucleotides mutated). We also prepared double mutants (A1/2-xnt) to create a pseudowild-type structure where base-pairing between the two arms is restored. In the context of both the 2 nt and 3 nt mutations, substitution of bases in arm 1 abolished terminationreinitiation, however substitution of the bases at the bottom of arm 2 had little effect on BM2fluc synthesis relative to the BM2-204 control. The double pseudowild-type mutation also demonstrated abolition of BM2fluc expression ( Figure 8A ). Taken together these results suggest that the structure presented in mfold 1 is unlikely to play a role in termination-reinitiation as disruption of arm 2 of stem 2 has little effect on BM2 translation, and no restoration of BM2 synthesis was observed in the pseudowild-type construct.
As opposed to mfold 1, the structure presented for mfold 2 would still be able to form when the ribosome has translated through the upstream ORF and is in the process of termination. We tested this structure in the same way, by introducing destabilising mutations independently in both arms at the base of stem 29 (creating mutants p2luc-BM2A1' and p2luc-BM2A29) and preparing a double mutant that would yield a pseudowild-type structure, which would be expected to exhibit BM2fluc synthesis (p2luc-BM2A19/29). It should be noted that the A19 mutation is very similar to that of the A1-2/ 3 nt mutant, given that this arm base pairs to form both stem 2 and stem 29 in the two putative folds. As expected, this mutation dramatically inhibited synthesis of BM2fluc similarly to that observed with the A1 mutation ( Figure 8B ). However, as before, no inhibition of BM2 synthesis was observed when stem 2 was destabilised in the 39 arm ( Figure 8B ). Nevertheless, the partial restoration of BM2 expression (to around 50% of wild-type) seen with the A19/29 double mutant, indicates that base-pairing in this region may play some role in termination-reinitiation. The simplest conclusion from these experiments, however, is that neither mfold 1 nor mfold 2 represent the sole active confirmation, although it is possible that these mutants may form another structure that can present the TURBS motif 1 to helix 26 of 18S rRNA.
Previous work has revealed that termination-dependent reinitiation on the BM2 ORF of segment 7 of influenza B virus is dependent on a relatively short (45 nt), largely unstructured, TURBS containing a typical motif 1 element towards the 59 end [11] . In the present study, we investigated features of the TURBS required for efficient reinitiation, focusing primarily on the proposed interaction between TURBS motif 1 and helix 26 of 18S rRNA. Whilst the effects of point mutations within motif 1 are consistent with such an interaction, direct evidence is lacking in the BM2 system. We began by attempting to block the interaction by targeting the binding partners with antisense oligonucleotides. Such an approach was successfully employed in studies of the IRES-like properties of the Gtx leader (which recruits ribosomes de novo by virtue of interactions between the 18S rRNA and the mRNA) and revealed that the IRES activity could be blocked by AONs that bind either the mRNA or the rRNA [36] . Whilst we were able to observe specific inhibitory effects with an oligonucleotide that targeted motif 1, no effect was observed with an oligonucleotide targeting the ribosomal RNA ( Figure 2 ). Our failure to observe an effect of the AON complementary to the apical loop of helix 26 may be due to a failure of the AON to access the target. The Gtx mRNA:rRNA interaction occurs at a In the p2luc-BM2Gtx mutants, the 7 nt core of motif 1 (bold italics in p2luc-BM2wt) was substituted for nucleotides complementary to the region of 18S rRNA where the Gtx IRES is believed to bind. Note the p2luc-BM2Gtx Rabbit sequence is altered from that of the p2luc-BM2Gtx Mouse reporter such that 100% complementarity with the different rabbit 18S rRNA could be achieved. Nucleotides differing between Gtx Mouse and Gtx Rabbit are underlined. RNAs were translated and analysed as in the legend to Figure 1 . A series of previously described motif 1 mutants [11] were also translated as controls. doi:10.1371/journal.pone.0016822.g003 different region of helix 26 than the putative segment 7 mRNA:rRNA interaction, and this may be more accessible. Indeed, termination-reinitiation could not be reconstituted in reporter mRNAs in which motif 1 was replaced by the Gtx 18S rRNA binding motif ( Figure 3C) , and thus different responses to oligonucleotides targeting the rRNA may be expected. It may also be that the 18S rRNA-targeting oligonucleotide may need to adopt a similar structure to the mRNA if it is to effectively bind to the 18S rRNA and block reinitiation. The inhibition of termination-reinitiation by the AON that targeted motif 1, in contrast, is highly consistent with its proposed role in binding to helix 26, although effects via RNA conformation or binding to an unknown molecule cannot be ruled out. However, the weight of evidence from mutational analysis, the yeast data described below and the AON titrations strongly suggest that the inhibitory effect of AONs directed against motif 1 are likely due to their blocking of the interaction between mRNA and 18S rRNA.
Subsequently, we sought to confirm the interaction by investigating termination-reinitiation in yeast cells, exploiting the fact that the helix 26 equivalent in yeast 18S rRNA has a primary sequence distinct from that of the rabbit. Using a dicistronic reporter mRNA with variant motif 1 sequences, we found that increasing the complementarity of the motif to that of yeast 18S rRNA stimulated termination-reinitiation, supporting strongly the view that intermolecular interactions are important in reinitiation on the BM2 ORF. However, two aspects of this experiment require comment. First, a high background activity was observed in control assays (Figure 4) , the origin of which is uncertain Secondly, in yeast cells, the BM2wt reporter mRNA, whose motif 1 is not fully complementary to the helix 26 target, showed an efficiency of termination-reinitiation only two-fold lower than the BM2yeast mRNA. In RRL, a single base mismatch between mRNA and rRNA is sufficient to inhibit BM2 synthesis 10-20 fold [11] . One possible explanation for this difference is that the higher concentrations of mRNA and rRNA in an intact cell can act to stabilise the (presumably) weakened mRNA:rRNA interaction. During preparation of this manuscript, Luttermann and Meyers (2009) published a similar, more sophisticated, investigation of FCV motif 1:18S rRNA interactions using a yeast expression system in which both mRNA and rRNA partners could be The rlucM1/BM2fluc overlap window was changed from UAAUG to CAAUG such that termination occurs at the next in-frame stop codon, some 63 nt downstream of the original stop site (+63). New stop codons were then inserted, preserving the stop codon context, at +12, +24, +36 and +48 nt downstream of the natural termination site. Messenger RNAs derived from these plasmids and a control mRNA (ps) were translated and analysed as detailed in the legend to Figure 1 . This experiment is a repeat of that found in [11] and is shown here as a control. (B) The rlucM1/BM2fluc overlap window was changed from UAAUG to CAACU, and the UAAUG sequence reintroduced at +12, +24, +36, +48 or +63 nt downstream preserving both the initiation codon and stop codon context. Messenger RNAs derived from these plasmids were translated and analysed as above. doi:10.1371/journal.pone.0016822.g005 Figure 6 . Effect on termination-reinitiation of altering the BM2 start codon position. (A) The termination-reinitiation overlap was mutated from AUAAUG to AUAAGC and the BM2fluc start codon reintroduced at +12,+24, +36, +48 or +63 nt, preserving the start codon context at the -3 and +4 positions. The codon immediately 59 of the native reinitiation site is denoted -1, the reinitiation site is shown as +1. Messenger RNAs derived from these plasmids were translated and analysed as above. (B) Left panel: Translations were performed as in Figure 6A and the samples were separated by SDS-PAGE and autoradiographed. The putative AUA reinitiation product is marked with an asterisk. In addition, the three right hand modified. This work provided strong evidence in support of a requirement for mRNA:rRNA interactions in termination-reinitiation in the expression of FCV VP2 [16] .
Since the reinitiation mechanism shows a fairly strong preference for an AUG codon, it seems a certainty that initiator Met-tRNA is involved. This, in turn, implies that the ribosome lanes of the left panel show translations of mutant mRNAs in which the codon -1 of the AUG start codon and the start codon (+1) were mutated to the indicated codons in the context of the BM2start +63 RNA. Lane numbers are shown below the figure. Right panel: Translations of RNAs containing stop and start codons at the indicated positions. C) Capped BMV (BMV) RNAs, BM2 wt reporters (Cap-wt), CrPV-IRES driven BM2wt (CrPV-wt) and BM2start +63 (CrPV+63) RNAs were translated in control (C) and eIF4G-depleted (D) RRLs in the absence or presence of ,1 mM dominant-negative eIFA (4A-R362Q). RNAs were translated as described in [26] and analysed as above. doi:10.1371/journal.pone.0016822.g006 which has just terminated at the stop codon must dissociate into subunits prior to reinitiation, in order to allow eIF2/GTP/Met-tRNA i ternary complex binding to the 40S subunit. So it is a 40S subunit rather than an 80S ribosome that interacts with TURBS motif 1. The fact that reinitiation efficiency decreases quite abruptly with increasing distance of displacement of the stop [11] ) is shown with motif 1 shown in red, the termination-reinitiation overlap shown in cyan and the sequences likely to be occupied by a ribosome terminating at the M1 ORF are 39 of the purple line. Mutations were introduced at the bottom of stem 2, with both the 2 nt and 3 nt substitution mutations shown next to arm 1 (A1) or arm 2 (A2). Messenger RNAs derived from these plasmids and control mRNA (204, ps) were translated and analysed as detailed in the legend to Figure 1 . RRF denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). (B) The mfold 2 structure (from [11] ) is shown with motif 1, the termination-reinitiation overlap, and the ribosome protected region marked as above. Mutations were introduced at the bottom of stem 29 and are shown next to arm 1 (A19) or arm 2 (A29). Messenger RNAs derived from these plasmids and a control mRNA (204) were translated and analysed as above. RRF denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). doi:10.1371/journal.pone.0016822.g008 codon further downstream ( Figure 5 ) favours a model in which this 40S subunit is transferred directly from the termination site to the TURBS, rather than dissociating from the mRNA after termination followed by reassociation with the TURBS.
Recent work has implicated eIF3 in the termination-reinitiation process [17] , which is of interest because eIF3 plays an important role in disassembly of ribosomes following the termination event which leaves the 80S ribosome with bound deacylated tRNA and still associated with the mRNA [37, 38] . At low (sub-optimal) Mg 2+ , this disassembly requires eIF1 and eIF3, but no other protein factor. First, eIF3 binds to the solvent face of the 40S subunit, promoting dissociation of the 60S subunit, and leaving the 40S subunit (with bound deacylated tRNA) still associated with the mRNA. Then eIF1 ejects the deacylated tRNA, while the eIF3j subunit promotes dissociation of the 40S/eIF1/eIF3 complex from the mRNA [37] . At higher, more physiologically relevant Mg 2+ , as would pertain in our translation assays, there is also a requirement for ABCE1 and ATP [38] , which catalyses the first step of dissociation of the ribosomal subunits, ejecting the 60S subunit and again leaving the 40S subunit (plus bound deacylated tRNA) still associated with the mRNA. Then eIF3 binds to the solvent face of the 40S subunit, thereby preventing any ribosomal subunit reassociation, and events thereafter are exactly as at low Mg 2+ [37, 38] .
Initiation factor eIF3 has been shown to crosslink to the FCV TURBS [17] . More important, supplementary eIF3 has been shown to stimulate reinitiation at the wild-type FCV TURBS, but more especially at TURBS derivatives which are partially defective due to deletions or point mutations [17] , just as has been observed here (Figure 7) . These results, particularly the stimulation of the partially defective TURBS, have suggested a model in which it is specifically the eIF3 associated with the TURBS that binds those 40S subunits which are destined to reinitiate translation [17] . There is no conflict between this hypothesis and the model in which tethering the 40S subunit to the TURBS is due to base-pairing between TURBS motif 1 and 18S rRNA. eIF3 binds predominantly to the back or solvent face of the 40S ribosomal subunit, and is known to make direct contacts with the 18S rRNA component of the small ribosomal subunit [39, 40] . A bridging interaction in which eIF3 binds simultaneously to the 40S subunit and to the TURBS could help stabilise the binding of the 40S subunit to the TURBS for sufficient time as is required to acquire an eIF2/GTP/Met-tRNA i ternary complex, and other necessary initiation factors. After all, the analogous Shine-Dalgarno interaction of prokaryotic 30S subunits is generally considered to be very transitory unless it is accompanied and stabilised by a P-site fMet-tRNA base-pairing with an AUG (or GUG) at an appropriate distance further downstream.
An important question that remains to be resolved is whether there is a role for TURBS RNA secondary structure. We previously proposed that the TURBS may fold into two main configurations, in the first of which (mfold 1) motif 1 is sequestered in a base-paired region [11] , which may explain why it does not have significant IRES activity (as is seen with Gtx [28] [29] [30] ). Translation through and termination at the M1 ORF would remodel mfold 1 to a structure similar to that shown in mfold 2 ( Figure 8B ) such that motif 1 would then be presented to the 18S rRNA on the apical loop of a stem-loop structure. The experiments described in Figure 1 agree well with dependence on a structure similar to that presented in mfold 2, in that the substituted nucleotides in the 6.1 and 6.4 replacement mutants would act to disrupt the stem, whereas substitution of nucleotides in the 6.6 replacement mutants would be expected to have no effect as they would lie within the mRNA channel of the terminating ribosome. However, we also carried out a mutational analysis of the main stem regions present in mfold 1 and mfold 2 but the data did not corroborate our structural predictions. Whilst disruption of either of the putative stems in one arm inhibited reinitiation, little effect was observed when stem formation was disrupted in arm 2 of either stem-loop structure, although some restoration of BM2 synthesis was observed in an mRNA containing a pseudowild-type mfold 2 structure. It should be noted that RNA structure mapping of the TURBS of BM2 [11] , FCV (Brierley et al. unpublished observations) and MNV [20] TURBS reveals that they are largely single-stranded, and probably metastable. It may be that TURBS are able to adopt a variety of conformations, some of which are able to facilitate terminationreinitiation, for example the 6 nt replacement mutant 6.4r2 ( Figure 1B) . Alternatively, the requirement for translation through the TURBS may not be due to a dependence on translational remodelling and 'unzipping' of motif 1 from paired regions, but rather just to place the ribosome in proximity to motif 1 (similar to the case for reinitiation in bacteriophage where the ribosome is placed close to the vestigial Shine-Dalgarno [SD] motif [41] ). It is clear that further work is required to understand the putative role of RNA secondary structure in termination-reinitiation.
Previous studies on the termination-reinitiation process have suggested the importance of the close proximity of the stop codon of the upstream ORF and the start codon of the downstream ORF [8, 11, 15, 17, 18, 32] . This is believed to reflect a restricted mobility of the tethered ribosome; that is, it may be able to undergo only limited movement following termination. However, we show here that the distance between the terminating ribosome and the TURBS is more critical to reinitiation efficiency ( Figure 5 ) and that when the start codon of the BM2 ORF is placed downstream of the stop codon, reinitiation is detectable even when the start codon is moved up to 63nt downstream of its original position ( Figure 6 ). This suggests that it is not solely the distance between the start and stop codons per se that affects reinitiation efficiency but rather how well the ribosome can be tethered (by virtue of the distance between where the ribosome terminates and the TURBS). However, whilst efficient reinitiation requires an AUG at, or very near, the wild-type site ( Figure 6A and 6B) , other reinitiation sites can be used when the native reinitiation codon is absent, albeit at lower efficiency. Under the latter circumstance, ribosomes can reinitiate at a downstream AUG codon ( Figure 6B ), or at a near-cognate initiation codon (such as the -1 AUA codon, Figure 6 ) close to the native reinitiation site. However, if given a choice of a wild-type AUG and one located downstream, the ribosome always selects the wild-type AUG, even if the ribosome artificially terminates downstream of the reinitiation window ( Figure 6B) , suggesting that the TURBS may cause the tethered 40S subunit to 'snap back' to the proper site of reinitiation. Importantly, we show that there is no requirement for eIF4G in location of either wild-type or downstream reinitiation sites. As such, location of downstream AUGs is likely due to the 40S subunit being transferred directly to the AUG whilst tethered to the TURBS (perhaps in complex with eIF3 and other factors) by an RNA-looping mechanism. Figure S1 Recombinant 4EBP1 has no significant effect on reinitiation on the BM2 ORF. Motif 1 and TURBS deletion mutants were translated in the absence or presence of 250nM 4EBP1 (details as in Figure 7) . The fold-stimulation over the reinitiation levels observed in the absence of 4EBP1 are shown below the autoradiographs. (EPS) RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids. Real-time (rt) PCR and reverse transcription (RT) PCR techniques are rapid and versatile diagnostic procedures broadly used in clinical virology where there are mostly considered as diagnostic ''gold standards'' [1] . Monitoring rt-PCR and rt-RT-PCR assays and validation of the results rely on the use of relevant external or internal controls (ECs or ICs) [1, 2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as HIV, hepatitis viruses, influenza viruses etc.. However, one of the main strengths of rt-PCR is versatility, which provides the opportunity to set-up ''in-house'' protocols for specific pathogens. The scientific literature now includes an impressive number of 'home made'' assays for various viral agents. Whilst most commercial kits include both ICs and ECs allowing accurate validation of the results [3] , ''home made tests'' are frequently performed in the absence of ICs and therefore without any possible individual monitoring of each diagnostic reaction. For example, the detection of technical errors or PCR amplification inhibitors is intrinsically impossible if only ECs are used. In addition, ECs are usually undistinguishable from the native genome.
Here, our objective was to develop and test on a large number of clinical samples a bacteriophage-based IC system suitable for a standard laboratory of medical virology. We present results obtained by using T4 and MS2 bacteriophages as ICs in a routine-based evaluation including 
Important criteria for the design of ECs include (i) the possibility to use quantified or semi quantified controls (in order to manage the detection level of the diagnostic test used), (ii) the possibility to distinguish amplicons obtained from ECs from those obtained from the detected pathogen (in order to detect false positives due to accidental amplification of EC) and (iii) the use of relevant nucleic acids (ie, RNA and DNA molecules for RNA and DNA viruses, respectively).
Simple protocols for preparing plasmid DNA or synthetic RNA quantified controls are described in Supporting Information S1 and schematised in figure 1. Such controls include specific sequences for the hybridisation of detection primers and probe, but also an exogenic ''Not I'' sequence (detectable by a specific probe or cleavable by the Not I restriction enzyme).
2a. rt-PCR assays for the detection of T4 and MS2 bacteriophages. The criteria to be addresses regarding ICs were: (i) to monitor all steps of the diagnostic procedure (extraction, RT, PCR); (ii) to be amenable for DNA and RNA viruses ; (iii) to rely on a detection system specific of the phage(s) to avoid complex molecular constructs; (iv) to be usable in either simplex or multiplex format, and in one-step or two-step RT-PCR reactions.
We used freeze-dried E. coli Enterobacteria phage T4 (T4) and Enterobacteria phage MS2 (MS2) obtained from the American Type Culture Collection (ATCC ref. 11303-B4 & 15597-B1, respectively). Protocols for real time PCR detection of phages TA and MS2 were elaborated in various formats and are described in Supporting Information S2. Briefly, primers and probes targeting T4 phage (T4F CCATCCATAGAGAAAATATCAGAACGA, T4R TAAATAATTCCTCTTTTCCCAGCG, T4probe VIC-AACCAGTAATTTCATCTGCTTCTGATGTGAGGC-TAM-RA) and MS2 phage (MS2F CTCTGAGAGCGGCTCTATTGGT, MS2R GTTCCCTACAACGAGCCTAAATTC, MS2probe VIC-TCAGACACGCGGTCCGCTATAACGA-TAMRA) were designed from genomic sequences. Optimised oligonucleotide concentrations were 10pmol for primers and 4pmol for probes for both T4 and MS2 detection assays.
2b. Spiking and validation procedures. A suspension of T4 and MS2 phages was used for spiking clinical samples. Both phages were diluted to provide a similar level of detection by rt-PCR.
The following format was used: Similarly, the influence of spiking on the sensitivity of rt-PCR detection was evaluated by comparing detection of enterovirus (EV) and CMV in serial dilutions of cell culture supernatant media and a series of positive clinical samples spiked with T4-MS2 mix in either Two-step or One-step rt-PCR format (see Supporting Information S4). Table 1 were included in the study.
The approval of the relevant Ethics Committee (IFR48, Marseilles, France) was obtained for investigating the benefit of using phage spiking, but individual consent from patients was not required since French national regulations under the term of Biomedical Research (Loi Huriet-Sérusclat (loi 881138)) indicate that the signature at the hospital entrance office warrants that all samples taken during hospitalization for diagnostic purposes are accessible for research (excluding human genetic research) without the specific consent of the patient.
2b. Spiking and rt-PCR assays. A 200ml volume of each 8,950 clinical specimens was spiked (according to the procedure described above) before extraction which was performed onto the MagNA Pure LC instrument (Roche) using the High Pure Viral Nucleic Acid Kit (DNA extraction) or the MagNA Pure LC RNA isolation High Performance kit (RNA extraction) according to manufacturer's recommendations. CSF, amniotic fluids, biopsy tissues, effusions and aqueous humor were processed for DNA+RNA extraction using the BioRobot EZ1 with the Virus Mini Kit v2.0 (both from Qiagen). Reverse transcription was performed for RNA viruses using the TaqMan Reverse Transcription Reagents kit (Roche) and random hexanucleotides as per manufacturer's instructions. For each specimen: (i) rt-PCR reactions were carried out according to medical prescription (Table 1) , and (ii) distinct rt-PCR reactions for detection of T4 or MS2 were performed under a 15 mL reaction format (7,5 mL of mastermix, 3 pmol of each primer and 1,2 pmol of probe) and a standard cycling protocol (50uC for 2 min, 95uC for 10 min and 45 cycles 95uC for 15 sec, 60uC for 1 min). 2c. Interpretation of results. For each series of T4 and MS2 rt-PCR, the mean Ct value and the standard deviation within the series were calculated. Each individual reaction was subsequently analysed as follows:
(i) If the Ct value was equal to or lower than the mean Ct value of the series +1SD, it was recorded as ''correct detection of the phage'' (CDP), and associated with the absence of detectable inhibitor or technical problem while processing the corresponding sample. (ii) If the Ct value was higher than the mean Ct value of the series +1SD (or undetectable), it was recorded as ''inefficient detection of the phage'' (IDP), and associated with the presence of amplification inhibitor(s) or technical problem while processing the corresponding sample.
N When IDP was associated with a positive PCR (detection of a pathogen), this result was validated despite the presence of inhibitors (this would not apply to the case in which quantification of viral load is necessary).
N When IDP was associated with negative PCR detection results, a new assay was performed using a tenfold dilution of the nucleic acid extract. All negative results were considered unresolved (UNR). Positive results were validated.
The detection of EV and CMV in serial dilutions of supernatant cell culture media or in series of positive clinical samples spiked with T4-MS2 mix was performed and the comparison of the Ct values showed that neither the addition of the phage mix itself (Wilcoxon test, p = 0.18 for CMV and 0.45 for EV), nor the presence of phage-specific primers and probe in the reaction mix (Wilcoxon test, p = 0.77 for CMV and 0.18 for EV) did interfere with the Ct value associated with viral detection in Two-step (Table 2 and 3) and One-step rt-PCR.
During the period of study, 8,950 clinical samples were spiked with phages and tested: 7,397 for the presence of DNA viruses only, 337 for the presence of RNA viruses only and 1,216 for both, corresponding to a total of 45,530 results transmitted to the prescribers (see details in Figure 2 ).
Inhibitors could be detected for all types of samples, but with highly variable rates (analysis performed for series including a minimum of 10 samples): less than 10% in the case of pleural effusions and sputum samples, genital and pharynx swabs, lymph nodes and placentas; 10% to 30% for EDTA whole blood, white blood cells, sera, biopsies, pericardial and peritoneal effusions, bronchial aspiration, CSF, amniotic fluids, others swabs, urine, aqueous humor and culture cells; 30% to 50% for plasma, BAL and stools; more than 50% for heparinized plasma and bone marrow.
IDPs were significantly more frequently detected for MS2 (RNA) than T4 (DNA) in plasma, BAL and CSF samples (p,0.05, Khi2 test, Yates corrected). Conversely, IDPs were significantly more frequently detected for T4 than MS2 in stools (p,0.05, Khi2 test, Yates corrected).
Finally, it was noted that clinical samples stored at 280uC prior extraction exhibited an IDP rate lower than 30%.
Rt-PCR and rt-RT-PCR techniques are now widely used for the molecular diagnosis of viral infections. Our study was focused on the importance of quality controls for such diagnostic assays. We proposed a simple protocol for synthesizing specific external controls which combines standard techniques for obtaining quantified DNA or RNA positive controls. Importantly, these controls were designed to include an extrinsic sequence that contains a (very rare) cutting site for the NotI restriction enzyme. This provides a simple tool for using adapted and reproducible amounts of positive controls, and also for identifying PCR contamination due to carry over of the positive control. This detection can be performed by real time amplification using the specific Not I probe (Figure 1 [4, 5, 6, 7] . Our study confirms that the performance of 'home made' tests can be significantly improved by the used of phage-based internal controls, but, most importantly, shows that such controls can be used for routine virological diagnosis and usable for a variety of clinical samples. Here, clinical samples were spiked with both T4 and MS2 phages, allowing the detection of inhibitors for both DNA and RNA viruses. Thirty-six different types of clinical samples were tested (including various blood samples, cerebrospi- nal fluids, stools, respiratory samples, swabs, biopsies or effusion fluids) and a large number of samples were tested in the context of an hospital routine molecular virology laboratory. The use of phages as internal controls proved to be extremely versatile and could be adapted to a broad range of methods and pathogens. It was validated for both PCR and RT-PCR real time techniques, in simplex or multiplex format and, in the case of RT-PCR assays, one-step or two-step amplification formats. In addition, whilst the current report relies on probe-based real time amplification techniques, the method could also be conveniently adapted to a real time SYBR Green detection assay [8] . This is important and suggests that a strategy including phage-based internal controls can be implemented in diagnostic laboratories irrespective of the technical characteristics of the amplification methods used for routine tests. In our experience, the simplex format strategy (i.e. based on testing phages and viral pathogens in distinct amplification reactions) proved to be the most simple and costeffective for routine molecular diagnosis since it does not require the specific development of multiplex reactions and relies on a unique control reaction for DNA viruses and another for RNA viruses.
Our study identified different frequencies of inhibitors according to the nature of the clinical samples. In samples such as CSF, sera or pharynx swabs, inhibitors were identified in ,10% and ,15-20% for DNA and RNA virus detection, respectively. These values are unexpectedly high, and imply that a significant number of samples with results believed to be ''negative'' in the absence of an internal control should be considered ''unresolved''. Specific samples such as heparinized blood (72,9% of inhibitors for DNA virus detection), bone marrow (73.8% of inhibitors for DNA virus detection) and stools (36.6% and 20.8% of inhibitors for DNA and RNA viruses detection, respectively) may also benefit from the detection of inhibitors and the identification of ''unresolved'' tests.
It should be noted that the frequency of amplification inhibition not only relates to the nature of the sample tested, but is also intrinsically linked with the technical protocols used for the extraction and amplification of nucleic acids. In our experience, no major differences were observed when different silica column-or magnetic beads-based extraction techniques, or different commercialized PCR or RT-PCR kits, were tested (data not shown). However, a detailed analysis may reveal that specific techniques give better results when used for testing specific samples. We suggest that, in the future, phage-based internal controls may constitute cost-effective tools with which to measure the frequency of amplification inhibition in specific samples. Estimation of this parameter may become a major criterion for the evaluation of extraction (and to a lesser extent amplification) techniques.
Finally, in the context of diagnostic virology laboratories, our study shows that standard extraction and amplification techniques used for the molecular diagnosis of human pathogens led to a significant proportion of 'unresolved' results, which cannot be identified if an internal control is not used. In the absence of an internal control, such samples are commonly identified as 'negative', which is with hindsight incorrect: in our hands, detecting amplification inhibition using phages as internal controls, and testing tenfold dilutions of the nucleic acid extracts demonstrated that some of these samples were actually positives. This cost-effective and convenient strategy can therefore be used for enhancing the quality of routine molecular diagnosis, but it may also be adapted in other contexts such as testing of large numbers of animal or environmental samples.
Supporting Information S1 Preparation of DNA and RNA synthetic ECs.  Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness BACKGROUND: Resistance to the neuraminidase inhibitor oseltamivir can be conferred by a well-characterized mutation in the neuraminidase gene, H275Y. In human H1N1 viruses that circulated in the first years of the 21st century, this mutation carried a fitness cost and resistant viruses were rare. During the 2007–08 influenza season, oseltamivir-resistant viruses of H1N1 phenotype emerged and predominated. March 2009 saw the emergence of a novel H1N1 influenza pandemic. We examined whether the H275Y mutation affected neuraminidase enzyme activity or replication of the pandemic influenza virus. METHODS: Using reverse genetics we engineered the H275Y mutation into the neuraminidase of a 2009 pandemic H1N1 virus and assessed the ability of this enzyme to desialylate mono- and multivalent substrates. The growth kinetics of wild-type and mutant viruses were assessed in Madin–Darby canine kidney (MDCK) and fully differentiated human airway epithelial (HAE) cells. RESULTS: The presence of H275Y was associated with a 1.3-fold decrease in the affinity of the neuraminidase for a monovalent substrate and a 4-fold compromise in desialylation of multivalent substrate. This was associated with a fitness cost to viral replication in vitro, which only became apparent during competitive replication in the mucus-rich HAE culture system. CONCLUSIONS: The neuraminidase protein of pandemic influenza isolates tolerates the H275Y mutation and this mutation confers resistance to oseltamivir. However, unlike seasonal H1N1 viruses isolated since 2007, the mutation is not associated with any fitness advantage and thus is unlikely to predominate without further antigenic drift, compensating mutations or intense selection pressure. March 2009 saw the emergence of a novel strain of influenza with the ability to transmit readily between humans. The rapid global spread of the new influenza A/H1N1 virus resulted in the first influenza pandemic in 40 years. 1 Two therapies are currently licensed to treat influenza infections: the M2 ion channel blocking adamantane drugs (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (oseltamivir and zanamivir). Pandemic influenza A/H1N1 2009 virus (pH1N1) crossed into humans carrying a well-characterized amantadine-resistance mutation, S31N, within the M2 ion channel protein, rendering the adamantane class of antiviral drug ineffective against the virus. Initial isolates were susceptible to NA inhibitors, including oseltamivir and consequently this drug was used extensively in the treatment and prophylaxis of pandemic influenza. 2 Oseltamivir resistance emerged infrequently in pH1N1 2009 influenza viruses. 3, 4 In contrast, seasonal H1N1 influenza viruses from the 2007-08 season onwards were predominantly resistant to oseltamivir with resistance conferred by the H275Y mutation in the viral NA. 5 Traditionally, the H275Y mutation was associated with compromised viral fitness amongst H1N1 isolates. 6 However, isolates from the 2007 -08 season with this mutation suffered no attenuation. 7 It is likely that certain other sequence variations, such as the D344N (N1 numbering) change found within the NA gene of oseltamivir-resistant viruses from the 2007-08 season, counteract the decrease in enzyme function that H275Y confers. 8, 9 Residue 344 is tyrosine in most avian influenza NA genes but mutated to asparagine in two N1 viruses, which crossed from birds into mammals shortly thereafter (the 1918 pandemic virus and the Eurasian lineage swine H1N1 viruses). 10 During circulation in humans, an aspartic acid at residue 344 was eventually selected in seasonal H1N1 viruses. Mutation back to asparagine occurred in circulating human H1N1 viruses prior to the 2007 season and was associated with increased NA activity. 9 The NA enzyme of pH1N1 virus is derived from a Eurasian lineage H1N1 swine virus, and harbours asparagine at residue 344. This suggested that it may tolerate mutations such as H275Y that would concomitantly decrease NA activity and confer oseltamivir resistance. This study aimed to assess in vitro the effect of the H275Y mutation in the NA of a prototypic pH1N1 2009 isolate.
Seven RNA segments of laboratory-adapted strain [A/Puerto Rico/8/34 (H1N1)] were combined with the NA of a pH1N1 strain [A/England/195/09 (pH1N1)] to rescue a pair of isogenic viruses that differed only at position 275 (wild-type PR8+E195 and mutant PR8+E195 H275Y ). Similarly a pair of isogenic viruses based on the whole genome of A/England/195/09 were rescued according to published methods. 11 The sialidase enzyme properties of both wild-type NA and the mutant H275Y NA were assessed using a fluorescent substrate [2 ′ -(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MuNANA)] as previously described. 12 Enzyme kinetic experiments were performed on a standardized amount of each virus for which the NA metabolized 10 nmol substrate/h. A chicken erythrocyte elution assay, where NA is required to cleave sialic acid from multivalent cell surface moieties of the red blood cell, was established. Equal haemagglutination titres of each virus were diluted in 2-fold steps and mixed with PBS containing 1% chicken red blood cells in a V-bottomed microtitre plate and left at 48C for 1 h to determine the haemagglutination endpoint titre. The plate was then incubated for 2 h at 378C. The last dilution of virus at which haemagglutination was lost was defined as the desialylation endpoint titre.
To assay virus growth kinetics, two isogenic viruses containing eight segments of pH1N1 (A/England/195/09) were grown on either Madin-Darby canine kidney (MDCK) cells (originally sourced from the European cell culture collection) or MucilAir TM human airway epithelial (HAE) cells (Epithelix-Sà rl) at a multiplicity of infection (moi) of 0.01. At 12, 24, 48 and 72 h post-infection (MDCK) or 24, 48, 72, 96 and 120 h post-infection (HAE), viruses were collected from the apical surface and subsequently plaqued on MDCK cells to assay for viral titre.
In competition assays, the two viruses were mixed at a defined ratio of either 50 :50 or 80 : 20 (mutant:wild-type) and used to infect MDCK cells or HAE cells at a total moi of 0.01. After 72 h, the released virus was pyrosequenced to quantify the proportion of each genotype.
Mutation H275Y in A/England/195/09 NA confers resistance to oseltamivir
The N1 NA of pH1N1 that carried the H275Y mutation showed an 300-fold increase in oseltamivir IC 50 values (Table 1 ), but the mutation did not confer resistance to zanamivir. There was a 1.3-fold decrease in the binding affinity of the enzyme for the small MuNANA substrate resulting from the resistance mutation as indicated by the difference in K m values (Table 1) . Furthermore, the EC 50 of oseltamivir for virus containing wild-type NA was between 10 and 100 nM, whereas for the H275Y mutant this value was .10 mM since the virus still formed plaques at this concentration of drug (data not shown).
Both recombinant viruses grew to equivalent high titres in MDCK cells and haemagglutinated chicken red blood cells with a geometric mean titre (GMT) of 128. Following incubation of the microtitre plates at 378C, wells containing high titres of virus showed haemagglutination reversal. This is accounted for by the digestion of the sialic acid receptor from the erythrocyte surface by active NA enzyme. The ability of the wild-type pH1N1 to elute virus from red blood cells was 4-fold greater than that of the H275Y isogenic mutant ( Table 1) .
The ability of isogenic A/England/195/09 recombinant viruses that differed only at residue 275 in the NA to infect and spread in MDCK or in differentiated HAE cells was assessed by Recombinant influenza viruses containing the NA from representative pandemic influenza A/England/195/09 were assessed for their ability to catabolize mono-and multivalent substrates. The MuNANA substrate was used to determine IC 50 values for two NA inhibitors and assess enzyme K m values. IC 50 and K m values are given as the mean of two determinations, and the SEM is indicated for K m values. For red cell elution, chicken erythrocytes were first mixed in equal volumes with 2-fold serially diluted virus and incubated on ice to determine haemagglutination titres. The microtitre plate was then moved to 378C to allow NA to cleave sialic acid residues and reverse haemagglutination. The geometric mean titre derived from duplicate wells shown is the last dilution at which an effect was seen. A lower desialylation titre indicates a reduced ability of the NA to cleave sialic acid and reverse haemagglutination.
Oseltamivir-resistant pandemic influenza A H1N1 virus 467 JAC performing infections at low multiplicity. Both viruses replicated efficiently in either cell culture system. Although there was an 0.75 log 10 decrease in the amount of mutant virus released from HAE cells at 72 h post-infection, this was not statistically significant (Figure 1a and b) .
The two viruses were used to co-infect triplicate wells of either MDCK or HAE cells at defined ratios and after 72 h released virus was analysed for the presence of H275Y mutation. Pyrosequence analysis showed that in MDCK cells there was no growth advantage for virus with either H or Y at residue 275 ( Figure 2 ). The output virus from all three wells of MDCK cells contained the same mixture of wild-type and mutant genomes as the input. Thus, the mean percentage of wild-type genotype (H275) following inoculation of the 50: 50 mixture was 50.3% and after inoculation of the 80 : 20 mutant:wild-type mixture this value was 21.4%. In contrast, after 72 h of propagation in HAE cells where mucus could accumulate, the wild-type oseltamivirsusceptible H275 genotype accumulated to a higher degree in two out of three wells than did the drug-resistant variant (Figure 2c) . The mean percentage of wild-type genome in the mixture after 72 h was 59%. The standard deviation of the pyrosequencing assay using this primer set was calculated by performing triplicate runs on six different extracted mixtures and was measured at ,1% (data not shown). Thus, the observed enrichment of wild-type genome after propagation in HAE cells was not a result of assay variation but rather indicative of a selective advantage of H275 in this system.
The biological consequences of the H275Y mutation in the NA gene of pH1N1 influenza virus, which confers resistance to oseltamivir, are important because the drug is a first-line treatment for patients who present with pandemic influenza infection. Drug resistance was already observed in infected individuals in the community and in the clinic during the first and second waves of the swine flu pandemic 4,13 although it did not spread widely. Whether oseltamivir-resistant pH1N1 viruses might disseminate in subsequent waves through the community is key to future public health planning.
The NA gene of the new pandemic H1N1 virus was acquired from Eurasian swine influenza H1N1 virus, a lineage of virus that crossed from bird to pigs in the late 1970s. In this background, the small compromise in enzyme affinity for sialic acid substrate (observed by a 1.3-fold increase in the NA K m value) and the decrease in cell surface expression that results from the H275Y mutation, 14 had no effect on virus growth in MDCK cell culture. On the other hand, in vivo NA must cleave complex substrates to mediate virus release from an infected airway cell and gain access through a complex layer of mucins to the new target cell. Subtle decreases in NA activity or cell surface expression may have more profound consequences in the airway than in monoculture. To probe this, we tested the NA activity of the mutated virus in assays that presented large sialylated substrates: in a red cell elution assay we detected a 4-fold compromise in the ability of the virus with H275Y mutation to mediate desialylation of chicken erythrocytes, although the biological significance of this assay is not entirely clear. In differentiated cultures of human airway cells, the difference detected in replication of wild-type and mutant virus was not statistically significant on either of two separate occasions (Figure 1 and data not shown) . However, in competition assays in HAE cultures, the wild-type virus out-competed growth of the drug-resistant strain suggesting that, in the absence of drug, the 275Y motif carries a fitness cost in the environment of the human airway.
In both recent seasonal H1N1 strains and in H5N1 highly pathogenic viruses, mutations that increase the NA activity, protein stability or cell surface transport likely compensate for the effects of the mutation at 275 that would otherwise decrease the function of the enzyme. 8, 9, 14, 15 Examples of such mutations are D344N, 9 and V235M and/or R223Q. 14 Neither of Brookes et al. the latter two mutations currently exist in the NA protein of pH1N1 isolates, but further circulation of the pH1N1 virus in humans may select for these or other NA or haemagglutinin (HA) mutations that better prime the virus to accommodate or even select for the H275Y mutation.
For contemporary pH1N1 viruses, the cost or advantage of drug resistance is so subtle that different groups have come to different conclusions about its relevance. In a hospital setting there have been reports that suggest that patient-to-patient transmission of drug-resistant virus has occurred amongst immunocompromised individuals. 16 Hamelin et al. 17 showed that oseltamivir-resistant pH1N1 virus was equally virulent as its wild-type counterpart in mice and ferrets and did transmit to co-housed animals, though they did not assess droplet transmission. Seibert et al. 18 used guinea pigs and ferrets in both contact and droplet transmission studies and concluded that the drug-resistant mutant could potentially circulate in the community. However, a detailed analysis of their data reveals that for one of the viruses they tested, there was a 2 day delay in transmission to half the contact-exposed guinea pigs and for the other strain of pH1N1 there was a reduction in droplet transmission to 88% rather than 100% of exposed sentinels. The ferret experiments were conducted with n¼ 1 so it is difficult to be sure of their significance. Similarly, in the manuscript from Kiso et al., 19 the conclusion is again that the H275Y mutant transmits through the air between ferrets, but there is a 2 day delay in transmission of one of the mutant strains of virus studied. Conversely, Duan et al. 20 found that the drug-resistant virus did not transmit between ferrets by the respiratory droplet route and that in co-infected animals, the wild-type virus outgrew the resistant mutant and was uniquely transmitted to contact animals. Thus, the current picture from animal experiments is confused and discrepant. This might be partly due to the use of different strains of pH1N1 virus as well as different experimental protocols used by the various investigators. Anecdotal evidence from the clinic shows that, in most instances, contemporary drug-resistant variants of pH1N1 were replaced by drug-susceptible variants when the selective pressure of oseltamivir was removed, suggesting that wild-type isolates are fitter in vivo in humans. 21 Whether the subtle fitness deficit reported here for one particular strain of drug-resistant mutant pH1N1 virus explains the epidemiological observation that mutant virus has not circulated through the community is not clear, since many other factors including heterogeneous mixing of populations and stochastic effects may influence whether a particular virus mutant predominates. Nonetheless the virus competition assay conducted in HAE cultured cells described here offers an alternative biologically relevant model as a useful adjunct to animal studies and this system may more accurately reflect the environment in which virus replicates in otherwise healthy humans. Information from a variety of model systems should be combined to guide the appropriate use of oseltamivir. Such knowledge clearly needs to be revised specifically for each novel influenza virus that emerges either as a seasonal strain by drift or as a pandemic virus by antigenic shift.  Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus BACKGROUND: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 10(5 )to 10(7 )CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA. RESULTS: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion. CONCLUSIONS: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis. Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) present a major public health problem because of recent increases in the incidence of these infections [1, 2] . In a 2007 report, the Centers for Disease Control concluded that Staphylococcus aureus is now the most important cause of serious and fatal infection in the United States [3] . The prototypical USA400 strain, MW2, (CDC nomenclature for this strain of MRSA) was first isolated in 1999 from a Midwest child with fatal CA-MRSA pneumonia [4] . In 2003, the prototypical USA300 CA-MRSA strain, LAC, was isolated from Los Angeles County patients with skin and soft tissue infections, severe pneumonia and sepsis. Recently, concerns about CA-MRSA infections were heightened after reports of severe invasive staphylococcal infections in some patients infected with the novel 2009 H1N1 influenza A virus [5, 6] .
CA-MRSA isolates express many virulence factors [7, 8] , including several cytolysins: α-toxin, γ-toxin, Panton-Valentine leukocidin (PVL), phenol-soluble modulins (PSMs), δ-toxin and, unlike traditional hospital-associated (HA-MRSA) isolates, may express superantigens such as TSST-1 [9] . These bacterial components can stimulate massive cytokine release and lead to septic shock, acute respiratory distress syndrome (ARDS) and death. It is likely that strategies designed to modulate the excessive and prolonged host inflammatory response could improve the outcome of fulminant MRSA infections.
Monocytes and macrophages play important roles in host defense against staphylococci and other pyogenic bacteria [10] , but excessive systemic or local production of inflammatory mediators by macrophages could be deleterious in patients with severe staphylococcal infections. We previously reported that RAW264.7 murine macrophages exposed to any of a series of six pediatric clinical isolates of S. aureus (two CA-MRSA, two HA-MRSA, and two methicillin-susceptible strains) in the presence of daptomycin (vs. vancomycin) secreted less TNF and accumulated less inducible nitric oxide synthase (iNOS) protein [11] . Vancomycin is a cell-wall active antibiotic that triggers bacterial lysis; it is the antibiotic most commonly used to treat severe MRSA infections in children [12] . Daptomycin is a novel antibiotic that is rapidly bactericidal against staphylococci but does not appear to cause rapid bacterial lysis; the mechanism of its action is not certain but it is reported to trigger depolarization of the bacterial membranes and inhibition of both DNA and RNA synthesis [13, 14] . The rapid lysis of staphylococci, streptococci and other pyogenic bacteria exposed to cell-wall active antibiotics such as beta-lactams and vancomycin results in exaggerated release of bacterial products and an augmented and potentially harmful host inflammatory response [15, 16] . Therefore, optimal treatment of sepsis and other severe bacterial infections might include the use of antibiotics and/or other medications that blunt the host inflammatory response and dampen the cytokine cascade [16] .
Ketamine is one of the recommended anesthetics in pediatric septic shock [17] [18] [19] , which is frequently caused by staphylococci [12, 20] . The reasoning for ketamine's use in staphylococcal septic shock is its blood pressure supporting effect. It increases cardiac output and blood pressure, possibly via a catecholamine release mechanism [17, 21] . Some data suggest that ketamine has anti-inflammatory effects [22] [23] [24] [25] . For example, it has been reported that ketamine suppresses macrophage TNF secretion in response to Gram-negative bacterial LPS in vivo and in vitro [22, 23, 25] . There is also one report that ketamine suppresses TNF production by human whole blood in vitro after exposure to staphylococcal enterotoxin B [24] . The mechanisms responsible for the anti-inflammatory effects of ketamine are not known [22] [23] [24] [25] .The present study examined the hypothesis that ketamine could suppress macrophage TNF production in response to whole bacteria, in this case clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Given the important role of TNF in sepsis [26] [27] [28] [29] , and the importance of staphylococcal sepsis in children, such suppression could have a therapeutic impact.
Although membrane-bound Toll-like receptors (TLR2 and TLR4) are essential for lipopolysaccharide (LPS)induced TNF production [30] , this is not the case for Staphylococcus aureus. Because S. aureus is able to "attack" or form pores in macrophages, TNF secretion occurs even in the absence of TLR 2 and TLR4 sensors (possibly via Nod1 and Nod2, intracytoplasmic sensors of peptidoglycan-derived muropeptides) [31] . Therefore, another mechanism independent of Toll-like receptors must exist for ketamine's anti-inflammatory action, at least in staphylococcal infections.
We also tested the effects of two chemically unrelated NMDA receptor antagonists, the anti-convulsant MK-801 (dizocilpine) [32, 33] , a non-competitive inhibitor of NMDA receptors, and APV (D-2-amino-5-phosphonovalerate), a competitive NMDA receptor antagonist [34, 35] , as well as the NMDA substrate itself, on macrophage TNF secretion in response to antibiotic-treated CA-MRSA bacteria.
For these studies, we utilized two well-characterized clinical isolates: LAC (Los Angeles County), representative of the USA300 group of organisms and closely related to the dominant CA-MRSA clone associated with soft tissue infections and serious invasive disease in the Memphis area [1] , and MW2, a clinical isolate from a midwestern child with fatal CA-MRSA sepsis [4] , representative of the USA400 group of organisms that constitute the other main lineage of CA-MRSA isolates in the United States.
Bacteria were grown to late logarithmic phase at 37°C in tryptic soy broth (Becton Dickinson and Co., Sparks, MD) and washed three times in endotoxin-free phosphate-buffered saline. Concentrations were determined by colony counts. A range of concentrations of bacteria (10 5 -10 7 CFU/mL) was studied, based upon our previously published data with other CA-MRSA strains [11] and our preliminary experiments using LAC and MW2 (data not shown).
Minimum inhibitory concentrations (MICs) for these strains were determined by the microbiology laboratory at Le Bonheur Children's Hospital using the E-test method: both strains were fully susceptible to vancomycin and daptomycin (LAC: MIC vancomycin 1.0 μg/mL; daptomycin 0.75 μg/mL; MW2: MIC vancomycin < 0.5 μg/mL; daptomycin 0.75 μg/mL).
Cell culture RAW264.7 murine macrophage-like cells were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium (Mediatech Inc., Herndon, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 2 mM glutamine (GIBCO, Carlsbad, CA). Experiments were done in 24-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) with 1 × 10 6 cells per well.
Either vancomycin or daptomycin was added to the cell cultures immediately before the addition of live staphylococci (10 5 -10 7 CFU/mL). Cells were then incubated for 18 hours. Daptomycin was obtained from Cubist Pharmaceuticals (Lexington, MA). Vancomycin was purchased via the Department of Pharmacy at Le Bonheur Children's Hospital (LBCH) from Hospira (Lake Forest, IL). Clinically achievable concentrations of each of the antibiotics, as previously tested in our laboratory [11] , were used (20 μg/mL).
These experiments were repeated in parallel in the presence of ketamine (100 μM) and/or MK-801 (dizocilpine, 150 μM), APV (D-2-amino-5-phosphonovalerate, 300 μM ("low") or 3 mM ("high"), or NMDA (30 μM). The modulation of MRSA-stimulated macrophage TNF production by ketamine was subsequently examined also at a range of concentrations of 10 μΜ, 50 μΜ, 100 μΜ and 150 μΜ. The selected concentration (100 μM) is based on the achievable anesthetic concentrations [36] [37] [38] [39] and on the pre-existing literature related to ketamine's TNF suppressive effect on murine macrophage models when stimulated by LPS [23] [24] [25] 27] . The concentrations for the other factors were selected from the available literature, MK-801 [40] [41] [42] , APV and NMDA [32] have previously been studied in cell culture models and have been shown to not cause cytotoxicity at the tested concentrations. Ketamine and/or MK-801 or APV or NMDA were added to the macrophage cultures one hour prior to bacterial challenge. The source of ketamine was Ketalar ® , a racemic mixture (1:1) of optically active isomers (R and L) of this drug, purchased from the LBCH pharmacy. Emphasis in the experiment was placed on correlation with the clinical situation; thus racemic ketamine, the most commonly clinically used product, was selected. Dizocilpine (MK-801), APV and NMDA were purchased from Sigma Chemical Co. (St. Louis, MO).
After incubation, cell-free supernatants were collected and assayed for TNF concentrations by using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer (eBioscience, San Diego, CA). TNF is a key cytokine produced by macrophages during MRSA stimulation. In our preliminary studies, we also measured secretion of other cytokines and found that IL-1, IL-6, and NO secretion were strongly correlated with TNF secretion in response to these bacteria (r 2 = 0.84, 0.87 and 0.93, respectively). We focused on TNF secretion for these studies.
The tested concentrations of vancomycin, daptomycin, ketamine, MK-801, APV, and NMDA had no effect on the viability of the RAW264.7 cells, as determined by visual inspection of the monolayer, low power microscopic inspection of the monolayer and exclusion of 0.2% trypan blue dye.
For the single comparison experiments (ketamine or MK 801 or APV), TNF secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times (a total of at least nine samples). The four preliminary runs and all the exposures (total of 16) where the inocula were different from 10 5 to10 7 CFUs/mL at the verifying colony count were excluded from the final analysis. Experiments with different exposure times (6, 10, 14, 24 hours) were conducted to determine whether the inhibition increased over time. In the multiple comparison experiments (ketamine and MK 801 synergistic action), TNF was measured from at least four well replicates. All experiments were performed separately for LAC and for MW2 MRSA strains. There is an intrinsic experimental variation of absolute values of TNF production (up to 25%) because of cell culture and macrophage growth characteristics.
The design was composed of factorial multiple measurements and the results were analyzed according to a mixed linear model, (GLIMMIX) SAS 9.2 (SAS Institute, Cary, NC) and R 2.9.1 and ggplot2 software. We set pre-planned (a priori) contrasts, i.e., we set all our comparisons in advance of multiple setting experiments. Significant differences were presumed at a probability value of p < 0.05. The results were graphed using error bars with 95% confidence intervals. Differences in the means were estimated either with asymptotic techniques for normally distributed data or bootstrapping techniques for non-normally distributed data.
CA-MRSA strains MW2 and LAC stimulated less TNF secretion by RAW264.7 murine macrophages in the presence of daptomycin than in the presence of vancomycin As previously observed with two USA300 CA-MRSA strains isolated from Memphis children with invasive staphylococcal infections [11] , macrophages exposed to either of the two prototypical CA-MRSA strains studied (the USA300 strain, LAC, or the USA400 strain, MW2) secreted significantly less TNF in the presence of daptomycin as compared with vancomycin (more than 50% reduction in each strain; Figure 1 ). Macrophage TNF secretion in response to MW2 was 34,535 ± 1,536 pg/ mL in the presence of vancomycin and 15,377 ± 1,267 pg/mL in the presence of daptomycin, a reduction of 55%, significant at p < 0.05. Similarly, macrophage TNF secretion in response to LAC in the presence of vancomycin was 33,345 ± 1,535 pg/mL, and 14,432 ± 1,536 pg/mL in the presence of daptomycin, a reduction of 57%, significant at p < 0.05. We previously reported similar findings in six S. aureus clinical isolates (including two pediatric CA-MRSA isolates of the USA300 group), suggesting that this effect of daptomycin is conserved in many different S. aureus isolates.
The addition of ketamine (100 μΜ) to macrophage cell cultures inhibited TNF secretion in response to vancomycin-or daptomycin-exposed CA-MRSA isolates ( Figure 2) . The effect was similar on both strains, LAC and MW2, in the presence of vancomycin (upper panel) or daptomycin (lower panel).
In the initial experiments we analyzed the effect of one hour pre-incubation with ketamine on the macrophage response to vancomycin-exposed CA-MRSA bacteria (MW2 and LAC). In response to vancomycinexposed MW2, pre-incubation with ketamine reduced macrophage TNF secretion by approximately 29% (p < 0.05), i.e., from 33,085 ± 867 pg/mL to 23,347 ± 862 pg/mL. Pre-incubation with ketamine led to a similar reduction (25%; p < 0.05) in macrophage TNF secretion response after stimulation with vancomycinexposed LAC (from 28,365 ± 735 pg/mL to 21,432 ± 736 pg/mL).
We next studied the effect of ketamine pre-incubation on macrophage TNF secretion after stimulation with daptomycin-exposed MW2 or LAC. Once again, the addition of ketamine resulted in significant inhibition of macrophage TNF secretion in response to MW2 (23,185 ± 1,267 pg/mL to 17,354 ± 853 pg/mL, a reduction of approximately 25%; p < 0.05) or LAC (approximately 18% reduction, p < 0.05; Figure 2 ). Adding ketamine after the MRSA inocula did not alter the response. Figure 1 The CA-MRSA isolates LAC (USA300) and MW2 (USA400) stimulated less TNF secretion by RAW264.7 murine macrophages when exposed to daptomycin (DAP) than when exposed to vancomycin (VAN). LAC or MW2 were added to RAW264.7 cells at a final concentration of 10 5 to 10 7 CFU/mL (retrospective confirmation) in the presence of either vancomycin or daptomycin at 20 μg/mL. Cells were incubated for 18 hours; supernatants were collected and analyzed for TNF content by an enzyme-linked immunosorbent assay (ELISA). Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF macrophage production by macrophages not stimulated with bacteria.
MRSA MW2 and LAC exposed to Ketamine One hour prior to stimulation, ketamine (100 μM) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA.
Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF macrophage production by macrophages not stimulated with bacteria. The NMDA inhibitor MK-801 (dizocilpine) inhibited macrophage TNF secretion after stimulation with antibiotic-exposed CA-MRSA strains
Pre-incubation of RAW264.7 cells for one hour with the NMDA receptor antagonist, MK-801 (150 μΜ), also inhibited TNF secretion by these cells after stimulation with antibiotic-exposed CA-MRSA strains (MW2 or LAC, Figure 3 ). In response to stimulation with MW2 in the presence of vancomycin, pre-incubation with MK-801 significantly inhibited TNF secretion by these cells, i.e., from 32,407 ± 1,188 pg/mL to 23,337 ± 1,272 pg/mL (approximately 28% reduction; p < 0.05, Figure 3 , upper panel). MK-801 also inhibited macrophage TNF secretion in response to vancomycin-exposed LAC, causing a 34% reduction (Figure 3, upper panel) . Pre-incubation with MK-801 also significantly inhibited macrophage TNF secretion in response to daptomycin-treated MW2 or LAC (Figure 3 , lower panel). In response to stimulation with MW2 in the presence of daptomycin, pre-incubation with MK-801 inhibited TNF secretion by these cells by approximately 26% (from 22,305 ± 648 pg/mL to 16,437 ± 642 pg/mL, p < 0.05). MK-801 inhibited macrophage TNF secretion in response to daptomycin-exposed LAC by approximately 33% (from 22,164 ± 864 pg/mL to 14,647 ± 832 pg/mL, p < 0.05).
Pre-incubation of RAW264.7 cells with combinations of MK-801 and ketamine did not affect the magnitude of inhibition of macrophage TNF secretion observed in the presence of ketamine (or MK-801) alone. Figure 4 depicts results for macrophages stimulated with vancomycin-or daptomycin-exposed MW2; responses to antibiotic-exposed LAC were similar (data not shown). One hour prior to stimulation, either ketamine at 100 μM, MK-801 at 150 μΜ, or both were added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Lane 1 (control) represents the mean TNF production by macrophages not stimulated with bacteria. The mean includes wells exposed to ketamine, MK-801, both ketamine and MK-801, and neither. In the absence of bacteria, TNF secretion was minimal and was not affected by ketamine and/or MK-801. NMDA augments macrophage TNF secretion in response to antibiotic-treated CA-MRSA bacteria: both ketamine and a competitive NMDA receptor antagonist, APV, block this effect
We further examined the role of NMDA receptors in modulating the macrophage TNF response to the CA-MRSA bacteria by studying the effects of a competitive NMDA receptor antagonist, APV, and the effects of the NMDA substrate itself ( Figure 5 ). We found that APV (at either 300 μM or 3 mM) also inhibited macrophage TNF secretion in response to vancomycin-exposed MW2 (p < 0.05, Figure 5 ). The magnitude of the inhibition was comparable to that observed with either ketamine or MK-801 (and, as in the case of MK-801, was not additive or synergistic with ketamine). Furthermore, the addition of the NMDA substrate (30 μM) resulted in a marked augmentation of the macrophage TNF response to the antibiotic-treated CA-MRSA bacteria (p < 0.05), and this effect was blocked by ketamine and by the competitive NMDA receptor antagonist, APV ( Figure 5 ).
We next studied the effects of a range of concentrations of ketamine and found that inhibition of macrophage TNF secretion in response to vancomycinexposed LAC or MW2 was consistently observed at concentrations of ketamine at the lowest concentration tested (10 μM) and was greater at concentrations of 50 -150 μM ( Figure 6 ). We also examined the kinetics of inhibition of macrophage TNF secretion by incubating RAW264.7 cells for 6, 10, 14, 18 and 24 hours after exposure to ketamine at a concentration of 100 μM 1 hour prior to stimulation with vancomycin-exposed LAC or MW2. We found that the magnitude of suppression of TNF secretion was similar at all times studied (Figure 7 ).
We found that exposure of murine macrophages to ketamine inhibited TNF secretion by 18-34% after stimulation with CA-MRSA bacteria in the presence of antibiotics. The magnitude of the effect was comparable in response to both MW2 (USA400) and LAC (USA300) bacteria and was similar in the presence of either vancomycin (a lytic antibiotic associated with a greater TNF response to the bacteria) or daptomycin (a non-lytic antibiotic associated with a blunted TNF response to the bacteria). Our data suggest that ketamine administration to macrophages stimulated by CA-MRSA is associated with blunting of the TNF response to these virulent pathogens, and suggest that these findings may have therapeutic significance in MRSA sepsis. Furthermore, these data confirm and extend our previous observations that CA-MRSA bacteria exposed to daptomycin (versus vancomycin) trigger less TNF secretion by macrophages. The potentially beneficial antiinflammatory effects of daptomycin and ketamine were additive (Figures 2, 3) . Bacteria were added at a final concentration of 10 5 to10 7 CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. One hour prior to stimulation, APV ("low" concentration of 300 μM or "high" concentration of 3 mM), ketamine (100 μM), or NMDA (30 μM) were added, alone or in combination, as indicated. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. The control lane represents the mean TNF macrophage production by macrophages not stimulated with bacteria. The mean includes wells exposed to APV, ketamine, or NMDA alone or in combination. In the absence of bacteria, TNF secretion was minimal and was not affected by APV, ketamine, or NMDA. Lanes 0-9 depict mean TNF secretion by macrophages exposed to vancomycin-treated MW2 alone (lane 0) or in the presence of the indicated concentrations of APV, ketamine, and/or NMDA (lanes 1-9). TNF secretion was reduced by approximately 30-40% when macrophages were pre-incubated with APV, ketamine, or APV + ketamine (lanes [1] [2] [3] [4] [5] . The magnitude of inhibition by ketamine and high-dose APV was similar and there were no additive or synergistic effect observed with combinations of ketamine and APV. Addition of NMDA (30 μΜ) led to a substantial increase in the amount of TNF secreted in response to the MW2 strain (lane 9), and this augmented response was blocked by both APV and ketamine. The "*" on "0.Control" and "8.NMDA+lo_APV" bars indicates significance at p < 0.05. The "**" on "0.Control_MW2" and "9.NMDA" bars indicates differences between the pretreated wells, and that TNF production after MRSA stimulation with NMDA substrate (9.NMDA) is significantly higher than that at the baseline MRSA stimulation (0.Control_MW2) at p < 0.05.
An improved understanding of the pathogenesis of sepsis and other life-threatening infections caused by CA-MRSA bacteria could expedite the development of novel strategies for the diagnosis, treatment, and/or prevention of these serious infections. CA-MRSA infections often are associated with severe and prolonged host inflammatory responses [43] [44] [45] [46] . Prompt antibiotic treatment of these and other serious bacterial infections is indicated, but paradoxically has the potential to trigger excessive release of bacterial products and the subsequent augmentation of the host inflammatory response [15, 16] . Macrophages are important sources of many of the proinflammatory cytokines (including IL-1β, IL-6, IL-8, IL-12, and TNF) secreted in response to staphylococci and other Gram-positive bacteria [15, 16, 41] . Although the cytokine cascade is essential for normal host defense, excessive or inappropriate inflammation can be harmful. Therefore we need an improved understanding of these interactions in order to develop better adjunctive therapies for patients with severe bacterial infections.
In a previous study, we found that exposure of either of two CA-MRSA strains isolated from Memphis children (or any of four other S. aureus isolates from children with invasive staphylococcal infections) to daptomycin (compared with vancomycin) led to a less pronounced macrophage inflammatory response, characterized by diminished secretion of TNF and reduced accumulation of the inducible nitric oxide synthase (iNOS) [11] . In this study, we found that this differential effect of daptomycin (versus vancomycin) was also observed when macrophages were stimulated with either of the two prototypical CA-MRSA strains most widely studied today: the USA400 isolate, MW2, and the USA300 isolate, LAC. Importantly, ketamine pre-incubation inhibited macrophage TNF secretion in response to both CA-MRSA strains in the presence of daptomycin as well as in the presence of vancomycin, and the greatest suppression of TNF secretion was noted in the presence of both daptomycin and ketamine.
The mechanism(s) responsible for the anti-inflammatory properties of ketamine are not known, but its neurological and psychotropic actions are believed primarily to be mediated by antagonism of NMDA receptors [21, 47] . Glutamate is the brain's primary excitatory neurotransmitter. NMDA receptors are found in many cell Bacteria were added at a final concentration of 10 5 to10 7 CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. The ketamine concentration was 100 μM. Results are depicted as percentile reduction with 95% confidence intervals, i.e., the percent of TNF reduction at the specific exposure time that occurs in comparison to inoculation without ketamine at the same time. The "*" indicates statistically significant difference at p < 0.05.
types, including blood lymphocytes, lung macrophages, and multiple hematopoietic precursors in bone marrow cells [40, 42, 47, 48] . Both ketamine and the chemically unrelated anticonvulsant dizocilpine (MK-801) are noncompetitive antagonists of the NMDA receptor, one of the three known glutamate receptors [32, 33, 47] . APV is a competitive inhibitor of the classical NMDA receptor and acts on the NR2 component of the receptor (30, 33) . We found that MK-801 and APV also inhibited macrophage TNF secretion in response to antibiotictreated MW2 or LAC cells. The magnitude of the inhibition by MK-801 (approximately 30%) and APV (25-35%) was comparable to that observed with ketamine (18-34%), and combinations of MK-801 and ketamine or of APV and ketamine did not exhibit additive or synergistic inhibition of TNF secretion. Furthermore, adding NMDA led to augmented macrophage TNF secretion in response to antibiotic-treated CA-MRSA bacteria, and the NMDA receptor antagonist, APV, blocked this effect. The suppression of TNF induced by ketamine was observed across a range of concentrations and throughout the incubation period.
Our study has its limitations. To translate the present findings, we are currently working on a clinical model to assess the clinical significance of ketamine's anti-inflammatory effects in patients with bacterial sepsis. Although studies of the effect of ketamine on macrophage responses to purified bacterial components such as Gram-negative lipopolysaccharide (LPS) or Gram-positive lipoteichoic acid (LTA) are instructive [23, 24, 49] , we argue that characterization of the macrophage responses to whole organisms is more likely to provide clinical insights. Indeed, the pioneering experiments of Carswell and Old that identified TNF used whole bacteria as stimuli in macrophage sepsis simulation settings [49] , and we have previously demonstrated that macrophage responses to live, antibiotic-treated staphylococci serve as a powerful model system. Furthermore, the model examines the effect of ketamine only in the presence of antibiotics (either vancomycin or daptomycin). In practice, this is a common clinical scenario. Our data suggest that clinically achievable concentrations of both ketamine and daptomycin could potentially inhibit the excessive macrophage inflammatory response that is observed in patients with severe staphylococcal infections.
In the battle of sepsis everything counts. Adjunctive therapies of sepsis are greatly needed. Studies in animal models and clinical trials will be required to determine whether the anti-inflammatory effects of ketamine and/or other agents that block NMDA receptors could be beneficial in the treatment of severe staphylococcal infections. Avipoxviruses: infection biology and their use as vaccine vectors Avipoxviruses (APVs) belong to the Chordopoxvirinae subfamily of the Poxviridae family. APVs are distributed worldwide and cause disease in domestic, pet and wild birds of many species. APVs are transmitted by aerosols and biting insects, particularly mosquitoes and arthropods and are usually named after the bird species from which they were originally isolated. The virus species Fowlpox virus (FWPV) causes disease in poultry and associated mortality is usually low, but in flocks under stress (other diseases, high production) mortality can reach up to 50%. APVs are also major players in viral vaccine vector development for diseases in human and veterinary medicine. Abortive infection in mammalian cells (no production of progeny viruses) and their ability to accommodate multiple gene inserts are some of the characteristics that make APVs promising vaccine vectors. Although abortive infection in mammalian cells conceivably represents a major vaccine bio-safety advantage, molecular mechanisms restricting APVs to certain hosts are not yet fully understood. This review summarizes the current knowledge relating to APVs, including classification, morphogenesis, host-virus interactions, diagnostics and disease, and also highlights the use of APVs as recombinant vaccine vectors. Avipoxviruses (APVs) are among the largest and most complex viruses known. APVs belong to the Chordopoxvirinae subfamily of the Poxviridae family [1] . They infect and cause diseases in poultry, pet and wild birds of many species which result in economic losses to the poultry industry. Infections have also been reported in a number of endangered species or species in captivebreeding recovery programs [2] [3] [4] . APVs are transmitted via biting insects and aerosols and are usually named on the basis of the bird species from which the virus was first isolated and characterized [4] . The disease, which is characterized by proliferative lesions of the skin and diphtheric membranes of the respiratory tract, mouth and oesophagus has been described in avian species [4, 5] . Although APV infections have been reported to affect over 232 species in 23 orders of birds [6] , our knowledge of the molecular and biological characteristics of APV is largely restricted to fowlpox virus (FWPV) and canarypox virus (CNPV) for which fullgenome sequences are available [7, 8] . Currently, only ten avipoxvirus species are listed under the genus by the International Committee on Taxonomy of Viruses (ICTV) [1] ; Table 1 . Thus, it is safe to assume that many APVs have yet to be characterized. Recombinant APVs have been evaluated for use as vaccine vector candidates against infectious diseases [7, 9] . APV-vectored vaccines are already in use in veterinary medicine [10] [11] [12] [13] [14] , and it is likely that such vaccines will also be used against human diseases in the future. This fact emphasizes the need to learn more about the molecular characteristics of APVs, which underpins the development of safe APV-vectored recombinant vaccines. This review summarizes current knowledge of APVs as avian pathogens, including classification, morphogenesis, hostvirus interactions, diagnosis, as well as issues relevant to their use as recombinant vaccine vectors.
Avipoxviruses are large, oval-shaped enveloped viruses whose genome consists of double stranded DNA ranging in size from 260 to 365 kb [8] . Unlike most other DNA viruses, APVs replicate easily in the cytoplasm of infected avian cells which results in a characteristic cytopathic effect (CPE) 4 to 6 days post infection depending on the virus isolate [4] . APVs also multiply on the chorioallantoic membrane (CAM) of embryonated eggs, resulting in the formation of compact, proliferative pock lesions that are sometimes focal or diffuse [15] . However, some isolates, especially from the host species great tit (Parus major), have failed to multiply on CAM of chicken embryos [16] . APVs are the etiologic agent of disease characterized by skin lesions in both wild and domestic birds [4, 5] . Histologically and ultrastructurally, APVs undergo morphologic stages that are similar to other chordopoxviruses, including the formation of intracytoplasmic inclusions bodies, a characteristic which has been observed in some epithelial and mononuclear cells of permissive hosts. APV particles can be detected and further characterized by use of transmission electron microscopy (TEM) [17, 18] .
Great discoveries made in the mid-nineteenth century facilitated major advances in pox virology. Based on the report by Bollinger [5] on poxvirus infected cells in chickens, and subsequent work by Fenner and Burnet [19] , APVs and other poxviruses were classified on the basis of original host, growth and morphological characteristics in the CAM of embryonated eggs or cell cultures and on clinical manifestations in different diseases of humans, birds and animals [20] rather than on genetic identity, which may provide both rapid and reliable virus identification [21] [22] [23] . These criteria have remained the basis for subsequent classification of APVs despite development of new molecular tools that have the capability of resolving the issue of species specificity of APV.
Members of the genus Avipoxvirus belong to the subfamily Chordopoxvirinae which shares several biological features with other poxviruses [7, 8] . Currently, little is known of the number of species within the genus. While only ten strains have so far been identified and classified Worldwide as APV [1] , avian poxvirus infections have been reported to affect a wide range of bird species [6] . These strains vary in virulence and host specificity, demonstrating an urgent need for further analyses and characterization of new isolates.
Avipoxviruses share several morphological, biochemical and physiochemical features with other poxviruses. Virus particles measure 270 × 350 nm and are composed of an electron dense, centrally located core and two lateral bodies that are visible in fixed and stained ultra-thin sections. In negative stained preparations, such as phosphotungstic acid (PTA) the membrane displays an outer-coat composed of a random arrangement of tubules [24] ; Figure 1 . APV particles have been shown to be resistant to ether, but sensitive to chloroform treatment [25] , although resistance to both chloroform and ether have been reported for pigeonpox virus and two pigeonpox virus mutants [26] .
Avipoxviruses have low G+C content (30 to 40%) and consist of a single linear molecule of double-stranded DNA of between 260-365 kb. The central region of the genome is flanked by two identical inverted terminal repeats (ITRs) which are covalently linked by hairpin loops and contains several hundred closely spaced open reading frames [7] . The central region contains about 90-106 homologous genes that are involve in basic replication mechanisms, including viral transcription and RNA modification, viral DNA replication, and proteins involved in structure and assembly of intracellular mature virions and extracellular enveloped virions [8] .
In general, genes located in this region have common molecular functions and are relatively conserved among poxviruses [8] . This is in contrast to the more variable, terminally located genes that have been shown to encode a diverse array of proteins involved in host range restriction [8] .
Complete genomes of the two most studied APVs, FWPV US (FP-challenge virus; Animal Health Inspection Service Centre for Veterinary Biologicals, Ames Iowa, USA), FWPV-FP9 (a plaque-purified tissue cultureadapted attenuated European virus) and a CNPV virulent strain (Wheatley C93, American Type Culture Collection; ATCC VR-111) have been sequenced [7, 8, 27] . Although nucleotide and amino acids sequences for these two viruses are known, the functions of some putative genes and proteins remain to be fully assigned. Comparison of the FP9 strain with FWPV US revealed 118 differences; of which 71 genes were affected by deletion (26 of 1-9334 bp), insertion (15 of 1-108 bp), substitution, termination or frame-shift [27] . FP9 strain is a derivative of European FWPV HP1 which was obtained through over 400 passages in chicken embryo fibroblast (CEF). Analysis of FWPV HP1 sequences at the loci in which differences exist between FP9 and FWPV US show that 68 of 118 loci differ from the FWPV US, but were identical to FP9. This thus indicate that more than half of the differences between the two geographic FWPV lineages represented differences between the parent virulent viruses FWPV HP1 and FWPV US [27] . Further comparison of molecular data; show that FWPV and CNPV share high aminoacid identity, significant gene-sequence rearrangements, deletions and insertions [8] . The CNPV genome is about 80-100 kbp larger than the FWPV genomes. Both FWPV and CNPV express cellular gene homologues with immunomodulatory functions, which might be responsible for their different virulence and host-range [8] , but CNPV shows a broader tissue tropism in permissive avian hosts [17] than FWPV. CNPV has additional sequence of over 75 kbp, 39 genes lacking in FWPV homologues and approximately 47% amino-acid divergence [8] . These divergences are primarily found in the terminal nonconserved regions [7, 8] . Genes located in the non conserved regions are more prone to mutation and recombination and are implicated in host range, immunomodulation and pathogenesis [28] , and may be responsible in some aspects of cell and/or tissue tropism or perform other cellular functions [8] .
Virulence genes are generally of non-conserved nature and influence the pathological profile of viruses in an infected host. These genes are important in viral evolution and have been used in studies to provide insight into how some poxviruses evolve strategies to ensure their replication [29] . Many of these strategies can be traced back to discoveries made with knockout (KO) viruses, in which a targeted disruption of a specific viral gene produced phenotypic changes reflective of the normal biological function of its protein product. Deletions of some non-conserved genes have also resulted in conditional replication defects in specific cell types [30] , such as the demonstration of spontaneous deletion of host range genes of vaccinia virus resulting in the compromised growth in the mammalian cell [31] . The K1L and C7L genes of vaccinia virus have been shown to be essential for completion of the replication cycle of vaccinia virus in human cells [32, 33] . In a knockout experiment, vaccina virus was unable to complete its replication cycle in Chinese hamster ovary (CHO) cells, since replication was aborted shortly after virus binding and entry, at the stage of intermediate gene expression [34] . But the insertion of another host range gene, CHOhr from cowpox virus into vaccinia virus allowed vaccinia virus to grow in CHO cells in which they are normally restricted [35] [36] [37] . Through use of these techniques, we now have a better understanding of the biology of vaccinia and other poxviruses, including their host range restriction. Although, some major advances have been made in genome sequencing and in vitro characterization of APVs [7, 8, 38, 39] , studies on APV host range genes are scarce. A wide array of gene homologues with likely host range functions such as NK-cell receptors, chemokines, serine protease inhibitors and homologues of genes involved in apoptosis, cell growth, tissue tropism and avian host range, have been identified in APVs, which suggests significant viral adaptation in the avian host [7] . Molecular knockout studies that target identification and further characterization of viral genes involved in regulation of cell proliferation, chromatin remodelling, virulence and apoptosis, in different APV-infected mammalian and avian cells are needed to better understand the tissue tropism and host range characteristics of APVs, including the abortive infection in mammalian cells.
Compared to other poxviruses, such as vaccinia virus, mechanisms that account for APV pathogenesis is poorly understood. APVs have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into host cells. Like many other DNA viruses, APV probably devotes much of its genes to allow it to evade host immune responses. Such viral genes commonly encode proteins that are critical for the virus to undergo molecular transformation that leads to successful membrane fusion, penetration and intracellular transport. These includes genes that encodes proteins which act on early innate pathways such as pathways involving interferon [40] , pattern recognition receptors as Toll-like receptor (TLR) [41] , chemokines [42] and cytokines [43] , as well as pathways that act on subsequent adaptive responses [44, 45] . The infection of a cell by a virus is a complex process, during which the virus must overcome several host factors restriction points and the host immune response. Host protein interaction networks and biochemical pathways are in most cases altered by the viral proteins that free the virus from normal cellular controls and allow nucleotide metabolism in cells that have shut down DNA synthesis [46] . Hence, understanding viral protein functions and their interactions with host proteins is a prerequisite, not only to understand the infection biology of the virus-host system in question, but also for the rational development of target vaccines, based on specific antigens and possibly immunomodulatory factors, as well as antiviral compounds.
Since the first isolation of APVs in cell culture, these viruses have been recognized as highly host specific. They are believed to replicate only in avian cells, notably chicken embryo fibroblasts (CEF; American Type Culture Collection; ATCC, Rockville, Maryland, USA; CRL-1590) [47] . CEF cells have a good split ratio compared to other cell lines, and are thus useful for large-scale propagation of virus, such as antigen production for vaccines or as a diagnostic tool. APVs have also been shown to replicate in chicken embryo kidney, chicken embryo dermis [48, 49] and quail cell lines, such as QT-35, although the presence of viable endogenous herpesvirus and Marek's disease virus (MDV) in QT-35 cells, limits their use for preparation of vaccines [4, 50] . APV have been isolated once from a mammal. In 1969, viable FWPV was isolated from a terminally ill rhinoceros [51] . The isolate was identified as atypical FWPV, based on pathological, virological and serological characteristics [51] . Nelson (1941) [52] reported mild pathology in mice following intranasal inoculation with FWPV, with no virus replication. Recent studies have also shown replication of APV in mammalian cell cultures, such as embryonic bovine tracheal cells [53] and baby hamster kidney cells [54] that are defined by the presence of infectious viral particles and CPE. These studies raise questions about the species specificity and mechanisms that restrict these viruses to certain hosts, and challenge the hypothesis that APV cannot undergo a full replication cycle in mammalian cells.
The cellular entry and exit of APV is complicated by the existence of at least two distinct forms of virus that can productively infect cells, namely the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). These two forms are surrounded by different lipid membranes and surface proteins that are yet to be fully characterized.
After virus binding to cellular membranes, a fusion step, which is generally poorly understood, results in the release of the virion core into the cytoplasm of the cell [39] . The released core, which contains the endogenous RNA polymerase and transcription factors, initiates the first wave of early viral gene transcription by synthesizing viral mRNA under the control of early viral promoters. This is followed by the uncoating stage, the release of viral DNA into the cytoplasm where it serves as a precursor for viral DNA replication as well as the source of intermediate and late viral gene transcription. As a late viral gene product accumulates, the virus undergoes assembly and morphogenesis of infectious virus particles. During morphogenesis, APVs induce the formation of inclusion bodies in the cytoplasm of infected cells (Figure 2A and 2B). The inclusions, which may also be termed viral factories, viroplasms, or viral replication complexes, are generally believed to be the sites of active viral replication and particle assembly within infected cells [17] . One model for the function of viral inclusion bodies is that they act to concentrate and sequester proteins, nucleic acids, and other small molecules essential for viral processes.
In permissive cells, the first viral structures detectable by electron microscopy are the crescent-shaped forms ( Figure 3A ), consisting of a membrane with spicules on the convex surface [39] . These structures develop into non-infectious spherical immature viruses (IV) ( Figure 3B ) from which the intracellular mature virus (IMV) is formed by a series of maturation steps ( Figure 3C ). The IMV represents the majority of infectious progeny from each infected cell [17, 39, 55] . There are three possible mechanisms by which poxviruses are released from host cells depending on the strain of virus, cell type and the post-infection time [54] [55] [56] . They can be released by cytolysis, in which case IMV are released when the cell undergoes lysis as a result of CPE at the advanced stage of infection. It can also be released via virus-induced exocytosis. The third way of release is by budding, in which case IMV migrates out of the virus factory through the plasma membrane. Budding is shown to be the main exit route for APV [39] in contrast to the orthopoxviruses which exit by exocytosis of intracellular enveloped virus (IEV) [57] . These exit processes all result in the acquisition of an additional double membrane [39, 55] . In non-permissive African green monkey cells (CV-1, Vero) and in a human cell line (MRC-5), there is a blockade of the APV morphogenesis cycle. This occurs in steps following the formation of immature virus and is shown to be devoid of an alteration in early gene expression [47, 58, 59] , indicating that this blockade may not be associated with cell receptors.
Poxvirus tropism may not be dependent upon specific cell surface receptors, but rather upon the ability of a given cell to provide intracellular complementing factors needed for productive virus replication, and on the ability of the specific virus to successfully manipulate intracellular signaling networks that regulate cellular antiviral processes following virus entry [28] . APVs have large genomes that would enable them to express unique collections of viral proteins that function as host range factors, which specifically target and manipulate host signaling pathways to establish optimal cellular conditions for viral replication. However, in some cells, especially mammalian cells, APV replication is blocked. This may be due to the ability of APV to specifically activate signalling pathways or mediators, for example interferon pathways, in those cells. The role of mediators and immunopathology of APV is complex and not well understood. However, considering the numerous steps involved in APV morphogenesis, it is relevant to note that these viruses induce several mediators that allow them to survive and interact with the host cells. Some potential mediators have been identified [7] and are awaiting proper functional characterization. These molecules alone may not fully explain the events that have been documented in mammalian cells that have supported the replication of APV [54] . Hence, identification of new mediators that are up or down-regulated in response to APV infected mammalian and avian cells could help advance our knowledge of immune responses against APV and the related immune-mediated pathology and cell tropism. It would be of vital importance to investigate these characteristics further, especially for the cell types that were recently shown to support APV replication [53, 54] .
APV infections are associated with significant levels of morbidity and mortality in domestic and wild bird populations [6, 60] . Most of the investigations and reported cases are based on single APV isolates, which makes it difficult to address the pathogenicity of different APVs in different bird species. Chickens are commonly used to determine the pathogenicity of new isolates, but chickens may not be the ideal host, since APVs from wild birds may not multiply in chickens. In an attempt to identify and characterize the pathogenicity of APVs, Tripathy and others [4] found that wild isolates of Hawaiian crowpox virus had a generally mild pathogenicity in domestic chickens, characterized by relatively minor lesions of short duration at the sites of inoculation, which were in contrast to the general ability of FWPV strains to produce extensive proliferative lesions [4] . In another experimental study, two APV isolates obtained from endangered Hawaiian wild birds, the Hawaiian Goose (Branta sandvicensis) and the Palila (Loxioides bailleui), were compared with FWPV in specific-pathogen-free chickens. Immune responses were measured by ELISA before and after immunization with Hawaiian APVs and after challenge with FWPV. Both isolates from Hawaiian birds developed only a localized lesion of short duration at the site of inoculation in chickens and did not provide protection against subsequent challenge with virulent FWPV, in which severe lesions were observed. In contrast to high antibody response in chickens immunized with FWPV, birds immunized with either of the two Hawaiian isolates developed low to moderate antibody responses against viral antigens [61] . Pathogenicity studies of APVs in parrots [62] , turkeys, pigeons and canaries have also been reported. Canaries were highly susceptible to CNPV, but showed resistance to turkeypox virus, FWPV and pigeonpox virus [4, 63, 64] . A poxvirus from a Canada goose (Branta canadensis) was transmissible to domestic goose, but not to chickens or domestic ducks [15] . Pigeonpox virus produced mild infection in chickens and turkeys, but was more pathogenic for pigeons [62] . Poxvirus isolates from magpies (Pica pica) and great tits (Parus major) did not infect young chickens [16] , however, poxvirus isolated from blackbacked magpie (Gymnorhina tibicen) produced lesions in chickens. These studies were based on clinical manifestations in the chickens and suggest host specificity and pathogenicity.
Despite the worldwide prevalence of APV infections, experimental infection studies in birds using APVs have centred on relatively few viral isolates. Analyses of variation have essentially focused on a FWPV strain termed the prototype, while a minority of experimental studies have been reported on CNPV, quailpox, juncopox, and pigeonpox virus isolates [4, 48, 49] . In fact, in the last twenty years, approximately 50% of published studies on APVs have been on the FWPV isolate directly (based on a PubMed search on APVs). The important nature of APVs which has been used successfully for vaccine development mandates that a larger pool of viral strains should be analyzed both for consideration of pathogenesis and determination of immune correlates of protection.
Our present understanding of the antigenic variation of APVs has been based on a limited number of virus isolates in assays that includes complement-fixation, passive hemagglutination, agar-gel precipitation, immunoperoxidase, virus neutralisation and immunofluorescence [48, 49, 65] . In addition to the immunological assays, variation of APVs has also been addressed through genetic assays, such as restriction enzyme analysis. Genomes of FWPV and quailpox virus isolates were compared by using BamHI, EcoRI, and HindIII endonucleases and distinct fragment patterns were observed between the isolates. The patterns of three quailpox virus isolates were similar to each other with a high proportion of comigrating fragments. However, when immunogenic proteins of three FWPVs, two quailpox viruses, a juncopox virus, and a pigeonpox virus isolates were examined by immunoblotting, shared as well as unique antigens were detected. The greatest disparity was observed between quailpox virus and FWPV [48, 49] , indicating extensive variation between the quailpox virus and FWPV, which would predict differences in immunogenicity and antigenicity, including neutralization sensitivity. Nucleotide sequence based studies for rapid identification of poxvirus species by PCR with specific primers and hybridisation are well established [21] . These approaches have concentrated on single genes or portions of genes that exhibits variations in their sequence and are important for quick analysis of genetic variability [22] .
Understanding the phylogenetics of APVs is essential to the understanding of host specificity and virulence, but also to provide insights into the variation of different viruses. Although the complete genome sequences of FWPV and CNPV are available [7, 8] , little is known about APV phylogeny. This is probably because of the difficulty in identifying pan-genus or species-specific PCR primers that can be used to amplify different genes. The most common PCR locus used until now has been the P4b locus [21] . Recent phylogenetic studies of APV isolates based on this locus [22, 23] indicated that most isolates clustered around either CNPV or FWPV, while another study based on the same locus demonstrated a third cluster, from psittacine birds [66] . Amano and coworkers [38] showed that the CNPV thymidine kinase locus was highly diverged from that of FWPV. The extent of this divergence was further illustrated by the fact that the amino acid similarity between CNPV and FWPV orthologue P4b was only 64.2% [23, 38] . A recent study, based on three different genes including the P4b, revealed that penguinpox virus, isolated from lesions around the eyes of African penguins (Spheniscus demersus), was most closely related to turkeypox virus, ostrichpox virus and pigeonpox virus [67] .
During avipox outbreaks, mortality can reach 80 to 100% in canaries and other finches. This is in contrast to a generally lower mortality seen in chicken and turkey [60] . Transmission of virus can occur through a break in the skin or, more commonly, when vectored by biting insect such as mosquitoes and mites [68] . Aerosols generated from infected birds, or the ingestion of contaminated food or water have also been implicated as a source of transmission [69] . The disease is most commonly characterized by cutaneous proliferative lesions consisting of epithelial hyperplasia of the epidermis that resulting in proliferative, wart-like projections. They are primarily confined to unfeathered parts of the body, such as legs, feet, eyelids and the base of the beak (Figure 4 ). Scars are usually visible after recovery and healing of skin lesions. The mortality in wild birds is usually low, depending on the number and size of the proliferative lesions. However, if infection occurs in feather-free areas of the skin, with secondary bacterial infection, mortality may be high. The other and less common form of APV infections is the diphtheritic or wet form [70] which occurs as fibrino-necrotic and proliferative lesions in the mucosa of the digestive and upper-respiratory tracts, and generally has a higher mortality than the cutaneous form [60] . In some instances, birds display both cutaneous and diphtheritic forms and in those cases, mortality rates are often higher compared to the cutaneous form alone. Despite the variety of hosts and virus strains, associated pathology remains the same in infected domestic birds, although clinical signs vary depending on the virulence of the virus, susceptibility of the host, distribution and type of lesions [60] . There exist a relationship between FWPV and the avian retrovirus, reticuloendotheliosis virus (REV) (see section on APVs and REV). However, the possible roles that simultaneous REV infection arising from the provirus integration into the FWPV genome might play in the expression FWPV during disease outbreak remain unresolved. It is well known that REV infection leads to immunosuppresion [71] in affected birds. Thus, it is plausible to suggest that the presence of REV in FWPV infection may exacerbate disease progression. In spite of the fact that some mammalian cell lines seems to be able to support the replication of APVs, there is no evidence that APVs have caused clinical disease in humans, in contrast to what is known for other poxviruses, such as several parapox and orthopoxviruses.
Clinical features of infected birds show multiple skin lesions varying from papules to nodules. Gross lesions in both the cutaneous and the diphtheritic forms, seen on birds and during necropsy, are usually sufficient to suspect APV infection [60] . However, these signs are sometimes not sufficient for definitive diagnoses of APV infection as other agents, such as papilloma virus, scaly leg mites [72] and mycotoxins may produce similar lesions in the skin [60] , and conditions like candidiasis, capillariasis and trichomoniasis may give lesions in the oral cavity similar to the diphtheritic form of APV infection [73] . It is therefore crucial to secure samples and confirm the viral etiology of the condition.
Suspicion of clinical signs of APV infection can if possible be supported by necropsy, especially if the oral cavities to reveal the diphtheritic form. Further, histopathology on tissue sections using the classic Wright's Giemsa stain may reveal typical large, solid or ring-like, eosinophilic intracytoplasmic inclusions known as Bollinger bodies [5] ; Figure 2A and 2B. Transmission electron microscopy (TEM) may also reveal definite proof of APV infection, demonstrating the typical APV particles within inclusion bodies. APV identification may also be carried out by negative staining electron microscopy with 2% phosphotungstic acid (PTA) on infected cells (Figure 1 ). This method has typically been used by national reference or research laboratories to identify APV [18] .
Demonstration of infectious virus by inoculation of homogenates of clinical samples of typical APV skin lesions onto the CAM of embryonated hen's eggs is the gold standard method for diagnosis of APV, although some strains of APV do not grow readily on chicken embryos [16] . Eggs are first swabbed with 70% alcohol and a pore is made in an area over the air-cell and another one on the other side of the egg to make a false air sac and lower the CAM by negative pressure using a rubber bulb. Inoculation of infectious samples by the CAM route is performed with sterile disposable 1 mL syringe with approximately 0.1-0.2 mL of inoculum. Eggs are incubated at 37°C for 5 days with daily candling to check for embryo death. Pock lesions measuring in size 0.5-1.5 mm are observed on the membrane 3-5 days after inoculation, depending on the virulence of the virus [15, 16] . Another method of isolation of APV requires the excision and homogenization of clinical skin lesions and inoculation of a homogenate supernatant onto a permissive cell culture, such as CEF cells. This results in the formation of CPE within 4-6 days post inoculation, depending on the virus isolate and on the multiplicity of infection (MOI) [4] .
APV are increasingly being detected and characterized by PCR, Restriction fragment length polymorphism (RFLP), Southern blot hybridization, and cycle sequencing, directed at specific genes such as the 4b core protein gene [22, 23] . PCR allows for sensitive and specific detection of viral nucleic acids and has been shown to increase the diagnostic sensitivity for many viral pathogens when compared to culture. A PCR amplicon sequence allows a rapid search for homologous sequences in gene databases, to verify and identify the virus in question and to address phylogenetic relationships. Detection by realtime PCR has been used to identify recombinant APV from individual plaques [74] . This method eliminates the need for amplification and hybridization from the transient dominant protocol and results in significant savings of time at each round of plaque purification [74] .
The conventional serological techniques of passive neutralization and agar-gel immunodiffusion are in continued global use for surveillance and disease control efforts in domestic poultry species [75, 76] , despite the availability of modern molecular and immunoassay techniques. The tests are time consuming, especially when carried out with large numbers of sera, and sensitivity appears to be low when compared with other detection method, such as enzyme linked immunosorbent assay (ELISA) [77] . ELISA has been described as a non-species specific test approach for birds [78] . It is a faster and easier method to detect antibodies against APV, particularly when large numbers of sera are to be tested. The technique is also more sensitive than the neutralization test [18, 78] . ELISA protocols have also been developed and used to test the efficacy of FWPV vaccines in commercial and wild bird species where agar-gel immunodiffusion is ineffective due to lack of precipitating antibodies [61, 79] .
The challenges of controlling APV disease in poultry are driven by economics, and require strategies that keep cost low while maintaining treatment efficacy. Prophylaxis can be achieved by vaccination [39] . Doyle [80] reported the use of live FWPV or Pigeonpox virus for vaccination against APV infection. Since then, recombinant and live modified vaccines have been developed and used to prevent APV infections in chickens, pigeons, turkeys and quails [79, 81, 82] ; Table 2 . These vaccines are very effective and have undoubtedly contributed immensely to the prevention of the disease in commercial poultry farming [47, 81] . Since different APVs are isolated from a wide range of bird species and since only a few isolates have been characterized, development of a taxon-specific vaccine, directed to all species, has been difficult. Thus, available vaccines are often applied on the basis of experimentation, and more knowledge of molecular biology, pathology and epidemiology of these viruses is necessary to develop vaccines that effectively can protect a range of bird species. As in most viral infections, there is no specific treatment for avian poxvirus infections in birds [39, 83] . Available treatments include the use of iodine-glycerin application on proliferating skin lesions to aid healing [84] , antibiotics to control secondary bacterial infections and vitamin A to aid healing [85] .
In the poultry industry, prophylactic measures against FWPV are achieved primarily by vaccination with live FWPV or antigenically similar pigeonpox virus strains produced in CEF cells [60] . In the past two decades, numerous outbreaks have been reported in vaccinated flocks, suggesting that vaccines used against the disease were not effective. In the United States a commercial FWPV vaccine was shown to be contaminated with REV and caused lymphoma among broiler chickens [86] . It has been shown that sequences of REV have been integrated into the DNA of FWPV vaccines as well as in field FWPV isolates [81, [87] [88] [89] [90] . The integration site is constant, while the size of the integrated fragments differs between various isolates and strains. Two different types of integrated sequences are reported; long terminal repeats (LTRs) with size of approximately 200 to 600 bp and the near-full-length REV provirus of about 800 bp [87, 90, 91] . Most vaccine strains carry only an LTR remnant while most FWPV field isolates carry the nearfull-length provirus. Singh and others [81] , however, detected REV LTRs of various lengths in the genome of two commercial FWPV vaccine strains and four field isolates, while several studies have shown that the source of REV infection was REV-contaminated FWPV [86, [92] [93] [94] and herpesvirus of turkeys vaccines [92, [94] [95] [96] [97] . Reticuloendotheliosis is a tumorigenic and immunosupressive disease. REV strains have been reported to cause diseases characterized by chronic lymphoma, non-neoplastic lesions and a runting-stunting syndrome in chickens, turkeys, and quails [98, 99] . REV are group of avian retroviruses and representatives include the defective REV-T and the non-defective REV-A, spleen necrosis virus (SNV), duck infectious anemia virus, and chick syncytial virus (CSV) [98] . The presence of REV in FWPV vaccines and the failure of currently used FWPV vaccines to evoke high level immunological protection against field challenge of FWPVs are of major concern to the poultry industry [100] , which emphasizes the need for research into alternative vaccines.
In 1796 Edward Jenner [101] published his landmark findings that vaccination of humans with cowpox virus could prevent infection with variola virus, the causative agent of smallpox [101, 102] . This traditional vaccine technology, based on live viruses and immunological cross protection, has given rise to a wide range of effective vaccines against a wide variety of infectious agents, both in veterinary and human medicine. However, the emergence of new deadly human pathogens and cancers, have proven less amenable to the application of traditional vaccine platforms, indicating the need for new approaches. The use of a live virus vector represent an attractive way to deliver and present vaccine antigens that may offer advantages over traditional platforms, by improving the quality and strength of the immune response, such as in the case of HIV-1 where two different strains of vaccinia virus have been used as vectors. The NYVAC vector has been shown to induce the CD4 + T cell-dominant response, whereas modified vaccinia virus Ankara (MVA) induces a stronger CD8 + T cell response with accompanying CD4 + T cell responses that are required for protection [103] . Although this assertion remains unproven (there are to date no virally vectored vaccines licensed for human use), virally vectored vaccines offer an avenue of possibilities, either as homologous regimens, or as heterologous (prime-boost) regimens in which different serotypes of a given vector, different vectors or vectors and traditional technologies such as recombinant protein in adjuvant are administered sequentially.
Currently, representatives of a wide range of virus families are under intensive development as vaccine vectors for human or veterinary use. Of these, FWPV and CNPV appear to be of great interest as vectors, and some veterinary APV-vectored vaccines are already licensed and in commercial use in North America, South America and Europe ( Table 2 ). The most important characteristics of APVs as vaccine vectors are that unlike most other DNA viruses, APV replicate in the cytoplasm of the infected cell and enzymatic functions used for transcription and replication are provided by the virus itself. This has several consequences regarding the use of these viruses as vaccine vectors. For example, APV promoters must be used for efficient transcription of recombinant genes and as APV transcripts are not spliced, genes cloned into APV vectors cannot contain introns [70, 104] . Other reasons include (1) their ability to accommodate and effectively express large amounts of foreign DNA or multiple genes that encode antigens [47] , (2) their inability to conduct a full replication cycle in non-avian species [105] [106] [107] , (3) antisera against orthopoxviruses do not neutralize APV and thus, prior Table 2 . Notably among them is the Trovac AI H5, a recombinant FWPV that express the H5 antigen of avian influenza virus. This product has had a conditional license for emergency use for chickens in the United States since 1998 and has been widely used in Central America, with over 2 billion doses administered [110] . ALVAC vectored vaccines have recently been registered for veterinary use in the European Union (Proteq-Flu) [111] and the United States (Recombitek). The equine influenza virus vaccine with CNPV vector expresses the hemagglutinin genes of the H3N8 Newmarket and Kentucky strains and contains a polymer adjuvant (Carbopol; Merial Ltd.). With the induction of both cell-mediated and humoral immunity, it is claimed that the vaccine produced sterile immunity 2 weeks after the second of two doses. The new vaccine is also designed to protect horses against the highly virulent N/5/03 American strain of equine influenza virus and to prevent the virus from spreading through the elimination of viral shedding.
Despite these notable advances in APV-vectored vaccine development, the list of licensed viral vectored vaccines for human medicine is short, with only a few vaccines that have entered clinical trials [112] ; Table 3 .
This may be in part owing to stringent safety requirements that must be met for viruses, that in their natural state have the potential to be human pathogens, to be used as viral vaccine vectors that may replicate in vivo in a manner similar to their wild-type parental viruses. Another reason may be fear of risk of spontaneous recombination between virus vectors and naturally occurring viral relatives in the ecosystems in which the vaccine is used. Even if APVs are not generally expected to replicate in mammals, the vaccine vectors may reach bird populations via animal populations. It is also possible that the vector, through spontaneous recombination and mutation events, may restore its replication competence. To cater for this, the aim during design and development of a virus vector is always to introduce at least two gene deletions crucial for viral to undergo a full replication cycle to assure a very low probability that replication competence could be restored. To our knowledge, such reversions have not been identified in clinical trials of APV-vectored vaccines. In addition to concerns regarding reversion or recombination, another safety signal was recently identified in an in vitro experiment that showed APV replication in cell clones derived from embryonic bovine trachea [53] and Syrian baby hamster kidney (BHK) cells. In this experiment, infectious IMV was observed; indicating complete virus replication had taken place [54] . These findings are in contrast to the general dogma that APVs are restricted to infection of cells of avian origin, and are an indication that there is still more to learn about the replication mechanisms and virus-host interactions of these viruses, including evasion of immune responses, cell tropism and host range mechanisms.
APVs cause disease of economical importance for the poultry industry, and also in pet and wild birds. Thus, prophylactic measures, such as vaccination, will always be required, and there is a need for more efficient and safe vaccines. One promising approach is the use of APVs as vectors for recombinant vaccines, increasing the efficacy and avoiding the potential contamination with REV and other agents. Many recombinant APV constructs are already licensed for use in veterinary medicine, and a range of vaccine candidates are currently being tested for use in vaccines against numerous infectious diseases in animals and man. Thus, it is likely that recombinant APV-vectored vaccines in the near future will also be used against human diseases. APVs have many advantages as vaccine vectors, including a large genome which allows for the inclusion of many heterologous genes, such as genes coding for antigens, cytokines and other immuno-modulating factors. The major safety argument for using APVs rather than vaccinia virus or other mammalian viruses as vectors, is that APVs are not zoonotic and are not able to conduct a full replication cycle in mammals. However, it was recently shown that FWPV was able to replicate and produce progeny virions in some established mammalian cell lines. This illustrates the fact that general knowledge of APVs is scarce. Indeed, only a few isolates have been characterized and classified. New molecular tools have led to a greater resolution of factors and mechanisms that restrict viruses to certain hosts, for example HIV and SARS. Mechanisms of host restriction, pathogenicity, host immunity and viral immune evasion strategies are of crucial importance regarding use of APVs as vectors in multispecies-targeted vaccines. A good understanding of the molecular properties of APVs underpins the development of safe APVvectored vaccines.  Apolipoprotein M Gene (APOM) Polymorphism Modifies Metabolic and Disease Traits in Type 2 Diabetes This study aimed at substantiating the associations of the apolipoproein M gene (APOM) with type 2 diabetes (T2D) as well as with metabolic traits in Hong Kong Chinese. In addition, APOM gene function was further characterized to elucidate its activity in cholesterol metabolism. Seventeen APOM SNPs documented in the NCBI database were genotyped. Five SNPs were confirmed in our study cohort of 1234 T2D and 606 control participants. Three of the five SNPs rs707921(C+1871A), rs707922(G+1837T) and rs805264(G+203A) were in linkage disequilibrium (LD). We chose rs707922 to tag this LD region for down stream association analyses and characterized the function of this SNP at molecular level. No association between APOM and T2D susceptibility was detected in our Hong Kong Chinese cohort. Interestingly, the C allele of rs805297 was significantly associated with T2D duration of longer than 10 years (OR = 1.245, p = 0.015). The rs707922 TT genotype was significantly associated with elevated plasma total- and LDL- cholesterol levels (p = 0.006 and p = 0.009, respectively) in T2D patients. Molecular analyses of rs707922 lead to the discoveries of a novel transcript APOM5 as well as the cryptic nature of exon 5 of the gene. Ectopic expression of APOM5 transcript confirmed rs707922 allele-dependent activity of the transcript in modifying cholesterol homeostasis in vitro. In conclusion, the results here did not support APOM as a T2D susceptibility gene in Hong Kong Chinese. However, in T2D patients, a subset of APOM SNPs was associated with disease duration and metabolic traits. Further molecular analysis proved the functional activity of rs707922 in APOM expression and in regulation of cellular cholesterol content. The human apolipoprotein M gene (APOM, Gene ID: 55937) is located on chromosome 6p21.33 and contains six exons spanning a region of 2.3 kb in length with gene structure conserved across species [1, 2] . In human and mice, APOM mRNA is highly expressed in liver and kidney [2] . The human apoM protein (MIM 606907) of 188 amino acids is mainly associated with HDL and to a minor degree with LDL, very low density lipoprotein, and chylomicrons [2] . Plasma apoM has been positively associated with plasma total cholesterol (TC), LDL cholesterol (LDL-C), and HDL cholesterol (HDL-C) [3] . APOM knockdown in mice by siRNA revealed its anti-atherosclerotic effect by participating in pre-b HDL formation and reverse cholesterol transport [4] . Kruit et al., recently reported the effect of cellular cholesterol accumulation on beta cell dysfunction in type 2 diabetes [5] . Such finding implies that factors (i.e., apoM) affecting the balance of cellular cholesterol content are likely to modify beta cell function and thus the susceptibility to or progression of type 2 diabetes.
Several additional lines of evidence also indicated the possible involvement of APOM in the development of diabetes and metabolic disturbances: 1) the human APOM gene is located within a high susceptibility region (6q21-q23) to type 2 diabetes (T2D) in genome-wide linkage analyses [6] . 2) SNP rs805296 (T-778C) in APOM promoter has been associated with the levels of plasma total cholesterol (TC) and fasting plasma glucose (FPG) in non-diabetic participants, 3) SNP rs805296 has also been associated with the susceptibility to T2D and coronary artery disease among the Northern Chinese [7, 8] .
In 2010, China became the country with the largest diabetic population in the world. The Northern and Southern Chinese populations are distinct in genetic marker analyses [9] , meaning disease markers identified in northen populations may not be shared by the Southern populations. The primary aim of the current study is to establish the association between APOM and T2D susceptibility in a Southern Chinese cohort in Hong Kong. By assuming the same effect size (OR = 1.934) and disease allele frequency as observed in the studies of Northern Chinese [8] , the power of the current case-control study is over 95% with 1234 cases and 606 controls. The secondary aims are to examine for association between APOM and component metabolic traits as well as to further assess the function of the gene.
The pilot cohort consisted of 103 male and 95 female controls (average age = 43 yrs). They were Hong Kong Chinese adults recruited from a community health screening program of cardiovascular risk factors with normal response at a 75 g oral glucose tolerance test [10] .
The study cohort had 1234 unrelated T2D patients and 606 controls. All participants gave written informed consent at the time of blood sampling. Ethics approval was obtained from the Clinical Research Ethics Committee of Chinese University of Hong Kong, Shatin, NT, Hong Kong. All T2D participants were selected from the Hong Kong Diabetes Registry. Control participants were recruited in a community health screening program for cardiovascular risk factors and some were hospital staff (3.1%, n = 19). No subdemographic differences were detected in control participants. All control participants had no known history of diabetes and had fasting plasma glucose (FPG) , 6.1 mmol/l. Clinical assessments of participants had been described elsewhere [11] . Body mass index (BMI), blood pressure (BP) as well as fasting blood biochemical and metabolic profiles were measured. Among the 1234 T2D patients, 9.8% (n = 121) were on diet treatment only, 41.3% (n = 510) were on oral anti-diabetic drugs only, 12.5% (n = 154) were on insulin only, 9.5% (n = 117) on both oral anti-diabetic drugs and insulin, and 7.3% (n = 90) were treated for dyslipidemia.
2.1. SNP selection and genotyping analyses. Genomic DNA was prepared from whole blood as previously described [12] . Seventeen APOM SNPs including rs6921907, rs1266078, rs9267528, rs805297, rs4947251, rs9404941, rs805296, rs805264, rs3117581, rs34490746, rs11462733, rs2273612, rs707922, rs707921, rs28432254, rs3132449, rs3178094 enlisted in the NCBI database [13] were selected for genotyoping in the pilot cohort of 198 controls by multiplex reactions using the Mass ARRAY system (Sequenom, San Diego, CA, USA) at the Genome Quebec Innovation Centre, McGill University (Montréal, Quebec, Canada). Six of the seventeen SNPs were confirmed in the pilot cohort: rs1266078(T-1628G), rs805297(C-1065A), rs9404941(T-855C), rs805264(G+203A), rs707922(G+1837T) and rs707921(C+1871A). These six SNPs were further genotyped in the study cohort of 1840 participants (1234 cases and 606 controls). Case and control DNA samples were genotyped in parallel on the same plates. Two hundred ninety one duplicate samples (15.8%) were used to assess intra-plate and inter-plate genotype quality. No genotyping discrepancies were detected. The overall call rate was 98.0%. Five out of the six SNPs (except for rs1266078) were successfully genotyped in the study cohort.
2.2. Plasma lipids and apoM levels. Plasma apoM concentration was estimated by dot-blot analysis using monoclonal mouse anti-human apoM antibody (ABNOVA, Taipei, Taiwan) following previously established protocols [14, 15] . Recombinant human apoM (ABNOVA, Taipei, Taiwan) was used as protein standard after serial dilution. The mean signal densities of each specimen and protein standards in triplicate measures were determined by ImageJ 1.42q software (http://rsbweb.nih.gov/ij/). ApoM concentration was derived from the standard curves developed using the recombinant apoM protein.
ectopic APOM1 and APOM5 expression. WRL-68 and HepG2 hepatic cell lines were purchased from American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS and 100 units/ ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere containing 5% CO 2 at 37uC. Prior to the expreiments measuring cellular and medium cholesterol content, cells were switched to serum free and phenol red free RPMI medium (Invitrogen, Carlsbad, CA, USA).
Ectopic expression of APOM was achieved by transient transfection of APOM1 or APOM5 cDNA (cloned into the pCMV-Myc vectors) into cultured cells at 70% confluence using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufactorer's protocols [16] .
Cellular lipids were extracted as previously described [17] . Total cellular cholesterol was measured using the InfinityTM Cholesterol Liquid Stable Reagent (Thermo Fisher Scientific Inc., Middletown, VA, USA) following the manufacturer's instructions. The measured amount of total cholesterol was normalized by cell protein concentration.
3.1. Comparative genomic and protein sequence analyses. APOM transcript and gene sequences were obtained from the NCBI human Genome Browser [18] , the Ensembl Genome Browser [19] and the human Expressed Sequence Tags (EST) databases. The Evolutionary Conserved Regions Browser [20] and the Ensembl Genome Browser were used to identify sequence conservation.
3.2. Rapid amplification of cDNA ends (RACE). The Human Liver FirstChoice RACE-ready cDNA kit (Ambion, Austin, Texas, USA) was used to amplify the 59 and 39 ends of novel APOM transcripts (Supplementary Figure S1 , and Supplementary Table S1).
3.3. Semi-quantitative RT-PCR analysis. Human normal adult tissue RNA samples were purchased commercially (Stratagene, La Jolla, CA, USA or Millipore Chemicon, Billerica, MA, USA). cDNA was synthesized using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA, USA) in combination with RNase inhibitor (Roche Applied Science, Indianapolis, IN, USA) and M-MuLV reverse transcriptase. The subsequent PCR amplification of cDNA was performed using the AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA) following standardized protocols [21, 22] . The vulgate transcript APOM1 (Ensembl: ENST00000375916) and the novel transcript APOM5 were amplified using primer RT-PCR-YY12 paired with RT-PCR-YY13 and RT-PCR-YY12 paired with RT-PCR-YY14, respectively (Supplementary Table S4 ). GAPDH was amplified as a house-keeping gene control for RNA integrity and equal loading using the GAPDH primers, RT-PCR-Tao1 and RT-PCR-Tao2 [23] (Supplementary Table S1 ).
Continuous variables were compared using Student's t test or one-way analysis of variance (ANOVA) for traits with normal distribution. Plasma triglycerides (TG) were skewed and logarithmically transformed. Association tests between genotypes and quantitative traits were performed in T2D patients and nondiabetic controls separately. Categorical variables, including genotype distributions were compared by x 2 tests.
Genotype distributions were tested for Hardy-Weinberg equilibrium using goodness-of-fit test (1 df). Informative missingness was checked by coding successful genotypes into one group and failed genotypes into another group followed by 262 Chi-square test for T2D and t-test for the quantitative traits of interest. One SNP (rs805264) with significant result (p,0.001) indicative of informative missingness (IM) was excluded for further T2D association analyses (Supplelmentary Table S2 ).
Pairwise LD of D' and r 2 analyses were performed using Haploview (Broad Institute of MIT and Harvard, USA, version 4.0). 262 contingency tables were used for comparing the differences of allele frequencies and 263 contingency tables were used for detecting the differences of genotype frequencies between cases and controls. Allelic, dominant, recessive and additive genetic models were used to test the association between each SNPs and T2D. Multivariate logistic regression analysis was used to assess the significance of covariates and adjusted for confounders in the association of genetic factors with T2D.
The independent contributions of all traits, covariates, SNPs, and haplotypes were determined by multiple regression analysis. Association between haplotypes and T2D or metabolic traits were tested by Haploview (version 4.0, Broad Institute of MIT and Harvard, USA) and PHASE software (version 2.1, UW Tech-Transfer Digital Ventures, University of Washington, Seattle, WA, USA) [24] . When using PHASE software, the probability thresholds were set at 90% for haplotype inference to deal with ambiguous haplotypes. It was only used to infer the haplotype of each individual and thus the case-control permutation test was not conducted. The PHASE-imputed haplotypes were counted 20 times using different seed numbers. No difference between runs was detected.
To account for multiple testing, we used the Bonferroni correction and a statistical significance was considered only when an SNP association with T2D/metabolic traits was p,0.017 (equivelent to 0.05/3), and a haplotype association with T2D/ metabolic traits was p,0.0125 (equivelent to 0.05/4). The human plasma apoM concentrations determined by dot-blot assays were compared by a nonparametric Kruskal-Wallis H test. A value of p,0.05 was considered significant.
Results from functional analyses were analyzed by Student's ttest for two-group comparison, and one way ANOVA for multiple group comparisons. A statistical significance is considered at p,0.05 level. All statistical analyses were performed using the SPSS program (SPSS version 15.0, Chicago, IL, USA) unless otherwise specified.
Seventeen APOM SNPs enlisted in the public databases were selected for genotyping in a pilot cohort of 198 control participants. Six SNPs were confirmed polymorphic in this pilot cohort of Hong Kong Chinese. These six SNPs were further genotyped in the full study cohort of 1840 participants. Five of the variants were successfully genotyped: rs805297(C-1065A), rs9404941(T-855C), rs805264(G+203A), rs707922(G+1837T), and rs707921(C+1871A) with genotype distributions fitting Hardy-Weinberg equilibrium. Table 1 summarized the allele and genotype frequencies of SNPs in non-diabetic controls and T2D patients. SNP rs805264 was removed from further association analysis for T2D due to informative missingness (Supplementary Table S2 ).
2.1. APOM SNPs and T2D susceptibility. No significant association was detected between individual SNPs and T2D (Table 1) . Further multiple logistic regression analysis adjusting for age, BMI, SBP (systolic blood pressure), DBP (diastolic blood pressure), TC and TG again detected no significant association between individual SNPs and T2D (data not shown). 2.2. APOM SNPs and T2D duration. We next examine the association between APOM SNPs and T2D disease duration. T2D patients were subgrouped into disease duration of # 10 years (n = 583) and disease duration of .10 years (n = 586). As shown in Table 2 , the C allele of rs805297 was associated with T2D duration of longer than 10 years (odds ratio = 1.245, p = 0.015).
2.3. APOM SNPs and metabolic traits. We next analyzed the association between SNPs and metabolic variables in patients and controls separately. SNP rs805297(C-1065A) was not associated with metabolic traits in either patients or controls. Since rs707922(G+1837T) was in near perfect LD with rs805264(G+203A) and rs707921(C+1871A) (Supplementary Figure S2B) , similar association results were expected and observed. Table 3 showed the representative results using rs707922 as the marker SNP. Under recessive model, homozygous minor allele TT of rs707922 was associated with significantly higher TC (p = 0.006), LDL-C (p = 0.009) in T2D patients. In controls, no association between SNPs and metabolic traits was detected. When plasma apoM concentration was measured in T2D patients and controls subgrouped by their rs707922 genotype, the TT genotype was found associated with significantly higher apoM level as compared to the GT (and GG) genotype(s) (p = 0.002).
As mentioned above, rs805264, rs707922, and rs707921 are located within the same LD block. Therefore, in the subsequent haplotype analysis, rs707922 was used to 'tag' the three SNPs. Haplotype construction was conducted for rs707922(G+1837T) with other independent SNPs rs805297(C-1065A) and rs9404941(T-855C). Four haplotypes (A-T-G, C-C-G, C-T-G, and C-T-T) accounting for 99.9% of all possible haplotypes were detected in our Hong Kong Chinese population (Supplementary  Table S3 ). No significant association was found between these haplotypes with T2D. Homozygous C-T-T was significantly associatiated with elevated TC, LDL-C and HbA1c in T2D patients (p,0.0125, Supplementary Table S4 ). Figure S3) . SNPs rs707922 (G+1837T) and rs707921 (C+1871A) which associated with plasma levels of TC, LDL-C and apoM in T2D patients fell within the evolutionary conserved region. Figure 1 (top panel) illustrated the cross-species sequence conservation of the rs707922-and rs707921-flanking region. BLAST search using this conserved sequence as the template returned unique human EST clones BI757556 (human brain) and AA975560.1 (human kidney) which are likely other APOM transcripts. The Ensembl Browser also displayed three APOM transcripts (APOM1: Ensembl-ENST00000375916, APOM2: Ensembl-ENST00000375920 and APOM3: Ensembl-ENST00000375918). 4.2. Molecular cloning of APOM transcripts. Since the sequences of EST clones BI757556 and AA975560.1 were different than the known APOM transcripts, we proceeded with cloning alternative transcripts of APOM. A novel transcript, designated APOM5, was identified by 59 RACE and 39 RACE. As shown in Figure 1 , the 39 end of APOM5 was identical to the 39 end of APOM3. The 59 end, however, was similar to that of the vulgate APOM transcript (designated APOM1) except that the transcription start site of APOM5 was 21 nucleotides downstream that of the APOM1. The full-length sequence of APOM5 is provided in Supplementary Figure S4 . It is important to note that the two SNPs rs707922(G+1837T) and rs707921(C+1871A) associated with metabolic traits in T2D are located to the exon 5 of APOM5. On the contrary, when reference to the vulgate APOM1 transcript, rs707921 and rs707922 are located to intron 5. These observations support the cryptic nature of exon 5 of the gene. 
The results of this study did not support an assoiation between APOM and T2D suseptibility in Hong Kong Chinese. For a subset of SNPs, we presented evidence of association between APOM and disease duration as well as metabolic traits in T2D patients. Further characterization of rs707922, one of the metabolic traitassociated SNP at molecular level lead to the discoveries of a novel transcript APOM5 and its SNP-dependent effect on cellular cholesterol content.
The LD block formed among rs805264, rs707922, and rs707921 in our cohort agreed with the LD structure reported in the Northern Chinese [25] . It is currently unknown whether this subset of SNPs is also associated with metabolic traits in Northern Chinese with T2D. Among the four common haplotypes constructed from rs805297, rs9404941, and rs707922, only homozygous haplotype C-T-T was significantly associated with higher TC, LDL-C and HbA1c levels in T2D patients. Given the established association between rs7070922 and plasma TC and LDL-C levels, these association results did not support additional effects of the haplotypes on serum cholesterol levels. Interestingly, the association between the homozygous haplotype C-T-T with HbA1c indicated the interaction among the three alleles to control systemic glucose level in T2D patients. It would have been ideal if the previously reported association between SNP rs805296(T-778C) and T2D in Northern Chinese were reproduced in this Hong Kong Chinese population. Unfortunately, genotyping of this SNP failed to produce results in this study, precluding it being used for discussions attempting to reconcile the current findings with prior results. Although rs805296 is physically close to rs9404941, the existing information/data does not allow the relationship between SNP rs805296 and T2D/T2D metabolic traits to be predicted in our cohort.
It is noteworthy that the case-control study design adopted by the current and other studies tend to be limited by the heterogeneity of the prevalent cases with regards to T2D ascertainment, i.e., both those have developed T2D and those have survived in the setting of T2D were included as cases. Therefore, those 'susceptible to' the disease were not distinguished from those 'survived' the disease'. One possibility to circumvent such issue is to examine for similar duration of diabetes across studies being compared and test for difference in duration of T2D by SNP. Interestingly, while our results did not support an association between APOM and T2D susceptibility, stratification of our cases by disease duration allowed us to detect an association between rs805297(C-1065A) and T2D duration. This result implied the possibility that relative to the rs805297-A carriers, the rs805297-C carriers better survived the diabetic condition over the long term and such possibility can be further tested. Interestingly, Zhao et al., recently reported a positive association between rs805297-A and the risk of stroke in Norhtern Chinese (OR = 1.38, p = 0.002) after adjusting for other risk factors including history of diabetes [26] . Whether such association is present among the Southern Chinese requires further investigation. Nevertheless, losing rs805297-A carriers with T2D to stroke over time provides a plausible explanation for the observed higher frequency of rs805297-C allele in the T2D duration .10 years subgroup (relative to T2D duration #10 years subgroup).
Previous studies attempting to correlate plasma apoM and cholesterol levels have generated inconsistent results [3, 27] . In this study, rs707922 homozygous minor allele (TT) was associated with elevated TC and LDL-C as well as plasma apoM levels in diabetic cases (average BMI of 25.26). These observations are consistent with previously reported positive association between plasma apoM and plasma TC and LDL-C in overweight-obese individuals [3] . Results presented by Han et al. from the study of a Northern Chinese cohort showed significant association between rs707922 T allele and increased risk of cerebral infraction (OR = 1.78, p = 0.000). In parallel they also confirmed hypercholesterolemia as an independent risk factor for cerebral infraction [25] . These results implied the possibility that rs707922 is also a modifier of serum cholesterol in Northern Chinese. The association between rs707922 TT genotype and elevated serum total-/LDL-cholesterol levels in type 2 diabetes found in the current report deserves to be further substantiated in strict replicate studies.
The mechanism underlying the effects of rs707922 on plasma TC and LDL-C levels in diabetes remains elusive. Richter et al., reported HNF-1 alpha being a potent transcription activator of APOM [28] . The decreased serum apoM level in maturity-onset diabetes of the young subjects as compared to the controls could be explained by the HNF-1 alpha mutations in these patients [28] . Given the association between APOM and metabolic traits found in this study, one may speculate that SNP rs707922 (G+1837T), in the capacity of an intronic SNP (reference to the APOM1 transcript), may modify APOM expression through SNP-specific recruitment of transcription factors (i.e., PAX 6 showed an allelespecific interaction with rs707922 T by computer prediction as presented in Supplementary Figure S5 ) and subsequently affect cellular cholesterol homeostasis in liver and possibly other tissues. More interestingly, we found that rs707922 can also assume the capacity as an exonic SNP (i.e., reference to the APOM5 transcript). While the function of APOM5 requires further elucidation, the high renal and hepatic expression levels of APOM1 and APOM5 indicated the possibility of these transcripts coordinate to regulate cholesterol homeostasis in these tissues. Such possibility is further supported by the results showing the activities of ectopically expressed APOM5 in modifying hepatic cell cholesterol content. With regards to systematic cholesterol homeostasis, we observed that homozygous rs707922-T allele associated with elevated total-and LDL-cholesterol levels. One possible mechanism of such elevation is through reducd hepatic and/or pheripheral clearance of circulating cholesterol. Consistent with this notion, our in vitro data showed that hepatic cells overexpressing APOM5-T transcript had lower cholesterol content relative to cells expressing the APOM5-G counterpart.
In conclusion, the APOM SNP frequencies and the LD structure reported in this study of Hong Kong Chinese population will facilitate future population genetics studies. While our results did not support an association between APOM and T2D susceptibility in Hong Kong Chinese, subgroup analyses found SNP as well as haplotype associations between APOM and metabolic traits in T2D. Bioinformatics/molecular analyses revealed the cryptic nature of exon 5 responsible for the expression of a novel transcript APOM5, predominantly in liver and kidney. The activity of APOM5 on modifying cellular cholesterol content revealed another layer of regulation underlying the expression and function of APOM. (Mus musculus; chr17) , rat (Rattus norvegicus; chr20), cow (Bos Taurus; chr23) and dog (Canis familiaris; chr12) genes are shown. Conserved sequences were defined as coding exons (blue), The Evolutionary Conserved Regions (ECRs) were indicated by pink lines (on top of the panel for each species) with a default value of 70%. The human APOM was depicted as a horizontal blue line above the graph, with strand/transcriptional orientation indicated by arrows. APOM coding exons were shown as blue boxes along the line, while untranslated regions (UTR) were indicated as yellow boxes. Peaks within the conservation profile which corresponded to these five exons of APOM were similarly coloured within the plot. Peaks within the conservation profile that did not correspond to transcribed sequences were highlighted in red colour. Regions of transposable elements and simple repeats were highlighted in green color. Figure S5 Computer-predicted transcription factor interaction sites in nucleotide sequences spanning SNPs rs707922(G+1837T) and rs707921(C+1871A). This figure is generated by the MATCH program. Top panel: The transcription factors predicted to interact with the nucleotide sequences spanning the major allele of SNPs rs707922 (G allele) and rs707921 (the C allele). Bottom panel: The transcription factors predicted to interact with the nucleotide sequences spanning the minor allele of SNPs rs707922 (the T allele) and rs707921 (the A allele). The predicted transcription factors are marked by blue text with scores of matrix match indicated in parentheses. The locations and orientations of the binding sites for these predicted transcription factors are marked by black horizontal dashed lines with arrows. Highlighted in pink boxes are allele-specific transcription factors (PAX6 and AREB6 for rs707922-T; HNF4 and OCT1 for rs707921-A) and their corresponding binding sites. The vertical dashed lines indicate the locations of SNPs rs707922 and rs707921. The precise nucleotide positions of SNP rs707922 and rs707921 are also highlighted with grey boxes in the DNA sequences represented by red colored text. (TIFF)
Table S1 Sequences of primers used in this study. (PDF) Table S2 Summary of data quality of the five successfully genotyped APOM SNPs in the full cohort (n = 1840). The clinical traits tested for IM include T2D, TG, HbA 1c , FPG, TC, HDL-C, and LDL-C. (PDF) Table S3 Frequencies of common haplotypes constructed by APOM SNPs rs805297, rs904941, and rs707922. (PDF) Table S4 Haplotype C-T-T formed from SNPs rs805297(C-1065A), rs9404941(T-855C), and rs707922 (G+1837T) and clinical characteristics of T2D patients. Values are either number of subjects, mean 6 SD, or geometric mean (95% confidence interval). p values here represent the comparisons between subgroup of homozygotes of C-T-T haplotype vs. subgroup with one C-T-T haplotype and without C-T-T haplotype (a recessive model). p values are adjusted for age, sex, BMI and disease duration in T2D. In non-diabetic controls, the p values without parentheses are adjusted for age, sex and BMI. ''+/+'' represent the homozygote of haplotype C-T-T, ''+/2'' represent the heterozygote of haplotype C-T-T, ''2/2'' represent the subgroup who do not have the haplotype of C-T-T. Individuals on lipid lowering medications (n = 90) were excluded for association analysis with lipid traits. * statistical significance (p,0.0125). (PDF) Non-Invasive Microstructure and Morphology Investigation of the Mouse Lung: Qualitative Description and Quantitative Measurement BACKGROUND: Early detection of lung cancer is known to improve the chances of successful treatment. However, lungs are soft tissues with complex three-dimensional configuration. Conventional X-ray imaging is based purely on absorption resulting in very low contrast when imaging soft tissues without contrast agents. It is difficult to obtain adequate information of lung lesions from conventional X-ray imaging. METHODS: In this study, a recently emerged imaging technique, in-line X-ray phase contrast imaging (IL-XPCI) was used. This powerful technique enabled high-resolution investigations of soft tissues without contrast agents. We applied IL-XPCI to observe the lungs in an intact mouse for the purpose of defining quantitatively the micro-structures in lung. FINDINGS: The three-dimensional model of the lung was successfully established, which provided an excellent view of lung airways. We highlighted the use of IL-XPCI in the visualization and assessment of alveoli which had rarely been studied in three dimensions (3D). The precise view of individual alveolus was achieved. The morphological parameters, such as diameter and alveolar surface area were measured. These parameters were of great importance in the diagnosis of diseases related to alveolus and alveolar scar. CONCLUSION: Our results indicated that IL-XPCI had the ability to represent complex anatomical structures in lung. This offered a new perspective on the diagnosis of respiratory disease and may guide future work in the study of respiratory mechanism on the alveoli level. Many lung diseases alter the morphology of the lung tissue [1] [2] [3] [4] [5] [6] . Unfortunately, due to the limitation of resolution and contrast, at the early stage of the disease, minor pathological changes can not be discerned by conventional absorption-based imaging techniques. For example, lung cancer is one of the most common diseases with low survival rates worldwide; early detection of the lung cancer is known to improve the chances of successful treatment. But in most cases, cancer has been detected in the terminal stage and is impossible to be cured [1, 5, 7] . As a result, it is of great importance to image micro-structures in lung to enable early detection.
Histological biopsy is the most commonly used method for micro structures observation. But this is invasive and usually nonrepeatable [8] [9] [10] . As one of the non-invasive methods, conventional radiography has been based purely on absorption contrast. However, the differences in X-ray absorption of biological soft tissues are quite small; this technique may lead to very low contrast and poor spatial resolution.
Phase contrast imaging (PCI) is a relatively new imaging technique, which can provide high contrast images by using phase shift of the X-ray. It is well known that X-ray is a form of electromagnetic wave. When it propagates through an object, both the amplitude and the phase of the wave are modified. It can be described by a complex reflective index [11] , given by n = 12d2ib. The imaginary part b is related to the attenuation based on absorption of the object, which we use in the conventional X-ray imaging. Unfortunately, the variations in Xray absorption between different biological soft tissues are quite small; this technique may lead to very low contrast and poor spatial resolution. The real part d is responsible for the X-ray phase shift. For biological soft tissues, the phase shift is almost one thousand times greater than the absorption term. Therefore, the aim of phase contrast imaging is to use the information of X-ray phase shift and convert it into image contrast [12] . This technique can greatly improve the image quality of soft tissues, particularly at the interface of tissues where the refractive index changes significantly. During recent years, it has been widely used by researchers for imaging of small animals. Among all the main PCI methods, in-line X-ray phase contrast imaging (IL-XPCI) is the simplest and the most straightforward, making it more suitable for clinical applications than other methods [13] . IL-XPCI has previously been used to study soft tissues of both human and small animals such as mice, rats, and rabbits. The results are satisfactory: high resolution images were obtained [14] [15] [16] . It may be an alternative method for lung observation and it is totally noninvasive.
To date, only a few studies have involved in-line X-ray phase contrast imaging to investigate lung of small animals. Due to the significant difference in density between air and tissues, the inner edges of the airway can result in marked edge enhancement in the phase contrast images. This has previously been proven by Suzuki et al [16] . Nevertheless, for the large number of alveoli, the fine structures were averaged out. Other researchers like Parsons and Sera et al. have utilized IL-XPCI to study lungs, and successfully generated the three-dimensional models of mouse airways [7, 17] . Despite advances in lung imaging using IL-XPCI, threedimensional visualization of the alveoli in the intact animals has remained elusive. Nevertheless, the alveoli are important tiny structures through which gas exchange occurs. The morphology and distribution of alveoli can reveal information regarding lung health. For the diagnosis of drowning or other types of respiratory disorder like pneumonia, asthma, chronic bronchitis and emphysema, the shape of alveoli can be of considerable value [3, 18] . Furthermore, the morphology and growth of alveoli in embryos can reveal the secret of lung development, which has long fascinated biologists and mathematicians [19] . Therefore, the aim of this study is to explore these small alveoli.
In this study, IL-XPCI technique was used to image a 1-day-old mouse in situ. For the demonstration of complex microstructures, we combined IL-XPCI with computed tomography (CT) technique to provide three-dimensional images of an intact mouse lung. Moreover, we described our investigations on the anatomical structures of alveolar duct, alveolar sacs, and alveoli. Quantitative assessment was carried out on the morphology of the alveoli.
The lung is an essential respiration organ with complex threedimensional configuration. About 90% of the lung is filled with air. IL-XPCI Computed tomography study was performed on a 1-dayold mouse. The results showed that the major features of the lung, including bronchi, bronchioles, and alveoli can be clearly displayed. Moreover, it is remarkable that the alveoli can be observed in three dimensions.
A CT projection image of the mouse lung is shown in Figure 1A . Although in this image, all the tissues overlapped with each other, some bronchi, lobes and many alveoli can be discerned. Figure 1B and C are two slices of the CT reconstruction images. Compared to conventional X-ray CT, in-line X-ray phase contrast CT had a much higher resolution (about 13 mm in our experiment). Bone, bronchi and alveoli were easily discernible with clear edges, which made it possible to separate different anatomical structures using the image segmentation technique. In order to extract the lung tissue and remove background noise, we applied a threshold-based image segmentation method to the CT slices. A comparison between original CT slice and threshold-based segmentation result is presented in Figure 2 . The threshold was chosen at the valley of the histogram ( Figure 2B ). Other pixels with intensity lower than the threshold were set to zero. After gray scale transformation, the resulted image was shown in Figure 2D . From the profiles at the white line drawn in the same place of the two CT slices, the background noise in the image was considerably reduced.
Surface rendering method was used to visualize the lung, which allowed a clear depiction of complex spatial relationships and provided a strong space sense. By manually selecting the value of the reconstruction iso-surface, 3D models of tissues in the mouse chest cavity were obtained. All 3D models can be varied in size and rotated in real time to facilitate a detailed assessment of each structure (Video S1). As shown in Figure 3 , a surface rendering view of lung is displayed. The three-dimensional relationships among bronchiole, alveolar duct and alveoli were all visible. Through the segmentation of the CT slices, three models were acquired. Figure 3A and B give an overview of chest which combines ribs, bronchi, bronchioles, and alveoli. Figure 3C focuses on the display of bronchi and alveoli. Figure 3D is the result of image segmentation; it highlights the bronchial tree. The virtual endoscope video is also provided in the supporting information (Video S2).
Moving down the respiratory tract from the trachea, the air ducts divided more into smaller branches. The alveoli are the final branching of the respiratory tree, where the gas/blood exchange occurs. The distribution of the alveoli reveals information regarding lung health. Immature 1-day-old mouse alveoli range in diameter from 100 to 150 mm [15] . The diameter of most alveoli in our reconstruction image was in this range. Figure 4 is a detailed demonstration of one small section of the lung. These images provided a magnified view of the alveolar sacs and the alveoli. The model could be cut at any angle, varied in size, and rotated in real time. As shown in Figure 4 , it was easy to measure the morphological characteristics, which was of great importance in the diagnosis of diseases related to alveolus and alveolar scar.
In order to illustrate the alveoli more precisely, we only focused on several of them. Twenty-one single alveoli were chosen from different part of the lung. The three-dimensional images of the alveoli were shown in Figure 5A . The maximum diameter and the minimum diameter were measured in Figure 5B . The ratio of maximum and minimum diameters reflects the morphology of the alveoli to some extent. If this ratio approximates one, the shape of the alveolus is closer to a spheroid. The alveoli provide an enormous surface area for respiratory exchange; the change of alveolar surface area is closely related to lung health. For this reason, we calculated the surface area of each alveolus, as shown in Figure 5C .
The lungs are composed of sponge-like soft tissues. Detection of soft tissues using conventional absorption-based radiography is limited by the variation in tissue density. Since PCI is a phasesensitive technique, it facilitates the visualization of fine structures, particularly in soft tissues. This technique has opened a new window for the visualization of lung. As a current imaging modality, clinical thin-section CT can be used to visualize airways with an internal diameter larger than 2 mm. It is better than conventional radiography and can provide more detailed information [6] . However, the terminal airspaces cannot be detected via this technique. Micro-CT for laboratory studies has a much higher resolution on the order of microns. Nevertheless, it is still based on the absorption information of the X-ray. The alveolar wall and the air space can not provide sufficient contrast to be observed [20] . A few studies have applied magnetic resonance imaging (MRI) to the lung imaging. Although MRI can provide excellent soft tissue imaging and functional imaging, the limitations lie in the low spatial resolution and long acquisition times [21] . The measurement of bronchial diameter will be hampered by these limitations, because normal spatial resolution of MRI is in the range of 4-6 mm and only a few can reach up to 1 mm [22] . The lung is an air-filled organ. At the air-tissue boundary, the refractive index changes significantly, so the lung becomes highly visible in PCI images. The lung images in our experiment have shown dramatic improvement in quality, and the alveoli can be clearly discerned. The theoretical spatial resolution of our experimental system is about 1 mm, while the diameter of mature mouse alveoli range from 38 to 80 mm and human alveoli are larger at 200-250 mm [15] . Thus, the resolution of PCI is adequate for micro-structures observation in lung. Though the radiation dose in our experiment was relatively larger than that used in clinical imaging, it can be reduced by the use of high energy X-ray, since contrast is not dependent on absorption of the beam [12, 15] . In addition, compared to other existing PCI methods, IL-XPCI requires relatively simple instrument. Most importantly, conventional laboratory X-ray source can be used as the light source, although longer exposure times are required [13] . The equipments are similar to conventional radiography. In this regard, it has the potential to be used in clinical diagnosis. When it comes to studying the architecture of micro-structures in lung, histological biopsy is the most commonly used method [9] . But it is invasive and can not be repeated [10, 23] . During the process of invasive sampling, anesthesia is often required and tissue deformation frequently happen which is a major disadvantage when studying the morphology of the organs. There has long been a desire to explain breathing mechanism at the alveolar level. Unfortunately, a full understanding of these fine structures' original morphology is not available, because the lung will collapse when opening the thoracic cage at autopsy, due to the loss of negative pleural pressure [18] . Some researchers used confocal microscope to evaluate alveolar dynamics in the mouse lungs. However, this technique is limited by the depth of imaging (about 50 mm) [24] . How lungs develop has long fascinated biologists and mathematicians. Nevertheless, this process cannot be visualized in living embryos with current techniques [19] . Therefore, we adopted IL-XPCI to observe the lung non-invasively. An intact mouse was used to the maximum extent to preserve the organ's original morphology. In addition, previous studies have shown that deceased lungs can maintain a sufficient aeration level to be visualized [15] .
In conventional radiography, it is impossible to conduct alveoli imaging without contrast agent. Although contrast agents are generally safe, adverse effects do sometimes occur [25] . In PCI experiment, some researchers instilled saline into the nasal airway of the mouse in order to see the alveoli [17] . Actually, the alveoli in human lungs had been visualized by conventional radiography. Some researchers used micro-CT to image human lungs [20] . In this case, an autopsy lung from a dead person was used and silver nitrate was needed as the contrast agent. However, in practice, when small pathological changes happen, it is impossible for doctors to open a live patient's chest or stain his/her alveoli to see where the lesion is. Therefore, non-invasive and non-staining technique is preferable.
In our study, the imaging of alveoli has shown promising results without any contrast agent. The 3D reconstruction technique provided an excellent view of lung and revealed structural details that were invisible to conventional radiography. The surface rendering results gave a perfect description of complex spatial relationships among bronchi, bronchioles and alveoli. The 3D volume data allowed virtual endoscope of the lung airways which helped the doctor visualize the 3D model of lung and perform a diagnosis without having to operate on the patient. Moreover, the measurement of alveoli showed the ability of PCI to observe the morphology of lung. Therefore, this technique has the potential use in disease diagnosis, such as asthma, chronic bronchitis and emphysema, which are associated with the size or morphology of lung airways. There are also potential uses in pharmacology when determining the optimum diameter of aerosolized drugs in lungs.
One limitation in our research was the study sample. Live animals were not imaged. Despite the advances in spatial resolution and contrast of phase contrast imaging, real-time Xray phase tomography of the live animals has remained difficult. Live animals can only be imaged in two dimensions. The major obstacle in realizing this is the motion artifacts caused by breathing and cardiac motion of the animals, which will lead to serious blur in the reconstructed CT images. It is well known that the alveoli in lung are very small; any noise can influence the accuracy of the imaging result. Some attempts have been made to live animals imaging by taking the projection images at a specific breathing phase in synchrony with ECG signals [26] . But the animals have to be anaesthetized and with their breathing controlled by a ventilator. High-speed X-ray phase tomography may be another possible solution to live animal imaging by substantially reducing the imaging time. In fact, finishing a CT in one respiration phase is possible but the rotation speed of the sample stage and the time resolution of the detector need to be improved [27] . Another limitation was the lack of pathological samples. In this study, a normal 1-day-old mouse was used. The results proved that IL-XPCI is possible for lung micro-structures observation. Lung disorder models were missing, such as asthma, emphysema and lung cancer. We plan to evaluate these disorder models in our IL-XPCI imaging experiment as next steps, which are in progress now.
In this study, in-line X-ray phase contrast imaging was used to visualize a lung of an intact mouse. Our findings showed that IL-XPCI had the ability to represent complex anatomical structures in lungs. The three-dimensional model of lungs has been successfully established which provided an excellent view of lung airways. Thanks to the high resolution of the imaging, deep study was carried out on the tiny alveoli. A full understanding of the alveoli's architecture can facilitate the study of respiratory mechanism on the alveoli level. Single alveolus was displayed and measured. This offers a new perspective on the diagnosis of respiratory disease. Pathological changes can be detected by measuring the size of the lung airways, although further research and experimentation on lung is required to test this hypothesis.
All experiments and procedures carried out on the animals were approved by the animal welfare committee of Capital Medical University and the approval ID is SCXK-(Army) 2007-004. The study sample was a 1-day-old mouse, provided by Laboratory Animal Science, Capital Medical University. Before imaging, the mouse was humanely sacrificed by intraperitoneal (ip) sodium pentobarbital injection overdose. The mouse was imaged after four hours of its death in order to avoid the small shifts of the body caused by the development of rigor mortis [7] . And then a piece of Kapton (Dupont, DE, USA) was used and rolled up into a tube. This kind of film is an electrical insulation material with outstanding thermal, mechanical and chemical properties. The mouse was constrained in the tube, and placed on the sample stage in a vertically up-side-down position.
The principle of IL-XPCI is based on Fresnel diffraction theory which can provide an edge-enhancement effect. Pogany, Gao and Wilkins first established the theoretical formalism for phase contrast image formation of weakly absorbing thin objects [28] . When X-ray beams travel through the object, the downstream beams carry the information of absorption and phase shift. The interaction between the object and the beam can be described by this transmission function
where (x, y) is the spatial coordinates in the plane perpendicular to the propagation direction z, m and Q are the attenuation and phase shift induced by the object. They are given by
where l is the wavelength of the X-ray, b and d are the imaginary and real parts of the refractive index, respectively. After propagating a sufficient distance, the phase shifts in the downstream beams are transformed into measurable intensity variations by means of Fresnel diffraction. An image detector records them as the phase contrast image. According to Fresnel diffraction theory and the wave front function, the Fourier transform of the recorded intensity can be approximated bỹ
where d(u, v) is unit impulse function, (u, v) is the coordinate in Fourier domain, M and W are the Fourier transform of m and Q respectively at the distance z. From Eq. (4) one can find that the recorded intensity of the image is determined by the phase shift and the absorption of the wave. Apparently, the optimal contrast depends on the spatial frequency, wavelength, and the objectdetector distance [14] . The in-line X-ray phase contrast imaging experiment was performed at X-ray imaging and biomedical application beamline (BL13W1) of Shanghai Synchrotron Radiation Facility (SSRF). SSRF is the third-generation synchrotron radiation source in China. Figure 6 shows the schematic image of the experiment setup. It consisted of two monochromator crystals, one automatic rotation sample stage and one X-ray sensitive CCD detector. The incident white synchrotron X-ray beam was first monochromatized by two Si (111) prefect crystals. The tunable energy range was from 8 to 72.5 keV, with the energy resolution of about 0.5%. In our experiment, it was adjusted to 18 keV. The theoretical spatial resolution of the system was about 1 mm. Subsequently, the highly parallel and monochromatic beam projected on the object was imaged. The CCD detector then recorded the transmitted beam at a distance of 1.2 m from the sample. During this distance, the downstream image was enhanced by Fresnel diffraction and the phase modulation was transformed via amplitude modulation. An X-ray sensitive CCD camera, which had maximum resolution of 4008 pixels62672 pixels with 13 mm613 mm each, was used as a two-dimensional detector to transform the beam into an image. During the CT data acquisition, the specimen was rotated around its cylinder axis for 180u. The number of projections was 1296, with exposure time of 80 milliseconds for each projection, and the total scanning time was 208 seconds. The surface dose was about 8 mGy for each projection. All the parameters were selected in order to obtain high quality images of the mouse lung.
Although IL-XPCI can provide high-resolution images of soft tissues, image processing is required during the analysis of the image. Two-dimensional projection images always suffer from spatial superimposition of the lesions; small lesions inside the tissue can not be detected. Therefore, 3D visualization method is preferable.
First of all, the background image was used for normalization of the projection images. For the reconstruction of the CT slices, conventional filtered back projection (FBP) algorithm was used [29] .
The ribs are bone structures, which had a distinct gray scale in PCI reconstructed CT slices. Threshold-based method was used for the segmentation of ribs. Then an image segmentation method was applied to separate the lung tissues from the background. Since the histograms of our lung images had two peaks and a valley between them, the threshold for image segmentation can be chosen automatically at the bottom of this valley [30] . The other pixels with intensity lower than the threshold were set to zero. After this step, the ribs associated with many background signals considered as noise were removed. The whole lung airways can be segmented from this volume dataset by manually set a gray-level threshold. Due to the complexity of the lung structures and their irregular shapes and similar gray levels on the images, threshold-based image segmentation method was inadequate to separate the bronchial tree from the whole lung airways. In this study, we developed an image segmentation method based on 3D region growing to separate bronchial tree which was a semi-automatic procedure. It started in manually placing the seed point in the section of bronchus as well as setting the intensity threshold. Under the control of the intensity threshold, the growing would stop when the bronchial tree were separated.
After the above steps, three volume datasets of the ribs, the whole airways of the lung and the bronchial tree were obtained. Finally, the 3D models were generated by the use of surface rendering method. The surfaces are reconstructed by an isosurface detection algorithm which allowed a clear visualization of the complex spatial relationships of anatomical features.
Video S1 Animated view of rendered bronchi and alveoli. This is the same 3D model shown in Figure 3C . The rotation of the model permits the viewers to observe the lung from different angles. The model can be cut from any angle in order to give a detailed view of the inner part of the lung. The bronchi, bronchioles, and alveoli are all visible. (AVI) Video S2 The virtual endoscope from the bronchi to one alveolar duct. This is the same model shown in Figure 3D . The volume data of the lung airways is separated from CT slices using 3D image segmentation method. 360u rotation of the model displays the spatial relationships of different lung airways. And the virtual endoscope of the model reveals the internal lung airways. (AVI) Evolution of size and pattern in the social amoebas A fundamental goal of biology is to understand how novel phenotypes evolved through changes in existing genes. The Dictyostelia or social amoebas represent a simple form of multicellularity, where starving cells aggregate to build fruiting structures. This review summarizes efforts to provide a framework for investigating the genetic changes that generated novel morphologies in the Dictyostelia. The foundation is a recently constructed molecular phylogeny of the Dictyostelia, which was used to examine trends in the evolution of novel forms and in the divergence of genes that shape these forms. There is a major trend towards the formation of large unbranched fruiting bodies, which is correlated with the use of cyclic AMP (cAMP) as a secreted signal to coordinate cell aggregation. The role of cAMP in aggregation arose through co‐option of a pathway that originally acted to coordinate fruiting body formation. The genotypic changes that caused this innovation and the role of dynamic cAMP signaling in defining fruiting body size and pattern throughout social amoeba evolution are discussed. BioEssays 29:635–644, 2007. © 2007 Wiley Periodicals, Inc. The social amoebas or Dictyostelia represent one of nature's several independent inventions of multicellularity. The dictyostelia are members of the amoebazoans, a genetically highly diverse group that is the closest sister group to the clade containing the animals and fungi. (1) Except for the myxomycetes, all other known amoebazoans are microscopic unicellular organisms. The myxomycetes alternate a trophic amoeboid stage with a syncytial form. Here, a single cell with millions of nuclei, can grow up to several meters across. (2) Social amoebas also have a trophic amoeboid stage, but they achieve macroscopic dimensions by aggregation. (3) This occurs in response to starvation, which triggers regulated secretion of chemoattractant by the amoebas (Fig. 1) . Cellular agglomerates are formed, which can consist of up to a million amoebas. Sophisticated cell-cell signalling mechanisms between the amoebas orchestrate the differentiation of up to five different cell types and coordinate an intricate progression of cell movements. In combination with the synthesis of a flexible skin-like matrix, cell differentiation and cellular movement first generate the formation of a motile structure, called the ''slug''. The slug responds to chemical gradients and to light and warmth, which cause it to move to the soil's top layer. Here, the slug projects upwards and forms the fruiting body. This again involves highly ordered movement and differentiation and yields a slender column of stalk cells that bears aloft a global mass of spores. Depending on the species, the stalk can show different patterns of side branches and/or be decorated with disc, root or cup-shaped support structures (Fig. 2) . Unlike the ontogeny of sessile organisms like plants and fungi, which depends largely on series of directional cell divisions, the formation of fruiting bodies in social amoebas is more similar to the ontogeny of animal form. Both depend strongly on an intertwined program of cell movement and cell differentiation.
Seminal work of Raper showed similarity of principle in the establishment of the body plan in Dictyostelium and vertebrate development. In vertebrate development, a small group of cells known as ''the organizer'' releases signals that coordinate cell movement during gastrulation and neurulation and thereby generate the animal's head-to-tail body axis. (4) Raper demonstrated that, in Dictyostelium aggregates, small groups of cells, recognizable as tips, secretes signals that generate anteroposterior polarity of all or a subpopulation of cells in the aggregate, yielding one or several slugs with a distinct head and body region. (5) More recent work shows that animals and Dictyostelia share many conserved pathways for processing external signals. Particularly the elucidation of the processes that control chemotaxis in Dictyostelium have become a paradigm for understanding cell migration in animals. (6) (7) (8) However, the external signals that trigger movement or differentiation are rarely conserved. For instance, growth-factor-like peptides and their tyrosine kinase receptors that play such crucial roles in animal development are not present in Dictyostelium. The homeobox-containing transcription factors that specify segment identity in arthropods and vertebrates have only a minor function in Dictyostelium development. (9) (10) (11) The signaling repertoire of the Dictyostelia is rather different. The model species, D. discoideum makes extensive use of cyclic AMP (cAMP), the ubiquitous intracellular messenger for hormone action in vertebrates. In D. discoideum, cAMP not only mediates the effect of a number of external signals, but is also secreted to act as a chemoattractant and inducer of cell differentiation. (12) Secreted peptides trigger maturation of spores, but are detected by sensor-coupled histidine kinases. (13) Polyketide-based metabolites and adenine-based cytokinins are secreted to regulate cell-type proportioning and spore germination respectively. (14, 15) Animals, plants and Dictyostelia evolved from different unicellular ancestors, supposedly the filter-feeding choanoflagellates for animals, (16) green algae for plants (17) and solitary amoebas for the Dictyostelia. (18) These unicellular progenitors used sensory signaling to monitor their environment and to find food and mates. The different developmental strategies that are now used by their multicellular descendants reflect how evolutionary forces differentially selected, duplicated and adapted these environmental sensing mechanisms for increasingly complex communication between cells.
Historical reconstruction as a tool to understand developmental signalling By acting on alleles that are generated by random mutation, organic evolution is intrinsically opportunistic. It does not create optimal design, but selects combinations of traits that provide the highest probability of reproduction in a specific ecological niche. Consequently, there are no unifying schemes to explain organismal development. The underlying molecular mechanisms only truely make sense in the context of their evolutionary history. What mechanisms were used by the ancestors and how were these mechanisms modified to improve functionality in the better adapted descendants?
The evolution of novel forms in multicellular organisms requires alteration of the developmental mechanisms that shaped the earlier form. Evo-devo, short for evolutionary developmental biology, is a relatively young discipline that sets out to retrace how gene and genome modifications have altered existing developmental mechanisms to produce novel forms. Evo-devo has been predominantly applied to the development of animals (19) (20) (21) and, to a lesser degree, of higher plants. (22, 23) However, an understanding of development of other multicellular organisms, such as the social amoebas or fungi will equally benefit from this approach. In the model organism D. discoideum, starving amoebas secrete cAMP pulses, which trigger chemotactic movement and aggregation of cells. Once aggregated, the amoebas differentiate into prestalk and prespore cells in a regulated ratio. The organizing tip continues to emit cAMP pulses, which shape the cell mass by coordinating cell movement. The cAMP pulses also cause the prestalk cells, which are chemotactically most responsive, to move towards the front. At the onset of culmination, the cells synthesize a cellulose tube, the apical prestalk cells move into the tube and mature into stalk cells, the remaining prestalk cells form support structures, such as the upper and lower cup and the basal disk. The prespore cells move up the stalk and mature into spores.
Moreover, the greater genetic tractability of these organisms will greatly aid in establishing how gene modification caused novel forms to appear. The social amoebas provide other opportunities to retrace the evolution of multicellular development. All known species can be grown under laboratory conditions and complete their multicellular life cycle within a 28 hour period. They show a broad range of different morphologies, with terminal structures varying in size between 0.1 mm and several centimeters. The genome of the model species D. discoideum is completely sequenced (10) and sequencing of four other Dictyostelia genomes is in progress.
About 7 years ago, we initiated research into the evolutionary history of developmental signaling in the social amoebas, concentrating on cAMP signalling. A primary requisite for this project was the availability of a family tree that shows the relatedness of social amoeba species relative to the more ancestral solitary amoebazoans. We joined forces with the teams of Sandra Baldauf, an expert in protist molecular phylogeny, Thomas Winckler, who had already prepared an SSU rRNA tree of a subset of Dictyostelia species and two Dictyostelium field biologists, Jim Cavender and Hiromitso Hagiwara, to construct a molecular phylogeny of all known Dictyostelia. (24) We next mapped morphological traits of all species to the tree in order to determine the directionality of morphological evolution in the social amoebas. In parallel studies, the presence, regulation and function of genes that are essential for various aspects of cAMP signalling were investigated in social amoeba species that span the phylogeny. (25) This review presents a synthesis of the outcome of these studies with current insights in developmental signaling in the model species D. discoideum. It highlights major trends in the evolution of multicellular complexity in the social amoebas, and correlates these trends with elaboration of function of deeply conserved cAMP signalling genes.
In traditional systematics, the Dictyostelia were grouped with the acrisid amoebas in the division of mycetozoans in the kingdom of fungi. (3, 26) However, recent molecular evidence shows that none of these groups are fungi; the acrasid amoebas are members of the discicristates, while the Dictyostelia and the mycetozoans are members of the amoebozoans. (1, 18, 27, 28) Based on fruiting body architecture, the social amoebas were subdivided into three genera: the dictyostelids with unbranched or laterally branched fruiting structures, the polyspondylids with regular whorls of side branches and the acytostelids with acellular stalks.
Comparison of conserved DNA or protein sequences is a more direct and reliable method to establish genetic relationships. Two family trees of the social amoebas were constructed by comparing the DNA sequences of their small subunit ribosomal RNA (SSU rRNA) gene on one hand and the amino-acid sequences of their a-tubulin protein on the other. Both trees show that the similarities in fruiting body architecture only partially reflect an underlying genetic similarity. (24) Instead, both the a-tubulin tree and the SSU rRNA tree shown here (Fig. 3 ) subdivide the 75 known species of social amoebas into four major groups. There are dictyostelids in all four groups. The acytostelids and all white polysphondylids are members of group 2, but the purple polysphondylid P. violaceum occupies a position between groups 3 and 4, and forms a small clade with the dictyostelid D. laterosorum. This indicates that at least two out of the three previously proposed genera are polyphyletic. Multiple origins for the polyspondylids were also predicted by a family tree that was based on 18 combined morphological traits. (29) The DNA-based family tree was subsequently used to investigate trends in the evolution of morphology. The multicellular stages of different species of Dictyostelia show a large variety of shapes and sizes, which have been carefully quantitated and noted in the original species diagnoses, along with differences at the cellular level (see Refs 3, 30 for overviews). Species use different chemoattractants and aggregate as single cells or as inflowing streams (Fig. 4) . Once formed, aggregates may either produce one or several organizing tips, giving rise to solitary or clustered fruiting bodies. Secondary tips may appear in characteristic positions on rising sorogens, giving rise to secondary body axes and a range of different fruiting body architectures. Fruiting body stalks may develop a variety of support structures, such as discs, crampons or triangular supporters, while their tips can vary from thinly pointed to bulbous. Many species form motile slugs, which may optionally form a stalk while migrating. At the cellular level, spores can be round or oblong and, in the latter case, display conspicuous granules at their poles, which are either loosely grouped or consolidated. Some species have retained the ancestral survival strategy of encystation, or display the capacity to mate and form sexual macrocysts. A mapping of all these characters to the molecular tree indicates which characters are shared between close relatives, and yields information about the order in which characters evolved (Fig. 5) . Amoeba size shows no strong group-specific trend, but spores are consistently smaller in the most basal group 1. Of all traits, spore morphology follows the phylogeny most strongly. Spores of the evolutionary youngest group 4 species have no polar granules, in group 2, polar granules are loosely grouped, and in groups 1 and 3, they are consolidated. The acellular stalk evolved only once. The shape of the stalk tip also marks relatedness; species in group 2 usually have pointy or blunt tips, while in groups 1 and 4 stalk tips tend to be club-or head-shaped. Species with similarly coloured stalks and spore heads are often related, but no color is specific for any of the four major groups.
Encystation of individual cells is lost from group 4 species, but retained in the evolutionary older groups, while sexual macrocysts are made by species scattered over all four groups. The chemoattractant that is used for aggregation is known for only a few species. It is cAMP for all investigated group 4 species while, in the other groups, at least three other compounds are used. Most species aggregate as inflowing streams of amoeba, but groups 1-3 contain some species that aggregate as individuals. Slug migration also occurs in all groups, but is most common in group 4. Stalkless migration is shown by a small cluster of group 4 species and a single nongroup 4 species, D. polycephalum. Fruiting structures of most but not all species throughout the phylogeny veer towards light (phototropism).
Fruiting bodies (sorocarps) tend to be clustered or grouped in groups 1 to 3 and solitary in group 4, while branched structures are also more common in the basal groups. Specific branching patterns do not show strong group-specific trends and laterally branched, rosary-type and whorled morphologies appear, respectively, six, two and five times across the tree. There is a modest trend towards taller sorocarps in the more-derived members of groups 1-3, and a very strong trend towards sorocarps with long thick stalks and large spore heads (sori) in group 4. These large sorocarps are usually buttressed by cellular support structures, such as basal disks and supporters. The crampon-base is almost uniquely associated with a tight cluster of group 3 species.
In summary, the most-obvious trend in the evolution of social amoebas appears to be related to size. Evolutionary younger species both have larger sized spores and larger sized fruiting bodies. The latter is particularly evident in group 4 where large stalk and sori size is correlated with a tendency to form solitary and unbranched fruiting bodies. In addition to large size, group 4 displays other distinguishing features, such as formation of cellular support structures, loss of individual encystation, loss of spore granules, and the use of cAMP as attractant. The correlation of the latter two traits was also noted earlier by Traub and Hohl. (31) These workers also associated the presence of polar granules with a tendency to form clustered and/or branched sorocarps and a tendency for those sorocarps to be smaller than in species without polar granules, both of which are borne out by the recent analysis.
The adaptive advantage of larger spores and fruiting bodies can be surmised. Larger spores may store more nutrients to survive dormancy, while larger fruiting bodies may aid spore dispersal, both contributing to species propagation. Individual encystation may have become redundant after the sporulation mode of survival became more robust. It is less easy to envisage how loss of spore granules and use of cAMP as attractant improved fitness. In the following paragraphs, we explore how the latter character may have been a means to achieve an end.
The appearance of new morphologies in multicellular organisms requires alteration of existing developmental pathways. In the social amoebas, developmental pathways have only been studied in detail in the model organism D. discoideum, where cell-to-cell communication is largely mediated by secreted signaling molecules.
cAMP plays a primary role; it is secreted in periodic waves by aggregation centres to mediate the aggregation of starving cells. (32) Later, organizing tips become the sources of cAMP waves (Fig. 1) , which direct the movement of cells in multicellular structures. (33) Secreted cAMP also triggers the differentiation of the prespore cells. (34, 35) In turn, the prespore cells secrete a chlorinated polyketide, DIF, that induces regulated redifferentiation of prespore cells into prestalk cells. (14) Ammonia, which is produced by protein degradation in the starving cells, represses terminal spore and stalk cell maturation during slug migration, (36, 37) and is implicated in cell sorting, slug phototaxis and fruiting body phototropism. (38, 39) Two secreted peptides, conditioned medium factor (CMF) and prestarvation factor (PSF) induce the growth to development transition. (40) Other peptides, the spore differentiation factors (SDFs), trigger the maturation of spores. (13) There is no information on the conservation of the peptide signals in other social amoeba species. Ammonia is always produced by starving amoebas, and at least some of its roles are therefore likely to be conserved. DIF was identified in another group 4 species, D. mucoroides, which also has the DIF-degrading enzyme, DIF dechlorinase. D. minutum and P. violaceum, which reside in group 3 and between groups 3 and 4, respectively, synthesize chlorinated factors that induce stalk cell differentiation in D. discoideum. However, they do not have DIF dechlorinase, indicating that the DIF signaling pathway is at best partially conserved. (41, 42) More information is available on the conservation of cAMP signalling. All tested group 4 species use cAMP to aggregate, but no species outside group 4. However, early biochemical work showed that species that use other attractants to aggregate, nevertheless display cAMP binding sites and cAMP phosphodiesterase on their cell surface after aggregation. (43, 44) The group 3 species, D. minutum, aggregates by continuous release of folic acid, but shows cAMP waves emerging from the tip region after aggregates have formed. (43, 45) This suggested that the role of the tip as a pacemaker of cAMP waves is deeply conserved in the social amoebas.
Cell surface cAMP receptors (cARs) mark the use of cAMP as an extracellular signal. D. discoideum has four homologous cARs. cAR1 is expressed shortly after starvation, while cAR3, cAR2 and cAR4 are expressed at progressively later stages. (45, 46) Recent studies show that the cAR1 gene is deeply conserved in social amoeba evolution and is present in all four taxon groups. (25) The gene duplications that gave rise to cAR2, cAR3 and cAR4 only occurred in group 4 (Y. Kawabe and P. Schaap, unpublished results). The basal cAR1s are functionally identical to D. discoideum cAR1, but there is a marked difference in their developmental regulation. The cAR1s from the basal groups are expressed as a single mRNA after aggregation while, in group 4 species, a second cAR1 mRNA is expressed before and during aggregation. (25) Transcription of this early mRNA is driven by a second promoter that is more distal from the cAR1 coding sequence than the promoter that drives expression after aggregation. (47) Also in the gene encoding the extracellular cAMP phosphodiesterase, the promoter that drives late expression is proximal to the coding sequence and the promoters that drive expression before and during aggregation are more distal. (48) This arrangement suggests that the use of cAMP as chemoattractant by the evolutionary younger group 4 species was achieved by addition of distal promoters to existing cAMP signalling genes.
Loss of cAR1 function in group 4 species blocks aggregation and further development. In the basal groups 1-3, aggregation is unaffected, but the subsequent formation of slugs and fruiting bodies is disrupted. (25) This indicates that, in the more basal species, extracellular cAMP signalling is required for slug and fruiting body morphogenesis.
cAMP signaling and size regulation In addition to using cAMP for aggregation, group 4 species also stand out by having large solitary unbranched fruiting structures, as opposed to the clustered and branched smaller structures that are common to the other groups. Are cAMP signalling and size related?
The segmentation of aggregates into clusters of fruiting bodies and the formation of side branches all represent the formation of multiple body axes that are initiated by newly emerging tips (Fig. 4) . Analogous to the phenomenon of apical dominance in plants, where lateral shoots are suppressed by the primary shoot, (49) D. discoideum tips suppress the formation of ancillary tips. D. discoideum tips are selforganizing pacemakers for cAMP waves. The waves are propagated through the cell mass by cAMP-induced cAMP production, also known as cAMP relay.
Tip dominance can be established in different manners: (1) higher frequency oscillators entrain cells that oscillate at lower frequency, (50) and (2) tips produce a diffusible inhibitor that reduces the excitability of surrounding cells. (51) The cAMP hydrolysis product adenosine was proposed to fulfill this role. (52, 53) Dominance will break down if there are physical or biochemical barriers that prevent propagation of cAMP waves or diffusion of the inhibitor.
Irrespective of the exact mechanism, dominance is intrinsic to oscillatory cAMP signalling. Group 4 species have larger fruiting bodies because their cAMP oscillators are better at suppressing competitors. Two aspects of cAMP signalling are specific to group 4: (1) oscillatory cAMP signalling occurs much earlier in development than in groups 1-3, and (2) the cAMP receptor gene was duplicated three times, and both expression and affinity of the daughter cARs were altered. It is conceivable that either of these novelties may have ''improved'' cAMP signalling to allow it to control larger numbers of cells and make it generally more robust.
The plasticity of fruiting body architecture The DNA-based phylogeny of the social amoeba did not reproduce the earlier classification into three genera that was based on fruiting body architecture. In fact, it appeared that many similar fruiting body branching patterns evolved several times independently (Fig. 5 ). This implies that specific architectures cannot be under extensive genetic control.
As discussed above, fruiting body branching patterns reflect how and when competing pacemakers for cAMP waves appear on multicellular structures. The production of cAMP waves by D. discoideum cells consists of a positive feedback loop where extracellular cAMP acting on cAR1 stimulates further cAMP production by adenylyl cyclase A (ACA), and a negative feedback loop where cAMP inhibits ACA and stimulates its own hydrolysis. (54) (55) (56) Variation in a range of parameters, such as the relative expression levels of the component proteins, diffusion or cell movement barriers generated by structural components, and the relative motility or cohesiveness of responding cells can potentially affect the dynamics of the signalling process in a such a way as to allow competing pacemakers to arise in a variety of configurations.
For instance, D. gloeosporum, which owes its name to its extremely sticky spore matrix, (57) is the single dictyostelid member of the clade of white polysphondylids (Fig. 3) . The branched whorls of the polysphondylids are formed when a group of cells detaches from the rear of a rising cell mass and then forms new tips (Fig. 4) . D. gloeosporum may owe its consolidated single spore head to the fact that, due to the highly adhesive matrix, detachment of cell masses does not occur. Similarly, other fruiting body architectures are likely to result from interaction of the cAMP signaling network with different biophysical environments, rather than being controlled by architecture-specific genes.
Does branching have any adaptive value at all? I believe it does. Within groups 1-3 there is a trend towards taller fruiting bodies in the more-derived species. Being carried in the air on tall stalks may not only aid spore dispersal but also contribute to spore preservation, away from the decomposing agents in wet humus. However, the construction of a robust stalk comes at a cost of reducing the spore-to-stalk ratio. Group 4 species resolve this problem by additional cell-type specialization to form support structures. For the basal groups, branching and particularly whorl formation may provide a solution for the problem of building tall well-balanced fruiting bodies without sacrificing too many spores.
This review summarizes the recent construction of a systematic framework to study causal relationships between genotypic and phenotypic change during evolution of the social amoebas. The foundation of this framework is the first DNAbased phylogeny for all known species of social amoebas. The phylogeny, which is based on SSU rDNA sequences and confirmed by a-tubulin protein sequences shows subdivision into four major groups and a molecular depth that is equal to that of all animals. (24) A plotting of the most consistently noted species characters onto the phylogeny shows unexpected trends in character evolution with the greatest changes occurring at the transition between the youngest group 4 and the evolutionary older groups 1,2 and 3. Group 4 species are characterized by large solitary and unbranched fruiting structures as opposed to smaller, clustered and branched structures in the other groups. Group 4 species have also lost the ancestral survival strategy of encystation and gained the use of cAMP as chemotactic signal for aggregation.
A study into the evolutionary origins of extracellular cAMP signalling revealed that this strategy is used by all social amoebas to coordinate the process of fruiting body formation. Group 4 species have recruited this mechanism to additionally control the aggregation process. This occurred by adding aggregation-specific promoters to existing cAMP signalling genes.
Many intriguing questions remain unresolved. Social amoebas are the only known organisms that use cAMP as extracellular signal. How did this role of cAMP originate in the first place? cAMP signals are produced by oscillating pacemakers. Are these dynamics unique for cAMP or are other chemoattractants, such as glorin, also released in an oscillatory manner? Are other D. discoideum signal molecules, such as DIF, ammonia, SDF, PSF and CMF conserved throughout the phylogeny?
Thus far, only those features were plotted to the phylogeny that were observed by standard light microscopy. One cellassociated character, the presence of granules in spores proved to be the strongest group-defining determinant. This suggests that there are other characters at the cellular level that define species within groups. More detailed (ultra)microscopic analysis would be required to identify such features.
In D. discoideum, the proportion of prespore and prestalk cells in slugs are regulated to the approximate proportions of stalk and spores in the fruiting body. However, in other species such as P. violaceum, P. pallidum and D. lacteum, cells first differentiate into prespore cells only to dedifferentiate into stalk cells at the tip. (58, 59) Apart from stalk cells and spores, D. discoideum has three more cell types, the basal disc, upper cup cells and lower cup cells, that each display specific patterns of gene expression. When and how did cell-type proportioning and greater cell-type specialization evolve?
In addition to these development-related aspects, the molecular phylogeny provides a framework to investigate conservation and divergence of any protein with an important function in the cell biology of D. discoideum. This is useful for identification of conserved domains and/or amino-acids in proteins that are thus far not well characterized, but also to outline how protein modification gave rise to novel protein functions. Projects are now in progress to sequence the genomes of at least four group-representative social amoeba species. Combined with detailed information of phenotypic evolution, this information on the evolution of genotype will provide tremendous opportunities to retrace how this particular form of multicellular life evolved. Plant Plastid Engineering Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation. Genetic material in plants is distributed into nucleus and the chloroplast and mitochondria in the cytoplasm. Each of these three compartments carries its own genome and expresses heritable traits [1, 2] . The chloroplast is one of organelles known as plastids in plant cells and eukaryotic algae [3] . According to Verhounig et al. [4] , plastids and mitochondria are derived from formerly free-living bacteria and have largely prokaryotic gene expression machinery. The plastid (biosynthetic centre of the plant cell) carries out photosynthesis, in plant cells and eukaryotic algae, which provides the primary source of the world's food [3] . There are other important activities that occur in plastids. These include sequestration of carbon, production of starch, evolution of oxygen, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism [5] . In spite of the prokaryotic past of the plastids, their gene expression has very different regulatory mechanisms from those operating in bacteria [6] . There are up to 300 plastids [7] in one plant cell. The plastid genome (plastome or plastid DNA, ptDNA), 1,000-10,000 copies per cell [8] , contrasts strikingly with the nuclear DNA. In most species, plastids are usually strictly maternally inherited [9] in most (80%) angiosperm plant species [10, 11] . It is also not influenced by polyploidy, gene duplication and recombination that are widespread features of the nuclear genomes of plants [12, 13] . Therefore, ptDNA varies little among angiosperms in terms of size, structure and gene content [14] . Currently 170 DNA. The authors, therefore, developed a simple and inexpensive method to obtain plastid DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Plastid engineering involves the targeting of foreign genes to the plastid's double-stranded circular DNA genome instead of chromosomal DNA [31] , and as a consequence the production of the foreign protein of interest (Fig.  1) . The advancement in the particle gun mediated transformation has enabled targeting the plastid genome for developing transgenic plants against many biotic (e.g. insects and pathogens) and abiotic stresses (e.g. drought and salinity), which reduce the plant productivity. Improving the quality of fruits has been another main target in plastid transformation [32] .
Plastid transformation was first achieved in a unicellular alga, Chlamydomonas reindhartii [33] . In 1990, Sváb et al. [34] reported the first successful chloroplast transformation of a higher plant (tobacco). This was followed by transformation of the plastid genome in tobacco by many researchers [35, 36] . Recently, tobacco plastid has been engineered to express the E7 HPV type 16 protein, which is an attractive candidate for anticancer vaccine development [37] . Similarly, a protocol for plastid transformation of an elite rapeseed cultivar (Brassica napus L.) has been developed [38] . Brassica napus - [132] Citrus sinensis 155,189 [133] Coffea arabica - [134] Cucumis sativus 155,293 [135] Daucus carota 155,911 [136] Ficus sp.
- [137] Glycine max 152,218 [138] Gossypium barbadense 160,317 [139] Gossypium hirsutum 160,301 [140] Helianthus annuus 151,104 [141] Hordeum vulgare, Sorghum bicolor - [142] Lycopersicon esculentum 155,460 [21] Manihot esculenta - [143] Morus indica 156,599 [144] Musa acuminata - [145] Nicotiana tabacum 155,943 [146] Oryza sativa 134,551 [15] Oryza nivara 134,494 [16] Phaseolus vulgaris 150,285 [147] Saccharum officinarum 141,182 [18] Solanum tuberosu 155,298 [148] Spinacia oleracea 150,725 [149] Triticum aestivum 134,545 [19, 20] Vitis vinifera 160,928 [150] Zea mays 22,784 [17] Jatropha curcas 163,856 [151] Vigna radiata 151,271 [22] More recently, a method for plastid transformation in eggplant (Solanum melongena L.) has been reported with pPRV111A plastid expression vector carrying the aadA gene encoding aminoglycoside 300-adenylyltransferase [26] . The authors believe that this may open up exciting possibilities to introduce and express novel genes in the engineered plants via plastid transformation for agronomic or pharmaceutical traits. Up to date, plastid transformation has been extended to many other higher plants, such as Arabidopsis thaliana [39] , potato [40, 41] , tomato [1, 42] , Lesquerella fendleri, a kind of oilseed Brassicaceae [43] , oilseed rape, [38, 44] , petunia [45] , lettuce [46] , soybean [47] , cotton [48] , carrot [49] , rice [50] , poplar [51] , tobacco, [28, 52, 53] , mulberry , [54] and eggplant [26] (see review written by Wang et al. [3] ).
Two interesting applications of plastid transformation were carried out by (i) [55] for the construction of a tobacco master line to improve Rubisco engineering in plastids, and (ii), [4] who explored the possibility of engineering riboswitches (natural RNA sensors that regulate gene expression in response to ligand binding) to function as translational regulators of gene and transgene expression in plastids.
The dominant trait that attracted the most attention for plastid transformation has been herbicide tolerance [28, 35, 56, 57] . Roudsari et al. [28] revealed the production of high level glyphosate tolerant plants (N. tabacum) through biolistic transformation of plastids by introduction of a mutated herbicide-tolerant gene coding for EPSP synthase. Plastid transformation is routine, however, only in tobacco and the efficiency of transformation is much higher in tobacco than in other plants [58] . Lee et al. [50] discussed the major obstacles to the extension of plastid transformation technology to other crop plants which includes regeneration via somatic embryogenesis.
Production of transgenic plants, at laboratory level or commercially, has traditionally been mainly through expression of transgenes in the nucleus [24, 25] . Among the ecological concerns raised about genetically engineered organisms is that transgenes could move ("transgene flow", the process of transgene movement by recurrent hybridisation) via pollen from the crop and into relatives growing in natural or semi-natural communities [59] . Such concerns have led to a new field of transgene containment [60, 61] .
Since plastids are inherited maternally in the majority of angiosperm species, they would therefore not be found in pollen grains of corps. Insertion of transgenes, therefore, into the plastid genome has the potential of preventing gene flow via pollen. Hence, genes expressed in the plastome will not be transferred through pollination to weedy or wild relatives of the transgenic crop. Bansal and Sharma, [62] believes that there is little risk of any transgene flow via pollen from transplastomic plants to the neighboring weedy or wild relatives since plastids are almost always maternally transferred to the next progeny. The authors suggested that plastid transformation could be a method of choice for generating improved transgenics in crops that grow along with their weedy or wild relatives in the same geographical region, such as rice, sorghum, cucurbits, solanaceous crops, Vigna and Cajanus species, and various Brassica crops. Therefore, the focus of many researchers has shifted to plastid engineering [26] , rather than nuclear transformation. Singh et al. 2010 [26] reported that engineering of the plastid genome is gaining momentum as an attractive alternative to nuclear transformation.
Ruf et al. [60] believe that plastid transformation is considered as a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. However, they pointed out that plastid transformation would only be effective as a biocontainment measure when applied on a landscape scale if it were combined additional mechanisms such as mitigating genes genetic use restriction technology, and/or male sterility [63] . In a recent study, it has been demonstrated that the use of plastid transformation would provide an imperfect biocontainment for GM oilseed rape (Brassica napus L.) in the United Kingdom [64] . In another study, Allainguillaume et al. [65] revealed that chloroplast transformation may slow transgene recruitment in two settings, but actually accelerate transgene spread in a third. Plastid transformation has become an attractive alternative to nuclear gene transformation due to several other advantages [3] . The high ploidy number of the plastid genome allows high levels (up to 1-40% of total protein) of protein expression or expression of the transgene [28] . Daniell et al. [66] and Hou et al. [44] reported that while nuclear transgenes typically result in 0.5 -3% of total proteins, concentration of proteins expressed by plastid transgenes is much higher; up to 18%. The greater production of the expressed protein is possible because plastid transgenes are present as multiple copies per plant cell, and they are little affected by phenomena like pre-or post-transcriptional silencing. Other advantages of plastid engineering are the capacity to express multiple genes from polycistronic messenger RNA (mRNA) [31] , and the absence of epigenetic effects and gene silencing [40] .
Wang et al. [3] believe that transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. Added to that, plastid transformation is more environmental friendly than transformation of the nuclear DNA for plant engineering because it eliminates the possibility of toxic transgenic pollen to nontarget insects [67] . Adverse effects of toxic proteins might be minimized by plastid compartmentalization but in case of nuclear transformation, toxic proteins accumulating within the cytosol might result in serious pleiotropic effects. Further, the expression of the transgene in case of plastid transformation is more uniform compared to that of trangenes inserted into the nuclear genome. Although there is a major drawback in the engineering of plastid gene expression, which is the lack of tissue-specific developmentally regulated control mechanisms [3] , the many advantages of plastid engineering stated above attracted researchers to engineer the plastid genome to confer several useful agronomic traits, and hence the number of species whose plastome can be transformed continues to expand [68] .
Gene delivery into the plastome was initially done by Agrobacterium mediated method [Block et al.1985 ] [69] . The discovery of biolistic DNA delivery led to plastid trans-formation via particle gun [Sanford 1990 ] [70] . In this method, the Escherichia coli plasmids contain a marker gene and the gene of interest is introduced into plastids. The foreign genes are inserted into plasmid DNA by homologous recombination via the flanking sequences at the insertion site [66] . Polyethylene glycol (PEG) mediated transformation of plastids was also utilized [71, 72] . PEG-mediated transformation of plastids requires enzymatically removing the cell wall to obtain protoplasts, then exposing the protoplasts to purified DNA in the presence of PEG. The protoplasts first shrink in the presence of PEG, then lyse due to disintegration of the cell membrane. Removing PEG before the membrane is irreversibly damaged reverses the process. Biolistic delivery is the routine system for most laboratories, as manipulation of leaves, cotyledons, or cultured cells in tissue culture is a simple practice than the alternative PEG treatment of protoplasts [58] . Recently, particle gun mediated plastid transformation has been demonstrated in rapeseed using cotyledons as explants [38] .
The major difficulty in engineering plastid genome for production of transplastomic plants is in generating homoplasmic plants in which all the plastids are uniformly transformed, for that takes a long process of selection, thus hampering the production of genetically stable transplastomic plants (e.g. rice). This is due to the presence of about 10-100 plastids, each of which has up to 100 copies of the plastid genome, in one cell, that does not allow achieving homoplastomic state [73] . It was also stated that getting high level of protein expression, even though the gene copy number is high, is another problem. In 2005, however, Nguyen et al. [41] described the generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.) using two tobacco specific plastid transformation vectors, pZS197 (Prrn/aadA/psbA3 ) and pMSK18 (trc/gfp/Prrn/aadA/psbA3 ). Similarly, Liu et al. [74] were able to develop homoplasmic fertile plants of Brassica oleracea L. var. capitata L. (cabbage). Among other higher plants of which fertile homoplasmic plants with genetically modified plastid genomes have be been produced are Nicotiana tabacum (tobacco), Nicotiana plumbaginifolia (texmex tobacco), Solanum lycopersicum (tomato), Glycine max (soybean), Lesquerella fendleri (bladderpod), Gossypium hirsutum (cotton), Petunia hybrida (petunia), and Lactuca sativa (lettuce) [68] . The amino glycoside 3adenylyltransferase (aadA) gene, which confers dual resistance to spectinomycin-streptomycin antibiotics, is still the selectable marker that is routinely used efficiently for plastid transformation [58, 75, 76] . Since the antibiotic resistant genes used in transformation are not desirable in the final products, different strategies have been developed to eliminate the necessity of using such selectable markers [56, 77] .
Apel & Bock, [42] demonstrated the potential of plastids genome engineering for the nutritional enhancement of food crops when they enhanced carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion. The transplastomic technology could also be useful for engineering agronomic traits including phytoremediation [78] reversible male sterility [79] , and toler-ance/resistance of stresses such as diseases, drought, insect pests, salinity and freezing that can severely limit plant growth and development [3] .
Since plastids are transferred mostly through the "maternal inheritance" as identical copies, and hence a female plant transfers identical copies to all the seeds it produces without changes from one generation to the next, an important promise for applying plastid transformation for industry is the stable passing on to the next generation of the foreign DNA [80] . Therefore, the plastid genome has also been utilized for metabolic pathway engineering and in the field of molecular farming (the production of drugs and chemicals through engineered crops) [27, 68] for the expression and production of biomaterials and biopharmaceuticals in plants, human therapeutic proteins, and vaccines for use in humans or animals (reviewed in [26, 27, 50] . Singh et al. [26] believe that for such applications, plastid transformation technology offers solutions to the ecological and technical problems associated with conventional transgenic technologies such as outcrossing and transgene silencing.
Transformation of the nuclear genome of plants with genes (e.g. Bt genes) to confer insect resistance gives very low levels of expression unless extensive modifications are carried. Whereas, introduction of the same genes into the plastid genome results in high levels of toxin accumulation as the plastid genome is bacterial in origin [81] . Therefore, the insect resistance genes were investigated for high-level expression from the plastid genome [3] . Hence, when insect resistance genes are expressed into the plastid genome, leaves of these transplastomic plants proved highly toxic to herbivorous insect larvae.
One of the major advantages of introducing the Bt toxin into the plastid genome is the high levels of toxin accumulation (3% -5% of total leaf protein as compared to > 0.2% of total soluble protein through nuclear genome transformation) [82] . Achievement of stable transformation of the plastid genome and transforming plastids in species other than tobacco (Nicotiana tabacum) are some of the hurdles for widespread adaptation of this technique [81] . Despite of overwhelming odds, many attempts have been made to produce transplastomic plants expressing Bt toxin for increased resistance against insect pests. [83] generated soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin. Similarly, Chakrabarti et al. [84] reported the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. The authors observed high-level expression (about 10% of total soluble protein) of the cry gene from the plastid genome which resulted in severe growth retardation.
Over-expression of the cry2Aa2 operon in plastids was proved effective in allowing a broad-spectrum of protection against a range of pests [81] . In cabbage, the cry1Ab gene was also successfully transferred into the plastid genome [85] . Expression of cry1Ab protein was detected in the range of 4.8-11.1% of total soluble protein in transgenic mature leaves of the two species. Insecticidal effects on Plutella xylostella were also demonstrated in cry1Ab transformed cabbage. In an attempt to increase insect resistance in trans-genic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for plastid-targeted expression [86] . Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25-and 100-fold, respectively, with the accumulated protein in plastids comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. It was concluded that targeting of cry1Ac protein to the plastid using the rbcS:tp system confers a high level of plant protection to insects. Several other cry proteins have also been expressed in plastids of tobacco [83, 84, 87, 88] and rice [50] (see Table 2 ).
Plastid engineering offers a new and effective option in development of plant varieties which are resistant to various bacterial and fungal diseases. In tobacco, introduction of MSI-99 gene, an antimicrobial peptide, into plastids resulted in transplastomic plants resistant to fungal pathogen Colletotrichum destructive [89] . The plastids expressed MSI-99 at high levels and showed 88% (T1) and 96% (T2) inhibition of growth against Pseudomonas syringe, which is a major plant pathogen. In another study, Agrobacterium mediated transformation was used to develop tobacco plants carrying argK gene, which encodes ROCT [90] . Since OCT in plant cells is produced in the plastid, argK was fused to the plastid transit sequence of the pea rubisco small subunit (rbcS) gene for localized expression of the enzyme. The ROCT enzyme produced by the transgenic tobacco showed greater resistance (83-100%) to phaseolotoxin compared to the wild-type OCT (0-22%). When phaseolotoxin was applied exogenously to the leaves of plants, chlorosis was observed in 100% of wildtype tobacco, but not seen in the leaves of the transgenic tobacco plants carrying the argK gene from P. syringae pv. phaseolicola. Transgenic tobacco plants that constitutively expressed both entC and pmsB in the plastid have also been reported [91] where transformation was accomplished through biolistic methods. The transgenic tobacco plants expressing these bacterial genes showed accumulation of salicylic acid that were up to 1000 times higher than that observed in wild-type tobacco. When challenged with the fungus Oidium lycopersicon, the transgenic tobacco plants showed increased levels of resistance compared to the wildtype plants. It was revealed that the transgenic plants generated did not show any adverse effects due to the high level expression of salicylic acid. Thus gene transfer in plastids can provide a significant protection from various bacterial and fungal diseases.
Transgenes that confer tolerance to abiotic stress may permanently transfer from transgenic crops to the nuclear genome of their weedy relatives which may result in drought tolerant superweeds [92] when the gene is inserted into the nuclear genome and the transgenic plant outcross with relative weeds. There is a great potential, therefore, for the genetic manipulation of key enzymes involved in stress metabolism in plants within plastids. Because plastid genomes of major crops including cotton and soybean have been Nicotiana tabacum nptII [153] Nicotiana tabacum uidA [154] Nicotiana tabacum Human somatotropin (hST) [155] Nicotiana tabacum cry [88] Nicotiana tabacum cry9Aa2 [84] Nicotiana tabacum Bar & aadA [156] Nicotiana tabacum Cor 15a-FAD7 [157] Nicotiana tabacum rbcL [55] Nicotiana tabacum DXR [158] Nicotiana tabacum aadA & gfp [60] Nicotiana tabacum Delta(9) desaturase [159] Nicotiana tabacum AsA2 [160] Nicotiana tabacum PhaG & PhaC [161] Nicotiana tabacum gfp [4] Nicotiana tabacum A1AT [162] Arabidopsis thaliana aadA [39] Solanum tuberosum aadA & gfp [40] Oryza sativa aadA & gfp [50] Solanum lycopersicon aadA [1] Solanum lycopersicon Lyc [42] Brassica napus aadA & cry1Aa10 [44] Brassica napus aadA [38] Lesquerella fendleri aadA & gfp [43] Daucus carota dehydrogenase (badh) [48] Gossypium hirsutum aphA-6 [49] Glycine max aadA [47] Petunia hybrida aadA & gusA [45] Lactuca sativa gfp [46] Brassica oleracea gus & aadA [163] Lettuce gfp [164] Populus alba gfp [51] Brassica oleracea aadA & uidA [74] Beta vulgaris aadA & uidA [165] Crocus sativus CstLcyB1& CstLcyB2a [166] Solanum melongena aadA [26] Arabidopsis thaliana pre-Tic40-His [167] Zea mays ManA [168] successfully transformed, this offers an exciting new approach to create transgenic plants with abiotic stress tolerance [93] . Therefore, the authors believe that there appears to be tremendous potential for increasing tolerance in plants to a number of stresses by expression of appropriate genes within plastids due to the maternal inheritance of transgenes that confer tolerance to abiotic stress. Plastid engineering had been successfully applied for the development of plants with tolerance to salt, drought [94] and low temperature [reviewed in 3]. Djilianov et al. [95] demonstrated that enhanced tolerance to abiotic stresses has been achieved when the gene was directed to plastid genome.
Among many strategies used for development of abiotic stress tolerance in plants, the over-expression of compatible osmolytes like glycinebetaine was found to be successful [24] . Initial attempts for producing transplastomic plants through the introduction of CMO (Choline monooxygenase) and betaine aldehyde dehydrogenase (BADH) pathway were made in tobacco. Tobacco plants were transformed with cDNA for BADH from spinach (Spinacia oleracea) and sugar beet (Beta vulgaris) under the control of CaMV 35 S promoter. The BADH was produced in plastids of tobacco. Betaine aldehyde was converted to betaine by BADH, thus conferring resistance to betaine aldehyde. In another attempt, cDNA for choline monooxygenase from Spinacia oleracea was introduced into tobacco and the enzyme thus synthesized was transported to its functional place i.e., plastids. But the leaves of tobacco accumulated betaine at a very low concentration i.e., 10-100 folds lower [96] . The reason for insufficient synthesis of betaine most probably was the absence of engineered BADH activity in plastids. Therefore, both CMO and BADH need to be present in the plastids for efficient synthesis of betaine in transgenic plants which do not accumulate glycinebetaine. In carrot (Daucus carota), homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis [48] . BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50-to 54-fold more betaine than untransformed cells grown in liquid medium containing 100 mM NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mM), the highest level of salt tolerance reported so far among genetically modified crop plants. Further, a gene for CMO, cloned from spinach (Spinacia oleracea) was introduced into rice through Agrobacterium mediated transformation. The level of glycinebetaine in rice was low to the expectations. The author has given several reasons for the low productivity of rice and low glycinebetaine accumulation. Firstly, the position of spinach CMO and endogenous BADH might be different and secondly the catalytic activity of spinach CMO in rice plants might be lower than it was in spinach [97] . Transplastomic plants constitutively expressing BvCMO under the control of the ribosomal RNA operon promoter and a synthetic T7 gene G10 leader were able to accumulate glycinebetaine in leaves, roots and seeds, and exhibited improved tolerance to toxic level of choline and to salt/drought stress when compared to wild type plants. Transplastomic plants showed higher net photosynthetic rate and apparent quantum yield of photosynthesis in the presence of 150 mM NaCl [98] . Thus, it can be concluded that glycinebetaine has a role as compatible solute and its engineering into non-accumulations will be a success only if both CMO and BADH pathways are introduced and if the localization of both CMO and BADH is in plastids. Very recently, George et al. [99] demonstrated how a chloroplastlocalized and auxin-induced glutathione S-transferase from phreatophyte Prosopis juliflora conferred drought tolerance on tobacco. For more examples of conferring tolerance to abiotic stress to plants via plastid engineering, see review by [3] .
Mulesky et al. [100] pointed out two reasons that make using crops to produce drugs interesting for industry. These are (i) crops can be employed more efficiently in this process than animals or bacteria, with a larger output achieved with fewer resources, and (ii) the oral delivery of the drugs produced to people and animals is easier. A third reason is the high-level production of antigens for use as vaccines and their tests for immunological efficacy in animal studies [3] . The hyper-expression of vaccine antigens or therapeutic proteins in transgenic chloroplasts (leaves) or chromoplasts (fruits/roots) and antibiotic-free selection systems available in plastid transformation systems made possible the oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus, [101] [102] [103] [104] and reviews of [102, 105, 3 and 106] explained why plastid engineering can be regarded as an attractive strategy and environmentally friendly approach for the production of vaccines, therapeutic proteins, and biomaterials, and provided some examples. Kumar & Daniell [107] described various techniques for creating plastid transgenic plants and their biochemical and molecular characterization. They also provided suitable examples for application of chloroplast genetic engineering in human medicine.
Wang et al. [3] also discussed applying plastid transformation for metabolic pathway engineering in plants, the production of biopharmaceuticals, and marker gene excision system and how plastid transformation can be applied to study RNA editing. Similarly, Hefferon [67] , considers plastid engineering as a valuable tool that gives enormous promise for the production of biopharmaceuticals and vaccines, because higher level of the protein expressed by the transgene inserted into the plastid genome can be achieved. Many vaccine antigens, which played a key role for the prevention of infectious diseases, and biopharmaceutical proteins, have been expressed at high levels via the chloroplast genome [108] and they proved to be functional using in vitro assays in cell cultures. [109] stated that production of therapeutic proteins in plastids eliminates the expensive fermentation technology, and that the oral delivery of plastid-derived therapeutic proteins eliminates cold storage, cold transportation, expensive purification steps, and delivery via sterile needles, and hence decrease their cost. The main goal for applications of plastid transformation by the biotech industry is molecular pharming, and food production is considered as only a secondary target [80] .
To create an edible vaccine, selected desired genes should be introduced into plants and then inducing these altered plants to manufacture the encoded proteins. Like conventional subunit vaccines, edible vaccines are composed of antigenic proteins and are devoid of pathogenic genes. Plastids of green plants as bioreactors for the production of vaccines and biopharmaceuticals are of great potential as indicated from a number of published studies [110] [111] [112] [113] [114] . The significance of using plants as production platforms for pharmaceuticals is due to the low production and delivery costs, easy scale-up and high safety standards regarding less risk of product contamination with human pathogen [27] . Keeping in view the high efficiency of plastids to express foreign genes, it is meaningful to explore this property of plastids for the production of proteinaceous pharmaceuticals, such as antigens, antibodies and antimicrobials. The candidate subunit vaccine against Clostridium tetani, causing tetanus was the first plastid-produced antigen that proved to be immunologically active in experimental animals [111] . In this initial attempt, fragment C of the tetanus toxin (TetC), a non-toxic protein fragment, was expressed from the tobacco plastid genome which resulted in high levels of antigen protein expression (30% of the plant's total soluble protein (TSP)). Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Significant development has been achieved towards the production of plastid-based vaccine for this infectious disease. Expression (14% of the plants TSP) of the pagA gene encoding the protective antigen (PA) from the tobacco plastid genome gave rise to stable antigen protein [112, 113] . The plastid-derived PA was equally effective in cytotoxicity assays as the bacterial protein produced in B. anthracis. The potential of plastid transformation as an alternative tool to produce high levels of HIV-1 Nef and p24 antigens in plant cells have been also demonstrated [115] . Different constructs were designed to express the p27 Nef protein either alone or as p24-Nef or Nef-p24 fusion proteins. All constructs were utilized to transform tobacco (cv. Petite Havana) plastids and the transplastomic lines. Analysis of p24-Nef and Nefp24 fusion proteins showed that both can be expressed to relatively high levels in plastids. As the best results in terms of protein expression levels were obtained with the p24-Nef fusion protein, the correspondent gene was cloned in a new expression vector. This construct was introduced into the tobacco and tomato plastid genomes. Transplastomic tobacco and tomato plants were analyzed and protein accumulation was found to be close to 40% of the leaf's total protein. Transcript and protein accumulation were analyzed in different ripening stage of tomato fruit and green tomatoes accumulated the fusion protein to 2.5% of the TSP [115] . Recently, a strategy for plastid production of antibiotics against pneumonia Streptococcus pneumonia has been outlined [116] . The authors describe it as a new technique for high level expression (to up to 30% of the plant's TSP) of antmicrobial proteins that are toxic to E. coli. It was also shown that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants. In 2007, Chebolu and Daniell [117] achieved a stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis. Shao et al. [118] reported the expression of the structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes CFSV E2 gene in tobacco chloroplasts. In another study on tobacco, plastid transformation of the high-biomass tobacco variety `Maryland Mammoth` has been assessed by McCabe et al. [119] as a production platform for the human immunodeficiency virus type 1 (HIV-1) p24 antigen. Similarly, Meyers et al. 2008 [120] revealed the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. Transgenic plastids were also proved efficient for high-yield production of the vaccinia virus envelope protein A27L in plant cellsdagger by Rigano et al. [121] , who revealed that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines. Very recently, Youm et al. [122] were able to produce the human beta-site APP cleaving enzyme (BACE) via transformation of tobacco plastids. The authors argued that the successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease. Because recombinant extra domain A from fibronectin (EDA) could be used as an adjuvant for vaccine development, [104] aimed to express EDA from the tobacco plastome as a promising strategy in molecular farming. Tobacco plastids transformation was also evaluated by Lentz et al. [123] for the production of a highly immunogenic epitope containing amino acid residues 135-160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). The authors concluded that this technology allows the production of large quantities of immunogenic proteins.
In spite of this huge success in applying plastid engineering for molecular pharming, Ho & Cummins [73] referred to several risks of plastid engineering in producing GM pharmaceuticals that are associated with its advantages. For more details on the use of plastid engineering for biotechnology applications, see review by Verma & Daniell [5] . Other valuable reviews are available on (a) generation of plants with transgenic plastids, summary of our current understanding of the transformation process and highlights on selected applications of transplastomic technologies in basic and applied research [124] , (b) Progress in expressing proteins that are biomedically relevant, in engineering metabolic pathways, and in manipulating photosynthesis and agronomic traits and the problems of implementing the technology in crops [125] , (c) plastid transformation in higher plants [56] , (d) the characteristics, applications of chloroplast genetic engineering and its promising prospects, [126] , (e) Engineering the chloroplast genome [127] (f) exciting developments in this field and offers directions for further research and development, [128] (g) the expression of resistance traits, the production of biopharmaceuticals and metabolic pathway engineering in plants [27] , (h) chloroplasts as bioreactors, and whether we can replace plants with plants [129] , (i) how plastid transformation played an important role in understanding the RNA editing [3] and (j) comparison of opportunities and challenges between nuclear and plastid genetic engineering of plants [130] .
Up to date, many transgenes have been successfully introduced into the plastid genome of model plant tobacco and many other important crop plants for various agronomic traits. Initially, this technology was limited to model plant species, but now it has been extended to some other important crops. Still there are many agronomically important cereals crops in which plastid engineering has not yet been standardized. Plastid transformation has been proved to result in high levels of transgene expression. It has also provided a baseline for production of proteinaceous pharmaceuticals, such as antigens, antibodies and antimicrobials in a cost effective manner. Bock [27] believes that a great progress has been achieved over years in investigating the mechanisms that govern transgene expression from the plastid genome and in using this technology for biotechnological applications. We agree with the author that "the routine use of plastid engineering in biotechnology is still a long way off, but would surely benefit the humanity in the near future". There is no doubt that plastid engineering holds a great potential in plant biotechnology; but like every new technology, there are some challenges which need to be addressed before its widespread adoption. Among other important factors to be solved are the protein purification and expression level control. Role of glutathione in immunity and inflammation in the lung Reactive oxygen species and thiol antioxidants, including glutathione (GSH), regulate innate immunity at various levels. This review outlines the redox-sensitive steps of the cellular mechanisms implicated in inflammation and host defense against infection, and describes how GSH is not only important as an antioxidant but also as a signaling molecule. There is an extensive literature of the role of GSH in immunity. Most reviews are biased by an oversimplified picture where “bad” free radicals cause all sorts of diseases and “good” antioxidants protect from them and prevent oxidative stress. While this may be the case in certain fields (eg, toxicology), the role of thiols (the topic of this review) in immunity certainly requires wearing scientist’s goggles and being prepared to accept a more complex picture. This review aims at describing the role of GSH in the lung in the context of immunity and inflammation. The first part summarizes the history and basic concepts of this picture. The second part focuses on GSH metabolism/levels in pathology, the third on the role of GSH in innate immunity and inflammation, and the fourth gives 4 examples describing the importance of GSH in the response to infections. It is now thought that oxidative stress is implicated in the pathogenesis of several diseases and conditions, from aging to inflammation and carcinogenesis, if not as a primary cause of disease at least as an aggravating mechanism.
The reality of oxidative stress is demonstrated by the existence, in all aerobic organisms, of several antioxidant enzymes devoted to ROS detoxification, such as peroxidase, catalase, superoxide dismutase, and peroxiredoxins. A number of small molecules also act as ROS scavengers. Unlike antioxidant enzymes, which catalyze the transformation of ROS into less toxic molecules, the concept of scavenger is that it must be an easily oxidizable target for ROS, and it must be present at concentrations high enough that the probability that a ROS reacts with the scavenger is higher than that of reacting with another target. When dealing with lipid oxidation (such as in rancidification of butter), the most important scavenger is probably vitamin E. As proteins with a sulfydryl group are often target of oxidation (-SH groups can be easily oxidized), small-molecular-weight thiols are potent scavengers.
The small thiol present in highest concentration in the cytoplasm is glutathione (GSH), a tripeptide glycine-cysteineglutamic acid ( Figure 2 ): the -SH group of its cysteine is extremely sensitive to oxidation, mainly by peroxides. Its importance is supported by the existence of a molecular machinery that makes it particularly effective. In fact, a scavenger, including any thiol antioxidant such as the common laboratory reagent beta-mercaptoethanol, would normally act as a suicidal decoy, being oxidized and thus becoming useless. However, when GSH is oxidized, it forms GSH disulfide (GSSG), and this can be re-reduced by a specific enzyme, glutathione reductase. Not only can GSH be enzymatically regenerated from GSSG, but also the reaction of GSH with the ROS (a peroxide), which already takes place with high reactivity, is catalyzed by GSH peroxidases that facilitate the inactivation of a wide range of peroxides ( Figure 3 ).
The key role of GSH as an antioxidant is demonstrated by many studies showing that experimental depletion of GSH levels -which can be achieved with various chemicals the most used of which is buthionine sulfoximine (BSO) an inhibitor of GSH synthesis -has a worsening effect in 
GSH in immunity and lung inflammation many disease models. Conversely, repleting GSH levels with precursors of its synthesis such as N-acetyl-cysteine (NAC) or 2-oxothiazolidine-4-carboxylic acid has protective effects. Cysteine precursors, rather than cysteine itself, are used because they are more cell-permeable, and can be given orally.
Innate immunity and inflammation, two faces of the same biological coin
The first line of immune defense against pathogens, before adaptive immunity (antibodies, T cell responses) develops, is called innate immunity. This is a complex set of responses triggered when specific cells (macrophages, phagocytes, dendritic cells) recognize even a yet unknown pathogen by some characteristics common to most pathogens (these "signatures" are called pathogen-associated molecular patterns) through a family of pathogen recognition receptors. This activates a response that involves production of ROS, a major bactericidal mechanism, and of soluble mediators (cytokines) whose role is to amplify the host response by recruiting and activating other immune system cells.
This aspect of the innate immune response is also known, from a different perspective, as the inflammatory response. Basically, the very same mechanisms and mediators of host defense are implicated in the pathogenesis of (noninfectious) inflammatory diseases, and inhibition of these mechanisms is the key to the mechanism of action of antiinflammatory drugs.
Many noninfectious diseases of the respiratory system, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, idiopathic pulmonary fibrosis (IPF), and oxygen toxicity, have an inflammatory component. Inflammation is also implicated in the lung toxicity of ozone, asbestos, silica, cigarette smoke, and particulate matter. An exaggerated inflammatory response is also involved in the pathogenesis or complications of pulmonary infections such as tuberculosis, severe acute respiratory syndrome, influenza, and acute respiratory distress syndrome (ARDS).
GSH is synthesized from its 3 amino acids with a biosynthetic pathway shown in Figure 4 . A study in humans, using radioisotopes, 3 has demonstrated that the availability of cysteine,and its precursor methionine, is the rate-limiting factor in GSH synthesis. In general, it is assumed that the two main limiting factors are the levels of cysteine and of the enzyme gamma-glutamyl-cysteine synthetase (also known as glutamate-cysteine ligase). 4
Glutathione synthetase (GS) deficiency (oxoprolinuria) is a rare autosomal recessive disorder affecting about 70 patients in the world. Patients with severe GS deficiency show, among other conditions, an increased susceptibility to bacterial infections. 5, 6 One case report showed that neutrophils from a child with GS deficiency undergo oxidative damage upon phagocytosis, indicating that GSH is important in defending the neutrophils from the ROS they produce as part of their microbicidal armamentarium. 7 Of note, neutrophils from GS-deficient patients, despite normal phagocytosis and increased release of hydrogen peroxide, are less efficient in killing bacteria, indicating a helper role for GSH in the bactericidal activity. 8
Many pathological conditions are associated with decreased GSH levels (Table 1 ). This could be due to several reasons. For instance, oxidative stress could cause GSH loss though oxidation. Another important aspect is nutrition, as it was shown that, even when dietary protein intake is sufficient for maintaining nitrogen balance, it may not be sufficient for maintaining cellular GSH, particularly in conditions of oxidative stress. 9 GSH deficiency could also arise from metabolic problems. For instance, in AIDS patients, a decrease in gamma-cystathionase activity in the liver was reported. 10 The use of stable radioisotopes allowed the characterization of cysteine metabolism and GSH synthesis in septic patients. This study showed that sepsis decreases blood GSH synthesis by 60%. 3 Of note, these patients had an intake of sulfur amino acids below that recommended by the World Health Organization. 3 Studies in animal models have reported an increased requirement for cysteine in sepsis, probably due to an increased turnover of GSH, as GSH synthesis accounts for 40% of the increased cysteine utilization. 11, 12 These and other studies show that even when the dietary intake of cysteine is sufficient for protein synthesis (nitrogen balance), it may not be sufficient to maintain adequate GSH levels. 9 One should also bear in mind that infectious and inflammatory conditions are associated with an increased production of acute-phase proteins by the liver, and it was estimated that they account significantly for the increased utilization of sulfur amino acids, thus competing with GSH. 13, 14 GSH as a regulator of innate immunity Anti-inflammatory role of GSH in lung diseases
The idea that GSH may play an anti-inflammatory role became popular in the 1990s with a study by Schreck et al 15 showing that antioxidants inhibit, whereas ROS activate, the transcription factor NF-κB that commands the transcription of several inflammatory genes, 16 a role that has been confirmed in the lung. 17 In fact, a protective role of GSH against inflammatory pathologies of the lung has been demonstrated by the protective effect of different GSH-precursors in various animal models ( Table 2) .
Along the same line, depleting endogenous GSH with BSO had a worsening effect in some of the models above, including chemically induced pulmonary edema, 21 cigarette smoke, 31 carrageenan-induced pleurisy, 32 and endotoxininduced pulmonary inflammation. 33 
It can bee seen that there is an overlap between the list of pulmonary diseases associated with inflammation and those where GSH repletion is protective, indirectly supporting the hypothesis of an anti-inflammatory role of GSH.
An increased susceptibility to sepsis-induced ARDS and lethality 34 was observed in mice lacking the transcription factor Nrf2, which has among its target genes the GSH biosynthetic enzyme gamma-glutamylcysteine synthase. 35 IPF is an example of lung disease in which normalizing the GSH deficit has positive effects. Patients with IPF have a 4-fold lower GSH concentration in the epithelial lining fluid of the normal lower respiratory tract; 36 moreover, the administration of the GSH precursor NAC not only restores GSH levels 37 but, in association with other drugs used for the treatment of IPF, it also improves end points such as vital capacity and single-breath carbon monoxide diffusing capacity. 22 
The functions of GSH are not only inhibitory as described above for the inflammatory response. In fact, GSH is essential for some functions of the immune system, both innate and adaptive, including T-lymphocyte proliferation, 38, 39 phagocytic activity of polymorphonuclear neutrophils (PMN), 40 and dendritic cell functions, 41 and is also important for the first step of adaptive immunity, consisting of the antigen presentation by antigen-presenting cells (APC). Indeed, cell-mediated immunity requires that protein antigens be first degraded in the endocytic vesicles of APCs (eg, macrophages, dendritic cells), so that the smaller peptides can be presented on the cell surface by the major histocompatibility complex to activate proliferation of antigen-specific T cells. One of the first steps in antigen degradation and processing is the reduction of disulfide bonds, 42 which requires GSH. 43 It should also be noted that although GSH inhibits the production of most inflammatory cytokines, it is required to maintain an adequate interferongamma (IFN-gamma) production by dendritic cells, 44 which is essential for the host defense against intracellular pathogens such as mycobacteria. 45 This essential role of GSH in immunity might explain why in many diseases, not only AIDS, decreased GSH levels are associated with an increased susceptibility to infection. These include COPD, 46 cystic f ibrosis, 47, 48 influenza infection, 49 and alcoholism, 50,51 as ethanol impairs Th1/Th2 balance via GSH depletion 52 (Table 3) .
The role of oxidative stress in the pulmonary damage by influenza virus is well characterized in the mouse model. Mice infected with influenza show pulmonary damage associated with a dramatic decrease in pulmonary GSH levels as well as an increase of oxidative stress markers such as oxidized glutathione (GSSG) and lipid peroxides. 53 In these mice, oxidative stress could be due to the induction of xanthine oxidase, 54 a well-known superoxide-generating enzyme. 55 Furthermore, influenza infection is associated with an induction of inflammatory cytokines, 56,57 which might represent a likely GSH-sensitive step in its pathogenesis. Among the antioxidants shown to be protective in animal models of influenza are the xanthine oxidase inhibitor allopurinol, 54 quercetin, 58 superoxide dismutase, 59 thioredoxin, 60 NAC (alone 61 or with ribavirin 62 or oseltamavir 63 ), and GSH itself. 64 A study reported that influenza virus M2 protein augments ROS production in human airway cells in vitro, resulting in toxic effects that are prevented by the addition of GSH ester. 65 Although these studies indicate a protective role of GSH at the level of the pathogenesis of pulmonary damage, there is a report that BSO increases viral replication, 66 implying a possible antiviral role of endogenous GSH. One clinical study has shown that NAC administration improves parameters of cell-mediated immunity in patients with influenza, 66 suggesting a possible clinical relevance of these observations.
Historically, the entire field of the role of GSH in immunity and its effect on NF-κB received the strongest boost by studies on AIDS, particularly by a study from the group of Wulf Droge who showed that AIDS patients have a low concentration 
Ghezzi of plasma cysteine. 67 The reasons for this are not clear, but AIDS patients have a deficit in the enzyme gamma cystathionase, which synthesizes cysteine from the methionine. 10 Later research showed that AIDS causes a decrease in intracellular GSH in CD4 T cells, and that low GSH is associated with decreased survival. 68 The lower GSH levels in AIDS patients could have various consequences. GSH depletion, at least in vitro, augments HIV replication, 69 while its precursor NAC blocks the stimulatory effect of tumor necrosis factor on HIV replication. 70 Furthermore, the neurotoxic effect of HIV proteins Tat and gp120 is associated with oxidative stress and antagonized by NAC. 71, 72 Example 3: bacterial infectionstuberculosis Mycobacterium tuberculosis is an intracellular pathogen that grows in the phagosomes, where it is protected from immune system effectors such as antibodies and T lymphocytes. Although the literature showing that GSH levels are lower in patients with tuberculosis dates back to the 1950s, it was not until the research of Venketaraman and colleagues that the effect of GSH on M. tuberculosis infection was studied in depth. Using a mouse macrophage cell line, the authors show that IFN-gamma and endotoxin increase both nitric oxide (NO) production and bactericidal activity; the paralleled decrease in GSH suggests that GSH reacts with NO to form S-nitrosoglutathione (GSNO). Under these experimental conditions, GSH depletion with BSO inhibited the microbicidal activity of macrophages while its precursor NAC increased intracellular killing of mycobacteria also from human macrophages, which are normally not very effective in killing mycobacteria. 73, 74 Other investigators have shown that the trans-sulfuration pathway, which converts methionine into cysteine and has a key role in maintaining cysteine, and hence GSH levels, is essential for mycobacterial killing. 75 In that study, it was found that not only are trans-sulfuration pathway enzymes increased in human monocytes as a response to mycobacteria, but their inhibitor propargylglycine lowered GSH levels and inhibited clearance of mycobacteria and phagolysosome fusion, while NAC increased them. 75 Similar results were obtained in whole human blood cultures. 76 In vitro, both GSH and GSNO have direct bactericidal activity against these pathogens. 77 This complex picture has been nicely reviewed by Connell and Venkataraman 78 and is summarized in Figure 5 . Depletion of GSH (by BSO or diethylmaleate) also inhibits macrophage leishmanicidal activity, as well as NO production, while the GSH precursor GSH-ethyl ester restores them, 79 suggesting that the requirement for the GSH/NO pathway might be a common feature of resistance to intracellular pathogens.
ARDS is one of the most serious complications in critically ill septic patients. Many studies have pointed out a role for oxidative stress in ARDS and shown a protective effect of GSH precursors. 80, 81 Most of the studies of ARDS in animal models, including those cited above, are based on the administration of lipopolysaccharide (LPS), a bacterial endotoxin. In mice, administration of LPS induces an acute lung injury similar to clinical ARDS, with production of inflammatory cytokines, leukocyte infiltration in the lung, and pulmonary edema. In this model, various thiol-based antioxidants are protective. [80] [81] [82] However, LPS administration is in fact a model of endotoxic shock, involving no live bacteria, to induce a state similar to what was once called septic shock and is now defined as systemic inflammatory response syndrome. [83] [84] [85] However, in septic patients, survival is affected by 2 opposite contributions of innate immunity: innate immunity is detrimental as systemic inflammation, which results in ARDS and shock, but on the other hand it is an essential component of the immune defense against infection. It is difficult to foresee how GSH affects this balance.
One more realistic animal model is that induced by cecal ligation and puncture (CLP), where puncturing the cecum causes the release of fecal material in the peritoneum that results in a polymicrobial peritoneal sepsis. This model allows studying the relevance of both arms of innate immunity, the detrimental and the protective one. 
We have used this model to study the role of endogenous GSH in sepsis. 86 In this model, CLP induced PMN infiltration in the peritoneal cavity, the site of infection, as well as in the lung, ultimately resulting in lung injury and death, with a concomitant decrease in endogenous GSH. 86 Lowering GSH further with BSO worsened the clinical settings. Not only did BSO increase PMN infiltrate in the lung, but it also diminished PMN infiltration in the site of infection, thus increasing bacterial growth. As a result we had more inflammation and less immunity, and survival was dramatically decreased. Repleting GSH with NAC had the opposite effect of reducing PMN infiltration to the lung but increasing that to the site of infection, thus decreasing bacterial colonies.
The picture that emerges from these experiments is that endogenous GSH is not just an inhibitor of inflammation, but it is required to allow a proper response to infection, and "direct" the migration of inflammatory PMN away from the lung, where they cause ARDS, and towards the site of infection, where they kill bacteria ( Figure 6 ).
The idea, therefore, is that GSH is not just an inhibitor of inflammation but also a regulator of innate immunity in a direction favorable to the host.
In parallel to the studies of GSH in immunity, more complex molecular and biochemical studies have pointed out a regulatory role of GSH. This was a result of an evolution from the concept of oxidative stress outlined above to that of redox regulation. The concept of redox regulation implies that some oxidative changes (such as changes in the redox state of protein cysteines) are not necessarily damaging but can have regulatory properties. While this idea shows a more complex picture from the popular one where free radicals and oxidants are bad and antioxidants are good, it implies that GSH is an essential molecule not only in acting as an antioxidant but also in the absence of oxidative stress, as an endogenous signaling molecule. The molecular mechanisms of GSH-mediated redox regulation are being actively investigated and have in part been identified. [87] [88] [89] Although the present review focused on the immuno pathogenesis of pulmonary diseases, the key concepts outlined here may help in interpreting the role of redox in other pathological conditions.
The author discloses no conflicts of interest. 
Submit your manuscript here: http://www.dovepress.com/international-journal-of-general-medicine-journal
The International Journal of General Medicine is an international, peer-reviewed open-access journal that focuses on general and internal medicine, pathogenesis, epidemiology, diagnosis, monitoring and treatment protocols. The journal is characterized by the rapid reporting of reviews, original research and clinical studies across all disease areas.
A key focus is the elucidation of disease processes and management protocols resulting in improved outcomes for the patient.The manuscript management system is completely online and includes a very quick and fair peer-review system. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.  We should not be complacent about our population-based public health response to the first influenza pandemic of the 21(st )century BACKGROUND: More than a year after an influenza pandemic was declared in June 2009, the World Health Organization declared the pandemic to be over. Evaluations of the pandemic response are beginning to appear in the public domain. DISCUSSION: We argue that, despite the enormous effort made to control the pandemic, it is now time to acknowledge that many of the population-based public health interventions may not have been well considered. Prior to the pandemic, there was limited scientific evidence to support border control measures. In particular no border screening measures would have detected prodromal or asymptomatic infections, and asymptomatic infections with pandemic influenza were common. School closures, when they were partial or of short duration, would not have interrupted spread of the virus in school-aged children, the group with the highest rate of infection worldwide. In most countries where they were available, neuraminidase inhibitors were not distributed quickly enough to have had an effect at the population level, although they will have benefited individuals, and prophylaxis within closed communities will have been effective. A pandemic specific vaccine will have protected the people who received it, although in most countries only a small minority was vaccinated, and often a small minority of those most at risk. The pandemic vaccine was generally not available early enough to have influenced the shape of the first pandemic wave and it is likely that any future pandemic vaccine manufactured using current technology will also be available too late, at least in one hemisphere. SUMMARY: Border screening, school closure, widespread anti-viral prophylaxis and a pandemic-specific vaccine were unlikely to have been effective during a pandemic which was less severe than anticipated in the pandemic plans of many countries. These were cornerstones of the population-based public health response. Similar responses would be even less likely to be effective in a more severe pandemic. We agree with the recommendation from the World Health Organisation that pandemic preparedness plans need review. The World Health Organization (WHO) declared that spread of the newly recognised quadruple reassortant influenza A H1N1 virus satisfied the criteria for a pandemic on June 11, 2009 , [1] although technically conditions for declaring a pandemic had been met some weeks earlier. The virus, generally referred to as pandemic influenza H1N1 2009 (pH1N1), had first been recognised in Mexico and the United States in late April 2009. More than a year later, WHO has declared the pandemic to be over and early assessments of the global response have commenced [2] .
When the pandemic was declared, Dr Margaret Chan, the Director of WHO, advised member states to implement their pandemic plans [1] and health agencies, other government agencies and businesses worked hard to do this. In most countries it may be correct to conclude, as did an evaluation of the UK response, that the "pandemic and the response it generated have provided confirmation of the value of planning and preparedness" [3] . It is also true that the apparent success of the response in 2009 must not lead to complacency. We now know that the relatively low virulence of pH1N1 meant we did not need to have implemented effective responses to get a good outcome.
The response to the pandemic included clinical and public health measures. In developed countries, such as Australia, the clinical response was effective for those whose illnesses were serious [4] . Clinical care will very likely have reduced the number of deaths due to pandemic influenza [5] , although the use of extra-corporeal membrane oxygenation was seen as a last resort and was not supported by the conclusions of a systematic review [6] . In developed and developing countries, the public health response focused on both the individual and the population. Individual responses promoted attention to personal hygiene, with an emphasis on cough hygiene and hand washing, which may not have been optimal [7] , and the use of personal protective equipment for those considered to be at increased risk of infection [8] . Population-based public health responses to the pandemic focused on two major elements: non-pharmaceutical and pharmaceutical interventions. The former comprised border control and various elements of social distancing, while the latter focussed on anti-viral medication for treatment and/or prophylaxis, and the development of a strain-specific vaccine.
Australia used the pharmaceutical and non-pharmaceutical interventions detailed in its pandemic plan [9] in an effort to delay entry of the virus into the country, contain the virus to limited areas once it had entered the country, sustain a response when widespread community transmission had been established and to protect the vulnerable [10] -the latter being a new response phase formulated once it was realised that the pandemic was not associated with the high case fatality ratios that had been anticipated [11] . We use Australia's experience to draw attention to issues related to the public health population-based pandemic response. The scope of this perspective does not allow us to consider other categories of response. Now is the time to acknowledge that a number of the strategies used in response to the 2009 pandemic could not control the spread of a novel influenza virus and their place in future pandemic response plans needs to be reconsidered in light of emerging new evidence. We examine four critical cornerstones of Australia's public health population-based response, namely border control, school closure (as an example of social distancing), the use of anti-viral medication and the development and use of a pandemic vaccine. We provide evidence from the pandemic experience in other countries to support our arguments.
In a very different world, Australia successfully applied maritime quarantine to delay the entry of a pandemic H1N1 virus into the country in 1918 and 1919. This was in contrast to many of Australia's Pacific neighbours. For instance, the virus reached New Zealand in October 1918 but did not enter Australia until January of the following year. Estimated death rates in countries where the entry of the virus had been delayed were lower than rates where earlier entry was documented [12] .
Prior to the 2009 pandemic, modelling studies had suggested a very limited role for border screening, providing an estimated delay of only 1-2 weeks without draconian measures that would be economically unacceptable in most countries [13] . A review of border control in Australia following the SARS epidemic pointed out the opportunity cost of screening. No case was identified despite 1.8 million passengers being screened, 794 referred for further evaluation and four identified as possible cases [14] . A similar lack of success was suggested in a review of the likely success of border control for influenza in other countries [15] . Indeed, it can be argued that prior to the pandemic there was only very limited scientific evidence for border control as an effective intervention. Despite this in 2009, as an island nation with history on its side, Australia, like many other (non-island) countries, embraced the concept.
Australia implemented a combination of approaches in an attempt to detect infected arriving passengers at international airports. These comprised notification of health status of passengers by airline staff (the pilot or crew identified passengers with respiratory symptoms), thermal scanning (infra-red cameras were installed in airport terminals), health declaration cards (the passenger reported current symptoms) and nurses at border entry points reviewed and tested passengers detected by one of these screening tools. In the Australian state of Queensland, although the number of passengers screened was not reported, and was likely to have been many tens of thousands, only four cases of confirmed pandemic influenza were found from 780 passengers identified by one or more of these border screening measures [16] . No cases were detected by similar screening at the busy international airport in Perth, Western Australia (unpublished data).
It has been suggested that Australia did well in its response to managing the pandemic, specifically in delaying establishment of community transmission [17] . During this time preparations were made to respond to a pandemic that was anticipated to result in many deaths. However, based on epidemiological and modelling evidence, we have demonstrated that community transmission was almost certainly established in the state of Victoria around the time the virus was first recognised in North and Central America in late April 2009 [18] . This followed one or more unrecognised silent importations, and spread of the virus in Australia came substantially from within its borders rather than from overseas. We now know that this is an entirely plausible scenario, given that a significant proportion of pH1N1 infections were afebrile [19] or entirely asymptomatic [20] and therefore impossible to detect at the border -or anywhere else.
This should not be surprising, as the finding that a high proportion of influenza infections are asymptomatic or afebrile was not new. Published experimental data from volunteer studies had previously shown that 33% of proven seasonal influenza infections were asymptomatic, but this varied by influenza type and subtype [21] . In particular, as few as 37% of experimental infections with influenza A(H1N1) were associated with fever recorded as >37.8°C, while 30% were completely asymptomatic [21] . Moreover, viral shedding in the presymptomatic phase of influenza infection has recently been confirmed to occur in approximately 1-8% of naturally acquired infections, in a study in which 14% of all influenza infections were asymptomatic and 31% of infections with influenza A (H1N1 or H3N2) did not have fever at the onset of other symptoms [22] .
Prodromal, asymptomatic and afebrile infections cannot be detected by temperature measurement, one of the main components of border control, whether by thermal scanning or by core temperature measurement of symptomatic travellers [23] . Moreover, the proportion of afebrile or asymptomatic people is likely to be higher in infected travellers, as more severely unwell people will be less likely to travel. Thermal imaging is therefore even less likely to have been effective at the borders than in other places where more severely ill patients are seen [24] . Indeed, China used intensive thermal screening for pH1N1 at airports and had a positive detection rate of only 14 cases per million passengers screened [25] .
The use of border control was evidently not based on a current understanding of influenza epidemiology and was not supported by modelling studies. In particular one modelling study, published two years before the identification of the current pandemic virus, showed that past pandemic patterns could not be adequately modelled without inclusion of asymptomatic infection (as well as varying degrees of pre-existing immunity) [26] .
Nonetheless, an early evaluation of the 2009 pandemic, with limitations acknowledged by its authors, suggested that border screening may have led to delays of 7 to 12 days in the establishment of local transmission [27] . We accept that border screening will have detected a limited number of influenza cases, but suggest that many more cases will have been missed than were detected. In Australia, at least, it is likely that border screening was implemented after the virus had entered the country [18] . On balance, we conclude that border screening was as ineffective as it should have been expected to be.
It is generally accepted that children, especially children of school age, are responsible for amplification of influenza epidemics [28] . An intervention targeting schools could therefore theoretically be effective in interrupting an epidemic. This assumption is supported by modelling studies, but only when all schools are closed early and remain closed for an unrealistically long period, up to the duration of the pandemic [29] . Modelling also shows that delay in closing schools, or partial closure of schools, are less effective interventions, [29] although, if school closures are timely, they may delay the peak and decrease the peak incidence of the epidemic [30] .
As expected, the pH1N1 infection rate was high among school aged children [20, 31] . Of the first 997 cases of confirmed pH1N1 infection in the state of Victoria in Australia, 67% were aged 5-17 years [32] . In Australia, school closure was intended to be associated with voluntary home quarantine. When a school -or class within a school -was closed, members of the class were asked to voluntarily quarantine themselves at home. This meant that parents of young children were frequently required to take time off work to care for children who would otherwise have been at school. Home quarantine has its own risks. We have recently shown that when an entire family was quarantined, the risk of secondary spread within households was increased by approximately 2.5-fold [33] .
Moreover, compliance with other social distancing measures needed to have been effective for school closures themselves to have been effective. A survey in Western Australia of parents of school children whose schools were closed at some stage during the pandemic indicated that 74% of home-quarantined children participated in outside activities at least once during the nominal quarantine period, recording an average of 3.7 activities per child. Most commonly reported were attendances at sporting events, parks, beaches and stores, places where it is likely other children would be exposed [34] . Public documentation of school closure during the pH1N1 epidemic in Australia is minimal, but the policy in the early phase of the pH1N1 response was to close only those classes with confirmed cases, escalating to whole schools where multiple classes across different age groups were affected. In the state of Queensland only 2.8% of all schools were closed for short periods [16] . In Western Australia school closures were only for one week and sometimes involved only closure of specific classes [34] . Too limited in scope and time, these strategies could not have been effective in interrupting the spread of the pandemic.
On the other hand, experience in Japan confirmed the conclusions from modelling [30] . Early widespread school closures in a defined area were successful in delaying pandemic spread in that area, but when the schools were re-opened, pandemic spread resumed [35] . In Hong Kong closure of kindergartens, pre-schools and primary schools appeared to decrease the attack rate in children aged less than 12 years for the weeks of closure [36] but the effect on the final attack rate in school children is yet to be evaluated. Indeed, it has been argued that the potential benefit of closing schools during a pandemic must be balanced against the enormous social disruption that ensues [37] . Only where schools were closed early and remained closed would there have been any significant interruption of the spread of the pandemic.
Countries around the world adopted different approaches to the use of neuraminidase inhibitors (NAIs) in their pandemic plans. In addition to treatment provisions, Australia opted for a stockpile of approximately 10 million courses of NAIs with the intention of implementing widespread prophylaxis, which has been shown in trials to be 58-84% effective in preventing laboratory proven influenza infection if given early following exposure [38] . However, even in the early phases of the response, when numbers of suspected and confirmed pH1N1 cases were low, those with responsibility for contact tracing were rapidly overwhelmed. The logistical difficulties of timely delivery of NAIs to those eligible for treatment or prophylaxis were such that it was likely only a minority received their medication in time for it to be effective. Lateness of NAI availability has been confirmed in a Victorian study of treatment doses. Oseltamivir was prescribed for only 207 (21%) of the first 1,000 confirmed cases. Of 690 cases confirmed not to have received oselatamivir, 670 were not eligible because more than 48 hours had elapsed since symptom onset (Unpublished data, James Fielding, epidemiologist, Victorian Infectious Diseases Reference Laboratory).
Other approaches to NAI distribution were used around the world, with varying effectiveness. For example, the UK National Health Service implemented an electronic checklist to allow patients rapid access to NAIs. Bypassing doctors and laboratory testing, this system aimed to speed up NAI availability. However, only 1932/16,560 (17%) of people who received NAIs using the electronic checklist subsequently tested positive for pH1N1 [39] .
On the other hand, in four outbreaks in Singapore military camps, when NAIs were able to be delivered effectively in conjunction with isolation of confirmed cases and quarantine of contacts, a beneficial effect could be demonstrated. These measures, which included ring prophylaxis with oseltamivir, resulted in a reduction of the infection rate in the outbreaks from 6.4% to 0.6% [40] . This study demonstrates the potential benefit of NAIs if available early in outbreaks, and when combined with social distancing. However extension of this strategy to large heterogeneous populations remains unproven and it may be feasible only in closed communities, such as boarding schools, military barracks and residential care facilities.
Another important consideration in setting out to provide mass treatment and prophylaxis with NAIs is the possibility of development of resistant strains. Surveillance studies during the first wave of the pandemic demonstrated a low frequency of resistance to oseltamivir and no reported resistance to zanamivir. Prior to 2007, it was rare to detect oseltamivir-resistant influenza strains in untreated patients, due to the compromised infectivity and transmissibility of many of the resistant mutants in the absence of drug pressure. But in 2007/2008 an oseltamivir-resistant seasonal A(H1N1) variant emerged that demonstrated viral fitness at least equivalent to the oseltamivir-susceptible strain. The resistant strain spread rapidly around the world and by 2009 had completely replaced the susceptible strain [41] . An oseltamivir-resistant pH1N1 virus might also retain viral fitness and subsequently spread throughout the community. Fortunately, to date, only a low frequency of oseltamivir-resistant pH1N1 strains have been identified [42] .
An anti-viral stockpile without a well-developed logistic strategy and resourcing for effective early delivery for treatment of cases and prophylaxis of contacts is not an adequate plan for successful limitation of viral spread in a population, especially considering that the high proportion of cases with asymptomatic and mild infections will not be identified. Moreover, as we have seen with seasonal H1N1 viruses, resistance may develop to NAIs and a resistant virus may retain viral fitness allowing it to become widespread. This would render stockpiles useless. Revised pandemic plans should therefore consider limiting the use of NAIs to treatment of those with more severe influenza infection or medical conditions that make them more vulnerable to complications. Dependent on the availability of NAIs and access to appropriate medical care, treatment should be commenced early in the course of the illness. In Germany the median delay between symptom onset and antiviral treatment was significantly longer in fatal cases than non-fatal cases [43] . Prophylaxis should probably be reserved for closed communities, with any plan for wide-scale use of prophylactic NAIs dependent on a large workforce able to perform contact tracing and a detailed logistics plan for early delivery.
After China, Australia was the second country in the world to roll out a population-based pandemic vaccine program, with monovalent pandemic vaccine available by 30 September 2009 [44] . The first wave of the pandemic in Australia had ended by this date. It was not expected that the vaccine would have been available in time to modify the first pandemic wave anywhere in the world. However, even in Australia, it was a case of 'too much too late'. An early estimate of 18% was made for population wide coverage for the vaccine [45] .
Most pandemic vaccines in Australia were formulated as multi-dose vials. Given recommendations that the vial contents should be used or discarded within 24 hours of first use, wastage was expected with this formulation. It has been estimated that around 40% of pH1N1 vaccine doses delivered to Australian general practices may have been wasted [46] . There was also concern among some immunisation providers, and within the general community, that the multi-dose vials contained the preservative thiomersal, which had been phased out of paediatric vaccines, and that use of the vials potentially increased the risk of contamination, including with blood-borne viruses [47] . Such concerns, whether ill-founded or not, were likely to have impacted adversely on vaccine uptake, even in identified high risk groups [48] . While it may be reasonable to assume that vaccine uptake would have been higher if the disease had indeed been more severe, future pandemic plans need to include greater flexibility in vaccine purchasing and contracting arrangements, and refinement of vaccine delivery protocols and public messaging, in order to minimise wastage and optimise uptake [49] .
The 21st century marks the first time pandemic-specific vaccines have been manufactured on a large scale. A preliminary report from Germany using the screening method estimated pandemic vaccine effectiveness for an adjuvanted pandemic vaccine of 97% in people aged 14-59 years [50] . A similar high level of protection has been reported for children in Canada [51] , although a more modest effectiveness of 72% has subsequently been reported from a pooled case control analysis from a number of European countries [52] . While vaccines were effective in protecting individuals, population coverage in Australia and other countries was unlikely to have been sufficient for the vaccine to have modulated the spread of the pandemic virus. However some European countries, such as Germany, experienced a very modest first pandemic wave [43] and, had they achieved high coverage with pandemic vaccine, may have been able to modify pandemic virus transmission in the next influenza season. Nonetheless, the experience with pH1N1 suggests that a pandemic vaccine will always be too late, at least for one hemisphere, using current vaccine manufacturing technology.
Control of pandemic influenza is a critical issue and one on which the world has already spent billions of dollars, both in planning and during the recent response to pH1N1. There are obvious lessons to be learnt from the first pandemic of the 21st century, a pandemic which was much less severe than many plans had anticipated [53] . If we think our response to this pandemic was adequate, we may be falsely reassured. A more severe pandemic may find us wanting. A mild pandemic may find us over reacting. However, with appropriate collection and analysis of data it should be possible to identify the severity of future pandemics early and to make a measured response [54] . The World Health Organization, governments and other agencies around the world are currently involved in reviews of the management of the pandemic [55] . It is vital that these reviews, while not diminishing the commitment and hard work of those who implemented the response plans in 2009, carefully assess the evidence base for those plans.
In addition, the widespread implications of the response to the pandemic -for policy makers, health professionals and the public -make it important for these reviews to be in the public domain. In Australia, where pandemic reviews are not yet in the public domain, there were examples where messages appeared to be mixed, and which confused both the public and healthcare professionals [56] . Partially closing some schools for short periods and not implementing other social distancing measures, such as cancelling public gatherings, is just one example.
Although we have provided examples from Australia, we believe our arguments will have relevance for many other countries. 'One size fits all', where authorities have only one response strategy for viruses with different infection rates and case fatality ratios, is not an appropriate response to pandemic preparedness. Revised pandemic plans should include different responses for different pandemic severities [57] . All areas of pandemic planning need to be re-examined, but perhaps by alternative processes to those that led to current plans. Certainly, new evidence about the practical difficulties and/or ineffectiveness of control measures, such as border control and school closures, needs to be considered seriously. The inadequacy of many plans has recently been publicly acknowledged by the head of the WHO's global influenza programme. Speaking at a United Kingdom Health Protection Agency conference on the international response to the H1N1 pandemic, Dr Sylvie Briand is reported to have said that the containment strategy during the last pandemic was 'not feasible' and that guidelines might have to be overhauled [58] . We believe this is sound advice.
National University for helpful comments on the manuscript. We acknowledge Dr Aeron Hurt from the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne for his expert contribution to the section on NAIs. Geoffry Mercer acknowledges partial funding from an NHMRC strategic influenza grant. We thank Kristina Grant and Francine Cousinery for help with preparation of the manuscript. The GLEaMviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale BACKGROUND: Computational models play an increasingly important role in the assessment and control of public health crises, as demonstrated during the 2009 H1N1 influenza pandemic. Much research has been done in recent years in the development of sophisticated data-driven models for realistic computer-based simulations of infectious disease spreading. However, only a few computational tools are presently available for assessing scenarios, predicting epidemic evolutions, and managing health emergencies that can benefit a broad audience of users including policy makers and health institutions. RESULTS: We present "GLEaMviz", a publicly available software system that simulates the spread of emerging human-to-human infectious diseases across the world. The GLEaMviz tool comprises three components: the client application, the proxy middleware, and the simulation engine. The latter two components constitute the GLEaMviz server. The simulation engine leverages on the Global Epidemic and Mobility (GLEaM) framework, a stochastic computational scheme that integrates worldwide high-resolution demographic and mobility data to simulate disease spread on the global scale. The GLEaMviz design aims at maximizing flexibility in defining the disease compartmental model and configuring the simulation scenario; it allows the user to set a variety of parameters including: compartment-specific features, transition values, and environmental effects. The output is a dynamic map and a corresponding set of charts that quantitatively describe the geo-temporal evolution of the disease. The software is designed as a client-server system. The multi-platform client, which can be installed on the user's local machine, is used to set up simulations that will be executed on the server, thus avoiding specific requirements for large computational capabilities on the user side. CONCLUSIONS: The user-friendly graphical interface of the GLEaMviz tool, along with its high level of detail and the realism of its embedded modeling approach, opens up the platform to simulate realistic epidemic scenarios. These features make the GLEaMviz computational tool a convenient teaching/training tool as well as a first step toward the development of a computational tool aimed at facilitating the use and exploitation of computational models for the policy making and scenario analysis of infectious disease outbreaks. The 2009 H1N1 influenza pandemic highlighted the importance of computational epidemic models for the real-time analysis of the health emergency related to the global spreading of new emerging infectious diseases [1] [2] [3] . Realistic computational models are highly complex and sophisticated, integrating substantial amounts of data that characterize the population and geographical context in order to attain superior accuracy, resolution, and predictive power [4] [5] [6] [7] [8] [9] [10] . The challenge consists in developing models that are able to capture the complexity of the real world at various levels by taking advantage of current information technology to provide an in silico framework for testing control scenarios that can anticipate the unfolding of an epidemic. At the same time, these computational approaches should be translated into tools accessible by a broader set of users who are the main actors in the decision-making process of health policy, especially during an emergency like an influenza pandemic. The tradeoff between realistic and accurate descriptions of large-scale dynamics, flexibility, computational feasibility, ease of use, and accessibility of these tools creates a major challenge from both the theoretical and the computational points of view [4, 5, 11, 12, 10, 13] . GLEaMviz is a client-server software system that can model the world-wide spread of epidemics for human transmissible diseases like influenzalike illnesses (ILI), offering extensive flexibility in the design of the compartmental model and scenario setup, including computationally-optimized numerical simulations based on high-resolution global demographic and mobility data. GLEaMviz makes use of a stochastic and discrete computational scheme to model epidemic spread called "GLEaM" -GLobal Epidemic and Mobility model, presented in previously published work [6, 3, 14] which is based on a geo-referenced metapopulation approach that considers 3,362 subpopulations in 220 countries of the world, as well as air travel flow connections and short-range commuting data. The software includes a client application with a graphical user interface (GUI) for setting up and executing simulations, and retrieving and visualizing the results; the client application is publicly downloadable. The server application can be requested by public institutions and research centers; conditions of use and possible restrictions will be evaluated specifically.
The tool is currently not suitable for the simulation of vector-borne diseases, infection transmission depending on local contact patterns such as sexually transmitted diseases and diseases with a time scale that would make demographic effects relevant. The tool, however, allows the introduction of mitigation policies at the global level. Localized intervention in space or time can be implemented in the GLEaM model and their introduction in the GLEaMviz computational tool are planned for future releases.
Only a few computational tools are currently available to the public for the analysis and modeling of epidemics. These range from very simple spreadsheet-based models aimed at providing quick estimates for the number of patients and hospitalizations during a pandemic (see e.g. FluSurge [15] ) to more complicated tools based on increasingly sophisticated simulation approaches [11, 16, 12, 10, 13, 5] . These tools differ in their underlying modeling approaches and in the implementation, flexibility, and accessibility of the software itself.
InfluSim is a tool that provides a visual interface to simulate an epidemic with a deterministic compartmental model in a single population [11] . The model includes age structure and explicit sojourn times with different stages in each compartment, extending an SEIR compartmentalization to include hospitalizations and intervention measures. The software provides the infectious disease dynamics and the user can set parameter values and add or remove interventions. However, no spatial structure or other forms of heterogeneity and stochasticity are considered in the model.
On the other hand agent-based models describe the stochastic propagation of a disease at the individual level, thus taking into account the explicit social and spatial structure of the population under consideration. CommunityFlu is a software tool that simulates the spread of influenza in a structured population of approximately 1,000 households with 2,500 persons [13] . User interaction with the software is limited to the spreadsheet portion of the program, where one can choose the type of intervention and other parameters describing the disease and the population.
A larger population is considered in FluTe, a publicly available tool for the stochastic simulation of an epidemic in the United States at the level of individuals [10] . The model is based on a synthetic population, structured in a hierarchy of mixing social groups including households, household clusters, neighborhoods, and nation-wide communities. FluTe comes with a configuration file in text format that can be modified by an expert user to set various parameters such as the initiation of the epidemic, the reproductive number, and the interventions considered. No GUI is provided, and the output of the simulations is given in the form of text files that must be analyzed through additional software.
EpiFast involves a parallel algorithm implemented using a master-slave approach which allows for scalability on distributed memory systems, from the generation of synthetic population aggregated in mixing groups to the explicit representation of the contact patterns between individuals as they evolve in time [5] . The Epi-Fast tool allows for the detailed representation and simulation of the disease on social contact networks among individuals that dynamically evolve in time and adapt to actions taken by individuals and public health interventions. The algorithm is coupled with a webbased GUI and the middleware system Didactic, which allows users to specify the simulation setup, execute the simulation, and visualize the results via plots. Epidemic models and interventions are pre-configured, and the system can scale up to simulate a population of a large metropolitan area on the order of tens of millions of inhabitants.
Another class of models focuses on the global scale, by using a metapopulation approach in which the population is spatially structured into patches or subpopulations (e.g. cities) where individuals mix. These patches are connected by mobility patterns of individuals. In this vein two tools are currently available. The Global Epidemic Model (GEM) uses a metapopulation approach based on an airline network comprised of 155 major metropolitan areas in the world for the stochastic simulation of an influenza-like illness [16] . The tool consists of a Java applet in which the user can simulate a hypothetical H1N1 outbreak and test pre-configured intervention strategies. The compartmentalization is set to an SEIR model, and the parameterization can be modified in the full or stand-alone mode, but not currently in the Java applet.
The Spatiotemporal Epidemiological Modeler (STEM) is a modeling system for the simulation of the spread of an infectious disease in a spatially structured population [16] . Contrary to other approaches, STEM is based on an extensible software platform, which promotes the contribution of data and algorithms by users. The resulting framework therefore merges datasets and approaches and its detail and realism depend on continuous developments and contributions. However, these are obtained from a variety of sources and are provided in different formats and standards, thus resulting in possible problems related to the integration and merging of datasets. Such issues are left to the user to resolve.
The existing tools described above thus offer the opportunity to use highly sophisticated data-driven approaches at the expense of flexibility and ease of use by non-experts on the one hand, or very simplified models with user-friendly GUIs and no specific computational requirements on the other. Our approach aims at optimizing the balance of complex and sophisticated data-driven epidemic modeling at the global scale while maintaining an accessible computational speed and overall flexibility in the description of the simulation scenario, including the compartmental model, transition rates, intervention measures, and outbreak conditions by means of a user-friendly GUI.
In the GLEaMviz tool the setup of the simulations is highly flexible in that the user can design arbitrary disease compartmental models, thus allowing an extensive range of human-to-human infectious diseases and intervention strategies to be considered. The user interface has been designed in order to easily define both features specific to each compartment, such as the mobility of classes of individuals, and general environmental effects, such as seasonality for diseases like influenza. In addition, the user can define the initial settings that characterize the initial geographical and temporal conditions, the immunity profile of the population, and other parameters including but not limited to: the definition of an outbreak condition in a given country; the number of stochastic runs to be performed; and the total duration of each simulation. The tool allows the production of global spreading scenarios with geographical high resolution by just interacting with the graphic user interface. While an expert input would be required to interpret and discuss the results obtained with the software, the present computational platform facilitates the generation and analysis of scenarios from intensive data-driven simulations. The tool can be deployed both in training activities as well as to facilitate the use of large-scale computational modeling of infectious diseases in the discussion between modelers and public health stakeholders.
The paper is organized as follows. The "Implementation" section describes the software application architecture and its major components, including the computational model GLEaM. The "Results and discussion" section presents in detail the GLEaMviz client and its components that allow for software-user interaction, including an application of the Simulator to an Influenza-like-illness scenario.
The top-level architecture of the GLEaMviz tool comprises three components: the GLEaMviz client application, the GLEaMviz proxy middleware, and the simulation engine. The latter two components constitute the GLEaMviz server, as shown in Figure 1 .
Users interact with the GLEaMviz system by means of the client application, which provides graphical userinterfaces for designing and managing the simulations, as well as visualizing the results. The clients, however, do not themselves run the simulations. Instead they establish a connection with the GLEaMviz proxy middleware to request the execution of a simulation by the server. Multiple clients can use the same server concurrently. Upon receipt of requests to run a simulation, the middleware starts the simulation engine instances required to execute the requests and monitors their status. Once the simulations are completed, the GLEaMviz proxy middleware collects and manages the resulting simulation data to be served back to the clients. A schematic diagram of the workflow between client and server is shown in Figure 2 .
This client-server model allows for full flexibility in its deployment; the client and server can be installed on the same machine, or on different machines connected by a local area network or the Internet. The two-part decomposition of the server in terms of middleware and engines additionally allows for advanced high-volume setups in which the middleware server distributes the engine instances over a number of machines, such as those in a cluster or cloud. This architecture thus ensures high speed in large-scale simulations and does not rely on the CPU-specific availability accessible by the user.
The GLEaMviz simulation engine uses a stochastic metapopulation approach [17] [18] [19] 2, [20] [21] [22] 16 ] that considers data-driven schemes for the short-range and
Design the compartmental model of the infectious disease in the Model Builder. 1 Configure the simulation of the world-wide epidemic spreading in the Simulation Wizard. 2 Submit the simulation for execution by the Engine on the server.
Inspect the results of a simulation in the interactive Visualization. 5 Inspect all simulations and retrieve results in the Simulations History. long-range mobility of individuals at the inter-population level, coupled with coarse-grained techniques to describe the infection dynamics within each subpopulation [6, 14] . The basic mechanism for epidemic propagation occurs at multiple scales. Individuals interact within each subpopulation and may contract the disease if an outbreak is taking place in that subpopulation. By travelling while infected, individuals can carry the pathogen to a non-infected region of the world, thus starting a new outbreak and shaping the spatial spread of the disease. The basic structure of GLEaM consists of three distinct layers -the population layer, the mobility layer, and the epidemic layer (see Figure 3 ) [6, 14] .
The population layer is based on the high-resolution population database of the Gridded Population of the World project by the Socio-Economic Data and Applications Center (SEDAC) [23] that estimates population with a granularity given by a lattice of cells covering the whole planet at a resolution of 15 × 15 minutes of arc.
The mobility layer integrates short-range and longrange transportation data. Long-range air travel mobility is based on travel flow data obtained from the International Air Transport Association (IATA [24]) and the Official Airline Guide (OAG [25] ) databases, which contain the list of worldwide airport pairs connected by direct flights and the number of available seats on any given connection [26] . The combination of the population and mobility layers allows for the subdivision of the world into geo-referenced census areas obtained by a Voronoi tessellation procedure around transportation hubs. These census areas define the subpopulations of the metapopulation modeling structure, identifying 3,362 subpopulations centered on IATA airports in 220 different countries. The model simulates the mobility of individuals between these subpopulations using a stochastic procedure defined by the airline transportation data [6] . Short-range mobility considers commuting patterns between adjacent subpopulations based on data collected and analyzed from more than 30 countries in 5 continents across the world [6] . It is modeled with a time-scale separation approach that defines the effective force of infections in connected subpopulations [6, 27, 28] .
On top of the population and mobility layers lies the epidemic layer, which defines the disease and population dynamics. The infection dynamics takes place within each subpopulation and assumes a compartmentalization [29] that the user can define according to the infectious disease under study and the intervention measures being considered. All transitions between compartments are modeled through binomial and multinomial processes to preserve the discrete and stochastic nature of the processes. The user can also specify the initial outbreak conditions that characterize the spreading scenario under study, enabling the seeding of the epidemic in any geographical census area in the world and defining the immunity profile of the population at initiation.
Seasonality effects are still an open problem in the transmission of ILI diseases. In order to include the effect of seasonality on the observed pattern of ILI diseases, we use a standard empirical approach in which Population layer Short-range mobility layer Long-range mobility layer The short-range mobility layer covers commuting patterns between adjacent subpopulations based on data collected and analyzed from more than 30 countries on 5 continents across the world, modeled with a time-scale separation approach that defines the effective force of infections in connected subpopulations. The long-range mobility layer covers the air travel flow, measured in available seats between worldwide airport pairs connected by direct flights.
seasonality is modeled by a forcing that reduces the basic reproductive number by a factor α min ranging from 0.1 to 1 (no seasonality) [20] . The forcing is described by a sinusoidal function of 12 months-period that reaches its peak during Winter time and its minimum during Summer time in each Hemisphere, with the two Hemispheres with opposite phases. Given the population and mobility data, infection dynamics parameters, and initial conditions, GLEaM performs the simulation of stochastic realizations of the worldwide unfolding of the epidemic. From these in silico epidemics a variety of information can be gathered, such as prevalence, morbidity, number of secondary cases, number of imported cases, hospitalized patients, amounts of drugs used, and other quantities for each subpopulation with a time resolution of 1 day.
GLEaM has been under continuous development since 2005 and during these years it has been used: to assess the role of short-range and long-range mobility in epidemic spread [30, 31, 6] ; to retrospectively analyze the SARS outbreak of 2002-2003 in order to investigate the predictive power of the model [22] ; to explore global health strategies for controlling an emerging influenza pandemic with pharmaceutical interventions under logistical constraints [21] ; and more recently to estimate the seasonal transmission potential of the 2009 H1N1 influenza pandemic during the early phase of the outbreak to provide predictions for the activity peaks in the Northern Hemisphere [3, 32] .
The GLEaMviz simulation engine consists of a core that executes the simulations and a wrapper that prepares the execution based on the configuration relayed from the client by the GLEaMviz proxy middleware. The engine can perform either single-run or multi-run simulations. The single-run involves only a single stochastic realization for a given configuration setup and a random seed. The multi-run simulation involves a number of stochastic realizations as set by the user and performed by the core (see the following Section), each with the same configuration but with a different random seed. The results of the multi-run simulation are then aggregated and statistically analyzed by the wrapper code.
The simulation engine writes the results to files and uses lock files to signal its status to the middleware component. The core is written in C++, resulting in a fast and efficient engine that allows the execution of a single stochastic simulation of a 1-year epidemic with a standard SEIR model in a couple of minutes on a high-end desktop computer. The wrapper code is written in Python [33] . The server components can be installed on most UNIX-like operating systems such as Linux, BSD, Mac OS X, etc.
The GLEaMviz proxy middleware is the server component that mediates between clients and simulation engines. It accepts TCP connections from clients and handles requests relayed over these connections, providing client authorization management.
A basic access control mechanism is implemented that associates a specific client with the simulations it launches by issuing a private simulation identifier key upon submission. Users can only retrieve the results of the simulations they launched, or simulations for which they have obtained the simulation definition file -containing the private simulation identifier key-from the original submitter.
Upon receipt of a request to execute a simulation, the middleware sets up the proper system environment and then launches an instance of the simulation engine with the appropriate configuration and parameters according to the instructions received from the client. For singlerun simulations, the daily results are incrementally served back to the client while the simulation is being executed. This allows for the immediate visualization of the spreading pattern, as described in "Visualization interface" Subsection. For multi-run simulations the results are statistically analyzed after all runs are finished, and the client has to explicitly request the retrieval of the results once they become available. The GLEaMviz proxy server component can be configured to keep the simulation data indefinitely or to schedule the cleanup of old simulations after a certain period of time. Multi-run metadata is stored in an internal object that is serialized on a system file, ensuring that authorization information is safely kept after a server shutdown or failure. The GLEaMviz proxy component additionally provides control features such as accepting administrative requests at runtime in order to manage stored simulations or to modify several configuration parameters like the number of simultaneous connections allowed, the number of simultaneous simulations per client, the session timeout, etc.
The middleware server is written in Python [33] and uses the Twisted Matrix library suite [34] for its networking functionality. Client and server communicate using a special purpose protocol, which provides commands for session handling and simulation management. Commands and data are binary encoded using Adobe Action Message Format (AMF3) in order to minimize bandwidth needs.
The GLEaMviz client is a desktop application by which users interact with the GLEaMviz tool. It provides GUIs for its four main functions: 1) the design of compartmental models that define the infection dynamics; 2) the configuration of the simulation parameters; 3) the visualization of the simulation results; and 4) the management of the user's collection of simulations. In the following Section we describe these components in detail.
The client was developed using the Adobe AIR platform [35] and the Flex framework [36] and can thus be deployed on diverse operating systems, including several Windows versions, Mac OS X, and several common Linux distributions. The GLEaMviz client has a built-in updating mechanism to check for the latest updates and developments and prompts the user to automatically download them. It also offers a menu of configuration options of the interface that allows the user to customize preferences about data storage, visualization options, the server connection, and others.
The software system presented above is operated through the GLEaMviz client, which provides the user interface: the part of the tool actually experienced on the user side. The GLEaMviz client integrates different modules that allow the management of the entire process flow from the definition of the model to the visualization of the results. In the following we will describe the various components and provide the reader with a user study example.
The Model Builder provides a visual modeling tool for designing arbitrary compartmental models, ranging from simple SIR models to complex compartmentalization in which multiple interventions can be considered along with disease-associated complications and other effects.
(An example can be found in previous work [37] .) A snapshot of the Model Builder window is shown in Figure 4 .
The models are represented as flow diagrams with stylized box shapes that represent compartments and directed edges that represent transitions, which is consistent with standard representations of compartmental models in the literature. Through simple operations like 'click and drag' it is possible to create any structure with full flexibility in the design of the compartmentalization; the user is not restricted to a given set of pre-loaded compartments or transition dynamics. The interactive interface provided by the Model Builder enables the user to define the compartment label, the mobility constraints that apply (e.g. allowed/not allowed to travel by air or by ground), whether the compartment refers to clinical cases, as well as the color and position of their representation in the diagram (see Figure 5 ). This allows the user to model many kinds of human-to-human infectious diseases, in particular respiratory and influenza-like diseases.
Transitions individuals is equal to  SI N , where N is the total size of the subpopulation. The GLEaM simulation engine considers discrete individuals. All its transition processes are both stochastic and discrete, and are modeled through binomial and multinomial processes. Transitions can be visually added by dragging a marker from the source to the target compartment. Spontaneous transitions are annotated with their rates, which can be modified interactively. Infection transitions are accompanied with a representation of the infection's source compartment and the applicable rate (i.e. b in the example above), which can also be modified in an interactive way. The rates can be expressed in terms of a constant value or in terms of a variable whose value needs to be specified in the variables table, as shown in Figure 4 . The value can also be expressed by simple algebraic expressions.
The client automatically checks for and reports inconsistencies in the model in order to assist the user in the design process (see bottom right window in Figure 4 ).
Models can be exported to XML files and stored locally, allowing the user to load a model later, modify it, and share it with other users. The diagram representation can be exported as a PDF or SVG file for use in documentation or publications. A few examples of compartmental models are available for download from the Simulator website.
The Simulation Wizard provides a sequence of panels that leads the user through the definition of several configuration parameters that characterize the simulation. Figure 6 shows some of these panels. The consecutive steps of the configuration are as follows:
•Choice of the type of the simulation (panel a)
The user is prompted with three options: create a new single-run simulation or a new multi-run simulation from scratch, or a new one based on a saved simulation previously stored in a file.
•Compartmental model selection and editing
The user can design a new compartmental model, modify the current compartmental model (when deriving it from an existing simulation), or load a model compartmentalization from a file.
•Definition of the simulation parameters (panel c) The user is asked to specify various settings and parameter values for the simulation, including, e.g., the number of runs to perform (only accessible in the case of a multi-run), the initial date of the simulation, the length of the simulation (in terms of days), whether or not seasonality effects should be considered, the airplane occupancy rate, the commuting time, the conditions for the definition of an outbreak, and others.
•Initial assignment of the simulation (panel d)
Here the user assigns the initial distribution of the population amongst compartments, defining the immunity profile of the global population on the starting date.
•Definition of the outbreak start (panel e) This panel allows the user to define the initial conditions of the epidemic by selecting the city (or cities) seeded with the infection.
•Selection of output results (panel f) Here the user selects the compartments that will constitute the output provided by the client at the end of the simulation. The corresponding data will be shown in the Visualization Window and made available for download.
When all the above configuration settings are defined, the user can submit the simulation to the GLEaMviz server for execution. This will automatically add the simulation to the user's Simulations History. It is furthermore possible to save the definition of the simulation setup to a local file, which can be imported again later or shared with other users.
The Simulations History is the main window of the client and provides an overview of the simulations that the user has designed and/or submitted, in addition to providing access to the Model Builder, the Simulation Wizard, and the Visualization Component. The overview panel shown in Figure 7 lists the simulation identifier, the submission date and time, the simulation type (i.e., single or multi-run), the execution status (i.e., initialized, start pending, started, aborted, complete, failed, or stop pending) and the results status (i.e., none, retrieve pending, retrieving, stop retrieve pending, complete, or stored locally). Additional File 1 provides a detailed explanation of all these values.
A number of context-dependent command buttons are available once a simulation from the list is selected. Those buttons allow the user to control the simulation execution, retrieve the results from the server and visualize them, clone and edit the simulation to perform a new execution, save the simulation definition or the output data to the local machine (in order to analyze the obtained data with other tools, for example), and remove the simulation. In addition to exporting the compartmental model (see the "Model Builder" Subsection) the user can export a complete configuration of a simulation that includes the compartmental model and the entire simulation setup to a local file, which can be imported again later or shared with other users.
Once the execution of a simulation is finished and the results have been retrieved from the server, the client can display the results in the form of an interactive visualization of the geo-temporal evolution of the epidemic. This visualization consists of a temporal and geographic mapping of the results accompanied by a set of graphs (see Figure 8 ). The geographic mapping involves a zoomable multi-scale map on which the cells of the population layer are colored according to the number of new cases of the quantity that is being displayed. Several visualization features can be customized by clicking on the gear icon and opening the settings widget. It is possible to zoom in and out and pan by means of the interface at the top left of the map. Dragging the map with the mouse (on a location where there are no basin marks) can also pan the visualization. All the widgets and the graphs displayed over the map can be re-positioned according to the user's preferences by clicking and dragging the unused space in the title bar.
The color coding of the map represents the number of cases on a particular day. The time evolution of the epidemic can be shown as a movie, or in the form of daily states by moving forward or backward by one day at a time. For single-run simulations it is also possible to show the airline transportation of the 'seeding' individuals by drawing the traveling edge between the origin and destination cities. In the case where the output quantity is a subset of the infectious compartments, the edges show the actual seeding of the infection. Note that the evolution of the epidemic depends strongly on the model definition. For example, it is possible that some basins are infected by a latent individual that later develops the disease. In this case no seeding flight will be shown if only infectious compartments are selected as output.
Beside the geographical map, the Visualization Window displays two charts. One chart shows the number of new cases per 1,000 over time (incidence), and the other shows the cumulative number of new cases per 1,000 over time (size). For multi-run simulations, median values and corresponding 95% confidence intervals are shown. The menu above each chart combo lets the user choose the context for which the corresponding charts show incidence and size data. This context is either: global, one of three hemispheres, one continent, one region, one country, or one city. The currently selected day is marked by a vertical line in these plots, and the day number, counted from the initial date selected for the simulation, is shown by side of the time slider.
Here we present an example application of the GLEaMviz tool to study a realistic scenario for the mitigation of an emerging influenza pandemic. Disease-control programs foresee the use of antiviral drugs for treatment and shortterm prophylaxis until a vaccine becomes available [38] . The implementation of these interventions rely both on logistical constraints [21, 39] -related, e.g., to the availability of drugs -and on the characteristics of the infection, including the severity of the disease and the virus's potential to develop resistance to the drugs [40] .
Here we focus on the mitigation effects of systematic antiviral (AV) treatment in delaying the activity peak and reducing attack rate [41] [42] [43] 7, 8, 39, 40, 3] , and assume that all countries have access to AV stockpiles. We consider a scenario based on the 2009 H1N1 influenza pandemic outbreak and feed the Simulator with the set of parameters and initial conditions that have been estimated for that outbreak through a Maximum Likelihood Estimate by using the GLEaM model [3] . The results provided by the present example are not meant to be compared with those contained in the full analysis carried out with GLEaM [3] due to the fact that in the Figure 8 The simulation results can be inspected in an interactive visualization of the geo-temporal evolution of the epidemic. The map shows the state of the epidemic on a particular day with infected population cells color-coded according to the number of new cases of the quantity that is being displayed. Pop-ups provide more details upon request for each city basin. The zoomable multi-scale map allows the user to get a global overview, or to focus on a part of the world. The media-player-like interface at the bottom is used to select the day of interest, or show the evolution of the epidemic like a movie. Two sets of charts on the right show the incidence curve and the cumulative size of the epidemics for selectable areas of interest. present example we do not consider additional mitigation strategies that were put in place during the early phase of the outbreak, such as the sanitary control measures implemented in Mexico [3, 44] , or the observed reduction in international travel to/from Mexico [45] . Indeed, the current version of GLEaMviz does not allow for interventions that are geographically and/or temporally dependent. However, these features are currently under development and will be available in the next software release. For this reason the simulation scenario that we study in this application of the Simulator does not aim to realistically reproduce the timing of the spreading pattern of the 2009 H1N1 pandemic. The results reported here ought to be considered as an assessment of the mitigating impact of AV treatment alone, based on the initial conditions estimated for the H1N1 outbreak, and assuming the implementation of the same AV protocol in all countries of the world.
We adopt a SEIR-like compartmentalization to model influenza-like illnesses [29] in which we include the systematic successful treatment of 30% of the symptomatic infectious individuals (see Figure 9 ). The efficacy of the Figure 9 Compartmental structure in each subpopulation in the intervention scenario. A modified Susceptible-Latent-Infectious-Recovered model is considered, to take into account asymptomatic infections, traveling behavior while ill, and use of antiviral drugs as a pharmaceutical measure. In particular, infectious individuals are subdivided into: asymptomatic (Infectious_a), symptomatic individuals who travel while ill (Infectious_s_t), symptomatic individuals who restrict themselves from travel while ill (Infectious_s_nt), symptomatic individuals who undergo the antiviral treatment (Infectious_AVT). A susceptible individual interacting with an infectious person may contract the illness with rate beta and enter the latent compartment where he/she is infected but not yet infectious. The infection rate is rescaled by a factor ra in case of asymptomatic infection [41, 46] , and by a factor rAVT in case of a treated infection. At the end of the latency period, of average duration equal to eps -1 , each latent individual becomes infectious, showing symptoms with probability 1-p a , whereas becoming asymptomatic with probability p a [41, 46] . Change in travelling behavior after the onset of symptoms is modeled with probability p t set to 50% that individuals would stop travelling when ill [41] . Infectious individuals recover permanently after an average infectious period mu -1 equal to 2.5 days. We assume the antiviral treatment regimen to be administered to a 30% fraction (i.e. pAVT = 0.3) of the symptomatic infectious individuals within one day from the onset of symptoms, reducing the infectiousness and shortening the infectious period of 1 day. [41, 42] .
AV treatment is accounted for in the model by a 62% reduction in the transmissibility of the disease by an infected person under AV treatment when AV drugs are administered in a timely fashion [41, 42] . We assume that the drugs are administered within 1 day of the onset of symptoms and that the AV treatment reduces the infectious period by 1 day [41, 42] . The scenario with AV treatment is compared to the baseline case in which no intervention is considered, i.e. the probability of treatment is set equal to 0 in all countries.
The GLEaMviz simulation results are shown in Figure 10 where the incidence profiles in two different regions of the world, North America and Western Europe, are shown for both the baseline case and the intervention scenario with AV treatment. The results refer to the median (solid line) and 95% reference range (shaded area) obtained from 100 stochastic realizations of each scenario starting from the same initial conditions. The resulting incidence profiles of the baseline case peak at around mid-November and the end of November 2009 in the US and Western Europe, respectively. These results show an anticipated peak of activity for the Northern Hemisphere with respect to the expected peak time of seasonal influenza. In order to make a more accurate comparison with the surveillance data in these regions, we should rely on the predictions provided by models that can take into account the full spectrum of strategies that were put in place during the 2009 H1N1 outbreak, viz. the predictions obtained by GLEaM [3] .
In the case of a rapid and efficient implementation of the AV treatment protocol at the worldwide level, a delay of about 6 weeks would be obtained in the regions under study, a result that could be essential in gaining time to deploy vaccination campaigns targeting high-risk groups and essential services. In addition, the GLEaMviz tool provides simulated results for the number of AV drugs used during the evolution of the outbreak. If we assume treatment delivery and successful administration of the drugs to 30% of the symptomatic cases per day, the number of AV drugs required at the activity peak in Western Europe would be 4.5 courses per 1,000 persons, and the size of the stockpile needed after the first year since the start of the pandemic would be about 18% of the population. Again, we assume a homogeneous treatment protocol for all countries in the world; results may vary from country to country depending on the specific evolution of the pandemic at the national level.
Computer-based simulations provide an additional instrument for emerging infectious-disease preparedness and control, allowing the exploration of diverse scenarios and the evaluation of the impact and efficacy of various intervention strategies. Here we have presented a computational tool for the simulation of emerging ILI infectious diseases at the global scale based on a datadriven spatial epidemic and mobility model that offers an innovative solution in terms of flexibility, realism, and computational efficiency, and provides access to sophisticated computational models in teaching/training settings and in the use and exploitation of large-scale simulations in public health scenario analysis.
Project name: GLEaMviz Simulator v2. 6 Project homepage: http://www.gleamviz.org/simulator/ Operating systems (client application): Windows (XP, Vista, 7), Mac OS X, Linux.
Programming language: C++ (GLEaMsim core), Python (GLEaMproxy, GLEaMsim wrapper), Action-Script (GLEaMviz)
Other requirements (client application): Adobe AIR runtime, at least 200 Mb of free disk space.
License: SaaS Baseline scenario Scenario with AV Figure 10 Simulated incidence profiles for North America and Western Europe in the baseline case (left panels) and in the AV treatment scenario (right panels). The plots are extracted from the GLEaMviz tool visualization. In the upper plots of each pair the curves and shaded areas correspond to the median and 95% reference range of 100 stochastic runs, respectively. The lower curves show the cumulative size of the infection. The dashed vertical line marks the same date for each scenario, clearly showing the shift in the epidemic spreading due to the AV treatment.
Any restrictions to use by non-academics: None. The server application can be requested by public institutions and research centers; conditions of use and possible restrictions will be evaluated specifically.
Additional file 1: The GLEaMviz computational tool: Additional File. This file includes information for installing the GLEaMviz Client and details of the features of its various components. Nursing heroism in the 21(st )Century' BACKGROUND: The Vivian Bullwinkel Oration honours the life and work of an extraordinary nurse. Given her story and that of her World War II colleagues, the topic of nursing heroism in the 21(st )century could not be more germane. DISCUSSION: Is heroism a legitimate part of nursing, or are nurses simply 'just doing their job' even when facing extreme personal danger? In this paper I explore the place and relevance of heroism in contemporary nursing. I propose that nursing heroism deserves a broader appreciation and that within the term lie many hidden, 'unsung' or 'unrecorded' heroisms. I also challenge the critiques of heroism that would condemn it as part of a 'militarisation' of nursing. Finally, I argue that nursing needs to be more open in celebrating our heroes and the transformative power of nursing achievements. SUMMARY: The language of heroism may sound quaint by 21(st )Century standards but nursing heroism is alive and well in the best of our contemporary nursing ethos and practice. Today's nursing heroism First, the more traditional concept of heroism as courage and providing service to others in the face of extreme personal danger is undoubtedly alive and well in nursing and in other human services. Firemen still enter burning buildings to save their occupants and nurses still join their health care colleagues in providing care to the hungry, the fearful, the injured and the dying in both natural disaster areas and man made conflict zones. Haitian nursing students and faculty from the Episcopal University of Haiti, were setting up first aid stations to help the victims of their city 30 minutes after that country's massive earthquake [4] . Military nurses and nurses from voluntary organisations such as Red Cross and Medicine Sans Frontiers are found in every war zone, every famine-blighted country, every dictatorial wasteland, every manifestation of 'hell on earth'. We fervently wish that the circumstances that draw them away from their own families and homes to these places did not exist, but they do, and thankfully, these nurses continue to respond.
Consider also, nurses' responses to the fear and danger surrounding the emergence of infectious outbreaks such as HIV/AIDS in the 1980s and SARS in 2003. In the early 1980s when first reports were emerging of young gay men in the USA dying of seemingly systemic immune system failure, we could not have realised that this thing called 'GRID' was the start of the AIDS pandemic that has claimed the lives of more than 25 million people worldwide and has left approximately 33.4 million people living with HIV/AIDS [5] .
During these times we saw the best and worst of nursing and health care. In an Oral History project: "The AIDS Epidemic in San Francisco: The Response of the Nursing Profession, 1981-1984 [6, 7] " we hear from nurses involved that some nursing and medical staff held the same fears and prejudices that were so widespread in the broader community. They would refuse to care for the AIDS patients and stigmatise them along with the other so-called "4H patients -Homosexuals, Haitians, Hemophiliacs and Heroin users" [8] .
Helen Miramontes, who later became one of the world's leading nurse advocates, specialists and educators in HIV/AIDS was then a clinician. She said that: "There was a lot of fear among health care providers about contagion, but there was also significant prejudice and discriminatory behavior because the new disease was identified in a population that was stigmatized by the larger society. Identification of the disease in people of color, especially African Americans and injection drug users, only exacerbated the biases, prejudices, and discriminatory behavior. Many nurses demonstrated the same attitudes, beliefs, and behaviors seen in the larger society. I was a critical care nurse working in an intensive care unit (ICU) in a large teaching facility. In the early years of the epidemic, it was not unusual to have two to three patients with Pneumocystis carinii pneumonia on ventilators in the ICU at any one time. Because some nurses avoided taking care of these patients, several of us volunteered to care for them on a regular basis. There were frequent breaches in confidentiality, not only among nurses but also among other health care workers" [7] . Gary Carr, who was a Nurse Practitioner at the AIDS Clinic at San Francisco General Hospital, described the perverse ambivalence of a wider community that lauds and praises nurses for their 'heroic efforts' in the face of such public health crises. Gary says: "I have no memories of being afraid or being brave. I just wasn't afraid. I just said to myself, this is what I want to do. This is important. The community needs this, and it's what I want to do. I remember there were people who stopped speaking to me. My mother for years didn't tell anybody what I did. My relatives for years thought I still worked in the trauma unit" [6] .
When, two decades later, SARS emerged as a potentially lethal viral infection, nurses and health care staff again faced considerable dangers as they strove to treat patients and protect their communities. Dr Dessmon Tai, who led the Singapore efforts against SARS, wrote that: "No other disease had such a phenomenal impact on healthcare workers." [9] But it seems that something in our professional ethos had changed over these two decades. There had been a rediscovery or reaffirmation of our professional ethic and mission as nurses, doctors and health professionals. In Hanoi, during the initial outbreak, rather than look for an 'opt-out' clause in their professional codes, doctors and nurses locked themselves into the hospital in isolation rather than risk spreading the disease [10] .
As Emmanuel notes: "More than half of the first 60 reported cases of SARS involved health care workers who had come into contact with SARS patients. Indeed, apart from the very first case, all of the people who died in Vietnam were doctors and nurses. Nearly a quarter of all patients with SARS in Hong Kong were health care workers. In Canada, of the 141 probable cases of SARS diagnosed between 23 February and 14 May 2003, 92 (65%) involved health care workers. Despite deadly peril, physicians and nurses tirelessly cared for patients with SARS". [8] However, the social stigma that surrounded HIV/AIDS twenty years earlier and the associated ambivalence of the community towards health care professionals was not so easily repressed. In Toronto, Canada, Hall and colleagues reported that: "children of nurses were barred from school trips and families were shunned by their neighbours. Other incidents included husbands of nurses being sent home from work, children of nurses shunned at school, nurses refused rides by taxi drivers, and singleparent nurses unable to get babysitters". [11] Similarly, in Singapore, nursing staff were reportedly shunned in public spaces, forbidden to use the lifts in their apartments, found that buses and taxis would not stop for them and as one review reported, "At any packed food court, there would always be a seat for a Tan Tock Seng Hospital nurse. Queue lines would quickly shorten when a nurse joined that queue". [10] These personal travails were compounded by what many saw as the unavoidable violence done to some of the best traditions of the nurse-patient relationship by the nature of the SARS virus and its containment. Nurses working with SARS patients were often isolated from collegial support, asked to eat meals alone and prohibited from attending meetings. Rigorous isolation and anti-infection procedures saw nurses, effectively in spacesuits, caring for patients in enforced cubicle isolation. If this was a terrible way to be ill, it was an even more solitary and disconnected way to die.
Yet despite these dangers and demands, nurses and our health care colleagues exemplified the best of who we are and what we do. They worked in a cauldron of contagion, initially unaware of what they were fighting, how the infection spread, how it killed or how it could be treated. As one French doctor commented, "We were not playing with fire. It was playing with us" [9] . Was this heroism and heroic actions on their part, or were they 'just doing their job'? If we accept that heroism is "providing service to others in the face of extreme personal danger", then I have no qualms in considering these nurses as exemplars of 21 st Century Nursing heroism.
Heroism and 'militarism': What's in a word?
Let me sidetrack slightly at this point to address a concern about the very legitimacy and appropriateness of the term heroism in nursing. For some critics, the very mention of 'wars against disease', 'defeating illness', 'the battle against SARS', or 'nursing heroism', is tainted. The concern is related to the militaristic or combative nature of such language and how this might shape not only our understandings of illness and disease but also our understanding of nursing itself and indeed the content and foci of our education, research and services.
The concerns are not new, having been articulated most forcefully and influentially by the late American essayist, critic and author, Susan Sontag in both her books: 'Illness as a Metaphor' and 'AIDS and its Metaphors' [12] . Sontag was deeply critical of the metaphorical language that scaffolds our understandings of, in these cases, Cancer and AIDS. She rejects metaphors of battle, war, magic bullets, invasion, surrender, attack etc and with AIDS is even more scathing of its damning metaphorical encumbrances around divine retribution, plague, sexual contagion and societal decay.
"The body is not a battlefield" says Sontag [12] , but when faced with illness or injury, I suspect that it may become one, if for no other reason that nurses, people and patients need something to fight against. People will often accept the most devastating of diagnoses with almost a gratitude that seems completely misplaced, until they explain to us that the previous uncertainty or not knowing had been far, far worse.
Author and doctor, Peter Goldsworthy depicts this phenomenon beautifully in his celebrated novella, 'Jesus Wants Me for a Sunbeam' which describes how a family reacts when their young daughter develops leukaemia:
"For Rick and Linda there was also, at the end of that terrible week of waiting and worry, an odd feeling of relief that it had happened to them, and theirs. Anything was better than uncertainty; the waiting had been intolerable, the fear of the unmentionable had almost come to be a desire for the unmentionable; its certainty, its mention, was at least a resolution. To finally hear the word (leukaemia) spoken aloud provided a focus for worry, a definite enemy that they could now face, and fight, together, as a family." [13] Before leaving the subject of language, let me touch on a recently articulated concern about the language and discourses of militarism and how these may influence Nursing.
In a new paper this year on the Politics of Nursing Knowledge, Perron and her colleagues [14] criticise what they call the 'militarization of nursing', claiming that: "Many accounts provide angelic portrayals of nurses faced with the devastating effects of war. Such nurses are described as loyal, beautiful, peaceful, healing, comforting, reliable, devoted, and courageous in the face of hardship. These romantic descriptions stand in sharp contrast to the organized killing and destruction of warmaking." [14] I would argue that what Perron and her colleagues have almost studiously overlooked are that these allegedly 'romantic descriptions' also stand in sharp contrast to any respectable historical account of the experiences of military nurses [1] . In such histories, and in particular the growing collection of oral histories of nurses' wartime and conflict zone experiences [3, [15] [16] [17] [18] [19] , we will find not saccharine, sentimentalized, spin-doctoring but vivid recollections and narratives from nurses, who, in addition to thankfully being 'loyal, comforting, devoted, healing, reliable and courageous', are also intelligent, skillful, determined, demanding, creative and resourceful.
Heather Höpfl, a Professor of Management, takes a quite different, and I believe more coherent and enabling view of women's heroism than Perron and her colleagues. She argues that: "The heroines of history are not impotent women. Quite the contrary, they are women who refuse to be put in their place. The stories of heroines offer a picture of women who were far from meek and far from conciliated into male reality definitions. They were real women not mythological constructions. They shared values rather than common backgrounds and they are, to use an unfashionable term 'indomitable'. (...) The stories of the heroines of 50 years ago and more are stories of recusancy. They are stories of opposition. They are in almost every case stories of gender politics. We are deceived when we are told they are stories of female oppression. (...) These women faced enormous obstacles but squared themselves off against them and responded with integrity and courage." [20] Of other 'quiet' and 'unrecorded' heroisms
Let me now speak of other heroisms, for I believe heroism in nursing to be a broad church, a continuum of courage rather than a zero-sum game. The nurse exhibiting what I might call, after Dickens' character, Sydney Carton, a "quiet heroism", or what Patricia Benner calls "unrecorded heroism" [21] , continues to be part of the fabric of health care today.
Nursing may not be among the first occupations that springs to mind when the word 'danger' is mentioned but a recent Forbes magazine feature in the USA did identify nurses and nurses' aides as one of the top two groups experiencing among the highest rates of injury at work, albeit usually from lifting and handling incidents [22] . In addition, Hall and colleagues in the US reported that: "Nursing assistants working in long-term care facilities have the highest incidence of workplace violence of any American worker". [23] Nurses in our Emergency Departments experience perhaps even more severe episodes of violence. In Scotland, we used to joke that any particularly rough place was: "Like casualty on a Friday night", but it is no joke now. In a recent Australian survey from WA, Rose Chapman and colleagues found that: "The majority (92%) of nurses said they had been verbally abused, 69% had been physically threatened and 52% had been physically assaulted". "Only 16% of the nurses completed an official incident report. Reasons for not reporting included the view that Work Place Violence is just part of the job, and the perception that management would not be responsive". [24] Would Emergency Department nurses see themselves as 'heroes'? Almost certainly not, as they seem to have internalized a workplace culture where abuse and violence is not an aberration but simply something that "comes with the job" [25] and where reporting such abuse is scarcely worth the trouble. Perhaps if we return to the definition of heroism as 'providing service in the face of extreme personal danger', then our Emergency Department nurses should allow themselves to feel, at least somewhat heroic.
When we define heroism as providing service in the face of personal danger, that danger is not always the danger of illness, injury or death. Sometimes, that danger emanates from inside the very organisations that we serve and what is under threat is not nurses' bodily integrity but their professional and personal integrity. I refer of course to the phenomena of whistleblowing in nursing and health care.
Nursing's official pronouncements, policy directives, mission and vision statements, strategic plans, nursing philosophies and the like invariably proclaim our commitment to the highest standards (indeed to excellence), to patient safety, to quality care, to collegiality, to mutual respect, to 'patient-centredness', to patient advocacy and more. But for many nurses, there is not merely a gap between these aspirations and clinical reality, there is a Katherine Gorge. Debra Jackson and her research team in Sydney have done some of the best Australian research in this area and it does not make for happy reading. As Debra notes: "Currently, whistleblowing represents a professional dilemma and a personal disaster." [26] This observation does not only apply to nursing. Medical whistleblowers fare equally badly at the hands of their own profession [27] . Indeed in all of the whistleblowing literature, it is difficult, if not impossible to find even one person whose principled actions have NOT resulted in huge personal and professional costs.
And yet we know that nurses are not only vital because of our numbers but because we are patient care, safety and quality where the rubber hits the runway. Nursing is not just some inert 'silo', needing to be dismantled. If a hospital or health authority does not understand the difference between nursing as a Silo and nursing as a Sentinel, they are in more trouble than they can possibly imagine. We are the world's best adverse patient experience early warning detection system. We are like the canaries down the mine or the frogs in the ecosystem. If nurses are metaphorically not singing or not croaking, if they are falling off the perch or disappearing from the ponds, then 'Huston -we have a problem"! This problem is a phenomena that blighted not only Bundaberg Hospital [28] , but other hospitals and health organisations across the world. This virulent, vocational virus -I'll call it: MRSA ('Management Resistance to Staff Alerts'), has been implicated in almost every hospital 'scandal' and health system failure inquiry in recent years. This strain of 'MRSA' seems endemic in health care systems and is as dangerous to staff and patients as any hospital acquired infection. In a nutshell, health organisations and their leaders are not only failing to listen to their front line nursing staff, but in the worst cases, they are actively and forcefully trying to silence them.
At Bundaberg Hospital, what stopped Dr Jayant Patel was not a new computer system, it was not an updated reporting mechanism, it was not visits from external regulators, it was not another reorganization, it was not a new management theory. It was the bulldog advocacy and persistence of ICU Nurse Toni Hoffmann in the face of managerial inactivity and intimidation [28] . Toni was recognised for her role with an Australian Local Hero Award in 2006 and an Order of Australia in 2007. Is Toni Hoffmann a 21 st Century nurse hero? Without a shadow of doubt.
What of the other contemporary meaning of heroism and heroes? Who are our nursing heroes? Who are the nursing giants upon whose shoulders we have stood and who continue to inspire us today? Who are the nursing heroes that we tell our students about so that they can understand the best of Nursing's history? Who are they to emulate if they want to be the best nurse possible?
Our hardwired and often confused sense of egalitarianism makes us uncomfortable in even talking in such a way. When I ask nurses to tell me something wonderful that they did recently that made a real difference to a patient, client or family, their embarrassment and discomfort is almost immediately palpable. 'I didn't really do anything', 'it was the team', 'I'm just a nurse', 'I don't like to 'big note' (brag about) myself', 'All I did was..., 'I don't know what you mean'....
Please, let us be more open and unabashed in celebrating our nursing heroes. If these were sportsmen or sportswomen, if they were musicians or actors, if they were business people: we would be lauding their finest performances, dissecting their latest work with relish and handing out awards and trophies by the cabinetful.
I thought of my heroes for this paper, the nurses who have inspired me and who continue to do so. So let me celebrate, in no particular order of merit:
Possibly the most lucid and coherent writer on nursing ever. I read her little 50 or 60 page book, 'Basic Principles of Nursing Care' as a young student nurse and could recite her definition of nursing, even at parties after a few drinks, as if it were a catechism. It is no exaggeration to say that I understood nursing in a completely different light after Virginia Henderson.
As a new PhD student grappling with philosophy, phenomenology, qualitative research approaches and new understandings of practice, reading 'The Primacy of Caring', followed by 'Novice to Expert' was to have the scales fall from one's eyes and to see and understand the world anew. The quality of Patricia Benner's thinking and scholarship is matched only by her graciousness and generosity of spirit.
Margaret was Dean and Head of School when I took up my first post-PhD Lecturing position at Glasgow Caledonian University and was simply the Dean from heaven. Her standards and expectations were exhilaratingly exacting and her work rate would have shamed a Chilean mine rescuer. Her enthusiasm for nursing, for education, for research and for innovation and creativity was boundless. Her guiding philosophy seemed to be: 'The answer's yes, now what's the question'. How could you fail to thrive and develop as a new Faculty member under such inspiring and utterly humane leadership?
Linda Aiken is undoubtedly the doyenne of contemporary nursing research and one of the most powerful voices in nursing, in that when Linda Aiken's research speaks, the world of healthcare listens. And so it should. Her exemplary international research programme at the Center for Health Outcomes and Policy Research at the University of Pennsylvania demonstrates clearly that nursing is not simply one of many factors involved in improving health outcomes and patient safety, it is THE KEY FACTOR. Get nursing right and you improve safety, quality and patient care. No ifs, no buts.
Dodie Bryce was an enrolled nurse at the intellectual disability hospital where I trained as a new nurse in the 1970s. These institutions could be grim places but Dodie Bryce's calm, humane, compassion and exemplary human caring skills made her truly, a light in the darkness. She was not just a great nurse but a presence. Older and wiser, I now understand the difference.
Was a ward sister at the Sick Children's Hospital in Edinburgh when I trained in pediatrics. I was simply in awe of her. She managed, as the sole charge nurse, a 30 bed surgical ward, the attached neonatal unit of around 6-10 cots and an attached 2 bed cardiothoracic surgery unit. She knew every child, everything about their condition and its care and seemingly all of their families as well. She was unflappable in any crisis and utterly respected and listened to by every doctor and health professional. That she managed to take time and trouble to help students on the ward like myself made her even more remarkable.
Is South Australia' first Nurse Practitioner and heads our Pediatric Palliative Care service. She was also my first clinical research collaborator when I took up my Joint Chair position at Women's & Children's Hospital. Sara is a human dynamo, possessed with vision, drive and absolute determination. Give a hospital half a dozen Saras in clinical leadership positions and they could rule the world.
Debra was my PhD student and the PhD student of every supervisor's dreams. Debra has a fierce intelligence matched only by her unstinting work ethic. When you combine these with a heart and personality that draws the best out of everyone around her, you can see why she now heads what is easily one of Australia's best research centers.
Who are the heroes on your list and why?
The business of Nursing?
We are told constantly that health care is a business and that nursing should follow more business-like principles. As a health service or hospital is indeed a multi-million dollar organisation that needs to be well managed, there are no arguments from me on that score. If we are in business however, then let us be absolutely clear about the nature of our business as nurses. We are in the transformation business and the 'making a difference' business. Nurses don't just make the tea and coffee they make decisions. We need to appreciate the importance of processes and structures, but more importantly, we need a laser focus and a near-reverence for tangible and valued outcomes that improve patients' experiences.
We are in the transformation business. As a clinician, you are not in the injections business or the dressingchanging business or the putting up IVs business or the bathing business.
Instead, as Kerfoot notes, we transform [29] "We transform a frightened 4-year-old girl in the emergency room into a little person who now feels she has some measure of control and can stop crying. We transform a 50-year-old father with out-of-control diabetes into a person who has the confidence to manage his condition. And we transform the frightening and painful experience of childbirth into a beautiful memory of ecstasy for a family that has created a new person. When life ends, we transform those final moments of life into sacred, beautiful transitions of passage for families to complete the circle of life."
As a nurse educator, you are not in the business of 'lecturing', 'marking', 'supporting students', or 'writing curricula'. You are in the transformation business. [30] We transform students into safe, skilled and self-confident practitioners.
We transform apathy and cynicism into enthusiasm and robust idealism.
We transform clinical, interpersonal and ethical problems from potential career-ending setbacks, into opportunities for deep learning and personal and professional mastery.
We transform patient and client experiences from everyday anecdote into the bedrock of clinical judgement and service quality.
We foster and build confidence and self-belief where this been eroded, damaged or has never developed while also challenging an equally dangerous overconfidence, arrogance or narcissism.
As a nurse researcher, you are not in the business of interviewing, administering surveys or managing data.
We transform the glib stereotype of the 'ivory-tower' academic by our meaningful, productive and mutually advantageous collaborations with clinical colleagues and service areas.
We challenge the prejudice that academics and their research has little relevance or use in the 'real world' of health policy and politics by our focus on knowledge translation, transfer and research impact and by the demonstrable profile and presence that our work has in numerous key areas of health policy and politics.
We are in the transformation business and the 'making a difference' business.
All over the world, nurses are making rhetorical notions of 'The Patient Experience', Quality & Safety and Improved Outcomes very, very real: Somewhere a nurse is helping a struggling and despairing new mum to learn all of the messages and nuances that her new baby is signalling, Somewhere a nurse is bearing witness to another mother's dying, and comforting her during her last moments on this earth, Somewhere a nurse is inserting a child's IV and helping them and their family begin their journey into the world of chemotherapy, Somewhere a nurse is listening to an Alzheimer's patient tell a story and trying to help them piece together who they really are, Somewhere a nurse is helping a new student learn from a patient encounter and is passing on the wisdom of our art, Somewhere a nurse is turning a hunch or a problem into a question that will eventually be researched and provide new knowledge and understanding, Somewhere a nurse is managing a service with the passion and enthusiasm that enables her staff to thrive and to appreciate why they wanted to become nurses in the first place, And somewhere a nurse is working in a war zone, helping service personnel and villagers alike.
These 'quiet' or 'unrecorded' heroisms surely deserve our acknowledgement and appreciation.
At the end of her classic novel 'Middlemarch' [31] , George Eliot writes an epitaph for her heroine Dorothea:
"But we insignificant people with our daily words and acts are preparing the lives of many Dorotheas...Her finely-touched spirit had still its fine issues, though they were not widely visible. Her full nature, like that river of which Cyrus broke the strength, spent itself in channels which had no great name on the earth. But the effect of her being on those around her was incalculably diffusive: for the growing good of the world is partly dependent on unhistoric acts; and that things are not so ill with you and me as they might have been, is half owing to the number who lived faithfully a hidden life, and rest in unvisited tombs."
So too, the health, wellbeing, safety and experiences of patients, clients and families are dependent upon the often invisible and overlooked caring practices of nurses. Today in the 21 st Century, they are worthy of sharing the term 'heroism' and I like to think that Sister Vivian Bullwinkel would agree. Implications of copy number variation in people with chromosomal abnormalities: potential for greater variation in copy number state may contribute to variability of phenotype Copy number variation is common in the human genome with many regions, overlapping thousands of genes, now known to be deleted or amplified. Aneuploidies and other forms of chromosomal imbalance have a wide range of adverse phenotypes and are a common cause of birth defects resulting in significant morbidity and mortality. “Normal” copy number variants (CNVs) embedded within the regions of chromosome imbalance may affect the clinical outcomes by altering the local copy number of important genes or regulatory regions: this could alleviate or exacerbate certain phenotypes. In this way CNVs may contribute to the clinical variability seen in many disorders caused by chromosomal abnormalities, such as the congenital heart defects (CHD) seen in ~40% of Down’s syndrome (DS) patients. Investigation of CNVs may therefore help to pinpoint critical genes or regulatory elements, elucidating the molecular mechanisms underlying these conditions, also shedding light on the aetiology of such phenotypes in people without major chromosome imbalances, and ultimately leading to their improved detection and treatment. One of the fastest-growing research areas in genetics in the past few years has been the investigation of structural variation in the human genome, with copy number variants (CNVs) found to be much more common than previously imagined. As well as contributing to interindividual phenotypic variation in the general population, these genomic variants may hold the key to understanding the differences in severity seen in people with chromosomal abnormalities. Aneuploidy, for example, can be regarded as a state of large-scale changed copy number resulting from loss or gain of an entire chromosome, arising due to non-disjunction during cell division in either meiosis I or II. Aneuploidies and other chromosomal abnormalities are a common cause of birth defects and are associated with significant morbidity and mortality. The spectrum of phenotypes seen in each affected individual is clearly dependant on the particular chromosomal region concerned, although there is still a great deal of variability in the presentation of phenotypes.
Individuals with Down's syndrome (DS), for example, like those with other aneuploidies or with chromosomal deletions or duplications, vary greatly in their clinical features, capabilities, disabilities and prognosis. Although many of these people, with the necessary social and clinical support, can and do lead active and fulfilling lives, some DS sub-phenotypes are associated with increased morbidity and mortality in infancy. Congenital heart defects (CHD), for example, occur in *40% of DS; 50% of these require surgical correction within the first year of life and survival to one year is only *76% compared with *91% for non-CHD DS patients (Yang et al. 2002 ). An appreciation of the molecular mechanisms behind this phenotypic variation and the identification of susceptibility loci may provide diagnostic and prognostic markers to enable us to predict the occurrence of clinically serious phenotypes, such as CHD, in DS. Additionally, analysis of these loci might help elucidate the mechanisms of CHD in non-DS children.
To assess the implications of genomic copy number variants in the context of chromosomal abnormalities, it is helpful to consider the evidence that they affect phenotype in euploid individuals. Until recently, it was assumed that there are two copies of every gene in euploid individuals, with one on the maternally-inherited chromosome and the other inherited paternally. Any two human genomes were thought to be 99.9% similar, with the main source of genetic variation due to single nucleotide polymorphisms (SNPs). Technological advances often result in paradigm shifts, however, and an unexpected amount of copy number variation was revealed through the advent of array comparative genomic hybridisation (array CGH) and, more recently, of second-generation sequencing technologies. There are currently over 14,000 CNV regions listed on the TCAG Database of Genomic Variants (DGV, updated March 2010) , predicted to overlap a significant proportion of the genome. Approximately 11,500 genes, including over 2600 listed in the Online Mendelian Inheritance in Man database, are reportedly overlapped by CNVs (DGV March 2010).
To understand the role of CNVs in human disease, it is important to consider how these genomic variants may affect phenotype. Deletion or duplication of dosage-sensitive genes, or their regulatory regions, could have adverse phenotypic effects, and copy number changes correlate with expression of affected genes (Henrichsen et al. 2009; Stranger et al. 2007 ). The majority of common CNVs are much smaller than the subset investigated by Stranger et al. (2007) , leading them to propose that a substantial proportion of heritable variation in gene expression may be explained by copy number variation. In addition to direct copy number effects, CNVs may also exert positional effects, for example by shifting them closer to or further away from heterochromatin. Furthermore, adverse phenotypic effects may result from interactions between CNVs and other variants, for example a deletion on one chromosome may unmask a harmful recessive allele on the other. Conversely, a genomic duplication that incorporates a harmful mutation may have a double gain-of-function effect: a duplication of the PRSS1 and PRSS2 genes along with a missense mutation was recently shown to cause hereditary pancreatitis (Masson et al. 2008) .
It is likely, therefore, that CNVs do significantly affect human phenotypes, and this is reflected by an increasing number of associations reported between CNVs and disease (Tables 1, 2). Several autoimmune disorders have been associated with CNVs, including systemic lupus erythematosus (Fanciulli et al. 2007 ), Crohn's disease (Fellermann et al. 2006; McCarroll et al. 2008 ) and psoriasis (Hollox et al. 2008) , and a range of other diseases have now been associated with CNVs (reviewed in Zhang et al. 2009 ). It has been postulated that CNVs could account for a large proportion of the missing heritability of common diseases that has emerged after the recent plethora of genome-wide Diskin et al. (2009) association studies (GWAS) (reviewed in Manolio et al. 2009 ), but this is the subject of heated controversy. In a recent large-scale genotyping survey of common CNVs, Conrad et al. (2010) found that the CNVs that they were able to genotype easily were only in linkage disequilibrium with 34 out of 1,554 trait-associated SNPs from GWAS, thus they suggest that common CNVs are unlikely to account for much of the missing heritability. There is, however, a particular challenge in analysis of amplified sequences (which may be remote from the original sequence copy (Conrad et al. 2010) ), multi-allelic variants (particularly VNTRs) and recurrent events, so further analysis of those CNV types is required before the full contribution of common CNVs to human disease can be assessed.
As an alternative to the common disease-common variants hypothesis (Chakravarti 1999; Lander 1996) , the rare variants hypothesis postulates that a collection of many, individually less frequent copy number changes may collectively significantly contribute to disease susceptibility. Although many CNVs are present in an appreciable proportion of the population, the majority are likely to be less frequent: for example, in our array CGH investigation of 50 apparently healthy French male samples, 809 out of 1469 (*55%) multi-probe CNV regions were identified in only one individual (de Smith et al. 2007 ). Rare copy number changes have been implicated in neurodevelopmental disorders, including autism (Marshall et al. 2008; Sebat et al. 2007 ) and schizophrenia (Wilson et al. 2006; Xu et al. 2008 ). Furthermore, a number of rare genomic structural variants have recently been associated with obesity (Bochukova et al. 2010; Walters et al. 2010) , and many more low frequency CNVs with strong effects may contribute to common disease phenotypes: large sample cohorts and different populations will need to be investigated to uncover these rare variants.
Thus, it is now clear that genomic structural variants may significantly impact phenotype in euploid individuals: these effects may be even more pronounced in people with chromosomal abnormalities. For example, a phenotype associated with a particular monosomy or sub-chromosomal deletion may be ameliorated by the presence of an amplification CNV on the unaffected chromosome, as gene expression within the CNV region could remain at a normal, euploid level. Conversely, a deletion CNV could cause a more severe phenotype.
An example could be Turner syndrome, which is the only whole chromosome monosomy (45, X) that is viable in humans, with approximately 1 in 2,000 female births having one copy of the X chromosome (Nielsen and Wohlert 1990) . In addition to the main features of short stature and ovarian failure, present in almost all cases, there are many other phenotypes that may present, including a short webbed neck, kidney malformations, hearing problems and various learning difficulties, as well as increased risk of type 1 diabetes (Gravholt et al. 1998) . It is possible that the extensive copy number variation on the X chromosome-37.8% according to the DGV (March 2010)-may contribute to phenotypic variability in this disorder. For example, a critical region for the neurocognitive deficits in Turner syndrome was mapped to Xp22.3, containing 31 genes (Ross et al. 2000) , and several CNV loci have been reported in this region.
Similarly, phenotypic variation in genomic disorders could also be a reflection of underlying copy number variation within the relevant genomic region. The most common microdeletion in humans is DiGeorge syndrome (or velo-cardio-facial syndrome) caused by deletion of a 1.5-3 Mb region at chromosome 22q11.2 (Scambler et al. Triplication (4 copies 1992) and occurring in 1 in 5,000 births (Botto et al. 2003) . This disorder is also associated with variable phenotypes, such as cleft palate, congenital heart disease (Shprintzen et al. 1978) , renal anomalies (Czarnecki et al. 1998 ) and increased risk of schizophrenia (Murphy et al. 1999) . Several CNV loci have been identified within the 22q11.2 deletion region in apparently healthy individuals: of interest, two of the candidate genes for the schizophrenia phenotype, PRODH (Li et al. 2004 ) and GNB1L (Williams et al. 2008) , are overlapped by a number of CNVs, and these could potentially modify this phenotype in DiGeorge patients.
In polyploidy or sub-chromosomal duplications where abnormalities result from extra copies of the chromosome regions, deletion CNVs may ''normalise'' copy number. Alternatively, there is potential for even greater amplification of copy number where amplification CNVs are present. A simple duplication CNV on the non-disjoining chromosome may lead to the presence of up to six copies of a gene in a trisomic individual (Fig. 1 ) and the subsequent phenotypic effects associated with increased gene expression levels. Conversely, a gene deletion on the non-disjoining chromosome may alleviate the expected effect of trisomy by reducing the local copy number state to one or two copies. Thus, the compounded effect of aneuploidy and copy number variation has the potential to generate a much wider range of phenotypes than would be expected on the basis of chromosome number alone. It has been suggested, therefore, that CNVs are likely to act as 'modifiers of the phenotypic variability of trisomies' (Beckmann et al. 2007 ) and the same is probably also true for smaller chromosomal imbalances.
Using Down's Syndrome as an example, an extra copy of chromosome 21 is not sufficient to cause the full range of phenotypes associated with this disorder, as individuals can present with a range of sub-phenotypes. Some features are almost always present, such as the characteristic facial appearance and mental retardation, although these can vary widely in the severity of their presentation (Kallen et al. e illustrates how a deletion on parent B and MII NDJ in parent A, however, that leads to a total of two copies of the gene in the trisomic child which could potentially ameliorate the pathological effects of trisomy 1996). Other features, however, are only present in a fraction of DS cases: for example, congenital heart defects (CHD) are present in *40% of cases (Park et al. 1977 ) and gastrointestinal (GI) defects in *8% of DS (Epstein et al. 1991) . Leukaemia is also common in DS, with an *20% increased risk of acute lymphoblastic leukaemia (ALL) (Hasle et al. 2000) , and transient myeloproliferative disorder occurring in *10% of DS newborns, of which 10-20% develop acute megakaryoblastic leukaemia (AMKL) before the age of 4 (reviewed in Hasle 2001) . A detailed knowledge of the genes on the affected chromosome is required to understand the phenotypic effects of aneuploidy. In addition, it is important to determine the overall effects of gene dosage imbalance, which may also be complex. Individuals with partial trisomy 21 resulting from unbalanced chromosomal translocations will only exhibit those features associated with the extra genomic material present. Molecular analysis of these patients enables 'phenotypic mapping' by defining the critical genomic regions that harbour genes associated with various phenotypes (Epstein et al. 1991) . As with all regions of the genome, many common CNVs and rarer genomic structural variants are found on chromosome 21. Two recent reports describe structural variations in which the presence of increased copies of genomic regions on chromosome 21 contributes to human phenotype. In one study, duplication of a region at 21q21 that included the amyloid precursor protein (APP) gene was found to cause familial Alzheimer's disease in five separate families (Rovelet-Lecrux et al. 2006) . The duplication varied in size from 0.58 to 6.37 Mb and contained from 5 to 12 annotated genes including APP. Despite the largest of these duplications being over 6 Mb, none of the families exhibited any clinical evidence of DS (Cabrejo et al. 2006) . A second duplication of a 4.3 Mb region at 21q22.13-q22.2, containing just over 30 genes, did help refine the map of the DS critical region (DCR) as it caused a DS phenotype in three family members (Ronan et al. 2007 ). These individuals had the facial characteristics of DS and mild cognitive disability indicating that this region includes part of the DCR but not all of it. It is apparent, therefore, that different sub-phenotypes of trisomy 21 are caused by increased copy number at different regions across chromosome 21. It is also evident that some DS individuals who are trisomic for a particular critical region do not always exhibit the associated phenotype, or display a much milder form.
A complex interplay of molecular factors is likely to be responsible for the trisomic phenotypes, possibly involving non-chromosome 21 genes. Furthermore, interaction of trisomy with the presence of certain embedded deleterious alleles or haplotypes may contribute to the variable presentation of the different phenotypes (including degree of mental retardation, congenital heart defects, Hirschprung and other gut diseases, and leukaemia). The increasing amount of copy number variation that has been discovered in the human genome, specifically on chromosome 21, adds another dimension to the molecular consequences of trisomy: it is possible that the phenotypic variability seen in DS may be due to CNVs on this chromosome.
On the DGV, there are 507 reported CNV calls covering 35.0% of chromosome 21, which is similar to the CNV coverage of other chromosomes (33.9%) (DGV March 2010). Many of these are common in the population. For example, our investigation of only 50 healthy French male samples revealed 20 multi-probe CNVs, and an additional 57 single probe CNV signals, on chromosome 21 (de Smith et al. 2007 ): almost half were detected in multiple samples (47%), and 34% had a frequency of [5% (Fig. 2 ). In addition, these CNVs overlapped 38 known genes. There were several CNVs within TIAM1 (T-lymphoma invasion and metastasis-inducing protein 1), for example, which is expressed in almost all analysed tumour cell lines, including B-and T-lymphomas, melanomas and carcinomas (Habets et al. 1995) . A CNV was also found within DSCAM¸which is a good candidate gene for the CHD phenotype seen in DS, as it lies within the predicted minimum critical region and is expressed in the heart during cardiac development (Korbel et al. 2009 ). Several other CNVs have since been discovered in this gene, as listed on the DGV, including a common deletion that incorporates 4 exons (Matsuzaki et al. 2009 ).
Other genes within the CHD critical region also overlap CNVs: a common intronic deletion (10%) was identified in C2CD2 (Conrad et al. 2010) ; 6 CNVs have been reported within PRDM15, including one deletion overlapping 5 exons that was found in 3/90 Yoruban individuals (Matsuzaki et al. 2009 ); and one deletion was identified in BACE2 overlapping the last exon of this gene (Mills et al. 2006) .
CNVs also overlap RUNX1, with one deletion removing the promoter region and first exon of this gene (Gusev et al. 2009 ). RUNX1 codes for a transcription factor involved in haematopoiesis (North et al. 2002) and is associated with AMKL, which is estimated to be 500-fold more frequent in children with DS than in the general population (reviewed in Zipursky et al. 1992) : CNVs in this gene may, therefore, protect against or increase susceptibility to leukaemia in DS. These CNV loci and other regions that could potentially impact variable phenotypes of trisomy 21 are listed in Table 3 .
Analysis of copy number variation on chromosome 21 may, therefore, lead to a better understanding of the etiology of DS sub-phenotypes and could possibly shed light on the origin of these conditions in the wider non-trisomic population. Knowledge of which genes are involved in the generation of CHD in DS, for example, and how they interact with other gene products, could have many implications for managing CHD in the non-trisomic population: for example, adult onset CHD may be prevented by therapeutic agents that target critical pathways controlling cardiogenesis, and new approaches may be developed for stem-cell guided cardiac repair (reviewed in Passier et al. 2008) .
Since the discovery of trisomy 21, it has been hypothesised that genes present in three copies are over-expressed Shaikh et al. (2009) by 1.5-fold relative to the euploid state, but this has subsequently been shown to not always be the case. Some genes are over expressed, some are expressed at euploid levels while other genes appear to be down-regulated (Li et al. 2006; Prandini et al. 2007 ). This may, of course, reflect adaptive regulatory control, but may also reflect the effects of local copy number variation on chromosome 21, which will result in deviations from the predicted three copies of each gene, as shown in Fig. 1 .
Copy number variation may, therefore, make a significant contribution to phenotypic variation in aneuploidy and other chromosomal abnormalities. Targeted analysis of such variation might, therefore, represent an effective strategy for refining critical regions and pinpointing genes central to key pathways (i.e. of cardiogenesis or oncogenesis). This would also help to enable the prediction of which individuals may develop serious phenotypes, such as CHD in DS, which may add an extra dimension to antenatal screening programmes in the future by contributing to the process of 'informed choice' by the potential parents of a DS child, as well as well as aiding the long term management of these patients. Additional benefits may also accrue from increased understanding of pathogenic mechanisms relevant to patients with similar phenotypes outside the context of chromosomal imbalance. In summary, recent technological advances, such as high-resolution array CGH and second-generation sequencing, have vastly and rapidly increased our knowledge of copy number variation in the human genome. As these techniques continue to improve, and greater numbers of samples and populations are investigated, so our understanding of how copy number correlates with phenotype will grow, yielding information not just relevant to individuals with chromosomal imbalances, but to similar conditions in the wider population. Hepatitis Associated Aplastic Anemia: A review Hepatitis-associated aplastic anemia (HAAA) is an uncommon but distinct variant of aplastic anemia in which pancytopenia appears two to three months after an acute attack of hepatitis. HAAA occurs most frequently in young male children and is lethal if leave untreated. The etiology of this syndrome is proposed to be attributed to various hepatitis and non hepatitis viruses. Several hepatitis viruses such as HAV, HBV, HCV, HDV, HEV and HGV have been associated with this set of symptoms. Viruses other than the hepatitis viruses such as parvovirus B19, Cytomegalovirus, Epstein bar virus, Transfusion Transmitted virus (TTV) and non-A-E hepatitis virus (unknown viruses) has also been documented to develop the syndrome. Considerable evidences including the clinical features, severe imbalance of the T cell immune system and effective response to immunosuppressive therapy strongly present HAAA as an immune mediated mechanism. However, no association of HAAA has been found with blood transfusions, drugs and toxins. Besides hepatitis and non hepatitis viruses and immunopathogenesis phenomenon as causative agents of the disorder, telomerase mutation, a genetic factor has also been predisposed for the development of aplastic anemia. Diagnosis includes clinical manifestations, blood profiling, viral serological markers testing, immune functioning and bone marrow hypocellularity examination. Patients presenting the features of HAAA have been mostly treated with bone marrow or hematopoietic cell transplantation from HLA matched donor, and if not available then by immunosuppressive therapy. New therapeutic approaches involve the administration of steroids especially the glucocorticoids to augment the immunosuppressive therapy response. Pancytopenia following an episode of acute hepatitis response better to hematopoietic cell transplantation than immunosuppressive therapy. Aplastic anemia, acquire or congenital anemia associated with hypoplastic "fatty or empty" bone marrow and global dyshematopoiesis, has been first described by Paul Ehrlich in year 1888 [1] . The pathophysiology is believed to be idiopathic [2] or immune-mediated phenomenon with active destruction of haematopoietic stem cells [3] . The abnormal immune response may be elicited by environmental exposures, such as to chemicals, drugs, viral infections and endogenous antigens generated by genetically altered bone marrow cells [4] . A small fraction of the genes involved in pancytopenia has been represented by the congenital BM failure syndromes (relatively rare) which lately develop in clinical syndromes as Fanconi's anemia, Dyskeratosis Congenita, and Shwachman-Diamond syndrome [5, 6] .
Hepatitis-associated aplastic anemia (HAAA) is a well recognized and distinct variant of clinical syndrome, acquired aplastic anemia, in which an acute attack of hepatitis leads to the marrow failure and pancytopenia [7] [8] [9] . HAAA has been first reported in two cases by Lorenz and Quaiser in 1955 [8] and the number of the cases increase up to value of 200 by the year 1975 [10] [11] [12] . However, this syndrome has been reported in 2-5% cases of west and 4-10% in area of more prevalent to hepatitis and Human Immunodeficiency Viruses (HIV) in the Far East [12, 13] , it belongs to the area of low socioeconomic status [14, 15] . HAAA is not considered relative to age, sex and severity of hepatitis [14] , predominantly it has been found in children [13] , adolescent boys and in young aged men [10, 16] .
The onset of syndrome, pancytopenia, usually takes two to three months [62 days: ranging from 14 to 225) after attack of acute hepatitis [12, 14] . Hepatitis associated with aplastic anemia may be acute and chronic [7] , mild and transient [16] , self-limiting and fulminant and the development of AA is always fatal if not treated on time [7, 10] . Majority of the cases have been found as fulminant where the mortality rate reaches up to 85% [17] . Aetiology of the syndrome has been attributed to various agents and factors [7] which may include pathogenic viruses, autoimmune responses, liver transplantation procedure [18] bone marrow transplantation, radiation [19] and drugs administered to control the viral replication [14] .
Several hepatitis viruses such as Hepatitis A [20] , B [21, 22] , C [23] , and E, G [24] have been anticipated to be associated with this set of symptoms [7] . No association has been found with blood transfusion, toxins and drugs [17] . In one study Safadi and co-workers (2001) found out that sera of eight of the patients had the existence of Hepatitis Bc IgG and/or anti-HBs antibodies which was suggested due to past exposure and immunizing effect and they were unable to establish any direct relation with acute hepatitis B [14] . As non-A, non-B hepatitis agents are usually responsible for the hepatitis associated aplastic anemia; the prevalence of anti-hepatitis C virus antibodies is similar in HAAA and aplasia of other origins [25] . HCV seropositivity has been observed in the patients developing cytopenia following non-A non-B hepatitis (NANBH). HCV viremia has been frequently observed without detecting anti-HCV antibodies in patients' blood reflecting the transfusion associated HCV infection [25, 26] . However, it has also been reported that HCV is not generally implicated as a causative agent of hepatitis preceding aplastic anemia [27] .
Hepatitis G virus (HGV) has been reported as a possible etiological agent of acute hepatitis, chronic liver dysfuntioning, and fulminant hepatitis and hepatitis associated aplastic anemia. A relation of Hepatitis G virus with hepatitis subsequently developing in the aplastic anemia has been seen in a 24 years old person by measuring the hematological, biochemical, serological and virological parameters and detecting HGV RNA in his serum by PCR and electro-immunoassay [24] .
The development of aplastic anemia generally found to occur in hepatitis not caused by the hepatitis A and hepatitis B viruses commonly known as the non-A and non-B hepatitis associated aplastic anemia which were reported in above than 80% of the cases of hepatitis preceding severe cytopenia [28, 29] .
Viruses other than the hepatitis viruses have also been implicated as a causative agent of AA [1, 5] which include parvovirus B19 [19, 30, 31] , Cytomegalovirus, Epstein bar virus [19, 31, 32] , Echovirus 3 [33] , GB virus-C [34] , Transfusion Transmitted virus (TTV) [35] , SEN virus and non-A-E hepatitis virus (unknown viruses) [7] .
Parvovirus B19, an under recognized hepatotrophic virus, is documented as an offending agent of HAAA. Its infection cause hepatic manifestation ranging from abnormal liver functioning to Fulminant Hepatic failure and aplastic anemia [30] . The primary site of infection of this virus is erythroid progenitor cell in which it halts the erythropoises and leads to the anemia in immunocompromised hosts. The DNA of this virus has been detected in liver of fulminant hepatic failure manifested with bone marrow aplapsia and in the serum of fulminant hepatitis children of unknown origin [36, 37] .
Association of Torque Teno virus, single stranded circular DNA has liver as a susceptible host as well as various other tissues including bone marrow. It has been firstly reported in 12 years old Japanese boy suffered from cytopenia following acute hepatitis by detecting Torque Teno virus DNA of genotype 1a and IgM antibodies against this virus in peripheral blood and bone marrow mononuclear cells which precludes that acute bone marrow failure majorly concerns with infection of TTV virus to haemopoietic progenitor cells. However, other studies have also been done on assessing the TTV as an etiological agent of HAAA [33, 38] .
In a study a 6-year-old boy was experienced aplastic anemia two months after the onset of acute hepatitis associated with echovirus-3 [33] . As idiopathic aplastic anemia is associated with the increased level of secretion of INF-γ and TNF-α from T cells which inhibit the hematopoietic cell proliferation [39] , similar increased level of serum INF-γ and TNF-α was found in the patient four weeks after the onset of aplastic anemia [33] . Similarly varying degrees of cytopenia has been related to the HIV infection and severe aplastic anemic conditions develop after subsequent attack of HSV-6 [17, 40] .
Epstein Bar virus infection, involved in hepatitis, manifests the pathogenesis of marrow aplasia [40] . The pathogenic mechanism involve in the EBV infection is direct cytotoxity or mediates the immune response of host [7, 17, 14, 41] .
Aplastic anemia can be acquired or congenital [1, 42] . As the aplastic anemia following the hepatitis has been elucidated as a severe bone marrow failure with an episode of acute hepatitis, following lymphocyte variations occur during the course of the syndrome: activation of circulating cytotoxic T cells increase, tend to accumulate in the liver, broad skewing patter of T cell reportrie in peripheral blood of the patient forms, a large number of T cell infiltration from liver parenchyma occurs [13, 26, 43, 44] defective monocyte to macrophage differentiation [45] and decreased circulating level of interleukin-1 occur [46] .
Various Immunological abnormalities have been accountable for the development of aplastic anemia following hepatitis. The immunological abnormalities with HAAA show that CD8+ kupffer cells detecting by liver biopsies appear as a mediator of this syndrome. In a study it has been reported that patient showed a decreased ratio of CD4/CD8 cells and a high percentage of CD8 cells which can be cytotoxic and myleopoietic during the in vitro study of aplastic anemia [10, 20] . The residing of CD8 cells in bone marrow during HAAA produces a high level of interferon gamma (INF-γ) and cells derived from bone marrow locating in liver may activate these cytotoxic T cells causing their intrahepatic accumulation strongly affected by the Tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) causing the onlooker damage to liver cell of genetically modified mouse model. However, it has also been shown in several studies that increased level of soluble IL-2 receptor forms the major reason of non specific inflammation of HAAA [47, 48] . The pathogenesis of HAAA in children has been suggested to relate with the interruption in balance of lymphocyte sub-populations and T lymphocyte activation [49] .
Being an acquired disease, severe HAAA has also been presented as a Familial Bone Marrow Failure Syndrome (FBMFS) in a study while finding the family donor of HSCs transplant for treatment of idiopathic fulminant liver failure patient who has developed myelodysplastic syndrome after onset of severe aplastic anemia. The donor sibling also found to be developed the acute lymphoblastic leukemia after diagnosing the hypocelluarity of bone marrow. The event of finding these two familial cases shows the bone marrow failure syndrome to be inherited [50] .
HAAA has not been clarified with any genetic tendency [18] . Mutation in genes of the telomere repair complex, TERC (the gene for the RNA component of telomerase) and TERT (the gene for the telomerase reverse transcriptase catalytic enzyme), reduce the marrow regenerative capacity, making genes mutation carriers susceptible to the development of aplastic anemia once it has been started [42] .
Most of the clinical features relating to aplastic anemia following the hepatitis include: Pallor and multiple skin bleeding [11] , lymphocytopenia, hypogammaglobulin [51] low number of CD8/T cell ratio [12] and increased number of cytotoxic cells [48] Neutropenia, fever [17] .
Bacterial and fungal infection may emerge as secondary in presenting the disease [2] . Later complications may develop especially involving myelodysplasia [4] . The victims of severe aplastic anemia following the hepatitis experience a severe immune deficiency that might be either due to the hepatitis or aplastic anemia that is yet to be discovered [51] .
On a course of HAAA, hepatitis can be detected on some of the following parameters: subsequent increase in serum Alanine Trasnaminase (ALT), Aspartate Transaminase (AST), by at least three times above the normal values which are 6 to 41U/l, 9-34U/l, 5-58U/L for ALT and AST respectively [12, 18, 30, 35, 36, 43, 52, 53] , increase in serum Alkaline phosphatase (ALP), gamma glutaryl transferase (GGT) and billirubin (39-117U/l, 5-58 U/l, and 2-7 micromol/L, respectively). Peripheral blood count can be determined by Flow cytometry analysis with directly conjugated monoclonal antibodies for CD2, CD3, CD4, CD8, CD19 and HLA-DR, whereas haematopoietic failure with bone marrow hypocellularity can be elucidated in terms of absolute neutrophil count (less than 500 per mm 3 ), Platelet count (less than 20,000 per mm3), Reticulocyte count (less than 60,000 per mm 3 ) [53] and Protrombin Index (%): normal value 70-100% [24] . To establish the onset of pancytopenia following hepatitis, hypocelluarity of bone marrow below 50% might be obtained by bone marrow aspiration [14] and trephine biopsy [11] .
Various virological and serological markers are available for the detection of hepatitis A, B, C, D, E, G, TTV and parvovirus. Among these tests, anti-HAV Ig total antibodies, HBsAg, HB core antigen, HBsIgG antibodies, various HCV recombinant antigens and hepatitis E virus IgM and IgG are being in use. However, to determine causative nature of all hepatitis viruses, RNA genome of RNA containing viruses such as HCV, HDV, HEV and HGV can be qualitatively detected by RT-PCR reaction and DNA of parvovirus B19 and TTV can be detected by Nested PCR [14, 53] . IgG antibody for Cytomegalovirus, EBV and parvovirus has been found a useful tool for the diagnostic purposes [18] . However, serological and virological parameters for hepatitis A, B, and C were found negative in majority of the HAAA cases reported in several studies [17, 23, 27] .
The standard therapy which is employed for the treatment of HAAA is allogenic bone marrow (BM) transplantation treatment from HLA matched siblings [13, 54] . HAAA is mostly occurring in children and it would be easier to find the HLA matched donor. As HAAA shows poor prognosis, most often it has been treated by hematopoietic stem cell transplantation [26, 55] . Immunosuppressive therapy has proved effective after BM transplantation. Various immunosuppressive drugs named Antithymocyte Globulin (ATG) and Cyclosporine have been administered without eliciting any acute side effects. Steroids such as Glucocorticoids have also been employed in combination with immunosuppressive medications for the treatment of the HAA patients [26] . A durable remission from HAA has been achieved by the administrating high dose of Cyclophosphamide (CY), a highly immunosuppressive, which elicits its effect by readily destroying the lymphocytes and committed myeloid cells. The haemopoietic stem cells are not susceptible to the toxic effects of the CY due to releasing the aldehyde dehydrogenase enzyme which inactivates the drug. However, restoration of haematopoesis process may achieved by high dose of CY which mediates autoimmune attack on haematopoiectic stem cells HSCs [13] .
Patients irresponsive to the IST are prone to be cured by unrelated donor bone marrow transplantation [18] . Immunosuppressive therapy might have proved as a safe and alternative treatment for HAAA after of bone marrow or haemotopoietic stem cell transplantation [55] . Several studies showed that Parvovius induced aplastic anemia improves by the administration of retroviral therapy [21, 56] . Antiviral therapy for treating hepatitis B associated HAAA has been unknown yet; however it has been tested by administrating the nucleoside analogs, lamiviudine, against the aplastic anemic secondary to hepatitis B virus infection and remission occurs from the severe aplastic anemia accompanied with the hepatitis B viral infection. Interferon, an effective therapy for the hepatitis B and C viral infections, cannot be employed as a potential approach for the HAAA because of its mylosuppressive effects [21] . Acyclovir has been used for the treatment of aplastic anemia caused by the Epstein Bar virus [32] . The blood count comes to the normal range after five years of treatment [55] , however, chances of recovery from hepatitis associated acquired aplastic anemia is rare [57] .
Growth factors deficiencies have been found to be responsible for the majority of the aplastic anemic cases [1] . As these growth factors released by stromal cells are essential for the survival, proliferation and differentiation of hematopoietic stem cells [58, 59] , a transient increase in granulocytes has been found effective in most aplastic anaemic trials by administrating the erythropoietin, growth factors, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 [1] and androgens [5] . The limiting factors in success of immunosuppressive therapy are found to be the extent to which organ has been destructed, tissue regeneration capacity and most importantly pharmacology effect of drugs that is not sufficient for uncontrolled potent immune response [1] .
The survival of the patients treated with hematopoietic cell transplantation and response rate to immunosuppressive therapy found to be 85% and 70% respectively [7] . Children response better than the adults to bone marrow transplantation and survival rate with Bone marrow transplantation from HLA matched donors is found to be similar as that for the non hepatitis associated aplastic anemia [14] .
It has also been reported that parameters of liver dysfunctioning tend to improve when pancytopenia starts presenting itself [18, 28] . Although majority of the patients survives after aplastic anaemia tends to have complete recovery, the mortality rate is yet very high [52] . The mean survival rate after developing the severe bone marrow aplasia has been 2 months and fatality rate ranges from 78-88% [28, 60, 61] .
HAAA is a well documented and diverse variant of clinical syndrome of aplastic anemia, in which an acute attack of hepatitis leads to the marrow failure and pancytopenia that may be acute or chronic. This disorder has been reported in 2-5% cases in West, 4-10% in Far East and high in area of low socioeconomic status. HAAA is not related to age, sex and severity of hepatitis, predominantly it has been found in children, adolesecent boys and in young aged men. A number of hepatitis viruses manifest the disease symptoms. Amongst the hepatitis viruses, HBV, HCV and HGV seropositivity has been mostly commonly observed in reported cases of HAAA. The causative agent of HAAA can be detected using hematological, biochemical, immunological and virological markers. The clinical features relating to HAAA are pallor and multiple skin bleeding, lymphocytopenia, hypogammaglobulin, low number of CD8/T cell ratio and increased number of cytotoxic cells, neutropenia and fever etc. For HAAA, immunosuppressive therapy is more effective after BM transplantation.  Factors Affecting Intention to Receive and Self-Reported Receipt of 2009 Pandemic (H1N1) Vaccine in Hong Kong: A Longitudinal Study BACKGROUND: Vaccination was a core component for mitigating the 2009 influenza pandemic (pH1N1). However, a vaccination program's efficacy largely depends on population compliance. We examined general population decision-making for pH1N1 vaccination using a modified Theory of Planned Behaviour (TBP). METHODOLOGY: We conducted a longitudinal study, collecting data before and after the introduction of pH1N1 vaccine in Hong Kong. Structural equation modeling (SEM) tested if a modified TPB had explanatory utility for vaccine uptake among adults. PRINCIPAL FINDINGS: Among 896 subjects who completed both the baseline and the follow-up surveys, 7% (67/896) reported being “likely/very likely/certain” to be vaccinated (intent) but two months later only 0.8% (7/896) reported having received pH1N1 vaccination. Perception of low risk from pH1N1 (60%) and concerns regarding adverse effects of the vaccine (37%) were primary justifications for avoiding pH1N1 vaccination. Greater perceived vaccine benefits (β = 0.15), less concerns regarding vaccine side-effects (β = −0.20), greater adherence to social norms of vaccination (β = 0.39), anticipated higher regret if not vaccinated (β = 0.47), perceived higher self-efficacy for vaccination (β = 0.12) and history of seasonal influenza vaccination (β = 0.12) were associated with higher intention to receive the pH1N1 vaccine, which in turn predicted self-reported vaccination uptake (β = 0.30). Social norm (β = 0.70), anticipated regret (β = 0.19) and vaccination intention (β = 0.31) were positively associated with, and accounted for 70% of variance in vaccination planning, which, in turn subsequently predicted self-reported vaccination uptake (β = 0.36) accounting for 36% of variance in reported vaccination behaviour. CONCLUSIONS/SIGNIFICANCE: Perceived low risk from pH1N1 and perceived high risk from pH1N1 vaccine inhibited pH1N1 vaccine uptake. Both the TPB and the additional components contributed to intended vaccination uptake but social norms and anticipated regret predominantly associated with vaccination intention and planning. Vaccination planning is a more significant proximal determinant of uptake of pH1N1 vaccine than is intention. Intention alone is an unreliable predictor of future vaccine uptake. Influenza contributes significantly to worldwide morbidity and mortality [1] . Periodically, influenza viruses mutate into antigenically-different strains leading to global pandemics [2] . The 2009 influenza pandemic (pH1N1) was caused by a triple reassortment of human, swine and avian influenza viruses [3] . Vaccination is the most effective intervention for preventing influenza [4] and a core part of national pandemic plans for pandemic mitigation. Lead times of at least 6 months in producing a vaccine against a novel strain means that while vaccines may be unavailable in time to prevent the first wave of a pandemic [5, 6] , effective public uptake of a vaccine may mitigate subsequent waves [7] .
Significant health promotion activities regarding influenza prevention have been prominent in Hong Kong since well before the onset of pH1N1, arising largely from the Severe Acute Respiratory Infection (SARS) epidemic and A/H5N1 Bird Flu outbreaks. Seasonal influenza vaccination is widely promoted each year. Hong Kong's pH1N1 epidemic started on 11 June 2009, peaking in September, and by early November had petered out ( Figure 1 ). By the end of December 2009, the Hong Kong government had recorded 37,174 human pH1N1 cases [8] in a population of ,7 million. To minimize any potential second wave, significant televised and other publicity was given to the launch of a pH1N1 vaccination programme on 21 December 2009, initially for five priority groups: healthcare workers, persons with chronic illness and pregnant women, children aged 6 months to 6 years, adults aged 65 years or above, and pig farmers and slaughtering industry personnel [9] . On 26 January 2010 pH1N1 vaccination was extended to the general public. The vaccination was free for priority group members [10] , but cost HK$100-150 (US$13-20, 1-1.5% of Hong Kong's median monthly income of HK$10,000/ US$1,286/J991) per dose for the general population. A study in July 2009 of 301 respondents projected that vaccine uptake would be influenced by end-user cost, with 45%, of Hong Kong's general population being ''highly likely'' to take up pH1N1 vaccine if free, and 24% if costing HK$100-200 (US$ [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [11, 12] .
From November 2009 onwards, television, radio, newspaper and official websites strongly encouraged priority groups to have pH1N1 vaccination [13] . However, the Hong Kong government did not make recommendations for the general population, who were asked to judge for themselves whether to be vaccinated or not. Shortly after the vaccine launch for priority groups, local media prominently attributed several adverse events to pH1N1 vaccination, including, a case of Guillain-Barre Syndrome (GBS) diagnosed a week after pH1N1 vaccination, reported on 6th January 2010, and an intrauterine death (IUD) 3 weeks following the mother's vaccination, reported on 20th January 2010 ( Figure 2 ). In both cases local health agencies presented convincing evidence challenging the link between vaccination and the two adverse events but were largely ignored. Retrospectively, a drop in pH1N1 vaccination uptake among priority groups was observed [14] . It seems probable that the adverse media reports had impeded vaccination uptake among general population. We collected baseline data between 12-25 January, 2010, immediately before pH1N1 vaccine was made available to the general population and then two months later (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) March 2010) we recorded their reported vaccination status ( Figure 2 ) with the intention of modelling how general population decision-making regarding pH1N1 vaccination might predict subsequent vaccine uptake.
Empirical studies have found that history of seasonal influenza vaccination [12, [15] [16] [17] [18] , perceived risk of pandemic influenza [17, [19] [20] [21] [22] [23] [24] [25] [26] , worry [17, 22, 26, 27] , and attitudes towards vaccine, such as vaccine efficacy and side-effects [12, 15, 20, [24] [25] [26] were significantly associated with intention to receive a vaccine against the influenza pandemic. This is consistent with the findings related to determinants of vaccination against seasonal influenza [28] [29] [30] [31] [32] . However, there are some common and significant limitations to these empirical studies. First, all except one [24] relied on vaccination intention to predict the actual vaccination uptake. In one study, since only a few respondents reported having received the pH1N1 vaccine, the authors combined those intending to get vaccinated with those who had already received the vaccine into one ''intending'' group and examined factors associated with this 'vaccination intention' [20] . This is problematic because factors associated with vaccination intention and actual vaccination receipt probably differ. Moreover, the reliability of intention as a predictor of actual behavior remains controversial. Harris et al. found that only about half of ''intending'' recipients of seasonal influenza vaccination actually take it and almost all those who do not intend to take it remained unvaccinated [33] . Moreover, most studies conducted before the pandemic occurred or before the vaccine was available [11, 12, [16] [17] [18] [19] 21, 23] of Dutch respondents reported intending to take pH1N1 vaccination prior to or at the onset of the (potential) pandemic phase, respectively [23] . Similarly, in Hong Kong 45% of 301 respondents in July 2009 reported being ''highly likely'' to receive pH1N1 vaccine if offered for free [11, 12] . However, by the time vaccination became available intention appeared much lower with only 10-15% of study respondents in France and in Turkey intending to take the pH1N1 vaccine [20, 24] . Second, all the studies are cross-sectional rather than longitudinal; none assessed subsequent actual vaccination status. Thus, although associations have been identified, there is no way to infer causality. Third, most of the studies are atheoretical. Although some of the studies developed their study questions based on theoretical framework such as HBM [17, 23, 24] , none have conducted model analysis and evaluated the model fit. Therefore, due to these three reasons, there remains a significant concern about how valid such results are and a significant knowledge gap about how the observed pattern of influences could be explained.
A major limitation of previous empirical studies [12, [15] [16] [17] [18] [19] [20] [21] [22] [23] 25, 26] is failure to accommodate the intention-behaviour gap. Although several behavioral theories such as Protection Motivation Theory (PMT) [34, 35] , Theory of Reasoned Action (TRA) [36, 37] and Theory of Planned Behaviour (TPB) [38, 39] propose that intention is the proximal determinant of behaviour, intention does not necessarily translate into actual behaviour. Empirical studies of the intention-behavior relationship showed that intention had a medium effect (a correlation of ,0.4-0.5) on behavior [40] [41] [42] , but a recent review including 47 experimental studies found that a medium-to-large change in intention induced by manipulated interventions caused only a small-to-medium change in behavior [43] , where an effect size of 0.5 is medium and one of 0.2 is small [44] . Sheeran found that about 47% of those intending to take action fail to act [42] , consistent with Harris et al's findings [33] . Factors that are prime contenders to moderate/ mediate the relationship between intention and behaviour include behavioural control/efficacy, action planning and anticipation of consequences [41] [42] [43] .
Perceived behavioural control/self-efficacy. The TPB is an extension of the TRA incorporating the concept of perceived behavioural control (PBC) as an intervening variable predicting both intention and also actual behavioural change directly [38, 39] . The direct effect of PBC on actual behavioural change partly explains why not all intention translates into behaviour. Previous reviews suggested that intention-behaviour relationships could be moderated by perceived behavioral control, with higher levels of perceived behavioural control improving prediction of intention on behaviour [42, 43] . Although some researchers suggested that PBC differs from self-efficacy because selfefficacy emphasized perceived internal control more while PBC also considers external control factors [45] , a systemic review on the efficacy of TBP found that PBC and self-efficacy had comparable effects on intention and behaviour [41] . Despite being a dominant theory of behavioural change, because the TPB is limited in predicting behaviour we sought to enhance its predictive power by replacing PBC with self-efficacy and incorporating enhanced social effects to accommodate external control factors.
Implementation of intention/planning. Implementation of intention, termed ''planning'', is a potentially important factor facilitating translation of intention into behaviour [42, 43, 46, 47] . Planning is specific to situations (e.g., when, where, and how) within which one will perform the behaviour [46] . It activates the situational context for goal attainment and thereby makes the goal become more accessible [46, 47] . A meta-analytic review showed that implementation of intention as planning consistently caused a medium-to-large effect on behavioural change [47] .
Anticipated regret. Anticipated regret is the expectation of feeling regret or upset if one does or does not conduct certain behaviours. Anticipated regret has been found to be a strong predictor of vaccine uptake against seasonal influenza [31, 32] , playing the lottery [48] and exercise [49] . Anticipated regret might also moderate the intention-behavior relationship: the higher anticipated regret for inaction, the better the prediction of intention on behaviour [48, 49] .
A robust theoretical framework comprehensively explaining behavior change that elucidates population decision-making for health protective and promoting behaviour has long been sought. As the main contender, the TPB explains ,34% of variance in health behavioural change related to addictive behaviour, automobile-related behaviours, clinical and screening behaviour, eating behaviour, exercising behaivour, HIV/AIDS-related behaviour and oral hygiene behaivour [40] . The standard version of TPB proposes that attitudes towards the behaviour, subjective norm and PBC predict behavioral intention while intention and PBC predict the actual behavioural change [38, 39] . Additional predictors that significantly improve the model's predictive power are needed [39] . Two previous studies have examined modified versions of TPB to predict vaccination uptake against seasonal influenza [50, 51] . One study used TPB plus two additional factors: influenza vaccination history and anticipated regret, to predict intention to receive vaccine against seasonal influenza among elderly from social clubs [50] : vaccination history and anticipated regret respectively accounted for an additional 10.7% and 13.7% of total variance in influenza vaccination intention [50] . However, again the study was cross-sectional and actual vaccination uptake was not assessed. A second study of healthcare workers [51] adopted an extended version of TPB that included additional elements of anticipated regret, moral norm, descriptive norm and professional norm. The study found that controlling for the original TPB variables, moral norm and anticipated regret were significant determinants of actual receipt of seasonal influenza vaccine [51] . The study provides useful information for future application of the extended version of TPB. However, since the study was conducted among healthcare workers, some of the variables such as moral norm and professional norm which emphasize obligation and professional convictions may not be applicable among the general population.
Factors influencing pH1N1 vaccine uptake at the later stage of a pandemic might be more cognitively driven unlike behavioral responses during the early stage of a pandemic which might be more affect driven [52] . Therefore, taking into account prior work on seasonal influenza vaccination uptake [50, 51] , extending the TPB could provide theoretical utility for understanding public decision on taking pH1N1 vaccination. Starting with TPB and existing literature, we therefore built a conceptual model of public decision-making for pH1N1 vaccination ( Figure 3 ). In addition to the original TPB components, seasonal influenza vaccination history, anticipated regret and vaccination planning were included in the model. The model proposed that attitudes towards vaccination (perceived benefits of pH1N1 vaccination and concerns regarding possible adverse effects of pH1N1 vaccination), perceived social pressures from significant others and other people around regarding pH1N1 vaccination (social norms regarding pH1N1 vaccination), perceived self-efficacy in taking vaccination (perceived self-efficacy), anticipated regret for not taking the pH1N1 vaccination (anticipated regret) and seasonal influenza vaccination history would predict vaccination intention, which in turn predicts vaccination planning and future vaccination uptake; anticipated regret and perceived self-efficacy could also predict vaccination status directly; finally, vaccination planning was proposed to bridge the intention-behavior gap and predict vaccination status directly ( Figure 3 ).
We conducted a longitudinal study of influences on pH1N1 vaccination behaviour in Hong Kong to test this model ( Figure 3) , and subsequently followed up participants to record their selfreported receipt of pH1N1 vaccine. In this study, we aimed to answer the following research questions: How well does intention predict future uptake of pH1N1 vaccine? Does vaccination planning mediate the relation between intention and future vaccination uptake? And do the original TPB components and the additional components (extended social norms, anticipated regret and seasonal influenza vaccination history) contribute to peoples' decisions on vaccination uptake? 
The study obtained ethics approval from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Written informed consent was waived by the IRB because all the data were analyzed anonymously, but verbal consent was obtained from all the subjects before the interview started.
Hong Kong has 99% landline telephone penetration, local calls are free and telephone interviews are common and representative methods of survey data collection [53] . We conducted 13 main cross-sectional telephone surveys of psychological and behavioural responses to the first wave of the 2009 influenza A/H1N1 pandemic in Hong Kong from April through November 2009 (the parent study) [53] in order to monitor these variables. As an extension, the present study re-contacted subjects from some of these surveys and sought to understand public decision-making regarding pH1N1 vaccine uptake for mitigating the potential second wave of the pandemic. Between 12-25 January, 2010 a baseline assessment for the present study was performed, immediately prior the local pH1N1 vaccination campaign extending to the general community (vaccination for high risk groups started from December 21, 2009), and we again contacted participants for follow-up two months later, between 15-30 March 2010.
Sample size determination. We estimated that a sample of at least 500 was required to achieve 80% power at an a = 0.05 to reject a model of the specified complexity ( Figure 3 ) if the model fit index Root Mean Square Error of Approximation (RMSEA) exceeded 0.08 [54, 55] . To allow for a response rate ,60% in the follow-up and the baseline surveys, we need to target at least 1,389 subjects in the baseline survey.
Subject selection and inclusion criteria. A flow chart showing subject selection is provided in Figure 4 . A total of 12,965 subjects participated in the parent study [53] . All these subjects were Cantonese-speaking adults (aged$18) selected within households using a Kish Grid methodology, who were capable of and willing to answer a telephone interview. Additional details about inclusion criteria are available elsewhere [53] . Respondents in the 7 th , 9-12 th surveys of the 13 surveys comprising the parent study who, in the parent study agreed to be re-contacted and who had not received pH1N1 vaccine were invited to complete the baseline assessment for the present study. These five surveys (the 7 th , 9-12 th surveys) were selected because participants in these surveys had not had any follow-up contact either in the parent study or otherwise. This minimizes interview fatigue thereby improving response rates. These surveys were all of a comparable sample size, between 1,000-1,007 [53] . The five selected surveys were conducted between 21 July and October 23, 2009, and generated a representative [53] pool of 5,014 respondents of whom 61.4% (3,079/5,014) gave consent for further contact. From a list of the 3,079 subjects who agreed to be re-contacted, 1,648 calls were randomly selected and successfully made by a university telephone polling organization. Unanswered calls were tried at least four times at different hours and weekdays before being replaced by new numbers. Finally, a total of 1,511 (92%, 1,511/1,648) respondents agreed to participate in the baseline survey. Of these 78 (5%, 78/1,511) reported already having received pH1N1 vaccination and were therefore excluded as ineligible, leaving 1,433 respondents who completed baseline interviews.
The interview questionnaire for the baseline survey was derived from literature review, our previous cross-sectional surveys [53] and the theoretical framework constructed for this study (Figure 3 ). Specialists in health psychology, statistics, infectious disease and public health jointly determined the measures comprising the final questionnaire, guided by the need to maintain low assessment load and parsimony to ensure good response rates. The finalized questionnaire consisted of five sections: Section 1 addressed respondents' self-rated health and their experience of influenzalike illness in the past six months; Section 2 addressed risk perceptions regarding pH1N1; Section 3 addressed perceived trust in information related to pH1N1 and pH1N1 vaccination from different information sources; Section 4 addressed attitudes, beliefs and social norms regarding pH1N1 vaccine/vaccination, vaccination intention and planning; Section 5 addressed key respondent demographics. Overall, the baseline assessment consisted of 44 questions, which took less than 15 minutes to complete. Other demographic data were obtained from the parent study [53] . Prior to baseline assessment for the present study, subjects were reminded of their prior participation and that they had agreed to participate in a further study. The study was introduced as a survey of attitudes towards swine flu vaccination. We sought their willingness to participate. Those agreeing were asked about their vaccination status. Subjects who reported that they had already received pH1N1 vaccination were excluded. The remaining interview was performed. A follow-up survey was conducted 2 months later wherein respondents were reminded of the earlier survey and asked about their vaccination status and reasons for having had or not having vaccination. All the data were collected through telephone interview in both Baseline and Follow-up surveys.
The measures comprising the study instruments were used to build the conceptual model ( Figure 3 ) and are described below and in Table 1 .
Perceived benefits of pH1N1 vaccination, and, Concerns regarding adverse effects of pH1N1 vaccination. These two constructs assessed attitudes towards pH1N1 vaccination. Perceived benefits of pH1N1 vaccination was assessed by measuring agreement on five-point ordinal scales (from 1 ''strongly disagree'' to 5 ''strongly agree'') with three statements (Table 1) . A Cronbach's alpha (a) of 0.71 indicated an acceptable internal consistency for this scale and these two items were treated as the indicators of a latent scale (Perceived benefits of pH1N1 vaccination). Concerns regarding adverse effects of pH1N1 vaccination were assessed by measuring agreement, using fivepoint scales, with two statements. The Cronbach's a for these two items was 0.64, considered acceptable by some researchers [56] , though clearly less than desirable. We therefore treated the items as reflecting a latent variable (Concerns regarding adverse effects of pH1N1 vaccination). Social norms regarding pH1N1 vaccination. While TBP considers the influence of solely coercive social pressure from significant others to perform a behaviour, previous studies suggest that it is also important to consider the generalized tendency to adopt behaviours demonstrated by others encountered in daily life for imitative reasons [48, 57] . We use the term Social norms rather than subjective norm to represent these broader coercive and imitative social influences. Social norms were assessed by agreement on a 5-point scale with two statements. The internal consistency for these two items was weaker, with a = 0.53, which suggests each item appropriately measures different social influences. We initially incorporated these items separately in the structural equation model but except for the path weights dividing almost equally between the two items, no difference was otherwise seen. We therefore retained them as indicators of a combined latent construct in the model for purposes of model parsimony [58] .
Anticipated regret. Anticipated regret was assessed with two statements asking about respondents' likelihood of feeling regret. Responses of these two items were on a 7-point categorical scale (from 1 ''definitely not'' to 7 ''certain''). The internal consistency a for these two items was 0.68. The two items were used to indicate the latent variable ''anticipated regret'' in the modeling analysis.
Perceived self-efficacy. One item was used to measure selfefficacy, asking about respondents' agreement on a 5-point scale with the statement ''I am confident that I can go independently to get human swine flu vaccination''. A standard scale of self-efficacy was not adopted to minimize assessment load. However, a single item for self-efficacy has been shown elsewhere to have validity in predicting behavioural change [59, 60] .
Seasonal influenza vaccination history. Respondents were asked whether they had received any seasonal influenza vaccination in the past three years (Yes/no/don't know).
Vaccination intention. Respondents were asked how likely it was that they would get vaccinated against pH1N1 during the winter flu season, using a 7-point Likert scale (from 1 ''definitely not'' to 7 ''certain'').
Vaccination planning. We measured vaccination planning by assessing respondents' agreement on a 5-point scale with three statement items, such as ''I have planned when and where to get my human swine flu vaccination this winter''. The internal consistency a for these three items was 0.59, though less than the most common acceptable level of above 0.7, remaining at the minimal acceptable level (a ranged between 0.5-0.6) of reliability for preliminary research [56] . These items were also treated as indicators of a latent variable for modeling purposes.
Reported vaccination uptake. In the follow-up survey, respondents were asked to confirm if they had received pH1N1 vaccine within the past three months. Respondents were also asked to indicate their major reasons for having or not having taken the pH1N1 vaccination using open-ended questions. Multiple reasons could be given by each respondent.
We first compared demographic differences between follow-up and lost-to-follow-up respondents with Pearson chi-square test while demographic differences of the respondents who completed both the baseline and follow-up survey and the general population [61] were assessed using Cohen's effect sizes [44] . Proportions were calculated to describe patterns of vaccination intention, reported vaccination uptake, and major reasons for taking or not taking pH1N1 vaccination. Structural equation modeling was then applied to examine the determinants of pH1N1 vaccination, vaccination intention and vaccination planning based on the extension of TBP. Mplus 6.0 for Windows (Muthén & Muthén, 1998 -2010 was employed because the model comprised dichotomous (vaccination status) and ordinal (vaccination intention) outcome variables. Before testing the full structural model, zeroorder correlations between the measures of related constructs were calculated. Confirmatory factor analysis was performed to assess the adequacy of the measurement model including perceived benefits of pH1N1 vaccination, concerns regarding adverse effects of pH1N1 vaccination, social norms regarding pH1N1 vaccination, anticipated regret and vaccination planning. To test the full structural model, all variables were entered into the model simultaneously. Mean and variance adjusted weighted least squares estimation was applied to evaluate the standardized parameters (beta, b). Since chi-square test is very sensitive to sample size and non-normally distributed data, several other model fit indices were evaluated including the Comparative Fit index (CFI), the Tucker Lewis index (TLI), and RMSEA. A CFI.0.90 and TLI.0.90 indicates a good fit. RMSEA less than 0.05 and one ranging between 0.05-0.08 respectively indicate a good and acceptable model fit [55] . Misfitting models were respecified guided by theoretical soundness and modification indices [55] . Missing proportions ranged from 0.1% for seasonal flu vaccination history to 5.5% for the item ''I have planned when and where to get my pH1N1 vaccination this winter''. There was no missing data for reported vaccination uptake. Missing data were handled with multiple imputation [62] .
Of the 1433 respondents who completed the baseline assessment, 896/1433 (63%) respondents agreed to participate and completed the March follow-up survey (Figure 4 ). Demographic characteristics of respondents in the baseline and followup surveys are shown in Table 2 . Compared to respondents completing both baseline and follow-up surveys, respondents lost to follow-up were younger (x 2 = 14.24, p = 0.001) and more likely to be single (x 2 = 20.26, p,0.001). Overall, the low Cohen effect sizes (,0.3) showed that the demographics of respondents who completed both the baseline and follow-up surveys were comparable to those of the general population of Hong Kong [61] .
Of the 1,433 respondents who completed the baseline survey, 36% (510/1,433) reported that they would ''definitely not'' take pH1N1 vaccination during the winter flu season; 36% (521/1,433) reported being ''very unlikely/unlikely'' to take it; 19% (278/ 1,433) reported their pH1N1 vaccination likelihood as ''evens'' (50:50/equal likelihood); and only 8% (119/1,433) reported vaccination likelihood as ''likely/very likely/certain''. Within the subset of 896/1,433 respondents who completed both baseline and follow-up surveys, 7% (67/896) had reported at baseline that they would be ''likely/very likely/certain'' to receive pH1N1 vaccination. However, in the follow-up survey, only 7/896 (0.8%) respondents reported having received pH1N1 vaccination in the intervening period, 4 of whom had reported being ''likely/very likely/certain'' to receive pH1N1 vaccination at baseline.
Reporting higher intention to receive pH1N1 vaccination in the baseline was associated with greater likelihood to vaccinate by follow-up (Fisher's exact test, x 2 = 24.24, p,0.001).
The 7 respondents who reported taking pH1N1 vaccination gave the major reasons for deciding on vaccination as follows: Three choose vaccination because of the 'high risk of swine influenza' characterized by statements like ''swine flu is serious'', ''I am worried that swine flu will become more serious'', and ''I feel vulnerable to swine flu''; two reported that their decision was due to 'doctors' advice' and two reported 'belief of the vaccine efficacy'. Other reasons provided by one respondent only were 'belief in the vaccine's safety', 'government recommendation', 'convenient availability', and 'protection of patients'.
Reasons for not having vaccination given by the 889 respondents who did not receive pH1N1 vaccination ( Figure 5) were, most frequently 'low risk of or from swine influenza' (529/889, 60%) and 'concerns regarding adverse effects of the vaccine' (328/889, 37%). Around 11% (100/889) of the respondents reported both 'low risk of/from swine influenza' and 'concerns regarding adverse effects of the vaccine'. Table 3 For the final full structural model (Figure 6 ), two additional paths were added and estimated based on the modification indices including a path from social norms to vaccination planning and path from anticipated regret to vaccination planning while the path from perceived self-efficacy to vaccination and the path from anticipated regret to vaccination were removed, coefficients for these two paths being nonsignificant and too small to be meaningful. The final model indicated a good fit with CFI = 0.96, TLI = 0.93 and RMSEA = 0.06 ( Figure 6) .
The model showed that respondents perceiving greater pH1N1vaccination benefits (b = 0.15), less concerns regarding vaccine adverse effects (b = 20.20), greater sensitivity to social norms b = 0.39), higher anticipated regret if not vaccinated (b = 0.47), higher perceived self-efficacy in taking pH1N1 vaccination (b = 0.12) and receiving seasonal influenza vaccination in the past three years (b = 0.12) reported greater intention to take pH1N1 vaccination, and accounted for 59% of variance in vaccination intention scores. Greater adherence to social norms (b = 0.70), higher vaccination intention (b = 0.31) and higher anticipated regret (b = 0.19) were associated with more vaccination planning, together accounting for 67% of variance in vaccination planning. Both vaccination intention (b = 0.30) and vaccination planning (b = 0.36) significantly predicted actual pH1N1 vaccination, accounting for 36% of variance in pH1N1 vaccination ( Figure 6 ). 
The World Health Organization recommended a stepwise use of pH1N1 vaccines for protecting people against the pH1N1 influenza pandemic in July 2009 [63] . However, a vaccination program's efficacy largely depends on the public's compliance. Our study found that only 5% of 1,511 subjects reported having received pH1N1vaccination and of 1,433 subjects remaining unvaccinated, only 8% reported intending (being likely/very likely/certain) to take the pH1N1 vaccine. Two months later in the follow-up survey, an even smaller proportion, 0.8% of the respondents who completed both the baseline and follow-up survey reported having been vaccinated against pH1N1. Perceived low risk of pH1N1 and concerns regarding vaccine-related adverse effects were the two most frequently cited reasons for refusing the vaccination. The extended TPB model suggests that both the original TPB components and the additional components contribute to people's decisions on vaccination uptake but that social norms and anticipated regret for not taking vaccination were the strongest determinants of vaccination intention and vaccination planning. Finally vaccination planning partially-mediated the relation between intention and reported vaccination uptake.
Compared to previous studies, vaccination intention was much lower in our study than that found in surveys conducted prior to the influenza pandemic [23] or before the vaccine was available [11] , but was comparable to the findings of surveys conducted in France [20] and Turkey [24] after pH1N1 vaccination programmes were launched there. An earlier Hong Kong study that relied on expressed intent to predict vaccination uptake [11] failed to accurately predict the subsequent meager population uptake of pH1N1 vaccination by, at best, an order of magnitude [64] , suggesting that intention alone is insufficient for predicting future vaccination uptake, consistent with empirical findings in other areas [33, 42] .
Despite predictions that intended pH1N1 vaccination uptake would decline if there was insufficient data on novel vaccine safety and efficacy [11] , safety issues were not the predominant barrier to vaccination in the present study. While 37% of our study respondents who remained unvaccinated cited vaccine safety concerns, despite good evidence that the vaccine is effective with a risk profile similar to that of seasonal influenza vaccine [65] , almost twice as many, 60%, cited 'low risk of/from swine influenza' as their reason for not getting vaccinated, suggesting that these respondents felt no advantage would be gained by vaccination. Around 11% of respondents, cited both 'low risk of/ from swine influenza' and 'concerns regarding adverse effects of vaccine' as the reasons for not getting vaccinated, seemingly adopting a risk-benefit approach to vaccination decision-making. However, in the setting of low influenza risk, with the reports of vaccine related adverse events in the media after the vaccine was available for the priority groups (Figure 2) , people may shift their perceived risks away from influenza and towards vaccination, suggestive of availability bias (risk distortion by easily recalled events) [66] . We believe that perceived vaccine risk would become progressively less of a barrier to vaccination as perceived influenza risk increases, and vice versa.
Moreover, despite recent reports that Hong Kong residents would be sensitive to vaccination pricing when considering whether to vaccinate [11, 12] , only 2.5% of our respondents cited high vaccine cost as the reason for rejecting vaccination.
Major reasons for taking pH1N1 vaccination corresponded to reasons for not taking it, with perception of pH1N1 risk most frequently cited. However, the few respondents receiving pH1N1 vaccination prohibited meaningful comparison.
The extended version of TPB model fits well to the survey data. The model showed that an expanded social norms and anticipated regret accounted for most of the variance in vaccination intention, rather than the more core elements of TPB. In turn, social norms independently accounted for more than twice the variance in vaccination planning than did intention, and vaccination planning accounted for more variance in vaccination uptake than did intention. Thus it seems that social norms comprise the major influences on vaccination uptake through modifying vaccination intention and planning. A meta-analytic review of TPB efficacy concluded that the TPB variable subjective norm (perceived coercive social pressure from significant others) weakly predicted intention compared to other TPB components, mainly due to poor measurement [41] . ''Descriptive norm'' (perception of what other people do, imitation or conformity behaviour) is reportedly a more important predictor for intention [48, 57] . Here we combined Table 3 . Correlations, means, standard deviations, and standardized factor loadings for the measurement model. measures of subjective and descriptive norms, treated as a latent variable (social norms), because they were found to have much the same predictive direction and weight. Multiple item measures of norms should have better predictive power than single item measures [41] . This model importantly informs public health approaches to population behaviour during respiratory epidemics. First, information uncertainty or untrustworthiness, for example regarding vaccine safety, is likely to prompt people look to others for their cues to action: the social environment, namely what other people believe and do powerfully influences decisions to action [45, 67] . People often tend to imitate others, so establishing a ''vaccination trend'' may help uptake. For example, it could be effective to encourage those who remain unvaccinated with feedback from vaccinated peers and by providing an updated total of numbers vaccinated. What the general public think and do may prove to be as influential as information from scientists or health professional [68, 69] . Second, encouraging uptake of a new vaccine will be problematic if the associated threat element is low, irrespective of vaccine pricing, particularly for novel and untested vaccines. Vaccine safety and efficacy data should be provided wherever possible at all levels including through health-care providers, media and the general public. To effectively communicate the risk and benefit of a novel vaccine, it is important to establish an effective surveillance system to monitor vaccination progammes and rapidly respond to any reported adverse events [70] . The media have an important influence and both reactionary and opinionated news items should be recognized as potentially detrimental to vaccination uptake. In particular, the need to develop stories that generate revenue increasingly overrides balanced reporting in contemporary media. Hence risk amplification remains a problem. Public health agencies need to improve their liaison with influential media outlets to minimize this, where possible. Third, omission bias, a phenomenon where people view vaccination as more risky than remaining unvaccinated, could be a barrier for vaccination [71] . Omission bias arises when there is anticipation of greater regret about adverse effects of vaccination, if taken, than the regret about being infected with influenza if vaccination is rejected [72] . Therefore, social marketing emphasizing the far greater likelihood of regret for consequences due to refusing vaccination than the regret over an improbably low adverse event due to taking vaccination may help to reduce this bias. For example, previous studies found that simply asking two questions about feeling regret for inaction could increase respondents' intention to play a lottery or do exercise [48, 49] . Finally, vaccination planning is a key intervening variable between vaccination intention and actual vaccination. This is to be expected given that it is more proximal to actual behaviour than intention is. In those who may be undecided, interventions facilitating planning may prompt action. This could include suggesting where, when and how to get vaccination, improving and publicizing accessibility of vaccination centres and opening times. Even so, intention and planning explained only 36% of the variance in the reported vaccination behaviour, suggesting that other factors, such as intention stability [42] , influencing vaccination behaviour await identification.
Study limitations include baseline attitudes/beliefs, vaccination intention and planning being measured at the same time point, prohibiting exploration of causality in observed associations. Some study measures were constrained due to length of telephone interviewing, and while sub-optimal were necessary methodological compromises. Although most researchers recommended Cronbach's a of 0.7 as the minimal acceptable for internal consistency of multi-item scales, others accept 0.6 or 0.5-0.6 for preliminary research as the cut-off point [56] . Other than dimensionality concerns, lower a can reflect too few items comprising the putative scale [73] . This is more likely for complex variables, such as social norms which have a broad spectrum of elements. Though less than perfect, measurement errors can be reduced by incorporating the items as a latent variable in SEM [55] , an approach we adopted. Additionally, collinearity between exogenous indicators, such as social norms and perceived benefit can be potentially problematic, perhaps lowering the accuracy of SEM estimation. However, since high associations between measures of the constructs were not observed (Table 3) then collinearity-related error is probably small [74] . Despite being randomly selected for the parent study, subjects of this study were not randomly selected from the general population, although demographics suggest the current sample is comparable to the Hong Kong general population [61] (Table 2 ). Moreover, subject recruitment was based on voluntariness and all data were selfreported. All could cause social desirability and selection bias, so caution is needed before extrapolation to the general population. Also refusal at follow-up could have influenced patterns of responses. Our study examined public decision-making regarding a novel influenza pandemic vaccine. Our findings may not apply to vaccination against seasonal influenza due to numerous differences in beliefs towards the vaccination. For example, although perceived low risk remains the major reasons for refusing vaccination against seasonal influenza as in our study, vaccine safety is seldom cited as a barrier [28, 29] whereas we found that about one third of respondents had vaccine safety concerns. Cultural differences in influenza and vaccination-related beliefs are possible [75] , but these differences may gradually diminish with the increasing identical news information available through the three dominant news agencies and common public health strategies being increasingly universal. Related stories, such as use of preservatives and adjuvants in vaccine manufacture may enhance knowledge and reduce trust in product safety [76] . The role of media remains much under-researched in this regard. Finally, data was insufficient to reliably report the reasons for pH1N1 vaccination uptake among the population.
Nonetheless, compared with other cross-sectional studies [12, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] , the longitudinal design of this study strengthens understanding of influences on population decision-making for pandemic influenza vaccination uptake and represents a step forward in this area of research. This study is novel in linking theoretically derived, vaccination-related cognitions to subsequent influenza vaccination behaviour, and exemplifies that within the Hong Kong Chinese culture, social norms and action planning are far more influential than intention in predicting vaccination behaviour. Dengue Virus Virulence and Transmission Determinants The mechanisms of dengue virus (DENV) pathogenesis are little understood because we have no models of disease; only humans develop symptoms (dengue fever, DF, or dengue hemorrhagic fever, DHF) and research has been limited to studies involving patients. DENV is very diverse: there are four antigenic groups (serotypes) and three to five genetic groups (genotypes) within each serotype. Thus, it has been difficult to evaluate the relative virulence or transmissibility of each DENV genotype; both of these factors are important determinants of epidemiology and their measurement is complex because the natural cycle of this disease involves human-mosquito-human transmission. Although epidemiological and evolutionary studies have pointed to viral factors in determining disease outcome, only recently developed models could prove the importance of specific viral genotypes in causing severe epidemics and their potential to spread to other continents. These new models involve infection of primary human cell cultures, “humanized” mice and field-collected mosquitoes; also, new mathematical models can estimate the impact of viral replication, human immunity and mosquito transmission on epidemic behavior. DENV evolution does not seem to be rapid and the transmission and dispersal of stable, replication-fit genotypes has been more important in the causation of more severe epidemics. Controversy regarding viral determinants of DENV pathogenesis and epidemiology will continue until virulence and transmissibility can be measured under various conditions. models can estimate the impact of viral replication, human immunity and mosquito transmission on epidemic behavior. DENV evolution does not seem to be rapid and the transmission and dispersal of stable, replication-fit genotypes has been more important in the causation of more severe epidemics. Controversy regarding viral determinants of DENV pathogenesis and epidemiology will continue until virulence and transmissibility can be measured under various conditions.
Dengue virus (DENV) pathogenesis seems to be determined by numerous, interacting factors: viral virulence, host immunity and immune status, host genetics and possibly others (e.g., preexisting diseases). Because we have no models of severe dengue disease (DHF), all associations of viruses with increased pathogenesis have been indirect and painstakingly slow in being developed. Transmissibility has also been measured indirectly: the successful isolation of viruses from patients and the preponderance of one serotype over another have been documented in numerous countries but this has also introduced biases in our sampling. We do not have available a fully representative set of DENV genomes to study and understand what truly constitutes the natural range of DENV variation; many samples from mosquitoes or less-ill human infections are missing, in addition to those from countries lacking the laboratory and public health infrastructure necessary for detecting and isolating viruses. Virus-mosquito interactions also add a layer of complexity to the determination of which genotype is being transmitted and we are only beginning to measure the effects of this selection. However, many new methods have been applied to the study of DENV genetic variation, replication fitness and their effects on transmissibility and pathogenesis in humans. None of these methods are perfect and we must still regard them as surrogates for measurements of the natural viral determinants of disease. Thus, understanding this complex system will probably require multidisciplinary approaches to solving the mysteries of the interaction of the many factors that contribute to DENV epidemiology. Other, nonviral factors contributing to DENV pathogenicity and transmission are discussed in accompanying chapters.
The first descriptions of DENV virulence differences came from epidemiologic and entomologic studies done in the South Pacific by Rosen and Gubler, in the 1970s (Gubler et al. 1978; Rosen 1977) . It was noted that some outbreaks in this region had fewer or no cases of DHF and the transmitted viruses were considered of low virulence; other outbreaks had many cases of DHF, after primary infection and these viruses were therefore more virulent. However, it took the development of RNA nucleotide sequencing techniques and the use of these sequences to generate phylogenetic trees of evolutionary relationships among viruses to discover that specific variant groups, or genotypes, were more frequently associated with dengue epidemics and severe disease (Chungue et al. 1995; Lanciotti et al. 1997; Lanciotti et al. 1994; Messer et al. 2003; Rico-Hesse 1990; Rico-Hesse et al. 1997 ). More recently it has been shown that some genotypes associated with DHF have been introduced and become established (endemic) in other continents, sometimes displacing the less-virulent DENV already being transmitted in those regions (causing DF only). "Virulent" genotypes have been described for serotypes 2 and 3 and it remains to be seen if further evolutionary studies will pinpoint similar groups in serotypes 1 and 4 (Rico-Hesse 2003) .
In the case of DENV, there is no evidence for rapid evolution and selection as in HIV, influenza or SARS viruses; for DENV, man-made ecologic disruption or increments in the number of mosquitoes or hosts are more important than evolution towards more virulent genotypes. There has yet to be evidence for the circulation of a recombinant DENV and those recombinant genomes described to date have been the product of enzymatic amplification techniques and are thus probably lab artifacts (Aaskov et al. 2007; Holmes and Twiddy 2003; Worobey et al. 1999) ; no one has isolated and fully characterized a recombinant DENV that is being transmitted in nature, causing disease. Although there is evidence for recombination in other, positive-strand viruses, specific steps in DENV replication might keep this event from occurring, although ample opportunities seem to exist in multiplyinfected humans and mosquitoes (Monath et al. 2005) . Also, there is no evidence for the epidemic transmission of the sylvatic genotype viruses from West Africa or Malaysia. These older, seemingly less-virulent viruses are transmitted mainly by canopy-dwelling mosquitoes to monkeys and they do not cause outbreaks in the human populations inhabiting those areas. The viruses isolated from dengue patients during outbreaks belong to genotypes imported from other continents (in the case of serotype 2, a genotype originating in the Indian subcontinent was introduced to Africa) (Rico-Hesse 2003) . Some researchers, with ample field experience in tropical areas, believe that these zoonotic cycles will eventually disappear, because of the constant reduction of natural forests; thus, these cycles have practically no importance as reservoirs of human dengue (Rodhain 1991) .
Although we have not detected increases in replication fitness for any given genotype, some of the evolutionary events leading to more virulent strains seem to have already occurred and by the time these viruses rapidly spread from Southeast Asia to other areas of the world (1940s), they were already virulent or replicatively fit (Gubler 2002) . We have yet to measure any specific genetic changes that are fixed in the viral population as virulent genotypes are successfully dispersed to other continents and we do not know whether there have been any changes imposed by selection in their new environments (e.g., for serotype 2, Southeast Asian genotype introduced into the Americas; for serotype 3, Sri Lankan genotype III introduced to the Americas). That is, these viruses had increased fitness at their origin, in that they are directly linked to the appearance of DHF and they are transmitted more efficiently by mosquitoes. However, some researchers believe differences in clinical presentation and severity of epidemics are a function of only immunologic and genetic differences between the human populations in both continents (Southeast Asia and Americas) (Halstead 2006) or that nonneutralizing antibodies formed during DENV infection play a role in gradually selecting for more pathogenic viruses in humans (Morens and Fauci 2008) .
Of special concern lately has been the effect of global warming on the incidence and spread of dengue disease. Although there are probably no effects on DENV replication in humans, if environmental temperatures rise, many investigators presume there might be an effect on virus transmission by mosquitoes. This is derived from the fact that increases in temperature (along with increases in rainfall) directly affect mosquito development (from larval to adult stages) and their populations can increase dramatically. Also, increases in average temperatures in new climes might make conditions favorable for mosquito breeding and the establishment of new populations. This could surely lead to more chances of exposure to mosquito bites for the human population and thus for infection by mosquito-borne viruses. However, the reason why many mosquito-borne diseases have yet to affect large populations in developed nations seems to be a lack of exposure to mosquito bites; air-conditioning and human behavior have been shown to reduce DENV transmission in the southern United States (Reiter et al. 2003) . That is, human activities and their impact on local ecology have generally been more significant in increasing dengue prevalence; thus, dengue disease seems to be influenced more by economic than climatic factors (Gubler et al. 2001; Reiter 2001 ). Also, research described below has shown that increases in temperature might have more of an impact on selecting for those virulent genotypes that are already being transmitted by mosquitoes. Mosquito survival rates and the time it takes for a virus to infect and be transmitted by the mosquito seem to be more important in this context.
Another subject that has received renewed attention is the possibility of human modification or management of viral virulence by impacting transmission dynamics. This is important for the application of rapid public health measures in the event of the emergence of new strains of parasites, bacteria or viruses (Lipsitch and Moxon 1997) . We presume we can influence virulence by the application of changes in human habits or by the use of control factors such as vaccines -and there are numerous precedents of how public health measures have changed the population dynamics of microorganisms (e.g., there are now more cases of vaccine-induced polio or yellow fever in some countries). Efforts to understand the relationship between parasite adaptation to hosts, virulence and transmission have developed into a small industry in evolutionary biology (Bull and Dykhuizen 2003) . Although most discussion is still theoretical, the relationships between virulence and transmission have been weighed, presuming that there is an evolutionary trade-off for optimizing either factor within an organism (Ebert and Bull 2003) . That is, to increase its chances of transmission to another host, an organism will limit its replication or virulence in so far as to not kill its host. For example, highly virulent, zoonotic viruses such as Ebola and rabies are less transmissible than measles or common cold viruses, which rarely kill their human host. However, this is overly simplistic when it comes to the evolution of viruses that have multiple strains (multiple infections, with some cross-protection), where virulence involves immunopathology, or where there is another host or an amplifying vector involved in transmission (Day et al. 2007 ). Thus, the main goal for disease control is to understand how we can reduce virulence in a virus population without making its level of transmission higher. However, this seems a daunting task if we include the effects of evolution by individual versus group selection, bottlenecks and changes in fitness trade-offs. The concern here is whether we will shift DENV evolution and population dynamics by applying incomplete control strategies (e.g., nonsterilizing vaccination or vector transformation).
The controversies mentioned above point to factors we should consider or understand in the control of dengue disease: evolutionary studies of DENV have helped us concentrate on detection (diagnosis and sampling), analysis (genetic and phenotype) and control (transmission dynamics in host and mosquitoes) of those genotypes already shown to be the culprits (Rico-Hesse 2007). The methods described below could help us reach these goals.
The determination of viral RNA sequences from different areas of the genome has now become routine and numerous laboratories around the world have this capability; this has added exponentially to the number of DENV samples available for comparison in GenBank. The comparison of these nucleotides and their encoded amino acids can be done with sophisticated computer algorithms that can tell us much about the rates and sites of mutation or evolution in the viral genome. These data can then be matched with patient viral loads, diagnoses, outbreak characteristics and transmission distribution, to look for specific associations. The RT-PCR technique has allowed for the enzymatic amplification of these sequences from very small amounts of viral RNA from almost any type of tissue but many researchers have now avoided virus isolation and characterization, thus introducing mistakes in some of the banked information (e.g., from amplification, sequencing, or cloning artifacts) and without information on viability or antigenicity. Therefore, it is also important to have access to classic virology techniques, especially if one is to derive information about virus phenotype.
The comparisons of full genome sequences of many DENV have helped pinpoint differences that could be involved in virulence: the comparison of viruses from two different genotypes associated with DHF or DF only (Southeast Asia and Americas, respectively) showed that there were consistent differences in the 5 0 -untranslated region (UTR), one envelope protein site (aa390) and the 3 0 -UTR of the DENV serotype 2 genome (Leitmeyer et al. 1999) . Comparisons within the Southeast Asian genotype did not identify any specific nucleotides associated with producing DHF (Mangada and Igarashi 1998; Pandey and Igarashi 2000) , so we assume all viruses of this genotype have the potential to produce severe disease. This has also been the case with other DENV, where recent studies have suggested that differences in 5 0 -and 3 0 -UTR in the genome can alter levels of replication (Miagostovich et al. 2006; Sirigulpanit et al. 2007; Tajima et al. 2007) , which can be extrapolated to viral load in blood or disease presentation (Wang et al. 2006) . Another chapter in this volume describes how these influences may occur.
The first targets of DENV replication, after mosquito bite, were postulated to be monocytes or macrophages and numerous studies focused on these cell types. However, more recent studies, using newer technologies for cell identification (mainly flow cytometry) have shown that DENV infects human monocytes poorly compared to dendritic cells, including Langerhans cells and monocyte-derived dendritic cells (Marovich et al. 2001; Wu et al. 2000) . Primary dendritic cell cultures can be derived from human peripheral blood donations to banks and this is the usual source for studies to compare the replication of low-passage DENV from patients. Because these samples are obtained anonymously, we must be careful to obtain them from blood banks in areas where there is no DENV transmission (i.e., no antigenic priming of cells) and we cannot determine the human genetic background that might lead to differences in virus replication. However, studies reported in 2003 (Cologna et al. 2005 Cologna and Rico-Hesse 2003) were able to show consistent differences in replication of DENV of two genotypes of serotype 2, demonstrating that there is an ex vivo correlation to the virulent phenotype derived from evolutionary studies. Although there seem to be innate, probably genetic differences in the yields of virus produced by cells from individual donors, this variation could be accounted for statistically and the correlations with virulence of patient-derived viruses were established (and note that these differences occur in the absence of antibodies). These primary cell cultures were also used to test recombinant viruses, to determine the influence of specific genome regions on virus replication and yields from human cell targets. These studies confirmed that the exchange of three genomic regions (5 0 and 3 0 UTRs, and E390) could reduce the levels of replication and virus yields of a Southeast Asian virus to those of wild-type, less virulent viruses of the American genotype (Cologna and Rico-Hesse 2003) . Other uses for cultured primary human cells include the identification of specific cells that are producing more virus (tropism) and whether virus replication is even required for pathologic effects.
Another step in determining if a virulent genotype had an increased transmission fitness phenotype was to study differences in replication and dissemination (i.e., the possibility of transmitting virus by bite) in the natural mosquito vector, Aedes aegypti. Laboratory-reared colonies of mosquitoes (e.g., Rexville or Rockefeller strains) seem to have lost their selectivity for infection and it is recommended that mosquitoes used in these experiments be from the F4 generation or lower (F0 ¼ field-collected eggs). The virulent, Southeast Asian strains of serotype 2 were shown to infect a larger proportion of mosquitoes than the less virulent, American genotype strains, after feeding mosquitoes on blood containing the same titer of virus (Armstrong and Rico-Hesse 2001) ; also, a greater proportion of mosquitoes develop disseminated infections with the virulent genotype (Armstrong and Rico-Hesse 2003) . If mosquitoes were fed both genotypes simultaneously, they were much more likely (sevenfold) to develop an infection with the virulent strains (Cologna et al. 2005) . When the dynamics of virus replication and dissemination were compared for both genotypes, the virulent strains had reached the salivary glands up to 7 days earlier than the less virulent viruses (Anderson and Rico-Hesse 2006) . This means that virulent strains may replicate and be transmitted much sooner to human hosts, outcompeting the less virulent viruses and causing many more cases of disease, thus ecologically displacing those that cause less severe dengue. This efficiency of transmission by the vector could explain how certain genotypes have displaced others, shifting the evolution of dengue disease towards more virulence (i.e., more DHF).
During this decade, major advances have been made in the development of new mouse breeds and their transplantation with human stem cells (from umbilical cord blood cells) that may effectively mimic the human immune system or show human signs of disease upon infection. The combination of studies in these mice with those in mice that are defective in interferon production or receptors have led to insights into the mechanisms of dengue pathogenesis (Bente et al. 2005; Kuruvilla et al. 2007; Kyle et al. 2007; Shresta et al. 2006) . Thus far, none of these models develop DHF and the production of DENV-specific antibodies has been low or undetectable. However, others have shown that mice engrafted with human hematopoietic cells can be effectively used to study pathogenesis of viruses for which no other models exist (Melkus et al. 2006; Watanabe et al. 2007) . It is anticipated that after adaptation of this system to DENV infection by multiple strains, with the acquisition of DENV-specific, functional B and T cells, that the signs of DHF might appear in these "humanized" mice. This would finally allow for the measurement of the many effects of immunopathogenesis, including the protective cross-immunity created by serial infection and the evaluation of many basic questions, such as dosedependence of infection, the relevance of mosquito factors to infection (e.g., salivary gland proteins) and the role of other cells as primary targets of infection (e.g., endothelial cells). Most importantly, this model could allow for the immediate testing of antivirals and vaccine candidates, where effective systems for testing products before human use have been lacking.
Another promising new field has been the development of mathematical models of DENV transmission, including "evolutionary epidemiology" and virulence management. These models are being used to estimate the effect of changing host immunity or mosquito transmission on the amount of virus circulating and the risks to a hypothetical human population and epidemic "topology." These analyzes have suggested that DENV cross-serotype immunity and mosquito demographics, rather than immune enhancement, are the most important determinants in the dynamics of specific serotype cycles or genotype replacement during epidemics and that the application of incomplete control strategies might actually increase the incidence of severe disease (Adams et al. 2006; Cummings et al. 2005; Nagao and Koelle 2008; Wearing and Rohani 2006) . Although these models are complex and still require many basic measurements for their refinement, some of the details are being added as they become available from laboratory or ecological studies (e.g., quantification of cross-protection by various DENV strains and many different measurements of mosquito transmission dynamics, including vector genetics and their effect on competence and capacity, etc.). Other applications of computer modeling have involved measuring the importance of host genetics over parasite contributions to virulence. For virology, the importance of host genetics in disease pathogenesis has been discussed for many years but recent technologies have allowed researchers to weigh the influence of virus virulence over human host genetics, in the case of pandemic influenza A (Gottfredsson et al. 2008; Pitzer et al. 2007 ). However, these approaches are extremely controversial at this point, as other investigators have reached opposing conclusions when using similar methods and results (Albright et al. 2008 ).
Only recently have public health officials in developed countries become concerned about the increased transmission and geographic spread of DENV. In many cases this is due to the increase in cases imported by tourists from less-developed tropical regions. For the United States, there has been a marked increase in imported cases and autochthonous transmission in Texas and Hawaii during this decade (Morens and Fauci 2008) and for the first time we have been able to document the introduction of a virulent DENV genotype into this country (CDC 2007; Rico-Hesse 2007) . This has added a sense of urgency to research using some of the models described here and a hope for additional support for studies of this long-neglected tropical disease. Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection Tylosema esculentum (marama) beans and tubers are used as food, and traditional medicine against diarrhoea in Southern Africa. Rotaviruses (RVs) are a major cause of diarrhoea among infants, young children, immunocompromised people, and domesticated animals. Our work is first to determine anti-RV activity of marama bean and tuber ethanol and water extracts; in this case on intestinal enterocyte cells of human infant (H4), adult pig (CLAB) and adult bovine (CIEB) origin. Marama cotyledon ethanolic extract (MCE) and cotyledon water extract (MCW) without RV were not cytotoxic to all cells tested, while seed coat and tuber extracts showed variable levels of cytotoxicity. Marama cotyledon ethanolic and water extracts (MCE and MCW, resp.) (≥0.1 mg/mL), seed coat extract (MSCE) and seed coat water extract (MSCW) (0.01 to 0.001 mg/mL), especially ethanolic, significantly increased cell survival and enhanced survival to cytopathic effects of RV by at least 100% after in vitro co- and pre-incubation treatments. All marama extracts used significantly enhanced nitric oxide release from H4 cells and enhanced TER (Ω/cm(2)) of enterocyte barriers after coincubation with RV. Marama cotyledon and seed coat extracts inhibited virion infectivity possibly through interference with replication due to accumulation of nitric oxide. Marama extracts are therefore promising microbicides against RV. Tylosema esculentum (Burch.) (marama) A. Schreib. (family Caesalpiniaceae or Leguminosae) [1] , also known as "the Green Gold of Africa," is a creeping plant found in the southern parts of Africa, namely South Africa, Namibia, and Botswana. Tylosema esculentum bean and tuber extracts have been used in traditional African medicine to treat diarrhoea and for general upkeep of human health [2] . Only little chemical characterization has been done to date on the T. esculentum plant. Research on chemical and health benefits of T. esculentum plant is part of an ongoing research project under EU-INCO Marama II FP6 programme (contract number 032059).
Various bioactive constituents are expected to be present in T. esculentum plant. These may include phenolic constituents, carbohydrates, and certain fatty acids, among many components. Large amounts of gallic acid have been detected in T. esculentum plant [3] . Gallic acid esterifies with glucose, the resultant hydrolysable tannins (HTs), secondary metabolites widely distributed in the plant kingdom, are known to be effective antagonists against viruses [3] . Tylosema esculentum tubers contain yet to be determined phenolics [3] . High amounts of phytosterols have also been detected in T. esculentum oil (about 75% of all phytosterols being 4-desmethylsterols and about 15.72% of the total being 4,4-dimethylsterols) [4] . Rotavirus (RV) infections are a major cause of acute gastroenteritis in infants and young children, accounting for about 611,000 deaths each year worldwide, mostly in developing countries [5] . Rotavirus is also an important pathogen in many agricultural species, inducing serious diarrhoeal diseases in neonatal and postweaning pigs and calves [6] [7] [8] . Rotavirus infection is limited to the mature enterocytes of the tips of the intestinal villi of humans, domestic animals, and mice, leading to severe gastroenteritis in the young [9] . Some strains of bovine and human RV have high sequence homology and interspecific infectivity, both implying phylogenetic relatedness [10, 11] . Previous in vitro tests of the bovine RF strain of RV on human, pig, chicken, and goat intestine epithelial cells in our laboratory have shown interspecific infectivity of rotavirus [12] , and, therefore, the bovine strain of rotavirus was used to infect human and pig intestinal cells in the current study.
It is known that infection of intestinal epithelial cells with RV causes disruption of tight junctions and loss of transepithelial resistance (TER), even in the absence of cell death [13] . Loss of TER involves alteration of tight junction proteins, mainly claudin-1, occludin, and ZO-1 as determined in human small intestine carcinoma cells (Caco2) [13] , although use of cancer cells for such mechanistic studies is not appropriate. Rotavirus infection is also associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP, conditions known to reduce the integrity of epithelial tight junctions [13, 14] .
While vaccines such as RataTeq and Rotatrix have been made available for prevention of RV infections [15, 16] , their effectiveness remains to be verified [17] . Use of a few synthetic compounds against simian RV, such as ribavirin [18] and isoprinosine [19] , and natural products against human and bovine RV [20] , such as theaflavins, has been reported. Unfortunately, these compounds are not available for human use, which necessitates alternative methods to control RV infection [21] . A promising alternative strategy to reducing the burden of diarrhoea caused by RV may lie in identifying and developing costeffective nutritional or phytomedical solutions; which can be applicable especially in children and immunocompromised persons.
With this information, and related empirical supporting information from other legume plants, it was hypothesised that T. esculentum bean and tuber extracts can inhibit RV infection. The proposed mechanisms of antiviral effects of T. esculentum are summarised in Figure 1 . It was herein projected that extracts from T. esculentum beans and tubers can be included in various medicine mixtures or whole beans and tubers be used as functional foods, with the aim to combat diarrhoea caused by RV.
In this study we sought to examine the antirotaviral activity of water and ethanol extracts from bean cotyledons and bean seed coats and water extracts from T. esculentum tubers by investigating their ability to increase survival of human foetal, pig, and calf intestinal epithelial cells.
All research on the plant was done with full authorisation granted to Marama II INCO (EU-FP6) research programme. T. esculentum (marama) bean samples were obtained in Botswana in 2005 and 2007, and the T. esculentum tuber sample was obtained in Botswana in 2007. Seeds were stored in plastic bags at room temperature until time for extraction. The T. esculentum tuber (approximately 20 kg wet mass) was dug out from a site in Jwaneng, then ground and air-dried at University of Botswana, and then further ground to fine powder before the extraction was done at University of Maribor, Slovenia. A voucher specimen of T. esculentum plant is kept in the Namibian National Herbarium (collecting number: GM 1063 and herbarium number: 59520). The following water extracts from T. esculentum beans and tuber were obtained: tuber water extract (MTW), cotyledon water extract (MCW), and seed coat water extract (MSCW) ( Table 1 ).
Procedure. Crude bean ethanolic extracts were prepared as described by Bolling and Parkin [22] . Briefly, 1 litre of ethanol was used to extract 100 g of shade-dried T. esculentum bean at 78 • C using a Soxhlet apparatus (Carl Roth WHLG2/ER-Serie, Karlsruhe, Germany) for extraction. The evaporation procedure ensured the exclusion of ethanol from the extracts; the resultant extract was a thick paste; these extracts were labelled MSCE (seed coat ethanolic extract) and MCE (cotyledon ethanolic extract) and stored at −20 • C until use. Ethanolic extracts are known to contain mainly lipids and other ethanol soluble contents such as isoflavones [22] [23] [24] . The following extracts from T. esculentum beans and tuber were obtained: cotyledon ethanolic extract (MCE) and seed coat ethanolic extract (MSCE) ( Table 1) .
Below are the yields of each extract (mg) per g of T. esculentum bean and tuber material used.
Cell cultures. The following cell lines were used in the antiviral tests: human foetal small intestine epithelial cells (H4) [25] (a generous gift from Dr. Tor Savidge, USA), pig small intestine epithelial cells (CLAB), isolated in our laboratory [26] and maintained by University of Maribor, Slovenia, and bovine calf small intestine epithelial cells (CIEB), isolated in our laboratory [26] and maintained by University of Maribor, Slovenia. The cells were grown in advanced Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland). Cell lines were routinely grown in 25 cm 2 culture flasks (Corning, New York, USA) at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air until confluent monolayers attained. Culture medium was changed routinely.
Propagation. An RF RV strain, as previously described in [27, 28] , was used in all tests. Confluent cultures (in 25 cm 2 Corning flasks) of H4, CLAB, and CIEB cells were washed twice in single strength phosphate buffer (1 × PBS) to remove the foetal bovine serum (FBS) before infection with RV (previously passaged 5 times on H4, CLAB, and CIEB cells in rotation; at 1 × 10 7.5 /mL TCID 50 in the presence of 1 μg of trypsin (Sigma Chemical Co., St. Louis, Mo) per mL. After a 1-hour adsorption period at 37 • C, DMEM, containing 0.5% FBS (Sigma Chemical Co., St. Louis, Mo.) and 1 μg of trypsin per mL, was added to each flask. The flasks with cells were incubated for 24 to 48 h at 37 • C, under 10% CO 2 until cytopathic effect (CPE) was observed by microscopy. When the cells showed extensive cytopathic degeneration, the flasks with cells and virus were subjected to two freeze-thaw cycles to detach and break the cells. The supernatant fluids containing detached cells and extracellular virus were removed, and then the cellular debris was removed by centrifugation at 3500 g for 10 min. Virus was stored at −70 • C until time for tests.
Tissue culture infective dose (TCID 50 ) was determined using the method of Reed and Muench [29] , as previously described in [30] . Clarified supernatants of RV titrating 1 × 10 7.5 TCID 50 /mL (tissue culture infectious dose 50%, TCID 50 ) were subsequently used for the antiviral tests with T. esculentum extracts. Similar TCID 50 values were obtained by Bae et al. [31] (1.27 × 10 6 per mL) when they titrated RV on Macacus Rhesus monkey kidney cells (MA104).
Cells. Tylosema esculentum extracts were dissolved in DMEM (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland) using a rotary magnetic stirrer (Yellowline, BigSquid, Kika-Werke GMBH & Co, Germany). As control, DMEM (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland), was used. The extracts were added diluted 10-fold to monolayers of cells in 96 well plates, then maintained at 37 • C in a humidified atmosphere of 4 Evidence-Based Complementary and Alternative Medicine 5% CO 2 and 95% air for 24 h. Following incubation, cells were carefully rinsed with PBS to remove T. esculentum extracts and other debris. Upon washing, plates were stained with 0.01% crystal violet for 5 minutes and then rinsed with water. Plates were then dried, and then crystal violet incorporated in viable cells was resuspended with 10% acetic acid (100 μL per well). Photometric quantification of crystal violet previously retained in living cells was done at 595 nm with a microplate reader (Multiscan, Finland) as described by Agelis et al. [32] . After this test for cytotoxicity, certain concentrations of T. esculentum extracts were selected for antiviral tests as shown in Table 2 .
RV with T. esculentum Extracts. Tylosema esculentum ethanolic extracts, namely, seed coat ethanolic extract (MSCE) and cotyledon ethanolic extract (MCE) were dissolved using a rotary magnetic stirrer (Yellowline, BigSquid, Kika-Werke GMBH & Co, Germany) in DMEM (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland). Rotavirus at 1 × 10 7.5 /mL TCID 50 was combined with concentrations of T. esculentum extracts (see Table 2 ). As controls, DMEM (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), Lglutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland), dilutions of Combivir GlaxoSmithKline-150 mg lamivudine and 300 mg zidovudine (AZT) as antiviral agent as described by Agelis et al. [32, 33] and T. esculentum extract at below IC 50 concentrations and RV solutions at 1 × 10 7.5 /mL TCID 50 were separately used. The plant extract and virus, Combivir, and virus mixtures were added to monolayers of cells in 96-well plates and then maintained at 37 • C in atmosphere of 5% CO 2 for 24 h.
Cytopathic effects of RV alone and in coculture with T. esculentum extracts or Combivir added at 0.75 mg/mL {modified from Agelis et al. [33] } on cell monolayers after treatment as listed above were measured after 24-hour coincubation of cell monolayers (1 × 10 6 cells/mL) at 37 • C in atmosphere of 5% CO 2 . It was previously shown that T. esculentum extracts alone were not cytotoxic at the concentrations used in these tests; hence the observed reduction in survival of cells upon coincubation of extract RV solutions was attributed mainly to the virus. Following incubation, relative viability of cells was determined photometrically in terms of amount of crystal violet incorporated in viable cells as described previously.
Human small intestinal epithelial cells (H4) were introduced into inserts in 12-well plates (Corning Transwell, 0.4 μm pore size, New York, USA) at concentration of 1 × 10 6 cells/mL. Transepithelial resistance (TER-Ω/cm 2 ) was measured periodically using Millicell-ERS Electrical Resistance System (Millipore, Bedford, USA) until values of about 1000 Ω/cm 2 were attained, such high values indicating maximum attainable polarity in H4 cells, as previously determined (results not shown). Rotavirus suspensions (at 1 × 10 7.5 /mL TCID 50 ) were mixed with T. esculentum extracts at below their predetermined IC 50 concentrations ( Table 2 ) and then introduced onto H4 monolayers in insert wells. Tylosema esculentum extracts diluted in DMEM, DMEM alone, and Combivir diluted in DMEM were introduced as respective controls. The effect of the extracts on the cell polarity was evaluated by measurement of transepithelial electrical resistance (TER) as previously described in [34] . TER values of monolayers exposed to combinations of marama/Combivir treatments, DMEM, and RV controls were plotted.
Rotavirus (at 1 × 10 7.5 /mL TCID 50 ) was incubated with T. esculentum water and ethanolic extracts at room temperature for 1 h. Following the incubation, dilutions of the incubated virus were added to CLAB, CIEB, and H4 cell monolayers in 96well plates and then incubated for 1 h at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air, after which the unattached virus and the extracts were washed off with PBS. DMEM (Sigma-Aldrich, Grand Island, USA), supplemented with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma), penicillin (100 units/mL, Sigma), and streptomycin (1 mg/mL, Fluka, Buchs, Switzerland), was then introduced on the cells and then incubated for 24 h at 37 • C in atmosphere of 5% CO 2 until viral cytopathic effects were observed in control wells. Cell viability was then determined with quantification of crystal violet as described previously. As control, viral only, T. esculentum only and cell culture medium only treatments were plated onto the epithelial cells. Relative protection by T. esculentum extracts against RV infection was determined by crystal Tests were done in triplicate wells. Tylosema esculentum extracts used were as follows: cotyledon ethanolic extract (MCE), seed coat ethanolic extract (MSCE), tuber water extract (MTW), cotyledon water extract (MCW), and seed coat water extract (MSCW). Tylosema esculentum extracts were introduced at and below IC 50 or at 1 and 0.1 mg/mL of MCE (the highest concentration of MCE was not cytotoxic).
Cells after Exposure to T. esculentum Extracts. Release of NO after cell exposure to T. esculentum extracts was detected using a microplate reader (Multiscan, Finland) at 540 nm after introduction of Griess reagent (Sigma, Germany) into supernatants after 24-hour incubation. Results were expressed as percent difference between nitric oxide release of test from control. Nitric oxide is proportional to the amount of nitrate accumulating in supernatants as shown by the change of colour at 540 nm.
Correlation coefficients for data obtained on level of protection, nitric oxide release, and transepithelial resistance (TER) were calculated using Statsoft Statistica version 7 software.
Intestinal Epithelial Cells. To determine concentration of RV in the stock solution, the method of Reed and Muench [29] was adopted with modifications. Titration results showed that the RV strain had high and variable but not cell-typespecific cytopathic effects on all the cells used ( Figure 2) . Rotavirus, when incubated without T. esculentum extracts on cells, showed dose-dependent cytopathic effect on human small intestine epithelial cells (H4 cells), pig small intestine epithelial cells (CLAB), and bovine calf small intestine epithelial cells (CIEB) (Figure 2 ). For subsequent antiviral testing an RV concentration at and/or below 1 × 10 7.5 /mL TCID 50 was used to avoid cotoxicity with extracts.
Cells. Tylosema esculentum bean water and ethanolic extracts and T. esculentum tuber water extract were added on cell monolayers of CLAB, CIEB, and H4 cells at concentrations as shown in Table 2 and Figures 3 and 6 . Tylosema esculentum extracts, at the concentrations used, resulted in dose-dependent enhancement of the three types of cells. Exposure to Tylosema esculentum seed coat water and ethanolic extracts (MSCW and MSCE, resp.) generally led to enhanced survival of CLAB, CIEB, and H4 cells. This result could show that exposure to cotyledon extracts, besides the projected inhibition of RV cytopathic effects, could ensure cell survival. Figure 3 . All T. esculentum extracts, especially cotyledon ethanolic extract (MCE), led to significant protection of CIEB, CLAB, and H4 cells from cytopathic effects of rotavirus. Tylosema esculentum seed coat water extract (MSCW) offered significant protection at the lowest concentration (0.001 mg/mL), making it the most potent extract. Tylosema esculentum cotyledon water extract (MCW) also led to highly significant protection of CLAB cells. There was largely highly significant protection of H4 cells exposed to MSCE, MSCW, MTW, and MCE. Combivir (control) was shown to have highly significant protection of cells against RV. Exposure to MCE was shown to enhance survival of CIEB cells (56%) and H4 cells (275%) more than that to Combivir. On H4, there was higher protection of cells with all extracts (91%-275%) depending on concentration than Combivir alone (Figure 3 ). Some extracts, mainly seed coat and cotyledon water and ethanolic, led to higher protection at concentrations lower than the determined IC 50 .
(% Ω/cm 2 ) of H4 Cells over Time. Figure 4 shows changes in TER of H4 cells exposed to T. esculentum extracts, Combivir (AZT), or DMEM with and without RV over time. Over the whole 67 hours, coincubation of RV with bean seed coat extracts (ethanolic extract (MSCE), and water extract (MSCW) or AZT led to higher TER across the cells than when the virus was coincubated with cotyledon extracts (MCE and MCW). Incubation of H4 cells with individual extracts alone or AZT alone resulted in lower TER than extract/AZT-RV coincubation treatments. Incubation of cells with RV alone from the start of the experiment until about 40 hours resulted in TER lower than the rest of the treatments. However between 40 and 67 hours of exposure, wells with RV only treatments generally had higher TER (though sporadic) than the rest of the treatments. All T. esculentum extracts generally had better enhancement of TER than AZT over the initial 67 hours.
NO 2 release was significant from human intestinal epithelial cells (H4) exposed to all extracts used {seed coat water and ethanolic extracts (MSCW and MSCE, resp.), cotyledon water and ethanolic extracts (MCW and MCE, resp.), and tuber water extract (MTW)}, while only MCE and MTW significantly increased NO in CLAB cells ( Figure 5 ).
Results in Figure 6 show that pre-exposure to T. esculentum extracts can significantly reduce infectivity of RV. This finding broadly points to inhibition of RV cytopathic effect by T. esculentum extracts prior to or during virus entry (specific mechanisms were not determined). Exposure of RV (at 1 × 10 7 Table 3 shows correlation of antiviral effects of different treatments with T. esculentum extracts on different cell cultures. There was high positive correlation between CLAB and H4 cells (r = 0.94), moderately positive correlation between CLAB and CIEB cells (r = 0.57) in survival from rotavirus cytopathic effects after coincubation with T. esculentum extracts, while a weak negative correlation between H4 and CIEB cells (r = −0.02) was observed after coincubation. Highly positive correlation between survival of CLAB cells after coincubation compared to preincubation with T. esculentum extracts (r = 0.91) and moderately positive correlation between CIEB cells pre-and coincubated with the extracts (r = 0.59) were observed. Poorly positive correlation between H4 cells co-and pretreated with the extracts was observed. Moderate positive correlations were also observed between survival of and nitric oxide release from CLAB (r = 0.43) and H4 cells (r = 0.79), as well as highly positive correlation in levels of nitric oxide release from H4 and CLAB cells (r = 0.9). Negative correlation was observed between survival rates and TER of cells (H4, CLAB and CIEB) after coincubation of rotavirus with T. esculentum extracts.
We show herein that the RV strain used (RF strain) could be adapted to pig and human cells, additional to bovine cells. T. esculentum bean cotyledons had low or no cytotoxicity, as shown by high IC 50 concentrations ( Table 2; Figures 3  and 6) , while seed coat and tuber extracts, especially seed coat ethanolic extract (MSCE) and tuber water extract (MTW), had elevated cytotoxic effects (low IC 50 concentrations lying between 0.01 and 0.001 mg/mL) on all the cells used (Table 2 ; Figures 3 and 6) . This study has identified T. esculentum bean ethanolic cotyledon extract (MCE) and bean seed coat extract (MSCE) as having highly significant inhibitory effects against cytopathic effects of RV, both with coincubation ( Figure 3 ) and preincubation ( Figure 6 ) of the extracts and RV on human, pig, and bovine intestinal epithelial cells. Tylosema esculentum seed coat ethanolic extract (MSCE) showed the best antirotaviral activity, as shown by the lowest IC 50 concentrations (1 μg/mL).
Marama cotyledon ethanolic extract (MCE) offered similar and/or higher protection against cytopathic effects of RV than that offered by antiviral drug Combivir on all intestinal epithelial cells, with higher effect on CIEB and H4 cells. Exposure of CLAB cells to seed coat and cotyledon water and ethanolic extracts at concentrations lower than IC 50 resulted in higher survival of the cells. This could be due to potentiated toxic effects due to combined presence of RV and extract (at IC 50 concentration) and diluted cytotoxicity at lower extract concentrations. This effect was cell culture dependent, implying possible different interspecies responses.
There was positive correlation between levels of protection of intestinal cells from cytopathic effects of RV (crystal violet assay) with coincubation and preincubation and enhancement of polarity (TER) of H4, CLAB, and CIEB cells by T. esculentum extracts and Combivir ( Table 3) . Survival of CLAB and H4 cells was moderately to highly correlated to release of nitric oxide (r = 0.79 and r = 0.43, resp.); similarly very high positive correlation was observed in levels of nitric oxide release from H4 and CLAB cells (r = 0.9) exposed to T. esculentum extracts (Table 3 ). These findings show that there is a positive link between effects of T. esculentum extracts (pre-and coincubated) against rotavirus on the different species of cells and the release of nitric oxide. Coincubation of RV and marama seed coat ethanolic extract (MSCE), cotyledon ethanolic extract (MCE), or tuber water extract (MTW) on H4 cells resulted in generally higher enhancement of TER than Combivir throughout 67hour test period. Enhancement of polarity across intestinal epithelial cells can be an important mechanism to control entry of RV across the intestinal epithelial barrier. Rotaviruses are known to disrupt tight junctions, resulting in loss of transepithelial resistance (TER) [13] , but without cell death during viral replication [35] ; hence T. esculentum bean seed coat and cotyledon ethanolic extracts (MSCE and MCE, resp.) and T. esculentum tuber water extract (MTW) can reduce entry of RV across the intestinal barrier in vitro. Effects of T. esculentum extracts on transepithelial resistance of H4 cells, however, could not be linked to survival of H4 cells, as shown by negative correlation between survival of cells and TER in presence of the extracts.
Another mechanism of inhibition of RV by T. esculentum extracts, especially in seed coats, may lie in the inactivation of RV, as shown with enhancement of cell survival after preincubation of rotavirus with T. esculentum extracts. It is herein suggested that the observed inactivation of RV may be through interference with either viral replication or capacity to bind to permissive cells. Clark et al. [36] have shown that some phenolic acids, which were expected to dominate the ethanolic extracts, have inactivation effect against RV. Similar observations were also made with extracts from peppermint, sage, and lemon balm leaves, which were highly inhibitive against HIV [37] . The inhibitive effects of T. esculentum extracts, especially seed coat and cotyledon ethanolic extract (MSCE and MCE) and tuber water extract (MTW) (≥100%), could additionally be due to enhanced release of nitric oxide (NO) from intestinal cells. Nitric oxide has been shown to inactivate RV, mainly by inhibiting replication of the virus [38, 39] . The observed enhancement of nitric oxide release from the human and pig cells was viewed as one of the mechanisms T. esculentum crude extracts can induce a nonspecific immune response in intestinal epithelium.
Certain plant phytochemicals have been shown to inhibit RV activity through inhibition of viral penetration and viral replication. Some flavonoids were shown to inhibit penetration of RV in rhesus monkey epithelial cell line (MA104) [31] , while others have an effect on the viruses or on the enzymes responsible for their replication [40] . These effects could not be singly demonstrated in this study; further work on these possible mechanisms of antiviral effects of T. esculentum extracts is, therefore, encouraged. The observed antirotaviral effects can be attributed to the high phenolic acid content (especially gallic acid), in bean cotyledons, among other components (Marama II report, unpublished). Gallic acid was previously shown to prevent viral replication, inhibition of virus attachment to and penetration into cells, and virucidal effects [41] . Interference with attachment and penetration into cells could not be proved in this study. Tylosema esculentum tubers may contain some yet to be determined phenolics (Marama II report, unpublished). Ethanolic extracts had notably higher antiviral activity than aqueous bean and tuber extracts (Figures 3  and 6 ). Some essential oils as found in T. esculentum beans may contribute towards the observed antiviral effects; similar findings were reported by Astani et al. [42] using essential oils extracted from Melaleuca alternifolia. Moreover, recently, a Kunitz-type inhibitor, distinct from other known plant serine protease inhibitors and shown to be specific for elastase, was isolated in T. esculentum beans [43] . The quantity of this elastase inhibitor present in T. esculentum beans is many times greater than in soybean or any other bean or nut source reported to date [43] . Certain protease inhibitors have been shown to impart high antienteroviral effects through interference with proteolytic cleavage during the replication process [44] . The high phytosterol content of T. esculentum bean oil 4-desmethylsterols (75%) and 4.4-dimethylsterols and 4-monomethylsterols (15.72%) [4] could also contribute to the high antiviral activity in bean extracts. Phytosterols have been determined to have antihuman cytomegalovirus (HCMV) and antiherpes simplex virus (HSV) effects [45] . The antiviral activity of phytosterols is through the blocking effect on immediate-early antigen expression in fibroblast cells and blocking of virus-cell interaction and/or virus multiplication [45] .
Our results have shown high in vitro inhibition of RV by T. esculentum extracts, especially seed coat ethanolic extracts (0.01 to 0.001 mg/mL) and cotyledon ethanolic extracts (≥0.1 mg/mL), and tuber water extract (0.1 to 0.01 mg/mL). Mechanisms of antiviral action may include release of nitric oxide and may also interfere with viral cytopathic effects (as shown by moderate to high positive correlation between survival from rotavirus and release of nitric oxide), inactivation of RV (inhibition of virus replication or entry into cells may be responsible), possibly through interference with replication, and enhancement of tight junctions (though not directly related to levels of survival) (Figure 1 ). Our findings suggest that T. esculentum beans can be an important source of microbicides against RV. Further phytochemical characterization of T. esculentum extracts, identification of the responsible bioactive compounds, and the elucidation of other modes of action and quality standard studies are essential.
MCE: Tylosema esculentum (marama) cotyledon ethanolic extract MSCE: Tylosema esculentum (marama) seed coat ethanolic extract MTW: Tylosema esculentum (marama) tuber water extract Sphaeranthus indicus Linn.: A phytopharmacological review Sphaeranthus indicus Linn. (Asteraceae) is widely used in Ayurvedic system of medicine to treat vitiated conditions of epilepsy, mental illness, hemicrania, jaundice, hepatopathy, diabetes, leprosy, fever, pectoralgia, cough, gastropathy, hernia, hemorrhoids, helminthiasis, dyspepsia and skin diseases. There are reports providing scientific evidences for hypotensive, anxiolytic, neuroleptic, hypolipidemic, immunomodulatory, antioxidant, anti-inflammatory, bronchodialatory, antihyperglycemic and hepatoprotective activities of this plant. A wide range of phytochemical constituents have been isolated from this plant including sesquiterpene lactones, eudesmenolides, flavanoids and essential oil. A comprehensive account of the morphology, phytochemical constituents, ethnobotanical uses and pharmacological activities reported are included in this review for exploring the immense medicinal potential of this plant.  Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression. Similarly, the subscript j denotes SAC j, c j is the average number of sexual partners per
year in SAC j, and N j is the sum of all sexually active individuals in SAC j.
In this Because a TIP shares all the same viral coat proteins as HIV-1, the TIP transmission probability per partnership is expressed as a function of identical form with the same parameter values as . 
' [ &%.(9-'&&'()* 3+R* '(* &@'9* >2%'L.&'$(4* * I$%* 9'-#<'1'&=7* &@2* %'9`* 9&%"1&"%2* '(* &@2* -$>2<* '9* %2#<.12>* B=* $(2* !MO* D'&@* 5# P* T4[X* #.%&(2%9b=2.%* U&@2* D2')@&2>* .L2%.)2* $,* &@2* ,$"%* !MO9V4* * g$&2* &@.&* @2&2%$)2(2'&=* '(* 1$(&.1&* %.&29* '9*`($D(* &$* '(1%2.92* &@2* %2#%$>"1&'L2* ("-B2%* '(* .* ($(<'(2.%* ,.9@'$(* hW[i7* 9$* &@2* 29&'-.&29* >2%'L2>* B=* &@'9* .##%$.1@* D'<<* B2*  Screening of Random Peptide Library of Hemagglutinin from Pandemic 2009 A(H1N1) Influenza Virus Reveals Unexpected Antigenically Important Regions The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins. The 2009 A(H1N1) influenza virus, which is also referred to as the swine-origin influenza virus (S-OIV), caused the first global influenza pandemic in recent decades [1] . Given continuous antigenic drift and reassortment of heterotypic influenza viruses circulating in human and animal reservoirs, global concerns have been raised regarding an increasing threat of an influenza pandemic [2] . Current treatment strategies for influenza-A viruses, such as vaccines and drugs, have not provided broad and lasting protection, partly due to the constantly evolving nature of the viral surface glycoprotein, hemagglutinin (HA) that allows it to avoid host immune attack.
HA is the key viral antigen in determining host specificity and inducing neutralizing antibody since it plays a major role in binding to host cell receptors and fusing with host cell membranes [3] . For many challenging diseases caused by viruses, the recognition of certain neutralizing epitopes by the immune system can indeed provide broad and potent protection [4, 5] . The antigenic structure of HA and the corresponding antibody response are not fully understood, complicating rational design of vaccines aimed at modulating antibody responses for targeting key epitopes.
Our previous knowledge of viral antigenic structure was based mainly on the structure of antibody-antigen complexes or mutational analysis of related antigenic drifts [6] [7] [8] . Recently, many new approaches have emerged for the rapid extraction of monoclonal antibodies toward target antigens from antibody phage display libraries [5, 9, 10] , using direct sorting of memory B cells [11] , or by immortalization of IgG expressing B cells that reflect the antibody repertoire [12, 13] . These methods provide much more information on viral epitopes, although they are often laborious. The major alternative approaches for epitope mapping are derived from scanning with antigenic peptides displayed on the surface of bacteriophage, bacteria and yeast [14] [15] [16] [17] [18] [19] [20] [21] , which in recent years have increasingly incorporated fluorescence-activated cell sorting (FACS). In these efforts, short defined or random peptides with a narrow length range (,50 amino acid residues (aa)) are used to screen against monoclonal or polyclonal antibodies.
Inspired by advances in cell surface display technology [22, 23] and peptide fragment library construction [24] , we devised a highthroughput scheme that utilizes yeast display and FACS that allows for direct screening of random viral peptide libraries against complex antisera instead of isolated antibodies (Zuo T, Shi XL, Xu WH, Lin Z, and Zhang LQ, unpublished results). The peptide library is generated by random digestion of the gene encoding the target viral protein, followed by a PCR-based reassembly step that results in fragments with controllable lengths (normally in the range of 100-500 bp) [24] . These peptides are then expressed on the yeast cell surface by fusion to the yeast adhesion receptor AGA 2 protein, which has previously been used to display defined short antigen fragments of various viral and non-viral proteins [19] [20] [21] . From the sequences of the antigenic peptides after screening, it is possible to determine the relative frequency of each amino acid residue involved in recognition by antisera, and how these residues are distributed in the three-dimensional structure of HA. Using this approach, we analyzed the 2009 A(H1N1) influenza virus HA protein using mouse, goat and human antisera. Unexpectedly, different antigenic profiles were seen for different antisera. Moreover, five antigenic regions were identified, four of which are located in the conserved HA stem region responsible for membrane fusion. ELISA binding assays and absorption experiments using peptides that encompass these regions have confirmed their antigenic activities.
The procedure for construction, expression and screening of random viral peptide libraries using yeast surface display is shown in Fig. S1 (see also Materials and Methods for details). In step 1, the full length HA gene of the pandemic 2009 A(H1N1) influenza virus (including the core HA protein of 519 aa, the signal peptide of 16 aa, and the inter membrane domain of 36 aa) was digested and re-assembled by PCR to form a pool of fragments enriched in 100-500 bp [24] . In step 2, the fragment library was ligated into the yeast display vector pCTCON-T (derived from the yeast surface display vector pCTCON-2 [23] shown in Fig. 1 ), transformed into yeast EBY100 cells and induced for expression, generating a typical library of 1610 5 -10 6 clones to cover most of the possible fragments [25] . Because there are 3 stop codons on pCTCON-T downstream of the peptide inserts to terminate every ORF, the theoretical possibility for in-frame fusions is 1/3 for the N-terminal end of the peptide inserts. Thus, the theoretical yield for cells expressing in-frame peptides is 1/6 (the fragments can be inserted in forward and reverse directions). Subsequently in step 3, the cell library was incubated with antisera, labeled with secondary fluorescent antibody and subjected to FACS in step 4, after which the cells harboring antibody-binding peptides were distinguished from those with non-binding peptides. In step 5, we isolated plasmids directly from the pool of yeast cells containing positive peptide sequences, which were retransformed into E. coli for sequencing. This reduced laborious plasmid extraction from individual yeast clones. Plasmids containing in-frame sequences were transformed back into yeast for individual flow cytometry (FCM) verification. Retransformation and FCM were also performed for plasmids containing out of frame sequences (false positive). Antigenic peptide sequences were aligned with the HA sequence in step 6 (see also Figs. 2 and 3). In step 7, statistical analyses both on the sequence and structural levels were then performed to map out the antigenically important regions of the HA protein, by summarizing the frequency of each amino acid residue appearing in the antigenic peptides (see also Fig. 4) .
The HA polypeptide is cleaved into two subunits linked by a disulfide bond, HA1 and HA2, to form the mature HA trimer. The majority of the HA1 subunit forms the viral membrane-distal globular head responsible for receptor binding, whereas the HA2 subunit together with the N-and C-termini of HA1 forms the membrane-proximal stem region that plays a major role in membrane fusion. Thus two plasmids, pCTCON-HA1, pCTCON-HA2, respectively, were constructed to display the HA1 subunit of 328 aa (residues 17-344 of the HA protein) and the HA2 ecto-domain of 192 aa (residues 345-536 of the HA protein) as positive controls for FCM (Fig. 1) . The HA1 and HA2 displayed on the yeast surface were recognized by the mouse, goat and human antisera, and confirmed by FCM (Fig. S2 , panels B, E, and, H; and C, F, and I, respectively). Screening of antigenic peptides recognized by mouse antisera immunized with HA protein
We first screened the yeast library displaying the H1N1 HA peptides to study the murine immune response to recombinant HA protein of the 2009 A(H1N1) Influenza virus. Freshly induced library cells were incubated with mixed sera from immunized mice and subsequently labeled with fluorescent secondary antibodies. To avoid potential bias introduced by growth advantage of the yeast cells, only one round of sorting was performed. Plasmids isolated from positive yeast cells were transformed into E. coli cells and plated. Single clones (100) were picked randomly and sequenced, of which 82 (82%) were shown to contain in-frame inserts. These results indicate that the sorting process is able to distinguish in-frame sequences from the random library that contained only 1/6 (16.7%) in-frame inserts efficiently. After verification of individual peptides by FCM detection, the ratio of recombinant antigen expressed on the yeast surface (RAYS) was determined by dividing the mean fluorescence intensity of peptide expressing yeast cells by the mean fluorescent intensity of the nonexpressing (pCTCON-2) yeast cells [26] . 56 positive clones with a RAYS ratio $2 were considered as antigenic and analyzed at both the sequence and structural level [26] . Alignment of positive peptides with the original HA sequence shows that 87% are in the HA2 region (95627 aa), with just 13% in the HA1 region (259626 aa) which cover nearly the entire HA1 protein (Fig. 2, panel A). The FCM histograms of several representative yeast clones are shown in Fig. 3 , panel A. Clones M-5 (corresponding to residues 55-313 of HA, or 70 aa shorter than HA1) and M-7 (residues 69-275, or 120 aa shorter than HA1) were strongly positive (RAYS ratio .5), and contained the entire receptor binding domain (RBD) and the vestigial esterase domain in the globular HA head. Clones M-33 (residues 387-489) and M-52 (residues 423-492) displayed much shorter peptides but also showed strong fluorescent FCM signals (Fig. 3 , panel A), albeit with relatively lower RAYS ratios of 3-4. Judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally weaker than the whole HA1 (Fig. S2,  panel B) .
The sequencing results of the antigenic peptides alone are less informative (Fig. 2) . Nevertheless, when we perform a statistical analysis by summarizing the normalized frequency of each amino acid residue in all positive antigenic peptides (defined as the residue frequency map), a major peak with the full width at half maximum (FWHM) from residues 387-480 is revealed (Fig. 4 , panel A). We also modify the residue frequency map by taking into account the respective RAYS ratios of positive peptides, which however results in an overall rather similar frequency map (Fig.  S3, panel B) . Furthermore, even if we combine all the in-frame sequences from sorted yeast cells, a similar map (Fig. S3 , panel C) is again obtained, implying that the noises from the in-frame but The residue frequency map is then overlaid onto the crystal model of the trimeric HA of the 2009 A(H1N1) influenza virus (PDB ID: 3LZG) [27] , and annotated by colors from red (high frequency) to blue (low frequency) (Fig. 4, panel B ). This structural frequency map reveals that the major peak (corresponding to residues 387-480 within the FWHM) on the residue frequency map can be divided into two dominant antigenic regions since they have independent structural features. One corresponds to the central coiled-coil helix CD in the HA2 subunit responsible for membrane insertion, designated as R1 (residues 424-480, indicated by red, see also Fig. 5 ), while the other contains helix A in the HA2 subunit that is important for conformational change upon low pH exposure, designated as R2 (residues 387-423, indicated by faint red and white, see also Fig. 5) [28] . R1 is present in a relatively recessed surface area while R2 is located near the exterior surface area of the stem region. For the HA1 subunit, no distinct antigenic region is identified, because of the limited number of peptides (7) with relatively longer lengths (all .200 aa), which are insufficient to form peak areas.
We also screened the same yeast library against serum samples from goats immunized by the 2009 A(H1N1) influenza virus to examine whether different animal models would yield a similar profile of antigenic peptides. Following sorting and sequencing, 78/100 clones were confirmed as in-frame, among which 55 tested positive by the goat anitsera in FCM (RAYS ratio $2). Surprisingly, alignment of the positive antigenic peptides with the full HA protein shows a distribution strikingly different from the alignment obtained using the mouse antisera in several aspects. First, 78% of the antigenic peptides are located in the HA1 region and have shorter lengths (54632 aa), while only 9% are in the HA2 region (96619 aa). Second, 13% of the peptides are in the region across HA1 and HA2 (66631 aa) (Fig. 2, 
Representative antigenic clones were subjected to FCM. As shown in Fig. 3 , panel B, clones G-15 (residues 38-136 of HA) and G-29 (residues 70-162) in the HA1 region, G-46 (residues 308-339) near the HA1-HA2 cross region, and G-55 (residues 413-479) in the HA2 region, all shorter than 100 aa, demonstrated strong antigenicity (RAYS ratio .5). Judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally stronger than the whole HA1 (Fig. S2 , panel E).
By analyzing the residue frequency map, more peaks are revealed than those obtained from the mouse anitsera, with the largest one with its FWHM spanning residues 22-125 with a small split around residue 60, and a medium one with its FWHM at residues 303-350 (Fig. 4 , panel C, and Fig. S4 ). In the structural frequency map (Fig. 4 , panel D), the largest peak is composed of two independent regions: residues 22-60 at the N-terminus of HA1 adjacent to helix A on HA2, designated as R3; and residues 61-125 located in the vestigial esterase domain of the globular head, designated as R5 (indicated by two red regions in the globular head and stem region, respectively, see also In order from most membrane proximal to distal: R1 (orange, residues 424-480 of HA) and R2 (yellow, residues 387-423) in HA2 were determined by screening against mouse and human antisera; R3 (purple, residues 22-60), R4 (green, residues 303-350), and R5 (cyan, residues 61-125) in HA1 were determined by screening against goat antisera. The HA monomer cartoon view is shown on the right and follows the same coloring scheme, with the third monomer shown in the back and colored in grey. doi:10.1371/journal.pone.0018016.g005 should be noticed that R1 and R2 identified in the mouse serum screening also appear in this residue frequency map, although in a much less dominant manner as illustrated by the small plateau between the end of R4 and residue 479 (Fig. 4, panel C) . Taken together, the antigenic peptides recognized by the goat antisera are different from those recognized by the mouse antisera, and in particular present a more diverse picture of antigenic sites in the HA1 region.
The screening against human plasma immunized by the 2009 A(H1N1) influenza vaccine was carried out essentially as described above. Among the 100 sequences obtained after FACS analyses, 74 peptides were found to be in-frame, and 51 were confirmed to be antigenic (RAYS ratio $2). It is interesting to see that the distribution of theses peptides is similar to that obtained using the mouse antisera, with only 4% of peptides in the HA1 region (87 and 37 aa in length), 4% across HA1 and HA2 (96 and 39 aa in length), and 92% in the HA2 region (100630 aa) (Fig. 2 , panel C). Using FCM, three representative clones, H-11 (residues 359-479 of HA), H-26 (residues 390-480) and H-45 (residues 423-480), all in the HA2 region, showed strong signals (RAYS ratio .5). Although only 2 antigenic peptides are in the HA1 region, a clone expressing H-1 (residues 108-194), which harbored the Sa antigenic site in RBD of the HA protein, yielded a RAYS ratio greater than 3 (Fig. 3, panel C) . Again, judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally weaker than the whole HA1 (Fig. S2,  panel H) .
The residue frequency map also reveals a major peak (FWHM at residues 392 to 480). This peak position is similar to that obtained using the mouse antisera (FWHM at residues 387-480), resulting in an almost identical structural frequency map (Fig. 4 , panels E and F, and Fig. S5 ). Thus, these antigenic regions are considered identical to R1 and R2 obtained from the mouse serum screening (Fig. 5 ).
The accessibility of short antigenic peptides displayed on the yeast cell surface [19] [20] [21] was confirmed in this study with fluorescence confocal microscopy, as exemplified by the antigenic clones G-29 (93 aa) and G-46 (32 aa), from the screening against the goat antisera (Fig. S6) . As shown in the figure, the antigenic peptides were localized on the outer surface of the membrane, and well accessible to the antibodies in the complex antisera.
Three representative yeast clones displaying antigenic peptides G-15, G-55, and H-11, obtained from the screening against the goat antisera and human plasma samples, were characterized for neutralization titers (IC 50 ) by testing protection efficacy to MDCK cells from the A/reassortant/NYMC X-179A/California/07/ 2009(H1N1) exposure. Cells containing pCTCON2 were used as absorbent controls for evaluation of background IC 50 values. HA1 and HA2 were also incorporated to determine the ability of the globular head and stem regions to absorb antibodies in the antisera. As summarized in Table S1 , all single clones reduced the IC 50 of the corresponding antisera, demonstrating qualitatively the neutralization efficacy of these antigenic peptides.
Binding affinities of purified peptides to mouse, goat and human antisera
To further confirm the antigenic activities of the identified R1-R5 regions, five newly designed short peptides encompassing regions R1-R5 (Fig. 6, panel A) were expressed as C-terminal fusions to thioredoxin (Trx) and purified from E. coli [29, 30] . The binding affinities of these peptides to the mouse, goat and human antisera were characterized by the method of ELISA, with thioredoxin as the control (Fig. 6, panels B and D) . As can been seen from the figure, the ELISA results generally correspond well to the screening results shown in Figs. 4 and 5 . Specifically, the peptide P1 (corresponding to the R1 region identified in all three screenings, Figs. 4 and 5 ) was reactive to all three antisera. The peptides P3, P4 and P5, corresponding to the R3-R5 regions identified only in the screening against the goat antisera (Fig. 4 , panels C, D, and Fig. 5) were only reactive to the goat antisera (Fig. 6, panel C) . There is one exception in that the peptide P2 (corresponding to the R2 region identified in the screenings using the mouse and human antisera, Figs. 4 and 5) showed no measurable affinity to either mouse or human antisera, suggesting that the R2 region might be less conformation-independent than the other four regions.
It should be noted that, after subtraction of the background binding of the negative control protein (Trx), the binding affinity of the peptide P1 to the human antisera after vaccination increased only about one fold compared with the antisera before vaccination (Fig. 6, panel D) . This might be contributed to cross-reactive antibodies to other influenza subtypes pre-existing in the human serum subjects, given the conservative nature of the P1 sequence or the R1 region [13] .
We report here the identification of antigenic peptides of the 2009 A(H1N1) influenza HA protein from a combinational library of viral protein fragments displayed on the surface of yeast cells and sorted by FACS. The peptide fragments were constructed in a way to have a broad but limited range of lengths [24] , and screened using antisera from mouse, goat and human. We then used a novel statistical approach to identify antigenically important regions of the viral protein based on the frequency of each residue appearing in the antigenic peptides at both the primary sequence and structural level.
This approach reveals interesting antigenic features of the HA protein. First, the antibody responses to the 2009 A(H1N1) viral protein HA appear to vary significantly depending on the species, with the goat response being strikingly different from those of mouse and human (Fig. 4) . These results imply possibly different immune responses among animal species, and in the case of human, the specific immunization background may also play a role [13] . Moreover, the antigenic peptides obtained from the screening against the mouse or human antisera are predominantly located in the HA2 region, whereas those from the screening against the goat antisera are predominantly in the HA1 region. Second, five antigenically important regions, R1 to R5 in the order from most membrane proximal to distal (Fig. 5) , are identified. Moreover, except for R5 which is located in the vestigial esterase domain of the HA globular head, all of the other regions are in the HA stem region. Among them, R1 and R2 come together to form a major peak with a FWHM range from residues 387 to 480 (Fig. 4, panels A and E) . Although the peptide P2 corresponding to the R2 region showed no measurable affinity to either mouse or human antisera (Fig. 6) , which suggests that this region might be more conformation-dependent than the rest four regions, it is PLoS ONE | www.plosone.org assigned as a separate region for the following reasons: (1) structurally it is distinguishable from the R1 region (Fig. 5) , and (2) several recent studies have reported the epitopes of monoclonal antibodies targeting H1 subtype HA proteins, which fall into the R2 region of this study [10, 31, 32] (see below).
Previously most influenza HA antibodies have been found to recognize epitopes in the globular head and interfere with binding to target cells [6] [7] [8] [33] [34] [35] [36] , for example, the classical five antigenic sites (Sa, Sb, Ca 1 , Ca 2 , and Cb) [6, 7] . However, technically the monoclonal antibodies used in these previous studies were obtained from murine hybridoma cells by radioimmunoassay (RIA) or hemagglutination inhibition (HI) assay. The nature of these assays favored the isolation of antibodies that bound predominantly to the globular head of the virus and inhibited binding of the virus to the host cells. In this current study, however, complex antisera were used, which should contain antibodies recognizing other regions of the HA protein. On the other hand, only one antigenic region (R5) is identified in the globular head in this study, possibly because the epitopes in this head region are more conformation-dependent and may require longer peptides or even a complete domain of HA1 to be displayed for full antigenicity (at least for the mouse serum and human plasma samples). It is then noteworthy that the head region, especially the area around the receptor binding site, contains mostly beta structures, which more likely require stabilization by long-range interactions than the helical structures in other regions of HA (Fig. 5) . However, the peptide library constructed in this study was enriched at 100-500 bp (or less than 170 aa), which was likely insufficient to map the conformational epitopes. Nonetheless, the current screening method is a valuable complementation to the more classical approaches that are biased toward full HA1 or HA2 domain.
Several recent studies support our findings regarding antigenic regions R1-R4 in the stem region of HA. In efforts aimed at characterizing antibodies with cross-neutralizing activity for various influenza virus subtypes [10, 13, 31] , it was found that most of these antibodies bind to the HA stem region and interfere with conformational changes of the protein critical for membrane fusion. For example, in the case of antibody CR6261 (binding to HA of the SC1919 virus, H1 type), two antigenic regions on HA stem were revealed: helix A in the HA2 region, and the adjacent HA1 region [32] . The critical sites in helix A (corresponding to residues 390-391, 393-395, 398, 401-402, 405 in H1N1 HA) fall within the R2 region identified in this study, whereas the hydrophobic interaction sites in the adjacent HA1 region (corresponding to residues 38-42, 306-307 in H1N1 HA) fall into regions R3 and R4 of this study, respectively. More recently, it was reported that for a different cross-neutralizing antibody that targets a broad range of H3 subtype virus strains, 12D1, the dominant contacts on the antigen (corresponding to residues 425-455 in H1N1 HA) [37] correlate well with the R1 region found in this study. Furthermore, vaccination in mice using a HA2-based synthetic peptide mimicking the 12D1 epitope was shown to provide protection against influenza viruses of H3N2, H5N1, and H1N1 subtypes [38] . Taken together, our approach should be useful for dissecting antigenic regions on the HA stem, and providing clues for designing more potent peptide vaccines in inducing cross-neutralizing antibodies than the entire HA protein [32] .
Compared with other labor-intensive epitope identification methods that rely on identifying critical residues in escape mutants and crystal structures, the method used here gives a direct and panoramic mapping of the antigenic regions of viral proteins recognized by antisera in a facile and high-throughput way. Our approach likely contains an inherent bias for shorter peptides, but nonetheless complements the more classical approaches that favor longer antigenic peptides or full antigenic domains. The scanning of antigenic peptides based on phage, bacterial or yeast surface display methods has been increasingly used in recent years. Our approach presents two advantages. First, technically we adopt an easy-to-implement scheme for generating a controllable size of peptides and our screening typically involves only one round of FACS-based sorting, in contrast to multiple rounds of panning or sorting used in the literature [15, 16, 18] . Although the latter results in many repeat sequences, we argue that much information is lost in the enrichment process. Second and more importantly, since simple alignment of antigenic peptides with the corresponding viral protein yields only limited information [15, 17, 39] , we employ a novel statistical means of summarizing the frequency for each residue appearing in the antigenic peptides at both the primary sequence and structural level, which enables a direct mapping of the antigenic structure of a viral protein. Along this line, it is interesting to note that, using our statistical approach, we reevaluate the highly repeated antigenic peptides identified for the H5N1 HA protein in a recent report [15] . Several major peaks are clearly revealed (Fig. S7) , which should be useful for further characterization of the antigenic peptides for H5N1 HA. (H1N1) influenza vaccine, and a mixture of 9 samples with the highest activity among 110 samples as judged by ELISA. These samples were provided by Dr. Boping Zhou of Shenzhen East Lake Hospital (Shenzhen, China), and written informed consent was obtained from all study participants. All samples were de-identified prior to analysis, and the protocols were approved by the Institutional Review Board (IRB) of Shenzhen East Lake Hospital. All viral strains are closely related.
Restriction enzymes and DNA polymerases were purchased from New England Biolabs (Beverly, MA) or Takara (Dalian, China). DNase I was obtained from Worthington (Lakewood, NJ). Fluorescence-labeled secondary antibodies were purchased from Invitrogen (Shanghai, China) or Santa Cruz Biotechnology (Santa P5 (blue arrows), in relation to the five antigenic regions R1-R5. The coordinate of the HA protein is indicated by the grey bar, with the five antigenic regions represented in the same color as in Fig. 5 . The peptides P1-P5 were expressed as C-terminal fusions to the thioredoxin (Trx) tag. Binding activities of the peptides against the mouse (B), goat (C) and human (D) antisera before and after immunization were characterized by the method of ELISA, with the Trx protein as the negative control. The x-axis shows the dilution ratios of corresponding antiserum samples. The y-axis shows the absorbance at 450 nm after development with the substrate 3,39,5,59-tetramethylbenzidine (TMB). doi:10.1371/journal.pone.0018016.g006
Cruz, USA). Oligonucleotides were synthesized by Invitrogen or Takara. The kits for DNA purification, gel recovery, plasmid miniprep and A-Tailing modification were obtained from Tiangen (Beijing, China) or Takara (Dalian, China). The kit for yeast plasmid DNA isolation was from Omega Biotech (Victoria BC, Canada). Sequencing was performed by Invitrogen or by SinoGenoMax (Beijing, China). Escherichia. coli DH5a was obtained from Takara. Yeast strain Saccharomyces cerevisiae EBY100 was obtained from Invitrogen.
The pCTCON-2 yeast display vector was kindly provided by Prof. Dane Wittrup [23] . The DNA fragments coding the HA1 and HA2 proteins were amplified from plasmid CMV-R-Cali Two Xcm I restriction sites (CCANNNNN/NNNNTGG) were introduced between Nhe I and BamH I sites of pCTCON-2 to create pCTCON-T. Digestion of pCTCON-T by Xcm I restriction nuclease and gel extraction yielded the T-vector with two 39 T overhangs. Random fragments with 39 A tails were inserted between the two Xcm I sites by T-A ligation.
The gene coding for the full HA protein of A/California/04/ 2009(H1N1) virus was amplified from plasmid CMV-R-Cali-04-09 with the forward primer 59-CGCCACCATGAAGGC-TATCC-39 and reverse primer 59-TTAGATGCAGATTCTG-CACTG-39. Detailed procedures for fragmentation and reassembly were described previously, except that PhusionH high-fidelity polymerase (NEB) was used in reassembly [24] . The reassembled DNA samples were purified and modified to add 59 A overhangs by an A-Tailing Kit (Takara). The backbone vector pCTCON-T was digested with Xcm I and purified with a Tiangen gel purification kit to reduce self-ligation. The gene fragments and backbone vector were ligated at 16uC for 16 h and then electroporated into E. coli DH5a competent cells for propagation. The plasmid library was then isolated and used to transform yeast EBY100 cells by the Li-Ac method [40] . Expression of peptide libraries on yeast EBY100 cells was performed as reported by Wittrup's group [23] . Transformed yeast cells were grown for 24 h at 30uC with shaking in SDCAA medium (yeast nitrogen-based casamino acid medium containing 20 g?L 21 glucose) and passed one time into fresh medium to eliminate dead cells. The library cells were then centrifuged, resuspended in SGCAA medium (yeast nitrogen-based casamino acid medium containing 20 g?L 21 galactose) to an OD 600 value of 0.5-1 and induced at 20uC for 36-48 h with shaking.
Prior to yeast library screening, nonspecific interactions of serum samples (i.e., mouse, goat or human antisera) with yeast proteins were removed by incubation with induced yeast cells carrying the control vector pCTCON-2. To label cells for FACS, 1610 7 (For yeast EBY1001 cells, OD 600 <1610 7 cells/mL) freshly induced library cells were pelleted at 8,000 g for 1 min in a 1.5 mL microcentrifuge tube and washed two times with 1 mL of 16Phosphate Buffered Saline (PBS). Cells were then incubated with 100 mL of pre-absorbed serum (1:50 diluted in PBS) at 4uC for 1 h. Unbound antibodies were removed by washing two times with 1 mL of PBS. The library cells were incubated with 1-2 mg of fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled secondary antibodies in 100 mL at 4uC for 45 min. After washing two times with PBS, cells were resuspended in 1 mL of PBS and loaded onto a BD-FACS AriaII TM machine (Shanghai, China) for sorting. Yeast cells harboring the pCTCON-2 vector were processed as described above and used as a negative control. The positive gate was set to exclude all negative control cells (,0.1% leakage). The total amount of cells used for sorting was adjusted to ensure that more than 1610 5 cells could be harvested, which were then collected in 5 mL of SDCAA medium and grown overnight at 30uC with shaking.
After one passage of library cells into fresh medium, heterogeneous plasmid DNA was isolated from the library and transformed into E. coli DH5a cells. For each library, a total of 100 colonies were randomly picked from the plates and sequenced using a pair of primers (pCTCON2-Seq-For: 59-GTTCCAGAC-TACGCTCTGCAGG-39 and pCTCON2-Seq-Rev: 59-GTTC-CAGACTACGCTCTGCAGG-39). The plasmids carrying gene fragments inserted in-frame with the original HA gene were retransformed into yeast EBY100 cells, induced, and labeled as described for FCM detection on a BD-FACS Calibur cytometer (Shanghai, China). The ratio of recombinant antigen expressed on the yeast surface (RAYS) was determined by dividing the mean fluorescence intensity of peptide expressing yeast cells by the mean fluorescence intensity of the non-expressing (pCTCON-2) yeast cells. Clones with a RAYS ratio $ 2 were considered positive and the corresponding peptide sequences were selected for further analysis [26] .
Yeast cells containing plasmids for displaying antigenic peptides were grown at 30uC for 24 h in SDCAA medium and induced at 20uC for 36 h in SGCAA medium. 1610 7 cells were harvested by centrifugation, incubated with goat antisera (1:100 diluted in 100 mL of PBS) and labeled with 1 mg of FITC conjugated antigoat IgG secondary antibody as described above. Labeled cells were resuspended to 5610 7 cells/mL, fixed with 4% paraformaldehyde and photographed on a Zeiss 710 laser scanning confocal microscopy (Carl Zeiss, Germany) at the Center of Biomedical Analysis, Tsinghua University. Yeast cells containing nonexpressing vector pCTCON-2 and HA1-expressing vector pCTCON-HA1 were treated in the same way as negative and positive controls, respectively.
ELISA assays for purified representative peptides against three antisera Five peptides newly designed to encompass the regions R1-R5 were expressed as fusions to the C-terminus of thioredoxin (Trx) and purified from E. coli, using a modification of the self-cleaving elastin-like polypeptide tag method (ELP) (with purity over 80% as judged by SDS-PAGE) [29, 30] . For comparison, the Trx tag was used as the negative control. When mouse and goat antisera were employed as the first antibody, the wells were coated with 100 ng of peptides or Trx. But for human antisera, 10 ng of peptides or Trx control was used to decrease the background signal caused by unspecific interactions between the human antisera and the trace impurities from E. coli cells. After blocking with 10% Fetal Bovine Serum (FBS) diluted in 16PBS with 0.25% Tween-20 (PBST), serial dilutions were added to each well and incubated for 1 h at 37uC, followed by addition of 1:5,000 diluted HRP-conjugated secondary antibody. Assays were developed by adding 100 mL of 3,39,5,59-tetramethylbenzidine (TMB) substrate solution and the reactions were stopped with 50 mL of H 2 SO 4 (1 M). The assays were carried out in duplicates and each well was washed 4 times with PBST between steps. The absorbance at 450 nm was recorded on a SpectroMAX 190 Microtiter reader (Molecular Devices, CA).
Prior to the Neutralization assay, 500 mL antisera (diluted 1:50, and pre-absorbed with induced yeast cells carrying the control vector pCTCON-2) were mixed with freshly induced yeast cells (1610 8 ) displaying specific antigenic peptides on surface. After incubation at 4uC for 12 h, the mixtures were centrifuged at 8,000 g for 1 min and supernatants mixed with another batch of freshly induced yeast cells. This process was repeated four times, and then the supernatants were used for determining the neutralization titers, using a microneutralization assay with A/reassortants/NYMC X-179A/California/07/ 2009(H1N1) virus and MDCK cells according to standard procedures [41] . Briefly, triplicate serial dilutions (1:50 to 1:1600) of antiserum samples were incubated with 100 50% tissue culture infective dose (TCID 50 ) of virus for 2 h at 35uC prior to adding MDCK cells. Cells were incubated at 35uC for 72 h, and the half maximal inhibitory concentration (IC 50 ) was determined. All the neutralization assays were performed at the AIDS Research Center, School of Medicine, Tsinghua University following standard procedures. Figure S1 Schematic outline of the screening approach. In Step 1, the gene encoding the viral protein H1N1 HA was amplified, digested and re-assembled to generate the fragment library (the red and grey segments indicate the antigenic and nonantigenic peptides, respectively). In Step 2, the fragment library was ligated into the display vector pCTCON-T, transformed into yeast cells and induced for expression. In Step 3, the yeast cells expressing random peptides were incubated with antisera and fluorescence-labeled second antibodies and subjected to FACS in Step 4. Afterward in Step 5, the gene fragments were isolated from the sorted cells and sequenced. The in-frame sequences were retransformed into yeast cells and verified by FCM detection individually. In Step 6, an antigenic peptide profile was extracted for the sequences corresponding to the antigenic peptides, based on which the residue frequency and structural frequency maps were derived in Step 7. (TIF) [15] . The x-axis represents all H5N1 HA amino acid residues. The y-axis shows the normalized frequency of individual residue appearing in the 784 antigenic peptides (39 unique sequences) obtained from panning against H5N1 avian influenza convalescent sera. The six clusters (I-VI) defined by Khurana et al. are graphically represented below the x-axis. Several representative antigenic peptides are also shown as green arrows (numbered according to Khurana et al.) . Even though the antigenic peptides were enriched multiple times during the screening process and thus these peptides are less diverse and might be biased in sequences, several peaks are clearly identifiable, and predominantly in the HA2 region. (TIF)  New Insights of an Old Defense System: Structure, Function, and Clinical Relevance of the Complement System The complement system was discovered a century ago as a potent defense cascade of innate immunity. After its first description, continuous experimental and clinical research was performed, and three canonical pathways of activation were established. Upon activation by traumatic or surgical tissue damage, complement reveals beneficial functions of pathogen and danger defense by sensing and clearing injured cells. However, the latest research efforts have provided a more distinct insight into the complement system and its clinical subsequences. Complement has been shown to play a significant role in the pathogenesis of various inflammatory processes such as sepsis, multiorgan dysfunction, ischemia/reperfusion, cardiovascular diseases and many others. The three well-known activation pathways of the complement system have been challenged by newer findings that demonstrate direct production of central complement effectors (for example, C5a) by serine proteases of the coagulation cascade. In particular, thrombin is capable of producing C5a, which not only plays a decisive role on pathogens and infected/damaged tissues, but also acts systemically. In the case of uncontrolled complement activation, “friendly fire” is generated, resulting in the destruction of healthy host tissue. Therefore, the traditional research that focuses on a mainly positive-acting cascade has now shifted to the negative effects and how tissue damage originated by the activation of the complement can be contained. In a translational approach including structure-function relations of this ancient defense system, this review provides new insights of complement-mediated clinical relevant diseases and the development of complement modulation strategies and current research aspects. The complement system was first recognized in the late 19th century when leading microbiologists such as Paul Ehrlich, Jules Bordet and George Nuttall discovered a bactericidal function of blood on anthrax bacilli (1) (2) (3) (4) . They noted that this bactericidal function was inactivated when blood was heated up to 55°C or kept at room temperature and named it "alexin."
Research on guinea pigs demonstrated that the bactericidal activity of blood not only depended on the already described heat-labile alexin, but also on a heat-stable bactericidal factor. In 1899, Paul Ehrlich renamed alexin as complement and called the heat-stable substance amboceptor (3) .
By 1920, four components of complement (C1, C2, C3 and C4) had already been detected, each factor being assigned a number in the order in which it had been discovered. Although the order of their discovery did not represent their activation sequence, the names were kept to avoid confusion. The antibodydependent pathway of complement activation was named the "classical pathway." Although it had already been discovered in 1913 that some bacteria and yeast as well as cobra venom factor could induce the complement system independently of antibodies, it was not until 1954 that Pillemer discovered the "properdin pathway." Now known as the "alternative pathway," it is able to induce the complement cascade independently of antibody interaction by binding directly to bacteria and yeast (5) .
Two decades ago, the mannosebinding lectin (MBL), or "lectin activation pathway," was discovered. Kawasaki et al. (6) found the MBL protein in 1978, but its function remained unclear until 1989, when Super et al. (7) recognized that reduced serum levels of MBL correlated with an opsonic defect in children. Matsushita et al. then detected the proteolytic activity of the MBLassociated serine proteases (MASP-1 and MASP-2), leading to the formation of the classical C3 convertase (8) (9) (10) (11) .
antibody-independent binding of danger signals such as bacteria, yeast and virusinfected cells, but also protein A, C-reactive protein, cobra venom factor, polysaccharides and damaged tissue (14, 16) . Because constant activation of the alternative pathway is due to spontaneous hydrolysis of the highly reactive C3, constant control by complement regulators is required (17) . Healthy cells are capable of various control mechanisms that prevent the spontaneous activation of C3 and protect the host from undesirable complement activation. These control mechanisms exist both in the fluid phase and membrane bound (see below).
The spontaneous hydrolysis of C3 produces C3(H 2 O), which functionally resembles C3b. C3(H 2 O) associates reversibly with factor B, while plasmatic protease factor D cleaves factor B. This event, as well as the small fragment Ba, produces the C3 convertase of the alternative pathway C3(H 2 O)Bb. Binding of the protein properdin stabilizes the fragment, extending the half-life 10-fold. The C3 convertase then splits C3 into C3a and C3b, with C3b being capable of creating a new C3 convertase with the aid of factor B and D. This amplification loop is highly important not only for the alternative pathway but also for the two other activation pathways.
Binding of more C3b to the C3 convertase now creates the C5 convertase C3(H2O)BbP3b. This initiates the terminal enzymatic cascade of the lytic membrane attack complex (13) (14) (15) 18) .
The lectin pathway. The lectin activation pathway has been rather less intensely studied. Activation takes place when MBL binds mannose-containing surface proteins on pathogenic surfaces. In its ultra-structure, MBL closely resembles C1q, and along with the serine proteases MASP-1 and -2 (which themselves resemble C1r and C1s, respectively), forms a potent multi-enzyme complex ( Figure 1 ).
Upon activation, MASP-2 catalyzes the cleavage of C2 and C4 in a similar manner to the classical pathway and forms a C3 convertase named C4b2a. MASP-1 is capable of C2 and C3 cleavage, although to a much lesser extent (11, 19) . Subsequently, C3 is cleaved into C3a and C3b, and by accretion of C3b to the C3 convertase, the C5 convertase is formed. A third serine protease MASP-3 has a distinct function compared with MASP-1 and -2, exerting inhibitory actions against MASP-2 (20) .
In addition to the established activation of the lectin pathway via MBL and MASPs, it was demonstrated that ficolins were also capable of initiating the lectin pathway by forming active complexes with MASPs. There are three distinct ficolins named ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen). Structurally homologous to the collectin MBL as well as C1q, ficolins are soluble collagen-like proteins that bind to sugar structures presented on microorganisms and dying host cells and consequently activate the innate immune system (8, 10, (21) (22) (23) .
Surfactant protein A and D (SP-A, SP-D), such as MBL, belong to the collectin family (24) , but unlike MBL, they are not able to activate the complement directly.
The impact of the MBL pathway remains to be completely elucidated. It is suspected that its major role takes place during early childhood and in particular during the translational period from the passive immunity provided by the mother's antibodies to the development of the body's own mature immunity (25) .
The lytic membrane attack complex. The final stage of all three activation pathways is the formation of the lytic membrane attack complex (MAC). In contrast to the three different upstream paths forming a C5 convertase, only the cleavage of C5 into the anaphylatoxin C5a and the active C5b represents an enzymatic step, while the rest of the cascade is solely an accretion of stable proteins. In detail, C5b remains bound to the target cell followed by association of C6, resulting in a hydrophilic complex. By accretion of C7, a conformational change occurs-facilitating a stable linkage by exposure of lipophilic groups. Attachment of C8 with its binding component, C8b, induces the penetration of C8a-g into the lipid double layer of the target cell membrane. The final step toward formation of a stable transmembrane pore with a diameter of 10 angstrom is the binding of 10-15 C9 proteins, which generate a cylindrical structure ( Figure  2) . Assembly of such a pore may lead to osmotic imbalance through the constant flow of ions, small molecules and water along their concentration gradient, resulting in the lysis of the target cell (26) .
It is noteworthy that the importance of these transmembrane pores should not be overrated, since, for instance, the blockage of the MAC only leads to a small increase in bacterial Neisseria infection (25, 27) . The upstream effects of the complement system, such as the anaphylactoid reaction and the opsonization, appear to play a more important role (14, 28) .
Effects of the complement system. The main effect of the complement system is the induction of a pathogen-associated and modulated enzymatic cascade that, once triggered, ends with the lysis of the target cell and protects the host from infection. In addition to this apparent effect, the complement system also displays crucial additional activities that appear to be even more relevant.
One effect is the opsonization of the pathogen. Cleavage products such as C3b and C4b as well as C5b opsonize the surface of recognized pathogenic substances and therefore facilitate phagocytosis. Additionally, opsonization is also important for the clearance of soluble, circulating antigen-antibody complexes. After the attachment of C3b and C4b to these complexes, they are bound to com-plement receptor 1 (CR1) on erythrocytes and are subsequently transported to the spleen and liver, where the immune complexes are eliminated.
C3 cleavage products also bridge the innate and the adaptive immune systems. Opsonized antigens are bound to the complement receptor 2 (CR2) on B-cells via the C3-fragment C3d, initiating the production of specific antibodies as well as the differentiation of B-memory cells.
Presumably, the most important function is the induction of an anaphylactoid reaction. The small activation products C3a, C4a and particularly C5a are potent anaphylatoxins, capable of inducing the migration of phagocytes (29) , smooth muscle relaxation, degranulation of mast cells and basophile granulocytes and therefore unleashing vasoactive substances such as histamine, prostaglandins, kinins and serotonin. All can cause vasodilation and capillary leakage (30) and induce the migrated cells to release eicosanoids, oxygen radicals and lysosomal enzymes, which cause damage to the pathogens (31) (32) (33) . It is noteworthy, that complement acts far beyond "inflammation," as indicated by its close interaction with the coagulation cascade (34) (35) (36) and its involvement in the regulation of apoptosis (37) (38) (39) (40) and cellular growth (41) . The anaphylactoid functions are mediated by the interaction of C3a and C5a with their corresponding seventransmembrane-spanning receptors C3aR and C5aR (CD88), respectively (see Figure 2 ). The role of the second C5a receptor named C5L2 is not fully understood and is still controversially discussed. However, there is increasing evidence that C5L2 represents a functional receptor acting as a negative regulator of the inflammatory response. For example, it was shown that inflammation in C5L2 knock-out mice was amplified (42) and that blockage of C5L2 increased serum interleukin (IL)-6 (43).
In summary, complement is highly capable of inducing all classical signs of inflammation, with the occurrence of pain, swelling, reddening, hyperthermia and impaired function.
In addition to the established pathways, new pathways of complement activation were recently discovered ( Figure 3 ).
During the last decade, more and more interaction sites between the two major serine protease systems of the human body, namely the coagulation and the complement cascades, were found.
The potent serine protease thrombin is able to directly cleave C3 as well as C5 in a dose-and time-dependent manner, leading to biologically active C3a/C5a (44) . In addition to thrombin, investigations by our group indicated proteolytic cleavage of C3 and C5 also by FXa, FXIa and plasmin (35) . Interestingly, FVIII and tissue factor failed to directly interact with C3 and C5 (35) . Furthermore, FXIIa activates the classical complement pathway via C1q. Crosstalk between the lectin pathway and the coagulation cascade has only recently been ascertained by the observation that the complex of FVIII and von Willebrand factor possesses lectin activity (45) .
Vice versa, complement factors also interact with the coagulation system. C1 inhibitor not only blocks all three established complement pathways but also the endogenous coagulation path (46). Ikeda et al. (47) found evidence that C5a induces tissue factor activity on endothelial cells. Furthermore, crosstalk between the anaphylatoxin receptor C5aR and tissue factor was recently found (36, 48) .
Another possibility for complement activation exists via direct cellular interactions. Huber-Lang et al. demonstrated that phagocytic cells (macrophages, polymorphonuclear leucocytes [PMNs]) are able to cleave C5 into biologically active C5a. This cleavage was conducted by a cell-bound serine protease that was inducible for alveolar macrophages, being more constitutively active on PMNs (49) .
The complement system can exert manifold detrimental effects not only on pathogenic or damaged tissue but also on healthy host tissue. To protect against a complement attack, the human body has developed various strategies. Principally, there are both membrane-bound and fluid phase complement regulators that are briefly covered ( Figure 4 ).
The best known regulatory protein is the C1 inhibitor (C1-INH). C1-INH controls the activity of the classical pathway by binding to the C1 complex and initiating the diffusion of the fragments C1r and C1s. This process leads to an irreversible inactivation of the initiating serine protease. As the classical and the lectin pathway resemble each other in many ways, the C1-INH also inactivates MASP-1 and -2, thereby also inhibiting the lectin pathway. As well as inactivating complement components, C1-INH also blocks certain parts of the kinin, fibrinolytic and coagulation systems, such as coagulation factors XII and IX.
Factor I is a serine protease catalyzing the cleavage of the α-chain of C3b and C4b, leading to their permanent inactivation. Cofactors for this enzymatic activity are factor H, C4-binding protein (C4-bp), CD35 and CD46.
Factor H is a protein that hinders the formation of the C3 convertase by competing for the binding site with factor B. Additionally, it facilitates the dissociation of already active C3 convertases and supports the proteolytic cleavage of C3b by factor I.
C4-bp also facilitates the proteolytic cleavage of the α-chain of C4b by factor I in a complex together with protein S. Serumprotein S (Vitronectin) and clusterin (Sp-40, 40) hinder the formation of the lytic membrane attack complex by adhesion to the lipophilic groups of C7, therefore leading to impaired anchorage in the cell membrane.
Carboxypeptidase N inactivates anaphylatoxins of the complement system as well as other factors such as kinins and creatinine kinase MM through cleavage of terminal arginine and lysine residues of the peptides (17, 50) .
CD35 (complement receptor 1 [CR1]) is found on the surface of erythrocytes as well as on leukocytes and on podocytes in the glomerula of the kidney. CD35 facilitates the decay of the C3/C5 convertase and also acts as a cofactor for factor I. CD46 (membrane cofactor protein [MCP]) also acts as a cofactor for factor I-mediated cleavage of C3b and is broadly expressed, except on erythrocytes.
CD55 (decay accelerating factor [DAF]) is widely expressed, except on natural killer cells and on a special subgroup of T-cells. The protein is glycosylphosphatidyl-inositol (GPI) anchored in the cell membrane and accelerates the decay of the classical as well as the alternative C3 convertases by replacing C2a/Bb in these complexes.
CD59 (protectin) is expressed ubiquitously and is similarly integrated into the cell membrane by GPI anchors. It regulates the formation of the terminal lytic membrane attack complex by inhibiting the interaction of the C8α-subchain and the first molecule of C9 so that integration into the cell membrane and the creation of a transmembrane pore is prevented (50) (51) (52) (53) .
In contrast to all the beneficial effects for the host organism, the complement system can also be detrimental for the host tissue ( Figure 5 ).
Many distinct pathogenetic mechanisms may lead to the expression of an excessive and uncontrolled immune response. Depending on the individual's immune status, this immune response leads to a proinflammatory systemic immune response syndrome or to compensated antiinflammatory response syndrome. Clinical complications of these reactions can be progressive sepsis and the development of multiple organ dysfunction syndrome, with enhanced susceptibility to infections.
Sepsis is defined as a systemic immune response syndrome with signs of infection. Excessive inflammation is induced by the recognition of pathogen-associated molecular patterns on invading microorganisms or danger-associated molecular patterns of damaged tissue by the cellular "first line of defense" and the complement system. This result consequently leads to the robust release of cytokines from phagocytes ("cytokine storm") to fight the infection. Although of benefit to the organism when acting locally, this pattern leads to a dramatic life-challenging event when occurring systemically (54) (55) (56) (57) (58) .
Various studies proposed excessive complement activation during sepsis in humans (59) . Because of its potent inflammatory profile, C5a appears to be the most detrimental molecule and has been described as "too much of a good thing" (54) . When activated, C5a may lead to immune paralysis, multiorgan dysfunction and thymocyte apoptosis (37, 40) as well as disturbance of the coagulation and fibrinolytic cascades (34) .
In accordance, some protection of septic mice has been shown by the application of a C5a receptor antagonist (60) . Furthermore, C5aR antagonism during sepsis led to a changed cytokine profile, such as decreased levels of tumor necrosis factor-α and IL-6, suggesting a direct or indirect role in the synthesis of these factors (40) . Czermak et al. (61) showed an enhanced survival rate of septic rats treated with a C5a antibody. Additionally, these authors found that C5a binds to neutrophils, which led to inactivation of their functions.
In a study by Flierl et al. (62) , the effects of complement on sepsis using C3 -/and C5 -/deficient mice were examined. In the absence of either C3 or C5, a reduced production of proinflammatory mediators was found.
On a cellular level, it has been shown that C5a effectively interacts with cells and modulates their apoptosis rate. Interestingly, the effects on programmed cell death seem to be cell dependent, with a higher rate of apoptosis in thymocytes (37,40) but decreased apoptosis in neutrophils (39, 63, 64) . Overall, the C5ainduced changes point toward an enhanced susceptibility toward infections, as well as to a prolonged presence of neutrophils resulting in an exaggerated inflammatory response and host damage. 
In the emerging field of osteoimmunology, the role of complement in bone biology in general and fracture healing in particular has started to raise interest. Although some direct interactions between the immune system and bone cells have been found (65, 66) , few studies demonstrated the presence of complement components in bone cells. In particular, the expression of various complement factors might depend on the cell differentiation state. Murine osteoblastic cells were shown to produce C3 in response to vitamin D3 (67, 68) . During osteoblastic differentiation in murine osteoblasts and in human cell lines, the complement components C1q, C4, C1 inhibitor, C3a receptor (C3aR), properdin and the complement factor H were upregulated (69), whereas the expression of the subcomponents C1r and C1s together with factor H was decreased (70) . The expression of functionally active C5a receptor (C5aR; CD88) that modulated IL-6 production was described in a human osteoblastic cell line (71, 72) . Recently, mRNA and protein expression of C3aR and C5aR in human MSC were reported (73) .
Immunohistochemical studies clearly indicated that complement was activated during enchondral ossification, possibly for the modulation of apoptosis (74, 75) . These studies indicated that some interaction of the complement system with bone cells exists. However, the resulting function to date has been minimally investigated. With the exception of our unpublished data and the immunohistochemical studies in enchondral ossification, in vivo data on the expression of complement components in bone are lacking, particularly with regard to fracture healing.
Recently, a major role of an excessively stimulated complement system in the posttraumatic inflammatory response was postulated (16, 76, 77) . Prospective studies with polytraumatized patients showed a consumption and massive activation of complement products, positively correlating with the mortality rate (78) (79) (80) . Fracture healing is delayed in particular when additional severe injuries stress the organism (81) . The massive trauma-induced inflammation correlates with consequent organ dysfunction (79) and may possibly be involved in the delayed fracture healing.
Studies with experimental blunt thoracic trauma found that the complement system contributed to the inflammatory reaction, and additionally successful inhibition of various inflammatory mediators occurred upon application of an antibody against C5a (76). Ganter et al. reported an earlier complement activation after major trauma, for which magnitude correlated with the mortality rate (16) . Unpublished data from our group underscore the role of complement in fracture healing, reflected by the enhanced C5aR immunostaining of bone sections.
The anaphylatoxins C3a and C5a have been reported to exert both protective and detrimental effects in the central nervous system (82, 83) . It is noteworthy that many cells of the central nervous system are more susceptible to a complement attack, since they lack important complement regulators such as CD59 (84) . Complement products were demonstrated to be dramatically upregulated in models of cerebral ischemia in rats (85) , and evidence for deposition of C3d and C9 after experimental cerebral contusion was found (86) . In agreement with these experimental findings, systemic complement activation has been shown in stroke patients (87) . A negative role for complement has been proposed in traumatic brain injury, since systemic depletion of complement by infusion of the cobra venom factor improved blood flow and neurological function and reduced cerebral edema during experimental intracerebral hemorrhage (88, 89) . Moreover, C1 deficiency (90) and complement inhibition by infusion of the C1 inhibitor revealed some neuroprotective effects (91, 92) . In contradiction to these results for traumatic/ischemic injuries, C1q has a strong neuroprotective effect in neu-rodegenerative diseases (93) . Traumatic brain injury induces C5aR upregulation in an experimental model in rats with enhanced C5a serum levels for >1 week. The functional consequences of traumatic brain injury-induced systemic C5a generation have still to be elucidated (94) .
After ischemia/reperfusion injury, complement is activated via all three established pathways (classical, alternative and MBL) (95) (96) (97) . Endothelium damaged by hypoxic stress activates complement, leading to elevated vascular permeability, release of anaphylatoxins and the accumulation of neutrophils (98) . Experimental studies indicated complement activation after ischemia/reperfusion in several organs, including lung, liver, gut, kidney, myocardium and skeletal muscle (99) (100) (101) (102) (103) (104) (105) .
Cardiac surgery and cardiopulmonary bypass surgery are known to cause a considerable immunologic impact to the body, resulting in a systemic inflammatory reaction (106, 107) . Various factors contribute to the extent of the immunological impact. In addition to general surgical trauma, hypothermia and blood loss, the main mechanisms are exposure of blood to foreign surfaces of the cardiopulmonary bypass, the ischemia/reperfusion injury by aortic cross-clamping and splanchnic hypoperfusion leading to mucosal damage and consequently endotoxemia (108).
An important pathogenetic role for complement in ischemia/reperfusion of the myocardium was indicated experimentally, where inhibition of the complement cascade greatly reduced myocardial damage after myocardial infarction (109) (110) (111) .
In translational studies, C5 inhibitor pexelizumab was capable of reducing mortality in patients undergoing coronary artery bypass surgery (112) but not myocardial infarction (113) . Recently, serum levels of C3a and C5a were found to be significantly elevated in patients suffering from stent restenosis after im-plantation of drug-eluting stents in patients with coronary heart disease (114) .
It was recently found that high MBL levels and low plasma levels of sC5b-9 were associated with an increased risk of dysfunctional cardiac performance after myocardial infarction, possibly owing to the increased complement activity during ischemia/reperfusion, which generally triggers an inflammatory response (115) .
However, activation of the complement cascade after ischemia/reperfusion injury or exposure to foreign surfaces is not the only cause of harmful effects of the complement system. Several studies alluded to a role for complement in cardiovascular diseases such as atherosclerosis or vasculitis.
Kawasaki disease, a systemic vasculitis in childhood causing coronary artery aneurysmata, appears to be related to MBL deficiency according to genetic family studies by Biezeveld et al. (116) . Rugonfalvi-Kiss et al. (117) found significantly lower restenosis rates in females with low MBL levels undergoing thromboendarteryectomy of the carotid artery because of atherosclerotical stenosis. Additionally, patients with MBL deficiency had a greater risk of myocardial infarction than individuals with normal MBL serum levels (118, 119) . The proposed protective effect of complement in the pathogenesis of atherosclerosis was experimentally underlined by C3 -/mice exhibiting accelerated development of atherosclerosis (120) . Clinical analysis of C2-deficient humans revealed a significant increase in cardiovascular diseases (121) . In particular, classical pathway involvement was demonstrated by Bhatia et al. (122) when C1q protected against the development of atherosclerosis in combined C1q and LDLr (low-density-lipoprotein receptor) knockout mice.
In agreement with these results, deficiency of complement inhibitors such as CD59 promoted atherogenesis (123) . However, it is noteworthy that both downstream complement defects and activation of the terminal pathway were as-sociated with inherited proatherogenic effects and an increased risk of cardiovascular disease (124) .
In conclusion, tightly regulated complement activation seems to protect against atherogenesis, possibly through the clearance of apoptotic cells and other debris, whereas inhibition of the terminal pathway by complement regulators appears to hinder proinflammatory effects (123, 125) . Once an atherogenic plaque has been formed, increasingly leading to ischemia and reperfusion injury, complement is excessively activated and may lead to harmful tissue damage.
Generally, a major complication of complement insufficiency is an enhanced susceptibility toward infection. Several studies have shown low MBL serum levels (genetically derived) are correlated with enhanced development of systemic immune response syndrome/sepsis in patients admitted to the intensive care unit (126, 127) . Low MBL serum concentrations were also associated with the development of pneumonia after surgery in colorectal cancer patients (128) . Similarly, MASP-2 deficiency caused increased infection rates with Streptococcus pneumoniae (129) . Furthermore, there is evidence for a higher rate of chorioamnionitis in patients with low MBL levels and preterm birth (130) . Low MBL serum levels appear to be associated with recurrent spontaneous abortion based on in utero infection. This was proposed by Kruse et al. (131) , who demonstrated that patients with MBL levels <100 ng/mL had a higher abortion rate. Some urogenital infections such as vulvovaginitis, herpes simplex virus-2 infection, vulvovaginal candidiasis and vestibulitis appear to be linked to low MBL levels in comparison to healthy controls (132) (133) (134) . Downstream complement deficiencies are typically associated with recurrent invasive infections of Neisseria meningitidis and gonorrhea (135) .
In defects further upstream, such as factor D or properdin, N. meningitidis also appear to be involved in most infections (136) . Deficiencies of the classical pathway may also cause enhanced infection rates, since invasive infection was the predominant clinical manifestation in patients with C2 deficiency, especially with S. pneumoniae (121) .
Many efforts have been undertaken to effectively modulate the activity of complement, thereby trying to ameliorate or completely abolish the symptoms of complement-mediated diseases. It is tempting to speculate that the effective modulation of the broad spectrum of internal diseases (as listed below) may also reflect an effective future target of various complement-dependent surgical diseases.
C1 inhibitor provided protective effects in myocardial cell injury (137) , transplantation (138) and various other diseases (139) . C1-INH was effective in a randomized, double-blind clinical study in septic intensive care unit patients. Whereas the renal dysfunction was reduced, the mortality rate remained unchanged in both groups (140) . The only clinical application for C1 inhibitor so far is for C1 inhibitor deficiency leading to hereditary angioedema (141) .
Nafamostat/FUT-175 is an unspecific serine protease inhibitor that, besides complement activation, also blocks abundant serine protease of the coagulation system (142) . It is currently used clinically for the treatment of acute pancreatitis and for the prevention of thrombosis in disseminated intravascular coagulation and extracorporal circulation (143, 144) , underscoring the crosstalk of the complement and coagulation cascades (35) .
Because of a lack of specificity and short half-life, other serine protease inhibitors, such as factor D, were not transferred to clinical trials, and further development was stopped (145, 146) . sCR1/TP10 is a soluble complement regulator inhibiting both the classical and alternative pathways and therefore is rather promising regarding ischemia/ reperfusion injury and various other conditions. The substance revealed some protective effects in male patients undergoing coronary artery bypass grafting but not in females (145, 147) . Therefore, clinical trials have been stopped (145) . Similar substances such as sCR1-sLe x / TP20 with an improved structure (148) and Microcept/APT070 (149) are currently under investigation for the treatment of diseases such as acute myocardial infarction, stroke and inflammatory diseases but have not entered clinical evaluation (150) .
A hybrid form of the complement regulators DAF and MCP was designed, named complement activity blocker 2 (CAB2), and entered clinical trials under the name MLN-2222 for the treatment of coronary artery bypass grafting (151) .
The complement regulator CD55 (DAF) was recently shown to ameliorate the hepatic inflammation in experimental chronic hepatitis C (152) and autoimmune posterior uveitis (153) .
The complement regulator CD59 was investigated in the context of cancer research. Neuroblastoma cells are known to escape cell lysis by abundantly expressing CD59. Therefore, a peptide was generated that was capable of suppressing CD59 expression that resulted in complement-mediated killing of the neuroblastoma cells (154) . Experimental studies for the treatment of paroxysmal nocturnal hemoglobinuria with the substitution of CD59 have recently been conducted (155) , but definitive results are pending.
C4BP as well as factor H have both been experimentally used to successfully abrogate complement activation on artificial surfaces and to inhibit the development of arthritis. However, these substances have not yet been clinically evaluated (156) (157) (158) .
Because of their specificity, antibodies against various complement factors appear to be a more reliable treatment strategy.
Eculizumab is a monoclonal antibody against C5 investigated for the treatment of paroxysmal nocturnal hemoglobinuria (159) and is currently in a phase I clinical trial for the treatment of systemic lupus erythematosus (160) . The first clinical studies in patients suffering from atypical hemolytic uremic syndrome were conducted and were partly promising, with the optimal dose and timing still to be determined (161) .
Pexelizumab is also a monoclonal antibody against C5 and has revealed beneficial effects after cardiopulmonary bypass surgery, but not after myocardial infarction in clinical studies (112, 113) . Various other antibodies are currently in developmental progress. Neutrazumab and TNX-558 are both directed against C5aR. TNX-234 is an antibody against factor D. TA106 blocks factor B from targeting, for example, in age-dependent macular dystrophia and asthma (162) . At least one antibody directed against properdin is currently undergoing testing (145) . In experimental studies, this antiproperdin monoclonal antibody was shown to be beneficial during coronary artery bypass grafting, reducing the activation of platelets and neutrophils (163) .
Ofatumumab is a monoclonal antibody against CD20 and exerts its effects not by inhibition, like most other complement therapeutics, but by stimulation of complement-dependent cytotoxicity. It is currently in clinical trials for the treatment of rheumatoid arthritis, B-cell chronic lymphocytic leukemia and follicular lymphoma (164, 165) . A similar approach that has not yet been clinically tested is the efficacy of the related substances HuMax-CD38 for multiple myeloma and HuMax-ZP3 for the treatment of colon, pancreatic and prostate cancer.
PMX-53 is a peptidic C5aR antagonist and has proven to be advantageous in experimental animal studies for neurodegenerative diseases, rheumatoid arthritis, ischemia/reperfusion and inflammatory bowel disease (102, 166, 167) . It is currently being evaluated in clinical trials, but a recent study in humans failed to show significant effects on synovial inflammation (168) .
Compstatin, a peptidic C3 inhibitor, is considered a promising drug and is currently in clinical trials for the treatment of age-dependent macular dystrophia (169) . Since compstatin has proven to be effective in various animal models for other conditions (170) , its application does not appear to be limited to agedependent macular dystrophia.
It is estimated that 30% of the human population present decreased plasma levels of MBL owing to genetic mutations. Therefore, a recombinant human form of MBL as a substitution therapy for MBL-deficient people was developed and found to be safe (171) . The substance is currently under clinical investigation for people suffering from multiple myeloma and undergoing high-dose chemotherapy (145, 172, 173) .
ARC1905 is an aptamer-based C5 inhibitor that inhibits the cleavage of C5 into C5a and C5b. It is intended for intravitreal application in age-dependent macular dystrophia and is currently undergoing phase I clinical trials (174) . With JPE-1375 and JSM-7717, there now exists even more C5aR antagonists that are in preclinical evaluation for the treatment of inflammatory, renal and ocular diseases.
Although complement research has been in the center of interest for many years, our understanding and insight into the cascade mechanisms and their complex interaction with other protein cascades such as the coagulation cascade is still in its nascent phase. Currently, complement activation is not divisable into three canonical pathways with separate activation patterns. The exact role of complement in many diseases needs to be further clarified because successful complement interventions need to be matched to the individual patient and be as specific as possible.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper. Targeting vaccination against novel infections: risk, age and spatial structure for pandemic influenza in Great Britain The emergence of a novel strain of H1N1 influenza virus in Mexico in 2009, and its subsequent worldwide spread, has focused attention to the question of optimal deployment of mass vaccination campaigns. Here, we use three relatively simple models to address three issues of primary concern in the targeting of any vaccine. The advantages of such simple models are that the underlying assumptions and effects of individual parameters are relatively clear, and the impact of uncertainty in the parametrization can be readily assessed in the early stages of an outbreak. In particular, we examine whether targeting risk-groups, age-groups or spatial regions could be optimal in terms of reducing the predicted number of cases or severe effects; and how these targeted strategies vary as the epidemic progresses. We examine the conditions under which it is optimal to initially target vaccination towards those individuals within the population who are most at risk of severe effects of infection. Using age-structured mixing matrices, we show that targeting vaccination towards the more epidemiologically important age groups (5–14 year olds and then 15–24 year olds) leads to the greatest reduction in the epidemic growth and hence reduces the total number of cases. Finally, we consider how spatially targeting the vaccine towards regions of country worst affected could provide an advantage. We discuss how all three of these priorities change as both the speed at which vaccination can be deployed and the start of the vaccination programme is varied. Vaccination has long been viewed as a vital tool in the armoury against infectious diseases. However, this perspective is largely based on our experience with endemic infections ( [1, 2] , where a routine policy of vaccination can be used to increase the level of herd immunity [3] ) and hence reduce or even eliminate the infection [4] . Examples of such approaches abound, from the eradication of smallpox and the virtual eradication of polio, to the long-running campaigns against childhood diseases such as measles, mumps and rubella [5, 6] , to the recently introduced schemes such as vaccination against human papillomavirus [7] . However, the recent experience with novel infections, such as SARS in 2003 and H1N1 pandemic influenza in 2009, have illustrated how vaccines are not a panacea due to the time needed to develop, manufacture and deploy the vaccine [8] . The UK, for example, had contracts to provide up to 132 million doses in the case of an influenza pandemic being declared, and 90 million doses were ordered in May 2009; however immunization did not begin until October. In total, only around five million doses [9] (approx. 400 000 to healthcare workers, about 37% of the 11 million people deemed in risk groups and about 20% of the three million children between 6 months and 5 years [10] ) had been administered to people in priority groups by the end of February 2010, when the pandemic had effectively died out; therefore in February and April 2010 the orders were substantially reduced. Such statistics mean that we must carefully assess whether mass or targeted vaccination has a role in controlling future pandemics or large-scale outbreaks, and develop a range of robust models that can rapidly assess the benefits of vaccination and the best ways by which it can be targeted [8, [11] [12] [13] [14] [15] .
Here, we extend a relatively simple model for vaccination (of previously unvaccinated individuals) at a constant rate in three main directions to consider how different forms of heterogeneity can impact optimal vaccination. In particular, we focus on the trade-off between vaccinating those at risk of severe complications (if they become infected) compared with vaccinating individuals who are more epidemiologically active; we consider who this epidemiologically active set are in terms of age groups within the population; and we consider whether vaccination should be deployed randomly or if there are benefits from geographical targeting. Throughout, our aim is to develop relatively simple models, where assumptions and parameters are transparent, and where it is feasible to rapidly perform large sweeps over parameter space. Therefore, while all results are formulated based on the UK experience of the 2009 H1N1 pandemic (assuming R 0 ¼ 1.4 and a doubling time of around one week [16] ), the model structure is sufficiently flexible that the qualitative results could pertain to a range of novel infections. ( We believe that the methodology and results outlined here are likely to hold for any rapidly transmitted infection, with a short infected period and life-long immunity, and where a vaccine can be rapidly developed and manufactured.) Obviously, once a new infectious disease is identified, specialist models are required that can accurately capture the known dynamics and can incorporate the appropriate economic and logistical facets [8] . Producing accurate results from all types of model (including the very complex and relatively simple) relies on the availability of high-quality surveillance and detailed case records. In the initial stages of an epidemic, before these data are available, policy-makers need to know what range of scenarios are consistent with the initial data. Additionally, for many parts of the world, detailed data are never available. For simple models with fewer basic parameters, a more comprehensive sweep of parameter space is feasible allowing the rapid assessment of various targeting vaccination strategies for ranges of parameters that are broadly consistent with early qualitative information. We therefore believe that the results developed here provide generic insights into the optimization of vaccine deployment during a novel outbreak of a directly transmitted pathogen with lifelong immunity.
We first introduce the most basic model of vaccination in response to an infection that conforms to the simple SIR-type (susceptible -infectious -recovered) paradigm ( [17] [18] [19] ); this model will form the basic template for all the work that follows. We assume that the uncontrolled epidemic obeys the simple SIR model dynamics, such that susceptible individuals (of which there are S) can become infected and infectious by interaction with infected individuals (of which there are I), infected individuals recover at a constant rate and enter the recovered class (of which there are R) after which they are assumed immune for life. (We stress that all results presented are qualitatively invariant to the precise model formulation, in particular using a model with gamma-distributed exposed and infectious classes more reminiscent of the 2009 H1N1 pandemic [16] .) In all the models that follow, we assume frequency-dependent mixing (such that the number of epidemiologically relevant contacts is independent of population size) and ignore the demographics of birth and death [17] -a reasonable assumption given the rapid time-frame of an epidemic. To this simple model, we add vaccination at a constant rate, v; we assume that individuals are vaccinated independently of their disease status but individuals are only vaccinated once, vaccination begins at time T, and a proportion p of vaccinated individuals are successfully immunized.
where N is the total population size. The precise way in which vaccination is implemented within the model ensures that a fixed number of individuals (v) are vaccinated per day, of which a fraction p are completely protected, and assumes that each person only receives one course of vaccine. If multiple doses of vaccine are required, or if protection only develops some time after vaccination, these can be included by delaying the time, T, at which immunization begins to take effect. Even this simplest of models confirms two simple rules-of-thumb regarding successful vaccination campaigns that seek to minimize the number of cases ( figure 1) . Firstly, that vaccination should begin as early as possible, so that susceptibles are depleted by vaccination before many cases arise; and secondly, that vaccination should be performed as rapidly as possible-both of which have been discussed before for a range of control measures [20, 21] . From figure 1 it is also clear that an early start to a vaccination campaign is far more beneficial than faster vaccination of the population. It should be stressed that when considering an ongoing epidemic, the critical vaccination threshold for the elimination of an endemic infection or prevention of epidemic invasion (¼1 2 1/R 0 ) [17] no longer plays such a clear role. Instead, the primary aim should be to immunize many people in as short a time as possible, subject to trade-offs from economic costs or adverse effects of vaccination (such as that observed for smallpox [12, 20] ). Here, and in all the figures that follow, we have considered a wide range of vaccination speeds ( y-axis in figure 1, line colours in figure 2, x-axes in figure 4 ); while some of these are extremely rapid and may be practically unachievable, the associated results are shown to provide a clearer picture of the most optimistic control scenario.
Given this absence of clear epidemiological trade-offs in this simple model, we need to consider a range of more structured models in which we can consider the trade-offs involved with the prioritization of different groups for vaccination.
When targeting a vaccination campaign (especially against the 2009 H1N1 influenza strain), there are often two competing priorities: minimization of transmission by immunizing those individuals that are most epidemiologically important; and minimization of the effects of the disease by immunizing those individuals that have the most severe health consequences when infected. (We note that for other infections these two groups may strongly overlap, in which case there is no conflict of priorities to resolve.) To tease apart these conflicting ideals, we extend the simple model above by having three groups [18, 19, 22, 23] : the dominant transmitter group (denoted by a subscript D), the group at highest risk of severe health complications if they become infected (denoted by a subscript H ) and the rest of the general population (denoted by a subscript G). These groups obey the basic equation (2.1) with two main modifications: firstly, the transmission dynamics are coupled through a 'who acquires infection from whom' matrix (b); and secondly, vaccination is prioritized so that either the dominant transmitter group (D) or the severe health risk group (H ) is vaccinated first, followed by the other group (either H or D), finally followed by the general population (G), (see electronic supplementary material). Here we have ignored the possibility that there is a group that are both dominant transmitters and at high risk of complications-obviously if such a group exists then it should be prioritized for vaccination before all others.
Obviously, an epidemiological model with three interacting groups has a large number of associated parameters, making a comprehensive sweep of the entire parameter space impractical and difficult to visualize. Instead, we show results from a relatively restricted scenario, but comment that these results are representative of all plausible scenarios that have been considered. In particular, we constrain the number of individuals in the three groups to be N D ¼ N H ¼ 0.1 N and H G ¼ 0.8 N, and constrain the transmission rates between all groups, except within the epidemiologically important group, to be equal (b XY ¼ g, except when X ¼ Y ¼ D). We note that different forms and parameters within this transmission matrix can potentially lead to different optimizations of vaccine [22] .
We now consider the optimal prioritization (either group D first or group H first), as four key parameters are varied: the transmission rate within the dominant transmitter group, b DD ; the relative adverse consequences of infection for the three groups, s H . s D ¼ s G ; the timing for the start of the vaccination programme, T; and the speed with which the population is vaccinated, v. Here the consequences of infection could capture a variety of measures, from risk of symptoms if infected, to concepts such as loss of QALYs (auality-adjusted life years), to risk of hospitalization, to risk of mortality associated with infection. The curves shown in figure 2 separate regions of parameter space where one form of prioritization is optimal, in terms of minimizing the total consequences of infection over the entire epidemic and across all three groups; regions above and to the left of the curves are where it is best to initially target vaccination towards the group with potentially severe health complications.
Two clear conclusions can be drawn. More rapid vaccination (larger v) generally favours prioritizing vaccination towards the group with potential health consequences, as does a later onset of vaccination (larger T ). However, it should be noted that for either extremely rapid or extremely slow vaccination (or extremely late start of the vaccination programme), the differences between the two prioritization schemes will be minimal. We can . The sequential deployment of vaccination that for each dose targets the age class that offers the greatest reduction in the epidemic growth rate. As such, at each point in the vaccinate delivery schedule that the distribution is optimal in terms of minimizing the rate at which new cases are produced. The surrounding seven sub-graphs show this deployment of vaccine at different points during the epidemic (as indicated on the central epidemic curve); the shaded grey areas represent the vaccine levels where the reproductive ratio (R) is less than one, and therefore the epidemic is in decline. The population is broken into eight age groups, mirroring those used by the Health Protection Agency to report age-structured case reports.
percentages, together with the fact that only 10 per cent of the general population are considered to have health problems, lead to estimates of s H : s G of atleast 9 : 1. Therefore, while there are a range of scenarios in which it would be optimal to target the dominant transmitters first, these tend to be in relatively extreme portions of parameter space, when the transmission rate b DD is very high and vaccination begins very early in the epidemic; for the vast majority of realistic scenarios, it is generally optimal to target vaccination towards those members of the population with underlying health problems first, before tackling the dominant transmitters and the rest of the general population.
Analysis of the 2009 pandemic to date in Britain, and elsewhere, indicates that there are some strong agedependent signatures. Most notably, school children have suffered the greatest per capita burden of infection as recorded by surveillance systems, whereas pre-school children have experienced the greatest per capita level of severe infection (as measured by hospital admissions), while the over 65 age group were most likely to suffer severe problems if they became infected [9, 16] . These different age-dependent effects are due to several interacting and conflicting factors: the highly structured mixing between age groups, the age-related I I I I I I I I I I I I I I I I   I I I I R I I I I I I I I I I I   I I R R R I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I R R I I I I I I I I I I   I I R R R I I I I I I I I I I I   I R R R I I I I I I I I I I I I   I R R I I I I I I I I I I I I I   districts I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I R I I I I I I I I I I I   I I R R R I I I I I I I I I I I   I R R R I I I I I I I I I I I I   I R R I I I I I I I I I I I I I   districts I I I I I I I I I I I I S S S S   I I I I I I I I I I I I S S S S   I R I I I I I I I I I I I S S S   I R I I I I I I I I I I I I S S   I R I I I I I I I I I I I I I S   I R I I I I I I I I I I I I I S   I R I I I I I I I I I I I I I I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I time to complete vaccination (days) I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I I   I I I I I I I I I I I I I I I susceptibility to infection and the age-dependent risk of severe symptoms following infection. To combine these factors require a mathematical model based on the available age-structured information. Here we use data from the POLYMOD study [24] to parametrize agerelated mixing patterns, where P a,b captures the estimated contact rate between individuals of ages a and b. In the electronic supplementary material we also show that an age-dependent susceptibility vector (q a ) can be estimated such that the early dynamics, as predicted by the dominant eigenvector of the transmission matrix, agree with the observed early age-structured distribution of infection. However, for greater generality, we set q ¼ 1 in figure 3 , although even using the estimated age-dependent susceptibility from the 2009 pandemic, together with the impact of school holidays, does not dramatically change the predictions (see electronic supplementary material). We use the age-dependent transmission matrix b to determine an optimal priority for a rapid age-dependent vaccination programme [23, [25] [26] [27] (figure 3). The methodology is as follows: for each single dose of vaccine, we consider which age class should be immunized such that the resultant growth-rate (as predicted by the dominant eigenvalue) is minimized; repeating this process successively generates a vaccination strategy that should rapidly control the epidemic for any given level of vaccine coverage. (We note that [27] provide an alternative, more analytical method of minimizing the eigenvalue, which is equivalent to our approach once the total level of vaccine exceeds a threshold.) The vaccination strategies given in figure 3 therefore inform about the instantaneous epidemiological significance of each age group at a particular point during an epidemic. We do not claim that these strategies are truly optimal (in terms of minimizing the predicted total number of cases across all possible distributions of vaccine), nor that such strategies are entirely relevant if vaccination is slow relative to the epidemic timescales (owing to the changes in the priorities we observe as the epidemic progresses, as shown in the sub-graphs). However, these age-specific vaccination profiles do provide an intuitive means of sequentially and efficiently increasing the vaccination coverage at any given point in the epidemic and have been found to agree with the optimal distribution of a fixed quantity of vaccine that minimizes the dominant eigenvalue [27] . What is crucial to note in these plots is that they represent a theoretical ideal when vaccine supply is limited rather than an achievable goal. If vaccine is not in short supply then it is clearly always better (both in terms of reducing growth rate and total epidemic size) to vaccinate someone than not, even if this leads to substantial deviation away from the optimal age profile. Therefore, these plots inform about possible prioritization of the vaccine campaign.
In the early stages of the epidemic, before there has been significant depletion of susceptibles, the predicted vaccination strategy initially targets the 5 -14 and 15-24 year old age groups; vaccination should then begin in the 25 -44 age group, with older ages (greater than 45) and the younger ages (under 5) not being targeted until vaccine coverage exceeds 50 per cent. What is somewhat counterintuitive is that the optimal deployment of vaccine could be partial in many age classes. For example, if 50 per cent of the population can be vaccinated, then the optimal strategy would be to attain highest levels of coverage in the 25-44 year old age group and less in the ages 5 -24, despite the fact that the 5 -24 year old age groups are favoured for early vaccination before the 25-44 year old age group. A second feature emerges as vaccination is begun later in the epidemic. Because the epidemic process has already depleted much of the susceptible population in the most epidemiologically active age classes (namely the 5 -14 year olds and 15-24 year olds), there is a decreased benefit from large-scale vaccination of these age groups. It should be stressed that both the distribution of optimal vaccination at a given time, and the way this changes as the epidemic progresses, are critically dependent on the number of individuals in each age group, the mixing matrix (b a,b ) and hence the age-dependent susceptibility (q a ). This means that precise details will depend on the detailed epidemiology of the infection under investigation, and are therefore likely to vary between different strains of influenza-for example, with models parametrized to match the 2009 pandemic in England (see electronic supplementary material), the 1 -4 year old age group plays a far more dominant role.
The final important question to address when delivering a vaccination programme, is whether there are any advantages in spatially targeting its deployment [28, 29] . Obvious choices could be to target regions with a high immediate burden (targeting based on current proportion of infectious cases), or to target regions that are likely to have many cases in the future (targeting based on current proportion of susceptibles), or simply to vaccinate randomly with a fixed per captia rate. Determining the optimal spatial targeting of vaccination is difficult because the impact of vaccination is long-lasting, cumulative and nonlinear, meaning that a generic understanding cannot be generated by considering the impact of low vaccination levels, nor can the action of vaccination be considered piece-wise as it was above. The only viable option is to simulate the dynamics with a variety of strategies and ascertain which performs the best numerically, however the issue now becomes the number of possible ways in which the vaccine could be distributed. We use a deterministic metapopulation model of the progress of an influenza-like infection in the 408 districts of Great Britain, linked by the commuter movements recorded in the 2001 census, and consider a wide range of epidemiological scenarios; with different onset times for the start of vaccination (T ), different numbers of districts initially infected and different levels of transmission heterogeneity, as captured by different b d in each district ( see electronic supplementary material). (Because of the added complexity of dealing with an explicitly spatial model, other forms of heterogeneity such as age or risk structure have been ignored, although their inclusion could be formulated in a similar manner to that described above. We have focused on modelling at the district scale as targeting could be practically achieved as such a resolution; finer scale targeting such as at the ward level would lead to a enhanced vaccination scheme but is unlikely to be practical.) The vaccination level in district d is given as a function of the current epidemiological conditions in that district:
As with equation (2.1), this formulation ensures the vaccination of a constant number (v) of ( previously unvaccinated) individuals each day, targeting unvaccinated individuals in each subpopulation at a per capita rate proportional to f. We have adopted the convention that variables and parameters with subscripts refer to specific districts, while those without subscripts refer to the entire population.
The initial assessment was to consider targeting ( f ) that was proportional to one of the standard model variables figure 4 . (Alternative more complex methods of targeting are shown in the see electronic supplementary material.) Each square is colour-coded and labelled according to which targeting consistently generates the lowest number of cases (from 100 replicate simulations with random initial distribution of infection). When there is no regional heterogeneity in epidemiological parameters (b d ¼ b, top row), then the best strategy depends on the speed of vaccination (x-axis), the time when the vaccination campaign begins (column) and to a lesser extent the number of districts initially infected ( y-axis). Early vaccination (begun as soon as cases are detected) generally favours targeting of vaccination proportional to the current proportion of infected cases ( f d ¼ I d /N d ); if vaccination takes more than three weeks to complete, then a later start to the vaccination programme (begun when 10% of the population have been infected and therefore approx. 20% of the way through the uncontrolled epidemic) favours targeting proportional to the proportion of remaining susceptibles in the population ( f d ¼ S d /N d )-therefore, areas that have seen relatively less infection are favoured. Finally, if vaccination is implemented, even later (begun when 20% of the population have been infected and therefore approx. 40% of the way through the uncontrolled epidemic) targeting according to the proportion of susceptibles is again favoured when the epidemic is relatively dispersed and vaccination is relatively prompt.
The top row of figure 4 corresponds to the unlikely scenario when each spatial district has identical parameters; in reality, there is likely to be significant heterogeneity in transmission between different areas owing to social and demographic factors. In the 2009 influenza pandemic in the UK, it was observed that the epidemic grew more rapidly in London and the West Midlands, possibly owing to such a mixture of socio-demographic factors. Unfortunately, the degree of such heterogeneity is difficult to estimate and is likely to vary between outbreaks; we therefore force the transmission rate within each district to be lognormally distributed about the mean, with the variance of the distribution controlled by the spatial heterogeneity (H ). (In particular, the transmission rate is given
where j d is a random variable, normally distributed with mean zero and a variance of one.) When such heterogeneity is added, in the overwhelming majority of scenarios (and certainly when H ! 0.2), targeting in terms of the proportion of individuals currently infectious ( f d ¼ I d /N d ) is the best strategy. This advantage for targeting in terms of current infectious cases is due to two main reasons: firstly, when there is significant heterogeneity in growth rates it will naturally target vaccination at those regions likely to suffer larger epidemics; secondly, by controlling infection in the worst affected area, the spread of infection to other areas is reduced and therefore more time is gained to vaccinate the remaining areas. (As a further refinement to targeting in the electronic supplementary material, we consider f ¼ (I d /N d ) a and seek the exponent, a*, that minimizes the predicted number of cases.)
The mass use of an effective vaccine clearly has the potential to provide major health benefits in terms of a reduction in the total number of infected cases, and therefore a reduction in the total number of adverse effects [1, 3, 7] . However, when used against an ongoing epidemic, the logistical constraints in terms of the speed with which vaccine can be manufactured and administered means that its deployment must be carefully targeted [8, [11] [12] [13] [14] [15] 23, 26, 27] . Many of these have focused on particular aspects of targeted vaccination (such as age-or risk-based) or have developed approximate [26] or analytical [27] methods for optimal targeting. Here, using standard differential equation models augmented to reflect particular heterogeneities, we have attempted to provide a relatively general framework that will be familiar with public-health epidemiologists and other non-specialist modellers. In general, our results agree with those of earlier studies although we have often attempted to consider a farwider parameter space, with the aim of spanning much of the parameter uncertainty that is likely to arise during the early stages of an epidemic.
Three forms of targeting have been considered, in terms of risk groups, age structure and spatial location, to derive general insights into the benefits of targeted vaccination. Throughout we have focussed on the development of highly parsimonious models, where assumptions and parameterization are relatively transparent; however, even for these models the parameter space is often too large to be visualized, in such cases model parameters are based on observations from the 2009 influenza pandemic in Great Britain. Despite Targeted vaccination M. J. Keeling and P. J. White 667 this large parameter space, several generic conclusions can be drawn about the optimal deployment of vaccine: -as predicted by even the simplest models, vaccination campaigns are most effective when they are applied rapidly and early in an epidemic before many cases arise. In fact, figure 1 shows that an early start to the vaccination campaign (and therefore rapid development of the vaccine) is of more advantage than administering the vaccine quickly; -owing to the natural levels of heterogeneity displayed within the population, it is generally better to initially target vaccination towards those groups likely to have severe symptoms rather than those groups that are most responsible for transmission ( figure 2 ). To some extent, this is due to the ease with which those groups that are likely to suffer complications can be identified. The parameter regime in which it is better to initially vaccinate the severe risk groups, is extended when the vaccination campaign begins later, or when the population can be vaccinated more rapidly; -once the groups with severe health risks have been protected (or it has been deemed better to target other groups), attention naturally focuses on which elements of the population are the most epidemiologically active and the benefits associated from vaccinating these individuals. Two main results emerge ( figure 3) ; firstly, any one group should not be targeted to the exclusion of all others, the best strategy is generally a mixed strategy. Therefore, while it is generally predicted (conditional upon age-dependent susceptibility) that school-age children should be the initial focus of vaccination, the optimal strategy does not concentrate on achieving complete coverage of this group, instead it is best to target other groups simultaneously; -in addition, the best groups to target for vaccination vary as the epidemic progresses. Most notably, age groups that play the dominant epidemiological role are rapidly depleted and therefore their importance wanes as the epidemic progresses, and hence the advantages of targeted vaccination compared with random vaccination also decline; and -although it is difficult (if not completely impractical) to calculate the true optimal spatially targeted vaccination policy, instigating at least some measure of targeting has significant advantages. Depending on the level of spatial heterogeneity in transmission within the population (which could reflect underlying demographic heterogeneity), targeting of vaccination towards regions that are currently experiencing high levels of infection generally reduces the total number of cases. The intuitive reason for this targeting is that by concentrating on centres of infections (and reducing the immediate growth rate), it buys extra time to vaccinate other regions. Achieving such a targeting in practice would require health services to be able to rapidly shift resources around the country, and only applies when there is a national limit to the deployment of vaccine. However, these results indicate two important points: firstly, that even when vaccination schemes are administered locally, there are likely to be strong advantages in spatially targeting any additional national resources; and secondly that the likely human reaction for there to be a greater demand for vaccine in regions with a higher proportion of cases would assist in control.
Because of the relatively simplistic nature of the models developed in this paper, several caveats should be made regarding the results. The first is that vaccination campaigns are unlikely to achieve a constant level of uptake over time; a more realistic assumption is that vaccination initially begins slowly due to logistical issues, builds to a plateau, but may finally decline if the epidemic begins to wane during the period of the vaccination programme and there is less incentive for individuals to be vaccinated. Associated with this is the fact that not all individuals are prepared to be vaccinated, and in particular parents in the UK are often anxious about vaccinating children; therefore, the optimal targeting of vaccination is unlikely to be possible and it may simply be better to vaccinate any individuals who wish to be vaccinated. The second issue, which applies to figures 2 and 3, is that it may not always be possible to identify or target relevant risk groups. For example, there may be considerable overlap between the age groups used in figure 3 , and the groups at risk of severe symptoms defined in figure 2 ; this was undoubtedly the case in 2009 (as well as previous pandemics), where age was often a key contributing factor to both the risk of infection and the risk of complications. Throughout, we have assumed a perfect vaccine, which offers 100 per cent protection to all those vaccinated. In practice, this is never realized, all vaccines fail to some degree; however, there are two ways in which this failure can be modelled. The first is an all or nothing approach in which a fraction of all vaccinations fails to generate any protection, while the remaining offers full protection; in this case, the results of our model natural extrapolate based on considering the numbers protected. The second approachis to consider 'leaky' vaccines that offer partial protection reducing either susceptibility or onward transmission; given the relatively low reproductive ratios considered within this paper, we believe that our results are likely to generalize to this case. A further issue related to figure 2 is the ethics of vaccinating the epidemiologically important groups in order to protect the group with severe health risks. While for influenza the vaccine has little associated risk and therefore it may be argued that the health benefits to the epidemiologically important (but healthy) groups outweigh the dangers, the same may not be true for other pathogens and the associated vaccine. Additionally, we have generally used deterministic models and hence assumed that vaccine would be used to mitigate the impact of infection rather than to prevent an epidemic occurring. This is particularly pertinent to the metapopulation model (figure 4) when we neglect the possibility of using vaccine to eradicate infection in the early stages and prevent its spread to the remainder of the country as exemplified in Ferguson et al. [11] . We feel this is a reasonable assumption given that most novel infections will be seeded continually by imports from abroad, and vaccine is unlikely to be available at the start of the epidemic. Finally, throughout we have assumed that there is a strong public demand for vaccine. Clearly, demand will vary both with disease severity and public perception of the infection; for example, the relatively mild nature of the 2009 pandemic meant that demand and uptake of the vaccine was low. However, if the infection has severe health implications, and therefore there is a clear need to optimize control measures, then demand for vaccination is likely to be substantial. The models in this paper, and therefore the results generated, are not designed to replace very detailed simulations parametrized to match epidemiological data from a given outbreak; however, they do provide a high degree of generality that is difficult to obtain with more case-specific simulations and hence provide a rapid assessment of conflicting methods of targeting vaccination. Often public-health action has to be taken in the absence of critical information: levels of prior immunity in the population affect the transmission patterns, but it takes time to develop the appropriate serological test; parameters such as casefatality ratios or the probability of hospitalization are difficult to estimate in real time as an epidemic progresses; and although clinical trials can measure the theoretical vaccine efficacy, vaccine effectiveness in practice can only be determined once the vaccination programme has begun. Given this range of uncertainties, models can be best used to assist policy-makers by examining a range of scenarios ahead of time; so decisions can be taken based on the range of scenarios that are consistent with the available data at the time that the decision has to be taken. Simple models often allow us to partition parameter space into clearly defined regions where a particular strategy is optimal; and while precise parameters may be difficult to estimate early in an epidemic, there may be sufficient evidence to suggest that the infection parameters lie within one of these prescribed regions. As such, the simple models developed here provide useful policy guidance before or during the early stages of an epidemic before there are sufficient data to parametrize more detailed simulations. Networks and the Epidemiology of Infectious Disease The science of networks has revolutionised research into the dynamics of interacting elements. It could be argued that epidemiology in particular has embraced the potential of network theory more than any other discipline. Here we review the growing body of research concerning the spread of infectious diseases on networks, focusing on the interplay between network theory and epidemiology. The review is split into four main sections, which examine: the types of network relevant to epidemiology; the multitude of ways these networks can be characterised; the statistical methods that can be applied to infer the epidemiological parameters on a realised network; and finally simulation and analytical methods to determine epidemic dynamics on a given network. Given the breadth of areas covered and the ever-expanding number of publications, a comprehensive review of all work is impossible. Instead, we provide a personalised overview into the areas of network epidemiology that have seen the greatest progress in recent years or have the greatest potential to provide novel insights. As such, considerable importance is placed on analytical approaches and statistical methods which are both rapidly expanding fields. Throughout this review we restrict our attention to epidemiological issues. The science of networks has revolutionised research into the dynamics of interacting elements. The associated techniques have had a huge impact in a range of fields, from computer science to neurology, from social science to statistical physics. However, it could be argued that epidemiology has embraced the potential of network theory more than any other discipline. There is an extremely close relationship between epidemiology and network theory that dates back to the mid-1980s [1, 2] . This is because the connections between individuals (or groups of individuals) that allow an infectious disease to propagate naturally define a network, while the network that is generated provides insights into the epidemiological dynamics. In particular, an understanding of the structure of the transmission network allows us to improve predictions of the likely distribution of infection and the early growth of infection (following invasion), as well as allowing the simulation of the full dynamics. However the interplay between networks and epidemiology goes further; because the network defines potential transmission routes, knowledge of its structure can be used as part of disease control. For example, contact tracing aims to identify likely transmission network connections from known infected cases and hence treat or contain their contacts thereby reducing the spread of infection. Contact tracing is a highly effective public health measure as it uses the underlying transmission dynamics to target control efforts and does not rely on a detailed understanding of the etiology of the infection. It is clear, therefore, that the study of networks and how they relate to the propagation of infectious diseases is a vital tool to understanding disease spread and, therefore, informing disease control.
Here, we review the growing body of research concerning the spread of infectious diseases on networks, focusing on the interplay between network theory and epidemiology. The paper is split into four main sections which examine the types of network relevant to epidemiology, the multitude of ways these networks can be characterised, the statistical methods that can be applied to either infer the likely network structure or the epidemiological parameters on a realised network, and finally simulation and analytical methods to determine epidemic dynamics on a given network. Given the breadth of areas covered and the ever-expanding number of publications (over seven thousand papers have been published concerning infectious diseases and networks) a comprehensive review of all work is impossible. Instead, we provide a personalised overview into the areas of network epidemiology that have seen the greatest progress in recent years or have the greatest potential to provide novel insights. As such considerable importance is placed on analytical approaches and statistical methods which are both rapidly expanding fields. We note that a range of other networkbased processes (such as the spread of ideas or panic) can be modelled in a similar manner to the spread of infection; however, in these contexts, the transmission process is far less clear; therefore, throughout this paper, we restrict our attention to epidemiological issues.
There are a wide number of network structures and types that have been utilised when considering the spread of infectious diseases. Here, we consider the most common forms and explain their uses and limitations. Later, we review the implications of these structures for the spread and control of infectious diseases.
We start our examination of network forms by considering the ideal network that would allow us to completely describe the spread of any infectious pathogen. Such a network would be derived from an omniscient knowledge of individual behaviour. We define G i, j (t) to be a time-varying, real, and high-dimensional variable that informs about the strength of all potential transmission routes from individual i to individual j at time t. Any particular infectious disease can then be represented as a function ( f pathogen ) translating this high-dimensional variable into an instantaneous probabilistic transmission rate (a single real variable). In this ideal, G subsumes all possible transmission networks, from sexual relations to close physical contact, face-to-face conversations, or brief encounters, and quantifies the time-varying strength of this contact. The disease function then picks out (and combines) those elements of G that are relevant for transmission of this pathogen, delivering a new (single-valued) timevarying infection-specific matrix (T i, j (t) = f pathogen (G i, j (t))). This infection-specific matrix then allows us to define the stochastic dynamics of the infection process for a given pathogen. (For even greater generality, we may want to let the pathogen-specific function f also depend on the time since an individual was infected, such that time-varying infectivity or even time-varying transmission routes can be accommodated.)
Obviously, the reality of transmission networks is far from this ideal. Information on the potential transmission routes within a population tends to be limited in a number of aspects. Firstly, it is rare to have information on the entire population; most networks rely on obtaining personal information on participants, and therefore participation is often limited. Secondly, information is generally only recorded on a single transmission route (e.g., face-to-face conversation or sexual partnership) and often this is merely recorded as the presence or absence of a contact rather than attempting to quantify the strength or frequency of the interaction. Finally, data on contact networks are rarely dynamic; what is generally recorded is whether a contact was present during a particular period with little consideration given to how this pattern may change over time. In the light of these departures from the ideal, it is important to consider the specifics of different networks that have been recorded or generated and understand their structure, uses, and limitations.
One of the few examples of where many of the potential transmission routes within a population have been documented comes from the spread of sexually transmitted infections (STIs). In contrast with airborne infections, STIs have very obvious transmission routes-sex acts (or sharing needles during intravenous drug use)-and as such these potential transmission routes should be easily remembered (Figure 1(a) ). Generally the methodology replicates that adopted during contact tracing, getting an individual to name all their sexual partners over a given period, these partners are then traced and asked for their partners, and the process is repeated-this is known as snowball sampling [4] (Figure 1(b) ). A related methodology is respondent-driven sampling, where individuals are paid both for their participation and the participation of their contacts while protecting each individual's anonymity [5] . This approach, while suitable for hidden and hard to reach populations, has a number of limitations, both practical and theoretical: recruiting people into the study, getting them to disclose such highly personal information, imperfect recall from participants, the inability to find all partners, and the clustering of contacts. In addition, there is the theoretical issue that this algorithm will only find a single connected component within the population, and it is quite likely that multiple disjoint networks exist [6] .
Despite these problems, and motivated by the desire to better understand the spread of HIV and other STIs, several pioneering studies were performed. Probably the earliest is discussed by Klovdahl [1] and utilises data collected by the Center for Disease Control from 19 patients in California suffering from AIDS, leading to a network of 40 individuals. Other larger-scale studies have been performed in Winnipeg, Manitoba, Canada [7] and Colorado Springs, Colorado, USA [8] . In both of these studies, participants were tested for STIs, and the distribution of infection compared to the underlying network structure. Work done on both of these networks has generally focused on network properties and the degree to which these can explain the observed cases; no attempt was made to use these networks predictively in simulations. In addition, in the Colorado Springs study, Interdisciplinary Perspectives on Infectious Diseases [3] ; squares refer to primary contacts. Given that the identity of contacts is known, they can be interlinked. (b) Caricature of a snowball sampling algorithm, squares are primary contacts, diamonds are secondary, and circles are tertiary contacts. Given that the identity of contacts is known they can be linked. (c) Example of a configuration model network. Each individual has a prescribed degree distribution, which gives rise to "halflinks" that are connected at random. (d) A household configuration network, consisting of completely interconnected households (cliques) with each individual also having one random link to another household. (e) Map showing Great Britain, together with the movements of cattle from six farms (each represented in a separate colour). Notice the heterogeneity between farms and the generally localised nature of movements. (f) Example of a small-world model based on a 2D lattice with nearest neighbor connections. The small-world property is given by the presence of rare random links that can connect distant parts of the network.
tracing was generally only performed for a single iteration although many initial participants in high-risk groups were enrolled, while in the Manitoba study, tracing was performed as part of the routine information gathered by public health nurses. Therefore, while both provide a vast amount of information on sexual contacts, it is not clear if the results are truly a comprehensive picture of the network and sampling biases may corrupt the resulting network [9] . In addition, compared to the ideal network, these sexual contact networks lack any form of temporal information; instead, they provide an integration of the network over a fixed time period and generally lack information on the potential strength of a contact between individuals. Despite these difficulties, they continue to provide an invaluable source of information on human sexual networks and the potential transmission routes of STIs. In particular, they point to the extreme levels of heterogeneity in the number of sexual contacts over a given period-and the variance in the number of contacts has been shown to play a significant role in early transmission dynamics [10] .
One of the few early examples of the simulation of disease transmission on an observed network comes from a study of a small network of 22 injection drug users and their sexual partners [3] (Figure 1(a) ). In this work, the risk of transmission between two individuals in the network was imputed based on the frequency and types of risk behaviour connecting those two individuals. HIV transmission was modelled using a monthly time step and single index case, and simulations were run for varying lengths of (simulated) time. This enabled a node's position in the network (as characterised by a variety of measures) to be compared with how frequently it was infected during simulations, and how many other nodes it was typically responsible for infecting.
A different approach to gathering social network and behavioural data was initiated by the Human Dynamics group at MIT and illustrates how modern technology can assist in the process of determining transmission networks. One of the first approaches was to take advantage of the fact that most people carry mobile phones [11] . In 2004, 100 Nokia 6600 smart-phones preinstalled with software were given to MIT students to use over the course of the 2004-2005 academic year. Amongst other things, data were collected using Bluetooth to sense other mobile phones in the vicinity. These data gave a highly detailed account of individuals behaviour and contact patterns. However, a limitation of this work was that Bluetooth has a range of up to 25 meters, and as such networks inferred from these data may not be epidemiological meaningful.
A more recent study into the encounters between wild Tasmanian devils in the Narawntapu National Park in northern Tasmania utilised a similar technological approach [12] . In this work, 46 Tasmanian devils were fitted with proximity loggers that could detect and record the presence of other loggers within a 30 cm range. As such, these loggers were able to provide detailed temporal information on the potential interaction between these 46 animals. This study was initiated to understand the spread of Tasmanian devil facial tumour disease, which causes usually fatal tumours that can be transmitted between devils if they fight and bite each other. Although only 27 loggers with complete data were recovered, and although the methodology only recorded interaction between the 46 devils in the study, the results were highly informative (generating a network that was far from random, heterogeneous, and of detailed temporal resolution). Analyses based on the structure of this network suggested that targeted measures, that focus on the most highly connected ages or sex, were unlikely to curtail the spread of this infection. Of perhaps greater relevance is the potential this method illustrates for determining the contact networks of other species (including humans)-the only limitation being the deployment of a suitable number of proximity loggers.
Given the huge logistical difficulties of capturing the full network of interactions between individuals within a population, a variety of methods have been developed to generate synthetic networks from known attributes. Generally, such methods fall into two classes: those that utilise egocentric information and those that attempt to simulate the behaviour of individuals.
Egocentric data generally consists of information on a number of individuals (the egos) and their contacts (the alters). As such the information gathered is very similar to that collected in the sexual contact network studies in Manitoba and Colorado Springs, but with only the initial step of the snowball sampling was performed; the difference is that for the majority of egocentric data the identity of partners (alters) is unknown and therefore connections between egos cannot be inferred (Figure 1(c) ). The data, therefore exists as multiple independent "stars" linking the egos to the alters, which in itself provides valuable information on heterogeneities within the network. Two major studies have attempted to gather such egocentric information: the NATSAL studies of sexual contacts in the UK [13] [14] [15] [16] , and the POLYMOD study of social interactions within 8 European countries [17] . The key to generating a network from such data is to probabilistically assign each alter a set of contacts drawn from the information available from egos; in essence, using the ego data to perform the next step in the snowball sampling algorithm. The simplest way to do this is to generate multiple copies of all the egos and to consider the contacts from each ego to be "half-links"; the half-links within the network can then be connected at random generating a configuration network [18] [19] [20] ; if more information is available on the status (age, gender, etc.) of the egos and alters then this can also be included and will reduce the set of half-links that can be joined together. However, in the vast majority of modelling studies, the egocentric data have simply been used to construct WAIFW (who-acquiresinfection-from-whom) matrices [15, 17, 21] that inform about the relative levels of transmission between different groups (e.g., based on sexual activity or age) but neglect the implicit network properties. This matrix-based approach is often reliable: for STIs it is the extreme heterogeneity in the number of contacts (which are close to being powerlaw or scale-free distributed; see Section 3.2) that drives the infection dynamics [22] although larger-scale structure does play a role [23] ; for social interactions, it is the assortativity between (age-) groups that controls the behaviour, with the number of contacts being distributed as a negative binomial [17] . The POLYMOD matrices have therefore been extensively used in the study of the H1N1 pandemic in 2009, providing important information about the cost-effective vaccination of different age-classes [21, 24] . The general configuration model approach of randomly linking together "half-links" from each ego [18, 19] has been adopted and modified to consider the spread of STIs. In particular, simulations have been used to consider the importance of concurrency in sexual networks [25, 26] , where concurrency is defined as being in two active sexual partnerships at the same time. A dynamic sexual network was simulated, with partnerships being broken and reformed such that the network density remained constant over time. The likelihood of two nodes forming a partnership depended on their degree, but this relationship could be tuned to make concurrency more or less common and to make the mixing assortative or disassortative based on the degrees of the two nodes. Transmission of an STI (such as gonorrhoea and chlamydia [25] or HIV [26] ) was then simulated upon this dynamic network, showing that increasing concurrency substantially increased the growth rate during the early phase of an epidemic (and, therefore, its size after a given period of time). This greater growth rate was related to the increase in giant component size (see Section 3.1) that was caused by increased concurrency.
Interdisciplinary Perspectives on Infectious Diseases 5 A slightly more general approach to the generation of model sexual networks was employed by Ghani et al. [27] . In their network model, individuals had a preferred number of concurrent partners and duration of partnerships, and their level of assortativity was tunable. A gonorrhoea-like infection was simulated on the resulting dynamic network. Regression models were used to consider the association between network structures (either snapshots of the state of the network at the end of simulation or accumulated over the last 90 days of simulation) and prevalence of infection. These simulations showed that increasing levels of concurrent partnerships made invasion of the network more likely and also that the mixing patterns of the most sexually active nodes were most important in determining the final prevalence of infection within the population [27] . The same model was later used to consider the importance of different structural measures and sampling strategies, showing that it was important to endeavour to identify infected individuals with a high number of sexual partners in order to correctly define the high-risk group for interventions [23] .
The alternative approach of simulating the behaviour of individuals is obviously highly complex and fraught with a great deal of uncertainty. Despite these problems, three groups have attempted just such an approach: Longini's group at Emory [28] [29] [30] [31] , Ferguson's group at Imperial [32, 33] , and Eubank's group at Los Alamos/Virginia Tech [34, 35] . The models of both Longini and Ferguson are primarily agent-based models, where individuals are assigned a home and work location within which they have frequent infection-relevant contacts together with more random transmission in their local neighbourhood. The Longini models separate the entire population into subunits of 2000 individuals (for the USA) or 13000 individuals (for South-East Asia) who constitute the local population where random transmission can operate; in contrast, the Ferguson models assign each individual a spatial location and random transmission occurs via a spatial kernel. In principle, both of these models could be used to generate an explicit network model of possible contacts. The Eubank model is also agentbased aiming to capture the movements of 1.5 million people in Portland, Oregon, USA; but these movements are then used to define a network based on whether two individuals occur in the same place (there are 180 thousand places represented in the model) at the same time. It is this network that is then used to simulate the spread of infection. While in principle this Eubank model could be used to define a temporally varying and real-valued network (where the strength of connection would be related to the type of mixing in a location and the number of people in the location); in the epidemiological publications [35] , the network is considered as a static contact network in which extreme heterogeneity in numbers of contacts is again predicted, and the network has "small world" like properties (see below). A similar approach of generating artificial networks of individuals for stochastic simulations of respiratory disease has been recently applied to influenza at the scale of the United States, and the software made generally available [36] . This software took a more realistic dynamic network approach and incorporated flight data within the United States, but was sufficiently resource-intensive to require specialist computing facilities (a single simulation taking around 192 hours of CPU time). All three models have been used to consider optimal control strategies, determining the best deployment of resources in terms of limiting transmission associated with different routes. The predicted success of various control strategies, therefore, critically depends on the strength of contacts within home, at work, within social groups, and that occuring at random.
Whilst smallpox has been eradicated, concern remains about the possibility of a deliberate release of the disease. The stochastic simulation models of the Longini group have predominantly focused on methods of controlling this infection [28, 31] . Their early work utilised networks of two thousand people with realistic age, household size, and school attendance distributions, with the likelihood of each individual becoming infected being derived from the number and type of contacts with infectious individuals [28] . This paper focused on the use of vaccination to contain a smallscale outbreak of smallpox and concluded that early massvaccination of the entire population was more effective than targeted vaccination if there was little or no immunity in the population. Later models [31] combined these subnetworks of two thousand people into a larger network of fifty thousand people (with one hospital), and the adult population were able to contact each other through workplaces and high schools. Here, the focus was on surveillance and containment which were generally concluded to be sufficient to control an outbreak. The epidemiological work of the Eubank group has also focused on a release of smallpox although these simulations showed that encouraging people to stay at home as soon as they began to feel unwell was more important than choice of vaccination protocol [35] ; this may in part be attributed to the scale-free structure of the network and hence the superspreading nature of some individuals.
The Ferguson models have primarily been used to consider the spread and control of pandemic influenza, examining its potential spread from an initial source in South-East Asia [32] and its spread in mainland USA and Great Britain [33] . The models of South-East Asia were primarily based on Thailand, and included demographic information and satellite-based spatial measures of population density. It focused on containment by the targeted use of antiviral drugs and suggested that as long as the reproductive ratio (R 0 ) of a novel strain was below 1.8, it could be contained by the rapid use of targeted antivirals and social distancing. However, such a strategy could require a stockpile of around 3 million antiviral doses. The models based on the USA and Great Britain, considered a wider range of control measures, including school closures, household prophylaxis using antiviral drugs, and vaccination, and predicted the likely impact of different policies.
Networks. An alternative source of network information comes from the recorded movements of individuals. Such data frequently describe a relatively large network as information on movements is often collected by national or international bodies. The network of movements, therefore, has nodes representing locations (rather than individuals) and edges weighted to capture the number of movements from one location to another-as such the network is rarely symmetric. Four main forms of movement network have played important roles in understanding the spread of infectious diseases: the airline transportation network [37, 38] , the movement of individuals to and from work [39, 40] , the movement of dollar bills (from which the movement of people can be inferred) [41] , and the movement of livestock (especially cattle) [30, 42] . While the structure of these networks has been analysed in some detail, to develop an epidemiological model requires a fundamental assumption about how the epidemic progresses within each locations. All the examples considered in this section make the simplifying assumption that the epidemic dynamics within each location are defined by random (mean-field) interactions, with the network only informing about the flow of individuals or just simply the flow of infection between populations-such a formulation is known as a metapopulation model [43] .
Probably the earliest work using detailed movement data to drive simulations comes from the spread of 1918 pandemic influenza in the Canadian Subarctic, based on records kept by the Hudson's Bay Company [44] . A conventional SIR metapopulation model was combined with a network model (the nodes being three fur trading posts in the region: God's Lake, Norway House, and Oxford House), where some individuals remained in their home locations whilst others moved between locations, based on records of arrivals and departures recorded in the post journals. Whilst this model described only a small population, it was able to be parameterised in considerable detail due to the quality of demographic and historical data available and showed that the movement patterns observed interacted with the starting location of a simulated epidemic to change the relative timings of the epidemics in the three communities, but not the overall impact of the disease.
The movement of passenger aircraft as collated by the International Air Transport Association (IATA) provides very useful information about the long-distance movement of individuals and hence how rapidly infection is likely to travel around the globe [37, 45, 46] . Unlike many other network models which are stochastic individual-level simulations, the work of Hufnagel et al. [37] and Colizza et al. [45] was based on stochastic Langevin equations (effectively differential equations with noise included). The early work by Hufnagel et al. [37] focused on the spread of SARS and showed a remarkable degree of similarity between predictions and the global spread of this disease. This work also showed that extreme sensitivity to initial conditions arises from the structure of the network, with outbreaks starting in different locations generating very different spatial distributions of infection. The work of Colizza was more focused towards the spread of H5N1 pandemic influenza arising in South-East Asia and its potential containment using antiviral drugs. However, it was H1N1 influenza from Mexico that initiated the 2009 pandemic, but again, the IATA flight data provided a useful prediction of the early spread [47, 48] . While such global movement networks are obviously highly important in understanding the early spread of pathogens, they unfortunately neglect more localised movements [49] and individual-level transmission networks. However, recent work has aimed to overcome this first issue by including other forms of local movement between populations [40, 50] . This work has again focused on the spread of influenza, mixing long-distance air travel with shorter range commuter movements and with the model predictions by Viboud et al. [40] showing good agreement with the observed patterns of seasonal influenza. An alternative form of movement network has been inferred from the "Where's George" study of the circulation of dollar bills in the USA [38] ; this provided far more information about short-range movements, but again did not really inform about the interaction of individuals.
A wide variety (and in practice the vast majority) of movements are not made by aircraft but are regular commuter movements to and from work. The network of such movements has also been studied in some detail for both the UK and USA [39, 40, 51] . The approaches adopted parallel the work done using the network of passenger aircraft, but operate at a much smaller scale, and again, influenza and smallpox have been the considered pathogens. As with the aircraft network certain locations act as major hubs attracting lots of commuters every day; however, unlike the aircraft network, there is the tendency for the network to have a strong daily signature with commuters moving to work during the day but travelling home again in the evening [52] . As such the commuter network can be thought of as heterogeneous, locally clustered, temporal, and with each contact having different strengths (according to the number of commuters making each journey); however, to provide a complete description of population movement, and hence disease transmission requires other causes of movement to be included [51] and requires strong assumptions to be made about individual-level interactions. The key question that can be readily addressed from these commuter-movement models is whether a localised outbreak can be contained within a region or whether it is likely to spread to other nodes on the network [39] .
Undoubtedly, one of the largest and most comprehensive data sets of movements between locations comes from the livestock tracing schemes run in Great Britain and being adopted in other European countries. The Cattle Tracing Scheme in particular is spectacularly detailed, containing information of the movements of all cattle between farms in Great Britain; as such, this scheme generates daily networks of contacts between over 30,000 working farms in Great Britain [42, [53] [54] [55] [56] (Figure 1 (f)). Similar data also exist for the movement of batches of sheep and pigs [57] although here the identity of individual animals making each movement is not recorded. This data source has several key advantages over other movement networks: it is dynamic, in that movements are recorded daily; the movement of livestock is one of the major mechanisms by which many infections are transferred between farms, and the metapopulation assumption that cattle mix homogeneously within a farm is highly plausible. In principle, the information in the Cattle Tracing Scheme can be used to form an even more comprehensive network, treating each cow as a node and creating an edge if two cows occur within the same farm on the same day-this would generate an individual-level network for each day which can then be used to simulate the spread of infection [52] .
The early spread of foot and mouth disease (FMD) in 2001 was primarily due to livestock movements, particularly of sheep [58] . Motivated by this epidemic, Kiss et al. [57] conducted short simulated outbreaks of FMD on both the sheep movement network based on 4 weeks' movements starting on 8 September 2004 and simulated synthetic networks with the same degree distribution. Due to the short time-scales considered (the aim being to model spread of FMD before it had been detected), nodes were susceptible, exposed or infected but never recovered, and network connections remained static. Simulated epidemics were smaller on the sheep movement network than the random networks, most likely due to disassortative mixing in the sheep movement network. Similarly, Natale et al. [59] employed a static network simulation of Italian cattle farms. Here, farms were not merely represented as nodes, but a deterministic SI system of ODEs was used to model infection on each node essentially generating a metapopulation model. The only stochastic part of the model was the number of infectious individuals moved between connected farms in each time step. This simulation model highlighted the impact of the centrality of seed nodes (measured in several different ways) upon the subsequent epidemics' course.
The use of static networks to model the very dynamic movement of livestock is questionable. Expanding on earlier work, Green et al. [53] simulated the early spread of FMD through movement of cattle, sheep, and pigs. Here, the livestock network was treated dynamically, with infection only able to propagate along edges on the day when that edge occurred; additional to this network spread, local transmission could also occur. These simulations enabled regional patterns of risk to a new FMD incursion to be assessed, as well as identifying markets as suitable targets for enhanced surveillance. Vernon and Keeling [55] considered the relationship between epidemics predicted from dynamic cattle networks and their static counterparts in more detail. They compared different network representations of cattle movement in the UK in 2004, simulating epidemics across a range of infectivity and infectious period parameters on the different network representations. They concluded that network representations other than the fully dynamic one (where the movement network changes every day) fail to reproduce the dynamics of simulated epidemics on the fully dynamic network.
Contact tracing and hence the networks generated by this method can take two distinct forms. The first is when contact-tracing is used to initiate proactive control. This is often the case for STIs, where identified cases are asked about their recent sexual partners, and these individuals are traced and tested; if found to be infected, then contact tracing is repeated for these secondary cases. Such a process is related to the snowball sampling that was discussed earlier, with the notable exception that tracing is only performed from known cases. Similar contact-tracing may operate for the early stages of an airborne epidemic (as was seen for the 2009 H1N1 pandemic), but here, the tracing is not generally iterative as contacts are generally traced and treated so rapidly that they are unlikely to have generated secondary cases. An alternative form of contacttracing is when a transmission pathway is sought between all identified cases [1, 60, 61] . This form of contact tracing is likely to become of ever-increasing importance in the future when improved molecular techniques and statistical inference allow infection trees to be determined from genetic differences between samples of the infecting pathogen [62] .
These forms of network have two main advantages but one major disadvantage. The network is often accompanied by test results for the individuals within the network, as such we not only have information on the contact process but also on the resultant transmission of infection. In addition, when contact tracing is only performed to define an infection tree, there is the added advantage that the infection process itself defines the network of contacts, and hence there is no need for human interpretation of which forms of contact may be relevant. Unfortunately, the reliance on the infection process to drive the tracing means that the network only reflects one realisation of the epidemic process and, therefore, may ignore contacts that are of potential importance and would be needed if the epidemic was to be simulated; therefore, while they can inform about past outbreaks, they have little predictive power.
Obtaining large-scale and reliable information on who contacts whom is obviously very difficult; therefore, there is a temptation to rely on alternative data sets, where network information can be extracted far more easily, and where the data is already collected. As such the movement networks and contact tracing networks discussed above are examples of such surrogate networks although their connection to the physical processes of infection transmission are far more clear. Other examples of networks abound [22, [63] [64] [65] ; while these are not directly relevant for the spread of infection, they do provide insights into how networks form and grow-structures that are commonly seen in surrogate networks are likely to arise in the types of network associated with disease transmission. One source of network information that would be fantastically rich and also highly informative (if not immediately relevant) is the network of friendships and contacts on social networking sites (such as Facebook); some sites have made data on their social networks available, and these data have been used to examine a range of sociological questions about online interactions [66] .
Given the huge complexity involved in obtaining large-scale and reliable data on realtransmission networks many researchers have instead relied on theoretically constructed networks. These networks are usually highly simplified but aim to capture some of the known (or postulated) features of real-transmission networks-often the simplifications are so extreme that some analytical traction can be gained. Here, we briefly outline some of the commonly used theoretical networks and identify which features they capture; some of the results of how infection spreads on such networks are discussed more fully in Section 4.2.
One of the simplest forms of network is to allow each individual to have a set of contacts that it wishes to make (in more formal language each node has a set of half-links), these contacts are then made at random with other individuals based on the number of contacts that they wish to make (half-links are randomly connected) [19] . This obviously creates a network of contacts ( Figure 1 (c)). The advantage of these configuration networks is that because they are formed from many randomly connected individuals, there are no short loops within the network and a range of theoretical results can be proved ranging from conditions for invasion [18, 67, 68] to descriptions of the temporal dynamics [69] . Unfortunately, the elements that make these networks amenable to theoretical analysis-the lack of assortativity, short loops or clusteringare precisely factors that are thought to be important features of real networks.
An alternative formulation that offers a compromise between tractability and realism occurs when individuals that exist in fully interconnected cliques have randomly assigned links within the entire population [69, 70] ( Figure 1(d) ). As such, these networks mimic the strong interactions within families and the weaker contacts between them. While such models offer a significant improvement over configuration networks and capture the known importance of the household in transmission, they make no allowance for clustering between households due to spatial proximity. Hierarchical metapopulation models [71] allow for this form of additional structure, where households (or other groupings) are themselves grouped in an ascending hierarchy of clustering.
Worlds. Both lattice networks and small world networks begin with the same formulation: individuals are regularly spaced on a grid (usually in just one or two dimensions), and each individual is connected to their k nearest neighbours-these connections define a lattice. The advantage of such networks is that they retain many elements of the initial spatial arrangement of points, and hence contain both many short loops as well as the property that infection tends to spread locally. There is a clear link between such lattice-based networks and the field of probabilistic cellular automata [72, 73] . The fundamental difficulty with such lattice models is that the presence of short loops and localised spread means that is it difficult (if not impossible) to prove exact results, and hence large-scale multiple simulations are required.
Small world networks improve upon the rigid structure of the lattice by allowing a low number of random contacts across the entire space (Figure 1(e) ). Such long range contacts allow infection to spread rapidly though the population and vastly reduce the shortest path length between individuals [74] -this is popularly known as six degrees of separation from the concept that any two individuals on the planet are linked through at most six friends or contacts [75] . Therefore, small world networks offer a step towards reality, capturing the local nature of transmission and the potential for long-range contacts [76, 77] ; however, they suffer from neglecting heterogeneity in the number of contacts and the tight clustering of contacts within households or social settings.
Networks. Spatial networks, as the name suggests, are generated using the spatial location of all individuals in the population, as such lattices and small worlds are a particular form of spatial network. The general methodology initially positions each individual i at a specific location x i , usually; these locations are chosen at random, but clustered spatial distributions have also been used [78] . Two individuals (say i and j) are then probabilistically connected based upon the distance between them; the probability is given by a connection kernel which usually decays with distance such that connections are predominantly localised. These spatial networks (especially when the underlying distribution of points is clustered) have many features that we expect from disease networks although it is unclear if such simple formulations can be truly representative.
In recent years, there has been growing interest in exponential random graph models (ERGMs) for social networks, also called the p * class of models. ERGMs were first introduced in the early 1980s by Holland and Leinhardt [79] based on the work of Besag [80] . More recently, Frank and Strauss studied a subset of those that have the simple property that the probability of connection between two nodes is independent of the connection between any other pair of distinct nodes. [81] . This allows the likelihood of any nodes being connected to be calculated conditional on the graph having certain network properties. Techniques such as Markov Chain Monte Carlo can then be used to create a range of plausible networks that agree with a wide variety of information collected on network structures even if the complete network is unknown [82, 83] . Due to their simplicity, ERGMs are widely used by statisticians and social network analysts [84] . Despite significant advances in recent years (e.g., [85] ), ERGMs still suffer from problems of degeneracy and computational intractability for large network sizes, which has limited their use in epidemic modelling.
Here, we have shown that a wide variety of network structures have been measured or synthesised to understand the spread of infectious diseases. Clearly, with such a range of networks, no clear consensus can be drawn on the types of underlying network structures that are generally present; in part, this is because different studies have focused on different infectious diseases and different diseases require different transmission routes.
However, three factors emerge that are key components of epidemiological networks: heterogeneity in the number of contacts such that some individuals are at a higher risk of both catching and transmitting infection, clustering of contacts such that groups of individuals are often highly interconnected, and some reflection of spatial separation such that contacts usually form locally, but occasional longrange connections do occur.
Three fundamental problems still exist in the study of networks. Firstly, are there relatively low-dimensional ways of capturing key aspects of a network's structure? What constitutes a key aspect will vary with the problem being studied, but for epidemiological applications, it should be hoped that a universal set of network characteristics may emerge. There is then the task of assessing reasonable and realistic ranges for these key variables based on values computed for known transmission networks-unfortunately very few transmission networks have been recorded in any degree of detail although modern electronic devices may simplify the process in the future. Secondly, there is the related statistical problem of inferring plausible complete networks from the partial information collected by methods such as contact tracing. This is equivalent to seeking an underlying model for the network connections that is consistent with the known partial information, and hence, has strong resonance with the more mechanistically motivated models in Section 2.3. Even when the network is fully realised (and an epidemic observed), there is considerable statistical difficulty in attributing risk to particular contact types. Finally, there are the key questions of predicting the dynamics of infection on any given network-and while for many complex networks, direct simulation is the only approach, for other simplified networks some analytical traction can be achieved, which helps to provide more generic insights into which elements of network structure are most important. These three key areas are discussed below.
Real networks can exhibit staggering levels of complexity. The challenge faced by researchers is to try and make sense of these structures and reduce the complexity in a meaningful way. In order to make any sense of the complexities present, researchers over several decades have defined a large variety of measurable properties that can be used to characterise certain key aspects [63, 65, 86] . Here, we describe the definitions of the most important characterisations of complex networks (in our view), and outline their impact on disease transmission models.
In general, networks are not necessarily connected; in other words, all parts of the network are not reachable from all others. The component to which a node belongs is that set of nodes that can be reached from it by paths running along edges of the network. A network is said to have a giant component if a single component contains the majority of nodes in the network. In directed networks (one in which each edge has an associated direction), a node has both an in-component from which the node can be reached and an out-component that can be reached from that node. A strongly connected component (SCC) is the set of nodes in the network in which each node is reachable from every other node in the component.
The concept of a giant component is central when considering disease propagation in networks. The extent of the epidemic is necessarily limited to the number of nodes in the component that it begins in, since there are no paths to nodes in other components. In directed networks, in the case of a single initial infected individual, only the out-component of that node is at risk from infection. More generally, the strongly connected component contains those nodes that can be reached from each other. Members of the strongly connected component are most at risk from infection imported at a random node, since a single introduction of infection will be able to reach all nodes in the component.
The degree is defined as the number of neighbours that a node has and is most often denoted as k. In directed graphs, the degree has two components, the number of incoming edges k in , (in-degree), and the number of outgoing edges k out , (outdegree). The degree distribution is defined as the set of probabilities, P(k), that a node chosen at random will have degree k. Plotting the distribution of degrees of nodes is one of the most basic and important ways of characterising a given network (Figure 2 ). In addition, useful characterisations are obtained by calculating the moments of the degree distribution. The nth moment of P(k) is defined as
with the first moment, k , being the average degree, the second, k 2 allowing us to calculate the variance k 2 − k 2 , and so on. The degree distribution is one of the most important ways of characterising a network as it naturally captures the heterogeneity in individuals' potential to become infected as well as cause further infection. Intuitively, the higher the number of edges a node has, the more likely it is to be a neighbour of an already infected node. Also, the more neighbours a node has, the more likely it is to cause a large number of onward cases. Thus, knowing the form of P(k) is crucial for the understanding of the spread of disease. In random networks of the type studied by Erdös and Rényi, P(k) follows a binomial distribution, which is effectively Poisson in the case of large networks. Most real social networks have distributions that are significantly different from the random case.
For the extreme case of P(k) following an unbounded power law and assuming equal transmission across all edges, Pastor-Satorras and Vespignani [87] showed that the classic result of the epidemic threshold from mean field theory [10] breaks down. In real-transmission networks, the distribution of degree is often heavily skewed, and occasionally follows a power law [22] , but is always bounded, leading to the recovery of epidemic threshold, but one which is much lower than expected in evenly mixed populations [88] .
The degree distribution provides very useful information on uncorrelated networks such as those produced by configuration models. However, real networks are in general correlated with respect to degree; that is, the probability of finding a node with given degree, k, is dependent on the degree of the neighbours of that node, k , which is captured by the conditional probability P(k | k). To characterise this behaviour, several measurements have been proposed. The most straightforward, and probably most useful measure, is to consider the average degree of the neighbours of a node
where the sum of degrees is made over the neighbours (Nbrs) of i. One can then calculate the average of k nn over all nodes with degree k which is a direct measure of the conditional probability P(k | k), since
When k nn (k) increases with k, the network is said to be assortative on the degree; that is, high-degree nodes have a tendency to link to other high degree nodes, a behaviour often observed in social networks. Other types of networks, such as the internet at router level, show the converse behaviour; that is, nodes of high degree tend to link to nodes with low degree [63, 89] . Characterising degree correlations is important for understanding disease spread. The classic example is the existence of strong correlations in sexual networks which were shown to be a key factor in understanding HIV spread [90] . More recently, mean field solutions of the SIS model on networks have shown that both the speed and extent of an epidemic are dependent on the correlation pattern of the substrate network [91, 92] .
In a network, the shortest path between two nodes i and j, is the path requiring the smallest number of steps to reach j from i, following edges in the network. There may be (and often there is) more than one shortest path between a pair of nodes. The distance between any pair of nodes d i, j is the minimal number of steps required to reach j from i, that is, the number of steps in the shortest path. The average distance, d is the mean of the distances between all pairs of nodes and measures the typical distance between nodes
where N is the number of nodes in the network. The diameter of the network is defined as the maximum shortest path distance between a pair of nodes in the network, max(d i, j ), which measures the most extreme separation of any two nodes in the network. Characterising networks in terms of the number of steps needed to reach any node from any other is also important. Real networks frequently display the small-world property; that is, the vast majority of nodes are reachable in a small number of steps. This has clear implications for disease spread and its control. Percolation approaches have shown that the effects of the small-world phenomenon can be profound [93] . If it only takes a short number of steps to reach everyone in the population, diseases are able to spread much more rapidly.
The notion of shortest distance through a network can be used to quantify how central a given node is in the network. Many measures have been used [94] , but the most relevant of these is betweenness centrality. Betweenness captures the idea that the more shortest paths pass through a node, the more central it is in the network. So, betweenness is simply defined as the proportion of shortest paths that pass through a single node
where N is the number of nodes in the network and the denominator quantifies the total number of shortest paths in the network. In terms of disease spread, identifying those nodes with high betweenness will be important. Central nodes are likely to become infected early on in the epidemic, and are also key targets for intervention [3] .
Clustering. An important example of an observable property of any network is the clustering coefficient, φ, a measure of the local density of a graph. In social network terms, this quantifies the likelihood that the friend of your friend is also your friend. It is defined as the probability that two neighbours of a node will also be neighbours of each other and can be expressed as follows:
where a connected triple means a single node with edges to a pair of others. φ measures the fraction of triples that also form part of a triangle. The factor of three accounts for the fact that each triangle is found in three triples and guarantees that 0 ≤ φ ≤ 1 (and its inclusion depends on the way that triangles in the network are counted). Locally, the clustering coefficient for each node, i, can be defined as the fraction of triangles formed through the immediate neighbours of i [74] φ i = # triangles centered on i # triples centered on i .
The clustering property of networks is essential to the understanding of transmission processes. In clustered networks, rapid local depletion of susceptible individuals plays a hugely important role in the dynamics of spread [95, 96] ; for a more analytic treatment of this, see Section 4.2 below.
Degree and clustering characterise some aspects of network structure at an individual level. Considering distances between nodes provides information about the global organisation of the network. Intermediate scales are also present, and characterising these can help in our understanding of network structure and therefore the dynamics of spread. At the simplest level, networks can be thought of being comprised of a collection of subgraphs. The simplest subgraph, the clique, is defined as a group of more than two nodes where all the nodes are connected to each other by means of edges in both directions. In other words, a clique is a fully connected subgraph, with the smallest example being a triangle. This is a strong definition and one which is only fulfilled in a limited number of cases, most notably households (see Figure 1 (d), Section 4.2 and House and Keeling [70] ). n-cliques relax the above constraint while retaining its basic premise. The shortest path between all the nodes in a clique is one. Allowing this distance to take higher values, one arrives at the definition of n-cliques, which are defined as a subgroups of the graph containing more than two nodes where the maximum shortest path distance between any two nodes in the group is n. Over the years, many variants of these basic ideas have been formalised in the social network literature and a good summary can be found in Wasserman and Faust [94] .
Considering higher order structures can be very informative but is more involved. Milo and coworkers began by looking for specific patterns of connections between nodes in small subgraphs, dubbed motifs. Given a connected subgraph of size 3, for example, there are 13 possible motifs. Statistically, some of these appear more often and are found to be overrepresented in certain real networks compared to random networks [97] . Understanding the motif composition of a complex network has been shown to improve the predictive power of deterministic models of transmission when motifs are explicitly modelled (see Section 4.2 and House et al. [98] ).
In the above definitions, a subgraph has been defined only in reference to itself. A different approach is to compare the number of internal edges to the number of external edges, arising from the intuitive notion that a community will be denser in terms of edges than its surroundings. One such definition, the definition of community in the strong sense, is defined as a subgraph in which each node has more edges to other nodes within the subgraph than to any other nodes in the network. Again, this definition is quite restrictive, and in order to relax these constraints, the most commonly used (and most intuitive) definition of communities is groups of nodes that have a high density of edges within them and a lower density of edges between groups. This intuitive definition is behind the most widely used approach for studying community structure in networks. Newman and Girvan formalised this in terms of the modularity measure Q [99] . Given a particular network which is partitioned into communities, the modularity measure compares the expected number of edges within communities to the actual number of edges within communities.
Although the impact of communities in transmission processes has not been fully explored, a few studies have shown it can have a profound impact on disease dynamics [100, 101] . An alternative measure of how "well-knit" a graph is, named conductance [102] , most widely used in the computer science literature has also been found to be important in a range of networks [103] .
3.6. Higher Dimensional Networks. All of the above definitions have concentrated on networks where the edges remain unchanged over time and all edges have equal weight. Both of these constraints can naturally be relaxed, but generally, this calls for a higher-dimensional characterisation of the edges within the network. It is a matter of common experience that social interactions which can lead to infection do change, with some contacts being repeated regularly, while others are more sporadic. The frequency, intensity, and duration of contacts are all time-varying. How these inherently dynamic networks are represented for the purposes of modelling can have a significant impact on the model outcomes [55, 104] . However, capturing the structure of such dynamic networks in a parsimonious manner remains a substantial challenge. More work has been done on weighted networks, as these are a more straightforward extension of the classical presenceabsence networks [105, 106] . In terms of disease spread, the movement networks discussed in Section 2.4 are often considered as weighted [37, 40, 107] .
In the sections that follow, we discuss how these network properties can be inferred statistically and the improvements in our understanding of the transmission of infection in networks that have come as a result.
One of the key advantages of the simulation of disease processes on networks is that it enables the study of systems that are too complex for analytical approaches to be tractable. With that in mind, it is worth briefly considering efficient approaches to disease simulation on networks.
There are two main types of simulation model for infectious diseases on networks: discrete-time and continuoustime models; of these, discrete-time simulations are more common, so we discuss them first. In a discrete-time simulation, at every time step, disease may be transmitted along every edge from an infectious node to a susceptible node with a particular probability (which may be the same for all extant edges or may vary according to properties of the two nodes or the edge). Also, nodes may recover (becoming immune, or reverting to being susceptible) during each timestep. Within a time-step, every infection and recovery event is assumed to occur simultaneously. In a dynamic network simulation, the network is typically updated every time step-for example, in a livestock movement network, during time-step x, infection could only transmit down edges that occurred during time-step x. Clearly, in a directed network, infection may only transmit in the direction of an edge.
Whilst algorithms for discrete-time simulations are not complex, some simple implementation techniques (arising from the observation that most networks of epidemiological interest are sparse) can significantly enhance software performance. In a directed network with N nodes, there are N(N −1) possible edges; in a sparse network with mean node degree k, there are Nk N(N − 1) edges. Accordingly, rather than representing the network as an N by N array, where the element in each array is 0 if the edge is absent, nonzero otherwise, it is usually more efficient to maintain a list of the neighbours of each node. Then, if a list of infected nodes is maintained during a simulation run, it is straightforward to consider each susceptible neighbour of an infected node in turn and test if infection is transmitted to that node. Additionally, a fast high-quality pseudorandom number generator such as the Mersenne Twister should be used [108] . The "contagion" software package implements these techniques (amongst others) and is freely available [109] .
The alternative approach to simulating disease processes on networks is to simulate a series of stochastic Markovian events-the continuous-time approach. Essentially, given the state of the system, it is possible to calculate the probability distributions of when possible subsequent events (i.e., recovery of an infectious node or infection of a susceptible node) will occur. Random draws from these distributions are then made to determine which event occurs next, the state of the system updated, and the process repeated. This approach was pioneered by Gillespie to study the dynamics of chemical reactions [110] ; it is, however, computationally intensive, so approximations have been developed. The τleap method [111] , where multiple events are allowed to occur during a time period τ, is clearly related to the discretetime formulation discussed above. However, the ability to allow τ to vary during a simulation to account for the processes involved [112] has potential benefits.
The continuous-time approach is clearly in closer agreement with the ideal of standard disease models; however, utilising this method may be computationally prohibitive especially when large networks are involved. Discrete-time models may provide a viable alternative for three main reasons. Firstly, as the time steps involved in the discretetime model become sufficiently small, we would expect the two models to converge. Secondly, inaccuracies due to the discrete-time formulation are likely to be less substantial in network models compared to random-mixing models, providing two events do not occur in the same neighbourhood during the same time step. Finally, the daily cycle of contacts that regulate most of our lives means that using time steps of less than 24 hours may falsely represent the temporal accuracy that can be attributed to any simulation of the real world.
In this section, we use the word "analytic" broadly, to imply models that are directly numerically integrable, without the use of Monte Carlo simulation methods, rather than systems for which all results can be written in terms of fundamental functions, of which there are very few in epidemiology. Analytic approaches to transmission of infection on networks fall into three broad categories. Firstly, there are approaches that calculate exact invasion thresholds and final sizes for special networks. Secondly, there are approaches for calculating exact transient dynamics, including epidemic peak heights and times, but again, these only hold in special networks. Finally, there are approaches based on moment closure that are give approximately correct dynamics for a wide class of networks.
Before considering these approaches on networks, it is worth considering what is meant by nonnetwork mixing and showing explicitly how this can derive the standard transmission terms from familiar differential equation models. Nonnetwork mixing can be taken to have one of two meanings: either that every individual in the population is weakly connected to every other (the mean-field assumption), or that an Erdös-Rényi random graph defines the transmission network, depending on context. To see how this determines the epidemic dynamics, we consider a population of N individuals, with a homogeneous independent probability q that any pair of individuals is linked on the network, which gives each individual a mean number of edges n = q(N − 1). We then assume that the transmission rate for infection across an edge is τ and that the proportion of the population infectious at a time t is I(t); then, the force of infection experienced by an average susceptible in the population is nτI(t) ≡ βI(t). The quantity β, therefore, defines a population-level transmission rate that can be interpreted in one of two ways as N → ∞. In the case where the population is assumed to be fully connected, the limit is that q is held at unity, and so τ is reduced to as N is increased to hold q(N − 1)τ constant. In the case where the population is connected on a random graph, q is reduced as N is increased to hold n constant.
In either case, having defined an appropriate populationlevel transmission rate, a stochastic susceptible-infectious model of transmission is defined through a Markov chain, in which a population with X susceptible individuals and Y infectious individuals transitions stochastically to a population with X − 1 susceptible individuals and Y + 1 infectious individuals at rate βXY/(N − 1). Then, the exact mean behaviour of such a system in the limit N → ∞ then has its transmission behaviour captured bẏ
where S, I are the proportion of individuals susceptible and infectious, respectively. The mathematical formalism behind deriving such sets of ordinary differential equations from Markov chains is given by Kurtz [113] , and a summary of the application of this methodology to infectious disease modelling is given in Diekmann and Heesterbeek [114] . However, it should be clear that (8) is familiar as the basis of all random-mixing epidemiological models.
In the case of exponentially distributed infectious periods and recovery from infection offering long-lasting immunity, the standard SIR equations provide an exact description of the mean behaviour of this system. Nevertheless, the existence of waning immunity, a latent period between an individual becoming infected and being able to transmit infection, and nonexponentially distributed recovery periods are also important for epidemiological applications [10, 42, 115] . These can often be incorporated into analytical approaches through the addition of extra disease compartments, which necessitates extra algebraic and computational effort but typically does not require a fundamental conceptual reevaluation. Sometimes, significant additional complexity does not even modify quantitative epidemiological results-for example, regardless of the rate of waning immunity, length of latent period, or infectious period distribution, if the mean infectious period is T, then the basic reproductive ratio is
The estimation of this quantity for complex disease histories, from data likely to be available, is considered by Wallinga and Lipsitch [116] . We, therefore, focus on the transmission process, since this is most affected by network structure, and other elements of biological realism typically act at the individual level. An important caveat to this, however, is when an infected individual's level of transmissibility varies over the course of their infectious period, which sets up correlations between the processes of transmission and recovery that pose a particular challenge for analytic work, especially in structured populations, as noted by for example Ball et al. [117] .
For nonnetwork mixing, the threshold for invasion is given by the basic reproductive ratio R 0 , defined as the expected number of secondary infectious cases created by an average primary infectious case in an otherwise wholly susceptible population. In structured populations, this verbal definition is typically altered to be the secondary cases caused by a typical primary case once the dynamical system has settled into its early asymptotic behaviour. As such, the threshold for invasion is R 0 = 1: for values above this, an infection can grow in the population and the disease can successfully invade; for values below it, each chain of infection is doomed to eventual extinction. Values of R 0 can be measured directly during the course of an epidemic by detailed contact tracing; however, there are considerable statistical issues concerning censoring and data quality. Provided there are no short closed loops in the network, R 0 can be defined through a next-generation matrix
where K km defines the number of cases in individuals with k contacts from an individual with m contacts during the early stages of the epidemic. Here and elsewhere in this section we use square brackets to represent the numbers of different types on the network; hence, [m] is the number of individuals with m edges in the network and [km] is the number of edges between individuals with k and m contacts, respectively. In addition, p is the probability of infection eventually passing across the edge between a susceptible-infectious pair (for Markovian recovery rate γ and transmission rate τ this is given by p = τ/(τ + γ)). The basic reproductive ratio is given by the dominant eigenvalue of the next-generation matrix
This quantity corresponds to the standard verbal definition of the basic reproductive ratio, and correspondingly the invasion threshold is at R 0 = 1.
Once an appreciable number of short closed loops are present in the network, exact threshold parameters can still sometimes be defined, but these typically depart from the standard verbal definition of R 0 . For example, Ball et al. [117] consider a branching process on cliques (households) connected to each other through configuration-model edgescliques are connected to each other at random (Figure 1(d) ).
By considering the number of secondary cliques infected by a clique with one initial infected individual, a threshold called R * can be defined. (For the configuration-model of households where each household is of the same size and each individual has the same number of random connections outside the household, the threshold R * is given later as (20) ; however, the methodology is far more general). The calculation of the invasion threshold for the recently defined triangular configuration model [118, 119] involves calculating both the expected number of secondary infectious individuals and triangles rather than just working at the individual level. Trapman [120] deals with how these sort of results can be related to more general networks through bounding. A general feature of clustered networks for which exact thresholds have been derived so far is that there is a local-global distinction in transmission routes, with a general theory of this given by Ball and Neal [121] , where an "overlapping groups" and "great circle" model are also analysed. Nevertheless, care still has to be taken in which threshold parameters are mathematically well behaved and easily calculated (e.g [122] ).
Size. The most sophisticated and general way to obtain exact results for the expected final size of a major outbreak on a network is called the susceptibility set argument and the most general version is currently given by Ball et al. [117] . We give an example of these kind of arguments from Diekmann et al. [123] , who consider the simpler case of a network in which each individual has n contacts. Where there is a probability p of infection passing across a given network link (so for transmission and recovery at rates τ and γ, resp., p = τ/(τ + γ)), the probability that an individual avoids infection is given by
Here, a two-step process is needed because in an unclustered, regular graph two generations of infection are needed to stabilise the network correlations and so the auxiliary variable S must also be solved for. Once this and S ∞ are known, the expected attack rate is R ∞ = 1 − S ∞ .
The main way to calculate approximate final sizes is given by percolation-based methods. These were reviewed by Bansal et al. [124] and also in [125] . Suppose that we remove a fraction ϕ of links from the network and can derive an expression for the fraction of nodes remaining in the giant component of the network, f (ϕ). Then,
and an invasion threshold is given by the value of p for which this final size becomes nonzero in the "thermodynamic limit" of very large network size. This approach is not exact for clustered graphs, but for unclustered graphs exact results like (12) are reproduced.
(where the probability of the population being in each possible configuration is calculated) on small, fully connected graphs as summarised in Bailey [126] . The rate at which the complexity of the system of master equations grows means that these equations quickly become too complex to integrate for the most general network. The presence of symmetries in the network, however, does mean that automorphism-driven lumping is one way to manipulate the master equations (whilst preserving the full stochastic information about the system) for solution [127] . At present, this technique has only been applied to relatively simple networks; however, there are no other highly general methods of deriving exact lower-dimensional systems of equations from the master equations. Nevertheless, other specific routes do exist that allow exact systems of equations of lower dimensionality to be derived for special networks. For static networks constructed using the configuration model (where individuals have heterogeneous degree but connections are made at random such that the presence of short loops can be ignored in a large network, see Figure 1 (c)), an exact system of equations for SIR dynamics in the limit of large network size was provided by Ball and Neal [69] . This construction involves attributing to each node an "effective degree", which starts the epidemic at its actual degree, and measures connections still available as routes of infection and is, therefore, reduced by transmission and recovery. Using notation consistent with elsewhere in this paper (and ignoring the global infection terms that were included by Ball and coworkers) this yields the relatively parsimonious set of equationṡ S k = −ρ τ + γ kS k − γ(k + 1)S k+1 ,
. (14) Here, S k , I k are the proportion of effective degree k susceptible and infectious individuals, respectively. Hence, for a configuration-network where the maximum degree is K, we require just 2K equations to retrieve the exact dynamics.
While R 0 can be derived using expressions like (11), calculation of the asymptotic early growth rate r requires systems of ODEs like (14) . If we assume that transmission and recovery are Markovian processes with rates τ and γ, respectively, two measures of early behaviour are
Interdisciplinary Perspectives on Infectious Diseases
where · informs about the average over the degree distribution. These quantities tell us that the susceptibility to invasion of a network increases with both the mean and the variance of the degree distribution. This closely echos the results for risk-structured models [10] but with an extra term of −1 due to the network, representing the fact that the route through which an individual acquired infection is closed off for future transmission events. For more structured networks with a local-global distinction, there are two limits in which exact dynamics can also be derived. If the network is composed of m communities of size n 1 , . . . , n m , with the between-community (global) mixing determined by a Poisson process with rate n G and the within-community (local) mixing determined by a Poisson process with rate n L , then in the limit as the communities become large, n i → ∞, the epidemic dynamics on the system areṠ
where S a and I a are the proportion of individuals susceptible and infectious in community a, and
Hence, we have a classic metapopulation model [43] , defined in terms of Poisson local and global connections and large local community sizes.
In the limit where n L → (n − 1) and m → ∞-such that there are infinitely many communities of equal size and each community forms a fully interconnected clique-"selfconsistent" equations such as in Ghoshal et al. [128] and House and Keeling [70] are exact. These equations evolve the proportion of cliques with x susceptibles and y infecteds, P x,y , as well as the proportion of infecteds in the population, I, as follows:
where β G = n G τ. Both of these two local-global models, the metapopulation model (16) and the small cliques model (18) , are reasonably numerically tractable for modern computational resources, provided the relevant finite number (m or n, resp.) is not too large. The basic reproduction number for the first system is clearly
while for the second, household model, invasion is determined by
where Z n ∞ (τ, γ) is the expected final size of an epidemic in a household of size n with one initial infected. Of course, the within-and between-community mixing for real networks is likely to be much more complex than may be captured by a Poisson process, but these two extremes can provide useful insights. These models show that network structure of the form of communities reduces the potential for an infectious disease to spread, and hence, greater transmission rates are required for the disease to exceed the invasion threshold.
Dynamics. While all the exact results above are an important guide to intuition, they only hold for very specialised networks. A large class of models exists that form a bridge between "mean-field" models and simulation by using spatial or network moment closure equations. These are highly versatile models. In general, invasion thresholds and final sizes can be calculated rigorously, but exact calculation of transient dynamics is only possible for very special networks. If one wants to calculate transient effects in general network models-most importantly, peak heights and times-then moment closure is really the only versatile way of calculating desired quantities without relying on full numerical simulation.
It is also worth noting that there are many results derived through these "approximate" approaches that are the same as exact results or are numerically indistinguishable from exact results and simulation. We give some examples below and also note that the dynamical PGF approach [129] is numerically indistinguishable from the exact model (14) above for certain parameter values [130] . What is currently lacking is a rigorous mathematical proof of exactness for ODE models other than those outlined in Section 4.2.4 above. While for many practical purposes the absence of such a proof will not matter, we preserve here the conceptual distinction between results that are provably exact, and those that are numerically exact in all cases tested so far.
The idea of moment closure is to start with an exact but unclosed set of equations for the time evolution of different units of structure. Here, we show how these can be derived by considering the rates of change of both types of individual and types of connected pair. Such pairwise moment closure model are a natural extension to the standard (random-mixing) models, given that infection is passed between pairs of infected individuals
Here, we use square brackets to represent the prevalence of different species within the network. We also use some nonstandard notation to present several diverse approaches in a unified framework: generalised indices κ, λ represent any property of a node (such as its degree), while arrows represent the direction of infection (and so for a directed network, the necessity that an edge in the appropriate direction be present), see Table 1 . Clearly, the system (21) is not closed as it relies on the number of connected triples, and so some form of approximate closure must be introduced to relate the triples to pairs and nodes, which will depend on underlying properties of the network. Most commonly, these closure assumptions deal with heterogeneity in node degree, assortativity, and clustering at the level of triangles. Examples include Keeling [95] and Eames and Keeling [96] , where the generalised variables κ, λ above stand for node degrees (k, l), the triple closure is symmetric with respect to the direction of infection, and the network is assumed to be static and nondirected. A general way to write the closure assumption is
where n ≡ n is again the average degree distribution and φ measures the ratio of triangles to triples as a means of capturing clustering within the network (see Section 3.4).
The typical way to analyse the closed system is direct numerical integration; however, some analytic traction can be gained. One example is the use of a linearising Ansatz to derive the early asymptotic behaviour of the dynamical system. Interestingly, when this is done for φ = 0 (such that there are no triangular loops in the network) as in Eames and Keeling [96] , the result for the early asymptotic growth rate agrees with the exact result of (10). In [95] , the differential equations for an n-regular graph were also manipulated to give an expression for final size that agreed with the exact result (12) Equation (22), however, is not the only possible network moment closure regime: Boots and Sasaki [131] and Bauch [132] considered regimes in which closure depended on the disease state (i.e., triples composed of different arrangements of susceptibles and infecteds close differently) to deal with spatial lattice-based systems and early disease invasion respectively. For example Boots and Sasaki [131] 
where O represents empty sites within the network that are not currently occupied by individuals, and the parameter ε = 0.8093 accounts for the clustering within lattice-based networks. House and Keeling [133] considered a model of infection transmission and contact tracing on a network, where the closure scheme for [ABC] triples was asymmetric in A and C-this allowed the natural conservation of quantities in a highly clustered system. The work on dynamical PGF models [129] can be seen as an elegant simplification of this pairwise approach that is valid for SIR-type infection dynamics on configuration model networks. The equations can be reformulated as S = g(θ),
where g is the probability generating function for the degree distribution, p S and p I correspond to the number of contacts of a susceptible that are susceptible or infected, respectively, and θ is defined as probability that a link randomly selected from the entire network has not been associated with the transmission of infection. Here, the closure assumption is implicit in the definition of S; that is, an individual only remains susceptible if all of its links have not seen the transmission of infection, and that the probability is independent for each link, which is comparable to the assumptions underlying the formulation by Ball and Neal [69] , equation (14) . The precise link between this PGF formulation and the pairwise approach is discussed more fully in House and Keeling [134] . There are many other extensions of this general methodology that are possible. Writing ODEs for the time evolution of triples and closing at a higher order allows the consideration of the epidemiological consequences of varying motif structure [98] . Sharkey et al. [135] considered closure at triple level on directed networks, which involved a more sophisticated treatment of third-order clustering due to the larger repertoire of three-motifs in directed (as compared to undirected) networks. It is also possible to combine stochastic and network moment closure [136] . Timevarying, dynamical networks, particularly applied to sexually transmitted infections where partnerships vary over the course of an epidemic, were considered using approximate ODE-based models by Eames and Keeling [137] and Volz and Meyers [138] . Sharkey [139] considered models appropriate for local networks with large shortest path lengths, where the generic indices μ, λ in (21) stand for node numbers i, j rather than node degrees k, l.
Another approach is to approximate the transmission dynamics in the standard (mean-field) differential equations models. Essentially, this is a form of moment closure at the level of pairs rather than triples. For example, in Roy and Pascual [140] the transmission rate takes the polynomial form
where the exponents, p and q, are typically fitted to simulated data but are thought to capture the spatial arrangement of susceptible and infected nodes. Also, Kiss et al. [141] suggest Transmission rate to S k from I l ∝ k(l − 1)(S k )(I l ), (26) as a way of accounting for each infected "losing" an edge to its infectious parent. Finally, a very recent work [142] presents a dynamical system to capture epidemic dynamics on triangular configuration model networks; the relationship between this and other ODE approaches is likely to be an active topic for future work.
This diversity of approaches leads to some important points about methods based on moment closure. These methods are extremely general and can be applied to consider almost any aspect of network structure or disease natural history; they can be applied to populations not currently amenable to direct simulation due to their size, and they do not require a complete description of the network to run-only certain statistical properties. However, there are currently no general methods for the proposal of appropriate closure regimes nor any derivation of the limits on dynamical biases introduced by closure. Therefore, closure methods sit somewhere in between exact results for highly specialised kinds of network and stochastic simulation, where intuitive understanding and general analysis are more difficult.
In the papers that introduced them, the differential-equation-based approximate dynamical systems above were compared to stochastic simulations on appropriate networks. Two recent papers making a comparison of different dynamical systems with simulation are Bansal et al. [124] and Lindquist et al. [130] . There are, however, several issues with attempts to compare deterministic models with simulation and also with each other.
Firstly, it is necessary to define what is meant by agreement between a smooth, deterministic epidemic curve and the rough trajectories produced by simulation. Limiting results about the exactness of different ODE models assume that both the number of individuals infectious and the network size are large, and so the early behaviour of simulations, when there are few infectious individuals, is often dominated by stochastic effects. There are different ways to address this issue, but even after this has been done, there are two sources of deviation of simulations from their deterministic limit. The first of these is the number of simulations realised. If there is a summary statistic such as the mean number of infectious individuals over time, then the confidence interval in such a statistic can be made arbitrarily small by running additional simulations, but agreement between the deterministic limit and a given realisation may still be poor. The second source of deviation is the network size. By increasing the number of nodes, the prediction interval within which the infection curve will fall can be made arbitrarily small; however, the computational resources needed to simulate extremely large networks can quickly become overwhelming.
More generally, each approximate model is designed with a different application in mind. Models that perform well in one context will often perform poorly in another, and this means that "performance" of a given model in terms of agreement with simulation will primarily be determined by the discrete network system on which simulations are performed.
The above considerations motivate the example comparisons with simulation that we show in Figure 3 . This collection of plots is intended to show a variety of different example networks, and the dynamical systems intended to capture their behaviour.
In Figures 3(a) -3(e), continuous-time simulations have their temporal origin shifted so that they agree on the time at which a cumulative incidence of 200 is reached, and then confidence intervals in the mean prevalence of infection are achieved through bootstrapping. The 95% confidence interval is shown as a red shaded region (although typically, this is sufficiently narrow it resembles a line). Six different deterministic models are compared to simulations: HomPW is the pairwise model of Keeling [95] with zero clustering, HetPW is the heterogeneous pairwise model of Eames and Keeling [96] , ClustPW is the improved clustered pairwise closure of House and Keeling [133] , PGF is the model of Volz [129] , Pair-based is the model of Sharkey [139] , integrated using the supplementary code from Sharkey [143] , and Degree-based is the model of Pastor-Satorras and Vespignani [87] . Figure 3 (a) shows a heterogeneous network composed of two risk groups, constructed according to the configuration model [18] . In this case, models that incorporate heterogeneity like HetPW and PGF (which are numerically indistinguishable in this case and several others) are in very close agreement with simulation, while just taking the average degree as in HomPW is a poor choice. In Figure 3 (b), assortativity is added to the two group model following the approach of Newman [89] , and HetPW outperforms PGF. Figure 3 (d) the rate of making and breaking links is much faster than the epidemic process. Models like HomPW and PGF are therefore better for the former and degree-based models are better for the latter-in reality the ratio of the rate of network change to the rate of transmision may not be either large or small and so a more sophisticated method may be best [137, 138] . Figure 3 (e) shows a graph with four links per node where clustering has been introduced by the rewiring method of Bansal et al. [144] sometimes called the "big V" [133] . In this case, ClustPW performs better than HomPW and PGF, but clearly there is significant inaccuracy around the region of peak prevalence and so this model captures qualitatively the effects of clustering without appearing to be exact for this precise network. Finally, Figure 3 (f) considers the case of a one-dimensional next-nearest-neighbour lattice (so there are four links per node). This introduces long path lengths between nodes in addition to clustering, meaning that the system does not converge onto a period of asymptotic early growth and so realisations are shown as a density plot rather than a confidence interval. ClustPW accounts for clustering but not long path lengths and so is in poor agreement with simulation while the pair-based curve captures the qualitative behaviour of an epidemic on this lattice whilst being quantitatively a reasonable approximation.
In order to be predictive, epidemic models rely on valid values for parameters governing outbreak dynamics, conditional on the population structure. However, obtaining these parameters is complicated by the fact that even when knowing the underlying contact network structure, infection events are censored-it is only when disease is detected either from symptoms or laboratory tests that a case becomes apparent. In attempting to surmount this difficulty, parameter estimates are often obtained by making strong assumptions as to the infectious period or through ad hoc methods with unknown certainty. Measuring the uncertainty in such estimates is as important as obtaining the estimates themselves in providing an honest risk prediction. Given these difficulties, inference for epidemic processes has perhaps received little attention in comparison to its simulation counterpart.
The presence of contact network data for populations provides a unique opportunity to estimate the importance of various modes of disease transmission from disease incidence or contact tracing data. For example, given knowledge of the rate of contact between two individuals, it is possible to infer the probability that a contact results in an infection. If data on mere connectivity (i.e., a 1 if the individuals are connected and 0 otherwise) is available, then it is still possible to infer a rate of infection between connected individuals. Thus, the detail of the inference is determined to a large extent by the available detail in the network data [145] .
Epidemic models are defined in terms of times of transitions between infection states, for example a progression from susceptible, to infected, to removed (i.e., recovered with lifelong immunity or dead) in the so-called "SIR" model. Statistical inference requires firstly that observations of the disease process are made: at the very least, this comprises the times of case detections, remembering that infection times are always censored (you only ever know you have a cold a few days after you caught it). In addition, covariate data on the individuals provides structure to the population and begins to enable the statistician to make statements about the importance of individuals' relationships to one another in terms of disease transmission. Therefore, any covariate data, however slight, effectively implies a network structure upon which disease transmission can be superimposed.
As long as populations are relatively small (e.g., populations of farms in livestock disease analysis), it is common for models to operate at the individual level, providing detailed information on case detection times and perhaps even information on epidemiologically significant historical contact events [146] [147] [148] . In other populations, however, such detailed data may not be available due to practical and ethical reasons. Instead, data is supplied on an aggregated spatial and/or temporal basis. For the purposes of inference, therefore, this can be regarded as a household model, with areas constituting households.
In a heterogeneous population, the behaviour of an epidemic within any particular locality is governed by the relationship between infected and susceptible individuals. For inference in the early stages of an epidemic, it is important to quantify the amount of uncertainty in the underlying contact networks as the early growth of the epidemic is known to be subexponential due to the depletion of the local susceptible population. This contrasts markedly to the exponential growth observed in a large homogeneously mixing population [10] . When the network is known and details of individual infections are available, contact tracing data may be used to infer the network; this data could also be used for inference on the epidemic parameters [116, 149] . Conversely, if the network is completely unknown, it would be useful if estimation of both the epidemic parameters and parameters specifying the structure of the network was possible. This is a difficult problem because the observed epidemic contains very limited information about the underlying network, as demonstrated by Britton and O'Neill [150] . However, with appropriate assumptions, some results can be obtained; the limited amount of existing work in this area is described in Section 5.3.1 below although clearly the problem is worthy of further study.
For homogeneous models the basic reproduction number, or R 0 , has several equivalent definitions and can be defined in terms of the transmission rate β and removal rate γ. For nonhomogeneous models, the definitions are not equivalent; see for example [122] .
Although inference for β and γ is difficult for real applications (see below), it turns out that making inference on R 0 (as a function of β and γ) is rather more straightforward. Heffernan et al. [151] summarise various methods for estimating R 0 from epidemiological data based on endemic equilibrium, average age at infection, epidemic final size, and intrinsic growth rate [114, 152, 153] . However, these methods all rely on observing a complete epidemic, and hence for real-time analysis during an epidemic, we must make strong assumptions concerning the number of currently undetected infections. An example of inference for R 0 based upon complete epidemic data is provided by Stegeman et al. [154] , where data from the 2003 outbreak of High Pathogenicity Avian Influenza H7N7 is fitted to a chain-binomial model using a generalised linear model.
Obviously, complete or near-complete epidemic data is rare and hence it is desirable to perform inference based upon partial observation. This is particularly relevant for real time estimation of R 0 . For example, Cauchemez et al. [155] attempt to estimate R 0 in real-time by constructing a discrete-time statistical model that imputes the number of secondary cases generated by each primary case. This is based on the method of Wallinga and Teunis [156] who formulate a likelihood function for inferring who infected whom from dates of symptom onset
where w(·) is the probability density function for the generation interval t j − t i , that is, the time between infector i's infection time and infectee j's infection time. Of course, infection times are never observed in practice so symptom onset times are used as a proxy, with the assumption that the distribution of infection time to symptom onset time is the same for every individual. Bayesian methods are used to infer "late-onset" cases from known "early-onset" cases, but large uncertainty of course remains when inferring the reproductive ratio close to the current time as there exists large uncertainty about the number of cases detected in the near future. Additionally, a model for w(·) must be chosen (see, [157] ). The tradeoff in the simplicity of estimating R 0 in these ways, however, is that although a population wide R 0 gives a measure of whether an epidemic is under control on a wide-scale, it give no indication as to regional-level, or even individual-level, risk. Moreover, the two examples quoted above do not even attempt to include population heterogeneity into their models though the requirement for its inclusion is difficult to ascertain in the absence of model diagnostics results. It is postulated, therefore, that a simple measure of R 0 , although simple to obtain, is not sufficient in order to make tactical control-policy decisions. In these situations, knowledge of both the transmission rate and removal rate are required.
Inference for households models is well developed in comparison to inference for other "network" models. In essence, this is for three main reasons: firstly, it is a reasonable initial approximation to assume that infection either occurs within the home or from a random source in the population. Secondly, entire households can be serologically sampled following an epidemic, such that the distribution of cases in households of given sizes can be ascertained. Finally, it is often a reasonable approximation that following introduction of infection into the household, the within-household epidemic will go extinct before any further introductions-which dramatically simplifies the mathematics.
The first methods proposed for such inference are maximum likelihood procedures based upon chain-binomial models, such as the Reed-Frost model, or the stochastic formulation of the Kermack-McKendrick model considered by Bartlett [158] . These early methods are summarised by Bailey [126] . They, and the significant majority of methods proposed for household inference to date, use finalsize data which can be readily obtained from household serology results. A simplifying assumption to facilitate inference in most methods is that the epidemics within the various households evolve independently (e.g., see the martingale method of Becker [159] , which requires the duration of a latent period to be substantial for practical implementation).
Additionally, fixed probabilities p C and p H , corresponding to a susceptible individual escaping community-acquired infection during the epidemic and escaping infection when exposed to a single infected household member, respectively, were initially assumed [160] . Two important, realistic extensions to this framework are to incorporate different levels of risk factors for individuals [161] and to introduce dependence of p H on an infectious period [162] . The latter inclusion was enabled by appealing to results of Ball et al. [163] . These types of methods are largely based upon the ability to generate closed form formulae for the final size distribution of the models.
The ability to relax assumptions further has been predominately due to use of Markov chain Monte Carlo (MCMC) methods as first considered by O'Neill et al. [162] for household models following earlier studies of Gibson and Renshaw [164] and O'Neill and Roberts [165] who focused on single, large outbreaks. This methodology has been used to in combination with simulation and data augmentation approaches to tailor inference methods for specific data sets of interest; for example, Neal and Roberts [166] consider a model with a spatial component of distance between households and data containing details of dates of symptoms and appearance of rash and has also resulted in a growing number of novel methods for inference, for example Clancy and O'Neill [167] consider a rejection sampling procedure and Cauchemez et al. [168] introduce a constrained simulation approach. Even greater realism can be captured within household models by considering the different compositions of households and, therefore, the weighted nature of contacts within households. For example, Cauchemez et al. [169] considered household data from the Epigrippe study of influenza in France 1999-2000 and showed that children play a key role in the transmission of influenza and the risk of bringing infection into the household.
Whilst new developments are appearing at an increasing rate, the significant majority of methods are based upon final size data and are developed for SIR disease models, perhaps due in part to the simplification of arguments for deriving final size distributions. One key, but still unanswered question from these analyses of household epidemics is how the transmission rate between any two individuals in the household scales with the total number of individuals in the household (compare Longini and Koopman [160] and Cauchemez et al. [169] ). Intuition would suggest that in larger households the mixing between any two individuals is decreased, but the precise form of this scaling is still unclear, and much more data on large household sizes is required to provide a definitive answer.
Perhaps the holy grail of statistical inference on epidemics is to make use of an individual-level model to describe heterogeneous populations at the limit of granularity. In this respect, Bayesian inference on stochastic mechanistic models using MCMC have perhaps shown the most promise, allowing inference to be made on both transmission parameters and using data augmentation to estimate the infectious period.
An analysis of the 1861 outbreak of measles in Hagelloch by Neal and Roberts [166] demonstrates the use of a reversible jump MCMC algorithm to infer disease transmission parameters and infectious period, whilst additionally allowing formal comparisons to be made between several nested models. With the uncertainty surrounding model choice, such methodology is vital to enable accurate understanding and prediction. This approach has since been combined with the algorithm of O'Neill and Roberts [165] and used to analyse disease outbreaks such as avian influenza and foot and mouth disease in livestock populations [146, 147, 170] and MRSA outbreaks in hospital wards [171] .
Whilst representing the cutting edge of inference on infectious disease processes, these approaches are currently limited by computing power, with their algorithms scaling by the number of infectives multiplied by the number of susceptibles. However, with advances in computer technology expected at an increasing rate, and small approximations made in the calculation of the statistical likelihoods needed in the MCMC algorithms, these techniques may well form the mainstay of epidemic inference in the future.
Tracing. In livestock diseases, part of the standard response to a case detection is to gather contact tracing information from the farmer. The resulting data are a list of contacts that have been made in and out of the infected farm during a stipulated period prior to the notification of disease [172] . In terms of disease control on a local level, this has the aim of identifying both the source of infection and any presumed susceptibles that might have been infected as a result of the contact. It has been shown that providing the efficiency of following up any contacts to look for signs of disease is high; this is a highly effective method of slowing the spread of an epidemic and finally containing it.
Much has been written on how contact tracing may be used to decrease the time between infection and detection (notification) during epidemics. However, this focuses on the theoretical aspects of how contact tracing efficiency is related to both epidemic dynamics and population structure (see, e.g., Eames and Keeling [173] Kiss et al. [174] , Klinkenberg et al. [175] ). In contrast, the use of contact tracing data in inferring epidemic dynamics does not appear to have been well exploited although it was used by the Ministry of Agriculture, Fisheries, and Food (now Defra) to directly infer a spatial risk kernel for foot and mouth disease in 2001. This assumed that the source of infection was correctly identified by the field investigators, thereby giving an empirical estimate of the probability of infection as a function of distance [148, 176, 177] . Strikingly, this shows a high degree of similarity to spatial kernel estimates based 22 Interdisciplinary Perspectives on Infectious Diseases on the statistical techniques of Diggle [178] and Kypraios [179] without using contact tracing information. However, Cauchemez et al. [155] make the point that the analysis of imperfect contact tracing data requires more complex statistical approaches, although they abandoned contact tracing information altogether in their analysis of the 2003 SARS epidemic in China. Nevertheless, recent unpublished work has shown promise in assimilating imperfect contact tracing data and case detection times to greatly improve inference, and hence the predictive capability of simulation techniques.
Qualitative results from simulations indicate that epidemics on networks, for some parameter values, show features that distinguish them from homogeneous models. The principal features are a very variable length slow-growth phase, followed by a rapid increase in the infection rate and a slower decline after the peak [180] . However, in quantitative terms, there is usually very limited information about the underlying network and parameters are often not identifiable. When the details of the network are unknown, but something is known or assumed about its formation, estimation of both the epidemic parameters and parameters for the network itself are in principle possible using MCMC techniques. All the stochastic models for generating networks described in Section 2.7 above realise a distribution over all or some of the 2 N(N−1)/2 possible networks. In most cases, this distribution is not tractable; MCMC techniques are in principle still possible but in practice would be too slow without careful design of algorithms.
However, with appropriate assumptions some results can be obtained, which provide some insight into what more could be achieved. When the network is taken to be an Erdös-Rényi graph with unknown parameter p and the epidemic is a Markovian SIR, Britton and O'Neill [150] showed that it is possible to estimate the parameters, although they highlight the ever-present challenge of disentangling epidemiological from network parameters. The MCMC algorithm was improved by Neal and Roberts [181] and the extension from SIR to SEIR has been developed by Groendyke et al. [182] . However, the extension to more realistic families of networks remains a challenging problem and will undoubtedly be the subject of exciting future research.
The use of networks is clearly a rapidly growing field in epidemiology. By assessing (and quantifying) the potential transmission routes between individuals in a population, researchers are able to both better understand the observed distribution of infection as well as create better predictive models of future prevalence. We have shown how many of the structural features in commonly used contact networks can be quantified and how there is an increasing understanding of how such features influence the propagation of infection. However, a variety of challenges remain.
Several open problems remain if networks are to continue to influence predictive epidemiology. The majority of these stem from the difficulty in obtaining realistic transmission networks for a range of pathogens. Although some work has been done to elucidate the interconnected structure of sexual encounters (and hence the sexual transmission network), these are still relatively smallscale compared to the population size and suffer from a range of potential biases. Determining comparable networks for airborne infections is a far greater challenge due to the less precise definition of a potential contact.
One practical issue is therefore whether new techniques can be developed that allow contact networks to be assessed remotely. Proximity loggers, such as those used by Hamede and colleagues [12] , provide one potential avenue although it would require the technology to become sufficiently robust, portable and cheap that a very large proportion of a population could be convinced to carry one at all times. For many human populations, where the use of mobile phones (which can detect each other via Bluetooth) is sufficiently widespread, there is the potential to use them to gather network information-although the challenges of developing sufficiently generic software should not be underestimated. While these remotely sensed networks would provide unparalleled information that could be obtained with the minimum of effort, there would still be some uncertainty surrounding the nature of each contact.
There is now a growing set of diary-based studies that have attempted to record the personal contacts of a large number of individuals; of these, POLYMOD is currently the most comprehensive [17] . While such egocentric data obviously provides extensive information on individual behaviour, due to the anonymity of such surveys it is not clear how the alters should be connected together. The configuration method of randomly connecting half-links provides one potential solution, but what is ideally required is a more comprehensive method that would allow clustering, spatially localised connections and assortativity between degree distributions to be included and specified.
Associated with the desire to have realistic contact networks for entire populations, comes the need to characterise such networks in a relatively parsimonious manner that provides important insights into the types of epidemiological dynamics that could be realised. Such a characterisation would allow for different networks (from different times or different locations) to be compared in a manner that is epidemiologically significant and would allow artificial networks to be created that matched particular known network features. This clearly relies on both existing measures of network structure (as outlined in Section 3) together with a robust understand of how such features influence the transient epidemic dynamics (as outlined in Section 4.2). However, such a generic understanding of all network features is unlikely to arise for many years. A more immediate challenge is to understand ways in which local network structure (clustering, cliques, and spatially localised connections) influence the epidemiological dynamics.
To date the vast majority of the work into disease transmission on networks has focused on static networks where all links are of equal strength and, therefore, associated with the same basic rate of transmission. However, it is clear that contact networks change over time (both on the short-time scale of who we meet each day, and on the longer time-scale of who our main work and social contacts are), and that links have different weights (such that some contacts are much more likely to lead to the transmission of infection than others). While the simulation of infection on such weighted time-varying networks is feasible, it is unclear how the existing sets of network properties or the existing literature of analytical approaches can be extended to such higher-dimensional networks.
For any methodology to have any substantive use in the field, it is important both to have effective data gathering protocols in place and to have the statistical techniques in place to analyse it. Here, three issues are perhaps most critical. Firstly, data gathering resources are almost always limited. Therefore, carefully designed randomised sampling schemata should be employed to maximise the power of the statistical techniques used to analyse data, rather than having to reply on data augmentation techniques to work around the problems present in ad hoc datasets. This aspect is particularly important when working on network data derived from population samples. Secondly, any inference on both network and infectious disease models should be backed up by a careful analysis of model fit. Although recent advances in statistical epidemiology have given us an unprecedented ability to measure population/disease dynamics based on readily available field data, epidemic model diagnostics are currently in their infancy in comparison to techniques in other areas of statistics. Therefore, it is expected that with the growth in popularity of network models for analysing disease spread, much research effort will be required in designing such methodology.
We have highlighted that the study of contact networks is fundamentally important to epidemiology and provides a wealth of tools for understanding and predicting the spread of a range of pathogens. As we have outlined above, many challenges still exist, but with growing interest in this highly interdisciplinary field and ever increasing sophistication in the mathematical, statistical and remote-sensing tools being used, these problems may soon be overcome. We conclude, therefore, that now is an exciting time for research into network epidemiology as many of the practical difficulties are surmounted and theoretical concepts are translated into results of applied importance in infection control and public health. Viral Encephalomyelitis  Encephalitic Viruses Can Use Neuronal or Non-Neuronal Pathways to Enter the CNS When a virus does invade the CNS, there are several routes by which infection of neurons can occur. The most common entry point is from the blood, and the level of viremia as a result of virus replication in peripheral organs often correlates with the likelihood of CNS infection. However, the blood-brain barrier (BBB), composed of vascular endothelial cells with tight junctions in contact with the foot processes of astrocytes, inhibits direct access to the brain parenchyma and neurons. Some neurotropic viruses can replicate in cerebrovascular endothelial cells, enter with infected leukocytes, or cross directly into the cerebrospinal fluid (CSF) through the porous capillaries of the choroid plexus. A specialized CNS entry pathway used by several viruses, most notably HSV, varicella zoster, and rabies viruses, is by way of nerve terminals in peripheral organs. These viruses can enter the nerve and then use neural transport mechanisms to transport the infecting virions to the neuronal cell body where replication occurs [6, 7] . A variation on this theme is infection through the exposed end processes of neurons in the nasal olfactory epithelium, followed by transport of the virus to the olfactory lobe within the CNS. Intranasal infection is commonly used to initiate infection of the CNS in experimental animals, but in humans this pathway may be important only in rare cases of aerosol exposure to a neurotropic virus [8] [9] [10] .
In tissue culture systems most of these viruses can infect many types of cells, in addition to neurons. Few neuron-specific virus receptors have been identified (e.g., p75NTR, NCAM, and AChR for rabies; nectin for HSV) but these do not always account for neuronotropism in vivo [11] [12] [13] . Recent studies with HSV suggest that receptors used to enter processes of peripheral neurons can be different from those used to infect neurons in the brain [12] . Therefore, the mechanisms by which encephalitic viruses target neurons to the exclusion of other cells within the CNS are poorly understood. Once within the nervous system, encephalitic viruses often follow synaptic pathways for spread to other neuronal populations. These viruses interact with motor proteins either directly or through accessory proteins to travel using both anterograde (kinesin motors) and retrograde (dynein-dynactin motors) neuronal microtubule transport systems [6] .
In addition to fever and headache, signs and symptoms of viral encephalomyelitis typically include evidence of neuronal dysfunction-seizures, cognitive impairment, ataxia, paralysis, etc. Virus replication can damage neurons directly by inducing cell death through apoptotic or necrotic mechanisms ( Figure 1 ) [14] . Many viruses cause more severe CNS disease in the young. For these Funding: Work from the author's laboratory was supported by the NIH/NINDS (R01 NS038932) and the Dana Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The author has declared that no competing interests exist.
* E-mail: dgriffin@jhsph.edu infections, immature neurons support more efficient virus replication and greater virus-induced cell death than mature neurons [15] . In humans, Venezuelan equine encephalitis and La Crosse viruses cause symptomatic neurologic disease almost exclusively in children, although adults are equally susceptible to infection [16, 17] . Conversely, for unexplained reasons, neurologic disease due to West Nile virus infection occurs primarily in people over the age of 60 years [18] .
Although neuronal virus infection per se is necessary for, and contributes directly to, neuronal dysfunction, the inflammatory response in the CNS is also a major contributor to neuronal damage and can even result in death of nearby uninfected neurons ( Figure 1 ) [19, 20] . The CNS inflammatory response to virus infection consists of activation and proliferation of resident astrocytes and microglial cells and of perivascular and parenchymal infiltration of activated monocytes and lymphocytes from the blood. The mechanism(s) by which the immune response causes neuronal damage are incompletely understood, but evidence exists for production of neurotoxins and reactive oxygen and nitrogen species by activated glial cells, increased levels of the excitotoxic neurotransmitter glutamate, and production of cytokines by activated lymphocytes [21] . The ability to control inflammation through induction of regulatory T cells and suppression of lymphocyte function by infected neurons can be an important determinant of outcome [22, 23] . In alphavirus encephalomyelitis, combined prevention of inflammation and glutamate excitotoxic- ity by treatment with glutamate receptor antagonists prevents paralysis and death despite continued virus replication [20] .
The immune response to infection can contribute to fatal disease, but is also necessary for recovery and virus clearance. Elimination of virus-infected cells from tissue requires elimination of all cells in which the virus is replicating. This can occur either by virus-induced or immune-mediated cytolysis. T cells are ideal for this purpose because they recognize viral antigen as processed antigen only in the context of cell surface-expressed MHC class I (CD8 + T cells) or of MHC class II (CD4 + T cells) and can possess cytotoxic properties. Neurons are long-lived essential cells that cannot be replaced, so a noncytolytic immune mechanism for virus clearance would be advantageous to the host to avoid death of surviving cells. If the immune clearance mechanism is damaging to the infected neuron, then the function of that neuron will be lost and the outcome for the host will be the same as if the virus infection had caused neuronal death.
Because mature neurons are relatively resistant to virus-induced cell death [15] , noncytolytic mechanisms for virus clearance can be employed to control or eliminate infection. If infected cells are allowed to survive, the clearance of virus must include mechanisms for inhibiting intracellular synthesis of virus nucleic acid and protein, and for removing virus genomes from cells or preventing their replacement after degradation. Alphavirus encephalitis has been most thoroughly studied, and two noncytolytic clearance mechanisms have been identified: IFN-c and anti-viral antibody ( Figure 1) . However, not all types of neurons are equally responsive to virus clearance by these mechanisms. IFN-c acts through a Jak/STAT signaling pathway to activate an antiviral response that suppresses virus replication in motor neurons without toxicity, but the relevant antiviral proteins have not been identified [24] . Antibody to the E2 viral glycoprotein present on the surface of infected neurons suppresses virus replication in all populations of neurons through a pathway that requires bivalent antibody, but does not require complement or effector cells [25, 26] .
Because the noncytolytic process for virus clearance does not completely eliminate viral RNA from neurons, a mechanism for long-term immunologic control of virus replication is needed to prevent virus reactivation or progressive disease [27, 28] . Antibody is likely to participate in control, as well as initial clearance. Maintaining adequate levels of antibody in the CNS for continued control of virus replication requires either passage of antibody from the blood into the brain parenchyma or local production by resident antibody-secreting cells. The BBB restricts the entry of proteins from the blood into the CNS, and although this function is compromised during the acute phase of infection, it is quickly repaired. Under normal conditions, levels of antibody in the brain are sustained at 1% of plasma levels that are likely to be inadequate for long-term prevention of virus reactivation. Therefore, resident long-lived antibody-secreting cells that can continue to produce antiviral antibody for a lifetime are a feature of recovery from most CNS virus infections [29] . Long-term immune control of virus replication is not always successful, leading to recurrent or progressive neurologic disease [30] [31] [32] .
Encephalomyelitis resulting from virus infection of neurons is a disease that can be fatal or result in permanent disability due to irreversible damage of infected neurons. The immune response to infection can enhance neuronal damage or can control virus replication by noncytolytic mechanisms and thus determine outcome. However, noncytolytic virus clearance results in persistence of viral nucleic acid in the CNS and thus establishes a need for long-term local immune responses to prevent reactivation of infection and progressive disease. Understanding these mechanisms is necessary for development of strategies for treating and preventing neurologic disease due to viral encephalomyelitis. Assessing the In Vitro Fitness of an Oseltamivir-Resistant Seasonal A/H1N1 Influenza Strain Using a Mathematical Model In 2007, the A/Brisbane/59/2007 (H1N1) seasonal influenza virus strain acquired the oseltamivir-resistance mutation H275Y in its neuraminidase (NA) gene. Although previous studies had demonstrated that this mutation impaired the replication capacity of the influenza virus in vitro and in vivo, the A/Brisbane/59/2007 H275Y oseltamivir-resistant mutant completely out-competed the wild-type (WT) strain and was, in the 2008–2009 influenza season, the primary A/H1N1 circulating strain. Using a combination of plaque and viral yield assays, and a simple mathematical model, approximate values were extracted for two basic viral kinetics parameters of the in vitro infection. In the ST6GalI-MDCK cell line, the latent infection period (i.e., the time for a newly infected cell to start releasing virions) was found to be 1–3 h for the WT strain and more than 7 h for the H275Y mutant. The infecting time (i.e., the time for a single infectious cell to cause the infection of another one) was between 30 and 80 min for the WT, and less than 5 min for the H275Y mutant. Single-cycle viral yield experiments have provided qualitative confirmation of these findings. These results, though preliminary, suggest that the increased fitness success of the A/Brisbane/59/2007 H275Y mutant may be due to increased infectivity compensating for an impaired or delayed viral release, and are consistent with recent evidence for the mechanistic origins of fitness reduction and recovery in NA expression. The method applied here can reconcile seemingly contradictory results from the plaque and yield assays as two complementary views of replication kinetics, with both required to fully capture a strain's fitness. Influenza is the most important respiratory disease in terms of mortality and morbidity. Each year, between 3 and 5 million severe cases and 250,000 to 500,000 deaths due to seasonal influenza are reported worldwide [1, 2] . Cyclic pandemics due to antigenic shifts constitute an important threat [3] as was demonstrated by the swine-origin pandemic of 2009 [4] . Since vaccines for novel influenza virus strains require approximately 6 months to develop and produce [5] , antivirals remain the first line of defense. There are only two classes of antivirals approved for treatment of influenza [6] . The adamantanes, such as amantadine and rimantadine, are ineffective against B-type viruses [7] and have recently become ineffective against most A/H3N2 and some A/H1N1 viruses due to a mutation in the M2 gene [8] . The neuraminidase inhibitors (NAI), which include zanamivir and oseltamivir, were approved a decade ago and have shown excellent activity against all influenza A subtypes and B viruses [9] . A recent rapid increase in resistance to oseltamivir, however, has become a cause for concern.
The H275Y mutation in the neuraminidase (NA) gene (H274Y in N2 numbering), first described in 2000 [10] , is the most frequent mutation associated with oseltamivir-resistance in the N1 subtype, but it had long been thought to critically reduce viral fitness [11] . With a location on the framework residue of the enzyme catalytic site [12] , the mutation has been shown to cause a reduced affinity for the substrate in enzyme activity assays [12, 13] , an impaired viral fitness in vitro [14] [15] [16] , and up to a 100-fold reduction in transmission efficiency in ferrets [14, 17] . For these reasons, strains carrying the H275Y mutation were not thought to be a great concern for public health [10, 18] . During the 2007-2008 influenza season, however, the A/ Brisbane/59/2007-like (H1N1) H275Y mutant emerged and rapidly disseminated worldwide in the apparent absence of antiviral pressure [8, [19] [20] [21] . Recently, our group performed a study on the replicative capacities of the A/Brisbane/59/2007 H275Y mutant strain where we showed that its fitness, based on in vitro and animal studies, was similar to that of its wild-type (WT), oseltamivir-susceptible, counterpart [22] . These observations, and those of others [23] , correlate with the clinical situation encountered in the 2008-2009 season where almost 100% of the A/H1N1 viruses isolated in North America and Europe were resistant to oseltamivir due to the H275Y mutation [1, 19, 23] .
Recent work suggests that the origin of both the fitness reduction conferred by the H275Y mutation and the unique fitness of the A/Brisbane/59/2007 mutant strain is found in the virus NA activity and surface expression. Specifically, the reduction of NA activity conferred by the H275Y mutation has been associated with a reduced expression of surface neuraminidase, possibly due to defects in the folding of the molecule or its transport through the cellular membrane [24] . It has been shown, however, that two other mutations in the NA gene (V234M and R222Q) can provide a compensatory effect by increasing NA surface expression, and that these two substitutions indeed occurred in the evolution of the H1N1 seasonal strain between 1999 and 2007 [24] . The neuraminidase of contemporary (A/Brisbane/59/2007-like) strains susceptible to oseltamivir have shown a higher affinity for the substrate in NA activity assays than older H1N1 seasonal strains (e.g., A/New Caledonia/20/99 and A/Solomon Islands/3/06); contemporary oseltamivir-resistant strains (with the H275Y substitution) have shown a decrease in that affinity, but remain above the level of older strains [25] . Thus, it seems that these pre-existing mutations led to an over-expression of NA and provided a favorable environment for the appearance of the H275Y mutation. The eventual dominance of the H275Y mutant may be due to a better balance between the hemagglutinin (HA) and NA activity [25, 26] . It remains an open question, however, precisely how these mechanistic changes lead to viral fitness changes. The answer to this should be found in the details of the infection kinetics in the interaction of virus and cell.
In this paper, we present a method to extract the values of viral kinetics parameters, specific to a particular strain, from parallel experiments of plaque and viral yield assays. Our previous study [22] assessed the in vitro replicative capacity and fitness of pairs of WT influenza virus strains and their H275Y mutant counterparts by use of viral yield assays and by qualitatively comparing plaque sizes. Here, we show that it is possible to use these experimental measures to quantitatively characterize the kinetics parameters responsible for the replicative efficiency of influenza virus strains. As a proof of concept, we apply our method for extracting the viral kinetics parameters to the oseltamivir-susceptible/-resistant pair of A/Brisbane/59/2007 influenza virus strains in order to determine how the known genotypic differences in these two strains map to quantitative changes in the viral kinetics parameters characterizing their replicative efficiency. The kinetics parameters extracted through our method suggest that the H275Y mutant has weaker NA activity compared to its WT counterpart -confirmed by NA activity assays -which manifests itself as a longer phase of latent infection before viral release -confirmed by single-cycle viral yield experiments. However, the results also indicate that this longer latent infection period for cells infected by the H275Y mutant is compensated for by a shorter infecting time required for that cell, once releasing virions, to successfully infect other cells.
In order to obtain two complementary views of the infection kinetics for the A/Brisbane/59/2007 WT and H275Y mutant strains, virus growth over time was observed in two different in vitro systems: the viral plaque assay and the multiple-cycle viral yield assay.
Viral plaque assays. Figure 1 shows representative plaques for the WT and H275Y mutant strains of A/Brisbane/59/2007 (H1N1) viruses at each time point. The average plaque radius of each strain over time, calculated by averaging three independent experiments of three such wells at each time point, is shown in Figure 2 . The plaque growth is characterized by an initial delay where no growth is observed, followed by a period of linear increase of the plaque radius over time. After 60 h, the rate of plaque growth declines and the linear approximation is no longer valid. The growth attenuation could be due to a number of factors including a hardening of the overlay, a depletion of nutrients required for viral production and cell maintenance, and the widespread destruction of the cell monolayer leading to holes and irregularities disrupting and limiting further growth.
The plaque assay is a long-standing and standard technique in virology [27, 28] and plaque sizes have been used in many in vitro studies to qualitatively evaluate the phenotypes of various viruses [22, 29, 30] . Plaque assays are often used for strain comparison and, in that context, plaque diameters at a single time point are reported. These plaque diameters are then typically used to conclude that, for example, if the plaques observed at 48 h for strain A are larger than those for strain B, then strain A must have a higher replicative fitness than strain B. However, one should question whether such conclusions are valid and robust to experimental variability. Looking at Figure 2 , one can see that at 36 h, the plaque radius of both A/Brisbane/59/2007 WT and its H275Y mutant counterpart are comparable in size. Yet at 60 h, the WT strain has significantly larger plaques than the H275Y mutant, and the situation is reversed at 96 h. Thus, relying on single time point measurements for comparing strains can be misleading.
Here, instead, we exploit the fact that the average plaque growth is approximately linear in time between 36 and 60 h. This allows us to extract a novel measure, the plaque velocity, which is the slope in the linear regression of the plaque radius to the 36, 48 and 60 h time points. The plaque velocity, unlike the plaque radius at a given time point, is a robust measure in that it takes into account plaque radius at several time points and is not affected by differences in the length of the delay period which precedes the period of linear growth. Using this method, the measured plaque growth was more rapid for the WT than the H275Y mutant, with a plaque velocity of 1:79+0:1d cell =h compared to 1:48+ 0:1d cell =h, where d cell is the diameter of one cell. Thus, using plaque velocity alone, it would appear that the WT strain has a replicative fitness advantage over the H275Y mutant.
Multiple-cycle viral yield assays. In order to complement the information provided by the plaque assay, namely the plaque velocity, we also conducted multiple-cycle viral yield experiments. The results of these experiments for the WT and H275Y mutant strains are shown in Figure 3 . The kinetics of the viral yield experiments can be broken into two different phases: an exponential growth of virus concentration, characterized by the viral titer growth rate, followed by an exponential decay of virus concentration, characterized by the viral titer decay rate, after the viral titer peak. The viral titer decay rate was the same for both strains at 0:19+0:02h {1 .
The viral titer growth rate of the H275Y mutant was 0:83+ 0:02h {1 , slightly greater than that of the WT which was 0:75+0:04h {1 . Thus, it would appear from the viral titer growth rate alone, that the H275Y mutant has a replicative advantage over the WT strain, in contrast with the findings using the plaque velocity extracted from the plaque assay alone.
This discrepancy between the conclusions drawn from each experimental measure points to a complementarity between the two assays: they appear to emphasize different aspects of viral replication. Thus, combining the information provided by these two assays is key to obtaining a complete and consistent picture of what shapes a particular strain's replicative fitness.
The plaque growth experiment yields a single experimental measure for each strain: the plaque velocity. The multiple-cycle viral yield assay provides two quantities: the viral titer growth rate, and the viral titer decay rate. It is the goal of this paper to associate these broad experimental measures to the values of fundamental infection parameters, specific to each strain, which quantitatively characterize replicative efficiency. To this aim, we have constructed mathematical models which allow us to simulate each in vitro assay in a computer experiment. The basic mathematical model used here is similar to other within-host models of viral infection [31] [32] [33] [34] [35] [36] . A cell can be in one of four states -target (uninfected), latently infected (infected but not yet releasing virus), infectious (releasing virus) and dead (no longer releasing virus) -and its passage through these states ( Figure 4 ) is determined by five infection kinetics parameters. Target cells interacting with virus become latently infected at a constant infection rate per virus, b. The average time a cell remains latently infected is called the latent infection period, t L , and the average time a cell releases virus is called the infectious lifespan, t I . Virus is produced by infectious cells at a constant viral production rate, p, and this free virus loses infectivity exponentially at a constant rate of viral infectivity loss, c (as is observed in experiments [35] ). In applying this model, it is assumed that the growth of a particular influenza virus strain in a particular cell line is determined by a single, unique set of values for these five parameters. Thus, although the mathematical structure of the models used for each experimental assay is different (see Materials and Methods for a detailed description of each), the parameter values for a particular virus strain are assumed to be constant from assay to assay. With only three experimentally measured quantities, it would be impossible to uniquely identify all five parameters for a particular virus strain. Fortunately, it is possible to reduce the number of parameters considered and obtain unique identification of a few key parameter values.
One parameter can be determined immediately from the multiple-cycle viral yield results. The viral titer decay rate, characterizing the decline of the virus concentration after the peak (Figure 3 ), corresponds to the slowest of the rate of loss of virus-producing cells and the rate of viral infectivity loss [37] . Since prior in vitro experiments have shown that infectious cell death is nearly complete shortly after the viral titer peak [38, 39] , we set the rate of viral infectivity loss, c, equal to the viral titer decay rate. Because this decay rate was determined to be 0:19h {1 for both A/Brisbane/59/2007 strains, we have fixed the rate of viral infectivity loss to this value for all simulations. This corresponds to a virion half life of approximately 3.6 h, which is consistent with prior measurements for influenza virus in the experimental literature (see, e.g., [35, 40] ). Having fixed the rate of viral infectivity loss, we are left with four undetermined parameters and two experimental measures.
For the experiments considered here, the infection rate per virus, b, and the viral production rate, p, can be combined into a single parameter, leaving only three parameters to be determined. The rationale for this simplification is the fact that, during an infection, the two parameters play equivalent roles: doubling the rate at which virus is produced by cells will have the same effect on new infections as doubling the rate at which virus infects cells. Therefore, the only identifiable quantity is the product of the two rates, pb. Since their product has units of inverse time squared, we have chosen to express this quantity as a new characteristic time, the infecting time, t inf~ffi ffiffiffiffiffiffiffiffiffiffiffiffiffi 2= pb ð Þ p , which corresponds to the average time it takes a single virus-producing cell to cause the latent infection of one more (see Materials and Methods).
We are left then with two experimental measures -the viral titer growth rate and the plaque velocity -whose values may depend on three unknown infection kinetics parameters: the infecting time, t inf ; the latent infection period, t L ; and the infectious lifespan of a cell, t I . To determine how each of these parameters affect the infection dynamics, we varied each individually about a base value and measured the effect on the simulated experimental quantities ( Figures 5A and 5B ).
One parameter, the infectious lifespan of a cell, t I , had very little effect on either the plaque velocity or the viral titer growth rate. In the latter case, this parameter was explicitly neglected in the derivation of the growth rate, because earlier viral yield experiments have shown little cell death prior to the peak of the virus concentration (see, e.g., [38, 39] ). The fact that, over a wide range of infectious lifespan values, the resulting plaque velocity remained unchanged, is perhaps more surprising. Indeed, a shorter infectious lifespan will lead to the earlier appearance of plaques, resulting in larger plaque sizes at any given time. We have shown, however, in earlier work where influenza virus plaques were observed by immunostaining [41] , that the same plaque velocity can be measured from both the progress of dead cells, as we consider here, and the progress of newly infected cells. This indicates that plaque velocity is established at the advancing edge of an infection wave, and is likely unaffected by cell death in the wake of that wave. In those experiments, the infectious lifespan of a cell appears only as a time-delay between the infected cell plaque growth and the dead cell plaque growth. This has also been observed for the plaques of other viruses [42] . Since the infectious lifespan has little effect on the experiments we consider, and is therefore not identifiable here, we have fixed its value for both strains and for all simulations to value of t I~1 2h, obtained from the literature ( Table 1) .
This leaves only two parameters, the infecting time, t inf , and the latent infection period, t L , to be determined from our two experimental measures, the plaque velocity and viral titer growth rate. The full dependence of each experimental measure on the two remaining parameters are presented as contour plots in Figures 5C and 5D .
Because the plaque velocity and the viral titer growth rate depend on both the infecting time, t inf , and the latent infection period, t L , the experimental measurement of either quantity alone is not sufficient to specify the values of these infection parameters for a given strain. The measurement of both, however, can provide enough information for this specification, provided that the dependence on the parameters is sufficiently different for the two quantities. To demonstrate this concept using the A/ Brisbane/59/2007 (H1N1) WT and H275Y mutant strains, we have plotted the experimentally-measured values of plaque velocity and viral titer growth rate as functions of the infecting time and latent infection period, using the model dependence determined above ( Figure 6 ). Figures 6A and 6D show the values of the kinetics parameters most consistent with the measured plaque velocities of the A/ Brisbane/59/2007 WT and H275Y mutant strains, respectively. Rather than plot a single line at the average measured value, we have accounted for the error in the measurement of the plaque velocities by plotting regions of contour denoting the one-and two-standard deviations (for a detailed description see Materials and Methods). We can see that while the plaque velocity does constrain the two parameters to a specific region, that region is too large to allow any useful comparison of the two strains. Similarly, Figures 6B and 6E show the values of the kinetics parameters most consistent with the measured viral titer growth rate.
The consistency of a particular pair of parameter values with each of the two experimental measures can be combined by finding the intersection of the two parameter regions. This region of intersection corresponds to those parameter values most consistent with the parallel plaque and viral yield experimental measurements for a particular strain. The extent of these regions, shown in Figure 6C for the A/Brisbane/59/2007 WT strain and Figure 6F for the H275Y mutant strain, is summarized in Table 2 . The region of intersection suggests that the latent infection period for the H275Y mutant (w7 h) is longer than that of the WT strain (1-3 h), while the infecting time of the mutant (v 5 min) is much shorter than that of the WT (30-80 min).
In order to test the predictions made in the previous section by applying the mathematical model to parallel plaque and viral yield assays, we performed two additional experimental tests which could provide some qualitative and quantitative confirmation.
To independently estimate the latent infection period for the two A/Brisbane/59/2007 influenza virus strains, we performed a single-cycle viral yield experiment. Single-cycle experiments were performed at an MOI of 1 such that most cells would be infected simultaneously and pass through the phases of latency and viral release at the same time. Therefore, the observed virus production of the cell culture can be considered roughly proportional to that of an individual cell. The results of two independent experiments for each strain are shown in Figure 7A ; one experiment shows the viral titer over one full day post-infection and the other over only 14 h but with more frequent sampling. For each replicate, the viral titer of each strain was observed to grow rapidly after 4 h postinfection, with the WT viral titer reaching a plateau at approximately 8 h post-infection and that of the H275Y mutant reaching a plateau between 10 h and 14 h post-infection. Although the viral titer data in each replicate followed a relatively smooth curve, the inter-replicate variation was quite large, with peak virus titer varying from 2|10 3 PFU mL to almost 10 5 PFU mL. It is also notable that all of these peak values were well below the values seen in the multiple-cycle viral yield assay (Figure 3) , by a factor of * > 1000. Both of these features could be explained by the action of a relatively large defective interfering particle population [43, 44] within the viral stock, which is not uncommon for the influenza virus [45] .
The delay in the peak of viral titer between the two strains is qualitatively consistent with the model predictions of the previous section: the H275Y mutant strain appears to have a longer latent infection period than the WT strain. To make this comparison more quantitative, we scaled each experimental data set such that the peak virus was equal to one and then performed a least-squares fit to the full set of normalized data for each virus strain ( Figure 7B ). We utilized a model similar to that used for the analysis of the multiple-cycle viral yield assay, but allowed for a normal distribution of the latent infection period among cells rather than a fixed value for all cells, as assumed previously (see Materials and Methods). The fitted value of the average latent infection period, t L , was found to be 5.6 h for the WT strain and 7.5 h for the H275Y mutant, with fitted values of the standard deviation in the normal distribution, s L , of 0.5 h and 1.2 h, respectively. These results are summarized in Table 2 , along with 95% confidence intervals determined by fitting 1000 bootstrap replicates [46] .
The longer latent infection period predicted for the H275Y mutant strain, could be the result of poorer NA activity. This would also explain the shorter infecting time for the mutant strain in that its virions would more easily bind to new cells with less interference from its NA activity. To investigate whether the H275Y mutant had poorer NA activity compared to the WT, we directly measured the enzymatic activity of the NA of each virus strain using the 
Through modeling and simulation of two common in vitro experiments, the plaque and viral yield assays, we have extracted We have shown that seemingly contradictory results from the two experiments -plaques of the susceptible strain grow more quickly than the resistant strain, while the reverse is true of their titer growth in viral yield assays -can be considered complementary views of the infection kinetics which allow for the determination of parameter values controlling the replication of each strain. Specifically, we have found that the latent infection period of the H275Y mutant strain -equal to the time elapsed between the successful infection of a cell by a virion and the significant release of virus progeny by the newly infected cell -is much longer than that of the WT strain (by 4-10 h). The infectivity of the mutant strain, however, was found to be much higher than the WT, as quantified by the infecting time -equal to the time for a single infectious cell to cause the latent infection of one other, within a completely susceptible cell population. Independent single-cycle viral yield assay results lend support to the hypothesis of a longer latent infection period for the mutant strain than the WT, but suggest a more moderate (*2h) difference between the two. These results are consistent with the larger NA activity of the susceptible (WT) strain compared to the H275Y mutant, reported here and by others [25] , and its increased NA surface expression [24] . Since neuraminidase is the viral surface enzyme responsible for cleaving the virus from its sialic-acid receptors at the cell surface [47] , it can be expected that an increase of its expression would lead to more rapid viral release (a shorter latent infection period for the WT strain) but may also hinder the subsequent attachment of virions to other cells, leading to decreased infectivity (longer infecting times).
A complete understanding of the viral kinetics requires investigation of the HA/NA balance. It has been shown that the A/Brisbane/59/2007-like strains of the 2007-2009 influenza seasons differ from earlier H1N1 seasonal strains by a few amino acid substitutions in the HA gene [25] , but none of these involve interaction with the receptor and are therefore not likely to have influenced the changes in fitness. We have recently sequenced the entire genomes of our A/Brisbane/59/2007 strains, and found three amino acid substitutions in the HA gene for the H275Y mutant compared to the WT strain. Two substitutions, G189V and L264F, do not involve interaction with the receptor, but the third, A193T, lies within the receptor-binding site. This latter substitution has been noted in earlier work in relation to oseltamivir-resistant strains of the influenza virus [48] and an investigation of its influence on viral kinetics is a necessary direction for future work.
Mathematical models have been successfully applied previously to characterize the in vivo virus replication kinetics of HIV [31, 32] , hepatitis B and C [33, 49] , and influenza [34, 36, 50] , as well as in vitro viral yield experiments studying the effects of antiviral drugs [35] and the optimization of vaccine production [51, 52] . Models of viral plaques have also been considered [53] [54] [55] , although these were primarily directed at phage growth in an agar suspension of bacteria, a slightly different system than the cell monolayers considered here.
The method presented here for the determination of the infection parameters differs from previous mathematical modeling approaches to viral dynamics in that we have considered the explicit dependence of two experimental quantities on the parameters, rather than fitting a full dynamical model to the time-course of an experiment. There are a number of benefits to this approach. First, we have been careful to determine that the two experimental quantities under consideration, plaque velocity and viral titer growth rate, depend on only two unknown infection parameters, the infecting time, t inf , and the latent infection period, t L . This ensures that the problem of parameter extraction is theoretically solvable, which is often not the case when fitting a multi-parameter model to experimental data (see, e.g., [56] ). Second, the experimental quantities themselves are robust and easily measurable in repeated experiments. The viral plaque is formed by the progression of an infection wave across the monolayer of cells [42] whose constant velocity is determined by the infection kinetics averaged over many thousands of cells. Therefore the measured plaque velocity depends on the average interaction of virus and cell, and is insensitive to stochastic effects on a small scale. Similarly, the viral titer growth rate is due to the collective infection of thousands of cells and is independent of the details of initial infection (i.e., the precise value of the multiplicity of infection) or the total number of cells in the system. Other quantities of in vitro experiments, such as the time and value of the viral peak in a yield experiment, are much more sensitive to experimental details. Finally, the method we have applied here is robust to changes in the construction of the mathematical model itself. We have, for example, performed the same analysis of the plaque and yield assays using a stochastic model with more general assumptions about the cell transitions from latently infected to infectious and found nearly identical results (not shown here).
It is important to note that the results we present here are preliminary, a proof of concept for the method which requires further verification and refinement. In particular, it would be useful to develop an experimental assay which could measure the infecting time for a given strain, in the same way that single-cycle viral yield experiments give an approximate measure for the latent infection period. It is also of interest to design a set of experiments which may be less expensive and laborious than those presented here, perhaps using fluorescent or photographic observations of cell cultures rather than virus titrations, and which can identify a fuller set of viral kinetics parameters. We are currently designing competition experiments for the A/Brisbane/59/2007 WT and H275Y mutant strains in which the predictions which follow from the parameters extracted here can be tested directly. When verified, the basic method of analyzing parallel plaque and viral yield experiments introduced here should be useful in other contexts. For example, the investigation of other drug-resistant viruses (e.g., that of the pandemic A/H1N1), the rapid characterization of fitness for emerging strains, and assays measuring the activity of new antivirals would all be enhanced through the application of our method.
The A/Brisbane/59/2007-like (H1N1) strains used were the oseltamivir-susceptible A/Québec/15230/08 (WT) and the oseltamivir-resistant A/Québec/15349/08 (NA-H275Y mutant). These clinical isolates were obtained from two distinct, untreated, immunocompetent patients during the 2007-2008 influenza season [22] . 
All experiments were performed on ST6GalI-expressing MDCK cells [29] which over-express the a -(2,6) sialic acid receptor predominantly found in the human upper respiratory tract. Prior to infection, cells were grown to confluence, achieving an average diameter of *20mm (used herein as a unit of length, d cell ). Plaque assays were prepared using a semi-solid overlay of 1.2% Avicel RC-581 (FMC Biopolymers, Newark, Delaware, USA) as described by Matrosovich et. al. [57] and stained with crystal violet. Six-well plates (Corning Life Sciences, Lowell, MA, USA) were infected with 25+10PFU=well, representing a multiplicity of infection (MOI) of approximately 10 {5 , and stained every 12 h for 96 h. The plates were then photographed using a DSLR camera (Fujifilm S2 with a 60 mm Nikkor macro objective) and the areas of viral plaques were measured using the Threshold and Analyze Particle features of ImageJ, an NIH opensource image analysis software [58, 59] . All plaque radii at one timepoint (three independent experiments of three wells each) were averaged and the standard error of the mean was calculated. The radial growth rate was determined by linear regression to the average radii at time points prior to 72 h. Multiple-cycle viral yield assays for the A/Brisbane/59/2007 WT and H275Y mutant were performed with MOI&10 {5 . Supernatants were harvested every 6 h for the first 36 h of infection and every 12 h subsequently, then titrated by plaque assay as previously described [22] . The geometric average and standard deviation was determined from three replicates at each time point. High MOI single-cycle yield assays were performed as described by Hurt et. al. [60] . Monolayers of ST6GalI-MDCK cells were grown to confluence in 12-well plates and infected with 10 6 PFU well (MOI = 1) in 1 mL of infection medium. Virus was adsorbed for 1 h at 37 0 C in a CO 2 incubator. The supernatant was then removed and cells were quickly washed once with acidic saline (0.9% NaCl in water, pH 2.2) and twice with PBS (pH 7.4). Fresh maintenance medium was added and plates were returned to the incubator. Supernatants were harvested every two hours for 24 h ( Figure 7A , Experiment 1) or every hour for 14 h (Experiment 2), in duplicate. Samples were frozen at {80 0 C until titrated in duplicate by plaque assay [57] .
The enzyme kinetics of the neuraminidase was measured in duplicate for each strain as described in [61] , using the MUNANA reagent (4-methyl-umbelliferyl-N-acetyl neuraminic acid (Sigma-Aldrich, St-Louis, CO, #M8639)). Briefly, 10mL of live viruses diluted to 1:2|10 6 PFU mL were incubated at 37 0 C in Opaque Black Microfluor B CS50 96-well plates (VWR, Montreal, QC, #62402-983) with 30mL of MUNANA reagent ranging from 0 to 3000mM final concentration and 10mL of enzyme buffer [1:1 mix of 325mM MES (2-[N-Morpholino]ethanesulfonic acid) pH 6.5 (Sigma-Aldrich, St-Louis, CO, #M8250) and 10mM CaCl 2 (Sigma-Aldrich, St-Louis, CO)]. Fluorescence was measured in a Viktor 3 Multilabel Counter (PerkinElmer, Waltham, MA) every 90 seconds for 45 minutes. The excitation wavelength was 365nm and the emission wavelength 450nm with a 2:5nm excitation slit and a 20nm emission slit. K m and V max were calculated using a homemade Excel macro, created following [62] , and confirmed using the built-in ''Enzyme Kinetics'' features of the GraphPad Prism 5.01 software (GraphPad Software, La Jolla, CA).
Plaque growth was simulated using a one-dimensional, timedelayed, partial differential equation (PDE) model:
where T(r,t) and I(r,t) are the densities of target and infectious cells, respectively, and V (r,t) is the virus concentration. s (20-fold smaller than the Stokes-Einstein value for a 100 nm particle in 37 0 C water); the rate of viral infectivity loss was fixed at c~0:19h {1 based on the observed viral titer decay rate for both A/Brisbane/59/2007 strains in the multiple-cycle viral yield assays (see Results); and the infectious lifespan was held fixed at 12 h (Table 1) . Simulations were initialized with a ''top-hat'' central region of infectious cell density with radius d cell =2, and with all other cells in the target state. All fields rapidly take the form of traveling waves T(z), I(z) and V (z), where z~r{vt, with the same velocity, v.
The multiple-cycle viral yield assay was modeled using a meanfield, delay-differential system of equations:
where T and I are now the number of target and infectious cells, N is the total number of cells, V is the homogeneous virus concentration and all parameters have the same meaning as in the PDE model above. These equations can be derived from Equation (1) by assuming spatial homogeneity and integrating over space. An expression for the exponential growth rate, l g , of viral titer in the multiple-cycle yield assay can be derived from this system by assuming that in the early phases of an infection (well before the viral titer peak): the number of target cells is approximately constant T(t)~T 0 &N; there is an exponential growth of infectious cells I(t)~I 0 e lgt and virus V (t)~e lgt with common rate l g ; and infectious cell death can be neglected (t I ??). Substituting these expressions into (2) yields a transcendental equation for the viral titer growth rate:
For any values of p, b, t L and c, this equation can be solved numerically for the viral titer growth rate, l g . The assumptions made in deriving the expression require that the viral titer growth rate be measured early in the course of infection, well before the time of peak, when the number of infected cells is small compared to the total number of cells.
Both the plaque velocity and viral titer growth rate depend on the infection and production rates only through pb. Since this quantity has units of inverse time squared (units of virus cancel), it is useful to rewrite the dependence on these rates as a characteristic time, ffiffiffiffiffi ffi 2 pb s . A physical meaning can be ascribed to this quantity by considering Equation (2) in the case of a single infectious cell (I 0~1 ), within a completely susceptible cell population (T 0~N ). If loss of viral infectivity, c, is neglected, the equations can be then integrated to show that t inf~ffi ffiffiffiffi ffi 2 pb s is the time for that single infectious cell to cause the (latent) infection of one more cell. Therefore, we call this characteristic time the infecting time.
The contour plots in Figure 6 were created using the functional dependence of the plaque velocity and viral titer growth rate on the infecting time, t inf , and the latent infection period, t L , as determined by model simulation, along with the experimentally measured values of these quantities and their associated measurement error, under the assumption that these errors are normallydistributed. For example, the function F v , plotted in Figure 6A and Figure 6D , takes values between zero and one, according to
where v exp is the experimentally-measured plaque velocity with measurement error s v and v mod (t inf ,t L ) is the theoretical dependence of the plaque velocity determined by model simulation. Contours for the one and two-s values are drawn at F v~0 :6065 and 0:1353. A function on the parameter space, F lg , for the viral titer growth rate is constructed analogously. The product of these two functions is plotted in Figures 6C and 6F to show the likely regions of viral kinetics parameters controlling growth for each virus strain.
In fitting the single-cycle viral yield data, a more biologicallyrealistic model was used which assumes that the set of latent infection periods for a collection of cells is normally-distributed about t L , rather than fixed [63] . In this model, target cell and virus dynamics are identical to that of Equation (2) 
where L 0 and I 0 are the number of cells latently infected and infectious, respectively, at the start of the experiment, f L (t) is the probability density function for the latent infection period and P I (t) is the probability that a cell remains infectious for at least a time t after the latent-infectious transition. If a Dirac delta function is used for f L (t) and a Heaviside step function for 1{P I (t), then the infectious cell dynamics of Equation (2) are recovered. In the fits to the single-cycle data (Figure 7 ), f L (t) was taken to be normal (truncated at t~0 and renormalized) with parameters t L and s L ; the function P I (t) was also derived from a normal distribution f I (t)
with P I (t)~1{ d dt f I (t)
, with fixed parameters t I~1 2h and s I~1 h. Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation. Bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to B. subtilis. For more than six decades, this organism has played an integral role in the biodefense community as a simulant for biological warfare and bioterrorism events (BW) and is commonly referred to by its military two-letter designation ''BG'' [1, 2] . B. atrophaeus has served in studies of agent dispersal [3] , decontamination simulations [4, 5] and large-scale process development [6] . In addition to its historical use as a BW simulant, it is currently in widespread commercial use as a surrogate for spore-forming bacteria [5, 7] and is the basis of numerous assays for spore inactivation [8, 9] . In addition to its role as a simulant, the organism plays an important role in the biotechnology industry as a source of restriction endonucleases and of the glycosylation inhibitor nojirimycin [10] .
The taxonomic placement of B. atrophaeus has changed dramatically over the years. Originally isolated as B. globigii in 1900 (Migula) as a variant of B. subtilis, it was originally distinguished from B. subtilis by the formation of a black-tinted pigment on nutrient agar and by low rates of heterologous gene transfer from B. subtilis [11] . It has been alternately known as B. subtilis var. niger, B. niger, and has been confused with B. licheniformis [12] . Other than the formation of the dark pigment, it is virtually indistinguishable from B. subtilis by conventional phenotypic analysis [13] , and the lack of distinguishing metabolic or phenotypic features has contributed to the confusionin the taxonomic placement of this organism. Low interspecies DNA transfer frequencies suggested substantial divergence [11] . Based onanalysis of comparative DNA hybridization, phenotypicand biochemical tests, Nakamura advocated that pigment-producing B. subtilis-like isolates should be classified as a distinct species termed B. atrophaeus [13] . Recently, more sensitive typing methods such as amplified fragment length polymorphism analysis showed that B. atrophaeus strains could be classified into two major biovars: var. globigii encompassing the classical, commonly used BG isolates, and var. atrophaeus encompassing other closely related yet genetically distinct strains [14] .
Here we report the definitive molecular typing of several BGstrains using whole-genome sequences, and develop a plausible microevolutionary history of a commonly used lineage based on the accumulation of mutations over time and during transfer between laboratories.The selected strains span more than six decades of development, use, and transfer of BGbetween various institutions and laboratories and offer an unparalleled opportunity to investigate mutation under selection and drift over time. Phenotypic analysis revealed substantial heterogeneity both between and within strains, even in type strains, while highthroughput metabolic profiling revealed metabolic ''enhancements'' to a population that had returned to the University of Wisconsin (UW) from Camp Detrick in 1952. Whole-genome comparisons of single-nucleotide polymorphisms (SNPs), small insertion/deletion motifs (indels), and large-scale genomic architecture analysis by optical maps are combined to generate a plausible history of acquisition and use of operationally relevant strains by the American Type Culture Collection (ATCC) and by several laboratories within the biodefense community.
Finally, our analysisof mutation profiles revealed potential signatures of the deliberate selection of strains with properties of enhanced growth and spore yields, properties that were deemed desirable in a simulant [6] . We also report genetic differences between strains in use in the biodefense community and the commercial sector that argue for adoption of a more uniform standard for B. atrophaeus as a simulant.
Strains and growth conditions B. atrophaeus strains and their sources are indicated in Table 1 . Archival strains were maintained as spores in sterile soil at the University of Wisconsin (Figure 1 ). The 1013 lineage, originally founded from the 1942 strain, was extensively passaged by serial transfer every 12-18 months on agar slants for 30 years. Unless otherwise indicated, strains were grown using LB agar plates, LB agar brothor Tryptic Soy agar containing 5% sheep's blood (SBA, HealthLink) at 37uC.
Spores were germinated by plating on LB media at 37uC. Plates were examined by stereomicroscopy using indirect lighting and imaged usinga Nikon SMZ1500 with a total magnification of 166. Colonies exhibiting distinct morphologies were repeatedly streaked to confirm stability of the phenotype.
Genomic DNA was prepared from all isolates using the Blood and Cell Culture DNA Midi Kit for Bacteria (QIAGEN) from 10 ml overnight cultures in LB. BACI051-N was sequenced at the Naval Medical Research Center, while all other isolates were sequenced to .25-fold coverage at the US Army Edgewood Chemical Biological Center by massively parallel pyrosequencing on the Roche/454 GS-FLX using the Titanium reagent package. Draft genome sequences of all isolates were assembled de novo using Newbler [15] (Roche) and analyzed using both Newbler and Lasergene (DNAStar, Madison, WI). The 1942 Vogel isolate was designated as the reference strain and was brought to completion using standard finishing techniques.
The draft genome of Bacillus atrophaeus var.globigii was finished at the Department of EnergyJoint Genome Institute (JGI) using a combination of Illumina [16] and 454 datasets [15] . For this genome, we constructed and sequenced an Illumina GAii shotgun library which generated 15120217 reads totaling 544 Mb, which was combined with 454 Titanium standard library which generated 387327 reads totaling 137 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. The initial draft assembly contained 25contigs in 25scaffolds. The 454 Titanium standard data were assembled with Newbler, version 2.3. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data wereassembled with VEL-VET, version 0.7.63 [17] , and the consensus sequences were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and using parallel phrap, version SPS -4.24 (High Performance Software, LLC). The software Consed [18, 19, 20] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [21] , or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 79additional reactions and 10shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. The total size of the genome is 4 168 266 bp and the final assembly is based on 137 Mb of 454 draft data which provides an average 33.46 coverage of the genome and 544 Mb of Illumina draft data which provides an average 1336 coverage of the genome. The complete sequence and WGS were deposited at DDBJ/ EMBL/GenBank under accession numbers listed in Table 2 . The WGS versions described in this paper are the first versions, e.g. AEFM01000000.
Templated assembly of the remaining strains were mapped to the 1942 finished sequence using the GSMapper tool in Newbler (Roche). High-confidence mutations were selected from Newbler ''HCDiffs'' calls (Table S1 ) by applying additional selection criteria that mandated high quality scores in both reference and templated assemblies with .80% of the sequencing reads differing from the reference, elimination of mutation calls associated with homopolymer tracts (with the exception of tracts that were formed by a deletion -see below), and a minimum coverage depth of 56 with bidirectional sequence reads. Finally, the raw 454 reads from the 1942 isolate were mapped to the finished sequence to assess error bias in the 454 process and to correct for residual sequencing errors in the finished sequence. Accession numbers of the relevant wholegenome shotgun sequences are found in Table 3 . Phylogeny was calculated using PAUP 4.0b10. Fifty-eightnucleotide positions were used with gaps being treated as a ''5th base'' and all characters assuming equal weight. One thousandbootstrap replicates were computed using a heuristic search with the optimal criterion set to ''parsimony''. The tree was created using stepwise addition.
Nineteen loci in which putative mutations were identified from the 454 dataset were re-sequenced from PCR products by standard Sanger dye-terminator methods. No false-negatives or false-positives were identified among the re-sequenced loci; however resequencing of the apparent mutation at position 1486408revealed mixed genotypesin several isolates that are artifacts of a large duplication in the 1942 chromosome. Therefore, this signalcannot be considered a true SNP.
Annotation, comparative genomic analysis, and multiple alignments Preliminary annotations were generated using a combination of the RAST [22] algorithm (rast.nmpdr.org). Loci containing mutations were used to query the non-redundant (nr) databases and Refseq protein databases at NCBI using directed BLASTx and BLASTp. The comparative BLAST tool from RAST was utilized for genome-wide protein sequence comparisons to B. subtilis. Results were filtered for bidirectional hits. Multiple alignments were generated by MegAlign from the LaserGene software package using the CUSTALW algorithm.
Genomic DNA was prepared from live bacteria on agar slants to maximize the yield of extremely high-molecular weight DNA. Optical maps were generated by digestion with NcoI of DNA arrayed linearly on glass slides and the resulting maps were aligned and compared with the MapSolver software package (OpGen, Inc., Gaithersburg MD).
Using an information-based method for genomic classification [23] , the sequence contigs from BG isolates 1942, 1013-2 and 49822 were analyzed in order to map the phylogenetic relationships of these isolates to other Bacillus species. In this method, genomic content is characterized by the frequencies of occurrence of short n-mers contained within each sequence (n typically from 3 to 16). These n-mers are then rank ordered by genome. The pair-wise comparison of the rank of n-mers within two different genomes is then used to compute an informationbased genetic distance (IBGD), where the sum of the differences in rank for all possible n-mers is weighted by an entropy factor that depends on the frequencies of occurrence of the respective n-mers in the two genomes. The pair-wise IBGD values are then used to construct a phylogenetic network [24] . Bacilli genomes were obtained from Genbank. This method for phylogenetic characterization enables computation even with the unassembled reads, and it can be applied to draft or partial genome sequence data, which was the case for the three B. atrophaeus genomes studied here.
The first seven BGstrains listed in Table 1 were streaked for single colonies on BHI plates and incubated at 33uC overnight, followed by subculturing a second time under the same conditions. Subsequently, cell suspensions were prepared according to Biolog specifications, with OD readings ranging between 0.35-0.45 at 600 nm. Biolog phenotypic microarray plates PM1 through PM20, were inoculated according to the manufacturer's specifications, and incubated at 37uC for 72 hours. Readings were taken every 15 minutes, and data processed by OmniLog Phenotype Microarray File Management/Kinetic Plot and Parametric modules. Two biological replicates of the experiment were conducted for each strain. PM1-10 contain single wells for each growth condition whereas PM11-20 contain quadruplicate wells for each condition.
The area under the curve (AUC) values were computed by adding all OmniLog values at all time points for each of the 1200 distinct phenotypes produced from the OmniLog software. The AUC values from the two different biological replicates for each unique phenotype were averaged. The ratio for each AUC was calculated between the 6 query strains (Detrick-1, Detrick-2, Detrick-3, 1013-1, 1013-2, and Dugway) and reference parent strain (1942) . For the purpose of visualization, 1920 phenotypes were included in the heatmap (i.e. this better represents the locations of the phenotypes which correspond to different modes of action categories). The same ratios were used for the phenotypes that have replicates. The ratio values were formatted as PM1 to PM20 for each strain across the columns and wells A i to H i , where i = 1 to 12 for the rows. The results were plotted in a heatmap using R [25] . Positive growth wells are represented by greenblocks while negative growth wells are represented by red blocks.
Catalase activitywas assayed by spotting drops of hydrogen peroxide (3%) onto isolated colonies on LB agar plates. Colonies were monitored for bubble formation, signifying the release of water and oxygen. A colony was considered to be catalase positive by observation of bubbles.
Streaks of Detrick 1, Detrick 2, and 1013 strains were grown for two days on TSA plates containing SBA.Bacterial cell mass was scraped using an inoculating loop (1 ml) from the streak and resuspended in PBS. Sporulation was evaluated by bright field phase-contrast microscopy. Phase-bright free sporesand phasedark vegetative cellswere counted. Five representative viewing fields were counted from each strain for each experiment. This experiment was completed in triplicate by repeating once per day over the course of three consecutive days.
In order to compare the percent sporulation between Detrick 1 and Detrick 2, and Detrick 1 and 1013, a mixed analysis of variance (ANOVA) was used to complete the analysis. Strain and viewing field were evaluated as fixed factors, and replicate was included as a random factor. The natural log of the percent sporulation was taken to obtain a normal distribution of the residual error. Tukey's method was applied to compare the difference between the mean log percent sporulation.
We traceda potential provenance of the commonly used BGstrains through an exhaustive search of the open literature and the archives of the University of Wisconsin,which suggested a possible lineage from which the ''military'' BGstrains were derived. The original source of the strains were the collections at the University of Wisconsin during the 1930s and 1940s, from which the strains were transferred to Camp Detrick at the initiation of the US Army's BW program at the beginning of the Second World War [6, 26] . At Camp Detrick, BG was used as a non-pathogenic surrogate in process development for sporeforming bacteria It is tempting to speculate that the University of Wisconsin supplied BG to Porton Down: A note found in the archive of Dr. Baldwin's papers, dated February 19, 1943 , contained an order from Dr. Fildes (presumably Sir Paul Fildes, a noted bacteriologist active in the British BW program at the time), for a batch of B. subtilis spores. It is not clear whether BG or B. subtilis subsp. subtilis was supplied, or whether this material was actually delivered. Unfortunately, original records describing in detail the maintenance of the strains during the period 1942-1955 were destroyed as per US Army policy at the time (Dr. Mark Wolcott, USAMRIID; personal communication), and the personnel who had first-hand knowledge of the strain passage histories and methods are deceased. Therefore, the actual source of the Camp Detrick isolates must be inferred from published work [6] , limited available documentation (e.g. ATCC 9372) and the genome sequences presented hereFrom Camp Detrick the isolates were eventually transferred to ATCC as B. subtilis var. niger ''red strain.'' The desire to maintain a phenotypically and genotypically uniform simulant throughout the biodefense communityprompted us to elucidate whether significant phenotypic and/or genomic differences had accumulated in any of the commonly used isolates during the growth and transfer of strains to different institutions and to compare the isolates in broad use today to the so-called ''Mil-Spec'' strain (ATCC 9372).In contrast, the origin of ATCC 49822 prior to acquisition F. Young's laboratory (the depositor) is unclear.
We obtained isolates from archival spore suspensions in sterile soilfrom the University of Wisconsin with legible labels dating back as far as 1942 ( Figure 1 ; Table 1 ). These isolates included an archival stock dated 1942 that likely predated the transfer to Camp Detrick, as well as material that had been returned to the University of Wisconsin from Camp Detrick in 1952. A derivative of the 1942 strain that had been repeatedly passaged in vitro on agar slants over a period ofapproximately 30 years allowed us to compare the genomic signatures of deliberate selection with the effects of long-term in vitro passage. In addition, a sample of strain NRS-356 [13] , which is mentioned as a possible parent strain in correspondence between various academic laboratories and Camp Detrick, was also obtained from the same source as the 1942 ''Vogel'' strain. These isolates were subsampled, germinated on LB plates, screened for colony morphology variation (see below). Genomic DNA was prepared from these isolates for sequencing.
Upon initial plating of the archival and modern-dayBG stocks, we noted distinct colony morphotypes for many of the strains, with some strains containing multiple variants ( Figure 2 , Table 2 ). Some of these morphotypes were consistent with those observed by Hayward et al. [6] whooriginally described the emergence of colony variants in ''B. globigii.'' As in the earlier report, individual morphotypes were stable and did not interconvert with high frequency (data not shown), suggesting that these morphotypes were the result of relatively rare chromosomal mutations, although 1013-1 occasionally threw off papillae in heavier streaks (not shown). Multiple morphotypes were noted for ATCC9372, ATCC 49822, Detrick, and 1013, while the archival 1942 isolate, the isolate obtained from Dugway Proving Ground (Dugway) and BACI051 appeared to be pure populations on LB. All strains tested positive for BGusing Real Time-PCR primers specific to the recF gene (Methods S1) [27] . The appearance of multiple colony morphotypes even within single ''strains'' strongly suggested an asyet undescribed level of genetic diversity within these samples that likely affected the expression of cell-surface components and/or sporulation. The intra-strain colony morphology variation was particularly dramatic in the in vitro passaged 1013 and ATCC9372 isolates, in which one variant of each lineage had lost the production of color on LB orSBAplates (Figure 2 ), suggesting more dramatic alterations to the genome.
Draft genome sequences were generated from several BGstrains in our collection. A summary of the results from the sequenced isolates is indicated in Table 3 . All of the ''military'' isolates (Detrick clones1through 3, BACI051, Dugway) were extremely closely related to each other and to both ATCC9372 variants. The ATCC isolates possessed additional mutations that were absent in the ''military'' isolates. The size of the finished and closed genome of B. atrophaeus var. globigii 1942 was 4,168,266 bp, and annotation using RAST [22] revealed 4433 features, including 4343 proteincoding sequencesand 90 RNA molecules [28] . The preliminary annotations derived from RAST are available as Genbank .gbk files in the supplementary material.
On average, the genome of B. atrophaeus is approximately 86% identical to B. subtilis on the nucleotide level,supporting its delineation as a distinct species and agreeing well with previous estimates [29] . Analysis of the IBGD using whole-genome sequences (N-mer length .4) supported the identification of B. subtilis 168 as the closest relative among sequenced bacterial genomes ( Figure 3 ). For this particular case, n = 5 (i.e., there were 4 5 = 1024 total 5-mers used to compute the IBGD). The IBGD values were relatively insensitive to the choice of n over the range of 4-8. Thethree BGgenomes analyzed grouped closely together, and our analysis of the Bacillus-wide phylogeny using IBGD revealed the phylogenetic distance of that B. subtilis/B. atrophaeus species from B. anthracis, supporting the inferences published elsewhere from rRNA sequence analysis ( Figure 3 ) [30] . Primary amino acid sequences of RAST-annotated proteins are on average 72% (median 83%) identical between B. atrophaeus and B. subtilis. When only the proteins that yielded bidirectional BLAST hits in RAST are examined, the predicted proteome of B. atrophaeus is, on average, 83% identical (86% median) to B. subtilis.
We utilized the finished sequence of the 1942 isolate as a reference strain for templated assembly of the remaining BG draft sequences. Two additional ATCC isolates of B. atrophaeus (49337 and 6537) were distinguishable from var. globigii on the basis of very high SNP/indel counts, lower coverage ofand percentage of reads mapping to the 1942 reference, and unique genomic features which supported their proposed classification as var.atrophaeus [14] . The distinguishing genomic features of var.atrophaeus strains and the delineation of the B. atrophaeus clade from B. subtilis will be published elsewhere.
Optical restriction mapping [31, 32, 33] was used to compare the overall genomic structure of selected isolates. No differencesin overall genome architecture between the ''military'' BG isolates, the archival 1942 isolate, or 1013-1 were observed (Figure 4 , data not shown), suggesting that the global architecture of these isolates is relatively stable, even over 30 years of serial in vitro passage.However, the optical maps and sequence coverage analysis of 1013-2 and 9372-1revealed substantial deletions of approximately 72,727and 23,678 bases, respectively, of genomic materialspanning from positions 3,992,613 to 4,065,341 (1013-2) or 4,022,138-4,045,817 (ATCC 9372-1) (Figure 4 ; Table S2 ). The genes within this deleted region are listed in Table S3 but notably contain genes encoding for nitrite reduction, germination (gerKABC), and biosynthesis of the lipopeptide surfactin (srfCAB) [34, 35] . A defect in surfactin production is a particularly intriguing candidate for the morphology and pigmentation variations in 1013-2 and ATCC 9372-1, since disruption of srfA has been shown to have dramatic effects on spreading motility on semisolid media, on biofilm formation [34, 36] , and low-grade hemolytic activity.
Using the de novo assembled draft sequence from the 1942 isolate as a template for subsequent analysis of SNPs and small indels in the other ''military'' isolates, we generated a list of highconfidence, discriminatorymutations that differentiate the strains ( Figure 5A ). The nature and annotation of the mutations are found in Table 4 and can be assigned an approximate temporal order in which they occurred ( Figure 5B ). Based on this analysis, 1942 is the most likely parental strain for all of the isolates in this study, with the 1013 lineage diverging earliest, followed by 49822, then the ''military'' lineage prior to the transfer to Camp Detrick. This conclusion is based on the observation that 49822 shares three SNPs with Detrick-1. The latter is the most likely progenitor of the other ''military'' isolates, since it has the fewest mutations relative to strain 1942. Detrick-1 can be differentiated from other ''military'' isolates by possessing the parental allele of spo0F rather than the H101R allele (position 3231470) that is characteristic of all of the other ''military'' BGisolatesand the ATCC9372 strains. The two colony morphology variants of ATCC9372 each exhibited distinct mutation profiles indicating that the reference strain is in fact a mixed population of at least two genetically distinct substrains.
The 72 kb deletion in 1013-2 included the structural genes for biosynthesis of surfactin, a cyclic lipopeptide with a mild hemolytic activity [34] . To test whether the ''military'' and in vitro passaged strains possessed low-grade hemolytic activity, we streaked these variants on rich agar media containing 5% sheep's blood and looked for hemolysis. To our surprise, all strains exhibited striking variation in their coloration (Figure 2A) , with the 1942, 9372-1 and Detrick-1 isolates considerably darker on blood agar than the other ''military'' and in vitro passaged isolates. In addition, on LB the 1013-2 and 9372-1 isolates appeared white and off-white, respectively. Pigmentation of B. subtilis colonies is associated with production of a melanin-like pigment by the CotA protein, a major component of the spore coat [37] . In addition to the variations in pigmentation, streaks of the 1942 and Detrick-1 isolates were consistently translucent under transillumination ( Figure 2B ). These zones of translucency are suggestive of weak b-hemolysis, which has previously been observed in B. subtilis strains that produce high levels of surfactin [34, 35, 38] . The other strains exhibited either weak a-hemolysis or none at all, with the exception of the strongly hemolytic 49822-1 variant. At least in the ''military'' lineage, the quasi-hemolytic phenotype and darkbrown colony pigmentation correlated with the presence of a wildtype spo0F allele, suggesting that the ability of B. atrophaeus to lyse red blood cells may be regulated in part by spo0F. However this was not universally the case; the BACI051 strain had two discernible variants on SBA (not shown), one of which appeared to have recovered partial hemolytic activity ( Figure 2B ).
To gain insight into the effects of genetic divergence of the adapted isolates on their metabolic capacity, the Detrick isolates and the separate 1013 isolates were compared by multiphenotype analysis using the Omnilog system, which allows the highthroughput comparison of 96620 discrete growth conditions, including carbon, nitrogen, phosphorus, sulfate, nutrient supplements, pH, osmolytes as well as a broad class of growth inhibitors. The growth of the 1942 strain was used as a reference for determining relative growth rates of the other strains. The results of Mutations exhibiting high quality scores in both reference and query sequences and with differences from the template exhibited in .85% of the individual sequencing reads are indicated as a blackened box. In one case (position 259001 in ATCC 9372-1) an initial false-negative due to the formation of a homopolymeric tract was found by direct inspection of the assemblies. The genes whose functions are altered by the given mutation are indicated in Table 4 these experiments are summarized in Figure 6 and Table S4 . In general, growth of the 1013 isolates was significantly diminished relative to the 1942 in many different growth conditions, most notably in the ability to use amino acids and peptides as carbon and nitrogen sources, to withstand osmotic stress, and to grow under reduced pH. In addition, the strains had developed sensitivity to beta-lactams, quinolones, and membrane-disrupting activities. These results suggested broad combined effects of several mutations on the phenotype of the strains. In addition to the spo0F(A98P) allele, which is a likely candidate for highly pleiotropic effects on the decision to sporulate under many different conditions, both strains contain substitutions in the yetF and yqgE genes that may be contributing to the phenotypes observed. The more pronounced defect in 1013-2 may be attributable to defects in the gerAB and gerAC genes and the large 72 kb deletion which contains several genes involved in germination. By contrast, the Detrick isolates in general grew more robustly than the 1942 strain under multiple growth conditions. Increased relative growth rates were particularly pronounced for acidic conditions and media containing osmolytes, but particularly for wells containing sodium lactate [6] .
Another isolate in the ''military'' lineage, Dugway, is clearly derived from the Detrick lineage by SNP/indel profiling yet has a metabolic profile that is much closer to the parental strain. Like the Detrick isolates, the Dugway strain grows better at low pH, but many of the other conditions do not promote elevated growth relative to 1942. Only one mutation differentiates that isolate from the Detrick-2 isolate -a 2-bp insertion in the yojO gene encoding a putative activator of nitric oxide (NO) synthesis. Again, the physiological role of this mutation is unclear, although nitric oxide synthesis plays a critical role in modulating antibiotic resistance in Bacillus spp. [39] . In addition to its role in promoting resistance to antibacterial drugs, NO is known to modulate B. subtilis genes involved in nitrate respiration when oxygen is limited [40] ; thus the lowered growth in this strain may reflect the inability to grow to higher densities and overcome the resulting lower oxygen tension. An additional isolate, BACI051 is clearly derived from Dugway, yet two variants have accumulated additional mutations in sigH (spo0H), hpr/scoC, and ebrB. Notably, the phenotype of BACI051-E on plates more closely resembles the 1942 strain ( Figure 2B ).
sequencing of the ''military'' isolates revealed a frameshift mutation in the katA gene encoding the major vegetative catalase [41] . The absence of catalase activity in ''military'' isolates was confirmed by adding a solution of 3% H 2 O 2 to smears of various strains. In contrast to the 1942 strain, which exhibited immediate and robust catalase activity, the strains containing the frameshift lacked this activity. A small amount of bubbling could be seen, probably due to the presence of a second catalase normally packaged in spores [42] .
To test whether the phenotype observed on blood agar was associated with differences in sporulation, selected strains were grown for two days as patches on blood agar, resuspended in PBS Annotations are a combination of RAST and directed tBLASTn and BLASTp searches vs Bacillus databases. 4 Forms part of a large polypeptide synthase containing highly homologous regions. 5 Also shared with strain ATCC 49822. 6 The conserved start codon of the radA gene (BG3899) of B. atrophaeus and B. subtilis falls within the BG3898 ORF. Therefore BG3898 as called by RAST is not deemed likely to be a protein-coding gene. 7 In putative transmembrane region. doi:10.1371/journal.pone.0017836.t004 Table 4 . Cont.
and counted directly. Strain Detrick-2exhibited significantly higher percentages of phase-bright spores than the Detrick-1 strain (Figure 7 , Mean +/2 standard error of the mean). Similar results were observed for the 1942 and Dugway strains (not shown). The 1013-1 strain exhibited an even higher degree of sporulation than the Detrick-1 strain under identical conditions ( Figure 7 ).
Bacillus atrophaeus has historically been grouped with B. subtilis, and is usually described as a black-pigmented variant (var. niger) because of its many phenotypic similarities to the bettercharacterized B. subtilis. Both organisms are soil-dwelling, nonpathogenic saprophytes, but have been differentiated by the ability to produce pigment on nutrient media containing an organic nitrogen source [13] . The orange pigmentation of B. atrophaeus var.globigii spores made it an attractive simulant for B. anthracis, facilitating the detection of dispersed spores in complex environmental samples. Recently, more sensitive phylogenetic approaches using AFLP have delineated B. atrophaeus as a separate species [13, 14] . The taxonomic confusion has arisen due to inadequately sensitive typing methods, and has led to misattribution of pathogenic qualities associated with some B. licheniformis strains to the B. atrophaeus strains currently in use as simulants [12] , for which no direct evidence of pathogenicity exists. This report defines the genomic composition of B. atrophaeus var.globigii and clearly separates the species by wholegenome phylogenetic analysis.
In this study, we generated a high-quality, closed reference genome for the 1942 isolate using a combination of 454, Illumina, and directed Sanger sequencing. We expect the final genome to have an error rate of ,1 in 50,000 basepairs. When we mapped the 454 datasets for all of the isolates back to the finished sequence that was generated using the same DNA, we noted several putative SNPs that were common to all datasets (Table 4) . We believe these represent errors introduced during generation of the final consensus sequence, as they did not appear when the isolates were mapped against draft sequence generated exclusively using the 454 platform; these are currently being verified and the final sequence will be updated.
Our sequences of multiple, closely related strains of this organism allow us to trace the derivation of the ''military'' BG isolates currently in use to a culture present at Camp Detrick during the 1940s and 1950s. The origin of ATCC 49822 is not as clear, but a publication from that era suggests a possible common origin at the University of Wisconsin [43] . While that strain is unlikely to be NRS-356 itself, given the presence of several strain-specific SNPs in our sequence, the SNPs common to both 49822 and the ''military'' lineage suggest a common ancestor that is not represented among the strains sequenced for this study. Given the lack of original records, it is unclear whether the NRS-356 variant in this study might have passed through Camp Detrick and been returned to the University of Wisconsin. However, given the date on the label and the general secrecy of operations at Camp Detrick during the Second World War [26] we consider this possibility unlikely.
During development of BGas a simulant for B. anthracis, strains were selected that exhibited the most desirable characteristics, those being rapid growth, high spore yield, and experimental reproducibility. Without being aware of the nature of the genetic alterations in their ''optimized'' strains, BW workers at Camp Detrick selected a mutant that provided dramatically higher total and relative spore yields, and generated consistent experimental results [6] . These strains were adopted into the inventories of numerous biodefense laboratories and have been used for many Figure 7 . The spo0F(H101R) and spo0F(A98P) alleles are associated with hypersporulation. Phase-contrast microscopy of BG strains after two days of growth on SBA. Vegetative cells appear as phase-dark rods, while spores appear as round, phase-bright globules. The mean percentage sporulation of each strain in a representative experiment is given 6SEM. The experiment was repeated on three consecutive days; representative results of a single experiment are shown. Statistical significance was determined by mixed ANOVA (Tukey's method, p,0.05). doi:10.1371/journal.pone.0017836.g007 Figure 6 . Omnilog phenotypic arrays of B. atrophaeus subsp. globigii strains. Six strains were each inoculated into twenty 96-well Omnilog plates and grown at 37uC. Reduction of tetrazolium dye by respiring cells was measured every 15 minutes by optical density. Dye reduction relative to the 1942 strain is shown; the red ratio values indicate less respiration while the green ratio values indicate more respiration as compared to the 1942 strain. Individual arrays or strains are displayed in each of the six major columns labeled Detrick 1, Detrick 2, Detrick 3, 1013-1, 1013-2, and Dugway. A) Heat map of all conditions for each strain. Each of the twenty plates for each strain is represented by the notation PM01-PM20 (left-toright for each strain) along the x-axis. The rows represent the well position, and are denoted as A i to H i (i = 1 to 12) from the bottom to the top of the plot in each array along the y-axis. Each cell ratio value represents the average of two biological replicates for each strain. Plates PM01-PM10 contains single wells for each growth condition, while plates PM11-PM20 contain quadruplicate wells for each growth condition. Solid circle indicates wells containing sodium lactate; dotted circle indicates well containing L-serine at pH 4.5. The details of the 1920 growth conditions can be found in the first worksheet labeled ''All strain AUC data'' in Table S4 . B) Most significant phenotypes for each of the six test strains as compared to the 1942 strain. The phenotypes with statistically significant increases and/or the decreases in ratio values for each of the six strains are presented. For the 1013 isolates only the conditions giving the five largest changes are presented. The number in each color block indicates the ratio for the test strain relative to the parent strain for the phenotype specified. The details of all significant phenotypes for each test strain can be obtained in Table S4 . Bold Italic font indicates p,0.05. doi:10.1371/journal.pone.0017836.g006 decades in simulations of decontamination and dispersal [12] . By applying a combination of genomic and biochemical profiling techniques, our data demonstrate that the BG isolates were ''enhanced'' by researchers at Camp Detrick during the development of the organism as a simulant.
The selection of a strain with the desired properties appears to have occurred in at least two discrete steps, as shown by the genome sequences and metabolic profiles. The initial step appears to have been the adaptation of a strain to growth in corn steep liquor, an acidic medium rich in protein and lactate [44] . The robust growth of the Detrick strains relative to 1942 in low-pH medium containing high lactate levels is likely due to mutations in mmgD (2-methylcitrate synthase, position 2029530), or a short-chain 3-oxoacyl-[acyl-carrierprotein] reductase (position 3437350), or both. The most likely candidate for a mutation in the Detrick isolates that increases growth is the frameshift in mmgDthat occurred following the divergence from the 49822 lineage and results in an altered C-terminus ( Figure S1 ). The mmgD geneencodes a 2-methylcitrate synthase that is expressed in the mother cell at the intermediate stages of sporulation [45] . A null mutation in mmgD had no perceptible effect on sporulation, although other TCA-cycle enzymes when mutated led to a loss of sporulation [45] . The effects of the frameshift mutation on sporulation and cellular physiology on the function of the enzyme are not clear at this time. We speculate that the frameshift mutation alters the substrate specificity of MmgD in favor of citrate, thus increasing the flux of lactate-derived intermediates through the tricarboxylic acid cycle. Evidence for this possibility includes the observations that 2-methylcitrate synthases can have partial citrate synthase activity [45] and that the B. subtilis mmgD gene can complement a gltA (citrate synthase) mutant of E. coli [46] . Alternatively, alteration of function of mmgD may have predisposed the lactate-adapted strain to acquisition of a hypersporulating phenotype, which is not readily isolated or stable in B. subtilis (see below); however the presence of a hypersporulating phenotype in an independently evolved lineage (1013) of BG indicates that the species may have an intrinsic predisposition to evolving such a phenotype in vitro.
The ''military'' strains also grow more readily on media containing D,L-diaminopimelic acid (meso-DAP), a major component of bacterial peptidoglycan. Corn steep liquor is derived from the incubation of corn in water at 42-55uC, during which a lactic fermentation by a community of wild organisms including numerous uncharacterized Bacillus spp. occurs. Total bacterial counts at the conclusion of CSL production can be quite high [44] , thus the availability of such compounds for growth is not surprising. Another potential source of meso-DAP could be bacterial autolysis during sporulation. The relative roles of each of the alleles in growth on lactate and/or meso-DAP is the subject of current investigation in our laboratory.
The second step in the development of BG as a simulant appears to have been the deliberate selection of a hypersporulating variant [6, 47] . Importantly, the selection of a strain optimized for spore yield resulted in the fixation of a new spo0F allele that has no counterpart among the available spo0F sequences (Figure 8 ). The sole Spo0Fsequence that differs at position 101 is that of B. clausii, in which tyrosine replaces histidine. Notably, the spo0F(H101R) mutation is distinct from a separate spo0F(A98P) mutation present in the in vitro passaged 1013 isolates. Given that the amino acid sequence of B. atrophaeus Spo0F is identical to that of B. subtilis but for two conservative substitutions, it is likely to have very similar if not identical biochemical properties. Detrick-1 and 1942 likely represent one of the two R colony morphotypes described by Hayward et al. [6] , whereas the hypersporulating F morphotypes likely arose due to the emergence of the spo0F(H101R) mutation. However, the possibility that Detrick-1 represents a reversion mutant at this locus from Detrick-2 cannot formally be excluded, but since it represented the dominant morphotype in the 1952 Detrick vial we believe this is unlikely. The presence of the spo0F(H101R) allele in the ATCC 9372 strains suggests that these strains were acquired by ATCC after this mutation appeared within the Detrick lineage. Experiments to verify the roles of each allele in modulating sporulation are currently in progress. Preliminary results indicate that transformation of B. subtilis Dspo0F with B. atrophaeus DNA and selection of spo+ cells dramatically alters colony morphology independently of the spo0F allele introduced; additional studies to verify the effects of each allele are currently in progress (James Hoch, personal communication).
The H101R and A98P allelesare likely to alter the response to signals promoting sporulation. Aspo0F(H101A) allele results in a sporulation-proficient strain that throws off sporulation-deficient papillae [48] , and the same mutation has been shown to suppress the spo 2 phenotype of a strain containing a defective kinA allele. H101 has been proposed as a potential metal-binding site with particular affinity for Cu 2+ [49] . Binding of Cu 2+ (or another divalent metal) at this site may modulate interaction with one or more sensor kinases that promote sporulation. Substitution of positively charged arginine at this position could potentially mimic the binding of a metal cation in the loop containing H101, resulting in altered sporulation of the strains due to a change in the interaction with the kinases governing sporulation. It is unclear why, given the proposed role of divalent Cu 2+ in suppressing sporulation, H101R would result in a hypersporulation phenotype. The mechanistic relationship between spo0F(H101R) and the hypersporulation phenotype will be tested in future experiments.
Both variants in the 1013 lineage possess an A98P allele in spo0F. Although the presence of several other mutations within this lineage confounds the attribution of the hypersporulating phenotype to this allele at this time, the presence of a mutation in the same gene as another hypersporulating mutant is highly suggestive. The effect of proline substitution at position 98 on Spo0F functionis not immediately obvious, but the relatively inflexible proline residue can disrupt alpha-helices in protein structures. The 1013-1 lineage exhibits a hypersporulating phenotype even more pronounced than spo0F(H101R) strains in the ''military'' lineage. The observation that hypersporulating phenotypes have emerged during cultivationof two independent B. atrophaeus lineages point to the possibility that certain in vitro selection pressures may actually favor hypersporulating variants.
The selection pressures acting on the sporulation pathwayare highlighted by the sheer number of mutations discovered within the entire data set that occur in proteins known to play roles in sporulation. Nine of the 38 mutations (23%) found in all lineages were in genes that directly or indirectly regulate either entry into stationary phase or sporulation; this number exceeds the number that would be expected if mutations were to occur by chance, since less than 5% of B. subtilis genes are dedicated to regulatory processes of any kind [50, 51] . In addition to the mutations found within the ''military'' lineage, the two variants of ATCC 49822 shown in Figure 2 differ by mutations in rpoB (Table S5) which also plays a role in entry into sporulation [52] . Null mutations in spo0F resulting in asporogenous phenotypes contribute to colony morphology variation in B. anthracis, B. thuringiensis and B. subtilis [53, 54, 55] . Enhanced in vitro ''fitness'' is also a likely driver behind the recovery of asporogenic B. anthracis mutants that were discovered during the investigation into the B. anthracis attacks of 2001 [56] . Because the process of sporulation is highly energy-intensive and irreversible once commenced, mutants that delay sporulation (or fail to sporulate altogether) to take advantage of remaining nutrients would out-compete wild-type cells during repeated passage in vitro in the absence of other selection pressures, as has been demonstrated in extended in vitro evolution studies with B. subtilis under relaxed sporulation conditions [57] . This may not be universally the case, since gain-of-function mutations in sporulation such as those observed in this studymay compete favorably with wild-type cells if cannibalism of vegetative cells by sporulating bacteria is the dominant selective pressure [58] . Finally, horizontally transferred genetic elements can have dramatic effects on sporulation: for example, recent studies of phage lysogeny in B. anthracis have revealed the ability of several integrated phages to positively affect the kinetics of sporulation upon lysogeny of commonly used B. anthracis strains [59] .
This study identifies the spo0F(H101R) allele as the signature of a deliberate selection during the development of B. atrophaeus as a simulant. However, without the knowledge of the history and the analysis of the phenotypes of the strains originating from ''Camp Detrick'' as published in the open literature, attribution of this genotype to a deliberate selection event would not have been definitive, since a similar phenotype is observed in the 1013 lineage which to our knowledge was not deliberately selected for any specific trait. Any study designed to determine genomic ''signatures'' of deliberate enhancement or selection is likely to require an analysis of the baseline likelihood that mutations conferring a similar phenotype would emerge and become fixed by natural processes within an evolutionary timeframe consistent with a known time interval or number of passages.
Available evidence suggests that hypersporulation is not easily evolved in vitro. Maughan and coworkers attempted to evolve populations of a laboratory strain of B. subtilis with a hypersporulating phenotype by repeatedly heat-shocking cultures. While their efforts to enrich for hypersporulators failed, other studies revealed that asporogenous mutants evolved readily [60, 61] , confirming many early studies ( [62] and references therein). With the exception of the studies by Maughan et al., most ofthese investigators applied selections intended to inhibit sporulation rather than to enrich for strains with elevated sporulation rates. The 1013 lineage was never heat-shocked during its many transfers; thus the adaptations seen in this work are the result of balancing sporulation versus vegetative growth for prolonged periods on agar slants. However, because undomesticated isolates were observed to sporulate to 98-100% [60] , we cannot formally exclude the possibility that in vitro culture of the 1942 strain following its isolation for an unknown period by the University of Wisconsin might have selected for a hyposporulating variant. In this scenario, the H101Rand A98P mutations would represent suppressor mutations. We consider this possibility unlikely, given the phenotypic similarity of two environmental isolates in the UW collection (1942 and NRS-356). Furthermore, a progression toward darker pigmentation and greater hemolysisis evident in the ''military'' lineage ( Figure 2B ). These phenotypic changes are associated with the accumulation of additional mutations including a P145L substitution mutation in sigH, a positive regulator of sporulation [63, 64] and an A13P mutation in scoC, a negative regulator of sporulation [65] . Together, the strains analyzed in this study suggest strong selective pressures on the genes in the sporulation pathway, and more carefully controlled studies should be carried out to determine the dynamics of in vitro evolution and adaptation of spore-forming organisms, as has been done extensively in E. coli [66, 67, 68, 69, 70] .
Unexpectedly, the ''military'' lineages were also marked by the loss of catalase activity, whose presence is an identifying feature of both B. subtilis and B. atrophaeus [13] . This activity was present in a separate lineage of in vitro passaged organisms, so it is not immediately clear why ''military'' isolates, i.e. those subjected to selection within the early days of the development of BG as a simulant organism, would have lost the catalase activity characteristic of the parental isolate. Because the KatA gene product is not found in spores [41, 71] , we consider it unlikely that the absence of this activity would impact the resistance of spores to decontamination reagents, and thus any antioxidant resistance phenotype exhibited by spores of ''military'' isolates would likely have gone unnoticed. However, direct comparisons of the ''military'' B. atrophaeus lineages to the progenitor strains have not been done, and pleiotropic effects of a spo0F mutation on spore physiology cannot currently be excluded.
Whole-genome approaches are becoming critical components of microbial forensics. The SNPs and indels identified in the analysis of evidentiary materials currently become the basis for higherthroughput assays to screen large numbers of samples [56, 72] . Decreasing costs of whole-genome sequencing, and the comprehensive nature of the analysis, may make this the preferred method of forensic analysis of microbial samples in the future. With recently developed techniques of allele quantitation within populations by mass spectrometry [73] , real-time PCR [74] , and census-bysequencing [68, 75] , it may be possible to quantitate accurately rare alleles within any given microbial population. We are particularly intrigued by the possibility that, given a mixture of different variants and sufficient sequencing power, ultra-high coverage sequencing may prove to be a more quantitative means of enumerating the relative populations in a sample even before the presence of variants has been established. The results from sequencing two strains of BACI051 in this study provide evidence of such hidden diversity.
The genomic basis of interlaboratory strain variation is only beginning to become evident, with recent studies tracing the histories of commonly used lab strains of B. subtilis 168, E. coli, Salmonella enterica serovar Typhimurium 14028s, Pseudomonas aerugi-nosaPA01 and Mycobacterium tuberculosis H37Rv [76, 77, 78, 79, 80, 81] . These have revealed significant divergence of putatively identical strains from one laboratory to another, largely arising from mutations that accumulate during serial passage. Like the earlier work, our study highlights the utility of approaches based on wholegenome sequencing for the discrimination of closely related strains, especially when investigating the provenance for a given isolate. Tragically, at least 13 institutions are known to have destroyed archival collections of Select Agents [82] following the implementation of mandatory monitoring and reporting requirements, representing an incalculable loss of phenotypic and genomic diversity. This report underscores the importance of maintaining the genetic heritage preserved in the culture collections of individual investigators and institutions.  Differential Induction of Functional IgG Using the Plasmodium falciparum Placental Malaria Vaccine Candidate VAR2CSA BACKGROUND: In Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta. PRINCIPAL FINDINGS: We have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein. CONCLUSIONS/SIGNIFICANCE: Binding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes. Animal models are required for preclinical development of new generation vaccines against infectious diseases [1] [2] [3] . The ideal animal model mimics the human immunological response, the pathogen infection pathway, and allows analysis of the mechanism of the vaccine-induced protective immune response. However, despite the availability of humanised animal models such as transgenic mice [4] , immunological responses in most animal models are only indicative of what to expect in humans. The development of a recombinant vaccine is initiated with identification and selection of an antigen that induces a desired immune response. At this step, multiple antigens are tested, which requires an inexpensive and easy to handle animal model. Following selection of antigen, the optimal route of vaccine administration together with a proper adjuvant formulation has to be evaluated. The design of the individual vaccine components and their delivery thus relies on the performance in these pre-clinical animal tests, emphasizing the importance of the animal model.
Vaccines are one of the future strategies to prevent and control malaria [5] , one of the most widespread, pathogenic and deadly parasitic diseases in the world. Immunity is only acquired after successive infections in areas of high and stable malaria transmission [6] . Pregnant women become susceptible to placental malaria (PM) independent of any pre-existing immunity acquired during childhood. Placental malaria can have serious consequences for both mother and child such as maternal and infant anaemia, premature labour, low birth weight, and increased neonatal mortality [7] . However, after successive pregnancies women rapidly acquire immunity to PM, indicating that a vaccine strategy against PM may be feasible [8] . PM is caused by the binding of Plasmodium falciparum infected erythrocytes (IE) to chondroitin sulfate A (CSA) present on placental syncytiotrophoblast cells located in the intervillous space [9, 10] . Placental parasites express on the IE surface VAR2CSA, which is a Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that mediates binding of the IE in the placenta [11, 12] . VAR2CSA is a large (350 kDa) polymorphic protein with six Duffy-binding-like (DBL) domains, which complicates the development of a vaccine. A potential animal model for studying protection induced by immunizations with recombinant domains of PfEMP1 could be chimpanzees (Pan troglodytes), whose natural malaria parasite, Plasmodium reichenowi, is closely related to P. falciparum [13] . However, for ethical and economic reasons this is not a feasible model for malaria vaccinology. To date, most malaria antigens are produced by recombinant technology and tested in lower mammalian species, such as rodents and rabbits. These models have allowed analysis of antibody responses to different combinations and boundaries of the six DBL-domains of VAR2CSA. Initially, we expressed all DBL-domains fromVAR2CSA-3D7 and found that all domains except DBL4e, could induce antibodies against native VAR2CSA expressed on the surface of IE, with limited differences in immunizations of mice and rabbits [14] . However, these VAR2CSA-3D7 specific sera did not show significant inhibition of IE binding to CSA (unpublished data). Recently, in a large screening of the immunogenicity of different VAR2CSA antigens from the parasite line FCR3 using rat immunizations, we found that the DBL4e domain of VAR2CSA could elicit the desired adhesion-inhibitory antibodies [15] . However, the induction of functional inhibitory antibodies to certain DBL-domains is poorly reproducible and the immune response tends to focus on non-adhesion blocking epitopes [16, 17] . In addition higher levels of inhibitory antibodies are acquired using full-length VAR2CSA (FV2) as compared to single DBLdomains [18] . To further investigate the induction of adhesion blocking antibody responses, all single recombinant domains of VAR2CSA-FCR3 were used in immunizations of mice, rats and rabbits, and compared to responses raised against the full length recombinant protein.
To examine the antigenicity of the recombinant proteins the levels of antigen-specific IgG from immunized animals were measured using ELISA. All animal species immunized with individual DBL-domains or FV2 produced an IgG response, with a typical sigmoid shaped dose response curve of the antibody titrations ( Figure 1 ). However, the levels of antibodies to each of the DBL-domains as well as to FV2 differed depending on the animal species used (for all statistical analysis of antigenic differences see Table S2 ). In mice, the DBL6e immunizations resulted in the most potent IgG response with a significantly higher titre than that of FV2 and the other DBL-domains. Following DBL6e in potency was DBL5e and thereafter a lower response induced by DBL1X, DBL2X, DBL3X, DBL4e and FV2 of similar potency. As observed in mice, immunizations of rats with DBL6e resulted in a significantly higher IgG response compared to all other immunogens. DBL5e and FV2 induced in rats a more potent response than DBL1X, DBL2X, and DBL3X ( Figure 1B ) but also DBL4e induced a high antibody response. In rabbits, the seven tested antigens produced IgG responses that can be grouped in two according to the levels of responses ( Figure 1C ). The DBL6e, DBL5e and FV2 produced a high antibody response whereas IgG responses to DBL1X, DBL2X, DBL3X and DBL4e domains were lower and essentially similar.
In summary, the tested VAR2CSA derived recombinant proteins induced medium to high levels of antibodies. FV2, DBL6e and DBL5e immunizations resulted in the highest antibody titers. FV2 appeared more antigenic in rabbits and rats compared to mice, and only rats appeared to induce high levels of antibodies to DBL4e.
Antibody reactivity against native VAR2CSA protein expressed on the surface of IE Epitopes exposed in the recombinant single-domain proteins may not be surface exposed in the native full-length protein, primarily due to the proposed globular folding of the VAR2CSA molecule [19] . Therefore we tested the reactivity of the induced antibodies against native VAR2CSA exposed on the surface of the IE.
Erythrocytes infected with the FCR3 parasite line were used in flow-cytometry to test the IgG reactivity in animal sera against native VAR2CSA. The parasite cultures were continuously selected for binding to CSA by panning on BeWo cells, resulting in surface expression of VAR2CSA and sex specific recognition by IgG from human female serum, compared to male and Danish serum (data not shown).
In line with the ELISA results measuring the reactivity against the recombinant proteins, we found that the antibody reactivity against native VAR2CSA on the surface of the IE differed both with respect to animal species, and with antigen type. For mice and rabbits (Figure 2A and Figure 2C ), the reactivity against native VAR2CSA on the IE surface was higher in sera from animals immunized with DBL5e and DBL6e, the reactivity against DBL1X, DBL2X, DBL3X and DBL4e was low. In general, it appeared that rats acquired higher levels of antibodies specific to the native protein compared to mice and rabbits, the difference being especially marked for DBL4e. However, as for mice and rabbits, the FV2, DBL5e and DBL6e immunizations resulted in high specific reactivity towards native VAR2CSA expressed on the IE ( Figure 2B ).
To test whether the reactivity against the native protein on the IE surface was associated with the apparent antigenicity of the recombinant domains, as measured by ELISA, the mean fluorescence values (MFI) was correlated to the EC50 titer values for all single domains. There was a highly significant linear correlation for mice and rabbits (data not shown) (r = 0.847, P,0.0001 & r = 0.809, P = 0.0008, respectively, Pearson), whereas the linear dependency for rats was somewhat lower (data not shown) (r = 0.476, P = 0.01, Pearson).
In summary, we found high levels of concordance between antibody reactivity against the recombinant domains and reactivity against the native protein in serum from rabbits and mice, but less so in serum from rats.
The proposed mechanism of protection against PM is that antibodies block adhesion of IE to CSA in the placenta [8] . Therefore, the functional capacity of the induced antibodies was measured in inhibition of CSA-binding assays. IE were incubated on decorin, either with or without specific serum or purified IgG. The specific inhibition was compared to the binding of IE incubated with negative control samples. Only sera from rabbits and rats were attained in sufficient volumes allowing three independent CSA binding experiments.
The inhibitory capacity of the sera was tested prior to IgG purification, since crude purification of IgG may introduce a bias due to differential efficacy of purification of different antibody isotypes. Only FV2 and DBL2X specific rabbit serum appeared to inhibit more than the negative control rabbit serum ( Figure 3A ). This was in contrast to the responses measured using FCM against the native protein in DBL5e and DBL6e specific rabbit sera ( Figure 2C ), where high reactivity was found. The inhibitory capacity of rat serum was also not determined by the surface reactivity, for instance there was prominent inhibition using DBL4e and FV2 specific serum and lack of inhibition by DBL6e specific antibodies ( Figure 3A ), although DBL6e specific antibodies also were highly reactive against native VAR2CSA ( Figure 2B ). To rule out that IgM or unspecific effects of serum did not mediate the inhibitory capacity, we purified IgG from both inhibitory and noninhibitory sera and tested them in inhibition of binding assays at 0.5 mg/ml. The level of inhibition was essentially sustained in the IgG preparations ( Figure 3B&C ).
In summary, differential induction of functional antibody responses against DBL4e, but not to the FV2 protein, were demonstrated in rats compared to rabbits.
To examine how the immune response against the full-length recombinant protein is focused with respect to individual DBL domains, we tested the levels of antibodies against individual DBLdomains in serum from animals immunized with FV2.
In general, we found that the pattern of antigenicity when immunizing with FV2 was similar to immunizations with single domains (Figure 4 ). In rats, FV2 antibodies targeted all DBLdomains with similar reactivity, with exception of DBL5e and DBL2x, which showed highest and lowest reactivity, respectively. The levels of reactivity were more or less in line with the reactivity against the native protein of single-domain specific antibodies ( Figure 2B ). In rabbits, the reactivity against the domains appeared to be overall lower than for rats. Furthermore, the rabbit-FV2 reactivity was more focused towards DBL5e and DBL6e, as was also the case for the reactivity of single-domain specific antibodies towards the native erythrocyte surface exposed protein ( Figure 2C ). In summary, it appeared that the pattern of antigenicity found when immunizing with single domains was essentially similar to immunizations with the FV2 protein in the different animals.
The DBL4e recombinant protein appeared previously to be the only single domain that induces highly inhibitory antibodies [15] . However, the inhibitory antibodies were not induced in rabbits. Therefore, we tested the specificity of the antibody response in rabbits and rats against linear epitopes using a DBL4e peptide ELISA.
IgG induced by DBL4e immunizations reacted with the DBL4e peptides and no reactivity was detected with IgG from control IgG (data not shown). The overall level of reactivity was lower in rabbit serum (n = 4) compared to rat serum (n = 10) and a lower proportion of the rabbits reacted with a given peptide (data not shown). In general, the same areas of the linear sequence were targeted by IgG from the rats and rabbits, with the most distinct reactivity in both species of animals observed against peptide 2 to 5 ( Figure 5A ). However, it appeared that the rat antibody reactivity was skewed one to three peptides N-terminally compared to rabbit antibodies. Hence, the major peaks of reactivity recognized by rabbit-antibodies were peptides: 1-5, 14-19, 21-28, 31-33, 35-36 & 58-61, whereas rats recognized peptides 2-6, 14-20, 22-26, 31-38 & 60-63. Other peptides in the array were recognized by one species only, albeit some of them with quite low OD values. The major peak of reactivity (peptides 3-6) recognized by both rabbit-antibodies and rat-antibodies and one minor peak with distinct skewed reactivity (peptides [33] [34] [35] [36] [37] [38] were mapped on the structure model ( Figure 5B ). Both these areas of the model were predicted to contain unstructured loops. In addition, peptide 3-6 contained a b-sheet hairpin motif and peptide 33-38 contained several a-helixes. The other minor peak primarily targeted by rat antibodies (peptides 61-62) was also characterized by unstructured amino acid sequences (data not shown). In summary, the average reactivity to linear peptides covering the amino acid sequence of DBL4e was, apparently slightly skewed, but otherwise remarkably similar between rabbits and rats.
The VAR2CSA protein is a vaccine candidate against PM due to its surface localisation, function as an adhesin for placental CSA and its role in immunity against PM [11, [20] [21] [22] [23] [24] [25] [26] [27] . Although the fulllength protein can be produced, a vaccine based on a single domain is more likely to be feasible, due to the complexity and size of the antigen. To analyze if the immune responses towards singledomains and the full-length protein were comparable, we tested all single VAR2CSA domains as well as the full-length protein in three different animal species.
In general, there was a direct association between antigenicity and reactivity to the surface of the infected erythrocyte. The reactivity against native VAR2CSA on the IE surface was higher in sera from animals immunized with DBL5e, DBL6e and FV2 compared to other domains (Figure 2) , which was similar to the observed reactivity against the recombinant domains measured by ELISA (Figure 1 ). The correlations between the antigenicity of the domains and reactivity to the native protein on the surface of IE were highly significant in immunization of mice and rabbits, whereas the levels of rat antibodies reacting with the native protein in general were higher, which resulted in a less significant correlation. However, taken together the reactivity increased towards the C-terminal end of the external part of VAR2CSA, both in immunizations with single domains and FV2. This may reflect both an inherent antigenicity of single DBL domains of VAR2CSA and/or the accessibility of single domains in the quaternary structure of the native VAR2CSA molecule. This is in line with data showing that the functional CSA binding region is located N-terminally allowing for non-blocking antibodies to react with the C-terminal located domains (Dahlbä ck et al submitted) .
The inhibitory capacities of the induced rat and rabbit antibodies following FV2 immunizations were very high. However, the different species-specific inhibitory FV2 antibodies do not necessarily target the same epitopes, but further analysis of the epitope targets of FV2 specific antibodies are needed to elucidate the mechanism of inhibition. For immunizations with single domains, the inhibition of IE binding to CSA varied between animal species. Only rats immunized with DBL4e induced IgG, which efficiently inhibited IE binding, whereas the rabbit sera specific against DBL4e were non-inhibitory. The inhibitory effect of the IgG was not an effect of antibody titer, since immunizations with DBL5e and DBL6e also induced high titers, but these were in general non-inhibitory. These findings are in contrast to what others have found [21, 28] . Differences in observations could be due to different domain boundaries or to the variance in posttranslational protein modifications introduced for example by the baculovirus transfected T. ni as opposed to the Pichia pastoris expression system, such as differences in glycosylation pattern.
The above findings are not likely to be caused by differences in the MHC repertoire of the rat and rabbit strain used, since both strains used were outbred, reducing the likelihood that all of the individual rats and none of the rabbits induced functional antibodies. In addition, fair levels of antibodies was induced against the recombinant form of DBL4e in rabbits, indicating appropriate T-cell responses during the immunizations, yet the DBL4e specific rabbit sera was non-inhibitory even at rather high concentrations. Differential induction of binding-inhibitory antibodies is probably caused by species-specific differences in immuno-dominance of Bcell epitopes in different species of animals, similar to observations with antigens from other organisms [29, 30] . Responses to FV2 appeared more homogenous between species, at least with respect to the adhesion blocking properties, possibly due to presentation of more epitopes in a native form compared to single domains. Since the VAR2CSA molecule appears partly globular, immunodominant B-cell epitopes, not present in the native protein, may be exposed in individual DBL domains. This could also be the cause of the finding that antibodies induced against the DBL1X and DBL3X single domains, when based on heterologous sequences, can inhibit the binding of FCR3-IE [17] , as opposed to antibodies induced against DBL1X and DBL3X, based on the FCR3 sequence [15] .
Attempting to identify epitopes targeted by inhibitory antibodies, we compared differences in rabbit and rat antibody reactivity to linear peptides covering the entire DBL4e domain. We previously did this with DBL4e-FCR3, DBL4e-3D7, DBL1X-3D7 and DBL3X-HB3 specific sera from rats [17] . The average reactivity to DBL4e linear epitopes was apparently slightly skewed when comparing rat and rabbit antibodies. Otherwise, the majority of responses in rats and rabbits were surprisingly similar. This similarity, combined with the lack of functional activity of the rabbit antibodies, both with regard to surface activity and inhibitory capacity, probably shows that surface-reactive and binding-inhibitory rat antibodies are not targeting linear epitopes in the native protein. The most pronounced areas of reactivity against the linear sequence were mapped to regions in the DBL4e model containing unstructured loops. This is in line with the findings that patches of high sequence diversity in VAR2CSA are concentrated in flexible loops [20, 31, 32] and that naturally acquired antibodies also are directed against these regions [33] . However, partly denatured antigen induced by Freund's adjuvant could increase the response to flexible loop regions in experimental immunizations [34] . Interestingly, a fraction of the expressed PfEMP1 during the maturation of the parasite in the IE remains intracellular [35] . Whether this intracellular pool of protein is denatured at the point of schizont rupture, promoting responses in natural infections to flexible loops more tolerant to mutations, as seen in other organisms [36] , remains to be investigated.
It should be noted that DBL4e and DBL4e-ID4 specific antibodies are cross inhibitory on a panel of maternal parasite isolates [37] , showing that a conserved region of this recombinant protein can be targeted by antibodies interfering with the interaction between VAR2CSA and CSA. We have not analyzed the cross-reactivity of the FV2 induced IgG, consequently it is unknown if the inhibitory response is directed towards conserved or variable regions. It is possible that selection pressure can increase exposure of immuno-dominant variable epitopes targeted by non-inhibitory antibodies.
In summary, it appears that the immunogenicity of the VAR2CSA protein is highest in the C terminal end, both immunizing with single domains and with the full-length protein.
Identification of the epitopes targeted both by FV2 and DBL4e specific binding inhibitory antibodies may serve as a tool to generate second-generation antigens by removing or masking redundant/ unwanted epitopes. We do not know how the immune response will be focused in humans upon immunization with DBL4e and the results from a phase 1 clinical trial in human volunteers with DBL4e will be decisive for further clinical development. Retrospectively, we would probably not have identified the binding inhibitory capacity of DBL4e specific antibodies had we only tested the antigen in rabbits and mice. This emphasises the value of testing different animal species as vaccination models, specifically for antigens like PfEMP-1 and EBA, which have evolved to generate variations in humoral immune responses. Plasmodium falciparum culture P. falciparum, FCR3 parasite (laboratory strain) were cultured in RPMI-1640 supplemented with 25 mmol/L sodium bicarbonate (Sigma-Aldrich), 0.125 mg/ml gentamycin, 0.125 mg/ml Albumax II (Invitrogen), 2% normal human serum and with 5% hematocrit of group 0+ human blood. To select for VAR2CSA expression, IE were repeatedly panned on BeWo-cells as described [38] . All isolates were mycoplasma negative and were regularly genotyped using nested GLURP and MSP-2 primers in a single PCR step.
All VAR2CSA single domains were cloned from genomic P. falciparum FCR3 parasite DNA (GenBank accession no. AY372123). Full length var2csa was based on a codon optimized synthetic DNA fragment as described [18] . Gene fragments were cloned into the Baculovirus vector, pAcGP67-A (BD Biosciences) modified to contain a V5 epitope upstream of a histidine tag in the C-terminal end of the constructs. Linearized Bakpak6 baculovirus DNA (BD Biosciences) was co-transfected with pAcGP67-A into Sf9 insect cells for generation of recombinant virus particles. Histidine-tagged recombinant protein was purified on Ni 2+ sepharose columns from the supernatant of baculovirus infected High-Five insect cells using an Ä KTA-express purification system (GE-Healthcare). The full-length protein VAR2CSA (FV2) and the six VAR2CSA DBL-domains were produced corresponding to the following amino acid number in 
The animals were subcutaneously immunized with either single VAR2CSA-domain constructs (DBL1x-6e) or with FV2. Control groups were immunized with the DBL3c-VAR1-FCR3 in the same concentration as the tested VAR2CSA proteins [15] . Groups of BALB/c mice (n = 8) (inbred) (Taconic, Denmark) were subcutaneously immunized with 15 mg per animal of recombinant protein in Freund's complete adjuvant, followed by three followup immunizations of 10 mg of protein in Freund's incomplete adjuvant at two-week intervals. However, due to insufficient amounts of protein only four mice received DBL2x, and six mice received DBL6x. For immunizations with FV2 (done on a later time point) the mouse strain used was CB6F1 mice (F1 hybrid between BALB/c females and C57BL/6 males) (Harlan, USA) following the same protocol as previously described for the BALBc mice. Seven groups of Wistar rats (outbred) (Taconic, Denmark) were immunized with 40 mg per animal of recombinant protein in Freund's complete adjuvant, followed by three booster injections of 20 mg of protein in Freund's incomplete adjuvant at 2K-week intervals. The rat groups consisted of three animals except the group receiving the DBL4e (n = 8) and DBL3c (n = 2). New Zealand White rabbits (outbred)(HB Lidköping Kaninfarm, Sweden) were immunized with 40 mg of recombinant protein in Freund's complete adjuvant, followed by three booster immunizations of 20 mg of protein in Freund's incomplete adjuvant at 3 week intervals. For each recombinant protein one rabbit was immunized with the exception of rabbits receiving DBL2x & FV2 (n = 2) and DBL4e & DBL5e (n = 3). For all animals sera were collected 8 days after the final immunization. Sera were stored at 220uC until use.
ELISA plates (Nunc Immunoplate) were coated with protein overnight (4uC) at a concentration of 1 mg/ml in 16 PBS. The plates were blocked with ELISA dilution buffer (1% BSA and 1% Triton x-100) before the serial diluted serum was added to the plate. Secondary horseradish peroxidase-conjugated (HRP) antibodies added in the following dilutions: anti-rabbit-IgG 1:2000 (P0448, Dako), anti-mouse-IgG 1:2000 (P0260, Dako), and antirat-IgG 1:3000 (A9037, Sigma). The optical density was measured at 490 nm in an ELISA plate reader (VersaMax, Molecular Devices). Antibody titration curves were determined using GraphPad Prisma5.
The FV2-specific IgG from both rats and rabbit's sera were affinity purified on HiTRap TM NHS-activated HP columns (GE Healthcare), according to the manufacturer's instructions. Briefly, each column was coated with 0.5 mg/ml of a DBL-domain resulting in six different columns for the individual six DBLdomains. The affinity purified IgG was verified using ELISA as previously described in this material and methods section. The assay was performed twice with similar results.
The reactivity of IgG in animal sera to the VAR2CSA protein expressed on IE surface was measured by flow-cytometry (FCM) using a protocol modified from [39] . Briefly, the parasite culture was enriched for late trophozoite and schizont stage parasites in a strong magnetic field (MACS, Miltenyi). Aliquots of 2610 5 IE in a total volume of 100 ml were labelled by ethidium bromide and sequentially exposed to (1) 15 ml animal serum and (2) 1:100 dilutions of FITC labelled secondary antibodies specific for IgG from the individual species: Mouse (FL2000, Vector); rat (62-9511, Invitrogen) and rabbit (FL1000, Vector). As a negative control, IE were incubated both with sera from non-immunized animals and without animal serum and then exposed to secondary antibodies specific against IgG from different animals spec. Data from 5000 infected cells (ethidium bromide positive) was collected on a FC500 flowcytometer (Beckman Coulter). The median FITC fluorescence intensity was determined using Winlist Software (Verity Software House).
For analyses of the capacity of serum to inhibit binding we used 2610 5 tritium-labeled late-stage IE and 15 ml serum in a total volume of 120 ml which were added in triplicates to wells coated with 2 mg/ml of the commercially available chondroitin sulfate proteoglycan Decorin (D8428; Sigma-Aldrich). Decorin was used as a source of CSA as this has a protein core resulting in more efficient coating to plastic than CSA. After incubation for 90 min at 37uC, unbound IE were washed away by resuspension performed by a pipetting robot (Beckman Coulter). The proportion of adhering IE was determined by liquid scintillation counting on a Topcount NXT (Perkin-Elmer).
The inhibitory capacity of the induced VAR2CSA domainspecific IgG was measured using a standardized Petri dish method both due to the smaller volume in this assay, and because this a more commonly used assay [40] [41] [42] . IgG was purified from the animal sera using HiTrap Protein G HP kit (GE Healthcare) and dialyzed against PBS 16 buffer. Briefly, 20 spots in each Petri dish (Falcon 351005) were coated overnight with 20 ml of decorin (2 mg/ml in PBS). Spots were blocked with 3% bovine serum albumin. Parasite cultures were enriched for late trophozoite and schizont stages in a strong magnetic field (MACS, Miltenyi) and adjusted to reach a 20% parasitemia at 0.5% hematocrit. The parasite suspension was pre-incubated with both animal sera and purified IgG at different concentrations at 37uC for 30 min. Duplicate spots were incubated for 15 min at 37uC in a humid chamber. Unbound erythrocytes were removed by washing with PBS on a gyro-turntable (Stuart) until spots were clear of blood. Bound erythrocytes were glutaraldehyde-fixed (1.5%, 5 min) and Giemsa stained. Five fields were counted in each spot using a magnification of 206 using a Leica Microscope. As negative controls, anti-DBL3cVAR1 serum and IgG were used. Parasite binding to decorin was abrogated by soluble CSA (Sigma-Aldrich) and by chondroitinase (Sigma-Aldrich) treatment of decorin (data not shown).
Epitope mapping was done using a peptide array covering the DBL4e domain. The peptide array consisted of 66 synthetic 16-28 amino acids long overlapping peptides, spanning amino acids 1607-1989 of the P. falciparum-FCR3 var2csa sequence (Table S1 ). The peptides were prepared by Schafer-N Copenhagen and the purity of the peptides was expected to be 70% or higher. Peptide binding was determined using ELISA. ELISA plates (Nunc Immunoplate) were coated overnight (4uC) with DBL4e peptides at a concentration of 3 mg/ml in 16PBS. Plates were blocked with ELISA dilution buffer (1% BSA and 1% Triton x-100) before animal serum samples were added. The serum samples were diluted 1:100. The following secondary horseradish peroxidaseconjugated (HRP) antibodies were used: anti-rabbit P0448 (DAKO), dilution 1:2000; and anti-rat A9037 (Invitrogen) and diluted 1:3000. The optical density was measured at 490 nm in an ELISA plate reader (VersaMax, Molecular Devices). The level of reactivity in this ELISA based peptide array was directly associated to the level of reactivity in an assay where identical peptides were synthesised directly on to a chip, confirming homogeneous coating of the peptides [17] .
The structure model of the DBL4e sequence (C1576-C1910) was created using the same method as described for other VAR2CSA DBL-domains [20] , but using as a template the newly published structure of DBL3X-VAR2CSA (3BQK) [43] .
Best-fit curves and mean EC50 values to determine compare antibody titres were done using Gradhpad Prism5 (Gradhpad Software Inc.). Sigma Stat 3.11 (Sysstat software Inc.) was used in the following analysis: one way ANOVA tests followed by multiple pair wise comparison procedures (Holm-Sidak method), which were used to determine differences between Log EC50 values to measure the antigenicity of single domains and FV2; Pearson correlation test, which was used to assess the correlation between MFI values and OD-EC50 values.  Severe pneumococcal pneumonia: impact of new quinolones on prognosis BACKGROUND: Most guidelines have been proposing, for more than 15 years, a β-lactam combined with either a quinolone or a macrolide as empirical, first-line therapy of severe community acquired pneumonia (CAP) requiring ICU admission. Our goal was to evaluate the outcome of patients with severe CAP, focusing on the impact of new rather than old fluoroquinolones combined with β-lactam in the empirical antimicrobial treatments. METHODS: Retrospective study of consecutive patients admitted in a 16-bed general intensive care unit (ICU), between January 1996 and January 2009, for severe (Pneumonia Severity Index > or = 4) community-acquired pneumonia due to non penicillin-resistant Streptococcus pneumoniae and treated with a β-lactam combined with a fluoroquinolone. RESULTS: We included 70 patients of whom 38 received a β-lactam combined with ofloxacin or ciprofloxacin and 32 combined with levofloxacin. Twenty six patients (37.1%) died in the ICU. Three independent factors associated with decreased survival in ICU were identified: septic shock on ICU admission (AOR = 10.6; 95% CI 2.87-39.3; p = 0.0004), age > 70 yrs. (AOR = 4.88; 95% CI 1.41-16.9; p = 0.01) and initial treatment with a β-lactam combined with ofloxacin or ciprofloxacin (AOR = 4.1; 95% CI 1.13-15.13; p = 0.03). CONCLUSION: Our results suggest that, when combined to a β-lactam, levofloxacin is associated with lower mortality than ofloxacin or ciprofloxacin in severe pneumococcal community-acquired pneumonia. Streptococcus pneumoniae is the leading causative agent of community-acquired pneumonia (CAP). Despite new antimicrobial agents and advances in supportive measures, attributable mortality linked to pneumococcal pneumonia remains unchanged and dramatically high when patient are admitted in intensive care units (ICU) [1] .
Most guidelines have been proposing, for more than 15 years, a combination of a β-lactam with either a quinolone or a macrolide as empirical, first-line therapy of severe CAP requiring ICU admission [2] [3] [4] [5] [6] [7] [8] . Although a recent study demonstrated combination antibiotic therapy to be associated with a higher survival rate than monotherapy in patients with severe CAP and shock [9] , the rationale for this combination was not to increase efficacy but rather to routinely provide coverage of all common pathogens causing severe CAP and particularly, S. pneumoniae and Legionella species.
In our ICU, we followed until 2003 the 1991 French recommendations [2] . Most patients received an empirical therapy based on a β-lactam-fluoroquinolone combination. Before 2003, fluoroquinolones used were ofloxacin and ciprofloxacin. Levofloxacin replaced these quinolones since its 2003 addition to the hospital formulary. Such a replacement was comforted by the ERS, French and IDSA guidelines published between 2005 and 2007 [6] [7] [8] . We wished to determine outcomes of patients treated with a combination of β-lactam plus fluoroquinolone for severe pneumococcal pneumonia. This homogenous modification of severe CAP antibiotic management in our ICU gives us the further opportunity to assess the influence of a fluoroquinolone with enhanced activity against S.pneumoniae. 
Firstly, we retrospectively collected all consecutive patients aged > 18 years who were admitted into our ICU (16-bed medical and surgical intensive care unit in a 450-bed general hospital) between January 1996 and January 2009 for severe community-acquired pneumonia (CAP) and who received a definite diagnosis of pneumococcal pneumonia. Secondly, we selected patients who received, as initial antibiotic treatment, a β-lactam plus a fluoroquinolone, used with an appropriate dosage by IV route. Thirdly, patients were divided into two groups according to the fluoroquinolone used, Group A for ofloxacin or ciprofloxacin, Group B for levofloxacin. The study protocol was submitted to the Institutional Review Board for University Hospital of Lille which gave an approval with waiver of informed consent, in agreement with French regulations concerning such retrospective studies.
CAP was defined by the following criteria observed at initial presentation or occurring within 48 h following hospitalization: acute onset of signs and symptoms of lower respiratory tract infection and a new pulmonary infiltrate found on the hospital admission chest radiograph. We excluded patients coming from nursing homes or hospitalized within 90 days prior to developing pneumonia or hospitalized > 48 h in general medical wards before ICU admission, and those with radiographic abnormalities attributed solely to any other known cause (i.e., pulmonary embolus, lung carcinoma or congestive heart failure). The decision for admission to our ICU was made, in all cases, by the attending physicians. However, only patients having a Pneumonia Severity Index (PSI) score ≥ 4 were included in this study [10] .
Streptococcus pneumoniae was considered as the causative agent of CAP when a S. pneumoniae strain was isolated from > 1 blood culture or when validated sputum (< 10 squamous epithelial cells and > 25 polymorphonuclear cells per low-power field) or tracheobronchial aspirates cultures grew with > 10 5 cfu/mL S. pneumoniae. Patients having CAP due to a penicillinresistant strain of S. pneumoniae (MIC > 2 mg/l) were excluded from our study.
Appropriate drug dosages were defined in the French recommendations as: amoxicillin > 50 mg/kg/d, cefotaxime > 50 mg/kg/d, ceftriaxone > 20 mg/kg/d, piperacillin > 200 mg/kg/d, ofloxacin = 200 mg/12 h, ciprofloxacin = 400 mg/12 h, levofloxacin = 500 mg/12 h [2, 3, 7] . These drug dosages for β-lactams, ofloxacin and ciprofloxacin were unchanged during the study period. Thus, doses used in both groups were similar.
Within 24 h of admission, all patients underwent clinical, radiological and biological tests. Briefly, we recorded age, gender, underlying clinical characteristics and initial vital signs. Chronic respiratory insufficiency was assessed combining the usual clinical and radiological criteria and the coexistence of ventilatory impairment assessed either before or after ICU stay. Immunosuppression was defined as recent use of immunosuppressant or systemic corticosteroids (i.e., prednisolone > 0.5 mg/kg/day for more than 1 month), human immunodeficiency virus infection, neutropenia (absolute neutrophil count < 1.000 cells/mm3), organ transplantation with ongoing immunosuppressant, cancer chemotherapy within the past 3 months, or asplenia. Shock was defined as a sustained (> 1 h) decrease in the systolic blood pressure of at least 40 mm Hg from baseline or a resultant systolic blood pressure < 90 mm Hg after adequate volume replacement and in the absence of any antihypertensive drug [11] . Severity of illness at admission to ICU was assessed using the Simplified Acute Physiology Score II (SAPS) II [12] , the Sepsis-related Organ Failure Assessment (SOFA) score [13] and the logistic organ dysfunction (LOD) score [14] . We also calculated the PSI at ICU admission [10] . For all patients, information on the following therapeutic topics instituted within 48 hours following ICU admission was recorded: supportive measures such as mechanical ventilation or hemodialysis, use of vasopressor drugs, hydrocortisone, drotrecogin alfa (activated), or intensive insulin therapy. The effectiveness of initial antimicrobial therapy was assessed within 72 h after treatment as follows: A lack of clinical improvement 3 days after treatment initiation (worsening or persistent fever or hypothermia, worsening of pulmonary infiltrates or of respiratory function assessed by PaO 2 /FiO 2 ) defined an ineffective treatment. On day 3, day 5 and day 7, body temperature, and SOFA score were determined. During the patient's stay in the ICU, occurrence of complications was recorded. We distinguished sepsis-related complications (secondary septic shock, acute respiratory distress syndrome or development of multiple organ failure), hospital-acquired lower respiratory tract (HA-LRT) superinfections and ICU-related complications (i.e., upper gastrointestinal bleeding, catheter-related infection, deep venous thrombosis and pulmonary embolism). Multiple organ failure (MOF), acute respiratory distress syndrome (ARDS) and HA-LRT were defined according to usual criteria [15] [16] [17] . Durations of mechanical ventilation, treatment with vasopressor drugs, and ICU length of stay were noted.
Finally, patient mortality was evaluated on D-15, and at the time of ICU discharge.
Descriptive analyses were performed in order to check and resume data. Characteristics of patients in each group were compared. Continuous variables were compared using the Student's t test. Categorical variables were compared using Chi-square test or Fisher's exact test when Chi-square was not appropriate. Differences between groups were considered to be significant for variables yielding a p value < 0.05. A stepwise logistic regression including variables collected within the first 48 hours of ICU stay and associated with a p value < 0.15 in bivariate analysis was performed. Adjusted odd-ratios were computed using a logistic regression analysis including the independent predictors of mortality. The Kaplan-Meier product limit method and the log-rank test were used to construct and compare survival curves for patients in each group.
All statistical analyses were performed using the SAS Software, V9.1.
During the study period, 378 patients with severe CAP were admitted in our unit. Among them, 83 (22%) patients exhibited a severe pneumococcal pneumonia and, finally, we identified 70 patients treated with a β-lactam combined with a fluoroquinolone, including 53 men (75.7%) and 17 women (24.3%). The mean age was 63.8 ± 16.8 years.
S. pneumoniae was identified in blood cultures in 25 patients (35.7%). Infection was polymicrobial in 18 patients (25.7%). Causative pathogens associated with S. pneumoniae were Haemophilus influenzae (n = 7), methicillin susceptible Staphylococcus aureus (n = 4), enterobacteriaceae (n = 4), Streptococcus spp. (n = 2) and Moraxella catarrhalis (n = 2). All pathogens were susceptible to at least one drug (β-lactam and/or fluoroquinolone) received by the patients. Thirty-eight patients (54.3%) were classified as Group A. β-lactams used were a third generation cephalosporin (n = 20; 52.6%), amoxicillin ± clavulanic acid (n = 16; 42.1%) and piperacillin-tazobactam (n = 2; 5.3%) combined with ofloxacin (n = 33; 86.8%) or ciprofloxacin (n = 5; 13.2%). Thirty-two patients (45.7%) were classified as Group B. β-lactams used were a third generation cephalosporin (n = 26; 81.3%), amoxicillin ± clavulanic acid (n = 5; 15.6%) and piperacillin-tazobactam (n = 1; 3.1%) combined with levofloxacin.
Main patients' characteristics on ICU admission are reported Table 1 . Most characteristics were similar in the two groups. However, underlying chronic respiratory insufficiency and bacteremia were more frequent in Group B patients.
Main therapeutics instituted during ICU stay, evolution of severity scores, and occurrence of complications are reported Table 2 . The most significant differences between the two groups of patients were the more frequent use of drotrecogin alpha, intensive insulin therapy and hydrocortisone in Group B patients.
On Day 15, 14 (20%) patients had died, 12 (31.6%) in Group A and 2 (6.3%) in Group B (p = 0.02). Overall, 26 patients died in the ICU, 17 (44.8%) in group A vs. 9 (28.1%) in group B (p = 0.15). So, difference in mortality rates was only significant during the first 15 days of ICU stay (Figure 1 ). In Group A, in-ICU mortality was 45% (9/20) when ofloxacin or ciprofloxacin were combined with a third generation cephalosporin and 44.4% (8/18) when combined with another beta-lactam, respectively (p = 0.97). In group B, it was 26.9% (7/26) when levofloxacin was combined with a third generation cephalosporin and 33.3% (2/6) when combined with another beta-lactam (p = 1).
Results of ICU-discharge survival prognosis bivariate analysis, including factors present on ICU admission, are reported Table 3 . All underlying diseases (excepted chronic heart failure), mechanical ventilation, use of a third generation cephalosporin combined with a fluoroquinolone, and bacteraemia on ICU admission did not appear as significant prognostic variables in this analysis. Among the 25 bacteremic patients, mortality was higher in group A patients (66.6%) than group B patients (31.3%), but the difference was not statistically significant (6/9 vs. 5/16; p = 0.11). Among the 34 patients with septic shock on ICU admission, mortality was higher in group A patients (71%) than in Group B patients (47%), but the difference was not statistically significant (8/17 vs. 12/17; p = 0.30).
Among variables collected during the ICU stay, use of hydrocortisone, intensive insulin therapy, haemodialysis and occurrence of HA-LRT superinfections did not appear as significant prognostic variables. Conversely, improvement on D3, SOFA > 8 on D3, D5, and D7, and occurrence of sepsis-related complications were significantly associated with outcome at ICU discharge (Table 4) .
According to the results of the bivariate analysis, the following variables were entered in the stepwise analysis: chronic heart failure, age > 70 yrs, acute respiratory failure requiring mechanical ventilation, septic shock on ICU admission, use of hydrocortisone, haemodialysis, PSI score = 5, SAPS II > 50 on D1, LOD > 8 on D1, 
The main finding of this retrospective analysis is that levofloxacin plus a β-lactam appears to be associated with improved survival compared to ofloxacin or ciprofloxacin plus a β-lactam in severe pneumococcal CAP.
Empirical antibiotic regimen for ICU-treated severe CAP has long been recommended to cover the 3 most common severe CAP pathogens (S. pneumoniae, S. aureus and H.influenzae), atypical pathogens and most relevant Enterobacteriaceae species. Levofloxacin is a fluoroquinolone active against most of these pathogens, especially S. pneumoniae with or without decreased penicillin susceptibility [18, 19] . Its clinical activity in CAP has been well documented in various clinical trials in Europe and the USA [20, 21] . Some studies demonstrated the efficacy of levofloxacin used as monotherapy in severe CAP, compared to ceftriaxone plus erythromycin or cefotaxime plus ofloxacin [22, 23] . Nevertheless, experts continue to propose, for ICU-treated severe CAP, an empirical antibiotic regimen based on an anti pneumococcal β-lactam combined with either a macrolide or a fluoroquinolone. Since respiratory fluoroquinolones with enhanced activity against S. pneumoniae (levofloxacin, moxifloxacin or gemifloxacin) became available, they replaced second generation fluoroquinolones (ofloxacin or ciprofloxacin) in the guidelines [6] [7] [8] . This fluoroquinolone generation shift has never been clearly justified and, to our knowledge, no clinical study has compared these different quinolones combined with a β-lactam in severe CAP. Our results suggest that, when severe CAP causative agent is S. pneumoniae, a combination levofloxacin plus β-lactam is associated with lower mortality than a combination ofloxacin or ciprofloxacin plus β-lactam.
These results could be surprising as all patients received an appropriately dosed β-lactam active against S. pneumoniae and as numerous strains of S. pneumoniae remain in vitro susceptible to ofloxacin or ciprofloxacin. However, there might be bacteriological and clinical data explaining our results. A synergy between β-lactams and levofloxacin against S. pneumoniae has been reported [24] . Conversely, synergy was rarely observed between the combination of cefotaxime and ofloxacin [25] . Recent clinical studies suggest that combination therapies could improve the prognosis of pneumococcal pneumonia: Waterer et al. retrospectively studying 225 patients with severe bacteremic pneumococcal pneumonia demonstrated that a single effective therapy was an independent predictor of mortality (AOR = 6.2) [26] . Baddour et al. performed a prospective, multicenter, international study including 844 adult patients with S. pneumoniae bacteremia [27] . Although the 14-day mortality was not significantly different for all patients receiving monotherapy versus combination (11.5% vs. 10.4%), a combination of in vitro active agents was associated with a significantly lower mortality than a single active agent (19.4% vs. 60%; p = 0.0006).
The present work has numerous limits. The most important is probably major treatment differences among the two groups. Patients were recruited during a long period (1996-2009), during which therapies such as hydrocortisone, drotrecogin alfa (activated), or intensive insulin therapy were introduced. Management of septic shock and ARDS has changed following results of large international studies [28, 29] . As most changes in management of patients with multiple organ failures overlap with our antibiotic policy changes, our results might be biased. Indeed, hydrocortisone use and intensive insulin therapy were more frequent in group B than in Group A. However, these factors were not significantly associated with ICU survival in bivariate analysis and hydrocortisone use, in multivariate analysis, was not an independent prognostic factor. Moreover, there is no evidence suggesting a survival benefit by most adjunctive therapies in patients with CAP [30] and the benefit of intensive insulin therapy in medical ICU and/or low-dose steroids is now highly questionable [31, 32] . Similarly, the use of cephalosporin is more frequent in group B than in group A. However, the use of a third generation cephalosporin rather than amoxicillin has no impact on prognosis. This is not surprising as, to our knowledge, no clinical study demonstrated a third generation cephalosporin to be superior to amoxicillin for non penicillin-resistant S. pneumoniae CAP as far as drug dosage is adequate. Finally, some important prognostic parameters such as the time elapsed between admission and the first dose of antibiotic were not taken into account in our study. Before 2006, we did not have computerized data charts thus, exact time of admission and antibiotics admission, particularly for patients transferred from other departments/hospitals cannot be obtained.
Our study suggests that levofloxacin combined with a β-lactam is associated with improved survival in comparison with ofloxacin or ciprofloxacin combined with a β-lactam in severe pneumococcal patients admitted in the ICU. This combination, proposed by current guidelines as empirical treatment of severe CAP patients admitted in ICU could improve their prognosis. Obviously, only a prospective, randomized, double-blind trial could confirm this result.
List of abbreviations AOR: adjusted odd ratio; ARDS: acute respiratory distress syndrome; CAP: community-acquired pneumonia; CI: confidence interval; HA-LRT superinfections: hospital-acquired lower respiratory tract superinfections; ICU: intensive care unit; LOD score: logistic organ dysfunction score; LOS: length of stay; MOF: multiple organ failure; MV: mechanical ventilation; PSI: pneumonia severity index; SAPS: simplified acute physiology score; SOFA: sepsis-related organ failure assessment; SD: standard deviation. Is Generalized Maternal Optimism or Pessimism During Pregnancy Associated with Unplanned Cesarean Section Deliveries in China? This research examines whether maternal optimism/pessimism is associated with unplanned Cesarean section deliveries in China. If so, does the association remain after controlling for clinical factors associated with C-sections? A sample of 227 mostly primiparous women in the third trimester of pregnancy was surveyed in a large tertiary care hospital in Beijing, China. Post-delivery data were collected from medical records. In bivariate analysis, both optimism and pessimism were related to unplanned c-section. However, when optimism and pessimism were entered into a regression model together, optimism was no longer statistically significant. Pessimism remained significant, even when adjusting for clinical factors such as previous abortion, previous miscarriage, pregnancy complications, infant gestational age, infant birthweight, labor duration, birth complications, and self-rated difficulty of the pregnancy. This research suggests that maternal mindset during pregnancy has a role in mode of delivery. However, more research is needed to elucidate potential causal pathways and test potential interventions. Worldwide, Cesarean section rates are increasing [1] [2] [3] . Despite recommendations that cesarean section rates not exceed 15% [4, 5] , many countries have rates double or even triple that threshold [3] . China-home to one-fifth of the world's population and 12 percent of all births annually [6, 7] -is no different. Data from hospital-based studies in urban China showed c-section rates ranging from 26% to 63% during the late 1990s [8] , while a more recent WHO study combining urban and rural populations reported overall c-section rates of 46.2% [3] .
Although cesarean section deliveries can be lifesaving for both mothers and their infants when indicated, their overuse is cause for concern due to their association with increased maternal morbidity and mortality, cost, and utilization of sometimes scarce health system resources [3] . Numerous researchers have investigated the predictors of higher than normal cesarean section rates [9] [10] [11] [12] [13] [14] [15] . Principal among these include including physician-related factors, insurancerelated factors, hospital and health-system factors, and maternal preferences. Additionally, cesarean section rates have also been found to vary by male versus female provider [15] , public versus private hospital setting [16] [17] [18] [19] , adoption and use of clinical guidelines [20] , public versus private insurance status [18] , and even day of the week and time of day [17, 19, 21] that women present for delivery. Patient race [16] , age [22] , income [22] , and preferences [23] have also been linked to increased c-section rates.
Although the literature is replete with clinical factors associated with elective and emergency cesarean section, such as advanced maternal age, short maternal stature, heavier infant birthweight, fetal dystress, preeclampsia, prolonged/obstructed labor, or shoulder distocia [8, 24] , less is known about psychological factors that affect women who intend to delivery vaginally but ultimately deliver via csection. It is probable that the vast majority of unplanned cesarean sections are attributable to clinical indications. However, are there potential psychological variables at play as well? And in a country like China, with exceedingly high rates of cesarean section, might the impact of those psychological variables be observable?
This exploratory study was designed to examine the psychological characteristic of dispositional optimism and pessimism in a woman's likelihood of undergoing an unplanned cesarean section delivery in urban China. Dispositional optimism is seen as a relatively stable personality characteristic (a "trait" rather than a "state") that is associated with general assumptions about positive future outcomes. Dispositional pessimism is the converse: it is a tendency to expect the worst when looking toward future outcomes. A meta-analytic review of the optimism literature from 2009 [25] that examined 83 separate studies found a persistent relationship between optimism and positive health outcomes [25] . In addition, women with higher levels of optimism during pregnancy have been found to have to lower levels of stress, anxiety, and peripartum depression than women with lower levels of optimism [26] [27] [28] [29] . Optimism has also been linked to birth outcomes, with one study finding that optimistic women gave birth to larger babies [30] , and a second study finding that when gestational age was controlled for, women who were least optimistic during pregnancy when compared to women with higher levels of optimism delivered smaller infants [31] .
It may seem logical to conclude that if optimism can lead to better health outcomes, pessimism might be detrimental. However, pessimism has been shown to have prophylactic effects in certain circumstances. In particular, pessimism can insulate people from the psychological consequences of failure, including anxiety, depression, and diminished self-esteem [32] . Norem and Cantor [32, 33] found that individuals who expect the worst can sometimes use those expectations to help them better meet the demands of stressful challenges. These "defensive pessimists" engage in active and constructive coping efforts-which may mediate the relationship between pessimism and outcomes [34] . For example, Moyer et al. found that among pregnant women in Ghana, those who were the most pessimistic were more likely to get tested for HIV whereas their optimistic counterparts were less likely to get tested [35] .
This research aimed to address the following research questions. (1) Is generalized maternal optimism or pessimism (assessed during pregnancy) associated with unplanned cesarean section among women giving birth in a tertiary care hospital in Beijing? (2) If optimism or pessimism is associated with unplanned cesarean section, which is more strongly associated, optimism or pessimism? And (3) if there is a significant relationship between optimism, pessimism, and unplanned cesarean section delivery, is that relationship robust enough to remain significant when clinical factors are included in the model?
Site. Data were collected from pregnant women presenting for prenatal care at the obstetric outpatient clinic at the Peking University First Hospital between May and July 2006. As one of the largest and most well-known academic medical centers in Beijing, Peking University First Hospital draws both public and private patients from in and around Beijing. Clinics see an average of 600 pregnant women per week and 3000-3500 deliveries per year.
All research protocols and survey instruments were reviewed and approved by the institutional review boards at the University of Michigan and Peking University.
Pregnant women in their last trimester of pregnancy who were 18 years old or older attending antenatal care clinic were eligible. Women facing an imminent health crisis, those in active labor, or those being admitted to the hospital were excluded (despite the generally stable nature of optimism and pessimism, those women in active labor were excluded because of concerns about disproportionate reporting of a pessimistic attitude if it was assessed during painful, active labor when compared to assessments obtained during a routine prenatal visit). After describing the study and obtaining verbal approval to continue, research assistants talked patients through an informed consent form, answering any questions the women may have had. All participants signed a written informed consent document and were given a copy to keep. Women were then given a self-administered survey to complete before their appointment. Translators were used when necessary. Surveys were designed to be selfadministered, but women were given the option to have the survey administered verbally.
Data were gathered using paper and pencil forms. Hospital registration numbers were collected from participants to allow for postdelivery followup. Hospital registration numbers were removed from the original survey and replaced with a unique ID number once the registration number was recorded in a separate location for follow-up purposes. Responses from the hard copies of the self-administered surveys were entered into an Excel spreadsheet and cleaned.
The survey included administering a demographic and health questionnaire and the Life Orientation Test (LOT-R).
The Demographic and Health Questionnaire measured patient characteristics including age, number of pregnancies, other medical conditions, and self-perceived health status.
Women were asked to enumerate any pregnancy complications or symptoms they had during pregnancy, including such things as vaginal bleeding, headaches, swollen hands, troubled vision, preeclampsia, dizzy spells, swollen face, abdominal/belly pain, eclampsia, or other problems. For the purposes of this analysis, these were combined into a single dichotomous variable, and termed maternal complications. Women were also asked to rate their perception of the difficulty of their pregnancy on a scale of 1 to 4, with 1 being "extremely easy" and 4 being "extremely difficult."
The Life Orientation Test (LOT), developed by Sheier and Carver in 1985 [36] and revised in 1994 [37] (Life Orientation Test-Revised, or LOT-R), was used to assess dispositional optimism. The LOT-R is one of the most commonly used measures of optimism/pessimism. It uses generalized outcome expectancies to measure dispositional optimism. The LOT-R has been widely validated [38] and used in China [39] [40] [41] [42] [43] [44] [45] [46] . It includes 6 scored items and 4 fillers that generate an overall score, as well as two possible subscales: an optimism subscale and a pessimism subscale. The items that make up the optimism subscale are (1) in uncertain times, I usually expect the best; (2) I'm always optimistic about my future and (3) overall, I expect more good things to happen to me than bad. The items that make up the pessimism subscale are (1) if something can go wrong for me, it will; (2) I hardly ever expect things to go the way I would like them to go; (3) I rarely count on good things happening to me. The participant answers each item based on a 5-point scale, with response options ranging from strongly disagree to strongly agree. The pessimism items are reverse scored and then added to the optimism items to create the overall score whereas the subscales are created by summing the items for pessimism and the items for optimism separately. For these analyses, the optimism and pessimism subscales were used separately.
The instrument was pilot tested, and minor modifications were made to ensure comprehension. The survey was translated into Mandarin and back-translated into English by native bilingual speakers. The original and backtranslated versions were compared for consistency, and any inconsistencies were resolved by discussion and consensus among the research team.
Chart Review. was used to collect data after women had delivered their babies. Mode of delivery was determined, which indicated vaginal delivery with and without forceps, vaginal delivery with and without vacuum extraction, planned cesarean section, or unplanned cesarean section. For the purposes of this analysis, a single dichotomous variable was created to reflect unplanned cesarean section yes/no. Thus women who delivered vaginally or via planned cesarean section were treated as one group, and women undergoing an unplanned or emergency cesarean section were treated as a separate group. Additional data collected from the medical record included gestational age of the infant at delivery, birthweight, labor duration, use of pain medication, 1-minute and 5-minute Apgar scores, and any of a number of delivery or birth complications, including such things as hemorrhage, preeclampsia, intrauterine infection, breech presentation, or delayed labor. For the purposes of this analysis, all of those factors were combined into a single dichotomous variable termed birth complications.
Chart review Data were entered into a spreadsheet and cleaned. All data were analyzed using SPSS statistical software, Version 17.1 (SPSS Inc, Chicago, IL). Frequencies and basic descriptive statistics were calculated for all variables. Women with complete baseline and chart data (and could thus be included in the larger regression analysis) were compared against those women with incomplete baseline or chart data using Student's t-test for continuous variables and Chi Square analysis for categorical variables.
To address Research Question 1, (is optimism or pessimism associated with unplanned cesarean section delivery?), bivariate statistics were calculated to determine if optimism or pessimism were independently associated with unplanned cesarean section. Additional demographic and clinical variables were examined to determine if there were factors aside from optimism and pessimism and expected clinical correlates that might be associated with unplanned cesarean section in this population. Bivariate analysis included Student's t-tests, ANOVAs, and Chi-Square analyses.
To address Research Question 2, (which is more strongly associated with unplanned cesarean section delivery, optimism or pessimism?) Binary logistic regression analysis was conducted with both optimism and pessimism regressed on unplanned cesarean section (yes/no). Area under the curve analysis was conducted to judge the strength of the model.
To address Research Question 3, (if there is a significant relationship between optimism, pessimism, and unplanned cesarean section delivery, is that relationship robust enough to remain significant when clinical factors are included in the model?), binary logistic regression analysis was conducted with optimism and pessimism regressed on unplanned cesarean section (yes/no) with the additional clinical factors of labor duration, birth complications, previous abortion, previous miscarriage, pregnancy complications, gestational age, infant birth weight, and self-rated difficulty of the pregnancy added into the model. Area under the curve analysis was conducted to judge the strength of the model.
For all analyses a P value of .05 was taken as statistically significant.
Two hundred fifty-one women were asked to participate, and 227 met our eligibility criteria and agreed to participate (90.4% response rate). Of the 227, 86 had missing items on their surveys or their birth outcomes data were not available in the hospital medical records system. Table 1 illustrates our sample demographics, comparing the 141 women who were ultimately included in our analysis with the 86 who were excluded. Overall, our sample is one of well-educated Han women in their last trimester of pregnancy who are married and working outside the home. They do not differ significantly from the 86 women excluded from the analysis 76 (91.5) P = .300 (NS) Missing = 3 * Women with incomplete baseline data were excluded from the regression analysis. Key variables for inclusion were age, education, income, number of previous deliveries, originally from Beijing (y/n), car ownership (y/n), work before pregnancy (y/n), intend to work after pregnancy (y/n), insurance status, previous abortion (y/n), previous miscarriage (y/n), and experience of this pregnancy. on any variable aside from education, with excluded women more likely to have lower levels of education (P = .012). Table 2 illustrates the health-related variables reported at enrollment. Again, there were no significant differences found between women included in our analysis and those excluded due to missing data. More than half of our sample has had at least one previous pregnancy that was either spontaneously or electively terminated, and only 2.8 percent of women report having anything other than mild complications in this current pregnancy. The vast majority of women in this study were primiparous. Table 3 reflects delivery data obtained via chart review. Mean gestational age at delivery was 39.6 weeks. Mean duration of labor-defined as the time from first documentation of regular contractions plus cervical dilation to vaginal delivery-was 9 hours, with a range of 1 to 21 hours. Slightly more than half of women delivered vaginally, with the remaining having planned, emergency, or posttrial-of-labor (PTOL) cesarean sections. Infants had a mean gestational weight of 3406 grams, and most had five-minute Apgar scores of 10.
Forty-one percent of women had at least one birth complication. The most common complications were fetal distress (41%), preterm membrane rupture (26%), umbilical cord issues such as prolapsed, entanglement or nuchal cords (17%), and delayed labor (7%). With regard to Research Question 1, (is optimism or pessimism associated with unplanned cesarean section delivery?), bivariate analyses comparing optimism and pessimism against unplanned cesarean section indicated that both were significant: optimism (P = .047, 95% CI. 012, 1.81), pessimism (P = .003; 95% CI −2.42, −.529) (See Table 4 ). In addition, labor duration (P = .004, 95% CI 1. 009, 5.16) and the presence of birth complications (P = .01, Chi Square = 6.65) were also found to be significant. No other demographic or clinical factors were significantly associated with unplanned cesarean section. Table 5 Model 1, illustrates the findings with regard to Research Question 2 (which is more strongly associated with unplanned cesarean section delivery, optimism or pessimism?).
In an unadjusted model in which both optimism and pessimism were regressed against unplanned cesarean section, pessimism remained statistically significant while optimism failed to meet the threshold for statistical significance. (pessimism OR = 1.28, 95% CI: 1.06, 1.56, P = .01; optimism OR = 0.88, 95% CI: 0.71, 1.08; P = . 22) . When the same model was then adjusted for a variety of clinical factors (see Table 5 Model 2) to answer Research question 3 "if there is a significant relationship between optimism, pessimism, and unplanned cesarean section delivery, is that relationship robust enough to remain significant when clinical factors are included in the model?", pessimism remained significantly associated with unplanned cesarean section (OR = 1.42; 95% CI: 1.11, 1.81; P = .004). Of note, 
This study showed an association between higher levels of generalized maternal pessimism during pregnancy and an increased likelihood of an unplanned c-section delivery among women presenting for prenatal care and delivering their infants at a tertiary care hospital in Beijing, China. This association was robust enough to remain, even when adjusted for clinical factors likely to be linked to a risk of unplanned cesarean section delivery. Interestingly, pessimism not optimism remained significant throughout the analysis. However, what is not clear, and what the cross-sectional study design of the study does not allow us to explore, is the mechanism of action. What is it about being pessimistic that is related to unplanned c-section delivery? It is possible that pessimists have qualitatively different or less effective coping skills than their less pessimistic counterparts [47] [48] [49] [50] . Additionally pessimists, by virtue of believing that negative outcomes are likely, may be more fearful during labor. Emotional factors such as fear of delivery or fear of pain [51] have been linked to increased risk of c-section. Pessimists may also be more likely than their optimistic counterparts to abandon a traditional vaginal delivery and opt for a csection if given the opportunity. Conceivably pessimism may serve as a proxy for another latent variable. Previous studies have linked optimism and pessimism to age, spirituality, and even SES, [52] [53] [54] but an additional, as yet undescribed and measured variable could explain the relationship between pessimism and unplanned cesarean section rates.
By contract, optimists have been found to be more likely to adopt active coping strategies and reappraise a situation in a positive way if an important goal is blocked [50] . It is possible that such coping strategies may allow optimists to relax during delivery more easily than their more pessimistic peers, reducing the likelihood of "failure to progress." Our findings do not support this possibility: pessimism showed a significant association with unplanned cesarean section deliveries, while levels of optimism did not. This is not only useful in reaffirming that optimism and pessimism are two separate constructs rather than poles on a continuum [55, 56] , but is also instructive in potential interventions during pregnancy. Encouraging positive thoughts may not be nearly as helpful as discouraging negative ones.
The idea that cognitive predispositions that precede delivery may be associated with type of delivery is worthy of further exploration-including whether interventions can be designed to influence women's predispositions. For example, could cognitive behavioral therapy be used to reframe pessimists' negative thoughts, and might that result in lower cesarean-section rates? Perhaps more fundamentally, can pessimism be unlearned?
Despite a dearth of information on pessimism, research suggests that optimism can be learned and practiced [57] . Avoiding negative environments, seeking the company of positive individuals and reframing challenges as opportunities are some of the ways experts suggest "activating" one's optimism [57] . Yet it is unclear whether such techniques would be effective enough to impact health outcomes.
Nevertheless, our findings are noteworthy for two reasons. First, they demonstrate the potential relationship/association between psychological factors assessed during pregnancy and eventual delivery outcomes, and second, they illustrate the potential strength of psychological factors such as pessimism.
That birth complications were significantly associated with unplanned cesarean sections is to be expected-given that complications such as fetal distress, preeclampsia, prolonged/obstructed labor, or shoulder dystocia are primary indications for cesarean section delivery [8, 24] . It is also not surprising that duration of labor is associated with unplanned cesarean section. We also observed that women who delivered vaginally in this sample had longer labors than those who had unplanned cesarean sections (data not shown). It was interesting to note that in this study the length of time women were allowed to attempt labor before a cesarean section was chosen was much shorter than in the United States (average unplanned cesarean section labor duration in this study was 3.5 hours, compared to 16.0 hours among nulliparas and 12.4 hours among multiparas in the United States [58] ).
There are several limitations to this study. First, the use of the LOT-R has not been formally validated among Chinese pregnant women. However, the instrument has been used repeatedly in China [39] [40] [41] [42] [43] [44] [45] [46] 59] and it was carefully pretested in this population prior to study implementation. Our focus groups and pilot testing did not indicate any difficulties in interpretation of these items. Nonetheless, the instrument may benefit from a more rigorous validation study in this population. Also, the use of a cross-sectional convenience sample that includes mostly primiparous women limits inference to a wider population of pregnant Chinese women. In this study, all women presenting to the clinic were asked to participate, and it is possible that the women presenting during this study period were different from the larger population of pregnant women in Beijing. Future studies would benefit from a design that includes random selection at a variety of institutions across Beijing and across China.
This study also includes women in their last trimester of pregnancy. Although optimism/pessimism is considered a stable construct, it would be valuable to determine the potential impact of earlier recruitment.
This study also reveals what some would call excessively high episiotomy, cesarean section, and forceps rates, limiting its generalizability to settings without similar rates. Nonetheless, we believe these findings reflect clinical practice at one large tertiary care center in China, and as such provide valuable insight.
Finally, this study asked women to self-report their pregnancy complications. It was not possible to verify these self-reports against medical records data. We were able to elicit birth complications from the medical record, but this study relies upon self-reported complications during the gestation period. We do not believe this to be a significant limitation, however, given the high probability that women will know whether they are experiencing nausea, vomiting, or abdominal pain, or whether they have vaginal bleeding or swollen hands and feet. We also expect that women will remember if a doctor has told them they have high blood pressure, gestational diabetes, or other more serious pregnancy complications.
Implications. This research has several important implications. First, it confirms what many women and practitioners may have believed anecdotally: that a woman's mindset during her pregnancy may have an impact on her delivery. It also raises questions about the value of positive thinking-the predominant advice given to pregnant women-versus the value of not thinking negatively. Second, it raises important questions about whether inexpensive cognitive behavioral therapy or other mindsetaltering interventions among pregnant women could be used to reduce unplanned cesarean section rates.
More research is needed to elucidate the relationship between pessimism and pregnancy outcomes. Is this study replicable? Is the finding real, or is it masking some other yet to be determined variable? Is a negative outlook merely associated with a risk of unplanned cesarean section delivery, or can a causal pathway be identified? In addition, is it possible to change women's levels of pessimism? And would interventions to decrease pessimism translate to reduced rates of c-sections?
These are just some of the questions in need of answers as researchers continue to explore the relationship between psychosocial variables and pregnancy outcomes.
(i) Cesarean section rates are rising, due, in large part, to nonclinical factors.
(ii) Physician factors, insurance status, hospital policies, and maternal preferences are all non-clinical factors that influence csection rates.
(iii) Maternal cognitive predispositions during pregnancy (specifically optimism/pessimism) have not been examined in relationship to unplanned cesarean section deliveries.
What This Study Adds.
(i) Pessimism during pregnancy appears to be associated with an increased risk of unplanned cesarean section delivery in this population.
(ii) Pessimism during pregnancy remains associated even when clinical factors are controlled.
(iii) Pessimism appears to be a stronger correlate than optimism-suggesting that having positive thoughts/expectations may not be as helpful as not having negative thoughts/expectations during pregnancy.
The project/study described was supported by Grant no.T37 MD001425-08, from the National Center of Minority Health and Health Disparities, National Institutes of Health. Its contents, including the design and conduct of the study, the collection, management, analysis and interpretation of the data, and the preparation, review, and approval of the paper, are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. No author has a financial conflict of interest in this research or in its publication. In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA) and γ-cyclodextrin (γ-CyD) (pDNA/γ-CyD/AF-liposomes) showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes. The principle of somatic gene therapy is that genes can be introduced into selected cells in the body in order to treat genetic or acquired diseases. The liver may be potentially an important target for gene therapy, because crucial diseases such as amyloidosis, primary biliary cirrhosis, familial hypercholesteremia, phenyl ketonuria, and virus hepatitis occur in this organ [1] . In addition, the liver has the ability to synthesize a wide variety of proteins, to perform various posttranslational modifications, and to secrete them into the blood.
Of various nonviral methods, the lipofection method, by which cationic lipids (cationic liposomes) are used for transfection and interact with plasmid DNA (pDNA) to give a lipoplex, has recently attracted attention [2] . Cationic liposomes have great advantages as gene delivery carriers such as (1) low cytotoxicity and immunogenicity [3] , (2) regulation of the pharmacokinetics through the modification of particle size or lipids components of liposomes [4] , (3) entrapment of pDNA into inner water phase of liposomes and suppression of DNA degradation by DNase [5] , and (4) delivery of gene to target cells by the addition of target ligands and/or antibody [6] .
Asialofetuin (AF) is a glycoprotein that possesses three asparagine-linked triantennary complex carbohydrate chains with terminal N-acetylgalactosamine residues. The protein displays affinity to asialoglycoprotein receptor (ASGP-R) on hepatocytes and enters the cells through the receptor [7, 8] . Thus, AF has been used as a ligand to deliver drugs to hepatocytes and a competitive inhibitor to ASGP-R [9, 10] . In fact, the widespread use of AF-appended liposomes (AFliposomes) as a hepatocyte-selective gene transfer carrier has been reported [11, 12] .
Cyclodextrins (CyDs) have recently been applied to gene transfer and oligonucleotide delivery [13] [14] [15] [16] . CyDs are 2 Journal of Drug Delivery cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose containing a hydrophobic central cavity and hydrophilic outer surface, and they are known to be able to act as novel host molecules by chemical modification [17] . Davis and his colleagues reported that the ternary complex of a water-soluble β-CyD polymer with 6 A ,6 D -dideoxy-6 A ,-6 D -di-(2-aminoethanethio)-β-CyD and dimethylsuberimidate (βCDP6), galactosylated, or transferrin polyethylene glycol conjugates with adamantane, and pDNA possesses higher transfection efficiency in hepatoma or leukemia cells, respectively, through receptor-mediated endocytosis [18, 19] . Recently, we reported the potential use of PAMAM dendrimer functionalized with α-CyD (α-CDE) [20] and lactosylated α-CDE (Lac-α-CDE) as a hepatocyte specific gene delivery in vitro and in vivo [21] . Meanwhile, Lawrencia et al. reported that lipoplex transfection of pDNA with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) in the presence of cholesterol, which is solubilized by methyl-β-cyclodextrin (methyl-β-CyD), has significantly improved transfection efficiency in urothelial cells due to change in membrane fluidity by methyl-β-CyD [22] . In addition, we previously demonstrated that intravenous injection of the pegylated liposomes entrapping the doxorubicin (DOX) complex with γ-CyD in BALB/c mice bearing Colon-26 tumor cells showed DOX accumulation in tumor tissues and the potent antitumor effect, compared with those of DOX solution and pegylated liposomes entrapping DOX alone [23] . These lines of evidence suggest that transfection efficiency and pharmacokinetics of pDNA can be altered by the association of CyDs with AF-liposomes.
Based on these backgrounds, the purpose of this study is to evaluate in vitro gene delivery of AF-liposomes associated with CyDs as a hepatocyte-selective nonviral vector in HepG2 cells. In addition, the mechanisms by which γ-CyD enhanced transfection efficiency of pDNA/AF-liposomes were investigated in the view of a receptor recognition, physicochemical properties (particle size, ζ-potential, and encapsulation ratio), membrane fluidity, cellular uptake, and cytotoxicity of AF-liposomes.
2.1. Materials. Dilauroylphosphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and diacylphosphatidylethanolamine-N-lissamine rhodamine B sulfonyl (RH-PE) were obtained from Avanti Polar-Lipid (Alabama). N-(α-Trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG) was purchased from Sogo Pharmaceutical (Tokyo, Japan). Asialofetuin (AF) and 2-mercaptoethanol were obtained from Sigma Chemical (St. Louis, MO). Nhydroxysulfosuccinimide (Sulfo-NHS) was purchased from Fluka (Buchs, Switzerland). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was from Dojindo (Kumamoto, Japan). 2-(N-morpholino) ethanesulfonic acid (MES) and 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) were purchased from Nacalai Tesque hydroxylamine HCl, AF-liposomes were obtained. To remove the free AF, we performed gel filtration using Sepharose CL-4B column (Amersham Pharmacia Biotech, Freiburg, Germany) and determined phospholipids and AF by the Bartlett method [24] and the Bradford method [25] , respectively. The fractions number 26-30, which eluted both phospholipids and AF, were collected as the AF-liposomes fraction (Figure 1(b) ). The ζ-potential value and particle size of AF-liposomes were 31.2 ± 0.1 mV and 230.7 ± 4.2 nm, respectively. The amount of AF modification in 1 μmol of AF-liposome lipids was 35.8 μg/μmol lipids, indicating that AF modification rate against DPPE in liposomes was 0.75%. Liposomes without AF modification (N-liposomes) was prepared by the solution without Sulfo-NHS-AF, and the other procedure was the same as that of AF-liposomes.
Preparation of pDNA/CyD/AF-liposomes was performed according to the method reported by Hara et al. with some modifications [12] . Briefly, 2 μL of the solution containing pDNA (1 μg/μL) and CyDs (1 μM/μM lipids) dissolved in TE buffer were added to AF-or N-liposomes suspension. After mixing, the solution was freeze-dried. Then, the sample was rehydrated with THBS (10 mM, pH 7.5) for 30 min. After freezingthawing for three times, the vesicles were extruded through PVDF membranes (Nucleopore, Plesanton, CA) with pores of diameter 450 and 200 nm. The filtrates were used for further experiments as pDNA/CyDs/AF-liposomes or pDNA/CyDs/N-liposomes. The entrapment ratios of CyDs were evaluated by the anthrone-sulfuric acid method [26] . Briefly, 3 mL of anthrone reagent was added to 0.5 mL of the suspension containing CyDs/liposomes. The tube was covered with a glass ball and was heated for 10 min in boiling water. After quenching with cold water, absorbance of the suspension was measured by a U-2000A spectrophotometer (Hitachi, Tokyo, Japan) at 620 nm. The encapsulation ratios of pDNA were determined by a fluorescent spectrometer F-4500 (Hitachi, Tokyo, Japan). Briefly, 350 μL of the suspension containing pDNA/CyD/AF-liposomes in 10 mM THBS (pH 7.5) were mixed with 200 times diluted Picogreen dsDNA reagent (350 μL). After incubation for 30 min at 25 • C, fluorescent intensity (F po ) was determined. Next, after addition of 20% of Triton-X (20 μL) to the sample, fluorescent intensity (F pt ) was determined and the encapsulation ratio of pDNA was calculated as follows: encapsulation ratio (%) = [(F pt · r − F po )/F pt · r] · 100, where r is compensation coefficient (r = 1.03). Particle size and ζ-potential value of liposomes in 10 mM THBS (pH 7.5) were measured by a submicron particle analyzer N4 Plus (Beckman Coulter, Fullerton, CA) and ELS-8000 (Otsuka Electronics, Osaka, Japan), respectively.
AF-liposomes encapsulating calcein were prepared by the freezing and thawing method after addition of 0.1 mM calcein in 10 mM THBS (pH 7.5). The vesicles were extruded through two stacked polycarbonate membranes (Nucleopore, Plesanton, CA) with pores of diameter 1 μm. The sample was subjected to 10 passes through the filter at 40 • C. The filtrates were extruded through the polycarbonate membranes (pore size 0.2 μm) as described above. Two milliliters of CyDs solution adjusted at the appropriate concentration (5-20 mM) using 10 mM phosphate buffer were added to 20 μL of the liposomal suspension, and then the resulting suspension was incubated for 30 min at 25 • C. The fluorescence intensity of calcein (F t ) was measured with a fluorophotometer (Hitachi F-4500, Tokyo, Japan) at 25 • C; excitation and emission wavelengths were 490 and 520 nm, respectively. After addition of 20 μL of cobalt chloride solution (10 mM) to the sample to quench the fluorescence of nonencapsulated calcein, the intensity of fluorescence of encapsulated calcein (F in ) was also determined. Then, the liposomes were completely disrupted by the addition of 20 μL of Triton X-100 (20%) solution, and the intensities of fluorescence after quenching by cobalt chloride (F q ) were measured. Calcein encapsulation ratio was calculated by the equation as follows: encapsulation ratio
2.5. Cell Culture. HepG2 cells, a human hepatocellular carcinoma cell line, A549 cells, a adenocarcinomic human alveolar basal epithelial cells, and NIH3T3 cells, a mouse embryonic fibroblast cell line, were obtained from Riken Bioresource Center (Tsukuba, Japan). HepG2, A549, and NIH3T3 cells were grown in DMEM, containing 1 × 10 5 U/L of penicillin, 0.1 g/L of streptomycin supplemented with 10% FCS at 37 • C in a humidified 5% CO 2 and 95% air atmosphere.
Transfer. In vitro transfection of the pDNA/CyDs/AF-liposomes was performed utilizing the luciferase expression of pDNA (pRL-CMV-Luc or pGL3control vector) in HepG2, A549, and NIH3T3 cells. The cells (2 × 10 5 cells per 24 well plate) were seeded 6 h before transfection and then washed twice with 500 μL of serum-free medium. Two hundred μL of serum-free medium containing pDNA/CyDs/AF-liposomes in the absence and presence of AF as a competitor protein or BSA as a control protein were added to each dish and then incubated at 37 • C for 3 h. After washing HepG2 cells with serum-free medium twice, 500 μL of medium containing 10% FCS were added to each dish and then incubated at 37 • C for 21 h. After transfection, the gene expression was measured as follows: Renilla and firefly luciferase contents in the cell lysate were quantified by a luminometer (Lumat LB9506, EG&G Berthold Japan, Tokyo, Japan) using the Promega Renilla and firefly luciferase assay reagent (Tokyo, Japan), respectively. It was confirmed that CyDs and AF-liposomes had no influence on the luciferase assays under the present experimental conditions. Total protein content of the supernatant was determined by Bio-Rad protein assay kit (Bio-Rad Laboratories, Tokyo, Japan). EGFP-expressing cells were determined by a confocal laser scanning microscopy (CLSM, Olympus FV300-BXCarl Zeiss LSM-410, Tokyo, Japan) with an argon laser at 488 nm after fixation. Briefly, the cells (2 × 10 5 cells per 35 mm glass bottom dish) were seeded 6 h before transfection and then washed twice with 500 μL of serum-free medium. Transfection with pEGFP N1 DNA was performed using the same protocol as described above. The EGFP expression ratio was determined by the number of EGFP-expressing cells per 100 cells. To observe the cellular uptake of Rhodamine-labeled AF-liposomes (RH-AFliposomes) and Alexa-labeled pDNA (Alexa-pDNA), HepG2 cells (2 × 10 5 cells/dish) were incubated with the Alexa-pDNA/CyDs/RH-AF-liposomes for 3 h. After incubation, the cells were rinsed with PBS (pH 7.4) twice and fixed in methanol at 4 • C for 5 min prior to observation by a CLSM. 
CyDs have been reported to interact with cell membrane constituents such as cholesterol and phospholipids, resulting in the induction of hemolysis of human and rabbit red blood cells at high concentrations of CyDs [28] [29] [30] . In addition, CyDs are well known to disrupt liposomal membranes, depending on CyD cavity sizes and membrane components [31, 32] . Then, we evaluated the interaction of CyDs with AF-liposomes These results suggest that the interaction of γ-CyD and HP-CyDs with AF-liposomes was weaker than that of α-CyD and DM-β-CyD.
CyDs. Next, we evaluated in vitro gene transfer activity of pDNA/CyDs/AF-liposomes in HepG2 cells, ASGP-R positive cells ( Figure 3 ). Here, we used pRL-CMV (CMV promoter) as pDNA and the charge ratio (AF-liposomes/pDNA) of 1.6 optimized by our previous study (data not shown). The gene transfer activity of pDNA/γ-CyD/AF-liposomes in HepG2 cells was significantly higher than that of pDNA/HP-α-, HP-β-, and HP-γ-CyDs/AF-liposomes ( Figure 3 ). Next, we measured the Renilla luciferase mRNA level after transfection of pDNA/CyDs/AF-liposomes in HepG2 cells by the RT-PCR method ( Figure 4 ). As predicted, γ-CyD significantly increased the luciferase expression, but HP-γ-CyD did not, suggesting that γ-CyD is involved in the enhancing effect on luciferase expression at or prior to a transcription process.
To evaluate the enhancing effects of γ-CyD on gene transfer activity of AF-liposomes associating pDNA encoding EGFP controlled by a CMV or SV40 promoter, we examined EGFP and firefly luciferase gene expression after transfection of pDNA/γ-CyDs/AF-liposomes in HepG2 cells ( Figure 5) . The charge ratio of AF-liposome/pDNA was 1.6. γ-CyDs were added to AFliposome suspension before freeze-drying. The concentrations of γ-CyDs were 1 μM/μM lipids. The ζ-potential was measured by a light-scattering method. The particle size was determined using a photon correlation spectroscopic analyzer. Each value represents the mean ± SEM of 3 experiments. * P < .05 versus without CyD.
The extent of EGFP-expressing cells in the pDNA/AFliposomes system without CyDs was found to be 8%, while that with γ-CyD and HP-γ-CyD were 19% and 10%, respectively ( Figure 5(a) ). Additionally, the enhancing effect of γ-CyD was observed in the pGL3-control vector encoding firefly luciferase and having a SV40 promoter ( Figure 5(b) ). These results suggest that the enhancing effect of γ-CyD on gene transfer activity of AF-liposomes is a geneand promoter-independent manner. To confirm whether pDNA/γ-CyD/AF-liposomes have ASGP-R-mediated gene transfer activity, we performed transfection experiments in HepG2 cells in the presence and absence of AF, as an ASGP-R competitive inhibitor. Here, we confirmed that ASGP-R are expressed in HepG2 cells by the RT-PCR method (data not shown), which is consistent with previous findings [33] [34] [35] . As shown in Figure 6 , gene transfer activity of AF-liposomes was markedly inhibited by the addition of AF, but not BSA, a control protein. These results suggest that AF-liposomes had the ASGP-R-mediated gene transfer activity. Interestingly, the similar enhancing effects of γ-CyD on Renilla luciferase protein expression after transfection of pDNA/AF-liposomes were, however, observed in A549 cells and NIH3T3 cells, ASGP-R negative cells (Figure 7) . These results suggest that γ-CyD enhances the transfection efficiency of pDNA/AFliposomes in ASGP-R-independent manner.
To reveal cytotoxicity of pDNA/CyDs/AFliposomes, we examined the WST-1 method (Figure 8 ).
Although the cell viability after treatment with pDNA/AFliposomes and pDNA/CyDs/AF-liposomes for 3 h slightly decreased as a charge ratio of AF-liposomes/pDNA was increased in both HepG2 and NIH3T3 cells, that is, more than 80% of cell viability after application of pDNA/CyDs/AF-liposomes was observed at the charge ratio of 1.6 used in the transfection study as described above. These results suggest that pDNA/CyDs/AF-liposomes have great advantages as a nonviral vector, that is, superior transfection efficiency and less cytotoxicity, and the enhancing effect of γ-CyD on gene transfer activity of pDNA/AFliposomes is not associated with cytotoxicity.
To clarify physicochemical properties of the pDNA/AF-liposomes, we determined the particle sizes and ζ-potential values of the pDNA/γ-CyD/AFliposomes at the charge ratio of 1.6. The mean diameter of the pDNA/γ-CyD/AF-liposomes was smaller than that of pDNA/AF-liposomes or pDNA/HP-γ-CyD/AF-liposomes (Table 2) . Meanwhile, the ζ-potential values of pDNA/AFliposomes, pDNA/γ-CyD/AF-liposomes, and pDNA/HP-γ-CyD/AF-liposomes were almost comparable ( Table 2 ). These results indicate that γ-CyD reduced the particle size of AF-liposomes but did not change the ζ-potential value of pDNA/CyD/AF-liposomes. The charge ratio of AF-liposome/pDNA was 1.6. γ-CyDs were added to AFliposome suspension before freeze-drying. The concentrations of γ-CyDs were 1 μM/μM lipids. The encapsulation ratios of pDNA were determined using Picogreen assay. The encapsulation ratios of γ-CyDs were determined by an anthrone-sulfuric acid method. Each value represents the mean ± SEM of 3 experiments. * P < .05 versus without CyD.
Next, we examined the effects of γ-CyDs on encapsulation ratios of pDNA into AF-liposomes ( Table 3 ). The encapsulation ratio of pDNA in pDNA/γ-CyD/AF-liposomes was significantly higher than those of pDNA/HP-γ-CyD/AFliposomes and pDNA/AF-liposomes. Meanwhile, the encapsulation ratios of γ-CyD and HP-γ-CyD into AF-liposomes were approximately 10.2% and 11.0%, respectively. These results suggest that γ-CyD improves the encapsulation of pDNA into AF-liposomes, although the extent of γ-CyD encapsulation into AF-liposomes is not high. In addition, both the encapsulation ratio of pDNA into AF-liposomes and gene transfer activity of pDNA/AF-liposomes were raised, as the number of the freeze-thaw cycle was increased, suggesting that the encapsulation of pDNA in AF-liposomes is correlated with the gene transfer activity of pDNA/AFliposomes (Table 4) .
It is known that membrane fluidity of liposomes affects the release profiles as well as a retention time of drug encapsulated into liposomes. Therefore, we investigated the effects of γ-CyD on membrane fluidity of AF-liposomes using a Microcal MC2 scanning calorimeter. In the present study, we utilized N-liposomes to eliminate the effects of the heat degeneration of AF in a DSC thermograph. Figure 9 shows the effects of γ-CyD and HP-γ-CyD on DSC thermograms of N-liposomes (5 mM of total lipids). The peak derived from gel-to-fluid state transition in N-liposomes was observed at 55 • C, while new peak was appeared at 42 • C in the presence of 50 mM of γ-CyD (Figure 9(a) ). On the other hand, no significant change in DSC thermographs was observed in the presence of HP-γ-CyD (Figure 9(b) ). These results suggest that γ-CyD may affect the membrane fluidity of Nliposomes.
Generally, membrane fluidity and phase transition of liposomes are determined by the intensity of lipid-lipid interactions such as hydrophobic interaction, van der Waals forces, and hydrogen bond. Therefore, to evaluate the effects of γ-CyD on lipid-lipid interactions in AF-liposomes, we performed DSC analysis of DLPC-liposomes in the presence and absence of γ-CyD. The reason why we used DLPC-liposomes is due to clear observation of lipid-lipid interaction using DLPC composed of AF-liposomes. Figure 10 The charge ratio of AF-liposomes/pDNA was 1.6. pDNA/AF-liposome was prepared using a freeze-thaw method which was repeated from 0 to 5 cycles. Cells were incubated with pDNA/γ-CyDs/AF-liposome for 3 h in FCS-free medium. After washing twice, the cells were incubated for 21 h in culture medium supplemented with 10% FCS. The luciferase activity in cell lysates was determined using a luminometer. Each value represents the mean ± SEM of 3 experiments.
effects of γ-CyD and HP-γ-CyD on DSC thermograms, phase transition temperature, and enthalpy (ΔHcal) of DLPCliposomes (5 mM of total lipids). The phase transition temperature (Tc) of DLPC-liposomes in the presence of γ-CyD was shifted to high temperature as the concentration of γ-CyD was increased (Figures 10(a) and 10(c) ). The ΔHcal value of DLPC-liposomes in the γ-CyD system was drastically elevated at 20 mM of γ-CyD (Figure 10(d) ).
Meanwhile, in the case of HP-γ-CyD, there was no significant change in DSC thermograms, phase transition temperature, and the ΔHcal values (Figures 10(b) , 10(c) and 10(d)). Taken together, these results strongly suggest that γ-CyD enhances the lipid-lipid interaction of DLPC-liposomes, leading to the membrane stabilization of DLPC-liposomes.
3.6. Cellular Uptake of pDNA/AF-Liposomes. Next, we examined the cellular uptake of pDNA/γ-CyD/AF-liposomes into HepG2 cells using a CLSM. Figure 11 shows the CLSM images for distribution of RH-AF-liposomes and Alexa-pDNA in HepG2 cells at 3 h after transfection. The strong fluorescence derived from RH-AF-liposomes and Alexa-pDNA in the presence of γ-CyD was mainly observed in cytoplasm of HepG2 cells. Meanwhile, the fluorescence RH-AF-liposomes and Alexa-pDNA in the absence of CyD and with HP-γ-CyD was mainly observed on cell surface. Hence, these results suggest that pDNA/γ-CyD/AF-liposomes can be internalized into HepG2 cells to a larger extent, compared to pDNA/AF-liposomes and pDNA/γ-CyD/AF-liposomes.
In this study, we clarified that pDNA/γ-CyD/AF-liposomes have potent hepatocyte-selective gene transfer activity and negligible cytotoxicity, compared to pDNA/AF-liposomes and pDNA/HP-CyDs/AF-liposomes. In cationic liposome-mediated gene transfection, lipid composition and lipid type are the most important physicochemical factors, because they affect not only the interaction with pDNA but also the affinity to target cells [36, 37] . In the present study, we prepared AF-liposomes with a lipid composition of TMAG/DOPE/DLPC/DPPE (2/4/3/1, molar ratio). DOPE is known to enhance the endosomal escape of pDNA due to its structural change into hexagonal II form at pH 5-6, an endosomal pH range, resulting in destabilizing endosomal membranes [38, 39] . TMAG, a cationic lipid, makes it possible to interact with pDNA in AF-liposomes. Additionally, DPPE was used as a binding lipid with AF. Actually, cationic liposomes composed of TMAG/DOPE/DLPC (1/2/2, molar ratio) are commercially available transfection reagents as GeneTransfer, which have already been utilized in a clinical trial for a nonviral vector to deliver the interferon-β gene for the treatment of brain tumor in Japan [40] . Therefore, we used AF-liposomes composed of these lipids in the present study.
The most important finding found in the present study is that γ-CyD enhances transfection efficiency of pDNA/AFliposomes in HepG2 cells (Figures 3-5 ) with negligible cytotoxicity (Figure 8 ). The enhancing mechanisms of γ-CyD presumed are discussed as follows.
In the present study, we revealed that transfection efficiency of the pDNA/γ-CyD/AF-liposomes, not N-liposomes, was inhibited by the addition of AF in HepG2 cells ( Figure 6 ). Meanwhile, in NIH3T3 cells, transfection efficiency of the pDNA/AF-liposomes was not suppressed by the addition of AF. These results strongly suggest that pDNA/γ-CyD/AFliposomes can be entered HepG2 cells through ASGP-Rmediated endocytosis, consistent with Aramaki and his colleague's report [41] , and the enhancing effect of the γ-CyD may by associated with the ASGP-R-mediated endocytosis. However, γ-CyD also enhanced gene transfer activity of pDNA/AF-liposomes even in A549 cells and NIH3T3 cells, ASGP-R negative cells (Figure 7) . These results suggest that the enhancing effect of γ-CyD on transfection efficiency of pDNA/AF-liposomes is in an ASGP-R-independent manner. The charge ratio of AF-liposomes/pDNA was 1.6. γ-CyDs were added to AF-liposomes suspension before freeze-drying. The concentrations of γ-CyDs were 1 μM/μM lipids. Cells were incubated with Alexa-pDNA/γ-CyDs/RH-AF-liposomes for 3 h in FCS-free medium. After washing twice, the cells were observed using a confocal laser scanning microscopy.
The particle sizes and encapsulation ratio of pDNA/γ-CyD/AF-liposomes should be involved in the enhancing effect of γ-CyD on gene transfer activity of AFliposomes. The particle size of pDNA/γ-CyD/AF-liposomes was decreased in the presence of γ-CyD, although the ζpotential value of pDNA/γ-CyD/AF-liposomes was almost equivalent to that of pDNA/AF-liposomes and pDNA/HP-γ-CyD/AF-liposomes ( Table 2 ), suggesting that γ-CyD inhibits the aggregation of pDNA/AF-liposomes, because the particle size shows more than 200 nm, despite the fact that the liposomes were extruded through a filter membrane having a pore size of 200 nm. Meanwhile, the encapsulation ratio of pDNA was significantly increased by adding γ-CyD to pDNA/AF-liposomes (Table 3) . Thus, these lines of evidence speculate that addition of γ-CyD enhances cellular uptake of pDNA/AF-liposomes. In fact, the CLSM study demonstrated that cellular uptake of pDNA/γ-CyD/AF-liposomes was higher than that of pDNA/γ-CyD/AF-liposomes ( Figure 11 ). Furthermore, we confirmed that transfection efficiency of pDNA/γ-CyD/AF-liposomes was increased, as the encapsulation ratio of pDNA into pDNA/γ-CyD/AF-liposomes was augmented (Table 4 ). In view of the findings, the particle size of pDNA/γ-CyD/AF-liposomes and encapsulation ratio of pDNA into pDNA/γ-CyD/AF-liposomes are crucial role for enhancing transfection efficiency of pDNA/AF-liposomes.
The important question regarding the enhancing effect of γ-CyD on transfection efficiency of pDNA/AF-liposomes still remains, because three types of HP-CyDs did not have the enhancing effect. To address this question, the DSC analysis was performed. This study indicated that γ-CyD, but not HP-γ-CyD, changed membrane fluidity and stabilized the N-liposomal membranes (Figure 9 ). In fact, γ-CyD increased the Tc value of DLPC-liposomes, although HPγ-CyD did not increase anymore ( Figure 10 ). This increase in the Tc value induced by γ-CyD could be attributed to a compactness of lipid bilayer of DLPC liposomes. It is thereby possible that γ-CyD may increase Tc values of the other liposomes such as DMPC, DPPC, and DSPC. Here, it is well known that γ-CyD is highly hydrophilic and surface inactive [17] . Therefore, we presumed that γ-CyD encapsulates the aqueous compartment of liposomes rather than in the bilayer of liposomes. Anyhow, it is clear that the magnification of the interaction of liposomal membranes containing DLPC with HP-γ-CyD is weaker than that with γ-CyD, possibly due to the steric hindrance of the HP group in a HP-γ-CyD molecule. Taken together, it is likely that the stabilizing effects of γ-CyD on AF-liposomal membrane may lead to inhibition of pDNA leakage from AF-liposomes and increase in cellular uptake of pDNA, eventually leading to the enhancement of in vitro transfection efficiency of pDNA/γ-CyD/AF-liposomes in cells.
Finally, we investigated the role of free γ-CyD on transfection efficiency of pDNA/γ-CyD/AF-liposomes in HepG2 cells. The physical mixture of pDNA/AF-liposomes and γ-CyD in culture medium had no enhancing effect on transfection efficiency of pDNA/AF-liposomes (data not shown). Therefore, encapsulation of γ-CyD into AF-liposomes may be pivotal for enhancing gene transfer activity. To reveal the detailed mechanism for the enhancing effect of γ-CyD associated in AF-liposomes on transfection efficiency of pDNA/AF-liposomes, further elaborate study should be necessary.
In the present study, we demonstrated that γ-CyD enhanced gene transfer activity of pDNA/AF-liposomes in not only HepG2 cells but also A549 and NIH3T3 cells, probably due to various effects of γ-CyD on AF-liposomes such as inhibition of aggregation of the liposomes, high encapsulation of pDNA into the liposomes, and stabilization of lipid bilayer of the liposomes. Consequently, the potential use of γ-CyD could be expected as an enhancer of gene transfer activity of AFliposomes. Also, these data may be useful for design of cellspecific cationic liposomes as a nonviral vector. Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation. Establishment and maintenance of cell polarity is chiefly orchestrated by a tightly regulated interplay between three multi-protein complexes: i) Scribble (SCRIB)/Discs Large (Dlgh1)/Lethal giant larvae (Lgl) complex, ii) partitioning-defective (PAR) 3 and PAR6/ atypical protein kinase C (aPKC) complex, and iii) Crumbs (CRB)/ Protein Associated with Lin Seven 1 (PALS1)/ PALS1-associated tight junction protein (PATJ) complex [1, 2] . However, each complex is not exclusive, as PAR6 links PALS1 to PAR3/PAR6/aPKC [3] . In T lymphocytes, cell polarity proteins were shown to partition the leading edge from the uropod at the cell rear, and therefore participate to cell migration, homing, and scanning [4, 5, 6] . In addition, SCRIB and Dlgh1 are transiently recruited to the nascent immunological synapse formed with an antigen-presenting-cell (APC) [4] . Their depletion in lymphocytes has been associated with an alteration of antigen receptor-mediated activation [7, 8, 9, 10] .
The adaptor PALS1 is crucial for cellular architecture as it maintains the apico-basal polarity in epithelial cells and authorizes indirect interactions between CRB and PATJ [11, 12] . Interestingly, Dlgh1 and PALS1 share a COOH-terminal part composed of a PSD-95/Dlg/ZO-1 (PDZ) domain followed by an SH3 domain adjacent to an inactive Guanylate kinase (GK) homology region [2] . This unique sequence of PDZ/SH3/GK defines the so-called membrane-associated guanylate kinase (MAGUK) proteins family, a group of molecules that serve as scaffolds to organize multi-protein signalosomes through their protein-protein interaction domains [13] . For example, the MAGUK-containing CARMA1 emerges as a central regulator of lymphocytes activation and proliferation downstream of antigen receptor stimulation [14] . Indeed, CARMA1 operates as scaffold to recruit the heterodimer BCL10/ MALT1 (CBM complex), a key step for conveying NF-kB signaling [14, 15, 16] . In addition to its established role in polarity, Dlgh1 was shown to modulate lymphocyte proliferation upon T-cell receptor ligation, possibly through p38 recruitment or via the transcription factor NF-AT [9, 10, 17, 18] .
Although the MAGUK PALS1 plays a central role in the establishment of cell polarity, its contribution to lymphocyte activation remains elusive [8] . Here we show that PALS1 mRNA and protein is expressed in human lymphocytes. Furthermore, knocking down of PALS1 with small interfering RNAs (siRNAs) led to a decreased proliferation of human T lymphocytes, resulting from a reduced activation of the transcription factor NF-kB.
Although several cell polarity proteins have been characterized in lymphocytes [4, 5] , PALS1 expression in T cells remains to be determined [8] . To address this question, we first performed RT-PCR analysis on resting human CD3 + T cells and Jurkat lymphocytes extracts, and detected mRNA for PALS1 ( Figure 1A ). These mRNA were efficiently translated into protein, as antibodies against PALS1 detected a band, which was absent from PALS1-siRNA transfected primary T lymphocytes lysates ( Figure 1B ). Similar results were obtained with Jurkat T cells ( Figure 1B ). Of note, PALS1 levels remained unchanged in cells stimulated with antibodies to CD3 and CD28, or with PMA and ionomycin ( Figure 1C ). We next investigated PALS1 subcellular location by confocal microscopy. In contrast to epithelial cells where it accumulate to tight junctions [12] , PALS1 did not reach membrane domains and remains essentially cytosolic with punctuate structures. Additional staining revealed that these structures coalesced with the Golgi apparatus ( Figure 1D and Figure S1 ). Accordingly, Brefeldin A-triggered disassembly of the Golgi apparatus also disrupted PALS1 punctuate structures ( Figure 1D ). This is reminiscent of PALS1 relocation to the Golgi apparatus in cells infected with SARS coronovirus [19] . Last, we observed that TCR-mediated stimulation only promoted a discrete redistribution of PALS1 within the cytosol of Jurkat cells ( Figure S1 ). Altogether, our results suggest that similarly to Dlgh1, SCRIB, CRB3, and PKCf [4, 5] , the cell polarity protein PALS1 is expressed in lymphocytes at both mRNA and protein level.
Because SCRIB and Dlgh1 were proposed to modulate lymphocyte proliferation [7, 8, 9, 10] , we evaluated whether PALS1 might also participate to T cell activation. To this end, peripheral blood lymphocytes (PBL) were purified on Ficoll-isopaque gradients. Primary human T cells were nucleofected for three days with siRNA targeting PALS1, prior stimulation with anti-CD3 and anti-CD28 antibodies. PALS1 knockdown led to a significant decrease in TCR-mediated induction of the activation markers CD69 and CD25 on cell surface (Figure 2A , B). This was accompanied by a reduction in Carboxyfluorescein Succinimydyl Ester (CFSE) dilution, which reflects cell proliferation ( Figure 2C ). Collectively, these data suggest that PALS1 participates to the optimal lymphocyte activation and subsequent proliferation upon TCR stimulation.
To further explore how PALS1 impacts on lymphocyte proliferation, early signaling pathways emanating from the TCR were examined in Jurkat cells transfected with PALS1 siRNA. We did not detect major alteration in the general pattern of tyrosine phosphorylation, or mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinases (ERK) 1/2 phosphorylation upon TCR stimulation ( Figure 3A ). Only a slight but consistent increase in TCR-mediated phosphorylation of p38 was noted ( Figure 3A ). Moreover, CD3-induced calcium mobilization was largely normal in PALS1-knockdown Jurkat cells ( Figure 3B ).
We next analyzed TCR-mediated activation of NF-AT and NF-kB transcription factors. siRNA-treated Jurkat T cells were cotransfected with firefly luciferase constructs driven by NF-AT or NF-kB binding sequences and with a renilla luciferase control. PALS1 knockdown had only a marginal effect on NF-AT activity following stimulation with PMA and ionomycin, or with antibodies to CD3 and CD28 ( Figure 3C and Figure S2 ). In sharp contrast, NF-kB activity was significantly reduced without PALS1 ( Figure 3D ). Interestingly, tumor necrosis factor-a (TNFa)induced NF-kB activation remained essentially unaffected, underscoring the selective involvement of PALS1 in the TCR-NF-kB pathway ( Figure S3 ). Altogether, our data unveiled an unexpected role for PALS1 in TCR-mediated NF-kB activation.
To gain insights on how PALS1 modulate NF-kB, we first investigated the transcription factor binding ability by electrophoretic mobility shift assay (EMSA). Less NF-kB bound to its specific probe in nuclei extracts from PALS1-siRNA transfected cells following TCR stimulation ( Figure 4A ). As expected, Oct-1 binding remained unchanged without PALS1. Consistent with a diminished NF-kB activity, both the phosphorylation and subsequent proteasomal degradation of NF-kB inhibitor, IkBa, were severely decreased in the absence of PALS1 ( Figure 4B ). Because TCR-induced NF-kB activation relies on the assembly of the CBM complex [15] , BCL10 was immunoprecipitated from nonspecific (NS-) and PALS1-siRNA transfected Jurkat cells. MALT1, which forms an heterodimer with BCL10, coprecipitated with BCL10 regardless of stimulation. Although PALS1 was not found bound to BCL10, its absence diminished CARMA1 recruitment ( Figure 4C , and data not shown). Hence, our data suggest that PALS1 participates to the optimal translocation and activation of NF-kB upon TCR stimulation, possibly by favoring the CBM assembly.
Since PALS1 nucleates a ternary complex containing CRB3 and PATJ, and further binds PAR6 to maintain cell polarity [3, 20, 21] , their contribution to TCR-mediated NF-kB was evaluated. Similarly to PALS1, mRNA for PATJ, CRB3, PAR6, were efficiently detected by RT-PCR ( Figure 5A ). The same hold true for the unrelated cell polarity protein SCRIB ( Figure 5A ). siRNA-based knockdown of PALS1 and CRB3 significantly decreased NF-kB activation in cells treated by antibodies against CD3 and CD28, or with a mixture of PMA and ionomycin. Although less dramatic, similar results were observed with PAR6 or PATJ knockdown. By contrast, NF-kB was normally activated in the absence of SCRIB ( Figure 5B ). In agreement, IkBa phosphorylation was diminished in lysates from CRB3-depleted cells, and to a lesser extent from PATJ-or PAR6-siRNA transfected cells, and not from SCRIB-depleted cells. Again, ERK phosphorylation occurred normally ( Figure 5C , D, E, and F). Altogether, our data suggest that PALS1 implication in the TCR-NF-kB pathway is inextricably linked to its cell polarity partners.
In summary, our data show that the cell polarity protein PALS1 is expressed in lymphocytes and contributes to their optimal activation. Although Dlgh1 and SCRIB were proposed to modulate NF-AT or p38 [9, 10, 17, 18] and NF-AT [7] respectively, a distinct scenario likely occurs for PALS1. Our results support a model in which PALS1 participates to NF-kB activation, upstream of IkBa phosphorylation and degradation. However, how precisely PALS1 modulates NF-kB remains unclear. Because MAGUK function as scaffold units to organize and integrate multi-molecular signaling complexes [13] , it is tempting to speculate that PALS1 nucleates its own signalosome. For example, CARMA1 anchors a .900 kDa complex including the heterodimer BCL10/MALT1 [22] , and Dlgh1 was reported to bind to Lck, Zap70, Wasp [17] , and p38 [10] . In our hands, PALS1 did not integrate the CBM, but its absence reduced CARMA1 binding to BCL10. It will therefore be interesting to identify PALS1 partners in the context of lymphocyte activation. In line with this, CRB3, PATJ and PAR6, which all bound PALS1 to maintain cell polarity [2] , also participate to NF-kB signaling upon TCR ligation in lymphocytes, and might therefore complex with PALS1 in lymphocytes. Altogether, our results strengthen the unexpected function of cell polarity proteins in lymphocyte proliferation [7, 8, 9, 10] , and unveil an original role for PALS1 during TCR-mediated NF-kB activation.
Jurkat T cells E6.1 were purchased from ATCC. CD3 + human T lymphocytes from healthy donors (Etablissement Francais du Sang) were isolated with the MidiMacs system (Miltenyi Biotec). Cells were activated with a mixture of soluble anti-CD3e (HIT3a, BD Biosciences) and anti-CD28 (BD Biosciences), or with 20-40 ng.ml 21 phorbol 12-myristate 13-acetate (PMA, Sigma) and 300 ng.ml 21 ionomycin (Calbiochem). Carboxyfluorescein Succinimydyl Ester (CFSE) and Brefeldin A were purchased from Sigma, and the calcium-sensitive dye Fluo-4 was from Invitrogen.
Cells were washed twice with PBS 1X and lysed with 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Igepal, 2 mM EDTA, supplemented with complete protease inhibitors (Roche). Lysates were cleared by a centrifugation at 10,000g at 4 o C, and protein concentration determined (micro BCA kit, Pierce). Samples were resolved on 5-20% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham). For Immunoprecipitations, samples were precleared with protein G-sepharose beads (Roche) for 30 min prior to overnight incubation with antibodies and additional protein G-sepharose beads at 4uC, as previously described [23] . Antibodies to BCL10 (A-6), IkBa (C-21), MALT1 (B-12), Tubulin (TU-02), PALS1 (H-250), SCRIB (C-6), PAR6 (G-9) and p65 (C-20) were purchased from Santa Cruz. Phosphospecific antibodies against IkBa, ERK, p38, and antibodies to CARMA1 and to ERK were from Cell Signaling Technologies. Anti-phosphorylated Tyrosine (4G10, Millipore), anti-GAPDH (Sigma), and Immobilon (Millipore) chemiluminescent substrates were also used.
Firefly luciferase constructs downstream of promoters for NF-kB or NF-AT were co-transfected with renilla luciferase pRL-TK (Int -) plasmid (Promega). Luciferase activities were analyzed using the Dual-Luciferase Kit (Promega), with firefly fluorescence units normalized to renilla luciferase fluorescence units (BMG microplate reader). were purified from blood on Ficoll-isopaque gradients. PBL were nucleofected with the Nucleofactor system and T cell solution (Amaxa, program U14), and left for three days in culture medium prior treatment.
Nuclear protein extraction and electrophoretic mobility shift assay (EMSA) 4 mg of nuclear extracts from Jurkat cells were examined for NF-kB-and Oct1-binding activity by electromobility shift assay (Panomics kit). Samples were resolved on a 6% native polyacrylamide DNA retardation gel in 0.5X TBE buffer and analyzed using a FUJI LA4000 system. 
Cells were left for 10 min on poly-lysine coated slides (Thermo Scientific) prior fixation with PBS1X containing 4% paraformaldehyde. For TCR crosslinking experiments, cells were incubated with 5 mg.ml 21 anti-CD3 at 4uC for 15 min. After two washes, cells were incubated with 5 mg.ml 21 of goat antimouse (Jackson) for 20 min either at 4uC or 37uC. To disassemble Golgi apparatus, cells were treated with 10 mg.ml 21 Brefeldin A for 60 min. Samples were permeabilized with 0.05% Triton-X100 in PBS1X for 5 min, and non-specific sites blocked with 10% FCS in PBS1X. Antibodies used were: PALS1 (Millipore), 58K Golgi (Abcam), Alexa-488 conjugated goat anti-rabbit IgG or Alexa-594 conjugated goat anti-mouse IgG (Invitrogen). Samples were analyzed using a Leica confocal microscope SP6.
Cells were incubated for 30 min at 4uC with FITC-and PEconjugated antibodies against CD25 and CD69 (ImmunoTools) and the respective isotype controls in PBS containing 0.5% BSA. After one wash with ice-cold PBS-BSA, cells were analyzed by flow cytometry with a FACSCalibur (BD Biosciences).  A Canadian Critical Care Trials Group project in collaboration with the international forum for acute care trialists - Collaborative H1N1 Adjuvant Treatment pilot trial (CHAT): study protocol and design of a randomized controlled trial BACKGROUND: Swine origin influenza A/H1N1 infection (H1N1) emerged in early 2009 and rapidly spread to humans. For most infected individuals, symptoms were mild and self-limited; however, a small number developed a more severe clinical syndrome characterized by profound respiratory failure with hospital mortality ranging from 10 to 30%. While supportive care and neuraminidase inhibitors are the main treatment for influenza, data from observational and interventional studies suggest that the course of influenza can be favorably influenced by agents not classically considered as influenza treatments. Multiple observational studies have suggested that HMGCoA reductase inhibitors (statins) can exert a class effect in attenuating inflammation. The Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial sought to investigate the feasibility of conducting a trial during a global pandemic in critically ill patients with H1N1 with the goal of informing the design of a larger trial powered to determine impact of statins on important outcomes. METHODS/DESIGN: A multi-national, pilot randomized controlled trial (RCT) of once daily enteral rosuvastatin versus matched placebo administered for 14 days for the treatment of critically ill patients with suspected, probable or confirmed H1N1 infection. We propose to randomize 80 critically ill adults with a moderate to high index of suspicion for H1N1 infection who require mechanical ventilation and have received antiviral therapy for ≤ 72 hours. Site investigators, research coordinators and clinical pharmacists will be blinded to treatment assignment. Only research pharmacy staff will be aware of treatment assignment. We propose several approaches to informed consent including a priori consent from the substitute decision maker (SDM), waived and deferred consent. The primary outcome of the CHAT trial is the proportion of eligible patients enrolled in the study. Secondary outcomes will evaluate adherence to medication administration regimens, the proportion of primary and secondary endpoints collected, the number of patients receiving open-label statins, consent withdrawals and the effect of approved consent models on recruitment rates. DISCUSSION: Several aspects of study design including the need to include central randomization, preserve allocation concealment, ensure study blinding compare to a matched placebo and the use novel consent models pose challenges to investigators conducting pandemic research. Moreover, study implementation requires that trial design be pragmatic and initiated in a short time period amidst uncertainty regarding the scope and duration of the pandemic. TRIAL REGISTRATION NUMBER: ISRCTN45190901 Background: Swine origin influenza A/H1N1 infection (H1N1) emerged in early 2009 and rapidly spread to humans. For most infected individuals, symptoms were mild and self-limited; however, a small number developed a more severe clinical syndrome characterized by profound respiratory failure with hospital mortality ranging from 10 to 30%. While supportive care and neuraminidase inhibitors are the main treatment for influenza, data from observational and interventional studies suggest that the course of influenza can be favorably influenced by agents not classically considered as influenza treatments. Multiple observational studies have suggested that HMGCoA reductase inhibitors (statins) can exert a class effect in attenuating inflammation. The Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial sought to investigate the feasibility of conducting a trial during a global pandemic in critically ill patients with H1N1 with the goal of informing the design of a larger trial powered to determine impact of statins on important outcomes.
Methods/Design: A multi-national, pilot randomized controlled trial (RCT) of once daily enteral rosuvastatin versus matched placebo administered for 14 days for the treatment of critically ill patients with suspected, probable or confirmed H1N1 infection. We propose to randomize 80 critically ill adults with a moderate to high index of suspicion for H1N1 infection who require mechanical ventilation and have received antiviral therapy for ≤ 72 hours. Site investigators, research coordinators and clinical pharmacists will be blinded to treatment assignment. Only research pharmacy staff will be aware of treatment assignment. We propose several approaches to informed consent including a priori consent from the substitute decision maker (SDM), waived and deferred consent. The primary outcome of the CHAT trial is the proportion of eligible patients enrolled in the study. Secondary outcomes will evaluate adherence to medication administration regimens, the proportion of primary and secondary endpoints collected, the number of patients receiving open-label statins, consent withdrawals and the effect of approved consent models on recruitment rates.
Discussion: Several aspects of study design including the need to include central randomization, preserve allocation concealment, ensure study blinding compare to a matched placebo and the use novel consent models pose challenges to investigators conducting pandemic research. Moreover, study implementation requires that trial design be pragmatic and initiated in a short time period amidst uncertainty regarding the scope and duration of the pandemic. Trial Registration Number: ISRCTN45190901 Background Influenza and Swine Origin Influenza A/H1N1 Infection (H1N1)
On June 11, 2009 , the World Health Organization (WHO) declared that infection with the Swine Origin Influenza A/H1N1 virus had reached pandemic proportions [1] . Cases were recorded in more than 180 countries and outbreaks that strained national resource capacities were documented in Canada, Australia, Chile, Argentina, and elsewhere.
Throughout history, pandemic influenza has posed a recurrent threat to human populations. Seasonal influenza is responsible for more than 50,000 deaths per year in the United States [2] . The capacity of the influenza virus to mutate and spread from animals to humans has resulted in intermittent pandemics. The 1918 pandemic was the largest in recent history and caused between 40 and 50 million deaths worldwide [3] . Smaller pandemics in 1957 and 1968 were associated with mortality spikes but their effects were mild at the population level [4] . Experts believe further pandemics will certainly occur, but are uncertain about when. Several years ago, the avian H5N1 influenza virus threatened to be the vector of the next pandemic. While highly virulent when transmitted from infected chickens to humans, the absence of human-to-human transmission resulted in a small number of cases worldwide [5] .
In early 2009, a novel strain of influenza, swine origin influenza A/H1N1 infection (H1N1) emerged in swine and rapidly spread to humans [6] . Originating in Mexico, the strain proved highly infectious and was spread by person-to-person contact with a predilection for younger hosts. While early epidemiologic data suggested that although H1N1 was highly infectious, it was less virulent [7, 8] than anticipated with a case fatality rate of approximately 0.5% of infected individuals. For the majority of infected individuals, symptoms were mild and selflimited; however, a small percentage of infected individuals developed profound respiratory failure requiring extraordinary means of oxygenation support including high frequency oscillation (HFO) ventilation and extracorporeal membrane oxygenation (ECMO) [9] . Caring for the most severely ill patients during a pandemic results in an increased need for intensive care unit (ICU) resources and strains available personnel and equipment. There is little excess capacity to care for critically ill patients in most developed countries, and minimal capacity in developing countries.
Supportive care and antiviral agents, especially neuraminidase inhibitors (such as oseltamivir and zanamivir), are the mainstay of treatment for influenza. While efficacious in reducing viral load and abating symptoms in ambulatory patients, their effects on outcomes in critically ill patients have not been established. Their utility in treating severe pandemic H1N1 influenza may be compromised by widespread use and emergence of viral resistance [10, 11] , limited supplies, policies regarding treatment strategies, and cost and availability, especially in developing countries [12] . Human and animal data suggest that the course of influenza may be favorably influenced by certain agents not classically considered as treatments for influenza [13] [14] [15] [16] that are comparatively inexpensive and readily available. Such agents may provide independent benefit in treating viral infection and are attractive as adjuvant treatments.
Statins lower plasma lipid levels by inhibiting HMG CoA reductase, the enzyme responsible for converting HMG CoA to mevalonate, a rate-limiting step in cholesterol biosynthesis. Multiple observational studies have suggested that statins may be of benefit in patients with a variety of severe infections [17] [18] [19] [20] [21] [22] by exerting an effect in attenuating inflammation [23, 24] . The mechanism of this activity is uncertain but may involve their ability to restrict cholesterol availability in cell membranes of the innate immune system. Cholesterol is a key component of lipid rafts, membrane-associated microdomains that support cell signaling in response to exogenous inflammatory stimuli. Raft disruption may attenuate the cellular response to inflammatory stimuli [25, 26] .
Experimental studies show that pre-treatment with statins attenuates the severity of acute lung injury (ALI) following intestinal ischemia-reperfusion [27] and the inflammatory response to intravenous lipopolysaccharide challenge in human volunteers [28] . The combination of a statin and caffeine inhibits viral replication and attenuates lung injury in murine influenza models [29] . Population-based studies suggest that statins are associated with reduced inflammatory morbidity in critically ill patients receiving them [20] and reduced mortality in patients with influenza and chronic obstructive pulmonary disease (COPD) patients [30] . Reviewing 3,921 hospitalized patients with laboratory confirmed influenza, of whom 1,019 were receiving a statin at the time of admission, Vandermeer and colleagues found that statin use was independently associated with a reduced risk of death (adjusted OR = 0.34, 95% CI: 0.16-0.70) in a multivariable logistic regression model [31] . The benefits of statins appear to be best established in patients receiving them prior to the onset of infection. Cohort studies report conflicting results on the ability of statins to attenuate organ dysfunction with Schmidt and colleagues finding that statins reduce mortality in critically ill patients with multiple organ dysfunction syndrome [32] and Kor et al reporting that statin use was not beneficial in resolving organ dysfunction [33] .
Statins are widely used and well-tolerated medications. Rosuvastatin differs from other HMG CoA reductase inhibitors in that only up to 10% of the parent compound is metabolized by cytochrome P450 2C9 and 2C19 and not by P450 3A4 [34] . As a result, it exhibits fewer drug-drug interactions, and serum concentrations are not affected by CYP2D6 gene polymorphism. Adverse events seen with rosuvastatin are generally mild and may include muscle symptoms, however, myopathy and rhabdomyolysis occur infrequently in patients taking 40 mg/day or less. As with other HMG-CoA reductase inhibitors, a dose-related increase in liver transaminases and creatinine kinase (CK) has been observed in small numbers of patients taking rosuvastatin. The overall occurrence of clinically significant transaminase increases is low (< 1%) and similar across rosuvastatin doses ranging from 5 mg/day to 40 mg/day [35] . Patients with severe liver disease may have increased exposure to rosuvastatin [35] .
The International Forum of Acute Care Trialists (InFACT) is an informal alliance of investigator-led clinical trials networks whose remit is to improve the care of critically ill patients through the promotion of scientifically rigorous clinical research. Based on our preliminary understanding of H1N1 influenza, members of the Canadian Critical Care Trials Group (CCCTG), working in collaboration with members of InFACT, designed the Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial to investigate the feasibility of conducting an international therapeutic trial during a global pandemic and the potential for adjuvant rosuvastatin, in addition to standard treatment, to influence clinical outcomes in influenza A (H1N1) associated critical illness.
A multi-national feasibility RCT involving adult ICUs in Canada, Saudi Arabia, Mexico, Argentina Australia/ New Zealand. An overview of the study design is provided in Figure 1 (see Figure 1 ).
[1] The ability to recruit the desired patient population under pandemic conditions (i.e., the proportion of eligible patients enrolled in the CHAT Pilot Trial).
[2] Adherence to the medication administration regimen as outlined in the study protocol.
[3] The ability to collect the required primary and secondary endpoints for the planned full CHAT trial.
[4] The number of patients who receive open-label statins.
[5] The number of consent withdrawals.
[6] The impact of approved consent models on recruitment rates.
A dedicated research coordinator will screen patients for eligibility on a daily basis in the ICUs at participating sites. If the study inclusion criteria are fulfilled and no exclusion criteria are present, the research coordinator will identify the patient as a potential study participant. The attending physician or intensivist will confirm eligibility for participation in the CHAT Pilot RCT.
Criteria to identify potential candidates for study inclusion will include: 1) Critically ill adult patients ≥ 16 years of age admitted to an adult ICU for any reason with suspected, probable or confirmed novel swine origin influenza A/H1N1 infection (see Additional File 1). 2) Requiring mechanical ventilation (invasive or noninvasive) 3) Receiving antiviral therapy (any medication at any dose and for any intended duration) for ≤ 72 hours 4) Attending physician or intensivist must have a 'moderate' to 'high' index of suspicion for H1N1. 3) Weight < 40 kg 4) Unable to receive or unlikely to absorb enteral study drug (e.g., incomplete or complete bowel obstruction, intestinal ischemia, infarction, short bowel syndrome) 5) Rosuvastatin specific exclusions: a. Already receiving a statin b. Allergy or intolerance to statins. c. Receiving niacin, fenofibrate, cyclosporine, gemfibrozil, any protease inhibitor (including but not limited to lopinavir and ritonavir) or planned use of oral contraceptives or estrogen therapy during the ICU stay. d. CK exceeds 5,000 U/L or ALT exceeds 8 times the upper limit of normal (ULN). 6) Severe chronic liver disease (Child-Pugh Score 11-15) (see Additional File 2) 7) Previous enrolment in this trial 8) Pregnancy or breast feeding 9) At the time of enrolment, receipt of > 72 hours of antiviral therapy. 10) Known or suspected clinically significant myositis or myopathy.
We will record reasons why eligible patients are not randomized into the CHAT Pilot Trial under the following categories: a) Substitute Decision Maker (SDM) consent refusal b) Physician refusal of consent c) SDM not available to provide consent and waived/ deferred consent not permitted d) SDM does not exist and waived/deferred consent not permitted e) Coordinator workload f) Coordinator not available during eligible timewindow g) Enrolment in a competing trial h) Other (specification required)
To enhance trial feasibility and given the need to conduct a pragmatic trial, co-enrolment in other prospective observational studies or RCTs (not investigating similar or alternative H1N1 treatments) in operation in the ICU setting will be permitted and recorded in sites where permitted by the local Research Ethics Boards (REBs) [36] .
To preserve allocation concealment, participants will be randomized centrally. Randomization lists will be distributed by the study methods centre to the research pharmacies of participating centres. Stratified variable block randomization, based on centre alone, will be performed to take into consideration differences in patient characteristics at participating ICUs. Day one will be considered the day of study treatment initiation, which may or may not be the same day of randomization.
A waiver of consent is the preferred option for participant enrollment given the context of a global pandemic. We have constructed a consent algorithm to direct the consent process at sites where the local REB has not approved a waiver of consent (see Additional File 3). In this trial, we will request that patients be enrolled in the study, and consent be deferred to SDMs or to the patient (whomever is able to provide consent first), when it is not possible to obtain consent within 24 hours. Consent may be obtained in person or by telephone as per local practices. In the event that patients die before providing consent, we request permission from REBs to include data collected during study participation.
Using randomization lists provided by the study methods centre, research pharmacists will assign critically ill adults to once daily enteral administration of rosuvastatin or matched placebo for 14 days. Only the research pharmacy staff will be aware of the assigned treatment arm. The site investigators, research coordinator, clinical pharmacist involved in the care of the patient and all other study personnel will remain blinded to treatment assignment. An oral placebo for nasogastric administration, identical in appearance (colour and consistency matched) to crushed rosuvastatin, will be prepared by Pharmacy 1 (Toronto, Canada) and supplied to the study sites. All other aspects of patient management will be left to clinician discretion as per pragmatic trial design. The Applied Health Research Centre of the Keenan Research Centre and Li Ka Shing Knowledge Institute (St Michael's Hospital, Toronto, Ontario) will be the Study Methods Centre.
Study drug will be administered once daily through an enteral feeding tube or orally if the patient is able to safely take oral medications. The type and placement of the enteral feeding tube (nasogastric, nasoenteric, percutaneous endoscopic gastrostomy, orogastric, oroenteric, etc.) will be at the discretion of the attending clinical team. The ability to safely take oral medications will be determined by the patient's primary care team.
The first study drug dose (rosuvastatin or placebo) will be administered within 4 hours of randomization as a loading dose of 40 mg unless the subject is of Asian (Chinese, Korean, Japanese, Phillipino, Vietnamese, or Asian-Indian) descent, age <18 years or has serum creatinine greater than or equal to 248 umol/L (2.8 mg/dL) (see requirements for dose adjustments below).
Thereafter doses of 20 mg will be administered at 22:00 hrs daily (+/-4 hours) starting on the next calendar day (study day 2) as a maintenance dose. If the patient is of Asian descent, is <18 years, or serum creatinine is greater than or equal to 248 umol/L (2.8 mg/dL) dose adjustments will be required according to the dose adjustment algorithm (see requirements for dose adjustments below). Dose adjustment is only necessary for patients with renal impairment and not receiving dialysis. Once dialysis is started and serum creatinine remains elevated, dose adjustment is not required.
If for any reason a maintenance dose is not administered at the intended time, it may be administered subsequently but not more than 12 hours after the intended time of administration. If greater than 12 hours has elapsed since the last scheduled dose, the patient will receive another loading dose, and then maintenance dosing will resume on the next calendar day as outlined above. A missed dose, for reasons other than outlined under medication discontinuation will be considered a protocol violation.
The loading (40 mg) and daily maintenance (20 mg) doses will be reduced by 50% for patients: a) who have at least one parent of Asian descent (loading 20 mg and daily 10 mg), b) whose age < 18 years (loading 20 mg and daily 10 mg), c) whose serum creatinine concentration is greater than or equal to 248 umol/L (2.8 mg/dL) who are not on renal replacement therapy (loading 20 mg and daily 10 mg), and by 75% for patients: d) who have at least one parent of Asian descent and/or age < 18 years and serum creatinine is greater than or equal to 248 umol/L (2.8 mg/dL) (loading 10 mg and daily 5 mg).
Intermittent oral antacids should be administered no closer than 6 hours before or after administering rosuvastatin to avoid influencing study drug absorption [37] Duration of Treatment Given the substantial potential for false negative influenza results, we will continue adjuvant treatment administration, regardless of H1N1 testing results (positive or negative), for 14 days or until a criterion for cessation is met. If patients are liberated from mechanical ventilation (invasive or non-invasive) and discharged from the ICU between days 1 and 9 they will be advanced to day 10 of study drug administration. In the event that patients remain in the ICU for observation (e.g., possible reintubation or initiation of non-invasive ventilation) then study drug administration will NOT be advanced to day 10 until they are discharged.
Study drug administration will be stopped when one of the following conditions is met, whichever comes first:
1. 14 days after randomization 2. Hospital discharge (including transfer to an alternate care facility) 3. Death 4. CK noted to exceed 5,000 U/L (in the absence of an alternative diagnosis) or patient is determined to have clinical myositis or myopathy (at the discretion of the primary care team, patient may be re-challenged with study drug if CK or clinical findings no longer meet this criterion). 5. Alanine aminotransferase (ALT) exceeds 8 times the ULN (in the absence of an alternative diagnosis) 6. Co-administration of any of the following: niacin, fenofibrate, cyclosporine, gemfibrozil, lopinavir, ritonavir, oral contraceptives or estrogen 7. Attending physician or intensivist or SDM request to stop treatment.
We will request for data collection to continue in patients withdrawn from therapy prematurely. Clinicians will be encouraged to continue study medication despite negative H1N1 testing due to the potential for false negative results and the potential role for an antiinflammatory agent (rosuvastatin) in severe lung disease. Decisions regarding continuation or discontinuation of antiviral treatment will be left to the discretion of the attending physician or intensivist.
It will not be feasible to protocolize ventilator and general clinical management under pandemic conditions. Adjunctive non-antibiotic, non-interventional management of sepsis, acute respiratory distress syndrome, and glycemic control will be at the discretion of the patient's primary clinicians. Key aspects of clinical management (such as choice of antiviral, dose of administration, duration of treatment, ventilator strategy, treatment with inhaled nitric oxide, HFO, prone positioning, ECMO, additional antiviral/anti-inflammatory treatments and treated episodes of infection) will be documented either as part of the Influenza A H1N1 (Swine Flu) ICU (Registry) Study for participating centres or on separate data forms for centres not participating in the registry. Antibiotic therapy may be prescribed for suspected or confirmed concomitant bacterial infection at the discretion of the attending physician.
Local testing procedures may be used to facilitate diagnosis of H1N1. Where no local testing procedures exist, we recommend using the following initial and repeat testing procedures. For all new admissions to adult ICUs meeting study inclusion criteria and having no exclusion criteria with non-confirmed H1N1 (i.e., all suspected or probable cases) or where uncertainty exists regarding prior testing, we request the following sequence of laboratory tests:
Initial Diagnostic Testing (assuming diagnosis not confirmed at ICU admission)
Repeat Testing (for patients with one positive test)
1. Repeat both tests (PCR and viral culture) from the site that was positive previously (i.e., ET or BAL or NP swab). If both ET and NP swab were positive, send repeat ET aspirate specimen at day 7 and at weekly intervals thereafter.
2. After the first negative specimen(s) from a previously positive site, send a repeat specimen from the same site at 48 hours after the first specimen was collected.
To verify adequate absorption of rosuvastatin, we will draw venous blood for peak and trough plasma rosuvastatin concentrations (total of 2 specimens) on day 7 (+/-1 day). Day 7 will be the preferred day for trough and peak specimen collection. A trough level specimen will be drawn prior to day 7 (+/-1 day) dose. A peak concentration specimen will be drawn 3 to 5 hours after the dose of study drug is administered. A maximum of 80 patients will have blood drawn for drug concentration analysis; however, only patients randomized to rosuvastatin will have their samples analyzed. Samples will be labeled and batched at the site, for shipment to St Michael's Hospital (Toronto, Canada) for analysis after the study is unblinded.
Research staff will assess participants daily for adverse effects for the duration of treatment. CHAT study participants will have daily CK and liver function [aspartate aminotransferase (AST) and ALT] levels collected as part of the study protocol. Study drug will be discontinued if hypersensitivity is suspected (see Criterion 7 Completion of Study Drug Administration).
We will record the number of patients receiving full treatment and reasons for the inability to complete the assigned treatment duration (i.e., death, transfer to an alternate care facility, study withdrawal, etc). The study Data Safety and Monitoring Board (DSMB) will review all patients withdrawn from the study, safety data, and deaths.
All patients will be followed until death or hospital discharge. We will record the vital status of all patients at 90 days and hospital discharge, whichever occurs first. At day 60 patients remaining on the ventilator will be deemed ventilator dependent. Randomized patients will be considered successfully extubated when they remain off positive pressure ventilation (invasive or non-invasive ventilation) for 48 consecutive hours. If patients are re-intubated within 48 hrs following extubation, they will be followed until they achieve one of the aforementioned outcomes. Patients discharged from the ICU and requiring readmission and re-initiation of mechanical ventilation (invasive or noninvasive) will be treated according to usual practice and will not be randomized on a second occasion to this study.
[1] Proportion of eligible patients enrolled in the CHAT pilot study.
[2] Adherence to the medication administration regimen as outlined in the study protocol.
[3] Proportion of completed primary and secondary endpoints for the planned full CHAT trial that are collected.
[4] Number of patients who receive open-label statins.
[5] Number of consent withdrawals.
[6] Recruitment rates by approved consent model.
Estimates are not available to allow precise sample size estimation of the primary outcome for the proposed CHAT pilot RCT. We propose to undertake a pilot study in a convenience sample of 80 patients with suspected, probable or confirmed H1N1 infection to assess trial feasibility.
We will collect CHAT specific data starting at ICU admission using paper-based versions of the electronic data collection forms developed for the Influenza A H1N1 (Swine Flu) ICU (Registry) Study [7] . The forms will document baseline characteristics, enrolment into concurrent influenza research studies, co-morbidities, illness severity (see Additional File 4), vaccination status, co-interventions, feasibility outcomes and clinical outcomes for the planned definitive trial (primary: the proportion of patients successfully weaned from mechanical ventilation in less than 10 days; secondary: impact of rosuvastatin on ICU, 60 and 90 day, and hospital mortality and on ICU free days at day 60). In addition to the data forms developed for the Influenza A H1N1 (Swine Flu) ICU study, we developed 13 additional forms including an (i) eligibility and randomization form, (ii) severity of illness form, (iii) consent form, (iv) drug administration form, (v) H1N1 diagnostic test results form, (vi) laboratory data form, (vii) drug level (serum) specimen collection form, (viii) 60 day and 90 day outcomes form, (ix) comments and end of study investigator sign off, (x) protocol violation form: biochemistry, (xi) protocol violation form: medication administration/discontinuation, (xii) adverse event form, (xiii) serious adverse event form in randomized patients. For centres not participating in the registry, we drafted 10 additional forms to capture necessary demographic, treatment and outcomes information. In addition, we drafted a form to capture demographic data and outcomes on eligible but not randomized patients.
Descriptive statistics will be used to summarize the data. For univariate analyses, we will use the Chi-square test (alternatively, Fisher's exact test when the expected cell size is ≤ 5) and Student's t-test (alternatively, the Mann-Whitney U-test, if normality assumptions are not satisfied) for binary and continuous outcomes, respectively. All analyses will be conducted on an intention-to-treat basis. Feasibility for the pilot study will be assessed by metrics that reflect our capacity to ultimately recruit a representative sample of 1,050 patients in the planned full CHAT trial. We will consider the study to be feasible if we recruit at least 30% (commonly used threshold in ICU studies) of all eligible patients in participating ICUs through careful review of site screening logs. Additionally, we expect that: (i) less than 10% of medication doses will fail to be administered in the absence of meeting one of the medication discontinuation criteria; (ii) less than 5% of data forms will be missing important primary and secondary outcomes data required for the planned full CHAT trial and (iii) no more than 10% of enrolled patients will be withdrawn prematurely due to open label use of statins or withdrawal of consent. We will describe recruitment rates based on approved consent models. Since centres in Australia and New Zealand will be permitted to use Atorvastatin and matching placebo (instead of Rosuvastatin/matching placebo), we propose to conduct the planned primary and secondary analyses (i) using the pooled data (rosuvastatin plus atorvastatin) and (ii) using rosuvastatin (as the predominantly used statin in the CHAT Trial) data alone.
A DSMB will oversee the trial and will consist of 3 individuals with expertise in viral infectious diseases, statistics and clinical critical care of which one will be international. The DSMB will hold a teleconference after either 30 patients have evaluable data or approximately 8 months after study initiation. Should the trial be completed, feasibility data from the pilot study will be analyzed by the DSMB at the end of the study. This information will be conveyed to the Steering Committee. Together the DSMB and Steering Committee will formulate a decision whether to proceed with the full trial. Clinical outcomes will remain blinded by study group assignment with a view to including them in the planned larger trial.
Investigators will evaluate any changes in laboratory values and physical signs and will determine if the change is clinically important and different from what is expected in the course of treatment of critically ill patients requiring mechanical ventilation for suspected, probable or confirmed influenza. If clinically important and unexpected adverse experiences occur, they will be recorded on an adverse event case report form. We will characterize adverse events (see Additional File 5) as expected, serious unexpected and study related or unanticipated.
We considered other factors (see Additional File 6) including patient withdrawals, consent (including telephone consent and waivers of consent) (see Additional Files 7 and 8), eligible non-randomized patients, equitable selection of subjects, justification for including vulnerable subjects, women of childbearing age, justification for excluding pregnant women, trial oversight and the trial data safety and monitoring board (see Additional File 6) in designing the CHAT Trial protocol. The investigators plan to make changes to the larger study protocol based on their experience in implementing the pilot trial. Regardless, we will publish the findings of the CHAT Pilot Trial, either alone or pooled with another trial evaluating the role of statins in a similar population, if recruitment ensues even if the study protocol is modified in important ways following conduct of the pilot trial or the planned larger trial never comes to fruition. Pilot trial data may also be combined with data from the larger trial if the latter trial comes to fruition, study personnel (including the data analyst) remain blinded to treatment assignment and no important modifications are made to the study protocol following the pilot trial.
Global concern arose from the threat of the H1N1 influenza pandemic. Despite the potential virulence of the illness, little is actually known about how severe disease develops or what treatments may confer benefit to critically ill patients. Even less is known about how to conduct clinical research in the setting of an evolving pandemic. Severe H1N1 infection primarily affects young and often previously healthy individuals. Early reports supported that aboriginal populations in Canada and Australia, obese individuals and women, especially pregnant women, appear to have a predilection for severe disease. Unlike the pandemic of 1918, the availability today of antiviral agents, antibiotics for secondary infection, and ICU supportive care interventions holds promise that the majority of patients with severe illness can be saved. The burden of severe H1N1 disease falls prominently on the ICU [8, 38] . Consequently, the opportunity to learn about treatments for severe H1N1 disease and how to conduct pandemic critical care research rests within the ICU community.
Data from observational studies in humans and interventional studies in animals, suggests that the course of influenza may be favorably influenced by relatively inexpensive and readily available agents, such as rosuvastatin, that are not classically considered to be treatments for influenza. These agents are attractive as adjuvant treatments amidst emerging reports of oseltamivir resistance and threatened drug supply shortages. However, the ability to administer and test the efficacy of an adjuvant agent as a treatment for severe H1N1 infection remains to be established.
The CHAT pilot trial is designed to evaluate the feasibility of implementing a randomized controlled trial of adjuvant rosuvastatin for treating severe H1N1 infection under a pandemic. We aim to evaluate whether centres can adhere to the study treatment regimens, collect the required primary and secondary endpoints for the subsequent planned full CHAT trial, and document patients who receive open-label statins and consent withdrawals. We also seek to evaluate the impact of approved consent models on recruitment rates.
Several time-honored aspects of RCT design including the use of central randomization, preservation of allocation concealment, multi-level study blinding, and use of a matching placebo posed challenges to us in designing a pandemic protocol. We contemplated the necessity of including each of these study design features. After careful deliberation, we decided to include central randomization (using lists distributed by the study methods centre to participating centres), preserve multi-level blinding (by involving pharmacies at participating centres) and contract a local pharmaceutical company to prepare crushed drug and matching placebo (in the absence of industry supply of study drug and an available placebo). Recognizing that trial initiation may be delayed and recruitment curtailed if a priori in-person SDM consent was required, we considered use of alternative consent models. A priori in-person SDM consent not only hinges on the existence and availability of SDMs [39] , but also the ability of SDMs to access hospitals during a pandemic.
Strengths of the proposed pilot trial design include the use of central randomization, allocation concealment, multi level blinding, standard criteria for medication discontinuation, and 90 day follow up. To ensure feasibility during a pandemic, we did not protocolize H1N1 testing, ventilator and sedation management or the clinical use of antibacterial agents. By merging study data with an Influenza Registry and capturing data on unique forms for centres not participating in the registry, we will, however, document key aspects of clinical management.
The conduct of an RCT during an evolving pandemic poses unique challenges not encountered during other forms of clinical research [40] . First, it is necessary to initiate studies quickly. The normal time interval from concept to first patient enrolment for a new RCT is typically of the order of two years or more. Second, the scope and duration of the pandemic is unknown and unpredictable. Third, the mitigating effects of large-scale vaccination programs and changes in H1N1 infectivity resulting from virus mutation are unknown. Fourth, the practicalities of conducting clinical research during a pandemic are unknown. For example, research personnel may be seconded to provide clinical care, pharmacists may face challenges in dispensing drug and placebo, REBs may not permit alternative consent models and it may be difficult to obtain consent from patients who may lack decision-making capacity and families, who may be unwell themselves, unable to visit the hospital or requested to stay away during the pandemic. Finally, faced with the clinical imperative of treating gravely ill and previously well, young patients, clinicians may opt to use open label treatment rather than permit enrolment into a blinded RCT. Because we believe that it is important to develop the capacity to initiate RCTs under pandemic conditions and to test study procedures prior to implementing a large scale RCT, we propose to conduct a multi-centre pilot trial to assess the feasibility of our clinical protocol and study procedures. Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. Influenza A virus (IAV) is an enveloped, segmented negativestrand RNA virus infecting a wide variety of birds and mammals. As its first step in infection IAV attaches to host cells by the binding of its major surface protein, the hemagglutinin (HA), to sialic acids, which are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. Where the structural requirements for this interaction have been studied in great detail, much less is known about whether and how the attachment to specific sialylated receptors (e.g. to N-linked glycoproteins, Olinked glycoproteins or gangliosides or even to specific receptors within these groups) affects the subsequent endocytic steps. Obviously, knowledge about the repertoire of endocytic pathways that can successfully be used by IAV will increase our insights into cell and species tropism of IAV. In turn, this will contribute to our understanding of the requirements for the generation of novel viruses with pandemic potential that can arise by exchange of RNA segments between currently circulating human serotypes and an animal virus during occasional co-infection in a human or an animal host.
Clathrin mediated endocytosis (CME) has for long been identified and studied as the major route of IAV cell entry [1, 2] and is, by far, the best characterized endocytic pathway. Evidence obtained from live cell imaging has revealed the de novo formation of clathrin-coated pits at the site of virus attachment [3] and the requirement for the adapter protein epsin 1, but not eps15, in this process [4] . Still, specific transmembrane receptors linking viral entry to epsin 1 or to other adapters have not been identified although a recent study performed in CHO cells indicated the specific requirement for N-linked glycoproteins in IAV entry [5] .
Some recent papers provided indications for the utilization of alternative entry pathways by IAV. Studies in which CME was obstructed by pharmacological or genetic intervention indicated the ability of IAV to enter host cells via alternative endocytic routes [4, 6, 7] . Also live cell imaging revealed the simultaneous availability of entry routes involving non-coated as well as clathrin-coated pits [4] . However, this alternative IAV entry route has not been characterized in any detail and requirements for any specificity in receptor usage apart from the need for the proper sialic acid moiety have not been established.
During the past decades quite a variety of endocytic pathways have been identified in eukaryotic cells [8, 9, 10] . Their occurrence, abundance and mechanistic details appear to vary between cell types, tissues and species and their utilization by viruses as a route of entry makes them an important factor in host and cell-type permissiveness for infection [11, 12] . Besides by CME, different viruses have been shown to enter cells via caveolae, macropinocytosis or other, less well described, routes [11, 12] . Most often, the selection of a specific endocytic route is linked to the utilization of a specific receptor that facilitates traveling via that particular route. Nevertheless, many receptors allow flexibility by their capacity to enter through multiple pathways. For IAV, an additional level of complexity to the dissection of potential entry routes is added by the apparent lack of an IAV-specific protein receptor.
A full experimental characterization of the IAV entry pathways will benefit from separation of the IAV entry pathways into routes that can be studied independently. Whereas co-localization with clathrin is an established marker for endocytosis via this route, the complete lack of unique markers for macropinosomes or most other endocytic compartments [13, 14] complicates such studies. Furthermore, crucial to any study concerning endocytic pathways is the abundantly documented fact that such pathways are highly dependent on experimental cell culture conditions [15] [16] [17] [18] [19] . Pathways that are constitutive in one cell type may be absent or inducible by specific experimental conditions in other cell types. Moreover, the manipulation of specific endocytic pathways may result in up or down regulation of other specific pathways.
Here we have established entry assay conditions that allow dissecting cell entry of IAV into a dynamin-dependent (DYNA-DEP) and a dynamin-independent (DYNA-IND) component. Dynamin is a large GTPase forming multimeric assemblies around the neck of newly formed endocytic vesicles. GTP hydrolysis is required for pinching off of the vesicles [20] . Whereas CME is completely dependent on dynamin, several other endocytic routes do not require dynamin [21] . We performed an extensive characterization of the dynamin-independent IAV entry route using pharmacological inhibitors as well as by expressing dominant-negative mutants and applying siRNA induced gene silencing as tools. Taken together the results identify a pathway that closely resembles macropinocytosis as a novel entry pathway for IAV.
To identify and characterize potential non-CME entry routes taken by IAV, we adapted a luciferase reporter assay [22] to enable the quantitative determination of infection or entry by measuring the activity of secreted Gaussia luciferase. Twentyfour hours prior to infection HeLa cells were transfected with a plasmid (pHH-Gluc) allowing constitutive synthesis (driven by the human PolI promoter) of a negative strand viral RNA (vRNA) encoding a Gaussia luciferase under control of the untranslated regions (UTRs) of the NP segment of Influenza A/WSN/33 (H1N1) (hereafter called IAV-WSN) NP segment. Upon IAV infection, the combined expression of the viral polymerase subunits and NP will drive transcription of luciferase mRNA from the negative strand vRNA and subsequent synthesis of Gaussia luciferase.
A dose-response curve demonstrating the applicability of the assay to inhibitor screening (Fig. 1A) was obtained for Bafilomycin A1 (BafA1), a known inhibitor of IAV entry [23] . BafA1 acts upon the vacuolar-type H(+)-ATPase, thus preventing endosomal acidification and thereby trapping IAV in peri-nuclear immature endosomes with a lumenal pH that does not permit viral membrane fusion. Remarkably, dynasore, a small molecule inhibitor of the GTPase dynamin 2 that is crucial for endocytic vesicle formation in clathrin-and caveolin-mediated endocytosis [8] as well as in a poorly described clathrin-and caveolin-independent endocytic pathway [8, 19] , did not give significant inhibition (Fig. 1B) .
BafA1 specifically inhibits IAV during the entry phase as demonstrated in Fig. 1C . The continuous presence of 10 nM BafA1 (added to the cells 1 hr prior to infection) for 16 hrs completely prevents infection. In contrast the addition of BafA1 at 1 hr or 2 hrs post infection resulted in high levels of luciferase activity (again measured at 16 hrs p.i.) that were 63% or 90% respectively of the control to which no BafA1 was added, indicating that entry was essentially completed within 2 hrs. The last bar of Fig. 1C shows that the inhibition by BafA1 is reversible as withdrawal of the inhibitor after 2 hrs resulted in high levels of infection. The specific effect of BafA1 on IAV entry was confirmed by confocal microscopy demonstrating that BafA1, as expected, traps IAV particles in a peri-nuclear location, presumably in nonacidified endosomes (Fig. 1D) . BafA1 was subsequently exploited to establish a specific IAV entry assay (hereafter further referred to as the Gluc-entry assay). HeLa cells transfected with pHH-Gluc were inoculated with IAV at a range of MOIs and incubated for 2 hrs after which the entry medium was replaced by complete growth medium containing 10% FCS and 10 nM BafA1 to prevent any further entry of virus. Entry was indirectly quantified by determination of luciferase activity after further incubation for 14 hrs demonstrating a quantitative correlation between infection dose and luciferase activity across a wide range of MOIs (Fig. 1E) . The indirect Gluc-entry assay was next tested for its capacity to examine the effects of inhibitors on IAV entry. Dynasore or BafA1 (Fig. 1F) were included in the medium (DMEM containing 10%
Attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. Influenza A virus (IAV) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. IAV subsequently enters cells of birds and a wide variety of mammals via receptormediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). The elucidation of the endocytic pathways taken by IAV has been hampered by their apparent redundancy in establishing a productive infection. By manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of IAV. Collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative IAV entry route. As the dynamindependent and -independent IAV entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative IAV entry route. A similar strategy might be applicable to the study of endocytic pathways taken by other viruses.
FCS) during entry (the first 2 h of infection) and were removed when the inoculum was replaced by growth medium containing BafA1. Concentrations up to 80 mM dynasore did not inhibit entry which is in agreement with the result shown in Fig. 1B . In contrast, 1.25 nM BafA1 already inhibited entry for more than 60% (Fig. 1F) . As a control, dynasore was also added at 2 hrs post infection to analyze whether the drug affected IAV replication during the post entry phase. As expected, 80 mM dynasore did not significantly inhibit IAV replication when present from 2 to 16 hrs p.i. (Fig. 1F ). Thus, with the Gluc-entry assay we can study the effect of specific inhibitors on IAV entry in a quantitative manner, at least as long as the inhibitors do not irreversibly affect IAV replication during the post entry phase.
Furthermore, the lack of inhibition of IAV entry by dynasore demonstrates that under these experimental conditions IAV is able to enter cells via a pathway that is fully redundant to any dynamindependent (DYNA-DEP) entry route, including the classical CME pathway. Also when IAV travels via this novel dynamin-independent (DYNA-IND) route, IAV apparently enters via low pH compartments as entry is fully sensitive to BafA1.
As factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel DYNA-IND IAV entry pathway (using the Gluc-entry assay) by inoculating cells in PBS in the presence of increasing concentrations of fetal calf serum (FCS). Whereas dynasore completely inhibited entry in PBS, inclusion of 5% and 10% FCS resulted in increasing levels of dynasore resistant entry ( Fig. 2A) , suggesting the existence of a serum-inducible DYNA-IND IAV entry pathway. This effect was not caused by inactivation of dynasore during the experiment as vesicular stomatitis virus (VSV), which enters cells by CME [24, 25] , was still sensitive to 80 mM dynasore in the presence of 10% FCS (Fig. 2B) . In agreement herewith, the uptake of transferin, known to occur via CME, was inhibited by dynasore regardless of the HeLa cells were grown on glass cover slips and infected with IAV (strain WSN; MOI of 10) and fixated after 30 min, 3 hrs or 6 hrs (column 1, 2 or 3 respectively). Infection was performed in 0.2% DMSO (upper row panels) or in the presence of 10 nM BafA1 (lower row panels). The nucleus was visualized by DNA staining with TOPRO-3 (red). IAV infection was visualized by staining with monoclonal antiserum directed against NP (green). In the absence of inhibitor, IAV localized to the nucleus after 3 hrs, while new virus particles spread to the cytoplasm after 6 hrs. BafA1 (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (E) Quantitative determination of IAV entry by a single-cycle Gluc-entry assay. HeLa cells (10,000 cells/well in DMEM supplemented with 10% FCS) were transfected with pHH-Gluc 24 hrs prior to infection with a serial dilution of infectious IAV particles (plotted on the x-axis). Two hours after infection 10 nM BafA1 was added to block any further entry. Cells were incubated for a further 14 hrs to allow expression of luciferase activity (y-axis; Relative Light Units, RLU). (F) Effect of Dynasore and BafA1 on IAV entry in the Gluc-entry assay. Dynasore (DY, dark grey bars; 20, 40 or 80 mM) or BafA1 (light grey bars; 1.25, 2.5 or 5 nM) were present from 1 hr prior to infection (strain WSN; MOI 0.5) to 2 hrs p.i. after which the inhibitor-containing medium was replaced with medium containing 10 nM BafA1 to block any further entry. Cells were incubated for a further 14 hrs to allow the quantitative expression of luciferase activity (y-axes; RLU relative to the control infection without inhibitor). Whereas BafA1 displayed dose-dependent inhibition of IAV entry, dynasore did not significantly inhibit IAV entry. presence of FCS (Fig. S2, panel A) . As expected, both DYNA-DEP entry in PBS and DYNA-IND entry in the presence of 10% FCS and 80 mM dynasore required sialic acid receptors for efficient entry as pre-treatment of HeLa cells with neuraminidases almost completely abolished entry via either pathway (Fig. 2C ). The kinetics of the DYNA-DEP and DYNA-IND entry pathways were compared by performing a time-course experiment in which IAV entry was terminated by the addition of 10 nM BafA1 at different time points (Fig. 2D) . In comparison to entry via the DYNA-DEP pathway (the only pathway available in PBS) entry in the presence of FCS (when presumably both the DYNA-DEP and DYNA-IND entry pathways are available) showed similar kinetics. In contrast, entry via the DYNA-IND pathway (which is the only pathway that is active in the presence of 10% FCS and 80 mM dynasore) was slower. The difference was most prominent after 15 min, while after 4 hrs similar levels of entry were reached.
To validate and extend these results we visualized the reduction of the number of infected cells by immunoperoxidase staining using an antibody against NP (Fig. 3) . A number of different cells of mammalian and avian origin were infected for 2 hours at an MOI of 1 in PBS with or without serum. After 2 hours the inoculum was replaced by growth medium containing 10% FCS and 10 nM BafA1 and the expression of NP was examined after 14 hours later. After incubation in PBS, staining was completely prohibited by the presence of 80 mM dynasore whereas in the presence of serum dynasore had no effect. A serum-inducible, DYNA-IND route of entry was thus functional in all five cell lines, including the human epithelial airway carcinoma cell line A549.
To confirm our results and to obtain further proof for the utilization of DYNA-DEP and DYNA-IND entry routes by IAV, we additionally used an IAV virus-like particle (VLP) direct entry assay [26] . These VLPs contain IAV HA and NA in their envelope and harbor a beta-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1), which allows the rapid and direct detection of entry, independent of virus replication. Upon fusion of viral and endosomal membrane, BlaM1 gains access to the cytoplasmically retained fluorigenic substrate CCF-2 that, after cleavage by BlaM1, shifts to a shorter fluorescent emission wavelength that can be detected by flow cytometry. Entry into HeLa cells was performed in the absence or presence of 10% FCS using VLPs containing HA and NA either from IAV-WSN (having a strict alpha 2-3 linked sialic acid binding specificity) or from the pandemic 1918 IAV (HA from A/ NewYork/1/18, binding to alpha 2-3 and alpha 2-6 linked sialic acids; NA from A/BrevigMission/1/18). Entry of VLPs of both IAV strains was severely inhibited by dynasore when no serum was added to the inoculum (Fig. 4A, 4D) , whereas the presence of 10% FCS rendered entry completely dynasore resistant. (Fig. 4B, 4E ). Quantification of VLP entry is shown in Fig. 4C and F. Importantly, to confirm the existence of the serum-inducible entry pathway by a method that is independent of dynasore, we used siRNA induced silencing of dynamin 2. Fig. 4G shows that two different siRNAs had a significant inhibitory effect (48 hrs after siRNA transfection) on entry of the Renilla luciferaseencoding pseudovirus WSN-Ren [27] in HeLa cells in the absence of FCS, whereas the presence of 10% serum no reduction in entry levels was observed, confirming the results obtained with dynasore. Knockdown of dynamin 2 protein levels (48 hrs after siRNA transfection) was analyzed by western We conclude that a DYNA-IND entry pathway can be induced by serum in different cell types from several species. The evidence was obtained using both replication-dependent (Gluc-entry assay and immunodetection of infected cells) and replication-independent assays (entry of VLPs), the latter allowing immediate detection of the fusion-mediated delivery of viral M1 protein into the cytoplasm. Cascade Blue). In the histograms entry is displayed by a shift to higher fluorescence (the grey area represents background fluorescence of noninfected cells). (C and F) Quantification of FACS results. Background fluorescence was subtracted from each measurement (geometric mean) and data were normalized to VLP entry in Optimem without dynasore (DY) (red curve of panel A and and D). VLP entry was not inhibited by dynasore in presence of 10% FCS whereas the access of BlaM1 to its CCF2 substrate in the cytoplasm was blocked by dynasore in PBS. VLP entry was more efficient in the presence of serum. (G) Effect of downregulation of dynamin 2 by siRNA silencing. Serum-inducible DYNA-IND entry was analyzed in HeLa cells that were transfected 48 hrs prior to infection with two different siRNAs targeting dynamin-2 (dyna). siRNA treated cells were infected with the pseudovirus WSN-Ren in PBS (grey bars) or in PBS containing 10% FCS (black bars) and luciferase activity was determined after 16 hrs post infection (y-axis; RLU relative to infection of cells transfected with a scrambled siRNA). Entry of pseudovirus WSN-Ren (MOI 0.5) was reduced by 50% to 70% when entry was performed in PBS (grey bars) whereas entry was not significantly affected in the presence of 10% serum (black bars). (H) Western blot showing the knockdown of dynamin 2 (in comparison to tubulin) at 48 hrs after transfection with siRNAs. (I) Quantification of the residual levels of dynamin 2 (dyna) mRNA (determined by quantitative RT-PCR) and protein (determined by densitometric scanning of the western blot) 48 hrs after siRNA transfection. Data were normalized to 18S RNA (RT-PCR) or tubulin protein levels and calculated relative to the levels obtained after transfection with a scrambled siRNA that served as a control. doi:10.1371/journal.ppat.1001329.g004
Inhibitors of growth factor receptor tyrosine kinases and actomyosin network dynamics reduce DYNA-IND entry of IAV The DYNA-IND entry pathway was further characterized by inhibitor profiling using an 80-compound kinase inhibitor library. Serum-induced DYNA-IND entry was examined in 10% FCS using the Gluc-entry assay. 80 mM dynasore was added in order to block CME and any other potential DYNA-DEP entry pathways. This allowed the independent inhibitor profiling of the novel pathway by avoiding the potentially masking effect of the presence of redundant entry pathways. Cells were preincubated with the kinase inhibitors (10 mM) for 1 h at 37uC and then inoculated with virus (MOI 0.5) in the presence of 10% FCS and 80 mM dynasore for 2 h at 37uC (DYNA-IND entry). In parallel, inoculations were also done in PBS to compare the effects of the inhibitors on DYNA-DEP entry. After 2 hr the medium and inhibitor were replaced by full growth medium containing 10% FCS and 10 nM BafA1 to allow the subsequent expression of Gluc activity under identical conditions for the DYNA-IND and -dependent entry assay. Six kinase inhibitors appeared to act non-discriminatively, inhibiting both DYNA-DEP and DYNA-IND entry (Fig. 5A ): the protein kinase C (PKC) inhibitors Ro 31-8220, rottlerin (both displaying moderate cytotoxicity, result not shown) and hypericin, which have all three been previously identified as IAV inhibitors [28, 29] ; the highly cytotoxic pan-specific serine/threonine protease inhibitor staurosporine; the irreversible PI-3 kinase inhibitor wortmannin and the receptor tyrosine kinase inhibitor TYR9. In order to investigate whether some of these inhibitors affect IAV replication during the post-entry phase, we performed the same experiments but now adding the kinase inhibitors after viral entry. Four of the inhibitors thus appeared to induce significant inhibition of post-entry processes (Fig. 5A ). Although unlikely, we cannot formally exclude that post-entry processes specific for only one of the two entry pathways are affected. Interestingly, whereas no specific DYNA-DEP entry inhibitors were identified, 15 inhibitors (none displaying cytotoxic effects, data not shown) caused significant (p,0.05) inhibition (.5-fold) of DYNA-IND entry (Fig. 5B ). This included inhibitors of the calmodulin dependent kinases myosin light chain kinase (MLCK) and CaMKII and seven inhibitors of different growth factor receptor tyrosine kinases. In contrast to the three non-specific PKC inhibitors mentioned above, the PKC inhibitors BIM-1 and HBDDE appeared to have a specific inhibitory effect on DYNA-IND entry. The specific effect of these drugs on DYNA-IND entry is not only shown by the lack of inhibition of DYNA-DEP entry in PBS, but also by the observation that none of the fifteen compounds induced .2-fold inhibition when added post-entry (at t = 2 hr post infection). The kinase library screen was repeated on A549 human epithelial lung carcinoma cells in order to confirm the results in a potentially more natural host cell line. The inhibition profiles obtained were very similar to those found for HeLa cells with the exception of the strong effect of AG879 (99% inhibition) and moderate effects of AG825 (39% inhibition) and Tyr51 (68% inhibition) on DYNA-DEP entry. (Fig. 5C) .
MLCK inhibitors ML-7 and ML-9 have been reported to be highly specific for their target kinase [30] . Phosphorylation by MLCK activates non-muscle myosin II light chain, indicating that a functional actomyosin network might be essential for DYNA-IND entry of IAV. This was further examined by testing the effect of Blebbistatin, an inhibitor of myosin II heavy chain activity, and of several inhibitors that affect actin dynamics by disrupting actin microfilaments (Cytochalasin B and D), by enhancing actin polymerization (Jasplakinolide) or by inhibiting actin polymeriza-tion (Latrunculin A). Actin inhibitors were used at the minimal concentration required to induce clearly visible changes in the actin cytoskeleton as pre-determined by staining with FITCphalloidin (results not shown). Whereas the inhibitors did not affect DYNA-DEP entry (Fig. 6A) using Gluc-entry assay, all inhibitors as well as ML-7 and ML-9 significantly inhibited DYNA-IND entry (Fig. 6B) .
Next, HeLa cells were transfected with plasmids encoding dominant negative or wildtype Rab5 fused to green fluorescent protein (Rab5 DN and Rab5 wt in Fig. 6 ) 24 h prior to infection with IAV. Rab5 is a small GTPase found in association with several endosomal compartments and crucial for the function and maturation of early endosomes. It is required for the trafficking of a wide range of endocytic cargo following different routes, including DYNA-DEP as well as DYNA-IND routes [31] . Entry of IAV has been shown to require Rab5 [32] . Consistently, we found that HeLa cells expressing Rab5 DN (as identified by GFP fluorescence, Fig. 6C ) were much less susceptible to productive IAV infection (as judged by indirect immunofluorescence using Alexa-488 labeled NP antibodies) than cells transfected with 
Several dynamin-independent endocytic pathways have been described [8, 19] . Of these, macropinocytosis has been demonstrated to be stimulated by growth factors present in serum and to depend on actin dynamics [12] [13] [14] . Yet, studies on macropinocytosis are hampered by a lack of specific inhibitors, cargo, membrane markers and characteristic morphology. Amiloride and the more potent derivative EIPA are inhibitors of epithelial sodium channels (ENaC) as well as of several other Na+/H+ antiporters. EIPA has often been used as a hallmark inhibitor that specifically inhibits endocytosis via the macropinocytic pathway [14] . Whereas DYNA-DEP entry of IAV was not inhibited by EIPA (Fig. 7A) , DYNA-IND entry was fully blocked EIPA (Fig. 7B) . The existence of redundant entry pathways in the presence of 10% FCS is clearly demonstrated by the marginal inhibition by either EIPA or dynasore whereas the combination of EIPA and dynasore resulted in strong inhibition both in the Gluc-entry assay (Fig. 7C ) and in the direct VLP entry assay ( Fig. 7D and E) . Supplementary  Fig. S1 shows that other cell lines, including the human lung epithelial cell line A549, display similar IAV inhibition patterns for EIPA and dynasore. Consistently, virus production displayed a similar inhibitor sensitivity profile (Fig.7 F and G) as virus entry indicating that the entry pathways we characterized lead to a productive infection. Clearly, VLPs and viral particles follow similar redundant entry pathways, distinguishable in a DYNA-DEP and a DYNA-IND pathway, the latter being sensitive to EIPA and dependent on actomyosin function.
One characteristic of macropinocytosis is the nonselective uptake of large amounts of extracellular solutes [33] . Therefore, the uptake of soluble FITC labeled dextran (Fdx) into relatively large vesicles (0.3 to 5 mM) has often been applied as a morphological marker for macropinosomes. Using this marker we found that the addition of 10% FCS to the culture medium slightly increased the uptake of Fdx into HeLa cells (Fig. 8A) . Notably, the distribution of Fdx changes in response to serum from a random distribution into a more granular pattern. At high magnification and at color settings adjusted to higher intensity it could be seen that these Fdx granules were free of actin staining (by phalloidin) indicating that they were in the lumen of vesicles (result not shown). Interestingly, in the presence of IAV (MOI of 10) the uptake of Fdx into vesicles was clearly enhanced. At a higher magnification viral particles could be found to co-localize in Fdx loaded vesicles as well as outside these vesicles (Fig. 8B) . Phalloidin staining of actin was used to demonstrate that many virus particles localized to actin-rich protrusions at the periphery of the cell. The uptake of Fdx was studied in a quantitative manner by flow cytometry (Fig. 8 C) . A moderate, but reproducible shift to higher Fdx fluorescence was observed at 37uC when virus was added in presence of 10% FCS whereas such a shift was absent when no serum or virus was added. This result confirms the observations by confocal microscopy (Fig. 8A) which showed that the combined presence of FCS and IAV increases the uptake of Fdx as compared to FCS alone. In a control experiment the uptake of Fdx in 10% FCS in presence of IAV was shown to be specifically inhibited by EIPA, but not by dynasore (Fig. S2, panel  B) . In contrast, transferrin uptake, which serves as a specific marker for CME, was affected by dynasore, but not by EIPA (Fig.  S2, panel A) .
In conclusion, serum induces the uptake of Fdx into large vesicles, which can be further enhanced by the addition of IAV particles that, after entry, co-localize in part with these vesicles. These results indicate the utilization of a macropinocytic pathway for entry of IAV, which is consistent with the observed sensitivity of the seruminducible DYNA-IND entry of IAV and VLPs to EIPA.
Macropinocytosis has been implicated in the entry of several viruses [12, 14] . However, differences in susceptibility to inhibitors suggest that distinct forms of macropinocytosis might be used by different viruses [34, 35] . By screening specific inhibitors in the Gluc-entry assay using DYNA-IND entry conditions we evaluated the possible involvement of a few signaling cascades that have been implicated in the induction of macropinocytosis. Serum-inducible macropinocytosis has been shown to be activated via a myriad of signaling cascades initiated by growth factors binding to transmembrane tyrosine kinase receptors [14, 17, 36, 37] , consistent with the results shown in Fig. 5 . A prominent downstream effect of these signaling cascades is the activation of p21 associated kinase 1 (PAK1) which in turn can activate a number of different pathways leading to actin network rearrangements that can ultimately lead to the induction of macropinocytosis [38] . Fig. 9A -B shows that 20 mM IPA3, an inhibitor of PAK1 [39] , specifically inhibits Background fluorescence from Fdx binding to the outside of cells was determined by performing the same experiment at 4uC (at which no endocytosis takes place) and was subtracted from the mean fluorescence intensity obtained at 37uC to determine the amount of fluorescent FITC-dextran that was internalized at 37uC. Data were plotted relative to FITCdextran uptake in PBS in absence of IAV. doi:10.1371/journal.ppat.1001329.g008 DYNA-IND entry of IAV. Activation of PAK1 in response to growth factor stimulation often involves upstream signal transduction by members of the Rho sub-family of small GTPases like CDC42 and/or Rac1 [34, 40, 41] . Alternatively, activated CDC42 and Rac1 can induce actin rearrangements independently of PAK1 [34, [40] [41] [42] [43] by direct interaction with WASP or WAVE family proteins, respectively [44, 45] . However, inhibitors of CDC42 (Pirl1 [46] ), Rac1 (NSC23766 [47] ) or N-WASP (wiskostatin [48] ) did not display inhibitory effects on DYNA-IND or DYNA-DEP entry of IAV (Fig. 9C-D) . Instead, Pirl1 and wiskostatin induced a significant, concentration dependent increase of entry. This stimulatory effect was not observed for the control vaccinia virus strain WR, which enters cells via a Rac1dependent, macropinocytotic pathway [43] (Fig. 9E) , indicating that this effect is specific for IAV. The results suggest a requirement for PAK1 in DYNA-IND entry of IAV that does not require activation by either CDC42 or Rac1. Growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [49] [50] [51] ; consistent with this observation the src inhibitor PP2 [52] specifically inhibited DYNA-IND entry of IAV (Fig. 9A-B) . Remarkably, 17-AAgeldanamycin, a specific inhibitor of the chaperone protein HSP90 [53] , also caused specific inhibition of DYNA-IND entry (Fig. 9A-B) . HSP90 affects the folding and activity of many proteins but the recent demonstration of direct activation of the catalytic activity of src by HSP90 [54] provides another indication of the involvement of src in DYNA-IND endocytosis of IAV.
In conclusion, like for other viruses utilizing a macropinocytic entry pathway, PAK1 seems to play a crucial role in DYNA-IND entry by IAV. However, this pathway is independent of Rac1 or cdc42 but may require src, either upstream and/or downstream of PAK1.
The data presented in this study demonstrate for the first time that IAV can enter cells via DYNA-IND macropinocytosis in addition to the previously described DYNA-DEP classical CME pathway [1, 2] . Several lines of evidence indicate that the DYNA-IND entry route of IAV that we identified corresponds with macropinocytosis. First of all, the entry pathway is dependent on the presence of serum, a well-known inducer of macropinocytosis. Second, IAV colocalized in vesicles with soluble FITC-dextran, a marker for macropinocytosis. Third, DYNA-IND IAV entry was sensitive to the amiloride-derivative EIPA, the hallmark inhibitor of macropinocytosis [14, [55] [56] [57] [58] . Fourth, this IAV entry pathway is sensitive to inhibitors or dominant-negative mutants affecting actomyosin dynamics. Fifth, the specific inhibition of DYNA-IND IAV entry by a number of inhibitors of growth factor receptor tyrosine kinases as well as downstream effectors thereof also points at the involvement of macropinocytosis. Finally, macropinocytosis is independent of dynamin [12, 14, 19] .
Despite this extensive list of arguments, viral entry by macropinocytosis needs to be considered with caution. The characteristics of the DYNA-IND route of cell entry by IAV are similar, but not identical to the macropinocytic entry routes taken by other viruses, like two different strains of vaccinia virus and by coxsackie virus B [34, 35] . As is shown in Table 1 and discussed in more detail below, the macropinocytic pathways used by each of these viruses have a few unique characteristics. This may very well reflect the growing notion that macropinocytosis represents a number of differentially induced and regulated processes, rather than being a single endocytic pathway [13, 14] . Macropinocytosis has collectively been described as an inducible form of endocytosis by which fluid-phase cargo travels via non-coated, relatively large and heterogeneous organelles that have emanated from extensive protrusions (e.g lamellar ruffles, circular ruffles or retracting blebs) of the plasma membrane [13] . In the case of DYNA-IND IAV entry more extensive studies using electron microscopy will be required to study the morphology of membrane protrusions with which IAV may associate. In addition, live cell imaging microscopy will be required to characterize the exact itinerary that is taken by IAV virions traveling via a macropinocytic process. This is especially important as different routes of IAV entry are likely to converge at some point in the endocytic pathway. Although unlikely, co-localization of IAV particles with fluid-phase dextran as shown in Fig. 8B may thus represent a situation occurring after convergence of several different routes. The use of microscopy to study macropinocytosis is however complicated by the lack of specific membrane-associated markers for any early step of this endocytic process.
A model (Fig. 10 ) based on our results explains the key steps involved in the macropinocytic entry pathway of IAV, which are described in more detail below.
By manipulating the inoculation conditions we were able to experimentally dissect IAV entry into a DYNA-DEP and DYNA-IND route. The DYNA-IND route required the presence of 10% FCS in the entry assay medium. Previously, a strict dependency on a DYNA-DEP entry route for IAV was concluded from experiments with a cell line expressing an inducible dominantnegative mutant of dynamin 2 [59] . In that study, as well as in other entry studies of IAV, entry was performed in DMEM containing 2% serum or BSA. Also in our hands 2.5% serum ( Fig. 2A) or 0.2% BSA (result not shown) was not sufficient to allow DYNA-IND entry. We are currently investigating which serum component is responsible for the observed effects on IAV entry. Dialysis of FCS (MW cut off .10 kDa) did not affect its capacity to induce DYNA-IND endocytosis (result not shown), indicating that low molecular weight solutes are not responsible for the observed effect.
Our evidence for a DYNA-DEP and a serum inducible DYNA-IND entry route is based on the use of pharmacological (dynasore, a highly specific inhibitor of dynamin) as well as genetic (siRNA directed against dynamin 2) tools, ruling out the possibility that the inhibitory effect of dynasore was due for instance to absorption of the inhibitor by serum components. Whereas dynasore resulted in near 100% inhibition of DYNA-DEP entry, only 65% inhibition was observed upon siRNA induced silencing of dynamin 2 indicating that the residual levels of dynamin 2 that remain after 48 hrs of silencing still support a low level of DYNA-DEP entry (Fig. 4H) . Reversible inhibitors like dynasore [60] offer a major advantage for characterization of IAV entry pathways. They can be applied for a limited period thus preventing the secondary adaptive effects of cells that may occur in response to long-term down regulation of a gene product by genetic methods like siRNA interference. Both entry routes were consistently identified by a viral entry assay quantified by virus induced expression of a luciferase reporter as well as by a VLP entry assay allowing direct analysis of the membrane fusion mediated entry step. The consistent performance of an HA with a strict preference for binding to a2-3 linked sialic acids (from IAV-WSN; our unpublished data) and an HA also binding to a2-6 linked sialic acids (from 1918 IAV [61] ) in the VLP entry assay indicates that both pathways can be utilized by HAs of different specificity and may therefore be relevant to avian as well as human IAV infections. Consistently, serum-inducible DYNA-IND entry was observed both in avian DF1 cells and in a human lung epithelial carcinoma cell line A549 (Fig. 3) . The DYNA-DEP and DYNA-IND IAV entry pathways were found by our quantitative assays to be fully redundant. In the presence of serum, the combination of dynasore (inhibiting DYNA-DEP entry) and EIPA (inhibiting DYNA-IND entry) completely abolished entry whereas either drug alone had no effect. EIPA, an inhibitor of plasma membrane Na+/H+ exchangers, has been shown to invariably inhibit macropinocytosis [14, [55] [56] [57] [58] . As other routes of endocytosis are generally not affected, EIPA is considered as a hallmark inhibitor of macropinocytosis [14] , although results obtained with EIPA should be considered with care as long as a mechanistic explanation for its effect on macropinocytosis is not yet fully clear [62] . Occasionally, a moderate two-to three-fold inhibition by dynasore alone was observed (result not shown) indicating that the capacity of the serum-inducible entry pathway is somewhat variable, possibly depending on slight variations in serum quality and factors like cell distribution in the wells that have been reported to influence viral infection [63] . A redundancy in the utilization of CME as well as a clathrin-independent route for entry of IAV has been visualized previously by quantitative live cell imaging [4] . Both routes were operative simultaneously in the same sample and the specific down-regulation of CME did not affect the total number of entry events.
In response to specific extra-cellular signals (e.g. serum induction), changes in the actomyosin network occur that give rise to membrane protrusions required for macropinosome formation [13] . Compounds inhibiting actin polymerization (cytochalasin B and D), depolymerization (jasplakinolide) or sequestering soluble actin (latrunculin A) all specifically inhibited DYNA-IND IAV entry. In addition, the requirement for myosinII activity was established by a specific inhibitor (Blebbistatin) of myosin II ATPase activity and by the expression of a dominant negative mutant of myosinIIA heavy chain. Also, the regulation of myosinII activity by phosphorylation of myosin light chain through the action of MLCK is suggested by the inhibitory effect of MLCK inhibitors ML-7 and ML-9 as well as by the similar effect of an expressed MLCK dominant negative mutant. Recently, a function for the actin cytoskeleton in IAV entry was reported to be required for the entry into polarized epithelial cells but not for entry into non-polarized cells [64] . When using the low-serum conditions used in that paper (2% FCS), we only observed DYNA-DEP entry that was not affected by actin dynamics inhibitors. Perhaps, the polarized cells permit DYNA-IND entry at lower serum concentrations.
The changes in actin network dynamics that can lead to the formation of macropinosomes can be triggered by a number of signaling cascades. Actin dynamics are induced by the activation of growth factor receptor tyrosine kinases by their respective Figure 10 . A model for IAV entry by macropinocytosis. The model summarizes the inhibitory (red boxes) or stimulatory (blue boxes) effects of compounds on dynamin-independent IAV entry. The effect of over-expression of dominant-negative mutants is indicated by red-lined boxes. The pathway requires the presence of serum factors in the entry medium and results in the enhanced uptake of dextran and its co-localization with IAV in large vesicles (green boxes). We hypothesize that the interaction of serum factors and/or IAV with receptor tyrosine kinases (RTKs) is the primary signal for the induction of macropinocytosis. A number of RTKs have been shown to be involved in this process in different cell lines. Remarkably, a recently published genome-wide siRNA screen of IAV infection identified the FGF receptor as a host factor required for influenza virus replication [22] . Activation of Rho family GTPases CDC42 and/or Rac1 has been shown to be essential for signal transduction leading to macropinocytosis in many cases [13, 14] but inhibitors are without effect or are stimulatory in the case of IAV entry. Downstream effectors of Rho family GTPases include scaffold proteins like N-WASP and WAVE and protein kinases like PAK1. Macropinocytic entry of IAV however seems to require a Rho family GTPaseindependent PAK1 activation mechanism. In addition, src family kinases, which can be directly activated by RTKs, play a role. PAK1 and src have previously been linked to the activation of macropinocytosis via their effect on changes in actomyosin dynamics, a process which is crucial to any form of macropinosome formation [13, 14] . Apart from N-WASP-or WAVE-containing macromolecular assemblies other actin binding proteins can induce such changes (e.g. cortactin, which can be activated by src [81] ) and thereby induce the formation of one of the different plasma membrane protrusions that can result in the formation of macropinosomes. In addition to an effect on the formation of plasma membrane protrusions and subsequent macropinosome formation, inhibitors can also affect downstream trafficking and maturation of macropinosomes which might be actindependent, but this is not depicted in the scheme. doi:10.1371/journal.ppat.1001329.g010 growth factor ligands that are normally present in serum [12] [13] [14] 17, 36, 37] The signal transduction cascades that link activation of growth factor receptor tyrosine kinases to actin remodeling and macropinocytosis are only beginning to be revealed. The specific inhibition of DYNA-IND entry of IAV by IPA3, an inhibitor of PAK1, provides proof for the involvement of these cascades. PAK1 is a key serine/threonine kinase regulating actin network dynamics but its crucial function in several pathways of endocytosis as well as numerous other cellular processes does not make it a very specific marker [65] . Even so, macropinocytosis has consistently been demonstrated to require PAK1 activation, both in the induction of the process and/or in further downstream trafficking events of macropinosomes [13, 14] .
Growth factor dependent activation of PAK1 has most often been demonstrated to depend on upstream activation of small GTPases Rac1 or cdc42 [34, 40, 41] . Different strains of vaccinia virus were recently shown to induce their uptake by macropinocytosis via activation of either Rac1 or cdc42 [34] . Activation of Rac1 has been linked to the induction of macropinocytosis via actin network-mediated formation of lamellipodia and/or circular ruffles whereas cdc42 has most often been implied in the formation of filopodia [44] . An inhibitory effect of the Rac1 inhibitor NSC23766 or the cdc42 inhibitor pirl1 on IAV entry, however, could not be demonstrated. Remarkably, cdc42 inhibitor pirl1 enhanced IAV entry and a similar effect was observed by wiskostatin, an inhibitor of N-WASP which functions directly downstream of cdc42 as a scaffolding complex required for the activation of actin polymerization leading to filopodia formation. Similarly, the macropinocytosis-like entry pathway taken by Coxsackie B virus was also shown to require PAK1 activity that was independent of Rac1 activation [35] . Direct examination of the magnitude and timing of the activation of PAK1 will be required to obtain more insight in the involvement of this complex pathway. The induction of macropinocytosis by a PAK1dependent mechanism has been associated with ruffling at the cell membrane [12, 14, 15, 37] . The identification of sub-membranous regions with increased actin staining by phalloidin has been interpreted as evidence for ruffling. This was not unambiguously identified by confocal microscopy in the experiments presented in Fig. 8 and Fig. S2 and needs to be investigated in depth by life cell imaging techniques.
In agreement with our observation that the DYNA-IND entry of IAV was inhibited by PP2, an inhibitor of src family kinases, the non-receptor tyrosine kinase c-src has been shown to function as a key signaling intermediate in the induction of macropinocytosis via a mechanism independent of Rac1 or cdc42 [49] [50] [51] . Downstream effects of c-src on actin networks proceed, amongst others, via phosphorylation of cortactin by c-src resulting in accelerated macropinosome formation [50] . C-src has been shown to associate with macropinosomes [49, 51] , both during their formation and their trafficking, while c-src kinase activity is required for macropinocytosis following EGF stimulation of HeLa cells [49] . Interaction of HSP90 with c-src was recently shown to induce c-src kinase activity [54] . Also HSP90 has been demonstrated to associate with macropinosomes, while its specific inhibitor geldanamycin reduced the membrane ruffling that preceded macropinocytosis [66] . Thus, the inhibition of IAV entry via macropinocytosis by AA-geldanamcyin may very well involve the effects of HSP90 on c-src.
As detailed above, the DYNA-IND entry pathway of IAV shares many characteristics with the endocytic pathway macropinocytosis. This is corroborated by the observation that IAV particles and dextran colocalize in large vesicles in the presence of FCS. Several viruses have recently been reported to enter cells via macropinocytosis [12, 14] . Apart from common factors like the requirement for PAK1 activation, actin dynamics and independence of dynamin, virus specific details have been described [34, 35] (Table 1 ). In part these might be contributed to differences in experimental conditions (e.g. cell types tested) but diversity in the molecular mechanisms by which macropinocytosis can be induced and executed is likely to exist and to be exploited by viruses. Whereas vaccinia virus is able to trigger its own macropinocytic uptake [34, 43] , we have described a macropinocytosis pathway that is operational under conditions that are activated by components in serum. Still, this does not exclude signaling induced by virus-host cell interactions, which are for instance suggested by the significant increase of FITC-dextran uptake in the presence of IAV. The possible requirement for costimulatory signals from serum components and virus imposes an additional layer of complexity on the analysis of IAV entry via DYNA-IND pathways.
Influenza viruses cause respiratory infections by targeting the epithelial cells lining the respiratory tract. These surfaces are covered by a mucous layer composed of a variety of small solutes and glycoproteins derived among others from goblet cells [67] . This semi-fluid layer in turn conditions the underlying cells and determines their physiological state, including the activities of their uptake and secretion pathways. It will be important to determine to what extent the DYNA-DEP and DYNA-IND IAV entry pathways are operational under the conditions prevailing along the respiratory tract. Current knowledge on the protein composition of the fluids covering the respiratory epithelium is rapidly expanding by the application of proteomic methods to determine the protein composition of bronchial alveolar lavage fluids (BALF). These studies have extended the previous notion that BALF is highly similar in composition to serum. For example, just as for the serum proteome more than 85% of the total protein mass of the BALF proteome is accounted for by albumin, immunoglobulins, transferring, a1-antitrypsin and haptoglobin. In addition, many other proteins have been identified both in serum and in BALF including growth factors that can bind to growth factor receptor tyrosine kinases [68] [69] [70] . Thus, BALF is likely to harbor, just as serum, the protein factors that can activate signaling pathways that are crucial for the induction of DYNA-IND entry of IAV. In agreement herewith, macropinocytosis has been described as a functional entry pathway of Haemophilus influenzae into primary human bronchial epithelial cells [71] although the factors involved in signaling the process have not been identified yet.
In addition to infecting the respiratory tract, IAV has been shown to be able to cause systemic infections involving multiple organs. This has mainly been studied in avian infections [72, 73] or by infection of mice with human-derived H1N1 or H3N2 IAVs [74] but is poorly documented for human infections and may have been underestimated thus far. Obviously, during potential systemic spreading of IAV, the serum-rich conditions that we have demonstrated here to enable the use of alternative entry pathways will be encountered and may contribute to such spreading.
MDCK, A549, DF-1 and HeLa cells were maintained in complete Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Biowittaker) containing 10% (v/v) fetal calf serum (FCS; Bodinco B.V.), 100 U/ml Penicillin, and 100 mg/ml Streptomycin. Chinese Hamster-E36 cells were maintained at 37uC in a-Minimal Essential Medium (Gibco) supplemented with 10% (v/v) FCS, 100 U/ml Penicillin, and 100 mg/ml Streptomycin. Cells were passaged twice weekly. Influenza A/WSN/33 (H1N1) (IAV-WSN) was grown in MDCK cells. Briefly, ,70% confluent MDCK cells were infected with IAV-WSN at a MOI of 0.02. Supernatant was harvested after 48 hr of incubation at 37uC and cell debris was removed by centrifigutation (10 min at 2000 rpm). Virus was stored at 280uC and virus titers were determined by measuring the TCID 50 on HeLa cells. The IAV-WSN luciferase pseudovirus (WSN-Ren) system has previously been described [27] . Briefly, WSN-Ren pseudovirus harbors a HA segment in which the HA coding region is replaced by Renilla luciferase. The pseudovirus is produced in a MDCK cell line that stably expresses the HA of IAV-WSN. WR-LUC, a firefly luciferase encoding vaccinia virus (strain WR) was previously described [75] . VSV-FL, a firefly luciferase encoding VSV virus was also previously described [76] .
Stocks of bafilomycin A1 (BafA1), dynasore, cytochalasin D, cytochalasin B, Blebbistatin, 17-AA-geldanamycin, ML-7, ML-9, PP-2, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), IPA-3 (all obtained from Sigma-Aldrich), Latrunculin A (Enzo), jasplakinolide, wiskostatin, NSC23766 (all obtained from Calbiochem) and pirl1 (Chembridge) were prepared in dimethylsulfoxide (DMSO). All stocks were stored at 220uC. A kinase inhibitor library composed of 80 kinase inhibitors was obtained from Biomol (2832A[V2.2]).
HeLa cells (10,000 cells/well in 96-well plates) were treated with 2 mUnits of Vibrio cholerae neuraminidase (Roche) in 50 ml phosphate-buffered saline (PBS) for 2 hr. After washing with PBS cells were infected with IAV as described.
Virus-like particles (VLPs) were produced as described [26] . Briefly, 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with pCAGGS-BlaM1 (encoding a beta-lactamase reporter protein fused to the influenza matrix protein-1), pCAGGS-HA (encoding HA derived from either A/NewYork/ 1/1918 or IAV-WSN) and pCAGGS-NA (encoding IAV neuraminidase [NA] derived from either A/BrevigMission/1/18 or IAV-WSN) and maintained in OptiMEM. Supernatants were harvested 72 h after transfection and centrifuged to remove debris. VLPs were used for inoculation of cells without further concentration. VLPs were incubated for 30 min at 37uC with trypsin/TPCK for activation of HA. MDCK or HeLa cells grown to near confluency in 24-well plates were inoculated with 250 ul of VLPs after pre-treatment of the cells with inhibitors as indicated. Infection was synchronized by centrifugation at 1500 rpm for 90 min at 4uC and was performed by further incubation at 37uC for 2 h in the absence or presence of 10% FCS and inhibitors as indicated. Detection of beta-lactamase activity was performed as described [25] by loading cells with CCF2-AM substrate (InVitrogen) and subsequent analysis by flow cytometry on a LSRII flow cytometer (Becton Dickinson). Typically 10,000 events were collected and analyzed using FlowJo 8.5.2 software.
The reporter construct pHH-Gluc was derived from plasmid pHH-Fluc [22] by replacing the firefly luciferase coding region with the Gaussia luciferase coding region of pGluc-basic (New England Biolabs). Unique SpeI and XbaI restriction sites were introduced into pHH-Fluc using the Quikchange XL Site-directed mutagenesis kit (Stratagene) and oligonucleotides Spe4262 (5-9GCCTTTCTTTATGTTTTTGGCACTAGTCATTTTACCG-ATGTCACTCAG), Spe4263 (59-CTGAGTGACATCGGTAA-AATGACTAGTGCCAAAAACATAAAGAAAGGC), Xba4260 (59-GTATTTTTCTTTACAATCTAGACTTTCCGCCCTTC-TTGG) and Xba4261 (CCAAGAAGGGCGGAAAGTCTAG-ATTGTAAAGAAAAATAC). A SpeI site was introduced by sitedirected mutagenesis in pGluc-basic directly following the start codon of the Gaussia luciferase coding sequence. The unique SpeI -XbaI fragment of pGluc-basic was subsequently cloned into the SpeI-XbaI site of pHH-Fluc resulting in plasmid pHH-Gluc.
Cells were seeded in 96-well plates at a density of 10,000 cells/ well and transfected the next day with 10 ng pHH-Gluc using Lipofectamine 2000 (InVitrogen) according to the manufacturer's protocol. After 24 hrs the transfected cells were treated with inhibitors and infected as indicated. At 16 hr p.i. samples from the supernatant were assayed for luciferase activity using the Renilla Luciferase Assay system (Promega) according to the manufacturer's instructions, and the relative light units (RLU) were determined with a Berthold Centro LB 960 plate luminometer. WR-LUC and VSV-FL were used to inoculate HeLa cells (10,000 cells/well) at an MOI of 2, in complete Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Biowittaker). After 7 hr the luciferase activity was detected using the SteadyGlo assay kit (Promega). The addition of 10% (v/v) FCS did not change infection levels for both viruses.
Cells were fixed with 3.7% paraformaldehyde (PFA) in PBS and subsequently permeabilized with 0.1% Triton-X-100 in PBS. After blocking with normal goat serum IAV-infected cells were incubated for 1 h with a monoclonal antibody directed against the nucleoprotein (NP) (HB-65; kindly provided by Dr. Ben Peeters). After washing, the cells were incubated with a 1:400 dilution of Alexa Fluor 488-or 568-labeled goat anti-mouse IgG (Molecular Probes) secondary antibody for 1 h. Nuclei were subsequently stained with TOPRO-3 and after three washing steps, the coverslips were mounted in FluorSave (Calbiochem). Actin was stained using phalloidin labeled with Alexa Fluor 633. The immunofluorescence staining was analyzed using a confocal laser-scanning microscope (Leica TCS SP2). FITC, GFP or Alexa Fluor 488 were excited at 488 nm, Alexa Fluor 568 at 568 nm, and TOPRO-3 at 633 nm.
HeLa cells were grown in 24-well plates on glass coverslips (50,000 cells/well). Prior to FITC-dextran uptake cells were serum-starved for 2 hr in PBS. FITC-dextran (MW70,000, Sigma-Aldrich) was incubated with HeLa cells (final concentration of 0.5 mg/ml) in 500 ml PBS or in PBS containing 10% FCS in the absence or presence of IAV (strain WSN; MOI 10; concentrated and purified by centrifugation through a 15 to 30% sucrose gradient with a 50% sucrose cushion at the bottom) at 37uC. After 15 min cells were washed 4 times with PBS at 4uC, fixed with 3.7% PFA in PBS and subsequently permeabilized with 0.1% Triton-X-100 in PBS. Slides were stained for examination by confocal microscopy as described above. For quantification of FITC-dextran uptake 1.5610 5 HeLa cells were infected with IAV-WSN (MOI 10) in suspension in a volume of 1 ml in the presence of FITC-Dextran (1 mg/ml). Infections were performed for 15 min in PBS (containing 2% BSA to reduce unspecific binding of FITC-Dextran) or in PBS containing 10% FCS at 37uC or at 4uC (control for binding of FITC-Dextran to cells in the absence of endocytosis). Mock-infected samples were analysed in parallel. Infection was terminated by addition of 3 ml ice-cold PBS followed by three washes with cold PBS and fixation with 3.7% PFA. 20,000 cells were analyzed by FACS and results were represented as the mean fluorescence which was plotted relative to the uptake in the mock-infection in PBS (after subtraction of background fluorescence obtained at 4uC).
The effect of dynasore and EIPA on dextran and transferrin uptake HeLa cells (grown on glass cover slips) were incubated at 4uC for 1 hr with 50 mg/ml Alexa633-labeled Transferin (InVitrogen) in PBS. After 1 hr the medium was replaced by PBS or PBS supplemented with 10% FCS containing IAV (strain WSN; MOI 10) and 0.5 mg/ml FITC-Dextran (Sigma; 70 kDa) and cells were transferred to 37uC for 15 min. After 15 min cells were fixed and stained as described above and examined by confocal microscopy.
Cells were fixed with 3.7% PFA in PBS and subsequently permeabilized with 0.1% Triton-X-100 in PBS. Peroxidase was visualized using an AEC substrate kit from Vector Laboratories. IAV-positive cells were detected using bright-field light microscopy.
Two siRNA duplexes targeting different sites within the coding sequences of dynamin 2 were obtained from Ambion Inc (15581 (Dynamin 2 siRNA 1) and 146559 (dynamin 2 siRNA2)). A scrambled siRNA (Ambion Inc.) was taken along as a control for non-specific effects of the transfection procedure and was used for normalization. One day after seeding in 96-well plates (6,000 cells/well), the HeLa cells were transfected with a final concentration of 10 nM siRNA using oligofectamine (Invitrogen). 48 h after transfection, the cells were inoculated with the WSN-Ren pseudovirus (MOI 0.5) in PBS or in PBS containing 10% FCS. After 2 h of infection the entry medium was replaced by complete growth medium containing 10 nM BafA1 to prevent further entry. At 16 h post infection intracellular Renilla luciferase expression was determined as described above. Each siRNA experiment was performed in triplicate. Cell viability was not affected as determined by performing a Wst-1 cell-viability assay (Roche). Functional knockdown of dynamin 2 mRNA levels was performed by quantitative RT-PCR. using a TaqMan Gene Expression Assay for DNM2 (Hs00191900_m1, Ambion) and using 18S RNA (Hs03928985_g1, Ambion) as a control for normalization. The comparative Ct-method was used for quantification of the results [77] . Reduction of dynamin 2 protein levels was determined by western blotting using polyclonal goat-anti-dynamin 2 C18 (Santa-Cruz SC-6400). A monoclonal against alpha-tubulin (DM1A, Sigma T9026) was used to detect tubulin for normalization. Results were quantified by Densitometric scanning of the dynamin 2 and tubulin signals displayed in Fig. 4H .
HeLa cells were grown in 24-well plates on glass coverslips (50,000 cells/well) for 24 hrs. Cells were then transfected (1 mg of DNA with lipofectamine 2000 as described above) with plasmids encoding wild-type or dominant-negative (DN) human MLCK fused to GFP [78] , wild-type or DN Rab5 fused to GFP [79] , or MyoII-tail or MyoII-head domain fused to GFP [80] . 24 hr after transfection cells were inoculated with IAV-WSN (MOI 1) in PBS or in PBS containing 10% FCS and 80 mM dynasore. 4 hr after infection cells were fixed and stained for examination by confocal microscopy as described above.
An unpaired Student's t-test was used for detemination of statistically significant differences. The use of the term significant in text refers to a comparison of values for which p,0.05.  Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells BACKGROUND: One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation. METHODOLOGY/PRINCIPAL FINDINGS: Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta'), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked. CONCLUSIONS: alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases. Blood meal digestion takes place in the midgut of hematophagous arthropods and provides the source of nutrients for vitellogenesis and oogenesis, as well as the entry point for pathogen transmission [1] . Mosquito midgut epithelial cell ultrastructures have previously been characterized by electron microscopy in several species, including Aedes aegypti [2, 3, 4, 5] , Anopheles gambiae [6] , An. darlingi [7] , Culex quinquefasciatus [8] , and Cx. tarsalis [9] . One of the most striking findings from these studies was that the rough endoplasmic reticulum (RER) in midgut epithelial cells of adult female Aedes and Anopheles mosquitoes is organized into large whorl-like structures prior to blood feeding. With the onset of feeding, the RER whorls undergo a dramatic reorganization, presumably to facilitate the synthesis and secretion of digestive proteases and components of the peritrophic matrix [2, 3, 5, 7, 10, 11] . Interestingly, male mosquitoes, which do not blood feed, lack RER whorls in their midgut epithelial cells [12] . The identity and functional contribution of protein components localized to mosquito midgut RER whorls have not been investigated.
ER whorls have been shown to form in vertebrate cells exposed to inducers of the ER stress response [10, 13] , and as a result of pathological conditions [14] . Whorl-like smooth ER structures have also been shown to form in mammalian tissue culture cells that over express transfected ER resident proteins or fluorescent protein gene fusions containing heterologous transmembrane domains [15, 16, 17] . Immunofluorescent staining has been used in these transfection experiments to show that weak proteinprotein interactions between the cytoplasmic domains of highly abundant ER imbedded proteins are sufficient to induce membrane stacking and whorl formation. The absence of some ER proteins can also induce whorl formation, suggesting that mechanisms are in place to prevent ER whorls from forming in most cell types [18] .
Plasma free amino acids and protein-bound amino acids in vertebrate blood provide the raw materials for energy conversion and reproduction [19, 20, 21] , and also function to initiate signaling through the target of rapamycin (TOR) signal transduction pathway [22, 23] . Moreover, an artificial amino acid meal is sufficient to induce translation of early trypsin mRNA using the TOR signaling pathway in midgut epithelial cells, however amino acids alone do not induce the transcription and translation of the late phase trypsin protein [24] . It is possible that amino acid signaling through the TOR pathway also stimulates RER reorganization in mosquito midgut epithelial cells.
In order to investigate mechanisms controlling RER whorl unwinding and the initiation of blood meal digestion in female Ae. aegypti mosquitoes, we performed proteomic studies using mass spectrometry and identified midgut proteins associated with microsomal fractions in unfed and amino acid fed mosquitoes. The 139 kDa alpha subunit of the COPI coatomer complex was selected as one of eight candidate RER whorl-forming proteins based on bioinformatic analyses. We used RNAi-mediated knockdown to determine if loss of alpha-COPI expression had any effect on RER whorl formation in unfed mosquitoes or blood meal digestion in fed mosquitoes. Our data show that alpha-COPI functions are indeed required for RER whorl formation in unfed mosquitoes, and moreover, that a deficiency in alpha-COPI blocked the expression of three late phase midgut proteases in Figure 1 . Amino acid feeding is sufficient to induce RER whorl unwinding. Three-day-old Ae. aegypti females were kept unfed (sugar fed), or fed an amino acid meal for 30 min., and then sacrificed and dissected 30 min. or 120 min. later. The dissected midguts were fixed for electron microscopy preparation as described in Materials and Methods and representative electron micrographs of midgut sections are shown here. A) Unfed mosquito (25,0006magnification) . B) Unfed mosquito (8,8006magnification) . C) Amino acid fed mosquito dissected at 30 min. post-feeding (8,8006 magnification). D) Amino acid fed mosquito dissected at 120 min. post-feeding (8,8006 magnification). doi:10.1371/journal.pone.0018150.g001 blood fed mosquitoes (AaSPVI, AaSPVII, AaLT). Interestingly, the early phase of blood meal digestion, which is characterized by the synthesis and secretion of the early trypsin protease (AaET), was not dependent on alpha-COPI expression.
To determine if amino acids in the midgut lumen are sufficient to trigger RER whorl unwinding in midgut epithelial cells of Ae. aegypti mosquitoes, females were fed an artificial amino acid meal and dissected midguts were characterized for ultrastructural changes at the subcellular level using electron microscopy (EM). As seen in representative electron micrographs (Figure 1 ), large whorls of stacked RER membranes were observed in midgut epithelial cells from unfed (sugar only) mosquitoes, with some RER whorls containing .50 stacked membranes. The whorls appeared to consist entirely of tightly packed RER membranes, with no evidence of a core lipid droplet as seen in other organisms [25] . Quantitative measurements of whorl size in 20 randomly chosen EM fields revealed that the RER whorls ranged in size from ,5-35 mm 2 in unfed mosquitoes ( Figure 2 ). However, following a 30 minute amino acid feeding period, and another 30 minutes of recovery, midgut epithelial cells were found to contain significantly fewer RER whorls of .5 membrane stacks (p,0.001) ( Figure 2 ). Moreover, most of the RER contained in the midgut epithelial cells of amino acid fed mosquitoes was arranged in short linear stacks ( Figure 1C ). Quantitative measurements of RER whorl size and number in midgut epithelial cells following a recovery period of 120 minutes after amino acid feeding gave similar results, suggesting that RER whorl unwinding is rapid and complete by 30 minutes post feeding ( Figures 1D and 2 ).
Reorganization of the ER in midgut epithelial cells of amino acid fed mosquitoes may be associated with differential abundance of KDEL-containing ER resident proteins, which could provide a clue as to possible whorl-enriched proteins. To test this possibility, we prepared total protein extracts from midguts of unfed and amino acid fed (30 min and 120 min post feeding) mosquitoes, and analyzed the extracts by Western blotting using a KDELspecific antibody as shown in Figure 3 . Four distinct protein bands were observed, two of which appeared to decrease in abundance after feeding (KDEL-2 and KDEL-4), relative to the internal control protein (GAPDH). The KDEL-1 protein was only present in the 30 min sample, whereas the KDEL-3 protein was present in all three protein samples. Since Ae. aegypti protein disulfide isomerase (PDI) has a predicted molecular mass of ,56 kDa, and is a KDEL-containing ER protein known to be expressed in Ae. aegypti midgut cells [26] , we analyzed these same protein extracts with a PDI antibody that recognizes Drosophila melanogaster PDI. Results from these experiments were negative (data not shown), however we could not rule out that antigenic differences between Drosophila and Aedes PDI proteins contributed to the negative result.
To more directly identify midgut proteins that might be contributing to whorl formation in unfed adult female mosquitoes, we isolated microsomal proteins from midgut tissues of unfed and amino acid fed mosquitoes (30 min post-feeding) using a modified procedure that minimized protease activity in the sample and enriched for RER associated proteins (see Materials and Methods). The protein samples were separated by molecular weight using SDS PAGE and four gel slices from each lane were processed for LC-MS/MS analysis and protein annotation ( Figure 4 ). The SDS PAGE analysis revealed that intact proteins of up to ,130 kDa were present in both samples, suggesting that protease activity was minimal in the midgut protein sample from fed mosquitoes. Moreover, no major differences were observed in the distribution of proteins isolated from the unfed or amino acid fed mosquitoes, indicating that the 30 min time point preceded feeding-induced protein synthesis.
Following in-gel trypsin digestion, each of the eight protein samples was analyzed by LC-MS/MS. Bioinformatic analysis of the mass spectrometry data led to the identification of 127 proteins using the Ae. aegypti genome database in a Sequest-based search (Table S1 ). Gene Ontology (GO) analysis revealed that ,30% of Figure 2 . Quantitation of whorl size and number in unfed and amino acid fed mosquitoes. Whorls with a minimum of five stacked ER membranes were counted and the total area of each whorl was determined using NIH Image software. Data are shown for whorl size and number in 20 random EM fields covering 3335 mm 2 of mosquito midgut epithelial cell area. A) Size and number of RER whorls in 20 random fields from unfed and amino acid fed mosquitoes after 30 min. and 120 min. B) Total whorl in the same sample as shown in A. Statistical analysis using Pearson Chisquare test revealed that the total whorl area in amino acid fed mosquitoes at both 30 min and 120 min post-feeding was significantly less than the total whorl area in unfed mosquitoes (bars with different letters signify significance at p,0.001 comparing 30 min and 120 min to unfed mosquitoes). doi:10.1371/journal.pone.0018150.g002 the identified proteins could be assigned to microsomes (including ribosomal proteins and protein synthesis related proteins), ,19% were mitochondrial proteins, ,37% were cytoplasmic proteins, and ,14% were from other cellular fractions, including the nucleus. Eight of the microsomal proteins that were present in the protein sample from unfed mosquitoes, were chosen as candidate whorl-associated proteins based on their known role in ER or Golgi processes (Table 1 ). Based on peptide frequency, the most often represented protein was SND1 (Staphylococcal nuclease domain-containing protein 1), which is also known as Tudor domain-containing protein 11 [27] . Although SND1 is assigned to the Golgi apparatus cellular component by GO analysis, its primary function is gene regulation and RNA processing [28] . The peptide frequency of SND1 was similar in protein samples from unfed and fed mosquitoes, even though EM analysis revealed that the majority of RER whorl unwinding had already occurred by 30 min post amino acid feeding ( Figure 1C ). This result suggests that SND1 is most likely not associated with whorl maintenance since its presence in microsomal fractions is independent of whorl formation.
Besides PDI, we also identified three of the seven known coatomer subunits of the COPI vesicle transport system. As shown in Table 1 , more peptides corresponding to the alpha-COPI, beta-COPI, beta'-COPI, subunits were identified in the microsomal fractions isolated from unfed mosquitoes than fed mosquitoes ( Table 1 ), indicating that endosomal membranes in these fractions were depleted of COPI subunits. Since COPI subunits are soluble proteins that transiently cycle between vesicle membrane bound and unbound forms as a function of interactions with Arf proteins [29, 30] , the observed differential abundance of COPI subunits in the two midgut microsomal fractions suggests that they could be associated with RER whorls. We tested this idea by knocking down alpha-COPI expression using RNAi to determine if this COPI coatomer subunit is required for RER whorl structures in mosquito midgut epithelial cells.
In order to test the role of alpha-COPI in RER whorl formation, we used an efficient dsRNA based RNAi protocol we previously developed for knocking down expression of abundant midgut proteases in Ae. aegypti [31] . As shown in Figure 5 , the mean level of alpha-COPI transcripts in midguts of individual dsRNA-injected mosquitoes was .90% lower than the mean level in both uninjected mosquitoes and mosquitoes that had been injected with a control dsRNA from the firefly luciferase gene (Fluc). Since 100% of the alpha-COPI dsRNA injected mosquitoes we analyzed showed a similar decrease in alpha-COPI transcript levels ( Figure 5 ), we used this dsRNA injection protocol to knock down alpha-COPI expression in mosquitoes that were subsequently maintained on sugar alone (unfed), or fed an amino acid meal and sacrificed 120 min post-feeding.
As shown in Figure 6 , EM analysis of representative midguts from alpha-COPI and Fluc dsRNA injected mosquitoes showed that while large characteristic RER whorls were present in the midguts of unfed Fluc dsRNA injected mosquitoes ( Figures 6A, 6B) , the RER present in unfed alpha-COPI dsRNA injected mosquitoes was disorganized and RER whorl structures were absent ( Figures 6C,  6D) . RER reorganization at 120 min post-feeding in Fluc dsRNA . Western blot analysis using a KDEL-specific antibody shows differential expression of four ER resident midgut proteins in response to amino acid feeding in Ae. aegypti mosquitoes. Total midgut protein extracts were prepared from three day old female mosquitoes that were unfed, or amino acid fed and dissected at 30 min. or 120 min. post-feeding. Proteins were resolved by 12% SDS-PAGE gel and Western blotted as described in Materials and Methods. The four unique protein bands were labeled KDEL-1 through KDEL-4 to denote their relative molecular weight, with KDEL-1 being the largest protein. Western blotting with an antibody that recognizes the ubiquitously expressed glyceraldehyde-3P dehydrogenase protein (GAPDH) was performed to control for equal protein loading. doi:10.1371/journal.pone.0018150.g003 injected mosquitoes looked similar to that of uninjected mosquitoes at the same time point, in that few if any whorls were present (compare Figures 7B and 1D ). As shown in Figure 8 , by quantitating the number and size of whorls in midgut epithelial cells from alpha-COPI and Fluc dsRNA injected mosquitoes, it is clear that loss of alpha-COPI expression in unfed mosquitoes was associated with a significant decrease in RER whorl formation. Moreover, a similar quantitative analysis of midgut epithelial cells from amino acid fed alpha-COPI dsRNA injected mosquitoes, showed highly disorganized endosomal structures and extended regions of swollen RER ( Figures 7C and 7D ). Taken together, these data indicate that functional alpha-COPI protein is required for normal whorl formation in the midgut epithelial cells of unfed mosquitoes.
Blood meal digestion in Ae. aegpti mosquitoes requires the synthesis and secretion of numerous proteases, the best characterized of which are serine proteases. The early trypsin protease (AeET) is synthesized from pre-existing mRNA and secreted into the lumen within the first 6 hr PBM, whereas the late phase serine proteases AaSPVI, AaSPVII, and AaLT, are transcribed, translated, and secreted between 18-30 hr PBM [31] . To determine if alpha-COPI functions are required for the synthesis and secretion of early and late phase serine proteases, we injected mosquitoes with Fluc and alpha-COPI dsRNA 3 days prior to blood feeding and used Western blotting to detect protease protein expression at 3 hr PBM (AeET) and 24 hr PBM (AaSPVI, AaSPVII, AaLT). As shown in Figure 9A , the pattern of AeET protein expression is similar in Fluc and alpha-COPI dsRNA injected mosquitoes, suggesting that alpha-COPI functions are not required for early phase blood meal digestion. However, as shown in Figure 9B , 100-400 ng of alpha-COPI dsRNA blocks expression of all three abundant late phase proteases at 24 hr PBM in a dose-dependent manner. Lack of late phase serine protease expression in alpha-COPI deficient mosquitoes was associated with ejection of the undigested blood meal between 12-30 hr PBM (data not shown).
Blood feeding is required by Ae. aegypti mosquitoes to obtain the necessary protein-derived nutrients for completion of the gonotrophic cycle [32] . Understanding the biochemical and cellular regulation of blood meal metabolism will provide insights into this critical process in Ae. aegypti, as well as other blood-feeding arthropods that function as vectors of blood borne pathogens. Our objective in the studies reported here was to first determine if amino acids were sufficient to induce RER whorl unwinding, and if they were, to use a combination of proteomic and RNAi approaches to identify proteins that may be required for maintenance of RER whorls in midgut epithelial cells. It is possible that RER whorls function by providing a mechanism to rapidly activate midgut secretion pathways upon feeding, rather than expend energy maintaining them in the absence of feeding.
Biochemical studies have shown that in addition to globin, albumin, and immunoglobulin proteins, which make up 80% of the soluble proteins in human blood, free amino acids are also present in blood [21] . Based on physiological studies showing that artificial amino acids meals are sufficient to induce early events in the blood digestion process [33] , it has been proposed that amino acids could play a signaling role by inducing protease expression [24] . Consistent with this idea, we have recently shown that amino acid feeding stimulates translation of pre-existing early trypsin mRNA through activation of the TOR signaling pathway [23] . Since large ER whorl structures have been shown to exist in the midgut of Ae. aegypti and other mosquito species [2,3,4,5,6,8,9], we used EM ultrastructural analysis to test if amino acid feeding induces ER whorl unwinding. As shown in Figures 1 and 2 , our data clearly demonstrate that amino acid feeding does induce ER whorl unwinding, and moreover, that the most abundant whorls consist of rough ER membranes based on the high density of ribosomal particles.
Recently, several studies have reported the presence of ER whorl-like structures in cultured cells following different treatment conditions. For example, treatment of mouse Leydig cells with the piperazine derivative diethylcarbamazine citrate (DEC), led to the appearance of large lipid droplets, degenerative mitochondria, and giant smooth ER whorls in some cells [25] . Since DEC has been shown to disrupt ER and golgi vesicle transport processes, it could explain the formation of ER whorls. A similar loss of function study leading to the formation of ER whorls was seen in human HeLa cells in which expression of the ER protein Yip1A was knocked down by RNAi [18] . Therefore one of the functions of Yip1A could be to maintain proper ER/golgi membrane networks under normal conditions, but when it is absent, ER membranes collapse into whorl structures.
Another explanation for whorl formation is the presence of one or more abundant ER-associated proteins that stabilize stacked ER membranes in the absence of ongoing protein synthesis. Support for this model comes from studies in cultured cells showing that protein overexpression can induce ER whorl formation. Snapp et al. (2003) [15] reported that overexpression of cytochrome b5 induced the formation of smooth ER whorls, they called organized smooth ER (OSER), in transfected CV-1 cells. These same OSER structures could be observed when b5-GFP fusion proteins were over expressed, as long as they retained dimerization function in either the b5 or GFP protein domains. The authors proposed that weak noncovalent interactions between ER resident proteins on apposing stacked membranes are sufficient to maintain smooth ER whorl structures. Similar OSER structures were shown to form in HeLa cells that were infected with an adenovirus vector expressing the ER resident protein LAT linked to a heterologous protein dimerization domain [16] , or overexpression of a viral protein that associates with ER membranes in infected cells [17] .
In order to identify candidate RER whorl forming proteins in Ae aegypti midgut epithelial cells, we took advantage of the fact that an amino acid meal is sufficient to induce whorl unwinding, which simplifies both EM analysis and protein identification by mass spectrometry because it eliminates interference from abundant blood meal proteins. Moreover, since we found that RER whorl unwinding occurs rapidly once feeding begins (Figure 1 ), we were able to use a short recovery time of only 30 min to enrich for microsomal proteins that are present in the mosquito midgut prior to feeding. The LC-MS/MS analysis identified 127 proteins using microsomal midgut protein samples from unfed and amino acid fed mosquitoes. Eight proteins were considered candidate whorl associated proteins based on their known location and function in ER and Golgi membrane compartments (see Table 1 ). Since three of the candidate proteins encoded COPI coatomer subunits, and the peptide frequency was lower for each of the proteins in samples from fed mosquitoes compared to unfed mosquitoes, we chose alpha-COPI as a representative coatomer subunit and performed RNAi knockdown experiments. Data presented in Figures 6, 7 , and 8 show that loss of alpha-COPI expression is associated with the absence of RER whorls in unfed mosquitoes, as well as membrane disorganization and ER swelling in midgut epithelial cells of amino acid fed mosquitoes. These data suggest that alpha-COPI, and likely two or more of the other seven COPI coatomer subunits, are involved in RER whorl formation and maintenance in midgut epithelial cells of unfed female mosquitoes. Based on the association of RER whorls with untranslated AeET mRNA transcripts in unfed mosquitoes, we predicted that RER whorls inhibited the translation of AaET transcripts. However the data in Figure 9A clearly show that this is not the case since sugar fed alpha-COPI dsRNA injected mosquitoes lacked both RER whorls and AaET protein expression. Surprisingly, while synthesis and secretion of AeET was not altered in blood fed alpha-COPI deficient mosquitoes, expression of three abundant late phase proteases (AaSPVI, AaSPVII, AaLT) was inhibited ( Figure 9B ), indicating that COPI vesicle transport is required for later events in the blood digestion process.
The COPI vesicle transport system has been shown to function in most cells as an retrograde transport mechanism that returns golgi-modified proteins back to the ER where they function in cell signaling and metabolism [29, 30] . However, the COPI system has also been shown to function in anterograde transport in some secretory cells where it was found to be required for exocytosis of specific proteins [34] . The COPI vesicle transport system consists of seven coatomer subunits (alpha, beta, beta', gamma, delta, epsilon, zeta), which function as structural components that promote vesicle formation, a G protein that facilitates coatomer assembly and membrane budding (Arf), guanine nucleotide exchange factors (GEFs) that activate Arf proteins and thereby initiate coatomer assembly, and GTPase activating proteins (ArfGAPs) that stimulate GTP hydrolysis in Arf proteins and induce coatomer disassembly, which is required for vesicle membrane fusion. Based on our finding that loss of alpha-COPI expression in midgut epithelial cells of unfed mosquitoes disrupts RER whorl formation, without decreasing the total amount of RER membrane in cells ( Figure 6C and 6D) , we propose that COPI coatomer proteins directly contribute to whorl formation through subunit assembly. Further experiments are needed to directly test this idea, for example, by using immunogold EM analysis to determine if COPI coatomer proteins are tightly associated with RER whorls, and if so, which coatomer subunits are colocalized.
Aedes aegypti (L.) (Rockefeller strain) mosquitoes were used for all studies. Larvae were maintained on a diet consisting of equal proportions of rat chow (Sunburst Pet Foods, Phoenix, AZ), lactalbumin hydrolysate (USB, Cleveland, OH), and yeast hydrolysate (USB, Cleveland, OH). Female pupae were separated Figure 8 . Quantitation of whorl size and number in dsRNA injected unfed and amino acid fed mosquitoes. Whorls with a minimum of five stacked ER membranes were counted and the total area of each whorl was determined as described in figure 2 legend. A) Size and number of RER whorls in 20 random fields from unfed and amino acid fed mosquitoes after 120 min. that were injected with Fluc or alpha-COPI dsRNA. B) Total whorl in the same sample as shown in A. Statistical analysis using Pearson Chi-square test revealed that the total whorl area in alpha-COPI dsRNA injected unfed and amino acid fed mosquitoes was significantly less than the total whorl area in Fluc dsRNA injected unfed mosquitoes (bars with different letters signify significance at p,0.001). In addition, the total whorl area in Fluc dsRNA injected fed mosquitoes was found to be significantly less than in Fluc dsRNA injected unfed mosquitoes using the Pearson Chi-square test (bars with different letters signify significance at p,0.001). doi:10.1371/journal.pone.0018150.g008 from males using a mosquito separator. Adult mosquitoes were routinely maintained at 28uC, 70-80% relative humidity and a photoperiod of 16:8 h (L:D), on 10% sucrose ad libitum.
Preparation of amino acids meal or feeding buffer for mosquito feeding Amino acid meals was prepared according to Noriega et al. [33] with modifications. Briefly, the amino acids meal consisted of 40 ml of amino acid-deficient M199 media (dM199) (Invitrogen Corporation, Carlsbad, CA), 1.6 ml of 1006 MEM nonessential amino acid solution (Mediatech, Inc., Herndon, VA), 3.2 ml of 506 MEM amino acid solution (Mediatech, Inc.), and 10 mg of HEPES (Sigma, St. Louis, MO) (pH 7.2), with a final concentration of 2.3 mg/ml total amino. Just before feeding mosquitoes, ATP (Sigma) was added to the meal to a final concentration of 5 mM. The feeding buffer was composed of 100 mM NaHCO 3 and 150 mM NaCl, pH 7.2, as described by Kogan [35] .
Dissected midgets were fixed in 2.5% glutaraldehyde+2% formaldehyde in 0.1 M PIPES buffer (pH 7.4) for 1 hr at room temperature, washed in buffer, post-fixed in 1% osmium tetroxide in buffer for 1 hr, washed in deionized water, and stained with 2% aqueous uranyl acetate for 30 mins. Specimens were dehydrated through an ethyl alcohol series, infiltrated with Spurr's resin and flat embedded at 60uC. Longitudinal sections (50 nm) were cut on a Leica UC2T ultramicrotome onto uncoated 150 mesh copper grids, counter-stained with lead citrate, and viewed in an FEI CM12S electron microscope operated at 80 kV. TIFF mages (8 bit) were collected via an AMT 4 M pixel camera and used for image analysis.
Twenty sequential and adjacent visual fields from each EM slide were imaged at a magnification of 8800. Total area of the ER whorls in 20 visual fields of electron microscope was measured with ImageJ (NIH), which covered a total area of 3335 mm 2 . Only ER membrane structures containing more than four membrane stacks were considered to be whorls and used for area measurements. The data were statistically analyzed as pixels per 20 visual fields using Pearson Chi-square test with SPSS for Windows (v11.5).
Western blots of early phase (AeET) and late phase (AaSPVI, AaSPVII, AaLT) protease expression in unfed and blood fed dsRNA injected mosquitoes were performed as previously described [31] . Analysis of KDEL containing proteins by Western blotting was done by dissecting fifty midguts from amino acid fed or unfed (sugar fed) female mosquitoes in pre-cold PBS buffer, dipped into 76Protease Inhibitor Cocktail in 100 mM phosphate buffer (pH 7.0) (Roche Applied Science, Germany) with a forceps, and transferred into an ice-cold 1.5-ml Eppendorf tube containing 60 ml of PBS/TDS buffer (1% Triton X-100, 12 mM Na deoxycholate, 0.2% SDS in PBS, and Protease Inhibitor Cocktail). The dissected midguts were homogenized using a blue Kontes pestle. The homogenate was incubated on ice for 10 min and then spun at 10000 rpm for 10 min at 4uC. The supernatant was transferred to a pre-chilled 1.5-ml tube and 56SDS sample loading buffer was added. The mixture was boiled for 4 min, chilled on ice for 2 min, and then spun at 13000 rpm for 5 min at room temperature. The yielded supernatant was stored at 220uC for SDS-PAGE.
Protein samples normalized to equal midgut equivalents were separated on 12% SDS-PAGE using standard procedures. PageRuler TM Prestained Protein Ladder (Fermentas, USA) was used as a protein standard. The proteins were transferred onto Odyssey Nitrocellulose Membranes (LI-COR Inc. Lincoln, Nebraska, USA). The membranes were dried in the air for 1 h and blocked at room temperature with 4% nonfat dry milk in 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 10% Tween-20 (TBST), and then incubated with mouse monoclonal antibody against the peptide sequence SEKDEL conjugated to KLH (Abcam Inc. Cambridge, MA, USA). Loading controls were performed using rabbit polyclonal antibody against full length native GAPDH protein from human erythrocytes (Abcam, USA) at 1:1000 dilution in 4% nonfat milk TBST solution at 4uC overnight. After washing with TBS containing 0.1% Tween-20 (TBST), the membranes were incubated with goat anti-rabbit IRDyeH 800CW or goat anti-mouse IRDyeH 800CW secondary antibody (LI-COR Biosciences, USA) at a dilution of 1:10000 in TBST/4% nonfat dry milk for 1 h at room temperature. Labeled proteins were visualized using Odyssey Infrared Imaging System (LI-COR Biosciences, USA).
About 40 midguts from the fed or unfed female mosquitoes were dissected with pre-cold PBS buffer, dipped into 76Protease Inhibitor Cocktail in 100 mM phosphate buffer (pH 7.0) (Roche Applied Science, Germany) with a forceps, and transferred into an Figure 9 . A deficiency in alpha-COPI inhibits feeding-induced expression of late phase midgut proteases. A) Representative Western blot of AeET protein expression in the midguts of mosquitoes injected with 400 ng of Fluc or alpha-COPI dsRNA and maintained on sugar for 3 days and then dissected (unfed), or blood fed after 3 days of sugar feeding and dissected at 3 hr PBM (fed). The GAPDH antibody was used as a protein loading control. Each lane contains the same midgut equivalents obtained from pooled mosquitoes. B) Representative Western blots of protein extracts prepared from pooled mosquito midguts dissected at 24 hr PBM and analyzed with antibodies against late phase serine proteases (AaSPVI, AaSPVII, AaLT) or GAPDH. Mosquitoes were injected with the indicated amount of dsRNA 3 days prior to blood feeding. doi:10.1371/journal.pone.0018150.g009
ice-cold 1.5-ml Eppendorf tube containing 100 ml of 16Isotonic Extraction Buffer (5 mM HEPES;pH 7.8, 0.25 M sucrose, 1 mM EGTA, 25 mM KCl), and an appropriate amount of Protease Inhibitor Cocktail. Mosquito midgut ER protein extraction was performed according using the Endoplasmic Reticulum Isolation kit (ER0100; Sigma, St. Louis, USA) with modification. Briefly, the dissected midguts were homogenized with a blue Kontes pestle and spun at 1000g for 10 min at 4uC. The post nuclear supernatant was transferred into a clean 1.5-ml tube and spun at 12000g for 15 min at 4uC. The post mitochondrial supernatant was transferred into a clean 1.5-ml tube and mixed with 7.5 volumes of 8 mM CaCl 2 by vortexing. The mixture was incubated on ice for 10 min and then spun at 8,000g for 10 minutes at 4uC. The supernatant was discarded, and the pellet containing the microsomal fraction, was resuspended in 20 ml of 16 Isotonic Extraction Buffer followed by a 10-min of incubation on ice. A 5 ml portion of the extracted ER protein fraction was used for measuring protein concentration with the BCA TM Protein Assay Kit (Pierce, USA). The remaining ER proteins were mixed with an appropriate amount of 56SDS sample loading buffer, boiled for 4 min, and spun at 130006 rpm for 5 min. The supernatant was stored at 220C for SDS-PAGE.
Equal amounts of ER proteins isolated from midguts of unfed and fed mosquitoes were separated by 12% SDS-PAGE and visualized using the GelCodeH Blue Staining Kit (Thermo Scientific, Rockford, IL, USA). Each gel lane was cut into 4 equal slices using a scalpel, and labeled as A, B, C, and D from high to low molecular weight regions. The gel slices were stored individually stored in a clean Eppendorf tube for in-gel digestion. Each gel slice was washed first with ddH 2 O for 15 min, then twice with 50% acetonitrile (ACN), and finally with 100 mM ammonium bicarbonate (Ambic, pH 8.0) containing 50% ACN for 15 min. After drying, the gel pieces were subjected to the standard in-gel digestion protocol. Briefly, proteins were reduced by 10 mM DTT/100 mM Ambic at 56uC for 45 min., and alkylated by 55 mM iodoacetamide (IAA)/ 100 mM Ambic at room temperature in the dark for 30 min. Gel pieces were washed with 100 mM Ambic dehydrated with ACN and dried. Proteolytic digestion was performed with 12.5 ng/ml trypsin dissolved in 100 mM Ambic and incubated on ice for 45 min. The digested mixture was acidified with 2% trifluoroacetic acid (TFA) in water for 1-2 minutes and then the supernatant was collected in a clean 1.5-ml tube. The peptides were extracted from the gel slice using 0.1% TFA in water, 0.1% TFA in 30% ACN, and 0.1% TFA in 60% ACN, respectively, with ultrasonication. The peptides extracted in the four steps were combined together, concentrated by a SpeedVac to a desired volume and subjected to LC-MS/MS analysis.
Mass spectrometry analysis was performed by the Chemistry & Biochemistry department proteomics core facility using in-house protocols. Briefly, trypsin digested protein samples were acidified with TFA and diluted to 20 ml prior to separation by C18 column (75 um61 mm, LC Packings, Amsterdam, Netherlands) at a flow rate of 30-500 nl/min and introduced into Finnigan LTQ (Thermo electron corporation) through nano spray (2.8 kV). Liquid chromatography mobile phases consisted of Solution A (90% water, 10%methonal, 0.5% formic acid, 0.01% TFA) and Solution B (98% methanol, 2% water, 0.5% formic acid, 0.01% TFA). A 120min linear gradient from 0 to 90% B was typically used. Samples were subjected to nanoelectrospray mass spectrometry using standard procedures and data were analyzed using Sequest (ThermoFinnigan, San Jose, CA; version27, rev. 12) and X! Tandem (www.thegpm.org; version 2007.01.01.1) using the Aedes aegypti database. Scaffold (Proteome Software Inc., Portland, OR) was used to validate MS/ MS based peptide and protein identifications. Sequest identifications required at least DCn scores of greater than 0.08 and XCorr scores of greater than 1.8, 2.5, 3.5 for singly, doubly, triply charged peptides. X! Tandem identifications required at least 2Log(Expect Scores) scores of greater than 3.0.
ER proteins listed in Table 1 and Table S1were RNAi-mediated knockdown of alpha-COPI expression and validation by QRTPCR alpha-COPI gene was subjected to a RNAi according to our previous study [31] . Briefly, a DNA fragment of alpha-COPI gene was amplified by PCR (forward primer containing the T7 promoter sequence: 59 TAATACGACTCACTATAGGGAGA TGCTGACAAATTTCGAAACCAA 39; and reverse primers containing the T7 promoter sequence: 59 TAATACGACTCAC TATAGGGAGATCCGTCGCCGTAGGATTCTT 39) and cloned into the pGEM-T easy vector (Promega). Subsequently, a double-strand RNA (dsRNA) was synthesized in vitro transcription using the MEGAscript RNAi Kit (Ambion). Two-day old female mosquitoes were injected with 400 ng of dsRNA using a Nanoject II microinjector (Drummond Scientific). The knockdown efficiency of mRNA encoding alpha-COPI was determined using mosquitoes injected with Fluc (firefly luciferase) dsRNA as a control. QRT-PCR was performed using PerfeCTa SYBR Green FastMix (Quanta BioSciences) by Real-Time PCR (7300 Real-Time PCR System, Applied Biosystems) with alpha-COPI forward primer: 59 GTGTCCGCATCGTTGGATCA 39, alpha-COPI reverse primer: 59 ACAACAGCATCAGCTTGCCCAA 39. Ribosomal protein S7 mRNA levels were used as an internal control for normalization. Statistical analysis of the gene expression was done by unpaired student t test using GraphPad Prism software (GraphPad Software, Inc.).
Table S1 Proteomic analysis of Ae. aegypti midgut microsomal proteins isolated from unfed (sugar fed) and amino acid fed (30 min. post-feeding).
(DOC) paper: GZ JI RLM. Edited the manuscript: GZ JI WAD RLM. Contributed equally in all aspects: GZ JI. Interaction of a Specific Population of Human Embryonic Stem Cell–Derived Progenitor Cells with CD11b+ Cells Ameliorates Sepsis-Induced Lung Inflammatory Injury Human embryonic stem cells differentiated under mesoderm-inducing conditions have important therapeutic properties in sepsis-induced lung injury in mice. Single cell suspensions obtained from day 7 human embryoid bodies (d7EBs) injected i.v. 1 hour after cecal ligation and puncture significantly reduced lung inflammation and edema as well as production of tumor necrosis factor-α and interferon-γ in lungs compared with controls, whereas interleukin-10 production remained elevated. d7EB cell transplantation also reduced mortality to 50% from 90% in the control group. The protection was ascribed to d7EB cell interaction with lung resident CD11b+ cells, and was correlated with the ability of d7EB cells to reduce it also reduced production of proinflammatory cytokines by CD11+ cells, and to endothelial NO synthase–derived NO by d7EB cells, leading to inhibition of inducible macrophage-type NO synthase activation in CD11b+ cells. The protective progenitor cells were positive for the endothelial and hematopoietic lineage marker angiotensin converting enzyme (ACE). Only the ACE+ fraction modulated the proinflammatory profile of CD11b+ cells and reduced mortality in septic mice. In contrast to the nonprotective ACE-cell fraction, the ACE+ cell fraction also produced NO. These findings suggest that an ACE+ subset of human embryonic stem cell–derived progenitor cells has a highly specialized anti-inflammatory function that ameliorates sepsis-induced lung inflammation and reduces mortality. Lung inflammatory injury from septic shock is the leading cause of death in patients in the intensive care unit, 1 with mortality remaining at ϳ40%. 2 The disease is characterized by progressive respiratory failure with bilateral alveolar infiltrates and lung edema. 3 Transplantation of adult bone marrow-derived mesenchymal stromal cells, endothelial progenitor cells, and bone marrow-derived progenitor cells has been studied in models of sepsis 4 -11 ; however, the results have varied, and specific cell populations responsible for the protection have not been characterized. Although in some cases transplanted cells differentiated into specialized parenchymal cells, 7,10 the lung repair observed may also be secondary to immunomodulatory effects of the transplanted cells. 4, 6, 8 Previous studies have not addressed the effects of a well-defined progenitor population derived from embryonic stem cells (ESCs) in resolution of sepsis-induced lung injury. Because ESCs are pluripotent, it was surmised that specific progenitors derived from ESCs could effectively mitigate sepsis-induced lung inflammation and injury. Using blast progenitor cells from human ESCs (hESCs) cultured in conditions favoring development of mesoderm, 12 the present study addressed the role of a purified population of progenitor cells in the lung response to polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that transplantation of hESC-derived progenitor cells after induction of sepsis reduced lung inflammation and edema formation, and it also reduced production of proinflammatory cytokines tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) without affecting production of the anti-inflammatory cytokine interleukin (IL)-10. Recipient mice also demon-strated marked reduction in mortality. Dampening of lung inflammation was the result of progenitor cells enriched with the endothelial and hematopoietic progenitor cell marker angiotensin-converting enzyme (ACE) and was largely ascribed to the interaction of these cells with CD11bϩ cells in lungs. This interaction in turn mediated reduction in production of proinflammatory cytokines and high-output NO production by CD11bϩ cells.
hESCs (H1, XY, WiCell, and National Institutes of Healthapproved WA01) were maintained on mitomycin-blocked mouse embryonic fibroblast feeders in hESC growth medium (Dulbecco's modified Eagle's medium and Ham nutrient mixture F-12) supplemented with 15% knockout serum replacement enriched with 4 ng/ml of human basic fibroblast growth factor-2, 1ϫ nonessential amino acid, 1ϫ glutamax-I, and 1ϫ ␤-mercaptoethanol (all from Invitrogen Corp., Carlsbad, CA). Half of the medium was changed every 48 hours until the colonies were close to confluence. For differentiation induction, 2 to 2.5 ϫ 10 6 hESCs were resuspended in 3 ml of stem cell medium (HEScGro; Millipore Corp., Billerica, MA) supplemented with 50 ng/ml of vascular endothelial growth factor and 50 ng/ml of bone morphogenetic protein-4, plated in one well of a six-well plate (Ultra-Low; Corning Inc., Corning, NY), and incubated at 37°C with 5% CO 2 . After 24 hours, 40 ng/ml of stem cell factor, 40 ng/ml of thrombopoietin, and 40 ng/ml of Fms-related tyrosine kinase-3 (Flt3) ligand (R&D Systems, Inc., Minneapolis, MN) were added to the cultures, followed by 25 ng/ml each of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-6, and IL-3, and 3 U/ml of human erythropoietin at day 3½ of differentiation culture.
d7EB cells were fractionated using fluorescein-activated cell sorting (FACS) for ACE and kinase insert domain receptor (KDR) expression. The isolated fractions were subcultured on fibronectin-coated plates in the presence of endothelial cell basal medium and 20 ng/ml of stem cell factor, 20 ng/ml of thrombopoietin, 20 ng/ml of Fmsrelated tyrosine kinase-3 ligand, 25 ng/ml each of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-6, and IL-3, and 1 U/ml of human erythropoietin.
Studies were performed using 6-to 8-week-old male CD1 mice (Jackson Laboratory, Bar Harbor, ME), which were housed in pathogen-free conditions at the University of Illinois animal care facility. Experimental sepsis was induced via CLP performed as previously described. 13 In brief, after proper sterilization, the cecum was exposed via a midline abdominal incision and ligated at 75% of the distance between the distant cecal pole and the base of the cecum, followed by a 21-gauge needle puncture in a mesenteric toward antimesenteric direction. Wound closure was achieved using separate sutures (6-0 nylon) to the abdominal musculature and the skin. Immediately after the procedure, 500 L of warmed normal saline solution was administered subcutaneously. For survival studies, the mice were monitored every 6 hours for 24 hours, and thereafter every 12 hours for 5 days. All cell injections were administered 1 hour after CLP via i.v. administration through the facial vein. Studies were performed in accordance with institutional guidelines, and approval was obtained from the institutional review board.
In some xenotransplantation studies, cyclosporine A (CsA) was administered as an oral solution diluted with corn oil at a concentration of 10 mg/ml. The CsA was administered p.o. via gavage at a dose of 75 mg/kg/d, beginning 1 day before CLP and continuing for another 24 hours after the procedure. 14 Pilot experiments demonstrated efficiency of this dosage in inhibiting production of IL-2 and IFN-␥ by T cells isolated from mouse spleens.
Lung tissue neutrophil (polymorphonuclear leukocyte) uptake was assessed via determination of myeloperoxidase activity. Lungs were dried and homogenized in 1.0 ml of PBS (50 mmol/L, pH 6.0) with 5% hexadecyl-trimethylammonium bromide and 5 mmol/L of EDTA. Homogenates were sonicated, centrifuged at 4 ϫ 10 4 g for 20 minutes and were frozen and thawed twice, followed by homogenization and centrifugation. The supernatant was mixed 1:30 (v/v) with assay buffer (0.2 mg/ml of o-dianisidine hydrochloride and 0.0005% H 2 O 2 ), and absorbance change was measured at 460 nm for 3 minutes. Myeloperoxidase activity based on dry lung weight was calculated as the change in absorbance over time. 15 
Lung capillary filtration coefficient was measured to quantify lung vascular permeability, as described previously. 16 Venous outflow pressure was elevated using a computer-controlled two-way electronic pinch valve (P/N 98301-22; Cole-Parmer Instrument Co., Vernon Hills, IL) that channeled venous fluid into silicon tubing (1/16-inch i.d.). The weight change resulting from the venous pressure increase of 6 cm of water was recorded for 5 minutes. The weight recording has two exponential components that reflect the rapid expansion of vascular volume and the slower phase of transvascular fluid filtration. The amount of fluid filtered in 5 minutes was determined via logarithmic extrapolation of the slower component to time 0. The lung capillary filtration coefficient, a measure of lung vascular liquid permeability, was computed as milliliters per minute per centimeter of water per gram dry weight by normalizing the estimate of filtered fluid by time, venous pressure change, and lung dry weight.
At 6 and 12 hours after CLP, lungs were removed from euthanized mice, minced into small pieces, and incubated in RPMI-1640 medium with 1% penicillin-streptomycin and 1% glutamine for 30 minutes at 37°C and at 5% CO 2 in the presence of 1 mg/ml of collagenase type 1 and 50 U/ml of deoxyribonuclease I. After incubation, cells were filtered through a 70-mm cell strainer and washed with RPMI medium. The cells were incubated for 15 minutes at 4°C with CD11b magnetic beads (Miltenyi Biotek, Inc., Auburn, CA) and subsequently applied to MS columns (Miltenyi Biotek, Inc.) for their positive selection. Negatively selected cells were also collected.
For CD11bϩ depletion studies, 24 hours before experimental procedures, mice were injected i.v. with gadolinium chloride, 10 g/g body weight; anti-Gr-1, 50 g per mouse; and anti-NK1.1, 50 g per mouse. Depletion of CD11bϩ cells was verified using flow cytometry.
Mouse lungs were inflated with 10% formalin and embedded in paraffin. Formalin-fixed paraffin-embedded tissue samples were cut into 5-m sections, mounted on slides (Starfrost Plus; Maenzel Glaeser, Braunschweig, Germany), and hydrated using an alcohol gradient. Slides were rinsed in distilled water, followed by antigen unmasking using a 10ϫ concentrated retrieval solution (antigen decloaker solution; Biocare Medical Inc., Concord, CA) according to the manufacturer's instructions, and rinsed in PBS for 5 minutes. For detection of human laminin A1, tissue sections were blocked with H 2 O 2 blocking reagent for 10 minutes at room temperature. Slides were treated with a protein-blocking solution for 10 minutes at room temperature, rinsed, and incubated with human anti-laminin monoclonal antibody (L8271, clone LAM 89; Sigma Aldrich Corp., St Louis, MO) at a titer of 1:1000 for 30 minutes at room temperature, followed by anti-mouse horseradish peroxidase (EnVision FLEXϩ autostainer kit; Daco A/S, Glostrup, Denmark). Slides were rinsed and treated using the mouse-on-mouse polymer kit (MM510; Biocare Medical Inc.) for 30 minutes at room temperature. Laminin staining was detected using a betazoid diaminobenzidine kit (Biocare Medical Inc.). Human tonsil tissue stained with anti-laminin monoclonal antibody was used as positive control, and the same tissue stained only with the secondary anti-mouse horseradish peroxidase served as the negative control.
Immunocytochemistry d7EB cells were fractionated using FACS according to their cell surface expression of ACE and KDR and were subcultured on 35-mm fibronectin-coated dishes on top of No. 1.5 coverslips (0.16Ϫ0.19 mm). After 2 weeks, the cells were washed twice with PBS, fixed with paraformaldehyde 4% in PBS for 30 minutes, washed with 100 mmol/L of glycine in PBS, and incubated for 1 hour with blocking buffer containing 2% to 5% normal donkey serum, 0.2% bovine serum albumin, and 0.1% Triton X-100. Subsequently, they were incubated for 2 hours at room temperature with primary antibodies against human vascular endothelial cadherin, von Willebrand factor, and CD43 at 1:200 dilution in 2% normal donkey serum, 1% bovine serum albumin, and 0.1% Triton-X in PBS. Control cells were incubated with goat IgG, rabbit IgG, and mouse IgG 1 , respectively, under the same conditions. After washing with PBS and repeat blocking for 1 hour, the cells were incubated with secondary antibodies Alexa 488 anti-goat IgG, Alexa 568 anti-rabbit IgG, or Alexa 568 anti-mouse IgG1 at 1:700 dilution for 1 hour at room temperature.
Single cell suspensions were created from d7EB cells via brief trypsinization. The cells were then fluorescently labeled via incubation with 10 mol/L of carboxy-fluorescein diacetate (Green Tracker, C2925; Invitrogen Corp.) in serum-free medium for 30 minutes at 37°C. Labeling was confirmed at fluorescence microscopy, and cells were kept on ice until use. Carboxy-fluorescein diacetate-labeled green fluorescent cells, 5 ϫ 10 5 , were injected i.v. through the facial vein. For cell tracking studies, mice were sacrificed at 6, 18, and 24 hours after injection, and 5-m snap-frozen lung sections were cut. Tissues were counterstained with DAPI (4=,6-diamino-2phenylindole) and examined for native green fluorescence at confocal microscopy (Zeiss LSM 510 META confocal microscope; Carl Zeiss AG, Oberkochen, Germany). Sections were graded using a semiquantitative scale established by counting the number of fluorescent cells per 100 nuclei per power field examined at 40ϫ magnification (0 ϭ no florescence, 1 ϭ Յ1 fluorescent cell, 2 ϭ 1 to 5 fluorescent cells, 3 ϭ Ͼ5 fluorescent cells). Ten power fields per slide were examined by two independent reviewers blinded to the animal group.
Extravascular lung water content was measured via determination of lung wet-to dry-weight ratios in which intravascular lung water was corrected using a hemoglobin assay of lung homogenates and peripheral blood. 4
The following antibodies were used for flow cytometry studies and FACS: mouse anti-CD11b Alexa Fluor 488 -conjugated (clone M1/70; isotype, rat IgG 2b -488); mouse anti-F4/80 (clone 6F12; isotype, rat IgG 2a ); mouse anti-NK1.1 PE-conjugated (clone PK136; isotype, mouse IgG 2a -PE); mouse anti-Gr1 PE-conjugated (clone RB6-8C5; isotype, mouse IgG 2a -PE); rat anti-mouse IFN-␥ (clone XMG 1.2; isotype, rat IgG 1 ); rat anti-mouse IL-10 (clone JES5-16E3; isotype, rat IgG 2b ); biotin mouse anti-human TLR4 (clone HTA 125; isotype, mouse IgG 2a -biotin); and biotin anti-Annexin V (all from BD Biosciences Pharmingen, San Di-ego, CA); mouse anti-KDR PE-conjugated (isotype, mouse IgG 1 -PE) and mouse anti-ACE fluorescein isothiocyanateconjugated (isotype, goat IgG-fluorescein isothiocyanate) (both from R&D Systems, Inc.); rat anti-mouse TNF-␣ (clone MP6-XT22; isotype, rat IgG 1 ; Invitrogen Corp.); rabbit polyclonal anti-NOS 2 , (clone N-20; isotype, goat IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and NO-Cu-Fl, final concentration 10 mol/L (Intracellular Nitric Oxide Sensor Kit, catalog No. 96 -0293; Strem Chemicals, Inc., Newburyport, MA). For intracellular staining, mouse lung cells were first fixed via incubation using 100 L of fixation buffer (eBioscience, Inc., San Diego, CA) for 20 minutes at room temperature, and washed twice with 1 ml of permeabilization buffer (eBioscience, Inc.). Consequently, 1 to 5 g/10 6 cells of antibodies against TNF-␣, IL-10, IFN-␥, or inducible NO synthase (iNOS) were then added in 100-L volume of permeabilization buffer, and cells were incubated for 30 minutes at 4°C. After an additional washing with 1 ml of permeabilization buffer, the cells were stained with secondary anti-rat-fluorescein isothiocyanate or anti-rabbit-PE (iNOS) conjugated antibody for 20 minutes at 4°C. The cells were resuspended in flow cytometry buffer and analyzed immediately. Intracellular NO staining with fluorescentbound copper was performed per the manufacturer's instructions and in accordance with published protocols. 17, 18 Surface and intracellular marker expression were analyzed using software (LSR and CellQuest Pro; Becton Dickinson & Co., San Jose, CA).
Enzyme-linked immunosorbent assays were performed on supernatants of lung homogenates and cell culture supernatants using kits for mouse-specific TNF-␣ (Mouse TNF-␣/TNFSF1A Quantikine ELISA Kit, MTA00); IFN-␥ (Mouse IFN-␥ Quantikine ELISA Kit, MIF00); and IL-10 (Mouse IL-10 Quantikine ELISA Kit, M1000) (all from R&D Systems, Inc.) according to the manufacturer's instructions.
Single-cell suspensions of mouse lung CD11bϩ and CD11bϪ cells were prepared and plated in 24-well plates at a concentration of 10 6 cells in 1 ml of complete RPMI-1640 for 1 hour. The supernatants were removed, and 2 ϫ 10 5 d7EB cells in fresh medium were added either directly with the mouse cells or on the insert membrane of the Transwell system (HTS Transwell 0.4-m pore size polycarbonate membrane; Corning, Inc.). As a control, CD11bϩ cells were plated without d7EB cells. The co-cultured cells were or were not stimulated with 1 g/ml of lipopolysaccharide (LPS) for 6 hours. At the end of the stimulation period, the supernatants were collected, and mouse-specific ELISAs (R&D Systems, Inc.) were performed on samples to detect the released mouse TNF-␣, IFN-␥, and IL-10. This protocol was repeated for the experiments with ACE and KDR sorted d7EB cells.
Concentrations of NO and nitrite were determined using an NO analyzer (NOATM280; Sievers Instruments, Inc., Boulder, CO). NO concentrations were determined via analysis of nitrite accumulation in the culture supernatants of co-cultured d7EB cells and CD11bϩ cells or CD11bϩ cells alone. Media aliquots were collected at the indicated times, and were injected directly into the analyzing cell containing the reducing solution (saturated sodium iodide in 50% acetic acid). The analysis was conducted at room temperature. Authentic NaNO 2 solutions of known concentrations were used as standards. For determination of NO production from human d7EB cells, cultures were washed twice with fresh serum-free media. Medium containing 1 g/ml of LPS was replenished, and cells were undisturbed for the duration of the experiment. Media samples were collected at selected times, and NO production was assessed as nitrite accumulated in the media.
Single-cell suspensions of CD11bϩ cells were prepared from septic mice as described above. The cells were plated in 24-well plates at a concentration of 10 6 cells in 1 ml of complete RPMI-1640 medium for 1 hour. The supernatants were removed, and fresh medium was added in the presence or absence of LPS, 100 ng/ml, plus a series of concentrations of the NO donor diethylenetriamine (DETA) NONOate for 12 hours. The range of concentrations was chosen to mimic approximately the amount of NO produced by d7EB cells as determined by direct measurements as described above. At the end of the experiment, supernatant free of cells was collected for cytokine measurements via ELISA.
Human d7EB cells were plated in 24-well plates at concentration of 2 ϫ 10 6 cells in 1 ml of growth medium. Cells were cultured overnight in the presence or absence of LPS, 1 g/ml. The supernatant was collected, spun down for 10 minutes at 500g to remove possible cell contamination, and injected i.v. into septic mice 1 hour after CLP at a volume of 200 L per mouse.
CD11bϩ cells were either co-cultured with d7EB cells as described above, exposed to DETA NO, or cultured alone. After stimulation with LPS, 1 g/ml, for 12 hours, the cells were lifted by gentle scraping. Human cells, which do not express CD11b, were removed via positive selection using CD11b magnetic beads (Miltenyi Biotek, Inc.). Live CD11bϩ cells were lysed in 1ϫ radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail, 60 L/10 ml PBS (Sigma-Aldrich Corp.). Lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. Supernatants were collected, and the protein concentration of each sample was measured using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Chemical Co., Rockford, IL). For each sample, 50 g of protein was loaded onto lanes of Nu-PAGE 4% to 12% Bis-Tris gel (Invitrogen Corp.). Proteins were transferred to nitrocellulose membranes (Millipore Corp.). After incubation in blocking solution (5% dry milk in Tris-buffered saline solution with Tween 20) at room temperature for 1 hour, membranes were immunoblotted (24 hours at 4°C) with anti-iNOS rabbit polyclonal antibody (ab3523) and anti-eNOS mouse monoclonal antibody (ab76199) (both from Abcam Inc., Cambridge, MA), followed by secondary horseradish peroxidaseconjugated goat anti-rabbit or mouse (1:1000). Peroxidase labeling was detected using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ).
Five NOD/SCID mice were injected subcutaneously with 2 ϫ 10 6 d7EB cells in the dorsal region, and the animals were observed for 6 months for development of teratomas. At the end of the observation period, the injection areas were dissected and stained with H&E. No evidence of teratomas was observed in any of the animals.
Data were analyzed and figures generated using commercially available software (Prism version 4.03; Graph-Pad Software, Inc., San Diego, CA). Quantitative data are given as mean (SEM). Comparison between transplant and control groups was made using the nonparametric Mann-Whitney test. P Ͻ 0.05 was considered significant. Survival between intervention and control groups was compared using the log-rank test.
First, the effects of d7EB cells on sepsis-induced lung inflammatory injury and death induced via CLP were determined. Because the studies involved transplantation of human progenitor cells in mice, the question of xenotransplantation was considered. In mice treated with CsA, transplantation of human d7EB cells (500,000 cells in 200 L of PBS) reduced mortality after lethal CLP, from 10% in control mice receiving mitomycin-blocked mouse embryonic fibroblasts (MEFs) to approximately 40% in the transplant group ( Figure 1A ). Although the CsA-immunosuppressed septic model proved useful in establishing a therapeutic role for human cells, there remained the important bias of pharmacologic immunosuppression in the sepsis model. Based on findings that ESCs and adult bone marrow stromal cells 19 -22 might not be strongly immunogenic because they generally lack major histocompatibility complex class II antigens, 23 the effectiveness of these cells in immunocompetent septic mice without the complicating effects of previous immunosuppression was evaluated. Human d7EB cells in these mice were as protective against CLP-induced death as demonstrated in CsA-treated mice; that is, survival improved from less than 10% at 48 hours after CLP in control mice to 50% in the transplant group ( Figure 1B) . Also investigated were alterations in lung vascular permeability and edema formation at 24 hours after CLP. d7EB cell transplantation significantly reduced neutrophilic lung inflammation and lung edema and prevented lung endothelial barrier dysfunction in treated mice compared with the control group (Figure 2, A-D) . The improvement in lung injury and survival was associated with decreased production of TNF-␣ and IFN-␥ in septic lungs, whereas there was no change in IL-10 production ( Figure 2E ). Despite the absence of immunosuppression, d7EB cells were not immediately rejected, and their presence in recipient mouse lungs was verified up to 24 hours after cell transplantation. Increased numbers of cells were observed in the recipient lungs at 1 and 3 hours after transplantation, as demonstrated by human-specific laminin staining ( Figure 3A ). The number of cells in the lungs gradually decreased, but cells were still detectable during the first 24 hours ( Figure 3B) ; however, no cells were visible at 48 hours after administration (data not shown).
To determine whether the protection induced by d7EB cells could be ascribed to secreted factors, cultured d7EB cells were stimulated overnight with LPS, 1 g/ml, or medium alone. Addition of LPS-conditioned medium obtained from d7EB cells did not alter mortality in mice that underwent CLP compared with controls injected with nonconditioned medium or PBS ( Figure 3C ), indicating that d7EB cells were essential for protection.
Because the primary source of TNF-␣ and IFN-␥ in inflamed lungs is CD11bϩ cells, that is, resident macrophages, blood monocytes, granulocytes, and natural killer cells, 24 the possibility that d7EB cell interaction with host CD11bϩ cells might induce an anti-inflammatory cytokine profile was investigated. CD11bϩ cells were isolated from lungs of septic mice treated with either d7EB cells or MEFs. These cells were then stimulated with LPS in culture in the presence of the intracellular protein transport inhibitor Golgi stop. Cytokine staining showed that lung CD11bϩ mouse cells obtained after d7EB cell transplantation produced significantly less TNF-␣ and IFN-␥ ( Figure 4A ) compared with lung CD11bϩ cells from mice receiving control MEF. These responses were not observed in CD11bϪ cells ( Figure 4A ).
Whether d7EB transplantation alone influenced the CD11bϩ cells by affecting the composition of the CD11bϩ cell population isolated from lungs of septic mice undergoing d7EB transplantation was addressed. No significant difference was observed in the number of monocytes (F4/80-positive), polymorphonuclear leukocytes (Gr-1-positive), and natural killer cells (NK1.1-positive) in the CD11bϩ populations of the d7EB recipient and control lungs ( Figure 4B) .
To examine whether cell-cell interaction mediated the lung protection induced by d7EB cell transplantation, studies were performed in mice depleted of macrophage and monocyte populations using GdCl 3 in combination with anti-Gr1 and anti-NK1.1 antibodies injected i.v. 24 hours before CLP challenge. Mortality was higher in these mice after CLP, which could not be prevented by d7EB cell transplantation ( Figure 4C) . Thus, d7EB cell interaction with macrophage or monocytic cells is important in the mechanism of protection.
To address whether direct interaction of mouse CD11bϩ cells with d7EB cells was required for the shift to anti-inflammatory lung cytokine profile observed in mice in the cell transplantation group, lung CD11bϩ cells were isolated from septic mice 12 hours after CLP and cocultured with d7EB cells either directly or indirectly via separation using a 0.4-L Transwell microporous filter. Both direct and indirect co-cultures were stimulated with LPS for 6 hours, and the supernatants were analyzed for TNF-␣ and IFN-␥ production using a mouse-specific Tissues were snap-frozen, cut into 5-m sections, and examined for native fluorescence at confocal microscopy. Nuclei have been counterstained with DAPI. Magnification ϫ40. Image is representative of at least 3 experiments. C: d7EB-conditioned medium has no effect on sepsis-induced mortality. In contrast to d7EB transplanted cells, conditioned medium from d7EB cells injected 1 hour after CLP did not prevent death. In this experiment, 2.5 ϫ 10 6 d7EB cells in 1 ml of medium were stimulated or not stimulated in vitro with LPS, 1 g/ml, for 12 hours. The supernatant was collected, centrifuged to remove any cells, and injected i.v. in mice 1 hour after CLP at a volume of 200 L per mouse. The two control groups received an equal volume of either DMEM/F-12 growth medium not exposed to d7EB cells or sterile PBS. N ϭ 25 in each group. ELISA. Only direct co-culture reduced TNF-␣ and IFN-␥ production by the CD11bϩ cells ( Figure 4D ).
The observation that direct interaction of d7EB and CD11bϩ cells was required for the anti-inflammatory phenotype of CD11bϩ cells led to consideration of the involvement of NO, an important inflammatory mediator of sepsis, 25 in the response. A time course study of NO production in cultured d7EBs over 6 hours demonstrated that d7EB cells constitutively produced NO and that they also strongly expressed eNOS ( Figure 5, A  and B) .
Evidence suggests that NO supplementation in the form of NO donors exerts an anti-inflammatory effect in animal models of endotoxemia through inhibition of nu-clear factor B activation and iNOS expression, and decreased production of proinflammatory cytokines. 26, 27 Therefore, whether the NO-producing d7EB cells exerted a similar anti-inflammatory effect on CD11bϩ cells was determined. d7EBs were co-cultured with CD11bϩ cells, and nitrite concentration was measured in the supernatant after LPS stimulation. Supernatant NO concentrations at 6 and 12 hours after LPS stimulation of cocultured d7EB and CD11bϩ cells were significantly lower than the supernatant NO concentration from CD11bϩ cells alone ( Figure 5C ). This effect could not be attributed to increased apoptosis because apoptosis was not significantly different in the co-cultured CD11bϩ cells compared with the CD11bϩ cells cultured alone (see Supplemental Figure S1 at http://ajpamjpathol.org). Analysis of iNOS expression in co-cultured CD11bϩ cells showed decreased protein expression in LPS-stimulated CD11bϩ cells interacting with d7EB cells (Figure 5D ). This finding was supported by measurement of iNOS expression Co-culture of d7EB cells with CD11bϩ lung cells reduces production of inflammatory cytokines. Lung CD11bϩ cells from septic mice were co-cultured with d7EB cells (ratio, 5:1) either directly or separated by a 0.4-m pore size Transwell filter. Non-co-cultured CD11bϩ mouse cells from septic mice were used as controls. The cells were stimulated with LPS, 1 mg/ml, for 6 hours, and supernatant was collected for cytokine measurement using an ELISA specific for mouse cytokines. Directly co-cultured cells demonstrated a decrease in TNF-␣ and IFN-␥ production, whereas production of these cytokines was not significantly different from that in controls when the cells were separated using Transwell filters. N ϭ 3 experiments. *P Ͻ 0.05; error bars represent SEM.
AJP January 2011, Vol. 178, No. 1 in CD11bϩ cells isolated from the lungs of septic mice 12 hours after CLP, where decreased expression of the enzyme was again observed ( Figure 5E ). To further test the hypothesis that NO production by d7EB cells contributes to the anti-inflammatory phenotype of the mouse CD11bϩ cells, an NO donor (DETA NONOate) was added directly to LPS-stimulated CD11bϩ cells, this time without d7EB cells, at concentrations ranging from 0.5 to 2 mol/L, and cytokine concentrations in the supernatant were measured. Reduction in TNF-␣ and IFN-␥ production by the LPS-stimulated CD11bϩ cells was observed ( Figure 5F ). In addition, iNOS expression in these cells after stimulation with LPS was reduced to levels comparable to those observed after co-stimulation with human d7EB cells ( Figure 5G ).
ACE and KDR expression in d7EB cells was monitored because these markers are associated with differentiation of hematopoietic and endothelial progenitor cells. 12, 28 Neither ACE nor KDR were expressed in undifferentiated stem cells; however, increased cell surface expression of these markers was observed after onset of mesodermal differentiation, starting at day 3 and reaching maximum at day 7 to 8 when 23% of d7EB cells were ACEϩKDRϩ and 23% were ACEϩKDRϪ ( Figure 6A ). To determine whether these two markers identified distinct ) in supernatants of directly co-cultured human d7EB and mouse CD11bϩ cells (ratio, 1:5) isolated from septic mice. The co-cultured cells were stimulated with LPS, 1 g/ml, for either 6 or 12 hours. At both times, NO production was significantly lower in the co-cultured wells compared with CD11bϩ cells alone. N ϭ 3 for each experiment. *P Ͻ 0.05; error bars represent SEM. D: Decreased iNOS expression in CD11bϩ cells co-cultured with human d7EB cells. After 12 hours of co-culture, CD11bϩ cells were selected using CD11b magnetic beads, and lysed. Western blot for iNOS protein was performed. Blot is representative of 3 separate experiments. E: Decreased intracellular iNOS expression in CD11bϩ cells isolated from lungs of mice transplanted or not with d7EB cells 12 hours after CLP. CD11bϩ cells were selected using magnetic beads, and permeabilized for intracellular staining for iNOS. Plot representative of 3 separate experiments. Open lines represent isotype-matched control. F: Exposure of CD11bϩ cells to NO alters subsequent inflammatory profile. CD11bϩ cells isolated from septic mice were stimulated with LPS, 1 mg/ml, and the indicated amounts of the NO donor diethylenetriamine NONOate (DETA NO) for 12 hours. Supernatants were collected, and TNF-␣ and IFN-␥ concentrations were measured using an ELISA. lumen-containing tubes. The ACEϩKDRϪ cells gave rise to colonies of hematopoietic precursors that stained positive for hematopoietic markers such as CD43 ( Figure  6B ). The endothelial and hematopoietic colony-forming capacity of the FACS-sorted cells was quantified by counting the number and types of colonies formed per 10,000 ACEϩ/KDRϩ, ACEϩ/KDRϪ, ACEϪ/KDRϪ, and ACEϪ/KDRϩ cells. Endothelial colonies were formed almost exclusively by the ACEϩ/KDRϩ fraction, with a small contribution from the ACEϪ/KDRϩ cells, whereas the ACEϩ/KDRϪ fraction showed great efficiency in production of hematopoietic colonies ( Figure 6C ). To determine the importance of ACE and KDR markers in the observed protective phenotype against sepsis, fractionated d7EB cells were co-cultured according to their expression of ACE and KDR with the CD11bϩ cells from septic mice as described above. The co-cultured cell fractions were stimulated in vitro with LPS for 12 hours, and mouse TNF-␣ and IFN-␥ production was monitored. Both the ACEϩ/KDRϪ fraction and the ACEϩ/KDRϩ fraction significantly reduced TNF-␣ and IFN-␥ production ( Figure 6D ), indicating the critical role of the progenitor cells expressing only ACE in reducing TNF-␣ and IFN-␥ production by CD11bϩ cells. In addition, a strong cell surface co-expression of ACE and toll-like receptor 4 (TLR4) was observed in d7EB cells, suggesting an association between ACE expression and the capability to respond to LPS ( Figure 6E ). To further validate these observations, the ACEϩ cells were fractionated and used for survival studies comparing them with the ACEϪ fraction. Only ACEϩ d7EB cells reproduced the protective effect of the mixed d7EB population in survival against sepsis, whereas ACEϪ cells were not protective ( Figure 6F ). To examine whether the reduced production of cytokines was associated with differential NO production by the ACEϩ progenitor cells, flow cytometry was used to determine co-expression of ACE and KDR with NO production. NO was produced only by the ACE-expressing cells, both ACEϩ/KDRϩ and ACEϩ/KDRϪ, whereas ACEϪ cells did not produce NO ( Figure 6G ).
hESCs, derived from the inner cell mass of the pre-implantation blastocyst, are defined by their ability to selfrenew and to differentiate into all types of mature cells. 29, 30 Although these cells and their derivatives in different stages of differentiation have been used for cell therapy applications in animal models of cardiovascular disease, 31-33 peripheral vascular disease, 12, 34 and central nervous system disorders, 21, 22, 35, 36 their potential in preventing sepsis-induced lung inflammatory injury characteristic of adult respiratory distress syndrome has not been addressed. The present study demonstrates for the first time the role of a population of progenitor cells derived from hESCs in preventing lung inflammatory injury induced by sepsis and improving survival in mice. The results show that the protection is the result of the subset of cells that are ACEϩ to respond to LPS by producing eNOS-derived NO. These cells functioned by moderating the pro-inflammatory cytokine production of the host immune CD11bϩ cells and reducing the high output of iNOS-derived NO cells and thereby mitigating lung inflammatory injury.
The protective effects of hESC-derived progenitor cells were associated with decreased production of the proinflammatory cytokines TNF-␣ and IFN-␥ and maintenance of the production of the major anti-inflammatory cytokine IL-10. It was demonstrated that protection by hESC-derived progenitor cells was the result of the interaction of these cells with resident lung CD11bϩ cells. Direct interaction of hESC-derived progenitor cells with CD11bϩ cells was required to create the anti-inflammatory environment in lungs because neither injection of septic mice with d7EB-conditioned medium nor indirect co-cultures reproduced the salutary changes in the cytokine profile.
Because of the need for cell-cell interaction, the possibility was considered that paracrine factors with sufficient diffusing capacity and instability in solution such as NO 37 might be involved. d7EB progenitor cells constitutively produced eNOS-derived NO in amounts comparable to those of immune cells, and strongly expressed eNOS. Co-culturing CD11bϩ cells with d7EB cells significantly reduced NO production in the culture supernatant after stimulation with LPS. This reduction in NO production was coupled with inhibition of iNOS expression in the co-cultured CD11bϩ cells and in CD11bϩ cells isolated from lungs of septic mice that received transplanted d7EB cells.
eNOS-derived NO is beneficial in maintaining vascular endothelial integrity, 38 and eNOS-derived NO production suppresses nuclear factor B activity, decreases the transcription of iNOS and intercellular adhesion molecule-1, and prevents lung injury and death due to endotoxin. 39 Thus, NO produced by d7EB cells may protect against iNOS activation and resultant high NO production, which has known deleterious effects on the host. 40 To test this hypothesis, CD11bϩ cells were exposed to an NO donor at a concentration range that induces NO release equivalent to the amount of NO produced by d7EB cells. This experiment reproduced the decreased generation of pro-inflammatory cytokines and reduced the iNOS expression observed in the CD11bϩ cells cocultured with human d7EB cells. Thus, the results suggest a critical role of eNOS-derived NO by d7EB cells in down-regulating iNOS activation in CD11bϩ cells and, thereby, dampening the production of pro-inflammatory cytokines.
These studies provide a mechanistic basis by which d7EB cells prevent sepsis-induced lung inflammatory injury. hESC-derived d7EB cells responsible for the protective phenotype were identified and found to be enriched in two cell surface markers, ACE and KDR. It was also demonstrated that the population consisting of ACEϩKDRϩ and ACEϩKDRϪ progenitor cells gave rise in culture to endothelial and hematopoietic colonies, respectively. In addition, the ACE-expressing cells expressed TLR4, the receptor sensing LPS, and produced NO. In contrast, ACEϪ cells did not exhibit these char-acteristics. The functional importance of these observations was reinforced by the ability of ACEϩ fractions of d7EB cells to modulate the inflammatory cytokine production profile of CD11bϩ host cells through direct cellcell interaction. ACE is constitutively expressed on the surface of endothelial and hematopoietic precursors, 41, 42 and ACE has been identified as a marker of hematopoietic stem cells present at all stages in the ontogeny of the human hematopoietic system 43 and as a novel marker of hemangioblasts differentiating from hESCs. 28 Evidence also points to an important role of ACE in sepsis. ACE knockout mice exhibited improved lung injury scores after acid aspiration, endotoxin challenge, and peritoneal sepsis. 44 Cohort studies in humans have shown a correlation between ACE polymorphisms and susceptibility to and death from adult respiratory distress syndrome, 45 and decreased plasma concentrations of ACE have been observed in patients with adult respiratory distress syndrome and sepsis. 46 A close homologue of ACE, ACE 2, has been identified as a key factor in protection from adult respiratory distress syndrome, and it also functions as a critical in vivo receptor for severe acute respiratory syndrome. 47, 48 A limitation of the present study is that the immunologic properties of human progenitor cells were studied in a xenograft model, introducing biases related to cross-species barriers. However, there is no reason to believe that species incompatibility affects the validity of the proposed mechanism, which involves interaction with host CD11bϩ cells and modulation of NO production. Human cells were not acutely eliminated by the immune-competent mice because cells were detected in lungs for 24 hours after injection. These data are in accord with observations in syngeneic mouse mesenchymal stem cells 6 and may be related to the immunosuppressive nature of the human cells. 49 It is likely, however, that some degree of immunosuppression will be required for longer term xenotransplantation studies. 50 Although, to our knowledge, the present study is the first to describe the function of mesodermally differentiated hESCs in a model of lethal sepsis and lung injury, other studies have used bone marrow mesenchymal stromal cells in similar models. 4,6 -11 These studies demonstrated an anti-inflammatory benefit of these cells but showed variable effects on the production of anti-inflammatory and pro-inflammatory cytokines. In a polymicrobial sepsis model, the beneficial effect of bone marrow stem cells was attributed to stem cell-induced production of IL-10 by host macrophages, 6 sphingosine-1-phosphate production by stem cells, 11 and homing of stem cells to lungs through integrin expression. 7,11 hESC-derived progenitor cells represent a much earlier developmental stage than bone marrow mesenchymal stem cells, and they have demonstrated ability to differentiate into niche-specific mature cells, a characteristic that distinguishes them from adult mesenchymal stromal cells. Although the importance of the interaction of hESC-derived cells with host CD11bϩ cells and the association of ACE expression with the protective phenotype were observed, factors such as homing to a niche and the engraftment potential of hESC-derived cells that may also be impor-tant in the observed protection cannot be ruled out. Findings of the present study demonstrate that d7EB cells, a population of hESC-derived progenitor cells, constitute a novel immunomodulatory cell population with therapeutic potential in sepsis that may have clinical application in cell-based therapy. Wide Prevalence of Heterosubtypic Broadly Neutralizing Human Anti-Influenza A Antibodies (See the editorial commentary by Donis and Cox, on pages 1010–1012.) Background. Lack of life-long immunity against influenza viruses represents a major global health care problem with profound medical and economic consequences. A greater understanding of the broad-spectrum “heterosubtypic” neutralizing human antibody (BnAb) response to influenza should bring us closer toward a universal influenza vaccine. Methods. Serum samples obtained from 77 volunteers in an H5N1 vaccine study were analyzed for cross-reactive antibodies (Abs) against both subtype hemagglutinins (HAs) and a highly conserved pocket on the HA stem of Group 1 viruses. Cross-reactive Abs in commercial intravenous immunoglobulin were affinity purified using H5-coupled beads followed by step-wise monoclonal antibody competition or acid elution. Enzyme-linked immunosorbent assays were used to quantify cross-binding, and neutralization activity was determined with HA-pseudotyped viruses. Results. Prevaccination serum samples have detectable levels of heterosubtypic HA binding activity to both Group 1 and 2 influenza A viruses, including subtypes H5 and H7, respectively, to which study subjects had not been vaccinated. Two different populations of Broadly neutralizing Abs (BnAbs) were purified from intravenous immunoglobulin by H5 beads: ∼0.01% of total immunoglobulin G can bind to HAs from both Group 1 and 2 and neutralize H1N1 and H5N1 viruses; ∼0.001% is F10-like Abs directed against the HA stem pocket on Group 1 viruses. Conclusion. These data—to our knowledge, for the first time—quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels. Influenza remains a major medical problem and is a constant threat to human health. Of the 3 influenza virus genera (A-C), influenza A is generally associated with more severe disease and is further subtyped by 2 surface proteins, hemagglutinin (HA) and neuraminidase (NA). Various combinations of the 16 HA (2 phylogenetic groups) and 9 NA subtypes define all subtypes of influenza A viruses. The Group 1 HA subtypes are H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16; the Group 2 HA subtypes are H3, H4, H7, H10, H14, and H15. Seasonal viruses, such as influenza A H1N1 (Group 1) and H3N2 (Group 2), and influenza B viruses cause infection in 5%-15% of the population worldwide and 250,000-500,000 deaths annually. In addition to frequent annual epidemics, influenza viruses periodically cause pandemics, the most recent example being the 2009 pandemic caused by the swine-origin H1N1/2009 influenza virus.
Vaccination is the principle means of preventing seasonal and pandemic influenza and it's complications. A universal vaccine that induces broad immunity against multiple subtypes of influenza A viruses is a long-sought goal in medical research. Recently, we and others identified a family of human broadly neutralizing ''heterosubtypic'' antibodies (BnAbs) that bind to a highly conserved pocket on the stem of HA present in all Group 1 influenza viruses [1, 2] and block virus-host cell membrane fusion. These BnAbs were identified by recombinant H5 panning from antibody (Ab)-phage libraries that are constructed from nonimmune B cells [1] , immunoglobulin (Ig) M memory B cells of seasonal vaccinees [3] , or bone marrow of H5N1-infected ''bird-flu'' survivors [4] . BnAbs with similar properties have also been recovered from immortalized IgGexpressing memory B cells of seasonal vaccinees [5] . An unexpected finding from these studies is the frequent contribution of VH1-69 heavy chain genes to these BnAbs, suggesting that a large fraction (up to 10%) of the human naive B cell repertoire has the capability of responding to this conserved epitope [5, 6] . These observations raise additional questions as to whether such BnAbs are present in human serum and at ''protective'' levels, whether they exist as ''natural'' Abs, and/or whether are they generated during the immune response to influenza virus infection or vaccination [5, 7] . To explore these questions, we analyzed serum samples from H5N1 vaccinees and commercial intravenous immunoglobulin (IVIG) samples for their crossreactive binding and neutralization activity against different influenza A subtypes. We also utilized a representative BnAb, F10, for which the precise epitope was determined crystallographically [1] , to probe for heterosubtypic BnAbs directed against the highly conserved pocket on the HA stem.
Serum samples from 77 healthy volunteers, matched before and after vaccination (1-4 months), had been collected and stored at a single center, from a dose-escalating clinical trial (Clin-icalTrials.gov identifier: NCT00383071) on an inactivated H5N1 vaccine, conducted at the National Institute of Allergy and Infectious Diseases, National Institutes of Health [8] . The vaccine (manufactured by Sanofi Pasteur) used in the trial was a monovalent, inactivated subvirion H5N1 vaccine (rgA/ Vietnam/1203/04 X A/PR/8/34). The study was conducted in accordance with institutional review board-approved protocol.
Enzyme-linked immunosorbent assays (ELISAs) were performed to test the cross-reactivity of the serum samples against multiple influenza A subtypes. Recombinant HA proteins, including H1 (A/New York/18/2009(H1N1), H1-NY18), H3 (A/ Aichi2/68 (H3N2), H3-A2/68), H5 (A/Vietnam/1203/04 (H5N1), H5-VN04), and H7 (A/Netherlands 219/03 (H7N7), H7-NL03), were expressed in insect cells as trimers of HA ectodomains [1] . HA proteins (0.2 lg) were coated onto 96-well Maxisorb ELISA plate (Nunc) at 2 lg/mL in phosphate-buffered saline (PBS) at 4°C overnight. The plate was washed with PBS for 3 times to remove uncoated proteins. Serially diluted serum samples were applied to the HA-coated plates, followed by Horseradish Peroxidase (HRP)-anti-human IgG or IgM (Pierce Biotechnology), to detect the IgGs or IgMs against various HA subtypes in the serum samples. The optical density at 450 nm was measured after incubation of the peroxidase tetramethylbenzidine substrate system.
A competition ELISA assay was conducted to determine the level of F10-like Ab in the serum samples. F10 Ab (human IgG1) was biotinylated with Sulfo-NHS-SS-Biotin (Pierce Biotechnology) in accordance with the manufacturer's instructions. Biotinylated F10 (Bio-F10; 3 ng/mL) was mixed at 1:1 (vol) ratio with serum samples at various dilutions and added to ELISA plates coated with H5-VN04 trimer. The competition of serum samples for the binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin (BD Bioscience).
To isolate H5-bound Abs and F10-like Abs from intravenous immunoglobulin (IVIG; 100 mg/mL, Gamunex IVIG; Talecris Biotherapeutic), we first immobilized H5-VN04 proteins on magnetic beads (Dynabeads M-270 Epoxy; Invitrogen) in accordance with the manufacturer's manual, and then used step-wise Bio-F10 elution and acid elution to separate the F10-like Abs and H5-bound Abs present in the IVIG. Specifically, 5 3 10 8 of H5-beads were incubated with 1.5 mL of IVIG (100 mg/mL) overnight at 4°C, washed extensively with 0.1% BSA/PBS, followed by Bio-F10 elution (100 lg/ml, 0.5ml, total 50 lg) overnight at 4°C. The remaining H5-bound Abs on the H5-beads were then isolated with acid elution (pH 5 2.8; buffer, 500 lL/10 9 H5beads). Next, the Bio-F10 eluents and the acid eluents were incubated with Strep-T1 beads (250 lL of 4 3 10 8 beads; Invitrogen) at 4°C for 4-8 h each time for 2 and 3 times, respectively. ELISA was performed to verify the Bio-F10 in both samples was completely removed after Strep-T1 beads absorption. The purified H5-bound Ab and F10-like Abs, as well as unpurified IVIG, were quantified by ELISA using known concentrations of F10 monoclonal Ab (mAb) as standards. The aforementioned purification procedure was scaled up proportionally to obtain enough H5bound Abs and F10-like Abs to test their cross-reactivity against different influenza A subtypes by ELISA and pseudotyped virus neutralization assay, as described elsewhere [1] .
Statistical analyses were performed using the paired t test for the comparisons of prevaccination and postvaccination Ab-binding levels and Bio-F10 Ab inhibition activities. The neutralization activity was compared using log 2 microneutralization assay (MN) [8] titers. The proportions of .2-fold increase in Ab level were compared by McNemar's test for paired binary outcomes. All P values were 2-sided, and P values ,.05 were considered to be statistically significant. The correlation between F10-like IgG Abs and MN titer against H5N1 in postvaccination serum samples (n 5 77) was analyzed by Spearman rank correlation coefficient analysis.
In the H5N1 vaccine group, pre-vaccine IgMs against recombinant H5-VN04 protein were detectable in the majority of study subjects. The IgM binding level increased significantly after vaccination (P 5 .006); however, only 8 (10.4%) of 77 subjects demonstrated a .2-fold increase (as shown by a .2-fold increase in optical density at 450 nm by ELISA) ( Figure 1A ). All of the study subjects had pre-immune IgG Abs that bound to H5-VN04 ( Figure 1B ). As expected, the binding to H5-VN04 (P , .001) ( Figure 1B ) and neutralization activity (MN titer) against H5N1 (the vaccine strain; P , .001) ( Figure  1E ) significantly increased after vaccination. IgGs against recombinant H3-A2/68 protein ( Figure 1C ) and H7-NL03 protein ( Figure 1D ) were also detected in the pre-immune serum samples. In contrast to H5-binding, the postvaccination IgG binding to H3 did not increase significantly (P 5 .11); however, it increased to reach statistical significance for H7 (P , .001), but the proportion of specimens with a .2-fold increase in H7 binding was significantly lower than that for H5 binding (1.3% vs 76.6%; P , .001).
To investigate further the presence of heterosubtypic BnAbs in serum, we probed serum samples for the presence of F10-like IgG Abs, as determined by inhibition of the binding of biotinylated F10 (Bio-F10) to H5-VN04 in a competition ELISA assay. We found that F10-like Abs that could compete for Bio-F10 binding were also present in prevaccination serum specimens, with 23 (29.9%) of 77 samples demonstrating .30% inhibition of Bio-F10 binding at 1:90 dilution ( Figure 1F) . Furthermore, the F10-like IgG titers increased significantly after vaccination (P , .001), and the majority of vaccinees (54 of 77) had a 1.5-fold increase. In addition, there is a weak but significant correlation between the F10-like IgGs and the MN titer against H5N1 in post-immune serum samples, as determined by Spearman rank correlation analysis (co-efficient 5 .44); however, these stem pocket-directed BnAbs do not reach high enough levels to render a strongly significant correlation.
To determine whether these BnAbs were unique to the subject population tested or are present more broadly, we quantified the anti-H5 and F10-like Abs in a commercial IVIG and determined their breadth of heterosubtypic binding and neutralization activity. The IVIG contained pooled IgGs from thousands of donors and is representative of the pre-immune IgG Ab composition in the general population. Although it cannot be formally ruled out that IVIG is truly H5 naive, this was the best representative sample available for this study. H5-immobilized magnetic beads were used to affinity-purify F10-like and anti-H5 Abs from the IVIG. F10-like Abs were isolated by Bio-F10 competition elution from the total H5-beads bound Abs, and the remaining H5-bound Abs on the H5-beads were released with acid elution. By quantitative ELISA, we found that, from 100 mg of IVIG, 10 lg of Abs bound to the H5-coated plate (0.01% of the total). Of these, 10% could be purified using affinity purification with H5-beads by acid elution. The final yields were 1-1.4 lg acid-eluted anti-H5. The Bio-F10 competitive elution yielded 0.1 lg of F10-like Abs per 100 mg of IVIG (0.001% of the total Ig level) and with similar or higher efficiency of recovery as compared with acid elution.
These anti-H5 and F10-like Abs were further tested for their heterosubtypic HA binding and neutralization activity against Group 1 and 2 viruses. The acid-eluted anti-H5 Abs are not H5-subtype specific: they showed cross-binding activity to H1-NY18, to H3-A2/68, and weakly to H7-NL219 ( 
In this study, we show that prevaccination serum samples have baseline heterosubtypic HA Ab binding activity to both Group 1 and 2 HA subtypes, including H5 and H7, to which these subjects are most likely unexposed because of their US geographic location. The IgM and IgG Abs to H5 increased significantly after the H5N1 vaccination, whereas this did not happen for IgG Abs to H3. F10-like IgG Abs are also detected in pre-immune serum samples and increased significantly in H5N1 vaccinees. A low level of serum anti-HA Abs that bind and neutralize H5N1 viruses has been reported to be age and influenza exposure dependent [9, 10] . Other investigators have reported enhanced levels of HA-directed anti-H5N1 neutralizing Abs in healthy donors after boosting with unrelated human influenza H1N1/ H3N2 seasonal vaccines [5, 11, 12] . These serum Abs may be directed to the Group 1 stem pocket on HA. Indeed, Corti et al [5] showed that a majority of 2007 and 2008 seasonal vaccinees had preexisting neutralizing Ab titers against pseudotyped H5N1 viruses that markedly increased after seasonal vaccination and that the HA stem-pocket directed Abs were present at relatively low levels, compared with Abs that bind to the globular head of HA.
We also observed an increase of H7 (Group 2) reactivity after H5N1 (Group 1) vaccination ( Figure 1D ). The precise location of the cross-binding epitope(s) is currently not known.
Observations that support the possible presence of heterosubtypic Abs of the broader type that bind Group 1 and 2 viruses have also been seen in children after primary influenza infection [13] and mucosal vaccination of experimental animals [14, 15] . In one study, a cross-neutralizing murine mAb with The binding levels are shown as optical density at 450 nm (OD 450). E, microneutralization assay (MN) titer against H5N1 virus; y-axis shows the Log 2 MN titer. F, Competition ELISA. Pre-and post-immune serum samples from H5N1 vaccinees were tested for their competition activity against a Group 1specific BnAb, F10, binding to H5-VN04. Serially diluted serum samples were mixed with 3 ng/mL Bio-F10 and applied to H5-coated ELISA plates. The serum competition for binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin. The serum competition activity is shown as percentage of inhibition. For all panels except panel E, data at 1 representative serum dilution are shown, as follows: panel A, 1:270; panel B, 1:5120; panels C and D, 1:2430; and panel F, 1:90. For all panels, data are shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles, respectively. The dots above and below the whiskers are data points beyond the 95th and 5th percentiles. hemagglutination inhibition activity against Group 1 (H1, H2, H5, H9, and H13) and Group 2 (H3) was recovered from an intranasally vaccinated mouse. In contrast to the F10-like Abs, this mAb was directed to the globular head, and escape mutants were readily obtained [16] . Other animal vaccine studies have shown that antibodies to the fusion peptide and an HA2 linear peptide can have broad reactivity to Group 1 and 2 HAs [17] [18] [19] and neutralizing activity [20, 21] , respectively. Whether different quantitative amounts of Group 1-specific BnAbs and/or Group 1 and 2-directed BnAbs are elicited by vaccination or natural infection remains an important but unanswered question of our study.
To quantify the baseline levels of anti-influenza heterosubtypic Abs in human serum, we used IVIG as representative of pre-immune IgG Ab composition in the general population, which includes individuals who are likely exposed to seasonal Figure 2 . Heterosubtypic antibodies against influenza A viruses in intravenous immunoglobulin (IVIG). H5-VN04 was immobilized on magnetic beads (H5-beads) and the beads were used to affinity purify antibodies (Abs) from the IVIG sample. F10-like Abs and the remaining H5-bound Abs were purified separately. F10-like Abs were purified by Bio-F10 competition elution followed by multiple steps of streptavidin-beads absorption to eliminate Bio-F10 completely. The remaining H5-bound Abs on the H5 beads were eluted with standard acid elution method followed by a complete absorption of Bio-F10 using Streptavidin-beads as well.An enzyme-linked immunosorbent assay (ELISA) detecting Bio-F10 was used to confirm that no residual Bio-F10 remained in either Bio-F10-eluted or the acid-eluted samples. The binding activity of these purified Abs to H1-NY18 (A), H5-VN04 (B), H3-A2/68 (C), and H7-NL03 (D) was measured by ELISA at different serially diluted concentration. The neutralization activity of these samples was measured using neutralization assay with pseudotyped viruses of H1-1918 (E) and H5-TH04 (F); 80R was used as a negative control mAb that is specifically against the spike protein of severe acute respiratory syndrome coronavirus [24] . OD 450, optical density at 450 nm.
influenza A virus infection and/or vaccinations (H1N1 and H3N2). Other investigators have shown that a low level of heterotypic anti-HA Abs that bind and neutralize H5N1 viruses is present in IVIG from diverse geographic locations [9, 10] . These investigations further showed that these heterosubtypic anti-H5N1 Abs cross-react with H3N2 and H1N1; however, efforts to purify and characterize these Abs (beyond neutralization titers) have not been reported. Our data show that there are 2 populations of heterosubtypic Abs with different HA binding ability in IVIG: one can bind to HAs from both Group 1 and Group 2 viruses; the other is specifically directed against Group 1 stem pocket. Approximately 0.01% of IVIG (most certainly derived from H5-and H7-naive donors), which is purified by acid elution from H5-beads, has heterosubtypic binding activity to both Group 1 and Group 2 HAs. This fraction of IVIG Abs also demonstrated neutralizing activity against the H1N1 and H5N1 pseudotyped viruses. The F10-like stem pocket-directed Abs were also detected in IVIG and were recovered at 10% of the levels of H5 binding Abs. As expected, these F10-like Abs displayed similar binding and neutralization profiles among the tested HAs and viruses as the Group 1 specific mAbs that are directed to the stem pocket of HA [1, [3] [4] [5] . For both Ab fractions, a broader range of other subtypes were not tested due to limited amount of the Abs that we could purify from IVIG (1 lg and 0.1 lg/100 mg IVIG, respectively), larger-scale purification of H5 protein and other materials will be required to characterize these heterosubtypic BnAbs in more detail.
Although we quantitatively show that BnAbs that bind to Group 1 and 2 HAs are present at very low levels, as well as that stem pocket-directed F10-like BnAbs exist at even lower levels, the question of whether serum or IVIG has protective levels of either type of heterosubtypic BnAbs is not answered in our study. However, our quantitative data support the notion that the levels of these BnAbs are borderline or below titers that would traditionally be considered protective. For example, there is up to 1 lg of F10-like Abs/100 mg IVIG; assuming 10 mg/mL IgG in normal human serum, then concentrations up to 0.1 lg/ ml of F10-like Ab could be present. Likewise, the fraction of acid eluted BnAbs with activities against Groups 1 and 2 could also be in this range. Furthermore, we did observe variability in these levels in our study patients (Figure 1) , and it remains possible that host factors, including VH polymorphism, may impact the baseline and inducible BnAb levels. In addition, the origins of these 2 populations of heterosubtypic anti-HA Abs are unknown. The possibility that they may be a component of ''natural'' polyreactive Abs cannot be excluded [22] . However, it is most likely that both our H5N1 vaccine study subjects and the IVIG donors had prior exposure to other Group 1 (H1N1) and Group 2 (H3N2) influenza A viruses, either through seasonal vaccinations and/or natural infection, and this may have given rise to heterosubtypic H5 and H7 binding Abs, respectively.
In summary, our findings show that the human immune system is capable of making BnAbs-not only to the conserved pocket on the HA stem of Group 1 viruses, but also to another unknown epitope(s) that are shared by Group 1 and 2 influenza A viruses. These observations provide the basis for further investigations aimed at obtaining a better understanding of these BnAbs, their origins, and the host genetic factors that restrict or enable their induction [23] . These additional studies should bring us closer to developing a universal influenza vaccine that provides durable protection beyond seasonal vaccines and mitigates that ability of the viruses to undergo neutralization escape. Indeed, a recently reported vaccine regimen-which induced protective level of the Group 1 stem-pocket directed BnAbs in animals-provides experimental evidence that the same may be possible in man [23] . Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00249-010-0645-4) contains supplementary material, which is available to authorized users. Abstract Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed b-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution q(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide's reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer.
Introduction A quantitative, dynamic, and functional understanding of novel natural or synthetically modified peptide species in the native membrane environment requires information about the reconstitutability, positioning, and orientation with respect to the lipid bilayer matrix. Compared to protein complexes, membrane active peptides present relative structural simplicity and are frequently used to validate experimental approaches, techniques, and applications (Bechinger 2001; He et al. 1993) . With the presented study, we are interested in evaluating the biophysical properties of a recently published motif of straightforward designed, aromatic, b-type polypeptides. This includes analysis of peptide positioning, orientation, and changes in bilayer thickness as well as the effect of peptide reconstitution on lateral lipid packing and ordering. Furthermore, we want to introduce a general and applicable X-ray-based approach that provides combined access to significant and quantitative parameters of peptide/lipid interaction on a molecular scale within the physiological membrane state. In this way, structural information, both in the bilayer plane and along the normal direction, can be determined.
While spectroscopic methods in their ''standard'' application, such as fluorescence emission (Bong et al. 2000; Krishnakumar and London 2007; Wimley and White 2000) , circular dichroism (CD) (Chen and Wallace 1997; Koeppe and Andersen 1996) , and transmission infrared (IR) spectroscopy (Küsel et al. 2007) , produce qualitative information on the structure and localization of the peptide species, more advanced applications, such as oriented CD (Wu et al. 1990 ), attenuated total reflection (ATR) IR spectroscopy (Tamm and Tatulian 1997) , Förster resonance energy transfer (FRET) experiments (parallax method) (Chattopadhyay and London 1987; Chung et al. 1992) , solid state nuclear magnetic resonance (NMR) (Andronesi et al. 2004; Strandberg and Ulrich 2004) , and electron paramagnetic resonance (EPR) techniques (Dzikovski et al. 2004; Marsh 1996) , usually account for quantitative values in one dimension of the lipid bilayer without the need for complementary simulations or geometrical models (Naik and Krimm 1986a, b) . Some of these techniques require membrane states or parameters that are far from physiologically or biologically relevant. In addition, sample preparation for some applications, e.g., frozen state samples for EPR spectroscopy, are often prone to uncertainties and artifacts. Therefore, the sample preparation method itself has to be carefully evaluated, usually with significant effort.
It is obvious that no single technique can give complete structural information for each component of a fluid membrane system. However, in recent years the potential of X-ray scattering methods in probing peptide/lipid complexes, i.e., the membrane as well as the peptide structure, has been shown to be almost solely limited by the nonuniformity or patchiness of the peptide/lipid system itself (Qian et al. 2008; Salditt et al. 2006) . Therefore, if samples of suitable quality can be obtained, the potential of a simultaneous analysis at the molecular scale of the incorporated peptide species, the surrounding lipid matrix, and the peptide/lipid interactions underlines the power of this analytical method. In addition, X-ray scattering is compatible with the fluid membrane state and capable of providing information for both dimensions of the lipid bilayer space.
The alignment of multilamellar membranes is quantified by the distribution function of the membrane normal vectors, which are directed along the z axis perpendicular to the solid substrate on which the membranes are deposited. The central width of this distribution is called mosaicity. A narrow distribution or equivalently small mosaicity allows for a precise distinction between the scattering vector components, vertical (q z ) and parallel (q k ) to the lipid bilayer. Thereby, the multilamellar state of the samples, which can be almost fully hydrated by controlled relative humidity (Aeffner et al. 2009 ), provides a tremendous amplification of the scattering signal compared to single bilayer preparations. However, compared to single bilayer reflectivity, the multilamellar stacks usually impede the determination of the electron density q(z) on an absolute scale, unless a full q-range fit of the electron density is performed (Constantin et al. 2003) .
In principle, an ideal small-angle scattering experiment with respect to distribution, concentration, and structural integrity of the peptide species can yield multiple structural parameters (Fig. 1) . The reflection geometry for aligned lipid films (Fig. 1i ) is suited to determine the vertical density profile q(z) of the bilayers, averaged in the xy plane, e.g., by least-square fitting of the specular reflectivity curve (Constantin et al. 2003) . The specular peaks are accompanied by diffuse scattering in the form of so-called Bragg sheets extending along q k . This scattering contribution can be assigned to lateral inhomogeneities at mesoscopic length scales (nanometers to a few micrometers) due to membrane fluctuations-always present in the fluid/ hydrated state-and peptide insertions causing local orientational disorders (Fig. 1ii) , which break the lateral translation invariance.
While the vertical electron density distributions may also be obtained by fitting the diffuse Bragg sheet intensities of the reciprocal space for a single angle of incidence, we have used full scans with background correction (Fig. 1iii ) in reflectivity mode (Fig. 1iv) .
The lateral membrane structure at molecular length scales is accessible via the grazing incidence diffraction (GID) scattering geometry, at which the 2D detector (CCD camera) is moved out of the plane of incidence at grazing incidence and exit angles. The lipid chain correlation peak (Fig. 1v) is usually stretched around a circle with the radius q = (q k 2 ? q z 2 ) 1/2 % 1.4 Å -1 reflecting the distribution of acyl chain tilt angles with respect to the bilayer normal. The radial profile of the peak has a Lorentzian lineshape with a peak position corresponding to the inverse nearestneighbor distance of chains and its width reflecting the lateral correlation length (see below). This peak is often broadened upon peptide insertion. A lateral correlation of membrane-inserted structures, e.g., a regular oligomerization as known from pore arrangements, can be detected and characterized from ''superstructure peaks'' (Constantin et al. 2007 ) in the small-angle region at low q z (Fig. 1vi ). In addition, the conformation of the reconstituted peptide's structure can in some cases be deduced from the low q k signal of molecular form factors, the so-called helix maximum (Fig. 1vii) , or from helix peaks revealing intrahelical distances at high q k (not shown) as it has been revealed for the regularly organized and conformationally constrained alamethicin single helix (Spaar and Salditt 2003) .
As we are interested in a general approach including a side-directed iodine-labeling for the reflectivity studies (Arbely et al. 2004) , the present work focuses on data recording and analysis, as can be applied for any peptide/ lipid sample. Fig. 1 Schematic representation of the reciprocal space for multilamellar membrane stacks (i) as a function of the parallel (q k ) and normal (q z ) components of the momentum transfer (q). The scheme shows the reflection geometry known as grazing incidence X-ray scattering (GISAXS) at small q k or grazing incidence diffraction (GID) at high q k . The recorded patterns include the diffuse Bragg sheets (ii) in the vicinity of the (specular) q z axis. In this study the q z components (iii) were measured by line scanning under specular conditions (iv). At low q z , the lipid acyl chain correlation maximum (v) is observed. Lorentzian fits yield the lateral lipid chain distance. The width of the acyl chain peak along q k gives information about the lipid ordering (correlation length); the angular width of the peak corresponds to the acyl chain tilt. In addition, superstructures (vi) and peptide geometries (helix maximum, vii) can be observed in some cases Eur Biophys J (2011) 40:417-436 419 Design of the b-type peptide helix motif
In contrast to well established, commercially available peptide species, we concentrate our studies on a recently reported peptide model that was designed for spanning the membrane hydrophobic core and anchoring at the membrane/water interface. Therefore, this motif holds a tunable, conformationally stable peptide structure that is open for multiple functionalization ). The D,L alternation of the peptide backbone concordant with a double-helical b-type shape is related to natural peptide antibiotics such as gramicidin A (Kelkar and Chattopadhyay 2007) or feglymycin (Bunkóczi et al. 2005) . In addition, the studied sequences are unique on their own due to their almost purely aromatic side-chain composition (Alexopoulos et al. 2004; Küsel et al. 2007 ).
On the basis of the crystallographically elucidated watersoluble nonameric sequence H-(Tyr-Tyr) 4 -Lys-OH (note: underlined amino acids indicate the D-conformer), the homodimeric dodecamer H-(Phe-Tyr) 5 -Trp-Trp-OH (1) (Fig. 2a, b ) and the covalently linked hairpin species H-(Phe-Tyr) 5 -Trp-Trp-Gly-Lys-Pro-Gly-(Phe-Tyr) 5 Trp-Trp-OH (2) and H-Lys(NBD)-Tyr-(Phe-Tyr) 4 -Trp-Trp-Gly-Lys-([C 2 H 4 O] 2 -CH 2 -CO-gcgtgg-Lys-Lys)-Pro-Gly-(Phe-Tyr) 5 -Trp-Trp-OH (3) (Fig. 2a) (note: gcgtgg represents the nucleobase sequence of aminoethylglycine peptide nucleic acid monomers) were derived via synthetic modification applying solid-phase peptide synthesis (SPPS) and loop design . The transmembrane helices of the latter structures (1-3) are well adapted to the membrane environment as optimized by sequence variation in length and composition (Küsel et al. 2007 ). Through the execution of design studies of the peptide substrates, the preservation of the b 5.6 helical structure ( Fig. 2b ; the superscript denotes the periodicity of the helix), as found for the homodimeric species 1, was maintained for the final hairpin prototype 2 via constant comparison of the respective circular dichroism data resulting from several tested constructs .
As there has been strong evidence for a conformational and monomer-dimer equilibrium of the homodimeric structure 1 involving antiparallel oriented b 5.6 double and b 6.3 single helices (Fig. 2c , i-iii) (Küsel et al. 2007 ), a covalent linkage of both strands seemed to be mandatory in order to circumvent the described equilibria. This goal was fulfilled through the synthesis of the peptide hairpin 2 (Fig. 2c , iv) allowing for further studies that address peptide oligomerization within lipid model membrane complexes. The helix assembly is, thereby, driven by molecular recognition of the peptide nucleic acid (PNA) moieties at the membrane's exterior (Fig. 2c, v-vi; . The attachment of fluorescence probes and peptide nucleic acid (PNA) recognition moieties led to functional constructs, such as compound 3, that have already been tested for their dynamic dimerization within lipid bilayer structures via Förster resonance energy transfer (FRET) assays ). a b c Fig. 2 Molecular representation of the different D,L-alternating peptide types (a) ranging from the homodimeric species 1 to the designed hairpin 2 to the functionalized construct 3. Furthermore, the molecular structure of the homodimer 1 (b) (Küsel et al. 2007 ) as well as proposed equilibria (c) are sketched, either for homodimeric structures (i)-(iii) or the PNA-equipped transmembrane domains of 3 (v)-(vi) that undergo oligomerization driven by molecular recognition at the membrane's exterior Here, we probe the membrane reconstitution of the three presented kinds of b-helical species via GID and reciprocal space mapping (RSM) and evaluate their influence on the lateral lipid packing by addressing the lipid chain correlation peak. In the respective studies, parameters such as the sample composition, the relative humidity (RH), and the peptide-to-lipid (P/L) ratio are varied. The obtained results can be explained by a qualitative model that predominantly takes into account a hydrophobic mismatch situation as well as a lateral packing and ordering of the annular lipid shell that is correlated to the extent of peptide-lipid contact surface. Furthermore, we analyze the differentially (double) iodine-labeled hairpin species of type 2 via anomalous and in-house reflectivity elucidating their positioning with respect to the membrane normal (z axis) and confirming their transmembrane orientation. These experiments created the basis for a functional FRET assay that was reported elsewhere .
Solid supported stacks of typically 1,000 aligned DLPC bilayers with a cholesterol (Chol) content of 5% and differing amounts of the respective peptide species were prepared from stock solutions following procedures described in the literature (Seul and Sammon 1990) . Polished and cleaned (sonication in MeOH and ultrapure water, 15 min each) Si wafers with h100i orientation and a thickness of 625 lm (Silchem, Freiberg, Germany) were used as substrates. Stock solutions of DLPC and Chol in chloroform were prepared at concentrations of 40 and 3 mg/ml, respectively. Peptide stocks were composed of MeOH/DCM/EtOH 4/3/3 (v/v/v) at 6 mg/ml concentration. For P/L ratios between 1/10 and 1/50 in a definite volume of 80 or 150 ll, mixtures of stock solutions were spread onto cleaned, horizontally mounted silicon substrates with dimensions of 10 9 15 mm (80 ll, beamline experiments) or 15 9 25 mm (150 ll, in-house experiments), respectively. The coated Si wafers were covered with a watch glass, and the solvent was carefully evaporated in a flowbox overnight to prevent film rupture and fast dewetting. Subsequently, reduced pressure was applied for an additional 12 h. The resulting film-covered substrates were stored at 4°C until use.
For measurements, the prewarmed (40°C, 1 h) and rehydrated (saturated water vapor atmosphere) samples were placed in home-built sample cells with Teflon sealing and either PE foil or kapton windows. A setup was applied at which the RH could be adjusted by PID control, remaining stable within 0.1% (Aeffner et al. 2009 ). The temperature (T) of the sample chamber, the water reservoir, and the pipings was controlled as well. The RH/T sensors and mass flow controllers were interfaced with the diffractometer controls (in-house and beamline), enabling the usage of long and fully automated scan macros including RH and T as parameters. Unless otherwise stated, GID experiments were carried out at RH = 94%, and the reflectivity experiments were performed at RH = 90%, both at 20°C. The chambers were mounted to the respective goniometers with the sample oriented either horizontally (beamline) or vertically (in-house) depending on the diffractometer setup. The X-ray beam enters and exits the chamber through kapton (in-house) or PE foil (beamline) windows.
GID and anomalous reflectivity experiments were performed with the insertion device 01 (ID01) undulator beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), while in-house reflectivity measurements were carried out with a home-built diffractometer.
In GID mode the sample was tilted at a fixed angle of incidence close to the critical angle of total external reflection a c in order to optimize the lateral scattering intensity and to minimize background scattering caused by the substrate. The vertical scattering depth along z is, thereby, tuned by the angle of beam incidence a i and the angle of the scattered beam a f (compare to Fig. 3) . GID experiments were performed at 17 keV radiation using a Peltier cooled 2D CCD detector (Princeton Scientific Instruments, Princeton, NJ, USA) providing a resolution of 1,340 9 1,300 pixels (pixel size: 48 9 49 lm 2 ; active area: 64.3 9 63.7 mm 2 ) that was mounted at approximately 19.5 cm from the sample. The beam was cut by the entrance slits to 0.7 9 0.7 mm 2 in front of the sample; no beam restrictions were set on the detector side. Three different types of measurements were performed:
1. After placing the sample roughly horizontally and adjusting the specular axis to the center of the CCD, four images were taken along the q z direction under stepwise increase of the angle of beam incidence (Da i = 0.45°) enabling the calculation of the actual a i as well as the exact sample detector distance and allowing for scanning the low q k space for peaks resulting from superstructures (Fig. 3 ). 2. Adjustment of the specular axis at one side of the CCD and coverage by a lead stripe facilitated scanning the Eur Biophys J (2011) 40:417-436 421 reciprocal space far from the specular axis yielding combinations of exposures that provide a partial overlap. With this data collection an overview of the reciprocal space was achieved in which peaks representing intrahelical distances might occur (data not shown). 3. Quantitative images including the nonattenuated specular axis (q k = 0) on one side of the CCD plate up to q k = 2.5 Å -1 were taken with accumulated exposure series of 20-600 images applying single exposure times of 0.3-5.0 s. In analysis of the chain correlation peak using the same geometry, images were taken with exposure times of 20-30 s under attenuation of the primary beam and the specular axis.
For the anomalous scattering experiments (ID01 beamline, ESRF) the polychromatic X-ray beam was monochromatized downstream from the undulator by a double crystal Si(111) monochromator with an energy resolution of DE = 1 eV yielding Dk/k B 10 -4 . The beam was cut to a size comparable to the GID experiments by the entrance slits directly in front of the sample. The reflected beam was recorded by two wide-opened slits of 4.0 9 4.0 mm 2 and 3.0 9 3.0 mm 2 . For line scanning under specular conditions, an avalanche photodiode (APD) was used as a point detector and placed directly behind the last slits reducing parasitic air scattering. The sample-to-detector distance was 28 cm. In order to reduce sample degradation (beam damage) during motor movement and in between scans, especially when applying low X-ray energies, a ''fast shutter'' was implemented in front of the entrance slits/absorber box and synchronized with the detector (Giewekemeyer and Salditt 2007) . Therefore, no automated absorbers could be used during reflectivity or offset scans; absorbers had to be calibrated in advance of data collection. A monitor, mounted directly in front of the sample, was used to control the beam intensity. To further reduce dose and avoid beam damage, the reflectivity curves and offset scans were recorded only in the vicinity of the Bragg reflections at calculated positions.
In resonant reflectivity experiments, the scattering power of iodine labels was used to retrieve selective structural information of the iodine positioning with respect to the membrane normal by varying the incidence of the X-ray radiation close to the iodine L III absorption edge around E = 4.5575 keV (Gullikson 1995 (Gullikson -2008 . The scattering length becomes explicitly dependent on the X-ray energy E near the absorption edge, according to
where f 0 is the number of electrons of the ion (nonresonant term) and f 0 and if 00 are the real and imaginary corrections of the scattering factor, respectively (Evans and Pettifer 2001 ). Both f 0 and f 00 , connected by the Kramers-Kronig relations, strongly vary in the vicinity of an absorption edge and can be measured (f 00 ) or obtained from databases. At an absorption edge the absolute values of f 0 and f 00 are the highest with respect to nonresonant energy regions, where f & f 0 . Thus, in a resonant diffraction experiment it is very important to determine the energy value of the absorption edge accurately to a resolution of about 1 eV. It is known that the scattering factors f 0 and f 00 of a chemical species within a sample, here iodine, depend very sensitively on its environment. Since there are many influences on these values, their accurate calculation by theoretical consideration is not feasible. Therefore, it is the most reliable strategy to measure f 00 and calculate f 0 during the experimental setup (Als-Nielsen and McMorow 2001). Experimentally, it is possible to access f 00 by absorption or fluorescence measurements. In order to Fig. 3 GID scattering geometry (left) and exemplary results from low q k scanning at different angles of a i in search of ''superstructure peaks'' (right) also required for determination of parameters concerning the scattering geometry. As the angle of incidence increases, the specular beam reflex (sb) moves along the q z axis. The primary beam (pb) is located at the origin of the q z axis, here shadowed by the sample horizon mimic the hydrophobic environment within a lipid membrane where peptides should be placed, absorption measurements were performed using quartz capillaries (700 lm diameter) filled with 0.5 M iodoform dissolved in chloroform. Instead of measuring f 00 and calculating f 0 the distinct iodine L III edge was found by varying the beam energy about ±0.025 keV around the tabulated L III edge by means of modulating the undulator gap and detecting the absorption. The iodine L III edge was found at E = 4.5578 keV. For the contrast variation in the anomalous scattering experiments, the reflectivity measurements were performed at energies of 4.5578 and 5.8000 keV.
In-house X-ray reflectivity (nonisomorphic samples)
For comparison and because of higher resolution (less absorption at higher energies) and enhanced contrast variation [see tabulated atomic scattering factor f 0 of iodine (Gullikson 1995 (Gullikson -2008 ], in-house reflectivity experiments have been performed with the same sample composition as applied in synchrotron experiments. As the beam energy cannot be varied at in-house experimental stations, nonisomorphic samples, with respect to the iodine labels, were used for the home-built diffractometer. The in-house experimental station is a stationary H/2H diffractometer with a Seifert long fine focus X-ray tube holding a Cu anode (U = 35 kV, I = 40 mA) and a Cyberstar point detector. The X-ray beam is monochromatized and parallelized by a Göbel mirror selecting the Cu-K a line (E = 8.048 keV, k = 1.541 Å ). The primary beam intensity is on the order of 10 9 counts per second (cps). Automatized absorbers are used to avoid detector saturation for 2H close to zero and at first Bragg reflexes.
The sample is mounted to a Huber goniometer for which three linear stages for x, y, and z translation are used to place the sample in the center of rotation. The sample-todetector distance is 400 mm. The lateral dimensions of the primary beam (1 9 5 mm 2 ) are defined by two slits in the horizontal and the vertical direction (S1). A vertical slit behind the sample (S2) screens scattering that does not stem from the sample; additional slits in front of the detector (S3) define the resolution of the instrument. All scans are performed at slit widths of 2 mm (S2) and 0.5 mm (S3). For the given sample-detector distance, the latter yields a resolution of 0.07°or 0.01 Å -1 . With these settings, the profile of the primary beam typically has a full width at half maximum (FWHM) of 0.2°. The diffractometer motor and the detector controls as well as the monitor counter readout are accomplished by the SPEC software (Certified Scientific Software, Cambridge, MA, USA).
Two-dimensional resolved maps of the q space were derived from the CCD images via data treatment that includes (1) positioning of the beam center, (2) polarization correction, (3) elimination of dead pixels, and (4) scaling. Data treatment was performed by applying a self-written MATLAB (The MathWorks, Natick, MA, USA) tool (Weinhausen 2010) . This tool further allows for sectioning through the reciprocal space at a definite angle (/) between q z and q k leading to extraction of the intensity courses along these sections via averaging over an angular ROI five pixels wide. The respective intensity profiles were linearized. Lorentzians with linear backgrounds were fitted to the extracted functions:
where x is the half width at half maximum, q 0 is the peak center, I 0 is the maximum of the Lorentzian without the linear offset, m is the slope of the linear background, and b is the constant offset of the Lorentzian. The correlation length n = 1/x and the average chain distance in real space were calculated:
For analysis of the resulting reflectivities, a self-written MATLAB software tool was applied ). The reflectivity data were plotted as a function of the vertical momentum transfer q z after subtraction of the diffuse scattering (offset scan) and illumination correction. The electron density profiles were calculated by applying an empirical Fourier Synthesis (FS) scheme, exploiting the area under Bragg peak intensities I n , as it is used for such multilamellar lipid membranes (Münster et al. 2000; Spaar et al. 2004; Wu et al. 1995) . In simple terms the onedimensional electron density profile q(z) was obtained via Fourier synthesis method from the integrated peak intensities via Gaussian fitting, applying the Lorentz correction factor 1/q z 2 and phases -, -, ?, -, -, -, -in accordance with the number of observed Bragg reflexes. The phases t n are reduced to positive/negative signs due to the point symmetry and were reconstructed in accordance with the 1D swelling method approach using Eq. 3 to obtain the continuous form factor F(q z ) with its relative amplitude |F(q z,n )| and phase t n : Eur Biophys J (2011) 40:417-436 423
The scattering vectors are represented by q z and q z,n , and d is the membrane periodicity (Aeffner et al. 2009 ). The phases have been extracted by the 1D swelling method of pure DLPC-lipid samples (Schneggenburger et al. 2009 ). The electron density profile q(z) normal to the interface is computed by N 0 Fourier coefficients f n = I n q z (Eq. 4):
The factor n in front of the Bragg peak intensity I n follows from an empirical correction factor to calculate the nth Fourier coefficient. The curves have been normalized by scaling higher-order Bragg peaks to the area under the first Bragg peak. General aspects of reflectivity experiments are discussed elsewhere (Salditt et al. 2002) .
Insertion and lateral lipid response by reciprocal space mapping
The homodimeric representative of the applied peptide motif H-(Tyr-Tyr) 4 -Lys-OH has already been shown to enable intermolecular interaction via its phenolic sidechain pattern within the aqueous phase (Alexopoulos et al. 2004 ). The homodimer species 2 adopts a membrane spanning orientation in DLPC bilayers (Küsel et al. 2007) and likewise tends to aggregation (Schneggenburger et al. 2009 ). Therefore, the GID approach was undertaken to evaluate if the latter observation of a membrane insertion and transmembrane alignment is likewise true for the designed hairpin species (2) and the recognition system (3) (Fig. 2) . Furthermore, it was our aim to screen the samples for any evidence of lateral peptide interaction, i.e., the formation of higher-order structures (low q k , q z ), as sometimes indicated by superstructure peaks (Constantin et al. 2007 ) as well as for regular intrahelical distances that can be revealed from the occurence of so-called helix peaks (Spaar et al. 2004 ).
Highly aligned membrane stacks of either a pure lipid matrix (DLPC/Chol) or differentially composed peptide/ lipid compositions (Fig. 2) were analyzed. The lipid phase of each sample contained 5% cholesterol to enhance the surface mosaicity and reduce bilayer fluctuations allowing for pronounced Bragg reflections and correspondingly higher resolution in the reflectivity experiments (Chen and Rand 1997; Mouritsen and Zuckermann 2004) . The particular sample composition, the variation of parameters, and the obtained data are shown in Table 1 .
The direct comparison between undisturbed DLPC/Chol membranes and membranes with either homodimeric or hairpin structures was carried out via analysis of samples 1-3 (Table 1 ). The homodimer 4 [=H-(Phe(4I)-Tyr-(Phe-Tyr) 4 -Trp-Trp-OH] was embedded at a P/L ratio of 1/10 while the hairpin system 5 [=H-(Phe-Tyr) 3 -Phe(4I)-Tyr-Phe-Tyr-Trp-Trp-Gly-Lys-Pro-Gly-(Phe-Tyr) 2 -Phe(4I)-(Tyr-Phe) 2 -Tyr-Trp-Trp-OH] was embedded at P/L = 1/20, both leading to a peptide-to-helix ratio of 1/20. The equimolar mixture of species 3 and 6 [= H-Lys(TAMRA)-Tyr-(Phe-Tyr) 4 -Trp-Trp-Gly-Lys-([C 2 H 4 O] 2 -CH 2 -CO-gcgtgg-Lys-Lys)-Pro-Gly-(Phe-Tyr) 5 -Trp-Trp-OH] representing the recognition system could at least be incorporated into multilamellar DLPC stacks at P/L = 1/40 avoiding precipitation. A second hairpin sample (sample 4) including compound 5 was prepared at a P/L ratio of 1/40. Therefore, samples 3 and 4 were analyzed to account for the concentration dependency of the observed lipid response. In order to evaluate the applied methodology and due to the fact that the recognition system (3/6) should be most amenable to changes in RH because of its polar PNA moieties facing the water layers, a series of experiments was performed at different RHs ranging from 94% to 25%. All other samples were analyzed at RH = 94%.
In searching for potential superstructure peaks in the low q z range and the helix maximum at higher q z , first, scans of the small q k space along the q z axis were performed in GID mode under variation of the angle of beam incidence (Fig. 3) . These scans were followed by addressing the far q k space under attenuation of the specular axis, yielding a combination of exposures that provide a partial overlap (data not shown).
As neither reflections resulting from superstructures nor from helix peaks could be observed for the reconstituted peptide species 3-6, quantitative images (accumulations) of the q space were taken, focusing on the lipid chain correlation peak (cc peak). In analyzing the lateral lipid bilayer response of different samples upon peptide insertion, the cc peak gives indirect information on the peptide reconstitution and peptide/lipid interactions. Attenuation of the specular axis at low angles of incidence, e.g., a i & 0.4°, avoiding overexposure of the detector provided sufficient access to the cc peak at low q z (Fig. 4a) . Taking images of the reciprocal space at higher a i (e.g., a i & 2.5°) without attenuation led to unacceptably high sample horizons (shadowed low q z ) that were not applicable for analysis of the cc peak.
Corrections of the scattering distribution (dark image, polarization), normalization (monitor signal, counting time), and coordinate transformation to (q k , q z ) coordinates based on the calculated sample-to-detector distance led to the 2D intensity distribution in the reciprocal space (Fig. 4a) . Intensity profiles (Fig. 4b) of the cc peak were extracted by interpolation along radial sections through the reciprocal space (black lines in Fig. 4a ) for different angles / corresponding to the tilting of lipid acyl chains in real space (Fig. 4a, inset) . The root mean square deviation of the intensity was estimated via extraction of five neighboring profiles within a distance of D/ = 0.2°( 1-2 pixels). A Lorentzian function (Eq. 1) was fitted to the intensity traces via a nonlinear least squares method using a trust region.
The Lorentzian function fits the intensity profiles [I(/)] very well for all /. For calculation of the average lipid acyl chain distance a (Eq. 2) and the correlation length n only intensity traces at small / were used in order to minimize the q z component. Depending on the angle of beam incidence the sample horizon was partially shadowing the intensity profiles at / \ 8°for some of the samples. Therefore, the Lorentzian fits for / = 8° (Fig. 5a) were used for the calculations of a and n (Fig. 5b) . In addition to the acyl chain distance a and the correlation length n, the extension of the cc peak in D/ gives information about the tilt homogeneity of the acyl chains (Fig. 4a, inset) . Quantitative fits of the profiles along this dimension, as performed for pure lipids (Weinhausen 2010), were not applicable for the present data for reasons of the signal-tonoise ratio.
First, it is important to keep in mind that the next neighbor distance a between lipid acyl chains as well as the In addition to the peptide component, the P/L ratio and the relative humidity ( Table 1 and visualized in Fig. 5b .
As we were interested in the changes of the lipid bilayer structure upon peptide insertion we had to exclude influences on the ordering and packing of lipids exerted by other parameters. These are, on the one hand, the constitution of the lipid species in terms of chain length and saturation (Seelig and Seelig 1977) , chain branching (Perly et al. 1985) , and headgroup structure (Cullis et al. 1986) , and on the other hand, the lipid phase state (Lafleur et al. 1990b; Perly et al. 1985; Sternin et al. 1988 ) and the cholesterol content (Dufourc et al. 1980; Oldfield et al. 1978; Stockton and Smith 1976) . The use of only one lipid component with a fixed cholesterol content eliminates the mentioned impacts. The uniformity of physical parameters, namely the temperature (Davis et al. 1980 ) and the relative humidity, was assured by the controlled sample environment.
Therefore, the experiments carried out with the isomorphic sample 3/6 (equimolar)/DLPC/Chol (1/38/2) at different values for RH (samples 5-9) served as a control for the suitability of the experimental protocol as well as the scattering setup and the data treatment. Furthermore, it should be tested whether a variation of RH is detectable by changes in a and n and if these changes are within an appropriate regime and follow expected trends.
In general, lowering the RH promotes the gel phase character of the lipid membranes causing a reduced fluidity and increased inter-bilayer potentials (Ho et al. 1995; Long and Hruska 1970) . Upon reducing the water content of the system, the fraction of gauche bonds within the lipid acyl chains is diminished and leads to lipid chain stretching. As a consequence, the lipid chain alignment and ordering (Cevc and Marsh 1985) as well as a lateral compression or packing (Bryant et al. 2001; Chen and Hung 1996) are enhanced. Such behavior was confirmed by the results obtained from measurements for samples 5-9. In line with the literature, a lowering of the acyl chain distance of about Da max = 0.097 ± 0.006 Å and an increase in the correlation length of Dn max = 0.8 ± 0.2 Å were observed when comparing the results for RH = 94% to RH = 25% and to RH = 60%, respectively. The revealed effect that at low water content with RHs between 40 and 25%, the correlation length n seems to reach saturation and even slightly decreases for sample 9, can be associated with the steric demands exerted by the peptide and cholesterol inclusions (Fig. 5b) . Steric demands of the relatively rigid peptide helices become more critical in the more condensed phases than in the fluid phase (Marčelja 1974) . This condition of so-called intermediate fluidity (Oldfield and Chapman 1972) describes the fact that the interaction between inclusions such as cholesterol or peptides cannot be as strong as interactions among ordered all-trans lipid chains (Marčelja 1974 ). We did not observe the phenomenon of an interchain hydration at high RH that is concomitant with an increased lipid chain-chain distance and results from defect structures caused by inserted peptides (Ho et al. 1995) .
As a general trend upon reconstitution of the different peptide species, we observed a significant decrease in the acyl chain distance a while the values for the lipid chain correlation length n increased (Fig. 5b) . One may initially expect the opposite effect, i.e., an increase in a and a decrease in n. This could appear conclusive, since the occupation of the lipid bilayer area by peptide inclusions would lead to additional and longer trans-helix chain-chain distances for so-called annular lipids, which are in direct contact with the peptide helix. Furthermore, the absent chain-chain correlation between those lipids could reduce n. From the experimental results it is obvious that this is not the case, or at least other phenomena are outweighing such assumed effects leading to an increase in lipid packing (density) and chain ordering.
Changes in the lateral bilayer structure upon peptide insertion are mostly discussed in theoretical studies and appear contradictory with respect to their conclusions. The intensity profiles (Fig. 4b) at a certain constant / were fitted to Lorentzian lineshapes (Eq. 2). The resulting fits at / = 8°(a) were applied to calculate the mean lipid acyl chain distance a and the acyl chain correlation length n (b) Already in early work, motivated by fluorescence data, a distinction is made between lipids not in contact with protein or peptide surface and lipids 'coupled' to the reconstituted protein, which are, thereby, disturbed in their interaction (Träuble and Overath 1973) . These so-called annular lipids were shown not to undergo lipid phase transitions. The postulated protein-derived disturbance was calculated to extend up to the third lipid neighbor of a protein inclusion (Marčelja 1976) . Addressing the structure of annular lipids in greater detail by theoretical molecular field approximations revealed an increased ordering of the lipid chains in proximity to membrane-incorporated structures within the fluid lipid phase (Marčelja 1974) . With increasing fraction of ''foreign molecules'' on the phospholipid bilayer, the membrane order parameter was reported to increase and the dependency of the order parameter on temperature to become less pronounced. We will denote this scenario as the (annular) lipid ordering scenario. In the lipid condensed phase, this effect was described to be the opposite, which we denote as the lipid disordering scenario. Other authors claim that rigid inclusions such as cholesterol might cause straightening and ordering of lipid chains, while the fluid-like surface of embedded proteins does not influence the motional freedom of the lipid chains (Lafleur et al. 1990a) . Molecular dynamics simulations of three different peptide a helices, published at the same time, likewise revealed an ordering of the lipid chains close to the peptide helices for all peptide species (Edholm and Johansson 1987) . Even if the overall effect was assessed as ''not drastic,'' the annular lipid ordering could be estimated as ''much less pronounced'' for lipids with bulky side chains. More recent theoretical considerations applying statistical mechanical integral equation theories (Lagüe et al. 1998 ) predict an expansion of the area per lipid for the annular shell upon peptide insertion into different phosphatidylcholine (PC) bilayers. In these studies, peptides are treated as soft cylinders (Lagüe et al. 2001 ). The authors postulate effective long range repulsion between lipids and peptides due to the formation of a lipid depletion layer around a protein leading to an increased cross-sectional area per lipid molecule. According to this view, which also supports the lipid disordering scenario for annular lipids, a lipid molecule in close contact with the peptide must significantly reduce its order. Effective lipid-protein repulsion would arise due to its entropic disadvantage. These theoretical results were further supported by MD simulations of a hydrated 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC) bilayer containing an a-helical peptide bundle of four transmembrane domains (Husslein et al. 1998 ). Comparable to the results of Lagüe et al., Husslein and coworkers revealed an increase in the area per lipid from 7.46 nm 2 for the unperturbed bilayer to 8.50 nm 2 in the vicinity of the peptide species. Finally, for the sake of completeness, we note that a pronounced annular lipid effect is not reported by all studies. Using spin labeling techniques, Marsh and Horváth found order parameters of protein-associated lipid chains to be very similar to those in the bulk liquid-crystalline phase regions (Marsh and Horváth 1998) . Considering these opposing scenarios, it is interesting that we observed a very clear indication of the lipid ordering scenario. The decrease in the average lipid chain distance a as well as an increase in the lipid chain correlation length n for the peptides studied here can be ascribed to a reduced number of lipid gauche rotamers (Fig. 6) . For different molecular systems imposing different molecular boundary conditions, the second scenario could be valid instead. It should be taken into consideration that for the sake of simplicity, the theories supporting either one of the scenarios neglect the individual chemical properties of the incorporated molecular species that are the bases for interaction at the peptide/lipid interface.
However, the particular molecular properties of different species may be the decisive parameters that determine whether a peptide/lipid interaction is attractive or repulsive. For the presented peptide species 1-6 these parameters are (1) the unique b-helical secondary structure caused by the D,L-alternation and bulkiness of the peptide side chains, (2) the exclusiveness of the side-chain composition with almost entirely aromatic amino acid residues, and (3) the presence of flanking interfacial anchoring moieties. Our findings are also in line with literature studies that report higher order parameters for hydrocarbon chains of lipids adjacent to peptide channels composed of aromatic amino Fig. 6 Schematic representation of the conclusions drawn from GID results, suggesting a positive hydrophobic mismatch and, most likely, an enhanced highly ordered packing of the annular lipid shell. These effects may be attributed to the specific properties of the peptide species, i.e., their sequences, side-chain orientations, and secondary structures Eur Biophys J (2011) 40:417-436 427 acids (Chiu et al. 1999 ). This should be particularly pronounced in the case of tryptophan-containing peptides, such as species 1-9, as tryptophan is known as an amino acid with high preference for lateral interaction (Adamian and Liang 2001; Ridder et al. 2005) . The planarity and hydrophobicity/amphipathicity of the aromatic side chains have already been shown to provide potential for a perfect alignment and interaction with the lipid bilayer core via intercalation between the lipid chains (Hite et al. 2008; Palsdottir and Hunte 2004) . Hydrophobic interaction might further direct lipid chains to approach closer contact to incorporated peptide species via lowering entropic costs for acyl chain deformation. This lipid/peptide interaction might play a predominant role for b-helical peptide species due to the fact that all peptide side chains are pinpointing radiantly outside the helix . Proper vertical alignment for the reconstituted peptide in the bilayer was concluded already in a previous reflectivity study exhibiting up to six lamellar orders nearly independent of the applied peptide concentration (Küsel et al. 2007 ).
Owing to the nature of bilayer elasticity, the effects of lateral packing cannot be discussed without addressing the vertical structure and density profile, which are closely interrelated. Namely, the effects of hydrophobic matching and interfacial anchoring (Killian and Nyholm 2006) interfere with the lateral forces. Hydrophobic matching and interfacial anchoring are especially important for the presented peptide species, as all of them hold two flanking tryptophan residues. The magnitude of hydrophobic mismatch is considered to be a function of the distance between interfacial anchoring residues, here tryptophans, and not the overall peptide length (De Planque and Killian 2003) . In this respect, tryptophan residues are known to provide the highest tendency for localization at the interface between the lipid bilayer core and the polar lipid headgroup. Hydrogen bonding and electrostatic interactions are discussed to account for the interfacial anchoring and z positioning of membrane-incorporated peptides (De Planque and Killian 2003; Doux and Killian 2010) .
A stretching of the lipid bilayer in the z direction caused by hydrophobic mismatch (Killian and Nyholm 2006) may alone result in pronounced lateral effects on annular lipids. Huang and coworkers observed a stretching of a DLPC lipid bilayer upon incorporation of gramicidin A that likewise adopts b-helical structures, similar to the D,L-alternating peptides applied in the presented approach (Harroun et al. 1999) . In previous studies of the homodimeric species 1, comparable effects were indicated by a thickening of the lipid bilayers of 1.4 Å (DMPC = 1,2-dimyristoyl-sn-glycero-3-phosphocholine) and 4.0 Å (DLPC), respectively (Küsel et al. 2007 ). Finally, the reflectivity experiments of the designed peptide hairpin structures 2, 5, 7, 8, and 9 unambiguously prove a positive mismatch situation, also for the hairpin structures, as presented in Table 2 . Such mismatching promotes stretching of the lipid chains and reduction in gauche conformations in the lipid acyl chains. The mismatch situation as well as the described lateral effects may be directly coupled or, alternatively, may independently contribute to the observed changes in a and n upon peptide insertion, as addressed in the following section.
Differences in peptide species: the cross-sectional area and the characteristics of peptide/lipid contacts If the ordering of the lipid chains is solely a function of hydrophobic mismatch, any of the studied peptide species should yield the same values for a and n due to sequence similarity. Obviously, this is not the case (Fig. 5b) .
For the sample solely composed of DLPC and cholesterol (sample 1) the acyl chain distance is a = 4.94 ± 0.01 Å . The shortening of the acyl chain distance a appears more pronounced for the homodimeric species 4 (sample 2) at a helix-to-lipid ratio of 1/20 (P/L = 1/10) but is even stronger for the hairpin species 5 at the same helix-to-lipid ratio (P/L = 1/20). Considering the recognition system 3/6 (equimolar) at RH = 94% (sample 5), the shortening of a is less distinct but can also be assigned to a lower helix-to-lipid ratio of 1/40 (P/L = 1/20). The concentration effect can be factored out by comparing different samples (3 and 4) of the hairpin species at P/L = 1/20 and P/L = 1/40, respectively, showing that higher peptide concentration yields shorter lipid chain distances. It can further be concluded that the recognition system 3/6 (equimolar ratio) at RH = 94% (sample 5) has less impact on a than the respective hairpin peptide 5 (sample 4) at identical P/L = 1/40. The values for the lipid chain correlation length n follow an almost opposing trend as that observed for a. Peptide incorporation generally leads to an increase in n. In principle, the homodimer (4), hairpin (5), and recognition system (3/6) structures are designed to adopt an identical secondary structure with respect to their TMDs. In the case of the hairpin structure 2, due to a decline in helix propensity compared to the homodimeric species 1 as revealed by CD spectroscopy , it was assumed that the hairpin structure exhibits steric clashes in the reverse-turn region leading to a less perfect alignment of the double strands and a broadening of the helix diameter. Therefore, the self-associated homodimer 4 should provide a closer alignment enabled by hydrogen bonding of the two antiparallel-oriented strands. The PNA recognition system 3/6 leading to an aggregation of two of the double helices in a close contact state would yield more than a doubled lateral occupied area within the lipid bilayer (Fig. 2c) . Under these circumstances, the lateral dimensions of the peptide species are supposed to increase in the order: homodimer (4) ? hairpin (5) ? recognition system (3/6).
As the differences in the values for a and n when comparing the different peptide species cannot be solely attributed to a hydrophobic mismatch situation (see above), lateral peptide parameters such as the peptide cross-sectional area or the peptide/lipid contact surface are assumed to be the primary cause for the observed differences. In line with the lipid disordering scenario discussed above, larger peptide cross-sectional areas and peptide lipid contact surfaces would probably cause higher values for the lipid acyl chain distance a and smaller values for the lipid correlation length n. This is true if peptide/lipid interactions are neglected or a repulsive potential between lipids and peptides is assumed (Lagüe et al. 2001) . For the present sample sequence: homodimer (4) ? hairpin (5) ? recognition system (3/6), this would imply an increase in a and a decrease in n. The fact that the observed results point to the opposite may be taken as evidence for attractive peptide lipid interactions. Due to the sequence identity of the antiparallel TMD strands of all peptide species, and therefore, the constitutional similarity of the postulated attractive peptide/lipid interactions, only the peptide secondary structure, i.e., the lateral sizes of the peptides, and the size of the lipid/peptide interfacial plane should affect a and n. Therefore, a monotonous decrease in the lipid chain distance would be expected for samples 1-3, which is in line with the obtained results (Fig. 5b) . On the contrary, a monotonous increase in the chain correlation length was not observed. The value for n of the homodimeric species 4 (sample 2) appears higher than that for the hairpin 5 (sample 3). This might be explained by the fact that oligomer 4 is known to experience a monomer-dimer equilibrium (Küsel et al. 2007 ) including a structural change from a membrane-spanning b 5.6 helix to two reasonable broader and shorter b 6.3 single helices (Fig. 2) . This would actually change the peptide dimensions in the z direction as well as the molecular constitution of the helix surface and therefore cause different hydrophobic mismatch situations as well as changed lateral peptide/lipid interactions.
In the case of the recognition system 3/6, a close contact state along the outer helix shells results from PNA pairing and TMD assembly . The adopted peptide complexes can be considered as one reconstituted peptide species surrounded by annular lipids (Fig. 2c) . The recognition process, therefore, diminishes the peptide/lipid contact area by shielding the inner helix flanks towards the lipid matrix. In this respect, the recognition system species 3/6 (sample 5) provides lower values for a and higher values for n than the hairpin species 5 at the same P/L ratio.
Peptide positioning and orientation from reflectivity using iodine labels
The membrane response in terms of changes in the lipid correlation length and lipid chain distance addressed above can neither reveal the insertion depth of a particular peptide side chain nor does it allow distinguishing between a membrane-spanning state or insertion perpendicular to the membrane normal. FRET experiments (parallax analysis) are well suited to studying membrane proteins in vivo but are often limited in resolution caused by large Förster radii of the fluorescence probes. In contrast, X-ray reflectivity in combination with contrast variation by heavy atom labeling can provide structural constraints down to subnanometer resolution for fluid or condensed peptide/lipid samples. For a fast in-house approach, the analysis of the electron density difference is a unique method to obtain highly resolved information about the localization of a certain structural element within a lipid bilayer (Fig. 7) . As shown in previous studies for the double helical homodimer 1, this method is even capable of producing evidence of a transmembrane peptide orientation within the lipid bilayer via iodine labeling at several positions (Küsel et al. 2007 ). With the iodine-labeling approach, the alignment of the membrane-spanning helical hairpin formed by the severe acute respiratory syndrome (SARS) coronavirus E protein could be elucidated by localizing the Phe23 adjacent to the lipid headgroup region (Arbely et al. 2004; Khattari et al. 2006a, b) . Instead of determining the label position by a comparison of the deduced electron densities q(z) resulting from unlabeled and labeled samples, this can also be accomplished using an isomorphic sample and applying different photon energies (Khattari et al. 2005) .
In this work, we used both approaches, the label replacement by a sample series for in-house reflectivity and the contrast variation via anomalous reflectivity for the synchrotron studies. In both cases, the commercially available building block Fmoc-Phe(4-I)-OH served as iodine label, substituting the native phenylalanine.
Within the centrosymmetric electron density profiles deduced from reflectivity scans (Fig. 7b) , maxima correspond to the lipid headgroup regions, while the global minimum reflects the acyl-acyl contacts of opposing hydrocarbon chains. The adjacent water layers are represented by the side minima. For anomalous scattering, the electron density profiles obtained at the L III absorption edge (E = 4.5578 keV) were subtracted from the electron density profiles at E = 5.8000 keV to estimate the position of the iodine label with respect to the z direction, indicated by rises in the electron density difference curve (Fig. 7b , blue curve). In in-house experiments such curves result from subtraction of the electron density q(z) of the samples containing iodinated peptides from the sample with unlabeled peptide species.
The detection of single iodine labels via difference analysis usually requires high peptide-to-lipid ratios in the range of P/L & 1/10 ( Arbely et al. 2004; Küsel et al. 2007) . With the synthesis of the novel double iodinated amino acid building block Fmoc-5,5-diiodoalyllglycine-OH, the use of an enhanced in-house reflectivity set up including substrate sizes of 15 9 25 mm and the addition of 5 mol% cholesterol to the lipid phase enabled recording up to seven lamellar orders from samples with a P/L = 1/50. The analysis of the electron density difference profiles then allowed for the determination of the iodine positions with high accuracy (Schneggenburger et al. 2009 ). Even for the Fmoc-Phe(4-I)-OH single label, P/L ratios of 1/50 turned out to be sufficient. Therefore, in-house reflectivity studies and anomalous reflectivity using synchrotron radiation were both performed at P/L = 1/50. Three differentially labeled peptide species, 5, 7, and 8 (Fig. 8) , were used to test for a transmembrane orientation. To avoid beam damage during anomalous reflectivity experiments, the respective curves were solely scanned around the Bragg reflections.
Four to five lamellar orders (Fig. 9a) were recorded by specular and offset (longitudinal diffuse) scans. Unfortunately, the specular condition (angular alignment) was lost in some of the scans, so that the diffuse offset scans, which by nature are less sensitive to angular misalignment and which show strong lamellar reflections, were used for the Fourier synthesis analysis. Due to the difficulties associated with low energies around the I-L III edge and the intrinsically small anomalous difference signal, all experiments were repeated on an in-house diffractometer without any risk of radiation damage during long scanning and with most careful alignment procedures. The one-dimensional swelling method was used to yield the phases t n .
In in-house experiments, six intensive equidistant Bragg peaks were obtained for the labeled (7) as well as the unlabeled (2) hairpin species (Fig. 9b ) due to the higher radiation energies (Cu-K a = 8.048 keV) and improved alignment. For peptides labeled with the Fmoc-Phe(4-I)-OH building block, species 2 served as reference structure. In case of hairpin 8, labeled with Fmoc-5,5-diiodoallylglycine-OH, analog 9 with the sequence H-(FY) 5 WW-Gallylglycine-PG-(FY) 5 WW-OH was applied as unlabeled species. As the electron density profiles of the lipid bilayer structures obtained from in-house experiments were more resolved, these data were used to estimate the respective changes in hydrophobic thickness (see below).
The X-ray reflectivity curves (Fig. 9 ) and the corresponding electron density profiles (Fig. 10a, b) are exemplarily shown for compound 7 and for its unlabeled analog 2 (in-house), respectively. More data are shown in the Electronic Supplementary Material. The derived electron density difference curves for all studied constructs 5, 7, and 8 and unlabeled compounds 2 and 9 are depicted in Fig. 10b, c. a b water-layer water-layer water-layer Si-substrate Fig. 7 Schematic representation of the reflectivity scattering geometry for multilamellar P/L complexes (a) with highlighted iodine positions (red arrows) and deduced electron density profiles (right, b) . The comparison of electron density profiles originating from samples containing labeled and unlabeled peptide species or from scattering at different photon energies yields the difference curve (blue)
The reflectivity curves and deduced electron density profiles reveal a stretching of the lipid bilayers upon peptide insertion. This was found for all examined peptide species (2, 5, 7, 8, 9) and is in line with the obtained GID results confirming a positive hydrophobic mismatch. The repeat spacing and the peak-to-peak distance (d pp , compare to Fig. 7) values (Table 2 ) evidence a small thickening of the lipid bilayer up to 1.4 Å , which is more pronounced for the mono-iodinated species 5 and 7. In case of the doublelabeled species 8 and its reference analog 9, this effect is inverted.
In general, the electron contrast variation resulted in changes in the headgroup densities, which were too pronounced to be explained by the contrast variation itself and therefore indicate systematic errors. These probably stem from misalignment or drift when changing the photon energies. Thus, the two approaches of contrast variation (in-house and synchrotron) are not in quantitative agreement. For the in-house measurement, the effect of a decrease in electron density contrast of the side minima compared to the global minimum is observed in most peptide/lipid systems. The decrease can be explained by a beginning lipid disorder that evens the density profile by increased density in the water layer and/or an enlarged area per headgroup ; this effect may be more pronounced for a peptide carrying a bulky iodine label. However, we also cannot rule out systematic errors for the in-house measurements, and beyond the thickening effect, which appears to be robust, the small differences between curves with and without labels may impede an unambiguous localization of the iodine position. Finally, we must be cautious since iodine-labeling itself may influence peptide/lipid interactions. However, there is no obvious reason why attachment of the iodine labels at different positions should cause different peptide positioning, especially as the iodine labels have been shown to have only minor effects on the peptide secondary structure (Schneggenburger et al. 2009 ).
Notwithstanding the problems mentioned above, we will now address the difference curves in view of a tentative interpretation of label position and peptide orientation in the next subsection, step by step for each peptide and labeled construct.
Species 5 The center maximum of the density difference curve for the middle-labeled oligomer 5 (both intertwined strands) indicates that a significant fraction of the iodine labels are buried deeply in the hydrophobic core of the lipid bilayer (Fig. 10b, d) . In both data sets, in-house and synchrotron, the membrane thickness (peak-to-peak distance) is approximately 32 Å (see the Electronic Supplementary Material). The distribution of the central maximum of the difference curve appears relatively broad, especially for the in-house experiment (Fig. 10c) .
The synchrotron data do not only illustrate a rise in intensity at the bilayer center but also at approximately z = ± 11.1 Å , which does not occur in the higher resolution in-house study (Fig. 10b, d) . These peak positions may still be commensurate with more than one orientation, for example either a membrane-spanning orientation of the b-hairpin (Fig. 11i) or an orientation of the peptide species parallel to the membrane surface (Fig. 11ii) at the bilayer center. The latter is, however, not very likely for energetic reasons. (1/47.5/2.5) from diffuse anomalous scattering (a) and inhouse reflectivity (c). In the inhouse experiments the samples containing the iodine-labeled compound 7 were compared to the samples lacking the iodine probes (compound 2, black curve). The electron density difference curves (blue, shifted for clarity) are depicted for all peptide constructs 5, 7, and 8 either studied by synchrotron radiation (b) or by in-house reflectivity (d). The curves derived from in-house measurements are multiplied by a factor of 3 for clarity; the headgroup region is highlighted (green area)
Species 7 Anomalous reflectivity of terminally labeled peptide species 7 in peptide/lipid complexes (Fig. 10a) showed relatively weak minima in the density corresponding to the water layer. If this is not an artifact of the rather problematic synchrotron experiment, it could be possibly attributed to increased lipid fluctuations or beginning lipid disorder upon localization of the rather bulky iodine labels at the lipid headgroup region. The electron density difference curve shows only two symmetrically distributed maxima at approximately z = ± 15.0 Å indicating a localization of the iodine labels at the lipid headgroup region tending to the membrane interior. This observation would perfectly fit with a transmembrane orientation of the b-hairpin (Fig. 11i) , although an orientation parallel to the membrane surface at the headgroup region (Fig. 11iii ) cannot be ruled out. Taking into account the observations made for the peptide/lipid complex 7/DLPC/Chol (1/47.5/ 2.5), this appears rather unlikely, since the depth of the insertion should not depend on the position of the label along the helix. The distance between the maxima of the electron density difference curve, associated with the distance of the two iodine labels is about 32 Å (see the Electronic Supplementary Material). This is in good agreement with the expectation based on a peptide length of 26 Å and the additional spacing of the Phe(4-I) side chains attached at both ends of the double helix and pointing to opposite directions (Schneggenburger et al. 2009 ).
The electron density profiles derived from the in-house measurements of compounds 7 and 2 (Fig. 10c) show reasonable lower contrast between labeled and unlabeled samples, which is much closer to the expectation. Four small bumps in the electron density difference curve appear at z = ±17.0 and ±7.0 Å (Fig. 10d ), corresponding to possible inter-iodine distances of approximately Dz = 34, 14, 10, or 24 Å . Compared to b-structures of gramicidin A that are likewise assumed for peptide 7 or the unlabeled analog 2 with a periodicity of 5.6 or 6.3, the investigated b-hairpin with a strand length of 12 residues would give a peptide length of 28.8 or 20.8 Å , respectively (Fahsel et al. 2002) . This would not directly match a transmembrane orientation without assuming additional effects from distortions, tilt (Fig. 11iv) , or a coexisting population (Fig. 11v) . The rather flat difference curve of the in-house data for species 7 appears less conclusive than difference curves with more clearly identifiable maxima.
Species 8 In case of the molecular species 8, holding a 5,5-diiodo-allyglycine label at the loop position, five lamellar orders could be obtained in the synchrotron experiment (see the Electronic Supplementary Material). The peak-to-peak distance of the electron density curves reveals a headgroup spacing of approximately 33 Å (4.55 keV) and 32 Å (5.80 keV), respectively. The corresponding electron density difference curve is pronounced due to the double signal intensity resulting from the double iodinated label, but differences in resolution at the different X-ray energies may also play a role. The profile shows strong maxima at ±15.4 Å and additional inflection points around ±7.1 Å (Fig. 10d) . The maxima at ±15.4 Å may indicate a transmembrane orientation of the b-hairpin structure 8 nearly spanning the same distance as the terminally labeled sample 7/DLPC/Chol. The electron density difference curve of in-house reference measurements does not show significant rises in the headgroup region, but only at the membranes outside (±21.4 Å ) in the membrane water layer (see above). A rather diffuse and less intense maximum appears spread out over the bilayer hydrophobic core.
Summarizing the results of the iodine-labeling experiments, the electron density difference data from synchrotron experiments and in-house data are not in agreement. While the synchrotron experiment faced technical challenges related to the relative low photon energy (L III edge) and sample realignment after beam drift when changing the energy, as well as intrinsically lower contrast, the comparison of two different, not necessarily isomorphic, samples in the in-house study may also pose a challenge. In other words, the differences from one sample to the next may be on the same order as the label effect itself. Note that one needs a much higher reproducibility to interpret the difference curve than the rather gross features of the profiles before subtraction. Therefore, we consider the results regarding the membrane repeat spacing to be more reliable and consistent than conclusions drawn from the peptide labels.
Fortunately, however, the reflectivity results for the different peptide species 5, 7, and 8 need not be judged alone to make conclusions about the peptide orientation Fig. 11 Exemplary schematic representation of orientations that could be adopted by labeled peptide species in lipid multilayers and fit the reflectivity data of one or the other peptide species (Fig. 11) . The observed changes of the inner hydrophobic core upon peptide insertion, as revealed by GID/reciprocal space mapping, together with the sum of the reflectivity results may not completely rule out a simple surface anchoring or surface adhesion of the peptide species 2-9, but they make an inserted orientation parallel to the membrane normal, possibly with some tilt distribution, much more likely. This would be further supported by band shifts of the fluorescence emission spectra , indicating a localization of flanking tryptophan residues in the lipid headgroup regions.
The lipid response upon the insertion of peptide constructs systematically varied in the oligomerization state and secondary structure was addressed by an X-ray scattering study. The approach included grazing incidence diffraction and X-ray reflectivity and is, in principle, applicable to any multilamellar peptide/lipid complex.
The preparation of multilamellar lipid samples is pretty much straightforward and only requires minor adjustments due to solvent compositions at high P/L ratios. In contrast, the requirements for the sample environment are rather high in terms of a stable and reproducible RH. Nevertheless, an advanced setup is not mandatory, since very distinct and stable RHs can also be generated by applying saturated salt solution reservoirs in a sealed sample chamber. (Greenspan 1977; Winston and Bates 1960) .
It has been shown that reflectivity experiments can be carried out with an in-house diffractometer with high performance and advanced resolution. This is especially true for an analysis of the hydrophobic membrane thickness variation upon peptide insertion. For a labeling approach, presented herein, in-house reflectivity holds the advantage of a higher contrast variation compared to energy variation around the I-L III absorption edge but includes the application of nonisomorphic samples. However, in-house reflectivity in combination with heavy-atom labeling is generally suited to generate reliable results for peptide side-chain positioning within the lipid bilayer (Küsel et al. 2007; .
GID experiments are normally not only carried out at beamline experimental stations; a more extended sample screening appears only reasonable for taking advantage of the relatively short exposure times resulting from usage of a high-brilliant synchrotron X-ray beam. The methods presented herein are amenable to users that are not closely related to the X-ray field. This is notably true, since macro scanning and automated data evaluation can be applied that is even recommended if it is dealt with an increased quantity of samples.
In this study GID yielded quantitative values for the lipid chain correlation length n and the average lipid chain spacing a via analysis of the lipid chain correlation peak. Reflectivity studies provided additional information about the variation in the lipid spacing d and bilayer thickness d PP (Fig. 7b) . This rather general approach of monitoring the collective lipid response to peptide interaction was combined with multiple site-directed iodine labeling, extending previous studies (Küsel et al. 2007; ) through a larger variety of constructs, labels, and label positions. While a good approach in principle, this part of the presented work showed results that were less consistent and thus less conclusive due to experimental difficulties. Future improvements could include the use of the I-K a edge at 33.17 keV rather than the low energy L edges or the application of a more dedicated setup for this spectral range. Despite some of these problems, the data seem to point to a membranespanning orientation of the designed b-helical peptide hairpins.
Without relying on the contrast variation, a thickening of the bilayer by stretching of lipids in the z direction could be clearly and consistently evidenced. Thus, membrane insertion of b-helical structures is characterized by a positive mismatch situation. This hydrophobic stretching of the membrane and a good accommodation of the peptide species, which might be based on their extraordinary sidechain composition, also result in an increased membrane packing and lipid ordering of annular lipids. These findings are in contrast to theoretical studies that describe peptide helices as soft cylinders (Lagüe et al. 1998 (Lagüe et al. , 2001 . Two effects could possibly contribute to the enhanced lateral packing, the hydrophobic mismatch and/or hydrophobic, attractive lipid/peptide interactions resulting from sidechain intercalation into the lipid matrix.
Future experiments can be designed to evaluate the separate effects of lateral packing and ordering, systematically varying the mismatch by using longer chained lipids such as DMPC or reducing the aromatic content of peptide side chains via sequence mutation.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Vaccine Potential of Nipah Virus-Like Particles Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease. Since it was first recognized in 1998, Nipah virus (NiV) has caused several outbreaks in humans of encephalitic disease associated with high lethality. In the first outbreak, which was in Malaysia and Singapore, 265 humans became sick and some ,40% of them died. Epidemiological links pointed to human contact with sick pigs in commercial piggeries, and the outbreak was brought under control through culling of approximately ,1.1 million pigs [1, 2, 3, 4] . Since then, the virus has re-emerged in Bangladesh and neighboring India, starting in 2001, and between then and now, has caused several smaller but even deadlier disease outbreaks with case fatality rates ranging between 60 and 90% [5, 6, 7, 8] . Unlike the Malaysian outbreak, the route of transmission in these outbreaks was considered to be bat-to-human via food contaminated with bat saliva [9] . In some cases, nosocomial transmissibility and person-to-person spread was also noted [5, 10, 11, 12] . An additional concern is that NiV is also potentially an agent of agro-terror since the rate of transmission of this virus in the pig population is close to 100% [13] . Effective vaccine and therapies are needed to combat the threats posed by NiV.
NiV is a member of the genus Henipavirus in the subfamily Paramyxovirinae, family Paramyxoviridae. It has several distinctive genetic and biologic features [14, 15, 16, 17, 18] although its morphology and genome organization is similar to that of other members of the subfamily. NiV has six genes arranged in tandem, 39-N, P, M, F, G and L-59 [15, 16] . The N, P and L are required for reconstituting viral RNA polymerase activity, the matrix protein M is required for particle formation and budding, and the two surface glycoproteins G and F are required for attachment and entry into the host cell [19, 20] . EphrinB2 and B3 have been identified as the NiV entry receptors [21, 22, 23] . After fusion of the virus and the cell membrane, the viral ribonucleoprotein is released in to the cell cytoplasm. Following transcription and replication, the viral components migrate to the plasma membrane for assembly and budding of progeny particles [24, 25] .
Two vaccination strategies for NiV disease prevention have already been explored experimentally: A canarypox virus-based vaccine vector approach was effective as veterinary vaccine [26] , and it is in the process of further development. The same approach for human use vaccines is undergoing extensive evaluation, largely for HIV and AIDS [27] . A soluble NiV G protein approach has also shown promise [28, 29] . However, subunit approaches are in general less effective than particulate immunogens, and can suffer from suboptimal presentation to the immune system [30, 31, 32] . Immunogenicity in mice to NiV glycoproteins has been reported recently using two vectored approaches for gene delivery; one using Venezuelan equine encephalitis virus replicons [33] and the other involving inoculation of a mix of two complementing defective vesicular stomatitis virus (VSVDG) vectors, one for expressing each of the two NiV glycoproteins [34] . The latter approach is new and seems promising but its regulatory approval as human vaccine might be problematic [34, 35] .
In this study we have explored the potential of NiV virus-like particles (VLPs) as a vaccine. Plasmid-mediated expression of selected viral proteins results in the spontaneous assembly and release of VLPs. These particles make highly effective immunogens because they possess several features of the authentic virus such as their surface structure and dimensions [31, 36] . They are also safe because they do not contain any viral genetic material. VLPs, where one or more of the constituent proteins serve as immunogens (native VLPs), are particularly effective as vaccines for infectious disease. The fact that two such vaccines [Gardasil (Merck & Co) for human papillomavirus (HPV), and Sci-B-Vac (SciGen) and Bio-HepB (GlaxoSimthKline) for Hepatitis B virus (HBV)] have already been approved for human use, and many, for non-enveloped and enveloped viruses [31, 32, 37, 38, 39, 40, 41, 42] are at various stages of development, attests to the desirability of this approach for vaccine development.
The budding capacity of virus proteins as VLPs, the proteinprotein interactions that facilitate this process, and the central role of M protein in VLP assembly and release has been described for several paramyxoviruses such as Sendai virus (SeV), Newcastle disease virus (NDV), respiratory syncytial virus (RSV), paramyxovirus simian virus 5 (PIV-5) and human parainfluenza virus type 1 (hPIV1) [20, 43, 44, 45, 46, 47] : The efficiency of VLP formation in virtually all these studies was based on M protein release in the supernatant. NiV virus-like particles have also been described [48] ; the results of this study showed 1) that NiV G and F proteins individually retained some budding capacity although it was far less efficient than that of the M protein and 2) NiV N, M, F and Gcontaining VLPs resembled the virus in some respects but differed significantly from it with respect to ratio of VLP-incorporated F protein; most of it was present in precursor F 0 form. Recently, the vaccine potential of native VLPs of NDV [49] has been described: these particles, composed of HN, F, M and NP proteins, had several virus-like properties. However, since the F protein in this formulation was modified by design to ablate the cleavage site, it remained in its precursor form; consequently, the NDV VLPs were non-fusogenic, and therefore incapable of inducing syncytia formation.
Here we describe NiV VLPs composed of the two surface glycoproteins G, and F, and the matrix protein M. The G and F proteins were included because they mediate attachment and entry into the host cell [50, 51, 52] , both are major targets of neutralizing antibodies, and both are major players in vaccine induced protection [52, 53, 54] . NiV G and F together are also the most effective as immunogens; this was elucidated in a canary pox virus vector-based experimental protective efficacy study [26] . The M protein was included in our formulation because it is required for particle formation and release [20, 25, 45] . Under optimized conditions, we were able to make substantial, quantifiable amounts of NiV VLPs composed of these three NiV proteins. This has allowed us to characterize their properties in detail to show that they possessed many virus-like/vaccine desirable properties in vitro. It has also allowed us to test for immunogenicity in vivo in Balb/c mice; note that although NiV does not cause disease in these animals, NiV proteins injected in them are known to induce robust neutralizing antibody response [29, 33, 34] . Importantly, NiVspecific mouse monoclonal antibodies are protective in the hamster model of NiV disease [55] .
In this study, careful assessment of immunogenicity has shown for the first time, that these NiV VLPs are able to induce neutralizing antibody response. We have also provided a detailed methodology to optimize production of the VLPs for research purposes. Beyond this, we have provided the first CryoEM study of NiV VLPs and thus provide a careful assessment of their morphology. We further demonstrate that NiV VLPs can trigger ''fusion from without'' upon addition to cells. To our knowledge this is a first for an enveloped VLP. Finally, we have shown that NiV VLPs activate innate immune signaling in ''infected'' cells and provide a transcriptional profile of this response. Based on all these attributes, NiV M, F and G-protein-containing VLPs show promise as vaccine and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.
NiV expression plasmids pCAGGS-G, F, and M are all under the control of chicken beta actin promoter [56] , and they were constructed in the laboratory of one of the co-authors of this study (CB) as described previously [20] . Human embryonic kidney 293 cells (ATCC, CRL-1573) and 293T cells (ATCC, CRL-11268) were grown in Dulbecco's minimum essential medium supplemented with10% fetal bovine serum (FBS) and penicillin and streptomycin, and maintained in the same medium containing 2% FBS. The minigenome that was used for optimizing VLP formation has been described previously [57] . All the initial minigenome-based optimization steps were done in BHK-T7 cells (a gift from Dr. N. Ito). The same conditions were applicable to produce VLPs in 293T cells and they were used throughout to generate the VLPs used for the work described in this study.
293T cells were grown in Dulbecco's complete medium to achieve semi-confluent (80-90% density) cell monolayers. The cells were transiently transfected with the plasmids constructs using the lipid reagent Lipofectamine 2000 according to the general guidelines provided by the manufacturers' instructions (Invitrogen Inc). At 48 hrs post-transfection, the VLP-containing cell supernatants (SUP) were harvested for concentration and purification of the VLPs. Because of the fusogenic property of our VLPs, there was widespread syncytia formation at this time point although the cells were still adherent.
VLPs released in the transfected-cell SUP were harvested and clarified by centrifugation at 3,500 rpm for 30 minutes at 4uC and concentrated by sucrose density gradient centrifugation based on previous descriptions [44, 45, 58] . Briefly, the clarified SUPs were concentrated by ultracentrifugation through 20% sucrose cushion in TN buffer (0.1 M NaCl; 0.05 M Tris-HCL, pH 7.4) at 200,0006 g for 8 hours at 4uC. The resulting VLP pellet in ,0.5 ml volume was purified on a discontinuous sucrose gradient formed by layering 80%, 65%, 50% and 10% sucrose in TN buffer. After centrifugation at 186,0006 g for 8 hours, the top ,1.5 ml of the gradient (which included the VLP-containing band at the interface between the 10% and 50% sucrose layers) was resuspended in 20% sucrose buffer and centrifuged once more at 160,0006 g for one hour. The resulting pellet was resuspended in 20% sucrose solution in endotoxin-free TN bufffer and stored at 4uC for subsequent analysis. Supernatant of 293T cells transfected with empty pCAGGS plasmid and processed similarly (referred to as ''mock'' particles) served as negative control when needed.
Since the ratio of the protein expression plasmids used at transfection and the time of harvest may have a bearing on the level of VLP formation, a minigenome-based VLP infectivity assay, similar to those described previously [59, 60] was used to determine the relative concentrations of the constituent plasmids, and to determine the kinetics of VLP formation for optimal production. This assay provides only a comparative assessment of VLP formation since it only accounts for VLPs that are able to incorporate and passage minigenomes. However, based on the assumption that the ratio of empty and minigenome-containing VLPs will be equivalent in each reaction, the method provides an indirect means to determine the optimal set of conditions for VLP production as determined by VLP-incorporated minigenomeencoded CAT enzyme activity. Briefly, the steps involved in the VLP infectivity assay were 1) transfection of NiV minigenome construct and co-transfection with full complement of the NiV protein expression plasmids, N, P, L, M, F and G, using Lipofectamine 2000. 2) following replication (48 hours posttransfection), passage of equal volume of VLP-containing transfected cell SUP on to fresh cells previously transfected with N, P and L plasmids and 3), determination of CAT activity in the VLP infected cells 48 hours later. Replication of the VLPincorporated incoming mingenomes based on reporter gene activity indicates the level of particle formation and release, VLP infectivity, and successful minigenome packaging.
FAST CAT Assay kit (Molecular Probes) was used according the manufacturer's instructions and allowed accurate quantification of CAT enzyme levels over a wide linear range.
VLPs were purified as described. The particles were adsorbed on Formvar carbon coated copper grid by floating it on a drop of VLP suspension for 15 minutes, the grids were blotted, and then negatively stained with 2% aqueous uranyl acetate for viewing by transmission electron microscopy.
The VLPs were vitrified as reported previously [61] on holey carbon film grids (C-flat TM , Protochips, Raleigh, North Carolina). VLPs were imaged at 40,000x indicated magnification using a 4k64k slow-scan CCD camera (UltraScan 895, GATAN, Inc., Pleasanton, CA) using a low-dose imaging procedure.
Unfixed VLPs were used for immunogold labeling to limit antibody reactivity to the cell surface proteins. The particles were adsorbed on formvar coated nickel grids, stained with NiV specific primary antibody (hyper immune mouse ascites fluid, HMAF, obtained from Dr. P. Rollin, CDC) diluted in buffer (1% BSA in 0.05 M tris buffer) rinsed in wash buffer (0.1% BSA in 0.05 M tris buffer), stained with colloidal gold labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), washed, and then negatively stained with 2% uranyl acetate for viewing by EM.
The total protein concentration of the purified VLP preparations was measured by the BCA (Bicinchoninic Acid) method (Thermo Scientific Laboratories).
VLP composition was determined by western blot analysis. Briefly, purified VLPs resuspended in endotoxin free PBS were lysed by resuspending them in equal amount of 2x SDS proteinloading buffer and loaded into a 12% SDS-polyacrylamide gel with a 4% stacking gel. 293T cell lysates processed similarly were run in parallel as negative cell control. Following electrophoresis to resolve the protein bands, and transfer to membrane, the blot was incubated with NiV-specific HMAF primary antibody at a dilution of 1:1000 dilution, overnight at 4uC, and HRP-conjugated antimouse secondary antibody (from GE Healthcare) at a 1: 20,000 dilution for one hour at room temperature. The proteins were revealed using western blot detection reagents according to instructions provided by the manufacturer (GE Healthcare).
These studies were undertaken with the approval of the Institutional Biosafety Committee (Protocol# #01/08-2010-1) and the Institutional Animal Care and Use (IACUC) Committee (Protocol # 0904028). Five to six week old female Balb/c mice (Harlan Laboratories) were housed in microisolater cage for 4 days in the Animal Resource Center at the University of Texas Medical Branch before beginning the immunization protocol. Mice in groups of five were immunized by subcutaneous inoculation of four different concentrations of VLPs (1.75, 3.5, 7 or 14 mg/ mouse, referred to subsequently as treatment groups A through D respectively) prepared just prior to use in sterile endotoxin free PBS. No adjuvant was used. A group of five mice inoculated with sterile endotoxin free PBS served as negative control group. Mice in the four treatment groups (A through D) were boosted (6 mg/ mouse) on days 15 and 29; the negative control group received PBS. Blood was collected from the submandibular vein of the animals on days 21, 14, 21, 28 and 35; they were euthanized on day 35.
Plaque Reduction neutralization test (PRNT). Two-fold dilutions of test sera were made in 50 ml cell culture medium. Under biohazard level 4 conditions, each of the diluted sera were mixed with 50 ml of NiV diluted to generate ,30 plaque forming units and incubated for 30 min at 37uC. The pre-incubated virusantibody mix was added to Vero cell monolayers grown in 96 well plates and incubated for 30 min at 37uC when the inoculum was removed and replaced 150 ml of cell media. After incubation at 37uC for 24 h, the cells fixed in 100% ice-cold methanol and staining by indirect immunofluorescence assay as follows: The wells in the plate were blocked with BSA/PBS and stained with rabbit sera raised against the G protein of HeV, and goat antirabbit Alexa Fluor 488 conjugate (Invitrogen) diluted 1:1000 in blocking buffer. Viral plaques were visualized and counted, and neutralizing antibody titers were reported based on reduction in plaque count by 50% relative to the untreated control (PRNT 50 ). Antibody levels measured by Immunofluorescence assay (IFA). For IFA, NiV-specific total antibody levels were measured by using NiV G, F and M expressing 293T cells as target antigen. Thirty six hours post-transfection, the cells were harvested, fixed in paraformaldehyde, cytospun (Cytocentrifuge, Thermoscientific) on glass slides to obtain monolayered preparations and then stored at 4uC, and used as antigen within three weeks of preparation. On the day of use, the slides were washed in PBS, permeabilized with Triton-X-100 and blocked with BSA/PBS. After incubation with two fold dilutions of the test sera, the cell monolayers were washed and stained with Alexa fluor 488-conjugated goat anti-mouse antibody according the manufacturer's (Molecular Probes) instructions. Negative and positive controls were run in parallel with each batch.
Gene expression profile by Real-time PCR VLP-mediated transcriptional activation was tested for eighty four genes involved in Toll-like receptor (TLR)-mediated signal transduction using RT 2 Profiler PCR array (SABiosciences). The 96 well array format included mediators of TLR signaling including adaptors and proteins that interact with the TLRs, and members of NFKB, JNKp38, NF/IL6 and IRF signaling pathways downstream of TLR signaling. Briefly, 293 cells grown overnight in 60 mm dishes were exposed to 10 mg of purified VLPs suspended in 1 ml of OPTI-MEM (Invitrogen). ''Mock particles'' (see Methods, VLP harvest and purification) resuspended similarly and exposed to 293 cells served as negative control. The inoculums were adsorbed on the cell monolayers for 3 hours at 37uC when additional 1.5 ml of OPTI-MEM was added and the dishes further incubated. Twenty fours post VLP exposure, total cell RNA was extracted according to the manufacturer's (SABiosciences) instructions. The integrity of the RNA was verified by agarose gel electrophoresis and the same concentration of total cell RNA from the VLP-stimulated and ''mock'' stimulated cells were used for gene expression profiling by Real-time PCR using Eppendorf Mastercycler unit. The array plate included positive and negative controls for quality assurance, and three sets of housekeeping genes for normalization for data analysis. The fold-change in gene expression in the VLP stimulated 293 cells relative to the ''mock'' stimulated 293 cells was calculated by the DDCt method according to the manufacturer's instructions.
In preliminary studies it was found that co-expression of NiV G, F and M proteins in 293T cells resulted in the formation of VLPs that bud out into the transfected cell SUP and that they can be harvested, concentrated and purified as described under Methods. However, the VLP yield was low. To improve the efficiency of VLP formation we proceeded to optimize the ratio of the three expression plasmids used at transfection. We speculated that this would be important based on the fact that a), during replication, paramyxoviruses form a transcription gradient where the 39 proximal genes are transcribed more abundantly than the successive downstream genes [52] and b), the stoichiometry of interaction of the viral proteins has proved to be critical in plasmid-driven minigenome and full-length rescue systems [62] .
The importance of protein ratios for VLP formation was alluded to in a previous NDV study where the expression plasmids were co-transfected at ''pre-determined concentrations'' to produce VLP-incorporated protein ratios analogous to those in virus infected cells [49] . In a study by Patch el al [48] , equivalent amounts of NiV N, M, F and G were initially used to produce the VLPs. In that study, VLPs were subsequently also made by adjusting NiV expression plasmid concentrations by experimental variations similarly to that in the NDV study [49] . The efficiency of particle formation and budding in both these, and many other paramyxovirus VLP formation systems was based on M protein release [43, 44, 45, 46, 48] .
We have chosen a minigenome-based functional assay, the VLP infectivity assay (described under Materials and Methods), to determine optimal expression plasmid ratios for efficient VLP formation based on reporter gene readouts. Briefly, 293T cells were transfected with plasmids as shown in Figure 1 . For titrating NiV G, F and M plasmids, increasing concentrations of either G, or F or M expression plasmids were, in turn, co-transfected with fixed concentrations of the other two plasmids. The minigenome and N, P and L plasmids were transfected using a predetermined ratio [57] . The VLP-containing cell SUP was harvested 48 hours post-transfection, clarified by centrifugation, and equal volume from each was passaged onto fresh cell monolayers (VLP-infected cells) previously transfected with the core proteins required to support the incoming packaged minigenomes. The VLP infected cells were harvested 48 hours later and tested for optimal particle production based on incoming minigenome-encoded CAT activity. This time point was chosen because maximal VLP formation was also found to be time dependent and optimal at 48 hours post-passage (data not shown). The reproducibility of the results was verified in an independent repeat experiment. Results presented in Figure 1 show that within the given range, and based on the levels of minigenome-encoded CAT activity, varying the concentrations of G, F and M plasmids had a bearing on VLP formation. CAT activity in the VLP infected cells appeared optimal in the boxed lanes 7 and 8 but further analysis to ensure reporter activity in the linear range (data not shown) indicated that the largest amount of minigenome-containing NiV VLPs were produced when the cells were transfected with the NiV M, F and G plasmid ratios of 3:1:1 as in lane 7. This ratio was used for making all our VLP preparations.
The optimized conditions were applied to transfect G, F and M expression plasmids in 293T cells grown in 10 cm dishes. The VLP-containing culture SUPs were harvested 48 hours later, and concentrated and purified as described. Briefly, the clarified SUPs were concentrated by ultracentrifugation through 20% sucrose cushion, and then purified on a discontinuous sucrose gradient. The VLP pellet was resuspended in TN buffer and viewed by EM after negative staining. The result presented in Figure 2A shows a VLP-containing band in the sucrose gradient. Viewing of the negatively stained purified particles by transmission electron microscopy ( Figure 2B) showed numerous virus-like particles. The size variation of these VLPs was consistent with the parental virus: NiV is a pleomorphic virus ranging in size from 40-1900 nm [63, 64] ; the sizes of the VLPs ranged from ,40-500 nm. The particles also resembled authentic NiV morphologically, and this is seen more clearly in the magnified images presented in Figure 2C ; here, the fringe of the glycoproteins is clearly visible on the VLP surface. An occasional VLP had what appeared to be a double fringe (shown with an arrow), a feature more frequently associated with Hendra rather than NiV virus particles [64] . The image in Figure 2D is a cryoelectron micrograph of one of our VLPs; the overall surface appearance is virus-like, which is described as dense, ordered and repetitive [31] , and it shows the surface glycoproteins and their spatial arrangement even more definitively.
To verify whether the NiV proteins were incorporated into the VLPs as designed, purified particles were analyzed by western blotting using NiV-specific mouse antibody, and HRP-conjugated anti-mouse secondary antibody as described under Methods. The right hand panel in the Figure 3 shows VLP-incorporated proteins in two different preparations of NiV VLPs. The protein bands are consistent in size to NiV proteins G, F 0 , F 1 and M proteins [17, 48] . The relative amounts of the VLP-incorporated G and M proteins appeared to be similar to that reported in NiV virions also [17] . This was in spite of the fact that the viral proteins in that study were revealed using rabbit sera raised against bacterially expressed Hendra virus proteins. However, the ratio of the VLPincorporated F 1 to F 0 was different from that in the virions. This difference is more likely to be a reflection of timing and protein turnover rather than the reagents used to reveal them since in a previous study, pulse chase experiments have shown that similarly to the VLP-incorporated F 1 to F 0 , intracellular cleavage of the precursor NiV fusion protein by cathepsin L results in near equal mix of mature fusogenic, and the precursor forms [65] . Absence of the NiV-specific bands in two different 293T cell lysate preparations processed similarly (and shown in the left hand panel in Figure 3 ) confirms specificity of the VLP-incorporated proteins.
The immunoreactivity of the VLP surface glycoproteins was verified by staining purified unfixed VLPs by the immunogold labeling technique using NiV-specific mouse antiserum and 6 nm colloidal gold particle-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories Inc). The particles were viewed by EM after negative staining. The use of unfixed particles assured that only the surface-exposed antigens would be reactive. Numerous VLPs with the gold particles decorating their surface were seen; Figure 2E shows two such VLPs.
Syncytium formation is a classical feature of NiV and other paramyxovirus-induced cytopathology that can be blocked by virus-specific neutralizing antibody. A similar observation was made when 293 cells were ''infected'' with the NiV VLPs. Briefly, the VLPs were pre-incubated with NiV-specific antibody, Junin virus (JV)-specific antibody, and with OPTI-MEM I (Invitrogen Inc) medium only for one hour at 37uC before inoculating onto near confluent 293 cell monolayers grown overnight in 60 mm dishes. The inoculum was removed after incubation for 3 hours at 37uC, replaced with OPTI-MEM I, and the plates were further incubated overnight at 37uC overnight. The monolayers were then viewed for the formation of syncytia after staining with crystal violet. The results in Figure 4 show that 293 cells exposed to NiV VLPs induced syncytium formation and that this process was neutralized by NIV-specific antibodies; prior incubation with the unrelated JV antibodies failed to block this process.
Mice in groups of five were inoculated subcutaneously with four different concentrations of purified VLPs and boosted as described under Methods. The negative control group of five mice were inoculated with sterile endotoxin free PBS for each inoculation. The mice were bled from the submandibular vein on the day before primary inoculation, and then on days 14, 21, 28 and 35. For initial evaluation, sera from each treatment group were pooled, and the IFA method used to determine levels of NiV-specific antibodies. The results in Figure 5A show that titers (reciprocal of the highest serum dilution showing reactivity) increased with time post primary inoculation, i.e., the highest titers (1:2560) were seen on day 35. Titers also increased with VLP dosage although by day 35, the three higher treatment groups seemed to produce similar titers. As expected, the mice in the negative control group remained nonresponsive.
All sera were tested individually by plaque reduction neutralization method by doubling dilution of each sample (1:5 to 1:80) as Figure 5B ) showed distinct association between VLP dosage and the ability to mount a neutralizing antibody response. Mice inoculated with the two highest VLP doses (treatment groups C and D) were each able to induce neutralizing antibodies by day 35. When samples from mice receiving the two lower concentrations of VLPs (3.5 ug/dose and 1.75 ug/dose, corresponding to treatment groups B and A respectively) were similarly tested, 3 of 5 and 1 of 5 mice respectively induced neutralizing antibody response; the titers ranged from 1:5 to .1:80. As expected, the control mice did not induce neutralizing response.
NiV VLP-induced activation of genes involved in signaling innate immune response A PCR array format (SABiosciences) was used to investigate modulation in transcription profile of 84 genes involved in innate immune responses to include TLR signaling family and members of the downstream signaling pathways, NFKB, NF/IL6, IRF and JNKp38. These genes represent key sensors of non-self that signal, and ultimately shape the nature of innate immune response that modulates the type and duration of adaptive immune responses [66, 67] . The differential expression of genes in VLP-exposed 293 cells relative to the ''mock'' infected 293 cells was measured by real-time PCR. Same concentration of total cell RNA from the VLP-stimulated and the ''mock'' stimulated control cells were used for first strand synthesis and Sybr green PCR amplification of the relevant genes as described under Methods. The integrity of RNA in each sample was confirmed by gel electrophoresis (Figure 6A ). Data representing the differential transcription profile of VLP exposed vs. ''mock'' stimulated cells is shown as a heat map ( Figure 6B) . A 4-fold cutoff threshold was used to determine modulation in gene expression. We noted significant VLP-stimulated up-regulation (89 fold and 7 fold) in the expression of NFKB2 and TBK1 genes respectively. Close to four fold (3.9 fold) up-regulation was noted also in IL-8 and MAPK8 genes. NFKB2 and IL-8 are target genes in the downstream NFKB pathway, and TBK1 which are in the IRF and JNK/p38 pathways respectively.
Using a minigenome-based functional assay, we have established conditions (described under Results and shown in Figure 1 ) that have allowed us to produce substantial quantities of NiV VLPs to be able to undertake the studies described in this manuscript. We have shown that these particles are functionally assembled, biologically active and are able to induce innate immune responses, and a neutralizing antibody response. Native VLPs have been used to study various aspects of the virus lifecycle, as carriers to deliver heterologous proteins for vaccination, and to deliver small molecules for gene therapy purposes. Particularly importantly, they have been used highly effectively as vaccines in their native form [31, 32, 39, 40, 41] .
No vaccine for NiV disease has been developed so far that would be both safe and protective for humans. The two vaccination strategies that have already been explored are the canary pox-based vector approach [26] and soluble subunit approach [28, 29] . NiV vaccine by the former method is undergoing development as a veterinary vaccine [26] . The same approach is being evaluated for human use vaccines, mainly for the prevention of HIV and AIDS [27] . The subunit approach has limitations as already mentioned above [28, 29, 30, 31, 32] . One particular challenge revealed by studies that tested a soluble NiV G protein-based subunit vaccine formulated with adjuvant is the potential difficulty of eradicating infection in the central nervous system. In that study [28] , live virus was present in the brain of one cat, and viral RNA was present throughout the 21 day postchallenge period in the brains of the remaining challenged animals. A recently reported vaccination strategy [34] requires simultaneous inoculation of two VSVDG vectors, one expressing NiV G, and the other expressing NiV F proteins. It was of interest to note that supernatants of cells co-infected with these two defective viruses were infectious and could be passaged indefinitely in the absence of VSV G trans-complementation. This vaccination approach seems promising since self-propagated stock of these two viruses induced robust neutralizing antibody response in mice. However, potential pathogenicity of VSV-based vaccine vectors remains a concern [34, 35] . The potential of a recombination event resulting in a single VSV vector virus expressing both these NiV proteins is unlikely, but it may still be problematic for a human use vaccine.
Native VLPs like the ones we have produced allow the viral proteins to be presented to the immune system in the same conformation as in the virion for effective B and T cell response [31] . VLPs are particularly effective in producing a protective antibody response because of their virus-like size range, their particulate nature, and their virus-like dense, repetitive and ordered surface structure [31, 36] . The spacing of the antigenic epitopes on the VLP is also optimal for B cell activation [31] : EM analysis showed that our particles resembled the real virus in terms of size and surface structure [63, 64] . The image in Figure 2D is the first elucidation of VLP structure of any paramyxovirus imaged by CryoEM, and it provides a careful assessment of their morphology; it alludes to a surface similar to that revealed for measles virus by the same imaging technique (Dr. Elizabeth Wright, Emory University). The proteins on the VLP surface are clearly visible here; the average distance between the spikes was 9.13 nm and standard deviation was 1.72 nm. This is of interest given that epitopes spaced between 5 and 10 nm are known to be sufficient to drive optimal B cell activation [36] .
NiV M, F, G and N protein-containing VLPs consistent in size and morphology to the parental virus have also been reported in a previous study which evaluated protein-protein interaction that facilitate VLP formation [48] . However, in that study, most of the particle-incorporated NiV F protein was predominantly in the uncleaved precursor form. This finding is clearly distinct from ours since our VLPs contained substantial amounts of cleaved F protein, and this may have been related to ratios of the interacting proteins expressed in 293T transfected cells. In a recent study of NDV VLPs [49] , the particle-incorporated proteins were reported to have virus-like protein ratios, but the F protein remained in its precursor form because the cleavage site required to produce the fusion competent form was mutated by design. What effect a VLP-incorporated non-fusogenic F protein may have, relative to the fusogenic form, on the level and quality of VLP-induced immune response is not clear at present since difference in immunogenicity between fusion-competent and fusion-defective VLPs has not been experimentally evaluated so far. However, a recent report suggests that viral fusogenic membrane glycoproteins may enhance vaccine potency [68] .
Immunogold labeling of our unfixed NiV VLPs confirmed that the surface proteins in our VLPs were functionally assembled and they were biologically active ( Figure 2E ). We could deduce the presence of biologically active G and F proteins on the VLP surface by the fact that they were able to induce the formation of syncytia in 293 cells ( Figure 4 ); this is a process that requires the interaction of both the surface glycoproteins, the attachment protein G, and the fusion competent F protein, when they come in contact with the cognate receptor-bearing cells. Formation of syncytia or multinucleated cells in replication competent enveloped viruses, especially paramyxoviruses, is induced by a process that is described as ''fusion from within'', and it can be blocked or neutralized by prior treatment of the virus with specific antisera. In contrast, ''fusion from without'' is induced by non-replicating viruses at high multiplicities of infection, and it too can be blocked by pretreatment with virus-specific antibodies ( [69] , and references therein; [70] ). Our non-replicating particles likewise induced syncytia formation in 293 cells that could be neutralized with NiVspecific antibodies (Figure 4 ). To our knowledge, this is the first study describing fusion from without induced by VLPs of any paramyxovirus, or any other enveloped viruses, although it has been described for the VLPs of the non-enveloped rotavirus [71] . The mechanism(s) of fusion from without is not clear but two models have been proposed [72, 73] ; one proposes that particles connecting adjacent cells effectively promote fusion between them, and the other is that when particles decorated with the surface glycoproteins fuse with the target cell membrane, the glycoprotein complexes diffuse freely in the lipid bilayer, and mimic fusion from within. The type of VLP-induced syncytia formation and eventual cell death is also not known. We are in the process of investigating it.
Neutralizing antibody response is the critical correlate of protection mediated by prophylactic vaccines [53, 54] and native VLPs promise to be highly effective prophylactic vaccines for paramyxoviruses like NiV, and others like NDV and measles where neutralizing immune response is known to play a pivotal role in protection against disease [53, 54, 55, 74] . Our VLPs were highly effective immunogens, and all, especially in the three higher treatment groups produced high levels of response by day 35 ( Figures 5A) . Importantly, NiV VLPs were able to induce neutralizing antibodies. This response was clearly dose-dependent ( Figure 5B ). All ten mice receiving a primary inoculation of 7 or 14 mg VLPs (subgroup C and D) were able to produce such response; but even of those animals that received a first dose of only 3.5 or 1.75 ug/mouse (treatment group B and A respectively), 3 of 5 and 1 of 5 produced neutralizing antibodies. Neutralization antibody response was first seen on day 28, and increasing titers were seen in some animals within a week of it; we believe that this response, induced by our non-replicating and potentially safe particles, formulated without adjuvant, compares favorably with the levels of such response induced at an equivalent time point by some replication competent pseudotype viruses [34] .
Immunogenicity to native VLPs has been reported previously for one other paramyxovirus namely NDV [49] . In that report, immune response to NDV VLPs was evaluated by primary inoculation of mice intraperitoneally with VLP concentrations ranging between 10 and 40 mg, and a booster dose of 10 mg, without adjuvant. NDV-specific titers by ELISA were high in each mouse in each treatment group. Neutralizing antibody response to 20 and 40 mg of these particles was also detected.
The nature of innate immune response dictates the type and duration of adaptive immune response [67, 75] . The mechanism by which NiV VLPs are recognized by host cells and trigger the induction of innate immune response, and how this translates into effective adaptive immunity is not known. Here we have taken the first step ( Figure 6 ) towards understanding this process. With the experimental conditions as described, we observed VLP-induced activation of some of the genes that are known to be involved in the induction of an effective innate immune response [75] . Results presented in the heat map in Figure 6 show that relative to the ''mock'' treated cells, NFKB2 gene (in the NFKB pathway) was up-regulated 89 fold as a result of VLP exposure, and TBK1 (in the IRF pathway) was 7 fold higher. In the light of these findings, we are testing PCR array expression profiles of the same set of 84 genes in 293 and other cells at earlier and later time points to identify their upstream effectors, and NiV VLP-responsive signaling networks. In this respect, the murine system, with the many available immunological reagents and knockout strains may provide the best system to identify these host sensors. Currently there is minimal information on live NiV infection-responsive cellsignaling changes [76] and there is none on array-based transcriptional alterations for comparative analysis. Likewise, it has not been possible to compare the NiV VLP-induced transcription modulation with those induced by other paramyxovirus VLPs since to our knowledge, such studies have not been undertaken so far. Lastly, a growing number of reports point to viral surface glycolproteins as relevant in host cell signaling and triggering of innate immune response. We believe that particles like NiV VLPs, with many virus-like properties (including their surface glycoproteins organized to resemble the parental virus, Figure 2D ) would induce an effective innate immune response for the promotion of the desired adaptive immunity [67, 76] .
Finally, as described above, our VLPs were highly effective as immunogens, able to induce neutralizing antibody response in all animals with primary inoculation of as little as 7 mg VLP protein each. Fusogenic property of our VLPs may be critically relevant in this regard in the light of recent findings, and would need to be experimentally verified by comparing the potency of fusioncompetent and fusion-defective VLPs as vaccine [68] .
In conclusion, we have been successful in producing substantial quantities of NiV VLPs needed to characterize NiV VLPs, we have demonstrated their many virus-like properties, and their effectiveness as immunogens in Balb/c mice. These findings are the basis on which we will be undertaking future challenge studies in the hamster model of NiV disease [77] . Antibody-Mediated “Universal” Osteoclast Targeting Platform using Calcitonin as a Model Drug PURPOSE: To generate and characterize a specific monoclonal antibody (mAb) against recombinant human RANK receptor and to develop an antiresorptive strategy using this mAb as an osteoclast-targeting platform that selectively targets osteoclast cells whilst delivering an attached (i.e. chemically conjugated) active drug cargo. METHODS: Using hybridoma technology, we generated a specific monoclonal antibody (mAb) against recombinant human RANK receptor and characterized by SDS PAGE, ELISA, Western Blot and immunocytochemistry, then synthesized osteoclast-targeting bioconjugates of salmon calcitonin (sCT) using this antibody by generating thiol groups on mAb using 2-Iminothiolane and subsequently reacting them with sCT-PEG-MAL synthesised from sCT and NHS-PEG-MAL. To test the efficacy of the conjugate in vitro, osteoclasts were generated from precursor RAW 264.7 cells by dosing with the cytokines macrophage-colony-stimulating factor (M-CSF), and RANK Ligand (RANKL) and TRAP activity assay, Resorption Pit Assay, TRAP staining were performed. Cytotoxicity of the mAb-sCT conjugate was also evaluated in RAW 264.7 cells; sCT bioactivity and CTR binding potential were evaluated by in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. RESULTS: Generation of antibody against human RANK receptor was confirmed by SDS PAGE, ELISA and Western Blot. Immunocytochemistry confirmed the osteoclast targeting potential of the antibody. Successful conjugation of the antibody with sCT was confirmed by SDS PAGE and ELISA.Multinucleated osteoclast formation was confirmed by staining for tartrate-resistant acid phosphatase (TRAP). Conjugate functionality was confirmed by TRAP activity and Resorption Pit assay, showing the inhibitory effect on osteoclast differentiation. cAMP assay confirmed the retention of calcitonin bioactivity after conjugation. CONCLUSIONS: Our strategy offers the potential for a “universal” osteoclast-targeting platform—one that targets the RANK receptor on osteoclast cells by simply altering the conjugated cargo in order to affect the specific regulation of osteoclast cells. Osteoclasts are the cells uniquely responsible for dissolving both the organic and inorganic components of bone during development and are part of the bone remodeling cycle throughout life. These cells originate from hematopoietic precursors of the monocyte/macrophage lineage that are present both in the bone marrow and peripheral circulation, and their numbers and/or activity are frequently increased in a wide range of clinical disorders that are associated with excessive bone loss and which affect millions of people (1) .
Osteoporosis is a major metabolic bone disease that predisposes patients to fracture in up to 40% of aging women and 15% of aging men (2) . The increase in bone resorption is due both to increased osteoclastogenesis and to decreased osteoclast apoptosis (2, 3) . Osteoclasts may also play a role in the etiology of post-traumatic osteoarthritis (PTOA), the most common form of arthritis. At an early stage of disease pathogenesis, there is a phase of increased bone resorption and turnover in periarticular subchondral bone volume and an increased number of osteoclasts, prior to later stage sclerosis and eburnation (4) . Similarly, increased osteoclast functional activity is directly responsible for the generalized bone loss that occurs in rheumatoid arthritis (5) . Thus, we hypothesized that an antiresorptive strategy that selectively targets osteoclasts and/or carries an active drug to osteoclast cells directly would be highly desirable as a therapeutic to treat bone disease involving upregulated osteoclast activity.
Osteoclast cells express the RANK receptor (receptor activator of nuclear factor Kappa B) and in effect serve as ideal molecular targets. RANK is expressed on osteoclast precursors as well as mature osteoclasts. RANK is the essential signaling receptor for osteoclast differentiation during the process of osteoclastogenesis, as triggered by the osteoclast differentiation factor known as RANK-ligand (RANKL) (6, 7) . RANK signaling, with additional signaling through c-Fms, the receptor for macrophage-colony-stimulating factor (M-CSF), triggers the proliferation and fusion of mononuclear cells and the formation of multinucleated, mature osteoclasts (7, 8) .
Mature osteoclasts will also express the calcitonin receptor upon its surface, which has been known for decades as a prominent negative controller of bone resorption (9) . Calcitonin receptors are of the G proteincoupled receptor family (comprising seven transmembranespanning receptor domains) whose signaling will inhibit osteoclast activity both in vitro and in vivo (10) . Calcitonin receptor activation upon osteoclasts, by its ligand calcitonin, will rapidly induce the loss of ruffled border and immobility followed by cell retraction and arrest of bone resorption. Calcitonin receptor signaling will also alter ion transporter distribution, impair enzyme activity (11) and inhibit the osteoclastogenic effects of RANKL (12) .
Since antibodies have exquisite specificity of target recognition and, thus, generate highly selective outcomes following their systemic administration, our purpose was to develop a "universal" osteoclast-targeting platform as a drug-delivery strategy in order to deliver antiresorptive drugs using an anti-RANK mAb capable of localizing to the cells responsible for bone resorption. We have chosen salmon calcitonin as a model drug, as it acts on the calcitonin receptor also found on bone-resorbing osteoclasts.
Monoclonal antibody (mAb) to the RANK receptor was generated using Hybridoma technology, as previously performed in our laboratory (13, 14) . Briefly, 6-8-week-old female BALB/c mice were immunized intraperitoneally three times with 25 μg of recombinant human sRANK receptor (Peprotech, USA) on days 0 and 14 using complete and incomplete Freund's adjuvant, respectively, and once with 10 μg of antigen on day 28 using phosphate-buffered saline (PBS, pH 7.3). The immune response to the antigen was assessed by measuring the titer of polyclonal antibody in mouse serum using an indirect enzyme-linked immunosorbent assay (ELISA). Mice with the highest antibody titer were euthanized and splenectomized 3 days after the final injection of antigen. Spleen cells were fused with SP2/0 myeloma cells at a ratio of 4:1 using 50% (w/v) polyethylene glycol (PEG) (Sigma, USA) according to the technique described previously by Köhler & Milstein (15) . The hybridoma cells were suspended in culture medium (RPMI 1640) supplemented with penicillin, streptomycin, L-glutamine (PSG), hypoxanthine aminopterin thymidine (HAT), and 20% fetal bovine serum (FBS) (Sigma, USA). Cells were seeded in 96-well tissue culture plates and incubated in a humidified 37°C, 5% carbon dioxide incubator for 2 weeks. Clones were maintained in HAT medium for a further 2 weeks.
Hybridoma cell lines were screened by an indirect ELISA. The cell lines producing specific antibodies were recloned successively 3-7 times by limiting dilution to ensure monoclonality and stability of the cell line. Hybridoma cell lines were then propagated in large 175 cm 2 tissue culture flasks, and the conditioned supernatant collected. Purification of IgG mAbs was achieved by affinity chromatography using Protein G agarose (Sigma, USA). The IgG immunoglobulin subclass was determined using the mouse hybridoma isotyping reagents according to instructions from the manufacturer (Sigma, USA).
All ELISAs were performed in flat-bottomed 96-well plates (Nunc-Immuno MaxisorbTM plates, Nunc). Antibody secretion by hybridoma cells was detected by indirect ELISA. Briefly, 100 μl of antigen (human sRANK receptor) was used for coating at a concentration of 1 μg/100 μl, overnight at 4°C. The wells were washed three times with PBS (pH 7.3) and, to avoid nonspecific binding, incubated with 3% BSA for 1 h at room temperature. After washing, the wells were incubated with 100 μl supernatant from each hybridoma clone for 1 h at room temperature. After washing, bound antibodies were detected using secondary goat anti-mouse IgG conjugated with horseradish peroxidase (GAM-HRPO) at a 1:5000 dilution for 1 h at room temperature. After final washing, 100 μl of 3,3′,5,5′tetramethylbenzidine (TMB substrate) was added to each well and incubated for 15 min at room temperature. The optical density (OD) was measured at 650 nm using an ELISA Vmax kinetic microplate reader (Molecular Devices Corp., California, USA). The clones showing ELISA values five times higher than the negative control were considered positive. Sera of unimmunized mice and irrelevant antibody were used as negative controls. RPMI media and positive sera from hyperimmunized mice were used as blank and positive control, respectively.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on purified antibody under both reducing and non-reducing conditions. Antibody was mixed with loading buffer and run in 10% polyacrylamide gel, with the thiol reagent 2-Mercaptoethanol added to the loading buffer for reducing conditions (to cleave the disulfide bonds between the polypeptides). Gels were stained with Coomassie blue for protein band detection of individual heavy and light chains of the antibody.
ELISA was performed as above, using 100 μl RANK receptor for coating at a concentration of 1 μg/100 μl, overnight at 4°C. To avoid nonspecific binding, the wells were incubated with 3% BSA for 1 h at room temperature. After washing, the wells were incubated with different concentrations of antibody for 1 h. The wells were washed with PBS, and the bound antibodies were detected using secondary antibody (goat anti-mouse IgG conjugated with horseradish peroxidase) for 1 h. After final washing, 100 μl of TMB substrate was added to each well and incubated for 15 min. The optical density (OD) was measured at 650 nm.
For Western blot analysis, RANK receptor (19.3 kDa) was electrophoresed on SDS-PAGE using 10% acrylamide gel and transferred onto nitrocellulose membrane using mini trans-blot apparatus. The membrane was blocked with 5% skim milk in PBS-T for 1 h, washed with PBS-T, cut into strips, and incubated with purified mAb solution for 1 h. After washing, strips were reacted with GAM-HRPO for 1 h, and the binding of mAbs to RANK receptor was detected using enhanced chemiluminescence. For the negative control, the primary antibody was omitted, and the antigen strip incubated with PBS.
To confirm the RANK receptor targeting potential of generated mAb in vitro, osteoclast-like cells were generated in Lab Tek II chamber slide system (Nunc) from RAW 264.7 cells, as described below for generation of osteoclasts. The MG-63 osteoblast-like cell line known to lack RANK receptor (16) was used as negative control. Likewise, osteoclast cell culture omitting primary mAb was also used as a method negative control. Cell cultures were washed with PBS, fixed in 4% paraformaldehyde in PBS (pH 7.4) for 5 min and rinsed thoroughly. After blocking with 3% BSA in PBS for 1 h, the cells were incubated with our anti-RANK mAb (4 μg/ml) overnight at 4°C. Antibody detection was carried out using rabbit anti-mouse IgG-FITC (1:100; Sigma, USA) for 60 min at 4°C. To visualize cell nuclei, slides were counterstained with 1.5 μg/ml 4-6diamidino-2 phenylindole (DAPI, Sigma) diluted in PBS. Culture slides were separated from their chambers, mounted and photographed using confocal microscopy (Zeiss LSM 710 with ZEN software and the microscope the Observer.Z1).
In order to generate anti-RANK mAb-salmon calcitonin conjugate (mAb-sCT), free thiol groups were generated on our anti-RANK mAb by reacting it in a 10 molar excess of Traut's reagent at room temperature for 1.5 h (Fig. 1a ). Traut's reagent (2-Iminothiolane or 2-IT, Sigma USA), a cyclic thioimidate compound, reacts spontaneously and efficiently with primary amines at pH 7-9 resulting in sulfhydryl addition (17, 18) . No free sulfhydryl moieties were found to be present in our IgG mAb, as determined by Ellman's assay (19) .
To generate thiol-reactive calcitonin analogue, synthetic salmon calcitonin (sCT, Calbiochem, USA) in DMSO (13.72 mg/ml) was mixed with NHS-PEG-MAL (Creative Biochem, USA) in DMSO (51 mg/ml) (Sigma,USA) in 1:3 molar ratio. DMSO was chosen as the reaction medium because of the instability of NHS and sCT in aqueous solutions. sCT is further highly soluble and highly stable in DMSO (20) . The reaction between the primary amines in sCT and NHS group of NHS-PEG-MAL was allowed to proceed at room temperature with constant stirring for 45 min. sCT has three primary amines at residues Lys 11, Lys 18 and the N-terminus, which can react with the NHS functional group of NHS-PEG-MAL to generate three intermediate conjugates: mono-, di-and tri-substituted thiol reactive sCT analogues (Fig. 1b) . 
Functionalized thiol-reactive sCT analogue was added intermittently with constant stirring to thiol-mAb solution at 10:1 molar ratio, and the reaction between the thiolreactive MAL groups in functionalized sCT and SH group of thiol-mAb was allowed to proceed at room temperature in the dark with constant stirring for 2 h. Unreacted sCT-PEG was removed by dialysis at 4°C (Fig. 1c) .
Anti-RANK mAb-sCT conjugate was examined under both reducing and non-reducing conditions of SDS-PAGE analysis, as described above. ELISA was performed, using 100 μl RANK receptor for coating at a concentration of 1 μg/100 μl, overnight at 4°C. To avoid nonspecific binding, the wells were incubated with 3% BSA for 1 h at room temperature. The wells were incubated in triplicate with either mAb alone, functionalized sCT-PEG, or mAb-sCT conjugate for 1 h. The wells were washed with PBST followed by incubation with rabbit anti-salmon calcitonin antibody (US Biologicals) for 1 h. The wells were washed with PBST three times and incubated with HRPO conjugated goat antirabbit IgG (R&D Systems) for 1 h. After final washing with PBST, TMB substrate was added to each well, and the OD measured at 650 nm.
Osteoclast-like cells were generated in culture from RAW 264.7 cells (transformed murine monocytic cell line), purchased from the American type culture collection (ATCC, VA, USA) (21) . RAW 264.7 cells were cultured to confluence in a 75 cm 2 flask in GIBCO High Glucose 1X Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, CA, USA) containing 4.5 g/L D-Glucose, Lglutamine, and 110 mg/L sodium pyruvate, with the addition of 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin added (10,000 U/mL; Invitrogen, USA). Confluent cells were harvested by scraping, centrifuged at 1,500 RPM, and resuspended in 25 mL of culture medium. The number of cells in the suspension was measured using a Bright-Line Hemocytometer (Hausser Scientific, PA, USA) and seeded at a concentration of 2×10 3 cells/well in 96-well culture plates (Corning) and placed in the CO 2 incubator overnight to allow the cells to attach to the surface. After 24 h, the culture medium was replaced with media containing 25 ng/ml macrophage-colony-stimulating factor (M-CSF) and 50 ng/mL RANKL (PeproTech, NJ, USA). Osteoclasts were successfully generated by dosing with M-CSF and RANKL every 48 h over the course of 7 days. To confirm the generation of multinucleated osteoclast-like cells, the cultured cells were stained for the enzyme tartrate-resistant acid phosphatase (TRAP) using the Leukocyte Acid Phosphatase TRAP Kit from Sigma-Aldrich (St.Louis, MO, USA), according to the manufacturer's instructions. TRAP-positive multinucleated osteoclasts were visualized by light microscopy and photographed.
To quantify the total TRAP activity from in vitro osteoclast cultures, RAW264.7 cells were seeded at a concentration of 2×10 3 cells/well in 96-well culture plates and incubated for 24 h. Peptide factors 50 ng/ml RANKL and MCSF 25 ng/ml were added to the culture, with or without the addition of 100 nM of mAb, 100 nM of mAb-sCT, or 100 nM of sCT. The medium and factors were replaced every 48 h. Osteoclastogenesis was assessed by the spectrophotometric measurement of TRAP activity on day 7. Briefly, the medium was aspirated, and the cell monolayer washed twice with PBS. The cells were then lysed with 100 μl of 0.2% Triton X-100 in water (v/v) for 10 min. TRAP activity in the cell lysate was determined using an Acid Phosphatase Assay Kit (Cayman chemical, USA). The assay utilizes para-nitrophenyl phosphate (pNPP) as a chromogenic substrate for the TRAP enzyme. In the first step, acid phosphatase dephosphorylates pNPP. L-tartrate, an inhibitor of nontartrate resistant acid phosphatase, provided in the kit was used to measure TRAP enzyme activity. In the second step, the phenolic OH-group was deprotonated under alkaline conditions, resulting in p-nitrophenolate that yields an intense yellow color, which was measured at 405 nm using a microplate reader.
To quantify the mineral resorptive activity of osteoclast-like cells in culture, RAW264.7 cells were suspended in cell culture medium and seeded in a 16-well BD Biosciences Osteologic® Slide (BD Biosciences, MA, USA) at the density of 2×10 3 cells/well and incubated for 24 h. Osteoclast-like cells were successfully generated by dosing with 50 ng/ml RANKL and 25 ng/ml M-CSF every 48 h over the course of 7 days. The wells were incubated with 100 nM of either mAb alone, sCT alone, or mAb-sCT conjugate. Half the volume of the medium and factors were replaced every 48 h. After 7 days of culture, the medium was aspirated, the slide placed in a petri-dish and washed with double-distilled water. To remove the adherent cells, the slide was then soaked in bleach for 10 min and washed with double-distilled water. Slides were air-dried, viewed under an inverted light microscope, and low power images of the calcium phosphate mineral remaining on the slide acquired. To quantify the resorptive effect of osteoclasts, the images were analyzed using the javabased image-processing program ImageJ (NIH, USA).
To ensure that the mAb-sCT conjugate was not cytotoxic to cells other than osteoclasts, we employed the MTT assay, in which mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring of MTT, yielding purple formazan crystals. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
RAW 264.7 cells were seeded on 96-well plates at an initial density of 2×10 3 cells/well and incubated for 72 h at 37°C, 5% CO 2 until the cells were 80% confluent. The culture medium was replaced by 200 μl basic DMEM media without FBS and incubated for 30 min. Then the culture medium was replaced by 100 μl basic DMEM media containing 50, 100 and 200 nM of mAb, mAb-sCT and sCT, respectively (in quadruplicate). The cells were incubated for 4 h at 37°C. The medium was then replaced with 100 μl basic medium containing (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) at a concentration of 100 μg/well and incubated for a further 4 h at 37°C. After removing the supernatant and washing twice with phosphate-buffered saline, the newly formed purple formazan crystals were dissolved in 200 μl solubilization solution using an in vitro toxicology assay kit (TOX-1, Sigma Aldrich, St. Louis, USA), and the absorbance was measured at 570 nm using a microplate reader.
To confirm that calcitonin bioactivity was not lost after conjugation to mAb and to confirm the ability for mAb-sCT conjugate to trigger the calcitonin receptor, T47D human breast cancer cells (known to contain calcitonin receptors) were used, as previously described (22) . Briefly, T47D cells (ATCC, VA, USA) were cultured in triplicate in RPMI-1,640 culture medium containing 1% penicillinstreptomycin, 10% fetal bovine serum, and insulin (0.2 IU/ mL). Cells were seeded on 48-well plates at an initial density of 5×10 4 cells/well and incubated in 95% air and 5% CO 2 at 37°C for 2 days. Cells were then washed with HBSS and preincubated in RPMI-1640 culture medium devoid of FBS, insulin and antibiotics. Cells were dosed with the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX, 1 mM) and incubated at 37°C for 30 min. One hundred nM of mAb, mAb-sCT and sCT were then added to the wells except the control where T47D cells were treated with IBMX only. The plate was incubated for 20 min at 37°C.
After removing the supernatant, cells were washed three times in cold phosphate-buffered saline and resuspended in 500 μl of cell lysis buffer. Cells were frozen at −20°C and thawed with gentle mixing. The freeze/thaw cycle was repeated three times, and the mixture was centrifuged at 600 g for 10 min at 2-8°C to remove cellular debris. The supernatant was collected and stored at −20°C. cAMP concentrations were then measured using the cyclic adenosine monophosphate (cAMP) Enzyme Immuno-Assay (EIA) kit (KGE002B, R & D systems, USA).
Statistical analysis was conducted using Graph Pad Prism 5 (GraphPad Software Inc., CA). Results are presented as the mean ± standard deviation of three samples. Unpaired t-tests were used to assign significance between groups, with a P-value of less than 0.05 as the threshold for significance. Two-way ANOVA was used for the MTT assay.
A band at 150 kDa was seen for unconjugated anti-RANK IgG, with separate bands for heavy chain at 50 kDa and light chain at 25 kDa observed under reducing condition. For mAb-sCT conjugate, a band above 150 kDa was observed, confirming the conjugation to have taken place. Under reducing condition of SDS-PAGE for conjugate, some bands above 50 kDa and 25 kDa were observed, suggestive of conjugation of PEGylated calcitonin to the heavy and light chains of the antibody. (Fig. 2) 
The optical density was seen to linearly increase with the concentration of mAb (Fig. 3a) . ELISA carried out for the conjugate showed the binding of the conjugated antibody to the coated RANK receptor. The sCT attached to the conjugate was detected using an anti-calcitonin antibody, thus confirming the intactness of mAb-sCT conjugates. Optical density for mAb alone was negligible, as it did not have calcitonin to be detected by anti-calcitonin antibody. Similarly, OD for sCT-PEG was also significantly low, as it did not have the anti-RANK mAb targeting moiety necessary to bind to the RANK coated plate (Fig. 3b) . ELISA-derived spectrophotometric absorbance values for mAb, sCT-PEG and mAb-sCT conjugate were 0.002, 0.025 and 0.153, respectively.
Recombinant human sRANK receptor is a 19.3 kDa polypeptide. It was electrophoresed on a 10% polyacrylamide gel and transferred to nitrocellulose membrane. On incubation with generated mAb, a band was detected between 15 and 20 kDa, after the chemiluminescent detection of anti-RANK IgG (Fig. 4) .
Osteoclasts were generated by dosing the RAW264.7 cells with RANKL and MCSF. The staining for TRAP is a technique commonly used to visualize osteoclasts. The principle behind staining of TRAP involves the use of napthol AS phosphates in conjunction with fast garnet GBC salts for the detection of acid phosphatase. This diazonium fast garnet GBC salt was selected because it couples rapidly at acid pH, forming insoluble dye deposits. Napthol AS-BI, released by enzymatic hydrolysis, couples immediately with fast garnet GBC salt, resulting in the Fig. 3 (a) ELISA conducted on RANK receptor-coated plate. The optical density was seen to be proportional with the concentration of anti-RANK antibody. (b) ELISA of the mAb-sCT conjugate where anti-Calcitonin secondary antibody reagent was used to detect immobilized Calcitonin residues that were part of the primary anti-RANK conjugates and that were binding to the RANK-coated wells. Optical density for mAb alone was negligible as it did not have calcitonin to be detected by anti-calcitonin antibody. Similarly, OD for sCT-PEG was also significantly low as it did not have anti-RANK antibody to bind to RANK-coated plate.
formation of insoluble maroon deposits at sites of activity. Tartaric acid was used in order to demonstrate the presence of tartrate-resistant acid phosphatase. Hence, cells containing tartrate-sensitive acid phosphatase are devoid of activity, and only the cells containing tartrate acid-resistant phosphatase show maroon dye deposits at the sites of activity. Staining confirmed that the presence of TRAP-positive multinucleated osteoclastlike cells was limited to those wells dosed with exogenous M-CSF and RANKL, under the cell culture conditions described (Fig. 5) .
Confocal microscopy confirmed that the anti-RANK receptor mAb staining (green color) was limited to surface receptors present on osteoclastic precursors as well as mature osteoclast-like cells (Fig. 6a) . Counterstaining with DAPI (blue color) confirmed the multinucleated phenotype of osteoclast-like giant cells. Nucleoli were also witnessed in the images. There was no demonstrable fluorescent fluorescein isothiocyanate (FITC) staining in negative control slides (Fig. 6b and c) . A commercially available anti-RANK mAb was used as a positive method control to confirm that RAW 264.7 osteoclastic precursors as well as mature osteoclast-like cells indeed stained for surface RANK receptors using immunocytochemistry.
RAW 264.7 cell cultures treated with exogenous RANKL and MCSF showed a 9.9-fold increase of TRAP activity compared to controls without cytokine treatment. We found that the release of this osteoclast-associated enzyme was potently inhibited by our mAb-sCT conjugate (and by our unconjugated mAb alone), as compared to sCT alone (Fig. 7) . We measured a 5.47-fold decrease in TRAP activity after treatment with mAb-sCT conjugate. Also, the conjugate showed a 2.82-fold greater inhibition of TRAP compared to sCT treatment alone.
The osteoclast-like cells generated on Osteologic® calcium phosphate-coated culture wells were shown to be capable of resorbing the immediate calcium phosphate layer surrounding them. That result was in direct contrast to precursor RAW 264.7 cells (cultured in the absence of M-CSF and RANKL) that were shown not to affect the calcium phosphate coating in any manner. Osteoclast-like cell cultures treated with the mAb-sCT conjugate, or mAb alone, demonstrated a significant inhibitory effect on the resorptive ability of the osteoclast-like cells, as measured by the conservation of calcium phosphate layer on the Osteologic® slide. The integrated density value (IDV) of the remaining calcium phosphate layer for the culture treated with the mAb-sCT conjugate was significantly greater than that treated with sCT alone (Fig. 8 ). Protein was transferred to nitrocellulose membrane which was blocked with 5% skim milk, followed by the incubation with anti-RANK mAb. Reaction was detected by using GAM-HRPO, and the membrane was developed using ECL reagent and exposure to X-ray film. 
To test for potential cytotoxic effects of our mAb and/or mAb-sCT conjugate, we tested RAW 264.7 cell viability using the MTT assay. Anti-RANK monoclonal antibody, mAb-sCT conjugate and sCT showed no demonstrable cytotoxicity at the concentrations tested (up to 200 nM), as measured by the absorbance of formazan solution formed after 4 h incubation (Fig. 9) . Thus, the viability of these cells was not perturbed by our mAb and/or conjugate treatments.
Using an in vitro competitive binding assay, cAMP activity was determined in TD47 breast cancer cells known to contain the calcitonin receptor. The assay confirmed that sCT and the mAb-sCT conjugate were capable of stimulating cAMP production after binding the calcitonin receptor. The significantly lower absorbance values measured (due to the competitive nature of the assay) indicated the ability for sCT and/or mAb-sCT to generate intracellular cAMP in the presence of a phosphodiesterase inhibitor. Conversely, the mAb-treated and control wells exhibited negligible intracellular cAMP generation, and, accordingly, high absorbance values in the competitive binding assay were observed (Fig. 10) . Fig. 7 Tartrate-resistant acid phosphatase (TRAP) activity assay. mAb-sCT conjugate showed an inhibitory effect on TRAP activity, an important marker of osteoclasts. RAW: cells cultured in media with 0 ng/ml RANKL+0 ng/ml MCSF; OC: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF; mAb-sCT: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+100 nM conjugate; mAb: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+100 nM mAb; sCT: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+100 nM sCT . * P<0.05 versus OC; ** P<0.05 versus sCT. No statistically significant difference was observed between naked antibody and the conjugate.
associated with differing classes of bisphosphonates, the most commonly prescribed medication for osteoporosis (23) . Estrogen hormone replacement therapy stimulates uterine and breast tissue, which predisposes the patient to neoplastic disease (24) . Leg muscle cramps, joint aches and deep vein thrombosis has been associated with selective estrogen receptor modulator therapy (25) . Similarly, conventional salmon calcitonin has shown limitations in antiresorptive efficacy, likely in part because of poor bioavailability to bone cells and the undesired uptake of the drug by calcitonin receptors present in tissues other than bone (26, 27) .
Since the pharmacological arrest of the osteoclast is the mainstay of treating systemic bone loss, drug delivery strategies that target the osteoclast directly would provide a potential therapeutic advantage at reducing systemic bone loss by selectively delivering an antiresorptive drug "cargo" to the osteoclast. Antibodies are renowned for their exquisite specificity of target recognition and generate highly selective outcomes following their systemic administration. As the RANK receptor is predominantly expressed on the surface of mature osteoclasts, we hypothesized that an anti-RANK antibody would facilitate an effective osteoclast targeting platform for the delivery of antiresorptive drug directly to the resorbing bone cells.
Salmon calcitonin, a single chain polypeptide hormone consisting of 32 amino acids, was chosen as a "model" drug for this study, as sCT therapy is known to inhibit and/or slow osteoclast-mediated resorptive bone loss, whilst positively influencing osteoinduction and bone formation (28) . Hence, we designed a conjugative strategy whereby calcitonin would be coupled to an antibody directed predominantly against osteoclast-specific receptors, in order to impart osteoclast specificity to the attached calcitonin "cargo." Delivery of sCT to the osteoclast cells by virtue of an antibody-mediated targeting platform would ensure that the calcitonin drug remained primarily localized to bone tissue after systemic administration. This would directly contrast with the passive interaction of calcitonin with other tissues that express the calcitonin receptor, such as kidneys, liver, lungs, spleen, heart and thyroid. Hence, a delivery system capable of improving sCT targeting and localization to osteoclast could have a potential to positively impact Fig. 8 Demonstration of resorption pit formation on osteologic substrate plate. After 7 days of culture, adherent cells were removed, and integrated density value of the remaining osteologic layer was calculated using alphaimager densitometer. RAW: cells cultured in media with 0 ng/ml RANKL+0 ng/ml MCSF; OC: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF; mAb-sCT: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+ 100 nM conjugate; mAb: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+ 100 nM mAb; sCT: cells cultured in media with 50 ng/ml RANKL+25 ng/ml MCSF+100 nM sCT. *P <0.05 versus OC; ** P<0.05 versus sCT. No statistically significant difference was observed for antibody versus conjugate and antibody versus calcitonin. Fig. 9 In vitro cytotoxicity of mAb, mAb-sCT & sCT on RAW 264.7 cells was determined by MTT assay. No demonstrable cytotoxicity was seen as measured by the absorbance of formazan solution formed after 4 h incubation with compounds, compared to that seen with untreated media. Samples were not statistically significant compared to the control at all concentrations. sCT therapy, whilst reducing the drug concentration in non-bone loci containing the calcitonin receptors.
Using hybridoma technology, we generated and characterized a specific monoclonal antibody against recombinant human RANK receptor, and chemically coupled synthetic sCT to that anti-RANK mAb carrier. We further PEGylated sCT in the process of conjugation by using the polymer polyethylene glycol, which is known to improve the major disadvantages of conventional sCT by increasing solubility, stability, and efficacy and reducing the immunogenecity. It has also been established that PEGylation enhances the enzymatic stability of calcitonin (29) against proteolysis, by forming an effective shield against degradative enzymes (30) whilst improving its efficacy in vivo by increasing molecular size, thus contributing to reduced renal filtration (31) allowing less frequent administration (32) .
The synthesized bio-conjugate was initially characterized using SDS-PAGE analysis and ELISA. Protein band above 150 kDa was observed for the conjugate under nonreducing condition of SDS-PAGE analysis, and some bands were seen above 50 kDa and 25 kDa under reducing condition, thus suggesting that conjugation of PEGylated sCT with mAb had occurred. An ELISA further verified the successful conjugation of sCT to our mAb, as the anticalcitonin secondary antibody reagent would only detect immobilized calcitonin residues that were part of the primary anti-RANK conjugates that were binding to the RANK-coated wells of the ELISA plate. A slight signal was observed for PEGylated sCT, and we assume that this could be due to the reaction of thiol reactive MAL functional group of PEGylated sCT with the free thiol group in BSA used as a blocking agent. In the case of conjugate, such MAL groups were utilized in the reaction with free thiol groups generated in antibody.
In order to verify the efficacy of mAb-sCT conjugates, we employed in vitro osteoclast-like cell cultures. It is well established that osteoclasts are derived from cells of the monocyte/macrophage lineage and are formed after the cellular fusion of their mononuclear precursors (33) . Hence, we utilized the RAW 264.7 cell line that can be differentiated in vitro into osteoclast-like cells after exposure to M-CSF and RANKL cytokines. RANKL is the essential factor necessary for osteoclast differentiation, whilst M-CSF (also known as CSF-1) has also been identified as an essential factor necessary to initiate osteoclastogenesis (34) . The main role of M-CSF is to act as a survival factor for osteoclast precursor cells and to induce the expression of the RANK receptor for stimulation by the RANKL.
Immunocytochemistry confirmed the osteoclast targeting potential of our generated anti-RANK mAb in vitro. Fluorescent staining of our anti-RANK mAb was specifi-cally localized to cellular surface receptors. DAPI staining showed the successful generation of multinucleated osteoclast-like cells. No demonstrable FITC staining was seen for negative controls. These results support the evidence that osteoclast-like cells generated from RAW 264.7 precursor cells express the RANK receptor, and our generated anti-RANK antibody can be used as a potential osteoclast targeting platform. The successful generation of osteoclast-like cells was also confirmed by TRAP staining, which confirmed multinucleated cells with maroon-colored dye deposits due to the presence of TRAP, the enzyme that has been used as a marker of osteoclast function for more than 20 years (35) .
To determine the in vitro efficacy of the anti-RANK mAb-sCT conjugate on osteoclast activity, we quantified osteoclast activity using a spectrophotometric total culture TRAP activity assay and a mineral resorption assay. TRAP has been shown to be a specific and sensitive indicator of bone resorption (36) and contributes to the intracellular processing of primary bone matrix degradation products prior to its final release through the basolateral membrane of resorbing osteoclast cells (37, 38) . Osteoclasts, as the bone-resorbing cells of the body, further show the ability to dissolve the mineralized inorganic phase of bone matrix known as hydroxyapatite (39) . We found that the use of our conjugate in culture significantly reduced TRAP enzyme activity as compared to sCT alone, and use of the mAb alone also showed inhibition of TRAP enzyme activity. Although the cause for this effect shown by our anti-RANK antibody alone has not been tested, we speculate that in binding the RANK receptor, the antibody may be functioning as a receptor antagonist. That competitive binding would interfere with the interaction of RANKL with RANK, an essential step of osteoclast differentiation. Nonetheless, further antiresorptive and/or anabolic effector functions could be attributed with the development of a targeted delivery strategy to osteoclast cell receptors. We further evaluated if the mAb-sCT conjugate had an effect on the ability of osteoclasts to resorb bone. We cultured osteoclast-like cells on calcium phosphate-coated culture plates in the presence of conjugate, antibody or sCT. We found that both the conjugate and the mAb alone were capable of significantly reducing the resorption of mineral layer as compared to the sCT alone. Then, we performed cyclic AMP assay to confirm that bioactivity of conjugated calcitonin. Human breast carcinoma T47D cells known to express the calcitonin receptor were used for this purpose. Binding of sCT with its receptor activates adenylyl cyclase, an enzyme responsible for the generation of cAMP. Our in vitro cAMP assay results confirmed that the modification of sCT by conjugation did not interfere with its ability to bind the calcitonin receptor and trigger its biological activity. We measured the conjugate to have increased activity over calcitonin alone, and the exact reason for that effect is still not clear. It is likely our conjugation strategy allowed one mole of antibody to become conjugated with greater than one mole of calcitonin. It has been reported that when IgG is treated with a 10 molar excess of Traut's reagent, 3-7 thiol groups are generated, which may result in the attachment of 3-7 PEGylated calcitonin molecules per molecule of IgG.
The results obtained so far have been quite encouraging from the generation of antibody and to the synthesis and in vitro evaluation of the conjugate. The conjugate and the antibody alone showed good efficacy in TRAP activity inhibition and resorption pit assay. However, cAMP assay showed that the conjugate served the purpose of delivering active drug cargo. The conjugate showed the ability to bind the calcitonin receptors and generate intracellular cyclic AMP, thus showing an advantage over the antibody alone.
With the advent of novel RANKL-scavenging antibodybased antiresorptive strategies (such as Denosumab-Pro-lia®, Amgen Inc.), it may further prove efficacious to target the RANK receptor directly with an antibody, in order to successfully antagonize receptor signaling and osteoclast function. However, neither the "scavenging" nor "antagonist" motives of those antiresorptive strategies focus on the targeted delivery of a given drug cargo (such as our anti-RANK mAb-conjugate design) which would function as a universal osteoclast targeting platform.
One potential limitation of our osteoclast targeting platform remains that RANKL-RANK signaling is not entirely restricted to osteoclasts and their precursors, but is known to be involved in the regulation of other cell types. Studies have that RANKL-RANK signaling controls the development of lactating mammary glands in pregnancy, with the highest levels being at day 15.5 in mice (40, 41) . Several studies that looked at transgenic Rankl-/-and Rank-/-mice found that there was a complete absence of lymph nodes (42) . This led to further studies involving patients with an osteoclast-poor form of the autosomal recessive osteopetrosis (ARO), where they found the cause of the disease to be various mutations in RANKL (43) . The same research then found that these patients showed no palpable signs of lymph nodes, further strengthening the theory that RANKL-RANK signaling is involved in lymph node formation in humans. RANK is further known to be expressed by monocyte/macrophages and dendritic cells, specialized immune cells programmed to capture and process antigens in the body and present them to naïve T-cells (44) . However, the targeting of drug "cargo" to the RANK receptors of those cells may not necessarily result in drug action with those cells if the cargo is carefully chosen not to affect those cell types. For example, it is highly unlikely for any cell (other than the mature osteoclast) to express both the RANK receptor and calcitonin receptor at the same time. Thus, the Boolean "and/or" logic can be applied to the selection of the differential cargo targeting to cellular phenotypes expressing the RANK receptor at any given time. The extent of this limitation remains to be fully determined, however, should not necessarily preclude the further exploration of RANK receptor targeting strategies, which may in turn provide a more comprehensive understanding of RANK signaling and crosstalk with other signaling pathways.
This study details the generation of an antibody-mediated osteoclast-targeting platform. This drug-delivery strategy may find utility as "a universal osteoclast-targeting platform" in order to directly target and deliver antiresorptive agents, anti-inflammatory agents, cathepsin K inhibitors, disintegrins, H + -ATPase inhibitors, and so on, directly to osteoclast cells. We have shown this platform is capable of being employed as an antiresorptive strategy, and our efforts will now center on the in vivo evaluation of this targeting strategy. Insights from Modeling the 3D Structure of New Delhi Metallo-β-Lactamse and Its Binding Interactions with Antibiotic Drugs New Delhi metallo-beta-lactamase (NDM-1) is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotic drugs. This is because it can inactivate most beta-lactam antibiotic drugs by hydrolyzing them. For in-depth understanding of the hydrolysis mechanism, the three-dimensional structure of NDM-1 was developed. With such a structural frame, two enzyme-ligand complexes were derived by respectively docking Imipenem and Meropenem (two typical beta-lactam antibiotic drugs) to the NDM-1 receptor. It was revealed from the NDM-1/Imipenem complex that the antibiotic drug was hydrolyzed while sitting in a binding pocket of NDM-1 formed by nine residues. And for the case of NDM-1/Meropenem complex, the antibiotic drug was hydrolyzed in a binding pocket formed by twelve residues. All these constituent residues of the two binding pockets were explicitly defined and graphically labeled. It is anticipated that the findings reported here may provide useful insights for developing new antibiotic drugs to overcome the resistance problem. The rapid growth of antibiotic resistance has become a main clinical and epidemiological problem for human health [1] . In bacteria, b-lactam antibiotics are primarily hydrolyzed by blactamases in an acylation-deacylation-based process [2] . Thus, it is believed that b-lactamases play an important role in leading to resistance of bacteria to b-lactam antibiotics. These enzymes are capable of cleaving the amide bond of the b-lactam ring so as to inactivate the b-lactam antibiotic drugs. According to their sequence similarities, b-lactamases can be generally divided into four classes, named as A, B, C, and D. Classes A, C, and D of blactamases contain serine groups in their active sites, while the enzymes in class B are metalloproteins, or called ''metallo-blactamases'', that require one or two zinc ions for their activity. Among all the b-lactamases, metallo-b-lactamases are the major culprit causing bacteria to resist antibiotics, due to the reason that they can degrade all b-lactams except monobactams and that they are special for their constant and efficient carbapenemase activity [3] .
In December 2009 a novel metallo-b-lactamase was identified in a patient hospitalized in New Delhi with an infection caused by klebsiella pneumonia [4] . This b-lactamase was later detected in bacteria in India, Pakistan, United Kingdom, United States, and Canada. The New Delhi metallo-b-lactamase (NDM-1) has the ability to make bacteria resistant to a wide range of b-lactam antibiotics, including the carbapenem family antibiotics that are a mainstay for the treatment of antibiotic-resistant bacterial infections [5, 6] . According to the report of the United Kingdom's Health Protection Agency, most isolates with NDM-1 are resistant to all standard intravenous antibiotics for the treatment of severe infections. The most common bacteria that make this enzyme are Gram negative such as Escherichia coli and Klebsiella pneumonia, but the gene for NDM-1 can spread from one strain of bacteria to another by horizontal gene transfer.
To reveal the resistance mechanism of bacteria to b-lactam antibiotics due to the existence of NDM-1, an indispensable knowledge is of the 3D (three-dimensional) structure of NDM-1. Since so far no 3D structure whatsoever has been determined by experiments for NDM-1, we have to resort to the approach of structural bioinformatics [7] . Recently, growing evidences have indicated that various tools in structural bioinformatics, such as homology modeling [8, 9, 10, 11, 12, 13, 14, 15, 16] , molecular docking [17, 18, 19, 20, 21, 22] , as well as molecular dynamics simulations [23, 24, 25, 26, 27, 28, 29, 30, 31] , can timely provide very useful information and insights for biomedical science and drug development and hence are quite rewarding [14, 22, 29, 32, 33, 34, 35, 36, 37, 38, 39] . In view of this, the present study was initiated in an attempt to develop a homology model for NDM-1, based on which the molecular docking operations and molecular dynamics simulations were performed in hopes that the information thus obtained may provide useful insights or clues for designing new drugs to overcome the antibiotic resistance problem.
The entire sequence of NDM-1, which contains 158 amino acids, was taken from NCBI Protein database with an accession of AB571289. According to the score of BLAST search, the crystal structure of VIM-2, a Zn-b-lactamase from Pseudomonas aeruginosa [40] , was selected as a structural template to perform homology modeling to develop the 3D structure of NDM-1. The PDB code of the crystal structure is 1ko3, which was released in 2008 with a resolution of 2.20 Å [40] . The entire sequence of 1ko3 contains 230 amino acids. The sequence alignment between NDM-1 and 1ko3 was performed by the Molecular Operating Environment (MOE), and the alignment result thus obtained indicates that the two proteins have a sequence identity of 43%.
Meanwhile, using the web-server EzyPred [41] at http://www. csbio.sjtu.edu.cn/bioinf/EzyPred/ and the protein sequence information, it was identified that NDM-1 is a member of hydrolases enzyme family (acting on carbon-nitrogen bonds other than peptide bonds), and so is 1ko3. Since both NDM-1 and 1ko3 belong to a same enzyme family with the same action mechanism, and their sequence identity is higher than 40%, it is quite reasonable to use the crystal structure of 1ko3 [40] as a template to develop the 3D structure of NDM-1 via homology modeling.
Based on the sequence alignment ( Fig. 1 ) as well as the atomic coordinates of 1ko3, the 3D structure of NDM-1 was derived by using the I-TASSER algorithm, an extension of the previous TASSER (Threading/Assembly/Refinement) method [42, 43, 44] .
The homolog-modeled 3D structure was subject to a short-time molecular dynamics simulation (,3 ns) for further refinement. The final 3D structure thus obtained for NDM-1 is shown in Fig. 2 . Meanwhile, some assessments by the Swiss Model Server indicated that the computed structure is quite reasonable and creditable.
Subsequently, molecular docking operations were carried out with Monte Carlo simulated annealing [45] to get the favorable binding interaction modes for NDM-1 respectively with Imipenem and Meropenem, two typical b-lactam antibiotic drugs. It was reported recently [6] that NDM-1 showed a comparatively high resistance to Imipenem and Meropenem. The binding pocket was identified using Q-SiteFinder [46] . Because binding of inhibitors in the active site may induce a conformational change to closing some flaps [47] , we adopted a flexible docking procedure to construct the binding modes of NDM-1 with Imipenem and Meropenem. Before the docking procedure, we extracted 1000 conformations of NDM-1 from the aforementioned 3-ns molecular dynamics simulation. The ligand (Imipenem or Meropenem) was then docked to all of these conformations to search for a favorable binding mode. The docking program [48] used in this study would automatically generate a diversified set of configurations by randomly changing the ligand's coordinates. When a new configuration of the ligand was generated, the search for the favorable binding was operated within a specified 3D box by the simulated annealing to optimize the purely spatial contacts as well as electrostatic interactions. Finally, the favorable binding mode thus obtained was further optimized by a short time molecular dynamics simulation (,5 ns). The binding energy was calculated using a scoring function London dG [49, 50, 51] . In all our calculations, the Merck force field (FFMM94) parameters were adopted. The most favorable binding interactions thus obtained for NDM-1 with Imipenem and Meropenem are given in Fig. 3 and Fig. 4 , respectively.
Homology-Modeled 3D Structure of NDM-1 A total of 100 homology-modeled structures for NDM-1 were derived with each having a C-score. The C-score is a ''confidence score'' for estimating the quality of a computed model: a high Cscore signifies a model with a high confidence and vice-versa. Shown in Fig. 2A is the structure with the highest C-score that was used as a receptor for further docking studies. Meanwhile, we employed PROCHECK [52] to estimate the quality of our models. It was indicated by PROCHECK that there are 93.1% residues located in the ''core'' and ''allowed'' regions, 4.6% in the ''general'' region and only 2.3% in the ''disallowed'' regions ( Fig.  S1 ). In the computational structure, all the bond lengths for the main-chain residues and 91.9% bond angles for the main-chain residues are within the allowed limits. To further examine our computational model of NDM-1, we also used the tool of QMEAN, which is a scoring function of a linear combination of 6 structural descriptors [53] . The QMEAN score ranges between 0 and 1 with higher value to reflect a better quality of the model. The QMEAN score for the current computational model is 0.67, and the density plot of this QMEAN score is shown in Fig. S2 . To evaluate the absolute model quality, we also calculated a Z-score of the computational model in comparison with the scores of the reference X-ray structures of similar size from the Protein Data Bank (Fig. S3) . For each of the QMEAN components, a Z-score was computed in comparison with the average X-ray structures (the average value is 0, as shown in Fig. S4 ). Based on these analyses, we further examined the per-residue error (Fig. S5) , visualized using a color gradient from blue (reliable regions with estimated error below 1 Å ) to red (potentially unreliable regions with estimated error above 3.5 Å ). We found that the residues in the potentially unreliable regions were mainly located on the loop regions in the computational model, indicating that further molecular dynamics optimizations are needed, as described below.
However, after a 3-ns molecular dynamics simulation, all the residues in our model were in the ''core'' and ''allowed'' regions. It can be seen from Fig. 2A that, like most of the other metallo-blactamases, the NDM-1 protein belongs to the a/b structural class [54] , with 3 helices and 7 b-strands. The 3 helices were exposed to the solvent. The N-terminal and C-terminal regions of NDM-1 can be superposed by a 180 degrees rotation around a central axis, indicating that the entire structure may have arisen from the duplication of a gene. It was found after superposing NDM-1 to its template structure 1ko3.pdb that the two structures share a quite similar backbone (Fig. 2B ) owe to their high sequence identity percentage. However, some a-helices and b-strands in 1ko3.pdb are not seen in the NDM-1 model because 1ko3.pdb contains 5 helices and 11 b-strands. This is due to the sequence deletion of NDM-1 in its C-terminal and N-terminal regions. It is instructive to note that such sequence deletion might make the b-lactam antibiotic drugs easier to access the binding pocket of NDM-1.
Similar with other metallo-b-lactamases, the active site of NDM-1 presents two metal ion binding sites: His site and Cys site. Thus, two zinc ions can be detected in our homology model at both the His and Cys sites with a distance of 4.20 Å apart. According to some experimental data, the one located in the His site has a higher occupancy (1.3 fold) than that in the Cys site (Fig. 3A) . The zinc ion in the His site possesses a tetrahedral coordination sphere and is coordinated by His56, His58, and His125. The other zinc ion in the Cys site has a trigonal-pyramidal coordination sphere that involves Ser11, Asp60, and Cys144.
Since metallo-b-lactamases can inactivate the b-lactam antibiotic drugs by cleaving the amide (carbon-nitrogen) bond of the blactam ring [2] , it can provide us very useful insights about the essence of the drug resistance problem by analyzing the binding interaction modes obtained by docking Imipenem and Meropenem to NDM-1 receptor, respectively. Shown in Fig. 3B is a close view of the binding interactions obtained by docking the antibiotic drug Imipenem to the receptor NDM-1. Its binding pocket is formed by nine residues that have at least one heavy atom with a distance within 5 Å [55] to Imipenem, as labeled in Fig. 3B . Interestingly, the constituent residues thus defined for the binding pocket are quite consistent those identified by the online web-server tool Q-SiteFinder for forming the extended cleft active site of NDM-1. In the NDM-1/Imipenem complex, Met3 and Phe6 are located on the brink of the active site, providing some van der Waals interactions to the drug molecule. It can be seen from Fig. 3B that, in addition to the van der Waals interaction of the Imipenem drug with the surrounding binding pocket residues, there are remarkable hydrogen bonds that have tightly tethered the drug to Glu88 and Thr126 of the receptor, making NDM-1 able to recognize the antibiotic drug followed by cleaving the amide bond of its b-lactam ring so as to inactivate Imipenem. The detailed information for these hydrogen bonds is listed in Table 1 .
For the case of Meropenem, it can interact with almost the same residues as Imipenem. As can be seen from Fig. 3C , the binding pocket for Meropenem involves six residues, of which Glu88 and Thr126 form three hydrogen bonds with the antibiotic drug. Since there is one more five-member ring in Meropenem (Fig. 3C) , some additional hydrogen bonds are needed to stabilize the ligand during the process of cleaving its amide bond and inactivate the antibiotic drug. Based on these structural findings and previous theoretical studies [56, 57, 58] , we proposed a catalytic mechanism for NDM-1, as illustrated in Fig. 4 . In our model, the metalbinding Asp60 acts as the general base that activates the water nucleophile, while the protonation of Asp60 results in the cleavage of its bond to the metal ion. Figure S4 The QMEAN score components calculated based on the Z-score of each component in comparison with the average xray structures.
(TIF) Figure S5 Per-residue error visualized by using a color gradient from blue (reliable region, estimated error below 1 Å ) to red (potentially unreliable regions, estimated error above 3.5 Å ).
(TIF) Production of IFN-β during Listeria monocytogenes Infection Is Restricted to Monocyte/Macrophage Lineage The family of type I interferons (IFN), which consists of several IFN-α and one IFN-β, are produced not only after stimulation by viruses, but also after infection with non-viral pathogens. In the course of bacterial infections, these cytokines could be beneficial or detrimental. IFN-β is the primary member of type I IFN that initiates a cascade of IFN-α production. Here we addressed the question which cells are responsible for IFN-β expression after infection with the intracellular pathogen Listeria monocytogenes by using a genetic approach. By means of newly established reporter mice, maximum of IFN-β expression was observed at 24 hours post infection in spleen and, surprisingly, 48 hours post infection in colonized cervical and inguinal lymph nodes. Colonization of lymph nodes was independent of the type I IFN signaling, as well as bacterial dose and strain. Using cell specific reporter function and conditional deletions we could define cells expressing LysM as the major IFN-β producers, with cells formerly defined as Tip-DCs being the highest. Neutrophilic granulocytes, dendritic cells and plasmacytoid dendritic cells did not significantly contribute to type I IFN production. Interferons (IFN) were first discovered over 50 years ago by Isaacs and Lindenmann [1] . Due to its complexity, the IFN system is still little understood and remains a subject of intensive research. Currently, three types of IFNs are distinguished -type I, type II consisting of only IFN-c, and the recently discovered type III also called IFN-l. Here, we will focus on type I IFN. This family of cytokines comprises a single IFN-b, more than 13 IFN-a, and several other less well characterized members [2, 3] . All these subtypes signal through a common cell surface receptor (IFNAR) to activate IFN-inducible genes that exert a wide range of effects central to innate and adaptive immunity [2] . As a rule, IFN-b is produced as the earliest of type I IFN initiating a cascade of IFN-a via autocrine and paracrine loops [4, 5] . In addition, during viral infections type I IFN may be induced in plasmacytoid dendritic cells (pDCs) also named ''natural interferon producing cells''. Due to constitutive expression of the transcription factor IRF-7, pDCs are able to immediately produce large amounts of IFN-a [6] . Nonviral pathogens (i.e., bacteria, protozoa, fungi and helminthes) may also induce type I IFN. Interestingly, in contrast to viral infections where IFNs are normally protective, in non-viral infections IFN production might be defensive or deleterious [7, 8] .
The prototype of a deleterious type I IFN response during bacterial infection is experimental listeriosis elicited by the grampositive rod-shaped intracellular bacterium Listeria monocytogenes that was discovered in 1926 (reviewed in [9] ). L. monocytogenes induces type I IFN synthesis via triggering a still uncharacterized pattern recognition receptor in the cytosol of host cells [10, 11] . Cyclic diadenosine monophosphate (c-di-AMP), secreted by L. monocytogenes, might be one of the cytosolic activators of type I IFN [12] . In addition, lymphotoxin-a might be involved in the triggering of type I IFN responses [13, 14] and lymphotoxin b receptor was shown to be crucially involved in the controlling of Listeria monocytogenes infection [15] .
Secretion of type I IFN increases susceptibility to Listeria by inducing apoptosis of T cells and macrophages [16] [17] [18] . In accordance, mice deficient of IFNAR are more resistant to Listeria and show decreased T cell apoptosis in the spleen during infection [17, 19, 20] . Moreover, type I IFN are able to down regulate IFN-c receptor thereby suppressing macrophage activation and increasing host susceptibility to Listeria infection [21, 22] .
Intravenously inoculated L. monocytogenes is taken up by the spleen and removed from the blood predominantly by mononuclear phagocytes in the marginal zone of the white pulp [23] . Myeloid cells, especially monocytes and macrophages are rapidly recruited to sites of bacterial infection and are required for initial control of L. monocytogenes infection [11, [24] [25] [26] . During bacterial infections, circulating monocytes are known to differentiate into tissue macrophages and dendritic cells (DCs) [27] . Additionally, inflammatory Gr1 + /Ly6C high monocytes are able to differentiate into tumor necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS) producing so-called Tip-DCs at sites of infection [11] .
According to recent publications, different myeloid cell populations were shown to be responsible for IFN-b production during Listeria infection. Stockinger et al. defined CD11b + CD11c 2 PDCA1 2 B220 2 macrophages to be the major IFN-b producers and pDCs were claimed to make no contribution [28] . On the other hand, Dresing et al. observed IFN-b exclusively in the myeloid cell population called Tip-DCs [29] . In the present study, we used a genetic approach to address the question which cells produce IFN-b during experimental listeriosis.
Previously, we generated a conditional mouse line, in which the IFN-b coding sequence is replaced by the reporter luciferase upon cell specific Cre expression [30] . Here we explored CD19cre, CD4cre and LysMcre mice to activate the reporter function in B cells, T cells, monocyte/macrophages and neutrophils. As reporter mice, we used mice that were heterozygous for the targeted mutation. This allowed IFN-b production from the functional wild type allele. To demonstrate the physiological relevance of IFN-b produced by the cell types mentioned above, we included mice homozygous for the conditional deletion and compared them with mice in which IFN-b was deleted in germ line. Overview of transgenic mice used in this paper is presented in Figure S1 .
Using these genetically modified mice, we highlight the importance of IFN-b during the deleterious action of type I IFN in the course of Listeria infection. In addition, the novel reporter mice revealed a maximum of IFN-b induction 24 hours post infection (p.i.) in spleen and, surprisingly, 48 hours p.i. in cervical and inguinal lymph nodes after high dose intravenous infection. Low dose infection followed the same pattern of IFN-b production, although with delayed kinetics. The luciferase signal in spleen and lymph nodes mirrored the presence of Listeria in these organs. Moreover, our results showed that LysM-expressing cells are the main producers of IFN-b during murine listeriosis, especially the cells previously defined as Tip-DCs. Finally, neutrophils, DCs and pDCs and did not significantly contribute to type I IFN production under our conditions of L. monocytogenes infection.
During Listeria monocytogenes infection, IFN-b is produced in spleen and lymph nodes Different strains of Listeria monocytogenes vary in their ability to activate type I IFN production [31] . Here we compared LO28 and EGDe, two commonly used strains of Listeria monocytogenes. First, we asked when and where type I IFN is induced. To address this question, we used a previously described IFN-b reporter mouse, which allows whole body in vivo imaging of IFN-b induction using firefly luciferase as a reporter [30] . Figure 1A shows induction of IFN-b in albino IFN-b +/Db-luc reporter mice after intravenous (i.v.) injection of Listeria monocytogenes LO28 and EGDe strains. At 24 hours post infection (p.i.), IFN-b induction occurred almost exclusively in the spleen. Interestingly, 48 hours p.i. a bright luminescent signal appeared in the cervical and in the inguinal lymph nodes, especially when using the LO28 strain. At this time, no production of IFN-b was detectable in the spleen any longer. Loss of the signal from the spleen 48 hours p.i. could be due to complete destruction of spleen together with the massive apoptosis of lymphocytes [32] . Quantitation of luciferase activity in the selected regions of interest (Fig. 1A , circles 1 and 2 for spleen and cervical lymph nodes, respectively) showed that LO28, induced a stronger signal compared to EGDe (Fig. 1B) , although colonization by bacteria was comparable (Fig. 1C) . This confirmed the findings by Reutterer et al. that LO28 is the most potent type I IFN stimulator amongst the commonly used Listeria strains [31] . Of note, there was no significant signal from the liver at any time point, although this organ is highly colonized by Listeria.
Systemic application of L. monocytogenes is used in many studies related to host-pathogen interaction [9, 33, 34] , and spleen and liver were defined as the target organs of these bacteria [23] . Little attention has been paid to lymph nodes until now. Our finding that IFN-b is produced in the lymph nodes lead us to test whether lymph nodes are also colonized by Listeria after i.v. infection. Indeed, already 24 hours p.i. we could observe bacteria in cervical and inguinal lymph nodes independent of the bacterial strain used (Fig. 1C) . Between 24 and 48 hours p.i., bacterial burdens in lymph nodes increased about 100 fold. Colony forming units of Listeria in spleens and livers also increased although not as dramatic.
To investigate the influence of type I IFN on lymph node colonization, we compared wild type C57BL/6, IFN-b 2/2 and IFNAR 2/2 mice. Like in spleen, a defect in the type I IFN system resulted in lower bacterial numbers (Fig. 2) confirming a detrimental role of these cytokines also in lymph nodes. These differences were only detectable at 48 hours p.i., consistent with the peak of IFN-b production at this time point.
To rule out whether lymph node colonization is specific and is not due to the high dose of infection, we first monitored albino IFN-b +/Db-luc reporter mice for IFN-b production after low dose Listeria monocytogenes infection (Fig. 3A) . Luciferase expression at 24 hours p.i. peaked in spleen. At 48 and 72 hours p.i. we observed the signal both in spleen and in cervical lymph nodes. At 96 hours p.i. the IFN-b production pattern was almost identical to the 48 hour time point after high dose infection (Fig. 1A) , although the overall luminescent signal was lower (note the different scales). Quantification of luminescence intensity in regions of interest ( Fig. 3B ) in spleen and cervical lymph nodes (Fig. 3A , selected circled areas 1 and 2, respectively) confirmed qualitative in vivo data presented in Fig. 3A .
As expected, colonization of lymph nodes paralleled IFN-b production (Fig. 3C) . These observations suggested that lymph node colonization by L. monocytogenes with subsequent IFN-b production is general phenomenon and does not depend on the bacterial dose, strain or type I IFN signaling.
pDCs do not contribute to the production of type I IFN during Listeria monocytogenes infection In response to a wide variety of enveloped DNA and RNA viruses (e.g., Influenza virus, vesicular stomatitis virus) as well as parasites (Plasmodium falciparum) and CpG oligonucleotides, IFN type I is produced by pDCs [35] . L. monocytogenes was shown after intragastric administration to activate pDCs in spleen and in mesenteric lymph nodes as detected by an increased expression of MHC II and CD86 [36] . By extrapolation, a contribution of pDC to the type I IFN response after systemic Listeria infection could be expected, although in vitro stimulation of splenic pDC with Listeria did not induce type I IFN [28] . To evaluate the contribution of pDC to the overall IFN production in vivo after Listeria monocytogenes infection, we performed depletion experiments. To this end, 24 hours prior to infection mice were intravenously injected with the depleting antibody anti-mPDCA-1. 24 hours p.i. spleens were analyzed for the presence of pDCs. Figure 4A demonstrates gating strategy for pDCs and depletion efficiency.
RT-PCR of spleen cells 24 hours p.i. showed that mRNA expression of IFN-b and total IFN-a were not altered regardless of pDC depletion (Fig. 4B) . Similarly, serum levels of these cytokines did not reveal any significant differences between mice depleted of pDCs or control mice (Fig. 4C) . Moreover, bacterial loads in spleens and livers of such mice were also similar (Fig. 4C) . Hence, we conclude that pDCs do not significantly contribute to type I IFN production and also do not have a physiological relevance with regard to colonization of spleen and liver during murine listeriosis.
To determine the contribution of other myeloid or lymphoid cell populations to the IFN-b production after L. monocytogenes infection, we employed tissue specific reporter mice since these mice allow tissue specific replacement of the IFN-b gene by the luciferase reporter in the IFN-b +/floxb-luc mice [30] . These mice were crossed with mice expressing Cre recombinase under various tissue specific promoters. CD19cre mice were used to implement the reporter activity in B cells, CD4cre mice in T cells and LysMcre mice in monocytes/macrophages and granulocytes. All reporter mice were heterozygous for both markers to allow normal cellular development as well as potential production of IFN-b in all cells. Comparison of such tissue specific reporter mice after Listeria infection with the ''global'' IFN-b reporter mouse (IFN-b +/Db-luc ) by whole body in vivo imaging demonstrated that T-and B-cells did not have any apparent impact on the IFN-b production after infection by L. monocytogenes (Fig. 5A) .
Moreover, about 80% of pDCs are supposed to exhibit recombination in CD4cre mice [37] . Nevertheless, these mice did not show any luciferase production, confirming our data from above. Thus, we conclude that pDCs do not contribute to the type I IFN production during Listeria monocytogenes infection. Only cells, in which the LysM-promoter is active, significantly contribute to the IFN-b signal in spleen at 24 and in lymph nodes at 48 hours p.i. (Fig. 5A ). Of note, the bioluminescence signal in such mice is lower than in the mice displayed in Fig. 1A . Obviously, the signal is quenched by the black skin and fur of the recombinant C57BL/6 mice.
To overcome the restrictions of in vivo imaging with black mice, the results were corroborated by ex vivo quantification of luciferase activity (Fig. 5B ). Using this assay, we could confirm that T-and Bcells did not contribute to luciferase activity in spleen and lymph nodes at both time points. In contrast, LysMcre specific IFN-b reporter mice contributed with similar levels of luciferase activity compared to IFN-b +/Db-luc mice. Hence, LysM-expressing cells almost exclusively produce IFN-b in both spleen and lymph nodes upon infection with L. monocytogenes. Confirming the role of LysM-expressing cells during Listeria monocytogenes infection
To confirm the contribution of LysM positive cells to the type I IFN response against L. monocytogenes as well as to characterize their role during infection with this pathogen, we analyzed homozygous mice, in which the gene encoding IFN-b was cell-specifically deleted on both alleles. As a control, we included IFN-b floxb-luc/floxb-luc mice to demonstrate that the genetic changes in the targeted locus have no impact on their own on the course of infection. C57BL/6, IFNb floxb-luc/floxb-luc , IFN-b floxb-luc/floxb-luc x LysMcre and IFN-b 2/2 mice were infected with Listeria monocytogenes and 24 hours p.i. serum levels of type I IFN were determined by ELISA (Fig. 6A) . Importantly, IFN-b production was identical in C57BL/6 and IFN-b floxb-luc/floxb-luc mice, while the extent of IFN-b expression was dramatically reduced in IFN-b floxb-luc/floxb-luc x LysMcre mice. As a consequence, since type I IFN induction in macrophages depends on feedback signaling, total IFN-a levels were also markedly reduced in IFN-b floxb-luc/floxb-luc x LysMcre mice and they were similar to the levels in IFN-b 2/2 mice (Fig. 6A) .
Consistent with these results, the CFUs isolated from spleen and liver of C57BL/6 and IFN-b floxb-luc/floxb-luc mice did not differ significantly, while IFN-b floxb-luc/floxb-luc x LysMcre and IFN-b 2/2 mice showed strongly reduced bacterial numbers in the organs 24 hours after infection (Fig. 6B) . Moreover, comparison of IFNb floxb-luc/floxb-luc mice with IFN-b floxb-luc/floxb-luc x CD11ccre, where IFN-b production is ablated in dendritic cells, showed no significant differences in bacterial loads in spleen and liver, as well as in serum levels of type I IFN ( Figure S2 ).
Taken together, our results showed that preventing LysMexpressing cells from IFN-b production is sufficient to avoid the detrimental effects of type I IFN during L. monocytogenes infection. Thus, such cells are apparently the major source of IFN-b during murine listeriosis.
The LysM gene in mice is active mostly in monocyte/ macrophages and neutrophils [38, 39] . Neutrophils are essential during the early innate response to Listeria monocytogenes and are known to be capable to produce various cytokines, such as IL-1b, IL-6, TNF-a, IL-12, MIP-1a, MIP-1b, IL-18 and reactive oxygen intermediates in response to different stimuli [40] [41] [42] [43] . However, the contribution of neutrophils to type I IFN production is still unclear. Therefore, we wanted to determine whether neutrophils are involved in IFN-b production after L. monocytogenes infection.
To answer this question, we depleted granulocytes 24 hours prior infection after carefully titrating the antibody. Depletion of neutrophils was close to complete (Fig. 7A) , while changes in other cell populations were negligible ( Figure S3 ). Ex vivo analysis of luciferase activity in IFN-b +/Db-luc mice depleted of neutrophils revealed, that these cells did not have a significant impact on luciferase expression (Fig. 7B ). In addition, similar amounts of IFN-b and total IFN-a in serum were found in C57BL/6 mice despite of the neutrophil depletion (Fig. 7C) . Hence, LysM-positive monocyte/macrophages are the population that is responsible to almost entirely produce type I IFN after Listeria monocytogenes infection.
It is known, that the LysM promoter is active not only in monocyte/macrophages and neutrophils, but also in part of the CD11c + DCs, while no expression is observed in T and B cells [44] . Since it was recently shown that CD11b + CD11c int Ly6C + cells previously defined as Tip-DCs are the major IFN-b producers upon Listeria infection [29] , we wanted to know whether Tip-DCs belong to LysM-expressing monocyte /macrophage population and whether we could confirm the production of IFN-b by such cells. Therefore, we sorted CD11b + CD11c 2 macrophages, CD11c + CD11b 2 dendritic cells (DCs), CD11b + CD11c int Ly6C + Tip-DCs and B220 + CD11c 2 B cells as a negative control from IFN-b +/floxb-luc x LysMcre mice 24 hours after Listeria infection and tested for LysM, LysMcre as well as IFN-b expression by RT-PCR. A strong signal was observed for LysM in Tip-DCs and macrophages and, to a lesser extent, in dendritic cells (Fig. 8) . Cre recombinase was expressed in macrophages and in Tip-DCs, but not in DCs. IFN-b mRNA was highly pronounced in Tip-DCs and weakly in DCs. Faint bands were also detectible for IFN-a in these cells. In contrast, CD11b + CD11c 2 macrophages failed to produce any detectable amount of IFN-b or IFN-a. Taking together, our results indicate that cells defined as Tip-DCs are within LysM-expressing cells and are responsible for the major IFN-b production upon Listeria monocytogenes infection.
Type I IFN are extremely pleiotropic cytokines. For instance, they are involved in defense against many viruses [45] [46] [47] , could be either protective or detrimental during non-viral pathogens [7, 8] , act in cancer surveillance [48, 49] , but they are also effector molecules in toxic shock elicited by LPS [50] and TNF [51] . In addition, recombinant type I IFN is used in the clinics as therapy against certain cancers as well as for treatment of chronic infections caused by HBV [52] , HCV [53] and multiple sclerosis [54] . Thus, these molecules could be of great health benefit but could also elicit serious detrimental effects. Therefore, knowledge about the cells involved in production of type I IFN and their regulation is of utmost importance for the understanding of the IFN system.
Listeria monocytogenes is an intracellular pathogen, broadly used as a model to investigate host-pathogen interactions. Different groups have shown the production of type I IFN in vitro and in vivo upon infection with Listeria monocytogenes [10, 11] . We followed IFN-b induction in the mouse using non-invasive whole body in vivo imaging. This way we derived a spatiotemporal resolved picture of the early IFN-b response during Listeria infection. We focused on production of IFN-b, because IFN-b appears to be the primary member of type I IFN family that is induced after infection with Listeria [16, 17] and initiates the cascade of type I IFN in most cell types [4, 5] .
By a genetic approach we investigated the cells that are responsible for IFN-b production during murine listeriosis. We could show that IFN-b is almost exclusively produced by LysMexpressing cells in the spleen as well as in colonized lymph nodes. LysM promoter is highly expressed in monocytes/macrophages and neutrophils [44] . However, the use of the LysMcre mouse does not allow distinction between myeloid cell populations, although lower expression in CD11c + cells was reported [55] .
Macrophages have been identified as one of the target cell population in the murine spleen [26] . Besides, macrophages play a major role in the early innate defense against Listeria monocytogenes via secretion of different cytokines [20, 56] . However, DCs were also observed to be directly infected by L. monocytogenes [57] . Since IFN induction requires intracellular Listeria, we would have expected that DCs, in addition to monocytes/macrophages, would be involved in IFN-b production. This is apparently not the case, because i) only negligible amounts of IFN-b are produced in infected mice, in which IFN-b is conditionally deleted by LysMcre. This also excludes a major contribution of a non-lymphoid tissue associated cell. ii) Similar amounts of reporter luciferase are produced in the global reporter mice and in the mice reporting production of IFN-b by monocytes/macrophages and neutrophilic granulocytes. Although LysMcre is supposed to be expressed by a small population of DCs [44] , CD11ccre mice experiments confirm that conventional DCs do not significantly contribute to IFN-b production. We also could exclude the contribution of pDC. The depletion of this cell population had no effect on the overall production of type I IFN. This extends the in vitro data of Stockinger et al. [28] to the in vivo situation. Finally, the involvement of neutrophils that are expressing LysMcre was also excluded by a depletion experiment.
Stockinger et al. [28] defined cells with surface antigens characteristic of macrophages as major producers of type I IFN during Listeria infection. According to the recent publication of Dresing et al. [29] , TNF-a and iNOS producing Tip-DCs, that share markers with macrophages, were the main cellular source of IFN-b in the course of Listeria infection. Our data showed that a LysM-expressing cell population is responsible for IFN-b production in murine listeriosis. Testing sorted macrophages, DCs and Tip-DCs for type I IFN and LysM expression revealed that the LysM promoter was active in all three cell populations. However, only Tip-DCs were able to express vast amounts of IFN-b in agreement with Dresing et al. [29] . Interestingly, such Tip-DCs showed strong expression of LysM. This sheds some doubts on the DC nature of Tip-DCs, which was originally defined by their T cell priming capacity in mixed lymphocyte reaction [29] . Thus, Tip-DCs might as well be defined as inflammatory monocytes.
DCs might be involved only at the very early time points after infection and IFN-b production might be below our detection limit. We think this is unlikely given the extreme sensitivity of our reporter system. Alternatively, infected DCs might undergo apoptosis before they are able to significantly contribute to IFN production or IFN production in DCs might be inhibited by L. monocytogenes. The latter notion would be in agreement with the fact that pDCs also do not produce IFN-b.
A similar argument we would use for the absence of IFN-b production by hepatocytes. Such cells are known to be severely infected by L. monocytogenes during the complete course of infection. Nevertheless, no significant signal was observed in the reporter mice from liver. The absence of a contribution of Kupffer cells was expected since such cells are known to only bind Listeria but are not directly infected [58] . IFN-b induction requires cytosolic residence of Listeria.
The colonization of lymph nodes after i.v. infection with L. monocytogenes has been ignored so far, although it is known that such bacteria are able to infect lymph nodes after subcutaneous [59] or oral application [36] . Here we have shown that Listeria monocytogenes successfully colonizes cervical and inguinal lymph nodes also after i.v. infection. We noticed lymph node colonization due to the activation of the IFN-b reporter in such tissues. This impressively demonstrates the importance of using reporter systems that allow holistic unbiased observations. Colonization of lymph nodes could have taken place via dissemination of bacteria from the ''classical'' target organs spleen and liver. The delayed production of IFN-b in such organs would be in agreement with this notion. However, production of IFN-b correlated with the number of bacteria found in the colonized lymph nodes, which also showed a delayed increase. Thus, lymph nodes obviously are also primary target organs for L. monocytogenes, although they do not show the same penetrance as spleen and liver.
Together, our work compellingly shows that LysM-expressing Tip-DCs are the major producers of IFN-b during murine listeriosis, which is responsible for the induction of the type I IFN cascade in most cell types. The expression of LysM sheds some doubt on the DC nature of such cells. We also show in vivo that pDCs, the cell type that is able to produce type I IFN independent of IFN-b and neutrophils do not contribute at all to the overall production of type I IFN during murine listeriosis. Having settled this, the question now arises: why cDCs, that are infected at least early after i.v. application or hepatocytes that are known to be heavily infected by L. monocytogenes, do not produce type I IFN.
Mouse care and experimental procedures were performed under the approval of local authority Niedersä chsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES). Permit numbers for this study are 33.11.42502-04-067/07 and 33.42502-071/06.
All mice were bred at the animal facility of the Helmholtz Centre for Infection Research (HZI) and maintained under specific pathogen-free conditions. For experiments, female mice 8 to 12 weeks of age were used. All mice used in this study were on the C57BL/6 background. IFN-b 2/2 [60] mice were backcrossed onto C57BL/6 for more than 15 generations. Conditional deletion/reporter mice IFN-b floxb-luc were generated using C57BL/6 ES-cells (Bruce4). To replace the IFN-b CDS by luciferase in germ line (IFN-b Db-luc ), IFN-b floxb-luc mice were crossed with K14cre mice [61] . To receive tissue specific IFN-b deletion as well as reporter expression, IFN-b floxb-luc were crossed to CD19cre [62] , CD4cre [63] and LysMcre [44] mice respectively (kindly provided by Dr. Angela Schippers, HZI). CD11ccre mice [64] were kindly provided by Prof. Dr. Ulrich Kalinke, Twincore. Additionally IFN-b +/Db-luc mice were crossed with albino C57BL/6 (C57BL/6-Tyr,c-2J.) kindly provided by Thomas Blankenstein (MDC, Berlin) to improve in vivo imaging.
Spleen cells were prepared by gently flushing the spleen with IMDM supplemented with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 10% FCS, 50 mM b-ME, and 2 mM Lglutamine. Erythrocytes were lysed for 2 min in ACK buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM EDTA) and cells were washed two times with PBS. Cell clumps were removed by passaging through a 50 mm nylon filter. Preparation was conducted strictly at 0uC.
Single cell suspensions were treated with anti-mouse CD16/ CD32 BD Fc Block (2.4G2, Becton Dickinson, NJ, USA) for 10 min followed by staining with appropriate mAbs for 20 minutes on ice. Abs used in this work included PE anti-mPDCA-1clone JF05-1C2.4.1 (Miltenyi Biotec), APC CD11c clone N418 (eBioscience, San Diego, USA), Pacific Blue CD11b clone M1/ 70.15 (Invitrogen), APC-Cy7 B220 clone RA3-6B2 (BD Pharmingen, New York, USA), anti-mouse Ly6G PE-Cy7 (Gr1, eBioscience, San Diego, USA), biotynylated anti-mouse Ly6C (Pharmingen, New York, USA), streptavidin APC-Cy7 (Pharmingen, New York, USA). Flow cytometric analysis and sorting was performed using LSRII (Becton Dickinson, NJ, USA. The data were analyzed using FACSDiva (Becton Dickinson) software.
Total RNA was extracted from spleen cells using RNeasy mini kit (Qiagen) according to the manufacturer's instructions. DNA contamination in the total RNA preparation was eliminated using DNase I (Qiagen). RevertAid First Strand cDNA Synthesis Kit (Fermentas) with oligo(dT) primers was used for reverse transcription of purified RNA. PCR was performed using the Promega kit according to the instructions of the supplier. 
Listeria monocytogenes strain LO28 serotype 1/2c [65] (kindly provided by Thomas Decker, Vienna, Austria) and EGDe serotype 1/2a [66] was grown in Brain Heart Infusion (BHI) infusion or on BHI-plates (Difco, Detroit, MI) at 37uC overnight. The next day, suspensions were diluted and grown until reaching log-phase. Bacteria were then centrifuged, washed several times and resuspended in sterile PBS. Concentrations of bacteria were determined by measurement at OD 600 and confirmed by plating serial dilutions on appropriate agar plates.
For the determination of the enzymatic activity of luciferase, cells were lysed in Reporter Lysis Buffer (Promega). For luciferase activity assays from tissue, weight of tissue fragments was determined and fragments were homogenized in proportional volumes of Reporter Lysis Buffer using Lysing Matrix A on a FastPrep-24 (MP Biomedicals). Lysates were mixed with LARII (Promega) and measured in a luminometer (Berthold). For in vivo imaging, mice were injected i.v. with 150 mg/kg of D-luciferin (Synchem) in PBS, anesthetized using Isofluran (Baxter) and monitored using an IVIS 200 imaging system (CaliperLS). Photon flux was quantified using the Living Image 3.2 software (CaliperLS).
Sera of infected mice were collected and levels of IFN-b and total IFN-a were measured by ELISA according to manufacturer's protocols (PBL InterferonSource).
Mice were infected intravenously (i.v.) with Listeria monocytogenes LO28 or EGDe strains. 5610 5 CFU/mouse is lethal dose (256LD 50 ), mice start dying at day 2-3 after infection. 2610 3 CFU/mouse is a sublethal dose (0.16LD 50 ), all mice are able to clear the bacterial around day 7 after infection. For determination of bacterial loads, spleens, livers and lymph nodes of sacrificed mice were removed and homogenized in 1 ml PBS supplemented with 0.2% NP-40. Serial dilutions of homogenates were plated on BHI agar plates and colonies were counted after overnight incubation at 37uC.
Plasmacytoid dendritic cells and granulocytes were depleted in vivo by intravenous injection of 100 mg anti-mPDCA-1 mAb (clone JF05-1C2.4.1, functional grade; Miltenyi Biotec) or 10 mg anti-Gr1 mAb (clone RB6-8C5) 24 hours prior Listeria monocytogenes infection. As an isotype control, rat IgG (Jackson ImmunoResearch Laboratories, Inc.) was used. Successful depletion of pDCs and granulocytes was determined by FACS analysis 24 hours after infection. Figure S1 Overview of transgenic mice used in this work. The generation of the global IFN-b reporter mouse has been previously described [30] and is since then maintained independent of cre expression. In brief, the targeted locus contains a luciferase gene (dark blue arrow) with a preceding polyA signal (black box) to avoid unspecific reporter activity. Two loxP sites (black arrowheads) allow cre-dependent replacement of the ifnb coding sequence by the luciferase reporter (light blue arrow) which is then driven by the endogenous ifnb promoter. In all reporter mice we keep one wt ifnb allel to allow IFN-b expression. Tissue specific reporter mice were obtained from breeding IFN-b floxb-luc/floxb-luc mice with mice expressing cre (red arrow) in a cell type specific manner. Cre activity (light red) in the given cell population (within the red circle) then allows ifnb promoter dependent reporter activity while the reporter cannot be activated in cells without cre expression (dark red). Tissue specific knockout mice carry two cre dependent alleles. Therefore in cells expressing cre both ifnb coding sequences are deleted. However, in cells without cre expression the IFN-b production is normal despite the genomic alterations introduced into the locus.  Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast Although first discovered in viruses, previous studies have identified operational −1 ribosomal frameshifting (−1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast −1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five −1 RF signals. Ablation of the −1 RF signals or of NMD stabilizes this mRNA, and changes in −1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous −1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that −1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence −1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that −1 RF is a broadly used post-transcriptional regulator of gene expression. Programmed ribosomal frameshifting (PRF) is has historically been associated with the study of viruses. PRF signals stochastically redirect ribosomes into new reading frames and viral PRF promotes synthesis of C-terminally extended fusion proteins. The most well defined PRF signals direct ribosomes to slip by one nucleotide in the 5 0 (À1) direction. À1 PRF signals typically contain three elements: a 'slippery site' composed of seven nucleotides (X XXY YYZ, incoming zero-frame indicated by spaces) where shifting occurs; a short spacer sequence and a downstream stimulatory structure, typically an mRNA pseudoknot (1) (2) (3) . Current models posit that the pseudoknot directs ribosomes to pause with their aminoacyl-(aa-) and peptidyl-tRNAs positioned over the slippery sequence, where re-pairing of the non-wobble bases of both tRNAs with the À1 frame codons occurs (4) (5) (6) (7) .
It is now clear that PRF is employed by organisms representing every branch in the tree of life, suggesting an ancient and possibly universal mechanism for controlling the expression of actively translated mRNAs (8) . The past few years have witnessed several reports describing in silico identification of recoding signals using a variety of computational approaches (9) (10) (11) (12) (13) (14) (15) (16) . While the methodologies of each study covered a broad range of bioinformatics techniques, the general goal with the exceptions of (9, 15) was to first find out-of-frame ORFs followed by the identification of PRF signals in the overlapping region between them. While this can identify new classes of PRF signals, it is based on the assumption that PRF outcomes should mimic those observed in viral genomes and thus cannot identify new operational outcomes of frameshifting.
While 'outcome-neutral' approaches using mRNA motifs known to promote efficient PRF cannot identify new classes of frameshift signals, they enable an expansion of our understanding of operational uses for PRF. The seminal study in this field searched the yeast genome for operational À1 ribosomal frameshift (À1 RF) promoting motifs resembling well characterized examples of viral À1 RF signals, identifying $260 putative such elements (9) . This work was limited by incomplete annotation of the yeast genome and insufficient computational resources available at the time. New bioinformatics tools were subsequently developed and applied using faster and more robust computational platforms. The results showed that: pattern matching approaches coupled with a predictive method for folding RNA sequences provided a dramatic improvement in the results; À1 RF motifs are widespread in the genome of Saccharomyces cerevisiae and many have predicted secondary structures with statistically significant measures of free energy (15) . This analysis showed that $11% of yeast genes contain at least one high probability À1 RF signal. Furthermore, we demonstrated that nine putative À1 RF signals selected from a variety of S. cerevisiae genes/genome promoted efficient recoding in vivo. More recently, this bioinformatics protocol has been applied to additional genomes. Currently, more than 25 genomes have been analyzed and it appears that 8-12% of genes contain at least one potential À1 RF signal (see PRFdB at http://prfdb.umd.edu/) (17) .
A key finding was that the outcome and function of À1 RF differs significantly between the viral and 'cellular' contexts. In viruses, PRF controls the stoichiometries of structural versus enzymatic proteins (18) . In contrast, 'cellular' RF events redirect elongating ribosomes to premature termination codons, suggesting that À1 RF is used to control cellular mRNA abundance and stability through the nonsense-mediated mRNA decay (NMD) pathway. While PRF is required for the production of functional products, in the cellular context RF appears to operate in a different manner. Thus, in the current work, PRF is used to connote frameshift signals whose function is to produce C-terminally extended proteins with novel functions, while RF is used to refer to frameshift signals that operate to direct ribosomes to premature termination codons. A proof-of-principle experiment demonstrated that a viral À1 PRF signal can function as an mRNA destabilizing element and that mRNA destabilization required NMD (19) . Here, rapid degradation of a reporter mRNA through NMD is demonstrated for four cellular yeast À1 RF signals. Further, genetic evidence suggests that the presence of the RF-stimulating pseudoknot may promote mRNA destabilization through the no-go decay (NGD) pathway (20) . The EST2 gene, encoding the catalytic subunit of telomerase (21) , was used to delve deeper into the relationships between À1 RF and mRNA stability. The EST2 mRNA is destabilized by À1 RF primarily via NMD. Ablation of its five À1 RF signals resulted in stabilization of the EST2 mRNA, and an inverse correlation between À1 RF efficiency and EST2 mRNA steady-steady state abundance was observed.
Escherichia coli DH5a was used to amplify plasmid DNA. Transformations of E. coli were performed as described previously using the calcium chloride method (22) . Yeast cells were transformed using the alkali cation method (23) . Yeast strains used in this study are shown in Supplementary Table S1 . Yeast were grown on YPAD and synthetic complete media (HÀ) (24) . yRP2056, yRP2077 were kind gifts from R. Parker. YJB2659 (generously provided by Judith Berman) was sporulated and strains JD1276, JD1281, JD1287 and JD1288 were obtained by tetrad dissection.
Dual luciferase and mRNA stability plasmids have been previously described (15) . Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA, USA) and are shown in Supplementary  Table S2 . Computationally identified putative À1 RF signals were amplified from yeast genomic DNA using PCR using Oligonucleotide primers which terminated in a SalI restriction site at the 5 0 and BamHI at the 3 0 . The zero-frame dual-luciferase reporter plasmid (pJD375) along with the -1 RF signal containing dsDNA fragments were digested using these restriction enzymes and ligated together to generate endogenous -1 RF signal containing dual-luciferase vectors. Oligonucleotide primers were chosen to terminate in KpnI restriction sites and amplify 41 and 30 bases of Renilla and firefly luciferase derived sequences respectively. The resulting amplicons were cloned into the KpnI site 492 bases into the PGK1 open reading frame of the unmodified PGK1 containing vector (pJD741). A premature termination codon vector (pJD828) was generated by cutting the readthrough (pJD753) with BamHI and backfilling with Klenow fragment. Plasmids so generated are described in Supplementary Table S3 .
Full length EST2 in a centromeric plasmid and the diploid S. cerevisiae EST2 deletion strain were generously provided by the Berman lab and have been previously described (25) . Individual mutant strains were obtained by tetrad dissection. Five potentially significant À1 RF signals were identified in the EST2 open reading frame using the Predicted Ribosomal Frameshift Database (17) . The wobble bases of five slippery heptamers were mutagenized to synonymous codons by oligonucleotide site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene). Oligonucleotide design and reaction conditions were performed as recommended by the manufacturer with minor modifications. All mutations were confirmed by sequencing. Plasmids so generated are described in Supplementary Table S3 .
Steady state and time course RNA blot analyses of PGK1 harboring endogenous À1 RF signals mRNA stability vectors were transformed into wild-type yeast (JD1158), upf1D or upf2D (JD1181 or JD1367), xrn1Á (JD1170), dcp1Á (JD1122), ski2Á (JD1345), ski3Á (JD19) and dom34Á (JD1363) cells. The EST2 mRNA stability vector (pJD754) was transformed into rpb1-1 (JD977) and rpb1-1/Upf À (JD978) cells and time courses were performed as described previously (26) . Total RNA was extracted with acid phenol/chloroform (pH = 4.5) from mid-logarithmic cell cultures (27) , or with Trizole ß Reagent following the manufacturer's directions (Invitrogen, Carlsbad, CA, USA). RNA (northern) blotting was performed as previously described (19) . Equal amounts of RNA (1, 2 or 4 mg) were separated through 1% agarose-formaldehyde gels. RNA samples were transferred and UV cross linked to Hybond-N-membranes (Amersham). Blots were hybridized with g[ 32 P] 5 0 -end-labeled oligonucleotides specific for U3 snoRNA (loading control) and the exogenous Renilla fragment (experimental). Messenger RNAs were identified using a GeneStorm phosphoimager (Bio-Rad) and quantified using QuantifyOne (Bio-Rad). Each experiment was repeated three or more times and averaged to generate graphs. Error bars for calculations including ratios of ratios may be approximated using either of the two following calculations. 'Average Ratio' is defined as the ratio of the two calculated values. Value ctrl is the value of the control (the denominator of the ratio) while Stdev ctrl is the calculated standard deviation of the control. Similarly, value exp and stdev exp are the value and standard deviations of the experimental (the numerator of the ratio). Rep ctrl and rep exp are the number of replicates performed for the control and experimental respectively. The error bars in the graphs of ratios of ratios use the approximated standard error.
Full length EST2 expression vectors (pJD641), EST2 mutant vectors (pJD796) and null plasmids (pJD315) were transformed into WT (JD1281), EST2 deletion (JD1287), UPF2 (JD1288) and EST2/UPF2 (JD1276) deletion strains. Total RNA was extracted with acid phenol/chloroform (pH = 4.5) from mid-logarithmic cell cultures. In parallel, total RNAs were extracted from isogenic rpl3D strains expressing wild-type RPL3 (JD1228), the down-frameshifting rpl3-R247A allele (AM-L3R247A), or the up-frameshifting rpl3-W255C/ P257S allele (JD1229). To prevent amplification from contaminating cellular DNA, RNA was treated with DNase I before reverse transcription using Turbo DNase (Ambion). cDNA was generated using the Bio-Rad iScript cDNA synthesis kit and used in the LightCycler real-time PCR system. PCR reactions were performed with 2 ml of cDNA in 20-ml reaction mixtures containing $10 nM each sense and antisense primer, and 1x LightCycler 480 SYBR Green I Master Mix (Roche). PCR cycles were run as follows: 1 cycle of 95 C for 10 min; 40 cycles of 95 C for 10 s, 54 C for 20 s and 72 C for 20 s. U3 snoRNA was chosen as a reference gene.
The SPR6, EST2, BUB3 and TBF1 orthologs from the genomes of S. paradoxus, S. mikatae, S. bayanus, S. castellii, S. kudriavzevii and S. kluyveri were extracted from the Yeast Gene Order Browser (http://wolfe.gen.tcd .ie/ygob/) (28) . Orthologs were identified for all genes. The nucleotide sequences were analyzed for the presence of potential À1 RF signals as previously described (15, 17) . Results are compiled in Supplementary Table S4 .
Cellular À1 RF signals are mRNA destabilizing elements
Four operational yeast cellular À1 RF signals derived from the BUB3, EST2, SPR6 and TBF1 genes were employed to test the hypothesis that À1 RF signals function as mRNA destabilization elements. The slippery heptamers for these À1 RF signals begin at nucleotides 858, 1653, 279 and 1521 of their respective ORFs. These were cloned into a yeast PGK1 reporter gene so that frameshifted ribosomes are directed to PTCs. All inserts were flanked by sequences derived from Renilla and firefly luciferase genes, providing unique exogenous sequences for specific detection of the reporter mRNAs. Two additional PGK1 reporters without À1 RF signals were used as controls: a readthrough reporter encoded a continuous ORF, while a PTC control contained an in-frame UAA termination codon ( Figure 1 ). Reporters were introduced into wild-type yeast cells; their steady state mRNA abundances were determined by RNA blot analysis and normalized to U3 snoRNA controls ( Figure 2) . A Figure 1 . Schematic of PGK1 reporter vectors used to monitor the effects of À1 RF signals on mRNA stability. The indicated Renilla and firefly luciferase derived sequences from pJD375 were cloned into the unique KpnI restriction site in a high copy PGK1 expression vector to create the readthrough control (pJD753). The indicated À1 RF signals derived from BUB3, EST2, SPR6 and TBF1 were cloned into SalI/BamHI digested pJD753. Colored arcs depict computationally predicted base-paired stems (17) . The premature termination control (PTC) was constructed by mutagenizing pJD753 to create an in-frame TAA codon. minimum of three independent blots were performed for all experiments. We note that the blots shown in Figure 2 are simplified for the purpose of publication and that the strains are not all isogenic with one another. In contrast, the bar graphs shown in Figure 2 represent data summarized from multiple blots using isogenic strains.
In wild-type cells, all four of the cellular À1 RF signals and the in-frame PTC containing control were less abundant than the PGK1 reporter mRNA (Figure 2A ). The decrease in mRNA steady-state abundance varied from $100-fold of the readthrough control (EST2) to $0.19-fold of wild-type (TBF1). Experiments were also performed in upf1D and dom34D strains, and the U3 snoRNA-normalized signal intensities were compared among the same signals between wild-type and mutant strains to determine the relative contributions of NMD and NGD on steady-state abundance of the À1 RF signal-containing reporters ( Figure 2B and C). The PTC containing mRNA was only affected through the NMD pathway: 28-fold increased abundance in upf1D cells relative to wild-type cells, but no change in dom34D cells. The TBF1 À1 RF signal similarly affected the reporter signal only through NMD ($4-fold).
In contrast, the steady-state abundance of the EST2 and BUB3 À1 RF signal-containing reporter mRNAs were increased in both the upf1D and dom34D mutants: the EST2 signal was 35-fold less effective in decreasing mRNA abundance in upf1D cells when compared to the WT strain and $14-fold less effective in dom34D cells, while the values for the BUB3 signal were $7-fold and 8-fold, respectively. The steady-state abundance of the SPR6 À1 RF signal containing reporter mRNA was primarily increased in dom34D cells ($2.0-fold). Deletion of DCP1, XRN1 and SKI3, all of which function downstream of UPF1 or DOM34, also generally increased the abundance of the reporter mRNAs ( Figure 2D -F). We note however that, in the case of the dcp1D cells, the reporter mRNAs were relatively abundant, most likely because of the presence of the 3 0 ! 5 0 exonuclease activity of the exosome, and/or due to decapping activity contributed by other factors, e.g. by the presence of the L-A virus (29) . These results establish that endogenous cellular À1 RF signals can decrease mRNA steady-state abundance in yeast through the NMD pathway. In addition, the data are consistent with the hypothesis that a subset of these signals may also affect mRNA abundance through NGD, although substantiation of this claim requires further studies, e.g. to monitor the abundance of the endonucleolytic cleavage products and mRNA stability assays in NGD À strains.
The EST2 À1 RF signal at nucleotide 1653 is primarily destabilized by À1 RF induced NMD Figure 2 suggests that À1 RF induced NMD is the major cause of decreased mRNA steady-state abundance by the EST2 À1 RF signal beginning at nucleotide 1653. To confirm this, a series of time course mRNA decay assays were performed employing the PGK1-EST2 À1 RF reporter, the readthrough control, and the PTC containing construct in cells harboring the temperature sensitive rpb1-1 allele of RNA polymerase II. At the zero time point, cells were shifted to the non-permissive temperature (42 C) to arrest transcription of mRNAs, total cellular mRNAs were extracted at 0, 1, 2, 4, 8 and 16 min. subsequent to the temperature shift, and RNA blots were hybridized with the firefly luciferase and U3 snoRNA probes. While the readthrough control was stable in wild-type cells ( Figure 3A and D) , both the PTC containing control and the reporter containing the EST2 À1 RF signal promoted rapid exponential decay of the reporter mRNA, thus demonstrating that this À1 RF signal can operate as an mRNA destabilizing element ( Figure 3B-D) . In a parallel experiment using rpb1-1 upf1D cells, all of the reporter mRNAs remained stable ( Figure 3E-H) . The rapid decay kinetic profile of the EST2 À1 RF containing reporter, and its stabilization in NMD-deficient cells are consistent with NMD being the major decay pathway triggered by this element (19) . To independently test of this, the A AAA AAT slippery site was partially inactivated by mutating it to G AAG AAC. This silent mutation stabilized the reporter mRNA $19-fold compared to the wild-type slippery site in wild-type, i.e. DOM34 cells ( Figure 3I ). Interestingly, this is less than the 35-fold stabilization in upf1D cells. One would expect that, since NMD is dependent of À1 RF, then inactivation of À1 RF should be quantitatively the same as inactivation of NMD. To address this, the steady state abundance of the G AAG AAC slippery site containing PGK1 reporter was assayed in an isogenic dom34D strain ( Figure 3I, dom34D lane) . This combination increased the steady-state abundance of the reporter mRNA to near wild-type levels.
Ablation of À1 RF signals increases the steady-state abundance of the yeast EST2 mRNA, and À1 RF efficiency inversely correlates with EST2 mRNA abundance
The EST family of yeast genes is named after their 'Ever Shortening Telomere' phenotype (30) . EST2 encodes the catalytic subunit of telomerase and the other three EST genes either encode protein subunits of telomerase (EST1 and EST3) or a telomere-associated regulator of telomerase (CDC13/EST4) (31) . Telomere elongation occurs in late S phase, although Est2p is associated to varying extents with telomeric chromatin throughout the cell cycle, and telomerase defects result in chromosome instability and rapid senescence (32) . The very low abundance EST2 mRNA is stabilized in NMD-deficient cells (33, 34) . Computational analyses revealed that EST2 contains four additional high confidence À1 RF signals beginning at positions 72, 1215, 1326 and 1995 (Supplementary Figure S1 ). The positions of the five predicted À1 RF signals in the EST2 ORF are shown in Figure 4A . Silent protein coding changes were introduced into the slippery sites of all 5 of the À1 RF signals in a full-length EST2 clone expressed from a low copy vector (pEST2ssÁ, Figure 4A ). Clones expressing either wild-type EST2 (pEST2) or pEST2ssÁ were introduced into isogenic est2D or est2D upf1D cells, and qRT-PCR analyses were performed. These silent mutations resulted $8.5-fold increase in the abundance of the full-length EST2ssÁ mRNA relative to wild-type EST2 mRNA ( Figure 4B) . Similarly, abrogation of NMD increased the abundance of the wild-type EST2 and EST2ssÁ mRNAs $5.8-fold and $7.0-fold, respectively. To independently monitor the influence of À1 RF on mRNA abundance, the steady-state abundance of the EST2 mRNA was monitored in isogenic cells expressing up-and down-frameshift promoting alleles of RPL3 (which encodes ribosomal protein L3) by qRT PCR. EST2 mRNA abundances were normalized to U3 snoRNA in cells expressing wild-type RPL3, the rpl3-R247A allele which decreases À1 RF from the L-A frameshift signal to $55% of wild-type levels (35) , and the rpl3-W255C/P247S allele which increases À1 RF by $1.6-fold (36) . Relative to wild-type cells, steady-state abundance of the EST2 mRNA was increased by 1.29 ± 0.04 fold in cells expressing rpl3-R247A, and decreased to 0.55 ± 0.02 in cells expressing rpl3-W255C/P247S ( Figure 4C ). Taken together, these experiments demonstrate that À1 RF induced NMD plays a significant role in destabilizing EST2 mRNA.
Programmed À1 ribosomal frameshifting, but not specific À1 RF signals appears to be conserved and rapidly evolving in budding yeasts If regulation of gene expression through À1 RF is biologically significant, then À1 RF signals should be present in orthologous mRNAs from other budding yeast species. To address this, the BUB3, EST2, SPR6 and TBF1 orthologs were identified in S. paradoxus, S. mikatae, S. bayanus, S. castellii, S. kudriavzevii and S. kluyveri, and analyzed for potentially significant À1 RF signals as previously described (15) . At first glance, Positions of the slippery sites of five predicted À1 RF signals and their sequences are indicated. The full-length gene including native 5 0 and 3 0 UTR sequences were cloned into a low-copy yeast vector to create pEST2. Silent coding mutations that are predicted to inactivate À1 RF were introduced to produce pEST2ssÁ. (B) pEST2 or pEST2ssÁ were introduced into est2D or est2D upf1D cells and EST2 mRNA steady state abundances were determined by quantitative real-time PCR. (C) Quantitative real-time PCR was used to monitor steady-state abundance of the endogenous EST2 mRNA in isogenic cells expressing three different forms of ribosomal protein L3: Wild-Type RPL3 (WT); the R247A mutant which promotes decreased rates of À1 RF and the W255C/P257S mutant which promotes increased À1 RF efficiency. EST2 mRNA abundances were normalized to U3 snoRNA abundance for each sample, and the values shown are relative to wild-type cells.
these analyses reveal that no single À1 RF signal is completely conserved among the budding yeasts (Supplementary Table S4) . However, closer analysis shows that strong candidate À1 RF signals can be identified in the orthologs of all of these genes, although not in every species. For example, as noted above, the S. cerevisiae EST2 mRNA contains five potential À1 RF signals. Similarly, the S. paradoxus ortholog contains five potential À1 RF signals, although none are identical to the S. cerevisiae elements. S. mikatae EST2 appears to harbor two potential À1 RF signals, S. bayanus has three, and S. castelli contains two, and S. kluyveri has three. None were identified in the S. kudriavzevii EST2 ortholog. Turning to SPR6, the S. cerevisiae mRNA contains a second potential À1 RF signal beginning at nucleotide 348 in addition to that identified beginning at nucleotide 279 (see http://cbmgintra.umd.edu/prfdb/index .cgi/detail?id=1755&accession=SGDID:S0000917& slipstart=348). Both the S. paradoxus and S. kudriavzevii SPR6 orthologs contain three potential À1 RF signals, but none were identified in the S. mikatae S. bayanus or S. castelli orthologs. Interestingly, the S. kluyveri SPR6 ortholog contains a slippery site followed by a strong stem-loop structure; while this may or may not constitute a À1 RF signal, it does suggest the presence of a rapidly evolving cis-acting element (see Discussion below). S. cerevisiae BUB3 contains the operational À1 RF signal at nucleotide 858, plus potential À1 RF signals beginning at nucleotides 27 and 732. The orthologous mRNAs in S. paradoxus, S. bayanus, S. castelli and S. kudriavzevii each appear to have one potential À1 RF signal, but none were identified in S. mikatae or S. kluyveri. Lastly, the S. cerevisiae TBF1 mRNA has only the single confirmed À1 RF signal. The S. castelli, S. kudriavzevii and S. kluyveri orthologs contain two each, and the S. mikatae has one. No potential À1 RF signals were identified in either S. paradoxus or S. bayanus. As a control, six S. cerevisiae genes lacking predicted À1 RF signals were selected (PGK1, HHT1, TEF2, MIC14, CMD1 and GRX1), orthologs from the six other yeast species identified, and these were in turn queried for the presence of putative À1 RF signals. These analyses revealed that none of the orthologs of these six genes contain predicted À1 RF signals (Supplementary Table S5 , and hyperlinked data therein). The potential evolutionary significance these observations are discussed below.
In a prior proof-of-principle experiment, we utilized the well characterized À1 PRF signal from the yeast L-A dsRNA virus to demonstrate that these elements can generally function as mRNA destabilizing elements through the NMD pathway (19) . Subsequently, a bioinformatics approach was used to determine that potential À1 RF signals are widely found in all genomes examined, and that the great majority of these are predicted to direct elongating ribosomes to premature termination codons (15, 17) . Here, we show that these chromosomally encoded, endogenous À1 RF signals can also function as cis-acting mRNA destabilizing elements, both in the context of a reporter mRNA, and also in one case in a natural context. Further, we demonstrated that À1 RF signals can differentially affect mRNA abundance through the NMD pathway, and the data are also consistent with destabilization through NGD. These are modeled in Figure 5 . In support of this idea, the EST2, BUB3 and SPR6 mRNAs were all stabilized in upf1D, upd2D/nmd2D, upf3D, dcp1D and xrn1D cells (37) , and the half-lives of these mRNAs were less than the mean in wild-type cells (38) . Interestingly, TBF1 is not represented in these databases. In the case of a ribosome shifting reading frame into a PTC, the surveillance complex led by the Upf proteins signals rapid decapping by Dcp1p/Dcp2p, followed by deadenylation and exonucleolytic decay via Xrn1p and the exosome. In parallel, the NGD pathway can be activated by ribosomes that are stalled at strong secondary structures in mRNAs. Stalled ribosomes are freed from mRNAs by Dom34p/Hbs1p, promoting exonucleolytic cleavage at unpaired nucleotides near the pause, thus resulting in two mRNA fragments which become substrates for decapping and exonucleolytic decay [reviewed in (39) ]. The findings presented here suggest that cells are not only well equipped to deal with aberrant messages which contain premature termination codons and to clear stalled ribosomes from mRNAs, but have also evolved to capitalize upon these functions to post-transcriptionally regulate gene expression.
The strength of these signals to function as mRNA destabilizing elements should be equal to a combination of (i) their strengths as À1 RF signals and (ii) their abilities to block ribosome progression, i.e. their thermodynamic stability. The EST2 signal is both highly efficient at promoting À1 RF [$55%, see (15) ], and is predicted to be quite stable ($À27 to À24 kcal/mol depending on the particular folding solution, see http:// prfdb.umd.edu/). It is important to note however that the software used to predict mRNA pseudoknots can neither identify base triples, which make major contributions to frameshifting (40) (41) (42) (43) (44) , let alone calculate their contributions to thermodynamic stability. Regardless, this combination of high frameshifting and thermodynamic stability results in very strong destabilization via NMD ( Figure 2B ), and perhaps NGD as well ( Figure 2C ). As discussed previously (19) , the exponential decay profile suggests that NMD can occur beyond the 'pioneer round' of translation.
In contrast to EST2, the TBF1 signal promoted $5% frameshifting (15), but is not predicted to be highly stable (À9.5 kcal/mol). Thus, all of its mRNA destabilization activity was through NMD (compare Figure 2B with C). The thermodynamic stability of the BUB3 signal is predicted to have an intermediate value to EST2 and TBF1 ($À12 kcal/mol), and hence the potential contribution of NGD to the stability of its reporter was significant. Interestingly, this signal only promoted $1% frameshifting (15) , yet the contribution of NMD to its destabilization was greater than observed for TBF1. One possible explanation for this apparent discrepancy may stem from the fact that, in order to measure frameshifting, one base had to be deleted from the spacer region between the slippery site and the stimulatory pseudoknot. Changes in the length and composition of this spacer are known to affect rates of À1 PRF (45) , and thus the À1 RF values so determined cannot be taken as absolute. In contrast, the reporters used to monitor mRNA stability contained the native sequences. In light of this, it is likely that the native BUB3 À1 RF signal promotes more frameshifting than the TBF1 signal. Lastly, the SPR6 À1 RF signal is predicted to be quite stable ($À20 kcal/mol), yet promoted very low levels of frameshifting ($0.5%) (15) . Accordingly, destabilization via NMD was negligible for this element, while NGD appeared to be the major contributor.
Beyond the pro forma demonstration that À1 RF signals can decrease cellular mRNA abundance, it is important to begin to understand the biological function of this phenomenon. As a first step in this direction, we showed that silently mutating the slippery sites in 5 predicted À1 RF signals within a full-length clone of EST2 significantly stabilized its encoded mRNA ( Figure 5B) . Similarly, abrogation of NMD stabilized this message, while changes in À1 RF efficiency inversely correlated with EST2 steady-state mRNA abundance. Est2p is the reverse transcriptase subunit of the telomerase holoenzyme (21) . Interestingly, prior studies have demonstrated that this mRNA, along with other mRNAs encoding proteins having telomere-associated functions, are stabilized in NMD À yeast cells (33, 37) . Analysis of the Programmed Ribosomal Frameshift Database (http:// prfdb.umd.edu/) reveals that, along with the other four putative À1 RF signals in the EST2 mRNA, the mRNAs encoding Est1p, Stn1p, Cdc13p and Orc5p, all components or regulators of telomerase that are stabilized in NMD À cells, also contain high confidence À1 RF signals (Supplementary Figure S2 ). In addition, the EST3 mRNA contains a +1 PRF signal (46) . Intriguingly, telomerase is limiting in cells: while a yeast cell contains 64 chromosome ends, there are only $29 telomerase molecules per cell, and telomerase is preferentially recruited to short telomeres (47) . Additionally, Tbf1p is a telobox containing general regulatory factor that binds to TTAGGG repeats within subtelomeric anti-silencing regions (48) . Intriguingly, ablation of NMD (49) or overexpression of single components of telomerase-associated proteins, i.e. the TEL1 RNA, Est2p, Stn1p or Cdc13p resulted in changes in telomere length (47, 50, 51) .
We hypothesize that yeast cells use À1 RF to limit the expression of these proteins in order to maintain the correct stoichiometric balance among telomere associated components. Corollary to this, mutations that alter À1 RF and/or NMD should affect telomere function, and should thus show phenotypic defects similar to those observed in telomerase mutants, e.g. cell cycle progression defects. Indeed, we have isolated numerous such mutants [reviewed in (52) ], and have reported that the mof2-1 and mof5-1 mutants, which affect both NMD and À1 RF tend to accumulate large mother-daughter cells, and/or multiply budded cells, typical of G2/M cell cycle defects (53) . Similarly, upf1D cells have abnormally elongated buds, and decreased telomere lengths (54, 55) . Intriguingly, mof6-1 mutants, which only affect À1 RF, arrest as large, unbudded cells, typical of M-phase exit defects (53) . These observations suggest that stabilization of the mRNAs encoding multiple telomere-associated proteins may have dominant negative effects on telomere homeostasis, and that NMD and À1 RF may regulate different aspects of the cell cycle. Additionally, the central role of Bub3p at the mitotic cell cycle spindle assembly checkpoint and the progeroid phenotypes caused by Bub3p deficiency (56) suggest a more general role for À1 RF in control of cell growth and division. Lastly, the expression of Spr6p during sporulation (57) suggests a role for À1 RF in this developmental process as well. Future studies will dissect the roles of the À1 RF signals in these mRNAs.
Finally, if À1 RF is widely used to regulate gene expression, then it should be well conserved. The major problem associated with attempting a phylogenetic analysis of À1 RF signals is the inherent limitations of the software used to predict them. In short, it is not well enough developed to automatically identify matching motifs. In an attempt to begin to address this issue, the orthologous Bub3p, Est2p, Spr6p and Tbf1p's in six closely related yeast species were identified, their nucleotide sequences extracted from the Yeast Gene Order Browser (28) , and analyzed for the presence of potential À1 RF signals. These analyses revealed that while specific À1 RF signals do not appear to be evolutionarily conserved, À1 RF itself may be relatively well-enough conserved as a mechanism to post-transcriptionally regulate the expression of these genes across many but not all species examined (see Supplementary Table S4 ). Importantly, the control experiment showed that putative À1 RF signals were not detected in any of the orthologs of six S. cerevisiae genes that themselves were not predicted to contain À1 RF signals. These observations are in agreement with current evolutionary theory based on analyses of 5 0 UTR sequences of Drosophila species proposing that rapid rates of mutation in cis-acting regulatory elements drives speciation because they confer very specific effects on gene expression, as opposed to mutations affecting protein structure, the pleiotrophic effects of which impose very high penalties on fitness (58) (59) (60) . The findings presented in this work, i.e. that while À1 RF signals are not conserved in orthologs from other yeasts, these ORFs contain generically contain À1 RF signals, and that the absence of À1 RF signals in other orthologous ORFs, agrees very well with this theory of molecular evolution. Time to revise the paradigm of hantavirus syndromes? Hantavirus pulmonary syndrome caused by European hantavirus Hantaviruses have previously been recognised to cause two separate syndromes: hemorrhagic fever with renal syndrome in Eurasia, and hantavirus pulmonary syndrome (HPS) in the Americas. However, increasing evidence suggests that this dichotomy is no longer fruitful when recognising human hantavirus disease and understanding the pathogenesis. Herein are presented three cases of severe European Puumala hantavirus infection that meet the HPS case definition. The clinical and pathological findings were similar to those found in American hantavirus patients. Consequently, hantavirus infection should be considered as a cause of acute respiratory distress in all endemic areas worldwide. The present paradigm is that hantaviruses in the Americas cause hantavirus pulmonary syndrome (HPS), while hemorrhagic fever with renal syndrome (HFRS) is caused by hantaviruses present in Eurasia [1] [2] [3] . Hantaviruses associated with HFRS and HPS are primarily transmitted by inhalation of viral particles shed by infected rodents [4, 5] . In both syndromes there is a local immune reaction in the lungs, mainly in terms of a CD8 + T lymphocyte response [2, 6, 7] . Another important feature is endothelial dysfunction and capillary leakage [1] [2] [3] 8] . After the prodromal phase, including fever, nausea, myalgia and headache, patients with HFRS commonly develop renal failure whereas in HPS the kidneys are often spared and instead the patient frequently presents with severe cardiopulmonary dysfunction [1, 3, 9] .
Although usually less severe and sometimes overlooked, pulmonary symptoms are common in European HFRS caused by Puumala virus (PUUV). Frequent clinical findings include cough, dyspnoea, interstitial lung infiltrates, pleural effusion and impaired pulmonary function [10] [11] [12] . Pronounced lung involvement in HFRS has previously been reported [13] [14] [15] [16] . However, none of those reported patients had a fatal outcome that could be attributed to the acute infection, and there is no description of histopathological findings.
Our hypothesis is that also European hantaviruses can cause HPS. The aim of this study was to investigate whether HFRS patients with severe cardiopulmonary distress fulfil HPS criteria and have similar clinical and pathological features.
In a prospective study, during the latest hantavirus outbreak in 2007 in the county of Västerbotten, Sweden [17] , HFRS patients with cardiopulmonary failure and need of invasive assisted ventilation, i.e. intubation and mechanical ventilation, were selected. We identified three patients, two of which had fatal outcome and one survived. The two patients who died both underwent autopsy three days post-mortem. Organ samples were investigated using immunohistochemistry to describe the immunological response and detect presence of viral antigen. Quantitative real-time RT-PCR was used to detect viral RNA in organ samples, plasma and bronchoalveolar lavage [18] .
For defining HPS we used the case definition criteria published by the U.S. Centers for Disease Control and Prevention (CDC) [19] . The project was approved by the Research Ethics Committee of Umeå University. Informed consent was obtained either from the patient, or a close relative when not possible.
A 73-year-old woman was admitted to the intensive care unit (ICU) with acute respiratory distress. She was a lifelong non-smoker living with her husband in a rural area, and had a medical history of hypertension and type II diabetes. The day prior to admission she fell ill with malaise and fever. Nausea, vomiting and pronounced shortness of breath ensued. The initial findings at admission included fever, tachycardia, tachypnoea, hypoxia and somnolence. Laboratory findings during hospitalisation indicated coagulopathy, elevated levels of lactate dehydrogenase (LDH), and development of renal failure (Table 1 ). There was release of cardiac enzyme troponin T (peak value 1.6; normal value <0.01 μg/L), indicating myocardial tissue damage.
Lung computer tomography (CT) on admission revealed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, dependant atelectasis, and moderate pleural effusions (Fig. 1 ) which were later drained (>800 ml). Echocardiography showed inferior hypokinesia, moderate mitral valve insufficiency, normal sized left ventricle and atrium, and systolic pulmonary arterial pressure estimated at 55 mm Hg. Despite non-invasive positive pressure respiratory support and furosemide, her respiratory distress progressed and she was intubated and mechanically ventilated on the first hospital day. The preliminary diagnosis was acute respiratory distress syndrome of uncertain cause, and she was treated with broad-spectrum antibiotics and corticosteroids without improvement. Vasopressor and inotropic support was required to maintain adequate circulation. Repeated echocardiogram showed no overt signs of cardiac failure, though several days into her illness the systolic pulmonary arterial pressure increased to >65 mm Hg. Maximal inspiratory pressures (40 cm H 2 O) were required to maintain minimally adequate oxygenation. She suffered a pneumothorax, and received a large bore thoracostomy with negative pressure drainage. The patient developed multiple organ dysfunction engaging the central nervous, respiratory, cardiovascular, renal and coagulatory systems, and she died after 13 days of ICU care.
PUUV serology was initially negative, but seroconversion occurred during the first week with development of positive immunoglobulin M (IgM) and IgG. No PUUV RNA could be detected in serum or bronchoalveolar lavage fluid, sampled two days after onset of disease. At autopsy PUUV RNA was detected in lung tissue, but not in samples from heart, brain, spleen and liver. Sequencing of the PUUV RNA from lung tissue showed that it was homologous to PUUV strains circulating in northern Sweden. No PUUV antigen could be detected in the tissue by immunohistochemistry using PUUV specific monoclonal antibody. Relevant bacterial cultures were all negative. Notably, three weeks prior to ICU admission the patient had sought medical treatment at our clinic for a urinary tract infection caused by E. coli. In the serum collected at that time PUUV RNA (1,700 copies/ml) was later detected. WBC white blood cell count (normal range 3.5-8.8×10 9 /L), TPC thrombocyte particle count (normal range for women 165-387 and for men 145- Autopsy revealed oedematous and atelectatic lungs with no normal aerated tissue. Pulmonary histopathological features consisted of diffusely oedematous parenchyma with interstitial and intraalveolar fibrosis. The alveoli contained exudates of fibrinous fluid, high number of alveolar macrophages (CD68 + ), proliferative epithelial cells and thick septa but without characteristic hyaline membranes. Interstitial mononuclear cell infiltrates were common, consisting mainly of CD3 + T lymphocytes whereof a vast majority was CD8 + . Many mononuclear cells were expressing cytotoxic markers granzyme B and T cell restricted intracellular antigen-1, TIA-1 (Fig. 2) . In pulmonary vessels, focal thrombosis was evident. Microscopy of the heart showed thrombosis in small vessels and focal massive infiltrates of granulocytes and macrophages. In the brain there was focal vasculitis, perivascular infiltration of CD8 + T lymphocytes and non-occlusive thrombosis. Infarctions were seen in the brain, lungs and spleen. Notably, the kidneys had no prominent inflammatory infiltrates.
A 65-year-old woman was admitted to the ICU with acute respiratory distress and circulation insufficiency. Besides a history of Waldenstrom's macroglobulinemia that had not required active treatment, she had no history of health problems, infections or recent hospitalisations. She was a lifelong non-smoker, lived in a rural home with her husband, and handled firewood for home heating. Four days prior to admission the patient noted fever, chills, dyspnoea with dry cough and diarrhoea. On the day of admission, these symptoms were more severe, and an ambulance had been called because of syncope. Her ability to oxygenate deteriorated progressively during the first hospital day, and she was intubated and mechanically ventilated.
Chest X-ray on admission showed bilateral diffuse lung infiltrates and signs of interstitial oedema. Large bilateral pleural effusions were noted, and >1000 ml were drained. Echocardiographic examination identified normal left ventricular wall motion, and no signs of structural abnormalities or of pulmonary hypertension. Thin-cut CT images of the lungs on the third ICU day showed diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation (Fig. 1) . The clinical course during the first seven days was dominated by respiratory insufficiency requiring maximal ventilatory support with high levels of inspired oxygen, as well as circulatory shock requiring treatment with vasopressor and inotropic infusions. Other important clinical aspects included coagulopathy with diffusely spread petechiae, progression of renal failure with anuria requiring dialysis, and elevated levels of LDH (Table 1) . She was treated presumptively for bacterial pneumonia and sepsis with a series of broad spectrum antibiotics and corticosteroids, without apparent response.
Hantavirus infection was verified with the detection of PUUV RNA in plasma (630,000 copies/ml) on the day of admission, while IgM and IgG were negative. Seroconversion with positive IgM and IgG occurred two and seven days later, respectively. Consecutive plasma samples were analysed for PUUV RNA with declining viral copy numbers until negative 16 days post onset of Fig. 1 Chest CT-scans of two European patients with hantavirus pulmonary syndrome. The examination showed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, together with bilateral dependent atelectasis and moderate pleural effusions in patient 1 (a) and diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation in patient 2 (b) Fig. 2 Immunohistochemistry results in lung sections from two fatal cases of hantavirus pulmonary syndrome. The findings were condensed oedematous pulmonary parenchyma with alveolar fibrinous exudate and infiltrates of mononuclear cells (a), whereof a vast majority were CD8 + T lymphocytes (b), and many were holding granules containing granzyme B (c) and T cell restricted antigen-1, TIA-1 (d); this immunophenotype is characteristic of activated cytotoxic T lymphocytes. In contrast, CD4 + helper T lymphocytes (e) were uncommon. Viral antigen was detected in capillary vascular endothelium (f) and in mononuclear cells, here represented by a monocyte (f; inset), using Puumala hantavirus nucleocapsid protein monoclonal antibody (A1C5, Progen Biotechnik GmbH, Heidelberg, Germany). For viral antigen, lung samples from two non-hantavirus patients were used as negative controls (data not shown). Panels (Patient 1 in a-e; original magnification, x 400; and Patient 3 in f; original magnification, x 600) display lung sections from paraffin embedded material. Staining was performed using hematoxylin and eosin (a), and immunoperoxidase technique (b-f) disease (data not shown). PUUV RNA was found in bronchoalveolar lavage fluid (11,000 copies/ml) nine days after onset of disease. Bacterial cultures were all negative. The patient remained ventilator-dependent for 13 days, but was finally extubated. She developed critical illness myopathy and needed six weeks in-hospital rehabilitation. At follow-up six months later, she complained of muscle ache during exercise and lowered general fitness, but was steadily improving.
A 63-year-old male construction worker was admitted to the ICU with acute respiratory distress, confusion and hypotension. He was a current and long-time smoker with a medical history of mild chronic obstructive pulmonary disease and hypertension. Three days prior to admission he had fallen ill with fever, chills, diarrhoea, dry cough, and dyspnoea. In the emergency room, physical examination was notable for fever, somnolence, tachycardia and hypotension. Arterial blood gas analysis on room air revealed hypoxia and respiratory alkalosis. The patient was taken to the ICU and therapy was started with broad spectrum antibiotics due to suspicion of pneumonia and sepsis. As with the other patients, coagulopathy, increased LDH levels, and renal failure were detected (Table 1) .
Initial bedside chest X-ray showed no findings to explain the respiratory distress. During the two days ICU course, the patient complained mostly of dyspnoea. He received supplemental oxygen and non-invasive ventilator support initially, but was eventually intubated and mechanically ventilated. CT findings which included only the lower lung lobes revealed bilateral pleural effusions and bibasilar atelectasis (data not shown). During the first day blood pressure was maintained with i.v. fluid, but on the second day his circulatory condition rapidly deteriorated. Despite massive efforts with i.v. fluid, corticosteroids and vasoactive drugs, the patient died in refractory circulatory shock.
PUUV serology on the first hospital day was IgM positive, while IgG was negative. The patient had 130,000 copies of PUUV RNA/ml in plasma. Bacterial cultures were all negative. At autopsy, PUUV RNA was detected in samples from lungs, heart, liver, kidney, brain and spleen. The organs were examined for viral antigen, which was found in the vascular endothelium and in mononuclear cells (Fig. 2) .
Post mortem examination showed that pulmonary architecture was preserved but with oedema and focal non-occlusive thrombosis. Similar to patient 1, there were pulmonary infiltrates of lymphocytes with the same immunophenotype as described (data not shown). Kidneys showed no prominent inflammation.
Growing evidence show that there are similarities between HFRS and HPS, as both syndromes can give rise to hemorrhagia, renal impairment and cardiopulmonary dysfunction, which have been attributed to thrombocytopenia and capillary leakage [1, 3, 8, 9, 11, 20, 21] .
Severe pulmonary involvement has not been generally perceived to be a significant feature of HFRS. Previously, respiratory symptoms were commonly attributed to fluid overload as a result of renal failure. However, increasing evidence show that lung and heart involvement is common during the acute phase of HFRS [10, 11, 22, 23] . Concerning the cases of European hantavirus infection in our present report, there was only mild or no renal impairment at the time of admission, whereas the respiratory involvement was early and severe, consistent with acute respiratory distress syndrome (ARDS), fulfilling criteria of HPS according to CDC case definition [19] . As described in HPS, the three reported patients suffered from cardiovascular dysfunction, requiring treatment with inotropic and vasopressor drugs [9] . Autopsy results support the cause of death to be cardiopulmonary distress due to hantavirus infection. The lungs of both the deceased patients were oedematous and contained mononuclear cell infiltrates with predominantly CD8 + T lymphocytes, as described in HPS patients [2, 7] . Many cells expressed granzyme B and TIA-1, indicating a cytotoxic activity, which could further be supported by high levels of LDH. We chose to demonstrate the immune response in a non-smoker's lungs (patient 1), but a similar picture was seen in patient 3.
In the first two patients, the IgM response was delayed, while the quantitative real-time RT-PCR for PUUV RNA was positive, suggesting it as a valuable tool to hasten the aetiological diagnosis [18] . Notably, in patient 1 PUUV RNA was found in serum from three weeks prior to the acute onset of disease, that is, in incubation phase and consistent with hantavirus incubation time ranging from one to four weeks [3, 24] .
Pulmonary involvement is well documented in European Puumala hantavirus infection. From a single outbreak in our county, we report on three cases of life-threatening hantavirus pulmonary syndrome. Arguably, additional cases are likely to either go undiagnosed or unreported. A clinical implication of this observation is that hantavirus infection should be included as a differential diagnosis for patients with febrile illness and acute respiratory distress of uncertain cause in endemic areas in Eurasia.
Conflict of interest The authors declare that they have no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Antigen-Specific Monoclonal Antibodies Isolated from B Cells Expressing Constitutively Active STAT5 BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5). Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells. In general, vaccination is a safe and efficient way to protect the human body against specific pathogens. However, vaccination is only applicable as a preventive measure, and development of new vaccines is a slow and expensive process. As an alternative, the use of sera enriched for pathogen-specific antibodies has been suggested [1, 2] . Treatment with pathogen-specific antibodies could then be applied in a prophylactic as well as in a therapeutic setting. However, non-human derived sera often provoke an immune response, thereby limiting the maximum number of treatments. Other possible caveats are the fact that it is difficult to obtain large amounts of sera of the same quality, and the risk of contamination with pathogens, in particular with viruses such as but not limited to Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV).
An alternative is the use of monoclonal antibodies [3] . Mouse monoclonal antibodies such as OKT3 have been used to treat humans but with limited success due to the immune response these antibodies provoked. An alterative approach could be the use of fully human antibodies. For this, novel technologies have been developed, including humanization of mouse antibodies, phage display of human B cell libraries, single cell PCR technologies and the creation of mice that express human immunoglobulin genes [4, 5, 6, 7, 8, 9] .
All of these technologies have resulted in clinical relevant antibodies but most methods do not directly tap the potential of the human immune system. Indeed, the human immune system itself can safely be assumed to be the best in generating highly efficacious antibodies and these antibodies are most likely superior to those generated from mice or using phage display. In addition, such antibodies may have a better safety profile than antibodies derived from mice. Novel technologies to obtain monoclonal antibodies from human B cells include EBV transformation of antibody-producing B cells activated by TLR9-agonists [10, 11] and single cell PCR to obtain immunoglobulin genes from individual ex vivo isolated B cells [12, 13] . We have previously shown that with forced expression of BCL-6 in human B cells stable human monoclonal antibody secreting cell lines can be produced [14, 15] . We moved on to describe that ectopic expression of a constitutively active mutant of the transcription factor Signal Transducer of Transcription 5 (STAT5) in human memory B cells resulted in a differentiation block of activated B cells, preventing them to mature into plasma cells. STAT5 transduced cells resemble activated germinal center centrocytes and show enhanced survival and expansion [16] .
In the present paper we exploited this enhanced survival and expansion of human memory B cells that express an active form of STAT5 in order to obtain antigen specific immunoglobulin. We established a series of cloned lines of human B cells that expressed an inducible STAT5 construct. By turning off STAT5 the clones regained their capacity to produce antibodies allowing identification of clones that produced specific antibodies.
Previously we have published that ectopic expression of active STAT5 mutants in human primary B cells results in a block in B cell differentiation and that these cells show enhanced survival and expansion [16] . We extended these findings by showing that constitutive activation of STAT5 in B cells led to loss of antibody surface expression when cultures were maintained for more than 6 weeks in the presence of IL-2 and IL-4 [17] . We then investigated whether we could exploit the immortalizing capacity of active STAT5 mutants in order to obtain human monoclonal B cell lines, which secrete antigen specific antibodies. For this, peripheral blood CD27 pos memory B cells were cultured in the presence of irradiated CD40-Ligand expressing L cells (CD40L-L cells) and interleukin (IL)-21 prior to retroviral transduction with constitutively activated (CA) STAT5b (CA-STAT5b). Pre-stimulation with IL-21 induced strong proliferation, which resulted in transduction efficiencies of 40 to 80% [15, 18] . Following transduction, in-active CA-STAT5b is expressed as a chimera with the hormone-binding portion of the Estrogen Receptor (ER) (CA-STAT5bER) [16] , thereby STAT5b is expressed in the cytoplasm complexed by heat-shock proteins. Incubation with tamoxifen (4-HT) leads to dissociation of the heat shock proteins from constitutively active STAT5bER, which results in receptor dimerization and subsequent translocation to the nucleus where it initiates STAT5 specific transcription [16, 19] . First we studied the effect of STAT5 transduced cells cultured with IL-2, IL-4 and tamoxifen or with IL-21 and tamoxifen. IL-2 and IL-4 induce proliferation of human primary B cells similar to IL-21 but IL-21 has been shown to also induce strong differentiation of B cells [20, 21, 22, 23, 24, 25] . The secreted immunoglobulin levels in the supernatant of the CA-STAT5bER B cells were measured over time. Around 3 to 4 weeks after transduction the level of secreted IgG started to decrease for the tamoxifen/IL-2 plus IL-4 and tamoxifen/IL-21 cultured cells (Figure 1a ). We also determined the long-term effect of constitutively active STAT5 expression on IgM secretion. IgM expressing peripheral blood B cells were transduced with CA-STAT5bER and cultured with tamoxifen, IL-2 and IL-4. Similar to the IgG secretion levels, IgM secretion disappeared in CA-STAT5bER B cells after prolonged culture (Figure 1b) .
The decline in immunoglobulin secretion corresponded with a decline in surface immunoglobulin expression [17] . This shows that with time, constitutive expression of STAT5 in human B cells leads to diminished expression and secretion of immunoglobulins. We questioned whether these effects could be reversed by deactivation of constitutively active STAT5 through removal of tamoxifen from the culture. We cultured the cells with tamoxifen, IL-2 and IL-4 for weeks to months until immunoglobulin production had vanished and then removed tamoxifen from the culture. In these cultures, when treated with IL-2 and IL-4 immunoglobulin secretion remained low or absent ( Figure 2 ), but when they were treated with IL-21, IgM or IgG immunoglobulin secretion resumed (Figure 2 and Figure S1 ). The immunoglobulin secretion lasted for about 7 days ( Figure S2 ). After approximately 14 days antibody production came to an end and the transduced B cells died [16] . It has been shown previously that IL-21 induces differentiation of B cells into plasma cells, but this was not observed in our B cells when STAT5 was active [18, 24, 25] . Together these data support the idea that IL-21 (but not IL-2 in combination with IL-4) induces strong plasma cell differentiation, which can be prevented by activation of STAT5 [16, 18, 24, 25] . Interestingly, our B cell lines maintained the capacity to secrete immunoglobulin after prolonged culture (weeks to months) and even after freeze thawing ( Figure S3 ).
Next we determined whether we could obtain stable cell lines from STAT5bER pos memory B cells isolated from clonal density cultures. For this we isolated CD27 pos memory B cells from the peripheral blood of two different donors, cultured these cells with CD40L-L cells plus IL-21 for 36 hrs, transduced them with STAT5bER-IRES-GFP and subsequently cultured these cells with CD40L-L cells, IL-2, IL-4 and tamoxifen. After seven days GFP pos B cells were plated out at a density of 1000, 100, 10 and 1 cell per well using high speed cell sorting and cultured with CD40L-L cells, IL-2, IL-4 and tamoxifen. Within two weeks multiple poly-and monoclonal STAT5bER memory B cell clones expanded. The frequency of outgrowth was 84%, 42%, 8% and 4% for the 1000, 100, 10 and 1 cell per well cultures respectively. These data show that stable STAT5bER pos memory B cell clones can be obtained from clonal cultures.
Since transduction of human B cells with STAT5bER can be utilized to develop immunoglobulin producing B cells lines cultured at clonal density, the next step was to determine whether we could make antigen specific immunoglobulin producing B cells lines. TT is produced by the gram-positive bacterium Clostridium tetani. Infants are routinely vaccinated with a combination vaccine against diphtheria, pertussis, tetanus, and poliomyelitis (DPTP) [26] . As a consequence, most adults have memory B cells specifically directed against TT in their blood that can be detected using PE-conjugated recombinant TT C-Fragment (rTT.C) ( Figure S4 ) [15, 27] . Using flow cytometry rTT.C pos CD27 pos memory B cells were isolated from the peripheral blood of healthy donors and cultured on CD40L-L cells in the presence of IL-21. After 36 hrs the cells were transduced with STAT5bER-IRES-GFP and cultured again on CD40L-L cells, now in the presence of IL-2, IL-4 and tamoxifen. Seven days after transduction the cells were checked for TT binding (Figure 3a) , GFP sorted and maintained in microcultures of 1, 5 and 10 cells per well, again in the presence of CD40L-L cells, IL-2, IL-4 and tamoxifen. Within two weeks a number of rTT.C pos STAT5bER pos memory B cell clones could be selected (Table 1) and expanded further. Next we determined whether these rTT.C sorted cultures still had the capacity to produce immunoglobulins. After removal of tamoxifen from the cultures, rTT.C sorted STAT5bER pos clones resumed production of either IgM or IgG (Figure 3b and c). Next we determined the antigen specificity of the rTT.C pos STAT5bER pos memory B cell lines. The majority of clones expressing either IgM (54%) or IgG (69%) were specific for TT as determined by IgM and IgG specific TT ELISA (Figure 3d and e). Together these data show that the immortalizing capacity of STAT5bER can be used to obtain B cell lines with the capacity to secrete antigen specific immunoglobulins.
It should be mentioned that we also found high numbers of IgA expressing cells in our STAT5bER transduced memory B cell populations, which indicates that these IgA expressing cells can be transduced and maintained in culture like the IgG and IgM expressing cells ( Figure S5 ).
rTT.C sorted STAT5bER pos clones are monoclonal and have undergone somatic hypermutation A crucial hallmark of memory B cells is that the cells have undergone somatic hypermutation [28] and affinity maturation, the mechanisms via which they become able to develop high affinity antibodies. For this reason we determined the mutation status of TT specific STAT5bER pos memory B cell lines from one donor (Figure 3d and e) . Monoclonality of memory B cell lines that were cultured at clonal density (Table 1 ) could be confirmed by VDJ sequencing per cell line, as only one VDJ sequence per cell line was detected ( Figure 4 ). From this one donor, we obtained three independent immunoglobulin sequences -one IgG and two IgM sequences ( Figure 4 ). Interestingly, one IgG and one IgM had a similar basic pattern of somatic hypermutation, with the IgM sequence containing 6 additional mutations, one of which led to an amino acid change. This suggests that these memory B cells arose from the same clone, and that the IgM clones had undergone additional somatic hypermutation and affinity maturation.
The results presented in this study demonstrate that the immortalizing capacity of an inducible active STAT5 mutant can be used to obtain human, antigen-specific, monoclonal B cell lines, which have the capacity to undergo plasma cell differentiation. After turning off STAT5 these cells can then be induced to secrete immunoglobulins. By making use of memory B cells isolated from healthy donors we could screen for B cells that have been positively selected for high affinity against a specific antigen.
Recently we published evidence that continued activation of STAT5 plays a role in the lymphomagenesis of classical Hodgkin Lymphoma [17] . We showed that long-term culture of primary B cells expressing constitutive active STAT5 mutants eventually led to a phenotype closely resembling classical Hodgkin Lymphoma cells, including the loss of immunoglobulin expression and other B cell specific markers as CD20 [29, 30, 31] . The absence of immunoglobulin expression is an important hallmark of classical Hodgkin lymphoma [32] . In approximately 25% of the cases mutations leading to a crippled immunoglobulin expression can be found [32, 33] . However, in the majority of the cases no crippled mutations can be found and epigenetic events on the promoter region of the immunoglobulin heavy chain have been proposed as an alternative explanation for the absence of immunoglobulin expression by Hodgkin Lymphoma cells [28] .
In this paper we show that inactivation of STAT5 (by removing tamoxifen from the cultures) in the presence of IL-21 results in reappearance of immunoglobulin expression, although levels are still relatively low compared to levels normally described for ex vivo derived plasma cells. This suggests that the loss of immunoglobulin expression by primary B cells transduced with STAT5bER is dependent on activation of STAT5. We have found previously that activation of STAT5 in primary B cells inhibits the expression of Blimp-1. Blimp-1 is a transcription factor crucial for plasma cell differentiation [16, 34, 35] , and we here postulate that inactivation of STAT5 in our cell cultures leads to re-activation of Blimp-1 and subsequent differentiation of these cells into antibody secreting plasma cells.
The STAT5b-ER transduced B cells only secrete antibody when STAT5b-ER is inactivated and in the presence of IL-21. IL-21 induces proliferation and plasma cell differentiation in B cells, both in mice as well as in humans [23, 24, 25, 36, 37] . This occurs at least partly through upregulation of Blimp-1 by STAT3 activation [18, 38, 39] . IL-21 can also activate STAT5 and thus counteract STAT3 induced differentiation but the activation of STAT5 by IL-21 is only transient (30 hrs) compared to STAT3 (days). Stimulation with IL-2 and IL-4 did not lead to antibody secretion, which is expected since both cytokines induce proliferation but do not plasma cell differentiation of B cells [16, 40, 41, 42] .
The extended lifespan induced by forced expression of inducible STAT5 mutants described in this paper provides a tool to expand in vitro cultured single human memory B cells to obtain monoclonal cell lines. However these cell lines do not express any immunoglobulins when the inducible active STAT5 mutant is ''on''. By subsequently inactivating STAT5 the cells regained the capacity to produce immunoglobulin offering an opportunity to screen the clones for secretion of specific antibodies. It should be noted that antigen specificity was not selected for by a functional assay. Instead, we used a binding assay (ELISA) to screen for antitetanus toxoid specific antibodies. It has been described previously however that TT specific antibodies selected through binding assays (ELISA) are functional as shown in a Toxin Binding Inhibition test (ToBI) and in in vivo TT challenge models in mice [43] [44] . Following selection of the antigen-specific clones the immunoglobulin genes can be retrieved from the clones. An obvious next step would be to scale up the production of antibodies through recombinant expression of the identified immunoglobulin genes in suitable cell lines. These recombinant antibody proteins can then be tested for other effector functions like antigen dependent cellular cytotoxicity (ADCC), complement activation or in vivo in mice for toxin inactivation properties.
The relevance of the technology we present here is that it can be used to obtain monoclonal antibodies that are antigen specific, even when present only in low frequencies in humans. Indeed, direct isolation from the blood of influenza virus-specific B cells with a highly restricted B cell receptor repertoire has been described recently. However, healthy volunteers in this study had received booster vaccination against Influenza, inducing high percentages of antibody secreting plasma cells in the peripheral blood of these individuals [12] . Indeed, such abundance of antigen-specific plasma cells is unique. Much more often the frequency of antigen specific memory B cells is low, for example in cases where appropriate vaccines to induce such overt antibody response are lacking. In those instances the method described here may be applied to isolate human antigen specific monoclonal antibodies.
The use of human materials was approved by the Medical Ethical Committee of the Academic Medical Center of the University of Amsterdam (project MEC 07/248) and was contingent on written informed consent.
B cells were obtained from buffy coats prepared from the peripheral blood of adults (Sanquin, Amsterdam, Netherlands) by Ficoll separation and CD19 MACS microbeads as described by the manufacturer (Miltenyi Biotech). CD19 MACS-selected B cells were subsequent sorted for CD3 neg CD19 pos CD27 pos , CD3 neg CD19 pos CD27 pos sIgG pos or CD3 neg CD19 pos CD27 pos TT pos .
The following mAbs against the human molecules CD3 (SK7), CD19 (SJ25C1), IgG (G18-145) (BD-Pharmingen, San Diego, CA), CD27 (O323; eBioscience) and IgA (F(ab) 2 , DAKO), were directly labeled with either fluoroscein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin cyanine 5, (PE-Cy5), allophycocyanin (APC), phycoerythrin-indotricarbocyanine (PE-Cy7), phycoerythrin-cyanine 7 (PC7) or allophycocyanin-indotricarbocyanine (APC-Cy7). Recombinant TT C-fragment was purchased from Boehringer Mannheim (Mannhein, Germany) and conjugated to PE (Cyanotech Corporation, Kaila-kona, HI) as previously described [27] . Stained cells were sorted using a FACSAria (BD) and analyzed using a LSR-II (BD) and flow cytometry data were processed with FlowJo computer software (Treestar, Ashland, OR).
The STAT5bER retroviral construct has been described previously [16] . In brief, cDNA encoding STAT5bER was expressed in the Lazarus (LZRS) vector upstream to a internal ribosomal entry site (IRES) and a marker gene (a signaling- Indicated is the total number of clones isolated and at which cell density they were seeded. One 96 well plate was used for each condition (1, 5 or 10 cell per well). Wells contained 2500 CD40L-expressing L cells, IL-2 and IL-4. J to K of the medium was replaced twice a week with fresh cytokines and 5000 L cells. The horizontal bar of each clone represents the sequenced region. The FR1 to CDR3 region was sequenced for clone 5D1 and for clones 10E1 and 10B7 region FR2 to CDR3 were sequenced. We were unable to obtain reliable sequence data of the FR1-CDR1 region of clone 1E10. Cell culture B cells (2610 5 ) were co-cultured on c-irradiated (100 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L-L cells, 10 5 well) and recombinant human IL-2, IL-4 or IL-21. Cells were routinely tested by PCR for the presence of mycoplasma and EBV (data not shown).
Plates were coated with anti-human IgG (Dako) at 5 mg/ml in PBS or Tetanus vaccine (Dutch vaccine institute, The Netherlands) diluted 1:10 for 1 hr at 37uC or o/n at 4uC and washed in ELISA wash buffer (PBS, 0.5% Tween-20). 4% milk in PBS was used as blocking agent, before serial dilution of cell culture supernatants and enzyme-conjugated detection Abs were added (dilutions 1:2500 for HRP-conjugated anti-IgG (Jackson Immu-noResearch Laboratories) and 1:250 for AP conjugated anti-IgG (DAKO)). TMB substrate/stop solution (Biosource) or Alkaline Phosphatase Substrate (Sigma-Aldrich) was used for development of the ELISAs. To detect Tetanus Toxin we used the ELISA Ridascreen Tetanus (r-biopharm AG, Darmstadt, Germany).
Total RNA was isolated from approximately 5610 5 B cells with Trizol (Invitrogen). cDNA was generated and subjected to PCR to produce heavy chain fragments using 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems Inc.). PCR products were run on agarose gels, purified and cloned into the pCR2.1 TA cloning vector according to manufacturers' recommendations (Invitrogen). Sequence analysis was performed using BigDye Terminator chemistry (Applied Biosystems Inc.) and Vector-NTI software (Invitrogen). Consensus sequences were determined with Codon code (CodonCode Corporation). Figure S1 IL-21 induced Ig secretion in STAT5bER immortalized memory B cell cultures. A) STAT5bER transduced polyclonal memory B cells were cultured in the absence of tamoxifen (4HT) and with IL-2 and IL-4 or IL-21. IgG secretion was determined at day 7. Data from two representative donors out of 5 donors is shown. B) STAT5bER transduced monoclonal memory B cell lines were cultured in the absence of tamoxifen (4HT) and with IL-2 and IL-4 or IL-21. IgG secretion was determined on day 7. Data from two representative cell cultures is shown. All B cells in these cultures had lost surface immunoglobulin expression and were in culture for at least 2 months. (TIF) Figure S2 IgG and IgM secretion in time after removal of tamoxifen (4HT). IgG and IgM secretion was determined in cells cultured with CD40L and IL-21 for 7 days and for 14 days. Days 0 indicates the day when the culture conditions were changed from CD40L, IL-2, IL-4 plus tamoxifen (4HT) to CD40L and IL-21. The black and grey bars represents two different donors. (TIF) Figure S3 IgG and IgM expression in long term cultures. Multiple TT specific clones which were in culture for more then 5 months and subsequent frozen. They were then thawed and taken in culture again with CD40L, IL-2, IL-4 and tamoxifen (4HT). When a stable culture was obtained, the cells were cultured with CD40L and IL-21. An ELISA was performed to determine IgG and IgM concentrations. (TIF) Figure S4 rTT.C staining on freshly isolated memory B cells. Phycoerythrin (PE) labeled rTT.C was added to freshly isolated B cells. Cells were subsequently sorted using flow cytometry (FACSAria). Shown are the results of 5 different donors. (TIF) Figure S5 IgA and IgG expression on polyclonal CA-STAT5b transduced and non-transduced B cells. A polyclonal mixture of total CD27+ selected human memory B cells from two donors were transduced with caSTAT5b-IRES-NGFR and cultured for a maximum of two weeks before they were frozen. After thawing cells were stained immediately for the IgA and IgG isotype. Cells expressing NGFR indicates they were transduced with CA-STAT5b. (TIF) Genomic Characterization and High Prevalence of Bocaviruses in Swine Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ∼2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45–75% and 55–70% respectively, with 30–47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses. Members of the Parvoviridae family are small non-lipid enveloped viruses with a diameter of 18-26 nm, icosahedral symmetry (T = 1), encoded by a single-stranded linear DNA genome of approximately 4,000 to 6,000 nucleotides (nt) [1] . The family includes two subfamilies: Densovirinae, Parvovirinae. The subfamily of Densovirinae contains four genera: Densovirus, Iteravirus, Brevidensovirus and Pefudensovirus, which infect only invertebrates [1] . The Parvovirinae subfamily is currently subdivided into five genera: Parvovirus, Erythrovirus, Dependovirus (adeno-associated virus), Amdovirus, and Bocavirus, infecting vertebrates [1] .
The bocavirus genus was recently assigned by the International Committee on Taxonomy of Viruses (ICTV) [1] to parvovirus genomes containing a third ORF (labeled NP1) between the NS1 and VP1/VP2 genes [2] . Bocaviruses were first identified in bovine and canine [3, 4] , samples from which it derives its genus name [1, 5] . Presently, the bocavirus genus contains eight members: bovine parvovirus, canine minute virus (CnMV), human bocavirus 1-4 (HBoV1-4), a gorilla bocavirus and a partially sequenced chimpanzee bocavirus [1, 6, 7] .
The first human bocavirus (HBoV) was found in the nasopharyngeal secretion of a child with respiratory problems using a methodology closely related to that used here [8] . HBoV has been associated with lower respiratory tract symptoms and possibly diarrhea [5, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , and shows a very low degree of genetic variability worldwide [6, 23, 24] . HBoV2 was first reported in the stool of Pakistani children with non-polio acute flaccid paralysis (AFP) [10] , and then in Australian children and Chinese children with diarrhea [9, 25] . HBoV3 was first reported in the stool of Australian children with diarrhea [9] and then in stool from Nigerian, Tunisian, Nepalese and US children [5] . HBoV4 was reported in the stool of children with AFP from Nigeria and Tunisia [5] . HBoV 1/2/3 were also detected in untreated sewage water from throughout the US [26] . Recently, a novel bocavirus was identified in the feces of captive gorillas with diarrhea [6] and from wild gorillas and chimpanzees [7] . In 2009, a porcine bocalike virus (PBo-likeV) was reported in swine feces with postweaning multisystemic wasting syndrome in Sweden and 1854 bp of its partial genome sequenced [27] . In 2010, the nearly complete genomes of distinct porcine bocaviruses provisionally named PBoV1 and PBoV2 were characterized from feces of swine in China [28] . Finally, partial genome sequences of 2.4 Kb from another clade of porcine bocaviruses labeled 6V and 7V where also identified yielding three major bocavirus groups in swine (PBo-likeV, PBoV1/PBoV2, and 6V/7V).
Random amplification and sequencing has been used to discover novel virus in human and animal [8, 10, . In this study, we found in swine feces highly distinct bocavirus whose genome we tentatively named porcine bocavirus 1 (PBoV1-H18), and porcine bocavirus 2 (PBoV 2-A6). The nearly complete genomes of both viruses were acquired and are described here. PBoV1-H18 and PBoV2-A6 were also screened for in 340 stool samples of asymptomatic swine from five provinces of China.
A total of 340 porcine stool samples from different aged swine were collected from 17 middle or large-scale porcine farms (200-2,000 sows each) in five provinces of China from April 2008 to October 2009, of which 80 were collected from four farms located in Shanghai, 60 from three farms located in Jiangsu province, 120 from six farms located in Anhui province, 20 from a farm located in Shandong province, and 60 from three farms located in Guizhou province and stored at 280uC (Table 1 ).
One stool sample was randomly selected from each of the 17 farms. The samples were suspended in PBS (0.01 M phosphate, pH7.2-7.4, 0.15NaCl), vortexed, centrifuged at 15000 g, and filtered through a 0.22-mm filter to remove eukaryotic-and bacterial-cell-sized particles [45, 46, 54, 55] . The filtrates were then treated with benzonase, DNase and RNase to digest non-particleprotected nucleic acid as reported [54, 55] . Viral nucleic acids were then extracted using the TIANamp virus DNA/RNA Kit (TIANGEN BIOTECH, BEIJING, CO., Ltd.). Viral cDNA synthesis was performed by incubation of the extracted viral RNA/DNA with 100 pmol of primer K-8N [56] with a degenerate 39 end and the use of Superscript reverse transcriptase, and the opposite strand of the cDNA was generated after melting and reannealing and primer extension using Klenow DNA polymerase [45, 46, [54] [55] [56] . PCR of extension products was performed as reported previously using the K-8 primer (K-8N without the degenerate 39 end) [54] . This protocol amplifies both viral RNA and DNA genomes [54, 55] .
Random RT-PCR DNA products ran as smears on agarose gel and were gel purified (Axygen, CA, USA), then subcloned into pMD-18T plasmid vector (TaKaRa, Japan) for sequencing. The sequences were then screened for sequence similarities using tBLASTx and BLASTn against the nr database in GenBank. 
Sequences of each PCR product were assembled using SeqMan II program (DNASTAR, Inc). The identification of open reading 
The near-full genomes of PBoV1-H18, PBoV2-A6 and the partial NS1 and VP1 sequences from the diagnostic nPCR have been deposited in GenBank under accession numbers HQ291308-HQ291309 and HQ291310-HQ291343.
Seventeen porcine samples stool supernatants from 17 farms were analyzed using a generic viral particle-protected nucleic acid enrichment procedure followed by random amplification of extracted RNA and DNA (see materials and methods) [46, [54] [55] [56] . Amplified DNA was then subcloned and 1190 plasmid inserts were sequenced (70 for each of 17 samples). Nine samples (totaling 82 subclones) showed the presence of fragments whose virtual translation products were related to canine and human bocaviruses using BLASTx. Twenty-four clones from pig sample H18 and 26 clones from pig sample A6 showed significant similarity with bocaviruses. H18 derived sequences showed high (.99%) identity with the recently described porcine bocavirus-like virus (PBo-LikeV), the only porcine bocavirus reported at the time of these experiments (GenBank GU902971) [27] . Sequences from porcine sample A6 showed low identity with those of H18 and PBo-likeV. The H18 and A6 samples were selected for targeted viral genome amplification and sequencing.
The 24 sequences from H18 were assembled to form a continuous sequence of approximately 2700 nucleotides that appeared to lack .1300 and 1000 nucleotides from the 59and 39 ends of its genome. PCR primers based on the available H18 sequences and regions highly conserved between HBoV2 (FJ170278) and canine bocavirus (FJ214110) were used to amplify the nearly complete bocavirus genome we provisionally called PBoV1-H18 (5267 nt). To confirm this genome sequence, this sequence was re-amplified using 6 sets of PCR primers generating overlapping fragments of the genome which were directly sequenced. Using the same method, the nearly complete bocavirus genome (5117 nt) from sample A6 was also sequenced and was provisionally labeled PBoV2-A6.
Using an open reading frame (ORF) finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html), three ORF were found in both genomes (Figure 1 ). The ORFs of PBoV1-H18 were 636 aa for NS1, 219 aa for NP1 and 621 aa for VP1/VP2. The ORFs of PBoV2-A6 were 703 aa for NS1, 221 aa for NP1 and 704 aa for VP1/VP2. The possible splicing of bocavirus NS1 transcripts recently shown to extend the length of NS1 proteins was not investigated here [6, 57] . 
Nucleic acids were extracted from 340 porcine stool samples. PBoV1-H18 related sequences were screened for using nested PCR with primers amplifying a 530-bp fragment of the NP1/VP1 region. The electrophoretic bands of the expected size were subcloned and sequenced. The results showed that the prevalence of PBoV1-H18 related viruses was high in China with 215 out of 340 (63.2%) porcine samples positive ( 
To determine the genetic relationship of PBoV1-H18 and PBoV2-A6 with recently described porcine bocaciruses and bocaviruses from other host species, both nucleotide and amino acid alignments were generated and used for phylogenetic analyses. When the whole genomes were considered porcine bocaviruses as a group (except for the V6/V7 variants with only NP1 sequences available), were most closely related to the canine bocavirus CnMV (Figure 2A ). Phylogenetic analyses of the 3 ORFs -NS1, NP1 and VP1/VP2 -were also performed ( Figure 2B-G) . In all three regions, PBoV1-H18 was most closely related to the Chinese PBoV1 and PBoV2 recently reported by Cheng et al [28] . PBoV2-A6 was closely related in NP1 to the first reported partial porcine bocavirus sequence PBo-likeV from Sweden, the only region available for comparison (Fig. 2C, F ) [27] . Table 2 numerically shows the protein similarities between PBoV1-H18 and PBoV2-A6 and other porcine and non-porcine bocaviruses.
The partial VP1 sequence of PBoV2-A6 related variants from different farms was also phylogentically analyzed and fell into two major clades we named PBoV2 genotype 1 and 2 (PBoV2-G1 and PBoV2-G2). The two previously described Chinese PBoV1 and PBoV2 ''species'' grouped within these two genotypes.
PBo-likeV was originally found in swine with postweaning multisystemic wasting syndrome (PMWS) in 2009 when approximately 35% of its genome sequence was reported [27] . The nearly full genomes of two distantly related porcine bocaviruses labeled PBoV1 and PBoV2 as well as two partial genomes labeled V6 and V7 were then reported in 2010 [28] . These bocaviruses grouped into 3 phylogenetic clades containing PBo-likeV, PBoV1/PBoV2, and V6/V7. In the present study, we characterize two nearly complete bocavirus genomes, one of which (PBoV1-H18) grouped with the PBo-likeV clade while the second (PBoV2-A6) fell with the PBoV1/PBoV2 clade. We provisionally named the genome from the H18 sample PBoV1-H18 since its closest homologue, PBo-likeV, was the first reported porcine bocavirus [27] . The virus from sample A6 was provisionally named PBoV2-A6 since it phylogenetically groups with the second reported set of porcine bocaviruses containing both PBoV1 and PBoV2 [28] . No close homologues of the V6/V7 clade were identified in this study. Under this proposed classification scheme, the viruses labeled PBoV1 and PBoV2 by Cheng et al, therefore both belong to the PBoV2 clade, the second reported clade of porcine bocaviruses [28] . Under this proposed taxonomic classification, the V6 and V7 bocaviruses [28] belong to the still only partially characterized PBoV3 clade.
Sequence analysis of the PBoV2 clade showed that their VP1/ VP2 genes were highly diverse and could be classified into two genotypes ( Figure 3 ). The partial NP1 and VP1 genes of PBoV1-H18 related viruses detected in this study were more highly conserved (99-100% identity), consistent with a recent report by Zhai et al reporting PBoV1 in Chinese pigs using partial VP1/ VP2 nested PCR and sequencing [58] . Zhai et al also found a high prevalence of PBoV1 (69% in weanling piglets) with a higher frequency of PBoV1 in animals also infected with PCV2, PRRSV, PTTV or CSFV and in pigs with respiratory symptoms versus healthy pigs [58] .
Members of the bocavirus genus contain an 3 rd ORF of unknown function labeled NP1 gene. Recently another parvovirus (PPV4) was identified in porcine feces that also contained a central 3 rd ORF, although unrelated in sequence to the bocaviruses NP1 [59] . None of the ORFs of PPV4 clustered phylogenetically with the bocaviruses (data not shown) but instead clustered with members of the parvovirus genus [59] . PPV4 is therefore unrelated to the bocaviruses reported here. HBoV1 has been associated with respiratory symptoms while other HBoV may be associated with diarrhea and acute flaccid paralysis [5, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] 25] . A gorilla bocavirus was detected in captive animals in the US experiencing severe diarrhea [6] . PBoV1 was found in pigs with PMWS in Sweden [27] and in pigs with respiratory tract symptoms in China [58] . In the present study, both PBoV1 and PBoV2 were highly prevalent in both asymptomatic swine from five provinces of China. Further studies are needed to examine possible associations between infections with these different porcine bocaviruses, the viral loads excreted, the presence of co-infections and various porcine diseases.  Analysis of codon usage and nucleotide composition bias in polioviruses BACKGROUND: Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and a member of the family of Picornaviridae and among the most rapidly evolving viruses known. Analysis of codon usage can reveal much about the molecular evolution of the viruses. However, little information about synonymous codon usage pattern of polioviruses genome has been acquired to date. METHODS: The relative synonymous codon usage (RSCU) values, effective number of codon (ENC) values, nucleotide contents and dinucleotides were investigated and a comparative analysis of codon usage pattern for open reading frames (ORFs) among 48 polioviruses isolates including 31 of genotype 1, 13 of genotype 2 and 4 of genotype 3. RESULTS: The result shows that the overall extent of codon usage bias in poliovirus samples is low (mean ENC = 53.754 > 40). The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in those polioviruses. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage among three genotypes. Geographic factor also has some effect on the codon usage pattern (exists in the genotype-1 of polioviruses). No significant effect in gene length or vaccine derived polioviruses (DVPVs), wild viruses and live attenuated virus was observed on the variations of synonymous codon usage in the virus genes. The relative abundance of dinucleotide (CpG) in the ORFs of polioviruses are far below expected values especially in DVPVs and attenuated virus of polioviruses genotype 1. CONCLUSION: The information from this study may not only have theoretical value in understanding poliovirus evolution, especially for DVPVs genotype 1, but also have potential value for the development of poliovirus vaccines. The result shows that the overall extent of codon usage bias in poliovirus samples is low (mean ENC = 53.754 > 40). The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in those polioviruses. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage among three genotypes. Geographic factor also has some effect on the codon usage pattern (exists in the genotype-1 of polioviruses). No significant effect in gene length or vaccine derived polioviruses (DVPVs), wild viruses and live attenuated virus was observed on the variations of synonymous codon usage in the virus genes. The relative abundance of dinucleotide (CpG) in the ORFs of polioviruses are far below expected values especially in DVPVs and attenuated virus of polioviruses genotype 1. Conclusion: The information from this study may not only have theoretical value in understanding poliovirus evolution, especially for DVPVs genotype 1, but also have potential value for the development of poliovirus vaccines.
When molecular sequence data started to be accumulated nearly 20 years ago, it was noted that synonymous codons are not used equally in different genomes, even in different genes of the same genome [1] [2] [3] . As an important evolutionary phenomenon, it is well known that synonymous codon usage bias exists in a wide range of biological systems from prokaryotes to eukaryotes [4, 5] . Codon usage analysis has been applied to prokaryote and eukaryote, such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Caenorhabditis elegans and human beings [6] [7] [8] . These observed patterns in synonymous codon usage varied among genes within a genome, and among genomes. The codon usage is attributable to the equilibrium between natural selection and mutation pressure [9, 10] . Recent studies of viral codon usage has shown that mutation bias may be a more important factor than natural selection in determining codon usage bias of some viruses, such as Picornaviridae, Pestivirus, plant viruses, and vertebrate DNA viruses [9, [11] [12] [13] . Meanwhile, recent report also showed that the G+C compositional constraint is the main factor that determines the codon usage bias in iridovirus genomes [11, 14] . Analysis of codon usage can reveal much about the molecular evolution or individual genes of the viruses.
Polioviruses belong to the family Picornaviridae and are classified as human enterovirus C (HEV-C) species in the genus Enterovirus according to the current taxonomy [15, 16] . Polioviruses can be divided into three different genotypes: 1, 2 and 3. The genome of each genotypes contains a single positive-stranded RNA with a size of approximately 6 kb consisting of a single large open reading frame (ORF) flanked by 5' and 3' untranslated region [17] .
As we known, the Sabin oral poliovaccine (OPV) was among the best known viral vaccines [18] . It has saved the lives and health of innumerable people, in particular children. However, poliovirus is highly genetically variable. OPV viruses may undergo transformation into circulating highly diverged VDPV, exhibiting properties hardly distinguishable from those of wild polioviruses [19] . So far, little information about synonymous codon usage pattern of polioviruses genome has been acquired to date. To our knowledge, this is the first report of the codon usage analysis on polioviruses (including wild strains, attenuated live vaccine strains and VDPV strains). In this study, we analyzed the codon usage data and base composition of 48 available representative complete ORFs of poliovirus to obtain some clues to the features of genetic evolution of the virus.
A total of 48 poliovirus genomes were used in this study ( Table 1 ). The serial number (SN), genotype, length value, isolated region, GenBank accession numbers, and other detail information about these strains were listed in Table 1 . All of the sequences were downloaded from NCBI http://www.ncbi.nlm.nih.gov/Genbank/, and 48 poliovirus genomes were selected in the study. The other sequences with >98% sequence identities were excluded.
The ENC is used to measure the degree of departure from the equal use of synonymous codons of coding regions of polioviruses. The values of the effective number of codon (ENC) range from 20 to 61. In an extremely biased gene where only one codon is used for each amino acid, this value would be 20; if all codons are used equally, it would be 61; and if the value of ENC is greater than 40, the codon usage bias was regarded as low. The values of ENC were obtained by EMBOSS CHIPS program [20] .
Genes, whose codon choice is constrained only by a mutation bias, will lie on or just below the curve of the predicted values. The predicted values of ENC were calculated as
where s represents the given (G+C) 3 % value [21] .
The calculation of the relative synonymous codon usage (RSCU)
To investigate the pattern of relative synonymous codon usage (RSCU) without the influence of amino acid composition among all polioviruses samples, the RSCU values of codons in each ORF of polioviruses were calculated according to the formula of previous reports [22, 23] .
where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codon with RSCU value more than 1.0 has positive codon usage bias, while the value <1.0 has relative negative codon usage bias. When RSCU value is equal to 1.0, it means that this codon is chosen equally and randomly.
Because dinucleotide biases can affect codon bias, the relative abundance of dinucleotides in the coding regions of polioviruse genomes was assessed using the method described by Karlin and Burge [24] . A comparison of actual and expected dinucleotide frequencies of the 16 dinucleotides in coding region of the 48 polioviruses genomes was also undertaken. The odds ratio r xy =ƒ xy /ƒ y ƒ x , where ƒ x denotes the frequency of the nucleotide X, ƒ y denotes the frequency of the nucleotide Y, ƒ y ƒ x the expected frequency of the dinucleotide XY and ƒ xy the frequency of the dinucleotide XY, etc., for each dinucleotide were calculated. As a conservative criterion, for r xy > 1.23 (or < 0.78), the XY pair is considered to be of over-represented (or under-represented) relative abundance compared with a random association of mononucleotides.
Principal component analysis (PCA) was carried out to analyze the major trend in codon usage pattern in different genomes of polioviruses (excluding non-coding regions). It is a statistical method that performs linear mapping to extract optimal features from an input distribution in the mean squared error sense and can be used by self-organizing neural networks to form unsupervised neural preprocessing modules for classification problems [6] . In order to minimize the effect of amino acid composition on codon usage, each ORF is represented as a 59-dimensional vector. Each dimension corresponds to the RSCU value of one sense codon excluding Met, Trp and three stop codons. Linear regression analysis was used to find the correlation between codon usage bias and gene length. Correlation analysis is used to identify the relationship between codon usage bias and synonymous codon usage pattern. This analysis is implemented based on the Spearman's rank correlation analysis way.
All statistical analyses were carried out using the statistical analysis software SPSS Version 17.0.
In order to investigate the extent of codon usage bias in polioviruses, all RSCU values of different codon in 48 polioviruses strains were calculated. There is only two preferred codons UUG (Leu) and GUG (Val), choosing G at the third position, and most of preferred codons are ended with A ( Table 3) . The values of ENC among these polioviruses ORFs are similar, which vary from 52.609 to 55.105 with a mean value of 53.754 and S.D. of 0.545. The data showed that the extent of codon preference in polioviruses genes was kept basically stable.
The values of A, U, C, G and C+G were compared with the values of A 3 , C 3 , G 3 , U 3 , (G+C) 3 , respectively. An interesting and complex correlation was observed. In detail, the (C+G) 3 have highly significant correlations with A, U, C, G and C+G, respectively, indicating C+G may reflect interaction between mutation pressure and natural selection. However, the A have no correlation with A 3 , G 3 and C 3 , and U have no correlation with A 3 (Table 4 ). Both cases suggested that the nucleotide constraint possibly influence synonymous codon usage of polioviruses. In addition, the correlation between the Axis 1 (calculated by PCA) and the values of A, C, G, U, A 3 , C 3 , G 3 , U 3 , (G+C), (G+C) 3 of each strain was also analyzed. The significant correlation was found between nucleotide compositions and synonymous codon usage to some extent excluding Axis 1 and the value of A ( Table 4 ). The analysis revealed that most of the codon usage bias among ORFs of polioviruses strains was directly related to the base composition. Finally, the ENC-plot [ENC plotted against (G+C) 3 %] was used as a part of general strategy to investigate patterns of synonymous codon usage and all of the spots lie below the expected curve (Figure 1 ). These imply that the codon bias can be explained mainly by an uneven base composition, in other words, by mutation pressure rather than natural selection. Effect of other potential factors on codon usage
Principal component analysis was carried out to identify the codon usage bias among ORFs. From which we could detect one major trend in the Axis 1 which accounted for 20.815% of the total variation, and another major trend in the Axis 2 for 16.273% of the total variation. A plot of the Axis 1 and the Axis 2 of each gene was shown in Additional file 1, Figure S1 . Obviously, those polioviruses belong to the same genotype tends to come together (except strain 48, isolated from Finland). Compared with the scattered groups of polioviruses genotype 1, genotype 2 and 3 strains aggregated more tightly to some degree. Although this graph is a little complex, it seems that there is a clear geographical demarcation in the polioviruses genotype 1 such as the VDPV strains isolated from USA, Dominica, China mainland and Taiwan. These may indicate that geographic is another factor on codon usage bias.
The frequencies of occurrence for dinucleotides were not randomly distributed and no dinucleotides were present at the expected frequencies. And the frequency of CpG and TpA was significantly low at all codon positions for coding region of 48 polioviruses genomes (mean ± S. D. = 0.490 ± 0.012; and mean ± S.D. = 0.748 ± 0.034. both < 0.78). The relative abundance of CpA and TpG also showed slight deviation from the ''normal range'' (mean ± S.D. = 1.253 ± 0.032 and 1.423 ± 0.023, respectively) ( Table 5 ). In addition, the RSCU values of the eight codons containing CpG (CCG, GCG, UCG, ACG, CGC, CGG, CGU, and CGA) were analyzed, to reveal the possible effects of CpG under-represented on codon usage bias. All of these eight codons were not preferential codons and were markedly suppressed. The six codons containing TpA (UUA, CUA, GUA, UAU, UAC and AUA) were suppressed too. Conversely, the RSCU values of the eight codons containing CpA (UCA, CCA, ACA, GCA, CAA, CAG, CAU, CAC) and five codons containing UpG (UUG, GUG, UGU, UGC, CUG) are high, and most of them (8 out of 13) were preferential codons ( Table 2 and Table 5 ). In addition, compared with DVPVs and live attenuated strain of polioviruses genotype 1, the wild viruses has higher frequencies of dinucleotides including CpG (Figure 2 and Table 5 ).
Furthermore, we also performed a linear regression analysis on ENC value and gene length of ORFs of 48 polioviruses genomes. However, there was no significant correlation between codon usage and gene length in these virus genes (Spearman P > 0.05).
Studies of synonymous codon usage in viruses can reveal much about viral genomes [25] . The overall codon usage among 48 ORFs of polioviruses was analyzed in this study. First, the ENC values of all the poliovirus samples were analyzed, and the results showed that the majority of polioviruses do not have a strong codon bias (mean ENC = 53.754 > 40). In addition, together with published data on codon usage bias among some RNA viruses, such as BVDV, H5N1 Table 4 Correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 influenza virus and SARS-covs with mean values of 51.43, 50.91 and 48.99, respectively, one possible explanation for this is that the weak codon bias of RNA virus is advantageous to replicate efficiently in vertebrate host cells, with potentially distinct codon preferences [26] [27] [28] . Natural selection and mutation pressure are thought to be the main factors that account for codon usage variation in different organisms [29] [30] [31] . In this study, the general association between codon usage bias and base composition suggests that mutational pressure, rather than natural selection is the mainly factors on codon usage pattern of polioviruses.
Codon usage can also be strongly influenced by underlying biases in dinucleotide frequency, which differs greatly among organisms. Specifically, after accounting for dinucleotide biases, the proportion of codon usage bias explained by mutation pressure often increases, as seen in human RNA viruses [25] . Our study revealed that CpG and the eight CpG-containing codons are notably deficient in ORFs of 48 poliovirus genomes. The explanation for the CpG deficiency is immunologic escape. A high CpG content may be detrimental to small DNA (or RNA) viruses, as unmethylated CpGs are recognized by the host's innate immune system (Toll-like receptor 9) as a pathogen signature [32] . As with vertebrate genomes, methylated viral genomes would face a high chance of mutation at CpGs, that would result in a reduction of this dinucleotide [9, 33] . We found that DVPVs and live attenuated virus of genotype 1 have lower frequencies of CpG dinucleotide compare with wild viruses of polioviruses genotype 1. The most popular explanation for lower frequencies of CpG in ORFs of DVPV genomes is that when OPV viruses turning into VDPV genotype 1, a lower frequencies of CpG dinucleotide maybe help VDPV out of the host immunity.
Although it seems speculative and complex, some researchers have found that reduction of the rate of poliovirus protein synthesis through large-scale utilization of codons that are not optimal has caused attenuation of viral virulence by lowering specific infectivity [34] . Therefore, the information from this study may not only have theoretical value in understanding poliovirus evolution (especially for DVPVs genotype 1), but also have practical value for the development the poliovirus vaccine. However, a more comprehensive analysis The range of coding region of 48 polioviruses's relative dinucleotide ratios. b Mean values of coding region of 48 polioviruses's relative dinucleotide ratios ± S.D. Figure 2 Comparison the relative dinucleotide abundance in polioviruses DVPVs genotype 1, live attenuated virus genotype 1, wild viruses genotype 1, DVPVs genotype 2, DVPVs genotype 3 and live attenuated virus genotype 3.
is needed to reveal more information about codon usage bias variation within poliovirus and other responsible factors.
The information from this study may not only help to understand the evolution of the poliovirus, especially for DVPVs genotype 1, but also have potential value for the development of poliovirus vaccines.
Additional files 1: Figure S1 . A plot of the values of the Axis1a (20.82%) and the Axis2a (16.27%) of each ORF in principle component analysis. Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing BACKGROUND: Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda. METHODOLOGY/PRINCIPAL FINDINGS: The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other arterivirus. Both new variants nevertheless have the same 3′ genomic architecture as the type strain, containing three open reading frames not present in the other arteriviruses. CONCLUSIONS/SIGNIFICANCE: These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3′ genetic architecture of SHFV relative to the other arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any arterivirus. These results expand our knowledge of the natural history and diversity of the arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens. Simian hemorrhagic fever virus (SHFV) was discovered during an ''explosive'' outbreak of hemorrhagic disease in rhesus macaques (Macaca mulatta) in 1964 [1] . Since then, sporadic epidemics have occurred in macaques, likely as a result of contact with, or iatrogenic transmission from, infected but asymptomatic captive African monkeys [2, 3, 4] . Viruses related to SHFV have not yet been found to infect humans, but the clinical manifestations of SHFV resemble those of other primate-associated hemorrhagic fever viruses significant for human health (e.g. Ebola, Marburg, Yellow fever), making SHFV of interest with respect to emerging infectious disease surveillance and biodefense [4, 5] . Although asymptomatic SHFV infections in captive patas monkeys (Erythrocebus patas), vervet monkeys (Cercopithecus aethiops), and baboons (Papio sp.) suggest that such species could be natural SHFV reservoirs [2, 3] , the virus has never, to our knowledge, been found in a primate in the wild.
SHFV is in the family Arteriviridae, which also contains equine arteritis virus (EAV), lactate dehydrogenase elevating virus of mice (LDV), and porcine reproductive and respiratory syndrome virus (PRRSV) [6] . Similar to the other arteriviruses, SHFV has a positive-sense RNA genome approximately 15 kb in length, consisting of a short 59 untranslated region, two large open reading frames (ORFs 1a and 1b) encoding the replicase plus various nonstructural proteins, a number of smaller downstream ORFs encoding structural proteins, and a short 39 untranslated region [7] . However, SHFV is unusual compared to the other arteriviruses in that it contains three additional ORFs (2a, 2b, and 3) immediately downstream of the replicase-encoding ORFs, perhaps as the result of a gene duplication event [8] . As of the time of this writing, only one full SHFV genome was available in GenBank (accession number NC_003092), representing prototype strain LVR 42-0/M6941, which was isolated on MA-104 cells from a moribund rhesus macaque during the original SHFV outbreak in 1964 [9] . Because the genome organization of SHFV was determined from this single strain, and because this sequence was generated from an isolate obtained from a macaque after growth in cells derived from embryonic rhesus macaque kidney tissue, it is unclear whether the unusual 39 genomic architecture of SHFV is typical or whether it might be an artifact of passage in a non-natural host or in cultured cells.
Here we report the discovery of two novel and highly divergent SHFV variants co-infecting a single male red colobus monkey (Procolobus rufomitratus tephrosceles) from Kibale National Park, Uganda. Our finding of novel, divergent SHFV variants dually infecting a wild primate sheds new light on the natural history and diversity of SHFV. In addition, using direct pyrosequencing, we were able to recover nearly complete viral genome sequences with only minimal PCR amplification. Our results therefore also confirm the authenticity of SHFV's unique genomic architecture during natural infection.
All animal use followed the guidelines of the Weatherall Report on the use of non-human primates in research and was approved by the Uganda Wildlife Authority, the Uganda National Council for Science and Technology, and the University of Wisconsin Animal Care and Use Committee prior to initiation of the study. Biological materials were shipped internationally under CITES permit #002290 (Uganda).
Kibale National Park, Uganda, is a semi-deciduous, midaltitude forest of 795 km 2 located in western Uganda near the foothills of the Rwenzori Mountains and is notable for having one of the world's highest species diversities (13 primate species) and densities of primates, including the world's densest population of red colobus [10, 11] . On February 5, 2010, a wild adult male red colobus in Kibale was captured as part of a larger study of primate ecology, health, and conservation. The animal was anesthetized with a combination of Ketamine (5.6 mg/kg) and Xylazine (1.7 mg/kg) administered intramuscularly using a variable-pressure air rifle (Pneudart, Inc, Williamsport, PA, USA). Blood was drawn from the femoral vein into an evacuated plasma collection tube (Becton, Dickinson and Company, Inc, Franklin Lakes, NJ, USA) and kept cool until processing. The animal was given a reversal agent (Atipamezole, 0.6 mg/kg), and released after recovery back to its social group without incident. Blood was separated using centrifugation in a field laboratory and frozen immediately in liquid nitrogen for storage and transport to the United States. Samples were shipped in an IATA-approved dry shipper to the USA for further analysis at the Wisconsin National Primate Research Center.
One ml of plasma was centrifuged at 5,0006g at 4uC for 5 min with subsequent filtration of the supernatant through a 0.45-mm filter (Millipore, Billerica, MA, USA) to remove residual host cells. Viral RNA was isolated using the Qiagen QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, except that carrier RNA was omitted. The eluted RNA was treated with DNase I (DNA-free, Ambion, Austin, TX, USA), and double stranded DNA was generated using the Superscript double-stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA) primed with random hexamers. DNA was purified using the Agencourt Ampure XP system (Beckman Coulter, Brea, CA, USA) and approximately 1 ng of DNA was subjected to simultaneous fragmentation and adaptor ligation (''tagmentation'') with the Nextera DNA Sample Prep Kit (Roche Titanium-compatible, Epicentre Biotechnologies, Madison, WI, USA). DNA was subsequently cleaned using the Agencourt Ampure XP system, briefly PCR amplified with primers to add Roche/454 titanium-based adaptors onto each fragment (15 cycles) , and cleaned again with the Agencourt Ampure XP system. DNA fragments were then sequenced using the GS Junior pyrosequencing system (Roche 454 Life Sciences, Branford, CT, USA).
To analyze pyrosequencing data, raw sequences were first screened to remove redundancy using Galaxy software (http:// usegalaxy.org). The remaining reads were then imported into CLC Genomics Workbench (CLC bio, Aarhus, Denmark), trimmed to remove Nextera-specific transposon sequences as well as short and low quality reads, and assembled using the CLC de novo assembler. Both singleton and assembled contiguous sequences (contigs) were queried against the GenBank database (http:// www.ncbi.nlm.nih.gov/GenBank) using the basic local alignment search tools blastn and blastx [12] , with an e-value cut-off of 10 and word sizes of 11 and 3 for blastn and blastx queries, respectively.
Once initial non-host contigs were assembled, a short sequence gap between two aligned fragments was filled by conventional PCR and Sanger sequencing. Briefly, PCR amplicons were generated with the SuperScript III One-Step RT-PCR System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, with specific primers designed based on pyrosequence data. Thermocycling conditions included cDNA synthesis at 50uC for 30 min and denaturation at 95uC for 2 min, followed by 40 cycles of denaturation at 94uC for 2 min, annealing at 55uC for 30 s, and extension at 68uC for 1 min. A similar sequence gap at the 39 terminal open reading frame of one contig was filled by 39 RACE according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Briefly, first-strand cDNA synthesis with an oligo(dT)-containing adaptor primer was performed at 42uC for 50 min, followed by amplification of the target cDNA with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) using a specific primer designed based on pyrosequencing data as well as the abridged universal amplification primer (AUAP) (59 GGCCAGGCGTCGACTAGTAC). Thermocycling conditions included an enzyme activation step of 3 min at 94uC followed by 35 cycles of denaturation at 94uC for 30 s, annealing at 55uC for 30 s, and extension at 72uC for 1 min. Specific primer sequences are available upon request. Amplification products were run on 1% agarose gels, purified (MinElute, Qiagen, Hilden, Germany), and directly sequenced with the DYEnamic ET Terminator Cycle Sequencing Kit (GE Healthcare, Little Chalfont, United Kingdom) on an ABI 3730 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).
To estimate the similarity of newly discovered SHFV variants to the type strain and to prototypical arteriviruses, nucleic and amino acid sequences of individual ORFs were aligned using the MAFFT method [13] , alignments were concatenated, and uncorrected percent sequence identities for the resulting full-length coding genomes were calculated using the computer program MEGA4 [14] . To determine the phylogenetic relationships of newly discovered SHFV variants to known arteriviruses, nucleotide sequences of homologous ORFs in SHFV, EAV, LDV, and PRRSV (1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7, with reference to the EAV genome) were compiled from the literature, with strains chosen from available full-length genome sequences to represent the known within-species diversity of each virus. Non-overlapping coding regions were aligned using a codon-guided version of the MAFFT method [13] with poorly aligned sites removed using the Gblocks alignment cleaning method [15] implemented in the computer program TranslatorX [16] . Phylogenetic analyses were then conducted on concatenated, non-overlapping regions of aligned, cleaned ORFs, using first and second codon positions only, to avoid error associated with third-codon position saturation. Phylogenetic trees were constructed using the maximum likelihood method implemented in the computer program PAUP* 4.0 [17] . The substitution model used in the analysis was estimated using jModelTest [18] and was of the form GTR+I+C with the following parameters: nucleotide frequencies of A = 0.2386; C = 0.2413; G = 0.2633; T = 0.2568; substitution rates of AC = 2.5732, AG = 2.7382, AT = 1.4815, CG = 1.6751, CT = 2.1065, and GT = 1; I (proportion of invariant sites) = 0.1330; and C (gamma distribution of among-site rate variation) = 2.144, with 4 rate categories. Robustness of phylogenetic groupings was assessed in PAUP* using 1000 bootstrap replicates of the data [19] . New SHFV sequences were deposited in GenBank under accession numbers HQ845737 (SHFV-krc1) and HQ845738 (SHFV-krc2).
Direct pyrosequencing of RNA extracted from a blood plasma sample of a wild adult male red colobus from Kibale National Park, Uganda, generated 77,891 reads of 280 bp average length, consisting of 19,285 host reads (24.8%) and 58,606 non-host reads (75.2%). De novo assembly of singleton non-host reads resulted in a 15.5 kb contiguous sequence (contig) that aligned to the SHFV type strain LVR 42-0/M6941 (GenBank accession number NC_003092.1). A reference assembly against the 15.5 kb contig yielded 35,120 sequence reads with full coverage across the entire contig. Two additional contigs with lengths of 13 kb and 1.9 kb also aligned to the SHFV genome but were highly divergent from the initial 15.5 kb contig. The positions of these additional aligned contigs relative to the SHFV genome suggested that the two fragments might belong to the same viral variant. Using one step RT-PCR and 39 RACE, respectively, we were able to fill the gap between these two contigs and extend the 39 end of the contig to complete the terminal open reading frame (ORF9), yielding a new complete contig of 15.2 kb. A reference assembly against this 15.2 kb contig yielded 1,068 sequence reads with full coverage across the entire contig. Initial alignments indicated that both contigs covered all ORFs of the SHFV type strain and extended partially into the 39 and 59 untranslated regions. The 15.5 kb and the 15.2 kb contigs were designated SHFV-krc1 and SHFV-krc2, respectively, to indicate their origins in Kibale red colobus and to reflect nomenclature previously used to describe novel simian retroviruses in this same population of red colobus [20] .
Because we generated near full-length viral genome sequences, we were able to reconstruct the genomic architecture of SHFV-krc1 and SHFV-krc2 (Figure 1 ). SHFV-krc1 and SHFV-krc2 both possess two large putative replicase-encoding ORFs and several smaller downstream ORFs, all comparable in size to the published ORFs of type strain LVR 42-0/M6941 but all highly divergent at the amino acid level from the type strain (Table 1 ). Consistent with previously described arteriviruses, the putative ORF1a and ORF1b coding regions in both new SHFV variants contain a canonical heptanucleotide ''slippery sequence'' (UUUAAAC) and predicted downstream pseudoknot structure [7] . Importantly, both new viral genomes contain homologs of the type strain SHFV ORFs 2a, 2b, and 3 in the same genomic positions, indicating conservation of the unusual 39 genomic architecture of SHFV even among highly divergent variants.
Across the coding genome, the SHFV-krc1 and SHFV-krc2 showed only 51.9% nucleotide identity with each other and only 52.0% and 51.8% nucleotide identity, respectively, with type strain LVR 42-0/M6941, which is roughly equivalent to the nucleotide percent identity between the prototypical PRRSV and LDV strains (51.1%; Table 2 ). A maximum-likelihood phylogenetic tree constructed from concatenated nucleotide sequence alignments of homologous ORFs from representative arteriviruses is shown in Figure 2 . The tree is consistent with established phylogenetic relationships among the arteriviruses [21, 22] , supporting with high statistical confidence the close relationship between PRRSV and LDV relative to SHFV and EAV. Within the SHFV clade, SHFV-krc1 and SHFV-krc2 are highly divergent from each other and from the type strain SHFV LVR 42-0/ M6941. Although SHFV-krc1 and SHFV-krc2 cluster together, statistical support for this relationship is relatively weak even with near-complete genome sequences. Overall, the degree of phylogenetic diversity within SHFV is remarkably high and is approximately equal to that between PRRSV and LDV.
Using direct pyrosequencing, we have demonstrated infection of a male red colobus monkey from Kibale National Park, Uganda, with two novel SHFV variants. To our knowledge, this is the first report of SHFV in a wild primate. These new viruses, SHFV-krc1 and SHFV-krc2, are highly divergent from each other and from the type strain (approximately 52% nucleotide sequence identity among the three variants). Both new viruses nevertheless contain homologs of the type strain SHFV ORFs 2a, 2b, and 3 in the same genomic positions.
Our results shed light on the natural history of SHFV. Asymptomatic infections of captive patas monkeys, vervet monkeys, and baboons support the notion that African primates are the natural hosts of SHFV [2, 3] . However, SHFV has, to our knowledge, only been isolated from these species upon their arrival in primate colonies, raising the possibility of infection during transport. The two new SHFV variants that we have discovered were isolated from a wild Ugandan red colobus monkey in a natural setting, providing conclusive evidence that wild African primates are natural hosts of SHFV.
Phylogenetic analysis of SHFV-krc1 and SHFV-krc2 demonstrate the diversity of SHFV to be higher than has been documented for any other arterivirus. Indeed, the degree of phylogenetic divergence between the two new SHFV strains and the type strain is approximately equivalent to the degree of divergence between PRRSV and LDV, which are considered different viral species. We caution, however, that phylogenetic diversity within the other arteriviruses may be underestimated due to small numbers of currently available full genome sequences. For example, divergent EAV strains have been documented [23, 24] but, at the time of this writing, full genomes of such EAV isolates were not available. Unfortunately, our attempts to align the available hypervariable partial ORF5 sequences of these divergent EAV strains with homologous regions from other arteriviruses failed due to low sequence similarity. Future studies of EAV and other as-yet undiscovered arteriviruses may demonstrate levels of within-species diversity that match or even exceed what we have documented for SHFV.
Particularly noteworthy was our finding of natural co-infection of a single primate host with two highly divergent SHFV variants. To our knowledge, no instance of co-infection of a single host with such highly divergent arteriviruses has previously been reported. Kibale red colobus may simply harbor a very diverse population of SHFV variants. However, it is also possible that SHFV-krc1 or SHFV-krc2 may have been transmitted to this animal from another species. For example, red colobus in Kibale form polyspecific associations with other primates, most frequently with red-tailed guenons (Cercopithecus ascanius) but also with black-andwhite colobus (Colobus guereza), blue monkeys (Cercopithecus mitis), and grey-cheeked mangabeys (Lophocebus albigena) [25] . Because such interactions can involve close proximity and direct contact, polyspecific associations could facilitate the cross-species transmission of viruses that require close contact, such as SHFV.
Our results highlight the utility of direct pyrosequencing for detecting novel viruses and for characterizing viral genomic architecture during natural infection. The combination of random hexamer-primed reverse transcription and double-stranded cDNA synthesis used in this study obviated the need for PCR amplification to generate suitable amounts of DNA for pyrosequencing libraries. This method also prevented the potential introduction of amplification-induced error and template bias associated with whole genome amplification [26] . Despite marked divergence from each other and from type strain LVR 42-0/M694, SHFV-krc1 and SHFV-krc2 share the same 39 genomic architecture as the type strain, consisting of additional ORFs 2a, 2b, and 3, which are not present in the other arteriviruses ( Figure 1 ). Because our methods did not involve tissue culture, which can introduce genetic anomalies as viruses replicate in, and potentially adapt to, cells or cell lines derived from non-natural host species, we conclude that the unique 39 genomic architecture of SHFV in comparison to the other arteriviruses is authentic and characteristic of SHFV. Future studies of isolated viruses should help clarify the stability of SHFV in cell culture and should facilitate investigations of biological differences among SHFV variants, including their ability to be detected by available serologic diagnostics tests [27] . Finally, our results have implications for primate health and conservation. The animal sampled was apparently healthy at the time of capture and, as of the time of this writing, continues to behave normally within its social group. Preliminary data (unpublished) indicate that this particular animal is also infected with the recently discovered simian immunodeficiency virus SIVkrc [20] , and that other apparently healthy red colobus in the same location are infected with SHFV-krc. This evidence supports the idea that wild primates can harbor SHFV subclinically, although long-term field observations of infected and uninfected animals will be required to detect any negative effects of infection and co-infection with other viruses on host fitness. Nevertheless, our findings underscore that wild primates can be reservoirs for unknown pathogens of potential concern for global health, and that extreme care should therefore be taken when introducing primates into new environments. Indeed, we suggest that direct pyrosequencing or other similar technologies for broad viral screening might prove useful in settings such as zoos, primate colonies, sanctuaries, and primate reintroduction programs where asymptomatic carriers of novel viral pathogens might come into contact with clinically susceptible hosts.  Live Bird Markets of Bangladesh: H9N2 Viruses and the Near Absence of Highly Pathogenic H5N1 Influenza Avian influenza surveillance in Bangladesh has been passive, relying on poultry farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, the results of an active surveillance effort focusing on the live-bird markets are presented. Prevalence of influenza infection in the birds of the live bird markets is 23.0%, which is similar to that in poultry markets in other countries. Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also observed. The highly pathogenic H5N1-subtype virus was observed at extremely low prevalence in the surveillance samples (0.08%), and we suggest that the current risk of infection for humans in the retail poultry markets in Bangladesh is negligible. However, the high prevalence of the H9 subtype and its potential for interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and attenuation of host morbidity, highlight the importance of active surveillance of the poultry markets. Influenza A is a negative-strand RNA virus that uses aquatic birds as reservoir hosts [1, 2] and is classified by the surface proteins hemagglutinin (HA) and neuraminidase (NA). There are 16 HA and 9 NA subtypes, but not all combinations have been recovered from aquatic birds [2] . In the aquatic bird host, replication primarily occurs in the intestinal tract [3] . As such, most avian influenza viruses (AIV) cause only limited morbidity and mortality in birds. However, several subtypes, including H5N1, H7N7, H9N2, can infect the lungs and respiratory tracts of birds (and other animals), resulting in significant levels of disease and death [4, 5, 6, 7, 8, 9] .
The first human outbreak of the highly pathogenic (HP) H5N1 subtype occurred in Hong Kong in 1997 [4, 9] . Since then, surveillance efforts to detect the HP H5N1-subtype virus in birds have increased in Asia. HP H5N1 has been detected in animals in Bangladesh, and in the nearby countries of Bhutan, China, India, Myanmar, Nepal, and Thailand. Human infections with HP H5N1 have been reported in Bangladesh and Myanmar (http:// www.oie.int/eng/info_ev/en_AI_avianinfluenza.htm). As of February 24, 2011, there have been 384 reported outbreaks of HP H5N1 subtype at either backyard or commercial farms in 49 of the 64 districts of Bangladesh [10] .
In addition to HP H5N1, the H9N2 subtype has been implicated in contributing to the influenza outbreak in 1997. Extensive surveillance efforts to discover the source of the Hong Kong pandemic revealed that influenza of subtype H9N2 was being isolated from chickens at the same markets that had HP H5N1-positive chickens [8] . Although the overall prevalence of the H9N2 subtype was low (4.4%), one market exhibited an unusually high prevalence (36.6%). During this outbreak, most chickens appeared healthy [8] . This observation and later experiments suggest that chickens that were previously infected by a H9N2-subtype virus may have been partially protected from the pathogenicity induced by a HP H5N1-subtype infection, which could have allowed the HP H5N1-subtype virus to 'silently' attain a prevalence where transmission to humans was probable [11, 12] . After the mass slaughter of poultry at these markets, HP H5N1 subtype was thought to be eradicated until it re-emerged in Hong Kong bird markets in 2001 [13] . Phylogenetic analysis later revealed that the HP H5N1 subtype was a reassortant virus that had obtained 6 internal gene segments from the H9N2-subtype virus A/Quail/Hong Kong/G1/97 (G1) [14] and the NA gene segment from the H6N1-subtype virus A/Teal/Hong Kong/ W312/97 [15] . These H9N2-subtype viruses that contain gene segments similar to those of the HP H5N1 subtype have become established and are circulating in the poultry markets in Southeast Asia and the Middle East [16, 17, 18, 19, 20, 21] . Thus, the internal genes of the HP H5N1 virus have remained in circulation in the poultry.
Most of the information regarding influenza infection in Bangladesh has focused on passive surveillance of backyard or commercial farms [22, 23] . This report primarily details influenza infection in several live bird markets of Bangladesh because of the hypothesized role that live bird markets play on the epidemiology of AIV [8, 19] . Active surveillance efforts were concentrated on the retail markets, with farms, pet bird markets, and migratory birds also being sampled. H9N2-subtype influenza is circulating in the bird markets, and HP H5N1 subtype has been found, albeit at extremely low levels. These findings highlight the need for continued, active surveillance in poultry markets.
Active influenza surveillance was performed in Bangladesh from November 2008 through April 2009, and from December 2009 through July 2010 ( Figure 1) . Because AIV has a fecal-oral transmission route, samples were collected by taking oropharyngeal or cloacal swabs of birds (host samples) or by collecting from feces, fecal digestors, water troughs, and standing water (environmental samples). Each month, 300-600 total samples were collected across several locations. The primary sampling locations were retail markets in Dhaka (Market-1 to Market-4); one pet bird market, a layer farm (Farm-1), and a natural lake were also included. After news of an outbreak in 2009, surveillance was expanded to several villages in the area of the outbreak. The sampling sites in the expanded area were mostly backyard (domestic) flocks (Village-1 to Village-5), but several samples were also obtained from retail markets in 2 villages (Market-5 and Market-6) and from a poultry farm that culled their flock of chickens (Farm-2). None of the birds that were swabbed or observed near the sampling location exhibited signs of disease, and none of the sampling sites were reported as an outbreak area during the study.
Samples were screened in 1 of 2 ways. Initially, all swab samples were injected into 10-day-old embryonated chicken eggs. After 72 hr of incubation at 35uC, eggs were chilled and harvested. Influenza-positive eggs were detected by testing for hemagglutination of 0.5% chicken erythrocytes according to standard procedures [24] . Shortly after the start of the surveillance program, real-time RT-PCR (rRT-PCR) was selected as an alternative to virus isolation in eggs to decrease the processing time of the samples. Viral RNA was extracted by using a KingFisher Flex Magnetic Particle Processor (ThermoFisher Scientific, Waltham, MA) and subjected to rRT-PCR using influenza Aspecific primers and probes [25] . The reactions were performed in an ABI 7500 Fast Real-Time PCR machine (Applied Biosystems, Carlsbad, CA). All rRT-PCR-tested positive samples, and some rRT-PCR-tested negative samples, were injected into eggs to confirm the presence, or absence, of infectious virus. The percentages of positive samples reported in this manuscript exclude those samples that were subtyped as Newcastle disease virus (NDV) unless otherwise noted in the text. Differences in prevalence were assessed by using the proportions test in R version 2.11.1 (www.R-project.org).
Where virus isolation was successful, sequence analysis [26] or hemagglutination inhibition (HI) assays [27] were performed to determine the subtype of influenza-positive samples. The Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital analyzed the sequences on Applied Biosystems' 3700 DNA analyzers by using BigDye Terminator (v.
3) chemistry and synthetic oligonucleotides. If cloacal and oropharyngeal swabs of the same host had more than one subtype of influenza, then the infection was identified as a mixed infection.
To assess the pathogenicity of the viruses, the intravenous pathogenicity index (IVPI) for several isolates was determined according to standards established by the World Health Organization [28] . Briefly, 0.1 mL of a 1:10 dilution of infective egg chorioallantoic fluid in sterile PBS was intravenously injected into each of 10 six-week-old, specific pathogen-free (SPF) chickens. The birds were examined twice daily for 10 days, and individuals were scored on the basis of the observed morbidity (0 = normal, 1 = sick, 2 = severely sick, 3 = dead). Chickens were used in accordance with protocols approved by the Institutional Animal Care and Use Committee at St. Jude Children's Research Hospital (protocol 081). The study was approved by the Institutional Biosafety Committee at St. Jude Children's Research Hospital (#03A-137 and #02A-221).
A total of 5715 samples from several locations in Bangladesh were tested for AIV. The results of the virus isolation and rRT-PCR detection methods on identical samples were in agreement for 86.4% of the samples. Comparing the percent positive between the 2 methods is not meaningful because rRT-PCR was used to test 84.1% of the samples, yet virus isolation was performed on most of the suspected positives and only a small portion of negatives. Therefore, the percentage of infection as identified by virus isolation will be higher than expected if all of the samples had been tested by this method.
For some of the samples, cloacal and oropharyngeal swabs were collected from the same bird. The results of the matched swab samples agreed for 77.8% (n = 239) and 78.6% (n = 714) of the hosts when tested by the virus isolation and rRT-PCR detection methods, respectively. Overall, oropharyngeal swabs were more likely to test positive than cloacal swabs were regardless of detection method. Furthermore, this observation holds true when swabs were analyzed by sampling site (Table 1) .
Hosts were considered positive for infection if at least one of the host samples, i.e., oropharyngeal or cloacal swabs, tested positive by virus isolation or rRT-PCR ( Table 2 ). The prevalence of influenza in hosts varied across the sampling sites, and analyses of prevalence revealed that differences exist across the sites for both virus isolation and rRT-PCR detection methods (p#0.0001 and p#0.00001, respectively). A significant difference in prevalence across the sites was also detected for the environmental samples (feces, fecal digestors, water, and water troughs) and when all samples, regardless of type, were tested by both detection methods (p#0.00001 for all comparisons). In Dhaka, the overall prevalence varied by site, but this difference was mostly due to low prevalence at the Pet bird market, Farm-1, and the Lake. That is, prevalence was approximately equal at the markets ( Table 2 ). Weighted average of prevalence for the markets in Dhaka was 47.3% and 23.0% for samples tested by virus isolation and rRT-PCR, respectively.
Nearly all of the host samples (84.3%) were collected from chickens (Phasianidae), the most abundant species at most of the sites. Exceptions include the lake, where the lesser whistling duck (Anatidae) is the primary inhabitant, and the pet bird market. The species list at the pet bird market includes species of the following: cockatiels (Cactuidae); doves (Columbidae); finches (Fringillidae); moorhens (Rallidae); mynah (Sturnidae); woodpecker (Picidae); love birds and parakeets (Psittacidae); munia and sparrows (Estrildidae); and pheasants and quail (Phasianidae). All birds in our dataset were grouped by family to increase the sample size of the host samples so that differences in prevalence of infection as detected by virus isolation could be examined. The major families that were infected included the Columbidae (11.4% prevalence, n = 114), Anatidae (25.0% prevalence, n = 24), and Phasianidae (33.2% prevalence, n = 754); prevalence differs by family (p#0.0001).
Subtyping of viruses by hemagglutination inhibition assays or by sequencing could only be performed on the 20.4% of samples that were influenza-positive by egg isolation. This represents 252 virus isolates from individual hosts or environmental samples (Table 3) . Twelve isolates were subtyped as containing only NDV. Most of the influenza isolates were H9-subtype viruses (94.2%). The H9 subtype was identified at most of the retail markets ( Figure 1 ). Based on the likelihood of obtaining the H9 subtype from rRT-PCR-positive host or environmental samples (76.3% of rRT-PCR positive samples were also virus isolation positive; 94.0% virus isolation positive isolates from the markets of Dhaka were H9-subtype viruses), the estimated prevalence of the H9-subtype in Dhaka is 16.5%. The H9-subtype isolates that were completely subtyped were all H9N2-subtype viruses. Other subtypes were observed in the markets including H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 ( Table 3 ). The HA2 region of the H1-subtype viruses, which was used for subtyping by sequencing, is most similar (.90%) to that of avian H1 viruses and not to the pandemic H1-subtype viruses, with highest homology (94%) to A/ goose/Italy/296426/2003 (H1N1). Although none of the birds that were sampled or in proximity to the sampling location were overtly sick, the IVPI was calculated for 5 isolates of the H9N2 subtype (A/Chicken/Bangladesh/559/2008, A/Environment/ Bangladesh/600/2008, A/Environment/Bangladesh/907/2009, A/Environment/Bangladesh/5473/2010, and A/Environment/ Bangladesh/5721/2010) and one H5N1-subtype isolate (A/ Chicken/Bangladesh/828/2009). All of the H9N2-subtype isolates were non-pathogenic (IVPI = 0), but the H5N1-subtype virus was highly pathogenic (IVPI = 2.95).
Seventeen H9N2-subtype isolates were tested using HI assays to determine the specific H9-antigenic group of the Bangladesh (12) Village-2 Ck 0 (7) 0 (7) 0 (7) 0 (7) 0 (7) 0 (7)
Village-4 Ck 0 (7) 0 (7) 0 (7) 0 (7) 0 (7) 0 (7) VIllage-5 Ck/Dk 0 (2) 0 (1) 0 (2) 0 (5) 0 (5) 0 (5) (12) Village-2 Ck 0 (7) 0 (7) 0 (7) 0 (7) Village isolates (Table 4 ). Included in the analyses were chicken a-H9 sera that was generated during the IVPI experiments by using A/ Chicken/Bangladesh/559/2008 (H9N2) and A/Environment/ Bangladesh/600/2008 (H9N2). The isolates from Bangladesh reacted strongly to all of the anti-H9 sera. Most of the isolates reacted less strongly to the a-G1 sera than to the remaining antisera.
Influenza surveillance in Bangladesh began in 2007 and was focused on the commercial and backyard farms [22, 23] . Here, we concentrate on the live bird markets because of the hypothesized role that live bird markets play on the epidemiology of AIV [8, 19 ]. An outbreak of AIV was reported in 2009 and surveillance was extended to villages in that area, but the primary focus remained on the markets.
Several conclusions can be drawn from this study. First, prevalence in the migratory waterfowl that visit the Lake is extremely low (1.7%), which suggests that AIV might not be maintained in birds at the lake ecosystem. Second, Farm-1 is a contaminated area, but the transmission within the flock appears to be at a minimum level as shown by the low prevalence at the site compared to that in the markets (rRT-PCR: 4.2% vs. 23.0%; Table 2 ). Third, although environmental samples collected near the birds (e.g., cockatiels, parrots, munia, quail) at pet bird markets tested positive for infection, only two samples were influenza A and the rest were subtyped as NDV. The 2 influenza-positive samples were subtyped as H9N2 and were collected from quail feces during the same visit to the pet bird market. Thus, the role of pet bird markets in perpetuating and spreading AIV remains unclear. Lastly, AIV is circulating in the 3 retail markets that were sampled in Dhaka. Chickens are the primary birds at these markets, but influenza has also been isolated from the ducks at all 3 locations. Therefore, there exists the possibility of interspecies transmission of influenza and an increased probability of reassortment between various lineages and subtypes. In fact, others have postulated that live bird markets are the ideal environment for influenza transmission because of the high density and variety of hosts within a localized area [19, 29, 30] . Indeed, reassortment of H9-subtype viruses in bird markets has been previously reported [18, 19, 20] .
The H9N2-subtype viruses were primarily isolated from chickens. In contrast, all but 1 of the H1, H3, H4, and H10 subtypes were recovered from ducks; a single H1N2-subtype virus was isolated from a water trough in a chicken cage. The ducks could have obtained these infections at the market, but it is more likely that the birds were infected prior to arrival at the market and are contributing to the diversity of subtypes that exist in these locations [19] . This diversity increases the probability that reassortment could occur between influenza subtypes and produce a novel pathogenic strain. Of particular importance was the isolation of several H5N1-subtype viruses (clade 2.2), 2 of which were isolated from chickens in the same bird market in January 2009 and the remaining 2 were from fecal samples of ducks in a different poultry market in January 2010. These H5N1-subtype isolates exhibit a polybasic cleavage site (-PQGERRRKKR-GLFG-) that is characteristic of highly pathogenic viruses. Moreover, one isolate (A/Chicken/Bangladesh/828/2009 [H5N1]) is also classified as highly pathogenic because of its IVPI. The isolation of H5N1-subtype viruses in the poultry markets could be related to outbreaks that were reported in commercial farms at approximately the same time. Specifically, an outbreak at a poultry farm in Mirpur, Dhaka, was reported on January 7, 2009 (http://www.oie.int/eng/info_ev/en_AI_avianinfluenza.htm), and HP H5N1-subtype viruses were isolated 2 days later from chickens at a poultry market in Dhaka. Although no outbreaks were reported at commercial poultry operations in the Dhaka district in Janurary 2010, an H5N1-subtype outbreak was reported at a farm in the Sirajgonji district, and another was reported in the Jaipurhat district (http://www.oie.int/eng/in-fo_ev/en_AI_avianinfluenza.htm). Infected birds could have been transported to the market, but it is unknown if the infected birds were from commercial farms or from backyard flocks. Thus, the source of HP H5N1-subtype viruses in the bird markets cannot be determined. Interestingly, the chickens that were shedding the HP H5N1-subtype virus and the ducks from which the H5N1-infected fecal samples were collected were apparently healthy at the time of sampling, and H9N2-subtype viruses were isolated from other birds at the same markets. This raises an important question regarding the epidemiology of viruses of the H5N1 and H9N2 subtypes in the bird markets: are the H9N2-subtype viruses contributing to highly pathogenic H5N1-subtype virus outbreaks? Before the H5N1-subtype outbreak in Hong Kong, no chickens and very few ducks tested positive for H5N1-subtype viruses, and H9N2-subtype viruses were only isolated from ducks in those markets [8] . Extensive surveillance during the outbreak in 1997 revealed high prevalence of the HP H5N1 subtype in apparently healthy chickens. This also corresponded with the surprising find of the H9N2 subtype in chickens, which had been reported only in ducks in the markets of Southeast Asia prior to the 1990's [8] . The prevalence of the H9N2 subtype in the Hong Kong markets in 1997 (4.4%) [8] was lower than its estimated prevalence in the Bangladesh retail markets (16.5%). Interestingly, a single retail market in Hong Kong in 1997 had an isolation rate of 36.6% for the H9N2 subtype, and the HP H5N1 subtype was isolated at the same market [8] . Later studies demonstrated that the H9N2 subtype might have allowed the HP H5N1 subtype to persist and circulate in poultry. Specifically, a previous H9N2-subtype infection could confer protection against an HP H5N1-subtype challenge [11, 12] . This may explain why the HP H5N1-infected chickens in the market were asymptomatic at the time of sample collection even though the virus killed all SPF chickens in the IVPI experiment by day 2 post-infection. Given that the primary influenza A subtype in poultry markets that we have been sampling in Bangladesh is H9N2, that HP H5N1-subtype outbreaks were reported in poultry in 2007 and continue today [22] , and that HP H5N1-subtype viruses were isolated in the markets, Bangladesh could experience an outbreak similar to the Hong Kong 1997 outbreak.
Molecular analysis of the H9N2-subtype viruses that were in circulation in 1997 in Hong Kong suggest that H9N2 viruses have established a stable lineage in chickens [14, 31] . A single H9N2subtype virus, A/Quail/Hong Kong/G1/97 (G1), was most similar to the HP H5N1 subtype from the 1997 outbreak in 6 of the 8 gene segments, and this lineage still circulates today [14, 32] . Serological evidence suggests that the H9N2-subtype isolates from Bangladesh are less like the G1-like reference virus, but the high degree of reactivity to the remaining reference sera obfuscates serological classification. The H9N2-subtype viruses from the United Arab Emirates exhibited a similar pattern of HI assay titers. In the original analysis, the authors concluded that these viruses were antigenically similar to the A/Duck/Hong Kong/ Y280/97 (H9N2) virus (Y280 lineage), yet the results of phylogenetic characterization suggest that these viruses are genetically similar to the G1-like viruses [16] . Genetic characterization of the viruses from Bangladesh is in progress to examine the molecular evolution of the H9N2 subtypes in that country.
A higher percentage of oropharyngeal compared to cloacal swabs were positive for influenza infection. This observation raises concern regarding the mode of transmission for these viruses. Several H9N2-subtype viruses can transmit through direct contact with infected chickens and ferrets [33, 34] . It is this route of infection that likely produced the H9N2-subtype infections that have been reported in humans [6, 7, 22] . However, aerosol transmission has been observed in chickens infected with a Beijing/1-like virus (A/Chicken/Shanghai/F/98 [H9N2]) [33] , and this could explain the high prevalence of the H9N2 subtype in the live-bird markets of Bangladesh. Aerosol transmission has not been observed between ferrets that were infected with H9N2subtype viruses, suggesting that transmission between mammals is currently limited [34] .
Influenza A of subtype H9N2 is established and circulating in poultry throughout the Middle East and Asia [16, 17, 18, 19, 20, 21] . Whereas previous studies in Bangladesh identified H9N2-subtype viruses on 3 farms [22] , current surveillance efforts demonstrate that H9N2 is the primary subtype circulating in chickens at the retail markets. In the markets of Bangladesh, the H9N2 subtype has an estimated prevalence of 16.5%, which is higher than the reported prevalence of 7-8% in the live-bird markets in South Korea [18, 20] . Overall prevalence of the H9N2 subtype is lower in the markets of southern China from 2000-2005, but prevalence peaked at 13% in chickens, 22% in other minor poultry species, and 3% in ducks [35] . The H9N2 subtype has been isolated sporadically from the swine population throughout China [36, 37, 38] . The results of the phylogenetic analyses of the swine isolates suggest that these viruses are reassortants between H5-and H9-subtype viruses [36, 37] . To date, the H9N2-subtype reassortant viruses have not established a stable lineage in the swine population, and it is unknown if these reassortants are currently circulating in the live-bird markets.
The H9N2 subtype in the markets is of concern to humans because many isolates, including all of those sequenced in this study, possess the L226 mutation in the receptor binding pocket of the HA1 protein. This mutation confers a higher degree of specificity to sialic acid residues that contain the human-like a-2,6 linkage [39] , and it has allowed the H9N2-subtype virus to grow in human airway epithelial cells [40] and be directly transmitted in ferrets [34] . The L226 mutation and the potential for aerosol transmission increases the risk of H9N2-subtype infection for humans that work in and visit the markets. Moreover, the H9N2 subtype may be modulating the morbidity and mortality that is associated with infection with the HP H5N1 subtype. Thus, early detection of the HP H5N1 subtype requires continuing surveillance efforts in the poultry markets. Monitoring Influenza Activity in the United States: A Comparison of Traditional Surveillance Systems with Google Flu Trends BACKGROUND: Google Flu Trends was developed to estimate US influenza-like illness (ILI) rates from internet searches; however ILI does not necessarily correlate with actual influenza virus infections. METHODS AND FINDINGS: Influenza activity data from 2003–04 through 2007–08 were obtained from three US surveillance systems: Google Flu Trends, CDC Outpatient ILI Surveillance Network (CDC ILI Surveillance), and US Influenza Virologic Surveillance System (CDC Virus Surveillance). Pearson's correlation coefficients with 95% confidence intervals (95% CI) were calculated to compare surveillance data. An analysis was performed to investigate outlier observations and determine the extent to which they affected the correlations between surveillance data. Pearson's correlation coefficient describing Google Flu Trends and CDC Virus Surveillance over the study period was 0.72 (95% CI: 0.64, 0.79). The correlation between CDC ILI Surveillance and CDC Virus Surveillance over the same period was 0.85 (95% CI: 0.81, 0.89). Most of the outlier observations in both comparisons were from the 2003–04 influenza season. Exclusion of the outlier observations did not substantially improve the correlation between Google Flu Trends and CDC Virus Surveillance (0.82; 95% CI: 0.76, 0.87) or CDC ILI Surveillance and CDC Virus Surveillance (0.86; 95%CI: 0.82, 0.90). CONCLUSIONS: This analysis demonstrates that while Google Flu Trends is highly correlated with rates of ILI, it has a lower correlation with surveillance for laboratory-confirmed influenza. Most of the outlier observations occurred during the 2003–04 influenza season that was characterized by early and intense influenza activity, which potentially altered health care seeking behavior, physician testing practices, and internet search behavior. The emergence of 2009 pandemic influenza A (H1N1) virus in the United States and Mexico, and its subsequent rapid global spread has underscored the importance of influenza surveillance for public health decision making [1] . Recently, Google.org developed Google Flu Trends, a model to estimate US influenza-like illness (ILI) rates from internet searches. The model was fit to CDC sentinel provider surveillance data for ILI from 2003 to 2007 and prospectively validated using the same surveillance system during the 2007-08 influenza season. During the 2007-08 influenza season, Google Flu Trends estimates were highly correlated to CDC surveillance for ILI, with a mean correlation coefficient over nine US Census Regions of 0.97 [2] .
For the purpose of CDC influenza surveillance, ILI is defined as a fever $37.8˚C and a cough and/or a sore throat without known etiology (http://www.cdc.gov/flu/weekly/fluactivity.htm, accessed 09/04/09). ILI is not specific to influenza, however. Prospective studies with laboratory sampling of persons with ILI have demonstrated a wide variability in the specificity of ILI for influenza disease, with the proportion of subjects testing positive for influenza ranging from 20% to 70% of those tested during the influenza season [3, 4] . ILI may also not be sensitive for influenza, particularly in certain age or risk groups where influenza may have atypical presentations [5, 6, 7, 8, 9] . Furthermore, even during peak periods of influenza circulation, a substantial number of cases of febrile respiratory illness may have non-influenza etiologies. In the United States, during the spring wave of the 2009 H1N1 outbreak from March through August 2009, the proportion of positive influenza laboratory tests did not exceed 45% (http://www.cdc.gov/flu/ weekly, accessed 09/04/09).
Because Google Flu Trends estimates of ILI may not necessarily correlate with actual influenza virus infections, we undertook this study to evaluate how Google Flu Trends influenza surveillance data compared with national surveillance data for laboratoryconfirmed influenza infections.
Methods National and regional estimates of the weekly percentage of persons seeking health care in the United States with ILI were obtained from Google Flu Trends on June 11, 2009 (http://www. google.org/about/flutrends/us-historic.txt). The remaining surveillance data were obtained from CDC. These data were from two separate surveillance networks: Outpatient Influenza-like Illness Surveillance Network (CDC ILI Surveillance) and the US Influenza Virologic Surveillance System (CDC Virus Surveillance). CDC ILI Surveillance consists of a network of health care providers who record the weekly proportion of patients who present with non-specific signs and symptoms that meet a case definition of influenza-like illness [10] . CDC Virus Surveillance consists of about 140 laboratories located throughout the United States that report the weekly total specimens tested and laboratory tests positive for influenza virus [10] . This is the only US surveillance system that provides national and regional data of laboratory-confirmed influenza virus infection. CDC Virus Surveillance is used in CDC statistical models in the estimation of influenza-associated morbidity and mortality [11, 12, 13, 14, 15, 16] . For this analysis, CDC Influenza Virus Surveillance was  used as the reference standard to which Google Flu Trends and  CDC ILI Surveillance were compared.  The study period was September 28, 2003 through May 17,  2008. These dates were chosen to include all available Google Flu  Trends historical ILI estimates and exclude the 2009 H1N1  pandemic which began during the 2008-09 influenza season. Analyses were performed by ''influenza season,'' defined as the period from July 1 through June 30 of the subsequent calendar year. As done in similar analyses [2] , we restricted our analysis to the period during which CDC influenza surveillance is intensified, from calendar week 40 through calendar week 20 of the subsequent year.
For the primary analysis, scatter plots with least square regression lines were constructed to compare Google Flu Trends and CDC ILI Surveillance to the standard reference surveillance (CDC Virus Surveillance). Pearson's correlation coefficients with 95% confidence intervals (95% CI) were then computed from these comparisons. Subsequently, additional correlation coefficients with 95% CI were calculated from surveillance comparisons by influenza season, US Census Region, and influenza season categorized by US Census Region. These subset analyses were for the non-specific complaint of influenza-like illness (ILI) based on internet key word searches. 2 CDC Influenza-like Illness Surveillance involves a network of health care providers who record the weekly proportion of patients seen with ILI. Google Flu Trends was created and validated using CDC ILI Surveillance data, explaining the similarity between the two curves. 3 CDC Influenza Virologic Surveillance consists of about 140 laboratories located throughout the United States that report the weekly total specimens tested and laboratory tests positive for influenza virus. This is the only US surveillance system that provides national and regional data of laboratory-confirmed influenza virus infection. 4 Because CDC surveillance is intensified from calendar week 40 through calendar week 20 of the subsequent year, we restricted our correlation analyses to this time period. doi:10.1371/journal.pone.0018687.g001 summarized with mean correlation coefficients and standard deviations (SD). Next, because Flu Trends was previously found to lead CDC ILI Surveillance observations by one to two weeks [2] , we undertook additional correlation analyses to determine whether Google Flu Trends or CDC ILI Surveillance had a stronger correlation with CDC Virus surveillance data for the subsequent one or two weeks. The unit of analysis was percentage of clinic patients with ILI (CDC ILI Surveillance and Google Flu Trends) and percentage of laboratory tests positive for influenza (CDC Virus Surveillance).
To ensure this assessment of surveillance data was comparable to the study that validated Google Flu Trends [2] , we performed a secondary analysis replicating methods from that study with our dataset. The mean coefficient of correlation between Google Flu Trends and CDC ILI Surveillance was calculated over nine US Census Regions for the 2007-08 influenza season. The mean coefficient of correlation between Google Flu Trends and CDC Virus Surveillance was calculated similarly for comparison.
We also performed a secondary analysis to determine the sensitivity of the primary analysis to high-leverage, outlier observations. First, we performed simple linear regression to evaluate the association between either Google Flu Trends or CDC ILI Surveillance rates with reference viral surveillance data as standard rates. The effect of outlier observations was assessed with differences in the beta statistic (DFBETA). Individual observations were considered influential if they had a DFBETA greater than the absolute value of 2 divided by the square root of the total number of observations in the model [17] . Subsequently, all influential observations were excluded and correlation coefficients were recalculated as had been done in the primary analysis. Correlation coefficients from the sensitivity analysis were compared to the same statistic from the primary analysis to determine whether any relevant changes in the strength of correlation had occurred with the removal of influential observations. Last, Spearman Rank correlation coefficients were employed for the primary analyses and noted to yield similar results.
This study received exempt review status from the Human Subjects Division at the University of Washington. Analyses were performed with STATA statistical software (version 10.1; STATA Corporation; College Station, TX).
Our analyses used 166 weeks of data from the 2003-04 through the 2007-08 influenza seasons obtained from three influenza surveillance systems used to monitor national and regional influenza trends. Data included five influenza seasons from 2003-04 through the 2007-08 influenza season. There was a strong temporal association among rates from each surveillance system ( Figure 1 ). Scatter plots of Google Flu Trends and CDC ILI Surveillance with the reference standard data showed high linear correlations ( Figure 2 and Figure 3 ). Pearson's correlation coefficient describing the strength of association between Google Flu Trends with CDC Virus Surveillance was 0.72 (95% CI: 0.64, 0.79) ( Figure 2 and Table 1 ). The strength of association between CDC ILI Surveillance and CDC Virus Surveillance was higher, with a correlation coefficient of 0.85 (95% CI: 0.81, 0.89) ( Figure 3 and Table 1 ). Google Flu Trends, which had been fit to CDC ILI Surveillance, was highly correlated to that surveillance data (R = 0.94; 95%CI: 0.92, 0.96).
Correlations among influenza surveillance systems differed by influenza season. The correlation coefficient describing the association between Google Flu Trends and CDC Virus surveillance ranged from 0.67 (95% CI 0.43, 0.82) during the 2003-04 influenza season to 0.94 (95% CI 0.89, 0.97) during the 2004-05 influenza season ( Table 2 ). The mean correlation coefficient for these comparisons was 0.79 (SD 0.13). The correlation between CDC ILI Surveillance and CDC Virus Surveillance ranged from 0.79 (95% CI 0.62, 0.89) during the 2005-06 influenza season to 0.94 (95% CI 0.88, 0.97) during the 2004-05 influenza season. The mean correlation coefficient for these comparisons was 0.86 (SD 0.07).
Correlations among influenza surveillance systems also differed by US Census Region. The correlation coefficients describing the association between Google Flu Trends and CDC Virus Surveillance over the study period ranged from 0.64 (95% CI 0.54, 0.72) in the East North Central Region to 0.80 (95% CI 0.74, 0.85) in the West North Central Region ( Table 3 ). The mean correlation coefficient for these comparisons was 0.70 (SD 0.05). The correlation between CDC ILI Surveillance and CDC Virus Surveillance ranged from 0.64 (95% CI 0.55, 0.73) in the East South Central Region to 0.86 (95% CI 0.81, 0.89) in the West South Central Region. The mean correlation coefficient for these comparisons was 0.76 (SD 0.07).
We assessed whether Google Flu Trends or CDC ILI Surveillance had a higher correlation with diagnostic tests positive With the exception of the 2003-04 influenza season, correlation coefficients decreased proportionately when seasonal Google Flu Trends or CDC ILI Surveillance were assessed against seasonal CDC Virus Surveillance for the subsequent one or two weeks (Table S1 ). The correlation over the 2003-04 influenza season between Google Flu Trends and CDC Virus Surveillance increased from 0.67 to 0.77 when assessed with a one-week lag and 0.82 when assessed with a two-week lag. CDC ILI Surveillance had a stronger increase from 0.80 to 0.89 with a one-week lag and 0.92 with a two-week lag.
The use of regional surveillance comparisons did not result in increased correlation with virologic surveillance of the subsequent one or two weeks. The mean Google Flu Trends correlation with CDC Virus Surveillance was 0.70 (SD 0.05) at baseline, 0.70 (SD 0.07) with a one-week lag, and 0.63 (SD 0.11) with a two-week lag ( Table S2 ). The mean CDC ILI Surveillance correlation with CDC Virus Surveillance decreased from 0.78 (SD 0.04) at baseline to 0.76 (SD 0.08) with a one-week lag and 0.68 (SD 0.11) with a two-week lag. Calendar week of peak influenza activity per influenza surveillance year for each of the three surveillance systems can be found in Table S3 .
We replicated the analysis that validated Google Flu Trends by assessing the correlation with regional CDC ILI Surveillance data during the 2007-08 influenza season ( Table 4 ). The mean correlation coefficient by US Census Region during the 2007-08 influenza season was 0.97 (SD 0.02), identical to the previously published result [2] . When regional Google Flu Trends data were compared to CDC Virus Surveillance, the mean correlation coefficient was lower, 0.87 (SD 0.04). Additional seasonal analyses by region demonstrated that the mean Google Flu Trends-CDC Virus Surveillance correlation coefficient was consistently below the mean correlation between CDC ILI Surveillance and the reference standard by US Census Region (Table S4) .
A sensitivity analyses was performed to investigate the possible effects of outlier observations and to determine whether the removal of influential outlier observations substantially affected tests of correlation. In the comparison of Google Flu Trends with CDC Virus Surveillance over the entire study period, 9 (5 
We compared data describing the proportion of subjects testing positive each week for influenza with data from CDC's ILI surveillance system and data from Google Flu Trends. A prior analysis compared Google Flu Trends data to US Census Region ILI data and demonstrated a strong correlation during the 2007-08 influenza season (R = 0.97) [2] . The correlation between Google Flu Trends data and national influenza test data was lower (R = 0.72) when assessed over five influenza seasons beginning in 2003. In terms of coefficients of determination (R 2 ), 88% of the variance is shared between Google Flu Trends and CDC ILI Surveillance, while only 51% of the variance is shared between Google Flu Trends and surveillance for laboratory-confirmed influenza. From September 2003 through May 2008, CDC ILI surveillance was more closely correlated with CDC Virus Surveillance (R = 0.85). Furthermore, the sensitivity analysis demonstrated that the Google Flu Trends correlation with the reference standard was more influenced by outlier observations than was CDC ILI Surveillance data.
Most of the influential observations occurred during the peak 2003-04 influenza season. This season was characterized by early and intense influenza activity, a large number of influenzaassociated pediatric deaths, and increased media attention to influenza [18] . It is possible that during this influenza season, physician laboratory testing patterns or patient health care seeking behavior differentially affected the relationship between ILI rates and laboratory confirmation of influenza. Additionally, internet search behavior about respiratory infections during this period could have been different than during subsequent, more typical influenza seasons. These findings are relevant to the applicability of surveillance using internet key word searches during the 2009 H1N1 pandemic and future anomalous influenza seasons.
The Google Flu Trends statistical model was created and validated using rates of ILI, which is a nonspecific syndrome that is not necessarily caused by influenza virus infection, but used for decades as an indicator of the burden of outpatient influenza illness. Any nationwide surveillance using internet key word search is likely to be most representative if the search engines being monitored has widespread use. As the popularity of a particular internet search engine wanes, so too may the overall accuracy of disease activity estimates using its data. Also, the stability of internet key word surveillance relies on consistency of internet search behavior [19] , as well as search term use between geographic regions and over time. Changing media trends, word search choices, and cultural make-up of regions and over time may also affect the representativeness of internet search surveillance.
Our analyses represent the first comparison of Google Flu Trends data with data on laboratory-confirmed influenza virus infections. Prior studies have demonstrated that Google Flu Trends can estimate rates of nonspecific ILI in New Zealand, Europe, and the United States [2, 20, 21] . We have demonstrated that Google Flu Trends performs less well when estimating surveillance data for laboratory-confirmed influenza, which is not surprising, as the Google Flu Trends algorithm was developed using only ILI data.
There are several US influenza surveillance systems [10] , and taken as a whole, they provide an excellent overview of influenza activity at any period during the influenza season. However, only CDC Virus Surveillance data tracks nationwide activity of laboratory-confirmed influenza. The original publication describing and validating the Google Flu Trends methods intentionally excluded specifics concerning the statistical model used out of concern that public knowledge of the search terms could alter its usefulness to track influenza activity [2] . Nevertheless, without the publication of the Google Flu Trends statistical model, further independent, prospective validation or improvements upon the model are not possible.
This study is subject to limitations. While US Influenza Virologic Surveillance System provides the best data source for following trends in laboratory-confirmed influenza infections, it is nevertheless a convenience sample of specimens sent to participating laboratories. In addition, health care seeking behavior, physician testing practices, and internet search behavior may change over time or through the course of an influenza epidemic, limiting the interpretation of correlation data from this analysis.
In conclusion, Google Flu Trends may make a useful contribution to public health given the timeliness of the data and its close association with traditional US ILI surveillance system data. However, CDC ILI Surveillance and positive influenza tests were more correlated during the five years of this study, including the unusual 2003-04 influenza season, than were Google Flu Trends and positive influenza tests. We hypothesize that differences in internet search behavior, patient health care seeking behavior, and physician testing practices may alter the correlation between influenza surveillance systems. In the absence of influenza virologic surveillance, sentinel surveillance for ILI may more accurately monitor influenza activity than Google Flu Trends during anomalous influenza seasons. Furthermore, given the non-specific nature of ILI, robust nationwide virologic surveillance remains critical to the understanding of influenza activity during inter-pandemic and pandemic periods alike.  Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. Malignant melanoma, the most fatal form of skin cancer, arises from malignantly-transformed melanocytes in the basal layer of the epidermis. The incidence of melanoma has been increasing at an accelerated rate in the past few decades amongst fair skinned populations [1] and advanced forms of the disease are highly resistant to treatment [2, 3] . Thus, an urgent need exists for novel therapies and earlier diagnosis.
Melanoma is widely thought to be immunogenic, supported by clinical observations such as the frequency of spontaneous tumor regressions, the prevalence of melanoma in immunosuppressed patients, and the partial success of clinically-available immune modulatory therapies such as the polyclonal immune activating cytokines IFNa-2b and IL-2 [4, 5, 6, 7] . Host adaptive immune responses have been described in melanoma with a main focus on melanoma specific T cell responses [8, 9] , and supported by successful case scenarios using immunotherapeutic strategies such as dendritic cell vaccines, adoptive T cell therapies, and CTLA4 monoclonal antibodies [7, 10, 11, 12, 13] .
Limited research has focused on B cells and the specificity of antibodies they produce in cancer. Promotion of cancer development by the creation of a pro-inflammatory environment [14, 15] and anti-tumor functions by activating mature T cell responses [16] have been proposed as potential roles for B cells in animal models of cancer. While there may be host immune responses to malignancy following immunization [17] , a variety of mechanisms involved in tumor escape have been described and understanding this complex relationship between immunosurveillance and tumor escape in patients is key to the design of effective immunotherapies [18, 19, 20, 21] .
Despite well-characterized tumor-induced immunomodulation, immunotherapies such as monoclonal antibodies are emerging as key diagnostic and therapeutic modalities and are now standard of care for the treatment of various cancers. Antibodies for the treatment of melanoma aimed at enhancing key pathways of T cell activation (Cytotoxic T Lymphocyte-Associated Antigen 4, e.g. Ipilimumab), targeting tumor vasculature (e.g. Bevacizumab), or tumor-associated antigens (e.g. High Molecular Weight-Melanoma Associated Antigen, HMW-MAA) have demonstrated promise in clinical studies [13, 22, 23, 24] . Antibodies therefore represent an attractive approach for the treatment of melanoma.
Reports of tumor-specific antibodies in the sera of melanoma patients date back over forty years [25] and have so far provided valuable insight into immune responses to cancer. Serological studies of individuals with melanoma have shown that patients expressing certain tumor-associated antigens have antibodies against these antigens, conversely, patients without the antibodies also lack the corresponding tumor antigens [26] . These studies have been restricted to few antibodies in sera against known tumor-associated antigens. Serological studies reported IgG antibodies recognizing intracellular melanocyte and melanomaassociated antigens such as tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and melanoma-associated glycoprotein antigen family (gp100/pmel17) in patients with melanoma. Serumresident antibodies to some of these antigens were enhanced following polyvalent melanoma cell vaccine immunization in patients with melanoma, suggesting that melanoma-associated antigens may be immunogenic and that humoral responses to melanocyte and melanoma antigens may constitute potential targets for immunotherapy [27] . New antigens, such as the NY-ESO-1, with restricted expression in normal tissues and wide distribution in various cancers including melanoma have been discovered using serological analysis of recombinant cDNA expression libraries (SEREX) techniques tested against tumor mRNA and autologous patient sera [28] . SEREX studies from human melanomas [29] and from one cell line [30] have led to the discovery of the human testis antigen HOM-MEL-40. Many of these antigens are primarily intracellular, making them less attractive targets as monoclonal antibodies. Furthermore, serological screens may also be limited by the temporal dynamics of sera antibodies. Evaluating the reactivity of antibodies secreted by circulating B cells may therefore provide additional insight to serological evaluations by interrogating the long-term memory anti-tumor systemic mature humoral response to cancer.
The production of tumor-specific antibodies in melanoma from patient-derived B cells in the peripheral blood and tumors has been reported and has yielded a few antibodies of the IgM and IgG class [31, 32, 33, 34] . In the past, such studies have been limited by poor EBV transformation efficiency of human B cells, low production of immunoglobulin, evaluation of few patients, and lack of effective, reproducible methods to rapidly screen for tumorspecific antibodies. To address some of these limitations, we took advantage of recent advances in growing and immortalizing memory B cells in culture [35, 36] , increased the number of patients evaluated, and developed a novel screening tool to specifically detect tumor-reactive antibodies against cell surface antigens on melanoma cells. Our approach entails culture of patient-derived circulating B cells and screening of the antibodies they secrete for their reactivity and specificity to melanoma cells versus melanocytes. Our strategy does not screen for antibodies against known antigens or evaluate antibodies secreted or sequestered in the serum at discrete times, but rather uniquely, the aim here is to monitor tumor cell-reactive IgG antibodies produced by B cell cultures, elucidating the breadth of the longterm mature B cell repertoire recognizing melanoma antigens expressed on the surface of cancer cells.
In this study, we screen for tumor-reactive and tumor-specific IgG antibodies produced by patient and healthy individual B cell cultures. This allowed characterization, beyond phenotype, of the circulating B cell repertoire of individuals with melanoma and clinical correlations of mature humoral responses and disease progression. We also provide an example demonstrating that this screen may facilitate the identification of antibodies able to target cancer cells.
We developed and optimized a cell-based ELISA for specific detection of tumor-reactive antibodies in order to obtain a robust and optimized system for the detection of anti-tumor antibodies from patients ( Figure S1 ). We first evaluated the sensitivity and specificity of an IgG antibody against a melanoma cell surface antigen (HMW-MAA), expressed on A-375 melanoma cells using immunocytochemistry (cytospins) and live cell flow cytometry. An anti-HMW-MAA antibody was observed to bind to A-375 cells, but not melanocytes over a range of concentrations as low as 20 ng/mL using both immunocytochemistry (Figure 1 , A) and flow cytometry (Figure 1, B) . Next, we compared the detection of this antibody bound to melanoma cells in our cell-based ELISA to the above methods.
Utilizing our novel ELISA, we detected tumor-specific antibodies at concentrations as low 10 ng/mL (Figure 1 , C), demonstrating comparable sensitivity to flow cytometric or immunocytochemical methods. Additionally, we validated our ability to identify tumor-reactive antibodies from our patient cultures, compared to equal amounts of non-specific IgG and culture media (Figure 1 , D). We also examined the potential applicability of this method to identify tumor-specific antibodies in other cancers using the mammary carcinoma cell line SK-BR-3, which highly expresses the cell surface tumor-associated antigen HER2/ neu [37] . Trastuzumab (Herceptin TM ), a humanized antibody specific for HER2/neu, was specifically detected compared to an equal amount of a control IgG employing our method (Figure 1 , D). Thus, we demonstrate that we can detect antibodies against tumor cell antigens in a sensitive, specific and reproducible manner.
We first established B cell cultures from the peripheral blood of melanoma patients to study antibody responses to cancer ( Figure  S1 ). In agreement with a previously published report, we detected a reduced memory B cell subset in melanoma patients. Melanoma patient and healthy volunteer B cells were cultured with B cell purity greater than 90%. Following EBV transformation and activation with a TLR9 agonist, patient B cells were observed to proliferate in culture for over eight weeks and 80% of the cells in these cultures were IgG positive. B cell cultures derived from healthy volunteers (n = 5) and melanoma patients (n = 5) had comparable mean antibody titers after 18 days, ranging from 1 to 7 mg from each individual, with an overall mean of 2.5 mg (95% CI = 2.3 to 2.7) per culture arising from 500 B cells per well. We therefore established antibody-secreting cultures from melanoma patients with comparable rates of IgG secretion to healthy volunteers.
We next investigated whether specific antibody responses to melanoma could be detected from circulating B cells of patients and healthy volunteers utilizing our cell-based ELISA. Antibodysecreting B cell cultures from 10 healthy volunteers and 10 patients (n = 4 stage II, n = 4 stage III, and n = 2 stage IV) were evaluated for reactivity to both metastatic and primary melanoma cells relative to non-specific human IgG control (calculated as fold increase above the non-specific human IgG control) employing our cell-based ELISA. We found a significant (P,0.0001) increase in the mean reactivity (fold increase) of patient-derived antibody cultures (n = 600) to metastatic melanoma cells (2.5 fold increase, 95% CI = 2.4 to 2.6) compared to antibody cultures (n = 600) derived from healthy volunteers (1.1 fold increase, 95% CI = 1.1 to 1.2) (Figure 2 , A). A significant (P,0.0001) increase was also seen in the mean reactivity of patient-derived antibodies to primary melanoma cells (2.3 fold increase, 95% CI = 2.2 to 2.4) compared to antibodies from healthy volunteers (1.0 fold increase, 95% CI = 1.0 to 1.1) (Figure 2 , B). From this patient cohort, we thus observed a significantly increased reactivity to primary and metastatic melanoma cells, compared to healthy volunteers.
To examine if antibody responses differ according to disease stage, we studied a cohort of 21 patients diagnosed with stage I, II, III and IV melanoma (Table 1 ) and evaluated the reactivity of antibody cultures (n = 1,800) from these patients to the metastatic melanoma cell line A-375 utilizing the cell-based ELISA. This patient cohort was almost exclusively Caucasian. Antibody reactivity against melanoma cells was quantified relative to a non-specific human IgG control and measured as fold increase above this negative control. Patients with local (non-metastatic, stages I and II) disease had a significantly (P,0.0001) higher mean antibody response (2.6 fold increase, 95% CI = 2.4 to 2.8) compared to those with confirmed metastatic disease (stages III and IV, 1.7 fold increase, 95% CI = 1.7 to 1.8) (Figure 3 , A). We also found an overall significant reduction (P,0.0001) in the mean reactivity of antibodies secreted in B cell cultures against melanoma cells from stage II (2.8 fold increase, 95% CI = 2.6 to 3.0, n = 660) to stage III (1.9 fold increase, 95% CI = 1.8 to 2.0, n = 540) and to stage IV patients (1.5 fold increase, 95% CI 1.5 to 1.6, n = 480) (Figure 3 , B). The stage I patient was not evaluated since samples from only one patient (n = 120 B cell cultures) from the cohort was available to include in this group. The highest mean antibody reactivity against melanoma cells was observed in Patients 5 and 6 diagnosed with stage II and III melanoma, respectively (Table 1 ). However, we observed variation in the antibody response among individual patients, with 19 out of 21 patients in our cohort having at least one antibody-producing culture with optical density values 2.5-fold above the negative IgG control (Table 1 ). These findings suggest that despite the significant reduction in the proportion of tumor-reactive antibody cultures as a function of disease progression, patients from each of stage groups had B cells with antibodies that recognized tumor cells.
We screened for tumor-reactive antibodies from patient B cell cultures and approximated frequency and specificity of selected cultures to tumor cells. For this we screened B cell culture supernatants against a stringent comparator using a positive control monoclonal antibody, Trastuzumab, that recognizes the HER2/neu tumor antigen, expressed on breast cancer cells and on some melanoma cells [38] . Trastuzumab was selected as a positive control because of comparable binding across melanoma cell lines and melanocytes, as shown by mean fluorescence intensities of antibody binding against a range of these cells (Figure 4, A) . Previous studies screening for tumor-specific antibodies have selected wells greater than the mean negative control optical density (OD) + three standard deviations as criteria for positive tumor-reactive antibodies [39] . Due to the inherent variability of cell-based assays, and the potential identification of false positive cultures, we chose more stringent criteria for antibody screening, by comparing antibodies produced by B cells to a positive control antibody (. 75% OD of positive control). Based on this antibody selection criteria (.75% OD of positive control), we estimate that 28% of B cell cultures (n = 1,800) derived from 21 patients, each arising from 500 B cells, produced antibodies that recognized metastatic melanoma cells, compared to 2% (n = 600) of cultures derived from 10 healthy volunteers (Figure 4 , B). From these 10 healthy volunteers, 2 individuals had one reactive culture (out of 60 cultures), 1 individual had 10 reactive cultures, and the rest of the cohort had no reactive cultures.
From our patient cohort, we can roughly estimate the frequency of B cells that produce an antibody that recognizes melanoma cells under the assumption that a reactive antibody culture, defined as having an OD .75% of the positive control, arises from only a single B cell (1 out of 500 plated per culture). By dividing the total number positive antibody cultures from the patient cohort by the total number of B cells evaluated from the patient cohort by the number of positive antibody cultures, we roughly approximate that from our patient cohort one out of 1,765 B cells produce an antibody that may recognize melanoma cells.
To estimate the frequency of melanoma-reactive antibodyproducing B cells in melanoma patients we performed limiting dilution analysis. We selected a stage II patient, who, we predict, may have a high antibody response to melanoma, based on our findings that the antibody responses were highest in this group (Figure 3 , B). For this stage II patient (Patient 15, see Table 1 ) from our limiting dilution analysis assays using the cell-based ELISA, we estimate that one out of 1,790 peripheral blood B cells produces antibodies that bind to A-375 melanoma cells (Figure 4 , C). For this same patient, the frequency of B cells producing antibodies that react with melanocytes was also evaluated at the same B cell densities as melanoma cells. We did not observe a comparable patient antibody response to melanocytes as we did to melanoma cells, suggesting a much lower frequency of antibodies that bind to normal cells of the same origin ( Figure 4 , C left). Limiting dilution analysis against two additional metastatic (SK-MEL-28, A-2058) and one primary (WM-115) melanoma cell lines for the same patient yielded different but comparable frequencies to A-375 for the metastatic cell lines (SK-MEL-28, 1 out of 1,650 B cells; A-2058, 1 out of 1,170 cells), and a much lower frequency of antibodies that bind to the primary melanoma line WM-115 which was similar to that observed with primary melanocytes (Figure 4 , C right). For this patient, the data suggest detectable circulating B cell humoral response frequency against metastatic melanoma cells and lower frequency for normal human melanocytes or primary melanoma cells. To further confirm the frequency observations for the patient-derived circulating B cell repertoire, we performed additional limiting dilution assays for another stage II patient (Patient 21, Table 1 ). For Patient 21, we estimate 1 out of 2,430 B cells that produces antibodies bind to the same melanoma cell line tested for Patient 15 (Figure 4 , D), suggesting lower but comparable frequency to those estimated for B cells from Patient 15. In summary, applying the above methodology, these results suggest that tumor-reactive antibodies from circulating B cells are more frequent in melanoma patients than healthy volunteers and more frequent against a range of metastatic melanoma cells compared to normal melanocytes.
We then selected patient-derived, tumor-specific antibodies in order to further evaluate their reactivity to melanoma cells, and conducted a preliminary assessment of the potential functional capabilities of a patient-derived antibody from this screen. B cell culture wells were selected based on stringent criteria (OD . 75% positive control antibody), using the cell-based ELISA. Tumor specificity of antibody cultures was evaluated by comparing binding of antibodies from these cultures against multiple melanoma cells (A-375, SK-MEL-2, WM-115) versus normal cells ( Figure 5 , A). We observed multiple antibody cultures with a higher degree of binding to some melanoma cells compared to melanocytes from the same patient (Patient 3, Figure 5 , A; a selection of five of these cultures is shown on Figure 5 , B). Similar results were obtained when we screened for tumor-specific cultures from different patients against melanoma cells and melanocytes. Positive cultures with different binding patterns against four melanoma cell lines (A-375, SK-MEL-2, SK-MEL-28, WM-115) and primary human melanocytes were detected (selected cultures derived from Patients 2, 3, 4 and 6 are shown as examples in Figure 5 , C), reflecting specificity and reactivity of different antibodies to a range of antigens expressed at different levels in a number of melanoma cell lines, and some reactivity to antigens lowly expressed on human melanocytes. Selection of a tumorpositive antibody culture for sub-cloning and limiting dilution was based on degree of reactivity to melanoma cells relative to melanocytes ( Figure 5 , C).
One B cell culture from Patient 6 was selected for further evaluation since cell culture supernatants were observed to have a higher degree of binding to A-375 and SK-MEL-28 cells compared to melanocytes by ELISA ( Figure 5 , C; right). After limiting dilution of this melanoma-reactive B cell culture, a monoclonal antibody (6_2G3) was further assessed for specificity to 6 melanoma cell lines, melanocytes and fibroblasts by live cell flow cytometry ( Figure 6 ). Since more antibody was available after monoclonal dilution, 2 additional melanoma cell lines along with dermal fibroblasts were evaluated. In concordance with the cellbased ELISA findings ( Figure 5 , C), the 6_2G3 clone bound to a range of melanoma cell lines, but not to melanocytes ( Figure 6 ). The antibody had no reactivity against primary human dermal fibroblasts. In summary, by evaluating the specificity of antibodies to melanoma cells versus melanocytes and fibroblasts we could identify a melanoma-specific monoclonal antibody clone 6_2G3. While we had limited amounts of monoclonal antibodies our B cell culture supernatants after evaluating melanoma-cell specificity, we were able to conduct a limited functional investigation of this antibody.
Using clone 6_2G3, we wished to assess whether a patientderived antibody has potential cytotoxic activity against tumor cells. We tested the tumor cell killing potential of this antibody using a real-time live-dead cell cytotoxicity assay using as targets metastatic melanoma cells recognized by this clone (Figure 7 & Supporting Videos S1 and S2). In these experiments, U-937 human monocytic cells which express Fcc receptors served as effector cells [40] and A-375 melanoma cells were used as target cells to evaluate antibody-dependent cellular cytotoxicity (ADCC) of tumor cells mediated by patient-derived IgG antibodies. We tested two monoclonal antibodies, both derived from Patient 6 ( Table 1) : (1) the 6_2G3 antibody, which bound to A-375 cells and not melanocytes and (2) the 6_2D10 antibody, which did not bind to A-375 cells or melanocytes in the cell based ELISA prior to limiting dilution, which served as a non-tumor-reactive control (Figure 7) . After 2 hours in culture, 18% (95% CI = -5 to 41%) of tumor cells given the melanoma-specific antibody were viable, compared to 95% (95% CI = 86 to 104%) of the tumor cells given the nonmelanoma specific antibody (P,0.0001) (Figure 7, A, left) . Relative to tumor cell fluorescence at the start of the assay, mean green/live tumor cell fluorescent intensity was reduced to 64% for the non-tumor specific antibody (6_2D10) compared to 18% for the tumor-specific antibody (6_2G3) (Figure 7, A, right) . These results highlight the potential of a patient derived tumor-specific antibody to kill tumor cells by antibody-dependent cell cytotoxicity (Figure 7 , B and see Videos S1 and S2). For tumor cells treated with the tumor-specific 6_2G3 antibody, we also observed a significant (P = 0.0002) reduction in the movement of monocytic effector cells in contact with tumor (13 mm, 95% CI = 10 to 17 mm) compared to effector cells not in contact with tumor (26 mm, 95% CI = 21 to 31 mm) (Figure 7, C and D) . Using the 6_2D10 non-specific antibody, no significant (P = 0.3) difference was observed for the movement of effector cells not in contact with tumor cells (20 mm, 95% CI = 15 to 25 mm) compared to those in contact with tumor cells (25 mm, 95% CI = 18 to 31). With this example, we demonstrate that a patient-derived tumor-specific antibody is capable of engaging immune effector cells in antibodydependent cellular cytotoxicity against tumor cells. Taken together, these data suggest that systemic melanoma-specific mature B cell responses may be present in patients with melanoma and may harbor the potential to be activated against cancer cells. Table 1 
We describe an approach to study the circulating B cell-derived humoral immune response to cancer and apply this to detect tumor-specific IgG antibodies from melanoma patient B cells. This strategy has the potential to be applied to any type of cancer. Findings presented herein complement previous serological studies, providing added insight into the mature systemic B cell response to melanoma.
As a first step to evaluate the tumor reactivity and specificity of patient B cell-derived IgG antibodies, we developed a mediumthroughput cell-based ELISA with melanoma cells to detect antibodies against tumor cell surface antigens (Figure 1 ). Cells were allowed to grow and adhere on to 96-well plates prior to being preserved by a light fixative (0.5% formaldehyde). While preserving the cells and allowing for storage and access to multiple plates at any one time, light fixation with formalin allows preservation of potentially-antigenic epitopes on the surface of target cells. Previous studies have reported cell-based ELISA methods to identify tumor-specific antibodies in melanoma [39, 41] , where tumor cells were preserved using strong fixatives such as glutaraldehyde, known to potentially mask antigenic epitopes, thus compromising the recognition of antigens by antibodies [42] . Furthermore, the specificity and sensitivity of such methods has not been reported using antibodies against known cell surface antigens. Although many intracellular melanoma associated antigens have been described (tyrosinase, TRP-1, TRP-2, gp100/pmel17), most are also expressed by normal melanocytes, only a few defined cell surface antigens such as the High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) are reported to be expressed on the surface of melanoma cells, and other antigens show heterogeneous expression among patients [43, 44] . Thus, this ELISA constitutes an attractive tool to evaluate broad responses to any naturally-expressed antigens on the surface of melanoma cells and melanocytes in this context. This screening methodology has additional potential advantages. Unlike assays screening against a single recombinant antigen or antigenic epitope, our method enables the evaluation of antibody repertoires of patients against a multitude of cell surface antigens in their native confirmation on the surface of both primary and metastatic melanoma cells and also melanocytes, providing more comprehensive information on the broad prevalence of tumorreactive and tumor-specific antibodies. Previous studies have shown concordance of cell line-associated antigens with antigens expressed on corresponding tumors, making them a suitable platform for tumor-reactive antibody screening [26, 45] . Thus, cell lines provide a promising alternative source of multiple tumor antigens in the absence of multiple well-defined, highly expressed, and readily available recombinant antigens. Unlike flow cytometric evaluations, the cell-based ELISA does not require the use of proteolytic enzymes such as trypsin, therefore better preserving cell surface antigens. Plates of target cells can be prepared, fixed and frozen in batches, thus allowing for higher throughput screening for tumor cell-reactive antibodies. It can be applied to evaluate . 300 culture supernatants against cell lines within a few hours. In principle, numerous ELISA plates for screening a range of cell lines with multiple supernatant samples can be processed simultaneously. Additionally, this methodology may be a potential tool for immunomonitoring tumor-specific humoral responses to therapies; selecting patients most likely to benefit from immunotherapy; or as a prognostic factor in linking tumor-reactive humoral responses to clinical outcomes. This assay may also be utilized to detect surface antigens in a range of cell types, and thus may be adapted to monitor the B cell-derived antibody repertoire in different disease contexts.
In agreement with a recent report [46] , we also observed a reduction in the peripheral blood memory B cell compartment of metastatic melanoma patients. We measured a reduction of the CD27+ subset of memory B cells in patients with both metastatic and non-metastatic melanoma compared to healthy volunteers ( Figure S2 ). Despite the reduction of circulating memory B cells in our cohort, patient-derived B cells were capable of secreting high amounts of IgG antibodies when activated in vitro with a TLR 9 agonist, with comparable antibody production to B cells from healthy individuals, and a high percentage of patient-and healthy volunteer-derived B cells expressed IgG antibodies within a few days in culture (80% of B cells from three patients with melanoma, Figure S2 ). Thus, while a reduced memory B compartment has been reported in cancer patients, we show that a melanomareactive portion of this compartment remains in our patient cohort.
We demonstrated a high prevalence of melanoma patientderived antibodies produced by circulating B cells in cancer patients that recognize melanoma cell lines (Figure 2 ). We observed that B cell culture supernatants from different patients displayed differential binding to each cell line, which reflects specificity and reactivity of different antibodies to a range of antigens expressed at different levels in a number of melanoma cell lines; these may also reflect binding to some antigens lowly expressed on human melanocytes ( Figure 4 and Table 1 ). Melanoma patients had a high percentage of melanoma-reactive antibody-producing B cell cultures, significantly higher than those from healthy volunteer-derived B cell cultures (Figure 2) , with 28% of melanoma patient-derived B cell cultures recognizing melanoma cells, compared to 2% of cultures from healthy volunteers (Figure 4) . Limiting dilution analyses of reactivity against melanoma cells versus normal melanocytes provided further evidence in support of the presence and frequency of tumor-reactive B cells in Figure 6 . Selection of a patient derived B antibody that recognizes melanoma cells but not melanocytes. A selected tumor-reactive culture from a stage III patient (Patient 6) which secreted antibodies that bound to tumor cell lines and compared to melanocytes by ELISA ( Figure 5 , C) was sub-cloned, and a monoclonal antibody 6_2G3 was selected and evaluated on live cells by flow cytometry (solid black line histograms) for reactivity to fibroblasts, melanocytes, and 6 melanoma cell lines. IgG isotype controls are shown in shaded grey histograms. doi:10.1371/journal.pone.0019330.g006 Figure 7 . A tumor-specific antibody derived from a patient with melanoma is able to induce tumor cell cytotoxicity. Two monoclonal antibodies were evaluated in vitro using a live cell imaging assay: 6_2G3 clone bound to A-375 cells compared to melanocytes; and 6_2D10, a clone also from Patient 6 did not, and served as a negative control antibody for these experiments. (A) Cell viability of A-375 melanoma cells incubated with patient blood (Figure 4) . For one stage II patient evaluated, the data indicate that metastatic melanoma cells are recognized by a higher proportion of B cells (estimated on average , 1 in 2,000 mature B cells) compared to primary melanoma cells or melanocytes, although in a cohort of 10 patients we measured reactivity of B cell cultures to both metastatic and primary melanoma cell lines ( Figure 2 ). Taking into consideration the expected variability in immune responses among patients, and the array of tumor antigens these patients may be exposed to, the observations that B cells from two patients with stage II melanoma yielded comparable reactivity to metastatic melanoma cells (estimated 1 in 1,790 for Patient 15, and 1 in 2,430 B cells for Patient 21) indicate the presence and support the prevalence of a circulating melanoma-reactive B cell compartment.
While tumor-reactive antibodies were detected from most melanoma patients studied, antibody responses derived from circulating B cells against melanoma cells decreased with more advanced disease stages (Figure 3 ). Previous serological studies report serum-resident antibodies against tumor cells in melanoma patients, with some evidence that serum antibodies are diminished in patients with advanced disease [25, 47] . It was unclear whether this was a consequence of the sequestering of antibodies into tumors with increasing tumor burden in these patients. Our findings provide further insight by demonstrating the presence of a circulating long-term mature B cell response to cancer at all disease stages, against a broad range of naturally expressed antigens on the surface of primary and metastatic tumor cells. We also report decreased frequency of tumorreactive antibody-producing B cells with advanced disease, thus supporting the premise that mechanisms of immune tolerance rather than adsorption of antibodies into tumors in advanced disease setting may also explain these reductions. One limitation may arise from screening for antibodies against mostly metastatic melanoma cells. It is possible that our observations may reflect reactivity to antigens present in primary disease, which may be preserved or upregulated in advanced disease setting. While our findings may not account for reactivity to tumor antigens that are lost with disease progression, this reduced reactivity to melanoma we observed may imply weakened immune responses to a subset of antigens on the surface of melanoma cells. Another explanation for these observations may be that with advanced disease, mature circulating B cells home into increasing tumor sites, thus reducing the circulating tumor-reactive B cell compartment in these patients. Future studies aimed at monitoring local B cell responses in tumors may provide further clues into the dynamics of mature B cell responses at the systemic and local levels in cancer. Thus, despite well-known weakened host immune response with disease progression [48] , we were able to detect melanoma-reactive antibodies from patient circulating B cells, implying that although mature humoral immune responses are weakened, responses in the form of mature memory B cells may persist. However, further work elucidating potential immunomodulatory roles of B cells and other immune cells in cancer, including the production of IL-10 by B cell population subsets [49] , merits consideration.
Although we report the presence of anti-tumor antibodies produced by patient memory B cells, and these cells were stimulated ex vivo to secrete antibodies, it is not clear whether tumor antigen-reactive B cells are activated in patients to secrete antibodies or whether these humoral responses are capable of exerting any beneficial anti-tumoral activities in the same patients in vivo. In the 21 patient cohort at different disease stages in this study, we were not able to draw any conclusions regarding the relationship between tumor cell reactivity and clinical disease progression in the short term (6 months to 2 year follow up) or associations with any particular disease treatment regimes. However, monitoring mature memory B cells and their antibody repertoires together with clinical outcomes in patients over a long period of time may help identify any correlations between melanoma-reactive mature memory B cell responses and disease progression. Additionally, future studies may help identify particular components of the humoral response which may hold clinical relevance, and elucidate the potential merits of monitoring these responses in relation to therapies, or of evaluating humoral responses as a prognostic factor to clinical outcomes.
An important question therefore relates to whether patientderived mature B cell responses have any functional capability to potently activate immune effector cells against cancer. For this, we measured the capacity of one antibody clone to kill tumor cells. Antibody clone 6_2G3 derived from a patient with stage III disease (Patient 6, Table 1 ) was not observed to bind to fibroblasts or melanocytes, but bound to a proportion of melanoma cell lines tested ( Figure 5 ). Antibodies against tumor-associated antigens can attack tumor cells via a number of mechanisms including induction of apoptosis in tumor cells and engaging Fc receptors on immune cells [50, 51, 52, 53] . Antibodies approved for the treatment of cancer have been shown to function through one or more of these mechanisms [54, 55] . While our strategy yields fully human monoclonal antibodies in a matter of a few months, we were limited in the amount of antibody we could produce from the B cells to perform functional studies and evaluate reactivity to patient-derived melanoma tumors. However, we had sufficient quantity to evaluate whether a patient-derived melanoma tumorspecific monoclonal antibody could mediate antibody dependent cellular cytotoxicity (ADCC) in the presence of monocytic effector cells and tumor cells using a real-time live cell imaging assay. We show that the tumor-specific 6_2G3 clone is capable of mediating ADCC in vitro and additionally measured the restricted movement of monocytic effector cells once in contact with tumor-specific antibody-coated tumor cells, providing further evidence of ADCC ( Figure 6 ). These preliminary assessments provide a promising clue that a potentially active mature B cell response against melanoma may be present in patients. An example of this possibility was recently reported by Yuan et al. who demonstrated that administration of the anti-CTLA-4 antibody ipilimumab led to serological enhancement of antibodies to the testis antigen NY human U-937 monocytic cells was compared between samples treated with 6_2G3 or 6_2D10 antibody after 2 hours at 37uC (*** = P,0.001) (left). Error bars represent 95% confidence intervals. Mean fluorescence intensity of A-375 tumor cells pre-labeled with the live cell dye Calcein AM, and incubated with U-937 cells and antibody 6_2G3 or 6_2D10 was measured at 0 and 120 min time points (right). (B) Fluorescent images of the live cell cytotoxicity assays at 30 minute intervals. Live Calcein AM-labeled melanoma tumor cells (green) were incubated with 6_2G3 or 6_2D10 antibody and U-937 cells (blue) and cell death was evaluated (red). Incorporation of Ethidium homodimer-1 (incorporation of red into tumor cells) was observed with 6_2G3 but not 6_2D10 (magnification 20x, Scale bar: 100 mm). (C) Movement of U-937 cells tracked and measured over two hours was compared for cells in contact and those not in contact with tumor cells (*** = P,0.001for 6_2G3 and P = 0. 3 
Movement is indicated by tracking lines (red to yellow) from the original position of U-937 cells at t = 0 to t = 2 hours (magnification 20x, Scale bar: 50 mm). doi:10.1371/journal.pone.0019330.g007 -ESO-1 in patients who responded to the antibody therapy [56] . It is therefore conceivable that the mature B cell compartment could be enhanced with immunotherapeutic approaches, and that monitoring humoral responses to therapeutics may have clinical relevance.
Harnessing the cancer-specific antibody repertoire of cancer patients using the methodology described herein may also potentially offer an alternate strategy to yield IgG antibodies against cancer antigens. Recent advances reported by Traggiai et al., evaluating monoclonal antibodies from human memory B cells have yielded fully-human virus-neutralizing antibodies of therapeutic relevance for infectious diseases and have contributed to the dissection of humoral memory responses to vaccinations [35, 57, 58] . Here, we focus on B cells from cancer patients such as melanoma patients, analyze systemic humoral responses to cancer and demonstrate the presence of tumor-reactive and tumorspecific antibodies. This approach may offer an advantage over other approaches such as phage display in that it yields in vivo affinity-matured human antibodies with naturally paired heavy and light chains. The patient-derived monoclonal antibody 6_2G3 bound to 2 out of 6 of the melanoma cell lines evaluated compared to melanocytes, suggesting that this antibody may be against a protein over-expressed or mutated on the surface of cancer cells. In light of the efficacy of Trastuzumab, against the HER2/neu antigen expressed on 20-30% of breast cancers, as a clinicallyvalidated therapeutic tool for the treatment of an equivalent proportion of breast cancer patients [59] , selection of antibodies that bind to a portion of cell lines may merit further characterization. Although the clinical significance of mature memory B cells expressing antibodies that recognize tumor cells in patients remains to be elucidated, antibodies derived from these cells, introduced by passive immunotherapy in therapeuticallyrelevant doses, such as those used for Trastuzumab to patients with breast cancer, merit investigation for any potential relevance in melanoma. Other potential future benefits of screening patientderived B cells from tumor-reactive antibodies may be identification of novel cell surface tumor antigens. Future evaluations of clone 6_2G3 will include sequence analysis and expression cloning to allow for further analyses of specificity to melanoma tumors, antigen identification, and for thorough functional assessments.
These data provide additional understanding of the mature B cell response to melanoma by evaluating antibodies derived from circulating B cells of cancer patients. The prevalence of mature humoral responses against cancer cells in patients, as well as the capacity of a patient-derived antibody to activate effector cells against melanoma cells indicate the potential functional significance of the humoral immune response against cancer.
Specimens from patients and healthy volunteers were collected with informed written consent. The work was conducted in strict accordance with study design approved by the Guy's Research Ethics Committee, St. Thomas' Hospital, London, UK.
After obtaining informed consent, peripheral blood was isolated from healthy volunteers (n = 10) and from patients with melanoma (n = 21). Patients were staged and classified according to the American Joint Committee on Cancer Melanoma Staging and Classification criteria [60] . B cells were isolated by negative selection using RosetteSepH B cell enrichment cocktail (Stem Cell Technologies, Vancouver, Canada) according to the manufacturer's instructions. B cell purity was assessed by flow cytometry by staining for mature B cells (CD22), T cells (CD3), monocytes (CD14) and plasmacytoid dendritic cells (BDCA3) using fluorescently-labeled monoclonal antibodies, all from BD Biosciences, Oxford, UK ( Figure S1 ). Flow cytometry experiments were conducted with either the FACSAria or FACSCanto (BD Biosciences) and flow cytometric data were analyzed using Flow Jo (Tree Star, Ashland, OR).
B cells were plated at 500 cells per well on 96 well U-bottom microplates (Nunc, Rochester, NY) along with 3x10 4 cells per well of irradiated (30 Gy) 
Qualitative detection of tumor-specific antibodies by immunocytochemistry was performed by centrifugation of 2610 5 cells at 300g using a Shandon CytospinH 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA) onto glass slides. Cells were fixed in 0.5% formalin and antibodies, such as those recognizing the human High Molecular Weight Melanoma-Associated Antigen (anti-HMW-MAA clone LHM2, Invitrogen, Carlsbad, CA), were incubated overnight at 4uC and detected following a 2 hour incubation at 4uC with a horseradish peroxidase-conjugated anti-IgG Fc-specific antibody (1:100 dilution in Tris Buffered Saline, Sigma, Dorset, UK). Slides were stained with DAB chromogenic substrate (DAKO, Ely, UK) for 5 minutes, washed and counterstained with Mayer's hematoxlin (Merck, Darmstadt, Germany) for one minute, dehydrated and mounted in DPX mountant (Sigma) prior to assessments.
Antibodies bound to cell surface antigens were also detected on live cells by flow cytometry. Adherent cells were detached using StemProH AccutaseH cell disassociation solution (Gibco) and incubated at 2610 5 cells per sample with antibody, isotype control or cell culture supernatants for 30 minutes at 4uC. Antibodies bound to cells were detected using a FITC-conjugated anti-IgG Fc-specific antibody (Jackson ImmunoResearch). The binding of tumor-specific antibodies to cells was compared to an excess of isotype control IgG 1 antibody (Jackson ImmunoResearch). Binding of Trastuzumab across melanoma cell lines and primary human melanocytes was evaluated by subtracting the mean fluorescence intensity (MFI) values of equal amounts of isotype control. Evaluations are representative of three experiments.
We developed and employed a novel cell-based ELISA to identify melanoma-reactive antibodies. Adherent cells of interest were plated at 3610 5 cells per in 200 mL of appropriate media well on 96-well flat bottom tissue culture plates (Corning, Corning, NY) and were grown in a monolayer at 37uC and 5% CO 2 to 80-100% confluence. Cells were then lightly fixed in 0.5% formaldehyde/ Hank's Buffered Salt Solution. Plates were then wrapped in foil and placed in a -80uC freezer until the day of the assay. On the day of the assay, plates were thawed for 30 minutes, washed 3 times with PBS and then blocked with a 5% non-fat milk/PBS solution for 2 hours. After removal of the blocking solution, 50 mL of culture supernatants or tumor-specific antibodies were diluted 1:2 in 1% non-fat milk/PBS solution and then added to each well, and plates were incubated for 90 minutes at room temperature on an orbital shaker. Plates were then washed 4 times with PBS/ 0.05%Tween (PBS-T). The binding of antibodies to cell surface proteins was detected following a 45 minute incubation with a goat anti-human horseradish peroxidase-labeled F(ab)' 2 Fc-specific antibody (Jackson ImmunoResearch, West Grove, PA) diluted 1:250 in 1% milk/PBS-T at room temperature on an orbital shaker. Wells were then washed 4 times with PBS-T. The color reaction was developed for 15 minutes with OPD (Sigma) and OD was measured in an ELISA reader (BMG Labtech, Offenbury, Germany) at 492 nm (reference wavelength, 650 nm). Each plate contained triplicate wells of a positive control antibody, Trastuzumab (Genentech, South San Francisco, CA), and a negative control antibody, non-specific human IgG 1 (Jackson Immunoresearch) at a concentration of 250 ng/mL both diluted in RPMI-1640 media supplemented with 10% fetal calf serum. Binding of Trastuzumab to cells and background OD values for the negative non-specific human IgG control antibody formed the criteria for inclusion of readouts in the study. Since we were limited by the volume of culture supernatants for each culture, assays were repeated only when sufficient culture supernatants were available to confirm reproducibility of readouts.
Using the Cell-based ELISA Patient and healthy volunteer antibody responses were assessed using the cell-based ELISA. We evaluated the reactivity of the supernatant from each B cell culture to tumor cells relative to negative and positive control antibodies. In order to compare antitumor antibody responses to metastatic and primary melanoma cells between patients and healthy volunteers, and among patient groups, optical densities (OD) were normalized using the following formula:
Fold increase~O ptical density of B cell culture supernatant Mean optical density of non{specific IgG 1 Additionally, this calculation was used to normalize ELISA results among multiple melanoma cell lines and primary melanocytes in order to evaluate the tumor specificity of antibodies.
To evaluate the presence and estimate the frequency of tumorreactive antibodies, we selected wells with OD values above 75% of the OD of the positive control antibody. To compare the percentage of positive cultures across patients, OD values were normalized against the positive control. For these evaluations, the mean positive control OD was assigned a relative absorbance of 1 for each plate and B cell cultures were converted from OD units to relative absorbance, and culture wells with relative absorbance values greater than 0.75 to melanoma cells but not melanocytes were selected. These criteria were also applied in limiting dilution assays to estimate the percentage of non-reactive B cell culture well. In these limiting dilution assays, B cells were plated at different densities (ranging from 125 to 2,500 B cells) and the percentage of non-reactive cultures was calculated for different patients and cell lines as a way to approximate the frequency of B cells producing melanoma-reactive antibodies using Poisson distribution.
The tumor-killing potential of 2 patient-derived monoclonal antibodies was assessed: one tumor-specific antibody (6_2G3), and another antibody that did not recognize tumor cells (6_2D10), both derived from the same patient (Patient 6). Both antibodies were simultaneously evaluated using a three-color fluorescent live cell imaging cytotoxicity assay. A-375 cells were plated overnight at 2x10 5 cells per well on 6-well culture plates (Corning). Using a LIVE/DEADH Viability/Cytotoxicity kit (Molecular Probes, Eugene, OR) live tumor cells were labeled with 2mM of Calcein AM 30 minutes prior to cytotoxicity assays, washed in RPMI 1640 supplemented with 10% FCS and 1% penicillin streptomycin, and re-suspended in media containing 4 mM Ethidium homodimer-1. Ethidium homodimer-1 incorporates into the DNA of dead cells and served as a label for cell death in this assay. U-937 monocytic cells expressing Fcc receptors were used as immune effector cells at a ratio of 3:1 (effectors: tumor cells) [40] . U-937 monocytes were incubated with the 6_2G3 or 6_2D10 antibody for 30 minutes, stained with the CellTracker TM Blue dye (4-chloromethyl-7hydroxycoumarin) (Molecular Probes), washed and added to the Calcein AM-labelled tumor cell cultures containing Ethidium homodimer-1. Samples were incubated and images were captured every 5 minutes for two hours in a humidified temperature controlled chamber using a Zeiss Axiovert microscope equipped with a LD-Plan-Neofluar 20x/0.4 Korr/Ph2 objective and AxioVision software system (Carl Zeiss, Jena, Germany). Following incubation, fluorescent intensities of Calcein AM-positive live tumor cells, as well as incorporation of Ethidium homodimer-1 into cells were measured and cell death was assessed with NIS-Elements BR 3 software (Nikon). The movement of effector cells in the cultures was tracked and analyzed using IMARIS software (Bitplane, Zurich, Switzerland).
Descriptive statistics were generated to examine the distribution of melanoma-reactive B cell cultures from each patient including the mean, 95% confidence interval and maximum reactivity to melanoma cells. A two-sided Student's t test was used to compare the mean reactivity of antibody cultures derived from melanoma patients to healthy volunteers to primary or metastatic melanoma cell lines and to compare antibody responses between patients with non-metastatic and metastatic disease. A one-way ANOVA was used to compare antibody reactivity to a metastatic melanoma cell line among B cell cultures derived from patients with stage II, III and IV disease with a Tukey's post hoc comparison test. A twosided Student's t test was used to compare antibody-mediated tumor cell killing between tumor-specific and non-specific monoclonal antibodies derived from the same patient. A twosided Student's t test was also employed to compare the movement of immune effector cells, pre-incubated with antibodies, in contact with tumor cells to the movement of immune cells not in contact with tumor cells. All statistical analyses were performed using GraphPad Prism software (version 5.03, GraphPad, San Diego, CA) and error bars in all figures represent 95% confidence intervals. Video S1 Real-time live-cell cytotoxicity assay for the 6_2G3 melanoma-specific antibody. (AVI) Video S2 Real-time live-cell cytotoxicity assay for the 6_2D10 non-melanoma-specific antibody (negative control).
Videos S1 and S2
These video files show our real-time cytotoxic assays. Video S1 shows real-time functional data of the 6_2G3 melanoma-specific patient derived antibody which was observed to kill melanoma cells. Video S2 shows the identical assays shown in Video S1 using a non-melanoma specific antibody derived from the same patient, as a negative control. In these assays, live tumor cells are labeled in green (live cell dye), U-937 monocytic cells are labeled in blue, and cell death is indicated by the incorporation of red (Ethidium homodimer-1 incorporation). Frames from these videos are also displayed in Figure 7 , B. The Multifaceted Poliovirus 2A Protease: Regulation of Gene Expression by Picornavirus Proteases After entry into animal cells, most viruses hijack essential components involved in gene expression. This is the case of poliovirus, which abrogates cellular translation soon after virus internalization. Abrogation is achieved by cleavage of both eIF4GI and eIF4GII by the viral protease 2A. Apart from the interference of poliovirus with cellular protein synthesis, other gene expression steps such as RNA and protein trafficking between nucleus and cytoplasm are also altered. Poliovirus 2A(pro) is capable of hydrolyzing components of the nuclear pore, thus preventing an efficient antiviral response by the host cell. Here, we compare in detail poliovirus 2A(pro) with other viral proteins (from picornaviruses and unrelated families) as regard to their activity on key host factors that control gene expression. It is possible that future analyses to determine the cellular proteins targeted by 2A(pro) will uncover other cellular functions ablated by poliovirus infection. Further understanding of the cellular proteins hydrolyzed by 2A(pro) will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell. A great variety of animal viruses encode for proteases that accomplish crucial functions during the biological cycle of the virus [1] . Usually, the main function of these proteases is to proteolyze viral polypeptide precursors to render mature viral proteins that form part of viral capsids or participate in virus vegetative processes [2] . Although both DNA and RNA viruses can encode proteases, the proteolytic tailoring of polypeptide precursors is most common among viruses with positive single-stranded RNA genomes, such as picornaviruses, flaviviruses, caliciviruses, and retroviruses [3] [4] [5] [6] [7] . This mechanism of gene expression by proteolytic processing serves to compress the genetic information of viruses in the limited space provided by the genome. In this manner, viruses reduce the genetic space occupied by 5 and 3 untranslated regions (UTRs), the signals devoted for mRNA transcription and to initiate translation are minimal, such that, for instance, in the case of picornaviruses or flaviviruses, only one 5 and 3 UTR is necessary for viral replication, transcription, translation, and morphogenesis, despite the fact that several viral proteins are synthesized by the infected cells. In addition, a number of polypeptide precursors may exhibit functions that differ from those present in their mature products. In the case of poliovirus (PV), eleven mature proteins are produced from a single translation initiation event, and at least two precursors, 2BC and 3CD, accomplish functions which are not present in their mature proteins. Taking together all these considerations, the "proteolytic strategy" provides the small RNA viruses with an advantageous and efficient mechanism for distribution of the genome to accomplish all the viral biological functions with the smaller genetic space.
Apart from generation of active viral proteins that participate in capsid morphogenesis and genome replication, viral proteases may also target a number of cellular proteins. Proteolysis of these cellular substrates can very much affect a variety of cellular processes and play an important role in virus-induced cytopathogenesis [8, 9] . In this regard, productive poliovirus infection induces rapid morphological alterations in host-cell. Among them, the most prevalent is the accumulation of numerous membranous vesicles in the cytoplasm, derived from endoplasmic reticulum where the viral proteins 2C and 2BC play a central role [10] . In addition, cellular shape is modified upon viral replication giving rise to cell rounding, which is most probably induced by disorders in the cytoskeletal network [11] . Finally, chromatin condensates at late times postinfection, associated with the nuclear envelope except for sites where nuclear pores are placed [11] . Interestingly, individual expression of the viral proteases 2A pro and 3C pro leads to the induction of most of these cytopathic effects, supporting the idea that these proteases actively contribute to the viral-induced morphological changes [12] . Indeed, long-term expression of either 2A pro or 3C pro triggers the activation of caspases and, thus, cell death by apoptosis [11, 12] , reflecting the strong cytotoxicity of both proteases. In addition to the cytopathic effects induced by 2A pro and 3C pro , hydrolysis of host proteins may impact on other cellular functions such as the antiviral responses to virus infection. Activation of innate immunity pathways, as well as the establishment of an antiviral response, is absolutely dependent on signals traversing the nuclear membrane through the nuclear pore complex. Therefore, many viruses block cellular gene expression at different levels, that is, translation, transcription or protein and RNA trafficking between nucleus and cytoplasm. The blockade of active trafficking can inhibit the nuclear import of antiviral signals or prevent the export of cellular mRNAs detrimental to virus processes. All these effects can be achieved by hydrolysis of specific cellular proteins. The precise number of cellular proteins degraded by a viral protease, which is known as the "degradome," still remains unknown for a given viral protease. Perhaps, one of the beststudied proteases in this respect is PV 2A pro . The discovery that PV 2A pro bisects the initiation factor of translation eIF4G leading to the regulation of translation in the infected cells has attracted much attention from many laboratories during the past three decades [13, 14] . More recently, 2A pro has been involved in the alteration of RNA and protein trafficking between the nucleus and the cytoplasm upon proteolysis of several nucleoporins [15] [16] [17] . The present paper focuses on the multifaceted activities of 2A pro and its regulation of different viral and cellular processes.
PV is a prototype member of the Picornaviridae family that infects cells of human or simian origin cytolytically or persistently and is responsible for poliomyelitis in humans [18] . The RNA genome is housed in a naked capsid formed by 60 copies of each of the four structural proteins: VP1, VP2, VP3, and VP4. The infectious cycle commences by the attachment of a viral particle to cellular receptors present at the cell surface [19, 20] . This interaction leads to virion internalization and destabilization of the capsid, which adopts a less compact structure. Once the RNA is released in the cytoplasm, it interacts with the translational machinery, directing the synthesis of viral proteins during the early phase of infection.
The PV genome is composed of a single-stranded RNA copy of positive polarity of about 7.4 Kb [21, 22] . This RNA molecule is uncapped and contains a poly(A) tail at its 3 end and a single open reading frame, which encodes for a polyprotein of about two thousands amino acid residues. This polyprotein is proteolytically processed giving rise to the mature viral proteins [23] (Figure 1(a) ). Three different cleavages can be distinguished on the viral polyprotein: (i) polysomal cleavages that are produced on the nascent polypeptide chain. The first of these cleavages is catalyzed by 2A pro at its amino terminus separating the P1 precursor that encodes for the structural proteins from the rest of nonstructural polypeptides (Figure 1(b) ). The second cleavage still on polysomes is performed by 3C pro , releasing the P2 precursor (2ABC) from P3 (3ABCD); (ii) cytoplasmic cleavages that are mostly exerted by 3C pro and (iii) hydrolysis of VP0 (VP4-VP2), which is concomitant with the morphogenesis of virus particles [2, 23] . All these hydrolytic events lead to the formation of eleven mature proteins and several precursors such as P1, P2, P3, VP0, VP3, VP1, 2BC, 3AB, and 3CD. This last precursor, 3CD, can be used as substrate by 2A pro or 3C pro . The alternative cleavage carried out by 2A pro renders the mature products 3C and 3D , whereas 3C pro generates the canonical proteins 3C pro and 3D pol . However, the biological significance of this alternative cleavage is obscure because PV mutated at 2A pro -cleavage site on 3CD does not exhibit defects in virus replication [24] .
The nonstructural proteins that are generated participate in the replication of viral genomes [25, 26] . To this end, the positive RNA genome is recognized at its 3 end by proteins of the replication complex to synthesize the complementary RNA strand of negative polarity. In this process, 3B protein, also known as VPg, acts as a primer to initiate viral RNA transcription [27] . This leads to the formation of a doublestranded RNA molecule, also known as the replicative form. The negative RNA synthesized serves in turn as a template to direct the synthesis of several copies of positive RNA, so this process leads to the production of several nascent RNA molecules with a VPg molecule bound to their 5 ends on the negative RNA molecule forming a replicative intermediate. The positive RNA molecules synthesized may participate in three processes (i) to serve as templates for synthesizing more negative RNA molecules; (ii) as mRNAs that will be engaged in translation, and (iii) as genomes that will be encapsidated in new viral particles. In picornaviruses, the only type of mRNA molecule known is exactly the same as the genome.
Once the synthesis of several thousands of positive RNA molecules is performed, the late phase of translation takes place, giving rise also during this period to a great amount of viral proteins, some of which will participate in virus morphogenesis. This late phase of infection is preceded by the abrogation of cellular mRNA translation, such that only viral proteins are being synthesized late in the PV life cycle [14] . In the case of picornaviruses, transcription is dependent on continuous viral protein synthesis [28] . Thus, inhibition of viral mRNA translation provokes the sudden blockade of viral RNA synthesis. Moreover, translation is coupled to transcription, such that viral RNAs transfected Journal of Biomedicine and Biotechnology Figure 1 : (a) Structure of poliovirus genome and proteolytic processing of its polyprotein. The PV genome consists of a single-stranded, positive-sense polarity RNA molecule, which encodes a single polyprotein. The 5 nontranslated region (NTR) is covalently linked to the viral protein VPg. The PV genome is polyadenylated (A n ) in its 3 end. The polyprotein contains four structural (P1) and seven nonstructural (P2 and P3) proteins that are released from the polypeptide chain by proteolytic processing mediated by the viral-encoded proteinases 2A pro and 3C pro /3CD pro . The intermediate products of processing 2BC, 3CD, and 3AB exhibit functions distinct from those of their respective final cleavage products. The alternative cleavage carried out by 2A pro rendering the mature products 3C and 3D is also shown. (b) Once the ribosome has synthesized the PV 2A pro sequence and continues translation on the P2 region, the autocatalytic activity of PV 2A pro is manifested by cleaving itself at its amino terminus still on the nascent polypeptide chain. This cleavage liberates the P1 precursor that will render the capsid proteins on subsequent proteolytic events catalyzed by 3C pro or 3CD pro . Cleavage at the carboxy terminus of PV 2A pro on the P2 precursor is accomplished by PV 3C pro , leaving free 2A pro and generating the 2BC precursor.
into picornavirus-infected cells are not able to direct protein synthesis [29] . Therefore, these two processes of viral macromolecular biosynthesis are tightly coupled, making it difficult to determine exactly the function affected in some PV mutants. Notably, continuous lipid and cellular membrane synthesis is also necessary for PV RNA synthesis [30] . The morphogenesis of progeny virions in PV-infected cells is observed concomitantly with viral RNA translation and replication. The release of new viral particles takes place by cell lysis, due to membrane permeabilization that occurs at the late phase of infection [31] . Viroporin 2B and its precursor 2BC are responsible for this permeabilization upon the formation of pore channels in cellular membranes [32, 33] .
PV has represented a useful model to gain insight into diverse aspects of molecular biology and gene expression. A number of discoveries concerning animal viruses with RNA genomes were initially made in PV. For example, the presence of uncapped mRNA, the sequencing and development of an infectious cDNA clone, the three-dimensional structure of a virus particle, the discovery of the IRES elements, the synthesis of an infectious virus in a cell-free system, the chemical synthesis of a complete viral genomes, among others, were initially reported in PV [34] [35] [36] [37] [38] . In addition, the first time that eIF4G was found proteolytically cleaved was in PV-infected cells [39] .
Picornaviruses encode different proteases depending on the virus species although it is common to all of them to encode 3C pro and its precursor 3CD pro (Figure 2 ). In PV, both these exhibit protease activity, and they execute most of the hydrolytic events on the viral polyprotein [2, 23, 40] . Apart from these two proteases, 3C pro and 3CD pro , picornaviruses also contain a 2A gene, whose product in some species exhibits proteolytic activity, as is the case for PV ( Figure 2) . 2A pro has a limited proteolytic effect on the polyprotein and its function is most probably one of altering cellular functions by the cleavage of a number of cellular proteins. In this regard, the best studied of these cleavages is the bisection of eIF4G ( Figure 3 ) [14] .
The general organization of the picornavirus genomes is to encode for P1-P2-P3 precursors giving rise to 4-3-4 mature products. Some picornavirus species, in addition, encode a leader protein (L) placed before P1 ( Figure 2 ) [22] . In the case of aphthoviruses, such as foot-and-mouth disease virus (FMDV), the L protein has proteolytic activity and it is known as L pro [42, 43] . During polyprotein synthesis L pro is the first protein synthesized and its autoproteolytic activity releases itself from the rest of the polypeptide chain. Thus, the only known hydrolysis executed by L pro on the polyprotein is to hydrolyze between its carboxy terminus and the amino terminus of VP4 ( Figure 2 ). Because L pro does not play a direct role in viral replication [44] and its protease activity has a limited impact in the viral polyprotein, this protease may be involved in the interaction with the hostcell [45] [46] [47] . Indeed, L pro also exhibits proteolytic activity on eIF4G acting at a position close to that of PV 2A pro (Figure 3 ) [48] [49] [50] . In the case of FMDV, the 2A protein is reduced to a small peptide of 18 residues that does not hydrolyze eIF4G; instead it induces the release of 2A from its carboxy terminus by a ribosomal skip mechanism [51, 52] . This model proposes that FMDV 2A modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage at the C-terminus of 2A, thereby releasing the polypeptide from the translational complex. However, not all L or 2A proteins from picornaviruses exhibit protease activity, since in the case of EMCV, which encodes both proteins, neither has been demonstrated to possess proteolytic activity [53] .
All known proteases have been classified in four classes and many subgroups according to three parameters: (i) their catalytic center, (ii) their substrate specificity, and (iii) their three-dimensional structure. The classification of the different picornavirus proteases initially relied upon the effect of protease inhibitors. Compounds that blocked sulphydryl groups abrogated the proteolytic activity of 2A pro and 3C pro , suggesting that the nucleophilic aminoacid in the active site was cysteine [54, 55] . However, another cysteine inhibitor such as E64 had no effect on these proteases, while L pro activity was inhibited not only by sulphydrylactive compounds but also by E64 [56, 57] . These findings together with structural observations imply that L pro belongs to the class of papain-like cysteine proteinases [43, [58] [59] [60] . Comparison of the structure of picornavirus proteases with prototypes of cellular ones revealed that both 2A pro and 3C pro are similar in structure to the chymotrypsin like group [61] [62] [63] . Picornavirus 3C pro reflects similarities to the staphylococcus aureus proteinase, whereas 2A pro is more akin to streptomyces griseus proteinase A.
PV 2A pro is a protein composed of 149 amino acids that belongs to the cysteine protease group [54] . PV 2A pro is autocatalytically processed at its amino terminus between the capsid protein VP1 and 2A [64] (see Figure 1 (b)). The determinants of substrate specificity of picornaviral PV 2A pro have been investigated in detail by identification of cleavage sites by N-terminal Edman degradation, mutational analysis and using synthetic peptides as substrates [34, [65] [66] [67] . PV 2A pro can recognize a wide variety of amino acid residues at the P1 position. The determinants of substrate specificity for PV 2A pro lie at positions P4, P2, P1 , and P2 , which are preferentially occupied with Ile/Leu, Thr/Ser, Gly, and Pro, respectively. Moreover, the determinants of substrate specificity of HRV and coxsackievirus 2A pro are very similar to those found for PV 2A pro [65] [66] [67] . The yeast two-hybrid system has been used to identify the substrate sequence interacting with PV 2A pro . All the sequences identified contain the Leu-X-Thr-Z motif (X for any amino acid; Z for a hydrophobic residue) in positions from P4 to P1 suggesting the presence of a common interacting site on PV 2A pro substrates [68] .
Several 2A pro variants have been generated in the entire PV genome or in the isolated 2A gene. Generation of PV 2A pro mutants was initially used to identify the Cys 106 , His 18 , and Asp 35 as the residues that form part of the catalytic triad of 2A pro [69, 70] . These data have been confirmed in the structure of HRV2 2A pro [71] . The role of the conserved Cys and His residues in the structure-function relationship has also been studied by mutagenesis. The residues Cys 55 , Cys 57 , Cys 115 , and His 117 play a critical role in the cis and trans proteolytic activity by maintaining 2A pro structure [72] . The structure of HRV2 2A pro shows that the Zn 2+ ion is coordinated tetrahedrally by the side chains of these conserved Cys and His residues. This Zn 2+ ion is tightly bound near to the C-terminal domain and may be important for the stability of the 2A pro [71, 73, 74] .
The yeast Saccharomyces cerevisiae has been used as a system to obtain PV 2A pro variants [75] . The fact that this protease is very toxic for yeast has been exploited to generate 2A pro variants devoid of this cytotoxicity. Using this approach, several PV 2A pro unable to cleave eIF4G have been obtained. The characterization of these mutants revealed a region in 2A pro involved in the interaction with substrates but none of the mutations were found in the catalytic triad. A parallelism has been observed between the ability of these PV 2A pro variants to block protein synthesis and to cleave eIF4G [76] . PV mutants in 2A gene that lack trans but not cis proteolytic activity have been also identified. Normal Journal of Biomedicine and Biotechnology processing of the viral polyprotein is observed upon infection with these PV variants, whereas eIF4G remains intact in these cells [77] . Interestingly, RNA replication of those mutant viruses is hampered, suggesting that there is a correlation between PV RNA replication and the trans activity of 2A pro . It is controversial whether PV 2A pro contributes directly to viral replication or not. Although for many years it was thought that PV 2A pro plays a direct role in PV replication, more recent studies have shown that a full-length dicistronic PV construct lacking 2A pro is capable to give rise to progeny viruses. Moreover, virus yields of PV variants lacking the P1 coding region is partially restored when P1 is expressed in trans, suggesting that cleavage of the viral polyprotein by PV 2A pro is not essential for viral replication. [78] . However, it is known that 2A pro is important for inducing the cytophatic effect and for avoiding the inhibition of PV replication in interferon (IFN) α treated cells [79] . In agreement with the idea that 2A pro participates in viral RNA replication, a fraction of this protease localizes in PV replicative foci although the majority of 2A pro is associated with the matrix structure in the cytoplasm of infected cells [80] . However, the presence of 2A pro in the proximity of replication complexes does not demonstrate that it participates directly in the replication process. 
5.1. Structure and Functioning of eIF4G. The process of translation can be divided in different steps: initiation, elongation, termination and ribosome recycling. The synthesis of cellular proteins is highly regulated, and in this sense, the most precisely controlled step is the initiation of translation (for a recent review, see [81, 82] ). For most eukaryotic mRNAs, the initiation of translation commences with the recognition of the cap structure ( 7 mGpppN) and the poly(A) tail by the heterotrimeric complex eIF4F and the poly(A)-binding protein (PABP), respectively, followed by the recruitment of the 43S preinitiation complex containing the 40S ribosomal subunit, the ternary complex Met-tRNA Met i -eIF2-GTP, and the eukaryotic initiation factors (eIFs), 1, 1A, 3, and 5. Then, the preinitiation complex scans along the 5 untranslated region until an AUG initiation codon is encountered in a favourable context. The perfect complementarity between the AUG start codon and the anticodon of Met-tRNA i leads to the arrest of scanning and the hydrolysis of GTP in the ternary complex. The release of eIF2-GDP and other factors triggers the interaction of the preinitiation complex with the 60S ribosomal subunit to form the 80S initiation complex, proceeding to translation of the mRNA coding region.
A number of eukaryotic initiation factors participate in both mRNA binding and scanning of the 5 UTR of the mRNA by the small ribosomal subunit. The cap-binding protein eIF4E, together with the DEAD-box helicase eIF4A and the translation initiation factor eIF4G, forms the protein complex eIF4F. The heterotrimeric complex eIF4F is required for recruiting the 43S preinitiation complex onto the cap structure located at the 5 end of the mRNA [83] . In this sense, eIF4G, the larger polypeptide of eIF4F, functions as an adaptor molecule that bridges the mRNAs to ribosomes via interactions with factors eIF4E (which binds the 5 cap structure), PABP (which binds the poly(A) tail), and eIF3, which interacts with the 40S ribosome subunit ( Figure 3 ) [81, [84] [85] [86] . In addition, eIF4G also contains binding sites for other polypeptides involved in translation, such as the RNA helicase eIF4A ( Figure 3 ) [87] , which is required to unwind the secondary structure within the mRNA 5 leader sequence that would otherwise inhibit ribosome scanning. The simultaneous interaction of eIF4G with eIF4E and PABP promotes circularization of the mRNA in a closed loop that facilitates the initiation of new rounds of translation by the proximity of the 5 and 3 ends [88, 89] . Furthermore, eIF4G also interacts with the mitogen-activated protein kinase 1 (Mnk1) (Figure 3 ), which phosphorylates eIF4E, although the role of this phosphorylation in the initiation of translation in still unclear [90] [91] [92] [93] [94] . Two forms of eIF4G, known as eIF4GI and eIF4GII, have been identified in mammalian cells. Both forms show only 46% amino acid sequence identity but they are thought to be functionally interchangeable due to the high homology in key domains that interact with other factors [88] . Evidence obtained by specific depletion of each eIF4G form or differential cleavage of each of them by specific proteases (see below) points to the idea that both factors should be lacking for complete abolition of protein synthesis [95, 96] . eIF4GI is the dominant form in HeLa cells, in which the ratio between eIFGI and eIF4GII is 9 : 1 [97] . However, the specific role of each form of eIF4G in the initiation of translation remains unknown. It was proposed that both eIF4G forms are differentially regulated by different kinases, supporting the hypothesis that eIF4GI and eIF4GII could drive differentially translation initiation [98] . eIF4GI is phosphorylated in response to serum and in a rapamycin-dependent manner at Ser 1148, 1188, and 1232, although the role of these posttranslational modifications is still under investigation [99] . In addition, eIF4GI is phosphorylated by p21-activated protein kinase (Pak-2) that is induced under stress conditions. This phosphorylation takes place in the eIF4E-binding site of eIF4G and avoids the interaction between these two factors, inhibiting cap-dependent initiation of translation [100] . On the other hand, eIF4GII is phosphorylated during mitosis [101] by calmodulin-dependent kinase I at Ser 1156 [102] . Therefore, activity of eIF4GI and eIF4GII might be tightly and reversibly regulated by phosphorylation under different physiological conditions. Nevertheless, this factor is also subjected to irreversible modifications such as caspasemediated proteolysis, which is triggered during apoptosis and leads to shutoff of protein synthesis. Caspase-3 cleaves directly eIF4GI in positions 532 and 1175 removing PABP, Mnk1, and one eIF4A-binding domain from the eIF4GI core ( Figure 3 ) [103, 104] . In contrast, eIF4GII is degraded during apoptosis with a delayed kinetics in relation to eIF4GI proteolysis, correlating with the shutoff of the protein synthesis [104] .
Furthermore, many eIF4GI isoforms have been detected in HeLa cells and these are synthesized from several distinct mRNAs via alternative promoter usage and alternative splicing [105] . The largest is the eIF4GI-a isoform, which contains 1,600 residues, while the eIF4GI-b, -c, -d, ande are shorter variants [106] . It has been described that the longer isoforms are more active in translation initiation, most probably because they contain the PABP-binding site [107] .
PV results in a rapid shutoff of host-cell protein synthesis, whereas viral mRNA translation takes place efficiently [108] . It was initially observed that the inhibition of host-cell translation in PV-infected cells correlated with the proteolysis of a component of the eIF4F complex with a molecular mass of about 220 kDa (later identified as eIF4G) [39, 109] . This cleavage is exerted by 2A pro and can be prevented by both insertion of mutations that abolish the protease activity and addition of 2A inhibitors [76, 110, 111] . Interestingly, this proteolysis is more effective when eIF4E is interacting with eIF4G, suggesting that PV 2A pro preferentially acts on the eIF4G pool involved in translation [112] . Cleavage of eIF4GI also occurs in cells infected with other picornaviruses such as HRV, coxsackievirus, and FMDV [48, 113, 114] . Interestingly, 2A pro from PV, HRV, and coxsackieviruses cleave eIF4GI at positions 681/682 (Figure 3 ), suggesting the conservation of the specificity of enterovirus 2A proteases for the substrate determinants present in eIF4GI [114, 115] . Cleavage at position 681/682 separates eIF4E-and eIF3binding sites of eIF4GI, contained in N-terminal and Cterminal fragments respectively, thus decoupling mRNA and ribosome recruiting activities. The PV 2A pro cleaves eIF4GI directly and does not require any additional proteins for this process to occur [116, 117] . However, the fact that PV 2A pro is not copurified with eIF4GI fragments from PV-infected cell extracts suggest that 2A pro induced the activation of a host protease, which in turn cleaves eIF4G during PV infection [112] . In addition, it has been proposed that eIF3 and an unknown host-cell protein could act as cofactors for eIF4GI cleavage by PV 2A pro [118] . In this sense, Zamora and colleagues suggested that PV infection activates at least two host-cell proteases, which together with PV 2A pro , cleave eIF4GI [119] . Nevertheless, no additional evidence has been put forward to support this hypothesis and the identity of these host proteases has not yet been determined.
Many reports have demonstrated that the kinetics of protein synthesis shutoff and eIF4GI cleavage are not correlated in PV-infected cells [120] [121] [122] . These data clearly indicate that additional translation factors may be cleaved to achieve an efficient inhibition of cellular mRNA translation. In this regard, additional reports showed that eIF4GII is also proteolyzed by 2A pro during PV and HRV infections and that this cleavage is exerted between amino acids 699/700 leading to a proteolytic pattern similar to eIF4GI. Interestingly, eIF4GII is significantly more resistant to 2A pro -mediated cleavage than eIF4GI and the kinetics of protein synthesis shutoff close correlates with eIF4GII cleavage in PV-and RHV-infected cells [122, 123] . In those studies, Gradi and colleagues proposed that hydrolysis of both eIF4GI and eIF4GII is required for achieving PV-and HRV-mediated inhibition of host-cell mRNA translation and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host-cell translation after infection with those viruses [122, 123] .
A variety of approaches have been devised to express PV 2A pro in order to cleave eIF4G in culture cells or in cellfree systems. Of these approaches, the most straightforward system has been the addition of the purified PV 2A pro , usually as a hybrid protein such as MBP-2A pro , to cellfree systems such as rabbit reticulocyte lysates (RRLs), HeLa and Krebs-2 extracts [76, 118, [124] [125] [126] [127] . In this sense, the addition of about 1 to 5 μg MBP-2A pro suffices to hydrolyze eIF4G in those cell-free systems [76, [125] [126] [127] . An alternative method to cleave eIF4G in an in vitro system is the translation of an mRNA encoding PV 2A pro in translation competent extracts [50] . This assay has the advantage of providing genuine and freshly made PV 2A pro , leading to total cleavage of eIF4G in the test tube after several minutes of translation. Many different approaches have been explored in culture cells, the most popular being transfection of plasmids encoding PV 2A pro in different eukaryotic cell types [75, 76, [128] [129] [130] [131] [132] [133] [134] . Several plasmids have been utilized in this respect, and perhaps the most successful one is pTM1-2A, which is transfected in mammalian cells that transiently express T7 RNA polymerase by infection with a recombinant vaccinia T7 virus [76, 130, 131, 133, 134] . The amount of protease synthesized in this system is similar to that found in PV-infected cells at late times of infection, but these amounts are reached 1-2 hours after transfection. Similar results have been obtained in cells constitutively expressing T7 polymerase, which comprises a less pleiotropic system, because vaccinia virus proteins are not expressed (unpublished data). Since PV 2A pro targets a number of different cellular proteins, which affect several cellular functions depending on the amount of protease synthesized (see below), in some instances, it is useful to express 2A pro at low levels. We have explored many alternative methods trying to get a system that allows us to control the levels of PV 2A pro into the cells. Novoa and colleagues developed a protocol based on the addition of hybrid proteins bearing PV 2A pro . These recombinant proteins enter into the cytoplasm on cell membrane permeabilization by different methods such as addition of MBP-2A pro mixed with replicationally inactive chicken adenovirus particles [135] . Cleavage of eIF4G following these protocols takes place after incubation for 8-10 hours, suggesting that the amount of protease internalized is probably low but sufficient to hydrolyze eIF4GI in virtually all culture cells [136] . Probably one of the most attractive systems is a stable cell line that inducibly express PV 2A pro , obtained in two different laboratories including ours [137, 138] . In these cell lines, PV 2A pro is synthesized when tretracycline is removed from the culture medium, leading to low expression of PV 2A pro that induces efficient cleavage of eIF4G after 13 h post induction correlating with a potent inhibition of cellular translation. Finally, long term expression of PV 2A pro in Tet off cell lines triggers apoptosis [12, 137, 138] . The main drawback of this cell line is the low PV 2A pro escape under repression conditions that gives rise to a basal cytotoxicity. Probably, the most efficient method is electroporation of an mRNA encoding 2A pro under the control of EMCV leader sequence (IRES-2A) [95] . The biggest advantage of this method is the capacity to regulate levels of 2A pro expression by controlling the amounts of IRES-2A transfected. For example, electroporation of 9 μg of IRES-2A into ∼ 1.5 · 10 6 HeLa cells leads to total cleavage of both eIF4GI and eIF4GII in only 2 h, resulting in an almost complete shutoff of cellular protein synthesis. In contrast, electroporation of low amounts of IRES-2A (1 μg) into HeLa cells induces efficient cleavage of eIF4GI, whereas eIF4GII remains largely intact. Therefore, 9-fold more IRES-2A mRNA is required to cleave eIF4GII compared to eIF4GI [95] . Based on the IRES-2A mRNA electroporation method we were able to induce the differential proteolysis of eIF4GI and eIF4GII in a timeand dose-dependent manner. Kinetics of protein synthesis shutoff and eIF4GII cleavage is closely correlated in HeLa cells, resembling what was found in PV-infected cells [122] .
In agreement with what was observed with the addition of exogenous recombinant proteins [136] , translation of de novo synthesized mRNAs showed higher susceptibility to low doses of PV 2A pro than mRNAs already engaged in translational machinery [95] . These results suggested a possible specific role of eIF4GI in the pioneer round of translation in agreement with a previous report [139] . However, specific ablation of eIF4GI using siRNAs induced a moderate inhibition of luciferase synthesis from de novo synthesized and preexisting mRNA (about 40% in both cases) [96] . These findings reported by Welnowska and colleagues indicated that the higher susceptibility of de novo synthesized mRNA translation to low doses of IRES-2A might be produced by an additional effect of PV 2A pro on another gene expression step. In this regard, further studies demonstrate that the stronger impact of 2A pro on de novo synthesized mRNAs is due to the concomitant inhibition of RNA nuclear export by Nucleoporin 98 cleavage, which is also achieved under these conditions (see below) [17] . Interestingly, cellular mRNAs are able to initiate translation after a polysome runoff with high salt treatment when eIF4GI is totally cleaved by PV 2A pro , whereas it is completely abolished when both forms of eIF4G are proteolyzed [95] . Taken together these set of data from cell expressing 2A pro [95, 136] as well as from PV-infected cells [120] [121] [122] we can conclude that complete shutoff of the protein synthesis induced by PV 2A pro is achieved when both eIF4GI and eIF4GII are completely cleaved. Therefore, when the levels of one of the two populations of eIF4G remain unaffected either because it is not cleaved by PV 2A pro [95] or it is not depleted by siRNAs [96] , extensive host protein synthesis takes place.
The infection of PV and coxsackievirus also leads to hydrolysis of PABP [140, 141] . This cleavage is carried out by PV 3C pro and coxsackievirus 2A pro and 3C pro and it might actively contribute to the host translational shutoff induced by these viruses [142, 143] . In conclusion, the proteolysis of different components of the translation initiation machinery by picornavirus proteases can account for the shutoff of host translation induced after infection although the specific contribution of hydrolysis of eIF4GI, eIF4GII, and PABP remains still unclear.
Infection of animal cells with FMDV also leads to proteolysis of eIF4G and to rapid inhibition of cellular translation [48] . The proteolysis of eIF4G is carried out by the two virally encoded proteases L pro and 3C pro [144, 145] . L pro cleaves both eIF4GI and eIF4GII extremely rapidly at positions 674 ( Figure 3) and 700, respectively, located seven and one amino acids upstream of the 2A pro cleavage sites on eIF4GI and eIF4GII [50, 146] . The cleavage of eIF4G by FMDV L pro results in the rapid shutoff of host-cell protein synthesis [145] . Although the initial cleavage of eIF4GI can be carried out by FMDV L pro in the absence of virus replication [145] , a sequential cleavage of the C-terminal fragment of eIF4GI by FMDV 3C pro also occurs in BHK cells at early stages of infection concomitant with the shutdown of viral translation. The 3C pro cleavage site on eIF4GI has been located at position 712, 38 amino acids downstream of the L pro cleavage site [147] although the role of this sequential cleavage is still unclear. The amino acid segment of eIF4G located between the L pro and 3C pro cleavage sites binds RNA and was suggested to be critical for mRNA scanning by the preinitiation complex [148] . Interestingly, this secondary cleavage does not occur in human cell lines due to an amino acid substitution at the cleavage site on eIF4GI [147] .
Infection of cells with other picornaviruses such as cardioviruses (EMCV and mengovirus) leads to a shutoff of host-cell protein synthesis. However, eIF4G remains unaffected in these cells. These findings indicate that apart from eIF4G cleavage, there are other mechanisms that may block host translation by picornaviruses.
In addition to picornavirus L pro and 2A pro , proteases from other viruses can also cleave eIF4G. The protease of human immunodeficiency virus type-1 (HIV-1) hydrolyzes eIF4GI during infection of human CD4+ cells [149] . The cleavage of eIF4GI takes place at positions 718, 721, and Journal of Biomedicine and Biotechnology 9 1125, separating it in three domains ( Figure 3 ) [149, 150] . Interestingly, HIV-1 protease efficiently cleaves eIF4GI, but not eIF4GII, both in cell-free systems and in mammalian cells [151] . The differential sensitivity of eIF4GI and eIF4GII to HIV-1 protease is more selective than that observed with picornaviral 2A proteases [122, 123] . HIV-1 protease also cleaves PABP at positions 237 and 477 separating the two first RNA-recognition motifs from the C-terminal domain of PABP [152] . Cleavage of eIF4GI and PABP by HIV-1 protease is sufficient to inhibit the translation of capped and polyadenylated mRNAs in cell-free systems, as well as in transfected cells [127, 151] . In contrast, IRES-driven translation is unaffected or even enhanced by HIV-1 PR after cleavage of both eIF4GI and PABP [127, 151] . Moreover, the translation of capped and polyadenylated HIV-1 genomic mRNA remains unaffected in HeLa extracts under these conditions suggesting that viral protein synthesis might persist at late phases of HIV-1 infection where those factors are cleaved [127] . In contrast, a previous report claimed that the hydrolysis of eIF4GI impaired the translation of both capped and IRES-driven mRNAs in reticulocyte lysate assays [150] . However, the different effect observed by these authors on IRES-driven translation can be due to differences already reported between HeLa extract and RRL [143, 153] . In addition, eIF4G cleavage is executed by proteases from other retrovirus species, such as HIV-2, simian immunodeficiency virus (SIV), human T-cell leukemia virus (HTLV-1), Moloney murine leukemia virus (MoMLV), and mouse mammary tumor virus (MMTV). These proteases hydrolyze eIF4GI and eIF4GII with different cleavage patterns and kinetics [134] . And indeed, several retroviruses, including HIV, SIV, and MoMLV, promote the translation of their gag gene products by internal ribosome entry, indicating that eIF4G cleavage could be compatible with viral protein synthesis in infected cells [154] [155] [156] . Furthermore, cleavage of eIF4GI and eIF4GII also occurs in feline calicivirusinfected cells although the cleavages occur at different sites to those observed for picornavirus proteases [157] . In addition, the 3C-like protease of two caliciviruses, like PV 3C pro , cleaves PABP perhaps as a complementary strategy to inhibit cellular translation [158] . The fact that proteases from many picornaviruses, retroviruses and caliciviruses, target eIF4G, and, in some cases, PABP, strongly suggest that those viruses may share a common mechanism to regulate cellular and viral translation. However, further investigation is required to determine the specific contribution of eIF4GI, eIF4GII and PABP to the shutoff of host-cell translation and virus protein synthesis.
The biological cycle of picornaviruses is confined to the cytoplasm of infected cells. However, some of the PV proteins are able to target nuclear proteins such as transcription and splicing factors and proteins involved in nuclear-cytoplasmic trafficking. One of the best studied cases in this regard is the cleavage of nucleoporins (Nups), components of the nuclear pore complex (NPC), by PV and HRV 2A pro , which directly impacts on nuclear-cytoplasmic trafficking of proteins and RNAs [15, 16, 159] . Complementarily, EMCV L protein affects on the phosphorylation status of Nup62 and induces similar effects to those described for PV 2A pro in the transport of macromolecules through NPC [160] . Nevertheless, Nups are not only targets for picornavirus proteins but also for matrix (M) protein from Vesicular stomatitis virus (VSV) [161] and nonstructural protein 1 (NS1) from influenza virus [162] , which also impair components of the nuclear export-import host machinery following analogous mechanisms. Taking into account that proteins from different positive and negative strand RNA viruses target Nups, we highlight in this paper NPC as a key target for viral proteins, although the possible role of NPC in virus biological cycle is still under intensive research.
Nucleus and cytoplasm are physically separated by a semipermeable barrier known as the nuclear envelope. Due to this compartmentalization, a large number of macromolecules traverse the nuclear envelope to reach their biological destination. For example, proteins are synthesized by ribosomes in the cytoplasm, but some of them such as polymerases, transcription factors, nucleosome components and splicing factors, have to traverse the NPC to reach the nucleoplasm. Conversely, all RNA species are transcribed and processed in the nucleus and later, most of mature RNAs are exported through the NPC to the cytoplasm, where their biological roles take place. Therefore, the regulation of RNA and protein trafficking between nucleus and cytoplasm directly impacts on gene expression [163, 164] . NPC forms large structures (∼125 MDa) embedded in the nuclear envelope with a polarized eightfold symmetrical core. It is sandwiched by a cytoplasmic and nuclear ring, which projects eight filaments of about 50 nm into the cytoplasm and a basket-like structure of about 100 nm into the nucleoplasm [165, 166] . The NPC is composed of multiple copies (8, 16 or 32) of ∼30 different proteins, called Nups, that are grouped in three major classes: (i) the phenylalanineglycine (FG)-containing nucleoporins that actively work in the nuclear-cytoplasmic trafficking of macromolecules; (ii) the structural components, which lack FG-rich domains and (iii) the membrane integral proteins, which anchor the NPC to the nuclear envelope [163, 167] . Whereas the two last groups of nucleoporins play a role in the architecture and localization of the NPC, the FG-nucleoporins directly regulate the transport of RNAs and proteins through the NPC, and they are the main nucleoporin class targeted by viruses (Figure 4) .
Movement of ions, metabolites and other small molecules between nucleus and cytoplasm takes place by passive diffusion; however, transport cargos larger than 40 KDa require the participation of specific receptors and carriers [168, 169] . FG nucleoporins are placed on both cytoplasmic and nucleoplasmic sides of the NPC and play a central role in the active transport of macromolecules. The FG domains of nucleoporins are unfolded regions that participate in energy-independent transient interactions with the cargo receptors during the docking and translocation processes [170] . Nevertheless, the delivery of the cargo and some of the directional steps require hydrolysis of GTP. Trafficking of proteins through NPC is mediated by a family of conserved transport receptors named karyopherins, which recognize short peptides known as nuclear localization signal (NLS) and nuclear export signal (NES) [169, 171] . In addition, Karyopherins also recognize nucleotide sequences during the export of some classes of RNAs [163] . Due to their key role, Karyopherins involved in cargo import are known as importins and those involved in export are known as exportins. The RanGTP cycle also plays a central role in karyopherin activity and, therefore, in trafficking of macromolecules through the NPC. Importins bind the cargo in the cytoplasm and release it on binding to RanGTP in the nucleus. In contrast, exportins bind the cargo in the nucleus together with RanGTP; and then the Ran-associated GTP is hydrolyzed in the cytoplasm by RanGAP and cargo is liberated [163, 164] . Proteins containing constitutively active NLS are predominantly nuclear; but in some cases, the accessibility of NLS or the NLS itself is modified to selectively regulate the localization of the protein [172] . A similar regulatory mechanism is also exerted for control of NES activity [173] . For example, the NLS of the transcription factor NFκB is masked by the interaction with the inhibitor IκBα. However, IκBα is degraded on proinflammatory stimuli, exposing the NLS of NFκB for importin recognition. Therefore, under proinflammatory conditions, NFκB is imported to the nucleus, where it triggers a specific gene response [172] . In addition to "masking strategies," phosphorylation, ubiquitination or methylation of NLS or NES also influences (negatively or positively) their recognition by importins or exportins. Therefore, trafficking of proteins between nucleus and cytoplasm could be finely regulated by posttranslational modifications in the NLS and NES [164, 174] .
Conceptually, export of most of nuclear RNA follows a similar mechanism to that described above for protein trafficking. This process also involves cargo receptors, export factors, and nucleoporins to deliver mature RNA to the cytoplasm, but in this case, structure and function of nuclear export signals are not well understood. Aminoacylated tRNAs are necessary in the cytoplasm for protein synthesis, but tRNAs are transcribed in the nucleus. tRNA export to the cytoplasm is mediated by exportin-t, which belongs to the karyopherin family. Exportin-t forms a complex together with RanGTP in the nucleus, and once the exportin-t-RanGTP-tRNA complex reaches the cytoplasm, RanGAP induces the hydrolysis of the Ran-associated GTP and the release of the tRNA [175, 176] . It has been proposed that exportin-5 could act as an auxiliary protein in this process [177] . However, later reports in yeast have opened the possibility of alternative pathways for tRNA nuclear export [163] .
Traffic of snRNA follows a complex mechanism involving adaptor proteins. snRNAs are transcribed by the RNA polymerase (Pol) II (with the exception of U6 snRNA which is produced by PolIII) and then they are capped but not polyadenylated. Cap-binding proteins (CBP) 80 and 20 interact with the cap of snRNAs in the nucleus and recruit an export adaptor known as PHAX [178] . This adaptor is phosphorylated and in this state, it is able to interact with CRM1, another member of karyopherin family. This interaction together with the joining of RanGTP is essential for snRNA trafficking [163] . Hydrolysis of RanGTP and dephosphorylation of PHAX lead to the release of the snRNA in the cytoplasm.
Ribosomal proteins are assembled together with the different rRNAs in the nucleolus following a complex process of maturation to give rise to the ribosomal subunits 40S and 60S. Although is known that preribosomal subunits are exported by separate routes that involve CRM1 and RanGTP, nowadays the exact mechanism followed by 40S preribosomal subunits to leave the nucleus remains unclear [163] . However, 60S preribosomal subunit relies on Nmd3 adaptor, which mediates the interaction with CRM1 [179, 180] . The release of the 60S preribosomal subunit requires two GTPase steps: (i) the hydrolysis of the Ran-associated GTP induces the liberation of CRM1; and (ii) the hydrolysis of GTP mediated by the cytoplasmic GTPase Lsg1, which induces the release of Nmd3 [181] .
Finally, mRNAs compose the most heterogeneous group of RNAs, varying in length and structure. Thus, different export factors and adaptor proteins associate with each subpopulation of mRNAs [163] . mRNAs are transcribed by PolII, and, concomitantly with this process, a number of RNA-binding proteins assemble with them. These RNAbinding proteins exert different modifications in immature mRNAs such as polyadenylation, splicing and capping. In addition, export factors and adaptor proteins are also recruited to nascent pre-mRNAs, playing a further function in nuclear export. TREX (transcription-coupled export) complex is recruited to the 5 end of nascent pre-mRNAs in a splicing-dependent manner by means of the interaction of one of its components, namely ALY/REF, with the subunit of the nuclear cap-binding complex CBP80 [182] . The TREX complex also recruits the conserved RNA-helicase UAP56 that is important for mRNP biogenesis [183] . TAP-p15, (also known as NXF1-NXT1) directly binds the mRNA immediately after splicing and actively participates in mRNA export. ALY/REF, TAP-p15 and UAP56 associate with exon junction complexes (EJC), which are deposited in a splicingdependent manner at 20-24 nt of every exon-exon junction [184, 185] . This interaction network makes spliced mRNAs more susceptible to export and couple splicing and mRNAtrafficking. In fact, unspliced mRNAs are exported by an alternative and less efficient pathway that involves CRM1 and RanGTP. This alternative route is also followed by mRNAs encoding some protoncogenes and cytokines [186, 187] . It is important to mention that although mRNA can follow different nuclear export pathways, in all cases the interaction of export receptors with nucleoporins plays an essential role in the transport of the mRNPs throughout the NPC [170] .
Protein and RNA Trafficking by PV 2A pro . PV infection strongly impacts on hostcell protein localization, giving rise to an unusual cytoplasmic distribution of nuclear proteins [188] . This particular effect has been characterized by different laboratories for a number of nuclear factors involved in several cellular processes and containing different types of NLSs [189] . Nevertheless, not all nuclear proteins are re-localized after PV infection, evidencing the presence of a viral-specific mechanism affecting protein subcellular distribution [188] . Gustin and colleagues proposed the inhibition of nuclear protein import machinery as the cause of the cytoplasmic accumulation of nuclear proteins in PV-infected cells (Figure 4 ). They demonstrated that PV impairs protein trafficking across the NPC by expressing GFP proteins encoding classical or transporting NLSs in mock and infected cultured cells [15, 16, 159] . These recombinant proteins accumulate in the cytoplasm after PV infection, being almost completely depleted from the nucleus. Nevertheless, PV does not affect GFP distribution when the NLS is mutated or deleted, since the small size of GFP allows its inefficient efflux throughout the NPC [15] . Interestingly, cell-free nuclear import assays demonstrated that NLS-containing GFP is unable to traverse the NPC when cells are previously infected with PV [15] . In agreement with these findings, shuttling endogenous proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNP K, are detected in the cytoplasm of infected cells from 3 hpi but are undetectable in the nucleus at 4.5 hpi. However, cytoplasmic accumulation of nuclear resident proteins such as hnRNP C requires longer times of infection [15] . These findings support the idea that the distribution of a protein that shuttles between nucleus and cytoplasm may be strongly altered by the disruption of protein trafficking pathways, as compared to nuclear resident proteins. However, not all the nuclear factors are redistributed to the cytoplasm of PV-infected cells. This is the case for SC35 (a serine/arginine-rich splicing factor), fibrillarin or TATA-binding protein (TBP), which remain in the nucleus for the duration of infection. The different behaviour of these groups of proteins could arise as a consequence of different turnover; thus, the distribution of highly stable nuclear proteins might be less affected by the inhibition of protein nuclear import than those proteins with low stability. Alternatively, PV might not impair all protein import pathways, and some might remain operative, thereby allowing the nuclear import of several families of nuclear proteins. Interestingly, some nuclear factors such as La antigen [190] , PTB [191] , Sam68 [192] , or nucleolin [188] have been shown to interact with PV RNA or viral proteins. These proteins accumulate in the cytoplasm of PV-infected cells and, consequently, their availability for viral replication is increased [188, [193] [194] [195] . Cytoplasmic accumulation of nuclear factors might be produced mostly by the blockade of nuclear-cytoplasmic trafficking. However, loss of the NLS after the cleavage of PTB and La proteins by PV 3C pro may also contribute to the subcellular relocalization of those proteins in PV-infected cells [193, 195] . Redistribution of nuclear proteins as well as impairment of cellular import machinery was also observed in cells infected with other picornaviruses such as HRV [159] or EMCV [160] . These findings indicate that most picornaviruses might share similar strategies to impair nuclear-cytoplasmic trafficking machinery.
An alternative hypothesis has been proposed as the cause of the redistribution of nuclear factors. Belov and colleagues reported that the nuclear envelope is permeabilized on PV infection, allowing nuclear proteins to diffuse across the nuclear membrane [196] . Indeed, electron microscopy revealed that PV 2A pro induces severe structural damage in NPC [196] . However, this hypothesis did not clarify why other proteins resident in the nucleus do not diffuse to the cytoplasm after PV infection. Both protein nuclear import and permeabilization of nuclear envelope could be integrated together as sequential steps in PV biological cycle. Protein import blockade is detected early after infection [16] , correlating with the first modifications in the NPC (see below). However, prolonged expression of viral proteins might induce nuclear membrane leakiness, reflected by stronger alterations in NPC architecture [196] . Nevertheless, what might the biological relevance of these events be for a cytoplasmic virus? The most evident answer, which was extensively commented above, is that inhibition of nuclear protein import and further nuclear envelope leakiness might increase the presence of nuclear proteins in the cytoplasm of infected cells, which has been proposed in some cases to play a relevant role in PV replication. In addition, many transcription factors are arrested in the cytoplasm of uninfected cells (e.g., NF-κB, IRF7, and IRF3), but they are immediately activated and imported to the nucleus after proinflammatory extracellular signals or on the activation of intracellular sensors as a consequence of the viral replication. Once in the nucleus, these factors trigger the transcription of a set of genes involved in the antiviral response [197] . Inhibiting the import of these transcription factors, PV might prevent or, at least attenuate, the establishment of a hostile intracellular environment. Further effort will be made in the future to explore this attractive hypothesis.
Interestingly, Nup98, Nup153 and Nup62, components of the NPC belonging to FG Nup family, were found to be degraded in PV-as well as HRV-infected cells (Figure 4 ) [15, 16, 159] . These proteins are essential factors of the nuclear-cytoplasmic trafficking machinery since their Nterminal FG-rich domains serve as docking sites for soluble transport factors [163, 198] . Nup98, Nup153 and Nup62 are proteolyzed in PV-infected cells following different kinetics. Thus, Nup98 is the cleaved early after infection (from 1 hpi), whereas Nup153 and Nup62 are targeted at late times after infection (from 4 hpi) [15, 16] . In agreement with these findings, cleavage of Nup98 is induced even in presence of inhibitors of PV replication, suggesting that small amounts of viral proteins are sufficient for this proteolysis to occur. However, cleavage of Nup153, and Nup62 are efficiently prevented on arrest of viral replication, probably because they are only efficiently achieved when large amounts of viral proteins are produced [16] . Therefore, Nup98, Nup153, and Nup62 exhibit different susceptibilities to PV replication, thus PV might have a gradual impact on the nuclear-cytoplasmic trafficking machinery. Nup153 is also proteolyzed by caspase-3 and caspase-9 during apoptosis induction; however, the involvement of these cellular proteases in PV-induced Nup cleavage has been ruled out by different laboratories. First, Nup153 cleavage Journal of Biomedicine and Biotechnology 13 products generated upon caspase activation differ to those found in PV-infected cells [159] . In addition, Nup62 is cleaved in PV-infected cells, but it remains intact despite caspase activation [199] . Most importantly, PV-induced NPC structural damage takes place in cells lacking caspase 3 and 9 [196] , and Nup153 and Nup62 are efficiently cleaved in PV-infected cells even in presence of the caspase inhibitor Z-VAD [159] . All together, these data support the idea that one or more viral proteins play a direct role in the cleavage of those Nups. In agreement with this hypothesis, PV-induced NPC damage is prevented by PV 2A pro inhibitors such as elastatinal, elastase, and MPCMK, suggesting an involvement of this viral protease in the alteration of NPC [196] . Indeed, individual expression of PV 2A pro in HeLa cells as well as addition of this protease to cell-free systems gives rise to Nup98, Nup62, and Nup153 cleavage [16, 17, 200] .
In agreement with the data obtained from PV-infected cells, on PV 2A pro expression in HeLa cells, Nup98 is cleaved faster than Nup62 and Nup153, which suggests the presence of optimal cleavage sites in this protein [17] . Proteolysis of Nup98 in PV-infected cells as well as in cell-free systems generates two different cleavage products of around 50-65 KDa and 35 KDa [16] . There are two optimal cleavage sites in Nup98 for PV 2A pro located between aminoacids 373-374 and 551-552, containing Gly at P1 , Thr at P2, and Leu at P4. Hydrolysis at both sites results in N-and Cterminal products with predicted molecular masses of 37 and 53 or 55 and 35 KDa, in good agreement with the size of the peptides detected experimentally in PV-infected cells and 2A-treated HeLa extracts [16, 17] . An explanation for the delayed kinetics of Nup62 and Nup153 with respect to Nup98 is that optimal PV 2A pro cleavage sites were not found in these Nups (unpublished data). Recently, Park and colleagues have reported that PV 2A pro directly cleaves Nup62 at six different positions rendering multiple proteolytic products. These cleavage sites are located between aminoacids 103 and 298, thus releasing the FG-rich region from the protein core [200] . Functionally, loss of the FG-rich region might make Nup62 inactive for interaction with cargo receptors. This hypothetical mechanism of Nup62 functional decoupling could be extrapolated to Nup98 and Nup153 (Figure 4) . However, it remains unknown whether PV 2A pro is able to directly cleave Nup98 and Nup153 or where cleavage might occur.
Nup98, Nup153 and Nup62 are also involved in RNA export from the nucleus and therefore, cleavage by PV 2A pro might also impact on this process (Figure 4) . However, oligo d(T) hybridization studies showed that PV infection does not affect distribution of the polyadenylated mRNA bulk after 3 hpi [16] . A more detailed analysis revealed that expression of PV 2A pro in HeLa cells induces a number of disorders in RNA location. Nuclear export of cellular mRNAs is inhibited in 2A pro -expressing cells in a dose dependent manner concomitantly with Nup98, Nup62 and Nup153 cleavage [17] . Interestingly, mRNA export of constitutively expressed mRNAs such as β-actin is less affected than that of newly synthesized mRNAs. For example, tetracycline-induced luciferase mRNA was almost totally retained in the nucleus when these Nups are cleaved by PV 2A pro . This effect was also observed for endogenous mRNAs such as IL-6, c-myc or p53 mRNAs which are induced on PV 2A pro expression. Therefore, PV 2A pro could counteract the induction of proapoptotic (c-myc and p53) and proinflammatory (IL-6) responses by accumulating the c-myc, p53 and IL-6 mRNAs in the nucleus [17] . This export blockage may prevent the establishment of a hostcell response against PV infection. These findings could explain why PV 2A pro is essential for replication of PV in cells pre-treated with IFN-α [79] . Furthermore, impairment of mRNA export strongly alters the localization of mRNAs with high turnover as compared to constitutively expressed and highly stable mRNAs such as β-Actin [17] . As observed in PV-infected cells, oligo d(T) hybridization revealed that PV 2A pro expression hardly affects the distribution of the polyadenylated mRNA pool after short times of expression (8 h). However, nuclear accumulation of polyadenylated mRNA bulk is detected when cells are exposed to PV 2A pro for longer times (16 and 24 h) . In this regard, the progression of the alterations of polyadenylated mRNA localization in 2A pro -expressing cells takes place as follows: (i) disruption of nuclear mRNA-containing foci (ii) appearance of mRNAcontaining granules in the cytoplasm (most probably stress granules) and (iii) depletion of cytoplasmic mRNAs. These events were more clearly observed when high amounts of PV 2A pro are synthesized, reflecting that nuclear accumulation of mRNAs is a time-and dose-dependent process [17] .
Nevertheless, PV 2A pro is not only able to block mRNA export, but also rRNA and snRNA transport. Both 18S rRNA and U2 snRNA accumulate in the nucleus of 2A proexpressing cells in a dose-dependent manner. Most probably, cleavage of Nup98, Nup62 and Nup153 is involved in these effects, since rRNA, snRNA, and mRNA are exported using different cargo receptors and auxiliary proteins (see above) but all of them relay in Nup activity to traverse NPC [163] . Importantly, tRNAs (val-tRNA) are exported normally despite PV 2A pro expression, indicating that some RNA nuclear export pathways are not affected by this viral protease. In fact, Nup98, Nup62, and Nup153 are not directly involved in tRNA export [163] , reinforcing the idea that nucleoporin cleavage plays a central role in the impairment of protein and RNA trafficking by PV (Figure 4) .
Notably, IFN-γ induced a specific increase of Nup98 levels in HeLa cells that counteracts the inhibition of mRNA export by PV 2A pro [17, 201] . Collectively, these findings reflect the central role of Nup98 in PV infection and in antiviral response, since its overexpression by itself prevents, at least in part, blockade of nuclear RNA export. Therefore, secretion of IFN-γ by immune cells might allow the induction of antiviral response by neighbouring cells by increasing the levels of Nup98 in order to protect nuclearcytoplasmic protein and RNA trafficking pathways. The physiological relevance of the crosstalk between Nup98-PV 2A pro in PV (and other viruses) infection might be studied in the future with cellular and animal systems.
That Target the Nuclear Pore. As mentioned above, HRV also induces cleavage of Nup62, Nup153 and most probably Nup98, leading to the impairment of nuclear protein import [159] . Nevertheless, PV and HRV 2A pro exhibit high homology and both proteases are therefore expected to share common targets. In contrast, the 2A gene of cardioviruses encodes a short peptide with autoproteolytic activity but lacks trans-protease activity. However, EMCV and mengovirus are able to damage NPC [202] . As occurs in PV-infected cells, these cardioviruses induce both protein nuclear import inhibition and late membrane leakiness but they do not induce the cleavage of Nups [160, 202, 203] . Cardioviruses encode an additional protein known as L protein, which is highly cytopathic although it lacks protease activity (in contrast to aphthovirus L pro ). L protein contains a Zinc finger domain, and an acidic region, which is proposed to be phosphorylated in infected cells [204] . Individual expression of this protein in cultured cells or in cell-free systems induces several cellular disorders including the inhibition of protein nuclear import that resembles that observed in EMCV-infected cells [160, 203] . Several studies reported EMCV L protein mutations that resulted in defective virus growth phenotypes in cell culture [202, 204] . In particular, mutations in the zinc finger domain (Cys19Ala and Cys22Ala) or in the acidic region (Thr47Ala) partially avoid blockade of protein trafficking between the nucleus and the cytoplasm [202] . Taken together, these data support the involvement of cardiovirus L protein in NPC damaging and in the inhibition of protein nuclear import, inducing Nups phosphorylation rather than their cleavage. Indeed, Nup62 is quickly and strongly phosphorylated after EMCV infection (2 hpi), and it was clearly detected by conventional Western blotting. Nevertheless, analysis with Pro-Q diamond phosphoprotein stain revealed that Nup153 and Nup214 are also phosphorylated to a certain extent upon EMCV infection. Notably, Nups phosphorylation was avoided when Cys19Ala mutation was inserted in the L protein sequence, suggesting that the Zinc finger domain is essential for this posttranslational modification to occur [160] . However, the exact role of Nups phosphorylation in nuclear-cytoplasmic trafficking is still unknown. The idea that Nups phosphorylation could regulate protein import and RNA export as a switch that turns the different pathways on/off should be pursued in more detail. As mentioned above, Ran is essential for the regulation of most of the nuclear export and import pathways, because it acts as a cofactor modulating the affinity of importins and exportins for the cargo. The RanGTP cycle is described in detail in Section 6.1. It has been described that EMCV L directly interacts with Ran, and this interaction is abrogated by the insertion of C19A mutation in L [203] . However, the potential role of L/Ran interaction in the modulation of RanGTP cycle and its impact in nuclear import and export pathways have not yet been studied.
Alteration of nuclear-cytoplasmic trafficking is not only restricted to picornaviruses but has also been observed with negative strand viruses such as vesicular stomatitis virus (VSV) and influenza virus. Her and collaborators reported that RNA export and protein import are strongly inhibited by VSV matrix (M) protein, by microinjection of oocytes with radiolabeled RNAs and proteins. Radiolabeled tRNAs, mRNAs, U snRNAs, and rRNAs were injected directly into the nucleus of Xenopus laevis oocytes, and the subcellular localization of those RNAs was monitored by autoradiography. The conclusion of this work is that mRNA, U snRNA, and rRNA but not tRNA trafficking is blocked by VSV M protein [205] , in agreement with our findings on 2A proexpressing HeLa cells [17] . In addition, protein nuclear import was monitored by microinjection of radiolabeled proteins containing NLS in the oocytes cytoplasm. This assay revealed that VSV M protein abrogates protein nuclear import to the same extent as treatment with specific inhibitors of this pathway such as WGA [205] . These interesting findings support the idea that PV 2A pro and VSV M protein could target similar host proteins to impair macromolecule trafficking between nucleus and cytoplasm. In agreement with this possibility, it was found that the VSV M protein interacts with Nup98 [161] , which is one of the primary targets of PV 2A pro [16] . The N-terminal domain of VSV M is sufficient to block RNA nuclear export and aa 52-54 may play an essential role in this blockade, because their mutations to Ala completely abrogate this inhibitory effect. Indeed, the N-terminal domain of M protein is involved in the interaction with Nup98 and, in particular, aa 52-54, because their mutation to Ala blocks the binding of VSV M to Nup98 [161] . In addition, binding of M to Nup98 requires active mRNA export pathways since, treatment with inhibitors such as WGA hampers this interaction. VSV M is also able to induce the accumulation of endogenous polyadenylated mRNAs in the nucleus of HeLa cells, and this effect is prevented again by mutations in aa 52-54 of M [161] . Furthermore, VSV M interacts with Rae1, which plays an essential role in mRNA nuclear export by its interaction with Nup98 and mRNPs. Overexpression of either Nup98 or Rae-1 prevents the nuclear accumulation of polyadenylated mRNAs, suggesting that both factors may play a role in the blockade of mRNA nuclear export by VSV M protein [206] . Interestingly, IFN-γ specifically increases the level of both Nup98 and Rae-1 and indicates a potential antiviral effect of these proteins. Indeed, overexpression of both proteins by IFN-γ treatment counteracts the inhibitory effects of VSV M protein on mRNA nuclear export, highlighting the possibility of a crosstalk between M and IFN-γ that might control the fate of the viral replication in infected animals [201, 206] .
Influenza virus replication also impacts on nuclearcytoplasmic trafficking and leads to the nuclear accumulation of host mRNAs [162, 207] . Influenza virus NS1 protein is a major virulence factor that is essential for pathogenesis, because it impairs innate and adaptive immunity by inhibiting host signal transduction and gene expression [208, 209] . NS1 forms a complex with NXF1/TAP, p15/NXT [162] , Rae1, and E1B-AP5, which are components of the mRNA nuclear export machinery (see above). Individual expression of NS1 in 293T cells induces the accumulation of polyadenylated mRNAs in the nucleus, suggesting that the interaction of NS1 with these export factors yields an inactive complex for mRNA export. Influenza virus also induces a strong reduction of Nup98 steady-state levels although the viral mechanisms involved in this process are still unknown [162] . Expression of reporter luciferase mRNA synthesized from a nuclear plasmid is inhibited by NS1. However, this inhibition is overcome by overexpression of NXF1, p15, Rae-1 or Nup98, evidencing the role of NS1 interaction with these factors in the impairment of mRNA nuclear export [162] . Furthermore, mouse cells expressing low levels of Nup98 or/and Rae-1 show greater susceptibility to influenza infection, resulting in a significant increase in cell death and virus production. In addition, mRNAs encoding antiviral factors or immunomodulators such as IRF-1, MHC I and ICAM1 accumulated more in the nucleus of those cells than in cells expressing normal levels of Nup98 or Rae-1 [162] . All these data support the physiological role of NS1 interaction with RNA export factors as well as the reduction of Nup98 levels in influenza pathogenicity. Interestingly, VSV M, influenza NS1 and PV 2A pro expression gives rise to similar effects on mRNA trafficking. All these viral proteins target Nup98 and other components of the cellular machinery involved in nuclear-cytoplasmic trafficking.
Survival of motor neurons (SMN) complex is composed by SMN and a class of proteins called Gemins, which localize in both cytoplasm and nucleoplasm [210, 211] . Gemin7 and Gemin8 constitute the core of the complex where the other Gemins associate by means of numerous proteinprotein interactions from the periphery. The SMN complex is involved in the biogenesis of uridine-rich small nuclear ribonucleoprotein (U snRNP) in the cytoplasm and then the U snRNP carries out the splicing of pre-mRNAs in the nucleus [212] [213] [214] . The snRNPs are composed of the major U snRNAs U1, U2, U4, U5, and U6 as well as a group of seven proteins known as Sm ribonucleoproteins that collectively make up the extremely stable Sm core of the snRNP. Gemins (except Gemin-2) associate with Sm proteins to form a heptameric ring structure in the presence of U snRNAs [211] . After Sm core assembly, the U snRNPs are imported to the nucleus, localizing in foci known as Cajal Bodies, where further maturation processes take place [215] . Gemin-3, one of the main components of SMN complex, is cleaved in PV-infected HeLa cells leading to a 50 KDa cleavage product. Scission of Gemin-3 negatively impacts on the kinetics of Sm core assembly, which is prevented in presence of inhibitors of PV replication [216] . These results indicated that high levels of PV proteins are required for this process to occur, as is the case of Nup153, Nup62, and eIF4GII, [16, 122] . PV 2A pro is able to hydrolyze purified Gemin-3 in vitro, rendering a cleavage product similar to that found in PV-infected HeLa cells [216] . Only one potential 2A procleavage site, between the amino acids Tyr 462 and Gly 463 (VHTYG), was found in this SMN complex component. Proteolysis of Gemin-3 at this position would render two cleavage products of about 50-30 KDa, in agreement with the polypeptide of about 50 KDa found in PV-infected and 2A pro -expressing cells. In addition, G463E mutation avoids direct hydrolysis of Gemin-3 exerted by PV 2A pro in vivo and in vitro [216] . Taken together, these findings support the notion that VHTYG is the cleavage site for PV 2A pro in Gemin-3. Although hydrolysis of Gemin-3 is exerted in cells transfected with plasmid encoding PV 2A pro , it does not take place when this protease is expressed from exogenous mRNAs; contrary to that found with eIF4GI, eIF4GII, Nup98, Nup153 and Nup62 [17] (and unpublished data). A probable explanation for this difference is that PV 2A pro is expressed at lower levels from transfected mRNAs than from plasmids [95] (Castello et al., 2006) . Thus, Gemin-3 cleavage may be a very late event in PV-infected cells because it requires expression of high amounts of PV 2A pro . Gemin-3 hydrolysis may directly impact on pre-mRNA splicing since this event reduces the availability of SMN complexes, which is involved in U snRNPs biogenesis. Nevertheless, Alstead and colleagues could not detect any apparent effect of Gemin-3 proteolysis in splicing of cellular pre-mRNAs [216] . Therefore, the physiological relevance of Gemin-3 cleavage in PV biological cycle remains unknown. In addition to eIFs, Nups and proteins from SMN complex, PV 2A pro is able to cleave proteins involved in other cellular processes, such as transcription. TBP is cleaved by PV 2A pro between amino acids Tyr 34 and Gly 35 in vitro, although this cleavage only removes the first 34 aa located at the N-terminus and does not inhibit transcription carried out by RNA Polymerase II [217, 218] . These findings are in agreement with the fact that host mRNA transcription takes place in 2A pro -expressing cells when both translation and RNA nuclear export are inhibited, upon cleavage of eIF4G and Nups [17] . One attractive hypothesis is that PV 2A pro could cleave specific initiation factors affecting specific rather than general mRNA transcription in order to modulate host-cell response to viral infection. Further studies in this direction can be carried out using microarray platforms to detect precise alterations in cellular transcriptome after PV-infection or 2A pro expression. These studies could be complemented by screening for new host factors cleaved by PV 2A pro using different in silico and experimental approaches.
The study of viral proteases is crucial to understand the mechanism used by animal viruses to replicate their genomes and to translate viral mRNAs at the molecular level. In addition, we wish to draw attention to the concept that viral proteases can be used as tools to reveal the exact functioning of their target cellular proteins. In this regard, PV 2A pro has been very useful for examining the requirements for eIF4G to translate different cellular or viral mRNAs. In addition to this, in this paper, we have highlighted the role of PV 2A pro not only in the processing of the poliovirus polyprotein but also in the interaction with the host-cell. Interestingly, a single viral protein is able to modulate many steps of gene expression in order to generate an optimal intracellular environment for the viral biological cycle. In particular, PV 2A pro cleaves cell proteins involved in transcription, pre-mRNA splicing, nucleus/cytoplasm transport and translation, in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny. For example, PV 2A pro inactivates host translational machinery for capped cellular mRNAs by cleaving eIF4G, whereas viral protein synthesis takes place under those conditions by IRES-driven translation [14] . Because of the decrease of cellular mRNA translatability in PV-infected cells, host ribosomes are available for viral protein synthesis. Probably, the inhibition of host protein synthesis may prevent the production of antiviral proteins. Similarly, the blockade of nucleus/cytoplasm transport of macromolecules might isolate nuclear processes from cytoplasmic cellular ones, hampering the arrival of specific proinflammatory transcription factors to the nucleus and of mRNAs encoding proinflammatory, antiviral or proapoptotic proteins to the cytoplasm of infected cells. Finally, transcription and pre-mRNA splicing could be also modulated by PV 2A pro and might reduce the availability of mature mRNAs. Nevertheless, very little is known about the potential role of PV 2A pro in transcription and pre-mRNA splicing, and further studies of these steps of gene expression in PV-infected and 2A-expressing cells should be carried out. Making use of the "omics" technologies it would be possible to identify changes in the host transcriptome in those cells, allowing us to understand the readjustment of host gene expression to viral infection and to the cytotoxic effect of PV 2A pro . Gene ontology tools can be used to cluster the pathways (KEGG), molecular activities and biological functions of the genes which are transcriptionally up-or downregulated or not affected after these unfavourable stimuli. In silico analysis could be carried out in order to identify whether these gene clusters belong to particular networks controlled by specific transcription factors. This approach will give us additional information about potential host targets for PV 2A pro . Complementarily, deep sequence (RNAseq), specific microarrays types and conventional RT-PCR could be used to screen for nonspliced or abnormal spliced mRNA variants in PV-infected and 2A pro -expressing cells. Finally, microarrays can be employed to identify the cytoplasmic and nuclear transcriptome in those cells to determine whether mRNA nuclear export inhibition induced by PV 2A pro impacts on the distribution of the entire host mRNA bulk or in specific mRNA pools. Taken together, all this information may provide us with a general and deep vision of the modification induced by PV 2A pro on the different steps of mRNA metabolism. Additionally, the physiological role of Nups and Gemin-3 cleavage might be studied using different models. One possible and interesting approach is to engineer stable cell lines expressing noncleavable versions of Nup98, Nup62, Nup153 and/or Gemin-3 and then to analyze the fitness of PV in the different cell types, especially in presence of extracellular antiviral stimuli such as IFNs or interleukins, which will activate different epigenetic programs. These studies will provide essential information to help us understand the specific role that those host factors play in PV infection. The total number of cellular proteins targeted by PV 2A pro (degradome) remains unknown, but it can be anticipated that with the expanding use of proteomic methodologies, this analysis will be known soon not only for PV 2A pro , but also for other viral proteases of interest. Furthermore, the analysis of the PV 2A pro -induced degradome in human cells will be of general interest for many researchers, including virologists and cellular biologists. This goal could be achieved combining in silico prediction of 2A pro cleavage sites and experimental tools such as proteomics. In the first case, Blom and collaborators developed a bioinformatics tool using neural network algorithms to predict cellular targets for picornavirus proteases [219] . This approach has been successfully used to predict the cleavage of dystrophin by coxsackievirus 2A pro [220] although most of the predicted human targets for rhinovirus and enterovirus 2A pro have not been proved yet. In addition, the algorithm did not predict the cleavage of cellular targets that have been later demonstrated to be proteolyzed by picornaviral 2A pro such as Nup98 and cytokeratin 8 [16, 17, 221] . Thus, it would be necessary to develop an improved algorithm able to find optimal cleavage sequences in the host proteome by implementing the proteolytic sites known for newly described 2A pro targets. Many parameters have to be taken into account, including the protein localization (cytoplasmic and nuclear protein will be considered, but not proteins resident in the lumen of other organelles such as RE or peroxisomes), the exposure of the cleavage site to the solvent (the sequence must be accessible to the protease), and the secondary structure in which the proteolytic site is included (optimally, unstructured regions). Potential targets could be ordered by their degree of homology with optimal cleavage sequences, as well as with the degree in which they fulfill the above prerequisites. On the other hand, novel PV 2A pro targets can be identified by proteomic tools such as two-dimensional differential gel electrophoresis (DIGE) or quantitative proteomics such as stable isotope labeling by amino acids in cell culture (SILAC) coupled to monodimensional electrophoresis. Following these two methods, it will be feasible to identify proteins with reduced levels on 2A pro expression and, in addition, to detect the cleavage products that will appear as lower size peptides. By the uncovering of novel 2A pro targets we will be able to map the cellular networks impacted by PV 2A pro and to integrate them in the context of PV infection. In fact, the role of 2A pro hijacking host processes could be potentially expanded to other cellular pathways with direct impact on control of viral infection. Such knowledge will provide more insight into our understanding of the cytopathogenicity of viral proteases at the molecular level. Transmission Potential of Chikungunya Virus and Control Measures: The Case of Italy During summer 2007 Italy has experienced an epidemic caused by Chikungunya virus – the first large outbreak documented in a temperate climate country – with approximately 161 laboratory confirmed cases concentrated in two bordering villages in North–Eastern Italy comprising 3,968 inhabitants. The seroprevalence was recently estimated to be 10.2%. In this work we provide estimates of the transmission potential of the virus and we assess the efficacy of the measures undertaken by public health authorities to control the epidemic spread. To such aim, we developed a model describing the temporal dynamics of the competent vector, known as Aedes albopictus, explicitly depending on climatic factors, coupled to an epidemic transmission model describing the spread of the epidemic in both humans and mosquitoes. The cumulative number of notified cases predicted by the model was 185 on average (95% CI 117–278), in good agreement with observed data. The probability of observing a major outbreak after the introduction of an infective human case was estimated to be in the range of 32%–76%. We found that the basic reproduction number was in the range of 1.8–6 but it could have been even larger, depending on the density of mosquitoes, which in turn depends on seasonal meteorological effects, besides other local abiotic factors. These results confirm the increasing risk of tropical vector–borne diseases in temperate climate countries, as a consequence of globalization. However, our results show that an epidemic can be controlled by performing a timely intervention, even if the transmission potential of Chikungunya virus is sensibly high. During summer 2007 Italy has experienced the first large outbreak caused by Chikungunya virus (CHIKV) documented in a temperate climate country [1] . CHIKV is an arthropod-borne virus which can be transmitted to humans by Aedes mosquitoes [2] , widespread in some tropical regions [3] [4] [5] [6] [7] [8] . Aedes albopictus is highly competent for CHIKV [9, 10] . In Italy, the presence of this mosquito was first documented in Genoa and Padua (Northern Italy) in the earliest 1990's [11, 12] . Over the following years, Aedes albopictus expanded its distribution and is now well established in Northern and Central Italy [13, 14] . Having the potential to colonize the Mediterranean basin [15] , the species has been reported from most Mediterranean European countries [16] . Samples of Aedes albopictus from the two villages were found to be positive for CHIKV sequences [1] .
Sustained transmission of CHIKV was mainly observed in two neighboring villages in Emilia-Romagna region (North-Eastern Italy), namely Castiglione di Cervia and Castiglione di Ravenna [1] , comprising 3,968 inhabitants in a built-up area of about 70 ha. The two villages are separated by a river with relatively stagnant water resulting from the presence of a lock. Houses are typically low (two storeys), surrounded by small gardens with many flowers, plants and flower pots. During the outbreak in the streets, drainage systems were visible, indicating open stagnant water underground [17] .
A total of 161 laboratory confirmed cases were reported to the enhanced surveillance system developed in the two villages [1] . Sporadic cases, probably due to travel towards the most affected villages and not leading to sustained transmission, were also observed in other areas of the same region [1] .
Moreover, a seroprevalence study, conducted on a random sample of residents in the village with the largest number of reported cases, shows a 10.2% of protected individuals [18] . Specifically, 82% were symptomatic -similar to 72.3% estimated in Mayotte, Indian Ocean [19] 285% of which satisfied the surveillance case definition, 63% of which were identified by the active surveillance system [18] . Higher prevalences were observed in La Reunion Island and in Mayotte, Indian Ocean, 38.2% and 37.2% respectively [19, 20] .
The index case was recorded on June 23 2007 (a man who had arrived in Italy from India on June 21, [1] ). On August 23, after the identification of CHIKV as the pathogen responsible for the ongoing epidemic, a set of interventions were undertaken to control the epidemic spread [1] : breeding sites and eggs removal on August 23; use of adulticides from August 23 to August 25 (3 days) and antilarval measures. Breeding sites were attempted to be removed in the entire area (house-to-house interventions were performed and community participation was encouraged as well) while insecticide interventions were undertaken within a radius of 100 m of each suspected case's residence (300 m for clusters of cases).
In this study we investigate the transmission potential of CHIKV in Italy, to provide insight into the possible impact of future outbreaks in temperate climate regions, and the effectiveness of the interventions performed during the outbreak, to provide insight into the epidemic control. To such aim, we developed a model describing the temporal dynamics of the competent vector, known as Aedes albopictus, explicitly depending on climatic factors, coupled to an epidemic transmission model describing the spread of the disease in both humans and mosquitoes, which allowed us to reproduce several observed features of the epidemic.
In Italy the competent vector for the transmission of CHIKV is Aedes albopictus [1] . CHIKV can spread from human to human through bites of adult female mosquitoes. As the dynamics of the vector depends, among several abiotic factors, on meteorological parameters, a population dynamics model accounting for seasonal temperature variations was used to estimate vector abundance. In particular, temperature plays a very significant role as it affects development and mortality rates of Aedes albopictus [10, 21] , influencing vector abundance and distribution over time [22] . The population dynamics model was then coupled to an epidemic transmission model describing the spread of the epidemic in both humans and mosquitoes (see Fig. 1 ), allowing the estimate of the crucial parameters of the epidemic (e.g. basic reproduction number, effective reproduction number, probability that a major outbreak of the disease would occur after the introduction of a single infective host) and the assessment of intervention strategies.
The main purpose for modeling the dynamics of the vector is to give an approximate estimation of the abundance of female adult mosquitoes during the CHIKV epidemic outbreak in order to get a reasonable value of the ratio of mosquitoes to humans over time, a crucial factor for the calculation of the fundamental parameters of the epidemic and for the assessment of intervention measures. To achieve this goal, a differential equation model, structurally similar to those analyzed in [23] [24] [25] , was introduced.
The dynamics of the mosquitoes over a land surface of about 70 ha (the extension of the study area) is described by a homogeneous mixing model. Briefly, the model simulates the abundance of the vector in the four life stages of Aedes albopictus, namely eggs (E), larvae (L), pupae (P), female adults (A), as follows:
where d E , d L , d P and d A are the temperature dependent developmental rates; m E , m L , m P and m A are the temperature dependent mortality rates; n E is the average number of eggs laid in one oviposition; K E is the carrying capacity of eggs; the term 1=2 in the fourth equation accounts for the sex ratio (sex ratio is 1:1, as reported in [21] ). The four developmental rates correspond to egg hatching (d E ), pupation (d P ), adult emergence (d L ) and gonotrophic cycle (d A ). Length of the gonotrophic cycles subsequent to the first one and number of eggs laid at each gonotrophic cycles are not significantly different within a range of temperature between 20 0 C and 35 0 C [21] . Therefore, we consider only one equation for modeling adults (and not a set of equations, describing transitions through gonotrophic cycles of different length, as in [23] for Aedes Aegypti) and the number of eggs laid in one oviposition does not depend on temperature. 
Human host population is assumed to be constant during the epidemic outbreak, given the brief duration of the epidemic compared to the lifespan of humans. We indicate with N h~3 968 the number of humans [1] . As for the epidemic transmission model, hosts are classified as susceptible (S h ), latent (E h ), infectious symptomatic (I s h ) or asymptomatic (I a h ), and recovered (R h ). As only adult female mosquitoes are responsible for virus transmission, adult males are not explicitly represented in the transmission model. Female adult vectors are classified as susceptible (A), latent (A e ) and infectious (A i ). A susceptible vector enters the latent class after biting an infectious host at the per capita rate kx v , where k represents the biting rate of the vector (i.e., the number of bites to humans per mosquito per day) and x v is the susceptibility to infection of the vectors (i.e., the probability that a mosquito get infected after biting an infectious host). A latent vector enters the infectious class after an average latent period of 1=v v days and remains infectious for the rest of its life [26] . Thus, to account for the epidemic transmission process, the 4th Eq. of system (1) is replaced by the following three equations:
where
Moreover, as we assume that the infection does not affect oviposition, the first Eq. of system (1) becomes:
A susceptible host enters the latent class following the bite of an infectious vector at the per capita rate kx h , where x h is the susceptibility to infection of humans. A latent host becomes infectious after an average latent period of 1=v h days, develops symptoms with probability p s and then, after an average infectious period of 1=c days, recovers.
The epidemic transmission process in humans can be modeled by the following system of ordinary differential equations, which has been added to system (1) as modified above (see Eq. 2 and 4):
where
In what follows we refer to the full system coupling dynamics of the vector and epidemic transmission process as model M.
The basic reproduction number R 0 of host-vector infectious diseases is the number of secondary infections that arise when a single infective host is introduced into a fully susceptible host population through pathogen transmission by the vector [27] . The average number of hosts directly infected by the introduction of a single infective vector into a fully susceptible host population is given by the transmission probability kx h multiplied by the adult mosquito infectious lifespan (that is, the entire lifespan) 1=m A :
The average number of vectors directly infected by the introduction of a single infective host into a fully susceptible vector population is given by the transmission probability kx v multiplied by the initial number of mosquitoes per human N v =N h (N v~A zA e zA i is the sum of all female adult mosquitoes, regardless of the epidemic status) that survive the latent period (probability: v v =(v v zm A )), multiplied by the human infectious period 1=c:
Thus, the number of secondary infections generated by an infective host in a fully susceptible host population over the entire transmission cycle is:
Eq. (8), however, is the threshold parameter of a simplified model M (with constant N v ). Therefore, to compute R 0 we assume a constant population of vectors, equal to the average value as predicted by the model in the initial phase of the epidemic, i.e. from June 21 to July 26 2007. By employing the next-generation matrix method [28] [29] [30] , one obtains the number of secondary cases generated either in hosts or vectors [31] , that is the square root of Eq. (8) .
Moreover, as shown in [32] , the probability that a major outbreak of the disease would occur after the introduction of a single infective host is given by
where the terms R VH 0 and R HV 0 are defined in Eq. (6) and (7).
On the basis of data presented in [21] , we estimated the length of the developmental stages (egg hatching, larval and pupal development) and of the gonotrophic cycle as a function of temperature. To estimate, for instance, the length of the egg hatching period we used the following procedure: let e T be the length of the egg hatching period for temperatures T [ T ? :f15 0 C,20 0 C,25 0 C,30 0 C,35 0 Cg, as reported in [21] . We assume that e T~lE (T; P)ze T where l E (T; P) is a parametric function of the temperature T (P indicate the set of parameters) in a suitable set of functions, comprising exponential and parabolic functions, and e T is a random sample of a 0 mean normal distribution with unknown variance s 2 . The square error Err between predicted and observed length of the egg hatching period is defined as Err~P T[T ? (e T {l E (T; P)) 2 . ParametersP P were estimated by minimizing Err. The variances s 2 was computed as the average of the estimated residuals of the model (i.e., the average of the quadratic differences of (e T {l E (T;P P)) 2 between the observed data and the best model fit l E (T;P P)). The uncertainty of the parameters was estimated by using a technique similar to that used in [33] . Specifically, we simulated 1000 different fe T g T [T ? , obtained by perturbing the best-fit l E (T;P P) by adding a simulated error sampled from a normal distributed N(0,s s 2 ) and we repeated the optimization procedure described above. Finally, the rate of eggs hatching is defined as d E (T; P)~1=l E (T; P). The same technique was used to estimate the length of larval and pupal development and the length of the gonotrophic cycle. Results are reported in Table 1 . Fig. 2b shows a comparison between observed and modeled data.
We estimated mortality rates of eggs, larvae and pupae as a function of temperature on the basis of data on the survival rates presented in [21] . To estimate, for instance, the mortality rate of eggs we used the following procedure: let s 19 T be the survival rates of eggs (19 days after oviposition) for temperatures T [ T ? , as reported in [21] . For a fixed value of temperatureT T, the following differential equation system describes the transition from eggs to larvae:
where d E (T T) is the development rate as estimated above and m E (T T) is the (unknown) mortality rate at the chosen temperaturẽ T T. We chose m E (T T) in such a way that, after 19 days, the survival rate as estimated through simulation of model (10) (i.e. the fraction of eggs that successfully develop into larvae) coincides with s 19 T T . This procedure allowed the estimation of the mortality rate of eggs at temperatures T [ T ? . Fig. 2c shows that the estimated mortality rates of eggs at different temperatures lead to values of the survival rates compliant with the observed ones. The same procedure was used to estimate the mortality rates of larvae and pupae for temperatures T [ T ? . Mortality rates of adults for temperatures T [ T ? were directly available from [21] . The procedure described above for estimating the length of the developmental stages was used to estimate the mortality rates of all stages as a function of temperature in the range ½15 0 C{35 0 C. Results are reported in Table 2 . Fig. 2d shows a comparison between observed and modeled data.
Mortality rates as computed above depend only on temperature. Since parasitism and deficient nutrients have been found to cause a 35% increase in the rate of larval mortality [34] and adult Aedes albopictus females have been found to survive an average of only 8:2 days (probability of daily survival = 0:8 days {1 ) in the natural environment [35] , mortality rates for immature stages (m L (T) and m P (T)) and adults (m A (T)) were multiplied by a factor 1:35 and 4 respectively.
The average number of eggs n E per oviposition is not significantly different at each gonotrophic cycle between 20 0 C and 35 0 C and in our simulations was uniformly chosen in the interval ½50 eggs{75 eggs, according to [21] .
The carrying capacity of eggs K E was estimated on the basis of data collected in the 2008 in the study area on the number of eggs per ovitrap per week as resulting from the analysis of 2741 ovitraps from week 21 to week 42 [36] . The mean egg density for the region of interest was found to be in the interval ½46:6{63:2 per ovitrap per week. Firstly, we estimated the carrying capacity of a single breeding site as the value K b giving rise (through simulation of model (1), where all other parameters are known) to an estimated weekly incidence of eggs in the observed range (½46:6{63:2) at temperatures observed in June and July. We estimated K b to be 19 in average (95% CI [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . The carrying capacity of the study region can be computed as K E~B K b H, where B is the density of breeding sites (number per ha) and H~70 ha is the surface of the study area. The exact number of breeding sites (public and private catch basins, stagnant pools of water, etc.) in the area at the time of the epidemic is unknown. Hence, in this study we considered different values of B, namely 50, 100, 150 and 200 ha {1 , in order to describe different (high) densities of mosquitoes, as those observed in the study area [1] . These different scenarios are thus characterized by average values of of the carrying capacity K E in the range ½66500{266000.
As for the dependence of rates on temperature, the developmental rates of the aquatic stages, namely egg hatching, larval and pupal developments, and the mortality rates of eggs, pupae and larvae, are daily calculated as a function of the water mean temperature, while the length of the gonotrophic cycle and the mortality rate of adults are calculated as a function of air mean temperature. Since 2008 a monitoring activity has been carried out in order to estimate the water temperature (a key parameter in the developmental stages) of breeding sites. In fact, most of Aedes albopictus life stages develop in aquatic micro-environment. Specifically, a linear regression model was used to estimate the daily mean water temperature as a function of daily mean, maximum and minimum air temperature and daily mean air relative humidity (see Fig. 2a ). This allowed us to get estimates of the water temperature for the 2007 season in order to get a more truthful calculation of developmental rates for eggs hatching and immature stages (larvae and pupae), impossible to obtain otherwise.
Plausible ranges for parameters most related to the epidemic transmission process were taken from literature (see Table 3 ), except for the biting rate k -it may vary a lot depending on human and mosquitoes populations, climatic and environmental factors [37] . We explored values of k in the range ½0,1 days {1 .
To account for the stochastic nature of the processes regulating both dynamics of mosquito and epidemic transmission, we used a discrete-time stochastic version of model M, with time-step where l is the force of infection (see for instance [38] [39] [40] All model parameters were calibrated to minimize the score function F~ffi
, where y t is the observed daily number of notified cases at time t and z t is the daily number of notified cases as predicted by the model at time t (times t represent days before the intervention). Model M outputs the daily incidence of symptomatic cases,Ĩ I s h (t), and asymptomatic cases, I I a h (t). Notified cases z t at time t were estimated by sampling from a binomial distribution of size n~Ĩ I s h (t) and probability p~p n , where p n is the notification ratio (p n~0 :54 [18] ).
Latin Hypercube Sampling (LHS) allows an efficient sampling of the parameter space which requires a smaller sample size than simple sampling to achieve the same accuracy [41] . LHS was used to build n Q~1 000 sets of parameters Q:fq 1 , . . . ,q nQ g (uniform sampling was used to determine the n Q values of each model parameter) and thus, through model simulations, n Q time series of predicted notified cases fz t (q)g q[Q . The optimal parameter set q ? was chosen as the one minimizing the score function, i.e. q ?~a rg min q[Q F (q). We repeated the above described procedure 100 times. This allowed us to estimate distributions of model parameters and, consequently, of the other quantities of interest (e.g. R 0 , probability of major outbreak, attack rate). The index case was recorded on June 23 2007 (a man who had arrived in Italy from India on June 21, [1] ) and thus we initialized all simulations with 1 infected individual on June 23. Results of the optimization procedure are shown in Fig. S1. Fig. S2 shows that 100 simulations are sufficient to obtain meaningful distributions of parameters and quantities of interest.
In summer 2007, an outbreak of chikungunya fever affected the Italian provinces of Ravenna, Cesena-Forli, Rimini and Bologna [1, [42] [43] [44] . Health authorities identified 214 laboratory-confirmed cases with date of onset from July 15 to September 28 2007. Most cases (161) occurred in the two neighboring villages of Castiglione di Cervia and Castiglione di Ravenna, but five smaller clusters of local transmission were also detected in five towns in the same region (i.e., Cervia, Cesena, Ravenna, Rimini, and Bologna) which are located 9 to 75 km from the initially affected villages [1, [42] [43] [44] , see Fig. 3 .
Model M was parametrized to describe epidemic spread only in Castiglione di Cervia and Castiglione di Ravenna. Daily estimates of the number of vectors over time N v were obtained by the vector dynamics model. The ratio of mosquitoes to humans was estimated to be in the range of 10-35 during the peak mosquito activity (Fig. S3 show the predicted dynamics of the vector for different numbers of breeding sites B). By fitting model M to notification data up to August 23 (the day before intervention) and by assuming B = 200 ha {1 we estimated k to be 0.09 days {1 (95% CI 0.05-0.16 days {1 , see Fig. 4b ). We recall that the explored range for k through the LHS procedure was ½0,1. Good fit to data were obtained for values in the entire range explored for all the other model parameters (see Fig. S4 ). x h Human susceptibility to infections 50%-80% [58] p s Symptomatic ratio 82% [18] p n Notification ratio 54% [18] 1=v v Latent period in mosquitoes 2-3 days [26, 58] x v Mosquito susceptibility to infections 70%-100% [62] doi:10.1371/journal.pone.0018860.t003 The uncertainty of k depends on the uncertainty of all model parameters. Unfortunately, no field data are available for the study area to validate these results. In [37] , plausible values for the biting rate and the ratio of mosquitoes to humans in Europe are considered to be k~0:25 days {1 and N v =N h~2 0 (based on published and non published data, e.g. [10] ). However, we acknowledge that it might be misleading to compare these results with others carried out in other localities. In fact, abundance and biting rate of Aedes Albopictus are strongly affected by abiotic factors, both climatic and environmental (e.g. presence of other hosts).
Estimates of the biting rate and its uncertainty allowed us to estimate R 0 and its uncertainty from Eq. (8). Besides parameters more strictly related to the infectious process, R 0 is an increasing function of the square of k, as the biting rate controls transmission from humans to mosquitoes and from mosquitoes to humans, and the ratio of vectors to humans N v =N h (see Fig. S5 ). However, it should be considered that Eq. (8) depends on the number of vectors N v which substantially varies over time as a results of seasonal meteorological factors. Thus, as for models explicitly considering seasonal variations in transmission, it is difficult to precisely define R 0 . Therefore, we computed R 0 by considering the average value of N v from June 21 to July 26 2007 (i.e. the initial phase of the epidemic), as predicted by the vector dynamics model. We estimated R 0 to be 3.3 on average (95% CI 1.8-6, see Fig. 4e ). Fig. 4d shows that R VH 0 is below the critical threshold for all vales of B and thus the epidemic is mainly determined by R HV 0 , i.e. by transmission from humans to vectors. As for the effective reproduction number R e (i.e., the average number of secondary cases generated per primary case at a given time), which accounts for both depletion of susceptible individuals and mosquito dynamics, its value over time is shown in Fig. 4c and Fig. 4f (by considering or not interventions) . It emerges that R e , which does not change much by varying the number of breeding sites, can vary substantially over time as an effect of mosquito dynamics. This suggests that R 0 could have been even larger, depending on the time of epidemic seeding.
Recently, it has been demonstrated using mathematical modeling in the context of dengue that it is possible to generate outbreaks even in cases when R 0 v1 provided that the vector-tohuman component of R 0 is greater than one and that a certain number of infected vectors are introduced into the affected population [45] . However, it has been demonstrated that the index case was a man of Indian origin from Kerala living in Castiglione di Cervia, without history of traveling during the previous year [1] . He only reported contact with a relative of his, who had arrived in Italy on June 21 2007 from Kerala, India (a region of India affected by the CHIKV epidemic), and visited him (see Eq. 6 and 7). e As a but for the basic reproduction number. f As c but by assuming reference interventions, resulting in the following reductions: 40% as for breeding sites and eggs, 90% as for larvae and 95% as for adults. doi:10.1371/journal.pone.0018860.g004
in Castiglione di Cervia village on June 23, while feverish. Therefore, having the human index case being identified, we can reasonably exclude the contemporaneous introduction of infected vectors in the two villages. Moreover, our estimates show that R VH 0 is well below the critical threshold.
As several cases were reported in Italy among travelers returning from endemic areas [46] (only one, however, in the study area; additional imported cases throughout the duration of the outbreaks were not detected), the question arises why no previous outbreaks of CHIKV occurred in other Italian regions. By assuming B = 200 ha {1 , we estimated the probability p (see Eq. 9) that a major outbreak of the disease would occur after the introduction of a single infective host to be 0.59 (95% CI 0.35-0.76, see Fig. S5 ) and, by assuming the same density of mosquitoes, epidemic outbreaks are more likely in rural areas with respect to urban areas -as the human population density is much lower in the former. This could explain why cities like Cesena (96,000 inhabitants), Rimini (141,000 inhabitants) and Bologna (377,000 inhabitants) and Ravenna (157,000 inhabitants) located in the same region of the two most affected villages did not experience any epidemic outbreak, though sporadic CHIKV cases were recorded in the same period [1, [42] [43] [44] . These results support the hypothesis that outbreaks of Chikungunya virus in those temperate climate countries characterized by high density of Aedes albopictus are probable after the importation of an index case from abroad.
The potential epidemic trajectory in the absence of interventions by assuming B~200 ha {1 is shown in Fig. 5a . The resulting cumulative attack rate (i.e., the percentage of symptomatic cases in the population at the end of the epidemic) was estimated to be 73.4% of the population (95% CI 57.8%-81.5%, see also Fig. 5b) . Results do not change much by varying B (B = 50 ha {1 : 74%, 95% CI 55.3%-81.6%, see also Fig. S6 ; B = 100 ha {1 : 73:9%, 95% CI 55.8%-81.5%; B = 150 ha {1 : 75%, 95% CI 57.3%-81.3%). Much lower prevalence values have been estimated in La Reunion Island and in Mayotte, namely 38.2% and 37.2% respectively. However, these estimates are hardly comparable with our model predictions as these territories have benefited from high resource allocation to mitigate the epidemic [19] .
As for the undertaken interventions, breeding sites and eggs were removed on August 23 2007, larvicides were used on August 23 (effect lasting 30 days), and adulticides were used from August 23 to August 25 2007. Through model simulations, we evaluated the effects of strategies mimicking the timing of the actual interventions undertaken in Italy. As for the effects in terms of reduction of breeding sites, eggs, larvae and adults, likely values Figure 5 . Baseline simulations and reference interventions. a Average daily number of symptomatic notified cases as predicted by the model in the absence of interventions (baseline scenario, blue line, scale on the left) and 95% CI (grey area) by assuming B~200 ha {1 , compared to the actual daily number of symptomatic notified cases (black points). Red line represents the overall average daily number of symptomatic cases as predicted by the model. Green line represents the average density of mosquitoes (scale on the right). b Histogram of the cumulative number of symptomatic cases as predicted by the model in the absence of interventions. c and d As a and b respectively but for assuming an intervention resulting in the following reductions: 40% as for breeding sites and eggs, 90% as for larvae and 95% as for adults (reference scenario). doi:10.1371/journal.pone.0018860.g005
are: 20% to 60% as for reduction breeding sites and eggs, 80% to 95% as for reduction of larvae and 80% to 95% as for reduction of adults. We discuss first results obtained by assuming 40% as for reduction of breeding sites and eggs, 90% as for reduction of larvae and 95% as for reduction of adults. The effects of such an intervention are shown in Fig. 5c . The resulting cumulative attack rate, by assuming B~200 ha {1 , was estimated to be 8.7% (95% CI 5.6%-12.7%, see Fig. 5d ), in good agreement with the observed value, namely 8.4%, computed by multiplying the overall observed prevalence, 10.2% [18] , by the symptomatic ratio p s~0 :82 [18] . Results are similar for other choices of B (for instance, for B = 50 ha {1 the figure becomes 8:5%, 95% CI 5.3%-13%; see also Fig. S6 ). To keep track of the number of symptomatic cases identified by the active surveillance system, we assume that human symptomatic cases are identified with probability p n and this allows fitting notification data in model simulations. According to model estimates, the number of notified cases would have been about 185 on average (95% CI 117-278), in good agreement with the number as reported to the surveillance system, namely 161 cases [1] . The number of cases drastically decreased in late August while the effective reproduction number, in the absence of interventions, would have fallen below the epidemic threshold in late September (see Fig. 4c and Fig. 4f) . This proves that a combined strategy resulted in a drastic reduction of the epidemic impact, despite the relatively large value of R 0 .
Let us now consider two aspects of the control strategy. Firstly, we assume different efficacy in terms of reduction of breeding sites, eggs, larvae and adults to evaluate the robustness of the estimated effects of the interventions undertaken in Italy. As shown in Fig. 6b , results are robust for small variations of the efficacy of the vectors control. In a fully susceptible population the time from primary index case to secondary infections was estimated to be 11 days on average (95% CI [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . This allows public health authorities to gain time to put in place control measures.
Secondarily, we investigate the efficacy of the single interventions (breeding sites removal, larvicides, adulticides). Results are shown only for B~200 ha {1 . As shown in Fig. 6a , reduction of eggs and breeding sites could be effective only by hypothesizing a massive intervention (cumulative attack rate is reduced on average from 73% to 55% by reducing eggs and breeding sites of 60%); adulticides do not contribute much to reducing the overall number of cases (cumulative attack rate is reduced on average from 73% to 60%); larvicides contribute to a substantial reduction of the overall number of cases (cumulative attack rate is reduced on average from 73% to 40%); In fact, larvicides are effective for a prolonged period of time and thus can contribute to decrease systematically the number of adults for a long period of time and, consequently, to substantially reduce the overall attack rate. Quite the contrary, adulticides were used for a very limited period of time (3 days) and thus their effect is limited due to the rapid increase of adults suddenly after the intervention. Overall, these results suggest that only a combined intervention, as the one performed during the outbreak, can result in a drastic decrease of the number of cases.
Five smaller clusters of local transmission were detected in five towns in the same region (i.e., Ravenna, Cervia, Cesena, Rimini, and Bologna). Cervia and Ravenna are the main towns of the municipalities where the two most affected villages (Castiglione di Cervia and Castiglione di Ravenna) are located. The two affected villages account for the 2.1% of the population of the municipalities of Cervia (27,000 inhabitants) and Ravenna (157,000 inhabitants). By analyzing commuting data of the Emilia-Romagna region [47] , we found that the number of individuals traveling daily to Cervia and Ravenna for work or study is 8,787 (from 249 different municipalities), and the number Figure 6 . Sensitivity analysis. a Red: distribution (2.5%, 25%, 50%, 75% and 97.5% percentiles) of the cumulative attack rate (only symptomatic cases are considered) by assuming no interventions (baseline scenario) as in Fig. 5a and Fig. 5b , and B~200 ha {1 . Green: as in the baseline scenario but for reductions of breeding sites and eggs. Blue: as in the baseline scenario but for reductions of larvae. Cyan: as in the baseline scenario but for reductions of adults. The horizontal dashed red line represent the observed attack rate (symptomatic cases, obtained by multiplying the observed prevalence, 10.2% [18] , by the probability of developing clinical symptoms, 0.82 [18] ). b Red: distribution (2.5%, 25%, 50%, 75% and 97.5% percentiles) of the cumulative attack rate (only symptomatic cases are considered) by assuming the same intervention as in Fig. 5c and Fig. 5d (reference scenario), namely reduction of 40% as for breeding sites and eggs, 90% as for larvae and 95% as for adults, and B~200 ha {1 . Green: as in the reference scenario but for different reductions of breeding sites and eggs. Blue: as in the reference scenario but for different reductions of larvae. Cyan: as in the reference scenario but for different reductions of adults. The horizontal dashed red line represents the observed percentage of symptomatic cases as resulting from survey data [18] . doi:10.1371/journal.pone.0018860.g006 of persons traveling daily from Cervia and Ravenna to other municipalities is 10,861 (towards 139 different municipalities). The exact number of commuters for Castiglione di Cervia and Castiglione di Ravenna is unknown but it should not exceed 2.1% of the overall number of commuters. However, the probability of traveling from/to a certain municipality should be similar to that observed for the two municipalities as a whole and we found that clusters of local transmission were recorded in municipalities well connected with the municipalities of Ravenna and Cervia (see Table 4 ). For at least four of the five clusters, population movement (i.e., persons who visited the area that was primarily affected or persons from the primarily affected area who visited one of the four towns) can be reasonably assumed to have been the main determinant of local transmission. Another possible explanation is passive vector mobility (e.g. infected mosquitoes transported by car from the initial cluster), since the flight range (active mobility) is usually considered to be less than 1 km [35, 48, 49] .
Our results suggest that the transmission potential of CHIKV in Italy was similar to the one observed in tropical regions where Chikungunya fever is widespread (e.g., Reunion Island, where the best estimate for the initial R 0 was 3.7 [3] ). Specifically, we estimated R 0 to be in the range of 1.8-6. However, being the reproduction number strongly dependent on the density of mosquitoes, which in turn varies a lot over time as a consequence of seasonal meteorological effects, different (even larger) values of R 0 could be observed in future outbreaks, depending on the time of epidemic seeding. In [3] , by adapting a method originally introduced in [50] for human-to-human infections, R 0 was estimated from the generation interval probability distribution function and the number of gonotrophic cycles of the mosquito. This method can not be applied in our study, as the undertaken control measures have contributed to alter the gonotrophic cycles of the mosquito in a indeterminable manner. We found that the probability of observing a major outbreak after the introduction of an index case depends on the ratio of mosquitoes to humans and was estimated to be in the range of 32%-76%. These results confirm the high risk to Europe of tropical vector-borne diseases as a consequence of globalization, which has been modifying the mobility of humans and vectors. Climate changes could have been playing a role, as the geographical limits of mosquito-borne diseases can be influenced by climate [51, 52] , but this is still debated [53] [54] [55] .
Moreover, our analysis strongly support the efficacy of the disinfestation strategy performed during the Italian outbreak, which drastically contributed to reduce the cumulative attack rate (of about 88%), though the application of self-protection preventive measures (insect repellents and window screens) could also have played a role [18] . Therefore, even if the transmission potential of Chikungunya virus could be sensibly high also in temperate climate countries, the epidemic can be controlled by performing timely interventions.
The proposed model has several limitations. We assume exponential distribution for all parameters of model M related to the length of the different periods (e.g. latency, infectiousness, etc.), though it would be preferable to use multiple classes within each group to give more realistic gamma distributed lifetimes (see for instance [56] ). We assume density-dependent growth only in eggs, though other density-dependent regulating processes should be considered for other lifestages of the mosquitoes, e.g. larvae and pupae [25] . These modeling choices are due to the lack of data for parametrizing the model. The lack of information on the actual number of breeding sites -it could be assessed only by performing a field study -prevent us to give precise estimates on the density of mosquitoes over time in the study area. However, we would note that our estimates of R 0 , attack rates and probability of major outbreak are robust with respect to assumptions on the number of breeding sites. Moreover, the temporal dynamics of the vector is qualitatively well captured by model M (though not in terms of absolute abundance) and this allowed us to clarify whether or not the sharp decrease in the number of cases observed after the intervention was due to the intervention itself or to the spontaneous reduction of adults due to decrease of temperature. Definitely, a key lesson learnt from the analysis of the Chikungunya outbreak in Italy is the necessity to improve tools for obtaining reliable, though costly, estimates of the vector density (see for instance [57] ) -thus bypassing the necessity of developing ad-hoc models. The proposed model, describing the temporal dynamics of Aedes albopictus, provides a valid alternative in the absence of reliable field data.
Figure S1 Parameters optimization. a Green points represent the values of the score function F plotted versus the number of notified cases as predicted by the model (with B~200 ha {1 ) before intervention for Q different values of the model parameters as obtained by the LHS procedure. Red point represents the minimum of F . Black points represent the values of the score function F plotted versus the number of notified cases as predicted by the model before intervention as obtained by repeating 100 times the optimization procedure. The inset shows the minimum of F for the 100 replicates (red points). The blue vertical line represent the number of notified cases reported to the surveillance system before intervention, namely 132. b As a but for B~50 ha {1 . (TIF) Figure S2 Results for increasing number of simulations. a Mean (red points), median (blue points) and 95% CI (shaded grey area) of R 0 for increasing number of simulations with B~200 ha {1 in the absence of interventions (baseline scenario). b As a but for probability of observing a major outbreak. c As a but for the cumulative number of symptomatic cases. d As a but for the biting rate. (TIF) Figure S3 Temporal dynamics of the mosquito. a Average density (number per ha) of adult female mosquitoes over time as predicted by the model by assuming B~200 ha {1 (green line) and 95% CI (grey area). b As a but for B~150 ha {1 . c As a but for B~100 ha {1 . d As a but for B~50 ha {1 . (TIF) Probability of origin and destination (and rank over all possible origins/ destinations) of individuals commuting daily for work or school from/to other municipalities were local clusters of transmission were observed. The two most affected villages are located in the municipalities of Cervia and Ravenna. doi:10.1371/journal.pone.0018860.t004 Figure S4 Range of the optimal parameters values. Distribution of the model parameters (2.5%, 25%, 50%, 75% and 97.5% percentiles) after LHS optimization. Numbers below and over the boxplot represent the explored range of values. (TIF) Figure S5 Epidemic threshold and probability of major outbreak. a Epidemic threshold R 0 in relation to biting rate k and ratio of mosquitoes to humans N v =N h . The black line represents the average threshold condition and the shaded blue area represents 95% CI, as resulting from uncertainty of model parameters. The red rectangle identifies the likely range of the two parameters in the two Italian villages affected by CHIKV. b Probability of observing a major outbreak as a function of the ratio of mosquitoes to humans N v =N h for two extreme values of the biting rate k, namely k~0:1 days {1 in red (solid line black represents the average probability and the shaded area represents 95% CI) and k~0:2 days {1 in blue.
(TIF) Figure S6 Baseline simulations and reference interventions. a Average daily number of symptomatic notified cases as predicted by the model in the absence of interventions (baseline scenario, blue line, scale on the left) and 95% CI (grey area) by assuming B~50 ha {1 , compared to the actual daily number of symptomatic notified cases (black points). Red line represents the overall average daily number of symptomatic cases as predicted by the model. Green line represents the average density of mosquitoes (scale on the right). b Histogram of the cumulative number of symptomatic cases as predicted by the model in the absence of interventions. c and d As a and b respectively but for assuming an intervention resulting in the following reductions: 40% as for breeding sites and eggs, 90% as for larvae and 95% as for adults (reference scenario).
(TIF) Clustering Heart Rate Dynamics Is Associated with β-Adrenergic Receptor Polymorphisms: Analysis by Information-Based Similarity Index BACKGROUND: Genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-AR) have a pivotal role in the functions of the autonomic nervous system. Using heart rate variability (HRV) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-AR gene polymorphisms and heart rate dynamics. METHODS: A total of 221 healthy Han Chinese adults (59 males and 162 females, aged 33.6±10.8 years, range 19 to 63 years) were recruited and genotyped for three common β-AR polymorphisms: β(1)-AR Ser49Gly, β(2)-AR Arg16Gly and β(2)-AR Gln27Glu. Each subject underwent two hours of electrocardiogram monitoring at rest. We applied an information-based similarity (IBS) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects. RESULTS: With the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β(2)-AR Arg16Gly genotype. Furthermore, the non-randomness index, a nonlinear HRV measure derived from the IBS method, was significantly lower in Arg16 homozygotes than in Gly16 carriers. The non-randomness index was negatively correlated with parasympathetic-related HRV variables and positively correlated with those HRV indices reflecting a sympathovagal shift toward sympathetic activity. CONCLUSIONS: We demonstrate a bottom-up categorization approach combining the IBS method and hierarchical cluster analysis to detect subgroups of subjects with HRV phenotypes associated with β-AR polymorphisms. Our results provide evidence that β(2)-AR polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control. Instantaneous heart rate in response to physiological perturbations often exhibits remarkable oscillations at multiple time scales. These oscillations, known as heart rate variability (HRV), are mainly mediated by the autonomic nervous system via parasympathetic and sympathetic innervations. Analysis of HRV has been suggested to reveal subtle patterns of heart rate dynamics that are relevant to the underlying physiological state and autonomic nervous system function [1] . Prior studies have shown that HRV measures are highly heritable traits that can be used to support genetic association and linkage studies [2, 3, 4, 5] . Family and twin studies have shown a significant genetic influence on a variety of HRV measures [6, 7] . Genetic polymorphisms related to cardiovascular functions have been associated with altered HRV [8, 9, 10, 11] . Recently, we have also made progress in identifying variations in two genes related to neuropsychiatric function that are associated with altered heart rate dynamics in samples of healthy adult and elderly subjects: those encoding brain-derived neurotrophic factor (BDNF) [12] and apolipoprotein E [13] , respectively.
Despite increasing focus on investigating the genetic influence on autonomic functions, current approaches to genotype-HRV associations have largely been characterized by a top-down approach involving a direct comparison of continuous HRV variables among pre-defined groups of subjects (i.e., healthy vs. ill or groups of known genotypes), yet it is unclear how a particular genetic polymorphism may determine a similar pattern of autonomic heart rate control from one subject to another. Specifically, heart rate dynamics is a phenotypic ''expression'' of the autonomic nervous system, so comparing similar heart rate oscillation phenotypes among individuals may reveal a global profile of autonomic function relevant to genetic variants. With these considerations in mind, in the present study, we introduce a bottom-up genotype-phenotype analysis to investigate the association between genetic polymorphisms and autonomic control of heart rate dynamics, using three common polymorphisms in genes encoding b-adrenergic receptor (b-AR) as an example.
b-AR has a pivotal role in the functions of the cardiac autonomic nervous system. Activation of b 1 -AR provides strong stimulus to increase the frequency and contractility of the heart, whereas the activation of b 2 -AR results in smooth muscle relaxation and increased cardiac output with less extent compared to b 1 -AR. Thus, the connection between b-AR and HRV is plausible and warrants evaluations. Limited evidences suggest that variations in genes coding subtypes of b-AR may be associated with heart rate or HRV. For example, Ser49Gly polymorphism in b 1 -AR gene has been found to be associated with resting heart rate [14, 15] , and an association of b 2 -AR gene polymorphisms with spectral components of HRV measures has been reported in a relatively small healthy adult male sample [11] .
We applied an information-based similarity index (IBS) [16, 17] to measure the pairwise dissimilarity of interbeat interval time series among a sample of healthy adult volunteers. The IBS method is based on rank-order frequency analysis of acceleration/ deceleration patterns of heart rate fluctuation. Because stimulus of b-AR results in acceleration of heart rate, the functional changes in genetic polymorphisms of b-AR may affect acceleration/deceleration patterns of heart rate, which can be detected by the IBS method. The analyses of the present study were two-fold: 1) a nonrandomness index [17] derived from the IBS method was applied to quantify the nonlinear aspect of HRV according to b-AR genotype and to test the correlation of this index with standard HRV indices; and 2) using agglomerative hierarchical cluster analysis, we unsupervisedly categorized these subjects into clusters based on pairwise dissimilarity among heart rate dynamics, and then we investigated the association of these clustering patterns with b-AR gene polymorphisms. We show that this bottom-up, categorization approach combining the IBS method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b-AR gene polymorphisms.
Two hundred forty-seven healthy Han Chinese adult volunteers were recruited from two medical centers: Taipei Veterans General Hospital and Kaohsiung E-DA Hospital, Taiwan. Subjects were recruited by advertisement among medical employees, research laboratory staff working at both hospitals, and their relatives. All subjects gave informed consent before commencement of the study. The protocol was approved by the institutional review boards of the Taipei Veterans General Hospital (Taipei, Taiwan) and E-DA Hospital (Kaohsiung, Taiwan). Each subject was given an interview using a standard questionnaire to carefully review the history of medical disease, psychiatric illness, and medication use. Subjects included in the study did not have a personal history of medical conditions (e.g., malignant tumors, heart failure, or diabetes mellitus), pregnancy, psychiatric illnesses or substance abuse/dependence. None of the subjects was taking any medication. The collected demographic data included age, sex, body mass index, and smoking. Of note, most volunteers were hospital colleagues, and the rate of smoking was low (n = 2, 0.9%).
Of these subjects, 228 were successfully contacted for ambulatory electrocardiogram (ECG) monitoring. Holter recordings (MyECG E3-80 Portable Recorder, Microstar Inc., Taipei, Taiwan) were used to obtain two hours of ECG signals. The E3-80 device continuously recorded three channels of ECG signals at a sampling rate of 250 Hz. All ECG monitoring took place in the daytime, and participants were asked to avoid smoking or drinking alcoholic beverages and to stay in a resting state while being monitored. Valid DNA samples were obtained in 221 subjects by drawing blood or by buccal swabs. The final study sample therefore consisted of 221 healthy adult subjects (59 males and 162 females, aged 33.6610.8 years, range 19-63 years). Among the present study sample, 211 subjects have been included elsewhere in a previous report on the altered sympathovagal balance associated with Val66Met polymorphisms of the BDNF gene [12] .
Each subject was genotyped for three polymorphisms (rs1801252, rs1042713 and rs1042714), and genomic DNA was isolated using the PUREGENE DNA purification system (Gentra Systems, Minneapolis, MN, USA). The genotypes of rs1042713 were determined using polymerase chain reaction and restriction fragment length polymorphism analysis. Briefly, primers and probes were designed with SpectroDESIGNER software (Sequenom, San Diego, CA, USA). PCR was then performed, and unincorporated double-stranded nucleotide triphosphate bases (dNTPs) were dephosphorylated with shrimp alkaline phosphatase (Hoffman-LaRoche, Basel, Switzerland) followed by primer extension. The purified primer extension reaction product was spotted on to a 384-element silicon chip (SpectroCHIP, Sequenom) and analyzed in a Bruker Biflex III MALDI-TOF Spectro-READER mass spectrometer (Sequenom). The resulting spectra were then processed with SpectroTYPER (Sequenom). All samples were genotyped for eight unrelated SNPs for DNA quality examination. The samples were diluted onto 96-well plates, and only the plates on which each of the eight unrelated SNPs had a successful genotyping rate greater than 95% were used for further study. All experiments were performed by investigators who were blind to phenotype. Failure in genotyping for rs1801252 polymorphism was noted in 6 cases.
The ECG signals were automatically processed and analyzed by open-source HRV algorithms [18] . The standard HRV analysis has been well reviewed [19] . Briefly, time domain measures of HRV include the mean heart rate and standard deviation of the normal interbeat intervals (SDNN), the root mean square successive difference between adjacent normal interbeat intervals (RMSSD), and the percentage of adjacent intervals that varied by greater than 50 ms (pNN50) [20] . The SDNN assesses the overall variability of interbeat intervals. The RMSSD and pNN50 measure the short-term variation of interbeat intervals, which is mainly modulated by parasympathetic innervation [21] . Standard spectral HRV measures [19] include high-frequency power (HF; 0.15-0.40 Hz), low-frequency power (LF; 0.04-0.15 Hz), and very low-frequency power (VLF; 0.003-0.04 Hz). LF power is suggested to be modulated by both sympathetic and parasympathetic activities, whereas HF power is mainly modulated by parasympathetic activity [22, 23] . The LF/HF ratio is considered a measure of the shift of sympathovagal balance toward sympathetic activity [19, 24] . The physiological mechanism underlying VLF power is disputed but has been suggested to be mediated partly by the renin-angiotensin-aldosterone system or parasympathetic modulation [19, 25, 26] .
In addition, we incorporated two nonlinear HRV indices: detrended fluctuation analysis (DFA) [27, 28] and multiscale entropy (MSE) [29] . DFA quantifies the presence of long-range (fractal) correlations whereas MSE measures the entropy over multiple time scales inherent in physiologic signals and is therefore a complexity measure. Both methods are available at Physionet (http://physionet.org), a research resource for complex physiologic signals [18] .
In the DFA method, the root-mean-square fluctuation of integrated and detrended time series is measured at different observation windows and plotted against the size of the observation window on a log-log scale. The scaling exponent a is then derived from the slope of line fitting to the obtained log-log plot. The short-term exponent a 1 (4 to 11 heartbeats) and the longterm scaling exponents a 2 (.11 heartbeats) were also calculated [27, 30, 31] . Low-exponent values represent reduced fractal propertiy of heart rate dynamics and have been implicated in the risk of fatal cardiac arrhythmia, increased mortality, or poor prognosis in cardiovascular diseases [32, 33, 34, 35] .
MSE has been proposed as a biologically meaningful complexity measure by quantifying the entropy over multiple time scales inherent in physiologic signals. The procedure and calculation of the MSE is summerized as following three steps: 1) construction of coarse-grained time series, 2) quantification of the sample entropy of each coarse-grained time series, and 3) summation of the sample entropy values over a range of scales. In the present study, sample entropy was calculated using a pattern length (m) of 2 and a similarity factor (r) of 0.15. The sum of sample entropy over all scale factors from 1 to 20 was computed to represent the overall MSE measure. In addition, the sum of sample entropy over scale factors from 1-5 and 6-20 was calculated to represent short-term and long-term MSE measures, respectively [36] .
Several methods of symbolic dynamic analysis of HRV have been proposed previously [37, 38, 39, 40, 41] . The IBS method was developed to effectively categorize symbolic sequences according to their information content. The method has been fully described and validated [16] , with applications to heart rate time series, literary texts, and genetic sequences [17, 42, 43] .
An interbeat interval time series (or heart rate time series) is mapped to a symbolic sequence, according to a mapping rule that accelerated heart rate in consecutive heartbeats is designated as 0 and a deceleration of heart rate is designated as 1 ( Figure 1A ). This way of mapping captures the essential dynamics of the autonomic nervous system's control of heart rate and is less sensitive to noisy fluctuations in interbeat interval time series commonly caused by ectopic heartbeats [17] . A binary, symbolic ''word'' is then defined as a n-tuple sequence derived from n+1 consecutive interbeat intervals. We determined the frequencies of each pattern of n-tuple sequences by applying a sliding window (moving one interbeat interval/step) across the entire interbeat interval time series and then ranked each n-tuple sequence according to its frequency in descending order.
To compare the similarity between symbolic sequences, we plotted the rank number of each n-tuple sequence in the first sequence against that of the second sequence ( Figure 1B ). If two sequences are similar in their rank order of n-tuples, the scattered points will be located near the diagonal line (e.g., comparison between healthy subjects) [17] . Therefore, the average deviation of these scattered points away from the diagonal line is a measure of the dissimilarity index between these two sequences.
We defined the distance (D n ) using n-tuples between two sequences, S 1 and S 2 , as
Here R 1 (w k ) and R 2 (w k ) represent the rank of a specific n-tuple, w k , in sequences S 1 and S 2 , respectively. N = 2 n is the number of different n-tuple sequences (or patterns). The absolute difference of The mapping procedure for a n-tuple binary symbolic sequence (here n = 6 for illustrative purposes) from part of an interbeat interval time series. (B) Rank-order comparison of two interbeat interval time series from the two healthy subjects, using 6tuple binary symbolic mapping. In this case, frequencies of 2 6 = 64 6tuple symbolic patterns were determined and ranked accordingly. For each 6-tuple symbolic sequence (black dot), its rank in subject A is plotted against its rank in subject B. The dashed diagonal line indicates the case where the rank order of 6-tuple symbolic sequence for both subjects is identical. doi:10.1371/journal.pone.0019232.g001 ranks, R 1 (w k ){R 2 (w k ) j j , is weighted by summing Shannon's entropy H for w k in sequences S 1 and S 2 [44] . Shannon's entropy measures the information richness of each n-tuple in both sequences. Thus, the more frequently used n-tuples contribute more to measuring similarity among symbolic sequences. Of note, the IBS is an empirical index which does not necessarily obey the triangular inequality criterion of a distance measure. Therefore, the triangular inequality test is required before generating a cluster [16, 17] . The IBS algorithm was available at Physionet (http:// www.physionet.org/physiotools/ibs/).
The applications of the IBS method in the present study were two-fold: 1) The pairwise distance of heart rate dynamics among individuals was calculated by an average of IBS distance using ntuple symbolic sequences (n = 2-10) and was used in subsequent hierarchical cluster analysis. 2) To quantify the nonlinear aspect of heart rate time series, a non-randomness index was defined to measure the symbolic patterns of heart rate dynamics away from complete randomness (i.e., absence of ordered control of the autonomic nervous system) [17] . The non-randomness index is independent of conventional entropic measures (i.e., sample entropy or approximate entropy) and has been evaluated in other studies [17, 45, 46, 47] . The calculation of the non-randomness index is based on estimating the average n-tuple distance between raw interbeat interval time series and its randomly shuffled surrogates using the IBS method, where the number of surrogates was 100 in the present study. The averaged non-randomness index derived from each n-tuple non-randomness index (n = 2-10) was then used in this study for comparisons among b-AR genotypes.
We calculated allele and genotype frequencies and performed Hardy-Weinberg equilibrium tests for each b-AR genotype. The spectral HRV indices were log-transformed to produce normalized distributions. To control for the effects of non-genetic factors, differences in HRV variables were compared for individual genotypes using a general linear model (GLM) followed by the Bonferroni post hoc test for corrections of multiple between-group comparisons., using age, gender, and body mass index as covariates. Effect sizes were calculated with Cohen d, defined as the difference between the means of two groups, divided by the pooled standard deviation of these groups. Partial correlation analysis was applied, controlling for age and BMI, to determine the associations between standard HRV indices and nonrandomness index derived from the IBS method. A p value of less than 0.05 (two-tailed) was required for all statistical comparisons.
A complete-linkage, hierarchical clustering tree was estimated using the generalized association plot (GAP) (http://gap.stat. sinica.edu.tw/). GAP is implemented here as an unsupervised clustering algorithm to visually categorize all subjects based on the dissimilarity matrix, which is derived from calculating the pairwise dissimilarity of heart rate dynamics among subjects using the IBS method. Cluster-specific association was analyzed by the chisquare test to assess the frequency of b-AR polymorphisms and by the GLM model to test the differences in standard HRV indices among resulting clusters. To validate cluster-specific associations, we tested the differences in HRV and b-AR genotypes in estimated clusters based on first-and second-half ECG data.
Demographic and clinical data for subjects with each b-AR genotype are presented in Table S1 and S2. The subgroups in three genotypes did not differ in age, gender ratio, smoking status, or body mass index. There was no detectable deviation from Hardy-Weinberg equilibrium in b 1 -AR Ser49Gly genotype (x 2 = 0.169, P = 0.681), b 2 -AR Arg16Gly genotype (x 2 = 0.824, P = 0.364) or b 2 -AR Gln27Glu genotype (x 2 = 2.188, P = 0.139).
Tables 1, 2, 3 summarize the association of b-AR genotypes with HRV indices determined by a GLM model using age, gender, and BMI as covariates. There was no significant effect of interaction on HRV indices detected between b-AR genotypes and demographic variables.
b 1 -AR Ser49Gly genotype was associated with mean heart rate and SDNN with borderline significance (Table 1 ; p = 0.087 and p = 0.064, respectively). No statistical association was found between b 2 -AR Gln27Glu genotype and HRV indices ( Table 2) . For the b 2 -AR Arg16Gly genotype (Table 3) , a significant codominant association was found with LF% (F = 3.636, p = 0.028) and the non-randomness index derived from the IBS method (F = 5.642, p = 0.004). Multiple comparisons showed that LF% was significantly lower in homozygous Arg16 carriers compared to subjects with the Arg16/Gly16 genotype (p = 0.016), but not to homozygous Gly16 carriers (p = 0.985). Likewise, a significantly lower non-randomness index was found in homozygous carriers of the Arg16 allele (p = 0.016) compared to subjects heterozygous for Arg16/Gly16 (p = 0.001), and a borderline significance was seen in the comparison to homozygous Gly16 carriers (p = 0.057).
There was no significant difference in HRV indices between heterozygotes Arg16/Gly16 and homozygous Gly16 carriers, Correlations between non-randomness index and standard heart rate variability
To ascertain the relationship of non-randomness index with autonomic function, we estimated the partial correlation between non-randomness index and standard HRV variables, controlling for age and BMI (Table S3) . A weak but significant positive correlation of the non-randomness index with the LF/HF ratio was found (r = 0.234, p = 0.001), whereas negative correlations were found with RMSSD (r = 20.274, p,0.001) and pNN50 (r = 20.279, p,0.001).
Association of heart rate dynamic clusters with b-AR gene polymorphisms Next, we investigated the b-AR genotype distributions in an unsupervised cluster tree based on the dissimilarity (distance) measure of heart rate dynamics. We first estimated the pairwise distance between interbeat interval time series of all subjects using the IBS method. No violation of the triangular inequality was observed. We then applied a hierarchical clustering algorithm to cluster these subjects based on the obtained distance matrix. Two major clusters were identified in the resulting dendrogram ( Figure 2 ). There was no significant difference in age, gender, or other demographic variables between the two clusters (data not shown). Table 4 shows b-AR genotypes and standard HRV characteristics according to two major clusters. A significant deviation in the distribution of homozygous b 2 -AR Arg16 carriers was detected between the two clusters (p = 0.011), whereas the other genotypes showed no deviation in genotype distribution. In terms of standard HRV characteristics, cluster 1 (with a higher rate of homozygous b 2 -AR Arg16 carriers) showed significantly reduced LF power (p,0.001) and significantly increased HF power (p = 0.001). The clusters did not differ in other HRV indices. We verified the genotype distribution with clusters based on first-and second-half ECG data, and the results were consistent (Table S4 ).
The data presented in this study demonstrate a significant association of a common b 2 -AR polymorphism, Arg16Gly, with the non-randomness index, a nonlinear HRV measure derived from the IBS method. Moreover, we illustrate that a bottom-up approach using the IBS method was able to measure the dissimilarity of heart rate dynamics among individuals, and we show that the resulting clusters were associated with b 2 -AR Arg16Gly genotype, indicating an impact of this b 2 -AR polymorphism on acceleration/deceleration patterns of heart rate oscillations.
Our study offers several advantages for studying genetic associations with physiological parameters and might be generalizable to other genotype-phenotype studies based on different types of time series (e.g., brain electroactivity or time series derived from functional magnetic resonance imaging). First, our method enables us to cluster heart rate dynamics by an IBS method based on their acceleration/deceleration patterns of heartbeat sequence. The IBS method was not exclusively developed for the analysis of heartbeat sequence but also for other generic datasets consisting of repetitive elements [42, 43] . With an appropriate mapping rule that is meaningful to a target dataset, the IBS method can be applied to other types of phenotypic data derived from the time series [16] . Second, we employed a visually aided hierarchical analysis by a software tool called a generalized association map, which enables a bottom-up approach to unsupervisedly identify heart rate clusters to study genotype-phenotype associations. This bottom-up approach may help reduce the false-positivity commonly seen in conventional association studies. b 1 -AR is the predominant b-AR subtype in the heart. Compared to activation of b 2 -AR, stimulation in b 1 -AR can result in more Figure 2 . Unsupervised, hierarchical cluster tree of subjects according to the pairwise dissimilarity matrix among heart rate dynamics using an information-based similarity index method. Dissimilarity matrix data were visualized and clustered by a generalized association plot algorithm [58] . The bars on the left indicate the b-adrenergic receptor (b-AR) gene polymorphisms rs1801252 (green: homozygous Ser49 carriers; blue: Gly16 allele carriers; red: unknown genotype), rs1042713 (green: homozygous Arg16 carriers; blue: Gly16 allele carriers) and rs1042714 (green: homozygous Gln27 carriers; blue: Glu27 allele carriers). doi:10.1371/journal.pone.0019232.g002 significantly increased frequency and contractility of heart. Although substantial efforts have been made to link b 1 -AR polymorphisms with cardiovascular function, studies have been mixed in this regard. For example, b 1 -AR Ser49Gly polymorphism was found to be associated with increased blood pressure and heart rate [14, 15] but data are inconsistent in our findings and other reports [48, 49] . Because we studied only one polymorphism in b 1 -AR, further research is needed to identify other possible b 1 -AR polymorphisms associated with HRV.
This study found that b 2 -AR Arg16Gly genotype was associated with clusters based on acceleration/deceleration patterns of heart rate. Although b 2 -AR is expressed in the heart at lower concentrations than is the b 1 -AR subtype, it also expresses on the presynaptic sympathetic nerve terminals and expresses abundantly in vascular and bronchial smooth muscle. Therefore, changes in b 2 -AR function may not only alter sympathetic activity [50] , but also respiration and vascular responses [51, 52] . It can be reasonable to speculate that these factors have influences in HRV. Of note, the association between b 2 -AR Arg16Gly polymorphism and heart rate fluctuation patterns may simply reflect a functional genetic component nearby due to linkage disequilibrium and warrants future genetic research targeting on this region.
The non-randomness index was developed initially as a nonlinear HRV index [17] and its connection with other traditional HRV indices needs to be explored. Though the nonrandomness index was weakly associated with HRV indices (rsquare,0.1; Table S3 ), it did correlate negatively with HF power and positively with the LF/HF ratio, suggesting that the non-randomness index is a marker related to sympathovagal balance. Our findings of lower non-randomness index and LF% seen in homozygous b 2 -AR Arg16 carriers are in line with prior studies showing that the b 2 -AR Arg16 polymorphism is associated with lower sympathetic activity, manifested as lower blood pressure [53, 54] , lower plasma norepinephrine [54, 55] , and enhanced agonist-mediated desensitization in the vasculature [56] .
Strength of this work include a larger sample size, matched gender distribution, and long recording of ECG signals using a Holter recorder, compared to prior genetic association study of HRV. Several limitations influence the interpretation of the findings presented in this study. First, we are unable to repeat findings of spectral HRV indices seen in a smaller male sample [11] . This may be due to different gender distributions [57] and means of ECG recording. Twenty-four hour Holter recording could validate and reinforce the association of b-AR genetic polymorphism with HRV seen in this study. Second, we were unable to evaluate the correlation of the non-randomness index with other commonly used sympathetic indicators, such as blood pressure or plasma catecholamine levels. Third, our findings are based on a healthy population and may not be generalizable to medically ill patients, such as those with cardiovascular diseases. The cross-sectional nature of our study also limited us examining the age modification of the effect of b-AR receptor polymorphism on autonomic function.
In conclusion, this study shows that a bottom-up, categorization approach combining the IBS method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b 2 -AR gene polymorphisms. Further research should aim to identify the physiological mechanisms underlying these findings.  The Infection of Chicken Tracheal Epithelial Cells with a H6N1 Avian Influenza Virus Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1–3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry. Sialic acids (SAs), consisting of a core of nine-carbon monosaccharide, are usually linked to the outermost capping position of glycans that are conjugated to cell-surface glycoproteins or glycolipids [1] . Sialyltransferase adds SA to the terminal sugar residues, such as galactose (Gal), N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) [1] . The conjugation between Gal and SA can be either in the form of an a2,3 or an a2,6 glycosidic linkage. In mammals, N-acetylneuraminic acid (Neu5Ac) and Nglycolylneuraminic acid (Neu5Gc) are the two most common types of SA, but Neu5Ac is the major type of SA in birds [2] .
Plant lectins extracted from Maackia amurensis (M. amurensis leukoagglutinin, MAL) and Sambucus nigra (S. nigra agglutinin, SNA) are usually applied for the detection of Neu5Aca2-3Gal (SAa2-3Gal) and SAa2-6Gal glycans in tissues, respectively [3] . Two types of M. amurensis lectins were discovered: one that can agglutinate erythrocytes (hemagglutinin) (MAH, also known as MAL-II) and one that can agglutinate leukocytes (MAL, also known as MAM, MAA, MAL-I). Although both MAL-I and MAL-II recognize the SAa2-3Gal glycan, previous studies [4] and recent glycan microarray data [5] demonstrated that subterminal sugars affect their binding affinity to these two lectins. For example, MAL-I, rather than MAL-II, showed the highest affinity to the SAa2-3Galb1-4GalNAc and did not bind to this oligosaccharide when the subterminal b1,4-linkage was replaced by a b1,3-linkage [6] .
Cell entry by influenza virus depends on the recognition of a terminal SA-capped glycosylated molecules by the viral hemag-glutinin (HA) protein [3] . Generally, human influenza viruses preferentially bind to cell surface oligosaccharides that have the SAa2-6Gal linkage, while avian influenza viruses (AIVs) prefer SAa2-3Gal [3] . Especially, the glycans containing the SAa2-3Galb1-4GalNAc or SAa2-3Galb1-4(6OSO3)GalNAc, show a high affinity to MAA/MAL-I [4] and AIVs [7, 8] . Tracheal/ bronchial epithelium, mainly consisting of ciliated cells, goblet cells and basal cells, is the initially attacked tissue by the invading influenza viruses. The infected chicken trachea shows necrosis and detachment of ciliated cells, suggesting that ciliated cell is one of the cells targeted by AIV [9] . Previous analyses of the expression of SAs in chicken have revealed that a2,3-linked SA was localized on tracheal ciliated cells and a2,6-linked SAs was also present in the tracheal tissues [10, 11, 12] . However, the detailed distribution of SAs among chicken tracheal epithelial (CTE) cells remains unclear. Clarification of the distribution and the expression intensity of influenza receptor on CTE cells will help us to understand the viral tropism, viral spreading and the pathogenesis of avian influenza viruses. Here, the SA distribution on CTE cells were identified by the staining of biotin-labeled plant lectins. A Taiwan-isolated AIV H6N1 was applied to characterize the cell tropism of AIV and the correlation of the cell tropism of AIV to the SA distribution on CTE cells was also explored.
The SAa2-3Gal expression of primary CTE cells
The distribution of SAa2-3Gal expression on the primary CTE cells was determined by the staining with a plant lectin, MAA (EY Laboratories). The ciliated cells were revealed by the intense fluorescent microvilli, labeled by the b-tubulin specific antibody [13] . Mucin 5AC glycoprotein and cytokeratin-14 (K14) were the specific markers for the goblet and basal cells, respectively [14, 15] . Immunocytochemistry (ICC) staining illustrated that most btubulin + cells expressed SAa2-3Gal terminal glycan (ratio of MAA + /b-tubulin + cells, 0.8760.06, n = 317) (Fig. 1A) . In a high magnification field, the intense spots of MAA signal were surrounded by cilia bundles on the ciliated cells (Fig. 1B) . In addition, MAA lectin could also be detected on the primary K14 + basal cells (ratio of MAA + /K14 + cells, 0.4660.12, n = 267) (Fig. 1G) .
Interestingly, the MAA signals did not colocalized with the mucin-expressing cells (ratio of MAA + /mucin + cells, 0.00760.01, n = 223) (Fig. 1D) , indicating that goblet cells may not express the SAa2-3Gal glycans which show high affinity to the MAA lectin. However, for the immunohistochemistry in tissue sections, the distribution of SAa2-3Gal expression may show inconsistent distribution when different MAA/MALs were applied or the same lectins were from different providers [16, 17] . To further address the SA expression on goblet cells, MAL-I, same as MAA, was given by Vector Laboratories and applied to stain the CTE cells. We found that abundant MAL-I was detected on the b-tubulin + cells (ratio of MAL-1 + /b-tubulin + cells, 0.8260.10, n = 301) (Fig. S1), rather than the mucin + cells (ratio of MAL-1 + /mucin + cells, 0.0260.02, n = 201) (Fig. 1E ), similar to the result for MAA.
Subtypes of SAa2-3Gal glycans with different subterminal sugars present varied affinity to MAA/MAL-I and MAL-II. Whether the SAa2-3Gal glycan is not expressed on goblet cells was further evaluated by MAL-II (Vector Laboratories). Surprisingly, 0.3760.12 mucin + cells (n = 594) were double-stained with MAL-II (Fig. 1F ), indicating that a glycan subtype that shows a high affinity for MAL-II only, was expressed on the goblet cells. We also found that only 0.3360.09 b-tubulin + cells (n = 230) can be labeled with MAL-II (Fig. 1C ). In addition, about half basal cells were MAL-II positive (0.5360.12, n = 265) (Fig. 1H ).
The SAa2-6Gal expression of primary CTE cells
The distribution of SAa2-6Gal on the CTE cells was determined by staining with SNA (Vector Laboratories). In contrast to the finding for MAA/MAL-I, abundant SNA signals were mainly restricted to the mucin + cells (ratio of SNA + /mucin + cells, 0.8560.09, n = 368) ( Fig. 2A) , indicating that the goblet cells expressed SAa2-6Gal terminal glycan. In addition, SNA lectin can also be detected in a one-third proportion of K14 + cells (ratio of SNA + /K14 + cells, 0.3660.09, n = 284) (Fig. 2B) .
Especially, the SNA signal did not localize on the b-tubulin + cells (ratio of MAA + /b-tubulin + cells, 0.0160.01, n = 237) (Fig. 2C ). Testing the SNA-I, which was provided by EY Laboratories, showed no difference to the result of SNA, evidenced by the undetectable binding by SNA-I on the ciliated cells (Fig. 2D ).
The SAa2-3Gal and SAa2-6Gal expression on basal cells
Although both MAA and SNA bound to the surfaces of a subpopulation of the K14 + cells ( Fig. 1G and Fig. 2B ), their detected signals were relatively weak (Figs. S2A and S2B). To determine whether a single basal cells can express both SAa2-3Gal and SAa2-6Gal, CTE cells were triple-stained with K14 primary antibody (Cy5-2u Ab, purple), FITC-conjugated MAA (green) and biotin-labeled SNA (detected by Cy3-Streptovidin, red) (Figs. 3A, 3B and 3C). Their cell nuclei were revealed by DAPI staining (blue). When viewed in the same field under a confocal microscope, the ICC result indicated that MAA and SNA can be co-expressed on single K14 + cells (indicated by the arrows).
The distribution and relative expression levels of SAa2-3Gal and SAa2-6Gal are summarized in Table 1 . It should be noted that because the relative affinities of the lectins to their targeted glycans may differ, comparing the intensity of staining between the MAA and SNA cannot be used as a quantitative comparison of the relative SAa2-3Gal and SAa2-6Gal expressions. In addition, the H6N1 viral antigens detected in the infected cells at a MOI of 0.1 were mostly restricted to the b-tubulin + cells at 6 h.p.i. (Fig. 5A ). Few H6N1 antigens were detected in the goblet cells or basal cells, possibly due to the low expression of AIV receptors (Figs. 5B and 5C). These results indicate that the ciliated cell, rather than the goblet or the basal cells, is the primary target cell for the AIV. In addition, these data also illustrate that the distribution of SAa2-3Gal expression among cells determines the cell tropism of the AIV H6N1 virus (as summarized in Fig. 6 and Table 1 ).
The infectivity of H6N1 in CTE cells was further characterized at a MOI of 1. ICC results showed that the ciliated cells (Fig. 5D ), goblet and basal cells were all susceptible to infection by H6N1 as measured at 6 h.p.i. (Figs. 5E and 5F ). Although SAa2-3Gal expression was relatively low in goblet and basal cells, still 21.360.05% of goblet cells and 51.1610.9% of basal cells were infected. We speculated that the low infection of goblet cells by influenza virus at a high MOI might mediate through the binding to MAL-II specific SA or through a SA-independent pathway [18] . However, this hypothesis still requires the support from further experiments.
In the human tracheal/bronchial epithelium, ciliated cells display mainly a2,3linked SAs and goblet cells express a2,6linked SAs [19] . About one third of ciliated cells also display the a2,6-linked SAs and goblet cells also express the a2,3-linked SAs at a low degree [19, 20] . The distribution of the a2,3and a2,6-linked glycans was primarily on the apical surface of both ciliated and goblet cells, respectively [19, 20] . As revealed by specific lectins and ICC staining in the primary CTE cells, our results clearly illustrate that all three types of CTE cells express SAa2-3Gal in a varied degree. SAa2-3Gal showed abundant expression on chicken ciliated cells but only a detectable level on basal cells ( Fig. 6 and Table 1 ).
Revealing the distribution and the expression intensity of influenza virus receptor on the tracheal basal cells will help us to understand the cell tropism of influenza virus and the pathogenesis of AIV infected tissues. Our previous study indicated that the replication of infectious bronchitis virus (IBV) is restricted to ciliated cells and goblet cells, but not basal cells [21] . The tracheal/bronchial basal cells, assumed as multipotent stem cells, are responsible for epithelial recovery and reestablish normal respiratory function after desquamation of the ciliated and goblet cells [14, 15] . In an uncomplicated IBV-infected chick, clinical signs persist for only 1 week and the unaffected basal cells may be responsible for epithelial reconstruction of the injured respiratory tract [22] . In contrast, basal cells are susceptible to the infection of avian influenza virus at a MOI of 1. The susceptibility of basal cells to AIV infection suggests that infection with AIV alone may cause the cell death of basal cells, and consequently affect basal membrane integrity and severe inflammation in the AIV infected trachea.
Interestingly, MAA/MAL-1 did not bind to the surface of goblet cells, suggesting that certain SAa2-3Gal glycans that show high affinity for MAA/MAL-I lectins, such as SAa2-3Galb1-4GlcNAcb and SAa2-3Galb1-4(6OSO3)GlcNAcb [4, 5] , are not present to a significant extent on the cell membranes of goblet cells. However, one-third of the goblet cells showed strong MAL-II staining, suggesting that the MAL-II specific glycans, such as SAa2-3Galb1-3(SAa2-6)GalNAca and SAa2-3Galb1-3(6OSO3)-GalNAca are highly expressed on goblet cells [4, 5, 6, 23] .
The expression profiles of SAs on CTE cells exhibits distributions that are distinct from those of human. In chickens, MAL-I bind to the surface of ciliated cells and basal cells, but not goblet cells. The goblet cells express a MAL-II specific SAa2-3Gal glycan. In addition, both SNA and SNA-I failed to label the btubulin + cells, indicating that SAa2-6Gal is exclusively expressed on non-ciliated cells. In humans, by contrast, both ciliated and goblet cells can be labeled with MAA/MAL-I and SNA [19, 20] , indicating that these two epithelial cells have both types of influenza viral receptors. Moreover, both ciliated and goblet cells cells are permissive to infection by human influenza viruses [19, 20] . Interestingly, using duck influenza A viruses to infect human airway epithelial cells, the viral antigen could only be detected in the ciliated cells, but not in the goblet cells, possibly due to the low SAa2-3Gal expression on the surface of goblet cells and a low MOI infection [19, 20] .
Although the SAa2-6Gal glycans was detected in human tracheal/bronchial goblet cells [19, 20] and was capped on mucin protein [24] , it was also proposed that tracheal mucin in the airway contains SAa2-3Gal [25] (Fig. 6A) . Especially, this secreted mucin was shown to prevent infection by influenza viruses with a SAa2-3Gal preference in the airway [26] . This false-receptor effect may mask the HA of the AIV, disable the viral access to the goblet cells and also account, at least in part, for the hindrance of AIV infection to human goblet cell [26] . In chicken trachea, goblet cells exhibit high affinity for SNA, suggesting that the mucin may contain SAa2-6Gal glycans. The viral neutralization effect of mucin in humans may be recapitulated in chickens to aid the clearance of the invading influenza viruses with a SAa2-6Gal preference.
In addition to the SA2,6-linkage glycan, SA2,3-linked oligosaccharides with Galb1-3GalNAc subterminal residues, which show a MAL-II preference, may be present in the goblet cells (Table 1) [4, 5, 6, 23] . Notably, although AIVs can bind to the terminal SAa2-3Gal, duck and chicken influenza viruses exhibit different affinities for glycans with different subterminal residues [7, 8] . For example, duck influenza viruses prefer Galb1-3GlcNAc or Galb1-3GalNAc, but chicken influenza viruses prefer Galb1-4GlcNAc or Galb1-4(6OSO3)GlcNAc [7, 8] . Our study suggests that SAa2,3-capped oligosaccharides following a Galb1,3-linkage [6] are candidate sugars conjugated to the chicken tracheal mucin protein. Determining the SA composites of chicken mucin protein and the neutralizing effect of chicken mucin on duck influenza viruses will be an interesting task that will provide important information about the innate defense mechanisms in the chicken trachea and about the interspecies transmission of influenza viruses between ducks and chickens.
Both the hindrance of the mucin barrier and the limited number of proper receptors on the epithelial cells of the host restrict cross-species infection by influenza viruses [6, 20] . Nevertheless, genetic mutations or recombinations in the HA of an influenza virus can render interspecies transmission by altering the receptor tropism [27, 28, 29] . The 226 and 228 residues in the HA of the influenza H3 and H2 viruses are particular important for determining the receptor specificity and host range restrictions [30, 31] . The Ser-to-Gly mutation at HA position 228 and the Leuto-Gln mutation at HA position 226 shift the receptor preference from SAa2-6Gal to SAa2-3Gal and enhance the infectivity of human influenza virus in duck intestinal cells [31] .
The HA of the chicken H6N1 virus we applied possesses the GQRSRI sequence, which corresponds to the 225 to 230 positions in H3 HA [32] . We showed that most MAA + cells, but few SNA + cells, were permissive to infection by the H6N1 virus. In addition, at a low MOI, most H6N1 proteins were detected in the SAa2-3Gal-rich ciliated cells, and few in the goblet and basal cells. These results indicate that the chicken H6N1 virus, even with a Ser228 (using H3 HA numbering) in its HA, still possesses a SAa2-3Gal preference. Similarly, the HA 226 residue, critical to cell tropism in human H3 and H9N2, has been shown to be an insufficient binding site for determining the receptor tropism of the human H1, H2, H5 and the avian H6, H9 and H5N1 viruses [4, 33, 34, 35] . These results emphasize that receptor tropism is determined by several critical residues in the HA proteins, but these sites in HA are not highly conserved among different influenza viruses.
A Taiwan AIV H 6 N 1 strain, 2838V (virulent), was obtained by serial inoculation of an original field isolate, H 6 N 1 2838, into specific pathogen free (SPF) chickens [36] . The virus was further amplified by passage into the amnion of SPF eggs. Cell debris in the collected fluid was clarified by centrifuging at 1000 rpm for 10 min and passing through a 0.45 mm filter. The viral titer (50% of the egg infectious dose, EID 50 ) of 2838V was 1610 8 /ml.
Tracheas were obtained from one-day-old SPF chicks (Animal Health Research Institute, Tansui, Taipei, Taiwan) and rinsed in a DMEM medium (Invitrogen, Carlsbad, CA, USA) under a sterile condition. The procedure for removing epithelial sheets from the tracheas and the detailed culture conditions for the CTE cells have been described previously [21] . Briefly, tracheas were digested with dispase I solution (2.5 U/ml dispase I, Roche) for 2 h at 37uC. The detached cell sheets of tracheal epithelium from the tracheal lumen were harvested and further digested with collagenase I (1 mg/ml, Roche) for 5 min at 37uC. The disrupted tracheal epithelial sheets were gently pippeted and homogenized into small cell clumps. The cell pellets were resuspended in a CTE medium [21] and were seeded on 2% matrigel-coated 24-well plates (Corning). The cells were cultured at 37uC with 5% CO 2 for 3 days. The animal use protocol in this study had been reviewed and approved by Institutional Animal Care and Use Committee in National Chung Hsing University. The approval number is 99-09.
The tested lectins for the SAa2-3Gal capped glycan were MAA (EY Laboratories, San Mateo, CA, USA), MAL-I (Vector Laboratories, Burlingame, CA, USA) and MAL-II (Vector Laboratories). The lectins for the SAa2-6Gal were SNA (Vector Laboratories) and SNA-I (EY Laboratories). The MAA lectins were conjugated with biotin or a green fluorescent dye, fluorescein isothiocyanate (FITC). The signal of the biotin-labeled lectins was enhanced and visualized by staining with a red-fluorescent Cy3conjugated streptavidin (Rockland, Gilbertsville, PA, USA).
The CTE cells were fixed in 4% cold paraformaldehyde and blocked using the Carbo-free TM blocking solution (Vector Laboratories). Immunocytochemistry (ICC) was performed using the biotin-labeled lectins (MAA, MAL I, MAL II, SNA and SNA I, 1:500) or the FITC-conjugated MAA (1:50, EY Laboratories). The used primary antibodies were b-tubulin (1:500, Sigma-Aldrich, St. Louis, MO, USA), mucin 5AC (1:100, Sigma-Aldrich) and cytokeratin 14 (K14, 1:100, Convance, Princeton, NJ, USA). The specificity of the anti-AIV 2838 polyclonal antibodies (1:500, from infected chickens) has been evaluated in our previous report [37] . Appropriate fluorescence-tagged secondary antibodies (2u Ab, Jackson ImmunoResearch, West Grove, PA, USA) and Cy3conjugated streptavidin (Rockland) were used for visualization.
Blue 49,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. The number of ICC-staining positive cells in a 24-well plate was manually counted from five individual fields. Images of the immunostaining were captured using a confocal microscope (LSM510 Meta, Zeiss). For comparing the expression intensity of SA among CTE cells, the images were collected with the same setting and same objective (406) under the confocal microscope. Figure S1 The SAa2-3Gal expression of ciliated cells (btubulin + ) was characterized by the double-staining of biotin-labeled MAL-1 (Vector laboratories, 1:500). Scale bar, 50 mm. (EPS) Figure S2 Expression levels of SAa2-3Gal (labeled by MAA)(A) and SAa2-6Gal (labeled by SNA)(B) in tracheal basal cell (K14 + ) were shown by immunocytostaining. The arrows in A and B indicate cells with high-intensity MAA and SNA staining, respectively. The cell nuclei were stained with DAPI (blue). Scale bar, 50 mm. (EPS) Classification of viral zoonosis through receptor pattern analysis BACKGROUND: Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell. RESULTS: We analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species. CONCLUSIONS: This study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains. Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins [1] . Zoonosis can occur not only through direct transmission, but also through intermediate reservoirs or other environmental factors [2] [3] [4] . The zoonotic viruses can be categorized according to genotype; of the various classes of viruses, the RNA viruses exhibit the highest mutation rates [5] . Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species [6, 7] . The high mutation rate of envelope proteins [5] hinders the development of accurate vaccines, as does the great ability of the RNA viruses to infect host species in order to exploit host proteins for viral reproduction [8] .
Lacking the ability to self-replicate, viruses must utilize the replication apparatus of their host cells [9] . Viral infection of a cell begins with attachment of the virus to the cell surface [6, 10, 11] . During attachment to the cell membrane, the viral envelope protein (a structural protein) interacts with the host-cell receptor protein(s) [12] . In non-envelope viruses, the capsid plays this role. The cell receptors that play a major role in viral attachment are predominantly membrane proteins of the immunoglobin superfamily [13] [14] [15] . The identification of virusbinding cellular receptors was rapidly accelerated in the late 1980s owing to developments in the use of monoclonal antibodies and molecular cloning techniques [15] . The various receptors that have been found are surface matrix structures containing carbohydrate, lipid, and protein moieties [1, 16, 17] . In some cases, viral attachment also exploits co-receptors. For example, HIV, which uses the CD4 molecule as its receptor, uses the CXCR4 and CCR5 co-receptors to strengthen the effectiveness of infection [1, 14, 18, 19] . Similarly, hepatitis C virus utilizes CD81 as a receptor and LDLR as a coreceptor [20] .
Since the host-cell range of a specific virus is predetermined by its ability to recognize specific receptors, the similarities between the receptors of its primary reservoir host cell and the potential human host cell play a major role in determining the likelihood of viral zoonosis. Here, we analysed zoonotic and non-zoonotic RNA viruses along with their cellular receptors in human and (non-human) primary reservoir species to extract the receptor characteristics common to zoonosis. Viruses not previously reported to infect humans were classified as non-zoonotic viruses. We excluded all viruses known to utilize co-receptors; i.e., only virus-receptor interactions occurring through virus tropism and pathogenesis were considered [5, 21] . The receptors and viruses examined in this study are listed in Table 1 . We hypothesized that the major barrier to the transmission of viruses between species is the difference in cellular receptor sequences. In other words, the specific amino acid sequence of the receptor should be the major determinant of the ability of the viral envelope protein to attach to the cell. Ordinary sequence alignment protocol tells us overall sequence similarity which we thought useful but insufficient because most receptors are membrane proteins and membrane proteins consist of distinctive hydrophobic and hydrophilic parts. Therefore, we analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species to predict the likelihood of transmission across species, including humans. To our best knowledge, this study is the first attempt to predict zoonosis through a simple analysis of receptor sequence similarities and differences. This method may be useful in predicting the zoonotic potential of newly discovered viral strains.
The pair-wise receptor sequence similarities ( g S i,1 , g S i,2 , and g S i,3 ) between host-species pairs for each virus family are shown in Table 1 . For logical comparisons, each virus contains at least one infected host (the primary reservoir, designated as "#" in Table 1 ). As shown in Table 1 , the similarity scores for the infected group (g = 1) were high, ranging from 0.790 to 0.988 for 1 S i,1 , from 0.841 to 0.996 for 1 S i,2 , and 0.794 to 0.962 for 1 S i,3 . All pair-wise comparisons in group 1 (human vs. primary reservoir, primary reservoir vs. host, and human vs. host) yielded high similarity scores, indicating a high similarity among receptor sequences. The similarity scores were comparatively low in the non-infection group (g = 2), ranging from 0.092 to 0.440 for 2 S i,1 , from 0.108 to 0.432 for 2 S i,2 , and from 0.130 to 0.416 for 2 S i,3 . For group 2, both the primary host species and non-infected species are listed to illustrate the differences in similarity. In pair-wise comparisons, all the non-infection cases yielded low similarity values, i.e., the receptor sequences differed significantly from each other.
We assume that a low similarity in receptor sequences disfavors infection despite the existence of a common receptor. For example, enterovirus infects only Sus scrofa (pig); it does not infect Rattus norvegicus (rat) or Homo sapiens (human) because of the high transmission barrier. Similarly, for leukovirus, only Gallus gallus (chicken) is infected as a primary reservoir; because of the high transmission barrier, R. norvegicus and H. sapiens are not infected. These results imply that for non-infection cases, species barriers exist, and the propensity to cross the barrier is determined by the sequence similarity between the potential and primary host receptors.
Similarity scores for rabies virus were low between Canis lupus familiaris (domestic dog) and Bos Taurus (domestic cow) ( 2 S i,1 = 0.280, 2 S i,2 = 0.373, and 2 S i,3 = 0.366) and also between B. taurus and H. sapiens ( 2 S i,1 = 0.267, 2 S i,2 = 0.371, and 2 S i,3 = 0.416) but were high between C. l. familiaris and H. sapiens ( 1 S i,1 = 0.947, 1 S i,2 = 0.985, and 1 S i,3 = 0.962). Clearly, C. l. familiaris is the primary reservoir, and transmission of the disease to H. sapiens is possible only because of the high human/ dog receptor similarity. Thus, for particular viruses, transmission of disease may be species-selective, although common receptors exist among species. Furthermore, infection specificity may be determined by the species barrier, which results from receptor differences.
The values in Table 1 are plotted in Figure 1 to illustrate the differences among groups. The x-and y-axes denote g S i,1 and g S i,2 , respectively, where "g" is the group classification. All pair-wise similarity scores are shown. Groups 1, 2 and 3 are each well separated in the colour-coded two-dimensional space. The results provide clear evidence that the receptor sequences from cases of cross-species infection are well separated from those of other infection cases. From these observations, we conclude that receptor differences are a major contributing factor to the potential of a specific viral strain to cross species barriers for transmission. In other words, the species dependence of infection is indirectly related to the receptor sequence similarity. This finding implies that once the receptor sequences of the primary reservoir and possible hosts are known, we might be able to predict the likelihood of viral disease transmission. The accuracy of these classifications can be judged Figure 1 Similarity scores of among groups. Three kinds of pairwise similarity scores ( g S i,1 , g S i,2 , g S i,3 ) are plotted in two dimensional space to show clear differences among groups. Groups 1, 2 and 3 are each well separated; the results show clearly that the receptor sequences from cases of cross-species infection are well distinguished from those of other infection cases.
by subsequent assessment of cases of actual zoonotic transmission to humans.
Our analysis revealed significant differences in receptor similarity between infection and non-infection cases.
The similarity values, and the experimentally determined group categories were fed into a statistical discriminant analysis to logically predict infection (or zoonosis, in the case of human infection). As described in the Materials and Methods section, the values D i 2 (i = 1, 2, 3) were calculated from the data in the Table 1 to yield results of a specific discriminant analysis.
The statistical discriminant analysis was verified using a test set of four viruses that were deliberately excluded from the training set. The viruses whose groups were predicted using the discriminant analysis are shown in Table 2 . The first virus, feline immunodeficiency virus (FIV), uses Felis catus (domestic cat) as its primary host and CD4 as its receptor. According to the literature [22, 23] , FIV infection of humans is rare but has been reported. Our method categorized this case as nearinfection (G = 3). The second virus, classical swine fever virus, is known to be non-zoonotic and was classified as such by our method (G = 2). Thirdly, the encephalomyocarditis virus infects S. scrofa but has been known to cause sporadic infections in H. sapiens; it was classified as group 1 (G = 1) by our method. Finally, the Lass virus is known to be zoonotic and was classified as group 1 (G = 1) by our method.
In Table 2 , the hydrophilic similarity scores (S 1 ) show less consistency, comparing to the hydrophobic scores (S2), with the predictive values (G). From the result, it could be said that the hydrophobic characteristics of receptor sequence might be the key contributor to the prediction. However, this observation should only be carefully interpreted because the variables (S1, S2, S3) are complementary in the statistical process.
Our analysis of viral receptor sequences shows that the likelihood of viral infection correlates with the similarity in sequence of the primary and host receptors. This result is not surprising, because viral infection also inversely correlates with the inhibition of viral coat protein binding to the receptors. Importantly, we were able to establish this relationship at the amino acid sequence level, allowing for the prediction of possible human infection at an early stage of a viral outbreak, before the structures of viral coat proteins and receptors are known. Therefore, once the receptor sequences of primary reservoir and the potential host are known, the likelihood of viral infection can be predicted if the virus does not mutate too abruptly. Our simplistic approach needs further refinement because the complex processes of host tropism of viruses are largely ignored in our current method. For example, the process of host immune response could be included for better prediction of zoonosis. Although further refinements of our methods and analyses of larger databases are needed, this simple conceptual approach may be useful, even now, as a basic tool for the classification of zoonosis of new viral species.
Viral infection requires the insertion of viral genes into host cells. Such a process begins with the binding of coat proteins to host receptors, and in some cases, coreceptors [24] . Ten RNA viruses (seven zoonotic viruses and three non-zoonotic viruses) were investigated. Viruses that use co-receptors were excluded from the study. Receptor sequence data for each virus were collected from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/, and the research literature was examined to determine the specific species tropism of each virus [[25] , http://www.ictvonline.org/]. The viruses, host species, receptors, receptor sequences, and infection information for each host are shown in Table 1 . We selected viruses that are each a representative of a different family, with different primary reservoirs. Viruses with unknown or poorly defined host receptors (particularly human receptors) were excluded from the study. Orthologues of the where N tot is the total number of amino acids in one sequence string; n tot is the total number of matched amino acids in the sequence; N phi and N pho are the numbers of hydrophilic and hydrophobic amino acids in the sequence, respectively; N others is the number of deleted amino acids (gaps/insertions in sequence) plus the number of amino acids with undetermined properties; n phi and n pho are the numbers of hydrophilic and hydrophobic amino acids matched, respectively; and g S i,1 is the similarity score for hydrophilic residues of the i th row of infection group g. Here, there are only three groups: g = 1, 2, or 3, which are the infection, non-infection, and near-infection groups, respectively. The interspecies infection information was identified and classified among three infection states: group 1 (g = 1) represents infection; group 2 (g = 2) represents non-infection; and group 3 (g = 3) represents nearinfection. By definition, if a group 1 species pair includes humans, then the infection is zoonotic. Decisions for grouping were made on the basis of experimental and epidemiological studies reported in the literature [4, [30] [31] [32] [33] .
The variables (shown in Table 1 ) were arranged in matrices to allow for discriminant analysis, a method of multivariate analysis that can determine the group related to variables [34] . Each group has three columns and l, m, or n rows, depending on the numbers of variable sets. Here, the matrix for group 1 is defined as:
Similarly, 2 S and 3 S were defined as: All of the related variables were tabulated as shown in Table 1 . From the above matrices, three averages were found for each group:
The averages 2 S m,1 , 2 S m,2 , and 2 S m,3 for group 2 and 3 S n,2 , 3 S n,2 , and 3 S n,3 for group 3 were calculated similarly.
Three covariant matrices were constructed as:
1 C = ⎡ ⎣ 1 C 1,1 1 C 1,2 1 C 1,3 1 C 2,1 1 C 2,2 1 C 2,3 1 C 3,1 1 C 3,2 1 C 3,3 ⎤ ⎦ 
where X 1,i = 1 S i,1 − 1 S l,1 2 X 2,i = 1 S i,2 − 1 S l,2 2 and X 3,i = 1 S i,3 − 1 S l,3 2 Similar treatments yielded the 2 C and 3 C matrices, resulting in three covariance matrices ( 1 C, 2 C, and 3 C). We then created a pool-within-class covariance matrix P. If we define L = 3l-1, M = 3m-1, and N = 3n-1, then: P = ⎡ ⎣ P 1,1 P 1,2 P 1,3 P 2,1 P 2,2 P 2,3 P 3,1 P 3,2 P 3,3 ⎤ ⎦ where P 1,1 = ( 1 C 1,1 L + 2 C 1,1 M + 3 C 1,1 N)/(L + M + N) P 2,2 = ( 1 C 2,2 L + 2 C 2,2 M + 3 C 2,2 N)/(L + M + N) P 3,3 = ( 1 C 3,3 L + 2 C 3,3 M + 3 C 3,3 N)/(L + M + N) P 1,2 = ( 1 C 1,2 L + 2 C 1,2 M + 3 C 1,2 N)/(L + M + N) P 1,3 = ( 1 C 1,3 L + 2 C 1,3 M + 3 C 1,3 N)/(L + M + N) P 2,3 = ( 1 C 2,3 L + 2 C 2,3 M + 3 C 2,3 N)/(L + M + N) also P 2,1 = P 1,2 P 3,1 = P 1,3 P 3,2 = P 2,3
We next found the inverse matrix I, where I = P -1 . Because there were three groups in our study, we predicted the likelihood of infection for a virus of unknown infection condition by calculating the Mahalanobis distance (generally D 2 = d 1 × C -1 × D i ).
Here, expansion of D 2 yielded three equations:
D 1 2 = σ 1,l I 11 + σ 2,l I 21 + σ 3,l I 31 σ 1,l + σ 1,l I 12 + σ 2,l I 22 + σ 3,l I 32 σ 2,l + σ 1,l I 13 + σ 2,l I 23 + σ 3,l I 33 σ 3,l D 2 2 = σ 1,m I 11 + σ 2,m I 21 + σ 3,m I 31 σ 1,m + σ 1,m I 12 + σ 2,m I 22 + σ 3,m I 32 σ 2,m + σ 1,m I 13 + σ 2,m I 23 + σ 3,m I 33 σ 3,m D 3 2 = σ 1,n I 11 + σ 2,n I 21 + σ 3,n I 31 σ 1,n + σ 1,n I 12 + σ 2,n I 22 + σ 3,n I 32 σ 2,n + σ 1,n I 13 + σ 2,n I 23 + σ 3,n I 33 σ 3,n where σ 1,l = S 1 − 1 S l,1 σ 2,l = S 2 − 1 S l,2 σ 3,l = S 3 − 1 S l,3 σ 1,m = S 1 − 1 S m,1 σ 2,m = S 2 − 1 S m,2 σ 3,m = S 3 − 1 S m,3 σ 1,n = S 1 − 1 S n,1 σ 2,n = S 2 − 1 S n,2 σ 3,n = S 3 − 1 S n,3
where S 1 , S 2 , and S 3 are the input variables; here, they were similarity variables of a virus of an unknown infection group.
Group classification (G) was identified using the criterion:
For example, if D 1 2 is the minimum among three values from the above set of three equations, then G = 1; i.e., "group 1" is the group classification. To automate the mathematical process described above, we developed a Java computer program named ZOO. To evaluate the accuracy of our method and software, we analysed a test data set (described in the Results & Discussion section). A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings BACKGROUND: Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. Clinical diagnostic assays targeted to nucleic acid (NA) markers are becoming an increasingly important part of the clinician's toolbox. Many disease states are difficult to diagnose due to the lack of specific and well-characterized biomarkers in an accessible specimen. These generalizations apply in particular to infectious disease diagnostics. The clinical signs of infection are often nonspecific (e.g., inflammation or fever) and may originate from many possible sources, yet the treatments are more often specific and require an accurate diagnosis to be effective. There are many infectious diseases endemic in LRS where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to effective treatment, in part because of co-morbidities that confound a differential diagnosis. These diseases include malaria, human immunodeficiency virus (HIV-1), tuberculosis (TB), influenza, and many others. [1] Millions of lives are lost and a huge morbidity burden incurred through inadequate diagnosis and treatment of these diseases. [1] In many cases the need for rapid diagnostics appropriate for these LRS is so severe that mediocre performance tests such as RDT are preferred to less accessible but better performing NA tests. [2] Clearly, any technology that can increase the practicality and availability of NA assays in LRS could have a significant impact on global public health.
Nucleic acid detection, to date, has mainly been confined to wealthy, developed countries or to the large centralized facilities in the developing world that can marshal the resources required to perform these techniques. Like many molecular diagnostic assays, nucleic acid amplification techniques (NAATs) typically require a significant investment in equipment, training, and infrastructure. Economic and infrastructural realities dictate that diagnostics for the developing world need to be foremost inexpensive; but also, accurate, reliable, rugged, and suited to the contexts of these lowresource settings (LRS). [3] [4] [5] Recent guidelines published by the World Health Organization recommend that diagnostic devices for developing countries should be ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipmentfree, and Deliverable to end users. [6] In some diagnostic contexts in LRS, rapid diagnostic tests (RDT) based on the immunochromatography strip (ICS) fit the ASSURED model, albeit with limited sensitivity and specificity. [7] [8] [9] NAAT assays that use polymerase chain reaction (PCR) amplification are capable of providing excellent sensitivity and specificity but generally fail to meet the ASSURED guidelines for affordability, rapidity and robustness, equipment-free operation, and deliverability. [10, 11] Appropriate, low-cost, equipment-free, pathogen-specific NA marker assays that characterize medical care in much of the developing world remain unavailable in LRS.
One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity of PCR amplification. PCR is inherently impractical in LRS where reliable electrical power, complex equipment, training, reagent storage, quality programs and clean water, are intermittent or absent. [1, 12] Recently, there have been significant developments in a class of NAATs that do not require temperature cycling. [13] [14] [15] [16] A comprehensive review of these techniques, and their application in LRS has recently been published. [17] These isothermal amplification techniques vary in amplification temperature and duration, as well as complexity of reagents required-and many are proprietary-but all have the potential to be simpler and require less complex equipment than PCR-based assays. These methods use a variety of reaction principles to specifically amplify NA targets through isothermal melting, exponential amplification and intermediate target generation; and which, in several cases, can be detected directly without the need for an instrument. [17] [18] [19] [20] Nevertheless, almost all investigators and manufacturers currently use some type of electrically powered equipment to achieve and maintain the temperature required for amplification, although this equipment can be much simpler than the typical PCR thermocycler. This inherent simplicity makes isothermal amplification more appropriate for diagnostics in LRS.
One of the letters in the acronym ASSURED -the guideline for providing diagnostics to LRS -represents ''equipment free.'' We are currently developing a non-instrumented nucleic acid (NINA) platform that requires no detection instrument, no electrical power, no batteries, and no external reagents. We believe this can be achieved by combining isothermal amplification with a novel method for generating the required temperature profile without electrical power in a simple disposable that contains the lyophilized assay reagents. Our first prototype of this platform uses loop-mediated amplification (LAMP) [21] as the model for an isothermal amplification technique and malaria as a model diagnostic target. The amplification protocol requires incubating the reaction mixture at ,65uC for at least 60 minutes. This temperature requirement is sufficiently flexible that small excursions (+/21.5uC) around this target are tolerable. [22] [23] [24] [25] [26] LAMP (and several other isothermal techniques) have been shown to far less sensitive to inhibitors than PCR, to the point where direct assay of whole blood and other unpurified specimens is feasible. [18, 19] In those cases, no power or instruments are required for NA purification, as is the case with PCR. In addition, recent advances in protein stabilization make it likely that the reagents can be dried-down in the reaction tubes with sufficient stability to avoid the need for a cold-chain during delivery and storage. Thus, another power consuming ''instrument'' is eliminated. We have not yet attempted to package all of these features and advances into a single prototype device; however, the successful demonstration of electricity-free temperature-controlled heating in a disposable format reported here is an important first step toward the long-term goal.
The prototype NINA platform exploits exothermic chemical heating, as used in ''ready-to-eat'' meals and camping hand warmers. Table S1summarizes the prior history of prototype development. Hatano and coworkers recently described a crude heater that was able to perform a qualitative LAMP assay for anthrax using off-the shelf pocket hand warmers and a Styrofoam box. [27] Dominguez et al. used a similar container with an unspecified phase change material to maintain a stable incubation temperature for a commercial interferon gamma release assay at 37C (although the heat source was conventional). [28] While these interesting approaches are compelling in their simplicity, the bulky apparatus displayed slow warm-up (.30 min.); and for LAMP, significant temperature variation within incubation time, and a lack of run-to-run repeatability was observed. To meet the performance goals implied in the ASSURED guidelines, an optimized heating unit should be engineered to eliminate or minimize all sources of variation. When combined with the temperature-moderating characteristics of engineered phase change materials (EPCM), we demonstrate that an engineered exothermal chemical heating unit can produce a consistent constant-temperature incubator for isothermal NA amplification suitable for a variety of isothermal techniques.
Heat Production and Temperature in the NINA Heater Ten replicate runs of the optimized prototype displayed minimal variation in temperature from run to run within the reaction tubes ( Figure 1 ). The heater reached the optimal incubation temperature in 15 minutes, and maintained the target temperature with minimal drift over 60 minutes. (Drift from minimum to maximum temperature within run, mean over all runs = 2uC.) Comparison of the temperature plots for the CaO, EPCM, and reaction tubes in Figure 1 to Figure 1B in Hatano et al. [27] illustrates the beneficial effect of having the EPCM component in the heater. The CaO temperature traces show rapid and poorly controlled heat generation, with maximum temperatures exceeding 100uC. The traces of the EPCM at the interface with the CaO have a pattern similar to the CaO, but the initial temperature excursions are reduced in magnitude, and the plots are far more repeatable. Finally, the reaction tubes display only a uniform ramping to the target temperature followed by a prolonged stable isothermal phase. The temperature in the NINA reaction appears more uniform than that shown by Hatano et al. [27] for their hand-warmer device.
These results evince the potential of EPCM in an optimized design for controlling exothermic reactions in a simple NINA. This level of temperature control is important to enable conformance to the ''sensitive'', ''specific'', and ''robust'' aspects of the ASSURED guidelines. Once the abundant heat from the CaO reaction begins to melt the EPCM, the additional heat produced by the exothermic reaction is converted into the latent heat of fusion of the EPCM. Thus, the temperature in the EPCM remains constant at the selected melting temperature until the solid to liquid transition is complete (provided heat transfer within the EPCM is rapid). Once the CaO reaction has reached equilibrium, the energy stored as latent heat keeps the two-phase EPCM at the target temperature until complete solidification.
In our optimization work we observed that the purity of the CaO need not be high, although it should be consistent to yield consistent heat profiles (data not shown). The ability to use less pure CaO is important for minimizing the cost per amplification, addressing the ''affordable'' aspect of the ASSURED guidelines.
Other key physicochemical parameters of raw CaO (particle size, particle porosity, presence of unoxidized calcium carbonate ''grit'', etc.) that result from variation in kiln calcination of limestone [29] (the industrial manufacturing process) also must be kept consistent for consistent heat profiles. However, we were able to produce precise heat profiles in our prototypes with commodity grades of CaO. This makes the only disposable materials (CaO, water, and PCR tubes) in the device very inexpensive. The reaction of CaO and water can be tuned somewhat to control the steepness of the temperature ramp and the maximum temperature for a given reaction chamber, although flexibility and precision is greatly improved by including the EPCM.
The EPCM used here is tunable for many of its important characteristics (melting temperature range, specific heat capacity, thermal conductivity, etc.) making this device a flexible incubation platform potentially applicable to a number of isothermal amplification techniques. When evaluated by differential scanning calorimetry, the EPCM melts over a range of temperatures around the target (62uC), and displays some hysteresis in the phase change, presumably due to polydispersity in polymer chain length and supercooling of the EPCM (personal communication from Renewable Alternatives). It is unclear at this time how this behavior contributes to variation seen in the results of the LAMP assay; however, the manufacturer of the EPCM is confident that further development of the EPCM for this application will mitigate this behavior. The EPCM is a fully hydrogenated fat product, so it is resistant to environmental oxidation and should be very stable. While the EPCM is not currently as readily available as CaO, and is not a commodity product like CaO, similar materials have been used in consumer products in the US. These EPCMs are made mainly from bio-based fats -namely beef tallow, palm oil, coconut oil and soybean oil -so local, low-cost production of the EPCM in the developing world should be feasible.
Portable energy for heat production could, of course, be supplied with conventional batteries, so a comparison seems appropriate. A cost analysis indicates that on a per calorie per test basis, using CaO as a thermal battery is several times less expensive than mass-produced, disposable, dry-cell batteries. Costs are scaled by the projected number of analytical runs possible and include both energy source and control hardware. CaO disposables are single use, while dry cells are expected to last five runs based on their energy density (four D-cells would be required). Two grades of CaO (reagent grade and soap grade), with an EPCM are compared to three possible dry-cell implementations (with an EPCM, with microprocessor closedloop control, with thermostat closed-loop control). With a projected cost per run of US$0.56, the soap-grade CaO/EPCM combination is clearly the least-expensive alternative (compared to $1.40, $1.17, $1.21, and $1.16 for reagent-grade CaO/EPCM, Dcell/EPCM, D-cell/microprocessor, and D-cell/thermostat, respectively.) Costs were estimated from MSRP. Increased value of CaO over the alternatives could be realized at increased production volumes. Any special disposal or recycling required does not seem any more onerous than what is required for common batteries.
The data shown here were not gathered under any stringent external environmental control; therefore, given that testing was performed in an air-conditioned laboratory, it is likely that the system was not challenged in the same way as it would be at its intended point of use. The wide external temperature ranges found in LRS could significantly change the ramp time and/or duration at the desired temperature of the heater, possibly significantly, but the characteristics of the EPCM will ensure that the desired temperature is held for some period of time, regardless without calibration to the ambient conditions or closed-loop control. First principles of heat transfer dictate that the effects of ambient on ramp time and/or duration should be greatest when the desired temperature is furthest from ambient. Thus, the problem should be appropriately non-dimensionalized to identify states of similitude. We plan to explore these phenomena and to evaluate their effects once we have improved our understanding of the intrinsic variation in the assay chemistry sufficiently to evaluate those effects. This evaluation will include trials under actual field conditions.
Representative images of the qualitative results ( Figure 2 ) shows 1) the NINA heater is capable of supporting LAMP, 2) that samples incubated in the NINA heater give results that are virtually identical to those incubated in parallel in the GeneAmpH 9600. For both incubators the turbidimetric readout method (Figure 2A ) is difficult to interpret, but turbidity due to accumulating LAMP product is observed (relative to the notemplate control, or NTC). The fluorescence of the Calcein reagent when illuminated with a UV lamp ( Figure 2B ) is more easily seen as an increase in intensity (relative to NTC) for the dilutions that are .1 pg/mL. Note that there is some background Figure 3B ) reveals a mean difference (ESE -NINA) of 226 FIU, no dependence of difference on mean, and all differences lie within the 62s interval that indicates the differences are random, not systematic. Although these experiments were intended to quickly assess the agreement between heater types and were not designed to rigorously define the dose response relationship of a nascent assay, closer inspection of the FIU for each concentration ( Figure  3A ) reveals a general increase in response with increasing dose, within the experimental noise limits of this admittedly small sample set. As with the qualitative assay demonstration, considerable background fluorescence was observed in NTC reactions in both heaters ( Figure 3A and 3B) .
These results clearly show that the NINA heater can incubate isothermal reactions predictably and precisely with no electricity and without any form of closed-loop control. We also demonstrate that it can be used for LAMP assays, with no discernable difference when compared to two reference heaters, the GeneAmpH 9600 and the ESE-Quant Tube Scanner. There is a bias between the NINA heater and the ESE-Quant (NINA higher), but this is not a significant finding considering we are comparing FIU without any assay calibration. This bias would be easily removed by applying a standard curve. Although we did not intend to rigorously qualify the LAMP assay for malaria here, these results suggest that a quantitative assay with a clinically significant lower limit of detection and three decade dynamic range might be possible with further development of the protocol. Planned work will comprehensively compare incubation of several isothermal assays with the NINA heater to incubation with conventional, electrically-powered instruments by many metricssensitivity, specificity, accuracy, precision, and other standard figures of merit must all be assessed before equivalence can be rigorously inferred. However, these prelimary results are very encouraging.
We have also explored heaters with temperature profiles suitable for other isothermal amplification techniques requiring different incubation temperatures, e.g., the Exponential Amplification Reaction (EXPAR), Nicking Enzyme Amplification Reaction (NEAR), or Recombinase Polymerase Amplification (RPA), could be integrated with this method. These prototypes are not significantly different in form, but use different EPCMs, and in one case a different exotherm. A CaO heater with a different EPCM formulation has been shown to yield a temperature profile suitable for EXPAR with a nominal temperature of 55uC ( Figure 4A ). Evaluation with EXPAR reactions are in process. We have also explored a similar heater approach with sodium acetate (NaAc, Figure 4B ). Hand warmers based on the crystallization reaction of NaAc are common. In a purified form, at typical ambient temperatures, liquid NaAc is thermodynamically unstable but kinetically stable due to the absence of nucleating sites for crystal formation. The application of a mechanical shock initiates the exothermic crystallization, and when mixed as a 25% aqueous solution the phase change occurs repeatably at ,37uC. In this system, NaAc acts as both the exothermic reactant and the EPCM. This system has the advantage of being regenerable (immersion of the NaAc in boiling water is sufficient). For isothermal amplification methods operating at temperatures below 45uC as well as for other diagnostic applications requiring heating (e.g., smart-polymer-based analyte pre-concentration [30] ), NaAc is the preferred exothermic/ phase change system. These results establish that the heater is a flexible platform for a number of isothermal detection techniques.
We have shown results for an instrument-free LAMP assay with a simple qualitative visual readout. As operated here, LAMP is an exponential rate assay being assessed with an endpoint measurement. Thus, the timing of reaction interrogation and/or a reliable ''stop'' reaction are required for quantitative precision. If quantitative results are required, improvements to the entire assay system to facilitate precise timing will be necessary. This could include, for example, a different heater-lid or incubation-vessels to facilitate access, or a ''reading window'' in the heater to enable visual interrogation while the vessel is still in the heater. An elevated temperature ''stop'' (.80uC) is generally used for LAMP. In our experimental work here, we used an electrical heat block for this purpose; however, this could be accomplished with the electricity-free heater by the inclusion of a parallel heating unit at a higher temperature (essentially, by including a second incubation chamber that uses a different EPCM, or no EPCM at all). Alternatively, a chemical ''stop'' could be developed, or a boiling water bath could be kept at hand. Other assays perform best with a pre-amplification, high-temperature denaturation step (''hot start''). A second incubator chamber could facilitate this feature even more readily.
These data were gathered on contrived samples diluted in buffer. It has already been demonstrated that LAMP assays can be performed on clinically relevant specimens without NA extraction/purification and without a pre-amplification, high-temperature denaturation step. [31] [32] [33] [34] [35] Recent results of an HIV assay on the NINA platform with clinical samples from HIV-positive infants will be reported elsewhere.
Furthermore, neither turbidity nor Calcein reactions are sequence-specific signals-as a result non-specific amplification will also produce a strong signal-a possible cause of the NTC background fluorescence noted above. Greater analyte specificity should be possible by incorporating a fluorescent molecular beacon probe specific to an internal region of the target amplicon, thus minimizing non-specific signal. [28] Alternatively, the amplified product could be the input to a lateral flow strip test with a visual readout. [36] [37] [38] [39] Any of these potential improvements should be approached with a secondary aim of minimizing the potential for amplicon contamination from previous tests. Wherever possible, opening of the amplicon container after amplification should be avoided. This may be challenging if molecular beacon quenchers need to be added, or aliquots for ICS testing need to be removed. Regardless of how the system and assays are improved, we have clearly demonstrated that the NINA heater is an effective device that can facilitate the electricity-free amplification of NA using an isothermal technique.
There are several applications of this technology that could have an impact on diagnostics for LRS. One application is as a modular amplification unit where a sample and the required reagents would be introduced to the heater and amplified product withdrawn for subsequent analysis by any simple detector. In this embodiment a standard PCR tube can be the reaction chamber and could be used later as a cuvette for fluorometric analysis to resolve the presence of amplicons. This would free the user from the high power requirements of electrical heating but would still require some sort of detection instrument or device with its attendant requirements. One could also imagine how a properly tuned, stand-alone heater unit could be applicable to any field analytical or preparative method that requires a constant heat source; e.g., cell lysis or temperature-responsive polymer mediated concentration. More compelling is the potential of the NINA heater as the core component of a stand-alone assay kit, capable of providing a result without external electrical power, a reader instrument, or any complex ancillaries. Such a device might include the NINA heater, reaction chambers containing lyophilized reagents, sample metering devices, a readout chamber or lateral flow strip for visible interrogation, and an LED ''penlight'' for fluorescence excitation (if required). We envisage versions of the kit that are fully disposable (for high-value applications in developed countries such as for home testing and for first-responder biothreat detection) and partially reusable (primarily for LRS use). Most of the components necessary to create such a NINA kit already exist. We are currently working to combine them into a field-ready, instrument-and electricity-free, sample-toresult, molecular diagnostic test system ( Figure S1 ).
We have demonstrated the ability of an optimized NINA heater prototype, based on exothermic chemical reactions and EPCM, to support isothermal NA amplification assays and established its equivalence to commercially available PCR instruments. The disposable heater described is a component of an instrument-free point of care molecular diagnostics system under development. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that enables electricity-free NA testing for many important analytes. Replicate temperature profiles display minimal variation between runs and far less variation than any similar devices, highlighting the advantages of including an EPCM in the design. Versions of the prototype for several isothermal techniques have been presented, clearly evincing the potential of the NINA heater.
In the NINA heater for LAMP, we used the exothermic reaction of calcium oxide (CaO, or quicklime; Science Stuff, Inc., Austin, TX, USA, Cat # C1450) and water to generate the necessary heat. To keep the isothermal device within the temperature band required for LAMP, the reaction chambers were surrounded with an engineered fat-based compound with a high specific heat capacity and specific melting range centered around 65uC (Renewable Alternatives, Inc., Columbia, MO, USA). While several prototype heater designs have been explored, the optimized heater uses an off-the-shelf insulated food storage container (a ''thermos'') to provide an insulated housing with two chambers ( Figure 5 ). The bottom chamber contains the exothermic reaction, and the upper chamber contains the EPCM and reaction wells. To facilitate directed heat transfer to the reaction wells, an aluminum ''honeycomb'' material (Plascore, Inc., Zeeland, MI, USA) was added to the upper chamber prior to introduction of the EPCM. The machined reaction wells, sized to closely fit a standard 200-mL PCR tube, are embedded in the EPCM. Three reaction wells were used for most prototypes (one for a positive control, one for a negative control, and one for an unknown specimen); however, the existing prototype could easily be modified to accept several times this number without significant loss of performance, based on the available space in the EPCM and first principles of heat transfer. An inexpensive spring timer (manufacturer's suggested retail price [MSRP] <10 US$) with an audible ''ready'' indicator was affixed to the lid of the heater unit for added electricity-free functionality.
LoopampH DNA LAMP kits were purchased from Eiken Chemical Co. Ltd (Tokyo, Japan, Code No.: LMP206). The Eiken LoopampH Fluorescence Detection Reagent (Code No.: LMP221), a Calcein-based reagent that indirectly indicates the progress of DNA amplification in the LAMP reaction, was used for fluorescence experiments. LAMP primer sequences for P. falciparum (Integrated DNA Technologies , Coralville, IA, USA) were as described by Poon et al. [40] Genomic DNA from P. falciparum for preparing contrived samples was obtained from the PATH laboratory specimen collection.
Either an ESE-Quant Tube Scanner (ESE GmbH, Stockach, Germany) or a PE GeneAmpH Thermocycler 9600 (Applied Biosystems, Carlsbad, CA, USA ) was used as a quantitative reference instrument for temperature incubation. The ESE-Quant Tube Scanner also served as a reference for quantitative fluorescence measurement. A SpectraMax M2 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) was also used (as noted in individual experiments). Physitemp IT-23, T-type thermocouples (Clifton, NJ, USA) and an Omega DaqPRO 5300 Data Recorder (Omega Engineering, Stamford, CT, USA) were used to monitor temperature.
Thermocouples were installed in a reaction well (below its microcentrifuge tube) at the bottom of the chamber containing the EPCM and in the exothermic reaction chamber for experiments performed to characterize the temperature profiles of the heaters. The temperature acquisition rate was 1 Hz. Initial experiments were focused on refining the dimensions of the heater, optimizing the quantity and quality of CaO and water, and testing EPCM formulations-with the goal of minimizing initial pre-heating time and variability during the specified incubation period. In the optimized device, 20 gm of CaO and 6.8 mL of water were added to the bottom chamber and mixed by rotary stirring for five strokes to initiate the heating and then the components were assembled as discussed above.
To verify that the device could incubate a LAMP assay, the Eiken kits were used as per package insert instructions, except where noted below. Clinically relevant dilutions of genomic DNA were made in Eiken kit buffer to yield the DNA concentration and approximate parasite count noted for each experiment. All dilutions were prepared as single solutions and then aliquoted across treatment conditions to minimize preparation variation. No template controls (NTC) were prepared first and immediately sealed to reduce the possibility of contamination. Mineral oil was layered on the tops of the samples to minimize evaporation. All reactions were incubated at 63uC.
Qualitative readout experiments were performed both with and without the Calcein reagent to determine if turbidimetric readouts were possible. To compare the performance of the NINA heater to a reference heater, these experiments were also performed in parallel with reactions incubated in both the test device and in the GeneAmpH thermocycler, programmed for a constant incubation at 63uC.
Quantitative fluorescence experiments were performed in parallel with reactions incubated in both the test device and in the ESE-Quant Tube Scanner, programmed for a constant incubation at 63uC. For NINA incubated reactions, LAMP was terminated at the time (,36 min.) when the signal from the parallel reactions on the Tube Scanner began to indicate detectable amplification to avoid signal saturation in all dilutions. Termination was accomplished by flash chilling and later inactivating the reaction by heating at 80uC for 5 minutes. The fluorescence signals of the NINA incubated samples were then read on the SpectraMax M2 plate reader with l ex = 485 nm and l em = 515 nm. Figure S1 The workflow of a proposed NA amplification assay kit. The kit will be an instrument-free, electricity-free nucleic-acid amplification test that is compatible with whole blood, is temperature stable and contains contamination. 1) Initiate NINA heater by installing heater cartridge (a) into insulated housing (b), add EPCM module (c) and lid (d). 2) Set up for assay by opening single assay subkit. 3) Sample blood to calibrated line on collection capillary. 4) Transfer blood and blister contents to ''S'' tube and prefilled diluent to ''NC'' and ''PC'' tubes and mix all. 5) Amplify. Verify temperature ''ready'' indication on the NINA device through transparent view port in the lid, remove the lid, add the three tubes to the NINA heater, and replace lid. Incubate 45 minutes. Verify temperature is still in range through transparent view port (process control). 6) Quench to all three tubes by pushing cap to burst frangible seal and transfer ,10 mL diluted quencher to the amplified mixture. (TIF)  Quantitative Subcellular Proteome and Secretome Profiling of Influenza A Virus-Infected Human Primary Macrophages Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X(7) receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages. Influenza A viruses are negative-stranded RNA viruses that are capable of infecting a variety of avian and mammalian species. These viruses are responsible for the annual epidemics that cause severe illnesses in millions of people worldwide. Influenza A virus and secondary bacterial infections can cause lethal pneumonia and encephalopathy especially in elder people. Host defence against influenza A virus infection is initiated by the innate immune system. The principal effector cells involved in innate immunity are macrophages, and dendritic cells (DC) that kill microbes through phagocytosis, present antigens to T cells, and produce cytokines. These innate immune responses are essential for the development of later adaptive immune responses, which provide specific cell-mediated and humoral protection, and are often necessary for a complete clearance of infection.
In viral infections innate immune responses are initiated when viruses or their genetic material is recognized by cellular pattern recognition receptors (PRRs) of the innate immune system [1] . This recognition results in an interferon (IFN)-a/b-mediated antiviral response, as well as activation of pro-inflammatory response and programmed cell death, apoptosis, of infected cells. PRRs involved in activation of antiviral response include Toll-like receptors (TLRs) and Retinoic acid-inducible gene I-like receptors (RLRs) and the coordinated activation of TLRs and RLRs results in proper activation of antiviral response during influenza A virus infection [2, 3] . Influenza A virus infection of human macrophages also results in production of pro-inflammatory cytokines IL-1b and IL-18 [4, 5] . Both of these cytokines have to be cleaved by cysteine protease caspase-1 to generate the secreted, biologically active forms of these cytokines [6] . Caspase-1 is in turn activated in a cytosolic protein complex called inflammasome [7] . The inflammasomes consist of an adapter molecule called apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC) and a PRR that belongs to either the nucleotidebinding and oligomerization domain like receptors (NLRs) or the PYHIN receptor gene family. Caspase-1 activating structures include NLRP3, NOD2/NLRP1, NOD2/NLRP3, IPAF/NAIP5, and AIM2 inflammasomes [8, 9] . Recent studies in experimental mice models have shown a critical role of NLRP3 inflammasome in the host response against influenza A virus infection [10] [11] [12] . These studies highlight the importance of IL-1b and IL-18 as mediators of host response to influenza A virus infection. However, the exact mechanism of inflammasome activation during viral infection is not known.
Proteomics combined with bioinformatics has emerged as an important tool to extract detailed information of cellular signaling mechanisms [13] . With modern mass spectrometry (MS) -based approaches it is possible to identify and quantify thousands of proteins from cellular samples, as illustrated by Luber et al. who studied subset-specific viral recognition in dendritic cells [14] . Most of the large scale quantitative proteomics experiments, however, have focused on studying changes in protein abundances in whole cell lysates or individual organelles [14, 15] . The use of subcellular proteomics provides a deeper insight into cellular events as protein abundancies can be studied on the level of different subcellular compartments and also protein translocations between different cell parts can be detected [16, 17] . Moreover, pathway and network analyses can provide mechanistic insights by subsequently linking the proteins found to be differentially regulated to the underlying cellular functions and other key players known to be involved in these events. Here, we have used quantitative subcellular proteomics combined with bioinformatics to provide a global view of host-pathogen-interactions during influenza A virus infection of human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection.
For subcellular proteome and secretome analysis (Fig. 1A) human primary macrophages were first infected with influenza A virus for 6 h, 12 h and 18 h (intracellular fractions) or 6 h, 9 h and 12 h (secretome). After this the cells were fractionated into mitochondrial, cytoplasmic and nuclear fractions, and macrophage growth media were collected for secretome analysis. The enrichment of mitochondrial, cytoplasmic and nuclear proteins into different fractions was confirmed with Western blots (Fig. 1B) .
Protein identification and quantification from each subcellular fraction and secretome was done using 4plex iTRAQ (isobaric tag for relative and absolute quantitation) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Fig. 1A) . A total of eight iTRAQ sample sets were analysed, including two biological replicates of mitochondrial, cytoplasmic and nuclear cell fractions as well as secretomes. Additionally, each iTRAQ sample set was analysed twice with LC-MS/MS to improve the quality of protein identifications and quantifications. Based on the LC-MS/MS data we identified 1999, 1423, 1230 and 627 proteins from the mitochondrial, cytoplasmic and nuclear cell fractions and secretome, respectively, with false discovery rates (FDR) of the sample sets varying from 0.5 to 1.6%. More than one thousand proteins were identified from more than one cell fractions and altogether we identified 3477 distinct proteins. From the identified proteins, we reliably quantified 2466 proteins, and of these proteins 1321 were differentially expressed in the intracellular fractions (fold difference $1.5 or #0.67) and 544 in the secretome (fold difference $3) as a result of infection ( Fig. 1C , Tables S1 and S2). To confirm iTRAQ quantification results, Western blot analyses were performed for a selected set of proteins ( Fig. S1A-D) .
To get an overview of our proteomic data the identified and quantified proteins were analysed further using different bioinformatics tools. The proteins were first classified using the gene ontology (GO) annotations of their known subcellular locations (Fig. 1D ) and biological functions (Fig. 2, Fig. S2 ). Most of the known mitochondrial proteins identified in this study were found from the mitochondrial fraction. In addition to mitochondrial proteins, most of the identified lysosomal and endoplasmic reticulum (ER) proteins were located in the mitochondrial fraction implying that these subcellular organelles are also enriched in the mitochondrial fraction. Proteins with a known cytosolic localization were most abundant in the cytoplasmic fraction. Many cytosolic proteins, however, also have a nuclear annotation which can be seen in our data as a large number of 'nuclear' proteins in the cytoplasmic fraction. In the nuclear fraction, proteins with a nuclear annotation were clearly the most abundant.
A large number of differentially expressed proteins were identified from the nuclear cell fraction at 12 and 18 h postinfection (Fig. 1C ). In the nuclear fraction 335 and 376 proteins were overexpressed, and 344 and 368 proteins underexpressed at 12 and 18 h post-infection, respectively. The overexpressed proteins contain several mitochondrial, ER, Golgi and cytosolic proteins. Functionally the overexpressed proteins form a heterogeneous group of proteins including several metabolism, gene expression, calcium ion binding, transport and signaling related proteins ( Fig. 2A) . Here, proteins related with gene expression are mainly nuclear or ribosomal proteins whereas proteins related with metabolism, transport, signaling and calcium ion binding originate from several different subcellular compartments. Proteins that are underexpressed in the nuclear fraction at 12 and 18 h postinfection are mainly known nuclear proteins. Almost 200 of them are involved in gene expression, especially in nucleotide metabolism and mRNA processing (Fig. 2B) . Host mRNA splicing machinery has been suggested to be important for influenza virus gene expression [18] , and interestingly, 40 proteins related with nuclear mRNA splicing were underexpressed in our data. Finally, our proteomics data showed that the amount of several nuclear histones increased clearly in the cytoplasmic fraction at 12 and
Influenza A viruses are negative-stranded RNA viruses that are capable of infecting a variety of avian and mammalian species. These viruses are responsible for the annual epidemics that cause severe illnesses in millions of people worldwide. The initial innate immune responses to influenza A viruses have to restrict virus spread before the adaptive immune responses fully develop. Macrophages are the key players of innate immune system and they have a central role in the activation of host response during viral infections. However, the host factors that are involved in the activation of innate immune response during influenza A virus infection are incompletely understood. Here, we have characterized in detail the nuclear, mitochondrial and cytoplasmic proteomes, as well as the secretome from influenza A virus-infected human primary macrophages to get a global view of host factors that are affected by the infection. Our approach allowed us to identify several novel host factors that contribute to innate immune system during influenza A virus infections. These include lysomal proteases cathepsins, P2X 7 receptor, src family tyrosine kinases as well as several dangerassociated molecular patterns.
18 h post-infection implying that there are major changes in the nuclear architecture after influenza A virus infection.
The iTRAQ quantitation results and gene ontology analyses show that the number of differentially expressed apoptosis-related proteins increases in macrophages as the infection proceeds (Fig. 2, Fig. 3A , Fig. S2 ). Our proteomics data showed, for example, that the amount of Bax decreases in the cytoplasmic fraction and increases in the mitochondrial fraction, and the amount of cytochrome c decreases in the mitochondrial fraction and increases in the cytoplasmic fraction upon infection (Fig. 3A) . These data together with Western blot analysis of cytochrome c (Fig. S1 ) are well in accordance with classical signs of mitochondrial apoptosis: translocation of Bax onto the mitochondria and leakage of cytochrome c into the cytoplasm.
APOPercentage apoptosis assay was used to confirm the progression of apoptotic events in influenza A virus infected macrophages (Fig. 3B) . No apoptotic cells were detected from control samples or influenza A virus infected samples at 6 h post-infection. At 12 h after the infection 19% of the cells were apoptotic, and at 18 h after the infection already 73% of the cells were apoptotic demonstrating the rapid progression of apoptosis at later timepoints.
It is known that influenza A virus infection triggers the production of IFN-a/b which is followed by the activation of IFN-inducible genes. Accordingly, a clear increase in the expression of several interferon-inducible proteins was seen in the cytoplasmic fraction of influenza A virus infected macrophages (Table S1 ). These proteins included three members of the interferon-inducible protein with tetratricopeptide repeat (IFIT) family, a group of proteins functioning as antiviral sensors that inhibit protein synthesis through translational arrest. Finally, the expression of several influenza A virus proteins such as hemagglutinin (HA), neuraminidase (NA) and nucleoprotein , cytoplasmic (C) and nuclear (N) fractions using mitochondrial, cytoplasmic and nuclear markers (voltage-dependent anion-selective channel protein 1 (VDAC1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H1, respectively). C. Numbers of reliably quantified and differentially regulated proteins (fold difference $1,5 or #0,67 for intracellular fractions and $3 for secretome) in each subcellular fraction and secretome at different timepoints. D. Gene ontology-based classification of all the proteins identified from intracellular fractions. Mitochondrial, cytoplasmic, nuclear, lysosomal and endoplasmic reticulum (ER) annotations for the identified proteins are shown. doi:10.1371/journal.ppat.1001340.g001 (NP) was clearly increased in the cytoplasmic fraction of the infected cells (Table S1 ).
Several proteins with mitochondrial, lysosomal and ER annotations were upregulated in the cytoplasmic fraction already at 6 h post-infection indicating rapid changes in these compartments upon infection (Fig. 1D ). Based on clustering analysis, these proteins belong to a group where the amount of protein in the cytoplasmic fraction increases rapidly at 6 h post-infection and starts to decrease at later time points after infection (Fig. 3C) . Surprisingly, the mitochondrial proteins in this group contain several components of the inner mitochondrial membrane including components of the electron transport chain and oxidative phosphorylation. Lysosomal proteins belonging to this cluster contain for example lysosomal proteases, cathepsins (Fig. 3C ).
To complete our high-throughput proteomics analysis, we performed secretome characterization of influenza A virus-infected macrophages. Influenza A virus infection of human macrophages clearly induced protein secretion already at 6 h postinfection, and more robust protein secretion was seen at 9 h and 12 h time-points ( Fig. 1C , Table S1 ). Our data shows that influenza A virus infection of macrophages activates both conventional as well as unconventional protein secretion [19] . Examples of the conventionally secreted proteins include e.g. C-C motif chemokine 3, C-C motif chemokine 24, complement C1q subcomponent subunit B, epididymal secretory protein E1, macrophage colony-stimulating factor 1, macrophage metalloelastase, matrix metalloproteinase-9, metalloproteinase inhibitor 2, and plasminogen activator inhibitor 1. The unconventionally secreted proteins include several danger-associated molecular pattern molecules (DAMPs) like amyloid beta A4 protein, annexins, galectins, heat shock proteins, high-mobility group box proteins (HMGBs), and S100 proteins (Table 1) . Enhanced secretion of amyloid beta A4 protein, galectin-3, HMGB1, and S100-A9 protein in response to influenza A virus infection was verified by Western blotting (Fig. S1D ). In addition to DAMPs, secretion of several other proteins known to be secreted through unconventional secretory pathway was enhanced. These include cystatins A and B, macrophage migration inhibitory factor, and thioredoxin. Interestingly, influenza A virus infection of macrophages also activated the secretion of Ras-related proteins Rab- 1A, Rab-2b, Rab-10, and Rab11, as well as several components of vacuolar ATPases (Table 1) . Furthermore, enhanced secretion of several lysosomal proteases, cathepsins, was seen in cell culture supernatants of influenza A virus-infected macrophages.
Cytoplasmic leakage of cathepsins has been associated with the activation of NLRP3 inflammasome-associated caspase-1 [20] . Our proteome analysis showed a rapid increase in the amount of cathepsins in the cytoplasm as a result of virus infection. In addition, secretome analysis showed that there is a major increase in the secretion of cathepsins and their regulators in macrophages infected with influenza A virus. To characterize the role of cathepsins in influenza A virus-induced inflammasome activation we performed Western blot analysis which showed that mature 25 kDa cathepsin B and 34 kDa cathepsin D are secreted simultaneously with biologically active p10 form of caspase-1 in response to influenza A virus infection (Fig. 4A ). Next, macrophages were infected in the presence and absence of cathepsin B inhibitors, Ca-074-Me and z-FA-fmk, after which IL-18 secretion was analysed by ELISA. Both Ca-074-Me (Fig. 4B ) and z-FA-fmk (Fig. S3A ) almost completely abolished IL-18 secretion in response to influenza A virus (Udorn/72) infection. This result was confirmed by Western blot analysis of concentrated cell culture supernatants which showed that the appearance of biologically active form of IL-18, IL-18 p18, is abolished in macrophages that have been infected with influenza A virus in the presence of Ca-074-Me (Fig. 4C ). In accordance with these results, we did not detect any caspase-1 p10 in cell culture supernatants after Ca-074-Me treatment (Fig. 4C ). In addition, Ca-074-Me clearly inhibited IL-18 secretion in response to another influenza A H3N2 strain, Beijing 353/89 (Fig. S3B) . Collectively, our data shows that cathepsin B activity is essential for inflammasome activation in response to influenza A virus infection.
In addition to inflammasome activation, cytoplasmic leakage of cathepsins is associated with cell death. Activation of caspase-3 is the hallmark of programmed cell death, apoptosis, and we have previously shown that caspase-3 is activated in human macrophages during influenza A virus infection [5] . To study the role of cathepsin B in influenza A virus-induced activation of apoptosis, macrophages were infected in the presence and absence of cathepsin B inhibitor, Ca-074-Me, and caspase-3 activation was analysed by Western blotting. Like caspase-1, caspase-3 is a latent zymogen, which is processed upon activation into smaller polypeptide chains: p17 and p12, which in turn form the bioactive enzyme. Influenza A virus (Udorn/72) infection clearly activated the formation of caspase-3 p17/p19 (Fig. 4D) . Furthermore, Ca-074-Me inhibited the formation caspase-3 p17/p19 in response to infuenza A viruses Udorn/72 (Fig. 4D) and Beijing 353/89 (Fig.  S3C ) confirming the role of cathepsins in the activation of apoptosis during infection. This effect was not dependent on diminished viral replication since Ca-074-Me treatment had only little effect on influenza A virus protein expression (Fig. 4D ).
Influenza A virus-induced inflammasome activation is dependent on P2X 7 receptor and src tyrosine kinase activity Several proteins related with inflammatory response were identified from influenza A virus-infected macrophages. Thus, we created a network based on known protein-protein interactions between all the inflammation-related proteins identified from the intracellular fractions of the infected cells (Fig. 5A) . NLPR3 was added to our protein interaction network since it has been shown to be essential for the activation of inflammatory response during influenza A virus infection. Protein interaction network showed that purinenergic P2X 7 receptor is directly linked to NLRP3. P2X 7 receptor has been shown to be involved in NLRP3 activation in response to extracellular ATP [21, 22] , and host tissue damage during influenza A virus infection may result in extracellular leakage of ATP. Therefore we studied the role P2X 7 receptor in virus-induced inflammasome activation. Specific inhibitor for P2X 7 receptor, AZ11645373, clearly diminished IL-18 secretion in infected macrophages (Fig. 5B ). In addition to IL-18, NLRP3 inflammasome regulates the secretion of IL-1b. We have previously shown that influenza A virus infection is not able to induce production of proIL-1b in human macrophages [23] . Therefore we stimulated macrophages with LPS for 18 h to activate proIL-1b production, after which the macrophages were infected with influenza A virus for 9 h in the presence and absence of AZ11645373. This P2X 7 receptor inhibitor almost completely abrogated influenza A virus-induced IL-1b secretion (Fig. 5C ). In addition to pharmacological inhibition, we used small interfering (si)RNA approach to study the role of P2X 7 receptor in influenza A virus-induced inflammasome activation. Human macrophages were treated with control siRNA and P2X 7 receptor specific siRNAs for 24 h after which the cells were left unstimulated or infected with influenza A virus for 9 h. Silencing of P2X 7 receptor clearly reduced influenza A virus-induced IL-18 secretion (Fig. 5D) and Western blot analysis confirmed that P2X 7 receptor protein expression was decreased in P2X 7 receptor siRNA-treated macrophages (Fig. 5E ). In conclusion, our results show that P2X7 receptor is essential for influenza A virus-induced inflammasome activation.
The inflammation-related proteins identified included also two NADPH oxidase subunits, gp91phox (CYBB) and p22phox (CYBA). These proteins have been linked to NLRP3 inflammasome activation through reactive oxygen species (ROS) formation in response to various stimuli [24] [25] [26] . Interestingly, also src tyrosine kinase is known to interact with CYBB and CYBA [27] .
To study the role of tyrosine phosphorylation in influenza A virusinduced inflammasome activation we infected human macrophages in the presence and absence of PP2 which is a highly specific inhibitor of src tyrosine kinases. PP2 completely inhibited infuenza A virus Udorn/72-and Beijing 353/89-induced IL-18 secretion ( Fig. 5F and Fig. S3D, respectively) . Furthermore, PP2 also abrogated IL-1b secretion in response to influenza A virus infection (Fig. 5G) demonstrating that influenza A virus-induced inflammasome activation is dependent on src family tyrosine kinase activity. 
Influenza A virus genome contains eight pieces of segmented negative-sense RNA which encode 11 proteins. Therefore the virus has to exploit host cell factors to promote its replication and suppress antiviral response. The function of viral proteins during influenza A virus infections is rather well characterized. However, the host response activated by viral infection is less well understood. The development of genome-wide screening techniques such as RNAi has resulted in the identification of hundreds of new host factors that are involved in influenza A virus replication in different cell lines [28] [29] [30] [31] [32] [33] . The cellular responses to influenza infection as well as other respiratory viruses have also been studied using proteomics in human cell lines with both two-dimensional gel electrophoresis (2-DE) based approach [34] [35] [36] and newer MS-based strategies [37] [38] [39] . These studies have shown that similar cellular pathways are activated in response to different viruses. However, these studies have also pointed out that the cellular responses to influenza infection are in part cell-type specific and more studies using human primary cells are needed to elucidate the host response in detail.
We have previously characterized host-response to influenza infection in human primary macrophages using traditional 2-DE based proteomics and shown that actin and RIG-I/MAVS signaling components translocate onto mitochondria upon infection [40] . However, 2-DE based proteomics suffers from certain drawbacks, most importantly it shows systematic bias against certain protein groups including membrane proteins as well as very big/small proteins and proteins with extreme pIs. Also, it requires a lot of manual work and is therefore not suitable for high-throughput studies. Here, we have used unbiased highthroughput subcellular proteomics approach to analyse the host response of human primary macrophages during influenza A virus infection in a global manner. We provide evidence that the expression and/or subcellular localization of more than one thousand host proteins is affected at the early phases of infection. Up-and downregulation of proteins in all subcellular fractions reflects the dynamic interplay between these compartments and highlights the importance of subcellular proteome characterization as many of these changes cannot be seen at total proteome level.
To defend against virus infection, the host activates antiviral machinery. Interferons and IFN-inducible genes (ISGs) are the central components of this response. We detected the upregulation of many IFN-inducible proteins in the cytoplasmic fraction of influenza A virus-infected macrophages (Table S1 ). Interestingly, A. Protein-protein interaction network of all inflammatory proteins identified from intracellular fractions. B. Human macrophages were infected with influenza A virus for 9 h in the presence and absence of AZ11645373 (1 mM), after which the cell culture media was collected and analyzed with IL-18 ELISA. C. Human macrophages were primed with LPS for 18 h, after which the macrophages were infected with influenza A virus for 9 h in the presence and absence of AZ11645373. After this cell culture supernatants were analyzed with IL-1b ELISA. D. Human macrophages were treated with these proteins included three proteins of the IFIT family. It was very recently shown by siRNA approach that interferon-inducible transmembrane proteins (IFITM) restrict an early step of influenza A virus replication in A549 lung epithelial cells [29] . With our proteomics approach we did not detect any inducible expression of IFITM proteins in human macrophages in response to influenza A virus infection. It may be that IFITMs and IFITs function in antiviral response in tissue specific manner and similar to IFITMs, IFIT proteins may be a novel family of antiviral restriction factors that mediate cellular innate immunity to influenza A viruses.
Influenza A virus infection begins with the binding of viral hemagglutinin to sialyated host plasma membrane glycoprotein. Following endocytosis, viral particles are trafficked through early endosomes to late endosomes/lysosomes. In these compartments the conformation of HA is changed resulting in fusion of host-viral membranes and entry of viral ribonucleoproteins into the cytosol. We detected major changes in subcellular localization of Rasrelated small GTPases and vacuolar ATPases, which are involved in the regulation of endosomal recycling pathway and have recently been shown to be essential for influenza A virus replication [28] . Furthermore, it was shown that small molecule inhibitor of vacuolar ATPase can antagonize influenza A virus replication probably by inhibiting the entry of viral ribonucleoproteins to the cytosol. Interestingly, our secretome analysis showed that several Ras-related proteins, Rab-10, Rab-11A, Rab-1A, and Rap-2b as well as components of vacuolar ATPases, Vtype proton ATPases, are rapidly secreted in response to influenza A virus infection. The results suggest that these proteins are involved in protein secretion during influenza A virus infection.
We have previously shown that inflammasome-associated caspase-1 is activated in human macrophages in response to influenza A virus infection resulting in the secretion of proinflammatory cytokines [4, 5] . Subsequently, it has been shown that the inflammasome structure that activates caspase-1 during influenza A virus infection is NLRP3 [10] [11] [12] 41, 42] . Our present results show that influenza A virus infection of human macrophages is associated with endolysosomal leakage of cathepsins to the cytosol. This was followed by the activation of inflammasomeassociated caspase-1 and secretion of IL-18, caspase-1, and mature forms of cathepsins. Furthermore, cathepsin specific inhibitor Ca-074-Me completely inhibited secretion of IL-18 and caspase-1 demonstrating that inflammasome activation during influenza A virus infection is completely dependent on cathepsin activity. These results indicate that lysosomal proteases, cathepsins, are an essential part of pro-inflammatory innate immune response during viral infections.
NLRP3 activators are chemically and structurally different suggesting that they are not directly recognized by the NLRP3 inflammasome. It is more likely that they activate inflammasome indirectly by inducing changes in endogenous proteins that are recognized as danger signals. In addition to cathepsin activity, potassium efflux and ROS production are the common features associated with NLRP3 inflammasome activation. It was very recently shown that thioredoxin-interacting protein links oxidative stress to inflammasome activation [43] . Our proteomic data shows that there is re-localization of mitochondrial proteins to the cytoplasm already at 6 h after influenza A virus infection. These proteins contained several components of the inner mitochondrial membrane including proteins involved in electron transport chain and oxidative phosphorylation. The results suggest that there is a substantial change and/or damage in mitochondrial structure at early phases of influenza A virus infection which may contribute to reactive oxygen species production during infection. This is likely to contribute to the inflammasome activation and apoptosis seen in influenza A virus-infected macrophages. Our current results clearly show that src tyrosine kinase activity is essential for inflammasome activation in response to influenza A virus infection: src kinase specific inhibitors PP2 (Fig. 5 F and G) and src kinase inhibitor II (data not shown) abolished influenza A virusinduced secretion of IL-1b and IL-18. It has also been recently shown that src tyrosine kinase Lyn plays a critical role in the activation of NLRP3 inflammasome in response to malarial hemozoin [44] . Src tyrosine kinases are responsible for the regulation of ROS formation through NADPH oxidase complex [27] . Activation of src tyrosine kinases results in phosphorylation of its substrates Tks4 and Tks5. These proteins act as molecular organizers that specifically activate NADPH oxidases resulting in ROS formation [45] . In conclusion, our results demonstrate a novel link between src tyrosine kinase activity, ROS formation, and inflammasome activation during viral infections.
Apoptosis is an innate immune response by which infected and other harmful cells are eliminated from the inflamed tissue. In this way intracellular danger signals are not released to extracellular space and inflammation is not further enhanced. We have previously shown that influenza A virus infection of human macrophages is associated with activation of caspase-3 [5, 23] which is the hallmark of apoptosis. Lysosomal proteases cathepsins are known to act in concert with caspases in apoptotic cell death [46] . Our current data shows that influenza A virus infection of human macrophages is associated with rapid cytoplasmic upregulation of cathepsins indicating lysosomal rupture upon infection. Furthermore, we show that cathepsins act upstream of caspase-3 and that their activity is essential for progression of apoptosis.
Most proteins are secreted through conventional protein secretion pathway. These proteins contain signal peptides that direct their transport to the plasma membrane through the ER-Golgi pathway. In our experiments, many classically secreted proteins were detected in cell culture supernatants of macrophages in response to influenza A virus infection. In addition to classical protein secretion, activated immune cells secrete proteins lacking signal peptides through unconventional secretory pathway [47] . DAMPs are nuclear or cytosolic proteins with defined intracellular functions [48] . They are usually hidden in intact cells and released during tissue damage through unconventional protein secretion pathway. Our quantitative high-throughput secretome analysis revealed secretion of several DAMPs in response to influenza A virus infection. These included 50 kDa cleavage product of amyloid precursor protein, HMGBs, galectins, and S100 proteins. Amyloid precursor protein is processed by a-, band c-secretases to generate inflammatory amyloid b peptide [49] . Fibrillogenic amyloid b peptide 42 (Ab 42 ) is a known activator of NLRP3 inflammasome in migroglial cells [50] and it is involved in the pathogenesis of Alzheimer's disease [51] . We were not able to control siRNA and P2X 7 receptor specific siRNAs for 24 h after which the cells were left unstimulated or infected with influenza A virus for 9 h. IL-18 secretion was analyzed from cell culture supernatants with IL-18 ELISA. E. P2X 7 receptor expression was analyzed by Western blotting with anti-P2X 7 specific Abs. F. Macrophages were infected with influenza A virus for 9 h in the presence and absence of PP2 (5 mM), after which the cell culture media was collected and analyzed with IL-18 ELISA. G. Macrophages were primed with LPS for 18 h, after which the macrophages were left untreated or treated with PP2 and infected with influenza A virus for 9 h. The cell culture supernatants were analyzed with IL-1b ELISA. doi:10.1371/journal.ppat.1001340.g005 detect secretion of Ab 42 by ELISA in influenza A virus-infected macrophages (data not shown). However, the secretome data showed that amyloid precursor protein is expressed, processed, and secreted in response to influenza A virus infection. In addition, subcellular proteomics data demonstrates that nicastrin and preselinin, which are components of c-secretases are expressed in human macrophages. This finding suggests that b-amyloid protein and its processing machinery have a specific function in antiviral response.
Taniguchi and coworkers have shown that HMGBs function as universal sentinels for nucleic acid mediated innate immune response [52] . HMGBs operate upstream of cytoplasmic RLRs and endosomal TLRs and they are essential for nucleic acidinduced activation of innate immune response. In our experiments both HMGB1 and HMGB2 were detected in cell culture supernatants of influenza A virus-infected macrophages. These results suggest that HMGBs have an important role in the activation of innate immune response to influenza A virus infection. Our proteomics approach showed also increased secretion of galectin-3 in response to influenza A virus infection. This finding is especially interesting since galectins are known to bind extracellularly carbohydrate structures and the envelope of influenza A virus contains two glycoproteins, hemagglutinin and neuraminidase. Hemagglutinin is required for influenza A virus entry, and it can be speculated that the extracellular galectins restrict viral entry. It is easy to envision that secreted galectins may have also other functions in antiviral response since there is ample evidence about the role of galectins in innate immunity to bacterial and fungal infections [53] . Clearly, future studies are required to characterize the importance of galectins in viral infections.
In conclusion, our high-throughput, unbiased quantitative proteomics study provides important new insight into hostresponse against influenza A virus infection in human primary macrophages (Fig. 6) . First, our data shows that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. Secondly, our data demonstrates that there is rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, upon infection which contributes to inflammasome activation and apoptosis seen in infected macrophages. Thirdly, our data demonstrates that P2X 7 receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different DAMPs suggesting an important role for DAMPs in antiviral response.
Human primary macrophages were obtained from leukocyterich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Monocytes were isolated as described previously [4] and differentiated into macrophages by maintenance in Macrophage-SFM medium (GIBCO) supplemented with 10 ng/ml GM-CSF (Biosource International) and antibiotics. After 7 days of culture, the resulting macrophages were used in experiments. Macrophages were infected with influenza A virus in complete Macrophage-SFM medium. The studied cells were lysed or fractionated into mitochondrial, cytoplasmic and nuclear fractions. Mitochondrial and cytoplasmic fractions were isolated by Qproteome Mitochondria Isolation Kit (Qiagen) and the cytoplasmic fractions were further purified with 2-D Clean-Up Kit (GE Healthcare). Nuclear fractions were isolated by Qproteome Nuclear Protein Isolation Kit (Qiagen) and the insoluble and soluble nuclear protein fractions were combined before analysis. About 1610 7 cells were used for all isolations. For secretome analyses, the cells grown in complete Macrophage-SFM medium were washed tree times with PBS after which the cells were stimulated in RPMI growth media supplemented with 1 mM HEPES, L-Glutamine and antibiotics (GIBCO). The growth media were collected and concentrated in Amicon Ultra centrifugal filter devices (Millipore Corporation, Billerica, MA).
Human pathogenic H3N2 influenza A virus strains, Udorn/72 and Beijing/353/89, were cultured in embryonated hen eggs and stored at 270uC. The hemagglutination titer of both influenza virus strains was 256, as measured by standard methods. In infection experiments, virus dose of 2.56 hemagglutination U/ml was used. The experiments were performed with strain Udorn/72 unless otherwise stated. The protein amount of cell lysates was analysed by SDS-PAGE followed by silver-staining and Western blotting to confirm that viral infection did not decrease the total protein expression level in our experiments (Fig. S1E ).
Mitochondrial, cytoplasmic or nuclear fractions or secretomes of uninfected control cells and influenza A virus infected cells at given timepoints were labeled with 4plex iTRAQ. The samples were first dissolved into 43 ml of iTRAQ dissolution buffer and 2 ml of each sample was run into an SDS-PAGE gel. For the intracellular fractions, equal protein amounts of each sample were taken for the iTRAQ analyses based on the silver stained gels. For secretome analyses, the whole samples were labeled. Protein alkylation, trypsin digestion and labeling of the resulting peptides were done according to manufacturer's instructions (AB Sciex). After labeling, the samples were pooled, dried and dissolved into 20 mM KH 2 PO 4 (pH 3). Labeled peptides were fractionated by strong cation exchange chromatography (SCX) on an Ettan HPLC system (Amersham Biosciences). Each SCX fraction containing labeled peptides was analysed twice with nano-LC-ESI-MS/MS using Ultimate 3000 nano-LC (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (AB Sciex) with nano-ESI ionization (approximately 22 SCX fractions for intracellular samples and 13 fractions for secretome). MS data were acquired automatically using Analyst QS 2.0 software. Information-dependent acquisition method consisted of a 0.5 s TOF-MS survey scan of m/z 400-1400. From every survey scan two most abundant ions with charge states +2 to +4 were selected for product ion scans. Once an ion was selected for MS/ MS fragmentation, it was put on an exclusion list for 60 s.
Protein identification and relative quantitation was performed using ProteinPilot 2.0.1 software (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. The search database was a self-built combination of Uniprot human protein sequences and Uniprot ssRNA negativestrand viruses sequences (both form the release 55.0, 02/08). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore  .1,3) . Additionally, automatic bias correction was used for intracellular fractions to correct for uneven protein loading. ProteinPilot identification and quantitation results were also manually checked: for each identified protein at least two unique peptides with good quality MS/MS data were required, and MS/ MS spectra with all reporter ion peak heights below 10 counts were manually removed from quantitation results. False discovery rates were calculated using a concatenated normal and reversed sequence database and a previously reported method [54] .
Proteins identified from each subcellular fraction were classified based on their Gene Ontology annotations using GeneTrail [55] . Additionally, k-means clustering analysis was performed for the differentially regulated proteins in each subcellular fraction using Chipster, an open source data analysis tool (http://chipster. sourceforge.net). Clustering was done based on the relative quantitation results from the iTRAQ experiments, and a suitable number of clusters for each subcellular fraction was determined by studying the cluster profiles. Protein-protein interaction networks for a selected group of proteins was created using String [56] .
Isolated mitochondrial, cytoplasmic and nuclear fractions from influenza A virus infected and untreated macrophages were dissolved in Laemmli sample buffer. Equal amount of protein from each sample was loaded into an SDS-PAGE gel and transferred onto PVDF-membrane. Membranes were blocked with 5% nonfat milk, stained with different Abs overnight and detected by ECL. The primary antibodies used in this study were cathepsin B (Calbiochem), caspase-3 and HSP90 (Cell Signaling), IFIT3 (BD Transduction Laboratories), actin, annexin A1, b-Amyloid, APOE, caspase-1, cathepsin D, cathepsin Z, cytochrome c, galectin-3, GAPDH, histone H1, HMGB1, LAMP-1, P2X7 receptor, S100-A9 and VDAC1 (Santa Cruz Biotechnology Inc.). IL-18 and influenza A virus (H3N2) antibodies have been previously described [4, 40] .
The IL-1b and IL-18 cytokine concentrations were determined by ELISA according to manufacturer's instructions. Human IL-1b and IL-18 ELISAs were purchased from Diaclone and Bender Medsystems, respectively.
The cells grown in complete Macrophage-SFM medium were washed tree times with PBS and the media was changed to RPMI growth media supplemented with 1 mM HEPES, L-Glutamine and antibiotics. The RPMI media has lower content of initial media proteins than Macrophage-SFM medium. The cathepsin B inhibitors, Ca-074 Me (Calbiochem) and z-FA-fmk (Calbiochem) were added 0.5 h before influenza A virus infection and used at final concentration of 40 mM and 50 mM, respectively.
The P2X 7 receptor inhibitor AZ11645373 and src tyrosine kinase inhibitor PP2 were purchased from Sigma, and they were added to macrophages 0.5 h before infection with influenza A virus. AZ11645373 and PP2 were used at final concentration of 1 mM and 5 mM, respectively.
After five days of cell culture in 12-well plates, macrophages were transfected with 200 nM non-targeting control siRNA (AllStars Negative Control siRNA, Qiagen, Hilden, Germany) and with 100 nM of each of two different P2X 7 receptor siRNAs (Hs_P2RX7_1, Hs_P2RX7_2; Qiagen) by using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer's instruction. After 4 h of siRNA treatment, fresh macrophage media was added to the cells. On the following day, the cells were left unstimulated or infected with influenza A virus for 9 h, after which the cell culture supernatants were collected and total proteins were isolated for ELISA and Western blot analyses, respectively.
The percentage of apoptotic cells was assayed with APOPercentage Apoptosis Assay according to manufacturer's instructions (Biocolor Life Science Assays). Photographs were taken with an Olympus DP70 Digital microscope camera connected to an Olympus IX71 light microscope using DP Controller (version 2.2.1.227) and DP Manager (version 2.2.1.195) softwares. The stained (apoptotic) and unstained cells were manually counted, and the percentage of apoptotic cells was calculated.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function Angiotensin-converting enzyme 2 (ACE2) is a new component of the renin-angiotensin system (RAS). Accumulating evidence shows that ACE2 provides protective effects in peripheral tissues and has great potential for the treatment of RAS-related diseases. The role of ACE2 in the central nervous system is not well established. However, in recent years, much more progress has been made on the studies of this carboxypeptidase in the central regulation of blood pressure and cardiovascular function in general. It has been shown that brain ACE2 interacts with the other components of the RAS (ACE, angiotensin II, and angiotensin II type 1 receptor), protects baroreflex and autonomic function, stimulates nitric oxide release, reduces oxidative stress, and prevents the development of or attenuates hypertension. These data support the critical role of ACE2 in the central regulation of cardiovascular function. This review summarizes recently published data on the central effects of ACE2 in the regulation of cardiovascular function. The renin-angiotensin system (RAS) plays a regulatory role in cardiovascular function and a pathogenic role in the development of hypertension and associated cardiovascular disorders. In addition to the classic endocrine system, local RASs are present in various tissues throughout the body, interacting with each other and the endocrine RAS.
Angiotensinogen (AGT) is the unique known precursor of angiotensin (Ang) peptides, which is transformed by renin to generate the decapeptide Ang I. Ang I is then converted by angiotensin-converting enzyme (ACE) into the octapeptide Ang II. Ang II is one of the major players in the RAS, acting on various receptors such as Ang II type 1 (AT1R) and type 2 receptors (AT2R). By binding to AT1R, Ang II promotes vasoconstriction, cell proliferation, and fibrosis, whereas when binding to AT2R it promotes the inhibition of cell growth. Most of the effects of Ang II have been shown to be mediated by AT1R. Increased activity of the RAS is associated with various cardiovascular diseases such as hypertension and heart failure. Classical treatment of RAS-associated diseases focuses on the blockade of Ang II formation or effects through the use of ACE inhibitors and AT1R blockers [1] .
ACE2 is a new component of the RAS that was discovered in 2000 [2, 3] . It is the first known human homologue of ACE but differs from ACE in substrate specificity. Although ACE2 was first shown to cleave Ang I to Ang (1-9) [3] , which can be converted by ACE into the heptapeptide Ang-(1-7), another study showed that ACE2 hydrolysis of Ang II into Ang-(1-7) has a much higher efficiency (∼400-fold) than that for Ang I to Ang (1-9) [4] .
It has been established that Ang-(1-7) acting on the Mas receptor mediates vasodilation, antiproliferation, and apoptosis [5] , therefore opposing the effects of Ang II. Using genetic and pharmacologic approaches, such as gene knockout, knockdown, gene transfer, activators and inhibitors, ACE2 has been shown to have protective effects in various tissues and to prevent overactive RAS-associated diseases, including hypertension [6] . Thus, the new arm of the RAS, the ACE2-Ang-(1-7)-Mas axis, has been shown to be effective at counter-regulating the effects of the classic ACE-Ang II-AT1R axis.
Brain RAS contains the same elements as the other tissue RAS. Besides Ang II and AT1R, all the other components of RAS such as AGT, renin, AT2R, ACE, Ang IV, Ang-(1-7), Mas, and new elements such as prorenin/renin receptor and Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , have been identified in the central nervous system [7, 8] . However, the distribution of the new element ACE2 in the brain was at first controversial because original reports failed to identify the carboxypeptidase in this tissue [2, 3] . A few years later, ACE2 mRNA and protein were found in the brain by our group and others [9] [10] [11] . Using a selective antibody, we reported that ACE2 is widespread throughout the mouse brain, present in nuclei involved in the central regulation of cardiovascular function such as the subfornical organ, paraventricular nucleus (PVN), nucleus of the tractus solitarius (NTS), and rostral ventrolateral medulla (RVLM), as well as in noncardiovascular areas such as the motor cortex and raphe ( Fig. 1 ). Shortly after our observation, Lin et al. [12•] confirmed the presence of ACE2 mRNA and protein in the mouse brainstem and reported the interaction between ACE2 and AT1R in this region. These data suggest that ACE2 is actively involved in the brain RAS function.
In the central nervous system, Ang II acting on AT1R results in cardiac baroreflex desensitization and resetting, increased sympathetic outflow, vasopressin release, and stimulation of the water intake, leading to the increase in blood pressure [13] . It has been shown that upregulated AGT, ACE, Ang II, and AT1R in the brain are involved in the development and maintenance of hypertension and heart failure [7, 14] . Similar to the observations in the periphery, Ang-(1-7) shows opposite properties to Ang II in the central nervous system, serving as an important neuromodulator in the central control of cardiovascular function. It has been well demonstrated that central Ang-(1-7) increases cardiac baroreflex sensitivity [15, 16] , reduces norepinephrine release [17] and decreases blood pressure [18, 19] in hypertensive rats. The depressor effects of Ang-(1-7) in the brain have also been shown to be mediated by increase in bradykinin levels [20] , potentiating the hypotensive effects of bradykinin [21] and stimulating nitric oxide (NO) release [22] . ACE2 provides an important route for the metabolism of Ang II and, as the principal forming enzyme for Ang-(1-7), shifts the balance between the two biological active peptides.
However, compared with the well-established central effects of Ang II and Ang-(1-7), there are very few studies supporting the central nervous system effects of ACE2. Most of the studies have focused on the role of ACE2 in the kidney, heart, lung, liver, and other peripheral tissues, as evidenced by the numerous reviews on ACE2 [23] [24] [25] [26] [27] . However, with the discovery of ACE2 in the brain, the role of ACE2 in the central nervous system gained increasing attention from the scientific community. In recent years, growing evidence has demonstrated the important role of ACE2 in the central nervous system, such as in blood pressure control [28, 29•, 30 , 31••].We previously reviewed the central effects of ACE2 and discussed the potential for this carboxypeptidase as a new target in the treatment of cardiovascular diseases [6] . In this report, we updated this information by reviewing the recent discoveries of the past 1 year to 2 years, specifically focusing on the role of ACE2 in the brain RAS and its effects on central regulation of cardiovascular function.
Over the past 1 year to 2 years, more studies from various groups have demonstrated that ACE2 can affect or be affected by other components of the RAS in the central nervous system. An in vitro study from Xiao et al. [32] showed that overexpression of ACE2 in neurons attenuates the Ang II-induced upregulation of AT1R, which could result from inhibition of the well-known feed-forward effect of Ang II on its receptor. In vivo, our group showed that overexpression of ACE2 in the mouse brain not only reduces AT1R expression as previously suggested [30] but also increases Mas and AT2R [33••], whereas blockade of AT1R increases brain ACE2 activity in hypertensive mice [31••]. Using a gene-silencing approach, Lin et al. [12•] reported that downregulation of AT1R mRNA is associated with downregulation of ACE2 mRNA in the mouse brainstem. In addition, downregulated ACE2 is accompanied by upregulation of ACE levels in the brain of a heart failure animal model [34] and in multiple sclerosis patients [35] , indicating a special link between ACE2 and ACE in the brain. These data confirmed and extend previous findings that ACE2 interacts with other RAS components in the brain and strengthens the concept that ACE2 is a significant player in the brain RAS.
Alteration of ACE2 expression or activity in the brain has been found in pathologic conditions related to cardiovascular diseases. Kawajiri et al. [35] reported decreased ACE2 and increased ACE concentration in cerebrospinal fluid from patients with multiple sclerosis compared to those with non-neurologic diseases. Similarly, downregulated ACE2 and upregulated ACE protein and mRNA expression were observed in various brain nuclei (including PVN, NTS, and RVLM ) from pacing-induced chronic heart failure (CHF) rabbits with increased sympathetic nerve activity [34] . These data suggest that the alteration of ACE/ACE2 in the brain is involved in the development of cardiovascular diseases. Moreover, we recently showed decreased ACE2 activity in the brain of a chronically hypertensive mouse model with increased Ang II level [31••]. These observations support the critical role of ACE2 in the central nervous system in the maintenance of normal cardiovascular function.
Because decreased ACE2 in the central nervous system is associated with multiple cardiovascular diseases, compensation by ACE2, as a result of increasing or activating this enzyme, in the brain could provide beneficial effects on these diseases. Several studies showed the effects of ACE2 on prevention or attenuation of hypertension. Using adenovirus coding for ACE2, Sriramula et al. [36] showed that PVN-targeted ACE2 overexpression attenuates the Ang II-induced pressor response in rats. Using a new transgenic mouse model (syn-hACE2) with neuron-targeted ACE2 overexpression, we found that the development of Ang IIinduced neurogenic hypertension was blunted [33••]. In additional experiments, the syn-hACE2 mice were bred with chronically hypertensive transgenic mice expressing both human renin and AGT genes [37] , to generate a tripletransgenic model (SARA) with elevated Ang II throughout the body and overexpression of human ACE2 selectively in the brain. Following chronic measurement of blood pressure in SARA mice, we observed that central overexpression of ACE2 significantly decreases baseline blood pressure in these chronically hypertensive mice [31••]. However, the blood pressure was not normalized due to the chronic and widespread expression of Ang II in the periphery. Nevertheless, our observations are consistent with previous reports from Yamazato et al. [28] and our group [30] showing the antihypertensive effects of ACE2 in specific brain nuclei, such as RVLM and subfornical organ. In addition to these gene transfer approaches, Cangussu et al. [38] showed that increases in ACE2 and Mas mRNA IML-intermediolateral cell column; NA-nucleus ambiguus expression in the hypothalamus following exercise training in 2K1C rats are associated with a reduction of the increased systolic arterial pressure in these animals, although ACE2 levels were not altered in these hypertensive rats before exercise training compared with sham controls. Besides the role of ACE2 in the regulation of blood pressure, Lima et al. [39] showed that central activation of ACE2 by intracerebroventricular injection of XNT, an activator of endogenous ACE2, attenuates air jet stress-induced tachycardia in rats, suggesting the beneficial effects of ACE2 on cardiac function.
Several studies have focused on the mechanisms involved in ACE2 regulation of blood pressure and other cardiovascular functions. Baroreflex sensitivity and autonomic function are important central mechanisms modulating blood pressure and other cardiovascular functions. It has been shown that inhibition of ACE2 activity in the NTS, by injecting a specific inhibitor in this region, reduces the sensitivity of the baroreceptor reflex control of heart rate to increases in arterial pressure [29•] . Similarly, we previously showed that ACE2 gene deletion on a C57bl/6 background mouse resulted in impairment of the spontaneous baroreflex sensitivity (SBRS), increased sympathetic tone, and decreased parasympathetic tone [40, 41] . Moreover, exercise training-induced increase in endogenous ACE2 in the brain of CHF rabbits was associated with an attenuation of sympathetic nerve activity in these animals [34] . Also, overexpressing ACE2 in the PVN, by virus-mediated gene transfer, improves renal sympathetic nerve activity in coronary ligation-induced CHF rats [42] . Global overexpression of ACE2 in the brain also restored the impaired SBRS, normalized the increased sympathetic tone, and decreased parasympathetic tone in transgenic hypertensive mice, contributing to the attenuation of hypertension in these animals [31••]. In low-dose Ang II infusion, a model of neurogenic hypertension, although ACE2 overexpression in the brain did not alter the increased sympathetic tone, it significantly prevented the decrease in both SBRS and parasympathetic tone and attenuated the development of hypertension [33••]. Similar observations were made by Gao et al. [43] showing that global overexpression of exogenous ACE2 in the brain prevents impairment of baroreflex sensitivity and sympathoexcitation in the CHF state.
These data suggest that ACE2 is involved in baroreflex control mechanisms and is able to modulate sympathetic and parasympathetic tones, thus participating in long-term and short-term regulation of blood pressure as well as cardiac function. The modulation of baroreflex and auto-nomic function by ACE2 could be independent of Ang II level, since measurement of Ang II in the brain and plasma showed no change in adult ACE2-deficient mice in which baroreflex and autonomic function were impaired [41] . However, the ACE2-mediated changes in baroreflex and autonomic function could also be resulting from, at least partially, metabolism of Ang II in the brain in some conditions, as decreased water intake following ACE2 overexpression in the brain was observed in both of the two hypertensive models mentioned above [31••, 33••]. Moreover, similar to the effects of Ang-(1-7) [15, 16] , intracerebroventricular infusion of a novel ACE2 product peptide, Ala1-Ang-(1-7), increases baroreflex sensitivity for reflex bradycardia [44] , suggesting the modulation of ACE2 on baroreflex function could also be triggered by its products.
In the central nervous system, it has been shown that NO reduces, whereas oxidative stress increases, sympathetic activity [45] [46] [47] . Overexpressing ACE2 in the PVN normalized the decreased neuronal nitric oxide synthase (NOS) protein levels in this region in CHF rats and was accompanied with improved sympathetic nerve activity, suggesting the inhibitory effects of ACE2 on sympathoexcitation involve an NO mechanism [42] . Similarly, Gao et al. [43] showed that overexpression of ACE2 in the brain prevented the downregulation of NOS expression in the CHF mice, contributing to the improvement of baroreflex sensitivity and the reduction of sympathoexcitation. This is confirmed by our observation showing that overexpression of ACE2 in the brain upregulated NOS expression and increased NO levels in the cerebrospinal fluid [33••]. In addition, ACE2 overexpression in the brain prevented the Ang II-mediated decrease in NOS expression in regions modulating blood pressure regulation [33••]. Furthermore, we observed that ACE2 gene deletion leads to increased superoxide levels in various brain nuclei of aged mice [48] , supporting the antioxidant capacity of ACE2 in the brain. These data indicate that the protective effects of ACE2 could be linked to an increased NO availability and decreased oxidative stress in the brain.
Clear progress has been made in our understanding of the role of ACE2 in the brain. The many beneficial effects of this carboxypeptidase on the central regulation of cardiovascular function could be independent or at least partially result from the modification of angiotensin peptides. The potential therapeutic implications of ACE2 suggest that it could be used to treat hypertension, heart failure, and other cardiovascular diseases. Increasing brain ACE2 by stimulating endogenous ACE2 activity and/or expression, or administration of exogenous ACE2 may provide beneficial effects in some pathologic conditions and this could be a new strategy for future development of novel therapeutic agents. Further understanding of its role in the central nervous system is needed because it would lead to the exploration of novel therapies for neurogenic hypertension and other cardiovascular as well as neurologic diseases.
that inhibition of ACE2 activity in the NTS impaired the baroreflex sensitivity for control of heart rate in response to increases in arterial pressure. Moreover, there was no further reduction of these effects following combined Ang-(1-7) receptor blockade and ACE2 inhibition, suggesting that ACE2 is the main enzyme forming Ang-(1-7 Using a chronically hypertensive model and a triple-transgenic model with chronic overexpression of Ang II and brain ACE2, the authors found that AT1R exerts an inhibitory effect on ACE2 activity in the brain of hypertensive mice, and that brain selective overexpression of ACE2 attenuated the hypertension by improving arterial baroreflex and autonomic function.  Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity. Myxoviruses are enveloped, negative-strand RNA viruses that are transmitted through the respiratory route. The orthomyxovirus family comprises five different genera of which the influenza viruses are clinically most relevant. Of the paramyxoviridae, respiratory syncytial virus (RSV), measles virus (MeV), mumps virus (MuV), human parainfluenzaviruses (HPIV) and the recently emerged, highly pathogenic zoonotic henipaviruses constitute major human pathogens [1] . Although clinical complications associated with some myxoviruses involve persistent infections, the viruses predominantly induce acute respiratory or systemic disease.
Collectively, myxoviruses are responsible for the majority of human morbidity and mortality due to viral respiratory illness globally [2, 3] . In particular, influenza virus is the leading cause of morbidity and mortality from respiratory disease in North America despite the existence of vaccine prophylaxis. This is due to the fact that the vaccines currently in use reduce illness in approximately 70% of healthy adults when homologous to the prevalent circulating virus, but protection in the elderly reaches only approximately 40%. Vaccine efficacy is reduced substantially when the circulating strains differ from those constituting the vaccine [2] .
Despite extensive research and in contrast to, for instance, MeV and MuV, no vaccines are currently available against several major pathogens of the paramyxovirus family such as RSV or different HPIVs. Infection with RSV is the leading cause of pneumonia and bronchiolitis in infants, both associated with significant mortality, while HPIV types 1 and 2 are the primary cause of croup syndrome and can likewise result in serious lower respiratory diseases such as pneumonia and bronchiolitis [4, 5] .
The availability of effective antiviral therapy for most clinically significant myxovirus infections is limited. Licensed neuraminidase inhibitors for influenza therapy, Zanamivir and Oseltamivir, show efficacy when administered within a 48-hour window after the onset of symptoms, but are increasingly compromised by preexisting or emerging viral resistance [6, 7, 8] . Ribavirin, although approved for RSV treatment, shows limited utility due to efficacy and toxicity issues [9] . The polyclonal immunoglobulin RSV-IVIG [10] and the humanized monoclonal antibody Synagis [11] provide RSV prophylaxis, but use is limited to high-risk pediatric patients. Considering the high mutation rates seen in particular with RNA viruses [12, 13] , the development of novel types of myxovirus inhibitors that circumvent the rapid development of resistance is highly desirable.
Of the strategies conceivable towards this goal, targeting host factors required for completion of the viral life cycle rather than pathogen-encoded factors directly has received heightened interest in recent years [14, 15] . This approach is expected to establish a significant barrier against spontaneous viral escape from inhibition, since individual viral mutations are less likely to compensate for the loss of an essential host cofactor than to prevent highaffinity binding of a conventional, pathogen-directed antiviral. Given some degree of overlap of host cell pathways required for successful replication of related viral pathogens, host-directed antiviral approaches also have the potential to move beyond the one-bug one-drug paradigm by broadening the pathogen target range of a chemical scaffold.
Naturally, targeting host factors for antiviral therapy bears an inherently higher potential for undesirable drug-induced side effects than conventional pathogen-directed strategies. While the approach is nevertheless under investigation for the treatment of chronic viral infections such as HSV-1 and HIV-1 [16, 17] , an application to the inhibition of infections by pathogens predominantly associated with severe acute disease, such as most members of the myxovirus families, is anticipated to render drug-related side effects tolerable to some extent, since the necessary treatment time and concomitant host exposure to the drug remain limited. In the case of influenza infections, for instance, typical neuraminidase inhibitor regimens consist of twice daily administration for a five-day period for treatment, or a 10-day period for prophylaxis [18] .
Relying on a broadened anti-myxovirus target spectrum as the main selection criterion in secondary screening assays, we have mined results of a recently completed high throughput chemical library screen [19] to identify hit candidates with a possible hostdirected mechanism of action. This has yielded a compound class with broad anti-viral activity, which was subjected to synthetic scaffold optimization, quantification of active concentrations for a select group of clinically relevant ortho-and paramyxovirus family members, testing against a panel of exposed host cells of different species origin, and characterization of the compound-induced point-of-arrest in viral life cycle progression. Viral adaptation to growth in the presence of inhibitor has been employed to compare escape rates from inhibition by this new compound class with those from a well-characterized, pathogen-directed antiviral.
To identify small-molecule hit candidates that block the myxovirus life cycle through a host-directed mechanism, we analyzed the results of a high-throughput cell-based anti-MeV screen of a 137,500-entry library of the NIH diversity set that we recently reported [19] . The primary screening agent, serving as the myxovirus representative, was the wild type MeV isolate MVi/ Alaska.USA/16.00 (MeV-Alaska). It was chosen based on its ease of growth and readily quantifiable cytopathic effect in the automated system [19, 20] . In search of candidates with a hostdirected antiviral profile, we anticipated three distinct features of desirable compounds: a) potent inhibition of virus replication at the screening concentration (3.3 mM); b) a primary screening score, representative of the selectivity index (CC 50 /EC 50 ), close to the cut-off value for hit candidates due to some anticipated hostcell interference ( = 1.9); and c) a broadened viral target spectrum in counterscreening assays that extends to other pathogens of the myxovirus families.
When inhibition of paramyxovirus family members was assessed, six compounds efficiently blocked the closely related canine distemper virus (CDV) and the more distantly related human parainfluenzavirus type 3 (HPIV3) in addition to MeV-Alaska, while leaving cell metabolic activity essentially unaffected [19] . Of these independent hits, three share a common molecular scaffold ( [19] and figure 1A ). Since HTS scores of these analogs best matched the target criteria and antiviral activity was highest in this group [19] , we subjected them to further characterization and developmental efforts. Synthetic optimization and structural confirmation of the scaffold returned a lead analog JMN3-003 (figures 1B and S1), which showed potent activity against MeV, a selection of clinically significant members of the para-and orthomyxovirus families, and, albeit to a lesser degree, representatives of positive strand RNA virus (sindbis virus of the Alphaviridae) and DNA virus (vaccinia virus of the Poxviridae) families (figure 1C, inhibitory concentrations for a larger panel of myxovirus family members are summarized in table 1). As observed for the primary hit compound, metabolic activity of different established cell lines exposed to JMN3-003 was unchanged at 75 mM, the highest assessable concentration based on solubility of the substance in growth media (figure 1D and  table 1 ). Of different primary human cells examined, metabolic activity was unaffected (PBMCs, smooth muscle cells) or only slightly affected (bronchial epithelial cells) by the compound ( figure 1E ). These data support potent anti-myxovirus activity of the compound with active concentrations ranging from 10 to 80 nM depending on the target virus.
To further explore whether JMN3-003 meets the profile of a host-directed antiviral, we examined whether the extent of inhibition is determined by the species origin of the host cell used for virus propagation. Based on its broad host cell range, inhibition of influenza A/WSN replication was monitored. In addition to higher mammalian (HT1080 (ATCC CCL-121), HeLa (ATCC CCL-2), MDCK (ATCC CCL-34)) cell lines, cells of rodent (NIH-3T3 (ATCC CRL-1658), MEL B16 (ATCC CRL-6322), BHK-21 (ATCC CCL-10), CHO (ATCC CCL-61)) and avian (DF-1 (ATCC CRL-12203)) origin were tested, which are all permissive for influenza A/WSN infection (table 2) . While inhibitory concentrations obtained for all higher mammalian cell lines examined were similar, A/WSN inhibition by JMN3-003 was found inactive on some rodent cell lines and when virus was propagated on murine or avian cells (table 2) . However, inhibitory activity extended fully to primary human PBMCs (figure 2). For the latter, inhibition of MeV-Alaska was monitored due to efficient growth of MeV isolates on PBMCs [21] . The host cell species effect of antiviral activity of JMN3-003 is consistent with specific targeting of cellular factors by the compound, while arguing against docking to conserved viral factors or an undesirable promiscuous, unspecific mode of activity.
The central 2-thio-connector found in the chemical scaffold of JMN3-003 may render the compound susceptible to rapid phase I oxidation in vivo [22] , thus possibly compromising its developmental potential. To test metabolic stability of the substance early in development, we exposed JMN3-003 to human S-9 hepatocyte subcellular fractions as an in vitro indicator for phase I metabolism.
After a 60-minute exposure, approximately 80% of the input material remained intact, corresponding to an extrapolated halflife of approximately 200 minutes (figure 3A). Unstable analogs of JMN3-003, JMN5-165 and JMN5-166 (figure S1), returned half lives of 38 and 5 minutes in this assay, respectively, confirming metabolic competency of the S9 fractions used.
Assessment of JMN3-003 stability in human plasma in comparison with unstable Procaine and stable Procainamide Table 1 . Active (EC 50 ) and toxic (CC 50 , determined on Vero-Slam cells) concentrations of JMN3-003 against a selection of clinically relevant para-and orthomyxovirus family members in comparison with active concentrations of AS-136A, a previously characterized, MeV-specific inhibitor of the viral RdRp complex [20, 36] . [23] corroborated these results, since JMN3-003 integrity was virtually unaffected after a 120-minute incubation period ( figure 3B ). Taken together, these findings suggest desirable metabolic stability for the JMN3-003 scaffold, recommending it for further mechanistic characterization. The data are corroborated by the good metabolic stability reported for the structurally similar compound RDEA-806 (figure S2), a non-nucleoside inhibitor of HIV reverse transcriptase and clinical precedent [24] , which shares the 2-thio-connector of JMN3-003 but lacks MeV inhibitory activity in our assays (data not shown).
Since direct cytotoxicity of JMN3-003 was low for all cell lines examined, we next tested the effect of the substance on cell cycle progression. Analysis of the DNA content of cells continuously treated with JMN3-003 for 36 hours by flow cytometry revealed accumulation of cells in a single population with 2N DNA content, which closely resembled the profile of a reference cell population exposed to hydroxyurea but markedly differed from the 4N DNA content of nocodazole-treated cells (figure 4A). Nocodazole interferes with microtubule polymerization [25] , resulting in a G 2 /M arrest, whereas hydroxyurea is thought to lead to an arrest in the G 1 /S-phase through depletion of cellular dNTP pools [26, 27] . To further explore the effect of JMN3-003 on cell cycle progression, we monitored the phosphorylation status of the cdc2-cyclin B kinase after exposure of cells to either the compound, hydroxyurea, nocodazole, or alsterpaullone, a nanomolar small molecule inhibitor of cyclin-dependent kinases that reportedly induces a potent G 1 /Sphase cell cycle arrest [28] . Pivotal in regulating the G 2 /M transition, cdc2-cyclin B kinase is inactivated through phosphorylation during the G 2 -phase. Accumulation in its phosphorylated form thus indicates a G 1 arrest. As in hydroxyurea-and alsterpaullone-treated controls, exposure of cells to JMN3-003 resulted in increased steady state levels of phosphorylated cdc2cyclin B kinase, supporting a G 1 -phase arrest (figure 4B).
To test whether this JMN3-003-induced arrest is permanent or temporary, we next incubated cells in the presence of compound or vehicle alone for 30 hours, followed by removal of the substance and reseeding of cells at identical densities. Monitoring cell growth over an additional 72-hour incubation period in the absence of JMN3-003 revealed that proliferation rates resumed those of untreated control cells after removal of the compound (figure 4C), indicating reversibility of the growth arrest.
In contrast to members of the orthomyxovirus family, paramyxovirus replication takes place in the cytosol and, thus, is considered not to be immediately dependent on active cell proliferation [1] . In fact, MeV itself has been shown to induce a G 1 /S arrest in infected T lymphoyctes [29, 30] , confirming that cell cycle progression is not required for virus replication. To directly test whether the JMN3-003-mediated growth arrest per se is causal for the antiviral effect of the compound, we generated MeV-Alaska inhibition curves of JMN3-003 in comparison with the cyclin-dependent kinase inhibitor alsterpaullone. Even at the highest concentration assessed (50 mM), alsterpaullone caused only a marginal reduction in MeV yields (figure 4D). These findings indicate that the antiviral effect of JMN3-003 is based on an upstream effect of the compound rather than being a consequence of the cell cycle arrest itself. Cellular mRNA production and protein biosynthesis are unperturbed by JMN3-003
To explore whether growth arrest of treated cells coincides with reduced host cell RNA synthesis or overall cell protein biosynthesis, we next assessed the effect of JMN3-003 on host mRNA and protein production. Relative levels of three signature host mRNAs with short half lives, MCL1, ASB7 and MKP1 [31, 32] , were determined by real time PCR after incubation of cells in the presence of different JMN3-003 concentrations ranging from 0.01 to 10 mM. In all cases, mRNA levels of JMN3-003exposed cells were similar to those of the vehicle-treated references, while exposure to Actinomycin D, which blocks RNA synthesis through arrest of the transcription initiation complex [33] , resulted in a major reduction in relative mRNA levels (figure 5A).
Immunodetection of cellular GAPDH and plasmid-encoded MeV F protein under the control of the CMV promoter demonstrated that productive transcription in the presence of the compound furthermore coincides with uninterrupted translation and, in the case of F, co-translational insertion into the host secretory system (figure 5B). Furthermore, equivalent levels of proteolytically processed F 1 material in JMN3-003 and vehicleexposed cells indicated that intracellular vesicular transport remains intact in the presence of JMN3-003, since cleavage is mediated by the cellular protease furin in a late-Golgi compartment [1] . In contrast to host-encoded or transiently expressed proteins, expression of virus-encoded proteins in the context of paramyxovirus or orthomyxovirus infection was fully blocked by 100 nM JMN3-003 (figures 5C and D). Thus, these observations demonstrate that the compound efficiently suppresses the expression of virus-encoded proteins, but that this is not due to general interference of the inhibitor with cellular mRNA synthesis or translation. This phenotype suggests possible interference of JMN3-003 with early steps of the viral life cycle, such as entry or viral RdRp activity, as the basis for antiviral activity.
To differentiate between those alternatives and identify the point of arrest in the viral life cycle induced by JMN3-003, we first examined whether the compound blocks membrane fusion and thus viral entry. Expression of plasmid-encoded paramyxovirus envelope glycoproteins in receptor-positive cells typically results in extensive cell-to-cell fusion, the hallmark cytopathic effect associated with most paramyxovirus infections in vitro [1] . Transient membrane fusion assays allow a quantitative assessment of whether an inhibitor blocks viral entry or post-entry steps of the viral life cycle [20, 34] . When we examined MeV glycoproteinmediated cell-to-cell fusion microscopically (figure 6A) and in a luciferase reporter-based quantitative cell-to-cell fusion assay (figure 6B) in the presence of JMN3-003, we observed extensive membrane fusion indistinguishable from that seen in vehicletreated controls, indicating that the compound does not act as an entry inhibitor.
To determine whether JMN3-003 predisposes host cells against viral infection by inducing an antiviral state, we pre-treated cells with the compound, followed by wash-out of the substance and virus infection after different time periods. Independent of incubation time after removal of the compound, we could not detect any substantial inhibitory effect in this set-up (figure 6C), arguing against priming of the innate antiviral response by JMN3-003. Likewise, preincubation of viral particles with JMN3-003 prior to removal of the article and infection lacked any appreciable antiviral effect (figure 6D), excluding direct virucidal activity of the substance.
When added in a time-of-addition experiment at distinct time points post-infection in comparison with two previously characterized, pathogen-targeted antivirals, the inhibition profile of JMN3-003 was distinct from that of the entry inhibitor AS-48 [34] but very closely resembled the profile of the AS-136A RdRp blocker class ( [20] , figure 6E ). Thus, these data point towards inhibition of the viral RdRp activity by JMN3-003 as one possible underlying mechanism for antiviral activity of the compound.
For myxovirus infection, the viral RdRp complex mediates both genome transcription and replication to express viral proteins and generate progeny genomes, respectively. Replication occurs through generation of an antigenome of positive polarity, which then serves as template for negative strand genome synthesis [1] . To directly test whether JMN3-003 affects viral RdRp activity in the context of virus infection, we determined the copy numbers of MeV-Alaska mRNA and antigenome in infected, compoundtreated cells relative to vehicle-treated controls by quantitative RT-PCR. Presence of JMN3-003 caused a dose-dependent reduction in viral RNA levels (figure 7A). At a concentration of 100 nM, for instance, we observed a .100-fold reduction of viral [35] , was required to achieve comparable mRNA and antigenome reduction levels ( figure 7A ).
When this assay was applied to orthomyxovirus infection, we likewise observed a dose-dependent inhibition of influenza A/ WSN antigenome levels relative to vehicle treated controls (figure 7B). Parallel quantification of genome copy numbers of released progeny virus demonstrated that an approximate .100fold drop in relative viral antigenome levels correlates to a .10,000-fold reduction in genome copies of released progeny virions ( figure 7B) .
Assessment of viral RdRp activity in a plasmid-based minireplicon reporter system confirmed dose-dependent inhibition of RdRp by JMN3-003 also in a sub-infection setting, since luciferase reporter expression was fully blocked at compound concentrations of approximately 100 nM (figure 7C). Taken together, these data suggest indirect inhibition of the viral polymerase complex through interaction of the compound with a cellular cofactor required for RdRp activity as the basis for the antiviral effect of JMN3-003.
It has been suggested for different viral pathogens that a hostdirected antiviral approach has the potential to reduce the frequency of viral escape from inhibition compared to direct targeting of pathogen components [14, 15] . To explore whether resistance to JMN3-003 could be induced experimentally, we attempted stepwise viral adaptation to growth in the presence of the compound in comparison with the pathogen-specific MeV RdRp inhibitor AS-136A [36] . Following an escalating dose scheme, inhibitor concentrations were doubled when virusinduced cytopathicity became detectable microscopically. While robust resistance to the pathogen-targeted AS-136A control emerged rapidly in an approximate 15 to 20-day time window (tolerated dose at the end of adaptation was $30 mM, equivalent to $100-fold resistance), only marginal increases in the tolerated dose could be detected for JMN3-003 after 90 days of continued viral incubation in the presence of the substance (figure 8). These results are consistent with a host-directed mechanism of action of JMN3-003 and suggest the existence of a systemic barrier that prevents rapid viral escape from inhibition by the article.
In recent years, host cell-directed antivirals have experienced growing recognition as a new concept for the development of advanced generation antivirals with the potential to counteract the challenge of preexisting or rapidly emerging viral resistance [14, 15] . Novel automated genomics and proteomics analyses have greatly advanced our insight into host-pathogen interactions [37, 38, 39, 40, 41, 42, 43, 44] . These studies have underscored the notion that several cellular pathways are exploited for virus replication [45, 46] , supporting the hypothesis that a host-directed antiviral may enjoy an expanded viral target range, rendering it effective for the treatment of several related viral diseases.
Technologies applied for host-directed drug discovery include cDNA and siRNA-based microarray analyses combined with pathway-guided data mining [47, 48, 49, 50, 51] , loss-of-function screens using aptamers or small oligonucleotides [52, 53, 54, 55, 56, 57, 58] , protein array analyses [59] and chemical library screening [60, 61] . By combining automated library screening [19] with counter screens against a variety of related viral pathogens of the myxovirus families, we have identified a candidate scaffold that, after moderate hit-to-lead chemistry, adheres to the profile of a host-directed antiviral based on several lines of evidence: I) antiviral activity is host cell species-dependent, indicating specific interaction with a distinct host factor rather than a viral component. Host cell-specific activity is incompatible with compound docking to conserved viral factors. For example, carbohydrate structures exposed on viral envelope glycoproteins that are targeted by antiviral lectins such as pradimicin A [62] . Furthermore, it is incompatible with an undesirable unspecific, promiscuous mode of action of the compound [63] ; II) affinities against a panel of human pathogens of the paramyxovirus family as well as laboratory adapted and wild type influenza virus isolates were very similar throughout (average EC 50 concentrations are ,40 nM). Equivalent active concentrations argue against compound docking to distinct viral components and suggest that inhibition of distinct myxovirus families follows the same mechanism of action; III) in vitro adaptation attempts to induce viral resistance were unsuccessful even after extended exposure times to the drug. A full assessment of the frequency of viral escape from inhibition by JMN3-003 will certainly need to include in vivo virus adaptation attempts in suitable animal models, since the rate of resistance build-up may vary between tissue culture and in vivo settings. We nevertheless reliably induced resistance in less than 30 days to a pathogen-directed MeV RdRp inhibitor that was analyzed in parallel, which is fully consistent with our previous experience [36] and provides confidence for the validity of our overall experimental design for viral adaptation.
Mechanistic analysis of the bioactivity of the JMN3-003 compound class through characterization of exposed cells and time-of-addition experiments revealed two distinct phenotypes, a temporary cell cycle arrest in the G 1 /S phase and an arrest in the myxovirus life cycle at a post-entry step. Current libraries of chemical analogs of JMN3-003 do not yet permit a definitive conclusion as to whether both activities adhere to discrete structure-activity relationships or are causally linked, but a bulk of experimental data demonstrate that host cell cycle arrest per se has no inhibitory effect on replication of paramyxoviruses such as MeV. Not only does the virus itself induce a G 1 /S-phase arrest in infected T lymphocytes [29, 30] , we also found that exposure of infected cells to alsterpaullone, a potent blocker of G 1 /S-phase cell cycle progression through nanomolar inhibition of cellular cyclindependent kinases [28] , did not affect the extent of virus replication even at concentrations exceeding reported alsterpaullone EC 50 values by more than 1,000-fold. Likewise consistent with the notion that the antiviral activity of JMN3-003 is not based on cell cycle arrest itself, virus inhibition was not restricted to the context of immortalized, rapidly dividing tissue culture cell lines but extended with equal potency to primary human PBMCs.
Reversible cell cycle arrest and block of virus replication indicate non-covalent docking of JMN3-003 to its target structures, which is corroborated by the compound's stability, low chemical reactivity profile and the complete absence of virucidal activity in pre-incubation settings. An inhibition profile of JMN3-003 closely mimicking that of AS-136A, the pathogen-directed blocker of MeV RdRp targeting the viral L polymerase protein [36] , and the block in viral RdRp activity in the context of viral infection and minireplicon reporter assays by JMN3-003 consistently point towards interaction of the compound with a host cofactor essential for RdRp function as the basis for its antiviral activity. While viral RdRp depends on a variety of host cell components [1] , unperturbed cellular mRNA synthesis and, thus, uninterrupted host RNA polymerase function in the presence of compound exclude interference of JMN3-003 with essential transcription initiation factors.
Recently, accumulating evidence has implicated host cell kinases as regulators of the activity of RdRp complexes of different negative-strand RNA viruses [64] : host cell kinases of the PI3K-Akt pathway manipulate paramyxovirus RdRp activity through Akt-mediated phosphorylation of the viral phosphoprotein, an essential component of the RdRp complex. Furthermore, Akt activity itself is upregulated through activation of PI3K during influenza A infection via direct interaction of the viral NS1 protein with PI3K [65, 66] . In the case of MeV, however, published data [67, 68] and our own observations (Krumm and Plemper, unpublished) demonstrate that Akt inhibition causes a moderate reduction in virus release, whereas titers of cell-associated progeny particles remain unchanged. While this rules out the PI3K-Akt pathway as a direct target for JMN3-003, it illuminates the intricate regulatory interactions between pathogen and host, which provide a wealth of possible points of entry for antiviral intervention. Future identification of the molecular target of JMN3-003 carries high potential to further our understanding of these interactions and may conceivably provide a basis for pharmacophore extraction and structure-driven scaffold optimization.
We note that the central sulfur in the JMN3-003 chemical scaffold could potentially render the molecule vulnerable to rapid phase I oxidation and thus compromise both metabolic stability and bioavailability. For instance, it has been reported that flavincontaining monooxygenases [69] , dioxygenases [70] and cytochrome P-450 enzymes [71] catalyze oxidation of alkylaryl sulfides to sulfoxides (R 2 S = O). However, the high stability of JMN3-003 in the presence of human hepatocyte subcellular fractions and human plasma argues against an undesirable short in vivo half-life of the substance. This is corroborated by good metabolic stability of the structurally similar HIV reverse transcriptase inhibitor RDEA-806 [72, 73] , which shares the central 2-thio-acetamide connector with JMN3-003 and has achieved success in clinical trials: the compound was well tolerated in both Phase 1 and 2a studies after single or multiple oral doses and showed no drugrelated CNS toxicity [72, 73] , creating a clinical precedence for the applicability of the broader scaffold. Although RDEA-806 follows a different mechanism of action than JMN3-003 and lacks any anti-paramyxovirus activity, the structural similarities provide sufficient confidence for the overall developmental potential of the JMN3-003 class to recommend it as a promising candidate for advanced synthetic optimization towards preclinical validation and development.
In toto, we have identified a novel chemical class of viral inhibitors that block viral RdRp activity with a host factormediated profile. A complete activity workup after synthetic identification of a clinical lead analog will be required to fully appreciate the range of the different viral families inhibited by the substance. However, we consider human pathogens of the myxovirus families that are primarily associated with acute disease among the most suitable for host-directed antiviral efforts due to anticipated short treatment regimens. While we cannot exclude that resistance to JMN3-003 may eventually emerge in in vivo settings, our in vitro adaptation efforts support the hypothesis that the mechanism of action of this compound class establishes a strong barrier against rapid viral escape from inhibition.
All cell lines were maintained at 37uC and 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Vero (African green monkey kidney epithelial) cells (ATCC CCL-81) stably expressing human signaling lymphocytic activation molecule (CD150w/SLAM), called in this study Vero-SLAM cells [74] , and baby hamster kidney (BHK-21) cells stably expressing T7 polymerase (BSR-T7/5 (BHK-T7) cells [75] ) were incubated at every third passage in the presence of G-418 (Geneticin) at a concentration of 100 mg/ml. Lipofectamine 2000 (Invitrogen) was used for cell transfections. Peripheral blood mononuclear cells (PBMCs) were prepared through overlay of whole blood samples from mixed, healthy human donors (Emory University Institutional Review Board approval IRB00045690, Phlebotomy of Healthy Adults for Research in Infectious Diseases and Immunology) on Ficoll Hypaque solution, followed by centrifugation at 2406g for 30 minutes at room temperature and removal of the interphase material. Red blood cells were lysed with RBC lysis solution (Sigma), followed by repeated washing of extracted PBMCs with phosphate buffered saline and transfer to tissue culture plates pre-coated with poly-L-lysine (Sigma). Other primary human cell lines were obtained from PromoCell, Germany. Virus strains used in this study were MeV isolate MVi/Alaska.USA/16.00, genotype H2 (MeV-Alaska) [76] , HPIV3, MuV strain South Africa, RSV strain Long, laboratory adapted influenza A strains WSN (H1N1) and PR8/34 (H1N1), swine-origin influenza virus isolates S-OIV Texas and Mexico, vaccinia virus and sindbis virus. To prepare virus stocks, cells permissive for the virus to be amplified (Vero-Slam, Vero, HepG2 (ATCC HB-8065), and Madin-Darby canine kidney (MDCK)) were infected and incubated at 37uC. Cell-associated paramyxovirus and vaccinia virus particles were harvested by scraping cells in OPTIMEM (Invitrogen), followed by release of virus through two consecutive freeze-thaw cycles. Influenza virus and sindbis virus particles were harvested from cell culture supernatants. Titers of MeV and MuV were determined through 50% tissue culture infective dose (TCID 50 ) titration according to the Spearman-Karber method [77] as described [78] , titer of all other viruses were determined by plaque assay on permissive cells.
To determine genome copy numbers of released progeny influenza A particles (strains WSN, PR8/34, S-OIV Texas and Mexico), culture supernatants of infected MDCK cells (4610 5 cells/well in a 12-well plate format) were harvested and total RNA prepared using a QIAcube automated extractor and the QIAamp viral RNA mini kit reagent. Purified RNA was then subjected to quantitative real time (qRT) PCR analysis using an Applied Biosystems 7500 Fast real-time PCR system and the qRT-PCR TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). Primers and probe are based on recent reports [79] and universally reactive with all influenza A strains including the recent S-OIV (H1N1) isolates. To generate a qRT-PCR standard, genome segment seven of influenza A/WSN was subcloned into pCR2.1-TOPO vector (Invitrogen) and copy numbers of the resulting standard calculated using Promega's BioMath Calculator tools (http://www.promega.com/ biomath/). For each TaqMan reaction, 10-fold serial dilutions of the linearized plasmid ranging from 10 7 to 10 1 were amplified in parallel.
Chemical synthesis of compounds AS-48, AS-136A and RDEA-806 was achieved as previously described [24, 34, 36] . Synthesis of JMN3-003, N-(4-methoxyphenyl)-2-nitroaniline (substance (3) in figure S1), and analogs JMN5-165 and JMN5-166 was achieved as outlined schematically in figure S1. To prepare inhibitor stocks, compounds were dissolved at 75 mM in DMSO.
Vero-SLAM cells were infected with MeV-Alaska at an MOI of 0.4 pfu/cell in the presence of the inhibitor analyzed ranging from 75 mM to 293 nM in two-fold dilutions. At 96 hours post-infection, cell monolayers were subjected to crystal violet staining (0.1% crystal violet in 20% ethanol), and the absorbance of dried plates at 560 nm determined. Virus-induced cytopathicity was then calculated according to the formula [% rel. CPE= 1002(experimental-minimum)/ (maximum-minimum)*100], with minimum referring to infected, vehicle-treated wells and maximum to mock-infected wells.
Cells were infected with the specified myxovirus at an MOI = 0.1 pfu/cell (all paramyxoviruses assessed), 0.05 pfu/cells (influenza viruses), 1.0 (vaccinia virus), or 10 sindbis virus) in the presence of a range of compound concentrations or equivalent volumes of solvent (DMSO) only, and incubated in the presence of compound at 37uC. When vehicle treated controls approached the end of the logarithmical growth phase, progeny viral particles were harvested and titered by TCID 50 titration, plaque assay or TaqMan real-time PCR, respectively, as described above. Plotting virus titers as a function of compound concentration allowed quantitative assessment of resistance. Where applicable, 50% inhibitory concentrations were calculated using the variable slope (four parameters) non-linear regression-fitting algorithm embedded in the Prism 5 software package (GraphPad Software). In vitro assessment of metabolic and plasma stability JMN3-003 was mixed with liver S9 fractions (protein concentration 2.5 mg/ml) from pooled mixed gender humans (Xeno-Tech) at a final concentration of 1 mM and reactions initiated by the addition of cofactors (1.14 mM NADPH, 1.43 mM glucose-6phosphate, 1.43 mM uridine 59-diphosphoglucuronic acid, 9.42 mM potassium chloride, 2.28 mM magnesium chloride) in 100 mM potassium phosphate buffer, pH 7.4. Samples were incubated at 37uC with mixing, aliquots removed after 0, 15, 30 and 60 minutes and subjected to reversed-phase LC-MS/MS (Applied Biosystems API 4000 QTRAP with heated nebulizer; Turbo IonSpray for JMN5-166) analysis. Peak areas were measured to calculate half life and percent of input compound remaining according to the formulas t 1/2 = (20.693/slope of linear regression analysis of log transformed peak area versus) and % input remaining = (peak area of test compound at t x /peak area of test compound at t 0 )*100. Positive controls to assess the metabolic competency of the liver S9 fractions were 7-Ethoxycoumarin, Propranolol, and Verapamil (Sigma), which were analyzed in parallel to the article. To determine compound plasma stability, articles were mixed with freshly prepared human plasma at a final concentration of 0.5 mM and incubated at 37uC for up to 120 minutes. Aliquots were removed at distinct time points as indicated and analyzed by LC-MS/MS with detection of the compound at 254 nm. Values are expressed as percent of compound remaining at each time relative to the amount of that compound present at the starting time point.
Actively proliferating HeLa cells were exposed to JMN3-003 (10 mM), hydroxyurea (4 mM), or nocodazole (200 ng/ml) for 36 hours, followed by resuspension in buffer I (20 mM citrate/PO, pH 3.0, 0.1 mM EDTA, 0.2 M Sucrose, 0.1% Triton X-100) and staining in buffer II (10 mM Citrate/PO, pH 3.8, 0.1 M sodium chloride, 20 mg/ml acridine orange) as described [80] . Green fluorescence at 525 nm resulting from DNA intercalating acridine orange was then measured using a BD LSRII flow cytometer and FlowJo software (Tree Star) for data analysis. For comparison, unstained and stained, solvent-only exposed cells were examined in parallel.
Cells were lysed with RIPA buffer (50 mM Tris/CL, pH 7.2, 1% deoxycholate, 0.15% sodium dodecylsulfate, 150 mM sodium chloride, 50 mM sodium fluoride, 10 mM EDTA, 1% NP-40, 1 mM PMSF, protease inhibitors). Aliquots with equal total concentrations of cleared lysates (20,0006g; 10 min; 4uC) were mixed with 2x-urea buffer (200 mM Tris, pH 6.8; 8 M urea; 5% sodium dodecyl sulfate (SDS); 0.1 mM EDTA; 0.03% bromphenol blue; 1.5% dithiothreitol) and denatured for 25 min at 50uC. Samples were then fractionated on 10% SDS-polyacrylamide gels, blotted to polyvinylidene difluoride (PVDF) membranes (Millipore) and subjected to enhanced chemiluminescence detection (Pierce) using specific antisera directed against phosphorylated or non-phosphorylated cdc2-cyclin B kinase (Cell Signaling Technology), GAPDH (Abcam), the cytosolic tail of the MeV F protein [81] , or influenza A/WSN virus M2 (Thermo Scientific). Immunostained PVDF membranes were developed using a ChemiDoc XRS digital imaging system (Bio-Rad) and horseradish peroxidase conjugated anti-species IgG (mouse or rabbit) antibodies. For densitometry, signals were quantified using the Quanti-tyOne software package (Bio-Rad).
Vero cells were seeded at a density of 6610 5 cells and incubated in the presence of 10 mM JMN3-003 or vehicle only for 30 hours at 37uC. Cells were then washed extensively and reseeded at a density of 1610 5 cells per well, followed by continued incubation at 37uC and assessment of life/dead cell numbers every 24 hours using a Countess automated cell counter (Invitrogen). Cells were reseeded as before when fastest growing cultures approached confluency. Growth rates were calculated for each 24-hour time interval using the Prism software package (GraphPad Software Inc.) based on the formula Y = Y 0 *exp(K*X) with Y equaling life cell numbers, Y 0 the Y value at the starting time (t 0 ), and K the growth constant equaling ln(2)/doubling-time.
Cells were infected with either recombinant MeV Edmonston (recMeV-Edm) [82] (Vero cells, MOI = 1.0) or influenza A/WSN (MDCK cells, MOI = 0.05), followed by removal of inocula one hour post-infection and addition of JMN3-003 in growth media at 0.1 mM or 1 mM. All MeV infected wells received in addition fusion inhibitory peptide (FIP, Bachem) at 100 mM to prevent premature breakdown of the monolayer through viral CPE in the vehicle control wells prior to RNA extraction. Twenty-four (influenza A/WSN) or forty (recMeV-Edm) hours post-infection, total RNA was prepared from all wells using the QIAcube automated extractor and the RNeasy Mini Kit (Qiagen), and subjected to reverse transcription using Superscript II Reverse Transcriptase (Invitrogen). For RNA samples originating from recMeV-Edm infected cells, antigenomespecific primer 5-GGCTCCCTCTGGTTGT or oligo-dT primer (viral mRNA and GAPDH quantification) were used for cDNA priming. In the case of samples originating from influenza A/WSN infected cells, primers for cDNA synthesis were 5-AGTAGAAA-CAAGGTAGTTT (antigenome) or oligo-dT (mRNA and canine GAPDH). Real-time reactions were carried out using an Applied Biosystems 7500 Fast real-time PCR system and iQ Fast SYBR Green Supermix with ROX (Bio-Rad). Probes were a fragment at the N/P junction (MeV antigenomic RNA, 5-AACCAGGTCCACACAG and 5-GTTG TCTGATATTTCTGAC), a fragment of MeV F mRNA (5-GTCCACCATGGGTCTCAAGGTGAACGTCTC and 5-CAGTTATTGAGGAGAGTT), a fragment of human GAPDH (SABiosciences proprietary primers), a fragment of influenza A/WSN segment seven (influenza A/WSN antigenomic RNA, 5-tagctccagtgctggtct and 5-AAGGCCCTCCTTTCAGTCC), and a fragment of canine GAPDH (Qiagen proprietary primer). Melting curves were generated at the end of each reaction to verify amplification of a single product. To calculate DDC T values, CT values obtained for each sample were normalized for GAPDH as reference and then DC T values of JMN3-003-treated samples normalized for the FIP-treated controls. Final quantification was based on three independent experiments in which each treatment condition and RT primer setting were assessed in triplicate. To assess the relative quantities of cellular mRNA, 9610 5 HeLa cells were incubated in the presence of JMN3-003 (0.01, 0.1, 1.0, 10.0 mM final concentration), AS-136A (25 mM), Actinomycine D (5 mg/ml), or vehicle only for six hours at 37uC, followed by preparation of total RNA as described above. Quantitative TaqMan RT-PCR was again achieved using the TaqMan Fast Master Mix (Applied Biosystems) combined with proprietary primer and probe sets specific for Induced myeloid leukemia cell differentiation protein 1-(MCL1), MAPK phosphatase 1 (MKP1), and ankyrin repeat and SOCS boxcontaining protein 7-(ASB7) encoding mRNAs (Applied Biosystems). Samples were standardized for GAPDH as before and normalized values expressed relative to the equally analyzed vehicle-treated controls.
An effector cell population (3610 5 cells/well) was cotransfected with 2 mg each of MeV H and F expression plasmids. To inhibit fusion until the cell overlay, the effector cells are incubated in the presence of 100 mM fusion inhibitory peptide (Bachem). Single transfections of plasmids encoding MeV F served as controls. Target cells (6610 5 cells/well) were transfected with 4 mg of the reporter plasmid encoding firefly luciferase under the control of the T7 promoter. Two hours post-transfection, modified vaccinia virus Ankara expressing T7 polymerase at an MOI of 1.0 PFU/ cell was added to the effector cells. Following incubation for 16 h at 37uC, target cells were detached and overlaid on washed effector cells at a 1:1 ratio and incubated at 37uC in the presence of different JMN3-003 concentrations as indicated. Four hours post-overlay, cells were lysed using Bright Glo lysis buffer (Promega), and the luciferase activity determined in a luminescence counter (PerkinElmer) after addition of Britelite substrate (PerkinElmer). The instrument's arbitrary values were analyzed by subtracting the relative background provided by values of the controls, and these values were normalized against the reference constructs indicated in the figure legends. On average, background values were ,1% of the values obtained for reference constructs. For qualitative assessment, transfected Vero-SLAM cells were photographed 18 hours post-transfection at a magnification of 6200.
For virus pre-incubation assays, 10 7 infectious MeV-Alaska particles were incubated for 60 minutes at 37uC in the presence of JMN3-003 (1.0 mM final concentration) or vehicle only, followed by 1,000-fold dilution in growth media and transferred to 3610 5 Vero-Slam cells/well (corresponding to final compound concentrations after pre-incubation of 1 nM and an MOI = 0.033). Reference wells were kept at 1.0 mM JMN3-003 for the duration of the experiment. Cell-associated viral particles were harvested 24 hours post-infection and infectious titers determined by TCID 50 titration. To assess cell priming, Vero-Slam cells (3610 5 /well) were incubated in the presence of JMN3-003 at 1.0 mM for one hour at 37uC at the indicated time points pre-infection, followed by washing and further incubation in growth media. Immediately before infection, cells were reseeded at a density of 2.5610 5 per well and infected with MeV-Alaska at an MOI = 0.2 pfu/cell. Inocula were replaced with growth media four hours post-infection and cells incubated for approximately 20 hours. Cell-associated viral particles were then harvested and infectious titers determined by TCID 50 Minireplicon assays BSR T7/5 cells (5610 5 /well) were transfected with plasmid DNAs encoding MeV-L (0.24 mg), MeV-N (0.94 mg) or MeV-P (0.29 mg) and 2 mg of the MeV luciferase minigenome reporter plasmid [83] . Control wells included identical amounts of reporter and helper plasmids but lacked the L-encoding plasmid. At the time of transfection, JMN3-003 was added as specified, while control wells received vehicle only for comparison. Thirty-six hours post-transfection, cells were lysed with Bright GLO lysis buffer and relative luciferase activities determined using the Britelite substrate and a luminescence counter as outlined above.
Adaptations were carried out essentially as we have previously described [36] . Briefly, Vero-SLAM cells were infected with MeV-Alaska at an MOI of 0.1 pfu/ml and incubated in the presence of gradually increasing JMN3-003 concentrations starting at 0.5 mM. Equally infected cells treated with the virus polymerase targeted RdRp inhibitor AS-136A were examined in parallel. When cultures became over confluent, cells were reseeded for continued incubation in the presence of the same compound concentration as before. At detection of extensive cell-to-cell fusion, cellassociated viral particles were harvested, diluted 10-fold and used for parallel infections of fresh cell monolayers in the presence of compound at unchanged and doubled concentrations. Cultures treated with the highest compound concentrations in which virusinduced cytopathicity became detectable were used for further adaptation. The approach was terminated after 90 days of continued incubation or when virus-induced cytopathicity was readily detectable in the presence of 30 mM compound in accordance with previous results [36] .  Insight into the Interaction of Metal Ions with TroA from Streptococcus suis BACKGROUND: The scavenging ability of sufficient divalent metal ions is pivotal for pathogenic bacteria to survive in the host. ATP-binding cassette (ABC)-type metal transporters provide a considerable amount of different transition metals for bacterial growth. TroA is a substrate binding protein for uptake of multiple metal ions. However, the function and structure of the TroA homologue from the epidemic Streptococcus suis isolates (SsTroA) have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here we determined the crystal structure of SsTroA from a highly pathogenic streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis in complex with zinc. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that apo-SsTroA binds Zn(2+) and Mn(2+). Both metals bind to SsTroA with nanomolar affinity and stabilize the protein against thermal unfolding. Zn(2+) and Mn(2+) induce distinct conformational changes in SsTroA compared with the apo form as confirmed by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra. NMR data also revealed that Zn(2+)/Mn(2+) bind to SsTroA in either the same site or an adjacent region. Finally, we found that the folding of the metal-bound protein is more compact than the corresponding apoprotein. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a mechanism for uptake of metal ions in S. suis and this mechanism provides a reasonable explanation as to how SsTroA operates in metal transport. Streptococcus suis serotype 2 (S. suis 2) is a gram-positive coccus that causes diseases in pigs and humans, and is therefore a zoonotic pathogen [1, 2] . Since the first human case of S. suis 2 infection was described in Denmark in 1968, over 400 human S. suis infection cases have been recorded thus far, covering nearly 30 countries [2, 3] . In 1998 and 2005, S. suis 2 human infections, with the new disease form of streptococcal toxic shock syndrome (STSS), had been reported in China, following the outbreaks in swine stocks initially, which caused a total of 240 human infections and claimed 52 lives [4] . Our group, in collaboration with other groups, has sequenced the entire genomes of the representatives of these two virulent S. suis 2 isolates (05ZYH33 and 98HAH12) in 2007 [5] and laid the foundation for the study of the STSS-causing S. suis 2 at the molecular level to reveal its pathogenicity [1, 6, 7] .
Almost one third of proteins in nature depend on a particular metal for their diverse functions [8] and divalent metal cations are essential for bacteria [9] . For instance, manganese plays a primary antioxidant role in bacteria [10] and affects bacterial pathogenesis [11] . Zinc is one of the most abundant metals in bacteria and is an essential co-factor of many metabolic enzymes and transcription factors [12] . The Mn 2+ ion is characterized as hard metal and tends to prefer hard ligands, while zinc prefers soft ligands [13] . To obtain appropriate cellular concentrations of transition metal ions, bacteria have evolved elaborate machineries (such as ABCtransporters and ion channels) to transport these ions across biological membranes.
ABC transporters and metal ions are significant for bacteria growth and virulence in Streptococcus [14] . However, only recently have insights emerged into the metal metabolism of S. suis. The adcR gene (which encodes the regulator of the Adc operon) was isolated by divalent cation deprivation [15] . Furthermore, the extracted cell surface proteins of S. suis from mutants defective in genes regulating metal ion uptake are able to confer significant protection against S. suis 2 infection in mice [16] . Previously, we identified a global zinc-response regulator of the Zur family (zinc uptake regulator) from S. suis 2 [17] , unveiling its relationship with zinc homeostasis in this organism. Whole-genome sequence analysis led to the proposition that S. suis expresses two putative transition metal transport systems, encoded by the Adc operon [15] and Tro operon (Table S1) , consistent with the situation in many other bacterial pathogens that harbor several metal transporters.
TroA was isolated from outer membrane preparations of Treponema pallidum (TpTroA) [18] . This protein belongs to a wellstudied family, formerly designated as cluster 9 substrate binding protein (SBP) [19] , but recently reclassified into the A-1 family of prokaryotic SBPs [20] . The SsTro operon contains five genes, an ATP-binding cassette metal transport system (troABCD) (encoding four proteins) and a transcriptional regulator (troR) (Fig. 1A) . The crystal structures of some A-1 family SBPs have been determined over the past decade. These structures include Mn-specific SBPs (e.g.,TpTroA [21] - [22] , Streptococcus pneumoniae PsaA [23] , and Synechocystis 6803 MntC [24] ), Zn-specific SBPs (e.g., S. pneumoniae AdcAII [25] , Synechocystis 6803 ZnuA [26, 27] , and S. pyogenes Lbp [28] ) and Fe-specific SBPs (Streptococcus pyogenes MtsA [29] ). They share a common structure of two (a/b) 4 domains linked by a long helix, with the metal bound at the interface between the domains. Hazlett et al. [30] and Desrosiers et al. [31] revealed that TpTroA has high binding affinity for both Zn 2+ and Mn 2+ , but Zn 2+ is considered to be the primary substrate of TpTroA [32] .
Although the A-1 family SBPs has been extensively characterized they have been the subject of considerable controversy with respect to their metal binding properties. When bound to metal, by CD spectra TpTroA does not undergo structural changes, however, T. pallidum ZnuA undergoes conformational changes when Zn 2+ is loaded [31] . Synechocystis 6803 ZnuA displays concomitantly large conformational changes in two of its three chelating histidines due to the release of Zn because it possesses two Zn binding sites [27] . However, the second binding site is absent in other transporters (e.g., TpTroA), and therefore its exact biological function remains unclear [33] . To obtain further insight into the function of SsTroA, we determined the X-ray crystal structure of the Zn 2+ -bound form of the SsTroA at a resolution of 2.6 Å , and further characterized the recombinant SsTroA. Binding of divalent metal cations to SsTroA, induces a substantial conformational reorganization to a more ordered state. Our structural and functional data reported here lay out the scenario of SsTroA participating in S. suis divalent metal homeostasis.
In silico analyses, cloning, purification and biochemical characterization of SsTroA
In silico analysis revealed that SsTroA is a putative lipoprotein (Fig. 1B) . The amino-terminal signal sequence of such lipoproteins is distinguishable by the presence of a lipobox motif at the signal sequence cleavage site. The conserved lipobox motif is usually characterized as L 23 -[A/S/T] 22 -[G/A] 21 -C +1 and lipid modification is achieved through a covalent linkage of a diacylglyceride to the conserved cysteine residue. Indeed, the cysteine residue is absolutely conserved in all bacterial lipoproteins [34, 35] . SsTroA contains a typical type II signal peptide for Sec-dependent transport. Its presumptive lipobox sequence is LGAC, containing the indispensible cysteine in position 30, indicating that SsTroA is not released into the environment but rather inserted into the plasma membrane via a diacylglyceryl anchor. Alignment of sequences also revealed that the metal binding site of SsTroA is very similar to Mn-specific SBPs (Fig. 1B) . These data suggested that the SsTroA should physiologically bind Mn 2+ .
We cloned the mature SsTroA domain (residues 36-317), and the protein was purified using GST-affinity column and a subsequent gel-filtration chromatography (Fig. S1A ). Samples collected from elution fractions were separated by 12% SDS-PAGE (Fig. S1A ) and the results show that SsTroA has a molecular mass of ,34 kDa, in consistence with the theoretical monomer molar mass of 32 kDa. An analytical FPLC SuperdexH 200 profile revealed that SsTroA exists as a mixture of monomer and dimer in solution, though mainly as a monomer (Fig. S2A) . To determine the native mass of SsTroA (unaffected by protein shape), quantitative hydrodynamic analyses were performed by analytical ultracentrifugation. Analysis of the sedimentation velocity profiles using the SEDFIT program yielded the weight average molar mass of the major peak is 31.8 kDa ( Figure S2B ), which accounts for 90% of the total protein (i.e., nearly identical to the theoretical molar mass of 32 kDa). Another peak at 62.6 kDa (6.8% of the total) is believed to correspond to dimers. The other ,3.2% of the protein has a significantly-higher sedimentation coefficient, but the amount of this subpopulation did not change significantly as a function of protein concentration. Thus, our data suggest that under the solution conditions used, a small portion of SsTroA is involved in the formation of stable complexes. This is consistent with the evidence that periplasmic SBPs exist in a monomer/dimer equilibrium, with monomers having higher affinity for the substrate [36, 37] .
To determine if SsTroA is expressed in S. suis 2, total cellular proteins from S. suis 2 cells, purified SsTroA, and negative control protein (Ss1661 protein from S. suis 2) were separated by SDS-PAGE, transferred to a nitrocellulose membrane (GE Healthcare), and probed with anti-SsTroA serum. A 34-kDa protein band was detected in the S. suis 2 cell lysate (Fig. S1B) , indicated that SsTroA is expressed in the bacteria.
To investigate the presence of divalent metal ions in the purified SsTroA and assess the ability of SsTroA to bind metal ions, the recombinant protein was subjected to a test of inductively coupled plasma mass spectrometry (ICP-MS) analysis. The metal contents of SsTroA as isolated or after reconstitution were shown in Table 1 . The stoichiometry was found to be 0.9660.2 Zn 2+ /SsTroA monomer and 0.8960.2 Mn 2+ /SsTroA monomer, which suggests that recombinant SsTroA protein binds Zn 2+ and Mn 2+ roughly in a 1:1 ratio. The ICP-MS data provided support for one metal binding site per SsTroA monomer, as observed in TpTroA and Neisseria gonorrhoeae MntC [31, 33] . This led us to evaluate the metal interactions with the SsTroA protein in more details.
We applied isothermal titration calorimetry (ITC) to monitor the energetics of Zn 2+ and Mn 2+ binding to the apo-SsTroA. ITC measures the heat directly released or absorbed upon an interaction triggered by mixing two components, and is capable of calculating both the extent of the ligand binding affinity and the free energy values (DG) and enthalpy(DH) changes, from which entropy (DS) is determined [38] . The isotherms for loading with Zn 2+ and Mn 2+ appeared in normal sigmoidal titration curves, in agreement with our ICP-MS data. Again the binding stoichiometry (n) was determined to be ,1 in both cases (Fig. 2) , confirming that a specific 1:1 complex is formed in each case. The association constants (Ka) for each metal ion binding to SsTroA were around 10 7 M 21 (Fig. 2) . SsTroA affinities for Zn 2+ and Mn 2+ were calculated from duplicate measurements with K D s ( = 1/Ka) of 43469 nM and 254613 nM, respectively, indicating the formation of tight complexes. It should be noted that SsTroA binds Mn 2+ with a higher affinity than that of Zn 2+ . These values are of a similar magnitude to those described for binding of Zn 2+ and Mn 2+ to N. gonorrhoeae MntC [33] . In each metal ion titration with both metal ions, we observed negative enthalpy and entropy values, demonstrating that binding of each ion to SsTroA is an exothermic event. The thermodynamic profiles for metal binding by SsTroA are not identical to those previously reported for TpTroA [31] . Intrinsic metal binding to proteins is usually entropically driven (DH.0) due to the very high dehydration energies of divalent cations. However, the binding enthalpy can become exothermic (DH,0) if metal binding is coupled to a protein conformational change [39, 40] . Our ITC data are consistent with a significant conformational change observed in The four metal-coordinating residues are highlighted in green. The LXXC lipoprotein motif (type II signal peptides for Sec-dependent transport) [34] is highlighted in red. The two conserved residues predicted to form the salt bridge are marked in yellow. Secondary structure elements for SsTroA are shown above the sequence. TpTroA, TroA from Treponema pallidum; SpPsaA, PsaA from Streptococcus pneumonia; SyMntC, MntC from Synechocytis 6803. doi:10.1371/journal.pone.0019510.g001 our nuclear magnetic resonance (NMR) and CD spectra. Thermodynamic parameters for the binding of Zn 2+ /Mn 2+ to SsTroA are summarized in Table S2 .
Room temperature Electron Paramagnetic Resonance (EPR) monitoring of Mn 2+ -SsTroA complex EPR spectroscopy is a useful tool that allows one to probe differences in the metal centers of proteins in solution that may not be apparent in the crystal structures [41] . To further investigate the SsTroA metal center, sample of 100 mM apo-SsTroA was loaded with 100 mM MnCl 2 . The X-band EPR spectra of Mn 2+ -SsTroA at room temperature displays a typical six-line pattern (Fig. 3A) . High-spin Mn 2+ centers typically exhibit EPR resonances with characteristic six-line splitting patterns due to hyperfine interactions between the unpaired electrons and the I = 5/2 55 Mn nucleus, indicating the binding of the Mn 2+ to the protein [42] . Upon entrance of the Mn 2+ into the binding site, the Mn 2+ bound protein reduces the coordination of weakfield water, increasing spin-orbit interactions and zero-field energies relative to that of Mn(H 2 O) 6
. As observed, both titration traces had similar spectra but with varying intensities of the signal. These data clearly illustrate that high-affinity Mn 2+ binding by SsTroA makes SsTroA sensitive to scavenging low levels of free Mn ions in the surrounding environment.
Decreased fluorescence intensity of 1-anilino naphthalene-8-sulfonic acid (ANS) with SsTroA upon metal loading
To examine whether ligand binding induces a variation of exposed hydrophobic protein regions, we applied fluorescence spectroscopy to monitor the binding of the hydrophobic probe ANS to SsTroA. ANS is extensively used to detecting the Table S2 . doi:10.1371/journal.pone.0019510.g002 equilibrium and kinetic folding intermediates of proteins, especially for a partially folded protein [43] . Interaction of the probe with a hydrophobic site yields an enhancement of fluorescence intensity, usually accompanied by a blue shift of the maximum. SsTroA significantly enhances the emission intensity of ANS and promotes a blue shift (Fig. 3B) . Meanwhile, the fluorescence intensity of ANS clearly decreases in the metal bound form compared to the apo-SsTroA. As indicated by the diminished fluorescence, ANS displays stronger binding to Mn 2+ -SsTroA than Zn 2+ -SsTroA. The spectra clearly provide evidence that the addition of metal ions induces a large change in the nonpolarity of the ANS-binding site, and the Mn 2+ causes a greater change than Zn 2+ does. These data suggested that the decreased plots observed in the spectra (Fig. 3B) can be attributed to a more compact and ordered structure for SsTroA in the presence of metal.
As protein aggregation occurs after demetallization, we suspected that apo-SsTroA is unstable compared to the native SsTroA at room temperature, i.e., we assumed that SsTroA displays metal-dependent conformational stability of SsTroA. To obtain further information on the metal sensitivity of the SsTroA and details on possible structural changes occurring upon its interaction with metal ions, we applied CD spectroscopy to monitor these processes. Proteins were prepared by incubation with Zn 2+ and Mn 2+ as previously described [31] . In the far-UV regions (Fig. 4A) , the CD spectra are generally characterized by distinct minima at 208 and 223 nm respectively, which are the features of proteins that contain a-helix conformational elements, consistent with the crystal structure data. The metal bound SsTroA samples both displayed spectra similar to apo-SsTroA, only with minor differences, though both has deeper 208 nm and 223 nm minima relative to apo-SsTroA. The intensity of the 223-nm band of SsTroA is in the order: apo-SsTroA.Zn 2+ -SsTroA.Mn 2+ -SsTroA, suggesting a decrease of the content of secondary structure in the apo form compared to the metal-bound forms. While previous work demonstrates that TpTroA does not manifest changes discernible by CD upon metal binding, in contrast, T. pallidum ZnuA does undergo conformational changes when Zn 2+ is loaded [31] . These data indicated that TpTroA and SsTroA present differential binding behaviors upon metal binding.
Thermal denaturation transitions were assessed by monitoring the temperature dependent change in ellipticity at 223 nm to determine apparent thermal denaturation midpoints in the presence or absence of metal ions. As expected, thermal denaturation proceeded, as expected, with a sharp decrease in ellipticity at 223 nm at the melting temperature and a complete loss of secondary structure (Fig. 4B) . The data are fit very well to a twostate model between the folded and unfolded states. Nonetheless, the unfolding profiles are obviously different, the heat denaturation analysis revealed that the apoprotein begins to dramatically lose CD signal at 60-65uC, whereas the Zn-bound or Mn-bound protein starts to significantly lose CD signal at a temperature as high as 65-70uC. The apoprotein exhibits a half-denaturation (T m ) of 71.560.1uC, and addition of Zn 2+ and Mn 2+ significantly stabilizes SsTroA as judged by a substantial enhancement in the Tm value (DTm = 4.660.2uC and 560.1uC, respectively), which agrees with the presence of Zn 2+ or Mn 2+ as seen in CD spectroscopic changes (Fig. 4A) . These results combined with CD spectrum data imply that the ternary protein-metal complex appears to form a compact domain organization, i.e., the metalligand interactions in the SsTroA protein indeed play an important role in imparting extra stability to the metal binding site of the protein. Further, as we proposed that Mn 2+ has a more significant effect on the conformation and thermal stability of SsTroA than Zn 2+ .
To gain structural insight into SsTroA function, we determined the crystal structure of Zn-bound SsTroA. The X-ray crystal structure was solved by molecular replacement using TpTroA as the search model (PDB code: 1K0F) [22] . The final structure was refined to a resolution of 2.6 Å and consists of 278 amino acids (residues 40-317). Phasing and refinement statistics are reported in Table S3 . The overall structure of SsTroA is typical of SBPs in the A-1 family [19] . The peptide chain crosses between the N-terminal and C-terminal domains only once linking the two (b/a) 4 domains via a 33-residue kinked rigid a-helical backbone that runs the full length of the molecule (Fig. 5A ) and the metal binding site is buried in the interdomain interface. The N-terminal domain (residues 40-164) is a b/a sandwich-like domain, consisting of a four-stranded b-sheet with 2-1-3-4 linking topology, and with an a-helix connecting each strand. The C-terminal domain (residues 196-317) is also a b/a sandwich composed of a four stranded parallel b-sheet with 2-1-3-4 linking topology, with both sheets sandwiched between layers of a-helices. The two independent domains interact with each other across a large interface that creates the binding pocket for the metal ligand.
In our structure, we clearly observed the metal binding pocket provided by the buried surface between the N-terminal and Cterminal domains (Fig. 5B) . Independent experimental evidence has demonstrated that the ion bound in the metal binding pocket of recombinant SsTroA is Zn 2+ (see metal content analysis). Zinc is pentacoordinated by His 76 (bond length, 2.14 Å ), His 139 Zn coordination in our structure is best described as distorted square pyramidal [21] . The N e2 nitrogen atoms of His 76 and His 139, with the O e1 an O e2 atoms of Asp 289 generated the square planar. While the N e2 atom of His 205 is the summit of the square pyramid. The binding site is buried ,7 Å below the molecule surface and the four coordinating residues are also completely buried. This coordination geometry and the amino acid types are very similar among the Mn-binding SBP family [21, 23, 24] .
Despite the determination of several SBP structures, the structural difference between Zn-specific and Mn-specific proteins remain unclear [44] . There exists difference with SBPs, specific either for zinc or manganese. In the Zn-specific ZnuAs, the metal is coordinated by 3 histidines plus a water or glutamate [44, 45] . In the case of Zn-specific AdcAII, the residues involved in Zn binding are three histidines and one glutamate [25] . This is similar to the observation in Zn-specific Lbp, the zinc ion is coordinated by the side chains of three histidines and one glutamate in a slightly distorted tetrahedral geometry [28] . In the Mn-specific SBPs (PsaA and MntC), the metal is coordinated via two hisitidine residues, the third histidine is replaced by a glutamate, and the fourth position is occupied by an aspartate [23, 24] . The coordination geometry of Zn is usually tetrahedral or distorted tetrahedral (coordination number = 4) in the protein, although the coordination number can increase to five to stabilize high-energy intermediates and their flanking transition states. In contrast, the Mn 2+ ion tends to prefer hard ligands such as the carboxylate oxygens of aspartate or glutamate, and the carboxamide oxygens of asparagine or glutamine. The nitrogen atoms of histidine imidazole groups are occasionally observed as metal ligands in Mn 2+ metalloenzymes despite their borderline hardness, the metal coordination geometry is usually square pyramidal or trigonal bipyramidal (coordination number = 5) [13] . Accordingly, our structure indicated that Mn 2+ is likely to be the natural ligand. However, despite great efforts, we failed to obtain any crystals of SsTroA containing Mn 2+ .
Sequence comparison of SsTroA with TpTroA, S. pneumonia PsaA and Synechocytis MntC revealed amino acid sequence identities of 37%, 26% and 24%, respectively (Fig. 5) . Although the A-1 SBPs have a similar topological structure, subtle differences in the metal-coordinating residues are observed. For instance, His 205 in SsTroA is replaced by Glu 205 at the homologous position in PsaA and Glu 220 in MntC (Fig. 1B) .
Intriguingly, in the structure of SsTroA, we observed two distinctive flexible loops mainly composed of charged residues, and these two flexible loops were designated as Loop 1 (Asn 126-Pro 38) and Loop 2 (Tyr 279-Glu 285) ( Fig. 5C and D) . Loop 1 is locateed between the b2-1 and b2-2 (refer to the TpTroA [21] topology nomenclature). The TpTroA and S. pneumoniae PsaA structures display a short loop compared to Loop 1 of SsTroA. Among the ZnuA proteins examined, this loop is longer (histidinerich) and it is proposed that this region is possibly involved in the capture of the Zn around the metal binding site or plays an important role in the process of docking with permeases [26] . Nevertheless, the significance of this loop remains unclear because there are limited experimental results to confirm these hypotheses. Regardless, it is speculated that the mobility of this loop may possess a biological function in SsTroA [26] .
It should be noted that Loop 2 is unique to SsTroA because it is absent from known SBP homologues. Moreover, the loop is surface-exposed and contains several charged residues, suggesting that it would be available to mediate higher order interactions with a ligand or protein, However, determining the significance of Loop 2 requires further structural and functional data to explain.
NMR reveals that Zn 2+ and Mn 2+ are bound to nearby sites (or the same site)
To probe the extent of protein conformational changes in apo-SsTroA induced by Zn 2+ /Mn 2+ binding, we recorded NMR spectroscopy at 25uC. Full 1 H-15 N spectra of SsTroA were acquired in the absence of metal and loaded with Zn 2+ or Mn 2+ . A superposition of the results is presented in Figure 6 . All of the cross-peaks represent backbone and side chain amide group. The spectral region encompassed is commonly referred to as the ''fingerprint'' for the protein conformation, and it is a known probe of minor conformational changes. As shown in Figure 6 , the apoprotein (purple) maintains a similar tertiary structure compared with the metal bound proteins, suggesting that the apo-SsTroA is not randomly unfolded, but seems to fluctuate among several stable states on the intermediate time scale. Such a result is consistent with the observation of T. pallidum, as suggested by Lee et al [22] .
The addition of Zn to apo-SsTroA results in substantial changes in the NMR spectrum. The spectrum of Zn 2+ -SsTroA (green) is shown in Figure 6A , displaying a large number of peaks shifts.
These dramatic spectral changes indicate that the binding of Zn 2+ induces conformational changes, demonstrating the high affinity of the apoprotein toward Zn, consistent with the ITC results described above. Also noteworthy is that many of the peaks in each protein form have similar chemical shifts, implying that the protein regions adopt similar tertiary structures. Specifically, we observed a downfield-shifted peak near 13.5 ppm in the ordered region and significant movement of multiple resonances (highlighted in circles, Fig. 6 ), suggesting that the involvement of the amide proton close to the Zn binding site.
In the presence of Mn 2+ , the complex solution leads to selective proton line broadening (data not shown). Two dimensional Heteronuclear Single Quantum Coherence (HSQC) NMR spectra of Mn 2+ -SsTroA are shown in Figure 6B . The number of peaks was less than the expected number of amide groups, presumably due to the paramagnetic broadening effects (Mn 2+ is a paramagnetic agent). The cross peaks, still visible after incubating with Mn 2+ , belong to protons distal from the Mn 2+ binding site. By contrast, the broadening and disappearance of the cross-peak strongly indicates the involvement of the amide proton close to the Mn 2+ coordination sphere. It should be noted that almost all of the peaks in the presence of Zn 2+ and Mn 2+ have similar chemical shifts (Fig. 6C) , indicating that the protein regions adopt identical structures when bound to Zn or Mn. This suggests that Zn 2+ and Mn 2+ could bind to the SsTroA in similar positions, perhaps even the same site. A similar result is observed in N. gonorrhoeae MntC protein, where the Zn and Mn ions compete for the same site [33] .
ABC transport systems that contain an A-1 family SBP have emerged as key transporters that are required for virulence in several bacterial pathogens [33] . To better understand the significant role of the Tro operon for metal transition in S. suis, we screened the S. suis 2 genome [5] for orthologs of known metaldependent enzymes and metal transporters as described previously [30] (Table S1 ). We identified 23 Zn-dependent proteins and three Mn-dependent proteins. Mn plays a key role in the growth of S. suis [47, 48] , whereas S. suis does not encode orthologs of either Mn 2+ -specific MntH transporter or the P-type ATPase, MntA. Moreover, another putative transition metal system in S. suis, the Adc operon was found to exhibit homology to the S. pneumonia Adc operon, the latter is an ABC-type Zn permease [49] . These data imply that SsTroA maybe plays an adaptive role in vivo, likely modulated by SsTroR expression in response to specific environmental conditions.
The metal binding properties of TpTroA have been studied in detail [30, 31] . One puzzling observation that was unclear until now is whether or not Zn and Mn bind to the same site in TpTroA. Nuclear spin relaxation properties of resonances from protons close to the ion-binding sites can be selectively perturbed in the presence of paramagnetic ions [50] . The unpaired electrons of Mn 2+ can interact with protons close to Mn 2+ and the effect of the strong paramagnetic interaction of the Mn cation leads to specific changes in the corresponding NMR signals, which helps to locate the Mn 2+ binding site [51] . Our NMR data demonstrated that Zn 2+ -SsTroA and Mn 2+ -SsTroA have very similar tertiary structures. Introduction of Mn induced the disappearance of some cross-peaks and the loss of intensity of others. Moreover, the binding of metal ions induces chemical shift changes for some of the cross-peaks (highlighted in circles, Fig. 6 ). However, the Zn 2+ -SsTroA and Mn 2+ -SsTroA spectra are nearly identical (except for the cross-peaks that disappear from spectrum), indicating that Zn 2+ and Mn 2+ share the adjacent binding region or even the same site in SsTroA.
Metal binding to the SsTroA has been proposed as a major event leading to conformational changes. From our combined NMR and ANS fluorescence data, we speculated that the metalbinding pocket of apo-SsTroA and other locally disordered folding adopts a loosely folded structure in solution. This behavior explains why metal-bound states induced the decreased ANS fluorescence intensity and changes in the NMR spectra of the metal-loaded forms. These loosely ordered structures may enhance an inducible response to the changing environmental conditions or may play a pivotal role in recognizing different divalent metal ions. In some cases, binding of a target molecule or ligand drives disordered folding to a more ordered state [52] . It should be noted that the crystallographic studies on TpTroA [21, 22] have referred to a comparatively minor folding movement upon the metal binding, and structural analysis of Escherichia coli ZnuA suggests that binding and release of Zn drive small yet significant conformational changes [45] . One explanation may be that the nature of protein in solution is shown to be more dynamic than suggested by crystal structures.
SsTroA presents high topological identity with lipoprotein PsaA in S. pneumoniae, the latter is a surface-exposed multi-functional protein detected on all known serotypes of S. pneumoniae. It is noteworthy that PsaA is immunogenic and being actively evaluated as a candidate vaccine component [53] . Thus far however, no data about SsTroA involvement in adhesion and/or invasion of human host tissues is yet available. There is increasing evidence that surface-related proteins are involved in bacterial pathogenesis, as exemplified by adhesion, invasion, and bacterial defense mechanisms [54] . Surface-displayed SBPs likely present an apparent dual function, metal ions transport and interaction with host cells [25] . Assessment of the potential immunogenicity of SsTroA is currently under way in our laboratory.
Currently, it is premature to speculate on metal import mechanism of the SsTroABCD transporter, but it is nevertheless very tempting to hypothesize a scenario (Fig. 7) . BtuF is a periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD. BtuF and TroA are classified in the same class of SBPs, which both exhibit very little domain movement upon ligand binding [20] . The complex structure of BtuCD-BtuF revealed that the conserved glutamate residues Glu 74 and Glu 202 from BtuF form salt bridges with Arg56 residues from two BtuC subunits [55] , which are also found in other ABC importers [56] . Moreover, these salt bridges are critical for molecular interaction in vivo [56] . Intriguingly, we found that SsTroA also has two conserved glutamate residues Glu 107 and Glu 230 that are positioned on the surface of the N-terminal and C-terminal domains (Fig. 5A) . Based on the findings described above, we predicted that SsTroA docks to the periplasmic face of SsTroCD, and charged amino acid residues probably are likely involved in salt bridge formation and contribute to the interface.
Taken together, the structural and functional data we have presented here clearly demonstrate that SsTroA binds both Zn 2+ and Mn 2+ with high affinity. In addition, the binding of metal ions increased the protein stability and induced conformational changes. Specifically, further analysis revealed that Zn 2+ and Mn 2+ bind to the SsTroA in either the same site or very nearby. The result of this work provides novel insight into the S. suis 2 divalent metal uptake process and establishes a framework for understanding the divalent metal homeostasis in S. suis. Additionally, we propose a functional model for the transport of metal ions Figure 6 . NMR spectra of SsTroA complexed with Zn 2+ or Mn 2+ . (A) Superimposition of two-dimensional 1 H 15 N-HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Zn 2+ -SsTroA (green). (B) Overlay of 2D HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Mn 2+ -SsTroA (red). (C) Comparison of 0.5 mM Zn 2+ -SsTroA (green) with 0.5 mM Mn 2+ -SsTroA (red). All spectra were acquired at 25uC in 20 mM sodium acetate (pH 6.5), and spectra were recorded at a 600-MHz 1 H frequency. A number of the cross-peaks exhibiting significant shifts are highlighted in circles, indicating local conformational changes in the metal binding pocket. doi:10.1371/journal.pone.0019510.g006 by SsTroBCD based upon similar substrate transporters. However, further structural and functional experiments are necessary to provide evidence to support our proposed model. Studies to address these issues are underway, and will lay a solid foundation for future studies of the metal ion transport mechanism.
The mature SsTroA domain (residues 36-317) was PCRamplified from genomic DNA extracted from S. suis 05ZYH33. Primers, plasmids and bacterial strains used for gene cloning are all summarized in Table S4 . The PCR product was cloned into the BamHI/XhoI restriction sites of the pGEX-6P-1 expression vector (Amersham) to generate the pGEX-SsTroA plasmid. This construct was then transformed into E. coli BL21 (DE3). E. coli cells were grown at 37uC to mid-log-phase in LB broth supplemented with 100 mg/ml ampicillin, and then SsTroA expression was induced with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 4 hours at 37uC. Cultures were harvested by centrifugation, and the cells were homogenized in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH 7.4) prior to sonication. The cell lysate was then centrifuged for 20 minutes at 35 000 g, the clarified supernatant was loaded onto a glutathione Sepharose 4B column (GE Healthcare) for 2 hours, and bound proteins were cleaved from the resin by incubating with PreScission Protease in accordance with the manufacturer's instruction for batch purification. The resulting supernatant was directly loaded onto a Superdex 200 column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The purified protein was verified by SDS-PAGE and high purity fractions containing SsTroA were concentrated using Amicon Ultra 15 centrifugal filters (Millipore) with a 10-kDa molecular mass cut-off. Protein concentrations were determined by the BCA assay (Pierce) according to the manufacturer's protocol using bovine serum albumin as the standard. To produce uniformly 15 N-labeled SsTroA samples for NMR studies, E. coli BL21 (DE3) were grown in M9 minimal medium containing 0.1% 15 NH 4 Cl (Cambridge Isotope Laboratories) as the sole source of nitrogen. The other steps of protein expression and purification were the same as described above.
A Beckman Optima XL-I analytical ultracentrifuge (Beckman Coulter Inc.) equipped with both absorbance and interference optical detection systems was used in the analytical ultracentrifugation experiment. Protein at an initial concentration of 1 mg/ml dissolved in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl was loaded into six-sector cells. Samples were centrifuged at 50,000 rpm to equilibrium at 20uC. Data were collected in continuous mode at a wavelength of 235 nm using a time interval of 300 s. The results were analyzed by the SEDFIT program (http://www.analyticalultracentrifugation.com) as described by Schuck [57] .
To generate the apo-SsTroA protein, SsTroA protein was treated as described previously with minor modification [31] . Briefly, the purified protein was dialyzed against 20 mM sodium acetate buffer (pH 6.5) and 20 mM EDTA for 3 days. The protein was then dialyzed against EDTA-free buffers for 3 days. All dialysis steps were performed at 4uC with stirring, and the buffer was replaced every 12 hours. Removal of metal was confirmed by ICP-MS (Inductively coupled plasma mass spectrometry). Clear plastic tips and containers were employed to prevent metal crosscontamination. All plasticwares were treated for .2 hours with 0.2 M EDTA to remove the contaminating metals, and all buffer solutions were passed through chelating Sepharose resin and stored in plastic beakers.
The purified recombinant SsTroA proteins were screened for reactivity with antisera by western blotting. Antisera directed against recombinant SsTroA was produced in rabbits according to the standard protocol [58] . Total bacterial protein and purified protein were then resolved by 12% SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare) for the western blot-based detection of protein SsTroA as previously described [17] . Animal experiments were conducted in compliance with the regulations of the Beijing Administration Office of Laboratory Animal.
Purified protein was concentrated to 10 mg/ml in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. Crystals with quality diffraction were obtained using the hanging drop vapor diffusion after 2 weeks at 4uC by mixing 2 ml of the protein with 2 ml of the reservoir solution containing 2.1 M ammonium sulfate and 6% v/v iso-propanol. Prior to data collection, crystals were transferred to the mother liquor containing 30% (v/v) glycerol as cryoprotectant and flash frozen in liquid nitrogen. X-ray Data were collected using a Rigaku MicroMax007 rotating-anode Xray generator equipped with an R-AXIS IV image-plate detector. Data were processed and scaled using MOSFLM [59] and SCALA [60] .
The structure of Zn-bound SsTroA was determined using the molecular replacement method as implemented in Molrep [61] using TpTroA as the search model (PDB entry 1K0F). The structure was rebuilt and refined using COOT [62] and REFMAC5 program [63] . The crystals belong to the P4 3 space group (a = b = 102.4Å , c = 107.3 Å , a = b = c = 90u). The final refined structure without water molecules was used as an initial model for the complex structure. REFMAC5 [63] was again used in conjunction with COOT [62] to refine and build the complex crystal structure. The data collection and final refinement statistics are given in Table S3 . Final models were validated using PROCHECK [64] , and structure figures were generated by PyMOL, unless otherwise noted. Zn-SsTroA was deposited in the Protein Data Bank (PDBID: 3MFQ).
Reconstitutions with Zn 2+ and Mn 2+ were performed as described previously [31] with minor modifications. Briefly, 10 monomer equivalents of Zn 2+ or Mn 2+ were added to apo-SsTroA protein at 4uC for 12 hours. The samples, were then subjected to three rounds of concentration with Amicon Ultra 15 centrifugal filters (Millipore) followed by dilution to remove unbound metal. The metal content of recombinant SsTroA samples exchanged into metal-free 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl, reconstituted SsTroA samples, and buffer controls were determined. Metal ion analysis was determined by ICP-MS, at the Analysis Center of Tsinghua University (Beijing, China). Metal content was analyzed on an XSeries II (Thermo-Fisher) apparatus. Each sample was quantified three times and averaged.
Zn 2+ /Mn 2+ Titration into apo-SsTroA by ITC Binding of Zn 2+ and Mn 2+ to apo-SsTroA was determined by Isothermal Titration Calorimetry (ITC) at 25uC using a NANO ITC 2G MicroCalorimeter (TA Instruments). The metal and protein solutions were prepared in 20 mM sodium acetate buffer, pH 6.5. Prior to experiments, the reference cell was filled with deionized water, and both metal solutions and protein solutions were clarified for 10 minutes at 16,000 g and degassed for 20 minutes to eliminate air bubbles, using the ThermoVac accessory of the microcalorimeter. Upon experimental setup, the protein solution present in the sample cell was stirred at 200 rpm. After a stable baseline was achieved the titration was initiated with 25 consecutive 10 ml injections of 500 mM Zn 2+ into the sample cell (volume = 1.5 ml) containing 90 mM apo-SsTroA,and then 200 mM Mn 2+ was injected into 30 mM apo-SsTroA. The background heat of dilution is collected by titration with buffer alone, and the integrated data were subtracted from data obtained with apo-SsTroA present. Integrated heat data obtained for Zn 2+ and Mn 2+ titrations were analyzed using the NanoAnalyze software (TA Instruments) fitting them to an independent binding model. The free energy (DG) and entropy (DS) of the binding reaction can be determined with Equations 1 and 2, respectively:
Far-UV CD spectra were recorded at a protein concentration of 10 mM in 20 mM sodium acetate (pH 6.5) buffer on a Jasco 810 spectropolarimeter (Jasco Inc.). CD spectra were measured for apo-SsTroA alone and in the presence of 100 mM Zn 2+ or Mn 2+ at room temperature using a 0.1 cm quartz cell, 1 nm band width, 50 nm min 21 scan speed, and 1 nm step. Each curve represents the average of three accumulations and a blank containing the same buffer and detergent, buffer spectra were always subtracted, and ellipticity results were expressed in millidegree. Calculations of the fractional percentage of secondary structures were performed using the computer programs Spectra-Manager (Jasco) and K2d2 [65] . The thermal reactions were monitored on the Jasco 810 spectropolarimeter (Jasco Inc.) connected to a digitally controlled water bath (Julabo). The data were monitored for apo-SsTroA alone and loaded with Zn 2+ or Mn 2+ at 223 nm with a constant heating rate of 0.5uC/min,at temperatures between 25uC and 90uC. The results analysis yielded the transition temperature (Tm) as described previously [66] , and the data were fit using SigmaPlot software.
X-band EPR measurement was performed on an ER 200D SRC spectrometer (Bruker Instruments) equipped with a rectangular microwave cavity, operating at a microwave frequency of 9.53 GHz at 25uC. Three scans were collected at a microwave power of 20 mW. The center field of the EPR scans was set to 3,500 G with a sweep width of 1,000 G. EPR samples were prepared by titrating Mn 2+ into a solution of apo-SsTroA. Stock solutions of MnCl 2 were diluted in protein buffer with doubly distilled water. To monitor Mn 2+ binding, one syringe was loaded with a Mn 2+ -saturated SsTroA solution (100 mM SsTroA and 100 mM MnCl 2 ) in protein buffer and the other was loaded with 100 mM ZnSO 4 freshly prepared in the same buffer. After each addition the sample was incubated for 10 minutes at 4uC prior to recording the EPR spectrum.
All NMR experiments were performed at 25uC on a Bruker DMX 600MHz spectrometer with a triple resonance cryo-probe. Uniformly 15 N-labeled SsTroA was prepared as described above. Sample of apo-SsTroA were prepared at 0.4 mM protein concentration in 20 mM sodium acetate buffer (pH 6.5). Samples of metal bound SsTroA were prepared by the addition of 100 mM zinc acetate or manganese chloride stock to a final concentration of 1 mM metal ions in 0.4 mM protein, and the samples were then subjected to three rounds of concentration with an Amicon Ultra 15 centrifugal filters (Millipore) followed by dilution to remove unbound metal. Samples for NMR measurements contained 0.4 mM 15 N labeled SsTroA, 95% H 2 O/5% 2 H 2 O in 20 mM sodium acetate buffer (pH 6.5). All NMR spectra were processed and analyzed using Felix software (Accelrys Inc.).
Fluorescence measurements were performed with a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer). Emission spectra were collected at 25uC using an emission wavelength of 400-600 nm with an excitation wavelength of 380 nm [66] . Apoprotein (5 mM) was mixed with 500 mM ANS in 20 mM sodium acetate buffer (pH 6.5). Zn 2+ and Mn 2+ were added to the mixture of 5 mM apo-SsTroA and 500 mM ANS to 10 mM final concentration, and the mixtures were incubated at 25uC for 15 minutes prior to collecting the excitation spectrum.  The complement system The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Complement activation results in opsonization of pathogens and their removal by phagocytes, as well as cell lysis. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases such as systemic lupus erythematosus and asthma. This review represents an overview of the complement system in an effort to understand the beneficial as well as harmful roles it plays during inflammatory responses. Complement was first discovered in the 1890s when it was found to aid or "complement" the killing of bacteria by heatstable antibodies present in normal serum (Walport 2001 ). The complement system consists of more than 30 proteins that are either present as soluble proteins in the blood or are present as membrane-associated proteins. Activation of complement leads to a sequential cascade of enzymatic reactions (known as complement activation pathways; see below) resulting in the formation of the potent anaphylatoxins C3a and C5a that elicit a plethora of physiological responses that range from chemoattraction to apoptosis. Initially, complement was thought to play a major role in innate immunity where a robust and rapid response is mounted against invading pathogens. However, recently, it is becoming increasingly evident that complement also plays an important role in adaptive immunity involving T and B cells that help in elimination of pathogens (Dunkelberger and Song 2010; Molina et al. 1996) and in maintaining immunologic memory preventing pathogenic re-invasion. Not only is complement involved in innate and adaptive immunity but it is also involved in tissue regeneration, tumor growth (Qu et al. 2009 ) and human pathological states such a atypical hemolytic uremic syndrome, age-related macular degeneration, etc. (Wagner and Frank 2010) .
C3 convertase C3bBb, which is stabilized by the presence of plasma properdin (Kemper et al. 2010) . Properdin is a protein released by activated neutrophils (properdin is also found in macrophages and T cells) and which stabilizes the convertase by binding to C3b and preventing its cleavage by Factors H and I. Recent studies suggest that properdin can directly bind to apoptotic and necrotic cells and initiate complement activation (Kemper et al. 2010 ).
The lectin pathway (LP) is activated when either mannose binding lectin (MBL) or Ficolin bind to carbohydrate moieties on surfaces of pathogens including yeast, bacteria, parasites and viruses. Both MBL and Ficolin circulate in the serum as complexes with MBL-associated proteins (MASPs; Wallis 2007; Sørensen et al. 2005) . There are four structurally related MASPs: 1, 2 and 3 and a truncated MASP2 known as MAP19 (Sørensen et al. 2005) . Binding to pathogens induces conformational changes resulting in autoactivation of MASP2 which cleaves C4 to form C4a and C4b. C4b attaches to the surface of the pathogens inducing C2 to bind, which is in turn cleaved by MASP2 to form C2b and C2a. C4b together with the attached C2a has enzymatic activity and forms the LP C3 convertase C4bC2a. The exact role of the other MASPs is presently unknown although MASP1 can cleave C2 but not C4 (Wallis 2007) and thus help in enhancing complement activation by the bound complexes.
The classical pathway (CP) is initiated when immune complexes are formed after IgG or IgM binding to pathogens or to other foreign and non-self antigens. The C1 complex, a multimeric complex consisting of C1q, C1r and C1s molecules, then binds to the Fc portion of the IgG or IgM immune complex. Activation of C1s and C1r occurs as a consequence of C1q binding to the exposed Fc portion of IgG or IgM. C1s then cleaves C4 and C2 to form the CP C3 convertase, C4bC2a.
In addition, pentraxins (PTX) can recognize pathogens and eliminate them by directly binding to C1q. Based on their subunit structure, PTX are divided into two subfamilies: the short PTX family to which belong the acute phase proteins SAP and CRP and the long PTX family to which belongs the prototype protein PTX3 (Sjöberg et al. 2009; Bottazzi et al. 2010) . Pentraxins are synthesized in the liver and other tissues in response to an infection (Bottazzi et al. 2010) . The C3 convertases C4bC2a of CP and LP and C3bBb of the AP further cleave C3 to release C3a and C3b. C3b acts as an opsin and helps to further amplify complement activation as well as help in phagocytosis. In addition, C3b complexes with the C3 convertases to form the C5 convertases: C3bBbC3b and C4bC2aC3b (Fig. 1) . The C5 convertases cleave C5 to form C5a and C5b. The membrane attack complex (C5b-9, MAC), also called the terminal complement complex (TCC), is then initiated by C6 and C7 binding to C5b and then C8 and multiple molecules of C9 binding to the C5bC6C7 complex. The MAC complex forms a pore by inserting itself into cell membranes, resulting in cell lysis.
In addition to the above 3 pathways, proteases released by neutrophils and macrophages (Ward and Zvaifler 1973; Huber-Lang et al. 2002) , factors such as Kallikrein, plasmin and factor XIIa (Hageman factor) can generate complement activation products. Thrombin, a member of the coagulation pathway, can locally generate C5a in in vivo in C3deficient mice which are unable to generate the conventional C5 convertase .
The anaphylatoxins C3a and C5a, which consist of 77 and 74 amino acids, respectively ), exert a multitude of effects in inflammatory responses. They act as potent chemoattractants for cells such as phagocytes (neutrophils, monocytes) to sites of injury or inflammation. They act as vasodilators and induce smooth muscle contraction. They cause histamine release from mast cells and induce oxidative bursts (consumption of O 2 ) from neutrophils. They are implicated in the production of the cytokine, TNFα, during liver regeneration (Markiewski et al. 2006 ). C3a and C5a exert pleiotropic effects by binding to their respective receptors, namely C3aR and, in the case of C5a, to two receptors, C5aR and C5a receptor-like 2 receptor (C5L2). C5L2 was discovered in 2000 (Ohno et al. 2000) and, although it shares homology with C5aR, its exact biologic function remains unclear.
The classical C5a receptor (C5aR, CD88) belongs to the group of seven-transmembrane spanning rhodopsin family of receptors that signal through G-protein-dependent path-ways (reviewed in Guo et al. 2004) . Originally, C5aR was presumed to be expressed exclusively on myeloid cells but now there is ample evidence that it can be expressed on a variety of non-myeloid cells (Wetsel 1995) . Under inflammatory conditions, expression of C5aR increases in lung endothelial, alveolar and bronchial epithelial cells and macrophages Riedemann et al. 2002) . The recently identified C5L2 receptor for C5a is also a seven-transmembrane spanning rhodopsin family receptor. Abundant expression of C5L2 has been found on neutrophils and dendritic cells (Ohno et al. 2000) . Unlike C3aR and C5aR, C5L2 is uncoupled from G-proteins due to an amino-acid replacement of arginine by leucine in the DRY region at the end of the third intra-cellular transmembrane loop (Cain and Monk 2002) . Following binding of C5a, C5L2 does not induce classical signaling (induction of intracellular calcium transients) or cause biological cellular responses. The role of C5L2 is largely unknown. It may act as a decoy or scavenger receptor for C5a (Okinaga et al. 2003; Scola et al. 2009 ). However, recent studies suggest that C5L2 acts as a functional receptor especially in the setting of sepsis where release of high mobility group box 1 protein requires the presence of C5L2 receptor on macrophages. Blockade of C5L2 along with C5aR greatly improves survival in sepsis when compared to blockade of either receptor alone (Rittirsch et al. 2008a ). Under such conditions, there is a broad suppression in levels of cytokines and chemokines in plasma.
Given the multitude of effects that complement can exert, there are mechanisms in place to limit complement activation where and when it occurs. For example, the phlogistic potential of both C3a and C5a is quickly reduced by plasma carboxypeptidases that cleave the C-terminal Arginine, resulting in C3a des-Arg and C5a des-Arg, each of which has less than 10% of their original biological activity. C3a des-Arg, which is also known as acylationstimulating protein (ASP), is thought to induce triglyceride synthesis in adipocytes (Kalant et al. 2005) . C3b and C4b are also quickly inactivated by proteolytic cleavage into fragments iC3b, C3dg, C3c, C4c, C4d by the serine protease Factor I in the presence of cofactors: membrane cofactor protein (MCP) and complement receptor 1 (CR1, CD35) that are membrane bound and Factor H which is bound to host surfaces. CR1 promotes phagocytosis, helping to clear immune complexes by binding to C3b in immune complexes. Failure to clear these complexes leads to deposition of complexes in tissues and activation via Fc receptors, resulting in tissue injury. CR2 (CD21) binds iC3b, C3dg and C3d (Holers and Kulik 2007) . Further-more, C1 inhibitor (C1-INH) inactivates C1r, C1s and MASP2 (Davis et al. 2008) . Complement activation is also regulated either by preventing the assembly of the C3 convertase or, once it is formed, by inhibiting its activity due to actions of decay acceleration factor (DAF, CD55), C4 binding protein (C4BP), Factor H and a membranebound protein found only in rodents, Crry. In addition, the MAC complex formation on cell surfaces or lysis is negatively regulated by S protein (a plasma glycoprotein synthesized by endothelial cells), vimentin (a cytoskeletal protein), and CD59 by interfering with the assembly of the MAC (Huang et al. 2006 ).
As early as the 1970s it had become evident that complement not only played a role in pathogen recognition and elimination but also played a role in B cell biology when it was observed that B cells could bind C3 (see Carroll 2004 for a review). Subsequently, it was recognized that the complement receptors CR1 and CR2 mediate complement-associated B cell functions. These receptors are expressed on B cells and follicular dendritic cells as well as on a subset of T cells. Furthermore, the CR2-CD19-CD81 complex modulates B-cell receptor signaling, thus affecting the amplitude of B cell responses when presented with antigens. CR1 and CR2 also appear to play a role in B cell differentiation, selection, maintenance and elimination of self-reactive B cells (Carroll 2004) .
As in the case for B cell biology, it has become increasingly evident that complement also plays an important role in T cell biology. It affects T cell activation, T cell responses and in induction of regulatory T cells (for a review, see Kemper and Atkinson 2007) . For example, during T cell activation, absence of DAF on T cells and on antigen-presenting cells (APC) enhances T cell proliferation (Heeger et al. 2005) . Furthermore, APCs and T cells express C3aR and C5aR and locally produced C3a and C5a interact with their respective receptors and participate in maintaining T cell viability, proliferation and differentiation Lalli et al. 2008) .
Hemolytic uremic syndrome Mutation of the factor H gene results in impaired C3 convertase activity and is associated with atypical hemolytic uremic syndrome (aHUS) wherein hemolytic anemia, thrombocytopenia and acute renal failure develop. It is thought that the mutant Factor H protein binds less efficiently to C3b and C3d on endothelial cells and, as a result, there is increased vascular damage, especially to endothelial cells, with resultant intravascular deposition of fibrin. However, recently, mutations in Factor I, Factor B and CD 46 have been implicated in aHUS patients (Botto et al. 2009; Pettigrew et al. 2009 ).
Hereditary angioedema C1 inhibitor (C1-INH) not only inhibits C1r, C1s, and MASP2 of the complement system, but it also inhibits factor XIIa and kallikrein. Mutations and deficiency of C1-INH and factor XIIa results in dysregulated bradykinin production, which increases vascular permeability, leading to angioedema as seen in hereditary angioedema (HAE; for a review, see Cugno et al. 2009 ). HAE also underscores the interaction of the proteins involved in the complement, coagulation and contact proteolytic cascades.
Complement regulators, CD59 and DAF, are both membrane bound glycosyl phosphatidylinositol (GPI)-linked molecules that are involved in inhibiting the MAC complex and causing dissociation of the C3 and C5 convertases, respectively. The paroxysmal nocturnal hemoglobinuria (PNH) mutation in the gene PIG-A results in decreased expression of GPI-linked proteins, including CD59 and DAF. As a result, intense complement-mediated lysis of red blood cells occurs in PNH patients with consequent hemolytic anemia. White blood cells are also susceptible to lysis, and tissue factor release from these damaged cells may contribute to thrombosis often seen in PNH patients (Liebman and Feinstein 2003) .
Systemic lupus erythematosus (SLE) syndrome is often found associated with deficiency in C1q, C1r and C1s (Table 1) . SLE in humans is characterized by fever, rash, glomerulonephritis and, sometimes, hemolytic anemia. SLE is associated with the presence of anti-nuclear antibodies and extractable nuclear antigen (ENA) and absence of anti-DNA antibodies. It is thought that absence of key complement proteins results in: (1) in defective immune complex clearance and consequent deposition of the complexes in various organs, especially in the kidney and in arterial walls (causing vasculitis); (2) defective recognition of self by B cells leading to autoimmunity; and (3) defective disposal of dying cells including B and T cells, again leading to autoimmunity. C2 and C4 deficiency is also associated with development of SLE, although the correlation is not as strong as with C1q deficiency. C3 deficiency is very rare in humans and does not usually lead to SLE. However, deficiency in C2, C3 and C4 increases the susceptibility to infections by bacteria Haemophilus influenzae and Neisseria (for a comprehensive review on complement deficiencies and resultant clinical manifestations, see Botto et al. 2009; Pettigrew et al. 2009 ).
There are hardly any known deficiencies of Factor B and Factor D. However, properdin deficiency is known and is associated with mortality from Neisseria meningitides. Factor I deficiencies are rare and lead to recurrent bacterial infections. MBL deficiency increases susceptibility (Eisen 2010) to respiratory tract infections and MASP2 deficiency is associated with recurrent infections (Stengaard-Pedersen et al. 2003) and has been reported to be found in a number of different populations including Caucasians, Africans, Brazilians and Chinese (Thiel et al. 2007 ). Instances of C5, C6, C7, C8 and C9 deficiencies are known and are associated with increased susceptibility to Neisseria meningitides. However, mortality due to infection is much lower than that found with properdin deficiency.
Pathogens appear to have evolved strategies to circumvent the capabilities of the complement system to eliminate them by dampening complement activation. Pathogens accomplish this evasion by affecting every facet of complement activation, including regulation of activation, amplification, opsonization, phagocytosis, chemoattraction, cell lysis (for a review, see Lambris et al. 2008; Rooijakkers and van Strijp 2007) by either proteolysis of complement proteins or mimicking the action of complement proteins or interacting with complement proteins. As will be seen below Staphylococcus aureus is a good example of a pathogen that subverts all these processes. Pathogens also take advantage of complement by binding to membrane bound complement proteins to gain entry into cells. For example, measles virus binds the membrane cofactor protein (MCP) and Epstein virus protein gp350/220 binds to CR2.
Staphylococcus aureus expresses two proteins on its surface: staphylococcal protein A (SPA) and staphylococcal immunoglobulin-binding protein A (Sbi) (Zhang et al. 1998 ) that can bind to the Fc portion of IgG and thus prevent complement activation and Fc receptor-mediated phagocytosis. Sbi can also block binding of C1q and thus prevent activation. Similarly, group C and G streptococcal bacteria express protein G that can bind the Fc portion of IgG. Furthermore, Staphylococcus aureus expresses receptors that bind plasminogen, and Staphylokinase secreted by Staphylococcus aureus cleaves bound plasminogen to plasmin. Bacterial surface bound plasmin can degrade IgG and the opsonin C3b, and thus evade the complement system. Further, Staphylococcus aureus-secreted staphylococcal complement inhibitors (SCINs) inhibit complement activation. These SCINs bind to C3 convertases and prevent subsequent steps involved in complement activation, and thus prevent opsonization, phagocytosis and cell lysis.
Bacteria also express surface proteins that can bind C4BP and Factor H, thereby preventing their cofactor function in Factor I-mediated cleavage of C3b/C4b and the subsequent downstream complement activation. A number of bacteria target C4BP for binding and inactivation including the M protein family members Arp and Sir, expressed by group A Streptococci and outer membrane protein OmpA expressed by Escherichia coli (for a comprehensive review, see Blom et al. 2009 ). Factor H is similarly a target for binding by proteins expressed by a number of bacteria including M protein expressed by Staphylococcus aureus and Streptococcus pyogenes and GNA1870 expressed by Neisseria meningitides ).
Similarly, complement-inhibiting proteins expressed by viruses such as vaccinia, cowpox and smallpox block C3 convertase formation (for a review see (Tortorella et al. 2000) . These inhibitory proteins contain short consensus repeats (SCR) that are also found in complement proteins (such as Factor B) (Ricklin and Lambris 2007 (CRIT) protein that inhibits C3 convertase by binding to C2 (Cestari Idos et al. 2009; Inal and Sim 2000) . Pathogens can also prevent recruitment of phagocytic cells by inhibiting C5a and C5aR interaction. For example, the Staphylococcus aureus product, chemotaxis-inhibiting protein of Staphylococcus aureus (CHIPS), can bind to C5aR and prevent C5a-mediated signaling, although CHIPS and C5a share hardly any homology but are similar in overall structure and size. Another Staphylococcus aureus product, SSL-7, can bind C5 and inhibit its cleavage preventing MAC formation (Rooijakkers and van Strijp 2007) .
As described above the complement system plays a critical role in both innate and adaptive immune responses. Clearly pathogens go to great lengths to avoid complement activation. Deficiencies in complement proteins can lead to serious consequences resulting in diseases such as lupus, HUS, etc. On the other hand, excessive complement activation or dysregulated modulation may contribute to several diseases and pathological conditions such as multiple sclerosis (Ingram et al. 2009 ), Alzheimer's (Kolev et al. 2009 ), asthma (Wills-Karp 2007), COPD , sepsis (Ward 2008) , hyperacute organ rejection (Wasowska 2010) , etc. In sepsis, excessive C5a generation is thought to contribute to increased thymocyte apoptosis and consumptive coagulopathy, decreased innate immune functions of neutrophils, cardiomyopathy and multiple organ failure (for review, see Rittirsch et al. 2008b) .
Complement proteins C3 and C1q are thought to bind to amyloid-beta proteins in the brain in the early stages of the Alzheimer's syndrome, and subsequent complementmediated lysis of neurons may further exacerbate the syndrome. In ischemia/reperfusion injury of tissues, complement activation during the reperfusion phase is thought to contribute to the ensuing inflammation. Age-related macular degeneration is thought to occur due to excessive complement activation (Zipfel et al. 2007 ) and appears to be related to defective Factor H attachment to the Bruch's membrane in the retina, leading to protein deposits on the membrane known as Drusen. Excessive complement activation has also been associated with disease severity in patients infected with malaria (reviewed in Silver et al. 2010) . Interestingly, C5 deficiency or intercepting C5a and C5aR interaction has a protective effect in an animal model of cerebral malaria (Patel et al. 2008) .
Asthma and the acute response distress syndrome (ARDS) are two of the more well-known examples of dysregulated complement activation. It is now well established from both experimental models of sepsis and clinical studies in humans that the complement system plays a key role in the pathophysiology of asthma and the acute respiratory distress syndrome (reviewed in Wills-Karp 2007; Sarma et al. 2006) . Asthma is a chronic inflammatory upper airway disease with increasing prevalence throughout the world and especially in young children in the developed world. In asthma, hyper-secretion of mucus occurs in the airways along with smooth muscle proliferation in upper airway (bronchial/bronchiolar) walls together with increased numbers of mast cells, CD4+ T cells and eosinophils. This leads to obstructed airways and accentuated upper airway contractile responses leading to shortness of breath, wheezing and coughing. In ARDS, there is a severe impairment of gas exchange due to damage to the alveolar and vascular epithelium resulting in increased permeability of plasma contents and influx of neutrophils into the interstitial and alveolar spaces.
Clinical studies show that elevated levels of C3a and C5a occur in bronchoalveolar lavage (BAL) fluids of individuals with asthma and ARDS when compared to healthy individuals (Wills-Karp 2007) . Elevated levels of C3a and C5a may also be found in the serum of ARDS patients. Similarly, in septic patients, elevated levels of the MAC complex precede the development of ARDS (Langlois and Gawryl 1988). There is substantial evidence suggesting that varied environmental factors trigger complement activation at the airway interface, leading to recruitment of cells that release inflammatory mediators resulting in further exacerbation of the inflammatory process in asthma and ARDS (Wills-Karp 2007) . For example, TNFα and IL-1 generated from inflammatory cells induce up-regulation of adhesion molecules (ICAM-1 and E-selectin) on vascular endothelial cells and in pulmonary epithelial cells, which results in tethering of neutrophils to these surfaces. Neutrophils can exert further tissue damage by releasing myeloperoxidase, elastase, etc. Examples of environmental triggers that activate complement are seen in elevated BAL C3 levels in humans exposed to ozone, and elevated serum C3 levels in children with chronic exposure to cigarette smoke. It has been suggested that proteases released from infiltrating cells into the airways can directly cleave C3 and C5, thereby generating C3a and C5a. Alternatively, antibodycontaining immune complexes as well as carbohydrate moieties and macromolecular structures present on pathogens and allergens can activate all three complement pathways. It is likely that complement gets activated by a number of different pathways in asthma and ARDS, although the exact mechanisms are not fully understood.
Thus, given above are a few examples of the pathology of dysregulated complement activation. For more examples of the role of complement in allergic asthma, transplant rejection, cancer and autoimmune diseases, see Huber-Lang et al. (2002) .
Given the growing recognition of complement in several pathological conditions, there has been a concerted effort recently in designing therapeutics aimed at complement proteins (Ricklin and Lambris 2007) . Understanding subversion tactics of pathogens can help in designing therapeutic interventions when unwanted or runaway complement activation does more harm than good. It has been a challenge to design effective therapeutic interventions, although a number of soluble and membrane-bound complement proteins are available as potential therapeutic targets. One of the main problems is the lack of understanding of the exact molecular mechanisms involved in complement-mediated disease pathology (reviewed in Ricklin and Lambris 2007) . For instance, a good example is the use of compstatin, a synthetic compound that blocks the C3 convertase. An ideal use of compstatin would be for treatment of patients experiencing age-related macular degeneration, using intravitreal injection into the eye, much as is now done with blocking mAb to VEGF. On the other hand, systemic treatment of septic patients with compstatin runs the risk of excessive blocking of the C3 convertase, which would compromise the production of the vital opsonic factors, C3b and iC3b. Accordingly, this presents the dilemma for using complement inhibitors that target the C3 convertase. The lack of sufficient knowledge about details of complement activation (including the complement regulatory proteins) makes it difficult to predict the efficacy of complement inhibitors that would be given systemically.
Another example of a dilemma related to in vivo blockade of complement comes from recent studies involving mice with CLP-induced sepsis (Flierl et al. 2008 ). Use of a blocking antibody to C5a was highly protective. However, when C6 was depleted in order to prevent formation of the MAC (C5b-9), it was found that the bacterial burden in blood was much higher in CLP mice compared to complement-intact mice, consistent with the knowledge that C5b-9 has lytic activity for bacteria. Accordingly, since complement is both a potent inflammatory-inducing system as well as critically important for innate immune defenses, we need to have a much better understanding of how the system really works.
Further, therapeutic strategies need to take into account the delicate balance between suppression of complement-mediated disease pathology and compromise of complement-mediated defense and immunity. After several attempts, several small molecules inhibitors such as compstatin that prevents C3 cleavage, PMX-53 that is a C5aR antagonist are in either preclinical or clinical phases of testing (Qu et al. 2009 ). Only eculizumab (trade name Soliris), which is a humanized monoclonal antibody directed against C5, has been approved by the United States Food and Drug Administration for use in patients with PNH. This antibody prevents the cleavage of C5 to C5a and C5b. The antibody does not seem to activate complement or bind to the host Fc receptor.
The complement system is composed of a network of proteins that play an important role in innate and adaptive immunity that range from opsonization of pathogens and chemoattraction to removal of apoptotic and necrotic cells. Activation of the system is exquisitely regulated, and inappropriate activation either due to deficiencies in key complement proteins or due to dysregulated activation has adverse consequences. There is also a growing appreciation that there is cross-talk between the complement system and other systems, especially the coagulation system (Kourtzelis et al. 2010) . For example, C5amediated tissue factor release from neutrophils has been implicated in the pathogenesis of thrombosis in patients on hemodialysis. Chest imaging features of patients afflicted with Influenza A (H1N1) in a Malaysian tertiary referral centre This is a retrospective descriptive study of the chest imaging findings of 118 patients with confirmed A(H1N1) in a tertiary referral centre. About 42% of the patients had positive initial chest radiographic (CXR) findings. The common findings were bi-basal air-space opacities and perihilar reticular and alveolar infiltrates. In select cases, high-resolution computed tomography (CT) imaging showed ground-glass change with some widespread reticular changes and atelectasis. In April 2009, a new global outbreak of a novel Influenza A virus, a swine virus with confirmed human infection, occurred. What is new about this virus is that it is unrelated to any swine influenza virus previously identified in North America. There is sustained humanto-human transmission in this new subtype of Influenza A virus [1] . It was termed Influenza A(H1N1) and 2009 H1N1 influenza by the United States Centre of Disease Control and Prevention (CDC) [2] and Pandemic H1N1/09 by the World Health Organization (WHO) [3] . The respiratory system is predominantly affected. Initial reported chest findings of A(H1N1) infections were bilateral diffuse lung opacities on chest radiographs (CXR). On computed tomography (CT), there were patchy ground glass opacities in both lungs with axial predominance and in the four lung quadrants consistent with acute respiratory distress syndrome [4] . To date, there has been no published data on CXR findings of patients with A(H1N1) in Malaysia. The authors report the CXR findings of 118 patients with confirmed A(H1N1).
Waiver of informed consent was obtained from the Hospital Ethics Committee due to public health care concern for information of this disease. A cross-sectional retrospective review of CXR and CT of the thorax of 166 patients with influenza symptoms who tested positive for Influenza A(H1N1) at the University of Malaya Medical Centre was done from end July 2009 to early September 2009. The majority of these patients were walk-in patients to the Accident and Emergency Unit who presented with constitutional influenza-like symptoms of fever, myalgia and headache. Laboratory confirmation of pandemic influenza A(H1N1) was carried out using a real-time polymerase chain reaction (PCR) protocol and primers from the CDC [5] , and virus isolation in Madin-Darby canine kidney cells.
The decision to perform chest imaging in these patients was entirely at the discretion of the attending doctors at the Accident and Emergency Unit and, subsequently, doctors from the Respiratory and Infectious Disease Units based on the clinical severity of the patients. Out of the total, 48 patients including pregnant women were deemed not necessary to have any form of imaging. The chest radiographic images obtained from the 118 patients were reviewed by two staff radiologists, who have more than 10 years postgraduate experience, via consensus reading and the patterns recorded. Serial radiographs of patients with progressive clinical signs were also reviewed to assess disease progression and response to treatment by the Infectious Disease Unit team of doctors. Chest CT, which was performed on select cases based on severity and treatment response, were also read and the findings noted. The clinical data of the afflicted patients were obtained from the Hospital Infectious Disease Unit and compiled using Microsoft Office Excel (2003).
Of the patients presenting with influenza-like symptoms, 73 males and 93 females tested positive for Influenza A(H1N1) from end July to early September 2009 at the centre. Of the 118 patients who had chest radiographs, 56 were males and 62 were females. More than half of the patients (69/118) had normal chest radiographs. One of the patients who had a normal chest radiograph twice but remained symptomatic subsequently underwent a high-resolution CT (HRCT) examination a day after the second chest radiograph and the HRCT showed bi-apical fibrosis and ground-glass changes in the left lower lobe.
Among the abnormal chest radiographs (49/118), bilateral involvement (28/49) was more commonly noted. The commonest presentations were bi-basal air space infiltrates (12/49), perihilar mixed reticular and alveolar infiltrates (13/49) and unilateral basal air space opacity (6/49). Less common findings were round opacity (1/49), scattered focal air space opacity (4/49), linear opacities (2/49), linear opacities that progress to alveolar opacities (3/49), plethoric lungs (2/49), bronchiolitis (2/49) and pleural effusion (4/49). In the majority of patients (43/49), there was radiological improvement in the follow-up chest radiographs. Five had progressive alveolar consolidation ranging from 2 to 13 days with one developing acute respiratory distress syndrome (ARDS) according to the American-European Consensus Conference (AECC) criteria. Five patients had CT done 2 days to 1 week after the last chest radiograph because they showed poor clinical response to initial treatment. Three were HRCT and the common findings were widespread interstitial shadowing, ground-glass change and atelectasis. These were not detected on plain chest radiographs. [6] . In Malaysia, the first confirmed case was a 21-year-old student who returned from Newark, USA, on a Malaysia Airlines flight MH091 on May 15, 2009. He presented with typical influenza symptoms -fever, sore throat and body aches. The first death occurred on July 24, 2009, where a 30-year-old Indonesian student who returned to Malaysia from a holiday break in Medan on July 5 had progressive fever and cough and eventually died [8] . As of October 17, 2009, there had been 77 H1N1-related deaths in Malaysia [7] . This made Malaysia the 36th country worldwide to be affected by the virus.
This short-term review highlights the chest radiographic findings in 118 patients with positive Influenza A(H1N1) from the centre. The bi-basal air space opacities seen correlate well with the findings of Influenza A(H1N1) in Mexico [4, 9] . Similar CXR findings are also seen in avian influenza such as sudden acute respiratory syndrome (SARS) and H5N1 [10, 11] .
Currently, radiographic criterion is not included in the WHO diagnosis of Influenza A(H1N1). In this series, only 42% of patients demonstrate positive findings and this is likely due to the range of clinical presentation of the study population. Several studies have a similar initial CXR positive findings of 42-50% for this disease [12, 13] .
In previous avian influenza outbreaks, such as SARS and H5N1 [10, 11, 14] , CXR has been shown to be useful in the prognostication of the disease. According to the authors' clinical and radiological observation of the Influenza A(H1N1), serial CXR revealed that lung infiltration increases with progression of the disease. This is similar to the findings of Ajlan et al where alveolar infiltration was predominant in cases that progressed to ARDS and showed near complete to total resolution in clinically resolving cases [14] .
In CT, the most common reported change in Influenza A(H1N1) was ground glass shadowing. Other changes recorded on CT were focal or multifocal consolidation, interstitial changes and pulmonary embolism [4, 13, 14] . CT was valuable in detecting positive findings in the presence of normal CXRs and showed more extensive pattern of involvement when compared to CXR [14] . These findings were similar to the authors' series although they did not find any evidence of pulmonary embolism in their sample population.
The Influenza A (H1N1) CXR findings are similar to those reported for SARS and H5N1. Although imaging will play an important role in detecting lung changes, and to monitor disease progression as well as response to treatment; knowledge of the current epidemic status is essential for correct diagnosis. Noninvasive ventilation in acute respiratory failure due to H1N1 influenza We present a case of severe H1N1 influenza with hypoxemic acute respiratory failure necessitating mechanical ventilation benefited from noninvasive positive pressure ventilation (NIPPV). The NIPPV may be of great use in treating patients with H1N1-related acute respiratory distress syndrome in a resource poor setting or when invasive ventilator is unavailable.  Temporal Anomalies in Immunological Gene Expression in a Time Series of Wild Mice: Signature of an Epidemic? Although the ecological importance of coinfection is increasingly recognized, analyses of microbial pathogen dynamics in wildlife usually focus on an ad hoc subset of the species present due to technological limitations on detection. Here we demonstrate the use of expression profiles for immunological genes (pattern recognition receptors, cytokines and transcription factors) as a means to identify, without preconception, the likelihood of important acute microbial infections in wildlife. Using a wood mouse population in the UK as a model we identified significant temporal clusters of individuals with extreme expression of immunological mediators across multiple loci, typical of an acute microbial infection. These clusters were circumstantially associated with demographic perturbation in the summertime wood mouse population. Animals in one cluster also had significantly higher individual macroparasite burdens than contemporaries with “normal” expression patterns. If the extreme transcriptional profiles observed are induced by an infectious agent then this implicates macroparasites as a possible player in mediating individual susceptibility or resilience to infection. The form of survey described here, combined with next generation nucleic acids sequencing methods for the broad detection of microbial infectious agents in individuals with anomalous immunological transcriptional profiles, could be a powerful tool for revealing unrecognized, ecologically important infectious agents circulating in wildlife populations. The role of infectious agents in the dynamics of natural vertebrate populations has been a growing interest for ecologists. Endemic infections have been shown to have important effects on survival [1, 2] and reproduction [3, 4, 5, 6, 7] and the theoretical potential to regulate populations and drive abundance cycles [8, 9] . At the same time there are many documented instances of epidemics associated with catastrophic mortality in nature [10, 11, 12] , although the observation of such events may often be serendipitous or depend on the focus of the researcher. Increasingly it is also appreciated that co-occurring pathogens should be regarded as a potential community [13, 14] , whose interactions may define the consequences of infection for the individual host and host population [15] . In general, however, ecological studies of microbial infectious disease in nature are constrained to the analysis of a single pathogen or an ad hoc panel of pathogens for which there is a priori knowledge, a preconception of possible importance and specific diagnostic reagents.
An alternative is to focus on non-pathogen-specific aspects of the host's immune response [16] , detecting the presence of significant microbial infections through perturbations caused in the expression of immunological genes in the host [17] . In the present study we present an example of such an approach, revealing spatio-temporal patterns of immunological expression in a summertime wood mouse population in the UK that may point to epidemic infections with an unknown microbial infectious agent.
We sampled wood mice (Apodemus sylvaticus) on 12 occasions through May-December 2008 at Cotgrave Forest (+52.891, 21.041242) a ,135 ha area of coniferous and mixed forestry plantation in Nottinghamshire, UK (total n = 141). Mice were caught by overnight live-trapping using Longworth traps. The traps were placed at permanently marked stations located at 20 m intervals (1 trap/station) along a 1600 m curvilinear transect that traced the margins of contiguous forestry blocks. These blocks were separated along their edges by narrow clear areas ,10-40 m wide. The use of an extended trapping line was intended to distribute trapping effort over a very large area and avoid local demographic effects. Traps were set in the afternoon (16.00 h BST) and collected the following morning (08.00 h) throughout the study period. Females that were lactating or heavily pregnant at the time of capture were released. At the last two sampling points, where numbers of mice captured exceeded the capacity to process them, some randomly selected individuals were also released. Other individuals were processed and infection variables quantified as previously described [18] . This was with the exception that in the present study lice were recorded in different infection categories (absent, ,50 egg cases present, $50 egg cases present). Additionally, in the present study, thin blood smears were prepared from cardiac blood and examined for the presence of Bartonella spp. and Babesia microti (counts of infected cells in one hundred 1006 microscope fields). Eyes from each specimen were stored in 4% formalin and then lenses were removed and dried in an oven at 80uC for 72 h and weighed for use as an index of age (combined weight, g) [19] . In the present study the same set of macroparasites was found as in a previous study along the same transect in 2007 [18] , with the exception that Heterakis spumosa was absent.
Use of animals in the present study complied with relevant UK legislation. Mice were killed by a listed competent person using a Home Office approved method as detailed in schedule 1 of the Animal (Scientific Procedures) Act 1986 (ASPA). In consultation with the Home Office ASPA inspectorate, the work was not considered to involve any scientific procedures on living animals (merely housing for a short period before killing [18] ) and was therefore not subject to regulation by ASPA.
For each mouse we established five sets of splenocyte cultures. One of these was left unstimulated, to provide ''constitutive'' measurements and three others were respectively stimulated with agonists for toll-like receptor (TLR) 2, TLR4 and TLR9, which are receptors involved in the innate recognition of, and the triggering of immune responses against, invading microorganisms [20] . Cells and supernatants harvested from these cultures at 24 h were used to measure mRNA and protein expression of the proinflammatory cytokine tumour necrosis factor alpha (TNF-a) [18, 21] , mRNA expression of TLR2, TLR4 and TLR9, and mRNA expression of TGF-b1 and IL-10, which are antiinflammatory cytokines involved in the regulation of immune responses [22, 23, 24] . In the unstimulated cultures alone we measured mRNA expression of FoxP3, a transcription factor associated with the anti-inflammatory regulatory T-helper cell (T reg ) subset [25, 26] . TNF-a protein expression was also measured in 96 h supernatants from a further set of cultures stimulated with Heligmosomoides bakeri somatic antigen (HbAg).
Spleens were disaggregated through a 70 mm cell strainer into RPMI 1640. Following erythrocytic lysis (Sigma R7757), leucocytes were washed three times in RPMI 1640 and then cultured (37uC, 5% CO 2 ) on 96 well plates at 2610 6 cells/ml in RPMI 1640 supplemented with 24 mM Na HCO 3 , 10% heat inactivated foetal calf serum, 2 mM L-glutamine, 100 u/ml penicillin, 100 mg/ml streptomycin and 60 mM monothioglycerol. Triplicate sets of individual cultures (150 ml volume) were assigned to each experimental condition. Three cultures sets were stimulated with different TLR agonists: heat-killed endotoxin-free Listeria monocytogenes (TLR2 agonist) at 0.6610 8 cells/ml, or a 50:50 mix of Ultrapure Escherichia coli K12 lipopolysaccharide and Ultrapure Salmonella minnesota lipopolysaccharide (TLR4 agonists) at 3 mg/ml, or oligonucleotide ODN2006 (TLR9 agonist) at 6 mg/ml. All TLR agonist reagents utilized were tested for functional induction of the target TLR and for endotoxin contamination by the manufacturer (InvivoGen) and were used at concentrations previously determined to be optimal [18] . Further culture sets were stimulated with HbAg antigen at 50 mg/ml (pretreated with 500 U/ml polymyxin B for 4 h at 4uC) or left unstimulated. Culture supernatants and cells were harvested after 24 h for the unstimulated and TLR cultures and after 96 h for the HbAg cultures. Supernatants were immediately stored at 280uC and cells were placed overnight at 4uC in RNAlaterH (Ambion) and then stored at 280uC.
Accumulated TNF-a protein was measured in 24 h culture supernatants by ELISA (R&D, DY410) as previously described [18] , except that all samples were analysed in triplicate wells.
Relative mRNA accumulations in cells harvested at 24 h were measured by two-step reverse transcription quantitative real-time PCR (Q-PCR) using SYBR Green chemistry. Total RNA was extracted from cells stored in RNAlaterH using an Ambion RNAqueousH-Micro kit (AM1931) and converted to cDNA using an Applied Biosystems High capacity RNA-to-cDNA kit (4387406). All samples were DNase-treated and periodic controls lacking a reverse transcriptase enzymatic step indicated that DNA contamination was negligible. Real-time PCR primers for the cytokines, transcription factor and endogenous control genes were designed from conserved regions identified in multiple alignments of rodent sequences in the NCBI database. For TLR genes de novo amplification and sequencing of a larger (300-500 bp) fragment was first carried out using conserved primers (designed as above). Real-time PCR primers were then designed from the resulting Apodemus sylvaticus TLR sequence. All primers were designed with Primer 3 v. 0.4.0. For each target and endogenous control gene 2-6 primer pairs were designed and trialled and the optimal set selected for further use. All selected primers (Table 1) were confirmed to be specific by re-sequencing of the target fragment, did not produce confounding primer dimers under assay conditions and had approximately equal efficiencies close to 100%, based on the average of 5-fold dilution series repeated on multiple different plates. Target genes were normalized against the endogenous control gene b-actin. This was the most stable endogenous control gene amongst a set of 9 candidates considered (including c-actin, G6PDH, GAPDH, HPRT1, Tubulin, YWHAZ, 18 s and Albumin) as determined by stepwise exclusion of those with low stability estimated by the M statistic [27] . This procedure was conducted separately for 10 LPS-, HKLM-or ODN 2006-stimulated cultures randomly selected out of a pool of 300 culture samples. In all three cases b-actin was identified as the joint most stably expressed gene. The primers used for b-actin were sense 59-AGATCAAGATCATTGCTCCTCCT -39 and antisense 59-TCCTGCTTGCTGATCCACATC -39 which gave a 102 bp amplicon. The 96-well real-time PCR assay was designed so that all target genes for one animal were run on a single plate, except for Foxp3 amplifications which were all run on a separate plate. Each target gene reaction and the endogenous control gene reaction were run in duplicate for samples and in triplicate for a reference cDNA that was included on each plate. A no-templatecontrol was included in duplicate for each primer pair. Real-time PCR assays were run using default settings and the manufacturerrecommended enzyme, buffer and SYBR Green reagents on an ABI 7500 Fast machine (Applied Biosystems). ABI 7500 v 2.0.4 software was used to calculate the expression of each target in each sample as a fold difference relative to the reference cDNA by the DDCt method [28] .
In total 30 immunological expression values were measured per individual. Initial examination of the data indicated a small number of individuals in which most or all of the readings were extremely high. In order to characterize this group each variable was log transformed (log10 [x+1]) and standardized ([x2mean]/ standard deviation) and then outlying observations in each variable were determined as those occurring .1.56 the interquartile range above the third data quartile. Individual mice with $10 such outlying values were classified as part of an ''extreme expression'' group. Examining the subset of 22 variables in which there were few missing values and considering animals with a full set of observations for these, we also used hierarchical cluster analysis (HCA) (average link) and ordinations against the principal axes derived from principal co-ordinates analysis (PCO) and principal components analysis (PCA) to assess the distribution of individuals in phenotypic space. Data were log-transformed and standardized prior to HCA and PCO and log-transformed prior to PCA on the correlation matrix.
To analyze clustering in the occurrence of ''extreme expression'' status we coded extreme (1) vs non-extreme (0) expression as a binary variable and carried out retrospective spatial, temporal and spatio-temporal scans [29] with the software SaTScan TM v9.1.0, specifying a Bernoulli model. Following identification of a most significant cluster, subsequent clusters were identified by adjusting for the presence of more significant clusters.
Variations in the macroparasite community were summarised by a PCA of individual parasite variables on the correlation matrix [18] . This was based on data for the common macroparasite species at Cotgrave Forest [18] : Skrjabinotaenia lobata, Brachylaemus recurvum, Calodium hepaticum, Syphacia nigeriana, Heligmosomoides polygyrus, Ixodes ricinus, laelapid mites and Polyplax serrata. As the PCA of macroparasite variables produced a significant (P,0.001) first component with coefficients of the same sign for most species, scores for this component (PC1 P ) were taken as an overall indicator of macroparasite infection.
An estimator of body condition was produced by taking residuals (BW resid ) from a regression of body weight (BW) on SVL, incorporating different slopes for males and females and quadratic terms to allow for a curvilinear association.
We used variations in trapping catch rate as a measure of demographic fluctuation in the host population as this rate would be expected to reflect either changes in population density or changes in spatial behaviour that relate, in some way, to the condition of individuals. Initial pairwise comparisons between trap success on different sampling occasions were based on contingency table analysis (Fisher exact tests). Overall variation in capture rates was then analysed by a linear mixed model (LMM) allowing for correlation between successive trapping intervals (AR1 covariance model).
Variations in body condition and PC1 P were assessed with general linear models (LMs) initially including terms for cluster membership, body size (SVL) and host stage (classified as in Jackson et al. [18] ), with deletion of non-significant terms. For analyses of individual macroparasite species, LMs with normal errors or equivalent generalized linear models (GLMs) with Poisson errors or negative binomial errors were used as appropriate (again with deletion of non-significant terms). A Poisson distribution was used as a surrogate distribution in the case of ordinal data for P. serrata. In the case of negative binomial errors, the dispersion parameter k was estimated from that of the entire host population and any significant results reported are also consistent with an analysis of log-transformed data in an LM.
We analysed splenocyte expression of immunological genes (TLR2, TLR4, TLR9, TNF-a, TGF-b1, IL-10, FoxP3) in a time series of A. sylvaticus at Cotgrave Forest, UK from May to December 2008. Preliminary analyses of individual variation indicated that a small proportion of individuals were associated with very extreme expression values for most or all of the measurements taken (Figure 1 ). In order to categorize this variation we initially identified outlier values in log-transformed, standardized data as occurring above the third data quartile cutoff by more than 1.56 the interquartile range. Individuals with .10 outlier readings from the 30 variables measured were considered as an ''extreme expression'' group ( Figure 2) . The individual composition of this group was broadly supported by HCA, PCA and PCO for the subset of 22 expression variables without missing data values (Figure 3) .
It was not possible to identify a candidate infectious aetiological agent for the extreme immunological expression syndrome described above amongst the microparasites (Eimeria spp., Babesia microti and Bartonella spp.) and macroparasites whose presence we 
We categorized individuals into ''extreme'' and ''normal'' expression groups (as above) and scanned for temporal clusters of extreme expression using a Bernoulli model in the programme SaTScan TM . A significant temporal cluster (cluster 1) was detected in late June 2008 (log likelihood ratio = 20.37, P = 0.001). Adjusting for the occurrence of the most significant cluster, a significant secondary temporal cluster (cluster 2) occurred in early September 2008 (log likelihood ratio = 13.10, P = 0.001). Analyses for spatial clusters and spatial-temporal clusters failed to detect any purely spatial clustering, but did find two significant space-time clusters (P = 0.014) within the time frame of the most significant temporal cluster. The areas included and not included in these clusters appeared to broadly correspond to distinct forestry blocks.
Against a background of apparent demographic increase during May-December 2008, significant decreases in trapping rate with respect the immediately preceding trapping occasions (either 1 or 2 intervals distant) were only recorded immediately after the detection of extreme expression clusters (Figure 2 ). LMM analysis of trapping success data against time (days), allowing for positive autocorrelation between successive trapping sessions, suggested a highly significant increasing trend across May to December (LMM, P,0.001; parameter estimate = 0.0013060.00035 catch rate day 21 ). A 2-level factor coding the two time intervals following the first detection of each extreme expression cluster (vs Using eye lens weight as an index of age, plots of lens weight with respect to time allowed unambiguous identification of overwintered (1 + ) and young-of-the-year (0 + ) cohorts in 2008 and also in a retrospective analysis of animals sampled at Cotgrave Forest by Jackson et al. [18] in 2007 ( Figure 4 ). Cohort dynamics differed in 2008 compared to 2007 by an abrupt earlier disappearance of 1 + animals, in late June, that was followed by a very large proportional contribution to the population by new recruits (0 + individuals with the lightest eye lenses, ,0.014 g) (Figure 4 ).
Although a small number of divergent individuals occurred within cluster 1 (Figure 3 ), significant overall variation in expression patterns occurred between cluster 1 and cluster 2 ( Figure 5 ). Most individuals in cluster 1 could be characterized by comparatively very elevated constitutive and LPS-stimulated TNF-a expression. Individuals in cluster 2 were characterized by relatively high levels of constitutive and LPS-stimulated expression of TLR4, TLR9 and FoxP3.
A comparison of contemporaneous cluster 1 and non-cluster animals was not possible (insufficient individuals occurred in the same time intervals) but cluster 2 individuals had significantly greater body condition (BW resid ) (LM, P = 0.036) and grouped macroparasite infection (PC1 p ) (LM, P = 0.048) than non-cluster individuals from the same sampling points ( Figure 6 ). For individual macroparasite species, there were significantly greater infections in cluster 2 animals for the louse, P. serrata (GLM, Poisson errors, P = 0.048) and the catenotaeniid cestode, S. lobata (GLM, negative binomial errors, P = 0.035).
The analysis of immunological expression profiles may provide an unbiased strategy for identifying important microbial infection pressures acting on a natural population. In this study we attempted to demonstrate such an approach using a time series of wood mice from a habitat in the east midlands, UK.
Acute infections with highly pathogenic viruses, bacteria or protozoa are often associated with extreme expression of cytokines and TLRs [30, 31, 32, 33] . We discovered significant temporal clusters of individuals with extreme expression of immunologicallyrelevant genes, including cytokines and TLRs, in late June (cluster 1) and early September 2008 (cluster 2). This is consistent with the presence of an acute, highly immunogenic microbial infection in the mouse population at these times. Other possible causes for the ''extreme expression'' clusters are considered less likely. Thus, autoimmunity, allergy or malignancy not involving infection does not well explain the clustered temporal epidemiological pattern and poisoning is unlikely because the habitat is managed for pheasant shooting (pheasants would be likely to take poisoned bait placed for rodents) . The pattern seen is also difficult to attribute to handling stress or to ontogenetic or genetic variation. Compared to the non-cluster animals in the present study and to hundreds of other ''normal'' wild rodents (A. sylvaticus and Microtus agrestis) recently analysed in our laboratory in a similar way [16, 18] , the relative mRNA expression ratios for immunological genes and the amount of constitutive TNF-a protein secretion in some cluster animals are unprecedentedly high. The variation in expression between cluster and non-cluster animals also seems to be much greater than constitutive variation in expression reported amongst wild-derived Mus inbred laboratory lines [34, 35] .
The occurrence of the clusters in time was circumstantially associated with significant reductions in A. sylvaticus trapping rate that were contrary to a prevailing trend of demographic increase during the period of study. An additional, circumstantial, corollary was that an abrupt turnover between 1 + and 0 + A. sylvaticus cohorts occurred in the weeks following cluster 1, consistent with a perturbation affecting the population at this time. Over an equivalent time interval in the preceding year (2007), clusters of individuals with extreme TNF-a protein expression did not occur ( [18] ; unpublished data) and the turnover between 1 + and 0 + cohorts proceeded much more gradually (Figure 4) .
Whilst the identity of any putative aetiological pathogen(s) is unknown, the two ''epidemic'' clusters differed in the nature of the immune responses observed. Members of both clusters expressed high levels of all immunological genes studied, but cluster 1 was characterized by relatively higher expression of TNF-a at the protein and mRNA level and cluster 2 by higher expression of TLR4 and TLR9 and the regulatory T-helper cell-associated transcription factor FoxP3. These differences may be indicative of more than one infectious agent stimulating different immune responses. Alternatively, different immune responses to the same pathogen may have occurred because the animals involved in the respective clusters were themselves different. Cluster 1 individuals were exclusively over-wintered (1 + ) animals, whilst cluster 2 animals were exclusively young-of-the-year (0 + ) individuals. Phenotypic differences might therefore have arisen from ontogenetic stage variation, or from a different history of programming by environmental cues in the respective cohorts. A further possibility is that animals in the second cluster might have had some level of acquired immunity, following the outbreak earlier in the year, and produced more highly regulated responses indicative of a degree of immunological tolerance. This latter possibility is supported by the fact that some atypical individuals occurred in cluster 1 that were either phenotypically intermediate, or, more similar to cluster 2 individuals (Figure 3 ).
Given the highly focussed temporal occurrence of the clusters (appearing and disappearing over the timescale of a few weeks), it might be inferred that if a putative infectious agent is involved this is highly transmissible and produces acute infections that rapidly result in death or recovery and the development of resistance. It is likely such a pathogen could only be maintained by rapid spatial movement through the wider population, via locally unstable dynamics, or by inactive latent infections in some hosts acting as a reservoir and intermittent source of infection through time. One possibility consistent with some of these considerations is cowpox virus, infections with which can show fine-scale temporal clustering in wood mouse populations [36] . However, cowpox virus is known to suppress TNF-a responses [37] , which were expressed at very high levels in cluster 1 mice. Furthermore, field studies of cowpox in UK wild rodents [38, 39] generally indicate that infection levels within populations persist with a much coarser temporal grain than was the case for the tightly focussed episodes of infection recorded here.
A further interesting characteristic of the second cluster is that cluster members, controlling for life history stage and size, showed significantly better body condition and greater macroparasite infection than non-cluster individuals from the same time interval. This indicates that healthier individuals with more macroparasites might either be more susceptible to any putative infection or more resilient to the effects of infection. Increased susceptibility could arise because individuals in better condition eat more and range farther, exposing themselves to greater infection risks from macroparasites and microbial infections. Macroparasites might also contribute to susceptibility due to their anti-inflammatory activities [18] . Increased resilience could occur because animals in better starting condition might be more likely to survive infection, thus raising their representation in infected classes by differential mortality. The immunoregulatory influence of macroparasites might also be important in this context: dampening potentially lethal immunopathogenic inflammatory responses. A further possibility is that individual mice respond to infection by adjusting life history traits in a way that produces an overcompensatory response in body condition. For example, A. sylvaticus infected with cowpox virus in summer may show positive effects on survival due to a diversion of resources from reproduction [3] .
We have shown here that very distinct anomalies in immunological gene expression patterns can be detected in wild mammal populations if these are surveyed over time. We suggest that such patterns will often be caused by infectious agents and can be used to monitor important infection influences operating on a natural host population. The general approach used here is technically accessible to a wide range of researchers. An obvious extension would also be to combine the present methods with next generation sequencing techniques for the broad detection of microbial pathogens [40, 41] in animals with anomalous immunological transcriptional profiles compared to ''normal'' animals. In the present example, we focussed on a population of wood mice and were able to detect the probable signatures of epidemic infectious disease outbreaks and their demographic and individual correlates. Given the long standing interest in the dynamics of A. sylvaticus populations in the UK [42, 43, 44, 45] , in particular the apparent slow growth during the earlier part of the breeding season, this study might point towards a possible, destabilizing, role for microbial infectious disease in addition to the factors identified by other authors. There is also a wider significance for the ability of ecologists to analyze the dynamic interaction between pathogen communities and their wildlife hosts and, particularly, to identify unsuspected acute microbial infectious disease outbreaks, whose effects might otherwise be dismissed as generalized environmental noise.
We are very grateful to Till Hill Ltd for allowing access to the study site. . Differences in body condition and macroparasite burden between cluster 2 and non-cluster animals captured at the same time. A. Cluster 2 animals (dark bar) had significantly better body condition (BW resid ) than non-cluster animals, adjusting for size and stage. B. Cluster 2 animals (dark bar) had greater grouped macroparasite burdens (PC1 P ) than non-cluster animals, adjusting for size and stage. PC1 P represents first principal component scores from a principal components analysis of common macroparasite species at Cotgrave Forest. This principal component accounted for 23% of total variation and showed high loadings of the same sign for most species. It was thus taken to represent the major pattern of positive covariation within the assemblage. Variable coefficients on PC1 P were: Skrjabinotaenia lobata 0.25, Brachylaemus recurvum 0.28, Calodium hepaticum 0.24, Syphacia nigeriana 0.52, Heligmosomoides polygyrus 0.50, Ixodes ricinus 20.040, laelapid mites 0.102, Polyplax serrata 0.52. There were individually significant associations with cluster 2 membership, in the same direction as PC1 P , for P. serrata and S. lobata, adjusting for host size and stage. doi:10.1371/journal.pone.0020070.g006 Spliced Leader RNAs, Mitochondrial Gene Frameshifts and Multi-Protein Phylogeny Expand Support for the Genus Perkinsus as a Unique Group of Alveolates The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80–83 nt in P. marinus, and ∼83 nt in P. chesapeaki, significantly larger than the typical ≤56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized ‘marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata. Perkinsus marinus is a pathogenic alveolate causing ''dermo'' disease in oysters in estuaries of the north and central American Atlantic and Gulf of Mexico coasts. Other species of Perkinsus cause similar afflictions in a wide variety of other marine molluscs worldwide, all of which yield serious losses for shellfish industries [1] . This genus belongs to the crown group of eukaryotes known as Alveolata, but its exact phylogenetic position is debated. Based on the presence of cell surface micropores and an apical complex, P. marinus is historically considered to be a member of the Apicomplexa (for review see [2] ), an exclusively parasitic lineage responsible for malaria and other infectious diseases in humans and animals. However, P. marinus shares cytological features with dinoflagellates, such as flagellar spurs and closed mitosis [2] . Phylogenetic studies based on small subunit ribosomal RNA (18S rDNA) and some conserved proteins such as actin and tubulin also conclude that P. marinus is closer to dinoflagellates than to apicomplexans (e.g. review by [2] - [4] ), and thus are an early branch of dinoflagellate [4] , [5] . These results challenge a proposition that both Perkinsus spp. and related Parvilucifera spp. parasites should constitute an independent phylum named Perkinsozoa [6] , [7] .
Since spliced-leader (SL) trans-splicing occurs throughout the phylum Dinoflagellata (e.g. [8] - [11] ) yet has not been found in apicomplexans and ciliates, SL trans-splicing appears to be unique to the dinoflagellates within the Alveolata [12] . Under this scenario, the presence of SL trans-splicing in Perkinsus spp. [8] , [13] allies Perkinsus spp. with dinoflagellates. While dinoflagellates use a 22-nt conserved SL (DinoSL), P. marinus harbors a longer (22 nt) and a shorter (21 nt) SL, with sequences varying slightly from the canonical DinoSL [14] . In addition, the genome of P. marinus (,86 million base pairs; Project ID: 12736, http://www.ncbi.nlm. nih.gov/genomeprj/46451) is closer in overall size to apicomplexans (9-60 million base pairs; http://www.ncbi.nlm.nih.gov/genomeprj), but orders of magnitude smaller than dinoflagellates (3-250 billion base pairs; e.g. [15] , [16] ), and P. atlanticus chromosomes are more like typical eukaryotic chromosomes than dinokaryotic chromosomes [17] . Thus, whether Perkinsus spp. should be considered dinoflagellates remains unresolved.
Mitochondrial (mt) mRNA editing is a distinct characteristic of dinoflagellates within Alveolata and can be a useful marker to assess whether a lineage of alveolate is a dinoflagellate [12] . RNA editing is a sequence re-tailoring process that can be recognized by changes in an RNA sequence compared to that of its encoding DNA. Initially used to describe the insertion or deletion of uridine residues in mitochondrial (mt) transcripts in the kinetoplastid protozoans, the term ''RNA editing'' now also refers to nucleotide (nt) substitutions in RNA that occur in a wide variety of organisms (see [18] , [19] for review). In Alveolata, mt gene mRNA editing only occurs in dinoflagellates, displaying the greatest diversity of modifications yet described in the context of a single genomic environment. The frequency of editing events decreases from high levels in the late-branching lineages to none in the ancient lineages such as Oxyrrhis and Noctiluca (e.g. [12] , [19] ). It is unclear if Perkinsus spp. mt gene mRNAs undergo editing, but the mt cox1 of P. marinus is not translatable by the standard or mitochondrial codon table. The reading frame must be shifted 10 times by an unknown mechanism to yield a consensus COX1 protein [20] . Once verified, this bizarre process may be used as another molecular feature to demarcate Perkinsus spp. from dinoflagellates.
In this study we investigated the Perkinsus genus for the SL RNA gene structure, intron prevalence, full-length mt cox1 and cytochrome b (cob) genes and their transcripts, and multiple-protein phylogenetic position. With the help of the GenBank database for six species and 33 unidentified Perkinsus sp. strains, and the P. marinus genome sequence, we performed thorough phylogenetic analyses and identification of introns in P. marinus. We paid special attention to histones because these proteins were thought to be absent in dinoflagellates until recently (for review see [21] ). We used deduced full-length amino acid (aa) sequences of 41 genes to reconstruct phylogenetic trees. Genomic structures and corresponding RNA sequences of the SL gene were analyzed. Sixtyeight Perkinsus full-length cDNAs obtained in our previous studies [8] , [14] were mapped to genome sequences to identify corresponding genes, and combined with 36 other reported genes to determine the frequency of introns. Although the Perkinsus clade shares commonalities with dinoflagellates, our data show that it is a unique lineage basal to the monophyletic clade of dinoflagellates.
Perkinsus marinus and dinoflagellate cultures, RNA isolation and cDNA construction Perkinsus marinus isolate ATCC 50439 and P. chesapeaki ATCC PRA-65 were grown in tissue culture flasks with liquid media, samples (3-4610 6 cells) were collected by centrifugation and total RNAs were isolated as reported previously [14] . Dinoflagellates Amphidinium carterae (CCMP1314) and Karlodinium veneficum (CCMP2778) were grown in f/2 seawater medium at 20uC at a 12 h:12 h light:dark photocycle with a photon flux of approximately 50 mE?m 22 s 21 . When the cultures were in the exponential growth phase, ,1610 6 cells were harvested and total RNAs isolated according to Zhang et al. [8] . These RNAs were used for cDNA synthesis as described previously [8] .
Identification of the SL RNA genes from the P. marinus genome project Perkinsus spp. were suspected to possess a SL sequence similar to that of dinoflagellates (DinoSL; [8] ). Two types of SL sequences were detected at the 59 end of P. marinus full-length cDNAs of pcna and cyclins [14] , PmaSL1, 59-ACCGTAGCCATCTTGGCT-CAAG-39 (22 nt) and PmaSL2, 59-ACCGTAGCCATCTGGCT-CAAG-39 (21 nt). These two Perkinsus SL sequences were used to query P. marinus whole-genome shotgun reads [http://www.ncbi. nlm.nih.gov/genomeprj/46451] to identify SL RNA genes. For hits with 85-100% identity to the queries, the genome sequences were collected for alignment with one another and with SL RNAs from dinoflagellates. Type-specific primers were designed for amplifying the putative SL RNAs (Table 1) .
Total RNA from ,10 6 cells of both Perkinsus species and four strains of dinoflagellates in our previous studies [8] , [9] , including Prorocentrum minimum (CCMP696), Polarella glacialis (CCMP2088), Karenia brevis (CCMP2228) and Karlodinium veneficum (CCMP1975) were used for RNA blots. RNA samples were loaded onto an 8% acrylamide/8 M urea gel, a medium resolution gel optimal for RNAs below 350 nt, electrophoresed, and transferred to nylon membranes [22] . Oligonucleotide probes used for hybridization included dinoSLa/s for detection of the general dinoflagellate SL RNAs and the two types of Perkinsus SL RNAs (PmaSL-La/s and PmaSL-Sa/s hybridizing to exons and PmaSL-Li and PmaSL-Si to introns) ( Table 1 ). The cDNA clones containing the two P. marinus SL RNAs were dot blotted to serve as positive controls for detection of the specific substrate SLs on RNA blots. Total RNA from Leishmania tarentolae cells was included to provide size markers. Oligonucleotide probes were labeled with cP 32 -ATP for hybridization [22] .
Poly (A) mRNA was depleted from P. marinus total RNA and a poly (A) tail was added to the remaining population using Escherichia coli Poly (A) Polymerase (Takara Mirus Bio) as reported [8] . First-strand cDNA synthesized using GeneRacer Oligo dT primer (Invitrogen) was used as PCR template. Two rounds of touch-down PCR were carried using the same conditions as above, with the extension time of 5 sec at 72uC. The first round of PCR was performed using PmaSL-LSF1 and GeneRacer3 primers. The PCR products were diluted 100-fold and used in the second round PCR with PmaSL-LSF2, PmaSL-LNF2, PmaSL-LNF3, PmaSL-S2F2, or PmaSL-S2F3, each paired with GeneRacer3, as the nested primers (Table 1) .
Structures were modeled for the two dominant types of SL RNA transcripts using the MFOLD online program [http://mobyle. pasteur.fr/cgi-bin/MobylePortal/portal.py?form = mfold]. Folding was performed using the default setting except that the temperature was set at 27uC to match the P. marinus culture conditions.
The mt cox1 and cob sequences were PCR-amplified from both genomic and cDNA templates using universal and Perkinsusspecific primers designed in this ( Table 2 ) and previous studies [12] , [23] , [24] . PCR was performed with 30 cycles of 95uC for 15 sec, 50-58uC for 30 sec, and 72uC for 40 sec. PCR products were sequenced either directly or after cloning into a T-vector, with 5-10 clones randomly chosen for sequencing. To obtain the ends of the mt genes, we designed Perkinsus-specific primers for both P. marinus and P. chesapeaki (Table 2 ) based on the mt cox1 and the partial cob sequences obtained from the newly released P. marinus genome shotgun sequence.
Ribosomal proteins (RPs) from dinoflagellates [21] were used to query the P. marinus genome and GenBank databases to retrieve RPs from P. marinus, apicomplexans, ciliates, diatoms and other eukaryotic representatives. Since many of the dinoflagellate RP cDNAs available were not full-length, to maximize phylogenetic information from these genes, 22 full-length cDNAs of RPs from dinoflagellates Amphidinium carterae CCMP1314 and K. veneficum CCMP2778 were cloned using dinoflagellate-specific SL coupled with 39 RACE as described previously ( [8] ; GenBank accession # GU372975-GU373034). To diversify the gene markers for phylogenetic analyses, another 12 conserved gene sequences were collected from our ongoing cDNA sequencing project for these two species, and their 59 and 39 ends achieved using RACE as necessary. Using these as queries, homologs were collected from GenBank for P. marinus and other species mentioned above. The absence of histones, long considered a benchmark of typical dinoflagellates, is erroneous (see [21] for review); thus, histone genes were retrieved from the Perkinsus genome project database. Full-length or nearly full-length mt cox1 and cob sequences were also obtained from P. marinus and P. chesapeaki. The 39 end of cob for both Perkinsus spp. was obtained using the 39 RACE technique with Perkinsus cob primers paired with GeneRacer3 primer (Invitrogen). All of these genes were used in phylogenetic analyses.
Predicted aa sequences of each gene were aligned with homologs from related organisms using CLUSTAL W (1.8) and inspected manually. Phylogenetic relationships of P. marinus with alveolate relatives and other eukaryotes were inferred using Neighbor Joining (NJ), Maximum Likelihood (ML), and MrBayes (MB) analyses. NJ analysis was performed online [http://clustalw. ddbj.nig.ac.jp/top-e.html] with the default setting. For ML tree reconstruction, the datasets were run through ProtTest [25] to identify the best-fitting aa substitution models (Table 3) , which were then employed in the phylogenetic analysis using PhymLv3.0 [26] . MB analysis was carried out with 20,000-1,000,000 MCMC generations depending on when the average standard deviation of split frequencies reached below 0.01, a tree sampling frequency of 10-100, and 25% of generations discarded as burn-in [27] . To verify the reliability of the tree topologies, branch support was estimated based on bootstrap (1,000 resamplings) in NJ, approximate Likelihood Ratio Test (aLRT) in ML, and posterior probability in MB.
Thirty-six and 37 unique full-length cDNAs from P. marinus and P. chesapeaki, respectively [8] , were used as the queries to nBLASTsearch against P. marinus genomic sequences to obtain the corresponding genomic DNA. The recently published full-length cDNAs and genomic DNAs for proliferating cell nuclear antigen (pcna) and two types of cyclins from P. marinus [14] , as well as 36 other common protein-coding genes of P. marinus such as tubulins, gapdh, centrin, hsp90 and ribosomal proteins reported in GenBank were compared (Table S1 ). Canonical GT/ AG intron/exon boundaries validated the deduced intron start and end positions. The percentage of genes within this cohort that contained introns was determined.
From the reported P. marinus genome database we identified two major types of SL RNA genes: PmaSLRNA-L or L-type, and PmaSLRNA-S or S-type ( Figure 1A) , with the SL exons corresponding to the two SL sequences found previously in pcna and cyclins [14] . These sequences were similar to DinoSL ( Figure 1B ). For the L-type, we identified seven sequences (Table 3) , and all but one (AAXJ01000089, containing two units of SL RNA) are 1-1.8 kb in length containing a single SL RNA gene. For the S-type, 42 sequences were identified with lengths ranging 1 to .14 kb (Table 4) ; of these, some were arrayed as tandem repeats or as a single unit clustered with both or either of the U2 and U4 snRNA genes downstream of the SL RNA gene; others were single or 2-unit tandem-repeat sequences not associated with U2 or U4 snRNA genes ( Table 4 ).
The sequences containing the two types of P. marinus SL RNA genes (PmaSLRNAs) were conserved in the first 82-83 nt, with the SL exon of the L type 1-nt longer than that of the S type. Sequence similarity diminished in the downstream intron region. The sequence upstream of SL was more complex: for the L-type PmaSLRNAs, upstream sequences were uniform, whereas those of the S type were diverse, with some resembling the L type ( Figure 1A ). When PmaSLRNAs were aligned with the representatives of known dinoflagellate SL RNAs, PmaSLRNAs showed similarity in the exon (i.e. the 21/22-nt SL region) and moderate similarity in the beginning of the intron region (i.e., immediately downstream; Figure 1B ). As in dinoflagellates, the predicted Smbinding sequence was located in the SL exon of PmaSLRNAs, and the 39 termini of the majority of substrate transcripts mapped within poly-T tracts, reminiscent of the termination element in SL RNAs of kinetoplastid [22] , some dinoflagellates [9] , and of other small RNA genes. The SL RNAs of two Perkinsus spp. and four dinoflagellates were analyzed by gel electrophoresis and hybridization. Ethidium [9] ; the number of identical clones retrieved for each type is indicated by ''@number'' following the species abbreviation and type number). The SL region (boxed) is shown in uppercase letter, intron and the flanking regions are shown in lowercase letters, * indicates the conserved nucleotide (nt). The first 'A' of SL is numbered as nt 1. SL RNA transcripts mapped by 39 RACE analyses are denoted by arrows to indicate the terminal positions, thickness with darkness of the arrows denote relative frequency of clones that ends where it is indicated. Note that the PCR-amplified Amoebophrya sp. genomic sequences contain only one unit of SL RNA gene, the partial SL sequence is of the primer used. Per, P. marinus, Amo, Amoebophrya sp.; Har, Heterocapsa arctica; Kbr, Karenia brevis; Kve, Karlodinium veneficum; Ppi, Pfiesteria piscicida; Pgl, Polarella glacialis; Pmi, Prorocentrum minimum. SL refers to SL RNA sequences obtained from SL-only repeats; SL-5S indicates SL RNA sequences from genes associated with 5S rRNA genes. *: sequences from [8] ; **: sequence from [46] ; #: sequences from [9] , $1-4: GQ178071-GQ178074; N: sequences missing in the original reports. Shaded are conserved positions defined as identical in over six sequences in at least three species. A non-canonical C in the splice donor site of KbrSL-3 is boxed. Gaps introduced in the sequence alignment are shown as '-'. doi:10.1371/journal.pone.0019933.g001 bromide staining revealed that the two Perkinsus species have similar small RNA molecule profiles with commonalities to the dinoflagellate P. minimum (Figure 2A) . Hybridization of an RNA blot of this gel with the 19-nt dinoflagellate SL probe DinoSLa/s (including 14 nt of SL and 5 nt of intron; Table 1 ) showed the dinoflagellate SL RNA pattern with major transcripts of ,56 nt for the four dinoflagellates as reported previously [8] , [9] ; no hybridization was detected for the two Perkinsus species ( Figure 2B ). Probing the blot with P. marinus L-type or S-type SL probes (PmaSL-La/s and PmaSL-Sa/s respectively; Table 1 ), strong bands of .72 nt appeared in both Perkinsus species for both probes, with a minor band of slightly shorter length in the P. marinus sample for probe PmaSL-Sa/s; neither probe hybridized to dinoflagellate SL RNA ( Figure 2C, 2D) , indicating that the .72-nt bands are specific to the genus Perkinsus, and that Perkinsus SL RNAs are longer than those of typical dinoflagellates. Consistent with the similar RNA levels seen on the gel for the two Perkinsus species, probe PmaSL-La/s detected equivalent levels of this SL RNA variant ( Figure 2C ) in the two species. However, the band of P. chesapeaki was weaker than that of P. marinus with probe PmaSL-Sa/s ( Figure 2D ), possibly reflecting reduced expression or impaired hybridization due to a nucleotide alteration(s) in the exon region in P. chesapeaki. The minor band in the P. marinus lane may represent degraded SL RNA products. To further distinguish the two types of PmaSL RNA transcripts and to explore whether P. chesapeaki SL RNAs have similar introns to those of P. marinus, additional probes were designed for the PmaSLRNA L-type and S-type intron sequences (PmaSL-Li and PmaSL-Si; Table 1 ). Both Table 4 . Genomic sequences containing SL RNA genes identified from P. marinus genome data. intron probes revealed bands at .72 nt and some minor bands of ,72 nt in P. marinus ( Figure 2E, 2F ), but no bands in P. chesapeaki, suggesting that P. chesapeaki SL RNAs have different intron sequences than P. marinus. An additional band appeared at ,150 nt with PmaSL-Si for both Perkinsus spp. (Figure 2F ), a likely result of non-specific hybridization to the abundant 5.8S ribosomal RNA (Figure 2A) . To validate the specificity of the probes, 39 RACE cDNA clones of the L-and S-type SL RNA were used to create dot blots that were hybridized separately with each probe. Each yielded a positive signal only when the corresponding probe was used ( Figure 2G, 2H) . A 39 RACE analysis gave an assortment of 39 ends for both PmaSLRNAs. Of the 48 PmaSLRNA cDNA clones mapped, 25 ended at the 2 nd T, 11 clones at the 1 st T, and 4 clones ended at the 3 rd T of the poly-T tracts present in both SL genes, representing 83% of the ends obtained. Thus, most PmaSLRNA transcripts were 80-83 nt in length, corresponding to the major band observed in the RNA blots. The minor end classes of ,72 nt may have contributed to the minor products seen by RNA blotting, possibly representing degraded or misprocessed SL RNA products.
BLAST analysis using PmaSL1 and PmaSL2 hit some cDNA or genomic DNA sequences apparently coding for proteins (e.g. EH076923, EH059894, EH059894, EH059894). In addition, over 100 genomic sequences were retrieved from the genome data that contained recognizable PmaSL1 (.60, e.g. AAXJ01000048, AAXJ01000335, AAXJ01000111, AAXJ0100359, AAXJ01004662, AAXJ01000077) and PmaSL2 (.40, e.g. AAXJ01000111, AAXJ01000162, AAXJ01000192, AAXJ01000237, AAXJ01000370) but no recognizable intron downstream. Most of these SL sequences started with T, and were arrayed in tandem repeats, and their downstream regions were variable. To investigate whether those SL RNA-like genomic sequences were also expressed, we designed primers (Table 1 ) containing a partial SL and downstream nucleotides or the downstream sequences alone and applied them to 39 RACE and RNA blotting analyses. Neither of the approaches yielded clear products, indicating that these SL-like sequences are not functional SL RNA genes.
Similar to the situation in dinoflagellates, no apparent Smbinding site sequence was found in the predicted intron regions of either of the PmaSLRNAs. Instead, AUUCUGG (L-type) or AUCUGG (S-type) found within the SL was the only recognizable candidate Sm-binding site, as in the DinoSL (AUUUUGG). The predicted intron region was similar between the two Pma-SLRNAs, in contrast to the conserved intron in DinoSL RNAs, with the exception of the ancient parasitic genus of dinoflagellates Amoebophrya that showed considerable variation ( Figure 1B ). In the structural simulation using the default conditions for all but temperature, which was adjusted to the culture temperature of 27uC, the splice-donor dinucleotide ('gu' in 'Gguag') was doublestranded and the putative Sm-binding site (AUUCUGG/AU-CUGG) single-stranded, forming a small terminal loop. The simulation yielded one comparable structure for both types of PmaSLRNAs ( Figure 3 ). The predicted structures were similar to typical dinoflagellate SL RNA structures, having two stem-loops [8] , [9] , with the 'extra' intron region situated in a bulge of unpaired sequence connecting the two stem loops.
All the possible combinations for cob primers designed based on dinoflagellate cob (Table 2 ; [12] , [23] , [24] ) were tested but failed to PCR amplify any products. BLAST searching using cob aa sequences from apicomplexans and dinoflagellates against the P. marinus whole genome shotgun sequencing database (tblastx) hit one contig (860 bp, AAXJ01022806) containing the 59 end of a cob-like sequence. The corresponding mRNA of this sequence and its 39 end were obtained for both species of Perkinsus by PCR and 39 RACE using Perkinsus-specific primers paired with the GeneRacer3 primer (GenBank accession numbers HQ670239, HQ670241; Figure 4 , Table 2 ).
Using dinoflagellate cox1 primer sets dinocox1F5-R3 [24] and universal cox1 primer set cox1_5b-3a (Table 2) , DNA fragments were amplified from genomic and cDNA templates of P. marinus (0.96 kb) and cDNA of P. chesapeaki (0.33 kb), respectively. Direct sequencing of these fragments proved that they were cox1 sequences with 50-60% identity to that of dinoflagellates and apicomplexans. When the 0.96-kb P. marinus cox1 sequence was used to BLAST against the P. marinus genome database, one 3147-bp sequence (AAXJ01004741) was obtained with 100% identity to the P. marinus DNA fragment we found. Nearly full-length cDNAs of cox1 were generated by PCR amplification using Perkinsus-specific cox1 primers for both Perkinsus species (GenBank accession numbers HQ670238, HQ670240; Figure 5 , Table 2 ). Both the cob and cox1 cDNA sequences matched the corresponding genomic DNAs, indicating that no mRNA editing events occurred in either transcript.
Comparison of nt and deduced aa sequences of Perkinsus cob and cox1 with counterparts in other alveolates revealed that correct translation of Perkinsus mt genes required the Mold/Protozoan/ Coelenterate mt codon table (TGA codes for tryptophan) in general. To be fully translatable without internal stop codons, however, frameshifts had to be introduced at every AGG and CCC codon, the equivalent of using AGGY to code for glycine (six sites in cob and 7-8 sites in cox1) and CCCCU for proline (twice in cox1) (Figures 4, 5 ). An analogous result was reported by Masuda et al. [20] for the P. marinus cox1. Multiple cDNAs and genomic sequences substantiated these unusual reading frames, as well as the direct sequencing of PCR products. An interesting difference was found between the two Perkinsus species: at one site in cox1, glycine was encoded by an AGGU codon in P. marinus, but by a standard GGU codon in P. chesapeaki ( Figure 5 ). With the introduction of these invoked anomalous quadruplet and quintuplet codons, the deduced aa sequences of the two Perkinsus COX1 were 98% identical to each other, 46-50% similar to the homologs in apicomplexans, 42-49% to dinoflagellates, 29-31% to ciliates, and 38-42% to other organisms ( Figure 6 ). For cob (Figure 4) , besides the quadruplet codon AGGY, glycine was also encoded by the quintuplet codons UAGGC (for P. marinus) and UCGGU (for P. chesapeaki). After these adjustments, the deduced COB aa sequences of the two Perkinsus spp. shared 97% similarity to each other, 34-36% to apicomplexans, 22-44% to dinoflagellates, 15-17% to ciliates, 27-33% to other organisms (Figure 6 ).
The corresponding P. marinus genomic sequences of 39 and 29 full-length cDNAs from P. marinus and P. chesapeaki, respectively [8] , [14] , were obtained. Comparison of these 68 cDNAs with the genomic DNA sequences revealed the presence of introns in 42 genes, yielding a 61.8% intron rate. Through GenBank database searches, we obtained an additional 36 common genes with known genomic structures, 30 of which have intron(s) (Table S1 ). Overall, the intron rate for P. marinus genes was 69.2% (72 out of 104). The intron-containing genes harbored between one and ten introns with the lengths ranging from 39 to 1622 bp, the majority of which were ,100 bp.
Twenty-two ribosomal proteins were obtained for Perkinsus and various organisms; Maximum Likelihood (ML) trees inferred from the individual sequences gave varied tree topologies (Figures S1, S2, S3, S4). In general, P. marinus, dinoflagellates, apicomplexans, and ciliates formed a monophyletic group, while in several cases the heterokont diatoms, the closest relative of the alveolates, branched with some of the alveolate lineages, but without bootfostrap support. Perkinsus spp. allied with dinoflagellates in some cases (e.g. Figures S1C, 1F, S2E) , and with apicomplexans (e.g. Figure S1D ) or the diatoms (e.g. Figure S2D ) in others, often with weak or no bootstrap support in these cases, indicating an unstable phylogenetic affinity. In contrast, NJ trees based on the 12 conserved protein sequences (actin, b-tubulin, GAPDH, atubulin, centrin, HSP90, EF1-a, ADP ribosylation factor, TIF5A, SmD1, cytochrome C and 14-3-3) produced similar tree topology, with P. marinus clustering with dinoflagellates in most of the cases (Figures S5, S6) . For mt genes, Perkinsus spp. clustered with ciliates in COB tree, while allied with dinoflagellate/apicomplexan cluster in COX1 tree ( Figure 6 ). When the concatenated RP sequence data (3,142 aa) was used, analyses using NJ, ML, and MB produced trees of similar topologies in which P. marinus branched at the base of the dinoflagellate clade (Figure 7) . This was true for the analyses both without ( Figure 7A ) and with ( Figure 7B ) the ancient dinoflagellate lineage Oxyrrhis marina. The only exception was the MB tree in which P. marinus was allied with the clade of apicomplexans ( Figure 7A) . Similarly, when the other 12 protein sequences were concatenated (3,879 aa) the consensus tree inferred from the three algorithms showed the close relationship between P. marinus and dinoflagellates ( Figure 7C ). In most of these concatenated trees, the alliance of P. marinus and dinoflagellates was supported.
Multiple sequences were obtained for each of the P. marinus histones; in most of the phylogenetic trees, these sequences clustered together and allied with apicomplexans except for the H3 tree, in which one P. marinus H3 grouped with the apicomplexan Toxoplasma gondii, the other with dinoflagellate/ ciliate clade (Figures 8, 9 ). Histone 2A in many organisms has acquired an isoform referred to as H2A.X. In both dinoflagellates and P. marinus, H2A.X seems to be the dominant, if not the only, form. The homolog retrieved from the P. marinus genome was clustered with H2A.X in the clade of apicomplexans (Figure 8 ).
To understand the evolution of parasitism in the Alveolata, the phylogenetic relationship among the major lineages in this crown group must be resolved accurately. No consensus exists for the relationship between the Perkinsus genus with other alveolates, particularly the partition between apicomplexans and dinoflagellates. Taking advantage of SL RNA, mt gene characteristics, gene structure (e.g. intron density), and the increasing availability of functional protein sequences, robust evidence is provided in support of a relatively close relationship between Perkinsus spp. and dinoflagellates, in addition to a distinct non-dinoflagellate position of this alveolate pathogen.
Perkinsus SL RNAs mark earlier emergence and more complex evolution of trans-splicing in alveolates PmaSLRNA sequences are similar to those of dinoflagellate SL RNAs, including the location of an apparent Sm-binding domain in the exon rather than in the intron, as is the case typically in other SL trans-splicing eukaryotes (see [8] , [9] for review). The SL has left its footprints in other parts of the dinoflagellate genome in the form of single and tandem exon repeats located adjacent to the 59 UTRs of protein coding genes [28] . This apparently unproductive phenomenon is postulated to occur when SLcontaining mRNA are reverse-transcribed and integrated to the genome [28] but could also be a result of chromosome cross-over recombination [16] . Likewise, SL exons in single or multiple units were found in some P. marinus genes. The S-type SL with L-type intron was also suggested to exist based on PCR-amplified cDNA sequences of P. marinus SL RNA [29] , although it requires verification by further genomic analysis.
PmaSLRNAs are distinct from dinoflagellate SL RNAs. In the apparent Sm-binding site, instead of a ''TTTT'' motif conserved in dinoflagellates, PmaSL has ''TCTT'' or ''TCT''. The intron region of the SL RNA in dinoflagellates is conserved, but the similarities diminish in Amoebophrya, a parasitic lineage currently considered to represent the most ancient dinoflagellate [30] . SL RNAs in P. marinus display similar divergence from dinoflagellates, with a substantially longer intron relative to the core dinoflagellates and Amoebophrya, suggestive of an earlier divergence for P. marinus. The SL RNAs in other SL trans-splicing eukaryotes range from 46 nt in the urochordate Ciona intestinalis to 142 nt in Trypanosoma brucei. The SL RNA transcripts in P. marinus range from 80-83 nt, and are ,83 nt in P. chesapeaki. Thus, Perkinsus SL RNAs have unique features in comparison to dinoflagellates, and Perkinsus spp. may represent the earliest trans-splicing lineage within Alveolata, separated from the non-trans-splicing Ciliophora and Apicomplexa [8] , yet distinct from the Dinoflagellata. Given the high diversity of the parasitic Syndiniales class of dinoflagellates [31] , the uncharacterized marine alveolate group I that lies between Perkinsus and the core dinoflagellates ( [7] and references therein) should be examined for the presence of additional types of SL RNA.
Perkinsus is a distinct pre-dinoflagellate taxon As SL trans-splicing occurs in both basal (e.g. Amoebophrya and Oxyrrhis) and advanced (e.g. Alexandrium) lineages of dinoflagellates but not in apicomplexans and ciliates [8] , the two closest relatives of dinoflagellates, the occurrence of this distinct mRNA processing mechanism is considered a defining indicator for dinoflagellates [12] . The presence of SL RNA trans-splicing in Perkinsus spp. indicates its inclusion in or alliance with the phylum of dinoflagellates, in accord with previous molecular phylogenetic studies (e.g. [2] - [4] , [30] ). Likewise, our multi-protein phylogenies consistently show that P. marinus is related to dinoflagellates among other representative eukaryotes with moderate-to-strong bootstrap support. Among the many single-gene phylogenetic trees, the majority is in agreement with the concatenated protein trees. In all trees, P. marinus was positioned as the earliest divergent even when Oxyrrhis, a genus hypothesized to be a pre-dinoflagellate [32] or an ancient lineage [12] , was included. In addition, P. marinus was always placed basal to Amoebophrya, another ancient lineage of dinoflagellates.
Yet some degree of uncertainty exists in the phylogenetic position of Perkinsus. Contrary to the long-held notion that dinoflagellates did not possess nucleosomes and canonical histones, genes of all four major histones have recently been found in dinoflagellates (for review see [21] ); however, dinoflagellate histones usually have unique sequences with insertions/deletions in several places, resulting long branches in the phylogenetic trees (Figures 8, 9 ). Comparing to dinoflagellates, P. marinus histones have typical eukaryotic sequences and group with apicomplexans in the phylogenetic trees. Besides histone trees, some other individual protein trees ( Figures S1D, S2A , 2B, 2C, S3B, S5C) also show an alliance of Perkinsus spp. with apicomplexans, in agreement with earlier morphological and cytological studies [2] . In rare cases, P. marinus is clustered with diatoms, apparently because the protein sequence was too short to provide strong support of any topology.
The current analysis is limited in that only the sequences from one or two species of Perkinsus were available. Perkinsus appears more distant from apicomplexans than from dinoflagellates; however its generally close relationship with the clade of dinoflagellates could be due to the absence of taxa from intermediate lineages such as marine alveolate group I, additional taxa from the Perkinsozoa (e.g. Parvilucifera spp.), and dinoflagellates of the class Syndiniales.
Cis-splicing is thought to be uncommon in dinoflagellates [2] ; however, only a few dinoflagellates have been examined for the presence of introns (e.g. form II Rubisco in Symbiodinium [33] , luciferase C in Pyrocystis lunula [34] ). We have examined more than 30 genes such as pcna, form II Rubisco, 14-3-3, and centrin for several dinoflagellates ( [35] , [36] and our unpubl. results), and did not find introns. A relatively high intron density for a dinoflagellate is found in Amphidinium carterae, in which a survey of 31 genes yields a 48% cis-splicing rate [37] . Our analysis of 104 Perkinsus genes yielded a 69.2% cis-splicing rate, a level contrasting those found in most dinoflagellates, and closer to the .50% level found in apicomplexans [38] , [39] .
The unique reading frame shifting and the lack of mRNA editing for mt genes again mark P. marinus as distinct from typical dinoflagellates. Both P. marinus cob and cox1 mRNAs are identical to their genomic DNAs, indicating that no mRNA editing occurs to correct the frameshifts in these mt genes. Masuda et al. [20] reported the full-length mt cox1 mRNA from P. marinus, showing that this mRNA was not translatable with standard codon usage, due to a reading frame that had to be shifted a total of 10 times at every AGG and CCC codon to yield a consensus COX1 protein.
One or two sites of +1 frameshifting have been documented in animal mt genes (for review, see [40] ), but such extensive +1 and +2 frameshifts are unique. In retroviruses, a -1 frameshift is corrected by tRNA back-slippage over homopolymeric codons adjacent to a local secondary structure that may include a pseudoknot (for review, see [41] ). Masuda et al. [20] suggest two feasible mechanisms for the translational frameshifts in Perkinsus: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons (similar to the model hypothesized by Beckenbach et al. [42] ), or specialized tRNAs recognizing non-triplet codons AGGY and CCCCU to code for glycine and proline, respectively. In this study, we add cox1 for P. chesapeaki and cob sequences for P. marinus and P. chesapeaki, which share the unusual AGGY codon with cox1 and use other unusual codons (UAGGC and UCGGU) to encode glycine as well. Specialized tRNAs in the Perkinsus mitochondrial system recognizing non-triplet AGGY and CCCCU codons, and likely UMGGY as well, may be more likely than the ribosomal frameshifting scenario, as naturally occurring tRNA mutants suppress +1 frameshifts via an extended anticodon loop in Escherichia coli (e.g. [43] ), and quadruplet codons are used in protein mutagenesis [44] .
The Perkinsus lineage is remarkably distinct from, while close to, dinoflagellates, and is most likely an independent lineage, supporting the postulate that Perkinsus spp., along with Parvilucifera spp., constitutes an independent phylum dubbed Perkinsozoa, the fourth phylum in Alveolata [6] . Although not addressed directly, a number of recent phylogenetic trees containing taxa from marine alveolate group I and Perkinsus-related parasitic alveolates such as Parvilucifera spp. reinforce grouping of Perkinsus spp. as an independent phylum [7] , [45] , [46] . Future phylogenies with broader taxon sampling that include species from Parvilucifera spp., Syndiniales in addition to Amoebophrya, and marine alveolate group I representatives will refine the phylogenetic relationships among Perkinsus, dinoflagellates, and other alveolates.  One-year molecular survey of astrovirus infection in turkeys in Poland The presence of turkey astrovirus (TAstV) was monitored in meat-type turkey flocks in Poland in 2008. Clinical samples (10 individual faecal swabs/flock) from 77 flocks aged 1-19 weeks were collected from different regions of the country. RT-PCR experiments were performed for detection and molecular characterization of TAstV using four sets of primers within the RdRp gene (ORF1b). The prevalence of astrovirus was 34/77 (44.15%) in the flocks tested. TAstV type 2 was associated with 30 of 77 infections (38.9%), either alone or in mixed infections; TAstV type 1 was detected in 9 of 77 flocks (11.6%), either alone or in mixed infections; ANV was detected only in one flock (1.29%) by sequence analysis during this study. Phylogenetic analysis revealed genetic variability in the TAstV strains that were isolated. Some of Polish TAstV-2 strains were genetically related to the North American isolates; however, most of them formed a distinct subgroup of “European” isolates, suggesting their separate origin or evolution. Additionally, due to the high variability of the TAstV sequences, the most suitable method for TAstV typing seems to be sequencing. The history of studies on astroviruses (AstV) is rather short, as they were first described by Appleton and Higgins in 1975 in the context of gastrointestinal problems in children [1] . Subsequently identified astroviruses were also associated with enteritis and affected a wide range of host species, such as lambs, calves, piglets, dogs, turkey poults, chickens, guinea fowl, red and roe deer, cats, mice, and mink [2, 4, 14, 16, 26] . Additionally, some astroviruses have also been associated with non-intestinal illness. In ducklings, the virus known as duck virus hepatitis type 2 (DVH2) causes fatal hepatitis, and in chickens, avian nephritis virus (ANV) results in mild growth depression, kidney lesions and mortality [8, 10] .
The effects of enteric diseases are especially visible in the intensive poultry industry. The consequences, such as decreased weight gain, increased morbidity and mortality, poor feed conversion and increased use of therapeutic antimicrobial treatments, all contribute to important economic losses. Different terms have been used to describe enteric disease syndrome: runting-stunting syndrome of broilers (RSS), poult enteritis complex (PEC), and poult enteritis mortality syndrome (PEMS). However, none of these descriptions relates to the specific infectious agents, and numerous viruses have been associated with them. However, astroviruses seem to be the most frequently involved in enteritis [2, 8, 12, 13, 24] . Among five different astroviruses identified in avian species, three types have been detected in turkeys. Diarrhoea and increased mortality in turkey poults associated with the presence of an astrovirus, now called turkey astrovirus type 1 (TAstV-1), were described for the first time in 1980 in the United Kingdom [16] and, five years later, in the USA [24, 25] . Turkey astrovirus type 2 (TAstV-2), which is antigenically and genetically distinct from the previously identified TAstV-1, was isolated in the USA in 1996 [13] . With regard to ANV, initially antibodies against this virus were only found in turkey flocks in the UK and Japan [17, 28] . Subsequently, the virus was detected using molecular techniques in intestinal contents of healthy and diseased poults in the USA, but its prevalence is low [5, 20, 21] .
Astroviruses are small, round, non-enveloped particles, typically 28-30 nm in diameter, having a star-like appearance in electron microscopy. AstV has a 6.8-7.9-kb positive-sense single-stranded RNA genome, which encodes three open reading frames (ORF1a and ORF1b, which are linked by a translational frameshift, and ORF2). ORF1b encodes the RNA-dependent RNA polymerase, and ORF2 encodes the capsid protein [3, 7, 11] . The ORF1b region is the most conserved gene in mammalian and avian astroviruses as well as in avian astroviruses [13] . On the other hand, ORF2 is the most variable region of the astrovirus genome and is possibly responsible for the antigenic and pathogenic diversity of astrovirus strains [23] . The family Astroviridae is divided into two genera: Mamastrovirus (mammalian astrovirus) and Avastrovirus (avian astrovirus). Recently, the Astroviridae Study Group of the International Committee for Taxonomy of Viruses (ICTV) has proposed a classification system based on genetic criteria instead of the host of origin. The genus Mamastrovirus includes19 astrovirus genotypes, and the genus Avastrovirus includes three genotypes that are distinguished based on the amino acid sequence of full-length ORF2 (http:// talk.ictvonline.org/files/proposals/taxonomy_proposals_ vertebrate1/m/vert01/2358.aspx).
The aim of the present study was to investigate the prevalence of TAstV infections in commercial meat-type turkey flocks in Poland and to estimate their genetic relatedness, based on the ORF1b region, to those available in the web database.
In 2008, a total of 770 fecal swabs were collected from 77 turkey flocks (10 individual faecal swabs/flock) located in different regions of Poland. Samples were collected from poults aged 1 to 19 weeks. Most of the flocks tested showed one or more of the enteritis symptoms, most frequently diarrhea, thermoregulatory disorders, depression and growth retardation. In 20 flocks, the birds were in good health, while PEMS symptoms (with increased mortality) were observed in 10 flocks. All samples were stored below -65°C until processing. After slow thawing, each individual swab was hydrated in phosphate-buffered saline (PBS) supplemented with antibiotics (100 U of penicillin with 100 mg of streptomycin/ml), incubated for 1 h at room temperature, clarified by centrifugation at 1500 g for 20 min and used for RNA extraction.
Total RNA was extracted from 250 ll of supernatant from 5 pooled swabs (2 pools/flock) using an RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. Each RNA was eluted in 50 ll RNase-free water.
Four different RT-PCR assays directed toward various regions of ORF1b were used for astrovirus detection and differentiation. Conventional RT-PCR was performed using a QIAGEN OneStep RT-PCR kit (QIAGEN, Germany), and the products were separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized using ethidium bromide stain. Real-time RT-PCR was performed using a QuantiTect Probe RT-PCR Kit (QIAGEN, Germany) in a 7500 Real Time PCR System (Applied Biosystems, USA).
Amplicons 601 bp in length were obtained in the RT-PCR assay aimed at detecting all TAstV types. Amplicons were purified using a NucleoSpin Extract II Kit (Macherey Nagel, Germany) according to the manufacturer's instructions and sequenced at the commercial service Genomed (Poland) . Sequencing was performed in both directions with the same primers that were used for RT-PCR reactions. Using the Seqman program (DNASTAR, Madison, WI), the forward and reverse nt sequences were edited, cured and aligned as one consensus sequence. Multiple alignments of nt and aa sequences were performed using the MegAlign application (DNASTAR, Madison, WI) using the Clustal W method. Phylogenetic analysis of aligned sequences was performed with MEGA 4.1 using the neighbor-joining method with the maximum-likelihood model. Bootstrap scores were generated from 1000 replicates. The nt sequences were translated to deduced amino acid (aa) sequences, which were also compared to detect any changes at the aa level.
The astrovirus strains used were as follows: Sequences determined in this study have been submitted to GenBank with accession numbers from HQ317705 to HQ317733.
Fecal swabs obtained from all 77 turkey flocks were tested for the presence of RNA of any type of TAstV. RNA of turkey astrovirus was detected in swabs from 34 flocks (44%). For differentiation between different types of turkey astrovirus, three further tests were performed with published primers targeting various type-specific regions of the polymerase gene (Table 1) . Mixed infection with TAstV-1 and TAstV-2 was observed in swabs from six flocks (17.6%), and TAstV-2 was detected in 20 flocks (58.8%).
However, in eight flocks, no TAstV was detected by RT-PCR (23.5%). All flocks were negative for ANV by this technique (Table 2) .
The 601-bp amplicons obtained by RT-PCR with primers TAPG-L1 and TAPG-R1 from 29 TAstV-positive flocks were sequenced. In eight cases where RT-PCR was unsuccessful, sequencing showed that three samples contained TAstV-1 (flocks 4, 5, 31) , four contained TAstV-2 (flock 8, 10, 18, 20) and one contained ANV (flock 21). Of the six flocks in which RT-PCR showed coinfection with TAstV-1 and 2, sequencing experiments indicated that three of them were infected with TAstV-1 (flock 9, 14, 16) , and the others were infected with TAstV-2 (flocks 7, 11, 33) . Fifteen flocks tested positive for TAstV-2 by RT-PCR, and this was also confirmed by sequence analysis ( Table 2) .
Polish avian astrovirus isolates are divided into three groups ( Fig. 1 ): TAstV-1-like, TAstV-2-like and ANVlike. Most of them belong to the TAstV-2 type. These isolates formed two subgroups (subgroups 1 and 2), with the majority being in subgroup 2 (17 isolates). In subgroup 1, three subclades (a, b, c) could be differentiated, and all six Polish isolates clustered in one subclade (a) together with astroviruses previously reported from the USA. Subclade b included strains from the USA, Italy and France that were isolated either from turkey or guinea fowl; however, the latter formed separate sublineages. Subclade c included strains only from the USA. A comparison of nucleotide and amino acid sequences of six Polish turkey astrovirus type 2 strains from subgroup 1, showed 93. 5-99.5% and 96.8-98 .9% similarity to each other and 86.9-91.2 and 93.5-96.8% to the prototype TAstV-2 isolate CA/00. Subgroup 2 TAstVs did not show any distinct subclades and included only European isolates Primer or/and probe positions refer to the sequences for A TAstV-1 (GenBank accession no. Y15936), B ANV Japan/CK/G-4260 (GenBank accession no. AB033998) and C TAstV-2 strain TAstV/CA/00 (GenBank accession no. EU143844) from Poland and Italy. Comparison of a 366-nucleotide region of ORF1b (from nt 4136 to 4502 of the TAstV-2 genome) revealed that, among these isolates, nt identity was between 96.9 and 100%, and deduced amino acid identity was between 97.6% and 100%. In turn, nucleotide and amino acid similarity of this subgroup to the prototype TAstV-2 was 89.5-90.6% and 95.3-96.5%, respectively. Indeed, these isolates formed a separate subgroup, but its distinction was rather weakly supported statistically (bootstrap value 60).
Among the five Polish TAstV-1 isolates, nucleotide identity was 97.4-99.8%, and amino acid identity was 98.2-100%. They were also closely related to the prototype TAstV-1 isolate, with identities of 95-95.5% at the nucleotide level and 98.2-98.8% at the amino acid level. The group of TAstV-1-like isolates was rather homogenous and included isolates from Poland, Brazil and the USA.
The nucleotide and amino acid identity between the Polish turkey ANV isolate and the ANV prototype Japan/ CK/G-4260 isolate was 86.7% and 97.1%, respectively. In however, it appears that the isolates from turkeys were more closely related to strains isolated from chickens in Japan and the UK than those from the USA.
As with other poultry diseases, many molecular methods are used for diagnosis of etiological factors in enteric diseases of birds. For detection of chicken and turkey astroviruses, RT-PCR-based methods have been developed that target conserved regions of ORFs1b and ORF2, which encode the viral polymerase and capsid protein, respectively [4, 5, 29, 30] . Because mixed infections with various types of astrovirus were identified in some cases, there was a need to distinguish between them. At first, the differentiation of four types of astroviruses, TAstV-1, TAstV-2, CAstV and ANV, relied on sequencing the amplicons. More recently, a new multiplex RT-PCR assay with primers specific for each of the four types of astroviruses was developed and used for survey of enteric viruses in the USA [5] . Overall, this method was unsuitable for differentiation of Polish isolates of astrovirus. Some isolates had additional changes in their nt sequences that prevented correct identification of astrovirus types (e.g., in the ORF1b sequence of PL/TK/G085/08, targeted by the T1pol1F primer, next to the three degenerate nucleotides, there were three additional nt mismatches; in the ORF1b sequence of PL/TK/G145/08 targeted by the ANVpolF primer, next to the four degenerate nucleotides, there were two additional nt changes). On the other hand, the usefulness of the primer set developed by Tang et al. [29] for detection of astrovirus seems to have been confirmed, as it was possible to detect astroviruses with many sequence variations. The presence of astrovirus in 44.15% (34/77) of Polish turkey flocks studied during 2008 is reported. The most frequently identified was TAstV-2, with samples positive from 30 of 77 flocks (38.9%), either as single or mixed infections. TAstV-1 was detected in only nine flocks, either as single or mixed infections (11.6) . ANV was detected in one flock (1.29%). These figures indicate that the prevalence of TAstVs in Poland was lower when compared with a prevalence of 84% reported in commercial turkey flocks during a survey in Minnesota, USA, during 2007-2008 by Jindal et al., or of 59%, reported in the UK; however, the prevalence in Poland was comparable to reports from Brazil (41.1%), as reported by Villarreal et al. [9, 12, 32] . However, it should be noted that in all the previous studies, astrovirus detection was focused only on the TAstV-2 type.
In another study from the USA, astrovirus was detected in 100% of the turkey flocks tested, among which TAstV-2 was the most frequently identified (69.6%), followed by TAstV-1 (28%) and ANV (12.5%) [21] .
Astroviruses were mainly detected in flocks suffering from enteric disorders; however, a few were detected in flocks without any symptoms of disease. This is in accordance with previous findings, and to date, the factors influencing astrovirus pathogenicity are unknown [22, 31] . Astrovirus was seldom detected in flocks older than 4 weeks. Recently, Jindal et al. observed astroviruses in poults up to 9 weeks of age [12] . In Poland, the maximum age of astrovirus-positive poults was 14 weeks, suggesting long-lasting astrovirus infection in turkeys or susceptibility to infection among older poults.
TAstV-1 was first described in 1980 and mainly detected in the USA [18] . Recently, the presence of TAstV-1 was also confirmed in Brazil (data based on available sequences in GenBank: TAst-1/Brazil/2009/USP350-3B: GU938473, TAst-1/Brazil/2009/USP351-1A: GU938474). Regarding ANV, at first, only antibodies against ANV were detected in turkeys in Japan and the UK, and the first isolation and sequencing was achieved in 2006 in the USA [6, 17, 18] . In our study, the presence of an ANV-like isolate in a Polish turkey flock was detected, and this is, to the authors' knowledge, the first time this virus has been detected outside the USA.
Genetic variability among astroviruses was observed, and this confirms previous results from the USA and Europe [5, 18, 19] . For ORF1b of TAstV-2, two subgroups could be distinguished. In subgroup 1, three subclades were identified. All six Polish TAstV-2 isolates clustered in subclade a together with North American isolates of group 1 identified by Pantin-Jackwood at al. and astroviruses from group 2 identified by Jindal at al. [4, 12, 18] . Subclade b included North American isolates from group 2 identified by Pantin-Jackwood et al. and from group 1 identified by Jindal et al. as well as TAstV from Italian group G1. In our comparison, AstV isolates from guinea fowl from Italy that were previously assigned to the G3 group and isolates from France (sequences available in GenBank) also clustered in subclade b, but in separate sublineages. Subgroup 2 included only European isolates originating from Italy (isolated in 2004/05) and Poland (2008) . Italian isolates from this subgroup were previously described as a distinct genetic lineage, G2 [4] . A similar subgrouping of European and ''American-like'' isolates of TAstV-2 was previously indicated by Maurel at al. [15] . However, the comparison of sequences of Polish and French TAstV strains was not possible, since the sequences were not available in the public domain.
The results of phylogenetic studies have suggested a separate origin/evolution of European and North-American isolates. Taking into account that the ORF1b region, encoding RNA-dependent RNA polymerase, is the most conserved in the astrovirus genome and seems to vary according to geography, the presence of European isolates in ''American'' subgroup 1 might be the result of international/continental commercial exchange of birds [19] .
In this study, we identified three types of astroviruses, TAstV-1-like, TAstV-2-like and ANV-like, circulating in turkey flocks in Poland. The most frequently identified were TAstV-2, with TAstV-1 occurring rarely and ANV occasionally. Isolated astroviruses were found to be genetically variable based on the sequence analysis reported here. This diversification was particularly noticeable among Polish TAstV-2 strains, as some of them were genetically related to the North American isolates. However, most of them formed a distinct subgroup of ''European'' isolates, suggesting their separate origin or evolution. In spite of the fact that some isolates were from healthy flocks, it could not be ruled out that astroviruses caused the observed health problems and poor performance of meat-type turkey flocks in Poland. It should be also mentioned that in the light of the new classification rules, none of the Polish turkey astroviruses studied here could be assigned to any of the genotypes. In order to enable classification of these viruses, a phylogenetic analysis of the full-length ORF2 gene is needed, and according to the new taxonomy proposal, they are classified as '' related viruses that might be members of the genus Avastrovirus but have not been assigned to a species'' [23] .
The results presented here also have some diagnostic implications. The conventional RT-PCR method developed by Tang et al. seems to be useful for detection of all TAstV infections, but additional methods for differentiation between astrovirus types failed to distinguish between them. For this purpose, the most suitable method still is sequencing. On the other hand, methods for differentiation of astrovirus types might reveal mixed astrovirus infections.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. A New Approach to Monitoring Dengue Activity  Among the world's most vexing emerging infectious diseases, dengue continues to spread, and in its many endemic areas is a major public health problem [1] [2] [3] . There is no vaccine available, and the immunology of dengue, whereby immunological ''priming'' can result in extremely severe manifestations (e.g., dengue hemorrhagic fever) complicates vaccine development [4] . Thus dengue control is dependent on controlling the mosquito vector, and resistance to insecticides, environmental and social disruption, climate change, and global movement of goods and people (and incidentally, vectors) provide ongoing hurdles to effective vector control [5, 6] .
Accurate risk analysis and allocation of resources for dengue control depends on disease surveillance. Dengue surveillance is similarly complex and depends in most areas on formal surveillance systems that capture case counts (or via syndromic surveillance at sentinel sites) [7, 8] . Laboratory reporting of serology can confirm not only individual cases but identify the viral serotypes found in a given area at a point in time [9] . Analysis of mosquito populations can also confirm dengue circulation and provide information on viral types [10] [11] [12] . Formal surveillance has many advantages: precise counts of case numbers, good geographic localization and the potential to identify precise disease etiology among them.
However, formal infectious disease surveillance systems have important limitations, including lags between case occurrence and reporting. Sentinel sites may report cases only periodically or fail to report altogether for a variety of reasons. Delays in reporting may occur when governmental organizations charged with surveillance aren't able to adequately collect and analyze data or publish reports in a timely manner. These problems may be particularly daunting in developing countries with limited resources to devote to strengthening surveillance systems: robust formal public health surveillance is expensive, requiring major investments in trained personnel, communications, buildings and equipment. Indeed, the economic conditions that prevent development of robust surveillance systems may also be those that potentiate dengue transmission: for example, a seroprevalence study performed in a city straddling the Texas-Mexico border found marked differences in dengue seroprevalence on the Mexican side of the border in association with economic disadvantage [13] .
The hierarchical nature of formal public health surveillance also poses challenges to surveillance. Hierarchical reporting structures can lose data at any point of interaction, for example when a regional authority fails to report to a national one. Finally, in some situations there can be short-term disincentives for the timely and transparent reporting of disease activity: governments may fear that surges in disease activity may chase away tourists or visitors, or may undermine government credibility [14] .
To address these drawbacks, a complementary system of informal surveillance tools have been developed, some by governmental agencies, but many by non-governmental organizations and/or researchers. Event-based surveillance systems such as ProMED, GPHIN HealthMap and BioCaster rely on unofficial reports of disease, for example from clinicians or web-based healt-related news media, to report on disease outbreaks [15] [16] [17] . Such systems have proven reliable and timely and informal sources of information were even recognized in the 2005 revision of the International Health Regulations as important sources of epidemic intelligence [18, 19] . The rapid and accelerating growth of the Internet has improved the usefulness and sensitivity of these systems and they have likely improved the timeliness of outbreak reporting [18] , and the ever-expanding availability of electronic information has also led to the discovery of other types of analyses that detect disease outbreaks. ''Web-crawlers'' (software programs that search internet sites for specific terms, and then use these search terms to generate reports or maps of disease activity) can provide important information on disease outbreaks that may be published on nongovernmental websites, in online newspapers, and in blogs, and this approach powers the widely-used HealthMap system mentioned above [20] . In the context of the recent cholera outbreak in Haiti, there were inconsistencies in initial accounts of regional disease activity, but information from HealthMap proved useful in the construction of a mathematical model that predicted disease spread on the island [21] .
The analysis of real time search queries-the so-called ''searchstream''-has been shown to be a sensitive and timely means of evaluating geographically-specific trends in influenza; both Yahoo and Google search engines have proven to be powerful tools for influenza surveillance [22, 23] . More recently, evaluation of data from the microblogging website Twitter has been shown to provide useful information about both disease activity and disease concern related to the 2009 influenza pandemic [15] . Finally, the widespread availability of smartphone technologies makes it possible to interact with population members to elicit information on illness (so-called ''crowdsourcing''), and also (by using cellphone or smartphone network data) to evaluate the movement of populations, which may be a key predictor of how epidemics spread [24] [25] [26] . This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Readers unfamiliar with these approaches may wish to try a simple experiment using the Google Insights for Search tool, which provides a graphical depiction of both search term volumes and online media reports of disease (http://www.google.com/ insights/search/#). Searches on terms such as ''norovirus'' or ''pneumonia'' produce seasonally oscillating patterns of searches as one might anticipate in diseases with strong wintertime seasonality (Figure 1) , and which is presumably generated by individuals who have, or know someone who has, this diagnosis seeking to learn more about it online. However, the pitfalls of this approach can be appreciated in a similar manner: a search on the term ''influenza'' produces a graph with a tremendous spike in 2009 ( Figure 2) ; indeed a spike so large that it obscures influenza activity in all other years. This reflects the difficulties that searchstream-based surveillance methods may encounter when evaluating diseases that generate extreme public concern or media attention.
Chan et al., in this edition of PLoS Neglected Tropical Diseases [27] , apply searchstream surveillance techniques to the monitoring of dengue. In this case, search queries appear to closely track (rather than lead) dengue activity as measured by traditional systems. The authors have limited their model to certain locations defined in part by the extent of Internet use in these areas (Bolivia, Brazil, India, Indonesia and Singapore). Their findings are exciting: when evaluated in a ''testing set'' of data not used to derive initial models, they found extremely strong correlation between denguerelated query volumes and case counts reported by traditional surveillance systems, but their approach has the advantage of both timeliness and transparency (including the availability of the system on the Google.org website).
As with any prediction-oriented surveillance tool, a major concern relates to model ''over-fitting'' such that the prediction model performs well in the dataset that was used in its creation but fails to work well in the ''real world''. Reassuringly the authors divided their data into a derivation set and a testing set (or ''holdout set'' as they call it), with the former used for model construction. As can be seen in the table and figure they present, their derived models perform extremely well in both sets in all countries, in the derivation set as expected, but also in the testing set. Perhaps less straightforward is the authors' decision to ''smooth out'' unusual spikes in search volumes in candidate queries; as demonstrated by the influenza example above, extreme surges in public interest in a disease can cause surges in query volumes, as can surges in interest related particular subject that is unrelated to the disease under surveillance but shares attributes that would be the subject of searches. By smoothing search volumes, the authors may have incorporated into their models terms that have the potential to ''misbehave'' in the future. For example, one imagines that if a novel (and frightening) new hemorrhagic fever unrelated to dengue emerges in one of these countries in coming years, one would imagine that the correlation between the search term ''haemorrhagic fever'' and dengue volumes would decline. As we don't have access to the precise query terms that were included in each country-specific model, it is difficult to know whether or not the terms included in the model would be vulnerable to such effects. The authors note that the expanding range of a clinically similar illness (Chikungunya) may confound the utility as well [28] .
It would also be helpful to see to what extent there is overlap in components of models across countries, as this may help us understand whether these models can be applied to other jurisdictions or whether they are applicable only in the country for which they were constructed. As dengue is a disease whose range may change under the influence of climate change, it is important to know whether such an approach is applicable in the face of novel emergence of dengue in a new region or jurisdiction, or whether it is only applicable in countries like these in which dengue is currently endemic.
Perhaps the greatest challenge for the use of the approach described here is the same that applies across surveillance modalities: the same geographic locations that lack public health resources to control dengue, and to perform traditional surveillance, are likely to lag in access to the Internet as well. Nonetheless, the application of web-query based monitoring to a major and growing health threat in the developing world represents an important step forward. The ability to inexpensively and reliably maintain situational awareness of dengue activity will be welcomed by those charged with the public health response.
Does the development of web-based surveillance tools represent a revolution in how we conceptualize surveillance? We think not: current high-quality public health surveillance already utilizes multiple sources of information to gain a more complete picture of the incidence and distribution of disease. For example, influenza surveillance may include laboratory-based virological surveillance, sentinel syndromic surveillance (e.g., school-based absenteeism reports) and evaluation of mortality trends for pneumonia and influenza, which taken together may provide a more complete picture of disease risk and impacts. Searchtermbased surveillance and other modalities mentioned above thus provide an additional tool in the surveillance toolbox, which has advantages over traditional surveillance as well as limitations. It should be noted, however, that limitations such as those described above are not absent from traditional surveillance systems either: estimates of incidence can change markedly with changing case definitions, incidence of laboratory-confirmed disease can change markedly with augmentation or restriction of clinical testing or changes in diagnostic test methodologies, and syndromic surveillance systems can be subject to poor specificity and frequent false alarms. Thus supplementary information derived using methods such as the one developed by Chan and colleagues should be welcomed by public health professionals. The transparency of such systems may also help demonstrate the value of openness in disease reporting, which may have ''spillover effects'' on traditional surveilance systems. Figure 2 . Screenshot of search performed on the term ''influenza'' using the Google Insights for Search tool (http://www.google. com/insights/search/#). Although influenza searches would expect to display similar wintertime seasonality to pneumonia searches, depicted in Figure 1 , the public concern and interest generated by the 2009 influenza pandemic generated a large spike in searches in that year, which obscures seasonal oscillation in other years. doi:10.1371/journal.pntd.0001215.g002 www.plosntds.org Dynamic variations in the peripheral blood lymphocyte subgroups of patients with 2009 pandemic H1N1 swine-origin influenza A virus infection BACKGROUND: Novel Influenza A (H1N1) is an acute respiratory infectious disease. Animal experiments indicated that when H1N1 virus infected early hosts, it showed strong CD4(+), CD8(+), and CD4(+)CD25(+ )T cell reactions. The aim of this study was to investigate the dynamic fluctuations of the peripheral blood lymphocyte subgroups in patients infected with H1N1 swine-origin influenza A virus (S-OIV). METHODS: The frequency of T cells, B cells, natural killer (NK) cells, and regulatory T cells (Treg) in 36 severe H1N1 and 40 moderate H1N1 patients were detected at different periods by flow cytometry. In parallel, serum cytokines were detected by enzyme-linked immunosorbent assay and C-reactive protein (CRP) was analyzed through an image-type automatic biochemical analyzer. In addition, 20 healthy volunteers, who were not infected with 2009 H1N1 virus, were selected as controls. RESULTS: The frequency of NK cells were decreased in all cases and CD19(+ )B cells were increased in severe cases than those of the controls. At 1-2d from onset, the frequency of CD4(+ )and CD4(+)CD25(+ )T cells in moderate cases was higher than in the severe cases. Serum cytokines, specifically IL-2, IL-4, IL-6, IL-10, and IFN-γ exhibited no significant change both in the moderate and the severe cases during the whole monitoring process. In the early stage of the disease, serum CRP levels in the severe and moderate groups were significantly higher than that in the control group. CONCLUSIONS: Patients showed different lymphocyte subgroup distributions between mild and severe cases, which might affect the incidence and development of 2009 H1N1. The latest public data from the Chinese Ministry of Health showed 120,940 confirmed H1N1 cases and 659 deaths were reported from the Chinese mainland as of January 2, 2010. Data analysis suggests that patients with chronic diseases, obese patients, and pregnant women are more liable to develop severe H1N1 influenza. After initial infection by the virus, the host innate immune system, as a first line of defense, takes protective measures earlier than the adaptive immune system to avoid further viral invasion or replication [1] . Animal experiments indicated that when the swine-origin influenza A virus (S-OIV) infected early hosts, it elicited strong CD4 + , CD8 + , and CD4 + CD25 + T cell reactions [2] . CD4 + and CD8 + T cells are related to viral immunity and the lack of these cells would lead to a delay in viral clearance and an increase in mortality [3] [4] [5] . On the other hand, CD4 + CD25 + cells are T cell subsets with an immune inhibition function and could play an important regulatory role in a variety of infectious diseases [6] [7] [8] . These complex molecular and cellular mechanisms are helpful in controlling and eliminating the acute stage of viral infection. Although successfully eliminating the virus is * Correspondence: ljli@zju.edu.cn † Contributed equally 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, PR China Full list of author information is available at the end of the article essential, the response of virus-specific T cells to the hosts could also result in tissue damage and autoimmune responses. Therefore, monitoring the variation of cellular immune functions in H1N1 patients have important clinical significance. The aim of this study is to analyze the dynamic fluctuations of T cells, B cells, natural killer (NK) cells, and regulatory T cells in patients infected with novel influenza H1N1, as well as serum cytokines and C-reactive protein (CRP).
Up to 76 H1N1 outpatients and inpatients were chosen from August to December 2009 in the Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University. Among these, 36 cases were severe and 40 cases were moderate (Table 1 ). In addition, 20 healthy volunteers who were not infected with 2009 H1N1 virus, were selected as controls. There were no statistical differences between their ages and genders compared with the H1N1 patients.
The diagnostic criteria for H1N1 were based on the Influenza A H1N1 Flu Clinic Program (pilot version of the second edition, 2009) released by the Chinese Ministry of Health in July 2009. Its main content includes the following:
I. Confirmation standard of Influenza A (H1N1) cases: Patients are considered to have contracted the virus if they present with influenza-like clinical manifestations and had one or more of the following laboratory test results: (1) Positive for S-OIV nucleic acid (through real-time RT-PCR and RT-PCR); (2) S-OIV is isolated and attained; and (3) Specific neutralizing antibody titers for serum S-OIV are increased four times or more.
II. Severe cases: When confirmed or suspected cases manifest one of the following signs, they are considered as severe: (1) Complicated pneumonia with or without hypoxemia and respiratory failure; (2) toxic shock; and (3) multiple organ dysfunction syndrome or multiple organ failure.
The study protocol was approved by the Ethics Review Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University. All subjects understood the procedures and consented participate in the study.
On day 1, 2, 3, 5, 7, 9, 11, 14, and 21, 2 mL of whole blood (EDTA-K2 anticoagulant) and 1.5 mL of serum were collected from H1N1 patients for the detection of lymphocyte subgroups, cytokines, and CRP. The same sample collection was carried out in the control group. Afterwards, 100 μL of anticoagulant was added into all flow tubes and 20 μL of CD3-PC5, CD4-FITC, CD8-PE, CD3-FITC-CD(16+56)-PE, CD19-FITC, and CD25-PE mouse anti-human fluorescence monoclonal antibodies (BD Bioscience) was added. After mixing, the samples were incubated for 15 min away from light at room temperature. Red blood cell lysis and cell fixation using Coulter QPREP specimen processing instrument (Beckman Coulter Inc., USA) was conducted, and the cells more than 1 × 10 4 were counted through a flow cytometer (Coulter Cytomics FC 500, Beckman Coulter). The results were expressed as the percentages of CD4 + CD25 + , CD4 + , and CD8 + T cells, as well as those of CD19 + B cells and NK (marked as CD3 -CD16 + CD56 + ) cells found positive for the antigen marker in the total lymphocytes population. The data were collected and analyzed using the EXPO32 software (Beckman Coulter Inc., USA).
Serum CRP detection was conducted using the imagetype automatic biochemical analyzer (Beckman Coulter Inc., USA). The kit was also provided by the same company. Serum cytokines, namely IL-2, IL-4, IL-6, IL-10, and IFN-γ were tested using an enzyme-linked immunosorbent assay (ELISA; Ex-cell). The experimental methods were in accordance with the instructions of the manufacturer in the reagent kit.
Statistical analysis SPSS 13.0 statistical package was used for data processing. Measurement data were indicated as mean ± standard deviation or percentages. Comparisons among groups were carried out using one-way ANOVA or χ 2 test. Differences with P < 0.05 were considered statistically significant. Correlation analysis among the detection indicators were conducted using Spearman's rank correlation analysis.
Up to 76 patients with confirmed laboratory diagnoses of influenza were enrolled in this study. The baseline characteristics of the patients are summarized in Table 1 . The samples were predominantly composed of full-grown adults with S-OVI infection.
As shown in Table 1 , the patients with severe H1N1 were significantly older than those that presented with moderate H1N1 (mean age, 40.1 years vs. 26.1 years; P < 0.01). The mean duration of symptoms before presentation was 4 days, with fever, cough, and myalgia as the most common in both groups. However, the patients with severe H1N1 had more prodromal symptoms (e.g., fever, dyspnea, and myalgia) and co-morbidities [e.g., chronic obstructive pulmonary disease (COPD) and coronary artery disease] than the patients with moderate H1N1 (p < 0.01). Although a relationship with pregnancy was more common in the severe H1N1 group (four patients vs. one patient with moderate H1N1), this difference was not statistically significant (Table 1) . Similarly, there was no difference in the other preexisting diseases such as diabetes mellitus and hypertension between the two groups.
Chest x-rays were taken from all patients, and abnormalities were detected in 23 patients, 3 with moderate H1N1 and 20 with severe H1N1. Ten patients presented with radiological abnormalities attributable to chronic lung conditions without evidence of concurrent pneumonia.
Up to 15 patients with severe H1N1 were admitted to the intensive care unit (ICU) with preexisting diseases, such as COPD. All patients were treated with Oseltamivir (Tamiflu) and 53% of the severe H1N1 patients were additionally treated with glucocorticoids (19/36). The duration of Oseltamivir treatment was based on the Pandemic Influenza A (H1N1) 2009 Clinical Guidelines (Second Edition, 2009). However, the subjects were not given immunomodulators. Treatment response did not differ significantly between the two groups and the mean duration of hospital confinement was 13.7 days (range, 0-51 days) and 3.3 days (range, 0-11 days) for the severe and moderate patients, respectively. Overall, only one severe H1N1 patient died during the course of the study. No patient dropped out and all completed the 21-week follow-up.
The frequency of peripheral blood CD19 + B cell count of patients with severe H1N1 gradually increased within the first 3-14 d of treatment. The NK cell count showed a gradual decline, whereas the T cell subtypes (CD4 + CD25 + , CD4 + , and CD8 + T cells) showed no significant changes. The frequency of peripheral CD4 + T and CD4 + CD25 + Treg cells of patients with moderate H1N1 were significantly higher than in patients with severe H1N1 within the first 1-2 d of treatment. The NK cell counts of patients with moderate H1N1 were similar to that of patients with severe H1N1, which demonstrated a gradual decline, whereas the frequency of CD19 + B and CD8 + T cells showed no significant changes.
The frequency of the different lymphocyte subsets at different periods were statistically compared and the results show that the frequency of peripheral CD4 + and CD4 + CD25 + T cells in the moderate H1N1 patients were higher than that of the severe H1N1 patients within the first 1-2 d (Figures 1a and 1c ; P < 0.05). The CD19 + B cell counts of the severe H1N1 patients were significantly higher than those of the moderate H1N1 patients and the control group for the same period (Figure 1e) , whereas the NK cell counts were significantly less than that of the control group (Figure 1d ; P < 0.05). The frequency of CD8 + T cells had no significant difference (P > 0.05; Figure 1b) among the three groups.
In the first 1-5 d of study, the serum CRP levels of both the patients with severe H1N1 and the patients with moderate H1N1 were significantly higher than that of the control group (P < 0.01; Figure 1f ). Correlation analysis of the CD4 + , CD8 + T, CD4 + CD25 + , CD19 + B, and NK cells with CRP was conducted and the results indicate that the various lymphocyte subgroups had no significant correlations with CRP.
The serum cytokines of patients, specifically IL-2, IL-4, IL-6, IL-10, and IFN-γ, whether the H1N1 infection was severe or moderate, showed no significant changes during the whole monitoring process (data not shown). The differences of the H1N1 patients with the control group had no statistical significance.
Influenza A (H1N1) is the greatest pandemic threat in 2009 [9] . The clinical course of H1N1 can be severe, particularly for very young patients with complications. The immune response affected by pathological damage to the body caused by S-OVI and clinical prognosis. Studies have shown that the incidence of patients with severe H1N1 was highest in the 15-to 19-year-old age group and the lowest for patients aged 65 or above [10] . Our clinical data also showed that the most severe infections occur in individuals younger than 25 years old or in middle-aged patients Figure 1 Analysis of the dynamic changes in the lymphocyte subgroup and C-reactive protein (CRP) in the peripheral blood of patients with Influenza A (H1N1) 2009. CD4 + T, CD8 + T, CD4 + CD25 + Treg, NK, and CD19 + B cells are presented as a percentage of total peripheral blood leukocytes (PBLs) in severe H1N1 (n = 36) patients, moderate H1N1 (n = 40) patients, and healthy volunteers (n = 20). †: denotes statistically significant differences compared with severe H1N1; *: denotes statistically significant differences compared with the healthy volunteers; **: denotes statistically significant differences compared with the moderate H1N1 patients and with the healthy volunteers.
older than 55 years old with comorbidities, especially lung disease.
Based on local guidelines, antiviral treatment would be considered for severe or critical cases and high-risk patients infected with the influenza A pandemic (H1N1) virus within 48 h of onset. For patients who presented later than 2 days, the managing physicians were allowed to make decisions regarding antiviral use. Oseltamivir, given to patients older than 12 years, was prescribed according to the standard dosing regimen (75 mg twice daily orally, for 5 days). Dosage adjustment, whenever necessary, were based on the patient's renal function (75 mg daily, if creatinine clearance is <30 mL/min). For children (1-12 years old), dosage adjustments were based on body weight (BW), that is: 30 mg twice daily for children with BW < 15 kg; 45 mg twice daily for BW 15-23 kg; 60 mg twice daily for BW 23-40 kg; and 75 mg twice daily for BW > 40 kg. Accordingly, patients with acute respiratory distress syndrome (ARDS) were treated with high dose glucocorticoids over a short period of time, which has been proven beneficial and safe.
Several lines of experimental evidence suggest that H1N1 infections accompanied by a characteristic impairment of the innate immune response. By monitoring the host functional response, patients with increased risks of developing severe influenza-associated complications could be identified immediately [11, 12] . The preliminary data from this study showed that the frequency of CD19 + B cells in patients with severe H1N1 was significantly higher than in the moderate and control groups (Figure 1e) , and the frequency of NK cells in the severe and moderate groups were less than that of the control group. In the host innate immune system, NK cells are the major effector cells during acute infection, rapidly killing infected cells in the process. Previous studies have shown that the significant decrease in peripheral NK cell count is mainly because the S-OVI directly infected and killed NK cells, thereby limiting their activity and leading to their apoptosis in vivo [13] .
Animal studies have found that CD4 + T, CD8 + T, and CD19 + B cells achieved peak values within 5-6 d after S-OVI infection, whereas the CD4 + CD25 + Treg cell count reached its peak at 24-48 h after infection [2] . Currently, the use of flow cytometric analysis to measure peripheral blood CD4 + , CD8 + , and CD4 + CD25 + T cells is a simple and common method. Reportedly, the mostly CD4 + CD25 + T cells were also positive for FoxP3 [14] . In this experiment, the peak values for all lymphocyte subgroups in patients with severe H1N1 could not be detected. At least two mechanisms could account for the lower effector T cell response detected after the S-OVI infection, and the peak response time for the effector T cells possibly missed. In addition, the virus could have also led to the immunotolerance of the T cells [15] .
As intercellular signaling molecules, cytokines regulate the immune response, mediate inflammatory responses, and participate in tissue repair. Experiments have shown that only the peak values of IFN-α and IL-6 could respectively be detected within 24 and 30 h in the serum of infected animals, as well as in the plasma of pediatric patients with severe influenza [16, 17] . In this experiment, no statistically significant changes were found during the continuous monitoring of serum IL-2, IL-4, IL-6, IL-10, and IFN-γ of patients with severe and moderate H1N1, as well as in the control group. This result might also be related to the immunotolerance of T cells, of which the peak detection might have been missed. The mechanisms responsible for maintaining these relatively constant levels remain unclear.
CRP is an acute phase protein produced by the liver and is a sensitive marker for the inflammatory response. It did not directly related to viral load [17] . However, CRP continuously increased in the initial stages of infection for both the patients with severe and those with moderate H1N1. The CRP of patients with severe H1N1 took longer to return to normal, which might be the result of underlying disease. Each lymphocyte subgroup has made correlatively analyzed with CRP and the results show no significant correlations.
In conclusion, S-OIV could stimulate the cellular immune response. When accompanied by CD19 + B cell increase and a continuous NK cell decline, it could indicate that the body is in a dynamic balance of the antiinfection immunity and autoimmune damage; this is particularly true for patients with severe influenza. Each lymphocyte subgroup in patients with H1N1 plays a more important antiviral role in the early stages of disease, but excessive immune response also leads to the increase and development of infection. Maternal Influenza Immunization and Reduced Likelihood of Prematurity and Small for Gestational Age Births: A Retrospective Cohort Study BACKGROUND: Infections during pregnancy have the potential to adversely impact birth outcomes. We evaluated the association between receipt of inactivated influenza vaccine during pregnancy and prematurity and small for gestational age (SGA) births. METHODS AND FINDINGS: We conducted a cohort analysis of surveillance data from the Georgia (United States) Pregnancy Risk Assessment Monitoring System. Among 4,326 live births between 1 June 2004 and 30 September 2006, maternal influenza vaccine information was available for 4,168 (96.3%). The primary intervention evaluated in this study was receipt of influenza vaccine during any trimester of pregnancy. The main outcome measures were prematurity (gestational age at birth <37 wk) and SGA (birth weight <10th percentile for gestational age). Infants who were born during the putative influenza season (1 October–31 May) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature compared to infants of unvaccinated mothers born in the same period (adjusted odds ratio [OR] = 0.60; 95% CI, 0.38–0.94). The magnitude of association between maternal influenza vaccine receipt and reduced likelihood of prematurity increased during the period of at least local influenza activity (adjusted OR = 0.44; 95% CI, 0.26–0.73) and was greatest during the widespread influenza activity period (adjusted OR = 0.28; 95% CI, 0.11–0.74). Compared with newborns of unvaccinated women, newborns of vaccinated mothers had 69% lower odds of being SGA (adjusted OR = 0.31; 95% CI, 0.13–0.75) during the period of widespread influenza activity. The adjusted and unadjusted ORs were not significant for the pre-influenza activity period. CONCLUSIONS: This study demonstrates an association between immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. However, no associations were found for the pre-influenza activity period. Moreover, during the period of widespread influenza activity there was an association between maternal receipt of influenza vaccine and reduced likelihood of SGA birth. Please see later in the article for the Editors' Summary Infections during pregnancy have the potential to adversely impact birth outcomes and fetal growth and development. Respiratory infections-particularly those resulting in pneumonia-have been associated with low birth weight and increased risk of preterm birth [1, 2] . Influenza virus is an important respiratory pathogen that causes substantial burden of diseaseincluding morbidity and mortality among pregnant women, with greater risk of influenza-related morbidity among pregnant women than among non-pregnant and postpartum women [3] .
Vaccination against influenza is the most effective tool to prevent morbidity and mortality due to influenza. Influenza vaccination during pregnancy provides protection for the infant as well as the mother. A randomized controlled clinical trial in Bangladesh demonstrated that vaccination of pregnant women with the inactivated influenza vaccine had 63% effectiveness in reducing laboratory-confirmed influenza in their infants [4] . Since there is evidence of adverse fetal/newborn outcomes after infection during pregnancy [5, 6] , including influenza infection [2] , it is plausible that influenza vaccination in pregnancy could mitigate adverse birth outcomes such as prematurity and small for gestational age (SGA) births. The potential impact of maternal influenza immunization on birth outcomes could have important public health implications for developed as well as developing countries and may be of particular interest during influenza pandemics.
The objective of this study was to evaluate whether there is an association between receipt of inactivated influenza vaccine during pregnancy and birth outcomes using a multiyear, populationbased, state-wide dataset from the state of Georgia (in the United States).
We conducted a retrospective cohort analysis of a large surveillance dataset. The primary exposure variable was receipt of inactivated influenza vaccine during any trimester of pregnancy by mothers of infants born between 1 
We analyzed pregnancy-and birth-related data from the Georgia Pregnancy Risk Assessment Monitoring System (PRAMS) and influenza activity information compiled by Georgia for the Council of State and Territorial Epidemiologists (CSTE) reports. PRAMS is a multistate surveillance system managed by the US Centers for Disease Control and Prevention and state health departments, including the Georgia Department of Community Health [7, 8] . The PRAMS sample is drawn monthly from the state's birth file and includes resident women who have experienced a live birth. Georgia PRAMS oversamples women based on race (black) and birth weight (,2,500 g). We adjusted for the oversampling by using analysis weights described elsewhere in the methods section. PRAMS data are collected primarily by mailed questionnaires, with telephone follow-up among nonresponders.
The Georgia PRAMS dataset contains information on maternal influenza vaccination (during any trimester); maternal attitudes, behaviors, and experiences before, during, and shortly after pregnancy; and newborn birth certificate data, including birth date and birth weight. Prematurity was defined as clinical estimate of gestational age (at birth) as less than 37 wk. Newborns with birth weight below the10 th percentile for their gestational age were considered to be SGA. We used gender-specific reference values for the US published by Oken at al. [9] to assign SGA (yes/no) categories.
In order to model the impact of the intensity of influenza activity in Georgia on the association between maternal influenza vaccination and birth outcomes, we used a modified version of the CSTE report categories of influenza activity. CSTE reports assess the spread of influenza within each state for each week based on lab-confirmed and syndromic data [10] . The influenza activity is considered local if there are influenza outbreaks or an increase in cases of influenza-like illness in a single region of the state along with recent identification of laboratory-confirmed influenza from that region. In the case of influenza outbreaks or increases in influenza-like illness with recent laboratory-confirmed influenza in at least two but fewer than half the regions of the state, the influenza activity level is considered to be regional. If the influenza outbreaks or increased numbers of influenza-like illness cases (plus laboratory-confirmed cases) are reported in at least half of the regions of the state, the influenza activity is considered to be widespread ( Figure 1 ). We defined the pre-influenza period as the period between the start of the putative influenza season (October 1) and the beginning of local influenza activity (per CSTE reports). The pre-influenza period is characterized by the availability of the vaccine and the absence of influenza activity ( Figure 1 ). The definition for the pre-influenza period was similar to the one used by other authors [11, 12] .
Stratified analysis was performed for the overall study period, the putative influenza season (1 October-31 May), the preinfluenza period (during the putative influenza season), the period of at least local activity, the period of regional activity, and the period of widespread activity ( Figure 1 ).
In Georgia PRAMS, live births to black women and those that are low birth weight are oversampled in order to provide enough statistical power for stratified analyses for groups of interest relevant to PRAMS objectives. For combined analyses, in accordance with standard practice, analysis weights developed by the Centers for Disease Control and Prevention [13] were used to adjust for the sample design (e.g., to account for oversampling of the two risk groups) and differential response rate across groups.
We used logistic regression to evaluate the association of maternal influenza vaccine and (a) prematurity and (b) SGA. Linear regression was used to evaluate the statistical significance of differences between infants born to vaccinated and unvaccinated women in terms of mean gestational age at first antenatal visit and mean birth weight.
Confounding due to differences between the vaccinated and unvaccinated individuals is a recognized issue in observational studies of influenza vaccination-particularly studies evaluating vaccine effectiveness for reducing all-cause mortality in the elderly [12] . One approach to account for confounding is to choose a period when the vaccine was available but the influenza virus was not circulating as the ''control'' pre-influenza period [11, 12] . In the pre-influenza period, there should be no vaccine effect, and observed effects during this period are assumed to be due to confounding. Therefore, as our primary strategy for confounder adjustment, we identified a group of covariates that would move the odds ratios (ORs) of association between maternal influenza immunization and birth outcomes during the pre-influenza period to 1.0 (i.e., no effect), hence arriving at a set of covariates that could effectively control for confounding due to the differences between the vaccinated and the unvaccinated women in analyses of all influenza activity periods. We selected covariates for the separate multivariate models for each of the birth outcomes (i.e., prematurity and SGA) using a modified version of the approach described by Jackson et al. [11] and Nelson et al. [12] . Briefly, variables for each birth outcome were evaluated for potential confounding by selecting the covariates that, in bivariate models, modified the association between maternal influenza vaccine and the birth outcome and moved the OR towards 1. From this initial group of variables, we arrived at a parsimonious model by sequentially dropping each covariate and observing a change in OR of the association between maternal immunization and the birth outcome. We excluded the variable whose removal moved the OR the most towards 1. If dropping a covariate resulted in moving ORs away from 1 and a change in magnitude of less than 1%, we removed the covariate that caused the least change and then repeated the process. If the change was more than 1%, we considered the current set of covariates as the smallest group of variables required to account for confounding.
In order to address the possibility that the confounders in the preinfluenza period were different from the confounding factors in the influenza activity period, we also developed secondary multivariate models using a traditional analytic approach for confounder adjustment. In these secondary models, the covariate list (for both the pre-influenza-period-based and traditional adjustment) was based on evidence in the literature regarding associations with birth outcomes and availability of data in the Georgia PRAMS dataset. The covariate list included the following: gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12 th grade, mother's marital status, and maternal weight before pregnancy. The ORs for association between the different covariates and receipt of influenza vaccine were computed using logistic regression.
We also computed the population prevented fraction of prematurity for the various periods of influenza activity using the formula:p p c (1{OR) p p c (1{OR)zOR wherep p c is the proportion of cases vaccinated and OR is the odds ratio approximating relative risk (we verified this assumption for our data). The formula, based on the approach described by Kleinbaum et al. [14] , is suitable for computing the population prevented fraction when adjusted measures of association are used. The population prevented fraction for a vaccine estimates the reduction in an outcome given the efficacy/effectiveness of the vaccine and the specific vaccine coverage. We used Stata version 10 (Stata Corporation) for statistical analysis. Identified associations were evaluated for statistical significance at a = 0.05 using two-tailed tests, and Taylor series linearization methods [15] were used to estimate variance.
The study was reviewed and approved by the Emory University Institutional Review Board and the Georgia Department of Human Resources Institutional Review Board.
A total of 4,326 women (and their newborns) were included in Georgia PRAMS during the 28-mo study period. Influenza vaccine information was available for 4,168 (96.3%) women in PRAMS (study population); of these, 578 women (14.9% [weighted]) had received the influenza vaccine during pregnancy. The vaccine coverage was 19.2% (weighted) among mothers of infants born during the putative influenza season. Out of the 122 wk of the study, at least local influenza activity was detected during 27 wk (22.1%)-including widespread activity in 8 wk ( Figure 1 ). There were 1,547 premature newborns (10.6% [weighted]) and 1,186 newborns with SGA (11.2% [weighted]) in our study population.
The odds of having received influenza vaccine during pregnancy were lower (a) for black women than for all other ethnic groups (OR = 0.78; 95% CI, 0.62-0.98), (b) for women with diabetes (OR = 0.30, 95% CI, 0.10-0.95), and (c) for mothers who used multivitamins in pregnancy (OR = 0.64; 95% CI, 0.50-0.83) ( Table 1) . Insured women were more likely to have received the influenza vaccine (OR = 1.41; 95% CI, 1.09-1.81). Likelihood of having received an influenza vaccine during pregnancy was not associated with any of the other binary covariates (Table 1) . Similarly, gestational age at first prenatal visit was similar for vaccinated women and unvaccinated women (mean: 5.2 wk versus 5.3 wk; p = 0.23), and maternal weight before pregnancy was similar for vaccine recipients and non-recipients (mean: 68.3 kg versus 68.1 kg; p = 0.88).
Based on the approach of identifying covariates that produce adjusted ORs of 1 during the pre-influenza period, the group of covariates in the prematurity multivariate models included gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. The covariates in the primary multivariate models for SGA included maternal age less than 19 y, maternal medical risk factors, labor/delivery complications, hypertension (treated or untreated), birth defects, and history of alcohol use during pregnancy.
In unadjusted models, and in models with covariates based on lack of effects in the pre-influenza season, infants born during the putative influenza season (1 October-31 May) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature than infants of unvaccinated mothers born in the same period (adjusted OR = 0.60; 95% CI, 0.38-0.94). The magnitude of effect of maternal influenza vaccine on prematurity increased during the period when there was at least local influenza activity in any part of the state (adjusted OR = 0.44; 95% CI, 0.26-0.73) and was the highest for those born during the period of widespread influenza activity (adjusted OR = 0.28; 95% CI, 0.11-0.74) ( Table 2 ). The adjusted and unadjusted ORs were not significant for the association between receipt of maternal influenza vaccine and prematurity for the pre-influenza activity period or for the analysis without consideration of influenza activity ( Table 2) .
Compared with newborns of unvaccinated women, those born to vaccinated mothers had lower odds of SGA (adjusted OR = 0.31; 95% CI, 0.13-0.75) during the period of widespread influenza activity (Table 3) . Although the magnitude of the ORs of the association between maternal influenza vaccine and SGA generally increased with the increase in the intensity of influenza activity, these associations were not statistically significant (other than for the period of widespread activity) ( Table 3 ). The associations observed in the secondary multivariate models using the traditional approach were qualitatively similar to the associations in the primary multivariate models (Tables 2 and 3) .
Newborns of vaccinated women were, on average, 96.7 g heavier than newborns of unvaccinated women (3,348 g versus 3,251 g; p = 0.002). During the putative influenza season, the difference between the two groups increased to 113 g (3,360 g for the vaccinated group versus 3,247 g for the unvaccinated group; p = 0.004). There were no significant differences in birth weights outside the putative influenza season (3,317 g [vaccinated] versus 3,255 g [unvaccinated]; p = 0.233).
There was no statistical interaction by specific influenza season (i.e., 2004-2005 and 2005-2006) for all analyses of prematurity, SGA, and birth weight (for all interaction terms: p.0.05-detailed data available on request). Moreover, the association between maternal influenza vaccine and birth outcomes was qualitatively similar for the two influenza seasons. For example, during the period of widespread influenza activity, the adjusted ORs for prematurity were 0.17 (95% CI, 0.03-0.86) for the 2004-2005 season and 0.32 (95% CI, 0.10-1.14) for the 2005-2006 season.
The fraction of prematurity prevented in the population during the study period (population prevented fraction of prematurity) was 0% for the pre-influenza activity period and 7.9% for the putative influenza season. The population prevented fraction increased during the periods of influenza activity (at least local activity, 15.7%; at least regional activity, 17.5%; widespread activity, 17.5%). 
This study demonstrates an association between immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. For births during the 8 wk of widespread influenza activity, the odds of prematurity were approximately 70% lower among the newborns of the vaccinated mothers compared to mothers who did not receive the influenza vaccine. During the period of widespread influenza activity there was also an association between maternal receipt of influenza vaccine and reduced likelihood of SGA. The magnitude of association between influenza vaccine and prematurity (as measured by the values of ORs) increased with the increase in the intensity of influenza activity and was higher for the 2004- [16] . Although the SGA-related ORs were not statistically significant for influenza activity periods except for the period of widespread activity, the overall ''gradient'' of effect in the point estimates of the ORs was qualitatively similar to that of prematurity. The increase in the impact of maternal influenza vaccines on birth outcomes by influenza activity, both in terms of ORs and population prevented fractions, supports the validity of our findings.
The possibility of confounding due to differences between vaccinated and unvaccinated individuals included in observational studies of influenza immunization is well recognized [12] . The most significant type of confounding in influenza studies is due to a higher likelihood of individuals with high functional capacity (i.e., healthier The primary adjusted models were based on the approach of identifying covariates that produce adjusted ORs of 1 during the pre-influenza period and included the following covariates: gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. b In the secondary adjusted models, the covariates included gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12th grade, mother's marital status, and maternal weight before pregnancy. The ORs for association between the different covariates and receipt of influenza vaccine were computed using logistic regression. c Ratio of the odds of prematurity in newborns of mothers who received influenza vaccine during pregnancy compared to mothers who did not receive the vaccine by intensity of influenza activity, e.g., in the analysis of all seasons/periods, the (unadjusted) odds of prematurity were 25% lower among the infants of mothers who received the influenza vaccine during pregnancy than among infants whose mothers who did not receive the vaccine. doi:10.1371/journal.pmed.1000441.t002 Table 3 . ORs of being SGA by maternal influenza vaccine status (ORs,1 imply a protective association of the vaccine). The primary adjusted models were based on the approach of identifying covariates that produce adjusted ORs of 1 during the pre-influenza period and included the following covariates: gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. b In the secondary adjusted models, the covariates included gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12th grade, mother's marital status, and maternal weight before pregnancy. The ORs for association between the different covariates and receipt of influenza vaccine were computed using logistic regression. c Ratio of the odds of being SGA among newborns of mothers who received influenza vaccine during pregnancy compared to those born to mothers who did not receive the vaccine by intensity of influenza activity, e.g., in the analysis of all seasons/periods, the (unadjusted) odds of being SGA were 84% lower among the newborns of mothers who received influenza vaccine during pregnancy than among infants whose mothers who did not receive the vaccine. doi:10.1371/journal.pmed.1000441.t003 individuals) to get vaccinated-a phenomenon often known as the ''healthy user effect.'' However, most observational studies where significant confounding has been documented were conducted in the elderly and included a relatively nonspecific end point of all-cause mortality. It is reasonable to assume that, compared to older individuals, women of reproductive age may be less likely to have significant functional limitation even in the presence of co-morbidities. Therefore, influenza vaccine studies in this age group may be less likely to suffer from bias due to the healthy user effect. Moreover, we found no statistically significant difference between the vaccinated women and the unvaccinated women in terms of gestational age at which they sought antenatal care. On the other hand, the possibility of other confounders cannot be discounted in studies involving pregnant women. In order to address confounding, we used the pre-influenza period (i.e., the season where vaccine was available but there was minimal circulation of influenza virus) as the ''control'' period. The use of the pre-influenza period for selecting confounders from a broad set of covariates is an approach suggested by Nelson et al. [12] and Jackson et al. [11] that takes advantage of the seasonality of influenza circulation. The associations observed in our study were robust to adjustment for confounders identified using this approach (and the more traditional approach)-supporting the validity of our findings. Influenza virus, particularly seasonal influenza virus, rarely crosses the placenta [3, 17, 18] . However, the effect of infection on prematurity is thought be at least partially mediated through inflammatory pathways [5, 6] . Increase in pro-inflammatory cytokines (e.g., IL-1, IL-6 and TNF-a) and reduction in antiinflammatory cytokines (e.g., IL-10) have been linked to preterm labor [6, 19, 20] . IL-1 stimulates the amnion and the decidua to produce prostaglandins and can stimulate myometrial contractions [20] . Prostaglandins are known to play an important role in the initiation and progression of labor [21] . Moreover, in animal models, administration of IL-1 results in preterm birth [20] . Similarly, TNF-a induces the amnion, the decidua, and the myometrium to produce prostaglandins, and administration of TNF-a to pregnant animals can induce preterm labor [19, 22] . Recent studies have shown that influenza virus infection induces gene expression of pro-inflammatory cytokines including IL-1b, IL-6, TNF-a, interferon (IFN)-b, IFN-a, and granulocyte macrophage colony-stimulating factor (GM-CSF) [19] .
In addition to biological plausibility, there is epidemiological evidence of an association between maternal infection and preterm birth [5] . The association is strongest for intrauterine viral infections and systemic and intrauterine bacterial infections [5, 6] . Viral infection outside the reproductive tract, including influenza infection, may also play a role in the etiology of prematurity. For example, in an analysis of 1957-1958 data, newborns of women who had serological evidence of ''Asian'' (pandemic) influenza during pregnancy were 50% more likely to be premature compared to newborns of uninfected women [2] . Moreover, a recent literature review found that SARS infection in the second or third trimester of pregnancy may be associated with spontaneous preterm delivery and early cesarean sections for deteriorating medical condition, although only 16 such cases were identified in the literature [23] . Moreover, in studies in China and Hungary, birth defects were associated with history of influenza [24, 25] .
However, a few observational studies have failed to demonstrate an association between influenza infection and birth outcomes [26, 27] . The lack of observed effect in some studies could be due to a true lack of association, small or difficult to measure effect size, challenges related to the study population (e.g., administrative datasets), or non-differential misclassification due to challenges in retrospectively identifying influenza infection. Although less than ideal, modeling receipt of influenza vaccine as the exposure/ independent variable reduces the likelihood and the intensity of non-differential misclassification bias.
Preterm births represent a significant burden to health care and society [5] . Like several developed countries, there has been an increase in the rate of preterm births in the US, which rose from 9.5% in 1981 to 12.8% in 2006 [28, 29] . Although the etiology of prematurity is complex [5] and not completely understood, our results suggest that at least a fraction of preterm births may be preventable through maternal influenza vaccination.
The association between maternal influenza vaccination and SGA was only statistically significant (and the highest in magnitude) for the period of widespread influenza activity. Possible reasons for the effect being limited to the period of highest influenza activity include the following: (a) in a developed country setting, the effect of maternal influenza infection on fetal growth is milder than the effect on prematurity; (b) SGA represents fetal compromise resulting from infection that is insufficient to trigger early parturition, but may result in the delayed observation of growth restriction (i.e., the observation in the widespread activity period may be the cumulative effect of previous periods). Moreover, in the vaccinated group, the birth weight distribution in the pre-influenza period was different from the distribution in the period of widespread activity (see Text S1). However, the difference in mean birth weights (in the vaccinated group) between these two periods was not statistically significant (p = 0.74). Since the ostensible increase in birth weight in the widespread activity period compared to the pre-influenza period in the vaccinated group cannot easily be explained by vaccine action, this difference-although non-significant-may suggest confounding vis-à-vis the birth weight outcome.
This study has a few limitations and strengths. Although we assessed and adjusted for many covariates, like any observational study, there is a possibility of residual confounding and selection bias. Moreover, data on influenza infection during pregnancy were not included in the PRAMS dataset. Although the primary explanation of the effects of influenza immunization in pregnancy on birth outcomes is through prevention of infection, having influenza infection data would have provided additional support for our findings. Another issue is that the information regarding maternal influenza immunization was based on recall and could be susceptible to information bias. However, the vaccination rates in our study are similar to the rates computed by other authors for Georgia, and to the United States national level coverage estimated by the National Health Interview Survey [30, 31] .
The PRAMS dataset does not contain information regarding the precise trimester of vaccination. Therefore, the effect of vaccination in a specific trimester could not be evaluated. Moreover, it is possible that mothers of premature infants had less time to receive influenza vaccine than mothers of term infants (i.e., reverse causality). On the other hand, since this was a population-based study with a sampling strategy aimed at producing representative estimates, the temporal distribution of influenza vaccination in pregnancy would be similar to that of the general population, hence adding to the generalizability of our findings. The results of this study, nevertheless, need to be replicated in other populations as it is plausible that the impact of vaccines on birth outcomes would vary with the underlying influenza epidemiology and demographic characteristics.
Text S1 Impact of maternal influenza immunization on likelihood of prematurity and SGA births. Editors' Summary Background. Maternal infections during pregnancy can have harmful effects on both mother and baby. For example, influenza is associated with increased morbidity and mortality among pregnant women compared to women who are not pregnant or who acquire influenza infection after delivery. And some respiratory infections, especially those that can cause maternal pneumonia such as influenza virus, are known to be associated with the baby being small-below the 10th percentile-for gestational age and with an increased risk of preterm birth-birth before 37 weeks of gestation. Previous studies have shown that inactivated influenza vaccination during pregnancy provides protection against influenza virus for both mother and baby. As there has been an increase in the rate of preterm birth the United States from 9.5% in 1981 to 12.8% in 2006, the impact of maternal influenza immunization on birth outcomes has important public health implications and is of particular interest during influenza pandemics.
Why Was This Study Done? Given that maternal vaccination can protect babies from influenza virus, it is plausible that influenza vaccination in pregnancy could mitigate adverse birth outcomes such as prematurity and the baby being small for gestational age. The researchers of this study set out to evaluate this hypothesis by investigating whether there was an association between women receiving inactivated influenza vaccine during pregnancy and positive birth outcomes for their babies in the population of the state of Georgia, in the United States.
What Did the Researchers Do and Find? The researchers conducted a retrospective cohort analysis of a large surveillance dataset (the Georgia Pregnancy Risk Assessment Monitoring System) to analyze the relationship between receipt of inactivated influenza vaccine during any trimester of pregnancy by mothers of infants born between June 1, 2004, and September 30, 2006 , and their baby being premature or small for gestational age. The study period encompassed the 2004-2005 and 2005-2006 influenza seasons-the two most recent seasons for which the data were available. The researchers did a stratified analysis for the overall study period, and various periods during it, and also weighted their analysis to adjust for possible oversampling. They used logistic regression to evaluate the association of maternal influenza vaccine and (a) prematurity and (b) small for gestational age, and also used linear regression to evaluate the statistical significance of differences between vaccinated and unvaccinated women for mean gestational age at first antenatal visit and mean birth weight. During the study period, 4,168 mother-baby pairs were included in the analysis. Local influenza activity was detected during 27 weeks (22.1%), and 578 women (14.9% [weighted]) had received the influenza vaccine during pregnancy, giving a vaccination coverage of 19.2% (weighted) among mothers of infants born during the assumed influenza season. In the study sample, 1,547 babies (10.6% [weighted]) were born premature, and 1,186 babies (11.2% [weighted]) were small for gestational age. Infants who were born during the assumed influenza season (October-May) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature than infants of unvaccinated mothers born in the same period, with an adjusted odds ratio of 0.60. The effect of maternal influenza vaccine on reducing prematurity was the highest for infants born during the period of widespread influenza activity, with 72% lower odds of prematurity in infants of vaccinated mothers than infants of unvaccinated mothers. Compared with newborns of unvaccinated women, babies of vaccinated mothers also had 69% lower odds of being small for gestational age during the period of widespread influenza activity, but the adjusted and unadjusted odd ratios were not significant for the pre-influenza activity period.
What Do These Findings Mean? These results show that there was an association between maternal immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. In addition, during the period of widespread influenza activity there was an negative association between maternal receipt of influenza vaccine and small for gestational age birth.  The role of non-formal education in combating the HIV epidemic in the Philippines and Taiwan The Philippines is experiencing a low but slowly growing prevalence of HIV, with a UN estimate of 6,000–11,000 cases out of a population of 91 million, and a 150% increase in new cases in 2008 compared to previous years. Earlier education programmes employed non-formal educational training techniques in the southern Philippines to target high-risk groups such as female sex workers and their establishment managers; the effort was expanded to target males in the community. In comparison, as of 2009, Taiwan has an estimated 40,000 cases of HIV/AIDS in a population of 23 million. It experienced a major increase in HIV infection among injecting drug users, from 77 newly reported cases in 2003 to 2,381 such cases in 2007. This article compares and contrasts the response to the epidemic in each country, describing non-formal educational programmes targeted and tailored to specific high-risk populations. The rate of HIV infection continues to rise in several regions of the world, despite declining incidence rates in other countries. Therefore, measures to prevent its spread remain an urgent global priority. India, for example, is now considered the epicenter of the epidemic and has committed 70% of its HIV spending to prevention (Claeson and Alexander 2008) .
Behavioural changes may prevent the spread of HIV in developing countries (Hearst et al. 1995) , but a continuing challenge is to accurately measure and influence behavioural change. Few national data are available that measure how behavioural changes reduce the risk of HIV infection (Parkhurst 2008) . Furthermore, process data such as the number of clients served or the number of condoms distributed are not very helpful measures (Davis et al. 2000) . Better predictors of HIV prevention are consistent condom use versus ''ever using a condom'' and the number of concurrent partners an individual has versus their number of lifetime partners (Parkhurst 2008) .
Culture and gender relations may also influence the acceptance of protective health measures even more than the physical characteristics of the prevention tool (Gollub 2008) . Therefore, assessing structural factors is especially important when targeting hard-to-reach populations (Mantell et al. 2008) . UNESCO (1997) defines the term non-formal education as follows:
Any organized and sustained educational activities that do not correspond exactly to the definition of formal education. Non-formal education may therefore take place both within and outside educational institutions, and cater to persons of all ages. Depending on country contexts, it may cover educational programmes to impart adult literacy, basic education for out-of-school children, life-skills, work-skills, and general culture. Non-formal education programmes do not necessarily follow the ''ladder'' system, and may have differing durations, and may or may not confer certification of the learning achieved. (p. 47) Some studies report that non-formal education programmes have mixed results (Vermund et al. 2009; Ellis 1995; Evans 1981) . Critics of non-formal education say its effectiveness is too often assumed without proper evaluation, and that untested causal assumptions are frequently made about education and societal change (Benavot 2002) . However, nonformal education serves as an alternative for populations that do not have readily available and accessible educational options (UNESCO 2006) . Globally, non-formal educators have addressed sexually transmitted infections (STI), especially in settings with limited resources (Latkin et al. 2009; Shah et al. 2007; Akani and Erhabor 2006) . For example, in a randomly controlled HIV prevention trial, researchers observed improvements in service delivery, including referrals for HIV testing and provision of condoms, when non-formal care providers received enhanced training in HIV case management (Shah et al. 2007) . Peer education, anti-AIDS clubs, drama, art, youth dialogues, music, and comic books are some examples of non-formal education. Non-formal educational activities were a key determinant in the tremendous decline in HIV prevalence in Uganda, where the Ministry of Education and Sports introduced national-level prevention campaigns in 1986 (Jacob et al. 2006) . These activities included media messages targeting youth and school theatrical performances that depicted real-life scenarios facing youth. Plays were presented in over 8,500 primary schools nationwide, also reaching a large proportion of out-of-school youth. Non-formal education is extremely important to HIV prevention, given that the majority of transmissions occur among young adults.
To some extent, Taiwan and the Philippines could be considered as low HIV impact countries in Asia in terms of their current reported number of HIV cases. At the beginning of 2009, the cumulative numbers of HIV cases in Taiwan and the Philippines were 16,977 and 3,654, respectively. However, like Taiwan, the Philippines must be very much aware of the potential for an outbreak of infection among injecting drug users (IDUs). Between 2000 and 2005, neighbouring Asian nations that previously had low HIV prevalence, such as Vietnam, experienced explosive spreads of the disease due primarily to injecting drug use, according to UNAIDS (2008) .
This article aims to address the non-formal educational strategies used in the two countries. It begins with an overview of the HIV epidemic in the Philippines and then in Taiwan, as these two nations face similar situations in their epidemics, with some very important distinctions. Following this overview, we describe the formal and non-formal educational activities being conducted in each country, which national and local organizations are directing to high-risk, vulnerable populations. We highlight social-structural and environmental approaches that have been integrated into the healthcare delivery systems.
The similarities that the two countries share can be extremely helpful for those designing and implementing innovative and targeted behavioural interventions for high-risk populations. Comparing the response to the AIDS epidemic in these two countries makes it possible to critically assess the strengths and weaknesses of various educational approaches to control the epidemic. Lessons learned can be shared and disseminated throughout governmental and non-governmental organizations to serve the most vulnerable populations.
The Philippines is considered to be a low HIV-prevalence country, but its epidemic is considered to be hidden and growing. Ever since the first AIDS case was reported in 1984, the prevalence among adults there has remained under 0.1%, according to the national Department of Health (Philippines DOH 2005) . From 1984 to 2009, the total number of reported cases was 3,654 (Philippines DOH 2009), but this figure may be inaccurate due to underreporting, as the United Nations estimates 6,000-11,000 cases (UNAIDS 2008). Table 1 shows the annual number of HIV and AIDS cases with combined numbers for 1984 to 2004, and annual numbers by major routes of transmission from 2005 to January 2009. Heterosexuals account for the highest number of cases in the Philippines; the second highest risk group is men who have sex with men (MSM)/bisexuals. The number of heterosexual transmissions has remained fairly stable during this period (about 137 new cases per year), but we see a significant and annually rising increase among MSM and bisexuals. According to the National Epidemiology Center at the Department of Health, 65 new HIV cases were reported in January 2009. This is the highest number ever reported in a month in the Philippines, and represents a 63% increase over the 40 cases reported in January 2008. Young people aged 15-24 are the age group most vulnerable to HIV infection in the Philippines (Alesna-Llanto and Raymundo 2005) , and sexual contact (at 89%) is the leading mode of transmission (Philippines DOH 2009). According to the Philippines National AIDS Council (PNAC 2008) , the prevalence of HIV is 0.08% among the populations identified as most at risk: registered and non-registered female sex workers (FSWs) and their clients, along with MSMs, and IDUs. However, consistent condom use among high-risk groups was low (under 30%) according to a 2003 Behavioural Sentinel Surveillance (PNAC 2003) .
In the Philippines, Cebu City has the highest number of IDUs; in 1995 it became the first site of a harm-reduction program (HRP) (PNAC 2008 ). An integrated HIV behavioural and serological surveillance revealed that 52% of IDUs in Cebu City had shared needles at their last injection (Philippines DOH 2007) , and this figure may be as high as 77% (Mateo et al. 2004; Philippines DOH 2003) . The risk appeared high though this population generally knows that this practice can lead to infection.
In the Philippines, the National AIDS and STI Prevention and Control Program began within the DOH in 1988. In 1992, the government formed the PNAC, a policy-making body with members from government agencies, nongovernmental organizations (NGOs), and professional groups, and representatives of people living with HIV. In 1998, Congress passed Republic Act No. 8504, the Philippines AIDS Prevention and Control Act, which mandates a nationwide implementation of policies and measures to prevent and control the disease (PNAC 2008; Republic Act No. 8504 1998) . A fourth AIDS Medium-Term Plan, 2005 -2010 , published by the PNAC (2003 , outlines five key strategies that focus on strengthening and institutionalizing interventions. They are: (1) strategies targeting highly vulnerable groups (sex workers and their clients, IDUs, and MSMs), and overseas Filipino workers (OFWs); (2) institutional and general public preventive interventions; (3) the quality of treatment, care, and support services for people infected with and affected by HIV/AIDS, (4) stigma-reduction measures and efforts to integrate them into preventive treatment, care, and support services and into the design of management systems; and (5) management systems that support the delivery of HIV/AIDS information and preventive services (p. 18).
In 1984, the first HIV infection, a foreigner in transit, was identified; in 1988, the first case of HIV infection contracted through injecting drug use was identified. In 1989, Taiwan established a nationwide surveillance system for HIV infection. Then, on 17 December 1990, the AIDS Prevention and Control Act was formulated and promulgated. With six amendments, the Act increased the number of components addressing human rights protections; on 11 July 2007, its name was changed to the HIV Infection Control and Patient Rights Protection Act.
As of 28 February 2009, 16,977 HIV-infected people (which excluded 687 HIV-positive foreigners diagnosed in the country) had been identified in Taiwan; of that number 5,338 had developed AIDS and 2,250 had died (Taiwan CDC 2009). Based on 2003 data, the ratio between the estimated number of adult (age 15-49) infections and the number of reported infected persons was 2.4:1 (Huang et al. 2005) . Hence, as of February 2009, Taiwan had an estimated 40,000 cases of HIV/AIDS out of a population of 23 million.
Further, an analysis using cumulative statistics from 1984 to February 2009 indicated that the major age groups for HIV infection are 20-29 (38%) and 30-39 years (35%). Of all those infected, 91% are males and 9% females, for a ratio of infected males to infected females of 10.6:1. In addition, 36% of all those who are infected are unemployed. The numbers contracting HIV were concentrated in several risk groups, including 6,053 IDUs (36%), 5,337 homosexuals (31%), 4,078 heterosexuals (24%), and 1,257 bisexuals (7%) (Taiwan CDC 2005) .
Since 1988, all people living with HIV (PLHIV) in Taiwan received free medical treatment; since 1997, they have received free highly active antiretroviral therapy (HAART). In 2004, medical treatment for PLHIV cost NT $250,000 (about US $7,500) per person on average; this is 70 times the average amount spent on health care by a person in the general population (Taiwan CDC 2005) .
Before 1993, the total number of HIV infections was under 500. The first 5-years national strategic plan for prevention and control of HIV/AIDS initiated in 1994 focused on disease surveillance and health education (Taiwan Department of Health [DOH] 2006) . Currently, the Department of Health is implementing the fourth national strategic plan (2007) (2008) (2009) (2010) (2011) . The plan's three major goals are preventing and controlling HIV infection, encouraging the use of screening and counseling to modify risky behaviours, and providing appropriate health care for infected people to improve their quality of life (Taiwan DOH 2006, p. 21) .
HIV-related NGOs have been established since 1992. In 2009, roughly 19 NGOs provided HIV/AIDS prevention education, counseling, anonymous testing, shelter, and care. One in particular, the Living with Hope Organization, a project of Taiwan's Society of Preventive Medicine, initiated HIV/AIDS prevention education for drug-using offenders in correctional institutions in 2004, the year when warning signs of an HIV epidemic among IDUs appeared.
The NGOs receive funding from government agencies to conduct HIV preventive education for vulnerable groups. Particularly in community settings, the NGOs reach IDUs and MSMs and train peer counselors to work with hard-to-reach populations. For instance, the NGOs reach out to the gay community, disseminate HIV/AIDS educational materials in gay venues, distribute condoms at gay bathhouses, gay bars, and public parks where gay men gather, and provide anonymous HIV testing and counseling (Twu et al. 2004) .
In this study, we compare HIV/AIDS prevention and education efforts and effectiveness in the Philippines and Taiwan. Below we outline our theoretical framework and data collection methods. Two research questions guide this work:
• What are the similarities and differences in non-formal educational activities with respect to HIV/AIDS prevention? • What are the challenges and best practices of non-formal education to promote prevention?
The non-formal educational interventions implemented in Taiwan and in the Philippines are based on a blend of social cognitive theory at the individual level and social influence
The role of non-formal education 339 theory at the organizational level. According to Social Cognitive Theory (Bandura 1987) , behaviour change is enhanced when individuals are trained to exercize influence over their own behaviours and their social environment. They can best make such changes when they experience interventions to heighten their awareness and knowledge, along with enhanced resources and social support. Bandura conceptualized the basic elements of social cognitive theory as consisting of personal determinants in the form of cognitive, affective, and biological factors, behaviour and the environment. In this article we examine how various programmes have applied this theoretical framework to behaviours related to HIV/AIDS, by exposing participants to interventions designed not only to promote safer sex guidelines, but also to develop individual skills and self-beliefs that would enable them to reduce risks in the face of counteracting influences (Bandura 2004) . The second theoretical framework used in this research combines power and social influence. The social psychological study of power and influence originated in the groundbreaking work of Kurt Lewin (1941) , who considered power to be the possibility of inducing force on someone else. More formally, it is the maximum force that Person A can place on Person B divided by the maximum resistance that B can offer. French and Raven (1959) further developed this conceptualization of power and social influence; they defined influence as a force one person (the agent) exerts on someone else (the target) to induce a change in the target, including changes in behaviours, opinions, attitudes, goals, needs, and values. Social power was later defined as the potential ability of an agent to influence a target. Raven (1965) further classified power according to the base from which it comes and listed six types: reward, coercive, legitimate, referent, expert and informational power.
For this study, data from Taiwan were collected from several major sources, including official HIV/AIDS surveillance statistics and the national strategic plan from the website of Taiwan's Centers for Disease Control, annual reports of prevention and education activities to the AIDS Prevention and Control Committee from 2005 to 2008, educational activities among correctional institutions provided by the AIDS NGOs, and a literature review. The current inter-ministry cooperative mechanism operates through the Executive Yuan's DOH AIDS Prevention and Control Committee, a communication platform chaired by the health minister (Taiwan CDC 2008). Since 2005, all HIV/AIDS prevention and control outcomes and activities have been reported to the committee twice a year.
Data collected in the Philippines included HIV/AIDS official surveillance statistics, country reports, and the national strategic plan from the Philippines National AIDS Council. In addition 56 interviews were conducted with key informants, including government health officials, university professors, NGO staff, establishment managers, and association representatives. Three focus groups were conducted with female entertainers in Metro Manila and Angeles City in 2006-2008.
Since 1990, mandatory HIV tests have been administered to each inmate in Taiwan, including drug-using offenders, when they are first incarcerated. Before 2001, no more than ten new infections among injection drug users (IDUs) were reported each year. In the past few years, however, the number of new infections among IDUs has been growing at an alarming rate (Chen and Kuo 2007 Fig. 1 . For Taiwanese IDUs, sharing needles/syringes and heroin diluents are major risk factors (Chen 2005) . After a harm-reduction programme was launched in 2005, the proportion of IDUs infected with HIV declined to 60.5% in 2006 and then dropped dramatically, to 36.7% in 2007 (Table 2) . The role of non-formal education 341
The success of HRPs in curbing the HIV epidemic among IDUs in Taiwan
The harm reduction programme in Taiwan consists of three major parts: (1) information, education, and communication (IEC); (2) a needle/syringe programme (NSP); and (3) drug substitution treatment (Taiwan DOH 2005; Taiwan CDC 2007) . The IEC programme expanded the HIV screening and monitoring of IDUs and provided timely health IEC to help them change their high-risk behaviours, especially the sharing of drug paraphernalia and diluents. The NSP enabled local health departments, in conjunction with neighbourhood pharmacies and residents, to decide on where to locate operations that could provide IDUs with clean syringe paraphernalia, counseling, and health information. In November 2005, four NSP pilot projects were launched in Taipei County, Taipei City, and Taoyuan County in northern Taiwan, and Tainan County in the south. Since July of 2006, the NSP has expanded island-wide. In total, 1,103 health education and counseling service centers were set up, along with 418 vending machines providing clean needles. Roughly 8.2 million needles were given out between 1 November 2005, when the NSP was implemented, and 31 December 2008. In addition, over 777 recycling trashcans for used needles were set up, and the return rate for used needles increased from under 1% at the beginning to 80% currently; the average return rate is 61%. About 5 million used needles have now been returned.
The major drug-substitution treatment programme uses methadone maintenance; it also offers follow-up counseling, education, and drug rehabilitation referrals. The first person received drug-substitution treatment on 10 February 2006, and by the end of 2008, the cumulative number of cases receiving substitution treatment was 25,683. Island-wide, a total of 87 hospitals and clinics now provide substitution treatment services.
The HRP has successfully reversed the increase in HIV among infected IDUs. As shown in Fig. 1 
In a programme to prevent and control the spread of STIs, city ordinances targeted women working as freelance sex workers or in entertainment establishments and saunas. In the Philippines, Quezon City's first ordinance required all job applicants to obtain health certificates and to attend HIV/AIDS awareness seminars. In 1999, a second ordinance created an STI/AIDS Council. A third ordinance focuses on strengthening the implementation of rules and regulations that require entertainers to get weekly checkups at social hygiene clinics. Sauna and massage parlor workers attend a 1-week seminar that includes HIV awareness, personal hygiene, principles of massage, and ways to prevent HIV. Local city health department task forces monitor employees weekly for health certificates, make certain they are at least 18 years old, and check to see that the establishments have condoms. If establishments do not comply, their managers receive one warning and are advised to allow staff to comply; otherwise they face closure through due process and recommendations by the licensing and health departments.
Other cities such as General Santos and Angeles have specific activities and policies to prevent and control the spread of HIV/AIDS and other STIs. These include a policy of 100% condom use, the creation of multi-sector AIDS councils, funding for HIV/AIDS prevention activities, and a monitoring system to ensure that the sex industry complies with a policy prohibiting the employment of minors. Legal action has been taken to curb child prostitution in and around entertainment establishments (USAID 2008) .
From 1994 to 2003, Morisky and colleagues in collaboration with the University of the Philippines (Morisky et al. , 2004 , collected longitudinal data from female bar workers, potential male customers, and establishment managers in the southern Philippines. The research initiative trained managers and peer educators from approximately 110 different establishments and implemented a 3-years longitudinal quasi-experimental study in four regions. The project had three parts. The teams implemented an educational policy on HIV/AIDS prevention in all intervention sites and met with city health officials at care sites to identify important recommendations that were found to significantly affect condom use behaviour and to reduce STIs. They also coordinated all intervention activities with the National AIDS Council of the Philippines, and worked closely with regional and city health departments in the four study sites. Finally, in a 3-years follow-up intervention using a crossover design for 18 organizations, they expanded this initial programme to address the educational needs of client-centered populations that female bar workers identified as their usual customers: military personnel, transportation drivers, factory workers, and members of high-risk communities. Morisky et al. (2002b) found that both personal and situational factors were important influences on whether female bar workers used condoms. Morisky et al. (2002c) also noted that female bar workers employed in establishments that had a written policy regarding 100% condom use were 3.5 times more likely to use condoms consistently compared to establishments who had no policy. Earlier studies found that over 50% of those female bar workers expressed concerns about pain when using condoms. Further, having male clients who were substance users increased the incidence of STIs among female bar workers, while having regular partners lowered their rate of STIs Chiao and Morisky 2007) . Morisky et al. (2002a) also found that STI incidence decreased when the city required karaoke bar workers to attend the SHC clinic. Sneed and Morisky (1998) discovered that attitudes and norms do predict condom use, but they are mediated by intentions.
As part of those studies, Morisky et al. (2006) conducted a multilevel analysis of the impact that a social influence intervention would have on condom use and STIs among female bar workers. The researchers contrasted the effects of peer influence, manager influence, and combined peer-manager influences on condom use and STIs; social influence models were used, which combined social cognitive theory and socio-structural theories. Female bar workers who received structural peer and establishment interventions reported more positive attitudes towards using condoms, more establishment policies favouring condom use, and fewer STIs, compared to those in either the peer-only intervention group or the control group. Those who only experienced interventions from their managers had fewer STIs, fewer negative attitudes towards condoms, and higher knowledge and perception of risk, compared to those in the peer-only intervention group. STI outcomes were measured as before-and-after comparisons (STI rates per 1,000 CSW clinic visits); those women who experienced the combined intervention (with both peer and manager) showed a significant drop in STIs, compared to those in other study groups (-21.8 difference, 36.4 to 14.6%) (Urada et al. 2008) . Also, HIV test results for the female sex workers who were registered in the study sites between June 1995 and December 2001 showed that the control group, those receiving the usual care (N = 6,157), had the highest number of HIV infections (10), compared to those in other study groups (0 for the peer intervention group, 2 for the manager intervention group, and 2 for the combined intervention group). After the intervention, it was found that those who were in the intervention The role of non-formal education 343 groups and had participated for at least 3 months kept significantly more of their appointments at the clinics, compared to those in the control group (p \ 0.05). As a result of these findings, the establishment managers formed an association and began a loan programme to increase the compliance of female bar workers to purchase and take the entire medical regimen for STIs (Morisky et al. 2002a, b, c) .
A follow-up study was implemented to evaluate the diffusion effects of interventions in other areas of the Philippines and to subsequent groups of workers. Interviews with key informants in Quezon and Angeles cities and with focus groups of female bar workers in Quezon City revealed that challenges remain in other areas, especially as new employees enter the workforce at entertainment establishments. Key informants described how some ordinances promote the policy of 100% condom use (no condom, no sex), but the informants tended to substitute phrases such as ''prophylaxis devices'' for condoms and ''preventing illness'' for ''family planning'' because of opposition from influential people, religious groups and because of cultural barriers.
According to these informants, freelance sex workers are especially challenging to reach. Because freelancers enter establishments to find customers, one way to identify them is to organize the registered workers and assume that the rest are freelancers. Internet cyber sex negotiations pose another challenge. The fact that freelancers can now negotiate their appointments on the Internet, using chat and text messages, makes it even harder to find them.
Another remaining challenge is to get sex workers to go to social hygiene clinics. Visits to one such clinic increased to 80 a day from the 25 a day that was standard 10 years earlier. Although fewer cases of herpes occur than in other countries such as Thailand, rates of chlamydia are high and those for gonorrhea have increased.
Key Informant interviews with staff at social hygiene clinics also revealed the difficulties of keeping track of workers' attendance at the clinics. If-but only if-a sex worker has a yellow registration card, the clinic can check if she comes back. She may also miss 1 month and return the next. Several factors account for this irregular use. First, not all managers cooperate because they are concerned about their business. Moreover, workers may not know about, or forget, their appointments. Or they may choose to see other doctors, especially if their managers arrange appointments for them.
Three focus groups with female entertainers revealed several continuing barriers to condom use and thus to preventing the spread of the disease:
• The women do not use condoms with boyfriends or regular partners.
• Clients offer to pay more to have sex without a condom.
• Condoms purchased at the clubs are expensive, at 100 pesos (US $3).
• The workers spend most of their income on clothing and jewelry; any income from ''ladies' drinks'' goes to the establishment. • Managers do not encourage workers to see doctors.
• The cost of a clinic visit (65 pesos) is too high; transportation is also costly. • Many fear the common disease chlamydia the most, but may not have money to buy medication and fear that they can no longer work (most have children).
• Entertainers are not organized among themselves; they are only registered with the City Health Department. • They have no opportunities for peer training.
In the focus groups, the entertainers made several recommendations.
• They need counseling.
• They want more information on HIV and want to learn more about all STDs.
• They would like to see violent clients put in jail.
• They would like peer trainings to create leaders and a point person in every establishment.
In the Philippines, NGOs play a vital role in implementing HIV prevention programmes in the general population and for high-risk groups. Government agencies partner with NGOs that have access to the community, especially hard-to-reach populations. NGOs also fill the gap where pressure from the Catholic Church makes it hard for government agencies to implement sex education and to distribute contraceptive information and condoms.
On the other hand, in Taiwan, where the HIV Infection Control and Patient Rights Protection Act was promulgated and implemented in July 2007, the responsibility for carrying out HIV education is no longer confined to health authorities. Article 7 states that ''competent authorities shall conduct education programmes and promotion campaigns on the prevention and control of HIV. The various central competent authorities shall devise annual plans detailing education programmes and promotion campaigns; the contents of such plans shall be sex-conscious and shall focus on anti-discrimination; the plans shall be implemented with the assistance of organizations, schools, groups and mass media.'' It appears that the Taiwanese government has a very positive attitude towards implementing a comprehensive campaign to educate people about HIV and AIDS and to promote prevention efforts.
In the Philippines, NGOs complement the life skills programme that the Department of Education offers in schools. The Girl Scouts of the Philippines (GSP) is the nation's largest association of girls and young women, with a membership of over one million. Through its 95 councils in six regions, it provides information and skill-building for HIV prevention in nearly all of the nation's public elementary and secondary schools (PNAC 2008; United Nations Global Coalition on Women and AIDS 2006).
Other NGOs, such as the UN Global Coalition on Women and AIDS (2006), emphasize life skills and peer education for school-aged youth in informal educational settings. The PRIME II project, run by IntraHealth International (2003), integrates family planning and peer education with HIV prevention for high-risk youth. An annual MTV Staying Alive Music Summit delivers messages about HIV prevention and transmission to thousands of youth (Rutstein 2005) .
Several other NGOs are listed in the Philippines HIV Initiatives Database, run by the Health Action Information Network (2007). For example, Advocates for Youth Philippines Foundation holds campus tours and road shows that promote education, prevention, Turning to Taiwan, in its school systems, education about the disease is part of the Chun-Huei (''spring sunshine'') Project, the substance abuse and HIV/AIDS prevention project which the Ministry of Education (MOE) has implemented since 1991. The project encourages schools at all levels, from primary to college, to conduct annual educational activities focused on the disease as well as on preventing substance abuse. Students' Chun-Huei clubs receive annual subsidies from the MOE to conduct extracurricular educational activities, like poster contests. In addition, the Taiwan AIDS Foundation has recently reached out to schools with education to prevent HIV.
In 2007, to coordinate with the HIV Harm Reduction Pilot Programme for IDUs, the DOH asked the MOE to establish drug abuse and HIV prevention as a learning indicator in the Health and Physical Education programme in the curriculum guidelines for Grades 1 through 9. For high schools, sessions on drug abuse prevention and control and on HIV prevention were incorporated into the course guidelines on Health and Nursing and Citizens and Society; these enable youth in the formal educational system to learn how to prevent the spread of the disease.
Four groups experience particularly high prevalence of the disease: Taiwanese users of injected drugs, Filipinos who work overseas, men who have sex with men, and women, especially in the Philippines.
In Taiwan, two campaigns of health education and promotion are targeted at IDUs, at both the community level and in the criminal justice system. The HRPs, mentioned earlier, serve IDUs in their communities. They are implemented through neighbourhood pharmacies, clinics, and 87 referring hospitals throughout the country which provide clean syringes, counseling, health information, and long-term oral methadone maintenance treatment. Since 2006, such programmes have also been directed at IDUs in the criminal justice system, provided by local health departments and NGOs. In 2008, local police focused on illegal prostitutes, sex consumers, pimps, and suspected drug users; when they found such people, they immediately notified the local health departments so that medical staff could conduct blood screening and teach them about the disease. Between January and September of 2008, the police recorded 28,648 arrests of suspected drug users.
The Philippines also has needle-and syringe-exchange programmes; they started in 1995 in Cebu City (PNAC 2008; Ball et al. 1998) . Currently, 800 IDUs receive prevention services and clean needles/syringes at three major outreach locations. Antiretroviral treatment has been provided free since 2005, and the STI AIDS Cooperative Central Laboratory offers a baseline CD4 count for free. Recently, 390 PLHIV were enrolled for drug treatment and were receiving extended care and support services (Mesquita et al. 2008; Asian Development Bank 2007) . Together, NGOs, local health departments, and stakeholders provide harm-reduction services, including outreach, peer education, psychosocial support, provision of clean needles and syringes, and treatment for STIs and other health-related concerns.
In the Philippines, overseas workers have a higher incidence of the disease than any other group: 1,196 cases, or 33% of all reported cases from 1984 to January, 2009, according to the Philippines DOH National Epidemiology Center (2009). Of these workers, 95% contracted HIV through sexual intercourse, and 75% are males. OFWs are required to attend a seminar on the cause, prevention, and consequences of HIV/AIDS before they can be certified for their overseas assignments. The DOH offers them voluntary counseling and testing after they return from abroad.
Founded in 2000, the Action for Health Initiatives (ACHIEVE), Inc., a non-profit organization based in Quezon City, targets migrant workers and their families, specifically addressing their sexual and reproductive health, rights, and HIV vulnerability. ACHIEVE is a member of the Coordination of Action Research on AIDS and Mobility in Asia (CARAM-Asia), a regional network of organizations working on migration and HIV/AIDS issues in the Asian region.
During a pre-departure orientation seminar run by the Foreign Service Institute of the Department of Foreign Affairs (DFA), ACHIEVE provides information about HIV and AIDS and guidelines for OFWs on how to handle cases of HIV and AIDS on the job. Seafarers are tested regularly. Another NGO, LIKHAAN, also conducts HIV/AIDS education in pre-departure seminars conducted by the Overseas Welfare Association.
The majority of people in conservative Asian societies like Taiwan do not accept sex between men, so men who have sex with men often face discrimination. Consequently, many MSM do not want to reveal their sexual orientation-which makes it very difficult for health authorities to reach them with their HIV-prevention programmes. NGOs can do so more effectively. From August to October 2008, the CDC sponsored the Light of Friendship Association of Taiwan to carry out rapid HIV-screening services at venues and locations where homosexuals tend to congregate in Taipei, Taichung, and Kaohsiung. In 1992, the Ministry of the Interior included testing for syphilis and HIV as part of the physical examination for those entering the military.
NGOs are also important in the Philippines, especially in carrying out outreach activities that target MSMs at the local level. The Butterfly Brigade, an NGO in the Boracay region of the Philippines, is described as a model for peer education groups for MSM communities. The gay community initiated the NGO themselves by holding informal group discussions. From these discussions, they discovered a need for health services targeting their community. The NGO influenced the regional health system to offer condom promotion, voluntary testing, counseling, and care, and to install condom vending machines in the tourist city's pickup spots. Gay-sensitive educational programmes incorporated participatory and interactive music, visuals, beauty contests, and timed interventions from 8 p.m. to 2 a.m., the time when many gay men cruise for sex. The number of MSMs seeking help at social hygiene clinics increased (PNAC 2008).
The role of non-formal education 347
Women Women are at high risk in the Philippines; they account for nearly 30% of all the cases of HIV since 1984 (Philippines DOH 2009). The highest prevalence is among women aged 20-39. Over 25% of women in the women's state penitentiary in Metro Manila were infected with STIs (Simbulan et al. 2001 ). In the general population, Avelino et al. (1997) found that residents in Quezon City, aged 15-49, had a high level of awareness of HIV/ AIDS, but only 32% had used a condom during their most recent intercourse with a nonregular partner, and 28% of IDUs shared their injection equipment with others. These findings indicate that women in the general population face some risk. Targeting high-risk females such as entertainment establishment workers, the Philippines government's Technical Skills and Development Authority office conducted livelihood programmes at some social hygiene clinics. Nurses and doctors at the clinics gave lectures on health education, and all NGOs were invited to provide services there. And, through its work at the clinic, an NGO called Belen hired a commercial sex worker who decided to find different work.
In Taiwan, as statistics from the CDC indicate, far more men than women were being infected: in 2003, the male-to-female ratio of newly reported cases was 20:1. Soon, however, infection among women increased rapidly. In 2006, the male-to-female ratio for newly reported cases rose to 7.4:1, but in 2008 it fell to 16.9:1. In Taiwan, most women who contract the disease do so through their sex partners, as sex workers, or through using injection drugs. At the end of February 2009, 41.57% of all HIV-infected women were between 20 and 29 years old (Taiwan CDC 2009). Women of childbearing age who are vulnerable to the disease need to get tested, especially if they have had an STD, are sex workers or IDUs, had multiple sex partners while pregnant or an HIV-infected sex partner, or if they engage in other high-risk behaviours. Early intervention and prevention of HIV infection among these vulnerable high-risk groups can prevent the spread of HIV, both horizontally and vertically. Consequently, since 2006, the DOH has included routine HIV screening in prenatal examinations. In addition, when police arrest illegal prostitutes, they immediately notify the local health department, which in turn conducts blood screening and connects the woman to a HIV health education and promotion campaign. Between January and September of 2008, police made 2,289 arrests of illegal prostitutes. Table 3 summarizes the comparative data on the targeted approaches, both structural and environmental, that both countries are implementing to stem the AIDS epidemic. Agencies of both governments were very active in promoting national strategic plans as the epidemic began. They also initiated sentinel epidemiological surveillance early on, in order to identify and implement educational approaches tailored to those specific high-risk groups. Minor differences can be seen in the handling of mandatory blood testing among high-risk groups: blood testing is mandatory for all those entering prisons in Taiwan, while the Philippines only has local government ordinances for testing and counseling.
The two countries differ in approaches for working with the highest targeted HIV risk groups. Taiwan focuses on IDUs and MSM, whereas the highest risk group in the Philippines is overseas contract workers and the disease is mostly transmitted through heterosexual contact. Both countries have harm reduction programmes, but Taiwan's is more widespread, given the larger numbers of infected IDUs. Taiwan focuses on pregnant Of the estimated 3 million drug users, roughly 15,000 are IDUs, and up to 77% of IDUs might share needles.
In 1995, the first harm-reduction project was introduced on the island of Cebu
The role of non-formal education 349 Looking at channels for non-formal services, both Taiwan and the Philippines rely heavily on government collaboration with NGOs at the local levels. Noteworthy is Taiwan's focus on mass media delivery. World AIDS Day is recognized in both countries, but antiretroviral medication and treatment for people who are HIV positive is offered more freely in Taiwan than in the Philippines. Taiwan does not yet have a dedicated evaluation component like the integrated evaluation system that the Philippines Department of Health conducts through its National Epidemiology Center.
Parkhurst (2008) examines ''what works'' in driving down the prevalence of the disease, and lists these five elements: falling HIV prevalence, fewer new infections, changes in behaviour, causes of behaviour change, and responsible policies.
Earlier studies involving sentinel surveillance in major regions of the Philippines provide an empirical basis for the very low HIV prevalence. However, national implementation of policy is devolved to local government levels, and many programmes are delivered by NGOs. This can be both beneficial and problematic, as community resources ensure that delivery systems continue, but few programmematic evaluations are conducted.
Over the past 5 years, the number of new cases reported to the Philippines HIV registry has begun to rise steadily. The government has placed a priority on outreach and services to overseas workers and IDUs as a result of the increased prevalence of unprotected sex among highly vulnerable groups (PNAC 2003) .
Service provision to overseas workers is an example of a best practice by the Philippines government. These workers, and their significant others, receive IEC, pre-departure seminars, and voluntary HIV testing and counseling upon their return. However, few empirical studies have examined the effectiveness of these approaches.
Also, efforts to reach the broader community have been made at various other worksites (Morisky et al. 2004) , and within both the army and schools. The government relies on non-formal educators and NGOs to implement many of these activities.
The PNAC aims to promote 100% condom use and behavioural change communication in every prevention effort, to encourage partnerships between local governments and NGOs, and to establish local AIDS councils. Some key cities have implemented the 100% condom use policy (UNAIDS 2008). But not all local governments have adopted these efforts. HIV has remained low among female sex workers; one explanation is the government's early and continuing strategy of monitoring and treating STIs through its social hygiene clinics. As in Taiwan, prostitution is illegal in the Philippines. However, the Philippines health system requires all workers at entertainment establishments to register with the government and to receive regular STI health check-ups at the government clinic. Outreach, treatment, and regular screening of sex workers in the Philippines have led to STI declines among sex workers and their clients (Wi et al. 2006 ). Rules on condom use at The role of non-formal education 351 entertainment establishments may also make a difference; in one study, workers were 2.6 times more likely to use condoms consistently when the establishment had such a rule (Morisky et al. 2002a, b, c) . Challenges remain for the Philippines. NGOs deliver psycho-social and medical services for IDUs. However, the Philippines could experience a rapid increase in HIV due to the use of injecting drugs, as happened in Taiwan. The Philippines National AIDS Council plans to expand HRPs to other areas where the numbers of IDUs are increasing. As part of a national surveillance system, 360 male IDUs were surveyed in Cebu City, Philippines, from 1997 to 1999. Although most of the men knew that certain behaviours put them at risk, more than two-thirds reported they shared needles and almost two-thirds of those who were sexually active said they never used condoms (Amadora-Nolasco et al. 2002) . Other challenges include a conservative culture that discourages open discussion of sexuality, inadequate social and behavioural research and monitoring, high numbers of STIs, and the weak integration of HIV/AIDS programmes into local governments (UNAIDS 2008) . Finally, the low rate of HIV testing among both the general and high-risk populations in the Philippines poses a major problem for detection and early treatment (PNAC 2008) .
In Taiwan, several concerns have arisen about the HRPs, although they are considered an example of good HIV prevention practice. Several studies have shown that HRPs are effective in decreasing HIV prevalence among criminal justice populations Chen 2007, 2008; Chen et al. 2008 ). Lan and Chen (2008) found that after an HRP was conducted in the northern region of Taiwan at the end of 2005, the HIV prevalence decreased from 37.6 to 18.7% in the detention center within 6 months. In contrast, the prevalence rose by 1.4 and 3.7%, respectively in the central and southern regions where no HRPs were conducted during that period. Significant differences were found between these regions in terms of the change in HIV prevalence. Moreover, the HIV antibodypositive rate among returned syringes also decreased significantly, from 18.0 to 9.4% (p \ 0.001) in 2007.
The significant decrease in the numbers of new cases among IDUs is evidence that HRPs have succeeded in curbing the epidemic. However, the role of non-formal education should not be ignored. The HIV epidemic began in 2004, but the pilot HRP was launched in November 2005 with limited access to drug substitution treatment, and then became nationwide after a 6-months trial. Needles and syringes are very easy to purchase in Taiwan, even without needle syringe programmes. Hence, non-formal education was also a key factor in this success. The rapid increase in the HIV epidemic caused greater media attention and increased reporting. Also, over 60% of the new reports of infections were among the incarcerated population and resulted from mandatory HIV testing. Segregating the HIV-infected prisoners prevented them from spreading the virus. Furthermore, NGOs began voluntary HIV prevention health education programmes in correctional institutions in 2004. Ko et al. (2009) found that, for drug-dependent inmates, a brief HIV education programme based on a transtheoretical model was effective.
At one time, up to half of IDUs commonly shared needles and syringes (Lyu et al. 1999) . A recent study indicated that following the SARS outbreak in Taiwan an increase in sharing of needles may have helped cause the rapid increase of infections among IDUs; they may have engaged in more of this risky sharing behaviour because of a perceived decrease in the drug supply during the outbreak (Lyu et al. 2008) .
Previous studies have found that marketing HRPs in correctional institutions requires a culturally sensitive approach to increase the incentives for participation. surveyed 2,239 new inmates in jails; they found that 694 were IDUs. Among those IDUs, 67 were PLHIV. In total, 56.6% of the IDUs had heard of harm reduction, but only 8% of the PLHIV had ever participated in a syringe-needle exchange programme. More than 51% had left their used needles in trash cans or gutters.
Education is the most important factor in behaviour change, especially in a situation as complex as the HIV epidemic. Ironically, in Taiwan, once the epidemic among IDUs was controlled, a hidden epidemic among homosexuals (including bisexuals) became apparent. The number of newly HIV-infected homosexuals increased steadily, from 472 (14%) in 2005 to 861 (49%) in 2008, as shown in Table 2 . In other words, approximately half of the newly reported infections occurred among homosexuals. This suggests the need to deliver appropriate messages to that group, using culturally sensitive and gender-sensitive approaches. Developing an appropriate non-formal educational approach to the MSM group might become the next challenge now that the high incidence among IDUs has been reduced.
In summary, the non-formal educational programmes in each country highlight the importance of environmental factors and their predictive ability in modifying HIV prevention behaviours among FSWs in the Philippines and IDUs in Taiwan. The results underscore the importance of developing structural and organizational interventions to prevent HIV/STDs. In addition to improving knowledge, efficacy, and skills related to needle use and safe sexual practices, health workers involved in these non-formal educational activities should work closely with community gatekeepers to create a supportive environment for HIV prevention.
Both countries have the special features of inter-ministry cooperation and HIV surveillance. Taiwan has several mandatory HIV testing practices among target groups such as inmates, and the Philippines has a sentinel surveillance programme. These serve as important bases for conducting needs assessments and for implementing non-formal education. The inter-ministry cooperation increases access to vulnerable groups and hardto-reach populations. The surveillance helps identify populations at risk. A notable strength of the Philippines in preventing HIV transmission is its system of registering sex workers, which enhances the non-formal education for this vulnerable group. A strength of Taiwan is its provision of free HAART to encourage voluntary testing. The mission of non-formal educational efforts should be disseminating knowledge and skills to prevent the disease's spread, and to increase service utilization.
Worldwide, most independent countries have implemented compulsory education; children are required to attend school for a given period of time (Benavot 2002) . With respect to HIV prevention, non-formal educational activities play an important role for children who drop out of school or do not have the opportunity to attend as well as for the adult population and vulnerable groups. The education of female bar workers in the Philippines (Morisky et al. 2005 ) and the HIV education in the correctional systems for IDUs in Taiwan (Ko et al. 2009 ) are examples of best practices for designing, implementing, and evaluating non-formal educational programmes. Both countries draw upon the assistance of NGOs to carry out non-formal educational activities.
Our analysis leads to six suggestions for policy:
1. Individual behavioural surveillance should be conducted regularly with at-risk groups to monitor behaviour that could lead to HIV infection as well as for priority setting.
The role of non-formal education 353 2. Organizational efforts at behaviour change must establish normative standards regarding regular attendance at local social hygiene clinics and training of peer educators in establishments and worksites, as well as in the prison system. 3. Non-formal education must focus on delivering appropriate messages to the target audience, using approaches that are both culturally sensitive and gender-sensitive. 4. More intervention research should be encouraged to target vulnerable groups, as in the testing of incarcerated injection drug users and condom use among female bar workers. 5. Indicators of programme effectiveness should be developed for implementation, followed by empirical studies to provide evidence of success, e.g., HRPs. 6. Highly motivated IDU inmates should be trained as peer counselors who can serve as outreach health education workers in their communities upon release. Standardization of Methods for Early Diagnosis and On-Site Treatment of High-Altitude Pulmonary Edema High-altitude pulmonary edema (HAPE) is a life-threatening disease of high altitude that often affects nonacclimatized apparently healthy individuals who rapidly ascend to high altitude. Early detection, early diagnosis, and early treatment are essential to maintain the safety of people who ascend to high altitude, such as construction workers and tourists. In this paper, I discuss various methods and criteria that can be used for the early diagnosis and prediction of HAPE. I also discuss the preventive strategies and options for on-site treatment. My objective is to improve the understanding of HAPE and to highlight the need for prevention, early diagnosis, and early treatment of HAPE to improve the safety of individuals ascending to high altitude. High-altitude pulmonary edema (HAPE) is a specific disease of high altitude. It has a high incidence and is often serious because of its rapid progresses. It is also life-threatening if treatment is not started in a timely manner [1, 2] .
Many studies [3, 4] have shown that HAPE is a major disease that often affects nonacclimatized healthy individuals who ascend to high altitudes. Therefore, early diagnosis of HAPE is essential to initiate early treatment of HAPE and maintain the safety of people who ascent to high altitude. Although international and Chinese criteria have been established for HAPE [5] , its early detection and diagnosis are difficult, except in the presence of typical pulmonary edema. Following the magnitude 7.1 earthquake that struck Yushu (Yushu Tibetan Autonomous Prefecture, Qinghai Province, China) on April 14, 2010 , approximately 50,000 rescuers assembled in the region. This region lies at an altitude ranging from 3700 m to 4900 m, the average altitude is 4,493 m. Although the incidence of HAPE is only 2-4%, the sudden influx of nonacclimatized rescuers to the region meant that HAPE was one of the most common life-threatening diseases at that time, even considering the effects of the earthquake, which caused 1944 deaths, with 216 people missing and 12,135 injured. HAPE can also induce psychological disorders in affected individuals. Therefore, it is essential to highlight the early diagnosis and on-site treatment of HAPE. Under the support of the National Science and Technology Program, we have conducted an extensive range of studies on HAPE intended to find effective methods for early diagnosis and clinical treatment of HAPE and, thus, improve the health and safety of individuals who rapidly ascend to high altitude.
It is generally not difficult for physicians to diagnose typical HAPE, which is based on medical history, symptoms, and signs including white, yellow, or pink frothy sputum, moist rales on pulmonary auscultation, and flocculent shadows on chest X-rays. However, atypical HAPE is much more difficult to diagnose. Patients usually develop atypical HAPE at altitude <3000 m, which may occur several days or longer after ascending to altitude. Symptoms differ from those of typical HAPE, such as less sputum or an absence of sputum and nonspecific findings on chest X-rays. Based on a previous study [6] , the development of HAPE can be classified into two stages from the onset to the presence of typical symptoms. The early stage is characterized by 2 Pulmonary Medicine interstitial pulmonary edema while the late stage is characterized by alveolar pulmonary edema. Patients with interstitial pulmonary edema do not usually exhibit serious dyspnea or typical signs such as pink frothy sputum, extensive moist rales, or wheezy phlegm. Thus, these patients are susceptible to misdiagnosis and delayed treatment. Li et al. [7] investigated the characteristics of HAPE in 482 patients. They examined the clinical symptoms and signs, and performed routine blood tests, electrocardiography, color Doppler echocardiography, chest X-rays, and computed tomography (CT) of the patients. They found that HAPE patients exhibited significant differences in these parameters compared with non-HAPE patients, and that the symptoms and signs were more obvious in HAPE patients than in non-HAPE patients. In addition, studies have revealed that oxygen treatment is more effective in HAPE than in other forms of lung injury. Therefore, some researchers have proposed that tentative oxygen treatment can be used for the early diagnosis of HAPE. Nevertheless, the most important examinations for the early diagnosis of HAPE are chest X-rays or CT. On chest X-rays, HAPE often presents as decreased pulmonary transmittance, increased or obscure lung markings, and ground glass-like changes in the lung, or patchy shadows. On CT scans, increased and enlarged lung markings, ground glass-like changes in the lung, nodule-like shadows, scattered or isolated alveolar edema of terminal bronchioles, and slim reticulate shadows can be observed.
According to the clinical symptoms, signs, and findings of blood routine tests, electrocardiography, color Doppler echocardiography, chest X-rays, and CT, the following criteria for HAPE have been developed [7] :
(1) recent ascent to high altitude (>3000 m); the presence of palpitations, chest tightness, dyspnea and cough with or without white foamy sputum;
(2) local, unilateral, or bilateral coarse breath sounds, with or without local moist rales, central cyanosis, tachycardia (>100/min), and tachypnea (>24/min);
(3) chest X-ray findings including decreased pulmonary transmittance, increased or obscure lung markings, ground glass-like changes or patchy shadows in the lung; CT findings including increased and enlarged lung markings, ground glass-like changes, nodulelike shadows, scattered or isolated alveolar edema of terminal bronchioles and slim reticulate shadows;
(4) routine blood test findings include an increased white blood cell count and an increased neutrophil count;
(5) arterial blood gas analysis showing continuous hypoxemia accompanied by mild respiratory alkalosis;
(6) electrocardiographic findings including sinus tachycardia, clockwise rotation and sharp P waves; (7) persistent pulmonary hypertension on echocardiography; (8) symptoms resolve rapidly following rest, oxygen treatment, pulmonary artery pressure lowering treatment, and diuresis.
For the diagnosis of HAPE, criteria 1, 2 and 3 must be met. These findings, together with those in 4, 5, 6, 7, and/or 8, are then used to confirm HAPE. Using these criteria, the severity of disease can be determined, which is critical for individualized therapy. With early diagnosis and early treatment, HAPE can be controlled at the early stage and the symptoms be markedly improved.
Once HAPE is diagnosed, its severity should be graded to provide individualized therapy. Therefore, when a patient with suspected HAPE is admitted, the grade of HAPE should be confirmed as soon as possible according to the criteria for early diagnosis [8] .
Dyspnea and cough with white foamy sputum may occur after intermediate manual labor. Lung auscultation shows local moist rales in a unilateral lung. The respiratory rate is often <24 breaths/min, and heart rate <100 beats/min. There is no arrhythmia. Chest X-rays shows the area of flocculent shadows occupies <1/4 of the lung. The shadows are confined to the right lower lobe and are spotty or patchy. CT scans reveal increased and enlarged lung markings. Routine blood tests are normal.
Moderate. Dyspnea, chest pain, chest tightness, and cough with a large amount of white foamy sputum occur after mild manual labor. Extensive moist rales are noted in the bilateral lower lung or unilateral lung on lung auscultation. The respiratory rate is >24 breaths/min while the heart rate is >110 beats/min and is accompanied by arrhythmia. Chest X-rays show patchy or flocculent shadows covering >1/2 of the lung, and CT scans show ground glasslike changes or nodule-like shadows. The white blood cell count and neutrophil count are slightly increased.
Patients can not lie in the supine or prone position. They may have a pale complexion, cold sweat on the forehead, serious dyspnea, and a heavy cough with a large amount of white or pink foamy sputum. Rales of small, intermediate, and large bubbles are extensive in the bilateral lungs and are accompanied by the sound of boiling water. The respiratory rate is >30 breaths/min, while the heart rate is >120 beats/min and is accompanied by arrhythmia. Chest X-rays reveal asymmetric cloudy shadows covering >1/2 of the bilateral lungs, and CT scans indicate scattered or isolated alveolar edema of the terminal bronchioles. The white blood cell count is 10 × 10 9 /L.
Severe. The symptoms and signs are more serious than those in severe HAPE. Patients are at high risk Pulmonary Medicine 3 of dying. They have a pale complexion, weak breathing, and a large amount of foam is discharged from the nose and mouth. A gurgling sound is audible in the bilateral lungs. The heart sounds are weak, and blood pressure is decreased. The patient is also likely to have high-altitude cerebral edema. Heart failure and secondary pulmonary infection are very likely. Chest X-rays reveal flocculent shadows in unilateral or bilateral lungs, enlargement of the heart, and the pulmonary artery is particularly clear. CT scans show diffuse alveolar edema in unilateral or bilateral lungs. The number of white blood cells is > 16 × 10 9 /L.
In terms of pharmacotherapy, there is no consensus on the types of drugs, their doses, or administration routes for HAPE. Thus, it is imperative to develop guidelines to standardize and optimize the treatment of HAPE. Based on the experience of several mountain sickness treatment centers and the efficacy of currently available drugs and strategies, in combination with recent advances in the treatment of HAPE, our research group proposed four regimens for early diagnosis and treatment of HAPE and conducted a prospective, randomized, controlled study to investigate their efficacy. A total of 400 patients with HAPE were divided into four groups and treated as follows: patients in group A received oxygen inhalation, dexamethasone, and aminophylline; patients in group B-C were treated as in group A, in addition to diuresis with furosemide in group B, blood pressure-lowering with nifedipine in group C, or L-arginine in group D. The efficacy and safety of these therapeutic regimens were compared. The treatment showing greatest therapeutic efficacy was subsequently used to prepare the guidelines for the treatment of HAPE. Overall, all four regimens were effective for the treatment of HAPE [9] . However, in terms of the time to resolution of symptom and signs, the time to resolution of chest X-ray findings, and duration of hospitalization, regimen B was superior to the other regimens. Interestingly, there were no marked differences between regimens A, C, and D. Furthermore, there were no differences in the incidence of adverse effects between regimens A, B, and D, or in the liver and kidney functions between all four regimens. These findings suggest that a regimen composed of oxygen inhalation, dexamethasone, aminophylline, and furosemide is effective, well tolerated, simple to follow, and thus offers a basic, standardized regimen for the treatment of HAPE Once the regimen has been selected, timely initiation of individualized treatment is a key factor that contributes towards its therapeutic efficacy. Our experience of on-site treatment of more than 300 patients with HAPE has shown that on-site individualized treatment is feasible, without increasing the risk for death [10] . Following the Yushu earthquake, the efficacy of this regimen was further confirmed as the prognosis after on-site treatment was significantly better than that after blind evacuation, which usually results in poor outcomes.
The principles of individualized treatment of HAPE are highly dependent on the severity of HAPE [11] .
Bed rest, intermittent oxygen inhalation or subcutaneous oxygen treatment, oral aminophylline (250 mg), prednisone (10 mg), furosemide (20 mg), atropine or anisodamine (5 mg) twice daily, and other treatments for specific symptoms are recommended.
Moderate. Absolute bed rest, continuous oxygen inhalation or subcutaneous oxygen treatment are necessary. Drugs are mainly administered intramuscularly, accompanied by oral and intravenous medication. Aminophylline (250 mg), prednisone (10 mg), furosemide (20 mg), atropine or anisodamine (5 mg) three to four times daily, and other treatments for specific symptoms are recommended.
Absolute bed rest, preferably in a semirecumbent position and continuous high-flow oxygen inhalation with defoaming agents in a humidifier bottle are necessary. Drugs are mainly administered intravenously and intramuscularly. Atropine (2-5 mg/0.5 h) or anisodamine (20-40 mg/0.5 h), dexamethasone (10 mg/4 h), furosemide (40 mg/8 h), gentamicin (80000 IU/8 h); or dexamethasone (200 mg), furosemide (40 mg), atropine or anisodamine (10 mg), and gentamicin (160000 IU) in 10% glucose solution (500 mL) three times daily are recommended. Symptomatic treatments may include intramuscular morphine (10 mg) for patients with dysphoria, intramuscular cedilanid (0.4-0.8 mg) for patients with heart failure, and vitamin C, ATP solution, coenzyme A, and cytochrome C, as deemed necessary.
On-site treatment is preferred, followed by escalation therapy during evacuation once the patient's condition has stabilized. The procedures for the treatment of extremely severe HAPE are similar to those of severe HAPE, except for the treatment of complications. For patients with cerebral edema, the doses of dexamethasone and diuretics should be increased and airway maintenance is necessary. Hyperbaric oxygen treatment or oxygen inhalation through a ventilator is also recommended. Hypothermia is beneficial for the brain. For patients with serious heart failure, half or two-thirds of the recommended dose of digitalis should be administered in combination with an appropriate dose of a sedative. The daily dose of glucose should be <400 g to maintain low energy consumption and to promote osmotic diuresis. The treatments should be escalated during evacuation once the patient's condition has stabilized.
The following factors are particularly important during evacuation. (1) Mode of transport: the patient should be transported in a vehicle that can provide rapid and steady evacuation. Such transportation options include helicopter, motor truck, heavy-duty medical car, and miniambulance. The vehicle's speed should be carefully controlled, particularly in undulating conditions, to ensure the patient remains in a horizontal position and excess movement is avoided.
(2) Accompanying personnel: the patient should be ideally accompanied by one nurse and one physician, or at least one medical staff who can provide effective on-site treatment, The personnel should continuously monitor and record the patient's vital signs and perform treatment as deemed necessary. (3) Patient position: the patient should be kept in a semirecumbent position, and airway maintenance is essential. However, the supine position is recommended for patients in a coma. Head vibration and bumps should be avoided to prevent cerebral hernia. (4) For deceased patients, the time of death and the patients symptoms and signs at death should be recorded, and the body should remain in a stable position.
For patients with suspected heart failure, treatment with digitalis (e.g., cedilanid and digoxin) is recommended to improve myocardial contractility. For patients with low blood pressure, dextran and hydroxyethyl starch 40 is recommended for blood volume expansion. For patients with secondary infections, penicillin or other antibiotics can be administered. For patients with dysphoria, sedatives can be given according to the disease state.
In summary, patients with mild to moderate HAPE should receive basic treatment plus any supplementary treatment required. Patients with severe and extremely severe HAPE should also receive treatment for any symptoms. Basic treatment consists of bed rest, oxygen inhalation, and intravenous administration of aminophylline, dexamethasone, and furosemide. Supplementary treatments include antibiotics, vitamin C, and cytochrome C, for example.
Treatment of HAPE 4.3.1. Bed Rest. Physical activity may lead to contraction of pulmonary vessels resulting in an increase in pulmonary artery pressure and a decrease in arterial partial pressure of oxygen. Therefore, absolute bed rest can reduce muscular activity and contraction of blood vessels and, thus, provide stable pulmonary artery pressure. In some studies [12] , bed rest alone was used for the treatment of mild HAPE.
Oxygen inhalation can decrease the pulmonary artery pressure. Evidence shows that inhalation of 100% oxygen can significantly decrease the increased pulmonary artery pressure and is accompanied by improvements in symptoms [13] . In addition, oxygen inhalation can increase arterial oxygen saturation and improve tissue oxygen supply, thus alleviating hypoxia. For patients with severe HAPE, hyperbaric oxygen is preferred. Some researchers [14, 15] have proposed mechanical ventilation for patients with respiratory distress. Hyperbaric oxygen treatment not only increases the oxygen concentration and subsequently the alveolar and arterial partial pressure of oxygen, but also increases pulmonary ventilation and alveolar pressure, which inhibit fluid exudation. Oxygen inhalation can also rectify respiratory alkalosis, which prevents the transportation of peripheral blood to the lung.
shown that hypoxia results in reduced nitric oxide levels, a principle cause of pulmonary vasoconstriction and that inhalation of nitric oxide can reduce hypoxic pulmonary hypertension, improve the patient's ventilation to perfusion ratio, improve hypoxemia, and improve HAPE in patients with signs and symptoms, as well as decrease hospitalization time with few adverse effects. Accordingly, we suggest that nitric oxide inhalation should be considered in the treatment of HAPE. It should be administered via a nasal catheter at a dose of 10 ppm (0.001%) at a flow rate of 3-5 L/min for 30 min in normal air or in oxygen.
Aminophylline is a classic bronchodilator and has long been used in the treatment of HAPE. Aminophylline can dilate the bronchus, decrease hypoxic pulmonary hypertension, enhance diaphragm function, suppress lipid peroxidation and hypoxia-induced pulmonary vascular inflammation, enhance cardiac function and diuresis, clear bronchial mucus, and subsequently resolve moist rales. Unlike other bronchodilators, aminophylline can dilate both the bronchus and vascular smooth muscle; hence, it is preferred over other bronchodilators for the treatment of HAPE.
Anticholinergics. Some hospitals use anisodamine (654-2) instead of aminophylline for the treatment of HAPE. Anisodamine can improve pulmonary vascular spasms, decrease pulmonary vascular resistance and hypoxic pulmonary hypertension, improve the pulmonary microcirculation, and maintain smooth pulmonary blood flow. As a result, anisodamine inhibits intravascular coagulation and, thus, reduces the risk of pulmonary embolism.
Nifedipine is a calcium channel blocker that inhibits release of catecholamines from sympathetic nerve endings. Therefore, it is an effective vasodilator, reduces hypoxic pulmonary hypertension, increases arterial oxygen tension, improves the symptoms and signs of HAPE, lowers right atrial pressure, and increases cardiac output. Therefore, nifedipine targets many of the effects of HAPE.
Hypoxia can lead to redistribution of systemic blood and abrupt increases in pulmonary blood volume. Furosemide and chlorothiazide not only promote diuresis and dehydration, but also increase renal blood flow and reduce left ventricular filling pressure. Thus, blood in the lungs is transported to the periphery, improving pulmonary congestion. In addition, oral acetazolamide can play a significant role in the treatment of HAPE because it can increase urine output in patients with HAPE or fluid retention. Acetazolamide is a carbonic anhydrase inhibitor that inhibits carbonic anhydrase activity in the brain, kidney, and blood. In the kidney, acetazolamide reduces the formation of hydrogen and bicarbonate ions and Pulmonary Medicine 5 inhibits sodium bicarbonate reabsorption to increase urine output and, thus, reduce sodium and water retention. In the brain, acetazolamide inhibits brain choroid plexus carbonic anhydrase activity to reduce cerebrospinal fluid production rate and hence reduce intracranial pressure.
Dexamethasone is an analog of adrenocortical hormones that can improve the functions of capillary endothelial cells and alveolar epithelial cells, decrease pulmonary capillary permeability, protect alveolar epithelial type II cells, promote the secretion of pulmonary surfactant, increase renal blood flow, and decrease the secretion of antidiuretic hormone. Thus, dexamethasone is a key drug in the treatment of HAPE.
HAPE is a disease that is very difficult to predict. Although much research has been done in attempts to predict HAPE, no consensus has been reached regarding markers for the early prediction of HAPE. However, many studies have suggested that HAPE could be predicted based on physiological functions, molecular biology, or genetic predisposition.
Respiratory gas exchange disorders may predict serious hypoxemia and even acute mountain sickness in individuals who rapidly ascend to high altitude. Thus, monitoring arterial oxygen saturation (SaO 2 ) can be used to predict HAPE. Studies have shown that 80-100% of patients with serious hypoxemia develop HAPE. Accordingly, Roach et al. [16] speculated that noninvasive measurement of SaO 2 was a simple and convenient way to predict the onset of HAPE. Similarly, Burtscher et al. [17] suggested that measurement of SaO 2 could be used to predict the onset of acute mountain sickness. In a prospective cohort study, Shen et al. [18] found that there were significant individual differences in the changes in oxygen saturation with increases in altitude. They found that when the SaO 2 decreased by >30% at low altitude, the susceptibility to HAPE increased markedly and about 62% of the participants developed HAPE. Thus, they speculated that a decrease in SaO 2 > 30% offers a biomarker to predict susceptibility to HAPE.
Zhang et al. [19] used submaximal bench stepping to directly calculate maximal oxygen uptake (VO 2max ), which was then applied to predict HAPE. They conducted this study in soldiers ascending to high altitude. They found that soldiers with a VO 2max ≥ 3 L/min at sea level were less likely to develop HAPE than those with a VO 2max < 3 L/min. [20] measured FVC, body surface area, and thoracic volume in an effort to predict HAPE in individuals ascending to high altitude. They found that a FVC/body surface area <3 L/m 3 , FVC/thoracic volume <400 L/m 3 , lung/body index >30, and lung/thorax index >0.24 were associated with increased risk for HAPE.
Geng et al. [21] investigated the diffusion of carbon monoxide (CO) in the lung of 27 subjects who rapidly ascended from 2260 m to 4700 m. They found that CO diffusion was correlated with the development of acute mountain sickness. In 10 patients with HAPE, the diffusing capacity of CO (DLCO) increased with increasing altitude, but the amplitude of this increase was greater in subjects without HAPE. Thus, they speculated that insufficient DLCO was a cause of acute mountain sickness and that DLCO is an objective parameter to predict adaptability to high altitude and the occurrence of HAPE.
Huang et al. [22] measured urine volume within 2.5 h after participants administered 1000 mL of water at sea level in an effort to predict HAPE. They found that urine volume in subjects who developed acute mountain response was significantly lower than that in the control group. In addition, the urine volumes at 1, 1.5, 2, and 2.5 h after hydration and the total urine volume within 1.5, 2, and 2.5 h after hydration were negatively associated with AMS. They concluded that greater urine volume within 2.5 h after hydration at sea level was associated with milder symptoms of acute mountain sickness after ascending to high altitude. Therefore, urine volume after hydration might be a marker for acute mountain sickness.
Zhou et al. [23] measured the pH of blood in individuals before they ascended to high altitude, in addition to PaCO 2 , and PaO 2 . In that study, the blood pH was significantly associated with susceptibility to HAPE, and there was a significant difference in blood pH value between subjects who developed HAPE and without HAPE. They found that during intense physical labor following a rapid ascent to 4000 m, the likelihood of developing acute mountain sickness was significantly greater among individuals with blood pH > 7.45 than among those with blood pH < 7.45. However, they found no significant differences in PaCO 2 or PaO 2 between these two groups of subjects. Thus, they concluded that blood pH, but not PaCO 2 or PaO 2 , could be used as a predictor of HAPE.
Zhou et al. [24] investigated the ventilatory functions in 113 newly recruited soldiers at sea level, and then the soldiers were transferred to 3658 m by airplane. Two and three days after ascending to high altitude, the investigators scored the symptoms of acute mountain sickness and evaluated the relationship between ventilatory functions and symptom scores. They found that FVC, peak expiratory flow, and forced expiratory volume in 1 second in soldiers with HAPE were significantly lower than those in soldiers without HAPE. Thus, they speculated that ventilatory functions at sea level could be used to screen subjects susceptible to HAPE. [25] investigated whether a cardiopulmonary exercise test could be used to predict HAPE. They found that subjects with poor cardiac and ventilatory responses to hypoxia were more susceptible to HAPE. They assessed several parameters, including the heart rate to oxygen saturation ratio (ΔHR/ΔSaO 2 ) and the minute ventilation to oxygen saturation ratio (ΔVE/ΔSaO 2 ), to establish a mathematical model that could predict HAPE.
They established a discriminant model based on Z = b 1 X 1 + b 2 X 2 , in which coefficients b 1 = 0.00769, b 2 = 0.0810, and Z 0 = 0.472 (discrimination threshold). In this model, Z > Z 0 is defined as non-AMS and Z < Z 0 is defined as AMS. The discriminant model had a high sensitivity (93.9%) and a high accuracy (92.2%) for the prediction of HAPE.
Endocrine parameters may also be used to predict HAPE. For example, Cai and Yang [26] used radioimmunoassays to measure the blood levels of 18 endocrine parameters, including thyroid stimulating hormone, adrenocorticotropic hormone, and corticotropin hormone, and found that plasma cortisol <20 µg/L and urine 17-hydroxy corticosteroid (17-OHCS) levels <17 µmoL/dL could predict HAPE with an accuracy >95%.
Luo et al. [27] [28] [29] compared the sequences of the mitochondrial genome in HAPE patients and rats at high altitude versus sequences from healthy individuals at high altitude and animals at sea level, and identified single-nucleotide polymorphisms in nine genes in mitochondrial DNA (T6680C, C3970T, G3010A, A13497G, c15508T, G4164G, G1598A, C16111T, and T7684C). All of the affected genes encoded proteins controlling mitochondrial electron transport. Based on singlenucleotide polymorphism of mitochondrial DNA T6680C, DNA C3970T, and DNA G3010A, they developed a kit that can be used to predict susceptibility to HAPE. This kit can be used to screen subjects for susceptibility to HAPE before ascending to high altitude. In another study, Qiu et al. [30] compared genetic parameters of patients with HAPE, as well as people living in Tibetan and Han Chinese migrants. They found that human leucocyte antigen (HLA) DR6, particularly DR6(1402), was associated with increased susceptibility to HAPE. Thus, they speculated that susceptibility to HAPE was not only related to genetic factors but was also influenced by some susceptibility genes.
Prevention of HAPE, as well as its associated symptoms and signs, is important to reduce its incidence among those ascending to high altitude. To achieve this, the following factors are particularly important. (1) Maintain body warmth with appropriate insulation and avoid upper respiratory tract infection. Several days before ascending to high altitude, bathing should be avoided to reduce exposure to cold temperatures. (2) Avoid fatigue. Physical activity at high altitude should be performed at an intensity of no more than 60-80% of that at sea level and duration of activity should be <6 h. Similarly, the body must be given sufficient time to rest both before and during the time at high altitude.
(3) Stop alcohol intake. Fifteen days before ascending to high altitude, the consumption of alcohol should be stopped. (4) Emotional stress. Unwanted emotional tension should be avoided, while optimism and aggressiveness should be maintained. (5) Patients with upper respiratory tract infection should be carefully monitored before and during the time at high altitude. However, delaying the ascent until the infection has been fully resolved is preferable. (6) Oxygen must be available at high altitude, either by maintaining high oxygen levels in living quarters or by supplying compressed oxygen tanks for use during physical activity. (7) Food and water sanitation. Diets containing high levels of carbohydrates and protein and low levels of fat are recommended. The intake of vitamins can also be increased. (9) The use of drugs such as Rhodiola rosea, Zangtianlu, Codonopsis compound tablets, Shenqi pollen tablets, and acetazolamide during the time at high altitude can also reduce the risk of HAPE.
For people working at high altitude, such as construction workers, preventative measures are particularly important. As outlined above, it is essential to provide sufficient nutrients, rest, and insulation against the cold, as well as to limit labor intensity and duration of labor to avoid fatigue. Shift-work is also recommended and may entail 20-30 days of work at high altitude followed by a period of rest at low altitude to provide more effective resting conditions. Shift-work also reduces the incidence of chronic mountain sickness induced by long periods of physically demanding work at high altitude. It is also essential to ensure there is an adequate medical team to enable early detection, diagnosis, and treatment, should mountain sickness of HAPE occur.
Finally, individuals planning on ascending to high altitude should receive education regarding the prevention and treatment of HAPE and take appropriate measures [31] to decrease the risk of developing HAPE: maintain a positive attitude after ascending to high altitude take measures to prevent HAPE, and consider the possible treatments be optimistic but do not fear high altitude maintain body temperature avoid hyperphagia stop smoking and alcohol consumption conduct appropriate excise but avoid strenuous exercise and fatigue do not worry about trouble sleeping but try to maintain good sleep quality pay attention to cough, particularly cough event of bloody sputum and consider seeking treatment consider bed rest and oxygen inhalation be aware that headache, vomiting, and unsteady walking are symptoms of HAPE and should be checked by a physician; treatment should be started as soon as possible, even if the symptoms are mild, as timely and comprehensive treatment favors positive outcomes. Noninvasive ventilation in acute respiratory failure due to H1N1 influenza: A word of caution   Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (∼10(11) viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January. Western honey bees (Apis mellifera) are highly social insects that live in colonies of ,30,000 individuals [1, 2] . Honey bees are essential pollinators of agriculturally important crops including apples, almonds, alfalfa, and citrus. Current agricultural practices, such as large-scale monocultures, demand a seasonal abundance of honey bees in geographic locations incapable of maintaining sufficient pollinator populations year-round. Migratory beekeeping operations fulfill this need. For example, each February in the Central Valley of California 1.3 million honey bee colonies (,50% of the U.S. honey bee population) are required for almond pollination [3, 4, 5] . Pollination of this and other U.S. crops is valued at ,$15 billion annually [5] .
There are numerous threats facing honey bee populations and the recent losses of honey bee colonies in the United States, Canada, and Europe is alarming. In the U.S., annual honey bee colony losses increased from 17-20% to 32% during the winter of 2006/07 with some operations losing 90% of their hives [6] .
Average annual losses have remained high, averaging 32.6% from 2007-2010 [6, 7, 8] . One factor contributing to increased losses is Colony Collapse Disorder (CCD), an unexplained loss of honey bee colonies fitting a defined set of criteria [9, 10] . While factors such as pesticide exposure, transportation stress, genetic diversity, and nutrition affect colony health, the most significant CCDassociated variable characterized to date is increased pathogen incidence [10] . Although greater pathogen incidence correlates with CCD, the cause is unknown in part due to insufficient knowledge of the pathogenic and commensal organisms associated with honey bees [10, 11] .
Parasitic threats to honey bee colonies include viruses, Nosema, bacteria, and Crithidia. The majority of honey bee infecting viruses are positive-sense single-stranded RNA viruses of the Picornavirales order. They include acute bee paralysis virus (ABPV) [12] , black queen cell virus (BQCV) [13] , Israeli acute bee paralysis virus (IAPV) [14] , Kashmir bee virus (KBV) [15] , deformed wing virus (DWV) [16] , sacbrood virus (SBV) [17] , and chronic bee paralysis virus (CBPV) [18] (reviewed in Chen and Siede, 2007 [19] ).
Several DNA viruses that infect honey bees have also been described [19] . Viral infections in bees can remain asymptomatic, or cause deformities, paralysis and/or death [19, 20] . Symptoms associated with specific viruses include wing deformities (DWV), hairless, dark, shiny bees (CBPV), swollen yellow larva and/or dark-brown larva carcasses in the cells of worker-bees (SBV) or queen-bees (BQCV), however accurate diagnosis requires molecular biology techniques as asymptomatic bees frequently test positive for one or more viruses [19, 21, 22, 23] . In addition to viral infections, honey bees are also readily parasitized by the microsporidia Nosema [19, 24] . Historically U.S. honey bees were predominantly infected by Nosema apis, but recently Nosema ceranae infections dominate [24, 25] . The effects of Nosema infection on individual bee and colony health are unclear [24, 26] . Some reports suggest infections decrease longevity and may lead to collapse [27, 28, 29] , but since Nosema is widespread and often detected in healthy colonies its role in colony health requires further investigation [10, 26, 30] . Another fungal pathogen Ascophaera apis, the causative agent of Chalkbrood disease, kills infected larvae, but does not typically cause colony loss [31, 32] . Bacterial pathogens of honey bees include Paenibacillus larvae and Melissococcus plutonius, the causative agents of American and European Foulbrood disease [33, 34, 35, 36] . In addition to microbial infections, mite infestation (Ararapis woodi, Tropilaelaps sp., and Varroa destructor) also weakens and kills honey bee colonies [37, 38] . Introduction of V. destructor mites, which feed on the hemolymph of developing honey bees and transmit viruses (DWV, KBV, IAPV), in the late 1980s was devastating to the U.S. honey bee population [39, 40, 41] . Notably, the restricted genetic diversity of the U.S. honey bee population may make it particularly susceptible to catastrophic and episodic losses [42, 43] .
To gain a more complete understanding of the spectrum of infectious agents and potential threats found in commercially managed migratory honey bee colonies, we conducted a 10-month prospective investigation. Our broad-scale analysis incorporated a suite of molecular tools (custom microarray, polymerase chain reaction (PCR), quantitative PCR (qPCR) and deep sequencing) enabling rapid detection of the presence (or absence) of all previously identified honey bee pathogens as well as facilitating the detection of novel pathogens. This study provides a comprehensive temporal characterization of honey bee pathogens and offers a baseline for understanding current and emerging threats to this critical component of U.S. agriculture.
Following devastating losses suffered by U.S. commercial beekeeping operations in 2006-2007, we initiated a prospective study monitoring a typically managed, large-scale (.70,000 hives), migratory commercial beekeeping operation over 10-months. Honey bees from 20 colonies were consistently sampled beginning with the introduction of a new queen in April 2009 (Mississippi (MS), through transport to summer foraging grounds in South Dakota (SD), and transfer to California (CA) for almond pollination (Figure 1 ). During our study, these colonies were exposed to antimicrobial treatments, transportation stress, different pollen and nectar sources, and three distinct geographic locations: MS, SD, and CA, (U.S.A.).
A molecular analysis pipeline consisting of custom microarray, polymerase chain reaction (PCR), quantitative PCR (qPCR) and ultra deep sequencing was employed to characterize the honey bee microbial flora. Pathogen screening was performed using the ''Arthropod Pathogen Microarray'' (APM) built on the same design principles used for human pathogen microarray screening [44, 45] . The array's design couples highly-conserved nucleic acid targets with hybridization-based detection to identify previously uncharacterized organisms [46, 47, 48, 49, 50, 51] . Specifically, the APM was designed to detect virtually all known microbial parasites of insects. Endpoint PCR provided sensitive detection while qPCR documented abundance of select pathogens. Ultra deep sequencing facilitated the discovery of novel and highly divergent microbes. Together the results from our monitoring study provide insight regarding the incidence of virus and microbe infections in honey bee colonies.
The Arthropod Pathogen Microarray (APM) is a custom DNA microarray capable of detecting over 200 arthropod associated viruses, microbes, and metazoans. This DNA microarray includes oligonucleotides representing every arthropod-infecting virus with published nucleic acid sequence in the International Committee on Taxonomy of Viruses database as of November 2008 [52, 53] . Design principles used for APM oligonucleotides (70-mers) were based on previous pan-viral microarrays using ArrayOligoSelector (AOS) [54] . In addition, non-viral pathogens including, Nosema (microsporidia), Crithidia (trypanosomatid), Varroa (mite), Tropilaelaps (mite) and Acarapis (tracheal mite) as well as Paenibacillus larvae and Melissococcus plutonius bacterial species [53] were represented on the microarray (Table 1 ). This new diagnostic tool is composed of 1536 oligonucleotides, including viral, non-viral and positive control targets (Table 1 ). Array analysis is performed computationally using e-predict [54, 55] . The sensitivity of the APM was estimated to be 1.9610 5 viral genome copies (1 pg Drosophila C virus in vitro transcribed genomic RNA) in an A. mellifera RNA (1 mg) background (see Materials and Methods). Array specificity was confirmed by performing pathogen-specific PCRs in conjunction with nucleic acid sequencing. Test samples included honey bees from managed and feral colonies, Vespula sp. (yellow jackets), and Bombus sp. (bumble bees) (Table S1) . A sample from a collapsed colony in Montana tested positive for the highest number of viruses (BQCV, DWV, KBV, IAPV) and documented the array's ability to simultaneously detect multiple pathogens. Analysis of symptomatic honey bees, such as hairless, shiny bees and bees with deformed wings, confirmed the presence CBPV and DWV, respectively [56, 57] . Likewise, analysis of Varroa destructor RNA validated the array's ability to detect mites and their associated viruses (DWV). Interestingly, pathogens normally associated with honey bees, DWV and ABPV, were also detected in a yellow jacket sample (Vespula sp.) obtained near a hive entrance from which the honey bees also tested positive for ABPV and DWV. We used the APM to detect several pathogens (BQCV, DWV, SBV and Nosema) in CCD-affected colony samples from an Oklahoma based migratory beekeeping operation (Feb. 2009). In total we detected and sequence confirmed ten previously characterized honey bee pathogens using the array including: CBPV, IAPV, DWV, ABPV, BQCV, SBV, KBV, Nosema apis, N. ceranae and Varroa destructor.
Honey bee samples were collected during their travels from Mississippi through South Dakota to California resulting in a prospectively collected 10-month time-course of 431 data points, each consisting of 50-100 bees isolated separately from both the entrance (older foragers) and brood comb (younger house bees). Hives # 10, # 14 and # 19 were lost in December due to queen death or infertility. We analyzed all the entrance samples (5 bees per colony each time-point) using the APM.
Nosema. There was an abundance of Nosema infections in our monitor colonies throughout the entire time-course. APM monitoring revealed that approximately half of the colonies in April and May were Nosema positive ( Figure 2A) . Notably, nearly every colony was infected during a surge in August and September. In order to determine which Nosema species was responsible for infections, each hive was analyzed at a single timepoint per month by species specific PCR. In April and May, N.apis was predominant whereas in June, July, and October through December, N. ceranae was exclusively detected ( Figure 2B ). During the highest incidence of Nosema (August-September), 75% of all colonies were infected with Nosema ceranae and less than 25% with Nosema apis, most of which were co-infected with N. ceranae. Quantitative-PCR data from pooled monthly samples confirmed that Nosema ceranae was prevalent throughout the time-course and peaked in August ( Figure 2C ). While seasonal variation may play a role, an anti-fungal (Fumagillan) was used to abrogate Nosema infection [58] and may be responsible for the observed decrease in Nosema abundance from November to January ( Figure 2 ). Viruses. The APM readily detected common honey bee viruses in samples collected throughout the time-course. In total, we report 69 virus incidences in 63 of 431 total samples ( Figure 3 ). Overall virus incidence was sporadic, which we attribute to cycles of acute infection in predominantly healthy monitor colonies. The majority of infections occurred during July, August, and September when the monitor colonies were in South Dakota. The most prevalent virus infections observed during our 10-month study were SBV, BQCV and ABPV; however the frequencies of specific viruses were insufficient for statistical tests regarding pathogen association (see Materials and Methods). Other viruses including DWV, IAPV, and KBV were infrequently detected in the latter half of our time-course. A total of six double virus infections were observed, frequently involving ABPV or SBV. There were only two cases in which the same virus (BQCV) was detected in consecutive time points from a particular monitor colony ( Figure 3A Hives #4 and #6). Typically a single virus was detected in multiple colonies at a given time-point and these infections did not persist. For example, there were waves of SBV infection in April and January and of BQCV in July and early August ( Figure 3A ). qPCR analysis of pooled monthly samples confirmed and extended APM findings. BQCV, SBV and ABPV levels peaked in mid-summer to early fall at 6.6610 9 -8610 10 genome copies per bee ( Figure 4 and Figure S1 ), consistent with previously characterized levels of these viruses [57, 59, 60] .
Ultra deep sequencing, discovery of novel viruses. A summer South Dakota time-point (August 5, 2009) was selected for deep sequencing due to high Nosema load and the presence of several common honey bee viruses, including ABPV, BQCV and SBV. All expected microbes (Nosema ceranae, Crithidia mellificae) and viruses were detected [ABPV (39,352 reads aligned by BlastN evalue,1610 27 ), BQCV (2,868 reads) and SBV (4,414 reads)]. In addition, we detected Spiroplasma sequences (70,407 reads) consistent with the presence of both Spiroplasma apis and S. melliferum (66 reads and 44 reads aligning to the RNA PolB gene of each, respectively).
Four distinct novel viruses were discovered via deep sequencing. Paired-end sequencing reads (2663 nt) of unknown origin were screened by tBlastx [61] against all known insect viruses present in GenBank [62] . Screening hits with an e-value greater than 1610 23 were used to target de novo contig assembly using the complete data set. Short contigs were screened by tBlastx against the non-redundant nucleotide database (NR) at an e-value threshold of 1610 25 . Hits to viral sequence, but not host sequences, were further assembled (see Materials and Methods). In each case, PCR primers were initially designed to bridge or confirm assembled contigs by Sanger sequencing. Confirmed contigs were extended with the PRICE assembler package (see Materials and Methods). In total, sequences from four novel viruses were recovered and Sanger validated. These include two members of Dicistroviridae, and two RNA viruses distantly related to Nodaviridae.
Investigation of contigs aligning to the Aphid Lethal Paralysis Virus genome, in the family Dicistroviridae, recovered a 4,125 nt contig (GenBank Q871932) spanning the RNA-dependent RNA Polymerase (RdRp) gene, the internal ribosome entry site (IRES) structure and the capsid coding region. The recovered sequence aligned with 83% nucleotide and 89% amino acid identity to the canonical ALPV genome over the RdRp gene. The two viruses shared 97% nucleotide homology along 171 nt of the IRES. The high sequence similarity between this new isolate and canonical ALPV makes it unclear whether this is a novel species or a new strain of ALPV. Regardless, ALPV has not previously been reported in association with honey bees. We propose the designation ALPV strain Brookings (after the SD county from which the virus was isolated). Specific PCR primers were designed for the Brookings strain and used to analyze additional time-course samples, resulting in detections on thirty distinct occasions, including in Mississippi, South Dakota and California. Incidence peaked in May, when 7 out of 20 hives were infected, whereas maximum abundance occurred in August albeit at a relatively low level, 4.42610 4 copies per 100 ng of RNA sample (approximately 2.21610 7 copies per bee), as compared to previously characterized honey bee viruses (Figures 3 and 4) . Frequent detection of ALPV strain Brookings throughout the time-course from multiple geographic locations suggests that this virus is not simply a ''passenger'' obtained from forage (nectar and pollen) shared with other insects. However, further investigation is required to determine whether ALPV strain Brookings is a honey bee pathogen.
Big Sioux River virus. A second novel dicistrovirus, designated Big Sioux River Virus (BSRV) after its place of discovery, is most similar to the Rhopalosiphum padi Virus (RhPV). Four contigs of size 1473, 861, 1164 and 1311 nt (GenBank JF423195-8) derived from the non-structural region, the IRES, and the capsid gene. BSRV shares low amino acid identity with RhPV; only 78% in the non-structural region and 69% in the capsid gene. This level of amino acid divergence is consistent with the taxonomic rank of a new species ( Figure S2 ). Twenty-eight incidences of BSRV were detected from 197 time-course samples by specific PCR with most individual colony detections occurring in samples collected from April to July 2009 in Mississippi and South Dakota. Incidence was low from October onwards ( Figure 3B ). Peak abundance was 7.64610 3 copies per 100 ng of RNA sample (approximately 3.8610 6 copies per bee) and occurred in August ( Figure 4 ). Of note, BSRV associated significantly with Nosema apis infections (p = 0.003, OR 6.0) and also with ALPV-Brookings (p = 0.014, odds ratio (OR) = 4.5).
Lake Sinai Virus strain 1 and 2. Three contigs had significant alignment to chronic bee paralysis virus (CBPV) and members of the family Nodaviridae. Both the individual reads and our initial contigs were further assembled and extended using the complete data set (see Materials and Methods). Two separate contig sequences (,5.5 kb each) were generated by de novo assembly. Both contigs were confirmed by specific PCR and Sanger sequencing. The first contig represents a novel RNA virus that we designate Lake Sinai virus (LSV1) (HQ871931), after Lake Sinai in Brookings County, South Dakota. The second contig also represented a related, yet divergent (71% nt identity), RNA virus which we designated Lake Sinai virus 2 (LSV2) (HQ888865). The 59 end of LSV1 was determined by RACE (rapid amplification of cDNA ends). The 59 end of the LSV2 assembly was within 57 nt of the LSV1 RACE results [18, 57] . The 39 ends of both viruses were refractory to traditional RACE methods and attempts at 59 RACE on the negative strand were also unsuccessful. Denaturing gel electrophoresis coupled with Northern bot analysis using three distinct LSV-specific probes indicated that only the monopartite genome and no subgenomic RNA species were present ( Figure S4 ). Both LSV genomes display similarities to the RNA1 molecule of chronic bee paralysis virus (CBPV) with predicted open reading frames (ORFs) of similar size and arrangement with the notable exception that LSV1 and 2 ORFs are contained on a single RNA rather than in the bipartite configuration of CBPV [18, 57] (Figures 5B and Figure S4 ). LSV1 and 2 possess the Orf1 gene, which is of unknown function, with predicted products (of 847 and 846 aa) previously unique to CBPV (853 aa). The Orf1 genes of LSV1 and CBPV share minimal (18%) amino acid identity. All three viruses encode an RdRp that partially overlaps and exists in a frame shift with respect to Orf1 [18] . Both LSVs possess a triple stop codon within 10 residues of the end of the Orf1 gene whereas CBPV has two adjacent stop codons. The RdRp genes are considerably more conserved with 80% identity between the two LSV strains and 25% amino acid identity between them and CBPV. Both LSV RdRp genes have the DxSRFD and SG amino acid motifs in the NTP binding pocket (residues 375-380 and 436-437 in LSV1) conserved between the families Nodaviridae,Tombusviridae and CBPV. An amino acid phylogeny of the Nodavirales superfamily RdRp places the LSV strains on the same branch as CBPV, and separated from the larger Nodavirus and Tombusvirus families ( Figure 5A ).
As previously noted, the capsid protein of LSV1 and 2 is encoded on the same RNA as Orf1 and the RdRp unlike that of CBPV, which possesses a bipartite genome ( Figure 5B ). The capsids of LSV1 and 2 have significant profile similarity to the capsid gene of Nudaurelia capensis beta-tetravirus by HHpred [63] (evalue 1.0610 226 ) and they exhibit weak direct protein alignment by Blastx (e-value 1.0610 204 ). Similarity to tetravirus capsid genes consistently outranked similarity to CBPV or nodavirus capsids by these methods. Tetraviruses are not close relatives of the Nodavirales superfamily, although Betatetraviruses have a similar monopartite genome organization to LSV ( Figure 5B ). LSV1 and 2 share 70% amino acid identity over the capsid. The LSV1 capsid overlaps the RdRp gene in the +1 reading frame for 125 nt before ending in a pair of stop codons (separated by two residues). The LSV2 capsid is in frame with the RdRp and separated by 18 nt without a redundant stop codon.
Seven of twenty hives sampled on August 5, 2009 were positive for LSV1 and an additional five hives in the time-course, from July (SD) and January/February (CA) were found to be positive for LSV1, all with greater than 95% nucleotide identity. LSV2 was more prevalent and was detected by PCR in 30 of 197 time-course samples from all three geographic regions. LSV2 incidence surged in April, July and January during which over a third of all 20 monitor hives were infected. Strain specific qPCR demonstrated high abundance ($2610 6 copies per 100 ng RNA) of both LSV strains in our monitor colonies throughout the majority of the time-course ( Figure 4 ). LSV1 copy number peaked in July, at 1.39610 8 copies per 100 ng of RNA sample (approximately 7.0610 10 copies per bee). Notably, LSV2 was the most abundant virus detected in this study (,10 11 copies per bee). Copy number peaked in both April and January, at 7.22610 8 copies per 100 ng of RNA sample (approximately 3.61610 11 copies per bee) and 1.42610 9 copies per 100 ng of RNA sample (approximately 7.1610 11 copies per bee), respectively.
Positive sense RNA viruses, like LSV 1 and 2, utilize a negative strand template to produce viral genome copies, therefore detection of the negative-strand intermediate is indicative of an actively replicating infectious virus [39, 64, 65] . We used negativestrand specific RT-PCR to detect the replicative forms of both LSV1 and LSV2 ( Figure S5 ). cDNA synthesis reactions were performed using tagged negative strand-specific LSV1 and 2 primers followed by exonuclease I digestion of excess unincorporated RT-primers [65] (Materials and Methods and Table S2 ). PCR amplification using a tag-specific forward primer and LSVspecific reverse primers confirmed the presence of the replicative forms of both LSV1 and LSV2 in the July RNA sample ( Figure  S5 ). Together, this data and the abundance of LSV1 and 2, compared to other significant honey bee viruses, suggests that LSV1 and LSV2 are novel honey bee viruses that may play significant roles in colony health.
Crithidia mellificae. The broad scope of our microarray platform enabled identification of an unexpected microbe, Crithidia mellificae, in our time-course samples ( Figure 6 ). Given that Crithidia bombi is a bumble bee pathogen and trypanosomatids were previously described in honey bees [9, 66, 67] , 5 unique oligonucleotides each from Crithidia oncopelti and C. fasciculata rRNA sequences were included on the microarray. Oligonucleotides from these two distantly related organisms were predicted to hybridize to all other Crithidia species with published sequence [53] . Three oligonucleotides and their reverse complements derived from Crithidia oncopelti were repeatedly detected in samples throughout the time-course. Pilot Sanger sequencing of randomly amplified genomic DNA from a honey bee intestinal sample yielded a 121 base-pair (bp) stretch of the kinetoplast minicircle with 74% homology to the Crithidia fasciculata kinetoplast (BlastN evalue = 3.5610 28 ). Specific PCR retrieved 593 nt of the GAPDH gene to confirm phylogenetic placement [68] .
We sought to further characterize this parasite by microscopy, PCR, culturing and DNA sequencing. Honey bee intestines were dissected in a sterile environment from which Crithidia mellificae was cultured. Light microscopy of these parasites enabled visualization of the flagella and motility ( Figure 6 ; Figure S6 and Figure S7 ). Fixed sample imaging facilitated DAPI visualization of the kinetoplast DNA, as well as nuclear DNA ( Figure 6 ). Previous studies describing trypanosomatids in honey bees lacked DNAsequencing data with the exception of Cox-Foster et al. (2007) who published a 715-nt sequence of 18S ribosomal RNA that was too conserved between trypanosomatids for precise taxonomic assignment [9] . Together, the features observed by microscopy (flagella and kinetoplast) and phylogenetic analysis unambiguously identify this species taxonomically. We have deposited the GADPH sequence (JF423199) for future molecular identification, and genomic sequencing of C. mellificae is underway.
In order to specifically monitor Crithidia mellificae, additional oligonucleotides complementary to the C. mellificae rRNA and kinetoplast sequence were designed and included on the APM beginning in October 2009. These additional oligonucleotides Figure 6 ). In addition, we screened samples throughout the time-course (April 2009-Jan. 2010) by PCR and qPCR specific to the C. mellificae rRNA gene. C. mellificae infection was detected by PCR at every time-point and in turn from every geographic location sampled in our study (MS, SD and CA). Likewise, C. mellificae was readily detected in pooled monthly RNA samples by qPCR throughout the year ( Figure 6C ). In contrast to BQCV, SBV, ABPV and Nosema ceranae, which exhibited peak levels in late summer and early fall, peak trypanosomatid levels occurred in January 2010. Despite this, C. mellificae infections statistically associated with N. ceranae infections (Chi Square p = 0.004, OR = 3.1). C. mellificae was also detected in numerous hobbyist and study hives in the San Francisco Bay Area (CA), as well as samples from a CCD-affected apiary in Oklahoma, indicating wide geographic distribution (Table S1) .
Spiroplasma melliferum and S. apis. Spiroplasma, a close relative of the genus Mycoplasma, are bacterial parasites that have been implicated as pathogens of insects, vertebrates and plants. Strains of spiroplasma similar to flower-associated parasites were identified as a pathogen of honey bees in France, Spiroplasma apis [69] , and the United States, Spiroplasma melliferum [70] . Pilot Sanger sequencing of a pooled honey bee sample (August 2009) identified an rRNA-derived sequence from a Spiroplasma. Pan-spiroplasma and pan-mycoplasma PCRs targeting the 16S rRNA gene detected sporadic infections over most of the time-points and a surge of 9 infections in August and 6 infections in September [71] . Sequence data indicates that these isolates have high homology to previously identified spiroplasma isolates (.98% nucleotide identity). Spiroplasma infections had strong associations with N. ceranae (Chi Square p = 0.015, OR = 7.2) and C. mellificae (p = 0.000076, OR = 16.3), however this may be an artifact of the short surge of Spiroplasma coinciding with a period of high Nosema load.
Phorid fly (Apocephalus borealis). Apocephalus borealis, phorid flies, have previously been associated with bumble bee parasitism [72] and have recently been described as a parasite of honey bees in the San Francisco Bay Area [73] . Phoridae family members (e.g. Pseudacteon sp.) are well-characterized parasites of ants and other insects. These flies lay eggs inside the insect hosts, which are in turn consumed by the larvae during development. Although, A. borealis parasitism of honey bees is uncommon, we analyzed our time-course samples for the presence of phorid rRNA by PCR. Pooled monthly samples were weakly positive for Apocephalus borealis in December and January ( Figure S3 ) and two individual hive samples produced robust amplicons. We sequenced PCR amplicons from two individual (September 2009 Hive # 7 and October 2009 Hive # 10) and one pooled-monthly (December 2009) samples and determined that the phorid rRNA sequences from our time-course shared 99% similarity to honey bee-parasitizing phorids captured in San Francisco. This is the first report of phorid flies in honey bee samples outside of California and thus expands their known geographic range (SD, CA), although the range A. borealis as a bumble bee pathogen extends across North America [74] .
The importance of honey bees to global agriculture and the emergence of CCD calls for increased longitudinal monitoring of infectious processes within honey bee colonies. The data presented herein represent the finest resolution time-course of honey bee associated microbes to date. We demonstrate the utility of an arthropod pathogen microarray (APM) for simultaneous detection of numerous pathogens and the power of ultra deep sequencing for viral discovery. Several previous studies examined honey bee samples from diseased or CCD-affected and healthy colonies [9, 10, 20, 75, 76] , but few have temporally monitored multiple pathogens [60, 77, 78] . Although these studies differed in sampling strategy, geography, colony management (e.g. migratory commercial versus stationary hobbyist, chemically treated versus organic), and pathogen monitoring technology (e.g. serology, PCR, spore counts, microarray) they provide a framework for our surveillance of previously characterized honey bee pathogens.
Nosema infection was prevalent in our 20 monitor colonies. N. ceranae was the predominant species. N. apis was detected in individual colony samples in April (Mississippi) and May (South Dakota), but was undetectable in pooled monthly samples, indicating relatively low levels. N. ceranae abundance peaked in early-spring and late-summer. Lower N. ceranae levels from November to January likely reflects antifungal (Fumagillan) treatments applied in the fall, but may also represent natural seasonal variation [58] . In comparison, another U.S.-based (Mississippi, Arkansas) study, which calculated Nosema levels using qPCR of genomic DNA calibrated to spore counts, also reported overall dominance by N. ceranae, but higher Nosema levels in November 2008 as compared to March 2009 [79] . Nosema spore count data from non-CCD and CCD-affected colonies in California and Florida was not significantly different and approximately 50% of the colonies assayed were infected [10] . Data from European studies indicate varying prevalence of N. apis and N. ceranae [25, 29, 80, 81] . For example, a retrospective analysis of honey bee samples from Spain, Switzerland, France and Germany indicated peak levels of Nosema (presumably N. apis) in early spring and mid-winter from 1999 to 2002, whereas from 2003 to 2005 Nosema incidences remained relatively high throughout the year, a result the authors attribute to increased prominence of N. ceranae associated with recent increased bee losses [29] . In contrast, a recent (2005-2009) time-course study in Germany demonstrated greater Nosema incidence in the spring, detected N. apis more frequently than N. ceranae, and found no correlation between colony loss and Nosema infection [80] . Variable Nosema species prevalence and abundance at both the apiary and individual colony level indicate that standardized, molecular biology-based monitoring of large sample cohorts is required in order to understand the dynamics of Nosema infection, which are likely influenced by multiple factors including host genetic variation, climate, exposure levels, and treatment regimes [79, 82] . Recently, higher levels of Nosema bombi were detected in North American bumble bee species experiencing population decline [83] . Although, like CCD, the causes of bumble bee decline are complex and not fully characterized, this report underscores the importance of further characterizing the epidemiology and pathogenicity of Nosema.
We monitored the incidence of all known honey bee viruses, discovered 4 new honey bee associated viruses, and quantified the relative abundance of select viruses in time-course samples. Overall, no chronic infections of previously characterized honey bee viruses were observed and our data suggest that healthy colonies are undergoing constant cycles of viral infection. The most prevalent, previously characterized viruses in our study were BQCV, ABPV and SBV. The peak incidence of BQCV (25%) occurred in July, whereas ABPV (6.3%) and SBV (12.5%) peaked in August. Summer peak virus incidence was also reported in a PCR based honey bee virus (BQCV, ABPV, and SBV) survey of 36 geographically distributed apiaries in France (BQCV, ABPV, DWV, SBV, CBPV, KBV) [78] , a qPCR time-course study of 15 colonies in England (BQCV and ABPV) [60] , and an unpublished East-coast U.S. based survey (BQCV) [19] . Another virus, invertebrate iridescent virus-6, claimed to be associated with CCD and prevalent (75%) in healthy colonies but not supported in subsequent analysis [84, 85] , was never detected by the APM (n = 431), end-point PCR (n = 197), or in any of the 20 samples that were deep sequenced [86] .
Seasonality of specific pathogens in our time-course study representing 2,155 individual bees from 431 samples varied, although many including BQCV, APBV, SBV, Nosema, exhibited reduced June and peak August levels. Peak incidences of these organisms in the spring and late summer are likely attributable to increased brood rearing [19, 78, 87] and foraging during these seasons [88] . Increased brood rearing during the summer, results in a greater number of bees capable of transmitting pathogens to other members of the colony living in very close proximity [19] . Honey bee viruses are transmitted vertically via infected queens and horizontally via the oral-fecal route or through the exoskeleton [19, 21] . Foraging activity also increases pathogen exposure [88] and may also stress the bees so that inapparent infections reach detectable levels. Although other sources of stress, such as transportation and poor nutrition, are hypothesized to increase pathogen levels [10] , these factors were minimal during the summer when the monitor colonies were stably situated in South Dakota foraging on diverse pollen and nectar sources, including alfalfa (Medicago sativa L.), sweet clover (Melilotus spp.) and a variety of other flowering plants in June with increasing availability of corn (Zea mays ssp.) and soybean (Glycine max) pollen later in the summer. Notably, these colonies were part of a typically managed commercial beekeeping operation and therefore received nutritional supplements, protein paddies and sugar syrup throughout the year (Materials and Methods). Adequate monitor colony nutrition may have played an important role in the rapid virus clearance observed in our study. Although further experimental validation is needed, recent work examining the effects of nutrition on DWV titer in caged-bee studies demonstrated that viral titer was reduced by pollen and protein supplementation [89] . In addition, anti-mite and antimicrobial treatments in the spring and late-fall may have accounted for the lower pathogen levels at those times of year and in turn for the relatively high levels during the summer (Materials and Methods). We did not observe either increased incidence or abundance of any of the microbes and viruses monitored in our study after long distance transport.
Although several monitor colonies were lost (n = 3; one unfertile (drone laying) queen, two queen-less colonies) and many (n = 8) had fewer than 6 frames of bees in February 2010, none exhibited CCD characteristics and none of the numerous viruses and microbes we surveyed correlated with the weak colonies. Interestingly, our sample cohort had very few incidences of IAPV and DWV. IAPV, a virus that has received much attention due to its correlation with CCD-affected samples in an early study [9] , although not in a subsequent expanded study [10] , was detected in our monitor hives in December. The colonies in our study cleared or reduced IAPV infection to levels below detection within one week, indicative of a mild infection (Figure 3 ). IAPV infection has been shown to cause paralysis and death in mini-colony and cage studies [14, 90] , although its role in CCD is unclear [10, 91, 92] . Likewise, DWV incidence in our time-course samples was very low (0.7%) and presumably cleared rapidly. In contrast, a French timecourse documented increased DWV incidence throughout the year (spring 56%, summer 66%, autumn 85%) [78] and two U.S. studies also report high DWV incidence [19, 76] . Our results are not indicative of poor DWV detection by the array or our sampling strategy, since DWV was detected in both entrance and interior samples from other colonies. In addition, DWV-specific PCR of pooled monthly time-course samples was negative ( Figure  S3 ). Therefore negligible DWV in our monitor colonies may be attributed to low exposure and/or good colony health. A thorough one-year investigation of virus (ABPV, BQCV, DWV) and V. destructor in England found a correlation between DWV copy number and over-winter colony loss [60] . Lack of DWV in our monitor colonies is consistent with low Varroa destructor incidence, since mites are known to transmit DWV [39, 93, 94] . Low incidence of both DWV and V. destructor in our study may be partially attributed to our analysis of entrance samples, which consist of actively foraging and/or guarding adult bees. Since Varroa mites parasitize larva they are more readily detected in larva and young bee samples as well as hive bottom boards. More significantly, monitor colonies received miticide treatments in order to reduce V. destructor burden.
Deep sequencing analysis revealed the presence of four novel viruses (ALPV-Brookings, BSRV, LSV1 and LSV2), illustrating the power of deep short read sequencing and de novo assembly for virus discovery. Significantly, LSV2 was the most abundant single component of the honey bee microbiome in our study, and it is likely that the reason this virus has previous gone undetected is the fact that the Lake Sinai viruses are extremely divergent from all known insect viruses in both amino acid identity and genome organization. The non-structural genes of LSV are most closely related to CBPV, a known pathogen of honey bees [57] , as well as other members of the Nodavirales. However, the capsid gene and monopartite genome structure resemble tetraviruses and together position this virus closer to species such as the Providence virus, which similarly has a monopartite genome, a Nodavirales-like polymerase and a tetravirus-like capsid. Since the presence of viral nucleic acid does not necessarily indicate infection, as pollen pellets of infected and non-infected workers are known to harbor honey bee viruses [88] , we confirmed the presence of the replicative forms of LSV1 and 2 in time-course samples. The enormous magnitude of LSV throughout the time-course also suggests that these are bona fide honey bee viruses. LSV2 was the most abundant virus in our study and exhibited a unique seasonality. It is intriguing that peak LSV2 copy number per bee occurred in April (,3.6610 11 ) and January (,7.1610 11 ) since colonies typically collapse during the winter months. In contrast, LSV1 copy number peaked in July, similarly to the previously described honey bee viruses monitored in our study. Frequent detections of both ALPV-Brookings and BSRV (,15% incidence in the time-course) by PCR screen in different geographic regions argues against simple carryover from other insects during foraging, but does not rule out potential re-infection from stored pollen (bee bread) [88] . Research to determine the potential pathogenicity of these four new viruses in honey bees is underway. There are a number of previously identified honey bee viruses described on the basis of serology and electron microscopy (Bee virus X [95] , Bee virus Y [96] Arkansas bee virus [97] and Berkeley bee virus [98] for which no nucleic acid sequence information is available in public databases. Without such data we cannot preclude the possibility that these previously described viruses overlap with our novel viruses, however we were not able to gain access to any nucleic acid or serological reagent to address the question directly. Regardless, the nucleic acid sequences of the viruses reported herein are attached to publically accessible records in the form of GenBank accessions (LSV1 -HQ871931, LSV2 -HQ888865, ALPV Strain Brookings -Q871932 and BSRV -JF423195-8), such that any future viral samples may be directly compared, or if historical samples can be found and analyzed, they too can be compared.
Crithidia mellificae was readily detected throughout the timecourse. In contrast to most other prevalent microbes and viruses, relative Crithidia levels peaked in the winter (January 2010). The effects of C. mellificae on the honey bee host remain relatively uncharacterized compared to those of C. bombi on bumble bee, which include reduced worker fitness and colony survival [66, 99] . To date, there are only a few reports of C. mellificae infection of honey bees in the literature including early work describing the first isolation and culture of this organism in 1967 from Australian honey bees [67] . This work tested the effect of feeding C. mellificae to honey bees and demonstrated similar mortality rates in infected and uninfected bees [67] . More recently, similar trypanosomatid prevalence and loads were reported in CCD-affected colonies and healthy controls [9, 10] . Although current data suggest that C. mellificae does not dramatically affect colony health additional pathogenesis research in honey bees is warranted considering the detrimental effects of C. bombi on bumble bee colonies.
The importance of honey bees in agriculture and the emergence of CCD underscores the need to monitor honey bee associated viruses and microbes in healthy colonies over time. The confinement of Spiroplasma infection to a two-month window demonstrates the value of time-course sampling as opposed to single-collection screens. The development of high throughput platforms, such as the APM, will facilitate monitoring of exogenous agents in order to better understand their effect on honey bee health and survival. Our discovery and genomic characterization of four new viruses will facilitate future monitoring. Temporal characterization of these and the other microbes described herein offers a more complete view of the possible microbe-microbe and microbe-environment interactions. Further studies examining any subtle or combinatorial effects of these novel microbes are required to understand their role in colony health. Increased analysis of prospectively collected samples is essential to address the hypothesis that either one or more viruses and/or microbes cause CCD. To our knowledge, this is the first U.S. honey bee pathogen monitoring study to report both comprehensive pathogen incidence and relative abundance of specific pathogens over time. Results from our molecular analysis pipeline (APM, PCR, qPCR, ultra deep sequencing) provide a basis for future epidemiologic studies aimed at determining the causes of CCD. . Honey bees colonies were periodically supplemented with sugar syrup and protein supplement. In April (1 gallon) and October (2 gallons) bees were fed 50% (weight/volume) sucrose; in November all colonies received 3 gallons of a 1:1 mixture of high fructose corn syrup-55 (HFCS-55, 55% fructose, 42% glucose) and sucrose syrup. Additional sugar syrup was given to colonies based on colony weight (,80 lbs -3 gallons, 80-90 lbs -2 gallons., 90-100 lbs -none). This operation experienced an average 18% colony loss from November 2009 to February 2010. Colonies with younger queens (#2 years old) experienced 11% loss, whereas colonies with older queens experience 21% loss.
Samples (,50-100 bees) were collected into 50 mL Falcon tubes using a modified hand-held vacuum cleaner from both the entrance and interior of the hive and immediately put on dry ice for overnight shipment to our laboratory. Samples were stored at 280uC until RNA extraction; excess bees were archived for longterm 280uC storage. Time-course samples were collected monthly from April 15 (week 1) through July 14 (week 14), 2009 and weekly samples were attempted thereafter, however due to inclement weather or shipping logistics the samples for weeks 15, [28] [29] [30] 32 , and 39-41 were not collected. A total of 864 samples were obtained and 431 exterior samples were analyzed.
We determined that analysis of five honey bees per sample was sufficient for our colony monitoring project. Arthropod pathogen microarray (APM) analysis of test samples revealed that combined analysis of 5 bees reproducibly detected most, if not all, of the pathogens detected from 10 or 15 independently analyzed bees from the same sample. In addition, we confirmed the consistency of APM results by performing multiple analyses of a single RNA sample. Based on our test results and practical sample handling considerations, we reasoned that repeated analysis of 5 bees from each colony over-time (115 bees per colony) was sufficient for this study.
Honey bee samples, 5 bees per colony each time-point, were homogenized in 1 mL 50% TRIzol Reagent (Sigma) and 50% phosphate buffered saline (PBS, UCSF Cell Culture) solution in a 2 mL micro-centrifuge tube containing one sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch), for 4 minutes at 30 Hz. RNA was isolated according to TRIzol Reagent (Invitrogen) manufacturer's instructions. In brief, TRIzol reagent honey bee homogenate was combined with 0.1 ml chloroform and mixed by vortexing for 5 seconds, samples were incubated at room temperature for 5 minutes, prior to centrifugation for 10 minutes at 13,2006g in a table top centrifuge. Next, 700 mL of the aqueous phase was transferred to a new microfuge tube containing 490 mL isopropanol. Following mixing, the samples were incubated at 220uC for 20 minutes and then either centrifuged (13, 2006 g for 15 min) or further purified utilizing Zymo-III RNA columns according to manufacture's instructions (Zymo). RNA was extracted from five bees collected from the colony entrance for each of the time-course samples.
Design principles used for APM oligonucleotides (70 nt) were based on previous pan-viral microarrays using ArrayOligoSelector (AOS) [54] . Briefly, array oligonucleotides were selected for uniqueness against an insect nucleic acid background, for ,50% GC content to maintain high complexity, and for cross-reactivity of highly-conserved nucleic acid features with evolutionarily related targets (,250 kcal/mol predicted binding energy). Arthropod pathogen oligonucleotides (GEO GPL11490) were synthesized by Invitrogen, suspended at 40 pmol/mL in 36 SSC and 0.4 pmol/mL control oligo and printed on poly-L-lysine slides (Thermo) with silicon pins as previously described [100] . Each oligonucleotide and its reverse complement were printed twice for redundancy. Arrays were allowed to air-dry and stored and room temperature. Prior to use, oligonucelotides were cross-linked to slides via UV exposure (600 mJ), washed with 36SSC/0.2% SDS and blocked using a methylpyrrolidone solution (335 mL 1methyl-2-pyrrolidinone, 5.5 grams succinic anhydride, 15 mL 1 M sodium borate).
(Reverse Transcription, CyDye Labeling, Hybridization, Scanning) For each sample, 5 mL (,15 mg nucleic acid) of extracted material was randomly primed and amplified as previously described [44, 45] . Briefly, an adapter-linked random nonamer (59GTTTCCCACTGGAGGATANNNNNNNNN) was used to prime the reverse transcription reaction using SuperScript II (Invitrogen). The same oligo is used for two rounds of secondstrand synthesis with Sequenase (USB) in order to produce adapter-flanked sequences from both RNA and DNA starting material. One-quarter of the random priming reaction is used in a 50 mLTaq PCR reaction for 25 cycles with a single primer (59GTTTCCCACTGGAGGATA). One-tenth of the amplified material was further amplified for 10-20 cycles with a Cy3-linked primer (59Cy3 -GTTTCCCACTGGAGGATA). Samples were purified with the Zymo DNA Clean and Concentrator (Zymo) and resuspended in a buffer of 36 SSC, 50 mM HEPES and 0.5% SDS, and hybridized on the APM overnight at 65uC. Arrays were washed and scanned with an Axon 4000A scanner. Samples were analyzed manually and scored as positive for a pathogen if at least three unique oligonucleotides hybridized with at least five times background intensity. Arrays were further analyzed by a second unbiased method using the E-Predict algorithm [54, 55] , wherein all virus genomes were computationally hybridized to the array oligos and array results are compared to expected binding profiles. The top 5 unique oligos were removed and the algorithm reiterated twice in order to improve detection of low titer target(s) during a co-infection. Known honey bee pathogens were called positive if they exceeded a similarity score of 0.001 and were the highest ranked call in any iteration. In the event of a disagreement between the two analysis methods, a specific PCR reaction was performed, using material from the first PCR step, to resolve the call.
In order to estimate the sensitivity of the arthropod pathogen microarray (APM) two positive control samples were prepared in the presence and absence of pathogen-free honey bee RNA. A fulllength (9,264 nucleotide) Drosophila C virus (DCV) clone was in vitro transcribed, serially diluted into honey bee RNA, reversetranscribed, amplified, dye-labeled and hybridized to the APM as described above. Detection of at least 3 of the 8 unique DCV oligonucleotides and their reverse complements resulted in an estimated DCV detection level of 1.9610 5 genome copies (1 pg DCV genomic RNA) in an A. mellifera RNA (1 mg) background. Similarly, detection of a BQCV genome segment (452 nt), corresponding to one array oligo and its reverse complement, diluted into either pathogen-free honey bee RNA (0.5 mg) or water indicated detection limits of 1.2610 5 genome segment copies (30 fg BQCV RNA segment) and 1.2610 4 genome segment copies (3 fg BQCV RNA segment) respectively.
Reaction conditions for polymerase chain reaction (PCR) amplifications of select samples were performed under the following conditions: 5 mL of 1:10 dilution of PCR-amplified DNA and 10 pmol of each forward and reverse primers were amplified with Taq polymerase with the following cycling conditions: 95uC for 5 min; 95uC for 30 s, 50-60uC for 30 s, 72uC for 1 min, 35 cycles; final elongation 72uC for 7 min, hold at 4uC. Select samples were Sanger sequenced directly from ExoI and SAP treated PCR product or from colony PCR of TOPO cloned (Invitrogen) gel-extracted bands. Bands produced by PCR assays for known honey bee pathogens were sequenced until each molecular weight product was unambiguously associated with either a true positive or non-target amplification of the honey bee genome or microbiome. All PCR results for the four novel viruses were confirmed by Sanger sequencing.
Quantitative PCR (qPCR) qPCR was performed on pooled samples from each month. Equivalent amounts of RNA (10 mg) from each hive sample (monitor hives [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] were pooled according to the month in which they were collected (April 2009 to January 2010). Pooled RNA was further purified using Qiagen RNAeasy columns, including on column DNase Treatment (Qiagen). cDNA synthesis reactions were performed with SuperScriptIII (Invitrogen) according to manufacturer's instructions. In brief, RNA from each pooled sample (5 mg), random hexamer (1.25 mg) and dNTPs (0.5 mM each) were combined in a 50 mL reaction volume, incubated at 65uC (5 min), cooled on ice (1 min) and subsequently combined with 50 mL of 26 First-Strand Buffer containing SSIII (1000 U), DTT (5 mM), and RNaseOUT (200 U). Reverse transcription reactions were incubated for 12 hours at 42uC followed by inactivation of the reaction (70uC, 15 min). qPCR was performed in triplicate wells using 2 mL of cDNA as template in 20 ml reactions composed of HotStartTaq 26 Mastermix (Denville), 16 SYBR Green (Invitrogen), MgCl 2 (3 mM), and forward and reverse primers (600 nM each) (Table S2 ) on a LightCycler480 (Roche). The qPCR thermo-profile consisted of a single preincubation 95uC (10 min), 35 cycles of 95uC (30 s), 60uC (30 s), and 72uC (30 s). No RT control reactions using pooled RNA as the template for qPCR were performed in triplicate on each plate. Target qPCR amplicons were cloned into pGEM-T (Promega) or TOPO CR 2.1 (Invitrogen) vectors and sequence verified. Plasmid standards, containing from 10 9 to 10 2 copies per reaction, were used as qPCR templates to assess primer efficiency and generate the pathogen-specific standard curves used to quantify the viral genome or rRNA copy number. The linear standard equations generated by plotting the crossing point (Cp) versus the log 10 of the initial plasmid copy number for each primer set were as follows: (Figure 4 ). Values obtained from the no RT control reactions, all below the detection limit of the assays, were subtracted from the total pathogen copy number for each month. An estimate of the number of viral genomes per bee can be obtained by multiplying the reported qPCR copy number values by 500. This estimate is based on the following: typical RNA yield was approximately 50 mg per bee, each qPCR reaction was performed on cDNA generated from 100 ng RNA, therefore each well represents 1/500 th of an individual bee. We choose to represent the raw data, since each monthly-pooled sample was composed of variable bee numbers due to differential sampling frequency each month. In addition, qPCR with a host primer set, Apis m. Rpl8, was performed using 1 mL cDNA template on each qPCR plate to ensure consistency and cDNA quality. qPCR products were analyzed by melting point analysis and 2% agarose gel electrophoresis ( Figure S1 ).
Honey bees from two LSV positive honey bee colonies were homogenized in 500 mL phosphate buffered saline (PBS, UCSF Cell Culture Facility) with a sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch) for 4 minutes at 30 Hz; 5 bees per micro-centrifuge tube. Lysates were centrifuged for 10 minutes at 12,0006 g and RNA was extracted from both the supernatant and the bee carcass containing pellet using TRIzol Reagent (Invitrogen) according to manufacturer's instructions. RNA was further purified using RNAeasy columns, including on column DNase Treatment (Qiagen). RNA (15 mg per lane) was combined with glyoxal-based loading dye (Northern-MaxH sample loading dye, Ambion) and denatured at 50uC for 30 min prior to gel electrophoresis on an ethidium bromide containing 1.5% agarose BPTE gel using BPTE running buffer. BTPE buffer is composed of 10 mM PIPES, 30 mM Bis-Tris, 1 mM EDTA, pH 6.5. The gel was imaged using a UV lightbox and then soaked in 0.05 N NaOH for 20 min prior to overnight transfer to a membrane using the NytranH SuPerCharge Turboblotter TM system and 206 SSC. Following transfer the membrane was washed in 26 SSC (265 min), dried and UV crosslinked using a Stratalinker (Stratagene). LSV specific primers were used to amplify sequences corresponding approximately to the 59, middle, and 39 regions of the viral genome (primers listed in Table S2 ). The PCR products were column purified using the MinElute Reaction Cleanup Kit (Qiagen), labeled with {á 32 P} dCTP using Ready-To-Go TM DNA Labelling Beads (-dCTP) (Amersham; GE Healthcare) and used as LSV-specific Northern blot probes. The membrane was cut into three pieces and incubated, while rotating, in ULTRAhyb buffer (Ambion) at 42uC for 30 min prior to the addition of the radiolabled probes (10 6 counts per minute per mL hybridization buffer). Following overnight hybrization at 42uC, the membranes were washed at 42uC (265 min in 26 SSC 0.1% SDS; 2615 min in 16 SSC 0.1% SDS, 2615 min in 0.16 SSC 0.1%SDS). Phosphoimaging was performed using a Typhoon 9400 imager (GE Healthcare) ( Figure S4 ).
Negative strand-specific RT-PCR LSV strain 1 and 2 positive samples were analyzed for the presence of negative-strand RNA, which is indicative of virus replication, using strand-specific RT-PCR [39, 64, 65] . RNA from select samples (e.g. pooled July sample) was further purified using Qiagen RNAeasy columns, including on column DNase Treatment (Qiagen). cDNA synthesis reactions were performed with SuperScriptIII (Invitrogen) according to manufacturer's instructions using negative strand-specific LSV1 and 2 primers tagged with an additional 21 nt of sequence (59-GGCCGTCA-TGGTGGCGAATAA) at their 59 end [65] ; the tag sequence shares no homology with LSV nor to the honey bee genome (primer sequences listed in Table S2 ). In brief, RNA from each sample (1 mg), tagged-negative strand specific LSV primer (10 pmole) or random hexamers (50 ng) and dNTPs (0.5 mM each) were combined in a 10 mL reaction volume, incubated at 65uC (5 min), cooled on ice (1 min) and subsequently combined with 10 mL of 26 First-Strand Buffer containing SSIII (200 U), DTT (5 mM), and RNaseOUT (40 U). Reverse transcription reactions were incubated for 1 hour at 50uC followed by inactivation of the reaction (70uC, 15 min). Unincorporated primers present in the RT reactions were digested with exonuclease I (Fermentas), 0.1 Units per reaction which corresponds to a 10-fold excess of enzyme relative to the initial primer concentration, at 37uC for 30 min followed by heat inactivation at 80uC for 15 minutes. PCR was performed using 2 mL of exonuclease I treated cDNA template in 25 ml reactions containing 10 pmol each of a tag-specific forward primer (TAGS) and an LSV-specific reverse primer using the following cycling conditions: 95uC for 5 min; 95uC for 30 s, 58uC for 30 s, 72uC for 30 s, 35 cycles; final elongation 72uC for 4 min, hold at 4uC. In addition to amplification and detection of the LSV replicative form using tagged-negative strand primed cDNA template and TAGS forward and LSVU-R1717 PCR primers, negative and positive controls were performed ( Figure S5 -labeled (1) ). Negative controls included utilizing unprimed RT reaction as a template for PCR amplification using TAGS forward and LSVU-R1717 primers (labeled (2)), LSV tagged negative-strand primed cDNA template in PCR reaction in which only the LSVU-R1717 primer was added in order to ensure that all of the unincorporated RT primer was digested with exonuclease I and thus not involved in priming the PCR reaction (labeled (5)), and no template PCR using LSV qPCR primer sets (labeled (6)). Positive controls included using random hexamer primed cDNA as template for PCR amplification using LSV1 or LSV2 -specific forward primer and LSVU-R-1717 (labeled (3)) and random hexamer primed cDNA amplified using LSV-specific qPCR primer sets (labeled (6)). PCR products were analyzed using agarose (2%) gel electrophoresis ( Figure S5 ).
Honey bees were collected from a San Francisco, CA (U.S.A.) colony previously identified to be Crithidia positive by microarray and PCR testing. Honey bees were immobilized by chilling at 4uC for 20 minutes, briefly washed in 70% ethanol, and decapitated prior to dissection. The SF strain was isolated from honey bee intestines dissected in a sterile environment, minced and placed in a T25 flask and cultured in BHT medium composed of Brain Heart Infusion (BHI) 28.8 g/L (DIFCO), tryptose 4.5 g/L (DIFCO), glucose 5.0 g/L, Na 2 HPO 4 0.5 g/L, KCl 0.3 g/L, hemin 1.0 mg/L, fetal bovine serum (heat inactivated) 2% v/v, pH 6.5, and containing penicillin G sodium (10 6 units/L) and streptomycin sulfate (292 mg/L) at 27uC [101] . Free active Crithidias were observed 24 hours post inoculation. Parasites were maintained by subculture passage every 4 days; stable liquid nitrogen stocks were archived. Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (49,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. Images of fixed Crithidia mellificae were obtained using both the Leica DM6000 microscope and a Zeiss LSM 510-M microscope equipped with both a 636 objective numerical aperture 1.4, and a 1006 objective numerical aperture 1.4.
For DNA purification, Crithidia mellificae (,10 6 trypanosomes/ mL culture medium) were pelleted by centrifugation (8006g for 6 min) and washed with PBS prior to DNA extraction. DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer's instructions. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 16 Micrococcal Nuclease Buffer (NEB).
Total nucleic acid from all twenty monitor hives at time-point 17 (August 5, 2009) was pooled (approximately 3 mg per hive). One quarter was treated with RNase A/T1 (Fermentas) and genomic DNA was isolated using a DNeasy column (Qiagen). 50 ng of genomic DNA was prepared for deep sequencing by Nextera recombinase (Epicentre) per the manufacturer's instructions. The remaining nucleic acid was treated with Turbo DNase (Ambion) and column purified (Zymo) before being split into thirds. One third was enriched for mRNAs with dT-linked Dynabeads (Invitrogen). RNA from this fraction and from a second unenriched fraction were primed for RT and secondstrand synthesis with an adapter linked oligo as above using oligo SolCommonN (59CGCTCTTCCGATCTNNNNNN). The third fraction of RNA was primed with an anchored oligo dT and subjected to two rounds of second strand synthesis with SolCommonN. Half of the initial material was amplified with primer SolCommon (59CGCTCTTCCGATCT) with KlenTaq (Sigma) at an annealing temperature of 37uC for 20 cycles. Reactions were cleaned by Zymo column, analyzed by NanoDrop spectrophotometer and 50 ng was used in a four-primer PCR reaction. In a 50 mL KlenTaq reaction, 10 pmol each of primers 5Sol1 (59AATGATACGGCGACCACCGA) and 5Sol1 (59CAAGCAGAAGACGGCATACG) and 0.5 pmol of Sol1 (59 AATGATACGGCGACCACCGAGATCTACACTCTTTC-CCTACACGACGCTCTTCCGATCT) and Sol2 (59CAAGCA-GAAGACGGCATACGAGATCGGTCTCGGCATTCCTGC-TGAACCGCTCTTCCGATCT) were incubated for 2 cycles annealing at 37uC and 10 cycles at 55uC. Products were run on an 8% native acrylamide TBE gel (Invitrogen) and a 300-350 nt smear was cut out and electro-eluted. The product was further amplified at an annealing temperature of 55uC with primers 5Sol1 and 5Sol2 for 5-10 cycles until at least 30 ng of material was produced, as determined by NanoDrop. Libraries were sequenced on an Illumina Genome Analyzer II with a V3 cluster generation kit and V5 sequencing reagent as per the manufacturer's instructions, producing paired-end 65 nt reads.
Six pools of sequence data were downloaded from GenBank: Nosema ceranae (draft genome), Spiroplasma (S. citri draft genome and all sequences longer that 500 nt), DNA viruses of arthropods (all complete genomes), all small RNA viruses of arthropods except dicistroviridae and iflavirus (complete genomes), all members of dicistroviridae and iflavirus except those infecting honey-bees (complete genomes), and all known honey bee RNA viruses (complete genomes). Each pool was converted into a Blast library and queried against the entire Solexa dataset by BlastN and tBlastx. Hits with an e-value greater than 1610 23 were extracted along with their paired end, regardless of similarity. Each pool was assembled using the Geneious sequence analysis package [102] . Contigs greater than 250 nt were queried again against the dataset by tBlastx with an e-value threshold of 1610 25 . Any positive hits were then queried against the NR database with the same parameters to eliminate spurious hits.
Contigs that appeared divergent or that were derived from nonhoney bee associated viruses were extended using the entire read dataset using a paired-end contig extension algorithm (''PRICE'' Graham Ruby, manuscript under preparation, software available at http://derisilab.ucsf.edu). The extended contigs were then independently confirmed by PCR recovery and Sanger sequencing. Individual paired-end reads that were discordant with the recovered contigs were used to further nucleate new contigs via contig extension. Primer3 [103] was used to design primers bridging adjacent contigs, as determined by mapping onto known virus genomes. Individual viruses or other microbes were queried with a BlastN threshold e-value of 1610 27 (W7) to determine read counts.
Associations were calculated treating each hive sample at each time-point as a distinct event. P-values (Chi-square values) and odds ratios (OR) listed were calculated by the OpenEpi statistical package v2.3 (http://www.openepi.com/OE2.3/Menu/OpenE-piMenu.htm). Only seven microbes with incidences in the study set of at least 10% (20 incidences in 197 samples) were examined for association, resulting in 28 discrete association tests and the corresponding Bonferroni multiple testing correction. Microbes occurring infrequently were not used in association tests and so did not contribute to multiple testing correction.
APM design and results have been submitted to GEO (design accession GPL11490 and array data accession GSE28235) and are MIAME compliant. All Sanger sequence-confirmed deep sequencing assemblies have been submitted to GenBank (accessions listed in text). Figure S1 Gel electrophoresis of RT-qPCR products from pooled-monthly samples. qPCR products were amplified using the primer sets listed in Table S2 Figure S4 Detection of the LSV genome by denaturing 1.5% agarose gel electrophoresis and Northern blots using three LSV-specific probes. RNA (15 mg) extracted from the supernatants of homogenized honey bees was transferred to a membrane and probed using LSV-specific probes corresponding to different regions of the genome (P1 -1482-1744, P2 -2289-2477, and P3 -4509-4714) as described in Materials and Methods. (TIFF) Figure S5 Detection of the replicative form of LSV1 and LSV2 by negative strand-specific RT-PCR. The pooled July RNA sample was analyzed for the presence of LSV negativestrand RNA, which is indicative of virus replication, using strandspecific RT-PCR as described in Materials and Methods; RT-PCR products from reactions were analyzed by agarose (2%) gel electrophoresis. (TIF) Figure S6 Crithidia mellificae, strain SF movies. Light microscopy of live parasites was performed using a Leica DM6000 microscope (1006 objective) equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). (MP4) Figure S7 Crithidia mellificae, strain SF movies. Light microscopy of live parasites was performed using a Leica DM6000 microscope (1006 objective) equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinElmer). (MP4) Figure 3B , ** denotes qPCR primer sets used to obtain the results in Figure 4 and Figure S3 . (DOCX)  Evaluation of Coseasonality of Influenza and Invasive Pneumococcal Disease: Results from Prospective Surveillance BACKGROUND: The wintertime co-occurrence of peaks in influenza and invasive pneumococcal disease (IPD) is well documented, but how and whether wintertime peaks caused by these two pathogens are causally related is still uncertain. We aimed to investigate the relationship between influenza infection and IPD in Ontario, Canada, using several complementary methodological tools. METHODS AND FINDINGS: We evaluated a total number of 38,501 positive influenza tests in Central Ontario and 6,191 episodes of IPD in the Toronto/Peel area, Ontario, Canada, between 1 January 1995 and 3 October 2009, reported through population-based surveillance. We assessed the relationship between the seasonal wave forms for influenza and IPD using fast Fourier transforms in order to examine the relationship between these two pathogens over yearly timescales. We also used three complementary statistical methods (time-series methods, negative binomial regression, and case-crossover methods) to evaluate the short-term effect of influenza dynamics on pneumococcal risk. Annual periodicity with wintertime peaks could be demonstrated for IPD, whereas periodicity for influenza was less regular. As for long-term effects, phase and amplitude terms of pneumococcal and influenza seasonal sine waves were not correlated and meta-analysis confirmed significant heterogeneity of influenza, but not pneumococcal phase terms. In contrast, influenza was shown to Granger-cause pneumococcal disease. A short-term association between IPD and influenza could be demonstrated for 1-week lags in both case-crossover (odds ratio [95% confidence interval] for one case of IPD per 100 influenza cases = 1.10 [1.02–1.18]) and negative binomial regression analysis (incidence rate ratio [95% confidence interval] for one case of IPD per 100 influenza cases = 1.09 [1.05–1.14]). CONCLUSIONS: Our data support the hypothesis that influenza influences bacterial disease incidence by enhancing short-term risk of invasion in colonized individuals. The absence of correlation between seasonal waveforms, on the other hand, suggests that bacterial disease transmission is affected to a lesser extent. Please see later in the article for the Editors' Summary The seasonal periodicity of pneumonia and influenza deaths in temperate countries is regarded as sufficiently commonplace that the term ''flu season'' is part of the English language vernacular. However, the widespread recognition of wintertime seasonality of severe respiratory disease obscures the fact that remarkably little is understood about the genesis of such seasonality [1, 2] . The wintertime co-occurrence of peaks in viral and bacterial respiratory disease, including severe invasive disease caused by Streptococcus pneumoniae, is well documented, but how and whether wintertime peaks caused by different pathogens are causally related is unknown. Existing work in this field has consisted largely of descriptive analyses that have evaluated week-by-week correlations between respiratory virus activity and invasive bacterial disease, with or without lags [3] [4] [5] .
However, seasonal oscillation in disease incidence implies a complex system that includes such elements as loss of immunity and seasonally enhanced transmissibility with the latter potentially attributable to environmental, microbiological, or social forcing factors. Such a complex system cannot be adequately characterized by evaluating cross-sectional correlation in risk, particularly where causal links are concerned. Indeed, statistically significant correlation in risk when evaluating disease processes with shared seasonality is to be expected, and such correlation could be casual, as seen with many human respiratory pathogens with a peak incidence in winter, rather than causal.
We aimed to investigate the relationship between influenza infection and invasive pneumococcal disease (IPD) in Ontario, Canada, using several complementary methodological tools. We evaluated the relationship between the seasonal wave forms for influenza and IPD using fast Fourier transforms in order to examine the relationship between these epidemic waveforms over yearly timescales, i.e., to test the hypothesis that influenza influences IPD by enhancing person-to-person transmission. We also used three complementary statistical methods (time-series methods, negative binomial regression, and case-crossover methods) to evaluate the short-term effect of influenza dynamics on pneumococcal risk, i.e., to examine whether the risk of IPD is increased in previously colonized individuals.
The Toronto Invasive Bacterial Diseases Network (TIBDN) study was approved by Research Ethics Boards at all participating institutions. Ethical approval was not required for the remainder of this work.
The Toronto Invasive Bacterial Diseases Network is a collaboration of all hospitals, microbiology laboratories, infection control practitioners, physicians, and public health units serving the population of metropolitan Toronto and Peel Regions (population 3.7 million), performing population-based surveillance for selected serious bacterial and viral infections [6] [7] [8] . All 25 hospitals, 19 laboratories, and 85 long-term care facilities serving residents of the population area participate in this network. All invasive pneumococcal infections identified in residents of Toronto/Peel from 1 January 1995 through 3 October 2009 were included in the analysis. Invasive pneumococcal infection was defined as illness in which S. pneumoniae was isolated from a normally sterile body site. When S. pneumoniae was isolated from specimens obtained from residents of the study area (defined by postal code), informed consent was obtained to collect isolates and detailed clinical data, including age, sex, and site of infection. Isolates were then sent to the central laboratory at Mount Sinai Hospital (Toronto), where they were confirmed as S. pneumoniae by standard methodology, including colonial morphology on blood agar, bile solubility, and susceptibility to optochin. Annual audits are conducted to ensure completeness of reporting.
A national network of hospital and provincial laboratories submit weekly reports of numbers of tests performed (using viral culture or direct antigen detection) and numbers of positive tests for influenza A and influenza B to the Public Health Agency of Canada. For the purpose of this study, the surveillance data of the province of Ontario from 1 January 1995 through 3 October 2009 were included in the analysis.
We obtained time series data on ultraviolet (UV) radiation and weather from Environment Canada monitoring stations in the Greater Toronto Area [9, 10] . Where daily readings were taken at multiple locations, the arithmetic means of environmental data were used as exposure variables.
Evaluation of seasonal waveforms. The seasonality of disease occurrence was evaluated through calculation of autocorrelations for weekly case counts [2] . As yearly periodicity was observed with IPD, we estimated seasonal trends in disease occurrence of IPD and influenza A and B using Poisson and negative binomial regression models that incorporated sine and cosine oscillators, with 52-wk (annual) frequencies (i.e., incorporated fast Fourier transforms) [2, 11] . Using these parameters, the expression for the expected number of case counts for a given week, E(Y) is given by: E(Y) = e a + b1Nsin(2p(week/52))+b2Ncos(2p(week/52)) . Accordingly, the phase-shift of the composite waveform generated by combining sine and cosine components can be approximated as tan 21 (b 1 /b 2 ) and can be used to estimate the timing of peak disease incidence [11] . Similarly, the amplitude (i.e., the maximum disease incidence) can be approximated as
). Standard errors for phase-shift and amplitude were estimated as sums of standard errors for each model coefficient minus covariance. We evaluated the correlation between phase and amplitude terms for influenza A and B (combined), influenza A, and influenza B and pneumococcal sine waves, by year, by calculating Spearman correlation coefficients. Heterogeneity of phase and amplitude terms for S. pneumoniae, influenza A and B (combined), influenza A, and influenza B was assessed through meta-analysis using Cochran's Q test, and quantified using I 2 statistics. Differences in the magnitude of heterogeneity of amplitude or phase by pathogen was assessed using Knepp and Entwisle's method for assessing difference between chisquared statistics [12] . As the 2003/2004 and 2008/2009 influenza seasons were outliers with respect to timing and amplitude, and we also performed sensitivity analyses in which these years were excluded.
Negative binomial regression models. As both deviance and Pearson goodness-of-fit statistics suggested over-dispersion of pneumococcal case counts, we evaluated the relationship between IPD rates, influenza, and environmental exposures using a series of negative binomial regression models [13] . Models incorporated population data derived from the 2001 and 2006 censes as offsets, with linear interpolation and extrapolation used to generate population denominators for noncensus years [14] . Model construction evaluating the impact of influenza A and B (combined), influenza A, or influenza B on IPD risk was performed by including viral infection incidence and environmental exposures considered a priori to be likely to influence pneumococcal disease epidemiology. Candidate exposures were based on prior observations by our group and previously published research by others [5, [15] [16] [17] . Environmental exposures included UV index, temperature, and relative humidity and both virological and environmental exposures were considered at lags of up to 4 wk prior to pneumococcal case occurrence, which we considered to be a biologically plausible window for effects. Models also incorporated seasonal and multi-year trend terms.
Once models had been fitted, we attempted to enhance model parsimony through model reduction, removing terms that had no influence on pneumococcal case counts, in a manner that minimized Akaike's information criterion (AIC), with final models representing those that best balanced parsimony and fit [18] . In models in which viral exposures were evaluated using either total influenza counts or influenza A counts, strong and statistically significant associations were observed between influenza burden, UV index, and relative humidity; we explored multiplicative interaction terms using categorical variables representing the highest quintile ranks of these exposures [19] . As noted above the 2004 and 2009 influenza seasons were outliers with respect to timing and amplitude, and we also performed sensitivity analyses in which models were reconstructed excluding these years.
Time series methods. Granger causality Wald testing was used to determine whether influenza A and B (combined), influenza A, and influenza B seasons can be used to forecast peaks in pneumococcal infections [20] . Originally, Granger methods were used in econometrics to evaluate the directionality of relationships between closely correlated time series; a causal exposure to an independent variable should be associated with a lagged change in the dependent variable, and this relationship should be absent when the temporal sequence of exposure and response is reversed. In other words, Granger ''causality'' can be used to postulate causality in linear prediction only if one thing happens before another, and not vice versa. It cannot be used to prove causation, but does provide important evidence that can support or refute causality.
Case-crossover analysis. We used a case-crossover approach to evaluate short-term associations between influenza A and B (combined), influenza A, and influenza B infection and IPD occurrence during influenza seasons in order to provide a means for evaluating the association between brief exposures and rare outcomes. The design is characterized by ''self matching,'' in that cases serve as their own controls. A ''case'' thereby is a day on which a case occurred, whereas a ''control'' is an adequately selected day on which a case did not occur [21, 22] . A time-stratified 2:1 matched case-crossover design in which hazard periods were defined as the reported date of IPD onset from the Toronto Invasive Bacterial Diseases Network database was incorporated. Beginning on January 1, 1995, the person-time at risk was divided into 3-wk time strata. Control periods were chosen by matching the hazard period by day of the week within the stratum, and could both precede, both follow, or straddle the hazard period [23, 24] . Random directionality of control selection was used in order to avoid biases occurring with unidirectional or uniform bidirectional control selection [24] . We evaluated the effects of influenza exposures and environmental covariates through construction of conditional logistic regression models with time lags from 0 to 4 wk.
Data were analyzed using Stata version 11.0 (Stata Corporation) and SAS version 9.1 (SAS Institute). p-Values of ,0.05 were considered statistically significant.
The period from 1 January 1995 through 3 October 2009 was available for analysis, including a total number of 38,501 positive influenza tests in Ontario and 6,191 episodes of pneumococcal disease in Metropolitan Toronto/Peel Region (Figure 1 ).
Annual periodicity (i.e., recurrence at annual intervals) could be demonstrated for pneumococcal disease by spectral decomposition and construction of autocorrelations, with a maximum autocorrelation coefficient (ac) = 0.4488 at week 52 (Figures 1 and 2) . Peak incidence could be observed in week 6 (mid-February).
Periodicity of influenza infections was less regular; the maximum ac could be identified at 59-week lags for influenza A and B (combined) (ac = 0.3514), at 59-week lags for influenza A (ac = 0.2922), and at 48-week lags for influenza B (ac = 0.2903).
Negative binomial regression models revealed strong statistical evidence for annual oscillation for both pneumococcal disease and combined influenza A and B infections (p for nonlinear combination of sine and cosine oscillators for both pathogens ,0.001). Whereas meta-analyses revealed substantial heterogeneity of phase (Cochran Q statistic: x 2 = 62810.15, df = 15, p,0.001; I 2 = 100.0%) and amplitude (Cochran Q statistic: x 2 = 5752.86, df = 15, p,0.001; I 2 = 99.7%) terms for influenza A and B (combined), there was less heterogeneity of amplitude (Cochran Q statistic: x 2 = 40.06, df = 15, p,0.001; I 2 = 62.6%) terms for S. pneumoniae, and no significant heterogeneity for phase (Cochran Q statistic: x 2 = 23.28, df = 15, p = 0.08; I 2 = 35.6%) terms. Differences in heterogeneity x 2 statistics between pathogens [12] were highly significant for both amplitude and phase terms (p,0.001). Heterogeneity of phase and amplitude terms of influenza A and influenza B was in line with influenza A and B (combined) (unpublished data). Findings were robust when outlier seasons (2003-2004 and 2008-2009 ) were excluded in sensitivity analyses. Accordingly, no significant association between amplitude and phase of oscillatory waves of pneumococcal disease and influenza A and B (combined), influenza A, or influenza B could be detected using Spearman correlation (Tables 1-3 ).
Negative binomial regression analysis. Results from negative binomial regression analysis suggest influenza A and B (combined) activity is associated with IPD (incidence rate ratio = 1.09 case of IPD per 100 influenza cases, p,0.001), after adjustment for seasonal oscillation, multi-year trends, humidity, temperature, and UV index. Furthermore, there is an inverse association between average clear-sky UV index, both during the prior week, and 3 wk prior, and IPD (Table 4 ). Increased relative humidity was also associated with reduced risk of case occurrence. Models constructed using influenza A counts rather than total counts were identical in covariates and model performance to those constructed using total influenza counts (consistent with the large predominance of influenza A infections identified by the FluWatch system). We identified no significant interactions between influenza counts, UV index, and humidity. There was no significant association between influenza B incidence and IPD risk in either univariable or multivariable models. Model results were robust when we excluded influenza seasons that appeared to be outliers with respect to incidence (2003-2004 and 2008-2009 ) (unpublished data).
Granger causality Wald test. Based on the Granger causality Wald test, there is evidence that influenza A and B (combined) Granger-cause pneumococcal disease (Chi-square = 23.28, df = 2, p,0.001). These results could be reproduced for influenza A (p,0.001) and influenza B (p = 0.017). Conversely, pneumococcal disease could not be shown to Granger-cause influenza A and B (combined) infection (Chi-square = 4.39, df = 2, p = 0.11).
Case-crossover results. We were able to identify a significant association between total influenza (A and B) and IPD with a 1-wk lag using a case-crossover approach (odds ratio [95% confidence interval] for one case of IPD per 100 influenza cases, 1.10 [1.02-1.18]) ( Table 5 ). There was no significant association between IPD risk and daily changes in clear-sky UV index, temperature, or relative humidity.
Although this observational study cannot prove epidemiological causality, our analysis of seasonal oscillation in IPD and influenza A and B incidence in Ontario, Canada, is strongly suggestive of a causal relationship between influenza and IPD. The fact that we did not find a relationship between seasonal dynamics of influenza and IPD suggests that this relationship likely results from effects of influenza on risk of invasive disease in individuals with pneumococcal colonization. We found no epidemiological evidence to support the concept that influenza has a strong effect on IPD risk via changes in pneumococcal transmission dynamics that would be caused by enhanced susceptibility to pneumococcal colonization, increased infectiousness of pneumococcus-colonized individuals, or increased duration of carriage. All such changes would be expected to perturb the dynamics of pneumococcal seasonality in such a way that pneumococcal seasonal waves would ''track'' influenza waves. While we found significant and temporally directional relationships between influenza burden in the population and IPD risk, the seasonal ''waves'' of influenza and IPD were independent of one another, with substantial variability in influenza contrasted with the far more regular seasonal occurrence of IPD. The nonstereotyped nature of wintertime seasonality of viral respiratory pathogens has been noted previously [17] .
While prior work has evaluated the impact of influenza infection on pneumococcal risk, much of this work has been performed in experimental rodent models that may not be generalizable to human populations [25] [26] [27] . A number of epidemiological analyses have been performed; many have failed to account for either confounding by other seasonal factors, or ''ecological fallacy'' resulting from the use of aggregated exposure and outcome data [3] [4] [5] 28] . More methodologically sophisticated studies have yielded conflicting results on the link between influenza and IPD [29] [30] [31] . The major clinical implications of our study are 2-fold: first, that dramatic increases in influenza incidence, as might be seen during a pandemic year, would not in and of themselves imply a marked surge in risk of IPD, but rather would do so only if there were overlap between flu and pneumococcal waves. This observation may help explain the absence of a marked increase in IPD risk in our jurisdiction during the (atypical) spring wave of the 2009 influenza A-H1N1 pandemic. Although public health agencies issued calls for stepped-up pneumococcal vaccination because of the pandemic [32] , the fact that the pandemic and pneumococcal season were out of phase may have made this unnecessary. Second, the short-term surge in IPD risk accompanying lagged increases in influenza, if causal, implies that some fraction of IPD is ''influenza-attributable.'' As such there may be important unrecognized population health benefits associated with influenza vaccination programs that include younger individuals. Traditionally, influenza immunization programs have focused on older individuals and sometimes the very young, as the risk of adverse outcomes of flu appear greatest in these age groups. Some modelers have recommended younger individuals be targeted with influenza vaccination in order to protect older individuals from influenza via herd effects, and these projections appear to have been borne out in at least one recent randomized trial [33, 34] . However, IPD causes important morbidity in those in age groups not included in traditional influenza immunization programs, and our analysis suggests that prevention of influenza-attributable pneumococcal disease could also be considered as a benefit of influenza vaccination targeting older children and younger adults.
If our findings are correct, and influenza increases the risk of IPD without influencing pneumococcal transmission dynamics, the question remains: what are the drivers of the remarkably stereotyped seasonality of IPD? Crude correlations between IPD risk and mean weekly temperature and mean weekly minutes of darkness, but not precipitation, have been reported by Dowell et al. [35] . A previous study by our group of environmental determinants of IPD risk in Philadelphia [22] , identified diminished ambient UV radiation as a significant predictor of increased IPD risk, an effect that could either be due to effects of UV radiation on 1,25-(OH) 2 -vitamin-D metabolism, or direct mutagenic effects on vegetative bacteria. The current study, performed in a different jurisdiction, replicates this finding.
Like any observational study, ours has several limitations. As it is not possible to randomize exposure to influenza, or to meteorological exposures, observational studies like this one are necessary to evaluate the impact of such exposures on invasive bacterial disease risk in real-world human populations. Limitations of observational designs like the one we have used here include difficulties with residual confounding, ecological fallacy due to aggregation of exposures, and measurement issues. While it is not possible to control for all residual confounding by unmeasured confounders in regression models, our use of seasonal smoothers, multivariable techniques, and case-crossover design should largely have controlled for confounding of influenza effects by seasonally varying factors including seasonal behaviors and meteorological effects. Our use of case-crossover design implicitly matches for all seasonal effects that would be constant over the 3-wk time block used for each risk stratum. The case-crossover approach assumes that the distribution of exposure is constant across referent times, which should be controlled by careful referent selection. It can be associated with bias if referent periods are not chosen a priori or if referents are functions of the observed event times [36] . In order to circumvent this so-called ''overlap bias,'' control periods in our analysis were chosen by matching the hazard period by day of the week within the 3-wk stratum, and could both precede, both follow, or straddle the hazard period [24] .
Although implied by its name, Granger causality testing does not prove true causation in the epidemiologic sense. It originally is an econometric method to infer predictive value of one time series Table 4 . Short-term effects of influenza dynamics on pneumococcal risk using multivariable negative binomial regression analysis. over another and thus, rather than prove causation, adds a valuable piece to the overall interpretation of our analyses towards potential causation. With respect to ecological effects, it should be noted that we effectively treat influenza as an aggregate ''environmental'' exposure, in that we don't have influenza status on the individuals who develop invasive bacterial disease, which creates the risk of ecological effects, including ecological fallacy, in our study. There are practical barriers to formulating an adequately powered study that actually captured data on the temporal relationship between influenza and invasive bacterial disease at the level of the individual (e.g., through ongoing evaluation of individuals in the population for influenza infection, with subsequent identification of the [rare] individuals who developed invasive bacterial disease). Finally, with respect to measurement issues, for influenza A and B, we had to rely on public health surveillance data that are incomplete both because of under-reporting, and also because a majority of individuals with influenza never undergo virological testing [37] . Other exposures may have been misclassified because of measurement or population mobility. This misclassification should not have introduced bias into our study in the absence of correlation between pneumococcal risk and influenza or meteorological reporting but may have reduced our statistical efficiency. Nondifferential misclassification of exposures would mean that the effects reported here are likely to be lower-bound effects, with true effects being larger. Furthermore, the strong degree of agreement in our findings, across multiple methodological approaches, suggests that they are robust and not artifacts of the analytic approach taken.
In conclusion, our data support the hypothesis that influenza influences IPD risk by enhancing pneumococcal invasion in colonized individuals, but has little effect on the transmission dynamics of pneumococcal infection. We suggest that the mechanism for such an effect might be influenza-related alterations at the level of the respiratory epithelium [27] . These effects appear distinct from those of ambient environmental effects, although the effect of increased UV radiation in reducing IPD risk that we had previously reported in a study performed in Philadelphia needs further study [22] . These findings have important implications for disease control policy, and suggest that improved efficacy of influenza vaccines [38] , and novel vaccination strategies that more effectively control influenza by vaccinating younger individuals [33] , could have important effects in reducing IPD as well. Although they found wintertime peaks for infections with both pathogens, there was no correlation between the seasonal wave forms for influenza and IPD. That is, there was no relationship between the seasonal patterns of the two infections. By contrast, two of the statistical methods used to test hypothesis 2 revealed a short-term association between infections with influenza and with IPD. Moreover, the third statistical method (the Granger causality Wald test, a type of time-series analysis) provided evidence that data collected at intervals on influenza can be used to predict peaks in IPD infections.
What Do These Findings Mean? These findings support (but do not prove) the hypothesis that influenza influences IPD incidence by enhancing the short-term risk of bacterial invasion in individuals already colonized with S. pneumoniae, possibly by increasing the permeability of the lining of the airways to bacteria. By contrast, the lack of correlation between the seasonal wave forms for the two diseases suggests that person-to-person transfer of S. pneumoniae is affected by influenza infections to a lesser extent. These findings have important implications for disease control policy. First, they suggest that the increased number of influenza infections in pandemic years may not necessarily be accompanied by a marked surge in IPD. Second, because the findings suggest that some cases of IPD may be influenza-attributable, the extension of influenza vaccination to school-age children and young adults (a group of people at particular risk of IPD who are not normally vaccinated against influenza) could reduce the incidence of IPD as well as the incidence of influenza.  Macrophage-Mediated Optic Neuritis Induced by Retrograde Axonal Transport of Spike Gene Recombinant Mouse Hepatitis Virus Following intracranial inoculation, neurovirulent mouse hepatitis virus (MHV) strains induce acute inflammation, demyelination and axonal loss in the CNS. Prior studies using recombinant MHV strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. A demyelinating MHV strain induces optic neuritis, but whether this is due to retrograde axonal transport of viral particles to the retina, or if it is due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. Using recombinant isogenic MHV strains, we examined the ability of recombinant MHV to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye following intracranial inoculation. Recombinant demyelinating MHV induced macrophage infiltration of optic nerves, demyelination and axonal loss whereas optic neuritis and axonal injury were minimal in mice infected with the non-demyelinating MHV strain that differs in the spike gene. Thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. These data indicate that MHV spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for CNS viral infections and associated diseases. Neurotropic mouse hepatitis virus (MHV) infection in mice causes meningoencephalitis, myelitis, and demyelination, with relative axonal preservation. Recent studies have additionally demonstrated that neurotropic MHV strains can also induce axonal loss (1, 2) ; direct virus-mediated axonal damage can occur concurrently with and independently of demyelination (2) . Thus, neurovirulent MHV strains provide useful tools for studying the neuroinflammation, demyelination, and axonal loss and as a virus-induced model of multiple sclerosis.
Recombinant MHV strains RSA59 (demyelinating strain; DM) and RSMHV2 (nondemyelinating strain; NDM) are isogenic except for the spike gene, which encodes the host attachment spike glycoprotein. Studies of these strains have elucidated mechanisms of axonal loss and demyelination (3) . RSA59 and RSMHV2 both cause hepatitis, encephalitis, and meningitis after intracranial inoculation. However, they differ in their ability to induce macrophage infiltration and subsequent demyelination and axonal loss in spinal cord (2) . There is a lack of viral antigen spread and subsequent inflammation extending into spinal cord white matter after intracranial infection with the NDM strain, whereas there is extensive macrophage-mediated white matter pathology secondary to DM strain infection. Thus, the spike protein plays a critical role in anterograde axonal transport of viral particles, an important mechanism mediating axonal damage and demyelination (2, 4) . Because both strains cause encephalitis after transcranial inoculation, the differences in spike protein between DM and NDM strains do not impair viral entry; however, differential neural cell tropism may contribute to the mechanism of demyelination (2, 4, 5) . Infection of brain neurons and oligodendrocytes occurs on inoculation with either strain, whereas in the spinal cord, oligodendrocyte infection is only seen with the DM strain. This is likely due to the route by which the virus gains access to white matter, that is, spinal cord infection does not occur as a result of direct trauma, whereas transcranial inoculation results in traumatic disruption of the brain gray-white matter interface. Viral particles that would require anterograde axonal transport from infected neurons to reach myelin are able to gain direct access to the myelin sheath and spread proximally to oligodendrocyte cytoplasm.
Anterograde axonal transport and spread of virus from neurons to oligodendrocytes have been documented, but retrograde axonal spread of virus from nerve ending to neural cell body also needs to be considered. Earlier studies suggested that MHV strains may spread via retrograde axonal transport (6, 7) , but the molecular mechanisms mediating such transport are not well defined. In the optic nerve, the parental demyelinating strain MHV-A59 causes inflammation, demyelination, and axonal loss (ie, optic neuritis), in contrast to the nondemyelinating MHV-2 strain (8) . Whether MHVinduced optic neuritis is dependent on retrograde axonal transport of viral particles or is due to local traumatic disruption of the intracranial portion of retinal ganglion cell (RGC) axons during inoculation is not known. Moreover, the immune response in MHV recombinant strain optic neuritis has not been well characterized. Here, we compared the incidence and phenotype of optic neuritis after inoculation with RSA59 and RSMHV2 and assessed the ability of spike protein to facilitate retrograde axonal transport and induce optic neuritis.
Recombinant isogenic DM strain of MHV (RSA59) and NDM strain (RSMHV2) have been described in previous studies (4, 9) . RSA59 and RSMHV2 strains of MHV are isogenic except for the spike gene, which encodes an envelope glycoprotein that mediates many biological properties of MHV including viral attachment to host cells and virus-cell and cell-cell fusion (10) . These recombinant strains also express enhanced green fluorescence protein (EGFP) (4, 9) .
MHV-free, C57BL/6 (B6) mice (Jackson Laboratory, Bar Harbor, ME) were inoculated intracranially at 4 weeks of age with 50% LD 50 dose of RSA59 strain (20,000 plaque forming units) or RSMHV2 (100 plaque forming units), as described previously (4) . Mice were monitored daily for signs of disease. Mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. Animals were killed (3Y5 mice per group) at day 3, days 5 to 7, and day 30 post inoculation (pi). All experimental procedures adhered to guidelines of, and were approved by, the Institutional Animal Care and Use Committee.
Mice were killed at days 5 to 7 pi (peak of inflammation) or at day 30 pi (during chronic demyelination) and were perfused transcardially with phosphate-buffered saline followed by phosphate-buffered saline containing 4% paraformaldehyde. Brain, spinal cord, eyes, and optic nerve tissues were collected, postfixed in 4% paraformaldehyde overnight, and embedded in paraffin; sections were then stained with hematoxylin and eosin (H&E) to evaluate inflammation and Luxol fast blue to evaluate demyelination. Experiments were repeated at least 3 times with 3 to 5 mice. Areas of demyelination and inflammation were quantified as previously described (8, 11) . All slides were coded and read in a blinded fashion. To confirm expected virulence of the strains used, livers from the infected mice were embedded in paraffin, sectioned at 5 Km, and stained with H&E (5, 12) . The degree of optic nerve inflammation was scored by 2 blinded investigators on a 0-(no inflammation) to 4-point (severe inflammation) scale, as described (8, 11) , during the time of peak inflammation (days 5Y7 pi). Any amount of inflammation (score of 1Y4) was considered positive for optic neuritis.
Serial sections from optic nerves were stained by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, CA) using 3, 3 ¶ diaminobenzidine as substrate, and antibodies against lymphocytic cell markers, antiviral nucleocapsid antiserum, or the axonal marker antineurofilament antiserum as primary immunoglobulin G antibodies. The sources and dilution of primary antibodies are listed in the Table. Control slides from mock-infected mice were incubated in parallel.
Fixed sections of optic nerve, brain, and eyes from mice at 3 or 6 days pi were stained by the previously described immunohistochemical methods using antiviral nucleocapsid antiserum. To visualize viral antigen by EGFP expression directly, frozen sections from infected mice were examined by fluorescence microscopy. Viral antigen was also assayed by Western blotting using antiYhepatitis virus nonstructural protein 9 monoclonal antibody (Rockland, Inc., Gilbertsville, PA), according to the manufacturer's instructions. Briefly, protein was extracted from the optic nerve and retina using RIPA buffer (Sigma, St. Louis, MO) in the presence of a complete protease inhibitor cocktail (Pierce, Rockford, IL) at 4-C. Protein concentration was determined with a Micro BCA Protein Assay Kit (Pierce). Protein (30 Kg) was electrophoresed on 4% to 15% SDS-PAGE, transferred to cellulose membrane, blocked, and probed with 1:1000 dilution of primary antibody. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined as a loading control using anti-GAPDH (Sigma) diluted 1:10,000. After incubation with horseradish peroxidaseYconjugated secondary antibodies, signals were developed with enhanced chemiluminescence agent (GE Healthcare, Buckinghamshire, UK), and intensity was determined using the NIH Image J program.
Longitudinal optic nerve sections were stained with antineurofilament antibody, and areas of axonal staining were quantified as described (11) . Briefly, 3 photographs were taken at 40Â magnification of each stained nerve at predefined locations (one each of the proximal, central, and distal portion of the nerve) covering a total area of 38,500 Km 2 of each nerve. The amount of tissue within this area that stained positively for neurofilament was calculated using ImagePro Plus 6.0 (Media Cybernetics, Silver Spring, MD) software. Data shown represent the cumulative area of positive staining/nerve.
Comparisons of optic neuritis incidence, demyelination, and axonal density were analyzed by one-way analysis of variance followed by Tukey multiple comparison test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Data represent mean (SD) percentage of eyes that developed optic nerve inflammation or demyelination or the mean (SD) density of axonal staining.
As in prior studies, RSA59-infected mice showed meningitis, encephalitis, myelitis, and concurrent axonal loss and demyelination as early as day 5 pi with an increase at day 30, whereas RSMHV2 showed only meningitis, encephalitis, and myelitis with no significant demyelination or axonal loss (data not shown) (2) . The livers of both strains showed moderate to severe hepatitis (data not shown), thereby confirming virulence.
As expected, mice infected with parental demyelinating strain MHV-A59 at days 5 to 7 pi had optic nerve inflammation ( Fig. 1C) , whereas optic nerves from mice infected with parental nondemyelinating strain MHV-2 did not (Fig. 1B) ; their optic nerves appeared similar to those of mock-infected mice (Fig. 1A) (8) . The inability of MHV-2 to induce optic neuritis was expected because MHV-2 does not infect the brain parenchyma and is unable to induce encephalitis (13, 14) . By contrast, the RSMHV-2 strain induces encephalitis (2, 4, 5) . RSA59 induced optic neuritis similar to the parental MHV-A59 strain at days 5 to 7 pi (Fig. 1F) , whereas most eyes of RSMHV2-infected mice exhibited no optic nerve inflamma-tion (Fig. 1D) ; however, there was mild inflammation in a few optic nerves (Fig. 1E) .
MHV-A59 induced optic neuritis in a mean of 55.6% of optic nerves in 4 experiments, whereas MHV-2 induced optic neuritis in only 3.7% of optic nerves (Fig. 1G) ; this result is similar to the incidence in prior studies (8) . RSA59infected mice developed optic neuritis with a high incidence (68.5%) similar to that in MHV-A59-infected mice, and significantly higher than the incidence in RSMHV-2 infected mice (25.1%) (Fig. 1H) . Serial sections from RSA59-and RSMHV2-infected mice were stained with anti-CD45 (leukocyte common antigen; LCA), antiYIba-1 (microglia/macrophage marker), anti-CD3 (T-cell marker), or anti-CD19 (B-cell marker) (Table) . LCA staining confirmed the presence of infiltrating inflammatory cells in optic nerves from RSA59-infected mice, whereas few LCA-positive cells were found in those of RSMHV2-infected mice (not shown). Among the LCA-positive inflammatory cells, the majority in the RSA59-infected mice were Iba-1+ microglia/macrophages (Fig. 2C) . The optic nerves of some control mice (Fig. 2) and RSMHV2-infected mice (Fig. 2B ) also had scattered Iba1+ cells that likely represent resident microglia, but there were far fewer than in the RSA59-infected samples. This result was confirmed by 2 blinded investigators. Significantly more optic nerves from RSA59-infected mice had increased Iba1+ cells versus nerves from RSMHV2-infected mice (14/23 total nerves from 4 experiments versus 4/20, respectively; p = 0.0124 by Fisher exact test).
Few or no CD3-positive T cells were present in optic nerves of RSA59-infected mice (Fig. 2D ) and no CD19stained B cells were observed (Fig. 2E ). Spleen sections from mock-infected mice stained with either anti-CD3 (Fig. 2F ) or anti-CD19 antibodies (not shown) served as positive controls. Thus, there was an increase of Iba-1Ypositive cells and few CD3-positive cells in optic nerves of RSA59-infected mice.
Optic nerves from RSA59-infected mice had areas of demyelination detected by Luxol fast blue staining both at day 7 and day 30 pi (Fig. 3) , whereas no demyelinating plaque was observed in day 7 pi RSMHV2 mouse optic nerve and little or no demyelination was observed at day 30 pi. These data are consistent with the previous result obtained from parental demyelinating strain MHV-A59 and nondemyelinating strain MHV-2 (8).
No axonal loss was identified at 5 to 7 days pi in optic nerves of RSA59-or RSMHV2-infected mice (Figs. 4AYC). At day 30 pi, RSMHV2-infected mice continued to show no axonal loss (Fig. 4D) , whereas optic nerves from RSA59infected mice showed regions of mildly reduced axonal staining, with focal areas of axon loss intermixed with areas of normal axons (Figs. 4E, F) . Quantification of the area of axonal staining across all sections of optic nerves of RSA59infected mice demonstrated a small but significant decrease compared with optic nerves of either RSMHV2-infected or mock-infected control mice (p = 0.0306) (Fig. 4G ).
The optic nerve inflammation, demyelination, and axonal loss observed after RSA59 infection, but not after RSMHV2 infection, demonstrate that the MHV-A59 spike protein is required for the induction of optic neuritis. This suggests that the spike protein may mediate retrograde axonal transport of the virus, allowing it to travel from brain regions containing RGC axonal projections along the optic nerve. Viral antigen was consistently detected within thalamic neurons in RSA59-infected mouse brains (Figs. 5C, D) , as well as in some RSMHV2-infected brains (Fig. 5B) . RSA59 viral antigen was also detected in the superior colliculi in some animals (data not shown). Light diffuse staining detected in optic nerves from RSA59-infected mice (Fig. 5G ), but not RSMHV2-infected mice (Fig. 5F ), suggests that only the RSA59 viral antigen is transported to the optic nerves. To further investigate this, the optic nerves were isolated 3 or 6 days pi and frozen sections were examined for the presence of EGFP. Viral antigenY positive EGFP signal was observed in optic nerves from RSA59-infected mice (Figs. 5I, J) but not RSMHV2-infected mice (Fig. 5H) . The punctate EGFP signal in optic nerve sections observed by fluorescent microscopy in RSA59-infected mice (Fig. 5J ) further suggests axonal transport through the optic nerve and was seen as early as 3 days pi.
To determine whether viral antigen is transported all the way to the RGC cell bodies, whole eyes were isolated and sectioned. Viral antigen immunostaining of retinal sections demonstrated no viral antigen in cells of the RGC layer of RSMHV2-infected mice (Fig. 6A) , whereas RSA59 viral antigen was detected in some cells within the RGC layer (Figs. 6B, C) at day 6 pi. Overall, viral antigen staining was detected in 60% of eyes from RSA59-infected mice, with 15 serial cross sections examined from each of 5 eyes. The timing of viral antigen spread and relative levels of antigen in retina and optic nerve were determined in protein extracts isolated at days 3 and 6 pi. Western blot analysis demonstrated presence of viral antigen in retinal tissue from RSA59infected mice at day 6 pi but not at day 3 and no viral antigen was detected in RSMHV2-infected mice (Fig. 6D ). Viral antigen was detected in protein extracts from optic nerves of RSA59-infected mice earlier than it was detected in retina. Antigen was detected on Western blots at both days 3 and 6 pi (Fig. 6E) . These results suggest that the viral antigen was able to enter the RGC axons and was transported retrograde to the cell bodies within the eye.
RSA59 induces optic neuritis at comparable levels of severity and incidence as that seen in mice infected with its parental strain MHV-A59 (8), whereas RSMHV2 has a limited ability to induce optic nerve inflammation. Accordingly, nerves from RSA59-infected mice at the chronic stage showed significantly decreased axonal density compared with nerves from RSMHV2-infected mice. This differential ability of MHV strains to induce optic neuritis and axonal injury is dependent on spike glycoprotein mediated retrograde transport of viral antigen along RGC axons. Prior studies demonstrated that RSA59 and RSMHV2 both can cause meningitis and encephalitis, but RSMHV2 did not induce subsequent demyelination and axonal loss in spinal cord. Our current results demonstrate that in addition to demyelination and axonal loss, RSMHV2 also has limited ability to cause significant optic nerve inflammation and RGC infection. However, RSMHV2 did induce some optic nerve inflammation, unlike the parental strain MHV2. This difference may be due to the ability of RSMHV2 to enter neurons in the brain parenchyma and replicate, as demonstrated previously (2, 4, 5) and shown again here, leading to a higher viral load that may allow some diffusion of the virus at an undetectable level. Alternatively, the higher viral load might trigger a more diffuse central nervous system (CNS) inflammatory response associated with mild, nonspecific inflammation in some optic nerves. MHV2, on the other hand, does not enter the brain parenchyma, and infection with it results in meningitis without inducing encephalitis (3). Prior studies demonstrated that one mechanism limiting the ability of the NDM strain to induce demyelination and axonal loss in the spinal cord involves impaired interneuronal spread of viral particles and defective translocation of viral antigen from gray matter to white matter (2) . Evaluation of axonal loss and demyelination in the spinal cord demonstrated that DM MHV infection begins in the neuronal cell body, propagates to the axon, and subsequently induces axonal degeneration and demyelination. The propagation of viral antigen from gray to white matter is dependent on anterograde axonal transport of virus particle mediated by the spike protein (2, 4) . Spike proteinYmediated axonal transport as an underlying mechanism is further supported by the current studies and demonstrates that it also plays a role in mediating retrograde axonal transport. It is especially intriguing that MHV uses similar molecular mechanisms to interact with cellular axonal transport machinery, suggesting that targeted disruption of this protein function might prevent all pathogenic spread of the virus.
Although it has been reported previously that neurotropic MHV strains can spread within neurons in a retrograde direction, the molecular interactions mediating this spread have not been fully examined (6, 7) . In cultured neurons, viral interaction with the microtubule network in neuronal processes has been shown to be important; based on cross-interaction of antimicrotubule antibodies and the nucleocapsid protein (N), it is possible that such interactions might be involved in axonal transport mechanisms (15) . However, in view of the fact that the DM and NDM strains used in the present study differ only in spike protein and contain identical N proteins, our data suggest more of a role for the spike protein.
How the DM strain of MHV initially infects neurons requires further study. One possible explanation could be that traumatic disruption to the nerve endings in the brain allows access of the virus through the damaged axolemma; or the virus may be capable of directly infecting intact axons either in the brain, or after diffusing into the optic nerve in the cerebrospinal fluid. Alternatively, virus may be engulfed into the axons at synaptic terminals. Although direct infection after diffusion in cerebrospinal fluid could potentially explain the presence of viral antigen in optic nerves, the fact that antigen is detected in RGC bodies in the retina demonstrates that retrograde transport does occur. Although the presence of viral antigen in RGCs alone cannot exclude hematogenous spread as an alternate mechanism from retrograde transport, the timing of its spread (ie, detected only at day 6 pi in the retina versus day 3 pi within optic nerves and within liver via hematogenous spread), the lack of hematogenous spread to the CNS after systemic infection with equivalent doses of MHV-A59 (6) , and the presence of viral antigen in areas of the brain containing RGC projections all suggest that retrograde transport is the more likely mechanism of viral spread. Retrograde transport may follow fusion of the spike protein with axonal membranes followed by loss of most of the viral structural proteins and then binding of the nucleocapsid via one or more viral proteins to the retrograde molecular motors. Such a mechanism has been seen in other neurovirulent viruses that follow a retrograde direction of axonal transport (16, 17) . Although it is not common that a single virus can use both directional transport mechanisms, this is not the first virus to demonstrate such properties. For example, herpesviruses can be transported rapidly along microtubules in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterograde in the opposite direction (18, 19) . Axonal transport is an important strategy used by several neurovirulent viruses, including herpes simplex, rabies, polio, influenza, and Borna disease viruses, which can be transported in axons either in anterograde or in retrograde direction (20Y22). In some instances, viruses can be transported within axons for a long distance, yet this journey occurs within the cell. Therefore, the virus cannot be inactivated by neutralizing antibody during its transit and may spread in the CNS without inducing an antivirus immune response while it is within the cell. When viruses can spread only within axons and/or via direct cell-tocell contact, they could potentially escape the attack of antiviral drugs or neutralizing antibodies. In the current studies, optic nerve inflammation induced by DM strain RSA59 consisted of predominantly macrophages/microglia, similar to immune responses in spinal cord (2) and without the marked T-cell infiltration seen in other demyelinating disease models (23Y26). Iba-1 staining was diffuse and was observed in cell bodies as well as cell processes, as has been seen previously with this macrophage/microglia marker (27, 28) . Although Iba-1 cannot distinguish between macrophages and microglia, we suspect most labeled cells represent infiltrating macrophages based on the overall increase in the number of cells observed. Macrophages have been shown to play both proinflammatory and anti-inflammatory roles in optic nerves (29) , and we suggest that the DM strain viral antigen might be moving within axons to avoid being cleared by infiltrating macrophages.
It is known that some viruses make use of the microtubules and/or the actin cytoskeleton for axonal transport (30Y32). Our studies have shown that a major mechanism of both retrograde and anterograde axonal transport of neurovirulent MHV is mediated by spike protein and further experiments will focus on identifying the molecular mechanisms by which the DM virus interacts with the axonal transport system and whether specific interventions targeting the transport system can delay or prevent the DM strain-induced axonal loss and demyelination. Analyzing the underlying principles of MHV axonal transport will be helpful in the design of viral vectors to be used in research, in human gene therapy, and in the identification of new antiviral therapies. Such therapies may also have the potential to prevent CNS demyelinating diseases that might be triggered initially by viral infection.  Non-Apical Membrane Antigen 1 (AMA1) IgGs from Malian Children Interfere with Functional Activity of AMA1 IgGs as Judged by Growth Inhibition Assay BACKGROUND: Apical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition. METHODS: Total IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children. RESULTS: AMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA. CONCLUSION: Children living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population. WHO estimates there were 243 million malaria cases and 0.9 million deaths in 2008; the vast majority of deaths occurred in African children less than 5-years old due to Plasmodium falciparum, which is the most virulent species of human malaria [1] . While the protective mechanisms remain to be elucidated, a passive transfer study has shown the importance of gamma-globulin against bloodstages of P. falciparum [2] . To control and eventually eradicate malaria, an effective vaccine is considered to be needed, in addition to the existing tools, such as drugs, insecticides, etc. [3] .
Apical membrane antigen 1 (AMA1) is the one of the beststudied blood-stage vaccine candidates and it is an essential protein for parasite invasion of an erythrocyte [4] . The invasion process is complicated (i.e., initial attachment, reorientation, tight junction formation and internalization), and different studies have suggested different roles for AMA1: binding to erythrocytes [5] [6] [7] , reorientation [8] , or internalization [9] . In addition to erythrocyte invasion, a recent study suggests that AMA1 is involved in sporozoite invasion of hepatocytes [10] . These results indicate the AMA1 protein may have multiple roles. Many studies have shown that AMA1 vaccination can induce protective immunity in animal models (reviewed in [11] ) and in an Aotus monkey challenge model (the monkeys were challenged with P. falciparum). In the monkey challenge model, anti-AMA1 antibody levels induced by a vaccine before challenge have shown to correlate with protection [12, 13] . In addition to the animal data, many, but not all, epidemiological studies suggest a high level of AMA1 antibodies is associated with a reduced risk of malaria [11] . Based on these findings, multiple AMA1 Phase 1 trials [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] and a Phase 2 field trial [25] have been conducted and published. However, to date no significant protective effects have been shown in the target population of African children.
An in vitro parasite Growth Inhibition Assay (GIA; also referred to as the Invasion Inhibition Assay, IIA) is one of the few widelyused biological assays that can measure the functional activity of antibodies against blood-stage malaria. While it is still controversial whether the activity measured by the GIA (IIA) reflects protective immunity induced by a vaccine, the assay has been used in preclinical and clinical studies as one of the immunological readouts. Not only anti-AMA1 antibodies induced by malaria infection [26, 27] , but also antibodies induced by AMA1 immunization in malaria naïve individuals show growth-inhibitory activity in the in vitro GIA [14, 16, 19, [21] [22] [23] .
In contrast, while the same AMA1 vaccine increased the anti-AMA1 antibody levels when it was administered to adults who lived in a malaria endemic area, the vaccine did not change the parasite growth-inhibitory activity [15] . In a previous study, we purified total IgGs from sera collected in an epidemiological study in Mali (majority of the sera were collected from adults) and separated the IgGs by affinity chromatography into an AMA1binding fraction (AMA1-specific) or an IgG fraction that does not bind to AMA1 (non-AMA1 IgG). Previously we have shown that the non-AMA1 IgG, more specifically a fraction of the non-AMA1 IgG which can bind to malaria extract, reduced the functional activity of the AMA1 antibodies from U.S. vaccinees [27] . Interference by the non-AMA1 IgG induced by malaria infections likely explains the reason why the AMA1 vaccine did not induce higher growth-inhibitory activity in the Malian adults in the vaccine trial. Because limited volumes of sera were collected from children in the epidemiological study, we could not investigate whether there was such ''interfering'' IgG in the children, who are the main target population of the blood-stage vaccine and who have less previous exposure to malaria.
Our recent Phase 2 trial in Malian children showed that there was a small, but significant, increase of growth-inhibitory activity when data from all AMA1-immunized children was analyzed (the GIA was performed with total IgGs in the study) [28] . The level of increase in the activity was not different from the level observed in U.S. adults who were immunized with the same vaccine formulation [28] . In the U.S. vaccine trial, all of the volunteers showed negligible levels of inhibition before vaccination, and after immunization the growth-inhibitory activity of total IgG was a function of anti-AMA1 titer. However, in the Malian children, the increase of growth-inhibitory activity was observed only in AMA1immunized children who had less than 20% inhibition before vaccination. None of the children who had more than 20% inhibition before vaccination (14/89 children in the AMA1immunized group) showed more than a 20% increase, while antibody levels measured by ELISA increased in most of the children. On the other hand, out of the remaining 75 children with less than 20% inhibition before vaccination, 25 (33%) children had more than a 20% increase after vaccination. In the present study we wished to investigate the effect of non-AMA1 IgGs in the children to determine whether similar interference effects could be identified. Therefore, we made multiple pools of total IgG, separated them into AMA1-specific and non-AMA1 IgGs, and tested them by GIA. The non-AMA1 IgGs showed an interference effect on growth-inhibitory activity.
The details of the U.S. adult Phase 1 trial [16] and the Phase 2 trial in Malian children [25] have been supplied elsewhere (NCT00344539 and NCT00341250). In brief, adults 18-45 years of age were enrolled in the U.S. trial and they were immunized on Days 0, 28 and 56 with 20 or 80 mg of AMA1-C1 (a mixture of the recombinant AMA1-FVO and AMA1-3D7 proteins) formulated on AlhydrogelH and mixed with CPG 7909. Plasma samples from individuals with high levels of anti-AMA1 antibody (as determined by ELISA) on Day 70 were collected 3 months after the final vaccination. For the Mali trial, Malian children 2-3 years old were enrolled and immunized on Days 0 and 28 either with 80 mg of AMA1-C1 on AlhydrogelH or a comparator vaccine (HiberixH). Blood samples were collected on Days 0 and 42. Both trials were conducted under Investigational New Drug Applications reviewed by the U.S. Food and Drug Administration. The U.S. Phase 1 trial was reviewed and approved by the Institutional Review Boards (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and by the University of Rochester Research Subjects Review Board. Written informed consent was obtained from all volunteers. The Mali Phase 2 trial was reviewed and approved by the IRB of NIAID at NIH and by the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry, University of Bamako. Community consent was obtained at a meeting with village leaders, heads of families, and other community members prior to the start of the Phase 2 study. Individual informed consent was then obtained after oral translation of the consent form into the local language. Understanding of the contents of the consent was confirmed by means of a multiple choice questionnaire. Parents or guardians unable to read placed an imprint of his/her finger in place of a signature; an independent witness also signed all consent forms.
Total, AMA1-specific and non-AMA1 IgG preparations Total IgGs were purified from individual plasma samples from the U.S. trial (n = 5) and normal U.S. sera (n = 2) using Protein G columns as described previously [14] . Similarly, total IgG was prepared from each plasma sample collected on Days 0 and 42 from the Mali Phase 2 trial. All of the total IgGs were dialyzed against RPMI 1640 and concentrated to 40 mg/ml. Because the volume of the total IgG from each individual Malian child was not enough to perform AMA1-specific IgG purification, the total IgG samples were ranked based on their anti-AMA1(3D7) antibody level as judged by ELISA and were divided into 3 or 4 groups to make pooled IgGs at each time point as follows: For Day 0 IgGs (regardless of immunization groups), D0-1, D0-2, D0-3 and D0-4; for Day 42 IgGs from HiberixH-vaccinated children, Hib-1, Hib-2 and Hib-3; for Day 42 IgGs from AMA1-vaccinated children, AMA1-1, AMA1-2 and AMA1-3. The number of individual total IgGs used to make each total IgG pool and the antibody level of the pool is shown in Table 1 . Although all of the anti-malarial antibody in Day 0 IgGs and Day 42 IgGs from the Hiberix group was induced by natural infection, it is possible that vaccination with HiberixH and/or the timing of blood collection during the malaria transmission season might alter the immune response. Therefore, we made separate pooled IgGs from Day 0 IgGs and Day 42 IgGs from HiberixH group in this study.
From these U.S. total IgG and Mali total IgG pools, AMA1(3D7) or AMA1(FVO)-specific IgGs and non-AMA1(3D7) or non-AMA1(FVO) IgGs were prepared individually using AMA1(3D7) or AMA1(FVO) protein immobilized on Sepharose 4 Fast Flow columns as described previously [27] . During the affinity purification process for each total IgG, the flow-through fraction was reloaded to the same AMA1-immobilized column three times to increase the efficacy of separation. Both AMA1-specific IgGs and non-AMA1 IgGs were dialyzed against RPMI 1640 and concentrated to 150,300 ml (AMA1-specific IgGs) or 40 mg/ml (non-AMA1 IgGs) of final product. Because of the limitations of volumes available, AMA1(3D7)-specific/non-AMA1(3D7) IgGs were not prepared from D0-2 or D0-3 total IgG pools.
The standardized methodology for performing the ELISA has been described previously [29] . The absorbance of each test sample was converted into ELISA units using a standard curve generated by serially diluting the standard in the same plate. The ELISA units of each sample were then converted to mg/ml using a conversion factor as described elsewhere [30] . The minimal detection level of the AMA1 antibody in this study was 4 mg/ml, and all responses below that limit of detection were assigned a value of 2 mg/ml for the analysis.
For the competition ELISA, a fixed amount of total IgG from a U.S. vaccinee (designated as US-total IgG), which gives approximately an O.D. value of 3 (approximately 30-40 mg/ml of total IgG in ELISA well), was mixed with 2-fold dilutions of non-AMA1(3D7) IgGs from Malian children (ranging from 2 to 133 mg/ml in ELISA well). The mixtures were tested by ELISA using an AMA1(3D7) or AMA1(FVO)-coated plate using the standard ELISA procedure, and the direct O.D. value was used as a final readout, instead of ELISA units.
The standard methodology for the GIA has been described previously [14] . The assay was performed with purified IgGs at indicated concentrations against the 3D7 or FVO strain of P. falciparum parasites.
For the mixture GIA experiment, 4 mg/ml of non-AMA1(3D7) or non-AMA1(FVO) IgGs were mixed with US-total IgG. The concentration of US-total IgG was determined at which the IgG was expected to give ,60% inhibition in the final mixture in the standard GIA. The growth-inhibitory activity of mixtures was determined using the standard GIA.
The correlation between the two data sets (e.g., anti-AMA1 antibody level and % inhibition in GIA, etc.) was tested by a Spearman rank correlation test. Best-fit formulations of the GIA data were calculated using logarithm-transformed antibody levels.
To evaluate interference effect of non-AMA1 IgG, delta % inhibition was calculated as follows:
Delta % inhibition = (% inhibition of the US-total IgG alone) -(% inhibition of a mixture of a non-AMA1 IgG and US-total IgG).
Data were analyzed using Prism 5 (GraphPad Software, Inc., CA, USA) and p values less than 0.05 were considered significant.
We began with the affinity purification of AMA1-specific and non-AMA1 IgGs in order to evaluate the growth-inhibitory activity of the AMA1-specific fractions and to assess the possibility of interfering antibodies in the non-AMA1 IgGs from the Malian children. The antibody levels of each total IgG pool and non-AMA1(3D7)/AMA1(FVO) IgG were measured by ELISA and the results are shown in Table 1 . The non-AMA1 IgGs had less than 10% of AMA1 antibody levels compared to the corresponding original total IgG pools. These results showed that the AMA1specific purification method used in this study was efficient.
We then investigated the biological activity of the AMA1specific IgGs from U.S. vaccinees and Malian children by GIA ( Figure 1 ). As in our previous study [27] , the AMA1-specific IgGs from U.S. vaccinees showed a significant correlation between antibody level and percent inhibition when they were tested against the homologous strain of parasites (Spearman rank correlation, p,0.001, r s = 0.96, 95% confidence interval (CI) 0.91-0.99 for 3D7; p,0.001, r s = 0.83, 95% CI 0.60-0.94, for FVO) and the relationship followed a symmetrical sigmoid curve when the antibody levels were log transformed (r 2 = 0.93 for 3D7 and 0.82 for FVO). The AMA1-specific IgGs from Malian children showed almost the same level of growth-inhibitory activity at the same level of antibody in the GIA well, regardless of which pools of total IgG were tested. When the data from all AMA1-specific IgGs from Malian children were combined, there were significant correlations between antibody levels by ELISA and percent inhibition in GIA (for 3D7, p,0.001, r s = 0.97, 95% CI 0.92-0.99; for FVO, p,0.001, r s = 0.94, 95% CI 0.86-0.98), and each relationship followed a symmetrical sigmoid curve (r 2 = 0.99 for 3D7, 0.94 for FVO). These results indicate that there was no obvious difference in biological activity of antibodies among AMA1-specific IgGs induced by an AMA1 vaccination (IgGs from U.S. vaccinees), by natural infection (IgGs from D0-1, 2, 3 & 4 and Hib-1, 2 & 3 pools) and by both natural infection and vaccination (IgGs from AMA1-1, 2 & 3 pools).
We next evaluated the activity of non-AMA1 IgGs from Malian children by GIA at two different concentrations in GIA well using homologous strain of parasites (i.e., non-AMA1(3D7) IgGs were tested against 3D7 strain of parasites and non-AMA1(FVO) IgG with FVO parasites). The % inhibition of non-AMA1 IgG was plotted against that of the original total IgG pool ( Figure 2 ). The slope of the best-fit line is 0.97 (95%CI: 0.81-1.14) for the 3D7 data set and 0.87 (95%CI: 0.64-1.10) for FVO. Since the slope was not significantly different from 1, it showed that the depletion of AMA1-specific IgGs from the total IgG did not materially change growth-inhibitory activity, even though AMA1-specific IgGs from the same total IgG pools also showed activity (Figure 1 ).
To assess interference effect in the non-AMA1 IgGs, non-AMA1 IgGs were tested by GIA either by themselves or in the presence of total IgG from a U.S. vaccinee (US-total IgG) against homologous strain of parasites (i.e., non-AMA1(3D7) IgGs were tested with or without US-total IgG using 3D7 strain of parasites in GIA, and non-AMA1(FVO) IgGs were similarly tested using FVO parasites). The US-total IgG was also tested alone as a positive control (black bar in Figure 3 ). The non-AMA1 IgGs showed less than 20 % inhibition at 4 mg/ml (Figure 3 ). The mixtures of non-AMA1 IgGs and US-total IgG displayed lower inhibition for both 3D7 and FVO parasites compared to the UStotal IgG alone. To evaluate the strength of the interference effect of each non-AMA1 IgG, the difference between US-total IgG alone and the mixture was calculated (delta % inhibition). As shown in Figure 4 , the non-AMA1 IgGs purified from total IgG pools with higher AMA1 antibody levels showed greater interference (larger delta % inhibition) than those from total IgG pools with lower titer. When all of the data were combined, there was a significant correlation between the AMA1 antibody level in the original total IgG pool and the interference effect of non-AMA1 IgG (Spearman rank correlation, p = 0.021, r s = 0.80 for 3D7; p = 0.003, r s = 0.82 for FVO).
To test whether the interfering effect of non-AMA1 IgGs was due to the blocking of binding between AMA1 antigen and anti-AMA1 antibody, competition ELISA was performed. A fixed amount of US-total IgG was mixed with serially diluted non-AMA1 IgGs which showed a higher interference effect in Figure 3 (i.e., D0-4, Hib-3 and AMA1-3). The mixtures were applied to AMA1(3D7) or AMA1(FVO)-coated ELISA plates as primary antibodies and the amount of antibodies which bound to AMA1 protein was measured. The mixture with 14 mg/ml of non-AMA1(3D7) IgG in this ELISA using AMA1(3D7)-coated plates had the same ratio of US-total IgG and non-AMA1(3D7) IgG as tested in Figure 3 by GIA using 3D7 strain of parasites, and 11 mg/ml of non-AMA1(FVO) IgG for FVO. As shown in Figure 5 , none of the non-AMA1 IgGs tested blocked binding of anti-AMA1 antibody in US-total IgG to the ELISA plates. The same assay was conducted using total IgG from another U.S. vaccinee and also showed no competition (data not shown). Thus it does not appear that the interfering effect is due to direct inhibition of the anti-AMA1 antibodies binding to the plate antigen.
In the present study, we have shown that the non-AMA1 IgGs from Malian children interfere with the growth-inhibitory activity of anti-AMA1 antibodies obtained from malaria naïve U.S. volunteers vaccinated with AMA1 (US-total IgG). Interestingly, the interference effect of non-AMA1 IgG from total IgG pools with higher titers of AMA1 was higher than those from total IgG pools with lower titers. However, while the non-AMA1 IgGs showed this interference in GIA, they did not block binding of anti-AMA1 antibodies in US-total IgG to AMA1 protein in a competition ELISA.
In the case of viruses, such as hepatitis C virus (HCV) or human immunodeficiency virus (HIV), there are studies showing either monoclonal or polyclonal antibodies can interfere with the effects of other monoclonal or polyclonal antibodies in a neutralizing assay [31] [32] [33] [34] [35] . In these studies, the antibodies recognize different epitopes on the same antigen and the mechanism of the interference may be explained by steric blocking [36] and/or conformational rearrangement induced by the non-neutralizing antibody. A similar phenomenon has been reported with the P. falciparum Merozoite Surface Protein 1 (MSP1), another bloodstage vaccine candidate. In the case of MSP1, a proportion of anti-MSP1 antibodies called ''blocking'' antibodies, which are found in people living in malaria endemic areas, competes with an anti-MSP1 monoclonal antibody capable of inhibiting merozoite invasion of erythrocytes in vitro, as judged by a competition ELISA and by an MSP1 processing assay [37] [38] [39] . However, it is not clear whether such ''blocking'' antibodies interfere with the activity of anti-MSP1 antibody, especially the activity of polyclonal antibodies, in a biological assay, such as GIA. It has been reported that human ''blocking'' antibodies interfere with the invasioninhibitory activity of mouse anti-MSP1 monoclonal antibody (mAb) 12.8, but the same human antibodies did not block the biological activity of another inhibitory mAb 12.10 [37] . To our knowledge, non-AMA1 IgG is the only reported antibodies which have been shown to interfere with the growth-inhibitory activity induced by human antigen-specific polyclonal antibodies. However, it is conceivable that similar functional interference effect exists in antibodies against other malarial antigens, including MSP1.
In contrast to the cases of HCV, HIV or MSP1, the non-AMA1 IgGs tested in this study showed negligible level of binding to the AMA1 protein as judged by ELISA (Table 1 ). In addition, the non-AMA1 IgGs did not compete with US-total IgG in a competition ELISA ( Figure 5 ). We tested up to ,10 times greater ratio of non-AMA1 IgG to US-total IgG compared with the ratio tested in GIA ( Figure 3) where non-AMA1 IgG showed this interference effect, but still there was no competition observed. Since the non-AMA1 IgGs had some residual anti-AMA1 antibodies in the final preparation, one may consider the possibility that the residual antibody in the non-AMA1 IgGs masked the competition effect. However, we think this is not the case. When non-AMA1 IgGs were tested at 11-14 mg/ml (which gave the same ratio of non-AMA1 IgG and US-total IgG as tested in Figure 3 ), the amount of anti-AMA1 antibody coming from the non-AMA1 IgG was less than 5% compared to the one from UStotal IgG. Even at the highest concentration of non-AMA1 IgG tested (i.e., 133 mg/ml), anti-AMA1 antibody from non-AMA1 IgG was less than 30% compared to the one from US-total IgG. Therefore, we concluded the competition effect of non-AMA1 was not obvious if any. These results suggest that either binding between AMA1 protein and non-AMA1 IgGs was too weak to detect by conventional ELISA and/or the mechanism of interference by non-AMA1 IgG is an indirect effect. Not only the function of AMA1 during the invasion process, but also the mechanism of invasion inhibition by anti-AMA1 antibodies is still controversial. Some studies show that a rabbit polyclonal growthinhibitory anti-AMA1 antibody disrupts proteolytic processing of AMA1 [40, 41] and other studies with inhibitory mAb or peptide show that they block complex formation of AMA1 and rhoptry neck proteins RON2, RON4 and RON5 [42, 43] . In our previous study [27] , we have shown that the interfering activity was due to the malaria-specific IgGs in the non-AMA1 IgGs population. However, because the growth-inhibitory activity of non-AMA1 IgGs in the previous study was high (the IgGs were collected mainly from Malian adults), we couldn't measure the strength of the interference effect. In this study with IgGs from Malian children, we could not perform malaria-extract-specific IgG purification from the non-AMA1 IgGs because of the limited quantity of blood samples available. On the other hand, as the intrinsic growth-inhibitory activity of non-AMA1 IgGs from Figure 4 . The correlation between anti-AMA1 antibody levels in the original total IgG pool and the interference effect of the corresponding non-AMA1 IgG. Anti-AMA1(3D7) (A) or anti-AMA1(FVO) (B) antibody levels (mg/ml) in the original total IgG pools (x-axis) are plotted against delta % inhibition of non-AMA1 IgGs (y-axis) tested with P. falciparum 3D7 (A) or FVO (B) parasites. Delta % inhibition of each non-AMA1 IgG was calculated using the data presented in Figure 3 as follows: delta % inhibition = (% inhibition of the US-total IgG alone (black bar in Figure 3 )) -(% inhibition of a mixture of the non-AMA1 IgG and US-total IgG). doi:10.1371/journal.pone.0020947.g004 Malian children is relatively low, we can measure the strength of the interference effect. We found that the interference effect of non-AMA1 IgG from total IgG pools with higher AMA1 titers was greater than that from total IgG pools with lower AMA1 titers. We believe the higher AMA1 titer reflects more malaria exposure, because the Malian children with higher AMA1 titer also display higher titers against other malaria antigens such as MSP1 (our unpublished observation). Therefore, the correlation between anti-AMA1 antibody levels in the original total IgG pool and the strength of interference effect in non-AMA1 IgG suggests that the interference effect is due to antibody against a malaria antigen other than AMA1. If the interference mechanism of the non-AMA1 IgGs is indirect, as the ELISA and competition ELISA results suggest, it is possible that the non-AMA1 IgGs may bind to RON2, RON4 and/or RON5 first and then may block the ability of the anti-AMA1 growth-inhibitory antibody to bind to the critical site of complex formation. Further studies are required to reveal the mechanism of the interference IgGs, and such studies may enhance our understanding of the invasion-inhibition mechanism by human anti-AMA1 polyclonal antibodies.
In the mixture experiment (Figure 3 ), we used non-AMA1 IgGs at 4 mg/ml, while the physiological concentration of IgG in human serum is 10-20 mg/ml. In the previous study where we prepared non-AMA1 IgGs from Malian adults, 4 and 0.4 mg/ml of non-AMA1 IgGs were tested. However, 0.4 mg/ml of non-AMA1 IgGs did not show a clear interference effect [27] . The result suggests that a certain level of non-AMA1 IgGs is needed to detect interference effect in this assay. On the other hand, if we use non-AMA1 IgGs at 10 or 20 mg/ml, several of them show .20% inhibition by themselves (Figure 2 ), so that it is difficult to calculate the interference effect, as the mixture cannot show lower inhibition than non-AMA1 IgG alone. Therefore, we decided to use the same 4 mg/ml concentration as the previous study [27] . The concentration of AMA1-specific IgG in the US-total IgG used for the mixture experiment was at 133 (for 3D7 parasites) or 202 (for FVO) mg/ml, and median level of AMA1-specific antibody in Malian children after immunization was 111.6 mg/ml [28] . Therefore, it is reasonable to assume than non-AMA1 IgGs interfere with growth-inhibitory activity of AMA1-specific IgG at physiological ratio (i.e., mix 10-20 mg/ml of non-AMA1 IgGs with 100 mg/ml AMA1-specific IgG).
This study clearly shows that there are interference IgGs in the 2-3 year old children who are the main targets of a blood-stage vaccine. From a vaccine development point of view, one of the other concerns is whether the AMA1 vaccination by itself induces such interfering IgGs. In our previous study, we did not detect any interference effect of non-AMA1 IgG from U.S. vaccinees [27] . That study suggests that the AMA1 vaccination per se is unlikely to induce the interference IgG at least in a malaria naïve population. In this study, the volume of plasma from each Malian child was too small to make sufficient amounts of AMA1-specific and/or non-AMA1 IgGs for experiments. Even if we collected a larger amount of plasma from each individual child, it is practically impossible to differentiate AMA1-specific and/or non-AMA1 IgG induced by the vaccination from those induced by a natural infection in children living in a malaria endemic area. For that reason, it is difficult to exclude the possibility that AMA1 vaccination induces interfering IgGs in the target population. However, as shown in Figure 4 , non-AMA1 IgGs from children immunized with the AMA1 vaccine showed the same (AMA1-3) or less (AMA1-1 and AMA1-2) interference than those from children without AMA1 vaccination with the same AMA1 levels in the original total IgG pools. Therefore, there is no evidence to suggest that AMA1 vaccination induced more interfering IgGs in this study, rather the data from AMA1-1 and AMA1-2 indicates that the interference antibody may be induced by natural infection, not by the AMA1 vaccination. AMA1 is a highly polymorphic protein [44, 45] and it is obvious that polymorphism is one of the major obstacles to make a broadly effective vaccine [11] . Indeed, when AMA1 vaccines are administered in malaria naïve individuals, the anti-AMA1 antibodies induced by vaccination show growth-inhibitory activity against the homologous strain of parasites, but weaker or no activity against heterologous strains of parasites, regardless of adjuvant used [16, 19, 22] . In this study, we investigated both 3D7 and FVO strains of parasites and the results demonstrate that the interference effect occurs in both allelic forms of AMA1. However, we did not test the heterologous combination, e.g., a mixture of non-AMA1 (3D7) IgGs and US-total IgG tested against FVO strain of parasites. Because the non-AMA1(3D7) IgGs still had anti-AMA1(FVO)-allele-specific antibody in the preparation, which was confirmed by ELISA and GIA (data not shown), it is very difficult to interpret the result of mixture experiments with FVO parasites. We tested a tandem purification method (i.e., total IgGs were applied onto an AMA1(3D7) purification column and an AMA1(FVO) column sequentially) using rabbit anti-AMA1 antibodies, but there were technical problems with completely depleting anti-AMA1(3D7) and anti-AMA1(FVO)-allele-specific antibodies from the total IgGs (data not shown). Further studies could be conducted to test the cross-reactivity of the interfering antibodies if enough volume of starting blood materials is available.
One of the major questions in malaria vaccine development is what assay can serve as a surrogate for clinical protection. At this stage, there is no immunological assay proven to be correlated with clinical protection. As noted above, anti-AMA1 antibodies induced both by a malaria infection [26, 27] and by an AMA1 immunization in malaria naïve individuals [14, 16, 19, [21] [22] [23] show growth-inhibitory activity, and a recent clinical challenge trial with AMA1 vaccine in a malaria naïve population has shown that there is a significant correlation between in vivo parasite multiplicationrate and growth-inhibitory activity measured by in vitro GIA in vaccine recipients (our unpublished observation). In addition, some epidemiological studies have shown that the total growthinhibitory activity (or invasion-inhibition activity) before the malaria transmission season is significantly associated with a subsequent malaria risk [46, 47] . However, other epidemiological studies have not shown such associations [48, 49] . Therefore, one may dispute the usage of this assay for vaccine development. These epidemiological studies did not test the specificity of antibodies. In addition, interpretation of growth-inhibitory activity of samples from epidemiological studies for a specific antigen is not straightforward. As shown in Figure 1 , when anti-AMA1-specific IgGs were separated from total IgG pools of Malian children, they showed similar activity as anti-AMA1 IgG from U.S. vaccinees. However, the AMA1-depleted IgG, i.e., non-AMA1 IgG, displayed the same level of inhibition as the original total IgG pool ( Figure 2 ). We also observed the same phenomenon in our previous study [27] where we separated non-AMA1 IgG from Malian adults' total IgGs. In addition, another of our studies [28] showed that pre-incubation of Malian children's total IgGs with AMA1 protein did not reduce growth-inhibitory activity induced by natural infection (even though the total IgGs had higher level of AMA1 titer), while the pre-incubation did diminish vaccineinduced activity almost completely. All of the data indicate that the overall growth-inhibitory activity induced by a natural infection is not simply the sum of growth-inhibitory activity of individual antibodies. Our preliminary study shows that there is no additive effect of growth-inhibitory activity between rabbit anti-AMA1 antibody and anti-MSP1 antibody (our unpublished observation). Furthermore, it is possible that malaria infection induces not only growth-inhibitory antibodies, but also interfering antibodies in humans, at least in the case of AMA1, as shown in this study. While it is difficult to prove whether such non-additive (and/or interference) effects really occur in vivo, or whether this is just a limitation of the in vitro assay, in either case the growth-inhibitory activity of antibodies from a malaria endemic area for a specific antigen should be interpreted with caution. However, the GIA is the only functional assay widely used for AMA1-based vaccine development. If the growth-inhibitory activity measured by the GIA reflects some mechanism of protection in vivo, the results of this study suggest that pre-existing anti-malaria immunity may modulate the efficacy of the AMA1 vaccine. At a minimum, we believe it is extremely important to take these findings into account in evaluating immunogenicity of AMA1-based vaccines when a study is conducted in populations exposed to malaria. Hepatitis B Virus Genotype G forms core-like particles with unique structural properties SUMMARY: We have determined the structure of the core capsid of an unusual variant of hepatitis B virus, genotype G (HBV/G) at 14 Å resolution, using cryo-electron microscopy. The structure reveals surface features not present in the prototype HBV/A genotype. HBV/G is novel in that it has a unique 36- bp insertion downstream of the core gene start codon. This results in a twelve amino acid insertion at the N-terminal end of the core protein, and two stop codons in the precore region that prevent the expression of HBeAg. HBV/G replication in patients is associated with co-infection with another genotype of HBV, suggesting that HBV/G may have reduced replication efficiency in vivo. We localized the N-terminal insertion in HBV/G and show that it forms two additional masses on the core surface adjacent to each of the dimer-spikes and have modelled the structure of the additional residues within this density. We show that the position of the insertion would not interfere with translocation of nucleic acids through the pores to the core interior compartment. However, the insertion may partially obscure several residues on the core surface that are known to play a role in envelopment and secretion of virions, or that could affect structural rearrangements that may trigger envelopment after DNA second-strand synthesis. Hepatitis B virus (HBV) is one of the most important causes of liver disease in humans. Although HBV infection is preventable by vaccination, it currently affects more than 350 million people worldwide, with many infected as long-term carriers [1] . HBV is a member of the Hepadnaviridae with a partially double stranded DNA genome of 3.2 kb in size [2] . The HBV nucleocapsid core is icosahedral and enveloped by a membrane containing the surface antigen glycoprotein (HBsAg). At the structural level, the nucleocapsid of the prototype HBV (genotype A) has been studied extensively by both cryo-electron microscopy (cryo-EM) and X-ray crystallography [2] [3] [4] . In addition to the presence of HBsAg, two other diagnostic markers can frequently be detected in the serum of infected patients: antibody directed against the core antigen (HBcAg, consisting of assembled capsids) and the e antigen (HBeAg) an alternate translation product of the core gene that is cleaved and secreted posttranslationally. The HBcAg is dimeric, and assembled cores exhibit oligomeric dimorphism: a proportion has T = 3 symmetry (with 180 subunits, consisting of 90 dimers of HBcAg) and the other proportion having T = 4 symmetry (240 subunits and 120 dimers) [5] . This icosahedral dimorphism is present both in cores that are purified from the serum of HBV-infected patients and in core-like particles which self-assemble upon in vitro expression of the core protein. When the HBV virion enters cells, the uncoated core is transported to the nucleus where HBV genomic DNA is repaired to the covalently closed circular DNA form. During replication, the RNA pregenome is incorporated into assembling cores, and reverse transcription to form the genomic DNA occurs within the assembled core [2] . The core protein dimers form spike-like protrusions which extend from the spherical surface of the core particle, and also form a network of pores or holes [2] .
The presence of different genotypes of HBV has been correlated with different geographical regions and with varying severity of disease [6] . Recently, a novel genotype of HBV (HBV/G) was described [7] , which has a unique 36-bp insertion downstream of the core gene start codon, resulting in a twelve amino acid insertion at the N-terminal end of the core protein. In addition, HBV/G has two stop codons in the precore region at positions 2 and 28, which prevent the expression of HBeAg [8] . The fact that HBV/G is almost exclusively detected in patients in association with one of the other seven HBV genotypes (usually HBV/A), and the frequent presence of HBeAg in HBV/G-infected individuals, suggests that HBV/G replication requires co-infection with a helper virus of another HBV genotype [9, 10] . The unique epidemiological and clinical profile of HBV/G suggested that it could be deficient in some other aspects of its function, perhaps as a result of the differences in the capsid structure and function caused by the presence of the N-terminal insertion in the core protein. Although HBV/G has been shown to be capable of replication in the absence of another co-infecting genotype in vitro, the envelopment of genotype G appeared to be less efficient than that of genotype A, while the presence of the 36-nt insertion enhanced core protein expression and genome replication [9] . The aim of this study was to investigate for the first time the structural morphology of the HBV/G core particles in comparison with HBV/A, and in particular to localize the 12 amino acid insertion. We used cryo-electron microscopy and structural modelling to investigate structural differences in the features of HBV/G core-like particles that could influence functions such as replication, genome packaging, or possibly HBsAg envelopment of the capsid and viral secretion.
Preparation and purification of core protein from HBV Genotype G HBV/G-infected individuals were identified through routine diagnostic testing and strain surveillance at the National Microbiology Laboratory (NML) using sequencing and phylogenetic analysis [10] . The gene coding for the HBV/G core protein was PCR amplified, cloned into the pET28b vector (Novagen, EMD Biosciences, Gibbstown, NJ, USA) and expressed in Escherichia coli strain BL21 (DE3). Clarified culture lysates were centrifuged on a 20-60% sucrose gradient at 30 000 rpm in a Beckman SW4OTi rotor for 14 h. Fractions were analysed by SDS-PAGE and Western blotting with anti-HBc antibody (MA1-21697; Pierce Antibody, Rockford, IL, USA). Cores were dialysed against PBS pH 7.4 using a 10 K MW cut off Pierce Slide-A-Lyzer cassette (Thermo Scientific, Waltham, Massachusetts, USA). The HBV/G core sequence was submitted to the National Center for Biotechnology Information GenBank database under accession number GU325783.
Cryo-EM and specimen preparation was performed as previously described [11, 12] . Briefly, specimens on glowdischarged Quantifoil grids (Quantifoil MicroTools GmbH, Jena, Germany) were plunge cooled in liquid ethane using a Vitribot Mark IV (FEI Company). Specimens were imaged at 200 kV in a FEI Tecnai 20G 2 electron microscope equipped with a 4K CCD camera (FEI Company, Hillsboro, Oregon, USA). Images were recorded at a magnification of 80 000·, (corresponding to 108 240· at the CCD detector, or 1.353 Å /pixel) using a total dose of 10 electrons/Å 2 and a defocus of 3.5-6 l. A total of 13 455 particles were analysed. Correction was made for the contrast transfer function using EMAN [13] . T = 3 or T = 4 capsids were separated in SPIDER [14] (Fig. 1b,c) . Two populations of 1887, and 11 568 images of T = 3 and T = 4 capsids, respectively, were analysed.
The T = 4 capsids were analysed, and class averages were calculated for the twofold, threefold and fivefold axes of symmetry. A preliminary icosahedral 3D model was visually inspected using the Chimera software package [15] . Data were then analysed using projection matching [14, 16] . Between each refinement cycle the software package SITUS [17] was used in parallel to select the contiguous mass which corresponded to a single T = 4 HBV/G capsid.
Resolution of the structure was estimated by plotting the Fourier shell correlation (FSC) against resolution, giving a value of 14 Å at an FSC of 0.5. The resolution of the HBV/A model 1QGT.pdb determined by X-ray crystallography [4] was then reduced to 14 Å using the EMAN program Ôpdb2mrcÕ [13] . Both maps were normalized for electron density, aligned and compared by creating a difference map. The contour level of the difference map was selected so that its mass matched the volume of the additional 12 residue N-terminal insert of the HBV/G core structure [17] . The tertiary structure of the 12 residues was predicted by the I-TASSER server [18] , and the resulting coordinates were modelled in to the difference map density HBV/G structure using the program COOT [19] .
Cryo-EM revealed that the HBV/G core protein was able to form T = 3 and T = 4 capsid structures similar to those formed by genotype A (Fig. 1) . Thus, we confirmed that the additional 12 N-terminal residues do not impair capsid formation. Furthermore, the insertion does not affect the basic structure of the core, other than presenting two additional regions of mass at the base of each of the spikes. Several groups have previously engineered N-terminal extensions to the HBcAg and found that this did not impair self-assembly of the expressed proteins into core-like particles [20, 21] . The majority of the HBV/G cores had T = 4 morphology, as previously reported in expressed core-like particles, and in cores isolated from patient specimens [22] .
Superficially, the three-dimensional structure of the T = 4 capsid formed by HBV/G core protein looks very similar to the crystal structure of the HBV/A capsid, PDB accession Fig. 2 Surface shaded representation of the HBV/A capsid structure solved by X-ray crystallography (yellow), and HBV/G capsid structure solved by cryoEM (green) are shown in (a) and (b), respectively. Structures are Fourier filtered to 14 Å resolution for comparison. A difference map (c, shown in blue) was created by subtracting the HBV/A capsid structure from the HBV/G core structure is presented with a mass threshold set to account for the 12 extra residues from the HBV/G cryo-EM map. Panel (d) shows the difference map (blue) superimposed over the HBV/A core crystal structure (yellow). In all four panels, a red oval has been superposed over the reconstruction to highlight a single dimer spike in the structures at the base of the spike.
1QGT (Figs 2a,b) [4] . The difference map created shows additional mass around the base of the spikes of the HBV core protomers (Figs 2c,d) . When the difference map is superimposed with the HBV/A atomic coordinates the additional mass of the HBV/G structure is found to be adjacent to the N-terminal residues of the core protein ( Fig. 2d and Figs 3a, b) . The extra mass protrudes from the surface of the core particle and sits beside the capsid pores and not over them, so this feature would not effectively reduce the diffusive movement of nucleotide triphosphates into the core interior, or affect the release of nucleic acids from the core (Fig. 3a, b) . When modelling the extra 12 residues by TASSER/COOT, it was apparent that there are a number of possible arrangements that 12 residues can take up and still be contained within the corresponding density (Fig. 3c) . Previously, a crystal structure of an HBV capsid protein engineered with an additional 11 residue extension to its N-terminal end has been determined, PDB accession 2QIJ [21] . Although the orientation was different from that observed in HBV/G, both additional masses were pointing away from the four-helix bundle as opposed to interacting with the main body of the core protein (Fig. 3c ).
The possible function or role that the additional N-terminal mass in the core protein of HBV/G may play in replication, and the reason for its retention only in the HBV/G strain is unclear. However, the 36-nt core insertion on HBV/G appears to result in less efficient envelopment of mature virions compared to that of HBV/A [9] .
The HBV capsid plays an important role in envelopment of the virion with membrane containing the HBsAg, which is a prerequisite for the secretion of viable progeny virions [2] . Amino acids in the core protein that are involved in envelopment competence, and which presumably make important contacts with the cytoplasmic domain of membrane-bound preS1 protein, have been mapped, both by mutagenesis [23] [24] [25] and by analysis of naturally occurring patient-derived HBV mutants that vary in their capacities to secrete mature virions [26] [27] [28] . There are thus 11 residues that have been shown to be essential for envelopment but were nonessential for capsid formation. A number of these 11 residues (shown in purple in Figs 3a, b) are found near the electron density made by the extra Fig. 3 (a) and (b) . Stereo images of the HBV/A core protein structure. The red ribbon is the HBV/A core crystal structure, PDB accession 1QGT, and the yellow density is the structure derived from the HBV/A core X-ray crystallography data displayed at 14 Å resolution, to match resolution of the difference map shown in blue. The additional mass found on HBV/G (blue density) was determined by subtracting the HBV/A structure from the HBV/G structure. (a) The mass from the extra 12 amino acids on the N-terminal end of HBV/G is near to the N-terminal end of the HBV/A core (the portion of the ribbon coloured green). (b) Top view looking down one of the spike 2-fold axes. Locations of the amino acids critical for the encapsidation of the mature HBV particle, and that interact with the cytoplasmic domains of the HBsAg, are shown as purple spheres. The extra mass from the 12 amino acid insert may stearically interfere with access to some of these residues (notably S17, F18, L60, L95). (c) Side and top views of HBV/G core protein model (red ribbon) with the12 residue N-terminal insertion (yellow ribbon) modelled into the difference map density (transparent blue). Only one copy of the core molecule within the dimer is shown for clarity.
N-terminal insertion peptide of HBV/G (Fig. 3b) . It is tempting to speculate that the 12 amino acid N-terminal insertion in HBV/G could be responsible for partially reducing the efficient envelopment of HBV/G, because of its proximity to several of these key residues that make contacts with the cytoplasmic domain of HBsAg. In particular, the N-terminal insertion partially covers several of these residues that were shown to be involved in secretion of mature particles, notably S17, F18, L60 and L95 (Fig. 3b) and thus might sterically hinder interactions between the L protein and core particle of HBV/G during envelopment. In addition, in previously published work, Hui et al. [20] showed that an insertion of 23 amino acids engineered on the N-terminal end of the HBV core protein could prevent core envelopment by membranes containing HBsAg. The additional N-terminal residues in HBV/G could interfere with one or more of the essential contacts that are important for core envelopment, leading to attenuated or partially deficient virion maturation. Our data show that the N-terminal insertion in the core of HBV/G partially covers the Ôhydrophobic pocketÕ which contains the residues that are essential for normal virion secretion [22] . This pocket has been shown to undergo structural changes after envelopment, which may act as a trigger, to ensure that envelopment does not take place until after DNA second-strand synthesis has completed [22] . In HBV/G the N-terminal insertion might therefore also interfere with these structural changes, as well as sterically hindering key amino acids that are thought to interact with the envelope. A reduced ability for virion envelopment and secretion might partially explain why the presence of HBV/G in patients is strongly associated with co-infection by another genotype.
Our structural analysis shows that specific antibodies or other ligands directed against the 12 amino acid insertion would be able to readily bind without stearic hindrance. This unique peptide that is exposed on the surface of the HBV/G core could be exploited as a possible site for epitope presentation in an HBcAg core-based vaccine. Because HBV/G already has an insertion in the N-terminus, it may tolerate even larger insertions than have been tried previously in HBV/A cores, while still maintaining the ability for efficient self-assembly. In addition, the possibility of detecting specific antibodies directed against the insertion peptide in sera from patients infected with HBV/G could be explored. Such testing might help to resolve the issue of whether HBV/G can replicate in vivo in the absence of a helper virus strain and would assist in diagnosing patients infected with HBV/G. In-Depth Analysis of the Antibody Response of Individuals Exposed to Primary Dengue Virus Infection Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization. Dengue virus (DENV) complex consists of 4 serotypes. People exposed to primary DENV infections develop robust antibody responses that cross-react with all serotypes (Reviewed in [1] ). Despite the extensive cross-reactivity, individuals only develop long term, protective immunity against the homologous serotype responsible for the primary infection [2, 3] . Indeed, the risk of progressing to DHF is greater during secondary compared to primary infection [4] . A prevailing theory that explains severe dengue during secondary infection is that pre-existing, non-neutralizing dengue specific antibodies enhance DENV entry and replication in Fc-receptor-bearing cells, which leads to a higher viremia and more severe disease [4] . Antibodies have been demonstrated to enhance DENV in cell culture [5, 6] and in animal models of dengue pathogenesis [7] [8] [9] .
Our current understanding of how antibodies interact with DENV and other flaviviruses is primarily based on studies utilizing mouse monoclonal antibodies (MAbs) (Reviewed in [10] ). The DENV envelope (E) protein is the principle target of neutralizing antibodies. Antibody neutralization occurs by blocking critical functions of the E protein, including attachment to host cells and low pH-dependent fusion of the viral and host cell membranes [11] . The crystal structures of the E protein of several flaviviruses have been solved [12] [13] [14] [15] . Individual subunits of E protein consist of three beta-barrel domains designated domains I (EDI), II (EDII) and III (EDIII), with the native protein forming a head-totail homodimer. Mouse MAbs that bind to all three domains of DENV E have been generated and characterized [16] [17] [18] [19] [20] [21] [22] [23] . Although neutralizing mouse MAbs have been mapped to all three domains of E, the most strongly neutralizing MAbs recognize epitopes on the lateral ridge and A strand of EDIII [24] .
Following a primary DENV infection, humans develop antibodies that cross-react with all 4 serotypes, but mainly neutralize the homologous serotype responsible for the infection (Reviewed in [3] ). Studies with human immune sera and, more recently, human monoclonal antibodies have demonstrated that the dominant antibody response is cross-reactive and weakly neutralizing [25] [26] [27] [28] [29] [30] . Multiple viral antigens including E protein, premembrane (prM/M) protein and non-structural protein 1 (NSP1) are recognized by the human humoral response [25] [26] [27] [28] [29] [30] . Nonetheless, few studies have defined the actual epitopes of DENV recognized by type-specific and cross-reactive human antibodies at the structural level and compared this to the epitopes defined using mouse antibodies. The target(s) of dengue type-specific, strongly neutralizing human antibodies remain unknown. The goal of this study was to study two subjects in-depth to define the major antigens and epitopes recognized by antibodies that develop following primary human DENV infection. Defining the human B-cell epitopes on DENV is a key step towards understanding how antibodies can both enhance and inhibit the severity of DENV infections.
Viruses, recombinant proteins and immune sera DENV1 WestPac-74, DENV2 S-16803, DENV3 CH-53489, and DENV4 TVP-360, provided by Dr. Robert Putnak (Walter Reed Army Institute of Research, Silver Spring, MD) were used in the present study [29] . Recombinant envelope (rE) proteins from the 4 DENV serotypes were kindly provided by Dr. Beth-Ann Coller (Hawaii Biotech, Inc) [12] . The recombinant proteins bind to conformational MAbs and X-ray crystallography studies have demonstrated that these proteins retained a native-like structure [12, 13] . Convalescent DENV immune sera were obtained from volunteers who had experienced natural DENV infections during travel abroad. The protocol for recruiting and collecting blood samples from people was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill. Written informed consent was obtained from all subjects before collecting blood.
ELISA plates were coated with 50 ng of purified virus or 100 ng of rE in carbonate buffer at pH 9.6 for 2 hrs at room temperature and incubated with blocking buffer (0.05% TBS-T containing 3% skim milk or 3% normal goat serum) at 37 o C for 1 hr. Human immune sera or hMAbs serially diluted in blocking buffer were added for 1 hr at 37uC followed by alkaline phosphataseconjugated goat anti-human IgG (Sigma) for 1 hr at 37uC. Finally, p-nitrophenyl phosphate substrate (Sigma) was added to each well and the reaction was allowed to develop for 15 minutes before recording optical density at 405 nm on a spectrophotometer.
DENV neutralizing antibodies was measured by a focus reduction neutralization test (FRNT) with Vero cells or using a flow cytometry-based neutralization assay with the U937 human monocytic cell line stably transfected with DC-SIGN as previously described [31] .
Dengue is a mosquito-borne viral disease of humans. The dengue virus complex is made up of four viruses designated as serotypes. People experiencing their first infection develop immune responses that prevent re-infection with the same serotype only. People experiencing a second infection with a new serotype face a greater risk of developing a severe disease known as dengue hemorrhagic fever. Although studies indicate that antibodies can prevent or enhance disease caused by DENV, few studies have explored the specific properties of human antibodies against DENV. The objective of this study was to conduct a detailed analysis of the antibody response of two individuals who had recovered from primary infections. Human antibodies bound to sites on the dengue virus particle including the viral pre-membrane (prM/M) and envelope (E) proteins. Our studies indicate that the human antibody response consists of a minor population of strongly neutralizing antibody and a major population of DENV serotype cross-reactive, non-neutralizing antibody with potential for enhancement of virus and disease. Further studies with more DENV-immune subjects are needed to determine if our findings are broadly applicable to primary infections. 
Peripheral blood samples were obtained from two healthy adult donors who were infected by DENV during foreign travel. The dengue neutralization profiles confirmed previous primary DENV2 (Donor 013) and DENV3 (Donor 033) infections (Table  S1 ). From both donor hMAbs were produced as previously described [32] . B-cells producing DENV specific antibody were identified by screening culture supernatants by flow cytometry for antibodies that bound to C6/36 insect cells infected with DENV2 (Donor 013) or DENV3 (Donor 033) [32] . A secondary screen to identify antibodies that bound to the rE was performed by ELISA as previously described [32] .
Epitope mapping of EDIII binding hMAbs DENV antibody escape mutant viruses were selected for by infecting Vero cells with DENV2 (strain S-16803) in the presence of hMAb concentrations estimated to neutralize greater than 99% of infectious virus (i.e. 1.0 mg/ml for DVC 3.7 and 1.5 mg/ml for DVC 10.16). Equivalent GC copy numbers of control and antibody treated viruses were repeatedly passaged in the presence of hMAbs until equivalent DENV genomic copy numbers were observed for MAb treated and control samples (4-6 passages under antibody pressure). Escape mutant viruses were plaque purified and amplified. E genes were amplified by RT-PCR and sequenced to identify mutations linked to antibody escape. Anti-body binding sites were also mapped by using yeast cells expressing a library of EDIII as previously described [33] . Mutations were mapped onto the DENV2 EDIII structure using the atomic coordinates of DENV2 EDIII (RCSB accession number 1OAN) and displayed using PyMOL Molecular Graphics System, Version 1.3 (Schrödinger, LLC).
The objective of the current study was to characterize the primary human antibody response to DENV by comparing immune sera and MAbs derived from two individuals previously exposed to primary DENV infections. Donor 033 had reported a high fever following a visit to India in 2005 and laboratory investigations confirmed a primary DENV3 infection (data not shown). One year later, when serum and peripheral blood mononuclear cells (PBMCs) were isolated, the subject had a neutralizing antibody response that primarily targeted DENV3 (Table S1) . Donor 013 developed a fever, clinically diagnosed as dengue, while visiting a Pacific Island in 1996. Eight years later when serum and PBMCs were isolated for the current study, the subject had a neutralizing antibody profile consistent with a past primary DENV2 infection (Table S1) .
Initially we characterized the binding properties of serum polyclonal antibodies in both subjects using purified DENVs and recombinant DENV E proteins from the 4 serotypes. We calculated endpoint titers (reciprocal of highest serum dilution that was positive in the assay) to estimate relative levels of antibody against whole virus and rE antigens ( Table 1 ). The quantity of antibody binding to E was only 10 to 35% of the quantity that bound to whole virus suggesting that antibodies bound to sites on the virus that were not present on rE protein (Table 1) . Thus, in both subjects the predominant antibodies binding to dengue virions were serotype cross-reactive and directed to epitopes on the virus and, to a lesser extent, to epitopes on rE protein.
We have previously reported that dengue reactive memory B cells are common following both primary and secondary DENV infection [30] . To further characterize the human B cell response in donor 033, we immortalized memory B cells. PBMCs were isolated and IgG+ memory B cells were immortalized with EBV and CpG as previously described [34] . The immortalized B cell culture supernatants were screened for antibodies that bound to C6/36 insect cells infected with DENV3. Thirty five percent of the B cell cultures generated from donor 033 were positive following this initial screen (Table 2) . From the dengue positive cultures only 7.5% of the cultures bound to rE protein (Table 2) . To further characterize the binding and functional properties of human antibodies, we isolated hMAbs from donor 033. Isolation and characterization of DENV-specific hMAbs from donor 033
To isolate hMAbs specific for DENV from donor 033, positive B cell cultures were cloned by limiting dilution and 16 clones were isolated and expanded. All the hybridomas produced IgG1 with the single exception DV20.10, which was IgG3 (Table S2 ). To identify antibodies that bound to structural viral antigens, the hMAbs were tested for binding to purified DENV3 particles in ELISA. Fourteen out of 16 hMAbs bound to DENV3 virus. Of the 14 hMAbs that bound to DENV3 particles, 13 cross-reacted with all four dengue serotypes dengue complex reactiveand one antibody (hMAb DV51.3) bound to serotypes 1 and 3, but not 2 and 4 (dengue sub complex reactive) (Table S2 ). Dengue virions were solubilized and subjected to Western blot analysis to identify the viral structural proteins recognized by hMAbs. Ten of the 14 hMAbs bound to prM and only a single hMAb (DV64.3) bound to E protein (Figure 1 ). Three hMAbs did not bind to viral antigens by Western blot, although they reacted with DENV3 virion. The 16 hMAbs were also tested for binding to rE protein in ELISA. Only hMAb DV64.31, which had also recognized E protein by Western Blot, bound to rE protein from all 4 DENV serotypes (Table S2) .
We next tested the ability of the 16 antibodies generated from donor 033 to neutralize DENV. Five hMAbs including the two that did not bind to the virion lacked neutralizing activity ( Figure 2 ). The remaining antibodies ranged from weak to moderately neutralizing and had DENV3 50% neutralization titers that ranged from 0.09 to 1 mg/ml (Figure 2A ). The neutralizing antibodies had similar 50% neutralization titers against all 4 serotypes, which was consistent with their broad binding specificity (data not shown). In general, prM antibodies had neutralization curves that were shallow and did not reach 100% neutralization, indicating that a fraction of the virus population was resistant to antibody neutralization ( Figure 2B ). The single E reactive antibody (DV64.31) exhibited a steeper neutralization curve and neutralized 100% of virus at high concentrations ( Figure 2C ). In summary, the hMAbs generated from donor 033, who had experienced a primary DENV3 infection, were broadly cross-reactive, weakly neutralizing, and mainly directed to epitopes on prM. None of the hMAbs mimicked the functional properties of immune serum from donor 033 that displayed strong type-specific neutralization of DENV3 (Table S1 ). A complete summary of the functional profiles of all sixteen hMAbs from donor 033 is included as supplementary material (Table S2 ).
Next, we characterized the dengue specific memory B cell response in donor 013, who had recovered from a primary DENV2 infection. We have previously reported on some of the properties of B cells and hMAbs generated from this donor [30] . Here we expand on these previous results by comparing properties of serum antibodies and hMAbs from this donor and by epitope mapping a subset of hMAbs from this donor. When immortalized B cell culture supernatants from donor 013 were screened for antibodies that bound to C6/36 insect cells infected with DENV2, 28% (567/2016) of the cultures were found positive for DENVspecific B cells following this initial screen ( Table 2) . From the DENV positive cultures only 2.9% of the cultures bound to rE protein (Table 2) . Thus, as in the case of donor 033, although dengue specific memory B cells were frequent, only a small fraction of the positive cultures from donor 013 produced antibodies that bound to rE protein.
Since a relatively unbiased selection scheme for producing hMAbs from donor 033 indicated that most hMAbs were crossreactive, weakly neutralizing and directed to antigens other than rE, we altered the selection scheme for donor 013 to enrich for hMAbs that recognized rE from DENV2. After the initial screening of memory B cell culture supernatants, the relatively rare rE protein binding cultures were selected for cloning and expansion. Ten rE binding hMAbs were produced (all IgG1). When the antibodies were tested for binding to heterologous serotypes, 5 hMAbs were DENV2 type-specific, 2 hMAbs were dengue subcomplexspecific and 3 hMAbs were dengue complex-specific ( Figure 3A) . Six of the ten hMAbs bound to EDIII from DENV2 ( Figure 3A ).
The hMAbs from donor 013 displayed variable neutralization properties ( Figure 3A -C), with 2 non-neutralizing hMAbs (DV1.6, and 21.5; 50% neutralization titers .1 ug/ml) , 4 weakly to moderately neutralizing hMAbs (DV14. 21, 35.3, 25.5 and 18.21 ; 50% neutralization titers between 0.1 and 1 ug/ml) and 4 strongly neutralizing hMAbs (DV3.7, 10.16, 13.6 and 23.13; 50% neutralization titers ,0.1 ug/ml). The strongly neutralizing hMAbs, including those that were cross-reactive in binding assays, neutralized DENV2 better than the other serotypes ( Figure 3) . Thus, by biasing the initial screen for hMAbs that bound to rE protein, we identified hMAbs with neutralization profiles that were more similar to the immune sera of donor 013, which strongly neutralized DENV2.
Several hMAbs from donor 013 bound to EDIII (Figure 3) . Two approaches were used to map the binding sites of these hMAbs on EDIII. Two strongly neutralizing hMAbs that bound EDIII, which were type-(DV3.7) or subcomplex-(DV10.16) specific, were mapped by identifying neutralization escape variants after passaging DENV2 in the presence of each antibody (ired a mutation on EDIII. The virus passaged in the presence of hMAb DV3.7 acquired the single point mutation V382G (Table 3 and Figure 4C ). This residue is located on the EDIII lateral ridge, which is a target of previously mapped type-specific strongly Human Antibody Response to Dengue Infection www.plosntds.org neutralizing mouse MAbs [17, 23] (Table 3 and Figure 4C ). The virus passaged in the presence of hMAb DV10.16 acquired the point mutation E311K (Table 3 and Figure 4C ). This amino acid is located on the A strand of EDIII and forms part of a dengue subcomplex epitope recognized by neutralizing mouse MAbs [18, 23] (Table 3 and Figure 4C ).
As an independent approach to mapping EDIII-reactive hMAbs, we used a yeast surface display assay that has been previously used to map numerous flavivirus antibodies [22, 23, 33] . Table 3 summarizes all mutations that resulted in loss of binding or neutralization of EDIII antibodies generated from donor 013. All the human EDIII-binding MAbs mapped to the lateral ridge Table 3 ) (orange). Ribbon diagram of DENV-2 EDIII was generated from a published X-ray crystallographic structure. The disulfide bond is highlighted in yellow. Human MAbs 3.7 and 25.5 are type-specific antibodies that bind to epitopes centered on the lateral ridge while 10.16 is sub-complex-specific and bind to an epitope centered on the A strand of EDIII. doi:10.1371/journal.pntd.0001188.g004
Human Antibody Response to Dengue Infection www.plosntds.org or the A strand regions that have previously been described as targets of mouse MAbs [23] ( Figure 4C ).
Our main objective was to define viral antigens and epitopes recognized by 2 individuals exposed to primary DENV infections. In both these subjects the dengue virion was a major target of the humoral immune response but many of these antibodies did not bind to rE protein. The DENV virion displays 180 E and 180 prM or M proteins that are arrayed with pseudo-icosahedral symmetry. Depending on the maturation state of the virus particle, the 180 E protein molecules are organized as 90 head-to-tail dimers that lie flat on the virion surface or 60 trimers that protrude as spikes from the surface [35] . Since the E protein, with ,500 amino acids, is considerably larger than the 166 amino acid prM protein, the majority of surface exposed viral protein consists of the ectodomain of E. Thus, our finding that many antibodies bound to epitopes on the virus that were not preserved on rE was somewhat unexpected. These findings are consistent with the results of another study from our group [30] as well as a study by Dejnirattisai and colleagues who observed that many hMAbs that bound to DENV particles did not bind to rE protein [25] . As the rE protein used in the current study lacked ,20% of the protein, which include the membrane proximal regions and the transmembrane domains, it is possible that some antibodies bind to these regions. Moreover, antibodies may recognize E protein epitopes that are only available in the context of the native oligomeric array on the virion. Several hMAbs generated from individuals infected with West Nile virus bound the virion but not rE protein; these hMAbs recognized epitopes that were created by adjacent E protein molecules on the surface of the virion or formed by hinge regions [36] .
When producing MAbs from donor 013, we increased the probability of identifying strongly neutralizing antibodies by biasing the selection scheme for MAbs that specifically recognized rE protein.
In contrast to the weak or non-neutralizing hMAbs isolated from donor 033, several hMAbs from donor 013 displayed strong typespecific DENV2 neutralization. Of note, even hMAbs from donor 013 that bound to more than one serotype (dengue subcomplex or complex MAbs) displayed type-specific neutralization of DENV2, which was the serotype that infected donor 013. Thus, by biasing the selection to enrich for rare rE protein binding antibodies, we generated hMAbs from donor 013 that were functionally similar to the polyclonal immune serum from the same donor that neutralized only DENV2. It is unclear how the results with hMAbs relate to the neutralization properties of polyclonal immune serum from these donors. One possibility is that the abundance of hMABs reflects the functional properties of antibodies in immune sera, where a small fraction of DENV-specific antibodies in immune sera are responsible for neutralization. Alternatively, it is conceivable that individual antibodies that are weakly neutralizing become strongly neutralizing due to cooperative effects that only occur in a polyclonal milieu. Further studies are needed to understand how the properties of hMABs from DENV immune donors relate to the properties of circulating serum antibody.
From the neutralizing MAbs from donor 013 that bound to rE, some bound to the lateral ridge and A strand epitopes on EDIII (ref) . We also identified several neutralizing hMABs (35.3, 18.21, 13.6, 23.13 ) that bound to rE but not EDIII and these antibodies most likely bind to epitopes on EDI or EDII. We are especially interested in mapping the binding sites of these hMABs as recent studies indicate that epitopes other than the lateral ridge and A strand of EDIII can be the targets of strongly neutralizing mouse, horse and primate antibodies [29, 37, 38, 7, 22] , Our results indicating that prM was a dominant target of the primary antibody response are in agreement with other recent studies [25, 30] . The prM protein is required for the proper folding and assembly of flavivirus particles in the endoplasmic reticulum [39] , while also preventing adventitious fusion of the virus [40] in the acidic environment of the trans-Golgi network. Recent studies have demonstrated that DENV virions produced in cell culture are often partially processed and contain a mixture of unprocessed prM and fully processed M [41] . The maturation state and relative amount of prM on a virion can alter the potency of antibodies that bind specific epitopes on E, including the cross-reactive antibodies that bind the fusion loop in DII [42] . Additionally, immature, noninfectious particles became infectious in the presence of prM antibody by enhancing the ability of immature dengue virions to infect Fc-receptor bearing cells in vitro [25, 43] . Given our findings and those of others [25] establishing that prM antibodies are common following primary and secondary DENV infections, more work needs to be performed to address their contribution to dengue pathogenesis in humans.
In summary, the studies reported here demonstrate an unexpected antibody profile in two individuals following primary dengue infection. In both individuals a majority of the DENVspecific human antibodies were broadly cross-reactive and weakly neutralizing. Many antibodies bound to prM and sites on the virus that were not preserved on rE protein. Only a minor fraction of the total dengue specific antibody response was responsible for potent neutralization of the homologous virus. Given the difficulty of identifying suitable donors and generating antigen specific hMAbs, we only characterized the antibody response in two subjects here. Further studies with more dengue immune subjects are needed to determine if our findings are broadly applicable to primary dengue exposure.  Spatial and Temporal Characteristics of the 2009 A/H1N1 Influenza Pandemic in Peru BACKGROUND: Highly refined surveillance data on the 2009 A/H1N1 influenza pandemic are crucial to quantify the spatial and temporal characteristics of the pandemic. There is little information about the spatial-temporal dynamics of pandemic influenza in South America. Here we provide a quantitative description of the age-specific morbidity pandemic patterns across administrative areas of Peru. METHODS: We used daily cases of influenza-like-illness, tests for A/H1N1 influenza virus infections, and laboratory-confirmed A/H1N1 influenza cases reported to the epidemiological surveillance system of Peru's Ministry of Health from May 1 to December 31, 2009. We analyzed the geographic spread of the pandemic waves and their association with the winter school vacation period, demographic factors, and absolute humidity. We also estimated the reproduction number and quantified the association between the winter school vacation period and the age distribution of cases. RESULTS: The national pandemic curve revealed a bimodal winter pandemic wave, with the first peak limited to school age children in the Lima metropolitan area, and the second peak more geographically widespread. The reproduction number was estimated at 1.6–2.2 for the Lima metropolitan area and 1.3–1.5 in the rest of Peru. We found a significant association between the timing of the school vacation period and changes in the age distribution of cases, while earlier pandemic onset was correlated with large population size. By contrast there was no association between pandemic dynamics and absolute humidity. CONCLUSIONS: Our results indicate substantial spatial variation in pandemic patterns across Peru, with two pandemic waves of varying timing and impact by age and region. Moreover, the Peru data suggest a hierarchical transmission pattern of pandemic influenza A/H1N1 driven by large population centers. The higher reproduction number of the first pandemic wave could be explained by high contact rates among school-age children, the age group most affected during this early wave. Although a few quantitative studies have started to shed light on the spatial, temporal, and age specific patterns of mortality and transmissibility levels of historical pandemic events [1] , relatively little is known about the spatial-temporal patterns of the 2009 A/ H1N1 influenza pandemic at different spatial scales. For instance, historical influenza pandemics have been characterized by a disproportionate impact on morbidity and mortality rates among young individuals, a feature that is in stark contrast with seasonal influenza epidemics [2] . Also, influenza pandemics have been found to disseminate in multiple waves occurring over short time periods. Furthermore, out-of-season influenza activity has been documented in spring and summer months in temperate countries during the 1918 influenza pandemic [3, 4, 5] and the recent 2009 influenza pandemic [6, 7] .
The 2009 A/H1N1 influenza pandemic represents a unique opportunity to increase our understanding of the spatial-temporal diffusion patterns of pandemic influenza at different spatial scales, which is crucial for improving public health interventions against future influenza pandemics. In particular, the role of school closure and environmental forcing on the transmission dynamics of pandemic influenza remains debated [8, 9] [10, 11, 12] . Peru is a particularly interesting case study in this respect as it covers a variety of climatic zones associated with diverse influenza seasonal patterns [13] and public health authorities moved the national winter school vacation period by 2-weeks, in an attempt to mitigate the impact of the 2009 A/H1N1 pandemic. Whether the winter school vacation period had any impact on the transmission dynamics of the pandemic in Peru has yet to be evaluated, and this could provide insight into the role of school closures on control of future pandemics [8, 9, 14, 15, 16] . Here we analyze the spatial patterns of age-specific time series of influenza-like-illness (ILI) and laboratory confirmed A/H1N1 influenza cases collected in Peru during 2009. We also quantify the association between local pandemic patterns, absolute humidity, and demographic factors.
Peru is a South American country sharing borders with Bolivia, Brazil, Chile, Colombia, and Ecuador, with a heterogeneouslydistributed population of 28 million (average population density ,22 per km 2 ). The country is divided by the Andes Mountains in naturally distinct regions (highlands, coastal desert and jungle). All regions span the entire length of the country from latitude 3uS to latitude 18uS, and experience different influenza seasonal patterns in inter-pandemic years [13] . Perú is divided into 24 administrative departments composed of 196 provinces ranging in population size from 7000 to about 7.5 million people [17] .
We relied on the epidemiological and virological surveillance network for influenza and other respiratory viruses conducted since 1998 by the Peruvian Ministry of Health (MoH) [18] . In 2006, the MoH of Peru expanded the surveillance network by joining efforts with the US Naval Medical Research Unit -6 (NAMRU-6) and scaled up its coverage to a total of 50 inpatients and outpatients Sentinel health care centers, uniformly distributed across geographic regions of Peru [13, 19] . Moreover, as a result of the global pandemic alert issued by the World Health Organization (WHO) on May 9, 2009, the MoH of Peru intensified surveillance efforts through a public health directive [20] and expanded the identification of influenza-like-illnesses (ILI) to include all public and private health care centers in the country. ILI was defined as a sudden onset of fever ($38uC) and cough or sore throat fewer than five days in duration, with or without general symptoms such as myalgias, prostration, headache, or malaise [21] . Nasal and/or oropharyngeal swabs were taken from a random sample of ILI patients and sent to the Instituto Nacional de Salud and NAMRU-6 for testing by reverse transcriptase polymerase chain reaction (RT-PCR) [18] . Case definitions and laboratory diagnostics did not change throughout the pandemic period covered in our study. However on July 7, 2009, public health authorities ordered prioritization of hospitalized ILI cases for A/H1N1 influenza testing in all health care centers, except for the 50 official sentinel units where testing of mild and severe ILI cases was maintained [22] . A chronology of events relating to the 2009 A/H1N1 influenza pandemic in Peru is given in Table 1 .
We obtained patient age (years), date of symptoms onset, and reporting department (n = 24) and province (n = 134) for ILI and laboratory-confirmed influenza A/H1N1 cases reported between May 1 and December 31, 2009 . We also obtained 2009 population size and density (people/km 2 ) estimates for Peru's provinces and departments, using the National Institute of Statistics and Informatics (http://proyectos.inei.gob.pe/mapas/ bid).
We compiled age-specific time series of ILI and A/H1N1 influenza cases by day of symptom onset for May-December 2009 at the refined spatial scale of provinces (N = 134), as well as the coarser scale of departments (N = 24) (Figure 1 ). For each spatial unit we recorded the cumulative number of cases and peak day, defined as the day with the maximum number of new cases. We also estimated the day of pandemic onset defined as the first day of the period of monotonously increasing cases leading up to the peak of A/H1N1 cases, as in [23] . We then investigated geographic variation in the timing of pandemic onset across departments and provinces and their association with demographic factors and distance from Lima.
Absolute humidity was shown to affect influenza survival and transmission in previous studies [24] , and was linked to the timing of onset of seasonal and pandemic influenza outbreaks in the US [11, 12] . Here we explored the relationship between daily variation in absolute humidity and the temporal profile of the pandemic in the 134 provinces of Peru. For this purpose, we compared daily variation in number of new A/H1N1 influenza cases and average specific humidity in Peru weighted by the total number of A/H1N1 cases reported in each province from May 1 to December 31, 2009. Specific humidity (g/kg) is a proxy for absolute humidity and was calculated from daily averages of temperature, relative humidity, and surface pressure obtained from the National Center for Environmental Prediction-National Center for Atmospheric Research (NCEP-NCAR) global reanalysis [25] .
We estimated the reproduction number, R, using a simple method that relies on the estimation of the growth rate, ''r,'' by fitting an exponential function to the early ascending phase of the epidemic curves in Lima and the rest of Peru [5, 23, 26, 27] . The early ascending phase was determined as the period between the day of pandemic onset (as defined earlier) and the midpoint between the onset and peak days. The reproduction number was calculated by substituting the estimate for r into an expression derived from the linearization of the classical Susceptible-Exposed-Infectious-Recovered (SEIR) transmission model [26, 28] :
where 1/b 1 and 1/b 2 are respectively the mean latent and infectious periods which are assumed to be exponentially distributed. Hence, the mean generation interval between two successive cases is given by T c~1 =b 1 z1=b 2 . As a sensitivity analysis, we also obtained an upper bound estimate for the extreme case of a fixed generation interval (delta distribution), using the following expression [26] :
We assumed a mean generation interval of three (1/b 1 = 1.5 days and 1/b 2 = 1.5 days) and four days (1/b 1 = 2 days and 1/b 2 = 2 days), which are within the range of mean estimates for the 2009 influenza pandemic [29, 30, 31, 32] . We assessed the sensitivity of our estimates to small variations in the definition of the ascending phase used to estimate the exponential growth rate (+/24 days).
The impact of the winter school vacation on the age distribution of cases and pandemic burden
We used two complementary approaches to assess the association between the transmission dynamics of the pandemic in Peru and the 2-week winter student vacation period, which started after the peak of the pandemic on July 16 and ended on August 6, 2009. First, we evaluated weekly trends in the ratio of incident A/H1N1 cases among the student population (ages 5-20 years) to incident cases among all other age groups in the Lima metropolitan area and the rest of Peru. A decline in the ratio of school-age to other cases is suggestive of an impact of school closure on transmission of pandemic influenza. However this approach does not take into account the natural dynamics of the pandemic and rapid depletion of susceptibles occurring in the first weeks of the outbreak, especially among highly connected individuals such as school-age children. To take into account susceptible depletion, we used a second approach based on an agestructured mathematical model of influenza transmission, tailored to the epidemiology of pandemic influenza and the population of Peru (Text S1). We quantified the expected reduction in the final pandemic attack rate as a function of the reduction of the transmission rate among the student population associated with school closing, the timing of the school closing period in relation to the pandemic peak, and R 0 values in the range 1.6-1.8 [29] .
A total of 22,994 ILI cases were reported to the Peruvian Ministry of Health from May 1 to December 31, 2009 . Of all reported ILI cases, 18,139 were laboratory tested for A/H1N1 influenza infection (78.9%), and 8,994 cases were confirmed as having A/H1N1 influenza (39.1%). The average RT-PCR positivity throughout the pandemic was 49.6% (see also Figure  A in Text S1 for time trends in RT-PCR positivity rate). Daily timeseries of ILI and A/H1N1 influenza cases in the greater Lima metropolitan area and the rest of Peru were highly synchronized (Spearman rho = 0.8-0.9, P,0.0001).
Overall testing rates were higher in the greater Lima metropolitan area (86%) than in the rest of Peru (56%), and rates remained relatively stable over the entire pandemic period with an average weekly testing rate of 73.1% (95% CI: 72.6, 73.7). There was an initial increase in average testing rates nationally from 60 to 80% during the first 6-7 weeks of the pandemic ( Figure B in Text S1), a peak testing rate of ,85% during the period of highest incident cases and then a gradual decline until it reached 60% on week 23 of the pandemic. There were no substantial differences in testing rates by age or geographic regions ( Figure C in Text S1).
A total of 134 provinces reported A/H1N1 influenza cases during the pandemic period, May-Dec, 2009. Only 50 provinces reported .50 A/H1N1 cases during the pandemic period and most cases were reported in the most populous departments of Lima and Callao (34%). The regional distribution of A/H1N1 influenza cases in Peru reveals that a first pandemic wave mainly affected the coastal region and peaked on June 22, 2009 . A second more widespread wave peaked in mid-July, 2009 and was synchronized in coastal, mountain, and jungle regions. The jungle region experienced a resurgence of A/H1N1 influenza later in the year, as the daily curve of new cases peaked on September 22, 2009 in this region (see Figure 2 for regional time series of A/ H1N1 cases and Figure 3 for a map). The first wave of the pandemic was concentrated in school-age children in the greater Lima metropolitan area and then disseminated across all age groups ( Figure 4 ). Overall, the median age of A/H1N1 influenza cases was 16 years (range: 0 to 98 years). The age distributions of cases was lower in the Lima metropolitan area than in the rest of Peru with a median age of 14 and 17 years, respectively (Wilcoxon test, P,0.009).
Because timing of pandemic onset was geographically asynchronous in Peru (Figure 1) , we explored the association between pandemic onset, demographic factors, and absolute humidity in the 134 provinces. At the province level, the timing of the pandemic onset was moderately and significantly associated with population density (Spearman rho = 20.44, P = 0.03; Figure D in Text S1). There was no significant association between the timing of the pandemic and population size (rho = 20.25, P = 0.23) or distance from Lima City (rho = 0.31, P = 0.13). Moreover, timing of pandemic onset at the province level was not associated with a decline in absolute humidity levels within 30 days of onset (Spearman rho = 0.16-0.30, P.0.14, Figure E in Text S1).
At the department level, timing of pandemic onset was moderately and significantly correlated with population size (rho = 20.51, P = 0.03) and population density (rho = 20.49, P = 0.03).
Estimates of the reproduction number and their corresponding confidence intervals were obtained for the greater Lima metropolitan area and the rest of Peru (Table 2 and Figure F in Text S1). Using a mean generation interval of three days and assuming exponentiallydistributed latent and infectious periods, the mean reproduction number was estimated to be 1.7 (95% CI: 1.6-1.7) for the greater Lima metropolitan area and 1.3 (1.3-1.3) for the rest of Peru. An upper bound of the reproduction number is also provided in Table 2 , in the extreme case of a fixed generation interval of 4 days, suggesting that R remained below 2.2 throughout the pandemic in Peru. There was no significant trend in testing rates during the exponential phase of the pandemic from which the growth rate was measured (P.0.45). R estimates varied little when the time period selected to estimate the growth rate increased or decreased by four days (difference of 0.1-0.2 for the R estimates for the greater Lima metropolitan area and less than 0.05 for the R estimate for the rest of Peru).
The impact of the winter school vacation on the age distribution of A/H1N1 influenza cases and the final pandemic attack rate
We analyzed the impact of the winter vacation period by monitoring temporal patterns in the ratio of incident student (defined as people 5-20 years of age) to non-student influenza A/ H1N1 cases. At the national scale, this ratio was significantly below 1.0 during the winter school vacations and exceeded 1.0 two weeks following the resumption of school activities on August 6 (Wilcoxon test for differences in ratio of student to non-student cases before and during the winter school vacation period, P,0.001, Figure 5 ). At the level of departments, the average ratio of student to non-student influenza A/H1N1 cases decreased by 46% during the 3-week school closure period as compared to the preceding 6 weeks (T-test for differences in mean ratio, P = 0.007, Figure 6 ). Moreover, the ratio was lower and stayed below 1 for longer in the Lima metropolitan area than in the rest of Peru, consistent with the early pandemic wave changing the age structure of susceptible individuals in Lima in June 2009 (Figure G in Text S1).
Next, we explored the impact of the 2-week winter school break implemented after the peak of the pandemic on July 16, using an age-structured mathematical model of influenza transmission tailored to the population of Peru (Text S1). The predicted reduction in the final epidemic size obtained by reducing the transmission rate within the student population (i.e., ,19 years) when R 0 = 1.6 and R 0 = 1.8 is shown in Figure H in Text S1 for various values of the percentage reduction of the transmission rate of the student population (20%-60%) and the timing of the start of the school closing period occurring a number of days after the epidemic peak. For an R 0 of 1.6, a reduction of 30% in the transmission rate among the student population yields a 4-10% reduction in the pandemic attack rate when the 22-day school closing period takes place 5-20 days after the epidemic peak. A higher R 0 value of 1.8 is associated with an even smaller reduction in the attack rate (4-7%). Overall we found that the reduction in the pandemic attack rate decreases significantly as R 0 increases.
We have conducted a detailed analysis of the spatial-temporal characteristics of the A/H1N1 influenza pandemic in May- December 2009 at two levels of spatial aggregation, relying on a large sample of 18,139 laboratory confirmed cases and 22,994 ILI cases collected by a national epidemiological and virological surveillance system. We found substantial spatial variation in pandemic patterns across Peru, with two pandemic waves of A/ H1N1 cases of varying timing and impact across geographic The serial interval is assumed to be exponentially distributed or fixed, with a mean of three or four days. The epidemic growth phase used to estimate the reproduction number consisted of 12 days for the greater Lima metropolitan area (May 31 to June 11) and 16 for the rest of Peru (June 10 to June 25). See Figure F in Text S1 for exact time periods considered as part of the epidemic growth phase. doi:10.1371/journal.pone.0021287.t002
regions and age groups. The 2-week winter school vacation period in the second half of July 2009 was associated with changes in the age distribution of A/H1N1 cases, and earlier pandemic onset was correlated with large population size in the 24 departments. By contrast there was no association between pandemic dynamics and absolute humidity. Our results suggest that pandemic virus activity began in Lima, consistent with the first case of pandemic A/H1N1 being detected in a Peruvian citizen returning to Lima from New York City on May 9th, 2009. We report a first wave of pandemic A/H1N1 transmission mostly focused on the Lima metropolitan area and peaking in late June, followed by a second wave affecting simultaneously coastal, mountain, and jungle regions of Peru and peaking in mid-July. It is likely that the movement patterns of infected tourists and traders disseminated the infection from the Lima metropolitan area to the rest of the country. A third increase in pandemic A/H1N1 case incidence was reported in the Jungle region in mid to late September 2009 and could be explained by lower population density and reduced connectivity in this region.
We found a significant reduction in the ratio of student to nonstudent cases during the winter school vacation period compared to the weeks before and after the winter break. Nevertheless, the winter school vacation period coincided with the downward phase of the pandemic in Peru. Our simulations based on a simple agestructured transmission model of influenza suggest that the winter school vacation period had a relatively small effect (,10%) on reducing the overall burden of the pandemic. In contrast, school closure interventions implemented during the early phase of the 2009 A/H1N1 pandemic have been associated with a reduction in influenza transmission rate, estimated at 25% in Hong Kong [33] and 29-37% in Mexico [34] .
While a previous study provided preliminary estimates of the reproduction number during the initial pandemic phase in Peru using an early series of confirmed A/H1N1 cases [35] , our study is based on a consolidated dataset of the pandemic with high spatial and temporal resolution. Our R estimate for the first wave in the greater Lima metropolitan area was estimated in the range 1.7-2.2. Estimates of the reproduction number for the second wave concentrated in other regions of Peru were significantly lower than those obtained for the first wave in Lima (R,1.3-1.5). The higher reproduction number of the first wave in Lima could be explained by the high contact rates characteristic of school age children, the most affected age group during this pandemic wave. Lower population density in other regions of Peru compared to the greater Lima metropolitan area could also support a lower reproduction number associated with the second pandemic wave. A recent review of reproduction number estimates for the 2009 A/ H1N1 influenza pandemic in 20 countries reported a range of R between 1.1 and 3.1 with a median value of 1.6 [36] . R estimates have been reported in the range of 1.2-2.4 for community-based settings in Mexico [29, 34, 37, 38] , Japan [39] , New Zealand [40] , Australia [41] , Chile [42] , Ontario, Canada [43] , and the United States [44] , while higher estimates ranging from 2.3 to 3.3 have been obtained during school outbreaks [6, 30, 45] . The variability in published estimates could be attributed to differences in estimation methods and assumptions, including different generation time distributions, inclusion of correction factors to adjust for case underreporting, and differences in the identification of the growth phase period [36] . Heterogeneity in the timing and intensity of intervention strategies, school activity periods, and climatic conditions [11, 24, 34] could have also contributed to differences in reproduction number estimates across locations. We assessed the correlation of the timing of pandemic onset across spatial units as a function of population size, population density and absolute humidity levels. Our results indicate that areas with larger populations experienced earlier pandemic onset at the scale of the 24 Peruvian departments. This observation suggests a hierarchical dissemination pattern of the pandemic driven by large population centers of Peru, reminiscent of the 1918-1919 influenza pandemic in England and Wales [23] and seasonal influenza epidemics in the United States [46] . While absolute humidity has been found to be significantly associated with the onset of seasonal and pandemic influenza epidemics in the United States [11, 12] , we did not find a significant correlation at the province level in Peru. Further analysis of the environmental or social factors influencing the transmission of seasonal and pandemic influenza is warranted in order to fully explain these patterns [47] .
The first pandemic wave in Peru affected mostly school age children in the Lima metropolitan area. This pattern was not the result of testing or age population composition differences since testing rates were consistent across age groups in this region ( Figure C in Text S1), and the population age distribution is very similar across geographic regions of Peru. The second major peak of cases affected all age groups and regions of Peru. Other countries experienced multiple pandemic waves including Mexico, the United States, and the United Kingdom, and Japan [45] [48, 49, 50] whereas a number of countries, particularly in the Southern Hemisphere, have experienced only a single pandemic wave in 2009, including Chile [51] , Argentina [52] , Australia [53, 54] , and New Zealand [53] . Other countries in Europe also experienced a single main wave in fall 2009 [55] .
Recent research has revealed that influenza circulation patterns differ across geographic regions in Peru in inter-pandemic periods [13, 19, 56] . Specifically, in the coastal region surrounding Lima, influenza circulates year round with particularly elevated numbers of cases in the winter time. In contrast, influenza transmission is focused in the cold season in the mountain region and is weakly seasonal in the jungle region where most transmission is limited to the rainy season during the first six months of the year [13, 19, 56] . The timing of the 2009 A/H1N1 influenza pandemic and contemporaneous winter influenza epidemics in Lima is in contrast with the timing of the 1918-1920 influenza pandemic in this city, which occurred during the summer periods of 1918-1919 and 1919-1920 [57] .
There are several strengths and limitations in our study. First, one shortcoming of sentinel surveillance is the potential for sampling and selection bias, which preclude us from calculating reliable incidence rates in our study. Most of the 50 sentinel sites comprising our surveillance network covered populations across all age groups except for six sentinel sites that mostly captured adult populations. These sentinel sites were set up in two hospitals in Lima (Hospital 2 de Mayo and Hospital Edgardo Rebagliati Martins), three military hospitals in Lima, and one military health center in Trujillo (northwestern Peru). On the other hand, sentinel surveillance data can allow the identification of spatial-temporal variations in disease trends and of the viruses associated with those trends using fewer resources than required by a population-based study [13, 19] . A second caveat is related to the assumption that the initial growth rate estimated from Sentinel data closely tracks the ''true'' growth rate of the pandemic in the community, which we use to estimate the reproduction number. In our study .60% of all ILI cases were consistently tested for influenza in all regions throughout the main pandemic period. It is also reassuring that our R estimate for the initial wave was in close agreement with estimates obtained during the early pandemic phase in other countries [29, 37] . Third, the practice of testing changed on July 7 when the Peru Ministry of Health prioritized hospitalized cases for influenza testing in all health centers, except for the 50 original sentinel units. Nevertheless, the resulting decline in testing rates took place after the pandemic reached the peak across all regions of Peru, which eliminates the possibility that our estimates of the reproduction number and correlation analysis could be biased by changes in surveillance methodology.
In conclusion, our work suggests that the 2009 A/H1N1 influenza pandemic in Peru exhibited a rich spatial-temporal pattern with two pandemic waves of A/H1N1 cases of varying timing and impact across age groups and geographic regions. Larger population areas experienced earlier pandemic onset, suggesting a hierarchical influenza transmission pattern reminiscent of past influenza epidemics and pandemics. Overall, our findings suggest that population size, population density, and school activity periods can account for some of the observed variability in influenza pandemic patterns.
Text S1 Supplementary information. (DOC)
We would like to express our gratitude to the people of Dirección General de Epidemiología, the Peruvian national network of epidemiology and the laboratory and database personnel of US NAMRU-6 in Peru for all their hard work during the 2009 A/H1N1 influenza pandemic. We also thank Kamal Barely for assistance with graphic enhancement. This research was conducted in the context of the Multinational Influenza Seasonal Mortality Study (MISMS), an on-going international collaborative effort to understand influenza epidemiological and evolutionary patterns, led by the Fogarty International Center, National Institutes of Health (http:// www.origem.info/misms/index.php).
Disclaimers
The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Ministry of Health of Peru, Department of the Navy, Department of Defense, nor the U.S. Government. NAMRU-6 participation was under protocol NMRCD.2002.0019 approved by the Ministry of Health of Peru and the Naval Medical Research Center Institutional Review Board. Disclosure: None of the authors has a financial or personal conflict of interest related to this study. The corresponding author had full access to all data in the study and final responsibility for the decision to submit this publication.  Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers. Human rhinoviruses (HRV) usually cause self-limited upper respiratory tract (URT) illness. However, they are increasingly reported to be associated with complications, such as asthma [1, 2, 3] , chronic obstructive pulmonary disease (COPD) exacerbations [4] , pneumonia, and bronchiolitis in young children [5] . HRVs have the ability to infect the lower respiratory tract (LRT) [6, 7] and can cause chronic infections in immunocompromised hosts [8, 9] . Unlike enteroviruses, most rhinoviruses replicate optimally at lower airway temperatures, which is thought to explain their URT tropism [10] . Rhinovirus strains infecting the LRT may require specific adaptative genomic changes that have not yet been identified.
For most viral infections, disease severity is the result of a close interplay between viral and host factors. The host immune status is known to play a critical role for viral clearance and resolution of infection. Abnormal innate immune responses to HRV have been observed in individuals presenting HRV-induced COPD or asthma exacerbation [11, 12] . By contrast, reports are rare on the implication of viral determinants in HRV disease severity. Palmemberg et al proposed that the rhinovirus 59UTR polypyrimidine tract may affect virulence [13] . At the serotype or species level, a clear association between the genotype and induced pathology remains to be demonstrated. HRVs were previously classified into two species, HRV-A and HRV-B. In 2006, a third species, HRV-C, was identified [14, 15, 16, 17, 18, 19] and appears to be highly prevalent and circulating worldwide [20, 21, 22] . Several studies have suggested that HRV-C types are prone to induce more severe illness in children [17, 21, 23, 24, 25] .
Due to their error-prone RNA polymerase, RNA viruses are subject to constant evolution. The error rate of picornavirus RNA polymerases has been estimated to range between 10 23 and 10 24 errors/nucleotide/cycle of replication [26, 27] . This variability is a driving force for virus evolution. The number of genera, species, and serotypes illustrates this diversity (http://www.picornaviridae. com) with at least 74 HRV-A and 25 HRV-B serotypes identified, and 61 HRV-C types proposed [22] .
While the evolution of rhinoviruses has been well characterized at the serotype or species level [13, 28, 29, 30, 31, 32] , little is known about the diversity generated during the course of a natural human infection. Several studies have investigated the genomic variability of enteroviruses and vaccine-derived polioviruses during chronic infections in immunocompromised patients, but there is no such report for rhinoviruses [33, 34, 35, 36] .
Our group analyzed recently HRV-39 genome evolution in experimental immunocompetent human infections [37] . We estimated an in vivo mutation frequency of 3.4610 24 mutations/ nucleotides over the entire open reading frame (ORF) during a five-day acute infection period, and identified regions of mutation hot spots in the viral capsid (VP1, VP2 and VP3), and 2C and 3C genes.
In the present study, we performed first a phylogenetic analysis to compare the relative distribution of HRV species or serotypes according to the respiratory site (URT versus LRT) and in protracted infection in hospital patients and lung transplant recipients. In the latter group, we evaluated the frequency of mutations and characterized mutation hot spot regions during the course of five natural human protracted infections, each caused by a different rhinovirus serotype. In one case, we followed intra-host rhinovirus genome variation at different time points in the URT and LRT over a period of 27 months using both classical and ultra-deep sequencing methods.
Written informed consent was obtained from all individuals prior to study participation. The studies were approved by the Institutional Review Board of the University of Geneva Hospitals.
Clinical specimens were extracted by the HCV Amplicor Specimen Preparation kit (Roche, Rotkreuz, Switzerland), TRIzol (Invitrogen, Carlsbad, CA, USA), or Easymag (bioMérieux, Geneva, Switzerland), according to the manufacturers' instructions. Reverse transcription (RT) and real-time PCR with the ''Panenterhino/Ge/08'' primers and probe assay were performed as previously described [38] . Picornavirus-positive samples were detected in patients enrolled in a cohort of lung transplant recipients (September 2008 to November 2010) [6, 39] and in hospital patients screened by the routine laboratory of the University of Geneva Hospitals (February to November 2009) (Table S1 ).
Complete genome sequences were obtained from the LRT and URT samples of five chronically-infected individuals collected at different time points during the course of infection. Fragments were amplified by PCR using degenerate primers designed to anneal highly-conserved sequences of the corresponding HRV reference strain. Overlapping fragments were assembled and, when necessary, specific non-degenerate primers were designed to fill the gaps between the original PCR products. All primers used in this study are listed in Table S2 . PCR (primers 46 and P1.204) and semi-nested PCR (primers 47 and P1.204, Table S2 ) were performed for VP4/VP2 sequences.
PCR products were purified with the microcon columns (Millipore, Zug, Switzerland) before sequencing. Each PCR product was sequenced at least twice. Nucleotide changes were confirmed by a second PCR and sequencing. Chromatograms produced with the ABI Prism 3130XL DNA Sequencer (Applied Biosystems, PE Europe BV, Basel, Switzerland) were directly imported for proofreading with the Geneious Pro 5.0.3 software (Biomatters Ltd., Auckland, New Zealand). All sequences are available at GenBank under accession numbers HM347236-727; JF285163-176; JF285179; JF285186; JF285192,193,195,197,198, 200,204,206-8,210 ,213-214,224-225, JF285228-307.
Alignments were constructed using Muscle [40] with a maximum of 64 iterations. Multiple FastA was converted into PHYLIP format (for tree-building) with the EMBOSS program 'seqret' [41] . Trees were built with PhyML [42] using the GTR model BIONJ for the initial tree and optimized tree topology and branch lengths. Trees with fewer than 50 species used 16 rate categories and larger trees used 8. Transition/transversion ratio, proportion of invariant sites, and the shape parameter (alpha) of the gamma distribution were estimated. Tree processing (including rooting, computation of support values, and displaying) was done with the Newick Utilities [43] .
All statistical data analyses were carried out using the SAS/STAT software version 9.1.3 (SAS Institute Inc., Cary, NC, USA). Associations between HRV species, infection site, and study cohorts were evaluated using chi-square tests when possible, or Fisher's exact tests when some expected values were smaller than 5.
URT and LRT full-length sequences were obtained by amplification of eight overlapping nested PCR products with forward and reverse primers (Table S2) . RT-PCR assays were performed in duplicate to discard any mutations introduced by polymerase errors. We used Platinium Taq DNA Polymerase High Fidelity (Invitrogen) for PCRs, according to the manufacturer's instructions. All amplicons were purified with the microcon columns (Millipore) before sequencing. The eight overlapping PCRs were pooled at equimolar concentrations and used for ultradeep sequencing for each individual sample.
Samples. Libraries were prepared according to the manufacturer's protocol (Illumina, Inc., San Diego, CA, USA) using indexed adapters designed by Fasteris (Fasteris SA, Plan-les-Ouates, Switzerland) (see Methods S1).
Genome analyzer run. Libraries were pooled and sequenced on an Illumina Genome Analyzer GAIIx (Illumina) single-read channel for 76 cycles using a version 4 sequencing kit. We performed base-calling using Illumina pipeline RTA SCS.2.6 and CASAVA 1.6, which produced over 24 million pass filter reads attributed unambiguously or 1.85G bases.
Bioinformatics data analyses. Mapping was performed using the MAQ software version 0.7.1 (http://maq.sourceforge. net/maq-man.shtml). An average of 95% reads were mapped on the ''F78 URT 1.4 m'' consensus sequence for each sample analyzed with a mean coverage of 21.2K reads per nucleotide. Mapping results were used to extract a list of single nucleotide polymorphisms (SNPs) throughout the genome (see Supplementary method: bioinformatics data analyses).
Mutation analysis along the whole HRV genome. Density of mutations was represented with a Gaussian kernel density function using a smoothing band width of 0.1 kernel standard deviation. Curves are shown at the same scale and normalized so that a value of 1 is the highest density found over all genomes. Graphs, including kernel estimates, were produced with the R statistical package.
HRV ORF amino acid conservation plot. The conservation plot was computed with the EMBOSS package Plotcon program using default parameters, except for the sliding window size, which was set to 30 aa. The same program was used in text mode to compute the mean and standard deviations.
We reported previously three cases of patients chronically infected with a unique rhinovirus strain [8] . Since then, we have identified two additional similar cases. All five patients were immunocompromised lung transplant recipients infected both in the URT and LRT (Table 1) . Three were infected with a HRV-A species (HRV-A64, -A24 and -A9), and two with a HRV-B species (HRV-B3 and -B27).
We genotyped strains found in nasopharyngeal specimens (URT), bronchoalveolar lavage fluids (LRT), and strains identified in cases with infections lasting more than three weeks (protracted infection) to assess whether members of the HRV-A, -B and -C species were equally represented in the LRT and/or in the case of protracted infection. Samples were collected from a prospective cohort of lung transplant recipients screened routinely from September 2008 to November 2010, as well as hospital patients screened at the routine laboratory of the University of Geneva Hospitals between February and November 2009 (Table S1 ). VP4/VP2 sequencing was performed on all positive cases whenever possible. Sequences were obtained for 50 HRV-positive specimens collected from the lung transplant recipient cohort, and 94 specimens collected from hospital patients (56 from children, 36 from adults, and 1 with age not available). Phylogenetic analysis was performed on all sequences obtained, as well as corresponding sequences of HRV-A and -B reference types and proposed HRV-C reference types [22] . Of the 144 sequenced samples, 127 represented different infectious episodes with 71 (55.5%) due to HRV-A types, 10 (7.8%) to HRV-B types, and 47 (36.7%) to HRV-C types ( Figure 1 and Figures S1A, B , and C).
No association was observed between a given rhinovirus species and the type of infection (URT, LRT, protracted Upper respiratory tract specimens (URT) are nasopharyngeal swabs or aspirates; lower respiratory tract specimens (LRT) are bronchoalveolar lavage fluids. 2 Partial VP1 sequence of these cases has been reported previously [8] . infection) in the whole study population (Fisher's exact test, 4DF; P = 0.41). HRV-A and -C were present at similar frequencies among LRT infections (22.45% and 19.15%, respectively) in lung transplant recipients and hospital patients (Figure 1) . We found also a good representation of different rhinovirus serotypes in all types of specimens (URT, LRT, protracted infection) ( Figure S1 ). By contrast, there was a clear association between the populations studied (lung transplant recipients versus adult and pediatric hospital patients) and the type of infection (URT, LRT, protracted infection) (Fisher's exact test, 2DF; P,0.05). Six protracted infections were observed; five in lung transplant recipients and one in a 3-month-old infant. Finally, we observed an association between the infecting HRV species and patient age (Fisher's exact test, 2DF; P,0.05). The proportion of HRV-C positive samples (LRT, URT, protracted infection) was significantly higher in pediatric hospital patients than in adult hospital patients (51% versus 17%, respectively) (chi-Square 1DF; P,0.05) ( Figure 1B) .
Complete genome sequences were obtained at different time points in the URT and/or LRT for the five chronically-infected lung transplant recipients ( Table 1 ). The number of substitutions per nucleotide and per day is reported in Table 1 for each patient, except for A46 for whom samples collected at different time points were not available. Calculated mutation frequencies ranged between 7.27610 26 and 3.88610 25 . No overlapping mutations in the URT or LRT were found between the five patients. However, superimposition of all changes along a reference genome (HRV-A2) (Figure 2A ) revealed that whereas synonymous changes were distributed along the genome without any apparent specific pattern, non-synonymous mutations were clustering mostly in the capsid genes VP2, VP3 and VP1, the first half of 2A genes, and in 3A genes. Cold spot regions were located in the viral capsid gene VP4, as well as in 2B, 2C, 3B, 3C, and most of 3D. Only three changes were found in the 59UTR and one in the 39UTR. The variation hot spots observed in the capsid genes were similar to the amino acid variability observed among HRV reference types ( Figure 2B ).
Genetic variation was extensively studied for patient F78 as several samples were available throughout his entire 27-month infection period, including samples collected the same day in the URT and LRT. A phylogenetic tree ( Figure 3A ) performed with 11 complete genome sequences showed that the URT and LRT (1). The mutation rate then slowed down shortly before the patient's death (4.3610 7 and 1.3610 8 viral copy/ml for the last URT and LRT, respectively), despite a high viral load at the end of our follow-up period. This may account for the overall slower mutation rate calculated for this patient when compared with those infected for a shorter period ( Table 1 ). The last three samples collected simultaneously in both the URT and LRT were further analyzed by high throughput sequencing to compare variation at the minority population level ($5% of the total population). Most minority variants present in the LRT after 26.6 months persisted in this site, but were absent in the corresponding URT viral populations ( Figure 3B) . A similar observation was made with the majority variants and demonstrates that the genomes of the URT and Figure 3C ). Specific synonymous changes were found in both the URT and LRT sequences. However, non-synonymous changes were observed only in the LRT where we identified one change in the 59UTR polypyrimidine tract [44] , one non-synonymous change in the VP2 immunogenic site 2, and one non-synonymous change in protein 2A [32, 45] .
Based on our phylogenetic analysis, the respective frequency distribution of strains infecting the URT and LRT did not reveal any apparent correlation between a given HRV serotype or species and their ability to infect the LRT. In both lung transplant recipients and hospital patients, HRV-A and -C strains presented a similar propensity to infect the LRT. No HRV-B strains were found to be associated with LRT, but the relatively small number of infected samples precludes us from drawing firm conclusions. In this respect, we have previously described chronic LRT HRV-B infections [8] . In our study, protracted infections were all found in hosts with a very low level of immunity (mostly lung transplant recipients and a 3-month-old infant). These results suggest that host factors are determinants of disease outcome, rather than virus type.
Five lung transplant recipients were chronically infected with HRV during periods of time ranging from three to 27 months, thus allowing us to study the intra-host genetic variation during natural chronic infections in immunocompromised hosts. Complete Sanger-based and ultra-deep genome sequencing were performed at different time points and sites during infection. Mutation mapping along the HRV genome pointed out that synonymous changes were roughly spread along the entire ORF, whereas non-synonymous changes clustered mostly in the capsid VP2, VP3, and VP1 genes. These capsid genes are also the most variable during acute infections in immunocompetent hosts [37] , as well as the most diverse among the 99 HRV reference types [13] . The VP2, VP3 and VP1 proteins form the outer shell of the virion and are under immune pressure, which explains their fast evolution. The localization of mutation hot spots in these genes in immunocompromised hosts may appear unexpected. However, although these patients are highly immunosuppressed at the time of transplantation, immunosuppressive therapy is then adapted to prevent graft rejection and to preserve a residual cellular and humoral immunity that may account for this observation. This residual immunity is also necessary for the control and recovery of viral and bacterial infections observed in these patients.
We calculated the mutation frequency occurring during these five chronic infections, each caused by a distinct HRV serotype and for various periods of time. Mutation frequency was different in each host and likely reflects different viral replication levels, different host environments, and different durations of infection. The calculated mutation frequency ranged between 7.27610 206 and 3.88610 205 change/nt/day. Recent data on HRV-39 genome evolution over a 5-day acute experimental infection in human immunocompetent volunteers revealed a mutation frequency of 3.4610 24 change/nt (equivalent to 6.83610 25 change/ nt/day) [37] . Therefore, the HRV mutation frequency observed in some immunocompromised hosts infected over months was of the same order of magnitude as that observed in acute 5-day infections.
Extensive analysis of rhinovirus genome modification was performed in the case of one patient (F78) infected for more than two years with a HRV-A9 strain. Phylogenetic analysis of 11 complete genome sequences (including ultra-deep sequences performed at three different time points) revealed that the URT and LRT populations were phylogenetically indistinguishable for a prolonged period of time, either because of co-variation or, more likely, constant viral population mixing. Once potentially adapted to the host through the accumulation of non-synonymous changes mainly in the capsid gene VP1, the viral populations appeared to evolve more slowly. Finally, we observed signatures of putative adaptation to lower airway conditions after several months of infection. Indeed, the last two LRT populations studied presented specific changes in the 59UTR polypyrimidine tract and two non-synonymous changes, one in the VP2 immunogenic site 2 and the second in protein 2A. These changes were absent in the URT population and, upon their appearance, the two populations remained separated as confirmed by ultra-deep sequencing analysis. Although the direct impact of these changes on virus growth ability at lower airway conditions remains to be demonstrated experimentally, the very high viral load observed at this stage suggests that both populations were adapted to their sites.
Taken together, our data suggest that immunocompromised patients cannot clear viral infections as immunocompetent individuals, and represent a potential reservoir for the emergence of new variants and inter-host transmission due to longer chronic viral infection. In addition, these patients may be co-infected by two viruses, thus opening the door to recombination, another putative driving force of rhinovirus evolution [30] . With the emergence of new therapies and progress in transplantation, the population of immunocompromised patients is constantly increasing. Our results suggest that this could accelerate the ability of viruses to adapt to the host, evolve, and propagate and may favor a more rapid emergence of new viral variants. Figure S1 Repartition of protracted and lower respiratory tract infections among the HRV-A (panel A), HRV-B (panel B), and HRV-C (panel C) reference serotypes. VP4-VP2 cladograms of rhinoviruses isolated from lung transplant recipients (blue) and routinely screened hospital patients (red) (Table S1), as well as the 74 HRV-A (panel A), 25 HRV-B (panel B), and 61 proposed HRV-C reference types (black) (panel C). SV1 (GenBank accession number AY064708) was used as an outgroup. LRT and PI are highlighted by arrows and black lines respectively. (PDF)
Table S1 Samples collected from the lung transplant recipient cohort and samples from hospital patients screened by the routine laboratory of the University Hospitals of Geneva. *Samples are designated by the first letter of their family name followed by the last two numbers of their birth year (eg. B48), the month and year of sample collection (eg. 0209), and the site of sampling (U, upper respiratory tract; L, lower respiratory tract).
Table S2 Primers used to amplify and sequence the genomes of the clinical strains described in Table 1 .
Methods S1 Ultra-deep sequencing analysis. (DOC) Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs. Mesenchymal stem cells (MSCs) are present in a variety of tissues during human development, and in particular, are prevalent in adult bone marrow (1) . MSCs isolated from bone marrow (BM) and expanded in vitro in their undifferentiated phenotype, retain an extensive capacity for multi-lineage differentiation into chondrocytes, adipocytes, osteoblasts, and tenocytes under appropriate environmental cues (2) . The presence of specific, distinct antigens identified by the monoclonal antibodies SH2, SH3, and SH4, on the surfaces of marrow-derived MSCs, that are not present on osteocytes and osteoblasts, suggests that these epitopes are developmentally regulated. The antigen which bound to SH2 antibody was identified as endoglin (CD105), a receptor for TGF-β3, which potentially plays a role in mediating the chondrogenic differentiation of MSCs and in their interactions with hematopoietic cells (3) .
Chondrogenesis, the differentiation of MSCs into chondrocytes, is crucial required for skeletal development and maturation, since the cartilage anlage is the model for bone formation. Cartilage development thus includes the differentiation of MSCs into chondrocytes, followed by their maturation, and eventual their hypertrophy and death (4) . Differential gene expression profiling has been widely performed to identify and characterize candidate genes that play potentially important roles in particular biological process (5) .
Although the amount of information regarding the role of growth factors and cytokines as inducers and mediators of MSC differentiation continues to increase, little is known about the gene expression profiling of MSC chondrogenic differentiation. In this study, we employed ABI GeneChips (representing > 30,000 genes) to identify genes differentially expressed during BM-MSC chondrogenesis. ABI is introduced technology based on nylonspotted 60 mer oligonucleotides, that uses on oligomers to detect each gene for most genes, chemiluminescence to measure gene expression levels, and fluorescence to grid to normalize and identify microarray features. The ABI gene list was compiled from information in public and Celera databases (6) . This study was designed to identify differential gene expression profiles and novel genes that might be involved in BM-MSC chondrogenesis. 
Mononuclear cells from BM aspirates were isolated by density Ficoll-Paque gradient separation. BM was placed in a 50 mL syringe containing 5,000 units of preservative-free heparin, diluted 1:1 with phosphate buffered saline (PBS), resuspended in PBS to a final volume of 10 mL, and layered over an equal volume of Histopaque-1,077 (Sigma Chemical Co., St. Louis, MO, USA). After centrifugation at 2,000 rpm for 30 min, mononuclear cells were recovered from the gradient interface, rinsed twice in PBS, adjusted to a concentration of 1.5 × 10 7 cells/10 mL, and seeded onto 100-mm culture plates in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG; 1 g/L glucose, JBI, Seoul, Korea) containing 1% penicillin-streptomycin (P/S; 10,000 units/mL, Gibco/BRL, New York, NY, USA) and 10% (v/v) heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Total numbers of nucleated and viable cells were determined using a hemocytometer and trypan blue (Gibco/BRL, Gaithersburg, MD, USA) staining. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere and allowed to adhere for 72 hr. Non-adherent cells were then removed. The medium was changed twice a week. When cells were 80%-90% confluent, adherent cells were trypsinized (0.05% trypsin, Gibco/BRL) at 37°C for 5 min and replated in 100-mm culture plates. After passage 3, a morphologically homogenous population of adherent cells was obtained. During this expansion, medium was changed every 4-5 days.
MSCs that adhered to spot slide bottoms were fixed with -20°C methanol (100%) for 5 min. Cells were then rehydrated in PBS for 5 min at room temperature, washed three times with PBS, blocked with 3% bovine serum albumin in PBS, and incubated overnight at 4°C with SH2 (American Type Culture Collection [ATCC], Rockville, VA, USA) as a positive control. Primary antibody (SH2) was removed by washing three times with PBS, and cells were then incubated with fluorescein isothiocyanate (FITC)labeled affinity-purified antibody to mouse IgG + IgM (H + L) (DiNonA Inc., Seoul, Korea) for 1 hr at room temperature. Secondary antibodies were removed by washing three times with PBS. Coverslips were mounted onto slides with a solution containing 50% PBS and 50% glycerol. Labeled cells were observed under an Axiovert 200 (Zeiss, Thornwood, NY, USA).
Flow cytometry was performed to determine MSCs positive for SH2. Cells were permeabilized with ice cold 75% methanol in PBS for 30 min at 4°C, and washed three times. A FITC-conjugated SH2 antibody (DiNonA), diluted 1:500 in PBS, was then added, and cells were incubated for 1 hr at 4°C. Cells were analyzed within 2 hr of staining using a flow cytometer (FACScali-bur, Becton Dickinson, Bedford, MA, USA). A total of 1 × 10 6 cells were collected for each measurement. Negative control samples were stained with an isotype-matched irrelevant mAb.
To induce chondrogenic differentiation, 200,000 MSCs were placed in a 15-mL polypropylene tube and centrifuged at 1,000 rpm for 5 min. Pellets were then cultured at 37°C in 5% CO2 and 300 μL of serum-free chondrogenic medium consisting of Dulbecco's modified Eagle medium-high glucose (DMEM-HG, JBI) supplemented with 10 ng/mL of transforming growth factor-β3 (TGF-β3, R&D Systems, Minneapolis, MN, USA), 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbate-2-phosphate, 100 μg/mL pyruvate, and 50 mg/mL ITS + Premix (Becton Dickinson Biosciences, Bedford, MA, USA; 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin [BSA], and 5.35 mg/ mL linolenic acid); the medium was replaced every 2-3 days for 3, 7, 14, or 21 days.
Total RNA was extracted from undifferentiated MSCs and from pellets after 3, 7, 14, or 21 days of differentiation using RNeasy kits (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. And 1,200 μL of RLT buffer supplemented with beta-mercaptoethanol (10 μL/mL) was added to the washed cells. RNA integrity was assessed by gel electrophoresis and RT-PCR and concentrations were determined by measuring absorbance at 260 nm.
Total RNA was processed using the Genesys Applied Biosystem Facility (Genesys, Munster, Germany), according to manufacturer's recommendations. Each RNA pool (2 μg) was labeled with Digoxigenin-UTP using the ABI Chemiluminescent RT-IVT Labeling Kit v 1.0. Double-stranded cDNA was prepared from total RNA. UTP-digoxigenin-labeled complementary RNA (cRNA) was synthesized by in vitro transcription. Labeled cRNA (10 μg) was hybridized to ABI Human Genome Survey Microarray v 2.0, which was then incubated with alkaline phosphatase-linked digoxigenin antibody. Phosphatase activity was then initiated to produce the chemiluminescent signal. Chemiluminescent (cRNA) and fluorescent (spot background) signals of the cRNA and standard control spots were then scanned. Chemiluminescent detection and image acquisition was performed using an Applied Biosystems 1,700 (AB1,700) Chemiluminescent Microarrays Analyzer, according to the manufacturer's instructions. Using the software supplied with the AB1,700 apparatus, the spot chemiluminescent signal was normalized over the fluorescent signal of the same spot to obtain normalized signal value. For inter-array normalization, global median normalization was ap-plied across all microarrays (7).
Data analysis and data normalization were performed using the method described by Quackenbush (8) . For background correction, the mean intensities of areas surrounding spots were subtracted from spot intensities (local area background). Data sets were normalized by dividing the mean intensity value of every spot (in duplicate) by sum of all spot intensities within a sample to eliminate experimental or data acquisition variations. Normalized data were used to calculate the gene expression level ratios of different culture stages. A two-fold expression cut-off was applied. For hierarchical gene cluster analysis, expression ratios were calculated for all genes as described by Eisen et al. (9) .
First strand cDNA was synthesized using reverse transcriptase (RT) and 1 μg of total RNA. Reactions were conducted in 20 μL of buffer containing; 0.5 μL oligo (dT)12-18 primer (Gibco/BRL, Grand Island, NY, USA), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 40 mM DTT, 0.5 mM deoxynucleotide triphosphate (dNTP) mixture (Invitrogen, Carlsbad, CA, USA), 10 unit RNase inhibitor (Gibco/BRL), and 200 units of MMLV reverse transcriptase (Invitrogen). After incubation at 37°C for 60 min, reactions were stopped by heating at 70°C for 15 min. To remove remaining RNA, 1 μL of E. coli RNase H (4 mg/mL) was added to reaction mixtures and incubated at 37°C for 30 min. cDNAs obtained were used as a template for PCR amplification using gene-specific primers for target genes and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are listed in Table 1 . 
The in vitro growth pattern of MSC is shown in Fig. 1 . Human bone marrow-derived MSCs were cultured and expanded. Dur- Table 1 . ing the log phase of growth, cells proliferated with a population doubling time of 30 hr, and this growth period was followed by a confluent growth-arrested phase. Colonies were examined approximately 7 days after initial plating. A morphologically homogeneous population of 90% confluent fibroblast-like cells was obtained after 2 weeks. The cells were replated into culture dishes and cultured for 2 weeks. The replated cells were used for subsequent experiments. The cultured MSCs were positive for SH2 by flow cytometry (Fig. 2) .
MSCs were pelleted into micromasses and differentiated in serum-free medium in the presence of TGF-β3 and dexamethasone. Immediately after centrifugation, the cells appeared as flattened pellets at the bottom of tubes. One day later, pellets had a thickened lip, and between days 2 and 3, pellet became spherical without any increase in size. Pellets then grew in size and pellet diameters increased to about 2-fold on days 7 and 14 (Fig. 3A) .
Using normalized microarray data, we identified 1,486 differen-tially expressed genes (Fig. 4A) , which included 1,303, 121, 40, 20, and 2 genes exhibiting minimum 2 to < 5, 5 to < 10, 10 to < 20, 20 to < 70 and > 70-fold changes, respectively. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes (2-48 fold changes, Table 2 ) were confirmed by RT-PCR.
The expression levels of the 10 genes selected (Hrad 6B, annexin A2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl, and IL-15) were analyzed by RT-PCR, by using total RNAs obtained from 5 samples (Fig. 4B) . The expression levels of 9 genes (Hrad 6B, annexin A2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) were low in undifferentiated cells and increased in differentiated cells by RT-PCR and microarray, but the expression pattern of IL-15 was different. Expression level of IL-15 tended to be decreased in microarray, but increased in RT-PCR (Fig. 5 ).
In this study, we determined gene expression profiles in differentiated chondrocytes and BM-MSCs. The microarray technology used did not allow quantitative comparisons between the expressional levels of different genes, but did allow us to compare fold changes with time and quantify differences in the expressions of multiple genes. Our results show the sequences for gene expressional changes during BM-MSC chondrogenesis. Microarray data showed that Axl, synaptotagmin IV, Hrad6B, peroxiredoxin-1, BMP-7, heat shock transcription factor-2, annexin A2, contactin-1 and serotonin receptor-7 expressions were maintained in differentiating BM-MSCs until day 14.
Axl is overexpressed in a number of tumors (10) , and IL-15 is known to mediate the transactivation and upregulation of Axl with subsequent activation of PI3K/Akt and upregulations of Bcl-2 and Bcl-XL (11) . On the other hand, synaptotagmin IV is required for the maturation of secretory granules in PC12 cells (12) . Human homologues of yeast Rad 6 (Hrad6B) encode ubiq-uitin-conjugating enzymes, and is highly expressed in lung cancer cell. It has been reported that DNA repair and UV mutagenesis are defective in Saccharomyces cerevisiae rad6 mutant (13) . Peroxiredoxin-1 is the most ubiquitously expressed member of the peroxiredoxin family, and is found in the cytoplasm, nucleus, mitochondria, and peroxisomes of many cell types (14) . Furthermore, recent studies have reported high levels of peroxiredoxin-1 expression in the bovine bladder, seminal vesicles, testes, adrenal gland (15) , and in the rat liver, skin, lungs and nervous system (14) . The role of peroxiredoxin-1 in cell differentiation and proliferation suggests that it has a possible role in growth and development.
Recent studies have confirmed that BMP-7 is a strong chemotactic component in cartilage cells produced by mesenchymal stem cells, and it can promote cartilage cells to secrete specific extracellular matrix (proteoglycans and collagen type II). And BMP-7 can induce the differentiation of BM-MSCs into cartilage cells, and that it offers a greater efficiency in repairing cartilage and subchondral bone defects (16) .
Heat shock transcription factor-2 (HSF-2) has been shown to be a transcriptional regulator of heat shock protein gene expression during the differentiation and development of eukaryotic cells in a tissue dependent manner (17) . HSF-2 plays an important role in FGF-2 stimulated osteoclast formation, and HSF-2 deficiency was found to modulate gene expression in stromal/ preosteoblast cells and affect osteoclastogenesis in the bone microenvironment (18) . Annexins bind to negatively charged phos- pholipids in a Ca 2+ -dependent manner and have a conserved structure. The human annexin, annexin A2 (alternative names: annexin II, p36, and lipocortin II) is expressed abundantly in various human organs, including the placenta, lungs, heart, and liver (19) . At the cellular level, annexin A2 is expressed on endothelial cell surfaces and acts as a co-receptor for plasminogen and tissue plasminogen activator (20) . Furthermore, annexins are commonly dysregulated in cancer (21) and annexin A2 is upregulated in a variety of tumors and cancer cell lines (22, 23) . Contactin-1 is a cell surface adhesion molecule, which is normally expressed by neurons, oligodendrocytes, and human astrocytic gliomas (24, 25) . Previous studies have reported that MSCs express IL-15, essential hematopoietic growth factor (26, 27) and IL-15 is also a potent apoptosis inhibitor and has many immunomodulatory activities (28) . The Serotonin receptor 7 is the most recently identified member of the serotonin receptor family and is found in brain, mainly in the hypothalamus, thalamus, hippocampus, and cortex (29) .
In the present study, we performed microarray analysis during BM-MSC chondrogenesis in vitro. We found that over 1,486 genes were expressed by BM-MSCs during chondrogenesis, and we identified genes that were differentially expressed. These data may provide novel information of the genes involved in chondrogenesis of human BM-MSCs. Identification of CD8(+ )cytotoxic T lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in BALB/c mice Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K(93)FITSRCRL and F(57)GYMTFVHF) as CD8(+ )cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2K(d)-restricted CTL epitope, and the latter is an H-2D(d)-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine viral pathogens, and has caused significant economic losses to the swine industry worldwide. Characterization of field isolates suggested that PRRSV are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. Based on significant sequence differencesPRRS viruses are grouped into two distinct genotypes, European isolate (Lelystad virus, LV) and North American isolate (VR-2332) [1] PRRSV has two major structural proteins, GP5 and M, encoded by ORFs 5 and 6, respectively. GP5, the most important neutralizing antigen of PRRSV, has the highest genetic diversity among isolates [2] . And, recent studies in Yorkshire × Landrace crossed and outbred pigs, showed that there are two immuno-dominant T-cell epitopes derived from the GP5 protein: L 117 AALICFVIRLAKNC and K 149 GRLYRWRSPVII/VEK [3] . The M protein, which contains highly conserved amino acid sequences, also has very good immunogenicity and is associated with protection against PRRSV infection. DNA vaccinations have also revealed that M is the most potent inducer of T lymphocyte proliferation [4] .
At present, effective vaccination strategies for the prevention and control of PRRSV infection are not available. Vaccines based on inactivated PRRSV virus have been ineffective at inducing protective immune responses. Live-attenuated PRRSV vaccines can provide protection against this pathogen, but have been observed to revert to virulence [5] , restricting the application of this vaccination approach. The rational development of future PRRSV vaccines will necessitate a systematic understanding of the protective humoral and cellular immune responses that occur during PRRSV infection, and should aim to induce a broad immune responses that accommodates the plasticity of the major antigenic sites. Recent research has indicated that cell-mediated immunity may play a very important role in the clearance of PRRSV [6] . Major histocompatibility complex (MHC) I restricted cytotoxic T lymphocytes (CTLs) can kill virus-infected cells and eliminate potential sources of new virus [7] . Hence, identification of CTL epitopes is crucial in the design of synthetic vaccines, and a number of studies have successfully identified pathogen-derived T cell epitopes [8] [9] [10] [11] .
Unlike many other pathogens, there is limited knowledge of the specific PRRSV-derived peptides targeted by T-cells. In the current study, we report the identification of CTL epitopes from the PRRSV (Ch-1a strain) M protein in a mouse model. We screened peptides derived from the PRRSV M protein for their ability to induce interferon (IFN)-γ in splenocytes harvested from BALB/ c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing M protein. The screen identified two peptides that elicited IFN-γ production in CD3 + CD8 + splenocytes of vaccinated mice. A multiple amino acid sequence alignment among different PRRSV M proteins indicates that these two peptides are strongly conserved across multiple PRRSV strains and therefore should be considered for further research.
The PRRSV Ch-1a strain, the vaccinia virus WR strain, and the Akabane virus OBE-1 strain were part of our laboratory collection. The former was propagated in Marc-145 cells, and the latter two were propagated in BHK-21 cells, respectively, and these two cell lines were cultured in DMEM (Invitrogen) supplemented with 10% of fetal calf serum (FCS, Invitrogen) in a humidified 37°C, 5% CO 2 incubator.
Total RNA was extracted from the PRRSV CH-1a strain and the spleens of BALB/c mice. Full length cDNAs were amplified based on the complete open reading frames (ORFs) of M and ubiquitin (Ub) following reverse transcription (GenBank accession numbers AY032626 and AZ033428) using the M-F and M-R primers pair or the Ub-F and Ub-R primer pair ( Table 1) . The full length cDNAs were cloned into the pMD18-T vector (TaKaRa) and are referred to as pMD-M and pMD-U, respectively.
An indirect enzyme-linked immunosorbent assay (iELISA) method was established to evaluate specific antibody against M protein. A truncated M protein (85-174aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. The truncated M gene cDNA was amplified from pMD-M using the truncated-M-F and truncated-M-R primers (Table 1) , ligated into pGEX-6P-1 (GE Healthcare), and subsequently named pGEX-M. For protein expression, pGEX-M was transformed into E.coli BL21 (DE3) and recombinant protein expression was induced with 0.5 mM IPTG. The samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline (PBS, PH 8.0). After being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot with anti-PRRSV-M monoclonal antibody (our laboratory collection), the fusion-expressed truncated M protein was purified by GST tag according to the manufacturer's instructions (GE Healthcare). Finally, the concentration of the purified protein was determined using a Bradford kit (Bio-Rad) according to the manufacturer's instructions. 
The Ub and M genes were fused using splicing by overlapping extension PCR (SOE-PCR). The Ub and M genes were amplified from pMD-U and pMD-M with the primer pairs Ub-F and Fusion-M-Ub-R and Fusion-M-Ub-F and M-R, respectively ( Table 1 ). The fusion gene product, referred to as Ub-M, was amplified from the purified PCR products with Ub-F and M-R and inserted into the eukaryocyte expression vector pVAX1, and was named pVAX1-Ub-M. The transferring vector pSC11 (our laboratory collection), which is composed of the early promoter P7.5 and late promoter P11 of vaccinia virus, and the LacZ and Amp genes controlled by the promoter P11 as the reporter genes, was used in this study. To construct the transferring vector pSC11-M, the complete M gene was amplified with the pSC11-M-F and pSC11-M-R primer pair (Table 1 ) and inserted into the pSC11. The primer P7.5-F (Table 1) , derived from promoter P7.5 sequence, was used for directional identification and the positive clones, which were subsequently named pSC11-M.
Homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid pSC11-M and the WR strain vaccinia virus into 80% confluent TK -143 cells cultured in MEM medium containing 25 μg/ml BrdU (Sigma). The viruses were collected after the appearance of cytopathic effect (CPE), and recombinant vaccinia virus was purified according to the expression of LacZ gene.
Western blot analysis was performed as described previously [12] . Briefly, a BHK-21 cell layer was infected with rWR-PRRSV-M recombinant vaccinia virus or the vaccinia virus WR strain at a multiplicity of infection (MOI) of 0.1. Cells were harvested 3 days post-infection, and total cell lysates were prepared with lysis buffer (10 mM Tris-Cl pH 7.4, 1 mM MgCl 2 , 0.5% NP40, 20 μg/ml DNase I). Cell lysates were separated by SDS-PAGE and were subsequently transferred to a membrane for Western blot analysis using an anti-PRRSV-M monoclonal antibody, HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad), and DAB substrate. The positive recombinant virus was named rWR-PRRSV-M.
The expression of M was further confirmed by IFA. Briefly, BHK-21 cells were infected with either rWR-PRRSV-M or the vaccinia virus WR strain when the BHK-21 cells reached 70-80% confluence in a 24-well plate. M protein expression was evaluated by IFA 3 days post infection using the anti-PRRSV-M monoclonal antibody followed by a FITC-conjugated rabbit antimouse IgG secondary antibody (Sigma). Specific fluorescence was observed using a fluorescence microscope (LEICA DM IRE2).
The sequences of M were screened for potential H2-K d / H2-D d /H2-L d 9-mer epitopes using the algorithms from the SYFPEITHI website [13] , the HLA peptide binding prediction website (BIMAS) [14] and PRED BALB/c [15] . The 27 peptides (Table 2) , with highest binding score (BS) as predicted by each algorithm, were synthesized by Scilight Biotechnology LLC (Beijing, China) and purified to a purity > 95% using high performance liquid chromatography (HPLC).
All animal experiments were performed according to national and institutional guidelines. One hundred and ninety female BALB/c mice (Vital River Laboratory Animal Technology Co., Beijing, China) were maintained in isolation cages at the Experimental Animal Center of Harbin Veterinary Research Institute (Harbin, China). Mice were divided into three groups: the pVAX1-Ub-M vaccination group (n = 120), the pVAX1 control vaccination group (n = 35) and the PBS control vaccinated group (n = 35). The plasmid DNA used for immunization was purified using the EndoFree Mega plasmid preparation kit (Qiagen). The pVAX1-Ub-M and pVAX1 cohorts were intramuscularly (i.m.) vaccinated with 100 μg pVAX1-Ub-M or pVAX1 plasmid DNA in 100 μl PBS, respectively. The PBS control group received an i.m. injection of 100 μL PBS. Each group was vaccinated four times at 3-week intervals. To enhance the specific CTL responses to M protein, the mice received an intraperitoneal (i.p.) injection containing 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth DNA vaccination. At the same time, five mice from the pVAX1 and PBS vaccination groups were also inoculated intraperitoneally with 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth vaccination in order to compare the specific antibody raised against M protein of different experimental groups following vaccination with rWR-PRRSV-M. All procedures were conducted with the protocols approved by Experimental Animal Center of Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS).
To investigate the specific antibody response, serum samples was obtained from vaccinated mice 7 days after each DNA vaccination and 3 days after boosting with rWR-PRRSV-M. An iELISA was performed according to methods described previously [16] . The purified M protein was used as the coating antigen, the tested sera applied at a 1:10 dilution, and an HRP-conjugated goat anti-mouse IgG antibody (Bio-Rad) used as the secondary antibody. The microplates were developed using orthophenylene diamine (OPDA, AMERSCO) and H 2 O 2 for 10 min, after which the reaction was stopped by the addition of 1 M H 2 SO 4 . Finally, the optical density (OD) was read at 492 nm. An anti-PRRSV-M monoclonal antibody was used as a positive control. Serum containing antibodies against Akabane virus and the sera of control group mice served as negative controls.
Isolated splenocytes were added to U-bottomed 96-well plates (Corning Inc) at 10 6 cells/well in 100 μL complete RPMI-1640 medium supplemented with 10% FCS. The cells were then mixed with 100 μl media containing Note: The 27 peptides with highest binding score (BS) as predicted by each algorithm are shown, starting with the peptide giving the highest score at the top for each protein. Sequences of 9 mers are given from BIMAS, SYFPEITHI and PRED BALB/c predictions, respectively. The N-terminal amino acid position is indicated for epitopes predicted from the whole M protein.
a nonamer peptide derived from PRSSV M protein at 20 μg/ml, or phorbol-12-myristate-13-acetate (PMA 10 ng/ mL) and ionomycin (500 ng/mL) as a positive control. Cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (IBV) H52 strain [17] were used as negative controls. Following a 12 h incubation at 37°C, 10 uM monensin (Sigma) was added and the splenocytes were incubated for an additional 4 h at 37°C before staining.
The number of CD3 + CD8 + T cells producing IFN-γ on days 3, 5 and 12 after boosting with rWR-PRRSV-M was determined using flow cytometric analysis. ICS analysis was performed using a FACSCalibur flow cytometer (BD) according to the methods described previously [11] . Twenty mice were tested from the group vaccinated with pVAX1-Ub-M, and five mice from the groups vaccinated with pVAX1 and PBS were tested at each time point. The splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with M protein-derived peptides, as detailed in section 2.12. Ten thousand CD3 + T lymphocytes were acquired per sample and the number of IFN-γ-secretion CD3 + CD8 + T cells were enumerated. The CD3 + CD8 + lymphocytes that expressed IFN-γ following peptide stimulation were considered to be peptide-specific CTLs. Specific CTL responses were evaluated as the increase in the number of CD3 + CD8 + IFN-γ + cells. Reagents used include PerCP-conjugated CD3e, PE-conjugated CD8a and FITC-conjugated IFN-γ, and BD Cytofix/Cytoperm™, all purchased from Becton, Dickinson & Co (BD).
To further confirm the results of the ICS, the other sixty mice from the group vaccinated with pVAX1-Ub-M and thirty mice from the groups vaccinated with pVAX1 and PBS were tested by the IFN-γ ELISPOT assay at three different time points (as detailed in section 2.13). Data are expressed as the number of IFN-γ-secreting cells per two hundred thousand splenocytes. Peptide-specific IFN-γ ELISPOT responses were considered to be positive if the response (minus media background) was ≥ 3fold above the media response and ≥ 50 spot-forming cells (SFC)/2 × 10 5 splenocytes were registered.
The iELISA results, the percentage of IFN-γ positive CD3 + CD8 + T lymphocytes and the number of spots per 2 × 10 5 splenocytes were analyzed using the analysis of variance (ANOVA), and a probability value below 0.05 was considered significant.
As the full-length M protein is difficult to express in vitro, we expressed a truncated version of M that lacks 84 hydrophobic amino acids at the N-terminus. SDS-PAGE analysis showed that cells transformed with the pGEX-M expression vector produced a large amount of protein with a molecular mass of approximately 36 KDa, consistent with the expected molecular weight of the truncated M protein fused with a GST tag (data not shown). Western blot analysis using an anti-PRRSV-M antibody confirmed the expression and identity of the truncated M protein fused with a GST tag (Additional file 1, Fig. S1 ).
The Ub Proteasome Pathway (UPP) is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. In order to enhance the Ub-mediated degradation efficiency of M protein, we expressed a Ub-M fusion protein in BHK-21 cells using the eukaryotic expression plasmid pVAX1-Ub-M, in which the Ub coding sequence was fused in-frame with the PRRSV M coding sequence. Following transient transfection of BHK-21 cells with the pVAX1-Ub-M plasmid, the Ub-M fusion protein was expressed and accumulated predominantly in the cytoplasm (Additional file 2, Fig. S2 ).
Immunization strategies that prime and boost with recombinant DNA vectors encoding antigens have been shown to elicit T-cell immunity against HIV in non-human primates [18] , and more recently, in humans [19] . Therefore, we used a DNA vector encoding the PRRSV M protein to immunize mice in order to generate and characterize M protein-reactive CD8 + T cells. The recombinant vaccinia virus rWR-PRRSV-M drove expression of an M protein with the expected molecular weight when transfected into BHK-21 cells (Additional file 3, Fig. S3 ). IFA confirmed expression of M protein following transfection of BHK-21 cells, and revealed M protein accumulation in the cytoplasm (Additional file 4, Fig. S4 ).
M protein-specific serum antibody increased steadily from the second to the fourth DNA vaccination with the pVAX1-Ub-M vector which drives M protein expression in vivo (Additional file 5, Fig. S5 ), indicating that DNA vaccination elicited M protein-specific immune responses as expected. A subsequent booster vaccination with the recombinant vaccinia virus, rWR-PRRSV-M, elicited a further increase in PRRSV-specific antibody (P < 0.01, Additional file 5, Fig. S5 ). In contrast, control mice vaccinated with pVAX1-only or PBS-only did not show significant increases in M protein-specific antibody titers after the booster vaccination with rWR-PRRSV-M (P > 0.05, Additional file 5, Fig. S5 ). Overall, mice vaccinated with pVAX1-Ub-M generated significantly higher M protein-specific antibody titers compared to mice vaccinated with pVAX1 or PBS (P < 0.01, Additional file 5, Fig. S5 ). These results indicate that rWR-PRRSV-M amplifies the protective effects of DNA vaccination and reveals the advantage of this priming-boosting strategy. DNA vaccination with pVAX1-Ub-M likely drives the differentiation of memory B cells which are subsequently activated by rWR-PRRSV-M following the booster immunization, resulting in increased M-specific antibody titers. Mice vaccinated with PBS and pVAX1 would not be expected to generate M protein-reactive memory B cells, accounting for a less pronounced increase in M protein-specific antibody titers following rWR-PRRSV-M innoculation.
In this study we identified potential CTL epitopes in BALB/c mice vaccinated with pVAX1-Ub-M and boosted with rWR-PRRSV-M. The frequency and number of CD3 + CD8 + T cells that produced IFN-γ following stimulation with peptides derived from PRSSV M protein was enumerated using ICS and ELISPOT assays. Using these approaches, we identified two peptides from PRSSV M protein that elicited IFN-γ production from the splenocytes of vaccinated mice. As shown in Figure  1 , intracellular IFN-γ staining following stimulation of splenocytes from vaccinated mice with the peptide K 93 FITSRCRL and F 57 GYMTFVHF revealed a population of IFN-γ producing CD8 + T cells comprising 4-5% of the total CD3 + splenocyte population. In contrast, unstimulated splenocytes and splenocytes exposed to an irrevelant peptide did not contain a population of IFN-γ producing CD8 + T cells (Figure 1 , panel B and C). Consistent with the ICS data, peptides K 93 FITSRCRL and F 57 GYMTFVHF elicited IFN-γ production from splenocytes of vaccinated mice when measured by ELISPOT, whereas unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide did not reveal IFN-γ producing cells (Figure 2 ). The K 93 FITSRCRL and F 57 GYMTFVHF PRSSV M protein peptides were identified bioinformatically as H-2K d and H-2D d restricted CTL epitopes (Table 2) . Specific increases in the number of cells producing IFN-γ following stimulation with the peptides "K 93 FITSRCRL" and "F 57 GYMTFVHF" was observed by day 3 after the booster vaccination with rWR-PRRSV-M (Figure 1 and 2) . Furthermore, splenocytes from mice vaccinated with pVAX1-Ub-M responded strongly to "K 93 FITSRCRL" and "F 57 GYMTFVHF", but not other M protein-derived peptides, at all time points tested following the booster vaccination with rWR-PRRSV-M. When the same pattern of reactivity on three different time points after boosting with rWR-PRRSV-M within each vaccination group were analyzed statistically using ANOVA analysis, statistically significant differences were noted for the peptides "K 93 FITSRCRL" and "F 57 GYMTFVHF" when compared to the other peptides tested among mice vaccinated and boosted with pVAX1-Ub-M and rWR-PRRSV-M, respectively (P < 0.01, Figure 3A and 3B). Conversely, and confirming the specificity of these responses, no difference among the stimuli was observed with mock-vaccinated (pVAX1) mice and naïve mice (data not shown).
It is important to recognize that the ICS assay calculates the percentage of IFN-γ + cells among CD3 + T cells in the spleen (4-5%), whereas the ELISPOT assay assesses the number of IFN-γ + cells among all splenocyte cell types (0.05%), and cannot definitively assign the production of IFN-γ to a particular cell type. Thus, the two assays use different denominators in calculating the frequency of IFN-γ + production by splenocytes. Importantly, each method clearly identified K 93 FITSRCRL and F 57 GYMTFVHF as the only two peptides from a panel of 27 that elited significant IFN-γ production from the splenocytes of vaccinated mice. In this study we used BALB/c mice as a model system to identify CTL epitopes in the M protein of PRSSV to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. The identification of PRSSV CTL epitopes in BALB/c mice allow for the future generation of reagents, such as MHC class I: peptide staining reagents, that will enable the in-depth investigations of CD8 + T cell responses during PRSSV infection and immuinization. Whether these two epitopes can bind the SLA class I molecules of pigs remains to be determined. In some cases, specific peptide epitopes are known to be recognized by cytotoxic T cells in different animal species. For instance, the core region of HCV contains an epitope that is recognized by cytotoxic T cells of both mice and humans [19] . Further research on the role of these peptide epitopes in different species is ongoing in our laboratory.
In conclusion, we identified peptides "K 93 FITSRCRL" and "F 57 GYMTFVHF" from the M protein of PRSSV as H-2K d and H-2D d restricted CTL epitopes, respectively. In this study, we also developed a mouse model of PRRSV infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to PRRSV. Network Clustering Revealed the Systemic Alterations of Mitochondrial Protein Expression The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ(0)) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ(0) cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions. Proteomics and expression profiling have been widely applied to understand the cellular processes [1] . Proteins with significant changes in expression have particular interest as markers for various diseases and cellular phenotypes [2] . While there are useful information encoded in the list of differentially expressed proteins, identifying the molecular mechanism of cellular processes from the long list of candidate proteins is challenging [3] . Data-integrative approaches have been successfully applied to address the challenge of interpreting lists of differentially expressed proteins by mapping them onto the protein functional network [4, 5] .
A protein functional network describes the functional and physical associations among proteins and provides a framework for understanding how individual protein work together to perform critical cellular functions and how protein compositions respond to changes in cellular environments [6] . Proteins rarely act alone but rather interact with other proteins and comprise specific functional modules in the network [7] . Functional module is a group of proteins which are related by one or more cellular and genetic interactions such as co-regulation, co-expression, and the member of a biological pathway or a protein complex [8] . Such module is the building block of cellular organization and carries out unique biological process [9] . Therefore, understanding the modular structure of protein functional network should be useful for characterizing the dynamic organization of cellular systems.
In eukaryotic cells, mitochondria are involved in many cellular processes including energy production, apoptosis, ion homeostasis, and the metabolism of glucose, lipids, and amino acids [10] . Although mitochondria possess their own DNA, it is estimated that at least 98% of the 1,500-2,000 mitochondrial proteins are encoded by nuclear genes and shuttled posttranslationally into the mitochondria [11, 12] . In addition, the majority of human mt disorders are known to be related with nuclear genome defects [13] . Thus, compiling a comprehensive list of mt proteins is essential to understand mitochondrial biogenesis and pathology. Large-scale approaches such as mass spectrometry (MS)-based proteomics [14] , epitope tagging combined with microscopy [15] , genome-wide predictions of protein subcellular localizations [16] , and comparative genomics analyses [17, 18, 19] have revealed the localization of the mt protein inventory.
The mt protein inventory is dynamically changed depending on the cellular state, tissue type, and species [11, 20] . For example, the compositions of mt proteins are different across various tissues and organs in mice [21, 22] and changed by fermentation states in yeast [18] or environmental stresses in plants [23] . Additionally, in humans, dynamic changes in the mt proteome affect the functional organizations of mt proteins and disease susceptibilities [24] . Genetic or biochemical abnormalities in mitochondria caused by complete or partial mtDNA depletion have been linked to a wide range of human diseases including metabolic syndrome, neurodegenerative disorders, mitochondrial myopathy, and cancer [12, 25] . We have previously reported that mtDNA depletion can lead to impairments of glucose metabolism [26] , insulin signaling [27] , and apoptosis [28] . However, the changes of the mt protein functional network in response to mt dysregulation remain to be revealed.
Here, we investigated the systemic alterations of human mt protein functional network under normal and dysfunctional mt states through a data-integrative computational biology approach and quantitative proteomic analysis. Specifically, a systematic data-integrative analysis was devised to evaluate the reliability of mt proteomics data and cluster the identified proteins into the modules of mt protein functional network. Our results revealed that human mt proteins comprise specific network modules to control unique biological processes in cells exposed to dysfunctional mt states. Furthermore, network clustering suggests that cells respond to pathological conditions by modulating the coordinated expression and transport of mitochondrial proteins. We believe that our results may provide critical information to gain better understanding of mitochondria function in the cell.
We investigated the systemic alterations of mitochondria by using network analysis of quantified proteomics data. A dataintegrative approach was devised to select reliable mt proteins for network analysis.
First, we applied the isotope-coded affinity tag (cICAT) quantitative analysis [29] to compare protein abundances in mitochondria isolated from normal (r + ) and mtDNA-depleted (r 0 ) osteosarcoma cells. MtDNA-depleted (r 0 ) osteosarcoma cell has been used as an important tool to investigate dysfunctional mitochondria. The r 0 cell was established by long-term treatment of ethidium bromide (EtBr) which intercalated into mtDNA without any detectable effect on nuclear DNA division [30] and led to the inhibition of mtDNA replication and transcription [31] . Thus, we examined a set of nuclear-encoded mt proteins as mt proteome. To quantify protein abundance ratios of mt proteins in r 0 versus r + mitochondria, we labeled mt proteins with cICAT containing light ( 12 C) and heavy ( 13 C) isotope signatures, which react with thiol groups of cysteines in proteins. The experimental scheme is summarized in Figure S1 (see Materials and Methods for details). From the cICAT analysis, we identified 1,121 proteins (Table S1 ). According to the mt protein abundance ratios (r 0 /r + ), we classified all proteins into three classes: up-regulated, downregulated, and not significantly changed proteins in r 0 mitochondria. The number of up-regulated proteins with r 0 /r + $1.5 was 201, while the number of down-regulated proteins with r 0 / r + #0.67 was 313.The thresholds of 1.5 represents the 1.5-fold increase, whereas the threshold of 0.67 represents 1.5-fold decrease under dysfunctional r 0 state. Meanwhile, 607 not significantly changed proteins were present in both r 0 and r + mitochondria with similar abundances (0.67,r 0 /r + ,1.5).
Next, the 1,121 proteins identified via cICAT analysis were evaluated by a systematic data-integrative approach to select more reliable mt proteins ( Figure 1A ). First, we examined the Gene Ontology (GO) cellular component annotation for the identified proteins and compiled 13 reference mt protein datasets from seven mt proteins databases and six mt proteomics datasets (see Materials and Methods for details). In total, 569 out of 1,121 proteins (50.76%) were annotated as mitochondrial proteins and observed from at least one reference mt protein dataset ( Figure 1B) . Then, we assessed the physical and functional links of reference mt proteins based on the assumption that protein pairs that interact or share similar functions tend to cluster within the same subcellular organelle [15] . A total of 201 proteins were physically (82 proteins, 7.31%) or functionally (119 proteins, 10.62%) linked to the 569 reference mt proteins ( Figure 1B ). Physical link represents proteinprotein interaction. Meanwhile, functional link represents a relationship between two proteins if they shared a substrate in a metabolic pathway, co-expressed, co-regulated, or involved in the same protein complex. We listed these 770 mt proteins (569+82+119) in Table S1 as a reliable mt protein dataset. Among the 770 mt proteins, the numbers of down-regulated, up-regulated, and not significantly changed proteins were 288, 122, and 360, respectively ( Figure 1C ). The remaining proteins (351 proteins, 31.31%) were assigned as non-referenced mt proteins that might be either novel mt proteins or proteins with localizations that were affected by mtDNA depletion.
Different cellular states change the expression and localization of proteins targeting mitochondria [22] . To analyze the systemic properties of the mt proteome under the dysfunctional r 0 state, we constructed a network of reliable mt proteins. By using information about the physical and functional links of these proteins, we could map 726 out of 770 reliable mt proteins into a major network (right panel in Figure 1A ; see Materials and Methods for details). The remaining 44 proteins disconnected from the major network were excluded from further network analysis. We discovered that the network is divided into two prominent subnetworks of upregulated (green) and down-regulated proteins (red) based on abundance ratios (Figure 2A ). Interestingly, more links were made
Mitochondria are dynamic organelles that are essential for energy production and cellular processes in eukaryotic cells, and their functional failure is a major cause of ageassociated degenerative diseases. To meet the specific needs of different cellular states, mitochondrial protein repertoires are adjusted. It is critical to characterize the systemic alterations of mitochondria to different cellular states to understand the dynamic organization of mitochondrial systems. In this study, we modularized the quantified proteomics data into protein functional networks to characterize gene expression changes under dysfunctional mitochondrial conditions. Our results demonstrate that mitochondrial protein repertoires changed to compensate for dysfunctional cellular states by reorganizing mitochondrial protein functional network. Through network clustering analysis, we discovered that cells respond to pathological conditions by modulating the coordinated expression/transport of mitochondrial proteins. Network analysis of mt proteins can advance our understanding of dysfunctional mitochondrial systems and elucidate the candidate mt proteins involved in human mitochondrial diseases.
within up-regulated or down-regulated proteins (intraregulatory links) than between up-and down-regulated proteins (interregulatory links). Among the 5,713 links in the network, the majority (4,854 links, 84.96%) were intraregulatory links ( Figure 2B ). When we measured the fraction of links per protein, intraregulatory links were 3-fold more common than interregulatory links (Whitney-Mann U test, p-value = 7.55610 278 ; Figure 2C ). Furthermore, the shortest path length of proteins connected within intraregulatory links was smaller than that of proteins connected within interregulatory links (Whitney-Mann U test, p-value = 7.55610 2156 ; Figure 2D ). Shortest path length is the minimum number of links connecting one protein to another protein in the network, thus a smaller shortest path length implies that two proteins are more closely related [6] . This result indicates that up-regulated and down-regulated proteins tend to cluster themselves and might have distinct functional roles in the protein functional network.
To examine whether up-regulated and down-regulated proteins participate in different biological processes, we examined the enriched function of proteins according to abundance ratios. We discovered that significant biases exist between the two groups when they were classified by GO biological process terms (Table S2 ). Functions of upregulated proteins were involved in mRNA metabolism and cytosolic ribosome-mediated translation, whereas down-regulated proteins were involved in mt ribosome-mediated translation, oxidative phosphorylation, and the TCA cycle (p-value ,1610 23 ).
To identify significantly altered functional groups under the dysfunctional r 0 state, we organized mt proteins into functional modules (right panel in Figure 1A ). We identified modules by examining whether a group of proteins was physically connected or functionally linked. We used hierarchical average-linkage clustering with the STRING confidence scores as a similarity measure (Materials and Methods for details). Then, we assigned the biological function to the module by examining the representative functional annotations from proteins in each module. Finally, we identified 13 functional modules that were enriched either within up-regulated or down-regulated protein groups (p-value ,0.01; Table S3 ). As shown in Figure 3A , five functional groups were up-regulated (shown in green boxes), whereas eight functional groups were down-regulated (shown in red boxes) under the dysfunctional r 0 state. The five up-regulated groups contain 59 out of 89 (66.29%) up-regulated proteins. They are linked to the function of cytosolic ribosome, ribonucleoprotein complex, protein folding on mt surface, DNA repair, and proteolysis, which are associated with the regulation of mt protein expression in response to mtDNA damage ( Figure 3B ). Conversely, the eight down-regulated groups include 138 out of 175 (78.86%) down-regulated proteins. They were associated with basal mt functions, such as mitochondrial energy production, metabolism, and protein folding in mitochondria ( Figure 3C ). Our result suggests that the expression control of mt proteins that are involved in different functional modules is regulated separately under dysfunctional mt states.
Expression profile analysis of mt protein and mRNA under a dysfunctional mt state Eukaryotic cells can monitor and respond to changes in mt conditions through alterations of nuclear gene expression [32] . To understand how cells respond to the dysfunctional r 0 state to facilitate survival, we analyzed the expression profiles of mt proteins and mRNAs. We found that 40% of mt proteins (274 = 127+147) exhibited a positive correlation between protein and mRNA abundances, while 43% of mt proteins (296 = 78+218) exhibited a negative correlation between protein and mRNA abundances ( Figure 4A ). By using the kmeans clustering algorithm, the mRNA and protein-expression profiles were divided into five groups depending on the abundance ratios of mt protein-mRNA: up-up (127), downdown (147), up-down (78), down-up (218), and unchanged (118) groups ( Figure 4A and Table S4 for protein list). We found that up-regulated and down-regulated functional modules exhibited distinctive patterns in expression profiles ( Figure 4B and C; details in Table S3 ). Specifically, both protein and mRNA abundances increased in the five up-regulated functional modules ( Figure 4B ). Conversely, mRNA abundances increased, but mt protein levels were decreased in the eight down-regulated functional modules ( Figure 4C ). The upregulated functional modules were involved in mtDNA maintenance and control of mt protein transport. The downregulated functional modules were associated with mitochondrial energy production and metabolism. These results suggest that cells actively modulate the expression and transport of mt proteins depending on the functions necessary to survival under pathological conditions.
To validate protein abundance ratios measured by cICAT, we performed western blot analysis by using isolated r + and r 0 mitochondria and confirmed that the tested protein abundances were largely consistent with those measured by cICAT ( Figure 5 ). We selected eight up-regulated and nine down-regulated proteins from each functional module, of which antibodies were commercially available. We found that the levels of seven out of the eight up-regulated proteins were increased in r 0 mitochondria ( Figure 5A ). The up-regulated proteins were eIF4A1, PLG, HNRPM, XRCC6 (Ku70), XRCC5 (Ku80), APEX1, and STUB1. Likewise, the levels of all tested down-regulated proteins were decreased in r 0 mitochondria, consistent with the cICAT quantifications ( Figure 5B ). The down-regulated proteins were GTPBP3, NDUFS6, ATP5A1, ALDH6A1, GLUD1, MTRF1, TFAM, HSPD1 (HSP60), and ALDH2. The change of PARP1 levels was not detected by western blot. Additionally, the reliability of the protein expression patterns of cICAT was further confirmed by comparing the expression patterns of previously reported 2DE proteomic analyses [33] . Thirty of the 33 mt proteins (90.91%) identified from the 2DE proteomic analyses showed similar expression patterns compared to those obtained from the cICAT analysis ( Figure S2 ). The difference between protein expression patterns of cICAT and 2DE was insignificant (p-value was only 0.08).
To verify the localizations of the identified mt proteins, we selected five proteins and cloned their cDNAs to express GFPhybrid proteins. We examined the localization annotations of 264 proteins involved in functional modules and found 20 proteins that have not been annotated their localization in GO database. Then, we chose five proteins that have available antibodies, which were significantly changed under r 0 mitochondria. Specifically, ZCD1 in mitochondrial ribosome function, GPT2 and PYCR2 in amino acid metabolism, CTSD in proteolysis, and HSPBP1 in protein folding on mt surface modules were tested. We confirmed that all five proteins localized in mitochondria and merged perfectly with the red fluorescence of the proteins with mt signal sequence ( Figure 6 ).
Mitochondrial dysfunction caused by mtDNA damage is involved in many diseases and is likely to be a driving force behind aging and apoptosis [34] . Here, we investigated the systemic alterations of mt protein expressions by using a dataintegrative network analysis. Respiratory-deficient r 0 cells have been studied to characterize retrograde signaling, which is a controlling mechanism of information flow from the mitochondria to the nucleus and cytoplasm [35] , but the systems property of dysfunctional mt state has not been studied extensively. Through network clustering analysis, we discovered that cells respond to pathological conditions by modulating the coordinated expression/transport of mt proteins.
Using a network analysis of proteomics data, we were able to find modules reflecting differentially regulated functions between normal and dysfunctional mt states. We found that up-regulated and down-regulated proteins of dysfunctional mt states were organized into two predominant subnetworks that exhibited distinct biological processes. It has been suggested that proteins cooperate with other proteins as a part of functional module which tend to physically associated or share similar expressions to deliver a certain biological function [36, 37] . Network analyses of molecular pathways or complexes elucidated the collective behavior of differentially expressed proteins and provided complementary information to conventional single gene-based analysis which routinely performed in proteomic analyses [38, 39] . We discovered that not only the relevant proteins changed their expression under dysfunctional mt state, but also the subnetworks composed of multiple functional protein groups changed their expression cooperatively to regulate biological processes. These network-module alterations are particular importance for relating an altered phenotype with dysfunctional mt state at molecular level because phenotypic alterations are more closely related with pathway remodeling than individual gene expression changes [38] .
We found that functional modules controlling mt protein translation, folding, proteolysis, and mtDNA repair were upregulated in the dysfunctional r 0 state (Figure 3 ). To properly respond to changes in cellular states, these processes may require retrograde regulation [35] . Retrograde signaling regulates many cellular activities under pathophysiological conditions by changing the protein inventories of subcellular organelles. It may be possible that up-regulated mt proteins shuttle between the mitochondria and other organelles for intracellular communication [40] . Indeed, we found that the proteins of up-regulated functional modules tended to have multiple annotations of subcellular localization and to be involved in processes occurring inside and outside of mitochondria (Table S1 ). For example, AP-endonuclease 1 (APEX1) of the DNA repair module is known to shuttle from mitochondria to the nucleus in response to oxidative stress [41] . Heterogeneous nuclear ribonucleoprotein K (HNRPK), a member of the ribonucleoprotein complex associated with mtDNA transcription, has been detected in the nucleus, cytoplasm, and mitochondria [42] . These observations suggest that up-regulated functional modules act as cross-talk components connecting mitochondria with other organelles to sense and propagate the We observed that up-regulated and down-regulated functional modules contain a small fraction of proteins with opposite protein expression patterns (Figure 3 ). On average, levels of 5.5% of the proteins in down-regulated functional modules were increased, while levels of 4.7% of the proteins in up-regulated functional modules were decreased under the dysfunctional r 0 state. Proteins with opposite expression patterns in the functional module could act as negative regulators of the module [43] . For example, in the TCA cycle module, acetolactate synthase (AHAS) was upregulated, while other proteins were down-regulated. It has been reported that upregulation of AHAS decreases the activity of the TCA cycle by reducing pyruvate flow into the cycle [44] . In addition, ATP6V1B2 and ATP6V1G1 in the ATP synthesis module were up-regulated, while other proteins were downregulated. Upregulation of these proteins is known to participate in ATP hydrolysis, leading to the downregulation of ATP synthesis [45] . Moreover, two heat shock proteins, HSP90AA1 and HSP90AB1, involved in protein folding on the mt surface were down-regulated, while other proteins were up-regulated. The heat shock proteins regulate chaperone activity in response to ATP concentrations in the cell [46] . Under the dysfunctional r 0 state, ATP reduction could induce the disruption of HSP90 chaperone activity. Thus, we suspect that oppositely expressed proteins may compensate for the function of other proteins in the same modules.
The expression of up-and down-regulated proteins was controlled in several different levels. From the comparisons between protein and mRNA abundance under the dysfunctional r 0 state, we found that most up-and down-regulated mt proteins had increased mRNA expression levels ( Figure 4 ). This indicated that up-regulated proteins were successfully recruited into r 0 mitochondria. It has been proposed that there is a compensatory mt protein import pathway independent from the dissipated membrane potential [47] . This pathway is known to facilitate the translocations of proteins involved in mt protein folding and mtDNA repair to the mitochondria [48] . It implies that ''emergency'' proteins are increasingly imported to dysfunctional r 0 mitochondria to repair pathophysiological mt conditions. The cross-talk properties of up-regulated functional modules support this idea.
One might expect that protein abundances in cytoplasm and mitochondria to be correlated with mRNA expression levels. However, we found that the functional modules of down-regulated proteins have up-regulated mRNA levels, suggesting that downregulated proteins might experience difficulties in protein import into r 0 mitochondria. To understand the differences of mRNA and protein expression of those proteins, we examined the levels of protein expression in r 0 cytoplasm and mitochondria by using western blot analyses. The tested proteins, GLUD1, GTPBP3, ATP5A1, TFAM, and NDUFS6, were selected from each downregulated functional module. We found that the cytoplasmic protein levels of GLUD1 (amino acid metabolism), GTPBP3 (tRNA metabolism), and ATP5A1 (ATP synthesis) were increased, whereas the mitochondrial levels of those proteins were found to be decreased ( Figure S3 ), suggesting that those proteins remained in the cytoplasm and did not properly transport into mitochondria. Meanwhile, both cytoplasmic and mitochondrial protein levels of TFAM (mtDNA replication) and NDUFS6 (electron transport) were decreased ( Figure S3) . It might be possible that these proteins degraded more rapidly than synthesized in r 0 cell, consequently showed down-regulated protein expression levels.
Mitochondrial protein compositions change to remodel the organization of mt protein functional networks in response to changes in cellular states. Analysis of mt protein functional networks elucidated the biological implications of mt regulatory mechanisms under dysfunctional mt states. Our efforts of systematic data-integrative analysis to evaluate the reliability of proteomics data and to identify important functional modules of mitochondrial proteins can be valuable to computational biology community working on gene expression and proteomics analysis. First, we applied a data-integrative approach to select protein list by using various databases, proteomics datasets, and protein functional network. Mitochondrial proteins were organized into functional modules to identify significantly altered biological processes under different cellular states. The framework of our systematic data-integrative analysis may be useful to reliable proteome analyses for other cellular systems. Second, organizing the thousand mitochondrial proteins into groups of up-or downregulated ones and identifying functional modules are necessary steps in getting an in-depth understanding of the complex molecular mechanism of mitochondria. Third, mapping both mitochondrial proteins and mRNA expression information together is critical to understand the cooperative expression regulation of mitochondrial functional modules. Such multidimensional data analysis can be a valuable asset to develop novel system level models and methods. Also, experimental biologists can utilize our dataset as a resource for target selection to elucidate regulatory mechanisms of mt proteins under dysfunctional mt states.
Human mtDNA-depleted (r 0 ) 143B TK 2 osteosarcoma cells and parental normal r + cells (provided from Dr. Wei YH, National Yang-Ming Univ., Taipei, Taiwan) were cultured in high glucose (4.5 g/L) Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics (100 mg/ml penicillin/streptomycin mix), and uridine (50 mg/ml) in a humidified atmosphere at 37uC with 5% CO 2 as described [49] . The absence of mtDNA in r 0 cells was verified by PCR and PicoGreen staining ( Figure S4 ). PicoGreen is a sensitive staining dye to visualize mtDNA [50] . Pure mitochondria from r 0 and r + cells (2610 8 ) were prepared by ultracentrifugation by using 30-50% (1.1 and 1.6 g/ml) Optiprep TM density gradient media (Sigma-Aldrich) as described previously [51] . The purity of mitochondria was confirmed by western blot analysis. Mt proteins, COX I, COX IV, and HSPD1 (HSP60), exhibited significant reductions in levels in r 0 mitochondria compared to their levels in r + mitochondria, supporting complete mtDNA depletion and good mt preparation. Meanwhile, nuclear fraction marker (HDAC1), cytoplasmic fraction marker (NF-kB and SOD1), ER fraction marker (GRP78), and lysosomal marker (LAMP1) were not detected in both r 0 and r + mitochondria ( Figure S5) . b-actin was used for equal loading verification. Then, we applied quantitative cICAT analysis to compare protein abundances in r 0 and r + mitochondria.
To validate whether the proteins identified from cICAT were associated with mitochondria, we examined the GO cellular component annotation of the proteins identified from proteomics. Then, we performed a systematic data-integrative analysis to examine whether proteins identified from proteomics were matched with 13 reference mt protein datasets representing complementary independent studies of mt proteome, which include seven mt protein databases (HMPDb (http://bioinfo. nist.gov/hmpd/index.html), Maestro [52] , MitoProteome [53] , MitoRes [54] , Locate [55] , MitoP2, and SVM-trained MitoP2 [56] ) and six mt proteomics datasets (two MitoCarta datasets [57] , and four mtDNA depleted mitochondria proteomics datasets [33, 58, 59, 60] ). Some of the proteins from the database might contain mitochondrial proteins from other species, thus we only selected human mt proteins from these datasets to construct a reliable mt protein list. We validated 569 human proteins annotated as mitochondrial proteins and observed from at least one reference mitochondrial protein dataset.
Next, the Human protein reference database (HPRD) [61] and Search Tool for the Retrieval of INteracting Genes/proteins (STRING) database [62] were utilized for detecting physical and functional associations of the resulting proteins. STRING provides a large set of known and predicted protein-protein associations by compiling experimental repositories, curated pathway database, literature-mining resources, and computational predictions. Two proteins were connected in the STRING network if they shared a substrate in a metabolic pathway, co-expressed, co-regulated, connected by protein-protein interactions, or involved in the same protein complex. We additionally identified 201 proteins, of which 82 proteins were physically associated and 119 proteins were functionally associated with the first 569 mt proteins. A total of 770 proteins were defined as reliable mt proteins.
We constructed the human mt protein functional network using physical and functional link information extracted from the STRING database (ver. 8.0) with the 770 reliable mt proteins. The resulting mt protein functional network contained 726 proteins with 13,618 links (Figure 2A) , which included 4,854 intraregulatory links and 859 interregulatory links ( Figure 2B ). Intraregulatory links were defined as the interactions among upregulated proteins or among down-regulated proteins, whereas interregulatory links were the interactions among up-regulated proteins and down-regulated proteins.
We separated the mitochondrial protein functional network into functional modules ( Figure 1A) . A functional module was determined by examining whether a group of proteins was physically connected or functionally linked. To identify modules, we used hierarchical average-linkage clustering by using the OC software (http://www.compbio.dundee.ac.uk/Software/OC/oc. html) with the STRING confidence scores as a similarity measure. STRING confidence score represented the probability of finding the proteins which were related by one or more cellular and genetic interactions. Then, we assigned biological function to the module by using Ontologizer 2.0, which collects GO representative functional annotations from proteins in each module [63] , and selected functional modules if the significance (p-value) of GO enrichment was less than 0.01. The enrichment of up-or downregulated proteins in a given functional module was used to check the consistency of protein expression in the module (hypergeometric tests, p-value ,0.01). Finally, we identified 13 functional modules consisting of five up-regulated and eight down-regulated modules.
Total RNA from r 0 or r + cells was isolated using TRIzol (Invitrogen) and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies, Inc.) (n = 3). Microarray analysis was performed in triplicate by using the Illumina Sentrix HumanRef-8 Expression BeadChip according to the Illumina Bead Array Technical Manual. Briefly, total RNA (500 ng) was used for cDNA synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA by using the IlluminaH TotalPrep RNA amplification kit (Ambion, Inc.). The cRNA sample (750 ng) was hybridized to the BeadChip and stained with streptavidin-Cy3. The chips were dried and scanned by the BeadArray reader. The raw scan data were subjected to logarithmic transformation (log 2 ratios of fluorescence intensities) and quantile normalization by using the Avadis 4.3 software (Strand Genomics). Statistical significances were adjusted by the Benjamini-Hochberg FDR multiple-testing correction. Genes were filtered out by using the detection p-value threshold (p.0.05).
All microarray data reported in this study are described in accordance with MIAME guidelines and have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) public repository, and they are accessible through GEO accession (GSE22970).
For cICAT labeling, protein extracts from the pure r 0 or r + mitochondria were prepared with lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl 2 , 0.5 mM dithiothreitol, 1 mM phenyl-methylsulfonyl fluoride [PMSF]). Proteins were concentrated, and other non-protein materials such as salts were removed by the acetone precipitation method. Precipitated proteins were denatured in labeling buffer (6 M urea, 0.05% SDS, 5 mM EDTA, 50 mM Tris-Cl, pH 8.3) for 30 min and reduced with 5.3 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 30 min at 37uC. After readjustment to pH 8.3 with 1 M Tris-Cl buffer (pH 8.3), the r + or r 0 protein samples were labeled with a 15-fold molar excess of cleavable cICAT light ( 12 C) or heavy ( 13 C) reagents (Applied Biosystems, Foster City, CA) relative to proteins for 2 h at 37uC. Each 110-mg aliquot of separately labeled samples was equally combined and digested by trypsin (Promega, Madison, WI). The cICAT-labeled peptides were selectively isolated by strong cation exchange (SCX) and avidin affinity chromatography on a manually programmed AKTA Explorer 100 system (Amersharm Pharmacia, Sweden). Biotin moieties from cICAT-labeled peptides were cleaved by incubation for 1.5 h with 95% trifluoroacetic acid at 37uC. Samples were then dried in a speed-vacuum dryer and dissolved with 0.4% acetic acid for LC-MS/MS analysis. A schematic summary of the cICAT analysis workflow is presented in Figure  S1 .
The cICAT-labeled peptides were loaded on a nanospray tip coupled to a capillary reverse-phase column (14 cm675 mm, Magic C18aq; Michrom BioResources, Auburn, CA) packed inhouse by using a helium pressure cell. Peptides were eluted with a linear gradient of 5-35% buffer B (running buffer A: 0.1% formic acid in H 2 O; elution buffer B: 0.1% formic acid in 100% acetonitrile) over 90 min at 200 nl/min using an Agilent 1100 capillary pump system. Eluting peptides from the column were analyzed using LTQ linear ion trap mass spectrometers (Thermo Finnigan, San Jose, CA). A MS survey scan from 300-2000 m/z was acquired with three mscans followed by three data-dependent MS/MS scans (isolation width, 1.5 m/z; normalized collision energy, 28%; dynamic exclusion lists, 100; dynamic exclusion duration, 3 min). Data from RAW MS/MS files (minimum ion counts, 15; minimum peak intensity threshold, 1; mass range, 600-4300 Da) were generated, and peptide sequences from the data were assigned against the International Protein Index (IPI) human database (Version 3.13; 57,034 proteins) including known contaminants (180 entries) by using a 4-node SEQUEST (version 27, revision 9) cluster with the following search parameters: specificity (no enzyme), number of missed cleavage (max three sites), cysteine mass (fixed, +227.13 for light cICAT), cysteine mass (variable, +9 for heavy cICAT), methionine mass (variable, +16 for oxidation), mass tolerance of precursor ion (3.0) and fragment ion (0.5), mass type of precursor and fragment ion (average mass), and subsequence (cysteine residue). To discriminate true proteins from false positives and to quantify the abundance of proteins, we used Trans-Proteomic Pipeline of Institute for Systems Biology (TPP; Version 1.7.2; INTERACT, PeptideProphet TM , ProteinProphet TM , XPRESS programs). The peptides with P$0.50 by PeptideProphet TM were applied to ProteinProphet TM . The proteins with P$0.95 by ProteinProphet TM were considered to have the correct identification. Single-and double-hit proteins of the correct identifications were further validated through manual inspections of MS/MS spectra (false positive rate, below 0.4%). Quantification of peptides and proteins was performed with XPRESS software, and the peptides with bad quality (e.g., below S/N#2) were not considered by quantification. Finally, we identified nuclear-encoded 1,121 mt proteins that included 313 down-regulated proteins (r 0 /r + #0.67), 201 up-regulated proteins (r 0 /r + $1.5), and 607 not significantly changed (0.67,r 0 / r + ,1.5) proteins. The thresholds of 0.67 represents the 1.5-fold decrease (r 0 /r + #1/1.5), whereas the threshold of 1.5 represents 1.5-fold increase (r 0 /r + $1.5). Protein abundance ratio smaller than 0.67 or larger than 1.5 were routinely-applied thresholds indicating significant changes in proteomic analyses [64, 65, 66, 67] . See Table S1 for further details on the list of identified mt proteins.
Cells were lysed with lysis buffer (50 mM Tris HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 10 mg/ml each of aprotinin and leupeptin, and 1 mM PMSF). In some cases, mitochondrial fractions were lysed with the lysis buffer. A portion of cells (20-30 mg) or mitochondrial lysates (10 mg) were separated on 12% SDS-PAGE, transferred onto nitrocellulose membranes (Schleicher and Schuell), and subjected to western blot analysis by using designated primary antibodies. Horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) followed by ECL (Amersham Biosciences Inc., Piscataway, NJ) were used for detection.
Antibodies against APEX1, STUB1, and ATP5A1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). GTPBP3 and HDAC1 antibodies were purchased from Abcam (Cambridge, UK), and anti-eIF4A1 was obtained from Cell Signaling Technology (Danvers, MA). Antibodies against ALDH2, ALDH6A1, MTRF1, NDUFS6, HNRPM, GLUD1, and PLG were purchased from Abnova (Taipei, Taiwan). Antibodies against COXI and COXIV were purchased from Invitrogen (Karlsruhe, Germany), and TFAM were prepared in our laboratory [51] . PARP1, HSPD1 (HSP60), NF-kB, SOD1, XRCC5 (Ku80), XRCC6 (Ku70), and b-actin were purchased from Santa Cruz Biotechnology.
SK-Hep1 cells were stably transfected with pDsRed2-mito vectors (Clontech) containing the mt signal sequence of COXVIII in front of red fluorescent protein (Red2). The cDNAs of several candidate proteins were synthesized from the total RNA of r + cells by RT-PCR, sub-cloned into the T-easy vector (Promega), and then cloned into the N-terminus of the pEGFP-N3 vector (BD Bioscience). The resulting GFP-hybrid plasmids of pZCD1-EGFP, pGPT2-EGFP, pPYCR2-EGFP, pCTSD-EGFP, and pHSPBP1-EGFP were transfected using Superfect (QIAGEN, Valencia, CA) into DsRed2-mito-SK-Hep1 cells. At 48 h post-transfection, the transfected cells were fixed with 4% paraformaldehyde, mounted, and imaged using a confocal microscope (Carl Zeiss LSM 5). The mt localizations of candidate proteins were determined by the overlap of EGFP and DsRed signals. Figure S1 Workflow of comparative cICAT analysis of mitochondria proteomes. See Materials and Methods for details. (TIF) Figure S2 The reproducibility of the changes in protein abundances detected by cICAT quantification. Thirtythree mt proteins were observed from both cICAT and 2DE proteome datasets. Similar expression patterns between our mt proteomics data (black) and 2DE analysis (light gray) are shown. (TIF) Figure S3 The level of protein expression in cytoplasm and mitochondria. Western blot analysis of five proteins in cytosolic and mitochondrial fractions isolated from r + and r 0 cells. b-actin was used as a loading control. (TIF) Figure S4 Confirmations of mtDNA depletion in r 0 cells. (A) PCR amplification of mtDNA. Genomic DNAs isolated from r + or r 0 cells were utilized as templates to amplify mtDNA and nuclear DNA-encoded b-actin control. (B) Cellular nucleotide staining. Cells were treated with PicoGreen for 1 h, washed with DPBS, and then stained with Mitotracker orange (Mito-T, 100 nM) for 10 min. The cells were fixed with paraformaldehyde for 20 min and visualized by conformal microscopy (61000). mtDNA was observed only in r + cells. (TIF) Figure S5 Identification of purified mitochondria. Mitochondria were isolated using gradient-based ultracentrifugation as described. Proteins from a total lysate (30 mg), and mitochondria (10 mg) were resolved using 12% SDS-PAGE and analyzed by western blot. Antibodies against the following marker proteins were used: COXI, COXIV, and HSPD1 (HSP60) for mitochondria, HDAC1 for nucleus, NF-kB and SOD1 for cytoplasm, GRP78 for ER, and LAMP1 for lysosome. b-actin served as a loading control. (TIF)  Claudins in lung diseases Tight junctions are the most apically localized part of the epithelial junctional complex. They regulate the permeability and polarity of cell layers and create compartments in cell membranes. Claudins are structural molecules of tight junctions. There are 27 claudins known, and expression of different claudins is responsible for changes in the electrolyte and solute permeability in cells layers. Studies have shown that claudins and tight junctions also protect multicellular organisms from infections and that some infectious agents may use claudins as targets to invade and weaken the host's defense. In neoplastic diseases, claudin expression may be up- or downregulated. Since their expression is associated with specific tumor types or with specific locations of tumors to a certain degree, they can, in a restricted sense, also be used as tumor markers. However, the regulation of claudin expression is complex involving growth factors and integrins, protein kinases, proto-oncogens and transcription factors. In this review, the significance of claudins is discussed in lung disease and development. Tight junctions are membranous structures present in epithelial, endothelial and mesothelial cells [1, 2] They form barriers between cells in cell layers regulating diffusion of molecules and ions through the intercellular space [3] . They also form a fence separating the apical part of the cell from other parts thus preventing membrane proteins from mixing up with each other [3] . They play a part in the formation of cellular polarity and attachment [2, 3] (Figure 1 ). The barrier formed by the tight junctions prevents pathogens from penetrating through the epithelial layers thus serving as components of innate immunity [4] . They also participate in the immune defense by forming secluded areas such as the brain, eye or testis, where they contribute to sealing these tissues from the immune system [5, 6] . Tight junctional proteins also participate in regulation of cell differentiation and proliferation [7] .
Tight junctions consist on one hand of membrane proteins mediating cell to cell contacts. They include occludin, claudins, tricellulin and JAMs (junctional adhesion molecules). Scaffolding proteins on the other hand mediate signals from the surface to cytoskeletal actin filaments and activate signaling cascades of the cell [1, 3] . They include ZO-1 (Zona occludens-1), ZO-2, ZO-3, MAGI-1 (membrane-associated guanylate kinase with inverted orientation-1), cingulin and MUPP1 (multi-PDZ domain protein 1) [1, 3, 7] . Membrane proteins are divided in two groups, those with one transmembrane domain (JAMs) and those with four (claudins, occludin, tricellulin) [7] . The membrane proteins contain sequences in their carboxyterminal end with which they can attach to the scaffolding proteins [7] . One such is the PDZ domain with which the membrane proteins bind to ZO-1, ZO-2, ZO-3 or MUPP1 proteins [7, 8] . Scaffolding proteins, like ZO-1 inhibit cell proliferation by binding ZONAB (ZO-1 associated nucleic acid binding protein) thus preventing its movement to the nucleus [7] . Apg-2 (Albino and pale green 2), a protein involved in heat shock reaction, may replace ZONAB from its association with ZO-1 thus promoting its movement to the nucleus resulting in increased proliferation [7, 9] (Figure 2 ). Also ZO-2 influences cell proliferation by binding to transcription factors AP-1 (Activator protein 1) and SAF-B (Scaffolding attachment factor B) [7] . on their sequence similarity they are divided into classic and non-classic claudins [1, 12] . The former include claudins 1-10, 14, 15, 17 and 19 and the latter claudins 11-13, 16, 18 and 20-24 [1] . Claudins have four transmembrane domains, between these there are two extracellular loops (EL1 and EL2) and between inner transmembrane domains there is a short 20-residue intracellular loop [1, 12] (Figure 3 ). The intracellular carboxyterminal part contains the PDZ domains by which scaffolding proteins attach to claudins [1, 12] . The larger EL1 loop influences paracellular charge selectivity and the smaller EL2 binds the claudin molecule to the corresponding one in the neighbouring cell [12] . Claudins can associate with the same claudin or another one on the same cell membrane or with the claudin of the neighbouring cell [1, 13] . Heterodimerization between claudins can take place only between specific claudins, for instance between claudin 1 and 4 but not between claudin 1 and 2 [13] . In general, claudins 2, 7, 10, 15 and 16 increase paracellular cation permeability by forming pores in the tight junctions whereas claudins 4, 5, 8, 11, 14 and 18 have a sealing function [1] . Claudins 1, 3, 4, 5, 7, 8 and 18 are expressed in human bronchi and bronchioles but there are discrepant reports of the expression of claudin 2 [14] [15] [16] [17] [18] . In immunohistochemical studies on formalin fixed human lung tissue bronchial epithelial cells expressed claudins 2 [17, 18] . Similarly, claudin 2 has been detected in mouse lung [15] . However, in mRNA analysis of bronchial tissues it was not found [14] . Claudin 2 has been detected in skin Figure 1 In an epithelial cell layer tight junctions (marked by green) regulate the permeability of solutes and ions through the paracellular space (a violet arrow pointing downwards). Thus they function as barriers of the paracellular space and also prevent pathogens from reaching the subepithelial tissues. Tight junction also takes part in determining the polarity of epithelial cells (red arrow). Since they are located in the apicolateral part of the cell membrane they separate the apical part of the cell membrane (red) from the lateral part (yellow) preventing membrane proteins from these parts of mixing up with each other (the fence function). Interaction of claudins with scaffolding proteins ZO-1, ZO-2, ZO-3, MUPP1, cingulin and ZONAB in tight junctional structures. The arrows show interactions between specific molecules. If ZONAB is released from the association with ZO-1 it moves to the nucleus and binds to cyclin dependent kinase 4 (CDK 4) resulting in cellular proliferation. Apg-2 competes with ZONAB for the same binding site in ZO-1 thus affecting the binding of ZONAB to ZO-1 and releasing it to the nucleus. This is one example how tight junction proteins may influence the functions of the cell squamous cells and in enterocytes where it takes part in absorption of D-vitamin from the intestine [19, 20] . High amounts of claudin 2 mRNA are found in kidney, pancreas, stomach and liver tissues while neural and lymphatic tissues lacked expression [21] . In the study of Aung et al trace amounts of claudin 2 were found in the lung but the source of the expression was not determined [21] . Claudin expression appears to be the same in larger and smaller bronchi and there is no variation in claudin expression as has been found in different parts of the gut or kidney tubular segments [14, 22, 23] . Quantitative differences may, however be present and claudins appear to be located not only in the apical region of cells but also on the lateral membrane [14] . Claudin 5 is considered to be expressed mainly in endothelial cells and it takes part in the formation of the blood brain barrier [24] . It appears to be involved in the sealing function of endothelial barriers in others sites such as blood retinal or blood testis barrier [5, 6] . When claudin 5 was overexpressed in bronchial tranfectant cells it, however, made the epithelium leakier while claudins 1 and 3 made it tighter [14] . In rat alveolar cells, EGF stimulation increased the transmembrane resistance with an increased claudin 4 or 7 protein expression while claudins 3 and 5 were associated with a leakier phenotype [25] . Paradoxically then, claudin 5 appears to induce loosening of the barrier both in bronchial and alveolar cells [14, 25] .
In immunohistochemical studies on human alveolar cells, claudins 3, 4 and 7 have been detected in type 2 alveolar cells while type 1 alveolar cells were negative [17, 18] . Rat alveolar epithelial cells expressed claudin 3, 4 and 7 mRNA but claudin 1 or claudin 5 mRNA were not found even though claudin 5 was found in western blot analysis in alveolar cells [25] . Rat type 2 alveolar cells express more claudin 3 and type 1 cells more claudin 7 [16, 26] . Thus there are inconsistencies regarding claudin expression also in alveolar cells detected by immunohistochemistry or mRNA analysis which may be species specific, partly due to the method used or may reflect extended half lives of proteins compared to RNA. Inconsistent results between protein and mRNA levels of claudins 1, 3 and 7 have also been detected in alcohol induced lung changes in rats where the protein expression of these claudins increased while the mRNA levels stayed the same [27] . In studies on claudin mRNA expression in human lung tissues claudins 6, 9, 10, 11, 15 or 16 have not been detected [14, 16] . Expression of claudins in lung cells has been compiled in Table 1 .
Claudins are involved in embryogenesis and organogenesis and changes in their expression can be seen in the organogenesis of the gut and kidney [23, 28] . Claudin 15 serves as a target for tcf2 gene in zebrafish creating one lumen instead of multiple ones [29] . Interestingly, claudin 15 knockout mice develop a megaintestine [30] . In organogenesis, claudin expression is turned off in cells taking part in epitheliomesenchymal transition [31] . In embryonic development claudins regulate cellular integrity, and create hydrostatic pressure differentials maintaining integrity of cystic compartments [31, 32] . Additionally, claudin 1 may have a role in right-left patterning of the embryonic tissues [31] . In Xenopus, claudin 5 is required for heart development [33] .
There are five stages in lung organogenesis. In the embryonic period, the tracheal bud forms. The pseudoglandular and canalicular periods are characterized by branching morphogenesis giving rise to the bronchial three while during the saccular and alveolar periods the alveoli form [17] . In investigations on claudin expression during these stages claudins 1,3,4, 5 and 7 can be detected in bronchial epithelium during the pseudoglandular and canalicular periods [17] . Pretype alveolar cells express claudins 3, 4, 5 and 7 but not claudin 1 [17] . In saccular and alveolar periods claudin 5 is lost from alveolar cells but weakly present in bronchial epithelial cells. Claudins 3, 4 and 7 show strong positivity in bronchial and alveolar cells while claudin 1 is negative in alveolar pneumocytes [17] .
Compared to lung development also kidney ureters develops by first forming an ureteral bud followed by branching morphogenesis. Like in the lung tracheal budding ZO-1 is required in ureter bud formation of rat tissues [28] . Branching morphogenesis is promoted by ezrin which is activated by growth factors EGF (Epidermal growth factor), HGF (Hepatic growth factor) or interleukin-1alpha by tyrosine phosphorylation. MT-MMP1 (Membrane type matrix metalloproteinase 1) and MMP2 (Matrix metalloproteinase 2) are secreted on tips of the epithelial branches and proliferation is higher in epithelial cells in those areas [28] . Proteins of adherence junctions such as E-cadherin, are expressed a bit Mesothelial cells 1,2, 3,5,7 [104, 107] earlier, however there is a gradual increase of claudin 3 and ZO-1 from day 3 to 5 in cultured ureteric buds [28] . Anatomically, the branching morphogenesis in the lung differs from the ureteric development in that in the ureteral development there is no lateral branching [28] . Also EGF (Epidermal growth factor) seems to be important for lung organogenesis, deletion of EGFR (Epidermal growth factor receptor) leads to impaired branching and deficient alveolisation, and pneumocytes remain immature [34] . Prealveolar fetal alveolar lung cells express claudin-1, 3, 4, 5, 7, 9, 10 and 18 mRNAs [16] . mRNAs for occludin, JAM-1, ZO-1, ZO-2, and ZO-3 are also present [16] . In experimental models on alveolar cell differentiation undifferentiated fetal alveolar epithelial cells increase their transepithelial resistance under the influence of cell culture medium containing dexamethasone, 8-bromo-cAMP and isobuthylmethylxanthanine and transform to type 2 alveolar cells at the same time increasing the claudin 5 and 18 mRNA levels and decreasing claudin 1 mRNA [16] . On the other hand, EGF stimulated fetal alveolar type 2 cells show a decrease in claudin 5 and 3 mRNA but an increase in claudins 4 and 7 mRNA while increasing their transepithelial resistance and transforming to type 1 cells [25] . While such in vitro models may differ from physiological conditions, changes in claudin expression apparently modulate or follow phenotypic changes in lung bronchial or alveolar cells during organogenesis. Such studies also emphasize EGF's role in lung alveolar development.
Tight junctions are important for lung defense since they function as a barrier for pathogens and other exogenous compounds preventing their penetration into the interstitial tissues. Tight junctions may thus be considered as a part of the innate immune system. The epithelial barriers of bronchi are, however, relatively leaky with a comparatively low transmembrane resistance of approximately 100 Ωcm2 compared to endothelial cell junctions of the blood brain barrier which have a 1500-2000 Ωcm2 barrier resistance [14, 35] . The thickness of the tight junctional belt in airway epithelia varies from 0.27 μm to 0.37 μm depending on the cell type [36] . There are reports of fragmentation and thinning of the tight junctional belt in diseases such as asthma and focal proliferations and thinnings are described in lung neoplasia such as bronchioloalveolar carcinoma [36] . Such changes are also seen in cell line studies induced by cytokines [37] Lungs are exposed to several pathogens and noxious stimuli which may influence paracellular permeability of bronchial airways, alveolar cells or endothelial barriers in alveolar walls. Tobacco smoke makes tight junctions leakier [36] . Carcinogenic compounds such as bentzpyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) influence claudin synthesis by upregulating transcription factors like twist, snail or ZEB1 [38, 39] resulting in downregulation of E-cadherin and claudins. Exposure of lung BEAS-2B cells and cancer cell lines to tobacco smoke leads to changes in claudin expression [40] . In bronchial cells sampled from smoking COPD patients and healthy smokers claudins and other tight junction genes are downregulated [41] . Tobacco smoke also induces the formation of reactive oxygen species. In kidney epithelial MDCK (Madine-Darby canine kidney strain) II cells, exposure to hydrogen peroxide leads to bimodal changes in the transepithelial barrier permeability with activation of ERK1/2 and p38 kinases, and a decrease in occludin, claudin 1 and claudin 2 levels [42] . Such changes could also make the bronchial and alveolar epithelium leakier and the airways more vulnerable to damage caused by pathogens causing irritation and chronic infection. On the other hand, an increase in claudin 2 expression leads to lower transepithelial resistance in MDCK I and II cells and formation of paracellular water channels [43, 44] . Generally air pollutants influence the barrier function of airway epithelium. Particulate matter of less than 10 μm in diameter decreases the transepithelial potential in airway epithelial cells causing internalization of occludin and its dissociation from ZO-1 [45] .
Inflammation may affect the barrier function of tight junctions. Several integrins and growth factors produced by inflammatory cells influence synthesis or organization of tight junction components leading to enhanced permeability and exposure of tissues to antigens [15, 46, 47] . Increased permeability of tight junctions is one factor lying behind the pathophysiology of colitis ulcerosa and Crohn's disease [47] . Also in chronic bronchitis and asthma airway epithelial cells are exposed to chronic stimulation by integrins and growth factors predisposing the bronchial wall to dysregulation of the epithelial barrier function further provoking the chronic infection [46, [48] [49] [50] .
Pathogens may influence tight junctions in two ways. Several bacteria and viruses lower the transepithelial resistance by decreasing the expression of tight junctional proteins making it easier for pathogens to penetrate the tissues. Rhinovirus downregulates the transepithelial resistance of nasal epithelial cells by lowering the mRNA expression of claudin 1, ZO-1, E-Cadherin and occludin [51] . HIV-1 decreases the transepithelial resistance in enterocytes and genital epithelial cells by disrupting claudins 1, 2, 4, occludin and ZO-1 in tight junctions and lowering mRNA expression of claudins 1, 2, 3, 4, 5, occludin and ZO-1 by cytokine involved mechanisms allowing penetration of bacteria and viruses through the leaky barriers [52] . Rotavirus in known to loosen tight junctions in gut epithelial cells by decreasing expression of claudin 3, occluding and ZO-1 [53] . Yersinia enterocolica causes a redistribution of claudins 3, 4 and 8 in colonic HT29/B6 cells with a lowering of transepithelial resistance and diminution of the protein expression of claudins 2, 3, 8 and 10 and ZO-1 [54] . Aggressive strains of E Coli cause dissociation of claudin 1, ZO-1 and occludin from cell membranes along with a decrease in electrical transmembrane potential [55] . Helicobacter pylori causes disruption of claudin 4 expression in tight junctions and its movement to the cytosol in gastric epithelial cells and lowering of claudin 4 protein levels, an effect which is dependent on IL-1β [56] . On the other hand, claudins may be used as receptors or co-receptors in the invasion of bacteria and viruses to cells and tissues. Claudins 1, 6 and 9 serve as a co-receptors for hepatitis C for invading liver and endothelial cells [57, 58] . Claudin 1 also promotes dengue virus entry to cells [59] . Claudins also serve as receptors for some bacterial toxins. A well known example is Clostridium perfringens enterotoxin (CPE) which binds to claudin 3 and 4 [60] . In experimental studies this has been used to destroy claudin 3 and 4 expressing cancer cells with chemical modifications of the toxin [60] . Interestingly, CPE has also been successfully used to destroy metastatic cells expressing claudin 4 in murine tissue [61] . Unfortunately, claudin 4 is many times downregulated in metastatic tissues such as metastatic breast carcinoma [62] or carcinoma metastasis to the lung [63] . The frequent associations of claudins as means and targets for spread of infections could predict putative polymorphisms in claudin molecules. Indeed, 50 variations have been found in the claudin 1 gene region some of which alter susceptibility to hepatitis C infection [64] .
Mechanisms of pathogen entry and its association with claudins have not been so extensively studied in the respiratory tract. It, however, appears probable that similar mechanisms apply to airway epithelia. Matrix metalloproteinase 9 (MMP 9)decreases epithelial barrier function in bronchial cells, and increases their vulnerability to adenovirus infection [65] . The cells show disruption of regular membranous staining pattern of claudin 1 and occludin and internalization of reactivity following MMP9 treatment which could be restored by tissue inhibitor of matrix metalloproteinase 1 (TIMP1) [65] . Pseudomonas aeruginosa has been shown to invade airway epithelial barriers by destroying tight junctions [66] . Interestingly, Plasmodium falciparum malaria affects lung endothelial cells by relocation of ZO-1 from the plasma membrane and downregulation of claudin 5 [67] . Thus also parasite infections may be associated with tight junctional changes in the pulmonary cells.
ARDS (Acute respiratory distress syndrome) is due to injury of alveolar cells the reasons of which may be multiple including septic infections and toxic chemicals. Such injury leads to edema due to leakage of alveolar epithelial cells. Since claudins regulate paracellular permeability of cell layers evidently changes in claudin expression are involved in such disease. There are no morphological studies on claudin expression by human tissues in ARDS. Evidence of tight junctional protein changes in ARDS can be obtained from animal experiments or cell culture studies. In alveolar cells cultured from septic rat lung a decrease of claudin 4, claudin 18 and occludin was found associated with a decrease in transmembrane resistance [68] . In ventilator-induced lung injury in mice the mRNA expression of claudin 4 increased after 3 hours [69] . Such a phenomenon appears to be an adaptive mechanism to make the alveolar layers tighter, since blocking specifically the mRNA synthesis of claudin 4 by siRNA or CPE lowers the transmembrane potential [69] . Similar results come from experimentally induced pancreatitis in rats where the mRNA levels of claudin 4, claudin 5 and occludin decreased due to pancreatitis induced lung injury but increased after administration of emodin, a chemical suggested to enhance epithelial barrier function [70] . Such data emphasize especially the role of claudin 4 in combating the abrogation of alveolar cell permeability in lung injury. Also the expression of claudin 5 is important for endothelial cell tightness and vascular permeability. Acrolein present in tobacco smoke may cause lung injury by influencing claudin 5 expression in endothelial cells and mice more resistant to acrolein have a better survival due to their increased expression of claudin 5 mRNA transcripts [71] .
Interestingly, protein kinase C delta inhibitor appeared to inhibit sepsis induced lung injury, such as disruption of lung tissue or development of edema [72] . In nasal epithelial cells treatment of cells with protein kinase C activator 12-O-tetradecanoylophospho-13-acetate led to an increased transepithelial electrical resistance and upregulation of claudin 1, ZO1, ZO2 and occludin [73] . It was shown that the upregulation of the tight junctional proteins was due to activation of PKC lambda and theta partly induced by the transcription factor GATA 3 [73] . In fact, phosphorylation of threonine sites in claudins by PKC or PKA may dislocate claudins from cellular membranes resulting in changed tight junctional properties [1] .
Also in lung inflammation alterations in alveolar cell permeability play a role. In experimental studies on mice where lung inflammation was induced by carrageenan disruption of claudin 2, claudin 4 and claudin 5 staining and ZO-1 was observed on cell membranes [15] . Such disruption was partly reversed by blocking TNFα suggesting that TNFα-induced chemotaxis of inflammatory cells might contribute to attenuation of tight junctional permeability in lung inflammation [15] . Additionally, alcohol which provokes patients to lung inflammation and ARDS was shown in experiments on rats to induce changes in claudin expression of alveolar epithelial cells [27] . Chronic alcohol ingestion of rats led to a decrease of claudin 1, 3 and 7 mRNA and protein expression and an increase in claudin 5 mRNA in alveolar epithelial cells resulting in an increased leakiness of tight junctions in the lung [27] . Additionally, inflammatory mediators like IL-1β or IL-6 decrease claudin 3 and 4 levels in amniotic membranes of mice making them more permeable [74] .
Respiratory distress leads to local hypoxia of the lung. Hypoxia induces cytoskeletal disruption of alveolar epithelial cells and decreases ZO-1 protein levels dislocating occludin from the cell membrane [75] . Alveolar epithelial cells do not show changes in claudin 3 or 5 protein expression, however [75] . Claudin 5 is known to influence the permeability of endothelial cells. In experiments performed on mice bEND.3 (brain-derived endothelial) and retinal endothelial cells, suppression of claudin 5 mRNA leads to a decrease in transepithelial resistance of these cells [76] . On the other hand, in experimental murine renal ischemia-reperfusion injury there was a significant upregulation of claudin -1, -3, and -7 genes and a slight downregulation of claudin-2 [77] . Additionally, when both of these cells suffer from hypoxia a decrease in claudin 5 mRNA and a lowering of transepithelial resistance was detected [76] .
There are only a few studies on the influence of inflammation on claudin expression in bronchial cells. In well differentiated human airway epithelial cells (HAE) from cystic fibrosis (CF) and non-CF patients, combined treatment with IL-1β and TNFα led to a decrease in transmembrane potential and tight junctional barrier function, the changes appearing more rapidly in CF cells [37] . In analysis of airway epithelial cells the expression of claudins 1 and 4 were not altered, however, there was a decrease in the protein expression of ZO1 and JAM and upregulation of hyperphosphorylated occludin and ICAM1 [37] . The influence of these cytokines appeared to be mediated by PKC/lambda [37] .
Interstitial lung diseases are characterized by chronic inflammation, fibrosis and damage to the lung parenchyma [78] . They consist of seven entities which are classified both by histology and clinicopathologic features [78] . In interstitial lung diseases, reports of claudin expression in UIP (Usual interstitial pneumonia) are present [18] . Changes in claudin expression mainly involve the regenerative alveolar cells or cells which have been replaced by metaplastic epithelia. In UIP regenerative metaplastic alveolar, squamous or bronchial epithelium shows strong reactivity for claudins 1, 2, 3, 4 and 7 compared to normal alveolar cells where only claudins 3, 4 and 7 are present [18] . There is a weaker reactivity for claudin 5 in such regenerative cells. In sarcoidosis, metaplastic regenerative epithelium displays a similar kind of reactivity in metaplastic cells as in UIP [18] . The formation of metaplastic epithelium in UIP probably is related to chronic inflammation and secretion of growth factors and integrins in the disease [78] . A different pattern of claudin expression in regenerative metaplastic cells compared to alveolar cells would lead to focal changes in the permeability of alveolar walls which is probably harmful for the function of the lung. There is also increased vascularity, neovascularisation and increased secretion of angiogenic factors in UIP [18] . Endothelial cells of blood vessels display intensive expression for claudin 5. According to some reports VEGF may upregulate claudin 5 [79] . Such increased staining may then be based on increased secretion of VEGF in UIP. Expression of claudins in different lung pathologies has been compiled in Table 2 .
Lung carcinoma cells contain tight junctions and their number is inversely associated with patient prognosis and aggressivity [80] . In lung tumor cell lines, variable expression of claudins 1, 2, 3, 4 and 7 is found [40] . In studies on lung tumor material different histological tumor types vary in their claudin expression showing up-or downregulation of different claudins compared to normal bronchial cells or lung tissue [40, [81] [82] [83] . In the study of Moldway et al small cell lung carcinomas show a 16 fold higher level of claudin 3 mRNA expression than normal lung tissue [82] . Claudin 4 mRNA is upregulated to 3-4 fold level in squamous, adenocarcinoma and small cell carcinoma compared to normal lung [82] . Both adeno-and squamous cell carcinoma showed a slight downregulation of claudin 1 mRNA compared to normal lung and squamous cell carcinomas had a 2.7 fold level of claudin 1 mRNA than adenocarcinomas [82] . Paschoud et al found an increase in claudin 5 and a decrease in claudins 3, 4 and 7 mRNA in squamous cell carcinoma compared to normal bronchial cells and an increase in claudin 1 and a decrease in claudin 5 compared to lung parenchyma [81] . Compared to bronchial cells claudins 1, 3 and 4 and 7 were decreased and claudin 5 mRNA increased in adenocarcinoma while when compared to lung parenchyma claudin 4 mRNA was increased but claudin 5 decreased [81] .
In immunohistochemical studies significant differences have been detected in claudin 3 expression between squamous cell and adenocarcinomas the latter displaying stronger expression [40, 82, 83] . Some studies also report significant differences in the expression of claudin 1, 4, 5 and 7 between these two tumor groups [81, 82] . In the study of Paschoud et al squamous cells and adenocarcinomas could be discriminated by the expression of claudins 1 and 5 because adenocarcinomas displayed strong positivity for claudin 5 and weak positivity for claudin 1, while the opposite could be detected in squamous cell carcinomas [81] . Chao et al studied claudin 1 expression in adenocarcinoma and found that a low expression of claudin 1 was associated with a worse survival in these tumors both by immunohistochemistry and mRNA expression [84] . Transfection of claudin 1 into CL1-5 lung carcinoma cells changed the cells to less invasive, less metastatic and less mobile, and their morphology changed to a more epithelial one [84] . Such changes could also be reversed by blocking claudin 1 mRNA expression by siRNA. Oligonucleotide microarray analysis showed a twofold change in 773 genes due to claudin 1 overexpression involving different cellular functions associated with signaling cascades, cellular associations, apoptosis and cytoskeletal regulation. They, however, found that claudin 1 overexpressing cells were able to activate MMP2 [84] . Similar results of claudin expression on MMP activities have been detected in other cell lines. In colon carcinoma CaCo-2 cells, invasion induced by claudin 4 overexpression was due to activation of both MMP2 and MMP9 the mRNA expression of which also increased at the same time [85] . Claudin 5 along with claudins 2, 3 and 4 has been shown to activate matrix metalloproteinases [86] . Claudin overexpression in cancer cells may thus be one factor promoting tumor spread.
The variable expression of claudins in histologically different lung tumor types may be related partly to the cell type it originates from [83] . On the other hand, several growth factors and integrins modify claudin expression and they expression may vary in different types of lung tumors. EGF, for instance, inhibits claudin 2 expression while simultaneously increasing claudin 1, 3 and 4 expression and the transepithelial resistance without affecting the levels of occludin or ZO-1 in canine kidney cells [87] . In pneumocytes EGF upregulates claudins 4 and 7 while 3 and 5 are decreased [25] . Activating EGFR mutations are known to exist in nonsmall cell lung carcinoma and especially in adenocarcinoma and bronchioloalveolar carcinoma [88] . In line with this, claudins 1 and 4 show a high expression in lung adenocarcinomas and bronchioloalveolar carcinomas and claudin 2 and 5 are lower [40, 82] . Small cell lung carcinomas also display a higher expression of claudin 2 than adenocarcinomas [83] . Another mutation common to lung cancer is K-Ras [88] . Such mutations are present 15-30% of non small cell carcinomas [88] . In kidney cells, overexpression of ras leads to claudin 1, 4 and 7 overexpression while claudin 2 decreases and claudins 3 and 5 remain the same [89] . Similarly, some non-small cell carcinomas have PTEN (phosphatase and tensin homologue) mutations [90] . Inactivation or mutations of PTEN leads to activation of PKB (Protein kinase B) resulting in increased cell proliferation and inhibition of apoptosis [91] . Knockdown of PTEN has been shown to lead to a loss of polarization in colon carcinoma cells and strong downregulation of claudins 1, 3, 4 and 8 [92] . Small cell carcinomas and large cell neuroendocrine carcinomas, however, express c-kit, and they harbor c-myc amplifications and p53 and ras mutations, and loss of p16 and RB expression and their influence on claudin expression is not known [93] . Additional, several cytokines influence claudin expression and barrier function of epithelial cells and may surely influence also the expression of claudins in lung carcinomas [47] .
EMT (Epitheliomesenchymal transition) is a process where tumor cells attain mesenchymal features which make it easier for them to invade and metastasize [94, 95] . It is regulated by transcription factors such as snail, slug, twist and zeb1 [94] [95] [96] . In mouse mammary epithelial cells snail induced EMT and at the same time [97] . In lung carcinoma there was an inverse association between zeb1 and claudins 1 and 2 and between twist and claudin 5 [63] . Metastatic tumors to the lung also showed an inverse association between claudins 5 and 7 and twist [63] . These results show that a part of claudin expression is regulated by EMT associated transcription factors and that claudins are involved in EMT. Also different PKCs which regulate claudin phosphorylation are overexpressed in cancer and may cause dysregulation or relocation of claudins in tumor cells [98] . PKCε is considered an oncogene and influences cellular motility and ras expression, and it is upregulated in lung and breast cancer [98] . PKC and PKA influence subcellular distribution of claudin 1 in melanoma cells [99] . PKCα phosphorylates claudin 5 causing its disappearance from cell membrane [100] . On the other hand, phosphorylation of serine 194 of claudin 4 by aPKC is required for tight junction formation in keratinocytes [101] . Claudin phosphorylation may also affect barrier junction permeability without changes in claudin distribution [101] .
Claudins are in contact with scaffolding proteins like ZO-1, ZO-2 and ZO-3 and through them with the cellular cytoskeleton [102] . ZO-1 has been shown to recruit the transcriptional factor ZONAB from cytoplasm thus preventing its movement to the nucleus where it promotes cell proliferation. ZO-2, on the other hand, is able to bind AP-1 [103] . How the expression of different claudins influences the functions of such zona occludens proteins is not known but it is possible that in cancer cells the relation between claudins and zona occludens and other scaffolding proteins is deranged leading to a dysregulation of transcription factors such as ZONAB or AP-1. Clearly more research is needed to understand the functions and consequences of claudin expression in lung cancer.
Because claudins 3 and 4 are overexpressed in adeno, squamous and small cell carcinomas [40, [81] [82] [83] they might be susceptible to CPE-mediated treatment. In a recent publication Yao et al created a modified CPE protein containing segments of pseudomonas aeruginosa enterotoxin A [104] . Treatment of cell lines containing claudin 4 induced apoptosis and cell destruction in them [105] . CPE also causes dislocation of claudin 4 from the tight junction in ovarian carcinoma cells and sensitizes them to chemotherapeutic drugs [105] . This effect is achieved by using a modulated C-terminal fragment of the CPE molecule in the treatment which is able to bind to claudin 4 and cause tight junctional loosening but harbours no toxic effects of the N-terminal part of the molecule thus enabling a better influx of chemotherapeutic agents to the cancer tissue (105) . Such CPE mediated treatment could also be one option in treatment of claudin 3 or 4 positive lung cancer.
Mesotheliomas are malignant tumors with an aggressive behavior. In pleural biopsies taken for malignant pleural disease, there is often diagnostic difficulty to distinguish mesotheliomas from metastatic adenocarcinomas or reactive mesothelial cells. Several markers, such as calretinin, DJ2-40, TTF1 and CEA, can be used in differential diagnosis [106] . Claudins have also proven to be one putative means of making a differential diagnosis between such entities [107] [108] [109] [110] .
In our study on metastatic adenocarcinomas and malignant mesotheliomas claudins 1, 3, 4, 5 and 7 had a lower expression in mesotheliomas suggesting that they could serve as differential diagnostic markers [107] . In mesothelioma subtypes, sarcomatoid and biphasic mesotheliomas showed less expression for claudins than epithelioid ones [107] . Non-neoplastic mesothelial cells showed expression for claudin 2 and weak expression for claudin 1, but no expression was found for claudins 3, 4, 5 or 7 [107] . In effusions claudins 3 and 7 significantly distinguished malignant Mesothelioma cells from metastatic adenocarcinoma cells [108] . Reactive mesothelial cells were negative for claudin 7 but showed infrequent claudin 1 and 3 expression. Thus also reactive mesothelial cells could be distinguished from metastatic cells in pleural effusions [108] . Metastatic ovarian carcinoma cells could also be distinguished from metastatic breast carcinoma cells by claudin 7, and generally, ovarian, endometrioid and cervical cancer metastases could be distinguished from lung or breast adenocarcinoma [108] . In the array study by Davidson et al serous mesotheliomas showed a significantly lower expression of claudins 3, 4 and 6 than ovarian adenocarcinomas speaking for a lower level of claudins in mesothelial derived neoplasms [108] . Facetti et al determined that especially claudin 4 could be used in differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma [110] . Since adenocarcinoma metastases many times express claudin 4, CPE could potentially be used in treatment of such adenocarcinoma metastases in pleural cavity since benign mesothelial cells do not express this tight junctional protein [110] .
Claudins have not been studied in other disease states of the mesothelium although it could be expected that they play a role in derangements of pleural fluid homeostasis and in pleuritis. Exposure of mesothelial cell monolayers to hydrogen peroxide lowers their transepithelial resistance with a simultaneous decrease in occludin and ZO-1 expression [111] . Thus oxidative stress, involved in many diseases, such as inflammation, may be one factor causing loosening of mesothelial barriers. In line with this, a recent article showed that the levels claudins 1, 3, 5 and 7 decreased and claudin 2 increased in mesothelial cells due to pleural inflammation [112] .
The permeability of tight junctions in epithelia is an important factor in several pulmonary diseases. Pathogens causing infections are many some of which may act by downregulating claudins or causing their deranged distribution inducing changes in tight junctional permeability allowing pathogens to invade through epithelial barriers. In lung disease the effect of such pathogens on claudin expression or cellular distribution is still mainly unelucidated and needs further research. Alteration of claudin expression also plays an important role in lung diseases such as COPD, asthma, and ARDS. Influencing alveolar or endothelial permeability by manipulation the expression of claudins (eg. claudins 4 and 5) might be future targets in the treatment of lung injury. Changes in the expression of claudins are also seen in pulmonary neoplasia reflecting complex changes in several genes related to tumor growth, spread and differentiation producing a characteristic expression patterns in histologically different tumor types. In adenocarcinomas a proposed new classification will surely also change our perspectives on assessment of claudins on the behavior of these tumors [113] . In addition to claudin 3 and 4 which may have therapeutic implications for tumor treatment in the future, claudin 18 might also be a new target for antibody mediated therapy in lung cancer [114] .
Authors' contributions YS is the sole author of the manuscript
The author declares that they have no competing interests. Human Bocavirus in Patients with Respiratory Tract Infection BACKGROUND: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. METHODS: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0×10(6) copies/mL as the threshold value of viral load. RESULTS: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78×10(5) copies/mL vs. 1.94×10(4) copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. CONCLUSIONS: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group. Human bocavirus (HBoV) was newly discovered by Allander et al. [1] and was stated to be the possible causative agent of respiratory illness. Phylogenetic analysis of the complete genome of HBoV revealed that the virus is most closely related to canine minute virus and bovine parvovirus, which are members of the Bocavirus genus of the Parvoviridae family [1] .
HBoV was found in 1.5-11.3% of respiratory samples investigated worldwide [2] [3] [4] [5] . HBoV may be a causative agent of respiratory tract infections. However, some investigators argue that the association between HBoV and respiratory tract diseases remains unproven because of the high rate of codetection of HBoV with other respiratory pathogens [5] . Its clinical epidemiology and role in respiratory infection have not yet been fully elucidated.
Although recently it was found that HBoV could be cultured in differentiated human airway epithelial cells [6] , routine viral culturing of HBoV remains difficult. Real-time PCR has been used to estimate viral load and its usefulness has been proved as an indicator of the degree of active viral infection, interactions between the virus and the host, and the role of viral reactivation or persistence in the progression of disease [7] . In this study, we investigated the epidemiology of HBoV and the clinical features of respiratory infection associated with HBoV, especially in terms of HBoV load. KJLM from 1,815 patients were randomly selected among the 5,891 samples collected from patients referred to the Korea University Guro Hospital for screening for respiratory viruses. Among the patients, 59.0% were men, and the ages of the patients ranged from 1 day to 86.0 yr (mean = 6.1 yr, median = 26 months); children aged 5 yr or less constituted 75.3% of the study population. The nasopharyngeal aspirates (NPAs) obtained from all patients were stored in a viral transport medium and quickly delivered to the laboratory by using wet ice. R-Mix culture system (Diagnostic HY-BRIDS, Athens, OH, USA) was used to screen the samples for presence of 5 respiratory viruses (influenza A and B viruses, parainfluenza viruses, respiratory syncytial virus, and adenovirus).
Total nucleic acid was extracted from 100 µL of the sample by using Instagene Matrix Kit (Bio-Rad, Hercules, CA, USA). PCR primers were designed using Beacon Designer software (Premier Biosoft International, Palo Alto, CA, USA) in the conserved region of the NS-1 coding region of the HBoV genome by using the HBoV ST2 sequence (Genbank accession number: DQ000496). The forward primer (5´-GCAAATCTCTTCTGGCTACACG-3´) and the reverse primer (5´-CCTCTGCGATCTCTATATTGAAGG-3´) were targeted at a portion of the HBoV NS-1 gene. The conventional PCR reaction mixture consisted of 0.2 pg/μL forward and reverse primers, 2.5 mM dNTPs, 50 mM KCl, 1.5 mM MgCl2, 5 U of Taq polymerase, and 3 μL of extracted DNA in a final volume of 25 μL. The PCR cycling conditions consisted of 35 cycles (involving reaction at 30 sec at 94˚C, 30 sec at 60˚C, and 30 sec at 72˚C) after the preheating step of 3 min at 94˚C. All the PCR products obtained from positive reactions were sequenced completely to confirm sequence specificity.
To obtain standard curves for absolute quantification, the PCR product of HBoV was cloned into a plasmid vector pGEM®-T Easy Vector (Promega, Madison, WI, USA), purified using a QIAprep mini prep kit (Qiagen Inc., Valencia, CA, USA), and quantified using UV spectroscopy (1 genomic copy = 5 × 10 -12 µg). Serial 10-fold dilutions of the cloned plasmid were prepared to generate the standard curves.
Real-time PCR for viral load was performed on the specimens positive for HBoV by conventional PCR. A TaqMan probe (5´-ATGTTGCCGCCAGTAACTCCACCC-3´) was labeled at the 5´ ends with the reporter molecule FAM and at the 3´ ends with Black Hole Quencher 1 (Biosearch Technologies, Inc., Novato, CA, USA). The assay was performed using Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) and the standard protocol of TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA, USA); each 25 µL sample of the reaction mixture contained 10 pg/ µL of the forward and the reverse primers and 3 µL of the extracted DNA. Amplification conditions consisted of reactions for 3 min at 50˚C, 3 min at 94˚C, and 40 cycles of 30 sec at 94˚C, 30 sec at 60˚C and 30 sec at 72˚C maintained for 3 min. Detection limit of real-time PCR for HBoV was 1.3 × 10 3 copies/mL, which corresponded to 33 copies per reaction.
Statistical analysis was performed using the SPSS software (version 10, SPSS Inc., Chicago, IL, USA), and graphs were prepared using Prism software (version 4.0, GraPad Software, Inc., San Diego, CA, USA). Mann-Whitney U test, Chi-squared or Fisher's exact test were performed to assess the significance. A P value < 0.05 was considered statistically significant for all the tests.
A total of 1,926 samples of patients with respiratory symptoms were included in the present study. Ninety-three (4. 8%) samples were found to be positive for HBoV by PCR and subsequent sequencing. Other respiratory viruses detected during the study period were as follows: influenza A virus (IFA), 56 patients (2.9%); influenza B virus (IFB), 50 patients (2.6%); parainfluenza viruses (PIV), 142 patients (7.4%); respiratory syncytial virus (RSV), 97 patients (5.0%); and adenovirus (ADV), 40 patients (2.1%). HBoV was more prevalent in men (73.1%) than in women (P = 0.005).
HBoV was detected in patients ranging from 3 months to 65.6 yr (mean = 36.2 months, median = 19 months) with a peak prevalence between the ages of 6 and 12 months (8. 6%, 22/257). Children aged 5 yr or less constituted 92.5% (86/93) of the HBoV-positive patients (Table 1) .
HBoV was detected in the samples obtained throughout the course of the study with the detection rate being the highest in June (16/129, 12.4%), followed by August (9/110, 8.2%) and May (13/162, 8.0%). From April to June 2006, 43% of the HBoV-positive cases were observed (Fig. 1) .
All the 93 HBoV-positive cases detected by conventional PCR were also confirmed by real-time PCR performed us-KJLM ing the TaqMan probe. The viral load detected in the HBoVpositive samples by using real-time PCR was in the range of 1.3 × 10 3 -4.6 × 10 9 copies/mL (median, 1.82 × 10 5 copies/ mL). The HBoV-positive cases were categorized in 2 groups: low-viral-load group (viral load ≤ 1.0 × 10 6 copies/mL, N = 58) and high-viral-load group (viral load > 1.0 × 10 6 copies/ mL, N = 35) ( Table 1 ). The HBoV-positive patients aged less than 3 yr had a significantly higher viral load than that in the patients aged more than 3 yr (P = 0. 001).
The other respiratory viruses found in 17 (18.3%) of the total HBoV-positive samples were as follows: IFA, 1 sample; IFB, 1 sample; PIV, 10 samples; RSV, 4 samples; and ADV, 1 sample. Most of the cases (88.2%, 15/17) belonged to the low-viral-load group. RSV was isolated from the remaining 2 samples with HBoV copy numbers of 1.09 × 10 6 copies/ mL and 2.65 × 10 8 copies/mL.
Patients positive for HBoV alone had a higher viral load than that in the patients who were positive for both HBoV and another respiratory virus (median 3.78 × 10 5 copies/mL vs. 1.94 × 10 4 copies/mL, P = 0.014). A high-viral-load was almost exclusively seen in the HBoV-positive patients alone (94.3%, 33/35) (Fig. 2) .
The high-viral-load group had a significantly higher pulse rate and respiratory rate than the corresponding rates in the low-viral-load group (P = 0.007 and P = 0.0231, respectively; Table 2 ). Although the duration of hospital stay was not significantly different between the 2 groups (Table 2), in cases of patients with less than 10 days of hospital stay, the highviral-load group had a longer hospital stay than the low-viral-load group did (5. Clinical examination of the HBoV-positive patients showed pneumonia, bronchiolitis, bronchitis, croup, asthma, sinusitis, and pharyngotonsilitis (Fig. 3) . HBoV was detected in 8.4% (N = 48) and 5.6% (N = 45) of the samples obtained from patients with and without pneumonia, respectively (N = 572 and 805, respectively; P = 0.049). However, no significant difference was observed in the viral load between 
Ninety-three (4.8%) of the 1,926 nasopharyngeal aspirates obtained from patients of all age-groups were positive for HBoV. Our detection rate is similar to that stated in other reports [2, 3, 8] . Generally, HBoV is detected in fewer than 8% of respiratory specimens [1, [8] [9] [10] [11] [12] [13] ; however, higher detection rates ranging from 10.3% to 19% have been reported [5, 14, 15] .
To investigate the epidemiological association of respiratory infection with viral load, HBoV-positive patients were categorized into low-and high-viral-load groups by using 1.0 × 10 6 copies/mL as a threshold value. The detection rate of HBoV infection was at its peak in the first year of life (rate of detection, 8.6% between the ages of 6 and 12 months), as in the cases of RSV or PIV infection. Most of the HBoVpositive patients aged less than 3 yr belonged to the high-viral-load group. The detection rate was lower (1.5%) in patients aged more than 10 yr, and these patients belonged exclusively to the low-viral-load group. HBoV is rarely de-tected in adults except in cases of immunosuppression [10, 16, 17] . The lower detection rate and viral load of HBoV in older patients may be attributed to immunity acquired from an infection at a younger age. A seroepidemiologic study of HBoV showed that 5.6-83.3% of children aged 6 months-3 yr were seropositive for HBoV [18] . Lau et al. [19] suggested that HBoV infection might develop only once because of the subsequent development of life-long immunity conferred by neutralizing antibodies produced in response to the infection.
The frequency of HBoV codetection with other respiratory viruses was 18.3% in the HBoV-positive samples and was lower than the previously published data [4, 20] . This difference in the codetection frequency is attributed to the different detection methods; molecular diagnostic methods were used for the detection of other respiratory viruses in the other studies whereas we used virus culturing. Among the 17 HBoV-positive patients who were also positive for infection with other viruses, 10 showed PIV infection. The high association of HBoV with PIV seems to be attributed to the high prevalence of PIV infection in 2006 (22.8% in May). Interestingly, almost all cases (except two) positive for both HBoV and another respiratory virus belonged to the low-viral-load group. As the virus culture was used for the detection of major respiratory viruses, the isolated virus could be the main causative agent of respiratory illness. Therefore, the presence of low copy number of HBoV, detected by molecular method, may indicate prolonged viral shedding or an asymptomatic infection. Recently, prolonged presence of HBoV in NPAs has been reported [21] . These results suggest that single HBoV infection in the high-viral-load group may play an active role in respiratory infection. These findings are consistent with a Norwegian study that reported detection of HBoV alone and a high-vi- Low-viral-load High-viral-load KJLM ral-load were associated with respiratory tract infection [20] . In that study, patients with a high-viral-load in NPAs developed viremia more frequently than the patients with a moderate-or a low-viral-load did. In our study, HBoV-positive patients in the high-viral-load group showed significantly higher pulse rates and respiratory rates than the in the low-viral-load group. These findings also support the idea that a high-viral-load may be associated with a respiratory infection.
Previous studies have reported that HBoV infection was more prevalent among individuals who had other respiratory viruses [10, 22] . In a study performed in Hong Kong [19] , a higher detection rate of HBoV was observed in NPAs positive for common respiratory viruses than in those that were negative for the same. However, in our study, similar detection rate of HBoV was observed in the samples positive and negative for other respiratory viruses in the R-mix culture (Data are not shown).
Previous studies showed that cases of HBoV infection were found throughout the year with a peak incidence rate in the winter season [10, 13, 23] . However, in our study, cases of HBoV infection were detected most frequently during the spring season. This finding is similar to those of reports from Korea [4, 24] . This seasonal difference in the incidence of HBoV infection may be attributed to regional and temporal differences.
Bastien et al. [8] suggested that risk factors for severe HBoV infection appear to be similar to those for RSV infection (prematurity, congenital heart disease, and asthma). Thirty percent (26/93) of the HBoV-positive patients had underlying conditions such as heart disease, asthma, allergy, preterm birth, and a history of convulsions; most of these patients (80.8%) showed a low-viral-load. Persistent HBoV shedding for more than 1 month is observed in both respiratory and fecal specimens obtained from patients with significant underlying diseases [19] . Although these finding were not fully understood, it is postulated to be a result of underlying immunosuppression [19] .
In summary, HBoV infection was more prevalent in young children. Patients positive for HBoV alone mainly constituted the high-viral-load group. Most of the HBoV-positive patients with infection caused by other respiratory viruses belonged to the low-viral-load group. These findings suggest that HBoV may be associated with a respiratory infection. Seasonal distribution of active systemic lupus erythematosus and its correlation with meteorological factors OBJECTIVE: To explore the characteristics of seasonal distribution of active systemic lupus erythematosus (SLE) and the influences of meteorological factors including temperature and humidity on active systemic lupus erythematosus. METHODS: The characteristics of seasonal distribution of active SLE and its correlation with meteorological factors were retrospectively analyzed in 640 patients living in the city of Zhanjiang, China and had active SLE between January 1997 and December 2006. RESULTS: In winter, when there are weaker ultraviolet (UV) rays, the ratio of patients with active SLE to total inpatients was 3.89 ‰, which is significantly higher than in other seasons with stronger UV rays, including 2.17 ‰ in spring, 1.87 ‰ in summer and 2.12 ‰ in autumn. The number of patients with active SLE had significant negative correlation with mean temperature and was not significantly related to mean humidity. CONCLUSION: Active SLE has the characteristics of seasonal distribution and is associated with temperature. The mechanism remains to be further studied. Systemic lupus erythematosis (SLE) is a common autoimmune disease. The pathogenesis of SLE and the induced factors of active SLE are not completely clear, but it is generally considered that SLE is caused by both hereditary and environmental factors that ultimately lead to an abnormal immune response. Sunlight and ultraviolet (UV) rays have been considered to be the most important environmental factors in the induction of SLE. 1 Based on this, a high incidence of SLE should occur in summer and autumn when there is stronger sunlight and more intense UV rays. However, in clinical practice, we have found that more patients with SLE visit hospital at the end of autumn and at the end of winter, as well as at the beginning of winter and the beginning of spring, while in the summer and the autumn, less patients with SLE visit hospital. In order to confirm that high incidences of SLE should occur in summer and autumn when sunlight and UV rays are strong, we retrospectively analyzed the characteristics of seasonal distribution of active SLE and its correlation with meteorological factors in 640 patients, who live in the city of Zhanjiang, China and had active SLE between January 1997 and December 2006.
All study methods were approved by ethics committee of Affiliated Hospital of Guangdong Medical College. All the subjects enrolled in the study gave written formal consent to participate.
The 640 patients with active SLE were attented the Affiliated Hospital of Guangdong Medical College between January 1997 and December 2006. All patients conformed to the diagnostic criteria for SLE published by the American College of Rheumatology in 1982. 2 Of the 640 patients, 567 were female and 73 were male, with a mean age of (28.8¡12.1) years (range: 5-69). Disease duration was between 2 days and 20 years. SLE disease activity index (SLEDAI) was evaluated with a SLEDAI score system in all patients. The patients with SLEDAI $10 were considered to have severely active SLE and were included in the study. Of the 640 patients, SLE occurred for the first time or recurred Copyright ß 2011 CLINICS -This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. as a result of stopping treatment when their condition improved. Of the patients who had been hospitalized several times as a result of a flare episode with SLEDAI $10, we only recorded the first data. The patients in whom SLEDAI score could not be calculated as a result of incomplete clinical data, infection, or who were living more than 100 km from the city center were excluded from this study.
Meteorological data between January 1997 and December 2006 were obtained from the Weather Bureau of Zhanjiang City, and the mean temperature and humidity of each month were calculated.
The absolute and relative numbers of the patients with active SLE (relative to the total number of inpatients) were calculated. The correlations between the number of patients with active SLE and meteorological factors including temperature and humidity were analyzed.
Statistical analysis was performed with SPSS (12.0) software. x 2 test was used for the comparison of ratios. Spearman analysis was used to evaluate the correlations between the number of patients with active SLE and meteorological factors. Statistical significance was established at p,0.05.
Between January 1997 and December 2006, there were 640 patients who conformed to the diagnostic criteria for SLE and had a SLEDAI score of $10. Results indicated that the number of patients with active SLE was highest in winter (December, January and February) and lowest at the junction between summer and autumn (July, August and September). Distribution of the 640 patients over 12 months is shown in Figure 1 and Table 1 .
Results indicated that the total number of inpatients was less in January and February, and there was no significant difference in the number of inpatients between other months. The ratios of patients with active SLE relative to total inpatients are shown in Table 2 . The trend of relative ratios of patients with active SLE was consistent with that of the absolute number of patients with active SLE. Namely, that the relative ratios of patients with active SLE were the highest in December, January and February, and lowest in July, August and September. The relative ratio of patients with active SLE was significantly higher in winter (December, January and February) than in spring (March, April and May), summer (June, July and August) and autumn (September, October and November) (p,0.001). There was no statistical significance in relative ratios of patients with active SLE between spring, summer and autumn (p.0.05).
The years between January 1997 and December 2006, were divided into 120 months with a 'month' categorized as a unit. Then the mean temperature of each month was calculated. The correlations between the number of patients with active SLE and the mean temperature in the same month were analyzed; it was found that there was a significantly inverse correlation between both (r = -0.352, p,0.001, Figure 2 and Table 1 ).
The years between January 1997 and December 2006 were divided into 120 months with a 'month' categorized as a unit, and then the mean humidity of each month was calculated. The correlations between the number of patients with active SLE and the mean humidity in the same month were analyzed; it was found that there was no significant correlation between either (r = -0.053, p.0.05, Figure 3 ).
Aside from hereditary factors and sexual hormones, 3, 4 environmental factors are also among the main factors in the induction of SLE. 5 Considering environmental factors, UVinduced SLE has attracted much attention. UV light may induce auto-antigenic apoptotic particles resulting in DNA peroxidation and up-regulation of some cytokines and chemotactic factors, resulting in SLE or its recurrence. 6 Based on this, the incidence of SLE in the summer and the autumn, when there is stronger sunlight and stronger UV rays, should be higher. However, there have been different reports regarding seasonal distribution of active SLE. [7] [8] [9] [10] [11] [12] Differing results may be due to the fact that patients lived in various geographical locations, the sample sizes were small, and SLE was stable in the patients who were being followed-up and being treated with drugs, which failed to objectively reflect seasonal distribution of active SLE.
The purpose of this study was to explore seasonal distribution of active SLE. Our hospital is the largest hospital in the local area (Zhanjiang City, Guangdong Province, China) and is also the control center for rheumatic autoimmune disease. The majority of patients with SLE in Zhanjiang City attend the hospital for diagnosis and treatment. Patients with active SLE in this study reside in Zhanjiang City. According to the SLEDAI score system, SLEDAI ,5 was considered as mild SLE, 5-10 as moderate and .10 as severe. 13 Hormones and immunosuppressive agents are generally given when the SLEDAI is $6. 14 After administration of hormones and immunosuppressive agents, most patients still have moderate SLE (SLEDAI: 5-10) for a lengthy period. Although therapy is effective, SLEDAI fails to decrease to 0. To ensure patients had active SLE, only patients with a SLEDAI of $10 were included in this study. These patients, therefore, basically reflect the distribution of active SLE in Zhanjiang City.
Our results indicate that the number of patients with active SLE was highest in winter (December, January and February) and lowest in summer (June, July and August).
To eliminate the influence of other factors, we also analyzed the total number of inpatients each month between January 1997 and December 2006 and calculated the ratio of patients with active SLE relative to the total number of inpatients. We also found that the relative ratio of patients with active SLE was significantly higher in winter than in other seasons. Zhanjiang City is located at east longitude 110.3˚and northern latitude 21.2˚, and has a subtropical climate regulated by the ocean. Based on collected meteorological data, the annual mean temperature is 23.2˚C. In July, the mean temperature is 29.1˚C, there is the most daylight and sunlight intensity is strongest. In January, the mean temperature is 16.6˚C, there is least daylight and sunlight intensity is weakest. In contrast with the traditional viewpoint that the highest incidence of SLE should occur when sunlight and UV rays are strong, our results show that the number of patients with active SLE is fewer in summer when UV rays are stronger, while there is a higher incidence in winter when UV rays are weaker. Our study failed to discover whether various lupus lesions have seasonal distribution; however, Schlesinger et al. 15 found that the incidence of membranous lupus nephritis increased in winter.
It is known that many infectious diseases have seasonal distributions, and high incidences of infectious diseases mostly occur in winter. 16 Some virus infections are positively correlated with serum anti-ds-DNA antibody titers of patients with SLE. 17 A high incidence of virus infection of the respiratory tract occurs in winter and spring. In order to eliminate the influence of infection factors on seasonal distribution of active SLE, patients who had active SLE combined with an infection were excluded from the study. Therefore, it can be discounted that infection causes a high incidence of active SLE in the winter and a low incidence in the summer.
Our study has found that there is no correlation between the absolute number of patients with active SLE and the mean humidity, while there is an inverse correlation between the number of patients with active SLE and the mean temperature, demonstrating that, based on genetic background, a low-temperature environment may be one of the causes to induce SLE. Some scholars speculate that a high incidence of SLE in winter may be caused by UV accumulation after exposure to strong sunlight in summer. 11 Leone et al. 18 found that the level of serum anti-dsDNA is increased, while the levels of C3 and C4 are decreased in patients with SLE from August to January. The mechanism of a high incidence of active SLE in winter and a low incidence in summer remains to be further elucidated.
In summary, our study indicates that active SLE has seasonal distribution and its mechanism remains to be further explored. Ceacam1 Separates Graft-versus-Host-Disease from Graft-versus-Tumor Activity after Experimental Allogeneic Bone Marrow Transplantation BACKGROUND: Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models. METHODS: We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1(−/−) T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25(hi), CD62L(lo)). Additionally, Ceacam1(−/−) CD8 T cells had greater expression of the gut-trafficking integrin α(4)β(7), though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1(−/−) recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1(−/−) mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1(+) lymphoma model was improved in animals receiving Ceacam1(−/−) vs. control T cells. CONCLUSIONS: We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation. Ceacam1 is a member of a large family of carcinoembryonic antigen proteins [2] . It is primarily a type I transmembrane protein with multiple splice variants [9] , though soluble forms also exist. Ceacam1 is widely expressed on a variety of tissues including endothelium [10] , epithelium [11] , hematopoietic cells [12] and both hematologic and solid tumors, and interacts in a homophilic and heterophilic fashion with physiological and pathogenassociated ligands, including carcinoembryonic antigen and the Neisseria spp. proteins [13] .
Some Ceacam1 isoforms contain intracellular ITIM motifs, and activation of Ceacam1 results in the recruitment of the SHP-1 and SHP-2 phosphatases [8, 14] , which dephosphorylate substrates across a range of signaling pathways. Ceacam1 thus inhibits T cell receptor (TCR) signaling and suppresses multiple aspects of T cell function. Ceacam1 agonists attenuate cytokine secretion, T cell polarization and cytolytic function. In vivo, ligation of Ceacam1 with soluble ligands or over-expression of ITIM-containing Ceacam1 isoforms on T cells attenuates experimental colitis [7, 8] . Additionally, Ceacam1 is also expressed on intestinal T cells in patients with Celiac disease [15] and ulcerative colitis [16] , and may represent an attempt by the immune system to negatively regulate these inflammatory processes.
In addition to immune regulation, Ceacam1 exerts a wide variety of other biological functions. It is a cell-cell adhesion molecule [17, 18] , and a receptor for a variety of commensal and pathogenic microbes in mouse and man [17, 19, 20, 21] . Ceacam1 also regulates angiogenesis [6] , energy homeostasis [22] , and tumor biology [23, 24, 25] . Ceacam1 regulates the tumorigenesis of colon cancers, and is a prognostic factor in lung adenocarcinoma. Tumor expression of Ceacam1 may regulate tumor angiogenesis and invasion, and the expression of both Ceacam1 and CEA by tumors may inhibit the functions of tumor infiltrating lymphocytes.
Allo-BMT is an established therapy with curative intent for a variety of hematologic malignancies and non-malignant conditions [26] . Alloreactive T cells of donor origin play a criticial role in both GVHD, a major complication of allo-BMT, and graft-versustumor activity, a major contributor to the efficacy of allo-BMT as a cancer therapy. Donor-recipient antigenic disparity, donor T cells, and tissue injury resulting in inflammation due to the conditioning regimen all contribute to GVHD, which primarily affects intestines, liver, skin and thymus [27] .
Ceacam1 is expressed both on leukocytes (especially T cells), as well as on epithelial and endothelial cells, which are prominent components of the parenchyma of the above-mentioned GVHD target organs. In addition, Ceacam1 is upregulated on many tumors. In this report, we assess the impact of Ceacam1 on alloreactive T cells in the donor allograft, as well as the effects of Ceacam1 deficiency on recipients of allo-BMT with respect to GVHD and GVT activity.
We assessed Ceacam1 regulation of GHVD on donor T cells or recipients in two well-described major histocompatibility complex (MHC) class I/II-disparate models C57BL/6 (B6, H-2 b )RBALB/ c (H-2 d ) and BALB/cRB10.BR (H-2 k ). We used Ceacam1 2/2 B6 mice [28] , Ceacam1-transgenic (Tg) B6 mice (described in Figure  S1 ), and Ceacam1 2/2 BALB/c mice [28] as the source of donor T cells or recipients. In all experiments, recipients received splitdose lethal irradiation (BALB/c: 8.5 Gy, B10.BR: 11 Gy) and a graft of 5610 6 allogeneic T cell depleted bone marrow (TCD-BM) of wildtype (WT) origin, with or without splenic T cells.
We first transplanted irradiated BALB/c mice with B6 TCD-BM with WT or Ceacam1 2/2 T cells, and observed that recipients of Ceacam1 2/2 T cells had significantly increased mortality compared to recipients of WT T cells ( Figure 1A , left). We confirmed this in a second MHC-disparate allo-BMT model, BALB/cRB10.BR ( Figure 1A, right) .
We next asked whether T cells overexpressing Ceacam1 would cause less disease, and transplanted BALB/c recipients with 0.5610 6 or 1610 6 donor WT or Ceacam 1-Tg T cells. At both doses, recipients of Ceacam1-Tg T cells showed attenuated mortality ( Figure 1B) . Finally, we assessed the role of Ceacam1 on tissues of allo-BMT recipients, and transferred TCD-BM+T cells into WT vs. Cea-cam1 2/2 BALB/c recipients. This revealed that Ceacam1 2/2 recipients had increased early (but not overall) mortality, with nearly 50% of mice succumbing within the first week ( Figure 1C ).
We next asked whether Ceacam1 regulated GVHD target organ damage, and again assessed effects of Ceacam1 deficiency or overexpression on donor T cells, and Ceacam1 2/2 allo-BMT recipients.
We observed that recipients of Ceacam1 2/2 T cells had more severe large intestinal GVHD (Figure 2A) . Surprisingly however, these mice exhibited less thymic GVHD, as determined by thymic cellularity and numbers of CD4 + CD8 + double-positive (DP) thymocytes ( Figure 2 ). Thymic cellularity from age/sex-matched non-transplanted animals are shown in Figure S2 . We also observed a modest trend towards less skin GVHD in recipients of Ceacam1 2/2 T cells ( Figure 2C ), suggesting that Ceacam1 2/2 T cells caused preferential damage to the (large) intestines.
In experiments comparing recipients of WT and Ceacam1-Tg T cells on day 21 post-transplant, we found that recipients of Ceacam1-Tg T cells demonstrated significantly less GVHD of the liver, intestines, and thymus compared to recipients of WT T cells, but similar skin GVHD ( Figures 2D-F ). This appears to suggest that Ceacam1-Tg T cells caused less GVHD overall, with relatively little organ specificity.
Finally, we assessed Ceacam1 2/2 allo-BMT recipients on day 14 post-transplant. In correspondence with increased early GVHD mortality, Ceacam1 2/2 allo-BMT recipients showed increased large bowel damage and thymic GVHD ( Figure 2G -H).
We next assessed the numbers of donor CD4 and CD8 effector T cells after transfer of Ceacam1 2/2 or Ceacam1-Tg T cellcontaining allografts, or in Ceacam1 2/2 allo-BMT recipients.
Comparing recipients of WT T cells with those receiving Ceacam1 2/2 T cells, we observed increased numbers of Ceacam1 2/2 donor alloactivated effector T cells in the spleen, MLN, and IEL of allo-BMT recipients ( Figure 3A) , which was associated with a concomitant decrease in the number of Ceacam1 2/2 alloactivated CD4 and CD8 T cells in the PLN and liver.
When we analyzed organs of recipients of allografts containing WT vs. Ceacam1-Tg T cells, we noted decreased numbers of donor effector T cells in the MLN, PLN, and liver ( Figure 3B ). Via histopathological analysis, we also observed decreased numbers of total lymphocytic infiltrates into the liver, small and large bowels by histopathology ( Figure 3B ), as well as decreased neutrophilic infiltrates in these organs (data not shown). Finally, we assessed infiltrating T cells in WT and Ceacam1 2/2 allo-BMT recipients, and observed increased numbers of donor alloactivated effector T cells in the MLN and IEL of Ceacam1 2/2 allo-BMT recipients, but decreased numbers of these cells in the PLN and liver ( Figure 3C ). Ceacam1 regulates the sensitivity of the small intestine to radiation injury
The accelerated early mortality of Ceacam1 2/2 allo-BMT recipients, together with increased accumulation of donor T cells in GI tract and mesenteric lymph nodes, but decreased numbers peripheral lymph nodes ( Figure 3C ), led us to ask whether Ceacam1 had differential effects in regulating GVHD target organ damage for various target organs and tissues. In the context of Ceacam1 2/2 recipients, we therefore tested the radiation sensitivity of Ceacam1 2/2 mice used as hosts, by irradiating WT and Ceacam1 2/2 BALB/c mice and assessing survival. Ceacam1 2/2 animals showed increased kinetics of mortality, and in some cases, overall mortality after radiation injury ( Figure 4A ). Similar results were obtained on the B6 background ( Figure 4C ). We then enumerated regenerating and surviving crypts in the small intestine (terminal ileum) at 84 hours after irradiation to assess intestinal radiation damage, and observed that Ceacam1 2/2 mice had fewer regenerating and surviving crypts as compared with WT counterparts ( Figure 4B and D), indicating greater damage to the small intestine across a wide range of radiation doses. It is thus quite likely that our radiation-containing conditioning regimen for transplant recipients also contributes in part to their survival kinetics, selective GVHD target organ damage ( Figure 2G -H), and the selective accumulation of donor T cells in lymphoid tissues and target organs ( Figure 3C ) in these recipients.
Donor Ceacam1 2/2 CD8 T cells express higher levels of integrin a 4 b 7 post-transplant Next, we studied a variety of possible mechanisms by which Ceacam1 may regulate donor T cell function. We analyzed donor WT and Ceacam1 2/2 alloactivated splenic T cells on day 14 after allo-BMT for trafficking molecules, and found that Ceacam1 2/2 CD8 + CD44 + CD62L 2 effector T cells expressed higher levels of integrin b 7 subunit and the gut homing integrin a 4 b 7 ( Figure 5A) , which is important for intestinal GVHD [31, 32, 33] . However, WT vs. Ceacam12/2 CD4 effector T cells had similar integrin b 7 subunit expression, yet also accumulated in greater numbers in the gut ( Figure 3A) , suggesting that regulation of target organ damage by Ceacam1 is very likely to involve multiple additional mechanisms beyond trafficking molecule expression. Additionally, levels of the a E subunit, which forms integrin a E b 7 , were similar, as were levels of CCR9, CD31, PSGL1, CCR7, CXCR3, and LFA1 (data not shown).
When we assessed the expression of trafficking molecules in recipients of WT vs. Ceacam1-Tg T cell allografts, we found no significant differences in levels of b 7 subunit, integrin a 4 b 7 ( Figure 5B ), or any other molecules. This is consistent with the systemic reduction in GVHD in recipients of Ceacam1-Tg T cells. Finally, we assessed trafficking molecules in irradiated WT vs. Ceacam1 2/2 recipients of identical donor allografts, and observed again that donor CD8, but not CD4 splenic T cells in Ceacam1 2/2 
Ceacam1 can be found on activated T cells [7, 8, 29 ] and, we thus performed a kinetic analysis of Ceacam1 expression on T cells during alloactivation. We adoptively transferred CFSE-labeled B6 T cells into irradiated BALB/c recipients, and observed transient expression only on day 2 after alloactivation ( Figure 6 and data not shown). Furthermore, only CFSE lo alloactivated T cells, which have divided $4 times in 48 hours, expressed low but consistently detectable levels of Ceacam1 ( Figure 6A ). These kinetics are consistent with a role for Ceacam1 in regulating early events in T cell alloactivation.
Because the expression of Ceacam1 on alloreactive T cells after adoptive transfer occurred in vivo with similar kinetics as T cell alloactivation [30] , we asked whether Ceacam1 on either donor alloreactive T cells or radio-resistant cells in allo-BMT recipients could regulate this process. We transferred CFSE-labeled purified B6 WT or Ceacam1 2/2 splenic T cells into irradiated BALB/c recipients and analyzed donor T cells in spleens on day 3.
We observed that relative to isotype control staining, an increased percentage of alloactivated CFSE lo CD4 Ceacam1 2/2 T cells were positive for the alloactivation marker CD25, and that a greater percentage of these cells downregulated CD62L than WT T cells ( Figure 6B-C) , suggesting that more of them became activated. Additionally, an increased percentage of donor Ceacam1 2/2 CD4 T cells had divided to a CFSE lo alloactivated state ( Figure 6D) , suggesting enhanced proliferation in the absence of Ceacam1.
We repeated these experiments with alloreactive Ceacam1-Tg T cells and as expected, observed a decrease in numbers of CFSE lo T cells as assessed by CFSE dilution ( Figure 6E ). This is consistent with an inhibitory role for Ceacam1 in the proliferation of alloreactive T cells. However, we did not observe significant differences in alloactivation between Ceacam1-Tg vs. WT donor T cells (data not shown).
Lastly, we assessed the role of Ceacam1 expression on radioresistant cells in allo-BMT recipients for donor T cell alloactivation. We transferred CFSE-labeled B6 T cells into irradiated WT vs. Ceacam1 2/2 BALB/c mice, and analyzed donor T cells in spleens on day 3. Here, we did not observe differences in proliferation (data not shown), but donor CD4 T cells in Ceacam1 2/2 allogeneic recipients did exhibit an increase in alloactivation as measured by CD25 ( Figure 6F ).
We measured serum cytokines in recipients of WT, Ceacam1-Tg and Ceacam1 2/2 T cells on days 7 and 14 post-transplant, and observed that levels of IFNc, TNF, IL-2, IL-4, IL-6, IL-10, and IL-12p70 were similar (data not shown). Percentages of FoxP3 + donor regulatory T cells and expression of T-bet were also similar between recipients of WT, Ceacam1-Tg and Ceacam1 2/2 T cells (data not shown and Table 1 ), and stimulation of splenocytes harvested on day 14 after BMT post-transplant from these three groups revealed essentially no IL-17 + donor T cells (not shown), and similar percentages of donor IFNc + T cells (data not shown and Table 1 ).
As Ceacam1 can regulate the cytolytic responses of lymphocytes [34, 35, 36, 37, 38] , we assessed the cytolytic function of WT vs. Ceacam1 2/2 alloactivated CD8 T cells from the spleens of allo-BMT recipients on day 14. Ceacam1 2/2 CD8 T cells and WT CD8 T cells demonstrated similar cytolysis against 51 Cr-radiolabeled allogeneic A20 B cell lymphoma cells and EL4 controls ( Table 1) . Lastly, we found no differences in DC numbers, activation state (CD80, CD86, MHC class II) from the infusion of Ceacam1 2/2 or Ceacam1-Tg T cells (Table 1) , or in Ceacam1 2/2 allo-BMT recipients.
Finally, we assessed the GVT activity of Ceacam1 2/2 donor alloreactive T cells against A20 lymphoma and RENCA renal cell carcinoma. Recipients of Ceacam1 2/2 donor T cells had improved survival in the A20 lymphoma model ( Figure 7A) , but both T cell replete groups showed comparable survival in the RENCA solid tumor model ( Figure 7B ). When we analyzed these two tumor lines for Ceacam1 expression, we noted that all A20 lymphoma cells uniformly expressed high levels, while only a subset of RENCA cells expressed some Ceacam1 ( Figure 7C ).
In this report, we show that Ceacam1, which is found on both donor alloreactive T cells as well as non-hematopoietic tissues such as gastrointestinal and hepatic epithelium, can regulate both donor T cell function and the sensitivity of allo-BMT recipients to radiation-containing preparative regimens. In addition, Ceacam1 on donor T cells and tumors may modulate GVT activity. Ceacam1 on both the donor allograft and recipient tissues thus appears to represent an important regulator of GVHD and GVT morbidity and mortality via both T cell dependent and independent mechanisms, suggesting that therapeutic approaches which modulate Ceacam1 may need to assess and balance GVHD vs. GVT.
Ceacam1 on T cells has previously been shown to restrain CD4 T cell polarization, cytokine secretion and cytotoxicity. In our GVHD model systems however, we found similar T cell polarization and cytokine secretion when we analyzed donor alloreactive T cells ex vivo ( Table 1) . We ascribe this to the strongly proinflammatory cytokine milieu found in recipients following myeloablative radiation treatment, as well as the ubiquitous presence of alloantigen, which together promote strong Th1 differentiation regardless of Ceacam1 expression. However, in our model systems Ceacam1 regulated T cell activation, and numbers of donor alloactivated T cells in both lymphoid tissues and GVHD target tissues, in patterns that generally correlated with levels target organ damage (Figures 2 and 3) .
We also assessed the role of Ceacam1 in allo-BMT recipients. In our model systems, WT T cells in a Ceacam1-deficient environment showed a phenotype similar to that of Ceacam1 2/2 alloactivated T cells: both showed increased activation, selective damage to the large intestines, and preferential accumulation in the MLN and intestinal parenchyma of mice with GVHD, and correspondingly decreased infiltration of the liver and PLN, ultimately leading to exacerbation of disease, with accelerated mortality in the first two weeks post-transplant. This suggests that Ceacam1 on donor T cells interacts with recipient tissues, and that Ceacam1 ''fraternal'' interactions between cells of the donor graft, were not sufficient to restrain GVHD in Ceacam1 2/2 recipients. However, the increased early mortality of Ceacam1 2/2 allo-BMT recipients with GVHD also led us to ask whether Ceacam1 2/2 mice were sensitive to radiation injury. While Ceacam1 2/2 mice were not significantly defective for hematopoiesis after sublethal irradiation at 3.5 and 4.5 Gy (data not shown), they did exhibit significantly increased damage to the small intestines after lethal irradiation ( Figure 4) .
Ceacam1 also directly regulates intestinal epithelia. Due to enhanced Wnt/b-catenin signaling, Ceacam1 2/2 jejunal and ileal enterocytes exhibit higher levels of the positive cell cycle regulators c-Myc and cyclin D1 [41] . Dysregulated c-Myc may sensitize cells to apoptosis [42, 43] , and higher levels of these proteins may render Ceacam1 2/2 enterocytes more sensitive to radiation injury. Finally, Ceacam1 also regulates cell-cell adhesion [17, 18, 44, 45] under normal and pathological conditions; it may therefore also be possible that loss of Ceacam1 regulates radiationinduced sloughing of intestinal epithelium.
It is difficult to directly assess the relative importance of gastrointestinal radiation sensitivity versus increased GVHD in Ceacam1 2/2 allo-BMT recipients, as radiation-induced gut damage may both be directly manifested in intestinal pathology, yet transmural migration of bacterial superantigens is an important first step for the initiation of GVHD [1, 39, 40] , and increased damage to the intestines of Ceacam1 2/2 mice may thus amplify the development of GVHD in these mice, and also explain in part the specifically increased large intestinal GVHD we observed.
In experiments with Ceacam1 2/2 donor T cells, we also observed a trend for splenic donor CD8 alloactivated T cells to express higher levels of a 4 b 7 . Although integrin a 4 b 7 is important for GVHD pathogenesis, and we have previously shown that b 7 2/2 T cells cause a sustained decrease in acute systemic and intestinal GVHD [31] , differential expression of integrin a 4 b 7 by Ceacam1 2/2 T cells is almost certainly only one part of how Ceacam1 regulates target organ GVHD. Indeed, donor alloactivated CD4 T cells expressed comparable levels of integrin a 4 b 7 as wildtype cells, yet were also found in increased numbers in the gut ( Figure 3A ). This suggests that other mechanisms, such as Ceacam1 regulation of donor T cell activation ( Figure 6 ) may also contribute to its regulation of GVHD target organ damage.
Moreover, recipients of Ceacam1-Tg T cells also had reduced intestinal infiltrates despite similar integrin a 4 b 7 expression, suggesting that Ceacam1 regulates the accumulation of donor T cells in target tissues via multiple mechanisms. Thus, our results on donor lymphocyte infiltrates into GVHD target tissues and secondary lymphoid tissues must be interpreted cautiously, as they must be influenced by T cell proliferation, retention and apoptosis, in addition to trafficking. Although Ceacam1 2/2 and Ceacam1-Tg T cells displayed overall symmetric and opposite phenotypes, we also noted differences. Ceacam1-Tg T cells primarily showed decreased proliferation, whereas Ceacam1 2/2 T cells showed changes in proliferation, but also trafficking and activation. Some of these differences may be due to our models: on WT T cells, Ceacam1 is only briefly and transiently upregulated during activation. Consequently, Ceacam1 2/2 T cells are ''missing'' Ceacam1 only transiently, while Ceacam1-Tg T cells constitutively over-express Ceacam1. Furthermore, while Ceacam1 2/2 T cells are effectively insensitive to all Ceacam1 ligands and interactions, Ceacam1-Tg T cells which over-express the protein may have increased fraternal Ceacam1 interactions with other donor T cells, but may not necessarily experience increased Ceacam1 interactions with donor BM or host hematopoietic and non-hematopoietic components. These differences may explain why their activation and trafficking phenotypes are not directly opposed.
We were interested to note that in our GVT experiments, recipients of Ceacam1 2/2 T cells had significantly improved survival when challenged with A20 lymphoma but not renal cell carcinoma. Although both A20 lymphoma and renal cell carcinoma express Ceacam1, A20 cells uniformly expressed Ceacam1 at high levels, while only a subset of RENCA cells showed (somewhat lower) expression. Indeed, a number of hematologic tumors, including EL4 leukemia, P815 mastocytoma, and C1498 myeloid leukemia all express substantial levels of Ceacam1 (data not shown), whereas some solid tumors, such as mouse 4T1 breast epithelial cancer and CT51 colon tumor normally express only lower or even minimal levels of Ceacam1, similar to the lower level of expression we found with RENCA (not shown).
Therefore, one possibility is that the GVT activity of T cells can be negatively regulated by tumors expressing high levels of Ceacam1, but is less important for tumors that express low levels or only on a subset of cells in the first place. However, RENCA in our GVT model systems is found primarily in the liver, and to a lesser extent, the lungs. Since donor allografts with Ceacam1 2/2 T cells showed decreased numbers of donor alloreactive T cells in the liver as compared with wildype in GVHD experiments ( Figure 3A) , interpretation of GVT activity against RENCA with respect to Ceacam1 on T cells must also consider this aspect of its biology.
In conclusion, our results show that Ceacam1 on both donor T cells and allo-BMT recipients controls the proliferation, activation, and trafficking of donor alloreactive T cells, and the sensitivity of gastrointestinal tissues to irradiation. Consequently, Ceacam1 may represent a viable target for reducing radiation-associated gastrointestinal toxicity, for the control of GVHD and GVT activity after allo-BMT.
All animal protocols were approved by the Memorial Sloan-Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (protocol #99-07-025).
Mice C57BL/6 (B6, H-2 b ), BALB/c (H-2 d ), and B10.BR (H-2 k ) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). B6 and BALB/c Ceacam1 2/2 mice, and B6 Ceacam1-Tg mice were generated at McGill University (B6 and BALB/c from Harlan (Montreal, Quebec, Canada)), and maintained at Memorial Sloan-Kettering Cancer Center. Mice used were between 8 and 12 weeks old.
BM cells removed from femurs and tibias were T cell-depleted (TCD) with anti-Thy-1.2 and low-TOX-M rabbit complement (Cedarlane Laboratories, Hornby, ON, Canada). Enriched splenic T cells were obtained by nylon wool column passage. Cells were resuspended in DMEM and injected into lethally irradiated recipients on day 0 after total body irradiation ( 137 Cs source) as a split dose 3 hours apart.
Purified splenic T cells were incubated with CFSE (Invitrogen, Carlsbad, CA) at a concentration of 2.5-5 mM in PBS (5610 7 cells/mL) at 37uC for 20 minutes, washed twice with PBS, resuspended in DMEM and infused intravenously into lethally irradiated allogeneic recipients. Splenocytes from recipients were harvested at varying time points and analyzed by FACS as described.
Survival was monitored daily, and mice were scored weekly for 5 clinical parameters (weight, posture, activity level, fur ruffling, and skin lesions) on a scale from 0 to 2. A clinical GVHD score was generated by summation of the 5 criteria scores; mice scoring 5 or greater were considered moribund and euthanized.
Small and large bowel, liver, and skin were assessed by experts in a blinded fashion. Organs were preserved in formalin, transferred to 70% ethanol, and then embedded in paraffin, sectioned, stained with hematoxylin and eosin, and scored with a semi-quantitative scoring system. Bowel and liver were scored for 19 to 22 different parameters associated with GVHD (detailed in Table S1 ); skin was evaluated for number of apoptotic cells/mm 2 of epidermis via terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL).
Histopathologic scores, median fluorescence intensities and cell counts were compared between groups using the nonparametric unpaired Mann-Whitney U test; the Mantel-Cox log-rank test was used for survival data.
Additional methods are described in Methods S1. Figure S1 Generation of Ceacam-1 transgenic mice and expression of Ceacam1 on transgenic T cells. A) The CC1-4L cDNA, expressing 4 Ig domains and the long cytoplasmic domain was inserted into the unique EcoR1 site within the VAhCD2 vector containing the hCD2 promoter and 2 polyadenylation sites (PolyA1,2) . B) The linearized construct was microinjected into C57Bl/6 oocytes to produce transgenic mice that were identified by Southern blot with a 1.3 kb 32P-labelled probe. This probe cross-reacts with the endogenous Ceacam1, Ceacam2 and Ceacam10 genes and also identifies the 1.7 kb EcoR1-digested transgene. C) Tg mice were identified by PCR amplification of a 320 bp fragment from tail genomic DNA with the CyT2 oligo within the CC1-L cytoplasmic domain and oligo CD2A2 within the hCD2 LCR region. D) Western blots of lysates from thymi and spleens from 5 and 8-week-old WT and Tg littermates with the rabbit polyclonal anti-Ceacam1 Ab 2457. E-F) Cell surface Ceacam1 on thymic (E) and splenic (F) CD3-gated T cells of WT and Tg mice (n = 4) was revealed with anti-Ceacam1 Ab 2457. Controls were normal rabbit serum. (TIFF) Figure S2 . Ceacam1 2/2 mice exhibit increased thymic and splenic cellularity compared with wildtype animals, but do not exhibit skewing towards particular leukocyte lineages or subsets, while Ceacam1-Tg transgenic mice have similar numbers of splenocytes, thymocytes, and bone marrow cells compared with WT animals. A) 2 month old Ceacam1 2/2 male BALB/c mice were analyzed with age and sex-matched wildtype BALB/c, N = 5. Similar results were observed with age and sex-matched female wildtype and Ceacam1 2/2 BALB/c mice (N = 5, not shown), and in age and sex-matched wildtype and Ceacam1 2/2 B6 mice (N = 5, not shown). B) Thymocytes from mice in (A) were analyzed by flow cytometry. N = 5. C) 8-week old B6 and Ceacam1-Tg mice were analyzed for splenic, thymic and bone marrow (BM) cellularity. N = 3/group. (TIFF) Table S1 Histopathological scoring scheme for gastrointestinal GVHD target organs.
Methods S1
(DOC) Programmed Death (PD)-1-Deficient Mice Are Extremely Sensitive to Murine Hepatitis Virus Strain-3 (MHV-3) Infection The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4(+), CD8(+) T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy. Although liver transplantation has emerged as an effective therapeutic approach for treating fulminant virus hepatitis (FH), mortality rates associated with FH remain very high worldwide [1] . The recent development of a mouse FH model, based upon infection with the murine hepatitis virus strain-3 (MHV-3), has provided insights into mechanisms underlying the disease pathogenesis and resulted in some novel treatment strategies [2] .
MHV-3 is a single-stranded, positive-sense RNA virus that belongs to the Coronaviridae family. In inbred laboratory mice, the virus produces strain-dependent disease profiles that depend on the infection route, age, genetic background, and immune status of the host. For example, Balb/c, C57BL/6 and DBA/2 mice develop acute fulminant hepatitis, while C3H mice develop a mild chronic disease and mice of the A strain exhibit no evidence of hepatitis [3, 4] . In contrast to the resistant A strain mice, FH induced by MHV-3 in susceptible mice is characterized by the presence of sinusoidal thrombosis and hepatocellular necrosis [2, 3] . These pathological findings occur concomitantly with expression of fibrinogen-like protein 2 (FGL2), a virus-induced procoagulant molecule in the sinusoidal lining cells in the liver. FGL2 has the capacity to promote fibrinogen deposition and subsequently directly induce the coagulation cascades by the expression of procoagulant activity (PCA) [5] . Thus, up-regulation of FGL2 is an essential component of the lethal effects of MHV-3-induced FH.
Programmed death (PD)-1 is an inhibitory receptor expressed on activated T cells, B cells and myeloid cells. PD-1-deficient mice (Pdcd1 2/2 ) develop various spontaneous autoimmune diseases, including glomerulonephritis and dilated cardiomyopathy, indicating that this receptor plays a critical role in maintenance of peripheral tolerance [6] . PD-L1 (B7-H1) and PD-L2 (B7-DC), two immunoregulatory molecules belonging to the B7 superfamily, were identified as ligands for PD-1, engagement of PD-1 with its ligands mediates negative signaling events via recruitment of phosphatases, such as SHP-2, and dephosphorylation of some effector molecules involved in downstream T cell receptor (TCR) signaling [7, 8] .
PD-1 signaling has also been shown to modulate the balance between antimicrobial immune defense and immune-mediated tissue damage. For example, PD-1-deficient mice develop more severe hepatocellular injury than their wild-type (WT) littermates in response to adenovirus infection [9] . In a herpes simplex virus (HSV) stromal keratitis mouse model, blockade of PD-1 signaling led to increased HSV-1-specific effector CD4 + T cell expansion, IFN-c production, and exacerbated keratitis [10] . A functionallysignificant high level of PD-1 expression has been found to be maintained by exhausted CD8 + T cells in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), in primates exposed to simian immunodeficiency virus (SIV), and in humans suffering from infection with human immunodeficiency virus (HIV), hepatitis B or C virus (HBV or HCV), or human Tlymphotropic virus (HTLV). However, blockade of the PD-1/PD-Ls pathway efficiently restored the virus-specific effector functions of the exhausted T cells, and lead to substantially reduced in viral load [11, 12, 13, 14, 15] . The PD-1 signal is also known to play a key role in the chronicity of infections with bacteria (Helicobacter pylori and Schistosoma mansoni) [16, 17] , pathogenic fungus (Histoplasma capsulatum) [18] , and parasitic worms (Taenia crassiceps) [19] . It appears that a number of pathogenic microorganisms exploit the PD-1 signal in order to evade host immune responses and to establish persistent infection.
Although the influence of PD-1 signal activity has been studied in several infection models, there are no data available concerning the role of this pathway in FH. To this end, we used the MHV-3induced mouse FH model to demonstrate that PD-1 signaling acts to limit the immunopathological damage during disease progression. Furthermore, our findings suggested that enhanced PD-1 signaling might represent a useful immunotherapeutic strategy for treating FH.
PD-1 expression has been previously described as being induced on specific cell subsets in response to viral or bacterial infection [20] . Thus, we first determined the status of PD-1 expression at 72 h after MHV-3 infection (10 PFU) by immunohistochemical techniques. PD-1-positive cells were observed in tissues from the thymus, spleen, lymph nodes and liver. Cellular expression was localized to the cell membrane and in the cytoplasma while was completely absent from the nuclear compartment. PD-1-positive cells were distributed throughout the medulla and cortex of the thymus and lymph nodes. In the spleen, PD-1-positive cells were restricted to the germinal center under normal conditions, but extended to the red pulp after infection. In infected liver, more PD-1-positive cells were present in the portal and parenchymal areas, as opposed to the relatively low presence of PD-1-positive cells in only the portal area in phosphate-buffered saline (PBS) treated-mice (Fig. 1A) . The amount of PD-1-positive cells in the different organs of infected and control mice were counted and compared, results showed that the number of positive cells was significantly higher in infected mice (Fig. 1B) . Furthermore, FACS analysis revealed that PD-1 expression was enhanced on multiple subsets of immune cells, including the CD4 + and CD8 + T cells, NK1.1 + NK cells and CD68 + macrophages (Fig. 1C) . PD-1positive cells were also observed in the lung, heart and kidney, however, the numbers of PD-1 positive cells in these tissues did not significantly increase in response to MHV-3 infection (Fig. S1 ).
To investigate the potential role of PD-1 signaling in regulating FH tissue pathology, organs from MHV-3 infected PD-1-deficient (PD-1 KO) and WT mice were assessed for morphological differences. Small and discrete foci of necrosis with sparse polymorphonuclear leukocyte infiltration were observed in liver tissues from PD-1-deficient mice after 24 h of infection. In contrast, WT mice exhibited normal liver architecture at this time point. Slight liver damage became apparent in WT mice after 48 h of infection, meanwhile, the damaged areas of PD-1-deficient mice had enlarged and confluent necrosis had become evident. By 72 h of infection, the damaged region in PD-1-deficient mice had extended throughout the entire liver, while WT mice suffered much less damage and up to 60% of their liver tissue remained normal at this time point ( Fig. 2A) . Likewise, higher levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed in serum from PD-1-deficient mice after 72 h of infection (Fig. 2B ). More interestingly, PD-1-deficient thymus, spleen and lymph node tissues infected with MHV-3 for 72 h exhibited severely disrupted architecture, loss of cellularity, and the presence of substantial amounts of karyorrhectic/apoptotic cell bodies. The histology of these organs from infected WT mice at 72 h was relatively normal (Fig. 2C ). In conjunction with the apparent tissue necrosis, higher levels of cell apoptosis were also evidenced in the organs from PD-1-deficient mice by TUNEL staining (Fig. 2D ). The architecture of other organs, including the heart, kidney and lung was relatively normal and only rare apoptosis events were observed in these tissues after infection (Fig.  S2 ). In all, these results demonstrated that PD-1 deficiency led to enhanced pathological damage by MHV-3 in the liver, spleen, lymph node and thymus, where higher levels of PD-1-positive cells were found after infection.
The earlier and increased organ damage suffered by PD-1deficient mice infected with MHV-3 instigated our monitoring of the mortality rates of PD-1-deficient mice and their similarlyinfected (10 PFU) WT littermates. As shown in Fig. 3 , all of the PD-1-deficient mice died within four days after infection, while
The principal characteristic of fulminant viral hepatitis (FH) induced by the murine hepatitis virus strain-3 (MHV-3) is severe hepatocellular necrosis, which is mediated by the fibrinogen-like protein 2 (FGL2), a molecule that has the capacity to promote fibrinogen deposition and activate the coagulation cascades. Here, we report that MHV-3 infection of program death-1 (PD-1)-deficient mice results in tissue damage throughout multiple organs, including the liver, spleen, thymus and lymph nodes. The liver damage, in particular, occurred earlier and was more severe in PD-1-deficient mice than in their wild type (WT) littermates. Further investigation determined that MHV-3 infection was associated with high levels of IFN-c and TNF-a in the damaged organs of PD-1-deficient mice. Conversely, intraperitoneal injection of a combination of anti-IFN-c and anti-TNF-a blocking mAbs led to inhibition of FGL2 expression, greatly attenuated tissue lesions and reduced mortality. Our results demonstrate that PD-1 signaling controls immunopathological damage following MHV-3 infection, indicating that manipulation of the PD-1 signal might represent a useful strategy for FH immunotherapy.
38% of the WT mice survived up to the end of the 15-day survey period (p = 0.007). These data indicated that PD-1 is likely a critical factor that controls MHV-3-mediated tissue damage and mortality.
To understand the mechanisms of PD-1 deficiency-mediated tissue damage and mortality, we performed a comparative genome-wide microarray analysis (NimbleGen) of genes expressed in liver tissues of PD-1-deficient and WT mice after 72 h of MHV-3 infection. The most notable finding was pronounced upregulation (3.75-fold) in the liver of PD-1-deficient mice of the fgl2 transcripts (Fig. 4A) , the protein product of which has been demonstrated to induce lethality of MHV-3-induced FH [5] . In addition, the enhanced fgl2 expression was confirmed by quantitative (q)PCR, results revealed a 2.84-fold and 5.72-fold higher level was present in liver from WT and PD-1-deficient mice, respectively, after 72 h of MHV-3 infection, as compared to their uninfected controls. Moreover, its level in PD-1-deficient liver was 2.01-fold higher than that in the WT group at this time point (Fig. 4B) . Immunohistochemistry was used to show that FGL2-positive cells were present in necrotic liver tissues in PD-1deficient mice at 24 h after MHV-3 injection. The protein expression was found to be enhanced rapidly upon infection, and the highest level occurred at 72 h post-infection. However, occasional FGL2-positive cells were detected in the livers of WT mice at 24 h post-MHV-3 infection and these cells were also present, and slightly enhanced in number, at both the 48 h and 72 h time point (Fig. 4C ). Western-blot was used to verify the higher FGL2 protein level in the livers of PD-1-deficient mice, as compared to WT littermates after 72 h of infection (Fig. 4D ).
FGL2 has the capacity to induce fibrinogen deposition, which then activates the coagulation cascades and finally induces procoagulant activity. Therefore, the expression of FGL2 and fibrinogen deposition in damaged liver tissues was measured. Dual fluorescent staining evidenced that substantial fibrinogen deposition occurred in the FGL2-positive damaged liver tissue (Fig. 4E ). Likewise, the level of fibrinogen deposition was more robust in livers from PD-1-deficient mice that in livers from WT littermates, at both the 48 h and 72 h time points (Fig. 4F) .
To determine whether FGL2-mediated PCA activity was also involved in inducing damage in the other organs of PD-1-deficient mice, the expression of FGL2 was analyzed in the thymus, spleen and lymph nodes. Immunohistochemistry evidenced that FGL2positive cells were also present in these organs. In thymus and lymph nodes, FGL2-positive cells were detected in both the medulla and cortex. In spleen, however, the positive cells were only found in the red pulp. Again, the expression of FGL2 appeared to be restricted to the cell membrane and cytoplasma. The distribution of FGL2-positive cells in PD-1-deficient mice had not changed after 72 h of MHV-3 infection, but the number of positive cells in the examined organs was enhanced significantly and the levels of expression were much stronger (Fig. 5A ). In addition, the transcription of fgl2 in the spleen of PD-1-deficient mice was also significantly increased in response to infection (Fig. 5B) . Meanwhile, higher levels of fibrinogen deposition were found in the spleen and lymph node tissues of PD-1-deficient mice (Fig. 5C) . Moreover, the level of FGL2 present in serum, as measured by ELISA, was found to increase rapidly after infection, and the level in PD-1-deficient mice was significantly higher than that in WT littermates (Fig. 5D ). To clarify the source of FGL2, fluorescent dual staining was performed on spleen tissues and results demonstrated that FGL2 was principally associated with CD11c-positive dendritic cells (DCs), CD68-positive macrophages and CD31-positive endothelial cells (Fig. 5E ). All of these results indicated that the absence of PD-1 signaling can result in enhanced FGL2 expression, consequently inducing stronger fibrinogen deposition and more severe tissue necrosis in PD-1deficient mice following MHV-3 infection.
We further examined whether FGL2 secretion was regulated by PD-1 directly or indirectly. FGL2/PD-1 dual fluorescent staining was performed and results indicated that FGL2 and PD-1 were not co-expressed on the same cells in the liver, thymus, spleen or lymph nodes (Fig. 6A) .
Previous studies have shown that the secretion of FGL2 can be triggered by the pro-inflammatory factors IFN-c and TNF-a [21, 22] . On the other hand, the production of IFN-c and TNF-a by activated T cells, NK cells and macrophages can be inhibited by the PD-1 signal [6] . Therefore, we compared the status of IFNc and TNF-a in PD-1-deficient and WT mice in response to MHV-3 infection. qPCR revealed that the transcription of the IFN-c gene in liver was significantly higher in PD-1-deficient mice than in WT mice at 72 h post-MHV-3 infection (Fig. 6B) . In PD-1-deficient spleen, the transcription of both IFN-c and TNF-a was found to be rapidly enhanced upon MHV-3 exposure (Fig. 6C ). FACs analysis indicated that IFN-c secretion from NK cells, but not from CD3 + T cells, in the liver was much higher in PD-1deficient mice at 72 h after MHV-3 infection (Fig. 6D ). The fact that IFN-c and TNF-a both have the capacity to initiate FGL2 expression may explain why higher FGL2 expression was observed in the PD-1-deficient mice.
To further demonstrate that IFN-c and TNF-a were responsible for the observed FGL2 up-regulation in MHV-3 infected PD-1deficient mice, PD-1-deficient mice were infected with MHV-3 and simultaneously treated with a combination injection of anti-IFN-c and anti-TNF-a blocking mAbs. The expression of fgl2 was measured by qPCR and protein detected by immunohistochemistry. The transcription of fgl2 mRNA (Fig. 7A ) and its protein levels (Fig. 7B) were completely inhibited by 48 h after injection of anti-IFN-c and anti-TNF-a mAbs, as compared to the control rat IgG1 isotype antibodies-treated group. Moreover, the tissue necrosis (Fig. 7C ) and liver damage (as indicated by ALT and AST levels) (Fig. 7D ) in PD-1-deficient mice were also significantly reduced, thus the MHV-3-mediated mortality rates were decreased as well (Fig. 7E ).
The PD-1 signaling is best known for its ability to inhibit or dampen the immune response. Most of the evidence for this function, however, comes from models of tolerance or chronic infections [11, 12, 13, 14 15] . Although some studies have indicated that this signal might also participate in regulating acute infections [23, 24, 25, 26] , its functions in this disease condition are much less clear. Here, we used a mouse FH model mediated by MHV-3 infection to describe the effects of PD-1 in this disease process. Firstly, PD-1 was found to be significantly up-regulated on T cells, macrophages and NK cells within the thymus, spleen, lymph nodes and liver in response to MHV-3 infection. To determine the exact role of PD-1 in the pathogenesis of FH, PD-1deficient mice were used to establish an infection model. Interestingly, MHV-3-induced liver damage in PD-1-deficient mice occurred rapidly and the lesion area was much larger than in their WT littermates. We then extended our investigation to the thymus, spleen and lymph nodes, where increased PD-1-positive cells were observed post-infection. Surprisingly, severe tissue necrosis and substantial apoptosis was observed in these organs of PD-1-deficient mice at 72 h after MHV-3 infection. In contrast, these organs from WT mice exhibited relatively normal histology, a finding in agreement with previously reported results [27] . Taken together, these results suggested that PD-1 deficiency promoted expansion of the pathological damage from the liver to the lymph organs, including the spleen, lymph node and thymus in this FH model, thereafter, the absence of PD-1 was associated with higher mortality rates in response to MHV-3 infection.
Murine FH induced by MHV-3 is a recognized and validated model for studying host resistance/susceptibility to human hepatitis virus, and several studies have shown that BALB/c or C57BL/6 mice have an innate susceptibility to the infection [3, 4] . FGL2 has been proposed as a critical mediating factor of lethality in the MHV-3-induced FH mice due to the fact that it has the capacity to induce fibrinogen deposition, which in turn activates the coagulation cascades and induces procoagulant activity [5] . To clarify whether the tissue necrosis we observed in PD-1-deficient mice following infection was also mediated by FGL2, the expression of FGL2 was analyzed. Results showed that the expression of FGL2 was principally associated with CD31-positive endothelial cells, CD68-positive macrophages and CD11c-positive DCs. Surprisingly, significantly higher levels of FGL2 were observed after infection in all of PD-1-deficient organs, including the liver, thymus, spleen, lymph nodes, and serum than that in those from WT littermates. In addition, increased fibrinogen deposition was observed in the organs of PD-1-deficient mice. Although we currently have no direct data to evidence that FGL2 directly mediates the mortality of our PD-1-deficient mice, data from other researchers have clearly shown that FGL2 promoted mouse mortality in response to MHV-3 infection [5, 28, 29, 30] . Considering this, our results strongly indicate that the mortality of PD-1-deficient mice post-MHV-3 infection is due to the higher level of FGL2 secretion and increased fibrinogen deposition.
Indeed, it has been reported that both FGL2 and PD-1 are expressed on T cells, macrophages, and DCs, and that targeted deletion of fgl2 or PD-1 leads to impaired T cell activity, and these events are related to the development of autoimmune diseases [6, 31, 32] . We here also observed PD-1 expression as being enhanced on T cells (both CD4 + and CD8 + T cells). It was reasonable to propose that the expression of FGL2 may have been directly regulated by PD-1 signals. Unexpectedly, our FGL2/PD-1 dual staining showed that PD-1-positive cells in the liver, thymus, spleen and lymph nodes did not co-express FGL2, indicating that the expression of FGL2 was not directly regulated by PD-1. On the other hand, the expression of FGL2 is believed to be induced by IFN-c and TNF-a [21, 22] , while PD-1 signaling has the capacity to inhibit IFN-c and TNF-a secretion from PD-1-positive immune cells [6] . Therefore, we evaluated and compared the status of IFN-c and TNF-a in both PD-1-deficient and WT mice. Definitively, the transcription of IFN-c and TNF-a genes was rapidly enhanced post-MHV-3 infection in PD-1-deficient mice, as compared to WT controls. In particular, a higher level of IFN-c was observed in NK cells but not in CD3 + T cells of PD-1deficient liver post-MHV-3 infection, indicating that the PD-1 signal can inhibit IFN-c secretion from NK cells under such condition. Conversely, injection of a the combination of anti-IFNc and anti-TNF-a blocking mAbs was able to successfully inhibit fgl2 mRNA transcription and protein expression, resulting in reduced tissue damage and significantly protecting against MHV-3-mediated mortality in these mice. These results demonstrated that up-regulation of FGL2 in PD-1-deficient mice after MHV-3 infection was controlled, at least partially, by IFN-c and TNF-a.
Recently, the secretion of FGL2 from naturally occurring CD4 + Foxp3 + regulatory T cells (Tregs) was demonstrated and it was reported that deficiency of Treg-produced FGL2 resulted in increased effector T cell proliferation [32] . More interestingly, Levy and colleagues showed that the frequency of FGL2 + Tregs was higher in lymphoid tissues of MHV-3 infected mice, and treatment with FGL2-specific antibodies reversed MHV-3induced liver injury and mortality in vivo. These findings demonstrated that FGL2 is an important effector cytokine of Tregs that contributes to MHV-3-induced FH [30] . PD-1 signaling has also been described as participating in regulation of Treg differentiation and function [33, 34] . In our study, we also analyzed the status of Foxp3 + cells in both PD-1-deficient and WT controls. However, the number of Foxp3-positive cells in the liver, spleen, lymph node or thymus was not significantly different between PD-1-deficient mice and their WT littermates after 72 h of MHV-3 infection (Fig. S3) . Therefore, Foxp3 + cells are unlikely to be involved in the mortality of PD-1-deficient mice. However, the functional status of these Tregs (for example, the level of FGL2 secretion) in PD-1-deficienct mice requires further investigation, and such studies are in progress in our lab.
In conclusion, we have determined that PD-1 signaling can limit the immunopathological damage induced by MHV-3 infection in a mouse FH model. Our results suggest that enhancing the PD-1 signal by an immunotherapeutic approach might be a useful treatment for FH.
All experiments were approved by and conducted in accordance with the guidelines of the Animal Care and Use Committee of the Third Military Medical University. All efforts were made to minimize animals' suffering.
Mice PD-1-KO-N10 (strain: BALB/cJ) mice were kindly provided by Prof. T. Honjo (Department of Immunology and Genomic Medicine, Kyoto University, Japan). The WT control mice were purchased from the Animal Center of Beijing University School of Medicine. All mice were maintained in micro-isolator cages and housed in the animal colony at the Animal Center, Third Military Medical University, standard laboratory chow diet and water was supplied ad libitum. Mice were used in experimental analysis at age of six weeks and at an average weight of 17 g (range: 16,18 g).
MHV-3 was kindly provided by Prof. Q. Ning (Institute of Infectious Disease, Tongji Hospital of Tongji Medical College, Wuhan, China). The virus was plaque-purified and then expanded in murine L2 cells. Virus-containing supernatants were collected and stored at -80uC until use. Mice were intraperitoneally (i.p.) injected with 10 PFU/mouse in a total volume of 200 ml. In some experiments, PD-1-deficient mice were infected with MHV-3 (10 PFU) and simultaneously treated with a infection was analyzed by immunohistochemistry. Blue color indicates nuclear DAPI staining. Scale bar = 20 mm. Magnification 6200. NS: not significant different. *p,0.05, ** p,0.01. doi:10.1371/journal.ppat.1001347.g004 combination injection of anti-IFN-c (200 mg/mouse per day, clone: R4-6A2, eBioscience, San Diego, CA, USA) and anti-TNF-a (200 mg/mouse per day, clone: MP6-XT22, eBioscience) mAbs, tissues were isolated for hematoxylin and eosin (H&E) staining to detect damage, and for fgl2 mRNA transcription measured by qPCR (see below). Serum ALT and AST levels were measured by an AU5400 automatic biochemistryanalyzer (OLYMPUS, Japan). In order to monitor the mortality, anti-IFN-c and anti-TNF-a blocking mAbs or rat IgG1 control mAbs were injected everyday for a total of 6 days.
Paraffin-embedded tissue blocks were cut into 5 mm slices which were mounted on polylysine-charged glass slides. Endogenous peroxidase activity was blocked by exposure to 3.0% H 2 O 2 for 15 min. Antigen retrieval was performed in a citrate buffer 
The expression of PD-1 on immune cells (CD4, CD8, NK and macrophages) from different organs was assessed by flow cytometry (FACsAria Cytometer; Becton Dickinson, Germany). Briefly, cell suspensions of liver, spleen, blood and thymus tissues were washed and resuspended in PBS. Cells were then incubated for 30 min at room-temperature in the dark using primary antibodies (PE-PD-1, FITC-CD4, FITC-CD8, FITC-NK1.1 and FITC-CD68. eBioscience). To analyze the source of IFN-c in the liver, PD-1-deficient and WT mice were treated with MHV-3 (10 PFU). After 72 h, liver tissues were isolated and mechanically homogenized, lymphocytes were collected thereafter. Cells were then treated with Brefeldin A solution (BFA) for 4 h, and FITC-NK1.1, FITC-CD3 or PE-IFN-c mAbs (eBioscience) were added and the solution incubated for an additional 1 h. For each analysis, 10000 cells were evaluated. Flow cytometric data were analyzed with CellQuest Pro Software.
The microarray experiment was performed under contact by Kangcheng Co. Ltd. (Shanghai, China). Briefly, total RNA was isolated by Trizol from liver tissue of PD-1-deficient and WT mice treated with 10 PFU MHV-3 for 72 h. RNA concentration was measured on the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA) and quality evaluated by denaturing gel electrophoresis. Samples were then amplified and labeled using a NimbleGen One-Color DNA Labeling Kit and hybridized using the NimbleGen Hybridization System (Roche Applied Science, Shanghai, China). After hybridization and washing, the processed slides were scanned by the Axon GenePix 4000B microarray scanner. Three independent experiments were performed, and for each test and control sample, two hybridizations were carried out by a reverse fluorescent strategy. Only genes whose alteration tendency was concordant between both microarray assays were selected as differentially expressed genes.
Total RNA from the liver and spleen of WT and PD-1-deficient mice was isolated by Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. RNA samples were quantitated by measurement of optical density at 260 nm. Total mRNA (2 mg) was reverse-transcribed to cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas China, Shenzhen City, China), in accordance with the manufacturer's instructions. qPCR was performed to quantitatively analyze the gene transcription levels of fgl2, IFN-c and TNF-a genes. The primers for fgl2 were: sense 59-TGGACAACAAAGTGG-CAAATCT-39 and anti-sense 59-TGGAACACTTGCCATC-CAAA-39. The primers for IFN-c were: sense 59-TCAAGTGG-CATAGATGTGGAAG-39, and anti-sense 59-CGCTTATG-TTGTTGCTGATGG-39. The primers for TNF-a were: sense 59-CACGCTCTTCTGTCTACTGAAC-39 and anti-sense 59-ATCTGAGTGTGAGGGTCTGG-39. The primers for b-actin (internal control) were: sense 59-CACTATCGGCAATGAG-CGGTTCC-39 and anti-sense 59-CAGCACTGTGTTGGCA TAGAGGTC-39. The qPCR was performed at 95uC for 10 s followed by 40 cycles of 95uC for 5 s, 60uC for 15 s, and 72uC for 15 s. The specificity of PCR product was examined by a dissociation curve, and results were analyzed by the 2 2DDCT method [35] .
The expression of FGL2 in liver from MHV-3 infected (72 h) PD-1-deficient mice or their WT littermates was determined by Western-blot; the protocol has been described previously [36] .
The serum FGL2 level from mice infected with or without MHV-3 was detected by using the mouse FGL2 ELISA Kit (Cat: E90512Mu; Uscn Life Science Inc., Wuhan, China) and following the manufacturer's instructions.
All results shown are representative of at least three separate experiments. Unpaired student's t-test (two-tailed) or the Mann-Whitney test was used for comparison of two groups where appropriate. Kaplan Meier curve with log-rank test (GraphPad Prism 4.03 software) was used to analyze the mortality rate. pvalue ,0.05 was considered as statistically significant. its protein expression in the liver, spleen and lymph node from PD-1-deficient mice after 72 h of MHV-3 infection in the presence of IFN-c and TNF-a mAbs or rat IgG1 isotype control antibodies was detected by qPCR and immunohistochemistry, respectively. (C) The IFN-c and TNF-a mAbs treatment resulted in decreased damage to the liver, spleen, lymph node and thymus after 72 h of MHV-3 infection. (D) Reduced FGL2 level by IFN-c and TNF-a mAbs treatment resulted in reduced liver damage (indicated by AST and ALT levels). (E) PD-1-deficient mice were infected with MHV-3 (10 PFU) and simultaneously treated with IFN-c and TNF-a blocking mAbs (n = 4) or rat IgG1 control (n = 6), the survival rate was monitored for a total of 15 days. p = 0.025,0.05 was considered significantly different. One representative of three experiments that yielded similar results is shown. Magnification 6 400. NS: not significantly different. *p,0.05 and **p,0.01. n = 5/group. doi:10.1371/journal.ppat.1001347.g007 Figure S3 The number of Foxp3-positive cells was not changed significantly in PD-1-deficient mice after MHV-3 infection. Foxp3-positive cells in the liver, thymus, spleen, and lymph nodes between PD-1-deficient and WT mice at 72 h after MHV-3 infection were detected by immunofluorescence staining (left). Statistical analysis of the number of Foxp3-positive cells in the indicated organs (right). Blue color indicates nuclear DAPI staining. Scale bar = 20mm. NS: not significantly different. Found at: doi:10.1371/journal.ppat.1001347.s003 (1.44 MB TIF) Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors. Arenaviruses have single-stranded, bisegmented ambisense RNA genomes and form enveloped virions [1] . Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and 'Lujo,' and the New World Clade B arenaviruses Machupo (MACV), Junín (JUNV), Guanarito (GTOV), Sabiá (SABV), and Chapare (CHPV) [2, 3, 4] . All of these viruses are US Select Agents and Risk Group 4 Pathogens [5, 6] . MACV, JUNV, and GTOV are also classified as US National Institute of Allergy and Infectious Disease Category A Priority Pathogens [7] . MACV causes human disease outbreak with high case-fatality rates. To date, at least 1,200 cases with <200 fatalities have been recorded [8, 9] .
Arenaviral genomes encode at least four proteins from two segments (Large and Small). The Large segment encodes a matrix protein (Z) and an RNA-dependent RNA polymerase (L); the Small segment encodes a nucleoprotein (NP) and the glycoprotein precursor GPC [2] . GPC is cleaved by a cellular protease to a stable signal peptide (SSP), and two subunits, GP1 and GP2. The three cleavage products form a stable complex and mediate virus attachment to host cells and fusion of the arenavirion envelope with that of the cell [10, 11, 12, 13, 14, 15] . GP1 mediates the binding of the virion to a cell-surface receptor, whereas the class I fusion protein GP2 mediates membrane fusion after internalization of the virion into an acidified endosome [16, 17] . Interrupting the interaction of GP1 with its cell-surface receptor is a current focus of antiviral development since it would prevent the first and pivotal step of arenavirus host-cell infection.
Transferrin receptor 1 (TfR1) is the principle cell-surface receptor of MACV, JUNV, GTOV, and SABV [17, 18] , and a major determinant of host adaptation. However, studies on receptor use and cellular tropism suggest that the non-pathogenic Clade B viruses Amapari (AMPV) and Tacaribe (TCRV) can enter human cells in a human TfR1-independent manner [18, 19, 20] . Nevertheless, AMPV and TCRV use TfR1 orthologs of their principal host animals to infect nonhuman cells [21] . These studies reveal a complex pattern of receptor use for New World arenaviruses, suggesting the existence of additional receptor molecules, and a possible relationship between receptor use and disease potential.
The GP1-binding site on human TfR1 (hTfR1) has been pinpointed to a prominent loop within the apical domain (residues 201-212) [22] . Conversely, until recently the TfR1-binding site on arenavirus GP1 has been poorly characterized. One study showed that the 20 N-terminal amino acids of MACV GP1 are dispensable for TfR1-binding as a MACV GP1 variant lacking these residues (MACV GP1D, residues 79-258) binds hTfR1 even more efficiently than wild-type GP1 [17] . A second study demonstrated that the central region of GTOV GP1 (residues 85-221) interacts with hTfR1 and that residues 159-221 are essential for this interaction [23] . A recently published crystal structure of a truncated MACV GP1 (region 87-239) [24] , as well as a co-crystal structure of the MACV GP1:TfR1 complex [25] provided structural insight of GP1 residues that contact TfR1. The latter study identified five interaction motifs in the interface between MACV GP1 and the apical domain of TfR1 involving some relatively conserved GP1 residues, such as D114 (motif 2), D123 (motif 4), and F226 (motif 3) (colored red, Fig. 1 ), some less conserved residues, such as Y122 (motif 4) and K169 (motif 4) (colored blue, Fig. 1 ), as well as some nonconserved residues, such as R111 (motif 1),and S116 (motif 2) (colored green, Fig. 1 ) [25] . To evaluate experimentally the functional significance of these residues and other conserved solvent-accessible MACV GP1 residues in TfR1-mediated entry, we created a panel of MACV GP1 variants replacing the mentioned amino acids with alanine, and tested the variants' ability to bind hTfR1 and mediate MACV entry in simian and human cells experimental systems.
We aligned the GP1 sequences of MACV (residues 59-258) with those of JUNV, GTOV, and SABV to identify conserved residues mapping to the surface of the determined MACV GP1 crystal structure (DNAStar Lasergene Software). We identified ten residues of interest that did not contact hTfR1 in the co-crystal structure yet are conserved in at least three of these viruses: D140, W147, D155, D159, P160, K167, N178, and K211 (colored red, Fig. 1 ).
We followed a strategy previously employed for virus receptor identification to evaluate the importance for TfR1 binding of these conserved residues, as well as GP1 residues recently indicated in the published MACV GP1:TfR1 crystal structure [17, 26] . Briefly, we designed codon-optimized open reading frames (ORFs) of MACV GP1D in which one or more of the chosen residues were mutated to alanine, and generated them by gene synthesis (DNA2.0). These ORFs were cloned into a pCDM8-like expression vector [17, 26] with the signal sequence of human CD5 upstream and the Fc region of human immunoglobulin G1 downstream of the ORF. Fc alone and wild-type MACV GP1D-Fc, which was described previously [17] , served as controls. Proteins were purified as described [17, 26] : the individual plasmids were transfected into HEK 293T/17 cells (ATCC) using the calcium-phosphate method and grown in 293 SFM II medium (GIBCO-Invitrogen). Media were harvested and clarified, and Fc fusion proteins precipitated with protein A-sepharose Fast Flow beads (GE Healthcare). Fc fusion proteins were eluted with 3 M MgCl 2 , dialyzed in PBS, and concentrated. Purified proteins were assayed by SDS-PAGE followed by Bio-Safe Coomassie staining (BIO-RAD), and measured using the Quant-iT Protein Assay kit (Invitrogen). All mutant plasmids expressed proteins ( Fig. 2A) . We evaluated the ability of the individual mutants to bind to hTfR1 using co-immunoprecipitation (co-IP) assays as described [17, 22, 26] . Briefly, HEK 293T/17 cells were transfected with plasmid encoding hTfR1 [17, 22] and lysed 48 h post transfection with RIPA Lysis and Extraction buffer (Thermo Scientific Pierce). Cleared lysates were added to equimolar amounts (200 nM) of Fc, MACV GP1D, or variants thereof, and bound proteins were immunoprecipitated with protein A-sepharose Fast Flow beads. hTfR1 in the cell lysate immunoprecipitates was analyzed by SDS-PAGE and western blot using the WesternBreeze Chromogenic kit (Invitrogen) and a murine monoclonal anti-TfR1 antibody (Invitrogen).
Four mutants, D114A, D140A, K167A, and K169A, precipitated hTfR1 as efficiently as wild-type (wt) MACV GP1D or at slightly lower levels. Reduction in GP1 interaction with hTfR1 was observed with mutants S116A, K211A, and D114A/S116A (motif 2 in [1] ), whereas mutants R111A, Y122A, D123A, W147A, D155A, D159A, P160A, N178A, F226A, and the triple mutant Y122A/D123A/K169A effectively inhibited MACV receptor interactions (Fig. 2B) . We performed cell-binding assays as described [17, 26] to determine the ability of these mutants to bind to the surface of MACV-permissive cells. Briefly, Fc constructs were added to 5610 5 Vero cells (ATCC) to a final concentration of 200 nM. Cells with bound proteins were incubated with a 1:40 dilution of FITC-conjugated anti-human Fc antibody (Sigma-Aldrich). Cell-surface binding of constructs was detected by flow cytometry. Baseline fluorescence was determined by measuring cells treated only with the secondary antibody, which was then subtracted from binding values of the tested constructs and control proteins. Overall, cell-binding of mutants supported the co-IP results as mutants that precipitated hTfR1 exhibited high cell-surface binding and vice versa (Fig. 2C) . Exceptions were Y122, S116A and D114A/S116A (motif 2), which bound the cell-surface of Vero cells as or almost as efficiently as wt MACV GP1D, yet exhibited lower hTfR1 levels in the co-IP assays. The K167A mutant exhibited an opposite trend, as it bound less efficiently to the cell-surface than wt MACV GP1D whereas it immunoprecipitated hTfR1 as effectively.
We next tested whether gross misfolding was the reason for the inability of some of the mutants to precipitate hTfR1 and to bind to the cell-surface of MACV-permissive Vero cells. We performed far-UV circular dichroism (CD) of the wt GP1D and selected mutants (Fig. 2D ). Far-UV CD spectra were taken in PBS at 5uC with a Jasco-810 spectropolarimeter and a 1 mm pathlength rectangular quartz cell. Protein solutions used were from 0.05 to 0.08 mg/ml. Ten scans were accumulated and averaged, without smoothing. The spectra all revealed a single broad minimum in the range of 108 to 216 nm. These results are consistent with similar folding of the wt and mutant proteins, although small changes in the conformation of the mutants cannot be ruled out (the Fc portion of the protein as well as GP1D contributed to the measured signal).
We then cloned ORFs encoding mutant GP1s into plasmids expressing full-length MACV GPC. Retroviral pseudotypes were created as described [17, 26] : HEK 293T/17 cells were transfected by the calcium-phosphate method with plasmid encoding 1) MACV GPC and variants thereof, together with 2) the pQCXIX vector (Clonetech) expressing enhanced green fluorescent protein (eGFP) flanked by the Moloney murine leukemia virus (MoMLV) long terminal repeats, and 3) plasmid containing the MoMLV gag/pol genes. Cell supernatants were cleared and measured using the Retro-X TM qRT-PCR Titration kit (Clonetech). Equivalent amounts of pseudotypes were adsorbed on MACV-permissive cells for 2 h. After 48 h, cells were stained with Hoechst 33342 (Invitrogen) and the percentage of eGFP-positive cells was determined by high-content quantitative image-based analysis using an Opera confocal imager (Perkin Elmer). Cells exposed to mock pseudotypes (media of cells transfected with the pQCXIX vector and the MLV gag/pol plasmid) were used as negative controls. Western blot analysis of MACV GP2 expression in the producer cells (data not shown) and in the various MoMLV pseudotype preparations ( Fig. 3 lower panel) was performed using an antibody against MACV GP2 (New England Peptide) to examine expression, processing, and incorporation levels of wt MACV GPC and variants thereof. In agreement with the co-IP and cell-binding data, MoMLV carrying GPC mutants D114A, S116A, D140A, and K169A had no effect on entry into human (HeLa) and nonhuman primate (Vero) cells (Fig. 3) . Mutants D123A, D155A, P160A, F226A, K167A, and K211A had an intermediate effect on MACV entry, with the first four amino acids playing a more prominent role. Interestingly, MACV GPC mutant F226A had a much more pronounced effect on HeLa cell entry (.80% reduction) than on Vero cell entry (.40% reduction).
D155A, P160A, and K211A mutants also exhibited decreased incorporation levels into MoMLV pseudotypes. However, as the cell-entry assay results are in agreement with our results using purified proteins (Fig. 2B and 2C) , we suggest that the observed phenotype is not due to decreased incorporation levels but due to the mutations themselves. MACV GPC mutants N178A and D159A were not incorporated into MoMLV pseudotypes and therefore were unable to transduce either cell line. MACV GPC mutant W147A behaved unexpectedly in that it had no effect on MoMLV transduction efficiency, despite its decreased precipitation of hTfR1 and attachment to the cell surface of MACVpermissive cells (Fig. 2) . We hypothesize that W147A might be misfolded in the context of MACV GP1D but folded correctly in the context of full-length GPC. The two mutants with the most significant effect on cell entry were the R111A mutant and the triple mutant Y122A/D123A/K169A, exhibiting more than 70% reduction in MoMLV transduction efficiency when compared to wt GPC. Finally, the Y122A mutant, while minimally affecting entry into Vero cells, decreased MoMLV entry into HeLa cells. This is in agreement with our assays using purified proteins (Fig. 2) , in which the Y122A mutant bound efficiently to the cell surface of Vero cells (Fig. 2C) yet was unable to immunoprecipitate hTfR1.
In summary, our results demonstrate that GP1 residues R111, D123, Y122, and F226 are important for hTfR1 binding and cellentry of MACV (Figs. 2 and 3) . These results are in accordance with a recent study demonstrating that the central region of GTOV GP1 (residues 85-221) interacts with hTfR1 and that residues 159-221 are essential for this interaction [17] . These results are also in agreement with the recently published structure of MACV GP1 bound to hTfR1 [25] . MACV GP1 R111, which is not conserved in any of the other New World arenaviruses, is part of interaction motif 1 and makes prominent contacts with hTfR1 bII-2 strand, namely V210 (Fig. 1, top panel, and Fig. 4) . F226, unique to MACV (W in other New World arenaviruses), is part of interaction motif 3 and has a hydrophobic interaction with hTfR1 V210, as well as a van der Waals contact with hTfR1 I201 and L212 (Fig. 1, top panel, and Fig. 4 ). D123 and Y122 are part of interaction motif 4 and are conserved between MACV, JUNV, and SABV. D123 forms a hydrogen bond with hTfR1 K344, whereas Y122 forms one with hTfR1 E343 and in addition contacts hTfR1 residues A293, E294, and A340 (Fig. 1, top into HeLa cells (Fig. 3) . In accordance, this mutant bound efficiently to the cell surface of Vero cells (Fig. 2C ) yet was unable to immunoprecipitate hTfR1 (Fig. 2B) . These results suggest that Y122 might be important for binding hTfR1 but dispensable for binding simian TfR1, or that additional receptors/attachment factors are facilitating binding and entry of MACV into Vero cells. Whereas TfR1 residues E343, A293, and A340 are conserved among human and simian TfR1, glutamate 294 is an aspartate in simian TfR1 and the proximal asparagine 292 is a lysine.
MACV GPC residues D114, S116, D140, K169, and most likely W147 are dispensable for cell attachment and entry of MACV. According to the crystal structure, D114 and S116 form interaction motif 2, which contacts hTfR1 residues N348, S370, and K371. K169, a part of interaction motif 4, contacts K344, and E294 of hTfR1. In our functional assays, using both purified proteins and MoMLV pseudotypes, mutating these residues had no or minimal effect on hTfR1 binding and cell-entry (Figs. 2 and 3), suggesting that although they might contact hTfR1, they by themselves play no significant role in hTfR1 usage by MACV.
Residues D155, P160, and K211 of MACV GPC had an intermediate effect on hTfR1 binding and cell-entry, with the first two amino acids being more predominant. These residues were not found to contact hTfR1 residues in the crystal structure and could exert their effect on cell-surface binding and entry by affecting overall or local folding of MACV GP1 and possibly by interacting with a yet unknown attachment or entry factor. P160 is conserved among all Clade B New World arenaviruses (Fig. 1, top  panel) , which use TfR1 as a receptor [1, 21] . However, D155 is conserved only among Clade B New World arenaviruses that cause hemorrhagic fevers in humans and bind hTfR1. Amapari (AMAV) and Tacaribe (TCRV) viruses, which are considered non-pathogenic for humans [2] and which do not bind hTfR1 but bind TfR1 of their mammalian hosts [21] , do not have an aspartate residue in this position (Fig. 1, top panel) .
Finally, it is difficult to establish the role of MACV GPC residues N178 and D159 in cell binding and entry of the virus. In the context of purified GP1D, mutation of these amino acids did not affect overall folding (Fig. 2D) , but in the context of GPC these mutants failed to be incorporated into MoMLV pseudotypes. These results suggest that mutagenesis of N178 and D159 might lead to small changes in folding of GP1 (not being detected by CD) that might affect accessibility of hTfR1-binding residues in their vicinity. Alternatively, trafficking, folding, or binding of GP1 to SSP or GP2 might be defective only in the context of full-length GPC. The MACV GP1-hTfR1 crystal structure revealed seven Nlinked glycans on Asn178 [25] . It is plausible that these glycans play an important role in MACV glycoprotein folding, trafficking, GP complex formation, or entry. N178 and D159 (with the exception of CHPV) are conserved among all Clade B New World arenaviruses (Fig. 1, top panel) , which use TfR1 as a receptor [1, 21] .
We have used purified MACV GP1 and MoMLV pseudotyped with the MACV glycoproteins in all experiments. Several studies suggest that retroviral vectors pseudotyped with the glycoproteins of arenaviruses adopt the receptor-binding characteristics of the corresponding viruses [17, 20, 27] . Therefore, we believe this surrogate system to be valuable for initial studies of viral cell binding and entry. However, it is important that the system is simplistic and uses only a few viral proteins in the absence of other viral-encoded proteins or genomic elements. Therefore, any results obtained with this system should be verified with infectious virus. Unfortunately, no reverse genetics system has been developed for MACV to date, making it impossible to evaluate the effect of the mutated residues on entry of infectious MACV.
Currently there is no FDA-approved treatment to stop the spread of MACV once humans are infected. Defining interactions between the viral glycoprotein and a cellular receptor may allow for drug development that could limit viral spread in the infected individual and reduce infection during an outbreak. Our mutagenesis analysis of MACV GP1 experimentally elucidates the structure and functional interaction motifs of MACV GP1 and its receptor hTfR1. These studies therefore provide a solid platform for the future design and development of small molecule inhibitors and antivirals specifically targeting viral entry. The advent of ECMO and pumpless extracorporeal lung assist in ARDS Despite advances in critical care facilities and ventilation therapies acute respiratory distress syndrome (ARDS) is associated with high mortality rates. The condition can stem from a multitude of causes including pneumonia, septicemia and trauma ultimately resulting in ARDS. ARDS is characterized by respiratory insufficiency with severe hypoxemia or hypercapnia. The treatment strategy depends on the knowledge of the underlying disease. But lung-protective ventilation with adjusted positive end-expiratory pressure remains the most effective therapeutic tool despite advances in prone positioning, inhalation of nitric oxide and the use of steroids. Newer modalities including extracorporeal membrane oxygenation (ECMO) and pumpless extracorporeal lung assist (PECLA) are being increasingly introduced in critical care settings as rescue therapies in patients who fail to respond to conservative measures. We describe here the introduction and advances of both ECMO and PECLA in the management of ARDS.  GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2 Pathogenicity islands (PAIs), a distinct type of genomic island (GI), play important roles in the rapid adaptation and increased virulence of pathogens. 89K is a newly identified PAI in epidemic Streptococcus suis isolates that are related to the two recent large-scale outbreaks of human infection in China. However, its mechanism of evolution and contribution to the epidemic spread of S. suis 2 remain unknown. In this study, the potential for mobilization of 89K was evaluated, and its putative transfer mechanism was investigated. We report that 89K can spontaneously excise to form an extrachromosomal circular product. The precise excision is mediated by an 89K-borne integrase through site-specific recombination, with help from an excisionase. The 89K excision intermediate acts as a substrate for lateral transfer to non-89K S. suis 2 recipients, where it reintegrates site-specifically into the target site. The conjugal transfer of 89K occurred via a GI type IV secretion system (T4SS) encoded in 89K, at a frequency of 10(−6) transconjugants per donor. This is the first demonstration of horizontal transfer of a Gram-positive PAI mediated by a GI-type T4SS. We propose that these genetic events are important in the emergence, pathogenesis and persistence of epidemic S. suis 2 strains. Streptococcus suis serotype 2 (S. suis 2) is a Grampositive, economically important, zoonotic pathogen that is a leading cause of contagious porcine diseases, including meningitis, arthritis, pneumonia, endocarditis and septicaemia Feng et al., 2010) . Further, S. suis 2 can also be transmitted to humans by direct contact with infected swine, causing meningitis, permanent hearing loss, septic shock and even death (Gottschalk et al., 2007) . Due to its high incidence, transmissibility, persistence in the environment and its ability to occur in explosive epidemic form, S. suis 2 continues to be a global public health concern. After two extensive human outbreaks of streptococcal toxic shock syndrome (STSS) caused by S. suis 2 in China in 1998 (Tang et al., 2006 , S. suis 2 has been recognized as a re-emerging zoonotic pathogen and was found to characteristically harbour a candidate pathogenicity island (PAI) designated 89K (Chen et al., 2007) . Recent genetic evidence showing that a two-component system (SalK/R) encoded within this island mediates a piglet-lethal phenotype has strengthened the case that 89K is a PAI (Li et al., 2008) .
As the name implies, 89K is approximately 89 kb in length, flanked by a perfect 15 bp direct repeat, and contains a putative phage-related integrase gene at its 3′ end. These architectural features strongly suggest that it may have been acquired by horizontal gene transfer (HGT). Meanwhile, the internal modular organization of 89K indicates that it was generated by multiple recombination events because it carries several unrelated gene blocks found in widely divergent species, making it difficult to ascertain its initial origin. A better understanding of the molecular biology of 89K will undoubtedly provide important insight into the emergence, virulence, and evolution of this important pathogen. Indeed, it may reveal some clues concerning the molecular pathogenesis of STSS caused by S. suis 2.
It is well recognized that type IV secretion systems (T4SSs) play a key role in HGT, thus contributing to the generation of genomic diversity, the evolution of new pathogenic strains, the dissemination of antibiotic resistance genes and other virulence traits (Christie et al., 2005; Juhas et al., 2008) . Based on the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs can be classified into three major types: (i) type IVA systems resemble the archetypal VirB/ VirD4 system of Agrobacterium tumefaciens and are considered to be the paradigm of T4SS; (ii) type IVB systems resemble the Dot/Icm system found in Legionella pneumophila and Coxiella burnetii; and (iii) other T4SSs bear little or no homology to Type IVA or IVB systems, and their functions await further investigation. Representative of the third group are the recently described genomic island (GI)-type T4SSs that are found in a broad spectrum of Gram-negative pathogens (Juhas et al., 2008) . Ultimately, it is the identification of the GI-type T4SSs that brings insight into the mechanisms by which GIs transfer between bacteria (Juhas et al., 2009) .
Here, we present a detailed molecular characterization of the function and evolution of the 89K PAI in epidemic isolates of S. suis 2. We provide genetic evidence showing that an 89K-borne integrase mediates sitespecific excision and circularization of 89K, with contribution from an auxiliary protein named excisionase. The 89K extrachromosomal circular excision product is selftransmissible and can conjugally transfer via a plasmidlike process into S. suis recipient cells through the 89Kencoded GI-type T4SS transport conduit. Thus far, while mobilization of genetic elements by T4SSs has been well studied in Gram-negative bacteria, the transfer of Grampositive mobile genetic elements by T4SSs is currently limited to conjugative plasmids and transposons. Without a previous comprehensive study, we present the first demonstration of the horizontal transfer of a PAI mediated by a GI-type T4SS in a Gram-positive bacterium. We suggest that this new mobile element is responsible, at least in part, for the recent STSS outbreaks in China and recent sporadic cases in neighbouring countries and areas including Vietnam and Thailand (Holden et al., 2009) , and it will possibly spread further in the future.
It is now well established that PAIs comprise a large family of mobile genetic elements and may undergo horizontal transfer to a new host species (Sakellaris et al., 2004; Qiu et al., 2006; Coburn et al., 2007) . To determine whether the 89K PAI is transmissible from a donor S. suis strain containing 89K to a recipient strain without this element, we performed conjugative mating experiments. To obtain a marked version of 89K, a 3′ single cross-over mutant of strain 05ZYH33 constructed in our previous work was used as the donor, which was tagged with a spectinomycin marker located downstream of the SalK/R two-component system within 89K (Li et al., 2008) . We designated this derivative 05ZYH33-89KSpc and the modified 89K island as 89K*. SS2-N, a virulent strain of S. suis 2 that does not naturally contain 89K, was engineered to be chloramphenicol-resistant by replacing the recA gene with a cat gene cassette (creating SS2-NDrecA) and used as the recipient. Medium containing both chloramphenicol and spectinomycin was used to select exconjugants that received 89K*. In the filter mating assay, double antibioticresistant transconjugants were obtained, and transfer of 89K* was detected at a mean frequency of 5.4 ¥ 10 -6 per donor (Table 1) . Using specific primers, the integrated 89K* was detected in 10 randomly selected transconjugants, providing further evidence that 89K* was transferred into the recipients. Transfer of 89K* was also observed at a similar frequency by using other S. suis 2 strains as recipients, including the virulent S735 strain and the avirulent (DNase I) SS2-NDrecA 4.9 ¥ 10 -6 05ZYH33-89KSpc (supernatant) SS2-NDrecA < 10 -8 05ZYH33-89KSpc S735::cat 6.0 ¥ 10 -6 05ZYH33-89KSpc 7996::cat 2.5 ¥ 10 -6 05ZYH33DmobA89K SS2-NDrecA < 10 -8 CDmobA89K SS2-NDrecA 9.6 ¥ 10 -5 05ZYH33DmobC89K SS2-NDrecA
SS2-NDrecA 8.7 ¥ 10 -8 CDxis SS2-NDrecA 2.9 ¥ 10 -6 05ZYH33Dhlc SS2-NDrecA < 10 -8 CDhlc SS2-NDrecA 6.5 ¥ 10 -6 05ZYH33 (pVA838-oriT) SS2-NDrecA 6.1 ¥ 10 -4 DmobA89K (pVA838-oriT) SS2-NDrecA < 10 -8
*These values are the means of three independent experiments.
T4SS-mediated transfer of 89K PAI in S. suis 2 1671 7996 strain, both isolated from the Netherlands (Table 1) . However, conjugation experiments using other serotypes of S. suis (serotypes 7 and 9) and other streptococcal species (S. sanguinis and S. agalactiae) as recipients were performed but without success, suggesting that the 89K transfer may be species-and serotype-specific.
To investigate the underlying transfer mechanism, we performed a systematic molecular dissection of the 89K PAI. Based on our previously reported comparative sequence analysis, 89K is located in the genome of S. suis strain 05ZYH33 at a position between 05SSU0902 (hyd) and 05SSU0983 (rplL), which encode a predicted hydrolase and the 50S ribosomal protein L7/L12 respectively (Chen et al., 2007) . A total of 80 open reading frames (ORFs) were annotated in the 89K island, most of them (n = 65) on the minus strand. The overall gene organization in 89K is presented in Fig. 1 . Notably, 89K contains a Tn916 element, which is likely associated with the tetracycline resistance phenotype because the S. suis 2 reference P1/7 strain without 89K is sensitive to tetracycline. Furthermore, 89K carries a large lantibiotic biosynthesis cluster, which was suggested to be incapable of producing a functional lantibiotic (due to mutation) but essential for full virulence (Phelps and Neely, 2007; Holden et al., 2009 ).
Due to the aberrant GC content of 89K and its absence in the reference P1/7 strain and a number of international virulent strains, it appears that 05ZYH33 acquired the 89K PAI horizontally. 89K then integrated into the chromosome at the attB site in the rplL gene, generating two direct repeats (attL and attR) at both ends of the island. It is well documented that integrative and conjugative elements (ICEs) are able to excise from the chromosomes to form circular intermediates prior to conjugative transfer into recipient cells (Burrus et al., 2002) . We confirmed that 89K did excise from the 05ZYH33 chromosome to produce a circular form and the host genome was repaired upon excision because PCR products were obtained for both primer pairs P2/P3 and P1/P4 (Fig. 2B) . Subsequent sequence analysis confirmed the expected structures of attP and attB generated upon site-specific excision ( Fig. 2C and D) . Noticeably, the amplicons obtained with primer pairs P2/P3 and P1/P4 showed a substantially weaker signal compared to that with P1/P2 and P3/P4, indicating that 89K excision is a rare event. To measure the rate of 89K excision, we developed a real-time quantitative PCR (qPCR) assay and determined that the frequency of 89K excision from an 05ZYH33 overnight culture was 0.032%, a rate similar to that observed for SLG excision from the Streptomyces lividans 66 chromosome (He et al., 2007) . These data demonstrated that 89K can spontaneously excise from the chromosome and form a circular A. Aberrant average G+C content of 89K, which is much less than that of the chromosome. B. Gene organization of 89K. The terminal direct repeats attL and attR are indicated by red triangles. Genes predicted to likely be involved in the mobilization of 89K are shown in yellow. For more gene details, see Chen et al. (2007) . A. Schematic representation of the site-specific integration and excision of 89K. The left and right junctions (attL and attR), which are shown as red rectangles, are formed by recombination between the chromosomal attB site and the 89K attP site. The flanking genes are shown by solid arrows indicating the direction of transcription. The location and orientation of primers used for detection of integrated and excised 89K are indicated by thin arrows. B. Detection of a circular extrachromosomal form of 89K and the empty chromosomal attB site following precise excision, by PCR analysis using primer pairs which are presented upon the lanes, with 05ZYH33 genomic DNA as the template. C. Printout of the sequencing chromatogram of PCR products amplified with primer pair P2/P3, showing the attP site (boxed) formed by the joining of the two ends of 89K. D. Printout of the sequencing chromatogram of PCR products amplified with primer pair P1/P4, showing the empty chromosomal attB site (boxed) upon 89K excision.
T4SS-mediated transfer of 89K PAI in S. suis 2 1673 excision product. Detection of both integrated and circular 89K suggests that the populations of 05ZYH33 carrying this island are heterogeneous. Most cells carry an integrated island, while others contain an extrachromosomal circular one. Essentially, the same results were obtained for 98HAH12, another highly pathogenic S. suis 2 strain, which is predominantly responsible for the earlier 1998 STSS outbreak in China (data not shown).
The int gene is essential for 89K excision A common feature of PAIs is the presence of a sitespecific recombinase that is presumably responsible for the integration of the PAI into the bacterial chromosome (Mantri and Williams, 2004) . The 89K int gene, located near the left end of the island (attL) at a distance of 63 bp, encodes a 403-amino-acid (aa) integrase belonging to the tyrosine recombinase family, which catalyses site-specific recombination between a chromosomal site (attB) and a similar or identical sequence (attP) found on mobile genetic elements including bacteriophages, insertion sequences and transposons (Esposito and Scocca, 1997; Nunes-Duby et al., 1998) . Functional studies of related members of this family give good reason to assume that 89K excision is mediated by the 89K-borne integrase. To determine if this is the case, we generated an isogenic int knockout mutant of strain 05ZYH33 (creating 05ZYH33Dint) and examined 89K excision in this mutant. PCR products were generated using primer pairs P1/P5 (primer P5 is located upstream of xis, as the int deletion removes the priming site for P2) and P3/P4. However, negative results were obtained with primer pairs P3/P5 and P1/P4 (Fig. 3B ). qPCR analysis of the Dint mutant also demonstrated that 89K excision was eliminated, with attP not being detected. Trans-complementation of int restored the excision capacity of 89K to a level comparable to that of the parental strain, with attP being present in~0.025% of cells. These results suggest that deletion of int in strain 05ZYH33 abolished the ability of 89K to excise from the host chromosome. Thus, Int is essential for 89K excision and circularization by mediating site-specific recombination between the attL and attR sites.
Based on most of the ICEs that have been examined, the excision function of an integrase is often stimulated by the presence of a closely adjacent recombination directionality factor (RDF), also termed an excisionase (Xis), that both facilitates excision and inhibits integration (Sam et al., 2004) . In our analysis, a small ORF (05SSU0904), located immediately upstream of the int gene and separated by a 14 bp intergenic region, was found to encode a possible Xis homologue with 32% aa identity to a putative excisionase (accession number AAL27447.1) from Enterococcus faecalis (Fig. 3A) . The predicted xis is preceded by a putative ribosome binding site (AGGAG), which is located 8 bp upstream from the initiation codon of xis. Further sequence analysis revealed a palindrome of 27 bp with the potential to form a stem-loop structure situated 85 bp downstream from the stop codon of int, which may function as a transcriptional terminator. The genetic organization of this locus suggests possible translational coupling between xis and int.
To ascertain whether xis is also involved in 89K excision, the excision of 89K was examined in an xis deletion mutant of strain 05ZYH33 (named 05ZYH33Dxis). As shown in Fig. 3B , PCR analysis of the excised circular 89K and the repaired chromosome using specific primers both gave faint but still detectable signals in the Dxis mutant, suggesting that there is a very low level of Xisindependent 89K excision. qPCR analysis revealed that xis deletion decreased the presence of attP to~0.0012%, while functional complementation of xis restored the excision frequency to~0.029%. These results demonstrated that xis stimulates, but is not essential for, 89K excision from the chromosome.
In general, HGT occurs in bacteria through three main mechanisms: transformation, phage-mediated transduc- tion and conjugation (Thomas and Nielsen, 2005) . The likelihood of transformation was excluded based on our observation that use of DNase I in the filter mating assay did not abolish the transfer ability of 89K (Table 1) . Furthermore, no 89K transfer was detected when the donor culture supernatant was incubated with the recipient, which also ruled out the possibility of phage-mediated transduction (Table 1) . Therefore, conjugation is the most likely mechanism for inter-strain transfer of 89K.
Extensive sequence analysis of 89K revealed several ORFs whose putative products show similarity to conjugation and mobilization proteins involved in conjugative DNA transfer (Fig. 1B) . On the 3′ half of the 89K island, a peptide encoded by 05SSU0913 showed similarities to various prokaryotic DNA relaxases that initiate conjugal transfer by strand-and site-specific cleavage at the transfer origin (oriT), such as the VirD2 component (accession number NP_059814.1) of the A. tumefaciens T4SS (36% aa identity) and the MobA relaxase (accession number AAA26445.1) of the broad host-range plasmid R1162 (39% aa identity). Peptide (05SSU0914), located adjacent to this relaxase, shared 36% aa identity to the accessory protein MobC (accession number AAA26444.1) of R1162, which is proposed to act as a molecular wedge, denaturing oriT strands and providing a single-stranded (ss) substrate for the relaxase (Zhang and Meyer, 1997) . Therefore, 05SSU0913 and 05SSU0914 were accordingly designated mobA89K and mobC89K respectively.
On the other half of 89K, four gene products display aa similarities to the Agrobacterium T4SS components. 05SSU0968 is a VirB1-type lytic transglycosylase (18% identity to the T4SS VirB1 component, accession number NP_059799.1), 05SSU0969 is a putative VirB4-like ATPase (41% identity to the T4SS VirB4 component, accession number NP_059802.1), 05SSU0971 is a hypothetical VirB6-like channel subunit (12% identity to the T4SS VirB6 component, accession number NP_ 059804.1), and 05SSU0973 is a putative VirD4-like coupling protein (40% identity to the T4SS VirD4 component, accession number NP_059816.1). Thus, 05SSU0968, 05SSU0969, 05SSU0971 and 05SSU0973 were accordingly designated virB1-89K, virB4-89K, virB6-89K and virD4-89K respectively. Based on these predictions, it was logical to assume that horizontal transfer of the 89K PAI may occur through a GI-type T4SS-mediated conjugation process.
To determine if the 89K PAI harbours a functional GI-type T4SS, mobilization experiments were performed to examine if the transferability of 89K decreased by making individual knockouts of the T4SS gene homologues. Table 1 shows the results of conjugation experiments between these T4SS gene homologue mutants and the recipient strain SS2-NDrecA. No transconjugants were obtained when DmobA89K, DmobC89K, DvirB4-89K, DvirB6-89K and DvirD4-89K were used as donors. However, a DvirB1-89K derivative of 89K was still able to transfer at a frequency of 9.2 ¥ 10 -7 , approximately sixfold less than that of 89K*. A similar phenomenon had been reported that the transfer capacity of the IncQ plasmid RSF1010 was reduced approximately 10-fold in a virB1 mutant (Bohne et al., 1998) . All of these transfer deficiencies could be fully or partially restored by transcomplementation (Table 1) . Thus, these results indicated that the GI-type T4SS genes are absolutely required for 89K transfer, with the exception of virB1-89K (which contributes to but is not essential for 89K transfer).
Parallel conjugative mating experiments were also performed to evaluate if the genes proposed to be involved in the excision and circularization of 89K are also involved in the transfer process. As shown in Table 1 , an int deletion mutant was transfer-deficient as expected, while the transfer frequency of the Dxis 89K derivative was reduced by almost two orders of magnitude, proximal to the lower limit of detection in our mating system. These observations suggest that the 89K-encoded integrase and excisionase likely cooperatively generate the extrachromosomal circular form of 89K, which represents an intermediate required for the transfer process itself.
The transfer of plasmids between bacterial cells by conjugation is the result of two processes: (i) mating-pair formation between the donor and recipient, and (ii) a DNA processing reaction that prepares the plasmid for transfer (Willetts and Wilkins, 1984; Pansegrau and Lanka, 1996) . Conjugation can proceed only after the formation of a protein-DNA complex (relaxosome) at the oriT site, which is often located in the intergenic region upstream of its cognate relaxase gene (Francia et al., 2004) . The oriT of the 89K PAI was predicted to be a sequence of~40 nt positioned within the 05SSU0915 coding sequence, which is predicted to encode a polypeptide of unknown function with 62% aa identity to a hypothetical protein (SMSK597-1197, accession number ZP_07642092.1) of S. mitis SK597. This segment contains a perfect 9 nt inverted repeat sequence (predicted by the DNA Strider program) that may be necessary to form a stable nucleoprotein complex during conjugation (Bhattacharjee and Meyer, 1993 ). The precise nic (nickase cleavage) site was predicted to reside at position 3′-(887140)CTCG/ CAAA(887147) on the negative strand, which is located 8 nt downstream of the inverted repeats within the oriT region based on an extensive sequence alignment with other oriT sites that have been experimentally determined previously. Figure 4 shows the overall gene organization of the DNA processing region of 89K. We noticed that MobA89K has an N-terminal relaxase domain but lacks a T4SS-mediated transfer of 89K PAI in S. suis 2 1675 C-terminal helicase domain, which usually possesses a processive 5′ to 3′ unwinding activity that provides the motive force for strand transfer (Llosa et al., 1996; Matson et al., 2001) . Instead, we found that ORF 05SSU0934 within 89K encodes a match to the DNA helicase (accession number ACF56805.1) of S. pneumoniae G54, with 93% aa identity. In vivo mutational analysis demonstrated that this gene (designated hlc, helicase) is not required for 89K excision and circularization (Fig. 3B ) but is essential for 89K transfer (Table 1) .
To test the hypothesis that the putative 89K oriT sequence plays a crucial role in the initiation of conjugal transfer, a 355 bp segment containing the putative oriT site was cloned into a non-transmissible Escherichia coli-S. suis shuttle plasmid, pVA838. The recombinant plasmid (pVA838-oriT) was found to be able to transfer at a frequency of about 6.1 ¥ 10 -4 transconjugants per donor, which is approximately two orders of magnitude greater than that of 89K*. PCR analysis confirmed the efficient mobilization of pVA838-oriT by using specific primers. However, no transconjugants were obtained when the DmobA89K mutant was used as the carrier donor. These results demonstrated that the nonconjugative plasmid pVA838 could be mobilized only by 05ZYH33 carrying an active relaxase after introducing a fragment encompassing the putative 89K oriT. In other words, the 89K PAI has a functional oriT site recognized by its cognate relaxase, MobA89K.
To gain insight into the relaxation process of 89K, His 6-tagged MobA89K, MobC89K and the first 258 aa of MobA89K (designated MobA89KN258) were expressed and purified. When overexpressed at low temperature (16°C), MobA89K and MobC89K were both mainly present in the soluble fractions. Although a majority of the MobA89KN258 protein was present in the inclusion body fractions, sufficient soluble material was produced to recover useful amounts of active protein. The molecular masses of the expressed recombinant proteins agreed well with those predicted. All proteins were purified to > 95% homogeneity after metal ion chelating chromatography and gel filtration (Fig. S1 ).
Relaxosomes are presumably composed of supercoiled plasmid DNA with transfer factors complexed at the oriT site (Lanka and Wilkins, 1995) . To confirm whether the MobA89K and MobC89K proteins can induce the site-and strand-specific DNA cleavage reaction, the relaxation activities of these purified proteins on supercoiled plasmid DNA were tested. The supercoiled oriT-containing plasmid pMD-oriT was used as substrate DNA in an in vitro cleavage reaction. As shown in Fig. 5B , no relaxation was observed in the absence of either protein component. However, MobA89K showed an obvious relaxation activity in the presence of the accessory protein MobC89K, yielding 56% nicked, open circular (OC) plasmid DNA compared with 10% in the untreated control. These findings suggested that both MobA89K and MobC89K are required for the nicking reaction. Meanwhile, MobA89KN258 also increased the conversion of the covalently closed circular (CCC) plasmid DNA into the relaxed form (OC) by the addition of MobC89K, yielding 45% nicked products (i.e. slightly less than that observed for the full-length MobA89K protein).
In another set of experiments, linearized plasmid DNA was subjected to nic site analysis using denaturing agarose gels. The oriT-containing plasmid pMD-oriT was A. Physical map of the pMD-oriT plasmid, which contains the oriT region of 89K and serves as substrate DNA in the relaxation analysis. The putative oriT nick site (nic) is indicated by a vertical arrow. B. Equivalent substrate pMD-oriT plasmid was incubated with (+) or without (-) the proteins of interest. Reaction products were analysed by standard agarose gel electrophoresis (0.9%). OC, open circular plasmid DNA; CCC, covalently closed circular plasmid DNA. C. Schematic representation of the expected single-strand species generated by a site-specific nick (arrow) on endonuclease-linearized pMD-oriT DNA. The size of each ssDNA fragment is shown. D. pMD-oriT was linearized with either ScaI or NdeI, and the relaxation mixtures were analysed on a 0.9% alkaline agarose gel. Lanes: 1, ScaI-linearized pMD-oriT with MobA89K and MobC89K; 2, NdeI-linearized pMD-oriT with MobA89K and MobC89K; 3, ScaI-linearized pMD-oriT with MobA89KN258 and MobC89K; 4, NdeI-linearized pMD-oriT with MobA89K and MobC89K. The 1 kb DNA ladder marker is shown to the right (M).
T4SS-mediated transfer of 89K PAI in S. suis 2 1677 linearized with a restriction endonuclease (ScaI or NdeI) and then co-incubated with the protein components of interest. The predicted oriT nick on each of these specifically linearized fragments is shown in Fig. 5C . As expected, three distinct bands corresponding to the predicted sizes (based on nicking at the predicted oriT site) were detected on an alkaline agarose gel, in which the dsDNAs were denatured into single strands (Fig. 5D ). The C-terminally truncated MobA89K protein showed a comparable nicking activity to that of the full-length protein. In all cases, the largest fragment corresponded to the fulllength plasmid ssDNA, and the different sizes of the two additional bands with higher mobility indicated sitespecific cleavage of one of the plasmid strands. Indeed, the sum of the sizes of these two fragments equals one full-length linear ssDNA plasmid. These results, together with the supercoiled plasmid DNA cleavage data described above, demonstrated that the in vitro nicking reaction was dependent on the presence of both MobA89K and MobC89K, and the MobA89K-dependent oriT nicking activity resides within the N-terminal 258-aa relaxase domain of MobA89K.
Integrase-mediated precise excision of mobile elements from the chromosome typically represents the first step in the lateral transfer process. Such excision events usually lead to the formation of circular episomal products that are intermediates in the packaging of some ICEs (e.g. P4) into phage particles (Bowden and Modrich, 1985) , or serve as substrates for conjugative transfer of other elements, such as PAPI-1 (Qiu et al., 2006) and SXT (Burrus and Waldor, 2003) . Sequence analysis revealed that 89K is not similar to an integrated prophage, suggesting that the excised circular 89K may undergo lateral transfer. Confirmation of this hypothesis was obtained by the observation that transfer of a Spc R -marked 89K occurred at a frequency of approximately 10 -6 per donor, similar to that of the Pseudomonas clc element (Sentchilo et al., 2003) and the Salmonella SGI1 island (Doublet et al., 2005) . Further, 89K transfer absolutely requires the 89K-borne integrase, while a Dxis derivative can still transfer at a low but measurable frequency. This suggests that 89K excision is required for its transfer, and Xis may exert its effect by reducing the number of copies of excised circularized 89K molecules. Notably, Gilmore and co-workers demonstrated that transfer of the enterococcal PAI did not require the PAI-resident integrase, excisionase or putative conjugation genes. Instead, transfer only occurred from donors possessing a pheromone responsive-type of conjugative plasmid via an Hfr-like and RecA-dependent mechanism (Manson et al., 2010) .
Chromosomal transfer mediated by this kind of Hfr-like mechanism was regarded as the major evolutionary force driving the genome dynamics of S. agalactiae (Brochet et al., 2008) .
A combination of bioinformatics and functional analyses revealed a novel member of the GI-type T4SS family that plays a key role in the conjugal transfer of 89K. Throughout the past half century, investigations of T4SSs have largely focused on defining the mechanisms of action of several model systems of Gramnegative bacteria, such as the F (IncF), R388 (IncW), RP4 (IncP) and pKM101 (IncN) plasmid conjugation systems and the A. tumefaciens VirB/VirD4 system. However, little is known about the architectures or mechanisms of T4SSs in Gram-positives (Alvarez-Martinez and Christie, 2009) . Although T4SS-mediated mobilization of genetic elements has been characterized in some species of Gram-positive bacteria, such elements are currently limited to conjugative plasmids (e.g. E. faecalis pCF10, S. agalactiae pIP501 and C. perfringens pCW3) and transposons (e.g. E. faecalis Tn916, B. subtilis ICEBs1 and S. agalactiae TnGBS2). Indeed, transfer of Gram-positive GIs (especially PAIs) by T4SSs has not yet been reported. Our findings expand the repertoire of Gram-positive ICEs that can be mobilized by T4SS, and this is the first characterization of horizontal transfer of a Gram-positive PAI by a GI-type T4SS. In contrast, GI-type T4SSs have been identified in various Gram-negative GIs such as pKLC102, PAPI-1, SPI-7 and the Pseudomonas clc element, as well as others derived from a wide variety of Gram-negative plant and animal pathogens, including Erwinia carotovora (Chen et al., 2008) , Xylella fastidiosa (Zaini et al., 2008) , Photorhabdus luminescens (Held et al., 2007) and Yersinia pseudotuberculosis (Collyn et al., 2006) . The complete set of 24 genes thought to encode the putative GI-type T4SS in H. influenzae ICEHin1056 has been fully or largely identified in the GIs of the above-mentioned Gram-negative pathogens, suggesting that GI-type T4SSs are highly conserved among these species (Juhas et al., 2008) . Comparative genomic and functional analysis suggested that 89K encodes a GI-type T4SS at its 5′ terminus (05SSU0961-05SSU0982), differing in gene content and organization with the Gramnegative counterparts (Fig. S2 ). This indicates that there may be an evolutionary divergence of GI-type T4SSs between Gram-negative and Gram-positive bacteria, perhaps due to the differences in cell wall structure.
Based on our experimental results presented here, in combination with previous studies on the Agrobacterium oncogenic T-DNA transfer system (Christie and Vogel, 2000; Atmakuri et al., 2004; Middleton et al., 2005; Guo et al., 2007) , we would like to propose a model for the transfer mechanism of the 89K PAI (Fig. 6) . In a small proportion of host bacteria, the 89K-borne integrase mediates precise excision of 89K from the chromosome with the help of an excisionase. Upon detection of a mating signal, the transfer of the excised circular intermediate of 89K begins (Llosa et al., 2002) . In the first step, MobA89K initiates the processing reaction by generating a strandand site-specific nick at the oriT site of 89K, with help from the auxiliary MobC89K protein. Next, the 05SSU0934 DNA helicase unwinds the nicked strand during relaxosome formation, and the VirD4-89K coupling protein then links the relaxosome and leads it directly to the mating channel (Llosa et al., 2003) . The VirB1-89K lytic transglycosylase, for which peptidoglycan cleavage activity has been previously demonstrated in A. tumefaciens (Zupan et al., 2007) , should play a crucial role in local opening of the peptidoglycan during the assembly of the transenvelope transporter The 89K-borne integrase mediates precise excision of 89K from the chromosome with the aid of excisionase. After receiving a mating signal, the transfer initiates with the oriT nicking reaction catalysed by the MobA89K and MobC89K proteins. The cleaved strand is then unwound by the helicase and delivered to the 89K-encoded GI-type T4SS machinery. The VirB1-89K component is thought to act as a lytic transglycosylase, locally opening the peptidoglycan with contributions from some other factor(s) during the assembly of the transport channel. VirD4-89K is a coupling protein required to deliver the DNA substrate to the transport channel, and together with VirB4-89K, required to energize the substrate transport across the channel. The highly hydrophobic VirB6-89K homologue constitutes the main portion of the transport channel.
T4SS-mediated transfer of 89K PAI in S. suis 2 1679 complex. However, it should be noted that some other factor(s) (e.g. muramidase) may also be implicated in the formation of the transmembrane pore because inactivation of virB1-89K did not completely abolish the conjugal transfer capacity of 89K. VirB6-89K is a highly hydrophobic protein containing six predicted transmembrane segments and is the best candidate for a channel-forming protein in A. tumefaciens (Christie, 1997) . Adjacent genes encoding peptides (e.g. 05SSU0970, 05SSU0972 and 05SSU0975) harbouring transmembrane domains may also contribute to the construction of the transport apparatus. After the conjugation machinery is manufactured, the VirB4-89K ATPase would work as an alternative translocation energy supplier, aside from VirD4-89K, to motor the DNA substrate through the mating channel and drive export as in A. tumefaciens (Dang et al., 1999) .
Depending on the selective advantages introduced in the recipient variety by the acquired GIs, the elements may undergo insertions, mutations or deletions (Kulasekara et al., 2006) , and the 89K PAI is no exception. We previously reported that three re-emerging S. suis 2 strains in China in 2007 all share the 89K PAI, and two of them had undergone gene losses/deletions . Further, using Roche NimbleGen microarray technology and PCR analysis, we found that several virulent (e.g. 606, 1941 and 8011) and avirulent (e.g. T15 and 2f) S. suis 2 strains carry only a portion of this island (unpublished data). The 89K element was also found to be partially included in strain 7996, one of the recipient strains used in our transfer experiments, albeit at a different location. Recently, published work suggests that S. suis strain BM407, which was isolated from a human meningitis case in Vietnam in 2004, contains two regions with extended similarity to 89K (Holden et al., 2009) . Structural and compositional analysis strongly suggests that both of these regions have the potential to undergo excision and transfer, just as 89K does.
Elucidation of the mechanisms involved in bacterial genome instability and, consequently, in the generation of new bacterial variants is still one of the principal goals that have not been completely achieved thus far. Understanding horizontal transfer of GIs is essential to elucidate how genome plasticity in S. suis is maintained and how it may contribute to the evolution of infectious diseases. Our work, which demonstrated the horizontal transfer of an important streptococcal PAI, provides an interesting insight into the molecular mechanism of GI transfer and may prove to be exemplary for the study of the evolutionary strategies of other pathogens. A better understanding of the molecular biology of PAIs will both greatly improve our knowledge of bacterial pathogenesis and may lead to new and improved vaccines, novel therapeutics and better environmental monitoring of epidemic strains.
The bacterial strains and plasmids used in this study are listed in Table S1 . S. suis strains were grown in Todd-Hewitt broth (THB) (Difco Laboratories, Detroit, MI, USA) supplemented with 2% yeast extract (THY), and E. coli strains were cultured in Luria-Bertani (LB) medium. Agar (1.5%) was included when solid medium was desired. If required, antibiotics were added at the following concentrations: spectinomycin, 100 mg ml -1 for both S. suis and E. coli; chloramphenicol, 5 mg ml -1 for S. suis and 10 mg ml -1 for E. coli; erythromycin, 1 mg ml -1 for S. suis and 250 mg ml -1 for E. coli; ampicillin, 100 mg ml -1 for E. coli; kanamycin, 50 mg ml -1 for E. coli.
To test if 89K can excise from the S. suis 05ZYH33 chromosome, a PCR strategy was designed to detect the unoccupied rplL locus and an extrachromosomal circular form of 89K using specific primers that face outward from the integrated island or anneal to chromosomal DNA flanking the insertion site ( Fig. 2A) . Primer combinations P1/P2 and P3/P4 were used to amplify the left and right junction regions respectively. Primer pair P2/P3 was designed to detect the presence of a circular 89K, and primer pair P1/P4 was used to amplify the repaired chromosomal junction formed upon excision. The primers used are listed in Table S2 , and PCRs were performed with a standard protocol consisting of 30 cycles. The resulting amplicons were analysed by electrophoresis and were cloned into the pMD18-T vector (TaKaRa) for sequencing.
To determine the frequency of 89K excision, real-time qPCR assays using SYBR Premix Ex Taq (TaKaRa) were developed to measure the percentage of cells in a culture that contained the 89K attP sequence. Amplification products were designed to be of similar length to facilitate quantitative comparison. The amount of attP DNA in each sample was normalized to that of the chromosomal hyd gene located immediately downstream of attB. The amplification specificity of each PCR was confirmed by determining melting curves and by electrophoresis of the final PCR products, and efficiency was determined using linearized pMDph plasmid DNA, which contains a single copy of attP (157 bp) and an internal region of hyd (156 bp) as a template. qPCR reactions and data analysis were performed as described by He et al. (2007) and Ramsay et al. (2006) respectively.
A 355 bp fragment containing the 89K oriT sequence was amplified from the 05ZYH33 chromosome using PCR primers oriT-F and oriT-R. The resulting PCR products were cloned into pMD18-T, creating pMD-oriT. Plasmid pMD-oriT was digested with EcoRI and BamHI to release the oriT fragment, which was then cloned into the E. coli-S. suis pVA838 shuttle vector, generating pVA838-oriT. Plasmids pET-MobA89K and pET-MobC89K were created by inserting the mobA89K and mobC89K PCR products (amplified with specific primer pairs mobA-F/mobA-R and mobC-F/mobC-R respectively) into the NdeI/XhoI sites of the pET-21a(+) expression vector, creating fusion proteins with a C-terminal His6 tag. A 774 bp mobA89KN258 fragment encoding the first 258 aa of MobA89K was also amplified using primers mobA-F/N258-R and cloned into the unique NdeI and XhoI sites of pET-28a(+), creating a fusion protein with His6 tags at both its Nand C-termini. All constructs were verified by DNA sequence analysis.
Streptococcus suis mutants were generated by allelic replacement with a spectinomycin (spc) or chloramphenicol (cat) resistance gene cassette, using the method we described previously (Li et al., 2008) . Genetic complementation was performed by transforming the mutant strain with an E. coli-S. suis shuttle plasmid (pSET1 or pVA838) construct containing the functional copy of the mutated gene fused in-frame to a cloned S. suis suilysin gene promoter (Lun and Willson, 2004) .
Mobilization assays were performed as previously described (Lorenzo-Diaz and Espinosa, 2009a,b) , with minor modifications. Briefly, cultures of donor and recipient cells were grown overnight at 37°C with shaking in 5 ml of THY containing the appropriate antibiotics. Cells were centrifuged and washed to remove the antibiotics, and then diluted 1:10 in pre-warmed fresh THY medium to ensure that cultures were in exponential phase during the matings. Two hundred microlitres of donor culture and 1 ml of recipient culture (1:5 ratio) were mixed in a 1.5 ml Eppendorf tube and spun for 2 min in a microfuge at 14 000 r.p.m. to pellet the cells. The pellet was resuspended in 50 ml pre-warmed THY supplemented with 10 mM MgCl2, 2 mg ml -1 BSA and 100 U DNase I. The cell mixtures were placed on sterile nitrocellulose filters (0.45 mm, Millipore) and incubated at 37°C for 6 h. Cells were removed by washing the filters in 1 ml of THY medium. Transconjugants were selected on THY medium with spectinomycin and chloramphenicol. Conjugative transfer frequencies were calculated as the number of transconjugant cells per donor. To confirm the occurrence of transfer, 10 transconjugants from each mating were randomly selected for PCR analysis with specific primers.
Escherichia coli BL21(DE3) cells harbouring the expression vectors were grown overnight in 50 ml LB broth containing ampicillin or kanamycin at 37°C. A 1:100 dilution of the overnight culture was used to inoculate 2 L fresh LB broth to an OD600 of 0.6 and then cooled to 16°C. IPTG was added to a final concentration of 1 mM, and incubations were continued at 16°C for 10 h. Cells were harvested by centrifugation at 6000 g for 10 min and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl and 5% glycerol. Lysis was completed by sonication on ice, the soluble fractions were collected by centrifugation, and then loaded onto a Ni-NTA affinity column (Qiagen). The proteins were eluted with an increasing concentration gradient of imidazole and then purified by gel filtration chromatography. Peak elution fractions were analysed by electrophoresis on 10% or 12% SDS-PAGE gels. Fractions containing pure proteins were pooled, concentrated in an Amicon apparatus (Millipore) with a 10 kDa molecular weight cut-off membrane, and then stored in 0.1 ml aliquots at -80°C. The concentrations of proteins were determined with a BCA protein assay kit (Pierce) according to the manufacturer's protocol.
In the assay for nicking supercoiled plasmid DNA, reaction mixtures containing 0.2 mmol MobA89K, MobA89KN258 or MobC89K protein, and 300 ng pMD-oriT plasmid substrate DNA were incubated in a total volume of 20 ml containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA and 2 mg ml -1 BSA (Pansegrau and Lanka, 1996) . After incubation overnight at 37°C, reactions were stopped by adjusting the mixture to 1% SDS and 0.1 mg ml -1 proteinase K, and the incubation was continued at 37°C for 30 min. The reaction mixtures were then subjected to electrophoresis analysis. Gel images were captured digitally, and band intensities were quantified with Quantity One software (Bio-Rad). The yield of nicked OC DNA was calculated after correction for the relative fluorescence of OC to CCC plasmid DNA.
In another set of experiments, linearized plasmid DNA was used as the substrate for the cleavage reaction. pMD-oriT plasmid DNA was linearized with an appropriate restriction endonuclease (NdeI or ScaI) that cleaves the plasmid only once before co-incubation with the proteins of interest. The resulting ssDNA fragments were separated on 0.9% alkaline agarose gels, and the amount of cleaved substrate DNA was determined.
Where appropriate, the data were analysed using Student's t-test, and a value of P < 0.05 was considered significant. G.F.G. is a leading principal investigator of the Innovative Research Group of the NSFC (81021003). A Conformation-Sensitive Monoclonal Antibody against the A2 Domain of von Willebrand Factor Reduces Its Proteolysis by ADAMTS13 The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13. Plasma von Willebrand factor (VWF) is a large multimeric glycoprotein that interacts with platelet surface receptors and is crucial for normal hemostasis. The adhesive activity of VWF is positively correlated with the size of the multimers in plasma [1, 2] . VWF multimer size is regulated by metalloproteinase ADAMTS13, which cleaves the central A2 domain of VWF at the Tyr1605-Met1606 bond [3] [4] . The importance of VWF proteolysis by ADAMTS13 is demonstrated in two syndromes, i.e., thrombotic thrombocytopenic purpura and von Willebrand disease type 2A. The former is associated with the deficiency of plasma ADAMTS13 activity, either due to congenital mutations or acquired autoantibodies [5] [6] [7] . The latter is mostly caused by mutations in the A2 domain of VWF that lead to the increased proteolysis of VWF multimers by ADAMTS13 [8, 9] .
Many factors modulate the proteolysis of VWF by ADA-MTS13. These ligands that bind the A1 domain of VWF such as platelet glycoprotein Iba, heparin and ristocetin promote VWF proteolysis by ADAMTS13 [10] . In addition, platelets also significantly enhance the cleavage of VWF multimers by ADAMTS13 under fluid shear stress [11, 12] . On the contrary, thrombospondin-1, an extracellular matrix adhesion protein, may compete with ADAMTS13 for binding to the A3 domain of VWF, which reduces the rate of VWF proteolysis by ADAMTS13 [13, 14] .
In this study, we investigated the effects of eight murine monoclonal antibodies (mAbs) against various domains of VWF on its proteolysis by ADAMTS13 under physiologically relevant conditions. Among those, mAb SZ34 dramatically decreased the susceptibility of VWF to ADAMTS13 under shear stress, but not under static/denaturing conditions. The epitope of SZ34 was mapped to the amino acid residues between A1555 and G1595 in the central A2 domain of VWF. Our findings highlight the critical role of this region for ADAMTS13-mediated proteolysis under shear stress.
Anti-VWF mAb SZ34 decreases the susceptibility of VWF to proteolysis by ADAMTS13 under shear stress
In this study, we used rADAMTS13 and pVWF as the sources of enzyme and substrate to determine the effect of SZ34 on VWF proteolysis under shear stress. The cleavage product was determined by SDS-PAGE under non-reducing conditions followed by Western blot. The results showed that the 350 kDa cleavage product of VWF under shear stress was reduced in the presence of SZ34 in a concentration-dependent manner ( Figure 1 ). The half maximal (50%) inhibitory concentration (IC 50 ) of the SZ34 was estimated to be approximately 50 mg/ml ( Figure 1 ). In contrast, other anti-VWF mAbs including 1C1E7 (an anti-VWF D'D3 mAb), SZ129 (an anti-VWF A1 mAb), SZ123 (an anti-VWF A3 mAb) and so on had no effects on the proteolytic cleavage of VWF by ADAMTS13 (Figure 1 and Table 1 ). The control experiments established the lack of detectable proteolysis in the absence of ADAMTS13 or in the addition of 20 mM EDTA to reaction mixtures ( Figure 1 ).
The proteolysis of VWF by ADAMTS13 in the absence and presence of anti-VWF mAbs was also determined by agarose gel electrophoresis visualizing VWF multimers. We showed that the decreased amount of the high and intermediate molecular weight multimers were dramatically reduced by mAb SZ34 in a concentration-dependent manner under shear stress (Figure 2 ), further confirming the role of SZ34 in decreasing the susceptibility of VWF to proteolytic cleavage by ADAMTS13 under physiologically relevant conditions. SZ34 had no effects on VWF proteolysis by ADAMTS13 under denaturing/static condition When pVWF was pre-denatured with guanidine-HCl before incubation with SZ34 and rADAMTS13, no obvious difference in the 350 kDa cleavage products was detected ( Figure 3 and Purified pVWF (150 nM) was preincubated with SZ34 (0-200 mg/ml) for 30 min at 37uC, and then incubated with 50 nM rADAMTS13. After 5 min of vortexing at 2,500 rpm on a mini vortexer, the 350 kDa cleavage products were visualized by 5% SDS-PAGE under non-reducing conditions and Western blot analysis. 1C1E7 (an anti-VWF D'D3 mAb), SZ129 (an anti-VWF A1 mAb) and SZ123 (an anti-VWF A3 mAb) were used as negative controls. (B) Changes in the cleavage products detected relative to that observed in the absence of mAbs were determined under shear stress by densitometry. The extent of cleavage was analyzed by detection of the intensity of the 350 kDa cleavage products. Results represent the mean 6 standard deviation of four independent experiments. doi:10.1371/journal.pone.0022157.g001 Table 1 ). Thus, SZ34 had no effect on the digestion of unfolded VWF by ADAMTS13. In the meantime, anti-VWF mAbs including 1C1E7, SZ129, SZ123, etc, exhibited no effects on ADAMTS13-mediated proteolysis of the unfolded VWF under static conditions either ( Figure 3 and Table 1 ).
To exclude the influence of denaturing condition on interaction between VWF and antibodies, we constructed and expressed VWF-R1597W mutant and investigated whether SZ34 inhibited its proteolysis by ADAMTS13. R1597W is the most frequent mutation in von Willebrand disease type 2A and the mutation site is adjacent to Tyr1605-Met1606 bond. This mutant VWF can be cleaved by ADAMTS13 under static condition and in the absence of denaturants such as urea and guanidine [8] . The results showed that with or without SZ34 R1597W VWF multimers were equally effectively cleaved by ADAMTS13 under static conditions (Figure 4 ), which suggested that SZ34 had no effects on the proteolysis of this VWF mutant by ADAMTS13. Preliminary experiments have shown that SZ34 binds to VWF-R1597W multimers by an ELISA method and the dissociation constants (Kd) is 0.6760.11 nM.
To determine the binding epitope of SZ-34, we prepared a series of recombinant VWF fragments, including A1A2A3, A1, A2, A3 and D'D3 ( Figure 5A ), and five GST fusion VWF-A2 fragments, i.e. A2-1, A2-2, A2-3 (VWF73), A2-12 and A2-23 (VWF114) ( Figure 5B ). We measured the binding capacities (dissociation constants, Kd) of SZ34 to native and guanidinedenatured VWF and various VWF fragments by ELISA method. SZ34 bound to native and pre-denatured full-length VWF, A1A2A3, and A2, but only native A2-12 and A2-23 (Table 2) . Furthermore, the binding of SZ34 to the pre-denatured VWF and VWF fragments was much weaker than the native counterparts ( Table 2 ). These data suggest that the epitope of SZ34 is conformationally sensitive. The binding site was mapped to the central A2 domain of VWF. Because SZ34 bound to both native A2-12 and native A2-23, the epitope of SZ34 appeared to be located within the A2-2 (A1555-G1595 region).
To further to testify that SZ34 is a conformational mAb against VWF, a western blot in combination with ELISA based on polystyrene microspheres was performed to compare the binding activities of SZ34 to native pVWF and denatured pVWF. Compared with native pVWF, the binding activity of SZ34 to heated pVWF or pVWF treated with 1.5 M guanidine-HCl was significantly reduced (Figure 6 ), further confirming that SZ34 is a conformation-sensitive mAb to VWF.
We found that SZ34, a mAb against the A2 domain of human VWF, reduces the susceptibility of VWF to proteolysis by ADAMTS13 under fluid shear stress. However, this effect was not observed under static and chemically denaturing conditions. Seven other mAbs against VWF including SZ129 and SZ130 (two mAbs against VWF A1), SZ29 (another mAb against VWF A2), SZ123 and SZ125 (two mAbs against VWF A3) and 1C1E7 and 75H4B12 (two mAbs against VWF D'D3) exhibited no effects on ADAMTS13-mediated VWF proteolysis under either shear stress or denaturing conditions. The epitope mapping shows that the epitope of SZ-34 is located within the A1555-G1595 region of the A2 domain of native VWF, suggesting that this region may directly interact with ADAMTS13. Furthermore, the mAb SZ-34 is a conformation-sensitive mAb because it preferentially binds to native VWF. VWF-D'D3 no effect no effect 75H4B12 25 VWF-D'D3 no effect no effect SZ129 26 VWF-A1 no effect no effect SZ130 26 VWF-A1 no effect no effect SZ29 28 VWF-A2 no effect no effect SZ34 27,28 VWF-A2 inhibition no effect SZ123 27 VWF-A3 no effect no effect SZ125 27 VWF-A3 no effect no effect doi:10.1371/journal.pone.0022157.t001 Only two reports have been published showing the inhibitory effects of VWF antibodies on the proteolysis of VWF by ADAMTS13, but the mechanism of inhibition by SZ34 may be different from that of the two other mAbs VP-1 and RU8 reported by Tsai et al [15] and Zanardeli et al [16] , respectively. The mAb VP-1, which was directly against residues 1591-1605 (inside VWF A2 domain) of VWF polypeptide, inhibited the susceptibility of the recombinant type 2A VWF mutants R1606W and R1606Q to proteolytic cleavage by ADAMTS13 under the static condition. But they did not examine whether VP-1 inhibited native pVWF or wild-type recombinant VWF proteolysis by ADAMTS13 [15] . Because VP-1 was raised against the synthetic VWF peptide (residues 1591-1605) and reacted poorly with the intact subunit (250 kDa fragment) of VWF in reduced SDS-PAGE followed by immunoblotting [3, 15] , we speculated that VP-1 could not bind to native VWF and could not affect its proteolysis by ADAMTS13 without denaturization or under shear stress. In addition, Zanardelli et al [16] reported the inhibition of VWF proteolysis by ADAMTS13 under shear by using RU8, a mAb directed against the VWF D4 domain. They suggested that a binding site in the Cterminal region (D4CK) of VWF is constitutively exposed and participates at the initial step of a multi-step interaction, ultimately leading to proteolysis of VWF by ADAMTS13. Thus, RU8 may inhibit VWF proteolysis by inhibiting the initial binding of the distal domains of ADAMTS13 to the C-terminal region of VWF. We found that the mAb, SZ34, bound to native VWF better than pre-denatured VWF at the central A2 domain, and decreased the cleavage of native VWF under shear stress, but not pre-denatured VWF under static conditions. These findings suggest that SZ34 binds native VWF-A2 domain, which may block the conformational change of VWF or block the access of ADAMTS13 to the ancillary binding site and cleavage bond located at the central A2 domain under fluid shear stress.
The ADAMTS13 cleavage site (the Y1605-M1606 bond) in the VWF A2 domain is buried within the native, folded structure of VWF multimers and is not accessible to cleavage by the metalloprotease [3] [4] [5] . Although a report found that recombinant VWF-A2 peptide (a.a.1481-1668) was sensitive to proteolysis by ADAMTS-13 under physiological pH and non-denaturing conditions [17] , most of studies have shown that even for the isolated A2 domain, unfolding by high fluid shear stress or chemical denaturants such as urea and guanidine-HCl is required for proteolysis by ADAMTS13 [10, 18] . Thus, for VWF with its multi-domain structure, not only the inter-domain but also the intra-domain A2 structural changes have regulatory roles in ADAMTS13-mediated cleavage of VWF. Our data demonstrate that the epitope of SZ34, a mAb raised against native VWF multimers, is located within the A1555-G1595 region, which comprises the a2-helix and a3-helix according to the crystal structure of VWF A2 domain [19] . It is possible that part or all of the a2-helix and a3-helix structure of A2 domain is constitutively exposed on the native VWF multimers and is involved in the regulation of VWF proteolysis by ADAMTS13. Previous studies, however, considered that the A2 domain of VWF is normally sandwiched between the much larger A1 and A3 domains [19, 20] , and the D1596-R1668 region in VWF A2 domain (the so called VWF73) is the minimal substrate for ADAMTS13 [21] .
We conclude that SZ34 is a conformationally sensitive anti-VWF mAb which can modulate proteolytic cleavage of VWF by ADAMTS13 under physiologically relevant conditions. This monoclonal anti-VWF antibody may be a useful tool for further investigating biological function of VWF in vivo.
Plasma-derived human VWF (pVWF) was purified from commercial VWF/FVIII concentrate by gel filtration with a Sepharose 4B-CL column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and heparin-Sepharose 6FF (Pharmacia) affinity chromatography, as reported [22] . VWF antigen (VWF:Ag) concentration was determined by an enzyme-linked immunosorbent assay (ELISA) kit from Dako (Glostrup, Denmark) and VWF multimers analysis was performed as reported [23] .
Plasmid ADAMTS13 containing human full-length cDNA sequence was generously provided by Dr. Jingfei Dong (Baylor College of Medicine, Houston, TX, USA) [24] . Recombinant ADAMTS13 (rADAMTS13) with the C-terminal His-tag was expressed in a stably transfected HeLa cell line. Expression medium was concentrated and purified using a Ni-NTA agarose column (QIAGEN GmbH, Hilden, Germany).
Eight murine mAbs against human VWF were used. The anti-VWF D'D3 domain mAbs 1C1E7 and 75H4B12 were kind gifts from Dr. Deckmyn (Laboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Kortrijk, Belgium) [25] . Others were all previously produced in our laboratory. SZ129 and SZ130 recognize VWF A1 domain [26] , whereas SZ123 and SZ125 interact with VWF A3 domain [27] . SZ29 and SZ34 are mAbs to VWF, whose epitopes are indefinite because of being directly raised against purified plasma-derived full-length human native VWF [27, 28] . Horse-radish peroxidase (HRP)-conjugated polyclonal rabbit antihuman VWF IgG was purchased from Dako (Glostrup, Denmark). 
Purified VWF (1.5 mM) was pre-denatured with 1.5 M guanidine-HCl in 20 mM Tris-HCl (pH 8.0) at 37uC for 2 h. After a 1:10 dilution with 20 mM Tris-HCl (pH 8.0), the denatured VWF was incubated with anti-VWF mAbs (0-200 mg/ml) at 37uC for 30 min and then treated with 25 nM rADAMTS13 that had been activated by incubation with 5 mM CaCl 2 at 37uC for 1 h. After incubation at 37uC for 1.5 h, the reaction was stopped by the addition of 20 mM EDTA.
Expression of VWF-R1597W mutant and its cleavage by ADAMTS13 in the presence of mAb SZ34 under static/ nondenaturing conditions The plasmid PSVHvWF1, which harbors a full-length cDNA insert of human VWF (kindly provided by JE Sadler, Washington University School of Medicine, St Louis, USA) [29] , was used to construct the R1597W mutant by PCR-based single-nucleotide mutagenesis. Recombinant VWF-R1597W mutant was expressed in transiently transfected Hela cell line using serum-free OPTI-MEM I (Invitrogen) for 48 h. The media were collected and added into EDTA-free 16 proteinase inhibitor Cocktail (Roche), followed by concentration using Amicon Ultra-15 Centrifugal Filter (Millipore). VWF antigen (VWF:Ag) concentration was determined by an ELISA kit (Dako).
Recombinant VWF-R1597W (150 nM) was incubated with 0 or 100 mg/ml SZ34 at 37uC for 30 min and then for 18 h with 25 nM rADAMTS13 at 37uC.
The cleavage reactions were measured by the distribution of proteolytic fragments (350 kDa) on a 5% SDS-PAGE under nonreducing condition or by VWF multimeric size distribution on a 1.5% agarose gel electrophoresis, and then analyzed by Western blotting with HRP-conjugated anti-VWF IgG (Dako) and visualized by chemiluminescence as reported [23, 30] . The intensity of the bands was analyzed using Image J software. Plasmids encoding different VWF fragments were all generated from pSVHVWF1 [29] .
Recombinant VWF D'D3, A1A2A3, A1, A2 and A3 all contained a 66His tag (NOVAGEN, San Diego, CA, USA) ( Figure 5A ). The preparation of VWF A1, A2 and A3 was described previously [26, 27, 31] . Plasmid construction, expression and purification of VWF A1A2A3 were similar to VWF A3 [27] . Two primers were used for amplifying VWF A1A2A3: 59-gga tcc GCA GGA GCC GGG AGG C-39 and 59-ctc gag TCC AGA GCA CAG TTT GTG-39 (Lowercase letters for BamHI and XhoI sites). Four recombinant VWF domains including A1A2A3, A1, A2 and A3 were all expressed in inclusion bodies. After purification, protein refolding was achieved using an 8 to 0 M urea linear gradient. Plasmids encoding A2-12, A2-23 (VWF114), A2-1, A2-2 and A2-3 (VWF73) ( Figure 5B ) were constructed similarly in the GST fusion vector pGEX-6P-1 (Amersham Biosciences, Piscataway, NJ). These recombinant plasmids were prepared by use of primers as follows: A2-12 (59-cgg gat cc GAC CTT GCC CCT GAA GCC CCT C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG ACC CTG GCT GAC CAA GAA GCT G-39), A2-23 (59-cgg gat cc GCA CAG TCC AAA GGG GAC ATC C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CCT CTG CAG CAC CAG GTC AGG A-39), A2-1 (59-cgg gat cc GAC CTT GCC CCT GAA GCC CCT C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CTC GCT GAA GGG GTA CTC CAC AG-39), A2-2 (59-cgg gat cc GCA CAG TCC AAA GGG GAC ATC C-39 and 59-cgg aat tc TCA GTG ATG GTG ATG GTG ATG ACC CTG GCT GAC CAA GAA GCT G-39) and A2-3 (59-cgg gat cc GAC CGG GAG CAG GCG CCC AAC C-39 and 9-cgg aat tc TCA GTG ATG GTG ATG GTG ATG CCT CTG CAG CAC CAG GTC AGG A-39). Lowercase letters indicate added restriction enzyme sites (BamHI and HindIII) and the underlined sequence was the inserted C-terminal 66His-tag. The GST fusion proteins were expressed and purified by chromatography on Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany) as described [21] . All five recombinant GST fusion proteins were mainly expressed in soluble fractions at low temperatures.
Heat treatment and denaturization treatment with guanidine-HCl were used to unfold recombinant VWF fragments and pVWF. Purified pVWF was denatured by heating at 80uC for 20 min in a thermo-block heater (ThermoStat Plus, Eppendorf) as reported [32] .
Recombinant VWF fragments and pVWF were denatured with guanidine-HCl [30] as following. Purified pVWF or various VWF fragments (10 mM) were incubated with 1.5 M guanidine-HCl at 37uC for 2 h. Then, guanidine-HCl treated VWF fragments and pVWF were diluted serially with 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl. The binding affinity of denatured VWF and SZ34 was determined by an ELISA. Because the initial concentrations of pVWF and VWF fragments were high, the final concentration of guanidine-HCl in solution was ,20 mM, which did not interfere with the binding activity of SZ34 (data not shown), consistent with what was reported by Tsai [30] in which he showed that guanidine-HCl at 24 mM did not interfere with the platelet aggregation.
Affinity measurements of SZ34 with full-length VWF and various VWF fragments using ELISA The native or denatured VWF fragments and pVWF at various concentrations were first incubated in solution with the antibody (SZ34 or SZ29) at constant concentration until equilibrium was reached, respectively. The denatured VWF fragments and pVWF were obtained by denaturization treatment with guanidine-HCl as above. The concentration of free antibody was then determined by an ELISA. The equilibrium dissociation constants (Kd) of antigen/antibody complexes in solution were obtained according to the equation [33] :
where A 0 and A are the absorbances measured for the antibody in the absence and presence of antigen, respectively, and a 0 and i 0 respectively the total concentrations of antigen and antibody. Increasing concentrations (0.5-100 nM) of the VWF fragments or pVWF with or without being denatured were mixed with a constant amount of SZ34 (0.3 nM), in 1% (w/v) BSA-PBS, respectively, until equilibrium were reached (15 h incubation at 22uC). Then the proportion of SZ34 which remained unsaturated at each concentration of various VWF fragments and pVWF was measured by an ELISA method as following. The antigen/ antibody complexes were added to the microtiter plates coated with 100 ml of pVWF (7.5 mg/ml) and incubated at 37uC for 2 h. After being washed, the wells were incubated at 37uC for 2 h with HRP-labeled goat anti-mouse IgG (Sigma) in 1% (w/v) BSA-PBS. The plates were washed 5 times and tetramethyl benzidine (TMB) solution was added for color development. Absorbance was read at 450 nm and A 0 and A were then obtained, respectively.
Binding of SZ34 to native and unfolded VWF by Western blot/ELISA based on polystyrene microspheres
To distinguish the linear from the conformational epitope of SZ34, we used an in-house Western blot/ELISA (Western blot in combination with ELISA) based on polystyrene microspheres. SZ129 (a mAb to VWF A1 domain) was used as a control. Briefly, 20 mg/ml of SZ34 or SZ129 in 0.05 M sodium carbonate buffer (pH 9.6) was mixed with 1:10 volume of 50 mg/ml polystyrene microsphere suspension (SDL37; Takeda Chemical Industries, Co., Ltd., Osaka, Japan). The mixtures were incubated at room temperature for 2 h with shaking. The microspheres were then centrifuged and resuspended in a two-fold dilution of the starting volume of 2.5% (w/v) BSA-PBS to block the free binding sites. This solution was incubated at room temperature for 2 h with constant shaking. After washing three times with PBS, the microspheres were incubated with 100 nM of pVWF with or without denaturing at room temperature for 1 h with gentle agitation. The microspheres were washed three times with PBS, added in sample buffer (0.0625 M Tris-HCl buffer, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) b-mercaptoethanol, 0.001% (w/v) bromophenol blue, 4 mM EDTA), and then heated at 99uC for 5 min. After centrifugation at 10,000 g for 3 min, the supernatants were subjected to 6% SDS-PAGE. The proteins were transferred to membranes and blotted with HRP-conjugated anti-VWF IgG (Dako). A Brief Review on Diagnosis of Foot-and-Mouth Disease of Livestock: Conventional to Molecular Tools Foot-and-mouth disease (FMD) is one of the highly contagious diseases of domestic animals. Effective control of this disease needs sensitive, specific, and quick diagnostic tools at each tier of control strategy. In this paper we have outlined various diagnostic approaches from old to new generation in a nutshell. Presently FMD diagnosis is being carried out using techniques such as Virus Isolation (VI), Sandwich-ELISA (S-ELISA), Liquid-Phase Blocking ELISA (LPBE), Multiplex-PCR (m-PCR), and indirect ELISA (DIVA), and real time-PCR can be used for detection of antibody against nonstructural proteins. Nucleotide sequencing for serotyping, microarray as well as recombinant antigen-based detection, biosensor, phage display, and nucleic-acid-based diagnostic are on the way for rapid and specific detection of FMDV. Various pen side tests, namely, lateral flow, RT-LAMP, Immunostrip tests, and so forth. are also developed for detection of the virus in field condition. Foot-and-mouth disease (FMD) is a highly contagious disease affecting artiodactylae, mostly cattle, swine, sheep, goats, and many species of wild ungulates [1] . FMD affects extensive areas worldwide and is included in the list of diseases notifiable to the World Organization for Animal Health (http://www.oie.int/eng/en index.htm). It is recognized as a significant epidemic disease threatening the cattle industry since the sixteenth century and till date it is a major global animal health problem. The history of FMD may be traced to era of Hieronymus Fracastorius, a monk who described a disease outbreak in 1546 A.D. that occurred in cattle near Verona, Italy [2] . Almost 400 years later, in 1897, Friedrich Loeffler and Paul Frosch demonstrated that a filterable agent is responsible for FMD [3] . This was the first demonstration that a disease of animal was caused by a filterable agent and ushered in the era of virology.
FMD generally involves mortality rates below 5%, but even so it is considered the most important disease of farm animals since it causes huge losses in terms of livestock productivity and trade. Although FMDV rarely causes death in adult animals, the virus can cause severe lesion in the myocardium of young animals, leading to high mortality rates [4] [5] [6] . The main constraints in controlling this disease and why it is considered as the most dreaded viral disease are its high contagiousness, wide geographical distribution, broad host range, ability to establish carrier status, antigenic diversity leading to poor cross-immunity, and relatively short duration of immunity. Poor surveillance and diagnostic facilities as well as inadequate control programs are major problems in control of this disease in the country. FMD is still a leading cause of loss of livestock economy in India. Outbreaks are still being reported from time to time around the year [7] . Besides causing direct losses to livestock economy it also causes indirect losses in terms of severe trade restrictions, impact which may be higher than direct losses.
The etiological agent, foot-and-mouth disease virus (FMDV) is classified within the genus Aphthovirus in the family Picornaviridae [8] . The virus exists in the form of 2 Veterinary Medicine International seven serologically and genetically distinguishable types, namely, O, A, C, Asia1, SAT1, SAT2, and SAT3, but a large number of subtypes have evolved within each serotype [9] . Serotype O and A reported in France by Valee and Caree [10] and in 1926, Waldmann and Trautwein [11] reported serotype C. Serotypes SAT1, SAT2, and SAT3 of FMDV was observed in sample collected from the FMD outbreak in South Africa. The seventh serotype, Asia 1, was reported from Pakistan [12] .
FMDV is a single stranded (ss) positive sense RNA virus with the whole virus particles having sedimentation coefficient of 146S [13] and genome of ∼8. 5 Kb size. The genome is polyadenylated at 3 end and carries a small covalently linked protein, VPg at 5 end [13] . The 5 untranslated region (UTR) contains a short fragment called S-fragment, a poly (C) tract followed by large (L) fragment of over 700 bases. Functionally, the genome can be categorized into three main regions: (a) 5 noncoding regulatory region, (b) polyprotein coding region (subdivided into L, P1, P2, and P3), and (c) 3 noncoding regulatory region. The translation initiation starts at two AUG codons separated by 84 nucleotides following the Internal Ribosome Entry Site (IRES). The viral genome is translated as a single polyprotein, which is posttranslationally cleaved by viral proteases [14] into four structural proteins (VP1, VP2, VP3, and VP4) and several nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D) [15] . The P1 region of genome encodes the 4 structural proteins VP1, VP2, VP3, and VP4 encoded by 1D, 1B, 1C and 1A genomic regions, respectively, [13] . Sixty copies of each structural protein (VPl-4) assemble to form the capsid [13] . Among which VP4 is internal whereas others are exposed on virion surface [16] . The 3 surface exposed capsid proteins carry the neutralizing antigenic sites [17] . Among the 4 structural polypeptides, VP1 is the most immunogenic protein of FMDV [18] having its G-H loop protruded from the surface [19] , and is maximally exposed on the capsid surface [16, 20] forming large part (54%) of virus surface [21] .
Although, the disease has been controlled successfully in many parts of the world by regular vaccination of susceptible animals and slaughtering of infected animals, no country has been considered safe, because of the highly contagious nature and rapid spread of the infection [22] . For the effective control of the disease, outbreaks should be detected at an early stage and persistent infections should also be recognized to prevent further transmittance. These can be achieved when vaccination is regular and effective and when diagnostic tools available are specific and sensitive and at the sametime rapid.
Lots of work has been carried out to develop and validate diagnostic tests in regard to this disease. Conventional techniques such as complement fixation test (CFT) [23] , serum neutralization tests (SNT) [24] and enzyme-linked immunosorbent assay (ELISA) [25] are still in use for the routine detection of FMDV in clinical samples. Sandwich ELISA is being carried out for the detection of specific FMDV antigens in epithelial tissue suspensions which is usually accompanied by concurrent cell culture isolation and the application of ELISA to any samples showing a cytopathogenic effect [26] . Virus isolation in primary cultures is laborious, expensive, and requires days/weeks (cell passages) before the results are obtained [27] . However, with the introduction of molecular techniques in the field of diagnosis, several techniques based on viral genome detection such as hybridization using DNA probes [28] and the advent of Polymerase Chain Reaction (PCR) technique in the recent past have led to development of several reverse transcription PCR (RT-PCR) procedures for specific detection of FMDV RNA [29] [30] [31] [32] [33] [34] . Because of the reported sensitivity and specificity, RT-PCR has been evaluated as a diagnostic tool for FMDV detection in parallel with ELISA and virus isolation [35] . Another form of PCR, multiplex PCR (mPCR), has also been evaluated for differentiating FMDV serotypes [27, 36, 37] as well as for differential diagnosis with other vesicular diseases such as Vesicular' Stomatitis, Swine Vesicular Disease [38] . The most recent development in the field of diagnosis by nucleic acid detection is the use of thermal cyclers capable of measuring fluorogenic PCR amplification in real-time have become available, making precise quantitation of nucleic acids possible over a wide concentration range. The fluorogenic RT-PCR provides relatively fast result, enables a quantitative assessment to be made of virus amount, and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure [39] .Therefore it is seen as a valuable tool to complement the routine diagnosis procedure for FMD virus diagnosis.
Currently, the FMD diagnosis in our country (India) is being carried out using techniques developed at Project Directorate on FMD (PD FMD), Mukteswar which includes Virus isolation (VI), Sandwich-ELISA (S-ELISA), Liquid-Phase Blocking ELISA (LPBE), Multiplex-PCR (mPCR) and recently DIVA test which is an indirect ELISA for detection of antibody against nonstructural proteins. LPBE is being used for the detection of antibody titers against the FMD vaccinated animals. The S-ELISA is being used for the antigen detection using the material from the lesions but because of its low sensitivity currently mPCR is being used. The real-time PCR-based detection method is used in the many reference laboratories in the world for the purpose [7] .
Foot-and-mouth disease virus (FMDV) was the first recognized viral pathogen [40] and is the sole member of the genus Aphthovirus belonging to the Picornaviridae family. Seven immunologically different serotypes of the FMD virus are known, namely, A, O, C, Asia-1, South-African Territories (SAT) -1, -2 and -3, which comprise more than 65 subtypes. Initially 2 types were named: type O for Oise in France and type A for Allemagne (Germany) [10] . Later type C was recognized as an additional type in Germany [11] . Some 30 years later, work at Pirbright laboratory in England demonstrated 3 novel serotypes of FMDV in sample collected from the FMD outbreak in South Africa and called SAT1, SAT2, and SAT3. The seventh serotype, that is, Asia 1 was first recognized in a sample from Pakistan [12] .
Veterinary Medicine International 3
The viral particle, or virion, contains a single-stranded RNA of positive polarity, approximately 8500 nucleotides long. It is an icosahedral particle with a smooth surface and a diameter of about 30 nm [13] . The fine structure of the virus has been described by X-ray crystallography [16, 41, 42] . There are 60 copies of each of the structural protein VP1, VP2, VP3, and VP4. While the first three structural proteins (MW≈24 kDa) have surface components, the fourth (MW≈8.5 kDa) is internal. The virion is also usually composed of one or two units of VP0, the precursor of VP2 and VP4 [16, 43] . The structural proteins, VP1-3 fold into an eight-stranded wedge-shaped 13-barrel which fits together to form the majority of the capsid structure [16] . The three-dimensional structure of FMDV has revealed a prominent surface feature formed by the loop between the G and H strands of VP1. The VP4 protein is located inside the capsid [44] . The strands of the 13-barrel of VP1-3 are connected by loops which form the outer surface of the virion [45] . Unlike other picornaviruses, FMDV lacks a surface canyon or pit which is the receptor binding site for entero-and cardioviruses [46] . Another feature of the virion is the channel at the fivefold axis which permits the entry of small molecules such as Cscl into the capsid resulting in FMDV having the highest buoyant density of the picornaviruses [42] .
The main cell attachment site and the immunodominant region of FMDV are both located on a solvent exposed region at the surface of the virion, namely, in trypsin-sensitive areas of VP1 [47, 48] . Earlier serological studies showed that different serotypes of FMDV shared a highly variable region of VP1, comprising residues 135 to 155 [49] , as one of the major antigenic sites of the virus.
The genome of FMDV is about 8.5 Kb in length enclosed within a protein capsid. It has four major parts: 5 Untranslated region (5 UTR), Coding region, 3 Untranslated region (3 UTR), and a poly "A" tail. The 5 UTR is linked with Vpg (3B) which serves as primer in replication. 5 UTR contains an S fragment at its 5 end which encompasses 360 bases, folds into long stem loop and plays a role in genome stability and the binding of protein involved in genome replication [50] . Following S fragment there is poly "C" tract comprising over 90% "C" residues. The length of this tract is extremely variable [51] . There are some evidences that length of this tract is associated with virulence and hence persistence [52] . After poly "C" tract, there is a series of RNA pseudo knot structures of unknown function [53] . Downstream to the pseudo knot there is cisacting replicative element (cre) [54] . The cre has conserved AAA sequences and is essential for genome replication [54] . Downstream to cre is internal ribosomal entering sites (IRES). The IRES has role in initiation of viral protein synthesis and shutting down of host cell protein synthesis [53] . Due to the presence of IRES, picornaviruses are able to bind to ribosomes to initiate the protein synthesis.The picornaviruses have no 5 cap (7-methyguanosine), so IRES serves as ribosomal entering site [55] . The 3 UTR follows the ORF (open reading frame) termination codon and contains a short stretch of RNA which folds into specific stem loop structures [56] followed by a poly "A" tract of variable length [57] . This 3 UTR also serves as some functions in genome replication [58] . 3 UTR is specific for each picornavirus. Poly "A" probably plays a role in FMDV translation [59] and replication [60] . The FMDV genome encodes a polyprotein from which four different structural and eight different non-structural proteins are formed by the viral proteases. After translation, the four primary cleavage products are formed: (i) the amino terminal L protease which cleaves at its own carboxy terminus, P1-2A, (ii) the precursor of the capsid proteins (iii) 2BC, and (iv) P3 which is cleaved to make the NSPs [61] . FMDV has two proteinases. The NSP leader proteinses (Lpro) located in the N-terminal region of the polyprotein acts both intra and intermolecularly. This protease initiates cleavage by separating itself from P1, the precursor of the capsid protein and the remainder of the growing polypeptide chain [62] . The 3C protease is responsible for the cleavage of P1 into 1AB (VP0), 1C (VP3), and 1D (VP1). The 1A/1B (VP4/VP2) cleavage occurs at the late stage in virus morphogenesis and is associated with maturation of capsid. The 2C/3A primary cleavage is cis and subsequent cleavage is also mediated by the 3Cpro and producing processing intermediates and mature proteins [63] . In addition to viral protein processing, the 3Cpro cleave the host cell protein histone H3 and may be involved with the shuting down of host cell transcription [64] . The cleavage between 2A/2B junctions is mediated by 2A polypeptide separating itself and P1 away from 2BC/P3 [61] . This change is independent of both L and 3C. The FMDV 2A region is very short (about 18 amino acids) and together with the Nterminal residues of protein 2B, represents an autonomous element capable of mediating cleavage at its own C terminus [65] .
FMD is a highly transmissible disease, and a limited number of infective particles can initiate host infection [66] . Contaminated animal products, nonsusceptible animals, agricultural tools, people, vehicles, and airborne transmission [67] can contribute to the mechanical dissemination of FMDV. The epidemiology of FMD is complex, and it is affected by different viral, host, and environmental factors, among them, variations in virus virulence (severity of lesions, amount, and duration of virus release), particle stability in different microenvironments, and chances of long-term persistence. FMDV multiplication and spread also depend on the host species, nutritional and immunological status, population density, animal movements, and contacts between different domestic and wild host species and animals capable of mechanical dissemination of the virus [68] . The environment can provide geographical barriers to virus dissemination or, alternatively, can promote virus transmission when appropriate atmospheric conditions prevail. In this multifactorial scenario [69, 70] , the high potential for FMDV variation and adaptation has modelled complex evolutionary patterns that are being revealed by molecular epidemiology analyses, mostly based on nucleotide sequencing of capsid protein genes.
Of the different serotypes prevalent in India, type "O" has been found to be predominant over other types [71] [72] [73] [74] [75] [76] [77] . Similarly in the North-Eastern region of India, the frequency of FMD is highest with type "O", which is always followed by other types including the type "Asia-1" [78, 79] . The agroclimatic and socioeconomic condition, mixed animal husbandry practices, unrestricted movement, and trade among animals and porous international border provide a conductive epidemiology niche for the FMDV to flourish, mutate, and persist over time and to affect the susceptible animal population [80, 81] .There are records of about 5,000 outbreaks to occur in India annually affecting nearly three lakh animals [82] with an estimated staggering loss of Rs. 4,300 cores to the economy annually [83] .The losses are mainly due to reduction in milk yield, draught power, and breeding capabilities. In India where world's largest livestock populations exist, it is a leading cause of loss of livestock economy (direct and indirect losses) due to its endemic nature.
Foot-and-mouth disease (FMD) is an acute systemic infection affecting even-toed ungulates, both domesticated and wild, including cattle, swine, sheep, and goats. Beside domesticated animals, other animals species affected with FMD includes elephant [84] , mithun [85, 86] , yak [87] , sambar, spotted deer, and wild buffalo [87, 88] .
FMDV produces an acute, systemic vesicular disease, which requires a differential diagnosis from other vesicular diseases [89] . In natural infection, the main route of virus entry is the respiratory tract. The initial virus multiplication usually takes place in the pharynx epithelium, producing primary vesicles, or "aphthae" [90] . The vesicles produced by FMDV generally affect cells from the epithelial stratum espinosum. The clinical outcome of the disease may vary among the host species considered and the infecting virus strain. In cattle and pigs, fever and viraemia usually start within 24-48 hr after epithelium infection, leading to viral spread into different organs and tissues and the production of secondary vesicles preferentially in the mouth and feet. The acute phase of disease lasts about 1 week and declines gradually coinciding with the emergence of a strong humoral response [91] .The morbidity and mortality in FMD depends upon the breed and age of the animal where mortality in adult animals is very low (two per cent) in comparison to 20 per cent in young stock [92] . The calves show prominent signs of myocarditis, whereas piglets manifest gastroenteritis [93] . In sheep and goats, symptoms are frequently less severe and may make the detection of the disease difficult [94] .
Asymptomatic, persistent infection can also be established in ruminants, during which infectious virus can be isolated from the oesophagus and throat fluids of the animals from a few weeks up to several years of the initial infection. There is epidemiological evidence to support the hypothesis that carrier animals may be the origin of outbreaks of acute disease when brought into contact with susceptible animals. This mode of transmission has been experimentally reproduced for serotype SAT isolates [95] .
The accurate diagnosis of infection with FMDV is of prime most importance for both control and eradication campaigns in FMD endemic areas and as a supportive measure to the stamping out policy in FMD-free areas [96] . The history of research and diagnosis in foot-and-mouth disease falls into several distinct areas. The search for experimental laboratory animals, producing the disease culminated in the demonstration by Waldmann and Pape [97] , the susceptibility of the guinea pig, and the suckling mouse by Skinner in 1951. Early work by Hecke and the Maitlands in the early 1930s, followed by the crucial demonstration by Frenkel in 1947 that large amounts of the virus could be produced in surviving tongue epithelium, formed the basis for the vaccination programmes initiated in Europe in the 1950s [98] . The subsequent development of cell lines has brought a remarkable degree of sophistication to the study of virus growth. The recognition of more than one serotype has led to the development of various techniques for serotyping of the virus. As early as 1927, Bedson et al. found that two isolates of a serotype "A" virus could be differentiated by cross-neutralization tests [99] . Earlier typing of FMDV was done by cross-immunity test in guinea pigs [11] and less frequently in cattle [13] . As this test was time consuming, expensive, and imprecise [100] , different serological tests like complement fixation test (CFT), virus neutralization test (VNT), and enzyme-linked immunosorbent assay (ELISA) were developed and the most recent is the development of molecular techniques, the polymerase chain reaction (PCR) method making diagnosis more rapid and precise. Presently we have demonstrated that multiplex PCR could detect FMD virus in the highest number of samples (65.47%) followed by sandwich ELISA (53.57%) and virus isolation (42.85%) [101] .
In 1929, Ciuca was first to use CFT for typing antiserum and FMDV of guinea pig origin [13] . Later, virus of bovine origin was successfully typed by CFT using guinea pig antiserum [23] . Since then CFT has been used extensively for distinguishing different strains of FMDV [24, 102, 103] . Subsequently, a modification of CFT, micro-CFT was developed, in which 96 well microtiter plates were used instead of tubes [104] . In the years 1964-1965 CFT (tube test) was used to replace the virus type identification by guinea pig cross-protection test [105] . Subsequently, the micro-CFT was adopted for this purpose [106, 107] . Although CFT was a fast method it needed high virus load and results were sometimes affected by pro-and anticomplementary activities of the test sample [26] .
Primary cell culture of bovine [108] [109] [110] , ovine and porcine [111] origin has exhibited susceptibility to FMDV from infected tissues. However, the most sensitive culture system for virus isolation is primary bovine thyroid cells [109] but cryopreservation of bovine thyroid cells directly after trypsinization results in the loss of susceptibility to FMDV [110] . Some stable cell lines, like IBRS-2 [112] , MVPK-1 clone 7 [112, 113] , LFBK cell line and Veterinary Medicine International 5 BHK-21 [114] are also susceptible to FMDV and so are most desirable for diagnostic system but these are less sensitive than primary cells for detecting low amount of infectivity [115] .
Revenson and Segura [116] reported that FMDV grew well on BHK-21 cell line enabling large-scale production of antigen with good complement fixing properties. The BHK-21 cell culture provides better growth for FMDV than the suspension culture [115, 117, 118] . It has also been reported that with subsequent passage in BHK-21 clone 13 cell line, the titre of FMDV increased significantly [108] . Nair [112] reported that the susceptibility and infectivity titers of IBRS-2 and MVPK cell lines were less as compared to BHK-21 cells, and thus had no advantage over BHK-21 cell line for vaccine production. Mishra et al. [119] also adapted FMDV field isolates to BHK-21 clone 13 cells in 3-7 serial passage.
Goel and Rai [120] reported that the field isolates of FMDV could be passaged in BHK21 clone 13 monolayer cell culture, which showed characteristic CPE and were readily adapted between 3rd and 5th passage. The CPE usually develops within 48 hours, if no CPE is detected the cells should be frozen and thawed, used to inoculate fresh cultures and examined for CPE for another 48 hours. An alternative to the virus isolation is cell suspension plaque test which also quantify the virus present in sample [121] . However, the cell culture system is laborious, time consuming, and relatively low sensitive. It also requires careful handling of specimens and a biosafety laboratory.
The availability of cell culture techniques and the realization that FMDV can be grown in in vitro cultured cells made possible the adaption of neutralization test for routine type identification of FMDV isolates and were found to be more specific than CFT [100] . In particular, virus isolation requires a laboratory cell culture facility, which can be difficult and expensive to maintain, besides requiring 4 to 6 days for test completion [122] . Subsequently microneutralization test (MNT) was used for the assessment of antigenic variation in field strains, as it correlated well with cattle protection test [123, 124] . But it was observed that, minimal heterotypic contamination in the sample could interfere with precise type identification of the virus [24] ; on the other hand VN test depends on tissue culture and is more prone to variability than ELISA and also require biocontainment facilities.
. ELISA came into use as diagnostic methods for many infectious diseases around the year 1975; till then it has been used as one of the most accepted serological techniques. The first report of use of an indirect ELISA to screen cattle for antibodies against FMDV was that of Abu Elzein and Crowther [125] . Subsequently, a sandwich ELISA using convalescent bovine immunoglobulin (Igs) as capture and anti-146S guinea pig sera as tracing sera was found suitable for detection and quantification of FMD virus in infected tissue culture fluid and epithelial tissue samples [126] . The use of anti-146S rabbit immunoglobulin in place of convalescent bovine immunoglobulin as capture antibody increased the sensitivity of sandwich ELISA [127] .
Later ELISA and its various modifications were applied for detection, typing, and strain differentiation of FMDV isolates with better sensitivity than CFT and, the results were comparable to that obtained with MNT [25, 125, [128] [129] [130] [131] [132] [133] [134] . Liquid phase blocking ELISA using bovine convalescent sera for characterization of field isolates was done and result tallied with conventional VNT [135] [136] [137] . ELISA results were much more reproducible than those obtained with VNT and are not influenced by variations in tissue culture susceptibility. At the FAO/WRL for FMD, the preferred procedure for the detection of FMDV antigen and identification of viral serotypes is ELISA [26, 138] . The results can be obtained within 3-4 hours after sample is received by the laboratory; a negative sample is confirmed by inoculation of sample into sensitive cultures followed by confirmation of the virus serotype by ELISA. Indirect ELISA was initially used for detection of FMDV antigen in infected cell culture fluid, mice carcass, and cattle tongue as well as antibodies in sera samples [129, 133] . Later a sandwich ELISA was used for subtype analysis of FMDV isolates [134] . Subsequently, a sandwich ELISA was developed for detection and typing of FMDV directly from field materials [139] . Although ELISA is far finer to CFT, a large number of samples failed to give positive results and such negative sample has to be confirmed by inoculation of sample into sensitive cultures [140] followed by confirmation of the virus serotype by ELISA taking 4 more days, a time frame compatible with the need to rapidly detect disease and initiate and appropriate disease control strategy. As a consequence, there is a need of an alternative assay system that allows more rapid confirmation of clinical diagnosis with more sensitivity and this has resulted in development of polymerase chain reaction (PCR) or the more recent real-time PCR.
Polymerase chain reaction was the most widely use nucleic-acid-based diagnostic techniques since its invention [141, 142] . With the development of RT-PCR to amplify RNA targets many workers have assessed the usefulness of it as a reliable tool for FMD diagnosis [29, 30, 32, 33] and in parallel with conventional assays [34, 143, 144] . A particularly high sensitivity was reported by RT-PCR ELISA [143, 145] . A specific RT-PCR was developed and validated for the detection of the polymerase gene (3D) of FMD with an analytical sensitivity equal to 1000 times higher than that of a single passage virus isolation [146] . In a study to compare the sensitivity of assays for the diagnosis of FMD, a cell suspension plaque test on BHK21-CT cells and a reverse transcription nested PCR (RT-nPCR) were used to examine nasal swabs and probang samples obtained from FMDV infected cattle, it has been observed that examination of nasal swab revealed a higher number of infected animals using RT-nPCR than by the used the plaque tests and for probang samples both test gave approximately equivalent result [147] . PCR products generated have currently only been analyzed by gel electrophoresis exposing to the chance of post-PCR contamination. However PCR offers potential advantages over other conventional tests. The risk of false negative associated with poor sample handling is limited. Because virus, if present, would be inactivated by RNA extraction, it would be acceptable to use lower level biosafety. Further, cell culture loses the sensitivity due to presence of inhibitors like interferons and presence of some enzymatic inhibitors. Several studies have compared Reverse Transcription (RT-PCR) methods with FMDV isolation [35, 143, [147] [148] [149] . The sensitivity of the methods reach similar to or greater than virus isolation techniques and these can supplement or go in parallel, but not replace, the routine procedures for diagnosis of FMDV. Detection of all seven serotypes of the virus with each of the serotype specific primers in selected RT-PCR protocols at OIE/FAO World Reference Laboratory for FMD (WRL), Pirbright demonstrated suitable specificity and detected cell culture passage isolate with some success but were not adequate for the serotyping of suspension prepared from clinical samples of epithelium. RT-PCR though has paved the way to more sensitive and rapid test in the field of molecular diagnosis, serotyping of FMDV has been of difficult and this has been solved with the advent of multiplex PCR (mPCR).
To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. This modified technique was originally developed to detect distinct/genetic alterations in large regions of human genome. Afterwards, mPCR was used for the detection and differentiation of multiple pathogens/different strains of the same pathogen. It has additional advantages such as costeffectiveness and rapidity. There are reports on the use of mPCR for differential diagnosis of FMDV serotypes [27, 36] . One such PCR [36] was evaluated in WRL FMD Pirbright and found to work on tissue samples with limited success [148] . To improve the overall diagnostic sensitivity and to provide tools for serotyping FMDV, multiplex PCR arrays have been developed to detect FMDV in infected tissue and food samples [150] .
The concept of using multiplex PCR to detect FMDV and its serotypes has also been examined by others [151, 152] . An advantage of multiplex PCR systems, compared with probebased assays, is that the intrinsic design does not rely on a probe for a single conserved gene or region of the genome. Rather, the multiplex system is designed to survey multiple regions of the genome simultaneously, thereby increasing the probability of detection [153] . To date, these mPCR assays are limited to partial serotyping of FMDV because no existing multiplex PCR assay offer complete coverage for all seven serotypes [154] . Multiplex PCR for differential diagnosis of various vesicular diseases like Vesicular Stomatitis, Swine Vesicular Disease, Vesicular Exanthema, and FMDV have also been described [38, 155] . Recently, mPCR for differentiation of Indian FMDV serotypes has been developed and evaluated on tongue epithelium and cell culture samples [37] . In the earlier studies, we have showed that multiplex PCR are more sensitive than sandwich ELISA and virus isolation for detection of FMDV from clinical materials [101] .
Reaction. The use of polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origin and it has become an essential tool in the research laboratory [156] . The potential of this format to provide sensitive, specific, and swift detection and quantification of viral RNAs has made it an indispensible tool for state-of-the-art diagnostics of important human and animal viral pathogens [157] . Real-time PCR has engendered wider acceptance of PCR due to its improved rapidity and sensitivity [156] overcoming poor precision, low sensitivity, low resolution, absence of automation, only size-based discrimination, absence of expression of results in numbers, poor quantitative performance (Ethidium bromide for staining is not very quantitative), and post-PCR processing, rendering the conventional PCR not very suitable for accurate diagnosis.
There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA-binding fluorophores, the 5 endonuclease, adjacent linear and hairpin oligoprobes, and the selffluorescing amplicons [156] . This approach is a highly sensitive technique enabling simultaneous amplification and quantification of specific nucleic acid sequences. In addition to enhanced sensitivity, the benefits of real-time PCR assays over conventional endpoint detection methods include their large dynamic range, a reduced risk of cross-contamination, an ability to be scaled up for high-throughput applications and the potential for accurate target quantification [158] [159] [160] . Real-time PCR assays recommended by the World Organization for animal health (OIE) for detection of FMDV incorporate universal primers and fluorescent-labeled probes that recognized conserved region within the 5 UTR [39] or conserved gene regions within the RNA-dependent RNA polymerase gene (3Dpol) [161] . The use of a specific probe facilitates an increase in specificity compared to conventional agarose-gel-based PCR assays [162, 163] .
Recently, TaqMan technology has combined the 5 nuclease activity of the Taq DNA polymerase and forster resonance energy transfer to detect and quantify amplification product in a closed tube format. Using this technology real-time PCR has been developed to detect the nucleic acid [164, 165] . This is most sensitive and rapid method to detect the nucleic acid. The viral RNA can be consistently detected over a seven log range, the lowest of which corresponded to as few as 10-100 RNA per volume tested. The test can be performed in 2 hours or less on a portable instrument and sample can be held at ambient temperatures. Real-Time chemistry allows for the detection of PCR amplification during the early phases of the reaction and as the name suggest, realtime PCR monitors the progress of a PCR reaction in the real-time. At the same time, a relatively small amount of PCR products (DNA, CDNA, or RNA) can be quantified. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use agarose gels for detection of PCR amplification at the final phase or end point of the PCR reaction which is associated with considerable hands on time, posses a serious hazard for amplification product Veterinary Medicine International 7 carryover, and limits the number of specimen that can be processed simultaneously. It is hard to differentiate the 5 fold changes on gel whereas real-time PCR is able to detect even twofold changes (e.g., 10 versus 20 copies). In addition to the widely exploited 5 -nuclease (TaqMan) system using duallabelled probes [39, 161, 166] and modified MGB probes [167] , assays have also been developed using other rRT-PCR formats such as those using hybridization probes [146] and PriProET [168] .
The greatest problem facing the diagnostic application of PCR is the production of false positive results. They are attributable to contamination by nucleic acids, particularly from the previously amplified material (carry over). Any contaminant, even the smallest airborne remnant carried over from the previous PCR procedure or from a strongly positive sample (contamination), may be multiplied and produce a false positive results. In the Real-time PCR, the problem of carry-over is significantly reduced because of the real-time measuring principle, which is based on closed tube system. In order to increase assay throughput and minimise operator errors, real-time PCR assays for FMDV have also been automated using robots for nucleic acid extraction and liquid handling equipment to setup the reaction mixes [146, 169] . The assay has also been applied for FMDV and it is capable of detecting all seven serotypes [39, 161, [169] [170] [171] [172] [173] [174] [175] [176] [177] . First interassay/laboratory equivalence investigation [178] , participated by five European reference laboratories for detection of FMDV has observed that the best of the Real-Time PCR assays used in each laboratory gave comparable results unlike the VI results which were highly variable. Performance of three real-time instruments: the LightCycler.2 (Roche), the SmartCycler I I (Cepheid), and the SDS 7900HT (AB) was compared for detection of FMDV and has successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples [167] . In addition to extensive testing of vesicular epithelium samples, the performance of rRT-PCR has recently been assessed on milk [179] and "probang" samples [180] . Automated real-time RT-PCR has been compared with VI and antigen-detection ELISA with result being more positive by real-time PCR indicating that the real-time PCR has higher sensitivity than VI for the detection of FMDV in epithelial samples [181] .
The development of multicolour real-time PCR cyclers and "ready-to-use" commercial multiplex real-time PCR kits has also made it possible to combine several assays within a single tube. Major advantages of multiplexing include a reduced sample requirement, which is especially important when sample material is scarce [182, 183] , and the ability to combine assays with an internal control system. However, it is important to optimize these assays in order to limit competitive interactions that may significantly impact upon assay sensitivity. The combined properties of high sensitivity and specificity, low contamination risk, and speed have made real-time PCR technology a highly attractive alternative to tissue culture-or immunoassaybased methods for diagnosing many infectious diseases [184] . Clinical diagnostic applications and the use of realtime PCRs are growing exponentially, and real-time PCR is rapidly replacing conventional PCR and other established diagnostic methods such as antigen-ELISA and cell culture isolation.
Antigen-Based Diagnosis. The 3AB protein of FMDV was expressed in E. coli [185] [186] [187] or in P. pastoris [188] and has been used for the diagnosis of FMD infection in cattle. Similarly, 3ABC proteins expressed in heterologous systems were used in ELISA (3ABC ELISA) for serodiagnosis of FMD [189, 190] . Further, four serotypes of FMDV structural proteins expressed in P. pastoris and its potential utility either as immunogen or antigen has been successfully assessed in animal model [191] [192] [193] . A recombinant FMDV polyprotein (P1) with 3C expressed in insect cells was evaluated for detecting antibodies to FMDV serotype Asia 1 in ELISA and has the potential to replace the liquid phase blocking (LPB)-ELISA using an inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype Asia 1 [194] .
Approach. The ability to identify and selectively delete genes from a pathogen has allowed the development of "marker vaccines" that, combined with suitable diagnostic assays, allow differentiating infected from vaccinated animals (DIVA) by differentiation of antibody responses induced by the vaccine (no antibodies generated to deleted genes) from those induced during infection with the wild-type virus [195] .
A number of antigenic non-structural proteins (NSPs) of FMD were identified and out of which 3ABC gene appears to be the most reliable marker of FMD virus replication [196, 197] . The deletion of NSP (3ABC) gene has been used for enabling DIVA approach for FMD (Cedivac-FMD inactivated vaccine). For detection of NSP antibodies, the Ceditest FMD-NS ELISA is commercially available. Indirect ELISA test for the detection of antibodies against nonstructural proteins will play an essential role in the serological survey of livestock herds in future postoutbreak situations. It has been demonstrated that protection against footand-mouth disease (FMD) could be achieved following vaccination with chimeric foot-and-mouth disease virus (FMDV) vaccines, in which the VP1 G-H loop had been substituted with that from another serotype. This indicated that the VP1 G-H loop may not be essential for the protection of natural hosts against FMDV. If this could be substantiated there would be potential to develop FMD marker vaccines, characterised by the absence of this region [198] . A mutant FMDV with deletion of immunodominant epitopes was evaluated to use as a marker vaccine recently, In that study, a B-cell epitope was identified in the 3A region of a nonstructural protein (NSP) by anti-FMDV cattle sera and a recombinant FMDV (rvAs-3A14D) was generated by selectively deleting 14 amino acids (position [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] in the 3A region of the NSP. Following in vitro transcription and transfection in BHK-21 cells, the rvAs-3A14D was rescued from BHK-21 cells. Characterization of the rvAs-3A14D revealed that the infectivity, antigenecity, and replication kinetics in BHK21 cells and virulence in mice of the rvAs-3A14D were similar to that of its parent virus. Those data 8 Veterinary Medicine International suggested that the recombinant FMDV with deletion of this epitope in the NSP may be potentially used as a candidate inactivated vaccine and therefore, the application of the marker vaccine and differential diagnostic tests may open a promising new avenue for the development of a vaccine for DIVA.
Microarray-Based Diagnosis of FMDV. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs) [199] [200] [201] . The application to discriminate among variants of FMDV is added to a number of microarray procedures used in virology to analyze multiple viral pathogens that belong to different virus families [202] , to detect specific viruses [203, 204] or to define genetic variations undergone by viruses [205, 206] .
The distinction among mutants of the same virus is becoming increasingly necessary in view of the extensive variation among representatives of most virus groups [207] , the quasispecies population structure of RNA viruses and some DNA viruses [208] , and the increasing recognition that one or a limited number of mutations in a viral genome can have a profound effect in its biological behavior [209, 210] .
Baxi et al. [211] developed a microarray-based test that used a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pairs (bp) long, and serotype-specific, designed from the VP3,VPl-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip [211] .
It was documented that DNA microarray technology can be used as a high-throughput method to analyze polymorphisms within a short region of the FMDV genome and have successfully devised a SVM-based method to classify the samples on the basis of their hybridization signal and to detect SNPs at a major antigenic site of the virus [212] .
An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot-and-mouth disease (FMD) diagnosis and virus typing [213] . Allosteric biosensors allow detection of antibodies against different viruses by accommodating peptide sequences from surface viral proteins, acting as antibody receptors, into permissive sites of allosterically responsive recombinant β-galactosidases. Among the advantages of such biosensors as diagnostic tools is the homogeneous nature of the assay, the short time required for the enzymatic reaction and antibody detection, and the potential for handling large number of samples and for automatic processing, as shown for human immunodeficiency virus [214, 215] . In the serological diagnosis of infectious diseases, the use of allosteric biosensors, namely, hybrid enzymes that respond enzymatically to antibodies directed to foreign peptides displayed on the enzyme surface [216, 217] , is highly promising [218] . Multiple insertions of a major FMDV Bcell epitope from the VP1 capsid protein near the active site of recombinant β-galactosidases dramatically increased the enzyme responsiveness to specific antipeptide antibodies, including sera from infected animals [219, 220] . It has been reported that recombinant β-galactosidases accommodating one or two different peptides from the FMDV NS protein 3B per enzyme monomer can be reactivated by anti-3B monoclonal antibodies (MAbs) and these recombinant βgalactosidases could be also efficiently reactivated by sera from infected animals that permitted differentiation between sera from infected animals and those from naïve and conventionally vaccinated pigs. These infection-specific FMDV biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals [221] .
. Nucleic-Acid-Based Diagnostic Methods. FMDV serotypes A, O, and C was possible, using cDNA probes from individual serotypes that corresponded to structural protein VP1, where thirteen complementary DNA (cDNA) probes labelled with 32P were used to detect the presence of footand-mouth disease virus (FMDV) enabled the detection of 1 pg of viral-RNA, or 1 virus copy per cell [222] .
A nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV) was developed. Two detection methods, NAS-BAelectrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzymelinked oligonucleotide capture (NASBA-EOC), were evaluated and compared with other laboratory-based methods, data analysis support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV [223] .
Due to the high antigenic variability of FMDV, it is important to undertake mutation analysis under immunological pressure. To study the bovine antibody response at a molecular level, phage display technology was used to produce bovine anti-FMDV Fabs where CH1-VH chains with FMDV specific binding was isolated after selection from a library made from vaccinated cattle [224] . Recently, screening a phage displayed random 12-peptide library, it was found that positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to 6ETTLLE11 at the N-terminus of the VP2 protein (structural protein) of the FMDV, a minimal epitopic region require to bind a monoclonal antibody of serotype independent FMDV (MAb 4B2) and thus can be used as a universal diagnostic candidate against.
Approach. Routine diagnosis of FMD is made at several laboratories by the combined use of enzyme-linked immunosorbent assay (ELISA), virus isolation techniques, supplemented by reverse transcriptase PCR (RT-PCR), and so forth, which has been already discussed. However, most of these diagnostic methods require the availability of a dedicated laboratory facility, highly trained laboratory personnel, stable reagents, multistep sample handling or preparation, and management of the logistical considerations associated with sample collection and transport is also required [122] . A rapid and easy-toperform test, which would allow for on-site diagnosis to be made in the case of a suspected disease outbreak, would circumvent problems associated with the transportation of samples to the laboratory and would be especially useful for a faster diagnosis in areas where the disease is endemic. Availability of "point of care" or "pen-side" diagnostic tests would have the advantage of rapid, user friendly, correct identification of a particular strain and economically feasible diagnosis of FMD in field condition. Development of a rapid chromatographic strip test, lateral flow device (LFD) for penside diagnosis based on a monoclonal antibodies that reacts against FMDV of all seven serotypes [170] .
The LFA is an appropriate technology on which to base a rapid assay. The technique permits rapid diagnosis, allowing time for the early implementation of control measures to reduce the possibility of spread of FMD. The LFA has been developed widely to support clinical diagnosis of different diseases [225] [226] [227] , including FMD [170, 228] . A rapid lateral-flow assay (LFA) based on FMDV antigen detection, which is easy to use and can be utilized on the farm to reduce the time required for transport and laboratory diagnosis. The detection of FMDV antigens by direct application of vesicular fluids and epithelial suspensions from animals of an infected farm may reduce the chances of diagnostic error arising from nonspecific reactions. Oem et al. showed that the diagnostic sensitivity of the LFA for FMDV types O, A, C, and Asia 1 was similar, at approximately 87.3%, to that of 87.7% obtained with antigen enzyme-linked immunosorbent assay (AgELISA). But the specificity of the LFA was 98.8%, compared to 100% for the AgELISA [122] . Recently a lateral flow device (LFD) for the detection of footand-mouth disease virus (FMDV) of the SAT 2 serotype was developed using a monoclonal antibody (Mab 2H6) for providing rapid and objective support to veterinarians in their clinical judgment of the disease and for specific confirmation of a FMDV type SAT 2 infection [229] .
Again a simple, rapid, colloidal gold-based immuno chromatographic strip tests were developed for easy clinical testing of serotype A of FMDV in field sites was developed with sensitivity and specificity 88.7% and 98.7%, respectively [230] . Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult [170] . In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.
An alternative molecular technique called Loop Mediated Isothermal Amplification (LAMP) was developed for detection of viruses, where target DNA or RNA specifically amplify using four specific primers under isothermal conditions [231, 232] . A new version LAMP called Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) was developed for rapid, specific and sensitive detection of viruses including FMDV in laboratory and in field condition [233, 234] .
Foot-and-mouth disease virus (FMDV) is an important veterinary pathogen which can cause widespread epidemics. Many works have been carried out to develop and validate diagnostic tests in regard to the FMD. Conventional techniques to detect FMDV infection are either not precisely specific or lack an optimum degree of sensitivity which cannot be overlooked when screening of a herd is concerned. With the critical need for improved diagnostic tests to detect viral infection, effort need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed without expensive laboratory equipment. In this context although various molecular tools are very promising but to make it a successful tool search for more rapid and accurate tests as well as an earlier detection system in preclinical state are needed for the hour. Renewed Global Partnerships and Redesigned Roadmaps for Rabies Prevention and Control Canine rabies, responsible for most human rabies deaths, is a serious global public health concern. This zoonosis is entirely preventable, but by focusing solely upon rabies prevention in humans, this “incurable wound” persists at high costs. Although preventing human deaths through canine rabies elimination is feasible, dog rabies control is often neglected, because dogs are not considered typical economic commodities by the animal health sector. Here, we demonstrate that the responsibility of managing rabies falls upon multiple sectors, that a truly integrated approach is the key to rabies elimination, and that considerable progress has been made to this effect. Achievements include the construction of global rabies networks and organizational partnerships; development of road maps, operational toolkits, and a blueprint for rabies prevention and control; and opportunities for scaling up and replication of successful programs. Progress must continue towards overcoming the remaining challenges preventing the ultimate goal of rabies elimination. Today, RNA viruses play an increasingly important role in emerging human diseases throughout the world [1] [2] [3] [4] [5] . One of the main reasons for this is their ability to evolve rapidly, adapting to new species of hosts and thus to expand their range [6, 7] , including humans (i.e., new zoonotic diseases). Social and environmental changes also contribute to providing new ecological niches and promoting the rapid selection of novel virus variants [8] [9] [10] . This group of viruses includes the causative agents of rabies. As with most generalist agents of a zoonotic nature, rabies is a 2 Veterinary Medicine International very complex disease, in part because of its wide global distribution, the diverse number of virus variants and host species, its extremely high case fatality rate, and for its existence at the border between animal and human health.
Rabies occurs across Africa, the Americas, Australia, and eastern Europe and Asia, from polar regions to temperate latitudes, and is endemic within tropical areas. Recognized etiological agents consist of an expanding range of species (previously genotypes) of negative-strand RNA viruses of the Lyssavirus genus [11] . Although susceptible natural hosts include all mammals, this zoonosis is perpetuated by domestic and wild carnivores, and by many species of bats, which all act as reservoirs for the eleven proposed Lyssavirus species and for emerging variants within species [12] . Humans are infected after being exposed to virus laden saliva or tissue from a rabid animal, usually through bites into open wounds or contamination of mucous membranes. There is no treatment and rabies almost always leads to death after the onset of clinical symptoms. A plethora of viral species and variants maintained by a diversity of hosts with potential for cross-species and cross-order transfers pose a formidable challenge to a strict concept of true disease eradication [13] . However, in any one geographic area, rabies virus (genotype/serotype/species 1), which is responsible for classical rabies and the most important cause of human cases, is typically maintained within a single terrestrial animal reservoir. Elimination of rabies virus variants is, therefore, a more realistic prospect.
The ultimate objective of controlling rabies is the prevention of human deaths. Human rabies mortality can be reduced through ensuring administration of vaccine and immunoglobulin (postexposure prophylaxis, PEP), following extensive washing of the wound, to all bite victims from infected animals. However, these life-saving biologics are often not accessible or affordable to the populations most at risk [14] . Furthermore, interventions focusing solely on rabies prevention in humans have no impacts on reducing infection in maintenance hosts, hence on achieving rabies elimination from an area-the so-called "incurable wound." For centuries, rabies control in reservoir hosts has constituted an essential measure to decrease the burden of human mortality. Effective vaccines for animals are available, and most developed countries have eliminated canine rabies [15] [16] [17] , responsible for the vast majority (99.9%) of all human cases [18] , by preventing the disease in dogs. However, lack of effective canine rabies prevention and control programs in poorly resourced countries continues to cause tens of thousands of human deaths every year [18] . In canine rabies endemic countries, rabies is a recurrent public health issue and also represents a threat to rabies-free areas through the movement of infected dogs-as was the case in Bali, where rabies was introduced recently [19, 20] . Despite the availability of modern and efficient prophylaxis tools for humans and animals, on average, rabies still kills one person every 10 minutes, with the highest burden of mortality in Africa and Asia and most of the deaths occurring in children less than 15 years old [18] .
Ineffective rabies prevention and control across most of the developing world has been driven by a lack of awareness about disease impacts and institutionalized attitudes about where the responsibility for development and enforcement of rabies control and prevention programs resides. From a public health perspective, rabies remains a "neglected" zoonosis largely because it occurs in communities suffering from poverty and inequitable human and animal health care [21] . Rabies predominantly affects the poorest segments of the population, living in remote, rural areas with insufficient access to life-saving interventions and the services to deliver them. These communities have little political voice and, therefore, limited impact on health policy making. As a consequence, rabies ranks low within national and international agendas, in spite of being present in more than 150 countries/territories and representing a public health threat to more than 3 billion people in Asia and Africa alone [18] .
In addition, from an animal health perspective, the species mostly involved in rabies maintenance and transmission to humans in rabies-endemic areas, the domestic dog, is often neglected by veterinary services serving the agricultural sector and/or the public health sector. In Asia, Africa, and Latin America, a substantial proportion of the dog population is unrestricted or semirestricted (stray), neither under direct control of their owners nor confined by a physical barrier [22] [23] [24] [25] [26] [27] [28] . This term encompasses both owned and unowned roaming dogs and does not distinguish whether the dog has an "owner" or "guardian". Indeed, in many countries, the majority of dogs defined as roaming are owned, but are allowed to roam on public property for all or part of the day. These dogs fall into an institutional vacuum. They are not included in any conventional health care system as compared to other species recognized as disease vectors (for example, insect vectors of malaria or dengue) that are controlled as part of public health control programs. Dogs are neither a species that the agricultural sector/veterinary services are inclined to take full responsibility for as, unlike livestock species, they are not a recognized economic commodity. Only fully owned and restricted dogs, which are a minority in countries where canine rabies is prevalent, are generally of interest, but mostly to private veterinary practitioners. As a consequence, in many rabies-affected countries, veterinary services and private veterinarians do not have the will nor the desire to prioritize and allocate the resources required to control a disease that is primarily seen as a public health issue with limited impact on production animal health or small animal private practice.
When we consider where the responsibility for rabies control resides, the issue becomes increasingly complex. Given the cosmopolitan nature of rabies, and the widely recognized global burden of mortality among humans, domestic animals, and wildlife [13, 29] , no one institution, agency, or country can bear the sole responsibility for effective rabies prevention and control. Rather, rabies is a "transboundary" disease, and basic intersectoral cooperation at any level requires a breadth of expertise, not residing in one single health ministry, agricultural department, academic endeavor, or private enterprise. In a true "One Health" context [30] , a wide variety of basic disciplines are necessary for a comprehensive rabies elimination program, including animal control and welfare, diagnostic, ecology, economy, education, epidemiology, health communication, anthropology, human and animal health services, virology, and wildlife biology. To be truly efficient, such multifunctional programs need to benefit from and begin with the essential support and commitment of country-based human and animal health authorities.
To this effect, significant milestones have been achieved motivated by the compelling need for alleviating the burden of rabies worldwide. Progress has been made towards uniting global expertise to promote a global campaign against rabies, bridging the gap between research-driven knowledge on rabies and its control and practical action towards its elimination, and creating a model that will stimulate further veterinary efforts in global health research and practice towards the control of zoonotic pathogens. Here we illustrate these achievements, including (1) global, "political" initiatives; (2) global, "applied" initiatives; and (3) regional, local, yet scalable and replicable initiatives. We conclude by discussing the challenges and future directions for additional initiatives on a global basis.
Prevention and Control 2.1. Building Global Partnerships. The building of public private partnerships is proving to be one of the most effective global strategies to address the needs of disadvantaged populations living in the midst of neglected diseases [31] that are no longer considered to be a priority in industrialized nations [32] [33] [34] . If established in a balanced manner, public-private partnerships can bring together the power, resources, and experience from both the industrialized and the developing world to create synergies, define priorities, and find solutions. There are many examples of successful public private partnerships that have pooled their resources and have coordinated global strategies to increase the availability of scarce and expensive medical interventions for those that need them most. For example, the Global Alliance to Eliminate Lymphatic Filariasis (GAELF) consists of a partnership between academic and research institutions, advocacy and resource mobilization partners, country representatives, international development agencies and foundations, international organizations, nongovernmental development organizations, and private sector companies [35] . This group retains a "light governance structure" that provides the flexibility to focus on specific needs at regional and national levels [35] . Several attempts at regional or international partnerships in rabies prevention and control have occurred throughout the 20th century. For example, once oral vaccination of wildlife became a reality during the 1980s, the World Health Organization (WHO) held multiple consultations involving its global network of WHO Collaborating Centers for Reference and Research on Rabies for an organized approach towards red fox rabies elimination in Western Europe. The breadth and longevity of several other partnerships, such as the Rabies in the Americas Association, Latin-America National Rabies Directors Network (REDIPRA), Southern and Eastern Africa Rabies Group (SEARG), Rabies in Asia Foundation (RIA), Asia Rabies Expert Bureau (AREB), Africa Rabies Expert Bureau (AFROREB), and the recently created Middle East and Central Eastern Europe Rabies Expert Bureau (MEEREB), all demonstrate the utility of coalitions in successful information flow and regional health promotion in bringing support to local scientific and public health communities worldwide. As consistently observed by all existing rabies networks, basic education of people exposed to the risk of infection, the appropriate training of human and animal health professionals, and necessary advocacy by local health decision makers, are major milestones towards global rabies control. The North American Rabies Management Team is another recent example of professionals in Canada, Mexico, and the United States working together, sharing surveillance data, prioritizing applied research needs, and cooperating across the continent, in training and technology transfer, which allows the implementation of human and animal rabies control programs, achieving a drastic decrease of rabies cases in this region of the world.
The establishment of these partnerships has been of critical importance in raising the profile of rabies at the regional level. However, a much wider (global) approach is needed in strategies aimed at rabies elimination, recognizing that the threat of rabies is evident on every continent throughout the world excluding Antarctica, disregards national borders and that disparity in knowledge and resource capacity should no longer be a reason for inequitable health. In the 21st century, the formation of the Global Alliance for Rabies Control (GARC) and the Partners for Rabies Prevention (PRP) groups perhaps best epitomizes this shift from a regional to a global approach and the modern concept of a global health fellowship [36] . Although these coalitions specifically focus on global rabies control and prevention, they have been established based on a number of broad principles relevant to health issues of zoonotic nature and aimed at addressing the needs of populations suffering from inequitable human and animal health systems. These elements include the following: (1) creating a working group integrating all disciplines/institutions relevant to a comprehensive approach to the control and eventual elimination of a given zoonosis. The inclusion of international organizations is of particular importance, especially as societal and political considerations determine the success of disease elimination [37] ; (2) identifying specific gaps concerning biological and technical feasibility, costs and benefits, and societal and political considerations [37] , which prevent the control, prevention, and eventual elimination of a given zoonosis; (3) working together to agree upon the most effective strategies to address these gaps; (4) identifying the partners within the partnership that can provide the expertise to deal with these gaps; (5) establishing programs to practically deal with these gaps; and (6) demonstrating success by delivering results with respect to these programs.
GARC is the first global nongovernmental organization focusing specifically on increasing awareness and advocacy for rabies prevention and control by supporting communityled interventions. Using the GAELF as an example, the PRP was established in 2008 and is comprised of an informal group of stakeholders working in the field of rabies prevention and control. This is the first time that a working group consisting of all key stakeholders in the field of rabies have joined to find workable solutions to rabies control, including timelines and deliverables. The PRP includes representatives from major international health organizations (WHO/Pan American Health Organization (PAHO), Food and Agriculture Organization (FAO), World Organization for Animal Health (OIE), European Commission (EC)), nongovernmental organizations including GARC and animal welfare organizations, representatives from the human and animal healthcare industry, and global health institutions, such as WHO Collaborating Centers for Rabies Research, and academic institutions working on new rabies prevention and control tools. The individuals in PRP are unpaid volunteers, comprised of the essential disciplines needed towards a common cause of advocacy, action, and research, in an international context. Together, members of the PRP continue to evaluate the global situation of rabies to find feasible, achievable solutions. The PRP thus provides a foundation by which partners can combine their resources, including expertise and experience, communications networks and outreach, and data and educational material to improve access to rabies prevention and control tools ( Table 1 ).
Public-private partnerships, such as the GAELF and PRP, provide a rich environment for brainstorming and optimizing the skills and knowledge required to generate new ideas, create realistic milestones and deliverables, and build novel systems around which global health strategies can be agreed upon. In the field of rabies, the PRP focuses on increasing global attention on rabies, enhancing educational awareness on all levels of society, and providing new tools for both poorly resourced and industrialized nations to improve access to rabies prevention and control, particularly for those living at greatest risk. Regarding increasing advocacy and awareness on local, national, and international levels, a "road map for rabies prevention and control" was initially developed including short-, medium-and longterm achievable goals. The network of the PRP agreed to utilize their resources to increase educational awareness for rabies prevention and to build a global network of rabies experts by supporting the World Rabies Day initiative, launched in 2007 [38] [39] [40] [41] [42] . One example of the successful collaboration provided by the PRP includes the coordinated efforts of the GARC, Centers for Disease Control and Prevention (CDC) in the US, the Universities of Washington and Pretoria, and FAO to distribute over 52,000 rabies awareness posters to 21 countries in Africa. World Rabies Day, observed annually on September 28th, the anniversary of the death of Louis Pasteur, has provided the vehicle by which rabies educational material can be transmitted through the global network set up through the PRP to reach more than 150 countries. Since its inception, lifesaving rabies prevention messages have been sent to more than 150 million people across the globe. The response of communities worldwide since the World Rabies Day inaugural campaign in 2007 is evidence that people living in rabies endemic areas have a desire for relevant educational material and are willing to support community programs aimed at increasing awareness about rabies prevention and control [40, 43] .
The PRP next combined their knowledge, efforts, and resources to define specific strategies that would enable canine-rabies endemic countries to design their own national programs for preventing human rabies. Despite the availability of several guidelines and publications on various aspects of controlling rabies in dogs and preventing rabies in humans, it was realized that there was no integrated standard operating procedure or "Blueprint" bringing together all necessary aspects for a "One Health" approach to controlling rabies. Rabies is a zoonotic disease and, in order for interventions to be successful, control programs must incorporate an integrated, or One Health, approach including human and animal public health principles, diagnostics and surveillance, education and advocacy, anthropology, social mobilization and community outreach, research and development, and program implementation and evaluation as well as funding options. The need for such comprehensive strategies has been clearly identified following the successful adoption of this approach in Latin America leading to dramatic impacts on human and animal rabies cases [44] . The PRP was able to draw on global expertise to incorporate all of these aspects into a Blueprint for rabies prevention and control which is currently available online. [45] . As a next step, the PRP is focusing on combining their efforts to re-evaluate the global burden of rabies in order to establish the cost effectiveness of intervention strategies.
Operational activities related to public-private partnerships are a challenge because strong partners may be restricted by their own institutions or companies as to their level of involvement and they often do not have the time to dedicate to the administering of required activities to reach agreed upon goals. However, one of the major strengths of a public private partnership is the ability to establish new approaches to overcome administrative barriers. The GARC acts as a secretariat for the PRP and is dedicated to bringing all partners and stakeholders together in a timely manner, without bias, and ensuring that operational activities run smoothly.
Other important considerations involved in setting up public private partnerships include establishing the correct balance between the variety of players that are involved as each may have different and often competing interests. If the correct balance is not initially established or is not continuously maintained, there is a potential to lose focus and the most urgent problems may not be addressed. The PRP has resolved these issues by setting up the organization in a similar manner as the GAELF [35] . The PRP maintains a light governance structure, meeting biannually to discuss global issues regarding rabies prevention and control, identify gaps and how best to address them, and concentrating on finding workable solutions that will benefit all of society (Table 1) . Table 1 : Key elements within current rabies partnership(s) based on: (1) identified gaps in areas related to canine rabies prevention, control, and elimination; (2) strategies agreed upon to address these gaps; (3) knowledge and resources provided by partners within the coalition; (4) programs established to address these gaps; (5) 
Perhaps the most important insight generated from decades of epidemiological and operational research on rabies is that the elimination of both canine and human rabies is a feasible objective in rabies endemic areas of the less developed world [12, 29, 44, [46] [47] [48] [49] [50] . We now have a very clear understanding of what constitutes the critical components of a successful rabies elimination program, and we also recognize that a fully integrated approach is critical for achieving this significant goal. One key scientific finding is that where rabies epidemiology is driven by cycles in domestic dogs, as in most of Africa, Asia, and Latin America, well-implemented and sustained rabies vaccination and control programs that target this species will lead to elimination of canine rabies in most areas [12] . Thus, a radical "paradigm shift" in strategic planning and implementation activities is required by the many countries focusing solely on human rabies prophylaxis to prevent human deaths. Dog rabies control should be the focus of efforts and resources also in areas where (occasional) rabies cases are detected in wildlife, since elimination of rabies in dogs should lead to the disappearance of the disease in all other species. Control efforts targeted at wildlife should only be considered where independent transmission cycles are detected in species other than dogs. Once canine rabies has been eliminated, rabies control strategies should aim at maintaining freedom from the disease. A sustainable rabies control and prevention program, therefore, consists of two essential components, an "attack/elimination" component and a "maintenance" component. Although the implementation of each of these two phases requires specific techniques, a number of "preparatory" steps are essential to both components. These include (1) increasing awareness about the local and global impacts of rabies as well as its burden on public health budgets, and establishing specific roles and responsibilities and cooperation amongst all parties involved in rabies control and prevention activities; (2) building capacity and allocating resources to laboratory-based surveillance, and control and prevention operations, including training of relevant professionals; (3) establishing a legislative framework relevant to rabies control and prevention; (4) ensuring engagement of local communities through raising awareness and education; and (5) obtaining information on the size and accessibility of the dog population to define the best strategy for canine rabies control compatible with local circumstances.
The "attack/elimination" component combines activities aiming at preventing the occurrence of human rabies by reducing virus transmission in the dog population. These include: (1) mass vaccination of the maintenance dog population, (2) management of the dog population, and (3) improved access to wound cleaning necessities, human rabies biologics, and information on available bite centers or emergency rooms. Throughout the elimination component, continuous epidemiological surveillance is essential to monitor the effectiveness of intervention in animal and human populations and to prevent overuse of human biologics.
Mass dog vaccination has long been recognized as the mainstay of successful dog rabies control and eventual elimination [12, 48] . In the most affected continents, Africa and Asia, generally characterized by very dense dog populations, adequate levels of coverage, of at least 70% [12, 51] , as well as sustained and frequent (usually annual) campaigns are essential for effective control aimed at achieving a "rabiesfree" status. Since dogs are often unrestrained and without any apparent evidence of ownership, local authorities often view costly oral vaccination of free-roaming dogs as the only solution to the rabies problem in the reservoir population. However, the vast majority of domestic dog populations have affiliations to households/communities and are, therefore, accessible for central-point parenteral vaccination, considered the most cost-effective strategy [29, [52] [53] [54] [55] . Participation in dog vaccination campaigns can also be increased by improved engagement of local communities through education and awareness, as discussed below, and delivery of "primary animal health care" against common infections (mange and internal parasites) to dogs brought to vaccination stations as in KwaZulu-Natal [56] . In some circumstances (e.g., very aggressive or truly unowned dogs, and dispersed communities) more intensive (i.e., house-tohouse) efforts [55] or alternative (oral) delivery strategies [57, 58] may, however, be required. In some circumstances, the effectiveness of mass dog vaccination campaigns can be increased through management of the dog population. Needs for this are determined at the start of the program through ecological surveys, generally conducted as part of the preparatory phase. A combination of approaches are available for dog population management programs: promotion of responsible dog ownership through community education and legislative measures (mandatory dog registration and identification, tie-up orders, abandonment legislation, etc.); reproduction control [59] ; temporary/permanent removal of dogs (shelters, foster homes, capture and release, euthanasia); and habitat control [60] . Elimination of dogs should be restricted to suspect rabid dogs, unvaccinated contact dogs, and dogs considered unsuitable for rehoming or release at the point of capture, and should always be implemented in conjunction with other approaches [60] .
Reducing human rabies incidence through correct utilization of human biologics, including preventive immunization of categories at risk, and wound care (through thorough washing of the wound with water and soap) and prophylaxis (vaccine and rabies immune globulin, RIG, when required) of individuals exposed to suspect rabid animals, is an essential component of the rabies elimination phase. Awareness about prevention behaviors among community members, particularly children, is critical and can be increased through appropriate communication channels, as illustrated below. While the integration of dog rabies control and human rabies prevention approaches is important at the start of the program to reduce animal and human rabies incidence, progressive declines of canine rabies should ultimately result in reduced demand for human biologics [61] .
Once freedom from canine rabies [12] has been achieved through the attack/elimination phase, the implementation Veterinary Medicine International of a "maintenance" phase is required to prevent reintroductions of disease and to ensure a continuous rabiesfree status. This component involves the maintenance of features established in previous phases including continual high political commitment and intersectoral cooperation, surveillance networks to uncover potential new cases or infected areas, regulations for in-country animal movements and animal trade, capacity for dog vaccination (e.g., maintenance/containment/emergency vaccination), policies on the judicious use of human rabies biologics, and educational channels linking government officials and local communities. The maintenance phase also comprises specific activities. An assessment of the rabies situation in neighboring areas is important, ideally followed by the establishment of rabies control and prevention efforts in these jurisdictions through close liaison and effective collaborations involving key stakeholders. Careful consideration also needs to be given to building sustainability in established programs to maintain freedom from rabies in the long term. When national rabies management efforts are developed as multisectoral programs, this could be achieved through integrated financial mechanisms across relevant sectors and parties, with costs and benefits shared across ministries.
Clearly, much progress has been made in the development of tools and strategies required for effective rabies prevention and control. The process of translating researchdriven interventions into better health outcomes for the populations living in rabies-endemic settings is, however, long and difficult. Yet, one recent achievement in this direction is to have gathered and synthesized all current scientific knowledge and available information on rabies prevention and control into the recently launched "Rabies Blueprint" [45] , an easy to use operational tool kit, which has the potential to contribute to evidence-based policies and action towards rabies elimination.
For the successful accomplishment of a canine rabies elimination program, the importance of engaging critical players in rabies prevention and control, that is, national and international policy makers as well as communities at risk, cannot be overstated. A higher level of awareness is a key to effective public and animal health policy and action at all levels, prioritization of rabies in national and international budgets, and increased intersectoral dialogue, hence collaborative rabies prevention and control initiatives. Enhanced awareness on rabies is also essential in preventing and ensuring adequate management of human exposures [12, 62, 63] , engaging personnel and communities in rabies prevention and control efforts, and increasing reporting of cases [13] .
To this effect, the involvement of global health communications (the study and use of methods to inform and influence individual and community decisions that enhance health) has gained increasing prominence in rabies elimination strategies worldwide. The World Rabies Day initiative is an example of the global impact that health communications can have on rabies prevention and control efforts [38] [39] [40] [41] [42] . Utilization of fundamental and innovative communication techniques as part of this initiative has increased awareness globally, enabled life-saving information to be communicated across the world instantaneously, galvanized support, empowered stakeholders at every level, and re-ignited rabies control efforts in countries that had previously abandoned national programs. The establishment of this global rabies network is not the only achievement of including health communications in the global campaign against rabies. Using modern health communications research [64, 65] , a comprehensive 8-step rabies communications plan, adaptable to the cultural, political, and behavioral needs of any location, has been developed for incorporation into a canine rabies elimination program [45] .
Since rabies epidemiology and the behavioral or cultural beliefs of the individuals most at risk will vary across localities, the first step of the communications plan is to identify the important points from an epidemiologic assessment as well as a comprehensive list of potential issues, challenges, and barriers to change that may affect the communication outreach. Challenges may be behavioral (e.g., beliefs on the use of medicine or vaccines), cultural (e.g., perceptions on dogs or other mammalian species), demographic (e.g., economic implications), or physical (e.g., access to healthcare or clean water). Another important aspect is to define the purpose of the communication, including identifying goals and objectives which should be adapted towards national needs. In addition, any outreach should be targeted to specific audiences, who can be segmented according to those most at risk, the primary audience (e.g., children and young boys, who in many areas take care of dogs), and influencers of the primary audience (e.g., healthcare providers, community leaders, and policy makers).
Once draft messages are developed, they should be tested with the target audience. Audience segmentation and message testing will ensure that messages resonate with target populations and can improve uptake of prevention behaviors [66] . In launching a communications campaign, choosing appropriate media channels and determining the best timing for release are also critical. There are numerous ways to disseminate rabies educational messages, and benefits and limitations to each of the channels [67] . After implementing any communications effort, evaluating its impact will help inform and improve future educational outreach, although funding limitations may discourage some localities from undertaking this task. Nonetheless, there are several ways in which rabies programs can be evaluated [68] , and options exist for evaluating communication efforts regardless of funding, such as process evaluation and outcome evaluation [45] .
The need for integrated approaches towards human rabies prevention, which incorporate disease control in the canine reservoir, has been widely recognized by major international health organizations. For such strategies to become truly applicable and universally accepted, successful pilot projects are essential to demonstrate the feasibility of canine rabies Veterinary Medicine International 13 control/elimination and direct benefits for human health. Such projects have the potential to reduce rabies incidence locally, and act as catalysts for encouraging larger-scale or national programs, engaging other areas/countries in similar initiatives, and attracting major international funding support. Building upon the success recognized in Latin America [44] , a number of pilot programs have been established recently across Asia and Africa, as described.
A "Humane and Sustainable Dog Population and Management" program has been initiated in Colombo (Sri Lanka) starting in 2007, including the establishment of a formal partnership between three primary partners: Colombo Municipal Council (CMC), Blue Paw Trust (BPT) and the World Society for the Protection of Animals (WSPA). The first phase of the program is planned for 2008-2012. In a second phase (after 2012), CMC will assume primary financial responsibility from WSPA. The current humane program is an evolution of the elimination and pound system of rabies control and dog population management that existed prior to 2007. Key components of the program include mass vaccination and targeted sterilization over the entire spectrum of the dog population; education of children and adults in bite prevention, rabies awareness, and responsible dog ownership; development of "Dog Managed Zones"; and training of CMC staff in relevant skills, including humane dog handling, recognizing dog behavioral signs, delivering responsible ownership messages to communities, and surgical neutering. Increased vaccination and dog management efforts resulting from existing national programs as well as the newly implemented pilot project have led to a considerable reduction in dog rabies cases [69, 70] . In addition, the combined education program on bite prevention, rabies awareness, and responsible dog ownership has led to greater primary knowledge and its retention. Specifically, 86% of primary school children and 90% of secondary school children gained the required knowledge immediately after education sessions, with 85% of primary and 78% of secondary school children maintaining this same level of knowledge after 6 months [70] .
Bali. The recent introduction of dog rabies in Bali, Indonesia (with >3.5 million people) during 2008 resulted in a major outbreak, which has killed more than 100 humans with a total of 31,000 dog bite injuries and administration of PEP to 28,000 people since November 2008 [19, 20] . The island had previously been "rabies-free," and the local veterinary authorities had little or no experience in rabies control. First attempts at vaccination and dog movement restrictions failed to stop rabies spread from the initial outbreak on the Bukit peninsular. Following this outbreak, the government began to cull dogs using strychnine, increasing the activity in late 2009. Reasons for this action were that (1) the Bali Animal Husbandry Agency did not believe that reaching sufficient vaccination coverage was possible; (2) vaccination campaigns were inadequate largely because of difficulties in handling many of the dogs, a result in part of the local dog owning culture (hands-off), which is common in most of Indonesia; and (3) the issue of roaming (stray or outside) dogs caused a distraction, leading to the diversion of limited resources and attention away from effective rabies control via dog vaccination towards attempts to reduce dog populations.
To assist the government in developing a coordinated and effective rabies control program, towards the end of 2009, WSPA started collaborating with local organizations, the Yayasan Bali Animal Welfare Association (BAWA) and the Bali Street Dog Fund Australia, to develop a pilot mass vaccination project in Gianyar, one of Bali's nine regencies. By the end of May 2010, 44,776 dogs had been vaccinated in 537 banjars (banjar = small subvillage of 100-200 dogs approximately), with an estimated 87% vaccination coverage obtained [69] . Although the average coverage at the end of the first one-day vaccination campaign was 81%, in 81 of the 537 banjars (15%) the coverage was estimated to be below 70%, and hence a second vaccination campaign was launched. In all cases, second campaigns resulted in over 70% coverage. While the rate of human rabies deaths has increased recently, with all nine regencies now infected, reflecting the continuation of the epizootic, confirmed human fatalities in Gianyar have been limited to one case since the start of the pilot mass vaccination project [71, 72] . These preliminary results of the pilot program confirm that mass vaccination of the reservoir species is a more effective and humane method of rabies control than culling. In the week before World Rabies Day 2010, the Balinese government signed an agreement with the BAWA to extend the mass vaccination project to Bali's remaining regencies, with cooperation and collaboration of the provincial and regency governments and utilizing a large donation of animal rabies vaccines funded by AusAID and procured by WHO.
Another example of a pilot program for the prevention of human rabies through reservoir control is the Bohol Rabies Program on the provincial island of Bohol with a population of 1.2 million. This program has been anchored on community-based initiatives, focusing on collaboration with the local government, empowerment of local communities to design, implement, and manage their own rabies control programs in accordance with the national rabies program, education of key target audiences (i.e., school children), and elimination of rabies in dogs. The Bohol project has been supported by the GARC and the provincial government, and has also been funded through other cost-sharing activities from the national government, provincial, municipal, and village or barangay local government units, dog owners, and other nongovernmental partners. Since its inception in 2007, the project has mobilized around 15,000 people including local government officers, animal and human health workers, school teachers, village leaders, and volunteers (village-based "rabies watchers"); introduced rabies education into school curricula in all public elementary schools on the island (reaching >182,000 children); generated $105,740 through community fund schemes to support dog rabies control programs; reduced the dog population by 24% (including the removal of unregistered, unowned, unmanageable, or unclaimed dogs according to the National Animal Welfare Act, and those that had either died of other causes or had been exported from the island); registered 53 [73] . While the effectiveness of mass dog vaccinations has been enhanced by dog population management in this case, measures are currently being taken to reduce the need to further eliminate dogs and improve animal welfare standards when removal is required. Measurable positive impacts of the program include increased awareness and enhanced animal and human health. Specifically, community awareness surveys revealed that >94% of local people heard about rabies, >61% had knowledge about rabies transmission, and >82% was aware and supportive of the Bohol Rabies Program [73] . Additionally, as of October 2010, Bohol will have had no reported rabies deaths in humans or in animals for two full years.
Rabies projects have also been supported by major international funding agencies to serve as a "hands-on" example to demonstrate the feasibility and effectiveness of an ideal "One Health" approach to human rabies prevention. Through the support of the Bill and Melinda Gates Foundation and coordination by WHO, three large-scale rabies elimination demonstration projects, based on mass vaccination of domestic dogs, have been implemented in Africa and Asia (KwaZulu Natal in South Africa, Tanzania, and Philippines) containing over 33 million people and aiming at vaccinating 3 million dogs [74] .
The KwaZulu Natal site (population of 9,500,000 inhabitants) extends from the international borders with Swaziland and Mozambique in the north, to the province of the Eastern Cape in the south, while inland it is bound by the provinces of the Free State and Mpumalanga, and by the Kingdom of Lesotho. The project aims at vaccinating about 700,000 dogs regularly during the project duration. The effective veterinary structure involved in rabies control measures and, more recently, the stronger commitment from provincial authorities alleviated many of the logistical, financial, and managerial difficulties in the implementation and maintenance of successful dog vaccination strategies and have been critical factors in successful implementation to date. Since 2009, the program has already achieved reductions in dog and human rabies prevalence through mass mobile vaccination campaigns, as well as targeted sterilization and dog management programs [56] . The program has also led to notable improvements in the delivery of PEP, with fewer human deaths recorded at present.
In the Philippines, the project includes only the Visayas group of islands covering 25% of the total number of animal rabies cases, 28% of the total cases of human rabies, and 27% of animal bites in the entire country based on the 2006 annual rabies report. Three of the 17 administrative regions of the country, 16 provinces and 31 cities of the country's 82 provinces, and 117 cities are, therefore, included. The project serves almost 19% of the country's human population (with 17 million inhabitants in the area) and an estimated 2 million dogs.
The project site in south-east Tanzania includes Dar es Salaam, Lindi, Mtwara, Morogoro, and Pwani Regions, comprising 24 districts, 459 wards, with approximately 6,200,000 people and 400,000 dogs. Infrastructure is being developed, and implementation of prevention and control programs has been initiated in Dar es Salaam and Morogoro regions.
Although the ultimate goal of these programs is to achieve local prevention of human rabies through the elimination of canine rabies from all sites within a 5-year period, the overall project is designed to be extended into neighboring regions and countries in an effort to achieve regional and international success towards broader canine rabies elimination.
There is no doubt that, from a veterinary and medical standpoint, considerable progress has occurred towards rabies prevention and control. Despite this achievement, obstacles remain that have impeded successful control. Here, we discuss these challenges, and suggest future strategies to overcome them.
One of the most important challenges is to reverse the cycle of neglect, which has created a low priority to rabies and its control in many poorly resourced countries. Global advocacy and enhanced awareness of the disease burden among national and international policy makers are essential to improve rabies prevention and control activities. Data on disability-adjusted life year (DALY) scores and economic burden models are required to compare a given condition in relation to other public health issues, although these elements are not the only criteria on which priorities are defined. Evidence-based estimates of the burden of rabies have been developed for Africa and Asia, as well as specific countries, such as Tanzania and Cambodia [18, 75, 76] , which indicate that canine rabies impacts human and animal health substantially, as well as local and national economies and wildlife conservation [29] . The global burden of rabies, including a more accurate evaluation of the global cost of postexposure prophylaxis as well as mass dog vaccination strategies, needs to be further documented and brought into the spotlight, particularly to the attention of national policy makers in rabies-affected countries and to the international health community. Therefore, there is an urgent need to gain a better sense of the burden of animal and human rabies in any given country. Given that poor surveillance and underreporting have dramatically contributed to a lack of data on disease impacts [77] , a focus on strengthening national capacity to better diagnose rabies and investigate human cases and animal outbreaks is essential for effective disease surveillance and a more accurate burden assessment.
Clearly, rabies is a primary public health issue, but is not without agricultural impacts. Major obstacles are the lack of priority given by the agricultural sector to the control of animal health problems unrelated to livestock species and poor awareness as to effective and ethically acceptable strategies to deal with dog-mediated health issues. This lack of priority is partially due to misconceptions about whose responsibility rabies control is. Veterinary services may usually handle dog-related problems in extreme situations (e.g., to "clean up" areas ahead of specific events, respond to complaints by the public about nuisance caused by "stray" dogs, including bites, or respond to major rabies outbreaks), usually through indiscriminate catch-and-kill operations. These operations are unpopular at the local and international level and counterproductive in the short and long terms. In addition, national and/or local organizations promoting animal welfare and responsible pet ownership, often supported by international animal welfare organizations (e.g., WSPA, Humane Society International, International Fund for Animal Welfare, etc.), are becoming more influential in an increasing number of poorly resourced countries. In many instances, these nongovernmental organizations will oppose catch-and-kill municipality actions, on ethical grounds and also on the basis of lack of effectiveness of these approaches, using evidence-based recommendations of humane alternatives [12, 60] .
In any plan for rabies prevention and control, there is a need to bring all disparate partners (national human health and animal health services, municipality services, nongovernmental organizations, etc.) together in a collegial, rather than adversarial fashion. In addition, there is a need to provide support for veterinary services to carry out annual or biannual mass dog vaccination campaigns for the multiple years required to eliminate dog rabies, particularly given other priorities relating to livestock production. As demonstrated in many Latin American countries, and in Bali and some Indian states, where the leading role in rabies control is assumed by the public health sector and local, national, and international animal welfare organizations, respectively, the additional intersectoral support provided to veterinary services can greatly improve dog rabies control program design, implementation, and sustainability [59] .
Rabies impact is mostly borne by bite victims and the health care system. In many countries the public health sector is often the first to advocate for dog rabies control, as it is usually responsible for the financial burden, in terms of health care and biologics costs. This sector has to assume responsibility for human rabies deaths following exposures to rabid animals when control strategies in the animal reservoir are not effectively implemented and when PEP is not administered correctly. In addition, the government and the Ministry of Health should supply necessary care and biologics to prevent the occurrence of rabies. Provision of vaccines and RIG needed to prevent rabies deaths is estimated theoretically to be around 200 PEP/100,000 inhabitants (based on figures from a range of poorly resourced countries) [78] , although this is likely to vary depending on the setting. While rabies vaccine costs remain in the range of other public health program expenses supported by national health services in several countries, in the absence of international funding these resources are usually beyond the capacity of most impoverished countries. These life-saving products are absolutely necessary and for obvious ethical reasons the considerable sums required to acquire them cannot be diverted from their emergency use. However, improvements in the long-term cost effectiveness of human rabies prevention will only be achieved by focusing on control of rabies in its animal reservoir. Yet, funds initially required to acquire human biologics cannot be diverted to another sector to help increase its capacity to establish effective animal rabies control programs [52, 54, 79, 80] . Any country willing to embark in rabies control initiatives must realize that additional investment will be required in all sectors, and that savings, particularly through reduced demand for PEP, will only occur after several years of implementation. Therefore, it is essential in this initial investment phase that resources can be mobilized among all sectors for human rabies prevention and canine rabies elimination.
Concerning human prophylaxis, an additional challenge is the lack of or limited availability and accessibility of human biologics for PEP, a reoccurring problem in many poorly resourced countries. These products should be provided free or at a subsidized price by the health care system, but in many places they are sold by government-owned and private clinics at a cost, which is often beyond the financial means of animal bite victims. The full cost of PEP alone (direct costs of vaccine and RIG range between $40 to $75, depending on country and product used [18] ) as well as additional costs (medical fees, transportation to hospital, accommodation, and income loss) can easily represent several months of a rural family revenue [18, 29] . Rabies biologics costs have been decreasing gradually mostly due to a more competitive market, with new vaccines and RIG being produced by manufacturers based in emerging economies (particularly in India and China). Although only a small number of vaccines are currently prequalified by WHO, more emerging products should fulfill WHO requirements and obtain this recognition, increasing market competition and further reducing costs. However, any increase in the availability of additional products must be balanced against the risk of biologics having poor quality [81] or counterfeit origins by greater regulatory scrutiny. To assist dog-bite victims and governments struggling to provide adequate quantities of rabies biologics locally, WHO has been promoting the use of the intradermal route for PEP, which provides a reduction by 60 to 80% of the vaccine needed for one PEP regimen [82] . Adoption of the intradermal route should be further encouraged [83] . Furthermore, research on new products, especially cocktails of monoclonal antibodies [84] to replace human and equine RIG, improved vaccines, reduced PEP regimens, and intradermal delivery devices should be a priority [59] .
Eradication of a disease (e.g., rabies) brings the greatest health benefit, which is the absence of the health threat. It is also the quintessential example of health equity, as all mankind reaps the benefits, leading to eternal cost savings. Rabies is undoubtedly the most feared zoonosis in the world, and takes a heavy toll on underprivileged communities living in the poorest countries. The expertise to reduce the global burden of rabies has been built, and significant strides have been made scientifically and practically towards eliminating canine rabies and reducing human rabies deaths in the industrialized world. Moreover, considerable milestones have been achieved towards taking this progress to the next level, by reducing the dramatic impact of rabies on human and animal communities in endemic areas of the developing world. In an effort to enact a "One Health" approach towards global rabies elimination, the creation of effective partnerships has focused on coordinating research and operational activities related to canine rabies elimination. A global rabies network, covering over 150 countries, and uniting stakeholders from the spectrum of veterinary, public health, pharmaceutical, animal welfare, and wildlife conservation agencies, as well as local communities, has been established. Strategies and tools to successfully prevent and control rabies have been formulated to assist rabies-affected areas in management efforts. Operational activities have been implemented in a diversity of Asian, African, and Latin American settings to create replicable and sustainable models, acting as exemplary showcases for others. More broadly, current strategies towards rabies elimination have the potential to be used as a modern example of integrated, evidence-driven, research-based approaches applicable to other major zoonotic pathogens affecting global health. While access to tools and knowledge has been increased, providing all countries with the potential to eliminate rabies from their territories, the ultimate achievement of this goal, which would lead to health equity, continues to face many challenges. Political and financial constraints remain, highlighting the need for further action towards building the political will to eliminate rabies and to create sustainable and economic solutions, to develop additional practical research tools, and to attract long-term national and international funding support. Interplay between SIN3A and STAT3 Mediates Chromatin Conformational Changes and GFAP Expression during Cellular Differentiation BACKGROUND: Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation- related genes for cell fate specifications are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte-like lineage, we revealed a novel role for Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 bound to the GFAP promoter and their occupancies were correlated with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte-like lineage, Sin3A- MeCP2 departed from the promoter and activated STAT3 simultaneously bound to the promoter and exon 1 of GFAP; meanwhile, olig2 was exported from nuclei to the cytoplasm. This suggested that a three-dimensional or higher-order structure was provoked by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruited CBP/p300 to exon 1 and targeted the promoter for histone H3K9 and H3K14 acetylation. The CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and recruitment of RNA polymerase II to activate GFAP gene transcription. CONCLUSIONS/SIGNIFICANCE: These results provide evidence that exchange of repressor and activator complexes and epigenetic modifications are critical strategies for cellular differentiation and lineage-specific gene expression. During embryonic development, the generation of three major neural cell types (neurons, astrocytes, and oligodendrocytes) in the central nervous system (CNS) sequentially occurs, whereby almost all neurons are generated before the appearance of glial cells [1, 2] . Recent findings demonstrated that glial cells are important in critical neuronal maturation processes such as axonal pathfinding, synapse formation, neurotransmitter transport, metabolic functions, and the response to CNS injury [3] [4] [5] [6] . Although rodent brain cultures and neuronal and glial cell lines have provided us with important information about the structure and function of the mammalian CNS, we have scanty understanding of astrocytic differentiation.
There has been longstanding interest in understanding how the process by which progenitors differentiate into different cell types is regulated. In a mouse model, the fate of progenitors in the developing brain is believed to be determined by external cues that involve various types of cytokines and internal cellular programs. External cues such as bone morphogenetic proteins, leukemia inhibitory factor, ciliary neurotrophic factor, Notch-Delta, and basic fibroblast growth factor promote astrocytic differentiation [7] [8] [9] [10] [11] [12] [13] , and most of these factors influence the essential astrogliogenic Janus kinase-signal transducer and activator of transcription pathway [14] [15] [16] [17] . A molecular basis for the cooperative action between these families of cytokines involves the formation of a STAT3-Smad1 complex with the coactivator, p300/CBP, that initiates astrocytespecific gene expression [15, [18] [19] [20] .
Intrinsic programs regulating cell fate determination of progenitors include epigenetic modifications such as DNA methylation and chromatin remodeling. Methylation of the STAT-binding element within the glial fibrillary acidic protein (GFAP) promoter in mice was shown to inhibit the association of activated STATs with the glial promoter, thereby repressing transcription of the GFAP gene [10, 16, 21] . Furthermore, conditional deletion of the maintenance DNA methyltransferase I from neural progenitor cells (NPCs) suggests that DNA methylation regulates the timing and magnitude of astrogliogenesis [22] . Another class of epigenetic modifications was found from FGF2, which regulates the ability of ciliary neurotrophic factor to enhance astrocyte differentiation by inducing H3 Lys4 dimethylation and suppressing H3 Lys9 dimethylation at the STAT3-binding site, resulting in access of the STAT/CBP complex to the GFAP promoter and activation of GFAP expression [16] . Those reports highlight the diverse epigenetic mechanisms that control lineage-specific gene expression; however, it remains unclear how the interplay among DNA methylation, transcriptional repressors or activators, and histone modifications contributes to regulation of the processes.
In this study, we used a human embryonal carcinoma cell line, NTera-2, to develop a model that induces the differentiation of these cells into an astrocyte-like lineage. NTera-2 is derived from a human teratocarcinoma which shares many characteristics of neuroepithelial precursor cells and is widely used as a tool to study the early development of the human CNS and identify new genes involved in neurogenesis [23] [24] [25] . We also used this system to investigate the mechanisms underlying GFAP activation. We identified components of the Sin3A-HDAC complex coupled with MeCP2 present at the GFAP promoter under undifferentiated conditions. Upon differentiation, the promoter underwent a conformational change triggered by STAT3 binding, which contributes to CBP/p300-mediated histone modification and assembly of a transcription preinitiation complex, resulting in GFAP gene activation.
NTera-2, 3T3, and 293T cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Durham, NC, USA) containing 10% fetal bovine serum (FBS; JRH Bioscience, Lenexa, KS, USA), 0.2 mM GlutaMax-1 (Invitrogen), and penicillin/ streptomycin (100 units/mL; Invitrogen). Cells were split every 2 days in a 0.5% trypsin-EDTA solution and maintained until used.
An oligonucleotide containing a stem-loop structure targeting the Oct-4 gene was designed using the RNAi program (http://athena. bioc.uvic.ca/). The targeted sequence was GCGAACCAGTATC-GAGAAC in the Oct-4/POU5F1 gene (515,534 nt; accession no. NM_203289). This sequence was first cloned into a pBS/U6 vector. Then the U6-RNAi cassette was subcloned into a lentiviral vector, pFUGW [26] , and the sequences were verified by DNA sequencing. 
Cells were resuspended in PBS and fixed with ethanol overnight at 220uC. Cells were then resuspended in PBS and treated with 100 mg/ml of ribonuclease A (bovine pancreas; Sigma), 0.1% Triton-X100, and 40 mg/ml propidium iodide (PI) for 30 min at 37uC. Cell cycles were detected with FACSCalibur, and analyzed by the ModFit LT program (Verity Software House, Topsham, ME, USA).
Total RNA was extracted from NTera-2 cells using the Qiagen RNeasy Mini kit following the manufacturer's protocol (Qiagen, Valencia, CA, USA). The target RNA was amplified by a one-step RT-PCR kit (GMbiolab, Taichung, Taiwan). Forward and reverse primers were as follows: GFAP (forward), 59-GTGGGCAGGTGG-GAGCTTGATTCT-39 and (reverse), 59-CTGGGGCGGCCTGG-TATGACA-39 [27] ; and internal control b-actin (forward), 59-TGGAATCCTGTGGCATCCATGAAAC-39 and (reverse), 59-TAAAACGCAGCTCAGTAACAGTCCG-39. The RT-PCR consisted of two programs. First was complementary cDNA synthesis: one cycle at 50uC for 30 min and one cycle at 94uC for 2 min. The other was second-strand cDNA synthesis and the PCR consisted of 35 cycles at 94uC for 30 s, 59uC for 30 s, and 72uC for 45 s; followed by a final extension step at 72uC for 10 min.
At different times after viral infection, cells were lysed in lysis buffer (4 M urea, 1 mM MgCl 2 , 50 mM HEPES, and 100 U benezonase/ 10 7 cells). Lysates containing the equivalent of 2610 5 cells per lane were separated by electrophoresis on 10% polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes overnight. The membrane was blocked with 5% fat-free milk in a TBST solution for 1 h and then probed separately with a mouse monoclonal antibody (mAb) against Oct-4 (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 0.2 mg/mL, 1:1000 of an anti-GFAP rabbit polyclonal antibody (pAb; Chemicon International, Temecula, CA, USA), and 1:500 of an anti-glutamine synthetase mouse mAb (Chemicon International). Mouse b-actin was used as an internal control (Sigma). Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG (Sigma) were used as the secondary antibodies. Signals were detected using an enhanced chemiluminescence (ECL) kit (Perkin Elmer, Wellesley, MA, USA).
Mock-, vector-, and RNAi-treated cells were solubilized in NP40 lysis buffer (10 mM Tris at pH 7.5, 150 mM NaCl, 0.5% NP40, 5 mM EDTA, 5 mg/ml aprotinine, and 3 mM pAPMSF) and then centrifuged at 12,000 rpm for 10 min at 4uC. The lysates were subjected to immunoprecipitation with an anti-STAT3 antibody (Santa Cruz Biotechnology). Then, these immunocomplexes were analyzed as described for the Western blot assay. These immunocomplexes were detected by an anti-p300 antibody (Santa-Cruz Biotechnology).
NTera-2 cells were grown on 12-well plates. These cells were washed with phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde. Then, cells were permeabilized in 0.25% Triton X-100 and incubated with the following primary antibodies overnight at 4uC: anti-Oct-4 rabbit Ab (1:200), anti-Pax6 mouse Ab (1:50), anti-Olig2 rabbit Ab (1:50; Santa-Cruz Biotechnology), anti-nestin mouse Ab (1:100), anti-A2B5 mouse Ab (1:200), antiglial fibrillary acidic protein rabbit Ab (1:1000), anti-MAP2 rabbit Ab (1:1000), anti-glutamine synthetase mouse Ab (1:500; Chemicon International). The next day, cells were washed with PBS and secondary antibodies of 1:500 of goat-mouse-594 (Molecular Probe, Carlsbad, CA, USA), 1:500 of goat-rabbit-cy3, and 1:200 of goat-mouse-cy3-IgM (Jackson ImmunoResearch, Suffolk, UK) which were used accordingly for 1 h at 37uC. Cells were then washed with PBS and counter-stained with Hoechst for 10 min. Cells were detected under florescence microscopy.
Preparation of Protein Extracts: Cell pellets were first washed three times with PBS and lysed in lysis buffer (0.25 M Tris-HCl at pH 6.8, 1% SDS). Protein concentration was obtained by BCA TM protein assay. Cell lysate was then dried and stored in 230uC.
Gel-assisted Digestion [28] : Acrylamide/bisacrylamide (40%, 29:1), 10% APS, and TEMED were then applied to protein solution to polymerize as a gel directly in the eppendorf. The gel was cut into small pieces and washed several times with TEABC containing 50% ACN. The gel samples were further dehydrated with ACN and the completely dried by vacuum centrifugation. Proteolytic digestion was then performed with trypsin (protein: trypsin = 50:1, g/g) in 25 mM TEABC with incubation overnight at 37uC and peptides were extracted by 5% FA in 50% ACN, dried in a SpeedVac and stored in 230uC.
Immobilized Metal Affinity Chromatography (IMAC) [29] : The IMAC column was first capping one end with a 0.5 mm frit disk enclosed in stainless steel column-end fitting. The Ni-NTA resin was extracted from spin column and packed into a 5-cm microcolumn (500 mm id PEEK column). Automatic purification of phosphopeptides was performed using the IMAC microcolumn connected with autosampler and HP1100 solvent delivery system with a flow rate 13 ml/min. First, the Ni2+ ions were removed with 100 ml EDTA (50 mM) in NaCl (1 M). Then the IMAC column was activated with 100 ml FeCl3 (0.2 M) and equilibrated with loading buffer for 30 min before sample loading. For optimization of the phosphopeptide enrichment, the loading/condition buffer (designated as loading buffer) was 6% (v/v) AA and the pH was adjusted to 3.0 with NaOH (0.1 M at pH 12.8). The peptide samples from trypsin digestion were reconstituted in loading buffer and loaded into the IMAC column that had been equilibrated with the same loading buffer for 20 min. Then the unbound peptides were removed with 100 ml washing solution consisting of 75% (v/v) loading buffer and 25% (v/v) ACN, followed by equilibration with loading buffer for 15 min. Finally, the bound peptides were eluted from the IMAC column with 100 ml NH4H2PO4 (200 mM at pH 4.4). Eluted peptide samples were dried under vacuum and reconstituted in 0.1% (v/v) FA for LC-MS/MS analysis.
LC-MS/MS Analysis: Purified phosphopeptide samples were reconstituted in 4 ml buffer A (0.1% FA in H 2 O) and analyzed by LC-Q-TOF MS (Waters Q-TOFTM Premier from Waters Corp). For LC-MS/MS analysis by Waters Q-TOFTM Premier, samples were injected into a 2 cm6180 mm capillary trap column and separated by 20 cm675 mm Waters1 ACQUITYTM 1.7 mm BEH C18 column using a nanoACQUITY Ultra Performance LCTM system (Waters Corp). The column was maintained at 35uC and eluted with a linear gradient of 0-80% buffer B (0.1% FA in ACN) for 80, 120, 210 and 270 min. MS was operated in ESI positive V mode with a resolving power of 10,000. NanoLockSpray source was used for accurate mass measurement and the lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/ml; Sigma) delivered through the NanoLockSpray source. Data acquisition was operated in the data directed analysis (DDA). The method included a full MS scan (m/z 400-1600, 0.6 s) and 3 MS/MS (m/z 100-1990, 1.2 s each scan) sequentially on the most three intense ions present in the full scan mass spectrum.
Database Search, Protein Identification and Quantitation: Raw data were processed using Mascot Distiller v 2.1.1.0 (Matrix science). The resulting MS/MS dataset was exported to *mzdata data file format. We performed the peptide identification and assignment of partial post-translational modifications using an inhouse version of Mascot v. 2.2 (Matrix science). The datasets were searched against International Protein Index (IPI_human v. 3.29, 68161 sequences) using the following constraints: only tryptic peptides with up to two missed cleavage sites were allowed; 0.3-Da mass tolerances for MS and 0.1-Da mass tolerances for MS/MS fragment ions. Phosphorylation (STY), oxidation (M) was specified as variable modifications. Only unique peptide with scores higher than 25 was confidently assigned. When unique peptides were identified to multiple members of a protein family, proteins having the highest sequence coverage were selected from the Mascot search output result. To evaluate the protein identification falsepositive rate, we repeated the searches using identical search parameters and validation criteria against a random database (from the 68161 sequence). Relative quantification of peptides was performed by IDEAL-Q software [30, 31] .
Genomic DNA sodium bisulfite conversion was performed using an EZ-96 DNA methylation kit (Zymo Research, Orange, CA, USA). The manufacture's protocol was followed using 1 mg of genomic DNA and an alternative conversion protocol (two-temperature DNA denaturation).
Seqqunom's (San Diego, CA, USA) MassARRAY platform was used to perform the quantitative methylation analysis. This system utilizes MALDI-TOF mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE kit). A detectable pattern is then analyzed to determine the methylation status. PCR primers were designed using EpiDesigner (Sequenom). Amplicons were designed to cover the CpG sites from an approximately 4500-bp region upstream of the GFAP transcription start site to the intron 1 region. For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10mer tag on the forward primer to adjust for differences in melting temperatures. The PCRs were carried out in a 5-ml format with 10 ng/ml bisulfite-treated DNA, 0.2 units of HotStart Taq DNA polymerase (Qiagen), 16 supplied HotStart buffer, and 200 mM PCR primers. Amplification of the PCR was as follows: preactivation at 95uC for 15 min, 45 cycles of 95uC denaturation for 20 s, 56uC annealing for 30 s, and 72uC extension for 30 s, with a final 72uC incubation for 4 min. Dephosphorylation of unincorporated dNTPs was performed by adding 1.7 ml of H 2 O and 0.3 units of shrimp alkaline phosphatase (Sequenom), followed by incubation at 37uC for 20 min and then at 85uC for 10 min to deactivate the enzyme. Next, in vivo transcription and RNA cleavage were achieved by adding 2 ml of the PCR product to 5 ml of the transcription/cleavage reaction and incubation at 37uC for 3 h. The transcription/cleavage reaction contained 27 units of T7 R&DNA polymerase (EpiCentre, Palmerston North, New Zealand), 0.646 of T7 R&DNA polymerase buffer, 0.22 ml T Cleavage Mix (Sequenom), 3.14 mM DTT, 3.21 ml H 2 O, and 0.09 mg/ml RNaseA (Sequenom). Reactions were additionally diluted with 27 ml of H 2 O and conditioned with 6 mg of CLEAN Resin (Sequenom) for optimal mass-spectrum analysis. Samples were then dispensed with the MassARRAY nanodispenser (Samsung, Irvine, CA, USA) on a 384-well SpectroChip (Sequenom). Mass spectra were acquired using a MassARRAY Compact MALDI-TOF (Sequenom), and the methylation ratios were calculated by comparing the difference in spectra intensity derived from methylated and non-methylated template DNA by the Epityper software version 1.0 (Sequenom).
Genomic DNA (500 ng) was bisulfite converted with the EZ DNA methylation-Gold kit (D5005; Zymo Research, Orange, CA, USA) according to the manufacturer's recommendations. Modified DNA was amplified with the primers listed in Table S1 . The PCRs were carried out with 25 ng bisulfite-treated DNA, 1 ml of HotStart Taq DNA polymerase (Qiagen), 106 supplied HotStart buffer, 4 ml of dNTP (25 mM), and 3 ml of PCR primers (5 mM). PCR conditions were 94uC for 15 min, then 40 cycles of 94uC for 30 s, 57uC for 30 s, and 72uC for 30 s. The PCR products were subjected to purification using the PCR cleanup kit (28160; Qiagen) following the manufacturer's instructions. The purified PCR products (150 ng) were then subcloned into a TA cloning vector (50 ng) (pGEM-T Easy vector; Promega, Madison, WI, USA). Several clones of each sample were verified by sequencing with the T7 universal primer.
ChIP was performed as previously described [32] with some modifications. Briefly, ChIP assays were carried out on 5610 5 NTera-2 and RNAi-treated cells. Cells were harvested by trypsinization, washed, and resuspended to 2610 6 cells/ml in PBS with 20 mM sodium butyrate. The protein-DNA complexes were fixed using 1% formaldehyde for 8 min, and crosslinking fixation was stopped by adding glycine to a final concentration of 125 mM for 5 min. Crosslinked cells were washed twice with cold PBS/20 mM butyrate, resuspended in 100 ml of lysis buffer (50 mM Tris-HCl at pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF, and 20 mM sodium butyrate supplemented with a fresh protease inhibitor cocktail (Sigma) and then sonicated to an average size of 250 bp by a MISONIX Sonicator 3000 for 30 min (with pulses of 30 s on and 30 s off). We used 5 mg of anti-Sin3A, 20 mg of anti-MeCP2 (sc-994X and sc-20700X; Santa Cruz Biotechnology), 5 mg of anti-STAT3 (05-485; Upstate-Millipore, New York, NY, USA), 5 mg of anti-CBP, 5 mg of anti-p300, 20 mg of anti-polII (sc-369X, sc-585X and sc-899X; Santa Cruz Biotechnology), 5 mg of anti-H3K9Ac, 5 mg of anti-H3K14Ac (06-942 and 06-911; Upstate-Millipore), and 2.5 mg of anti-H3K4me3 (ab8580; Abcam, Cambridge, MA, USA) for immunoprecipitation, which was performed at 4uC with the indicated antibodies by incubation with Protein A Dynabeads (Invitrogen) equilibrated with 1 ml RIPA buffer (10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 0.5 mM EGTA 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, and 140 mM NaCl) and protease inhibitors for 2 h. The immunocomplexes were further incubated with chromatin at 4uC overnight. The bound fraction was isolated by Protein A Dynabeads according to the manufacturer's instructions, and the immunoprecipitated complexes were eluted by using 300 ml of elution buffer (20 mM Tris-HCl at pH 7.5, 5 mM EDTA, 50 mM NaCl and 20 mM sodium butyrate) for 30 min at 65uC then for 10 min at room temperature on a rotator. Chromatin was eluted from the antibody and de-cross-linked by added of 30 ml pronase (20 mg/ml) and 1 ml CaCl 2 (1 M) then incubating it at 42uC for 2 h and 65uC for 6 h. The immunoprecipitated DNA was recovered by a PCR purification kit (Qiagen) according to the manufacturer's instructions, and the purified samples were analyzed by real-time quantitative PCR using an SYBR Green master mix and a LightCycler 480 sequence detection system (Roche Applied Science, Mannheim, Germany). PCR primers were designed using the software program Primer Express (Roche Applied Science), and the amplification primers are listed in Table S2 . For each sample, the PCR analysis was performed in triplicate, and the bound fraction was compared with 1:40 diluted input DNA of 1610 5 cells. The results are reported as the ratio of immunoprecipitated (IP) DNA to input DNA (IP/input). To obtain relative occupancy values, the IP/input ratio was further normalized to the level observed at a control region, h, which was defined as 1.0. Values of NTera-2 cells were compared to those of RNAi-treated samples.
NTera-2 cells are capable of differentiating into mixtures of neural and glial cells depending upon the induction conditions [33] [34] [35] . Since NTera-2 cells express high levels of Oct-4 [36, 37] , we used a lentiviral vector carrying a short hairpin (sh)RNA (Fig. 1A) to downregulate Oct-4 expression in order to develop a new protocol for cell differentiation. Three days after transduction with the shRNA lentivirus, Oct-4 expression was greatly diminished (Fig. 1B) . As shown in Fig. 1C , the cellular morphology remained the same as control NTera-2 cells at 3 days. However, dramatic morphological transformation was observed after 21 days of transduction, and cells had become flattened and enlarged, and showed a bushy morphology with numerous short and highly branched processes (Fig. 1C) . Such a cell morphology exhibited characteristics similar to protoplasmic astrocytes, distinct from ''fibrous astrocytes'' [38] . In addition to these morphological changes, the reduction in Oct-4 expression by NTera-2 cells was accompanied by a marked reduction in cell proliferation (Fig.  S1A ). The growth potential of NTera-2 cells after transduction was studied by flow cytometry, and these cells had almost ceased cell cycling with accumulations of cells in the G0/G1 phase on days 4 and 14 (40.6% and 69.1%, respectively, in Fig. S1B ). Thereafter, these cells remained mostly in the G0/G1 phase (e.g., on day 14) and underwent significant morphological changes, as described above. In addition, downregulation of Oct-4 expression often leads to upregulation of Cdx2 in ES cells [39, 40] . In NTera-2 cells, however, Cdx2 expression was not detected in RNAi-transduced cells (data not shown), indicating that this cell line may be derived from cells at a stage of embryogenesis later than the origin of the ES cell line. In addition, NTera-2 cells normally express the intermediate filament protein, nestin (Fig. 1C) , a marker of neuroglial progenitor cells. But, the expression of nestin disappeared 3 days after transduction (Fig. 1C) .
The morphological transformation and cessation of proliferation in RNAi-transduced cells suggested that NTera-2 cells might undergo a process of differentiation. To further verify that these changes were accompanied by differentiation toward an astrocyte-like lineage, cells were examined for the expression of astrocytic markers. The intermediate filament protein, GFAP, is considered a cell type-specific marker for astrocytes. GFAP expression was observed in differentiated cells examined by immunofluorescence and Western blot assays (Fig. 1D) . Another astrocyte-specific marker, glutamine synthetase (GS) [41] , was also detected by immunofluorescence and Western blot assays in differentiated cells (Fig. 1E ). In addition, we detected neither the oligodendrocyte markers, A2B5 [42] and Gal-C [43] , nor the neuron-specific markers, MAP-2 and Pax6 [44] , after 21 days of transduction (Fig. S1C) . Taken together, these differentiated NTera-2 cells produced by our newly developed protocol had an astrocyte-like morphology and expressed the astrocyte-specific markers, GFAP and GS, suggesting differentiation toward an astrocytic lineage. Therefore, these differentiated cells distinctly differed from previous reports of a mixture of different neuronal cell types after retinoic acid induction [35] .
Nuclear export of olig2 and STAT3 activation during cell differentiation Oligo 2 is a basic HLH transcription factor, and its expression in the nucleus is required for oligodendrocyte development by preventing the formation of the STAT3 and p300 complex in the nucleus [45, 46] . To confirm the cellular differentiation of NTera-2 cells, we examined the expression of olig2 and found that olig2 was expressed in nuclei of undifferentiated NTera-2 cells (Fig. 2A) . During specification for an astrocyte-like lineage, there was a striking translocation in the distribution of olig2 from nuclei to the cytoplasm ( Fig. 2A) . Thus, the translocation of olig2 from nuclei may suggest activation of STAT3 in nuclei. Therefore, we analyzed the phosphorylation status of STAT3 and found that there was significant accumulation of phosphorylated STAT3 in differentiated cells compared to undifferentiated cells (Fig. 2B) . Furthermore, specific antibodies against STAT3 were found to have co-immunoprecipitated p300 in differentiated cells, but not in undifferentiated cells, suggesting specific formation of STAT3-p300 complexes in differentiated cells (Fig. 2C) . These findings suggest activation of the STAT3 signaling pathway during the differentiation of NTera-2 cells.
In order to investigate the mechanism of suppression and desuppression of astrocyte differentiation-related genes in NTera-2 cells, we investigated and quantified differentially phosphorylated proteins using a newly developed label-free quantitation technol-ogy platform [30] . Briefly, phosphoproteins were digested in an optimized gel-assisted digestion and purified by a highly specific and reproducible automatic IMAC nanotechnology, followed by LC-MS/MS analysis. The relative quantification method was performed by software ''IDEAL-Q'' which was developed by SEMI alignment approach (Fig. 3A) .
Using NTera-2, it was found that the dynamic phosphoproteomic profile was observed after induction toward astrocyte differentiation. We found that there was a slightly increased in the overall phosphoproteomic profile levels at day 14 after induction for differentiation. In contrast, the upregulation and downregulation levels of phosphoproteomic profile increased dramatically and Figure 2 . Translocation of olig2, activation of STAT3 signaling and STAT3-p300 complex formation, and loss of Sin3A after differentiation. A. Immunofluorescence staining for olig2 (red, upper panel), Hoechst dye (blue, middle panel), and merged images (bottom panel) are shown for undifferentiated control NTera-2 cells and NTera-2 cells 21 days after differentiation. Magnification 4006. B. STAT3 activation was examined with a specific antibody against phosphorylated STAT3, and total levels of STAT3 in lysates from undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation (left panel) were determined. C. Lysates from cells similarly transduced were subjected to immunoprecipitation with an antibody against STAT3 and then were also probed with an anti-p300-specific antibody (right panel). doi:10.1371/journal.pone.0022018.g002 more than 50% phosphorylation sites have differential levels after stimulation for 21days, suggesting the dramatic change in phosphoproteomic pattern.
To gain insight into the putative biological functions of the identified proteins, differentially phosphorylated proteins were functionally annotated and classified by using the functional annotation tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) [47, 48] bioinformatics resources (http://david.abcc.ncifcrf.gov) on the National Institute of Allergy and Infectious Disease (NIAID). This software performed a statistical analysis based on gene ontologies to identify groups that were significantly overrepresented (enriched) in this filtered data set. Upon DAVID gene functional classification, there were two groups of annotated proteins identified that showed statistically significant enrichment scores; they included phosphoproteins and those that were posttranslationally modified by acetylation (Fig. 3B ). In addition, these proteins with significant enrichment scores were separated to nucleus and cytoplasm in location. And the proteins that function in the nucleus were remarkably represented by those that control mRNA processing, chromosome organization, chromatin assembly/disassembly, DNA packaging, etc, (Fig. 3B) .
Among several potential chromatin related functions, we chose to study epigenetic regulation and chromatin remodeling during cellular differentiation in more depth. Three important themes emerge from the shift in the expression of several known, classifiable proteins (Fig. 3C) . First, the data set contains histone modifying complexes such as Sin3A and SUDS3. Second group of proteins are the components of chromatin remodeling complexes including BAZ1B, SMARCC1 and CHD7. They are thought to regulate chromatin structures using ATPase activity. The third is a group of chromatin associating proteins, such that RB1, MCM2, TRIM24, HMGA1 and RCC1. They are involved in diverse functions such as transcriptional control, DNA replication and chromosome condensation.
Of particular interest is the significant reduction of Sin3A in the phosphoproteomic pattern after 3 days of transduction. Sin3A is a component of Sin3A/HDAC co-repressor complexes, which functions in transcription repression [49] [50] [51] , but its role in astrocyte differentiation has not been characterized. By a Western blot analysis of samples through the course of differentiation (control, 7, 14, and 21 days), we further confirmed that the protein expression of Sin3A decreased significantly and continuously after differentiation (Fig. 3D) . Reduction of the expression of Sin3A may lead to disintegration of Sin3A/HDAC complexes thus causing de-suppression of the transcriptional program that favors astrocyte-like differentiation, such as activation of GFAP.
Since there was a significant reduction in the expression of Sin3A at the cellular level after cell differentiation in NTera-2 cells, we next investigated regional alterations in chromatin around the STAT3-binding site at the promoter and transcription initiation sites of GFAP before and after astrocyte-like differentiation. To ascertain local changes in transcription factor binding, we performed ChIP assays with specific antibodies against Sin3A and MeCP2. In addition, the binding of these two co-repressors was quantitated by a real-time PCR using primers spanning from the STAT3 binding site at the promoter to exon 1 relative to the transcription start site of GFAP (primer sets a-g in Fig. 4A ). In addition, a pair of primer, h (Fig. 4A) , located between exons 6 and 7 near the end of GFAP gene and remote from the transcription regulatory region, served as a negative control. It was found that upon cellular differentiation, the binding of Sin3A significantly and specifically diminished around the STAT3 binding site about 1500 bp upstream of the GFAP start site in the promoter region (primer sets a-c in Fig. 4A) (Fig. 4B ). In addition, we also identified that Sin3A occupancy at primer set g in the vicinity of exon 1 was strongly reduced after cell differentiation, whereas binding levels of Sin3A across the exon 1 locus (primer sets e and f) were not altered. Since Sin3A lacks an intrinsic DNA-binding capacity, it must target itself to promoters via interactions with other DNA-binding or adaptor proteins, such as MeCP2, a methyl-CpG-binding protein, which was reported to interact with Sin3A in Xenopus laevis oocytes [52] . To test the possibility that MeCP2 may target Sin3A to the GFAP promoter, we performed a ChIP analysis to inspect the binding of MeCP2 around the STAT3-binding site at the GFAP promoter and in the proximity of exon 1 before and after cell differentiation. As shown in Fig. 4B , the level of specific binding between MeCP2 and the STAT3-binding site at the GFAP promoter (primer sets a-c) and in the proximity of exon 1 (primer sets d-g, except site e) regions of GFAP in undifferentiated NTera-2 cells was substantially reduced in differentiated astrocyte-like cells. As the binding patterns for MeCP2 and Sin3A at the GFAP promoter and exon 1 occurred in a coordinated manner, these results suggest that MeCP2 may be responsible for recruiting the Sin3A co-repressor complex to the area surrounding the GFAP promoter.
MeCP2 is capable of binding to methylated DNA. There is a possibility that the reduction in MeCP2 occupancy at the GFAP promoter was triggered by DNA demethylation of the same region; we therefore examined the DNA methylation status of the GFAP gene in NTera-2 cells and differentiated astrocyte-like cells. However, the bisulfite sequencing analysis showed that upon differentiation toward an astrocyte-like lineage, the CpG dinucleotide around the STAT3-binding site at the promoter (primer sets P1 and P2 in Fig. 4C ) and exon 1 (primer sets E1 and E2) was not demethylated (Fig. 4C) . To further confirm these findings, we performed a quantitative methylation analysis using MALDI-TOF/MS to examine the mass signal shift of methylated DNA compared to unmethylated DNA extracted from undifferentiated NTera-2 cells, differentiated cells, and control human monocytes. This study confirmed that CpG sites scattered in the approximately 4500-bp region upstream of the GFAP transcription start site were stably methylated before and after differentiation (Fig. 4D) . We concluded that the DNA methylation status of the GFAP promoter and exon 1 remained unchanged during cellular differentiation. Our results showed that reduced expression of Sin3A is accompanied by decreased occupancy of Sin3A and MeCP2 around the STAT3-binding site of the GFAP promoter, which was not necessarily accompanied by DNA demethylation in the same region for the induction of GFAP expression. These results suggest a critical role for Sin3A in regulating GFAP expression during astrocytic differentiation.
The binding of activated STAT3 to a STAT3 recognition element (21518 to 21510 bp in relation to the transcription start site) in the mouse GFAP promoter plays a major role in the transcriptional activation of GFAP [53] . Activation of STAT3 was also identified in astrocyte-like cells (Fig. 2B) , which suggested that STAT3 may be responsible for GFAP activation. To test this, the occupancy of activated STAT3 was examined by a ChIP assay, using similar primer sets a-g as shown in Fig. 4A . As anticipated, the occupancy of activated STAT3 was more abundant around the STAT3-binding site at the GFAP promoter (primer sets b and c, especially primer set b at the STAT3 binding site) in differentiated cells compared to undifferentiated cells (Fig. 5A) .
Interestingly, at the exon 1 coding region and the 39 end of the exon 1 region of the GFAP gene, we also detected a second peak of activated STAT3 binding signals (primer sets e-g) in differentiated cells but not in undifferentiated NTera-2 cells (Fig. 5A ). STAT3 is a sequence-specific DNA-binding protein; in addition to the binding site at 21500 bp in the promoter (75% identities), we had identified a second binding site at +250 bp (TTCCTGGAA) at exon 1 of human GFAP (85% identities), based on a detailed sequence comparison with mouse GFAP gene. These results argue that STAT3 may originally target the binding sequence that resides at the GFAP promoter, but due to the chromatin configuration between the promoter and exon 1 in differentiated cells, STAT3 is also cross-linked to chromatin at exon 1. Alternatively, the occupancy of activated STAT3 may cause a bridge between the promoter and exon 1 which induces a stereospecific interaction on surfaces around the GFAP promoter and transcription start site.
To further validate activation of GFAP induced by STAT3 binding, a ChIP analysis was performed to examine the recruitment of RNA polymerase II. A comparison of undifferentiated NTera-2 cells with differentiated cells revealed increased recruitment of RNA polymerase II to the GFAP gene transcription start site (primer set e) after differentiation, consistent with enhanced transcription (Fig. 5B) . Previous studies suggested that H3 Lys4 trimethylated (H3K4me3) nucleosomes occur near transcription start sites of actively transcribed genes. Moreover, several large-scale and genome-wide analyses revealed that about 91% of all RNA polymerase II-binding sites are correlated with H3K4me3-binding sites and were positively correlated with gene expression levels [54, 55] . Using a ChIP assay, we also found that H3K4me3 was enriched at sites of transcription initiation (primer sets e-g), with maximal enrichment immediately downstream of the start site (primer sets e and f) and overlapping the peak of RNA polymerase II at the transcription start site (primer set e) in differentiated cells compared to undifferentiated cells (Fig. 5C) . These results suggest a close correlation among STAT3-mediated chromatin remodeling, RNA polymerase II recruitment, and GFAP activation. Figure 2B and 2C show that activated STAT3 was coimmunoprecipitated with p300. Furthermore, it was reported that STAT proteins activate transcription by recruiting the transcription co-activator, CBP/p300 [56, 57] , and CBP/p300-induced acetylation on histone tails is coupled with chromatin remodeling, thereby enhancing target gene expression [58, 59] . We next examined whether the CBP/p300 co-activator was also associated with the activated GFAP promoter along with an increase in the histone acetylation level in the same region. Notably, we did not detect a significant association between CBP/p300 proteins and the STAT3-binding site at the GFAP promoter (primer sets a-c), as indicated by the ChIP analyses (Fig. 5D) . Conversely, upon cellular differentiation, robust recruitment of CBP/p300 proteins was observed in the vicinity of the exon 1 coding region (primer sets e-g with the CBP antibody and primer sets d, f, and g with the p300 antibody) (Fig. 5D ). Since there is no report of DNA-binding ability of CBP/p300 in these regions, this result suggested that CBP/p300 was brought to the vicinity of exon 1 by transcription factors such as STAT3 (Fig. 5A ) after cell differentiation. We further inspected the status of histone acetylation before and after differentiation to survey the influence of CBP/p300 deposition on exon 1. ChIP assays were conducted with specific antibodies against acetyl-H3K9 (H3K9Ac) and acetyl-H3K14 (H3K14Ac) in undifferentiated NTera-2 cells and differentiated cells. The results showed that there were significant increases in active markers of H3K9Ac (primer sets b and c) and H3K14Ac (primer sets a and b) around the STAT3binding site at the GFAP promoter region, particularly the STAT3binding site (primer set b), but not in the exon 1 coding region (Fig. 5E ). In addition, in a separate experiment (Liao, CH and Yu, J. unpublished observation), we found that treatment of NTera-2 cells with HDAC inhibitors led to an increase in the acetylated H3K9 and H3K14 using specific antibodies and also GFAP gene expression. Therefore, our results raise the possibility that the chromatin structure of the GFAP promoter may undergo dramatic changes in organization during cell differentiation.
Astrocytes are the most numerous cells in the CNS that provide important support to neurons and modulate synaptic activity [60] .
In the present study, we developed a new protocol to obtain pure population of astrocyte-like cells from human NTera-2. We revealed a novel role for Sin3A which coupled to MeCP2 and bound to the STAT3-binding site in the GFAP promoter and its occupancy was inversely correlated with de-suppression of GFAP transcription. Sin3A is thought to be devoid of intrinsic DNAbinding capacity, but is able to be recruited by MeCP2 that linked to methylated DNA with HDAC complex. Such formation of corepressor complex by chromatin-bound MeCP2 may lead to local deacetylation of core histones with subsequent transcriptional silencing [52, 61] . Recent reports proposed that another corepressor, N-CoR, controls differentiation of neural stem cells into astrocytes [62] because the complex containing N-CoR, Sin3A and HDAC mediated transcriptional repression [63, 64] . In fact, our phosphoproteomic analysis also confirmed the presence of N-CoR2 in the undifferentiated NTera-2, which declined significantly after cell differentiation (data not shown). These results support transcriptional repression by Sin3A/MeCP2 complex serving as one of the critical mechanisms underlying the inhibition of astrocytic differentiation. It seems that MeCP2 with Sin3A binds to the methylated STAT3-binding site of the GFAP promoter, thus making the site inaccessible to STAT3. Upon cellular differentiation, occupancy by MeCP2 and Sin3A was significantly and specifically diminished at the STAT3-binding site of the GFAP promoter, suggesting a new regulatory path to trigger GFAP activation, in addition to the regulation by STAT3.
On the other hand, in differentiated cells, activated STAT3, present at both STAT3-binding sites of the GFAP promoter and its exon 1 suggests that a conformational change may bridge both sides of the transcriptional start site during GFAP activation. This conformational change resulted in subsequent recruitment of CBP/p300 to the exon 1 coding region but not the promoter in the ChIP assay. Finally the specific increase of CBP/p300 occupancy at GFAP locus implies that the acetylation by these histone acetyltransferases play roles for gene activation of GFAP. Furthermore, CBP/p300 targeted histone H3 acetylation of the promoter but not exon 1 and induced chromatin remodeling, thereby enhancing recruitment of RNA polymerase II to activate GFAP transcription (Fig. 6 ). In addition, this notion of the change of the acetylation status accompanied with GFAP expression in NTera-2 cells was also supported by another independent assay in which specific HDAC inhibitors were used to increase the acetylated H3K9 and H3K14 in NTera-2 cells (Liao, CH and Yu, J., unpublished observation).
Processes that regulate gene transcription are directly under the influence of genome organization, and intrachromosomal interactions such as chromatin looping were shown to be involved in promoting transcriptional activation of genes in eukaryotes [65, 66] . Our ChIP experiments showed that activated STAT3 was present both in STAT3-binding sites of the promoter and in the exon 1 coding region of GFAP gene, and CBP/p300 was specifically recruited to the exon 1 coding region but not to the promoter. These findings imply that there are conformational changes in the GFAP brought about by STAT3 activation.
To investigate whether the interaction between STAT3 and CBP/p300 can result in the formation of a DNA loop between the promoter and coding region of GFAP, a 3C (chromosome conformation capture) assay [67] was performed. We also combined the 3C with ChIP (using p300 as a ChIP antibody) in a ChIP-loop assay [68] that presumably allowed us to determine which genomic sites would interact and how the candidate proteins mediate the interactions. Unfortunately, we failed to detect direct evidence of DNA looping at the GFAP promoter and thus conclusions must await further study. Although we had not yet establish a cause-and-effect relationship between DNA looping and GFAP gene transcription, our data of STAT3 and CBP/p300 binding suggest the possibility of DNA looping in the region surrounding the GFAP promoter. Alternatively, it was shown that during cell differentiation from a pluripotent to a committed state, there were global changes in the chromatin structure and interactions of chromatin-binding proteins [69] . Therefore, we cannot exclude the possibility that intrachromosomal interactions occurred in other regions where we did not examine in this study or that interactions between the promoter and coding region of GFAP may still occur in a more complicated manner [70] . Activation of GFAP without DNA demethylation It is known that binding of activated STAT3 to a consensus site in the GFAP promoter plays a role in the transcriptional activation of GFAP [53] . Our observations showed that GFAP expression was induced during astrocyte-like differentiation of NTera-2. ChIP experiments confirmed that there was a strong association of STAT3 with the GFAP promoter, suggesting the existence of mechanism that facilitates access of the STAT3 complex to the GFAP promoter. Methylation of CpGs in DNA constitutes one epigenetic mark that generally correlated with transcriptionally silent chromatin, and hypomethylated DNA in the promoter is a hallmark of vertebrate genes that are actively transcribed [71] . In mouse model, GFAP activation was associated with the loss of DNA methylation at the STAT3-binding site [22] . However, our investigation on the epigenetic regulation of GFAP expression in human NTera-2 cells did not observe changes of DNA methylation at the promoter (up to 4,500 bp upstream), indicating that regulation of the interaction of the STAT3 complex in the GFAP promoter was not mediated by DNA demethylation [21] . There seem to be several explanations that may account for the discrepancies between our results and previous data from the mouse study. First, since no enzyme has yet been identified that actively removes the methyl group from DNA, it is believed that DNA methylation is passively removed through multiple rounds of DNA replication [72] . In present study, the differentiated cells from NTera-2 underwent growth arrest on day 3 after induction. Therefore, it was not possible for these cells to have reduced the methylation level of DNA through cell division. Secondly, it was recently observed that DNA hypermethylation was completely maintained at the promoter region of the erythroid-specific carbonic anhydrase II upon hormone-induced activation [73] . Thus this finding challenges the paradigm that the methylation of promoter-containing CpG islands invariantly causes gene silencing. Finally, it was reported that parental allele-specific histone modifications in the promoter, rather than the differentiated methylated DNA, marked the imprinting status of imprinted genes [74] .
In other word, the promoter-restricted change of histone modifications is one of the governing epigenetic marks in transcriptional regulation. Studies in yeast have demonstrated Figure 6 . Model for GFAP gene activation for induced human astrocytic differentiation. In undifferentiated NTera-2 cells, the presence of the MeCP2 and Sin3A corepressor complex and possibly associated HDAC activity at the GFAP promoter maintain the deacetylated status of chromatin and repress GFAP transcription. Upon cellular differentiation, MeCP2 and Sin3A became dissociated from the GFAP promoter, and the subsequent recruitment of the phosphorylated STAT3 caused a conformational change in the region surrounding the GFAP transcription starting site. This, in turn, facilitated histone H3 acetylation of the promoter resulting from the recruitment of CBP/p300 to exon 1. The combination of chromatin remodeling and promoter conformational changes enabled the recruitment of RNA pol II. doi:10.1371/journal.pone.0022018.g006 that nucleosomes with histone H3K4me3 are associated with actively transcribed genes [75] [76] [77] [78] [79] [80] . Similarly, acetylation of histone H3K9 and H3K14 is critical for the recruitment of transcription factor II D, an initiation step in transcription, and therefore be associated with actively transcribed genes [77, 79, [81] [82] [83] . Histone acetylation is catalyzed by several evolutionarily conserved histone acetyltransferases, including Gcn5/PCAF, TAF1, and CBP/p300 [84] . Genome-wide analysis in human cells confirmed that H3K4me3, H3K9Ac and H3K14Ac are present together at actively transcribed genes [85] . Our ChIP experiments showed that upon GFAP activation after cell differentiation, there were significant increases in active markers of H3K9Ac and H3K14Ac around the STAT3-binding site at the GFAP promoter region. At the same time, H3K4me3 was enriched and overlapped with the peak of RNA polymerase II at the transcription start site. These results support the notion that chromatin remodeling regulated by histone modification is linked to active transcription and provide evidence that the acetylation status of chromatin at the GFAP promoter is likely to be predominant regulator of transcription. Altogether, our study has added a novel regulatory path to GFAP gene expression in addition to DNA methylation.
Human NTera-2 cells are widely used as models in human neurogenesis and differentiate into mixtures of various neuronal cell types [35, 86] ; but directed differentiation toward specific lineages such as astrocytic cells has not been accomplished. In this study, we developed an in vitro model of cellular differentiation in which human NTera-2 cells were differentiated into a homogenous population of cells with an astrocyte-like morphology and expressing astrocyte-specific, but not neural or oligodendrocyte markers. The advantages of this model over other in vitro systems for human astrocyte-like differentiation include its capability for robust differentiation and the formation of astrocyte-like cells but not other neuronal cell types. Such directed differentiation differs from those which induce differentiation into mixtures of mostly neurons and a few astrocytes after the 4-or 5-week treatment with retinoic acid [35] . Thus this in vitro human cell model can be used to examine a detailed mechanism underlying astrocytic differentiation. By now there is strong evidence that astrocyte development and regeneration play critical roles in repair after brain injury and future studies should clarify how these astrocytes are regenerated and how precursor cells can be manipulated to recover from brain injuries. Figure S1 Cell proliferation cessation and cell-cycle arrest induced during astrocytic differentiation. A. Growth of NTera-2 cells before and after differentiation as analyzed with a hemocytometer (n = 3; error bars indicate standard deviations). B. For the cell-cycle analysis, NTera-2 cells were similarly induced and then collected, stained with propidium iodide, and analyzed by flow cytometry. The ratios of cells in the G0/G1 phase on days 4, 7, and 14 days are shown. C. Immunofluorescent localization of A2B5, Gal-c, MAP-2, and Pax6 was examined in undifferentiated NTera-2 cells and cells 21 days after differentiation. Magnification 4006. (TIF)  The fertility quality of life (FertiQoL) tool: development and general psychometric properties(†) BACKGROUND: To develop the first international instrument to measure fertility quality of life (FertiQoL) in men and women experiencing fertility problems, to evaluate the preliminary psychometric properties of this new tool and to translate FertiQoL into multiple languages. METHOD: We conducted a survey, both online and in fertility clinics in USA, Australia/New Zealand, Canada and UK. A total of 1414 people with fertility problems participated. The main outcome measure was the FertiQoL tool. RESULTS: FertiQoL consists of 36 items that assess core (24 items) and treatment-related quality of life (QoL) (10 items) and overall life and physical health (2 items). Cronbach reliability statistics for the Core and Treatment FertiQoL (and subscales) were satisfactory and in the range of 0.72 and 0.92. Sensitivity analyses showed that FertiQoL detected expected relations between QoL and gender, parity and support-seeking. FertiQoL was translated into 20 languages by the same translation team with each translation verified by local bilingual fertility experts. CONCLUSIONS: FertiQoL is a reliable measure of the impact of fertility problems and its treatment on QoL. Future research should establish its use in cross-cultural research and clinical work. 'Quality of life' (QoL) was defined by the World Health Organization (WHO) as an ' . . . individuals' perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns . . . ' (WHOQOL, 1995) . The WHOQOL measures QoL broadly according to 29 facets (e.g. self-esteem, mobility and safety). QoL measurement is important to identify aspects of fertility problems associated with poor QoL and advance research in health service-evaluation, patient satisfaction and policy-making through the use of a standard measurement tool (Saxena et al., 2001) .
Psychosocial studies convincingly demonstrate a high incidence of negative reactions to infertility and its treatment (Verhaak et al., 2007) that impact on overall life satisfaction and well-being (Greil, 1997) , success of treatment (Boivin and Schmidt, 2005) , willingness to continue with treatment (Smeenk et al., 2004) , treatment evaluation (Dancet et al., 2010) and the long-term satisfaction people can hope to achieve if treatment is unsuccessful and they remain childless (Daniluk, 2001) . Therefore, the need to measure and take into account QoL in infertility is imperative and tackling this measurement hurdle could lead to improved patient outcomes.
The 14 existing self-report measures of infertility-specific distress, treatment reactions and QoL shown in Supplementary data, Table  S1 do not fulfill the need for a fertility specific QoL assessment tool (the table includes details of development sample, content and reference). The fertility problem inventory (FPI: Newton et al., 1999) is the most frequently used distress measure. However, the items were developed without consultation with people experiencing fertility problems and the validation sample comprised primarily Caucasian patients from a homogeneous socioeconomic category using assisted reproductive techniques. Further, the FPI assesses level of strain rather than the broader construct of QoL and does not separate effects due to infertility treatment from those due to childlessness, which is important given the emotional challenges of each. These issues apply to most measures listed in Supplementary data, Table S1 . The most frequently used QoL measure was developed for women suffering from polycystic ovarian syndrome (Cronin et al., 1998) . Several studies have examined its psychometric properties (Jones et al., 2008) and used it to investigate moderators of QoL (e.g. obesity) and cross-cultural effects. Results confirm its reliability and the importance of cultural background as a moderator of QoL (Schmid et al., 2004; Adamson, 2009 ). However, this and other quality of life measures for infertility were designed for specific sub-populations (e.g. endometriosis, male factor) and therefore cannot be used as generic measure for all people with fertility problems.
In summary, the need for a quality of life measure for infertility measure has not been fully met. Given the importance of addressing this need, the European Society of Human Reproduction and Embryology (ESHRE) and the American Society of Reproductive Medicine (ASRM) joined forces with Merck-Serono S.A. Geneva-Switzerland (an affiliate of Merck KGaA Darmstadt, Germany) to create the fertility quality of life (FertiQoL) measure (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) . The overall aim of the FertiQoL project was to develop an international instrument to measure QoL in men and women experiencing fertility problems. Secondary aims were to evaluate the psychometric properties of the tool and to translate FertiQoL in multiple languages. The development phase was carried out according to the protocol used for the development of the WHOQOL measure (WHO, 1998) and is briefly described in the present article. However, the main focus of this report is on the psychometric evaluation.
Men and women experiencing fertility difficulties with and without medical experience were sampled from one fertility clinic in Australia, Canada, New Zealand, UK and two clinics from the USA. Patient advocacy websites in these countries (i.e. ACCESS, American Fertility Association, Resolve, Infertility Awareness Association of Canada, International Consumer Support for Infertility, Infertility Network UK) hosted the online survey. The clinic sample consisted of 291 women and 75 men, and the online sample consisted of 1014 women and 34 men. The Ethics Committee of the School of Psychology, Cardiff University approved the online study and the Internal Review Board of each clinic approved the clinic studies.
The Background Information Form covered socio-demographic status (e.g. age, education), medical history (e.g. current illness) and fertility-related characteristics (e.g. duration of infertility).
FertiQoL prototype: The FertiQol items were designed to translate abstract concepts (e.g. commitment, sense of belonging) into quantitative items that could collectively indicate the impact of fertility problems on QoL. Full details of item generation for the prototype are described in the Supplemental file and briefly presented here. As shown in Table I , item-generation involved four stages: generating potential items; eliminating redundant, irrelevant and outlier items; validation among people with fertility problems, and cross-cultural survey of acceptability and feasibility. A comprehensive literature review and consultation with psychosocial infertility experts generated an initial pool of 302 items on consequences of fertility problems on QoL in the following 14 areas (e.g. marriage/partnership, social network, emotions, cognitions, coping, treatment, physical health etc.). The authors classified the 302 items into three levels of increasing concept specificity: dimensions (e.g. interpersonal), domains (e.g. partner relationship) and facets (e.g. intimacy) to form groups of items tapping into related aspects of QoL. Classification and subsequent focus groups reduced this pool to 102 items, which were submitted to the acceptability and feasibility study (see Table I ). The prototype evaluated in the present study included these 102 Core items and 27 optional treatment items identified through the feasibility and acceptability phase.
FertiQoL was produced in English and translated into 20 languages: Arabic, Chinese, Croatian, Danish, Dutch, English, Finnish, French, German, Greek, Hindi, Italian, Portuguese, Romanian, Russian, Serbian, Spanish, Swedish, Turkish and Vietnamese (see www.fertiqol.org to download FertiQoL; Korean and Hungarian versions in progress). Cardiff University professional translators carried out the first translation, and two local fertility experts reviewed it to ensure that it was appropriate to local customs and fertility word usage. Cross-cultural data will be presented in a separate paper.
The items in the prototype FertiQoL survey were randomly presented and rated on a scale of 0 -4, where higher scores indicated more favorable QoL. The online survey (prototype FertiQoL and Background Information Form) was designed using SurveyTracker software for Training Technologies, inc and the paper version for clinic distribution was designed using InDesign. Webmasters were provided with a hyperlink to the survey. In clinics, FertiQoL coordinators at each site distributed the study pack to consecutive patients who returned completed surveys anonymously in a marked collection box in the patient waiting room.
Data were screened and duplicate internet protocol (IP) addresses were eliminated unless of different gender and response pattern. Descriptive statistics and correlations were used to identify the best items for each a priori domain of QoL (e.g. emotional, mind/body, relational and social). This a priori work was done to ensure that conceptually similar groups of items were entered into the factor analysis. Factor analyses (orthogonal rotation) were computed (clinic, online) to ascertain relations among these items. Items with factor loadings less than 0.30 and eigenvalues less than one were eliminated. The FertiQoL total and subscale scores were computed and transformed to scaled scores and summary statistics (e.g. reliability coefficient, mean and standard deviation) produced. Scaled scores were computed to achieve a range of 0 -100, making comparisons between scales easier. For scaling, items were reverse-scored (where necessary); all items then summed and multiplied by 25/k, where k was the number of items in the desired subscale or total scale. Higher scores mean better QoL. For the sake of brevity, only final analyses are shown here. These analyses generated the final FertiQoL, which composed 24 core items, plus 10 optional treatment items). See www.fertiqol.org for final FertiQoL in all languages and for scoring instructions. Table II shows background characteristics and these show that the Clinic group were older, and included more men, single women, same-sex couples and people with a university-education, but fewer American and UK residents and people living in rural/ suburban areas compared with the Online sample. The Clinic sample was more likely to have at least one child, a shorter duration of infertility but less likely to have other health problems.
Descriptive and inferential statistics were used to screen for problematic items. distribution, high inter-correlations (of .0.80 among item set), poor scale coherence, interpretive issues]. Other items were deleted because they measured broad constructs (e.g. self-esteem) that could be better captured by measures designed for that purpose and that, if retained, would confound associations with those measures in future research. The final FertiQoL item set submitted for exploratory factor analysis was comprised of 24 items from the core set of items and 10 items from the optional treatment module. The 24 core items were conceptualized as reflecting QoL in the emotional, mind-body (i.e. cognitive and physical), relational and social domains. The 10 optional treatment items were conceptualized as indexing treatment environment and treatment tolerability. An additional two items measuring satisfaction with QoL and physical health were retained for the FertiQoL measure to indicate general physical and QoL satisfaction, but were not included in the factor analysis. Wording for these items changed as a result of psychometric evaluation and participant feedback.
factor analyses. Table III shows factor loadings for the Online and (in parenthesis) Clinic samples for the Core FertiQoL and Optional Treatment Module domains. The first factor explaining item variance in the Core FertiQoL was the Emotional subscale explaining 31.8% (Online) and 37.8% (Clinic) of the item variability. Other factors (mind/body, relational and social) explained 10% or less of the item variance but all eigenvalues were above one. Loadings showed that items conceptualized to tap into the same concepts all had high factor loadings (.0.30) on their designated factor. Cross-loadings were observed for items of the mind/body (i.e. concentration, life on hold) and social domains (i.e. isolation, shame) onto the emotional domains. For the Optional Treatment Module, the first factor was Treatment Environment, explaining 34.0% (Online) and 38.0% (Clinic) of item variance. There were no cross-loadings for the Treatment Quality and Treatment Tolerability subscales. Table IV shows summary information for all FertiQoL scales. Core FertiQoL and Treatment FertiQoL were normally distributed and individual subscales were normally distributed (data not shown), with only the relational subscale showing mild positive skew toward more favorable QoL in this domain.
Potential moderators of QoL (gender, parenthood status and recruitment source) were examined in relation to FertiQoL scores. Women had a significantly lower Core FertiQoL ( The relationship between treatment subscales and six treatment persistence items (e.g. likelihood of trying further treatment, couple agreeing to persist, thinking of ending treatment) was also examined. Greater intention to persist with treatment was significantly associated with better Treatment FertiQoL (r(1026) ¼ 0.172, P , 0.001), especially in the Clinic sample (r(206) ¼ 0.289, P ,0.001).
It is currently accepted that to effectively measure the impact of disease, one needs a disease-specific instrument (WHOQOL, 1995) . FertiQol is a reliable and sensitive measurement tool for QoL in individuals with fertility problems. More than 2000 people with fertility problems contributed to the creation of FertiQoL, and it was developed using an integrated mixed-method approach that included literature reviews, international expert consultations, patient focus groups, a cross-cultural feasibility and acceptability survey, and a psychometric survey evaluation. FertiQoL comprises of a Core module evaluating the impact of fertility problems on emotional, mind-body, relational and social domains and an optional Treatment module evaluating treatment environment and tolerability. Subscales and total scales show mainly high reliability and sensitivity of contribute to future research and clinical endeavors aimed at investigating and ultimately improving QoL in people with fertility problems. Certain methodological limitations need to be taken into account. First, despite the multi-disciplinary contributions from experts worldwide, focus groups and a feasibility and acceptability study in 10 countries, the final psychometric evaluation only occurred in five English-speaking countries. Second, targeted efforts to recruit a diverse group of people were not entirely successful in recruiting particular subgroups (i.e. secondary infertile, men). Indeed, more psychometric research on men is required to fully establish reliability and validity. Third, the major proportion of the final sample was recruited online, and differences between the Online and Clinic sample were observed. Although, data generated online have been shown to be as valid as data collected through traditional methods (Bunting and Boivin, 2007; Lieberman, 2008) , one would need to determine whether the differences observed warrant a more in-depth analysis-for example, a different set of norms for clinic samples. We eliminated records coming from the same IP address but it may be possible that the same person replied more than once to the survey. Finally, the subscales of the Core FertiQoL were not entirely orthogonal with cross-loadings on the social and mind/body domains. While these associations are expected, we have now modified the final wording of four FertiQoL items to reduce these crossloadings. Further evaluation of these changes and FertiQoL as a whole on a new sample is required for final validation. These main limitations should be addressed in future psychometric research evaluating FertiQoL. However, the strengths of our mixed-method approach, and consultation and evaluation from infertile people ensure that FertiQoL captures the key life domains affected by fertility problems. It is hoped that FertiQoL will become a gold standard for the measurement of QoL for individuals experiencing fertility problems (whether in treatment or not).
FertiQol will be useful to clinicians and researchers alike. FertiQoL can be used to identify people at risk of impaired QoL so that psychosocial resources can be offered and subscale scores could identify the specific domains where intervention might be most beneficial. Recent research has shown a close correspondence between Core FertiQoL and standardized measures of anxiety and depression in a Dutch sample (Aarts et al., 2011) . The availability of FertiQoL in 20 languages will facilitate essential cross-cultural research, particularly in developing nations (Ombelet et al., 2008; van Balen and Bos, 2010) . However, whether cross-cultural differences exist, whether different populations have different mean scores and whether separate cultural norms are needed are all important questions that need to be addressed in future research.
A unique aspect of FertiQoL compared with other QoL measures is the optional 10-item treatment module. This module measures QoL in respect of treatment quality (interactions with staff, quality of information), and treatment tolerability (effects on mood, disruptions daily life). These subscales can be used to assess effectiveness of new treatments/medications, to monitor quality of services and to optimize patient treatment experiences. Research has shown that quality of treatment and its tolerability are predictors of treatment satisfaction (Dancet et al., 2010) and willingness to persist with treatment (Olivius et al., 2004) , the latter also shown in the present study. Further a recent large, multi-centered study showed a strong association between a high level of patient-centered care and favorable Fer-tiQoL scores (Aarts et al., 2010) . However, the sensitivity of Treatment FertiQoL for these purposes needs to be investigated in clinical trials of new interventions.
In conclusion, the overall aim of the FertiQoL project was to develop an international instrument to measure QoL in men and women experiencing fertility problems, with the collaboration of individuals experiencing fertility problems and international experts in the field. This objective was accomplished and future use of FertiQoL will be essential to establish FertiQoL as an essential measurement tool for practice, research, health service-evaluation and policy-making.
Supplementary data are available at http://humrep.oxfordjournals.org/. Long-term respiratory follow-up of H1N1 infection BACKGROUND: The first case of 2009 pandemic influenza A (H1N1) virus infection was documented in our Hospital on 10th August 2009. METDODS AND FINDINGS: Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) testing was used to confirm the diagnosis. All patients were treated with oseltamivir from the first day of hospitalization. Upon admission 12/44 had local patchy shadowing in their chest x-ray and additionally antibiotic regimen was added to these patients as pneumonia was suspected based on clinical evidence. In total 44 patients were hospitalized 15/44 had asthma, 6/44 COPD, 5/44 leukemia. Lung function was evaluated with forced vital capacity, forced expiratory volume in 1 sec and diffused carbon monoxide upon discharge and every 3 months, until 6 months of observation was completed after discharge. The purpose of this retrospective cohort study was to evaluate whether influenza A (H1N1) had an impact on the respiratory capacity of the infected patients. CONCLUSIONS: An improvement of pulmonary function tests was observed between the first two measurements, implicating an inflammatory pathogenesis of influenza A (H1N1) to the respiratory tract. This inflammation was not associated with the severity or clinical outcome of the patients. All patients had a mild clinical course and their respiratory capacity was stable between the second and third measurement, suggesting that the duration of respiratory inflammation was two months. Early treatment with antiviral agents and vaccination represent the mainstay of management. The first human infections with the new influenza A (H1N1) virus were confirmed in April 2009 in America, but the infection had been rapidly spreading around the world and in June 2009 World Health Organization (WHO) declared a pandemic [1] [2] [3] [4] [5] . WHO had advised countries in the northern hemisphere to prepare for a second wave of pandemic spread [6] . To date, almost all countries in the world have confirmed a second wave of H1N1 virus [7, 8] . For this reason a working group of WHO member states agreed on a pandemic influenza preparedness framework [9] . The reason for creating this framework began with the need for real time virological data exchange among laboratories in different countries upon a pandemic outbreak [10, 11] . The H1N1 virus has resulted from a triple recombination containing genes from human, avian, and swine influenza virus [12, 13] . The incidence, clinical characteristics and factors affecting the patients outcome of the first wave have been well described in previous published studies [14] [15] [16] [17] . However, while the numbers of positive H1N1 patients were lower, the hospitalisation rates were higher in comparison to the numbers and rates of 2009. In addition, the proportion of hospitalised cases admitted to intensive care units (ICU) was higher in 2010. The reason for these differences are not yet completely clear. There has been no obvious change in the severity of pandemic influenza A (H1N1) 2009 disease thresholds for hospital and ICU admission [8] . Three possible explanations were given. First, a new pandemic influenza A (H1N1) genetic variant predominated in the winter 2010 influenza season [7] . The Influenza A (H1N1) 2009 strain had undergone mutation in the hemagglutin. This mutation was associated with severe clinical outcome in comparison to the usual influenza A (H1N1) 2009 strain [7, 18, 19] . Secondly, resistance to oseltamivir developing from the first wave reached a peak in the 2010 pandemic [20, 21] . Thirdly, it has been reported that this year's seasonal vaccine effectiveness was moderate, suggesting an insufficient protective effect against the second wave of influenza A (H1N1) [22] [23] [24] [25] [26] [27] .
In this retrospective study, we evaluated the respiratory capacity of 44 patients admitted to the Unit of Infectious Diseases (UID) of our hospital from 10 th August 2009 to 15 th November 2010. These were followed with Forced Vital Capacity (FVC), Forced Expiratory Volume in 1 sec (FEV1) and Carbon Monoxide Diffusing Capacity (DLCO) for a period of six months after their discharge. The rationale for the study was published data that H1N1 presents with hypoxemia and acute respiratory distress syndrome (ARDS) [14] [15] [16] [17] 28, 29] . In addition, we examined the clinical characteristics of these patients.
Pharyngeal or nasopharyngeal swabs were taken upon admission in accordance with the protocol from the U.S. Centers for Disease Control and Prevention, as recommended by WHO and the average time between obtaining the samples and testing was 8-48 hours. The laboratory of the University Hospital of Alexandroupolis is a reference center for infectious diseases for more than 350.000 residents and one of the most important reference centers in Nothern Greece. Confirmed case was defined by a positive result of a real-time reversetranscriptase-polymerase-chain-reaction (RT-PCR) [30] .
The pandemic (H1N1) 2009 influenza virus is thought to spread from person to person in the same way as seasonal influenza, where transmission occurs predominantly through droplets produced from coughing or sneezing. Indirect transmission may also occur through self-inoculation after contact with surfaces or objects contaminated with the virus from infected persons. The incubation period for pandemic (H1N1) 2009 influenza virus is understood to be approximately four days (range: 1-7 days). The period of communicability is estimated to be seven days in uncomplicated cases; however, it may be longer in children (up to 10 days) and other individuals in whom symptoms and virus shedding may persist (i.e. immuno-compromised and severely ill). Consistent with seasonal influenza, transmission of the pandemic (H1N1) 2009 influenza virus is most likely during the initial days of infection when cases are typically symptomatic and have higher viral loads [31] .
The following isolation precautions were recommended for healthcare personnel who are in close contact with patients with suspected or confirmed 2009 H1N1 influenza. Close contact was defined as working within 6 feet of the patient or entering into a small enclosed airspace shared with the patient (e.g. average patient room). The standard precaution for all patient care was use of non-sterile gloves for any contact with potentially infectious material, followed by hand hygiene immediately after glove removal; use of gowns along with eye protection for any activity that might generate splashes of respiratory secretions or other infectious material.
Respiratory protection recommendation, according to CDC continues to be the use of respiratory protection that is at least as protective as a fit-tested disposable N95 respirator for healthcare personnel who are in close contact with patients with suspected or confirmed 2009 H1N1 influenza. This recommendation applies uniquely to the special circumstances of the H1N1 pandemic during the fall and winter of 2009-2010 and CDC will continue to revisit its guidance as new information becomes available, within this season if necessary.
The current recommendation is based on the unique conditions associated with the current pandemic, including low levels of population immunity to 2009 H1N1 influenza, availability of vaccination programs well after the start of the pandemic, susceptibility to infection of those in the age range of healthcare personnel, increased risk for complications of influenza in some healthcare personnel (e.g. pregnant women), and the potential for healthcare personnel to be exposed to H1N1 influenza patients because of their occupation [32] .
Respiratory evaluation with FVC, FEV1 and DLCO was determined according to a joint statement based on the previous statements from the American Thoracic Society and European Respiratory Society [33] . Patients were evaluated the day of their discharge and every three months until six months all follow up were complete for each one. The concept was to evaluate whether these patients had clinical presentation of adverse effects of the virus on their respiratory capacity and to establish the duration of these effects.
Analysis was carried out with the use of SPSS statistical software package (SPSS version 17.01; SPSS, Chicago, IL, USA). Continuous variables were expressed as mean ± SD or median (with interquartile ranges). For categorical variables, the percentages of patients in each category were calculated. Unpaired t-test was used in normally distributed parameters to compare the mean values between the two groups. A p value of less than 0.05 was considered to indicate statistical significance.
Mean patient age was 36 years (14-65). The majority of the confirmed patients hospitalized were Caucasian male (28/44). All patients were treated immediately with oseltamivir (75-150 mg every 12 hours). Treatment with oseltamivir was continued for 4 to 10 days depending on the individual patient's clinical condition. In 12/44 patients, empiric antibiotic treatment (azithromycin +amoxicillin+clavulanic-acid or ceftriaxone+quinolone) was given upon admission due to suspected secondary bacterial pneumonia elevated C-reactive protein, WBC count and local patchy shadowing on x-ray as reported in previous studies [34] [35] [36] . C-reactive protein and WBC count were elevated in these patients with local patchy shadowing on radiography (12/44 pts 27.2%).
Patients with radiological evidence of pneumonia were tested for serum procalcitonin (PCT) (36 patients in total) and Legionella/Streptococcus pneumoniae urinary antigens. Pneumonia score index (PSI) was applied and empirical antibiotic treatment was initiated according the treating physician's clinical judgment. Patient's procalcitonin test was within normal range (0.05-0.1) in all patients. Additionally, urine antigen results for Legionella and Streptococcus pneumoniae and blood culture that were drawn were also negative. The pneumonia score index was evaluated in these patients and the range was between class II to IV for 12/44 patients [37] .
Coexisting conditions were as follows: asthma 15 [38] ) was noted in 11/44 and morbid obesity (BMI ≥ 40 kg/m 2 ) was noted in 12/44 patients. Patient characteristics are presented in Table 1 .
No further pharyngeal or nasopharyngeal swabs were taken during hospitalization or after treatment. The criteria for discharge were absence of hypoxemia and temperature ≤ 37°C for 1 day without antipyretic treatment [17] .
Patient's respiratory capacity was evaluated with FVC, FEV1 and DLCO upon discharge and every three months until six months of observation was completed for each patient. The evaluation is demonstrated according to four categories which the authors selected based on the same underlying disease of the patients: a) chronic obstructive pulmonary disease, b) asthma, c) comorbidities (diabetes mellitus, coronary heart disease, and cancer), d) previously healthy patients. Whenever a patient had both an underlying respiratory disease and co-morbidity he was selected to be included in a group according to his major factor affecting his clinical outcome, preferably his underlying respiratory background. There was no association between radiographic data and pulmonary function tests. The mean values for FVC, FEV1 and DLCO are presented in Figures 1. A significant difference was observed between mean values of FEV1, FVC and DLCO between the evaluations upon discharge and the sixth month follow up ( Table 2 ).
We described a retrospective cohort study of 44 patients who were hospitalized with the pandemic 2009/10 Influenza A (H1N1) in the University Hospital of Alexandroupolis, Thrace from 10 th August 2009 to 15 th November 2010. So far, there has been no evaluation of H1N1 respiratory function. The correlation between the mechanisms and the impact that viruses have on the respiratory system has been established in previous published studies [14] [15] [16] [17] 28, 29, [39] [40] [41] [42] [43] [44] . As might be anticipated, we found a major decreased respiratory capacity (FEV1 -10.205, FVC -9.909, DLCO -12.409) between the measurements taken upon discharge and in the sixmonth follow-up. This occurred due to the inflammatory effects of the virus through the respiratory tract. The differentiation of respiratory capacity also varied between the measurements taken upon discharge and the three-month follow-up, but it was observed that the variation was smaller in comparison to the sixth-month visit, which is possibly due to the time needed for the resolution of the inflammatory effects.
Patients with previously diagnosed respiratory disease such as COPD and asthma were well controlled on their treatment. H1N1 induced inflammation and was the factor that dysregulated their condition. WBC count and CRP were elevated in patients with suspected bacterial infection. Hypoxemia was more severe in patients with opacities on chest x-rays. The mean values in 7/44 patients with abnormal liver functions did not exceed 138 IU/L for ALT and 125 IU/L for AST. These patients did not present jaundice or other clinical signs of liver dysfunction. Patients with BMI ≥ 30 kg/m 2 were 20/44, implicating a mild clinical course overall, since BMI ≥ 30 is considered an adverse prognostic factor. The laboratory findings were not correlated with the respiratory functions, based on the fact that patients upon exacerbation or with opacities on chest x-ray would present a severe deterioration of their respiratory capacity. None of the patients had been previously vaccinated, but all patients received oseltamivir immediately on hospitalization and antibiotics upon suspicion of bacterial respiratory infection preventing an escalation of the inflammation and infection. The mechanisms of H1N1-induced respiratory effects have received considerable attention [39] [40] [41] [42] [43] [44] . Recent studies have shown that the high mortality rate of avian influenza virus infections is a consequence of an overactive inflammatory response and the severity of infection is closely related with virus-induced cytokine dysregulation. The most important feature of influenza A immune-pathogenesis is the appearance of "cytokine storm", which is characterized by the extreme production and secretion of numerous pro-inflammatory cytokines. This is responsible for the development of lethal clinical symptoms, such as massive pulmonary edema, acute bronchopneumonia, alveolar hemorrhage, reactive hemophagocytosis, and acute respiratory distress syndrome, associated with necrosis and tissue destruction. and IFN-gamma. It has been detected that mutations of some viral genes (NS1, PB2, HA and NA) are responsible for the "cytokine storm", by increasing the viral replication rate, expending the tissue tropism, as well as facilitating the systemic invasion and the development of resistance against the host antiviral response. Glu92 and Ala149 mutations and carboxyl-terminal ESEV/ EPEV motif of NS1 protein have been implicated as determinants of virulence for A/H5N1 strains. In addition, Lys627 mutation in PB2 protein, polybasic aminoacid mutations in the cleavage region of hemagglutinin (HA) polyprotein, and glycosylation and sialyzation mutations in HA and neuraminidase (NA) proteins were found to enhance the immune-mediated pathology of highly virulent A strains [40] [41] [42] [43] [44] [45] . Furthermore, in a preliminary analysis of macrophage gene expression data based on published studies, 64 genes in H1N1-infected cells showed at least 1.5-fold difference in expression level compared to mock infected cells in at least one time point [43] . Impressively, as many as 60 genes were thereby upregulated [43] .
Given these important inflammatory processes, it is important to attempt patient prophylaxis. Several antiviral agents were tested both in vitro and/or in vivo and presented results implicating that the early use of such agents suppress the inflammation apart from being a therapy [43] [44] [45] [46] [47] [48] [49] Antibiotics especially macrolides, which are well known for their anti-inflammatory and immunomodulatory properties were also tested. Clarithromycin inhibits the middle to late stage of the influenza virus replication cycle, resulting in inhibition of progeny virus production from the infected cells. Macrolides could mediate this effect by inhibiting intracellular hemagglutinin HA0 proteolysis. The inhibitory effect on influenza virus replication of macrolides has been known since the 80s [50] [51] [52] [53] [54] . N-acetylcysteine (NAC) is a thiol-containing compound which non-enzymatically interacts and detoxifies reactive electrophiles and free radicals. NAC has already been shown to effectively protect human bronchial fibroblasts against the toxic effects of tobacco smoke condensates and the isolated perfused lung against the glutathione (GSH)-depleting effect of tobacco smoke. NAC has also been demonstrated to reduce the reactive oxygen intermediate hydrogen peroxide (H 2 O 2 ) and to confer protection from the toxic effects of H 2 O 2 . In vivo studies, however, have demonstrated that orally administered NAC has very low bioavailability due to its rapid metabolism mainly to GSH but also other metabolites. Thus, even though NAC is very effective in protecting cells of different origins from the toxicity of reactive components in tobacco smoke and reactive oxygen species, a direct scavenging effect by NAC in vivo, does not seem likely. This holds especially true for oral administration. A more relevant mechanism in vivo for any protective effect of NAC may be attributable to its activity as a precursor of GSH, thereby facilitating its biosynthesis. GSH will then serve as the protective agent and detoxify reactive species both enzymatically and non-enzymatically [55, 56] . Finally, the efficacy of vaccination for H1N1 has been documented by assessing antibody formation in the vaccinated elderly subjects from the 2009 pandemic in contrast to the younger ones who were not vaccinated. Nonetheless, antibodies from older subjects previous encounter with pandemic influenza A strains may be a confounding factor [57] .
This study has a number of limitations. First, our patient series was small, but it should be borne in mind that since these H1N1 positive patients represent a population of 350.000 residents. Furthermore, diagnostic tests with procalcitonine, urine antigen and blood cultures were given only upon suspicion of an infection. The respiratory capacity tests represented a small number of patients in each group, and so no correlations could be preformed between the four subgroups. In addition, the baseline condition of each patient was not evaluated; this was either because they did not have a respiratory underlying condition or because they were patients that admitted in our hospital for the first time.
Respiratory capacity differed between categories of patients at least for the first three months of observation, probably due to the inflammatory factors that are released with the influenza A (H1N1) infection and underlying disease. Importantly, the respiratory inflammation lasted almost two months, as evidenced by the fact that respiratory capacity remained stable between the second and third measurement. Several treatment modalities can be administered to confer protection and suppress the inflammation. Vaccination and early treatment with antiviral agents represent the mainstay of management. Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate. We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight. Milk is a hallmark of mammals, providing the primary source of nutrition for their young until weaning. It is an emulsion of fat globules in a water-based fluid. The major proteins of this fluid are the caseins. The caseins are serine rich phosphoproteins which are almost exclusively expressed in the lactating mammary gland [1, 2] . In cows the casein proteins together constitute up to 80% of total milk protein and also 80% of total mRNA in the lactating mammary gland [3] . The caseins are members of a large family of serine rich phospho-proteins, which are clustered on chromosome 5 in the mouse (chromosome 6 in cattle, chromosome 4 in humans). There are 5 functional casein genes in the mouse whereas most other species (including ruminants) only express 3 or 4 functional genes [1] . The casein proteins show little homology with each other outside their signal peptide domain [2, 4] .
Inactivation of the b-casein gene has shown to have little effect on milk secretion and growth of the offspring in the mouse [5] . In contrast, deletion of the k-casein gene in mice completely abrogates milk production [6] . Exactly why this happens is unclear as neither the structure of the individual casein proteins nor the structure of the casein micelles is currently known in detail [7, 8, 9] . The available results, however, support a model in which k-casein (the casein protein which is present in milk in least abundance) plays a critical role in ensuring transport and solubility of casein micelles [7] . The sequences of a-(aS1 in ruminants) b-, and ccasein (aS2-casein in ruminants) contain one or more clusters of phosphorylated residues known as phosphate centres. Phosphate centres can sequester amorphous calcium phosphate, probably in the Golgi vesicles of mammary secretory cells, to form thermodynamically stable complexes of defined chemical composition which are then secreted through the apical membrane of the mammary epithelial cells [10, 11, 12, 13, 14] . Milk, like many other biological fluids, is supersaturated with respect to the crystalline mineral of bones and teeth (apatite) but due to the sequestration reaction, is under-saturated with respect to amorphous calcium phosphate. Since apatite only forms by maturation of the amorphous phase of calcium phosphate, it cannot form at all from milk and this helps to protect the mammary gland against soft tissue mineralization [15] . In most milks, the majority of the total calcium is sequestered within the casein micelles. In milks of different species, the total calcium and total casein concentrations are highly correlated [16] and provided this balance is maintained, the milks remain stable. If there is insufficient casein to sequester the secreted calcium and orthophosphate then the milk becomes unstable [15] . In mouse milk, 3 of the phosphate centres are in present a-casein and only one in b-casein. Thus the loss of a-casein is likely to have more serious consequences for milk stability than the loss of b-casein.
Deficiencies for b-casein and a-casein exist as naturally occurring genotypes in goats [17] but have no apparent detrimental effect on milk production. However, the aS1-Cn0 allele has been reported to decrease the efficiency with which other casein proteins are secreted [18] . We set out to define the role of acasein in milk secretion and its nutritional role by inactivating the corresponding gene in mice. Our results demonstrate that a-casein deficiency has a significant effect on milk protein secretion and growth of the offspring.
DNA PCR amplifications were done using Taq Polymerase from various suppliers. Oligonucleotides were purchased from MWG, Sigma-Genosys or Invitrogen. Primer sequences, amplicon size and annealing temperatures are given in table 1. Template DNA for PCR analyses was isolated as described [19] .
The targeting construct for the a-casein gene was generated using a short arm of homology of 679 bp corresponding to nucleotides 208 to 887 downstream of the transcriptional start site (which was isolated as a EcoRV/BssHII fragment from the bacterial artificial chromosome BAC 490H23 of a Research Genetics BeloBAC11 library derived from 129SV mouse DNA) and a long arm of homology of 6696 bp corresponding to nucleotides 1422 to 8117 downstream of the transcriptional start site (isolated as a StuI/EcoRV fragment). The targeting construct carries a hygromycin-thymidine kinase fusion gene under the control of the murine phospho-glycerol kinase (PGK) promoter as selection marker.
Transgenic mice were generated at Genoway (Lyon, France) as a SV129xC57BL/6 mixed background. After transfer to the transgenic mouse facility at the Roslin Institute the mice were maintained there in accordance with Home Office guidelines. This Table 1 . Primer combinations used for PCR analysis. 
Before establishing the mating couples, mice were kept in samesex groups of 2 to 6 littermates. The animals were kept in opaque M3 type cages with dimensions of 48(15(13(cm from North Kent Plastics, UK equipped with bedding ('Eco-bedding', B&K Universal Ltd, UK) and nesting material ('Nestlet', Datesand Ltd, UK). Cages were changed once per week and in case of the nursing females the nesting material was transferred to the new cage. The environment was kept at 21+/(2(C and 55+/(15% relative air humidity with a 12(hour light 12(hour dark cycle (lights off 8:00 pm) Food (BeeKay Transgenic Rodent Diet BK021E, B&K Universal Ltd, UK) and tap water were available ad libitum, water bottles being routinely changed twice per week.
Apparently pregnant females were separated from the male and checked daily in the morning for pups in the cage. The first day pups were found was considered the day of birth (day 1). On this day the pups were marked individually by foot pad tattooing and checked for the presence of a milk spot and signs of lack of maternal care. The offspring of 3(wt females were fostered onto 3 (-casein deficient females and vice versa on day 2 (Table 2) , the cross fostered litters being matched for day of birth. The pups were weaned on day 23 by separating them from the nursing female and placing them in cages as described above with same-sex litter mates. From weaning until the age of 6 weeks, water-soaked diet was provided ad libitum in Petri dishes on the cage to weaned pups in all groups to facilitate solid food intake by growth impaired animals.
Pre-weaning mortality was low in all groups and unaffected by dam genotype. One wild type female had 1 pup stillborn and 2 born alive but lost between day 1 and cross fostering. Two acasein deficient females lost 2 pups and one pup respectively between day 1 and cross fostering. No pups were lost from cross fostering until day 21, but on day 22 two pups were found dead in the largest litter (11 pups before day 22) nursed by an a-casein deficient female.
All monitoring took place in a testing room adjacent to the housing room and with similar environmental conditions. The entire housing cage was moved from the housing room to the monitoring room. Physical and social aspects of mouse housing are described in detail in table 3.
The pups were individually weighed and monitored for general behaviour and reactivity, health, sensory and muscle development, and reflexes on day 1, 3, 7, 14 and 21 of age following a protocol based on pre-weaning development stages and adapted from [20] and [21] . Additionally, from day 13 onwards pups were weighed every second day and screened daily to detect the day when both eyes were opened. During scoring the adult female was removed from the home cage and placed into an empty opaque cage, food and water being provided. Specifically for the respective monitoring days, the following records were taken: Day 3: presence/absence of a milk spot and the position of the animals regarding the nest (inside/outside). Righting reflex: the pup was positioned in dorsal recumbence and the time it took to turn to a ventral position was taken with a cut off time of 60 sec.
Day 7: presence/absence of a milk spot and the position of the animals regarding the nest (inside/outside). Presence/absence of the first fur was recorded.
Day 14: Pups were inspected for the presence of a fully developed fur and the opening of the eyelids, auditory canal and the presence/absence of the incisors. Grip reflex: The animals were stimulated at their 4 paws with a cotton swab and the reflex reaction (closing of paws) recorded.
Day 21: At the originally scheduled weaning day (day 21), the home cage was placed into the testing room for at least 10 minutes. After this the pups were weighed and placed individually into an empty opaque cage (observation cage; similar to the housing cage as described above) for close inspection. There each animal was screened for apparent deviations from fluid gait, horizontally extended tail position, normal body position (e.g. hunched back), normal body proportion (e.g. lumps) and normal respiration while moving freely in the cage. While being restrained in a dorsal position, signs of dehydration (lifting a skin flap between index finger and thumb), general fur condition (smoothness), the presence of wounds, trimmed whiskers, presence of discharge in nose and the anogenital region, general eye condition and skin colour were recorded. Muscle tonus in fore/hind legs and abdomen was recorded by applying slight pressure with the index finger of the free hand.
After the assessment under restraint, the following tests were performed (adapted from [20] and [21] ): Provoked biting: To assess reflexive biting reaction, the animal was stimulated with a cotton swab at the muzzle followed by a check for conspicuous teeth and mucosa condition. Grip strength: To assess the ability of the animal to hold its own bodyweight, the individual was placed on a cage lid which was then turned upside down. Animals being able to hold on for at least 15 sec. were recorded as successful. Vertical pole test: As a test of motor coordination and balance the animal was placed on a horizontally positioned pole (80 cm long, 1.55 cm diameter, taped) which was erected slowly to vertical position. Animals falling off before reaching vertical position were recorded. Postural reflex: The animal was placed in an empty observation cage which was then gently shaken 3 times vertically and 3 times horizontally. The presence/absence of the natural reaction to this treatment (extending all 4 limbs) was recorded.
Thereafter, while still being in the observation cage, the senses of touch and of hearing were measured. First a sudden airflow was 
The animals were weighed weekly after weaning and at 8 weeks of age the animals were individually screened using a protocol adapted from [22] , including parameters on health, general behaviour and reactivity, reflexes and muscle strength.
First the animal was placed into a transparent Perspex cylinder (height 18 cm, Ø 15 cm) and observed regarding general activity, respiration and occurrence of tremor. Then the animal was transferred into a Makrolon IV cage (42630627 cm, Allentown) and deviations from normal palpebral closure, piloerection, gait, pelvic elevation, tail elevation, and positional passivity were recorded. Touch escape response was assessed by slowly approaching the mouse with a finger sideways and attempting to stroke it.
In tail suspension, trunk curl and limb grasping were recorded with the mouse hanging and body tone, corneal/pinna/toe pinch reflex were observed while the animal was placed on a cage grid. Efficiency of grip was assessed by gently pulling the animal back by the tail. A wire manoeuvre was performed by letting the mouse hold on to a horizontal wire (length 45 cm, Ø 3.1 mm) with the forelimbs while being held by the tail and releasing it from a horizontal position.
Under supine restraint, skin colour, deviations from normal heart rate, limb/abdominal tone, lacrimation, salivation and signs of fear, irritability and aggression were recorded. Provoked biting response was tested as described above. A negative geotaxis test was performed by placing the animal on a horizontal grid and turning the grid to vertical position with the head facing down.
Milk was isolated from mammary tissue at peak lactation (day 10 of lactation) after cervical dislocation of the mice. The milk was harvested using a Pasteur pipette which was put onto the nipple while pressure was applied to the tissue. Milk was drawn up into the pipette transferred into a microcentrifuge tube and stored at 280uC.
After thawing at room temperature, milk samples were well suspended, diluted 1 in 4 in distilled water, and centrifuged for 5 sec. at 200 g in a table top centrifuge. The lower phase containing the defatted whole milk was removed from the upper lipid layer into a new tube and diluted with 2 volumes of distilled water. Then one volume of a 46 concentrated protein sample buffer was added. To separate the whey and casein fractions, defatted whole milk was spun for 15 min. at 200 g. The supernatant containing the whey fraction was diluted with 2 volumes of distilled water and one volume of a 46 concentrated protein sample buffer. The pellet containing the casein fraction was re-suspended in an equivalent of 3 volumes (of starting volume) of distilled water and then one volume of a 46
Score Definition Table 3 . Cont.
a-Casein Deficient Mice concentrated protein sample buffer was added. The samples were separated on a 10% polyacrylamide gel and stained with Coomassie Blue. Milk protein expression visible in the stained gel was then analysed on a Kodak Imaging Station using the Kodak 1D imaging software. Total protein extracts from tissues were isolated as described [23] . Briefly tissue fragments were dounced on ice at 100 mg/ml in a buffer containing 1% SDS, 1% NP-40 and 0.5% deoxycholic acid in 16 PBS supplemented with a broad spectrum proteinase inhibitor mix (SIGMA P-8340). The lysates were incubated on ice for 30 min. and then centrifuged at 4uC for 10 min. at 10000 g. The supernatants were then aliquoted and stored at 280uC.
Cytoplasmic protein extracts for the analysis of caspase activity were prepared as described [24] . Briefly, tissue fragments were dounced on ice in a buffer containing 25 mM KPO 4 pH 7.8, 8 mM MgCl 2 , 1 mM EDTA, 1% Triton X-100 and 15% glycerol. The extracts were incubated on ice for 5 min. and then centrifuged at 4uC for 1 min. at 10000 g. The supernatants were then aliquoted and stored at 280uC.
Western blots were done after semi-dry transfer of the proteins to a nitrocellulose membrane as described [25] . Mouse a-casein protein was detected using a rabbit-anti a-casein antiserum (Santa Cruz Biotechnology sc-98699) and a horse-radish-peroxidase linked goat anti-rabbit serum (Cell Signalling Technologies #7074). Mouse b-casein protein was detected using a goat-anti b-casein antiserum (Santa Cruz Biotechnology sc-17969) and a horse-radish-peroxidase linked rabbit anti-goat serum (Jackson Immuno-Research). Mouse grp78/BiP protein was detected using a goat-anti grp78/BiP antiserum (Santa Cruz Biotechnology sc-1051) and a horse-radish-peroxidase linked rabbit anti-goat serum. All antibodies were used at a dilution of 1:1000.
Milk proteins were separated on a 15% polyacrylamide gel and bands were excised manually. The proteins represented by these bands were trypsinised using a protocol of the Micromass MassPrep Station (Micromass Ltd, Manchester, UK) and analysed by electrospray LC-MS methods as described previously [26] .
Calcium and phosphate concentration were measured in milk derived from wild-type, a-casein deficient [2/2] and heterozygous [+/2] mice using a Konelab 30 Clinical analyser (Thermo Scientific) using the Konelab calcium (catalogue number 981367) and phosphorus (catalogue number: 981386) analysis kits as recommended by the supplier.
RNA was isolated from tissues using the Ambion RNAwiz reagent following the supplier's protocol (1 ml of RNAwiz per 100 mg of tissue). Reverse transcription of RNA was done with MLV RNAse(-) reverse transcriptase (Promega) following the suppliers recommendations. 2 mg of total RNA was used as template for the cDNA synthesis reaction. A one in 10 dilution of the reaction was subsequently used as template for quantitative PCR (Applied Biosystems 7500 Fast System).
Quantitative PCR amplifications were done with a final primer concentration of 0.5 mM. Primer design was done using the Primer Select program of the DNA Star software suite. The sequences, annealing temperatures and amplicon sizes of the oligonucleotides used in this study are provided in Table 1 . The PCR products obtained by quantitative PCR were evaluated by melting point analysis and agarose gel electrophoresis. Amplifications were done at 40 cycles of 15 sec. at 95uC, 15 sec. at the indicated annealing temperature and 30 sec. at 72uC. Data were collected at the end of each PCR cycle. Standard curves for all genes were generated from serial dilutions of a plasmid containing the cDNA for each gene. The crossing points obtained from the sample analysis was then correlated with the standard curves to provide a concentration of the individual PCR product. Expression of the casein genes was then correlated with expression of the reference genes (b-actin or GAPDH) in the same sample (expressed as pg of gene per pg of reference).
PCR arrays were carried out using a mouse apoptosis array (SABiosystems: catalogue number PAMM-012) following the recommendations of the supplier.
Tissues were collected from mice at day 10 of lactation,after schedule 1 cervical dislocation. The left lower mammary gland was dissected out and placed in 4% para-formaldehyde overnight at 4uC on a rocking platform. The para-formaldehyde was then removed and the tissues were washed, first, in PBS for 30 minutes at 4uC, then in 30% ethanol for 15 min. at room temperature. This was followed by two 30 min. washes in 70% ethanol at room temperature. The tissues were then transferred to fresh 70% ethanol until ready for processing for histology. The tissues were dehydrated through a series of alcohol dilutions followed by penetration by wax under vacuum (in a Shandon Hypercentre). The tissues were then embedded in wax blocks for sectioning. Each sample was sectioned at three intervals at least 100 mm apart and a 5 mm section mounted for immuno-histochemistry analysis at each interval.
Sections were de-waxed using Histo-clear (Lamb, Inc.) for 5 min. and then re-hydrated in a graded series of ethanol (100%, 95%, 70%; 2 min. per incubation). The slides were then washed in deionised water and 16 PBS (two 5 min. incubations). The endogenous peroxidase activity was blocked by a 5 min. incubation in 3% hydrogen peroxide. After two 5 min. washes in PBS the slides were blocked in 1% BSA in 16 PBS-Tween for 20 min. After addition of the primary antibody (rabbit-anti acasein antiserum; 1:100 dilution in 16PBS-Tween) the slides were incubated at 4uC overnight. The slides were then washed twice for 5 min. in 16 PBS and the secondary antibody was added (horseradish-peroxidase linked goat anti-rabbit serum; 1:1000 dilution in 16 PBS-Tween). After a 30 min. incubation at room temperature the slides were washed twice for 5 min. in 16PBS-Tween.
Horseradish peroxidise activity was detected using a diaminobenzidine dye-kit (Vector-Laboratories). The slides were incubated with substrate for 5 min. The reaction was stopped by washing the slides with distilled water.
The slides were then counterstained with Haematoxylin (Cell-Path) for 2 min. and washed in running tap water. The slides were then de-hydrated in a graded series of ethanol (70%, 95%, 100%; 2 min. per incubation), cleared in Xylene (Fisher Biochemicals) and coverslips were mounted in Pertex (Cell-Path). Following staining the sections were photographed using a Leica Stereomicroscope at a 20 fold fold magnification.
Mouse RAW264 cells (ECACC catalogue number 85062803) were grown in DMEM (4.5 g/l glucose) supplemented with 10% foetal calf serum, 2 mM glutamine and penecillin/streptomycin. To induce apoptosis the cells were treated with 10 mM staurosporin for 6 h. Cytoplasmic extracts for the analysis of caspase activity were prepared as described in section ''milk analysis'' above. Protein concentrations were measured using a Bradford assay (Sigma). Caspase activity of cell extracts was measured using the Caspase-3/7-Glo assay (Promega) as recommended by the supplier. The assay provides a pro-luminescent caspase-3/7 substrate, which contains the amino acid sequence DEVD in a reagent optimised for caspase activity, luciferase activity and cell lysis. Presence of caspase 3 or 7 in the cell extract leads to cleavage of the substrate and light emission which is detected in a luminometer (Berthold). Three or more parallel samples were analysed to measure caspase activity. 50 ml of protein extract (from cells and tissues) were mixed with 25 ml of the Caspase Glo reagent and incubated at room temperature for 20 min. after which the readings were taken. Apoptosis in RAW cells was also confirmed by measuring cell viability using the Cell-Titre Blu reagent (Promega) and DNA laddering on a 2% agarose gel.
Categorical data measured only once were analyzed using the Fisher's exact test (2-sided) . Count data were analyzed using the Mann Whitney U test. For comparison of body weight development we fitted a linear model for weight using time and group as covariates. Litter size was also tested as a covariate but no significant effect was observed and it was removed from the model. The interaction between time and treatment was included in the model so each treatment would have its own growth rate. We also considered a spline at day 23 (weaning threshold). The spline allows the growth rate to change after the day 23 for each treatment. This way we are able to test differences of the growth rate between treatments before and after weaning. The model was fitted using generalized estimation equations (GEE) with compound symmetry for the working correlation matrix because of the repeated measurements for weight. The comparisons of the average growth rates between the three groups were computed using the covariance matrix of the model coefficients. The data was analyzed using SPSS version 16 
In order to inactivate the a-casein gene (Fig. 1a) a targeting construct was established based on previous experience with constructs used to inactivate the band c-casein genes [5, 27] . The targeting event removes the complete second exon which includes the translational start codon and the 15 amino acid signal peptide (Fig. 1b) . In 3 independent transfections into ES cells the targeting frequency was found to be around 3%. Successfully modified ES cells clones were identified using a PCR analysing the 59 end of the targeted gene (Fig. 1c) . The targeting event was then confirmed by Southern blot analysis assessing the 59 end of the targeted a-casein gene (Fig. 1d) . The integration event was also confirmed using PCR analysis and Southern blotting with primer combinations and probes specific for the 39 end (Fig. 1e) . Two targeted cell clones were grown up and injected into blastocysts. Animals carrying a homozygous deletion of the a-casein gene are phenotypically normal prior to lactation. Fig. 2a alongside a wild-type control milk sample. Milk from control animals displayed a series of protein bands, with the most prominent proteins corresponding to molecular weights previously observed for a-, band c-casein (with apparent molecular weights of 42, 30 and 25 kDa, respectively) [28] . The identity of the protein bands was confirmed using mass-spectrometry. Table 4 details the data for the 8 most prominent protein bands indicated in Fig. 2c . These findings were also supported using Western blot analyses for a-casein and b-casein ( Fig. 3a and 3b ). Milk from acasein deficient mice shows some characteristic protein bands which are absent from milk of wild-type or heterozygous mice (Fig. 2d) . Mass-spectrometry analysis was used to identify some of these proteins. The major additional band with an apparent molecular weight of 78 kDa was identified as grp78/BiP. BiP is an ER resident protein which is involved in the assembly of multi-protein complexes like antibodies [29, 30] . The identity of the grp78/BiP protein was confirmed by Western blot analysis (Fig. 3c) . In addition two further ER-resident proteins, grp94 and PDIA6 (protein disulfide-isomerase associated protein 6), were detected in milk of the a-casein deficient mice (Fig. 2d, table 4) .
Milk protein abundance was then analysed quantitatively using a Kodak imaging system (Fig. 3d and e) . In heterozygous mice the amount of a-casein protein is reduced by around 50% when compared to albumin (Fig. 3e) . In contrast, the relative amounts of b-casein and c-casein compared to albumin remain constant. In the milk derived from homozygous a-casein deficient mice, no acasein protein can be detected (Fig. 2a, 3a and 3d ). In addition, the amounts of band c-casein are also significantly reduced (Fig. 2a,  3d and e) . Whereas the ratio of b-casein to albumin in control milk is 2:1, the ratio in milk from homozygous a-casein deficient mice is 1:1 indicating at least a 50% reduction in b-casein protein concentration in milk (Fig. 3d and e) . This reduction is also detected by Western blot analysis (Fig. 3b ) Analysis of the data using one-way ANOVA demonstrated that the changes in the concentration of band c-casein in the milk of a-casein deficient mice are highly significant (p.0.001; Table 5 ). The concentration of milk proteins (Fig. 3d) is consistent with published data [5] .
In a-casein deficient milk the reduction in protein secretion also affects the mouse WAP protein, which is secreted into the whey fraction of milk (Fig. 2b) . This suggests that all milk proteins secreted from mammary epithelial cells are affected by the absence of a-casein. In contrast, secretion of albumin, which is derived from serum, is not affected by the absence of a-casein (Fig. 2a) . Furthermore, the overall amount of milk secreted by a-casein deficient dams appears to be reduced (observation during harvesting). Thus, a-casein deficiency causes a generalised reduction in milk secretion and a reduction in milk protein concentration.
The presence of the ER proteins grp78/BiP, grp94 and PDIA6 in milk of a-casein deficient mice is unexpected. We therefore analysed whether the grp78/BiP, grp94 and PDIA6 proteins are up-regulated in lactating mammary tissue of a-casein null mice using protein gel analysis (Fig. 4a) and Western blotting (Fig. 4c) . In total protein extracts derived from mammary tissue it is evident that grp78/BiP and grp94 expression is indeed up-regulated in response to the absence of a-casein. In contrast, the PDIA6 protein can not discerned in Coomassie stained gels of mammary protein extracts, as it co-localises with other proteins of similar molecular mass. Grp78, grp94 and PDIA6 are all ER resident The number of amino acids (aa) and the expected molecular weight (MW) are given for the mature proteins (i.e. without the signal peptide) where appropriate. In addition, the reported molecular weights for the mouse caseins [28] and the molecular weights observed in the SDS-PAGE analysis are shown. The MS score is a measure of confidence of the detected protein species. A score higher than 39 is significant. The number of peptide sequences and the fraction of the total protein covered are also indicated. Protein bands #1-8 are the predominant protein species in the milk of wild-type mice. Protein bands #9-13 are detected in the milk of acasein deficient mice but not in the milk of wild-type or heterozygous mice. L-AAO-1: L-amino acid-oxidase1, PDIA6: protein disulfide isomerase associated 6. doi:10.1371/journal.pone.0021775.t004
proteins which are not normally secreted from the cells but act as chaperones, aiding protein folding. Their up-regulation is typically associated with ER stress [31, 32, 33] . This suggests that the lack of a-casein protein in mammary epithelial cells induces ER stress. Analysis of gene expression rates of grp78, grp94 and PDIA6 confirms that all three genes are significantly up-regulated in tissue samples of a-casein deficient mice with respect to control mice and heterozygous mice (Fig. 4d) . In contrast, another protein often upregulated under conditions of ER stress, REDD1 [34] is not significantly regulated by the absence of a-casein (Fig. 4d) , Several other protein bands with apparent molecular weights of 28, 27 and 22 kDa were identified in milk from a-casein deficient mice (Fig. 2d) . Mass-spectrometry analysis confirmed that these proteins are breakdown products of band c-casein (Table 4 ). The lower molecular weight b-casein products are also detected by Western blot analysis of milk samples derived from a-casein deficient mice (Fig. 3b) . Analysis of protein extracts from mammary gland tissue suggests that the breakdown products are the predominant intracellular forms of b and c-casein ( Fig. 4a and  b) but are not secreted as efficiently as the full length protein. The caseins are important for ion transport, mainly calcium and phosphate [35, 36] . Not surprisingly we therefore find that the lower casein protein levels in a-casein deficient mice are accompanied by a significantly lower concentration of calcium and phosphate in milk (reduced by around 90% compared to milk derived from wild-type animals; Fig. 5a ).
We analysed the expression of casein specific mRNAs in the lactating mammary gland from control and a-casein deficient mice (Fig. 5b and c) . Expression of the casein genes was correlated with expression of the reference genes GAPDH (shown in Fig. 5b and c) and b-actin (Fig. 5d) . Identical results were obtained for both reference genes. As expected the amount of a-casein specific mRNA in heterozygous mice is at 50% of control levels and absent in homozygous knock-out mice. Expression of the corresponding b-casein and c-casein casein genes is significantly reduced in acasein deficient mice (to around 10% of the expression levels detected in wild-type and heterozygous mice; Fig. 5b, c and d) . This suggests that the absence of the a-casein protein not only interferes with protein secretion from the mammary epithelial cells but that absence of both alleles of the a-casein gene also reduces the expression of the other casein genes. The expression of the kcasein gene is also affected but not to the same extent. Its expression is reduced to around 20% of control levels in a-casein deficient mice (Fig. 5c and d) . No consistent reduction of b-casein, c-casein and k-casein expression is detected in the heterozygous animals. Fig. 5d presents the expression of the casein genes as percentages of wild-type expression. The consistent reduction of b-, cand k-casein gene expression in the a-casein deficient mammary tissue is highly significant (p,0.001; table 6) whereas expression changes in heterozygous vs wild-type mice are marginally or not significant (all p values.0.01; table 6). Histological analysis of mammary tissue in a-casein deficient mice
In order to assess whether the reduction in protein and RNA expression is correlated with overall morphological changes, sections of mammary tissue were obtained from animals at peak lactation and analysed by haematoxylin/eosin (H&E) staining and immuno-histochemistry (Fig. 6 ). As expected a-casein expression was clearly detectable in tissue from wild-type control mice and heterozygous mice (Fig. 6a) . In contrast no a-casein protein could be detected in sections of a-casein deficient mice (Fig. 6a) . As expected sections analysed with pre-immune serum did not show any a-casein specific staining (Fig. 6a) . No gross morphological alterations were detected in the sections of the different genotypes (Fig. 6b) . This suggests that the absence of a-casein, although critical for overall milk composition, does not significantly impact on the survival of the mammary gland. To address this question further we measured the expression and activity of capase proteins in the mammary gland. Firstly we assessed the presence of cleaved caspase 3 in the protein extracts derived from control mice, heterozygous mice and a-casein deficient mice. No cleaved caspase 3 could be detected (Fig. 7a) . In contrast cleaved caspase 3 protein with the expected molecular weight of 19 kDa was readily detected in the extracts of RAW264 cells treated with 10 mM staurosporin (Fig. 7a) . Similarly, no specific signals for cleaved caspase 3 could be detected in immuno-histochemical analyses of tissue sections. In addition, caspase 3 and caspase 7 activities was measured in cytoplasmic protein extracts derived from mammary tissue of control, heterozygous and a-casein deficient mice. No significant differences in caspase 3 and 7 were detected (Fig. 7b) . In contrast a significant increase in caspase activity could be detected in RAW cells treated with 10 mM staurosporin (Fig. 7b) . cDNAs derived from representative samples of control and acasein deficient mice were analysed for characteristic gene expression changes related to apoptosis. Potential changes in candidate genes were assessed using a PCR array containing 84 apoptosis related genes. The expression of 16 genes was changed significantly (11 down-regulated and 5 up-regulated in a-casein deficient mice vs. control). Five genes, which were changed by more than 7 fold (1 down-regulated and 4 up-regulated), were further analysed in cDNAs derived from 3 control, 5 heterozygous and 5 a-casein deficient animals (Fig. 7c) . Only 3 genes showed consistent and significant expression differences between control and a-casein deficient animals. Expression of the anti-apoptotic genes survivin (baculoviral inhibitor of apoptosis repeat-contain- ing-5; Birc5) and nucleolar protein 3 (Nol3/ARC: nuclear apoptosis repressor with caspase recruitment domain) was increased by 22 and 2.5 fold respectively. In contrast expression of another anti-apoptotic gene, TNF receptor-associated factor 1 (Traf1), was reduced to 5% of control levels in a-casein deficient mice.
In order to distinguish between the effect of the genetic alteration in the offspring and the effect of the modified milk, the mice were put into 3 groups and cross-fostered such that wild-type offspring was nursed by a-casein deficient dams (3 litters with a total of 25 pups; 6 to 11 pups per litter) and heterozygous a-casein [+/2] offspring was nursed by wild-type dams (3 litters with a total of 22 pups; 4 to 10 pups per litter). Wild-type offspring nursed by wild-type dams were used as additional controls (3 litters with a total of 34 pups; 10 to 13 pups per litter; Table 2 ).
Weight gain during lactation was significantly reduced in offspring nursed by a-casein deficient dams (p,0.001 for all comparisons of offspring nursed by a-casein deficient dams with the other two groups after day 3). This effect was seen when acasein deficient dams nursed their own pups and if the offspring of wild-type mice was nursed by deficient dams. This demonstrates that the effect is mediated by the genotype of the nursing female. This effect was also seen in two further litters after breeding of the a-casein deficient mice onto a CD1 background (Kolb et al., unpublished) . All females were able to nurse their pups, as indicated by the presence of milk in the stomach ('milk spot') in all groups of pups (Table 3) . However, by day 6, pups nursed by acasein deficient dams were visibly emaciated (Fig. 8a) in comparison with pups nursed by control dams (Fig. 8b) . By midlactation offspring nursed by a-casein deficient mice are significantly smaller than offspring nursed by control dams (Fig. 8c) . Pre-weaning mortality was low in all groups and unaffected by dam genotype. Weighing of individual pups throughout lactation demonstrates that offspring nursed by acasein deficient dams only reach a weight of around 3 g by the end of lactation, whereas pups nursed by control dams weigh around 12 g (Fig. 8d and Fig. 9a ). After weaning mice were maintained on a standard chow diet ad libitum. Offspring nursed by a-casein deficient dams display a significantly accelerated growth with respect to the control group between weaning and day 50 of life ( Fig. 9b and 10) . However, this brief growth spurt is insufficient to bring the weight of pups nursed by a-casein deficient dams up to the weight of control animals in the long term (Fig. 9c) . The difference in weight is displayed in both sexes (Fig. 9d) .
Before the weaning day (day 23), wild-type pups nursed by acasein deficient dams had a significantly slower growth rate than pups (wild-type and heterozygous) nursed by wild-type dams (p,0.001 for both comparisons). The growth rate of wild-type and heterozygous pups nursed by wild-type dams 1 and 3 was not significantly different (p = 0.729). After weaning, wild-type pups nursed by a-casein deficient dams had a significantly higher growth rate than pups (wild-type and heterozygous) nursed by wild-type dams (p = 0.039 and p = 0.014 respectively). The growth rate of wild-type and heterozygous pups nursed by wild-type dams was not significantly different (p = 0.449) For wild-type pups nursed by a-casein deficient dams the average growth rate was 0.14 g/day for days 1-23 and 0.52 g/day for days 24-60 (cf. Fig. 9b ). For pups (wild-type and heterozygous) nursed by wildtype dams the growth rates were for days 1-23 0.64 g/day and 0.63 g/day, respectively, and for days 24-60 0.47 g/day and 0.45 g/day, respectively (Fig. 10 ).
In order to assess whether the deficient nutrition during lactation has an impact on general development, mice in the three experimental groups were exposed to an early phenotyping protocol including an adapted version of the general screen of the SHIRPA assay [22] . Pre-weaning observations indicated physical impairment in the 'small' mice. Measures of maturation (e.g. eye opening; tables 3 and 7) occur with a slight delay with respect to control animals. 60% and 50% of the pups nursed by wild-type dams had both eyes opened on day 14, all of the pups nursed by acasein deficient females still had their eyes closed at that age, opening them only around 3 days later (Table 7) . Physical abilities which rely on physical strength and coordination when tested on day 21 are significantly less developed in pups reared by a-casein deficient dams (Table 8 ). Significantly fewer of the growth impaired animals (36%, P-value,0.001 Fisher's exact test) were able to hold on to the pole in the vertical pole test, whereas none of the pups nursed by wild-type dams fell off before the pole reached a vertical position. Some animals nursed by a-casein deficient dams presented a rough condition of the fur (84%, P-value,0.001 Fisher's exact test) whereas all control animals had normal fur condition. Grip strength in growth impaired pups was significantly lower (P = 0.001), as 36% of the animals could not hold on to the cage lid for 15 seconds when it was turned upside down, compared to 3% and 0% in the two control groups. Animals nursed by knockout females defecated less during handling and scoring on day 21 (P-value,0.001, Mann Whitney U). Also significantly fewer (76%, P = 0.004) pups nursed by a-casein deficient females showed a provoked biting response at day 21 when compared to pups nursed by wild-type dams (100%). In summary, at preweaning the a-casein deficient milk suckled pups were smaller and physically weaker than those reared on wild-type milk.
Subsequently, post-weaning observations clearly indicated that the 'small' mice had, in-effect, caught up with respect to their relative physical characteristics. Specifically, when the animals were assessed again at 8 weeks of age, there were no significant differences between groups for most of the parameters observed (Table 9 ). This suggests that in offspring nursed by a-casein deficient mice, general development of pups is less impaired than what would be expected from their significant growth retardation which has persisted throughout their life so far (currently the mice are almost 2 years old).
The role of caseins in milk protein secretion Deficiency for a-casein severely impairs milk protein secretion in mice. In contrast b-casein deficiency does not result in a similar decline [5] . This suggests that, in contrast to b-casein, a-casein plays a more critical role in the establishment of a functional casein micelle thereby affecting secretion of all casein proteins. Alternatively or additionally due to the higher number of phosphate centres present in a-casein vs. b-casein (3 vs. 1), deficiency for a-casein (but not for b-casein) may decrease the stability of the biofluid resulting in a precipitation of calciumphosphate in the Golgi vesicles. These results argue against a functional redundancy of the calcium-sensitive caseins (a-, b-, cand d-casein). Whereas overall secretion of milk proteins is not adversely affected in heterozygous mice (apart from a 50% reduction in a-casein protein secretion), milk protein secretion from mammary epithelial cells is severely curtailed in homozygous a-casein deficient mice. This affects both, caseins and whey proteins like WAP. In contrast, secretion of albumin which is not secreted by the mammary epithelial cells is not affected by the absence of a-casein. These results suggest a functional role for acasein in casein micelle formation and/or stabilisation (similar to that of k-casein) [6] . We find that the glucose regulated protein 78 (grp78 or BiP) is significantly up-regulated in response to the absence of a-casein. Grp78/BiP is an endoplasmic reticulum (ER)resident protein whose expression is enhanced under conditions of ER stress [29] [31, 33] . Surprisingly the protein is also found in milk of a-casein deficient dams. This suggests that grp78/BiP may be critically involved in the assembly of the casein micelle. One can speculate that grp78/BiP is associated with the maturing casein micelle and is co-secreted with the immature micelle into milk. Two further ER resident proteins, grp94 and PDIA6 are also secreted into milk of a-casein deficient animals indicating that the lack of a-casein has significant impacts on protein processing and transport in the mammary epithelium. Interestingly, secretion of grp78/BiP and grp94 is also observed in mice over-expressing human protein C [37, 38] . This may suggest that perturbation of the balance of milk proteins invokes a common ER stress response resulting in the co-secretion of ER resident proteins into milk. Despite the clear evidence for changes to the expression and localisation of ER resident proteins there is no clear evidence for significant increases in mammary apoptosis in the absence of acasein. Firstly, lactation is not terminated prematurely in the deficient animals suggesting that at least a significant portion of the mammary epithelial cells is viable. Under physiological conditions mammary gland involution after weaning leads to a complete cessation of milk secretion, apoptosis and regression of the epithelial tissue [39, 40] . In homozygous protein C transgenic mice no successful lactation is established, suggesting that in that case ER stress is followed by loss of mammary epithelium function [38] . Secondly, we cannot detect any gross morphological alteration of the secretory mammary epithelium in a-casein deficient mice. Thirdly, we also failed to detect increases in caspase activity in a-casein deficient mammary tissue. Finally, the apoptosis related genes which showed consistent and significant increases in mRNA expression in mammary tissue of a-casein deficient mice include the anti-apoptotic genes survivin (Birc5) and ARC/Nol3, suggesting that the mammary cells are actively avoiding apoptosis. On the other hand a reduction in the expression of the Traf1 gene, encoding an anti-apoptotic gene in the context of the mammary gland [41] suggests a balance of proapoptotic and anti-apoptotic responses to a-casein deficiency. Taken together these findings may suggest that the ER-stress response in mammary epithelial cells of a-casein deficient mice is sufficient to rescue protein secretion, thereby preventing large scale apoptosis and loss of tissue function. Interestingly, we find that steady state levels of mRNAs encoding b-casein, c-casein and k-casein are also severely reduced in the mammary gland of a-casein deficient mice (cf. Fig. 5d ). In contrast no such reduction is observed in heterozygous mice. There could be (at least) two explanations for this. The deletion of cis-acting DNA elements in the a-casein gene may affect the expression of the entire casein gene locus. This genetic explanation would imply that the presence of one functional a-casein allele is sufficient to mediate the activation of both copies of the casein gene locus. Alternatively there may be physiological explanations. ER stress has been associated with an attenuation of protein synthesis [33] . One can speculate that in the mammary gland this leads to a decrease in the transcription of milk protein genes which account for most of the transcripts during lactation to prevent overloading of a compromised endoplasmic reticulum [33] . A decrease in the abundance of milk protein gene specific mRNAs was also observed in the transgenic mice over expressing human protein C [38] . Alternatively, the viability of the secretory mammary epithelial cells may be impaired to some degree by the ER stress response, leading to a lower relative number of casein producing cells in the mammary gland.
Not surprisingly a-casein deficiency and the reduced concentration of casein proteins is accompanied by a reduction in the concentration of both calcium and phosphate in milk. The effect of a-casein deficiency in mice is similar to that reported for a aS1casein deficient goat breed (Cn0) in that total casein secretion is reduced by around 75% [18] . While there are no data available which suggest a critical growth delay in the offspring of aS1-Cn0 goats, it is unlikely that an allele that would have a nutritional impact similar to the inactivation of the a-casein in mice could have persisted in the natural environment. In addition, the Table 9 . Comparison regarding general health and behaviour between pups nursed by wild-type dams and pups nursed by acasein deficient dams. absence of a-casein does not appear to have a critical effect on calcium secretion in goats [17] . This indicates that there may be species (and maybe also strain-) specific differences in the assembly of casein micelles.
The consequences of a-casein deficiency for the offspring
The most critical consequence of a-casein deficiency for the offspring is a sustained shortfall in body size. Pups nursed by acasein deficient dams only reach 25% of body weight of control animals at weaning and even at later stages in life remain around 33% lighter than control animals. The experiments described in this paper have been carried out with 3 litters of each genotype on a C57BL/6 background. However we have observed the same phenotype of reduced weight gain during lactation with two more litters on a CD1 background (Kolb et al., unpublished data) , suggesting that the phenotype is similar in different genetic backgrounds. The observed changes in calcium concentration in the milk of a-casein deficient animals are unlikely to play a role in this permanent reduction in body size as calcium restriction during lactation only exerts a transient growth delay in rodents [42] .
In contrast, protein deficient diets have been implicated as important regulators of metabolic programming and later health outcomes in mice and men [43, 44, 45, 46] . Gestational protein deficiency in the mother leads to intra-uterine growth delay in the offspring. However, when such offspring is then cross-fostered onto dams on a control diet, they show a phase of rapid catch up growth during lactation [47] . The combination of poor foetal and rapid post-natal growth leads to increased susceptibility for metabolic disease and reduces life-span [48, 49] . In contrast, nutritional restriction during lactation leads to a growth delay in rodent pups which is maintained throughout life [48] .
Milk supply in rodents can also be modulated by a number of approaches [48, 50, 51] . Variations in litter size lead to differences in adult body weight, although generally the effects in mice are less drastic (around 10% in adulthood) than those observed in offspring nursed by a-casein deficient dams. Alternatively, milk protein concentration can be manipulated by changing the protein content of diet of the nursing dams [45] . Specific alterations in milk composition have been more difficult to achieve technically and have relied on rearing rodent pups by gastrostomal nutrition away from their mothers [52] . However, this results in a radically different early environment as these pups are not exposed to the normal maternal behaviour [53] . Therefore the effect of specific protein restrictions during lactation has not been addressed in detail.
The molecular mechanisms by which early nutrition alters body size permanently are not fully understood at the moment. But the growth hormone/IGF axis and programming of appetite control have been implicated as key mediators. Attenuated growth rates in early post-natal life provide a significant protection against metabolic disease and extend health-and lifespan [45, 51, 54, 55, 56] . Many genetic mutations of the growth hormone/IGF axis result in both, small body size and an increased life-and health-span. Increased lifespan and increased resistance to metabolic disease is also observed in mice nursed by dams on a low protein diet [45] . This suggests that permanent changes in body size induced by altered protein supply during lactation are accompanied by an improvement in metabolic health. In humans formula feeding has been associated with both, a more rapid increase in body weight during lactation [57] and an increased susceptibility to metabolic disorders [46, 58, 59] .
Mice reared on a-casein deficient milk show a marked delay in the development of abilities which are related to physical capabilities. However, the deficiencies displayed at weaning had disappeared at 8 weeks of age. This indicates that these impairments are transient, whereas the changes in body weight are permanent.
Thus the phenotype of a-casein deficient dams and their offspring confirm the critical role of lactation in determining lifelong body size and underline the decisive role of protein supply during this developmental window. These mice represent a genetically defined model system of nutritional restriction with a significant impact on whole animal metabolism. This model can be exploited to study mechanistic aspects of metabolic programming of body size, resistance to obesity and long-term health outcomes. Geometry and Adhesion of Extracellular Domains of DC-SIGNR Neck Length Variants Analyzed by Force–Distance Measurements [Image: see text] Force–distance measurements have been used to examine differences in the interaction of the dendritic cell glycan-binding receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR (L-SIGN) with membranes bearing glycan ligands. The results demonstrate that upon binding to membrane-anchored ligand, DC-SIGNR undergoes a conformational change similar to that previously observed for DC-SIGN. The results also validate a model for the extracellular domain of DC-SIGNR derived from crystallographic studies. Force measurements were performed with DC-SIGNR variants that differ in the length of the neck that result from genetic polymorphisms, which encode different numbers of the 23-amino acid repeat sequences that constitute the neck. The findings are consistent with an elongated, relatively rigid structure of the neck repeat observed in crystals. In addition, differences in the lengths of DC-SIGN and DC-SIGNR extracellular domains with equivalent numbers of neck repeats support a model in which the different dispositions of the carbohydrate-recognition domains in DC-SIGN and DC-SIGNR result from variations in the sequences of the necks. T he human glycan-binding receptors DC-SIGN (dendriticcell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also designated as L-SIGN, CD209L, and CD299) share overall sequence identity of 77% and arose by a relatively recent gene duplication within the primate lineage. DC-SIGN is expressed on dendritic cells and some macrophages, while DC-SIGNR is expressed on endothelial cells in the liver and lymph node sinusoids and in cells lining placental capillaries. 1À3 Expression of these receptors on distinct cell types suggests that they have potentially different functions. Both bind to the T-cell surface receptor ICAM-3 1,2 in order to facilitate infection of T cells by human immunodeficiency virus (HIV 4 ) and act as primary receptors that mediate entry of Ebola virus into cells. 4, 5 However, they bind differentially to other pathogens. 6 For example, DC-SIGN plays a critical role in modulating the host response to schistosomal parasites, and it interacts with Helicobacter pylori, while DC-SIGNR facilitates infection of hepatocytes, by presenting hepatitis C virus on liver endothelial cells. 7 DC-SIGNR also promotes transmission of human immunodeficiency virus across the placenta. 8 These differences in the biological properties of DC-SIGN and DC-SIGNR are mirrored by differences in their molecular properties. For example, although both receptors bind to highmannose oligosaccharides, these are the only glycans that are detected in glycan array analysis of DC-SIGNR, while the presence of key residues in the carbohydrate-recognition domain (CRD) of DC-SIGN, particularly Val351, supports binding to fucosylated Lewis-type oligosaccharide structures as well. 9 Such differences probably explain why DC-SIGN binds to parasites that are rich in such structures, while DC-SIGNR does not. 10, 11 In addition to these differences in the ligand-binding domains of DC-SIGN and DC-SIGNR, there appear to be significant but less well understood differences in their overall architectures ( Figure 1A ,B). In both receptors, the carbohydrate-recognition domains are projected from the cell surface by a neck domain comprising multiple copies of a 23-amino acid repeat sequence, but the number of copies of the repeat is largely fixed fixed at 7.5 in DC-SIGN, while polymorphisms in the gene for DC-SIGNR result in variant forms of the neck containing between 4.5 and 9.5 repeats. 12, 13 The allele frequency of the 7.5-repeat variant is over 50% in the human population, but the frequencies of the 6.5and 5.5-repeat variants are 12À16% and 26À37%, respectively. Different neck-length genotypes have been associated with variations in susceptibility to certain viral infections. The 7.5-repeat form is more common in HIV-infected individuals than in uninfected individuals. 14 However, this correlation with HIV susceptibility has not been observed in all cases. 12 The presence of 4.5repeat and 9.5-repeat variants correlates with decreased viral load in hepatitis C virus infection. 15 The shorter necks are also associated with diminished rates of SARS coronavirus infection. 16 It is also noteworthy that the neck repeat adjacent to the CRD of DC-SIGNR differs significantly in sequence from the Biochemistry ARTICLE corresponding sequence in DC-SIGN. Recent structural analysis of a truncated form of the neck domain of DC-SIGNR, combined with a structure of the final neck repeat attached to the CRD, has indicated that a phenylalanine in the final neck repeat of DC-SIGNR causes the polypeptides forming the necks to splay apart at this point. 17 In DC-SIGN, the presence of a valine residue at this position suggests that the neck could remain packed as they are in the other neck repeats, thereby placing the CRDs closer to each other. Thus, differences in the neck regions of DC-SIGN and DC-SIGNR may also contribute to differences in their interactions with pathogens.
ForceÀdistance measurements have provided a means of looking at interactions of the intact extracellular domain of DC-SIGN with ligands. 18 In these experiments, an N-terminal histidine tag was used to anchor the protein and project the extracellular domain of DC-SIGN from the surface of one membrane, which was apposed to a second membrane bearing a neoglycolipid with a high-mannose oligosaccharide headgroup ( Figure 1C ). Measurement of the distance dependence of the interaction force between the membranes demonstrated that DC-SIGN undergoes a conformational change driven by engagement with ligand. This change reflects flexibility in the linkage between the neck and the CRD.
In the experiments described here, forceÀdistance measurements have been used to demonstrate that DC-SIGNR undergoes a conformational change, similar to that observed for DC-SIGN. Distinct from the prior studies, here we further investigated how differences in the final neck repeat as well as the genetic polymorphisms affect both the projected distance of the CRDs from the membrane surface and the magnitude of adhesion.
' MATERIALS AND METHODS Materials. 1,2-Dipalmitoyl-sn-glycero-3 phosphoethanolamine (DPPE) and 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC) were from Avanti Polar Lipids (Alabaster, AL). 6-[9-[2,3-Bis(dodecyloxy)propyl]-3,6,9-trioxanonyl-1-oxycarboxylamino]-2-[di(carboxymethyl)amino]hexanoic acid (NTA-TRIG-DLGE) was custom synthesized by Neuftech Chemicals (Vancouver, BC). HEPES was purchased from Fisher Bioreagents (Fair Lawn, NJ), and all other high-purity salts were from Aldrich (St. Louis, MO). The neoglycolipid Man 9 GlcNAc 2 -DPPE was synthesized according to described procedures. 18 DC-SIGNR Expression and Purification. Restriction fragments from vectors used for bacterial expression of the extracellular domains of three variants of human DC-SIGNR 19 were moved into a vector encoding an N-terminal His 6 tag previously designed for use with DC-SIGN. 18 The vector encodes the sequence Met-Ala-His-His-His-His-His-His-Gly-Glu-Leu, which begins at residue 101 of the full-length DC-SIGNR protein, and corresponds to the middle of the first 23-amino acid repeat in the extracellular domain. The tagged protein was purified by affinity chromatography on mannoseÀSepharose following the protocol used for the untagged extracellular domain. 20 The affinity purification confirms that the CRDs are folded and active. Biochemistry ARTICLE DC-SIGNR Immobilization on Planar Lipid Bilayers. The recombinant proteins with hexahistidine tags at the N-termini of the extracellular segments of DC-SIGNR were immobilized on asymmetric, planar lipid bilayers, which were supported on mica sheets affixed to hemicylindrical silica disks with 1À2 cm radii of curvature. 18 The first layer is a gel phase DPPE monolayer that was transferred at a surface pressure of 38 mN/m by LangmuirÀ Blodgett deposition onto the mica. DPPE was transferred from a pure water subphase at 21°C. At this temperature and pressure, the area per lipid is 43 Å 2 . The outer leaflet is a 100% NTA-TRIG-DLGE monolayer deposited onto the DPPE monolayer at 65 Å 2 per lipid, from a subphase of buffer A (20 mM HEPES, 50 mM NaNO 3 , 3 mM Ca(NO 3 ) 2 , and 50 μM NiSO 4 at pH 7.8) at 21°C. The deposition surface pressure was 35 mN/m, and the NTA-TRIG-DLGE monolayer is in the fluid phase at 21°C. The resulting supported bilayer was kept under buffer A at all times. It was then incubated for 1.5 h with 0.5 μM of either of the hexahistidine-tagged 7-, 6-, or 5-repeat DC-SIGNR variants in buffer A. Nonspecifically adsorbed protein was removed by rinsing the bilayer with buffer A.
Determination of the Surface Coverage of Immobilized 7-, 6-, and 5-Repeat Forms of DC-SIGNR. The amount of DC-SIGNR immobilized on the supported NTA-TRIG-DLGE monolayers was quantified by surface plasmon resonance. 21 For these measurements, the LangmuirÀBlodgett monolayer of NTA-TRIG-DLGE (65 Å 2 /lipid) was supported on a monolayer of 1-octadecanethiol that was self-assembled on a gold-coated glass slide. The sample was then assembled in the surface plasmon resonance cell, while maintaining it under solution at all times.
DC-SIGNR adsorption was quantified from the change in the plasmon resonance angle measured after incubating the NTA-TRIG-DLGE monolayer with 0.5 μM of either of the 7-, 6-, or 5-repeat DC-SIGNR for 1.5 h at room temperature. The 1.5 h incubation time was also used to prepare samples for all surface force measurements in this study. The total protein adsorbed was determined from the change in the resonance angle and use of the 5-phase Fresnel calculation program (R. Corn, University of California, Irvine). The DC-SIGNR surface coverage was calculated from the effective refractive index of the protein monolayer n eff . This value is obtained from the measured optical thickness (product of the refractive index and layer thickness, n eff  D T ). This measurement and the thicknesses D T determined from surface force measurements were used to obtain the effective refractive index n eff . Assuming a linear dependence of the refractive index on surface concentration, the surface coverage Γ (molecules/m 2 ) of the different DC-SIGNR variants was calculated from the refractive indices and use of a refractive index increment dn/dc of 0.187, where c is the surface concentration in mg/m 2 . 22, 23 The error in protein densities determined from refractive index measurements is ∼20%. 22, 23 This analysis differs slightly from that used previously (see Supporting Information). 18 The protein (tetramer) densities of the 7-repeat and 6-repeat forms are determined by dividing the monomer density by four. A similar calculation for the 5-repeat form is more complicated because it is expressed as a mixture of interconverting dimers and tetramers, with a solution dissociation constant of 0.14 μM, as determined by equilibrium analytical ultracentrifugation. 11 Whether this distribution persists on the membrane, particularly under applied force, cannot be easily determined. Nevertheless, the measured jumps into contact presented here indicate the presence of folded tetramers on the membrane.
Asymmetric bilayers displaying Man 9 GlcNAc 2 -DPPE were supported on a gel phase DPPE monolayer (43 Å 2 per lipid). The DPPE was in turn supported on a mica sheet that was affixed to a hemicylindrical, silica disk. The outer layer contains 5 mol % of Man 9 GlcNAc 2 -DPPE mixed with DTPC (fluid, T m < 21°C) ( Figure 1C ) and was transferred by LangmuirÀBlodgett deposition from a subphase of buffer A at 21°C at 65.4 ( 0.8 Å 2 per DTPC. These neoglycolipid/DTPC monolayers are in the fluid state at 21°C. The molar concentrations of DPPE, DTPC, and Man 9 GlcNAc 2 -DPPE in stock solutions were determined with the Bartlett phosphorus assay. 24 At 5 mol % Man 9 GlcNAc 2 -DPPE in the outer leaflet, the average area per neoglycolipid is 7.7 Â 10 16 glycolipid/m 2 .
The silica disks supporting the DC-SIGNR and the 5 mol % Man 9 GlcNAc 2 -DPPE bilayers were maintained under buffer A and transferred into the chamber surface force apparatus, which was filled with buffer A. All measurements were performed at 21 ( 0.2°C.
Surface Force Measurements. Surface force measurements were carried out with a Mark III instrument, and the spring constant used was 205 N/m. In surface force measurements between oriented monolayers of DC-SIGNR and 5 mol % Man 9 GlcNAc 2 containing lipid monolayers ( Figure 1C ), the separation distance D between the lipid bilayers can be determined within (0.1 nm by interferometry. 25 The difference in the total thickness, T, of the molecular assembly relative to contact between supporting DPPE monolayers was measured after the deposition of the outer lipid layers. The absolute membrane 26 where T NTA-TRIG-DLGE is the thickness of the NTA-TRIG-DLGE monolayer 27 and T DTPC is the thickness of the DTPC on the neoglycolipid-containing membrane. The DTPC layer thickness does not include the thickness of the Man 9 GlcNAc 2 headgroup, such that D = 0 is defined as contact between the NTA-TRIG-DLGE and bare DTPC membranes.
The normalized intersurface force (F c /R) was measured when first bringing the protein and ligand surfaces into contact and again upon separating them. The normalized force F c /R is determined as a function of the separation distance D. The adhesion was then measured during surface separation. In these measurements, adhesion was measured after 8 min in contact. Prior studies of DC-SIGN showed that adhesion to fluid neoglycolipid membranes increases with time up to a limiting value, due to the lateral diffusion of glycan ligands and corresponding increase in number of CRDÀglycan bonds. 18 Because the lateral lipid diffusion and lipid density are identical for all samples in this study and in the prior measurements, differences in adhesion energies measured at identical contact times would therefore reflect functional differences between the different length variants.
The adhesion energy per unit area (E Adh , mJ/m 2 ) measured between receptor and ligand monolayers is quantified from the pull-off force required to separate the adherent sample surfaces (F po ) 28 and use of the DerjaguinÀM€ ullerÀToporov model for adhesion between undeformed surfaces: E a = F po /2πR. Here, R is the geometric average radius of the two discs: R =(R 1 R 2 ) 1/2 . Further normalizing the adhesion energy per area by the protein surface coverage (number of proteins/area) accounts for differences in protein densities across experiments.
Because the pull-off forces can depend on the rate of separation, preliminary measurements were done at different separation Biochemistry ARTICLE rates to establish conditions where the pull-off forces were rate independent and would therefore report the equilibrium adhesion energy per area. In the present study, at typical separation rates of ∼20À40 Å/min, the pull-off forces were in the rate-independent regime.
Statistical Analyses of Distance Measurements. The determined initial contact position D C , equilibrium separation D eq , position of maximum adhesion D Adh , and thickness in the absence of ligand D T are averages of 7À20 measurements from 2À3 independent experiments. Standard deviations are the pooled standard deviations from separate sets of measurements. Averages were compared using the student's t test, and p-values <0.05 indicate a statistically significant difference, at the 95% confidence level. The standard deviation of the difference between pairs of values was determined by propagation of errors.
The surface densities of the immobilized proteins, as determined from surface plasmon resonance (SPR) measurements, were 2.9, 1.2, and 0.72 mg/m 2 for the 7-, 6-, and 5-repeat forms, respectively. In the absence of Ni 2þ there was no detectable nonspecific protein adsorption, indicating that all proteins bound selectively by their histidine tags. The molecular weights of the monomers of the length variants are 35 344 Da (7-repeat), 32 718 Da (6-repeat), and 30 119 Da (5-repeat), so that the calculated monomer surface densities were Γ 7 = 4.9 Â 10 16 , Γ 6 = 2.1 Â 10 16 , and Γ 5 = 1.4 Â 10 16 monomers/m 2 .
In the absence of ligand, the normalized force versus distance curves obtained with the surface force apparatus were reversible and exhibited intersurface repulsion due to steric interactions between the protein and bare lipid monolayers ( Figure S1 ). The thickness of the unbound protein D T was determined from the distance at which the repulsive force exceeded the measurement standard deviation of (0.03 mN/m. Thicknesses of 31.5 ( 0.5, 27.6 ( 0.5, and 22.4 ( 0.5 nm were thus obtained for the 7-, 6-, and 5-repeat DC-SIGNR monolayers, respectively.
Instead of increasing the range of the intersurface repulsion, the presence of the bulky neoglycolipid on the target membrane resulted in the protein monolayers jumping into contact with the membranes, at distances that were smaller than the thickness of unbound protein, D T . Figure 2 shows the forceÀdistance curve measured between the 6-repeat form of DC-SIGNR and a 5 mol % Man 9 GlcNAc 2 -DPPE monolayer. The surfaces jump into adhesive contact (left pointing arrow) from membrane separations D J . In force measurements, surfaces jump to contact when the gradient of the intersurface potential exceeds the spring constant. 26 The jump-in distance D J , trajectory, and final resting position at contact D C are determined by the spring constant, which was 205 N/m in these measurements. Here, the normalized force at D C is slightly negative (∼À0.1 mN/m). The equilibrium separation D eq is at F = 0 where the attractive and repulsive forces balance.
The DC-SIGNR length variants with 7-, 6-, and 5-neck repeats jumped to contact with the neoglycolipid layers from distances of respectively D J, 7 = 30.6 ( 1. 1 nm for the 7-, 6-, and 5-repeat proteins. The greater uncertainty in determinations of D eq is due to the low force values relative to the measurement uncertainty of (0.03 and the shallow slopes near D eq , especially with the 5-repeat variant (cf. Figure 3C ). In these measurements, Biochemistry ARTICLE the average value of D eq is less than D C by 2.1 ( 0.5 nm (Table 1) due to the relatively soft repulsion at D < D eq . In the majority of the measurements, at D < D C the force curve is nearly flat for ∼1.5 nm (Figure 2 ), before increasing more steeply. This jump-to-contact is evidence of functionally active protein because the mechanical instability, which underlies the jump-in requires an attractive force between the ligand and protein monolayers. Protein inactivation would ablate the attraction. All three DC-SIGNR variants exhibit this jump-to-contact behavior, which was also observed in comparable experiments with DC-SIGN. 18 Furthermore, the observation that both D C and D eq are significantly less than D T , despite the presence of the bulky glycan on the target membrane, is attributed to flexibility in the DC-SIGN structure that enables the proteins to undergo a conformational change upon binding the target neoglycolipid. 18 Such protein flexibility would increase the reaction cross section of the CRDs and hence the range of interaction of the CRDs as well as their ability to bind ligands at different, lateral spatial distributions on target surfaces. Both DC-SIGN and all three DC-SIGNR variants exhibit this flexibility, although it is less apparent with the 5-repeat form of DC-SIGNR, due to the weaker attraction.
Elasticity in the DC-SIGNR/glycolipid complex is also apparent from comparisons of approaching and receding forceÀ distance curves between the various forms of DC-SIGNR and Man 9 GlcNAc 2 membranes. During separation, the bonds fail and the surfaces pull out of contact at the position of the maximum adhesion (D Adh ) ( Figure 2B, right pointing arrow) . For the 7and 6-repeat forms of DC-SIGNR, D Adh exceeds D eq by 3.5 nm (Table 1 , Figures 2B and 3A,B) . This difference must result from stretching of the DC-SIGNR/Man 9 GlcNAc 2 complex such that, upon separation, the complexes extend relative to their equilibrium thickness D eq . This may be due, in part, to the reorientation of the carbohydrate ligand. Protein flexibility, which enables the DC-SIGNR variants to jump to contact at D C < D T , would also play a role in this force-dependent stretching.
Comparison of the D C or D eq values for the 7-repeat DC-SIGNR with an exactly equivalent construct for the extracellular domain of DC-SIGN 18 reveals that the average difference of 1.3 nm is larger for DC-SIGN than for DC-SIGNR (p < 0.05, DF = 13; Table 1 ). In pairwise comparisons, a p value less than 0.05 signifies a statistically significant difference at the 95% confidence level. As noted in Figure 1 , differences in the sequence of the neck repeat adjacent to the CRD in DC-SIGNR are postulated to result in the splaying apart of this repeat and the CRDs compared to DC-SIGN ( Figure 1B) , which would make the bound thickness of DC-SIGN slightly longer than DC-SIGNR. 17 Thus, the forceÀdistance measurements provide experimental evidence to support this model of the differences in the arrangement of the distal portion of the DC-SIGN and DC-SIGNR extracellular domains ( Figure 1B) .
At D < D eq , the repulsive force increases with decreasing membrane separation (Figures 2 and 3) , and the slope reflects the rigidity of the proteinÀligand complexes. The slopes of the curves obtained with the 6-repeat and 7-repeat forms, as well as with DC-SIGN, 18 are indicative of relatively rigid protein layers and hence relatively rigid necks ( Figure 3A,B) . By contrast, the forceÀdistance curve measured with the 5-repeat form at D < D eq is much shallower and does not increase steeply until D < 4À5 nm ( Figure 3C ), which is the approximate diameter of a folded CRD. 29 The forceÀdistance profile at D > 4 nm is similar to flexible polymers 30 and suggests that the neck region is unstructured in a significant population of the immobilized 5-repeat protein. The difference between the 5-repeat and other forms of DC-SIGNR and DC-SIGN is attributed to the loss of rigidity conferred by the tetramer. Tetramer dissociation into dimers could also lead to greater protein inactivation under applied force. This exception, relative to the behavior of the stable 6-and 7-repeat tetramers, provides evidence that the rigid neck region and relatively steep force profiles at D < D eq ( Figure 3A,B) requires stable tetramers.
The adhesion energy per area between DC-SIGNR and 5 mol % Man 9 GlcNAc 2 -DPPE monolayers is determined from the pull-off force F po at the minimum in the forceÀdistance curve D Adh ( Figure 2B ) and the DerjaguinÀM€ ullerÀToporov model. 28 At the position of bond rupture ( Figure 2B, D Adh ) , the surfaces jump out of contact (Figures 2 and 3 , right pointing arrows). There is no evidence of irreversible damage to the membranes due to, for example, lipid pull-out or the rupture of the NTAÀhexahistidine linkage. This would be evident in altered force curves measured at the same position after membrane separation, as documented previously. 31 In these studies, Biochemistry ARTICLE upon reapproach after detachment, the measured force curve, D J , and D C were unchanged (Figure 2 ). When separating the surfaces, D Adh and the pull-off force were similar within experimental error, even after as many as seven successive measurements at the same contact site. The average time between successive measurements was 10À15 min, which is too short for damaged membranes to heal 31 but more than sufficient to allow re-equilibration, as evidenced by the reversibility of the measurements. The reversibility is expected because this is a multivalent (number of bonds = 4) interaction, and the affinity of individual monomerÀglycan bonds is relatively low. Leckband et al. 31 showed that lipid pull-out occurs at receptorÀligand bond energies >∼15kT, which is much higher than the energies of the individual monomerÀglycan bonds. Each tetramer is also anchored to the membranes via four hexahistidine anchors.
The adhesion energies per area measured with the 7-repeat and 6-repeat variants are respectively 0.32 ( 0.04 and 0.13 ( 0.03 mJ/m 2 ( Table 2) . When normalized by the protein density on the membranes, the estimated adhesion energies per tetramer are E Adh,7 = 8.0 ( 1.3 kT/molecule and E Adh,6 = 6.9 ( 2.1 kT/ molecule, respectively ( Table 2) . Estimating the adhesion energy per DC-SIGNR molecule for the 5-repeat form is not feasible because of the decreased tetramer stability caused by the truncation associated with the addition of the histidine tag employed in these force measurements. 19 Comparing the estimated, population-averaged adhesion energy per monomer ( Figure 4A ) may provide a better basis for comparison of adhesion by the different DC-SIGNR forms. The calculated energies per monomer for the 7-repeat and 6-repeat forms are 2.0 ( 0.2 and 1.7 ( 0.4 kT/monomer, respectively. These values are statistically similar (p value = 0.7, DF = 12). They are also similar to the adhesion energy per DC-SIGN monomer of 2.0 ( 0.3 kT/monomer (p value = 0.3, DF = 18) after 8 min of contact. 18 However, the calculated adhesion energy per monomer for the 5-repeat DC-SIGNR is 0.2 ( 0.1 kT/ monomer.
The much lower adhesion mediated by the 5-repeat DC-SIGNR might at first be unexpected, since the CRDs fold autonomously and appear to be fully functional based on solution binding data and on affinity purification. 20 Hydrodynamic measurements show that this protein forms mixture of interconverting dimers and tetramers. However, some of the 5-repeat DC-SIGNR proteins may not be optimally oriented for binding, and it is possible that the dimers are more prone to unfolding under an applied force. Both factors would reduce the adhesion. The softer repulsion (shallow, repulsive force at D < D C ) is also attributed to the mixture of dimers and tetramers.
The 5-repeat DC-SIGNR shows completely different behavior in the force measurements relative to the 6-and 7-repeat forms. Viewed in this way, these findings provide important confirmation that the force measurements with the longer versions of DC-SIGNR reflect the interactions of rigid, stable tetramers that orient perpendicular to the membrane.
The forceÀdistance measurements quantify the molecular dimensions of the DC-SIGNR-Man 9 GlcNAc 2 complex and thus provide direct evidence for an extended configuration of the extracellular domain of DC-SIGNR. At initial contact, the length of the 7-repeat form of DC-SIGNR complexed with the glycan is D C = 26.6 ( 0.8 nm ( Table 1) . Modeling of the structure of the extracellular domain of DC-SIGNR bound to a Man 9 GlcNAc 2 oligosaccharide 17 suggests that the overall length of the ligand complex of the DC-SIGNR fragment used in the forceÀdistance measurements would be 26.5À27.0 nm, which agrees quite well with the measurements reported here. The crystallographic and forceÀdistance measurements also agree well with hydrodynamic measurement on the extracellular domains and the isolated neck domains. 19, 32 The equilibrium thickness is smaller at ∼25 nm but still comparable to the model prediction.
The slope of the line relating the distances at initial contact D C and the number of neck repeats gives a value of 3.87 ( 0.23 nm/ repeat (N = 22) ( Figure 3B ), which matches almost exactly the length of 3.7 nm for a single repeat determined from the crystallographic analysis of the isolated neck region. 17 A similar Biochemistry ARTICLE plot of D eq versus the number of repeats is shifted slightly, but the calculated slope is 3.6 ( 1.4 (N = 22). The larger error in the latter reflects the greater uncertainty in the estimated value of D eq , as discussed above. However, the comparative length measurements presented here provide exceptionally important direct experimental evidence that the neck repeat structure, which is observed essentially as a fragment out of context in the crystal structure, is an accurate reflection of the conformation of the neck in the intact molecule.
The force measurements show that, in the 6-and 7-repeat versions of DC-SIGNR, stable tetramers form a rigid neck region that projects the CRDs away from the membrane, ensuring strong, multivalent adhesion to the target membrane. These variants have comparable adhesion energies and solution binding affinities. However, disruption of the tetramer in the 5-repeat variant both produces a more flexible/compressible molecule and substantially reduces the adhesion. Further, loss of neck repeats in this variant reduces the extension of the CRDs from the membrane surface-a feature that could reduce access to pathogens.
The close correspondence between the measured 3.7 nm length of individual repeats in the neck domains obtained from the crystallographic and forceÀdistance measurements strongly reinforces proposed models for the organization of the extracellular domain with extended neck regions. 17 Hydrodynamic measurements and crystal structures of fragments of the extracellular portions of DC-SIGN and DC-SIGNR generated predictions that the CRDs in these two receptors would be positioned differently, in part due to differences within the final neck repeat adjacent to the CRD that would lead to splaying apart of the CRDs in DC-SIGNR. 17, 19, 32, 33 The forceÀdistance measurements provide experimental evidence that, although these receptors are very similar, they differ in overall length and thus in the disposition of the CRDs.
' ASSOCIATED CONTENT b S Supporting Information. Details of the protein surface coverage determinations from SPR data and the forceÀdistance measurements between protein monolayers and bare lipid membranes without neoglycolipid. This material is available free of charge via the Internet at http://pubs.acs.org.  Global mRNA Degradation during Lytic Gammaherpesvirus Infection Contributes to Establishment of Viral Latency During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3′ end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment. A number of viruses from diverse evolutionary lineages downregulate cellular gene expression through a variety of mechanisms [1, 2, 3, 4] . For certain viruses this host shutoff activity is a consequence of viral gene expression strategies, such as influenzavirus cap snatching [5] or picornavirus translation factor cleavage to facilitate alternate mechanisms of ribosome engagement [6] . For others, the contribution of host shutoff towards viral replication and gene expression is not as apparent, although proposed roles include resource reallocation and immune evasion [6, 7] . In many cases, the precise in vivo function has been difficult to delineate, in part due to the multifunctional nature of the viral proteins driving host shutoff and the lack of appropriate model systems. Within this latter category are the gammaherpesviruses, which direct host shutoff primarily through the induction of host mRNA degradation.
Gammaherpesviruses include the human pathogens Epstein-Barr virus (EBV/HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) [8] , the etiologic agents of some of the most common AIDS-associated cancers, such as Burkitt's lymphoma, nasopharyngeal carcinoma, and primary effusion lymphoma. The related murine gammaherpesvirus 68 (MHV68) is often used as a model for human gammaherpesviruses because, unlike KSHV and EBV, it is genetically tractable, readily replicates in tissue culture without the use of chemicals or overexpression to overcome latency, and infects small rodents including laboratory mice [9, 10, 11] . MHV68 has therefore been instrumental in the identification of factors that contribute to in vivo replication and pathogenesis of the gammaherpesviruses.
All herpesviruses exhibit a biphasic lifecycle. In the latent state few viral genes are expressed, and the viral genome is maintained in the nucleus of the host cell until stimulating signals induce lytic reactivation [12, 13, 14] . Most gammaherpesvirus-induced diseases are associated with latency. During the lytic cycle the vast majority of viral genes are expressed, and the virus undergoes active replication to produce progeny virions. At this stage during productive replication, gammaherpesviruses induce global mRNA degradation via the product of the alkaline exonuclease gene, termed SOX in KSHV, muSOX (ORF37) in MHV68, and BGLF5 in EBV [1, 15, 16] . SOX and its homologs are conserved in all subfamilies of herpesviruses, where they were first identified as DNA exonucleases critical for the resolution of branched structures that arise during viral DNA replication [17, 18, 19, 20] .
Only in members of the gammaherpesvirus subfamily does the SOX protein have an additional mRNA turnover function. Although the precise mechanism by which these proteins cause mRNA degradation remains unclear, biochemical evidence suggests that at least EBV BGLF5 and KSHV SOX possess some intrinsic ribonuclease (RNase) activity in vitro [21, 22] . In addition to mRNA turnover, SOX promotes the nuclear relocalization of cytoplasmic poly(A) binding protein (PABPC), which leads to aberrant polyadenylation and nuclear retention of mRNAs [23, 24] . The failure to repopulate the cytoplasm with newly transcribed mRNAs is thought to increase the overall magnitude of the host gene expression blockade [23] .
The contribution of host shutoff towards the gammaherpesviral lifecycle is unknown, and efforts to delete muSOX from MHV68 yielded virus unable to replicate, presumably due to an essential role for the DNase activity [16] . Previous studies found the DNase and host shutoff functions of SOX and BGLF5 to be genetically separable [25, 26] , making it possible to evaluate the role of each function of the protein in isolation. We report herein the identification of a functionally similar MHV68 muSOX point mutant that renders the virus selectively defective for host shutoff, enabling the analysis of the role of this phenotype during infection of cultured cells and in vivo. The mutant virus exhibited little to no defect during multi-step growth curves in tissue culture or during acute replication in the mouse lung following intranasal inoculation. Unexpectedly, the virus accumulated to significantly reduced levels in the lymph nodes at 10 days post infection and was highly attenuated at the downstream stage of latency establishment. Once trafficking was bypassed via an intraperitoneal infection, the host shutoff defective virus still was attenuated in establishing latency in the spleen. Our results identify for the first time a key role for the lytic mRNA turnover activity in establishing viral latency, emphasizing the important interplay between these seemingly disparate stages of the viral lifecycle.
We sought to evaluate the contribution of muSOX-induced mRNA turnover towards the gammaherpesvirus lifecycle through the generation of a muSOX mutant lacking mRNA turnover activity but retaining the conserved DNase function. We engineered mutations in the sites necessary for host shutoff activity in muSOX homologs from other gammaherpesviruses (EBV BGLF5 [25] and KSHV SOX [26] ), as well as in several sites selectively conserved amongst these homologs that lay outside the putative DNase domains [27] . However, none of the mutants generated had the desired single-function phenotype (Table S1) .
We therefore designed an unbiased screen to investigate a large number of mutants following the previous methodology used to identify the single-function SOX variants ( Figure 1A ) [26] . Briefly, the muSOX gene was amplified by error-prone PCR under conditions designed to introduce 1-4 mutations per gene. The PCR product was then cloned into a mammalian expression vector, and clones were screened for the ability to repress expression of a GFP reporter in a fluorescence-based assay in 293T cells. Mutants that did not deplete GFP fluorescence were categorized as host shutoff defective and further analyzed for wildtype protein expression levels by Western blotting, as even minor changes to the muSOX primary amino acid sequence often significantly reduced its protein expression. Finally, candidate mutants were tested for retention of DNase activity using an in vitro assay established previously for SOX and BGLF5 [25, 26] . After screening approximately 150 candidates, we identified a clone that lacked mRNA degradation activity of the GFP reporter, but retained wild-type protein expression levels and DNase activity. Sequencing revealed this clone to have only a single non-silent mutation, the amino acid substitution R443M, which we modified to R443I for purposes of screening by restriction digest. MuSOX R443I failed to suppress GFP protein expression in the fluorescent-based assay for host shutoff ( Figure 1B) , which was a consequence of its inability to degrade GFP mRNA ( Figure 1C ). R443I is expressed at slightly higher levels than wild-type (WT) muSOX ( Figure 1D ), as expected of a mutant that cannot promote turnover of its own mRNA. Importantly, WT and R443I muSOX both degrade linear DNA with very similar kinetics ( Figure 1E ). We conclude that the amino acid substitution R443I produces a single-function muSOX mutant selectively defective for host shutoff activity.
To investigate the role of muSOX-induced host shutoff during viral infection, we engineered a host shutoff defective MHV68 virus by replacing WT muSOX with muSOX R443I using bacterial artificial chromosome (BAC)-based homologous recombination [28] . The R443I mutation introduced an additional PsiI restriction site within the muSOX gene, allowing us to screen for successful recombination via restriction digest (Figure 2A) . We also generated a mutant rescue (MR) virus by replacing the R443I mutant with WT muSOX to ensure that any observed phenotypes could be attributed to the R443I mutation rather than to unexpected secondary mutations elsewhere in the viral genome. Restriction digests with PsiI and EcoRI confirmed that both recombinant viruses were successfully isolated and did not have any unexpected recombination events ( Figures 2B & 2C) . These viruses shall henceforth be referred to as MHV68.DHS (host shutoff defective virus) and MHV68.MR (mutant rescue virus). Sequencing of muSOX and the surrounding genomic regions in both recombinants confirmed that only the desired changes were introduced.
We next verified that the R443I mutation in MHV68.DHS conferred a host shutoff defect during viral infection. We assayed levels of several endogenous mRNAs by real-time quantitative PCR (RT-qPCR) following infection of NIH 3T3 fibroblasts with wildtype MHV68 (MHV68.WT), MHV68.DHS, or MHV68.MR. WT
Herpesviruses are ubiquitous pathogens infecting a wide range of hosts. They exhibit a biphasic lifecycle comprised of a lytic replicative stage and a relatively dormant latent stage. Members of the gammaherpesvirus subfamily include the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, the etiologic agents of some of the most common AIDS-associated malignancies. During a lytic gammaherpesvirus infection, cellular gene expression is severely compromised in a process termed host shutoff. In this study, we address the purpose of this activity in the viral lifecycle through the generation of a single amino acid point mutant selectively defective for host shutoff activity. Unexpectedly, host shutoff appears largely dispensable for viral replication, yet plays an important role in the ability of the virus to traffic and establish latency in the host organism. These findings link two distinct phases of the viral lifecycle and indicate that the global manipulation of gene expression may contribute to the establishment of a lifelong gammaherpesvirus infection. and MR infections reduced endogenous b-actin (actB), tubulin-b (tubb5), rplp2, and gapdh mRNA levels to 20-40% of the mock infected sample. In contrast, mRNA levels remained significantly higher in MHV68.DHS infected cells ( Figure 3A-3D) . Thus, the single amino acid change R443I in muSOX is sufficient to suppress virus-induced endogenous mRNA turnover. The somewhat lower endogenous mRNA levels in MHV68.DHS compared to mock infected cells could be due either to incomplete inactivation of the muSOX host shutoff function, or to the contribution of one or more additional viral genes towards host shutoff.
An additional phenotype linked to host shutoff caused by muSOX and its homologs is the relocalization of cytoplasmic poly(A) binding protein (PABPC) into the nuclei of infected cells [24] . We observed clear nuclear relocalization of PABPC in cells infected with MHV68.WT, whereas in mock infected or cells infected with MHV68.DHS, PABPC remained cytoplasmic ( Figure 3E ). Thus, cells were transfected with a plasmid expressing GFP alone or together with a plasmid expressing HA-tagged wild-type (wt) muSOX or muSOX R443I. GFP fluorescence was monitored 24 h post transfection, along with HA-muSOX, which was detected by immunofluorescence with anti-HA antibodies. Samples were co-stained with DAPI to visualize nuclei. (C) Cells were transfected for 24 h as described above, whereupon GFP mRNA and 18S rRNA levels were analyzed by Northern blot. Shown is a representative northern blot for GFP mRNA and 18S rRNA, and below is the normalized data from five independent experiments with means and standard deviations shown. (D) Total protein was harvested 24 h post transfection with the indicated plasmids, and Western blotted with anti-HA antibodies. The bottom band present in all samples is due to non-specific binding of the antibody. (E) In vitro translated GFP, wild-type muSOX, or muSOX-R443I was incubated with linear DNA for the indicated times. DNA was then extracted and separated by agarose gel electrophoresis. A representative figure from three independent experiments is shown. doi:10.1371/journal.ppat.1002150.g001 MHV68.DHS is defective for two hallmarks of host shutoff, mRNA depletion and PABPC relocalization. These results also demonstrate for the first time that muSOX-induced host shutoff is necessary to drive PABPC relocalization during a viral infection.
Previous studies with MHV68 mutants have shown that muSOX is critical for replication in cultured cells [16, 19] . To determine the contribution of host shutoff activity towards this defect, we performed a multi-step growth curve in murine fibroblasts. 3T3 cells were infected at a multiplicity of infection (MOI) of 0.1 plaque forming units (pfu) per cell and MHV68.WT, MHV68.DHS, and MHV68.MR all replicated to equivalent titers and with similar kinetics (Figure 4) . We sequenced the muSOX gene from MHV68.DHS samples at 5 days post-infection (dpi) and found no evidence for reversion or secondary mutations (data not shown). Therefore, muSOX-dependent mRNA degradation had little effect on viral titers in 3T3 cells through multiple replication cycles.
Interestingly, we noticed that plaques derived from MHV68.DHS infections differed morphologically from those obtained upon MHV68.WT or MHV68.MR infections ( Figure 5A ). To quantify these differences, we measured 75 plaques from 5 independent MHV68.DHS and MHV68.MR infections and found that, indeed, MHV68.DHS plaques were generally larger (p-value ,0.01) and had a broader frequency distribution ( Figure 5B ). This seemed unlikely to be a consequence of more rapid lytic replication, given the above growth curve results. In addition, the altered plaque size does not appear to be caused by enhanced cell-to-cell spread of the mutant virus, as we observed no significant differences in the ratio of extracellular to intracellular virus produced in 3T3 cells infected with MHV68.WT, MHV68.DHS, and MHV68.MR (data not shown).
We and others have consistently observed that plaques formed upon infection with wild-type (or mutant rescue) MHV68 are often quite heterogeneous in size. One possible explanation for this is asynchronous entry into the lytic cycle, in which smaller plaques arise from later entry into the lytic cycle. In this regard, we sought to determine whether the large plaque phenotype of MHV68.DHS might be due to enhanced entry into the lytic cycle, signified by an increased percentage of infected cells expressing early lytic proteins relative to the wild-type virus. To test this hypothesis, we acquired a version of MHV68 (MHV68-YFP) which constitutively expresses YFP from a CMV promoter, regardless of whether infected cells are in the lytic or latent phase [29] . Thus, YFP expression serves as a general marker of infection, whereas lytically infected cells are identified by immunofluorescence-based detection of the early lytic protein muSOX. NIH 3T3 cells were infected at an MOI of 1 with either MHV68-YFP or a version of MHV68-YFP bearing the muSOX R443I mutation (MHV68-YFP.DHS), and the percentage of cells co-expressing muSOX and YFP was calculated at 18 hours post infection. A similar number of YFP-expressing cells were detected in cultures infected with MHV68-YFP and MHV68-YFP.DHS, indicating equivalent efficiency of initial infection ( Figure 5C & data not shown). However, there was an approximately three-fold increase in the percent of cells coexpressing both YFP and muSOX following infection with MHV68-YFP.DHS compared to MHV68-YFP (30.1% versus 11.0%; Figure 5C ). A similar trend was observed when the YFPand muSOX-expressing cell populations were analyzed by flow cytometry following infection with these viruses ( Figure S1 ). Also in agreement with these findings were increased levels of the viral ORF54 transcript following a MHV68.DHS infection relative to MHV68.WT or MHV68.MR ( Figure S2 ). Thus, enhanced and/ or more rapid expression of lytic genes occurs in cells infected with MHV68.DHS, perhaps escalating entry into the lytic cycle and leading to larger plaques.
We also evaluated whether increased cell death might contribute to the larger plaques observed following a MHV68.DHS infection. We first measured necrosis by quantifying levels of lactate dehydrogenase released into the media at 16 hpi, but detected no measurable release following infection with any of the viruses ( Figure  S3A ). We next monitored apoptosis by several assays, including measuring caspase activity, and testing for loss of plasma membrane symmetry and increased membrane permeability. While there was a slight, but statistically significant, increase in caspase 3/7 activity following MHV68.DHS infection relative to MHV68.WT or MHV68.MR infection, in all three cases the level was below that observed in mock infected cells ( Figure S3B ), making it unlikely that this is a primary cause of the increased plaque size. Plasma membrane asymmetry was monitored using PE-conjugated Annexin V, which binds to a component of the plasma membrane normally found on the cytoplasmic surface, and membrane permeability was quantified by the nucleotide binding dye 7-Amino-Actinomycin (7-AAD). By flow cytometry we detected no significant increase in Annexin V binding or 7-AAD staining following infection with either MHV68.DHS or MHV8.MR relative to the uninfected control ( Figure S3C ). Collectively, these findings indicate that although a higher percentage of MHV68.DHS infected cells express viral lytic antigens, this does not appear to cause enhanced cell death, nor does it culminate in higher viral titers.
The presumed natural route of MHV68 infection is through the upper respiratory tract [30] , whereupon the virus undergoes lytic replication in the mucosal epithelial cells lining the mouse lung and nasal cavity. Following intranasal inoculation, viral loads in the lung peak 5-8 dpi before subsiding as the virus traffics to the draining lymph nodes and ultimately to the spleen [31] . A burst of lytic replication occurs in these sites of latency establishment [30, 32] which seeds subsequent latency, primarily in germinal center B cells, although also in macrophages and dendritic cells [33] . By 14-18 dpi splenomegaly develops, and as many as one out of 100 splenocytes harbor the latent viral genome [34] .
Host shutoff manifests with delayed early kinetics during lytic replication, consistent with the onset of muSOX expression [16] . We therefore hypothesized that MHV68.DHS would be attenuated during lytic viral replication in the lung. We infected C57BL/ 6 mice intranasally with 5610 4 pfu of MHV68.WT, MHV68.DHS, or MHV68.MR, and monitored viral titers in the lung at 3, 5, and 7 dpi by plaque assay. Viral titers were equivalent at 3 dpi for all three viruses, and at 5 and 7 dpi we observed only a very modest 2-3 fold defect in MHV68.DHS relative to MHV68.MR accumulation ( Figure 6 ). Although MHV68.WT reached higher titers than MHV68.MR at 5 dpi, the two viruses accumulated to equivalent levels at all earlier and later timepoints (see subsequent sections). To ensure MHV68.DHS had not reverted back to WT or incorporated any compensatory mutations, we sequenced the muSOX gene from infected lung homogenate but found no changes. Thus, contrary to our hypothesis, host shutoff appears largely dispensable for acute replication of gammaherpesviruses in vivo.
To evaluate the role of host shutoff during later stages of the viral lifecycle, mice were infected intranasally with 5610 4 pfu of MHV68.WT, MHV68.DHS, or MHV68.MR, and the spleens were harvested at 17 dpi, during the peak of latency establishment. Interestingly, mice infected with MHV68.DHS did not display the characteristic splenomegaly and had spleens three to four times smaller than MHV68.WT and MHV68.MR infected mice ( Figure 7A) , indicating a role for host shutoff in viral pathogenesis.
To evaluate the cause of MHV68.DHS attenuation, we first performed an infectious center assay to test whether the splenocytes from infected mice harbored latent virus capable of lytic reactivation. In this assay, equivalent numbers of splenocytes from infected mice are overlayed onto cultured mouse fibroblasts. Spontaneous lytic reactivation of the splenocytes leads to infection of the fibroblasts, which can be quantified by counting the resulting plaques [34] . Splenocytes from MHV68.DHS infected mice reactivated at a significantly lower frequency than those from MHV68.WT or MHV68.MR infections ( Figure 7B ). This reactivation frequency was independently assessed in a subset of the samples by a limiting dilution cytopathic effect assay, which yielded identical results (data not shown). Plaque assays on spleen homogenates confirmed that no preformed virus was present, indicating that the virus detected in these experiments originated from latently infected cells.
The reduction in lytic reactivation frequency could either be a consequence of a decreased number of latently infected splenocytes or, alternatively, a failure of latently infected cells to undergo lytic reactivation. To distinguish these possibilities, we first monitored viral DNA load in the spleens of infected mice using a previously described qPCR assay for the glycoprotein B (gB) gene [35] and found that MHV68.DHS infected splenocytes harbor fewer viral genomes than splenocytes infected with MHV68.WT or MHV68.MR ( Figure 7C ). We also analyzed the frequency of splenocytes harboring the viral genome by limiting dilution PCR. Briefly, serial dilutions of splenocytes were plated in a 96 well plate and subjected to nested PCR for the viral ORF50 gene [36] . Spiked template controls confirmed that in this assay we were achieving near single copy sensitivity for the viral genome, with a very low rate of false positives (data not shown). While approximately one out of 200 splenocytes latently harbor the MHV68.WT or MHV68.MR genome, the frequency of MHV68.DHS genome harboring splenocytes was less than one out of 10,000 splenocytes ( Figure 7D ). Thus, while host shutoff appears dispensable for acute replication in the lung, it plays an important role in the downstream events leading to efficient latency establishment in the spleen.
During the normal course of an infection, the virus traffics from the site of lytic replication in the lung to the draining lymph nodes and spleen where latency is established. We reasoned that the decreased frequency of latently infected splenocytes during MHV68.DHS infection could be a consequence of a defect in trafficking, persistence at these sites, or a latency establishment defect. We quantified levels of virus in the cervical lymph node at 10 dpi, an intermediate time point after peak virus replication in the lung but before peak latency establishment in the spleen. Following an intranasal inoculation, high levels of MHV68 have been found in the cervical lymph node at this time point [30, 32] . Interestingly, we detected significantly lower levels of MHV68.DHS at this site relative to MHV68.MR ( Figure 8A ), suggesting that muSOX-induced mRNA degradation contributes to the ability of the virus to traffic to or persist at the sites of latency establishment.
To determine whether this was the root cause of the defect in latency establishment upon MHV68.DHS infection, we next infected mice via the intraperitoneal route, which bypasses the requirement for the virus to undergo acute replication and traffic through the lymph before reaching the spleen [37] . At 19 dpi, we found significantly less MHV68.DHS virus in the spleen relative to MHV68.MR by qPCR ( Figure 8B ), indicating that a defect in viral trafficking cannot fully account for the latency establishment defect observed during an intranasal infection. Collectively, these data indicate that muSOX-induced host shutoff plays important roles both in the ability of MHV68 to traffic to the lymph nodes, as well as establish latency in the spleen.
All gammaherpesviruses studied to date block host gene expression through widespread mRNA degradation, yet the contribution of this function towards the viral lifecycle remained unknown. Here we analyzed the impact of host shutoff both in tissue culture and in vivo infections with the murine gammaherpesvirus MHV68, and we found this activity to be largely dispensable for acute lytic replication yet critical for the downstream viral accumulation in the lymph nodes and subsequent establishment of latency in the spleen. These results were unexpected given that MHV68-induced host shutoff is executed by muSOX, a viral protein whose expression has only been detected during the lytic cycle [38, 39, 40] . In addition, the role for muSOX-induced mRNA turnover clearly diverges from that of the alphaherpesvirus host shutoff factor vhs, which similarly induces global mRNA degradation but plays a critical role during acute replication in vivo [41, 42, 43] . The severe attenuation of HSV-1 and HSV-2 vhs mutants is hypothesized to be a consequence of ineffective immune evasion, as vhs blocks dendritic cell activation and down-regulates the type I interferon response [44, 45, 46] . In contrast, we find little difference between MHV68.MR and MHV68.DHS during acute replication in mice, suggesting that while alpha-and gammaherpesviruses block host gene expression via analogous mechanisms, the functional ramifications of this activity are distinct, and may relate to the unique in vivo biology of each class of virus.
Modeling the muSOX three-dimensional structure from the KSHV SOX or EBV BGLF5 crystal structure [21, 47] indicates that amino acid R443 is positioned on the outer surface, away from the catalytic core where nucleic acids are presumably cleaved (data not shown). Although SOX, BGLF5, and muSOX are functionally homologous and likely induce mRNA degradation through a similar mechanism, the identified amino acid mutations that selectively remove host shutoff activity are not conserved, nor are they positioned in the same three-dimensional region. Likely these different mutations alter the local structure of each protein thereby inhibiting or weakening the mRNA or co-factor interactions which contribute to overall mRNA degradation. In this regard, we anticipate that the residual host shutoff activity observed upon infection with the MHV68.DHS virus is at least partially due to incomplete inactivation of muSOX mRNA turnover activity by the R443I mutation.
Host shutoff has been hypothesized to play a role in the diversion of gene expression resources and machinery towards the virus during lytic replication [16, 48] , as well as general immune evasion through the down-regulation of immune stimulatory factors [49] . Our failure to detect a significant replication defect for MHV68.DHS during a multi-step growth curve in cultured murine fibroblasts or in the mouse lung implies that resource reallocation is unlikely to be the primary role for muSOX-induced mRNA depletion. Likewise, the similar viral titers in the lungs of mice infected with MHV68.MR or MHV68.DHS argues against a role for muSOX in the general suppression of host innate immune responses Because MHV68.DHS still retains residual mRNA degradation activity, it is possible that some degree of host shutoff could be important for viral replication. However, the appearance of significant defects in downstream viral events even with a partial host shutoff defect further strengthens our conclusion that host shutoff plays a vital role in the lifecycle and pathogenesis of MHV68 in vivo.
The first major defect observed in an in vivo MHV68.DHS infection is significantly lower levels of the virus in the lymph nodes at 10 dpi. Following an intranasal infection, wild-type MHV68 undergoes lytic replication in the lung and upper respiratory track, after which the virus drains to the lymph nodes and spleen where a variety of cell types are infected, including macrophages, dendritic cells, and B-cells [37, 50, 51, 52] . A burst of lytic replication occurs at these sites [30, 32] , after which replicating virus is cleared and long-term latency is established. Lower levels of MHV68.DHS in the lymph node could be caused by a cell-type specific role for muSOX-dependent mRNA degradation in viral replication or immune evasion during viral transport to, and maintenance in, the lymphatic tissue. The observation that the EBV muSOX homolog (BGLF5) down-regulates HLA class I molecules and CD8 + T cell recognition in cultured cells may support this model [25] . The means by which the virus traffics to the sites of latency establishment remain unclear, yet it is likely that latently infected B cells carry the virus given that viremia is undetectable during an infection, and latently infected B cells are present in the lung very early after infection [37, 53, 54] . A failure of MHV68.DHS to establish latency in these cells might also cause their selective immune-based eradication and decreased accumulation in the lymph nodes. Alternatively, muSOX could influence the ability of the virus to reactivate from latency, as this process has been linked to efficient latency establishment. Mutations in a number of genes alter the frequency of lytic reactivation and lead to lower levels of latency establishment following an intranasal infection, such as vcyclin (ORF72) [55] , M1 [56] , M2 [57, 58] , and LANA (ORF73) [59, 60] , but unlike muSOX these genes are all expressed during latency as well as during lytic replication [61] . Mutations in the lytic ORF36 gene encoding a protein kinase also lead to defects in latency establishment and reactivation [62, 63] , which have been tracked to the requirement for ORF36 to inhibit the IRF-3 mediated type I interferon response [62] and its modification of the DNA-damage response protein H2AX [63] . Experiments are currently underway to examine MHV68.DHS replication, reactivation, and latency establishment in specific cell types relevant to in vivo infection.
The second major defect observed upon MHV68.DHS infection is a marked reduction in the number of infected splenocytes present during peak latency establishment relative to mice infected with wild-type or mutant rescue viruses. This defect could arise simply as a downstream consequence of the aforementioned impairment in trafficking. However, significantly reduced levels of viral DNA are detected in the spleen even after an intraperitoneal infection, which bypasses the need for acute replication in the lung and subsequent trafficking through the lymph nodes [37] . Thus, MHV68.DHS appears defective in both trafficking and latency establishment in the spleen. Such a phenotype might occur if MHV68.DHS-infected cells preferentially entered the lytic cycle, perhaps enabling more efficient clearance by the immune system. This model is supported by our observation that an increased percentage of cultured 3T3 cells infected with MHV68.DHS express lytic markers relative to those infected with MHV68.WT. However, if this represents enhanced entry into the lytic cycle, there must be a downstream defect that tempers subsequent viral output, as the host shutoff mutant virus does not replicate to higher titers in 3T3 cells than the wild-type virus. In this regard, host shutoff could be important for optimizing the balance of host or viral proteins in an infected cell, as has been reported in an EBV mutant lacking BGLF5, where altered levels of proteins inhibited viral maturation and egress [64] . One mechanism that could shift the balance of infection away from latency is if muSOX down-regulates factors that inhibit latency establishment; such negative regulators would then accumulate in the presence of the R443I mutant, driving cells into lytic replication. MuSOX activity may also be required for the virus to achieve the appropriate level of host and or viral factors packaged into the viral particle in order to efficiently establish latency in newly infected cells rather than enter the lytic cycle.
The large plaque phenotype observed with MHV68.DHS is similar to what is observed following infection with MHV68 constitutively expressing (or over-expressing) the lytic transcription factor RTA [65] . Also similar to MHV68.DHS, these viruses replicate to near wild-type levels in the lung but establish latency at lower levels in the spleen. The constitutive RTA-expressing viruses have been found to efficiently traffic to the spleen but fail to be maintained at this site [66] . This attenuation has been ascribed to the viruses producing excessive amounts of the RTA-inducible lytic genes, creating an intracellular environment favoring immediate entry into the lytic cycle rather than entering latency. Thus, during an infection, an important role for muSOX may be to ensure that factors driving lytic replication are not overrepresented. While it is tempting to speculate a connection between the increased percentage of lytically infected 3T3 cells and the latency establishment defect in vivo with MHV68.DHS, at present the precise relationship between these observations remains unclear.
Given the broad effects of host shutoff, the mechanism of MHV68.DHS attenuation is anticipated to deviate from the genefor-gene interactions associated with many virulence factors. Further research in this area will uncover the likely means by which host shutoff influences latency establishment, as well as reveal novel connections between the lytic and latent stages of the gammaherpesviruses lifecycle.
A hemagglutinin (HA) tag was introduced at the 59 end of muSOX by PCR using the primers 59-CGGAATTCATGGCT-TACCCATACGATGTACCTGACTATGCGATGGAAG GG-TCGATTATTC-39 and 59-ATAGTTTAGCGGCCGCTTA-GGGGGTTATGGGTTTTCT-39. HA-muSOX was then cloned into the EcoRI/NotI sites of pCDEF3 containing a T7 promoter to generate pCDEF3-T7-HA-muSOX. MuSOX was randomly mutagenized by PCR with the Genemorph kit (Strategene) according to the manufacturer's protocol using 35 ng of pCDEF3-T7-HA-muSOX as template, the above primers, and 30 PCR cycles to generate a pool of random mutants. The mutants were then cloned into the EcoRI/NotI sites of pCDEF3-T7. R443I was generated by QuickChange (Stratagene) using the primers 59-GCTCATCATCACTCCTGT TATAATTCCATC-TACTGTGCTGC-39 and 59-GCAGCACAGTAGATGGAAT-TATAACAG GAGTGATGATGAGC-39.
HEK293T, COS7, NIH 3T3, NIH 3T12, and Vero cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). 293T cells were transfected with Effectene (Qiagen) following the manufacturer's protocol.
The green fluorescent protein (GFP)-expressing MHV68 bacterial artificial chromosome (BAC) infectious clone has been described elsewhere (RcHV68A98.01 [28] ), and mutants were generated by allelic exchange as previously described [67] . To generate the MHV68.DHS BAC, a targeting region consisting of 566 nt upstream and 568 nt downstream of the mutation site was ligated into pGS284 between BglII and NotI restriction sites and electroporated into the S17lpir strain of E. coli. The MHV68-YFP BAC infectious clone was a generous gift from Dr. Samuel Speck (Emory University) [29] , and MHV68-YFP.DHS was generated using the same methodology as MHV68.DHS. The targeting vector for the mutant rescue BAC (MHV68.MR) was generated by ligating the region around wild-type muSOX into pGS284. Targeting vector-containing cells were cross-streaked with BACcontaining GS500 cells and successful recombinants were identified by colony PCR and subsequent digest with PsiI. BAC DNA was isolated from positive clones using the Qiagen Large-Construct kit (Qiagen). BAC variants were verified by restriction digests with EcoRI and PsiI and sequencing of the region surrounding the recombination site. BAC-derived MHV68 virus was produced by transfecting 2 mg of BAC DNA into NIH 3T3 cells using SuperFect (Qiagen). Virus was then amplified in NIH 3T12 cells and titered by plaque assays on NIH 3T3 cells. Before infecting mice, the loxP-flanked BAC vector sequence was removed from the recombinant viruses by passaging the virus over Vero cells expressing Cre recombinase (kindly provided by Dr. Samuel Speck, Emory University) [59] , and BAC removal was confirmed by PCR analysis.
MuSOX expression and PABPC or HA-muSOX localization were analyzed as described previously [16, 23] . Briefly, NIH 3T3, HEK293T, or COS7 cells were grown on coverslips, and infected with MHV68 variants or transfected with HA-muSOX variants. Cells were then stained with rabbit polyclonal anti-muSOX (1:25 dilution), mouse monoclonal anti-PABPC 10e10 (1:25 dilution) (Santa Cruz Biotechnology), or mouse monoclonal anti-HA (1:500 dilution) (Abcam) and AlexaFluor 546-or 488-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (1:1500). Coverslips were mounted in DAPI-containing Vectashield mounting medium (Vector Labs) to stain cell nuclei.
For flow cytometry analysis, NIH 3T3 cells were infected with the indicated virus and, 18 hpi, cells were washed with PBS and harvested via trypsin digestion. Cells were fixed in a 4% formaldehyde solution, permeabilized in 1% Triton X-100 and 0.1% sodium citrate in PBS, and incubated with anti-muSOX antibodies at a 1:12.5 dilution in 1% Triton X-100, 0.5% Tween, and 3% BSA in PBS. Stained cells were then washed in PBS and incubated with goat anti-rabbit antibodies conjugated to PE-Cy5.5 (Invitrogen) at a dilution of 1:150. Data were collected on an EPICS XL cytometer (Beckman-Coulter) and analyzed using FlowJo software (Tree Star).
For Western blotting, cell lysates were prepared in RIPA buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (vol/vol), 0.1% SDS (w/vol)] containing Protease Inhibitor Cocktail (Roche) and quantified by Bradford assay (Bio-Rad). Equivalent amounts of each sample were resolved by SDS-PAGE, transferred to a PVDF membrane, and Western blotted with anti-HA 12CA5 monoclonal antibodies (Invitrogen; 1:5,000) and HRP-conjugated goat anti-mouse secondary antibodies (Southern Biotechnology; 1:5,000).
For Northern blotting, total RNA was harvested using RNA-BEE (Tel-Test) and resolved by agarose-formaldehyde gel electrophoresis. RNAs were transferred to a 0.45 mm nylon membrane and probed with 32 P-labeled GFP DNA probes generated using the Rediprime II random prime labeling system (GE Healthcare).
DNase activity of the muSOX variants was performed as described previously [16] . Briefly, muSOX variants were in vitro transcribed with the mMessage mMachine T7 Kit (Ambion) and translated using rabbit reticulocyte lysates (Promega). Translational product was incubated with linear DNA at 37uC for the indicated time period and then the DNA was phenol/chloroform extracted, and resolved by agarose gel electrophoresis. One-sixth each IVT reaction was also separated by SDS-PAGE. The gels were then fixed, dried, and visualized by autoradiography to verify equivalent protein expression.
To quantify RNA, we isolated RNA from transfected cells using RNA-Bee (Tel-Test) or the Zymo Mini RNA II Isolation Kit (Zymo Research). Samples were treated with Turbo DNase (Ambion) according to the manufacturer's protocol to remove genomic DNA contamination. To generate cDNA, RNA samples were reverse transcribed using AMV RT (Promega) and an oligo dT or 18Sspecific primer. 18S rRNA or GAPDH mRNA levels were quantified using TaqMan ribosomal RNA control reagents or TaqMan rodent GAPDH control reagents (Applied Biosystems). Other endogenous mRNA levels were quantified using Applied Biosystems Taqman Gene Expression Assays (actB-Mm01205647_g1, rplp2-Mm03059047_gH, tubb5-Mm00495806_g1). To quantify viral genomes, DNA was first isolated from spleen cells or cervical lymph node cells by the Qiagen QIAamp DNA Mini Kit following the manufacturer's protocol. Viral DNA levels were then measured using a previously described assay targeting the MHV68 ORF8 gene [35] . All qPCR reactions were performed with TaqMan Universal PCR Master Mix.
To measure levels of lactate dehydrogenase (LDH) released into the media during an infection, NIH 3T3 cells were infected at an MOI of 5 for 16.5 h. Infection supernatant (60 ml) was mixed with 60 ml LDH detection reagent in triplicate in a 96-well plate as described previously [68] . Absorbance was read on an Elisa-reader at 490 nm wavelength. To detect caspase activity, NIH 3T3 cells were infected at an MOI of 5 for 24 h, whereupon caspase activity was measured using the Caspase-Glo 3/7 Assay System (Promega) following the manufacturer's protocol. To monitor Annexin V-PE and 7-AAD staining by flow cytometry, NIH 3T3 cells were infected at an MOI of 10 for 18 h. Cells were then washed with PBS and harvested via trypsin digestion. Infection supernatant, PBS wash, and cells were combined and pelleted by centrifugation. Cells were washed in PBS and then stained for Annexin V-PE (BD Biosciences, Material # 556422) and 7-AAD (BD Biosciences, Material # 559925) via the manufacturer's protocol using 3 ml of each dye. Data were collected on an EPICS XL cytometer (Beckman-Coulter) and analyzed using FlowJo software (Tree Star).
Female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and infected when 4-6 weeks old. Mice were anesthetized with isoflourane and inoculated intranasally with 5610 4 plaque forming units (pfu) in 20 ml DMEM (Invitrogen). For intraperitoneal infections, 1610 3 pfu in 0.2 ml PBS were injected into the peritoneal cavity of mice. Lungs were harvested at 3, 5, or 7 dpi, homogenized with a tissue homogenizer for 1 minute at 24,000 rpm in 10 ml DMEM with 10% FBS, 100 U of penicillin per ml, and 100 mg of streptomycin per ml (Pen/Strep, Invitrogen). Spleens were harvested at 17 or 19 dpi. To identify any preformed infectious particles, half of each spleen was homogenized as above in 5 ml DMEM with 10% FBS, and Pen/Strep. The tissue homogenate was then assayed for viral particles by plaque assay on monolayers of NIH 3T3 cells overlaid with 1% agarose for 4 days. The cells were then fixed and stained with 0.04% methylene blue. Splenocytes were isolated from the other half of the spleen by dissociating the spleen and passing through a 40 mm cell strainer (BD-Falcon). Cells were then pelleted, resuspended in red blood cell lysis buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA), and incubated at room temperature for 5 minutes. Cells were again pelleted and resuspended in RPMI medium with 10%FBS and Pen/Strep before counting. Cervical lymph nodes were harvested at 10 dpi and isolated in the same manner as splenocytes, but without lysing the red blood cells.
The number of reactivating splenocytes was determined as described previously [34] . Briefly, 21,000 NIH 3T3 cells were plated per well in a 24-well plate the day before infection. 2.5610 6 , 5610 5 , or 1610 5 splenocytes were overlayed onto the NIH 3T3 cells and incubated for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.04% methylene blue to visualize plaques.
The frequency with which cells latently harbor the viral genome was determined as described previously [36] . Briefly, 4-fold serial dilutions of splenocytes were plated in 96-well plates with 16 wells per dilution. Nested PCR for the ORF50 gene was performed and the resulting PCR product was run on an agarose gel. Wells positive for a PCR product by ethidium bromide staining contain at least one copy of the viral genome. Single-copy sensitivity was confirmed using serial dilutions of MHV68 BAC DNA.
All graphs were designed and statistical analysis performed using the GraphPad Prism Software version 4 or 5. Data were analyzed for statistical significance using the Student's t-test or, for in vivo data, the Mann-Whitney nonparametric test. For limiting dilution PCR analysis, data were subject to nonlinear regression using the log(agonist) vs. response curve with a nonvariable slope.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California Berkeley (Permit Number: R292-0507). All animals were anesthetized prior to infection with isoflurane, and all efforts were made to minimize suffering.  A modified MS2 bacteriophage plaque reduction assay for the rapid screening of antiviral plant extracts INTRODUCTION: Traditional methods of screening plant extracts and purified components for antiviral activity require up to a week to perform, prompting the need to develop more rapid quantitative methods to measure the ability of plant based preparations to block viral replication. We describe an adaption of an MS2 plaque reduction assay for use in S. aureus. RESULTS: MS2 bacteriophage was capable of infecting and replicating in B. cereus, S. aureus and F + E. coli but not F- E. coli. Indeed, both B. cereus and S. aureus were more sensitive to MS2 induced lysis than F+ E. coli. When MS2 bacteriophage was mixed with Camellia sinensis extract (1 mg/ml), Scaevola spinescens extract (1 mg/ml) or Aloe barbadensis juice and the mixtures inoculated into S. aureus, the formation of plaques was reduced to 8.9 ± 3.8%, 5.4 ± 2.4% and 72.7 ± 20.9% of the untreated MS2 control values respectively. CONCLUSIONS: The ability of the MS2 plaque reduction assay to detect antiviral activity in these known antiviral plant preparations indicates its suitability as an antiviral screening tool. An advantage of this assay compared with traditionally used cytopathic effect reduction assays and replicon based assays is the more rapid acquisition of results. Antiviral activity was detected within 24 h of the start of testing. The MS2 assay is also inexpensive and non-pathogenic to humans making it ideal for initial screening studies or as a simulant for pathogenic viruses.  Equal Graph Partitioning on Estimated Infection Network as an Effective Epidemic Mitigation Measure Controlling severe outbreaks remains the most important problem in infectious disease area. With time, this problem will only become more severe as population density in urban centers grows. Social interactions play a very important role in determining how infectious diseases spread, and organization of people along social lines gives rise to non-spatial networks in which the infections spread. Infection networks are different for diseases with different transmission modes, but are likely to be identical or highly similar for diseases that spread the same way. Hence, infection networks estimated from common infections can be useful to contain epidemics of a more severe disease with the same transmission mode. Here we present a proof-of-concept study demonstrating the effectiveness of epidemic mitigation based on such estimated infection networks. We first generate artificial social networks of different sizes and average degrees, but with roughly the same clustering characteristic. We then start SIR epidemics on these networks, censor the simulated incidences, and use them to reconstruct the infection network. We then efficiently fragment the estimated network by removing the smallest number of nodes identified by a graph partitioning algorithm. Finally, we demonstrate the effectiveness of this targeted strategy, by comparing it against traditional untargeted strategies, in slowing down and reducing the size of advancing epidemics. Understanding and containing the spread of an infectious disease has always attracted a lot of interest from the scientific community, and even more so after the recent SARS and H1N1 outbreaks. Besides their social and healthcare impacts, severe infectious disease outbreaks also present an important burden to the economy through the decrease in productivity and high cost of treatment. As denser populations promote faster spreading, this problem can only grow in severity and magnitude with the increasing world population density. Motivated by this, many works have been done to predict [1, 2] or even contain [3] [4] [5] [6] the spread of severe epidemics. In spite of these efforts, effective control of infectious disease outbreaks continues to elude us.
A recent important advancement in the field is the application of network theory to study epidemic dynamics. Network-based models have been shown able to accurately explain complex phenomena in terms of the relatively-simple interactions between its small constituents, and are therefore broadly applicable to various different fields [7] . In the area of infectious diseases, numerous studies have been done on modeling epidemic using a network approach -mainly covering sexually-transmitted infections [8] [9] [10] [11] , respiratory and flu-like diseases [2] , and general features of infectious disease dynamics [12] [13] [14] [15] [16] [17] .
From the network point of view, we speculate that different infectious diseases might have very similar infection networks if they share the same mode of transmission. In particular, we believe that severe respiratory infections such as H1N1 and SARS share an infection network similar to that of the less severe common cold. Hence, infection network inferred from the latter can be useful in controlling rare outbreaks of the former. Estimating the infection network from common infections has the advantage of a large volume of daily incidences. As we will show in the Results section, the volume of incidences data gathered is critical in getting accurate estimation of the network.
Traditional epidemic intervention procedures, such as quarantine and other social distancing measures, involve weakening or cutting links around the infected nodes. However, these procedures are not systematic from the network point of view. A more effective intervention strategy would employ understanding the 'shape' of the infection network and applying that knowledge to efficiently tear the network apart. This can be done by targeting nodes that play important roles in the network (i.e. the 'hubs'), and the 'backbones' connecting one hub to another.
Based on the above ideas, we proposed a targeted method to effectively contain infectious disease epidemics. This strategy involves estimating the infection network of a severe disease using incidences data from common infections sharing the same infection network, and fragmenting the network into disconnected pieces using a graph partitioning method. To test the proposed strategy in principle, we first generate artificial social networks of different sizes but with roughly the same clustering characteristics to serve as our infection networks. Then we simulate multiple SIR epidemics on the networks to play the role of common infections circulating in society. We apply censorships on the incidence data collected to emulate the low reporting rates of common infections, and use the censored incidences to construct estimates of the original infection networks. To mitigate new epidemics, we fragment the estimated infection networks. To do this, we apply a graph partitioning method on the estimated networks to identify the smallest sets of nodes that, when removed, will efficiently break the networks up into isolated pieces. Finally, we evaluate the effectiveness of this targeted strategy by comparing against traditional untargeted methods. While each of the problems has been studied independently (see for example [8, 18] for network reconstruction and [19] [20] [21] [22] for graph partitioning), to the best of our knowledge no work has been done applying both methods to control epidemics.
At this point in time, we know of no available databases of common infections that (1) are comprehensive enough for reconstructing the infection network and (2) have relevant ongoing epidemics to test the proposed strategy. Hence, we used computer simulations to study the proposed method.
Many naturally-occurring networks like the Internet, the World Wide Web, and biological networks are scale-free and are thus well described by Barabasi's preferential attachment model [23] . Others argued, however, that social networks are different as they show strong clustering of nodes (also called community structures) and their degree distributions are not power laws [24] . To specifically reproduce the community structures seen in social and social-like networks, Holme and Kim modified the preferential attachment model to incorporate clustering [25] . Newman et al., on the other hand, started out with random graphs and progressively build the social-like degree distribution [26] , whereas Boguna et al. and Jin et al. proposed friendship-formation dynamics models to generate social-like networks from scratch [27, 28] .
For our proof-of-concept study, we generate social-like networks to act as our infection networks. We follow the three intuitive rules described by Jin, Girvan, and Newman (JGN) in Ref. [28] : (1) the probability of two individuals meeting is high if they have one or more mutual friends, and low otherwise; (2) the friendship between two individuals is reinforced by regular meetings, but decays with time if they rarely meet; and (3) there is a maximum number of friends one can have.
Following the discrete algorithm presented in Ref. [28] , we first start with a random network of N nodes and m links. The average degree of the network (average number of links per node) is given by SkT~2 m N . At each time step, we select n m pairs of nodes that have at least one mutual friend. This is done by first picking n m intermediate nodes at random, before choosing two neighbours of each to become the pairs. In addition to this, we choose n r other pairs of nodes uniformly at random, with n m .n r . For every pair that is not already connected, we form a new link between them provided that both nodes have not reached the maximum number of friends L. At the end of each time step, we randomly break r existing links to simulate the friendship decay. We then calculate the clustering coefficient c of the network using the method by Schank and Wagner [29] . After the clustering coefficient stops increasing and only fluctuates about a time-independent long-run average, we say the network has converged and stop the simulation. As expected, the clustering coefficients of the converged networks are much higher than c R & SkT N for random networks with the same N and ,k..
In generating the infection networks, we set L = 50 as the maximum number of friends a node can have. To produce high clustering coefficients, we ensure that the mutual friend formation is dominant over the random friend formation by choosing n m = 400 and n r = 100. For simplicity, we choose r = n r +n m = 500 such that the total number of links (hence the average degree ,k.) of the initial random network remains more or less constant. This way, we can generate social-like infection networks with arbitrary size and average degree by simply adjusting N and ,k. of the initial random network to the desired values.
After generating the infection network, we simulate S susceptible-infected-recovered (SIR) epidemics. To start the epidemic, we initialize the network so that all nodes are susceptible. One random node is then infected to act as seed of the epidemic. At each time step, every infected node will transmit the disease to its susceptible neighbours with probability q. After a certain time interval t R , the infected nodes will recover and become immune to subsequent infections. This way, the number of infected nodes grows from the single seed, peaks, and thereafter decreases as more and more nodes recover and become immune. When there are no more infected nodes in the network, the epidemic ends and all nodes are reset to the susceptible state for simulating the next epidemic.
In this SIR model, the probability q of infecting susceptible neighbours reflects the characteristic infection rate of the particular disease over the simulation time step Dt. For a given time step size Dt, a more infectious disease will have a larger q, whereas a less infectious disease will have a smaller q. For a given infectious disease, q will be smaller for a smaller Dt, and larger for a larger Dt. The simulation time step Dt itself is chosen, for simplicity, to be roughly equal to the typical incubation and recovery period of the disease (about 3-5 days for common cold). This implies that t R <Dt, i.e. the infected nodes recover after one simulation time step.
When running the simulations, we find that the infection rate q must be greater than a certain minimum value q min for the epidemics to be self-sustaining and cover a large fraction of the network. We also observe that q min depends on the network's average degree ,k.. A less-connected network with smaller average degree ,k. requires a larger value of q min to sustain an epidemic, as compared to a more highly-connected network with higher ,k.. Hence, we fine tune the infection probability q so that 50%-70% of the nodes are infected in one epidemic simulation. We find that q min < 0.80 in the case of ,k. = 3, q min < 0.20 for ,k. = 10, and q min < 0.08 for ,k. = 20. Finally, we note that while it is relatively easy to infect the majority of the nodes in the network with high ,k., infecting all nodes is extremely hard. We need q to be very close to one before the epidemic infects the entire network.
When collecting incidence data of common and mild diseases, we expect low incidence reporting rates because: (1) some infected individuals do not show symptoms (sub-clinical cases); and (2) some others choose not to consult doctors despite showing symptoms (unreported cases). In countries where consultation fees are high, we expect the latter to be dominant. Fortunately, the low reporting rates are compensated by the large volume of daily incidences generated by such diseases. We also note that voluntary reporting rates in Singapore are relatively high because of the low consultation fees, making the strategy for estimating the infection network presented below attractive.
To simulate different reporting rates, we censor the incidence data before using them to estimate the infection network. Here, the censor rate 0,C,1 is defined as the fraction of incidence data discarded randomly, and hence not used in the network estimation. For our study, we vary C from 50% to 85%.
Our network estimation algorithm is essentially a modified JGN algorithm. To estimate the unknown infection network from the incidence data, we note that all newly-infected individuals must have been infected by individuals that were infected one time step back, as shown in Figure 1 . For the epidemic shown in Figure 1 , the incidences are {9}, {5, 12}, {4, 6, 10, 11, 13}, {3, 8} at time steps t = 0, 1, 2, 3 respectively. Suppose some of these incidences were censored, and we ended up with the censored incidences {9}, {5}, {4, 11, 13}, {8} at time steps t = 0, 1, 2, 3 respectively. Based on these censored incidences, we then draw a tentative link between nodes 9 and 5, because the infection of node 9 precedes the infection of node 5. We then draw tentative links between the pairs (5, 4), (5, 11) , (5, 13) because the infection of node 5 precedes the infection of nodes 4, 11, 13. Finally, we draw a tentative link between the pairs (4, 8) , (11, 8) , (13, 8) , because the infection of nodes 4, 11, 13 precedes the infection of node 8.
As we can see, only the links (4, 5) and (5, 9) are correctly estimated. The rest of the estimated links are all wrong, partly because of the algorithm, and partly because of the censorship. To improve the accuracy of our estimated network, all tentative links are introduced with a weight of w = 1.00. Thereafter, the weight of all links decay by Dw = 20.01 every time step. Tentative links are removed when their weights fall to zero. In the above example, the links (5, 9) , (4, 5) , (5, 11) , (5, 13) , (4, 8) , (8, 11) , (8, 13) are introduced at t = 1, 2, 2, 2, 3, 3, 3, and so when the epidemics end at t = 4, their weights would be 0.97, 0.98, 0.98, 0.98, 0.99, 0.99, 0.99 respectively. We then repeat the estimation over more SIR epidemics. In subsequent epidemics, if an existing estimated link is reactivated, it is reinforced by adding Dw = 1.00 to its weight. We expect the algorithm to produce mostly wrong links at first. However, as the weights are decaying with time, most of the wrongly-estimated links will disappear at the end, because they will not consistently appear in the estimation process. On the other hand, correctly-estimated links tend to get constantly reinforced over multiple epidemics. Hence, we expect the population of correctly-estimated links to increase over time. More importantly, we note that existing estimation can always be further refined by incorporating new incidence data as and when they become available.
From the network point of view, efficient epidemic mitigation can be achieved by identifying and targeting the most denselyconnected nodes (also called 'hubs') in the infection network. However, targeting only hubs in the infection network may not be the most efficient mitigation strategy, because a strongly clustered network may not break up into fragments after these are removed.
Since we want to fragment the infection network to stop the epidemic from spreading across the entire network, we perform equal graph partitioning (EGP) [22] on the estimated network. EGP identifies the smallest set of nodes which, when removed, will break the network into isolated chunks of roughly equal sizes.
To start the EGP, we first randomly assign every node into two groups A and B. We then move all nodes in A that is connected to B, and all nodes in B that is connected to A, into a third group C (also called the separator group). Once this is done, there are no nodes in A that is connected directly to B and vice versa. Following this we minimize the size of group C by swapping nodes in C with those in A and B, without introducing direct connections between A and B. A swap is accepted if it results in a smaller group C. We perform repeated swaps until no further improvements can be made. If we then remove this optimal set of nodes in C, the network will be efficiently broken into two disconnected chunks. When necessary, the resulting chunks can be further fragmented into even smaller pieces by applying EGP recursively.
For our preliminary study, we tested our estimation algorithm on a small social network with N = 1,000 nodes, average degree ,k. = 3, and clustering coefficient c = 0.43 (random network with the same N and ,k. would have clustering coefficient c* < ,k./ N = 0.003). Besides allowing shorter simulations on a desktop computer, this smaller network has the advantage that the entire network, together with the simulated epidemics, can be easily visualized (see Videos S1 and S2).
We simulated S = 100 epidemics and censored 70% of the incidence data before estimating the infection network. The estimated network is shown in Figure 2 , superimposed onto the original network. The reconstruction is highly accurate in this test Figure 1 . An SIR epidemic spreading through a small network of N = 14 nodes. The epidemic starts on node 9 at t = 0. Thereafter, nodes 5 and 12 are infected at t = 1 by node 9, which recovers and become immune to subsequent infection. At t = 2, nodes 4 and 6 are infected by node 5 (which recovers), while nodes 10, 11, and 13 are infected by node 12 (which recovers). Following this, at t = 3, node 3 is infected by node 4 (which recovers), while node 8 is infected by node 6 (which recovers). Nodes 10, 11, and 13 recover without infecting any other nodes. Finally, at t = 4 (not shown), nodes 3 and 8 recover and the epidemic ends. In this figure, susceptible nodes are white, infected nodes are red, recovered nodes are green, and links responsible for the transmission are colored red for the time step they are activated. doi:10.1371/journal.pone.0022124.g001 case. From the total 1,584 links in the original network, the algorithm estimated 738 links correctly and made only 196 wrong estimates, resulting in 79% estimation accuracy. More importantly, the estimation found mostly links between highly connected 'hubs' that form the 'backbones' of the network. These backbones play an important role in disease transmissions as they link one highly-connected cluster (community) to another throughout the network.
After estimating the infection network, we performed EGP and found a set of 67 nodes that will efficiently fragment the estimated network. We then immunized the same 67 nodes on the actual infection network. To evaluate the effectiveness of the method, we picked 10 random seeds and ran 500 SIR epidemics each on the immunized network to find the average number of infected nodes as a function of time. Using the same seeds, we also ran 500 SIR epidemics each on the original infection network and found that EGP immunization reduced the average size of the epidemics by 58%-88% and lowered the average peak incidence rate by 51%-68% (see Videos S3, S4, S5, and S6).
Motivated by the promising result of small network estimation, we performed systematic studies of larger networks to see how well these can be estimated. We varied the number of estimated links n, the censor rate C, the size of the network N, the number of SIR epidemics S used for estimation, and the average degree of the network ,k.. The same clustering coefficient of c = 0.05 is maintained across the networks to ensure meaningful comparisons.
3.2.1 Accuracy versus number of estimated links. In a given estimation, there is a trade-off between accuracy and number of estimated links n. At the end of the estimation, frequently reinforced links (likely to be the correct ones) will have weights much higher than those infrequently reinforced links (likely to be the wrong ones). To determine which tentative links we would accept as estimated links, we set a cut-off value w C for the weight. Links with weights above w C will be accepted as estimated links, otherwise they are rejected. If w C is large, only a few estimated links are accepted, and most of these will be correct estimates. On the other hand when w C is small, more estimated links are accepted but a larger fraction of these will be wrong. Our simulation results confirmed this and showed that the accuracy falls off very slowly as a power law in the number of estimated links n, with power-law exponents much smaller than 1 (Figure 3 ).
3.2.2 Accuracy versus censor rate C. As common infections are typically under-reported, it is important to study how the estimation accuracy varies with censor rate. For a fixed network with N = 10,000 nodes and ,k. = 10, we simulated S = 100 SIRs and applied various censor rates C from 50% to 85%. The estimation accuracy is found to decrease as C increases (Figure 4a ). This is expected, as lower censor rate results in more data available for estimation and thus improves the accuracy.
3.2.3 Accuracy versus number of SIR S. Incidence data from a single epidemic will not provide a good picture of the network, because of censorship and history dependence of the epidemic. For different seeds, the epidemic progresses along different transmission routes. Thus, even if we could track all incidences for a given epidemic we are seeing an incomplete snapshot of the infection network. With censorship, this snapshot is even more incomplete. This is why we need to accumulate information from successive epidemics to make consistent and reliable estimates of the network links that participate in the transmission. In Figure 4b , we show the estimation accuracy as a function of the number of different SIR epidemics S used in the estimation with 50% censor rate. We found the accuracy to improve with S initially before saturating to 100% as more incidence data becomes available. It thus appears that faithful estimation of the infection network is asymptotically possible.
degree ,k.. Naturally, we expect larger and highly-connected networks to be more difficult to estimate. To study how the estimation accuracy varies with N, we performed estimation on networks with three different sizes N = 1,000, 10,000, and 100,000 using data from S = 100 epidemics. We also compared the estimation accuracies for networks with N = 10,000 nodes and two different average degrees ,k. = 10 and 20. In Figure 4c -d, we show that the accuracy falls with increasing network size N and increasing average degree ,k., as expected.
Summarizing the results of the systematic studies, the estimation accuracy for a given infection network depends strongly on the censor rate, the number of epidemics over which incidence data is accumulated, and the average degree of the network. The accuracy falls with increasing censor rate, network size, and average degree, but improves over time as more incidence data from new epidemics become available.
Since the preliminary EGP result in Section 3.1 is encouraging, we tried EGP mitigation on the larger networks despite the poor estimation accuracy.
We started by estimating an infection network (N = 10,000, ,k. = 10, and a total of 50,126 links) using incidence data from 100 SIR epidemics and 50% censor rate. The estimated network has a total of 2,561 nodes and 6,467 estimated links. 1,074 of these estimated links are correct (16.6% accuracy). Next, we applied EGP to this estimated network (not to the actual infection network) and obtained a set of 864 nodes that will efficiently fragment the estimated network. We then immunized these 864 nodes on the actual infection network to test how well this will mitigate subsequent epidemics. To obtain a non-targeted intervention benchmark for comparison, we also randomly removed 864 nodes in the actual infection network. We then ran 100 SIR epidemics each on the two immunized networks and plot the average incidence rate as a function of time, as shown in Figure 5a .
Considering the estimated network represents only a small fraction (0.13) of the actual number of links, and only 16.6% of the estimated links are correct, the EGP-based mitigation is surprisingly effective in both slowing down the progress and reducing the peak incidence rate of the outbreaks. We show that systematic isolation of only 8.6% of the total population reduced the peak infection rate by about 40% and delayed the peak infection time by about 20%, as compared to the unmitigated case. More importantly, even though random isolation also reduced and delayed the peak, the targeted intervention is still significantly better.
The 864 separator nodes identified by EGP are clearly special on the estimated network (and thus also on the actual network).
However, all the 2,561 nodes identified in the estimation process are important, because they are on the transmission paths for nearly all epidemics. As suggested by the referee, removing all these nodes may bring the infection network below its percolation threshold, thereby completely stopping all epidemics. To test this idea, we isolated the 2,561 nodes on the actual network and ran 100 SIR simulations. We found that in about 40 simulations, the 'epidemics' infected only 2-5 nodes and died off very quickly after 1 or 2 time steps after starting. For the other 60 simulations, we observe very slowly circulating epidemics that lasted more than 60 time steps and infected 2,580 nodes on average, with the number of incidences reaching a peak of 116 cases 28 time steps after the start.
3.3.2 In-epidemic EGP. The results above were obtained assuming we can partition the network before the start of each epidemic. Since it is more realistic to assume we intervene only after an outbreak has been detected, we also investigated the effectiveness of the EGP intervention enforced at different times during the epidemic (Figure 5b ). Our simulation shows that the strategy is still very effective even when it is applied as late as 10 time steps (roughly 30 days) after the start, just 2 time steps away before the peak. The peak infection rate is reduced by about 20% and total number of cases by 12%, as compared to the unmitigated case. Unfortunately, the time of maximum infection rate is not delayed when EGP is applied at t = 10, and is only delayed by about one time step when intervention is applied at t = 2 and t = 6 time steps after the start.
Based on the results reported in Section 3.3, we have shown that the proposed intervention based on estimated infection networks is very effective in limiting the scale and slowing down the spread of infectious disease epidemics. However, as discussed in Section 3.2, the estimated infection networks are not always accurate, especially when the proportion of unreported incidences is high. Figure 4a suggests that the estimation accuracy goes to zero at censor rate C T ,100%. This implies that it might be difficult to do network estimation in the US, where censor rates for mild illnesses are close to 100% because of the expensive medical insurance and co-payments. However, we stand a better chance of making the scheme work in Singapore as the low medical cost and the necessity to obtain medical certificates to stay away from work promote doctor consultations even for mild symptoms. To further improve the accuracy, the incidence data can be easily complemented with other types of relevant data. For instance, cell-phone and GPS co-location information can be used to rule out a large proportion of tentative links connecting pairs of individuals that have never been found in the same location over the infection time step of about three days. This will reduce the rate of forming false links significantly, thus improving the accuracy of the estimation.
Real infection networks are also likely to be dynamic and changing with time. There are seasonal variations to the infections, and also seasonal variations to social mobility patterns. For example, most temperate countries have an annual flu season that peaks during winter [30] [31] [32] , whereas tropical countries experience multiple flu seasons each year [33] . School holidays in June and December also change the social dynamics drastically, as students start hanging out more in shopping centers and families go on vacations [34, 35] . With real data, what we are estimating is effectively the infection network averaged over multiple epidemiological and social seasons. With enough data, it might also be possible to estimate these seasonal infection networks, and clearly interventions and mitigations might become more effective with the added knowledge of how the network varies with time. However, we believe that even with the averaged infection network, interventions and mitigations will still be effective enough to justify its study and estimation.
The N = 10,000 network estimated in Section 3.3 for demonstrating the effectiveness of EGP-based intervention is obtained from 100 SIR epidemics. In subtropical countries, there is typically one flu season each year. So if the estimation is based solely on flu, it would take 100 years to obtain the estimated network. In Singapore, because of the tropical climate and high population density, there is anecdotal evidence that epidemics of mild respiratory infections come around every one or two months. Assuming there are six epidemics a year, the estimation above will take a much shorter time of 17 years in this country. The main risk of using multiple infections to shorten the estimation time is the increased number of wrong estimates when two epidemics overlap. However, this is a problem that cell phone and GPS co-location can help to solve as well.
Finally, we highlight that the estimated network needs not to be very accurate to be useful. The results of Section 3.3 beautifully illustrate this, where targeted isolation based on an estimated network which is only 16.6% accurate is still very effective in mitigating epidemics (Figure 5a-b) . This unexpected effectiveness of EGP applied on an inaccurately estimated network prompted us to better understand why this is happening. A deeper look at the preliminary study which we can visualize suggests that, while the predicted links are inaccurate, the nodes that emerged from the estimation are mostly hubs and are concentrated along the backbones of the infection network. Hence, removal of these nodes will effectively slow the advancing epidemic. Naturally, we expect even better results with a more accurate estimation.
Effective epidemic control measure is important for mankind as the problems associated with severe infectious disease can only increase in severity along with global population growth. To put this into the proper context, we highlight two most important features that characterize severe epidemics: (1) the large number of cases that emerges in relatively short time (surge in hospital load), and (2) the fast propagation of the disease as compared to treatment time and development of cure. We will discuss how the proposed mitigation method addresses these two problems.
For highly-infectious epidemics, the capacities of hospitals are stretched due to the large number of infected individuals appearing near the peak of the epidemic. Lack of medical attention and insufficient resources (medics, vaccines, and healthcare apparatus) might delay the recovery process and result to a higher number of casualties than need be. This is why it is important for an epidemic mitigation strategy to consider peak load reduction as a primary goal. Delaying the peak infection time, thus stretching the overall time scale of the epidemic, is also crucial. When dealing with a deadly epidemic caused by a new infection agent, chances are we would not have vaccine at hand, or even a vaccine development program. As development of vaccines and drugs for new diseases can only begin after cases have been confirmed and analyzed, the race against the epidemic to develop and administer such drugs would be inevitable. Delaying the progress of the epidemic will not only buy time for drug development, but will also give governments more time in general to implement other crisis handling measures.
Furthermore, any epidemic mitigation procedure can only be enforced after the epidemic has started and positive cases have been confirmed. Mitigation measures applied at the start of epidemic is ideal but may not be practical. Hence, good performance when applied late into the epidemic is critical for a mitigation method to be successful. Figure 5b hints at a the promising potential our proposed targeted strategy can offer in this regard, where significant reductions of the peak infection rate and the total number of infected people are observed even when applied late into the epidemic. However, the result also suggests that early implementation is critical in slowing down the epidemic.
Here let us also discuss the impact of the proposed strategy on privacy as a potential reason to object to its implementation. In a country like the US, this strategy is unlikely to be adopted no matter how well it is shown to work in simulation due to privacy concerns when we selectively isolate individuals according to EGP. However, the proposed strategy may find a more receptive audience in a country like Singapore, given that home quarantine orders have been issued by the Ministry of Health during the SARS and H1N1 outbreaks. Here we must stress that node removals as suggested by EGP need not be home quarantine or any other physical isolation practices. If early vaccine stocks are available but limited, the EGP nodes should be the ones to receive it, because of the role they play in disease transmission. We must nevertheless be wary that this procedure should not be used as a mean of discrimination.
Finally, we highlight a crucial difference of the proposed method to traditional strategies. Unlike conventional quarantine, this intervention involves isolating healthy individuals from the rest of the infection network. At first this might sounds absurd: ''Why isolate healthy people and not the infected ones?'' From network point of view, however, the reason is clear: by isolating these people we are intercepting the path of an ongoing epidemic. As comparison, it is a standard practice in controlling forest fire to cut or burn down trees ahead of advancing fire to contain it. For infectious diseases no one has imagined this intervention possible, but with an estimated infection network our proof-of-concept results are impressive. If all nodes on the entire estimated network are isolated, the results are even more impressive: for our N = 10,000 network with ,k. = 10, we find propagating epidemics in only 60% of the simulations. In these propagating epidemics, the epidemic peak with intervention is less than 1/10 the epidemic peak without intervention, while the total number of infections with intervention is 1/3 that without intervention. Also, instead of peaking at t = 12 and ending at t = 20 without intervention, the epidemic peaks at t = 28 and ends at t = 60 with intervention. In terms of actual durations, removing all nodes on the estimated network stretches two-month epidemics to over half a year. However, about 1/4 of the population needs to be isolated, compared to only about 9% of the population for EGP intervention. A separate systematic study will be necessary to understand how effective and efficient this more aggressive intervention strategy can be.
In conclusions, we have presented in this work a proof-ofconcept study of a novel epidemic mitigation method based on equal graph partitioning of the estimated infection network. We used computer simulations to study and show the effectiveness of the method. First, we followed the intuitive JGN method to generate artificial social network of various sizes but with roughly the same clustering characteristic to serve as the infection networks. We then simulated SIR epidemics on the artificial infection networks, recorded the incidences, and applied censorship with rates between 50% and 85% to mimic low reporting rates for mild infections. We then used the remaining incidence data to construct an estimate of the infection network using a modified JGN algorithm. We found that the estimation accuracy falls with increasing censor rate, average degree, and size of the network, but improves as more data from multiple epidemics are incorporated.
With the estimated infection networks at hand, we applied the Equal Graph Partitioning (EGP) algorithm to remove the smallest sets of nodes that will efficiently fragment the estimated networks. We then immunized the same set of nodes on the actual infection network before simulating subsequent epidemics. We compared the effectiveness of the targeted strategy to an untargeted method that randomly isolates the same number of nodes, and showed that the former outperforms the latter in decreasing and delaying the peak of the epidemics, as well as reducing the total number of infections. We also applied the proposed strategy at different times after the epidemic has started and showed that it is still effective in decreasing the peak infection rate and total number of cases even when applied late into the epidemic. In particular, we demonstrated that the EGP based strategy is surprisingly effective in mitigating epidemics even when the estimated network is not very accurate. Through visualizing small network, we find that in spite of the large number of wrongly estimated links, the nodes appearing in the estimated network are concentrated along the backbones of the actual network and thus play important roles in the disease transmission.
Video S1 SIR epidemic on an artificial social network. In this video, an SIR epidemic spreads through an artificial social network with N = 1,000 nodes, 1,584 links, average degree ,k. = 3.168 and clustering coefficient C = 0.3928. The artificial social network is generated using the Jin-Girvan-Newman algorithm (Jin, Girvan, and Newman, 2001) . The transmission probability used for this SIR epidemic is p = 0.80, and infected nodes recover after t R = 1 time step. (WMV)
Video S2 Another SIR epidemic on the artificial social network. In this video, another SIR epidemic starts from a different seed on the artificial Jin-Girvan-Newman social network with N = 1,000 nodes, 1,584 links, average degree ,k. = 3.168 and clustering coefficient C = 0.3928. The transmission probability for this SIR epidemic remains at p = 0.80, and infected nodes again recover after t R = 1 time step. (WMV)
Video S3 SIR epidemic on the EGP-immunized artificial social network. I. In this video, an SIR epidemic spreads through the artificial Jin-Girvan-Newman social network after EGP immunization. The EGP immunization is based on the 475node, 934-link network estimated over S = 100 SIR epidemics from the 1,000-node, 1,584-link network, using the algorithm described in the text, and a weight cutoff of w c = 7. Of the 934 estimated links, 738 are correct, giving an accuracy of 79.01%. The EGP procedure then identifies 67 separator nodes, and when these are removed, the immunized network has 1,359 links remaining.
Video S4 SIR epidemic on the EGP-immunized artificial social network. II. In this video, we start an SIR epidemic from a second seed on the EGP-immunized artificial Jin-Girvan-Newman social network. The original network consists of 1,000 nodes and 1,584 links, whereas the estimated network EGP is based on consists of 475 nodes and 934 links. 67 separator nodes identified by the EGP procedure are removed from the original network, giving an immunized network with 1,359 links. (WMV) Video S5 SIR epidemic on the EGP-immunized artificial social network. III. In this video, we start an SIR epidemic from a third seed on the EGP-immunized artificial Jin-Girvan-Newman social network. The original network consists of 1,000 nodes and 1,584 links, whereas the estimated network EGP is based on consists of 475 nodes and 934 links. 67 separator nodes identified by the EGP procedure are removed from the original network, giving an immunized network with 1,359 links. (WMV)
Video S6 SIR epidemic on the EGP-immunized artificial social network. IV. In this video, we start an SIR epidemic from a fourth seed on the EGP-immunized artificial Jin-Girvan-Newman social network. The original network consists of 1,000 nodes and 1,584 links, whereas the estimated network EGP is based on consists of 475 nodes and 934 links. 67 separator nodes identified by the EGP procedure are removed from the original network, giving an immunized network with 1,359 links. (WMV) Strengthening systems for communicable disease surveillance: creating a laboratory network in Rwanda BACKGROUND: The recent emergence of a novel strain of influenza virus with pandemic potential underscores the need for quality surveillance and laboratory services to contribute to the timely detection and confirmation of public health threats. To provide a framework for strengthening disease surveillance and response capacities in African countries, the World Health Organization Regional Headquarters for Africa (AFRO) developed Integrated Disease Surveillance and Response (IDSR) aimed at improving national surveillance and laboratory systems. IDSR emphasizes the linkage of information provided by public health laboratories to the selection of relevant, appropriate and effective public health responses to disease outbreaks. METHODS: We reviewed the development of Rwanda's National Reference Laboratory (NRL) to understand essential structures involved in creating a national public health laboratory network. We reviewed documents describing the NRL's organization and record of test results, conducted site visits, and interviewed health staff in the Ministry of Health and in partner agencies. Findings were developed by organizing thematic categories and grouping examples within them. We purposefully sought to identify success factors as well as challenges inherent in developing a national public health laboratory system. RESULTS: Among the identified success factors were: a structured governing framework for public health surveillance; political commitment to promote leadership for stronger laboratory capacities in Rwanda; defined roles and responsibilities for each level; coordinated approaches between technical and funding partners; collaboration with external laboratories; and use of performance results in advocacy with national stakeholders. Major challenges involved general infrastructure, human resources, and budgetary constraints. CONCLUSIONS: Rwanda's experience with collaborative partnerships contributed to creation of a functional public health laboratory network. Communicable diseases remain the leading cause of illness, death and disability in African countries [1, 2] . Even though well-known, efficacious responses are available for the control and prevention of these diseases, the capacity for timely detection, confirmation and response actions needs reliable public health systems. To address the demand from countries for improved surveillance systems that provide relevant and accurate epidemiologic and laboratory information, the Member States of the World Health Organization (WHO) Regional Headquarters for Africa (AFRO) adopted a strategy in 1998 called Integrated Disease Surveillance and Response (IDSR) [2] . A major goal of IDSR is to strengthen district-level surveillance capacities for detecting, confirming and responding to priority diseases that afflict African communities. In the IDSR implementation framework, epidemiologic surveillance is linked with laboratory support in order to produce relevant information for taking public health action [3] .
* Correspondence: hap5@cdc.gov 3 Centers for Disease Control and Response, 1600 Clifton Rd., Atlanta, USA Full list of author information is available at the end of the article The World Health Organization (WHO) emphasized the role of public health laboratories in national surveillance systems through a resolution in 2008 which called for the organization of national public health laboratory networks that would link national laboratories with subregional, regional and international laboratories [4, 5] . Thus a public health laboratory network is a collection of laboratories that use standard operating procedures, carry out quality assessments, and report information for public health action in a systematic manner [4] .
With the adoption of the revised International Health Regulations (2005) and the commitment of countries to achieve disease reduction targets set by the Millennium Development Goals (MDGs), countries are facing increased demands to rapidly improve their public health systems. The IHR (2005) are especially clear in calling for strengthening core capacities for detecting and confirming public health threats, especially those with potential to extend beyond national borders [6] . Thus progress in countries with limited resources depends on identifying opportunities for creating and streamlining resources to achieve functional national laboratory networks that meet national priorities and support global disease program objectives [7] .
Disease-specific guidelines provide robust recommendations to create laboratory networks, but may not always include guidance on how to organize, mobilize and integrate the essential resources, procedures, and policies for creating and maintaining a laboratory network within a national public health surveillance system [8, 9] . Thus we wanted to examine Rwanda's eight-year (from 2000 to 2007) experience with IDSR to see how the country developed a public health laboratory network through coordination of multiple resources and technical support in order to meet national priorities and partner interests.
In 2005, the Rwandan Ministry of Health commissioned an independent assessment of the national laboratory network to determine its status in relation to the national reference laboratory and for disease prevention programs [10] . Subsequently, in 2006, a team from WHO/AFRO, the United States Centers for Disease Control and Prevention (CDC) and the Rwanda National Reference Laboratory (NRL) in the Ministry of Health met to review Rwanda's public health laboratory network. The intention was to use the review findings to inform guidance for other countries that would make explicit practical factors to consider when implementing a public health laboratory network.
A multi-agency team reviewed published and unpublished reports related to the implementation of Rwanda's laboratory activities, conducted site visits to observe how the laboratories functioned, and interviewed staff at the Ministry of Health and partner agencies. The National Reference Laboratory (NRL) coordinated the site visits to seven district hospitals and two health centers. These sites were selected as a convenience sample because each health center and district hospital in Rwanda offers the same standardized group of laboratory tests as specified by government policy. Interviews and follow-up discussions were conducted with laboratory personnel in charge of the district hospital and health center laboratories. Interviewees included district supervisors and representatives of disease-specific programs for tuberculosis (TB), malaria, and HIV/AIDS. We also met with personnel at the Ministry of Health's Epidemiology and Health Prevention unit, and with staff responsible for the Health Management Information System. Experiences with external support to laboratory services were gathered through interviews with locally-based partners who worked closely with the Ministry of Health and the NRL including WHO, CDC, Columbia University and the United States Agency for International Development (USAID).
To guide our review, we adapted an assessment protocol developed by WHO, CDC and USAID for use in IDSR programs in African countries [11] . Topics assessed included clarification of the disease surveillance goals and activities for the country, the role of the laboratory in surveillance activities, the coordination and structure of the laboratory network, and the collaboration of partners and resources for creating quality laboratory services for disease outbreak investigations and response.
Rwanda's implementation of IDSR began in 2000 with a national assessment of the country's communicable disease surveillance program. The results of the assessment were used to develop a strategic plan for improving the country's surveillance, laboratory and response capacities. The WHO/AFRO IDSR technical guidelines were adapted to ensure that Rwandan disease priorities would be emphasized in the streamlined system. Training of health workers on IDSR approaches began in 2001. Additional training in laboratory and data management was provided for relevant staff through national and regional training workshops. The initial IDSR strategy in Rwanda targeted 19 priority diseases and syndromes (Table 1 ) recommended by WHO-AFRO because they are among the leading causes of illness, death and disability in African countries and are relevant to Rwanda based on epidemiologic criteria [3] . Implementation of the full IDSR program within the Ministry of Health is still under development; the focus of this review is on the laboratory component.
The Rwanda health service consists of a referral system encompassing three levels of health care: referral, district and health center levels. In the whole country, there are four referral hospitals, 34 district hospitals, and 385 health centers. At the central level, the NRL's mandate is to provide referral laboratory services to all health-care providers in the country, to prepare and distribute laboratory specimen transport media, to develop policies and to enforce standards for all laboratories in the country. The NRL also oversees the licensing, certification and accreditation of private and public health laboratories.
The NRL has autonomous status thus highlighting its role in promoting stronger laboratory capacities and leadership in disease control activities. Specific responsibilities of the NRL involve improved diagnostic capacities for HIV, tuberculosis, and malaria. The NRL also has capacity to identify and isolate bacterial causes of outbreaks. Additionally, the NRL's capacity to diagnose other diseases was expanded with support from USAID and CDC to diagnose influenza subtypes A and H5N1 as part of the country's epidemic preparedness and control activities.
Within the NRL, the laboratory personnel participate in investigations of suspected disease outbreaks in several ways. They ensure that specimen transport media is available. They also process and test specimens and then ship specimens to regional or international laboratories for further characterization as required by disease-specific guidelines. Other activities include training, supervision, and quality assessments for laboratories at all levels of the national health system.
At the time of this review, the Government of Rwanda was involved in decentralization of administrative functions that also impacted the health sector. Decentralization was seen as an opportunity for expanding laboratory capacity, improving data management and maximizing the use of surveillance information at all levels of the health care system in line with the IDSR strategy. In the Department of Epidemiology and Public Hygiene (DEHP), decentralization focused on improving coordination of disease surveillance with the alert and response system at the district level. Among the changes fostered by decentralization, a Geographic Information System (GIS) was set up, and bacteriology laboratories were established at five district hospital laboratories.
The laboratory network in Rwanda is aligned with the overall three-tiered organization (central, district and peripheral levels) of the national health system. The network connects the central level's four reference hospital laboratories and the National Reference Laboratory with 34 district hospital laboratories at the intermediate level and 385 health center laboratories at the peripheral level.
Each level has a defined role supported by standard operating procedures (SOPs) that specify the safe collection, handling, storing, shipping and processing of laboratory specimens. Supervision and feedback are carried out using a level-specific checklist. The SOPs specify communication channels and procedures linking each level for the referral of laboratory specimens, reporting of data, supervision and quality assurance activities. For example, the district hospital laboratories submit weekly reports of selected priority diseases. The district level performs commonly requested laboratory tests such as hematology, clinical chemistry, urine analysis and parasitology. At the health center level, laboratories perform microscopy for detecting malaria, tuberculosis and other bacterial and parasitic agents. Almost all health center laboratories have the capacity to do human immunodeficiency virus (HIV) rapid testing. For some disease programs such as malaria, the network extends to the community level where volunteers are trained in detection and treatment of malaria including home-based management of malaria. The community volunteers contribute to surveillance activities by referring patients to health centers for laboratory services.
The laboratory capacity for the isolation and identification of bacterial pathogens has been established at the NRL. Specimens for bacteria such as Vibrio cholerae, Salmonella typhi, Shigella species, Neisseria meningitidis and Haemophilus influenzae are collected from the peripheral levels and transported to the NRL where they are analyzed. The results are fed back to the districts and also shared with WHO/AFRO on a monthly basis. Decentralization of services was also implicated in expansion of access to antiretroviral treatment (ART) at the national level. Following the Rwanda National Paediatric Conference in 2004, there was recognition that early infant diagnosis of mother-to-child transmission (PMTCT) and ART sites should be expanded at all levels of the health system. In July 2005, with support from CDC, three paediatric diagnostic sites were established for early infant diagnosis using either dried blood spots (DBS) or whole blood methods. By April 2008, the number of rapid diagnostic sites had expanded to 126.
The strengthening of laboratory capacity including advocacy, provision of laboratory equipment, training and research was achieved through partner resources for specific tests and procedures. The training topics included advanced techniques for tuberculosis diagnosis (for example, testing for multi-drug resistance), HIV/ AIDS testing (HIV serology, CD4 counts, and PCR for infant diagnosis of HIV), and techniques for hematology, biochemistry, and parasitology, including blood smears to detect malaria and intestinal helminths. Training topics also included good laboratory practices, biosafety, biological sample collection, and the safe transport, handling and storage of specimens. In 2005, the NRL trained 467 biotechnologists on laboratory detection of malaria, TB, HIV rapid testing, standard laboratory practices and biosafety. In 2007, 969 laboratory personnel completed an integrated laboratory training in malaria, TB, and HIV (471 participants), biochemistry and haematology (61 participants) as well as training in how to perform CD4 counts (34 participants), conduct testing with dried blood spots (180 participants) and carry out HIV-specific testing at new VCT sites (223 participants).
The national reference laboratory coordinates and carries out supervision for laboratory activities in referral hospitals, district hospitals, health centres and private clinics. The supervisory activities, which take place each trimester, are integrated across disease and subject areas and include: TB, malaria, HIV, biochemistry, and haematology. Supervision of bacteriology activities is carried out separately at referral and district hospitals only.
During the supervisory visits, a check list is used to assess performance in technical activities, the extent of compliance with SOPs, the quality and reporting of results, record keeping, internal quality controls, instrument performance (especially microscopes and spectrophotometers), availability and quality of reagents, and management of consumables. The extent of reliably available utilities (such as water and electricity), laboratory space and standard biosecurity capacities are also assessed. There are 420 facilities that receive at least one visit per year. Some may receive two or three visits during the year depending on the supervisory results. The number of actual supervisory visits to laboratory sites is maintained at a high level, e.g., 517 in 2005, 862 in 2006, and 689 in 2007. The supervisory visit data is analyzed and feedback is given to the respective health facilities. When results are not satisfactory, the NRL intervenes for corrective actions, in the form of retraining or amelioration of other causes of poor performance. Plans are underway to decentralize quality control and supervisory activities. In the new plan, the district hospital laboratories will take charge of the health centers while the NRL will supervise the referral hospitals, the district hospital and private laboratories.
Disease specific programs in Rwanda are managed by the Government of Rwanda in collaboration with partners such as WHO, the Global Fund and PEPFAR. Mapping of health facilities ensures that each partner has specific facilities to support so that all facilities are aligned in support of national goals and objectives for control and prevention programs targeting HIV/AIDS, TB, malaria, epidemic disease surveillance and opportunistic infections. In this way, partner resources complement each other to fill the gaps in terms of sharing equipment, reagents, consumables, human resources and technical support. Merging of the HIV/AIDS, TB and malaria programs into one institution called the Treatment and Research AIDS Center (TRAC Plus) allows for clinical planning and laboratory activities in addition to collaboration at high levels. This coordination aims to avoid overlapping or duplication of activities in any area.
We observed examples of collaboration between national disease programs and the national and external reference laboratory systems such as those for polio, measles, and multi-disease resistant TB activities. For example, a key component in many WHO disease eradication or elimination programs (such as the polio eradication activity within the Expanded Programme on Immunization or EPI), is the collaboration between national disease programs and the national and external reference laboratory systems. In the case of polio eradication in Rwanda, the transport of stool specimens from patients with acute flaccid paralysis (AFP) originates at peripheral levels through district hospitals and then to the NRL or to the WHO-EPI program in Rwanda. The NRL or WHO-EPI program in turn ships these specimens to the Ugandan Virus Research Institute in Entebbe, Uganda (UVRI) for confirmation and characterization of the organism. The results are reported back to Rwanda's NRL and EPI offices and then shared with the WHO. The results are communicated to the district health team and health facilities that use the results to inform timely and relevant public health response actions.
For monitoring of the measles program, serum specimens from patients with suspected measles are collected and transported from the health facilities to the NRL. The samples are analyzed for viral antibodies by ELISA. The results are given to EPI for their use in determining a timely response. The results are shared with WHO-AFRO on a monthly basis. Serum specimens also are sent to UVRI on a quarterly basis for external quality control. In addition, the NRL receives quality control panels every quarter from UVRI.
Monitoring for multi-drug resistant TB is done by the NRL, and some specimens are subsequently referred to the external laboratory at the Institute of Tropical Medicine (IMTA) in Antwerp, Belgium for quality control (QC) testing. The QC panel samples for epidemic bacteria, malaria and TB microscopy, CD4 counts, HIV ELISA and Western Blot are received from the National Institute of Public Health (NIPH) in South Africa every quarter. The QC panels for assessing PCR capacity are received from CDC in Atlanta, USA.
Laboratory results from the network Table 2 illustrates examples of data produced through the laboratory network in support of the communicable disease surveillance system. Specimens reflected in the table are referred specimens or specimens collected by the national reference laboratory for confirmation of suspected cases during outbreak investigation or surveillance. Once specimens are confirmed according to criteria specified by WHO, there is no need for more specimens for laboratory investigations. Specimens continue to be processed to inform clinical management.
The NRL conducts quality control (QC) and quality assurance (QA) activities for all levels of the network laboratories at regular intervals. The NRL also participates in the WHO-AFRO External Quality Assessment (EQA) scheme for enteric and meningitis pathogens, TB and malaria. External quality assurance for HIV, measles and selected bacterial diseases is carried out through designated international partner or disease-specific collaborating laboratories. As an example, in 2003, 100% concordance was reported for the unlinked, anonymous HIV testing in a total of 288 samples sent to CDC-Atlanta.
Health centres and district health facilities regularly send 10% of randomly selected HIV positive and 5% HIV negative specimens to the NRL for quality control assessment. Table 3 .
To ensure the quality of measles laboratory testing, each trimester the NRL sends 10% of the total measles and rubella samples received at the NRL to the Uganda Virus Research Institute (UVRI). The NRL also receives 20 panel specimens each year from the UVRI. The panels are tested and results are subsequently shared with UVRI. In 2004, the result showed good concordance and the NRL was subsequently accredited for measles laboratory confirmation by WHO/AFRO. In 2005, the Rwandan NRL obtained 95% concordance for measles and rubella testing.
Documented reports and participant responses suggested that achieving a public health laboratory network is not without considerable challenges in major areas. For example, written reports and interviewee responses noted the following obstacles: a delay implementing an integrated strategy for improving an integrated public health surveillance and response strategy, insufficient number of skilled human resources, weaknesses in the general infrastructure, and shortages of vehicles, equipment, supplies, and reagents.
One of the major limitations cited by participants has been the delayed implementation of IDSR in Rwanda. Without a functional IDSR strategy in place, health staff reported that the role of laboratory is not well understood in its context of a broader public health surveillance network. While there is a strategic plan with defined goals for IDSR in place, implementation of the plan has yet to be fully implemented. At the time of this review, the surveillance and reporting system was using multiple forms from a variety of implementing partners resulting in a duplication of effort and resources.
The shortage of skilled human resources at all levels is due to high attrition and turnover of trained personnel. Decentralization has resulted in a gap in human resources for specialized testing at the national level and routine functions at the district and health facility levels. The long distances between some health centers and the national level -in addition to difficult driving conditions in some areas -contributes to a situation where specimens arrive at the NRL in inadequate condition (for example, hemolyzed samples for CD4 and measles IgM testing). Computer and internet access for transmitting data is affected by the erratic supply of electricity. Finally, participants described working within the context of ongoing shortages of vehicles, fuel, laboratory safety equipment, laboratory supplies and reagents. 
The government of Rwanda, through collaboration with its partners, has begun to strengthen the organizational capacity of the public health laboratory network for identification and confirmation of priority diseases. The laboratory network has a hierarchal structure with the central referral laboratories as lead authorities and the health centre-based laboratories at the periphery. Other referral hospitals laboratories also link to the National Reference Laboratory. There is a defined role for each level with level-specific standard operating procedures. The NRL is the focal point for national public health laboratory leadership and coordination of partners to establish and expand the laboratory network. Reporting on specimens processed from the district and hospital laboratories is integrated and submitted along with the weekly disease reports for selected priority diseases. Supervision and feedback is done using a check list as applicable to the different levels.
The benefits of an organized laboratory network are evident in the increases in capacity and performance as illustrated in Tables 2 and 3. Table 2 illustrates an increase over time in the number of specimens received and isolates confirmed by the NRL between 2005 and 2007, and Table 3 highlights a 3-fold increase during the same time period in the number of health facility laboratories participating in quality control for TB microscopy.
The laboratory network in Rwanda is contributing to the successful strengthening of the disease surveillance and response system by ensuring involvement of the public health laboratory network in the planning for IDSR, laboratory participation in outbreak investigations, engagement in feedback communications, integration of laboratory indicators to monitor progress, and developing mechanisms for coordination of epidemic preparedness and response at all levels. The coordination and leadership role of the NRL and the commitment of multiple partners under its leadership provide unique opportunities for strengthening the laboratory networking in Rwanda.
Because a major focus in IDSR implementation is to strengthen the district level, the ongoing decentralization process provides a good opportunity for expanding IDSR implementation in all districts. Recently, a Field Epidemiology and Laboratory Training Program was established which will eventually result in considerable capacity building, especially if the applied training program is accompanied by a well-structured human resources plan for a career path through and retention of competent laboratory staff at all levels of the network.
The strengthening of the collaboration between the NRL and the Treatment and AIDS Research Center (TRAC Plus) through the Center for Communicable Disease Control in Rwanda will lead to improved coordination. As a first step, an integrated informatics system and a national integrated surveillance system are being developed, which will facilitate not only communication of data but also the sharing of results. A recommendation of the study team to the NRL was to consider implementation of international standards such as OECD's Global Laboratory Practices (GLP) [12] and to seek international accreditation.
Recommendations for creating a functional public health laboratory network usually highlight the need for: (1) defined communication channels between the various levels of the health system, (2) explicit linkages with relevant Ministry of Health divisions, (3) a structure for coordination of laboratory activities, and (4) collaboration with funding and technical partners [5, 7, 8] . These elements of a laboratory network were evident in our review. For example, Rwanda's laboratory network benefits not only from government ownership and partner commitment but also from the defined structures and communication linkages between levels of the health system and with partners. Responsibility for coordination of laboratory activities through the NRL and TRAC Plus provides a structure for avoiding duplication of resources. The example of the collaboration between the government of Rwanda and its technical and funding partners in creating a functional laboratory network that provides results is one that can be emulated by other countries. Immunity Traits in Pigs: Substantial Genetic Variation and Limited Covariation BACKGROUND: Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment. METHODOLOGY/PRINCIPAL FINDINGS: Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNα, TNFα, IL6, IL8, IL12, IFNγ, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.1<h2≤0.4) or high (h2>0.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits. Increasing robustness by improving resistance/tolerance to pathogens is an important selection objective in most livestock species, particularly in pigs. In the past 30 years, selection for growth, carcass leanness, meat quality and prolificacy, combined with stringent sanitary rules, vaccination and use of antibiotics, has been highly effective in pigs [1] . Since the early 2000's, prophylactic use of antibiotics as growth promoters has been forbidden by European legislation. As a result, the health status of numerous farms has deteriorated, leading to an increase in the therapeutic use of antibiotics. Indeed, animals highly selected for production traits may be more susceptible to pathogens or less able to maintain performance after infection. Deterioration of the global health status may also be due to environmental trends. In this context, including health traits in existing breeding schemes using direct and/or indirect strategies is an emerging trend in pig breeding. Direct strategies target animal resistance/tolerance to specific pathogens but may result in increased susceptibility to other diseases [2, 3] . Alternatively, an indirect and putatively more global approach focuses on immune traits (ITs) providing a measure of immune capacity (i.e. immunocompetence) and hopefully predicting the responses to pathogens in general [4] .
The choice of relevant ITs is further based on knowledge of the immune system. This highly interactive and cooperative system is classically separated into two arms referred to as innate and adaptive, which produce a combined response. Innate immunity is the first line of defence. Its activation is non pathogen-specific and depends on the recognition of evolutionarily conserved pathogenassociated molecular patterns such as lipopolysaccharides constituting bacterial cell walls [5] . Innate immunity involves physical barriers, innate immune cells such as dendritic cells (DCs), monocytes, natural killers (NK cells) or cd T lymphocytes, and inflammatory cytokines such as IL1B, IL6 and TNF. Adaptive immunity is antigen-specific and requires the recognition of specific ''non-self'' antigens via a process of antigen presentation and results in an immunological memory. Adaptive immunity is divided into cell-and humoral-mediated immunity with different effector functions [6] .
In order to include ITs in a breeding plan to improve pig immunocompetence, the genetic and phenotypic parameters of the different ITs need first to be estimated. Several studies in swine, mice, poultry and cattle demonstrated the possibility of selecting animals with high or low immune response (IR) as characterized by one or a few ITs [2, 7, 8, 9, 10] . A study on Yorkshire pigs selected for eight generations for high and low adaptive IR (HIR and LIR, respectively) on an index combining four standardized measures of specific antibodies and cellmediated IR, after stimulation with specific antigens (bacillus Calmette-Guérin and hen egg white lysozyme), has revealed that HIR and LIR animals differ in response to immunization and infection [2, 11, 12, 13, 14] . Other studies have also shown that various innate and adaptive ITs are genetically controlled. For example, variation in innate ITs, such as NK cells, monocytes, interferon a (IFNa) production or phagocytosis [15, 16, 17] is heritable and several adaptive ITs have moderate to high heritability values including total white blood cells (WBC), CD4 + T lymphocyte, CD8a + T lymphocyte and B lymphocyte subsets [15, 16, 17] , delayed-type hypersensitivity reaction [15, 18] , lymphocyte proliferative response [15] , interleukin-2 (IL2) production by lymphocytes [15] and antibody response [12, 15, 18, 19] . Clapperton and colleagues have also reported that variation in acute phase protein levels is heritable [16, 17] . Finally, several significant QTLs for total leukocyte count ( [20, 21] ; Animal-QTLdb, http://www.animalgenome.org/cgi-bin/QTLdb/index), mitogen-induced proliferation [20] , antibody response [20, 22] , cytokine production (IL10 and IFNc) [23] , complement activity [22] , and acute phase protein serum concentration [22] have been detected and mapped to different pig chromosomes. Taken together these data demonstrate that variation in some ITs is under genetic control. However, most of the results reported so far have targeted a limited number of traits and very few studies have combined innate and adaptive ITs.
Our global goal is to identify immunocompetence traits for inclusion in selection schemes aiming to improve both zootechnical performances and health traits in pigs. For this purpose, we have launched a genetic and genomic study of numerous ITs covering innate and adaptive IR [24] . In this report, we present the results of a global genetic study, combining principal component analysis (PCA), and genetic parameter estimation applied to a large number of innate and adaptive ITs in a pig population vaccinated against Mycoplasma hyopneumoniae (M. hyopneumoniae).
A set of 54 ITs was measured on a population of 443 pigs three weeks after vaccination against M. hyopneumoniae (Tables 1 and 2;  Table S1 ; Figure 1 ). These ITs comprise either traits related to IR (phagocytosis, lymphocyte proliferation, cytokine production after in vitro stimulations, levels of total and specific antibodies, levels of acute phase proteins) or traits related to total leukocyte and leukocyte subpopulation counts. The various characteristics and descriptive statistics of the traits measured on each animal of the studied population (n = 383 to 442) are summarized in Tables 1  and 2 . Among the cell-mediated ITs evaluated after diverse stimulations, higher responses in cytokine levels were observed after phorbol myristate acetate (PMA)-Ionomycin (PMAIONO) stimulation compared to lipopolysaccharide (LPS) and concanavalin A (CONA) stimulations, except for IL2 production. Conversely, a higher lymphocyte proliferation was detected after CONA and PMAIONO stimulations than after LPS stimulation.
Ample phenotypic variation was observed for most traits. The coefficient of variation (CV) was equal to 0.8 on average and ranged from 0.07 to 3.9 (Tables 1 and 2 ). Limited dispersion (CV#0.9) was observed for traits derived from hemograms, cell subsets characterized by fluorescence-activated cell sorting (FACS), phagocytosis capacity and non-specific immunoglobulins. Moderate dispersion (0.9,CV#1.5) was observed for most cytokines produced in vitro except for tumour necrosis factor a (TNFa) and mitogen proliferation-related traits. Finally, the CV for seric C reactive protein (CRP) and haptoglobin (HAPT) levels were close to 1.6 and 2, respectively. The highest CV (3.9) was obtained for the specific IgGs directed against M. hyopneumoniae. These data clearly indicate that the seric inflammatory protein levels and the specific IgGs had the greatest phenotypic variance in our study.
In order to analyse the factors causing the variation, we performed a normed PCA with 32 traits (Tables 1 and 2 ). For cellmediated adaptive IR, we included only those traits related to cytokine production and lymphocyte proliferation after PMAionomycin stimulation. For innate ITs, we included 10 FACScharacterized cell subtypes including the percentages of B lymphocytes (IgM + ), cd T lymphocytes (TCRcd + ), three subsets of ab T lymphocytes (CD4 + CD8 + , CD4 -CD8 + and CD4 + CD8 -), NK cells (CD16 + CD2 + ) and three monocyte subsets (CD16 + CD172A + , CD16 + MHCII + , MHCII + CD172A + ). We excluded from the analysis four haematological traits not directly involved in immunity: red blood cell count (RBC), hematocrit (HT), red blood cell distribution width (RDW) and platelet count (PLT). The percentage of variance (inertia) explained by the first five components was over 50% (Figure 2A ). Each of these five components explained more than 5% of the total variance and the first two components accounted for 16.4 and 10.8% of the total variance, respectively. Taking into account the 32 components from PCA, multivariate normal mixture modelling and modelbased clustering (see Materials and Methods) were used to identify clusters of ITs. The highest Bayesian Information Criterion (BIC) was obtained using the diagonal model with variable shape and variable variance (VVI in pink on Figure 2B ) and K = 3 (first factorial plan on Figure 2C ). No parameter was located near the correlation circle indicating that the phenotypic correlations between ITs are globally weak. A first cluster (K1 in blue on Figure 2C ) groups together four hemogram-derived cell counts: white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MON) and neutrophil count (NEU). This cluster is representative of total cell number traits despite the eosinophil count (EOS) not being included. A second cluster (K2 in green on Figure 2C ) groups together all the traits related to FACScharacterized leukocyte subpopulations (expressed as the percentage of cells with one or two surface antigens) except cd T lymphocytes (TCRcd + ), with a cell response parameter (IL10-PMAIONO) and the seric level of haptoglobin (HAPT). This cluster can be considered as representative of the leukocyte subsets. A third cluster (K3 in red on Figure 2C ) includes all other cell response traits, one hemogram-derived cell count (EOS) and one FACS-characterized leukocyte subpopulation. Note that K1 and K2 related traits, which mainly correspond to cell subsets and explain around 25% of the phenotypic variance, show clear clustering ( Figure 2C ). These traits are grouped on the first PCA axis and separated on the second axis. Traits belonging to K3, representative of cell activity (cytokine production, phagocytosis and antibody production), are more spread out on the other axes (data not shown). Interestingly, cluster analysis did not highlight any cluster of innate or adaptive ITs.
The estimation of phenotypic correlations (r r p ) with WOMBAT confirmed that the ITs are weakly correlated, except i) among a few cell count traits (WBC, LYM, MON, NEU), and ii) between cell count traits and a few leukocyte subsets (WBC, LYM, CD16 + CD2 + and CD4 -CD8 + ) for whichr r p greater than 0.4 were estimated (Table S2; Figure S1 ). Weakr r p were mainly positive (330) with 166 negative. No strongly negativer r p (#20.4) were found. Taken together, PCA and estimations of phenotypic correlations showed that the level of redundancy between the different immune parameters was limited.
Heritability estimates of the 54 analyzed ITs was equal to 0.45 on average (se = 0.20; Table 1 For the large set of adaptive ITs, the mean heritability was 0.48 (se = 0.21). No significant difference in average heritability values was detected either between group of ITs qualifying the innate and adaptive immunity or the humoral and cellular adaptive immunity. In addition, an equivalent proportion of innate and adaptive ITs had significant heritability values. Indeed, 40% (10/25), 40% (2/5) and Among the traits involved in cell-mediated immunity, variation in ab T lymphocyte (CD4 -CD8 + , CD4 + CD8 + and CD4 + CD8cells) counts was highly heritable. Heritability estimates of the three cytokine (IL4, IL10, IFNG) levels were moderate to high after PMAIONO and CONA stimulations and weak to moderate after LPS stimulation. For those cytokines induced by CONA or LPS stimulation, confidence interval (95CI) for the heritabilities overlapped zero, except for IL4-CONA. IL2 production after PMAIONO, CONA and LPS stimulations gave high estimates of heritability significantly different from zero for IL2-PMAIONO and IL2-LPS. Proliferation measurements after various stimulations (PROLIF-CONA, PROLIF-PMAIONO, PROLIF-LPS) provided moderate estimates of heritability not significantly different from zero. Among the traits involved in humoralmediated adaptive immunity, heritabilities for total IgG and IgA antibody levels were higher than for total IgM and specific antibodies, and weak h 2 values were obtained for B lymphocyte count (IgM + cells).
Heritability for innate ITs such as i) total cell number (EOS and NEU), ii) leukocyte subsets (CD16 + CD2 + cells, CD16 + CD172Acells, CD16 + MHCII + cells and TCRcd + lymphocytes), iii) cytokine production (IFNA and IL12), and iv) phagocytosis were high and significantly different from zero. In addition, several innate ITs showed weak to moderate heritability, including proinflammatory cytokines (IL1B, IL8, TNF and IL6), MON, CD16 -CD2 + cells, CD16 + CD2cells, MHCII -CD172A + cells, MHCII + CD172Acells, CD16 -CD172A + cells and CD16 + CD172A + cells. Variation in acute phase proteins was moderately (CRP) to highly (HAPT) heritable. Among the four traits, which measured the total number (MON) or proportions (MHCII + CD172A + , CD16 + CD172A + and CD16 + MHCII + ) of monocytes, moderate to high h 2 , but not significantly different from zero, were estimated, except for CD16 + MHCII + cells. Other haematological traits (RBC, HT, RDW and PLT) gave high h 2 estimates, of which HT and PLT were significant.
Pairwise genetic correlations are presented in Table S2 and illustrated in Figure 3 . Genetic correlation estimates among most ITs were generally weak but a few high genetic correlations were observed. The number of positive genetic correlations (310) was higher than that of negative correlations (183), as already observed for phenotypic correlations. Positive genetic correlations were higher (in absolute values) than negative ones (Table S2, Figures 3 and S1). Only 28 (2.7% of the total number of correlations) and five r ĝ (0.4% of the total number of correlation estimates) were higher than 0.4 or lower than -0.4, respectively.
The unsupervised hierarchical clustering distinguished two main clusters of traits ( Figure 3 ). The first cluster of 14 traits (Cluster A) could be divided into two groups: i) a group of four traits including one innate immunity cytokine (IFNA), two antibody levels (total IgG and specific IgG-Mh), and one FACScharacterized leukocyte subpopulation (CD16 + CD172A -) and ii) a group of 10 traits with nine hemogram-based cell counts or FACScharacterized leukocyte subpopulations (WBC, LYM, MON, NEU, EOS, CD16 + MHCII + , CD16 + CD2 + , CD4 -CD8 + cells) and two cell activity traits (IgM and IL10-PMAIONO). The second cluster of 18 traits (Cluster B) is also subdivided into two groups of traits: i) a group of three different cell response traits (IL6, IgA and IFNG-PMAIONO) and one FACS-characterized leukocyte subpopulation (IgM + cells), and ii) a group of nine cell response traits (IL12, IL4-PMAIONO, IL2-PMAIONO, TNF, PROLIF-PMA, HAPT, CRP, IL1B, IL8 and PHAG) and four FACS-characterized leukocyte subpopulations (CD4 + CD8 -, CD4 + CD8 -, TCRcd + and MHCII + CD172A + cells). In cluster A, the first group of 10 traits showed moderately to highly positive genetic correlations with each other. Indeed, r ĝ values greater than 0.4 were estimated between WBC, LYM, MON, NEU, EOS, CD4 -CD8 + and CD16 + CD2 + (NK) cells ( Figure 3 , Table S2 ). In cluster B, r ĝ values greater than 0.4 were estimated between i) TNF, IL8 and PHAG, and ii) CRP and HAPT ( Figure 3 , Table S2 ). Strong negative r ĝ values (,20.4) were found between a few traits from both clusters: i) TNF and three cell number traits (WBC, LYM, MON), ii) IL6 and CD16 + MHCII + , and iii) CRP and IgA.
The measured ITs globally cover innate and adaptive immunity
The large-scale study reported here allowed us to estimate the genetic and phenotypic parameters of numerous ITs measured on pigs bred in the same environment. Innate immunity is represented by 25 traits, humoral-mediated immunity by five traits and cell-mediated adaptive immunity by 18 traits. We have also considered the total number of white blood cells and lymphocytes, and four other haematological traits (RBC, HT, RDW and PLT). Within cell-mediated immunity, we explored both Th1 and Th2 responses by measuring cytokine production. Figure 1 summarizes the traits that we selected to cover immunity globally. These traits include in vivo measures on blood such as quantification of cell populations by hemogram, dosage of circulating immunoglobulins and acute phase proteins, as well as ex vivo measures obtained after in vitro tests such as lymphocyte proliferation, phagocytosis capacity and cytokine production after blood stimulation. All these ITs have been widely studied in humans [25, 26] . The trait typology we have used (Table 1 ) follows a model based on a clear distinction between innate and adaptive immunity, which may be over-simplistic since both immune systems are closely interconnected [27, 28] . Monocytes are involved in innate immunity but are also antigen-presenting cells required for adaptive immunity, and cytokines such as IL12 are at the interface between innate and adaptive immunity. Similarly, the cell-mediated and humoral adaptive immunity subdivision is artificial. For example, IL4 is a cytokine produced by Th2 lymphocytes that is usually classified as part of adaptive cellmediated immunity, whereas it is also involved in antibody production and thus adaptive humoral immunity. In addition, the conventional paradigm that CD4 + ab T lymphocytes differentiate into Th1 and Th2 lineages expressing specific cytokines is collapsing. Indeed, recent studies have revealed that cytokine production by the different CD4 + T cell subsets (Th1, Th2, Th, Th17 and iTreg) is highly flexible, providing new insight into the Th cell plasticity [29] . Nevertheless, although schematic, the approach used in our report provides a comprehensive overview of genetic variation and co-variation across the entire immune spectrum in pigs (Figure 1 ). Table 1 illustrates various ranges of phenotypic variation in the measured immune traits, with most having a CV over 0.5. Such variability has already been reported in large panels of healthy humans [26] , and in previous studies on pigs [2, 16, 17, 30] . For instance, we substantiated the high level of variation of cytokine production previously reported for IL2 production and virusinduced IFNa production in a Swedish Yorkshire population [20] . For innate immunity-related cytokines and IL12, stimulation was performed with a mixture of LPS, PMA and ionomycin. IL8 was the cytokine produced with the highest levels followed by IL1B, TNF, IL12 and lastly IL6. These four cytokines are not expected to be expressed at similar levels at all time points after stimulation and a kinetic study would help to improve comparison of cytokine production levels. Weaker levels of adaptive IR cytokines are observed after LPS stimulation compared to CONA or PMAIONO. These differences could be related to the distinct modes of action of these molecules. Indeed, PMA, a plant-derived functional analog of diacylglycerol, in conjunction with ionomycin, a calcium ionophore produced by Streptomyces conglobatus, and CONA, a lectin originally extracted from the jack-bean Canavalia ensiformis, are known to be potent mitogens of blood lymphocytes [31, 32] . LPS, a major structural component of the outer membrane of gram-negative bacteria, which binds the CD14/ The five first components, which explain more than 50% of the total variance, are in red. B. Plot of the Bayesian Information Criterion (BIC) calculated with different models according to number of clusters. Six models are compared: EII (spherical with equal volume and equal shape), VII (spherical with variable volume and equal shape), EEI (diagonal with equal shape and equal volume), VEI (diagonal with variable shape and equal volume), EVI (diagonal with equal shape and variable volume), VVI (diagonal with variable shape and variable volume). C. First factorial plan (1: first component, 2: second component) with three clusters identified by multivariate normal mixture modelling and model-based clustering taking into account the 32 components (clusters K1, K2 and K3 are in blue, green and red, respectively). doi:10.1371/journal.pone.0022717.g002 TLR4/MD2 receptor complex and promotes the secretion of proinflammatory cytokines, has been extensively used to study innate immune response [33] . We have already shown that transcriptome modifications in peripheral blood mononuclear cells (PBMCs) differ between PMAIONO and LPS stimulation and that PMAIONO and LPS target different cells and cellular pathways [34] . The combination of PMA and ionomycin induces a stronger stimulation that may be related to a higher production of cytokines as detected in the present study. In addition, the lymphocyte proliferation induced by LPS is weaker than that observed with other stimulants, as expected.
Our study provides the first heritability estimates for innate and adaptive cytokine production and for lymphocyte proliferation after PMA-ionomycin and LPS stimulations in pig. Pro-inflammatory cytokines appear to show less heritable variation than adaptive system-related cytokines. Among the adaptive system-related cytokines, estimated heritability was weakest for cytokines produced after LPS stimulation, except for IL2 production. Heritability estimates for lymphocyte proliferation after CONA, PMA and LPS stimulations were moderate and that of lymphocyte proliferation after CONA stimulation was comparable to the value obtained by Edfors-Lilja and colleagues [15] . Moderate to high heritability estimates for cell count traits from hemogram or FACS also confirmed those previously obtained for WBC, total lymphocytes, neutrophils, eosinophils and some leukocyte subsets (for CD4 + and CD8 + T lymphocytes, cd T lymphocytes, CD11R1 + , CD11R1 + CD8a -, CD11R1 + CD8a + and CD16 + MHCII + ) [15, 16, 17, 19] . Likewise, our results confirmed the high heritability estimate for phagocytosis [15] . Conversly, a lower heritability estimate for CRP than previously reported was observed [15, 16] . Overall, the heritability estimates for these traits appear robust regardless of populations, environments and protocols.
Some discrepancies exist between our heritability estimates and previous results for humoral-mediated adaptive ITs and some innate ITs. Indeed, in our study, B lymphocyte levels (IgM + cells) are not significantly heritable contrary to other results [16, 17] and specific IgGs (IgG-Mh) have lower heritability estimates (0.12, se = 0.19) than previously reported (range from 0.27 to 0.45) for specific antibodies directed against other antigens [12] . Our estimated heritability for total IgG is higher (0.92, se = 0.20) than in published reports [3, 15, 18, 19] . In addition, our estimated h 2 for IFNa production is moderate to high (0.60, se = 0.23), contrary to previous results (range from 0 to 0.08) [15] , and for haptoglobin (0.55, se = 0.21) is higher than previously published (range from 0.14 to 0.23) [17, 19] . These discrepancies could be due to differences in the pig breeds and in environment factors but also to the absence of common standardised protocols between laboratories. In order to better qualify the phenotypes, protocol standardisation is needed.
Overall, we show that variation in both innate and adaptive ITs is under substantial genetic control (Figure 1 ; Tables 1 and 2) . Similar heritability estimates for innate and adaptive ITs and also between cell number and cell response parameters were observed. Further, heritability estimates do not differ consistently between in vivo and ex vivo measures with no apparent bias due to phenotyping methods. These data also suggest candidate ITs for QTL mapping. Indeed, mapping studies have already started for total leukocyte count ( [20, 21] ; AnimalQTLdb, http://www.animalgenome.org/cgi-bin/QTLdb/index), mitogen-induced proliferation [20] , antibody response [20, 22] , cytokine production (IL2, IL10 and IFNc) [23, 35] , complement activity [22] , and acute phase protein serum concentration [22] .
Compared to the previously limited data on genetic correlations between ITs in pigs, our study provides a large-scale estimation of phenotypic and genetic correlations among 32 ITs. PCA results and correlation estimations highlight the weak phenotypic and genetic correlations between the different ITs, except mainly for cell subsets. No cluster of innate and adaptive ITs is revealed. These results illustrate that many of the ITs included in our study provide more or less independent potential clues for selecting for improved immunocompetence. Such complementarity is expected since innate immunity is in place or ready for activation prior to infection or antigenic stimulation and collaborates with adaptive immunity, which is induced by infection or antigenic stimulation. Nevertheless, a few highly positive genetic correlations have been detected between total number of white blood cells and some leukocytes subsets, and between some leukocyte subsets such as total number of lymphocytes, CD4 -CD8 + lymphocytes (which contains ab CD4 -CD8 + lymphocytes and NK), and NK cells (CD16 + CD2 + cells). Phagocytosis, production of IL8 and TNF, two pro-inflammatory cytokines produced by monocytes and macrophages, were positively correlated with acute inflammatory phase proteins produced by hepatocytes i.e. CRP and HAPT. A high correlation was also found between CRP and HAPT. Clapperton and colleagues [17] have shown that phenotypic correlations between leukocyte subsets and acute phase proteins are weak (,0.2) and not significantly different from zero in agreement with our results. In contrast to our study, Clapperton and collaborators have not detected any significant genetic and phenotypic correlations between different leukocyte subsets except when one subset was nested in another [16] .
Our results provide a framework for including ITs in multitrait selection for immunocompetence in pigs. Criteria for inclusion should take into account heritability, biological relevance, biological sensitivity and feasibility of measurement [25] . The weak genetic correlations between most ITs suggest that it will be difficult to choose only a few ITs to select for immunocompetence, and that a combination of many traits may be required [26] . The moderate to high heritabilities estimated for many traits together with the selection study on pigs carried out by Wilkie and colleagues a decade ago support the feasibility of selecting for immunocompetence [2, 13] . Chickens have also been successfully divergently selected for carbon clearance (phagocytic activity), high antibody response to Newcastle disease virus three weeks after vaccination (adaptive humoral IR) and wing web response to PHA (high cell-mediated immune response) for more than twelve generations [9, 36] .
In order to include immunocompetence in selection for improved health, a major challenge will be to correlate variation in heritable ITs in healthy animals with inter-individual variability in response to various pathogens. Testing this hypothesis will be a key point for further use of ITs as indirect selection criteria in multitrait selection to improve resistance to disease. Some results on genetic and phenotypic relationships between immunocompetence and susceptibility to specific pathogens have already been reported in the literature for pigs. Among pigs selected for eight generations for high (HIR) or low (LIR) response based on an index of four cell and humoral-mediated immunity traits, an increased specific antibody response and lower polyserositis were observed in the HIR pigs compared to the LIR pigs after challenge with a novel pathogen, Mycoplasma hyorhinis [12, 37] . Thus, animals with a high IR level to unrelated challenges, as defined by Wilkie and colleagues [12, 37] , have a better response to infection with Mycoplasma hyorhinis. However, HIR pigs develop more severe arthritis than LIR pigs. Indeed, the levels of humoral and cell-mediated adaptive ITs included in the Wilkie et al index induce the formation of immune complexes and/or the development of inflammatory responses, central to the pathogenesis of Mycoplasma hyorhinis-induced arthritis. Other correlation tests between ITs and response to various infections are needed. However, it is important to remain cautious with high responder animals, which could develop autoimmune pathologies or pathological iummne responses.
All the studies on immunocompetence and resistance to disease will have to be completed by estimation of genetic correlations with economically important traits already under selection. Negative genetic correlations have been reported between some ITs (monocytes, CD11R1 + cells) and average daily gain [16] . However a larger correlation study considering a higher number of ITs and pig performances is needed and is ongoing in our population. In the future, a more sustainable production system may require a compromise with a slight decrease in performance traded off for a gain in animal robustness. More studies are required to better understand the correlations between ITs and production traits and it is not established which levels of ITs would be good predictors for resistance to pathogens if any. In conclusion, our results show that variation in many ITs is under significant genetic control in pigs and these findings may provide insights in other species. Moreover, based on heritability and correlation estimations, some of the ITs that we have studied might be incorporated into selection schemes, provided they are associated with improved global health and do not exhibit strong antagonisms with other economically important traits.
Our experiment was conducted in accordance with the French national regulations for humane care and use of animals in research. No ethics approval was required for the vaccination and the collection of blood samples under the then current regulations. Experiments were performed under the individual license numbers 77-01 assigned to Marcel Bouffaud who was responsible for experiments in the test farm, and 78-16 assigned to a veterinarian, Dr Silvia Vincent-Naulleau. The experimentation agreement number for the test farm at le Rheu was A35-240-7.
A total of 443 Large White pigs (castrated males, dam line) tested for performance traits in a pig test station (UE450, INRA, Le Rheu, France) was included in the study. The pigs were distributed in seven contemporary groups and belonged to 307 nuclear families obtained from 106 boars, with an average of 4.1 (+/2 1.7) piglets per boar. Animals were born and weaned in 16 different selection herds and arrived in the test station at five weeks of age with no prior vaccination. They were placed into pens of 30 piglets in a post weaning unit and vaccinated against M. hyopneumoniae (Stellamune, Pfizer, one injection) one day after their arrival in the test station, when 36.3 days old in average. All pigs were apparently healthy with no clinical sign of infection. All animals were sampled three weeks after vaccination. Blood samples were collected via the external jugular vein into tubes with or without anti-coagulants, according to further use.
Blood collected in 10 mL tubes with no anti-coagulant was centrifuged at 3200 g for 15 min at 4uC. The serum was collected and stored at 220uC until use. Plasma was collected from blood sampled in heparinised tubes and stored at 220uC before use.
Hemograms were measured with an MS4-5 counter (Eli-techGroup, France) with blood sampled in EDTA tubes. Among a set of 18 traits, nine were included in the genetic analyses: total number of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, platelets and hematocrit (Tables 1 and 2 ).
Total concentrations of immunoglobulin subsets were measured by ELISA as previously described [38] . Plasma samples were diluted 1:6000, 1:4000 and 1:60,000 to detect IgM, IgA and IgG, respectively, in Tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (Bethyl laboratories Inc., Interchim, France). The different subsets were detected with the appropriate peroxidase anti-pig IgM, IgA or IgG (Bethyl laboratories Inc.) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. Anti-M. hyopneumoniae IgG titers were also measured by ELISA using a commercial kit (ELISA ID ScreenH M. hyopneumoniae Indirect, IDVET, France). Absorbance was read at 450 nm using an ELISA plate reader (Spectra thermo, Tecan, NC, USA) and the Biolise 2.0 data management software.
Haptoglobin and C reactive protein levels were measured in pig serum by colorimetric tests (Phase Haptoglobin Assay, ABCYS Biologie, France) and ELISA assays (Porcine C reactive Protein Assay, ABCYS Biologie, France), respectively. Absorbance was read at 450 nm using an ELISA plate reader (MRX revelation, Dynex).
PBMCs were purified by density gradient centrifugation. A volume of 13 mL heparinised blood was added to Leucosept tubes (Greiner Bio-one, France) pre-filled with 17 mL Ficoll (Lymphocytes Separation Medium, Eurobio, France) and centrifuged at 1200 rpm for 35 min. PBMCs were collected at the ficoll interface and washed in 50 mL D-PBS without MgCl 2 and CaCl 2 (GIBCO, Invitrogen, France). Cells were then incubated in 2 mL BD Pharmlyse 1X (BD Biosciences, France) at room temperature.
Purified PBMCs were washed in 50 mL D-PBS without MgCl 2 and CaCl 2 , incubated with 2 mL pig serum at 4uC for 20 min, washed again in 50 mL D-PBS without MgCl 2 and CaCl 2 and then washed in 50 mL S/W buffer (1 g/L NaN 3 , 10 g/L bovine serum albumin in PBS, pH 7.3) at a final concentration of 5.10 6 cells/mL. 10 6 cells were used for each antibody labelling. Single, double or triple staining was performed using monoclonal antibodies (mAbs) directed against i) CD2 (MSA4, isotype IgG2a, VMRD) and CD16 (MCA1971, isotype IgG1, Serotec), ii) CD4 (PT90A, isotype IgG2a, VMRD) and CD8a (PT81B, isotype IgG2b, VMRD) iii) TCRcd (MAC320, isotype IgG2a, BD Biosciences Pharmingen), iv) IgM (PIG45A, isotype IgG2b, VMRD) and v) MHCII (MSA3, isotype IgG2a, VMRD), CD16 (MCA1971, isotype IgG1, Serotec) and CD172a (74-22-15A, isotype IgG2b, BD Biosciences Pharmingen). Briefly, PBMCs were stained with primary mAbs for 25 min at 4uC, washed in S/W buffer and stained with allophycocyanin-conjugated anti-mouse IgG1 (BD Biosciences, France), phycoerythrin-conjugated antimouse IgG2a (Southern Biotech, France), FITC-conjugated antimouse IgG2b (Southern Biotech, France), APC-conjugated antimouse IgG1 (BD Biosciences, France), phycoerythrin-conjugated anti-rat IgG2a (BD Pharmingen, France), or phycoerythrinconjugated anti-mouse IgG2b (Southern Biotech, France). After washing in S/W buffer, cells were fixed in BD Cellfix solution (Becton Dickinson, Germany). Data acquisition and analysis were carried out with the FACScan and CELLQuest software (Becton Dickinson, UK).
Synthesis of IFNa by leukocytes of pigs was tested in vitro by incubating diluted total blood in the presence of pseudorabies virus (PrV, Suid Herpesvirus 1)-infected, glutaraldehyde-fixed, PK15 cell monolayers, according to a protocol previously described for transmissible gastroenteritis virus [39] . Confluent PK15 cell monolayers grown in 24 well plates were infected by PrV at a multiplicity of infection of 20, fixed with 0.05% glutaraldehyde 8 h post-infection and washed with D-PBS and RPMI1640 before the addition of blood samples. For each animal, monolayers were incubated with diluted heparinized blood (270 ml of blood diluted 1:5 in DMEM supplemented with antibiotics) for 18 h at 37uC. Plates were then centrifuged at 450 x g for 20 min at 4uC, and supernatants were collected and stored at -20uC. IFNa was assayed in the supernatants using a classical sandwich ELISA test as previously described [40] .
Production and dosage of IL1B, IL6, IL8, TNFa and IL12
Heparinized blood samples (400 mL) were fivefold diluted in 24well plates in 1.6 mL RPMI 1640 medium (BioWhittaker, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (QB perbio, UK), 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. For stimulation, a mixture of 10 ng/mL PMA (Sigma, France), 1 mg/mL ionomycin (Sigma, France) and 1 mg/mL LPS from Escherichia coli O111:B4 (Sigma, France) was added to the diluted blood. For mock stimulation, a volume of PBS equal to the volume of stimulation reagents was added to the diluted blood. After incubation at 37uC for 24 h, culture supernatants were collected by centrifugation at 450 g for 20 min and stored at 220uC before use.
The cytokines IL1B, IL6, IL8, TNF and IL12 were quantified using commercial ELISA tests (DuoSet ELISA development kits, R&D Systems, USA). For quantification of basal levels of cytokines in supernatants from mock-stimulated cells, the samples were not diluted for quantification. Supernatants collected from stimulated cells were diluted (1:22 for IL1B and IL8, 1:1 for IL6, 1:10 for TNF and 1:2 for IL12). All samples were tested in duplicates. Absorbance was read at 450 nm using an ELISA plate reader (MRX revelation, Dynex). Results were expressed as pg of cytokine/mL.
Heparinized blood diluted 1:5 in complete culture medium consisting of DMEM (Dulbecco's Modified Eagle Medium, Eurobio, France) supplemented with 5% fetal calf serum (Hyclone, Perbio, France), 2 mM L-glutamine, 100 U/mL penicillin and 50 mg/mL streptomycin (Eurobio, France) was stimulated with 10 mg/mL CONA (Sigma, France), or with 50 ng/mL of PMA (Sigma, France) and 1 mg/mL of ionomycin (Sigma, France) or 1 mg/mL LPS from Escherichia coli (Sigma, France). Cytokine content was measured in supernatants using ELISA tests as already described [41] . Briefly, purified fractions of anti-swine IL-2, IL-4, IFNc (clones A150D 3F1, A155B 16F2 and A151D 5B8 respectively, Biosource, France) and IL10 (clone 148801, R and D System, France) were used as capture antibodies, in conjunction with the biotinylated anti-swine IL-2, IL-4, IL10 and IFNc monoclonal antibodies (clones A150D 8H10, A155B 15C6 and A151D 13C5, respectively, Biosource, Clinisciences, France) or anti-swine polyclonal antibody (goat anti-porcine IL-10, R and D System, France). Streptavidin-horseradish peroxidase (Biosource) and TMB (Fermentas, MD, USA) were used for detection. Absorbance was read at 450 nm using an ELISA plate reader (Spectra thermo, Tecan, NC, USA) and the Biolise 2.0 data management software. Recombinant pig IL-2, IL-4, IL10 and IFNc were used as standards. The detection limits were 700 pg/ mL, 60 pg/mL, 90 pg/mL and 100 pg/mL for IL-2, IL-4, IL10 and IFNc, respectively. Results were expressed as pg of cytokine/ mL.
Lymphocyte proliferation was performed in 96 well plates as already described [42] . Briefly, heparinized blood samples were diluted 1:15 in complete culture medium consisting of DMEM (Dulbecco's Modified Eagle Medium, Eurobio, France) supplemented with 5% fetal calf serum (Hyclone, Perbio, France), 2 mM L-glutamine, 100 U/mL penicillin and 50 mg/mL streptomycin (Eurobio, France). For detection of unspecific lymphocyte proliferation, the diluted blood samples were seeded in 96 well plates (200 mL/well) and mock-stimulated for 48 h by incubation in culture medium (control wells), or stimulated for 48 h by incubation with the culture medium supplemented with either 10 mg/mL ConA (Sigma, France), or 50 ng/mL of PMA (Sigma, France) and 1 mg/mL of ionomycin (Sigma, France), or 1 mg/ml LPS (Sigma, France). Control wells remained unstimulated. After 48 h of incubation at 39uC, 0.5 mCi of 3 H-methylthymidine (ICN, France) was added to each well. After another 24 h incubation period, the cells were harvested through glassfiber filters (Whatman, United Kingdom) by means of an automatic harvester (Titerteck-Skatron, Molecular Devices, France). Incorporation of tritiated thymidine was measured with a Liquid Scintillation Beta Counter (Kontron Instruments, France). Results were expressed as a stimulation index of lymphocyte proliferation calculated as mean counts per min (cpm) of the triplicate cultures in stimulated culture/mean cpm in control non-stimulated culture.
Preliminary statistical analyses were performed using R software [43] . In order to test if trait distributions deviated from Gaussian, a D'Agostino normality test was used (p = 0.05). Since most traits were not sampled from a Gaussian distribution, they were all normalized using a Box-Cox transformation except for phenotypes reaching the value zero, which were normalized using ln(1+x) transformation. Significant effects of age at the time of vaccination, of time of vaccination, of breeding unit and of time of experiment were detected for most traits by variance analysis taking into account these effects. Normed Principal Component Analysis (PCA, [44] , dudi.pca function, ade4 package [45] , R software [43] ) on a subset of ITs adjusted for age at the time of vaccination, time of vaccination, breeding unit and experiment (Table 1) were performed using a linear model. Clusters of ITs were detected using the R package mclust for normal mixture modelling and model-based clustering [46] . It combines model-based agglomerative hierarchical classification, based on the classification likelihood, and the expectation-maximization (E-M) algorithm for maximum likelihood estimation of multivariate mixture models. Variance components, genetic parameters and their standard errors were estimated by the Restricted Maximum Likelihood (REML) method [48] , using the WOMBAT software [49] . This is the reference method to estimate genetic parameters with a mixed model. Univariate and bivariate mixed linear animal models were employed to estimate heritability and genetic correlations, respectively. For the univariate analyses, the fixed part of the model included experiment time, age at the time of vaccination, vaccination time and herd of origin effects and the random part included a common litter environmental effect and direct genetic effects. In matrix notation y~X b zW a azW c cze where y = the vector of observations; X b , W a and W c are known incidence matrix relating observations to fixed and random effects; ß = a vector of fixed effects and covariates; a = the vector of direct genetic effects; c = the vector of common litter environmental effects; and e = the vector of random residual effects. All random effects were assumed to follow a normal distribution with zero mean. For bivariate analyses, the same effects as for univariate analyses were taken into account in the fixed part of the model and a direct genetic effect was included in the random part of the model. 95% confidence intervals (95CI) were calculated for heritability (h 2 ) estimates (h 2 ). Heritability estimates have been classified: high (h 2 .0.4), moderate (0.1,h 2 ,0.4) or weak (h 2 #0.1). A graphical representation of the genetic correlations combined with a hierarchical clustering (euclidian distance, average link) was obtained with the heatmap function from the Bioconductor software [50] . Comparisons of heritability average between subsets of traits were tested using the Wilcoxon Mann-Whitney Test (significance threshold p-value = 0.05). Figure S1 Heatmap of the phenotypic correlations between 32 ITs. The correspondence between colour scale and genetic correlation levels are presented on the right-hand side of the heatmap. (TIF)  Demographic, etiological, and histological pulmonary analysis of patients with acute respiratory failure: a study of 19 years of autopsies INTRODUCTION: Acute respiratory failure has been one of the most important causes of death in intensive care units, and certain aspects of its pulmonary pathology are currently unknown. OBJECTIVES: The objective was to describe the demographic data, etiology, and pulmonary histopathological findings of different diseases in the autopsies of patients with acute respiratory failure. METHOD: Autopsies of 4,710 patients with acute respiratory failure from 1990 to 2008 were reviewed, and the following data were obtained: age, sex, and major associated diseases. The pulmonary histopathology was categorized as diffuse alveolar damage, pulmonary edema, alveolar hemorrhage, and lymphoplasmacytic interstitial pneumonia. The odds ratio of the concordance between the major associated diseases and specific autopsy findings was calculated using logistic regression. RESULTS: Bacterial bronchopneumonia was present in 33.9% of the cases and cancer in 28.1%. The pulmonary histopathology showed diffuse alveolar damage in 40.7% (1,917) of the cases. A multivariate analysis showed a significant and powerful association between diffuse alveolar damage and bronchopneumonia, HIV/AIDS, sepsis, and septic shock, between liver cirrhosis and pulmonary embolism, between pulmonary edema and acute myocardial infarction, between dilated cardiomyopathy and cancer, between alveolar hemorrhage and bronchopneumonia and pulmonary embolism, and between lymphoplasmacytic interstitial pneumonia and HIV/AIDS and liver cirrhosis. CONCLUSIONS: Bronchopneumonia was the most common diagnosis in these cases. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Further studies are necessary to elucidate the complete pathophysiological mechanisms involved with each disease and the development of acute respiratory failure. Acute respiratory failure (ARF) is a major cause of death in patients with a variety of primary underlying diseases. In addition, the prevalences of comorbidities and mortality have been reported to be higher than 40-50% in ARF patients, especially in those with diffuse infiltrates as seen on chest X-rays. [1] [2] [3] The clinical and radiological findings in ARF are nonspecific, [4] [5] [6] and prompt investigation and diagnosis are essential to improving patient survival. [7] [8] [9] In this context, the complexity of clinical presentations makes diagnosis a constant challenge for the clinician. Despite recent advances, most types of diagnostic support are still expensive. Clinicians often initiate treatment to avoid the rapid progression of the disease and to spare the patient from more invasive procedures. Therefore, it is important to determine the leading causes of death in this population to establish correct prophylactic actions, which is the least expensive strategy for preventing further pulmonary dysfunction and avoiding the need for lung biopsies. 10 We performed a retrospective study of 4,710 autopsies on patients whose cause of death was ARF to better describe the demographic and etiological data and the associated histological pulmonary findings and diseases.
Autopsies. This study was conducted at a tertiary health care center. From 1990 to 2008, 26,560 medical autopsies were performed at this center, with an annual mean of 1,889. We examined the autopsies of the 4,710 (17.7%) patients in whom ARF was the cause of death. In 980 of these patients, the histological pulmonary findings could not be reviewed because the pulmonary tissue was not available (it was classified as ''without analysis''); histological pulmonary analyses were performed in the remaining 3,730 (79.2%) cases. In this study, we reviewed all of the available microscopic and macroscopic diagnoses of death at autopsy, along with the patient medical records. The only inclusion criterion for the definition of ARF was based on arterial blood gases (PaO2,60 mmHg or PaCO2.50 mmHg with pH,7.30) while breathing room air. [1] [2] [3] We excluded patients younger than one year of age and individuals not diagnosed with ARF.
We also obtained data regarding each patient's age, sex, and major underlying associated diseases (as determined at autopsy).
The clinical data from the patients enrolled in the study were collected with the approval of the Internal Review Board for this study, and informed consents were obtained from family members to perform the autopsies.
After a complete review, the pulmonary pathological reports were categorized as follows:
Ndiffuse alveolar damage (DAD), which was defined as diffuse involvement and a uniform temporal appearance of alveolar collapse, hyaline membranes, obliterative fibrosis, neo-septal formation, and moderately organizing fibrosis;
Npulmonary edema (PE), which was defined as the accumulation of proteinaceous fluid in the alveolar spaces that gave the appearance of a granular, pink coagulate within such spaces; Nalveolar hemorrhage (AH), which was defined as the presence of blood in the alveolar spaces; and
Nlymphoplasmacytic interstitial pneumonia (LPIP), which was defined as widened and edematous alveolar septa, usually accompanied by a mononuclear inflammatory infiltrate of lymphocytes, histiocytes, plasma cells, and neutrophils.
All the lungs were analyzed by microscopy, even when the medical records indicated the patient's diagnosis. The lungs were fixed in 10% formalin prepared in 0.9% saline for at least four weeks. We studied a minimum of five sections per lung for a total of ten sections per person, regardless of the presence or absence of morphologically demonstrable lesions. The paraffin-embedded tissue sections were assessed following hematoxylin and eosin staining. To document the presence and distribution of the wide spectrum of infectious agents and neoplasms to which this population was susceptible, we prepared a variety of special stains (periodic acid-Schiff staining, immunohistochemical analysis, fluorescence, Ziehl-Neelsen acid-fast staining, Gram acid-fast staining, Mucicarmine acid-fast staining, and Gomori's methenamine silver acid-fast staining) for selected tissue sections. Bacterial bronchopneumonia (BBP) was defined as the presence of cell consolidation with polymorphonuclear leukocyte accumulation in bronchioles and adjacent alveoli. For the diagnoses of cytomegalovirus and fungal pneumonia, histological evidence of lung involvement was required, with or without tissue culture.
Severe sepsis and septic shock were defined as sepsis with the addition of organ dysfunction or the clinical diagnosis of arterial hypotension, which may or may not have been responsible for the aggressive fluid resuscitation. The diagnoses of Mycobacterium tuberculosis infection and atypical mycobacterial infection were confirmed using fluorescence, the Ziehl-Neelsen techniques and a Lowenstein-Jensen culture. The proportion method and biochemistry were used to identify all of the positive cultures.
Statistical analysis. The descriptive analysis of the data included median, minimum, and maximum values. The probabilities (odds ratios) that patients with underlying diseases and comorbidities would develop specific pulmonary histopathological patterns and die of ARF-related pulmonary alterations were determined by logistic regressions. The independent variables included the following major diseases and/or comorbidities: BBP, cancer, liver cirrhosis, HIV/AIDS, acute myocardial infarction, systemic arterial hypertension, dilated cardiomyopathy, pulmonary embolism, chronic obstructive pulmonary disease, diabetes mellitus, sepsis and septic shock, chronic kidney failure, and tuberculosis. All the statistical procedures were performed using the SPSS v10.0 statistical software. Statistical significance was set at a p-value of 5%.
ARF was described in 4,710 autopsies (17.7%) from 1990 to 2008. The patients' ages ranged from 1 to 99 years (with a median of 52). A total of 2,713 (57.6%) men and 1,997 (42.4%) women were included in the study. The demographic data are listed in Table 1 .
We observed a single associated disease in 1,793 (38.0%) of the cases, 2 diseases in 1,505 (32.0%), 3 in 797 (16.9%), 4 in 349 (7.4%) and 5 diseases in 184 (3.9%) of the cases. A diagnosis could not be determined after autopsy in 82 (1.7%) of the cases.
The pulmonary histopathological analysis showed DAD in 40.7% (1,917) of the patients, PE in 23.5% (1,107), AH in 10.4% (491) and LPIP in 4.6% (215) of the patients. The pulmonary histopathological findings and the most prevalent diseases in the 4,710 patients are shown in Table 2 .
The major underlying diseases and comorbidities in the ARF patients are shown in Table 2 . BBP was present in 33.9% of the patients (1,597 cases) and was the most frequent pulmonary complication found at the time of autopsy. Cancer was the second most frequent complication; it was observed in 28.1% of the patients (1,324 cases), followed by severe sepsis and/or septic shock in 14.3% (675), liver cirrhosis in 13.6% (639), HIV/AIDS in 10.4% (490), pulmonary embolism in 9.0% (426), acute myocardial The multivariate analysis, with the statistically significant associations between the most prevalent diseases/comorbidities and the different histopathological findings, is shown in Table 3 .
The early and accurate treatment of ARF remains an important problem in the management of critically ill patients. Despite recent technological diagnostic advances, the autopsy remains an important complementary tool for identifying and understanding diseases. Autopsy studies have shown important differences between autopsy findings and the antemortem clinical diagnoses. [11] [12] [13] Such diagnostic disagreement can vary from 10% to 90%, depending on the disease and the population involved. [4] [5] [6] [7] [8] [9] [10] [14] [15] [16] [17] [18] [19] These discrepancies may be attributable to different clinical manifestations of a single disease or to poor-quality medical care. 4 Recently, ARF was reported to be one of the leading causes of morbidity and mortality in critically ill patients. 4, 9, [14] [15] [16] In this study, we observed a high prevalence (17.7%) of ARF patients. Most of the study patients were males (57%), and the mean age was 52 years. Others studies have shown similar results. 4, 9, [14] [15] [16] A retrospective study carried out between 1996 and 2002 in 58 patients with DAD diagnosed by surgical lung biopsy found that 52% of the patients were male and that the mean age was 61 years. 20 We observed a single disease in 38% of the patients with ARF and two or more diseases in 62% of the patients. The high prevalence of multiple diagnoses illustrates the complexity and critical status of the patients that presented with ARF as the cause of death; it may indicate the need for a different therapeutic strategy with these patients. [21] [22] Our study specifically showed that the patients who developed ARF had underlying diseases such as BBP (33.9%), cancer (28.1%), sepsis and septic shock (14.3%), liver cirrhosis (13.6%), HIV/AIDS (10.4%), pulmonary embolism (9.0%), acute myocardial infarction (4.7%), brain stroke (4.4%), chronic kidney failure (4.4%), and diabetes mellitus (4.1%). Infectious diseases were the most common diagnoses, as has also been described by other authors. 6, 9, [14] [15] [16] [17] [18] [19] [20] 23, 24 Interestingly, these findings are not consistent with our previously published study in 2008 that reported related cases of ARF in autopsies from 1990 to 2000 only; it reported the following associated diseases or complications in descending order: HIV/AIDS (31.4%), BBP (21.8%), sepsis and septic shock (11.7%), liver cirrhosis (11.5%), pulmonary embolism (5.7%), acute myocardial infarction (5.5%), brain stroke (4.6%), tuberculosis (3.6%), cancer (2.3%), chronic kidney failure (1.9%), and leukemia (0.2%). 22 BBP was present in 33.9% of the patients and was the most frequent pulmonary complication found during autopsy. Frequently, the ARF patients had BBP as the initial cause of pulmonary disease, but BBP often occurs as a complication of other pathologies, usually in immunocompromised and intubated patients. Other studies have described similar findings and reported BBP as the most common disease in ARF patients. 20,23-25 Gross et al 25 analyzed 234 autopsies of elderly patients and found that 33% of them had BBP, which is exactly the same prevalence observed in our study but in an elderly-only population. 25 Cancer was the second most important complication diagnosis in our study; it was present in 28.1% of the autopsies. This incidence rate was higher than that observed in other studies. 25 The most interesting observation was the increase compared with the incidence found in our previously published survey (2.3%). 22 This finding suggests that the number of diagnoses and the severity of cancer presentations have increased. These patients had been exposed to immunocompromised conditions from the use of quimioterapic agents and anorexia. Both these conditions increase the risk of associated infections, which are one of the major causes of death in this population. 20, 26 Consequently, pulmonary manifestations are one of the most common causes of death in these patients; there is a high incidence of BBP, including atypical bacterias. 20, 25, 26 In this study, HIV/AIDS was present in 10.4% of the patients with ARF. Pulmonary involvement has been reported in between 80 and 94% of the patients with HIV/ AIDS. The classical evolution of patients with HIV/AIDS to ARF and its importance have been reported in several other studies. [11] [12] [13] 21, 22 As in patients with cancer, immunocompromised conditions substantially increase the risk of infection. Associated bacterial BBP is the major cause of respiratory failure, but tuberculosis, cytomegalovirus and Pneumocystis jiroveci pneumonia are also important causes of pulmonary commitment. [11] [12] [13] 21 In contrast to our previous publication, the rate of HIV/AIDS infection has declined over the last ten years from 16-31% to only 10.4%. 21, 22 Modern diagnostic methods, antibiotics and antiretroviral therapy have been reported to be the major causes of this modification of the HIV/AIDS prevalence in critically ill patients. 21 We observed liver cirrhosis in 13.6% of the autopsies in this study; a similar association in ARF patients was not found in the literature. We realize that liver cirrhosis can result in an immunocompromised or inflammatory status similar to that of other pathologies and predispose patients to infectious diseases.
Others diseases, such as pulmonary embolism and acute myocardial infarction, were significantly associated with ARF. The underdiagnosis of pulmonary embolism has been a significant problem in critical care units until recently. 22 Based on the pulmonary histopathological analysis, DAD was the most common pattern observed (40.7% of cases), which is consistent with our previous study. 22 No similar results have been previously found in other studies of ARF patients. Parambil et al 20 described a 90% prevalence of DAD in patients with acute respiratory distress syndrome who underwent surgical lung biopsies. 20, 27 However, these findings are consistent with the higher prevalence and the significant association with BBP of sepsis and/or septic shock, HIV/AIDS and other infectious and inflammatory diseases. Other contributing factors include impeded mechanical ventilation, which can accelerate the development of these histopathologies. 21, 22 Unexpectedly, DAD was also associated with conditions such as liver cirrhosis, acute myocardial infarction, pulmonary embolism, dilated cardiomyopathy and chronic obstructive pulmonary disease. We did not find that this association has been reported elsewhere in the literature, and it probably indicates that a common inflammatory response is present in these distinct pathologies.
As expected, PE was mostly associated with myocardial infarction and dilated cardiomyopathy; regardless, isolated PE was still observed and was associated with almost all of the major diseases studied. PE was the most specific pattern in the cancer patients, a finding that has not been previously reported.
AH was present in 10.4% of the patients and was associated with BBP and pulmonary embolism. We think that the association with myocardial infarction is not as relevant because of the small number of patients (four cases). AH was also associated with severe pulmonary complications and the presence of large necrotic areas and hemorrhaging, as is expected to result in cases of massive pulmonary embolism. 22 One group has described pulmonary embolisms in 5% of the patients who died from ARF. 14 Two other groups have reported pulmonary embolisms in 14% and 20% of the patients who died from ARF. 15, 16 Finally, LPIP showed an important association and was the most specific pattern in the HIV/AIDS patients, a finding that has been previously reported. [11] [12] [13] 21 These findings are consistent with the higher prevalence of opportunistic infections, mainly viral, fungal and mycobacterial, that frequently cause LPIP to develop. 21, 28 Liver cirrhosis was also associated with LPIP, although the possible reasons are unknown.
First and foremost, this was a retrospective study of medical records in which the quality of the information was inherently limited. The second limitation of our study is related to the interobserver variability, which is an issue even though all of the autopsies at our institution are performed by a resident pathologist and supervised by a senior pathologist, who also checks the histological analysis to prepare the final reports. Different observers can have different opinions; some studies have shown significant interobserver variability in about 15% to 35% of the autopsies analyzed. 4, 7 In addition, the accuracy of autopsy findings depends on the interest and skill of the pathologist.
Despite recent advances in diagnostic technological, the autopsy has remained an important complementary tool for identifying and understanding diseases in ARF patients. BBP was the most common diagnosis in our ARF patients, followed by cancer. The most prevalent pulmonary histopathological pattern was DAD, which was associated with different inflammatory conditions.
Further studies are necessary to elucidate the complete pulmonary pathophysiological mechanisms involved in each disease and in the development of ARF. Type 1 Interferon Induction of Natural Killer Cell Gamma Interferon Production for Defense during Lymphocytic Choriomeningitis Virus Infection Natural killer (NK) cells are equipped to innately produce the cytokine gamma interferon (IFN-γ) in part because they basally express high levels of the signal transducer and activator of transcription 4 (STAT4). Type 1 interferons (IFNs) have the potential to activate STAT4 and promote IFN-γ expression, but concurrent induction of elevated STAT1 negatively regulates access to the pathway. As a consequence, it has been difficult to detect type 1 IFN stimulation of NK cell IFN-γ during viral infections in the presence of STAT1 and to understand the evolutionary advantage for maintaining the pathway. The studies reported here evaluated NK cell responses following infections with lymphocytic choriomeningitis virus (LCMV) in the compartment handling the earliest events after infection, the peritoneal cavity. The production of type 1 IFNs, both IFN-α and IFN-β, was shown to be early and of short duration, peaking at 30 h after challenge. NK cell IFN-γ expression was detected with overlapping kinetics and required activating signals delivered through type 1 IFN receptors and STAT4. It took place under conditions of high STAT4 levels but preceded elevated STAT1 expression in NK cells. The IFN-γ response reduced viral burdens. Interestingly, increases in STAT1 were delayed in NK cells compared to other peritoneal exudate cell (PEC) populations. Taken together, the studies demonstrate a novel mechanism for stimulating IFN-γ production and elucidate a biological role for type 1 IFN access to STAT4 in NK cells. IMPORTANCE Pathways regulating the complex and sometimes paradoxical effects of cytokines are poorly understood. Accumulating evidence indicates that the biological consequences of type 1 interferon (IFN) exposure are shaped by modifying the concentrations of particular STATs to change access to the different signaling molecules. The results of the experiments presented conclusively demonstrate that NK cell IFN-␥ can be induced through type 1 IFN and STAT4 at the first site of infection during a period with high STAT4 but prior to induction of elevated STAT1 in the cells. The response mediates a role in viral defense. Thus, a very early pathway to and source of IFN-␥ in evolving immune responses to infections are identified by this work. The information obtained helps resolve long-standing controversies and advances the understanding of mechanisms regulating key type 1 IFN functions, in different cells and compartments and at different times of infection, for accessing biologically important functions.
from our group, examining responses in mouse spleens, has demonstrated that NK cells express high basal levels of STAT4 and that their exposure to type 1 IFNs activates STAT4 (9) . Nevertheless, it has only been possible to identify the type 1 IFN induction of NK cell IFN-␥ production during acute viral infections of STAT1deficient but not of STAT1-complete mice because the concurrent induction of STAT1 by type 1 IFN and/or IFN-␥ negatively regulates the response (6, 9) . These results leave open the intriguing question of why a pathway from type 1 IFN to STAT4 activation under basal NK cell conditions would be evolutionarily preserved when it is rapidly turned off at times of type 1 IFN production.
With the hypothesis that type 1 IFN induction of NK cell IFN-␥ is in place to access low, regional levels of the cytokine as infections are initiated, studies were undertaken to examine responses at the earliest times of viral infection under immunocompetent conditions. The system used for study was intraperitoneal (i.p.) infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus (LCMV) (7, 9, 16, 17) . This infection has been well characterized and is of relevance to the human condition because LCMV can cause significant morbidity and mortality, particularly in immunodeficient individuals (18) (19) (20) . The virus is a potent inducer of type 1 IFN, with systemic levels produced in serum and spleen for several days after i.p. infection (7, 9, 17) . In contrast, LCMV is a poor inducer of IL-12, with low to undetectable levels of the cytokine produced (8, 16) , and NK cell IFN-␥ expression is also low to undetectable in the serum and spleens of LCMV-infected mice (8, 9) . Because the initial site of virus entry is the peritoneal cavity, the studies focused on previously uncharacterized events at this location.
The results of the experiments reported here reveal unique compartmental innate cytokine responses, with IFN-␣ and IFN-␤ production and NK cell IFN-␥ expression detected at 24 h after infection and peaking at 30 h after infection. Studies under conditions of blocked type 1 IFN receptor access and/or deficiency in STAT4 conclusively demonstrated that NK cell IFN-␥ expression was dependent on the type 1 IFNs and STAT4. The pathway enhanced the antiviral state. Interestingly, in comparison to other peritoneal populations, STAT1 induction in NK cells was delayed and separated from the NK cell IFN-␥ response by 10 to 12 h. Thus, the results uncover a key role for type 1 IFN activation of NK cell IFN-␥ and mechanistically resolve important issues concerning the earliest pathways to, sources of, and functions for IFN-␥.
To characterize responses at the earliest times after infections, lavage fluids and cells were collected from peritoneal cavities of B6 mice that had been uninfected or infected i.p. with 5 ϫ 10 4 PFU LCMV at the indicated times prior to harvest. The fluids were tested to evaluate cytokines released at the site. The IFN-␥ levels were measured using a cytometric bead assay (CBA). Low but consistently detectable levels of IFN-␥ were found peaking at 30 h after LCMV infection (Fig. 1A) . Enzyme-linked immunosorbent assays (ELISAs) were used to measure IFN-␣ and IFN-␤. In contrast to the reported extended kinetics of systemic levels of these cytokines (7, 9, 17) as well as the type 1 IFNs detected through 48 h after infection in serum samples taken from mice used for these experiments (data not shown), concentrations of IFN-␣ and IFN-␤ in the peritoneum reached their peak values of 340 and 69 pg/ml, respec-tively, at 30 h after challenge and were sharply confined to the 6-h periods surrounding that point (Fig. 1A) . The levels of IL-12p70, a known inducer of IFN-␥, were acquired from the CBAs. All samples were below the limit of detection for the assay, with no detectable levels in a large majority of samples isolated after 20 to 48 h of infection (Fig. 1A) . Thus, the peritoneal cytokine responses to LCMV infection include early induction of IFN-␥, with kinetically overlapping IFN-␣ and IFN-␤ production, but undetectable IL-12.
To define the major cell populations contributing to IFN-␥ production, peritoneal excudate cells (PECs) from uninfected (0 h) mice and mice infected with LCMV for 20, 24, 30, 36, 40 , and 48 h were isolated, stained for NK1.1 and T cell receptor ␤ (TCR-␤), fixed, and permeabilized to stain for cytoplasmic IFN-␥ (see Materials and Methods for staining and gating strategies). Total lymphocytes were evaluated with an extended lymphocyte gate. The NK cell subpopulations were identified as NK1.1 ϩ TCR-␤ Ϫ and the T cell subpopulations as NK1.1 Ϫ TCR-␤ ϩ . Lymphocytes isolated from uninfected mice, including total, NK, and T cells, did not basally express detectable IFN-␥; the positive frequencies for all populations were Ͻ4% ( Fig. 1B and C) . By 24 h after challenge with LCMV, approximately 14 to 37% of the NK cells expressed moderate to high levels of intracellular IFN-␥. The peak of this NK cell response was at 30 h after infections, when 28 to 43% of the NK cells expressed IFN-␥. The response was resolving at 36 to 48 h. In contrast to the NK cells, neither total lymphocyte nor T cell populations had high IFN-␥ levels at any of the times examined ( Fig. 1B and C) . These results demonstrate that during LCMV infection, peritoneal NK cells respond with IFN-␥ expression and are the major producers of the cytokine in the peritoneal cavity.
Type 1 IFN responsiveness is required for induction of NK cell IFN-␥ expression. Because induction of the biologically active IL-12p70 heterodimer can be accompanied by higher-level IL-12p40 subunit production (8), representative lavage samples from 0, 24, 30, 36, 40, and 48 h following LCMV infection were tested in an ELISA for the subunit. Consistent with our earlier studies (8) , all samples had IL-12p40 levels at or below the limit of detection for the assay (data not shown). To determine if biologically relevant IL-12 was induced below detection limits, uninfected and LCMV-infected mice were treated with control antibody or anti-IL-12p40 antibody neutralizing the function of biologically active IL-12p70 (8) . As a positive control for an IL-12induced IFN-␥ response (21, 22) , mice were given lipopolysaccharide (LPS) i.p. at 6 h prior to harvest. In comparison to LCMV infection, LPS treatment induced higher levels of IFN-␥ in lavage fluids ( Fig. 2A) and higher proportions of NK cells expressing IFN-␥ (Fig. 2B) . The enhanced response to LPS was blocked by the anti-IL-12 treatment. In contrast, the IFN-␥ response to LCMV infection was resistant to the anti-IL-12 treatment ( Fig. 2A and B) . Thus, IL-12 is not required for the NK cell IFN-␥ response elicited by the virus.
Two different approaches were used to evaluate the role of type 1 IFNs in stimulating NK cell IFN-␥ within mice blocked in their responsiveness to the cytokines. The first examined responses in cells from uninfected and 30-h LCMV-infected immunocompetent mice that had been treated with control antibodies or antibodies blocking access to the type 1 IFN receptor, anti-IFNAR (23) . As shown in Fig. 3A , the infected control-treated mice had the expected 25 to 30% of their NK cells expressing IFN-␥, but this response was reduced to Ͻ1% in the infected mice treated with antibodies against IFNAR. The second approach examined responses in mice that were blocked in type 1 IFN responsiveness as a result of genetic mutation of the type 1 IFN receptor (IFNAR Ϫ/Ϫ ) (24) . In comparison to the up to 36% of NK cells expressing IFN-␥ in wild-type (WT)-infected mice, Ͻ1% of the NK cells in the infected IFNAR Ϫ/Ϫ mice expressed IFN-␥ at 30 h after LCMV infection (Fig. 3B ). Thus, blocking type 1 IFN respon- Because type 1 IFNs stimulate a wide range of effects, including those important for antiviral defense, the direct consequences of NK cell responsiveness to these cytokines required examination in the context of a WT environment. Therefore, experiments evaluated IFN-␥ induction in peritoneal WT (IFNAR ϩ/ϩ ) and IF-NAR Ϫ/Ϫ cells after their isolation and adoptive transfer into WT recipient mice (Fig. 3C ). Cells from recipient and donor mice were distinguished by expression of the CD45.1 or CD45.2 allele, with recipient mice being CD45.1 ϩ and donor mice being CD45.1 Ϫ (Fig. 3C ). For these studies, WT and IFNAR-deficient PECs were prepared and transferred into WT recipient mice prior to LCMV infection. None of the populations expressed IFN-␥ when recipients remained uninfected ( Fig. 3C ; 0 h after LCMV). When WT PECs were transferred into WT recipients, both donor and recipient NK cells expressed IFN-␥ at 30 h after infection, with overall mean expression of 23 and 21%, respectively (Fig. 3C ). In contrast, transferred NK cells isolated from IFNAR-deficient mice were significantly decreased in their ability to express IFN-␥, with expression averaging approximately 4% compared to 22% of WT recipient NK cells (Fig. 3C ). Taken together, the studies conclusively prove that type 1 IFN signaling within peritoneal NK cells is required for the induction of IFN-␥ expression during LCMV infection.
To determine the role of STAT4 in NK cell IFN-␥ expression, responses in mice lacking STAT4 as a result of genetic mutation (STAT4 Ϫ/Ϫ ) (13) were evaluated. Here, the PECs were isolated from uninfected mice and mice at 30 or 36 h after LCMV infections (Fig. 4A ). NK cells from infected STAT4-deficient mice were induced to express much lower levels of IFN-␥ and did so in lower frequencies than NK cells from infected WT mice (Fig. 4A ). To define the effects of STAT4 loss within NK cells in a WT environment of infection, adoptive transfer experiments were carried out using PEC donor cells isolated from WT or STAT4-deficient mice and then delivered into WT recipient mice. The donor cells were identified by lack of CD45.1 expression and the recipient cells by CD45. IFN-␥ at 30 h after infection. In contrast, when STAT4 Ϫ/Ϫ cells were transferred, donor NK cells produced significantly lower levels of IFN-␥ compared to WT recipient NK cells in response to infection (Fig. 4B) . Hence, STAT4 expression within peritoneal NK cells is necessary for optimal IFN-␥ induction.
The STAT1 and STAT4 levels are differentially expressed in cell subsets during LCMV infection. Because our previous studies have shown that a pathway from type 1 IFN through STAT4 for NK cell IFN-␥ expression in the spleen is associated with high basal STAT4 levels but blocked by increasing STAT1 levels during infections (9), intracellular STAT levels were examined in the peritoneal populations by flow cytometric approaches. The experiments demonstrated high basal STAT4 levels in NK cells (Fig. 5A) . The trait was unique to NK cells, with only low proportions of total lymphocytes and T cells expressing STAT4 and only doing so at lower levels, and NK cells retained their unique high levels of STAT4 throughout infection (Fig. 5A) . In contrast, all populations isolated from uninfected mice were low for STAT1, but expression in the NK cells was always slightly lower (Fig. 5B) . Following LCMV infection, the total lymphocyte and T cell pop-ulations rapidly elevated STAT1 expression, with 80 to 95% of the populations expressing high levels of STAT1 by 40 h after infection ( Fig. 5B to D) . The peritoneal NK cells also had STAT1 levels induced during the infection, but in comparison to the other populations, there was a delay, requiring upwards of 8 to 16 h longer than total lymphocytes and T cells to reach 50% high levels of STAT1-expressing cells (Fig. 5B to E) . Thus, the NK cell populations uniquely express high levels of STAT4 basally and after infection. Moreover, although all populations have elevated STAT1 after infection, induction in NK cells is delayed.
The peritoneal IFN-␥ response promotes resistance to LCMV infection. After i.p. infection, LCMV was difficult to detect in lavage fluids at 20 and 24 h, but titers rapidly increased by 30 h and subsequently decreased (Fig. 6A) . To determine the physiologic relevance of the peritoneal IFN-␥ response, viral burdens were measured in samples collected from WT mice and mice rendered unresponsive to IFN-␥ as a result of genetic mutation of the IFN-␥ receptor (IFN-␥R Ϫ/Ϫ ) (25) . The IFN-␥R-deficient mice had significant increases in LCMV titers of approximately 1 log at 30 h after infection (Fig. 6A) . Experiments evaluating the require- ment for NK cells demonstrated that in comparison to control antibody treatments, antibodies to NK1.1 depleted the following: (i) the NK cells from the PECs isolated from uninfected and infected mice (Fig. 6B) , (ii) the major population expressing intracellular IFN-␥ after infection (Fig. 6B) , and (iii) the levels of detectable IFN-␥ in lavage fluids collected at 30 h following challenge (Fig. 6C) . The effects were accompanied by proportional increases in viral titers (Fig. 6C ). Taken together, these studies demonstrate that the IFN-␥ responses have an impact on controlling the viral burden in the peritoneal cavity during LCMV infection and that NK cells are required for the optimal IFN-␥ response, and its effects.
In the work presented above, the interplay between cytokines and intracellular signaling molecules in the regulation of NK cell activation and function is defined at the earliest site of viral infection in immunocompetent mice. Production of IFN-␣, IFN-␤, and IFN-␥ in the peritoneal cavity was shown to peak at 30 h after LCMV infection. NK cells were the major producers of IFN-␥ and uniquely expressed high levels of STAT4. The pathway for induction of NK cell IFN-␥ production was dependent on responsiveness to type 1 IFNs and on the STAT4 signaling molecule. The elicited IFN-␥ enhanced defense because loss of responsiveness to the factor resulted in increased viral burdens. Thus, type 1 IFN induction of IFN-␥ production by NK cells, dependent on STAT4, is conclusively proven to occur and to lead to downstream effects under immunocompetent conditions. Previous work from this laboratory has established that under basal conditions, splenic NK cells express high STAT4 and low STAT1 levels and preferentially activate STAT4 over STAT1 after ex vivo exposure to type 1 IFNs (9). Nevertheless, it has only been possible to identify type 1 IFN activation of STAT4 and IFN-␥ in the spleens of STAT1-deficient mice during LCMV infection because the type 1 IFNs and IFN-␥ induce STAT1 levels to block the pathway to STAT4 (9). The results reported here answer the question of why a pathway from type 1 IFN to STAT4 for IFN-␥ expression would be maintained when it is rapidly turned off by concurrent STAT1 induction; it is used to locally access a short burst of IFN-␥ at very early times after infection. In this case, "locally" is the peritoneal cavity. In addition to the detectable NK cell IFN-␥ response, innate responses to LCMV at this site differed from the well-characterized responses in spleen and serum (7, 9, 17) with regard to the kinetics and magnitude of type 1 IFN production and the kinetics of STAT1 induction in NK cells (9) . Type 1 IFN production in the peritoneal cavity was very early and of short duration-detectable at 24 h, peaking at 30 h, and resolving by 36 h after infection (Fig. 1 )-and although total lymphocytes and T cells had dramatically elevated STAT1 levels early, induction was delayed by 8 to 16 h in the NK cells (Fig. 5) . Thus, in contrast to the spleen, the environment in the peritoneal cavity allows type 1 IFN to access STAT4 prior to STAT1 induction in NK cells (Fig. 7) .
Why was STAT1 elevation delayed in the NK cells compared to the other PEC populations examined? Promoter sequences in the STAT1 gene specific for STAT1 complexes may act to enhance STAT1 induction whenever STAT1 is activated (26) . Thus, any cytokine signaling through STAT1 would be expected to induce elevated STAT1 levels. In addition to type 1 IFN receptors, receptors for IFN-␥ also activate STAT1 (10). The results presented here suggest that NK cells might first respond to type 1 IFN expo-sure with IFN-␥ because of their high STAT4 levels, but once IFN-␥ is induced, it acts back on the cells to induce STAT1. This would explain the delay in STAT1 induction in NK cells compared to other subsets. Alternatively or additionally, it might just take longer for type 1 IFN to induce STAT1 in populations with high STAT4 levels. The NK cells in both the spleen (9) and peritoneum (Fig. 5) consistently have lower levels of STAT1. Because the spleen is a secondary compartment of infection, most of the NK cells may be experiencing cytokines produced earlier at other sites and/or not be synchronized to make a detectable short burst of IFN-␥ in this compartment. In any case, it is remarkable that there is a window of opportunity for IFN-␥ production under the conditions in the peritoneum given the link between this response and shutting it off. The pathway suggests that there will be a very early source of NK cell IFN-␥ whenever type 1 IFNs are induced.
Our studies are helping to resolve conflicting reports in the literature on potential type 1 IFN consequences for IFN-␥ expression (5, 6, 9) , adding information to the growing literature reporting cellular differences in type 1 IFN responses (27, 28) and advancing the mechanistic understanding of how the biological effects of STAT cytokines are regulated to access diverse and paradoxical effects as needed. Type 1 IFNs have long been known to exert antiviral effects and enhance NK cell cytotoxicity through the activation of STAT1 (4, 5, 11) . The NK cells, however, are positioned to respond to type 1 IFNs with STAT4 activation because high basal levels of STAT4 are associated with the type 1 IFN receptor (9) . Increasing the endogenous levels of total STAT1 protein negatively regulates STAT4 access in part by displacing STAT4 from the receptor, and this induction of STAT1, as observed in the spleen, is beneficial because it protects from dysregulated systemic IFN-␥ production and cytokine-mediated disease (9) . In contrast, the results reported here show that the type 1 IFNs infection. Shown is a schematic representation of the differences in the immune response to LCMV in the peritoneal cavity compared to the spleen. The results presented here studying the peritoneal cavity show that both type 1 IFNs and the viral burden peak at 30 h. This is correlated with an early peak in IFN-␥ production in NK cells. NK cell production of IFN-␥ occurs before STAT1 levels have risen. Previous work with the spleen has shown that a more vigorous peak in viral load and type 1 IFNs is seen between 2 and 3 days after LCMV infection with a concurrent rise in STAT1 (7). Type 1 IFNs are unable to access STAT4-dependent IFN-␥ production due to the earlier rise in STAT1 levels. The response in the peritoneum promotes antiviral defense. The response in the spleen protects from dysregulated cytokine production and cytokine-mediated disease.
have early access to STAT4 and the NK cell IFN-␥ pathway prior to STAT1 induction and that this pathway promotes resistance to infection. Recent studies have shown that similar STAT4-STAT1 regulation is in place in humans; type 1 IFNs induce STAT4 activation in NK cells isolated from healthy individuals, but the response is diminished in NK cells from individuals chronically infected with hepatitis C virus. The activation of STAT4 negatively correlates with STAT1 levels in these populations (29) . Thus, modulation of STAT4 and STAT1 concentrations in different cell types under different conditions of infections in mice and humans helps explain how type 1 IFNs can enhance or antagonize IFN-␥ stimulation, and the studies with mice provide explanations for how the balance, in both directions, works to the benefit of the host. A change in relative STAT ratios might also play a role in conditioning NK cells to modify type 1 IFN responses from promoting cytokine production to enhancing cytotoxic function. In addition to our earlier studies with STAT1-deficient mice, there have been a few other reports suggesting a role of type 1 IFN in NK cell IFN-␥ expression. One examined a number of activation responses, including intracellular IFN-␥, of type 1 IFN receptor-deficient compared to WT NK cells following in vitro exposure to vaccinia virus and dendritic cells (30) . Early in vivo IFN-␥ induction has been reported in the mouse peritoneum during different viral infections (31) , and intracellular IFN-␥ expression by splenic NK cells has been observed following treatments with the chemical analogue of viral nucleic acids, polyinosinicpolycytidylic acid [poly(I · C)] (32, 33) . These in vivo studies, however, have been done under conditions where the type 1 IFN effects were not delineated from those that might have been mediated by other cytokines induced under the experimental conditions used, including reported IL-12. In one study with poly(I · C) (32), the intracellular NK cell IFN-␥ expression was shown to be blocked by treatments with antibodies neutralizing type 1 IFN function, but the consequences of the treatments on other cytokine responses were not evaluated. Here, in vivo responses under which the type 1 IFN receptor was blocked were evaluated by different approaches, including adoptive transfer of peritoneal populations from WT or type 1 IFN receptor-deficient mice into WT-infected mice (Fig. 3) . This method allows direct comparison of donor and recipient cells in the context of the complete endogenous immune responses. Thus, to our knowledge, this is the first demonstration of type 1 IFNs inducing IFN-␥ production from NK cells under immunocompetent conditions of viral infection.
The studies do not exclude possible roles for accessory cytokines in enhancing type 1 IFN induction of IFN-␥. Certainly, IL-12 is an activator of STAT4 and a potent inducer of IFN-␥ (13, 34) . The cytokine IL-18 can enhance the stimulation of IFN-␥ by either type 1 IFNs or IL-12 (7, (35) (36) (37) , and new studies deciphering pathways from different sensors have demonstrated synergism between type 1 IFNs and IL-12 for IFN-␥ induction in human cells (38) . The results presented here, however, suggest that there may be a short burst of IFN-␥ induced by type 1 IFNs independent of IL-12, and our previous work examining murine cytomegalovirus infections of mice has shown that induced type 1 IFNs promote NK cell cytotoxicity but IL-12 is required for splenic and systemic NK cell IFN-␥ (3, 4) . The experiments carried out here neutralizing IL-12 in vivo show that IL-12 does not play a role in the LCMVinduced NK cell IFN-␥ response (Fig. 2) . Interestingly, the positive controls for these studies (i.e., effects on LPS induction of IFN-␥ in the peritoneal cavity) (Fig. 2) demonstrate that when it is elicited, IL-12 is important in eliciting a peritoneal NK cell IFN-␥, but also that there is residual NK cell IFN-␥ after neutralization. This is consistent with early reports of incomplete IFN-␥ blockade by IL-12 neutralization following LPS treatment in other settings (21) and the known LPS induction of IFN-␤ (39) . Taken together, the studies suggest that IL-12 is an important inducer of NK cell IFN-␥ if elicited and the critical cytokine for IFN-␥ induction once STAT1 levels are increased. There are likely to be additional regulatory factors in play, however, as the presence of STAT1 eventually results in reduced IFN-␥ induction by either type 1 IFNs or IL-12 (6) . Thus, much remains to be done to map the complex interactions between type 1 IFN and IL-12 for shaping NK cell responses, but eliciting different type 1 IFN and IL-12 responses may be part of directing the magnitude of, and continued access to, the NK cell IFN-␥ response.
The peritoneal responses reported here demonstrate that by being receptive to early type 1 IFN signaling with IFN-␥ production, NK cells can serve as sensors for and responders to the presence of viral pathogens. The studies surprisingly reveal a previously elusive role for NK cells in early defense against LCMV. In comparison to other viral infections in compartments outside the peritoneal cavity, LCMV has been shown to be relatively insensitive to the direct antiviral effects of NK cells (40) (41) (42) or IFN-␥ (43) . Our previous attempts to access NK cell IFN-␥ in defense against LCMV have shown that administration of IL-12 prior to infection can induce the response to result in under a 1-log decrease in splenic viral titers (44) . In this report, infection-induced IFN-␥, dependent on NK cells, was shown to proportionally limit viral burden at this first site of infection. Thus, though modest, there is a documented effect of the NK cells and IFN-␥ during LCMV infection. The mechanism for the antiviral effect mediated by IFN-␥ is not known. Given the rapid kinetics, it is likely to be a result of the activation of direct antiviral effects in infected cells or macrophages rather than enhancement of adaptive immune functions resulting from promoting major histocompatibility complex (MHC) class 1 expression or CD8 T cell effector functions (45) . It remains to be determined whether or not the NK cell IFN-␥ elicited by type 1 IFNs will be a more important first response in defense against other more sensitive viral infections and/or for promoting immunoregulatory functions.
In summary, these studies discover a novel pathway for NK cell stimulation at an initial site of infection. Moreover, they advance understanding of the regulation of type 1 IFN responses and provide critical insights into how the immune response is finely regulated to deliver maximal protection without being detrimental to the host.
Mice. Specific-pathogen-free C57BL/6 (B6) (C57BL/6NTac) and B6.SJL-Ptprca/BoyAiTac mice, which are C57BL/6 mice that express the congenic CD45.1 allele of the Ptprc (protein-tyrosine-phosphatase, receptor type c locus) gene, were purchased from Taconic Laboratories (Germantown, NY). Breeding pairs of the Stat4 Ϫ/Ϫ mice (13) on the B6 background were a gift from Mark H. Kaplan of Indiana University, B6 IfngR Ϫ/Ϫ mice (25) with control WT mice were purchased from the Jackson Laboratories (Bar Harbor, ME), and Ifnar Ϫ/Ϫ mice (24) on the B6 background were a gift from Murali-Krishna Kaja of the University of Washington. All mice except B6.SJL mice expressed the CD45.2 allele. Genetically mutated mice were bred and maintained in isolation facilities at Brown University through brother-sister mating. All mice used in experiments were 5 to 12 weeks old. Animals obtained from sources outside Brown University were housed in the animal care facility for at least 1 week before use. Handling of mice and experimental procedures were conducted in accordance with institutional guidelines for animal care and use.
Virus, infections and treatments. For LCMV infection experiments, mice were either uninfected (0 h after infection) or infected i.p. with 5 ϫ 10 4 PFU of LCMV Armstrong strain, clone E350 (7, 9, 17) . For IL-12 blocking experiments, animals were injected with either 750 g or 1 mg of anti-IL-12p40 (anti-IL-12/23, clone C17.8; BioXCell, West Lebanon, NH) or control rat IgG2a (clone 2A3; BioXCell) antibodies 12 h prior to LCMV infection and 36 h prior to treatment with 20 g of LPS (from Salmonella enterica serovar Enteritidis; Sigma, St. Louis, MO). Samples from LPStreated mice were harvested 6 h after treatment (22) . (The levels of efficacy of treatment with either 750 g or 1 mg of anti-IL-12 were similar.) For IFNAR blocking experiments, animals were injected with 250 g of anti-IFNAR1 (clone MAR1-5A3; Leinco Technologies, St. Louis, MO) or control IgG antibodies 12 h prior to LCMV infection. For NK depletion experiments, animals were injected with 500 g of anti-NK1.1 (clone PK136; BioXCell) or control IgG2a antibodies at 36 or 12 h prior to LCMV infection.
Sample preparation. PECs were extracted on the indicated days of infection. LCMV-infected and control uninfected (0) mice were anesthetized and bled retro-orbitally to collect serum samples. Mice were humanely sacrificed, by cervical dislocation after anesthetization, and the peritoneal cavity was lavaged with 5 ml of cold serum-free RPMI medium (Gibco, Invitrogen, Carlsbad, CA). Mice were gently agitated to increase PEC suspension in lavage medium. For analysis of surface marker expression or total STAT levels, PECs were resuspended in staining buffer containing 2% heat-inactivated fetal bovine serum (FBS) and then prepared for flow cytometric analysis as described below. For examination of intracellular IFN-␥, PECs were resuspended in complete media containing 5 g/ml brefeldin A (Sigma, St. Louis, MO), without additional stimulation, for 4 h at 37°C. Cells were then stained as described below. Peritoneal lavage fluids and serum samples were aliquoted and stored at Ϫ80°C for later use in cytokine and viral titer measurements.
Adoptive transfers. Total donor PECs from uninfected mice (CD45.2) were isolated as described above and resuspended in 300 l of RPMI medium. These cells were injected i.p. into uninfected recipient mice (CD45.1). Recipient mice were rested for 1 h and either left uninfected or infected with LCMV as described above. Staining to identify donor and recipient cell populations was performed as described below using an anti-CD45.1-fluorescein isothiocyanate (FITC) antibody.
Flow cytometric analysis. Detection of surface markers, intracellular IFN-␥, and intracellular total STAT1 and STAT4 was done with minor modifications as previously described (9, 46) . Briefly, PECs were incubated with 2.4G2 antibody (BioXCell, West Lebanon, NH) to block nonspecific binding to the Fc receptor. Cell surface staining was then performed with antibodies specific for the following: TCR-␤-FITC, TCR-␤peridinin chlorophyll protein (PerCP), NK1.1-phycoerythrin (PE) (all from eBioscience, San Diego, CA), TCR-␦-FITC, and CD45.1-FITC (BD Biosciences, Franklin Lakes, NJ). For cell surface staining only, cells were fixed with 2% paraformaldehyde. For intracellular staining of IFN-␥, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and then stained with IFN-␥-allophycocyanin (APC) (BD Biosciences). For intracellular staining of total STAT1 and STAT4, cells were fixed and permeabilized with Cytofix/Cytoperm and then further fixed and permeabilized with ice-cold pure methanol. Subsequent staining was performed with combinations of STAT1-PE and STAT4-APC (custom prepared by BD Biosciences). The following isotype controls were included in each experiment: mouse IgG2a, mouse IgG2b, mouse IgG1, rat IgG1, rat IgG2a, rat IgG2b, and hamster IgG (BD Biosciences or eBioscience). Samples were acquired using a FACSCalibur (BD Biosciences) with the Cell-Quest Pro software package (BD Biosciences). Laser outputs were 15 mW at 488-and 635-nm wavelengths. At least 120,000 events were collected within a live-cell gate that was determined by forward and side scatter.
Analysis of flow cytometric data was performed using FlowJo9 (Tree Star, Inc., Ashland, OR).
To ensure proper identification of the NK cell population in PECs, NK1.1-positive cells were costained with markers identifying TCR-␣/␤ and TCR-␥/␦ T cells. A lymphocyte gate was set to maximize inclusion of NK1.1 ϩ cells at different times after infection, and it was identified as an "extended lymphocyte gate." Using this scheme, Ͻ5% of the NK1.1 ϩ cells were TCR-␦ ϩ under naive conditions; after LCMV infection, the population was reduced to Ͻ2%, and the cells were not producing IFN-␥ (data not shown). NK1.1 ϩ cells that were TCR-␤ ϩ (NK T cells) represented approximately 30% of the total NK1.1 ϩ population at day 0. After LCMV infection, this population was again diminished to Ͻ5%, and the IFN-␥ produced by these cells represented only about 3% of the total IFN-␥ ϩ cells. Thus, all experiments defined NK cells to be NK1.1 ϩ TCR-␤ Ϫ .
Cytokine measurements. IFN-␥ and IL-12p70 were measured in the peritoneal lavage fluid or the serum using the mouse inflammation kit cytometric bead assay (BD Biosciences). The limits of detection were 2.5 pg/ml for IFN-␥ and 10.7 pg/ml for IL-12p70. IFN-␣ and IFN-␤ were measured in the peritoneal lavage fluid or the serum using the VeriKine mouse IFN-␣ or mouse IFN-␤ ELISA kits, respectively (PBL Interferon-Source, Piscataway, NJ). The limits of detection for these assays were 12.5 pg/ml for IFN-␣ and 15.6 pg/ml for IFN-␤. The IL-12p40 ELISA, with a limit of detection of 100 pg/ml, was done as previously described (8) .
LCMV plaque assay. LCMV titers were determined as previously described (7, 17) . Briefly, peritoneal lavage fluids were thawed from storage at Ϫ80°C and serially diluted. Samples were overlaid onto Vero cell monolayers and incubated at 37°C for 1.5 h. Plaques were allowed to form upon overlay of samples with a 50:50 mixture of agarose-medium 199 (Sigma) for 3 days and then visualized using neutral red staining (Sigma). Included in each assay were positive LCMV control samples and negative controls (uninfected animals).
Statistical analysis. All analyses were performed using Prism 5 (GraphPad Software, La Jolla, CA). Statistical significance was determined using the two-way analysis of variance (ANOVA) test. The Interaction of LFA-1 on Mononuclear Cells and ICAM-1 on Tubular Epithelial Cells Accelerates TGF-β1-Induced Renal Epithelial-Mesenchymal Transition The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-β(1) on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-β(1) stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-β(1) (0.1 ng/ml) (HK-2-TGF-β(1) (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-β(1) (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-β(1) (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-β(1) (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-β(1) (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-β(1) (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72–96 hrs) TGF-β(1) stimulation increased, that of HK-2-TGF-β(1) (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-β(1) induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-β(1.) Regardless of the underlying etiology, tubulointerstitial fibrosis is a common mechanism in the progression of chronic kidney disease (CKD) to end-stage renal disease [1, 2] . This progressive pathway involves interstitial infiltration by inflammatory mononuclear leukocytes [1, 3] . Integrin lymphocyte function-associated antigen 1 (LFA-1: a L b 2 integrin) is the predominant integrin on leukocytes and an important molecule in firm adhesion and migration of leukocytes to inflammatory sites [4, 5] . LFA-1 also plays pivotal roles as a signal transduction molecule by binding its ligand, namely, intracellular adhesion molecule 1 (ICAM-1) [6, 7] . Normally, LFA-1 is expressed in a low-affinity state for its ligand and, thus, cells do not make unnecessary adhesive contacts while in circulation [8, 9] . The affinity of LFA-1 for ICAM-1 is mediated by a conformational change of LFA-1 and they play essential roles in most inflammatory reactions [8, 9] . ICAM-1 has been reported to be expressed on renal tubular epithelial cells (RTECs) and the expression of ICAM-1 on RTECs was found to be associated with the infiltration of leukocytes in CKD [10, 11] . An experimental animal study showed that ICAM-1 was promptly up-regulated after renal injury and leukocyte infiltration subsequently occurred [12] . Kelly et al. reported that anti-ICAM-1 mAb mitigated leukocyte infiltration in tubulointerstitial space in an ischemic renal injury animal model [13] . Although these results suggested that ICAM-1 on RTECs and LFA-1 on leukocytes have some roles in the progression of renal diseases, the pathogenetic roles of their direct interaction in renal fibrosis remain unclear.
Epithelial-mesenchymal transition (EMT) plays pivotal roles in organ fibrosis including that of kidney [14, 15] . It has been reported that a large proportion of the interstitial fibroblasts in fibrotic kidneys originate from proximal tubular cells [16] . Therefore, it is important to identify the molecules involved in the induction and progression of EMT of RTECs. TGF-b 1 is up-regulated in the fibrotic kidney and is the main inducer of EMT of RTECs [17] [18] . In the present study, we investigated the roles of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on RTECs after stimulation of TGF-b 1 on the EMT.
ICAM-1 was highly expressed on HK-2 cells. ICAM-1 expression decreased with TGF-b 1 stimulation at concentrations of 10 .0 ng/ml, 1.0 ng/ml and 0.1 ng/ml after 24 hrs in a dosedependent manner ( Figure 1A ); its expression also showed a timedependent decrease at 24 hrs, 48 hrs and 72 hrs after TGF-b 1 (10.0 ng/ml) stimulation ( Figure 1B) . However, its expression was still maintained at a high level.
TGF-b 1 increased the expression of chemokines that mediate LFA-1 activation of PBMCs on HK-2 cells
The change of expression of chemokines that mediate LFA-1 activation on PBMCs, such as CCL2, CCL3, CCL4, CCL5, CCL17, CCL19, CCL20, CCL21, CCL22 and CXCL12, was investigated on HK-2 cells after stimulation of TGF-b 1 (10.0 ng/ ml, 1.0 ng/ml and 0.1 ng/ml). Although the expression of CCL2 decreased, the expression of CCL3 and CXCL12 increased on HK-2 cells after TGF-b 1 stimulation at concentrations of 10.0 ng/ ml, 1.0 ng/ml and 0.1 ng/ml for 24 hrs in a dose-dependent manner ( Figure 2 ). The expression of CCL21 was not changed on HK-2 cells after TGF-b 1 stimulation ( Figure 2 ). CCL4, CCL5, CCL17, CCL19, CCL20 and CCL22 were not detected on HK-2 cells either before or after TGF-b 1 stimulation (data not shown).
We investigated the conformational activation change of LFA-1 on PBMCs by HK-2 cells after stimulation with TGF-b 1 (0.1 ng/ ml). We employed flow cytometry using anti-LFA-1 mAb (clone: TS1/22) to detect total LFA-1 on PBMCs and anti-active form LFA-1 mAb (clone: AL-57) that reacted only with the active form of LFA-1 but not with the resting form of LFA-1 to detect conformational activation of LFA-1 on PBMCs. After co-culture with HK-2 cells stimulated by TGF-b 1 (0.1 ng/ml) for 30 minutes, AL57-positive cells were significantly increased , whereas TS1/22positive cells were not changed on PBMCs. AL57-positive cells on PBMCs did not change by co-culture with HK-2 cells without TGF-b 1 and TGF-b 1 alone ( Figure 3A ). Next, we investigated the effect of CCL3 and CXCL12 of HK-2 cells on the LFA-1 activation on HK-2 cells. CXCL12 and CCL3 on HK-2 cells were knocked down by transfection of CXCL12-siRNA or CCL3-siRNA, respectively. Non-treated (Mock) HK-2 cells and nontargeted-siRNA-transfected (Control) HK-2 cells served as controls. CXCL12 knockdown (CXCL12 KD) HK-2 cells did not increase CXCL12 expression regardless of TGF-b 1 stimulation. CCL3 knockdown (HK-2-CCL3 KD) HK-2 cells did not increase CCL3 expression regardless of TGF-b 1 stimulation ( Figure S1 ). PBMCs were co-cultured with CXCL12 KD, CCL3 KD, Control or Mock HK-2 cells after stimulation with TGF-b 1 (0.1 ng/ml) for 30 minutes. Significantly less AL57-positive cells were detected among PBMCs after co-culture with CXCL12KD HK-2 cells than among CCL3KD, Control and Mock HK-2 cells ( Figure 3B ).
Interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells accelerated TGF-b 1 -induced EMT of HK-2 cells
We investigated the impact of the interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells on the EMT of HK-2 cells. HK-2 cells after stimulation of TGF-b 1 (0.1 ng/ml) for 24 hrs were co-cultured with PBMCs for a further 24 hrs (HK-2-TGF-b 1 (0.1)-PBMCs). Then, the changes of epithelial cell junction markers and mesenchymal markers on HK-2-TGF-b 1 (0.1)-PBMCs were investigated by real-time RT-PCR and western blotting analysis. HK-2 cells after stimulation of TGF-b 1 (0.1 ng/ml) without coculture with PBMCs (HK-2-TGF-b 1 (0.1)) and HK-2 cells without stimulation of TGF-b 1 co-cultured with PBMCs (HK-2-PBMCs) were investigated as control experiments. The results of real-time RT-PCR showed that the expression of epithelial cell junction markers (E-cadherin and occludin) further significantly decreased and that of mesenchymal markers (vimentin and fibronectin 1) further significantly increased in HK-2-TGF-b 1 (0.1)-PBMCs compared with those of controls. These EMT marker changes on HK-2-TGF-b 1 (0.1)-PBMCs were blocked by anti-LFA-1 mAb or anti-ICAM-1 mAb, whereas they were not blocked by isotype control mAb (Figure 4 ). Phase contrast microscopy analysis showed that HK-2-TGF-b 1 (0.1)-PBMCs changed more, morphologically, from a cobblestone-like monolayer to spindle-shaped fibroblast-like cells, than the controls. These morphological changes of HK-2-TGF-b 1 (0.1)-PBMCs were mitigated by anti-LFA-1 mAb or anti-ICAM-1 mAb, whereas they were not mitigated by isotype control mAb ( Figure The interaction of HK-2 cells pre-stimulated with TGF-b 1 and PBMCs up-regulated TGF-b 1 level in co-culture media To investigate whether pre-stimulation of HK-2 cells by TGFb 1 contributes to EMT or just induces the CXCL12 that activates LFA-1 on PBMCs, we employed a transwell co-culture system as described above. HK-2 cells without TGF-b 1 stimulation and PBMCs were co-cultured in a lower plate and HK-2 cells prestimulated by TGF-b 1 (10.0 ng/ml, 1.0 ng/ml and 0.1 ng/ml) were cultured in an upper insert. After 24 hrs of incubation, TGFb 1 concentrations in the media in the lower wells were determined by TGF-b 1 ELISA. The concentrations of TGF-b 1 in the coculture media of HK-2 cells and PBMCs in the lower wells were about threefold higher than those of HK-2 cells alone (Fig. 6 ).
The interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK 1/2 signaling on HK-2 cells
We investigated the phosphorylation of smad2, smad3 and ERK1/2 in HK-2-TGF-b 1 (0.1)-PBMCs to investigate the signaling pathways that might be involved in the acceleration of TGF-b 1 -induced EMT on HK-2 cells by PBMCs. The phosphorylation of smad2 and smad3 was not detected (data not shown); however, the phosphorylation of ERK1/2 was detected in HK-2-TGF-b 1 (0.1)-PBMCs at 4 hrs and 12 hrs after co-culture by western blot analysis (Figure 7) . The phosphorylation of ERK1/2 Figure 9B ).
The present study showed that the TGF-b 1 -induced EMT on HK-2 cells was accelerated by PBMCs. The decrease of expression level of epithelial cell junction markers and increase of expression level of mesenchymal markers on HK-2 cells after stimulation of TGF-b 1 were accelerated by co-culture with PBMCs. These EMT marker changes on HK-2 cells were mitigated by anti-LFA-1 mAb or anti-ICAM-1 mAb. These results demonstrated that the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells contributed to accelerate TGF-b 1 -induced EMT on HK-2 cells. HK-2 cells stimulated by TGF-b 1 induced conformational activation of LFA-1 on PBMCs; however, HK-2 cells without stimulation by TGF-b 1 or TGF-b 1 alone did not induce conformational activation of LFA-1 on PBMCs. The locally secreted chemokines can trigger integrin-dependent adhesion of circulating leukocytes that leads to infiltration [19, 20, 21, 22] . Several studies have characterized the expression pattern of chemokines in animal models of tubulointerstitial disease [23, 24, 25] . They demonstrated that various chemokines such as CCL2, CCL3, CCL4 and CCL5 are expressed only in the diseased compartment of the kidney and renal chemokine expression has been found to correlate with the leukocyte accumulation area and renal damage [23, 24, 25, 26] . On the basis of these findings, we investigated the regulation of the chemokines that may mediate LFA-1 activation of PBMCs on HK-2 cells by the stimulation of TGF-b 1 . CCL2 decreased on HK-2 cells by stimulation of TGF-b 1. This result was consistent with a previous study [27] . CCL3 and CXCL12 increased on HK-2 cells by TGF-b 1 stimulation. CCL21 did not change on HK-2 cells by TGF-b 1 stimulation. CCL3, CCL4, CCL17, CCL19, CCL20 and CCL22 were not detected on HK-2 cells either before or after TGF-b 1 stimulation. CCL3 and CXCL12 were reported to activate LFA-1 on both monocytes and lymphocytes [28, 29, 30, 31, 32] . In the present study, not CCL3 but CXCL 12 contributed to induction of the conformational activation of LFA-1 on PBMCs, because less AL57-positive cells were detected among PBMCs after co-culture with CXCL12 knockdown HK cells compared with those among Mock, CCL3 knockdown and control knockdown HK-2 cells pre-stimulated with TGF-b 1. Furthermore, the EMT marker changes of HK-2 cells pre-stimulated with TGFb 1 and morphological changes upon co-culture with PBMC were inhibited by CXCL12 knockdown on HK-2 cells (Figures S2 and  S3 ). These results suggested that CXCL12 was a key chemokine for EMT of RTECs that induced TGF-b 1 accelerated by PBMCs.
Since TGF-b 1 is up-regulated in the fibrotic kidney and is the main inducer of EMT in RTECs [17, 18] , we determined TGFb 1 concentration in the co-culture media of HK-2 cells and PBMCs with non-direct contacted culture with HK-2 cells pre-stimulated by TGF-b 1 using transwell plate to investigate whether prestimulation of HK-2 cells by TGF-b 1 contributes to EMT on RTECs or just induces the CXCL12 that activates LFA-1 on PBMCs. TGF-b 1 concentrations of the co-culture media of HK-2 cells and PBMCs were about threefold up-regulated compared with those of HK-2 cells without PBMCs. These results suggested that the pre-stimulation of HK-2 cells by TGF-b 1 contributes to increase not only chemokine production but also up-regulation of TGF-b 1 that induced EMT in RTECs upon the interaction of RTECs and PBMCs. We conducted real-time RT-PCR to detect the change of the TGF-b 1 mRNA in the HK-2 cells that underwent EMT and PBMCs in the lower plate separately at several time points after co-culture; however, we could not detect up-regulation of TGF-b 1 mRNA in both these HK-2 cells and PBMCs (data not shown). Although further study will be needed to investigate the regulation of TGF-b 1 on the interaction of LFA-1 on PBMCs and ICAM-1 on RTECs, the up-regulation of TGF-b 1 concentrations of the co-culture media of HK-2 cells and PBMCs also suggested In the EMT of RTECs, smad2 and smad3 were mainly mediated by TGF-b 1 [33] . In the present study, the phosphorylation of smad2 and smad3 was not detected in HK-2 cells by the stimulation of TGF-b 1 for 24 hrs before and after co-culture with PBMCs. This lack of phosphorylation of smad2 and smad3 is considered to be due to the low concentration of TGF-b 1 (0.1 ng/ ml) because phosphorylation of smad2 and smad3 on HK-2 cells was detected after stimulation with higher concentrations of TGFb 1 (10.0 ng/ml) for 24 hrs (data not shown). Xie et al. reported that the activation of ERK pathway was required for TGF-b 1induced EMT of RTECs [34] . Schramek et al. reported that longterm ERK1/2 activation was an important mechanism involved in the EMT of HK-2 cells [35, 36] . In addition to the role of ICAM-1 as an adhesion molecule, ICAM-1 also has roles as a signal HK-2 cells co-cultured with PBMCs for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation (D). HK-2 cells co-cultured with PBMCs with pre-incubated anti-LFA-1 mAb following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (E). HK-2 cells pre-incubated with anti-ICAM-1 mAb co-cultured with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (F). HK-2 cells pre-incubated with isotype control mAb co-cultured with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (G). Magnification 1006. HK cells were co-cultured with PBMCs for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs. Then, the changes of the epithelial markers (E-cadherin and occludin) and the mesenchymal markers (vimentin and fibronectin 1) on HK-2 cells (HK-2-TGF-b 1 (0.1)-PBMCs) were assessed by western blotting analysis (H). For blocking the interaction of LFA-1 and ICAM-1, before co-culture, PBMCs were pre-incubated with anti-LFA-1 mAb for 20 minutes (Anti-LFA-1 mAb ) or HK-2 cells were pre-incubated with anti-ICAM-1 for 20 minutes (Anti-ICAM-1 mAb) or isotype control mAb for 20 minutes (Isotype mAb). HK-2 cells without co-culture with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (TGF-b 1 (0.1)) and HK-2 cells without stimulation of TGF-b 1 co-cultured with PBMCs (PBMCs) were investigated as control. One representative immunoblot is presented at the top of the graph. Values are expressed as mean 6 SEM of at least three experiments. *P,0.05. doi:10.1371/journal.pone.0023267.g005 transduction molecule [37, 38, 39] . With the interaction of LFA-1 with ICAM-1 and the formation of ICAM-1 multimers within the cell membrane, signal transduction is initiated [40, 41] . ICAM-1 with associated proteins such as Grb2, SOS and Shc can lead to downstream activation of the mitogen-activated protein (MAP) kinase pathway, including ERK1/2 [38, 42] . The ERK1/2 proteins were most commonly activated through ICAM-1 ligation [38, 40] . In the present study, phosphorylation of ERK1/2 was detected on HK-2 cells after stimulation of TGF-b 1 co-cultured with PBMCs. This phosphorylation of ERK1/2 was blocked by anti-LFA-1 mAb or anti-ICAM-1 mAb. These results suggested that ERK 1/2 may be activated through ICAM-1 on HK-2 cells by the interaction of ICAM-1 on HK-2 cells and LFA-1 on PBMCs. LFA-1 also acts as a signal transduction molecule when it binds ligands [7] . Although ICAM-1 expression still showed a high level in HK-2 cells by TGF-b 1 stimulation, its suppression by TGF-b 1 might be an inhibition factor for EMT of HK-2 cells by the interaction of ICAM-1 of HK-2 cells and LFA-1 of PBMCs. Further study will be needed to investigate the mechanism and significance of the regulation of ICAM-1 expression, the signaling pathway that affects EMT of HK-2 cells on PBMCs by the interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells.
Next The extent to which EMT contributes to renal fibrosis remains to be clarified; however, evidence for the role of EMT in renal fibrosis in various renal diseases is emerging. A large proportion of the interstitial fibroblasts in fibrotic kidneys have been reported to originate from proximal tubular cells in an animal model [16] . Clinical studies utilizing human kidney biopsies also suggest that EMT plays a role in the pathogenesis of renal fibrosis [43, 44, 45] . Furthermore, EMT of RTECs was well correlated with declining renal function [43, 45] . The renal fibrosis also involved interstitial infiltration of inflammatory mononuclear leukocytes [3] . Thus, our finding suggested that inflammatory mononuclear leukocytes contributed to renal fibrosis by accelerating EMT of RTECs.
In conclusion, HK-2 cells stimulated with TGF-b 1 induced activation of LFA-1 on PBMCs by increased CXCL12. The direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-b 1. These results suggested that drugs that can block the interaction of LFA-1 and ICAM-1 could be an option to treat renal fibrosis.
Cell culture HK-2 cells (human renal proximal tubular epithelial cells immortalized by transduction with human papilloma virus 16 E6/E7 genes) were purchased from American Type Culture . The effect of PBMCs on the activation of extracellular regulated kinase 1 and 2 (ERK1/2) of HK-2 cells. HK cells were cocultured with PBMCs for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (TGF-b 1 (0.1)-PBMCs). After 4 and 12 hrs of co-culture with PBMCs, aliquots of HK-2 cell lysate were subjected to western blot analysis. For blocking the interaction of LFA-1 and ICAM-1, before coculture, PBMCs were pre-incubated with anti-LFA-1 mAb for 20 minutes (Anti-LFA-1 mAbs) and HK-2 cells were pre-incubated with anti-ICAM-1 mAb for 20 minutes (Anti-ICAM-1 mAbs) or isotype control mAb for 20 minutes (Isotype mAb). HK-2 cells without co-culture with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (TGF-b 1 (0.1)) and HK-2 cells without stimulation of TGF-b 1 co-cultured with PBMCs (PBMCs) were investigated as controls. One representative immunoblot is presented at the top of the graph. doi:10.1371/journal.pone.0023267.g007
Collection (Manassas, VA). HK-2 cells were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY) and 1% penicillin and streptomycin (Invitrogen). PBMCs were obtained from three healthy donors, after obtaining informed consent, using Ficoll-gradient sedimentation. PBMCs were cultured with RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) and 1% penicillin and streptomycin (Invitrogen).
The sub-confluent cultures of HK-2 cells in 6-well plate (Falcon, Franklin Lake, NJ) were treated with recombinant human TGF-b 1 (R&D Systems, Minneapolis, MN, USA) at concentrations of 10.0, 1.0 and 0.1 ng/ml for 24 hrs. Next, the cells were washed three times with phosphate-buffered saline (PBS). Then, PBMCs (2610 5 ) were added to each well for co-culture for 24 hrs. For blocking the interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells, HK-2 cells were pre-incubated for 20 minutes at 37uC with 20 mg/ml anti-ICAM-1 monoclonal antibody (mAb) (clone: 6.5B5, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or naïve PBMCs were pre-incubated for 20 minutes at 37uC with mg/ml anti-LFA-1 mAb (clone TS1/22, kindly provided by Dr. M. Shimaoka, IDI Harvard Medical School, Boston) before coculture. Pre-incubation with HK-2 and PBMCs with mouse IgG 1 isotype control mAb (clone MOPC-31C, BD, San Jose, CA) was used for control experiments.
Total RNA from cells was isolated with RNeasy total RNA isolation kit (Qiagen, Valencia, CA). The isolated 1 mg of total RNA was reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen) according to the manufacturer's protocol. Real-time RT-PCR was performed using SYBR Green ER qPCR super mix (Invitrogen) and an Applied Biosystems Step One Plus Real-Time PCR system (Applied Biosystems, Carlsbad, CA). All reactions were carried out in a 20 ml reaction volume in triplicate. Primers for human GAPDH, E-cadherin, occludin, vimentin, fibronectin 1, a-smooth muscle actin, snail, slug, CCL2, CCL3, CCL4, CCL5, CCL17, CCL19, CCL20, CCL21, CCL22 and CXCL12 were as previously described [46,47,48,49,50, 51,52,53, Lin, 2009 
For western blotting, cells were extracted with lysis buffer [20 mM Tris-HCl (pH 7.4), 1% Triton, 10% glycerol and 0.1 mM phenylmethylsulfonyl fluoride] and phosphatase inhibitors (2 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate). Equal amounts of protein samples (40 g) were electrophoresed on NuPAGE sodium dodecyl sulphatepolyacrylamide gels (Invitrogen), and then transferred onto Immobilon-P membranes (Millipore Corporation, Bedford). After nonspecific protein binding was blocked with skim milk and bovine serum albumin, the membrane was incubated overnight at TGF-b 1 ELISA TGF-b 1 ELISA kit specific for human TGF-b 1 (TGF-b 1 ELISA, IBL, Flughafenstrasse, Hamburg) was used according to the manufacturer's protocol. Briefly, 100 ml of samples was added to the TGF-b 1 mAb pre-coated plate and incubated overnight at 4uC. After washing, 100 ml of antiserum was added and incubated for 2 hours at room temperature for blocking. After washing, antimouse IgG antibody conjugated to biotin was added and incubated for 45 minutes at room temperature. The color was developed with 100 ml of streptavidin peroxidase and substrate.
Cytoselect 24-well cell migration assay kit (Cell Biolabs, Inc., San Diego, CA) and Cytoselect 24-well cell invasion assay kit (Cell Biolabs, Inc.) were used according to the manufacturer's protocol. Briefly, 500 ml of RPMI 1640 supplemented with 10% FBS was added to each well of the plate. Then, 300 ml of cell suspension containing 3610 5 cells/ml or 1.5610 6 cells/ml was added to the 8 mm pore size polycarbonate membrane cell culture inserts for cell migration assay or to the 8 mm pore size polycarbonate membrane cell culture inserts coated with a uniform layer of basement membrane matrix solution for cell invasion assay, respectively. Then, cells were incubated for 24 hrs at 37uC in a 5% CO 2 atmosphere. Next, the medium was removed from the inside of the insert and the interior of the insert was gently swabbed with the wet end of cotton-tipped swabs to remove non-migratory cells. Then, each insert was transferred to a clean well containing 400 ml of cell stain solution and incubated for 10 minutes at room temperature. After washing, inserts were transferred to an empty well and 200 ml of extraction solution was added and incubated for 10 minutes. Then, the optical density (OD) was measured at 560 nm.
Transwell co-culture system Cytoselect 24-well cell migration assay kit (Cell Biolabs, Inc.) was used. HK cells without TGF-b 1 treatment with/without PBMCs were co-cultured in the lower well with complete media and HK-2 cells pre-stimulated with TGF-b 1 (10.0, 1.0 and 0.1 ng/ ml) for 24 hrs followed by three careful washes by PBS or HK-2 cells without TGF-b 1 stimulation were added to 2 mm pore size polycarbonate membrane cell culture inserts. Then, after 24 hrs of incubation, TGF-b 1 concentration in the lower well media was measured by TGF-b 1 ELISA.
Statistical significance was evaluated by Student's t-test or by one-way analysis of variance followed by Fisher's PLSD test. Statistical significance was defined as P,0.05. All values are expressed as means 6 SE. Figure S1 The change of CCL3 and CXCL12 expression in CCL3 and CXCL12 knockdown HK-2 cells by TGF-b 1 stimulation. CCL3 knockdown HK-2 cells (CCL3 KD) and CXCL12 knockdown HK-2 cells (CXCL 12KD) were induced by transfection of CCL3 siRNA or CXCL12 siRNA, respectively. Then, CCL3 KD and CXCL12 KD HK-2 cells were stimulated with TGF-b 1 (10.0, 1.0 and 0.1 ng/ml) for 24 hrs. Non-treated HK-2 cells (Mock) and non-targeted-siRNA-transfected HK-2 cells (Control) served as controls. The expressions of CCL3 and CXCL12 were analyzed by real-time RT-PCR. CCL3 was upregulated for CXCL12 KD, Mock and Control; however, it was not up-regulated for CCL3KD (upper graph) regardless of TGFb 1 stimulation. CXCL12 was up-regulated for CCL3 KD, Mock and Control; however, it was not up-regulated for CXCL12 KD (lower graph). Values are expressed as mean 6 SEM of at least three experiments. *P,0.05. (TIF) Figure S2 Effects of CCL3 and CXCL12 of HK-2 cells on the change of EMT markers on HK-2 cells by PBMCs. PBMCs were co-cultured with non-treated HK-2 cells (Mock), CXCL12 knockdown HK-2 cells (CXCL12 KD), CCL3 knockdown HK-2 cells (CCL3 KD) or non-targeted-siRNA-transfected HK-2 cells (Control) for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs. Then, the changes of the epithelial markers (E-cadherin and occludin) and the mesenchymal markers (vimentin and fibronectin 1) on those HK-2 cells were assessed by real-time RT-PCR. The expression of epithelial cell junction markers (Ecadherin and occludin) decreased and that of mesenchymal markers (vimentin and fibronectin 1) increased in Mock, CCL3 KD and Control, whereas these changes were significantly decreased in CXCL12 KD. Values are expressed as mean 6 SEM of at least three experiments. *P,0.05. (TIF) Figure S3 Assessment of the effect of CCL3 and CXCL12 of HK-2 cells for morphology and EMT marker change by PBMCs. PBMCs were co-cultured with non-treated HK-2 cells (Mock), CXCL12 knockdown HK-2 cells (CXCL12 KD), CCL3 knockdown HK-2 cells (CCL3 KD) or non-targeted-siRNA-transfected HK-2 cells (Control ) for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs. The cells were fluorescence-labeled by CM-Dil. Then, the cell morphology was observed using a fluorescent microscope. Magnification 1006. The phase contrast microscopy analysis showed that CXCL12 KD (C,D) changed less, morphologically, from a cobblestone-like monolayer to spindle-shaped fibroblast-like cells, than Mock (A,B), CCL3KD (E, F) and Control (G,H). Western blot analysis showed that the expression of epithelial cell junction markers (E-cadherin and occludin) decreased and that of mesenchymal markers (vimentin and fibronectin) increased in mock, CCL3KD and Control HK-2 cells, whereas these changes significantly decreased in CXCL12KD HK-2 cells (I). Values are expressed as mean 6 SEM of at least three experiments. *P,0.05. (TIF)
Conceived and designed the experiments: YM. Performed the experiments: YM MW EN. Analyzed the data: YM. Contributed reagents/materials/ analysis tools: YM EK. Wrote the paper: YM KI EK. Histological study of the biodynamics of iron oxide nanoparticles with different diameters The biodynamics of ultrasmall and small superparamagnetic iron oxide (USPIO and SPIO, respectively) particles that were injected intraperitoneally into 36 C57BL/6 mice were investigated chronologically. Their distribution was studied histologically at six time points by measuring iron-positive areas (μm(2)) in organ sections stained with Prussian blue. The uptake of the differently sized particles was also compared by cultured murine macrophages (J774.1). Iron-positive areas in the liver were significantly larger in the mice injected with USPIO than those injected with SPIO at the first three time points (P < 0.05). The amount of USPIO in the lung parenchyma around the airway was larger than that of SPIO at four time points (P < 0.05); distribution to the lymph nodes was not significantly different. The amount of iron was significantly larger in SPIO- than USPIO-treated cultured cells (P < 0.05). In conclusion, it is suggested that intra peritoneally injected USPIO particles could be used more quickly than SPIO to make Kupffer images of the liver and that both agents could help get lymph node images of similar quality. The uptake of superparamagnetic iron oxide (SPIO) particles by cells of the mononuclear phagocytic system (MPS) results in hepatic, splenic, bone marrow, and nodal iron accumulation. 1, 2 The selective accumulation of these particles by the MPS is exploited at organ-specific magnetic resonance imaging (MRI). [3] [4] [5] [6] [7] [8] [9] [10] The biodynamics of iron oxide nanoparticles vary depending on particle diameter. Unlike SPIO preparations comprised of large particles and magnetite microspheres (mean particle diameter, 72 nm and 1-5 µm, respectively), ultrasmall superparamagnetic iron oxide (USPIO) particles (mean diameter, 18 nm) are not immediately recognized by the hepatic and splenic MPS. 11, 12 The resultant prolongation of its intravascular half-life, together with its inherent T1 shortening properties, render USPIO a useful magnetic resonance angiography blood-pool agent. 13, 14 In contrast to large-particle superparamagnetic agents, the size of USPIO particles allows the extravasation of USPIO through capillary pores, the diameters of which range from 5 to 100 nm, and this capillary permeability facilitates the uptake of USPIO in MPS cells throughout the body. 15 Although information on the biodynamics of iron oxide nanoparticles of different diameters is important for various organ-specific MRI studies, to date this issue has not been addressed histologically. Therefore, a comparative histological study of the sequential biodynamics of USPIO and SPIO was performed. 
Shiga University of Medical Science's animal experimentation committee approved all experimental protocols and all experiments were conducted in accordance with the animal care guidelines of the university.
Two kinds of iron oxide were studied: USPIO (Meito Sangyo Co, Ltd, Kiyosu, Aichi, Japan) and SPIO (Resovist ® ; I'rom Pharmaceutical Co, Ltd, Shinagawa-ku, Tokyo, Japan). The mean particle diameters and iron concentrations of USPIO and SPIO were 30 nm and 56 mg/mL and 57 nm and 28 mg/mL, respectively. Average diameter of the magnetic core was the same (5 nm) in both particle types. The iron oxide core of both particle types was coated with dextran; their magnetic susceptibility was almost the same (about 0.027 cgs).
USPIO or SPIO was injected intraperitoneally (500 µmol Fe/kg) into 8-week-old female C57BL/6 mice (Japan SLC, Inc, Tokyo, Japan), each weighing approximately 30 g. Groups of three mice each were treated with USPIO or SPIO and sacrificed at time points of 30 minutes and 1, 3, 12, 24, and 48 hours afterward; two mice were the controls. Organs to be studied were fixed in 10% paraformaldehyde and examined histologically.
The mice were sacrificed at the indicated time points and their organs were removed for histological study. Sections of the right lobe of the liver were cut in the axial plane; splenic sections were also cut in the axial plane and included the hilum. Lung sections were cut in the coronal plane and included the hilum. Sections from the heart, great vessels, gastrointestinal tract, and kidney were cut in the coronal plane. Sections (4 µm in thickness) of paraffin-embedded organs were stained with Prussian blue to identify the accumulation of iron oxide. Macrophages were immuno histochemically stained with F4/80. Areas stained with Prussian blue and F4/80 were compared. For quantitative assessment, Prussian-blue-stained areas (µm 2 ) in a single field of view (magnification ×200) of three different lesions for each histological section were measured using Image-Pro ® Plus software (Media Cybernetics, Bethesda, MD) and average values were calculated.
To compare phagocytosis of USPIO and SPIO by cultured cells murine macrophage cell line J774.1 (RIKEN Cell Bank, Wako, Saitama, Japan) was used, which is widely used in research on macrophages. [16] [17] [18] Cells (5 × 10 4 ) were seeded in 12-well multiple well culture plates (Costar®; Corning Inc, Corning, NY) and grown at 37°C for 24 hours in 1 mL of growth medium (RPMI-1640; Nacalai Tesque, Inc, Kyoto, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen Ltd, Carlsbad, CA) and 1% (w/v) penicillin and streptomycin solution (Nacalai Tesque, Inc). After 24 hours, the medium was replaced, USPIO or SPIO was added (10 μg : 100 μL) and the plates were incubated for 1 hour for cell labeling. After this, the medium was again replaced and the wells then incubated for 24 hours. To identify intracellular iron oxide accumulation, the cells were stained with Prussian blue. The amount of iron contained in cell lysates by atomic absorption photometry (AA-6800; Shimadzu Co, Kyoto, Japan) was measured. Cell labeling was as described in a previous study. 19 
Statistical analysis was carried out using Dr SPSS II for Windows (SPSS Japan Inc, Tokyo, Japan). Differences in the uptake of USPIO and SPIO in murine tissues and the uptake of iron by cultured cells were determined with the one-tailed Student's t-test. A P-value , 0.05 was considered statistically significant.
Quantitative analysis based on the measurement of Prussianblue-stained areas revealed that iron-positive areas in the liver were larger in the mice injected with USPIO than those injected with SPIO at almost all time points except the last one (828 vs 0 µm 2 at 30 minutes, 1299 vs 297 µm 2 at 1 hour, 3647 vs 853 µm 2 at 3 hours, 2256 vs 1497 µm 2 at 12 hours, 1885 vs 1168 µm 2 at 24 hours, and 1948 vs 2483 µm 2 at 48 hours), as shown in Figures 1 and 2 . The difference was significant at 30 minutes, 1, and 3 hours (P , 0.05). USPIO was quickly distributed throughout the liver; its distribution increased until 3 hours postinjection and decreased thereafter. On the other hand, the distribution of SPIO occurred more slowly and increased over a longer period of time. Unlike USPIO, SPIO was not detected in the liver at 30 minutes. Iron-positive areas at the hepatic sinusoid corresponding to the area harboring Kupffer cells coincided with macrophage-positive F4/80-stained areas, as shown in Figure 3 .
In lung specimens, a few USPIO and SPIO particles were observed early; they were located in the parenchyma around There was no significant accumulation in the heart, great vessels, kidneys, or gastrointestinal tract. As the controls harbored abundant stores of iron, visualized as Prussianblue-positive areas, macroscopically it was observed that there was almost no difference between them and USPIOor SPIO-treated mice with respect to the spleen. Control mice manifested no significant iron deposits in organs other than the spleen.
While almost all cultured J774.1 cells phagocytized USPIO and SPIO, the amount of intracellular iron measured by atomic absorption photometry was significantly higher in cells treated with SPIO than with USPIO (695 vs 108 pg/cell, P , 0.05), as shown in Figure 8 .
To our knowledge, this is the first study comparing the biodynamics of intraperitoneally injected USPIO and SPIO. SPIO (Resovist) is the contrast agent used in MRI and generally its safety in the clinical setting has already been established. USPIO used in this study is different in particle size from that of SPIO, but very similar in composition. Ferumoxtran-10, similar to the USPIO used in this study, was well tolerated in phase I and II studies in adult humans. 20, 21 In addition, in a safety study conducted in adult rabbits, no inflammation or fibrosis occurred after intraperitoneal (IP) injection of NC100150, a USPIO similar to ferumoxtran-10. 22 IP irondextran supplementation is known to be effective and safe in correcting iron deficiency in rats and humans. 23, 24 In our study, IP injection was safe and never caused the death of any mouse. USPIO was distributed to the liver as early as 30 minutes postinjection; it peaked at 3 hours and decreased thereafter. SPIO appeared later and its distribution throughout the liver increased over time. At 12 hours postinjection, the hepatic distribution of both agents was comparable. A greater amount of USPIO than SPIO tended to be distributed to the lung parenchyma around the airway, possibly because USPIO was presented more easily to the small lymphatic vessels around the airway. There was no significant difference in lymph node accumulation between the two groups of mice.
The early distribution of intraperitoneally injected USPIO to the liver and the absence of significant differences in the lymph node distribution of USPIO and SPIO are primarily attributable to differences in the absorption route. The trapping of intravenously administered USPIO by reticuloendothelial cells in the liver and spleen has been reported to be difficult because the particle diameter is small and the particles persist in the blood. 11, 12 Lukas et al 25 who studied the absorption route of intraperitoneally administered compounds, suggested that particles absorbed by the subperitoneal capillary system reached the liver via the hepatic portal system. The earlier hepatic distribution of USPIO suggests that it is delivered via this pathway.
Intercellular spaces directly open to lymphatic vessels are present in the mesothelial layer of the peritoneum. The peritoneal cavity is the origin of two major lymphatic networks: the thoracic duct drains the retroperitoneal region and enters the left subclavian vein; the right lymphatic duct collects lymph from parasternal and intercostal-paravertebral lymph ducts and drains mainly through the diaphragm and, ultimately, into the right subclavian vein. In the former network, lymph passes through iliac, renal, splenic, posterior gastric, portal, superior mesenteric, and cisternal lymph nodes; in the latter network it passes through more distal (eg, parathymic) and left and right posterior mediastinal lymph nodes. 26, 27 The authors posited that there was not only USPIO present in abundant amounts in the lymphatic tracts due to these networks but also SPIO, which tended to be trapped in the liver with only slight distribution to the lymph nodes. Cultured macrophages absorbed SPIO more readily than USPIO. Under conditions where the same number of macrophages respond to iron oxide compounds containing the same amount of iron, larger particles are recognized more easily. This is consistent with the hypothesis that upon intravenous (IV) infusion, reticuloendothelial cells in the liver and spleen trap SPIO more readily than USPIO. This study had some limitations. First, the number of mice was too small for an accurate elucidation of the pharmacokinetics and prevented the exclusion of interindividual differences. Second, it could not be confirmed that the minute amounts of USPIO and SPIO used were injected accurately into the peritoneal cavity. In some instances, the agent might have been injected subcutaneously or into the bowel. Third, due to technical challenges, the pharmacokinetics of intravenously delivered USPIO or SPIO was not studied. However, considering the mechanisms by which USPIO reached the liver earlier than SPIO and the result that similar amounts of USPIO and SPIO were distributed throughout the lymph nodes, it is important to compare the differences of distribution between USPIO and SPIO via two different injection routes (IP and IV).
The findings suggest that the IP delivery of both USPIO and SPIO might be useful as a contrast agent for the study of lymph nodes in a clinical setting. The IP delivery of USPIO might also be used as a contrast agent for studying the liver. As IP delivery is not a popular injection route in clinical settings, drugs or contrast agents are currently intraperitoneally injected in limited cases of patients with cancer, peritonitis, or chronic renal failure requiring continuous ambulatory peritoneal dialysis (CAPD). 24, [28] [29] [30] Although clinical application is limited, the authors suggest, for cases of patients with ascites of unknown origin, that intraperitoneally injected iron particles may be very useful for getting Kupffer images of the liver and lymph node images to help diagnose metastases in these areas. Also, in cases of iron-deficient CAPD patients, intraperitoneally injected iron dextran has potential for filling both roles, as a route of entry in iron therapy and as a contrast agent for the liver and lymph nodes. In addition, depending on the diameter of the administered particles, it may be possible to deliver agents other than USPIO and SPIO intraperitoneally for the study of various organs.
This study suggests that intraperitoneally injected USPIO could be used more quickly than SPIO can to make Kupffer images of the liver and that both agents could help get lymph node images of similar quality.
The authors report no conflicts of interest in this work.
Submit your manuscript here: http://www.dovepress.com/international-journal-of-nanomedicine-journal
The International Journal of Nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. This journal is indexed on PubMed Central, MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine, Journal Citation Reports/Science Edition, EMBase, Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.  The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. A common method of choice for clinical pathogen identification is polymerase chain reaction (PCR) [1] which is a sensitive and specific method. However, each PCR reaction only provides identification of one specific virus, or a group of related viruses. Thus, a clinical hypothesis regarding the suspected pathogen needs to guide the decision as to which PCR analyses to perform, resulting in a large number of PCR protocols needed to facilitate the identification of all human pathogens.
DNA microarray testing has emerged as a promising new technology for broad-spectrum virus detection, making it possible to test for the presence of thousands of viruses simultaneously. Several microarray platforms have been developed for detection of all known viruses, as well as novel viruses related to known virus families [2, 3, 4] . Some of them have been tested on clinical samples, mostly respiratory samples [2, 3, 4, 5] . A microarray containing probes for all human pathogens could be used for diagnostic analysis, as well as for research purposes, environmental samples or epidemiological surveillance. Moreover, with some clinical syndromes or symptoms, it might be difficult to determine which viruses to test for in order to establish the correct diagnosis since many viruses can present similar symptoms. For this reason various multiplexed assays with virus panels, utilizing PCR or other methods, have been established. However, a microarray could be viewed as an extended pathogen panel, covering all viruses and bacteria, increasing the chance of the correct identification of one or several pathogens even in situations without any prior knowledge or suspicion about the etiology.
Without a pre-amplification of the clinical sample, such as PCR or virus culture, most microarrays are not sensitive enough for pathogen identification in clinical samples since virus titres normally are below their detection limit. For example, a diagnostic human Papilloma virus (HPV)-specific microarray utilizes an HPV-specific PCR-amplification prior to microarray hybridization [6, 7] . Different PCR-based random amplifications have been developed to achieve enough material for a successful identification [3, 4, 8] . However, studies have shown that PCR-based whole genome amplification (WGA) may generate incomplete coverage of genomes [9] . Instead we used multiple displacement amplification (MDA)-based WGA through isothermal amplification by the Phi29 polymerase [9] . The Phi29 polymerase has the highest processivity rate reported, ,70,000 bases every time it binds [10] and an error rate of only 1 in 10 6 -10 7 bases [11] , allowing for good coverage across genomes [9, 12, 13] . The average length produced is .10 kb compared to #3 kb by the Taq polymerase [9] . Phi29-amplification prior to microarray analysis has been used for human genomic DNA analysis [14, 15, 16] and for bacterial DNA analysis [17, 18] . However, the Phi29 polymerase cannot amplify RNA or short DNA fragments (,2000 bp), such as cDNA generated with random hexamers. To overcome this, the method Whole Transcriptome Amplification (WTA) has included a ligation step prior to amplification [2] resulting in cDNA fragments that are ligated into larger transcriptomes that can be efficiently amplified by the Phi29 polymerase.
Here we investigate real life utility and sensitivity of the microarray technique in a diagnostic set-up. We have established a protocol including pre-treatment, nucleic acid (NA) purification and Phi29amplification followed by microarray analysis of DNA and RNA viruses from a diverse set of clinical samples. We show that Phi29amplification generates enough material for successful identification by the Lawrence Livermore Microbial Detection Array (LLMDA) [19, 20] , demonstrating the potential of the microarray technique for broad-spectrum pathogen detection in clinical samples.
Exemption for review by the ethical committee system and for obtaining informed consent was obtained from the Committee on Biomedical Research Ethics for the Capital region in accordance with Danish law on quality control and assay development projects. [21] and Sapovirus [22] , and ClartH HPV2 microarray (Genomica) for HPV. PCR's were performed on Mx3005P (Stratagene) or ABI7900 (Applied Biosystems) thermal cyclers.
Five-to two-hundred microliters of clinical sample was centrifuged at 17,000 g for 10 minutes. The supernatant was filtered through a 0.22 mm Ultrafree MC spin filter (Millipore) at 2000 g for 2 minutes and DNase treated (6 U DNaseI Amplification grade, Invitrogen) for 1K hours at room temperature while shaken in a Thermomixer (AH Diagnostics). The viral NA was extracted using the PureLink Viral RNA/DNA kit (Invitrogen), without the addition of carrier RNA. The extracted viral NA was eluted with 20-30 ml DNase/RNase-free sterile water, and stored at 220uC or immediately used.
Reverse transcription on purified viral RNA was performed using the Superscript VILO cDNA synthesis kit (Invitrogen) according to protocol. Briefly, 14 ml of extracted viral RNA was put into a 20 ml-reaction containing Superscript enzyme and random primers, and incubated at 42uC for 1 hour. The enzyme was inactivated at 95uC for 5 minutes and the sample stored at 220uC or immediately used.
Viral DNA was amplified by Phi29 polymerase amplification using GenomiPhi V2 Amplification kit (GE Healthcare) or Repli-g Midi kit (typical yield ,40 mg from a 50 ml reaction, Qiagen), following manufacturer's protocols. Briefly, using GenomiPhi V2 Amplification kit, 1 ml of purified DNA was added to 9 ml of sample buffer, incubated at 95uC for 3 minutes and cooled on ice. Ten microliters of GenomiPhi reaction was added, containing Phi29 polymerase and random primers, and the reaction incubated at 30uC for 1 K hours. The reaction was terminated by incubation at 65uC for 10 minutes. The amplified viral DNA was stored at 280uC or immediately used. Briefly, using Repli-g Midi kit, 5 ml of purified DNA was mixed with 5 ml denaturation buffer, incubated at room temperature for 3 minutes and the reaction terminated by the addition of 10 ml stop solution. Thirty microliters of Repli-g reaction mix, containing Phi29 and random primers, was added and the reaction incubated at 30uC for 16 hours. The reaction was terminated by incubation at 65uC for 3 minutes. Amplified material was purified using QIAamp DNA mini kit (Qiagen) using a modified protocol (www.qiagen.com), and checked for purity and concentration using a NanoDrop spectrophotometer (Thermo Scientific) and agarose gel electrophoresis. The DNA was stored at 280uC or immediately used for microarray analysis.
For viral NA amplification (both DNA and RNA virus) we used the described WTA method [2] by using the QuantiTect Whole Transcriptome Amplification kit (typical yield ,40 mg from a 50 ml reaction, Qiagen) according to manufacturer's protocol, except for the reverse transcription step that was replaced by the Superscript VILO kit (Invitrogen) described above. The WTA protocol includes 3 sequential reactions: first a reverse transcription reaction to generate cDNA, secondly a ligation of generated cDNA into larger transcriptomes and thirdly a Phi29-amplification of the generated transcriptomes. Briefly, as described above, 14 ml of extracted viral NA was put into a 20 ml-VILO reaction and incubated at 42uC for 1 hour. The Superscript enzyme was inactivated at 95uC for 5 minutes, 10 ml of the VILO cDNA reaction was added to 10 ml of ligation mix and incubated at 22uC for 2 hours. Thirty microliters of amplification mix containing Phi29 polymerase and random primers was then added and amplification performed at 30uC for 8 hours. The reaction was terminated by incubation at 95uC for 5 minutes. Amplified material was purified using QIAamp DNA mini kit (Qiagen) using a modified protocol (www.qiagen.com), and checked for purity and concentration using a NanoDrop spectrophotometer (Thermo Scientific) and agarose gel electrophoresis. The DNA was stored at 280uC or immediately used for microarray analysis.
For the quantification of virus before and after Phi29amplification, virus-specific real-time PCR's were performed. These were in-house assays for HSV1, HSV2, HPV6, HPV16, BKV, Rota A, Astrovirus, EV and RSV. For the detection of human gDNA, an in-house assay for b-actin was used (95uC for 15 minutes, 40 cycles of 95uC for 15 seconds and 55uC for 1 minute; Forward primer: CTC TTC CAG CCT TCC TTC CT, Reverse primer: AGC ACT GTG TTG GCG TAC AG, Probe: Yakima Yellow-TGG AGT CCT GTG GCA TCC ACG A-BHQ. In addition, previously published assays were used for JCV [23] , HCV [21] and Sapovirus [22] . For the multi-HPV (6, 16, 53, 61) positive sample (determined in the routine by ClartH HPV2 microarray, Genomica), only HPV 6-and 16-specific PCR's were performed before and after Phi29-amplification. For the quantitative analysis of BKV, we used an in-house standard containing 20,000-2610 7 copies/ml. For the confirmation of additional viruses found by microarray analysis, in-house real-time PCR's were used for HPV103, Picobirnavirus, Rota C and Dengue subtypes (1, 2, 3 and 4), and previously published assays for JCV [23] , GB virus type C (GBV-C) [24] and Human Adenovirus C (HAdV-C) [25] . Thermal cycling was performed in a thermal cycler (Mx3005P (Stratagene) or ABI7900 (Applied Biosystems)). Fold difference was calculated from DCt obtained from real-time PCR results before and after Phi29-amplification, combined with dilution factors for each sample. In doing so, we also made the assumption that a change in Ct-value was equivalent to a doubling of target DNA.
The microarray used was the LLMDA version 2, developed at Lawrence Livermore National Laboratory (LLNL), USA and already described elsewhere [19, 20] . Briefly, the LLMDA contains 388,000 probes designed from all sequenced virus and bacteria, resulting in a large number of probes covering each genome. The microarray was designed for both detection and discovery, to both identify known organisms and to detect unsequenced or emerging organisms, while strain or subtype identification was not a goal. The array have both conserved and unique probes within each family, where oligos that were conserved, to the extent possible, within a family, and unique relative to other families and kingdoms, were prioritized [19] . Labelling and microarray hybridization was performed at NimbleGen Systems-Iceland, Iceland, or in-house at SSI, Denmark according to manufacturer's protocol (Gene expression analysis, Roche NimbleGen). Briefly, 1 mg of heat-denatured WTA-amplified sample was labelled using nick translation with Cy3-labeled random nonamer primers (TriLink Biotechnologies) and Klenow fragment (exo-) (NEB) at 37uC for 2 hours. Labelled DNA was iso-propanol precipitated and the pellet washed, dried, reconstituted and quantitated. For hybridization, the in-house assay used 12 mg of labelled DNA whereas NimbleGen Systems-Iceland used 6 mg. Hybridization was performed in a NimbleGen Hybridization system using the NimbleGen Hybridization kit (Roche NimbleGen).
Microarrays were scanned using a GenePix 4000B Scanner (Molecular Devices) and data analysed using the maximum likelihood method developed at LLNL, extensively described elsewhere [19] . Additional stringency criteria were applied to exclude bacterial sequences and viruses having fewer than 20% of probes detected out of those expected. Briefly, probes were classified as detected if the intensity exceeded a threshold equal to the 99 th percentile of intensities for negative control probes, except that one sample was analysed using the 95 th percentile. Targets in an internal database of 43 705 viral sequences, developed at LLNL [19] , were screened against the stringency criteria; an unconditional log-odds score for presence of each remaining target was computed, and targets having log-odds scores less than 5 were excluded. A greedy forward selection algorithm was applied to find the collection of targets most likely to be present in the sample. At every forward selection step, a conditional log-odds score was computed for each remaining target, representing its potential contribution to the log-likelihood conditioned on the presence of the previously selected targets; the target having the largest conditional log-odds was selected and added to the collection. The resulting conditional and unconditional log-odds scores were plotted in a bar graph format [19] , where the conditional log-odds scores shows the contribution from a target that cannot be explained by another, more likely target above it, and the unconditional score illustrates that some very similar targets share a number of probes, so that multiple targets may be consistent with the hybridization signals.
As a complement, another mode of data analysis was developed at SSI, where data was processed using the NimbleScan software program (NimbleGen, Roche). A mean of intensities for each probe-set was calculated. Outlier intensities within each probe-set were filtered out when signals were more than 3 standard deviations from the mean of the probe-set. A signal threshold was defined as the 99 th percentile of the random control intensities, and every probe-set mean containing more than 10 signals above this threshold was considered as a positive signal.
Microarray data has been submitted to the Gene Expression Omnibus (GEO) database http://ncbi.nlm.nih.gov/geo/ with the accession number GSE28597. All microarray data used in this study is MIAME compliant.
To remove contaminating human gDNA present in clinical samples, we designed a pre-treatment protocol including centrifugation, filtration and DNase treatment. It has been demonstrated that filtration, DNase and RNase treatment can increase the efficiency of downstream amplifications [26, 27] . During DNase treatment the viral NA should be protected inside the viral particle [28] . After pre-treatment, the viral particles were lysed, viral NA purified and later Phi29-amplified using GenomiPhi V2 Amplification kit (GE Healthcare). During purification, the addition of carrier RNA did not improve the yield and interfered with the downstream Phi29-amplification, and was therefore left out (data not shown). Quantitative analysis of viral DNA or human gDNA before and after pre-treatment or Phi29-amplification was done by real-time PCR (Figure 1 and Table S1 ). Only centrifugation and filtration left large amounts of human gDNA still present in a HSV1 + sample (Ct = 22), while DNase treatment reduced the ßactin signal 1000-fold (Ct = 32) ( Figure 1A) . The DNase treatment also had an effect on the HSV1, resulting in a 3-to 20-fold decrease in virus signal after DNase treatment (Table S1 ). However, this was accompanied by a much larger fold increase after Phi29-amplification compared to without DNase treatment (e.g. 88,292 compared to 1176), resulting in lower Ct-values and more viral DNA available for downstream analysis (Table S1 ). This was also seen for the RNA virus HCV (Table S1 ) as well as for other DNA and RNA viruses (data not shown). To investigate to what extent Phi29-amplification of remaining human gDNA would occur we performed a time-course experiment with the amplification-time extended up to 16 hours ( Figure 1B) . The ßactin signal was still at acceptable background level (3000-fold increase in signal), while the HSV1 signal showed an 80,000-100,000-fold increase in signal. In conclusion, our pre-treatment removed a significant amount of human gDNA, which allowed for good virus amplification.
To establish an amplification protocol suitable for both DNA and RNA viruses, we used the described Phi29-amplification method WTA [2] that has included a ligation step prior to the amplification step, thereby generating larger templates for Phi29. The WTA performed on a HCV + serum sample (RNA virus) resulted in a ,200,000-fold increase in signal (Figure 2A and Table 1 ), as compared to only 3-fold without ligation (data not shown). The WTA RT-reaction and ligation step did not have any adverse effects when performed on a DNA-virus such as JCV from a CSF sample ( Figure 2B and Table 1 ), enabling us to run both viral DNA and RNA through the same amplification protocol prior to microarray hybridization. The WTA was used on 12 different DNA or RNA viruses found in 7 different types of clinical samples (Table 1 and Figure 2 ). The fold increase in PCR signal comparing before and after WTA, ranged from 330 to 564610 6 . The highest fold increase was detected for the circular viruses JCV, BKV and HPV (Table 1 and Figure 2B ). Such circular genomes have previously been shown to be efficiently amplified by Phi29 polymerase, through rolling circle amplification (RCA) [29, 30] . The lowest increase in signal was seen for Rota A found in faeces (Table 1 ) due to large quantities present already before amplification (Ct = 12), which resulted in a Ct-value of 7 after Table 1 ). The fold increase in PCR signal for gDNA (b-actin) was analysed in samples representing all 7 types of clinical samples, and showed no or low levels of b-actin amplification (Table S2) . WTA-amplified samples were purified and typical yield after 8 hours of WTA was ,25 mg (data not shown) showing good size distribution ( Figure 2C ).
Samples amplified by WTA representing 7 different types of clinical samples were labelled and hybridized to the LLMDA ( Figure 3 and Table 2 ). Microarray data was analysed using the maximum likelihood method developed at LLNL [19, 20] , with additional stringencies applied. In 14 out of 14 clinical samples, the expected virus was detected: HSV1, HSV2, HPV16, HPV6/ 16/53/61, BKV, JCV, Rota A, Astrovirus, Sapovirus, Dengue 1, HCV in duplicate, EV and RSV (Table 2) . For the RSV + sample, a detection threshold equal to the 95 th percentile of control probes had to be used for detection. For this sample we also used the data analysis developed at SSI, where RSV was detected using a detection threshold equal to the 99 th percentile of random control probes (data not shown). Moreover, using this approach on all samples, we generated the same result as seen with the maximum likelihood method (data not shown). For the double-positive faeces sample containing both Astro-and Sapovirus, only Astrovirus was detected by the microarray analysis. In the negative control no viruses were detected.
In 10 out of 13 clinical samples (77%) representing all 7 types of clinical samples used in the study (skin lesion, urine, CSF, cervical smear, serum, faeces and TA), viruses from the Anelloviridae family were found (Torque Teno virus (TTV), Torque Teno Midi virus (TTMV) and TTV-like mini virus) ( Table 2 ). They were detected in samples containing DNA or RNA viruses, demonstrating that our pre-treatment and amplification protocol followed by microarray analysis can easily detect both types in one sample. Additional viruses were detected in 9 samples (Table 2 and data not shown), with log-odds scores ranging from 91.0 to 677.5. Some were clinically irrelevant since they were phages (Propionibacterium phage, Phage Gifsy-2, Lactococcus phage, Enterobacteria phage ST104, Mycobacteriophage and Pseudomonas phage) or plant viruses (Pepino mosaic virus), probably from digested or environmental sources (data not shown). Human endogenous retrovirus was detected in the Dengue 1 and the RSV sample ( Table 2 ). The rest of the additional viruses found (HPV103, Picobirnavirus, HAdV-C, GBV-C, JCV, Rota C and Dengue 2) were tested with virus-specific PCR for confirmation (Table 2 and data not shown). The HPV103 (cir dsDNA) detected in the HPV16 + sample is not present on the microarray used for routine HPV diagnosis (Genomica). Instead its presence was confirmed using a HPV103-specific PCR. The Rota A + faeces sample was found to be positive for Picobirnavirus (dsRNA) and negative for Rota C (dsRNA) by PCR. The Sapovirus + faeces sample was found to be positive for HAdV-C (linear dsDNA) in the microarray analysis, which was confirmed by PCR. Two separate microarray experiments detected GBV-C ((+)ssRNA) in the HCV + sample, which was confirmed by a GBV-C-specific PCR. The BKV + sample was detected as positive for JCV by microarray but tested negative by PCR. The Dengue 1 + sample was found to be positive for both Dengue-1 and Dengue 2 in the microarray analysis, but tested negative for types 2, 3 and 4 using Dengue subtype-specific PCR's.
To investigate the detection range of the microarray on clinical samples, we performed analysis on urine samples positive for the circular virus BKV (Table 3 and Figure 4 ). Samples ranging from 15,810 to 4.0610 8 copies/ml of BKV were amplified by Phi29 polymerase for 16 hours using Repli-g Midi kit (Qiagen). Also included were three dilutions of a BKV urine sample, containing 892, 119 or 73 copies/ml, respectively. Samples containing $1000 copies/ml of BKV (BKV1-BKV5) were efficiently amplified with yields from 1.0610 11 to 1.9610 8 copies/ml of BKV (Table 3 and Figure 4A ), and with ,25 mg of DNA generated after amplification (data not shown). The total input of viral copies per 50 ml Phi29-reaction for BKV1-BKV5, were 2610 6 , 4100, 2100, 80 and 5 genomic copies, respectively ( Table 3 ). Amplification of samples containing ,100 copies/ml (BKV6 and BKV7) resulted in loss of reproducibility and low yields (Table 3 and Figure 4A ). The total input of viral copies per Phi29-reaction for these two samples were 0.6 and 0.4, respectively (Table 3) . Theoretically, amplification of samples containing 200 copies/ml means that ,1 viral copy is added to the Phi29-amplification reaction, resulting in stochastic problems due to unequal distribution in highly diluted solutions. Four amplifications were selected for microarray analysis (BKV3, BKV4, BKV5 and BKV6) and hybridized to the LLMDA (Table 3 and Figure 4B ), and analysed using the data analysis method developed at SSI. BKV + urine samples containing $1000 copies/ ml were clearly detected by the microarray analysis after Phi29amplification ( Figure 4B ). The human genomic sequence SSX3 (synovial sarcoma, X breakpoint 3) was used as a hybridization control and had the same signal intensity in all 4 microarrays. Some cross-hybridization with JCV was seen in the 3 samples where BKV was detected, even if they all were negative in a JCVspecific PCR (data not shown). This is similar to the BKV sample presented in table 2. From this we conclude that, from as little as 5 copies input ($1000 copies/ml) of BKV genomes the Phi29amplification was able to generate enough material for a clear detection by microarray analysis.
By using WTA for viral amplification, both DNA and RNA viruses could be run through the same protocol. The correct DNA or RNA virus was detected in 14 out of 14 samples, representing a diverse set of clinical materials. Moreover, we can correctly detect multiple subtypes of a virus present in a sample, as seen for two clinical samples positive for multiple HPV subtypes (6/16/53/61 and 16/103, respectively), with no cross-hybridization towards other subtypes. Furthermore, EV-A (Coxsackievirus A6) was clearly detected without any cross-hybridization towards any other of the 6 species with several serotypes included in the large Enterovirus genus (EV-B/C/D and Rhinovirus A/B/C). It should be noted that subtype identification was not a goal in the probe design when developing LLMDA, nevertheless, the ability to combine the signals from multiple probes in the analysis made it possible to discriminate between different subtypes [19] . In several samples analysed, more than one virus was found, in some cases including both DNA and RNA viruses. Taken together, this demonstrates the potential and real life utility of the microarray technique for broad-spectrum pathogen detection in clinical samples.
In 4 samples we found viruses not previously tested for in the routine analysis, HPV103 in a HPV16 + cervical smear sample, Picobirnavirus in a Rota A + faeces sample, HAdV-C in a Sapovirus + faeces sample and GBV-C in a HCV + serum sample. Two of these were viruses within the same family as tested for before, HPV103 and GBV-C, while the other two were from different families, Picobirnavirus and HAdV-C. Picobirnavirus is a dsRNA virus (Picobirnaviridae) found together with the dsRNA virus Rota A (Reoviridae), while HAdV-C is a dsDNA virus (Adenoviridae) found together with the (+)ssRNA virus Sapovirus (Caliciviridae).
There was a false detection of virus in 3 samples, but they were all within the same family as previously tested for in the routine analysis, JCV in a BKV + urine sample, Rota C in a Rota A + faeces sample and Dengue 2 in a Dengue 1 + serum sample. They could all be explained by cross-reactivity between the probes targeting one species with other species in the same viral family. Future improvements to the current probe design can address crossreactivity issues such as these described. Regarding the failure to detect Sapovirus in the double-positive Astrovirus + /Sapovirus + faeces sample, we believe the reason was poor amplification due to low viral copy number rather than a specificity problem, since Sapovirus was detected in the Sapovirus + faeces sample ( Table 2) . Supporting this is the Sapovirus-specific PCR used before and after Phi29-amplification (Table 1) , which indicates that the singlepositive sample had more amplified Sapovirus (Ct = 11) after amplification, compared to the double-positive sample (Ct = 18) and thereby a sufficient amount for a successful detection by microarray analysis.
By microarray analysis, TTV and related viruses were detected in 77% of the samples, from 7 different types of clinical samples. We believe this is the first report regarding the prevalence of TTV and related viruses in clinical samples from Denmark. These TTV viruses are reported to frequently infect humans, with as much as 100% in certain populations, but no direct evidence links them to any specific clinical disease [31] . They have circular genomes (cir ssDNA) and are efficiently amplified by the Phi29 polymerase through RCA.
A microarray such as the LLMDA [19, 20] could be a useful tool in a number of ways. Since it enables simultaneous detection of viruses as well as bacteria, it could be useful in e.g. diagnosis of sexually transmitted diseases, or of respiratory illnesses with combined viral and bacterial components. Furthermore, many viruses cause symptoms that clinically are very similar, making it hard to choose the correct diagnostic analysis. In cases of clinical hypothesis failure, the microarray could be a useful tool for finding an etiologic agent in samples that are presumed negative or in samples with unknown etiology. Microarray could also play a dual role combining diagnostics and research, by being a suitable research tool for finding new pathogens. It could also be of great value for epidemiological surveillance, where clinically unimportant viruses or viruses with unknown consequence for the carrier (e.g. TTV, retroviruses or others) could be found, described and followed in both human and animal populations.
The protocol established here enabled us to get proof of concept that microarray analysis can be used to correctly identify viruses present in a diverse set of clinical samples. However, the protocol is at the moment to time and labour intensive to be used in a Figure 3 . Microarray analysis performed on WTA-amplified clinical samples. Results from the microarray data analysis of WTA-amplified clinical samples, using the maximum likelihood method developed at LLNL [19] , with additional stringency criteria applied. The lighter and darkercoloured portions of the bars represent the unconditional and conditional log-odds scores, respectively. The conditional log-odds scores shows the contribution from a target that cannot be explained by another, more likely target above it, while the unconditional score illustrates that some very similar targets share a number of probes. routine set up. We are currently focusing on reducing the overall assay time and the costs to develop a more suitable protocol for routine usage. Incubation times throughout the protocol might be shortened and some assays substituted or combined to make it both cheaper and faster to perform. Furthermore, the microarray design can be changed to multiplex, e.g. 12-plex as offered by the Roche NimbleGen system, instead of 1-plex as used now to increase the number of samples that can be run per slide. Future data analysis software needs to be customized for routine diagnostic analysis both regarding data handling and easy-to-use. The cost for the assay at the moment is in the hundreds of dollars per sample, but once established and refined as a technique, the cost is estimated to drop significantly but probably never to levels comparable to PCR. However, the benefit with this assay is that it replaces hundreds of PCR reactions with just one reaction, and often hospitals requests more than one PCR assay per patient in the search for a correct diagnose. Furthermore, since the microarray also contains probes for bacteria, it can combine both viral and bacterial diagnostics. Another benefit is that both DNA and RNA viruses can be analysed using the same protocol. In recent years WGA by MDA has been applied extensively, enabling researchers to get enough material from as little as a single cell for down-stream analysis [13, 32, 33] . Phi29 polymerase amplifies all accessible DNA present in a sample, including any exogenous DNA contamination. For viral or bacterial diagnostic purposes, where presence rather than abundance is important, the main concern is contaminating human gDNA that can compete with pathogen DNA. We show that our pre-treatment can reduce this impact from gDNA and thereby allowing for a greater amplification of the viral DNA. However, in some cases this is not enough due to large amounts of gDNA being present that can not be completely removed and this, combined with low viral copy number, might make certain types of clinical samples less suitable for efficient amplification and microarray analysis. This is currently under investigation. Our microarray analysis of BKV + urine samples indicated that as little as 5 genomic copies of a circular DNA genome could be enough to generate enough material by Phi29-amplification to enable successful identification by the LLMDA. Such a high sensitivity might not be achievable for other types of viral genomes, and is therefore under further investigation. Furthermore, to increase the chance of producing enough material from low copy number samples, we found that the WTA reaction should be allowed to run to completion (8 hours). The sample volume used for purification could also be increased.
Our results show that the protocol established for pre-treatment and amplification followed by microarray analysis is a useful method that potentially could optimize and simplify the current way of doing multiple analyses in a diagnostic laboratory. Supporting Information Table S1 Representative examples of the effect of DNase-treatment on virus before and after Phi29amplification.
(DOC)  B Cell Repertoire Analysis Identifies New Antigenic Domains on Glycoprotein B of Human Cytomegalovirus which Are Target of Neutralizing Antibodies Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133–343 of gB and domain II, a discontinuous domain, built from residues 121–132 and 344–438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization. Human cytomegalovirus (HCMV) is an important, ubiquitously occurring, human pathogen in immunocompromised hosts. The virus can cause severe disease in transplant recipients [1] . In large parts of the world HCMV is also the most common viral infection acquired in utero. In the USA and Europe an estimated 0.2%-1.2% of all live born infants are infected with HCMV [2, 3] . Congenital HCMV infection is a leading cause of sensorineural hearing loss in children and the leading infectious cause of central nervous system damage in children [4, 5] . As a consequence of the importance of congenital HCMV infection for public health, the Institute of Medicine of the National Academy of Sciences, USA, has ranked the development of a HCMV vaccine as a top priority [6] .
As with all successful antiviral vaccines, induction of an efficient antibody response will be crucial for the success of such a vaccine [7] . Importantly, an effective anti-HCMV vaccine will need to protect the vaccine from HCMV infection/disease as well as in the case of pregnant women, the developing fetus. Transfer of protective maternal antibodies to the fetus will be critical in this respect and a study of passive transfer of immunoglobulins to pregnant mothers has supported a role of antibodies in reducing the risk for congenital infection [8, 9] . Also, naturally acquired maternal immunity contributes to prevention of congenital HCMV infection [10] . Although correlates of protection from HCMV infection are poorly understood, it can be predicted that humoral immune responses to the envelope glycoproteins will be particularly important since antibodies directed against these antigens can neutralize virus infectivity directly and/or induce immunoglobulin Fc-receptor mediated effector functions such as antibody dependent cytotoxicity and/or complement mediated effects which can lead to elimination of infected cells [11] .
HCMV is a highly complex virus harboring more than 20 different glycoproteins in its envelope [12, 13] . With respect to induction of neutralizing antibodies during natural infection, the glycoprotein (g) B dominates, but additional antigens such as the gM/gN complex and the gH/gL complex have also been identified as highly immunogenic [14] [15] [16] . Antibodies directed against gB can be detected in all naturally infected individuals [17] . Moreover, a major fraction of neutralizing antibodies in human sera seems to be directed against gB and the overall neutralizing capacity in sera from HCMV-seropositive donors correlates with anti-gB antibody titer [18] . In addition, anti-gB antibodies are effective in preventing cell-to-cell spread [19] . In the guinea pig CMV model, immunization with gB DNA vaccines confers protection from infection [20] . In a recent study protection from brain pathology in murine cytomegalovirus (MCMV) infected mice was accomplished by passive transfer of a gBspecific monoclonal antibody (mab) [21] . Thus, gB is an attractive antigen for inclusion in a human vaccine and has been part of a number of experimental vaccines [22, 23] . In fact, a recent phase 2 trial using recombinant gB as vaccine has shown significant protection from infection [24] . However, the spectrum of anti-gB antibodies developing during infection remains poorly defined.
HCMV gB is an essential viral protein which is involved in the early events of infection. It has been shown to bind to a variety of cell surface molecules such as heparan sulfate proteoglycans, integrin heterodimers and platelet-derived growth factor-a receptor [25] [26] [27] . In addition, HCMV gB has been shown to mediate fusion of viral and cellular membranes [28] [29] [30] . The protein is essential for viral entry and cell-to-cell spread but not for virion attachment, assembly or egress [31] . Three antigenic domains (AD) have been described previously. AD-1 consists of approximately 80aa between positions 560 and 640 of gB of HCMV strain AD169 [32] . It is the immunodominant region of gB since nearly all sera from HCMV-infected individuals recognize AD-1 [33] . Antibodies that bind to AD-1 can have virus neutralizing capacity as indicated by the fact that a number of AD-1-specific human mabs have been isolated which show various degrees of neutralizing activity [34, 35] . Polyclonal AD-1-specific antibodies, purified from human serum, are incapable of completely neutralizing HCMV even at high concentration, indicating that AD-1 is bound by antibodies with widely differing neutralizing activity [36] . It has been suggested that the competitive binding of neutralizing and non-neutralizing antibodies to AD-1 may represent a mechanism to evade efficient neutralization of cell free virus [36] .
AD-2, located at the extreme amino terminus of the protein, consists of at least two distinct sites between aa 50 and 77 of gB. Site I is common to all HCMV strains and induces neutralizing antibodies, whereas the aa sequence of site II differs between strains and is recognized by strain specific antibodies which are incapable of neutralization in vitro [37] . The overall immunogenicity of AD-2 is lower than that of AD-1 since only about 50% of human sera from HCMV-infected donors have antibodies against this determinant [17] . An additional linear aa sequence, AD-3, recognized by gB-specific antibodies in human sera includes epitopes at the intraluminal/intraviral part of the molecule [38, 39] . However, antibodies binding to these determinants are non-neutralizing as can be expected from the localization of AD-3 within the molecule. Additional protein domains which are bound by murine mabs have been identified, but whether these regions are relevant in the context of antibody response during natural infection is unknown [40] . The gB-specific human monoclonal antibodies for which the binding sites has been identified react with either AD-1 or AD-2 [35, 41] .
Overall, there are significant gaps in our knowledge of antibody epitopes on gB. Given the size of the gB protein it seems highly likely that additional antigenic domains exist on gB. Defining these sites will not only provide an antigenic map of this important protein, it will also be helpful for monitoring the response to vaccination for production of antibodies with binding profiles similar to natural infection.
We comprehensively analyzed the human antibody repertoire against gB as it is developed during infection. To this end we isolated gB-specific memory B cells from different healthy HCMVseropositive donors and activated the cells at the clonal level to immunoglobulin production. The produced antibodies were tested for reactivity with gB and parts thereof and in in vitro neutralization assays. Our results revealed that most of the anti-gB antibodies produced during infection failed to neutralize cell-free virus. In addition, and perhaps more importantly, we find that the vast majority of anti-gB antibodies with potent neutralizing capacity recognize two protein domains which have not been identified previously as target sites.
The antibody repertoire against gB is dominated by antibodies that do not neutralize virus and those which bind to unknown protein domains We intended to comprehensively analyze the human IgG anti-gB memory B-lymphocyte repertoire established by healthy HCMV infected individuals in terms of epitope specificity as well as neutralizing capacity. To this end we used the complete extraviral part of gB, as it is used in vaccination trials [24] , for sorting of IgG positive memory B-lymphocytes (CD19 + /CD27 + ) binding to fluorochrome-labeled gB by flow cytometry (Fig. S1A) . In a first set of experiments we analyzed the possibility to identify
The development of antibodies is a major defense mechanism against viruses. Understanding the repertoire of antiviral antibodies induced during infection is a necessary prerequisite to defining the protective activities of an antiviral antibody response. The isolation of antigen specific memory B cells and subsequent stimulation to antibody producing cells provides a powerful tool to study the antibody repertoire in infected individuals. We have used this approach to analyze the antibody repertoire against glycoprotein B (gB) of human cytomegalovirus (HCMV), a major antigen for the induction of antiviral antibodies during infection and a constituent of experimental vaccines in humans. We find in different infected individuals that the vast majority of gB-specific B cells produce antibodies that cannot neutralize free virus. Antibodies with antiviral capacity target two domains of gB that have not been previously identified. The identification of these new antigenic domains was possible with the aid of a 3D molecular model of HCMV gB. Our results will be useful for vaccine development since comparison of the immune response after natural infection with that induced by vaccination can be readily accomplished. Moreover, neutralizing human monoclonal antibodies could constitute powerful therapeutics to combat the infection in populations at risk for HCMV disease.
gB-specific memory B cells in 15 seropositive individuals. Frequencies of gB-binding, IgG-positive memory B cells among all IgG-positive memory B cells ranged from 0.33 to 1.4%, being in the range of frequencies of IgG memory B-lymphocytes against other viral antigens [42] (Fig. S1B ). Among HCMV seronegative individuals, gB-binding memory B cells were detectable but with considerably lower frequency. These cells might bind gB unspecifically or may represent part of the natural antibody repertoire [43] . Sorted gB-binding B cells were activated and immortalized at the clonal level by an in vitro culture system using CpG oligonucleotides and EBV [44] . Among clonal cultures with IgG secretion 40-95% (mean 63%) of cultures showed IgG binding to gB in ELISA, substantiating a high degree of specificity in the cell sorting process (data not shown).
Seven donors were selected for further analysis. The anti-gB antibody titer was comparable in this group (Fig. S2A ) while the neutralization titer varied significantly, which is not uncommon for HCMV-infected individuals (Fig. S2B ). From these 7 donors we were able to analyze a total of 888 clonal IgG gB-binding culture supernatants for epitope specificity towards the well-known antigenic domains AD-1 and AD-2 [45] as well as neutralizing capacity against HCMV AD169 on fibroblasts. With respect to binding of gB protein domains we found a high frequency of antibodies binding to the AD-1 epitope (mean 38.1%) in all individuals correlating with earlier findings that up to 50% of gBspecific IgG in sera might be directed against the AD-1 epitope (Table 1 ) [38] . Interestingly, only few of the AD-1-specific antibodies were able to neutralize HCMV in vitro (mean 2.0%, range 0-6%; Table 1 ). The frequency of clones producing AD-2specific IgG was low (Table 1 ) and in only 3 out of 7 individuals were we able to retrieve AD-2-specific memory B cells, correlating with earlier data that this specificity is found only in approximately 50% of HCMV infected individuals [17] . None of the rare AD-2specific antibodies was neutralizing in vitro. A high frequency of clones from all individuals did not react with either AD-1 or AD-2 (range 40-86%; Table 1 ). Importantly, among these antibodies a significant number was able to neutralize HCMV in vitro (17% out of 429 clones; Table 1 ). A summary of the neutralizing capacity of the gB-specific B-cell supernatants of 5 donors from which we were able to isolate B cells that secreted neutralizing antibodies is shown in Fig. S2C . We conclude from this analysis that the memory B-cell repertoire against gB is dominated by antibodies that do not neutralize the virus and that most neutralizing antibodies bind to a so far unknown antigenic site.
Neutralizing human monoclonal antibodies bind to a region between residues 100-448 of gB Next, we attempted to map the target sites of antibodies that did not react with the known antigenic sites on gB. To obtain a consistent supply of mabs, the Ig-genes from 10 selected B-cell clones that secreted neutralizing antibodies unreactive with AD-1 and AD-2 were cloned and expressed in recombinant systems as IgG1 molecules (Table 2) . We choose the IgG1 subtype since 8 of the 10 selected B-cell clones secreted IgG1, whereas 2 secreted IgG3. The recombinant antibodies showed comparable neutralizing activity to the B-cell supernatants when tested on fibroblasts as target cells indicating that the Fc-part of the IgG is not significantly contributing to the in vitro neutralizing activity of the antibodies (Fig. 1 ). 50% neutralization was achieved at concentrations of 0.2-1.3 mg/ml (Table 2) . Importantly, the recombinant antibodies were also able to neutralize virus on endothelial, epithelial and dendritic cells with comparable activities. Representative results for Group A mabs (see next paragraph) on endothelial cells and Group B mabs on epithelial cells are shown in Fig. 1 and the data for all mabs are summarized in Table 2 .
The fact that all tested antibodies were not reactive in western blot analyses using extracellular HCMV particles as antigen indicated that the intact three-dimensional protein conformation was crucial for binding (data not shown). Therefore, we used indirect immunofluorescence of transiently expressed fragments of gB in Cos7 cells to obtain further information on the binding sites of the mabs. Two distinct reactivity patterns were observed for the mabs (Group A and Group B in Fig. 2 ). Full length gB 1-906 and fragments as short as gB 1-447 were reactive with the entire set of Table 1 . Epitope specificity and neutralizing activity of IgG HCMV-gB specific antibodies. (Fig. 2 ). For Group A mabs gB residues 100-342 were sufficient for binding whereas for Group B antibodies a larger fragment of gB comprising residues 100-448 was required for binding, indicating that the gB region between aa 100-448 harbors at least two distinguishable antibody target sites.
The generation of a structure model of gB reveals two globular protein domains within the antibody binding region of gB
In order to obtain further information on potential structural domains of gB in the region between aa 100-448, we generated a three dimensional model of the trimeric conformation of the ectodomain of HCMV gB strain AD169 based on the crystal structure of HSV-1 gB [46] (Fig. 3) . According to the model, both the overall structure of the HCMV gB monomer and the organization of the subunits in the trimer were highly similar to that of HSV-1 gB, as expected from the degree of sequence similarity of gB between human herpesviruses (28% identity and 40% similarity between HSV-1 gB and HCMV strain AD169 gB). The individual domains I to V, which were previously defined based on the HSV-1 gB structure, can be clearly identified from the homology model of HCMV gB. With respect to potential antibody binding structures, the protein domains (Dom) I and II were of particular interest since they are located within aa 100-448 of gB. Dom I (Ile 133 to Thr 343 ) constitutes part of the trimer interface and is located proximal to the membrane, potentially containing the fusion domain [47] . The discontinuous Dom II is composed of two separate segments comprising residues Leu 121 to Asn 132 and Cys 344 to Ser 438 (Fig. 3) . Either domain contains a single disulfide bond which helps to stabilize the conformation of the respective protein domain [48] .
To investigate Dom I and Dom II for antibody binding, expression plasmids were constructed which allowed for the synthesis of either domain in eukaryotic cells. In both cases the cloning strategy involved the attachment of a HA epitope tag at the amino terminus of the respective peptide in order to facilitate detection. Dom I comprised aa 133-343 of gB. The Dom IIspecific peptide consisted of the gB-specific residues 112-132 and 344-438 joined by a 5 aa (Ile-Ala-Gly-Ser-Gly) synthetic linker sequence. Following transient expression of the peptides in Cos7 cells, antibody binding was analyzed by indirect immunofluorescence. All four antibodies from Group A were reactive with the Dom I-specific peptide, whereas all Group B mabs recognized Dom II ( Fig. 4 and Table 2 ). The crystal structure of HSV-1 gB as well as the model of HCMV gB predict that for Dom I and Dom II discontinuous sequence stretches are essential for formation of either domain. Shorter protein fragments would be expected not to fold correctly. We tested this assumption by expressing a Dom II variant with five amino acids deleted at the carboxyl-terminus and observed complete loss of binding of all Group A-specific antibodies. Likewise, the continuous part of Dom I (residues 140-255) showed no antibody binding capacity (data not shown). Thus, further resolution of antibody epitopes will be possible only by generation of point mutants in Dom I and Dom II, respectively. Having identified Dom I and Dom II as new targets for neutralizing antibodies, we re-tested clonal antibody supernatants from 4 individuals to obtain information about the overall frequency of Dom I-and Dom II-specific antibodies in HCMV infected individuals. As shown in Table 1 , the frequency of Dom I and Dom II specific memory B cells was variable among different donors and in most cases considerably lower as compared to AD-1-specific B cells.
In summary, the repertoire analysis revealed that most anti-gB IgG antibodies derived from memory B cells are non-neutralizing. Among those antibodies that neutralized HCMV in vitro, most antibodies reacted against two previously uncharacterized regions of the gB protein. Addition of complement had no influence on the neutralizing capacity of the recombinant antibodies. Moreover, when a selected set (n = 10) of non-neutralizing antibodies directed against different antigenic domains of gB was tested in concentrations up to 5 mg/ml in the presence of complement we observed no significant increase in neutralization capacity which is in agreement with previous reports on gB-specific human mabs (data not shown) [35] . However, we cannot completely rule out a moderate enhancing effect of complement for some antibodies, especially those of the IgG3 subtype.
Dom I and Dom II mabs function during a postadorption step of the infection gB has been postulated to be involved in receptor binding of HCMV and entry [26, 49, 50] . We therefore analyzed at which stage of the infection the mabs exerted their action. To assay influence on attachment, virus/antibody mixtures were added to target cells at 4uC and the number of HCMV DNA copies attached to the cells was determined by quantitative real time PCR. Neither Dom I-nor Dom II-specific antibodies inhibited attachment of virions, indicating that the mabs did not block receptor binding of HCMV (Fig. 5A ). In accordance with previous reports, heparin almost completely prevented virus attachment, whereas the AD-2-specific antibody C23 had no effect on virus binding [27, 51] . The slight increase of bound virus in the presence of some antibodies as compared to control might reflect deposition of antibody/virus aggregates on the surface of cells. We also determined activity of the mabs towards virus that is already adsorbed to cells. To this end, virus was preadsorbed to cells for 1 h at 4uC before antibody was added. Both Dom I-and Dom IIspecific antibodies were capable of neutralizing HCMV at a postadsorption step, whereas non-neutralizing antibodies had no effect (Fig. 5B) . The higher antibody concentration that was required to completely neutralize adsorbed virus is explained by the need to block fusion of an already adsorbed virus particle.
Competitive binding of neutralizing and non-neutralizing human mabs has been described for gB [35] . We tested whether a similar phenomenon occurs for Dom I-or Dom II-specific neutralizing antibodies. A total of 14 non-neutralizing mabs, directed against different antigenic regions of gB was analyzed in neutralization assays for competition with Dom I-or Dom IIspecific mabs (SM10, 1G2, SM5-1). In no case did we observe a reduction in neutralizing capacity by addition of non-neutralizing mabs. The result of a representative analysis is shown in Fig. 6A . Although these data indicated that reduction of neutralizing activity by competing non-neutralizing mabs is not common for Dom I-and Dom II-specific antibodies, it is difficult to assess the relevance of this finding for the situation in human sera. For AD-1specific antibodies it has clearly been shown that the sum of antibodies, as present in human sera, is not capable of completely neutralizing infectious virus, indicating that non-neutralizing antibodies can constitute a significant fraction of the entire AD-1-specific IgG fraction [36] . To obtain more information on the neutralizing capacity of Dom II-specific antibodies in human sera, we purified Dom II as a GST fusion protein following expression in E. coli (Fig. S3A) . The bacterially derived peptide retained the capacity to bind all Group B mabs and the affinity of mab SM5-1 was similar for gB and Dom II (Fig. S3B ). Circular dichroism spectra indicated a homogeneous three-dimensional structure (Fig.  S3C ). Using this protein, polyclonal Dom II-specific antibodies were affinity-purified from pooled human sera (Fig. S4) . The affinity-purified IgG preparation was then tested in neutralization assays. 50% neutralization of input viral infectivity was achieved with an IgG concentration of approximately 0.2 mg/ml which is within the same concentration range as the Dom II-specific mabs (Fig. 6B) . In summary, these data provided evidence that gB Dom II not only represents an immunogenic domain on gB, but also that antibodies binding to it in general have potent neutralizing capacity. Similar experiments using Dom I could not be carried out since the Dom I domain does not fold correctly after prokaryotic expression and thus antigen for affinity purification of antibodies could not be generated.
Previous analyses of human sera for recognition of gB domains have revealed differential rates of antibody reactivity for the individual antigenic domains. Whereas almost 100% of infected individuals develop antibodies against AD-1, only 50% show reactivity against AD-2 [17] . To obtain information on the frequency of recognition of Dom I and Dom II we determined antibody reactivity against these domains in a larger serum panel of HCMV infected individuals and compared it to the known antigenic domains. A total of 80 randomly selected sera from HCMV seropositive individuals, as determined by a commercially available ELISA test, was analyzed. Ten sera from HCMV negative donors served as controls. Within the serum panel from HCMV-seropositive individuals, reactivity for gB was 100%, underscoring the antigenicity of this protein (Fig. 7) . In accordance with our previous observations, positive reaction with AD-1 was also 100% and 57% of the sera contained antibodies against AD-2 [17] . Dom I was recognized by 55% whereas 94% of the specimens reacted with Dom II. Thus, Dom I and Dom II represent antigenic domains on gB which induce antibodies with high frequency during infection. For the sake of consistency in nomenclature of gB antigenic domains, they were designated AD-4 (Dom II) and AD-5 (Dom I).
We have used recombinant gB to analyze the antibody repertoire derived from activated memory B cells of healthy HCMV seropositive individuals. The donors were not selected for hight titers of anti-gB or neutralizing antibodies against HCMV in order to obtain an unbiased result. Our results reveal a number of new findings with respect to the anti-gB response in humans.
The vast majority of anti-gB antibodies did not show antiviral activity in in vitro assays. Among the seven donors that were tested comprehensively, the percentage of neutralizing antibodies among the gB binders ranged from 0% to 11% (mean 3%). Previous studies using adsorption of antibodies to gB have noted that in some individuals the overall neutralization capacity cannot be reduced, indicating that in these cases non-gB specific antibodies are major components of the neutralizing antibody response [18] . Repertoire analysis in other viral systems have also noticed an excess of binding versus neutralizing antibodies [44, 52, 53] .
At this time we can only speculate on a potential role of the nonneutralizing antibodies on the infection in vivo. Besides being irrelevant for the infection, non-neutralizing antibodies might have positive or negative effects. Effector functions mediated via the Fcportion of the antibodies such as ADCC and/or complement fixation may contribute to elimination of infected cells and thus could lead to enhanced clearance of these cells. That such mechanisms are operative in vivo has been shown in different viral systems, including herpesviruses, but not for HCMV [54, 55] . On the other hand, infection-enhancing effects could be contributed by mechanisms such as competition of non-neutralizing antibodies for binding to neutralizing epitopes or enhanced infection of Fcreceptor bearing cells such as monocytes, which is one of the major target cell population for HCMV infection in vivo [56] .
Whereas all anti-gB mabs recognized mammalian cell expressed gB aa 1-687, only 40% to 50% of anti-gB antibodies were reactive with a shorter fragment expressing aa 1-447 indicating the presence of epitopes in gB between aa 448 and 687. These antibodies were negative for recognition of the bacterial fusion protein containing AD-1 (aa 484-650). Thus, the location of the antibody binding site(s) remains unknown. There is the possibility that residues 447-484 and 650-687 contain additional epitopes since they are not represented by the AD-1 fusion peptide. However, this seems unlikely. The more plausible explanation for our finding is that the region between aa 447-687 contains additional conformational epitopes that are not formed in the bacterial fusion protein. Our previous analyses have shown that AD-1 induces a multitude of antibodies with different binding characteristics, even when assayed as bacterial fusion proteins or synthetic peptides [33] . Thus, it would be not surprising that this region of gB contains additional epitopes which are present only on the native protein. The large fraction of antibodies falling in this category also supports this possibility. A corresponding protein region of HSV-1 gB was found to contain a ''pseudocontinuous'' epitope, further supporting our interpretation [57] . However, no matter what the underlying mechanism of this finding is, antibodies binding within the 447-687 region of gB were nonneutralizing.
Antibodies which show potent in vitro neutralizing activity were found to bind to two previously unknown protein domains, namely AD-4 and AD-5. Because this was demonstrated in most donors, it is unlikely a sampling artifact. The antigenicity of these domains is also indicated by the fact that in randomly selected serum samples from HCMV-seropositive donors we found positivity rates of .90% for AD-4 and .50% for AD-5, which identified both domains as strongly antigenic structures on gB. Comparable positivity rates among sera from HCMV infected individuals have been reported for AD-1 and AD-2, respectively, and were confirmed in the current analysis [17] . A distinct difference between AD-4/AD-5 and AD-1/AD-2 is the functional antiviral activity of the domain specific antibodies. AD-1 is bound by virus neutralizing and non-neutralizing monoclonal antibodies which can compete for binding to the domain [35, 58] . Affinity purified AD-1-specific IgG fractions from pooled human sera were shown to have neutralizing capacity not exceeding 50%, indicating that during natural infection a considerable proportion of competing non-neutralizing antibodies are induced [36] . The incomplete neutralizing capacity of polyclonal anti-AD-1 antibodies has been suggested to provide the virus with an effective mechanism to evade the humoral immune response. Likewise, AD-2 harbors two different antibody binding sites which are bound by neutralizing and non-neutralizing antibodies, respectively [37] . The situation seems to be different for AD-4 and AD-5 in that almost all human monoclonal antibodies that have been obtained so far have potent virus neutralizing activity. Moreover, the affinity-purified AD-4specific polyclonal human IgG fraction had a neutralizing titer that was comparable to the monoclonal antibodies with 50% neutralization in the low nanomolar range. Thus, it seems unlikely that AD-4 induces significant concentrations of non-neutralizing antibodies that compete with neutralizing antibodies for binding to the domain. Whether this correlation also holds true for AD-5 needs to be determined in further studies.
AD-4-and AD-5-specific mabs did not prevent virus attachment to fibroblasts indicating that neither type of antibody interferes with receptor binding of HCMV. However, they were capable of neutralizing infectious virus at a postadsorption step. In the case of HSV-1 it has been shown that neutralizing anti-gB murine mabs have different effector mechanisms. Antibodies that bind to the HSV-gB domain IV (corresponding to AD-1 in HCMV gB) block fusion of viral and cellular membranes but do not interfere with interaction of gB with gH/gL, the second constituent of the minimal herpesviral fusion complex. Murine mabs specific for the HSV-gB domains I and II (corresponding to AD-4 and AD-5 in HCMV gB) block interaction of gB with gH/ gL and thereby inhibit fusion [59, 60] . Whether similar effector mechanisms apply for HCMV needs to be determined in further studies.
HCMV isolates from clinical samples exhibit extensive genetic variation and HCMV reinfections have been demonstrated to occur in seropositive individuals. In immunocompromised hosts, when the development of a de novo humoral immune response is impaired, reinfection with a different HCMV isolate might result in clinical symptoms, due to unrestricted replication of the 'new' virus strain [61] [62] [63] [64] [65] . However, we have no information on whether reinfection of seropositive individuals is inevitable upon contact with a different HCMV strain or whether some infected hosts develop an immune response which prevents reinfection. It will be important to analyze whether such ''absolute controllers'' exist and what the correlate of protection is. In the HIV field it has clearly been shown, that a small fraction of infected individuals can develop broadly neutralizing antibodies which control the viral mutants that develop during infection and prevent progression to disease [53, 66] . In the case of HCMV, it will be interesting to determine whether production of antibodies against the individual antigenic domains of gB or, for that matter additional envelope glycoproteins, correlates with reduced susceptibility to reinfection. Of note, the gB protein region harboring AD-4 and AD-5 is highly conserved between different HCMV isolates (Fig. S5) and is situated well outside the polymorphic protein segments that have been defined [67] .
In summary, we have investigated the human antibody repertoire against gB using a recombinant protein that is currently used in vaccine trials. Our data reveal new antigenic sites on the protein, which are immunogenic during infection and, more importantly, target of potent antiviral antibodies. It will be interesting to compare our findings to the B-cell repertoire against gB as it is produced during infection since this may enable us to draw conclusions about the structural integrity of the gB vaccine antigen. Improving our understanding of the antigenic map of gB will be of value in the rational design of future vaccine antigens. Lastly, human mabs with potent and broadly neutralizing activity might be useful as biologicals in prophylaxis and/or therapy of HCMV infections.
Ethics approval for the sample collection has been obtained from the Ethics Committee of the Medical Faculty of the Friedrich-Alexander Universitä t Erlangen-Nürnberg. Written informed consent was obtained from all donors.
African green monkey kidney cells (Cos7) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Germany) supplemented with 10% fetal calf serum (FCS), glutamine (100 mg/l) and gentamicin (350 mg/l). HCMV was propagated in human fetal lung fibroblasts (MRC-5) or human foreskin fibroblasts (HFF) grown in DMEM supplemented with 10% FCS, glutamine and gentamicin as above. For immunofluorescence cells were grown on 13-mm glass coverslips in 24-well plates. HB15-UL84prluc represents a recombinant AD169-based HCMV which expresses the firefly luciferase gene under the control of the HCMV UL84 promoter.
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy, HCMV-positive volunteers using Ficoll-density gradient centrifugation. After B-cell enrichment using anti-human CD22-microbeads (Mitenyi Biotec, Bergisch Gladbach, Germany), B cells were labeled with the following reagents: 1. Anti-human CD19-PerCP (Dianova, Germany); 2. Anti-human CD27-PE (BD Bioscience Pharmigen, Switzerland); 3. anti-human IgG-FITC (Dianova, Germany); 4. Cy5-labeled glycoprotein B (Sanofi Pasteur, 100 ng per 1610 6 B cells). The gB protein was labeled with Cy5 using the FluoroLink-Ab Cy5 labelling kit (Amersham Pharmacia Biotech, Germany). gB-specific, IgG-positive memory B cells were either analyzed using FACSCalibur (Becton Dickinson, Germany) or isolated by sorting cells that fulfilled the criteria PerCP+/PE+/FITC+ and Cy5+. Cells were sorted at 5 cells/well, in 96 F-bottom microplates containing a confluent layer of irradiated feeder cells (HFF), using a MoFlo cell sorter (Cytomation, Germany). Sorted cells were grown in complete RPMI-1640 medium supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-mercaptoethanol and 10% FCS (heatinactivated) (PAN-Biotech, Germany) in the presence of EBV and CpG ODN 2006 as previously described [44] . After 3 weeks, the culture supernatants were screened for antigen recognition and/ or virus neutralization. Supernatants were classified as neutralizing if at a 1:1 dilution the neutralization capacity exceeded 70% of input virus.
To produce selected recombinant human monoclonal antibodies the Ig heavy and corresponding light chains were amplified by RT-PCR from clonally expanded activated memory B cells and cloned into eukaryotic expression vectors exactly as described by Tiller et al. [68] . The respective cloning vectors were kindly provided by H. Wardemann, Berlin. V gene usage and CDR sequences are supplied in Table S1 .
Fibroblasts (1610 4 ) in a volume of 100 ml were seeded in 96well plates. HB15-UL84prluc (275 pfu) was preincubated with serial log 2 dilutions of complement inactivated serum or mab in a volume of 50 ml in culture medium for 1 h at 37uC. The mixture was added to fibroblasts for 4 h. The inoculum was removed and the cells were incubated at 37uC for 48 h. Cells were lysed using 100 ml Glo Lysis Buffer (Promega, USA) per well. 30 ml of each cell lysate was placed in white 96-well LIA-plates. Per well, 50 ml assay buffer (15 mM KH 2 PO 4 , 25 mM glycylglycine, 1 M MgSO 4 , 0.5 M EGTA, 5 mM ATP, 1 mM DTT) was added. Luciferase activity was measured by injection of 50 ml D-luciferin (P.J.K., Germany) solution per well (in 25 mM glycylglycine, 1 M MgSO 4 , 0.5 M EGTA, 2 mM DTT and 0.05 mM D-luciferin) and detection of chemiluminescence was performed by an Orion Microplate Luminometer (Berthold Technologies, Germany). Neutralization assays using endothelial and epithelial cells were performed using the HCMV isolate TB40E as described [69] . The influence of complement on neutralization capacity of monoclonal antibodies was tested by inclusion of 5% rabbit complement (Cedarlane Labs, Canada) in the neutralization assay.
Fibroblasts were seeded at 3610 4 cells per well in 96-well plates. HB15-UL84prluc was preincubated with individual mabs for 1 h at 37uC at concentrations ensuring complete neutralization. Cells and the virus/mab mixture were cooled to 4uC and the virus/mab mixture was added to the cells at a multiplicity of infection (m.o.i.) of 0.5. Following incubation for 1h at 4uC, cells were washed three times with ice-cold PBS and cell lysates were prepared by freezing/thawing. DNA was extracted from the lysates using a MagNA Pure LC (Roche, Germany) instrument and quantitative real-time PCR was performed on an ABI PRISM 7500. To control for recovery of cells, copy numbers of albumine DNA was determined in parallel to HCMV and HCMV copies were calculated per 1000 copies albumine. Primers : CMV 59:GAG-CAGACTCTCAGAGGATCGG; CMV 59: AAGCGGCCTCT-GATAACCAAG; Albumine 59: GTGAACAGGCGACCATG-CT; Albumine 39: GCATGGAAGGTGAATGTTTCAG. For the penetration assay, precooled cells were preincubated with virus at a m.o.i. of 0.2 for 1 h at 4uC, washed twice with ice-cold PBS and incubated for 2 h at 37uC with log 10 dilutions of mabs. After incubation, remaining mabs were removed by washing twice with PBS before cells were incubated for 48 h at 37uC. Subsequent steps were carried out according to the virus neutralization assay. Chemiluminescence of virus only was set to 100%.
Construction of the expression plasmid coding for complete gB has been described previously [32] . Carboxyterminal truncated forms of gB were expressed using pcDNA3 as plasmid. Aminoterminal truncations were expressed using the vector pcUL132sigHA. This pcDNA3.1 based plasmid contains the coding sequence of the HCMV gpUL132 authentic signal sequence aa 1-27, followed by the coding sequence for the influenza hemagglutinin (HA) epitope YPYDVPDYA [70] . To express Dom II (AD-4), the nucleotide sequence coding for aa 112-132 and 344-438 was chemically synthesized by GeneArt, Germany. The two parts were joined by a nucleotide linker coding for the sequence Ile-Ala-Gly-Ser-Gly and inserted in pcUL132-sigHA to give rise to pcAD-4. To express Dom I (pcAD-5) the nucleotide sequence coding for aa 133-343 were inserted into pcUL132sigHA. To generate plasmids for expression of AD-4-GST (Glutathion-S-transferase) fusion proteins in E. coli we used the expression vector pGEX-6P-1 (Pharmacia Biotech, Germany).
Cos7 cells grown on glass coverslips in 24-well plates were transfected with 0.8 mg of plasmid DNA using Lipofectamine (Invitrogen, Germany). 48 hours after transfection the cells were fixed and permeabilized with ice cold methanol. Primary antibodies were then added. Unbound primary antibody was removed by three washing steps using PBS. Binding of the primary antibody was detected with the appropriate secondary antibody conjugated with FITC (fluorescein isothiocyanate) (Dako, Germany). Counterstaining of cell nuclei was done with DAPI (4',6diamidino-2-phenylindole). Images were collected using a Zeiss Axioplan 2 fluorescence microscope fitted with a Visitron Systems charge-coupled device camera (Puchheim, Germany). Images were processed using MetaView software and Adobe Photoshop. Antibodies: gB-specific human mab C23 (TI-23) [41] , gN-specific murine mab 14-16A [71] , gH-specific murine mab SA4 [72] , murine anti-HA (Sigma Aldrich, Germany) and murine anti-GST (BIOZOL, Germany).
Plasmid DNA was used to transform E. coli DH10B for expression of GST fusion proteins. The respective fusion proteins were induced and the soluble form of the protein was purified from E. coli lysates according to the manufacturer's instructions. To prepare an affinity matrix, 2.6 mg of purified AD-4-GST fusion protein was dialysed against coupling buffer and conjugated to AminoLink Plus Coupling Resin (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Four ml of a HCMV hyperimmune globuline preparation, diluted 1:3 (v/v) with PBS, was passed over 2 ml antigen-coupled beads, followed by extensive washing with PBS. Bound IgG was eluted with 0.2 M Glycin-HCl, pH 3.0, in 1 ml fractions and fractions were dialysed against PBS. Total IgG concentration was determined by an ELISA. In brief, polystyrene 96-well plates were coated with 100 ng AffiniPure goat anti-human IgG, Fcc-specific (Jackson Immuno Research, USA) in 0.5 M carbonate buffer, pH 9.6, overnight at 4uC. Serial log 2 dilutions of the eluted fractions in a volume of 50 ml were added and bound IgG was detected by using a polyclonal peroxidase-conjugated goat F(ab) 2 -fragment antihuman IgG, Fcc-specific (Jackson Immuno Research, USA). A human IgG preparation (Jackson Immuno Research, USA) with known concentration was used as standard.
The following gB-specific antigens were used: gB, AD-1, containing aa 484-650 of gB, AD-2, containing aa 68-80 and AD-4-GST. Proteins were diluted between 25 ng and 200 ng (depending on antigen) in 0.5 M sodium carbonate buffer, pH 9.6, or in 6 M urea (AD-1) and 50 ml was used to coat microtiter plates overnight at 4uC. All subsequent steps were carried out at room temperature. Reaction wells were rinsed with PBS supplemented with 0.1% Tween 20 and blocked for 2 h with PBS containing 2% FCS. Plates were again rinsed with PBS supplemented with 0.1% Tween 20 and incubated with mabs, human serum or polyclonal eluted antibody fractions (50 ml/well) for 2 h. Unbound antibody was removed by washing and peroxidase-conjugated anti-human or anti-mouse IgG (Dako, Germany) was added at an appropriate dilution for 1 h. The plate was washed and 100 ml TMB (tetramethylbenzidine) peroxidase substrate, diluted 1:1 in peroxidase substrate solution B (KPL, USA), was added for 5 min. The reaction was stopped by the addition of 100 ml 1 M H 3 PO 4 and the OD 450 was determined using Emax microplate reader (Eurofins MWG Operon, Germany). Dilution of all antibodies was done in PBS with 2% FCS. In all assays involving gB fusion proteins, the respective prokaryotic fusion partner was assayed in parallel and the optical density subtracted from values obtained with the gB fusion protein. AD-5-specific antibodies in human sera were measured in a capture ELISA. 96 well plates were coated with 125 ng/well of an anti-HA monoclonal antibody (Sigma, Germany) and blocked for 2 h with PBS containing 2% FCS. The plates were incubated with culture supernatant from cells that had been transfected with pcAD-5 six days before. Plates were rinsed and incubated with human sera in a 1:50 dilution for 2 h at 37uC. The plates were washed and developed as described above.
The model of the HCMV gB structure was generated by standard homology modelling procedures using the program MODELLER [73] , based on a sequence alignment with the template structure of HSV-1 gB [46] . Two loop regions (Val 306 to Glu 317 and Leu 439 to His 468 of HCMV gB) were not resolved in the reference structure and could therefore not be modelled. All images were generated with Accelrys DS Visualizer v2.0.1. The quality of the model was validated using ProSA [74] and PROCHECK [75] . ProSA analysis reveals that the overall model quality (Z-score = 26.22) is quite similar to that of the template crystal structure (Z-score = 27.84). Both values are within the range of z-scores typically found for crystal structures of proteins of similar size. In addition, the residue energy profiles of template and target structure are very similar indicating that the modelling did not place amino acids in an unfavourable environment. In addition, analysis of the backbone geometry shows that that 87.5% of all residues of HCMV gB are located in the most favourable regions of the Ramachandran Plot. This value corresponds to a crystal structure with 2.0-2.5 Å resolution. The HCMV gB model will be made available by the authors upon request.
GenBank accession numbers for the individual heavy and light chain nucleotide sequences of the recombinantly expressed IgG molecules are JF806449-JF806467. The protein was expressed and purified as described in Material and Methods. (B) Kinetic data for binding of SM5-1 to gB and Dom II-GST. SM5-1 was used as Fab-fragment generated by papain digestion. Kinetic experiments were performed at 25uC using a Biacore T100 (GE Healthcare, Germany). gB and Dom II-GST proteins were captured on the Series S Sensor Chip CM5 (GE Healthcare, Germany) using N-hydroxysuccinimide-N-ethyl-N-dimethylamino-propyl-carboimide chemistry. PBS, 0.05% P20 was used as the running buffer. Approximately 300 RU of Dom II-GST and GST as reference surface were captured with a contact time of 400 sec and a flow rate of 10 ml/min. Different concentrations of SM5-1 (33.33, 11.11 and 3.7 nM) were injected with a contact time of 90 sec, dissociation time of 600 sec and a flow rate of 30 ml/min. The sensor surface was regenerated between each binding reaction with 10 mM glycine (pH 2.0) with a contact time of 20 sec and a flow rate of 30 ml/min. The kinetics were fitted to a 1:1 binding model. k a , apparent association rate constant; k d , apparent dissociation rate constant; K D , apparent dissociation equilibrium. (C) Circular dichroism (CD) measurement of Dom II protein demonstrating structural folding. PreScission Protease (GE Healthcare, Germany) was used to cleave the GST-Tag. Dom II protein at a concentration of 7.8 mM was dialyzed against 20 mM sodium phosphate buffer (pH 7.0) and filtrated. CD measurement was performed at 20uC using a Jasco J-815 CD Spectrometer (Jasco, Japan) and a cuvette with 0.1 cm path length. Spectrum was registered from 185 to 260 nm and was corrected for the contribution of phosphate buffer. Spectrum was accumulated eight times with a band width of 1.0 nm and a sensitivity of 100 mdeg. The scan speed was 20 nm/min, the time response 1 sec and the data pitch 0.1 nm. (TIF) Figure S4 Quality controls of Dom II-specific polyclonal antibody preparation. (A) The affinity purified polyclonal antibody fraction is specific for Dom II of gB. ELISA plates were coated with gB, AD-1, AD-2 and Dom II, respectively, and tested with various antibodies. Dom II poly: affinity purified IgG fraction, Serum pool pre: Serum pool before affinity purification, Serum post: Serum pool after affinity purification, AD-2-specific mab: C23, anti-AD-1-specific mab: 89-104, anti-GST: murine mab specific for GST. (B) The affinity purified polyclonal antibody fraction does not contain detectable antibodies against additional envelope glycoproteins of HCMV. Cos7 cells were transfected with the plasmids indicated in the top row. 48 h later the cells were fixed and incubated with the affinity purified IgG fraction (upper panel) and control antibodies (lower panel). Binding of the primary antibody was detected by incubation with appropriate FITCconjugated secondary antibody. Anti-HA: mouse mab specific for HA, C23: anti-AD-2 human mab, 14-16A: mouse mab specific for the gM/gN complex, SA4: mouse mab specific for gH. Antibody purification was performed twice with similar results. (TIF) Figure S5 Sequence alignment of HCMV strains and clinical isolates. Full length HCMV protein sequences from Genbank and EMBL databases (accession numbers on the left) were aligned to the HCMV TB40 strain. The regions for AD-2, domain I, domain II as well as AD-1 are depicted. Two regions of hyper-variability lie close to the N-terminus and C-terminal from domain II in a linker region. The protein alignment was performed with the Geneious software v4. 8  Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease. The Chikungunya virus (CHIKV), an arthropod-borne virus (arbovirus), is a single-stranded positive-sense RNA virus with three genotypes. The virus is of the Alphavirus genus in the Togaviridae family [1, 2] . CHIKV has been shown to infect and be transmitted by Ae. aegyptii and Ae. albopictus mosquitoes. It was identified in East Africa in the early 1950s and since then has caused epidemics in continental Africa, the Indian Ocean region, and countries of Southeast Asia such as India, where since 2006 suspected cases have been estimated to be 1.39 million, and Singapore [3] [4] [5] [6] . The only reported outbreak outside these areas was in Italy in the Emilia Romagna region in 2007. Small nonepidemic imported cases have been reported in other regions such as North America, France and Japan, which were caused by travelers returning from affected areas [7] [8] [9] .
The epidemic occurring on La Reunion Island in the Indian Ocean remains the most devastating of all CHIKV outbreaks where over one-third of the population was affected [10] . During this outbreak, the CHIKV acquired a genetic mutation allowing the new vector Ae. albopictus mosquito to carry the virus where previously CHIKV only circulated in Ae. aegyptii mosquitoes [10, 11] . The Ae. albopictus differs in susceptibility to various genetically different isolates of the virus compared to the Ae. aegyptii [12] . CHIKV is now of global health concern since expansion of mosquito vectors has created potential for the Chikungunya virus to spread to temperate areas as Ae. albopitcus inhabits regions in North America and Europe [2, 13] .
CHIKV infection is clinically characterized by the sudden appearance of high fever, rash, headache, nausea, vomiting, myalgia and arthalgia or severe joint pain. Severe joint pain is the defining symptom of CHIKV disease [11] . The word Chikungunya originated from the Tanzanian and Mozambique region of Africa meaning that which bends up. A bent posture is often taken by those in severe joint pain after being infected with CHIKV. CHIKV symptoms start 4 to 7 days after exposure and most resolve within the acute phase of the disease. Although the acute phase lasts approximately 2 weeks, joint pain can persist for months or years following initial infection [1, 14, 15] . Minimal research has been done investigating the immune response following CHIKV infection. Currently, there is no CHIKV specific therapeutic available.
The Italian outbreak of CHIKV spread through communities surrounding the city of Ravenna during August to October 2007 and also involved the major Italian city of Bologna [15, 16] . A recorded 254 people were identified to be infected through the Ae. albopictus mosquito which has inhabited the Emilia Romagna region since 1990 [14, 17, 18] . The virus brought to the Emilia Romagna region by a traveler returning from a CHIKV affected country was of the Central/East African genotype and matched most closely (100% amino acid identity) with the IND-06 virus isolated from the Reunion Island outbreak [14, 17] . The amino acid identity confirmed that this virus included a substitution mutation in the E1 envelope protein (E1-A226V) [19] which is important for viral entry into host cells. This mutation was acquired during the 2005-2006 Indian Ocean CHIKV outbreak and increased the virus's infectivity to the Ae. albopictus mosquito [20] .
Cytokines are important immune mediators that conduct immune responses. Recently, cytokine profiles have been investigated in CHIKV infected humans by two groups [21, 22] . Ng and colleagues established cytokine profiles from 10 CHIKV patients that were infected during the Singapore 2007 CHIKV outbreak [22] . Although this study reported that IL-1b, IL-6 and RANTES were correlated with severe acute phase CHIKV disease, cytokine profiles were not determined for the progression and convalescence of the disease. Here we investigated cytokine profiles during the acute phase and 6-and 12-month follow-up of CHIKV infected patients of the Italian 2007 outbreak. Since CHIKV disease can have severe acute phase symptoms and be followed by persistent symptoms in the convalescence phase it was important to investigate the immune response responsible for these maladies. Furthermore, the Italian CHIKV included the A226V mutation and the Singapore virus did not. Furthermore, we analysed the relationship between cytokine levels and patient severity, and IgG levels linking high CXCL9, CXCL10 and IgG levels with disease severity. Therefore, the results presented here are virus specific and reflect previously unreported cytokine profiles which may be important for the development of future therapeutics for CHIKV outbreaks.
Patients all gave written consent to the participation in scientific studies. Permission to perform scientific studies was given by Comitato Etico di Area Vasta Romagna Et IRSTof the Servizio Sanitario Regionale Emilia-Romagna, Italy.
Since the immune response during CHIKV disease has not been extensively investigated, our objectives were to create a clear clinical picture of CHIKV disease at the acute phase and during convalescence at 6-and 12-month follow-up by cytokine profiling. To achieve this objective, we investigated the cytokine profiles from patients at the acute phase and at 6-and 12-month follow-up.
Included patients were from the region of Emilia-Romagna in north-east Italy suspected to be infected with CHIKV since they showed symptoms such as myalgia, severe back and joint pain, headache, and skin rash. Collaboration with the regional microbiology reference laboratory of Bologna University and the Department of Infectious and Parasitic Diseases of the National Institute of Health in Rome was initiated and identified the patients as having CHIKV. The clinical criteria was described as acute onset of fever (.38.5uC) and severe arthralgia not explained by other medical conditions. CHIKV infection was confirmed by one or more of the acute phase tests: virus isolation, reverse transcriptase-PCR (RT-PCR) positive for CHIKV nsp1 gene, seroconversion to virus-specific serum antibodies collected at least 1 to 3 weeks apart, or presence of virus-specific IgM antibodies in a single serum sample collected [15] . Acute CHIKV patient samples were determined to be in the viral stage if the sample was PCR positive for CHIKV (7 patients), in the IgM antibody initiation stage if the sample was PCR negative, IgM positive, IgG negative (6 patients) or in the seroconversion stage (22 patients) if the sample was PCR negative, IgM positive and IgG positive. The samples were considered to be high IgG if the IgG level was greater than 6400 (6 months) (31 patients high out of 50) or greater than 3200 (12 months) (20 patients out of 50). IgG levels below or equal to these thresholds were considered low IgG. CHIKV patients were considered to be non-symptomatic (15 patients out of 50 at the 6-month follow-up; 34 patients out of 50 at the 12 month follow-up), have mild symptoms (21 patients out of 50 a the 6 month follow-up; 14 patients out of 50 at the 12 month followup), or have severe symptoms (14 patients out of 50 at the 6 month follow-up; 2 patients out of 50 at the 12 month follow-up) based on their responses to a questionnaire at the time of sampling which was based on: articular pain, muscle pain, mono-arthritis, oligoarthritis, symmetric polyarthritis, asymmetric polyarthritis, tenosynovitis, arthralgia and fibromyalgia. Control samples were collected from 10 healthy volunteers screened for symptoms of viral infection.
Blood samples were collected from consenting CHIKV positive patients at the time of diagnosis. Viral infection was determined as described above. Two follow-up samples were then collected from each patient at the 6-month evaluation and the 12-month
Chikungunya virus (CHIKV) is transmitted by mosquitoes and causes a human disease clinically characterized by sudden appearance of high fever, rash, headache, nausea, and severe joint pain (the defining symptom). Chikungunya was identified in Africa and the word Chikungunya means that which bends up, describing the bent posture of CHIKV patients while in severe pain. CHIKV, a current problem in Africa, Indian Ocean region, and Southeast Asia, is now spreading to temperate regions of North America, France and Italy. Presently, the immune response for CHIKV infection remains largely uninvestigated and no treatment is available. We investigated cytokine profiles at diagnosis and follow-up of CHIKV infected patients during the Italian 2007 outbreak and associated cytokine levels with antibody level and symptom severity. Cytokines, important immune mediators, are often drug targets. Since CHIKV symptoms can persist for months or years following infection it is important to investigate possible drug targets to alleviate discomfort. We found cytokine profiles that describe the initial infection and recovery phase. We determined the cytokines CXCL9/MIG and CXCL10/IP-10 as well as antibody levels were associated with symptom severity. These results reflect previously unreported cytokine profiles which may be important for the development of future therapeutics for CHIKV outbreaks.
Inflammatory Cytokines Resolve Chikungunya Disease www.plosntds.org evaluation. After sampling, serum was extracted and immediately frozen at 280uC until serum analysis.
Serum samples were analyzed for cytokine levels using BD TM Cytometric Bead Array (CBA) Human Chemokine Kit, Human Inflammatory Cytokine Kit, and Human Th1/Th2 Cytokine Kit (BD Biosciences) according to the manufacturer's instructions for a total of 13 cytokines. Capture Beads were added to the serum sample followed by the PE detection reagent. The samples were then incubated for 3 hours at room temperature and washed with the assay Wash Buffer and resuspended again in Wash Buffer for analysis on the Flow Cytometer. CBAs were then run on a BD FACSCalibur Flow Cytometer.
CBA data was analysed using SPSS statistical software. Boxwhisker plots were created from the CBA FACS raw data. Sixmonth and 12-month samples were compared to the acute samples using the non-parametric two-tailed Wilcoxon signedrank test for related samples to determine statistical significance. Each acute phase, 6-month and 12-month cytokine sample sets were statistically compared to healthy control CBA data using the non-parametric two-tailed Mann Whitney Test for unrelated samples.
CHIKV resolution is associated with differential cytokine programs of increasing and decreasing trends CHIKV causes a disease of crippling joint pain that has affected most of Asia and has demonstrated the capability to spread to nontropical areas such as Europe and parts of North America [1] . Cytokines are inflammatory mediators and their balance is often associated with inflammatory disease [23] . Previously, the cytokine profiles of acute phase CHIKV patients have been examined [22] . Here we profiled cytokine levels in acute phase and 6-and 12month follow-up CHIKV patient serum samples to determine a cytokine signature that may correlate with acute symptoms, following persistent joint pain and/or disease resolution. Blood samples were collected from 50 patients suffering from CHIKV infections during the 2007 Italian outbreak. Serum separated from whole blood was analyzed by cytokine bead analysis (CBA) for 13 cytokines with the intention of determining a cytokine profile during CHIKV acute phase and disease convalescence. Three cytokine profiles emerged from our data: decreasing, increasing and no-trend.
The first trend showed cytokine levels significantly higher in the acute samples compared to the follow-up time points revealing a decreasing pattern as patients left the acute phase. CXCL9/MIG (CXCL9), IL-6, CCL2/MCP-1(CXCL2) and CXCL10/IP-10 (CXCL10) cytokines had significantly decreased at both 6-month and 12-month follow-ups ( Figure 1) . Interestingly, some patients had extremely high levels in the acute phase; CXCL9 and CXCL10 levels decreased 1000 fold to 10,000 fold during convalescence. Furthermore, the decreasing trends for IL-6, CXCL9, and CXCL10 reached similar levels as those of the community control levels (shown by dotted line). CCL2 levels decreased significantly lower than the control levels by 12 months. Taken together, this data demonstrated that CXCL9, IL-6, CCL2 and CXCL10 were initially increased with acute CHIKV infection and decreased over time.
The second cytokine trend that emerged described cytokines that significantly increased following the acute phase. Cytokine profiles that were markedly lower in the acute phase and subsequently increased included IL-1b, TNF-a, IL-12, IL-5, IL-10 and IFN-c ( Figure 2 ). The cytokine increase was more gradual than the previous decreasing trend, where fold changes were generally closer to 2. Both the 6-and 12-month follow-up were statistically increased compared to acute values for IL-5 levels. IL-1b, TNF-a, IL-12, IL-10 and IFN-c had significantly increased by 12 months. Even though the average for these cytokines had also risen by 6 months it was not significant. Furthermore, the increasing trends for TNF-a, IL-5, and IL-10 reached similar levels to the community control levels (shown by dotted line) and IFN-c reached significantly higher than controls at 12 months. Interestingly, although IL-1b and IL-12 increased through the observed time, these cytokines stayed significantly lower than those of the controls. This data showed that cytokines IL-1b, TNF-a, IL-12, IL-5, IL-10 and IFN-c increased in the convalescence phase of CHIKV infection.
No significant change was seen for IL-2, IL-4, and IL-8 from the acute phase to the 12-month follow-up ( Figure 3 ). Interestingly, IL-2 reached similar levels to those of the controls where IL-8 and IL-4 remained significantly raised and lowered, respectively. Figure 4) . The median of CXCL10 in the viral stage was approximately 7000 pg/ml and dropped to less than 1000 pg/ml after seroconversion. Interestingly, the IL-10 median decreased by 3 fold from the viral stage to the seroconversion stage.
Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG. The patients were put into an IgG high group (H) or an IgG low group (L) and the levels of each cytokine were statistically compared for each group using the Mann-Whitney U Test. In the 6-month follow-up phase CXCL9, CXCL10 and Il-6 were found to be statistically different between the high IgG group and the low IgG group ( Figure 5A ). High levels of all 3 cytokines were associated with high levels of IgG antibodies. IL-10 is also shown for comparison since it was statistically significant during the acute phase breakdown and the 12-month follow-up. Interestingly, in the 12-month follow-up phase, CXCL9 was found to be statistically higher in the IgG high group where IL-10 was significantly lower in the high IgG group ( Figure 5B ). CXCL10 is shown for comparison at 12 months although it was not significantly different between the IgG high and IgG low groups. In summary, the results suggested that CXCL9, CXCL10 and Il-6 were associated with IgG levels in the 6-month follow-up phase and CXCL9 and IL-10 in the 12month follow-up phase.
Inflammatory Cytokines Resolve Chikungunya Disease www.plosntds.org CXCL10, CXCL9 and IgG levels are possible biomarkers of CHIKV disease severity IL-1b, IL-6 and RANTES were found to be associated with symptom severity of the Singapore 2007 CHIKV outbreak [22] . After determining the cytokine profiles of our Italian 2007 CHIKV patients during their disease resolution, we next sought to determine the association between symptom severity and cytokine levels. Patients were determined to be non-symptomatic (N), to have mild symptoms (M) or to have severe symptoms (S) depending on their responses to a questionnaire. The cytokine levels were then grouped by symptom level and a Mann-Whitney U test was used to determine significance among the severity groups for each cytokine. CXCL10 and CXCL9 were found to be significantly increased in the patients with mild and severe symptoms at 6 months following initial infection compared to the patients reporting no symptoms ( Figure 6A) . A 2 fold difference was seen between the medians of the non-symptomatic and severe patients for CXCL10 and a 5 fold difference for CXCL9. No statistical difference was seen for any of the 13 cytokines profiled for the 12-month follow-up. CXCL10 and CXCL9 at 12 months are shown for comparison ( Figure 6B ). Moreover, we analyzed the IgG levels at the 6-month follow-up in patients with no symptoms, mild symptoms, and severe symptoms. The results showed IgG levels were statistically increased with Figure 1 . Acute phase CHIKV disease was associated with high levels of IL-6, CXCL9, CCL2, CXCL10. Cytokine Bead Array analysis of CHIKV patient serum samples showed high levels of IL-6, CXCL9, CCL2 and CXCL-10 are associated with acute disease phase and decreased with patient convalescence. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute levels. All samples were also analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value less than 0.05 for 6-and 12-month groups compared to acute values and star symbol indicates a p-value less than 0.05 for acute, 6-and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12 Figure 6C ). Taken together, these results suggested CXCL10, CXCL9 and IgG to be possible markers of CHIKV severity during early phases of disease resolution.
CHIKV disease is a self-limiting disease caused by an alphavirus of the Togaviridae family. Although historically the virus only caused a disease of mild symptoms, a recent outbreak on La Reunion Island caused significant mortality due to genetic alterations [1, 20, 22, 25, 26] . Here we have investigated the immune response of an Italian population infected with the Indian Ocean genotype of CHIKV and have generated a cytokine signature for the initial infection to convalescence phase of CHIKV disease, the signature of viral and antibody production phases, and identified cytokines raised in patients with severe symptoms. We found that initial Significance by comparing with acute phase values. As well, samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value (Wilcoxon test) less than 0.05 for 6-and 12-month groups compared to acute values and star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6-and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12 infection and the subsequent convalescence were described by a set of decreasing and increasing cytokines. Furthermore, we have shown that CXCL10 and IL-10 levels were associated with the viral stage of the acute phase and CXCL10 and CXCL9 with high IgG levels of the 6-month follow-up. As well, CXCL10 and CXCL9 were markers of symptom severity. Importantly, these identified signatures depict the immunological programs and may be key to the development of therapeutics for the frequently reemerging CHIKV disease.
Our analysis has indentified 2 cytokine profiles that followed disease onset and continued with disease progression/convalescence. We found CXCL9, CXCL10, CCL2 and IL-6 levels were high in the acute phase and decreased as patients convalesced. Conversely, the trend for TNF-a, IL-1b, IL-2, IL-5 and IL-12 were low initially and increased as patients began to recover from acute illness. High levels of CXCL9, CXCL10, CCL2, and IL-6 in the acute phase possibly indicated an inflammatory program initiating adaptive T-cell immunity [27] . CXCL9 and CXCL10 are both chemokines induced by IFN-c and are part of the chemokine program that regulates the migration of monocytes/ macrophages, memory T cells and NK cells and are associated with the polarization of T cells [27] . IL-6, a pleiotropic cytokine, has a destructive role in rheumatoid arthritis (RA), contributing to joint inflammation as well as joint pain [28, 29] . The increased IL-6 levels in the acute phase of our study may be the initiating factor of the severe joint pain symptoms reported in CHIKV patients which mimics RA. Furthermore, IL-6 is important during acute phases of the disease by acting as an important immune mediator of fever activating muscle metabolism to increase core body temperature [28] . Since fever is a common symptom of acute CHIKV disease, it is highly probable that the high IL-6 levels were contributing to the acute fever and the IL-6 decreasing trend followed patient core body temperature as it returned to resting temperature in the follow-up.
A second host immune response profile was characterized by TNF-a, IL-1b, Il-10, Il-12, IFN-c and IL-5, which increased from the acute phase into convalescence. Interestingly, TNF-a and IL-1b, which are known to co-induce the other's expression, are both involved in chronic inflammatory diseases such as RA, chronic hepatitis B and C infection [23, 29, 30] . Importantly, TNF-a and IL-1b are main contributors to joint pain, which is the major symptom of RA. An internal balance of TNF-a or IL-1b levels is imperative as mis-regulation of either has been shown to be a major proponent of chronic diseases (RA). Our data indicated that these cytokines increased significantly during patient convalescence above those of the control group, but were not statistically changed in patients reporting mild or severe symptoms. These data may imply that the increased levels of TNF-a and IL-1b in the convalescence phase were not a major contributor of chronic damage causing persistent severe joint pain symptoms during the Italian outbreak even though these cytokines have previously been found to play a destructive role in chronic inflammatory diseases. Furthermore, TNF-a immunomodulators have previously been used as a common treatment for RA and IL-1b immunomodulators are effective with other chronic diseases such as systemiconset juvenile idiopathic arthritis and in adult onset Still's diseases [29, 31] . Taken together, these findings suggest TNF-a and IL-1b therapies would not be effective controlling the prolonged symptoms of CHIKV disease since the raised levels during convalescence were not associated with patient severity. Previously, cytokine profiles have been analyzed from patients during an Asian outbreak of CHIKV [22] . Ng and colleagues, investigated 30 cytokines and growth factors from 10 acute phase CHIKV patients, determined that the levels of 8 cytokines, 2 chemokines and 3 growth factors were significantly raised in patients compared to those of the control group. In accordance with their data, IL-6, CXCL9 and CXCL10 were also increased in the acute phase of our patients as compared to control. Since the results from the previous study did not follow the patients during convalescence, our study added significant insight to the progression of CHIKV disease. We found that these three cytokine levels decreased as the patients convalesced as discussed above. Conversely, we found CCL2 also to be increased in the acute phase compared to that of the control group, which was unchanged in the Ng study. Furthermore, Ng et al. found IL-5 and IL-10 were significantly increased in the acute phase where our data indicated that IL-5 and IL-10 were initially low and below control levels and increased following the acute phase. These discrepancies can possibly be explained by the patient populations: the Ng study patients and our patients were from significantly varied genetic backgrounds (Asian and European, respectively). Therefore, differences in immune response may reflect variations in immunological genetic programs. As well, the virus that caused the two outbreaks also differed genetically. Even though the virus that caused the Singapore outbreak was the same genotype as the Italian virus, the virus that infected the Italian patients had the A226V mutation in the E1 gene which was acquired on La Reunion Island [19] . Although the impact of this mutation has been shown to increase vector infectivity [20] , the mutation has not been investigated on the human or mammalian immune response.
Previously, we have identified 3 stages of the acute phase of WNV; a viral stage, antibody initiation stage, and seroconversion stage [24] . As the viral load decreased in the WNV patients, IgM antibody levels were initiated and followed by IgM conversion to IgG, thereby marking the 3 stages of the acute phase. From this work we were able to identify the stages of the CHIKV patients and compare their respective serum cytokine levels. We found CXCL10 and IL-10 levels decreased as patients progressed through the viral stage to seroconversion. Since CXCL10 is often correlated with viral load, this observation was not surprising [32] . High IL-10 levels in the viral stage may act in an effort to control the IFN-c program [33] shown by high CXCL10 levels. Furthermore, plasma levels of CCL2, IL-6 as well as CXCL10 have all been correlated with viral loads in virus infected individuals [24, 34, 35] . It is possible that the decrease of CCL2 and IL-6 we observed subsequent to the acute phase [14] correlated with viral clearance, although not with antibody levels. Analysis of the cytokine/antibody response was carried on to the 6-and 12-month follow-up where we grouped the patients on their IgG levels. CXCL9, CXCL10 and IL-6 were raised in the patients with increased IgG levels at 6 months and CXCL9 at the 12month follow-up. These proinflammatory cytokine associations with high IgG levels may represent the persistence of an active immune system.
CXCL9 and CXCL10 as well as high IgG levels were found to be biomarkers of severe CHIKV symptoms. Previously, CXCL10 has been associated with severe viral disease supporting a role for CXCL10 in severe CHIKV disease [34] [35] [36] [37] . These studies did not find an association with CXCL9 and severity as seen in our CHIKV patients. Our findings suggest high CXCL10 and CXCL9 associated with severity to be a unique signature of CHIKV. Interestingly, not only are CXCL10 and CXCL9 expressed in RA and other arthritis related disease patients [38] [39] [40] [41] , but they have been shown to be biomarkers for RA symptoms, implying a similar mechanism for joint destruction in CHIKV disease [42] . Moreover, CXCL9 and CXCL10 may be contributors of persistent immune activation in CHIKV disease leading to chronic symptoms, which implies cytokine immunomodulation may significantly improve patient treatment and recovery. Importantly, CXCL10 also has prognostic value in the treatment of viral hepatitis where CXCL10 levels follow disease recovery [43] supporting our finding and proposes a prognostic role for CXCL10 in CHIKV. Furthermore, our data puts forth CXCL10 and CXCL9 as possible drug targets for treatment of CHIKV symptoms in the convalescence phase due to the association with severity; however, further investigation is needed on CXCL10 and CXCL9 efficacy. In addition, not only are the identified cytokines useful as possible drug targets but the cytokine signatures described can also be applied when testing newly developed CHIKV therapeutics. As CHIKV therapeutics are evaluated in the CHIKV disease model, cytokine profiles can be used as an output for determining the efficacy of the novel therapeutics.
The synovial mast cell remains an important component during RA joint destruction by the exocytosis of intracellular granules containing inflammatory mediators. Currently, mast cell activation through FccRs by high levels of circulating IgG antibodies is hypothesized to contribute to the pathological destruction of synovium in RA [44] [45] [46] . In addition, antibody immune complex formation within the joint stabilizing inflammatory mediators, such as chemokines and complement proteins, is another possible low IgG level group (L) and a high IgG level group (H). Cytokine Bead Array analysis of the serum samples showed a significant difference in CXCL9, CXCL10 and IL-6 between patients with high and low IgG levels at the 6-month time point. Twelve-month follow-up CHIKV patient samples showed a significant difference in CXCL9 and IL-10 between low (1) and high (2) IgG groups. A Mann-Whitney U test was used to determine significance among the IgG groups. The cross symbol indicates a p-value less than 0.05. doi:10.1371/journal.pntd.0001279.g005
Inflammatory Cytokines Resolve Chikungunya Disease www.plosntds.org Figure 6 . CHIKV disease severity is associated with high CXCL10, CXCL9 and IgG levels at the 6-month time point. CHIKV patients were determined to be nonsymptomatic (N), to have mild symptoms (M) or to have severe symptoms (S). The cytokine and IgG levels were then grouped by symptom level and a Mann-Whitney U test was used to determine significance among the severity groups. CXCL10, CXCL9 and IgG were found to be significantly increased in the patients with mild and severe symptoms at 6 months following initial infection. The cross symbol indicates a p-value less than 0.05. doi:10.1371/journal.pntd.0001279.g006
Inflammatory Cytokines Resolve Chikungunya Disease www.plosntds.org facet of pathogenesis during RA [47, 48] . We found high concentrations of IgG to be associated with symptom severity in CHIKV patients. Similar IgG mediated mechanisms leading to synovium destruction and severe pain experienced by CHIKV patients are possible. Taken together with the roles of IgG in RA, our findings support the need for further investigation into the contribution of IgG levels and mast cells to CHIKV symptoms.
We have found that the cytokines investigated had one of two immunologically important profiles during CHIKV disease onset and convalescence; furthermore, CXCL10 and CXCL9 were makers of disease severity. By identifying the immune profiles, we have created a clearer clinical picture of CHIKV disease. Importantly, further investigation is needed to correlate these profiles with disease onset and progression to use the cytokine profiles as biomarkers for severity and symptom persistence. The data presented here suggest that with further investigation, immunomodulators may significantly enhance patient recovery. The Failure of R (0) The basic reproductive ratio, R (0), is one of the fundamental concepts in mathematical biology. It is a threshold parameter, intended to quantify the spread of disease by estimating the average number of secondary infections in a wholly susceptible population, giving an indication of the invasion strength of an epidemic: if R (0) < 1, the disease dies out, whereas if R (0) > 1, the disease persists. R (0) has been widely used as a measure of disease strength to estimate the effectiveness of control measures and to form the backbone of disease-management policy. However, in almost every aspect that matters, R (0) is flawed. Diseases can persist with R (0) < 1, while diseases with R (0) > 1 can die out. We show that the same model of malaria gives many different values of R (0), depending on the method used, with the sole common property that they have a threshold at 1. We also survey estimated values of R (0) for a variety of diseases, and examine some of the alternatives that have been proposed. If R (0) is to be used, it must be accompanied by caveats about the method of calculation, underlying model assumptions and evidence that it is actually a threshold. Otherwise, the concept is meaningless. The basic reproductive ratio-also known as the basic reproductive number, the basic reproduction number, the control reproduction number, or R 0 -is one of the foremost concepts in epidemiology [1] [2] [3] . R 0 is the most widely used epidemiological measurement of the transmission potential in a given population [4] . It is a measure of initial disease spread, such that if R 0 > 1, then the disease can invade an otherwise susceptible population and hence persist, whereas if R 0 < 1, the disease cannot successfully invade and will die out. The concept is defined as the number of secondary infections produced by a single infectious individual in an otherwise susceptible population [5] .
Despite its place at the forefront of mathematical epidemiology, the concept of R 0 is deeply flawed. Defining R 0 proves to be significantly more difficult than it appears. Few epidemics are ever observed at the moment an infected individual enters a susceptible population, so calculating the value of R 0 for a specific disease relies on secondary methods. There are many methods to calculate R 0 from mathematical models, few of which agree with each other and few of which produce the average number of secondary infections. Methods to calculate R 0 from theoretical models include the survival function, the next-generation method, the eigenvalues of the Jacobian matrix, the existence of the endemic equilibrium, and the constant term of the characteristic polynomial. R 0 can also be estimated from epidemiological data via the number of susceptibles at endemic equilibrium, the average age at infection, the final size equation and calculation from the intrinsic growth rate. For an overview, see Heffernan et al. [2] .
Furthermore, there are many diseases that can persist with R 0 < 1, while diseases with R 0 > 1 can die out, reducing the utility of the concept as a threshold. R 0 is also used as a measure of eradication for a disease that is endemic, but issues such as backward bifurcations, stochastic effects, and networks of spatial spread mean that an invasion threshold does not necessarily coincide with a persistence threshold. This results in a reduction of the usefulness of R 0 . For example, it is possible that a disease can persist in a population when already present but would not be strong enough to invade. Finally, the threshold value that is usually calculated is rarely the average number of secondary infections, diluting the usefulness of this concept even further.
In this paper, we outline the problems with R 0 and examine a number of alternatives that have been proposed. We include a worked example of malaria to demonstrate the many different results that the various methods give for the same model. Finally, we survey some of the recent uses of R 0 in the literature. The number of articles that use R 0 likely numbers in the tens of thousands, so an exhaustive review is not feasible. We have restricted ourselves to articles published since 2005 and which include interesting or novel explorations of R 0 .
In this section, we identify some of the more popular methods (although by no means all) used to calculate R 0 . We also describe the limitations that each method presents and demonstrate one of the core problems with R 0 . Specifically, we address a key problem with R 0 : how do biologists make sense of it from mathematical models? (See, for example, the puzzled discussion in van den Bosch et al. [6] .)
Although the "R" in R 0 is derived from "reproductive", based on the original formulation of the concept as the average number of secondary infections, many thresholds have been denoted by "R 0 ", even when they are not related to the average number of secondary infections. Thus, in keeping with the notation, we will use the notation R 0,X to denote an R 0 -like surrogate associated with a particular method, symbolised by X. The survival function has the advantage that it always produces the average number of secondary individuals infected by a single infected individual, in the same class. Thus, in Figure 1 , where one human infects two mosquitoes, who each infect three humans, the survival function produces R 0 = 6. This is the number of humans infected by a single infected human via mosquitoes; or, equivalently, the number of mosquitoes infected by a single mosquito via humans. The survival function is a generalised method of calculating the basic reproductive ratio that is not restricted to ODEs.
However, determining the individual probabilities can be cumbersome, especially if multiple states are involved. For a vector-borne infection such as malaria, with two infection states (human and mosquito), calculating the first probability involves determining the probability that a human infected at time 0 exists at time t, the probability that a human infected for total time t infects a mosquito and the probability that an infected mosquito lives to be age a − t, where 0 ≤ t ≤ a [2] . For diseases with more states, such as Guinea Worm disease, where there is a waterborne parasite, which can attach itself to copepods, which in turn are ingested by humans and which subsequently grow into an internal nematode, the calculations of the survival probabilities become unwieldy.
Thus, although this method always produces the correct R 0 , in practice, it is difficult to use. This is especially true for models with sufficient complexity, which are often those encountered most frequently.
The Jacobian matrix is used to linearise a nonlinear system of differential equations. Around the disease-free equilibrium, the linear system will have the same stability properties as the nonlinear system if it is hyperbolic; that is, if no eigenvalues have zero real part. In particular, if all eigenvalues have negative real part, then the equilibrium is stable, whereas if there is an eigenvalue with positive real part, the equilibrium is unstable.
It follows that a threshold is λ max = 0, where λ max is the largest eigenvalue of the Jacobian matrix (or the largest real part if the eigenvalues are complex). However, for a system of n differential equations, this requires solving an nth order polynomial, which may be impossible. Furthermore, rearranging the condition λ max = 0 to produce a threshold R 0,J = 1 is not a unique process and does not always produce the average number of secondary infections.
When λ max = 0, the constant term of the characteristic polynomial will be zero. However, the reverse is not true, as the Computational and Mathematical Methods in Medicine 3 polynomial could have both zero and positive roots. If the characteristic polynomial is λ n + a n−1 λ n−1 + · · · + a 1 λ + a 0 = 0,
then a 0 = 0 is a threshold if a j ≥ 0 for all j. More generally, the Routh-Hurwitz condition allows the coefficients to take on other signs, under certain restrictions, but the nonconstant coefficients all being positive is a sufficient condition. Another sufficient condition is that a j ≥ 0 under the constraint a 0 = 0 (so that the largest eigenvalue at a 0 = 0 is 0). This method is significantly easier to use than finding the largest eigenvalue, although verifying that a 0 = 0 necessarily corresponds to the largest eigenvalue can become complicated for some models. However, similar to the above, rearranging a 0 = 0 to produce R 0,C = 1 is not a unique process and does not always produce the average number of secondary infections.
Method. The next-generation method, developed by Diekmann et al. [8] and Diekmann and Heesterbeek [9] , and popularised by van den Driessche and Watmough [10] , is a generalisation of the Jacobian method. It is significantly easier to use than Jacobian-based methods, since it only requires the infection states (such as the exposed class, the infected class and the asymptomatically infected class) and ignores all other states (such as susceptible and recovered individuals). This keeps the size of the matrices relatively manageable.
However, the next-generation method suffers from a lack of uniqueness. In order to determine the matrices F and V (where F accounts for the "new" infections and V accounts for the transfer between infected compartments), biological insight must be used in order to decide which terms count as "new" infections and which terms are transfer terms. While this may seem intuitive for most models, it is easy to construct a counterexample
Here, the term +5I might represent, for example, new infections arising from vertical transmission, whereas the term −5I might represent a disease-specific death rate. Although this construction is clearly arbitrary, it demonstrates that identifying "new" infections is not a unique process and relies on the modeller's judgement.
We would then have
and thus
This has the same threshold property as R 0,N = βS/μ, but is clearly not the average number of secondary infections. In essence, the next-generation method is a mathematical generalisation of putting the negative values on one side and dividing (this is what V −1 is) so that the eigenvalue threshold at zero is transformed into an R 0 -like threshold at one. However, as we have seen, this does not produce a unique result. van den Driessche and Watmough [10] note that other decompositions of F and V can be chosen, which lead to different values for R 0,N . They claim that only one choice of F is epidemiologically correct. However, this is not true, as the above example shows. Furthermore, it means that the definition of R 0 relies upon the judgement of the modeller as to what "epidemiologically correct" means.
Furthermore, the next-generation method does not produce the number of humans infected by a single human if there is an intermediate host, but rather the geometric mean of the number of infections per generation.
For example, consider a mosquito-borne disease where humans infect two mosquitoes, while mosquitoes infect three humans, as shown in Figure 1 . For convenience, label these R H = 2 and R M = 3. Then the number of humans infected from a primary human (via mosquitoes) is R 0 = 2 × 3 = 6. (This is also the value calculated by the survival function.)
However, the next-generation method would calculate R 0,N = √ 6, which is a weighted average (2 < √ 6 < 3) of the number of infectives each individual produces in the next infection event. While mathematically sound, it is questionable whether this is biologically meaningful.
This example could be extended. Consider a three-stage disease, such as tularemia, where ticks may transmit between humans and livestock, but humans may also be infected by eating livestock. Suppose that a single human directly infects two ticks (so R H = 2), each tick infects four animals (so R T = 4) and each animal infects three humans (so R A = 3). Then, a single human has resulted in
infected humans. However, R 0,N = 3 √ 24 ≈ 2.88, since there are three infection stages. As the number of infection stages increase, R 0,N becomes a progressively higher surd.
The next-generation method is likely the most frequently used method to calculate R 0 . It has been used extensively to calculate R 0 -like values from host-vector models (see Wonham et al. [11] , Gaff et al. [12] or Gubbins et al. [13] ). However, it does not produce the number of newly infected individuals in the same infection class and does not always produce the average number of secondary infections.
In de Camino-Beck et al. [14] , a graph-theoretic method for calculating R 0 is given. Starting from the definition of R 0 = ρ(FV −1 ), they derived a series of rules for reducing the digraph associated with Fλ −1 − V to a digraph with zero weight, from which λ = R 0 . The rules are as follows.
To reduce the loop −a ii < 0 to −1 at node i, every arc entering i has weight divided by a ii .
For a trivial node i on a path j → i → k, the two arcs are replaced by j → k with weight equal to the product
Creation of trivial nodes using Rule 1
Rule 2 Figure 2 : The graph-theoretical method of de Camino-Beck et al. [14] applied to a vector-host model may not produce a unique R 0 .
of weights on arc j → i and i → k. Weights on multiple arcs j → k are added.
The graph-theoretic method has the advantage that it avoids calculation of V −1 , which may be complicated for large systems. However, it always produces the same threshold value as the next-generation method. They considered the simple vector-host model
where I, W, S, and M are proportions of infective hosts, infective vectors, susceptible hosts, and susceptible vectors, respectively, b is the host birth and death rate, γ is the host recovery rate, c is the vector birth and death rate, and β s and β m are disease transmission coefficients. The disease-free equilibrium is (0, 0, 1, 1) T . They found matrices
From this, they produced a digraph of Fλ −1 − V and concluded that
However, this method still contains the same issues as the next-generation method. The R 0 value calculated is not the number of humans infected by a single human, but rather the (less biologically meaningful) geometric mean of the number of humans infected by the vector and the number of vectors infected by a human. Furthermore, the creation of F and V is not a unique process, as we have seen above, and does not always produce the average number of secondary infections.
For example, consider the mathematically equivalent model
with matrices
The graph reduction then proceeds as in Figure 2 . It follows (either by the graph-reduction method or using the conventional next-generation method) that
Thus, the graph-theoretic method inherits the existing problems from the next-generation method.
2.6. Existence of the Endemic Equilibrium. R 0 can also be calculated from the endemic equilibrium, in the case where there is a bifurcation at R 0 = 1 such that the endemic equilibrium does not exist for R 0 < 1. The existence of the endemic equilibrium is, thus, a threshold that can be rearranged to produce an R 0 -like surrogate. However, for many models, calculating the endemic equilibrium can be quite difficult. Furthermore, in the case of a backward bifurcation, the endemic equilibrium still exists for R 0 < 1 (in fact, two endemic equilibria exist in this case, one of which is stable and the other unstable). It follows that the endemic equilibrium is not a useful general method for calculating R 0 and does not always produce the average number of secondary infections.
van den Bosch et al. [6] use the endemic equilibrium to determine a threshold given by I = (1/α)(μq−μ+αK) so that I > 0 if μq − μ + αK > 0 (where I represents infected plants at the endemic equilibrium, α is the transmissibility, μ is the death rate, q is the fraction of infectious seeds, and K is the total plant population density). They show that two different rearrangements of this inequality give
and note that either would suffice as an R 0 value, but were unable to resolve the question of which was appropriate.
Summary of R 0 Methods. In summary, there are many methods available for calculating R 0 , but few of them agree with each other and almost none reliably calculate the average number of secondary infections in a wholly susceptible population. The only method which does is the survival function, but this method is too cumbersome to be used except for the simplest of models.
In this section, we take a sample model and apply the various methods for calculating R 0 to it. We show that each method can produce a different result, all of which have the property that they have an outbreak threshold at R 0 = 1 but otherwise bear little relation to one another. 
Humans may be susceptible or infected (H S and H I , resp.), while mosquitoes may be susceptible or infected (M S and M I , resp.). The birth rates are Λ H for humans and Λ M for mosquitoes. The background death rates are μ H and μ M for humans and mosquitoes, respectively, while the diseasespecific death rate for humans is σ. The transmission rate from mosquitoes to humans is β MH , while the transmission rate from humans to mosquitoes is β HM . See Figure 3 .
The Jacobian matrix at the disease-free equilibrium is
where H S and M S are the equilibrium values of susceptible humans and mosquitoes, respectively. Thus, the nontrivial part of the characteristic polynomial satisfies
Using the Jacobian method, the largest eigenvalue is
Rearranging λ max = 0, we have
Conversely, since the nonconstant coefficients of the characteristic polynomial are positive, all eigenvalues will be negative if the constant term of the characteristic polynomial is positive, whereas there will be an eigenvalue with positive real part if the constant term is negative. Rearranging, we have
This is not the only rearrangement possible, so, like the nonuniqueness of the next-generation method, it is possible to rearrange by adding and subtracting arbitrary constants. Thus, for example,
is also a measure of disease spread with the property that the disease persists if R 0,9 > 1 and is eradicated if R 0,9 < 1. Other rearrangements are possible, so long as the threshold at 0 is transformed into a threshold at 1. Thus,
has the same threshold property. A generalised version of this formulation was used in Smith? et al. [15] called T 0 . The endemic equilibrium satisfies
It follows that there will be a biologically meaningful endemic equilibrium if H I > 0, or
since M S = Λ M /μ M and H S = Λ H /μ H . As above, this is not the only rearrangement possible. The next-generation matrices are
since the next-generation method only considered the two infected classes. Then, using the next-generation method,
Thus, for the same model, R 0,N , R 0,J , R 0,C , R 0,9 , and R 0,e are all distinct. Although R 0,E = R 0,C in this case, this does not hold in general. Note that R 0,C and R 0,E produce the number of infected humans per infected human (or, equivalently, the number of infected mosquitoes per infected mosquito), but this is also not necessarily true in general. Figure 4 illustrates how these R 0 -like expressions vary with σ, when all other parameters are held constant.
Anecdotally, we have found that most biologists believe that there is one and only one R 0 value for a given disease and, in order to verify an R 0 is correct, all that is required is to check that it has a threshold at 1. We have demonstrated here that R 0 is not a unique concept. Indeed, it is straightforward to construct simple variations that satisfy the threshold criterion at 1. Furthermore, since multiple methods calculate multiple thresholds, it follows that the vast majority of them cannot be the average number of secondary infections. However, the nonuniqueness is not the only problem associated with the concept, as we will demonstrate. Figure 4 : The changing face of R 0 . All expressions for R 0 were generated from the same model, using standard techniques. As the death rate varies, all have the property that if R 0 > 1 then an outbreak will occur, whereas if R 0 < 1, then the disease will be eradicated. However, aside from the common threshold at R 0 = 1, none of these expressions are equal to each other. The curve labelled "Jacobian" illustrates R 0,J , as given by (18) . The curve labelled "Adding and subtracting 9" illustrates the value R 0,9 as given by (20) . The curve labelled "Next generation" illustrates R 0,N , as given by (25) . The curve labelled "Constant term of the characteristic polynomial" illustrates R 0,C , as given by (19) . The curve labelled "Exponential" illustrates R 0,e , as given by (21) . 
Backward bifurcations occur when multiple stable equilibria coexist for R 0 < 1. This presents a serious complication when a disease is already endemic, since lowering the basic reproduction number below 1 may no longer be a viable control measure; hence, different prevention and control measures may have to be considered. In particular, a backward bifurcation makes the system more complicated, since the behaviour now depends on the initial conditions.
A backward bifurcation at R 0 = 1 may result in persistence of the disease when R 0 < 1. In this case, the disease will always persist for R 0 > 1. However, there is a point R a < 1 such that the endemic equilibrium exists for R a < R 0 < 1 and a third, unstable, equilibrium also exists between the two stable equilibria. Hence, an endemic disease is only eradicated if 0 < R 0 < R a . For R a < R 0 < 1, the outcome depends on initial conditions. If the disease is still in its early stages-that is, if the initial conditions are sufficiently small-then the system will approach the disease-free equilibrium and the disease will be eradicated. However, if the initial conditions are large, then the system will approach the endemic equilibrium and the disease will persist. Thus, a backward bifurcation prevents the system from switching to the disease-free equilibrium as soon as R 0 < 1 is reached. See Figure 5 (a). (a) A backward bifurcation at R 0 = 1 may result in persistence of the disease when R 0 < 1. There is a point R a < 1 such that the endemic equilibrium exists for R a < R 0 < 1 and a third, unstable, equilibrium also exists. Hence, the disease-free equilibrium is only globally stable if 0 < R 0 < R a . (b) Backward bifurcations at other points may also affect the outcome. Although the disease persists for all R 0 > 1 and is eradicated when R 0 < 1 (due to the transcritical bifurcation at R 0 = 1), there is a region R m < R 0 < R n , where three equilibria coexist. In this region, the outcome depends on the initial conditions.
Several mechanisms have been shown to lead to backward bifurcation in epidemic models, but backward bifurcations in compartmental models have only recently attracted serious research attention.
Gómez-Acevedo and Li [16] investigated a mathematical model for human T-cell lymphotropic virus type I (HTLV-I) infection of CD4 + T cells that incorporates both horizontal transmission through cell-to-cell contact and vertical transmission through mitotic division of infected T cells. They assumed that a fraction σ of the infected cells survive the immune system attack after the error-prone viral replication. Under the biologically sound assumptions that the fraction σ should be very low and the rate of the mitotic division should be high, their model has a bifurcation that predicts persistent infection for an extended range of the basic reproduction R 0 > R 0 (σ 0 ), where R 0 (σ 0 ) < 1. This model undergoes a backward bifurcation as σ increases: multiple stable equilibria exist for an open set of parameter values, when the basic reproduction number is below one.
Safan et al. [17] studied an epidemiological model under the assumption that the susceptibility after a primary infection is r times the susceptibility before a primary infection.
They present a method for determining the control effort required to eliminate an infection from a host population when subcritical persistence may occur. This effort can be interpreted as a reproduction number but is not necessarily the basic reproduction number.
For r > 1 + μ/α, this model exhibits backward bifurcations, where μ is the death rate and α is the recovery rate. For such models, the authors presented a method for determining the minimum effort required to eradicate the infection from the endemic steady state if one concentrates on control measures affecting the transmission rate constant.
Garba et al. [18] presented a deterministic model for the transmission dynamics of a single strain of dengue by realistically adopting a standard incidence formulation and allowing dengue transmission by exposed humans and vectors. The model was extended to include an imperfect vaccine for dengue. A backward bifurcation was observed in both models. This makes R 0 < 1 no longer sufficient for effectively controlling dengue in a community. However, this phenomenon can be removed by replacing the standard incidence function in the model with a mass-action formulation.
Reluga et al. [19] proposed a series of epidemic models for waning immunity that can be applied in many different settings. With biologically realistic hypotheses, they found that immunity alone never creates a backward bifurcation. However, this does not rule out the possibility of multiple stable equilibria, which can be shown by a counterexample of the forward bifurcation at R 0 = 1. See Figure 5 (b).
When R 0 is considered in a spatial context, many of its properties fail to hold. In particular, diseases with R 0 > 1 can fail to persist, depending on the nature of the spatial transmission. Since many diseases are spatially dependent, this further limits the utility of R 0 . As R 0 increases beyond 1, the probability of disease invading the initially infected host group increases, but additional criteria are important to determining the probability of the spreading of the disease to other groups.
Green et al. [20] related deterministic meanfield models to network models, taking into account the manner in which the contact rate and infectiousness change over time. For a node with exactly m connections, the expected number of secondary infections at infection age u is given by
where β is the transmissibility and k is the mean number of connections per node. This model applies when the rate of infectious contact is independent of k.
If there is a constant rate of generation of new cases, then the expected number of secondary infections in an infectious period of length u is given implicitly by
8
where C IS (t) denotes the contact per susceptible neighbour and ψ(t) denotes the infectiousness at time t after infection. Kao [21] showed that novel pathogens may evolve towards a lower R 0 , even if this results in pathogen extinction. This is because the presence of exploitable heterogeneities, such as high variance in the number of potentially infectious contacts, increases R 0 ; thus, pathogens that can exploit heterogeneities in the contact structure have an advantage over those that do not. The exploitation of heterogeneities results in a more rapid depletion of the potentially susceptible neighbourhood for an infected host. While the low R 0 strategy is never evolutionarily stable, invading strains with higher R 0 will also converge to the low R 0 strategy if not sufficiently different from the resident strain. This is in contrast to the conventional belief that the emergence of novel pathogens is driven by maximisation of R 0 .
In a randomly mixed epidemiological network, R 0 can be approximated by
where l in and l out are, respectively, the number of inward and outward "truly infectious" links per node; the angled brackets represent the expected value of the relevant quantity [22] . Meyers [23] showed that in a contact network framework,
where T is the mean probability of transmission between individuals and k and k 2 are the mean degree and mean square degree of the network. Here, R 0 depends explicitly on the structure of the network (i.e., on k and k 2 ). A single pathogen may, therefore, have very different transmission dynamics depending on the population through which it spreads. If two networks have the same mean degree, k, then the one with the larger variance in degree, k 2 − k 2 , will be more vulnerable to the spread of disease. In compartment models, infected hosts are assumed to have potentially disease-causing contacts with random individuals from the population according to a Poisson process that yields an average contact rate of β per unit time. The mass-action assumption of compartmental models is tantamount to assuming that the underlying contact patterns form a random graph with a Poisson degree distribution. Estimates of R 0 that assume a mass-action model may, therefore, be invalid for populations with non-Poisson contact patterns and, in particular, will underestimate the actual growth rate of the disease in highly heterogeneous networks. [24] noted that if R 0 > 1 in an agent-based model then, due to the stochastic nature of interactions, it is possible that no epidemic occurs or that it ends early if, by chance, the few initially contagious individuals recover before generating new cases.
Schimit and Monteiro [25] showed that in an individuallevel model, R 0 cannot be uniquely determined from some features of transient behaviour of the infective group. The value of R 0 can be unambiguously determined from the asymptotical stable stationary concentrations, but this relies on waiting for the system to reach its permanent regime, which is not feasible in practice. The same value of R 0 can be associated to networks with distinct values of clustering coefficients and average shortest path length. This result can affect the evaluation of the effectiveness concerning different strategies employed for controlling a disease. Because distinct values of topological properties can produce the same value of R 0 in a model considering the spatial structure of the contact network, it is difficult to evaluate the effective contribution of each control measure. This is because the correspondences among R 0 and the topological properties of the contact network are not one-to-one.
Cross et al. [26] showed that when R 0 is based on data collected from simulated epidemics mimicking epidemiological contact-tracing data, R 0 can be substantially greater than one and yet not cause a pandemic. In populations with social or spatial structure, a chronic disease is more likely to invade than an acute disease with the same R 0 , because it persists longer within each group and allows for more host movement between groups.
Under the settings where the rate of host population turnover was negligible relative to the rate of disease processes of infection and recovery, they showed that R 0 > 1 was insufficient for disease invasion when the product of the average group size and the expected number of betweengroup movements made by each individual while infectious was less than 1.
Smith? et al. [15] examined a metapopulation model with travel between two regions, with reproductive ratios R (0) 0,1 and R (0) 0,2 for each region in the absence of travel, and R 0,1 and R 0,2 when only susceptibles travel, but infectives do not. They showed that if both R (0) 0,1 < 1 and R (0) 0,2 < 1, then there are conditions on the travel of susceptible such that R 0,1 > 1 and R 0,2 < 1. Thus, a disease which would otherwise be eradicated in both regions could be sustained in one of the regions if there were sufficient travel of the susceptibles (not infectives). Furthermore, if R (0) 0,1 < 1 and R (0) 0,2 > 1, then there are conditions on the travel of susceptibles such that R 0,1 > 1 and R 0,2 > 1. Thus, if one region sustains the disease on its own, while the other does not, then sufficient travel of susceptibles (not infectives) could sustain the disease in both regions.
. Althaus et al. [27] examined an age-dependent partial differential equation model of in-host HIV infection. They showed that
where the denominator is the Laplace transform of the generation time distribution g(a) and r is the growth rate. They found that estimates for R 0 were generally smaller than those derived from the standard model when the generation time was taken into account.
Computational and Mathematical Methods in Medicine 9
When stochastic effects, such as those inevitably found in nature, are included, the threshold at R 0 = 1 may be disturbed. This includes assumptions about the distribution of transition times (assumed to be exponential in most models) as well as variations in individual parameters. Heffernan and Wahl [28] derived improved estimates of R 0 for situations in which information about the dispersal of transition times is available to the clinical or epidemiological practitioner. Rather than rederiving R 0 for a number of models (SIR, SEIR, etc.), they introduce a "correction factor", φ: the ratio of R 0 when the lifetimes are nonexponentially distributed to the value of R 0 that would be calculated assuming exponential lifetimes. They were able to derive limiting values of φ and used this to gauge the sensitivity of R 0 to dispersion in the underlying distributions.
By combining the movement of hosts, transmission with groups, recovery from infection and the recruitment of new susceptibles, Cross et al. [29] expanded the earlier analysis of Cross et al. [26] to a much more broader set of disease-host relationships, exploring settings where the duration of immunity ranges from transient to lifelong or where the demographic processes occur on comparable (or faster) timescales to disease processes. The focus of this study was to investigate how recruitment of susceptibles affects disease invasion and how population structure can affect the frequency of superspreading events (SSEs). They found that the frequency of SSEs may decrease with the reduced movement and the group sizes due to the limited number of susceptibles available.
The hierarchical nature of disease invasion in host metapopulations is illustrated by the classification tree analysis of the model results. Firstly, the pathogen must effectively transit within a group (R 0 > 1), and then, the pathogen must persist within a group long enough to allow for movement between different groups. Hence, the infectious period, group size and recruitment of new susceptibles are as important as the local transmission rates in predicting the spread of pathogens across a metapopulation. It should be noted that in 35% of simulations when R 0 was greater than one, the disease failed to invade.
Tildesley and Keeling [30] examined whether R 0 was a good predictor of the 2001 UK Foot and Mouth disease. They concluded that R 0 explained just 29.3% of the standard deviation of the epidemic impact. They also noted that R 0 = 1 did not act as a threshold: at the value of R 0 = 1, only 20% of initial seedings generated epidemics; this probability increased to around 50% for the largest reasonable R 0 values. When heterogeneities exist in the population, infection is most likely to become focused within the high-risk individuals who are both more susceptible and more infectious. This highlights the stochastic nature of the disease in its early stages and the dependence of the ensuing epidemic on favourable local conditions in the neighbourhood of the initial infection.
The probability of extinction, assuming exponentially distributed infectious periods, was
when an epidemic began with a single infected individual. Thus, if R 0 ≤ 1, then extinction occurs with probability 1, but if R 0 > 1 then extinction occurs with some probability. See Chiang [31, Chapter 4] for more discussion.
In this section, we note a variety of problems with R 0 that are not covered in the previous sections. These include problems with the underlying structure of compartment models, the mismatch between an individual-based parameter and a population-level compartment model, and the failure of R 0 to accurately measure an outbreak of a new disease. Breban et al. [32] argued that in order to associate an R 0 to a model of ODEs, an individual-level model (ILM) which is compatible to the ODE model must be developed; only then can the R 0 of the ILM be unambiguously calculated. These ILMs are growing (not static) network models, with individuals added to a network of who infected whom based on global or local network rules. Then, R 0 is computed as the limit of the average number of outgoing links of individuals in a node that no longer accepts new links, as time goes to infinity. They showed that a broad range of R 0 values were compatible with a given ODE model.
For example, consider the basic model
where β is the transmissibility and μ is the disease death rate; note that the transmission term is equivalent to the standard term βSI/(S + I) when the depletion of susceptibles is negligible so that S/(S + I) ≈ 1. The expected R 0 value from this model using other methods (such as the next-generation method) is
The corresponding ILM consists of an infection rule, where an individual joining the infectious pool is infected by an infectious individual who is uniformly randomly selected, and a removal rule, where a uniformly randomly selected individual leaves the infectious pool. The flow of newly infected individuals is βI(t). Thus, the flow per alreadyinfected individual is β. Since the removed individuals are randomly sampled from the infectious individuals, the average length of the infectious period equals the time expectation of the infectious period, which is 1/β (rather than 1/μ, as calculated from the next-generation method). It follows that R 0 = 1, which is independent of the transmission and death rates from the corresponding ODE. Thus, in this case, R 0 does not signal epidemic growth as anticipated from other methods. Roberts [3] noted three fundamental properties commonly attributed to R 0 : (i) that an endemic infection can persist only if R 0 > 1, (ii) R 0 provides a direct measure of the control effort required to eliminate the infection, and (iii) pathogens evolve to maximise their R 0 value.
He demonstrated that all three statements can be false. The first, as we have noted, can fail due to the presence of backward bifurcations. The second can fail when control efforts are applied unevenly across different host types (such as a high-risk and a low-risk group), since R 0 is determined by averaging over all host types and does not directly determine the control effort required to eliminate infection.
The third can fail when two pathogens coexist at a steady state that exists and is stable whenever both single-pathogen steady states exist but are unstable. In this case, the order in which the pathogens are established in the host population matters. The established parasite has a role in determining a modified carrying capacity and the pathogen with the largest basic reproductive ratio does not necessarily exclude the other.
Breban et al. [33] showed that two individual-level models having exactly the same expectations of the corresponding population-level variables may yield different R 0 values. They showed that obtaining R 0 from empirical contact-tracing data collected by epidemiologists and using this R 0 as a threshold parameter for a population-level model could produce misleading estimates of the infectiousness of the pathogen, the severity of an outbreak, and the strength of the medical and/or behavioural interventions necessary for control. Thus, measuring R 0 through contact tracing (as generally occurs during an outbreak investigation) may not be a useful measure for determining the strength of the necessary control interventions.
Many different individual-level processes can generate the same incidence and prevalence patterns. Thus, assigning a meaningful R 0 value to an ODE model without knowledge of the underlying disease transmission network may be impossible. Only an epidemic threshold parameter can be used to design control strategies. Since R 0 fails to possess this threshold quality, its usefulness may be vastly overstated.
Meyers [23] compared the theoretical calculation of R 0 with observed SARS data from China and showed that estimates of R 0 seemed incompatible. The basic reproductive rate has two critical inputs:
(1) intrinsic properties of the pathogen that determine the transmission efficiency per contact and the duration of the infectious period,
(2) the patterns of contacts between infected and susceptible hosts in the population.
While the first factor may be fairly uniform across outbreaks, the second may depend significantly on context, varying both within and among populations. The problem with the SARS estimates stems from the mass-action assumption of compartmental models; that is, that all susceptible individuals are equally likely to become infected. When this assumption does not hold, the models may yield inaccurate estimates or estimates that do not apply to all populations. R 0 estimates for SARS in the field were based largely on outbreak data from a hospital and a crowded apartment building, with anomalously high rates of close contacts among individuals.
The author suggested that it might be inappropriate to extrapolate estimates for R 0 from specific settings such as these to the population at large. Conversely, since the population at large is also unlikely to satisfy mass-action requirements, it may also be concluded that R 0 is not a meaningful estimate of disease spread.
A number of alternatives have been offered over the years, due to recognised problems with R 0 . Older examples include the critical community size [5] , the group-level reproductive number, R * [34] , the type reproduction number [35] and the basic depression ratio [36] . See Heffernan et al. [2] for a summary of these methods. In this section, we review some of the more recent alternatives to R 0 .
The actual reproduction number, R a , is defined as a product of the mean duration of infectiousness and the ratio of incidence to prevalence [37] [38] [39] . R 0 coincides with R a when the transmission probability is constant but accounts for the more general situation when the transmission probability varies as a function of infection age (as happens in diseases such as HIV/AIDS).
The effective reproduction number R(t) measures the number of secondary cases generated by an infectious case once an epidemic is underway. In the absence of control measures, R(t) = R 0 S(t)/N, where S(t)/N is the proportion of the population susceptible [40, 41] . The estimation of reproductive numbers is typically an indirect process because some of the parameters on which these numbers depend are difficult or impossible to quantify directly. The effective reproductive number satisfies R(t) ≤ R 0 , with equality only when the entire population is susceptible [40] .
The effective reproduction number is of practical interest, since it is time dependent and can account for the degree of cross-immunity from earlier outbreaks. However, since it is based on the basic reproduction number, the effective reproduction number inherits many of the issues from R 0 .
Breban et al. [32] proposed Q 0 , the average number of secondary infections over the infectious population. The average number of secondary infections of actively infectious individuals, Q 0 (t), is computed as the average number of outgoing links of a node in the infected compartment at time t. Then,
when the limits exist. Unfortunately, R 0 (t) is never defined in the paper, limiting the usefulness of this formulation of R 0 . However, by analogy with Q 0 (t), R 0 (t) is the average number of outgoing links of a removed compartment at time t (Romulus Breban, personal communication).
For the SI model (32) , under the assumption that every infection is uniquely assigned as a secondary infection for either a removed or an infected individual,
where N i (t) = Their definition of R 0 evaluates the average number of secondary cases over removed individuals as the distribution of secondary cases becomes stationary. This definition does not imply a particular individual-level model; it depends exclusively on the structure of the disease-transmission network [42] . However, R 0 is not measured at the start of an epidemic, which may limit its usefulness during an initial outbreak.
Grassly and Fraser [43] demonstrated that standard epidemiological theory and concepts such as R 0 do not apply when infectious diseases are affected by seasonal changes. They instead define
where β(t) is the transmission parameter at time t and D is the average duration of infection. Thus, R 0 can be interpreted as the average number of secondary cases arising from the introduction of a single infective into a wholly susceptible population at a random time of the year. They noted that the condition R 0 < 1 is not sufficient to prevent an outbreak, since chains of transmission can be established during high-infectious seasons if Dβ(t) > 1. However, R 0 < 1 is both necessary and sufficient for longterm disease extinction.
Meyers [23] noted that in the case of networks, estimating the average transmissibility T may be more valuable than R 0 . This means reporting not only the number of new infections per case, but also the total estimated number of contacts during the infectious period of that case. Given the primary role of contact tracing in infectious disease control, these data are often collected. Unlike R 0 , T can be justifiably extrapolated from one location to another even if the contact patterns are quite disparate.
Instead of R 0 , the author offered a number of alternatives to determine whether an outbreak will occur, based on network modelling. These include the probability that an individual will spark an epidemic and the probability that a disease cluster will spark an epidemic.
The probability that an individual with degree k will spark an epidemic is equal to the probability that transmission along at least one of the k edges emanating from that vertex will lead to an epidemic. For any of its k edges, the probability that the disease does not get transmitted along the edge is 1 − T. The probability that disease is transmitted to the attached vertex but does not proceed into a full-blown epidemic is Tu, where u is the probability that a secondary infection does not spark an epidemic. Thus, the probability that an individual will spark an epidemic is 1 − (1 − T + Tu) k .
The probability that an outbreak of size N sparks an epidemic is
where p k is the relative frequency of vertices of degree k in the network.
Kao et al. [22] defined an epidemiological network contact matrix M whose elements m i j are either 1 or 0, depending on whether an infectious contact between nodes i and j is possible. The spectral radius of M is an alternative approximation for R 0 , which can be calculated via a weighted version of (28) .
This explicitly accounts for the full contact structure of the network, but the evaluation of extremely large, reasonably dense matrices (some highly active nodes may have hundreds of potentially infectious links) is difficult and time consuming, particularly when this evaluation process must be repeated multiple times. However, comparisons between the two approximations for subsets of a sheep network with several thousand nodes show little difference between R 0 and the spectral radius of M (typically less than 5%).
Kamgang and Sallet [44] used the special structure of Metzler matrices (real, square matrices with nonnegative off-diagonal entries) to define T 0 , an analytical threshold condition. T 0 is a function of the parameters of the system such that if T 0 < 1, the disease-free equilibrium is locally asymptotically stable, and if T 0 > 1, the disease-free equilibrium is unstable. T 0 has an association with R 0 , although it is a stronger condition; however, it has no direct biological interpretation. The algorithm for deriving T 0 , although highly mathematical in nature, allows computation of a threshold for high-dimensional epidemic models.
Huang [45] defined four reproductive numbers associated with four types of transmission patterns, each depending on z, the ratio of the mean infectious period to the mean latent period. These four reproduction numbers are the following:
(1) R I 0 , the minimal reproductive number associated with the slowest latency process and the fastest recovery process,
(2) R II 0 , the middle reproductive number associated with mean latency and recovery processes, (3) R III 0 , the maximal reproductive number associated with the fastest latency process and the slow recovery process, (4) R IV 0 , the largest reproductive number associated with the fastest latency process and the extremely slow recovery process.
All four reproduction numbers are strictly increasing functions of z and satisfy
These numbers allow a disease to be classified as mild (R I 0 < R 0 < R II 0 ) or severe (R III 0 < R 0 < R IV 0 ). Hosack et al. [46] noted that R 0 does not necessarily address the dynamics of epidemics in a model that has an endemic equilibrium. They used the concept of reactivity to derive a threshold index for epidemicity, E 0 , which gives the maximum number of new infections produced by an infective individual at a disease-free equilibrium. They also showed that the relative influence of parameters on E 0 and R 0 may differ and lead to different strategies for control.
If R 0 is derived from the next-generation operator so that
then the threshold for endemicity is
such that the system is reactive when E 0 > 1 and nonreactive when E 0 < 1. E 0 describes the transitory behaviour of disease following a temporary perturbation in prevalence. If the threshold for epidemicity is surpassed, then disease prevalence can increase further even when the disease is not endemic. This suggests that epidemics can occur even in areas where long-term transmission cannot be maintained. Reluga et al. [47] defined the discounted reproductive number R d . The discounted reproductive number is a measure of reproductive success that is an individual's expected lifetime offspring production discounted by the background population growth rate. It is calculated as
where δ is the discount rate, I is the identity matrix, and F and V are from the next-generation matrix decomposition. R d combines properties of both the basic reproductive number and the ultimate proliferation rate although it also inherits the nonuniqueness problems from the nextgeneration method. Nishiura [4] developed a likelihood-based method for estimating R 0 without assuming exponential growth of cases and offers a corrected value of the actual reproduction number. The author noted that R 0 is extremely sensitive to dispersal of the progression of a disease or variations in the underlying epidemiological assumptions.
Despite being a crucial concept in disease modelling, with a long history and frequent application, R 0 is deeply flawed. It is not a measure of the number of secondary infections, it is never calculated consistently, and it fails to satisfy the threshold property. Rarely has an idea so erroneous enjoyed such popular appeal. Why, then, are we so attached to the concept?
The answer is that R 0 , despite all its flaws, is all that we have. No other concept has so effectively transcended mathematics, biology, epidemiology, and immunology. No other concept is so general that it can be understood in terms of compartment models, network models, partial differential equations, and metapopulation models. "The number of secondary infections" has an intuitive appeal that outlasts even the inaccuracy of that statement when applied to the concept.
The threshold nature of R 0 is used to monitor and control severe real-time epidemics; control measures are often concluded if R 0 < 1 [48] , making the problems with R 0 more than just theoretical. Due to the inconsistencies in calculation, different diseases cannot be compared unless the same method was used to calculate R 0 ; if HIV has an R 0 of 3 and swine flu has an R 0 of 4, we cannot conclude that swine flu is worse than HIV if different methods were used to determine these values. All we can conclude is that both diseases have R 0 > 1; however, as we have demonstrated, this does not necessarily guarantee disease persistence.
Of the many different methods used to calculate R 0 , only the survival function reliably calculates the average number of secondary infections; however, this method is too cumbersome to use in most practical settings. The nextgeneration method is probably the most popular, yet it suffers from uniqueness problems and does not cope well with more than one disease state. Since R 0 is rarely measured in the field, it instead relies upon after-the-fact calculations to determine the strength of disease spread [2] . This limits its usefulness even further.
Policy decisions are being based upon the concept, with limited understanding of the complexity and errors that exist in the very structure of the concept. Funding decisions about where money should be best spent are based on estimates of R 0 , resources are directed towards one disease over another, and monitoring programmes are abandoned, their objectives only half-realised, because of R 0 . Lives may be saved or lost, based on this imperfect and inconsistent measure.
R 0 is a quantity that relates to the initial phase of an epidemic. This makes practical sense in terms of disease prevention. However, it is also used to guide eradication efforts when a disease is endemic. Some methods derive an eradication threshold from an equilibrium value that may not be attained for a very long time. This suggests that different measures are needed in different stages of an epidemic in order to characterise transmissibility and to guide intervention strategies. When a new pathogen emerges, a quantity describing the initial spread is useful. When a disease is endemic, a quantity that applies to the long-term equilibrium is more appropriate.
We note that although the definition of R 0 is broad, it is not a universal quantity that applies to all settings. In different settings, one should use a quantity that satisfies the following properties: that an endemic equilibrium only persist if R 0 > 1 and that R 0 provides a direct measure of the control effort required for eradication. The contact structure of the population, the variation of risk factors and the order of establishing parasites (if applicable) should accompany the identification of a meaningful R 0 .
What is urgently needed is a simple, but accurate, measure of disease spread that has a consistent threshold property and which can be understood by nonmathematicians. If R 0 is to be used, it must be accompanied by a declaration of which method was used, which assumptions are underlying the model (e.g. mass-action transmission) and evidence that it is actually a threshold, with no backward bifurcation. Without such caveats, the concept of R 0 will continue to fail.
illustrates the wide variety of values that are presented as being "the" R 0 value for a specific disease.
A.1. HIV. Bacaër et al. [49] developed a mathematical model of the HIV/AIDS epidemic in Kunming, China. They considered needle sharing between injecting drug users and commercial sex between female sex workers and clients. The basic reproductive ratio is expressed as
where the superscript "IDU" represents injecting drug users and "sex" represents sex workers. They estimate R IDU 0 = 32 and R sex 0 = 1.7. Abu-Raddad et al. [50] examined the population-level implications of imperfect HIV vaccines. They divided the population into two groups: unvaccinated and vaccinated, both of whom could become infected (albeit at different rates, depending on the efficacy of the vaccine). By forming two independent next-generation matrices, they derived R 0 and R 0V , the basic reproductive ratios for the unvaccinated and vaccinated populations, respectively. They note that the condition R 0V < 1 may not be sufficient for disease eradication, due to the possibility of a backward bifurcation. The two formulas are equivalent in the absence of the vaccine, assuming that risky behaviour does not change. They estimate R 0V = 1.4 in the absence of vaccine protection.
Gran et al. [39] argued that R 0 was inappropriate for long-lasting infections and instead defined the actual reproduction number R a (t). This is a time-dependent measure defined as the average number of secondary cases per case to which the infection was actually transmitted during the infectious period in a population. Thus,
where X(t) is the incidence rate at time t, Y (t) is the prevalence at time t, and D is the average length of the infectious period. They illustrated their results for a staged HIV/AIDS progression model for homosexual men in the UK and showed that R a was 13.81 and 17.01 in 1982 and 1983, respectively, but was much closer to 1 by the mid 1990s, due to decreases in risk behaviour. Smith? et al. [15] used the nonuniqueness of R 0 -like thresholds to evaluate the feasibility of eradicating AIDS using available resources. They developed a linear metapopulation model that has the same eradication threshold as more realistic models. They defined the threshold as
where s(K) is the maximal real part of all eigenvalues of K, the Jacobian matrix evaluated at the disease-free equilibrium; K represents the change of status of infected individuals in different regions. The model has the property that the disease will be eradicated if T 0 < 1 but will persist if T 0 > 1. The paper stresses that this quantity should be understood only as a threshold condition for eradication; the model from which it derives does not quantify the transient dynamics, the timecourse of the infection or the prevalence of the disease. However, they showed that the simpler version of the model has the same eradication threshold (T 0 ) as more realistic, nonlinear models. The mathematical analysis results, together with a simple cost analysis, used the threshold nature of R 0 to show that eradication of AIDS is feasible, using the tools that we have currently to hand, but action needs to occur immediately and globally.
A.2. Influenza. Chowell et al. [51] used four methods to calculate R 0 using data from the 1918 influenza pandemic in California. The four methods involved (1) an early exponential growth rate, (2) In particular, uncertainty during the early part of the epidemic leads to a wide range of estimates for R 0 (0.5-3.5, 95% confidence interval), but uncertainty decreases with more observations. Wallinga and Lipsitch [52] investigated how generation intervals shape the relationship between growth rates and reproductive numbers. For new emerging infectious diseases, the observed exponential epidemic growth rate r can indirectly infer the value of the reproductive number. The existence of a few different equations that relate the reproductive number to the growth rate makes such inference ambiguous. It is unclear which of these equations might apply to new infection. The authors showed that these different equations differ only with respect to their assumed shape of the generation-interval distribution. They found that the shape of the generation-interval distribution determines which equation is appropriate for inferring the reproductive number from the observed growth rate. By assuming all generation intervals to be equal to the mean, they obtained an upper bound on the range of possible values that the reproductive number may attain for a given growth rate. They also showed that it is possible to obtain an empirical estimate of the reproductive number by taking the generation-interval distribution equal to the observed distribution.
The authors illustrated the impact that various assumptions about the shape of the generation interval distribution may have on the estimated value of the reproduction numbers for a given growth rate using human infections with influenza A virus. Observed generation intervals for influenza A in a Japanese household study excluding possible coprimary and tertiary cases [53] estimated a mean generation interval of 2.85 days and a standard deviation of 0.93 days. Without specific assumptions about the shape of the generation-interval distribution, they found that the reproductive number of influenza A is larger than R = 1, but smaller than R = 1.77.
Chowell and Nishiura [54] reviewed quantitative methods to estimate the basic reproductive ratio of pandemic influenza. They use the intrinsic growth rate to estimate R 0 in the range from 1.36 to 2.07. They defined the effective reproduction rate
where A(t, τ) is the reproductive power at time t and infection age τ at which an infected individual generates secondary cases. If contact and recovery rates do not vary with time, then this simplifies to
where S(t) is the population of susceptibles at time t. They also outlined statistical methods of estimation and showed that the expected number of secondary transmissions is given by
where the pattern of secondary transmission follows a discrete probability distribution p k with k secondary transmissions. This approach accounts for demographic stochasticity, which can be crucial during the initial phase of an epidemic when the number of infected individuals is small. This has been applied to observed outbreak data for avian influenza, where extinction was observed before growing to a major epidemic [55] . Tiensin et al. [56] used flock-level mortality data and statistical back-calculation methods to estimate R 0 from an SIR model for the 2004 avian influenza epidemic in Thailand. They calculated R 0 to be between 2.26 and 2.64, depending on the length of the infectious period.
A.3. Malaria. Smith et al. [57] revisited the basic reproduction number of malaria and its implications for malaria control. Their estimates of R 0 are based on two more commonly measured indices called the entomological inoculation rate, which is the average number of infectious bites received by a person in a year, and the parasite ratio, which is the prevalence of malaria infection in humans. They made 121 estimates of R 0 for Plasmodium falciparum malaria in African populations. The estimates (which do not come from a mathematical model) range from around one to more than 3000; the median is 115 and the interquartile range is 30-815.
They then revised the formula of R 0 to adopt potential sources of bias when biting is heterogeneous and when there is some transmission-blocking immunity. Under the assumption that the transmission-blocking immunity develops, the estimates of R 0 ranged from below one to nearly 11,000, with a median of 86 and an interquartile range of 15-1,000. This is because transmission-blocking immunity and heterogeneous biting skew the probability that a mosquito becomes infected, per bite. They also showed that in small human populations, the classical formulas of R 0 approximate transmission when counting infections from mosquito to mosquito after one parasite generation, but they overestimate it from human to human.
A.4. West Nile Virus. Wonham et al. [11] examined the conflicting outbreak predictions for West Nile virus, showing how the choice of transmission term affects R 0 . They concluded that some transmission terms apply biologically only at certain population densities and showed that six common North American bird species would be effective outbreak hosts. All seven models considered shared a similar compartment structure, but with varying factors, such as incubation periods, loss of immunity, age structure, and so forth. They created a core model synthesised from all seven models and used the next-generation method to calculate
where β R is the transmissibility of the reservoir, d R is the death rate of the reservoir, d V is the death rate of the vector, N * V is the total vector population at the disease-free equilibrium and N * R is the total reservoir population at the disease-free equilibrium.
The first term under the square root sign represents the number of secondary reservoir infections caused by one infected vector. The second term under the square root sign represents the number of secondary vector infections caused by one infected reservoir. The square root gives the geometric mean of the two terms (see Section 2). Using reported minimum, mean and maximum estimates, 10,000 Monte Carlo estimates were run to generate an estimated distribution of R 0 . Mean numerical values for R 0 for house sparrows ranged from 25-1200.
However, these values are enormous, implying that a single sparrow would infect up to 1.44 million sparrows (i.e., 1200 × 1200) during its infectious lifetime. Cruz-Pacheco et al. [58] , for example, estimated R 0 = 5.08 for the house sparrow, using data from Komar et al. [59] .
A.5. Cholera. Hartley et al. [60] developed a compartment model of cholera incorporating both a hyperinfective state and a lower infective state. They used the average age of infection to estimate R 0 from data for four cholera outbreaks in developing countries during the nineties. R 0 values ranged from 3.1 in Indonesia (1993-1999) to 15.3 in Pakistan (1990 15.3 in Pakistan ( -1995 , with an average of 8.7. They then used the nextgeneration method to develop a theoretical estimate for R 0 that incorporates hyperinfectivity. They show that this raises R 0 from approximately 3.2 when there is no hyperinfectivity to 18.2 when the contact with hyperinfected water is similar to contact with nonhyperinfected water, but that R 0 could be significantly larger with more frequent contact with hyperinfected water.
A.6. Dengue. Nishiura [61] clarified the contributions of mathematical and statistical approaches to dengue epidemiology. This highlighted the practical importance of the basic reproduction number, R 0 , in relation to the critical proportion of vaccination required to eradicate the disease in the future. The author illustrated three different methods to estimate R 0 : (i) the final size equation, (ii) the intrinsic growth rate, and (iii) age distribution, with published estimates and examples. The author pointed out that, although the estimates of R 0 most likely depend on the ecological characteristics of the vector population, it would be appropriate to assume a serotype-nonspecific estimate for R 0 is approximately 10, at least when planning vaccination strategies in endemic areas.
A.7. Rift Valley Fever. Gaff et al. [12] modelled Rift Valley fever, a mosquito-borne disease affecting both humans and livestock that currently exists in developing countries but has the potential to be introduced to the western world. The disease can be transmitted both horizontally (from host species to host species via mosquitoes) or vertically (via parent-to-offspring transmission in mosquitoes). They expressed the basic reproductive ratio as
where R 0,V is the basic reproductive ratio for vertical transmission and R 0,H is the basic reproductive ratio for horizontal transmission. They used the next-generation method and uncertainty analysis to calculate R 0 = 1.19, with a range from 0.037 to 3.743.
A.8. Bluetongue. Gubbins et al. [13] examined bluetongue virus, an insect-borne infectious disease of ruminants in Europe. They used the next-generation method to determine R 0 and then Latin Hypercube sampling and partial rank correlation coefficients to assess the effect of parameter variation. Median values for R 0 were 0.81 (cattle only), 0.73 (sheep only) and 0.55 (both cattle and sheep), implying that the disease should not persist. However, the corresponding maximum values were 18.77, 15.48, and 12.82, respectively, suggesting that variation in parameters can have an enormous impact on the outcome, including persistence of the disease. The most significant changes in the estimation of R 0 were due to the mosquito biting rate, the extrinsic vector incubation period, the vector mortality rate, the probability of transmission from host to vector, and the ratio of vectors to cattle and sheep.
A.9. Plant Pathogens. van den Bosch et al. [6] described a systematic method to calculate the basic reproduction ratio from knowledge of a pathogen's life cycle and its interactions with the host plant. They developed a system of linear difference equations and rearranged the dominant eigenvalue to find R 0 . A fraction q of infected spores are deposited in location 1, while a fraction 1 − q are deposited in location 2. This results in R 0 = q γ 1 α 1 τ 1 ρH + 1 − q γ 2 α 2 τ 2 ρH , (A.9)
where the first term represents the number of spores that successfully germinate in location 1 and the second term represents the number of spores that successfully germinate in location 2. Here, γ i is the probability that a spore deposited on location i will germinate, α i is the number of spores produced per time unit on location i, τ i is the infectious period of a spore-producing lesion on location i, ρ is the probability that a spore is deposited on a susceptible site, and H is the density of susceptible sites in a host population.
They included a separate box explaining pitfalls in calculating the basic reproductive ratio from nonlinear models and noted its nonuniqueness, calculating two distinct values for R 0 . However, they were unable to decide which value was correct. The authors conclude by stating that "Although nonlinear differential equations are invaluable tools for studying epidemics, they should not serve as the primary basis for the determination of R 0 ." Cunniffe and Gilligan [62] considered the effects of host demography upon the outcomes for invasion, persistence and control of pathogens in a model for botanical epidemics. They used the next-generation method to calculate
partitioned into primary and secondary infection. These values are unaffected by host demography. However, the endemic level of infection is highly sensitive to changes in R 0 when R 0 > 1. If R 0 is increased by shortening the infectious period, then the endemic level of infection increases monotonically. However, if increases in transmission rates or decreases in the decay of free-living inoculum increase R 0 , then the endemic level of infection first increases (for 1 < R 0 < 2) but then decreases (for R 0 > 2). Consequently, increasing the intensity of control can result in more endemic infection. Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ(2) = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs. Influenza A viruses (IAVs), members of the Orthomyxoviridae family, are highly contagious to a variety of avian and mammalian species. IAVs cause seasonal influenza epidemics annually and recurring pandemics with severe consequences for public health and global economy [1, 2] . At least three IAV-pandemics emerged in the last century (1918 A/H1N1, 1957 A/H2N2, and 1968 A/H3N2). The 1918 Spanish flu was the most serious influenza pandemic that killed over 50 million people worldwide [3] . The latter two pandemics, although mild compared to the 1918 incidence, resulted in significant mortality, with close to 2 million and 1 million deaths, respectively [4] . The latest pandemic influenza, and newest global health challenge, occurred in 2009 due to the emergence of an A/ H1N1 pandemic IAV (H1N1pdm virus). The H1N1pdm virus has been detected in more than 214 countries and territories and has caused 18,389 deaths as of July 30, 2010 [5] .
The viral genome of IAV consists of eight single-stranded negative sense RNA segments that encode at least 11 viral proteins, including two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [6] . Based on the antigenic properties of HA and NA, IAVs have been classified into 16 HA subtypes and 9 NA subtypes [7] . All 16 HA subtypes have been identified in avian species, while only 6 HA subtypes (H1, H2, H3, H5, H7 and H9) are known to infect human beings [8, 9, 10] . H1, H2 and H3 subtypes have caused pandemics, while H1 and H3 also dominate seasonal epidemics together with influenza B virus.
HA, encoded by segment 4 of the IAV genome, is a glycoprotein of approximate 560 amino acid. The biologically active HA is a homologous trimeric molecule that is attached to the virion membrane through its carboxy terminus [11] . HA plays a critical role in the pathogenesis of IAVs. HA mediates IAVs' binding to the cellular receptor N-acetylneuraminic (sialic) acid as well as the subsequent membrane fusion process [12] . HA also stimulates host protective immunities, specifically the production of neutralizing antibodies. The generation of anti-HA neutralizing antibodies has been the major target for influenza vaccine development [11, 13] . Due to its specificity in immune response, HA is also an important target for IAV subtyping using immunoassays [7, 14] .
Active serological surveillance for viral antibodies is of great importance for influenza control and prevention. Several IAV subtype-specific serological tests have been developed. At present, subtyping of IAV mainly relies on a hemagglutination inhibition (HI) test using HA and NA subtype-specific reference sera [15] . However, there are a number of drawbacks to HI testing. This assay is 1) relatively laborious; 2) low in sensitivity; 3) requires preparation of antigen from viable viruses which are potentially hazardous and 4) contains low signal to noise ratio, e.g. the assay exhibits inter-variability and subtype cross-reactivity [16, 17] . Moreover, the HI test can be confounded by steric hindrance from NA antibodies, leading to nonspecific inhibition and misidentification [18] .
Microneutralizing test is an alternative method to type and subtype influenza viruses. However, due to the needs of cell culture process, this method is labor-intensive and requires biological safety containments (particularly for high pathogenic strains). As such, it is not suitable for large scale investigations [19, 20] . Recently, subtyping of IAV antibodies using different categories of ELISA assays have also been reported [16, 17, 21] . However, present ELISA assays mainly rely on an HA antigen, which can lead to nonspecific detection to some extent due to the possible cross-reaction of different subtypes [22, 23] .
Virus-derived epitopes are useful tools to accurately evaluate immune response and to differentiate which responses are specific or due to cross-reactivity [24, 25, 26] . Several studies have reported the existence of HA subtype-specific as well as inter subtypeconserved epitopes [27, 28, 29] . ELISA assays based on epitopes that are highly conserved and specific for one certain HA subtype will be useful for rapid and simple subtyping of IAVs. Such epitopes in IAVs have not been fully addressed although many studies have been performed. In the present study, we report the successful identification of a new epitope, which is highly conserved among the majority of IAV strains of H1 subtype. Moreover, we developed an ELISA assay for H1 antibody subtyping based on this epitope. Results derived from this new assay correlate with results obtained through the use of HI test.
To identify the immunodominant epitopes in the HA protein, a peptide scanning assay was performed. A set of 50 peptides with five residues overlapping with the adjacent peptides spanning the ectodomain sequences of the HA protein of the H1N1pdm virus strain A/California/04/2009 were synthesized. The binding between these peptides and the convalescent serum samples from 11 H1N1pdm patients were examined by ELISA using these peptides as coating antigens. Five of these peptides (P3, P5, P15, P16 and P31) were found to react well with the sera tested. These peptides corresponded to the sequences of amino acid (aa) residues 38-52, 58-72, 158-172, 168-182, and 318-332 in the HA molecule, respectively ( Fig. 1A and Table 1 ). Among them, the P3 peptide reacted with 54.5% (6/11) of the sera, the P15 and P16 peptides reacted with 81.8% (9/11) of the sera, while the P5 and P31 peptides reacted with 100% (11/11) of the sera. These data indicated that these peptides may contain H1N1pdm virus B cell epitopes.
To visualize the location of the peptides on the HA protein, we mapped the peptides on the crystal model of this protein (Fig. 1B) . The various colors in Figure 1B represent the different peptides.
Although P3 (residues 38-52, indicated by blue) and P31 (residues 318-332, indicated by red) are parts of HA1 in primary sequence, they are located in the middle of helix A and B in the trimeric structure and are partially surface exposed. P5 (residues 58-72, indicated by magenta) seems to be a dispatch that links the stem region and the globular region and is fully surface exposed (Fig. 1B) . P15 and P16 (residues 158-172 and 168-182, indicated by orange) are located in the receptor binding domain [11] .
To confirm the immunogenicity of these peptides in vivo, we analyzed sera derived from peptide-immunized mice. The five positive peptides and two control peptides (P6 and P30) were coupled with keyhole limpet hemocyanin (KLH) and were used to immunize BALB/c mice ( Table 1 ). The antisera were collected five days after the third immunization and titrated by ELISA using corresponding peptide as a coating antigen. Our results showed that all of the peptide conjugates except P15 induced potent antibody titers. The endpoint titers of antisera in ELISA from mice immunized with P3, P5, P6, P16, P30, and P31 peptides were 1:6,400, 1:51,200, 1:51,200, 1:12,800, 1:51,200, and 1:25,600, respectively ( Fig. 2A) . These data indicate that most of the positive peptides elicite humoral immunity and are highly immunogenic in mice.
To confirm that these antibodies can recognize the HA antigen, the reactivity of the anti-peptide sera were evaluated by Western blot and ELISA against the purified HA0 protein of H1N1pdm virus. Our data demonstrate that sera against P3, P5, and P31 but not those against P6 and P30 (controls) react to the HA0 protein ( Fig. 2B and 2C ). The anti-P16 sera did not react to the HA0 protein, although it exhibited a high ELISA reactivity to the HA0 protein ( Fig. 2B and 2C ). Taken together, our results demonstrate that P3, P5, and P31 peptides contain dominant epitopes of H1N1pdm virus. We then characterized these three peptides in the following studies.
To determine if the epitopes identified in this study can stimulate neutralizing antibodies, a HA-pseudotype neutralization test was performed against the anti-peptide sera using the H1N1pdm pseudotyped lentivirus. None of the sera against P3, P5, P16, and P31 could efficiently inhibit (90% inhibition [30] ) the entry of H1N1pdm HA pseudotypes ( Figure 2D ), indicating that these epitopes do not contain neutralizing activity.
Western blot analysis was used to determine the specificity of the epitopes present in the peptides P3, P5, and P31. The H1-H16 recombinant HA proteins were obtained by transient expressions of corresponding genes by the pCAGGS vector in 293T cells. The lysates of these cells were used to examine the specificity of antibodies elicited by peptide-conjugates. As shown in Fig. 3A , the anti-P3 serum reacted with H1 (including 07H1 and 09H1 viruses), H2, H5, and H6 HA proteins, while anti-P5 and anti-P31 sera only reacted with the H1 HA proteins. These findings indicated that P5 and P31 may contain H1-subtype specific epitopes.
To evaluate the subtype-specificity of epitopes in P5 and P31 further, additional HA proteins of three epidemic human strains from different years (1918, 1934 and 1977) as well as a swine strain were expressed by pCAGGS vector in 293T cells. The reactivity of anti-P5 and P31 sera with the cell lysates was determined by Western blot analysis. Our results showed that the anti-P5 serum strongly reacted with all of the six H1 HA proteins in a manner similar to an antibody against the HA1 of H1N1pdm virus (Fig. 3B ). We found that the anti-P31 sera reactivity was weak against HA proteins from the 1918 and 1977 virus strains (Fig. 3B ). These data indicated that the epitopes in P5 and P31 peptides are relatively conserved among H1-subtype IAVs though these viruses have circulated in the world for almost a century.
To test if anti-P5 sera cross-react with influenza type B virus, the reactivity of anti-P5 sera with two representative influenza type B virus strains (B/hubeiwujiagang/158/2009,Yamagata lineage and B/heilongjianghulan/116/2010, Victoria lineage) and an influenza type A virus strain (A/H1N1/PR8/34) was examined by Western blot analysis. The results showed that anti-P5 serum reacted well with A/H1N1/PR8/34 virus but not with influenza type B virus strains (Fig. 3C) , further confirming the specificity of the epitope in P5.
To determine the conservation of the identified epitopes among IAVs, the aa sequences of P3, P5, and P31 were aligned with the corresponding aa sequences of all the 16 subtype HAs available in the GenBank. Fig. 4 is a representative of the alignment analysis, showing that P5 is identical to HA of H1 subtype strains. P3 is identical to HA of the H1 subtype, as well as highly identical to HA of the H2, H5, and H6 subtypes. These data are consistent with the specificity analysis by Western blot (Fig. 3A) . Although anti-P31 antibody only recognizes the H1-subtype HA, it is similar to multiple subtypes.
To assess the identity levels of P5 and P31 sequences among the known IAV strains, in silico coverage analysis was performed. This analysis showed that the P5 peptide sequence could be identified in 91.6% (9860/10767) of the H1-subtype HA sequences available in the Influenza Research Database (http://www.fludb.org) ( Table 2) . Notably, this sequence scarcely presented (3.3% 792/23895) among the HAs of H2-H16 subtype IAVs. However, despite a high identity in the H1 HA proteins (93.1%), the peptide sequence of P31 also presented among the HAs of H2-H16 viruses (78.8%).
Taken together, these findings indicate that the P5 peptide is H1-subtype specific and is conserved among H1 virus strains.
To define the epitope contained in P5 precisely, a peptideinhibition ELISA was performed. This experiment is reliable and is a standard methodology to determine the fine specificity of antigen-antibody reactions [31, 32] . A panel of short peptides derived from P5 (N6-N14, with C-terminus truncation; and C6-C14, with N-terminus truncation) were used to block the binding of anti-P5 antibody to coated P5. As shown in Fig. 5 , antibody induced by the P5-KLH conjugate was inhibited by peptide N10-N14 and the parental peptide P5 to similar extents, whereas peptides N6-N9 only showed inefficient inhibition even at high molar concentrations. A similar pattern of inhibition was observed with the C-terminal conservative derivatives. Peptides C12, C13, C14, and the parent peptide P5 demonstrated comparable and efficient inhibition, whereas only slight inhibition was observed in peptides C6-C11 (Fig. 5B) . Since the amino acid sequence LRGVAPL overlapped both peptides N10 and C12 (Fig. 5) , we speculate that this sequence met the minimum requirements of binding to the anti-P5 antibody. However, the synthetic peptide LRGVAPL did not block the binding between P5 peptide and its antibody, nor did it directly bind to the P5 antibody (Fig. 6 ). As P5 (1918, 1934, 1977, 2007, 2009 ) and a swine H1N1 strain. 293T cells transfected with an empty vector was used as a control (Ctrl). b-actin was used as a loading control. For the backgrounds of various subtype IAV strains, see 
Inspired by the high specificity of P5 among the H1-subtype viruses, we developed an indirect ELISA assay using the P5 peptide to evaluate its performance as a diagnostic tool for H1 antibodies. The HI test was used as a reference method. As shown in Table 3 , the overall agreement of these two methods was 87%, showing that the two methods have good correlation (Pearson correlation coefficient R = 0.741). The sensitivity and specificity of peptide-ELISA versus HI test was 96.5% and 74.4%, respectively, indicating the potential of the peptide-ELISA method in detecting antibody against H1-subtype IAVs.
In the present study, we identified immunodominant linear B cell epitopes on the H1N1pdm virus HA protein by a peptide scanning approach using H1N1pdm patients sera. We confirmed that an unidentified epitope was highly conserved among H1 subtypes viruses and showed a good correlation with results obtained using the HI test. These findings demonstrate the potential of epitope-based antibody detection in IAV diagnosis and surveillance.
IAV escapes the human immune system by continuous antigenic drifts and occasional antigenic shifts [33] . Attempts to develop universal vaccines and reliable diagnostic tools based on conserved epitopes of IAV are big challenges. Several epitopes that can elicit broad spectrum neutralizing antibodies have been identified recently. For example, Sui et al. identified a universal neutralizing epitope for group 1 HA [34] . Yoshida et al. reported a universal epitope in antigenic site B shared by H1, H2, H3, H5, H9, and H13 subtypes [35] . All these epitopes are conformationdependent. In this study, we identified two epitopes (P5 and P31) which have not been identified previously (Fig. S1 ). The P5 (aa58-72) seems to be a dispatch that links the stem region and the globular region and is fully exposed on the surface, while P31 (aa 318-332d) is located in the middle of helix A and B on HA2. In contrast to previous studies, we found P5 to be a linear B cell epitope. Our data demonstrate that this epitope is highly conserved among H1 viruses (9860/10767, 91.6%). Because viral mutants that are resistant to conformational epitopes are more easily generated, the conserved linear epitope is more suitable for differentiating subtypes than conformational epitopes [33] . Hence, the epitope in P5 provides a new target for reliable diagnostics of H1-subtype IAVs.
Antigenic sites in IAV HA proteins of H1, H2, and H3 subtypes had previously been characterized by sequence analysis on antigenic variants and amino acid substitutions. These previously identified antigenic sites were mainly located in the globular head in the three-dimensional structure of the HA1 subunit of the HA molecule [36, 37, 38, 39] . For instance, five antigenic sites have been identified in HA of influenza virus A/PR/8/34, a well-known reference strain of H1N1 IAV [39] . Recently, several epitopes were identified in the HA2 unit [34, 40, 41] . Together with these reports, our results indicate that there are more epitopes than what we have imaged and the epitopes of IAV need to be further characterized.
The difference between our findings and previously identified epitopes can be explained by the difference of screening method used between our study and those of others. In previous studies, monoclonal antibodies from murine hybridoma cells were used to identify antigenic sites, while in this study we used a peptide scanning approach, which involves overlapping peptide library and human convalescent antisera-a strategy that is widely used for viral epitope identification [27, 42] . Given the fact that viral antigen can be recycled and presented as short peptides with different conformation during humoral immune response and these short peptides can be selected by B cell clones [43] , and that convalescent sera from patients were much more complex than monoclonal antibodies from mice and can reflect the real immune responses during viral infection [44] , our approach adds to the available techniques currently being used to identify linear epitopes in serologic tests. Because HA pseudotyped lentivirus has been widely applied in the study on neutralizing antibodies against IAVs [30] , we used this method to evaluate if the epitopes identified in this study could stimulate neutralizing antibodies. Our data showed that these epitopes could not elicit neutralizing antibodies in pseudovirion neutralizing assays due to their linear nature. Previous studies have shown that most neutralizing epitopes are conformation dependent [34, 35] .
The length of B cell epitopes can vary from 5 to 20 amino acids [45, 46] . To map the epitope contained in P5, we performed a peptide-inhibition ELISA using a series of N-terminal and Cterminal truncated peptides. However, we found that the fulllength P5 (15 aa in length) rather than truncated peptides showed strongest binding to the corresponding antibody ( Fig. 5 and Fig. 6 ). As peptides N10 and C12 are the shortest truncated P5 that can bind to anti-P5 antibody and share a core sequence of LRGVAPL, we tested whether this sequence could be the epitope. However, the synthetic peptide LRGVAPL did not block P5-antibody interactions nor bind P5 antibody (Fig. 6) . Thus, we speculate that adjacent amino acids to this sequence are also involved in the binding of antibody elicited by P5-KLH conjugates. The concept of using linear epitopes in influenza virus diagnostics and control has not been extensively investigated. In a recent study, an epitope-blocking ELISA, which can universally detect antibodies to human H5N1 virus, has been developed [17] . Our results show that a peptide-ELISA based on the highly conserved H1 subtype-specific epitope can also be used for the detection of H1 antibodies, displaying good correlativity with the HI test. Our results indicate the potential of the P5 epitope in H1subtype IAV diagnosis. However, the performance of this assay needs to be further evaluated in studies with large scale samples.
In conclusion, our data provide evidence that the H1 subtype HA harbors more epitopes than what has been found previously. The conservation of an epitope (P5, aa 58-72) in the H1-subtype HA of IAV and its near complete absence in other subtypes indicate that this epitope meets the critical requirements for diagnosis of H1 subtype influenza virus infections. The peptide-ELISA developed in our study may be applicable for serodiagnosis and may serve as a universal diagnostic tool for H1subtype IAV surveillance.
To screen the H1-subtype specific epitopes, a set of 50 peptides spanning the amino acid sequences of the HA protein ectodomain of pandemic A/H1N1 2009 (H1N1pdm) influenza virus strain A/ California/04/2009 were synthesized. Each peptide is 15 amino acids in length with five residues overlapping with the adjacent peptides [46] (Fig. 1) . The peptides selected for immunization experiments are shown in Table 1 . These peptides were conjugated with a carrier protein, the keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO), to improve their immunogenicity [47] . As the water solubility of peptides P5, P15, P16 and P31 were too low to conjugate with KLH directly, these peptides were first linked to 6-aminocaproic acid and then to the tripeptide KKC prior to being conjugated with KLH [48] . In addition, a family of short peptide homologs to the P5 peptide was also synthesized to fine map the epitope contained in the P5 peptide (Fig. 5) . All of the peptides and their conjugates used in this study were synthesized by Sangon (Shanghai) Biotechnol Co., Ltd. (Shanghai, China). Each peptide was purified to homogeneity (.95% purity) by highperformance liquid chromatography and verified by mass spectrometry.
The reference influenza virus strains A/PR8/34 (H1N1) (abbreviated PR8), B/hubeiwujiagang/158/2009 (Yamagata lineage, abbreviated BY) and B/heilongjianghulan/116/2010 (Vic-toria lineage, abbreviated BV) were kindly provided by the Beijing Center for Disease Control and Prevention. The viruses were grown in MDCK cells as described elsewhere [5] . The titers of virus strains were determined by hemagglutination tests and expressed as hemagglutinating units (HAU). For Western blot analysis, the inactivated viruses were lyzed in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, pH 7.4) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN).
Serum samples were collected from 11 convalescent patients during the early 2009 H1N1 pandemic in Beijing. The diagnostic criteria for H1N1 influenza virus infection of these patients fully followed the WHO's descriptions. Sera from 10 healthy blood donors were used as negative controls. In addition, serum samples collected from 100 blood donors were recruited to evaluate the peptide-ELISA assay developed in this study. All these samples were kindly provided by the Beijing Municipal Center for Disease Control and Prevention (Beijing, China) and written informed consent was obtained from all participants. All samples were coded prior to analysis to ensure anonymity and the procedures were approved by the Institutional Medical Ethic Review Board of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences (Beijing, China).
The full-length cDNA fragments corresponding to H2-H16 HA subtypes of IAV were inserted into the pCAGGS vector (purchased from Addgene) to express entire HA proteins (unpublished data). For H1 proteins, HA gene representing human IAV strains from different years (1918, 1934, 1977, 2007 and 2009 ) and a swine influenza virus strain were expressed by inserting the corresponding cDNA fragments into the pCAGGS vector in a similar manner. For the details of these influenza virus strains, please refer to Fig. 4 . Recombinant plasmids were transfected into 293T cells (ATCC Number CRL-11268) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cells were harvested and lyzed 72 hours after transfection. The expression of HA proteins was verified by Western blot analysis using murine antibodies against corresponding HA1 proteins (unpublished data).
The reactivities of the synthetic HA peptides or purified HA0 protein (eENZYME LLC, Montgomery Village, MD) with the convalescent-phase sera from H1N1pdm patients and the serum samples from mice immunized with peptide conjugates were determined by ELISA. Briefly, each peptide (1 mg/well) or protein (0.1 mg/well) was used to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 M carbonate buffer (pH 9.6) at 4uC overnight. After blocking with 1% bovine serum albumen (BSA), the plates were incubated with indicated diluted serum samples (human or mouse) at 37uC for 2 hr, then washed four times with PBS containing 0.1% Tween 20 (PBS-T). Bound IgG antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG or anti-mouse IgG (Sigma) at 37uC for 1 hr. After four washes with PBS-T, the reaction was visualized by addition of the substrate 3,39,5,59-tetramethylbenzidine (TMB, sigma). Color development was stopped by the addition of 50 ml/well of 2 M sulphuric acid after 15 min. The optical density at 450 nm (OD 450 nm ) was measured by an ELISA plate reader (Molecular Devices, Sunnyvale, California). To evaluate the reactivity of the P5-derived short peptides with the anti-P5 antibody, peptide-inhibition ELISA assays were performed by adding dilutions of the peptides to a constant amount of the antibody elicited by the P5-KLH conjugates (1:5000 dilution). Maximum binding to antigen-coated wells was observed in the absence of a peptide inhibitor. The antibody bound was expressed as a percentage of the maximum level of binding.
Female BALB/c mice of 6-8 weeks old were immunized subcutaneously with various peptide-KLH conjugates mixed with Freund's Complete Adjuvant (Sigma) at 100 mg per injection. Boost injections were given at 2-week intervals with 50 mg antigen in Freund's Incomplete Adjuvant (Sigma) [49] . The antibodies were collected five days after the third boost. All the animal experiments were carried out in the facilities of the Institute of Laboratory Animal Sciences (ILAS), Chinese Academy of Medical Sciences (CAMS). All the experimental procedures were approved (permit number SLXKJ2009-0007) and supervised by the Animal Protection and Usage Committee of ILAS, CAMS.
At 72 hr post-transfection, the cells transfected with HAexpressing plasmids were harvested, pelleted, and lyzed in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, pH 7.4) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). Aliquots of cell lysates (50 mg) or virus lysates were blotted after 10% SDS-PAGE onto nitrocellulose membranes (Pall, Port Washington, NY). The membranes were blocked with 5% non-fat milk and then incubated with the primary antibodies indicated in the figures at 4uC overnight. This was followed by incubation with the goat anti-mouse IRDyeH Fluor 800-labeled IgG secondary antibody (1:10, 000) (Li-Cor, Lincoln, NE). After washing, the membranes were scanned by the OdysseyH Infrared Imaging System (Li-Cor) and analyzed with Odyssey software. The molecular sizes of the developed proteins were determined by comparison with the pre-stained protein markers (Fermentas, Maryland, CA).
HI test was carried out as described elsewhere [5] . RDE treated serum samples were inactivated at 56uC for 30 min and two-fold serially diluted at an initial dilution of 1:10. Twenty five ml of the diluted serum were incubated with 25 ml of the four hemagglutination units from reference influenza strains for 30 min at room temperature. The reference H1N1 IAV strains for HI test were A/ Tianjinjinnan/15/2009(H1N1) and A/California/04/2009 (H1N1), respectively. 50 ml of 1% chicken erythrocyte suspension was added to each well and incubated for 30 min at 4uC. Positive reactions were recorded when the HI antibody titer was equal to or greater than 40.
H1N1pdm virus pseudotyped lentiviruses were produced in 293T cells co-transfected with pNL4.3-R 2 E 2 , HA and NA constructs using a polyethylenimine (PEI)-based transfection protocol [50] . Cell culture supernatants were collected 48 hr post-transfection, filtered through a 0.45 mm-pore size filter (Millipore, Billerica, MA ) and used in pseudotype neutralization test. Serum samples, heat inactivated at 56uC for 30 min, were diluted 40-fold in culture medium and mixed with an equal volume of diluted H1N1pdm influenza pseudovirions. After incubation at 37uC for 1 hr, 100 ml of pseudovirions (containing 50 ng/mL of HIV p24 gag protein) and serum mixtures were added into 96-well plates that contained 293T cells grown for 24 hr at initial 1610 4 cells. Infectivity was evaluated at 72 hr post-infection by luciferase assay. The percentage of infectivity of pseudovirions treated by tested serum to that of negative serum (as control) was calculated. 90% reduction in infectivity by serum addition is considered to be neutralizing activity [30] . Tests were run at least as duplicates.
To assess the identity of the HA epitopes in IAVs, in silico analysis was performed by utilizing bioinformatics tools at the Influenza Research Database (http://www.fludb.org) [51] . The two programs used in this study were Search for Protein Sequences and Identify Short Peptides in flu proteins. The former program was used to define the number of total sequences in HA proteins according to the Subtype parameter (H1 or H2-H16). The latter program defined the number of hits (P5 or P31) in the H1 or H2-H16 total sequences. Because there are no standards for evaluating short peptide sequence homology, we chose the fuzzy match analysis to represent the identical level of a peptide sequence to HA proteins. The analysis type was chosen as fuzzy match, which meant .50% of characters were identical to the searched aa string. For example, entering GILGFVFTL may also find AILGFVFTI but not ALIGFVFSI.
The Pearson correlation coefficient was calculated by Pearson chi square test for crosstab tables using SPSS software. The sensitivity and specificity of the peptide-ELISA versus HI test was determined by ROC curve analysis using SPSS software. Figure S1 Localization comparison between the identified peptides and the classical five antigenic sites in stereo view. The HA monomer surface view of influenza virus A/PR/8/34 (PDB ID:1RU7) is shown and colored to illustrate the five antigenic sites (Sa, Sb, Ca1, Ca2, and Cb) and the identified peptides. From most membrane distal to proximal: P3 (blue), P31 (red), P5 (black), Cb (green), Ca1 (magenta), Ca2 (rainbow), Sa (yellow), and Sb (cyan). (TIF) Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3′-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼150 nt 3′-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3′ RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis. There are two types of exceptions to universality of the genetic code. In one, the meaning of a codon is globally reassigned in a context independent manner (1) . In the other, codon redefinition is in competition with standard decoding and it is codon context dependent (2) . Though there is an example where the meaning of a sense codon is redefined (3) , most cases of codon redefinition involve one of the three stop codons of the standard code (UGA, UAG or UAA) specifying an amino acid at least a proportion of the time that it is decoded. Where the significant feature of stop codon redefinition is to allow ribosomes to continue translation into a downstream open reading frame (ORF), rather than the identity of the amino acid specified, then it is generally termed stop codon readthrough (RT) (4) . In contrast, when selenocysteine or pyrrolysine are specified by UGA or UAG, respectively, then the important features are the special properties of these non-universal amino acids (5) (6) (7) . Both types of non-global codon redefinition are just one aspect of the variety of ways (collectively referred to as 'recoding') in which genetic readout can be dynamically altered in a site-or mRNA-specific manner (8, 9) .
Numerous studies have shown that the identity of the 3 0 -adjacent nucleotide influences stop codon leakiness in both prokaryotes and eukaryotes and, correspondingly, there is considerable bias in the identity of the nucleotide at this position for natural gene terminators (10) (11) (12) (13) (14) (15) (16) . Of great interest was the discovery that RT of the coat protein (CP) gene terminator of the phage Qb yields a greatly extended protein that is important for viral propagation (17, 18) . Shortly afterwards, studies that utilized purified yeast suppressor tRNAs in in vitro experiments found that several plant viruses, including tobacco mosaic tobamovirus (TMV), also utilize RT to express their replicase proteins (19) (20) (21) . Similarly murine leukemia gammaretrovirus (MuLV), whose relevant sequence is identical to that in xenotropic MuLV-related virus (XMRV), utilizes RT of the gag gene terminator to allow ribosomes to enter the pol gene and synthesize the Gag-Pol polyprotein that is the source of viral reverse transcriptase (22, 23) . MuLV Pol binds to the translation release factor, eRF1, and non-interacting mutants of Pol failed to synthesize adequate levels of Gag-Pol to permit replication (24) . This raises the possibility of temporal control of RT (25) . The efficiency of RT in the Drosophila gene kelch also appears to be developmentally regulated (26) . Two other Drosophila genes are known to employ RT-headcase and out-at-first-though another approximately 150 candidate cases have recently been identified via comparative genomic approaches utilizing sequences from 12 Drosophila species (27) (28) (29) . Although some of these candidates may actually be cases of alternative splicing or RNA editing, the indication is that utilized RT may be significantly more common in cellular organisms than previously supposed.
Several alphaviruses, including Sindbis virus (SINV), utilize RT of a UGA stop codon in their replicase gene (30, 31) . For SINV, primarily on the basis of in vitro translation studies, the only contextual feature reported to be important for RT was the identity of the cytidine nucleotide immediately 3 0 of the stop codon, directly analogous to the results of the early stop codon leakiness studies (32) . Similarly, in the tobraviruses (specifically tobacco rattle virus) and, by implication, the pecluvirus, furovirus and pomovirus replicase gene, and the furovirus CP-extension gene, it has been reported that RT of the UGA stop codon might depend on just the three 3 0 -adjacent nucleotides (33) . For these plant viruses, and alphaviruses that utilize RT, the consensus motif in wild-type (WT) viruses is UGA-CUA or UGA-CGG (34) . In contrast, for TMV (where the RT codon is UAG), plant tissue culture experiments showed that the 6 nt immediately 3 0 of the stop codon are relevant, with the consensus motif for efficient RT being UAG-CAR-YYA (35, 36) . The same motif is utilized by a number of other plant viruses, while the motif UAG-CAR-NBA stimulates RT in yeast (37) . In terms of 5 0 stimulatory motifs, adenines at the À1 and À2 nucleotide positions have been shown to positively modulate RT in yeast and are a feature common to many virus RT sites, notably in the tobamoviruses, poleroviruses and luteoviruses (38) .
For a relatively small number of cases of utilized RT, the known stimulatory signals involve an mRNA structure 3 0 of the stop codon. In MuLV, in vitro translation studies showed that a compact pseudoknot structure 3 0 of the gag terminator, UAG, is essential for meaningful levels of RT, with the identity of certain nucleotides in the 8 nt 'spacer' region between the stop codon and the pseudoknot, as well as some of the nucleotides in loop 2 of the pseudoknot, being important (39) (40) (41) (42) . The location of the pseudoknot (8 nt 3 0 of the stop codon) may permit it to act at the mRNA unwinding site half-way through the mRNA entrance channel of the ribosome (43) . A very different stimulatory element is present in the plant luteoviruses, where RT at the end of the CP gene produces a much larger CP-extension protein that is important for aphid transmission (44) . In the best-studied of these viruses, barley yellow dwarf, both 3 0 -adjacent sequences and an element $700-750 nt 3 0 of the UAG stop codon have been identified as important for RT and long-range RNA base pairing between the 3 0 -proximal and 3 0 -distal elements has been suggested as a possible mechanism (45) . Similar results were found for beet western yellows virus (46) .
Although cytidine residues are under-represented at the position immediately 3 0 -adjacent to UGA (and other) terminators in eukaryotes, they are by no means absent (11, 12) . Thus we hypothesized that, at least in vivo, RT in SINV and other alphaviruses might be modulated by additional sequence elements. To test for the existence of such elements, we investigated the degree of phylogenetic conservation at synonymous sites downstream of known RT stop codons in alphavirus genomes, and then extended the analyses to other RNA viruses and selected cellular RT genes. Regions of enhanced conservation at synonymous sites are indicative of overlapping functional elements such as RNA secondary structures or primary nucleotide sequences with functions in addition to amino acid coding. In many cases, and in particular those cases where RT of a UGA codon had been previously assumed to be stimulated simply by the 3 0 -adjacent nucleotides CUA or CGG, we found considerably enhanced conservation at synonymous sites in the 3 0 -adjacent sequence, typically extending over a region of 100-200 nt 3 0 -adjacent to the stop codon. Here, we computationally and experimentally explore these conserved regions and their significance for RT.
The genus Alphavirus encompasses approximately 30 described species, many of which infect humans and livestock, causing rashes, painful arthritis, fever and potentially fatal encephalitis (reviewed in reference 47; see reference 48 for a phylogeny). Transmission is generally via arthropods such as mosquitoes. The single-stranded positive sense genomic RNA is about 11-12 kb long and contains two long ORFs separated by a short non-coding sequence ( Figure 1A ). The 5 0 -proximal ORF codes for the non-structural proteins nsP1-nsP2-nsP3-nsP4 while the 3 0 -proximal ORF, which is translated from a subgenomic RNA, codes for the structural polyprotein C-E3-E2-6K-E1 and, via programmed ribosomal frameshifting, C-E3-E2-TF (49) . In SINV, Venezuelan equine encephalitis (VEEV), eastern equine encephalitis (EEEV), western equine encephalitis (WEEV) and related alphaviruses, a UGA stop codon separates the coding sequence for nsP4 (RNA-dependent RNA polymerase, RdRp) from the coding sequence for nsP123 (30, 50) . In contrast, the salmonid alphaviruses lack the UGA stop codon while, for alphaviruses in the Semliki Forest complex, the stop codon tends to be present in some but not all strains even within a single species, possibly as a result of conflicting selective forces in alternating arthropod and vertebrate hosts (passaging in cell culture may also drive selection for or against a stop codon at this location; see ref. 51 and references therein).
Virus sequences were obtained from GenBank in May 2009, updated in October 2010, and processed using BLAST, EMBOSS and ClustalW (52) (53) (54) . The accession numbers of all sequences used are given in the Supplementary Data. Coding sequences were extracted, translated, aligned with ClustalW and back-translated to nucleotide sequence alignments, and manually adjusted in a few cases. For the synonymous site conservation plots, alignment columns in which the reference sequence ( Figure 4 ) contained gap characters were removed so that the plots are in reference sequence coordinates. RNA structures were predicted using a combination of Vienna RNA RNAfold and alidot, pknotsRG and manual inspection (55, 56) .
Conservation at synonymous sites was analyzed as described in ref. (57; a procedure inspired by the SSSV statistic of ref. 58 ). The procedure takes into account whether synonymous site codons are 1-, 2-, 3-, 4-or 6-fold degenerate and the differing probabilities of transitions and transversions. Briefly, for a given pair of sequences within an alignment, a codon position was defined as a synonymous site if the same amino acid was encoded in both sequences. A 'null' substitution model was defined such that the relative probability of each possible synonymous codon substitution (including substitution with itself) at such sites may be calculated by assuming that the component nucleotides evolve neutrally. Neutral evolution was modelled using a Kimura nucleotide substitution matrix with k = 3 (59) . For each sequence pair, the divergence parameter t was set so that the total expected number of nucleotide substitutions at synonymous sites under the null model was equal to the total observed number. Next, the difference between the expected number and observed number of nucleotide substitutions was calculated at each synonymous site in the pairwise comparison. The variance at each site was calculated from the expected probabilities of each possible synonymous codon substitution, assuming a multinomial distribution. Statistics were summed, at each alignment codon position, over a phylogenetic tree as described in ref. (60) . Finally the statistics were averaged over a sliding window. An approximate P-value (probability that the mean conservation in the sliding window would be as high as observed if the null model were true) was also calculated, under the assumption of a normal distribution as an approximation to the sum of many independent multinomial distributions.
The sequences encompassing the RT site and the predicted 3 0 stem-loop structure for VEEV and SINV were synthesized by GenScript and cloned into the XhoI and BglII sites of pDluc, a derivative of the p2luc vector (61, 62) . The firefly luciferase gene is in the same reading frame relative to the upstream renilla luciferase gene such that RT of the stop codon results in a renilla-firefly luciferase fusion product. Derivative constructs were generated by PCR using appropriate primers and recloning into pDluc. All plasmids were verified by DNA sequencing.
Plasmid DNAs (0.2 mg) were used as templates in 10 ml reactions of the rabbit reticulocyte lysate TNT Õ T7 Quick Coupled Transcription/Translation system (Promega). 35 S-methionine (Perkin Elmer) was included in the reactions and protein products were separated by SDS-PAGE. Dried gels were analyzed using a Typhoon PhosphorImager (GE Healthcare) and the amount of A   nsP1  nsP2  nsP3  nsP4  C E3 E2 6K E1   stop codon  readthrough  −1 frameshift site   n  i  e  t  o  r  p  y  l  o  p  l  a  r  u  t  c  u  r  t  s  n  i  e  t  o  r  p  y  l  o  p  l  a  r  u  t  c  u  r  t  s  n The plot depicts the probability that the degree of conservation within a 9-codon sliding window could be obtained under a null model of neutral evolution at synonymous sites. Note that the RT stop codon itself has been excluded from the conservation statistics. In order to map the conservation statistic onto the coordinates of a specific sequence in each alignment, all alignment columns with gaps in a chosen reference sequence were removed prior to calculation of conservation. The following reference sequences (GenBank accession numbers) were used: VEEV NC_001449, SINV NC_001547. radioactivity in each product was determined using the ImageQuant 5.2 program (Molecular Dynamics). After normalization for the number of methionine residues in termination and RT products (9 and 22, respectively), the RT efficiencies were calculated as [RT/ (RT+termination)].
Tissue culture RT assays RT assays were performed using the dual luciferase reporter constructs, as previously described (62, 63) . To control for possible differences in stability of specific mRNA sequences, each RT construct was compared with a control construct that was identical except that the TGA stop codon was replaced with a TGG codon. RT efficiencies were calculated as (firefly activity/renilla activity) for the RT sequence normalized by (firefly activity/renilla activity) for the corresponding TGG control sequence. Standard deviations were calculated based on six independent transfections.
Sequence alignments of coding sequences containing RT stop codons were generated for a number of RNA virus taxa and the degree of conservation at synonymous sites was analyzed as described in the 'Materials and Methods' section. For an alignment of 63 VEEV, EEEV and WEEV sequences, this analysis revealed significantly enhanced conservation in a region comprising the $140 nt 3 0 -adjacent to the RT stop codon and a 9-codon sliding window size clearly resolved the conservation into two distinct peaks ( Figure 1B ). Inspection of the sequence alignment demonstrated the potential for base pairing between the sequences corresponding to these two peaks to form a stem-loop structure. In VEEV, the 5 0 -end of the 5 0 component of the stem is separated from the stop codon by an 8-9 nt 'spacer' and the 5 0 and 3 0 components of the stem are separated by a less-conserved 'loop' region (which may nonetheless contain structured elements) of 101 nt ( Figure 2 ). The predicted stem has 11-12 bp with a 1 nt asymmetric bulge in the centre of the 5 0 component and, despite the enhanced conservation, is further supported by a compensatory A:U to G:C substitution that occurs in some strains at the fourth base pair from the 'top' of the stem. In EEEV and WEEV, the predicted stem has 10 bp with a 1 nt asymmetric 5 0 bulge, and is separated from the stop codon by a 9 nt 'spacer' (Figure 2) . Again, the predicted stem is supported by a compensatory G:C to A:U substitution in the related Fort Morgan virus (FMV). High conservation was also noted for the 1-2 codons immediately 3 0 -adjacent to the 3 0 component of the predicted stem in VEEV, EEEV and WEEV.
With respect to the non-structural polyprotein, SINV and Aura virus (AURAV) form a separate clade from VEEV, WEEV and EEEV but, again, the conservation analysis revealed striking tandem conservation peaks 3 0 of the RT site ( Figure 1B ) and, again, the conservation peaks corresponded to sequences with the potential to base pair to form an RNA structure-this time comprising an 11 bp stem with a 1 nt asymmetric 3 0 bulge, a 12 nt 'spacer' from the RT stop codon, and a 154 nt 'loop' region ( Figure 2 ). For those alphavirus species where there appears to be a constant flux between presence and absence of the RT stop codon, it is not unreasonable to suspect that the 3 0 structure, if any, will be present whether or not the stop codon is present in any particular sequence. However, although we found the potential for conserved RNA stems to form in a number of these species (e.g. Ross River, getah, Semliki Forest and chikungunya viruses; Figure 3 and Supplementary Data), the range of divergences in the available sequence data proved inadequate to obtain supporting evidence from an analysis of conservation at synonymous sites.
Curiously this phenomenon was not just limited to the alphaviruses. The potential to form an extended stemloop structure 3 0 -adjacent to a RT stop codon-phylogenetically conserved and supported by a pair of peaks in synonymous site conservation-was also found in a number of plant virus RT cases, for example, in the replicase gene in the genera (Figures 3 and 4) . Further, the predicted stem is well-supported by a large number of compensatory substitutions-i.e. paired substitutions that preserve the predicted base pairings-between the different species ( Figure 3 and Supplementary Data). The furoviruses and pomoviruses have a second RT site in the CP gene. Here, however, there is a marked dichotomy between the two genera in the RT context. In the furoviruses, the RT context is generally UGA-CGG (UGA-UGG in the highly divergent sorghum chlorotic spot virus, AB033692) and there was evidence for tandem synonymous site conservation peaks and an associated stem-loop structure that, together with a 7 nt 'spacer', covered 96 nt 3 0 -adjacent to the stop codon (Figures 3 and 4) . In the pomoviruses, however, the RT context is generally A-UAG-CAA-UYA (A-UAA-CAA-UUA in the highly divergent broad bean necrosis virus, D86637) and the synonymous site conservation analysis failed to reveal extended conservation in the vicinity of the RT site ( Figure 4 ). Thus the furovirus context and predicted structure is alphavirus-like while the pomovirus context and lack of predicted structure is tobamovirus-like (see below). The animal-infecting coltiviruses also have an alphavirus-like RT site (UGA-CGG) in the VP9/VP9 0 -coding sequence and, again, there is potential to form a 3 0 -adjacent RNA stem-loop structure (Figure 3 ; as noted previously in ref. 65) , which is tentatively supported by our conservation analysis ( Figure 4) .
Stop codon RT is also utilized by members of the plant virus taxa Tombusviridae, Luteoviridae, Benyvirus and Tobamovirus but the RT signals for these viruses had previously been grouped separately from those utilized by the alphaviruses, coltiviruses, tobraviruses, pecluviruses, furoviruses and pomoviruses (excepting pomovirus RNA2), and our analysis likewise supported this distinction at the level of extended 3 0 -adjacent synonymous site conservation (34) . In the case of the tobamoviruses, greatly enhanced synonymous site conservation is seen from codons À1 to +3 relative to the UAG stop codon, and the motif xxA-UAG-CAA-UUA-xxG is completely conserved in the 105 sequences analyzed (despite lack of amino acid conservation at the À1 and +3 codons). However, more extended conservation of the type seen in the alphaviruses was not observed (Figure 4 ). In the luteoviruses and poleroviruses (family Luteoviridae), the stop codon context AAA-UAG-GUA is completely conserved in all except one of 247 sequences analyzed (rose spring dwarf-associated virus, EU024678, has GAA-UGA-CGG), and enhanced synonymous site conservation was also observed over several further codons, especially codons À1 to +5. However, while this region may well interact with distal elements as discussed in ref. (45) , the extended 3 0 -adjacent conservation of the type seen in the alphaviruses was not observed in the Luteoviridae (Figure 4) . The highly conserved local nucleotide contexts of the different RT sites mentioned here have been noted, discussed and characterized in detail in a number of previous works (34 and references therein). A compilation of our own sequence analysis is given in the Supplementary Data and, to our knowledge, represents the largest such compilation to date.
In the benyviruses-which generally have a tobamovirus-like stop codon context (i.e. UAG-CAA-UUA; however, highly divergent rice stripe necrosis virus, EU099845, has UAG-GGG-UAC), the potential was observed for a local stem-loop structure (e.g. 9 nt spacer, 12 nt stem, 8 nt loop in beet necrotic yellow vein virus, D84411; Figure 3 ), but there was insufficient sequence data to obtain strong support from the synonymous site conservation analysis ( Figure 4 ). Previous deletion experiments in beet necrotic yellow vein benyvirus have shown, incidentally, that RT efficiency is considerably reduced when sequence corresponding to codons +3 to +34 from the RT codon is deleted, even though the immediately 3 0 -adjacent nucleotide context UAG-CAA-UUA is left intact (66) . In contrast, deletion of codons +28 to +61 had little effect on RT. These results indicate that there is an additional stimulatory element within the region defined by codons +3 to +27, consistent with the predicted stem-loop structure (codons +4 to +14).
In the Tombusviridae family (including genera Tombusvirus, Carmovirus, Necrovirus and others), RT occurs at a UAG stop codon followed by GGR, but enhanced synonymous site conservation was observed for approximately 200 codons 3 0 -adjacent to the UAG (Figure 4) . Some of this conservation, however, may be explained by other conserved elements in the region (see ref. 67 and references therein; see also refs 68, 69) . RNA folding software predicted alphavirus-like 3 0 -adjacent stem-loops in some species and more complex structures in other species, a detailed analysis of which is beyond the scope of this article. The RT site in gammaretroviruses has been studied in depth and our computational analysis supported the known stimulatory spacer sequence and pseudoknot structure but did not reveal further conservation in the vicinity (39) (40) (41) (42) . RT sites in enamoviruses, carrot red leaf luteovirus-associated RNA, Middelburg and Barmah Forest alphaviruses, Providence tetravirus and others, were not analyzed in detail due to lack of sequence data for useful comparative computational analysis (70) (71) (72) . In contrast, to our knowledge, no stimulatory RNA structure has been previously proposed for UGA RT in kelch. However, when we applied our computational analysis to kelch, we found tandem synonymous site conservation peaks 3 0 of the RT codon and the corresponding sequences were predicted to form an RNA stem (268 nt loop in D. melanogaster) that is conserved in all 12 Drosophila species (Figures 3 and 5; Supplementary Data) . The predicted stem has 14 bp with a 1 nt asymmetric bulge near the center of the 5 0 component, and is separated from the RT codon by an 8 nt 'spacer' sequence that is completely conserved in all 12 Drosophila species but, perhaps unusually, the RT codon context is UGA-AUG (UGA-AGC in Anopheles, Culex and Aedes mosquitoes).
In order to verify and investigate the functionality of the predicted RNA structures in VEEV and SINV, local sequences (15 nt 5 0 of the UGA stop codon and 156 nt 3 0 for VEEV or 204 nt 3 0 for SINV) were cloned in-frame between the renilla luciferase and firefly luciferase genes in vector pDluc. The firefly luciferase gene lacks an initiation codon and its expression is dependent on RT of the UGA codon. RT efficiencies were determined both in vitro using rabbit reticulocyte lysate, and in HEK293 tissue culture cell lysates. A positive control for RT, the MuLV gag-pol RT site and 3 0 -adjacent pseudoknot, was included in all assays ( Figure 6A and B, lane 11).
The WT VEEV and SINV constructs promoted RT in vitro at 2.9% and 1.6%, respectively ( Figure 6A, lanes  1 and 10) . The RT efficiencies in tissue culture cells were much higher: 7.6% for VEEV and 6.4% for SINV ( Figure 6B, lanes 1 and 10) . Substitution of the 6 nt immediately 3 0 of the UGA codon in VEEV with the tobamovirus-like RT stimulator, CAA-UUA, increased RT both in vitro (5.8%) and in tissue culture cells (10.1%; Figure 6A and B, lane 2). Derivative constructs lacking the sequences for the predicted structures were generated ( Figure 6C ). The VEEV derivative containing only 9 nt 3 0 of the UGA codon directed just 0.2% RT in vitro and 0.8% in tissue culture cells ( Figure 6A and B, lane 3), while the SINV derivative containing only 3 nt 3 0 of the UGA codon directed just 0.4% RT in vitro and 2.0% in tissue culture cells ( Figure 6A and B, lane 9). Thus the stimulatory effect of the stem-loop sequence is $10-to 14-fold for VEEV and 3-to 4-fold for SINV, depending on the assay system. This is in direct contrast to ref. 32 where no difference was found in vitro between an insert comprising just SINV UGA-CUA and an insert comprising the entire SINV nsP3+nsP4-coding sequences.
The VEEV stem-loop sequence was chosen for further analysis due to the higher RT efficiency and its greater stimulatory effect. When the 3 0 part of the stem was deleted, RT was reduced to 0.2% in vitro and 0.9% in tissue culture cells ( Figure 6A and B, lane 4) , thus demonstrating the importance of the sequence corresponding to the 3 0 component of the predicted stem, >120 nt downstream of the RT codon. To address base pairing within the predicted stem, two mutations were constructed that were predicted to disrupt Watson-Crick interactions: 3 G residues in the 5 0 part of the stem were changed to Cs or 3 C residues in the 3 0 part of the stem were changed to Gs ( Figure 6C ). In both cases, RT was drastically reduced in both in vitro and tissue culture cell assays ( Figure 6A and B, lanes 5 and 6) . However, when the two mutations were combined such that the predicted base pairings were restored, RT recovered to near the WT level ( Figure 6A and B, lane 7) . The importance of the sequence between the two halves of the stem (referred to here as the 'loop' region but without implication about internal structure) was tested by deleting all but 5 nt of its sequence. Interestingly, this resulted in substantially higher RT than the WT level in both assay systems ( Figure 6A and B, lane 8) . 
We have shown that the stimulatory elements for efficient RT in VEEV and probably also SINV include not just the immediately 3 0 -adjacent nucleotides, but also a stem-loop structure that spans $140 nt 3 0 of the stop codon. Computational analyses provide strong evidence that similar structures are relevant for RT in several other alphaviruses, and in plant viruses where RT occurs at a UGA codon. Although this RNA structure is clearly not essential for some level of RT to occur in some systems [as in many previous analyses the predicted WT structure was not present (32, 33, 74) and, in our own experiments, $1% RT was achieved in tissue culture without the WT structure], it does have a pronounced stimulatory effect on RT efficiency (3-to 4-fold for SINV, 10-to 14-fold for VEEV). As with the gammaretrovirus 3 0 pseudoknot, the precise mechanism by which the stem-loop affects RT remains to be determined. Possibilities include direct interaction with the ribosome (including pausing and/or promotion of conformational changes in the ribosome); provision of a physical block that preferentially occludes release factor from the A-site in favour of tRNAs; or an indirect action via some trans-acting factor. The function of the RNA structure may simply be to achieve a higher RT level that is optimal for the virus. Alternatively, the structure may provide a regulatory mechanism, perhaps allowing different RT levels to be achieved in different hosts or at different stages in the viral cycle.
The long 'loop' length of the predicted structures is noteworthy. While long-distance base pairings have been demonstrated to play important regulatory roles in RNA viruses (reviewed in ref. 75) , the distances involved in the RT base pairings identified here are very much smaller and a genome-scale regulatory role seems unlikely. Furthermore, our loop-deletion mutant promoted even higher RT than the WT construct, suggesting that the presence of a long loop region, or any sequence motifs therein, play little if any role in stimulating efficient RT. Thus, we hypothesize that evolutionary selection simply acts to place the 3 0 component of the stem in a convenient location (e.g. with regards to minimizing interference with the encoded amino acid sequence). Although we refer to the region between the two components of the stem as a 'loop', it should not be taken to imply that this region does not fold. In fact the region generally is predicted to fold, and the fact that it can fold may indeed be functionally important-perhaps just to provide stability to the basal stem. However, in most cases, the nature of the fold seems to be relatively unimportant as it is not well-conserved between related sequences.
How can our results be reconciled with previous results which indicated that only the immediately 3 0 -adjacent 1-3 nt were relevant for RT in these viruses? There are several possibilities. Previous analyses of the RT cassette in SINV alphavirus, and also tobacco rattle tobravirus, were performed in in vitro systems (32, 33) . However, RT efficiency may vary considerably between in vitro and cell culture systems, depending on the absence or presence and abundance of various relevant near-cognate tRNA species (34) , and potentially also on the concentration of various trans-acting factors, salt concentrations, temperature, ribosome loading density and intracellular architecture. Thus a high RT efficiency measured in vitro for a short insert does not mean that the full complement of elements that stimulate efficient RT in cell culture or in vivo has been recapitulated faithfully. Such factors may also explain why our in vitro experiments produced much lower RT efficiencies than previous in vitro experiments, and highlight the importance of our experiments in mammalian cell culture (32, 76, 77) . Although ref. (32) compared, in vitro, the RT efficiency for a short insert (that excluded the predicted structure) with a long insert (comprising the entire nsP3+nsP4-coding sequences), such comparisons between inserts of very different sizes are not always straightforward, in part because the different protein products may be degraded at different rates, and because chance base pairings with the construct sequence could affect RT efficiency differently for the long and short inserts. In contrast, guided by our computational analysis, we were able to make small but targeted substitutions that allowed for more precise comparisons in the context of a long VEEV insert that included the predicted RNA structure elements. Accurate measurements of the RT efficiency in alphavirus-infected cells are not readily obtainable due to the multiple cleavage products of the non-structural polyprotein and rapid degradation of excess nsP4 (31, 47, 78) . Nonetheless, in ref. (31) , 5-to 8-fold less nsP34 was found in WT SINV-infected cells than in cells infected with mutant viruses in which the UGA was replaced by a Ser, Trp or Arg codon, thus suggesting a WT RT efficiency in the range 12.5-20%. Our measurement of $7% for WT VEEV and SINV sequences in the dual luciferase construct suggests that there may be additional factors that affect alphavirus RT.
Interestingly, although RT for the VEEV and SINV cassettes was much more efficient in cell culture than in vitro, there was little difference between the two systems for the MuLV RT cassette ( Figure 6 ). While the action of some cellular trans-acting stimulatory factor cannot be ruled out (albeit presumably not interacting with the loop region, given the increased RT observed when the loop was deleted), other possible explanations include: (i) the different stop codons and nucleotide contexts involved (UGA-C in VEEV and SINV; UAG-G in MuLV) and hence the different pools of potential stop codon-decoding tRNAs and (ii) the nature of the 3 0 structure (a compact pseudoknot in MuLV but an extended stem-loop in VEEV and SINV) with possible consequences for the ease with which the structure may fold in different environments. Similar differences in RT efficiency between in vitro and cell culture systems were noted for Colorado tick fever coltivirus which, like SINV and VEEV, utilizes a UGA RT codon with a predicted 3 0 -adjacent stem-loop structure (65) .
Besides the known structure-stimulated RT cases discussed above, RNA structure also plays an integral role in the recoding of UGA codons for selenocysteine insertion. In eukaryotes, this process is dependent on an RNA stem-loop structure containing specific nucleotide motifs, known as the SECIS element, usually located in the 3 0 -UTR of the corresponding mRNAs (5,6). In certain cases, an additional stem-loop structure close to the recoded stop codon has also been identified (79, 80) . For example, in the human SEPN1 gene there is a phylogenetically conserved 16 bp stem (with a 1 nt symmetric bulge) and a 5 nt loop, separated from the UGA by a 6 nt spacer. Interestingly, this structure has been shown to stimulate RT in cell culture (but not in vitro) even when the SECIS element is absent. Howard et al. located potential 3 0 -adjacent structures for at least 5 of 36 human selenocysteine-encoding UGA codons analyzed. However, their initial computational selection involved RNA-folding of just nucleotides +1 to +60 of the human sequence-an analysis which would have missed most of the 3 0 RNA structures predicted in this report. Thus 3 0 -adjacent structures may be a feature of a larger proportion of selenocysteine RT sites than these, though it does not appear to be an essential feature for selenocysteine RT (61) .
The various motifs that stimulate RT in eukaryotic cells have been previously classified by Beier and Grimm and by Harrell et al. (34, 81) . Beier and Grimm define the classes Type I (generally UAG-CAA-UYA; includes tobamovirus replicase, and benyvirus and pomovirus CP extension), Type II (generally UGA-CGG or UGA-CUA; includes alphavirus replicase, tobravirus, pecluvirus, furovirus and pomovirus replicase, and furovirus CP extension), and Type III (generally UAG-G, plus a compact pseudoknot in gammaretroviruses and possible but as yet relatively uncharacterized structures in the luteoviruses and tombusviruses). There are exceptions to the rule (e.g. enamovirus UGA-G, various pomovirus cases with atypical stop codons, and so on). One reason for this may be that the required level of RT may vary between different viruses, and may also be modulated by other sequence elements (e.g. 5 0 nucleotide and/or amino acid context) so that, in certain cases, deviations from one of the 'canonical' RT motifs may be tolerated. With this proviso, our results suggest that the definition of the Type II motif should, in general though perhaps not ubiquitously, be modified to include a 3 0 RNA structure component. Our discovery in alphaviruses and phylogenetically supported predictions for many plant viruses and the Drosophila gene kelch, together with the small number of previously identified cases of structure-stimulated RT, now suggest that 3 0 RNA structures as a component of efficient RT cassettes in eukaryotes (especially those that lack a CAR-YYA tobamovirus-like stimulator), rather than being exceptional, may in fact be the norm. Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations. Synthetic biology is a rapidly emerging interdisciplinary research field that aims to construct new biological parts and systems with new functionalities through a process of engineering and standardization. Vaccines may also benefit from a synthetic biology-based design. With vaccination the aim is to delude the immune system with an antigenic formulation to make it believe it is dealing with a natural infection, however, without causing illness. At present, the majority of vaccines on the market consist of attenuated or inactivated pathogens. Although effective, these systems are poorly defined, suffer from batch-to-batch variation and can only be used for pathogens that can be readily cultivated in the lab at scales that permit vaccine production. Moreover, as the ratio of antigenic compounds within the pathogen is more-or-less fixed, there is poor control over the direction against which antigenic compound an immune reaction will be evoked. The bottom-up design of vaccines which consist of well-defined antigenic compounds (e.g. proteins or nucleic acids) offers better control over the evoked immune reaction, however, the design of such vaccines is often empirical and in general yields vaccines that are poorly immunogenic and rely on adjuvants in order to be effective. Moreover, most vaccine production schemes are rather time-consuming, and therefore not suitable for rapid response intervention, for example, to prevent the pandemic spread of a new influenza strain in the human population.
Cell-free synthetic biology may offer new ways to design potent and genetically programmable vaccines (Jewett et al. 2008; Tsuboi et al. 2008 Tsuboi et al. , 2010a Zichel et al. 2010; Kanter et al. 2007; Simpson 2006) . It is based on the in vitro transcription and translation of single or multiple gene constructs in order to obtain a functional part or system. Applications of cell-free biology include the production of membrane proteins that are difficult to express in heterologous hosts (Henderson et al. 2007; Junge et al. 2010; Beebe et al. 2010; Nozawa et al. 2010; Reckel et al. 2010; Ishihara et al. 2005; Berrier et al. 2004) , high-throughput screening of protein libraries by in vitro compartmentalization (Mastrobattista et al. 2005) , generation of artificial cells by encapsulation of these complex biochemical reactions into cell-sized compartments, like liposomes (Kita et al. 2008; Sunami et al. 2006 Sunami et al. , 2010 Yamaji et al. 2009; Murtas et al. 2007; Ishikawa et al. 2004; Oberholzer et al. 1999 ). Recently, we have shown that protein expression in liposomes can yield microgramquantities of a model antigen (Amidi et al. 2010) .
In this study, we propose the use of antigen-expressing immunostimulatory liposomes (AnExILs) as artificial microbes for vaccination (Fig. 1) . The potential advantages of such antigen-expressing immunostimulatory liposomes (AnExILs) over conventional vaccines are numerous: the specificity of the vaccine can be easily altered by simply changing the DNA templates without having to change the entire vaccine formulation. Moreover, compared to DNA vaccines AnExILs do not exclusively rely on the often inefficient transfection of DNA into cells of the vaccine recipient in order to be effective. Secondly, compared to vector-based vaccines, AnExILs will be unaffected by neutralizing antibodies against the vector. Thirdly, there is no limit to the number of genes that can be expressed inside AnExILs. Lastly, AnExILs mimic characteristics of viruses and bacteria without the safety concerns related to the use of attenuated pathogens for vaccination. Here, we show that AnExILs expressing b-galactosidase are well tolerated after i.m. injection and were capable of inducing strong systemic immune responses, which were superior to that of liposomal DNA or protein vaccines encoding the same antigen.
Egg-derived L-a-phosphatidylcholine (EPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (PEG) 5000 (DSPE-PEG 5000) and 1,2-dioleoyl-sn-glycero-3-([N(5-amino-1-carboxypentyl) iminodi-acetic acid] succinyl) (DOGS-NTA) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Luria Broth, 2mercaptoethanol, adenosine-5 0 -triphosphate (ATP), phosphoenol-pyruvate (PEP), cytidine-5 0 -triphosphate (CTP), guanosine-5 0 -triphosphate (GTP), 3 0 -5 0 -cyclic adenosine monophosphate (cAMP), folinic acid, cholesterol (CHOL) and b-galactosidase enzyme (400 IU/mg) and each of the 20 encoded amino acids were purchased from Sigma (Saint Louis, MO, USA). The Fluorescein di-b-D-galactopyranoside (FDG) was supplied from Marker Gene Technologies (Eugene, OR, USA). E-coli tRNA, creatine kinase and creatine phosphate were obtained from Roche (Basel, Switzerland). Uridine 5 0 -triphosphate (UTP) and T7 polymerase were supplied from Fermentas (Burlington, Ontario, Canada). Dithiothreitol (DTT), Lysogeny broth (LB) and pyruvate kinase (PK) were from Flucka (Seelze, Germany). Rabbit polyclonal anti-b-galactosidase IgG and Cy-5 conjugated goat IgG anti-rabbit immunoglobulin was from Abcam (Cambridge, UK). Horseradish Peroxidase (HRP)-labeled goat anti-mouse total IgG and HRP-labeled rabbit anti-mouse IgG1 were purchased from Invitrogen (Breda, The Netherlands). HRP-labeled Rat monoclonal anti-mouse IgG2a was obtained from Abcam (Cambridge, The United Kingdom). PEG 8000 was from Promega (Madison, WI, USA). All other materials used were of analytical or pharmaceutical grade.
Preparation of PEG-liposomes and Ni 2? NTA liposomes A mixture of 2.5 lmol of total lipids (EPC, CHOL and DSPE-PEG 5000 with a molar ratio of EPC:CHOL:PEG Fig. 1 Schematic representation of AnExIL formulations used in this study 5000 = 1.6:0.9:0.025) or (EPC, CHOL, DOGS-NTA) with a molar ratio of EPC:CHOL:DOGS-NTA = 1.55:0.9: 0.025) were dissolved in dichloromethane:diethylether (1:1, v/v) in a round bottom flask. A thin, dry lipid film was obtained after evaporating the solvents using a rotary evaporator under vacuum at 30°C and subsequently dried with nitrogen for 30 min. The lipid film was hydrated in distilled water by shaking using glass beads to form large multilamellar liposomes, further sonicated with a probe sonicator to produce unilamellar liposomes. The liposomes suspensions were divided into 100 ll aliquots in 1.5 ml tubes (6 lm of total lipids/batch), freeze-dried and the obtained lyophilized lipid cakes were stored in a desiccator at room temperature until used.
Volume-weighted mean diameters and size distributions of the liposomes were determined by single particle optical sensing (Accusizer TM 780, Santa Barbara, California, USA).
For cell-free protein expression, E. coli b-galactosidase was used as a model antigen. LacZ, encoding E. coli b-galactosidase was cloned into vector pIVEX2.2EM, which allowed T7 promoter-driven expression in prokaryotic cell-free transcription and translation systems. The vector appends a 6-histidine (6-HIS) coding sequence to the C-terminal end of lacZ for efficient purification of the b-galactosidase protein (Amidi et al. 2010) . The E.coli Tuner TM strain, which is devoid of endogenous b-galactosidase (Merck Chemicals Ltd., Nottingham, UK), was used to make S30 bacterial extract as described previously (Amidi et al. 2010) . A coupled in vitro transcription/ translation reaction mixture (further referred to as IVTT mix), consisted of 30% (v/v) S30 extract, 175 lg/mL tRNA, 250 lg/mL creatine kinase, 5.8 mM magnesium acetate, 260U T7 polymerase, and 50% (v/v) lowmolecular-weight mix (LM mix) containing: 110 mM HEPES, 3.4 mM DTT, 2.4 mM ATP, 1.6 mM CTP, 1.6 mM GTP, 1.6 mM UTP, 0.8 M creatine phosphate (CP), 0.65 mM cAMP, 0.05 mM folinic acid, 0.21 M potassium acetate, 27 mM ammonium acetate, 2 mM each of the 20 amino acids, and 8% (v/v) PEG8000, was used for protein synthesis. To initiate protein expression, plasmid DNA was added to the IVTT mix at a final concentration of 20 nM and the reaction mixture was incubated for 3 h at 30°C. Generation of b-galactosidase-producing AnExILs
For preparation of AnExILs with b-galactosidase expressed inside liposomes (further referred to as AnExIL-IN), 75 ll of IVTT mixture and pIVEX2.2EM-LacZ with a final concentration of 20 nM, was used to rehydrate a batch of 6 lM of PEG-lipid cakes in order to form liposomes encapsulating IVTT mix and pDNA. The liposomes were incubated on ice for 10 min to complete the rehydration process. To inactivate protein expression outside liposomes, RNase with a final concentration of 10 lg/ml, was added to the liposomal suspension. Samples were incubated at 30°C for 3 h to allow protein synthesis to complete.
To prepare b-galactosidase-bound onto Ni 2? -NTA liposomes (further referred to as AnExIL-ON), b-galactosidase was expressed in bulk and subsequently bound onto liposomal bilayers as described below. Ninety-ll of IVTT and pIVEX2.2EM-LacZ as a pDNA template with a final concentration of 20 nM was used for bulk expression of a b-galactosidase with a C-terminal 6HIS-tag. IVTT mix with DNA template was incubated at 30°C for 3 h. After the expression was completed, 75 ll of the reaction mix containing expressed b-galactosidase was added to the NTA-lipid cakes (6 lM lipids) and incubated for 10 min at room temperature to form liposomes. The expressed b-galactosidase has a 6-histidine residue which binds to Ni 2? ions of Ni 2? -NTA liposomes. To remove the non attached b-galactosidase, the liposomes were washed three times with 0.5-1.5 ml phosphate buffer saline (PBS, NaCl concentration was adjusted to make it isotonic with the IVTT mix) by centrifugation at 8,000-9,0009g for 10 min at 4°C. After washing, the liposomes were re-suspended in 50 ll PBS.
An aqueous solution of b-galactosidase protein (40 IU/ml) was prepared in PBS. Subsequently, 100 ll of each solution was slowly added to freeze-dried lipids (6 lM) and incubated for 10 min at room temperature until rehydration was completed and liposomes were formed. To remove nonencapsulated b-galactosidase, liposomes were washed three times with PBS by centrifugation at 8,000-9,0009g for 10 min at 4°C and re-suspended in 50 ll PBS. The amount of encapsulated b-galactosidase in PEG-liposomes was calculated from total amount of protein entrapped in Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine 23 liposomes. For some formulations pDNA encoding bgalactosidase was co-entrapped with the b-galactosidase enzyme at a concentration of 20 nM. The amount of encapsulated b-galactosidase was measured based its enzymatic activity.
Quantification and antigenicity of the produced b-galactosidase b-galactosidase activity assay
The amount of encapsulated b-galactosidase as well as expressed b-galactosidase in bulk, inside the PEG-liposomes or bound onto Ni 2? -NTA liposomes were determined based on enzymatic activity of b-galactosidase using fluorescence spectroscopy (Amidi et al. 2010) . Briefly, in this assay, FDG as a substrate was cleaved by b-galactosidase into fluorescein, and two galactose molecules in a twostep reaction. By using high FDG concentrations relative to enzyme concentrations, the concentration of formed bgalactosidase could be directly deduced from the substrate conversion rate as measured by an increase in fluorescein fluorescence in time (excitation/emission wavelengths: 488/ 512 nm). A calibration curve of 0-500 lg/ml (corresponding to 0-200 IU/ml) of commercially available purified E. coli b-galactosidase was used in all experiments.
To determine the amount of encapsulated b-galactosidase or formed by IVTT in liposomes, 10 ll of each liposome suspension was diluted in 90 ll Cell lytic B buffer (Sigma, Saint Louis, MO, USA) to disrupt the liposomes and release the produced protein. A same treatment was done with a blank liposomal formulation and standards to correct for possible interferences of lipid components and Cell lytic B buffer with generated fluorescent signal. Subsequently 100 ll of substrate (FDG, 1 mM) was automatically added to each sample and standard and generated fluorescent signal was measured every 0.5 s over a period of 120 s at 25°C using a fluorescent well-plate reader (FLU-Ostar OPTIMA, BMG-Labtech, Offenburg, Germany). To determine the concentration of encapsulated or expressed bgalactosidase, initial substrate conversion slopes (relative light unit (RLU)/s) were determined and compared to those of standards with known concentrations of b-galactosidase (Amidi et al. 2010) .
Western blotting was done to evaluate the antigenicity of the expressed b-galactosidase. Liposomes containing b-galactosidase were disrupted by adding 100 ll of Cell lytic B buffer to 55 ll of liposome suspension. After adding electrophoresis loading-buffer (60 mM Tris-HCl, pH 6.8, with 25% glycerol and 2% SDS containing 0.1% bromophenol blue solution, and b-mercaptoethanol), the samples were heated for 5 min at 95°C and electrophoresed at 125 V under reducing conditions, in a 10% SDS-polyacrylamide gel (Bollag and Edelstein 1991) . After gel electrophoresis, antigen bands were transferred to a nitrocellulose membrane by using a dry Western blotting system (iBlot, Invitrogen, Frederick, MD) in 6 min. After blotting the free sites were blocked with 1% non-fat milk-powder in PBS containing 0.05% Tween 20 (PBS-T). Next, the membrane was incubated with a solution of goat anti-b-galactosidase polyclonal antibody in PBS-T containing 0.1% non-fat milk powder. The membrane was then washed to remove the unbound antibody and incubated with Cy-5-labeled, rabbit IgG anti-goat immunoglobulin. The blotted antigen-antibody complexes were visualized by a fluorescence using a Typhoon imager (Amersham Corporation, Arlington Heigths, IL, USA).
Female Balb-c mice, 6-8 weeks old (Charles River, Netherlands), were housed in groups of 5 and maintained in the animal facility of Utrecht University with a 12 h day and night schedule, while food and water were ad libitum. The experiments were approved by the Ethical Committee for Animal Experimentation of Utrecht University. Mice (5 per group) were intramuscularly (i.m.) immunized twice with 2-week intervals. Mice received 100 ll of different formulations (Table 1 ) divided over two batches of 50 ll which were injected in each of the hind leg muscles. Before each immunization and 2 weeks after the last immunization, the mice were bled by cheek puncture and then sacrificed by inhalation of excess CO 2 . Individual serum samples were separated from blood cells and coagulated proteins by centrifugation for 5 min at 14,0009g and 4°C, and stored at -20°C.
b-galactosidase-specific antibody responses were determined by using an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates (high binding capacity, Greiner, Alphen a/d Rijn, the Netherlands) were coated overnight at ambient temperature with b-galactosidase (100 ng in 100 ll/well) in coating buffer (0.05 M carbonate/bicarbonate, pH 9.6). To measure antibody responses in mice vaccinated with liposomes containing expressed b-galactosidase, the ELISA plates were coated with recombinantly produced b-galactosidase in E. coli strain BL21 (DE3) (Invitrogen Carlsbad, CA) and purified by using an Akta Purifier equipped with 5 mL His-Trap HP columns (GE Healthcare, Uppsala, Sweden). For serum samples of mice vaccinated with control formulations containing b-galactosidase encapsulated in liposomes, the plates were coated with the same commercial source of b-galactosidase that was used for entrapment. After coating, plates were washed and blocked by incubation with 2.5% (w/v) milk powder in coating buffer (200 ll/well) for 1 h at 37°C. Subsequently, the plates were washed with PBS containing 0.05% Tween, pH 7.4 (PBS/Tween). Appropriate dilutions of sera of each individual mouse were applied to the plates, serially diluted two-fold in PBS/ Tween and incubated for 2 h at 37°C. Plates were then washed and incubated either with horseradish peroxidase (HRP)-conjugated goat antibody against mice IgG or HRPconjugated rabbit antibody against mice IgG1 or HRPconjugated rat antibody against mice IgG2a (all diluted 1:5000 in PBS/Tween, 100 ll/well) for 1 h at 37°C. Thereafter, the plates were washed twice with PBS/Tween and once with PBS. Specific antibodies were detected by adding 100 ll of 3,3 0 ,5,5 0 -tetra methyl benzidine (TMB, 0.1 mg/ml) in 10 mM sodium acetate pH 5.5 buffer also containing 0.06% (v/v) hydrogen peroxide to each well. After 10 min, the reaction was stopped by adding 50 ll 2 M H 2 SO 4 to each well. Total IgG, IgG1 and IgG2a antibody titers are expressed as the reciprocal sample dilution corresponding with an A 450 of 0.2 above the background (Amidi et al. 2007 ). Comparison between mice of different groups with positive titers was made by a oneway ANOVA test.
Characterization of AnExILs and liposomes loaded with b-galactosidase and/or pDNA Cell-free protein synthesis was used to transcribe and translate the lacZ gene encoding for E. coli b-galactosidase either in bulk, inside liposomal compartments (AnExILs-IN) or adhered onto the surface of liposomes (AnExIL-ON) (For details of liposome preparation see materials & methods section). The volume-weighted mean diameter of b-galactosidase-loaded liposomes and AnExIL formulations was approximately 1.5 lm. The dose of b-galactosidase was standardized to 800 mU per formulation (see ''Materials and methods''), which corresponds to approximately 2 lg of active b-galactosidase from a commercial source (Sigma). The S30 extract used for IVTT was derived from E. coli BL21 Tuner TM strain, which is devoid of endogenous b-galactosidase. This is important to avoid interference of endogenous b-galactosidase in the experiments (Amidi et al. 2010 ). SDS-PAGE and Western blot analysis of S30 extract made from E-coli Tuner TM strain proved absence of endogenous b-galactosidase in S30 extract (Fig. 2) . Importantly, Western blot analysis confirmed that the expressed b-galactosidase was recognized by polyvalent anti-bgalactosidase antibodies and the antigenicity of b-galactosidase was preserved in IVTT mix (Fig. 2) . Systemic antibody responses after i.m. immunization in mice
To investigate the potency of AnExILs as alternatives for conventional protein or DNA vaccines, we compared systemic humoral responses of mice immunized i.m. with AnExIL-IN, AnExIL-ON, pCMV-Lac-Z encapsulated in liposomes (further referred to as liposomal DNA vaccine), b-galactosidase encapsulated in liposomes (further referred to as liposomal protein vaccines) and b-galactosidase coencapsulated with pDNA (pIVEX-Lac-Z) in liposomes (further referred to as liposomal protein/DNA vaccines). The liposomal DNA vaccine was poorly immunogenic and induced very low serum IgG titers in only some of the vaccinated animals after single dose i.m. immunization (Fig. 3a) . In contrast, AnExIL-IN induced high serum antibody responses after i.m. immunization, which were significantly higher than those achieved after i.m. injection of liposomal protein and liposomal protein/DNA vaccines (Fig. 3b) (P \ 0.001). Single i.m. vaccination with AnExIL-ON could elicit substantially higher serum antibody responses than those elicited by other vaccines (Fig. 3a , b) (P \ 0.001). It has been shown that surfacelinked liposomal antigens could enhance presentation of antigens to APCs and induce stronger immune responses (Taneichi et al. 2006a; Naito et al. 1996; Uchida and Taneichi 2008) . Altogether, these results point to strong immunostimulating effects of AnExIL vaccines and robust impact of surface antigen presentation on systemic antibody response. Since there are usually needs for booster vaccinations in order to induce prolonged and strong immune responses, the effect of a booster immunization on the systemic antibody production was studied in mice.
After booster immunizations all group of mice showed higher IgG titers but not significantly higher than those after prime immunization (Fig. 3) .
To exclude that the observed anti-b-galactosidase serum titers were induced by components within the S30 bacterial extract, serum antibody titers of mice injected with AnExILs without the pDNA encoding b-galactosidase were measured. After i.m. immunizations with pDNAlacking AnExILs, mice showed a negligible b-galactosidase specific antibody response (Fig. 3b) . This clearly indicates that the antigen specific immune response is exclusively mediated by the genetic input.
To gain more insight into the nature and quality of the systemic immune response, IgG1 and IgG2a subclasses of b-galactosidase-specific antibodies (Fig. 4a, b) were determined. After i.m. immunizations with liposomal DNA vaccine, there were no detectable levels of IgG1 or IgG2a in the sera of the vaccinated mice. As compared to liposomal protein and protein/DNA vaccines, AnExIL-IN and the AnExIL-ON vaccines showed superior IgG1 (P \ 0.05) and IgG2a (P \ 0.001) responses and those induced by AnExIL-ON was the highest (Fig. 4a, b) . The IgG1 and IgG2a responses of control and tested formulations largely corresponded to the total IgG responses (Fig. 4a, b) . Remarkably, the AnExIL-ON induced significantly higher IgG2a antibody levels (Fig. 4a) and consequently notably higher IgG2a/IgG1 ratios after booster vaccination compared to the other formulations (Fig. 4d) . Sera were collected 14 days after each immunization. Antibody titers are expressed as mean of the responding mice; the bars represent the 95% confidence intervals. The numbers above the columns indicate the number of responders per group. Asterisks indicate titers that are significantly (* P \ 0.05; ** P \ 0.001) higher than those of the group immunized (booster vaccination) with liposomal protein/DNA vaccines. Circles indicate titers that are significantly (°P \ 0.05) higher than those of the group immunized (booster vaccination) with AnExIL-IN
The results presented here demonstrate that AnExILs can be used as a synthetic biology platform to construct vaccines which can be genetically programmed in order to obtain antigen-specificity. AnExILs mimic characteristics of natural pathogens, without being virulent, and were able to induce strong humoral immune responses.
The particle size of AnExILs is amenable for uptake by antigen presenting cells (Foged et al. 2005; Tabata et al. 1996; Xiang et al. 2006; Yoshida and Babensee 2006) . The amount of pIVEX2.2EM-LacZ-6HIS encapsulated in liposomes (20 nM) was sufficient to initiate in vitro protein expression in liposomal compartments. b-galactosidase was successfully produced and quantified in liposomes. Enzymatic activity was used as a fast and sensitive method for evaluating biological activity and quantification of expressed b-galactosidase in AnExILs and showed that the produced enzyme has a correct folding and is active in cellfree protein synthesis. Western blot analysis showed that antigenicity of the expressed b-galactosidase was preserved. These results indicate that cell-free protein synthesis can efficiently produce antigens avoiding complexity and maintenance of cell viability associated with recombinant and in vivo systems (Jewett et al. 2008 ). Furthermore, it was demonstrated that the use of AnExILs can circumvent current problems of low immunogenicity with non-adjuvanted DNA vaccines (Liu and Ulmer 2005; Rosenberg et al. 2003; Trimble et al. 2009 ). As opposed to i.m. vaccination with liposomal DNA vaccine, i.m. immunization with AnExILs resulted in strong humoral immune responses. This result is in agreement with other studies showing that DNA vaccination is incapable of inducing strong antibody responses most probably because of poor transfection of DNA in endogenous cells, which leads to low doses of antigens produced (Abdulhaqq and Weiner 2008; Lu 2008 ). Moreover, AnExILs were able to induce significantly higher antibody responses as compared to conventional liposomes encapsulating antigen with or without pDNA. These results demonstrate strong immunostimulating effect of AnExILs as alternative for DNA and protein vaccines. Factors that might contribute to the adjuvant effect of AnExILs include efficient delivery of antigen to APCs, possible adjuvant activities of pDNA (due to CpG methylation pattern) (Klinman et al. 2009 ) and the presence of pathogen-associated molecular patterns (PAMPs) in the bacterial S30 extract which can activate the innate immune system via pattern recognition receptors. AnExIL-ON with surfaced-linked b-galactosidase was tested in mice as an alternative to AnExIL-IN formulation, in which antigen is entrapped inside vesicles. From the results of the vaccination studies with AnExIL-ON, it can be concluded that antigen presentation on the surface of the liposomes could significantly enhance systemic immune responses as compared to the other tested formulations and even a single immunization was sufficient to strongly stimulate an immune response superior to those achieved after i.m. immunization with conventional liposomal vaccines. Several studies have previously shown that surfacelinked liposomal antigens could enhance presentation of antigens to APCs and induce stronger immune responses Matsui et al. 2010; Ohno et al. 2009; Takagi et al. 2009; Taneichi et al. 2006b ). The impact of the AnExILs and other control vaccines on the antibody subtype profile was also investigated. In most of infectious diseases protection against viral or bacterial infections are mainly mediated by neutralizing immunoglobulins that bind to the viral or bacterial antigens (Gissmann 2009; Ho et al. 2002; Montefiori et al. 2007; Montefiori and Mascola 2009; Sattentau 2008; Smith 2009 ). In mice IgG1 and IgG2a antibodies are known to contribute to virus neutralization. IgG2a has been reported to contribute in complement activation and antibody-dependent cell-mediated immunity and is more effective than IgG1 in protection against viral infections by preventing virus replication (Coutelier et al. 1987; Hocart et al. 1989 ). On the other hand, the absolute concentration of IgG is important for reducing the viral shedding (Hocart et al. 1989; Hovden et al. 2005) . Therefore, induction of a combined IgG2a/IgG1 (T-helper 1 /T-helper 2 ) response may improve vaccine efficacy especially against viral infections. In this study, i.m. immunizations with all vaccines resulted in a mixed Th 1 /Th 2 immune response and i.m. administered AnExILs were able to markedly enhance both the IgG1 and the IgG2a response after i.m. vaccination as compared to protein and DNA liposomal formulations (Fig. 4a, b) , which may be advantageous for protection against viral or intracellular bacterial infections. The IgG2a/IgG1 ratio of AnExIL-IN was the highest among those of other vaccines after prime vaccination (Fig. 4c) and that of AnExIL-ON was substantially increased after booster immunization (Fig. 4d) . These data suggest that the quality of the immune response to b-galactosidase vaccine is substantially affected by the characteristic of AnExIL formulations.
The work presented here shows for the first time that liposomes can be used as antigen-producing artificial-cells for vaccination. AnExILs combine antigen-production, delivery and adjuvanticity in one system, making them more potent in inducing antibody responses compared to liposomal DNA vaccines as shown here. Still, the specificity of AnExILs is only determined by its genetic input which offers great flexibility in vaccine production and formulation. Preliminary data from our lab show that it is possible to store AnExILs lyophilized. Upon hydration with a DNA solution, the DNA is being transcribed and translated inside the rehydrated liposomes (data not shown). Such a universal vaccine platform may solve some of the problems of production lag-time with conventional vaccines (Hinman et al. 2006; DesRoches et al. 2005; Ulmer et al. 2006 ). Furthermore, it may allow easy production of personalized vaccines for e.g. cancer vaccination, in which the antigens are optimized for each individual (Poland et al. 2008) .
Although not tested here, the injection of bacterial components as essential part of the AnExIL formulation may pose problems of reactogenicity. S30 extract derived from E. coli is an essential component of AnExILs and which is likely to contain the pyrogenic endotoxin LPS. Visual inspection of the mice after i.m. administration of the AnExILs did not show any discomfort or illness. Nevertheless, alternative sources of IVTT mixes that are free of endotoxins should be tested. For example, extracts prepared from gram-positive bacteria, wheat germ or the synthetic PURExpress system (Shimizu et al. 2001 ) may be substituted for the E. coli-based extract.
Although we have only used a single antigen to show proof-of-concept of our AnExIL vaccination platform, mixtures of antigens can be expressed inside AnExILs by simply mixing pDNAs encoding different antigens. Although different antigens will have different levels of expression in cell-free expression systems, in general, the expression levels are high enough for the purpose of vaccination, where only microgram quantities of proteins are required. In fact, membrane proteins which are often difficult to produce in bacteria show remarkably high levels of expression in prokaryotic cell-free systems in the presence of liposomes (Junge et al. 2010; Goren et al. 2009; Kuruma et al. 2010; Moritani et al. 2010; Nozawa et al. 2010; Reckel et al. 2010) . Besides DNA encoding antigens, a variety of genetic inputs can be used in order to control the type of immune response provoked. For example, genetic adjuvants, such as chemokines or cytokines may be coexpressed inside the AnExILs in order to enhance the influx of immune cells at the site of injection and uptake of AnExILs by antigen presenting cells. As such AnExILs form an excellent synthetic biology platform for genetic programming using standard biological parts (Canton et al. 2008; Knight 2003) .
In conclusion, we have shown proof-of-concept for a genetically programmable vaccine based on the in situ production of antigens from DNA templates. Further immunization studies focusing on co-administering of DNAs, encoding biologically relevant antigens and genetic adjuvants (e.g. cytokines) are needed to demonstrate the possibilities and limits of the AnExIL vaccination platform.
The authors declare that they have conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Chinese Herbal Formula Xiao Yao San for Treatment of Depression: A Systematic Review of Randomized Controlled Trials Objectives. To assess the beneficial and adverse effects of Xiaoyaosan for depression. Search Strategy. Electronic databases were searched until December 2009. Inclusion Criteria. We included randomized clinical trials testing Xiaoyaosan against placebo, antidepressants, or combined with antidepressants against antidepressants alone. Data Extraction and Analyses. Study selection, data extraction, quality assessment, and data analyses were conducted according to the Cochrane standards. Results. 26 randomized trials (involving 1837 patients) were included and the methodological quality was evaluated as generally low. The pooled results showed that Xiaoyaosan combined with antidepressants was more effective in comprehensive effect, the score of HAMD and the score of SDS compared with antidepressants alone. Xiaoyaosan was superior to antidepressants for the score of HAMD. However, Xiaoyaosan was not different from placebo for the score of SDS. There was no adverse effects reported in the trials from Xiaoyaosan. Conclusions. Xiaoyaosan appears to be effective on improving symptoms in patients with depression. However, due to poor methodological quality in the majority of included trials, the potential benefit from Xiaoyaosan need to be confirmed in rigorous trials and the design and reporting of trials should follow international standards. Depression is a common mental disorder that presents with depressed mood, loss of interest or pleasure, feelings of guilt or low self-worth, disturbed sleep or appetite, insomnia or hypersomnia, fatigue or loss of energy, and poor concentration or difficulty making decisions. These problems can become chronic or recurrent and lead to substantial impairments in an individual's ability to take care of his or her everyday responsibilities.
Depression is recognized as a major public health problem, which has a substantial impact on individuals and society. Depressive disorders are common in the general population. It affecting about 121 million people worldwide. At its worst, depression can lead to suicide, a tragic fatality associated with the loss of about 850 000 thousand lives every year. Depression is the leading cause of disability as measured by Years Lived with Disability (YLD). The World Health Organization has described depression as an "unseen burden" [1, 2] . It was the 4th leading contributor to the global burden of disease when measured by Disability Adjusted Life Years (DALYs) in 2000. By the year 2020, depression is projected to reach 2nd place of the ranking of DALYs calculated for all ages in both sexes. Today, depression is already the 2nd cause of DALYs in the age category 15-44 years for both sexes combined. Mood disorders have been shown to have a greater impact on quality of life compared with conditions such as hypertension and cardiac disease [3] .
In China, depression is now one of the top three public health problems. Statistics show that 5 per cent of Chinese people suffer from the disease and 13 out of 1,000 Chinese have mental health issues [4] . Until 2003, China has over 26 million depression patients, for which discrimination and neglect are the two major obstacles to curing them, thus incurring an annual loss of over 64 billion yuan, according to a reserved estimation made at the Seminar on Attention to the Socio-Economic Burden of Depression held [5] . . We used the search terms "depression," "xiaoyaowan," "xiaoyaosan," and "xiaoyaotang." Various combinations of the terms were used, depending on the database searched. The bibliographies of included studies were searched for additional references.
All the parallel randomized controlled trials (RCTs) of all the prescriptions based on "xiaoyaosan" including pills, powder, decoction dosage form compared with antidepressants in patients with depression were included. RCTs combined xiaoyansan with antidepressants compared with antidepressants and all the modified xiaoyaosan formula were included as well. There were no restrictions on population characteristics, language and publication type.
Outcome measures include Clinical Comprehensive Effect, Hamilton depression scale (HAMD) scores, self-rating depression scale (SDS) scores, self-rating anxiety scale (SAS) scores, Hamilton Anxiety Scale (HAMA), scores, clinical global impression (CGI) scores, the scale for TCM syndrome and symptom differentiation (TCM-SSD) scores and so forth, the criteria "recover, significant effective, effective, or not effective" was also include in the outcome measurement. Duplicated publications reporting the same groups of participants were excluded.
Two authors (J. P. Liu and Y. Q. Zhang) extracted the data from the included trials independently. The methodological quality of trials was assessed using the 6 criteria 6 election bias (study design, confounders, blinding, data collection methods, withdrawals and dropouts) to following 3 categories: Category A (strong quality): four strong ratings with no weak ratings above. Category B (moderate quality): less than four strong ratings and one weak rating. Category C (weak quality): two or more weak ratings.
Quality assessment of included randomized controlled trials: sequence generation, allocation concealment, blinding of participants personnel and outcome assessors, incomplete outcome data, selective outcome reporting, and other sources of bias.
The statistical package (RevMan 4.3.2) was used for data analyses, which was provided by The Cochrane Collaboration. Dichotomous data were presented as risk ratio (RR) and continuous outcomes as mean difference (MD), both with 95% confidence interval (CI). Meta-analysis was performed if the intervention, control, and outcome were the same or similar. The statistical heterogeneity was presented as significant when I square (I 2 ) is over 50% or P < 0.1. Random effect model was used for the meta-analysis if there was significant heterogeneity (I 2 > 50%) and fixed effect model was used when the heterogeneity was not significant (I 2 < 50%) [7] .
After primary search of 5 databases, 263 trials were screen out from electronic and manual searches (Figure 1 ), and the majority were excluded due to obvious ineligibility which including irrelevant titles and abstract (some papers being found from more than one database). 141 trials with full text papers were retrieved. 122 RCTs were excluded because of reporting the depression complicated with other disease such as stroke and postpartum, 60 trials were excluded because of duplicated publication, 22 trials were excluded due to the animal studies, and the rest 41 trials were noncontrolled [7, 17, 22, 25, 31, 32] used the China classification and diagnostic criteria for mental disorder (second edition CCMD-2-R) alone, six [8, 9, 11, 18, 20, 24, [26] [27] [28] 30] trials used the third edition (CCMD-3) alone, three trials combined international classification on the diagnosis of depression (ICD-10) with CCMD-2-R [19] or CCMD-3 [12, 15] , one trials [21] combined the depression standard in "internal medicine of Chinese medicine" and CCMD-3 together, one trial [16] used CCMD-3 and affective disorder in the western medicine and Chinese medicine classification and diagnostic criteria on depression breaks out, two trials [10, 29] used CCMD-3 and the diagnostic criteria of Chinese medicine on depression and stagnation of liver qi, one trial [23] used CCMD-3 and depression of liverqi stagnation and spleen deficiency, the last trial [13] used the depression standard in TCM on liver-qi stagnation and spleen deficiency.
The interventions included all the prescriptions based on "xiaoyaosan" including pills, powder, decoction dosage form alone, with Maprotiline placebo or with antidepressants. The controls included antidepressants alone or the combination of danzhiXiaoyaosan (DXS) placebo and antidepressants. Eight trials investigated the prescriptions based on "xiaoyaosan" using alone [9, 17-19, 23, 30] or plus placebo [12, 15] versus antidepressants, one three-arm trial and the rest sixteen trials [7, 8, 10, 11, 13, 14, 16, 20-22, 24-29, 31, 32] compared the prescriptions based on "xiaoyaosan" plus antidepressants versus antidepressants.
The total treatment duration ranged from 30 days to 3 month. The variable prescriptions are presented in Table 1 . The different composition of formula Xiaoyaosan are presented in Table 2 . Nineteen (19) of the 26 trials used the hamilton depression scale (HAMD) as the outcome measure, other 4 kinds of scales including self-rating depression scale (SDS), self-rating anxiety scale (SAS), the scale for TCM syndrome and symptom differentiation (TCM-SSD), the hamilton anxiety scale (HAMA) were also be used. Side effect was evaluated by asberg side effect scale and treatment-emergent symptom side effect (TESS) scale or described in details. Eleven (11) trials used four classes to evaluate treatment effects including cure, significant effective, effective, ineffective, while ten (10) trials used three classes (except of cure) according to the scores reducing rate.
Six [10, 12, 15, 16, 23, 26] out of 26 trials (23.08%) were evaluated as strong quality, the rest of 20 trials (76.928%) were evaluated as moderate quality. The majority of the included trials were assessed to be moderate methodological quality. The sample size of including trials varied from 24 to 200 patients. None of the 26 trials reported sample size calculation. Ten trials described the randomization procedure, six [10, 12, 15, 16, 23, 26] trials used random number table, four trials [8, 17, 25, 30] used visiting time to realize the randomization. One trial [23] used opaque envelopes to allocate concealment. Only four [12, 15, 23, 27] of the 26 trials employed a blinding procedure: three of them using patients blinding, doctors blinding, and assessors blinding, and the other one [27] mentioned single-blind without further details. Seven trials [8, 10-12, 15, 21, 25] reported the withdrawals/dropouts information. Three trials [13, 16, 26] mentioned follow-up, and neither of them used intention to treat method. The reporting quality of 26 trials according to quality assessment tool for quantitative studies varied among different trials (Table 3) . (Tables 4-6) 3 [9, 15, 18, 23, 30] used the percentage of HAMD scores reduced rate to measure the outcome: cure (HAMD scores reduced rate more than 75%), significant effective (HAMD scores reduced rate between 51% and 75%), effective (HAMD scores reduced rate from 25 to 50%) and ineffective (HAMD scores reduced rate less than 25%). None of the five trials showed significant difference between treatment and control group on the four criteria outcome measurement Two [17, 19] trials compared the effectiveness using the three criteria outcome measurement: significant effective (SDS scores reduced rate ≥ 50%), effective (50 > SDS scores reduced rate ≥ 30%), not effective (SDS scores reduced rate < 30%). The Total effective rate is the combination of "cure", "significant effective "and" effective rate". We put these two different kinds of measurements together to evaluate the general effectiveness. The meta-analysis showed no significant difference between xiaoyaosan and antidepressants on the Total effective rate Bupleuri 10 g, Paeoniae lactiflorae 12 g, Angelicae sinensis 12 g, Poriae cocos 20 g, Atractylodis macrocephalae 10 g, Moutan radicis 12 g, Gradeniae jasminodidis 10 g, Curcumae l2 g, Acori graminei 10 g, Fructus aurantii 10 g, Draconis 30 g, Ostreae 30 g, Polygalae tenuifoliae 12 g, Cizyphi spinosae 30 g, Tritici aestivi levis 30 g, Glycyrrhizae uralensis 10 g, Zizyphi jujubae 5. Blood stasis plus Ligustici chuanxiong 12 g, Salviae milgiorrhizae 20∼30 g; Phelgm and dampness plus Citri reticulatae 10 g, Pinelliae ternatae 10 g; Yin deficiency plus Lilii 30 g, Anemarrhenae asphodeloibis 10 g; Qi deficiency remove Gradeniae jasminodidis, plus Pseudostellariae heterophyllae 15 g; Astriction plus Cannabis sativae 10 g or Radix et rhizome 10 g
Li et al. [11] pill Chinese patent medicine Li et al. 2007 [12] powder Chinese patent medicine (bupleuri, angelicae sinensis, poriae cocos, atractylodis macrocephalae, gradeniae jasminodidis, moutan radicis)
Li et al. [13] decoction Bupleuri 15 g, Angelicae sinensis 15 g, Atractylodis macrocephalae 15 g, Paeoniae lactiflorae 10 g, Poriae cocos 10 g, Menthae haplocalycis 6 g, Glycyrrhizae uralensis 6 g Liu and Chen [14] decoction Bupleuri 8 g, Gradeniae jasminodidis 6 g, Ligustici chuanxiong 6 g, Glycyrrhizae uralensis 6 g, Moutan radicis 10 g, Taeoniae rubrae 10 g, Atractylodis macrocephalae 10 g, Draconis 30 g, Ostreae 30 g, Poriae cocos 15 g. Liver qi stagnation and abdominal distention plus Aucklandiae lappae 10 g, Citri reticulatae 10 g, Cyperi rotundi 6 g; Insomnia plus Albizziae julibrissin 15 g, Polygoni multiflory 15 g, Poriae cocos pararadicis 15 g, Polygalae tenuifoliae 10 g; Cizyphi spinosae 10 g; Spleen and stomach deficiency plus Pseudostellariae heterophyllae 10 g, Citri reticulatae 10 g; Phlegm and dampness and no appetite plus Pinelliae ternatae 10 g, Bambusae in taeniis 10 g, Citri reticulatae 10 g, Amomi Wang [18] d ec octio n Bupleuri 12 g, Angelicae sinensis 20 g, Paeoniae lactiflorae 12 g, Atractylodis macrocephalae 12 g, Lilii 15 g, Albizziae julibrissin 15 g, Citri aurantii 10 g, Pinelliae ternatae 12 g, Gradeniae jasminodidis 10 g, Scutellariae baicalensis 10 g, Bambusae textillis 15 g, Curcumae 12 g, Acori graminei 12 g, Glycyrrhizae uralensis 6 g, Zizyphi jujubae 6
Wang [19] d ec octio n Bupleuri 10 g, Paeoniae lactiflorae 12 g, Angelicae sinensis 12 g, Poriae cocos 20 g, Atractylodis macrocephalae 10 g, Moutan radicis 12 g, Gradeniae jasminodidis 10 g, Curcumae 12 g, Acori graminei 10 g, Fructus aurantii 10 g, Draconis 30 g, Ostreae 30 g, Polygalae tenuifoliae 12 g, Cizyphi spinosae 30 g, Tritici aestivi levis 30 g, Glycyrrhizae uralensis 10 g, Zizyphi jujubae 5. Blood stasis plus Ligustici chuanxiong 12 g, Salviae milgiorrhizae 20-30 g; Phlegm and dampness plus Citri reticulatae 10 g, Pinelliae ternatae 10 g; Yin deficiency plus Lilii 30 g, Anemarrhenae asphodeloibis 10 g; Qi deficiency remove Gradeniae jasminodidis, plus Pseudostellariae heterophyllae 15 g; Astriction plus Cannabis sativae 10 g or Radix et rhizome 10 g Wang et al. [20] pill Chinese patent medicine Wang and Liu [21] decoction Angelicae sinensis 10 g, Paeoniae lactiflorae 12 g, Bupleuri 12 g, Atractylodis macrocephalae 10 g, Poriae cocos 12 g, Zingiberis officinalis recens 10 g, Glycyrrhizae uralensis 10 g, Moutan radicis 10 g, Gradeniae jasminodidis 10 g Table 6) .
Other Outcomes (TCM-SSD Scores, SAS Scores, 5-HT, BDNF, etc.) One trial [15] showed that there are no significant differences on TCM-SSD and SAS scores. One trial [12] showed that after 6 weeks of treatment, the serum level of 5hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) increased (P < 0.01) and the Interleukin-6 (IL-6) level decreased in both groups without significant difference between two groups, the cortisol (CORT) level reduced significantly in the DXP group compared to Maprotiline group. Other Outcomes (HAMA Scores, CGI Scores). One trial [10] showed significant benefit on HAMA scores decreased in favor of combination group after 6 (MD: 
A funnel plot analysis of the 14 trials comparing xiaoyaosan plus antidepressants to antidepressants on Clinical Comprehensive Effect was generated, and it showed a significant asymmetry (Figure 2 ).
Twenty-four out of twenty-six trials mentioned the adverse effect except two trials [22, 24] . Twentyfour trials reported the twenty-seven specific symptoms including diarrhea, dizziness and headache, somnolence, dry mouth, Bloating, constipation, tachycardia, blurred vision, insomnia, prolonged QT, naupathia, fatigue, anxiety, tremor, anorexia, palpitation, asthenia, oscitancy, sweat, akathisia, tetter, excitation, hypertension, bellyache, dysuria, transaminase increased, and sexual dysfunction (Figure 3) .
Amitriptyline showed main side effect including dry mouth, constipation, dizziness, blurred vision, tachycardia, somnolence and so forth, [8, 9, 14, 18, 22, 25, 32] . Imipramine, chlorimipramine, doxepin showed main side effect including dry mouth, constipation and other symptoms in alimentary canal [11] . Fluoxetine, paroxetine, citalopram showed main side effect including anxiety, insomnia, headache, naupathia, sexual dysfunction, and tremor, [8, 13, 16, 19-21, 23, 26-28, 30] . Venlafaxine showed main side effect including dry mouth, sweat, insomnia, headache, and anxiety, [17, 31] .
Four trials [9, 17, 19, 23] reported no side effect in the herbal medicine group compared to the antidepressants 10 Evidence-Based Complementary and Alternative Medicine group. Three trials reported side effect in xiaoyaosan group including headache, dizziness, and slightly diarrhea [15, 18, 30] . Fifteen out of eighteen trials reported the combination group has less side effect compare to the antidepressants group. Twelve trials [7, 15-19, 21, 25, 29-31] mentioned the side effect are significant reduced in intervention group compared to control group. Seven trials [7, 16, 18, 21, 25, 30, 31] used treatment-emergent symptom side effect (TESS) scale scores, one trial [15] used asberg side effect scale scores, the rest three trials [17, 19, 29] did not mentioned the tools they used to evaluate the side effect.
A meta-analysis of four trials [7, 8, 16, 27] with five comparison using TESS scale scores showed less side effect (WMD: −2.51 [−3.18, −1.84]; P < 0.00001) using xiaoyaosan plus antidepressants compare to antidepressants using alone with significant heterogeneity (I 2 = 87.2%). There is another trial [18] showed modified xiaoyao decoction had less side effect compared to amitriptyline (WMD: 
Based on this paper and meta-analyses of the outcome on Clinical Comprehensive Effect, HAMD scores, SDS scores, HAMA scores, and CGI scores, the prescriptions based on "xiaoyaosan" including pills, powder, decoction dosage form using alone or combined with antidepressants may have beneficial effects on patients with depression. The prescription xiaoyaosan may have the same effectiveness as antidepressants at the end point of the treatment with fewer side effects. The combination group may have significant beneficial effect compared to the antidepressants group variable on onset time with less side adverse events. We tried to analysis the trend of "xiaoyaosan"'s effectiveness by different followup time points as well.
The SAS scales scores, TCM-SSD scales scores and the outcome of the four criteria outcome measurement "cure, significant effective, effective, or ineffective" showed that there are no significant differences between the prescription group and antidepressants group. Meanwhile the xiaoyaosan prescriptions [12, 15] using alone may not as effective as antidepressants after 2 weeks treatment but after 4 or 6 weeks treatment the effectiveness tend to be no significant difference between two groups. We could clearly tell the trend from the HAMD scores and the reduced rate [15] of HAMD scores [12, 15] . The SDS scores showed the xiaoyansan prescriptions are significantly more effective after 4 weeks [17] and 6 weeks [19] treatment compared to antidepressants.
The combination of xiaoyaosan prescription plus antidepressants group may have significant beneficial effect compared to the antidepressants group. The onset time are variable may depended on the form of prescription such as pills and decoctions. Yang and Xie [10] Li et al. [31] Ma [27] Zhang [14] Huo et al. [28] Luo et al. [25] Wang [23] Yang et al. [ Two meta-analysis on HAMD scores showed significant heterogeneity. It may due to the different intervention and treatment time or the methodology quality. The significant heterogeneity on TESS scales may due to the dosage of the antidepressants.
According to the twenty-six trials the xiaoyansan prescription group and the combination of xiaoyaosan and antidepressants group have less adverse events compared to antidepressants group with significant differences which were showed by the TESS scales and Asberg side effect scales.
We should consider several limitations before accepting the findings of this paper. First, the quality of the included studies is generally moderate according to the quality assessment tool for quantitative Studies (Effective Public Health Practice Project 2007) which was recommended on the Cochrane Handbook. It also indicated that there are moderate risk of bias in most of the trials. Due to inadequate reporting of the allocation sequence, allocation concealment, blinding, intention to treat analysis and drop outs account in the majority of trials, it was possible that there was performance bias and detection bias due to patients and researchers being aware of the therapeutic interventions for the subjective outcome measures. Most of the trials provided limited descriptions of study design, randomization were mentioned but without further details after randomly assignment of patients which do not allow a proper judgment of the conduct of the trials. Therefore, we canot draw a confident conclusion that there are significant beneficial effects in patients with depression on combined groups or xiaoyaosan prescriptions using alone comparing to antidepressants. The number of trials identified limits us to perform meaningful subgroup or sensitivity analyses to illuminate robustness of the results in the review. Sixteen out of twenty-seven trials didnot described the blinding in details, only two trials [12, 15] used double-dummy in their study design.
Second, Liu et al. [33] found that some Asian countries including China publish unusually high proportions of positive results, considering all of the nineteen trials included are in Chinese the publication bias possibly existed. We cannot explore quantitatively the possibility of publication bias due to the small number of trials.
Third, different modified xiaoyao prescriptions and different form of the prescriptions were used in the trials: eight trials [7, 8, 11, 16, 20, 27, 28, 31] used fixed xiaoyaowan throughout the treatment, five trials used modified xiaoyaowan, and one trial used modified xiaoyansan based on menstruation period of young female [9] . The rest thirteen trials [10, 13, 14, 18, 19, 21-24, 29, 32] used modified xiaoyao decoction according to syndrome differentiation based on Chinese medicine theory, the herbal compound varied from 7 to 17 herbs ( Table 2 ). The treatment duration varied from 30 days to 3 months.
Fourth, the use of composite outcome measures in 26 trials to evaluate overall improvement of symptoms limits the generalization of the findings. The classification of cure, significant effective, effective, or ineffective and the Total effective rate are not internationally recognized, and these outcome measurement are vague to interpret the effect. We suggest future trials to comply with international standards in the evaluation of treatment effect.
Most of the sample size in the including 26 trials is small and there is a moderate risk of bias. Further high-quality studies with larger sample size are needed to confirm the effectiveness of xiaoyaosan in treating depression. Proper randomization techniques need to be clearly described and fully reported. Blinding and double-dummy should be used and reported clearly although the double-dummy of the herbal decoction might be very difficult, blinding of patients and outcome assessors should be used to minimize performance and assessment biases. Intention-to-treat principle and appropriate method for including drop out into data analyses are also important in the design of the trials. Since different forms of xiaoyaosan prescriptions were used in patients with depression such as pills and decoctions, they are likely to have different onset time according to the existed trials. Therefore, future clinical trials may focus on particular subgroups or large sample size to evaluate the effect of different forms of xiaoyaosan prescriptions on treating patients with depression. Further randomized trials with well design and adequate sample size are warranted to support or refute the positive findings. Trials should be reported according to the CONSORT Statement [34] .
In general, comparing to three categories (tricyclictertiary amines, nontricyclic, specific serotonin reuptake inhibitors (SSRIs)) of antidepressant drugs such as Amitriptyline, venlafaxine and Fluoxetine, the prescription based on xiaoyaosan in different forms appears to improve the symptoms with less adverse event. The combination of xiaoyaosan and antidepressants may have shorter onset time compare to antidepressants using alone. The mechanism [12] may due to the regulating the levels of 5-HT, CORT, BDNF, IL-6. Since depression may occurred recurrently with or without treatment, a longer followup period with serial measurement of outcomes after the treatment is important to determine the effectiveness and long term effect of the xiaoyaosan prescription. Considering there are not sufficient amount of high-quality trials on xiaoyaosan prescription treating patients with depression, the effectiveness of xiaoyaosan prescription need further rigorous trials to prove. Preventing the next 'SARS' - European healthcare workers' attitudes towards monitoring their health for the surveillance of newly emerging infections: qualitative study BACKGROUND: Hospitals are often the epicentres of newly circulating infections. Healthcare workers (HCWs) are at high risk of acquiring infectious diseases and may be among the first to contract emerging infections. This study aims to explore European HCWs' perceptions and attitudes towards monitoring their absence and symptom reports for surveillance of newly circulating infections. METHODS: A qualitative study with thematic analysis was conducted using focus group methodology. Forty-nine hospital-based HCWs from 12 hospitals were recruited to six focus groups; two each in England and Hungary and one each in Germany and Greece. RESULTS: HCWs perceived risk factors for occupationally acquired infectious diseases to be 1.) exposure to patients with undiagnosed infections 2.) break-down in infection control procedures 3.) immuno-naïvety and 4.) symptomatic colleagues. They were concerned that a lack of monitoring and guidelines for infectious HCWs posed a risk to staff and patients and felt employers failed to take a positive interest in their health. Staffing demands and loss of income were noted as pressures to attend work when unwell. In the UK, Hungary and Greece participants felt monitoring staff absence and the routine disclosure of symptoms could be appropriate provided the effectiveness and efficiency of such a system were demonstrable. In Germany, legislation, privacy and confidentiality were identified as barriers. All HCWs highlighted the need for knowledge and structural improvements for timelier recognition of emerging infections. These included increased suspicion and awareness among staff and standardised, homogenous absence reporting systems. CONCLUSIONS: Monitoring absence and infectious disease symptom reports among HCWs may be a feasible means of surveillance for emerging infections in some settings. A pre-requisite will be tackling the drivers for symptomatic HCWs to attend work. In the aftermath of the recent global outbreaks of SARS and H1N1, public health professionals are evaluating the response and management of these in order to establish lessons learned for the development of future strategic response frameworks [1] . Key to improving the timely response to outbreaks of newly circulating infections is the early recognition of unusual illness and clusters of cases, whilst the numbers affected are low. To achieve this, current infectious disease surveillance and health systems need to be further developed and enhanced.
Healthcare workers (HCWs) are at the frontline of any public health outbreak response, and therefore among the most vulnerable to emerging infectious diseases (EIDs), evident by the extent of morbidity and mortality caused by SARS and other outbreaks [2] [3] [4] [5] [6] [7] . In the past, hospitals have become amplifiers of newly emerging infections due to transmission from patients to HCWs, owing to ineffective infection control measures and failing to recognise a new pathogen [4, 8] . HCWs may therefore be the first to experience secondary transmission of an EID, and consequently a population where clusters of cases are first to appear. Monitoring HCWs' health could provide an important opportunity to detect newly circulating infections earlier, enhancing current efforts for their identification and control.
The European Commission funded project REACT (Response to Emerging infectious diseases: Assessment and development of Core capacities and Tools) aims to improve and harmonise the response to public health threats throughout the European Union (EU) [9] . As part of this, a conceptual framework for a Europe-wide sentinel monitoring system for infectious diseases in HCWs is being developed. A number of studies have examined syndromic surveillance systems in hospital settings for timely recognition of nosocomial outbreaks in HCWs with positive results [5, 10, 11] . This study aims to explore the views and attitudes of European HCWs towards such surveillance systems, monitoring absence and symptom reports for the early detection of unusual illness and clusters of cases in previously healthy HCWs.
We used qualitative research methods to obtain a variety of attitudes, views and opinions and sought to bring together healthcare professionals from similar settings, sharing similar experiences, but from diverse cultural and professional backgrounds. From May to September 2010, we conducted six 60-90 minute focus group discussions in four European countries: two each in England and Hungary and one each in Greece and Germany. The research team, comprising of a group of international partners in the aforementioned countries [12] , employed a targeted recruitment strategy to include a group of expert staff of hospitals they either were affiliated to or were particularly relevant to the study, such as those with large infectious disease departments. Purposive sampling and snowballing techniques were used to recruit hospital-based HCWs from diverse professions, departments and responsibilities. As the aim of the research was to explore a specific topic in the context of a diverse range of views, we did not sample a representative group of the healthcare worker or hospital populations. The study intends to provide the ground work for highlighting which factors could be issues in a representative and or broader population. GE, GT, TE, ES and GP identified and emailed potential participants and distributed information sheets. In total 49 staff from 12 hospitals participated and all participants had experience of working in other hospitals. A description of participating hospitals and participants is provided in table 1. AA, GE and FN prepared a protocol, topic guide and information leaflets about the study which were translated by an external company. GE, GT, TE, ES and GP facilitated the sessions in their respective countries. All participants provided written consent, and were informed that they could cease to participate at any time. They were assured their participation would remain confidential and their answers analysed anonymously.
To avoid potential response bias, where possible participants were split into two groups dividing senior from junior staff. In countries where only one session was held, the groups were structured such that either line managers or their staff and not both participated.
Each of the discussions was conducted in the primary language of the host country and audio recorded. Discussions were conducted with the aid of the topic guide for which facilitators in each country had received briefing via a teleconference. We used open ended questions intending the discussions to be participant led (examples given in appendix 1). The recordings were transcribed verbatim and back-translated into English by an external company. The transcripts were coded and analysed by two independent authors, AA and GE. Analytic themes were developed inductively in consultation with all authors and used to organise the data. A constant comparative method was used to consecutively inform sampling, topic guide development and analyses [13] . As the study sample is based on purely qualitative research methods, aiming solely to identify possible themes, we did not measure the frequency of responses or attempt to weight them. Finally, all recordings were destroyed after the transcripts and analyses were verified for accuracy.
This study did not require ethical approval. An official waiver was granted from participating institutions.
Key findings from the discussions were HCWs' views on risk factors for occupationally acquired infectious diseases, their perceptions of illness and sickness-related absence, their views on monitoring absence and the structured collection of symptom data, factors affecting the willingness to report symptoms, factors affecting the validity of self-reported symptom data and knowledge and structural barriers for early recognition of nosocomial outbreaks. Healthcare workers' views on risk factors for occupationally acquired infections
All participants described experiences of nosocomial infectious disease transmission in terms of infections they had acquired or witnessed colleagues acquire, stating common infections to be gastrointestinal and respiratory viruses, and less common meningitis, hepatitis, TB and HIV. They identified risk factors for occupationally acquired infections to be (1) exposure to patients with undiagnosed infectious diseases, especially in accident and emergency wards, (2) break-down in infection control procedures due to lack of knowledge, resources and equipment, (3) immuno-naïvety and (4) symptomatic colleagues; all of which are likely to occur during an EID outbreak. In general, nosocomial disease transmission risk was perceived to be low, although UK participants considered healthy people to be more likely to contract infectious diseases in a hospital than in the community. A breakdown in infection control procedures was deemed acceptable in some situations as some participants in Hungary, who worked in intensive care departments, expressed the view that they have 'more pressing issues' than following infection control procedures, such as 'saving lives'. Other members of this group however felt that staff, in particular accident and emergency staff, needed to be 'more afraid of patients' and thought intensive care staff to be more at risk, but also more aware. It was generally accepted that it was the responsibility of the individual HCW to report suspicion of nosocomial transmission incidences, though they expressed the belief that there was a need for more policies to protect the most vulnerable, such as for accident and emergency and pregnant staff. Participants' statements on risk factors for occupationally acquired infectious diseases are presented in appendix 2.
Healthcare workers' perceptions of illness and sicknessrelated absence
Participants believed HCWs' health to be perceived of low importance by themselves, peers and employers, resulting in little sympathy towards their own health needs. It emerged that they often experienced anxiety when reporting absence from work with an illness and described common feelings of scepticism and distrust between themselves and line managers. Consequently, they were reluctant to stay at home with mild symptoms and even felt they were expected to come to work under such conditions to relieve managers from staffing pressures as, at times, managers were required to cover these shifts themselves. They felt they lacked understanding concerning the relationship between severity of symptoms and infectiousness and at which point they may pose a risk to others. The participants commented on the varying illness behaviour patterns and absence reporting practices across professions with doctors being those most likely to attend work with infectious disease symptoms (appendix 3). They stated that only severe symptoms such as high fever and acute pain would encourage higher level staff to be absent and highlighted a lack of clear guidelines for symptomatic staff in the work place. In addition, in Hungary, staff sick leave entitlements amount to less than their actual pay, further motivating HCWs to come to work when unwell or take sick leave as annual leave. Participants' statements on their perceptions of illness and sickness-related absence are presented in appendix 3.
Healthcare workers' views on monitoring their absence and symptoms for the surveillance of emerging infections and their willingness to report During the discussions, a framework for a syndromic surveillance system was described to participants whereby absent staff, who suspected themselves of having a contagious illness, would be encouraged to report symptoms for daily monitoring and identification of potential outbreaks [12] . All felt they had experienced activities similar to those suggested, having been asked to report symptoms, stay away from work and in some cases provide specimens when unwell during the recent H1N1 outbreak.
Whilst all HCWs agreed colleagues habitually reported symptoms informally when notifying their absence, a number of benefits of frequent monitoring were highlighted such as the ability to 1.) identify staff absenteeism, 2.) improve infection control monitoring, and 3.) prevent spread of infections resulting in reduced rates of illness. Willingness to disclose symptoms for routine surveillance however varied. UK participants were more open towards discussing symptoms and were of the view their colleagues would also be, in cases where the sickness was genuine and the rationale of the reporting system understood (appendix 4). They were nevertheless also concerned that detailed enquiries could be perceived as intrusive and provoke or increase feelings of anxiety and distrust. Further, they believed the sensitivities around disclosing this level of detail would vary depending on the individual, employer-employee relationships and the type of symptom, potentially undermining the validity of the data (appendix 4). Nevertheless, the concept was positively received as opportunities were identified for hospitals to give direction on how to manage symptomatic staff and, they in fact, anticipated reduced rates of absence.
In Hungary and Greece participants were also positive towards the concept of monitoring symptom reports and agreed this would lead to the earlier identification of outbreaks and improved management. However, they were sceptical as to how effective this would be for EIDs where the characteristics of the novel pathogen may be poorly understood.
In contrast, participants in Germany were less in favour of the suggestion that employers enquire about and formally record reported symptoms as part of absence data recording. They described the tight legislation around employer-employee relations, where currently it is unlawful for employers to request personal health details. They expressed the need for the protection of employee privacy and confidentiality and thought the risks associated with nosocomial transmission of EIDs were too small to compromise these rights. They were concerned about potential consequences for employment upon discovering an infectious HCW, and about the risk of victimisation, describing the existing controversy for hepatitis and HIV infected HCWs (appendix 4). However, they recognised a need for balancing staff and patient safety with confidentiality and the right to privacy, and identified situations where workers' councils needed to adopt a more proactive rather than reactive approach to identifying and preventing nosocomial transmission. HCWs' views on monitoring absence and symptom reports for surveillance of emerging infections are presented in appendix 4.
Beyond aspects relating to monitoring absence and symptom reports to detect outbreaks, participants in all groups voiced their concerns regarding local knowledge and structural barriers for earlier identification of EIDs.
Firstly, HCWs were concerned the lack of experience and low perceived risks of exposure to new pathogens would minimise awareness among hospital staff, leading to reduced suspicion and increased delay to the recognition of a new pathogen. Specialist input would be required for the monitoring process, introducing further labour and financial burdens on hospitals. In addition, the need for HCWs to be discriminating as to which symptoms to report, could result in wide variation in reporting practice, again potentially undermining the validity of the system. With EIDs viewed as rare events, participants felt that if the detection of EIDs was the sole rationale for reporting, adherence could decline as HCWs might question the usefulness of such a system.
Secondly, participants queried the heterogeneity in absence reporting across professions and working groups within the hospital. Nurses often had independent reporting systems to higher level health professionals and described experiences where the absence of doctors had been noticed only hours after they were due to attend work, if at all. They stated that often, there was a complete lack of structure in absence reporting among senior staff. With regular use of bank, temporary and contract staff, an accurate overview of absentee levels and reasons for absence within the hospital was deemed challenging.
Thirdly, all participants discussed the current role of occupational health departments and opportunities for their involvement in a surveillance system of this kind. Participants criticised the lack of funding and resources supplied to these departments, which currently played a minor role in managing HCWs' health beyond initial employment screening. They felt occupational health departments would be well placed to manage symptom and personal health data and organise diagnostic testing, and were keen to see their function broaden, assisting with the management of acutely as well as chronically sick staff, and in particular those symptomatic in the work place. Further, they felt independent assessments of symptomatic staff by occupational health departments would make decisions to send staff home 'more valid' and minimise anxieties and scepticism (appendix 5). In all countries, participants described the current practice for sick HCWs to visit their own general practice doctors, highlighting the potential inability for these to identify unusual symptoms in the first instance and make time-spatial links with other possible cases. There was consensus on the need to expand standard management and control guidelines of infectious disease outbreaks in the work place beyond those for gastrointestinal infections. However, with occupational health departments struggling with current functions, such as promoting and providing seasonal influenza vaccinations, participants lacked confidence in the ability of these departments to manage staff with acute infections and perform epidemiological investigations. HCWs' perceptions of knowledge and structural barriers are presented in appendix 5.
HCWs identified risk factors for occupationally acquired infections to be exposure to undiagnosed infectious patients, especially in accident and emergency wards, break-down in infection control procedures, immunonaïvety and symptomatic colleagues. A further concern was a lack of monitoring and guidelines allowing health professionals to work whilst infectious. All participants felt HCWs' health needs were perceived as of low importance and described feelings of anxiety when reporting sickness related absences which were at times received with scepticism. Staffing demands and loss of income were noted as pressures for symptomatic HCWs to come to work.
HCWs had mixed views on the feasibility and acceptability of monitoring their absences and symptom reports for the detection of unusual cases or clusters of cases of infectious disease. All felt they had experienced activities similar to those described in the suggested monitoring system during the recent pandemic. However, in the absence of a clearly perceived threat, issues concerning privacy, confidentiality and sensitivity were raised and, particularly in Germany, identified as barriers. Nevertheless, in the UK, Hungary and Greece, participants felt the disclosure of symptoms for monitoring would be appropriate, provided the rationale, purpose, effectiveness and efficiency of such a system could be demonstrated.
Beyond individual preferences for monitoring absence and disclosing symptoms, knowledge and structural barriers for early recognition of EIDs were recognised. The potential lack of suspicion and awareness among HCWs towards new infections was of concern as EIDs were perceived as rare events. Financial and labour resources required for implementation would impose a burden on already stretched services. The heterogeneity in absence monitoring across professions and lack of communication between working groups would hinder accurate data collection on absence. Furthermore, all participants criticised the lack of funding and minor role of occupational health departments in managing the health of hospital staff. Participants, especially in the UK, were keen for occupational health departments to increase their involvement and assume responsibility for staff with acute as well as chronic conditions. They felt occupational health departments maintained an independence making them well placed for the management of personal health data and symptomatic staff in the work place.
This is the first study to use a common methodology to examine HCWs' perceptions and attitudes in different European countries towards monitoring their absence and symptoms for the surveillance of EIDs. It is unique in exploring a diverse range of healthcare professionals' risk perceptions in relation to occupationally acquired infectious diseases and sensitivities around the disclosure of symptoms in light of the recent history of the emergence of new pathogens. The study has provided insight into knowledge gaps and structural barriers to the timely detection of newly circulating infections in hospital settings.
General limitations of this type of research are selection and response bias. The focus group participants may not be representative of healthcare worker and hospital populations and research in other settings may have produced different results. Group dynamics may have influenced individual participants' responses in a way perceived to be culturally desirable. Data from qualitative research is also generally at risk of decontextualisation and misinterpretation, perhaps more so in this case where the audio of four of six focus group sessions were back translated into English. Attempts were made to minimise this with the review of transcripts and analyses by the facilitators. Facilitators may have exhibited different interview techniques, potentially examining themes to varying extents, but they all had a common briefing and this approach permits identification of culturally specific factors. Not all EU countries were represented in this analysis and other cultural and structural differences may have generated different findings.
To the best of our knowledge, there are no previous reports of similar studies, particularly none that examined sensitivities around absence reporting and the disclosure of symptoms by HCWs. Some of the key findings here have been highlighted in the 2009 NHS Health and Well-Being Review Interim Report [14] . These include under-resourced occupational health services and uncertainty over the role and function of these departments (including the balance between supporting staff and managers), the issue of HCWs coming to work when feeling unwell, and the perception that senior managers or the employing organisations fail to take a positive interest in HCWs' health. Another study has also provided evidence that HCWs attend work with symptoms suggestive of communicable disease [15] . Similar to findings here, a study by Blake et al. found a loss in income was associated with the general working population's ability to comply with recommendations during an influenza pandemic in the US, resulting in symptomatic staff in the workplace [16] . A recent survey, also in the US, found that 55% of workers without sick pay compared to 37% with, would attend work with an infectious disease [17] .
There is evidence to support HCWs' fears of the increased risk in accident and emergency and intensive care departments as studies had found H1N1 attack rates among staff highest in these departments [5, 18] . Concepts for a surveillance system in this population could focus primarily in these departments where HCWs may have a higher invested interest. However, even during a pandemic, at varying World Health Organisation levels of alerts, it was difficult to motivate HCWs to take precautionary measures [19] .
The main findings from this study reveal that developing a European surveillance system for infectious diseases in HCWs using absence and symptom reports would need to overcome cultural and logistical barriers but would nonetheless probably be feasible if HCWs can be convinced that it has value. Differences in the perception of risk and need for monitoring will challenge any unified system. Economic and structural barriers for HCWs to manage their health, such as loss in income and staffing pressures will undermine the development of concepts for this type of surveillance, as it is based on encouragement and the responsibility of individual staff to report when unwell.
Grounds for variation in HCWs' perceptions on the appropriateness to ask to report symptoms seem to be cultural, with participants in Germany holding stronger views on the need to protect privacy and confidentiality. Any concepts developed for surveillance in this population will need to be sufficiently flexible to embrace a wide range of attitudes and practices. An example to circumvent barriers potentially predominantly in Germany may be to use aggregate data without identifiers and information on reasons for absence to highlight patterns. This concept has been explored in further detail in the REACT surveillance system framework model report [12] .
There is a need for more education on infectious disease epidemiology among hospital staff and further work to be done to increase adherence to recommended infection control practices. EIDs may be perceived as rare events; however their occurrence can be catastrophic on numerous levels. Early detection and response is crucial for control on a global level. Infections in HCWs pose risks to patients [15] and HCWs need to feel protected to deliver an efficient and effective response, which public health experts must consider for preplanning response frameworks [3, 20] . The resonance across countries in underfunded occupational health departments is worrying as is the poor understanding of the risk and consequences of nosocomial outbreaks.
Aside from the detection of newly circulating infections, there may be many additional benefits from monitoring absence and symptom reports of hospital staff. These data may be used to identify patterns of absence and their cause, highlight problem areas, provide transparency, pinpoint areas where infection control procedures break down and prevent sickness consequentially reducing costs. In the UK, sick leave currently costs the National Health Service (NHS) 10.3 million working days and £1.7 billion per year, with higher rates than anywhere else in the public or private sector [14] . Cutting sick leave even by a fraction could save the NHS millions of pounds, and may be achieved by increased vigilance and encouragement for symptomatic staff to stay home, reducing the spread of infection. Further, reducing the spread of infection will decrease the risk of the need to close wards which incurs huge opportunity costs for elective procedures as well as significant financial losses [21] .
Asking HCWs to report symptoms when absent from work with a suspected contagious illness may be feasible as a means of surveillance for EIDs in some settings. However, more fundamental issues need to be addressed for the further development of this concept. These include standardising a culture of safe healthcare with education and compliance in infection control, tackling staffing pressures, homogenising absence recording, reviewing sick pay entitlements and improving the understanding of nosocomial outbreaks. Pilot studies will need to be tailored to specific countries and hospitals to investigate how these initial concepts may be adapted.
What different experiences do focus group discussion members have in terms of outbreaks of infectious disease in the hospital and how common is it for hospital staff members to contract infectious diseases at work?
What are your views on the need for more measures to protect healthcare workers from work associated infections? Discuss the pros and cons of various methods and how it may impact on behaviours and reporting.
What are your views towards an ongoing surveillance system monitoring absence and symptom reports of HCWs? Discuss views on practicality, simplicity, acceptability, adaptability to different departments, concerns, suitability for prevention of outbreaks and protection of staff. Is there a place for a surveillance system of this kind in your hospital?
How would you feel about describing symptoms to your manager? As a manager how would you feel about staff describing their symptoms to you?
How would you feel if this were to be implemented in your hospital? Would you be willing to participate in a pilot study?
Higher risk in A & E wards (UK) "... around the time of the swine flu epidemic, I did feel a bit vulnerable at that stage because people would present to A&E and at that point, you don't know what they've come in with and then we get them the mask."
(Hungary) "...here [in emergency medicine] we investigate, send samples for testing, make a possible diagnosis of infectious illness, then get afraid that we may have been infected. THEN they put on a mask and gloves. " (Greece) "Where an [ER] patient with an airborneinfectious disease is present and coughs, it makes up an infectious situation in the sitting room and in the transmission department until the patient has been assigned."
(UK) "(Infection control may break down when) not having equipment ...not having the knowledge to know what to do and when to do it right." (Hungary) "'In an emergency we have to save lives, and if a few bacteria or viruses are transmitted in the process so be it."
(Hungary) "In everyday, real life, there is not sufficient supply of protective gear. We had difficulties in dealing with the financial departments to try to increase this stock."
Immuno-naïvety (UK) "I'm new to infectious diseases so from January I've had a chest infection, an ear infection and throat infection."
(Hungary) "I do not believe it [SARS] would just suddenly appear at an emergency department. Or if it was to suddenly appear nobody would know, and the patient would die." Current monitoring systems (Hungary) "Hopefully, as with H1N1, there would be an outbreak in some other country, which would be followed by the setting up of a monitoring system."
(Germany) "An epidemic alarm plan has been established, for pandemics, ... the more the topic is discussed, the more aware each person becomes... for example in emergency admissions, we have inspected the premises and doors, which have to be plastered up in urgent cases [...] but I can't envisage it leading to any kind of constant surveillance system, I mean what would they be reporting the whole time?" Appendix 3. Healthcare workers' perception of illness and sickness-related absence Barriers towards reporting: Perceptions of HCWs' health (Hungary): "Staff health has a lower importance, but this obviously depends on the given staff (occupational) health unit, how well organised it is, how much the employees can rely on it." (UK) "Is that because we all are a bit unsympathetic to our own needs as staff? We're very sympathetic to patients but probably to our own staff, we're not very good, we don't probably listen to them and think...like I've just said we think they're lying."
Barriers towards being absent from work when unwell: Staffing pressures (Hungary) "...they may be more concerned that there will be nobody to replace them, and there will be inadequate staffing." (UK) "But I think the issue with ward managers and that grey area is that if you send somebody home who are you going to replace? That member of staff, there's never a spare person." (UK) "...and then we curse them (the absent HCWs) because they've left you in a position where you've got no staff again." Sick leave (Hungary) "As long as sick leave does not pay a hundred percent of their usual salary, which is often the case, they will continue working or take some paid leave so they would not suffer a loss of income. Even in cases of MRSA..." (Hungary) "Self-employed staff are reluctant to report (sick)...following an illness with diarrhoea they should not resume work for 48 hours as they are still spreading the infection, but they often say they 'do not care' because they are not paid ..." (Germany) "There is this fact: Germany's employees do not take sickies, because the economy is now worsening and it tends to make people get their acts together..."
Expectations and habits for HCWs to work with infectious disease symptoms (UK) "With norovirus some people didn't want to go off duty so sometimes they could have been working and it was symptomatic. ...you had to physically make them go off duty."
(UK) "That happened with the flu though; doctors were the worst weren't they? You'd find doctors walking round sniffing and sneezing and have you been like that, you need to go home." (UK) "It's a bit ironic because the advice we gave to relatives was if you had any symptoms, if you had a cold, if you had anything then you shouldn't come and visit. ... Yet I suppose as managers I probably expected my staff to come in if they were slightly under the weather."
(Greece) "...we all have the tendency to come to work even if we don't feel well."
(Greece) "For the medical practitioners, it is their symptom of high fever which would keep him/her away from work. If gastroenteritis, as mentioned, is not at a risk stage, then they could technically function as long as they don't have diarrhoea 4-5 times per day, or acute pain." Appendix 4. Healthcare workers' willingness to report infectious disease symptoms for surveillance purposes Factors contributing to willingness to report symptoms Genuine illness (UK) "...If you're genuinely ill and you've got nothing to hide then I'd be comfortable, it wouldn't bother me, I'd tell everything."
Understanding the rationale (UK) "I think most people would be happy if they knew there was a risk, if you could tell them why you're asking the questions." (UK)" If all the staff were educated on that, like if you've got D&V, please report it, surely they should take a little bit of ownership and report it." Informal reporting (Hungary) " I feel that in case of a healthcare worker [...] if it is anything acute they will call in and say 'I have diarrhoea' or 'I have a fever' 'I can not get out of bed'."
(Germany) "Because everyone knows each other on the wards and there are many friends there, it's not just 'I am ill now' but 'I feel lousy'. 'I am retching all the time, and vomiting' and then you get the next person who says 'I also had that last week', who happened to have the same symptoms, so I think, it is informally but frequently fed back like that."
Reluctance to being monitored and consequences for the infected HCW (Germany) "Well, I certainly wouldn't tell you what kind of symptoms I have. And even if I did, it would only be informally. Peter's got diarrhoea just like I had last week. If the whole process were structured, it would be a refusal." (Germany) "In this period, the question has also been raised, what do we do with hepatitis positive staff, the idea that you would out them is almost a matter affecting the employment contract." Factors affecting the validity of self reported symptom data Accuracy of staff reports (UK) "...people will ring up and say I can't come in, I've got abdo pain, but really, they've just split up with their husband or their wife or something and that's something that then comes out when you're speaking to your line manager or something."
Accuracy of data collected (UK) "When you take phone calls off the staff, they just say they've got diarrhoea, some are embarrassed really so you don't really go into depth. Whoever takes the phone call, it's not necessarily yours, it could be whoever's on charge of that shift and whether they pass the message on correctly or do they ask them why they're off sick, some people don't, they just go 'oh okay' and put the phone down." (Greece) "Most of the times when (sick leave) notes are composed they are not real, so this inflicts distrustfulness and false information within the data given on files. When they received the note they are quite healthy."
Barriers to data collection Shift patterns (Greece) "...there is a way so that the absent shifts are covered in [a] different way where [the] illness is not declared."
Confidentiality, data protection and legislation (Hungary)" Well I don't see that [the proposed monitoring system] as so positive. I can't imagine it working well, because I think in many areas, the need for data protection and protection of identity of any kinds of diagnoses is much higher [...]" (Germany) "...but certainly, the workers' council would be the key issue and confidentiality would make it very difficult in Germany to find a regulation, which would also be acceptable from a data protection perspective, the question always looms in the background, which disadvantages would the employee, admitting to such a thing in this context, namely having such and such an illness, be subject to, perhaps suspended from service or not allowed to work with patients any longer."
Perceived benefits of monitoring Identify staff absenteeism (UK) "...you might find the person that's on a certain ward, every time they're put on that ward, they don't like it, they're off. So you can track like that, you know, what's the problem."
(Hungary)"It may be also useful to monitor appropriate hygiene, because if a certain ward has an increased number of an illness, there may be problems with staff hygiene." (Hungary) "I feel that if it would lead to more education about prevention, and it would be established with good intentions, then it would just be to the benefit of the nursing staff."
(Hungary) "Had the case been reported after the first two or three illnesses, we could have advised on preventative measures to be taken and the entire thing would not have spiralled so far. I do think that proper reporting and monitoring has an important role in reducing the length and the number of people affected by an epidemic." Appendix 5. Barriers for the timely recognition of outbreaks in the hospital environment Lack of knowledge (Hungary) "Infection control is not an incorporated subject in doctor training, and old habits and bad habits are much harder to change. It would be of utmost importance to have infection control taught to medical students and specialist trainees. To get an early concept of these matters."
Heterogeneity in absence monitoring Differences in reporting among staff (Hungary)" You have been talking about nursing unit managers and nursing staff, but these people are more aware of infectious matters and I always felt the 'weak link' was the doctors... Yes. Totally. Yes. Up to the point when they have an epidemic that completely stops the ward from functioning, this is their nightmare"
Staff based at multiple sites (Hungary)" In the case of self-employed doctors who work at several hospitals, it is virtually impossible to follow MRSA for example, because if during an outbreak we test doctors as well and it turns out their results are positive, I am convinced that 99 percent of them will not report to their other employers that they are carriers of MRSA: Because this would lead to a decrease in income."
Lack of absence reporting mechanism (Greece) "I have begun to file the absences of the staff under order of the Administration, and there is not one day of absence recorded by the medical practitioners. I believe that between us we certainly know of someone who has been ill, but because of our relationships with one another, the medics don't really take any sickness leave. They inform you, but they aren't declared and recorded." (Hungary) ".. for agencies who send nursing staff [...] if one person is ill, they will just send somebody else and nobody will know that the ill person has something infectious."
Financial and resource burdens Financial constraints (Hungary) "On the whole we see no obstacle to Hungary joining any unified European system, but the resources would not be made for this to happen. Isolation of patients or personal protective equipment is quite expensive."
Lack of skill and resources in occupational health departments (Hungary) "We also have a staff health service, with such minimal number of people involved that it is shameful, and they themselves do not really try to break out of their limitations, with their goal being the achievement of routine checks being completed and that is about it. We have turned to them on one occasion to please make a report of the illness of workers in the hospital kitchen and even that lead to complete chaos as to what they need to do and how. They do not really care about infectious diseases. When there is a mention of contagious illness the first thing they do is call us." Informational needs assessment of non‐Hodgkin lymphoma survivors and their physicians  . Given how well this series was received, we believed it would be worthwhile providing a similar review of relevant abstracts from this year's BMT meetings. There has been dramatic progress in this field that we believe is of great interest and importance to the hematology/oncology community. There was an incredible body of work presented and it is misleading to suggest that there is a ''top 10'' list. Many more abstracts than can be presented here are worthy of discussion and recognition and any exclusion of important studies is solely a reflection of space limitation rather than quality of work. We tried to pick abstracts that were important, novel, biologically interesting, and that address either changing or controversial areas in the field. All 10 abstracts are published in full in the February 2010 issue of Biology of Blood and Marrow Transplantation (volume 16, No. 2) and are identified here by their abstract number.
Frequency of CD4 1 CD25 hi FOXP3 1 Regulatory T Cells Has Diagnostic and Prognostic Value As a Biomarker for Acute Graft-Versus-Host-Disease (#5)
The diagnosis and severity of acute graft versus host disease (GVHD) is typically based on clinical and laboratory observations that are often subjective or inconclusive. For instance, a skin rash may be graded as GVHD by one observer and attributed to an allergic drug reaction by a second observer. Biopsies of involved organs may be useful but can be associated with significant risk and may also return conflicting or inconclusive results. For many years investigators have attempted to identify biomarkers to predict and diagnose GVHD [2, 3] and ideally herald the onset and severity before clinical symptoms develop allowing for early intervention and better, more effective, and targeted immunosuppressive therapies. To date, however there are no validated diagnostic or prognostic noninvasive tests for acute GVHD.
Regulatory T cells (Tregs) have been found to be important mediators of tolerance after experimental hematopoietic SCT, and have been used to prevent and treat GVHD in murine models [4] . While there is great interest in using Tregs therapeutically in clinical transplantation, just the presence and number of Tregs could serve as a useful biomarker for GVHD. The study by Magenau et al. uses flow cytometry to measure CD41/CD25hi/FOXP31 Tregs in 215 patients with (n 5 60) and without (n 5 155) GVHD.
Patients with GVHD (N 5 60) had a Treg frequency that was 40% less (0.66% ±0.07, P < 0.001) than those without GVHD and Treg frequency was identified as a significant independent biomarker of GVHD. Furthermore, Treg frequency correlated with the maximum overall grade of GVHD (r 5 20.33; P < 0.001), suggesting Treg values could predict severity and potential prognosis of GVHD.
To test this they then divided patients according to the median Treg frequency. Patients with Treg frequency below the median had significantly higher nonrelapse mortality (largely attributable to GVHD) compared to patients with Treg frequency above the median 41% vs. 8%, P 5 0.03. This translated into inferior 2 year survival of 38% versus 63% (P 5 0.03). There was no difference in relapse mortality according to Treg frequency. This analysis shows that the frequency of CD4 1 CD25 hi FOXP3 1 Tregs at the onset of GVHD correlates with ultimate severity, nonrelapse mortality, and overall survival. It will be of interest to determine whether Tregs can predict the onset of GVHD and whether therapy can be targeted depending on the frequency of regulatory T cells identified at GVHD onset. This relatively simple and rapid flow cytometry assay may have important diagnostic and prognostic value in patients developing clinical findings consistent with graftversus-host disease after hematopoietic SCT. Abstract Summary GVHD affects 20-70% of allogeneic SCT recipients and can result in significant transplant-related morbidity and mortality due to tissue injury and opportunistic infections from intensive and chronic immunosuppressive therapy [5] [6] [7] . GVHD is initiated by both host and donor antigen presenting cells [including dendritic cells (DC)] and mediated by memory and effector T cells of donor origin. A better understanding of the pathogenesis of GVHD is needed and could lead to more effective preventative or therapeutic interventions and better outcomes from SCT.
Sartor and colleagues report results of their investigations into the relationship between the severity of acute GVHD and the expression of the chemokine receptors CCR5 and CCR7 on dendritic cells, which are critical molecules for mediating DC trafficking into tissues. They evaluated serial peripheral blood samples from 32 patients after allogeneic SCT and 11 developed grade II-IV GVHD. The percentage of DC expressing either CCR5 or CCR7 was calculated and correlated with the development of acute GVHD. CCR7 expression showed no association with GVHD. In contrast, higher CCR5 expression was detected on DC in patients developing grade II-IV GVHD compared to DC in patients with grade 0-I GVHD (P < 0.0001). All 11 patients with grade II-IV GVHD expressed CCR5 in over 35% of their DCs while only 2 of 21 patients with grade 0-1 GVHD expressed CCR5 at this high a percentage. Most importantly, the expression of CCR5 on DC preceded the development of moderate to severe GVHD by a median of 19 days (range 2-47 days; samples drawn twice weekly). This is an exciting study which identifies a biologic marker which predicts for the development of GVHD; if verified, this could result in preventative modulation of immunosuppressive therapy for certain patients found to be at higher and lower risk for GVHD. In addition this study highlights a specific potential target for GVHD intervention (the CCR5 receptor), which has gained notoriety as the site of entry for HIV into lymphocytes. Other studies have suggested that CCR5 is important for development of GVHD [8, 9] . For this reason CCR5 antagonists such as maraviroc may be potential mediators of GVHD and should be studied in clinical trials.
Early infection with common respiratory viruses (RV) after lung transplant has been associated with both acute and chronic rejection. To determine if an infection with RVs was associated with alloimmune lung syndromes after hematopoietic SCT, Versluys et al. performed a prospective trial in 110 pediatric patients receiving a variety of graft sources for both malignant and nonmalignant diseases and conditions with either TBI (33) or chemotherapy based (77) conditioning regimens. They used quantitative PCR of nasopharyngeal aspirates to monitor for the most common RVs and found that 50% of patients had evidence of infection at a median of day 116 after transplant (range: 7-100). The most frequent infection was Rhinovirus (28), followed by Parainfluenzavirus 1-3, Coronavirus, Influenza A virus and Adenovirus. They reported that clinical symptoms were mild and all patients recovered spontaneously.
During this time, patients were monitored for pulmonary toxicity and 16% were diagnosed with IPS and 11% ultimately diagnosed with BO. Multivariable analysis showed that any RV infection was associated with subsequent development of an alloimmune lung syndrome (P < 0.0001) independent of the type of viral infection. Interestingly the development of acute GVHD was associated with a lower risk of an alloimmune lung syndrome (P 5 0.004), perhaps related to the protective effects of prolonged immunosuppression. Most striking was the finding that development of an alloimmune lung syndrome was the only predictor for mortality (P 5 0.04). Overall survival was 73% for all patients but 53% for patients who developed pulmonary toxicity.
Although tissue damage, inflammatory cytokines, and an allogeneic ''graft vs. lung'' effect have all been implicated in the development of IPS and BO, the precise etiology of these syndromes is not well-defined. This study implicated common RV as a major risk factor for alloimmune lung syndromes. It is possible that these infections enhance local inflammation or changes that make the lung a target for allogeneic recognition. This is supported by the protective effects of acute graft vs. host disease perhaps related to the prolonged use of immunosuppression. This study is important for several reasons. The high incidence of respiratory virus infection (50%) is concerning in itself and may support the use of reverse isolation precautions so common in many transplant centers. This study suggests a possible mechanism of pulmonary toxicity after allogeneic transplant and should lead to further prospective studies for early monitoring, protection, and intervention of patients undergoing transplant. It would also be of interest to determine if early common RV infections occur with this frequency and are associated with pulmonary toxicity and adult patients undergoing HSCT.
Prochymal Improves Response Rates in Patients with Steroid-Refractory Acute Graft Versus Host Disease (SR-GVHD) Involving the Liver and Gut: Results of a Randomized, Placebo-Controlled, Multicenter Phase III Trial in GVHD (41)
Treatment options for steroid-refractory GVHD (SR-GVHD) are unsatisfactory and prognosis is poor. The use of conventional intensive therapies to inhibit T-cells and inflammatory cytokines often result in high response rates ranging between 30 and 70%, but consistently long-term survival is limited ranging between 5 and 30% [12, 13] . Available therapies are limited both by lack of response as well as by high mortality from infectious complications related to intensive immunosuppression. There is immediate need to develop effective and safe therapies for SR-GVHD.
Martin et al. presented promising results of a phase III study comparing institutionally selected second line therapy with or without the addition of Prochymal, a mesenchymal stem cell (MSC) product derived from unrelated volunteer adult donors.
This was a large study enrolling 244 patients. Most patients have advanced GVHD; IBMTR severity index grade for Prochymal vs. placebo treated patients was B in 22 vs. 26%, C in 51 vs. 58%, and D in 27% vs. 16%. The groups were well matched and randomized in a 2: 1 fashion. Although the intention to treat analysis showed no difference in durable complete response (DCR) rates (35 vs. 30%, P 5 0.3), there was a trend toward better DCR in the treated population at 40% vs. 28% (P 5 0.08). For the 22 patients with involvement of three organs, the rate of CR/PR at day 28 was 63% for the treatment group compared to 0% for the placebo group (P < 0.05). Importantly there was no significant difference in infections, relapse (9% vs. 8%) or adverse events.
Various case reports and phase II trials have demonstrated potential efficacy with a good safety profile of MSCs for GVHD. This study can be com-mended simply for completing a randomized study in a large group of patients with SR-GVHD. Response rates in the MSC-treated patients were reasonable, and higher than placebo-treated patients. Nevertheless, despite including such a large number of patients, many questions remain unanswered. Would MSCs be most effective combined with a specific consistent second-line therapy? Numerous trials of SR-GVHD show high response rates but ultimately limited survival due to recurrent GVHD, infections, and even relapse; long-term outcomes beyond day 100 from this trial will be important to report for the transplant community. Finally, given the suggestion of important activity, further studying the role of MSCs for GVHD therapy earlier in the course of the disease will be important.
Second Solid Cancers After Allogeneic Hematopoietic-Cell Transplantation Using Busulfan-Cyclophosphamide Conditioning (#59)
The number of patients undergoing autologous and allogeneic SCT has continued to increase over time. This, in combination with improvements in short-term treatment-related mortality rates has led to an increased prevalence of SCT recipients in the population who have unique long-term health care needs related to their survivorship. A potentially devastating long-term complication of SCT is the development of secondary malignancies [14] . Secondary myelodysplastic syndrome or acute myeloid leukemia usually occur within the first 7 years after SCT [15] . The risk of developing solid tumors however continues to increase with time and patients are often no longer under the care of a transplant physician at the time of increased risk. Secondary solid tumors predominantly occur in the upper GI tract, oral cavity, brain, soft tissues and skin. A clear dose response has been found with the use of total body radiation and the incidence of solid tumors after SCT [16, 17] . In addition to increased risk from the direct cytotoxic effects of the conditioning regimen, chronic inflammation (as in the setting of GVHD), chronic immunosuppression (with increase risk of viral infection and decreased tumor surveillance) and increased genetic susceptibility may also play roles in the pathogenesis of these secondary tumors after allogeneic SCT [14, 17] .
The authors in this study report the incidence and risk factors associated with the development of solid tumors in 4349 pediatric and adult patients who underwent allogeneic stem cell transplant (SCT) for acute myelogenous leukemia (AML) in CR1 or chronic myelogenous leukemia (CML) in first chronic phase. All subjects received chemotherapy only conditioning regimens with busulfan and cyclophosphamide. The median time of follow-up for survivors was 8.2 years; 47% of patients were alive and evaluable at 5 years after SCT and 18% were alive and evaluable at 10 years after SCT. Sixty-six solid cancers were reported; the cumulative incidence of solid tumors at 3, 5 and 10 years after SCT were 0.7, 0.9, and 2.0% for AML patients and 0.5, 1.1, and 3.4% for CML patients. These rates were higher than expected rates in the general population. . On Cox-regression analysis, risk factors for developing solid tumors included older age, chronic GVHD and use of peripheral blood (as opposed to bone marrow) as a stem cell source.
This study supports the fact that SCT recipients are at significant longterm risk for the development of secondary solid tumors. Further longer term reports from larger cohorts of SCT recipients are needed to better define overall risk over time. It is important for patients and their physicians to be aware of these risks to modify screening and preventative techniques in this patient population. an effort to limit transplant related toxicity and broaden potential treatment options for older and sicker patient populations. The role of pretransplant consolidative chemotherapy may be different in the RIC-SCT setting than the myeloablative setting. One could hypothesize that with less intensive conditioning therapy, additional rounds of cytarabine-based consolidation chemotherapy before RIC-SCT would result in lower relapse rates by minimizing residual disease. Alternatively, RIC-SCT recipients tend to be older and sicker than myeloablative SCT recipients and increased rounds of therapy before SCT may contribute to more treatment-related mortality.
In this report, McCormack and colleagues retrospectively reviewed outcomes of 61 patients at their institution who underwent nonmyeloablative SCT and evaluated the impact of pretransplant consolidation chemotherapy on outcome. All patients had AML in CR1 at time of transplant and received either HLA matched sibling (n 5 18), cord blood (n 5 42) or unrelated (n 5 1) donor grafts. Of these patients, 26 received pretransplant consolidation (73% received 1 round; range 1-3) and 35 received none: 58% and 40% in each group respectively had high-risk cytogenetics. In univariate analysis, exposure to pre-SCT consolidation therapy had no impact on 2 year OS or relapse rates and did not increase treatment related mortality rates (See Table I ). In multivariate analysis (variables tested: comorbidity score, number of induction cycles to achieve CR, high risk cytogenetics) exposure to consolidation therapy still had no effect on relapse. The only variable significantly associated with treatment related mortality was comorbidity score.
This study suggests that in the RIC-SCT setting, pre transplant consolidation may not impact outcomes. It also further validates the value of the pretransplant comorbidity score on predicting treatment-related mortality. It is important to point out that the numbers of patients evaluated in this study are small and composed mostly of cord blood recipients. The study is likely underpowered to detect a significant difference in outcomes and outcomes from RIC-SCT cord recipients are likely different from recipients of RIC sibling or unrelated SCTs. Nevertheless, this is an important topic that warrants further investigation in the cord blood, sibling and unrelated setting using larger cohorts of patients.
Multi Tumor Antigen Specific Cytotoxic T Lymphocytes for Therapy of Hematologic Malignancies (#64)
Tumor-specific immunotherapy holds great promise, but in most cases tumor-specific antigens are not well defined and numerous strategies have been limited in part by the inability to generate functional tumor-specific T cells. In some cases this is due to the low frequency of tumor-reactive T cells. Furthermore, even when they can be isolated they may be anergized, tolerized, or after allogeneic SCT, they may be senescent and unable to expand [20] . The most successful use of targeted cellular therapy has been with the generation of viral-specific T cells to treat viral-associated malignancies such as EBV-associated Hodgkin's disease or post-transplant lymphoproliferative disorders [21] .
Gerdemann et al. reported on an important technical advance in the ability to generate tumor-specific T cells. T cells are activated in the presence of autologous dendritic cells (DCs) transfected with plasmids encod-ing multiple potential tumor-associated antigens or pulsed with tumor-associated antigen peptides. This permits generation of T cells specific for multiple antigens and multiple epitopes. Antigens and epitopes would not be restricted to a specific HLA polymorphism as occurs when tumor-specific T cells are generated against only 1 specific peptide that is expressed in an HLA-restricted manner. Polyclonal T cell activation occurs by exposure to a potent cocktail of cytokines developed to promote generation of Th1 cells (IL12), inhibit expansion of Tregs (IL-6) and provide enhanced survival and proliferative signals (IL15). To target lymphoma, they combined three potential lymphoma associated antigens, SSX2, Survivin, and MAGEA4 and generated ''multi-tumor associated antigen cytotoxic T cells'' (multiTAA CTL) against all three antigens from six healthy donors. They showed that these cells had activity against autologous antigen pulsed targets and whole antigen expressing fibroblasts and were not reactive against control targets. Similarly to target myeloid leukemia they combined WT1, Prame, PR3, and Survivin and generated multispecific T cells against all antigens from two healthy donors.
To date, tumor-specific cellular therapy has been successful primarily when well-defined viral associated antigens can be defined and targeted. Multiple potential tumor-associated antigens have been identified but development of targeted cellular therapy has been limited by inability to generate sufficient, HLA-restricted T cells, and in the case of patients who have had prior allogeneic SCT, donor T cell senescence has limited ability to expand tumor-specific T cells [20] . This technical advance is important since it permits generation of CTLs against multiple tumorassociated antigens and therefore may reduce the risk of tumor escape; it is likely that multiple antigens will need to be targeted for maximal efficacy. Furthermore, the ability to generate CTLs with multiple specificities markedly increases the efficiency of this approach and eliminates the need for specific polymorphic HLA-restriction. It will be of interest to see if activation by this method can reverse functional tolerance or senescence in patients with primary disease or after allogeneic stem cell transplantation.
Lymphomas in the HAART Era: A Meta-Analysis of Response and Survival Post Transplant (#114)
The prognosis for HIV-infected patients with non-Hodgkin lymphoma (NHL) has improved dramatically since the consistent use of highly-active anti-retroviral therapy (HAART) and conventional chemotherapy. Despite initial reports showing lower responses and higher mortality from combination chemotherapy in HIV-infected patients, in the modern era HAART-treated patients with diffuse large B-cell lymphoma treated with CHOP or rituximab-CHOP have similar outcomes to patients without HIV infection [22] . Nevertheless, many patients relapse and lymphoma remains a significant cause of death for patients with HIV-related lymphoma. In the past, HIV infection was felt to be a contraindication to autologous SCT out of concern for excessive toxicity and poor response rates. Currently, several reports suggest that HIVinfected patients may tolerate autologous SCT similar to non-HIV-infected patients [23] .
Sunil et al. performed a meta-analysis that ultimately included five studies and abstracts dealing with autologous SCT for patients with HIV related lymphoma. They were 35 patients with Hodgkin lymphoma and 83 with NHL. Outcomes were as impressive as would be anticipated in a similar non-HIV infected population. After transplant, 71% of patients (95%CI 60-80) achieved complete remission (CR) and 2 year overall survival was 71% (95%CI 61-79). A CR before transplant was highly predictive of a CR after transplant in 2 year overall survival. Overall this meta-analysis confirms the There are caveats to this report as there are in any meta-analysis. This is a retrospective analysis combining results of several case series. In addition there is likely to be significant reporting bias in this field. However the review of five studies including 111 patients support the conclusion that autologous transplant should be offered to HIV-infected patients as clinically appropriate and is not contraindicated. The authors appropriately conclude that prospective trials are necessary (and these data confirm that they are appropriate) and the community should anxiously await results from a planned multicenter prospective trial of autologous SCT in HIV-related lymphoma patients in development by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN).
The Graft-Versus-Myeloma Effect Using Nonmyeloablative or Reduced Intensity Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) (#204)
A graft vs. myeloma (GVM) effect has been suggested in transplant studies and has been demonstrated as a ''proof of principle'' concept using DLI to treat relapsed myeloma [24] . However outcomes after DLI for relapsed myeloma have been disappointing [25] calling in to question the potential efficacy of nonmyeloablative allogeneic stem cell transplantation. Large series of allogeneic SCT are not available to guide treatment decisions.
Dr. Ringden for the CIBMTR reported on outcomes for 177 recipients of matched sibling allogeneic SCT conditioned with a nonmyeloablative or reduced intensity conditioning (RIC) regimen for myeloma. The median age of patients was 50 (range 24-69) and 105 patients received a planned tandem autologous transplant followed by allogeneic SCT. With a median follow-up of 55 months (range 3-98) for all patients, and 25 months (range 3-76) for recipients of tandem autologous/allogeneic SCT, 3 year overall survival was quite reasonable at 45% (95% CI 33-58) for recipients of a single allogeneic SCT and 64% (53-75) for recipients of tandem autologous/allogeneic SCT. Treatment related mortality (TRM) at 3 years was 27 (17-38)% and 16 (10-25)% respectively and relapse rates were 48 (36-60)% and 41 (29-54)%. The 3 year progression free survival (PFS) was 25 (15-37)% and 42 (20-43)% demonstrating that long-term control was possible in some patients. The multivariate analysis showed that chronic GVHD decreased the probability of relapse (RR 0.43, P 5 0.012), though this effect was limited to patients without IgG myeloma for reasons that are difficult to explain. In addition, PFS was better with autologous 1 allogeneic HSCT compared to allogeneic SCT (RR 3.6, P 5 0.001), suggesting that dose intensive chemotherapy still has a major role in care of these patients. This report has many limitations. In particular it is a retrospective study and it is not known how patients were selected for specific therapies. Some findings are not easily explained, such as a benefit of chronic GVHD in terms of relapse noted only in patients without IgG myeloma. Nevertheless, it is a large study using available registry data demonstrating that at least in some patients, a GVM effect can be generated and long-term disease control achieved. Defining the proper patient population, timing of transplant, and the role for autologous/allogeneic SCT before RIC allogeneic SCT remain important subjects for future studies that hopefully will someday harness the GVM effect for maximal benefit.
Response to Acute Graft vs. Host Disease (GVHD) Treatment (#385)
Mycophenolate mofetil (MMF) is commonly used for GVHD prophylaxis, particularly in the setting of nonmyeloablative allogeneic SCT. It is also being used with increasing frequency to treat GVHD and may be more effective in combination with steroids than other therapies [26] . However, increasing evidence suggests that variable pharmacokinetics may be critical to efficacy of MMF.
As part of a randomized phase II trial from the Bone Marrow Transplant Clinical Trials Network (BMT-CTN), MMF 1 g twice daily plus corticosteroids was given as 1 of 4 possible therapies; MMF pharmacokinetics were studied in the context of acute GVHD response. This cohort included 32 patients and MMF pharmacokinetic sampling occurred in weeks 1 and 2. Targets for therapeutic concentration of the active metabolite mycophenolic acid (MPA) were set at 0.5 mg/mL or unbound MPA at 0.015 mcg/mL. One of the most significant findings was that approximately half of the patients did not achieve these drug levels, and drug levels had a direct impact on response rates.
The overall response rate of acute GVHD at day 28 and day 56 for all patients was impressive at 65.6 and 78% respectively as shown below. Single mycophenolic acid (MPA) pharmacokinetic measurements during weeks 1 and 2 after treatment did not correlate with CR at either day 28 or 56 (P > 0.07). However, as shown in Table II , if the mean of weeks 1 and 2 are taken together and the MPA trough was above the thresholds noted, more patients achieved CR 1 PR compared to cases where the MPA trough was low.
There is increasing use of MMF for the prevention and treatment of acute GVHD. Although direct comparative data is limited, there is an expectation that this drug has significant activity and a favorable toxicity profile compared to other immunosuppressive therapies for GVHD. However, the pharmacokinetics have not been well defined in this setting. This analysis from Jacobson and colleagues for the BMT-CTN is important since it shows that obtaining therapeutic levels is important to optimize response rates for acute GVHD without resulting in increased risk of infection or limited survival at least by day 180. This should directly impact how transplant physicians use MMF. Further pharmacokinetic studies will be needed to define the optimal dose and schedule, but consideration should be given to either close drug monitoring and/or altering dosing from 2 to 3 g/day or body weight dosing. Primary pulmonary non-Hodgkin's lymphoma (PPL) is a rare extranodal lymphoid neoplasm. The present knowledge of this entity is vague and minimal. A total of 29 patients diagnosed with PPL at Sun Yat-Sen University Cancer Center in China were retrospectively analyzed. Nineteen patients had mucosa-associated lymphoid tissue (MALT) lymphoma, nine had diffuse large B-cell lymphoma (DLBCL), and one had plasmacytic lymphoma. Six patients received PET/CT scans before treatment, and mean maximum standardized uptake values (mSUVs) were much higher in the DLBCL cases (n 5 3) than in the MALT lymphoma cases (n 5 3). The 3-year overall survival (OS) rate was 79.3%. Cox regression analysis showed that hilar/mediastinal lymphadenopathy (LAP) was an independent predictor of survival (P 5 0.029). The mean survival time (MST) was 75.5 months for patients who underwent surgery and 65.7 months for those who didn't (P 5 0.38). The MST was 77.4 months for patients treated with rituximab and 54.1 months for those not treated with rituximab (P 5 0.101). Clinical characteristics in our study were partly similar to those previously reported. However, we found that hilar/mediastinal LAP seemed to be an effective prognostic factor in Chinese PPL patients. The role of surgery and rituximab in the management of PPL warrants further investigation. PPL is quite rare and it is defined as clonal lymphoid proliferation involving the lung parenchyma and/or bronchi but without extrathoracic extension at the time of diagnosis or within the subsequent 3 months [1, 2] . Patients diagnosed with PPL may present with hilar or mediastinal LAP, but most of the tumor burden is in the lung [1] . PPL represents only 0.4% of all lymphomas, and less than 1% of NHL [2] [3] [4] . The treatment for primary pulmonary lymphomas (whether surgery, radiotherapy, chemotherapy, or targeted therapy alone or in combination) has not been clearly established. The role of rituximab in the treatment of PPL remains controversial [5] .
In this study, we retrospectively reviewed 29 cases of newly diagnosed PPL in the Sun Yat-Sen University Cancer Center in China from January 2001 to December 2008 to identify prognostic factors of PPL and optimal treatment for PPL in the rituximab era. The clinical features of all patients are presented in Table I . Nineteen patients (65.5%) had MALT lymphoma, nine (31.0%) had DLBCL, and one (3.4%) had plasmacytic lymphoma. In most of the patients in this series, surgery provided a definite diagnosis, whereas bronchoscopic biopsy had a low-diagnostic yield. Of the 16 patients with PPL who underwent bronchoscopic examination, only four had positive results. Prior to treatment, all 29 patients were evaluated by CT scans, and six patients underwent integrated 18 F-FDG-PET/CT scanning. The radiological findings for all patients are listed in Table I . Twenty patients (69.0%) had unilateral lung involvement and nine (31.0%) had hilar/mediastinal LAP. Of the six patients who underwent integrated 18 F-FDG-PET/CT scanning, three had DLBCL and three had MALT lymphoma. The maximal standardized uptake values (mSUVs) ranged from 2.0 to 7.6 (mean mSUV, 3.9 ± 1.72) in the three MALT lymphoma cases and from 10.5 to 16.9 (mean mSUV, 13.5 ± 2.74) in the three DLBCL cases.
Clinical characteristics of PPL (MALT vs non-MALT) are shown in Table I . Patients with MALT lymphoma had a higher frequency of elevated LDH level at diagnosis (15.8% vs 70.0%, P 5 0.011) and B symptoms (21.1% vs 70.0%, P 5 0.017). Moreover, a significant difference in the rate of hilar/ mediastinal LAP was found between the MALT type lymphoma group and non-MALT lymphoma group (10.5% vs 70.0%, P 5 0.002).
The primary treatment modalities of all patients were: (a) surgery alone (5 cases); (b) chemotherapy alone (6 cases); (c) surgery followed by chemotherapy (8 cases); (d) chemotherapy followed by radiotherapy (10 cases). A total of 13 patients received surgery, including two patients by VATS, one by wedge resection thoracotomy, and 10 by lobectomy. Twenty-four patients treated with chemotherapy (median: 5 cycles, range: 2-9 cycles). The most frequently used first-line treatment were CHOP (cyclophosphamide, doxorubicin, vincris- tine, and prednisone) or CHOP-like regimens. Thirteen patients received rituximab (median: 5 cycles, range: 2-9 cycles) in combination with chemotherapy: seven had MALT lymphoma, five had DLBCL, and one had plasmacytic lymphoma. The overall response rate to initial chemotherapy was 81%, including complete remission in four cases and partial response in nine cases.
Ten patients underwent radiation. The total dose of radiotherapy ranged from 30 to 46 Gy. Pneumonia related to radiation was observed in one case. In a median follow-up of 35 months (range: 5-95 months), 6 of 29 patients (20.7%) died due to disease progression (3 cases), pneumonia related to bone marrow suppression after chemotherapy (1 case), renal failure (1 case), and unknown causes (1 case). OS at 3 years was 79.3% and MST was 73.7 months (95% confidence interval, CI 59.4-88.0). MST was shorter in patients with hilar/mediastinal LAP than in those without hilar/mediastinal LAP (50.6 months vs 84.2 months, P 5 0.006) ( Fig. 1 ) and in patients with non-MALT lymphoma than in those with MALT lymphoma (48.8 months vs 83.1 months; P 5 0.009). Univariate analysis identified non-MALT type (P 5 0.009), B symptoms (P 5 0.012), high-LDH level (P 5 0.011), advanced Ann Arbor Stage (IIIE-IVE) (P 5 0.012), International Prognostic Index scores (IPI) 5 3-5 (P 5 0.040), and hilar/mediastinal LAP (P 5 0.006) as indicators of poor prognosis. Multivariate Cox regression analysis identified only hilar/mediastinal LAP as an independent predictor of OS (RR 5 11.0, 95% CI: 1.29-94.4, P 5 0.029). The MST was 75.5 months for patients who underwent surgery initially and 65.7 months for those who did not undergo surgery (P 5 0.38). The MST was 77.4 months for patients treated with rituximab and 54.1 months for those not treated with rituximab (P 5 0.101).
PPL is a very rare entity accounting for only 3.6% of all extranodal lymphoma cases [2] . The predominant pathologic subtype in our study was MALT lymphoma, followed by DLBCL, confirming previous data [6, 7] . PET/ CT imaging is a highly sensitive and specific tool for detection and localization of HL and aggressive NHL, and it also superior to CT in differentiating immunophenotypes of lymphoma [5, [8] [9] [10] . PET scans may be able to reliably distinguish between indolent and aggressive subtypes in lymphoma patients. In our study, the mSUVs were much higher in DLBCL cases than MALT lymphoma cases. DLBCL in contrast to MALT lymphoma often shows aggressive growth, which may increase FDG uptake by the lesions. Further clinical trials are warranted to determine whether the effectiveness of PET/ CT in PPL depends on the histological subtype.
MALT and non-MALT PPLs are reported to differ in clinical behavior and prognosis [7, [11] [12] [13] . In this series, hilar/mediastinal LAP, high-LDH elevation, and B symptoms were more frequent in non-MALT lymphoma. Our data demonstrated that hilar/mediastinal LAP was the only independent prognostic factor in PPL in multivariate analysis. Previous studies have presented little evidence indicating the prognostic significance of hilar/mediastinal LAP in PPL [6] . Previous reports focused mainly on MALT lymphoma and some studies did not include patients with hilar/mediastinal LAP [7, 14, 15] . Until now, no risk factors have been clearly identified in PPL. Our findings showed that patients with MALT type PPL survived longer than those with non-MALT PPL, but unlike other authors [11] [12] [13] 16] .
The optimal modality for the management of PPL has not been established so far [16] [17] [18] . In patients with MALT primary lung non-Hodgkin's lymphomas (NHL), surgical resection is commonly preferred for localized tumors. A review of 17 primary pulmonary B-cell lymphoma cases by Fadel and coworkers suggested that surgery was the treatment of choice in localized PPL cases [13] . However, in our study, the difference in survival between surgically treated and untreated groups did not reach statistical significance, similar to some of the previous reports [6, 19] .
Although the results of rituximab (a chimeric anti-CD20 antibody) plus chemotherapy for a wide variety of B-cell lymphomas, including DLBCL and MALT lymphoma, have been encouraging [20, 21] , the role of rituximab in PPL treatment is unclear. In this study, we did a preliminary evaluation of the efficacy of rituximab in the management of primary pulmonary B-cell lymphoma. Patients treated with rituximab tended to have better outcomes, but no statistical difference. Most patients in our study were in early stages. The impact of rituximab could not be demonstrated, possibly because the results of surgery and conventional chemotherapy for early-stage disease were excellent. Multicenter clinical trials will be needed to define the best therapeutic strategies.
In conclusion, the clinical characteristics of Chinese patients with PPL in our study were partly similar to those previously reported. PET/CT scan may be helpful in differentiating immunophenotypes and clinical behaviors. Furthermore, we found that hilar/mediastinal LAP could identify patients with significantly worse outcomes in Chinese PPL patients. The role of surgery and rituximab in the management of PPL patients warrants further investigation.
Patients and staging. The data from 29 patients with newly diagnosed PPL in the Sun Yat-Sen University Cancer Center in China from January 2001 to December 2008 were retrospectively analyzed. The study protocol was approved by the Institutional Review Board of the Sun Yat-Sen University Cancer Center. All patients consented to have their medical records reviewed for research purposes. The diagnosis of PPL was based on the following criteria [2, 11] : unilateral or bilateral lung involvement of NHL; no history of lymphoma; no evidence of extrathoracic lymphoma at the time of diagnosis by clinical staging workup. No extrathoracic lymphoma was detectable within 3 months after the initial diagnosis. Cases with hilar/mediastinal adenopathy or adjacent chest wall invasion were also included in our study, as described in previous reports [6, 11] . All patients were staged according to the Ann Arbor staging system [22] . IPI scores were also measured [23] .
All hematoxylin and eosin stained and immunohistochemically stained slides were reviewed, and pathological subtypes were identified according to the World Health Organization criteria by histopathologists [24] . Antibodies to the following antigens were used to confirm the diagnosis in suspected subtypes: leukocyte common antigen, cluster designation (CD)3, CD5, CD10, CD19, CD20, CD30, CD45, CD79a, Ki-67, Cyclin D1, kappa and lambda immunoglobulin, and anaplastic lymphoma kinase. For the cases with inconclusive morphological features and immunophenotypic findings, molecular genetic analysis was performed to detect rearrangements of immunoglobulin heavy chain genes and T-cell receptor gamma chain genes.
Response criteria and statistical analysis. Response to treatment was assessed according to the International Working Group Recommendations for Response Criteria for NHL [25, 26] . OS was calculated from the date of diagnosis to the date of death from any cause or to the date of last followup. Clinical characteristics of PPL cases were summarized by means, medians, and percentages, and were compared using the g 2 or Fisher's exact test. The primary endpoint of our analysis was OS. Survival curves were analyzed using the Kaplan-Meier method. The prognostic importance of different variables in OS was estimated by multivariate analysis using the Cox regression model. A two-tailed P < 0.05 assessed by the log-rank test was considered statistically significant. Statistical analysis was performed using SPSS 16.0 for Windows. 
The assessment of human organic cation transporter 1 (hOCT1) mRNA expression in patients with chronic myelogenous leukemia is affected by the proportion of different cells types in the analyzed cell population
Zdenek Racil, 1 * Filip Razga, 1 Lucie Buresova, 2 Tomas Jurcek, 1 Dana Dvorakova, 1 Daniela Zackova, 1 Shira Timilsina, 3 Petr Cetkovsky, 4 and Jiri Mayer 1
The monitoring of hOCT1 mRNA expression in patients with chronic myelogenous leukemia (CML) was used for predicting the response to imatinib treatment. However, different cell populations from patients who received various degrees of pretreatment were used for this analysis. Therefore, several biases in the results and their interpretation may arise. We investigated hOCT1 mRNA expression in different cell populations of peripheral blood (PB) from healthy volunteers and in imatinib naïve de novo CML patients by analyzing changes in hOCT1 mRNA expression during the first 6 months of imatinib therapy. The hOCT1 mRNA expression was significantly higher in PB polymorphonuclears compared to mononuclears. The hOCT1 mRNA expression in total PB leukocytes is, therefore, preferentially determined by the percentage of polymorphonuclears. Expression in each analyzed group of cells was always significantly lower in imatinib naïve de novo CML patients compared to healthy volunteers. This difference disappeared after the initiation of imatinib therapy, suggesting that CML tumor burden and the degree of pretreatment at the time of monitoring were both influencing factors. Imatinib (IMA) uptake is predominantly mediated by human organic cation transporter 1 (hOCT1) [1] . Decreased expression or function of this protein can lead to a reduction of the intracellular concentration of IMA and, there-fore, lead to pharmacokinetic resistance to the drug [1, 2] . There are several data suggesting a correlation between hOCT1 activity/expression and the therapeutic outcome of CML patients treated with IMA [3] [4] [5] . Unfortunately, this issue has several uncertainties in the literature that may lead to the misinterpretation of previously reported data and the interpretation of future studies. First, hOCT1 activity, which is measured by a functional assay [1, 2] , must be clearly differentiated from mRNA expression monitoring [1, [3] [4] [5] . Second, the cell populations used for analysis vary widely in cell type, especially in studies that monitor mRNA hOCT1 expression [1, [3] [4] [5] . Furthermore, while some studies have monitored a well-defined group of patients with de novo chronic phase CML [3] , others make analyses on heterogeneous study populations that contain pretreated patients [4, 5] .
We investigated differences in hOCT1 mRNA expression in different cell populations of PB leukocytes (LEU) from healthy volunteers and patients with de novo CML, a change in hOCT1 mRNA expression during the first 6 months of IMA treatment, and the relationship between hOCT1 mRNA expression and the percentage of immature myeloid cells as well as BCR/ ABL transcript levels in PB (as indirect markers of disease burden).
The hOCT1 mRNA expression was measured in PB LEU (n 5 66), PB PMNC (n 5 35), and PB MNC (n 5 33) from 66 healthy volunteers. Figure  1 summarizes these results and clearly shows a wide range of hOCT1 mRNA expression in total LEU as well as in PMNC and MNC. The hOCT1 mRNA expression was significantly higher in PMNC compared to MNC. Therefore, the total LEU hOCT1 mRNA expression was preferentially determined by the percentage of PMNC in the total LEU.
Analogous measurements were performed in 23 patients with IMA naïve de novo CML. Similarly, a large distribution of expression was seen from individual groups of cells (Fig. 1) . Total hOCT1 mRNA expression in PB LEU was again significantly influenced by the PMNC hOCT1 mRNA expression and PMNC percentage. The large variability in the percentage of PMNC from total LEU in patients with IMA naïve de novo CML (median: 75%; range: 33-93%) is, therefore, an important factor in the marked variability seen in hOCT1 mRNA expression in total LEU at the time of CML diagnosis. In addition, the hOCT1 mRNA expression from each analyzed group of cells was always significantly lower in IMA naïve de novo CML patients compared to healthy volunteers (Fig. 1 ). This suggested that CML cells have an additional effect on the observed hOCT1 mRNA expression level.
The hOCT1 mRNA expression was detected in total LEU and in MNC at monthly intervals from IMA naïve de novo CML patients from the time of diagnosis until the 6th month of IMA therapy. The median hOCT1 mRNA expression level from total LEU was significantly lower in the CML patients at the time of diagnosis compared to median expression levels in healthy individuals ( Fig. 1 ; P < 0.001; nonparametric Mann Whitney test). However, after 1 month of IMA treatment, the median hOCT1 mRNA expression level in total LEU significantly increased (P 5 0.031; nonparametric Wilcoxon test) and reached the expression levels from healthy volunteers. Therefore, the previously described difference in expression level disappeared during this observation period (P 5 0.769; nonparametric Mann Whitney test) ( Fig.  2A) . The same finding of hOCT1 mRNA expression was seen in the MNC samples from patients under the same criteria ( Figs. 1 and 2B) . The lower hOCT1 mRNA expression level in LEU from IMA naïve de novo CML patients compared to healthy volunteers as well as its progressive increase after the start of IMA therapy could be explained by the initial presence and subsequent gradual disappearance of tumor cells from the measured cell population. To confirm this hypothesis, we performed an analysis on the relationship of hOCT1 expression with the percentage of immature myeloid cells in the LEU samples (as a marker of disease burden) obtained from IMA naïve de novo CML patients. In addition, because of the fact that a majority of patients did not have immature myeloid cells present in PB LEU samples after the first month of IMA therapy, we analyzed the dependence of LEU hOCT1 mRNA expression on the amount of BCR /ABL transcripts from these samples. We found a statistically significant relationship between hOCT1 mRNA expression level and the percentage of immature myeloid cells at the time of diagnosis as well as the BCR/ABL transcript level at the first month after the start of IMA treatment (Fig. 3A,B) .
Very limited data concerning hOCT1 mRNA expression in different cell populations exist to date [6] [7] [8] . Bazeos et al. reported a higher hOCT1 mRNA expression level in PB PMNC compared to MNC from healthy individuals [6] . In this study, we have enhanced these findings and described the difference in expression level in IMA naïve de novo CML patients as well.
Our finding of lower hOCT1 mRNA expression in total LEU from IMA naïve de novo CML patients that quickly reached the expression levels of healthy volunteers is in accordance with observations from Bazeos et al. [6] . However, the authors from that study assumed that this finding was related to the achievement of a complete cytogenetic response. According to our findings, the increase in hOCT1 mRNA expression after the start of IMA treatment was not caused by the suppression of inhibitory effects of the BCR/ABL oncogene, as suggested by Bazeos et al., but rather was due to a significant effect of the lower hOCT1 mRNA expression in immature myeloid cells (mostly CML tumor cells), which rapidly disappeared from the PB within the first month of IMA therapy.
Several studies have monitored hOCT1 mRNA expression in the MNC fraction of the leukocytes [3, 9] . However, the MNC is composed of several types of cells (including immature myeloid precursors, mostly tumor cells in CML patients). Although not analyzed, because of a low number of available samples for hOCT mRNA measurement in MNC (required for statistical significance), we expected that the impact of the cell composition found in total leukocytes on the hOCT mRNA expression results would be similar when measured in MNC. Therefore, it is not surprising that Labussiere et al. and White et al. did not dem- letters onstrate a relationship between hOCT1 expression measured in IMA naïve de novo CML patients and the future therapeutic response [3, 9] . Although Wang et al. [5] was able to show this relationship, only 16 of the 70 CML patients from this study were not pretreated. Thus, it is unknown whether the high hOCT1 mRNA expression level seen in Wang et al. could be a real predictor of therapeutic response in the future, or whether patients with a high hOCT1 mRNA expression level had a differential leukocyte and mononuclear count (due to pretreatment) that was close to that of healthy individuals and therefore higher than other patients in the study. In addition, Wang et al. did not clearly define the cell population in which measurements was carried out, which creates additional uncertainty in their results. Similarly, Crossman et al. demonstrated a relationship between of hOCT1 mRNA expression and the achievement of a complete cytogenetic response [4] . However, all patients in that study were pretreated and this fact could explain why there was no observed difference between expression of hOCT1 mRNA in CML patients and healthy donors. Furthermore, Crossman et al. used bone marrow MNC for monitoring hOCT1 mRNA expression. The influence from the higher percentage of different myeloid precursors in these samples, as well as the greater heterogeneity in hOCT1 mRNA expression, is likely to exceed the influence of the immature cells in the PB samples that we have described in the present study.
In summary, our study has shown that monitoring hOCT1 mRNA expression in total PB leukocytes is controversial. However, we have shown that this level corresponds to the current differential count of leukocytes and the quantity of tumor cells at the time of testing, rather than being a prognostic factor that correlates with the future therapeutic response of patients with CML. Although we did not assess enough patients in this study for unequivocal evidence, it can be assumed that an analogous situation exists in monitoring the hOCT1 mRNA expression level in PB mononuclear cells, as it is also a mixture of cells (although less heterogeneous than the total leukocytes) with different expression levels of hOCT1 mRNA. Finally, the question remains of whether or not the dependencies of hOCT1 mRNA expression in total leukocytes described in this study also apply to functional assays that analyze the activity of the hOCT1 transporter. It would be of great interest to perform analogous analyses to those in this study for monitoring the activity of the hOCT1 transporter. These data would be essential for the evaluation of all studies that assess the importance of this parameter as a marker of therapeutic response to IMA in de novo CML patients.
A total volume of 10 mL of PB in ethylenediaminetetraacetic acid was obtained from 66 healthy volunteers and from a cohort of 23 IMA naïve de novo CML patients (month 0 5 M0 samples). CML patients were investigated monthly for a period of 6 months from the start of IMA treatment (M1-M6 samples). All volunteers and patients signed an informed consent form and the study was approved by the institutional review board.
The total volume of PB was used for the leukocyte (LEU) separation and for fractionation into polymorphonuclear cells (PMNCs) and mononuclear cells (MNCs) using gradient centrifugation Histopaque 1 1077/1119 (Sigma Diagnostics, St. Louis, USA). The fractionation was performed according to the manufacturer's recommendations. For verification of the composition of leukocyte fractions obtained by separation, microscopic analysis of individual cell groups (PMNC and MNC) was performed. Smears were processed using May-Grünwald and Giemsa-Romanowski solutions. Interestingly, the MNC fraction from CML patients whose PB was collected at the time of diagnosis also contained immature cells (e.g. myelocytes, promyelocytes, and metamyelocytes).
Extraction of total RNA from all selected cell fractions (LEU, PMNC, and MNC) was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's manual. A cDNA synthesis was performed using random hexamers and MuLV reverse transcriptase (Applied Biosystems, Foster City, USA).
To detect the expression level of hOCT1, GUS, BCR/ABL, and ABL mRNA from selected fractions at different time points of IMA therapy, the TaqMan based real-time reverse transcription PCR (RQ-RT-PCR) with absolute quantification was carried out on an ABI PRISM 7300 (Applied Biosystems Inc, Foster City, USA). The RQ-RT-PCR using the Absolute Q PCR Mix (ABgene, Epsom, UK) was performed at 958C for 15 min followed by 50 cycles of denaturation at 958C for 15 s and extension at 608C for 1 min.
For hOCT1 cDNA amplification, a TaqMan Gene Expression Assay (serial number Hs00427552_m1) (Applied Biosystems Inc, Foster City, USA) was used according to the manufacturer's recommendations. The final hOCT1 mRNA expression level was then calculated using the following formula: hOCT1 (absolute copies) /GUS (absolute copies) 3 100%. To detect the expression of the control gene GUS in all selected cell fractions, the primer set and probe from Beillard et al. were used [10] .
The BCR/ABL quantification was performed in LEU using standard methodologies with the primer set and probe previously published by Gabert et al. [11] . The final BCR/ABL expression was calculated according to BCR/ ABL (absolute copies) /ABL (absolute copies) 3 100%.
The RQ-RT-PCR product obtained from the hOCT1 TaqMan Gene Expression Assay (Hs00427552_m1; Applied Biosystems Inc, Foster City, USA) was purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and ligated into pCR 2.1 vector using the TA Cloning Kit (Invitrogen, Carlsbad, USA). Recombinant plasmids were subsequently used for Escherichia coli transformation. Plasmid DNA was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and used as the standard for hOCT1 absolute copy number quantification in 10-fold dilutions (range 10 6-10 1 copies). Standards for GUS, BCR/ABL, and ABL with serial numbers CGRS-03, FGRS-10, and CGRS-01, respectively (FusionQuant 1 Standards Kit, Ipsogen, Marseille, France), were used for the absolute quantification of these particular genes. 
To ensure reproducibility, all samples were performed and analyzed in duplicate. To obtain robust results, a cDNA from the KCL-22 cell line (DSMZ, Braunschweig, Germany), which has low hOCT1 mRNA expression, was used as an internal control for hOCT1 and GUS assessment. In addition, cDNA from the K562 cell line (DSMZ, Braunschweig, Germany) was used as an internal control BCR/ABL quantification.
For all statistical analyses, the software Statistica, version 9.0 (StatSoft, Tulsa, USA) and R (R Development Core Team, Auckland, New Zealand) were used.
Daphne R. Friedman, 1 * April D. Coan, 2 Sophia K. Smith, 3 James E. Herndon II, 4 and Amy P. Abernethy 5
Cancer Survivorship Care Plans (SCPs) are a recommended part of medical care for cancer survivors. We sought to identify the medical and psychosocial informational needs of non-Hodgkin lymphoma survivors and their physicians in cancer survivorship care to be included in SCPs. Questionnaires were mailed to eligible lymphoma survivors and their physicians, querying their preferences about informational needs in SCPs. The survivor cohort had a median age at diagnosis of 62, with 57% Female, 87% White, and 76% from North Carolina. The physician cohort was comprised of oncologists (27%) and nononcologists (73%), and 86% practiced in North Carolina. Greater than 60% of both survivors and physicians preferred an oncologist and primary care provider comanage cancer survivorship care. The most highly rated informational needs were medical issues, although there were differences between survivors and physicians for many of the informational needs queried. There was higher concordance between patient and physician responses for medical issues (63 to 100%) compared to psychosocial issues (25 to 63%). Important components of SCPs for lymphoma survivors and their physicians may go unrecognized. Tailoring SCPs to patient or physician users may better accommodate the differences in need for particular cancer survivorship care information.
As cancer is becoming more treatable and curable, the numbers of cancer survivors is increasing [1] . Medical complications, long-term and late effects of therapy, and psychosocial difficulties are among the issues with which cancer survivors must live [2] [3] [4] .
In recognition of this, SCPs were recommended, in the first comprehensive manner, by the Institute of Medicine [5] . Several templates exist (e.g. American Society of Clinical Oncology [6] , Lance Armstrong Foundation/ OncoLink [7] , and Journey Forward [8] ), but patients criticize these tem-plates as being too technical, lacking personalization, and being deficient in resource and wellness guides [2, 9] . Barriers to development and implementation of these care plans include difficulty in identifying evidence-based components [10] , insufficient reimbursement for record abstraction [11] , lack of specific medical knowledge by primary care physicians (particularly regarding hematologic malignancies) [12] , and poor communication between oncologists and primary care physicians [5, 13] .
Despite a growing body of literature regarding SCPs, the oncology community has not fully recognized that cancer survivors, oncologists, and primary care providers have varying levels of knowledge and different experiences, and thus may have different informational needs regarding cancer survivorship care [2] . By focusing survivorship care based on prioritized needs, cancer survivors may be more receptive to receiving continued medical care in all areas.
To address these issues, we focused on a group of survivors who are treated in a standardized manner, but who are often left out of the SCP development discussions, specifically survivors of diffuse large B-cell lymphoma, the most common type of non-Hodgkin lymphoma. We aimed to (1) identify the most important informational needs of survivors and their treating physicians responsible for cancer survivorship care, (2) define the effect of demographic factors on informational needs, and (3) assess how closely the informational needs of individual survivors link to that of their physicians.
A total of 178 surveys were mailed to cancer survivors. Fourteen were excluded because of death (4) or returned mail (10) . Sixty-seven of the 164 potential respondents responded, resulting in an overall response rate of 41%. Seventy-eight surveys were mailed to physicians. Two were excluded because of returned mail. Twenty-two of the 76 potential participants responded, resulting in an overall response rate of 29%. Baseline character-letters istics for survivor and physician cohorts are summarized in Table I . Survivor responders were older at diagnosis than nonresponders (P 5 0.024); otherwise responders did not differ significantly from nonresponders with respect to baseline characteristics. Thirteen survivors (19%) completed the survey less than 2 years since their diagnosis, 26 (39%) completed the survey 2 to 5 years since their diagnosis, and 28 (42%) completed the survey more than 5 years since their diagnosis.
The majority of survivors (63%) and physicians (64%) preferred an oncologist and primary care provider to comanage cancer survivorship care. Among survivors, there was no association between having a primary care physician and the preference of type of physician(s) to manage cancer survivorship care (P 5 0.143).
The queried components of SCPs and their ratings for survivors and physicians are presented in Table II . Compared to physicians, survivors were more interested in having a plan for screening of recurrence (P 5 0.012) and late effects of therapy (P 5 0.018) and a summary of treatments given (P 5 0.044), whereas physicians thought summaries of the treatment complications (P 5 0.005) were more essential parts of the SCP than survivors. Additionally, a plan to monitor overall health was a more important component of cancer survivorship care for survivors than for physicians (P 5 0.0002). Overall, physicians and survivors rated psychosocial issues as less important in SCPs than medical issues. However, comparing the responses obtained from survivors and physicians, survivors identified nutrition and exercise (P 5 0.002), insurance (P < 0.0001), and payment for providing cancer survivorship care (P 5 0.0006) as more important than physicians did.
There were no differences among the survivor subgroups with regards to who should provide cancer survivorship care, having a primary care physician or having an oncologist outside of our tertiary referral hospital. Male survivors rated sexuality and fertility as more important than women (P 5 0.004); otherwise there were no significant differences based on gender. Survivors who were younger than 60 years old at time of diagnosis identified a plan for monitoring overall health problems (P 5 0.03), sexuality and fertility (P 5 0.007), mental health services (P 5 0.044), and financial issues (P 5 0.042) as more important than survivors who were older than 60 years old at diagnosis. There were no differences in responses obtained from survivors greater than 5 years compared to those less than 5 years from the time of diagnosis.
Physicians of different specialties were equally likely to prefer both oncologists and primary care physicians comanage cancer survivorship care. Nononcologists thought that having a plan to screen for cancer relapse was more important than oncologists (P 5 0.004), whereas oncologists were more likely to find that providing insurance information and help with resolving financial issues were important components of cancer survivorship care compared to nononcologists (P 5 0.021 and 0.035, respectively).
There were 24 matched survivor physician survey pairs. One survivor had two physicians and one physician had three survivors; thus one survivor and one physician contributed more than once to the analyses. As seen in Fig.  1 , there was higher concordance between survivors and their physicians with regards to medical issues (62.5 to 100%) than for psychosocial issues (25 to 62.5%). 
The number of cancer survivors is increasing, and the burden of their diseases and late or long-term effects of cancer therapy has medical, psychosocial, and economic effects on the survivors themselves and society at large [3, 4, 14] . Providing comprehensive and efficient medical care to this population would help to address these problems. But, what should constitute this medical care?
Instead of moving directly into implementing a ''one-size-fits-all'' approach, we decided to query a defined subgroup of non-Hodgkin lymphoma survivors and their physicians, to identify who should provide cancer survivorship care and to determine the important components of such care. We chose this group of cancer survivors and their physicians because they have been evaluated in a limited fashion [12, 15, 16] , and not as thoroughly as breast cancer, childhood cancer, or Hodgkin lymphoma survivors. In addition, we wanted to begin our analyses with a homogeneous group of survivors with regards to curative intent and treatment regimens. Survivors with incurable malignancies or with cancers treated with a variety of different chemotherapy regimens or therapeutic modalities might have varying opinions about cancer survivorship care.
Using our mailed questionnaire, we confirmed prior studies' findings that survivors and physicians prefer cancer survivorship care coordinated by both the oncologist and the primary care physician [3, 13, 17] . We also found that medical issues were also highly rated as important components in cancer survivorship care for both survivor and physician groups. Psychosocial, alternative medicine, and fertility issues have been noted to be important to cancer survivors, particularly breast cancer survivors, and are increasingly being studied in the lymphoma survivor population [4, 18] . However, our results suggest that survivors of diffuse large B-cell lymphoma believe medical content is a more important component of SCPs.
We found that survivors thought that a plan to monitor overall health was a more important part of SCPs than physicians. This might imply that survivors view cancer survivorship care as part of their general health maintenance, whereas physicians compartmentalize survivorship care and noncancer related care.
Survivors rated nutrition, exercise, and insurance SCP components higher than their physicians. Whereas physicians might be more comfortable discussing a survivor's general health, they may have less immediate information and the resources to address these other issues. Notably, the results found in this cohort vary from those found in other studies, in which there was more agreement between physicians and a heterogeneous survivor cohort in expectations for cancer survivorship care [19] .
Our findings of differences between physicians and survivors regarding the relative importance of different cancer survivorship care issues confirmed our hypothesis that the different ''target audiences'' might need different SCPs. While providing the maximum available information to everyone might seem optimal, this approach risks overloading the user and diluting the impact of the most important content. Developing personalized SCPs could address these barriers and adjust the type, level, and amount of information for the end user's needs.
Limitations of this study include the small sample size, low response rate, exclusion of survivors with >1.5 years since last contact, and inclusion of survivors from a single institution. The questionnaire was not formally tested in a pilot cohort, and the wording of the questions varied slightly between physician and survivor questionnaires. Because many of our findings echo results obtained by other studies performed in other survivor groups, we feel that major improvements in the response rates would unlikely influence our findings.
In contrast to the currently available templates containing standardized information by cancer type, our findings indicate that SCPs should be individually tailored to reflect the prioritized needs of survivors, primary care physicians, and oncologists. Our findings should be confirmed and compared to other larger cohorts or cohorts of other cancer subtypes. Comparative effectiveness research is needed to evaluate whether the implementation of personalized survivorship care plans provide higher levels of survivor and/or physician satisfaction, knowledge, and quality of life than the use of a single standardized survivorship care plan.
Participants. Survivors were identified through the Duke Comprehensive Cancer Center (DCCC) Tumor Registry. Eligible participants seen at Duke for treatment or for consultation, were treated with curative intent, and had no evidence of lymphoma at their last visit at Duke. We further limited our cohort to lymphoma patients with a diagnosis of diffuse large B-cell lymphoma as treatment is more standardized, homogenous, and aggressive than for all subtypes of lymphoma taken together. To reduce the chance of questionnaires mailed to survivors who had died, moved, or otherwise lost to follow up, eligible patients were included in this study only if they had been seen within the Duke University Medical system within 1.5 years prior to the chart review.
Physicians were identified in two ways. The Duke oncologists' names were obtained from the DCCC registry. Local oncologists' and primary care physicians' names were obtained from survivors' questionnaires responses.
Questionnaires. The mailed questionnaires for survivors and for physicians were developed by the investigators. The questionnaires were designed after reviewing available SCP templates, exploring literature regarding medical and psychosocial issues relevant to cancer survivors and their physicians, and querying oncologists and internists. Questionnaires for survivors and physicians included the same components, but the wording was not identical. Participants rated the importance of each item in a set of medical and psychosocial informational needs (listed in Table I ) using a numeric scale of 1 to 10 for each question. A letter explaining the study was mailed to each survivor with the questionnaire. After 6 months, a reminder card was mailed to nonresponders. Following receipt of identified physicians from survivor questionnaires, survey packages were mailed to the identified physicians. Survivor surveys were received between June and December 2008. Physician surveys were received between November 2008 and January 2009. Informed consent was inferred by a response, per the Duke University School of Medicine Institutional Review Board (Duke IRB) mailed questionnaire policy. This study was approved by the Duke IRB.
Patient characteristics. Demographic information was obtained from the DCCC registry and chart review. Specifics regarding stage of disease, therapies received, and treatment outcomes were obtained from chart review.
Statistical analysis. Baseline demographics and characteristics for the survivor and physician cohorts were summarized by descriptive statistics. Within these cohorts, the characteristics of responders and nonresponders were compared using two-sample t-tests (for continuous variables) and Fisher's exact tests, Chi-square tests, or Cochran-Armitage Trend tests (for categorical variables) to assess selection bias.
Descriptive statistics (mean and standard deviation) were computed for survivor and physician ratings of individual informational needs. Two-sample t-tests and exact chi-square tests were used to assess the relationship between outcome (e.g., informational needs or type of health care provider) and survivor/physician subgroups. Gender, age at diagnosis (<60 years, =60 years), and time since diagnosis (<5 years, =5 years) were used as survivor subgroups and specialty (oncology, nononcology) was considered for physicians. Sixty years was chosen as the age cut-off, consistent with the International Prognostic Index [20] . Likewise, 5 years since diagnosis was chosen as a cut-off, as the 5-year time horizon from diagnosis often signifies ''cure'' from cancer.
Initially the survivor and physician cohort were treated as independent groups and two-sample t-tests were used to detect differences in mean ratings of each individual item between survivors and physicians. Survey responses from survivors were also matched with their self-identified primary care physician and/or oncologist to form survivor physician pairs. A physician may have cared for more than one survivor and a survivor may have had more than one physician. Thus, in all paired analyses, a survivor or physician may have contributed more than once to the analyses. Agreement in ratings of issues concerning survivorship care was assessed between survivors and their physicians using concordance. Concordance was defined as complete agreement in response categories. For this analysis, ratings were categorized as follows: 1-3 (low importance), 4-6 (moderate importance), and 7-10 (high importance) [19] .
All statistical analyses were conducted with SAS version 9.2 (SAS Institute, Cary, NC). A significance level of 0.05 was used for all statistical tests. P-values are presented for descriptive purposes.
Pain rate and social circumstances rather than cumulative organ damage determine the quality of life in adults with sickle cell disease Charlotte F.J. van Tuijn, 1 Eduard J. van Beers, 1 John-John B. Schnog, 2 and Bart J. Biemond 1 * Due to the significant morbidity associated with sickle cell disease (SCD), sickle cell patients have a reduced quality of life (QoL). Even though pain is considered an important determinant of QoL in sickle cell patients, factors such as organ damage and socioeconomic circumstances may also be important. Therefore, we determined the contribution of chronic organ damage and sickle cell-related complications to QoL and also analyzed the effect of vaso-occlusive crises and socioeconomic circumstances on QoL. Consecutive adult sickle cell patients were included. QoL was represented in a physical component scale (PCS) and a mental component scale (MCS) and assessed with SF-36 forms. Higher pain rates were related to lower QoL scores. Both occupation and the level of education were significantly related to PCS while no relation with MCS or pain rate was found. Thirty-five percent of the patients were unemployed when compared with 6% of the general population and 16% of immigrants without SCD. Neither genotype nor the presence of chronic organ damage were significantly related to QoL. In conclusion, a reduced QoL was mainly determined by pain rate, occupation, and educational level. Chronic organ damage, although a major factor determining life expectancy in SCD, was not a determinant of QoL.
SCD is a hereditary hemoglobinopathy characterized by recurrent microvascular vaso-occlusion and chronic hemolytic anemia resulting in progressive organ damage and reduced life expectancy [1, 2] . Given the chronic nature of SCD and the severity of its complications, patients are likely to have a significantly reduced QoL. Intuitively and historically, pain is widely regarded as the major factor in determining QoL in SCD. However, even though previous studies have indeed demonstrated a reduced QoL of sickle cell patients, this was often not or not only attributable to pain [2] . Factors such as anxiety, depression, and socioeconomic circumstances may also contribute to the reduced QoL of sickle cell patients [3] [4] [5] .
Sickle cell patients develop organ damage irrespective of their frequency of acute painful events, which has been related to significant morbidity and increased mortality, and it seems likely that SCD-related organ damage negatively impacts QoL of sickle cell patients [6] . However, to our knowledge, the impact of chronic organ damage on QoL in SCD has not been previously assessed. Therefore, we prospectively assessed the QoL in a cohort of consecutive sickle cell patients in whom chronic organ damage, pain rate, and the history of sickle cell-related complications in the last 5 years was systematically analyzed. In addition, the impact of occupation and educational level on QoL of these patients was studied.
One hundred seventeen adult patients with SCD (HbSS, HbSb 0 , HbSC, and HbSb 1 ) were eligible for our study. Twenty-three patients were excluded from analysis because they were not able to fill in the SF-36 questionnaire for different reasons (illiterate persons, mental retardation or repeated no show at their appointments). These excluded patients did not differ from the study population concerning outcome variables (pain, disease severity, education, and occupation). QoL was assessed in 51 patients with HbSS, 5 with HbSb 0 , 11 with HbSb 1 , and 27 patients with HbSC.
The QoL data of our population are presented in Table I . As expected, our patient group scored lower on QoL compared with the healthy Dutch population [7] (see Supporting Information Table IV) . No correlation between the MCS and PCS was found in the sickle cell population (spearman r 5 0.14; P > 0. 20) . When comparing male to female sickle cell patients, the overall PCS appeared to be significantly higher in males when compared with females (P 5 0.015). We found no significant difference in the overall MCS between males and females. Older patients scored lower on the PCS in comparison to younger patients (P 5 0.002) while no significant differences in QoL scores between genotypes was found (data not shown).
The QoL scores in relation to pain rate are depicted in Table II . With respect to the MCS, the overall score and all subscales, except for mental health, were significantly lower in patients with the highest pain rate (P 5 0.021). Although significantly lower scores were found on the role-physical scale (P 5 0.005) and the general health scale (P 5 0.033) in patients with the highest pain rate, the overall PCS was not determined by the number of admissions for painful crises (P 5 0.110). Also, analysis by linear regression confirmed that pain rate is significantly associated with MCS and is less likely to influence the PCS (data not shown).
The prevalence of different forms of organ damage in our study population was: pulmonary hypertension (PHT): 24.5%, renal failure: 3.2%, microalbuminuria: 21.3%, retinopathy: 31.2%, iron overload: 9.6%, avascular osteonecrosis: 11.7%, leg ulcers: 6.4%, Acute chest syndrome (ACS): 25.5%, priapism: 8.5%, and stroke: 4.3%. Except from lower QoL scores (for the PCS) in patients with iron overload (P 5 0.017), no association between any form of organ damage or sickle cell-related complications and QoL scores was found (Table III) . Moreover, when patients with organ damage and complication were divided according the pathogenesis (hemolysis-related complication versus vaso-occlusion/ischemia-related complications), no significant differences in QoL was observed when compared with the patients without organ damage at all.
With respect to the relation between occupation and educational level and QoL scores, the PCS was strongly associated with occupation and level of education (P < 0.001). The overall MCS was not related to occupation or the level of education (P 5 0.206 and P 5 0.177, respectively) (see Supporting Information Tables V and VI) . Occupation and the level of education were not different between different genotypes and were not related to pain rates.
SCD is heterogeneous in its clinical presentation with patients being continuously admitted for the management of disease-related complications at one end of the spectrum and patients rarely requiring medical care at the other [6] . Irrespective of the frequency of acute clinical complications, most patients develop accumulating organ damage throughout their lives as a result of chronic hemolytic anemia and ongoing vaso-occlusion [1] . With respect to QoL, previous studies have reported that sickle cell patients have lower QoL scores when compared with race-and age-matched controls, which were related to pain [8] [9] [10] . However, its relation to the presence of chronic organ damage has not been described before. In the present study, organ damage appeared not to be related to any of the QoL scores. If patients were grouped according the pathophysiology of their different forms of organ damage or a history of sickle cell-related complication as recently proposed by Kato et al. [11] , also no relation with QoL scores was demonstrated. Surprisingly, PHT, which has been related to early mortality in SCD, was not related to QoL in our patients. This might be explained by the fact that our study population consists of patients with mild PHT (regurgitation jet flow velocity < 2.9 m/sec), which is generally not related to severe complaints or significantly reduced exercise tolerance.
Factors significantly associated with a reduced QoL were pain rate and social circumstances defined as educational level and occupation. Interestingly, pain rate was only associated with a reduced MCS, whereas the social circumstances of the patients were related to the PCS. These data suggest that frequent painful and unpredictable crises are an important psychological burden rather than a physical burden to sickle cell patients as has been suggested previously [12] . In contrast, social circumstances as reflected by occupation and level of education were mainly related to the PCS, suggesting that only patients in good physical performance are able to finish school and find jobs independently of the frequency of admission for painful crises. This was further supported by the finding that no correlation between the MCS and PCS was found in the sickle cell population, which is in contrast to letters the findings of QoL assessments in healthy individuals [7] . Similar to previous findings of McClish et al. [10] , we did not find a correlation between genotype and QoL scores. Also, no difference in QoL scores was observed between patients on hydroxyurea and those who are not on hydroxyurea. This might be explained by the fact that in our study population, hydroxyurea was only prescribed to reduce painful crises and the occurrence of acute chest syndrome. By reducing these complications in patients with an otherwise more severe clinical presentation, hydroxyurea may have improved the QoL. This is in line with a previous prospective study of Ballas et al. [13] who demonstrated an improved QoL with the use hydroxyurea.
Certain limitations of our study should be taken into account. First, by defining pain rate by the amount of clinical admissions, the conclusions of our study may not be extrapolated for the number of painful crises experienced at home, which have been reported to occur frequently in sickle cell patients [14] . However, previous studies have shown that pain (during a painful crises as well as chronic pain) is related to QoL in sickle cell patients [8, 10, 13] . Second, most forms of organ damage and sickle cell-related complications were only present in a relative small group of patients, which may have underpowered our study to detect an effect of specific forms of organ damage on QoL. Mild PHT, as one of the most relevant forms of organ damage that has been associated with poor prognosis, was present in 24% of all sickle cell patients and was not related to QoL.
In conclusion, the QoL scores in consecutive sickle cell patients appear to be determined mainly by pain rate and social circumstances. Despite the contribution of organ damage such as (mild) PHT on prognosis and life expectancy, cumulative organ damage does not seem to be an important determinant of QoL in SCD. In terms of clinical presentation, pain rate is the most important factor for the QoL of adult sickle cell patients.
Adult sickle cell patients visiting the Department of Hematology of the Academic Medical Centre in Amsterdam were eligible for our study. Inclusion criteria were: high-performance liquid chromatography (HPLC) confirmed diagnosis of homozygous sickle cell anemia (HbSS), sickle-C disease (HbSC), HbSb 1 -thalassemia (HbSb 1 -thal), or HbSb 0 -thalassemia (HbSb 0 -thal), age 18 years or older and capable of filling in a questionnaire (Dutch or English). All data were collected during a routine outpatient clinic visit.
QoL was assessed by the use of SF-36 forms. The SF-36 is a short-form health survey that has been proven to be valid and reliable in the black population [15] . It has been previously used to determine QoL in adults with SCD [16] . Briefly, it yields eight different scales (physical functioning, role limitations due to physical problems, role limitations due to emotional problems, social functioning, mental health, vitality, bodily pain, and general health perceptions) of functional health and well-being as well as psychometrically based physical and mental health summary measures. The SF-36 is a generic measure, which is not age, disease, or treatment specific. Accordingly, the SF-36 has proven to be useful in surveys of general and specific populations, comparing the relative burden of diseases [7] . All patients were asked to complete this questionnaire during a routine outpatient visit. All scale scores are linearly converted to a 0-100 scale, with higher scores indicating higher levels of functioning or well-being. We analyzed the scores of our study population and compared the data with the scores of the general healthy Dutch population [7] .
Data on social circumstances represented by occupation and level of education were routinely gathered during the first routine visit for every patient. With respect to occupation patients were divided into: employed, unemployed, or student. Education level was divided into: high school or less, vocational education/community college, and tertiary education/ university.
Pain rate was defined as the number of admissions for treatment of a vaso-occlusive crisis from January 2002 until January 2007 and was determined by chart review [6] . Subsequently, three groups were arbitrarily defined: patients without, patients with <1 admission for painful crises per year, and patients with 1 or >1 admission for painful crisis per year.
Organ damage and sickle cell-related complications were assessed by systematic screening and medical record review and defined as follows: PHT: tricuspid regurgitation jet flow velocity (TRV) equal to or higher than 2.5 m/sec in rest detected by Doppler echocardiography. PHT was considered absent with no or only trace TRV [17] . Renal failure: an estimated creatinine clearance lower than 100 mL/min (Cockcroft and Gault). Microalbuminuria: urinary creatinine (mmol/L) to urinary albumin (mg/L) ratio higher than 3.5 for males and higher than 2.5 for females confirmed with 24 hr urine collection with microalbuminuria higher than 30 mg/24 hr [18] . Retinopathy: presence of at least mild nonproliferative retinopathy [19] . Iron overload: plasma ferritin level higher than 1,000 mmol/L (on at least three occasions during steady state) and a history of more than 20 transfused packed cells [20] . Symptomatic avascular osteonecrosis: local pain and reduced function with documented osteonecrosis of the femoral or humeral head (hip or shoulder X-ray) or a history of surgical intervention for osteonecrosis. Leg ulcers: chronic ulcers of the ankle not otherwise explained. Acute chest syndrome: defined as described by Stuart and Setty [21] occurring between January 2002 and January 2007. Priapism: spontaneous painful erection requiring hospital care. Stroke: history of stroke confirmed by magnetic resonance imaging or computerized tomography. We analyzed the relation between organ damage and QoL both for each form of organ damage or complication separate as well as for groups. Groups were divided in a group with no organ damage or sickle cell-related complications, a group with organ damage or complications due to hemolysis (leg ulcers, PHT, priapism, and stroke), or a group with organ damage related to vaso-occlusion (ACS, retinopathy, avasculair osteonecrosis, renal failure, and microalbiminuria) as proposed previously [11] .
Hematological and biochemical laboratory parameters were assessed at the same visit at which the patient completed the QoL questionnaire. Fetal hemoglobin percentage (HbF%) was determined by cation-exchange HPLC and a-thalassemia screening was performed with a multiplex PCR assay [22] . If not available, we used the results from the latest outpatient visit [23] .
All numbers in the tables are medians with corresponding interquartile ranges unless stated otherwise. Difference in continuous data between groups was tested with the Mann-Whitney U test. Difference in categorical data between groups was tested with the x 2 test or Kruskal Wallis test. Pvalues 0.05 were considered statistically significant. Multiple linear regression analyses were performed to analyze the interaction between the different scales of the SF-36 questionnaire and pain rate. All missing data were considered missing at random. In the case that one or more questions were not answered in a specific scale of the SF-36 questionnaire, the mean value of the remaining questions on that specific scale was used for the missing items. When half or more of the questions were not answered, the results of the specific scale were discarded. Statistical analysis was performed by using SPSS 12.0.2 (SPSS, Chicago, IL).
A specific linkage between the incidence of TP53 mutations and type of chromosomal translocations in B-precursor acute lymphoblastic leukemia cell lines Takeshi Inukai, 1 * Xiuru Zhang, 2 Takeshi Kameyama, 2 Yukiko Suzuki, 2 Kazuhito Yoshikawa, 2 Itaru Kuroda, 1 Atsushi Nemoto, 1 Koshi Akahane, 1 Hiroki Sato, 1 Kumiko Goi, 1 Kazunori Nakamoto, 3 Jun-ichi Hamada, 2 Mitsuhiro Tada, 2 Tetsuya Moriuchi, 2 
Chromosomal translocation plays an essential role in leukemogenesis of childhood B-precursor acute lymphoblastic leukemia (ALL) in combination with specific gene mutations. Although several reports suggest correlation of TP53 mutation with the type of chromosomal translocation in childhood B-precursor ALL, it has not been directly confirmed in a single cohort study due to the limited incidence of TP53 mutation in primary samples at diagnosis. Leukemia cell lines generally show higher incidence of gene mutations compared with primary samples. Thus, to clarify possible differences in cooperation of the p53 pathway for leukemogenesis with the type of chromosomal translocation, TP53 gene mutation was analyzed in 32 B-precursor ALL cell lines. TP53 mutation was observed in 40.6% (13 cell lines), and missense mutation of R248Q was the most commonly observed (5 cell lines). Of note, TP53 mutation was frequently observed among MLLrearranged and t(1;19)-positive ALL cell lines, but was very rare among Philadelphia chromosome-positive and t(17;19)-positive ALL cell lines. Although the number of samples analyzed is limited, this is the first direct observation of the correlation between the incidence of TP53 mutation and types of translocation in B-precursor ALLs, suggesting a functionally different fusion gene product in the p53 pathway for leukemogenesis.
Chromosomal translocation is one of the most important events in leukemogenesis of childhood B-precursor acute lymphoblast leukemia (ALL), and its significance in disease prognosis has been well documented. p53 is critical regulator of cell death, cell cycle progression, and DNA repair, and, recently, its critical involvement in the efficiency of induced pluripotent stem cell generation was documented [1] . Of note, TP53 mutation is widely observed in various tumors including leukemia. An early retrospective study of exons 5-8 of the TP53 gene in 50 B-precursor ALL cases demonstrated that only one case (2%) had mutation [2] , and a large retrospective study of 264 childhood B-precursor ALL cases at diagnosis demonstrated that only five cases (1.9%) had TP53 mutation in exons 5-8, which encode highly conserved regions during evolution [3] . The incidence of TP53 mutation in exons 4-9 among poor-outcome childhood ALL cases at diagnosis was 30% (3/10 cases) [4] . Later studies reported that TP53 mutation might be relatively frequent among ALL with MLL-rearrangement and t(1;19): the inci-dence of TP53 mutation in exons 5-9 in MLL-rearranged ALL at diagnosis was 23% (3/13 cases) [5] , and that in exons 2-11 in t(1;19)-ALL at diagnosis was 10% (2/20 cases) [6] . In contrast, TP53 mutation was rare in Philadelphia chromosome (Ph1)-positive ALL: none of 15 Ph1-positive ALL cases at diagnosis had TP53 mutation in exons 2-11 [7] . Although these reports suggest correlation of TP53 mutation with the type of chromosomal translocation in childhood B-precursor ALL, it has not been directly confirmed in a single cohort study due to the limited incidence of TP53 mutation in primary samples at diagnosis. Leukemia cell lines are usually established from leukemia in progressed disease status, and, thus, generally show higher incidence of gene mutations compared with primary samples [8] . In fact, Kawamura et al. [6] reported that the incidence of TP53 mutation was increased to 80% (4/5 cell lines) among t(1;19)-ALL cell lines, suggesting that the incidence of TP53 mutation might be augmented in cell lines. Accordingly, ALL cell lines would be useful tools to confirm the correlation of TP53 mutation with chromosomal translocations.
To clarify the TP53 gene status in B-precursor ALL cell lines with representative chromosomal translocations, we analyzed 32 B-precursor ALL cell lines including 10 MLL-rearranged ALL [9], 6 t(1;19)-ALL, 7 Ph1-positive ALL [10] , and 4 t(17;19)-ALL cell lines [11] using the yeast functional assay [12] , which is sensitive for detecting TP53 mutation. TP53 mutation was detected in 13 cell lines (40.6%) ( Table I ). The incidence of TP53 mutation in the present study is higher than that in previous reports that used primary ALL samples at diagnosis, in which TP53 mutation was identified in 2% of the cases [2, 3] , suggesting that acquisition of TP53 mutation might provide advantages for disease progression and/or sustained proliferation in vitro.
Among 13 cell lines with TP53 mutation, three cell lines had two types of mutations simultaneously; one cell line (KOPN36) had double mutation on the same allele, while two cell lines (KOPB26 and KOPN63) had two mutations on different alleles (Table I ). The distribution of the 16 mutations identified in 13 cell lines is shown in Fig. 1A and compared with that in previous reports on B-precursor ALL samples [2] [3] [4] [5] [6] [7] and T-ALL samples [18, 19] . Five (38.5%) of the 13 cell lines with TP53 mutation had missense mutation of c.743 G>A resulting in p.R248Q, and 2 cell lines (15.4%) had missense mutation of c.817C>T resulting in p.R273C; both are well known as hotspots. Among the 16 mutations, 11 mutations were located in highly con-served regions. This pattern of distribution was similar to that in B-precursor ALL samples previously reported, in which 5 (18.5%) of the 27 samples with TP53 mutation had R248Q. In contrast, R248Q mutation was reported in only one of 22 samples of T-cell ALL with TP53 mutation.
The incidence of TP53 mutation among MLL-rearranged ALL cell lines and t(1;19)-ALL cell lines was 60% (6/10) and 66.7% (4/6), respectively, while that among Ph1-positive ALL cell lines and t(17;19)-ALL cell lines was 14.3% (1/7) and 0% (0/4), respectively (Fig. 1B) . Of note, consistent with the series of clinical samples in previous reports [5] [6] [7] , the incidence of TP53 mutation among MLL-rearranged and t(1;19)-positive ALL cell lines was significantly higher than that among the other ALL cell lines including Ph1 and t(17;19)-positive ALL cell lines (P < 0.05, chi-squared test). Although the number of cell lines analyzed is limited, this is the first direct observation in a single analysis of the correlation between the incidence of TP53 mutation and types of translocation in B-precursor ALLs. The incidence of TP53 mutation among MLL-rearranged and t(1;19)-positive ALL cell lines observed in this study was higher than that in previous reports using clinical samples, suggesting that TP53 mutation plays a role in disease progression and/or the in vitro proliferative activity of MLL-rearranged and t(1;19)-positive ALL but not necessarily Ph1 and t(17;19)-positive ALL. It should be noted that E2A-HLF chimera derived from t (17;19) has the potential to protect cells from apoptosis induced by activation of the p53 pathway [20] , suggesting that E2A-HLF might overcome at least the proapoptotic activity of p53. Similarly, it was reported that BCR-ABL blocks apoptotic cell death induced by DNA damage without altering p53-dependent G1 arrest [21] . Thus, E2A-HLF and BCR-ABL may have the potential to overcome the pro-apoptotic activity of p53 and, thus, TP53 mutation is not necessary for disease progression. In other words, regardless of the TP53 gene status, Ph1-positive ALL and t(17;19)-ALL could be relatively resistant to therapeutic modalities that activate the p53 pathway such as irradiation. In contrast, without TP53 mutation, MLL-rearranged ALL and t(1;19)-ALL might be relatively sensitive to therapeutic modalities that activate the p53 pathway, and, thus, TP53 mutation could be one of the prognostic factors for therapeutic outcome in these leukemias.
In conclusion, TP53 mutation was observed in as high as 40% of B-precursor ALL cell lines, but was significantly less frequent among Ph1 and t(17;19)-positive ALL cell lines. TP53 mutation might be associated with the functional significance of the fusion gene product derived from translocation in the p53 pathway. (KOPN-30bi, 257bi, 266bi, 272bi, YAMN-73, 283bi, and 291) , 4 t(17;19)-ALL cell lines [11] (UOC-B1, HAL01 [14] , YCUB-2 [15] , Endo-kun), and 5 other ALL cell lines including 1 with t(12;21) (Reh) [16] and 4 with others (Nalm6, KOPN-32 [13] , 235 [13] , and 261 [17] ). Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA), reverse transcription was performed using random hexamer (Amersham Bioscience, Buckinghamshire, United Kingdom) by Superscript II reverse transcriptase (Invitrogen), and then incubated with RNase (Invitrogen). The state of the TP53 gene was tested by the yeast functional assay [12] as described previously [22, 23] . In brief, RT-PCR amplified TP53 cDNAs were cotransfected with a linealized gap vector carrying the 5 0 and 3 0 ends of the gene into the yIG397 strain containing an integrated plasmid with the ADE2 open reading frame under the control of TP53-responsive promoter. Yeast containing the mutant TP53 cDNA fails to transcribe ADE mRNA and formed red colonies, whereas yeast expressing the wild-type TP53 forms white colonies. Thus, the status of the TP53 gene is quantitatively analyzed by the color of colonies. When functional assay demonstrated the TP53 mutation, sequences of We describe here the clinical manifestations of 37 patients with combined coagulation factor (F) V and FVIII deficiency, which is the commonest multiple coagulation factor deficiency state. Only a few cases are reported in the literature from India. Prolonged bleed post injury/ surgery (62%) is the commonest manifestation in our series, and epistaxis (19%) is rare in our patients in comparison to other series described in the literature. Although the frequency of bleeding manifestations differs among various reports, there is no evidence for increased bleeding manifestation due to combined deficiency of two coagulation factors as against a single coagulation factor. To the best of our knowledge, this is the largest series of this disorder described so far. Combined Factor (F) V and VIII is the commonest combined hereditary coagulation factor deficiency with a prevalence of 1:1,000,000 [1] . It is an autosomal recessive disorder of blood coagulation [2] and the frequency of such autosomal recessive disorders increases 8-10-folds in populations where consanguinity is common. It is caused by a defect in lectin mannose binding protein 1 (LMAN 1) gene or in multiple coagulation factor deficiency (MCFD) gene 2 [3] [4] [5] . It was initially described by Oeri et al. [6] in 1954 and so far about 150 patients have been described from around the world [7] [8] [9] [10] [11] . The largest of these studies so far, describe the clinical manifestations of this disorder in 27 patients of Iranian origin [7] . Patients who are homozygous for this combined factor deficiency state maintain a factor level between 4% and 30% [12, 13] . We present herewith the clinical and phenotypic data of combined FV and FVIII deficiency in a large series of 37 patients of Indian origin seen at our center.
All patients with combined FV and FVIII deficiency seen at our center over a period of 20 years, between 1988 and 2008 were included in this analysis. History of bleeding was considered significant if i. After dental extraction bleeding persisted for more than 48 hr or required repeat surgical intervention.
ii. Menstrual period lasted 6 days or more with history of clots and required hormonal therapy for control.
iii. Epistaxis was spontaneous and persisted even after local measures to control. iv. Gastrointestinal bleed, presented as hematemesis or malaena.
v. Post-traumatic bleed if the bleeding was significant with minor trauma.
vi. Any other history of bleeding that required blood product support. For hematoma, the nature of the injury and for hemarthrosis, the radiological changes in the joint were documented.
FVIII coagulant (FVIII: C) activity was measured on citrated plasma samples from these patients by one-stage APTT method while FV coagulant activity was measured by a PT based method. From 1998 till 2001, the factor assay was done by Coag-a-mate; RA4 (Organon Teknika), from 2001 till 2006 by CA-1500 Sysmex coagulometer (Sysmex, Hyogo, Japan) and from 2006 onwards by ACL Advance (Beckman Coulter). For the diagnosis of combined FV and VIII deficiency, the cut-off values for FV (<40%) and FVIII: C (<45%) was considered as previously described [7] .
There were a total of 37 cases of combined FV and FVIII deficiency diagnosed during this time. There were 21 (57%) female and 16 (43%) male patients, with a mean age of 17 years (range: 1-49). Twenty-eight (76%) out of 37 cases were born of consanguineous marriages. A family history of bleeding was present in 14 (38%) cases. Nineteen patients (51%) were symptomatic prior to 5 years of age. The mean level of FV was 12.5% (range: 5-31), and the mean level of FVIII was 8.8% (range: 1227). In 11 out of 37 (30%) patients both FV and FVIII levels were 10% or lower. Seven out of 37 (19%) patients had both factor levels more than 10%.
Nineteen patients (51%) had one factor level above and the other below the 10% limit (five patients had FV level less than 10% and fourteen patients had FVIII level less than 10%). Median levels of FV and FVIII around 10% was typical of the homozygous state for the combined deficiency of FV and FVIII [11] [12] [13] .
Prolonged bleeding post injury/surgery was the commonest bleeding manifestation seen in 23 (62%) cases. The other significant symptoms were bleeding from tooth socket in 21 (57%) cases, gum bleed in 18 (49%), easy bruisability in 11 (30%), epistaxis in seven (19%), hemarthrosis in five (13%), gastrointestinal bleed in one (3%) case. No patient had presented with intracranial bleed. Menorrhagia was seen in 6/9 (66%) females who had attended menarche.
Our data shows that there are differences in the clinical manifestations of patients in India compared to those described in the only other large series reported so far in the literature from Iran. The clinical manifestations in our patients have been compared with that of other large series in Table I . Epistaxis (77% and 48%) was the most common symptom among patients from Iran [7, 9] . However, this occurred much less frequently (epistaxis, 19%) in our patients. Epistaxis was also significantly less in our patients when compared to that from Israel [11] ; (57% vs. 19%). Hemarthrosis was observed in 13% of our patients though this has not been reported from the other small series reported from India [10] . Though hemarthrosis has been reported from the series published from Iran [7, 9] , it has not been reported from that reported from Israel [11] . Bleeding following circumcision was a major feature in patient from Iran [7, 9] . But since this is not a common practice in this part of the world, only one of our patients had presented with this symptom. Gum bleeding, which occurred in almost half of our patients was not documented in the Iranian series but was shown to be present in a smaller group of patients from India [10] and from Israel [11] . Prolonged bleeding post trauma/surgical procedure was the most frequent (62%) symptom in our patients. Our data shows that the pattern of bleeding among patients with combined FV and FVIII deficiency in India is different from that reported from Iran and Israel, even though the mean factor levels are similar. At present, we are not able to explain the difference in clinical behavior in our patients but the change in the life style may be a factor because similar observation has also been documented from other small series India [10] .
When we compared between three groups of patients (1st group: both FV and FVIII <10%; 2nd group: both FV and FVIII levels >10%; 3rd group: either FV or FVIII <10%) there was no difference between different bleeding symptoms. The same has also been reported from both the series from Iran [7, 9] . When we compared between two groups of patients (1st group: both FV and FVIII <5%; 2nd group: both FV and FVIII >5%), there were no differences in the bleeding symptoms observed. This shows that presence of two defects do not make the severity of bleeding greater than that expected in patients with single coagulation defects of similar degrees.
We have reported here the clinical manifestations in 37 patients with combined FV and FVIII deficiency from India. This is the largest series in the world describing the clinical manifestations of patients with combined factor V and VIII deficiency reported so far. It is evident from this as well as previous studies [7, [9] [10] [11] that the bleeding symptoms in combined FV and FVIII deficiency is almost similar to the deficiency of single coagulation factor. There is no clear evidence to support increased bleeding manifestations due to the combined deficiency of two coagulation factors.
In combined FV and FVIII deficiency, our knowledge about the clinical manifestation so far is based on just one or two large series published in the literature. There is a need for careful documentation of clinical data from different population to see what the profile is for large number of patients and whether there are any ethnic differences. Further molecular studies to identify the causative mutations in patients with this disease will shed light on the basis for the clinical diversity seen among the patients. We studied CD1d-restricted invariant natural killer T (iNKT) cells in classical Hodgkin lymphoma (cHL). Tumor cells stained positive for CD1d in 21/44 cHL cases, whereas in non-Hodgkin lymphoma (NHL), only 9/31 stained positive. In contrast, CD1c expression was more common in NHL. The percentage of iNKT cells in cHL cell suspensions was similar to the percentage in reactive lymph nodes and was not related to the CD1d expression status of the tumor cells. In conclusion, we found expression of CD1d in HRS cells in half of the cHL cases, and also observed a substantial population of iNKT cells in cHL cell suspensions. Classical Hodgkin lymphoma (cHL) is a B-cell neoplasm characterized by a minority of neoplastic cells, the Hodgkin and Reed-Sternberg (HRS) cells, which are located within an extensive infiltrate of reactive cells, such as T cells, B cells, plasma cells, stromal cells, eosinophils, and macrophages. The HRS cells shape their microenvironment by attracting specific favorable T cell subsets, like regulatory T (Treg) and T helper 2 (TH-2) cells and producing factors such as CC-chemokine ligand 17 (CCL17 or TARC), transforming growth factor (TGF)-b and interleukin (IL)-10 [1] . Additionally, HLA class I is downregulated in HRS cells in 65% of the cHL cases and HLA class II in 40%, possibly as a strategy to escape from an effective immune surveillance [2] . All together, these factors may suppress the development of an effective antitumor response and provide a microenvironment that is favorable for the HRS cells.
Recent studies have suggested that T cells restricted to nonclassical MHC class I-like CD1 molecules may be involved in immune surveillance of hematological malignancies [3] [4] [5] [6] . CD1 molecules are nonclassical MHC class I-like molecules that present lipid antigens to T cells, triggering a specific immune response. Of the five CD1 isoforms (CD1a, CD1b, CD1c, CD1d, and CD1e) expressed in human tissue, only CD1c and CD1d are expressed in B cells [7] . The T cell receptors (TCRs) of T cells that recognize CD1c are indistinguishable from those that recognize MHC class I or II complexes. Most CD1c-restricted T cells appear to be TH-1-like cells that provide adaptive immunity to microbial-lipid antigens [8] . In contrast, CD1d presents lipid antigens to natural killer T (NKT) cells. The best characterized CD1d-restricted NKT cells in humans are the invariant NKT (iNKT) cells, also known as type I NKT cells. These cells are characterized by the expression of an invariant Va24Ja18 chain paired to a semi-invariant Vb11 chain [9] . Two distinct functional subsets of human iNKT cells are recognized in terms of cytokines production and cytotoxic activation. CD42 iNKT cells are more likely to be cytotoxic and produce TH-1 type cytokines, whereas CD41 iNKT cells are more likely to produce both TH-1 and TH-2 type cytokines after stimulation [10] . Several studies have shown the impor-tance of iNKT cells in B cell malignancies [4] [5] [6] . In chronic lymphocytic leukemia (CLL), CD1d1 tumor cells loaded with aGalCer can activate iNKT cells and in turn induce cell death of tumor cells [4] . Malignant multiple myeloma is characterized by a reversible functional defect in iNKT cells in comparison with nonprogressive myeloma and premalignant myeloma [5] . Furthermore, iNKT cells were shown to induce effective antilymphoma responses and were essential for the survival of mice in murine lymphoma models [6] . The role of iNKT cells in the immune surveillance in cHL is unknown. Here, we examined the expression of CD1c and CD1d in HRS cells and tumor infiltrating cells in 44 cHL cases, as well as in four cHL cell lines. Furthermore, we studied the presence of iNKT cells in cell suspensions of 10 cHL cases.
CD1c was undetectable in cHL cell lines L428, KMH2, L1236, and U-HO1 (data not shown). In tonsil tissue, CD1c stained positive in the mantle zone (MZ) and predominantly negative in the germinal centers (GC) of secondary lymphoid follicles (Fig. 1A) consistent with the previously reported expression pattern [11] . HRS cells in cHL were consistently CD1c negative in all cases (Fig. 1B) , whereas 14 out of 39 non-Hodgkin lymphomas (NHL) were positive (Table I) . The absence of CD1c expression in both GC B cells and HRS cells is consistent with the current assumption that HRS cells are derived from GC B cells [12] . CD1c expression has been reported in some other B-cell NHL subtypes, including GC B cell derived follicular lymphoma [11] . In B-cell NHL, an inverse correlation was noticed between CD1c and the proliferative activity assessed by expression of Ki-67 [11] . This is in line with the lack of CD1c staining in GC B cells that show a high proliferation index and high CD1c expression in the MZ B cells that are in a resting state. Ki-67 was shown to be highly expressed in HRS cells [13] correlating with the lack of CD1c expression. CD1c expression was found in reactive cells in all cases with a percentage varying from a few to the vast majority, similar to the CD1c expression pattern observed in interfollicular regions of tonsil tissue.
In contrast, CD1d was detected in all four cHL cell lines, varying from weakly positive in U-HO1 to strongly positive in the other three cHL cell lines ( Fig. 1C-F) . By flowcytometry, we validated that CD1d was present on the membrane in cHL cell lines (data not shown). In normal tonsil tissue, CD1d staining was observed mainly in the MZ and showed no positive staining in the GC (Fig. 1G ) consistent with the reported results [14] . CD1d was detected in HRS cells in 21 out of 44 cHL cases (48%) (Fig. 1H , Table I,  Supporting Information Table I) , showing both cytoplasmic and membranous staining. No correlation was found with EBV status and CD1d expression (Table I) . CD1d was also positive in the reactive background, usually in more than half of the reactive cells. Nine of the 39 NHL cases were positive for CD1d (23% , Table I ), i.e., in three out of 19 GC B cell derived NHL and in six out of 20 non-GC B cell NHL (Table I, Supporting Information Table I ). The CD1d expression in HL is remarkable since GC B cells and the majority of the GC B cell derived NHL are negative. Positivity of HRS cells indicates that these cells might have acquired CD1d expression during malignant transformation. CD1d expression has been detected in some B cell malignancies, such as CLL [4] and multiple myeloma [5] . In these B cell malignancies, iNKT cells activated by a-GalCer loaded CD1d1 tumor cells resulted in the induction of apoptosis of the tumor cells in vitro [4, 5] .
Since half of the cHL cases were positive for CD1d in HRS cells, we further investigated the iNKT cell population in cell suspensions of cHL and reactive lymph node (RLN). The mean percentage of iNKT cells was 4% (range 0.8-8%) in cHL and 4% (range 0.4-7%) in RLN (Table II) . iNKT cells were present at similar percentage in either CD1d positive or CD1d negative cHL cases. Two distinct iNKT subsets have been distinguished as Th1 type (CD4 2 iNKT) and Th2 type (CD4 1 iNKT) in terms of cytokine production and cytotoxic activation [10] . We observed a similar percentage of CD41 iNKT cells, i.e., around 50%, in cHL and RLN (Table II) . Given the high expression of CCL17 in HRS cells of cHL [15] and the high expression of the CCL17 receptor, CCR4, on CD41 iNKT cells [16] , it might be speculated that especially the CD41 iNKT cells are present in the close vicinity of HRS cells and might favor the survival and growth of HRS cells. Since it is known that the rosetting CD41CD262 T cells in HL have several features of anergy [17] , the CD1d-restricted iNKT cells might also be dysfunctional or anergic favoring HRS cells survival by escaping from an effective immunosurveillance in cHL. Taken together, we showed expression of CD1d in HRS cells of cHL cell lines and a significant proportion of cHL cases as well as the presence of a marked population of iNKT cells in the reactive background of cHL. The CD1d-iNKT cell axis might play a role in the disturbed immunoregulation in cHL.
Patient samples and cell lines. Frozen cHL samples consisted of 39 cases of nodular sclerosis (NS), four cases of mixed cellularity (MC), and one case of not otherwise specified cHL (NOS). Frozen samples of 39 NHL cases were used and consisted of 13 chronic lymphocytic leukemia, seven mantle cell lymphoma, nine follicular lymphoma, and 10 diffuse large B cell lymphoma cases. Cell suspensions were available of eight NS and two MC cHL cases. The cHL cell lines L428, KMH2, L1236, and U-HO1 [18] were cultured in RPMI-1640 medium (Lonza Walkersville, Walkersville, MD) supplemented with ultraglutamine-1, 100 U/ml penicillin/streptomycin, 10% fetal calf serum (5% for L428) (Lonza Walkersville).
Immunohistochemistry. Immunohistochemistry was performed with monoclonal antibodies against CD1c (L161, Abd Serotec, Oxford, UK) and CD1d (NOR3.2, Abcam, Cambridge, UK) (both 1:100) on frozen cHL tissue sections and cytospins using standard laboratory protocols and appropriate positive and negative controls. Cases were defined positive when more than 50% of tumor cells showed a clear staining.
Flow cytometry. Cells (1 3 10 6 ) were stained simultaneously with PE labeled Mouse Anti-Human iNKT Cell (clone 6B11, BD Bioscience, San Jose, CA), FITC labeled anti-TCR Vb11 (clone C21, Beckman Coulter, Fullerton, CA) and CyQ labeled anti-CD4 (clone Edu-2, IQ Products, Groningen, Netherlands). A total of 100,000 lymphocytes were collected. The percentage of iNKT cells was determined by positive staining for both 6B11 and Vb11, and the percentage of CD41 iNKT cells was determined by the percentage of CD41 cells among the gated iNKT cells by flowcytometer (Calibur, Becton Dickinson, San Jose, CA). We used an iNKT cell clone as a positive control, and samples without primary antibody were included as a negative control. cHL cell lines were stained with PE labeled CD1d (clone 51.1, eBioscience, Hatfield, UK), and a IgG2b isotype control antibody, to show the presence of membrane CD1d.
The genetic profile of chronic lymphocytic leukemia (CLL) is characterized by a pool of recurrent lesions, including trisomy 12, deletions of 13q14, 11q22-q23, 17p13, and 6q21, and t(14)(q32) translocations [1] . The aforementioned lesions can be detected by FISH and may harbor prognostic information [1] . Array-based comparative genomic hybridization (aCGH) has identified gain of multiple regions on the short arm of chromosome 2 as a recurrent CLL lesion, that contain the REL, BCL11A, and MYCN proto-oncogenes that are involved in hematologic malignancies [2] [3] [4] [5] . The exact prevalence of REL, BCL11A, and MYCN gains at the time of CLL diagnosis is currently unknown in consecutive series of the disease. Moreover, some studies indicate that 2p gains as a whole may herald progressive and/or poor risk disease, yet the prognostic contribution of the individual genes included in the amplicon, i.e., REL, BCL11A, and MYCN, remains undefined [4] [5] [6] . The aim of this study was twofold: (i) characterize the prevalence of REL, BCL11A, and MYCN gains in a consecutive CLL series at the time of diagnosis; (ii) define the prognostic relevance of REL, BCL11A, and MYCN gains in CLL.
The study is based on a consecutive series of 176 newly diagnosed and phenotypically typical CLL (median Matutes score: 5; range 4-5) [7, 8] , representative of all disease stages. The biological and clinical characteristics of the CLL series are reported in Table I . By FISH, gain of REL was observed in 6/176 (3.4%) cases, gain of BCL11A in 5/176 (2.8%), and gain of MYCN in 4/176 (2.3%). The median percentage of nuclei harboring gains was 46.8% (range: 18.0-85.0%) for REL, 66% (range: 14.0-77.4%) for BCL11A, and 78.2% (range: 45.0-90.0%) for MYCN. The number of additional copies was 1 for REL, 1 for BCL11A, and 1 for MYCN. REL, BCL11A, and MYCN were concomitantly gained in the same clone in 4/176 (2.3%) cases. REL and BCL11A, but not MYCN, were concomitantly gained in the same clone in 1/176 (0.5%) cases. In 1/176 (0.5%) cases, the sole REL gene was gained.
Analysis of survival in patients with 2p gains revealed that all but one patient with 2p gains including MYCN have died for CLL progression or transformation to Richter syndrome (Table II ). In contrast, the two patients harboring 2p gains not including MYCN were alive and treatment free after a follow-up of 88 and 105 months, respectively (Table II) .
On the basis of these observations, we analyzed the impact of MYCN gain on CLL presentation and outcome. By comparing biological and clinical features at diagnosis, no differences were observed between CLL with gain of MYCN compared with those devoid of such genetic lesion, with the possible exception of a higher absolute lymphocyte count in cases carrying MYCN gain (P 5 0.029; Table I ).
The impact of MYCN gain on CLL prognosis was estimated by utilizing OS as a clinical endpoint. In the whole CLL cohort, after a median follow-up of 72.1 months for alive patients, 39/176 patients had died, accounting for a 5-years OS of 84.1%. By univariate analysis, CLL harboring MYCN gain at diagnosis were at increased risk of death (Events/N: 3/4; HR: 5.35; 95% CI 1.62-17.68; P 5 0.006; 6-years OS: 25.0%; 95% CI 0-67.5%) compared with CLL without MYCN gain (Events/N: 36/172; 6-year OS: 80.8%; 95% CI 74.0-87.6%) (Table III and Fig. 1) . Other features at diagnosis associated with CLL survival by univariate analysis were those expected in a consecutive CLL series and included IGHV homology 98% (P 5 0.005), 112 (P 5 0.049), 11q22-q23 deletion (P 5 0.014), 17p13 deletion (P < 0.001), TP53 mutations (P 5 0.048), CD38 expression (P 5 0.006), age >70 years (P < 0.001), Binet stage B-C (P < 0.001), and beta-2-microglobulin > 2.5 mg/l (P 5 0.001) (Table III) .
By multivariate analysis, gain of MYCN was selected as an independent predictor of OS in this CLL cohort (HR: 4.79; 95% CI 1.31-17.39; P 5 0.017), along with 17p13 deletion (HR: 2.28; 95% CI 1.01-5.15; P 5 0.045), age > 70 years (HR 3.89; 95% CI: 1.88-8.05; P < 0.001), and Binet stage B-C (HR: 3.31; 95% CI 1.55-7.06; P 5 0.002) (Table III) .
This study suggests that: (i) gains of REL, BCL11A, and MYCN occur at low frequency at CLL diagnosis; and (ii) 2p gains including MYCN might have potential prognostic relevance in CLL.
Gains of REL, BCL11A, or MYCN have been reported by Chapiro et al. [5] to occur in 10% CLL, a prevalence that is higher than the 2-3% prevalence observed in our cohort. The discrepancy between our study and the study by Chapiro et al. [5] may be explained by the differences that distinguish the two CLL cohorts. Indeed, Chapiro et al. [5] analyzed REL, BCL11A, or MYCN in 86 CLL, all Binet stage B-C, at the time of treatment requirement. On the contrary, our study was based on a consecutive series of 176 CLL, that included all Binet stages and that in all cases have been analyzed at the time of diagnosis.
Some studies suggest that, in CLL, 2p gains as a whole may associate with poor risk features, such as advanced stage, unmutated IGHV genes, and 17p13 deletion [4] [5] [6] . Our results are consistent with these observations and suggest that, among individual proto-oncogenes included in the 2p amplicon, poor prognosis may be heralded by gain of MYCN. The relevance of MYCN gain in determining CLL prognosis is also suggested by the observation that, in our cohort, the negative prognostic impact associated with gain of MYCN appears to be independent of stage at presentation, IGHV mutation status, or presence of 17p13 deletion. Future studies are required to validate these results in larger patient populations. 
All patients
Normal P
Patients. The study was based on a consecutive series of 176 previously untreated CLL who presented for initial evaluation at the Division of Hematology of the Amedeo Avogadro University of Eastern Piedmont from June 1996 through June 2008. Patients provided informed consent in accordance with local IRB requirements and Declaration of Helsinki. CLL diagnosis was based on NCI criteria and confirmed by a flow cytometry score >3 [7, 8] .
The following biological variables were analyzed on peripheral blood mononuclear cells (PBMNC) collected at diagnosis: (i) IGHV gene homology to germline; (ii) FISH karyotype; (iii) TP53 mutations; (iv) CD38, ZAP70, and CD49d expression. Patients were managed according to NCI guidelines [7] .
Interphase FISH. Probes used for FISH analysis were as follows: (i) LSI13 and LSID13S319 for detection of 13q14 deletion; CEP12 for detection of aneuploidy of chromosome 12; LSIp53 for detection of 17p13 dele-  Prediction of Peptide Reactivity with Human IVIg through a Knowledge-Based Approach The prediction of antibody-protein (antigen) interactions is very difficult due to the huge variability that characterizes the structure of the antibodies. The region of the antigen bound to the antibodies is called epitope. Experimental data indicate that many antibodies react with a panel of distinct epitopes (positive reaction). The Challenge 1 of DREAM5 aims at understanding whether there exists rules for predicting the reactivity of a peptide/epitope, i.e., its capability to bind to human antibodies. DREAM 5 provided a training set of peptides with experimentally identified high and low reactivities to human antibodies. On the basis of this training set, the participants to the challenge were asked to develop a predictive model of reactivity. A test set was then provided to evaluate the performance of the model implemented so far. We developed a logistic regression model to predict the peptide reactivity, by facing the challenge as a machine learning problem. The initial features have been generated on the basis of the available knowledge and the information reported in the dataset. Our predictive model had the second best performance of the challenge. We also developed a method, based on a clustering approach, able to “in-silico” generate a list of positive and negative new peptide sequences, as requested by the DREAM5 “bonus round” additional challenge. The paper describes the developed model and its results in terms of reactivity prediction, and highlights some open issues concerning the propensity of a peptide to react with human antibodies. Given their key role in the immune response, antibody-protein interactions play a major role in a variety of clinical domains (infectious diseases, autoimmune diseases, oncology, vaccination and therapeutic interventions). For this reason, the prediction of antibody-protein interactions can be of critical importance [1] [2] . The antibodies have a wide range of heterogeneous structures generated by genomic recombination: the number of human antibodies is estimated to be around 10 10 and 10 12 [3] . The antibodies interact with proteins (called antigens) through their binding sites (called paratopes).
The region of the antigen bound with the paratope is called epitope. Two types of epitopes are typically distinguished in protein-antibody interaction studies: conformational and linear epitopes. A linear/sequential epitope is recognized by its linear sequence of amino acids (primary structure). In contrast, most antibodies recognize conformational epitopes with a specific threedimensional structure.
All potential linear epitopes of a protein are short peptides that can be synthesized and arrayed on solid supports, e.g. glass slides [4] . By incubating these peptide arrays with antibody mixtures, such as human serum or plasma, it is possible to determine specific interactions between antibodies and peptides.
The binding site of a linear epitope has a typical length ranging between 8 and 10 amino acids. An antibody binds to its epitope/ peptide independently of the physical position of the binding site within the peptide. Every amino acid has a different impact on the epitope reactivity; this is not only due to its physicochemical properties but also to its interaction with the neighboring residues within the whole peptide sequence.
It has been often assumed that a specific antibody selectively binds to a specific sequence. However, experimental data indicate that many antibodies bind to a panel of related (or even distinct) peptides with different affinities. The open question is whether there exist rules that enable the prediction of common peptide/ epitope sequences, which can be recognized by human antibodies.
In order to address this problem, the DREAM (Dialogue for Reverse Engineering Assessments and Methods) Consortium issued the Epitope-Antibody Recognition (EAR) Specificity Prediction Challenge (Challenge 1). In the experimental work leading to this challenge, 75534 peptides were incubated with commercially available intravenous immunoglobulin (IVIg) fractions. IVIg is a mixture of naturally occurring human antibodies isolated from up to 100000 healthy individuals. From this dataset, high-confidence negative and positive pools of peptides were determined. Training and test datasets were assembled from these peptide pools. The epitope-antibody recognition challenge consists of determining whether each peptide in the test set belongs to the positive or negative set starting from the data of the training set.
A so-called ''bonus round'' was proposed beside this main challenge. It consists of generating ''in-silico'' a list of positive and negative new peptide sequences, which should significantly differ from the ones contained in the training set. The lists provided by the best performing teams will be subsequently experimentally evaluated.
In the literature, epitope prediction has been focused primarily on sequence-dependent methods based on various amino acid properties, such as hydrophilicity, solvent accessibility, secondary structure and others [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . Several methods based on machine learning approaches have been applied, too [17] . They comprise hidden Markov models (HMM), artificial neural networks (ANN) and support vector machines (SVM) [18] [19] [20] [21] [22] . Machine learning methods have been frequently coupled with the so-called scale-based approach; this approach exploits one or more scales of amino acid properties to weight each residues of the sequence of interest. In particular it has been shown that the combination of different scales with several machine learning algorithms have better performances than single scale methods [23] .
We coped with the DREAM challenge by resorting to a classical supervised machine learning strategy with knowledge-based feature construction. After the definition of the problem features, we developed a logistic regression classifier that showed a very good performance on the test set.
Moreover, we developed a new method for dealing with the bonus round challenge and we generated a list of de-novo peptides that will be further experimentally assessed.
As mentioned in the introduction, one of the DREAM 5 challenges dealt with the prediction of the reactivity of peptides to bind intravenous immunoglobulin (IVIg) antibodies. The challenge organizers made available a dataset that comprises sequences of peptides, which either bind IVIg antibodies with high affinity/avidity or not.
In particular 75534 peptides were incubated with commercially available human IVIg fractions. A set of 6841 peptides with high affinity was identified (positive set). From the same original set, 20437 peptides were identified showing no antibody binding activity in any of the triplicate assays (negative set). Each of these peptides is unique in terms of its amino acid sequence.
Most of these sequences are 15 amino acids long; however, there are also sequences with different lengths (several of them were 13 amino acids long, while a few were long 9, 16, 18, 20 and 21 amino acids).
A reactivity value was calculated for each peptide. The reactivity values range from 1 to 65536. The reactivity of the positive peptides ranges between 10000 and 65536, while this value ranges from 1 to 1000 in the negative peptides case. The training and test datasets were assembled from these two peptide sets.
Training set. The training set contained 13638 peptides and was created by selecting 3420 peptides from the positive set and 10218 peptides from the negative set. Two features of each peptide were provided: the amino acid sequence and a measure of the peptide reactivity to the IVIg antibodies. The predictive model of the peptide reactivity was trained on this dataset.
Test set. The test set contained 13640 peptides and was formed by grouping the remaining 3421 positive peptides and the remaining 10219 negative peptides. Only the sequence of these peptides was provided for the initial phase of the challenge, while their class (positive or negative) was made available to us only when the results of the challenge had been published.
The main challenge consists of determining whether peptide reactivity with antibodies is strong or weak, i.e., whether a peptide of the test set belongs to the positive or negative set. The goal is therefore to exploit the training set to develop a predictive model, taking into account the available information (e.g., the information on amino acids and protein-protein interactions available in biological databases). Participants are required to submit a ranked list of the peptides in the test set, ordered according to the predicted probability that the peptide belongs to the positive set (predicted reactivity).
We have dealt with this challenge by applying a proper supervised learning pipeline. The approach consisted in feature selection, classification and cross-validation on the training set and finally evaluation of the model on the test set. These steps followed a crucial phase of knowledge-based construction of the initial set of features.
In the following sub-sections, we will describe, step-by-step, the procedure applied to develop and test the proposed predictive model.
Feature construction. The construction of a proper set of features is the most important step of the development of a successful predictive model.
In particular, we considered two sets of features for every peptide: the first set is computed from the peptide sequence, while the second set is generated taking into account the entire training set.
The values of all the features have been normalized between 0 and 1.
In order to generate the first set of features, we exploited information about the peptides and the epitopes reactivity.
In more detail, we used the following peptide attributes:
1. The sequence length, i.e. the number of residues of the peptide. 2. The isoelectric point, computed by using the iterative method described by Tiengo et al. [24] . 3. The amino acid frequencies (24 features), calculated as the occurrence of each amino acid along the peptide; the four ambiguous amino acid B (asparagine or aspartic acid), X (unspecified or unknown amino acid), Z (glutamine or glutamic acid) and J (leucine or isoleucine) have also been considered.
As mentioned in the introduction, several approaches have been used for epitope prediction; the so-called scale-based approach exploited one or more scales of amino acid properties to weight each residues of the sequence of interest [2, 18, [25] [26] [27] [28] . The use of multiple scales was essential to predict epitope location reliably, as reported by Blythe et al. [29] . Therefore, we considered some of the most promising amino acid properties reported in these studies, by resorting to a set of widely used scales (i.e. the five scales reported in Table 1 ) [9-13]:
1. The antigenicity was calculated as proposed by Kolaskar et al. [9] . The frequency of the residue in antigenic determinants (experimentally identified) was exploited to calculate the antigenic propensity of each amino acid. 2. The accessibility was calculated on the basis of the scale proposed by Janin et al. [10] . The importance of the accessibility information is widely reported in the literature; the hypothesis is that an accessible site is likely to be recognized by the antibodies [25, [30] [31] [32] . 3. The hydrophilicity was computed following the scale proposed by Parker et al. [11] . This scale was recently found to have slightly better results than the other ones [2, 33] . The hypothesis for hydrophilicity is that the antigenic sites are on the surface, so they are probably hydrophilic [5, 11] . 4. The flexibility was calculated with the scale proposed by Bhaskaran et al. [12] . A high flexibility of the structure is hypothesized to favor the propensity of a peptide to bind the antibodies [34] [35] . 5. The beta-turn prediction was calculated by exploiting an amino acid scale of propensities following the Chou-Fasman method [2, 8, 13 ].
The five attributes described above were computed on the basis of the correspondent amino acid scale, computing the maximum value within a sliding window of 9 residues. The size of the sliding window was chosen because it is known that the binding site covered by an antibody typically includes a stretch of 8 to 10 amino acids [36] [37] .
The second set of features has been generated taking into account the entire training set. To obtain such features, every peptide was aligned with all the others by both the Needleman-Wunsch algorithm (global alignment) and the Smith-Waterman algorithm (local alignment) [38] [39] . In this way, a scoring matrix [13638613638] has been computed. In this way, we have generated a set of additional features, as follows:
1. Global alignment. For every peptide we computed: the maximum score obtained by the global alignment with every negative peptides (MaxScore0_nw); the maximum score obtained by the alignment against the positive set (MaxScor-e1_nw); the difference between MaxScore1_nw and MaxScor-e0_nw (DiffMaxScore_nw).
2. Local alignment. For every peptide we considered the maximum score of the local alignment with the elements of the positive set and with the elements of the negative set (MaxScore0_sw, MaxScore1_sw), and the difference between these maximum values, as well (DiffMaxScore_sw).
The rationale for selecting the features mentioned above is related to the so-called classification for homology (sequence similarity), which consists of classifying a sequence (in terms of structure and function) looking at the most similar sequence in a dataset of available sequences [40] [41] . The principle is that similar sequences have similar structures and, thus, similar functions (in this case similar reactivities to antibodies) [42] .
In our case, for example, a peptide has a high value of MaxScore0_nw, if the negative examples contain at least another very similar peptide. Moreover, the MaxScore feature is used to check the importance of the absolute value of a good alignment, while the DiffMaxScore attribute takes into account the difference between class groups.
It is important to notice that the use of the information about the class (i.e. positive or negative example) during the feature generation phase requires to properly designing the crossvalidation phase in order to avoid overfitting.
Finally, the two types of alignments have been used to understand whether the reactivity depends on the entire sequence of the peptide (global alignment) or on a small portion (local alignment), as hypothesized.
Feature selection. Because the training set was made of 13638 examples and the generated features were 37, a features selection step was not mandatory. However, we decided to filter the features to obtain a more parsimonious model. We resorted to a filtering strategy because the use of wrapper methods would have made the cross-validation approaches (and in particular the leaveone-out strategy) computationally very demanding. We have applied three different procedures for feature selection, thus obtaining three different subsets of features. N Subset C. Feature selection with the LASSO method (least absolute shrinkage and selection operator) [45] .
Cross-validation of the classifiers. As mentioned above, the final aim of this challenge is to discover whether there exist rules that enable to predict that a peptide/epitope sequence is recognized by human antibodies. For this reason, we mainly considered classifiers that provide a predictive model easy to be interpreted.
N Linear regression. Even if linear regression is a simplistic model due to its strong assumptions, it gives the possibility to evaluate the contribution of each single variable to classification. The outcome variable we considered is the reactivity value, which ranges from 1 to 65536. The distribution of these values shows that the outcome can be easily binarized: in fact, as previously mentioned, the reactivity of the positive peptides ranges between 10000 and 65536, while this value ranges from 1 to 1000 in the negative peptide case. For this reason, we also tested this classifier by considering the binary classes 0-negative and 1-positive as continuous values.
N Logistic regression. Also this approach allows assessing the contribution of each variable to classification: in fact, the Columns 2-6 report five of the most promising amino acid properties for predicting the peptide reactivity: antigenicity [9] , accessibility [10] , hydrophilicity [11] , flexibility [12] and predicted beta-turn propensity [13] . doi:10.1371/journal.pone.0023616.t001 estimated regression coefficients provide an easy way to evaluate the reliability of the model. Moreover there are no assumptions about the probability distribution of the attributes. However, in the model that we have exploited we supposed that they were not strongly correlated.
N Naïve Bayes. It is a simple probabilistic classifier based on the Bayes' theorem under the attribute independence assumption, given the class [46] . The model allows an easy interpretation of the results, since each variable can be separately considered.
The main limits of this approach are the strong assumptions of conditional independence between variables and the need of choosing prior distributions.
N Decision tree. This method has the great ability to learn complex and non-linear relationships between variables and outcome. Decision trees, however, require the implementation of careful strategies in order to avoid overfitting. In particular, we used the J48 algorithm, an open source Java implementation of the C4.5 method [47] ; the dimension of the tree was limited by fixing the minimum number of instances for each leaf equals to 1% of the training set.
N Rules learner. This method permits, like decision trees, to extract complex rules; however the accuracy of the predictions is high only if the rules have a sufficiently large support. Moreover, it can be computationally demanding in case of large datasets. In this work we applied the PART method to generate a decision list. Such method is based on an iterative strategy. In each step, PART builds a partial decision tree and converts the best leaf into a rule [48] . The minimum number of instances for each leaf was fixed at 1% of the examples in order to limit the number of generated rules.
To evaluate the best classifier, the performances have been assessed applying the so-called ''leave-one-out'' cross-validation approach. This approach is particularly suited in our case, since, together with maximizing the size of the training set, it allows to properly generating the features related to the alignment scores.
Choice of final model and its interpretation. The model was assessed not only in terms of its predictive performance but also taking into account its interpretation, i.e. by considering the contribution of the different features included in the prediction.
Together with standard performance measures, such as accuracy, sensitivity and specificity, we also computed the Fmeasure of the predictive model. The F-measure is the harmonic mean of precision (positive predictive value) and recall/sensitivity. As a matter of fact, in order to develop a model that is useful to generate new reactive peptides, it is important to maximize both precision and sensitivity: it means to have a high probability that the peptide predicted to be positive is really reactive and that the reactive peptides are correctly classified.
As previously mentioned, we decided to select, among the best classifiers, the model with the clearest interpretation. In the case of logistic regression, we evaluated the reliability of the regression coefficients by comparing their values and signs with what was expected in the light of the available knowledge. The predictions of all the participants to this DREAM5 challenge have been evaluated and compared. Teams were ranked according to their performance score based on two metrics: the area under the precision versus recall (PR) curve and the area under the receiver operating characteristic (ROC) curve. P-value was defined as the probability that a given or larger area under the curve value is obtained by a random prediction. The overall final score was defined as minus the logarithm of the geometric mean of the ROC and PR p-values.
The final aim of this challenge is to discover whether there exist rules able to predict reactivity of peptides with human antibodies. These rules can be used to develop new reactive peptides. The ''bonus round'' was conceived to test the rules learned during the main challenge: each team was required to submit a list of de-novo peptides generated using their predictive models; the list generated by the teams that achieved the top performance in the main challenge will be experimentally validated by the DREAM5 organizers.
In particular, the bonus round challenge required the provided list to contain peptides with sequence length equal to 15, which must follow these specifications: Moreover, in order to ensure that the peptides of the generated list are different from the peptides of the training and test sets, the following conditions must hold:
1. All submitted peptide sequences should not have stretches of more than three amino acids in common with any of the amino acid sequences supplied in the training or test set. 2. The overall identity between any peptide sequence of the predicted peptides and the training set should not be higher than 5 within a stretch of 11 amino acid positions.
In summary, the final output of the bonus round should be a list of 1000 peptides for each of the three classes (i.e. H, L and M). In the next paragraph we describe the procedure we implemented to generate such a list. The main idea is to generate de-novo peptides by extracting from the training set the motifs that characterize the epitope. A schematic representation of the implemented procedure is shown in Figure 1 .
Clustering. The first step of our strategy is to obtain clusters of similar peptides. In particular we exploited the scoring matrix computed by aligning every sequence with all the others with the Smith-Waterman algorithm (local alignment). We chose local alignment because the results of the main challenge showed that it has higher predictive performance than the global one (see Results). We obtained a distance matrix by subtracting each element of the normalized scoring matrix to one. Then, we applied hierarchical clustering with complete linkage and we used a cut-off value equal to 0.7 to generate the clusters.
Cluster selection and multiple-alignment. We selected three types of clusters by exploiting the information about the peptides reactivity. A multiple-alignment was then performed on the sequences of each cluster. Thanks to this strategy it was possible to compute the conservation of each amino acid in a specific position.
Extraction of the motifs in a cluster. We generated a motif for every sequence 15 amino acids long and belonging to each cluster/multiple-alignment. In detail, we considered all the amino acids composing each of these sequences ordered by the conservation in the corresponding multiple-alignment (computed in terms of information as shown in Figure 2) . A residue was kept as constant in the motif if it satisfied the first constraint of the bonus round (no more than three consecutive amino acids already present in the training set). The remaining amino acids are less conserved and do not satisfy the constraint of the bonus round; so these residues were allowed to vary within their amino acid group or following the variation patterns in a specific position reported in the multiple-alignment results. The amino acids groups were obtained by clustering amino acids on the basis of the BLOSUM50 matrix. A motif was thus generated for every sequence in the clusters.
Generation of all the possible peptides and selection based on final model. All the possible sequences have been generated starting from the motifs extracted with the method described in the previous paragraph. Such new sequences were then filtered in accordance with the second constraint of the bonus round (identity with the other sequences not higher than 5 amino acids in a window of 11).
The predictive model used in the main challenge (model B) was exploited to predict the reactivities of the remaining new peptides. This prediction has been used to rank the new peptides in terms of predicted reactivity.
We selected the 1100 peptides with the highest predicted reactivity generated from the positive clusters and the 1100 with lowest predicted reactivity obtained from the negative clusters. Finally, we randomly selected 1100 elements from the uncertain clusters.
Feature selection. As described in the previous section, we generated 37 features to predict peptide reactivity to human antibodies. We applied three different procedures for feature selection: no selection (subset A), selection based on collinear attribute elimination and on the M5 method and (subset B) and selection based on the LASSO method (subset C). The subset B and C contain 28 and 27 remaining attributes, respectively. The subsets B and C are partially different (see Table 2 ).
Cross-validation of the classifiers. As explained in the Methods section, we learned five different classifiers on the three features subsets. Cross-validation was performed with a leave-oneout approach. The models obtained by applying decision tree and rules learner are reported in the supplementary material (see Text S1 and Text S2). Table 3 shows the results obtained in terms of mean accuracy, sensitivity, specificity, precision and F-measure:
N The results of the classifiers are in general quite good. This shows that the generated features contain useful information to predict the peptide reactivity. Each cluster of peptide is described by its multiple alignments (on the right top of each sub-figure) and by its representation through sequence logo [49] . This graphical representation displays the conservation of the amino acids in each position of the multi-alignment by their one-letter code. Different residues at the same position are scaled according to their frequency. In particular the height of the entire stack of residues is the information measured in bits (y-axis). doi:10.1371/journal.pone.0023616.g002 N Linear regression and logistic regression show the highest performance, even if the difference between the results of the different classifiers is not statistically significant.
N We tested linear regression by both considering discrete or continuous outcomes. This first alternative always gave the best results. N The best results are obtained after feature selection (subset B
and C). This shows that some redundant information is present in the original set of features.
N In terms of F-measure, the logistic regression had a performance clearly higher than all the others (71.15% and 71.17% on subset B and C, respectively). Moreover, considering the quality of the learned models in terms of their Brier Score, the following results were achieved: Lin.Reg. Choice of the final model. The logistic regression models obtained by considering feature subsets B and C have been evaluated in terms of their explanation capabilities.
First of all, we analyzed the two subsets of features by giving some explanations about the removed attributes (zeros are assigned to removed attributes in Table 2 , columns 4 and 5).
N By calculating the correlation among the features along the examples and also among the amino-scales used, we found that accessibility, flexibility and hydrophilicity were quite correlated. This is probably the reason why only flexibility was selected in subset B while only hydrophilicity was kept in subset C.
N Concerning the features derived from the alignments, DiffMaxScore attributes were based on MaxScore features, so they are functionally related. The DiffMaxScore feature was removed in subset B because it did not bring any additional information.
N Moreover in subset B all the features derived from global alignment have been removed, suggesting, as expected, that the reactivity depends on a small portion of the peptide, which probably corresponds to the binding site (information retrieved by local alignment). Also in the case of subset C, two out of the three remaining alignment-based features have been derived from local alignment. N As the amino acid frequencies are concerned, the attributes associated to the presence of X, B and J are present in both subsets B and C, since few sequences contain such residues. The features related to Alanine and Isoleucine have been removed only from subset C.
We then analyzed the estimated coefficients of the logistic regressions in order to further investigate which was the most reliable between the two models. In particular, the estimated coefficients of both models are reported in Table 2 , columns 4 and 5. These coefficients have been evaluated on the basis of the available knowledge but also on the basis of the correlation of each feature with the class, as computed in the training set (Single Regression Coefficients -SRCs). The second column of Table 2 reports the regression coefficient computed for each attribute, while column 3 reports its F-measure. N Every SRC corresponding to an alignment-based feature has the expected sign, given its definition: SRCs are negative for MaxScore_0 and DiffMaxScore features, while they are positive for MaxScore_1. This is confirmed by the corresponding values of the regression coefficients for model B (see Table 2 at column 4). However, model C has an unexpected negative value related to MaxScore_1_nw (see Table 2 at column 5)., Both models confirm that local alignment is more useful for classification than global alignment. Based on all these considerations, we selected model B as the best final model, even if model C had a higher F-measure.
Evaluation of the model. The selected model was used to generate predictions on the test set data (3421 positive and 10219 The Table 4 . Both models had a very high performance in terms of PR and ROC, as shows in Figure 3 . It is important to note that Model B achieved the highest score.
By analyzing the scores of all the participants, reported in Table 4 , it can be noted that two teams (our team and team 725) clearly over-performed all the others.
As explained in the previous section, the final output of the bonus round is a list of 1000 new peptide sequences for each of the three classes: high reactivity (H), low reactivity (L) and medium reactivity (M).
The procedure for the generation of these peptides follows the steps described in the Methods section and schematically reported in Figure 1 .
In the first phase we used the scores of local alignment to cluster the available sequences. As result of this first phase, about 7000 clusters with different size have been created.
Then we exploited the class information to select three types of clusters. By applying the rules described in the Methods section, we selected 23 positive clusters, 27 negative clusters and 4 uncertain clusters. An example of positive cluster and an example of negative one are shown in Figure 2 : the figure depicts the multiple alignments of the two clusters and their representation through sequence logos [49] .
The motif generation phase resulted in a few thousands sequences for each class group (i.e. H, L and M).
Finally, we computed the predicted reactivity for all the sequences generated from the positive and negative clusters. The final list was formed by: i) the 1100 peptides with the highest predicted reactivity generated from the positive clusters, ii) the 1100 with lowest predicted reactivity from the negative clusters and iii) 1100 peptides randomly selected from the uncertain clusters. As shown in Figure 4 , the distributions of the predicted reactivity clearly separate the peptides coming from the positive clusters and the negative ones. This demonstrates the validity of the strategy adopted to generate new peptides.
The experimental test of the real reactivity of these peptide sequences is still ongoing.
In the present work we described the procedure implemented to cope with the Epitope-Antibody Recognition (EAR) Specificity Prediction Challenge of the DREAM5 competition. The aim of the EAR challenge was to extract rules able to predict the binding of a peptide/epitope to a human antibody. A training set of peptides with experimentally identified high and low reactivity to human antibodies was provided. The challenge consists therefore in determining whether the peptides of an independent test set belong to the positive or negative set.
As mentioned in the previous section, we have exploited a machine learning approach to analyze the data, after a knowledgebased feature generation phase. In particular we extracted two types of features for every peptide: (i) sequence-dependent features, which are based on both general information about peptides and knowledge about the propensity of a peptide to interact (amino acid frequencies, antigenicity, accessibility, etc.); (ii) datasetdependent features, which are generated by exploiting the scores obtained by aligning every peptide of the training set with all the others with both global and local alignment. A total of 37 features have been finally generated.
We considered three different subsets of such attributes, based on different feature selection strategies. As a last step, we learned some simple classifiers, which have been evaluated with a leaveone-out cross-validation approach. Since the final aim of the EAR challenge is to extract rules able to explain the propensity of peptide to react, we selected classifiers able to provide a model easy to be interpreted (e.g. logistic regression, rule learners, decision trees, etc.).
The classifier finally selected was built with the logistic regression, one of the most widely used classifiers, able to predict the probability of the class on the basis of both continuous and discrete features. The best results were achieved by using a reduced subset of features; in particular, taking into account the model interpretation needs, we selected the logistic regression fitted with the features obtained by M5 method.
The evaluation of the prediction of the model on the test set showed the validity of the approach: the model had one of the best performances of the challenge. As a note, the performances of the model on the test set are higher than the one obtained with crossvalidation on the training set (e.g. F-measure metrics are 71.26% and 71.15%, respectively).
In general, this good performance has demonstrated that, even if the prediction of epitope reactivity is a difficult problem, there are ways to obtain promising predictive models based on the combination of prior knowledge and data analysis [50] .
Together with the high performance of the proposed reactivity prediction model, the present work highlights some open issues concerning the propensity of a peptide to react with human antibodies.
N The features based on local alignment are more predictive than the ones based on global alignment. This shows that, as expected, the reactivity depends on a small portion of the peptide, which probably corresponds to the binding site.
N In contrast to some hypotheses previously formulated, the features related to accessibility, flexibility and hydrophilicity are negatively correlated with the reactivity values of the dataset. Concerning accessibility and hydrophilicity, the hypothesis that the antigenic sites are on the surface, and thus probably hydrophilic, was recently confuted [9] . As a matter of fact, the analysis of the experimentally determined antigenic sites has revealed that the hydrophobic residues are more likely to be a part of antigenic sites if they occur on the surface of a protein.
Moreover, some studies hypothesized that the flexibility is inversely proportional to antigenic propensity [9, 51] : a relatively high positive correlation (i.e. Spearman correlation = 0.61 and p-value = 0.018) was found between the flexibility and the minimum concentration needed to inhibit the E.Coli growth with antimicrobial peptides. As a matter of fact, a small flexibility may be related to a compact structure, which could favor antigenic propensity. This result confirms the appropriateness of the scale used for its calculation [9] : this is defined as the ratio between the frequency of the residue in antigenic determinants (experimentally identified) and the frequency of the amino acid on the surface (predicted by using the average of hydrophilicity, accessibility and flexibility values reported by Parker et al. [11] ). N Finally, the high values of both SRCs and regression coefficients, as shown in Table 2 (see columns 2 and 4), demonstrate that some amino acids, like F and Y, favour the peptide reactivity.
The future developments of this work will concern the test of our model on other datasets related to the prediction of epitope reactivity. Preliminary encouraging results have been achieved on some peptides of the IEDB (Immune Epitope DataBase) [52] .
The final aim of the challenge was to elucidate the mechanisms of epitope reactivity with human antibodies; for this reason, a ''bonus round'' was proposed beside this main challenge. To this end, we have developed a method based on a clustering approach. We grouped the peptides of the training set in about 7000 clusters by using as distance the score of the local alignment. Then, we selected 23 positive, 27 negative and only 4 uncertain clusters by a-posteriori taking into account of the class of the peptides. It is worthwhile mentioning that the small number of negative and positive clusters demostrates that there are many rules underlying peptide reactivity. Each rule has thus a small support; this is probably related to the wide variability of the antibodies.
The clusters have been used to extract a set of motifs that were the basis to generate an initial list of potential new peptides. We predicted the reactivity of such new peptides relying on our model: the sequences with highest and lowest predicted reactivity formed the final list of de-novo peptides. The results of the experimental test of the real reactivity of these peptide sequences will be available in the near future.
Text S1 J48 pruned tree learned on the entire training set starting from the features subset B.  Description of two measles outbreaks in the Lazio Region, Italy (2006-2007). Importance of pockets of low vaccine coverage in sustaining the infection BACKGROUND: Despite the launch of the national plan for measles elimination, in Italy, immunization coverage remains suboptimal and outbreaks continue to occur. Two measles outbreaks, occurred in Lazio region during 2006-2007, were investigated to identify sources of infection, transmission routes, and assess operational implications for elimination of the disease. METHODS: Data were obtained from several sources, the routine infectious diseases surveillance system, field epidemiological investigations, and molecular genotyping of virus by the national reference laboratory. RESULTS: Overall 449 cases were reported, sustained by two different stereotypes overlapping for few months. Serotype D4 was likely imported from Romania by a Roma/Sinti family and subsequently spread to the rest of the population. Serotype B3 was responsible for the second outbreak which started in a secondary school. Pockets of low vaccine coverage individuals (Roma/Sinti communities, high school students) facilitated the reintroduction of serotypes not endemic in Italy and facilitated the measles infection to spread. CONCLUSIONS: Communities with low vaccine coverage represent a more serious public health threat than do sporadic susceptible individuals. The successful elimination of measles will require additional efforts to immunize low vaccine coverage population groups, including hard-to-reach individuals, adolescents, and young adults. An enhanced surveillance systems, which includes viral genotyping to document chains of transmission, is an essential tool for evaluating strategy to control and eliminate measles The World Health Organization Regional Office for Europe (WHO/EURO) has set 2010 as the target for elimination of measles in the region [1, 2] . This objective has already been achieved in some States through routine immunization programmes which maintain high coverage using a two-dose measles-mumps-rubella (MMR) vaccine schedule [3] . However, vaccination coverage still remains inadequate in several western European countries, including Italy, and although mass vaccination has successfully lowered the incidence of the disease outbreaks continue to occur, often affecting communities with low vaccination coverage [4] .
In Italy, measles vaccine (MV) was introduced in 1976 and routine administration of one dose of measles vaccine to all children aged ≥15 months was recommended since 1979. Combined MMR vaccines became available in the early 1990s and the two-dose schedule (first dose at 12-15 months and second dose at 5-6 or 11-12 years) was introduced in 1999 [5] . However, despite the existence of national recommendations, responsibility for implementation of measles vaccination activities lies within each of Italy's 21 regions. This has contributed to non uniform coverage across regions, with lower rates observed mainly in central and southern regions (including Lazio) with respect to northern regions [6] .
In the years 2002-2003 a large measles outbreak occurred in Italy with over 100,000 estimated cases among children below 15 years of age [7] . Following the outbreak, in November 2003, all 21 Italian regions signed an agreement with the Italian Ministry of Health to implement the "National Plan for the Elimination of Measles and Congenital Rubella" [8] . Strategies include among others, improving routine MMR coverage among children below 2 years of age, implementing supplementary vaccination activities for older children and adolescents (aged 6-13 years) and strengthening disease surveillance. Following implementation of the elimination plan, national vaccination coverage for first MMR dose in children at two years of age increased from 82.0% in 2003, to 89.6% in 2007. During the same period of time, incidence of measles in Italy decreased from 22.6/100,000 to <1/100,000 [4, 9] .
In the Lazio region, the measles elimination plan succeeded in increasing coverage for the first dose of MMR among children at two years of age from 83.9% in 2003 to 90.7% in 2007 [10] . During the same five year period, coverage among school-aged children (6-13 years of age) increased from 53% to 67% while coverage for the second dose at 15 years of age increased from 14% to 42%. In 2004 and 2005 a historically low incidence of < 1 case per 100,000 was reached in the Lazio region, with approximately 50 measles cases reported per year. However, sporadic cases continued to be reported, especially among susceptible adolescent and adults. Pockets of low coverage were also present in specific areas and among emarginated and hard-to-reach populations (HRP), such as the Roma/Sinti population, and illegal immigrants [11] .
Between June 2006 and August 2007, two measles outbreaks occurred in the Lazio region. The first outbreak (June-December 2006) initially involved the Roma/Sinti population, and subsequently spread to the rest of the population. The second outbreak overlapped with the first (October 2006-August 2007) and affected mainly the Italian adolescent and adult population. In this article we describe the two outbreaks and highlight the importance of pockets of low vaccine coverage in sustaining such outbreaks. Data from the mandatory infectious diseases surveillance system, field epidemiological investigations, and molecular characterization of measles virus by the national reference laboratory are presented.
Lazio, one of Italy's 21 regions, has a population of 5.3 million people (2006), 3.2 million of which living in the urban area of the capital city of Rome. It is divided into 5 provinces (Rome, Rieti, Viterbo, Latina and Frosinone) and 12 Local Health Units (LHUs).
The Public Health Agency of the Lazio Region (Agenzia di Sanita' Pubblica, ASP), is responsible for surveillance of infectious diseases and immunisation coverage in the region. The ASP monitored the two measles outbreaks and coordinated outbreak control activities in the 12 LHUs of the region.
In Italy, measles is a disease subject to mandatory notification, and according to the routine procedure, physicians must report suspected measles cases to their LHU within 48 hours of diagnosis. The local health authorities then report confirmed measles cases to the ASP monthly. At the beginning of the outbreaks this procedure was modified and physicians were asked to report suspected measles cases to both the local health authorities and ASP offices within 24 hours of diagnosis. Personnel of the LHUs performed epidemiological investigation of suspected cases including laboratory investigation and contact tracing.
A suspected measles case was defined as a subject with fever (≥38°C), generalised rash and at least one of the following symptoms: cough, coryza, or conjunctivitis. A confirmed case was defined either as a laboratory confirmed case (in which measles-specific IgM antibodies were present in serum or saliva samples or measles virus nucleic acid was detected in urine samples by PCR or as a case with an epidemiological link to a confirmed case.
For each confirmed case, demographic data characteristics (including whether cases belonged to a Roma/Sinti community), vaccination history, date of onset of symptoms, and hospitalisation, were collected. Information was conveyed to the ASP, which discarded non confirmed cases from the database, eliminated redundant records, performed quality check and contacted the local health authorities for any missing information. To assess presence of indigenous transmission or sources of imported virus the data set was integrated with information provided by the National Institute of Health (Istituto Superiore di Sanità, ISS), which conducted viral molecular characterization from urine samples of measles cases, utilising the PCR technique [12] . Data analysis regarding the age distribution and immunization status of cases, as well as the percentage of cases requiring hospital admission was performed on the total number of cases reported in the two outbreaks.
Phylogenetic analysis based on the available partial nucleoprotein gene sequences of measles virus and tree reconstructions were performed with MEGA software version 4.0 [13] . Virus isolates and genotypes were designated according to the new official WHO nomenclature [14] .
Data from confirmed cases was entered into Microsoft Excel and converted to SAS version 8 (SAS Institute Inc., Cary, NC) for analysis. The temporal and geographical distribution of cases, together with the age distribution, was calculated separately for the Roma/Sinti and rest of the population.
The present study did not require approval from an Ethics Committee. Laziosanità -the public health agency of the Lazio Region is the local government agency responsible for the collection of infectious disease notifications, hospital admission and discharge records and laboratory surveillance data. The management of these data for public health purposes does not require a patient's informed consent nor does it require any authorization regarding privacy laws.
From June 2006 to the end of August 2007, a total of 449 cases were reported, of which 302 in 2006 and 147 in 2007. The two outbreaks overlapped and not all cases were genotyped; therefore, it was not possible to determine the exact number of cases that occurred in each of the two outbreaks. Overall, 347/449 cases occurred amongst the Italian ethnic population and 102/449 amongst the Roma/Sinti population. Seventy-eight cases (17%) were laboratory confirmed while the remaining were epi-linked. The virus serotype was identified for fifty-seven cases (Serotype D4: n = 32; serotype B3: n = 25).
The first reported case was an eight year-old Roma child of Romanian nationality, living in a settlement located in the outskirts of Rome. The child was admitted to hospital with rash, fever, diarrhoea, conjunctivitis, rhinitis and otitis on 28 June 2006, reporting onset of symptoms on 24 June 2006. Analysis of routine data allowed the identification of an additional case that had been notified one week previously, in Rome by a different LHU. This case was an unvaccinated six-year-old Roma child, also of Romanian nationality, who developed symptoms on 16 June 2006. The child was not hospitalised and the source of his infection was not determined, since his parents refused to answer to questioning by the health officials and then moved away. He transmitted the infection to an eight-year-old child living in the same building, who developed symptoms on 1 July 2006 and was hospitalised. In the last week of June two additional Roma/Sinti children, in two different health authorities, developed measles symptoms on 23 June and 24 June respectively. By the end of July in Rome, an additional 25 measles cases had been reported in various settlements of the Roma/Sinti community and 16 cases among the rest of population. Two cases among the Italian ethnic population, who developed symptoms on 19 and 20 July, reported contact with Roma/Sinti patients with measles in a hospital waiting area, on 4 and 10 July 
In November 2006, a cluster of six measles cases was reported amongst adolescents and young adults attending a professional school in the outskirts of Rome (attack rate 0.7%). The infection subsequently spread outside the school and cases continued to be reported amongst the Italian ethnic population until the summer 2007. This second outbreak reached a peak in March 2007 and was considered over only in August 2007 (Figure 1 ). During July-August 2007 three measles cases were notified per month, bringing the incidence level to that reported in the Lazio region before the described outbreaks (≤4 cases per month, or <1/100,000 population per year).
The D4 genotype, grouped in two different clusters of common origin, was responsible for the first cases reported in the Roma/Sinti population and detected in several other cases, including the Italian ethnic population, up to December 2006. Starting in October 2006 the B3 genotype was isolated in a contact of a case from the school outbreak, overlapping for some time with genotype D4 (Figure 1 ).
The first outbreak (D4) started in the RM-B LHU (Figure 2 Rome, where the outbreak in the professional school occurred. (Figure 2 ).
Overall, the median age of cases was 11 years. Children aged 0-4 years were the most affected age group, with one third (152/449) of cases reported belonging to this age group (76/100,000 incidence for children <5 years). Measles incidence in the total population during the two outbreaks was 9/100,000. When analysed separately, the age distribution of measles cases was different among the Roma/Sinti population (median age two years) with respect to the rest of the population (median age 15 years). Seventy percent (72/102) of Roma/Sinti cases occurred in children aged 0-4 years and over 90% (93/ 102) were aged below 15 years. The age distribution of cases in the Italian ethnic population was more evenly distributed among all age groups. Only 23% of cases (80/ 347) were aged 0-4 years, and less than 50% (170/347) occurred in children below 15 years of age ( Figure 3 ).
None of the Roma/Sinti cases were vaccinated against measles, and only 16 (5.5%) cases from the Italian ethnic population had received one dose of measles-containing vaccine. Overall, over 57% (258/449) of cases required hospital admission. Fifteen cases (3.3%), 12 of whom from the first outbreak and three from the second, reported a hospital contact with children affected by measles, either in the waiting area or after admission for a different condition. Four of these contacts were healthcare professionals (two nurses and two physicians). Out of the 15 reported cases of nosocomial transmission, seven involved contacts with Roma/Sinti children, including the first two cases in the Italian ethnic population.
In response to the described clusters of measles cases, active tracing and vaccination of susceptible contacts was performed by local health authorities. A second dose of MMR vaccine was also offered to incompletely vaccinated contacts. In addition, MMR vaccine was offered to all susceptible or incompletely vaccinated children and adolescents attending any of the schools in which cases had been detected and to Roma/Sinti children up to 5 years of age. Vaccination sessions were conducted directly in the involved settlements; in total 493 persons in seven settlements were vaccinated in August 2006. Isolation of cases and susceptible contacts (in hospital for Roma/Sinti patients and at home for other subjects) was recommended, Local health authorities were urged to identify possible contacts of all suspected measles cases, and alert general practitioners, family paediatricians, and hospitals about the outbreaks. Physicians were asked to report cases within 24 hours of diagnosis. In addition, guidelines regarding respiratory isolation of patients with suspected measles, and vaccination of susceptible hospital staff were forwarded to all hospitals of the region. Before the start of the 2006/07 school year a meeting was organized by the ASP with staff in charge of the measles elimination campaign and the public health departments of the 12 health authorities. Local media were also informed of the outbreaks.
The two described outbreaks, which involved 449 cases (incidence 9/100,000) notified from June 2006 to August 2007, represent the most serious episodes after the 2002-2003 measles outbreak [7] . They confirmed that pockets of low vaccination coverage exist in some areas of the Lazio region, particularly among Roma/Sinti communities and adolescents Thanks to relatively high immunisation rates amongst new born children (90,7%) [10] and the work done by the local health authorities, conducting contact investigation of cases', vaccination of susceptible school and household contacts, and implementing isolation measures, the outbreaks did not affect the whole region and, in the city of Rome, was mainly limited to a few peripheral districts (Figure 2 ). Both outbreaks started in populations known to have low coverage, (Roma/Sinti community, and students of a secondary school). The subsequent spread to the rest of the population, at least for the first outbreak, was facilitated by nosocomial transmission.
Differences were found in the affected age groups among the Roma/Sinti and the rest of population. As expected in a susceptible population, the most affected age group in the Roma/Sinti population was the 1-4 year-old age group. Conversely, in the Italian ethnic population, which had a higher percentage of vaccinated subjects with respect to the Roma/Sinti population, especially among young children, most cases occurred in the 15-19 year-old age-group.
Molecular characterization of measles virus is an important surveillance tool for monitoring measles elimination [15, 16] . In this case it was fundamental in tracing the origin of both outbreaks and showing that two distinct chains of transmission took place in the region. It is highly likely that the first outbreak, due to the D4 measles serotype, which is not endemic in Italy [12] , was imported from Romania. In fact, first cases occurred in families of Romanian nationality with family and social links in Romania. In addition, the D4 sequence identified among the first cases in Lazio was found to be identical to the D4 isolated in Romania during the 2004-2006 outbreak [17] in which over 4000 cases and 10 deaths occurred [18] .
A D4 measles strain highly correlated with the one isolated in Lazio caused a smaller outbreak in northern Italy (South Tyrol), between 21 June and 11 August 2006 [19] . The first case was reported during the same week of the beginning of the first outbreak in Lazio, and 13 of 17 cases belonged to the Roma/Sinti population. In South Tyrol, a transit camp for Roma/Sinti travelling between Italy and Eastern Europe appeared to be the entry point for the D4 measles genotype in Italy (Figure 4) .
Between August 2006 and February 2007 other measles outbreaks linked to the Lazio outbreak (D4) occurred in Sardinia (Italy) and Barcelona (Spain), both affecting mainly Roma/Sinti communities. In Sardinia, nine cases, all aged below 15 years and three of which laboratory confirmed, with genotype D4 isolated, were reported from a Roma/Sinti settlement in the town of Alghero, between 12 August and 3 September 2006 [19] . Four of the children had travelled to Rome from 3 to 14 August 2006, to attend a funeral. In the Barcelona region (Spain) an outbreak occurred from October 2006 to February 2007. The first case, a six-year-old girl of Eastern European origin had attended a family gathering in Italy with her mother where other guests may also have had measles. Genotype D4 was identified [20] confirming the link with the outbreak in Lazio (Figure 4) .
Genotype D4 was no longer isolated after the end of 2006, being replaced by B3 genotype starting in October 2006. Despite an accurate epidemiological investigation, the origin of this genotype was not identified. B3 is not considered endemic in Italy and is most frequently detected in Sub-Saharan Africa, although transmission of this virus within Europe has been reported. The isolated B3 was similar, but not identical, to the strain circulating in UK in 2005 [12] . This serotype was first identified in Lazio and subsequently introduced in Puglia (Figure 4) , a region of South Italy, where it was responsible for an outbreak from November 2006 to January 2007 [21] .
The percentage of cases which required hospitalisation during the two outbreaks in Lazio was high (57%). This can be partly explained by the well known underreporting of cases by general practitioners and paediatricians, as compared to hospital physicians. The number of cases requiring hospitalisation, especially during July-September 2006, was sufficiently high to create problems to the hospital system. The inadequate number of isolation beds in hospitals may represent a serious problem in case of occurrence of an epidemic due to a more aggressive infective agent, such as SARS or pandemic flu.
Measles nosocomial transmission has been recently documented in several other outbreaks in Italy and other European countries [22] [23] [24] [25] and the public health importance of nosocomial measles transmission has been established in many situations.
The 15 cases reported in this paper represent only 3% of the total number of cases and therefore did not contribute significantly to the measles incidence during the outbreak. However, these cases most likely represent an underestimate of the real number of infections that occurred through hospital contacts and nosocomial transmission. It is likely that isolation measures and separate admission procedures were not always adopted in case of admission of a patient with signs and symptoms compatible with measles. In the first outbreak, nosocomial transmission may have been responsible for the spread of the infection from the Roma/Sinti to the Italian population. Also of concern is the fact that four healthcare professionals developed measles. As the incidence of measles declines, nosocomial transmission is likely to become an important source of infection and sustain the occurrence of outbreaks among non-immunised health staff and hospital contacts, representing a serious problem in the elimination of measles [26] . Strategies to minimize nosocomial spread of infection should become a priority for control and effectively implemented in the future.
The described outbreaks highlight the threat represented by pockets of susceptible populations, even in the presence of good coverage levels in the overall population [27] . These groups include hard-to-reach populations (HRP) such as the Roma/Sinti communities (estimated MMR coverage in Italy below 10%,) [11] , families who refuse vaccination for ideological or religious reasons, as reported in recent outbreaks amongst students in private religious schools and orthodox communities in Europe and Israel [28] [29] [30] , and families objecting to having their children vaccinated out of concern for vaccinerelated adverse events [31] [32] [33] . The risk for the community represented by HRP or organized groups of objectors should not be underestimated and represent a more serious treat than sporadic susceptible individuals. Susceptible population groups may reintroduce indigenous measles virus transmission even in countries confirmed as disease-free or in a population with high immunization coverage [34] facilitating the transmission of the disease to susceptible individuals still present in the region. Efforts are needed to improve methods to identify areas with low coverage and to develop specific strategies which target susceptible groups. An improvement in health services delivery may be needed to reach Roma/Sinti communities and new immigrants. At the same time, more effective communication strategies should be defined to address subjects objecting to vaccination either for religious or other reasons, involving these subjects in a wider discussion on their responsibility toward the community. The implementation of catch up campaigns targeting adolescents and young adults should also be considered, with the additional objective of protecting women of childbearing age against rubella. Systematic Identification of Novel, Essential Host Genes Affecting Bromovirus RNA Replication Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control. Viruses survive with limited genetic material by interacting with and exploiting host factors at essentially every replication step [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] . Identifying the host factors and pathways exploited in virus replication and the nature of their contributions and interactions with virus-encoded replication factors represent major challenges and opportunities for understanding and controlling viruses.
Positive-strand RNA viruses comprise over one third of all virus genera and include important human pathogens such as hepatitis C virus, dengue virus, chikungunya virus, and West Nile virus [15] . Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, plant, and animal viruses, has been used as a model system to study gene expression, RNA replication and virus-host interactions of positive-strand RNA viruses [1, 2, 8, 10, 16] . BMV has three genomic RNAs and one subgenomic (sg) mRNA. Genomic RNAs 1 and 2 encode the multifunctional replication proteins 1a and 2a polymerase (2a Pol ), respectively, which are required for RNA replication [17, 18, 19] . Genomic RNA3 encodes the 3a movement protein, required for infection spread in plants. A sg mRNA, RNA4, encodes the viral coat protein and is produced from the (2)RNA3 replication intermediate. BMV RNA replication and encapsidation can be recapitulated in the yeast Saccharomyces cerevisiae by expressing the viral replication and/or capsid proteins together with at least one genomic RNA template [20, 21, 22] . The ability of BMV to duplicate nearly all major replication features of its natural plant hosts in yeast, combined with yeast genetics, has advanced our understanding of BMV replication and virus-host interactions [1, 2, 9] .
Previously, we tested deletions of nearly all non-essential yeast genes (,80% of the yeast genome) and identified 99 genes, that when deleted, altered BMV replication, revealing the involvement of many novel host pathways in viral replication, transcription, and translation [9] . However, classical yeast genetics and other approaches have demonstrated that genes essential for cell growth also make major contributions to BMV RNA replication [23, 24, 25, 26] . To more globally identify additional essential host factors critical for BMV RNA replication, we assayed a doxycycline (dox)repressible library of ,900 yeast strains, each of which allows repressing the expression of a selected essential gene by adding dox to growth media [27] . Using this genome-wide approach, we identified 24 essential host factors whose repressed expression reproducibly altered BMV RNA replication. These host factors are involved in protein homeostasis, protein trafficking, and translation, among others. The results presented here, in conjunction with previously identified host factors [2, 8, 10, 24, 25] , provide a more complete understanding of cellular pathways utilized by BMV. Dissecting the role of these essential host genes in virus replication should significantly advance our understanding of basic virus biology and virus-host interactions. Additionally, these results may lay a foundation for extending such studies to other virus groups, thus potentially identifying common cellular pathways that could be targeted for the development of broad-spectrum antivirals.
The yeast Tet-Promoter Hughes Collection of essential yeast strains was purchased from Open Biosystems (Huntsville, AL). The tet-promoter mutant strains (designated here with the prefix P TET ) were provided in the haploid R1158 background (MATa URA3::CMV-tTA MATa his3-1 leu2-0 met15-0), which was constructed by a one-step integration of the cytomegalovirus (CMV) promoter-driven tTA* transactivator at the URA3 locus [28] . The kanR-tetO7-TATA was then integrated into the promoter of a different essential gene in strain R1158, allowing the repression of essential gene expression upon the addition of dox to growth medium [27] . pB12VG1 expresses BMV 1a and 2a Pol from the GAL1 and GAL10 promoters, respectively [9] . pB3BG29, based on pB3Rluc [9] , uses a truncated GAL1 promoter (GALL [29] ) to express RNA3 with the coat protein ORF replaced by the Renilla luciferase (Rluc) ORF (from pRL-null; Promega, Madison, WI). pB3BG29 also expresses the firefly luciferase (Fluc) ORF (from pGL3-Basic; Promega, Madison, WI) from the GAL10 promoter. To construct pB3BG29, the AgeI-AatII RNA3/Rluc-containing fragment of pB3Rluc [9] replaced the AgeI-AatII FHV RNA1/Rluc-containing fragment of pBDL250-Ren [B. Lindenbach, unpublished] . pB3MS82 expresses a BMV RNA3 derivative in which the coat protein gene has a four-nucleotide insertion and a point mutation, abolishing expression of the coat protein [30] . The use of pB3MS82 in this study, as in many other studies [8, 19, 30, 31, 32, 33] , allows analysis of RNA3 and RNA4 levels while avoiding any possible effects of coat protein expression and RNA encapsidation. pB12VG1 and pB3BG29 were used in reporter gene-based primary screens and pB12VG1 and pB3MS82 were used in secondary validation testing by Northern blotting.
96-well yeast transformations were based on a one-step procedure [34] . The P TET essential yeast strains were grown to saturation overnight at 30uC in 96-well plates (1 ml per well). The cells were pelleted, suspended in 100 ml of transformation mix (0.18 M LiAc, pH 5.5, 36% polyethylene glycol-3350, 90 mM DTT, 0.5 mg/ml sheared salmon sperm DNA, and 20 mg/ml of each plasmid) per well and incubated at 30uC for 60 min. Cells then were heat shocked at 42uC for 20 min, pelleted, re-suspended in 20 ml sterile water per well and 10 ml was plated on solid media. Transformants were selected by complementation of auxotrophic markers. 96-well plates of transformed yeast were re-formatted to contain 48 strains in duplicate per plate so that strains could be analyzed in the absence of dox (allowing essential gene expression) or in the presence of dox (repressing essential gene expression). Strains containing BMV expression plasmids were grown overnight in medium containing raffinose, subcultured to a starting OD 600 = 0.1 in medium containing raffinose 610 mg/ml dox, grown for 24 hr subcultured to a starting OD 600 = 0.1 in medium containing galactose 610 mg/ml dox. Cells were analyzed at 24 hr and 48 hr post gal-induction of virus expression. Strains were grown in 96-well plates for luciferase assays and 14 ml culture tubes for Northern analysis.
For 96-well Fluc assays, 2.5 ml of cells were lysed in 16 Passive Lysis Buffer (Promega, Madison, WI), 25 ml of Luciferase Assay Substrate (Promega, Madison, WI) was injected and read for 1 s with a 1.6 s delay using a VictorV (PerkinElmer, Waltham, MA). For 96-well Rluc assays, 5 ml of cells were lysed in 16 Passive Lysis Buffer, 25 ml of Renilla Luciferase Assay Substrate (Promega, Madison, WI) was injected and read for 1 s with a 1.6 s delay using a VictorV. To allow comparison between plates, the median of untreated samples and the median of doxtreated samples were calculated for each plate. Each untreated sample was then normalized to the untreated median whereas each dox-treated sample was normalized to the dox-treated median. For each pass of the 741 P TET strains, we calculated BMV-directed Rluc expression as [Rluc dox-treated /Fluc dox-treated ] normalized to the dox-treated median and [Rluc untreated / Fluc untreated ] normalized to the untreated median. The doxtreated to untreated ratio of ratios was calculated and converted to fold change. High-throughput isolation of total RNA from yeast cells was performed as previously described [35] . Northern blotting was performed as previously described [36] except that 2 mg RNA was separated in 1% (wt/vol) agarose-MOPS (morpholinepropanesulfonic acid)-formaldehyde gels. RNAs were detected using 32 P-labeled probes specific for positive-or negative-strand BMV RNA3 and RNA4 as previously described [24] . The 18S rRNA probe was derived from pTRI RNA 18S templates (Ambion, Austin, TX). Probes were synthesized using an Epicenter Riboscribe probe synthesis kit (Madison, WI) with the appropriate enzyme, i.e., T7 or or SP6 polymerase. Northern blots were imaged on a Typhoon 9200 instrument (Amersham Biosciences, Piscataway, NJ) and band intensities were analyzed with ImageQuant software (Molecular Dynamics, Piscataway, NJ).
Protein extraction, Western blotting, and total protein analysis Total protein was extracted as previously described [24] and equal volumes of cell lysates were separated on 4-15% Criterion TM TGX TM precast polyacrylamide gels (Bio-Rad, Hercules, CA). Proteins were transferred to PVDF membrane and expression of target proteins was detected with the following antibodies and dilutions: rabbit anti-BMV 1a at 1:10,000, mouse anti-BMV 2a Pol at 1:3,000, and mouse anti-Pgk1p (Molecular Probes, Carlsbad, CA) at 1:10,000 using HRP-conjugated secondary antibodies (Thermo Scientific, Rockford, IL) and Supersignal West Femto substrate (Thermo Scientific, Rockford, IL).
The tools in R statistical package (version R-2.11.1) (http:// www.r-project.org/) [37] was used for statistical analysis of BMV RNA replication data obtained by Northern blotting. Log transformation was applied to [RNA4/18S rRNA] dox-treated / [RNA4/18S rRNA] untreated ratios from Northern blot data, where 18S rRNA served as normalization standard. One-sided t-statistics were used to identify the dox-treated strains whose RNA replication was statistically significantly altered compared to untreated strains. p-values from t-statistics were converted to qvalues to control for false discovery rate [37] .
To systematically identify novel, essential host genes affecting BMV RNA replication, we screened a dox-repressible library of essential yeast strains [27] . This collection contains ,900 yeast strains, each with a single endogenous essential gene promoter replaced by a tetracycline-repressible promoter [27] . Upon the addition of the tetracycline derivative dox to growth media, expression of the essential gene is repressed, and the protein depleted. This library was used to identify changes in BMV RNA replication after repression (dox-treated) relative to continuous expression (untreated) of a specific essential host gene.
To assess the role of essential genes in BMV RNA replication, we co-transformed each of the ,900 strains with BMV expression plasmids pB12VG1 and pB3BG29 (Fig. 1A) . pB12VG1 expresses BMV RNA replication proteins 1a and 2a Pol [9] . pB3BG29 expresses an Rluc reporter-expressing BMV RNA3 cDNA derivative and, as an expression control, the Fluc reporter gene (Fig. 1A) . DNA-dependent transcription produces an initial (+)RNA3 transcript that serves as a template for 1a-and 2a Pol -dependent RNA3 replication and sgRNA4 synthesis via a (2)RNA3 intermediate (Fig. 1A ). Because sgRNA4, which encodes the coat protein, is derived from the (2)RNA3 intermediate, its synthesis depends on, and can serve as a reporter for, BMV RNA replication [20] . Accordingly, in pB3BG29 we replaced the coat protein ORF with the Rluc reporter gene, so that luciferase assays could be used as a rapid measure of RNA4 production and expression (Fig. 1A) . A similar approach was used for primary screening of the yeast non-essential gene deletion library [9] . Of the 892 doxrepressible strains transformed, 151 did not produce colonies after repeated transformation attempts (Table S1), suggesting that the transformation process or expression of the viral constructs significantly impacted the fitness of these strains.
The remaining 741 transformants were re-formatted on 96-well plates such that duplicates of each strain were present on the same plate ( Fig. 1B) , allowing untreated and dox-treated strains to be directly compared for changes in RNA replication. It is important to note for this library that expression levels from the substituted TET promoter are often different than expression levels from the endogenous gene promoter [27] . Therefore, to test and to control for possible changes in viral RNA replication and/or gene expression, each treated strain was compared to its untreated counterpart rather than to the parental wild type strain. Strains were grown in raffinose-containing selective medium lacking dox (allowing essential gene expression) or raffinose-containing selective medium containing 10 mg/ml dox (repressing essential gene expression) for 24 hr to allow for initial depletion of the essential gene mRNA and protein turnover in strains treated with dox. After this 24 hr treatment period, strains were sub-cultured into galactose-containing selective medium 610 mg/ml dox to induce the expression of BMV components and subsequent viral RNA replication in the continued expression (untreated) or repression (dox-treated) of essential host factors. At 24 hr and 48 hr postvirus induction, Rluc expression was measured as a readout of BMV RNA3 replication and sgRNA4 synthesis. Because of expected differences in the kinetics of gene product depletion and their specific and non-specific effects on cell growth and BMV RNA replication, we measured Rluc at 24 hr and 48 hr post-virus induction for every strain. To monitor any potential adverse effects of dox on the viability of these yeast strains and to ensure that GAL promoter induction was effective, GAL driven Fluc expression, which is independent of BMV RNA replication (Fig. 1A) , was also assayed at 24 hr and 48 hr post-virus induction (Fig. 1B) . Two independent analyses of the entire library were performed.
Since ,70% of the strains in this library exhibit growth defects in the presence of dox (,20% of which exhibit growth defects even in the absence of dox, presumably as a result of endogenous promoter replacement) [27] and because BMV RNA replication levels are often non-specifically enhanced in slow-growing cells, stringent growth requirements were employed to minimize false positives. Additionally, because we expected some strains to exhibit either immediate dox-induced growth defects and/or transcriptional defects from the GAL promoter, Fluc values in untreated vs. dox-treated strains were closely monitored. Accordingly, to be included in the final data analysis for potentially specific effects on BMV replication, each strain was required to: 1) double at least twice between 0-24 hr in galactose-containing media; 2) double at least one additional time between 24-48 hr in galactose-containing media; 3) and have an Fluc dox-treated value within 20% of its Fluc untreated value. As a reference, wild type strain R1158 doubled 3 times between 0-24 hr and 2 times between 24-48 hr and had comparable Fluc untreated and Fluc dox-treated values. Strains that did not meet these growth or Fluc value requirements in both passes were excluded from further analysis (Tables S2 and S3 ). The remaining strains that satisfied these growth and Fluc requirements and were included in analysis (Table S4) showed .90% overlap between pass 1 and 2 at the 24 hr time point and .80% overlap between passes at the 48 hr time point, confirming good reproducibility of screen conditions (e.g. growth, dox-treatment, etc.) and assay performance.
From the analyzed strains (Table S4) , we identified 42 essential yeast genes that, when dox-depleted, altered BMV-directed Rluc expression at least 6-fold in both passes at the same time point (Tables 1 and 2) . A more stringent 6-fold cutoff was used in the primary screen in response to our observation that some strains in this essential gene library showed more luciferase assay variability than in our previous screen of non-essential genes. This increased variability is likely due to the fact that these genes are essential for cell growth. Thus, upon dox-induced repression of these genes, the experiment is a race between specific effects of the relevant gene on virus replication and the nonspecific, general suppression of cell growth and viability that eventually occur with each strain. Therefore, the results are more subject to variations due to small changes in growth conditions or timing of the experiments. Accordingly, a 6-fold cutoff was employed to limit the inclusion of false positives.
In principle, altered BMV-directed Rluc expression observed in the 42 candidate genes might result from general defects in RNA4 translation or viral RNA synthesis, or from selective defects in replication, expression or function of the Rluc reporter gene. To identify genes that specifically affected BMV RNA synthesis and/ or accumulation, secondary validation testing of the 42 candidate genes was performed using Northern blotting to analyze RNA3 replication and RNA4 production, as described in the next two sections.
Of the 42 candidate genes identified in the reporter gene-based primary screens, 23 were genes whose repression enhanced BMVdirected Rluc expression relative to Fluc expression at least 6-fold in both passes at the same time point (Table 1) . For secondary validation tests, these 42 candidate strains were transformed with the 1a-and 2a Pol -expressing plasmid pB12VG1 and with a plasmid expressing RNA3 retaining the BMV coat protein ORF (Fig. 1A, pB3MS82) . The levels of RNA3 and RNA4 replication expresses Fluc and RNA3. BMV-specific RNA-dependent RNA replication and subgenomic mRNA synthesis is initiated from a cDNA derivative of RNA3. DNA-dependent transcription produces an initial (+)RNA3 transcript that serves as a template for 1a-and 2a Pol -dependent RNA3 replication and sgRNA4 synthesis via a (2)RNA3 intermediate. X, the BMV coat protein gene or any gene replacing it, such as Rluc (used here); GAL1/GAL10, yeast promoters, Rz, self-cleaving ribozyme. (B) 892 yeast strains, each with a single essential gene promoter replaced by a doxycycline (dox)-repressible promoter, were transformed with BMV expression plasmids. White wells indicate strains that did not transform. Transformants were re-formatted on 96-well plates with duplicates of each strain present on the same plate, allowing untreated and doxtreated strains to be directly compared. Strains were grown in raffinosecontaining selective medium lacking dox (allowing essential gene expression) or containing 10 mg/ml dox (repressing essential gene expression) for 24 hr to allow for initial depletion of the essential gene mRNA and protein turnover in dox-treated strains. After this 24 hr treatment, strains were sub-cultured into galactose-containing selective medium 610 mg/ml dox to induce expression of BMV components and subsequent viral RNA replication. Viral RNA replication was quantitated with a chemiluminescent Renilla luciferase assay at 24 hr and 48 hr post-virus induction. Cell viability and promoter launching were monitored with a chemiluminescent firefly luciferase assay at 24 hr and 48 hr post-virus induction. Two independent analyses of the library were performed. doi:10.1371/journal.pone.0023988.g001 Table 1 . Genes whose repression was associated with $6fold enhanced BMV-directed Rluc expression in both primary screen passes. (Fig. 1A) . Moreover, RNA4 level is the parameter most closely related to the Rluc expression measured in the primary screen (Fig. 1A) .
A statistically significant increase in BMV (+)RNA4 accumulation relative to 18S rRNA was confirmed for 19 of the 23 genes (,83% confirmation) at the commonly applied false discovery rate of 5% ( Fig. 2 and Table 3 ). False discovery rate analysis is a robust statistical method that controls for multiple test variables by calculating an adjusted, more stringent p-value, termed a q-value [37] . (+)RNA4 levels were enhanced 1.5-to 8-fold in the 19 confirmed strains (Fig. 2 and Table 3 ). Similarly, levels of (2)RNA3, the replication intermediate that serves as a template for sgRNA4, were increased 1.4-to 5-fold in these strains (Table S5) .
The 19 confirmed dox-repressed genes that enhanced BMV RNA accumulation encode proteins with functions in varied cellular processes, including ribosome biosynthesis (DHR2, ECM16, NOP7, PWP1, RIO2, RPA43, UTP4, UTP18, and YGR251w), cell cycle/ DNA maintenance (ADE13 and SPC29), mRNA metabolism (RNA15, SPT6, and SWD2), protein homeostasis (PFY1), translation (DED1), trafficking (NUP57) and lipid synthesis (ALG14) ( Table 4 ). Possible relations of these functions to viral replication are considered further in the Discussion.
The primary screens additionally identified 19 essential host genes that inhibited BMV-directed Rluc expression at least 6-fold in both passes at the same time point (Table 2 ). Northern blotting confirmed statistically significant decreases in BMV (+)RNA4 accumulation for 5 of 19 genes (,26% confirmation) at a false Table 2 . Genes whose repression was associated with $6fold inhibited BMV-directed Rluc expression in both primary screen passes. discovery rate of 5% ( Fig. 3 and Table 5 ). (+)RNA4 levels were inhibited ,1.5-to 8-fold in these strains ( Fig. 3 and Table 5 ). In addition to the severe inhibition of (+)RNA4 accumulation in P TET -HSF1, P TET -PRE1, and P TET -RPT6, (2)RNA3 was inhibited 5.5-, 1.8-, and 5.5-fold in these strains, respectively ( Fig. 3 and Table S6 ). Interestingly, all five genes that inhibited BMV RNA replication (ESS1, HSF1, JAC1, PRE1, and RPT6) perform cellular functions that, in various ways, contribute to modulating host protein levels ( Table 4) .
One possible reason for altered RNA replication is deregulation of viral protein accumulation. To test this, accumulation of BMV RNA replication proteins 1a and 2a Pol were assayed by Western blotting. Addition of dox to growth medium had no detectable effect on BMV 1a and 2a Pol accumulation in the wild type strain (Figs. 4 and 5) . For 20 of 24 confirmed hits, BMV 1a levels in doxtreated strains were comparable to their untreated sample (Figs. 4 and 5). However, in P TET -ADE13, P TET -DED1, P TET -PRE1 and P TET -RPT6 dox-treated cells there was a detectable increase in 1a compared to the untreated strains (Figs. 4 and 5) . Moreover, BMV 2a Pol levels were significantly increased in P TET -HSF1, P TET -PRE1 and P TET -RPT6 dox-treated strains (Fig. 5) . In P TET -JAC1 doxtreated cells, 2a Pol levels were elevated in the absence of dox, but reduced to near wild type levels in the presence of dox (Fig. 5) . These results suggest that, with the potential exception of HSF1, PRE1 and RPT6, viral protein regulation is unlikely to be the cause of altered viral RNA replication phenotypes upon depleting the products of the implicated genes.
We employed a high-throughput, systematic analysis of 741 dox-repressible essential yeast strains to identify 24 novel, essential host factors that alter BMV RNA replication. Our previous systematic analysis of ,4,500 non-essential yeast deletion strains identified 99 genes that inhibited or enhanced BMV RNA replication [9] . Collectively, we have analyzed ,93% of all yeast genes (,5,800), and the 123 host genes identified to date that affect BMV RNA replication represent 2.3% of the yeast genome. As observed with tomato bushy stunt virus (TBSV) [38] , another positive strand RNA virus, BMV replication is affected, directly or indirectly, by essential genes at a higher frequency (3.2%) than non-essential genes (2.2%).
For multiple reasons, our studies to date likely underestimate the number of host genes that contribute to BMV RNA replication. ,60% of non-essential yeast genes are genetically redundant, meaning that the functions of many gene deletions are partially compensated for by other genes [39, 40] . Additionally, although the dox-repressible library is a powerful tool for analyzing the effects of essential genes on virus replication, over 70% of the strains in this collection exhibit growth defects in the presence of dox and 14% exhibit growth defects in the absence of dox [27] . Moreover, expressing viral components in some of these strains resulted in no or poor growth, interfering with meaningful analysis of these strains. Finally, in both the screens for non-essential genes [9] and essential genes (here), virus dependence on some host functions was likely masked by continuous expression of BMV replication proteins and RNA templates, compared to natural infections resulting from a single viral RNA template. This is analogous to studies in which high multiplicity of infection overcomes antiviral resistance in some cell lines [39, 41] . The lower confirmation rate for the 19 candidate genes that inhibited BMV-directed Rluc expression may be because these primary screen candidates included false positives arising from the tendency of essential gene depletion to produce non-specific inhibitory effects and greater variability in results, as is also noted above in the Results section. Additionally, translation and protein expression are the basis of our primary screen luciferase assays, whereas secondary confirmation by Northern blotting analyzes RNA levels normalized to 18S rRNA. Subtle differences (e.g., growth conditions) not controlled for by Fluc in the primary screen or 18S rRNA in Northern analysis may also contribute to the lower confirmation rate observed. Despite such limitations, this study identified 24 novel, essential host genes from various cellular pathways with potentially diverse roles in BMV RNA replication.
Enhanced BMV RNA replication upon repression of an essential gene suggests that, when present, the host factor contributes to an inhibitory response in a cellular process/pathway that competes with the virus. For example, of the 19 genes whose repression stimulated BMV RNA replication, 9 genes (DHR2, ECM16, NOP7, PWP1, RIO2, RPA43, UTP4, UTP18, YGR251W) are functionally associated with ribosome biosynthesis ( Table 4 ). All of these genes perform or participate in ATP-dependent processes and depleting products of the implicated genes may result in an increased pool of energy and/or nucleotides available to the virus (Fig. 2, Tables 3 and 4) . Alternatively, depleting these genes may alter the competition between viral and cellular translation. UTP4 and UTP18 function in rRNA processing, a cellular pathway that enhances TBSV replication (as shown by the effects of UTP9 and UTP15) [38] , but has the opposite effect on BMV RNA replication. Depleting genes involved in processing cellular mRNA 39 ends (RNA15), regulating transcription (SPT6), modulating cellular gene expression (SWD2), and controlling nucleocytoplasmic trafficking (NUP57) may increase the availability of ribosomes and/or alter the levels of specific proteins, preferentially stimulating BMV RNA replication (Fig. 2, Tables 3 and 4 ) by disrupting cellular pathways that would compete with BMV RNA translation or replication under wild type conditions. Thus, experimental depletion of these genes is in some ways analogous to the global shutoff of host mRNA pathways, employed by many mammalian viruses through diverse mechanisms [42, 43, 44] . Additional studies are necessary to define the role of these mRNA metabolism genes in BMV RNA replication.
In previous studies, we have observed that significantly disrupting the cell cycle and extended doubling times can nonspecifically increase BMV RNA levels per cell, apparently because the virus has more time to accumulate RNA replication products prior to cell division (unpublished data). To avoid such false positives, we excluded from further analysis strains for which doxtreatment significantly slowed cell division (see Results above). Thus, although SPC29 and PFY1 encode proteins potentially related to cell division (respectively a spindle pole body protein and actin-binding protein) the doubling times of untreated and doxtreated P TET -SPC29 and P TET -PFY1 cells were comparable and likely do not account for the increased levels of BMV RNA4 observed (Fig. 2. and Table 3 ). Alternatively, disrupting the cell cycle in P TET -SPC29-and/or P TET -PFY1-repressed cells may alter cell cycle signal transduction pathways or the localization of cellular factors that normally inhibit viral RNA replication.
Mutating general translation initiation factor DED1 severely inhibits translation of BMV 2a Pol from BMV genomic RNA2 in a fashion dependent on specific sequences in the RNA2 59 noncoding region (NCR) [32] . Because the RNA2 59 NCR was not present in the BMV expression constructs used in this study, we did not expect a detectable change in BMV RNA replication in the dox-repressed P TET -DED1 strain. However, we observed an increase in RNA replication ( Fig. 2 and Table 3 ), suggesting that dox-repression of P TET -DED1 and global depletion of its gene products may have an alternative effect(s) on viral RNA replication compared to the previously analyzed ded1-18 point mutant [32] . For example, under our screen conditions, repressing P TET -DED1 may alter the production of viral or cellular proteins in ways that favor increased viral RNA accumulation. Further studies are necessary to define such potential additional role(s) for DED1 in BMV RNA replication. Reduced BMV RNA replication upon repression of an essential gene suggests that, when present, the host factor directly or indirectly facilitates viral RNA synthesis or accumulation. Interestingly, the five genes whose depletion inhibited BMV RNA replication (ESS1, HSF1, JAC1, PRE1, RPT6) have varied roles in protein stability or activation. For example, a small but reproducible (,20%) inhibition of BMV RNA replication resulted upon dox-repression of P TET -ESS1, a peptidyl-prolyl cis-trans isomerase (PPI) (Fig. 3 and Table 5 ). Changes in isomerase activity can alter the structure, stability, or intracellular localization of client proteins [45] , and deleting or mutating PPIs has variable effects on positive-strand RNA viruses [46, 47, 48, 49] . For example, knockdown of cyclophilin A and loss of its PPIase activity severely inhibits hepatitis C virus replication [46, 50, 51] . Conversely, siRNA-mediated knockdown of cyclophilin G mRNA stimulates hepatitis c virus replication [49] . Collectively, these findings suggest multi-faceted, complex roles for PPIs in positive-strand RNA virus replication.
JAC1, a member of the Hsp40/DnaJ family of proteins, encodes a specialized J-protein co-chaperone that assists Hsp70 in ironsulfur (Fe-S) cluster biogenesis [52, 53] . Fe-S clusters are among the most versatile protein co-factors in the cell and participate in electron transfer, ribosome biogenesis, regulating gene expression and enzyme activity, and nucleotide metabolism [54, 55, 56] . The inhibition of BMV replication upon dox-repressing P TET -JAC1 ( Fig. 3 and Table 5 ) may result from negatively affecting the activation and/or function of one or more cellular or viral factors required for BMV RNA replication. Previously, we showed that YDJ1, another J-protein co-chaperone of Hsp70 and Hsp90, is required to activate the BMV RNA replication complex, likely through modulating BMV 2a Pol folding or assembly into the complex [26] . Dox-repression of another heat shock protein, P TET -HSF1, inhibited BMV RNA replication by 84% (Fig. 3 and Table 5 ). Thus, our data suggest that BMV utilizes multiple members of the heat shock protein family to facilitate RNA replication.
Dox-repressing P TET -PRE1, a 20S proteasome core component, inhibited BMV RNA replication by 70% ( Fig. 3 and Table 5 ). Similarly, repressing P TET -RPT6, one of six ATPases of the 19S regulatory particle of the 26S proteasome, resulted in an ,90% reduction in BMV RNA replication ( Fig. 3 and Table 5 ). Additionally, BMV 2a Pol accumulation increased significantly in both P TET -PRE1 and P TET -RPT6 dox-repressed cells (Fig. 5) . These results are consistent with previous findings that multiple non-essential ubiquitin-proteasome system components contribute to BMV RNA replication and that cells lacking PRE9, the only non-essential 20S proteasome component, also exhibit a substantial increase in BMV 2a Pol levels [9] . Our prior data show that having a substantial excess of 2a Pol shifts replication compartments from small spherular compartments to double membrane layers, but does not inhibit viral RNA replication [57] . Thus, the increase in 2a Pol accumulation in dox-repressed P TET -PRE1 and P TET -RPT6 (Fig. 5) is not likely the cause of decreased RNA replication. However, prior results do not exclude the possibility that PRE1 and RPT6 depletion might affect BMV RNA replication by directly or indirectly modulating 2a Pol localization, post-translational modification or interacting partners. The 26S proteasome localizes predominantly to the nuclear envelope-ER network [58] , the site of BMV RNA replication [16, 59] , and numerous viruses utilize the ubiquitin-proteasome system to facilitate infection or replication [60, 61, 62, 63, 64, 65] . Studies to define the specific role(s) of the ubiquitin-proteasome system in BMV RNA replication are ongoing.
With the exception of NUP57, RNA15, SPC29 and YGR251w, each of the essential genes identified in this study have recognized orthologs in Arabidopsis thaliana (http://www.arabidopsis.org/ and Figure 3 . Dox-induced repression of five essential yeast genes inhibits BMV RNA replication. Total RNA extracts were obtained from wild type R1158 and untreated and dox-treated (10 mg/ml) essential yeast strains expressing BMV 1a, 2a Pol and RNA3. Accumulation of positive-and negative-strand RNA3 and subgenomic RNA4 was detected by Northern blotting using probes specific for BMV RNA3 and RNA4. Equal loading of total RNA was verified by probing for 18S rRNA. Values represent the mean of four independent experiments. doi:10.1371/journal.pone.0023988.g003 Table 5 . Essential genes whose repression was confirmed to inhibit BMV RNA accumulation in secondary validation testing. [66, 67] . Similarly, PRE1 (PBD1 and PBD2 in Arabidopsis) and RPT6 (RPT6a and RPT6b in Arabidopsis) are essential components of the highly conserved 26S proteasome and recent results from our laboratory show that BMV RNA replication in yeast and plant cells depends critically on the ubiquitin-proteasome pathway (B. Gancarz and P. Ahlquist, unpublished results) [9, 68] .
In summary, our high-throughput analysis of essential yeast genes identified a diverse set of host factors that affect BMV RNA replication and significantly expanded our knowledge of cellular pathways utilized by BMV. Additional studies both in yeast and in BMV's natural plant hosts should reveal how these host factors affect the virus and provide new insights to host cell function and virus-host interactions. Although targeting some essential genes may result in deleterious effects on cells or patients, focusing on the relevant cellular pathways, rather than only individual genes, may overcome such issues. For example, two of the essential genes with the most substantial inhibitory effect on BMV RNA replication, PRE1 and RPT6, are proteasome components, while other proteasome components are not essential (e.g. PRE9, identified in previous screen). Proteasome inhibitors have antiviral activity against multiple diverse viruses (including herpes simplex virus, hepatitis B virus, and HIV, among others), have been through multiple clinical trials, and are already approved for use in patients for some purposes. Many of the genes identified function in pathways utilized by other viruses and thus may present potential cellular targets for developing broad-spectrum antivirals.
Table S1 Dox-repressible essential yeast strains that did not transform with BMV expression plasmids after repeated attempts. (XLSX)  Unusual association of ST-T abnormalities, myocarditis and cardiomyopathy with H(1)N(1 )influenza in pregnancy: two case reports and review of the literature INTRODUCTION: Myocarditis is rarely reported as an extra-pulmonary manifestation of influenza while pregnancy is a rare cause of cardiomyopathy. Pregnancy was identified as a major risk factor for increased mortality and morbidity due to H(1)N(1 )influenza in the pandemic of 2009 to 2010. However, to the best of our knowledge there are no previous reports in the literature linking H(1)N(1 )with myocarditis in pregnancy. CASE PRESENTATION: We report the cases of two pregnant Caucasian women (aged 29 and 30), with no pre-existing illness, presenting with respiratory manifestations of H(1)N(1 )influenza virus infection in their third trimester. Both women developed evidence of myocarditis. One woman developed acute respiratory distress syndrome, almost reaching the point of requiring extra-corporeal membrane oxygenation, and subsequently developed persistent cardiomyopathy; the other recovered without any long-term consequence. CONCLUSIONS: While it is not possible to ascertain retrospectively if myocarditis was caused by either infection with H(1)N(1 )virus or as a result of pregnancy (in the absence of endomyocardial biopsies), the significant association with myocardial involvement in both women demonstrates the increased risk of exposure to H(1)N(1 )influenza virus in pregnant women. This highlights the need for health care providers to increase awareness amongst caregivers to target this 'at risk' group aggressively with vaccination and prompt treatment. Many previous studies have explored the link between influenza and myocarditis. Influenza virus (along with Coxsackie B, adenovirus, echovirus and cytomegalovirus) has long been a recognized cause of myocarditis. Myocarditis can manifest in varying severity, ranging from a mild rise in myocardial enzymes to presenting with profound cardiogenic shock. Previous studies investigating influenza pandemics have confirmed multiple organ involvement on autopsy, including myocarditis and pericarditis. A pandemic caused by the H 1 N 1 type influenza virus has been a topic of great interest of late. Treatment with osteltamivir shortened the period of infection.
To date, only one study has explored the association of myocarditis in H 1 N 1 infection in children. This highlighted that there should be a high index of suspicion for myocarditis in children with H 1 N 1 influenza A infection. It emphasized the importance of early detection and aggressive management. Timely intervention with circulatory support was said to perhaps decrease morbidity and mortality, with potential for a favorable cardiac prognosis [1] .
Two pregnant women were admitted to our hospital in 2009 with a history of an acute viral-like illness.
Our first patient was a 30-year-old Caucasian woman who presented at 28 weeks' gestation with a four-day history of pyrexia (spiking at 40°C) and shortness of breath. Aside from childhood bronchitis, there was no other relevant medical or surgical history. Examination revealed reduced breath sounds and bronchial breathing in the left base. Her C reactive protein (CRP) level was raised, with a mildly raised white cell count. A chest radiograph ( Figure 1 ) showed consolidation and collapse of the left lower lobe. Arterial blood gas levels taken at the time were consistent with a severe type 1 respiratory failure. As a result of her severe hypoxia, she was electively intubated and ventilated. In view of her deteriorating status, her baby was delivered by emergency Caesarean section with no immediate post-operative complications. From admission, she was treated with antimicrobials and osteltamivir. She was also swabbed and subsequently confirmed as being H 1 N 1 positive.
Post-operatively whilst in intensive care, she proved difficult to oxygenate and ventilate. Therefore, she was transferred to Glenfield Hospital (Leicester, UK) for consideration of extracorporeal membrane oxygenation (ECMO). However, she did not need ECMO and improved on conventional mechanical ventilation.
Our patient was transferred back to our hospital for further convalescence. An electrocardiogram was performed, which revealed sinusoidal and anteroinferior ST elevation. Her troponin levels returned negative. She was referred for an urgent echocardiogram, which demonstrated preserved overall biventricular systodiastolic function. She made a good recovery from this episode and was seen as an out-patient, where she was found to have persisting symptoms of myocardial dysfunction; namely Medical Research Council (MRC) class II to III dyspnea, chest pain and palpitations. She had a repeat echocardiogram, which confirmed preserved left and right ventricular function, and is awaiting further cardiac investigations.
Our second patient was a 29-year-old Caucasian woman who was admitted by our Obstetric team with a five-day history of pyrexia and vomiting. On admission she was 37 weeks' pregnant. She had no medical or surgical history of note. On examination, she had bronchial breathing in the entire left lung and the right mid and lower zones. Her CRP level was raised with a moderately raised white cell count. A chest radiograph at this point revealed dense multi-lobular shadowing and consolidation (Figures 2 and 3 ) and she was started on intravenous antibiotics and zanamivir. Osteltamivir was added at a later date. As in our first patient, she continued to deteriorate and developed severe type 1 respiratory failure requiring her transfer to our intensive care unit and invasive ventilation. In light of her deteriorating clinical condition, her baby was delivered by emergency caesarean section. She suffered no immediate post-operative complications and her child was healthy.
Whilst in the intensive care unit, our patient also suffered from a persistent left sided pneumothorax ( Figure  3 ) requiring an intercostal chest drain. Furthermore, she was noted to have T wave inversion in her anterior and lateral leads. A troponin test was negative. Her creatinine kinase levels were also within the normal range. She underwent an echocardiogram, which showed global hypokinesia and moderate to severely impaired left ventricular systolic function. Subsequent repeat echocardiograms confirmed persistent left ventricular (LV) systolic dysfunction. As a result, she was commenced on treatment with an angiotensin converting enzyme inhibitor (ACE-I). A repeat echocardiogram still showed moderately impaired LV function (ejection fraction estimated at 35%). Despite this, our patient made a good recovery and was discharged from hospital.
She was followed up as an out-patient by both the Respiratory and Cardiology departments and was . Complications in extra-respiratory tissues such as encephalopathy, myocarditis, and myopathy occur occasionally [2, 3] . The association of a severe influenzalike illness followed by the development of myocardial dysfunction or cardiomyopathy has been described in 20% of patients in epidemiological studies [4, 5] and also recognized via a rise in antibody titers in association with pregnancy [6] .
In patients with suspected viral myocarditis, echocardiography and electrocardiographic abnormalities are usually seen in 29% to 33% [7] . Physiological changes associated with pregnancy is recognized as one of the factors reducing the efficiency of T helper cells thus increasing the risk of mortality from influenza [8] . Murine studies indicate that the acute cardiac injury is related to cytotoxic immunologic interactions, virusinduced cytolysis and, to ischemia due to intra-capillary thrombosis [9] , while myocarditis is caused frequently by viral infections of the myocardium [10] .
In the past, enteroviruses (EV) were considered the most common cause of myocarditis in all age groups. Other viruses that cause myocarditis are adenovirus, influenza, parvovirus B19, members of the Herpesviridae family, cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) have all been associated occasionally with myocarditis [11] . Viral genomes are frequently detected by polymerase chain reaction enhancement in endomyocardial biopsies of patients with systolic left ventricular dysfunction and this may play a role in the pathogenesis of cardiomyopathy far more frequently [12, 13] .
Acute H 1 N 1 infections in pregnancy have been reported in the current pandemic leading to severe morbidity, as seen in our two patients, and mortality [14, 15] . The fact that this influenza A (H 1 N 1 ) can develop in healthy patients and evolve in few hours to a severe ARDS with a refractory hypoxemia needing recourse to ECMO in 5% to 20% of patients is novel [16, 17] . The first publications of patients admitted to intensive care units for severe influenza A (H 1 N 1 ) often associated to an ARDS reported a mortality rate from 15% to 40% [18] .
In California, data were reported for 94 pregnant women, eight post-partum women, and 137 non-pregnant women of reproductive age who were hospitalized with 2009 H 1 N 1 influenza. Most patients who were pregnant (95%) were in the second or third trimester, and approximately one-third (34%) had established risk factors for complications from influenza other than pregnancy. As compared with early antiviral treatment (administered before or at two days after symptom onset) in pregnant women, later treatment was associated with admission to an intensive care unit or death (relative risk, 4.3). In all, 22% required intensive care, and 8% died [19] . The estimated rate of admission for pandemic H 1 N 1 influenza virus infection in pregnant women during the first month of the outbreak was higher than it was in the general population. Between 15 April and 16 June 2009, six deaths in pregnant women were reported to the Centre for Disease Control, USA; all were in women who had developed pneumonia and subsequent acute respiratory distress syndrome requiring mechanical ventilation [20] .
Although influenza virus is a rare but recognized cause of myocarditis and pregnancy is a known risk factor for the development of peri-partum cardiomyopathy, the association of H 1 N 1-associated severe viral pneumonia combined with features of troponin negative myocarditis and cardiomyopathy in our two consecutive patients raises the novel and hitherto unreported association between H 1 N 1 infection and myocardial involvement which increases the risk significantly for pregnant women. The absence of an acute rise in cardiac enzymes and the low sensitivity of transthoracic echocardiography in recognizing myocarditis may be detrimental to early recognition and institution of appropriate treatment as may be seen in up to two out of three patients. Obstetric providers need to be prepared to provide the care necessary to address the increased morbidity, mortality, and pregnancy-related complications (including spontaneous miscarriage and pre-term birth) faced by pregnant women during an influenza pandemic [21] . Many obstetric health care workers often lack knowledge regarding the safety and importance of influenza vaccination during pregnancy. Misinformed or inadequately informed health care workers may represent a barrier to influenza vaccine coverage of pregnant women. This lack of knowledge among the health care workforce takes on added importance in the setting of the H 1 N 1 2009 swine-origin influenza pandemic [22] . Inactivated influenza vaccine can be safely and effectively administered during any trimester of pregnancy. No study to date has demonstrated an increased risk of either maternal complications or adverse fetal outcomes associated with inactivated influenza vaccination. Moreover, no scientific evidence exists that thimerosal-containing vaccines are a cause of adverse events among children born to women who received influenza vaccine during pregnancy [23] . Maternal influenza immunization is a highly cost-effective intervention at disease rates and severity that correspond to both seasonal influenza epidemics and occasional pandemics. These findings justify ongoing efforts to optimize influenza vaccination during pregnancy from an economic perspective [24] .
These two cases of H1N1 infection in relatively normal pregnant women illustrate the increased risk of lifethreatening complications (including myocarditis and cardiomyopathy) in this group and the multi-system involvement seen. Thus, increased awareness amongst patients and health care professionals and a higher uptake of prevention strategies may result in improved survival in future epidemics.
Written informed consent was obtained from both the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Dominating Biological Networks Proteins are essential macromolecules of life that carry out most cellular processes. Since proteins aggregate to perform function, and since protein-protein interaction (PPI) networks model these aggregations, one would expect to uncover new biology from PPI network topology. Hence, using PPI networks to predict protein function and role of protein pathways in disease has received attention. A debate remains open about whether network properties of “biologically central (BC)” genes (i.e., their protein products), such as those involved in aging, cancer, infectious diseases, or signaling and drug-targeted pathways, exhibit some topological centrality compared to the rest of the proteins in the human PPI network. To help resolve this debate, we design new network-based approaches and apply them to get new insight into biological function and disease. We hypothesize that BC genes have a topologically central (TC) role in the human PPI network. We propose two different concepts of topological centrality. We design a new centrality measure to capture complex wirings of proteins in the network that identifies as TC those proteins that reside in dense extended network neighborhoods. Also, we use the notion of domination and find dominating sets (DSs) in the PPI network, i.e., sets of proteins such that every protein is either in the DS or is a neighbor of the DS. Clearly, a DS has a TC role, as it enables efficient communication between different network parts. We find statistically significant enrichment in BC genes of TC nodes and outperform the existing methods indicating that genes involved in key biological processes occupy topologically complex and dense regions of the network and correspond to its “spine” that connects all other network parts and can thus pass cellular signals efficiently throughout the network. To our knowledge, this is the first study that explores domination in the context of PPI networks. A network (or a graph) is a set of nodes (or vertices), and edges (or links) between the nodes. Networks enable studying the properties of complex systems that emerge from interactions among individual parts. Hence, networks have been used to model and analyze many real-world phenomena in numerous domains. Examples include social, technological, transportation, information, financial, ecological, chemical, and biological systems. We focus on molecular interaction networks, with the goal of understanding complex cellular functioning by studying cells as inter-connected systems rather than as a collection of individual constituents [1] . Nodes in these networks represent biomolecules, such as genes, proteins, or metabolites, and edges connecting the nodes indicate functional, physical, or chemical interactions between the corresponding biomolecules. Since proteins execute the genetic code and carry out most biological processes, we focus on proteinprotein interaction (PPI) networks. In these networks, nodes correspond to proteins and undirected edges represent physical interactions between them.
We have been witnessing the exponential growth of the amounts of available PPI network data, along with the development of computational approaches for studying and modeling of these data. High-throughput screens for interaction bacterial or viral pathogens (e.g., HIV, herpesvirus, hepatitis, and influenza), exhibit some ''topological centrality'' compared to the rest of the proteins in the PPI network [1, [28] [29] [30] [31] [33] [34] [35] . Many approaches have focused on examining only simple topological properties of these proteins, such as their direct neighborhoods in a PPI network. For example, the key assumption of many studies is that proteins that are direct neighbors are more likely to perform the same function than those that are not [25, 26] , or that a neighbor of a disease-causing gene is likely to cause either the same or a similar disease [1, 34] . Another example is the observed correlation between a protein's essentiality and its degree centrality (the larger the degree of a node, the more ''degree-central'' the node) in a PPI network of baker's yeast [36] . However, the controversy arose in the light of newer and more complete PPI network data for which this correlation was not observed [37, 38] and it appears to hold only for literature-curated [39] and smaller in scope Y2H PPI networks [3] , possibly because these data sets are biased towards essential proteins [38] . Also, degree alone might be a weak measure of network topology, as it captures limited network topology, i.e., only direct neighborhood of a node [27, 31, 40] . A similar controversy arose when cancer genes were initially shown to have greater connectivities and centralities compared to non-cancer genes, indicating central roles of cancer genes within the interactome [33] , but it was later demonstrated that most of disease genes do not show a tendency to code for proteins that are hubs [29] , although a recent study again reached the conclusion that cancer proteins have different network topologies, e.g., higher degrees, than ''control'' genes [35] . Apart from this, general conclusions are that disease genes have high connectivity and are centrally positioned within the PPI network [1] . In addition, it has been suggested that aging genes tend to have higher degrees than non-aging ones [41, 42] , as well as that the majority of viral and bacterial pathogens show tendency to interact with high-degree proteins, or with ''bottleneck'' proteins that are central to many paths in the PPI network [43] .
Measures of network topology that are more constraining than degrees might help resolve these controversies. Hence, various topological centrality concepts have been formulated. Examples include the betweenness centrality [35] , according to which nodes that occur in many of the shortest paths in a network have high centrality, and the subgraph centrality, which counts the number of closed walks of different lengths in the network starting and ending at the node in question and according to which nodes that participate in a large number of such walks have high centrality [44, 45] .
In addition, we have recently designed a graphlet-based measure of network topology; graphlets are small induced subgraphs of a large network [46, 47] . As opposed to partial subgraphs (e.g., network motifs [48] ), graphlets are induced, meaning that they contain all edges between the nodes of the subgraph that are present in the large network. This measure generalizes the degree of a node that counts the number of edges that the node touches, where an edge is the only 2-node subgraph, into the graphlet degree vector (GDV) that counts the number of different graphlets that the node touches, for all 2-5-node graphlets. Hence, GDV of a node describes the topology of its up to 4-deep neighborhood. This is an effective measure: going to distance of 4 around a node captures a large portion of a network due to the small-world nature of many real networks [49] . For this reason, and since the number of graphlets on n nodes increases exponentially with n, we believe that using larger graphlets would unnecessarily increase the computational complexity of the method. We designed the similarity measure between GDVs of different nodes, GDVsimilarity, to quantify the topological similarity of the extended neighborhoods of two nodes. We used this constraining measure of network topological similarity to demonstrate that: in PPI networks, biological function of a protein and its local network structure are closely related [27, 50] ; from topology of PPI networks we can extract biological information that cannot always be extracted from sequence and hence, topology could be used as a complementary method to sequence-based methods for homology detection [51] ; topology around cancer and non-cancer genes is different and can be used to successfully predict new cancer genes in melanogenesis-related pathways [31, 40] ; purely topological network alignments can be used to extract protein function and species phylogeny [52, 53] .
Here, we present novel network-based approaches applied towards a deeper understanding of biological function and disease. We aim to further study and understand currently poorly described mechanisms by which ''biologically central'' genes interact with each other and with other genes in the cell. We define as biologically central (BC) the genes that belong to one of the following four gene categories: aging (A) genes, cancer (C) genes, HIV-interacting (HIV) genes, and pathogen-interacting (PI) genes. Our hypothesis is that BC genes, i.e., their protein products (henceforth, we use terms ''gene'' and ''protein'' interchangeably), will have a topologically central role in the human PPI network. We use two different concepts to define ''topological centrality'': graphlet degree centrality and domination (defined below).
Previously, we defined GDV-similarity of nodes' neighborhoods that is independent of the densities of these neighborhoods: nodes with identical GDVs have the maximum GDV-similarity, regardless of whether they reside in dense or sparse neighborhoods. Here, we propose a new centrality measure, graphlet degree centrality (GDC), to measure the density and complexity of nodes' neighborhoods by counting the number of different graphlets that the node touches. According to GDC, nodes in dense and complex 4-deep neighborhoods will have higher centralities than nodes in sparse 4-deep neighborhoods. GDC is a different and more constraining measure of network topology than the degree centrality (DC), as illustrated in Figure 1 : GDC ranks highly a low-degree gene if its 4-deep neighborhood is dense and gives a low rank to a high-degree gene if its 4-deep neighborhood is sparse (details are below). GDC is conceptually different than the betweenness centrality (BWC), which does not measure topological denseness at all. Subgraph centrality (SC) measures the number of closed walks (which can be thought of as partial subgraphs) that the node touches and it has been shown to be more highly correlated with the lethality of proteins in the PPI network of baker's yeast than DC [44] . Unlike SC, GDC counts induced subgraphs rather than partial ones and in a more rigorous way: while SC counts an edge that a node touches many times, as a 2-edge closed walk (going from node A to node B along edge AB and returning from B to A along the same edge), as a 4-edge closed walk (going from node A to node B and back to A twice), as a 6edge closed walk (going from A to B and back to A three times) etc., GDC counts the edge only once and only as an edge, rather than as different subgraph structures.
For each of the four centrality measures (DC, BWC, SC, and GDC), we identify the most central genes (explained below) in the human PPI network [28] and measure the enrichment of these genes in BC genes (i.e., the percentage of the most central genes that are BC genes), with the goal of finding the centrality measure that is the most discriminative in uncovering BC genes; ideally, the most discriminative measure would have all of the most central genes to be BC genes. We find that: (1) enrichments in BC genes of the most GDC-central genes are much higher than those of non-GDC-central genes, (2) the observed enrichments in BC genes of the most GDC-central genes are statistically significant, while those of non-GDC-central genes are not, (3) BC genes that are GDC-central have higher and statistically significant enrichments in known drug targets than BC genes that are non-GDC-central, and (4) GDC is at least as discriminative as the next best centrality measure.
Second, we hypothesize that genes that are vital for normal cellular functioning might correspond to the ''spine'' of the network that connects all parts of the network. The field of telecommunications and the domain of the efficient design of routing protocols for wireless networks in particular, uses the notion of a dominating set (DS) to find the most central set of nodes in wireless networks that would be used for efficient data routing and lead to bandwidth increase and energy savings; in wireless networks, nodes correspond to computers and routers, and edges correspond to links between them [54] [55] [56] [57] . A dominating set of a network is a set of nodes such that every node in the network is either in the DS or is a direct neighbor of a node in the DS. Hence, the nodes in the dominating set act as a ''gateway'' in the network, since all nodes in the network are at most one step away from them and the transfer of the information to all nodes can be quick and cheap. The challenge is to identify a minimum order DS, a DS of the minimum size (i.e., the minimum number of nodes). This problem is NP-hard. Thus, approximate (heuristic) algorithms are sought.
Given the topologically central role of nodes in a DS, we hypothesize that a good DS algorithm might capture a set of proteins in a PPI network that are involved in important biological processes and mechanisms crucial for cell vitality, i.e., that DSs of PPI networks might contain BC proteins and signaling pathways (SPs). We test this by constructing a connected dominating set in the human PPI network with an algorithm that is commonly used in telecommunications [57] . We are interested in connected DSs only since signaling pathways are connected. Other algorithms for finding connected DSs are used in telecommunications as well (e.g., [54, 56, 58, 59] ), but are not applicable to biological networks, because they require nodes to be assigned meaningful numerical IDs, e.g., IP addresses in computer networks; clearly, proteins in PPI networks do not have numerically meaningful labels. Also, several algorithms for finding disconnected (i.e., independent; see Methods) DSs exist [60, 61] , but they are inappropriate for our study for the above mentioned reasons. In addition to applying the existing DS algorithm of Rai et al. [57] , we design a new and simpler DS algorithm that outperforms the algorithm of Rai et al. on our data (explained below). Note that the main focus of this study is not to create a state-of-the-art algorithm for finding DSs, but instead, to demonstrate, as a proof of concept, that a DS of a PPI network found by a very simple algorithm indeed captures biologically vital proteins. Any further algorithmic improvements are likely to yield more optimal DSs and hence improve the biological results.
We apply DS algorithms to the human PPI network [28] and measure the size of the resulting DSs, as well as their enrichments in BC and SP genes. We find that: (1) the enrichments in BC and SP genes of nodes of DSs are much higher than the enrichments of nodes outside of DSs; (2) the enrichments in BC and SP genes of nodes of DSs are statistically significant, while those of nodes outside of DSs are not; and (3) BC and SP genes that are in DSs have much higher and statistically significant enrichments in known drug targets than BC and SP genes that are not in DSs. Hence, we confirm our hypothesis that DSs capture biologically vital proteins and also drug targets.
Furthermore, we demonstrate not only that each of the two measures of topological centrality, GDC and DS, captures a statistically significant biological signal, i.e., BC and drug target genes (as described above), but also that the combination of the two centralities is even more discriminative in capturing these genes. To our knowledge, this is the first study that uses dominating sets to analyze PPI networks.
We analyze the human PPI network of Radivojac et al. that contains 41,456 physical interactions between 9,141 proteins [28] , as well as the human PPI networks from BioGRID [24] , that contains 30,513 physical interactions between 8,581 proteins, and from HPRD [23] , that contains 36,811 physical interactions between 9,449 proteins (we downloaded them in June 2010). Since we obtained qualitatively similar results for all three networks, for simplicity we report only on the PPI network of Radivojac et al. [28] ; we chose this network, since it has the largest number of interactions.
As mentioned above, biologically central (BC) genes that we analyze include: aging, cancer, HIV, and pathogen-interacting genes. We obtained them from the following databases. Aging genes (A) are human genes implicated in the process of aging that are available from AnAge Databank -Human Ageing Genomic Resources (http://genomics.senescence.info/) [62] . Cancer genes (C) are human genes implicated in cancer that are available from: Cancer Gene Database (http://ncicb.nci.nih.gov/projects/cgdcp), Cancer Genome Project -the Cancer Gene Census (http://www. sanger.ac.uk/genetics/CGP/Census/) [63] , GeneCards (http:// www.genecards.org/) [64] , Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/disease/) [65] , and Online Mendelian Inheritance in Man (OMIM) (http://www. ncbi.nlm.nih.gov/sites/entrez?db = omim) [66] . HIV genes (HIV) are human genes known to interact with genes of the HIV virus [63] that are available from HIV-1-Human Protein Interaction Database (http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions/) [67] . Finally, pathogen-interacting genes (PI) are human genes known to interact with genes of pathogens [43] . The data are downloaded in 2009 and 2010.
In the human PPI network, there are 2,101 BC genes in total, of which 237 are aging genes, 887 are cancer genes, 1,132 are HIV genes, and 500 are PI genes. Figure 2 illustrates the overlap of different BC gene categories in the network. The overlap is low and there are only 20 BC genes that are simultaneously aging, cancer, HIV, and PI genes.
Signaling pathways (SPs) that we analyze include the human: MAP kinase interactome [68] , cancer and immune pathways from NetPath [69] , and all human signaling pathways from KEGG [65] . The data are downloaded in November 2010. In the PPI network, there are 2,253 SP genes, 911 of which are also BC genes. Given that there is a total of 2,101 BC genes in the network, the total number of BC and SP genes together is 2253z2101{911~3443.
The drug target data was downloaded from DrugBank [70] .
Related work. Several notions of node centrality have been used in the past. Degree centrality (DC) of a node is the number of its neighbors, i.e., its degree. Alternatively, DC can be normalized by dividing the degree with n{1, where n is the number of nodes in the network. Betweenness centrality (BWC) of a node is the sum, over all node pairs i and j in the network, of the percentage of all shortest paths between i and j in the network that go through the node of interest. Subgraph centrality (SC) of a node is a weighted sum of the numbers of all closed walks of different lengths in the network starting and ending at the node. These closed walks are related to partial subgraphs of a network, e.g., a closed walk with four nodes can ''go through'' different subgraphs on four nodes, such as along the same edge AB twice (as described above: from node A to node B along edge AB, then back to A along the same edge and then again from A to B and back to A along the same edge), or along a 4-node cycle ABCD that includes edge AB (along the ''square'' from node A to node B to node C to node D and back to A; this is regardless of whether edges CA and DB that ''go along the diagonal of the square'' exist) etc. The above mentioned sum is weighted so that the contribution of the closed walks decreases as the length of the walks increases, i.e., shorter walks (smaller subgraphs) have higher weight.
Graphlet degree centrality. We introduce a new node centrality measure as follows. Graphlets are small, connected, induced, non-isomorphic subgraphs of a large network (Figure 3 A) [46, 47] . Previously, we generalized the degree of a node, that counted how many edges the node touched, into the graphlet degree vector (GDV), that counted how many graphlets of a given type, such as a triangle or a square, the node touched (Figure 3 B) [27] . In Figure 3 B, this is illustrated by a node being touched by an edge (the leftmost illustration), a triangle (the middle illustration), or a square (the rightmost illustration). More precisely, coordinates of a GDV count how many times a node is touched by a particular symmetry group (automorphism orbit, see [47] for details) within a graphlet (Figure 3 B) . Clearly, the degree of a node is the first coordinate in GDV, since an edge is the only 2-node graphlet. There is a total of 73 orbits in all 2-5-node graphlets. Thus, the GDV of a node, describing its up to 4-deep neighborhood (i.e., 2-5-node graphlets around it), has 73 coordinates [27] . An example of a GDV of a node that contains all 73 orbits can be found in [52] .
We introduce a new node centrality measure, graphlet degree centrality (GDC), which measures the density of the node's extended network neighborhood. Hence, nodes that reside in dense extended network neighborhoods will have higher GDCs than nodes that reside in sparse extended network neighborhoods. In particular, we define GDC as follows. For a node v, we denote by v i the i th coordinate of its GDV, i.e., v i is the number of times node v touches an orbit i. Then, GDC of node v is computed as follows: 
where w i is the weight of orbit i that accounts for dependencies between orbits, as in [27] ; e.g., counts of orbit 3, a triangle, will affect counts of all orbits that contain a triangle. Hence, for each orbit, we count how many orbits affect it and assign a higher weight w i (w i [½0,1) to the orbits that are not affected by many other orbits (see [27] for details). We use log in the formula because the coordinates i and j of the GDV of node v can differ by several orders of magnitude and we do not want the GDC to be entirely dominated by orbits with very large values. We add 1 to v i in the formula to prevent the logarithm function to go to infinity for an orbit count of 0. Finally, we scale the value of the GDC(v) to (0,1] by dividing it with the maximum GDC(u) over all nodes u in the network.
Let G(V ,E) be a network, where V is the set of nodes of G and E is the set of edges of G. A dominating set (DS) of graph G is a subset S(V of the nodes such that for all nodes v[V , either v[S or a neighbor u of v is in S. A dominating set is said to be minimal if it contains no proper subset that is dominating and it is said to be minimum if it is of the smallest cardinality. The cardinality of a minimum dominating set of graph G, c(G), is called the domination number of G. It has been shown that for graph G with jV j nodes:
where d max (G) is the maximum node degree in G [60] . Identifying a minimum DS is NP-hard, and hence, approximate (heuristic) algorithms are sought.
Heuristic algorithms result in either an independent DS or a connected DS. A subset P of V is said to be an independent set if no two vertices in P are adjacent. A connected DS is a DS in which each node is connected to at least one other node that is in the DS. (Note that if a graph consists of several connected components, a DS of such a graph would be connected within each component, but disconnected across components.) In the context of biological networks, we are interested in connected DSs.
First, we implement an existing algorithm by Rai et al. for constructing a connected DS of graph G(V ,E) that is commonly used in telecommunications [57] . We call this algorithm ''DS-RAI''. It consists of three phases: (1) constructing an independent DS named S, (2) finding a set of nodes C(V \S to connect nodes in S by constructing the Steiner tree between the nodes in S, and (3) pruning the DS defined on nodes S|C to reduce the number of nodes in the DS. More specifically, the algorithm works as follows. In phase 1, each node is colored white. A white node u that is connected to most other white nodes is taken from V , colored black meaning that it is a ''dominator,'' and added to S. All neighboring nodes of u are colored gray meaning that they are ''dominatees'' and added to V \S. Previous steps are repeated on the remaining white nodes in V until all nodes of V are either colored black and added to S, or colored gray and added to V \S. In phase 2, a gray node from V \S that is connected to the largest number of black nodes in S is selected, colored dark gray meaning it is a ''connector,'' and added to C. The algorithm then checks whether node set S|C is connected and if so, it stops; otherwise, the algorithm selects the next gray node from V \S that is connected to the largest number of black nodes in S and repeats the entire process until node set S|C becomes connected. In phase 3, ''redundant'' nodes are deleted from the connected DS defined on S|C to reduce its size as follows. Let G½V ' denote a subgraph of G induced on a subset of nodes V '(V . The algorithm selects a node u with the minimum degree in G½S|C and checks whether the DS defined on S|C\fug remains a Figure 3 . Graphlets, automorphism orbits, and GDVs. (A) All 9 graphlets with 2, 3 and 4 nodes, denoted by G 0 , G 1 ,…,G 8 ; they contain 15 topologically unique node types, called automorphism orbits, denoted by 0, 1, 2, …, 14. In a particular graphlet, nodes belonging to the same orbit are of the same shade (see [47] for details). (B) An illustration of the GDV of node v; it is presented in the table for orbits 0 to 14: v is touched by 4 edges (orbit 0), end-nodes of 2 graphlets G 1 (orbit 1), etc. The figure is taken from [53] . doi:10.1371/journal.pone.0023016.g003 connected DS of G. If so, the node u is removed from S|C. Otherwise, it remains in S|C. This is repeated for all nodes in S|C, in the order of their increasing degrees. The node set resulting from node removals from S|C in step 3 is the final DS produced by DS-RAI algorithm. An illustration is presented in Figure 4 A.
The algorithm breaks all ties uniformly at random. Interestingly, the algorithm is robust to this randomness: we run the algorithm on the human PPI network 30 times using different random seeds, which results in 94.2% overlap between the resulting 30 DSs. The average DS size over the 30 runs is 1,817+1 nodes, out of which 1,711 (i.e., 94.2%) appear in all of the 30 DSs. Hence, given that such a large proportion of any DS is in all DSs, any DS is representative of all of them. Therefore, we continue further analyses of one of the DSs.
Next, we introduce a new, simple, one-step algorithm for constructing a connected DS, that we call ''DS-DC'': it starts with S~V , selects a node u with the minimum degree in G½S, removes u from S only if the DS defined on S\fug remains a connected DS of G, and repeats the above steps for all nodes in S in order of their increasing degrees. An illustration is presented in Figure 4 B. Clearly, DS-DC is much simpler than DS-RAI. Also, as illustrated in Figure 4 , DS-DC results in a smaller DS than DS-RAI (the same holds for real-world PPI networks, as demonstrated in Section 0). Finally, we introduce a modification of DS-DC in which nodes from S are visited in order of their increasing GDCs instead of degrees, which we call ''DS-GDC'' algorithm.
For a given protein set X of size jX j, we measure its enrichment in BC (and SP) genes. We compute the statistical significance (pvalue) of observing a given enrichment by measuring the probability that the same enrichment would be observed in a randomly chosen set of jX j proteins in the PPI network. This probability is computed as follows by using the following notation: the total number of proteins in the network is jV j; the number of proteins in set X is jX j; the number of proteins in set X that are BC (SP) genes is jf j; there are jFj proteins in the entire PPI network that are BC (SP) genes. Then, the enrichment is jf j=jX j, and the p-value, i.e., the probability of observing the same or higher enrichment purely by chance, is obtained by using the hypergeometric distribution formula for sampling without replacement:
Results and Discussion
For each of the four centralities (DC, BWC, SC, and GDC) and each of the four categories of BC genes (A, C, HIV, and PI), we find in the human PPI network the top k% of the most central genes (k~1,2,3,:::,100%) and measure how many BC genes they contain. For example, we measure how many cancer genes (C) are in the top 1%, the top 2%, the top 3% etc. most central genes with respect to each of the four centrality measures. We do the same for aging (A), HIV, and PI genes. For a given centrality measure, BC gene category, and k, we quantify the accuracy of the centrality measure in capturing BC genes by computing precision and recall. Precision can be seen as a measure of exactness: it is the percentage of the top k% of the most central genes that are BC genes. Recall can be seen as a measure of completeness: it is the percentage of BC genes of the network that are in the top k% of the most central genes. We need to determine a threshold for k that results in the best combination of precision and recall. Since when varying the values of k, every decrease in precision corresponds to increase in recall, we choose as the threshold for k the point where precision and recall cross ( Figure 5 ). We do this for each of the four centrality measures and each of the four BC gene categories. If the threshold is found to be K, we denote as ''central'' those genes that are amongst the top K% of the most central genes and as ''non-central'' all the remaining genes in the network. We find that the thresholds are 3, 10, 12, and 6, for A, C, HIV, and PI genes, respectively, for each of the four centrality measures.
We compute the BC gene enrichments of central and noncentral genes. We find that with respect to GDC, enrichments in each of the four BC gene categories are much higher for central genes, ranging between 23.5% and 36.4%, than enrichments for [57] , and the authors describe the algorithm as follows. In phase 1, nodes 1, 4, 8, 12, and 16 are colored black as members of an independent DS. In phase 2, nodes 2, 9, and 11 are colored dark grey as connectors that connect nodes in the independent DS resulting from phase 1. In phase 3, the connected DS resulting from phase 2 is pruned to reduce it size by removing node 16 from the DS (no other nodes can be removed without violating the requirement of producing a connected DS of the graph). In panel B, all nodes are initially in the DS and then nodes are visited in order of their increasing degrees and removed from the DS if the resulting DS is a valid connected DS of the graph. That is, nodes are removed in the following order: 3, 16, 2, 4, 7, 10, 13, 14, 15, and 9. The resulting DS therefore contains the remaining nodes: 1, 5, 6, 8, 11, and 12. Clearly, the DS produced by DS-DC (black nodes in panel B) is smaller than the DS produced by DS-RAI (black and dark grey nodes in panel A). doi:10.1371/journal.pone.0023016.g004 non-central genes, ranging between 1.6% and 9.5% (Figure 6 A) . These enrichments are statistically significant for central genes, with p-valuesƒ10 {11 , while for non-central genes they are not, with p-values~1 (see Methods). As expected, if we choose lower k, e.g., 1%, precision is even higher (although recall is lower): out of the top 1%~91 of the most GDC-central proteins in the network, 55% (i.e., 47 of them) are aging genes, 45% (i.e., 41 of them) are cancer genes, 71.5% (i.e., 65 of them) are HIV genes, and 42.9% (i.e., 39 of them) are PI genes (Figure 7) . Also, we measure the enrichment in drug targets of BC genes (i.e., of each of the four BC gene categories: ''A'', ''C'', ''HIV'', and ''PI'' defined above) that are GDC-central and of BC genes that are non-GDC-central. We hypothesize that higher GDC of nodes in the PPI network reflects their functional importance. Proteins that are targeted by drugs are clearly functionally important. Hence, we examine whether the sets of BC genes that are GDC-central contain more drug targets than the sets of BC genes that are non-GDC-central. Indeed, we find that enrichments in drug targets are higher for BC genes that are GDC-central than for BC genes that are non-GDC-central (Figure 6 B) . Furthermore, these enrichments in drug targets are statistically significant for GDC-central BC genes (with the exception of GDC-central HIV genes), with p-valuesƒ0:047, while for non-GDC-central BC genes they are not, with p-values §0:9 (see Methods).
In addition to the above demonstration that GDC captures statistically significant biological signal, we compare its performance against the performance of the three other centrality measures (DC, BWC, and SC). We do so by determining which measure is the most discriminative in the sense that it uncovers the largest number of BC genes amongst the top K% of the most central genes (K is computed as above) and hence results in the highest enrichments. As shown in Figure 6 C, GDC is at least as good as other centrality measures for all categories of BC genes, except for cancer genes, for which SC has a slightly higher enrichment, but GDC still outperforms DC and BC. GDC always outperforms DC, confirming our hypothesis that GDC, as a more constraining measure of network topology, could capture the biological signal better. SC also outperforms DC for aging genes, but interestingly not for HIV and PI genes. Hence, although GDC and SC both capture deeper network topology than DC and are conceptually similar in the sense that they both count a number of subgraphs that a node participates in, unlike GDC, SC is not always more discriminative than DC.
To evaluate whether GDC captures statistically significant biological signal and outperforms other centrality measures irrespective of the chosen thresholds k, for each centrality measure, we compute the area under precision-recall curve (AUPR) as the threshold is varied between 0% and 100% in increments of 1%. The results obtained from AUPRs corresponding to different centrality measures are mostly consistent with the results obtained at selected thresholds where precision and recall cross (described above): for HIV and PI genes, AUPRs for GDC are the highest, followed by AUPRs for DC, SC, and BWC, respectively; for A and C genes, AUPRs for SC are the highest, followed by AUPRs for GDC, DC, and BWC, respectively. Hence, as was the case for individual thresholds (see above), GDC always outperforms DC, while SC outperforms DC only for A and C genes. Hence, GDC is always more discriminative than DC, while SC is not always more discriminative than DC, even though SC captures a deeper network topology compared to DC. The values of AUPRs for GDC are: 0.27 for A, 0.2 for C, 0.34 for HIV, and 0.2 for PI genes. These somewhat law values are not surprising, since in biological applications, the number of positive examples (here, the known BC genes) is much smaller than the number of negative examples (here, all proteins in the network that are currently not known to be BC genes). Furthermore, we do not know true negatives (genes that are true non-BC genes). Since we expect that many currently unreported BC genes will turn out to be BC genes in the future, AUPRs are likely to increase as this happens. Moreover, the observed AUPRs are statistically significant: we compute, at each value of recall, the probability of observing a given precision and we find that the probabilities of observing a given number of BC genes among k% of randomly chosen genes are in the range 0:03{10 {13 for k up to 90% (clearly, for k close to 100%, results become statistically insignificant, which is expected, since we choose as GDC-central all genes in the network).
Dominating sets capture BC genes, signaling pathways, and drug targets We find DSs in the human PPI network by using the three DS algorithms described above, DS-RAI, DS-DC, and DS-GC (see Methods). We find that the overlap between the three resulting DSs is large, containing 1,720 nodes, out of the total of 1,834 nodes in DS-RAI, 1,815 nodes in DS-DC, and 1,828 nodes in DS-GC DSs (Figure 8 ). Both of our algorithms, DS-DC and DS-GDC, produce smaller DSs than DS-RAI. Also, each of them produces a DS that captures a huge portion of the DS produced by DS-RAI. Using GDC to guide our algorithm does not seem to result in a smaller DS then when we use DC and thus, we continue our analysis on the DS created by DS-DC.
For the DS created by DS-DC algorithm and for its complement (the set of proteins in the network that are not in the DS, ''non-DS''), we calculate their enrichments in BC genes, genes that are members of signaling pathways (SP), genes that are in the union of BC and SP genes (''BC or SP''), and genes that are both BC and SP genes (''BC and SP''). We find that the enrichments are much higher for the DS than for non-DS (Figure 9 A). Furthermore, the enrichments for the DS are statistically significant, with p-valuesƒ10 {11 , while for non-DS they are not, with p-values~1 (see Methods). Furthermore, we measure the enrichment in drug targets of BC and SP genes (i.e., of gene categories ''BC'', ''SP'', ''BC or SP'', and ''BC and SP'' defined above) that are in the DS, and of BC and SP genes that are not in the DS. If our hypothesis that the topological positioning of nodes in the DS indeed reflects their functional importance is correct, then BC and SP genes that are in the DS should contain more drug targets than BC and SP genes that are not in the DS, since proteins that are targeted by drugs are clearly important for normal cellular functioning. Indeed, we find that enrichments in drug targets are much higher for BC and SP genes that are in the DS than for BC and SP genes that are not in the DS (Figure 9 B) . Furthermore, these enrichments for the BC and SP genes that are in the DS are statistically significant, with p-valuesƒ10 {4 , while for SP and BC genes that are not in the DS they are not, with p-values~0:9998 (see Methods).
For each category of BC genes (A, C, HIV, and PI genes), we compute enrichment of GDC-central and non-GDC-central genes in each of the Gene Ontology (GO) terms [71] . We consider all GO terms belonging to each of the three GO categories: Hence, central genes appear to group by functions that are different than functions of non-central genes. Biological functions with the most significant enrichments that are present among all groups of central genes (but none of which is present among any of the groups of non-central genes) include many processes critical for normal cellular functioning, such as: regulation of cell cycle, , and pathogen-interacting (PI) genes). In all panels, the values of k where precision and recall cross (as illustrated in Figure 5 ) are used; k equals 3, 10, 12, and 6, for A, C, HIV, and PI genes, respectively, for each of the four centrality measures. doi:10.1371/journal.pone.0023016.g006 apoptosis, multicellular organism growth, telomere maintenance, innate immune response, regulation of cell differentiation, signal transduction, activity of many signaling pathway cascades (e.g., MAPK, I-kappaB kinase/NF-kappaB, EGFR, FGFR, IGFR, androgen receptor, nerve growth factor receptor, T cell receptor, toll-like receptor, etc.), phosphorylation, response to DNA damage, blood coagulation, regulation of cell proliferation, T cell activation and co-stimulation, response to tumor necrosis factor, response to drug, interspecies interaction between organisms etc.
GDC captures the density and topological complexity of up to 4-deep network neighborhood around a node. Since we have demonstrated significant enrichment of GDC-central proteins in BC genes, this means that genes that are involved in key biological processes occupy topologically complex and dense parts of the human PPI network. Similarly, since we have demonstrated significant enrichment of DSs in BC and SP genes, this indicates that proteins that are vital for normal cellular functioning reside on the ''spine'' of the network that dominates, i.e., connects, all other parts of the network. Hence, the notion of network domination seems to capture the topology required for passing cellular signals efficiently throughout the network.
We hypothesize that GDC-central proteins and proteins in DSs of PPI networks could represent potential candidates for therapeutic intervention, since targeting GDC-central proteins with drugs would have more significant impacts on the network than targeting proteins that reside in sparse and non-complex network regions and since the topology of a DS can enable quick propagation of drug effects through the entire network. Indeed, we find that the enrichment in drug targets of genes that are GDC-central or are in the DS (this is the union of the set of genes that are GDC-central and the set of genes that are in the DS) is 11.4% and it is statistically significant, with p-value of 1:3|10 {4 . Furthermore, the enrichment in drug targets of genes that are simultaneously GDC-central and are in the DS (this is the intersection of the set of genes that are GDC-central and the set of genes that are in the DS) is even higher, it is 31.7%; this enrichment is also statistically significant, with pvalue of 0. Hence, not only that each of the two concepts of topological centrality, GDC and DS, captures a statistically significant percentage of drug targets, but also when the two centralities are combined, the percentage of drug targets that they capture is significant and even higher.
We propose a new centrality measure, graphlet degree centrality (GDC), to simultaneously measure the density and complexity of a node's extended neighborhood by counting the number of different graphlets that the node touches. We find that: (1) the enrichments in BC genes are much higher for GDC-central genes than for non-GDC-central genes; (2) the observed enrichments are statistically significant for GDC-central genes, while for non-GDC-central genes they are not; (3) BC genes that are GDCcentral have higher and statistically significant enrichments in known drug targets than BC genes that are non-GDC-central; and (4) GDC outperforms other centrality measures in the sense that it uncovers the largest number of BC genes among the most central genes and is thus the most discriminative centrality measure.
Given the topologically central role of nodes in a DS, we apply to the human PPI network an existing DS algorithm that is commonly used in telecommunications, with the hypothesis that a DS might capture a set of proteins in a PPI network that are involved in important biological processes and mechanisms crucial for cell vitality. Also, we design a new and simpler DS algorithm that outperforms the existing algorithm on our data. We emphasize that our main focus is not to create a state-of-the-art algorithm for finding DSs, but instead, to demonstrate, as a proof of concept, that a DS of a PPI network found by a very simple algorithm captures biologically vital proteins. Indeed, we find that: (1) the enrichments in BC and SP genes are much higher for nodes of DSs than for nodes outside of DSs; (2) the observed enrichments are statistically significant for nodes of DSs, while for nodes outside of DSs they are not; (3) BC and SP genes that are in DSs have much higher and statistically significant enrichments in known drug targets than BC and SP genes that are not in DSs; and (4) GDC-central genes that are also in the DS contain the highest, statistically significant percentage of drug targets.
These results imply that nodes in dense and complex neighborhoods that dominate the network are vital for normal cellular functioning and signaling. Hence, they might be targets for new therapeutic exploitation. Further algorithmic improvements would aid in more precise identification of these new targets. Neglected Disease – African Sleeping Sickness: Recent Synthetic and Modeling Advances Human African Trypanosomiasis (HAT) also called sleeping sickness is caused by subspecies of the parasitic hemoflagellate Trypanosoma brucei that mostly occurs in sub-Saharan Africa. The current chemotherapy of the human trypanosomiases relies on only six drugs, five of which have been developed more than 30 years ago, have undesirable toxic side effects and most of them show drug-resistance. Though development of new anti-trypanosomal drugs seems to be a priority area research in this area has lagged far behind. The given review mainly focus upon the recent synthetic and computer based approaches made by various research groups for the development of newer anti-trypanosomal analogues which may have improved efficacy and oral bioavailability than the present ones. The given paper also attempts to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness. African sleeping sickness remains one of the most neglected life threatening diseases that have been left untreated till date. Two forms of human African trypanosomiasis (HAT) have been identified that are parasite dependent. The first one Trypanosoma brucei gambiense (T. b. gambiense) causes a human chronic infection, endemic in Western and Central Africa while the other Trypanosoma brucei rhodesiense (T. b. rhodesiense) has a vast animal reservoir and causes acute illness in people in eastern and Southern African countries. HAT has high occurance in the remote rural areas, where the surveillance is weak or nonexistent, with 50 to 70 thousand estimated cases. According to World Health Organization (WHO) estimation there are half-million cases of HAT or "sleeping sickness" resulting from infections with T. brucei rhodesiense and T. brucei gambiense. WHO has attributed 50.000 deaths annually to the disease [1] . By more recent estimates, up to 25.000 new cases occur per year, and 50 million people are at risk [2, 3] .
Out of six clinically approved drugs for the treatment of HAT, five (suramin, pentamidine, melarsoprol, eflornithine and nifurtimox) have had been discovered more than 30 years ago. Because suramin and pentamidine are ionized at physiological pH, they are unable to cross the blood brain barrier in therapeutic concentrations and are thus used for the treatment of hemolymphatic early stage HAT, caused by T. b. rhodesiense and T. b. gambiense infections, respectively. The treatment of the second or neurological stage, when the parasites invade the central nervous system (CNS), relies on the organoarsenical drug melarsoprol and the more recently registered eflornithine. The latter is ineffective against T. b. rhodesiense sleeping sickness and is used primarily to control CNS-involved HAT caused by T. b. gambiense. All the existing anti-trypanosomal therapies suffer from unacceptable toxicity, poor efficacy, difficulties of administration, and increasing treatment failures due to the development of parasite resistance [4] [5] [6] [7] [8] [9] . In the last couple of decades, no new drug has been developed for treatment of early-stage HAT, and only one drug has been developed for late-stage HAT [1, 3, 5] . The need is great for new orally active drugs for the control and eradication of this disease.
Novel medicines are typically developed using a trial-and-error approach, which is timeconsuming and costly but yet has the potential to yield new drugs. The application of computer-assisted drug design (CADD) methodologies to this problem has the potential to greatly decrease the time and effort required to discover new medicines or improve current ones in term of their efficacy.
This review focuses on the synthetic and computer-assisted drug design (CADD) approaches made by various research groups for the development of newer antitrypanosomal agents having improved efficacy and oral bioavailability. The current research also endeavors [10] to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness.
Safe, effective and affordable orally active therapies for trypanosomiasis capable of overcoming resistance are required, so the identification of new anti-trypanosomal drug candidates is an urgent priority. Compared to the last 15 years there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing [11] . In view of this, the researchers have endeavored to compile in the present section of the review, diverse series of compounds that have been recently synthesized by various research groups targeting DNA minor groove and many other new targets. Based on the mechanism of action this section has been divided into two sub-sections. The first sub-section includes the series of compounds acting as DNA minor groove binders while in the second sub-section are included all the other anti-trypanosomal compounds and their respective targets. Also are described herein prodrug approaches to provide oral bioavailability for the dication class.
The wide range of antifungal and antiparasitic activities related to aromatic diamidines and its excellent results in preclinical and clinical phase of drug development has made aromatic diamidines an interesting class for the development of newer anti-trypanosomal drug therapy. The aromatic diamidines i.e. pentamidine ( Figure 1) show their anti-parasitic action by binding strongly to AT-rich sequences in the minor groove of DNA. Therefore DNA minor groove has evolved as a productive target for designing new ant-parasitic drugs. In order to design newer analogues belonging to diamidine series, studies of the DNA complexes with diamidine compounds have been conducted and a number of diamidines have been crystallized with the DNA duplex d(CGCGAATTCGCG)2 which provide valuable models for drug development in the diamidine series. Structures of DNA complexes of furamidine, berenil, and pentamidine, for example, reveal that they all bind in the DNA minor groove at the central AATT sequence. These drugs penetrate deeply into the groove and fit snugly between the walls of the groove. Their amidines form H-bonds with thymine-O2 and/or adenine-N3 acceptor groups on the edges of the bases at the floor of the groove. The amino group of G protrudes into the minor groove and prevents the compounds from assuming their preferred orientation deep in the minor groove. This binding to DNA eventually leads to inhibition of one or more of the several DNA-dependent enzymes (e.g., topoisomerases and nucleases) or direct inhibition of transcription.
For more than 50 years aromatic diamidines and related dicationic molecules have been extensively used but so far only pentamidine [12] [13] [14] [15] has been widely employed as a drug in humans despite several adverse effects, such as hypotension, abdominal pain, vertigo, hypersalivation, hypoglycemia, nausea, and mild nephrotoxocity [5] [6] [7] [8] . Being a highly flexible molecule that can assume an array of linked conformations related through torsional rotation, changes can be made in the nucleus to produce improved analogous against HAT. In line to this with an aim to increase the efficacy and decrease the side effect related to pentamidine various research groups have made several changes in the pentamidine molecule [16] [17] [18] [19] [20] [21] [22] [23] 
Large numbers of structurally related congeners of pentamidine were synthesized by Tidwell RR et al [24] by introducing substitutions on the cationic groups, changing the position of dications from 4,4' to 3,3' position, changing the length of the aliphatic chain between the two aromatic rings, adding substituent on the aromatic rings at 2,2' and 3,3' position and replacing oxygen atoms in the alkyl linker with isosteric sulfur or nitrogen atoms (Figure 2a-g) . On comparing the activities of the synthesized compounds with pentamidine and melarsoprol, it was found that few compounds showed activity in the range or better than that of pentamidine and/or melarsoprol. The unsubstituted diamidine compound 64 ( Figure 3a ) with secondary amino group in place of oxygen atom exhibited subnanomolar activity (<0.001 µM) against T. b. rhodesiense and was nearly twice as selective against the pathogen as pentamidine. At the same time the diimidazoline compound 66 (Figure 3b ) exhibited the second highest anti-trypanosomal activity in the series with the IC 50 value of 0.001 µM and also had the highest parasite selectivity (SI T = 34500), being 63 times more selective against T. b. rhodesiense than pentamidine. The replacement of oxygen atom with sulphur atom as in compound 62 ( Figure 3c ) also generated active congeners having IC 50 (0.005 µM) value better than that of pentamidine (0.007 µM). The compounds 20-31 ( Figure 2c ) in which the cationic groups were present in the 3,3′-position of the aromatic rings and the length of the carbon linker was varied from 3-6 exhibited lower anti-trypanosomal activity compared to pentamidine whereas among the compounds 12-19 ( Figure 2b ) of the series, in which the amidine groups were present at 4,4' position and the length of aliphatic chain was varied from three carbon atoms to six carbon atoms, compound 12 ( Figure 3d , IC 50 =0.007 µM) with three methylene linker between the aromatic rings had same in vitro activity as that of pentamidine (0.007 µM). Further introduction of 2,2′-dichloro substituent in compound 12 improved the antitrypanosomal properties as evident from the in vitro activity of compound 32 (Figure 3e , IC 50 =0.004 µM).
These compounds having excellent in vitro activity were evaluated in vivo in the STIB900 mouse model of African trypanosomiasis. The screening was conducted using intraperitoneal dosing at 20 mg/kg daily for four days. The compounds 12, 62 and 64 showed very poor in vivo activity, whereas compound 32 and 66 exhibited excellent in vivo efficacies in the acute mouse model of trypanosomiasis, providing cures of all infected animals.
Thus, because of high selectivity, excellent in vitro and in vivo activity compounds 66 and 32 can serve as a novel lead for further pre-clinical and clinical trials, but its cytotoxcity profile needs to be monitored during its evaluation. Also compound 64 which showed excellent in vitro, in vivo activity and high selectivity index against the T. b. rhodesiense merits further SAR optimization in order to synthesized newer lead compounds with reduced cytotoxicity compared to pentamidine. 
Pentamidine analogues with activity better than that of melarsoprol and/or pentamidine
Increased efficacies of pyridyl analogues of 2,5-bis(4-amidinophenoxy)furan (furamidine) [25] [26] [27] encouraged Tidwell RR et al [28] to synthesize pentamidine analogues in which the phenyl rings of pentamidine were replaced with pyridyl fragments. This replacement resulted in series of 18 compounds (Figure 4a ), most of which had lower cytotoxicity than pentamidine. SAR study pointed out that the antiprotozoal properties of these compounds depended on the placement of cationic moieties on the pyridine rings as well as the nature of substituents on the amidine groups. The N-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the N-alkylation of cationic fragments reduces the activity of compounds against T. brucei rhodesiense compared to pentamidine. A same trend was observed in pentamidine analogues series. The 2, 6-substituted dications (compounds 11-13, figure 4a ) and 2,4-substituted dications (compounds 14-18, figure 4a ) displayed lower potencies against T. brucei rhodesiense than the corresponding 2,5-substituted isomers (compounds 1-10, figure 4a), while among the 2, 5-substituted dications, the compounds possessing cationic substituents adjacent to nitrogen atoms in pyridine rings displayed superior activities against parasites compared to pentamidine as evident from compound 6 (figure 4b) which showed promising anti-trypanosomal activity (0.001µM) and lower cytotoxicity (4.90µM) than pentamidine and melarsoprol, but had poor in vivo activity giving only 1/4 cures in the STIB900 mouse model. However diamidoxime compound 9 (Figure 4c ), an oral prodrug of diamidine compound 6 ( Figure  4b ), exhibited excellent in vivo activity curing four out of four animals upon oral administration in STIB900 mouse model. Although compound 9 did not provide cure in the CNS mouse model of infection but its BBB permeability could potentially be improved by developing prodrug using lipophillic substitutions in place of hydrophilic substitution as used in compound 9. This could provide higher concentration of compound 6 in CNS.
Thus excellent in vitro activity of diamidine 6 and high in vivo efficacy of its prodrug diamidoxime 9 warrant further investigation of these dications as potential antitrypanosomal drug candidates with improved oral efficacies. The structure activity relationship of the two series indicates that in both the cases the unsubstituted diamidines were more active than the N-substituted diamidines. The replacement of oxygen atoms in alkyl chain with secondary amino group improves the activity against T. b. rhodesiense of pentamidine analogues while in case of pyridyl analogue the compounds with oxygen atoms in the alkyl chain have been the active one. The most potent compound of these two series are compound 6 ( Figure 4b ) and compound 66 (Figure 3b ), which have excellent in vitro activity (0.001µM) better than pentamidine and melarsoprol and also have high selective against the T. b. rhodesiense parasite as evident from their selectivity index (SI of compound 6 = 4900 and compound 66 = 34500 µM). Initially it has been believed that the oxygen atom in the aliphatic linker and the amidine groups are important for anti-trypanosomal activity of pentamidine as these groups were part of recognition motif for P2 amino purine transporter in trypanosoma species. Thus the activity of compound 6 can be explained on this basis, but in case of compound 66 the amidine groups have been replaced by imidazoline and the oxygen atom has been replaced by secondary amino groups. Despite these replacements, the compound has shown excellent in vitro and in vivo activity. Thus this is in accordance to recently reported work [29] according to which there is no direct connection between the affinity for P2 carrier and anti-trypanosomal activity.
In view of this, these two compounds can serve as a lead structure for the development of newer analogues of pentamidine with reduced side effect and improved pharmacokinetic profile.
In the early 1970s Dann O et al [30] [31] [32] Fig. 6 .
Structure of two phenylbenzofuran dications with promising anti-trypanosomal activity Tidwell RR et al found that the in vitro anti-trypanosomal activities of bisbenzofuran derivatives against Trypanosoma brucei rhodesiense, and cytotoxicity against mammalian cells depended on the position and the type of cationic substituents as well as the length of the carbon linker between aromatic moieties. As observed in most of the dicationic molecules, the N-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the N-alkylation of cationic fragments reduced the activity of compounds against T. brucei rhodesiense compared to pentamidine. At same time the 5-substituted bisbenzofurans were generally less cytotoxic than compounds bearing substituents in the 4-or 6-positions where as the 4-substituted bisbenzofurans were significantly less active against T. b. rhodesiense than corresponding 5-and 6-substituted isomers. The in vitro activity of the bisbenzofuran series was not so promising and only lead compound 8, (Figure 5b ) exhibited in vitro anti-trypanosomal activity (0.008 µM) comparable to that of pentamidine and melarsoprol. However some compounds from Tidwell series (Figure 7b ) showed reduced cytotoxicity profile compared to pentamidine (for example compound 43 has cytotoxicity of 1.85 µM), but had very poor selectivity profile. Thus was proved that the selectivity of bisbenzofurans against T. b. rhodesiense decreases as number of methylene groups in the alkyl bridge increases.
However, how the substitution on the amidine groups affects the anti-trypanosomal properties of bisbenzofurans has not been explained. Thus in light of their reduced cytotoxicity compared to pentamidine these molecules require further investigation to study the influence of the type of cationic substituents and the distance between aromatic moieties on uptake and intracellular distribution of dicationic bisbenzofurans in order to understand better the mode of action and thus improve the efficacy of aromatic diamidines. Thus promising in vitro, anti-trypanosomal activity, excellent potency in the acute mouse model of trypanosomiasis and the reduced cytotoxicity (1.9 µM) of compound 1 compared to pentamidine warrants further pre-clinical and clinical trials of this molecule. While compound 32 and 20 can serve as lead compounds for further SAR optimization to derive congener with enhanced activity and lesser cytotoxicity. 
To combine the rigidity of 2-phenylbenzofurans with the flexibility of pentamidine congeners, Tidwell RR et al [35] incorporated the benzofuran ring into molecules of pentamidine-related analogues. A series of 48 pentamidine congeners containing benzofuran fragments (Figure 10a , b) were synthesized and tested in vitro against T. b. rhodesiense. Most of the compounds in the series showed cytotoxicity less than that of pentamidine, but had lesser potency and selectivity index as compared to pentamidine. The properties and cytotoxicities of these dications depended on the nature of the cationic substituents, the placement of the benzofuran motif, and the length of the carbon linker.
Within the series cytotoxicity of the compounds decreased with the substitution on cationic groups and increased with the elongation of the carbon linker.
Dications with the benzofuran motif in the 4'-position (compounds 25-48, Figure 10b ) and connected by the propylene linker are the most potent and more selective against T. b. rhodesiense among the series. Thus on the basis of SAR study further improvements can be made in order to produce newer anti-trypanosomal agents with improved pharmacological activity. 
The SAR of the three series clearly indicates that the nature of cationic substituents and their position is important in deciding the anti-trypanosomal activity of benzofuran derivatives. The introduction of phenoxy fragment into a benzofuran structural motif may result into retention of affinity for the aminopurine P2 transporter, which could be the reason for anti-trypanospomal activity of benzofuran derivatives. However the precise mechanism by which these compounds show their anti-trypanosomal activity in not known. Among the three series the 2-phenylbenzofuran derivatives are attractive molecules because of their high activity, low cytotoxicity and high selectivity against the HAT parasite. The introduction of phenoxy fragment into the aromatic ring protects the phenylbenzofuran dervatives from metabolic deactivation. This structural modification retains affinity for the aminopurine P2 transporter. The diamidine and the hydroxy or methoxy group may be involved in binding to the DNA minor groove. Thus multiple modes of action may be the reason for promising anti-trypanosomal propertries of 2-phenylbenzofuran derivatives. Thus these compounds require further assessment in order to develop as newer antitrypanosomal agents with promising pharmacokinetic profile.
Since a prodrug of furamidine, 2,5-bis[4-(N-methoxy)amidinophenyl]furan (pafuramidine), has shown promising results in clinical trials against HAT [36] , therefore Furamidine ( Figure 11a ) has gained attention for further evaluation to produce newer antitrypanosomal analogous. Two other analogues of furamidine (Figure 11b &c) have been evaluated against T. b. rhodesiense mouse model and have shown promising results. One approach to enhance the activity of furamidine has been the replacement of the central furan ring with other heterocyclic systems, including thiophene, pyrrole, oxazole, oxadiazole, thiadiazole, pyridazine, methylpyrimidine, and triazine [37] [38] [39] [40] [41] . Such structural modification in past has resulted into compound with good anti-parasitic activity. For example in 1977 Boykin DW and Das PB have reported the synthesis and antiprotozoal activity of eighteen substituted 2,5-bis(4-guanylphenyl)furans and related analogues, including "masked" amidines in which the guanyl function was incorporated into a heterocyclic ring. Among these, six compounds have produced cures in mice at submilligram dosage levels and have been somewhat more active in this screen than stilbamidine, hydroxystilbamidine, and pentamidine. . 11 . Structure of furamidine and its analogue that have shown promising antitrypanosomal properties
Tidwell RR et al [43] has recently synthesized a series of 3,5-diphenylisoxazole analogues ( Figure 12 ), in which the central ring of furamidine was replaced by isoxazole, and their activities with furamidine and melarsoprol were compared. Among 43 synthesized dications, the compounds with at least one p-amidine moiety (compound 22, Figure 13a and compound 3, Figure 13b ), displayed good IC 50 values (3.5 nM and 5.1 nM respectively) comparable to that of furamidine (4.3 nM) while loss in potency was observed in compounds with substituted diamidine at m-position (compound 32, IC 50 =29 nM, Figure  13c ). In general, the introduction of nitro, chloro, or methoxy substituents on either aromatic ring resulted in decreased anti-trypanosomal activity. However, the introduction of methoxy group on the aromatic rings of compound 32 resulted into compound 41 ( Figure 13b ) with comparable anti-trypanosomal activity (4.2 nM) as that of furamidine. Hence these compounds of the isoxazole series that showed good in vitro antitrypanosomal activity and less cytotoxcity profile relative to furamidine, could be a candidate for further evaluation against animal models of the diseases.
However in vivo evaluation of the present 3,5-diphenylisoxazole series has not been reported yet. [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] and also because 1,4-diphenyl-1,2,3-triazoles showed geometrical resemblance to furamidine. It was observed that the cytotoxicities of triazoles were as lower compared to that of pentamidine and were not affected by the alkylation on the amidine groups or the substitution on the aromatic rings. However, the placement of the cationic moiety in the 4-position increased the cytotoxicities of diamidines 46 ( Figure 15 ) with respect to the pentamidine. Except few, majority of unsubstituted diamidines exhibited higher in vitro activities against T. brucei rhodesiense than bis(N-isopropyl)amidines and diimidazolines.
The compounds with cationic fragments in the 5,5'-position (compounds 1-15, Figure 14a The replacement of central furan ring of furamidine either by isoxazole or 1,2,3-triazole resulted into compounds with better activity than that of furamidine. Position and the nature of the cationic substituent governed the anti-trypanosomal activity of the two series.
In general the para substituted diamidines in both the series were the most promising compounds with good activity. Thus these compounds should be further evaluated to produce better analogue of furamidine
The recent report on N,N'-bis(4-amidinophenyl)piperazine (Figure 16) , which was shown to be very effective in vivo anti-trypanosomal agent has attracted the interest in this compound [56] . Hence in search for new HAT chemotherapy Dardonville C et al [57] decided to carry out an in vitro screening of a total of 62 compounds against the parasite T. brucei rhodesiense taken from their in-house library. Based on this they showed that bisguanidine and especially bis(2-aminoimidazoline)diphenyl compounds displayed potent anti-trypanosomal activity in vitro and vivo against T. b. rhodesiense, the causative agent of acute HAT [58, 59] . Among the 62 compounds screened, compounds1c, 28b, 32b and 41b ( Figure 17 ) showed excellent in vitro activity (49nM, 69 nM, 22 nM and 118 nM respectively) as well as high selectivity (>5294, 3072, 29.5 and 881respectively) for the parasite. These studies revealed that compounds bearing 2-aminoimidazoline cations had higher selectivity for the parasite and similar activities with respect to their guanidine counterparts. In addition, a correlation between anti-trypanosomal activity and DNA binding affinity was observed, suggesting a possible mechanism of action for these compounds. Those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite represent new anti-trypanosomal lead compounds. Fig. 16. N,N'-bis(4-amidinophenyl) piperazine
In light of these promising results, bis(2-aminoimidazoline) derivatives deserve more investigation as anti-trypanosomal agents and DNA minor groove binders. The synthesis and study of new derivatives and prodrugs of these lead compounds is ongoing. 
Progress has also been made on targets other than DNA minor grove. In addition to newer targets some new lead compounds have also been identified with proper antitrypanosomal activity but uncertain mechanism of action.
The enzyme trypanothione reductase (TR) which restores the oxidized trypanothione to reduced state has evolved as an effective drug target. Many inhibitors of this enzyme have been developed but most of them combat with problems related to bioavailability, pharmacokinetics and metabolism. Despite this quinolines have evolved as most important class of compounds against TR due to their broad spectrum of activity, excellent pharmacological and pharmacokinetic properties such as high plasma levels, high clearance, oral and parenteral applicability, chemical stability and rare side effect.
In view of this Gilbert IH [60] reported the screening of 62000 compound libraries against T. brucei. High through put screening (HTS) which resulted into identification of two novel compound series active against the enzyme trypanothione reductase. Series 1 was based on the quinoline scaffold (Figure 18a ) in which compound 2 (Figure 19a) with methylfuran group at R 3 position, Br at 6 th position of quinoline ring and N-methylethanamine group at R 1 position was the most potent and significant TR inhibitor with TR inhibitory activity of 1.1 µM. According to SAR study the replacement of methylfuran with other groups like furan, phenyl, pyridinyl, thiophene led to decrease in activity. Also the replacement of Br with H or F reduced the activity while Cl group retained the activity. At 4 th position the NH and NMe groups were equally active. The alkylamines at R 1 were active whereas the simple alkyl or aryl groups led to inactive compounds. The second series was based on the pyrimidopyridazine scaffold (Figure 18b) , in which the compound 49 ( Figure 19b ) showed promising inhibitory activity of 2.6 µM. In case of series 2 replacement of methyl group at R 1 position by H led to the decrease in activity. At R 2 position methyl and ethyl group showed activity while the H and chain extensions to propyl, butyl and cyclopentane led to decrease in activity. Substituted phenyl and alkenylphenyl group at R 3 position gave the most active compound of the series. Thus these quinoline compounds with promising activity could serve as lead compounds for further development of targeted drugs against African trypanosomiasis. In addition to this synthetic optimization study based on the lead anti-trypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate ( Figure 20 ) was undertaken by Werbovetz KA et al [61] in an attempt to discover new trypanocides with potent in vivo activity targeting TR enzyme. In course of this a total of 53 compounds were evaluated in vitro for their anti-trypanosomal activity and cytotoxicity. The compounds with oxygen atom at 6 th position were the most active compounds in the series, for example compound 9 ( Figure 21 ) showed better anti-trypanosomal activity (0.007 µM) and low cytotoxicity (6.8 µM) than melarsoprol (IC 50 = 0.008µM and cytotoxicity = 7.9µM). Whereas compounds lacking the 6-oxygen atom or bearing an oxygen atom at the 7-position rather than the 6-position were far less potent than those containing an alcohol or acyloxy group at the 6-position. Compounds carrying aliphatic or a 2-phenylacetyl ester side chains were as potent and selective as their benzoylated or acetylated counterparts. However compound 9a was unstable due to auto-oxidation, so the unstable alcohols were esterified to generate prodrug 10a ( Figure 21 ) having promising activity of 0.014µM against these parasites and a selectivity index of 1700. The in vivo evaluation of compound 10a in a murine model of African trypanosomiasis showed good results as the prodrug extended the lifespan of mice infected with T. b. brucei.
Thus compound 10a can serve as lead compound for further investigation of this class of molecules as potential candidates against HAT. Efforts are also be undertaken to further elucidate the metabolism, pharmacokinetics, and the anti-trypanosomal mechanism of action of this novel and promising class of compounds. 
DNA topoisomerases have evolved as an effective drug target in prokaryotic and eukaryotic systems as these enzyme mediate mechanistic interactions such as supercoiling, relaxation, knotting or catenating of DNA double helices. Based on their mechanism of action, topoisomerases can be classified as type I enzymes, which break a single strand of the DNA helix during the catalytic cycle, and type II enzymes, which make double-stranded breaks. On the basis of primary sequence and reaction mechanism, type I topoisomerases are further subdivided into type IA and type IB.
Recently Shapiro TA et al [62] evaluated the activity of indenoisoquinolines (Figure 22) , originally known to have anti-cancer activity, against T. brucei and found that most of the compounds showed in vitro activity at submicromolar concentrations. The compound 12 ( Figure 23 ) with propylamino group at R 6 position and methoxy group at R 2 , R 3 and R 9 was the most active one with in vitro anti-trypanosomal activity of 0.05 µM. The compound also showed good in vivo activity as it delayed parasitemia and extended survival in infected mice. According to structure-activity analysis the compounds with enhanced potency included alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Testing of indenoisoquinolines with promising activity on L1210 mouse leukemia cells revealed all the compounds were more effective against trypanosomes than against mammalian cells. The indenoisoquinolines also showed appreciable water solubility indicating that these compounds have good quality for drug development. These compounds showed their anti-trypanosomal action by multiple mechanisms. The study indicated that they stabilize topoisomerase-DNA complexes in situ and may also impede topoisomerase binding to DNA. These agents markedly inhibited DNA synthesis by interfering with topoisomerase and possibly other DNA-metabolizing enzymes. concentrations in the range of 300 ng/ml up to 900 ng/ml. Some of the DuPont compounds, developed as anti-tumour drugs, were highly active but also showed high cytotoxicity on HT-29 cells. It was observed that the position R 1 and position R 2 in the quinolone core nucleus was not prerequisite for anti-trypanosomal activity and also substitution at R 8 position was not necessary for trypanocidal activity. Thus, based on the result obtained from SAR analysis special attention should be given to R 7 position and the tetracyclic derivatives. The in vivo results of these compounds were very poor, as none of the compounds evaluated produced cure of mice in dose escalation experiment up to 100mg/kg i.p. However no signs of toxicity were observed during the experiments. The in vivo ineffectiveness had not been explained and no drug level determination in the plasma of the treated mice was performed. 
Polyamines are generally involved in growth and differentiation [64] [65] [66] [67] within the cell and their analogs are also used as anticancer agents, antiparasitic agents, antidiarrhoeals, anti-HIV agents, metal chelators, and gene delivery agents. Since the inhibition of the initial polyamine biosynthesis enzyme, ornithine decarboxylase, by DL-α-difluoromethylornithine (DFMO) is toxic to African trypanosomes cells, [68, 69] polyamines can become a promising anti-trypanosomal compound.
DFMO [70] [71] [72] is the most recently developed agent for late stage T. b. gambiense and T. b. brucei sleeping sickness, but has not been active against all strains of T. b. rhodesiense. The major drawbacks of DFMO are its cost, the duration of treatment and its availability. Recent clinical studies have investigated that DMFO can be used in combination with clinically used trypanocides including suramin, nifurtimox and melarsoprol [73, 74] . These combinations result in significant reduction in DFMO dosage and time of administration. Initial clinical study has shown that DFMO + nifurtimox are superior to DFMO + melarsoprol and melarsoprol + nifurtimox. The DFMO + nifurtimox regimen (NECT regimen) allowed reduction in DFMO regimen from 14 to 7 days (56 versus 28 infusions) with a 94% cure rate and are associated with significantly reduced adverse side effects as compared to melarsoprol-based therapy. The success of the combined regimen has been most likely due to ability of DMFO to reduce trypanothione levels and resistance to oxidative stress and the ability of nifurtimox to generate oxidative stress. Recently Gilbert et al [76] designed, synthesized, and evaluated substituted polyamines, carrying 1,3,5-triazine units, as potential anti-trypanosomal drugs. Preliminary results indicated that this route might be successful, and lead structure A (Figure 26 ) was used as a starting point for the synthesis of two series of analogues. In the first series, the influence of structural changes of the central core unit was investigated while in the second series, the effect of additional methyl substituents on the 1,3,5-triazine was studied. The compounds were designed with the intention to selectively target the interior of T. brucei via the P2 amino-purine transporter. In the first series the compound containing the ndodecyl chain as core unit, showed weak activity against T. b. rhodesiense. The compound with n-nonyl chain was the most promising compound and its various analogues were designed by replacing NH 2 groups on the triazine ring with NHMe and NMe 2 groups. Introduction of one or two methyl groups per triazine unit resulted in a 10fold increase in anti-trypanosomal activity. When four methyl groups per triazine unit were introduced, an 80-fold increase in activity was observed. Similarly, replacement of NH 2 group in n-dodecyl chain led to 2-20-fold higher anti-trypanosomal activity for the methylated derivatives. Monosubstituted compounds showed a slight increase in activity against T. b. rhodesiense as compared to the disubstituted compounds. The methylamino substituted triazines attached to the C9-(compound 8c, Figure 27 ) or C12-(compound 8f, Figure 27 ) polyamine precursor via an additional CH 2 linker resulted in most active trypanocidal compounds(IC 50 of 8c = 0.27µM and 8f = 0.18 µM). Beside good activity, the compounds showed poor in vivo activity producing no cure to the infected mice and concentrations greater than 10 mg kg −1 induced severe acute toxicity. The actual mode of action for the reported triazine substituted polyamines remains unclear. So to understand and improve the activities of these compounds, further research has to verify intracellular drug targets and possible metabolic pathways.
Stanislaw FW et al [77] have reported the anti-trypanosomal activity of 5'-deoxy-5'-(E)-(iodomethylene)adenosine, which is a known inhibitor of AdoHcy hydrolase, [78, 79] The 5'-deoxy-5'-(E)-(iodomethylene)adenosine (EIDDHA) and its 6-N cyclopropyl analogue (Figure 28a , b) have shown promising in vitro inhibitory activity (IC50 at 9 and 12 μg/mL) against T. brucei. The utilization of adenosine analogues as anti-parasitics should be explored as a therapeutic paradigm, as it has been shown previously that inhibitors of AdoHcy hydrolase are also very potent inhibitors against the growth of Plasmodium falciparum [79] . This class of 6-N-cyclopropyl adenosine analogues modified at carbon 5', does not exhibit an inhibitory effect on human or parasite forms of the enzyme and displays only marginal antiviral activity in comparison to analogues which have been unmodified at 6-amino position (that are potent inhibitor of AdoHcy hydrolase). Therefore these compounds require further structural modification in order to develop newer analogues with improved activity against T. brucei.
The synthesis of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl] morpholine derivatives by Perozzo R et al [80] resulted in to the discovery of newer class of anti-trypanosomal compounds having stage specific action, as these compounds have shown moderate to very good activity against the blood stage of T. b. rhodesiense. The two compounds, 4-[3-(4-phenoxyphenyl)-1H-pyrazol-5-yl]morpholine (1.0µM) ( Figure 29a ) and 1-[3-(4-phenoxyphenyl)-1H-pyrazol-5-yl]piperazine (1.1 µM) (Figure 29b ) with a pyrazol ring, are the most potent anti-trypanosomals of the series and have same cytotoxicity (61.6 µM), indicating that the pyrazol ring is very important for anti-trypanosomal activity. The substitution of pyrazol ring with isoxazole derivative leads to a six fold reduction in activity as compared to the most potent compound. In addition, substitution with nitrophenyl or aminophenyl also results in strong reduction in activity. The phenoxy ring in the compounds is also important for activity as replacing it by an ethylene group results in nine fold reduction in efficacy. Further substitution with a nitro group or an amino group reduces potency up to 4-fold or 18-fold respectively. The stage specific action of these compounds is unknown. Further optimization of this class of compounds is required in order to lower the cytotoxicity profile of the compound. 
In vitro evaluation of a series of N-, S-, and COOH-blocked glutathione derivatives have been carried out by D'Silva and Daunes [81] against bloodstream form Trypanosoma brucei trypomastigotes, to identify the determinants necessary for activity and for further development into an active lead structure. The results shows that N, S-blocked glutathione diesters are the most active inhibitors of T. brucei parasites and that N-acetyl-S-benzyloxycarbonylglutathione dimethyl ester (compound 5) and the N,S-benzyloxycarbonyl-S-2,4dinitrophenylglutathione diester derivatives (compounds 17-19 & 21) (Figure 30 ) represent lead structures possessing minimal toxicity which potentially could be developed further to yield a therapeutically active agent for the treatment of trypanosomiasis.
Pentamidine, furamidine and its analogues lack oral bioavailability [82, 83] .In addition several analogues of furamidine show excellent activity on intravenous dosing but are ineffective on oral administration [83, 84] . Generally, oral administration is the preferred dosing regime, and hence, prodrug strategies for diamidines that have the potential to overcome their limited oral bioavailability merit attention. The following works have been performed by Boykin DW et al to develop prodrugs.
Boykin DW et al [85] syhthesized and evaluated five O-alkoxyamidine analogues of the prodrug 2,5-bis [4-methoxyamidinophenyl] furan against Trypanosoma brucei rhodesiense in the STIB900 mouse model by oral administration. It was observed that the size of the O-alkyl side-chain determined the metabolic stability of the prodrugs. The prodrugs with the O-methyl analogue were most susceptible to metabolism while the larger O-n-butyl and O-n-hexyl groups were least susceptible to metabolism. The in vivo studies in the STIB900 mouse model for T. b. rhodesiense showed that compounds with an O-methoxy-amidine or O-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. Therefore the O-alkoxyamidine prodrugs, where the alkyl chain is less than three carbons, could effectively be used as prodrugs for amidines. 
In addition to above mentioned prodrug synthesis Boykin DW et al [86] also reported that bis-amidoximes and bis-O-alkylamidoximes of a number of diamidine systems are effective prodrugs. In order to develop orally effective anti-trypanosomal agents, they synthesized these two types of potential prodrugs in the terphenyl series. It was found that compound 10b and 10d ( Figure 32 ) showed good activity in the range of 2 nM. Among these compounds 10b had lower cytotoxicity (6.4μM) profile but had very poor in vivo activity (cured none of the infected animal in STIB900 mouse model). Whereas compound 10d showed excellent in vivo activity, by curing all the infected animals upon oral administration in STIB900 mouse model. To capitalize on the efficacy of these potent dications, other prodrugs that rely on different bioconversion pathways need to be developed. 
Boykin DW et al [87] showed that some of the dicationic guanidine, N-alkylguanidine, and reversed amidine derivatives of fused ring systems have good in vitro activity against Trypanosoma brucei rhodesiense [87, 88] . The dicationic N-isopropylguanidino-9Hfluorene (12c, Figure 33 ) showed promising in vivo biological results by giving 4/4 cures of the treated animals in the STIB900 animal model for African trypanosomiasis. In addition the N-methyl analogue (12a, Figure 33 ) also showed high activity giving 3/4 cures of the treated animals in the STIB900 animal model for African trypanosomiasis. In order to enhance the oral bioavailability, two novel classes of potential guanidine prodrugs were prepared. The N-alkoxyguanidine derivatives 12d and 12e ( Figure 33) were not effective as prodrugs. Whereas the carbamate prodrugs (11c, Figure 33 ), gave promising results with 4/4 cures on oral administration in the STIB900 mouse model. The result showed that these compounds bind strongly to the DNA minor groove, but despite strong bonding these compounds do not have high antiparasitic activity. 
As compared to the last 15 years, there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing. However discovering lead compounds with anti-trypanosomal activity remains a crucial step to sustain the progress achieved till date. The use of Computer-assisted drug design (CADD), since their start, has become increasingly helpful in understanding many aspects of chemical-biological interactions in drug and other scientific research. The latest technological advances (QSAR, structure-based design, ligand-based design, cheminformatics & bioinformatics) are providing a much improved basis for the design of ligands and inhibitors with desired specificity. Recently, computerassisted drug design approaches based on ligand-based and structure-based drug design have been successfully employed to develop new drugs for the treatment of cancer, AIDS and other diseases [89] [90] [91] [92] [93] [94] [95] [96] . QSAR [97] has been widely used for years to provide quantitative analysis of structure and activity relationships of compounds. Our core research group has also performed QSAR analysis of dicationic diphenylisoxazoles [10] . In this study, attempt has been to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity of dicationic 3,5-diphenylisoxazoles that may be helpful in development of potent antitrypanosomal agents against sleeping sickness.
Several statistical expressions have been developed using stepwise multiple linear regression analysis (MLR) and partial least squares (PLS). The best MLR model showed good correlative and predictive ability as shown in following equation 84 has also been obtained. The developed model has been validated by leave-one-out method of cross-validation and prediction of test set. The study indicates that the anti-trypanosomal activity is largely explained by cosmic energy, log P and total lipole descriptors. The QSAR study has reported in present study provides important structural insights, related to anti-trypanosomal activity. Authors have developed a validated and highly predictive model sharing important structural requirement for effective binding of anti-trypanosomal compounds to minor groove of T. b. rhodesiense DNA. The model reported in the study should be helpful in development of new compounds with improved efficacy and oral bioavailability. In line to the developed model authors have also designed some molecules which showed good activity in silico. The further study of these compounds is in progress.
The poor pharmacokinetic profile and toxicity produced by the drugs currently used to treat trypanosomiasis requires an immediate attention for the development of safe, economical and high affinity chemotherapeutic agents to meet the need for this class of drugs. The unacceptable truth is the lack of attention of the government and less interest of pharmaceutical companies in light of less monitory gain in the area of protozoal infection since most of the affected people belong to poorer country. Surprisingly the endeavors of different research group discussed in this review belong to academic institutions. It is felt by the researchers that the treatment for HAT requires a combined approach by government organization, pharmaceutical companies and academic institutions. The most promising fact is that in the last decade several synthetic approaches have been made in the field of development of anti-trypanosomal therapies. Several compounds are being synthesized that yield new compounds for the treatment of HAT, as a result of these synthetic approaches newer leads have been identified, which are under different phase of drug development.
The promising in vitro and in vivo activities of dicationic molecules clearly indicate that aromatic diamidines are the most promising class of compounds for the development of newer drugs against HAT. The most important one is the newer analogous of existing drugs pentamidine (compounds 32, Figure 3e and 66, Figure 3b ) which shows excellent in vivo and in vitro activity (0.004 and 0.001 μM respectively) and also has very good selectivity index against the parasite but is cytotoxic. In an attempt to synthesise newer compounds with reduced cytotoxicity than pentamidine molecules, several benzofuran derivatives have been synthesized. However the replacement of phenoxy fragement of pentamidine with a benzofuran motif has resulted in poor analogues with lower antitrypanosomal activity but improved cytotoxic profile. However the strong activity against T. b. rhodesiense isolates indicates that steps should be taken to initiate further studies of compound 66 and compound 32 which can be further evolved as new lead compound. The newer analogues of furamidine also show promising anti-trypanosomal activity along with lower cytotoxicity than furamidine. Thus strong activity against T. b. rhodesiense and lower cytotoxicity of compound 3 (Figure 13b ) indicates that these compounds should be further evaluated. Thus the DNA minor grove binders are still the most interesting and potential target for the development of newer anti-trypanosomal agents.
Apart from diamidines, polyamines and guanidine also have the potential to give antitrypanosomal compounds. The success of DMFO as polyamine inhibitors has attracted researchers for synthesis and development of agents targeting polyamine metabolism. Recently polyamines carrying 1,3,5-triazine units have been synthesized and evaluated against T. brucei. These compound exhibited good activity but are cytotoxic. Morpholine and dihydroquinolines have also shown promising in vivo anti-trypanosomal activity. In addition to this development of prodrugs, the existing compounds also promise to address the pharmacokinetic related problems, in near future.
Thus excellent in vitro and in vivo activities and high selectivity of aforementioned compounds merit further investigation in order to reduce the cytotoxicity that may result in development of newer anti-trypanosomal drug with reduced toxicity, improved efficacy and pharmacokinetic profile. As the drug discovery and development process is expensive in terms of time and money, the cross application of existing series of compounds with selective trypanocidal activity may be the best prospect to new anti-trypanosomal drugs in the short term. More emphasis has to be put in the field of CADD approaches for development of anti-trypanosomal agents as CADD study reduces the time and cost required for development of newer analogues. NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins BACKGROUND: Nucleolar localization sequences (NoLSs) are short targeting sequences responsible for the localization of proteins to the nucleolus. Given the large number of proteins experimentally detected in the nucleolus and the central role of this subnuclear compartment in the cell, NoLSs are likely to be important regulatory elements controlling cellular traffic. Although many proteins have been reported to contain NoLSs, the systematic characterization of this group of targeting motifs has only recently been carried out. RESULTS: Here, we describe NoD, a web server and a command line program that predicts the presence of NoLSs in proteins. Using the web server, users can submit protein sequences through the NoD input form and are provided with a graphical output of the NoLS score as a function of protein position. While the web server is most convenient for making prediction for just a few proteins, the command line version of NoD can return predictions for complete proteomes. NoD is based on our recently described human-trained artificial neural network predictor. Through stringent independent testing of the predictor using available experimentally validated NoLS-containing eukaryotic and viral proteins, the NoD sensitivity and positive predictive value were estimated to be 71% and 79% respectively. CONCLUSIONS: NoD is the first tool to provide predictions of nucleolar localization sequences in diverse eukaryotes and viruses. NoD can be run interactively online at http://www.compbio.dundee.ac.uk/nod or downloaded to use locally. The nucleolus is a sub-nuclear cellular compartment that is accessible to a large number of proteins since it is not surrounded by a membrane. To date, over 4500 distinct human proteins have been identified from purified nucleoli [1] . The most well-characterized function of the nucleolus is the biogenesis of ribosomes [2] . However, nucleolar proteins are diverse and dynamic, reflecting the central role of this compartment in the cell through its involvement in numerous other key cellular processes and in the cellular response to changing conditions [3] [4] [5] [6] [7] . Indeed, many proteins have been found to localize cyclically or conditionally to the nucleolus [3, 4, 7, 8] .
Although such a large and dynamic volume of cellular traffic likely requires extensive regulation, proteins are often proposed to localize to the nucleolus simply through high-affinity binding to core nucleolar components [6, 9] . Despite this, numerous disparate reports of short nucleolar targeting sequences in proteins have been published over the past 20 years. Many of these sequences can localize non-nucleolar reporter proteins to the nucleolus when fused to them. In an effort to catalogue and systematically characterize these Nucleolar Localization Sequences (NoLSs), we have recently curated the literature and assembled a human NoLS dataset which we subsequently used to train an artificial neural network computational predictor [10] . The predictor considers the protein sequence and JPred predictions of protein secondary structure [11] . When applied to the entire human proteome, it identified thousands of candidate NoLSs, ten of which were experimentally tested and confirmed to target the nucleolus [10] .
Here, we describe NoD, a web server and a commandline program that provides computer predictions of NoLSs in proteins. We also investigate the application of the human-trained predictor in other eukaryotic and viral organisms, demonstrating that NoD can give effective NoLS predictions in a wide variety of species.
The NoD web server provides an easy way to predict NoLSs within a protein sequence. NoD predictions are obtained by entering a protein sequence in fasta format on the NoD webserver http://www.compbio.dundee.ac. uk/nod. Protein sequences are encoded as previously described [10] . Briefly, sliding windows of size 13 are sparsely encoded in a binary format using a reduced alphabet of size 12 for submission to an artificial neural network (ANN). The current implementation of NoD uses a local version of Batchman from the Stuttgart Neural Network Simulator [12] and the human-trained NoLS prediction model developed previously [10] to provide the prediction for each encoded subsequence. The Batchman output is then processed and NoLSs are predicted if the average score output by the ANN of 8 consecutive windows is at least 0.8 [10] . Finally, the prediction is displayed as shown in Figure 1 if at least one NoLS is identified. Otherwise, the user is informed that no NoLS is predicted in the input protein. As shown in Figure 1 Example of NoLS prediction returned by NoD. If at least one NoLS is predicted in a protein, NoD returns an output page that displays the sequence and position of the predicted NoLSs, the full-length protein sequence as entered by the user with the NoLSs in red and a graph showing the average NoLS prediction score for every 20-residue window in the protein. The region shown in pink in this graph is the NoLS candidate segment region and represents the range of scores within which a 20-residue segment is predicted to be a NoLS. -the sequences of the predicted NoLS(s) are first enumerated -the full-length protein sequence is displayed with the predicted NoLS(s) shown in red -finally, a graph is presented of the NoLS windowbased score [10] as a function of position in the protein sequence.
The NoLS window-based score graph can be useful to guide experimental design of nucleolar targeting. The graph gives an overview of the entire protein and shows the proportion of the protein with putative nucleolar targeting capabilities as well as regions of the protein that are near the cut-off threshold and therefore almost predicted as NoLSs.
When entering a protein sequence, the user is provided with the option of also running JPred secondary structure predictions [11] to include as input to the NoLS neural network. If JPred is selected, the accuracy of prediction is slightly higher [10] but the computation time is increased.
For users who want predictions for whole proteomes there is a command line version of NoD called clinod. Clinod produces the same results as a web server but it is more suitable for processing of multiple sequences and is convenient to use within software pipelines.
Clinod requires Java 6 and the Batchman executable from the Stuttgart Neural Network Simulator [12] to run. Clinod accepts the list of FASTA formatted sequences from an input file and outputs the predictions to a file or the console. By default the following output is produced for each sequence-the name of the sequence, the number of NoLSs predicted, the start and the end positions and the sequences of each predicted NoLS. However, for better integration with other bioinformatics tools, many more output options are supported. For example, the input sequences can be cleaned (stripped of ambiguous characters), and output along with the prediction results and sequences with no predicted NoLS can be omitted from the output. Various output options are described in Table 1 but for a detailed description of the clinod switches please refer to Additional file 1.
Finally, for users preferring to run and visualize their predictions locally, there is a virtual appliance version of NoD, which can easily be deployed on a variety of operating systems by a non-specialist user. The virtual appliance version of NoD offers the same functionality as our public server, with the exception of JPred predictions. However, in the near future we intend to release a version which will support JPred. 
Because more NoLSs have been reported in human than in all other organisms combined, the NoLS predictor was originally trained and tested only on human sequences [10] . More precisely, as described previously [10] , the predictor was trained on a manually curated positive set of 46 human experimentally validated NoLSs and a negative set consisting of several hundred human proteins chosen because they are believed not to localize to the nucleolus. After training, ten of the NoLS predictions were chosen for experimental validation and all were confirmed as positives [10] .
However, the prediction of NoLSs is relevant in all eukaryotes and in particular in their viruses, many of which encode proteins that localize to the nucleolus of their host cells [13] . To investigate whether the humantrained predictor can be applied to other organisms, we searched the literature to find examples of NoLSs that have been experimentally identified in other organisms. In total, we collated 31 eukaryotic or viral NoLSs (including 6 human NoLSs that had not been used for training or testing previously) which are listed in Table  2 , along with the position of the experimentally determined NoLSs. Sequences were filtered to remove redundancy within this dataset and redundancy with the original training set as described previously [10] . The full-length sequences of these NoLS-containing proteins were then passed through the NoLS predictor. As with the original NoLS paper [10] , only experimentally validated NoLSs of length less than 50 residues were considered for testing. This focuses the testing on those NoLSs that have been most confidently identified by experiment and reduces the likelihood that we are dealing with signal patches (ie signals formed from residues distant in the primary sequence but that come into close proximity in the folded molecule). We considered NoLSs as correctly-predicted if the region of overlap between the predicted NoLS and the experimentally determined NoLS covered at least 60% of the shortest of the two molecules. In many cases, the predicted NoLS region is entirely contained within the experimentally determined NoLS or vice versa. Details of the predictions, including the position of predicted NoLSs, are given in Table 2 and a summary of the prediction accuracy is given in Table 3 . As shown in Table 3 , mammalian NoLSs and their viral counterparts are well predicted, with sensitivity and positive predictive values ranging from 0.75 to 1.0. This is not surprising because of the close evolutionary distance between humans and other mammals and the constant adaptation of their viruses. Amongst the nonmammalian proteins considered, the Dictyostelium discoideum protein investigated has two NoLSs, one of which is well-predicted. The NoLS that was not correctly identified consists of only 7 amino acids and is likely too short for the predictor to find. The two mollusc NoLSs are entirely well-predicted but low numbers of examples in this group of organisms prevents robust conclusions. Similarly, plant and plant-infecting virus NoLSs are generally well-predicted but also suffer from small numbers of examples. However, the humantrained predictor is entirely incapable of identifying the NoLSs experimentally detected in trypanosomes. This agrees well with experiments in which the NoLS of a Trypanosome brucei protein, ESAG8, was fused to a fluorescent reporter protein and tested for nucleolar localization in human cells. With an intact trypanosome NoLS, the fusion protein was found to be nuclear but not nucleolar in human cells [14] . This observation and our predictions suggest that nucleolar targeting mechanisms differ significantly between humans and trypanosomes and are not interchangeable. Although a larger sample would be needed to confirm this difference, trypanosomal nucleolar targeting mechanisms might represent good drug targets. While no experimentally generated negative dataset is available for testing the predictor in non-human organisms, we note that the non-NoLS regions of NoLS-containing proteins provide such a set. For example, the human adenovirus 2 protein VII encodes three basic regions at positions 47-54, 93-112 and 127-141 which represent possible nuclear/nucleolar localization sequences [15] . Deletion constructs demonstrate that only the 93-112 segment targets a reporter protein to the nucleolus [15] . This segment matches very closely the NoD NoLS predictions (see Table 2 ), providing not only an accurate test example but also perfect negative controls (the two other basic regions are not predicted as NoLSs). Thus, the positive predictive values shown in Table 3 give an indication of the false positive rate of prediction in different organisms. However, while some false positives probably represent prediction errors, others might have occurred because NoLSs were not experimentally mapped with enough precision or more than one NoLS exists in the protein. Larger experimental datasets will undoubtedly help to clarify this problem.
Of the 31 eukaryotic and viral NoLSs considered for independent testing, 22 were correctly identified, resulting in an overall sensitivity of 71%. In addition, 6 non-NoLS regions were also identified as positives (and thus are considered here as false positives) yielding an overall positive predictive value of 79%. Finally, of the 27 proteins considered, 6 were predicted to encode NoLSs in regions not experimentally shown to harbour a NoLS resulting in a specificity of 78% (although we note that some of these false positives might represent NoLSs that have yet to be experimentally identified).
NoD is currently the only predictor capable of providing and visualizing NoLS predictions for protein sequences.
The web server takes a protein sequence as input and returns the positions and the sequences of the predicted NoLSs. It also draws a graph of the predicted scores for each residue of the sequence.
The command line NoD takes the list of FASTA formatted protein sequences as an input, and for each sequence outputs the number of detected NoLSs, their positions in the full-length sequence and their sequences. However, the command line predictor output is highly customisable and can be adjusted to the user's needs. Finally, the virtual appliance version of the predictor allows the deployment and running of the predictor locally, eliminating data privacy issues.
Cross-species testing shows NoD to perform best for mammalian and mammalian-infecting viral proteins but preliminary results suggest sequences from molluscs, amoebae, plants and their viruses are also wellpredicted.
• Project name: NoD (Nucleolar localization sequence Detector) • Project home page: http://www.compbio.dundee. ac.uk/nod • Operating system(s): Platform independent • Programming language: Java • Other requirements: The command line version requires Java 6 or higher, and the SNNS Batch Interpreter V1.0 [12] . The virtual appliance version requires freely available VMware Player 3.1 [16] or higher, commercial VMware Fusion (for Mac users) [17] or the freely available Oracle VirtualBox v3.2.12 [18] • License: Apache 2 • Any restrictions to use by non-academics: no restrictions
Additional file 1: NoD command line manual. The additional file describes the usage of the NoD batch predictor command line utility. Avian Influenza Risk Perception and Preventive Behavior among Traditional Market Workers and Shoppers in Taiwan: Practical Implications for Prevention BACKGROUND: Avian influenza (AI) can be highly pathogenic and fatal. Preventive behavior such as handwashing and wearing face masks has been recommended. However, little is known about what psychosocial factors might influence people's decision to adopt such preventive behavior. This study aims to explore risk perception and other factors associated with handwashing and wearing face masks to prevent AI. METHODOLOGY/PRINCIPAL FINDINGS: An interviewer-administered survey was conducted among 352 traditional market workers and shoppers in Taiwan between December 2009 and January 2010. Factors associated with the recommended AI preventive behavior (i.e., when in a traditional market, wearing a face mask and also washing hands after any contact with poultry) included: having correct knowledge about the fatality rate of AI (adjusted odds ratio [AOR] = 4.18), knowing of severe cases of AI (AOR = 2.13), being informed of local AI outbreaks (AOR = 2.24), living in northeastern Taiwan (AOR = 6.01), having a senior high-school education (AOR = 3.33), and having a university or higher education (AOR = 6.86). Gender interactive effect was also found among participants with a senior high-school education, with males being less likely to engage in the recommended AI preventive behavior than their female counterparts (AOR = 0.34). CONCLUSIONS/SIGNIFICANCE: Specific information concerning AI risk perception was associated with the recommended AI preventive behavior. In particular, having correct knowledge about the fatality rate of AI and being informed of severe cases and local outbreaks of AI were linked to increased AI preventive behavior. These findings underscore the importance of transparency in dealing with epidemic information. These results also have practical implications for prevention and policy-making to more effectively promote the recommended AI preventive behavior in the public. A total of 520 cases of avian influenza (AI) had been reported around the world, resulting in 307 deaths, with a case fatality rate of 59.0% [1] . The AI virus has been found in domestic poultry (e.g., chickens, ducks, geese), wild birds [2] , waterfowl and shorebirds [3] . An extensive review indicated that the AI virus could be transmitted through direct contact with infected poultry, including holding diseased or dead poultry, slaughtering, defeathering, or preparing sick poultry for cooking [4] .
Taiwan experienced sporadic AI outbreaks in 2004 and most recently in 2009, including major local outbreaks in Kaohsiung (a region located in southern Taiwan) in 2008. Limited information released in government reports [5] later confirmed that these outbreaks were caused by H5N2 virus. Although there had not been any highly pathogenic avian influenza (HPAI) H5N1 cases reported in Taiwan, yet considering the geographical location of Taiwan being an important stopover for migrating birds [6] and previous H5N1 outbreaks in neighboring Asian countries such as Thailand and China [7] , plus increasing travel and direct transportation links with other countries, Taiwan is at risk for HPAI outbreaks as well. The most worrisome scenario is that human-to-human transmissions may begin to take place if there is a change in the viral genome [8] , and according to a relatively conservative estimate by the World Health Organization (WHO), such transmissions may cause 2 million to 7.4 million deaths [9] .
In Taiwan, people have the habit of shopping at traditional markets for live poultry [10] , and such traditional markets with live chicken for sale provide a possible AI viral reservoir [11] , thereby placing traditional market workers and shoppers at risk for contracting AI. Poultry are usually selected and purchased by shoppers while they are still alive and slaughtered on site as shoppers in Taiwan commonly believe that live poultry preserves the freshness [11] . During the purchasing process, shoppers may come into contact with live poultry, thereby exposing shoppers to risk of contracting AI, and thus it is important that shoppers wash their hands after any contact with poultry. Normally, a poultry vendor would not sell other foods; however, in a traditional market, a poultry stand could be adjacent to any other food stands without any partitions separating them. Notably, AI viruses can also be transmitted to humans through the particles raised up by the movement of the poultry [12] , and therefore, regardless of whether the shoppers are purchasing live poultry, they are advised to wear a face mask while in a traditional market. Given the modes of AI transmission as described above, WHO [13] and Taiwan Centers for Disease Control (Taiwan CDC) [14] both recommended washing hands after any contact with poultry and using a face mask when coming into contact with poultry to prevent AI infection and its spread. Handwashing [15, 16] and wearing face masks [17] have been documented as effective preventive measures against respiratory disease in community settings. In addition, these two preventive behaviors are relatively easy to practice. Taken together, both handwashing and wearing face masks could be a cost-effective way of preventing AI in the general public. Therefore, an enhanced understanding of the factors associated with AI preventive behavior could inform renewed prevention efforts to more effectively promote the recommended preventive behavior in different target populations.
Previously, studies have been conducted in various countries in regard to AI. For example, a study in Turkey investigated AI knowledge and anticipated attitudes in the general population; however, this cross-sectional survey only examined the anticipated preventive measures rather than actual preventive behaviors [18] . Another study in Italy focused merely on poultry workers, exploring the relationships of their knowledge, attitudes, and compliance with precautions at work, such as self-reported use of face masks and gloves [19] . An earlier study reported its findings in a letter to the editor, presenting regional differences in AI knowledge, risk perceptions, and AI-related behavior changes among Laotians after HPAI outbreaks [20] . Further, a telephone survey in the Hong Kong general population examined participants' AI risk perception in relation to their live chicken purchasing behavior [21] . Similarly, a study in Taiwan conducted by marketing and business management researchers assessed consumer knowledge and risk perceptions of AI in association with chicken consumption behavior, instead of AI preventive behavior under the threat of AI [11] .
However, relatively little is known about the recommended handwashing behavior, especially in combination with face mask wearing behavior, to prevent AI. Therefore, considering the modes of AI transmission taking place at traditional markets in Taiwan as reviewed earlier, the current study aimed to estimate the prevalence of the recommended AI preventive behavior (i.e., when in a traditional market, wearing a face mask and washing hands after any contact with poultry) among traditional market workers and shoppers in Taiwan and examined their knowledge and risk perception in relation to AI preventive behavior.
Participants of this cross-sectional study were market workers and shoppers in traditional markets located in northeastern and central Taiwan. As described in the Introduction, there were major AI outbreaks in Kaohsiung (in southern Taiwan), and therefore, the current study aimed to explore the risk perception about AI, as well as the recommended AI preventive behavior, among traditional market workers and shoppers in central and northeastern Taiwan, where there have not been reported AI outbreaks. Accordingly, two traditional markets were selected in central Taiwan and two others were selected in northeastern Taiwan. Since this study sought to examine simultaneously both groups of participants (i.e., traditional market workers and shoppers), we decided to interview market workers and shoppers with a one-to-one ratio. With this particular purpose in mind, purposive sampling was employed in traditional markets: market workers were interviewed by trained interviewers during their breaks; because there were far more market shoppers than workers, market shoppers were randomly selected for an interview at the market entrance if they happened to step into the market when an interviewer just became available to conduct a survey after completing the previous interview. The interviewer-administered survey using a structured questionnaire was conducted in December 2009 through January 2010. A total of 352 anonymous interviews were completed with a response rate of 95.1%. Each interview was conducted by a trained interviewer and took the participant 5-10 minutes to complete the questionnaire. Traditional market workers and shoppers were first approached and informed of the study's goals and procedures by our interviewers to obtain verbal informed consent before each interview. Every attempt made to approach potential participants, be it successful or unsuccessful, was documented in order to calculate the response rate. We also provided a gift worth approximately US $1 as incentives to increase the response rate. The study protocol and verbal consent procedure were reviewed and approved by the Institutional Review Board of the National Taiwan University College of Public Health.
The survey collected sociodemographic information such as the participant's age, gender, region of residence, education, and whether the participant was a market worker (yes/no). This study also classified each participant's risk of AI by type of work: 1) nonmarket worker (i.e., shopper), 2) low risk market worker (e.g., flower vendor, vegetable vendor, cleaner, administrative staff), 3) medium risk market worker (e.g., pork vendor, beef vendor, seafood vendor, mutton vendor, cooked poultry vendor), and 4) high risk market worker (e.g., chicken butcher, chicken vendor, poultry organ vendor). It is noteworthy that the AI risk classification above takes into account the nearness in space to live poultry. For example, meat and seafood stands are normally located in the same section as poultry stands in a traditional market, and hence are closer to poultry vendors than are flower and vegetable vendors. As such, pork vendors, beef vendors, etc. are classified as medium risk market workers, whereas flower vendors and vegetable vendors are classified as low risk market workers.
Further, this study assessed the participant's knowledge about AI such as its transmissibility and fatality rate (Table S1 ) [31] . The survey also evaluated the participant's risk perception of AI, including whether the participant anticipated an AI epidemic in Taiwan, whether the participant knew about severe cases of AI, and whether the participant knew about AI outbreaks in Kaohsiung (a region located in southern Taiwan). The variables regarding knowledge and risk perception of AI were binary (e.g., yes/no, correct/incorrect). The outcome measure of recommended AI preventive behavior was assessed by asking: ''When you are in a traditional market, do you wear a face mask and also wash your hands after any contact with poultry (yes/no)?'' Notably, each participant's response to this behavioral outcome measure was validated by the interviewer through direct observation in regard to the face mask wearing aspect of the preventive behavior. In other words, since all interviews were conducted in traditional markets, only participants who were wearing a face mask at the time of the interview would be coded as adopting the recommended AI preventive behavior as defined in this study. Several theoretical models have been used for understanding protective health behaviors, including the Health Belief Model (HBM) [22] [23] [24] . The risk perception measures in this study were derived from theoretical constructs of the HBM, which posits that risk perceptions such as perceived severity and perceived susceptibility are associated with adoption of healthrelated behaviors [25] . This model has also been used in a recent AI study [26] .
Descriptive statistics were first examined for sociodemographic data. Sociodemographic variables, knowledge, and risk perception of AI were cross-tabulated with AI preventive behavior, and x 2 comparisons were performed to test for group differences between participants who practiced and those who did not practice the recommended AI preventive behavior. T-test was used for comparing the ages of participants who practiced the recommended AI preventive behavior and those who did not. Then, variables with significant x 2 or t-test results were included as candidates in subsequent stepwise logistic regression modeling. The final model was adjusted for age and gender as control variables. All statistical analyses were carried out with SPSS (version 17) and P,.05 indicated statistical significance.
The sociodemographic characteristics of the participants (n = 352) are as follows: the mean age was 43.9 years; 62.5% were female; 59.4% lived in central Taiwan; 18.2% had a degree from university or above, and 41.2% had a senior high-school diploma. About half (49.7%) of the participants were shoppers; 22.7%, 18.2%, and 9.4% were market workers at low, medium, and high risk for AI, respectively. Slightly more than half (52.6%) of the participants adopted the recommended AI preventive behavior. Table 1 shows the associations of sociodemographic characteristics and AI risk perception with the recommended AI preventive behavior. Younger participants were more likely than older participants (mean age = 42.0 vs. 45.6, t = 3.00, p = .003), females were more likely than males (50.0% vs. 43.2%, x 2 = 1.54, df = 1, p = .22), and participants who lived in northeastern Taiwan were more likely than those in central Taiwan (62.9% vs. 36.8%, x 2 = 23.19, df = 1, p,.0001) to practice the recommended AI preventive behavior. Furthermore, participants with a university or higher degree (70.3%) were the most likely to adopt the AI preventive behavior, followed by those with a senior high-school diploma (53.8%) and those with a junior high-school or less education (30.8%) (x 2 = 37.71, df = 2, p,.0001). Finally, compared with their counterparts, participants who had correct knowledge about AI fatality rate (68.8% vs. 39.8%, x 2 = 23.16, df = 1, p,.0001), who anticipated an AI epidemic in Taiwan (54.8% vs. 41.6%, x 2 = 6.08, df = 1, p = .01), who knew about severe cases of AI (55.0% vs. 26.1%, x 2 = 22.78, df = 1, p,.0001), and who knew about AI outbreaks in Kaohsiung (63.5% vs. 37.2%, x 2 = 23.20, df = 1, p,.0001) were more likely to practice the recommended AI preventive behavior. Table 2 presents the final multivariate logistic regression model for covariates of AI preventive behavior. Participants who lived in northeastern Taiwan were 6 times as likely as those in central Taiwan to practice the recommended AI preventive behavior (adjusted odds ratio [AOR] = 6.01, 95% confidence interval [95% CI] = 3.40-10.61). Gender did not have a statistically significant effect; however, male gender was found to interact with senior high-school education (AOR = 0.34, 95% CI = 0.12-0.98). Hence, females with a senior high-school diploma were more than 3 times as likely (AOR = 3.33, 95% CI = 1.56-7.07), and participants with a university or higher degree were nearly 7 times as likely (AOR = 6.86, 95% CI = 2.60-18.06) to adopt the AI preventive behavior, compared with their counterparts with a junior high-school or less education. Further, participants who had correct knowledge about AI fatality rate were more than 4 times as likely (AOR = 4.18, 95% CI = 2.25-7.75), those who knew about AI severe cases were approximately 2 times as likely (AOR = 2.13, 95% CI = 1.13-3.99), and those who knew about AI outbreaks in Kaohsiung were more than 2 times as likely (AOR = 2.24, 95% CI = 1.28-3.92) to practice the AI preventive behavior, compared with their counterparts.
An earlier study reported that greater knowledge of AI (i.e., knowing correctly the modes of AI transmission, occupational groups at risk for AI infection, and proper AI preventive measures) among poultry workers was associated with increased odds of adopting preventive measures, including wearing protective clothing and face masks [19] . Adding to the literature, our study further found that, compared with participants who misperceived that AI fatality rate is lower than that of pandemic H1N1, those with correct knowledge were more than 4 times as likely to practice the recommended AI preventive behavior. Another study, conducted among adults in the general population, also reported that participants who had correct knowledge about AI were more likely to practice AI preventive behavior [27] . While these prior studies also identified knowledge of AI, such as modes of AI transmission, to be a significant factor for increased preventive behavior, our study discovered that to promote the recommended AI preventive behavior, it is crucial to inform the public specifically of the AI fatality rate. In addition, unlike most previous research [18] [19] [20] which focused on poultry workers or the general public, this study expanded this line of research by examining AI preventive behavior and related factors among traditional market workers and shoppers.
This study also found that participants with greater risk perception of AI (i.e., those who knew about AI severe cases and those who knew about AI outbreaks in Kaohsiung) were more likely to practice the recommended AI preventive behavior. The greater adoption of precautionary measures among these participants with higher risk perception in the current study could be explained by their possibly elevated anxiety levels as posited in an earlier study [28] . These findings further underscore the importance of transparency in dealing with epidemic information, specifically, AI severe cases and local outbreaks, if any, as in this study. Accordingly, future public service announcements or pandemic control initiatives should consider disseminating the aforementioned specific information to the public in the face of an AI epidemic.
Participants living in northeastern Taiwan were found to be much more likely to practice the recommended AI preventive behavior than those living in central Taiwan in the current study. Such regional differences in preventive behavior were also reported in a Laotian population [20] and were attributed to different participant characteristics in urban and rural areas. In light of such findings, the present study also took into account gender, age, education, and other covariates in the multivariate regression model. However, the regional differences still remained. A possible explanation is that participants in northeastern Taiwan, which is more rural and has fewer healthcare resources than central Taiwan, may choose the relatively easy and cost-effective measures such as wearing face masks and washing hands to protect against AI. Another study in China also found differences between Guangzhou and Hong Kong in regard to participants' AI risk perception and live poultry purchase [29] ; while its outcome variable of interest was not AI preventive behavior, regional differences were present and were ascribed to cultural differences. Notably, the current study found that participants who lived in rural areas were more likely to practice the recommended AI preventive behavior (i.e., both face mask wearing and handwashing) than those who lived in urban areas; however, prior research has found that people living in rural areas were more likely to practice the AI risky behavior (i.e., live poultry purchase) than those living in urban areas [29] . Caution needs to be exercised in interpreting such inconsistency, because different behaviors could have different determinants, and therefore, it is possible that these two studies found inconsistent results due to the different natures of these behaviors in question. These findings suggest that future studies may investigate potential cross-national differences in AI preventive behavior, and that qualitative research is also needed to explore regional differences caused by cross-cultural differences as well as other possible causal mechanisms.
Gender differences in the practice of protective behavior against emerging infectious diseases, including SARS and AI, have been explored in prior research, although the results have been inconclusive. For example, a review article on handwashing practices during and after SARS outbreak indicated that females in general were more likely than males to adopt the protective behavior, suggesting that females might be more health conscious and risk averse, although the reported differences were not always statistically significant [16] . Similarly, in a limited number of AI studies which included gender as a variable, its effect on the AI preventive behavior was generally found to be not statistically significant [e.g., 19, 26] . On the other hand, higher education levels have repeatedly been reported to be associated with increased knowledge and intention to adopt the recommended AI preventive behavior [e.g., 18, 19, 26] . Consistent with these findings, our study also found participants with higher education levels to be more likely to practice the AI preventive behavior. Interestingly, while gender difference was not statistically significant in the current study, we found significant interactive effect between gender and education among participants with a senior high-school education, males being less likely to adopt the recommended AI preventive behavior than their female counterparts. To our knowledge, such findings have not been reported in previous research and warrant further investigations to elucidate possible mechanisms.
Moreover, the x 2 comparison in this study found an alarming pattern that market workers at higher risk for AI appeared to be less likely to adopt preventive behavior than shoppers and other market workers with lower risk for AI. Further, an ancillary analysis (data not shown) indicated that these high-risk market workers also had a significantly lower level of education, which was associated with lower compliance with recommended preventive behavior. Taken together, more attention should be paid to this group of high-risk market workers. It is worth noting that the aforementioned x 2 comparison of AI preventive behavior was not statistically significant, possibly owing to relatively smaller cell counts of high-risk market workers and hence reduced statistical power. Therefore, future studies may consider increasing not only the total sample size but also the number of high-risk market workers so as to confirm the above-noted pattern.
Limitations of this study include the potential reverse causality due to the cross-sectional design; however, a number of variables identified to be significantly associated with AI preventive behavior (e.g., region of residence and education) are likely to precede temporally the outcome measure, thereby lending additional support to our explanations discussed earlier. Also, combining wearing face masks and handwashing as the outcome variable without assessing them separately could be a limitation of this study because determinants of these two practices could be different. On the other hand, however, in view of the modes of AI transmission through contact and air particles, practicing both face mask wearing and handwashing behaviors could provide better protection against AI infection. Another potential limitation is that this study was not based on national data but on data from northeastern and central Taiwan; yet, regional differences were uncovered. Therefore, future studies should consider drawing a national sample to explore other possible cross-regional differences in Taiwan. In addition, cross-national comparisons may also reveal interesting differences across various countries and cultures in preventive behaviors associated with such emerging infectious diseases. Lastly, while self-efficacy (a construct borrowed from Bandura's Social Learning Theory) was not added to the HBM until 1988 [30] , it has increasingly become an important construct in the HBM but was not included in our study. Hence, future research should consider measuring all other HBM constructs, including self-efficacy, when examining AI preventive behavior. 
In conclusion, this study found that specific information concerning AI risk perception was associated with the recommended AI preventive behavior. In particular, having correct knowledge about the fatality rate of AI, and being informed of severe cases of AI and local AI outbreaks, were linked to increased AI preventive behavior. These findings have important implications for future practice as they could inform policy-making and renewed prevention efforts to more effectively promote the recommended AI preventive behavior in the public.
Table S1 Questions used to measure AI knowledge, risk perception, and preventive behavior. (DOC) In the name of the greater good? In the event of a pandemic that poses widespread infection and high death rates, the utilitarian mandate to ‘reduce harm’ is the relevant moral value that trumps other ethical considerations. The primacy of a utilitarian approach dictates that those who are in a position to assist the cessation of the most serious outbreaks in whatever role they may have, must be present to provide their services, and those who administer health care must also be present to ensure that all responders are supported and protected to the highest degree. In the event of a pandemic that poses widespread infection and high death rates, the utilitarian mandate to 'reduce harm' is the relevant moral value that trumps other ethical considerations. The primacy of a utilitarian approach dictates that those who are in a position to assist the cessation of the most serious outbreaks in whatever role they may have, must be present to provide their services, and those who administer health care must also be present to ensure that all responders are supported and protected to the highest degree.
Public health authorities in many economically advantaged nations are bracing themselves to face future pandemics that will harm large numbers of citizens. Modern medical horrors such as Monkeypox or the much-feared future mutations of Avian Influenza (H5N1) are mentioned in the same breath as virulent strains of influenza, as a danger to our 'way of living.' Far beyond sickness and large numbers of death, an outbreak of one of these pandemics poses a real threat to long-term health, as well as to the social and economic well being of significant percentages of our surviving population. 1 While confronting issues brought forth by a pandemic, the fundamental nature of 'public health' and its focus on the welfare of a population demands special attention to utilitarian considerations of promotion of the greatest goodFin this case, healthFas well as the limitation of illness and death in the 'worst-case' scenarios posed by the most lethal of pandemics. Of particular interest to this paper are questions related to the obligation of health-care workers (HCWs) to report to work in the face of heightened immunological threat and whether those same workers should have greater access to immunizations and treatments than should non-HCWs.
The fundamental feature of the ethical theory of utilitarianism states that moral behavior is that which promotes good and minimizes harm. 2 In writings based on public health, utilitarianism is widely recognized as a fragment in the ethical 'scheme' of public health, 3 but it is not afforded a stronger role for two primary reasons: first, considering its extreme position, utilitarianism is morally problematic, 4 as it could literally permit anything in the name of the 'greatest good to the greatest number,' and second it is virtually impossible to live a moral life under the most extreme forms of utilitarianism, because the obligations are too difficult to discern (the 'what' of promoting the good) and impossible to execute (the 'how'). 5 Utilitarianism, in a moderate form, used in public health ethics, means that our actions and policies should be focused on increasing the total 'net' goodness rather than an average 'net' good for each person. The institutions of individual rights and the recognition of patient autonomy are not contradictory to this, but are believed to serve the overall good, as individual benefit increases the total good, and serves as a preventative measure of unjustified majoritarian actions against smaller groups.
This model of utilitarianism is evident in many aspects of public healthFnot only through health-promotion projects that encourage the otherwise illness-free individuals to take up a more healthful diet and exercise regimen but also through harm-reduction programs, in which people with negative health behaviors such as abuse of drugs or dietary fats are aided to eliminate, or at least minimize the harm they cause to those around them.
In everyday practice, the force of this utilitarian aspect has a supportive role along with other ethical elements of public health practice, and presents a balanced moral justification for all actions undertaken in accordance with this practice. 6 However, I contend that there must be an 'escalator clause' in the utilitarian aspect that suggests that in the event of an extensive threat to the existence of a population, the force of this utilitarian aspect becomes the primary consideration in proportion to the threat. Therefore, the greater the threat, the greater the moral force of utilitarianism in making public health decisions. This also entails that the greater the threat, the greater the moral impetus to minimize the harm to the population.
Obligations to minimize harm and promote the goods of public health are not particularly controversial in times of relatively stable 'good-health' measures among the populace. The more troubling question emerges from the scenario in which promoting health and minimizing illness and death demands more from HCWsFhow can, or should, we compel HCWs to attend to their duties in the event that a highly lethal form of communicable disease should start spreading? 7 Although current debates focus on questions of duty, and how much personal risk invalidates that commitment, utilitarian aspects of that obligation are not given enough weight in the debate. In many of the debates, the question of risk is posed in terms of how we do not expect a trained 'first responder' to recklessly endanger his or her life to save the life of another. The classic story of the lifeguard is offered as exemplar: a lifeguard is not expected to rescue a drowning swimmer if a shark is clearly present. 8 Although this statement seems reasonable, it does not justify itself. By contrast, the consideration of the utilitarian aspect makes the point that in attempting to save a life, two are likely to be lost, thus propagating a greater total harm. The same holds true for the example of firefighters rushing into a house badly damaged by an active fire. Although there may be a life on that second floor to save, we do not expect any number of firefighters to sacrifice their lives for the doomed soul because the loss of many, including the original life in peril, is a maximization of harm, when harm should be minimized. When you control for the risks involved, such as using precautions to assure a level of safety for the rescuers, such as shark nets for the lifeguard, or safety gear for the firefighters, then the obligation to assist comes back into full force, as the potential for greater harm is manageable. 9 It is the variable of risk, which creates variable demands on those whose duty it is to care for the population in times of crisis. We consider not only the risk to the obligated but also a question of the scope of risk to the population. In academic and public debates regarding the compulsion to attend to duty in the face of danger, one fallacy has been allowed to stand: the notion that exposure to a pandemic can be avoided if one simply does not come to his or her job as a HCW. Although it is true that working in a hospital during times of influenza outbreak puts one at a greater risk for contracting the illness, 10 the more widespread the outbreak, the more people become sick, and the more likely the 'stayat-home' HCW will become sick even after having avoided contact in the course of his or her duties. We could reasonably state that, by virtue of staying home during a time of need for his or her service, the HCW improves the odds that he or she will contract this illness outside professional practice as part of the greater number who will be exposed. Another feature of the argument offered to defend dereliction of duty is to suggest that this risk that the HCW takes with his or her own health is a fixed variable, and thus should be considered as an exception to duty. On the contrary, it is a common feature of the infection-control literature that states that doctors and nurses are overwhelmingly neglectful toward their own basic infection-control protocols. 11 Therefore, the threat is not a fixed variable, but one that is actually quite within the scope of the control of a HCW. Ethically, one cannot willfully or negligently enhance the exceptions to duty. At the same time, it is an obligation of the management to ensure that diligent HCWs are equipped to do all they can to reduce their risks. During the SARS crisis in Toronto, health-care administrators did not effectively communicate which precautions should be undertaken by HCWs to protect themselves. 12 It bears mentioning that once clear direction could be given about the type and execution of masking procedures, the intrahospital transmission of SARS decreased to 0%. 13 This fact speaks to the issue of risk, as the non-transmission of SARS correlated with the increased attentions of management and staff to infection-control precautions and the provision and use of proper equipment. 14 When we speak in terms of risk and pandemics from the utilitarian perspective discussed herein, we recognize that it is completely nonsensible to sacrifice highly trained HCWs by rushing them ill equipped into dangerous situations. Much as with the example of firefighters and the unsafe burning house, we find it morally unacceptable to treat them as disposable, because of the singularity of their lives and their right to exist as individuals. There is also the detriment we would cause in an event such as a pandemic by losing the people trained to save us to the very threat they were trained to save us from. By that same logic, it could be argued that HCWs should have first access to available and medically accepted vaccinations by virtue of the fact that those HCWs are absolutely essential to our survival. The fear of an Avian Influenza outbreak brought with it much debate about scarce Tamiflu supplies and giving HCWs preferential access. 15 However, the added value of a HCW is the fact that he or she will be facing the greater risk by virtue of faithful and responsible execution of his or her duty, and if this is trueFand we have seen from the example of SARS that it is not always the case that HCWs exercise due diligence or face unmanageable risks of infection simply by being 'on-site'Fthen we should do more to protect them. Nevertheless, if the claim is that they can excuse themselves from duty because of risk, then we excuse ourselves from privileging their protection, through the preferential access to measures such as Tamiflu. The same should be true for access to vaccines or treatments: those who are compelled into service to defend the overall health of a society at tremendous risk should be first in line, as their opportunity for infectionFand to act as a vector for infection both within and outside their health-care facilitiesFmeans that the greater good is served by privileging their access to prophylaxis. A common objection to this comes from the perspective of social justice. The objection would point out that those who are least able to prevent their own infection, such as those from the lower socioeconomic classes, retirees and pensioners, and other vulnerable groups, would be denied access to the protections and treatments that are going to HCWs whoFto varying degreesFenjoy more comfortable socioeconomic positions. Although this question of access is valid in questions of many public health interventions, the preference of HCWs in questions of preferential access to vaccines and treatments is not unjust in these terms. Fundamentally, justice addresses unjustified imbalances in treatment. Aristotle famously mandated that equals should be treated as equals, and unequals as unequals. 16 The key point of justice is that there should be a valid justification for differential treatment, and in that light, in this context, we are describing pandemics that pose a unique and credible threat to the public in a manner that could fundamentally undermine our way of life. Preferential treatment of HCWs, in this limited context, is a just and defensible practice.
It is this same special status that we afford those who can save us from the most lethal and dangerous illnesses in times of public health emergency that also places greater demands on those same people. The greater the risk to society, the greater the responsibilities on those who can reduce the body count. The relationship between the duty of a HCW and the lethality of a disease is proportionalFdanger and obligation increase in step with each other, as opposed to other conceptions that suggest a threshold of exception as the risk of illness becomes too great. The fundamental flaw with this suggestion is that a negation of duty in such an outbreak simply allows the outbreak to pose an even greater threat to the populationFincluding that same derelict HCWFrather than confronting the illness in the relatively controlled environment of a hospital.
Utilitarianism in the form of promoting the good and diminishing the bad is a key moral belief in the realm of public health. It is one view in concert with others, all working to counterbalance each view to achieve a tenable moral equilibrium. In the extreme cases under consideration herein, such equilibrium dictates that the moral force of health promotion and harm minimization increases in relation to the threat posed to the well being of a larger society. In the case of widespread death or disability caused by a pandemic, this paper contended that an increased threat generates a heightened obligation on the part of HCWs, while also creating a reasonable expectation that those same HCWs will have preferential access to vaccines and treatments. Emerging viral threats in Gabon: health capacities and response to the risk of emerging zoonotic diseases in Central Africa Emerging infectious diseases (EID) are currently the major threat to public health worldwide and most EID events have involved zoonotic infectious agents. Central Africa in general and Gabon in particular are privileged areas for the emergence of zoonotic EIDs. Indeed, human incursions in Gabonese forests for exploitation purposes lead to intensified contacts between humans and wildlife thus generating an increased risk of emergence of zoonotic diseases. In Gabon, 51 endemic or potential endemic viral infectious diseases have been reported. Among them, 22 are of zoonotic origin and involve 12 families of viruses. The most notorious are dengue, yellow fever, ebola, marburg, Rift Valley fever and chikungunya viruses. Potential EID due to wildlife in Gabon are thereby plentiful and need to be inventoried. The Gabonese Public Health system covers geographically most of the country allowing a good access to sanitary information and efficient monitoring of emerging diseases. However, access to treatment and prevention is better in urban areas where medical structures are more developed and financial means are concentrated even though the population is equally distributed between urban and rural areas. In spite of this, Gabon could be a good field for investigating the emergence or re-emergence of zoonotic EID. Indeed Gabonese health research structures such as CIRMF, advantageously located, offer high quality researchers and facilities that study pathogens and wildlife ecology, aiming toward a better understanding of the contact and transmission mechanisms of new pathogens from wildlife to human, the emergence of zoonotic EID and the breaking of species barriers by pathogens. Despite intensive research and considerable effort from public health agencies to prevent or eradicate infectious diseases, emerging infectious diseases (EID) are currently the major threat to public health worldwide. 1 Indeed, many new infectious agents, characterized by a high pathogenic potential, have been recently identified. Furthermore, some well known pathogens have been expanding their territories, causing increasing concerns in the recent decades due to changing epidemiological patterns. 2 Most of these EID events have involved zoonotic infectious agents: more than 60% of EID affecting humans have a zoonotic origin 3, 4 and B75% of the diseases that have emerged over the past two decades have wildlife sources. 5 Therefore, zoonotic EID represent a major and increasing threat to global health. 1, 3, 6 Zoonoses refer to infectious diseases that are susceptible to be transmitted from animals to humans and are responsible worldwide for a great deal of pain, morbidity and even human fatalities. Two categories of zoonotic diseases have been described: 4 (1) diseases for which transmission events to humans are rare but once occurred, horizontal transmission from human-to-human maintains a more or less sustainable infectious cycle (for example: ebola virus, from naturally infected chiropteran to human epidemics); (2) diseases for which direct or vector-mediated animal-tohuman transmission remains the common source of human infection (for example: Rift Valley fever virus (RVFV), mosquito-transmitted from infected domestic ungulates).
Emerging and re-emerging zoonoses include recently identified infectious diseases, diseases that have recently evolved from a subclinical state to a clinical syndrome, and previously known diseases that have recently displayed an increase in incidence or that have spread to new regions, hosts or vectors. 4 However, a disease may not be recognized as zoonotic at the first outset. The disease can spread undetected for a period of time depending on the incubation period (weeks to years), the epidemiological pattern of a subclinical disease to a clinical picture (the emergence of the symptoms depending on host or pathogen factors) or, if the number of cases, in both human and animal populations, is too small and undetectable during the initial stages of transmission to suspect a link between the two events. 7 Zoonotic EID outbreaks result from a now classically accepted phenomenon of concurrency of fundamentals and territories of emergence. Fundamentals include factors related to the host, the vector (if any), the pathogen, and favorable environmental factors (climate) although territories at risk are the product of human activity and high-risk behavior among the human population. For instance, territories (that is: city, district) with an inefficient disease detection system or a failure to control vectors and other carriers of diseases as well as man-made environmental changes (breakdown of the water system, deforestationy) will force an increase in contact between the human population and wildlife. 2, 8 Tropical forests form the ecosystem harboring the highest species richness of all terrestrial ecosystems and shelter almost 50% of the total global biodiversity. 9, 10 This includes wildlife, flora, multi-cellular organisms as well as an immense diversity of pathogens including bacteria, parasites and viruses. Actually, there is a latitudinal spatial gradient of pathogenic species richness increasing towards the Equator. 11 After the Amazonian Basin, the Congo basin in Central Africa has the world's second largest contiguous block of tropical rainforest, which encompasses many areas that remain largely undisturbed, due in large part to low human population densities and the remoteness of interior rainforests. 12 As wildlife host species richness is a good predictor for the emergence of zoonotic EIDs with a wildlife origin, 3 Central Africa in general, and Gabon in particular are privileged areas for the emergence of zoonotic EIDs.
The potentialities of emerging zoonotic diseases from wild to domestic environments Latest reviews on EID show that nearly 75% of zoonotic EID have a wildlife origin. 3, 5, [13] [14] [15] In fact, the number of EID events caused by pathogens coming from wildlife has increased during the past six decades. 3 The majority of pathogens recorded were of viral origin. 16 Therefore viral zoonoses of wildlife origin represent the most significant and growing threat to global health among all EIDs. 3, 13 As anthropogenic activities have been identified as the cause of a significant majority of outbreaks, 16, 17 it is essential to fully understand the mechanisms driving contacts between wildlife and the human population as well as speciesjumping infections to set up public health information campaigns. On the contrary, efforts to conserve areas rich in wildlife diversity (13 National Parks were created in 2002 in Gabon) by reducing anthropogenic activity may have an added value in reducing the likelihood of future zoonotic disease emergence in these areas. 3 EIDs in free-living wild animals can be classified into three major groups on the basis of key epizootiological criteria: 18 (i) EIDs associated with 'spill-over' from domestic animals to wildlife populations living in proximity; (ii) EIDs related directly to human intervention, via host or parasite translocations; and (iii) EIDs with no overt human or domestic animal involvement. These phenomena have two major biological implications: first, many wildlife species are reservoirs of pathogens that threaten domestic animal and human health; second, wildlife EIDs pose a substantial threat to the conservation of global biodiversity, with for example the disappearance of the most great ape populations in protected areas in Central Africa after the 2002-2003 ebola virus outbreaks. [19] [20] [21] [22] Emerging zoonotic diseases in Gabon In Gabon, rainforests cover B80% of the territory. 23 These forests are known for their rich biodiversity of animal and plant species. 23 The human population, estimated at 1.5 million, 24 lives more in cities (54%) than in rural areas. 25 Gabon's economy has relied in the past mainly on petroleum exports, forest exploitation, and mining activities. Forest habitats are now exploited by logging and mining companies, tourism and hunting activities, which produce about 17,500 metric tons per year of game meat. Altogether these human incursions in Gabonese forests for exploitation purposes lead to intensified contacts between humans and wildlife and generate a risk of emergence of zoonotic diseases. 26 This risk is not strictly restricted to the forest but exists countrywide as the urban demand for bush-meat in Gabon is important (45 kg/per/year 27 ). Indeed, every Gabonese city has traditional and local food markets where fresh and smoked bush-meat coming from all around the country are available. Potential EIDs due to wildlife in Gabon are thereby plentiful and need to be inventoried.
At least 51 endemic or potential endemic viral infectious diseases have been reported in Gabon 28 (Table 1) . Among them, 22 are of zoonotic origin and involve 12 families of viruses. The most represented are Flaviviridae (dengue virus, yellow fever virus (YFV), zika fever virus), Poxviridae (monkeypox virus (MPXV)), Filoviridae (ebola and marburg Viruses), Arenaviridae (lassa fever virus), Bunyaviridae (RVFV) and Togaviridae (chikungunya virus).
During the past two decades, several outbreaks of these zoonotic viral diseases have been reported in Gabon. All of them had a major impact on the public health:
Zaïre ebola virus (ZEBOV): in Gabon, ZEBOV outbreaks occurred in 1994, 1996, 1997 and 2001; 29 primary human cases were generally contaminated by direct contact with 33 and concurrent infections of DENV and CHIKV have been reported in towns affected by the two outbreaks. 34 Dengue fever and the severe form of the disease, dengue hemorrhagic fever (DHF), are caused by the world's most prevalent mosquito-borne virus. 37 DENV is carried by Aedes aegypti mosquito, which is strongly affected by ecological and human drivers, but also influenced by climate (temperature, humidity and solar radiation). 37 Although DENV was known to circulate among mosquitoes within limited areas in West Africa and East Africa, dengue fever first emerged among the African population during the epidemic of Nigeria in 1964-1968, 38 then in Senegal in 1980 39 and Burkina Faso and Kenya in 1982. 40, 41 Since then epidemic manifestations were recorded in East Africa (Mozambique, Sudan, Djibouti, Somalia, Eritrea), in Senegal and more recently in Gabon. 34, 42 It seems that dengue fever is on the edge of emergence in Africa with the potential appearance of the devastating DHF that is yet to be observed on the continent. Yellow Fever Virus (YFV): Gabon is officially designated as an infected country. A YFV outbreak occurred in 1994 in Ogooue-Ivindo Province, North East of Gabon with 44 cases reported. 43 More recently, in 2009, Cameroon reported a laboratory-confirmed case of yellow fever (YF). 44 YF has become an important public health issue because of its case-fatality rate of 50% and the estimated 200,000 cases and 30,000 deaths that occur each year worldwide. Also, despite the efficiency of the YF vaccine and its inclusion in the national vaccination program, human populations situated in remote areas have a limited access to the public health system.
Based on serological evidence, several pathogens identified among wild or domestic animals, are suspected to infect the human populations of Gabon and therefore represent a potential threat to public health. Among them are the follows:
Foamy virus: simian foamy virus (SFV), a retrovirus in the Spumaretrovirinae subfamily, is widely prevalent in wildcaught and captive-born non human primates. 45, 46 Contamination between non-human primates and humans can occur via contact with infectious body fluids, through biting, 45, 47 or when manipulating fresh bush-meat. 48, 49 However, the potential for SFV to become a human disease and to spread among human populations after crossspecies transmission is not yet fully understood. 50 Human monkeypox: Human monkeypox, caused by the MPXV, a member of the genus Orthopoxvirus, is clinically almost identical to ordinary smallpox. 51 Humans become infected through direct contact with infected wild animals. It seems that monkeys are also incidental hosts as the reservoir species of MPXV remain unknown (most likely one or several rodents living in secondary forests of Central Africa). 51 Epidemiological surveys recorded 47 cases of human monkeypox (7 lethal) in Central Africa (RDC, Gabon, Congo, CAR, Cameroun, Ivory Cost, Liberia, Sierra Leone and Nigeria) 52 with possible secondary transmission in the human population. Since 1980, the large majority of cases to be reported from the DRC mainly concern children. 51 In 72% of those cases, an animal source of infection was suspected whereas secondary transmission from human source was presumed for the remaining cases. The longest documented chain of infection did not exceed four generations of person-to person transmission. There is only little probability of a large epidemic spread of MXPV. 53 Rift valley fever virus (RVFV): Rift Valley fever is an African disease that affects both livestock and humans. RVF outbreaks are associated with persistent heavy rainfall, sustained flooding and appearance of large numbers of mosquitoes, the main vector. Localized heavy rainfall is seldom sufficient to create conditions for an outbreak; 54, 55 Rift Valley fever is a good example of a disease that is well coupled with climatic anomalies; and Gabon is one of the African countries known to have some evidence of RVFV circulation as antibodies of the disease has been found in humans and livestock. 55 Impact of a changing environment Climatic variations and extreme weather events have a profound impact on infectious diseases: 56 for example, the emergence of vector-borne diseases is highly sensitive to changes in environmental conditions (rainfall, temperature, severe weather events). 56 Indeed arthropod vectors (that is mosquito, ticksy) are devoid of thermostatic mechanisms, hence reproduction and survival rates are strongly affected by fluctuations in temperature. 37 A rise in arthropodborne EID events due to climate anomalies has been observed during the 1990's. 3 Average global temperatures are predicted to be 1.0-3.51C by 2100. 57 Further epidemic events of vector-borne diseases due to climate change are, therefore, to be expected. Concerning other zoonotic infectious diseases like ebola, marburg or RVF, too little information is available concerning their ecology to be able to assess the impact of climate changes on the potential emergence or re-emergence of these diseases. However some preliminary reports show a strong association between wet environment (rainfall and hydrographic conditions directly dependent on climate and climate change) and the recent reemergence of ebola fever in Gabon and RDC. 58 Also El Niño/Southern oscillation (ENSO) is the strongest naturally occurring source of climate variability around the globe, 59 and more than 75% of RVF outbreaks between 1950 and 1988 occurred during warm ENSO event periods. 54 Moreover, RVF epidemics between 1950 and 1998 have coincided with unusually high rainfall in East Africa. 60 Epidemic prevention and control Existing structures in Gabon (data cited below was updated in 2007, ministry of public health, Gabon 61 ) Gabon has one of the highest expenditure on health per capita in Africa. In 2006, Gabon's total health care expenses were comparable to eastern European countries and most southern American countries: 62 This public Health system geographically covers most of the country allowing a good access to sanitary information and efficient monitoring of emerging diseases. However, access to treatment and prevention is much better in urban areas where medical structures are more developed, more organized with higher level of technicality and material 61 while the population is equally distributed between urban and rural areas ( Table 2) . A total of 60% of human and equipment resources from the Gabonese government are allocated to the main cities. In fact, all hospitals and clinics are found in Libreville, Port Gentil, Franceville and the surrounding areas, whereas in the distant rural areas, the numerous small health structures like Mother and Child Health Centers, Health Medical Centers, and Primary Care Health Center (Table 2 ) are often old with limited basic equipment and drugs. 61 Also the medical staff is more concentrated in urban areas (Table 3) . Indeed, more than 70% of the doctors and midwives are assigned to urban areas as well as 58% of the druggists. Only public nurses are equally distributed between rural and urban areas.
To fight against diseases, Gabon has developed 17 national health control programs. These programs monitor diseases such as HIV and sexually transmitted diseases, malaria, tuberculosis and also include a wide vaccination program, which covers 100% of the Gabonese territory. Despite the existence of 10 epidemiological stations distributed around the country in both rural and urban areas ( Table 2 ) that act as surveillance outposts, there is no other national health program to combat the more neglected EIDs.
In addition to public health Ministry activities, there are the following in Gabon:
The CENAREST (National Center for Scientific and Technologic Research) that evaluates and carries out research in Gabon, contributes to the application and promotion of research results and supports research training. The CIRMF (International Medical Research Centre of Franceville), a Gabonese world-renowned scientific research center, inaugurated in 1979, the CIRMF had for an initial focus to study the reasons for infertility in Central African populations. By the mid 1980s, CIRMF broadened its research to focus also on tropical diseases including HIV, trypanosomiasis and malaria. More recently CIRMF concentrated also on EIDs including the deadly ebola and marburg viruses, CHIKV and DENV. 63 Its central position in Africa and its world-renowned researchers ensure that CIRMF benefits from support and collaboration from several international institutions including the WHO (World Health Organization), the CIRAD (Centre de Coopération Internationale en Recherche Agronomique pour le développement), the IRD (Institut de Recherche pour le Développement), the Pasteur Institute, the CDC (Centers for Disease Control and Prevention), and several overseas universities from Europe, North America, South America and Asia. International NGOs (non-governmental organizations) are also involved in research on emerging diseases in Gabon. For example, the WCS (Wildlife Conservation Society) with its 'One World, One Health' Program and the Zoological Society of London and its 'Mikongo Conservation Centre' are working on great ape diseases, wildlife monitoring and the bush meat trade.
In Central Africa there are few regional organizations involved in public health:
The OCEAC (Organisation de coordination pour la lute contre les endémies en Afrique Centrale) is an organization of coordination and cooperation to fight major endemic diseases in Central Africa. Created in 1963, in Yaounde, by the determination of the health ministers of Cameroon, Congo, Gabon, CAR and Chad, it was originally known as the OCCGEAC until 1965. The Equatorial Guinea joined later the OCEAC. Its goals are to (1) coordinate public health policies and actions in Central African region, (2) participate in the training of the medical staff of member countries of the organization, (3) coordinate applied research projects undertaken by national institutions, (4) implement missions of expertise in the different areas of health sciences, (5) contribute to the public health promotion in the member countries and (6) support the actions undertaken in response to health emergencies. Today OCEAC is in charge of regional health programs and projects like the Sub Regional Program against HIV/AIDS, the Harmonization Program for Pharmaceutical Policy, the Regional Program to fight human African trypanosomiasis and research projects on malaria. The CEMAC (Communauté É conomique des É tats d'Afrique CentraleFEconomic Community of Central African States) is an economic community of the African Union for promotion of regional economic co-operation in Central Africa. Member countries include Gabon, Republic of Congo, Equatorial Guinea, CAR, Cameroon and Chad. It 'aims to achieve collective autonomy, raise the standard of living of its populations and maintain economic stability through harmonious cooperation'. It was established in 1983 and its ultimate goal is to establish a Central African Common Market. However CEMAC may have a role in the public health systems in Central Africa: in 2009, CEMAC signed a memorandum of understanding with Germany, which donated a 23 million euro grant for the prevention of HIV in Central Africa. The CIESPAC (Centre inter-états d'Enseignement en Santé Publique pour l'Afrique centrale) is a sub-regional public health training institution, originally located in Brazzaville. It was created to provide Central African countries with qualified health service staff and managers. It offers several courses, the most recent of which is recognized with a professional diploma in public health and targets mainly potential health district managers. 64 The civil war events that occurred in Brazzaville in the late nineties provoked the transfer of the institution to Yaoundé, Cameroon. The CAMES (Conseil Africain et Malgache pour l'Enseignement SupérieurFAfrican and Malagasy Council for Higher Education) exists to (1) promote and encourage the understanding and the solidarity between member States, (2) establish a permanent cultural and scientific cooperation between member states, (3) collect and diffuse all academic and research documents, (4) prepare agreement drafts between states concerned by Higher Education and Research and contribute to their implementation and finally (5) develop and promote dialogues to coordinate the higher education system and research so as to standardize programs and recruitment levels. This means that Gabonese university lecturers and researchers (in particular health researcher) are assessed by CAMES before obtaining a promotion.
Gabon is part of the Global Outbreak Alert and Response Network (GOARN), which contributes towards global health security by, 65 (1) fighting the international spread of 
Infectious diseases, including zoonoses, remain the major and increasing health threat in most developing countries. 1, 3, 6, 67 Even if in industrialized countries, cardiovascular diseases and cancers are considered to be the main causes of illness and death, special attention still needs to be paid to zoonotic EID. 67 This statement is now well described by the 'one healthFone medicine-one world' concept which is a worldwide strategy for expanding interdisciplinary collaboration and communications in all aspects of health care for humans and animals and the interaction with environmental factors. Also, viral hemorrhagic fevers, because of their high infectiousity and the dramatic outcome, have attracted the attention of the medical world and the public in Africa and around the world to this particular category of EID. 68 However, global effort in EID surveillance and investigation is inadequately allocated. Indeed, the majority of scientific resources focus on places from where the next important emerging pathogen is least likely to originate. 3 Jones et al. advocated for the re-allocation of resources to EID hotspots in lower latitudes, such as tropical Africa because of the critical need for health monitoring and identification of new potentially zoonotic pathogens in African wildlife populations, and this to be used as a forecast measure for EIDs. 3, 48, 67 Like other African countries, Gabonese resources for public health and health monitoring are unequally allocated; 60 % are spent at a central level. Public health services and clinical practitioners need more resources to be able to actively educate the public about the risks of repeated contacts with wildlife or other sources potentially harmful for health. 48 However, Gabon could be considered as a good model to investigate the emergence or re-emergence of zoonotic EID. On one hand, Gabonese forests are a hot spot for biodiversity (wild animals and unknown pathogens) and on the other hand there is a relatively small population (1.5 million of habitants), which is often in contact with surrounding wildlife. Also, the CIRMF, a research center advantageously located, offers high quality researchers and facilities that study pathogens and wildlife ecology. Altogether the combination of these factors should help to better understand the mechanisms of contact and transmission of new pathogens from wildlife to human, the emergence of zoonotic EID and the breaking of species barriers by the pathogens. Indeed the emergence of infectious diseases in wildlife is a continuous and ongoing process. The factors that give rise to zoonotic EID, such as ecosystem perturbations and modifications, climate changes, migrations of reservoirs species, pathogens or vectors, and intrinsic changes of pathogens may be of natural origin or due to human influences. 17 To understand the underlying mechanisms that govern relationships between reservoir species, ecological factors and environmental perturbations with the emergence, transmission and dissemination of viral diseases in tropical forests, the CIRMF wishes to set up permanent surveillance of the health of the population by the establishment of (1) a network reference laboratories (WHO based reference laboratories including CIRMF, Pasteur Institute Network and other National laboratories or universities based) and (2) a Health Ecology Observatory (that is,. The CIRMF's Scientific Station in la Lopé National Park). Such measures will compile data from the public health system with the monitoring of the emergence of new pathogens. The collected information would favor better outbreak risk appraisal in the Gabonese human population as well as for the entire Congo basin region.  Poverty, Global Health, and Infectious Disease: Lessons from Haiti and Rwanda Poverty and infectious diseases interact in complex ways. Casting destitution as intractable, or epidemics that afflict the poor as accidental, erroneously exonerates us from responsibility for caring for those most in need. Adequately addressing communicable diseases requires a biosocial appreciation of the structural forces that shape disease patterns. Most health interventions in resource-poor settings could garner support based on cost/benefit ratios with appropriately lengthy time horizons to capture the return on health investments and an adequate accounting of externalities; however, such a calculus masks the suffering of inaction and risks eroding the most powerful incentive to act: redressing inequality. income settings, such as Cuba or Kerala State, where India has an excellent performance on population health measures, these instances represent important exceptions to the general rule. What are the linkages between poverty and ill health? How can the vicious cycle of destitution and sickness be broken?
Poverty is arguably the greatest risk factor for acquiring and succumbing to disease worldwide, but has historically received less attention from the medical community than genetic or environmental risk factors. Several factors likely contributed to this oversight: first, being poor is not considered a disruption of normal physiologic function. Physicians and basic scientists viewed themselves as ill-equipped to understand or manipulate an individual's socioeconomic status. Second, unlike the largesse dedicated to finding technical solutions for population health problems, funding for research dedicated to understanding and alleviating poverty was sparse. Third, although some acknowledged that poverty plays a pivotal role in determining disease vulnerability and outcomes, the resultant solutions intended to redress poverty were often wrongheaded. For example, structural adjustment programs (SAPs) intended to increase gross domestic product (GDP) growth often involved austerity measures, such as cuts in government spending, currency devaluation, and privatization. These macroeconomic shifts involved intertemporal trade-offs (temporary pain for long-term gains) and completely ignored the path-dependent nature of health care. If a child does not get vaccinated, a pregnant mother lacks antenatal care, or a TB clinic goes without drugs, the health consequences can reverberate for generations.
The tide in public health and medicine has started to shift. The global HIV/AIDS crisis brought into sharp relief the vulnerability of financially strapped health systems to epidemic disease and revealed disparities in health outcomes along economic fault lines. The protestations of injustice regarding the withholding of life-sustaining antiretroviral treatment from the developing world, made first by patient-activists, then students, and (more gradually) academics and politicians, have provided a template for addressing other diseases linked to poverty. This hope was echoed in the preface of Farmer's 1 AIDS and Accusation: "If AIDS care becomes a right rather than a commodity.we have no more excuses for ignoring the growing inequality that has left hundreds of millions of people without any hope of surviving preventable and treatable illnesses.Taking on AIDS forcefully would allow us to start a 'virtuous social cycle,' long overdue." And if a rights-based approach to controlling communicable disease falls on deaf ears, enlightened self-interest might still invoke concern. Severe acute respiratory syndrome (SARS), multidrug-resistant TB, and H1N1 remind the developed world of its porous borders. Many investors view the developing world as a potential market for their goods, and military strategists foresee the danger of allowing states to collapse from pandemic disease.
Economic thought regarding the link between poverty and disease has also evolved. Sen's 2 landmark treatise, Development as Freedom, exposed the false dichotomy between political and social and economic rights. Sen 2 posited that development was broader than income: an affluent, stable democracy could not be achieved without an educated and healthy populace. "There is a deep complementary between individual agency and social arrangements," Sen 2 wrote. And more pointedly: "Development requires the removal of major sources of unfreedom: poverty as well as tyranny, poor economic opportunities as well as systematic social deprivation, neglect of public facilities as well as intolerance or overactivity of the repressive states." Such insight paved the way for the creation of the Human Development Index (HDI) 20 years ago. The HDI is a composite measure of health, education, and income and was designed by Mahbub ul Haq to counter the inordinate reliance on income alone as a measure of well-being. Building on the conceptual framework created by Sen and parameterized (albeit imperfectly) by ul Haq, Jeff Sachs became the next economist to make a broader concept of development operational by promoting the Millennium Development Goals (MDGs). The 8 MDGs, endorsed by 189 countries, are timelimited commitments to reduce poverty, expand educational opportunities, promote gender equality, and safeguard population and environmental health. 3 This article discusses the complex relationship between poverty and communicable disease, and draws on experience gleaned from working in solidarity with the destitute sick in Haiti, Peru, Rwanda, and elsewhere, as well as from anthropologic and economic theory and evidence. We conclude that the twin afflictions of poverty and disease cannot be treated in isolation and require a biosocial understanding to achieve lasting health gains.
One link between poverty and disease that is readily observable to most physicians is the increased vulnerability of the poor to communicable diseases, and the lack of medical care once infected. This link was eloquently documented by German pathologist Rudolf Carl Virchow, investigating an outbreak of typhus in the nineteenth century:
The population had no idea that the mental and material impoverishment to which it had been allowed to sink, were largely the cause of its hunger and disease, and that the adverse climatic conditions which contributed to the failure of its crops and to the sickness of its bodies, would have not caused such terrible ravages, if it had been free, educated and well-to-do. For there can now no longer be any doubt that such an epidemic dissemination of typhus had only been possible under the wretched conditions of life that poverty and lack of culture had created in Upper Silesia. If these conditions were removed, I am sure that epidemic typhus would not recur. Whosoever wishes to learn from history will find many examples. 4 To prevent typhus from recurring, Virchow announced a radical prescription: medicine must concern itself with the social condition of the population, and characterize efforts short of that as palliative. Although daunting, these words inspired the creation of social medicine, and the veracity of Virchow's observations are reflected in modern epidemics.
The recent cholera epidemic in Haiti provides a current example. In early December 2010, the US Centers for Disease Control and Prevention (CDC) Morbidity Mortality and Weekly Report announced that the outbreak had spread nationwide. At that time, the Haitian Ministry of Public Health and Population (MSPP) reported 91,770 cases of cholera, including 43,243 hospitalizations and 2071 deaths. Deaths occur "as rapidly as 2 hours after symptom onset and [identify] important gaps in access to life-saving treatments, including oral rehydration solution (ORS)." 5 Disentangling Haiti's dire health condition from historical, political, and economic concerns leads to the characterization of the epidemic as a medical disaster stemming from the twin natural disasters Haiti suffered in the last year: a 7.0 magnitude earthquake and subsequent flooding from Hurricane Tomas. However, a narrative that uses phrases such as medical and natural implies inevitability and general inculpability. A more careful reading of the context in which the cholera outbreak has occurred proves such ahistorical views misleading.
The Republic of Haiti is the only nation tracing its genesis to a successful slave revolt. After more than a decade of war that destroyed the country's infrastructure and cost tens of thousands of lives, the French relinquished military control in 1804. However, France maintained financial repression by demanding that the fledgling nation pay damages for property losses incurred during the revolution. These demands marked the birth of Haiti's longstanding debt burden. As historian von Tunzelmann 6 describes, Haiti was on the brink of humanitarian calamity even before the devastating earthquake of 2010:
. France gained the western third of the island of Hispaniola-the territory that is now Haiti-in 1697. It planted sugar and coffee, supported by an unprecedented increase in the importation of African slaves. Economically, the result was a success, but life as a slave was intolerable.After a dramatic slave uprising that shook the western world, and 12 years of war, Haiti finally defeated Napoleon's forces in 1804 and declared independence. But France demanded reparations: 150 [million] francs, in gold.
For Haiti, this debt did not signify the beginning of freedom, but the end of hope. By 1900, it was spending 80% of its national budget on repayments. To manage the original reparations, further loans were taken out-mostly from the United States, Germany and France. Instead of developing its potential, this deformed state produced a parade of nefarious leaders, most of whom gave up the insurmountable task of trying to fix the country and looted it instead.
Staggering debt obligations hampered Haiti's ability to provide basic sanitation and public health interventions to its population. According to the United Nations Development Program (UNDP) Human Development Report, Haiti ranks 145th out of 169 countries. 7 It has occupied the unenviable position of poorest nation in the Western Hemisphere for decades. Income per capita is US $560, 54% of Haitians live on less than US $1 a day, and 78% live on less than US $2 a day. In 2005, total external debt owed was US $1.5 billion, more than one-quarter of total GDP, 8 whereas Haiti spent only 1.2% of GDP on health care. 7 Widespread lack of access to clean water has been a chronic threat to the health of the Haitian population. 9 In 2007, only 63% had access to an improved water source, and only 17% had access to sanitation. 10 The earthquake on January 12, 2010 (which killed an estimated 250,000 people and displaced more than one-tenth of the Haitian population) turned the water situation in Haiti from bad to worse. It was in the context of hundreds of thousands of people living in refugee camps (1 million on the outskirts of Port-au-Prince alone) with intermittent access to drinking water and gross underprovision of sanitation facilities that cholera took hold. 11 Recent findings published in the New England Journal of Medicine and using third-generation single-molecule real-time DNA sequencing found that the clonal strain causing the Haiti outbreak was genetically similar to those previously isolated in Bangladesh. The study investigators concluded that, "Collectively, our data strongly suggest that the Haitian epidemic began with introduction of a V. cholerae strain into Haiti by human activity from a distant geographic source." 12 These results corroborated those from the CDC and the National Public Health Laboratory (NPHL) in Haiti. 13 The initial CDC findings were released in early November and sparked protests against the Nepali UN peacekeepers quartered near the river presumed to be the source of the outbreak. At least 3 Haitians were killed. 14 After a 100-year hiatus, Poverty, Global Health, and Infectious Disease cholera has now gripped Haiti and has started to spread to the Dominican Republic. 15 Given the increased virulence associated with this particular strain (the death rate in Haiti is 12 times higher than that of the 1991 Peruvian epidemic) there are calls from public health leaders to mass vaccinate the populations of the island and its closest neighbors. 16 Taking into account the long view of Haiti's history, from slavery to colonialism, debt, despotic leaders, and a woeful undersupply of public goods, the cholera outbreak seems less like an unforeseen catastrophe and more like an event that Virchow would have easily predicted.
Neglected tropical diseases (NTDs) provide another example of how economic position can interact with host susceptibility. According to the World Health Organization (WHO), NTDs are defined by their association with poverty: "though medically diverse, neglected tropical diseases form a group because all are associated with poverty." 17 Substandard housing, lack of access to safe water and sanitation, and inadequate vector control contribute to the efficient transmission of infection. Currently, of the world's 2.7 billion impoverished individuals, more than a billion people suffer from NTDs. 17 Thankfully, there is growing attention to this matter among the international community. The first annual report on NTDs was released by the WHO in 2010. Director-General Margaret Chan refers to the MDGs in her preface and provides several examples of how eliminating NTDs would foster economic development: "Onchocerciasis and trachoma cause blindness. Leprosy and lymphatic filariasis deform in ways that hinder economic productivity and cancel out chances for a normal social life. Buruli ulcer maims.Human African trypanosomiasis (sleeping sickness) severely debilitates before it kills. Chagas disease can cause young adults to develop heart conditions, so that they fill hospital beds instead of the labor force," and so on. 17 The emphasis on potential negative productivity implications associated with untreated NTDs is understandable. Like cancer and heart disease, NTDs do not travel widely. New justifications are needed to persuade the international community to intervene when the direct threat to the health of wealthy country inhabitants is muted. If the medical community is to be committed to global health equity and not simply to reducing morbidity or mortality from the cluster of diseases that most affect the wealthy world, a rights-based approach to health care must be adopted.
As discussed earlier, poverty and associated disease rarely arise de novo. Heavy burdens of disease predictably strike those places, most often resource-poor communities, where structural violence weighs most heavily. Moreover, structural violence (institutionalized biases and inequalities including racism, elitism, gender inequality, militarism, and economic policy that fosters inequity) often emanates from global centers of power and privilege, and increases the risk of encounter with communicable disease. 18 Rwanda's recent history makes these processes clear. The 1994 Rwandan genocide took an enormous toll on the population: at least 800,000 Rwandans massacred in 3 brutal months by approximately 15% of the population. 19 However, what commonly escapes our memories is that the Rwandan genocide was predicated on far more than physical violence alone. Structural violence played a significant role in setting the stage. Uvin 20 has argued that an uncritical development enterprise, dominated by foreigners, contributed to the creation of the processes that led toward genocide. In Aiding Violence, he summarizes:
[A]id financed much of the machinery of exclusion, inequality, and humiliation; provided it with legitimacy and support; and sometimes directly contributed to
Many weapons used in the genocide did not originate in Rwanda, but in many of the political and economic powers of the world. 21 The physical and structural violence of the Rwandan genocide directly affected the spread of communicable disease. Systematic rape during the genocide served as a vector for HIV transmission. 22 The exodus of Rwandans into refugee camps in the Democratic Republic of the Congo without adequate food, water, and sanitation gave rise to epidemics of infectious disease (such as cholera) that resulted in a crude mortality of 20 to 35 per 10,000 people each day. 23 Increased incidences of both malaria and tuberculosis have lasted far beyond the formal end of the genocide. 24 Careful attention to Rwanda's and Haiti's places in global history, economics, and politics shows that the forces of structural violence increase risk of communicable disease for resource-poor populations in ways that are distinct from the behavioral and cultural explanations uncritically circulated in academic literature and the popular press.
Conditions of poverty and structural violence facilitate disease acquisition. It is straightforward to extend this analysis to document the impact of poverty on access to care and health outcomes. Since the 2001 report by the WHO Commission on Macroeconomics and Health, chaired by Jeff Sachs, attention has shifted to exploring how investments in population health can spur economic growth. 25 Given that most of the developing world is engaged in physically demanding agricultural labor, health setbacks likely have a greater impact there than in the wealthy world. In economic terms, the marginal returns to good health in the labor force are higher in poorer countries. However, there are other, more nuanced channels by which ill health can affect economic prospects. These channels include the interactions among health and demography, cognition, and investment behavior.
There is a strong association between child survival and fertility. Total fertility rate (TFR; the number of children that would be born to a woman if she were to live to the end of her childbearing years and bear children in accordance with current agespecific fertility rates) and mortality before 5 years of age (child mortality rate [CMR], the probability per 1,000 that a newborn baby will die before reaching the age of 5 years, if subject to current age-specific mortality) have a correlation coefficient of 0.876. Table 1 shows the linear trend between these indicators. 10 Explanations Table 1 The association between fertility and mortality before 5 years of age Poverty, Global Health, and Infectious Disease abound as to why the relationship between child survival and fertility are so robust. One view espoused by many demographers and economists is that families in societies where child survival is low tend to compensate for expected and actual child deaths by having more children. Nobel laureate and economist Gary Becker famously modeled this quality/quantity trade-off with respect to family size. Another explanation is that places with high child mortality also lack access to contraception, educational opportunities for women, and gender equality. Although desired family size is a difficult concept to define and measure, the World Bank does attempt to collect data on access to contraception and indices of gender inequality. 10 Although the correlation between the gender inequality index and child mortality in the World Bank's indicators database is negligible (0.01), perhaps at least in part because of the small sample size (75 countries) and imprecise measuring, contraceptive prevalence is negatively correlated with child mortality (À0.32). Although a major focus of academic inquiry is the direction of causality in the fertility-CMR relation, it seems safe to assume that the relationship is bidirectional. These data imply that efforts to improve public health through the provision of either culturally appropriate family planning or pediatric care would also pay dividends in the other health sector. From an economic standpoint, household resources are divided among fewer individuals as family size shrinks. This division allows parents to invest more in their children's education and nutrition, potentially interrupting the intergenerational transmission of poverty.
As with the relationship between child survival and fertility, the interplay between health and education is complex. As mentioned earlier, more-educated mothers tend to have fewer and healthier children. Moreover, the returns from education in terms of increased wages or agricultural output give heads of households the financial opportunity to promote their own health and that of their children. David Barker, a British physician who noted a correlation between low birth weight and cardiovascular health in midlife, put forward the fetal origins hypothesis that the in utero environment has important consequences for health and cognitive abilities later in life. Several epidemiologists have since confirmed this association. 26 Almond, 27 an economist at Columbia, used the natural experiment of the 1918 Spanish influenza epidemic to assess the impact of fetal health on educational and labor outcomes. Pairing data from the pandemic with those from the 1960 to 1980 decennial US censuses, Almond 27 found that cohorts in utero during the pandemic displayed reduced educational attainment, increased rates of physical disability, lower income, and lower socioeconomic status compared with other birth cohorts. Similarly, Miguel and Kremer 28 analyzed a randomized control trial of deworming in Kenyan schools at the facility level designed to capture the positive externalities associated with reducing the transmission of helminths. Miguel and Kremer 28 found that the program reduced absenteeism from school by one-quarter, although there was no significant effect on test scores. A series of follow-up studies have shown that those who received deworming for a longer period of time enjoy higher wages years later. Taken together, these results suggest that health investments, especially early in life, affect educational attainment.
There is perhaps an even more subtle way in which health affects education and economic outcomes: through the channel of savings and investment. Economic growth theory has repeatedly underscored that savings and investment are engines of development, 29 but what explains why some countries invest more than others?
This question was first posed by John Rae writing in the early nineteenth century. As Frederick and colleagues 30 explain, "Like [his contemporary] Adam Smith, Rae sought to determine why wealth differed among nations. Smith had argued that national wealth was determined by the amount of labor allocated to the production of capital, but Rae recognized that this account was incomplete because it failed to explain the determinants of this allocation. In Rae's view, the missing factor was 'the effective desire of accumulation," which determines the "rate of time preference." The rate of time preference is a mathematical representation of the tendency individuals have to weight the present more heavily than the future when making decisions. It summarizes how willing consumers are to delay immediate consumption and instead invest or save their income. 30 Thus, the relationship between the rate of time preference and savings behavior at the individual level can be linked to income growth and disparities at the aggregate level. Studies by psychologists and economists have shown that the poor discount the future more steeply than the wealthy. 31 This result has given rise to the view that the poor are impatient or, more pejoratively, lack self-control. However, this rationale fails to account for the uncertainty and risks associated with living in poverty. In particular, how do health status and expectation of longevity influence one's willingness to make trade-offs over time? Recent evidence from Sri Lanka sheds light on this question. Jayachandra and Lleras-Muney 32 examined how a sudden drop in maternal mortality between 1946 and 1953 sharply increased the life expectancy of girls. This increase in turn led to greater investment in their education: Jayachandra and Lleras-Muney 32 found that, for every extra year of life expectancy, literacy among girls increased by 0.7 percentage points (2%) and years of education increased by 0.11 years (3%). At Partners In Health, we often use the phrase antidote to despair to describe our work. Translating the language of social justice into the language of economics, the antidote is the extended time horizon afforded by longevity. Knowing that a healthy and well-fed tomorrow awaits may affect the psychology of those living in poverty. The impact of this health-led hope on investments in microenterprise, education, and complementary health inputs has yet to be fully measured.
Having reviewed the ways in which poverty, structural violence, and infectious disease confine poor populations to vicious cycles of suffering and despair, we now examine the implications of these understandings on the design of health interventions. As shown by disease patterns in Haiti and Rwanda, social forces interact with human biology and affect who falls ill and who has access to care. Thus, use of a biosocial analytical framework provides a useful and effective tool for designing and implementing health interventions to address these inequalities. Failure to use a biosocial lens often gives rise to charity and development models of health intervention that replicate preexisting unequal structures. Such models localize blame for disease with the poor themselves. In contrast, a biosocial lens makes clear that disease among the poor results from the embodiment of structural violence and requires that any serious attempt to address disease in resource-poor settings incorporates efforts for social change. Through commitment to models built on the principles of social justice, we have found that advocacy and long-term partnerships with the public sector and the communities in which we work are indispensable to sustainable transformations in health that reduce suffering caused by infectious and chronic disease. Biosocial understandings of disease in Haiti and Rwanda reveal that a sustainable response must not only make available the fruits of modern medicine (ie, diagnostic tools, pharmaceuticals, and trained clinicians) but must also address the consequences Poverty, Global Health, and Infectious Disease of deep poverty: limited transportation, poor housing, and food scarcity, among others. In Haiti, Rwanda, and numerous other settings, Partners In Health and local partners provide care that integrates social and economic programs. These programs include constructing homes and schools, establishing potable water systems, and providing food and transportation stipends. In addition, paid community health workers are used to deliver top-quality health care to patients in their homes, rather than requiring sick, impoverished individuals to confront innumerable barriers to reaching clinics and hospitals. Such solutions, which privilege a biosocial approach to identifying and breaking down barriers to care, have resulted in remarkable successes in addressing epidemics of HIV/AIDS, TB, malaria, and other communicable and chronic diseases in some of the most challenging domestic and global settings. 33 
Poverty and infectious diseases interact in subtle and complex ways. Casting the problem of destitution as intractable, or epidemics that afflict the poor as accidental, erroneously exonerates us from responsibility for protecting and caring for those most in need. Our experience working in Haiti and Rwanda has shown that appropriately and adequately addressing the scourges of communicable diseases requires a biosocial appreciation of the structural forces that shape disease patterns. Although there is ample evidence that heath investments pay dividends in labor productivity, educational attainment, population control, and, potentially, capital investments, the idea that health is an instrument for development should complement, not supplant, a rights-based approach to health equity. It is plausible that most health interventions in resource-poor settings could garner support based on cost/benefit ratios with appropriately lengthy time horizons to capture the return on health investments and an adequate accounting of externalities; however, such a calculus masks the untold suffering of inaction and risks eroding the most powerful incentive to act: redressing inequality. Forecasting incidence of hemorrhagic fever with renal syndrome in China using ARIMA model BACKGROUND: China is a country that is most seriously affected by hemorrhagic fever with renal syndrome (HFRS) with 90% of HFRS cases reported globally. At present, HFRS is getting worse with increasing cases and natural foci in China. Therefore, there is an urgent need for monitoring and predicting HFRS incidence to make the control of HFRS more effective. In this study, we applied a stochastic autoregressive integrated moving average (ARIMA) model with the objective of monitoring and short-term forecasting HFRS incidence in China. METHODS: Chinese HFRS data from 1975 to 2008 were used to fit ARIMA model. Akaike Information Criterion (AIC) and Ljung-Box test were used to evaluate the constructed models. Subsequently, the fitted ARIMA model was applied to obtain the fitted HFRS incidence from 1978 to 2008 and contrast with corresponding observed values. To assess the validity of the proposed model, the mean absolute percentage error (MAPE) between the observed and fitted HFRS incidence (1978-2008) was calculated. Finally, the fitted ARIMA model was used to forecast the incidence of HFRS of the years 2009 to 2011. All analyses were performed using SAS9.1 with a significant level of p < 0.05. RESULTS: The goodness-of-fit test of the optimum ARIMA (0,3,1) model showed non-significant autocorrelations in the residuals of the model (Ljung-Box Q statistic = 5.95,P = 0.3113). The fitted values made by ARIMA (0,3,1) model for years 1978-2008 closely followed the observed values for the same years, with a mean absolute percentage error (MAPE) of 12.20%. The forecast values from 2009 to 2011 were 0.69, 0.86, and 1.21per 100,000 population, respectively. CONCLUSION: ARIMA models applied to historical HFRS incidence data are an important tool for HFRS surveillance in China. This study shows that accurate forecasting of the HFRS incidence is possible using an ARIMA model. If predicted values from this study are accurate, China can expect a rise in HFRS incidence. Hemorrhagic fever with renal syndrome (HFRS), or epidemic hemorrhagic fever (EHF) is an acute viral syndrome caused by infection with one of hantaviruses. HFRS is an important infectious disease in developing countries. In China, HFRS is caused mainly by 2 types of hantaviruses, Hantaan virus (HTNV) and Seoul virus (SEOV), each of which has coevolved with a distinct rodent host. HTNV is associated with Apodemus agrarius, whereas SEOV, which causes a less severe form of HFRS, is associated with Rattus norvegicus [1] . In hantavirus -endemic areas, HFRS is most common among farmers and others who may have close contact with excreta of infected rodents [2, 3] . In mainland China, HFRS remains a serious public health problem with approximately 20,000-50,000 human cases reported annually, approximately 90% of the total cases worldwide [4] [5] [6] . Currently, HFRS is endemic in 28 of 31 provinces in mainland China [4, 7] .
In response to the spread of HFRS in China, the Chinese Center for Disease Control and Prevention designed a surveillance system for HFRS and created educational programs for the general public. However, the impact of control efforts remains difficult to measure due to the inherent complexities of HFRS as a disease: multiple viral strains with identified genetic polymorphisms, complex disease manifestation, diverse animal reservoirs, and multiple routes of transmission [8] . Infectious diseases have certain characteristic features that lead themselves to modeling, such as: speed of pathogen variation, accumulation of susceptible hosts, and environmental indices [9] . Thus, epidemic modeling and forecasting can be essential tools to prevent and control HFRS. Recently, statistical methods including linear regression [10] [11] [12] , correlation coefficients [13] , grey swing model [14] , back propagation artificial neural network model [15] have been used for prediction of HFRS incidence. The variation of HFRS incidence, which is influenced and constrained by diversified factors, is characterized by tendency and randomicity. These statistical tools are inappropriate for analyzing the randomicity of HFRS. Autoregressive integrated moving average (ARIMA) models, which take into account changing trends, periodic changes, and random disturbances in time series, are very useful in modeling the temporal dependence structure of a time series. In epidemiology, ARIMA models have been successfully applied to predict the incidence of infectious diseases, such as influenza mortality [16] , malaria incidence [17] , as well as other infectious diseases [18, 19] . This study aimed to develop a univariate time series model for the HFRS incidence; specifically, a stochastic ARIMA model, for short-term forecasting of HFRS incidence (per 100,000 population) in China.
Chinese HFRS incidence data from 1975 to 2008 was obtained from the Chinese Center for Disease Control and Prevention. All HFRS cases were initially diagnosed by clinical symptoms. Patient blood samples were also collected and sent to local Centers for Disease Control and Prevention (CDC) laboratories for serological confirmation. Finally, data were collected by case number according to the sampling results. There might be admission rate bias in the disease report, but this has been reduced as much as possible. In China, HFRS is a nationally notifiable disease and hospital physicians must report every case of HFRS to the local health authority within 12 hours. Local health authorities later report monthly HFRS case totals to higher the national level CDC for surveillance purposes. Due to mandatory reporting, it is believed that the degree of compliance in disease notification over the study period was consistent.
We used the Box-Jenkins approach to ARIMA (p, d, q) modeling of time series [20] . This model-building process is designed to take advantage of associations in the sequentially lagged relationships that usually exist in periodically collected data [21] . The following were the parameters selected when fitting the ARIMA model: p, the order of autoregression; d, the degree of difference; q, the order of moving average. The annual data used in this study did not show seasonal pattern, so the series was differenced at the nonseasonal level to induce stationarity. Autocorrelation function (ACF) graph and Partial autocorrelation function (PACF) graph were used to identify the order of moving average (MA) and autoregressive (AR) terms included in the ARIMA model. Estimates of the model's parameters were obtained by the conditional least squares method. Diagnostic checking including residual analysis and the Akaike Information Criterion (AIC) was used to compare the goodness-of-fit among ARIMA models. The Ljung-Box test was used to measure the ACF of the residuals. In addition, we used the mean absolute percentage error (MAPE) and fitting effect diagram to assess forecast accuracy. 
The present study was reviewed by the research institutional review board of Shandong University and the China CDC, and found that utilization of disease surveillance and meteorological data did not require oversight by an ethics committee.
From 1975 to 1986, the HFRS incidence in China rose regularly with a peak in 1986 of 11.06 cases per one hundred thousand population. After 1986, the incidence descended sharply with a dramatic fluctuation until 2008 ( Figure 1 ). The lowest incidence could be seen in 2008, 0.68 per one hundred thousand.
According Figure 1 , the series showed a non-stationary mean, so we stabilized the mean of HFRS incidence by taking both second and third order differences. All further statistical procedures were performed on the transformed HFRS incidence. Based on the distribution characteristics, we conducted five models, ARIMA(0, 2, 1), ARIMA(1,2,1), ARIMA(0,3,1), ARIMA(1, 3, 1), and ARIMA (2, 3, 1) . Of all the models tested, the ARIMA (0,3,1) model was the best fit for the data ( Table 1 ). The transformation series by taking third-order differences is shown in Figure 2 . The plots of ACF and PACF ( Figure  3 ) described the temporal dependence structure in HFRS incidence. The slow decay in the PACF, associated with a ACF cutoff at lag 1 suggested a MA(q = 1).
The parameter estimates for the optimum ARIMA(0,3,1) model are shown in Table 2 
Time series analysis of surveillance data on incidence of various infections is very helpful in developing hypotheses to explain and anticipate the dynamics of the observed phenomena and subsequently in the establishment of a quality control system and reallocation of resources [22] . ARIMA model is one of the most widely used time-series forecasting techniques because of its structured modeling basis and acceptable forecasting performance [23] . In this paper, we applied an ARIMA (p, d, q) model to analyze the surveillance data of HFRS in China. Disease monitoring by public health department entails ongoing data collecting, processing, and updating. However, the national level China CDC is the appropriate level of organization for the implementation of an ARIMA predictive model, because reported data is continually received and updated. We found that model predictions are further improved by the assured availability of the Health Department data. In this study, we have obtained an ARIMA model that closely fits HFRS incidence in China. The autoregression and moving average parameters of our model imply the incidence of HFRS in a month can be estimated by the residual occurring one month prior. According to the results above, the conducted model is reliable with a high validity. Once a satisfactory model has been obtained, it can be used to forecast expected numbers of cases for a given number of future time intervals [24] . Thus, the fitted ARIMA(0,3,1) model can be used to predict the next three years' HFRS incidence in China.
The forecast results suggest that the HFRS incidence in China will experience a slight growth in the next three years (2009) (2010) (2011) . A rise in the number of HFRS incidence may also result from an increase in the number and size of natural foci [25] , climate change, especially the increase of mean temperature [26, 27] . Therefore, knowledge of HFRS forecasts is necessary to prompt health departments to strengthen surveillance systems and reallocate resources in anticipation of increasing HFRS incidence. Several studies have used ARIMA model to fit and predict changing trends in infectious disease. Luz et al applied an ARIMA(2,0,0)×(1,0,0) 12 model to predict dengue incidence in Rio de Janeiro [18] and found that ARIMA models were useful tools for monitoring dengue incidence. Earnest et al indicated that ARIMA models provided useful tools for administrators and clinicians in planning for real-time bed capacity during infectious diseases outbreaks such as SARS [28] . Li et al have applied an ARIMA model to monthly incidence of HFRS in Linyi City, China to predict HFRS incidence, and found that the ARIMA model could be used to predict HFRS incidence with high predictive precision in the short-term [29] . In the present study, we further confirmed the consensus that ARIMA model is a useful tool in monitoring and predicting changing trends in infectious diseases.
To the best of our knowledge, this is the first study to apply ARIMA model to fit the HFRS incidence in China with as many as 34 observations at year level. Some previous studies [30, 31] in China also used ARIMA model to fit and forecast HFRS incidence of some regions, but they had the same problem that the number of observations was not enough, which led to the instability of their forecast results. In order to conduct a stable and effective ARIMA model, we have to collect at least 30 observations [32] . Thus, parameter estimates of the fitted model would be more robust. The longer the series, the better; however, the series should not extend so far into the past as to include periods during which a different case definition was applied or in which any other reporting artifact resulted in a mean number of cases per interval that differs from the mean of recent intervals. As mentioned above, for adequate ARIMA modeling, a time series should be stationary with respect to mean and variance. If the mean increases or decreases over time, or if the variance does, the series may need to be transformed to make it stationary, before being modeled. Otherwise, the prediction effect of the model will be poor.
In order to improve the model, updating the forecasts is very important. A model without seasonal terms will need to be updated frequently. Confidence intervals that widen rapidly as time increase from the starting point of the forecasts also indicate a model that needs frequent updating. Generally speaking, there are two ways to implement the updating. The model can be reapplied to the original series with extra observations added at the end to give forecasts based on a later starting point. Alternatively, a new model can be fitted to the longer series. This is probably preferable, since fitting a model is quick, especially when the old model is used as a guide, and it makes better use of the additional observations. Some limitations of this study also need to be taken into account when interpreting the results. In this study, the interval of HFRS incidence is one year, so we could not analyze its seasonal characteristic. In further study, we would use monthly data to predict HFRS incidence in order to get seasonal pattern and higher predictive precision. In addition, the data are from a passive surveillance system, the possible biases in disease reporting and potential underreporting of HFRS cases might influence the precision of our analysis.
There is an urgent need for monitoring and predicting HFRS incidence to reduce the substantial morbidity and mortality caused by this disease [33] . ARIMA models applied to historical HFRS incidence data are an important tool for HFRS surveillance. Accurate forecasting of the incidence of HFRS is possible. Our modeling approach can be used to monitor and predict HFRS incidence in China. The ARIMA model could be used to optimize HFRS prevention by providing estimates on HFRS incidence trends in China. Delivery Systems for Intradermal Vaccination Intradermal (ID) vaccination can offer improved immunity and simpler logistics of delivery, but its use in medicine is limited by the need for simple, reliable methods of ID delivery. ID injection by the Mantoux technique requires special training and may not reliably target skin, but is nonetheless used currently for BCG and rabies vaccination. Scarification using a bifurcated needle was extensively used for smallpox eradication, but provides variable and inefficient delivery into the skin. Recently, ID vaccination has been simplified by introduction of a simple-to-use hollow microneedle that has been approved for ID injection of influenza vaccine in Europe. Various designs of hollow microneedles have been studied preclinically and in humans. Vaccines can also be injected into skin using needle-free devices, such as jet injection, which is receiving renewed clinical attention for ID vaccination. Projectile delivery using powder and gold particles (i.e., gene gun) have also been used clinically for ID vaccination. Building off the scarification approach, a number of preclinical studies have examined solid microneedle patches for use with vaccine coated onto metal microneedles, encapsulated within dissolving microneedles or added topically to skin after microneedle pretreatment, as well as adapting tattoo guns for ID vaccination. Finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. The prospects for bringing ID vaccination into more widespread clinical practice are encouraging, given the large number of technologies for ID delivery under development. microneedle pretreatment, as well as adapting tattoo guns for ID vaccination. Finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. The prospects for bringing ID vaccination into more widespread clinical practice are encouraging, given the large number of technologies for ID delivery under development.
The skin contains high concentrations of antigen-presenting cells, and is thus a site capable of inducing potent immune responses. The skin is composed of multiple layers, each with characteristic resident and transient immune cell subsets. Outermost is the thin layer of the epidermis (0.05-0.2 mm), which is primarily made up of epithelial cells as well as Langerhans cells, melanocytes, and Merkel cells. Beneath the epidermis, the dermis is a thicker layer (1.5-3 mm) consisting of a network of collagen fibers. Cells of the adaptive and innate system reside in or circulate through the dermis, including macrophages, mast cells, Langerhans cells, and dermal dendritic cells. Antigenpresenting cells in the skin perform an essential role in processing incoming antigens, resulting in immune system activation or immune tolerance of self or harmless antigens (Nicolas and Guy 2008) . For these reasons, it is possible that delivery of vaccines to the epidermis or dermis may result in superior immune responses compared to other anatomical sites (Glenn and Kenney 2006; Lambert and Laurent 2008; Nicolas and Guy 2008) . Alternatively, an equivalent immune response could be stimulated by delivery of a smaller quantity of vaccine antigen to the skin. Either of these mechanisms could be beneficial for developing vaccines against new disease targets, improving immune responses in hard-to-treat groups, or lowering the cost of vaccine antigens, and may be particularly valuable for improving access to vaccines in low-resource settings.
While a substantial number of clinical studies evaluating intradermal (ID) delivery of vaccines have been performed, the majority of studies have not been designed to evaluate whether ID delivery is immunologically superior to other routes. In most cases, to simplify administration, a reduced dose (10 or 20%) delivered ID was compared to the full dose delivered either subcutaneously (SC) or intramuscularly (IM) . Only a few studies have compared delivery of the same dose of vaccine ID and SC/IM. Further research will be needed to establish whether the potential for dose-sparing is unique to ID delivery (PATH 2009 ). However, some ID delivery devices in development offer additional desirable features such as needle-free delivery or improved ease of administration, which may be drivers for further adoption of ID vaccine delivery even if there is no net immunologic benefit.
Vaccines for smallpox have been delivered to the skin dating back to Edward Jenner's first experiments in 1796 demonstrating that exposure to cowpox could protect against smallpox infection. A variety of scarification techniques and devices have been used to allow virus introduction, including knives, needles, scalpels, and rotary lancets. During the global smallpox eradication campaign, both multi-dose nozzle jet injectors and bifurcated needles were used for ID vaccinia virus inoculation (Henderson et al. 2008 ).
Bacille Calmette-Guérin (BCG) vaccine for tuberculosis is globally the most widely delivered ID vaccine. ID injection by needle and syringe is the most commonly used method, but in some areas BCG is also delivered to the skin using a multipuncture device. New versions of BCG are under development in an effort to improve immune protection, and are also delivered ID (Hoft et al. 2008 ).
Rabies vaccines are conventionally delivered IM, but due to the high cost of cellculture-derived vaccines and the pressing need for affordable vaccination regimens in endemic regions, ID delivery has been extensively studied. Both post-exposure prophylaxis and pre-exposure prophylaxis ID regimens induce protective titers, and WHO has recommended ID delivery of reduced doses of rabies vaccines since 1991 (WHO 2005; . Given equivalent doses of antigen, delivery to the dermis appears to be either superior or equivalent to IM/SC (Bernard et al. 1982; Bernard et al. 1987; Fishbein et al. 1987; Phanuphak et al. 1990) . A detailed review on ID rabies vaccination can be found elsewhere in this special volume on ID immunization (Warrell 2011 ).
Multiple studies of reduced-dose delivery of influenza vaccines have been conducted, providing some of the most informative clinical data on the potential for dose-sparing through ID delivery. One study found that ID delivery of 6 lg HA per influenza strain was comparably immunogenic as the standard IM dose of 15 lg HA per strain (Belshe et al. 2004) . A later comparison of 3, 6, and 9 lg delivered both ID and IM found equivalent responses for the two delivery routes for each dose (Belshe et al. 2007) . Trials have also been conducted with influenza using novel microneedle devices to aid accurate ID delivery, as discussed in Sect. 3.2.
ID delivery of reduced doses of hepatitis B vaccine has been evaluated in healthy infant, child, and adult populations as well as in immuno-compromised patient groups. Meta-analyses have concluded that seroconversion rates are lower than full-dose IM delivery, although responses are higher in children and females (Chen and Gluud 2005; Sangare et al. 2009 ). When the same dose of hepatitis B antigen has been delivered ID and IM, immune responses were equivalent for both routes (Ayoola 1984; Milne et al. 1986; Heijtink et al. 1989; Coberly et al. 1994; Rahman et al. 2000) .
Hepatitis A vaccines have also been proposed as a possible target for reduced-dose ID delivery. Two studies found that reduced doses delivered ID produced comparable immune responses to IM delivery, while a third indicated that the ID route was inferior (Brindle et al. 1994; Carlsson et al. 1996; Pancharoen et al. 2005) . Local reactogenicity was observed for alum-adjuvanted formulations.
In a few countries, ID was originally the standard route of delivery for inactivated poliovirus vaccine, but injection depth was later shifted to IM (Weniger and Papania 2008) . Studies have found that ID delivery of reduced doses is capable of inducing seroconversion, which may help make this vaccine more affordable for use in developing countries (Samuel et al. 1991; Samuel et al. 1992; Nirmal et al. 1998 ). More recently, the WHO Global Polio Eradication Initiative has worked to determine the potential for this mode of delivery to be used in post-eradication settings after phase-out of oral polio vaccine.
Several studies have been conducted evaluating ID delivery of measles vaccine, with mixed results (Burland 1969; Kok et al. 1983; Whittle et al. 1984; de Moraes et al. 1994) . However, the vaccine dose and method used to deliver the vaccine varied, and it is possible that trials using older generation delivery technology did not deliver vaccine reliably to the dermis (PATH 2009). Transcutaneous immunization of measles vaccine on a coated patch has also been attempted. Although a salivary sIgA response was observed, the key marker of immunity, an increase in neutralizing serum IgG, was not detected (Etchart et al. 2007 ).
Studies were conducted delivering the 17D attenuated yellow fever virus vaccine by scarification, but this delivery mode was abandoned as efficacy was low. More recently, a clinical trial compared full dose SC delivery of a 17D vaccine to 1/5 dose delivered ID by Mantoux injection, which found equivalent seroprotection between the two routes (Roukens et al. 2008) . A more extensive description on ID vaccination against yellow fever is provided elsewhere in this special volume on ID immunization (Roukens et al. 2011 ).
A number of other vaccines have been considered for ID delivery. Research has shown that a reduced ID dose of vaccines for diphtheria-tetanus-pertussis, tetanus toxoid, and tick-borne encephalitis can generate a comparable immune response to the standard dose and way of injection (Stanfield et al. 1972; Zoulek et al. 1984; Zoulek et al. 1986; Dimache et al. 1990 ). ID delivery is also under investigation for a number of vaccines in development, including vaccines for tuberculosis, enterotoxigenic E. coli, and pandemic influenza, as well as DNA vaccines.
The traditional methods used for ID delivery of vaccines have limitations which may hinder adoption of ID delivery. Bifurcated needles and multipuncture devices have been used successfully for delivery of smallpox and BCG vaccines, but do not deliver reproducible quantities of vaccine antigen to the dermis and are therefore unlikely to be appropriate delivery devices for new vaccines (Lambert and Laurent 2008) . The Mantoux method of inserting a needle at a shallow angle into the skin can also be inconsistent, and requires additional training and skill to perform correctly (Flynn et al. 1994) . The perceived difficulty of performing an ID injection using this method may prevent development of vaccines for ID delivery. New generations of devices, such as those discussed in the rest of this article, may improve the reliability of ID delivery and enable adoption of the ID route for more vaccines.
Most vaccines are administered IM or SC using a hypodermic needle. To achieve ID vaccination, conventional hypodermic needles can be used by employing the Mantoux technique to inject into the skin. Simpler and more reliable ID injection is being pursued through adaptations of hypodermic needle technology, as well as novel hollow microneedle devices produced by microfabrication ).
The Mantoux technique is an ID injection method characterized by a needle inserted at a 5-15 degree angle, approximately 1 mm deep into the dermis, to inject a vaccine or drug (Fig. 1a) . This method was developed by Charles Mantoux in the early 20th century, and it has been used to identify tuberculosis infection by the ID injection of tuberculin (Mantoux 1909) . However, this technique requires training and is often considered an inconsistent delivery method, thus preventing vaccine makers or medical practitioners from using ID injection as a common immunization method (Lambert and Laurent 2008) . Also, age or elasticity-related skin conditions have a significant effect on adequate placement of the needle in the dermis for the traditional ID injection technique Laurent et al. 2007) , thus leading to inadequate vaccination. Other disadvantages of Mantoux technique injection include inaccurately delivered dosage of vaccine, vaccine wastage in dead space of the needle, and variable injection success when using different gauge needles (Flynn et al. 1994) . Moreover, success rate of ID injection by untrained personnel was found to be 80-90% (Howard et al. 1997) . In an effort to reduce training requirements and to improve the reliability of the Mantoux injection technique, an intradermal adapter is under development by PATH (Seattle, WA, USA), a nonprofit, international health agency that develops and advances health technologies for low resource settings, and SID Technologies. This device fits over a conventional hypodermic needle and syringe and limits the angle and depth of penetration of the needle into the skin in order to facilitate delivery to the dermis. 
To overcome these limitations of conventional ID injection, Becton-Dickinson (BD) has developed a micro-sized needle that can be inserted into skin vertically, unlike the angled injection of the Mantoux method. This novel microneedle device has been studied in animals and human subjects, and is currently used in approved influenza vaccines (INTANZA Ò and IDflu Ò ). BD's microneedle device (called Soluvia TM ) uses a 30 gauge microneedle that extends 1.5 mm beyond an insertion depth-limiting tip, which is connected to a prefilled syringe (Figs. 1c and 1e). The microneedle system was evaluated versus the conventional Mantoux technique to compare delivery efficiency and safety in human subjects. Using ultrasound echography analysis, the distribution of fluid delivered by the microneedle was seen to be larger than the Mantoux injection control. In addition, the microneedle system had a high ID administration success rate (95%) and, in a study of patient compliance and safety, the microneedle device showed promising results. This system also caused fewer occurrences of injuries to the papillary dermis, lesser pain than Mantoux injection and was administered easily by untrained personnel (Laurent et al. 2007 ).
An earlier prototype of the BD microneedle using a 1 mm, 34 gauge needle has been tested in rats for delivery of influenza vaccines which showed dose sparing effects compared to an IM control. ID microneedle administration of a low dose (0.01 lg) of inactivated virus vaccine induced similar serum antibody response as IM injection of a dose 100 times larger (1 lg). Additionally, using microneedles for ID immunization with split-viron vaccine (seasonal H1N1 strain) showed approximately ten-fold dose-sparing compared to IM immunization. In the same study, ID immunization using plasmid DNA vaccine encoding the hemagglutinin protein of influenza A virus showed similar dose-sparing effects after multiple immunizations (Alarcon et al. 2007 ). The BD microneedle was also used to deliver a live attenuated vaccine against Japanese encephalitis (ChimeriVax TM -JE) in non-human primates. In this study, ID microneedle injection was compared to SC injection and transcutaneous microabrasion (see Sect. 6.1). The microneedle ID injection provided the best and most consistent immune responses (i.e., neutralizing antibodies) of the three types of immunizations .
A further study compared anthrax vaccine delivery using ID microneedle immunization, IM injection, intranasal delivery, and epidermal delivery by microabrasion into mice (10 lg dose) and rabbits (50 lg dose). Microneedle ID vaccination showed slightly better response in the murine model than the other routes used, while all treatments in the rabbit had similar responses. A follow-up ID immunization was performed to compare ID injection with IM injection over a range of doses in a rabbit model: 10, 0.2, and 0.08 lg of anthrax vaccine (Mikszta et al. 2006) . After prime immunization, ID injection showed significantly higher immunogenicity than IM injection when using 10 and 0.2 lg dosages. Interestingly, ID injection with 0.2 lg showed a statistically equivalent response to IM administration of the 10 lg dose. After administration of a booster immunization, this dose-sparing phenomenon continued. Furthermore, an aerosol lethal challenge with anthrax spores showed that a 10 lg ID injection completely protected the immunized rabbits, whereas IM injection of the same dose protected only 71% of the rabbits.
An early prototype of the BD microneedle system was first tested in a clinical study examining influenza vaccine delivery in healthy adults (18-60 yrs) and elderly adults ([60 yrs) (Belshe et al. 2004) . In this study, 6 lg of hemagglutinin was delivered by ID injection and compared to a full dose (15 lg) delivered by IM immunization. It was found that in younger participants, ID immunization was not significantly different from immunization by IM, as shown by geometric mean hemagglutination inhibition (HAI) titers. However, ID administration showed lower HAI titers than IM in elderly patients. Further evaluation of ID microneedle vaccination against influenza was performed in clinical studies of healthy adults (18-57 yrs) (Leroux-Roels et al. 2008; Beran et al. 2009 ) and elderly persons ([60 yrs) (Holland et al. 2008; Arnou et al. 2009 ). For healthy adults, 9 lg of hemagglutinin (H1, H3, and B strains) was delivered by ID injection and was compared to a 15 lg IM immunization. This study confirmed previous results that reduced-dose ID injection was equally immunogenic as full-dose IM injection (Leroux-Roels et al. 2008) .
In a Phase II clinical trial, the effects of lower dose ID immunization was investigated using 3, 6, and 9 lg of hemagglutinin (ID) and 15 lg of hemagglutinin (IM). ID immunization using 3 and 6 lg of hemagglutinin induced inferior immune response as shown by HAI titer, but a dose of 9 lg showed comparable response compared to full-dose (15 lg) IM vaccination (Beran et al. 2009 ). For elderly subjects ([60 years old), a booster vaccine (15 lg) was administered due to the inferior immune system generally found in the elderly compared to younger adults (Goodwin et al. 2006) . Therefore, 15 lg of hemagglutinin was administered twice by either ID or IM routes in elderly subjects. In this phase II clinical trial, ID immunization showed significantly better immune response as determined by postimmunization GMT (geometric mean titer), seroprotection (% participants with HAI titers C40), GMTR (geometric mean ratio of post-immunization titer to pre-immunization titer), and rate of seroconversion (post-immunization titer in participants with a pre-immunization titer \10). Therefore, ID microneedle vaccination provided superior immunogenicity in a high priority population for protection from influenza due to high vulnerability (Holland et al. 2008 ). These findings were further confirmed in a phase III clinical trial for elderly persons ([60 years old), where ID immunization showed superior seroprotection, GMTR, and rate of seroconversion compared to IM after prime immunization.
After administration of two booster immunizations, ID immunization induced consistently higher seroprotection rates than IM immunization (Arnou et al. 2009 ).
As a final note, ID immunization caused more local inflammatory-like reactions than IM immunization. It is possible that because ID delivery occurs in the skin, inflammatory or immunologic reactions are more easily visible than those that may occur after IM immunization, which presents the antigen deep into the muscle layer where an inflammatory reaction would not be visible to the eye (Belshe et al. 2004; Holland et al. 2008; Arnou et al. 2009; Beran et al. 2009; Van Damme et al. 2009 ).
Hollow microneedles have also been developed as multi-needle arrays, which have involved shorter needles (\ \1 mm) produced by novel microfabrication techniques, including laser micromachining (Davis et al. 2005) , silicon-based MEMS technique using deep reactive-ion etching (Gardeniers et al. 2003; Roxhed et al. 2007) , integrated lithographic molding technique (Luttge et al. 2007) , deep X-ray photolithography (Perennes et al. 2006) , photolithography with micromolding technique ), drawing lithography with viscoelastic polymer ) and others. In addition, glass hollow microneedles have been fabricated by drawn glass micropipette techniques (Wang et al. 2006) . A recently developed hollow microneedle array (MicronJet from NanoPass Technologies) was used in a human clinical trial involving healthy adults ). This device consists of a row of four hollow silicon microneedles that are 450 lm in length (Fig. 1f) . In this study, ID injection with the array using 20 and 40% of the IM dose (15 lg) induced similar immune response as measured by GMT increase, seroconversion rate, and seroprotection rate.
ID delivery can also be achieved via jet injection or particle injection routes, which are needle-free methods of vaccine and drug delivery. There have been decades of clinical experience with jet injection, and more recent studies are being conducted with newer innovations in this technology.
Needle-free jet injectors create a fine stream of pressurized liquid that penetrates the skin. The depth of delivery-ID, SC, or IM-is largely determined by design variables such as the injection stream coherence, quality, and pressure; orifice size, skin and tissue thickness, and the angle of the injection relative to the skin (Schramm-Baxter and Mitragotri 2004; Weniger and Papania 2008) . Vaccines that have been shown to achieve immunity when administered via jet injection to conventional depths (i.e., ID, SC, or IM, depending on the vaccine) include typhoid, cholera, BCG, tetanus-diphtheria for adults, whole cell diphtheria-tetanus-pertussis (DTP), measles, meningococcal A and C, smallpox, yellow fever, hepatitis A, hepatitis B, influenza, plague, polio, and tetanus (Weniger and Papania 2008) .
Historically, multi-use nozzle jet injector (MUNJI) devices with reusable nozzles were used successfully worldwide in the latter half of the 20th century to deliver countless millions, or by some estimates billions of doses of vaccines to both adults and children over the course of several decades (Weniger and Papania 2008) . In response to the risks of disease transmission due to cross contamination from reuse of injection devices, a new generation of jet injector designs were developed starting in the late 1980s to address this safety concern. These new jet injectors utilize a sterile, disposable cartridge or syringe for each patient injection and a reusable hand-piece that relies on a power source, such as a manually powered spring or gas canister. A number of disposable-syringe jet injectors (DSJIs) have been developed and approved by national regulatory authorities for a variety of applications and uses, including vaccine delivery. Some of these are low-cost, manually powered DSJI technologies, developed specifically for application to developing countries' immunization requirements and needs, which include design features to prevent reuse ('auto-disable') of the needle-free syringes. DSJIs in clinical development for ID delivery include the Biojector Ò 2000 and Zetajet Ò (Bioject), and PharmaJet Ò (PharmaJet Inc.).
There is a long history of ID delivery via the jet injector route through the use of modified syringe orifice nozzles that can either have direct contact to the skin or can involve a setback feature or 'spacer' intended to introduce a gap between the nozzle orifice and the injection site, thereby weakening the injection stream and limiting deposition to the dermal space (Weniger and Papania 2008) (Figs. 2a, b) . MUNJI devices provided millions of ID smallpox doses during the implementation of the smallpox eradication program (Millar and Foege 1969; Weniger and Papania 2008) . Jet injectors have also been utilized historically for ID vaccination of rabies (Bernard et al. 1982; Bernard et al. 1987) , hepatitis A (Williams et al. 2000) , BCG (Paul et al. 1978; Parker 1984) , DTP combination vaccine (Stanfield et al. 1972) , measles (Burland 1969; Kok et al. 1983) , and influenza vaccine (Weniger and Papania 2008) .
A number of studies have been or will soon be implemented to address the application of DSJI ID delivery to vaccines of importance to global public health.
For example, the US Centers for Disease Control and Prevention is leading a study on seasonal influenza vaccine delivered ID via a DSJI technology in children of 6-24 months of age. This study compares full and fractional dose IM with ID vaccination. Results-to-date indicate that injections were generally tolerable with few study-related adverse events. Initial blinded assay results demonstrate comparable immune response rates. Final study results and analysis can be found in Gomez et al. (2010) .
The WHO Global Polio Eradication Initiative has worked to determine the potential for DSJI ID delivery of inactivated poliovirus vaccine (IPV) to be used in post-eradication settings after phasing out the use of oral polio vaccine. Studies have been conducted in Oman, Cuba, and India to evaluate reduced ('fractional') dose of IPV delivered with two different DSJI devices. Compared to IM, inferior seroconversion rates were found when ID doses were delivered at 6, 10, and 2000) with ID spacer (white portion at end of syringe), used for investigational use only (Courtesy of BioJect). b Jet injector applied to the skin for injection (Courtesy of PharmaJet). c Epidermal powder immunization device for ID projectile injection (Courtesy of PowderMed) 14 weeks of age, but non-inferior rates of protection ([95%) were seen using a later 2, 4, and 6 month schedule. When IPV was used as a booster to oral polio vaccine, inferior seroconversion rates were observed for ID compared to IM delivery (Sutter 2009; Mohammed et al. 2010; Resik et al. 2010) .
DSJI technology has also been used for the delivery of DNA vaccines for malaria in young adults (Epstein et al. 2002; Wang et al. 2006 ) and an HIV-vaccine candidate (PATH 2009). A pilot study assessment of human papillomavirus vaccine has also recently occurred (PATH 2009) . PATH is also working to implement a new study of purified Vero cell rabies vaccine for ID post-exposure prophylaxis using a DSJI technology in India. Results of this study are anticipated in 2012. Other vaccine trials of ID vaccine delivery are planned for other applications including BCG, IPV, varicella zoster virus, H1N1 and yellow fever (PATH 2009).
Epidermal powder immunization (EPI) and particle-mediated epidermal delivery (PMED) utilize helium gas to deliver powdered proteins, polysaccharides, inactivated pathogens, or DNA-coated particles into the epidermis at supersonic speeds (Weniger and Papania 2008) (Fig. 2c) . Companies involved in developing this technology include Powderject, PowderMed (acquired by Pfizer in 2006), and Iaculor Injection. It is not known if this device technology class is still in active development (PATH 2009 ). Conventional protein antigens must be specially formulated for delivery by EPI, and are spray dried into powders of suitable density and size (20-70 lm). A clinical trial has been conducted evaluating delivery of a powdered inactivated influenza vaccine by EPI injection, which found that immunogenicity was comparable to standard delivery by IM needle and syringe . EPI has also shown efficacy in preclinical studies with hepatitis B and HIV vaccines (Chen et al. 2002; Osorio et al. 2003) .
In PMED, gold beads 1-3 lm in diameter are coated with vaccine and delivered by needle-free jet injection into the epidermis. This approach may be particularly suited to DNA vaccines, as deposition of coated particles into the stratum corneum and epidermis may encourage DNA uptake and expression by resident antigen-presenting cells. DNA vaccines for hepatitis B delivered by PMED have induced protective antibodies (Roy et al. 2000; Roberts et al. 2005) . Clinical studies have also been conducted with DNA vaccines for seasonal influenza to evaluate the feasibility of this approach. Results have been promising, but immune responses are not yet equivalent to standard vaccine delivery methods (Drape et al. 2006; Jones et al. 2009 ). EPI and PMED delivery of DNA vaccines for a variety of other diseases have also shown immunogenicity preclinically, including malaria, avian influenza, herpes simplex virus, HIV, non-small cell lung cancer, Eurasian encephalitic viruses, hantaviruses, SARS coronavirus, and smallpox (Weniger and Papania 2008) .
For more than 200 years, various sharp instruments have been used for vaccination by creating small holes in the skin that allow vaccine to penetrate into the body (Weniger and Papania 2008) . Although most vaccine administration is currently performed by hypodermic needle injection, sharp tools such as bifurcated needles have historically been used for smallpox (Frey et al. 2002) and BCG (Darmanger et al. 1977) vaccination and remain in use to this day. Over the past decade, new skin piercing technologies for ID drug transport have been developed, and include techniques such as microneedles (Prausnitz 2004 ) and tattooing (Bins et al. 2005) . Recently these methods, especially microneedles, have shown promise for delivering vaccines to the skin, thereby enabling improved immunogenicity and simpler patient administration.
The bifurcated needle (Fig. 1b) was invented by Benjamin Rubin in 1961 for smallpox vaccination. It consists of two sharp prongs which hold vaccine fluid by capillary action between the two tines. The use of this device is simple and does not require trained personnel (Baxby 2002; Weniger and Papania 2008) . The needles are dipped into vaccine and then punctured perpendicularly into skin repeatedly over an area of about 5 mm diameter by a process called scarification (WHO 2010) . Although this method was effective for the smallpox eradication program, poorly controlled dosing, inefficient use of vaccine and needle-stick injuries were significant shortcomings that have limited the use of bifurcated needles for other vaccines.
In addition to hollow microneedles discussed in Sect. 2, solid microneedles can be used to pierce the skin and thereby deposit vaccine in the epidermal and/or dermal space ). Techniques for vaccination using solid microneedles include the use of microneedles that penetrate the skin to make a hole through which vaccine can be transported. Vaccine formulations may be placed on the skin after microneedle penetration, coated onto microneedles or embedded within microneedles and released into the skin after insertion. Solid microneedles can be prepared as patches that can be easily applied to the skin, perhaps by self administration.
Coated microneedles have been the most extensively studied technique for ID microneedle vaccination (Figs. 3a, b) . Using this approach, vaccine forms a solidstate coating on the surface of solid microneedles that dissolves off within the skin upon application. Typically, this method provides a bolus delivery of a sub-milligram dose of antigen within minutes of application, which is often suitable for delivery of vaccines. An effective microneedle coating process typically involves dip-coating metal microneedles in a coating solution containing the vaccine, a surfactant to promote wetting of the microneedle surface, and a viscosity enhancer to increase coating thickness (Gill and Prausnitz 2007b; Gill and Prausnitz 2007a) . Using this technique, compounds over a large range of sizes including small molecules, proteins, DNA, and virus particles have been coated onto microneedles. Novel coated microneedle designs for improved delivery have been demonstrated, such as the three-dimensional grooves-embedded microneedle (Han et al. 2009 ) and the pocketed microneedle (Gill and Prausnitz 2008) . The first ID vaccination using coated microneedles delivered ovalbumin as a model protein antigen to (Courtesy of University of Queensland). b Array of solid stainless steel microneedles coated with yellow dye. Each 12 mm by 12 mm device contains 50 microneedles measuring 700 lm tall. Inset shows magnified view of two coated microneedles (Courtesy of Georgia Institute of Technology). c Dissolving microneedles shown intact before insertion into skin, partially dissolved 1 min after insertion into skin and fully dissolved 5 min after insertion into skin (Reproduced from (Sullivan et al. 2010 ); Courtesy of Georgia Institute of Technology) hairless guinea pigs (Matriano et al. 2002; Widera et al. 2006) . In these studies, ID microneedle vaccination showed a better immune response than an equivalent SC or IM injection at low dose. The investigators also found that immune response by microneedle vaccination was dose-dependent.
Among the various vaccine candidates, influenza vaccine has received the most attention by ID immunization using small arrays of coated microneedles measuring approximately 700 lm in length (Zhu et al. 2009 ). Microneedles coated with 10 lg of seasonal influenza H1N1 inactivated virus vaccine induced complete protection against lethal virus infection in mice. However, subsequent studies showed that influenza vaccine lost more than 95% of its antigenicity during the coating process (Kim et al. 2010b) . In order to maintain antigenicity, the disaccharide trehalose was added to the coating formulation to serve as a stabilizer. This enabled successful immunizations requiring smaller doses of vaccine (0.4 lg) as compared to immunizations with similar immune responses by conventional IM immunization Kim et al. 2011) . Coated microneedles also showed improved thermal stability of vaccine compared to the liquid form of vaccine (Kim et al. 2010b ). More detailed studies showed that coated microneedle vaccination with inactivated influence virus vaccine induced similar antibody IgG response, HAI titer, and neutralizing activity as conventional IM immunization in mice Kim et al. 2010b ). To account for antigenic changes to the vaccine during the coating process, vaccine coated on microneedles was dissolved off the needles and then delivered IM by injection. In this case, vaccination using microneedles showed a better primary immune response than corresponding IM immunization using the same antigen formulation ).
Vaccination by coated microneedles induced robust immunity to influenza after challenge in a mouse model (Kim et al. 2011) . Notably, microneedle-immunized mice were shown to have undetectable levels of influenza virus titer in their lungs after challenge, unlike IM immunized mice, which had virus titers at least 100-fold higher. Additional assays for immune response from corresponding lung samples such as lung cytokine and lung IgG also consistently showed microneedle immunization to be superior to IM. As evidence for microneedle-enhanced immune system memory response, the microneedle immunized group was found to have significantly higher levels of total IgG and isotypes IgG1 and IgG2a postchallenge than pre-challenge, but antibody levels in IM immunized mice were lower post-challenge than pre-challenge (Kim et al. 2011 ). In addition to improved humoral immunity, coated microneedles also induced cellular recall response such as MHC II-associated CD4 + T helper cell response ). Finally, microneedle immunization performed using a different strain of influenza (H3N2) virus vaccine induced similar complete protection against lethal challenge (Koutsonanos et al. 2009 ). Studies using virus-like particle (VLP) vaccine coated on microneedles were also performed. The VLP dose was controlled using a coating formulation including antigen concentration and a number of coating dips (Kim et al. 2010a) . When a 0.35 lg dose of VLP was delivered, microneedle vaccination induced a stronger immune response than IM, as measured by IgG, IgG subtype (IgG1, IgG2a, IgG2b), HAI, neutralizing activity, lung IgG, lung cytokine, and more suppression of lung virus infection. Microneedle immunization by VLP showed complete protection from a lethal viral challenge without major body weight loss, unlike IM after the same dose, which partially protected mice from lethal viral infection (40%) and caused significant body weight loss (Quan et al. 2010) .
A novel approach to coated microneedles involved the use of polyphosphazene (PCPP), which served as both an effective coating excipient and an immune adjuvant ). ID microneedle immunization with hepatitis B surface antigen (HBsAg) in pigs using the PCPP coating formulation was superior in inducing antigen-specific IgG compared to ID injection by hypodermic needle with or without PCPP. Another study demonstrated effective generation of cellular immune responses to a hepatitis C DNA vaccine administered to mice using coated microneedles (Gill et al. 2010) . Other studies have sought to specifically target delivery to antigen-presenting Langerhans cells using extremely short (*100 lm) needles that penetrate only into the epidermis. These short needles were coated using a novel coating process involving gas-jet drying (Chen et al. 2009 ). In an initial study, vaccination with ovalbumin-coated needles induced similar immune response to IM immunization. In a follow-up study, microneedles coated with a low dose of hemagglutinin-based influenza vaccine generated a similar immune response as IM vaccination at a 100-times larger dose. The authors proposed that these short, densely packed microneedles could deliver more than half of the antigen directly to antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells (Fernando et al. 2010) .
Methods for long-term vaccine storage without significant immunogenicity loss, especially without refrigeration, are important for vaccination campaigns. Microneedles are coated with vaccine in the solid state, which is expected to confer thermal stability. In a stability study of microneedles coated with inactivated influenza vaccine, mice immunized with coated microneedles stored at room temperature for 1 month produced similar IgG responses to those of mice immunized by microneedles stored for 1 day. Furthermore, both groups were completely protected from lethal challenge after viral infection. In vitro assay of the microneedles, however, showed a decrease in antigenicity by about 80% (Kim et al. 2010c ).
As an improvement over coated microneedles, dissolving microneedles have been developed in order to eliminate sharp, biohazardous waste after vaccination (Fig. 3c) . Unlike non-dissolving (e.g., metal) microneedles coated with a vaccine formulation, dissolving microneedles are made solely of material such as polymers or sugars that will safely dissolve in the skin after insertion, which leaves behind only the microneedle patch backing. Typically, the vaccine is incorporated into the matrix of the microneedle and is released into the skin upon microneedle dissolution. Dissolving microneedles have been made using a number of different materials, including polyvinylpyrrolidone (Sullivan et al. 2008) , maltose (Kolli and Banga 2008) , carboxymethylcellulose (Lee et al. 2008 ), polylactic and/or polyglycolic acid (Park et al. 2005; Park et al. 2006 ) and dextrin (Ito et al. 2006 ). In a recent study, dissolving microneedles were prepared by encapsulating inactivated influenza vaccine in a polyvinylpyrrolidone matrix and used to immunize mice. The vaccine was gently encapsulated without significant damage to immunogenicity and was shown to generate similar antibody and cellular immune responses compared to IM injection of the same dose and provided complete protection against lethal challenge. Compared to IM injection, dissolving microneedle vaccination resulted in more efficient lung virus clearance and enhanced cellular recall responses after challenge (Sullivan et al. 2010 ). TheraJect has also developed biodegradable microneedles using carboxymethylcellulose containing various biomolecules including influenza vaccine (Oh et al. 2006 ).
As a simpler, albeit probably less efficient, method, microneedles can be used to pierce the skin to make it more permeable and thereby enable entry of topically applied vaccines. This method is attractive because the micro-scale pores made by microneedle insertion are generally too small for penetration of microorganisms (Donnelly et al. 2009 ), yet large enough for delivery of sub-unit and possibly viral vaccines. After insertion and removal of the microneedles, vaccine can be applied using a patch or other topical formulation for slow delivery by diffusion through long-lived pores (Kalluri and Banga 2011) . This approach was investigated for transcutaneous vaccination using diphtheria toxoid and influenza vaccine (Ding et al. 2009a; Ding et al. 2009b) . When diphtheria toxoid was applied to microneedle-pretreated skin in combination with cholera toxin adjuvant, a similar immune response was induced compared to SC injection. However, microneedle pretreatment did not enhance immune response for influenza vaccine. This vaccination approach has also been studied in an ex vivo human skin model to investigate skin immune cell responses (Ng et al. 2009 ). Using a related approach, blunt-tipped microneedles were used to scrape the skin, thereby making microtroughs in the skin through which a DNA vaccine encoding HBsAg was administered (Mikszta et al. 2002) . This approach generated stronger humoral and cellular immune responses than IM or ID injection.
Tattoo guns use high-frequency oscillating needles to make thousands of punctures in the skin, which is conventionally used to deposit tattoo ink in the dermis, but has been adapted to deliver ID vaccines (Fig. 1d) . In one study, hemagglutininexpressing DNA vaccine was administered to pigs and derived humoral and protective immunity as shown by methods including HAI titer and improved virus clearance from nasal swabbing (Eriksson et al. 1998) . To overcome the slow processing of an immune response induced by DNA vaccination, DNA tattooing was suggested for short-interval DNA vaccination (Bins et al. 2005) . In this study, it was shown that short-interval ID DNA tattoo immunization generated fast and stable T cell responses to human papillomavirus and complete protection from influenza virus challenge. When compared to the IM route, DNA tattoo vaccination elicited much stronger and quicker humoral and cellular immune responses. In addition, studies indicated that even IM immunization with adjuvant was inferior to DNA tattoo immunization (Pokorna et al. 2009 ). To determine the effect of the tattooing process on DNA vaccine stability, the DNA topology change was evaluated, including critical factors for antigen expression and immune response (Quaak et al. 2009 ). It was found that the DNA tattooing tool had negligible effect on DNA structure and activity. Other vaccines including an adenoviral vector vaccine against respiratory syncytial virus (Potthoff et al. 2009 ) and a peptide vaccine against human papillomavirus (Pokorna et al. 2009 ) were administrated by ID tattooing. In the case of the adenoviral vector vaccine, tattooing showed similar performance to ID injection. Tattooing of the peptide vaccine with CpG motifs adjuvant showed better response than IM vaccination with adjuvant.
DNA tattooing was evaluated in non-human primates, which have previously shown poor DNA vaccine immunization effect, but showed remarkable enhancement of immune response by this method administering an HIV vaccine (Verstrepen et al. 2008) . In order to advance this technique to human clinical trials, a human ex vivo skin model was tested, which showed that DNA concentration was the most critical factor for effective DNA vaccination by tattooing (van den ). A human clinical trial for treating melanoma is planned (Quaak et al. 2008) . A comprehensive review on DNA tattooing can be found in one of the accompanying papers in this special volume on ID immunization (Oosterhuis et al. 2010) .
Most of the ID vaccination methods described so far involve minimally invasive needle-based methods or non-invasive jet-based methods that actively deposit vaccine within the skin. Another set of approaches involve mostly non-invasive methods that increase skin permeability to enable vaccine transport into the skin in a transiently permeabilized state. The key to success using these approaches is disruption of skin's outer layer, called stratum corneum. Although the stratum corneum is only 10-20 lm thick, it provides a highly effective barrier to the permeation of xenogens, including topically applied vaccine formulations (Scheuplein and Blank 1971) . A number of methods to increase skin permeability have been developed, largely for drug delivery applications, many of which have been tested for vaccination (Mitragotri 2005; Prausnitz and Langer 2008) .
A number of studies have demonstrated that the skin barrier can be broken by abrasion. A variety of abrasion methods including rough surfaces (Frerichs et al. 2008) , tape-stripping (Takigawa et al. 2001; Peachman et al. 2003; Inoue and Aramaki 2007; Vandermeulen et al. 2009 ), and microdermabrasion devices ) have been shown to induce adequate removal of the stratum corneum. Repeated peeling by tape (for example, Scotch Ò tape) effectively removes the stratum corneum. Application of tumor epitope peptides on tape-stripped mouse skin primed tumor-specific cytotoxic T cells in the lymph nodes and the spleen, protected mice against a subsequent challenge with the corresponding tumor cells, and also suppressed the growth of established tumors (Takigawa et al. 2001) . Skin abrasion using a razor and a toothbrush followed by application of adenoviral vectors has yielded promising results in humans (Van Kampen et al. 2005) .
Skin abrasion using an abrasive paper is perhaps the most commonly used method of disrupting the stratum corneum for immunization. For example, abrasion with emery paper, after skin hydration, has been shown to induce adequate penetration of anthrax vaccine (Matyas et al. 2004 ) and influenza virus vaccine (Guebre-Xabier et al. 2003) , among others. This has led to the development of a Skin Prep System (SPS) to provide a controlled method of stratum corneum disruption for transcutaneous immunization currently under development by Intercell (Frerichs et al. 2008) (Fig. 4a ). This technique has been shown to be effective in humans. Specifically, the skin was prepared by use of two mild strokes with the skin preparation device containing a mild abrasive affixed to a pressure-controlled device. The device was a single-use, disposable system and was discarded immediately after use. Following skin preparation, the patch containing vaccine against traveler's diarrhea (LT patch) was applied within the marked area and worn for 6 h at each vaccination, then removed and discarded by the participant. 59 LT-patch recipients were protected against moderate-to-severe diarrhea (protective efficacy of 75%) and severe diarrhea (protective efficacy of 84%). LT-patch recipients who became ill had shorter episodes of diarrhea (0.5 vs 2.1 days) with fewer loose stools than placebo . In another study, a similar technique was used to boost response against influenza vaccine. In this case, prior to application, the patch area was lightly abraded with ECG-grade emery paper on skin wetted with 10% glycerol/70% alcohol to disrupt the stratum corneum. In weeks following vaccination, hemagglutination inhibition (HAI) responses in LT immunostimulatory patch recipients showed improvement over those receiving vaccine alone (Frech et al. 2005) .
Microdermabrasion is a common cosmetic procedure that has been adapted to remove superficial skin layers by sandblasting and thereby enable selective removal of the stratum corneum barrier. This approach has been shown to increase skin permeability and thereby enable topical application of live attenuated vaccinia virus on microdermabraded skin to generate virus-specific antibodies in the blood ). As mentioned in Sect. 4.2.3, a microneedle-based abrasion method has also been successfully used for vaccination.
Ultrasound, especially at low frequencies, is very effective in permeabilizing the skin (Tezel et al. 2001) . It is now understood that acoustic cavitation, which is formation, pulsation, and collapse of gaseous bubbles under the oscillating pressure field of ultrasound, is the principal mediator for ultrasound-induced enhanced skin permeability. Several studies have shown that during ultrasound exposure, transient cavitation is predominantly induced in the coupling medium (the liquid present between the ultrasound transducer and the skin) and is primarily responsible for skin permeabilization (Tang et al. 2002; Tezel et al. 2002; Tezel and Mitragotri 2003 ). An estimated 10 bubble collapses/s/cm 2 in the form of symmetric collapses Fig. 4 Skin permeabilization methods. a Skin abrasion device, in which a sandpaper device is placed on the skin (1), scraped across the skin in a controlled fashion (2) and then a vaccine patch is applied to the abraded skin (3) (Courtesy of Intecell). b Hand-held skin electroporation device, which uses microneedles as electrodes to cause highly localized electroporation in the skin to facilitate DNA vaccine delivery into skin cells (Courtesy of Cyto Pulse Sciences). c Heat-based device for thermal ablation of the skin. The microheater array (left side of inset) is used to ablate the skin and then a vaccine patch (right side of inset) is applied to the ablated skin (Courtesy of Altea Therapeutics) (generating shock waves) or asymmetric collapses (producing microjets) near the surface of the skin are sufficient to explain the experimentally-observed skin permeability enhancements by ultrasound-induced skin permeabilization. Ultrasound has been shown to enhance the delivery of vaccines into skin (Tezel et al. 2005) . Studies performed in mice have shown that the immune response generated by ultrasonically delivered vaccine was about 10-fold greater compared with SC injection per unit dose of the vaccine that entered the skin (about 1% of the topically applied dose entered the skin) (Tezel et al. 2005) . Compared to simple topical administration, ultrasound pretreatment showed increased vaccine delivery, thereby enabling sufficient vaccine to enter the skin to activate the immune response. Furthermore, application of ultrasound resulted in activation of Langerhans cells, the reasons behind which are not clear. In another study, it was shown that application of tetanus toxoid to skin pretreated with ultrasound generated anti-tetanus toxoid IgG and neutralizing antibody titers (Dahlan et al. 2009 ). Several parameters, including concentration of co-applied sodium dodecyl sulfate and ultrasound duty cycle, impacted the magnitude of antibody titers. The authors concluded that the main mechanism of ultrasound-assisted skin immunization involved factors in addition to enhancement of skin permeability to topically applied antigen.
Electroporation involves the application of high-voltage, short-duration electric pulses to transiently disrupt lipid barriers in the body. For vaccination, electroporation has been used to increase stratum corneum permeability and thereby enable vaccine entry into the skin. Electroporation has also been used to permeabilize cells within the skin and thereby drive, for example, DNA vaccines into epidermal and dermal cells (Fig. 4b) . Electroporation has been well established as a tool for delivering molecules across the stratum corneum (Prausnitz et al. 1993) or across the cell membranes (Bilitewski et al. 2003) . Many studies have focused on the use of electroporation for DNA vaccination. This is not surprising given the long history of use of electroporation for delivery of DNA into cells in vitro. However, many electroporation studies involve insertion of electrode needles into the skin. Some studies have demonstrated the use of electroporation for topical vaccine delivery (Zhao et al. 2006) . In one study, electroporation has been found to stimulate the exodus of Langerhans cells from the skin, which is likely to have an adjuvant-like effect (Zhao et al. 2006) . In this study, the efficacy of peptide delivery was found to be comparable to that of ID injected with Freund's complete adjuvant. Further, the peptide-specific CTL response to the vaccine delivered by electroporation was equivalent to that delivered by ID injection.
Electroporation has been shown to induce an effective immune response after delivery of DNA vaccines (Peachman et al. 2003; Foldvari et al. 2006; Medi and Singh 2008; Vandermeulen et al. 2009) . For example, studies in pigs have shown the ability of electroporation to deliver HBsAg gene using a single-needle or a six-needle electrode (Babiuk et al. 2002) . Studies have demonstrated that in vivo skin electroporation may be used to increase transgene expression relative to naked DNA injection (Drabick et al. 2001) . Transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. Transfected cells were also observed in lymph nodes draining electropermeabilized sites. A HBsAg-coding plasmid was used to test skin electroporation-mediated nucleic acid vaccination in a murine model. Applications for these findings include modulation of immune responses to pathogens, allergens, and tumor-associated antigens and the modification of tolerance. In another study, in vivo electroporation has shown protection against avian influenza in nonhuman primates (Laddy et al. 2009 ). A number of human clinical trials testing vaccination enhanced by electroporation are currently under way.
Several chemicals are known to interact with the skin and disrupt the highly ordered lipid bilayer structure in the stratum corneum. This observation led to the study of chemical agents to enhance transport across skin. More than 300 chemicals have been studied for their ability to increase skin permeability (Karande et al. 2004) . Chemical permeation enhancers are relatively inexpensive and easy to formulate, they offer flexibility in their design, are simple in application and allow the freedom of self-administration to the patient. Chemical enhancers comprise a wide variety of different chemical functional groups and facilitate drug transport across the skin by a variety of complex mechanisms. They can directly exert their effect on skin structure by acting on intercellular lipids or corneocytes. Chemical enhancers can extract lipids from the skin thereby creating diffusion pathways for transdermal permeation. Alternatively, they can partition themselves into the lipid bilayers thereby disrupting the highly ordered lipid lamellae and causing their fluidization. Chemical enhancers can also significantly increase skin transport of a drug by enhancing its thermodynamic activity in the formulation (Karande et al. 2005) .
Recently, chemical enhancers have been shown to possess the ability to deliver antigens and generate immune responses. This was achieved by designing formulations that possess the ability to enhance skin permeability as well as exhibit high adjuvanticity. The rational design of such multi-functional formulations from first principles requires in-depth knowledge of interactions between chemical enhancers and skin, which exist for a very limited pool of chemicals. Hence, combinatorial libraries of chemical mixtures were screened. Studies have shown that in a randomly selected population of chemical formulations, certain binary mixtures of chemicals are far more potent in permeabilizing the skin as compared to single chemicals (Karande et al. 2004) . In vaccination studies, a third chemical was added with the goal of enhancing the ability to offer adjuvanticity. The lead chemical formulations were tested in mice using the model antigen ovalbumin. The formulations that exhibited high permeation and adjuvanticity potential in in vitro screening also induced high IgG titers in mice (Karande et al. 2009 ). In another study, penetration enhancers and immunomodulators oleic acid and retinoic acid were used to enhance transcutaneous immunization with inactivated influenza virus across tape-stripped skin (Skountzou et al. 2006) . Pretreatment of mouse skin with oleic acid elicited increased levels of influenza virus-specific binding and neutralizing antibodies to levels equivalent to those induced by intranasal immunization with inactivated influenza virus. Oleic acid and retinoic acid treatments differentially affected the pattern of cytokine production upon stimulation with influenza viral antigen and provided enhanced protection.
Thermal poration of skin has been used to deliver vaccines into skin. Microporation systems are designed to porate the skin and are being developed by a number of companies. In this method, an array of micropores is created in the skin by removal of stratum corneum by the application of focused thermal energy based on resistive heating via the contact of electrically heated small-diameter wires to the skin surface (Bramson et al. 2003) (Fig. 4c ) or other methods based on radiofrequency or laser-based approaches. In this study, the microporation tip was comprised of a set of 80 lm diameter tungsten wires with control circuitry allowing for precise control of the electrical current pulses that were passed through each wire. The software user interface was designed to enable the control of various microporation parameters including micropore density, resistive element temperature, current pulse width, number of pulses, pulses pacing, and contact pressure. The temperature of the tip that was placed in contact with the skin was calibrated by an optical calibrator device. The study showed that microporation significantly increased the penetration of topically delivered vaccine. Microporation enhanced expression of luciferase upon placement of adenovirus vectors by 100-300-fold. The same procedure led to increased CTL response and increased IFN-c secreting cells. In a related study, the same technology has been shown to deliver influenza vaccine into mouse skin. Eighty micropores were created in 1 cm 2 area and the vaccine was placed on the porated skin. This procedure generated adequate protective response in mice (Garg et al. 2007 ).
ID vaccination offers potential immunologic advantages to public health. The skin is known to be a site rich in antigen-presenting cells, some of which are specific to the skin, including epidermal Langerhans cells and dermal dendritic cells (Glenn and Kenney 2006) . In addition, antigen may be taken up directly by lymphatic vessels for transport to antigen-presenting cells in the lymph nodes. At a minimum, the ID route of vaccination appears to follow different pathways to immunity compared to IM or SC routes. However, there is evidence that the ID route is not only different, but is also beneficial (Glenn and Kenney 2006; Lambert and Laurent 2008; Nicolas and Guy 2008) .
The possibility of dose sparing enabled by ID vaccination has been suggested by previous preclinical and clinical studies; however, the successful application of this approach has yet to be definitively confirmed for many vaccines (Glenn and Kenney 2006; Lambert and Laurent 2008; Nicolas and Guy 2008) . Although the conclusions vary between different studies and different vaccines, there is an indication that dose sparing may be possible. However, it is not currently clear under what conditions the skin's unique immune environment can be harnessed for optimal effect. In addition to dose sparing, there is preclinical study evidence of other beneficial differences of ID vaccination. Studies with microneedles showed improved influenza virus clearance from the lungs and enhanced memory responses compared to IM vaccination (Kim et al. 2011) . Studies with EPI showed a specific role for Langerhans cells to generate robust antibody responses . Studies with ultrasound-mediated vaccination suggested an adjuvant effect on the skin (Dahlan et al. 2009 ).
ID vaccination offers potential value to public health also in terms of possible logistical advantages. For comparison, IM and SC vaccination can only be carried out by hypodermic needle injection with few other options beside jet injection. ID vaccination opens the door to many other technologies because the skin is readily accessible at the surface of the body. As a result, ID injection may enable vaccination methods that generate no biohazardous sharp waste, can be administered by personnel with minimal training, and simplify transportation and storage logistics (Table 1) .
Mantoux technique injection requires specialized training by clinical personnel. Microneedle systems and patch-based delivery (accompanied by skin permeabilization technologies) offer the promise of simplified vaccination methods that require minimal training and may permit self-vaccination by patients in certain scenarios. This not only benefits routine vaccination scenarios, but is especially important to mass vaccination campaigns associated with disease eradication programs or pandemic emergencies. In contrast, some of the novel ID delivery methods, such as projectile delivery and tattoo guns, introduce new, sophisticated devices that require additional training of clinical personnel. Assuming the injection is done properly, the Mantoux technique can administer essentially all of the vaccine into the skin. Hollow microneedles and projectile delivery can be similarly efficient. However, solid microneedles typically retain some vaccine on the device and patch-based skin permeabilization methods are extremely inefficient, such that most vaccine typically remains on the skin surface. The efficiency of vaccine utilization will be of critical importance for new, costly vaccines, as well as in developing countries where vaccine cost can be a significant barrier to access.
Eliminating the hypodermic needle from vaccination is a major objective of public health, given that close to one million people die each year from disease transmission from contaminated needles (Miller and Pisani 1999; Kermode 2004) . Microneedles are a step in the right direction, but still generate biohazardous sharp waste, with the exception of dissolving microneedles. Projectile delivery and patchbased methods eliminate needles and therefore offer an improved safety profile. Thermal ablation +++ + +++ + + ++ a +++ requires little or no personnel training, ++ requires personnel training, + requires personnel training and maintenance of a dedicated device b +++ almost 100% in skin, ++ [50% in skin, + \50% in skin c +++ no biohazardous sharp waste, ++ microscopic biohazardous sharp waste, + macroscopic biohazardous sharp waste d +++ in widespread clinical practice, ++ published vaccination data in humans, + preclinical e +++ no reformulation required, ++ possible new liquid formulation required, + reformulation required to produce solid-state vaccine f +++ inexpensive disposable device, ++ specialty disposable device, + reusable device. Perinjection cost of reusable devices will depend on the number of times the device can be used and the cost of any disposable components However, some ID delivery methods can cause added tissue trauma to the skin (Bremseth and Pass 2001) . Most of the new methods of ID vaccination require significant technology development. While jet injection is already in widespread clinical use, many other technologies are only in the preclinical stage of development for vaccination. That being said, many of those technologies are in much later stage of development or use for non-vaccine applications, which will facilitate their adaptation to ID vaccination. Most of the new ID vaccination technologies also require vaccine reformulation. Hollow microneedle, jet and tattoo-based methods may use standard, currently available liquid formulation, but in some cases will need to be concentrated or otherwise modified. The other methods mostly use a solid-state vaccine formulation, which offers likely advantages in terms of vaccine stability during storage, but, however, requires significant reformulation, with associated research, regulatory, and manufacturing hurdles. Finally, device cost is a significant consideration, given that a hypodermic needle and syringe are extremely inexpensive, disposable devices. Microneedle systems and some of the patch-based methods are expected to have low manufacturing cost in mass production. However, many of the other technologies require multiple device components, which may be engineered into disposable devices with added cost or reusable devices with disposable components that require an initial investment that can be amortized over many patients.
ID vaccination has already made significant impact on public health as the primary means of immunization during smallpox eradication and continues to play a role in BCG and rabies vaccination in current clinical practice (Plotkin et al. 2008) . However, as discussed in this article, there are many more opportunities for ID vaccination to potentially improve immunogenicity and simplify logistics of the administration of other vaccines. A number of new ID vaccination technologies have been successful in human clinical trials. ID vaccination using the BD hollow microneedle was approved in Europe in 2009 for ID administration of the Sanofi Pasteur seasonal influenza vaccine and was introduced in Australia and New Zealand during the 2010 influenza season (Holland et al. 2008; Beran et al. 2009 ). This microneedle device may be adapted for use to administer other vaccines as well. Jet injectors have a long history of use for vaccination and are receiving renewed attention for ID delivery of vaccines in clinical trials, especially to address developing countries' needs, through support from WHO and US Centers for Disease Control and Prevention (see Sect. 3.1.2.). Skin abrasion as a pretreatment before applying a vaccine patch is also in clinical trials for prevention of influenza and traveler's diarrhea (Frech et al. 2005; Frech et al. 2008) . Projectile based delivery by EPI and PMED have been studied in a number of human clinical trials for both DNA and protein-based vaccines Jones et al. 2009 ), although it is unclear as to what extent this technology is under continued commercial development.
Other ID delivery devices are under advanced preclinical study. Solid coated microneedles have been the subject of numerous vaccination studies in mice and larger animals to administer influenza and other vaccines (see Sect. 5.2), and have been used in a Phase II clinical trial of a drug, parathyroid hormone (Cosman et al. 2009 ). Likewise, skin electroporation, in some cases in combination with microneedles, has been studied in animals for skin vaccination. As evidence for clinical feasibility, electroporation of skin for targeted delivery of chemotherapeutic agents to skin tumors is approved and used in Europe (Gehl 2008) . Tattooing is of course in widespread human use, and its application to vaccination has been studied preclinically. Other methods to increase skin permeability, such as ultrasound, chemical enhancers and heat, are also in clinical use or trials for transdermal drug delivery applications (Prausnitz and Langer 2008) , which compliment preclinical studies of their use for vaccination. Given the large number of technologies for ID vaccination under development, and the advanced clinical status of many of them, the future outlook for bringing ID vaccination into more widespread clinical practice appears encouraging. The optimal delivery method will depend on the specific application and other factors, such as immunologic response, logistical needs, and financial constraints. Recurrent Recruitment Manoeuvres Improve Lung Mechanics and Minimize Lung Injury during Mechanical Ventilation of Healthy Mice INTRODUCTION: Mechanical ventilation (MV) of mice is increasingly required in experimental studies, but the conditions that allow stable ventilation of mice over several hours have not yet been fully defined. In addition, most previous studies documented vital parameters and lung mechanics only incompletely. The aim of the present study was to establish experimental conditions that keep these parameters within their physiological range over a period of 6 h. For this purpose, we also examined the effects of frequent short recruitment manoeuvres (RM) in healthy mice. METHODS: Mice were ventilated at low tidal volume V(T) = 8 mL/kg or high tidal volume V(T) = 16 mL/kg and a positive end-expiratory pressure (PEEP) of 2 or 6 cmH(2)O. RM were performed every 5 min, 60 min or not at all. Lung mechanics were followed by the forced oscillation technique. Blood pressure (BP), electrocardiogram (ECG), heart frequency (HF), oxygen saturation and body temperature were monitored. Blood gases, neutrophil-recruitment, microvascular permeability and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) and blood serum as well as histopathology of the lung were examined. RESULTS: MV with repetitive RM every 5 min resulted in stable respiratory mechanics. Ventilation without RM worsened lung mechanics due to alveolar collapse, leading to impaired gas exchange. HF and BP were affected by anaesthesia, but not by ventilation. Microvascular permeability was highest in atelectatic lungs, whereas neutrophil-recruitment and structural changes were strongest in lungs ventilated with high tidal volume. The cytokines IL-6 and KC, but neither TNF nor IP-10, were elevated in the BAL and serum of all ventilated mice and were reduced by recurrent RM. Lung mechanics, oxygenation and pulmonary inflammation were improved by increased PEEP. CONCLUSIONS: Recurrent RM maintain lung mechanics in their physiological range during low tidal volume ventilation of healthy mice by preventing atelectasis and reduce the development of pulmonary inflammation. Mechanical ventilation (MV) of mice is increasingly used in biomedical research. While the mechanisms of ventilator-induced lung injury (VILI) have been explored intensively [1] , experimental conditions required to keep physiological parameters stable in mice during ventilation for several hours are not well defined. Major reasons for this are the focus on the mechanisms of VILI and the lack of comprehensive monitoring of pulmonary and cardiovascular key parameters in most studies.
Monitoring of key physiological parameters is standard during mechanical ventilation of humans and should be aimed also in experimental research. These key parameters need to reflect both the pulmonary (e.g. tidal volume, airway pressure) and the cardiovascular (e.g. heart rate, blood pressure) consequences of MV as well as oxygenation and acid-base status. Although MV may affect all these parameters, these entities have rarely been assessed together in the same study in mice (Table 1) . Table 1 summarizes ventilation studies, in which either lung impedance was measured or ventilation was performed for at least four hours. The table reveals that many studies that have focused on lung mechanics examined only a relatively short period of ventilation [2] [3] [4] [5] and only one study fulfilled both inclusion criteria [6] . Interestingly, studies in which mice were ventilated for more than three hours often provided cardiovascular parameters, but neglected the examination of lung functions [7] [8] [9] [10] . Some studies even completely lacked physiological parameters, although in several cases the authors referred to preliminary experiments that were not included in the published data [11] [12] [13] [14] . We believe that, without comprehensive information on physiological parameters, it is difficult to properly assess, standardize and compare the various ventilation strategies.
Studies on the mechanisms of VILI have identified several beneficial ventilation strategies, among them low tidal volume (V T ) ventilation and application of recruitment manoeuvres (RM) as well as high positive end-expiratory pressure (PEEP). Although RM have a sound physiological basis, it remains unclear how they should be applied [15] . In principal, RM may be used to reopen atelectatic lung areas in injured lungs or to prevent atelectasis in healthy lungs. The latter application requires lower recruitment pressures and hence can be applied more frequently. Without RM, pulmonary compliance is likely to decrease as shown already many years ago in anesthetized dogs both during spontaneous breathing and during mechanical ventilation [16] . In addition, regular short recruitment manoeuvres have proved to be useful in models of isolated rat and mouse lungs [17, 18] . In mechanically ventilated mice the usefulness of RM with the specific aim to keep pulmonary compliance and other lung functions constant has been explored only sporadically. Previous RM studies in healthy mice have focused on short periods of ventilation and potential lung injury, but did not compare ventilation with and without RM over several hours [4, 5] . In addition, the effect of RM on blood pressure in mice is not well defined, although increased intrathoracic pressure might decrease cardiac output [19] . The beneficial effects of PEEP have been demonstrated in several animal studies [20] [21] [22] . PEEP helps to prevent repetitive alveolar collapse and preserves surfactant function [1, 23] . While it is widely accepted that adequate PEEP together with low V T is a protective ventilation strategy, the effects of recurrent RM in this setting are unknown.
The present study had several aims: (1) In order to assess the consequences of MV properly, we established a set-up that permits the ventilation of mice under permanent monitoring of clinically relevant physiological parameters. (2) We used this set of parameters to define ventilatory conditions that guarantee stable lung mechanics, hemodynamics, acid base status and oxygenation over six hours. In particular, we studied the input impedance of the lung at low frequencies to distinguish mechanical properties of conductive airways and the distal lung [24] . (3) We investigated the physiological effects of recurrent RM on lung mechanics at two different tidal volumes (8 vs. 16 mL/kg) and at two different PEEPlevels (2 vs. 6 cmH 2 O). (4) Since inflammatory lung injury may occur even with 'non-injurious' ventilation [7, 8, 25] , we also assessed the extent of pulmonary inflammation, i.e. microvascular permeability, pulmonary sequestration of leukocytes, production of proinflammatory cytokines and lung histopathology. Our findings in healthy animals show that low V T ventilation will only maintain lung functions and gas exchange in a normal range if recurrent RM are used. In addition, recurrent RM reduced the extent of pulmonary inflammation, although mild inflammation was present in the lungs of all ventilated animals. The higher PEEP of 6 cmH 2 O was beneficial regarding lung mechanics, oxygenation and pulmonary inflammation. Thus, we suggest that a preferable ventilation strategy is one that combines low tidal volumes with sizable PEEP and recurrent recruitment manoeuvres.
Experiments were performed with female C57BL/6 N mice (Charles River, Sulzfeld, Germany) aged 8 to 12 weeks, weighing 20 
Mice were initially anaesthetized with an intraperitoneal injection of pentobarbital sodium [75 mg/kg] and fentanyl [40 mg/kg]. Anaesthesia was maintained with pentobarbital sodium [20 mg/kg] via an intraperitoneal catheter every 30 to 60 minutes. Mice were tracheotomized with a 20-gauge cannula and connected to the ventilator. A catheter was inserted into the carotid artery, which allowed blood pressure monitoring and permanent infusion of 0.9% NaCl (200 mL/h) to prevent hypovolaemia and thrombus formation. Pulsoxymetry was performed with a tail clip (MouseOx, STARR Life-Science, Oakmont, PA, USA). Blood pressure and ECG were recorded permanently (PowerLab, ADInstruments, Spenbach, Germany). Heart rate was calculated from the ECG. Body temperature was measured rectally and kept stable between 36.5uC and 37.5uC by a homeothermic blanket (Harvard Apparatus Holliston, MA, USA).
Mice were ventilated for six hours with the flexiVent ventilator (SCIREQ, Montreal, Canada). All mice survived the protocol. Mice were either ventilated with low tidal volume (lowV T ) of Figure 2 . Heart rate and blood pressure. Electrocardiogram (ECG) was recorded permanently. A. Heart frequency (HF) was calculated simultaneously from the ECG and is displayed in beats per minute (bpm). B. Mean arterial blood pressure (BP) was measured via a catheter in the carotid artery.(LowV T RM5 n = 6, highV T RM5 n = 6, lowV T noRM n = 5, highV T noRM n = 4, lowV T RM60 n = 5). doi:10.1371/journal.pone.0024527.g002 Figure 3 . Blood gas results. Arterial blood was analysed after six hours of ventilation. A. pO 2 /FiO 2 ratio was calculated, FiO 2 was 0.5. B. Comparison of pCO 2 levels. (LowV T RM5 n = 6, highV T RM5 n = 6, lowV T noRM n = 5, highV T noRM n = 4, lowV T RM60 n = 5). * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0024527.g003 8 mL/kg and a frequency of 180 min 21 or high tidal volume (highV T ) of 16 mL/kg and a frequency of 90 min 21 , so that both groups received the same minute volume. The highV T group was ventilated with 3% CO 2 to maintain normocapnia without further decreasing ventilation frequency. The fraction of inspired oxygen (FiO 2 ) was 0.5 in all experiments. A positive end-expiratory pressure (PEEP) of either 2 cmH 2 O or 6 cmH 2 O was applied. One recruitment manoeuvre (RM) with 30 cmH 2 O pressure and six seconds duration was performed after onset of ventilation to open airspaces and standardize lung volume. Resistance, compliance and impedance of the lung were measured by the lowfrequency forced oscillation technique every ten minutes.
Two different series of experiments were performed and analyzed separately (summarized in Table 2 ). The first series of experiments ( Fig. 1-2345678 ) was performed with a PEEP of 2 cmH 2 O. Repeated recruitment manoeuvres of 1 s duration and 30 cmH 2 O peak pressure were applied every five minutes (RM5) in one lowV T (lowV T RM5) and one highV T group (highV T RM5) or every 60 minutes (RM60) in one lowV T group (lowV T RM60). One lowV T and one highV T group were ventilated without RM (lowV T noRM and highV T noRM). One group of anaesthetized but not ventilated mice served as control. A second series of experiments ( Fig. 9-101112 ) was performed, in which mice were ventilated with a PEEP of 6 cmH 2 O and low V T . RM were performed every five minutes (PEEP6_RM5), every 60 minutes (PEEP6_RM60) or not at all (PEEP6_noRM).
Mice received 1 mg bovine serum albumin (BSA) intravenously 90 min before exsanguination for analysis of microvascular permeability. Mice were sacrificed by exsanguination via the carotid artery. Blood samples from ventilated mice were analysed for pO 2 , pCO 2 , pH, HCO 3 2 and standard base excess (SBE) by blood gas analysis (ABL700, Radiometer, Copenhagen, Denmark). Blood gas analysis from anaesthetized control mice was not representative due to reduced breathing activity.
Right lung: lung permeability, cytokine detection, differential cell count After thoracotomy lungs were perfused free of blood with ice cold phosphate buffered saline. Bronchoalveolar lavage of the right lung was performed by instilling two times 300 mL NaCl via tracheal tubing. About 500 mL BAL fluid was retrieved from each mouse. BAL fluid was centrifuged and the supernatant was frozen for quantification of proteins. The pellet was transferred to a cytospin preparation, followed by a modified Giemsa stain (Diff-Quick; Medion Diagnostics, Düdingen, CH) and differential leukocyte count. Cytokine levels in serum and BAL fluid were quantified with commercial enzyme-linked immunosorbent assays (ELISA) (R+D Systems, Abingdon; UK). BSA was quantified in blood serum and BAL fluid by ELISA (Bethyl Laboratories, Montgomery, USA). The ratio of BSA in serum and BAL fluid was calculated to determine microvascular permeability. Additionally, total protein levels were measured using a DC protein assay (BioRad, Hercules, CA, USA).
The left lung was filled with 4% formalin for fixation and embedded in paraffin for histopathological examination. Sections of 3 mm thickness were stained with hematoxilin and eosin (HE). Histopathology was evaluated in a blinded manner. The following scoring system based on four criteria was used for each histological section: neutrophils in the alveolar or interstitial space, alveolar septal thickening, alveolar congestion and formation of hyaline membranes. Each criterion scored one point, if present, thus scores ranged from 0 (none criterion found) to 4 (all four criteria observed). Nine to ten sections per lung were evaluated and the mean was calculated for every lung and every group.
Lung mechanics were analysed with the mixed model procedure followed by correction for false discovery rate (FDR). To evaluate lung mechanics from the groups lowV T RM60 and PEEP6_RM60 in a linear model, one time point every 60 minutes before and one after RM were analyzed separately, resulting in two separate linear slopes (lowV T RM60a and PEEP6_RM60a: before RM, lowV T RM60b and PEEP6_RM60b: after RM). BoxCox transformation was performed to achieve homoscedasticity and normal distribution, when necessary. Analyses of parametric data were carried out with One-Way Analysis of Variance and FDR correction. Non-parametric data were analysed with the Kruskall-Wallis test followed by Dunn's post-test. Data in figures are shown as mean 6 standard error of the mean (SEM). P-values,0.05 were considered as significant. Statistical analyses were carried out with JMP 7 or SAS 9.1 software (SAS Institute Inc., Cary, NC, USA).
A. First series of experiments: PEEP = 2 cmH 2 O Lung mechanics. The first series of experiments was performed at a PEEP = 2 cmH 2 O, a PEEP level that is commonly used in animal studies that aim to study VILI (see Table 1 ). Ventilation with 8 mL/kg and no RM (lowV T noRM) caused a continuous decrease of pulmonary compliance (C) over more than two hours that reached a plateau at approximately 30% of the physiological baseline value (Fig. 1 ). Within this time pulmonary resistance (R) nearly tripled. Resistance and compliance showed less dramatic trends in mice ventilated with 16 mL/kg and no RM (highV T noRM). In this group C plateaued earlier (60 min) at about 60% of the baseline value and R increased to a lesser degree (,25%). R and C differed significantly between the lowV T noRM and the highV T noRM group (R: p,0.01; C: p,0.001). Respiratory input impedance revealed major alterations in the periphery of the lung. Tissue damping (G) and tissue elastance (H) were elevated in both highV T noRM and lowV T noRM mice, reaching significantly higher values in the lowV T noRM group (G: p,0.01; H: p,0.001). In the latter group, airway resistance (R aw ) was increased as well. The increase in G, H and R aw indicates that low V T ventilation without RM leads to closure of peripheral areas of the lung and causes a small degree of airway constriction. In consequence, hysteresivity (G/H = g) decreased slightly over time, but was in a comparable range in all groups.
These findings indicate that ventilation without RM leads to impaired lung functions, which stabilize within two hours, though at a low level. We therefore examined the effect of repetitive RM (30 cmH 2 O) every 5 min. In both the lowV T RM5 and the highV T RM5 group, lung mechanics stayed in a physiological range and remained unchanged during the whole experiment. Depending on the different tidal volumes, C and H differed reciprocally between low-and highV T groups (p,0.05). The stability of the lung mechanics demonstrates that application of deep inflations of 1 s duration and 30 cmH 2 O are sufficient to prevent the deterioration of lung functions in healthy mouse lungs ventilated with 2 cmH 2 O PEEP and moderate tidal volumes.
Further, we examined whether it would suffice to apply RM only every 60 min instead of every 5 min. This was studied in the low V T group (lowV T RM60) only. In this group, respiratory conditions were instable. R and C worsened comparable to the lowV T noRM group, but improved significantly after each RM. These alterations were reflected in changes in G and H. However, even though each RM was beneficial, after six hours tissue elastance had increased permanently, indicating that one deep inflation per hour is not sufficient to maintain lung volume at base line values. This is illustrated by the finding that H (p,0.001), R, C, G (all p,0.01) and R aw (p,0.05) were significantly different between the lowV T RM60 and the lowV T RM5 group.
Heart frequency, blood pressure and oxygenation. Anaesthesia with pentobarbital sodium resulted in reduced heart frequency (300-400 min 21 ) and mean blood pressure (50-60 mmHg), compared to average data of unsedated mice. ECG (data not shown), HF and BP remained unchanged throughout ventilation and were not significantly different between the groups (Fig. 2 ). This demonstrates that ventilation and recruitment strategy did not affect the circulation. The fluid support with 200L NaCl per hour in all groups was adequate to keep blood pressure stable. Oxygen saturation, measured by pulsoxymetry, was over 90% in all mice at all times (data not shown).
Blood gas analysis. Blood gas analyses revealed that infusion of saline and application of 3% CO 2 in the high V T groups were adequate to keep the acid-base status stable and that ventilation resulted in normocapnia in the lowV T RM5 and highV T RM5 group, with pCO 2 levels ranging from 35 to 40 mmHg ( Table 3) . The pO 2 /FiO 2 ratio was between 530 and 620 mmHg in both RM5 groups and in the highV T noRM group (Figure 3 ). Most mice ventilated with highV T noRM had physiological pCO 2 levels after six hours. The pO 2 /FiO 2 ratio was decreased significantly to around 200 mmHg in the lowV T noRM group, indicating acute respiratory failure. In this group, impaired gas exchange was further indicated by an increase in pCO 2 levels to about 60 mmHg, leading to respiratory acidosis with a mean pH of 7.22. Mice in the lowV T RM60 group showed very heterogeneous pO 2 /FiO 2 ratios between 260 and 570 mmHg and markedly increased pCO 2 levels as well as initiating hypercapnic acidosis. Bicarbonate (HCO 3 2 ) was only slightly reduced, resulting in negative values for standard base excess, but did not indicate metabolic imbalance.
Pulmonary microvascular permeability. Total protein levels were increased in all ventilated mice, compared to unventilated controls. Protein levels were highest in the groups Figure 5 . Cytokine levels. Cytokines were quantified in blood serum or BAL supernatant with commercial ELISA kits after six hours of ventilation and in unventilated mice. (Unventilated: n = 5, lowV T RM5 n = 6, highV T RM5 n = 6, lowV T noRM n = 4 in BAL and n = 5 in serum, highV T noRM n = 4, lowV T RM60 n = 5). * p,0.05, ** p,0.01, *** p,0.001, 1 *** p,0.001 versus all other groups). doi:10.1371/journal.pone.0024527.g005 without RM (Fig. 4A) . The BSA BAL/serum ratio confirmed that ventilation augmented microvascular permeability in the lung, particularly when inspiratory pressures were high, as in the groups ventilated without repetitive recruitment (Fig. 4B) .
Pro-inflammatory cytokine levels. Interleukin-6 (IL-6) and Keratinocyte-derived chemokine (KC) were elevated in the BAL fluid and blood serum from all ventilated mice (Fig. 5A, B, E, F) . BAL fluid from mice ventilated with lowV T RM5 showed a less dramatic increase in these pro-inflammatory cytokines. In both RM5 groups serum levels of IL-6 and KC were clearly lower than in all other ventilation groups. Interestingly, Interferon-c induced protein (IP-10) and Tumour necrosis factor-a (TNF-a) were not significantly increased in the BAL from ventilated mice, compared to unventilated controls (Fig. 5C, D) .
Differential cell count in the BAL fluid. The only cell types found in BAL fluid of unventilated control mice were monocytes and macrophages. In contrast, all samples from ventilated mice also contained neutrophils (Fig. 6) . Numbers of neutrophils were highest in mice ventilated with high V T . Considerably less neutrophils were recruited to alveoli of lowV T mice, whereof the group lowV T RM5 had the lowest neutrophil count. Histopathology. Histopathological samples revealed that no severe lung injury was induced by the ventilation strategies applied (Fig. 7) . Nonetheless, significant histopathological alterations were present in all ventilated lungs and were most obvious in mice ventilated with high V T (Fig. 8) . Alveolar septal thickening was observed in most of the ventilated lungs, resulting in a score of one. A score of two was given, when neutrophils were observed additionally, as in most lungs from highV T mice. This is in line with the results from the differential cell count in the BAL fluid. Only some highV T noRM mice showed alveolar congestion, bringing the lung injury score up to three. Hyaline membranes were not observed in any sections, indicating that the applied ventilation strategies induced only moderate injury in the healthy lungs.
Lung mechanics. In the second series of experiments the ventilation strategies RM5, noRM and RM60 were applied to mice ventilated with low V T and a PEEP of 6cmH 2 O, to find out whether the impairment of lung mechanics during ventilation with noRM and RM60 could be prevented by a higher PEEP (Fig. 9 ). Ventilation with RM5 at a PEEP of 6 cmH 2 O (PEEP6_RM5) showed stable lung functions. Ventilation without RM (PEEP6_noRM) resulted, notwithstanding the increased PEEP, in a strong decrease in C during the first 180 min of ventilation until a plateau was reached at about 30% of the initial C. Correspondingly R, G and H increased and finally doubled in this group. Also ventilation with RM60 (PEEP6_RM60) did not suffice to keep lung functions entirely stable at a PEEP of 6 cmH 2 O. Although the decrease in C between the RMs was relatively moderate, the initial C was not maintained over six hours. R, in contrast, returned to the initial value after each RM. In line with this, lung impedance measurements revealed a moderate increase in H, but not in G after six hours. All three groups differed significantly from each other regarding C, R, H and G, except from the parameters measured in the PEEP6_RM60 group directly after the RM. R aw and hysteresivity were not different between the groups and R aw remained at baseline in all experiments (data not shown).
These results indicate that an elevation of PEEP, although it helps to stabilize lung mechanics, is not sufficient to replace repetitive RM.
Blood gas analysis. One aim of the second series of experiments was to investigate whether a higher PEEP can prevent the worsening of gas exchange in mice ventilated at a PEEP = 2 cmH 2 O and RM60 or noRM. The pO 2 /FiO 2 ratio was around 500 mmHg in the PEEP6_RM60 group and around 400 mmHg in the PEEP6_noRM group (Fig. 10) . Both groups differed significantly from the control group PEEP6_RM5, which showed unimpaired gas exchange with a pO 2 /FiO 2 ratio of about 600 mmHg This further demonstrates that regular RMs are necessary even with a PEEP of 6 cmH 2 O. In accordance, pCO 2 levels were significantly elevated in the groups PEEP6_RM60 and PEEP6_noRM, compared to the control group (data not shown).
Pro-inflammatory cytokine levels. In the second set of experiments IL-6 and KC levels in the BAL fluid were highest in the PEEP6_noRM group, followed by the PEEP6_RM5 group (Fig. 11A, B) . Both mediators were lowest in the PEEP6_noRM group. IL-6 in the blood serum was not significantly different between these three groups (Fig. 11C) . KC levels were highest in the group ventilated without RM, but differed only slightly between the groups ventilated with RM5 and RM60 Fig. 11D ).
These results indicate that a higher PEEP helps preventing the release of pro-inflammatory mediators induced by formation of atelectasis.
Differential cell count in the BAL fluid. In the second part of the study, in which all mice were ventilated with low V T , neutrophil numbers were clearly highest in the group PEEP = 6_noRM. Numbers of neutrophils were lower in the BAL fluid from PEEP6_RM5 mice and lowest in the PEEP6_RM60 group (Fig. 12 ).
There is an increasing demand for mechanical ventilation of mice, not only in pulmonary research, but also in many other areas such as neurology [26] , imaging [27] or cardiovascular research [28] . Monitoring of pulmonary and vital parameters is an essential prerequisite for correct interpretation of data from such models. The present study indicates that the general notion according to which low tidal volumes and sizable PEEP levels are preferable is -at least in healthy mice -only true when repetitive recruitment manoeuvres are applied.
The effects of repetitive RM are not well established neither in experimental animals nor in men, one reason being that trials are difficult to compare [19] . To date, application of RM in patients with healthy lungs has been explored only sporadically [29] , although there is some evidence that RM may support protective ventilation by improving oxygenation and lung mechanics, thereby allowing reduction of V T [30, 31] . Clinically, it has been shown that low V T ventilation improves the outcome of patients with injured lungs [32] , and also reduces inflammation in those with healthy lungs [33] . Taken together, it seems highly important to gain more information on the effects of repetitive 'sigh-like' recruitment manoeuvres during low V T ventilation.
We demonstrated that application of deep inflations of 1 s duration and 30 cmH 2 O peak pressure in five minute intervals was sufficient to keep respiratory mechanics stable and thereby lung volume constant in low and high V T ventilation, without increasing PEEP over 2 cmH 2 O (Fig. 1) . We initially selected a relatively low PEEP level, because we plan to use the same PEEP in future studies on the molecular mechanisms of VILI, where high PEEP levels are known to be protective (many VILI models use no PEEP at all). Since RMs can be performed with varying duration, peak pressure and end-expiratory pressure [34] , we chose a very short type of RM, which we considered to be noninjurious to the lung and not to impair cardiac output. Accordingly, blood pressure and heart frequency were stable, regardless of the ventilation strategy. Although blood pressure was in a low range due to anaesthesia with pentobarbital, so that small changes might have been masked, this did not result in reflex tachycardia or impairment of microcirculation, as the pH values indicate (Table 3) . These stable cardiovascular conditions were also guaranteed by several supportive measures such as constant fluid support (200 mL/h), regular administration of pentobarbital via an intraperitoneal catheter, and an automated system to keep the body temperature constant. Further, it can be speculated that the low blood pressure is protective in this model, since edema formation is generally aggravated by high blood pressure.
In order to keep blood gases stable, the low and high V T groups had to be ventilated with different respiratory rates and, based on other studies [35] , we cannot completely rule out that this might have affected the outcome of our study. Our conditions were chosen to provide the same minute volume at both tidal volumes. The supplementation of 3% CO 2 to the inhaled gas mixture in the high V T groups was adequate to prevent hypocapnia, which otherwise would have occurred at a respiratory rate of 90 min 21 in the highV T RM5 group. 3% CO 2 alone did not induce hypercapnia (see Table 3 ). Thus, a protective effect of hypercapnia during MV, which is described in several studies [36, 37] , seems unlikely.
A pressure of 30 cmH 2 O for each RM was necessary to maintain lung volume stable. In accordance with previous studies [4, 5] RM with 25 cmH 2 O peak pressure were not sufficient to avoid a loss of lung volume (data not shown). The double sigmoidal nature of the murine pressure volume curve [38] may explain the positive response to RM with 30 cmH 2 O pressure. According to Zosky et al. [38] , pressures above 20 cmH 2 O induce fundamental changes in the murine lung, resulting in increased compliance. These changes are proposed to be either due to reopening of collapsed alveoli or to a secondary population of alveoli that are present but not aerated below a critical transrespiratory pressure [39] . In the present study, measurement of lung input impedance revealed that the alterations in pulmonary resistance and compliance in RM60 and noRM mice were predominantly due to changes in the periphery of the lung. Although we observed significant differences in R aw between RM5, RM60 and noRM mice ventilated at the same V T , this effect was rather small, indicating that bronchoconstriction of large airways is of minor importance to explain our findings. The strong increase in G and H in mice ventilated without RM demonstrates a progressive loss in lung volume [24] . Increases in tissue damping reflect energy dissipation and are associated with an increase in tissue resistance and/or regional heterogeneity as result of peripheral airway constriction, whereas tissue elastance reflects energy storage in lung tissues and represents lung stiffness [5] . The small decrease in hysteresivity shows that H increased slightly stronger than G. Further, it underlines that impaired lung functions were not primarily due to heterogeneity caused by narrowing of small airways [40] . Thus narrowing of conductive airways played only a minor role in this model and we conclude that the loss of lung volume was mainly due to formation of atelectasis.
In fact, lung volume declined only to a certain threshold and a plateau phase was reached in all mice ventilated without RM. This supports the hypothesis that depending on the pressure applied to the lung some populations of alveoli are open and others closed [38] . The minimal lung volumes in the plateau phase differed according to the employed tidal volume, leading to the highest tissue resistance and elastance values in the lowV T noRM group. Formation of atelectasis was more severe in low V T than in high V T ventilation, as shown elsewhere [3, 4] , indicating that low V T ventilation without RM was injurious. These findings are in line with a previous work showing that low V T ventilation augmented lung injury in isolated rat lungs, proposing that the degree of lung injury is dependent on the end-expiratory lung volume [41] . Ventilation with high V T resulted in a higher compliance and thereby to some degree protected from the decline in lung volume. However, the application of one RM per hour was not sufficient to keep tissue resistance and elastance entirely stable over six hours, demonstrating that the beneficial effect of recruitment is transient and formation of atelectasis is reversible only to a certain point. The present data emphasize the importance of frequent deep inflations in mice in order to maintain lung functions in a physiological range.
Further evidence for the formation of atelectasis was given by blood gas analysis, which revealed impaired gas exchange in lowV T noRM and lowV T RM60 mice (Fig. 3 , Table 3 ). The present study demonstrates that impairment of gas exchange, to a level that would fulfil the clinical definition for ARDS [42] , can be caused by a ventilation strategy that leads to derecruitment of lung volume. On the other hand, our data on lung histopathology, neutrophil count and BAL levels of pro-inflammatory cytokines indicate that inflammatory lung injury was not severe even in the lowV T noRM group. Notably, neutrophil counts and histopathological alterations ( Fig. 6-8 ) did not correlate well with gas exchange and lung functions, further supporting our conclusion that atelectasis rather than inflammation was the major reason for the impaired gas exchange in the groups ventilated without sufficient RM. In fact, neutrophil infiltration was highest in the high V T groups, suggesting ventilation-induced inflammation (biotrauma) as a mechanism.
Nonetheless, protein leakage, indicating increased microvascular permeability, another hallmark of VILI, was highest in those low V T groups that developed atelectasis due to lacking adequate RM (noRM, RM60) (Fig. 4) . Collapse of lung units is accompanied by an increase in vascular filtration pressure, leading to edema formation, when vascular barrier functions are impaired [43] . This is in line with the observed alveolar septal thickening. Furthermore, microvascular leakage may also interfere with pulmonary surfactant, thus contributing to a reduction of compliance [44] , which was clearly present in this model. Finally, in atelectatic lungs shear forces between closed and open alveoli may develop [1] . Taken together we conclude that a lack of recruitment manoeuvres may cause what has been termed atelectotrauma.
The present study demonstrates that repetitive recruitment manoeuvres are not only beneficial for lung functions, gas exchange and barrier function, but also reduce the liberation of pro-inflammatory cytokines (Fig. 5) . This is an important finding, because in theory frequent deep inspirations might be a trigger for stretch-induced pro-inflammatory mediator release. IL-6 and KC were increased in the BAL and blood serum of all ventilated mice and it appears that ventilation will always cause mild inflammation in the lungs, both experimentally [7, 8, 25] and clinically [45, 46] . The underlying mechanisms may be related to the surgery or mechanotransductive processes, i.e. stretchactivation of signalling cascades, or to increased vascular shear stress in those groups ventilated with higher positive pressures [47] . IL-6 and KC were significantly lower in the BAL fluid of lowV T RM5 mice compared to all other groups, demonstrating that this ventilation strategy was the most protective one. It is well-established that protective ventilation, minimizing overdistension and recruitment/derecruitment of the lung, attenuates cytokine liberation in the lung and circulation [48] . Interestingly, the blood serum of both V T groups ventilated with RM5 contained significantly lower levels of IL-6 and KC than the corresponding groups ventilated with RM60 or noRM. This shows that the RM5 procedure was also protective at higher tidal volumes. The high levels of IL-6 and KC in the BAL of highV T RM5 mice indicate that the inflammatory response was initiated in the lung. Notably, neither levels of IP-10 nor of TNFa -two cytokines that are frequently found in severe forms of acute lung injury -were altered significantly in ventilated mice, compared to unventilated controls. The relatively high baseline values for TNF-a in unventilated mice are most likely due to the fact that BAL samples had to be diluted on order to gain enough volume for the assay. Concentrations were calculated by multiplication with the dilution factor, possibly leading to higher concentrations than actually present in the BAL. Nevertheless, all groups were analyzed at the same time and cytokine levels were in the same range in all groups, allowing a comparison. IP-10 is proposed to play a role in development of ARDS and is further considered as a useful biomarker for lung disease [49, 50] . TNF-a is considered as a mediator of pulmonary inflammation released by injurious ventilation [51, 52] . The finding that these mediators remained unaltered by ventilation further suggests that lung injury was only moderate in the current model. This study aimed to define settings for ventilation under stable conditions and therefore a second set of experiments was performed with a PEEP of 6 cmH 2 O, to address the question whether a higher PEEP could prevent the severe impairment of lung functions and the effects on oxygenation and inflammation, which were observed in the lowV T noRM and lowV T RM60 groups ventilated with a PEEP of 2 cmH 2 O. Lung mechanics and blood gas results revealed that a higher PEEP, even when combined with RM60, was not sufficient to prevent formation of atelectasis. C decreased and R, G and H increased strongly during the first 180 min of the experiments in the PEEP6_noRM group before parameters reached a plateau. A previous study demonstrated that an increase in PEEP from 2 cmH 2 O to 6 cmH 2 O alone did not prevent an increase in R aw , G and H during 150 min of ventilation, but was more effective during pressure controlled RM [5] . The present study shows that a combination of higher PEEP and short RM every 60 minutes does not suffice to prevent impairment of lung functions and that frequent RM are required, as with a PEEP of 2 cmH 2 O. Of note, the increase in R aw , which was observed in the groups lowV T noRM and lowV T RM60 ventilated with a PEEP = 2 cmH 2 O, was completely abolished in all groups ventilated with a PEEP = 6 cmH 2 O, indicating that a higher PEEP helps to prevent narrowing of large airways during MV.
It is well known that PEEP plays a critical role in preventing/ reducing lung injury [23] . The combination of low V T and high PEEP has already proved to be beneficial for the outcome of patients with ARDS [53] . Further, an experimental ventilation strategy using low V T , PEEP and RM resulted in a better outcome in ARDS patient than a strategy without RM, although differences were not significant due to limitations of the trial [54] . Given the significance of biotrauma [55, 56] , it is an important question whether a ventilation strategy that combines high PEEP with RM attenuates pulmonary inflammation during low V T . The second part of this work showed that differences in cytokine levels between mice ventilated with the same RM strategies were smaller with 6 cmH 2 O than with 2 cmH 2 O PEEP. IL-6 serum levels were even in a comparable range in all PEEP6 groups. Interestingly, the highest IL-6 and KC BAL levels were detected in the PEEP6_noRM group. This correlates well with the neutrophil count that was also highest in this group. Thus, the application of RM appears to be beneficial with respect to pulmonary inflammation also at a PEEP of 6 cmH 2 O.
We compared lung mechanics during ventilation with and without RM in healthy mice over six hours during close monitoring of many clinically relevant parameters including lung mechanics, cardiovascular functions and gas exchange. Stable cardiovascular conditions resulted amongst others from appropriate fluid support, emphasizing the need for careful monitoring and stabilisation of vital parameters in animal studies.
The present study shows that protective non-injurious ventilation requires the application of frequent non-injurious recruitment manoeuvres and low V T . A ventilation pattern including deep inflations is closer to the variable breathing pattern of spontaneously breathing subjects than monotonic MV without variation in V T or breathing frequency, supporting the finding that variable or 'noisy' ventilation improves protective ventilation in the porcine and human lung [57, 58] . Beyond that, variable ventilation, characterised by breath-to-breath variation of V T and breathing frequency, has been shown to be beneficial in terms of reducing VILI in mice with injured lungs [59] .
Furthermore, our data indicate that a PEEP level of 6 cmH 2 O has protective effects and is therefore advisable in studies that require MV. A low PEEP of 2 cmH 2 O combined with recurrent RM is less protective, but still suffices to gain stable respiratory conditions and may be preferable in models that aim to investigate VILI.
We conclude that ventilation with low V T , recurrent RM and sizable PEEP is the most protective ventilation strategy for healthy mice.  Virtual High-Throughput Screening Identifies Mycophenolic Acid as a Novel RNA Capping Inhibitor The RNA guanylyltransferase (GTase) is involved in the synthesis of the (m7)Gppp-RNA cap structure found at the 5′ end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed. The synthesis and maturation of eukaryotic mRNAs are crucial events for gene expression. During mRNA synthesis, eukaryotic mRNAs undergo a series of essential modifications before being exported to the cytoplasm where they are translated into proteins [1] . These processing events include the addition of a cap structure at the 59 terminus, the splicing out of introns, the editing of specific nucleotides, and the acquisition of a poly(A) tail at the 39 terminus. The RNA cap structure found at the 59 end of mRNAs is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs [2, 3] . The cap is synthesized by a series of three enzymatic reactions [4] . The first step involves the hydrolysis of the RNA 59-triphosphate end of the nascent RNA by an RNA triphosphatase to form a diphosphate end. An RNA guanylyltransferase then catalyzes a two-step reaction in which it initially utilizes GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the GpppN structure. The guanosine residue is finally methylated by an RNA (guanine-N7)-methyltransferase to form the typical m7 GpppN cap structure.
A number of different microbial pathogens code for their own enzymes involved in the synthesis of a cap structure [5, 6, 7, 8, 9, 10] . Although the RNA cap structures originating from human and microbial enzymes are often identical, the physical organization of the genes, subunit composition, structure and catalytic mechanisms of the microbial-encoded enzymes involved in the synthesis of the RNA cap structure are often significantly different from those of host cells [2] . As a consequence these pathogenic capforming enzymes are potential targets for anti-microbial drugs.
During the past few years, both the RNA triphosphatase and the RNA (guanine-N7) methyltransferase (N7-MTase) components of the RNA capping machinery have been major targets for the development of drugs directed against RNA cap synthesis [11, 12, 13, 14, 15, 16, 17, 18, 19, 20] . Of all the enzymes involved in RNA capping, the RNA guanylyltransferase (GTase) has traditionally been considered a poor candidate as an anti-microbial target because of the high mechanistic and structural conservation of this enzyme across species [21] . Based on various crystal structures of GTases, a general mechanism for phosphoryltransfer has previously been elucidated which involves conformational changes between an open and closed form of the enzyme [22, 23] . In the first step of the reaction, GTP binds to the open form of the enzyme which promotes closure of the Nterminal nucleotidyl transferase (NT) domain and the C-terminal oligomer-binding (OB) fold domain. This closure is stabilized by interactions between the residues of the NT domain, the bound nucleotide, and residues on the OB fold domain. Domain closure is then followed by hydrolysis of the GTP substrate to produce the enzyme-GMP covalent intermediate. Hydrolysis of GTP disrupts the interactions between the bound guanylate and the C-terminal OB fold domain, thus destabilizing the closed form of the enzyme, which opens up with the concomitant release of pyrophosphate. This exposes the RNA-binding site of the enzyme, thereby allowing the subsequent transfer of the GMP moiety onto the acceptor RNA. Figure 1 summarizes the mechanistic and structural pathway used by GTases.
Recent in vitro studies have shown that foscarnet is a potent inhibitor of the GTase reaction [24] . Its mechanism of action is purported to occur through substrate binding inhibition on account of its analogous nature to pyrophosphate (PPi), a product of the GTase reaction. Ribavirin, a broad-spectrum nucleoside analogue approved for the treatment of various viral infections, is another inhibitor of the GTase activity [25] . Biochemical studies have shown that ribavirin triphosphate can actually be used as a substrate by the vaccinia virus GTase to form a covalent enzyme-ribavirin monophosphate intermediate reminiscent of the covalent enzyme-GMP intermediate [25] . Furthermore, ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure [25] . However, RNA transcripts blocked with ribavirin are not recognized efficiently by the cap-binding protein eIF4E, and are not translated into proteins [26] . The use of guanine-N7 methylation-inert cap donor molecules could potentially prove to be an interesting line of research for the development of antimicrobial drugs. However, on account of the possibility of off-targets, the risk of major side effects upon tratment with GTase substrate/ product analogs remains. Several issues related to the specificity problem faced with these inhibitors can likely be partially resolved by the development of non-nucleoside inhibitors.
In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. We demonstrate that mycophenolic acid, a compound which is currently used both in cancer and immunosuppressive chemotherapy, is an inhibitor of the GTase reaction. The biological implications of these findings for the MPA-mediated inhibition of RNA capping are discussed.
To identify potential candidate compounds that can bind to GTases, the crystal structures of various GTases were retrieved from the Protein Data Bank (Chlorella virus: 1CKN and 1CKO; Candida albicans: 1P16; S. cerevisiae: 3KYH). The chemical databases used in our virtual screening included the Sigma-Aldrich, Ambinter, ASINEX, IS Chemical technology, MolPort, and Vitas M Laboratory catalogs. Collectively, these 6 databases offered a collection of 13,800,000 small-molecule compounds.
The molecular docking program DOCK (Version 4.0) was used to perform the virtual screening [27] . Componds displaying at least 80% of structural similarity to GTP or ATP (.25,000 compounds) were screened for the binding to the four GTases. The levels of similarities were measured using the Tanimoto equation [28] and the PubChem dictionary-based binary fingerprint (http:// pubchem.ncbi.nlm.nih.gov/). Based on the binding models of these compounds predicted by DOCK, the X-SCORE program (Version 1.1) was applied to obtain an estimate of the binding affinities of these compounds [29] . The compounds were then ranked according to their binding affinities as estimated by X-SCORE.
The RNA guanylyltransferases from S. cerevisiae (Ceg1), vaccinia virus (D1R), Chlorella virus (A103R), and human (HCE) were expressed and purified as described before [24, 25, 30, 31] . The ATP-dependent ligase DNA ligase from Chlorella virus (ChVLig) was also expressed and purified as described previously [32] .
The assay was performed by incubating the enzyme (0.1 mM) with 10 mM [a-32 P]GTP in a buffer containing 50 mM Tris-HCl, pH 8, 5 mM DTT, and 5 mM MgCl 2 for 5 min at 30uC. The reactions were stopped by the addition of EDTA to 10 mM and SDS to 1%. The reactions were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS. The radiolabeled proteins were visualized by autoradiography of the gel. The extent of covalent complex formation was quantitated by scanning the gel with a PhosphorImager (Amersham Biosciences).
An RNA substrate of 81 nucleotides was synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. The RNA [22, 23] . GTP (grey sphere) initially binds to the apo-enzyme (structure 1) which promotes closure of the N-terminal nucleotidyl transferase (NT) domain and the C-terminal oligomer-binding (OB) fold domain (structure 3). This is followed by hydrolysis of the GTP substrate to produce the enzyme-GMP covalent intermediate (structure 4). Hydrolysis of GTP disrupts the interactions between the bound guanylate and the C-terminal OB fold domain, thus destabilizing the closed form of the enzyme, which opens up with the concomitant release of pyrophosphate (structure 5). This exposes the RNA-binding site of the enzyme (exact location unknown), thereby allowing the subsequent transfer of the GMP moiety onto the acceptor RNA (structure 7). The capped RNA is then released and the enzyme can reinitiate the pathway. doi:10.1371/journal.pone.0024806.g001 transcript was synthesized from the pBS-KSII+ plasmid (Stratagene) that had been linearized with HindIII. The RNA substrate was purified on a denaturing 20% polyacrylamide gel and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantitated by spectrophotometry. Alternatively, radiolabeled RNA substrates were also synthesized by adding [a-32 P]ATP or [a-32 P]GTP to the transcription reaction.
The purified 59-triphosphorylated RNA was further processed to obtain a diphosphorylated 59 end using the S. cerevisiae RNA 59triphosphatase (Cet1) which was expressed and purified as described before [33] . The diphosphorylated RNA (ppRNA) was precipitated with ethanol, resuspended, quantitated by spectrophotometry, and stored at 220uC.
Docking calculations were carried out using the Docking Server software and the Dreiding force field was used for energy minimization of MPA using built-in Chemaxon tools in Docking Server [34] . PM6 semi-empirical charges calculated by MO-PAC2007 were added to the ligand atoms. Non-polar hydrogen atoms were merged and rotatable bonds were defined [35] . Docking calculations were carried out using the Chlorella virus and Candida albicans RNA guanylyltransferase crystal structures (Protein Data Bank 1CKN and 1P16). Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools [36] . Affinity (grid) maps of 20620620 Å grid points and 0.375 Å spacing were generated using the Autogrid program [36] . AutoDock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm and the Solis and Wets local search method [37] . Initial position, orientation, and torsions of the ligand molecules were set randomly. Each docking experiment was derived from two different runs that were set to terminate after a maximum of 2,500,000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å , and quaternion and torsion steps of 5 Å were applied.
UV-crosslinking between the internally 32 P-labeled RNA of 81 nt and the yeast GTase was performed in a crosslink buffer (50 mM Tris pH 7.5, 5 mM DTT, 5 mM MgCl 2 ). The protein (12 mM) was incubated with GTP (1 mM) and different concentrations of MPA for 10 minutes at 30uC. The radiolabeled RNA (3 mM) was added to the reaction mixture and incubated for 5 minutes at 30uC. The reaction mixture was exposed to UV light (254 nm, 20 Joules/cm 2 ) for 5 min at 30uC using a Stratalinker 2400 UV Crosslinker (Stratagene). The crosslink mixture was denatured (50 mM Tris-HCl, pH 7.0, 5% sucrose, 5% bmercaptoethanol, 2% SDS) and separated by electrophoresis on a 12% SDS-PAGE. The gel was analyzed by phosphorimaging.
The ligation reaction was performed as described previously [32] . Briefly, a 36 bp DNA duplex harboring a centrally placed nick was used as a substrate. The 18-mer constituting the 59phosphate-terminated strand 59-d(ATTCCGATAGTGACTA-CA)-39 was 59-radiolabeled and gel purified as described before. This labeled 18-mer was then annealed to a complementary 36mer in the presence of a 39-OH 18-mer strand 59-d(CAT-ATCCGTGTCGCCCTT)-39 [38, 39] . Ligation reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl 2 , 1 mM ATP, nicked duplex substrate and the Chlorella virus DNA ligase were incubated at 22uC for 15 min. The reactions were stopped by the addition of 1 ml 0.5 M EDTA and 5 ml formamide. The samples were heated at 95uC for 5 min and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea. The extent of ligation was determined by scanning the gel with a PhosphorImager (Amersham Biosciences).
Primer extension reactions were performed as described previously [40] using a 59 32 P-labeled 18-mer DNA oligonucleotide complementary to the 59 region of the SSA1 mRNA (positions +1 to +19). Total RNA was extracted form S. cerevisiae cells that were grown in the presence or absence of 500 mg/ml MPA for 3 h at 30uC. The primer extension reactions were analyzed by electrophoresis through a 8% polyacrylamide gel containing 7 M urea in TBE and visualized by autoradiography.
We initially performed a virtual screen of more than 25,000 purine-related compounds for their ability to bind to the GTases of albicans (open form). The ligands were docked and ranked according to their respective docking scores. Our initial screen indicated that very few compounds bound to the open forms of these three different GTases. In all cases, the highest scoring compounds were not predicted to bind with very strong affinities to the enzymes (predicted K d .2 mM). However, one compound (mycophenolic acid) docked efficiently in the cavity of the closed form of the Chlorella virus GTase (predicted K d of 280 mM). Mycophenolic acid (MPA, Fig. 2A) is a well-known inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key cellular enzyme required for biosynthesis of guanine nucleotides [41] . Because T-and B-lymphocytes are critically dependent for their proliferation on de novo synthesis of purines, whereas other cell types can utilize salvage pathways, IMPDH inhibitors have potent cytostatic effects on lymphocytes [42, 43] . Accordingly, MPA has been used both in cancer and immunosuppressive chemotherapy as wll as in antiviral and antifungal therapy [44, 45, 46, 47, 48, 49, 50, 51, 52] . Because GTP is required for the transcription and replication of cellular and microbial genomes, it has traditionally been assumed that the decrease in the cytosolic concentration of GTP could affect both cell growth and the multiplication of fungal and viral pathogens.
The ability of MPA to inhibit the GTase of the model organism S. cerevisiae (Ceg1 protein) was then investigated. The GTase activity is actually a two-step ping-pong reaction in which the enzyme first reacts with GTP to produce the enzyme-GMP (EpG) covalent intermediate with the concomitant release of pyrophosphate [2] . In the second step of the reaction, the GMP moiety is then transferred to a 59-diphosphate RNA. The ability of MPA to inhibit the complete GTase reaction (both steps) was monitored using a standard GTase assay in which the purified enzyme was incubated with [alpha-32 P]GTP and a 59-diphosphate acceptor RNA. The reaction products were then analyzed on a denaturing polyacrylamide gel. As can be seen in figure 2B , the presence of increasing concentrations of MPA significanlty decreased the transfer of the radiolabeled GMP moiety onto the acceptor RNA. MPA was shown to inhibit the overall GTase activity by 50% at 360 mM, and by 80% at 900 mM.
In order to characterize the inhibition of the GTase activity by MPA, we next set out to investigate which of the two catalytic steps of the reaction is inhibited by MPA. The first step of the reaction, i.e. the formation of the enzyme-GMP covalent intermediate, was monitored by incubating the purified enzyme in the presence of [alpha-32 P]GTP. The radiolabeled covalent enzyme-GMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Our results indicate that very high concentrations of MPA are required to inhibit the formation of the enzyme-GMP covalent complex (Fig. 2C) . We determined that a concentration of 3 mM of MPA is required to inhibit the first step of the reaction by 50%, a concentration that corresponds to 8-times the amount required to inhibit the overall GTase reaction (both steps). The effect of MPA on the second step of the RNA reaction (i.e. the transfer of the GMP moiety onto an acceptor RNA) was next evaluated by pre-incubating the enzyme with GTP to ensure formation of the covalent enzyme-GMP complex, followed by the addition of the acceptor 59-diphosphate RNA in the presence of MPA. Using such an approach, it was determined that a concentration of 640 mM of MPA is sufficient to inhibit 50% of the second step of the GTase reaction (Fig. 2D) . Taken together, these results indicate that MPA inhibits the GTase reaction mainly through inhibition of the catalytic transfer of the GMP moiety onto an acceptor RNA.
In the typical GTase reaction, the nucleophilic attack on the aphosphate of GTP by the enzyme during the first step of the reaction results in the formation of a covalent intermediate in which GMP is linked via a phosphoamide bond to a lysine residue of the enzyme [2] . Interestingly, it has previously been demonstrated that the nucleotide analog ribavirin triphosphate can be used as a substrate by the GTase to form an enzyme-RMP covalent intermediate [25] . Although MPA is not a nucleoside analog per se, we were still interested to monitor its ability to potentially be used as a substrate by the enzyme. The appearance of a slower migrating protein species is traditionally observed upon electrophoresis through a polyacrylamide gel when an RNA capping enzyme is incubated with GTP [25] . This slower migrating species corresponds to the enzyme with covalently bound GMP. We therefore incubated the enzyme in the presence of GTP or MPA and the polypeptide was analyzed by capillary electrophoresis. The appearance of a slower migrating protein species was observed repeatedly when the protein was incubated with GTP (Fig. 3A) . However, this was not observed when the enzyme was incubated with MPA (Fig. 3A) . We therefore conclude that MPA is not a substrate for the GTase. Accordingly, the transfer of MPA to an acceptor RNA could not be detected when the enzyme was incubated in the presence of MPA (Fig. 3B ). This was tested by incubating the enzyme with an RNA substrate (81 nt) synthesized in the presence of [alpha-32 P]GTP. This RNA substrate harbored a radiolabelled diphosphate 59-end (59 ppG-RNA 39, where the boldface indicates the radiolabelled moiety) and 27 internally labelled guanosine residues. The RNA was then incubated with the GTase in the presence of GTP or MPA. The products of the reaction were extracted with phenol/chloroform and the RNA acceptor molecules were recovered by ethanol precipitation. Aliquots of the RNA samples were then digested with nuclease P1 and alkaline phosphatase and analyzed by polyethyleneiminecellulose thin layer chromatography (Fig. 3B) . The transfer of radiolabeled GMP to RNA was confirmed by demonstrating the release of a GpppG structure following digestion of the RNA samples with both nuclease P1 and alkaline phosphatase. However, the transfer of MPA to the acceptor RNA could not be detected when the enzyme was incubated in the presence of MPA (Fig. 3B) . Overall, these results demonstrate that MPA is not a substrate for the GTase, and that it cannot be transferred to an acceptor RNA.
In order to better understand the mechanism by which MPA inhibits the transfer of GMP to RNA, we set out to use the power of molecular docking to provide information on the interaction between MPA and both the open and closed conformers of the RNA guanylyltransferase ( Fig. 4A and B ). Using extensive computational docking and structure optimizations, we generated models of MPA bound to the closed form enzyme-GMP complex of the Chlorella virus GTase. More than 2,000,000 energy evaluations were performed in order to provide an accurate description of the interactions. The models underwent 150 rounds of steepest descent energy minimization and did not contain energetically unfavorable bonds, angles or torsions.
Analysis of the molecular docking model predicts that MPA binds in the cleft created by the closure of the N-terminal and Cterminal domains. The space-filling model suggests that the molecular structure of MPA is sterically complementary to the cleft between the domains (Fig. 4B) . The molecular docking model provides instructive findings on the interaction between specific residues of the enzyme and MPA (Fig. 4B ). Arg106 and Asp213 of the N-terminal NT domain and Lys234, Asp244, Gln293, Arg295, Asp297, and Glu281 of the C-terminal OB fold domain would be involved in the coordination of the MPA through hydrogenbonding (Fig. 4C) . Ile86 (N-terminal) is predicted to make a hydrophobic interaction with MPA, which is located 4.0 Å away from the bound GMP (Fig. 4D) .
As observed in our initial virtual screening, the molecular docking analyses suggest that MPA only binds to the closed form of the enzyme. No significant binding of MPA was detected when the molecular docking experiments were performed on the open forms of the C. albicans, S. cerevisiae and Chlorella virus RNA guanylyltransferases bound with GMP. It is tempting to speculate that the opening of the active site of the enzyme that is normally observed following the hydrolysis of GTP to produce the enzyme-GMP covalent intermediate [23] is inhibited by MPA. Following the formation of the GMP adduct, the enzyme must open up to provide access for the incoming mRNA substrate, since this site is blocked off in the closed form of the enzyme [23] . The presence of multiple interactions between MPA and residues of both the Nterminal and C-terminal domains of the enzyme likely inhibits this critical conformational change that is required for the binding of RNA. In order to validate this hypothesis, cross-linking assays were used to monitor the binding of radiolabelled RNA to the enzyme-GMP complex with or without MPA. An RNA harboring a 59triphosphate end was used as a ligand instead of the classical RNA with a 59-diphosphate since the latter would lead to the transfer of the GMP moiety onto the RNA. Using this approach, an apparent K d of 30 mM could be estimated for the binding of the RNA to the enzyme-GMP complex (data not shown). However, the binding of RNA to the enzyme-GMP/MPA complex was severely decreased in the presence of MPA (Fig. 5A ). An IC 50 value of 410 mM could be estimated from the inhibition assays (Fig. 5B) . These results demonstrate that the presence of MPA inhibits the binding of the enzyme-GMP complex to RNA.
GTases are members of the RNA/DNA nucleotidyltransferase superfamily that share six conserved sequence motifs (Fig. 6A) and a similar three-dimensional architecture consisting of an Nterminal NT domain and a C-terminal OB fold domain [21] . As observed previously, the NT domain (aa 1-243) of the C. albicans GTase aligns to the Chlorella virus GTase enzyme with 1.9 Å rmsd over 210 amino acids (26% side chain identity) [53] . In addition, the C. albicans GTase OB domain (aa 244-390) aligns to the Chlorella virus GTase OB domain with 1.8 Å rmsd over 72 amino acids (28% identity) [53] . Because of the high level of structural conservation between members of this family (Fig. 6B) , we hypothesized that MPA should inhibit the GTases of various organisms. We therefore expressed and purified the RNA guanylyltransferases from vaccinia virus and Chlorella virus, as well as the corresponding human enzyme. Our results demonstrate that MPA inhibits all these GTases to the same extent than the S. cerevisiae homolog (Fig. 6C) . IC 50 values similar to the ones obtained for both the first (,3 mM) and second step (,620 mM) of the GTase reaction were observed when MPA was added to the reactions catalyzed by the vaccinia, Chlorella, and human enzymes. Overall, these results indicate that MPA inhibits the activity of different GTases through a similar mechanism that mainly prevents the catalytic transfer of the GMP moiety onto an acceptor RNA.
GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases [21] . The crystal structures of various family members revealed a common tertiary structure consisting of an N-terminal NT domain and a Cterminal OB-fold domain [22, 23, 53, 54, 55] . Based on both the sequence and structural similarities between GTases and DNA/ RNA ligases (Fig. 6A and B) , it is tempting to speculate that MPA might also be an inhibitor of DNA/RNA ligases. DNA/RNA ligation entails three sequential nucleotidyl transfer steps, and the first two steps are mechanistically related to the GTase reaction [56] . In the first step of the ligation reaction, nucleophilic attack on the a-phosphorus of ATP by ligase results in the formation of a covalent ligase-adenylate intermediate with the concomitant release of pyrophosphate. In the second step, AMP is transferred to the 59-end of the 59-phosphate-terminated DNA strand to form a DNA-adenylate complex. In the last step of the reaction, The the polynucleotides are joined with the concomitant release of AMP [56] . To verify the potential ability of MPA to inhibit DNA/RNA ligases, we have monitored the effect of MPA on the purified DNA ligase encoded by Chlorella virus. The ability of MPA to inhibit the complete ligation reaction was monitored by incubating the purified enzyme with ATP and a DNA duplex containing a centrally located nick. The reaction products were then analyzed on a denaturing polyacrylamide gel. As can be seen in Fig. 7A , the presence of increasing concentrations of MPA significanlty decreased strand joining by the Chlorella virus DNA ligase. MPA was shown to inhibit the strand-joining activity by 50% at 700 mM. We next set out to investigate which of the first two catalytic steps of the ligation reaction is inhibited by MPA. The first step of the reaction, i.e. the formation of the enzyme-AMP covalent intermediate, was monitored by incubating the purified enzyme in the presence of [alpha-32 P]ATP. The radiolabeled covalent enzyme-AMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Similarly to what was observed in the case of the GTase activity, our results indicate that very high concentrations of MPA are required to inhibit the first step of the reaction, i.e. the formation of the enzyme-AMP covalent complex (Fig. 7B) . We determined that a concentration of ,3.0 mM of MPA is required to inhibit the first step of the ligation reaction by 50%, a concentration that corresponds to 4times the amount required to inhibit the overall reaction. The effect of MPA on the second step of the ligation reaction (i.e. the transfer of the AMP from ligase-adenylate to a 59-phosphate terminus acceptor DNA) was next evaluated. In a typical ligation reaction, the adenylate-DNA intermediate would not be detected since the enzyme is highly efficient in ligating the two DNA strands [57] . However, as reported previously [58] , the adenylate-DNA intermediate can accumulate to high levels when the enzyme acts on a substrate that contains a 1-nt gap between the reactive 39-OH and 59-phosphate strands. Using such an approach, the incubation of the enzyme with ATP and a gapped DNA substrate resulted in the conversion of the 59-radiolabeled 18-mer strand into an adenylated species (AppDNA) that migrated 1-nt slower than the input 18-mer during polyacrylamide gel electrophoresis (Fig. 7C) . The ability of MPA to inhibit this second step of the ligase reaction was investigated by pre-incubating the enzyme with ATP to ensure formation of the covalent enzyme-AMP complex, and by incubating the complex in the presence of the ligation substrate that contains a 1-nt gap between the reactive 39-OH and 59phosphate strands. Using this strategy, it was determined that a concentration of 600 mM of MPA is sufficient to inhibit 50% of the second step of the ligation reaction (Fig. 7C) . Taken together, these results indicate that MPA inhibits the ligation reaction mainly through inhibition of the second step of the reaction, i.e. the catalytic transfer of the AMP moiety onto the 59-phosphate terminus of the nicked DNA substrate.
The effect of MPA on the capping of mRNAs was then assessed in vivo. A primer extension assay was used to monitor the effect of MPA on the formation of the RNA cap structure in S. cerevisiae. Previous studies have shown that during cDNA synthesis by reverse transcriptase, the presence of a cap structure results in the synthesis of products that harbor an extra 39 nucleotide [40, 59] . We therefore performed primer extension assays with a 59 P 32labeled 18-mer oligonucleotide complementary to the 59 region of the SSA1 mRNA. The oligonucleotide was annealed to total mRNAs extracted from cells that were grown in the presence or absence of 500 mg/ml MPA for 3 h, and extended with reverse transcriptase. Our results indicate that cDNA products with apparent chain lengths of 71 and 72 nucleotides, corresponding to uncapped and capped mRNAs, were synthesized using mRNAs extracted from untreated cells (Fig. 8) . However, treatment with MPA resulted in a marked reduction of the 72 nucleotides species, indicating that cap formation was inhibited in the presence of MPA. Quantitative analysis indicate that cap formation was reduced by 50% in the presence of 500 mg/ml MPA (Fig. 8) . We conclude that the addition of MPA to S. cerevisiae cells leads to an inhibiton of RNA cap synthesis.
The current study provides the first biochemical evidence that MPA can directly interact with an RNA guanylyltransferase and inhibit its activity. We demonstrated that MPA inhibits the RNA guanylyltransferase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. Moreover, our RNA binding studies demonstrated that the binding of the enzyme-GMP intermediate to RNA is inhibited in the presence of MPA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Several inhibitors of the RNA guanylyltransferase activity have previously been identified [60] . However, these inhibitors all target the formation of the enzyme-GMP complex. For instance, foscarnet inhibits the formation of the enzyme-GMP intermediate on account of its analogous nature to pyrophosphate (PPi), a product of the RNA guanylyltransferase reaction [24] . Ribavirin, a broad-spectrum nucleoside analogue used as an antiviral for severe respiratory syncytial virus, Hepatitis C and other viral infections, is another example of an RNA guanylyltransferase inhibitor that prevents the formation of the enzyme-GMP intermediate [25] . Non-nucleoside competitive inhibitors have also been generated against the RNA guanylyltransferase of respiratory syncitial virus [61] . MPA, with its ability to inhibit the second step of the RNA guanylyltransferase reaction, has the potential to serve as a template for the development of more potent inhibitors. In fact, a number of MPA derivatives have been developed in recent years [62, 63] . Analysis of the interaction between these derivatives and RNA guanylyltransferases could shed light on the chemistry of the RNA capping reaction and lead to the development of more efficient anti-proliferative/antimicrobial drugs.
What is the biological relevance of the present finding? MPA has been shown to cause a reduction of the cellular GTP pools through the inhibition of IMPDH which is required for the de novo biosynthesis of GTP [41, 42] . A decrease in GTP concentrations could potentially have a negative effect on the capping of mRNAs. Evidence for this mechanism comes from studies performed with both the Sindbis virus and the Borna disease virus that showed that the viruses cannot replicate in cultured cells treated with ribavirin because the level of GTP falls too low to permit the capping of viral RNAs [48, 64, 65] . However, mounting evidence indicates that the antiproliferative/antimicrobial effect of MPA is not mediated entirely through the reduction of the intracellular GTP pool. For instance, MPA is not always a potent viral inhibitor [66] . The second mechanism by which MPA could inhibit the capping of mRNAs is by directly inhibiting the activity of GTases, as we demonstrated in the current study. Moreover, we do not exclude the possibility that MPA may have an increased frequency of utilization in vivo when the levels of GTP are lowered through the inhibition of IMPDH. In the present study, we demonstrated that MPA can inhibit RNA guanylyltransferases and ligases. MPA therefore appears as a pleiotropic agent that may function through multiple targets (IMPDH, RNA guanylyltransferase, ligases), as recently suggested through proteomic analysis [67] . As such, MPA shares many properties with ribavirin. Both compounds lead to a reduction of the de novo synthesis of GTP through IMPDH inhibition, and both molecules can inhibit RNA capping, albeit through different mechanisms of inhibition. Long-term follow-up and treatment of congenital alveolar proteinosis BACKGROUND: Clinical presentation, diagnosis, management and outcome of molecularly defined congenital pulmonary alveolar proteinosis (PAP) due to mutations in the GM-CSF receptor are not well known. CASE PRESENTATION: A 2 1/2 years old girl was diagnosed as having alveolar proteinosis. Whole lung lavages were performed with a new catheter balloon technique, feasible in small sized airways. Because of some interstitial inflammation in the lung biopsy and to further improve the condition, empirical therapy with systemic steroids and azathioprin, and inhaled and subcutaneous GMCSF, were used. Based on clinical measures, total protein and lipid recovered by whole lung lavages, all these treatments were without benefit. Conversely, severe respiratory viral infections and an invasive aspergillosis with aspergilloma formation occurred. Recently the novel homozygous stop mutation p.Ser25X of the GMCSF receptor alpha chain was identified in the patient. This mutation leads to a lack of functional GMCSF receptor and a reduced response to GMCSF stimulation of CD11b expression of mononuclear cells of the patient. Subsequently a very intense treatment with monthly lavages was initiated, resulting for the first time in complete resolution of partial respiratory insufficiency and a significant improvement of the overall somato-psychosocial condition of the child. CONCLUSIONS: The long term management from early childhood into young adolescence of severe alveolar proteinosis due to GMCSF receptor deficiency requires a dedicated specialized team to perform technically demanding whole lung lavages and cope with complications. Pulmonary alveolar proteinosis (PAP) is characterized by a substantial and persistent increase in surfactant pool size [1, 2] . There are several causes of this rare condition; mouse models with deletion of granulocyte-macrophagecolony stimulating factor (GM-CSF) or the GM-CSF receptor (GM-CSFR) beta-chain showed the first evidence for involved molecularly mechanisms [3, 4] . Autoantibodies against GM-CSF, blocking GM-CSF signaling, are the cause for the most frequent form of PAP, mainly found in adults and also called autoimmune PAP [5] . In 2008 the first two families with congenital PAP and mutations in the alpha-chain of the receptor for GM-CSF were described [6, 7] and very recently another six families were reported [8] . The patients presented with progressive dyspnea of insidious onset between the ages of 1.5 and 9 years; some were asymptomatic. Short term responsiveness to whole lung lavage (WLL) treatment has been described, however not much information on the long term outcome of molecularly defined patients is yet available.
Other genetic conditions that lead to PAP include a recently identified mutation in the beta chain of the GMCSF receptor [9] , surfactant protein B or C deficiency [10, 11] , Niemann-Pick Type C2 disease [12] and lysinuric protein intolerance [13] . Secondary PAP develops in association with conditions involving functional impairment or reduced numbers of alveolar macrophages like inhalation of inorganic dusts, myeloic leukemia, myelodysplastic syndrome, immunosuppression related to organ transplantation, and some infections including Pneumocystis [1] .
Not much is known on the clinical spectrum, course and treatment options in patients with molecularly defined, congenital PAP due to mutations in the GM-CSF alpha chain. Also the role of long term WLL, which are be very demanding due to the small size of the airways, how to measure clinical response to lavage therapy and the relevance of glucocorticoid therapy have not been reported. Here we present the successful management of a child with a severe congenital PAP caused by the homozygous p.Ser25X mutation in exon 3 of the CSF2RA gene and its follow up for more than a decade. These data may be helpful for future treatment of infants and children with this rare condition.
The patient was the 2nd of 3 living children; born at term in 1998, with no immediate postnatal respiratory distress. The family history was unremarkable for pulmonary or other rare diseases; the parents were consanguineous and of Turkish descent. At age 2 1/2 years during an acute respiratory tract infection with productive cough and fever with no response to antibiotics, intermittent cyanosis occurred and the child was referred to our centre because of chronic tachypnoea and weight loss. Because of a typical chest computed tomography (CT) and chest x-ray ( Figure 1A , B), BAL macroscopic appearance ( Figure 1G ) and microscopy ( Figure 1C , H), and after exclusion of infectious or metabolic causes or malignancy, PAP was suspected and confirmed by histology ( Figure 1F , I). Figure 1 Diagnosis of alveolar proteinosis was suspected based on typical radiological picture in chest x-ray and CT with groundglass attenuation and interstitial thickening, resulting in crazy paving pattern ( Figure. 1 A, B) . Figure. 1 E shows CT after whole lung lavage of the right lung. Macroscopic appearance ( Figure. 1 G) and light microscopy of BAL fluid stained with PAS ( Figure. 1 C, magnification x400) showed extracellular positive proteinaceous material, lipid-laden macrophages ( Figure. 1 H, MGG stain, magnification x1000), and after exclusion of infectious or metabolic causes or malignancy, PAP was confirmed by histology (right lower lobe). HE stained tissue demonstrated alveolar filling with eosinophilic material ( Figure. At presentation the child had global respiratory insufficiency, combined with an elevated level of LDH and CEA ( Figure 2A ). Based on the clinical diagnosis of alveolar proteinosis we initiated whole lung lavages and some additional treatments.
Therapeutic bronchoalveolar lavages of either the right or left lung were performed under general anaesthesia and paralysis. From age 2 1/2 to 6 years we used a technique that isolated one lung with the help of a balloon catheter (5-7 Fr, Arrow, Reading, USA) placed into one Figure 2 Long term clinical course and treatments. Lactate dehydrogenase (LDH), height, weight for height (WfH) expressed as the current weight of the child as a percentage of the normal weight for a given height, capillary CO2 pressure, and carcino embryonic antigen, CEA in serum ( Figure. main stem bronchus through the cuffed endotracheal tube (ID 4.0-5.0) and blocked there (see also Figure 3A ). The position and fitting of the catheter was permanently monitored via an Olympus bronchoscope (BFN20, O.D. 1.8 mm), as described in detail [14] . From age 7 onward the size of the main bronchus was sufficient to allow double lumen tube and lavage of one lung, while ventilating the other. Sterile 0.9% NaCl warmed to body temperature was used as lavage fluid. The lavage was done by manual injection and withdrawal of saline from a 50 ml syringe, starting with smaller volumes of 4 ml/kg body weight (used for diagnostic purposes) and increasing to about 11 to 15 ml/kg under continuous control of the correct position and tightness of the balloon. The recovered fluid was collected via a 2-way cock stop into 500 ml bottles to allow judgement of turbidity and to follow the process of lavage [15] . In 2003, at age 5 y, we tried to increase the yield of lipo-proteins recovered from the lungs with the help of perfluorocarbons (PFC) [16, 17] . A total of 100 ml of Perfourodecalin (Pharmpur, Augsburg, Germany) was instilled in 3 aliquots after the 6th, 7th, or 8th 500 ml wash during three consecutive WLL sessions. This approach did neither improve the yield of phospholipids washed out ( Figure 4A , B) nor that of total protein (not shown).
Empirically, we found that WLL were the most efficient treatment. This was clearly shown for the short term; during 49 instances investigated until the age of 11, the amount of nasal oxygen flow was reduced in 40 after the lavages ( Figure 3B ). This effect could be sustained for many years demonstrating long term efficacy. However the extreme value of WLL was only very recently Figure 3 Whole lung lavages were performed, due to the small sizes of the airways until the age of 6 y, via a blocked endotracheal tube through which the child was ventilated ( Figure. 3 A, 1.) and through which also a pulmonary artery catheter ( Figure. 3 A, 3., blue) was inserted and blocked in the left or right main stem bronchus (small syringe) and the lavage was done ( Figure. 3 A, 4., large syringe). The tight fit of the blocked pulmonary artery catheter was continuously monitored via a 1.8 mm endoscope ( Figure. 3 A, 2., black). Figure. demonstrated unequivocally following implementation of our concept of very aggressive WLL. Up to the age of 10 years, WLL were done more or less to ameliorate partial respiratory insufficiency, i.e. to decrease the need for additional oxygen. From year 10 onward, we performed one lavage per month, in order to try to completely clear the lung from its proteinosis load. This approach was very successful and resulted in complete resolution of partial respiratory insufficiency for the first time. The patient started puberty, growth and weight were sustained by oral nutrition without need of using the percutaneous tube and the dependency on supplemental oxygen up to that point in time, could be finished. This also led to increased self-confidence and better integration at school. Also, the lung function improved very rapidly and chest radiograph cleared to almost normal ( Figure 1D , E; Figure  3C ). Together somato-psychosocial condition substantially improved. A consecutive brief trial to increase the time lag between the lavages failed and an interval of about 4 weeks was maintained.
Because at clinical diagnosis of the patient, both the exact cause of the PAP and effective treatments in small children were unknown, empirical high dose glucocorticosteroids in pulses were used ( Figure 2A ) and under the impression that they might be helpful, systemic corticosteroids were used for prolonged periods until the age of 7.5 years. During this time, azathioprine as a steroid saving agent was also used without any apparent benefit. However, we clearly observed that severe infectious complications were only observed during the time of increased immunosuppression by these agents (Figure 2A, B) . At 5 years of age an Aspergillus fumigatus infection with formation of a cavity, leading to severe cardio-respiratory failure and resuscitation followed by resection of the left lower lobe ( Figure 2B , Figure 5A -D). At age 7.5 years she suffered a pulmonary para-influenza infection, leading to ARDS and necessitating mechanical ventilation. Additionally, the child had many mild respiratory exacerbations, mostly believed to be induced by viral upper-and lower respiratory tract infections. Figure 4 At age 5 y, during four consecutive whole lung lavage sessions perfluorocarbon (PFC) was instilled into the lungs after the 6th, 7th, or 8th of the 500 ml wash in order to enhance the recovery of surfactant material. The concentration of phosphoplipids (and total protein (not shown)) washed out was not significantly altered compared to lavages done at that age without PFC ( Figure. 4 A, B) . From age 3 to 4 1/2 years therapy with inhaled and subcutaneous injections of recombinant GM-CSF was done (see also Figure. 2 B). Although the intervals between consecutive therapeutic whole lung lavages were increased with GM-CSF treatment the amount of protein removed was also increased, demonstrating no reduction of protein amount with GM-CSF treatment ( Figure. 4 C, D) . Note that not all lavages were available for total protein measurements.
Of interest, from age 3 to 4 1/2 years, well before the molecular nature of the PAP was determined, we used inhaled and subcutaneous recombinant GM-CSF ( Figure  2B) . A transient increase in peripheral blood eosinophils up to 17% of the neutrophils occurred [18] (data not shown), but clearly no improvement of the alveolar proteinosis (Figure 2A, B, Figure 4C , D). Due to the expectations of the treating physicians, the intervals between consecutive therapeutic WLL were increased during GM-CSF treatment: In parallel, this was associated with an increased load of protein, demonstrating a lack of an effect of GM-CSF treatment ( Figure 4C, D) .
Nutritional support was optimized with the help of a percutaneous gastrostomy (PEG, Figure 2B ) placed at the age of 3 years, which was used regularly; the gastrostomy was changed to a jejunostoma at the age of 61/2 years, to completely exclude gastro-esophageal refluxes, although no such events had been demonstrated in pHor impedance studies ( Figure 2B ).
At age 12 years (in 2009) analysis of the patient's CSF2RA gene revealed the homozygous Ser25X stop-mutation in exon 3 resulting in the almost complete absence of the GM-CSF receptor alpha chain and causing the alveolar proteinosis we observed ( Figure 6A ). The parents were heterozygous for the mutation (Figure 6A, B) . Mutations in SFTPC, SFTPB and ABCA3 were excluded.
GM-CSF level were increased in serum (106 pg/ml, normal < 6 pg/ml) of the child and normal in the parents. No anti-GM-CSF autoantibodies were detected in serum [19] . GM-CSF-Ra chain expression after stimulation with 50 ng/ml GM-CSF on peripheral mononuclear cells of the patient was markedly reduced and normal in both parents ( Figure 6C ). In the absence of GM-CSF stimulation, GM-CSF-Ra chain in the parents was only 50% of that of the controls ( Figure 7A ), whereas GM-CSF-Rb chain and CD11b were normal. After stimulation with GM-CSF, GM-CSF-Ra and CD11b remained low in the patient ( Figure 7A, lower panel) , whereas the parents' levels were in the normal range level. GM-CSF-Rb chain without and with GM-CSF stimulation was normal ( Figure 7A ). CD11b expression on the neutrophils was used to assess signal transduction after GM-CSF stimulation. In the patient, similar as blocked by auto-antibodies from a subject with auto-immune PAP, no dose-dependent stimulation of CD11b was observed, demonstrating interruption of signalling ( Figure 7B ).
Here we report a patient with molecularly defined severe congenital PAP due to a previously undescribed autosomal recessive mutation in the alpha chain of the GM-CSF receptor. This mutation leads to a stop of transcription and to a lack of functional protein. The GM-CSF induced responses are mediated through activation of the transcription factor PU.1 and include increased surfactant catabolism and CD11b expression [20] . Impairment of the latter was shown directly in mononuclear cells of the patient after stimulation with GM-CSF. Impaired GMCSF receptor activation of alveolar macrophages leads to decreased surfactant catabolism and accumulation of surfactant in the alveolar space, i.e. alveolar proteinosis.
Important messages from this study are related to the long-term management of this condition. First, persistent and aggressive removal of surfactant filling the alveolar space may eliminate gas exchange abnormalities and consecutive sequelae including developmental and growth failure, and restricted level of performance due to respiratory limitation. Second, immune insufficiency, a Figure. 6 B) . Flow cytometric analysis of peripheral blood cells demonstrates the absence of the alpha-chain ( Figure. 6 C) . Isolated neutrophils were incubated with antibodies, washed with Dulbecco's PBS twice and measured by means of flow cytometry. Ten thousand cells were counted and analyzed by the FACSDiva software. Fc blocking and isotype controls were applied to exclude unspecific bindings. CD11b (mouse monoclonal IgG1, PE conjugated), CD116/GM-CSF-R alpha (mouse monoclonal IgG1, PE conjugated), CD131w/GM-CSF-R beta (mouse monoclonal IgG1, purified) and secondary antibody for CD131w (rat antimouse IgG1, PE-conjugated).
problem also primarily resulting from abnormalities of the GM-CSF signal transduction pathway [20] , may be augmented by immunosuppressive therapy initiated to treat the condition empirically. Therefore, molecular genetic definition of the basic defect in all children with PAP is important. Lastly, we describe the successful use of outcome measures of the efficacy of therapeutic WLL, including oxygen demand, and amount of washed out protein and phospholipids.
A major strength of this study is to demonstrate the feasibility of technically demanding repetitive WLL in a very small child over extended periods of time. Although therapeutic WLL is generally accepted as the established treatment option for PAP in adults, its optimal method, frequency of application and many other details are currently not known in infants or children. Here we show that consecutive lavages via a small catheter located in a main stem bronchus ( Figure 3A) can be used to efficiently remove accumulated surfactant from the alveolar space in a very small child. Furthermore we show that it is helpful to monitor efficacy of the washing procedure by determination of proteins and lipids removed from the lungs [15] . These measurements allowed us to demonstrate only a marginal, but not clinically significant increase in the removal of surfactant material from the lungs, by the use of PFC for lavage. In a case report on an infant with alveolar proteinosis due Niemann Pick disease the usage of PFC was recently shown not to be of benefit as well [21] .
Although feasibility of the long term management of congenital PAP with WLL was demonstrated in this CD11b. The purity of neutrophils was greater than 95% as assessed by differential cell counts of Pappenheim cytospin preparations. Cell viability was greater than 95% using trypan-blue exclusion method. 106 neutrophils were treated with 50 ng/ml GM-CSF or the same volume of the vehicle buffer (aqua a.i.) for 30 min at 37°C. In the absence of GM-CSF stimulation, GM-CSF-Ra chain expression in the parents was only 50% of that of the controls ( Figure. 3 A) , whereas GM-CSF-Rb chain and CD11b expression were normal. After stimulation with GM-CSF, GM-CSF-Ra in the patient remained low, whereas the parents' level increased to almost control level. GM-CSF-Rb chain and CD11b stimulation were normal ( Figure. 3 A) . CD11b expression on the neutrophils was used to assess signal transduction after GM-CSF stimulation. In the patient, similar as blocked by auto-antibodies from a subject with auto-immune PAP, no dose-dependent stimulation of CD11b was observed, demonstrating interruption of signalling ( Figure. case of severe PAP, molecular diagnosing PAP as caused by a genetic deficiency of GM-CSFRa may have other important prophylactic and therapeutic implications. First, based on experiments in mice with PAP bone marrow transplantation may cure the disease [3] . Currently we believe however that the risks of a bone marrow transplant (chronic graft versus host disease, among others) outweigh its benefits (elimination of need for WLL). Second, if diagnosed early in a family with an index case, the opportunity of early intervention by lavages at times of good clinical condition will help to reduce complications.
Subcutaneous injections or inhalations of GM-CSF, which have been successfully utilized in adult patients with autoimmune PAP [22, 23] , were not helpful in our case to reduce alveolar filling as assessed by CT scanning (not shown) or improvement in gas exchange ( Figure 2 ). Treatment with 20 μg/kg of GM-CSF per day subcutaneously was also shown to be ineffective for the child with congenital PAP described by Martinez-Moczygemba et al. [7] .
Immunosuppressive treatment was used empirically and because of the presence of neutrophils and some lymphocytes in the lavage specimens and in the interstitial space of the lung biopsy sample of the patient ( Figure 2B ). Unfortunately severe and prolonged infections occurred, including a cavity forming infection with Aspergillus fumigatus which was treated by i.v. and inhaled amphotericin B and surgical resection of the cavity. Sustained withdrawal of the systemic corticosteroids from age 8 years onward did not alter the activity of the underlying PAP, but reduced the rate of infectious respiratory complications considerably.
Our study exemplifies detailed long term management of severe molecularly defined alveolar proteinosis from childhood into young adolescence. It is of interest that a dedicated specialized team may be advantageous to maintain the appropriate expertise of complex procedures such as e.g. whole lung lavages in small children [8, 15, 24, 25] . Therefore a centralized approach, as it has been employed for rare lung diseases and PAP in particular on a national basis in France [24] , may be warranted. A web-based system to collect these rare cases, follow them and also to receive support is available at the kids lung register (http://www.kids-lung-register.eu). The novel whole lung lavages technique using an inflatable balloon catheter was feasible in very small sized airways. Whereas empirical immunosuppressive therapy and inhaled and subcutaneous GMCSF were without significant benefit, a very intense treatment with WLL resulted in complete resolution of respiratory insufficiency, and a normalisation of lung physiology and overall somato-psychosocial condition of the child.
Abbreviations PAP: Pulmonary alveolar proteinosis; GMCSF: granulocyte-macrophagecolony stimulating factor; GM-CSFR: GM-CSF receptor; WLL: whole lung lavage; washing of a single right or left lung. Severe influenza cases in paediatric intensive care units in Germany during the pre-pandemic seasons 2005 to 2008 BACKGROUND: Data on complications in children with seasonal influenza virus infection are limited. We initiated a nation-wide three-year surveillance of children who were admitted to a paediatric intensive care unit (PICU) with severe seasonal influenza. METHODS: From October 2005 to July 2008, active surveillance was performed using an established reporting system for rare diseases (ESPED) including all paediatric hospitals in Germany. Cases to be reported were hospitalized children < 17 years of age with laboratory-confirmed influenza treated in a PICU or dying in hospital. RESULTS: Twenty severe influenza-associated cases were reported from 14 PICUs during three pre-pandemic influenza seasons (2005-2008). The median age of the patients (12 males/8 females) was 7.5 years (range 0.1-15 years). None had received vaccination against influenza. In 14 (70%) patients, the infection had been caused by influenza A and in five (25%) by influenza B; in one child (5%) the influenza type was not reported. Patients spent a median of 19 (IQR 12-38) days in the hospital and a median of 11 days (IQR 6-18 days) in the PICU; 10 (50%) needed mechanical ventilation. Most frequent diagnoses were influenza-associated pneumonia (60%), bronchitis/bronchiolitis (30%), encephalitis/encephalopathy (25%), secondary bacterial pneumonia (25%), and ARDS (25%). Eleven (55%) children had chronic underlying medical conditions, including 8 (40%) with chronic pulmonary diseases. Two influenza A- associated deaths were reported: i) an 8-year old boy with pneumococcal encephalopathy following influenza infection died from cerebral edema, ii) a 14-year-old boy with asthma bronchiale, cardiac malformation and Addison's disease died from cardiac and respiratory failure. For nine (45%) patients, possibly permanent sequelae were reported (3 neurological, 3 pulmonary, 3 other sequelae). CONCLUSIONS: Influenza-associated pneumonia and secondary bacterial infections are relevant complications of seasonal influenza in Germany. The incidence of severe influenza cases in PICUs was relatively low. This may be either due to the weak to moderate seasonal influenza activity during the years 2005 to 2008 or due to under-diagnosis of influenza by physicians. Fifty% of the observed severe cases might have been prevented by following the recommendations for vaccination of risk groups in Germany. Influenza belongs to the most frequent vaccine-preventable diseases. Clinical manifestations of influenza infections range from illness with asymptomatic, atypical (i.e., gastro-intestinal) or oligosymptomatic disease to severe toxic progression resulting in death [1, 2] . Elderly people and infants account for the highest morbidity [1, 3, 4] .
Complications of influenza may occur at any age, affecting most severely patients with chronic underlying diseases, as chronic pulmonary diseases, diseases of the circulatory system, metabolic diseases, or immunodeficiency. The most severe complication is peracute fatality in adolescents and young adults caused by acute circulatory failure due to myocarditis or fulminant necrotising influenza-associated pneumonia. A frequently reported complication is a secondary bacterial pneumonia. Other known complications include encephalitis, otitis, and neuromuscular diseases [1, 3, 4] .
Surveillance studies from several industrialized countries reported an incidence of paediatric hospitalisations associated with seasonal influenza of 11-154/100,000 children < 5 years of age, and an incidence of 1-13/ 100,000 children treated for influenza in paediatric intensive care units (PICUs) [5] [6] [7] . For the influenza season 2003-2004, 153 influenza-associated deaths among children were reported in the United States; 47% of those occurred in children without underlying chronic disease [8] .
In Germany, only limited epidemiological data on hospitalisations and complications of seasonal influenza in children is available. In the population-based PRIDE study, conducted in 11 paediatric practices and 4 hospitals on children < 3 years of age from November 1999 to October 2001, annual incidence of influenza-associated hospitalisations was estimated as 120/100,000 [9] . In another population-based study conducted 1996-2001 in two paediatric hospitals, incidence was calculated as 123/100,000 children < 6 years of age, and 53/100,000 in children < 17 years of age [10] . However, both studies were too small to detect rare severe influenza-associated complications and fatalities resulting in PICU admittance.
The objective of this study was the nation-wide detection of severe influenza virus infections and fatalities in children, and to identify possible risk factors for complications. These data are important in the current discussion of a possible extension of yearly influenza vaccination from a risk-based strategy to general vaccination of all children. The study was conducted before the occurrence of the new influenza A (H1N1) 2009 pandemic in Germany.
The study population included all children and adolescents < 17 years of age in Germany, with on average 13,166,000 children and adolescents during the years 2005-2008, including on average 3,497,000 children < 5 years of age (German Federal Statistical Office, Germany, 2006-2009).
Prospective, active surveillance to capture severe influenza-associated complications and fatalities was performed from October 2005 to May 2008, covering three influenza seasons. Patients fulfilling the study case definition were included through the Surveillance Unit for Rare Paediatric Diseases in Germany (ESPED), a nation-wide voluntary reporting system covering 448 paediatric hospitals and departments. These hospitals contain approximately 21,500 beds, including about 2,650 paediatric intensive care beds (National Hospital Statistics, 2008). ESPED actively collected monthly reports on the occurrence of severe influenza-associated cases requiring intensive care treatment from all hospitals. Monthly reporting was requested even if no cases were identified. For identified cases, the hospitals were asked to return anonymous, structured questionnaires on basic demographic characteristics, underlying chronic medical conditions, influenza vaccination status, and influenza diagnostics, symptoms, treatment, pre-defined complications, and sequelae. The reporting physician filled in the questionnaire according to the information from the medical chart. If feasible, hospitals provided additionally anonymous medical discharge letters or autopsy results.
All children and adolescents < 17 years of age fulfilling the following criteria for severe paediatric influenza-associated infections were included: i) laboratory-confirmed influenza (antigen test or PCR or virus isolate) within one week before or after start of influenza symptoms, and ii) hospitalisation to a PICU with need for intratracheal/CPAP ventilation and/or one of the following diagnoses: encephalitis/encephalopathy, bronchitis/bronchiolitis, complicated febrile seizure, acute respiratory distress syndrome (ARDS), influenza-associated pneumonia, secondary bacterial pneumonia, status asthmaticus, sepsis, myocarditis, apnoea/bradycardia, or iii) influenza-associated death.
All data were entered into a Microsoft Access 2000 database and, after plausibility checks, transferred into SPSS 15.0 for statistical analysis. Data were analysed descriptively (percentages or median with inter-quartile range, IQR; reported percentages refer to the number of available data per variable). Comparisons among groups were assessed for significance (p < 0.05, two-sided) using Pearson's Chi 2 -test or, where appropriate, Fisher's Exact test for categorical data. Continuous data were assessed using the Mann-Whitney U-test. Due to the small number of cases per subgroup, the evidence of the statistical comparisons was limited.
The study was reviewed and approved by the Bavarian Data Protection Office, Munich, Germany. The institution agreed that patient informed consent was not needed for this surveillance study.
Twenty (5%) hospitals reported 29 patients. The reporting hospitals were situated in 9 (56%) of the 16 federal states of Germany. Of the 29 reports, 9 (31%) had to be excluded from the analysis: for 3 patients, the hospitals did not provide information on the inclusion criteria, 3 patients had no laboratory-confirmed influenza diagnosis, 1 patient was older than 16 years of age, and 2 patients had been reported twice. Twelve (60%) of the 20 children were male. The median age was 7.5 years (IQR 0.5-12.75, range < 1 month to 15 years of age); 9 (45%) children were < 5 years of age, including 5 (25%) children < 1 year of age.
Duration of hospital stay varied from < 1 to 96 days (median 19, IQR 12-38), duration of PICU stay was between < 1 and 50 days (median 11, IQR 6-18). In 70% of patients, PICU admission occurred at the day of hospital admission or within one day afterwards (range 0-10 days). In one child, the influenza infection was assumed to be nosocomial, with start of influenza symptoms and transfer to the PICU 9 days after initial hospitalisation due to complicated pneumonia caused by Mycoplasma pneumoniae.
There were 14 (70%) cases of seasonal influenza A and 5 (25%) cases of influenza B; in one (5%) patient influenza infection was confirmed but the influenza type was not available. Influenza subtypes were determined for 3 samples, with seasonal influenza A (H1N1) in two patients and influenza A (H3N2) in one child. Influenza virus infection was determined by antigen testing in 8 (40%), PCR in 4 (20%), both antigen testing and PCR in 7 (35%) patients, and PCR combined with virus isolation in one (5%) patient. Most frequently, tracheal (30%) or pharyngeal (20%) secretions, nasopharyngeal swabs (15%) or a combination of these materials (20%) were used for diagnosis. In 2 (10%) children influenza infection was confirmed by serum diagnostics and in one (5%) patient from lung biopsy material after fatal outcome.
CRP values were available from 18 children (median 8.4 mg/dl, IQR 3.9-23.1). Laboratory-confirmed co-infections were reported in a total of 13 (65%) children (4 viral, 7 bacterial, 2 fungal; Table 1 ). Diagnostic analysis for simultaneous respiratory syncytial virus (RSV) infection was performed in 10 (50%) children; in 4 with positive results. All 4 children diagnosed with both influenza and RSV suffered from pneumonia and/or bronchitis/bronchiolitis and needed PICU treatment for a median of 9 days (IQR 4-13); three were < 1 year of age. Microbiological diagnostics were performed from blood cultures of 17 (85%) patients ( Table 1 ). Other positive results were mainly reported from tracheal aspirates in patients receiving intratracheal ventilation (Table 1) . Of seven patients with bacterial co-infections, three were infected with two different bacterial species. Patients with co-infection by bacteria or fungi were treated at the PICU for a median of 18 days (IQR 12-30), and, thus, twice as long compared to patients without such co-infections (median 9 days, IQR 5-11; p = 0.01).
A total of 158 clinical signs and symptoms at PICU admittance were reported from 18 patients (71 (45%) respiratory, 38 (24%) gastro-intestinal, 49 (31%) neurological; Table 2 ). The most frequently reported symptoms were tachypnoea (16) , feeding difficulties (15), and fever (15) .
A chest radiograph was performed in 17 (85%) and was abnormal in 16 (80%) patients. Reported radiological diagnoses were pneumonia (8), ARDS (2), pneumothorax (2), pleural effusion (2), mediastinal emphysema (1), pericardial effusion (1), and severe pulmonary haemorrhage (1) .
Oseltamivir was administered to 10 (50%) children for a median duration of 5 days (IQR 4.5-5.25); treatment started in 5 children at the day of PICU admittance, in one child one day and in 4 children 5-12 days after PICU admittance. The delay between start of symptoms and oseltamivir treatment was between 1 and 15 days. A total of 18 (90%) children were treated intravenously with antibiotics, for a median of 13 days (IQR 7-15). Catecholamines were administered in 8 (40%) children for a median of 4 days (IQR 2-14). Inhalation with bronchodilators was required by 12 (60%) children. A total of 10 (50%) children needed mechanical ventilation: 4 (20%) received solely intratracheal ventilation and 2 (10%) CPAP; 4 (20%) children received both types of ventilation.
Thirty-nine severe influenza-associated complications and two fatalities were reported among the 20 children ( Table 3 ). The most frequently reported complication was influenza-associated pneumonia in 12 (60%) patients, followed by bronchitis/bronchiolitis in 6 patients (30%). Secondary bacterial pneumonia was observed in 5 (25%) patients; in 4 cases in combination with influenza-associated pneumonia. ARDS was reported in 5 (25%) patients, all suffering from influenza-associated pneumonia, and in three patients together with sepsis. All 5 patients with ARDS had predisposing underlying chronic conditions. Encephalitis/encephalopathy occurred in a total of 5 (25%) patients (including one fatal case), in three cases in combination with bronchitis/bronchiolitis.
Of the two (10%) reported fatalities, one was an 8-yearold boy admitted to the PICU with suspected encephalitis and status epilepticus in 2007. Previously he was diagnosed with a delay in speech development and suspected rolandic epilepsy. He received antibiotics, catecholamines, acyclovir and intratracheal ventilation. He was diagnosed with influenza A one day after PICU admission and was started on oseltamivir treatment. He died 4 days after admission from massive cerebral edema. The autopsy report stated death due to cardiac and circulatory failure. Streptococcus pneumoniae was diagnosed from PCR analysis of cerebrospinal fluid and brain tissue, indicating encephalopathy following influenza infection complicated by bacterial superinfection of the CNS. In 2008, a second fatality was reported from a 13-year-old boy with several chronic underlying diseases (asthma bronchiale, cardiac malformation, Addison's disease). He was found unconscious by his parents after febrile infection during the previous week. After resuscitation he was transported to the hospital where he died in the emergency room from cardiac and respiratory failure. The autopsy report diagnosed cerebral edema and PCR-confirmed seasonal influenza A (H1N1) from lung biopsy tissue.
Permanent or possibly permanent sequelae were described in 2 (10%) and 7 (35%) patients, respectively. Permanent sequelae were worsening of an underlying chronic lung disease (BPD) in a patient after influenza Aassociated pneumonia, and post-encephalitic/organic brain syndrome in a patient with influenza A-associated encephalopathy. Possible permanent sequelae occurred in 5 patients after influenza A and in 2 patients after influenza B. Reported possible sequelae were pulmonary callosity following secondary bacterial pneumonia complicated by pleural effusion (2 patients), neurological sequelae (ataxia; severe illness polyneuropathy) following encephalopathy (2 patients), regression in locomotion development following PICU treatment for almost 2 months (1 patient), and postural damage after pleural decortication (1 patient); in one patient the sequelae were not specified.
Underlying chronic medical conditions were reported in 11 (55%) children, whereby 7 of these had more than Information about the influenza vaccination status was available from 17 (85%) patients; none had been vaccinated against influenza. Out of the 11 children from risk groups with chronic underlying disease the vaccination status was not available in 2 (18%) patients, and 1 (9%) child was < 6 months of age and therefore too young to be immunized.
We describe the results from a nation-wide surveillance study on severe paediatric seasonal influenza cases in Germany during three pre-pandemic influenza seasons between 2005 and 2008. A total of 20 laboratory-confirmed influenza-associated PICU cases (14 influenza type A, 5 type B, 1 type unknown), including two fatalities, were reported. This number was surprisingly low and corresponded to an estimated annual incidence for PICUadmitted children with seasonal influenza of 0.1/100,000 children < 5 years of age. In contrast, for the same age group incidences of PICU admissions associated with seasonal influenza were 4/100,000 in USA and 1-13/100,000 in South Australia [5, 7] . Apart from country-specific differences in access to health care as well as in criteria for ICU hospitalisation, the lower incidence found in our study may also be due to the weak to moderate influenza activity during the observation period in Germany [11] . Another important explanation is possible under-diagnosis of influenza in the hospitals, as not all physicians perform influenza testing of children admitted with severe acute respiratory infection. Low participation in the voluntary reporting system due to frequent changes of the medical staff in the PICUs is a further possible reason. Hence, the low reported incidence for PICU-admitted influenza cases in this study may underestimate the true incidence of severe paediatric influenza cases in Germany. With regard to fatal paediatric cases it should be noted that our study was not designed to capture influenza-associated fatalities that occurred outside PICUs, e.g. at home or during emergency treatment. For comparison, the national mortality register reported a total of 25 influenza-associated (ICD-10 code J10) deaths in children < 15 years of age during the years 2006 to 2008.
Our study showed that in Germany children < 1 year of age and children with chronic underlying diseases are the main paediatric risk groups for severe seasonal influenza infections. Influenza-associated pneumonia, secondary bacterial infections, ARDS and encephalitis/encephalopathy were frequent complications. The observed clinical characteristics were similar to the results from the U.S. and other countries [2, 4, 8, [12] [13] [14] [15] [16] [17] . However, children in our study were older (median 7.5 years) than in an ICU study in the U.S. (median 1.5 years) [12] . Fatalities also were reported only in two older children (8 and 14 years of age), whereas the majority of reported paediatric fatalities in the U.S. and Australia are below 5 years of age [14, 17] . In our study, ARDS occurred in 25% of the PICU children, in contrast to a retrospective chart review in an U.S. children's hospital with 27 PICU children diagnosed with seasonal influenza 2008/2009 [18] , which reported only 4% of ARDS.
In the current study, 10 (50%) patients were treated with oseltamivir, whereby in 5 (25%) patients treatment was started several days after the appearance of influenza symptoms or ICU admission. Antiviral treatment is most beneficial when started within 2 days after onset of symptoms. Nevertheless, oseltamivir treatment initiated later than 2 days after the first influenza symptoms is still considered useful in children with influenza pneumonia or to reduce viral shedding in the hospital [19] ; it also seems to reduce the risk of secondary bacterial super-infections, although the mechanisms are not fully understood [13] . In our study, co-infection with more than one bacterial species was found only in patients that did not receive oseltamivir treatment.
In total, 35% of the patients suffered from bacterial co-infection, including one fatality associated with Streptococcus pneumoniae. Patients with bacterial or fungal co-infections stayed twice as long (median 18 days) at the PICU than other influenza patients, thus indicating the high influence of those co-infections on the morbidity. Staphylococcus aureus was the most frequently observed bacterial species. The data from Germany indicate a similar situation as observed on severe paediatric influenza cases (fatalities) in the USA, which showed bacterial co-infections in 34% of patients in 2007, most frequently with Staphylococcus aureus [13] .
In Germany, influenza vaccination in children is currently recommended for risk groups, defined by chronic underlying diseases. In 2005, a regional survey in children < 24 months of age showed that 5% of all children had been vaccinated against influenza [20] . Among the severe cases reported in our study no child had been vaccinated. Eleven (55%) belonged to risk groups with chronic underlying disease, including eight (40%) of at least two years of age for whom influenza vaccines are considered to be efficacious [21] . One risk group child was below 6 months of age and, hence, too young to be immunized. Two other risk group children were under two years of age; for this age group, the efficacy of the inactivated influenza vaccines licensed in Germany is unclear, and data on safety are insufficient thus far [21] . Forty-five% of the patients with severe influenza were previously healthy children, who would not be covered by a risk-based vaccination strategy. A recommendation to vaccinate healthy children routinely against seasonal influenza is not widespread in Europe, but has been argued to have potential community benefits [22] .
Renewed interest in paediatric influenza arose with the occurrence of the new pandemic influenza A (H1N1) 2009 in Germany from April 2009 onwards [23] . An ongoing ESPED study on children with severe pandemic influenza A (H1N1) 2009 reported 93 cases below 15 years of age from 55 PICUs during a single season (August 2009 to April 2010), including 15 fatalities (11 within PICUs) [24] . This high number of cases reported in the ongoing ESPED study may be partly due to an increased awareness in physicians regarding the new subtype of influenza with unclear severity at the start of the pandemic, resulting in more frequent laboratory testing and diagnosis of influenza [5, 24] . Additionally, the higher number of PICU admissions may be due to a truly higher severity of the pandemic influenza A (H1N1) 2009 subtype in children compared to previous seasonal influenza subtypes [e.g., [18, 25] ], although not all studies report such a difference in severity [26, 27] , or found a difference only regarding risk groups [28] . Thus, a comparison of both ESPED studies on the absolute number of severe influenza cases [24] has to be regarded with caution, and may underestimate the importance of severe seasonal influenza caused by non-pandemic influenza virus subtypes in children in Germany. Further prospective, laboratory-confirmed surveillance in PICUs is needed to clarify the complication rates associated with influenza virus A and B and their different subtypes and lineages.
Almost half of the children with severe complications associated with seasonal influenza were previously healthy children, for whom there is currently no recommendation to vaccinate against influenza. Fifty% were children with chronic diseases for whom vaccination had been recommended but had not been followed. In these children the severe course of disease might have been prevented by vaccination. Oseltamivir treatment was often started several days after start of influenza symptoms or PICU admission, indicating that influenza is often diagnosed too late for effective antiviral patient treatment. The general incidence of influenza-associated severe disease in German PICUs was very low, but under-diagnosis and under-reporting during these prepandemic influenza seasons may be likely. Therefore prospective studies providing influenza subtype screening on PICU admissions of children with respiratory disease are needed to assess more accurately the burden of severe influenza-associated complications. These studies are necessary to provide data and evidence for the discussion on influenza prevention by general routine vaccination in children.  Epidemiological and clinical characteristics of childhood pandemic 2009 H1N1 virus infection: an observational cohort study BACKGROUND: There was a pandemic influenza around the world in 2009 including South Korea since last pandemic occurred four decades ago. We aimed to evaluate the epidemiological and clinical characteristics of this infection in childhood. METHODS: We evaluated the epidemiologic characteristics of all the subjects infected with the 2009 H1N1 influenza A virus (2,971 patients, ≤ 15 years of age), and the clinical and laboratory findings of the inpatients (217 patients, 80 had pneumonia) between 1 September 2009 and 31 January 2010 in a single hospital throughout the epidemic. RESULTS: The age distribution of all the subjects was relatively even. Over 90% of cases occurred during a two-month period. Two hundred and five patients (94.5%) received oseltamivir within 48 h of fever onset, and 97% of inpatients defervesced within 48 h of medication. The group with pneumonia included more males than females, and had higher leukocytes counts with lower lymphocyte differentials than the group without pneumonia. The white blood cell count and lymphocyte differential were associated with the severity of pneumonia. Corticosteroid treatment for severe pneumonia patients was highly effective in preventing disease progression. CONCLUSION: Children of all ages affected with even rates of infection, but males were predominant in pneumonia patients. Pneumonia patients showed lymphopenia and its severity was associated with the severity of illness. Our results suggest that the mechanism of lung injury in 2009 H1N1 virus infection may be associated with the host immune response. Although influenza virus infection has been a major global concern since the pandemic 1918 'Spanish flu', there have been no pandemic influenzas for near four decades after the 1968 'Hong Kong flu'. The pandemic 2009 H1N1 influenza A (2009 H1N1) virus infection was reported first in Mexico in February 2009, and then the virus spread rapidly worldwide, including in South Korea [1] . It has been reported that the majority of H1N1 patients were children and young adults and the mortality rate was not higher than that of seasonal influenza [2] [3] [4] . The majority of patients affected by the 2009 H1N1 virus infection recovered uneventfully, but some previously healthy patients developed a rapidly progressive pneumonia, leading to acute respiratory distress syndrome (ARDS), multi-organ failure, and death [5] [6] [7] . With this enigma, the pathogenesis of acute lung injury (pneumonia) in influenza infections remains unknown [1, 8, 9] .
In South Korea, the first patient with 2009 H1N1 virus infection was reported in May, 2009. The number of patients gradually increased until mid-October, when the number of patients was overwhelming for a month, and then gradually decreased, with few people becoming infected after February, 2010. Owing to sensational reports of childhood fatality in the mass media and a new diagnostic tool, the real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we had the opportunity to evaluate patients with 2009 H1N1 virus infection from the onset of their illness. In addition, during the study period we observed a dramatic effect of early treatment with corticosteroids and oseltamivir for patients with severe pneumonia including rapidly progressive pneumonia [9, 10] . In this study, we evaluated the epidemiological, clinical and laboratory features of children with 2009 H1N1 virus infections in a single hospital throughout the epidemic.
We retrospectively evaluated all patients with 2009 H1N1 virus infection during the pandemic (2,971 patients) for epidemiologic characteristics, and for clinical characteristics, we reviewed the medical records and chest radiographic findings of 217 children admitted to The Catholic University of Korea, Daejeon St Mary's Hospital between 1 September 2009 and 31 January 2010. The diagnosis of patients depended on positive results for the 2009 H1N1 virus RT-PCR (AccuPower ™ in Korea, BiONEER, Alameda, CA, USA) through throat swabs.
Although indications for admission were not clearly defined in this study, the majority of the inpatients were those who were suspected to have severe disease such as pneumonia and to have risk factors for severe disease such as infants and bronchial asthma. However, it might be possible that excessive concern of parents on fatality of this infection in part affected on admission of the uncomplicated cases. Among the 217 inpatients, we selected 80 patients with pneumonia and 137 patients without pneumonia, based on the chest radiographs. The chest radiographic patterns of admitted patients were reviewed and classified by two pediatricians (KY Lee and JW Rhim) and one pediatric radiologist (JC Kim). The patients with chest radiographic patterns that showed increased nodular densities along the bronchial trees unilaterally or bilaterally, were designated the bronchopneumonia group (49 patients). Patients with a distinctive large patch of infiltration, segmental or lobar consolidations were designated the segmental/lobar pneumonia group and regarded as having a severe pneumonia (31 patients). The first day of fever was regarded as the first day of illness. Among pneumonia patients, we tried corticosteroid treatment for 17 patients with severe pneumonia. As for indication of corticosteroids (methyprednisolone, MP or prednisolone), the subjects had severe respiratory distress with hypoxemia at presentation (12 cases) or during hospitalization (5 cases) requiring O 2 therapy (9,10). We compared the clinical and laboratory characteristics of the different groups.
The study was approved by the Institutional Review Board of the Catholic University of Korea, Daejeon St Mary's Hospital.
Statistical analyses were performed using the Statistical Package for the Social Sciences for Windows version 12.0 (SPSS, Chicago, IL, USA). Continuous variables are reported as means ± standard deviations. Statistical significance was assessed using the Student's t-test and the paired t-test for continuous variables, and using the χ 2 test for categorical variables. A P value of < 0.05 was considered significant for the statistical tests.
During the study period, 5,975 children (aged 2 months-15 years) with influenza-like illness were seen at our hospital. Among them, the 2,971 patients were positive by RT-PCR, and 217 patients were admitted to the isolation wards. The hospitalization rate was 7.3%. The mean age of the patients (2,971 cases) was 7.6 ± 4.1 years of age and the male-to-female ratio was 1.1:1 (1,581/1,390). The age distribution of the patients is shown in Figure 1 . Children of all age groups except infants had a relatively even rate of infection ( Figure 1 , gray bars). The numbers of new patients each week is shown in Figure 2 . There was an explosive pattern, with over three quarters of the cases occurring during a single month (the 43rd-47th weeks of 2009; 18 October to 14 November). The age distribution and the weekly case rate of the patients admitted to hospital (217 cases) are also shown on Figure 1 and 2 (black bars). These demonstrated similar patterns to those of the total patient cohort. For hospitalization rates, younger children (0-4 years, 9.8%, 75/762) showed higher admission rates than older children (5-9 years, 8.6%, 104/1216 and 10-15 years, 3.8%, 38/993). 
In the 217 hospitalized children, the mean age was 6.2 ± 3.7 years, and the male-to-female ratio was 1.6:1 (132/85). Almost all the patients had a high fever (97.2%) and cough (92.6%) suggestive of severe infection such as pneumonia at the time of admission. Most of the patients were previously healthy although some had underlying diseases and only 17 patients had underlying disorders (7.8%), including 11 chronic pulmonary diseases (10 patients with bronchial asthma and one with past history of brochopulmonary dysplasia), 4 neuromuscular disorders (two with epilepsy and two with cerebral palsy), one with chronic liver disease, and one with nephrotic syndrome. The outcomes for these patients were uneventful, except for two asthmatic patients who developed a mild pneumonia. All inpatients received oseltamivir of the recommended doses for body weight and a broad-spectrum antibiotic (ampicillin/sulbactam). Two hundred and five patients (94.5%) received oseltamivir within 48 h of fever onset. The mean duration of fever before admission (including the day of admission) was 2.0 ± 0.8 d and 167 patients (77%) defervesced on the next day. Only seven patients had fever that persisted > 48 h after oseltamivir treatment. During hospitalization, five patients showed progressive pneumonia despite early antiviral therapy.
According to the initial chest radiographs, 80 patients had pneumonia and were divided into two groups: the bronchopneumonia group (49 patients) and the segmental/lobar pneumonia group (31 patients). In pneumonia patients, pneumonic infiltrations appeared within 48 h of fever onset in 68 patients (85%). When we analyzed the inpatients according to age (0-4 years, 75 patients; 5-9 years, 104 patients; and 10-15 years, 38 patients), the rates of pneumonia in each age group were 36.0% (27/75), 45.2% (47/104) and 8.8% (6/38), respectively. The severe pneumonia (segmental/lobar type) was predominant in the older age groups (14.8% (4/27), 48.9% (23/47) and 50% (3/6), respectively).
Among the 80 pneumonia patients, 17 patients showed severe respiratory distress with hypoxemia at presentation (12 cases) and during their hospital stay (5 cases). Arterial blood gas analysis was done in 15 patients, and 12 patients showed hypoxemia (PO 2 < 60 mmHg in the room air). These patients received additional corticosteroids as soon as possible when indicated; 12 patients received intravenous MP (10 mg/kg/day, divided two doses, at presentation, 5 mg/kg/day at next day and then tapered off within a week) and 5 patients received oral prednisolone (1 mg/kg/day, divided 3 doses, for 3 days tapered off within a week). Six patients received early MP with osetalmivir which is recommended early use, before the positive RT-PCR results. Interestingly, all patients except one (age 4 years) were among the 5-9 years age group, and male patients were predominant (13/17). We performed serial chest radiographs of some cases in these patients. A 6-year-old male patient complained of one day fever and cough, and his initial chest radiograph showed few pulmonary infiltrations. On the following morning, he complained of severe dyspnea and showed chest radiographic infiltrations on right lower lung and left hilar regions. He had received two doses of oseltamivir before the MP treatment ( Figure 3A -C). A 7 year-old male patient was presented with a day fever and severe cough and had a rapidly progressive pneumonia in which initial patchy infiltrates on left upper lobe progressed to total left lung consolidation within 12 h after admission. He was given three doses of oseltamivir before the MP treatment ( Figure 4A-G) . These two patients showed dramatic improvements in their clinical symptoms and radiographic findings within 24 h after MP treatment. A 7-year-old male patient with lobar pneumonia was treated with oseltamivir, MP and intravenous immunoglobulin (IVIG) treatment. He was admitted with fever, cough and progressive dyspnea of 2 days, and after MP (10 mg/ kg/day, divided 2 doses) infusion, he showed persistent dyspnea and slightly aggravated pneumonic consolidation ( Figure 5C ). On the following morning, high-dose IVIG (1 g/kg) was infused for 6 hours. At the time of termination of IVIG, his clinical symptoms disappeared and a dramatic improvement of radiographic findings was observed within 6 hours after IVIG termination ( Figure  5A-F) . There was no adverse or rebound reaction in any patient treated with corticosteroids. The clinical symptoms of all patients improved within a day and their pneumonic infiltrations, regardless of severity, ceased immediately after corticosteroid treatment and disappeared within several days without adverse reactions.
Extrapulmonary manifestations (complications) of H1N1 infection were observed as four cases of febrile seizure, two cases of urticaria, one case of hepatitis and one case of myositis, and no cases of encephalopathy.
When we compared the patients with and without pneumonia, and the patients with segmental/lobar pneumonia versus those with bronchopneumonia, there were significant differences in certain parameters between the groups. Compared with the group without pneumonia, the males were over-represented in the pneumonia group (P = 0.001) and longer hospitalizations (P < 0.001), higher values for hemoglobin (P = 0.03), WBC (P < 0.001) and CRP (P < 0.001) and lower values for lymphocyte differential (P < 0.001) ( Table 1 ). The patients with the more severe type of pneumonia (segmental/lobar pneumonia) showed a higher mean age (P = 0.001) and leukocyte count (P = 0.01) and lower lymphocyte differential values (P < 0.001) compared with the group with bronchopneumonia (Table 2 ). In addition, the severe pneumonia patients who received corticosteroids (17 cases) had the highest leukocyte counts and CRP levels, and the lowest lymphocyte differentials (11 800 ± 3600/mm 3 , 3.0 ± 3.1 mg/dL, and 8.8 ± 6.3%, respectively) [10] .
In this study, we found that children of all ages except infants had a relatively even rate of infection with the 2009 H1N1 influenza virus. This is in agreement with other studies showing that few children and young adults have immunity against a new viral infection [2, 11, 12] . Our study of all 2009 H1N1 virus-infected children, in a single hospital, throughout the epidemic may have epidemiological implications, along with other studies based on data from admitted patients or gathered during a portion of the pandemic period [11] [12] [13] [14] . It has been reported that younger children (< 4-5 years old) with H1N1 virus infection tended to be admitted to hospital more frequently than older children or adults, and our results are compatible with these studies [2, 3, 6] . It has also been reported that certain risk factors are related to the likelihood of developing severe illness, including a younger age and particular underlying diseases, including obesity [6, 7, 15, 16] . In this study, we did not analyze obesity and young age as risk factors, and only 17 patients (7.8%) had underlying diseases. Although the hospitalization rate was higher among the younger children (0-4 years of age), in this study, pneumonia and severe pneumonia were more prevalent in the 5-9-year-old group than in younger children.
The explosive increase in the infection rate during two months (mid-October to mid-December) of the study period may be a typical pattern of spread of an acute respiratory viral infectious disease that has a short incubation period (1-3 days). This pattern is similar to those reported in other regions of Korea and other countries [3, 4, 17] . Vaccination against the 2009 H1N1 influenza virus in Korea started on 18 November, 2009 for school-aged children, on 7 December, 2009 for children aged 6 months to 5 years and on 21 December, 2009 for adults with risk factors, which was after the peak of the epidemic. The rapid disappearance of the spread of infection might be the effect of vaccination and the strengthened individual hygiene, but the epidemiologic pattern suggests that an unknown herd immunity against a new viral infection might also be responsible [3, 17] .
It is reported that patients with 2009 H1N1 virus infections had a relative leukopenia with lymphopenia [18, 19] . However addition to this finding, we found in the present study that the patients with pneumonia had a higher WBC count with lower lymphocyte differential, and the more severely affected patients had the highest WBC with the lowest lymphocyte differential in the early stages of the infection (within two days of fever onset). Previous human studies of H5N1 virus infections have revealed that a lower lymphocyte count was associated with a poor outcome [20] , and mice infected with influenza viruses showed a lymphopenia and the H5N1 subtype was associated with marked lymphopenia with total lymphoid depletion [21] . Therefore peripheral lymphocyte may be associated with the pathogenesis of acute lung injury in influenza virus infections [9] . Altered CRP and ESR values were not prominent in 2009 H1N1 virus infections, but higher CRP values were associated with a more severe illness.
The male-to-female ratio was 1.1:1 among all patients, and 1.6:1 among the admitted patients. Furthermore, male patients were predominant among those with pneumonia (3:1, 60/80) and those with respiratory distress who received corticosteroids (3.3:1, 13/17). Other epidemiologic studies on children have reported a male predominance [16] , but some studies on inpatients have reported a female predominance [4, 7] . These findings suggest that genetic factors and possibly environmental factors are Regarding the pathogenesis of lung injury in influenza virus infections, it has been believed that the viruses from upper respiratory tract spread to lower lung tissues and elicit the cytopathic reaction. However, some clinical and experimental studies have suggested that the innate and/ or cell-mediated immune reaction (T cell) with excessive production of cytokines of the host may also be responsible for the lung injury in influenza virus infection [9, [22] [23] [24] [25] . We experienced no intensive care patient in this series despite large subjects of the study, and it may be, at least in part, explained by a rapid use of corticosteroids on the patients with severe pneumonia [9, 10] . Because this pandemic occurred over 40 years after last pandemic (Hong Kong flu), there have been no controlled-clinical trials for the efficacy of corticosteroids on influenza virus infections, although yearly seasonal influenza and small cases of sporadic H5N1 avian influenza virus infection have occurred during inter-pandemic period. The beneficial effect of corticosteroids in pneumonia caused by influenza virus infections may have resulted from reduction of systemic inflammation caused by immune cells and cytokines [26, 27] . Our treatment policy which needs to be proven by controlled clinical studies in coming pandemics or in other influenza virus infections, may help to reduce the morbidity and possibly prevent the progression to fatal pneumonia [9, 10] . We have reviewed the rationale of our corticosteroid treatment and the host immune responses to viral insults in influenza virus infections, and proposed a new concept for the pathogenesis of acute lung injury in influenza virus infections, using a 'protein homeostasis system' of the host [9] .
It has been reported that antiviral therapy (oseltamivir) is effective in the acute stages of influenza infection, including H1N1 virus infection in humans and experimental animals [4, 28] . In agreement with this, we found that the majority of patients (97%) defervesced within 48 h after medication and most pneumonic infiltrations in pneumonia patients had improved at discharge.
There are some limitations to this study. Compared with other studies, we had many uncomplicated inpatients owing to flexibility of our admission policy. Because we did not perform extensive microbiological testing, such as viral cultures, paired-sample serologic studies and PCR for other pathogens, we cannot rule out the possibility of coinfection with other respiratory pathogens.
In pandemic 2009 H1N1 virus infections, children of all ages were evenly affected, and males were predominant in pneumonia patients. Early antiviral treatment was very effective in inducing rapid defervescence for the patients.
The patients with 2009 H1N1 infections showed lymphopenia, and its severity was associated with the severity of the illness in the early stages. Together this finding, the rapid improvement in clinical signs and the prompt resolution of severe pneumonic consolidations after immunemodulator (corticosteroid and IVIG) treatment suggest that the mechanisms of lung injury in this infection may be associated with the cell-mediated immune response of the host, rather than virus-induced cytopathies. Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury BACKGROUND: Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI. METHODS: Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry. RESULTS: EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy. CONCLUSION: The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes. ALI encompasses a spectrum of pulmonary disorders that is often accompanied by life-threatening hypoxemic respiratory failure and diffuse bilateral pulmonary infiltrates. Moreover, the origins of ALI are often complex (e.g., pneumonia, sepsis, or left atrial hypertension) and not easily attributed to a defined cause [1] [2] [3] [4] [5] [6] . In its most dramatic clinical form, acute respiratory distress syndrome (ARDS), precipitous impairment of gas exchange is associated with a high mortality rate (38.5%), especially among elderly patients [7] . Therapy for most patients with idiopathic ALI is limited to supportive care and infection prevention [6, [8] [9] [10] . Select studies have suggested a benefit of corticosteroid therapy in a subset of patients with ALI/ARDS (e.g., [11] [12] [13] [14] ). However, large prospective studies have not supported a consensus opinion for the routine use of corticosteroids [15] [16] [17] [18] .
The pathogenesis of ALI remains unclear, largely a consequence of the heterogeneity of patients coming into the ICU and the broad clinical features characteristic of ALI [1, 19] . The cellular mechanisms contributing to lung tissue injury in ALI are for the most part unknown, [20] although the potential involvement of neutrophils in the development of ALI remains the focus of many studies (see for example [21, 22] ). In particular, neutrophil-derived products (e.g., extracellular matrix degrading proteinases and reactive oxygen species), inflammatory fibrotic cytokines, and growth factors [23] [24] [25] [26] have been postulated as causative agents underlying the onset and progression of disease [27] .
For most patients with ALI, eosinophils have not been reported as a prominent histological feature, despite their potential tissue damaging capability [5, 28] and their presence in a number of other well defined pulmonary diseases such as asthma [29] and acute eosinophilic pneumonia [30] . However, among this larger literature there are some studies that have implicated this granulocyte as either a potentially important contributor to disease or, at the least, a diagnostic biomarker of events occurring in ALI patients [21, 27, 31] . Interestingly, these studies suggested a "predictive" and "discriminative" value of eosinophil activity assessments in designing an effective therapy for patients exhibiting clinical features of ALI. Unfortunately, the lack of an observable eosinophil infiltrate at the histological level remains a significant confounding issue, especially for those patients who may have been treated (even briefly) with systemic corticosteroids. In turn, the typical absence of visible eosinophils in most cases of ALI that come to biopsy has limited the further development of hypotheses and experimental studies investigating a role (s) of eosinophils in ALI.
In this study we demonstrate that standard histological evaluation of lung biopsies significantly underestimates eosinophil accumulation when compared to assessments using a novel eosinophil peroxidase (EPX)specific monoclonal antibody (EPX-mAb) visualized by immunohistochemistry. We employed this unique increase in sensitivity to detect eosinophils and evidence of tissue degranulation in a group of ALI patients. A retrospective assessment of ALI patients who had a lung biopsy taken during the course of their acute care was conducted, comparing the results of these assessments with lung tissue from control subjects. These blinded assessments revealed that the added sensitivity of EPX-mAb immunohistochemistry detected a significant increase in eosinophil accumulation/degranulation in ALI vs. control subjects. More importantly, EPX-mAb immunohistochemistry identified a subset of ALI patients who survived hospitalization, suggesting its use as a prognostic indicator may represent a previously underappreciated diagnostic strategy in the management of pulmonary patients.
The patient studies presented in this manuscript were performed in accordance with NIH guidelines and Mayo Foundation institutional policies (Institutional Review Board, 08-001908 Immunohistochemical Study of Eosinophil Degradation in Archived Lung Biopsies), and in compliance with HIPPA guidelines for patient privacy.
An overview of the demographic data and pathological/ clinical assessments of our ALI study subjects is presented in Tables 1 and 2 . These pulmonary patients were initially identified by study personnel from a search of the Mayo Clinic Arizona Pathology Database with the search key words: lung, biopsy, acute lung injury (ALI), diffuse alveolar damage (DAD), organizing pneumonia, and ARDS. Study personnel subsequently reviewed the available pathology reports and clinical medical records to identify the subset of patients who had received a diagnosis of ALI. Thus, to be included in this study a given patient had to have a diagnosis of ALI and have undergone a biopsy during their course of treatment. It is noteworthy, that this process was not discriminatory and all patients with available biopsy material that had received an ALI diagnosis as part of their standard-ofcare were included in this study. The ALI patients included in this study met AECC established clinical criteria [4] for an acute lung injury diagnosis and also displayed characteristic pathological changes linked with this disease. Specifically, assessments of all study patients upon admission with acute respiratory distress revealed the presence of diffuse radiographic abnormalities and hypoxia characterized by an A-a gradient (PaO2/FiO2) less than 300 [4, 32] . Indeed, the PaO2/ FiO2 ratio in a subset of these cases (11 of 20 total cases) was below 150 and thus would meet clinical criteria for ARDS [4, 32] . In addition, the twenty ALI patients in this study included thirteen patients (independent of PaO2/FiO2 levels) that required mechanical ventilation during their hospitalization. Review of patient medical records also revealed the absence of typical risk factors associated with ALI, including myocardial infarction, pulmonary embolism, infection/sepsis (e.g., pneumonia), and acute drug reaction. The pathology evaluations of all study-subjects confirmed the clinical indications of ALI. That is, each patient included in our study displayed three specific histopathologies in the available biopsies [33] : (i) The presence of fibrin in the alveoli; (ii) The demonstration of an organizing pneumonia (i.e., a prominent airway cellular infiltrate); and (iii) Evidence of reactive airway epithelial Type II cell hyperplasia. Among these twenty ALI patients, 45% (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Seven of the study group did not survive hospitalization, and of these non-surviving patients >71% (5/7) received an ALI-DAD diagnosis. A variety of co-morbid medical disorders were evident in our ALI patients, including several patients with connective tissue disorders. However, examination of the medical records and care-giver notes failed to identify elements of commonality regarding disease onset or progression. Nonetheless, the unresolved character of disease progression in these subjects was such that nineteen of the twenty patients received corticosteroid therapy during their course of treatment.
Control lung samples consisted of uninvolved areas of lung tissue recovered from patients undergoing resection for a diagnosis of lung cancer ( Table 1) . None of the control subjects were receiving systemic corticosteroids, although four control subjects were receiving inhaled corticosteroids.
The twenty lung specimens obtained from patients with ALI included sixteen surgical lung biopsies and four transbronchial lung biopsies considered adequate for histologic review (i.e., containing alveolated lung parenchyma). All control biopsies were taken from uninvolved areas of surgically resected lung tissue removed for treatment of lung cancer. Lung tissue biopsies were fixed in 4% buffered formalin, embedded in paraffin, and serial 4 μm thick sections were cut. Histopathological and immunohistochemical staining of lung tissue sections H&E staining was performed using an automated stain processing unit in the Mayo Clinic Arizona clinical histology unit. Immunohistochemistry was performed using a EPX-mAb as previously described [34] . Evaluations of the slides were performed with either an Olympus BX50 or a Zeiss Axiophot compound microscope.
Serial sections from each biopsy were coded by clinical histopathology laboratory personnel and in each case the middle (slide 2) of the three serial sections was stained with Hematoxylin -Eosin (H&E). Slide 1 of the series was subjected to immunohistochemical staining with EPX-mAb and slide 3 served as an isotype immunoglobulin negative control for the immunohistochemistry.
The eosinophil infiltration of lung tissue using H&E staining was performed in an investigator-blinded fashion independently by an experienced pulmonary pathologist with a specialty in lung diseases (KL) and a pathology resident (PG). The slides were evaluated by each individual as a numerical average calculated from 10 randomly selected hpf (high powered fields; 40x objective/10x ocular lens, 0.29 mm 2 field of view); that is, a total area of~3 mm 2 per biopsy-investigator. The reported values are the mean ± SEM of all investigatorderived counts.
Quantification of eosinophil tissue infiltration within each biopsy using EPX-mAb immunohistochemistry was independently performed in an intra/inter-blinded fashion that included investigators from pathology (KL, EJ, and PG -EPX-mAb based eosinophil counts in lung tissue); KL and EJ -EPX-mAb assessments of eosinophil degranulation), a hospital/clinic-based pulmonary fellow (KP -EPX-mAb based eosinophil counts in lung tissue), and a PhD graduate research fellow (LW -EPX-mAb based eosinophil counts and degranulation in lung tissue). All evaluations were done at a magnification of 400x. The number of positively stained eosinophils was determined in the alveolar lung parenchyma as a numerical average calculated from 10 randomly selected hpf; that is, a total area of~3 mm 2 per biopsy-investigator. The reported values are the mean ± SEM of investigator-derived counts.
The level and extent of eosinophil degranulation observed within each patient biopsy was also determined by scanning 10 randomly selected hpf (40x objective/10x ocular lens, 0.29 mm 2 field of view); that is, a total area of~3 mm 2 per biopsy-investigator. Each field examined was graded using a scale that permitted stratifying the available patients based on a relatively low resolution grading scale that was easily reproduced by multiple evaluators of varying levels of experience and expertise: Level 0 = No identifiable eosinophils and/or degranulation [35] ; Level 1a = The field shows evidence of eosinophil degranulation (i.e., extracellular release of EPX) that represents ≤10% of the field's total area and has <3 independent areas within the field displaying degranulation; Level 1b = The field shows similar evidence of eosinophil degranulation as Level 1a but instead displays ≥3 independent areas within the field with evidence of degranulation; Level 2a = The field shows evidence of eosinophil degranulation that includes extracellular release of EPX, enucleated eosinophils (i.e., cytoplasmic fragments), and/or the presence of free eosinophil granules. The extent of degranulation represents 10 -50% of the field's total area; Level 2b = The field shows similar evidence of eosinophil degranulation as Level 2a but instead has a level of degranulation representing >50% of the field's total area. Eosinophil degranulation was quantified for each of 10 randomly selected hpf of a given biopsy (i.e., patient) by initially applying an increasing numerical value to the level of degranulation evident in the field (Level 0 = 0, Level 1a = 1, Level 1b = 2, Level 2a = 3, and Level 2b = 4). The extent of eosinophil degranulation in the biopsy was then determined as the average of the numerical values assigned to each of the 10 hpf examined. This grading of eosinophil degranulation was performed independently by three outcome-blinded evaluators (an experienced pulmonary pathologist (KL), a pathology resident-fellow (EM), and a Ph.D. graduate student (LW)), all of whom were also unaware of the scores reported by the other evaluators. Degranulation scores are reported as the mean numerical value derived from all three evaluators ± SEM.
Data are expressed as the mean ± SEM. Statistical analysis for comparisons between groups was performed using either a Student's T test or a Wilcoxon Two-Sample Test for non-parametric data for comparisons between data sets that were not uniformly distributed. Differences between mean values were considered significant when p < 0.01. Intraclass correlation coefficients (ICC) were also determined between investigators reading slides as a measure of inter-rater agreement [36] .
Serial biopsy sections from either control or ALI subjects were stained for evaluations of eosinophil tissue infiltration. Representative photomicrographs of H&E stained slides as well as lung sections subjected to EPX-mAb immunohistochemistry are shown in Figure 1 . The quantitative evaluations of eosinophil density using both staining methods are presented as individual patient assessments in the histograms of Figure 2 . As expected, the evaluation of the H&E stained patient biopsies revealed little evidence of eosinophil infiltration in both control and ALI subjects (0.02 infiltrating eosinophils/ hpf and 0.04 infiltrating eosinophils/hpf, respectively). However, evaluation by the same pathology investigators of the serial slides subjected to EPX-mAb immunohistochemistry demonstrated an enhanced sensitivity to detect eosinophils in these lung tissue sections. Specifically, evaluations of the lungs of control subjects using EPX-mAb immunohistochemistry revealed a >40-fold increase in the ability to detect tissue infiltrating eosinophils relative to H&E staining (0.81 eosinophils/hpf vs. 0.02 eosinophils/hpf, p < 0.01).
Evaluation of serial lung sections following EPX-mAb immunohistochemistry demonstrated that the density of pulmonary eosinophils in the collective group of ALI patients is significantly higher relative to control subjects (3.6-fold, 2.88 eosinophils/hpf vs. 0.81 eosinophils/ hpf (p < 0.01), respectively). More importantly, further evaluations of the ALI patients ( Figure 2 , shaded histograms) surprisingly showed that EPX-mAb detection of infiltrating lung eosinophils divided these patients into subjects which survived vs. those that did not survive hospitalization (8.4 ±2.9 eosinophils/hpf vs. 1.9 ± 0.6 eosinophils/hpf, p < 0.01).
ALI patients surviving hospitalization display significant levels of eosinophil degranulation (i.e., extracellular matrix deposition of EPX) compared with non-surviving patients Assessments of lung sections following EPX-mAb immunohistochemistry revealed that ALI patients displayed significant and varying levels of degranulation that were quantifiable. This degranulation was often observed in these patients in the absence of identifiable intact eosinophils. The photomicrographs of Figure 3 are representative of the stratified levels of increasing degranulation observed in ALI patients from no evidence of degranulation (Level 0) in a given high powered field to >50% of the field evidencing eosinophil degranulation (Level 2b). Similar to the higher levels of eosinophil infiltration observed in the collective group of ALI patients, the collective group also evidenced a >2-fold increase in the level of eosinophil degranulation compared to control subjects (2.20 ± 0.15/hpf vs. 1.02 ± 0.38/hpf, respectively). More importantly, quantitative assessments of degranulation (i.e., mean numerical score ±SEM) based on EPX-mAb immunohistochemistry (Table 3 and Figure 4 ) revealed that ALI patients surviving their hospitalization also displayed significantly higher levels of degranulation compared to non-surviving patients (2.62 ± 0.18/hpf vs. 1.58 ± 0.10/hpf, respectively).
Independent of any conclusions regarding our evaluation of ALI vs. control subjects, two technical observations regarding our assessments of the lung biopsies using EPX-mAb immunohistochemistry relative to H&E staining are noteworthy: (i) EPX-mAb immunohistochemistry is an easily performed assessment that provided a >40fold enhancement to detect tissue infiltrating eosinophils. This increased sensitivity not only allowed for the greater detection of tissue infiltrating eosinophils but also corresponding increases in the speed, accuracy, and reproducibility of this determination. (ii) EPX-mAb immunohistochemistry provided a rapid and definitively quantitative assessment of eosinophil degranulation within the lung parenchyma, observable even in the absence of intact infiltrating eosinophils.
Unfortunately, studies of ALI patients are often incomplete and subject to ambiguities resulting from the broad and complex character of symptoms and contributing etiologies [2, 37] . Compounding these issues are the limited sample materials that are available for analysis (e.g., lung tissue or BAL fluid), including the timing of when the samples were recovered during the course of a given patient's care. In this respect, the study presented here is subject to these very same limitations. That is, our study is of a small heterogeneous cohort of patients (n = 20) whose disease origins and severity vary Figure 2 Assessment of individual patient biopsies revealed that unlike traditional H&E histopathology, EPX-mAb immunohistochemistry demonstrated that ALI patients have increased levels of eosinophils relative to control subjects and that within the ALI cohort this increase correlated with patient survival. Serial sections from either control subjects or acute lung injury patients were stained with H&E and subjected to EPX-mAb immunohistochemistry prior to evaluation for infiltrating eosinophil numbers per high powered field. Eosinophil counts per hpf were determined by individual investigators (n = 2) as the average count resulting from the examination of 10 randomly selected fields; investigators were blinded to both the clinical outcome and the scores of the fellow evaluator. The scatter plots presented represent values for each individual patient derived as the mean of the average eosinophil counts from these evaluators (ICC = 0.785 (95% confidence interval: 0.540 to 0.908). The scatter plots within the shaded area represent acute lung injury patients following EPX-mAb immunohistochemistry that were then stratified (following decoding of the data) on the basis of their hospital survival. The mean for each cohort is presented as a horizontal bar. *p < 0.01
considerably. Moreover, we did not have control over the general demographics of these ALI patients nor could we dictate why, when, or where within the lung the biopsies for study were taken relative to the course of disease and/or patient treatment. The ALI subjects of this study were also not selected on the basis of a defined and standardized medical history or a regimented treatment plan. Finally, the control subjects available to us were limited and did not include healthy volunteer biopsies or ALI patients prior to any medical interventions.
Given the limitations of the ALI study group and our control subjects noted above, we were surprised at the ability of EPX-mAb immunohistochemistry to distinguish ALI patients from control subjects. Specifically, eosinophils are not considered a reliable histopathological marker of ALI (reviewed in [5, 28] ). Yet in a completely patient-blinded fashion that was reproducible among 3-4 independent evaluators who had no knowledge of one another's assessments, our numerical results were able to identify a group of ALI patients relative to control subjects on the a basis of both increased numbers of tissue infiltrating eosinophils and increased levels of eosinophil degranulation. Furthermore, these evaluations allowed us in a completely clinical outcomeblinded fashion to stratify the ALI patients into those surviving their hospitalization relative to the non-surviving patients. It is clear that the design and power of this study precludes us from overly provocative conclusions regarding the role of eosinophils, including their link with specific symptoms or their part in pathways that exacerbate or attenuate disease pathologies. Nonetheless, this study does suggest that EPX-mAb immunohistochemistry may represent a previously unrecognized diagnostic tool providing prognostic information for the management of ALI patients. In addition, given the paucity of available therapeutic options and discriminatory testing modalities, [1, 6, 8, 10, 19] assays detecting the release of eosinophil products (e.g., ELISA based assessment of degranulation from biological fluids such as breath condensate, intratracheal tube secretions, and/or BAL fluid) may also represent rapid and minimally invasive biomarkers of disease to assess this difficult patient population [38] . Indeed, this initial report provides the Figure 3 Acute Lung Injury patients display quantitatively different levels of eosinophil degranulation that may occur even in the absence of intact infiltrating eosinophils. Representative photomicrographs of the five described levels of eosinophil degranulation within biopsies from ALI patients. Level 0: No evidence of eosinophil degranulation. Level 1a: Nominal levels of eosinophil degranulation representing <3 areas of granule protein release that is <10% of the field of view. Level 1b: Slightly elevated level of eosinophil degranulation representing ≥3 areas of granule protein release that again is <10% of the field of view. Level 2a: Significant level of eosinophil degranulation that includes 10-50% of the field of view. Level 2b: Significant level of eosinophil degranulation that includes extracellular release of EPX, enucleated eosinophils (i.e., cytoplasmic fragments), and/or the presence of free granules (i.e., EPX-containing secondary granules not associated with fragmented eosinophils). The extent of degranulation represents >50% of the field's total area. Scale bar = 50 μm.
rationale for future studies of increased design and complexity to expand this link between ALI patients and their survival based on evidence of pulmonary eosinophils and tissue degranulation.
The studies presented in this report have identified both significant technical insights and revelations regarding eosinophils in lung biopsy samples from control and ALI patients. These observations will likely have a direct impact on the assessment of eosinophils and their role (s) in lung diseases and, more important, may lead to previously overlooked or untried diagnostic and therapeutic strategies with which to treat these problematic patients.
• Immunohistochemical detection of EPX in lung tissue biopsies provides even a board certified pathologist (with a specialty in pulmonary diseases) a >40-fold increase in sensitivity to detect tissue infiltrating eosinophils in lung tissue sections
• EPX-mAb based immunohistochemistry allows for the unique assessment of eosinophil activation (i.e., degranulation events) within the lung even in the absence of a demonstrable eosinophil infiltrate
• The increased sensitivities afforded by EPX-mAb demonstrate that increases in lung infiltrating eosinophils and evidence of eosinophil degranulation are surprisingly characteristic features of biopsies from ALI patients relative to control subjects • Assessments of lung biopsies from ALI patients following EPX-mAb immunohistochemical staining may provide a potential basis to identify a subset of thẽ 40% of ALI patients who will not survive their hospitalization. Figure 4 EPX-mAb immunohistochemistry provides a quantitatively significant strategy to distinguish acute lung injury patients that survive their hospitalization vs. those patients that did not survive. Sections from acute lung injury patients were subjected to EPX-mAb immunohistochemistry prior to evaluation for evidence of eosinophil degranulation as described in the Materials amd Methods and the legend of Figure 3 . Eosinophil degranulation scores were determined by individual investigators (n = 3) as the average numerical score resulting from the examination of 10 randomly selected high powered fields (hpf -400x); investigators were blinded to both the clinical outcome and the scores of the fellow evaluator. The scatter plots presented represent values for each individual ALI patient stratified based on hospital survival. Patient eosinophil degranulation values are expressed as the mean of the average eosinophil degranulation score from all three evaluators. The error bars associated with each patient data point is the SEM linked with the mean value derived from each of the three evaluators. The mean for each cohort is presented as a horizontal bar. *p < 0.01 Overlapping signals for translational regulation and packaging of influenza A virus segment 2 Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1–4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1–4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging. Influenza A virus (IAV) is a major pathogen, capable of infecting a number of species including humans, birds, swine and horses. Its genome is contained on eight segments of negative sense viral RNA (vRNA), individually complexed with the trimeric viral polymerase (PB2, PB1 and PA) and nucleoprotein (NP) to form ribonucleoprotein (RNP) particles (1) . On infection, the RNPs migrate to the nucleus where the polymerase initially transcribes the vRNA templates to produce mRNA, and later replicates the genome using positive sense cRNA intermediates (2) . Subsequently, new vRNAs are exported from the nucleus (as RNPs) and packaged into progeny virus particles at the plasma membrane. As each segment encodes at least one essential gene product, a viable virus particle must contain one copy of each segment, which is facilitated via specific cis-acting packaging signals present in the terminal non-coding and coding regions of each vRNA (3) . IAV strains also show considerable variation in pathogenicity, and the molecular mechanisms underlying this have not been fully elucidated. Segment 2 encodes PB1, the core component of the viral polymerase, which has been linked to inter-strain differences in pathogenicity and host range (4, 5) . However, the single mRNA species transcribed from the segment also encodes two further proteins that are non-essential for virus replication: PB1-F2 and PB1-N40 (6, 7) . PB1-F2 is encoded by the+1 open reading frame (ORF) relative to PB1 and is initiated from AUG4 ( Figure 1A ). Depending on virus strain, the PB1-F2 ORF is up to 90 codons long, but in many viruses (including the recent pandemic H1N1 virus, where the gene is effectively absent), is truncated to variable extents by one or more stop codons (8, 9) . PB1-F2 polypeptides of 79 amino acids or longer can localize to mitochondria and the protein has been associated with pro-apoptotic and pro-inflammatory effects (6, (9) (10) (11) (12) . A proportion of the protein also localizes to the nucleus where it interacts with PB1 and may influence polymerase activity (13, 14) . In some strains of virus, manipulating the expression or sequence of PB1-F2 altered replication and/or pathogenicity, leading to its identification as a virulence factor (6, 10, (14) (15) (16) (17) . However, in many cases, the presence or absence of an intact PB1-F2 ORF had little or no impact on virus replication in vitro or in vivo (7, 8, 17, 18) . Overall the contribution the protein makes to IAV pathogenesis is imperfectly understood.
Recently, we showed that AUG5 of segment 2 is also used to initiate translation of a protein product called PB1-N40, made at $5% of the abundance of PB1 (7) . AUG5 is in frame with AUG1, and so N40 is a truncated form of PB1, lacking the first 39 amino acids of the longer polypeptide ( Figure 1A ). The 'missing' region is important for the interaction of PB1 with PA (19) , and therefore N40 should not be able to form the stable complex with PA necessary for efficient nuclear import and polymerase function (20, 21) . Indeed, N40 predominantly localized to the cytoplasm, and was not transcriptionally active (7) . A function for PB1-N40 has not yet been identified, although PB1-N40 null viruses retaining an intact PB1-F2 ORF displayed delayed single cycle growth kinetics (7) .
It has been suggested that leaky ribosomal scanning is responsible for PB1-F2 and PB1 N40 expression (6, 7, 17) . In the scanning model of translation initiation, ribosomes bind to the 5 0 end of mRNA and move along until they recognize a start codon (22) . The sequence context of the AUG affects the probability that it will be recognized as a bona fide initiation codon; the Kozak consensus GCC(A/G)CCAUGG, is thought to be optimal, with a purine at À3 and G at +4 exerting the strongest effects (23, 24) . In support of the ribosomal scanning hypothesis, AUG1 is set in a medium strength Kozak consensus, lacking a purine at À3 ( Figure 1A and B), while mutation of AUG4 has been shown to lead to upregulation of N40 translation from AUG5 (7) . However, the presence of two short ORFs (sORFs) initiated by AUGs 2 and 3 upstream of the PB1-F2 and N40 AUGs ( Figure 1A and B) is suggestive of additional regulatory Kozak consensus sequence (green, strong consensus, with A/G at À3 and G at +4; yellow, medium consensus with either A/G at À3 or G at +4; red is a weak consensus U at -3 and +4). Adapted from (7) . (B) Nucleotide sequence and site of mutations used in this study. The 5 0 -end of segment 2 mRNA is shown in positive sense and as cDNA, since all mutations were introduced into a plasmid clone of the segment. (C) Summary of the predicted effect of the mutations used in this study on AUG strength and ORF structure (non synonymous changes in PB1 are indicated after red asterisks).
complexities. Furthermore, a previous study that investigated the effect of improving the Kozak consensus of AUG1 found little effect on PB1 levels in virus infected cells, and the authors suggested that start codon selection was not the primary control element for segment 2 translation (25) . Thus unresolved questions remain over the control of segment 2 gene expression.
Here, we report a systematic investigation of the role of the first four AUG codons in segment 2 in directing viral protein synthesis. Our findings indicate a modified leaky scanning model in which translation initiation at internal start codons is influenced by upstream AUGs, but where sORF2 is a critical regulatory element that depresses PB1-F2 synthesis but promotes N40 translation through a reinitiation mechanism. Unexpectedly, we also found that the translational regulatory sequences surrounding AUGs 1 and 2 overlapped with sequences required for packaging of the segment into virus particles, providing an interesting insight into the evolutionary constraints acting on this section of the viral genome.
Human embryonic kidney 293T cells and Madin-Darby canine kidney (MDCK) cells were cultured by standard methods. For transfections, 293T cells were transfected in Optimem (Invitrogen) according to manufacturer's instructions using Lipofectamine 2000 (Invitrogen).
Plasmids pcDNA-PB2, -PA and -NP, containing cDNA copies of the influenza A/PR/8/34 (PR8) genes as well as plasmid pPolI-Flu-ffLuc containing an influenza virus-based luciferase minireplicon vRNA under the control of the human RNA polymerase I (Pol I) promoter have been previously described (7, 26) . Dual promoter reverse genetics plasmids for PR8 segments 1 and 3-8 and a pPol-I segment 7 clone were donated by Professor Ron Fouchier (27) . A similar construct for segment 2 cloned from the NIBSC strain of PR8 is described in (7) . To assess PB1-F2 expression in vitro, a CAT fragment was ligated into SmaI/XbaI digested pcDNA-PB1 in frames 1, 2 or 3. To analyse viral gene expression from transfected plasmids, nucleotides 1-380 of EF467819 were subcloned into pEGFPN1 (Clontech) as a AgeI/KpnI fragment. Site directed mutagenesis was then employed to position the green fluorescent protein (GFP) ORF into frame with either the PB1 or PB1-F2 reading frames while concurrently removing the GFP AUG codon. Additional segment 2 mutations as detailed in the results section were made using site directed mutagenesis with the wild-type segment plasmids as templates. For brevity, the sequences of the mutational oligonucleotides are not given but are available on request. All plasmids were sequence verified.
Rabbit polyclonal anti-PB1 serum V19 raised against amino acids 50-370 of PR8 PB1 has been previously described (28) , as has rabbit polyclonal antiserum A2915 against PR8 NP (29) . Rabbit antisera to the C-terminus of the PB1-F2 protein and to the full length PB1-F2 protein were kindly provided by Jonathan Yewdell. Rat monoclonal anti-tubulin YL1/2 was purchased from Serotec, anti-GFP mouse monoclonal JL8 from Clontech and IR800 or IR680 dye conjugated anti-rabbit IgG and anti-mouse IgG sera were purchased from LiCor.
Recombinant PR8 viruses were produced by transfection of plasmids into 293T cells in suspension as previously described (7, 30) . Rescued viruses were passaged once in MDCK cells at an input MOI of 0.001, and where indicated, once in 11-day-old embryonated eggs using an inoculum of 1000 PFU. Virus titres were determined by plaque assay on MDCK cells (30) , and the presence of the desired mutations in segment 2 were confirmed by sequencing. Multiple independent rescues were performed (minimum twice, mostly 3-6 times) to ensure that a given phenotype did not result from adventitious mutations elsewhere in the virus genome. Virus infections of MDCK cells were performed at an MOI of 3-5 in serum free media for 30 min at 37 C, after which cells were overlaid with serum-containing media. Haemagglutination (HA) assays were performed as previously described (30) .
Coupled in vitro transcription-translation reactions were carried out in rabbit reticulocyte lysate using the Promega TNT system according to the manufacturer's instructions. SDS-PAGE followed by coomassie blue staining (to ensure equal loading of samples) and autoradiography was performed according to standard procedures. Blots were imaged using infrared fluorescence of appropriately tagged secondary antibodies and quantified using a LiCOR Odyssey scanner and software. Transcriptional activity of reconstituted RNPs was assessed using pPolI-Flu-ffLuc or pPol-I segment 7 as reporter plasmids. An amount of 50 ng of 3PNP and 20 ng of the reporter were transfected into adherent 293T cells and 48 h later, either luciferase levels from passively lysed cells were measured using a Promega GloMax luminometer or total RNA was extracted and segment 7 mRNA levels determined by reverse-transcriptase primer extension.
The vRNA content of virus particles was determined by silver staining as previously described (30, 31) . Quantitative RT-PCR (qRT-PCR) for segments 2, 3, 5 and 7 was also performed on RNA extracted from equal PFU using the QIASymphony system (Qiagen) as previously described (30, 31) . Reverse transcriptase primer extension analysis of RNA from infected or transfected cells was performed as described (32) , with the exception that SuperscriptIII (Invitrogen) was used and reverse transcription was performed at 50 C. Reaction conditions, primer and probe sequences are available upon request. Quantification was performed by densitometry of scanned X-ray films using Image J (Research Services Branch, NIH). Values were corrected with respect to a loading control (cellular 5S ribosomal RNA) and normalized to those of WT virus.
Segment 2 mRNA is known to encode three polypeptides: PB1 and PB1-N40 in frame 1, and PB1-F2 in frame 2 (6,7), translated from AUGs 1, 5 and 4 respectively ( Figure 1A ). The sub-optimal Kozak consensus flanking AUG1 prompted the hypothesis that the PB1-F2 ORF is accessed by leaky ribosomal scanning (6) and consistent with this, we previously showed that there was increased translation from AUG5 in the absence of AUG4 (7). However, there are two intervening start codons in frame 2 between AUG1 and the PB1-F2 ORF that initiate short sORFs with minimal protein coding capacity; eight and two codons respectively ( Figure 1A and B). Nevertheless, both these AUGs are highly conserved, being present in >99% of the available segment 2 sequences, similar to the conservation shown by AUGs 4 and 5 for PB1-F2 and N40 respectively (Table 1) . Notably, the termination codons for these sORFs are also highly conserved in that although three of the four overlapping PB1 codons in frame 1 are not themselves highly conserved at the RNA level, a stop codon is almost always (>99.9% for both sORF1 and 2) maintained in frame 2 ( Table 2 ). This degree of conservation is suggestive of functional importance, potentially for the regulation of translation of the downstream PB1-F2 and N40 cistrons. Accordingly, we set out to further delineate the mechanisms controlling translation from segment 2 mRNA by systematically introducing mutations into AUGs 1-4 and their flanking regions that would be predicted to alter their usage.
The sequences surrounding AUG1 are highly conserved, and conform to a moderately strong initiation consensus (Table 1) . Upstream residues were modified to each of the other possibilities at the crucial À3 position in the T22A, T22G and T22C mutants ( Figure 1B) , with the A/G changes but not the U!C alteration expected to result in increased ribosome recognition of the AUG (23,24) ( Figure 1C) . A further set of segment 2 AUG1 variants were produced on the background of the previous mutants in which the upstream residues at positions À1 and À2 were changed to match the canonical Kozak consensus (ACC, CCC, TCC mutants). The initiation context of AUG1 was also weakened by mutating the G at the +4 position to an A (G28A); this change also resulted in a non synonymous D2N change in the predicted PB1 translation product ( Figure 1B and C). Similar approaches were taken to probe the function of AUG2 and 3 in regulating translation. The context of these initiation codons was improved by mutating the +4 nt to G (T32G and C74G respectively, which caused V3G and A17G changes in the PB1 ORF, respectively). Additionally, AUGs 2 and 3 were individually destroyed (T30C and T72C respectively) without altering the PB1 amino acid sequence. Two further mutations were made to examine the importance of AUG3/sORF2. A78T removes the stop codon from sORF2, and thus the predicted frame 2 protein product from this construct is an N-terminal fusion to PB1-F2. To investigate if the length and position of sORF2 was important (as in the case of a reinitiation event), a stop codon was re-introduced prior to AUG4, in the A78T+G101T mutant. This mutant left only 15 nt between the stop codon and AUG4, and also produced a G26V alteration in the PB1 sequence. Finally, AUG4 was removed by a T120C alteration, as used by many previous studies to ablate PB1-F2 expression (6, 10, 13, 14, 16, 17, 33, 34) . The positions of all mutations are shown on the PR8 segment 2 sequence in Figure and their predicted effects on AUG context and ORF structure are summarized in diagrammatic form in Figure 1C .
Initially, PB1 and N40 protein synthesis by the mutants was investigated using coupled in vitro transcription and translation (IVT) in rabbit reticulocyte lysate. As expected (7) wild-type (WT) PR8 segment 2 expressed both PB1 and N40, with preferential usage of AUG1 ( Figure 2A , lane 1). All changes tested in the À3 to À1 positions relative to AUG1 lead to notable increases in the expression of PB1 (lanes 2-7). Quantification of replicate experiments showed these increases to be at least 2-fold relative to the WT gene ( Figure 2D ). Although this was the predicted outcome when the U at the À3 position was swapped to a purine, a similar effect when it was replaced with another pyrimidine was unexpected (23) . Consistent with a role for leaky scanning in accessing downstream AUGs, a concomitant reduction in N40 levels was observed in all cases ( Figure 2D ). Conversely, weakening the Kozak consensus of AUG1 through replacement of the +4 G nucleotide downregulated PB1 expression, and although the absolute amount of N40 remained similar ( Figure 2A , lane 8, quantification in Figure 2D ), its ratio relative to PB1 was increased 1.7-fold compared to wild-type segment 2. In contrast, mutations affecting AUGs 2 or 3, whether by altering their context (T32G and C74G) or by destroying them (T30C and T72C) had little effect on the ratio of PB1 to N40 ( Figure 2B ; quantification in Figure 2D ). Similarly, increasing the length of sORF2 (A78T+G101T) or fusing it with the PB1-F2 cistron (A78T) did not substantially alter relative use of AUGs 1 and 5. However, loss of the PB1-F2 AUG4 through the T120C mutation increased N40 synthesis from AUG5 by nearly 3-fold ( Figure 2B , lane 8, Figure 2D ). This recapitulated our previous observation for a construct in which AUG4 was mutated and its surrounding Kozak consensus disrupted [ÁAUG; (7)]. The small size ($10 kDa) of the PB1-F2 polypeptide made it difficult to visualize directly in IVT reactions. Accordingly, we utilized a set of constructs in which the CAT gene was fused downstream of AUG5 to increase the size of the polypeptide products. Examination of IVT reactions programmed with plasmids containing the CAT gene inserted into WT segment 2 in each of the three reading frames showed the expected set of polypeptides: frame one produced PB1 and PB1-N40 fusion proteins, frame 2 produced a PB1-F2 fusion while frame 3 lacked an obvious polypeptide product ( Figure 2C , lanes 1-3 respectively). The quantity of the frame 2 product synthesized showed partial correlation with the presence and number of upstream AUG codons. For example, improving the Kozak consensus of AUG1 had only a small effect on PB1-F2-CAT synthesis (T22A: Figure 2C , lane 4; quantification in Figure 2D ) and nor was F2 synthesis increased by the G28A mutation that weakened the context of AUG1 ( Figure 2C, lane 5) . Similarly, alteration of AUG2 by the T30C and T32G mutations had little effect (lanes 6 and 7). In contrast, the level of PB1-F2 was up-regulated 2-fold when AUG3 was removed via the T72C change (lane 8). The C74G mutation, predicted to improve the Kozak consensus of AUG3 caused a slight (<2-fold) reduction in F2 expression that was not statistically significant (lane 9). These results suggested that while AUG2 was of little translational significance, AUG3 was recognized well by ribosomes and directly regulated the level of PB1-F2 expression. Supporting this, when sORF2 was fused to the F2 ORF through removal of the intervening stop codon in the +2 frame by the A78T mutation, increased levels of a slightly longer (presumed) fusion protein were seen (lane 10). Thus overall, the data suggested that expression of PB1-F2 and PB1-N40 in vitro could be partially but not wholly explained by leaky ribosomal scanning. AUGs 1, 3 and 4 were clearly functional and exerted a significant influence on use of downstream start codons. The poor context AUG2 however, was apparently not used.
Next, we wished to examine the behaviour of the mutant segment 2 genes in the context of virus infection. However, four of the mutants had non-synonymous changes in PB1 (G28A; D2N, T32G; V3G, C74G; A17G and A78T+G101T; G26V).
We therefore first tested the ability of the mutant PB1 polypeptides to support viral gene expression in 'minireplicon' assays (26, 35) . To reconstitute active viral RNPs, plasmids encoding the three influenza A virus polymerase proteins and nucleoprotein were co-transfected with a further plasmid that expressed a synthetic vRNA molecule encoding luciferase in antisense from an RNA polymerase I promoter. The luciferase levels in transfected cells therefore represent a measure of the transcriptional activity of the polymerase complex. When luciferase values were normalized to those obtained with WT PB1, all of the segment 2 mutants with unaltered PB1 sequences gave values that fluctuated around the 100% mark (between 62 and 129% of normal), while a sample from cells lacking PB1 gave <1% output ( Figure 3A ). However, three of the four non-synonymous changes in PB1 were deleterious to transcriptional activity. The T32G (V3G) and A78T+G101T (G26V) polymerases were the most impaired, with luciferase readings of 13 and 20% of WT respectively. The G28A mutant (D2N) was marginally impaired, producing luciferase activity at 54% of WT. Only the C74G mutant (A17G, a relatively conservative change) supported transcriptional activity in the range of that observed with the mutants with synonymous changes in PB1 (72%). The reduced ability of the T32G and A78T+G101T mutants to support virus gene expression was also seen when authentic segment 7 was used as a RNP substrate and M1 and M2 accumulation analysed by western blot (data not shown) or when unspliced segment 7 mRNA accumulation was measured by reverse transcriptase-primer extension assay ( Figure 3B ).
WT and the 13 mutant viruses were therefore generated by transfection of cells with plasmids encoding the eight segments in cDNA form (27, 30) . Notwithstanding the reduced transcriptional activity associated with some of the non-synonymous changes in PB1, it was possible to rescue all of the mutants. Virus stocks were amplified in MDCK cells and titred as a first assessment of virus fitness (7, 30, 31) . For the mutants with non-synonymous changes in PB1, the endpoint titres showed a strong correlation with polymerase activity in the minireplicon system. The most deficient virus in this system (T32G) rescued only once out of four attempts in 293T and MDCK cells, where it showed a six log 10 growth defect relative to wild-type virus ( Figure 3C ). The A78T+G101T mutant was successfully rescued five times out of seven tested and reached endpoint titres of $1% of WT. All other mutants rescued on every attempt (between two and six times each). The G28A mutant, which had a 2-fold reduction in transcriptional activity, had a growth defect of $10-fold. Only the C74G virus grew similar levels as WT virus. However, not all viruses with unaltered PB1 coding sequences grew normally. Three of the mutant viruses with alterations around AUG1 (ACC, CCC and TCC), showed growth defects of between 8-and 20-fold relative to WT virus. In contrast, the T22A, T22G and T22C viruses grew normally, despite also having mutations to the upstream Kozak consensus of AUG1 that showed similar perturbations to segment 2 translation in the IVT system to the ACC, CCC and TCC changes. Similar relative results were also obtained when virus stocks were passaged in embryonated eggs, although here the T32G virus grew better, to $10 6 PFU/ml or 0.5% of the WT control (data not shown).
Low level expression of the replicative machinery is a general feature of many viruses and in some cases, mutations that result in overexpression of the viral polymerase have been shown to be deleterious to virus fitness (36, 37) . It was therefore possible that the poor replication of the ACC, CCC and TCC mutants resulted from overexpression of PB1, although as noted above, the T22A, T22C and T22G viruses did not show growth defects. More generally, we wished to compare segment 2 protein expression from recombinant and authentic viral settings. Accordingly, PB1, N40 and PB1-F2 accumulation in MDCK cells infected with the panel of viruses was examined at 8 h post-infection (p.i.) by western blotting. To provide numerical data, the levels of each protein from replicate experiments were quantified and normalized to levels in cells infected with WT PR8. The results obtained could be divided into those that were in concordance the previous in vitro analysis, and those that differed. In agreement, weakening the Kozak consensus of AUG1 (G28A) reduced PB1 accumulation relative to N40 and F2 expression ( Figure 4A , compare lanes 2 and 9; quantification in Figure 4B ). Similarly, mutation of AUG2 had little effect on either the quantity (T30C) or the relative ratios (T32G) of the segment 2 polypeptides ( Figure 4A, lanes 12 and 13) , with the lower levels of all three polypeptides seen with the latter virus being plausibly ascribed to the reduced polymerase activity of the mutant PB1 protein, as NP accumulation was also reduced. Also in agreement, loss of AUG4 increased N40 synthesis by $6-fold (T120C; lane 20), as we previously observed for a similar mutant virus (7).
The first major discrepancy between the infection and in vitro data concerned the role of AUG3 in controlling expression of the downstream ORFs. The virus lacking AUG3 (T72C) showed a statistically significant 3-fold increase in PB1-F2 accumulation relative to WT (compare lanes 11 and 14) , and this was consistent both with the in vitro data and the proposed role for leaky ribosomal scanning in accessing AUG4. Unlike in vitro however, there was a concomitant (and statistically significant) $2-fold reduction in N40 levels, despite normal PB1 accumulation. This result was not possible to reconcile with a model where leaky ribosomal scanning was the sole contributor to N40 expression, because removal of an upstream ORF should, at worst, leave N40 levels unchanged. Instead, we hypothesized that in vivo, ribosomes terminating at the end of sORF2 are able to reinitiate at AUG5, bypassing AUG4 because of the time taken to reacquire the necessary initiation factors (22) . In the absence of AUG3, more ribosomes initiate at AUG4 due to leaky ribosomal scanning, and so F2 levels increase. However, no ribosomes are available to reinitiate at AUG5, and so N40 levels decline. Supporting this hypothesis, when sORF2 was fused to PB1-F2 (A78T), N40 levels were reduced >2-fold ( Figure 4A, lane 16) . This virus would also be unable to express N40 by reinitiation from sORF2, due to the removal of the stop codon. However, reintroducing a stop codon using the G101T mutation reinstated N40 levels to 90% of wild-type (lane 17).
To investigate the reinitiation hypothesis further, the AUG3/sORF2 mutations were made on a ÁAUG4 (T120C) virus background ( Figure 4C ). These viruses were rescued and grew to comparable titre to wild-type PR8 (data not shown). Western blotting was performed on cells infected with these viruses, as well as (for comparison) from cells infected with the 'parental' T72C, C74G, A78T and T120C. As before, preventing potential reinitiation from AUG3/sORF2, using either the T72C or A78T mutations in the presence of an intact AUG4 reduced levels of N40 ( Figure 4A , compare lanes 19, 21 and 25), while improving the predicted strength of AUG3 had little effect (lane 23). Also as before, removal of AUG4 (T120C) led to a large increase in N40 levels (lane 20). A very similar outcome was obtained when the Kozak consensus of AUG3 was improved by the C74G mutation (C74G+T120C; lane 24). When AUG3 and 4 were removed concurrently (T72+T120C), synthesis of N40 was increased even further, to $14-fold greater levels than with the WT virus ( Figure 4B lane 22, quantification in Figure 4B ). However, when sORF2 was fused to the F2 ORF in the absence of AUG4, only a small enhancement (on average, 1.4-fold over WT) of N40 synthesis resulted (lane 26). If N40 expression was purely dependent on leaky scanning to bypass AUGs 3 and 4, this combination of mutations should have behaved identically to T120C alone. Instead, the absence of AUG4 only makes a significant difference to N40 expression when either sORF2 terminates before AUG5 or AUG3 is also absent, consistent with a reinitiation mechanism for synthesis of N40.
There were also discrepancies between virus infection and in vitro data for the AUG3/sORF2 mutant A78T virus regarding PB1-F2 expression, as the mutation decreased accumulation of the protein to $50% of normal instead of increasing it. Equally however, there was no evidence of expression of a larger form of PB1-F2 ( Figure 4A, lane 16) . Here, we surmise that the larger product was unstable in infected cells, and that the remaining F2 expression came from ribosomes initiating normally at AUG4 via leaky scanning. Consistent with this hypothesis, combining the A78T and T120C mutations (the latter removing AUG4) led to the loss of all detectable PB1-F2 accumulation ( Figure 4A, lane 26) .
If PB1-F2 is accessed by leaky scanning but N40 is accessed by reinitiation after translation of sORF2 then the insertion of further AUG codons in the region between the end of sORF2 and the beginning of the PB1-F2 ORF at AUG4 would be predicted to decrease F2 expression (through 'soaking up' initiation competent scanning ribosomes) but to have little effect on N40 expression because they would be effectively invisible to scanning small subunits that had terminated after reading sORF2 but not yet had time to acquire new initiation factors. To test this hypothesis, we introduced mutations that created new strong context AUG codons in each of the three reading frames in this region ( Figure 5A ). To permit the analysis of mutations that were lethal to virus growth and to try and minimize the effects of differing protein stability on polypeptide accumulation, we created sets of chimaeric plasmids containing the 5 0 -end of segment 2 encompassing the PB1-F2 coding sequence followed by a GFP ORF such that either PB1 (frame 1) or PB1-F2 (frame 2) were fused in frame ( Figure 5B ). To validate this system, we first retested the effect of the key mutations affecting sORF2 and AUG4. Cells transfected with a plasmid encoding the WT segment 2 fused in frame 1 to GFP produced the expected ratio of PB1 and PB1-N40-derived fusion proteins, while the frame 2 fusion Figure 5D ). Similarly, fusion of sORF2 and the F2 ORF by the A78T mutation significantly reduced N40 production ( Figure 5C , compare lanes 10 and 11). In contrast to virus infection, this mutation also increased accumulation of the F2-fusion polypeptides, presumably because of the greater stability conferred by the GFP moiety. Both the decrease in N40 expression and the increase in F2 synthesis were reversed by reinstating the sORF2 stop codon by the further mutation G101T (lane 12). Thus this system successfully recapitulated the regulatory effects seen in the context of authentic virus infection.
Next, we tested the effect of introducing novel AUG codons between the termination codon of sORF2 and AUG4. Insertion of new AUG 'A' into the PB1 ORF by mutating glycine codon at position 26 to ATG resulted in the production of a prominent novel frame 1 product ('PB1-N26') as well as a significant reduction in the synthesis of the PB1-F2 fusion protein ( Figure 5C , compare lanes 2 and 5, quantification data in 5D). N40 synthesis was however unaffected. These effects were specific to the creation of a new AUG codon, since mutation of codon G26 to ATC left expression of PB1-F2 unaltered (lane 6). Similarly, when new AUG codon 'B' was introduced into frame 2 by mutation of PB1 codon T25 to TAT (with the 5 0 -A!T change avoiding the simultaneous introduction of a stop codon in frame 3; Figure 5A ), N40 expression was not significantly altered while F2 accumulation was substantially reduced, partially at the expense of a slightly longer N-terminally extended form (lane 7). Again, the effect was specific to the AUG codon rather than mutation of codon 25 per se, because its mutation to CGT (B ctr) left F2 expression unchanged. N40 expression was also insensitive to the introduction of an AUG codon into frame 3, whereas F2 accumulation was reduced >2-fold (codon 'C'; lane 13). Once again, the paired control mutation had no affect on PB1-F2 synthesis, although unexpectedly, this change increased N40 accumulation (lane 14). Overall therefore, PB1-F2 levels were sensitive to the presence of start codons in all three frames following sORF2, whereas PB1-N40 levels were not significantly affected. These data show a fundamental difference in how AUG codons 4 and 5 are accessed: ribosomes can be diverted away from AUG4 by the insertion of new upstream AUG codons in the 'UTR' following sORF2, but AUG5 is insensitive to this approach. The simplest explanation consistent with the data is that AUG4 is primarily accessed by leaky ribosomal scanning that bypasses AUGs 1-3, while AUG5 is reached by reinitiation of ribosomes that have recently terminated synthesis after translation of sORF2.
The other major source of divergence between the in vitro data and that observed from the virus infections was seen with the mutants where the Kozak consensus of AUG1 was up-regulated. While the levels of N40 and PB1-F2 were predictably reduced, in all cases, the cells infected with these mutants also underexpressed PB1 relative to the WT virus ( Figure 4A, lanes 2-8) , showing on average 20-80% reductions in PB1 accumulation ( Figure  4B ). This was most pronounced for the triple AUG1 mutants, ACC, CCC and TCC, which produced 18, 32 and 47% of the PB1 levels of WT virus respectively. This was in marked contrast to the in vitro translation data, where PB1 levels were increased $2-fold over WT ( Figure 2 ). However, it should be noted that when the ratios of the three segment 2 polypeptides were considered, their relative amounts changed as predicted for leaky ribosomal scanning: N40 to PB1 levels were reduced between 2-fold (T22G) and 1.5-fold (ACC) in the AUG1 up mutants while PB1-F2: PB1 ratios decreased on average by $3-fold. Furthermore, transfection experiments confirmed that the AUG1 up mutations produced elevated amounts of PB1 in a cellular environment when introduced via plasmid (data not shown). We therefore considered the alternative hypothesis that in the background of authentic viruses, the AUG1 mutations also perturbed segment specific packaging. It is well established that the terminal unique coding and non-coding regions of all segments (including the regions of segment 2 under investigation here) contain specific packaging signals (3, (38) (39) (40) (41) (42) . In this hypothesis, the growth defect of the AUG1 mutants and their failure to express normal, let alone elevated quantities of PB1 could be explained by reduced delivery of segment 2 to the infected cells because of underincorporation of the segment into virions.
First, we measured virus particle formation by the panel of mutant viruses by HA assay. This showed only small fluctuations in particle assembly and release, with even the most replication deficient virus, T32G, showing on average, only a 4-fold drop in HA titre ( Figure 6A ). The copy numbers obtained for the mutant viruses were normalized to that of the WT virus to derive a relative segment copy number:PFU ratio. Data plotted are the mean ± SEM from at least two independent extractions, and for each extraction, the qRT-PCR reaction was performed in triplicate, with the exception of T32G and A78T+G101T, where RNA was extracted from a single rescue (the mean of triplicate determinations is plotted).
These data were then used to derive the proportion of infectious virus particles by calculating the ratio of HAU to PFU. By this measure, most mutants possessed values similar to that of the WT virus; the ACC, G28A and T32G viruses however had notably higher particle to infectivity ratios, indicating a large number of defective virions ( Figure 6B ).
To examine genome packaging in the segment 2 mutant viruses directly, vRNA was extracted from equal plaque titres of virus. The segments were resolved and detected by Urea-PAGE and silver staining, and in all cases the expected pattern of seven vRNA segments were seen ( Figure 6C ; under these conditions, segments 1 and 2 comigrate). Obviously greater quantities of RNA were recovered from the ACC and TCC viruses (compare lanes 2, 6 and 8), a finding suggestive of an increased genome copy: PFU ratio and thus consistent with a raised virus particle: PFU ratio (30, 31) . However, the inability of this gel system to reliably separate the three largest genome segments hampered direct analysis of segment 2. In addition, the poor growth of the T32G virus made it difficult to extract sufficient vRNA to detect by this procedure (data not shown). We therefore used quantitative RT-PCR (qRT-PCR) to examine the copy number of segments in the mutant viruses. RNA was again extracted from equal PFU of virus and one step RT-qPCR was performed for segments 2, 3, 5 and 7. The amounts of each segment from the mutant viruses were normalized to that of the WT virus to derive a segment copy number:PFU ratio. The T30C AUG2 mutant and all AUG 3 and 4 mutants had similar levels of each of the segments tested to the WT virus, and also had equivalent amounts of each segment within each virus ( Figure 6D ). In contrast, most of the AUG1 up-mutants underincorporated segment 2. In addition, the ACC, CCC, TCC up-mutants as well as the AUG1 G28A and AUG2 T32G down-mutants showed several fold increases in the relative amounts of the other three segments. Since vRNA was extracted from equal numbers of infectious virus particles, these results are consistent with a specific packaging defect for segment 2 resulting in a higher number of defective virions and thus a higher segment copy number:PFU ratio of the other segments (30, 31, 42) . This is consistent with the hypothesis that the failure of the AUG1 mutants with an improved Kozak consensus to express elevated quantities of PB1 in infected cells results from lower delivery of the segment by infecting virions.
To further test this hypothesis, we analysed segment 2 RNA accumulation in cells infected with the ACC (as a representative of an AUG1 up-mutant), G28A and T32G viruses in comparison with WT and two mutant viruses (T30C and T72C) with no obvious packaging defects. All three RNA species (m-, c-and vRNA) were readily detectable in samples from cells infected with the WT, T30C and T72C viruses ( Figure 7A, lanes 2, 5 and 7) . However, the three viruses with apparent defects in segment 2 vRNA packaging produced much reduced quantities of vRNA and (with the exception of G28A), m-and cRNA also (lanes 3, 4 and 6). This defect was particularly apparent for segment 2, as more consistent levels of segment 7 vRNA were seen for all the viruses ( Figure 7A ). When replicate experiments were quantified, the three viruses with potential packaging defects produced <10% of the normal amount of segment 2 vRNA ( Figure 7B ).
Although the above data were consistent with reduced delivery of the vRNA by the infecting viruses, we also considered the possibility that the AUG1/2 Kozak mutations perturbed the function of the viral RNA promoter (either the 3 0 -end of vRNA or the 5 0 -end of cRNA). This could lead to a reduction in segment 2 vRNA levels with a potential secondary effect of reducing the quantity available to be packaged into virions. Although the mutations lie well outside of the conserved promoter region, there are precedents for sequence alterations in the non-unique regions of a segment affecting RNA synthesis (30, 43, 44) . To examine this possibility in isolation, the amount of segment 2 produced from RNPs reconstituted by transfection was measured. Wild-type PB2, PB1, PA and NP were transfected into cells with the reverse genetics plasmids encoding the mutant segment 2 vRNAs. The segment 2 plasmids would be transcribed by RNA Polymerase I to produce a negative sense segment 2 transcript that would be encapsidated, transcribed and replicated by the WT RNP proteins. In addition, mutant PB1 protein would be also be expressed from the vRNAs with non synonymous changes to the PB1 gene (G28A, T32G), but the addition of wild-type PB1 would be expected to at least partially compensate for this. Thus this system allowed us to examine viral RNA production from the mutant segments in isolation from potentially confounding issues of segment delivery and PB1 protein function. Forty-eight hours post-transfection, RNA was harvested and primer extension analysis for segment 2 v, m and cRNA was performed. Omitting PB2 from the transfections determined the baseline levels of segment 2 vRNA that were expressed from the pPolI promoter ( Figure 7C , lane 1). In the presence of the full 3PNP complex, the mutant segment 2 constructs were transcribed and replicated to broadly similar extents ( Figure 7C ). When replicate experiments were quantified, the AUG1 mutants and the T32G AUG2 mutant accumulated vRNA to >75% of the WT level, in clear contrast to the >10-fold reductions they exhibited in the context of virus infection ( Figure 7B) . Similarly, all mutants expressed mand cRNA to reasonable levels, with, on average, no change of >2-fold compared to the WT ( Figure 7D ). These data argue against a defect in the promoter sequence of the viral RNA being solely responsible for the reduced levels of viral RNA seen in the context of infection and support instead the hypothesis that mutations around AUG1 not only affect translation initiation of PB1 and downstream cistrons, but also affect genome packaging.
The single known species of segment 2 mRNA produces three proteins: PB1, PB1-F2 and PB1-N40. PB1 is an essential protein, encoding the potential antiviral drug target of an RNA polymerase, while PB1-F2 modulates pathogenicity in some host-virus combinations and the function of N40 is unknown. Despite representing the only known functionally tri-cistronic influenza virus mRNA, the mechanisms that control protein expression from the segment have not been fully elucidated. Here, we confirm the hypothesis that leaky ribosomal scanning has a role in mediating expression of PB1-F2 and PB1-N40. However, this mechanism does not fully explain segment 2 translation and we also identify ribosomal reinitiation after sORF2 as important for PB1-N40 expression.
Our data further refine the model for segment 2 protein expression. PB1 translation occurs via the canonical pathway of eukaryotic translation initiation (22) in which a preinitiation complex consisting of an eIF2aternary complex (eIF2-TC) attached to a 40S ribosomal subunit scans 3 0 -wards from the 5 0 -cap structure, recognizes AUG1 and commences translation after loss of the initiation factors and recruitment of the 60S subunit ( Figure 8A ). The simplest explanation for PB1-F2 expression is that it occurs via leaky ribosomal scanning, in which the preinitiation complex misses the moderate context AUGs 1 and 3 and the poor context AUG2 before initiating translation at the strong context AUG4 ( Figure 8B ). AUG3/sORF2 evidently plays an important role in down-regulating use of AUG4, as its loss through the T72 mutation substantially increased PB1-F2 accumulation, in vitro and in the context of virus infection. In contrast, the presence of AUG3/sORF2 up-regulated N40 expression in infected cells, a finding inconsistent with leaky scanning. Instead, we think this is best explained via leaky ribosomal scanning to bypass AUGs 1 and 2 followed by initiation at AUG3, almost immediate termination at the end of the two codon sORF2 and continued scanning of the 40S ribosomal subunit. The 40S subunit then scans past the strong context AUG4 but has time to reacquire an eIF2-TC before reaching the strong context AUG5 whereupon translation initiation occurs ( Figure 8C ). The distances between the sORF2 stop codon and AUGs 4 and 5 (40 and 63 nt respectively) are consistent with previously characterized instances of reinitiation (22, (45) (46) (47) . In some circumstances, changes in levels of eIF2-TC during conditions of cell stress (as for example when virus infection activates PKR) are known to regulate expression of downstream ORFs accessed via reinitiation strategies (22) . It is therefore interesting to speculate that segment 2 translation might be further regulated during the course of infection. The distance between the extended sORF2 in the A78T mutant and the N40 AUG (39 nt) is similar to that between the normal sORF2 and the PB1-F2 AUG, so we would not rule out the possibility that reinitiation after sORF2 translation also contributes to F2 expression. However, shortening the intercistronic distance between sORF2 and the F2 ORF to 18 nt (a distance predicted to be too short to allow efficient reacquisition of an eIF2-TC) in the A78T+G101T mutant did not significantly reduce F2 accumulation, so we do not think it plays a major role.
Another major conclusion from this study is that the 5 0 -end of segment 2 mRNA itself has a number of overlapping functions. These include coding sequences critical for PB1 function, regulation of expression of downstream ORFs and also regions important for vRNA packaging. This has practical implications by reinforcing that this region represents an attractive target for therapeutic intervention, either by anti-viral drugs [e.g. those targeting the PB1-PA protein interface; (48) ] or through T-cell epitope immunization (49) , because the chance of finding escape mutations that maintain all functions of the protein/RNA sequence is likely to be lower than in a less functionally intricate area of the virus genome. Understanding the overlapping functional requirements also provides an interesting perspective on the evolutionary selection pressures that could be operating in this region of the influenza genome.
Packaging signals have been previously mapped to the general area of the 3 0 -end of segment 2 vRNA (38-41) (summarized in Figure 9 ) but this is the first study to show that the same nucleotides also contribute to translational regulatory sequences. This finding echoes our previous finding that sequences important for directing packaging of segment 7 overlap other cis-acting signals for mRNA splicing (30) and further demonstrates the functional complexities contained within sections of the influenza A virus genome. Examining the sequence of the 5 0 -end of segment 2 (in mRNA sense) using the criterion of reporting sequences that are conserved in >95% of the available isolates makes it evident that the primary selection pressure acting on the region is PB1 function (50) . By this admittedly simple measure, only two amino acid residues (positions 12 and 14) are not conserved, in obvious contrast to PB1-F2 or sORF1 and sORF2. At the nucleotide level, as previously noted (50) , it is clear that the majority of sequence polymorphisms are found at the third base position of the PB1 gene ( Figure 9 ). Consistent with this, experimental evidence shows that the majority of the 14 N-terminal amino acids as well as (where tested) Figure 8 . Model for translation of segment 2 polypeptides. PB1 translation occurs by canonical initiation at the first AUG. The majority of PB1-F2 translation occurs via leaky scanning to bypass AUGs 1-3. In contrast, reinitiation after termination at the end of sORF2 is a major contributor to PB1-N40 translation. See text for further details. residues further downstream, are important for one or more functions of PA binding, polymerase activity and virus replication (21, (51) (52) (53) (and data presented in this study). However, although over half of the first 41 codons of PB1 show some variability at the wobble position, it is notable that the primary translational signals within this region are much more highly conserved, with all five start codons showing >99% conservation and only one of the two stop codons (that of sORF1) showing apparent variation (Figure 9 , Tables 1 and 2 ). Even here, as discussed, the variation is such that >99.9% of viruses maintain either a UGA or UAA stop codon (Table 2) .
That AUG1 is essential for PB1 expression is obvious; the moderate Kozak consensus surrounding it has presumably evolved to allow expression of one or more of the downstream ORFs via leaky scanning. This sequence element appears be additionally selected for via the contribution these nucleotides make to the segment 2 specific packaging signal. However, in light of the theory that RNA viruses gain additional genes through selection of unused or poorly expressed ORFs (54) and that a selective advantage for PB1-F2 or N40 is not always obvious (7) (8) (9) 17, 18, 55) , it is not clear which functional element came first. AUG2 or sORF1 seems to be of no significance as a translational element since modulation of the AUG made no difference to protein expression in vitro or in virus infected cells. Similarly, removal of the stop codon had no effect on segment 2 protein expression, genome packaging or virus replication (data not shown). The AUG may be retained because PB1 function requires an aspartate residue at position 2 [this study; (21, 51, 52, 56) ] and because the wobble position of codon 2 has become fixed through its secondary role in the segment packaging signal. Retention of the stop codon is more difficult to explain, although positions 10 and 11 require leucine and lysine respectively (21, 51, 52, 56) and of the twelve possible permutations of this codon pair, only two do not result in a termination codon.
AUG5 may be maintained either because methionine 40 is essential for PB1 function and/or because expression of N40 supplies a selective advantage in vivo, for reasons as yet unknown. However, an isoleucine change at position 40 does not obviously inhibit PB1 transcriptase activity or inhibit virus growth in vitro or in eggs (6,7), perhaps favouring the latter hypothesis. AUG4 is presumably conserved at least in part to allow expression of PB1-F2, although it is less obvious what maintains it in the large number of viruses (9,57) that do not possess an intact F2 ORF. It does not seem to contribute to a packaging signal, so one possibility is that it is retained as a 'ribosome sink' to prevent overexpression of N40. AUG3 and the stop codon for sORF2 also seem likely to be conserved for a regulatory role: depressing PB1-F2 synthesis and/or permitting N40 expression. Since neither PB1-F2 or PB1-N40 is required for virus replication in cell culture, elucidating which (if either) of these roles is more important for maintaining virus fitness (as well as the wider question of their function in virus pathogenicity) will require either animal experiments and/or more sophisticated model systems for virus replication in vitro. Understanding the mechanisms that underlie F2 and N40 expression informs the design of virus mutants that could answer these questions.  Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood. Acute and chronic infection, especially that induced by Gramnegative bacteria is associated with increased risk of thrombosis and atherosclerotic disease [1, 2, 3, 4, 5] . Little is known about the underlying cellular mechanisms responsible for these risks. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is an antigen which initiates inflammation and innate immune responses by interacting with Toll-like receptor 4 (TLR4). TLR4 is expressed on the surface of cells, including leukocytes and platelets [6, 7, 8] . Under physiological conditions, platelets and leukocytes circulate in quiescent state and do not interact with each other. However, once activated under pathophysiological conditions such as those associated with infection, platelets change shape, secrete prothrombogenic inflammatory and cellular adhesion molecules from alpha-and densegranules which cause the platelets to adhere to each other or to leukocytes and/or vascular endothelium [9, 10, 11, 12] . The physiological consequences of stimuli associated with infection, like LPS stimulation, are acute but can be sustained. For example, half-life of platelets was shortened and the activation state of newly formed platelets from bone marrow megakaryocytes increased within seven days following a single acute intravenous injection of LPS in mice [13, 14] . However, cellular events, specifically those occurring among blood elements, contributing to the shortened half-life and increased activation state of platelets remains to be clarified.
One mechanism offered to explain how infection contributes to the onset and progression of cardiovascular diseases is through increased production of proinflammatory cytokines [1, 3] . However, this explanation does not address how the production of inflammatory cytokines might proceed nor does it identity the cell types which are targets for the LPS stimulation. Platelets may represent one of the first blood borne elements to react to LPS stimulation as changes in platelet reactivity via TLR4 seems to occur prior to sustained changes in circulating levels of cytokines [14] . Alternatively, comparable activation of leukocyte as well as platelet result in formation of cell-derived microvesicles (MV) which may contribute to increased thrombogenic propensity of the blood, pro-inflammatory immune processes and thus cardiovascular risk [15, 16, 17, 18, 19, 20, 21, 22] . Clarifying the interactions of these blood elements (platelets and leukocytes) in the setting of TLR4 activation might provide insight into how infection initiates or facilitates progression of cardiovascular disease.
MV are cell membrane-derived vesicles ranging in size from 0.1 to 1 micron in diameter which are shed in response to cellular activation, cell-cell interaction and apoptosis [23, 24, 25, 26, 27] . These cell-derived vesicles are an interface of activation between cellular components of the blood with the vascular wall and between soluble components of the blood associated with immunity including response to infection [24, 28, 29] . For example, phosphatidylserine (PS) on the surface of MV provides catalytic sites for prothrombinase complex to generate thrombin needed for the conversion of fibrinogen to fibrin in formation of clots [25, 30, 31] . Furthermore, exposure of diluted blood to LPS in vitro increased production of platelet-derived as well as tissue factor positive MV within 3 to 6 hours [32, 33, 34] . While those experiments provide evidence that LPS modulates platelet activation, they do not provide any insight about the interactions of platelet with other blood elements within the earliest stages of activation especially at time points prior to the period when measurable changes in circulating cytokines are observed in vivo [14] . Therefore, the present study was designed to test the hypothesis that acute exposure to a sentinel dose of LPS would induce MV production and exchange of specific proteins/ receptors between platelets and leukocytes via TLR4 activation. MV transport of biologically active cell contents including cell surface receptors among cells were identified using antibodies specific for cell antigens (i.e. CD41 or CD45, which is platelet-or leukocyte-specific antigens respectively) [24] ; MV derived from platelets and/or leukocytes were distinguished by cell specific fluorescein conjugated antigen staining using calibrated flow cytometry.
Four to eight month old, male and female C57BL10SnJ mice (wild type, WT) and C57BL10ScN mice homozygous for deletion of TLR4 (dTLR4) were obtained from the Jackson Laboratory, Bar Harbor, Maine. These mice do not express the IL-12Rb2 mutation that was originally described for this strain [35] . Mice of each sex and age were used randomly in each of the various protocols. Mice were housed in a temperature-controlled environment (2262uC; 5565% relative humidity), 12/12 light/dark cycle, and fed standard chow. Experiments were approved by the Institutional Animal Care and Use Committee, Mayo Clinic, Rochester, MN.
Ultra-pure E.coli lipopolysaccharide (LPS, 0111:B4 strain-TLR4 ligand, product number tlrl-pelps), pepinh-MyD (product number tlrl-pimyd) or pepinh-TRIF (product number tlrl-pitrif) inhibitory peptide (InvivoGen, San Diego, CA) were prepared as suggested by the supplier. Mouse thrombin and bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, MO, USA. Cellular origin of antigens was determined using platelet (rat anti-mouse CD41 antibody) and total leukocyte (rat anti-mouse CD45 antibody) membrane specific fluorescein conjugated {Phycoerythrin (PE)-or fluorescein isothiocyanate (FITC)-} antibodies by flow cytometry. PE-or FITC-conjugated Annexin-V and matched isotype control antibodies were purchased from BD PharMingen International, San Diego, CA. All other reagents and solvents used in this study were of analytical/ reagent grade.
Blood was collected from the retro-orbital sinus plexus (250-300 mL/mouse) of wild type and dTLR4 mice through siliconized capillary tubes coated with hirudin (thrombin inhibitor) and soybean trypsin inhibitor (STI, Factor Xa inhibitor) into 1.5 mL polypropylene tubes containing 20 mL of 100 mM hirudin and 1 mM STI [13] . For in vitro experiments, anticoagulated blood was aliquoted into pairs of tubes within 30 min after collection so that measurements from a vehicle-treated, control tube and LPStreated tube could be analyzed from each mouse at each time point. Vehicle (saline) or LPS (500 ng/mL) was added to one of each paired aliquots of blood. At designated time points (5, 30, and 60 minutes) after addition of vehicle or LPS, 10 mL whole blood from each aliquot was diluted into 990 mL (1:100) HEPES/ HANKS' buffer (130 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2 , 0.8 mM MgSO 4 , 0.44 mM Na 2 HPO 4 , 20 mM HEPES, pH 7.4) with 1 mg/ml albumin and 1 mM STI for staining of platelets positive for leukocyte antigen and leukocyte positive for platelet antigen. The remaining blood was used to prepare platelet free plasma (PFP) for MV analysis.
For in vivo experiments, male mice (8 month of age) received a single injection of LPS (500 ng/mouse or 20 ng/gm body weight) into the tail vein. Seven days after the injection, blood was collected as described above. For analysis of platelet antigen positive leukocyte sub-populations, flow cytometry was triggered with total leukocyte marker CD45-conjugated with allophycocyanin (CD45-APC). All CD45 positive events containing granulocytes, monocytes, lymphocytes and apoptotic bodies were separated by light forward and side scatter blot analysis and gated each sub-type of leukocytes based on their size. Each gate was verified using cell specific antibodies (CD11b for granulocytes, CD14 for monocytes and CD3 for T-lymphocytes and CD19 for B-lymphocytes. Differential leukocyte populations were then subjected to two-color analysis to differentiate leukocytes positive for phosphatidylserine and platelet antigen (annexin-V-FITC vs CD41-PE) from leukocytes negative for phosphatidylserine and platelet antigen. Percentages of positive events were calculated above the set threshold from isotype control antibody.
A blood sample from each mouse was divided into two 100 ml aliquots; one was diluted with 100 ml saline (control), the other with 100 ml saline containing LPS (500 ng/ml final concentration). Samples were incubated at 25uC. At time-points 0 (prior to LPS or vehicle), 30 and 60 min, a 20 ml aliquot was removed from each sample, diluted in 20 ml 2% paraformaldehyde and incubated for an additional 30 min. Each sample was then diluted in 250 ml PBS (0.1 mm pore membrane-filtered) and centrifuged at 456g for 12 min. The supernate, which contained platelets, was removed, placed into a new vial and used for imaging analysis. An aliquot of each platelet suspension was diluted 1:4 in PBS, and a 25 ml drop was placed on a glass slide, cover-slipped and sealed with glue. Platelets were then imaged in dark-field mode using a light microscope (Olympus BX41 with a 1006 oil-immersion lens) coupled to a CytoViva illumination system (CytoViva, Inc., Auburn, AL). Scanning from a corner of each cover-slip, platelets in each field were counted until 100 were totaled. Platelets were categorized according to shape morphology (discoid, irregular, flattened, pseudopodia) or whether exhibiting membrane granules or in aggregates (each aggregate was counted as 1). The number of platelets in each category was expressed as percentage of total 100 platelets counted.
Diluted (1:100) whole blood (100 mL) was incubated with PEconjugated platelet-and leukocyte-specific antibodies (CD41 and CD45, respectively), or separately with annexin V-FITC (binding to phosphatidylserine) for 30 min, after which 1% paraformaldehyde (400 mL) was added. Matched fluorescein conjugated isotype control antibodies were used simultaneously staining to set the threshold and exclude nonspecific binding. Interactions between cell (platelets or leukocytes) and cell-derived MV and PS expression on both platelets and leukocytes were analyzed by flow cytometry (FACSCalibur TM and FACSCanto TM , BD Biosciences, San Jose, CA). Platelets were identified by forward and side scatter and with fluorescein conjugated CD41 antibody until 20,000 events gated for each sample, respectively ( Figure 1A ).
All buffers and antibodies were filtered twice through 0.2 mm membrane (Millipore) filters for MV analysis. Platelet free plasma (PFP) was prepared by double centrifugation at 30006g for 15 min. PFP was diluted (15 mL PFP+85 mL HEPES/HANKS' buffer) and incubated with 4 mL FITC-conjugated annexin-V and PE-conjugated CD41 or CD45 antibodies for 30 min in dark, at which time 350 mL HANKS' balanced salts buffer (pH 7.4) with 2.5 mM CaCl 2 was added. Matched isotype control antibodies were used to set threshold and exclude nonspecific binding. MV were quantified based on counts of calibration beads (TruCOUNT TM beads) added immediately to the samples prior to analysis by flow cytometry (FACSCanto TM , BD Biosciences, San Jose, CA) as previously described [25, 26, 31] . In this study, MV were defined as events of ,1 mm in diameter using size calibration beads and positive for annexin-V and platelet-and leukocyte-specific markers.
Analysis of TLR4 intracellular signaling pathway LPS stimulated TLR4 signaling was analyzed by introducing MyD88 or TRIF inhibitory peptides as described in other studies [36, 37] . Anticoagulated blood from WT mice was aliquoted into six tubes and treated with pepinh-control (vehicle), pepinh-MyD (100 mM, MyD88 inhibitory peptide which binds to MyD88 to block TLR4 stimulated MyD88 signaling) and/or pepinh-TRIF (100 mM which binds to TRIF to block TLR4 stimulated TRIF signaling) alone or in combination for 30 min after which either saline or LPS (500 ng/mL) was added for one hour. At this time, an aliquot was diluted for analysis by flow cytometry to measure cellular origin of MV and platelet positive leukocyte antigen and leukocyte positive platelet antigen.
Data are presented as percentage or fold increase from the paired vehicle and LPS treated blood samples at each time point. All data are presented as mean 6 SEM; n = number of animals used in each experiment. Statistical significance was evaluated by paired or unpaired two-tailed Student's t-test. Statistically significance was accepted at P,0.05. 
Prior to addition of LPS, platelets from WT mice were discoid, formed few aggregates and did not have extended psuedopodia (Figure 2 ). Surface expression of PS on platelets or leukocytes was similar in WT and dTLR4 mice. Within each group of mice, expression of PS was significantly lower on leukocytes than on platelets (Table 1) .
During the first hour of incubation of the blood with LPS, platelets from WT mice underwent a shape change, extended pseudopodia and formed aggregates (Figure 2 ). Within this first hour, neither surface expression of PS on either platelets or leukocytes or numbers of platelet-or leukocyte-derived MV changed significantly in WT mice or dTLR4 mice (Table 1) . However, the number of platelets positive for leukocyte-specific antigen (defined by the platelet-gate on the flow cytometer, Figure 1 ) increased significantly in a time-dependent manner in blood of WT mice, but not dTLR4 mice (Figures 1) . Using the total leukocyte specific antibody (CD45-APC) to define total leukocytes, numbers of leukocytes positive for platelet-specific antigen did not increase significantly in either WT or dTLR4 mice ( Figure 1) .
In blood from WT mice, the percentage of platelets positive for leukocyte antigen (CD45) was reduced significantly and to the same extent by MyD88 and TRIF (Figure 3 ). The combination of MyD88 with TRIF did not reduce the percentage of aggregates to a greater extent than either alone (from 3.8360.48 to 1.3860.08).
Seven days following a single intravenous injection of a sentinel dose of LPS (20 ng/gm body weight which is 500 ng/mouse), none of the platelets were positive for leukocyte antigen. On the contrary, compared to leukocytes obtained from animals treated with vehicle or a week before LPS injection in the same mouse, granulocytes and monocytes were significantly positive for platelet antigen seven days after the single LPS injection (Figure 4) . Apoptotic bodies also stained positive for platelet antigen (Figure 4 ).
Understanding how infection alters cell-cell interactions and release of MV from specific blood borne elements may help to identify new targets for reducing cardiovascular/thrombotic risk with infection. This study demonstrates the acute, immediate interaction of platelets and leukocytes after incubation of whole blood with a sentinel dose of LPS through TLR4 signaling. Exchange of antigens and associations of specific cell-derived MV among cells is a mechanism for the transfers signaling molecules to specific cells. For example, MV derived from neutrophils induced platelet activation by binding to platelets GP1ba (Glycoprotein 1b a) via activated a M b 2 on MV [38] , while platelet-derived MV mediate leukocyte-leukocyte aggregation, activate leukocyte phagocytic properties and amplify leukocyte-mediated tissue injury in thrombotic and inflammatory disorders [39] . Results from the present study demonstrate that the number of platelets positive for leukocyte antigen increased within 60 min of exposure to LPS. This increase was not accompanied by increased expression of PS on cell or MV surface. Because expression of the leukocyte antigen on platelets was defined using the platelet size gate and platelet specific marker CD41 on the flow cytometry, larger leukocytes would be excluded. Therefore, these leukocyte antigens may represent a membrane exchange during platelet-leukocyte adhesion or adhesion of leukocyte-derived MV to the platelets. The half-life of whole platelet-leukocyte aggregates may be shorter and therefore, we did not determine whole platelet-leukocyte aggregates in this study.
With agonist binding, the TLR4 dimerizes and undergoes a conformational change required for the recruitment of signaling molecules [40] , such as the adaptor molecules myeloid differentiation protein 88 (MyD88) and Toll-interleukin-1(IL-1) receptor Figure 2 . Representative dark-field micrographs of platelets from a WT mouse taken immediately before or after 30 and 60 min. incubation of the blood with 500 ng/ml LPS. Prior to LPS application, platelet aggregates (of more than 5 platelets) were absent, and most platelets were discoid shaped (Left panels). At 30 and 60 minutes incubation with saline, platelet morphology did not change (Top panels); whereas, large aggregates of irregularly-shaped platelets were observed in those incubated with LPS (bottom panels, inset shows platelets with pseudopodia). Each bar represents 5 mm. Similar results were observed with blood from two additional animals (n = 3). doi:10.1371/journal.pone.0025504.g002 domain containing adaptor inducing interferon-b (TRIF), which mediate MyD88 dependent or independent pathway respectively [41, 42] . LPS activates both MyD88 and TRIF pathways, which are important in the TLR4 mediated intracellular signaling [42] . The acute effects of LPS on platelet and leukocyte activation were most likely mediated through activation of TLR4 as platelet positive leukocyte antigen was not observed in blood from dTLR4 mice. Furthermore, leukocyte antigen expression on platelet was reduced by inhibition of MyD88-and TRIF-dependent pathways alone or in combination. These signaling pathways may be potential molecular targets to inhibit infection/inflammation induced interactions among formed elements in the blood.
A single TLR4 sentinel dose injection of LPS, such as used in this study, shortened platelet half-life and increased platelet production without increases in cytokine production within 3 hours of stimulation [14] . Although in vivo interaction of plateletand leukocyte-aggregates with the vascular wall could stimulate or exacerbate proinflammatory immune responses [43, 44] , the half-life of these cell aggregates is not known. A significant finding of the present in vivo study is that leukocytes sustain or retain platelet antigen seven days after an in vivo injection of LPS. This time point corresponds to turnover of the platelet pool and is consistent with observations that changes in platelet half-life and increases in platelet turnover are dependent on the concentration of agonist, i.e. LPS, activation [13, 14] . Quantification of dual-positive events is usually not considered in studies of cellular or MV quantification with LPS stimulation. Results from the present study, therefore, suggest that platelet-leukocyte antigen could be used as an additional biomarker of cellular activation for diagnostic or prognostic purposes in settings of sub-clinical or asymptomatic exposures to infective agents.
Monocytes showed the greatest expression of platelet antigen following LPS injection. Since monocytes are considered to be the primary leukocytic cell involved with development of atherosclerotic lesions [45, 46, 47] , the present results provide insight into a mechanism linking low to moderate levels of infection to progression of cardiovascular disease.
To our knowledge, results of the present study represent the first to examine time-dependent changes in production of MV/ exchange of cell-specific antigens in cells derived from whole blood incubated with LPS. In diluted blood, addition of LPS (500 ng or 1 mg/mL) increased the number of MV from platelets and those positive for tissue factor but only after four hours of incubation [34] . Because the blood was diluted, unlike the present study, cell-cell interactions were most likely attenuated and no evaluation was performed relative to interaction with leukocytes which could have indirectly affected platelet activation and production of cytokines.
Unexpectedly, PS expression on platelets (or leukocytes) did not increase significantly with acute exposure to LPS. PS is expressed in the inner leaflet of plasma membrane but rapidly inverts to outer surface following activating stimuli [48, 49, 50] . Exposure of PS to the outer membrane surface also occurs with release of MV [51] . Results of the present study are consistent with reports that production of MV is not accompanied by PS expression on cell of origin but might be restricted to that portion of the membrane undergoing MV blebbing [52] .
Within one hour of exposure of whole blood to a concentration of LPS that has threshold effect on cytokine production in vivo, platelets become positive for leukocyte antigen. Platelet-leukocyte interactions require TLR4 signaling as the dual antigen positivity of platelets was observed in blood derived from wild type but not dTLR4 mice. Furthermore, peptide inhibitors of TLR4 signaling molecules blocked the interaction. These events occur within 1 hour after the initial exposure to LPS. In addition, effects of LPS stimulation are sustained at least up to 7 days past the initial LPS exposure, as leukocytes express platelet antigen at this time point. Collectively, these results identify an acute and rapid signaling mechanism by which sentinel-grade acute infection through TLR4 alters blood hemostasis and sustained leukocyte activation which may contribute to progression of cardiovascular diseases.  NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain Activated murine cytotoxic T cells express the NKG2D natural cytotoxicity receptor. This receptor recognizes MHC class I-like molecules expressed on the surface of infected cells and serves to augment T cell-mediated cytotoxicity. The role of NKG2D-mediated augmentation in the clearance of central nervous system viral infections has not been explored. Using the Theiler's murine encephalomyelitis virus model we found that NKG2D-positive CD8+ cytotoxic T cells enter the brain, that NKG2D ligands are expressed in the brain during acute infection, and that interruption of NKG2D ligand recognition via treatment with a function blocking antibody attenuates the efficacy of viral clearance from the central nervous system. NKG2D is a natural cytotoxicity receptor expressed on natural killer cells (NKCs) and on activated CTLs in mice (Eagle and Trowsdale, 2007) . This receptor recognizes stress-and infection-regulated MHC class I-like molecules such as Rae1, Mult1, and H60 expressed on the surface of murine cells (Raulet, 2003) . While NKG2D is a direct mediator of cytotoxicity for NKCs, it serves as a co-stimulatory receptor for CTLs and augments cytotoxicity downstream from T cell receptor recognition of peptides presented on MHC class I (Markiewicz et al, 2005) . Because the mechanisms that direct CTL-mediated clearance of infected neural cells are unclear, we asked whether NKG2D-positive immune effectors are present in the CNS during acute TMEV infection, whether NKG2D ligands are expressed in the brain following infection, and whether interruption of NKG2D-mediated co-stimulation of CTLs alters viral clearance from the CNS.
Using flow cytometric analysis of a previously characterized preparation of brain-infiltrating leukocytes (BILs) (Howe et al, 2007) , we measured the number of NKG2D-positive immune cells present in the CNS at 3 and 7 days after TMEV infection (2×10 5 PFU i.c.; 4-6 week old C57Bl/6J mice; all experiments adhered to Mayo IACUC guidelines). We found that at 3 days postinfection (dpi) 6 ± 0.4% of CD45 hi BILs were CD8-positive, of which 20 ± 0.6% were also positive for the NKG2D costimulatory receptor. By 7 dpi the BILs were comprised of 24 ± 2% CTLs, with 76 ± 2% of these cells positive for NKG2D (n=4-6 mice per timepoint; mean ± 95% CI). Thus, between 3 and 7 dpi the phenotype of immune cells infiltrating the CNS shifted toward a population that was enriched in NKG2D + CD8 + CD45 hi CTLs ( Figure 1A -1F). The presence of NKG2D-positive CTLs within the infected hippocampus (Wada and Fujinami, 1993) was confirmed by simultaneously immunostaining cryosections from mice at 7 dpi with anti-CD8 (53-6.7, 1:100) and anti-NKG2D (CX5, 1:50) (Figure 2A -2E) (Howe et al, 2004) .
In order to determine the viral specificity of the NKG2D-positive CTLs at 7 dpi we costained BILs with anti-CD8, anti-NKG2D, and the TMEV VP2 121-130 -specific tetramer (Beckman Coulter Immunomics, San Diego, CA; 1:100) (Howe et al, 2007) . Flow cytometry revealed that 74 ± 2% of CD45 hi CD8 + cells were VP2-specific ( Figure 1G ), while none of these cells were stained by an irrelevant D b /E7 tetramer (data not shown) (Mendez-Fernandez et al, 2005) . Moreover, 81 ± 2% of the VP2-specific CTLs were also NKG2D-positive ( Figure 1H ), while only 12 ± 2% of the VP2 tetramer-negative CTLs were positive for NKG2D ( Figure 1I ) (n=4 mice; mean ± 95% CI). We conclude that a robust population of NKG2D-positive VP2-specific CTLs are present within the brain at 7 days after infection with TMEV.
Others have reported the upregulation of NKG2D ligands following infection in a variety of peripheral tissues (Eagle and Trowsdale, 2007) . However, the upregulation of these ligands by picornavirus infection has not been previously explored. We found that Rae1, Mult1, and H60 were all upregulated within the hippocampus during acute TMEV infection as determined by RTPCR analysis of RNA isolated from the excised hippocampus (see Table 1 for conditions). Of the three ligands we analyzed, Rae1 showed the most robust upregulation, with a peak induction of 10 ± 1 fold over the uninfected hippocampus at 7 dpi (F(4,9)=4.845, P=0.017 by one-way ANOVA; q(9,5)=5.841, P=0.012 with the SNK pairwise comparison between 0 and 7 dpi; mean ± 95% CI) ( Figure 3A ). H60 and Mult1 were also upregulated 2 ± 0.1 fold and 2 ± 0.2 fold over uninfected, respectively, at 7 dpi (n=3 mice; mean ± 95% CI) ( Figure 3A ).
In order to identify the cellular locus of NKG2D ligand expression in the hippocampus we attempted, unsuccessfully, to immunostain fresh frozen or paraffin-embedded sections of hippocampus after fixation with paraformaldehyde, methanol, or ethanol:acetone. We tested specific anti-Rae1 (clone 186107; 1:25; R&D Systems) and anti-H60 (clone 205326; 1:100; R&D Systems) antibodies as well as a pan-specific NKG2D ligand antibody (goat anti-Rae1; 1:100; Santa Cruz Biotechnology) and the pan-specific NKG2D-Fc chimeric molecule (R&D Systems 139-NK; 1:10). Likewise, we attempted to confirm the expression of the NKG2D ligands at the protein level within the hippocampus by western blot analysis of hippocampal lysates. Of the numerous antibodies tested (same as immunostaining, above), only the anti-H60 antibody (clone 205326; 1:100; R&D Systems) successfully identified a prominent band that was present within the hippocampus only after infection and which was also present in lysate from the YAC-1 NK cell target line ( Figure 3B ). The apparent molecular weight of this prominent band (ca. 50 kDa) matches that described by Hasan and colleagues (Hasan et al, 2005) . The relative difference in mobility between the putative H60 band in the hippocampal lysate and the YAC-1 cell lysate may reflect differential expression of one of the three recently identified isoforms of H60 that share high sequence homology but differ in molecular weight (Takada et al, 2008) . We conclude that NKG2D ligands such as H60 are upregulated at both the transcriptional and translational level within the hippocampus following acute TMEV infection, but we are unable to identify the cells that express these ligands with the currently available immunologic tools.
Finally, we asked whether interruption of NKG2D-mediated recognition of NKG2D ligands was sufficient to alter viral load within the hippocampus. We used the CX5 function blocking antibody following a therapeutic regime adapted from Lanier and colleagues (100 ug/mouse i.p. on −1, +1, and +4 dpi; kindly provided by Dr. Lanier, UCSF) . Control mice received an equivalent dose of polyclonal rat IgG . Plaque assay (Pavelko et al, 1998) of freshly excised brain homogenates at 7 dpi revealed that CX5 treatment led to 9 ± 2 times more infectious virus as compared to control IgGtreated mice (U(5,5)=15, P=0.008, Mann-Whitney rank sum test; mean ± 95% CI) ( Figure  3C ). Based on these observations, we propose that VP2-specific CTLs expressing NKG2D recognize NKG2D ligands such as Rae1 and H60 expressed on infected cells within the brain and thereby engage an enhanced cytotoxic response (Markiewicz et al, 2005 ) that contributes to the clearance of picornaviruses from the CNS.
In addition to expression on activated CTLs, NKG2D is also a primary cytotoxicity receptor for NKCs and NKC activity is certainly modulated by viral infection. We chose to specifically focus on the role of NKG2D on antiviral CTLs because the number of NKCs present within the brain during the peak of viral clearance is low compared to CTLs (ca. 10% of CD45 hi cells are NK1.1-positive at 7 dpi; data not shown). However, we certainly cannot rule out a role for NKC-mediated recognition of NKG2D ligands on infected neural cells as part of an early response required for viral clearance. Likewise, NKG2D is expressed by subpopulations of CD4 T cells (Saez-Borderias et al, 2006) , γδ T cells (Dandekar et al, 2005) , and macrophages (Baba et al, 2006) , suggesting that mutliple cells types may be inhibited by CX5 treatment. Regardless, our observations provide evidence that NKG2D-mediated recognition of virus-infected targets by CTLs participates in the clearance of picornaviruses from the CNS. These findings extend previous studies regarding the immunovirology of NKG2D ligand expression which have largely focused on viral evasion of immune surveillance (Lodoen et al, 2003; Lodoen et al, 2004; Vilarinho et al, 2007) and add to the potential repertoire of therapeutic strategies for reducing and controlling CNS picornaviral infections . NKG2D-positive antiviral CD8 + T cells infiltrate the brain during acute infection with TMEV. Brain-infiltrating leukocytes (BILs) were isolated from mice at 3 (A-C) and 7 (D-F) days postinfection (dpi) and analyzed by flow cytometry. The number of CD45 hi CD8 + cells increased between 3 (A) and 7 (D) dpi. Likewise, the number of CD45 hi cells positive for both CD8 and NKG2D increased from 20% at 3 dpi (B) to 76% at 7 dpi (E). Moreover, the intensity of NKG2D surface labeling increased from a mean fluorescence intensity (MFI) of 35 at 3 dpi (shaded histogram in C) to 89 at 7 dpi (shaded histogram in F). The MFI for the isotype control was 4 at 3 dpi (open histogram in C) and 5 at 7 dpi (open histogram in F). BILs were further analyzed for antiviral specificity by costaining with an MHC class I tetramer loaded with the VP2121-130 H-2D b -specific viral peptide (G-I). Of the cells that were both CD8 + and tetramer-positive (box "H" in G), 81% were also NKG2D-positive, with an MFI of 136 (H). In contrast, only 12% of cells that were CD8 + but tetramer-negative (box "I" in G) were also NKG2D-positive, with an MFI of only 18 (I). NKG2D ligands are upregulated in the hippocampus during acute infection with TMEV and interference with NKG2D recognition of these ligands increases viral load in the CNS. qRTPCR analysis of brain mRNA revealed a 10-fold upregulation of Rae1 at 7 dpi (A) as compared to the uninfected brain. Likewise, H60 and Mult1 were also upregulated throughout the acute phase of infection (A). Western blot analysis of hippocampal protein lysates showed the translational upregulation of H60, with peak expression at 7 dpi (B). The 50 kDa band (arrow) upregulated in hippocampus was also present in YAC-1 cell lysates (B). Treatment with the CX5 anti-NKG2D function blocking antibody throughout the acute phase of infection resulted in a 10-fold increase in viral load within the brain at 7 dpi (C). Bar graph shows mean ± 95% confidence interval. Five mice were treated in each group and individual viral titers are shown as black circles in (C). All CX5-treated mice showed increased viral titer. Discovery and Genomic Characterization of a Novel Ovine Partetravirus and a New Genotype of Bovine Partetravirus Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively). In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of ovine spleen samples. One of the bovine partetraviruses detected in the present samples is phylogenetically distinct from previously reported bovine partetraviruses and likely represents a novel genotype. The ovine partetravirus is a novel partetravirus and phylogenetically most related to the bovine partetraviruses. The genome organization is conserved amongst these viruses, including the presence of a putative transmembrane protein encoded by an overlapping reading frame in ORF2. Results from the present study provide further support to the classification of partetraviruses as a separate genus in Parvovirinae. The parvoviruses are a group of small, non-enveloped animal viruses with a single-stranded DNA genome between 4 and 6 kb in size [1] . At least 2 open reading frames (ORF) are present in the parvovirus genome, with ORF1 encoding the non-structural proteins and ORF2 encoding the viral capsid proteins. Some parvovirus genomes may contain an additional ORF encoding for other proteins, such as the non-structural protein NP1 found in human bocavirus and bovine parvovirus. Under the current International Committee on Taxonomy of Viruses (ICTV) classification system, the Parvoviridae are divided into two subfamilies based on their host range: the Parvovirinae which infect vertebrates, and the Densovirinae which mainly infect insects and other arthropods. The Parvovirinae are further subdivided into 5 genera: the amdoviruses, bocaviruses, dependoviruses, erythroviruses, and the parvoviruses. Novel parvoviruses discovered in recent years, such as the human partetravirus (previously known as human parvovirus 4 or PARV4) [2] , are not included in the current classification system, and will be addressed by the ICTV in an upcoming update. Some human parvoviruses are well-known pathogens associated with a range of diseases in infected patients. Parvovirus B19 (also known as erythrovirus B19) can cause syndromes ranging from erythema infectiosum in children to late intrauterine death in pregnant women. The human bocavirus, a parvovirus discovered only in 2005, was associated with respiratory disease and could be detected in stool of children [3, 4, 5, 6, 7, 8] , although clear assessment of its pathogenicity is confounded by the frequent codetection of other respiratory viruses [9, 10] and its presence in the stool of healthy children. On the other hand, the human partetravirus was initially discovered in patients suffering from acute viral syndrome [2] , but its association with clinical disease has yet to be confirmed despite positive PCR detection in blood products, HIV-infected patients, intravenous drug users, transplant patients and blood donors in different localities [11, 12, 13, 14, 15, 16, 17, 18] .
Since the discovery of the human partetravirus, related animal viruses have been found in various mammalian species. We first reported the discovery of porcine and bovine partetraviruses (also known as hokoviruses previously), which are novel related animal parvoviruses found in Hong Kong [19] . Closely related porcine partetraviruses have since been found in German wild boar populations [20] , while human partetravirus-like viral DNA was detected in the plasma samples of a chimpanzee and baboon in Cameroon [21] . As the genetic distances between human partetravirus and other known parvoviruses at the time of its discovery were relatively large, it was unclear if the human partetravirus had diverged from a human parvovirus ancestor early in the history of parvovirus evolution or if it had diverged from an undiscovered animal parvovirus. Parvovirus evolution is characterized by the development of host-specificity and congruent viral and host phylogenies [22] , which has led to the hypothesis of long-term co-evolution of parvoviruses and their hosts. In addition, evolutionary rates can vary significantly among different parvovirus species [23, 24] , which complicates evolutionary analysis between distant parvoviruses. Hence, the identification of related partetraviruses in animals has contributed to our knowledge of the evolutionary history of the human partetravirus.
As part of our ongoing program in the discovery of viruses associated with emerging infections, we continued our surveillance for novel partetraviruses in animals closely related to humans. In the present study, bovine and ovine samples were selected for targeted screening, as both domestic cattle and sheep are important food animals in Southern China. PCR screening of the animal samples identified a novel ovine partetraviruses as well as a new genotype of bovine partetravirus, and their nearly fulllength genome sequences were obtained. Genomic and phylogenetic analyses of the new viruses confirmed them to be closely related to the previously identified partetraviruses.
All specimens were collected over a one year period (Sept 2008 to Aug 2009). A total of 110 bovine liver samples, 14 ovine spleen samples and 9 ovine liver samples were collected from local food markets with the assistance of the Veterinary Public Health Section, the Food and Environmental Hygiene Department, and the Agriculture, Fisheries and Conservation Department, the Government of Hong Kong Special Administrative Region (HKSAR). It should be noted that ovine liver and spleen samples were not uniformly sampled throughout the study period due to supply issues. All animals from which the specimens had been obtained have passed the relevant health inspection to certify its meat and other products as fit for human consumption. Precautions taken to avoid cross contamination include the use of disposable scalpels during tissue dissection and the collection of samples only from the centre of each tissue block after surface decontamination. Disposable protective gloves were also used and changed after processing each tissue sample.
DNA was extracted from all samples using QIAamp DNA Mini kit (Qiagen) according to manufacturer's instructions, and then subjected to PCR using 2 different sets of screening PCR primers. The primer sequences are: forward primer A 59-CCCGCKAS-TACWGGNAARAC -39 and reverse primer A 59-CCGTAAYT-CKRCCYTCKTCCCA -39 (targeting a 148 bp fragment); forward primer B 59-TCTGCTATTGTAATHAARGAYGT-39 and reverse primer B 59-AAACACTCTGCRTCRTGRTGYTC-39 (targeting a 293 bp fragment). The primers were designed from multiple alignments of the nucleotide sequences of VP2 regions of PARV4 and related parvoviruses using our previously described strategy [19] . The PCR mixture (25 ml) contained DNA extracted from samples, PCR buffer (10 mM Tris/HCl pH 8.3, 50 mM KCl, 2 mM MgCl 2 and 0.01% gelatin), 200 mM of each dNTP and 1.0 U AmpliTaq Gold polymerase (Applied Biosystems). PCR cycling conditions were as follows: hot start at 94uC for 7 min, followed by 50 cycles of 94uC for 1 min, 50uC for 1 min and 72uC for 1 min with a final extension at 72uC for 10 min in an automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid PCR contamination and no false positive results was observed in negative controls.
PCR products were gel-purified using the QIAquick gel extraction kit (Qiagen). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA Analyser (Life Technologies) by using the PCR primers as sequencing primers. The sequences of the PCR products were searched against known VP2 sequences of parvoviruses in the National Center for Biotechnology Information (NCBI) GenBank database using BLASTN and TBLASTN.
Nearly full-length genome sequences spanning the entire protein-coding regions were determined for 2 bovine and 4 ovine strains of the PARV4-related parvoviruses identified in the present study by using our genome sequencing strategy [19] . Briefly, DNA extracted from the corresponding specimens was used as template and amplified by degenerate primers designed from multiple alignment of PARV4 and related sequences available in NCBI GenBank. Additional primers were designed from the first and subsequent rounds of sequencing. Non-overlapping regions were confirmed by independent PCR and sequencing reactions by using specific primers, and no sequence discrepancies were found between repeated sequencing of any region. Sequences of genome amplification and sequencing primers are available from the authors upon request. For sequencing of the terminal regions, a modified protocol for rapid amplification of cDNA ends was adopted [25] . Sequences were assembled and manually edited to produce final sequences of the viral genomes.
Open reading frames were located using the ORF Finder tool at NCBI (http://www.ncbi.nlm.nih.gov/projects/gorf/) and by comparison with the genome annotations of PARV4 and partetraviruses. Prediction of transmembrane domains was performed using TMHMM version 2.0 server (http://www.cbs.dtu.dk/services/ TMHMM/) [26] . Functional annotation of predicted proteins was performed by BLAST similarity search against annotations in the NCBI RefSeq database, as well as by using the InterProScan search tool (http://www.ebi.ac.uk/Tools/pfa/iprscan/) [27] . Multiple alignments of sequences for phylogenetic analysis were constructed using MUSCLE version 3.8.31 [28] , and phylogenetic informative regions were extracted using BMGE [29] . Maximum-likelihood phylogenetic trees were constructed using PHYML version 3 [30] , under the best-fit protein evolution model as selected by ProtTest 3 [31] . Recombination detection was performed using bootscan analysis (as implemented in SimPlot) [32] and GARD [33] . Sites under positive selection were inferred by consensus between the single-likelihood ancestor counting (SLAC), fixed effects likelihood (FEL) and random effects likelihood (REL) methods as implemented on the DataMonkey server (http://www.datamonkey.org) [34] . A p-value of ,0.1 is considered to be statistically significant for the positive selection analysis.
The nucleotide sequences of the nearly full-length genomes of the PARV4-related viruses have been submitted to NCBI GenBank, and are available under the accession numbers JF504697 -JF504702.
PCR detection of PARV4-like DNA using screening primer set A was positive for 2 out of 110 (1.8%) bovine liver samples, while PCR using screening primer set B was positive for 10 out of 14 (71.4%) ovine spleen samples and 6 out of 9 (66.7%) ovine liver samples. The rate of positive PCR detection is significantly higher in ovine liver samples than in bovine live samples (by Fisher's exact test; p,0.001), while no statistically significant difference was noted between ovine liver and ovine spleen samples (by Fisher's exact test; p.0.05). Sequencing of the PCR products was performed: sequences from the two positive bovine liver samples showed the highest nucleotide identities (100% and 89%) to the ORF2 of bovine partetravirus HK2 (GenBank accession no. EU200670), and sequences from the positive ovine samples showed the highest nucleotide identity (88%) to the ORF2 of human partetravirus PARV4 strain BONN-14 (GenBank accession no. EU175857). This suggested that two related but distinct strains of partetravirus are present in the two different bovine samples, while a novel partetravirus is present in the ovine samples.
The nearly full-length genomes of 2 strains of bovine partetravirus (5096-5240 nucleotides) and 4 strains of ovine partetravirus (2 from liver samples and 2 from spleen samples) (5249 nucleotides) were determined. Similar to our earlier attempt at sequencing the complete genomes of other PARV4-like animal parvoviruses [19] , the obtained genome lengths were shorter than expected, as sequencing of the ends of the genomes was hampered by the presence of extensive hairpin structures. Pairwise comparison of the genome sequences of the parvoviruses was performed. Among the presently identified bovine partetraviruses, HK4 was highly similar (99.3299.4% nucleotide sequence identity) to the bovine partetraviruses reported previously, while HK5 showed much greater sequence divergence (90.0-90.1% nucleotide sequence identity). The genome sequences of the ovine parvoviruses were 100% identical, and they exhibited 68.2-68.3% nucleotide sequence identities to that of the bovine partetraviruses and around 64.0% nucleotide sequence identities to that of the porcine partetraviruses. Results of further sequence comparison with related parvoviruses were shown in Tables 1 and 2. All sequenced virus strains possessed a genome organization typical of parvoviruses. There were two large non-overlapping ORFs, with ORF1 encoding a non-structural polyprotein NS1 and ORF2 encoding overlapping VP1/VP2 capsid proteins and a small conserved putative protein. A small non-coding gap of around 115 nucleotides was found between the two ORFs. Inverted terminal repeats were found at the 59 and 39 ends of the viral genome. Further sequence analysis was not performed for the bovine partetravirus HK4 due to its high genetic similarity with previously characterized bovine partetraviruses. For bovine partetravirus HK5, the predicted NS1 protein consists of 652 aa and is 73.9 kDa in molecular weight. Conserved sequence features including helicase and ATPase domains are present, in agreement with the non-structural functional role of NS1 in parvovirus replication. The VP1 protein is predicted to contain 931 aa and is 102.8 kDa in molecular weight, which is comparable to that of previously identified bovine partetraviruses and larger than the Similar to other partetraviruses, there exists a putative third ORF in a different reading frame within the VP1u region for both bovine partetravirus HK5 and ovine parvovirus. In agreement with previous studies, the phospholipase A 2 motifs of ORF2 are found in the VP1u region, which is also the most conserved region among partetraviruses [19, 35] . The putative third ORF encodes a small protein containing a single transmembrane helix spanning 20 aa in the centre, with predicted molecular weights of 9.5 kDa in bovine partetravirus HK5 and 9.7 kDa in ovine parvovirus. The aa sequences of this putative protein are conserved among human, bovine, ovine and porcine partetraviruses, with pairwise sequence identities ranging from 59.3% (between porcine partetravirus HK7 and ovine partetravirus) to 96.4% (between bovine partetravirus HK1 and bovine partetravirus HK5).
Phylogenetic inference was conducted on the multiple sequence alignment of full-length NS1 and VP1/2 sequences of the partetraviruses. The phylogenetic trees ( Fig. 1 and 2) showed a common topology, and placed the presently identified viruses in the same clade as known partetraviruses. Bovine partetraviruses HK4 and HK5 formed a cluster with known bovine partetraviruses, although the genetic distance between HK5 and the other bovine partetraviruses suggested that it should be considered as a different genotype of bovine partetravirus. The 4 identical strains of ovine parvovirus are clearly shown to be distinct from the other known partetraviruses, and are most closely related to bovine partetraviruses. Based on these results, we recognize these ovine parvoviruses as a new member of the partetraviruses and propose to describe them as ovine partetraviruses. The phylogeny is consistent with the hypothesis of virus-host co-evolution, as the sheep and cattle are both ruminants and are evolutionarily closer to each other than to the other hosts like pigs, chimpanzee and humans.
Bootscan analysis on the genome sequences of PARV4-related viruses highlighted some genomic regions that may have undergone past recombination events between ovine parvovirus and PARV4 (Fig. S1 ). GARD analysis on the DataMonkey server (http://www.datamonkey.org/) also revealed the presence of 11 potential recombination breakpoints (data not shown), suggesting a multitude of possible past recombination events. However, incongruence testing with the Kishino-Hasegawa (KH) test failed to confirm the presence of phylogenetic incongruence in the partitions proposed by the bootscan and GARD analyses. Hence, it is more likely that the potential recombination signals were falsepositives resulting from local differences in evolutionary rates among different genes.
Sequence analysis for sites under positive selection revealed 1 site under positive selection in ORF1 and 13 sites under positive selection in ORF2 ( Non-bold percentages indicate nucleotide identities and bold percentages indicate amino acid identities. All sequence identities are based on the full-length sequences and calculated using MatGat [54] . GenBank accession numbers are as follows: porcine PtV HK1 -EU200671 [19] ; bovine PtV HK1 -EU200669 [19] ; bovine PtV HK5 -; ovine PtV-; human PtV PARV4 C51-4 -DQ873387 [55] ; bovine PV 2 -AF406966 [25] ; bovine PV 3 -AF406967 [25] ; minute virus of mice -DQ196317 [56] ; human PV B19 -AY044266 [57] ; bovine bocavirus -DQ335247 [58] ; human bocavirus -FJ858259 [59] ; Aleutian mink disease virus -NC_001662 [61] ; bovine adeno-associated virus (AAV) -AY388617 [60] . doi:10.1371/journal.pone.0025619.t002
require further knowledge of the structure and function of the viral proteins for interpretation.
The present study extends our previous discovery of partetraviruses in domestic animals. Although no clear association has been found between clinical disease and human partetraviruses, the relatively high prevalence of PARV4 in human blood products has raised safety concerns, especially since available evidence appears to support possible parenteral transmission of the virus [14, 36, 37, 38, 39] . Similarly, animal partetraviruses have not been found to be associated with any clinical diseases in the hosts, and are detected frequently in apparently healthy animals [19, 20, 21] . So far, these viruses were mostly detected in liver, spleen, lymph node and bone marrow, suggesting significant tropism for lymphoid tissue, although there is also a report of detecting PARV4 DNA at relatively high frequencies in other organs such as the heart, lungs and kidneys [12] . The development of immune electron microscopy and related techniques can determine the intracellular location and cellular tropism of partetraviruses in infected tissues, and success have been reported with the visualization of PARV4 particles in a high-titre-positive plasma sample [40] . This will aid efforts to target screening for subclinical disease in patients or animals with positive partetravirus detection by PCR or serology.
The understanding of parvovirus evolution has changed greatly over the years. Initially, parvoviruses were thought to evolve slowly relative to RNA viruses due to the utilization of the high-fidelity host DNA polymerase during replication. This was also supported by the very limited sequence diversity of the human pathogenic parvovirus B19 that were known at the time. However, studies examining the evolutionary rate of different parvoviruses suggested that the evolutionary rates of parvoviruses are generally fast and comparable to those of RNA viruses. Moreover, a broad range of rates have been observed for different viruses: 1.7610 24 substitution/site/year (s/s/yr) in canine parvovirus, 9.4610 25 s/ s/yr in the closely related feline panleukopenia parvovirus [23] , 8.6610 24 s/s/yr in human bocavirus [41] , 1610 24 s/s/yr in parvovirus B19 [42] . In particular, it has been inferred that the evolutionary rate were as high as 0.727.1610 23 s/s/yr during the time interval from which canine parvovirus emerged. The discovery of additional genotypes of parvovirus B19 has also bolstered the view of parvoviruses as fast-evolving viruses [43, 44] .
Nonetheless, questions remain on certain aspects of parvovirus evolution. Although virus-host co-evolution had been proposed as an important mechanism in the emergence of new animal parvoviruses [22] , this hypothesis cannot be easily reconciled with the high evolutionary rates observed in diverse parvoviruses, as the evolutionary timescale of the genetic distance between the animal hosts would be several orders of magnitude greater than that of the genetic distance between the corresponding parvoviruses. While the effects of negative selection on parvoviruses highly adapted to their animal host may slow further evolution of the virus, it is unlike to account for the discrepancy between host and virus evolution to a significant degree. In comparison, observations on the emergence of canine parvovirus from feline panleukopenia parvovirus [45] and the experimental cross-species infection of rodent parvoviruses provide more support to the role of crossspecies transmission in the evolution of parvoviruses. This would also be more consistent with the degree of recombination seen in diverse parvoviruses [46] , which would have required frequent cross-species transmission and co-infection of different parvoviruses in the same host [47] . The present detection of these partetraviruses in organs of otherwise healthy animals suggests the possibility of viral persistence, although comprehensive sampling of other sites such as the respiratory tract would be needed to exclude acute infections. Relatively little is known about persistence of partetraviruses in animal hosts, although the phenomenon is well-described for other parvoviruses. Genomic DNA of human erythrovirus genotype 1 has been found to persist in the synovial membranes of both patients with chronic arthropathy and healthy individuals [48] . A larger study has additionally uncovered information on the distribution and persistence among different genotypes of human erythrovirus, which revealed the epidemiological history of these genotypes in the human population [49] . The exact mechanism for viral persistence has not been fully elucidated for many parvoviruses, though site-specific integration has been described for adeno-associated viruses [50] . Among the partetraviruses, the phenomenon of persistence is best studied in the human partetravirus genotype PARV4, which has been found in the blood, lymphoid tissue and bone marrow of HIV-infected patients [12, 18] . Results from interferon c enzyme-linked immunospot assays suggested that PARV4 persistence may be present at 26% of hepatitis C virus-positive individuals [51] .
From the results of the current and previous studies, it is shown that a diversity of partetraviruses could be found in humans and other animals. As these viruses were only recently discovered, only limited epidemiological and genetic data is available and they are unlikely to reflect their true prevalence and genetic diversity.
Nonetheless, at least 3 genotypes have been identified for the human partetravirus [52, 53] , which showed some degree of geographic segregation in their circulation. In contrast, the porcine partetraviruses identified in wild boars in Germany were highly similar to the porcine partetraviruses initially discovered in Hong Kong, though it remains possible to distinguish them as two close clusters of viruses on a phylogenetic tree constructed from discontinuous genome sequences [20] . The distinct cluster formation by these related viruses on phylogenetic trees argue for the consideration of the viruses to be classified in a new genus, which has already been suggested in a current ICTV taxonomic proposal. The present discovery of the ovine partetravirus and a new genotype of bovine partetravirus adds to the diversity of known partetraviruses, and should aid future efforts to characterize the evolution and transmission of these related viruses. Figure S1 Bootscan analysis on the genome sequences of ovine partetravirus and related viruses (porcine partetravirus HK7 (P), human partetravirus HK1 (H), and bovine partetraviruses HK4 and HK5 (B)) using Simplot version 3.5.1. Consensus threshold of 50% was employed for analysing the bovine partetravirus sequences. Parameters for the analysis are shown in the figure. (PDF) Reconstructing disease outbreaks from genetic data: a graph approach Epidemiology and public health planning will increasingly rely on the analysis of genetic sequence data. In particular, genetic data coupled with dates and locations of sampled isolates can be used to reconstruct the spatiotemporal dynamics of pathogens during outbreaks. Thus far, phylogenetic methods have been used to tackle this issue. Although these approaches have proved useful for informing on the spread of pathogens, they do not aim at directly reconstructing the underlying transmission tree. Instead, phylogenetic models infer most recent common ancestors between pairs of isolates, which can be inadequate for densely sampled recent outbreaks, where the sample includes ancestral and descendent isolates. In this paper, we introduce a novel method based on a graph approach to reconstruct transmission trees directly from genetic data. Using simulated data, we show that our approach can efficiently reconstruct genealogies of isolates in situations where classical phylogenetic approaches fail to do so. We then illustrate our method by analyzing data from the early stages of the swine-origin A/H1N1 influenza pandemic. Using 433 isolates sequenced at both the hemagglutinin and neuraminidase genes, we reconstruct the likely history of the worldwide spread of this new influenza strain. The presented methodology opens new perspectives for the analysis of genetic data in the context of disease outbreaks. The most effective strategy to avert infectious disease epidemics is to identify outbreaks at an early stage and identify their transmission routes. For example, wider spread of nosocomial bacteria could be best avoided by rapid identification of colonized medical personnel spreading the infection (Albrich and Harbarth, 2008) . Early identification of preferential transmission routes could also prove critical for containing wider epidemics, such as SARS or avian influenza (Cooper et al., 2006; Ferguson et al., 2006; Germann et al., 2006) . To be most effective, such prophylactic interventions must be implemented early on and will be based on only very preliminary scientific evidence. Thus, it is crucial that all sources of useful information be considered. Genetic sequence data can now be generated essentially in real time and the analysis of sequence data has already been added to the toolbox of infectious disease epidemiology (Rambaut et al., 2008; Russell et al., 2008; Cottam et al., 2008b; Garten et al., 2009; Sloan et al., 2009) . However, the statistical methodologies to harness the full potential of genetic sequence information are currently lacking. In particular there is a need for methods devoted to the reconstruction of the transmission tree of a set of isolates collected during the early stages of an outbreak.
Current state-of-the-art genetic methods for the reconstruction of pathogen genealogies rely on the phylogenetic paradigm (Grenfell et al., 2004; Templeton, 1998; Drummond and Rambaut, 2007; Cottam et al., 2008a; Lemey et al., 2009a) based on the reconstruction of ancestries between hypothetical common ancestors (most recent common ancestor) and sampled isolates. Unfortunately, such approaches may be inappropriate when ancestors and their descendents are both present in the sample analyzed, as is likely during the early stages of an outbreak. Phylogenetic methods consider that sampled strains are all tips of an unknown genealogy, making it impossible for a sampled strain to be (directly or indirectly) ancestral to another sampled strain. By definition, these methods are thus unable to uncover ancestries between sampled isolates, and can fail to reconstruct the transmission tree of an emerging pathogen (Figures 1a-c) .
To circumvent this problem, we introduce a new methodological approach for the analysis of genetic data collected during disease outbreaks. Contrary to phylogenetic methods, we consider that sampled isolates represent a fraction of the genealogy, including internal nodes and tips (Figure 1d ). Direct and indirect ancestries can therefore be inferred between the sampled isolates. Within this framework, we developed an algorithm called SeqTrack, which directly reconstructs the most plausible genealogy of a set of sampled isolates, allowing for a direct assessment of the spatiotemporal dynamics of the epidemic under study.
Using simple simulations, we illustrate the major difference between phylogenetic methods and our approach, showing the advantage of SeqTrack over traditional phylogenetic approaches for inferring transmission trees in densely sampled disease outbreaks. We then evaluate the performance of our method using spatially explicit simulations of outbreaks. Finally, we illustrate the novel methodology using sequence data from the early stages of the 2009 A/H1N1 influenza pandemic, and reconstruct the likely scenario of worldwide spread of this newly emerged pathogen.
SeqTrack algorithm Our method aims to reconstruct the transmission tree of pathogens during a disease outbreak, using genotypes and collection dates to uncover ancestries between sampled isolates. The fundamental innovation of our approach is to seek ancestors directly from the sampled isolates, rather than attempting to reconstruct unobserved and hypothetical ancestral genotypes. Because ancestries are in essence temporally oriented connections between isolates, it seemed natural to tackle the problem of inferring genealogies within graph theory.
SeqTrack relies on three fundamental, yet simple observations. First, in the absence of genetic recombination, each observed isolate has one, and only one ancestor. Second, ancestors always precede their descendents in time. And third, among all possible ancestries of a given isolate, some are more likely than others, and this likelihood can be inferred from the amount of genetic differentiation between the isolates considered. The purpose of SeqTrack is to identify the most likely genealogy. Technically, this problem translates into finding the optimum branching in a directed graph, where each node is an isolate, and where a given isolate is connected to all isolates occurring strictly later.
Let G ¼ (S, E, w) be a directed, weighted graph where S ¼ {s 1 ,y, s n } is the set of vertices corresponding to the n sampled isolates, with associated collection dates T ¼ {t 1 ,y, t n }. E is the set of directed edges of G modeling all possible ancestries in S, such that (s i , s j )A E if and only if t i ot j . The weight function w: E-R assigns a weight to each possible ancestry, which reflects how credible each ancestry relationship is. For instance, w could be defined as a genetic distance or similarity, or as the log-likelihood resulting from a probabilistic model of evolution. The weight of a subset A of E is computed as w A ¼ P e2A wðeÞ. The 'best' genealogy of the sampled isolates is the spanning directed tree (that is, 'branching' reaching all the nodes) J ¼ (S,B,w) with BDE optimizing (that is, minimizing or maximizing) w B . Whenever sampled isolates form more than one connected set, the algorithm is applied to each connected set separately (see example in Figure 3b ). This problem has been solved by Edmonds (1967) and Chu and Liu (1965) , who developed an algorithm to find J so that w B is maximum or minimum. The algorithm proceeds by identifying optimum ancestors for each node at the exception of the root (the oldest isolate), and then recursively removes possible cycles. However, in our case, cycles are impossible as ancestries cannot go back in time, which greatly simplifies computations.
The entire SeqTrack procedure has been implemented in the adegenet package (Jombart, 2008) for the R software (R Development Core Team, 2009 ). This implementation allows for specifying any weight w i and for choosing the type of optimization (minimization or maximization of total weight) to take advantage of the method's versatility.
Maximum parsimony genealogies using SeqTrack Outbreak genetic data are typically characterized by low genetic diversity, implying that descendents differ from their ancestors by very few mutations. When recombination and reverse mutations are unlikely, the most plausible genealogy is the one involving the fewest mutational steps. Such maximum parsimony genealogies can be recovered by SeqTrack by weighting edges according to the number of mutations between pairs of isolates, and seeking the branching of minimum weight. The resulting ancestry path connects all the sampled isolates while minimizing the required number of mutational steps, and respecting their temporal ordering.
Given the low levels of genetic diversity expected during the early stages of disease outbreaks, isolates with identical haplotypes but different collection dates and Figure 1 Illustration of possible reconstructions of genealogical relations. Panel a represents a hypothetical genealogy of six isolates from which three were sampled (highlighted by a blue glow and numbered from 1 to 3). Panels b and c illustrate phylogenetic reconstructions where the red dots represent inferred nodes; panel b corresponds to a classical phylogenetic reconstruction and c to a tree that accounts for heterochronous sampling (e.g., BEAST software; Drummond and Rambaut, 2007) . Panel d illustrates the direct ancestry reconstruction method implemented in SeqTrack.
locations are likely to occur, resulting in several possible choices for the most parsimonious ancestry. In such cases, SeqTrack can use an optional rule to select the 'best' ancestor, and select the closest ancestor according to a given proximity measure. For instance, this approach can be used to select, among all equally parsimonious ancestors, the one being geographically closest. Another possibility would consist in favoring ancestors whose haplotype is most frequent in the sampled isolates. By default, SeqTrack resolves ties in the choice of ancestors by maximizing the likelihood of the genetic differentiation observed between the ancestor and the descendent. The number of mutations n occurring between two isolates sampled D t time units apart follows a Poisson distribution:
where L and m are, respectively, the length (in number of nucleotides) and the mutation rate (per nucleotide and per time unit) of the considered DNA sequence. Note that this likelihood can also be used to assess the statistical support of each ancestry, as we do in Figure 5 .
Individual-based simulations were used to assess the efficacy of our method for reconstructing transmission trees from outbreak genetic data. All simulations were performed using the R software, and procedures that we implemented in the adegenet package. The simulation system is further described in Supplementary Information. We first used simulations to compare SeqTrack to classical phylogenetic reconstruction. Because the outputs of both approaches are very different (phylogenies do not provide ancestries between tips), we could not proceed to systematic comparisons over a large number of data sets. However, while not quantitative, the comparisons we performed can be used to show the fundamental differences between both approaches. We simulated two simple genealogies of isolates, using haplotypes of 10 000 nucleotides and a mutation rate of 10 À4 mutation per nucleotide and generation, over three and four generations, respectively (Figures 2a and 3a ). In the second data set (Figure 3) , only a portion of the genealogy was sampled, so as to show the behaviors of the methods for inferring ancestries with unsampled ancestors. Note that the two data sets were chosen randomly, and the results were qualitatively similar in all other simulations we examined. SeqTrack was used to obtain maximum parsimony genealogies (Figures 2b and 3b) . A classical phylogeny was obtained by neighbor-joining using pairwise nucleotidic distances between sampled isolates (Figures 2c and 3c) . To give the neighbor-joining phylogenetic trees the right direction, trees were rooted using the most ancient isolate sampled. We also analyzed these data using temporally informed phylogenetic reconstruction as implemented in the BEAST software (Drummond and Rambaut, 2007; Figures 2d and 3d) . We used the exponential growth model with strict molecular clock model, keeping other parameters to their default settings. A consensus phylogeny was computed on the 5000 last simulated trees.
Simulations were also used for a quantitative assessment of SeqTrack's performances. Data sets were obtained Reconstructing disease outbreaks T Jombart et al by simulating genealogies of haplotypes evolving stochastically in time and space. A detailed description of these simulations, along with scripts allowing to reproduce them, are provided in Supplementary Information. Two types of simulations, differing in the process governing the spatial spread of the isolates, were performed. The first scheme allowed isolates to disperse on a 5 Â 5 spatial grid according to a random Poisson process, with identical probabilities to move in all directions (Supplementary Figure S1 ). This scheme is later referred to as 'homogeneous diffusion'. The second type of simulations used stochastic dispersal based on predefined connectivity between locations, in which we specified the probabilities of migration between every pair of locations. This allowed us to recreate a spatial dynamic of 'sources' and 'sinks' with some locations seeding many other locations, including distant ones, and other locations attracting migration but hardly seeding other places (Supplementary Figure S2) . This type of simulation is later referred to as 'heterogeneous dispersal'. For each simulation scheme (homogeneous and heterogeneous dispersal), 10 genealogies of isolates simulated over 20 generations were obtained. Ten random samples of 800 isolates were then drawn from the genealogies, yielding 200 data sets that were analyzed. Simulations and analyses were performed on the computer cluster of the Centre de Calcul de l'Institut National de Physique Nucléaire et de Physique des Particules (CCIN2P3; http://cc.in2p3.fr/).
Genetic data were used to infer the spatiotemporal spread of the early stages of the swine-origin influenza A/H1N1 pandemic. We analyzed all isolates typed for both hemagglutinin (HA) and neuraminidase (NA) genes available from GenBank (Benson et al., 2007;  http://www.ncbi.nlm.nih.gov/Genbank/index.html) as of 20 August 2009. Genetic sequences and their annotation (including sampling dates and locations) were retrieved and processed using ad hoc scripts in the R language. We only retained isolates for which collection date and location were available. Influenza viruses are routinely amplified by serial passaging through cell cultures or chicken eggs before sequencing. Duplicate sequences have been submitted for some isolates that were multiply amplified, sometimes resulting in slightly different sequences for the same isolate. In these cases, significantly shorter sequences were discarded, as well as sequences obtained from amplification in eggs because this method is known to induce additional 'artificial' mutations (Bush et al., 2000) .
The alignment of all retained sequences was realized using ClustalW (Larkin et al., 2007) and refined by hand using Jalview (Waterhouse et al., 2009) . HA and NA segments were concatenated after verifying that analyses run with either segment yielded similar results (results not shown). The final alignment contained 433 sequences of 3025 nucleotides, with collection dates ranging from 30 March to 12 July 2009. Accession numbers, alignments, dates and locations of the isolates analyzed are provided in Supplementary Information. Raw pairwise genetic distances (in number of differing nucleotides) were computed using the R package ape (Paradis et al., 2004) . Substitution rates for the HA and NA segments were based on earlier work by Smith et al. (2009) . Note that the analyses presented in this paper are largely insensitive to the substitution rate, as this information is only used for the resolution of ties between genetically equally likely ancestors sampled at different dates.
Flight data were used to assess the role of air traffic in the dispersal of the pathogen. Passenger flows between countries were compiled from a list of all commercial flights that occurred between 5 May 2008 and 4 April 2009, purchased from OAG (http://www.oag.com/). Numbers of passengers between countries were then correlated to the number of inferred transmissions, and tested using standard Pearson correlation.
Simulation results showed fundamental differences between the SeqTrack algorithm and classical phylogenetic reconstruction (Figures 2 and 3) . In two randomly chosen examples, SeqTrack reconstructed very satisfyingly the transmission tree of the isolates (Figures 2b and  3b ). When all isolates were sampled, the method perfectly identified the transmission tree ( Figure 2b ). However, results remained very satisfying in the incompletely sampled genealogy (Figure 3b ), in which SeqTrack correctly inferred the most recent sampled ancestors, and therefore true branches of the transmission tree. In contrast, phylogenetic trees could hardly be used to draw inferences about the underlying genealogies (Figures 2c and 3c) . Indeed, the phylogenetic trees were not a good match to the transmission tree, and contained some oddities such as several identical internal nodes for a single polytomy (Figures 2c and  3c ). Even when considering tips with zero branch length as internal nodes, the resulting inferred ancestries were largely erroneous. Results obtained by BEAST were somewhat more satisfying, as the tips were ordered according to the sampling time. However, BEAST failed to identify any of the direct ancestries present in the simulated data set (Figures 2d and 3d) .
Further simulations were used to assess quantitatively the performance of our method at uncovering ancestries from genetic outbreak data. Most satisfyingly, our results indicated that the true ancestral haplotype was successfully retrieved in 93% of cases on average throughout all simulations (CI 99% ¼ [92.2-93.7%]; Figure 4 ). The correct location of the ancestor was also inferred in a majority of cases, although more frequently under source and sink dynamics than under homogeneous dispersal (76 and 71%, respectively; t ¼ 3.34, df ¼ 115, P ¼ 1.1 Â 10 À3 ; Figure 4 ).
Having tested the ability of the method at reconstructing transmission trees from outbreak genetic data, we used SeqTrack to infer the spatiotemporal dynamics of the early stage of the 2009 swine-origin A/H1N1 influenza pandemic. The data set analyzed consisted of 433 nearcomplete HA and NA sequences collected between 30 March and 12 July 2009. Although no sequences are available for the first confirmed cases (La Gloria, 15 February; Fraser et al., 2009) , the time window defined by the collection dates extends from the early stages of the outbreak to the global pandemic, which was officially declared by the WHO on 11 June 2009. In Figure 5 , we present snapshots of the spatiotemporal dynamics of the pandemic reconstructed by SeqTrack. Overall, most inferred ancestries displayed very few mutations between ancestor and descendent (Supplementary Figure  S3) , suggesting that the actual ancestral haplotype had often been sampled, and was correctly identified by the method. The inferred scenario of the early stages of the pandemic can be split into three main acts: (1) initial spread in Mexico and the United States (Figure 5a ), (2) sustained transmission within North America and spread to the rest of the world (Figure 5b) and (3) further worldwide spread and secondary outbreaks outside the Americas (Figure 5c ). For a day-by-day reconstruction of the pandemic, see Supplementary Movie S1 and Supplementary Information. Details of inferred ancestries are provided in Supplementary Table S2 .
The initial stage ending around the 20 April is characterized by numerous transmissions from Mexico to the United States, as well as some local transmissions within Mexico and the United States (Figure 5a ). The fact that two Mexican isolates trace their ancestry back to the United States was inescapable as the oldest sequences present in the data set were all sampled in the United States. However, most ancestries of early US isolates point to a Mexican origin (Figure 5a ), which gives further support for the A/H1N1 pandemic having originated in Mexico. The second stage (ending around 5 May) is characterized by the spread of the virus within North America, with several local transmissions (dots and sunflowers on Figure 5b and Supplementary Movie S1), indicating the existence of secondary outbreaks and the likely establishment of the pathogen in several cities in the United States and Canada. During the same time period, Mexico and the United States also started seeding the rest of the world (Figure 5b ). It is worth noting that the first case of within-country transmission outside the Americas (France on 1 May, Figure 5b , Supplementary Movie S1) is observed remarkably early after the spread of the new influenza strain within the United States. In the third and last stage, we observe a considerable number of transmissions inferred from the Americas to the rest of the world, alongside with transmissions between countries outside the Americas (Figure 5c ). Local transmissions with a high statistical support point to multiple secondary outbreaks occurring in several countries, which likely reflect the worldwide establishment of the new influenza strain as a global pandemic.
Flight traffic is widely recognized as an important determinant for the spread of seasonal and pandemic influenza at large geographic scales (Cooper et al., 2006; Ferguson et al., 2006; Germann et al., 2006; Viboud et al., 2006; Wu et al., 2009) . Thus, we evaluated the relationship between air travel passenger-flow and the number of transmissions between countries inferred by SeqTrack. Both quantities were fairly correlated (r ¼ 0.55; t-test: t ¼ 5.29; df ¼ 63; P ¼ 8.3 Â 10 À7 ), although this correlation was largely driven by the high connectivity of the United States with Mexico and Canada.
We have introduced a new methodological approach called SeqTrack for the analysis of genetic data sampled during disease outbreaks. Using simulated data, we showed the originality of this method compared to classical phylogenetic reconstruction, and its ability to infer correct genealogies of isolates in densely sampled disease outbreaks. The reconstruction of transmission trees from genetic data has recently received considerable attention (Gonzalez-Candelas et al., 2003; Hue et al., 2005; Cottam et al., 2008a; Cottam et al., 2008b; Harris et al., 2010) . All previous applications we are aware of have been based on the reconstruction of most recent common ancestors. Although this body of work has been fairly successful, the simulations presented in this paper suggest that our method outperforms phylogenetic approaches for the reconstruction of transmission trees in intensively sampled disease outbreaks. Even when taking temporal information into account, phylogenetic reconstruction seems unable to infer filiations between sampled isolates. This statement does not amount to a criticism of phylogenetics but simply reflects the fact that transmission trees and phylogenies are actually different entities. As such, transmission trees are likely to be inferred more successfully by a dedicated approach. Our method is also not meant to supersede phylogenetic approaches but rather to complement them. Although SeqTrack is better suited for reconstructing the transmission tree during early outbreaks, it does not include some useful features of modern phylogenetic tools, such as the possibility to infer underlying population dynamics parameters.
We illustrated our method with a reconstruction of a plausible scenario for the spatiotemporal spread of the A/H1N1 influenza strain during the early stages of the 2009 influenza pandemic. This data set might not be ideal for showing the full potential of the new methodology, which may be best suited for the reconstruction of transmission trees in more localized settings. Nonetheless, the results suggest that SeqTrack can contribute to a better understanding of the spatiotemporal dynamics of emerging pathogens, even at a worldwide scale. The results are largely in line with the general epidemiological understanding of the spatiotemporal spread of the 2009 A/H1N1 pandemic. We also recover individual Reconstructing disease outbreaks T Jombart et al ancestries that seem sensible and correlate with human flight data. It is not entirely straightforward to compare our reconstruction with previous phylogenetic work based on sequence data from the 2009 A/H1N1 influenza pandemic (Fraser et al., 2009; Parks et al., 2009; Rambaut and Holmes 2009; Smith et al., 2009; Lemey et al., 2009b) . These authors used a smaller set of isolates and did not specifically aim to reconstruct the spatiotemporal dynamics with the exception of Lemey et al (2009b) , who analyzed 242 isolates sequenced for the HA and NA genes. They also recovered a strong connection between Mexico and Texas and multiple instances of transmissions from the United States and Canada to outside the Americas. In contrast to their results, we infer a larger proportion of direct transmissions from North America to other continents. For instance, although we infer several direct transmissions from North America to China, their reconstruction points to China being only indirectly connected to the United States through either Russia, Europe or South America.
SeqTrack is a very versatile approach that allows for weighting the plausibility of individual ancestries in various ways. In this study, we used a maximum parsimony version of the method because the origin of A/H1N1 influenza virus is sufficiently recent for homoplasies (recombination or back mutations) to be very unlikely, and safely ignored. Nonetheless, SeqTrack genealogies could be optimized on other criteria, such as genetic distances or log-likelihoods based on complex models of sequence evolution. Another possible extension of this method relates to the use of heterogeneous genetic information, which occurs when the regions of the genome sequenced vary across isolates. Such heterogeneity is likely to occur as soon as the sequencing effort is undertaken independently by a large number of labs, as was the case during the early stage of the 2009 A/H1N1 influenza pandemic. As a result, sequence data of the new influenza strain consisted of essentially random combinations of segments, ranging from small fragments of specific genes to whole genomes. In such cases, the analysis of homologous sequences results in a possibly considerable loss of information. This issue could be overcome by averaging genetic differentiation across available segments, using appropriate weights to account for different substitution rates and segment lengths. However, including a large fraction of isolates with heterogeneous sequencing coverage would require accurate estimates of substitution rates, which can be difficult to obtain for emerging pathogens.
Some other developments would require more effort. In particular, it would be desirable to consider the various parameters of the model from a probabilistic perspective. For instance, the date of probable transmission could be better captured by a distribution of the time during which a specific sequence remains unaltered rather than a fixed collection date for the isolate. Moreover, a probabilistic framework would also allow incorporation of information besides genetic sequences and collection dates, such as prevalence data or spatial connectivity between locations. A final possible extension of SeqTrack relates to the inference of unsampled ancestral isolates. Although phylogenetic methods assume that no isolate in the sample is (directly or indirectly) ancestral to another one, our method considers that ancestries can be inferred between the sampled isolates. In practice, however, the ancestral population of a given isolate may not have been sampled. In such cases, SeqTrack is likely to identify the most recent sampled ancestor of the isolate as a substitute for the unsampled direct ancestor (Figures 1d, 2b, and 3b) . In other words, the algorithm is likely to retrieve an actual branch of the transmission tree by overlooking unsampled intermediate nodes. The possibility of inferring unobserved intermediate nodes based on a probabilistic model for sequence evolution and spatial dispersal (Lemey et al., 2009a) would represent an additional significant improvement in the present approach.
Understanding the underlying factors behind the spread of diseases is one of the core objectives of infectious disease epidemiology. By reconstructing the transmission tree of an outbreak, our method creates new opportunities for testing how various features of the connectivity between hosts shape transmission patterns. For instance, despite limited genetic information available for the early stage of the swine-origin A/H1N1 influenza pandemic, we observed a positive association between flight traffic and the routes of transmissions inferred by SeqTrack, in line with previous works on both seasonal and pandemic influenza (Brownstein et al., 2006; Cooper et al., 2006; Ferguson et al., 2006; Germann et al., 2006; Viboud et al., 2006; Fraser et al., 2009) . The versatility of the presented methodology makes it applicable over a wide range of scales, from localized outbreaks of a nosocomial infection within a hospital to the global spread of a newly emerged pathogen. In not requiring extensive genetic polymorphism, the method should prove effective in bacteria and fungi, in addition to viruses. We hope that the performance of the method, together with its wide applicability, flexibility and computational speed will convince infectious disease epidemiologists to adopt it as an integral part of the toolkit for infectious disease outbreak analysis. Human Bocavirus – Insights into a Newly Identified Respiratory Virus Human Bocavirus (HBoV) was discovered in 2005 using a molecular virus screening technique. It is often found in respiratory samples and is a likely cause for respiratory diseases in children. HBoV is distributed worldwide and has been found not only in respiratory samples, but also in feces, urine and serum. HBoV infections are mostly found in young children and coinfections with other respiratory viruses are often found, exacerbating the efforts to link HBoV to specific symptoms. The purpose of this review is to give an overview of recent HBoV research, highlighting some recent findings. Human bocavirus (HBoV) was discovered in 2005 by Allander et al. [1] in respiratory samples from children with suspected acute respiratory tract infection (ARTI) using a novel technique. This molecular virus screening is based on a random PCR-cloning-sequencing approach and was employed on two chronologically distinct pools of nasopharyngal aspirates (NPAs). It revealed a parvovirus-like sequence, with close relation to the members of the bocavirus genus.
A retrospective study revealed 17 (3.1 %) out of 540 NPAs positive for HBoV, with 14 specimens tested negative for other viruses, giving the suggestion that HBoV is a causative agent of respiratory tract infections [1] .
HBoV is a putative member of the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Before identification of HBoV, parvovirus B19 of the genus Erythrovirus was the only known human pathogen in the family of parvoviruses. Parvovirus B19 is widespread and manifestations of infection vary with the immunologic and hematologic status of the host. In immunocompetent children, parvovirus B19 is the cause for erythema infectiosum. In adults it has been associated with spontaneous abortion, non-immune hydrops fetalis, acute symmetric polyarthropathy, as well as several auto-immune diseases [2, 3, 4, 5] .
Based on its genomic structure and amino acid sequence similarity shared with the namesake members of the genus, bovine parvovirus (BPV) and canine minute virus (MVC), HBoV was classified as a bocavirus and therefore provisionally named human bocavirus [1] .
Other subfamily Parvovirinae members known to infect humans are the apathogenic adenoassociated viruses of the genus Dependovirus and parvovirus 4 [6, 7] . Parvovirus 4 has not yet been assigned to a genus, but it was proposed to allocate it to the genus Hokovirus as it shares more similarities to the novel porcine and bovine hokoviruses than with other parvoviruses [8] . Recently a second human bocavirus has been identified, HBoV2, with 75.6 % nucleotide similarity to HBoV [9] . HBoV2 was found in stool samples from Pakistani children as well as in samples from Edinburgh (1 of the 3 positive samples was derived from a patient >65 years old), indicating that it is not restricted to one region or to young children.
The parvoviridae are small, non-enveloped viruses. The isometric nucleocapsids with diameters of 18 to 26 nm contain a single molecule of linear, negative-sense or positive-sense, single stranded DNA with an average genome size of 5,000 nucleotides.
A study on the polarity of the packaged strand confirms that HBoV replication leads to packaging of single stranded DNA in the majority of cases. By using the NASBA methods, Böhmer et al. showed that negative strands were packaged in 87.5 % of the investigated samples [10] .
The complete genome of HBoV has yet to be determined. Until today at least 5,309 nt were identified (GeneBank Accession-Number EU 984244). The genome of other parvoviruses is flanked by palindromic hairpin structures essential for DNA replication and it can be assumed that this is also true for HBoV. The hairpin structures of HBoV could not be deciphered by sequencing methods so far and the complete sequence of the genome remains unknown until the flanking structures are elucidated.
Three open reading frames (ORF) can be found in the genome of HBoV, similar to BPV and MVC. One ORF encodes a non-structural protein (NS1) and a second one at least two capsid proteins (VP1 and VP2). The third ORF encodes a non-structural protein (NP1). The function of HBoV NS1 is unknown. In MVC and minute virus of mice, NS1 is a multifunctional protein, essential for viral DNA replication [11, 12] . Furthermore, a role in apoptosis, cell cycle arrest, and transactivation of cellular genes has been described for parvovirus B19 NS1 [13, 14, 15, 16] . NP1 is absent in other parvoviruses and, like for NS1, the function of HBoV NP1 is unknown. In MVC, NP1 plays an essential role in DNA replication [11] . Cross-complementation tests with NP1 of MVC, BPV, and HBoV showed that they all could increase DNA replication in NP1 knockout mutants, suggesting they all have analog functions [11] .
Alignment studies showed that amino acid variations seem to appear mostly in the genes of the capsid proteins while NS1 and NP1 represent the most conserved regions of the HBoV genome [17] , reflecting the more immunogenic character of the virion-associated proteins.
HBoV detection has been mostly performed on NPAs and swabs and relies mostly on classical [1, 18, 19, 20, 21, 22, 23] and real-time PCR [24, 22, 25, 26, 27, 28] . Real-time PCR sure has advantage over the conventional PCR, as it offers greater sensitivity, specifity, and reduced expenditure of time. PCR assays detecting the NS1 or NP1 gene are most common. Tozer et al. established a highly sensitive real-time PCR assay targeting the NP1 and the VP1 gene and were able to detect HBoV in respiratory samples, as well as in fecal samples and whole blood [29] .
Additional to the PCR assays, HBoV can be detected indirectly via detection of antibodies to HBoV. This method has also been performed with different ELISAs using virus-like-particles (VLP) of HBoV VP1 or VP2 [30, 31, 32, 33] . VLPs were produced by using an insect cell line infected with a baculovirus expression vector. These VLPs were then used to produce rabbit anti-serum with high titers of immunoglobulins specific for HBoV, which could be used in the ELISA. All established ELISAs were able to detect anti-HBoV antibodies in sera.
After the first description of HBoV, it has been reported from various countries, pointing to a worldwide endemicity. HBoV has been identified in Europe [34, 35, 36, 37, 38, 39, 40] , Asia [41,42,24,43], Australia [18, 44, 23] , Africa [25] and America [45, 19, 46, 20] . Until now, it is known that HBoV exists as a single lineage with two different genotypes [47] . The prevalence of HBoV ranges between 1.5 to 19.3 % [19, 48] . Primary infection with HBoV seems to occur early in life and children between the ages of six and 24 months seem to be mostly affected [31, 49, 43, 22, 50] , but older children can be infected, too. Newborns may be protected by antibodies against HBoV derived from the mothers A seasonal distribution of the virus has not yet been clearly demonstrated. HBoV has been detected throughout the whole year, with peak seasons varying from year to year and from study to study. However, HBoV has been detected mostly in fall and winter months [39, 55, 56, 57] .
As HBoV has been first identified in respiratory samples, it has been suggested as a respiratory tract infection agent [1] . The majority of the following studies in fact detected HBoV in children with respiratory tract infections. Clinical symptoms mostly described in conjunction with an HBoV infection are wheezing, fever, bronchiolitis and pneumonia [28, 49, 36, 50] . Studies including asymptomatic controls showed that HBoV is also detectable in these controls but with a lower incidence [20, 58, 59] . For example, HBoV was detected in 17 % of children hospitalized because of respiratory infection, while only 5 % of the surveyed asymptomatic children were HBoV positive [58] . This supports the assumption that HBoV in fact could be assigned to the respiratory viruses.
In contrast to other studies, in the study of Longtin et al. 43 % of asymptomatic children tested positive for HBoV [53] . Most of those children underwent myringotomies, adenoidectomies or tonsillectomies. Thus, Lu et al. suggested that HBoV may be present in tonsillar lymphocytes [60] . They tested DNA extracts of lymphocytes from nasopharyngeal tonsils or adenoids and palatine/lingual tonsils. 32.3 % of the tested extracts were HBoV positive, indicating that HBoV establishes latent or persistent infection.
Coinfections with other viruses are frequently observed in HBoV infections and often occur in more than 50 % of the tested samples [56, 46, 61] . Two recent studies report that the viral load of HBoV was significantly higher in children with monoinfections than in children with coinfections [57, 62] . The high rate of coinfections with other viruses may then be explained by the persistence of HBoV in the respiratory tract. DNA quantification in HBoV positive samples revealed that the viral load of 42.5 % of the positive patients was > 1.0 x 10 5 DNA copies/mL, suggesting that below this cut-off HBoV may be a persistant virus or a bystander [54].
MVC and BPV, the two other members of the genus bocavirus are also known to cause gastrointestinal infections in dogs and calves, respectively [63, 64] . Several studies detected HBoV in stool samples from children with acute gastrointestinal illness [65, 66, 67, 38] , but the role of HBoV infection in the gastrointestinal tract is still unclear. A study on the role of HBoV in gastroenteritis outbreaks in day care facilities detected HBoV in 4.6 % of 307 stool samples. Coinfections with Norovirus were frequent [68] . Another study on hospitalized children with acute gastroenteritis also reports a high coinfection rate with other gastroenteritic viruses [69] . Both reports could not link HBoV to gastroenteritis in children, indicating that HBoV may not be a causative agent. The gastrointestinal epithelium may instead be the place of HBoV replication.
Besides HBoV detection in respiratory samples and feces, HBoV DNA was also found in serum/whole blood [28, 59, 29] and one study reports detection in urine [70] . As there are no currently established methods to detect HBoV particles, it remains unclear if the detection of HBoV in serum indicates viremia or if HBoV targets blood cells. Parvovirus B19 infects erythroid progenitor cells in the bone marrow [2] , but HBoV DNA was not detected in bone marrow of HIV (human immunodeficiency virus)-infected and HIV-uninfected individuals, while parvovirus B19 was detected in both groups [71] .
As most of the published data regarding HBoV are prevalence studies, the immunologic response to HBoV remains unclear. The work of Chung et al. gives a first insight into the cytokine response to HBoV. They observed that interferon-γ, interleukin-2 and interleukin-4 are elevated in HBoV positive specimens compared to the controls [72] . In comparison to respiratory syncytial virus infected children, HBoV infected children showed lower levels of tumor necrosis factor-α and interleukin-10.
It remains unclear how far HBoV contributes to respiratory and/or gastrointestinal disease. More and more evidence supports the assumption that HBoV is indeed an infectious and contagious agent, but a chance remains that it solely synergistically increases the clinical severity of other infections. Consequently, well planned and designed clinical studies with sophisticated case controls need to be performed in order to finally rule out the role of bocavirus, unless animal or at least in vitro models demonstrate its pathogenicity.  Plasmacytoid Dendritic Cells and the Control of Herpesvirus Infections Type-I interferons (IFN-I) are cytokines essential for vertebrate antiviral defense, including against herpesviruses. IFN-I have potent direct antiviral activities and also mediate a multiplicity of immunoregulatory functions, which can either promote or dampen antiviral adaptive immune responses. Plasmacytoid dendritic cells (pDCs) are the professional producers of IFN-I in response to many viruses, including all of the herpesviruses tested. There is strong evidence that pDCs could play a major role in the initial orchestration of both innate and adaptive antiviral immune responses. Depending on their activation pattern, pDC responses may be either protective or detrimental to the host. Here, we summarize and discuss current knowledge regarding pDC implication in the physiopathology of mouse and human herpesvirus infections, and we discuss how pDC functions could be manipulated in immunotherapeutic settings to promote health over disease. Herpesviruses are enveloped viruses, with genomes consisting in double-stranded linear DNA. Nine herpesviruses have currently been associated with diseases in humans (Table 1 ). Three herpesviruses have been identified in mice. Herpesviruses establish life-long persistent infections in their natural hosts, with a very high seroprevalence ranging from 30% to 90% depending on the country and on the virus. The infection is usually asymptomatic in immunocompetent individuals. Upon resolution of the acute phase of the primary infection, herpesviruses establish latent infection in specific cell types. A variety of stress conditions, including immunosuppression or inflammation, promote herpesvirus reactivation which can eventually lead to severe pathology (for review see [1] ). Indeed, herpesviruses are some of the most common opportunistic agents encountered in AIDS patients and in recipients of bone marrow or solid organ transplantation. Herpesviruses harbor a very selective tropism for their host species, attesting to a very long history of co-evolution spanning several millions of years. The mouse cytomegalovirus (MCMV) cannot infect rats or humans, and reciprocally for the human or rat CMVs. The genome of herpesviruses is of considerable size and complexity as compared to that of many other viruses. Herpesviruses harbor a high number of genes that are not essential for their replicative cycle but that appear to have been selected for escape from, or hijacking of, the host immune system. However, reciprocally, the immune systems of the hosts have evolved counterstrategies to control the viruses. One of the earliest and most potent responses of the hosts to infections with herpesviruses is the production of type-I interferons (IFN-I).
IFN-I were the first cytokines discovered, a little over 50 years ago, based on their direct, potent and broad antiviral activity [14, 15] . More generally, IFN-I are now known to play an essential role in the global orchestration of antiviral immunity, by linking innate and adaptive immunity through multiple immunoregulatory functions [16] . For instance, IFN-I do not only play a crucial role in the control of the replication of many viruses, but they can also promote NK cell or CD8 T cell antiviral cytotoxic activity, either directly [17] [18] [19] [20] or through the licensing of accessory cells such as conventional dendritic cells (cDCs) [13, [21] [22] [23] [24] . In mammals, there are one IFN-β and more than ten different IFN-α, the exact number of which varies across species. All of these IFN-I exert their activities by engaging an ubiquitous receptor (IFNAR) composed of two chains (IFNAR1 and IFNAR2) [25] . Although any cell type could theoretically produce IFN-I (at least IFN-β) when infected by a virus, one cell type specialized in the production of high levels of multiple species of IFN-I in response to herpesviruses and many other viruses has been identified and characterized in the last decade. It has been named IFN-I producing cells (IPCs) or plasmacytoid dendritic cells (pDCs). [5, 6, 7] No (blood pDCs) [6] Yes (tonsil pDCs) [7] Partial [8] Human [12] No ± [13] Yes ± [13] * pDCs have been described to be the main IFN-I source upon herpesvirus stimulation. ± including in vivo in mice $ maturation of pDCs following stimulation with herpesviruses in terms of CD40, CD80, CD86 and MHCII up-regulation ND: not determined In humans, pDCs are characterized by selective high level expression of the C-type lectin CLEC4C (also referred to as BDCA2) and of the immunoglobulin superfamily member LILRA4 (also referred to as ILT7). In mice, there are no orthologs of CLEC4C or LILRA4. Instead, mouse pDCs selectively express high levels of the C-type lectin SIGLECH and of the bone marrow stromal cell antigen 2 (BST2/CD317) membrane marker. SIGLECH is also found on a subset of marginal zone macrophages in the spleen. BST2 is induced at low to intermediate levels on a variety of immune cell types upon stimulation with IFN-I, including cDCs, and is also constitutively expressed on plasma cells. Therefore, mouse pDCs can be rigorously identified as CD11c int BST2 high or as CD11b -SIGLECH + . Although it has been frequently used, it is important to be aware that the combined expression of B220 and CD11c is not adequate to identify mouse pDCs, as B220 + CD11c + cells also encompass other leukocyte populations including a subset of NK cells [26] [27] [28] [29] and a precursor of cDCs [30] . While mouse and human pDCs are identified by different markers, they share many functional characteristics [31] and selective expression of over 200 genes as compared to other leukocyte subsets [28] . Thus, there clearly is a very strong homology between mouse and human pDCs, such that the investigation of the functions of pDCs in vivo in the mouse model should be largely relevant for understanding their biology in humans. The initial discovery and the functional study of pDCs have been closely linked to the study of innate immune recognition of herpesviruses. Indeed, together with the influenza virus [32-34], herpes simplex viruses were the very first viruses used to identify human [2] or mouse [35] pDCs in vitro, based on the much older observation that a rare cell type had the unique ability to rapidly recognize herpesviruses for consecutive high level production of IFN-I [36, 37] . We reported the first evidence that pDCs actually constitute the major source of systemic IFN-I production during a viral infection in vivo using MCMV as a model [12, 32] . To the best of our knowledge, pDCs have been shown able to recognize, and respond to, all of the herpesviruses tested (Table 1 ). These observations suggest that pDCs may be amongst the earliest sentinels for detection of, and defense against, herpesvirus infections. The present work aims at reviewing the current knowledge regarding the role of pDCs in the orchestration of the immune responses against herpesviruses, not only focusing on their beneficial role for the host but also raising the question of their possible implications in immunopathology under defined conditions, leading to the discussion of whether and how pDC responses to herpesviruses could be used in antiviral immunotherapy or vaccination.
In humans, pDCs were demonstrated to be the major source of IFN-I production within total peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation with HSV-1 [2] . Indeed, on a per cell basis, purified pDCs produced up to a thousand fold more IFN-I than cDCs or monocytes. Moreover, depletion of pDCs led to over a hundred fold decrease in PBMC production of IFN-I. Resting human pDCs do not contain mRNA for IFN-I. Upon HSV-1 activation, human pDCs rapidly express mRNA of all the IFN-I family members, whereas cDCs do not [38] . Under these conditions, IFN-I mRNA constitute up to 60% of the total pool of pDC neo-synthesized mRNA, attesting to the tremendous energy allocation that pDCs dedicate to the production of these antiviral cytokines upon proper stimulation. Human pDCs also produce high levels of IFN-I in vitro in response to other herpesviruses including HCMV [5] [6] [7] [8] and EBV [11] . In mice, pDCs were likewise demonstrated to be the major source of IFN-I production within total splenic leukocytes upon in vitro stimulation with HSV- 1 [35] or MCMV [39] . Among all the cells producing IFN-I in response to herpesviruses, pDCs are characterized by the fact that their IFN-I production does not require endogenous virus replication [2, 40] . Moreover, pDCs are not productively infected by MCMV [13, 41] or HCMV [6, 7] . However, the resistance of pDCs to herpesvirus infection is not absolute. It depends both on the virus and on the source of pDCs. HHV-6 or HHV-7 productively infect blood pDCs [9, 10] . Tonsil pDCs have been recently reported susceptible to HCMV infection in contrast to blood pDCs [7] .
In summary, pDCs are the main IFN-I producers among total circulating leukocytes stimulated in vitro with herpesviruses. In most instances, pDC IFN-I production does not require endogenous viral replication. Indeed, human blood and mouse spleen pDCs appear resistant to productive viral infection by several herpesviruses.
Using MCMV as a model, we were the first to report that pDCs are the major producers of IFN-I in vivo during a viral infection [12, 32] . pDCs isolated from the spleen of d1.5 MCMV-infected mice produced high levels of IFN-I ex vivo without any restimulation, while the cytokines could not be detected in the supernatant from cDCs isolated from the same animals [13] . A dramatic decrease in ex vivo IFN-I production was observed in splenocytes depleted of pDCs. Moreover, systemic IFN-I production was dramatically reduced in infected animals upon anti-GR1 [12, 32] or anti-BST2 [39] antibody-mediated pDC depletion. These results thus demonstrated that pDCs are the major producers of systemic IFN-I in vivo during MCMV infection, even though CD11b + cDCs that are infected by MCMV in vitro at a high multiplicity of infection can also produce significant levels of IFN-I [42]. This conclusion was further confirmed by several other complementary approaches. First, pDCs isolated from the spleen of MCMV-infected mice expressed much higher amounts of the mRNA for all the different IFN-α and IFN-β subtypes tested as compared to other splenocytes [43] (Figure 1) . Second, the vast majority of IFN-I expressing cells in vivo in the spleen of infected mice were demonstrated to be pDCs, using intracellular staining with specific antibodies on cell suspensions or tissue sections at 30-36 hours post-infection [43] or YFP-IFNb reporter knockin mice at 12-24 hours post-infection [44] . We showed that splenic pDCs were the major producers of IFN-I in three mouse strains: 129S2, BALB/c and C57BL/6 mice. In all these instances, IFN-I production by splenic pDCs took place relatively rapidly and was very transient since it was easily detectable around 36 hours but not at 24 hours or 44 hours post-infection [43] . Third, a deficient IFN-I production upon MCMV infection was observed in a knockin mouse strain (Ik L/L mice) which harbors a selective block in pDC differentiation at an immature stage in the bone marrow consecutive to a hypomorphic mutation of the IKAROS transcription factor [45] .
The intravenous infection of mice with another herpesvirus, HSV-2, also leads to a systemic IFN-I production that is abrogated upon anti-BST2 antibody-mediated pDC depletion [3] . Thus, no doubt remains that splenic pDCs are the major source of IFN-I during systemic infections of mice with several herpesviruses in vivo. Interestingly, an intra-vaginal challenge with HSV-2 also leads to a pDC-dependent IFN-I production, but only locally as the cytokines can be detected in the vaginal wash but not in the blood, consistent with the lack of dissemination of the infection in immunocompetent animals [46] .
Taken together these data highlight the primordial role of pDCs as major IFN-I producers in response to systemic or local herpesvirus infections in vivo. However, this phenomenon cannot be generalized to all viruses as pDCs are not the major producers of IFN-I with certain viruses such as lymphocytic choriomeningitis virus (LCMV) [12] or upon local infections of the airways such as with intranasal Newcastle disease virus (NDV) [47] or influenza [48, 49] challenges. During reovirus infections, cDCs contribute as well as pDCs to IFN-I production locally in the intestine [50] . It is noteworthy that we could not detect any IFN-I production by pDCs from the blood, lymph nodes, liver or lung of MCMV-infected mice, despite high levels of viral replication in some of these organs [43] . This is consistent with a similar observation reported by the group of Shizuo Akira in the intravenous NDV infection model [47] . This suggests that splenic pDCs are especially prone to high level IFN-I production upon systemic acute viral infections as opposed to pDCs located in some other organs such as the liver or the lung. The mechanisms for this differential responsiveness to viral stimuli of pDCs from distinct tissues are not clear, although a role of the local cytokine milieu has been proposed [51].
Cytokine and chemokine mRNA expression by pDCs and cDCs isolated from MCMV-infected animals at 36 hours post-challenge. pDCs (red circles), CD8α cDCs (blue diamonds) and CD11b cDCs (green triangles) were purified from C57BL/6 mice injected with vehicle (open symbols) or infected with MCMV for 36 h (closed symbols). Pangenomic microarray experiments were performed with the total mRNA isolated from these cell populations [43] . The expression of mRNA (Y-axis, in arbitrary units in log10 scale) encoding various innate cytokines and chemokines (X-axis) in the three DC subsets studied is represented. Genes were classified in 5 groups accordingly to their pattern of expression across the 6 types of biological samples examined, using the GeneCluster software. Group 1 corresponds to the IFN-I genes which are undetectable or expressed only at very low level under steady state conditions, but which are induced to extremely high levels specifically in pDCs after infection. Group 2 corresponds to genes that are induced in all DC subsets but to a higher level in pDCs. Group 3 correspond to genes that are strongly induced to a comparable extent in all three DC subsets. Group 4 corresponds to genes induced to higher levels in cDCs as compared to pDCs. Group 5 corresponds to genes induced to higher levels specifically in CD8α cDCs.
A crucial function of IFN-I is their ability to inhibit viral replication and dissemination [16] . Indeed, pDC depletion leads to enhanced local HSV-2 replication in mice infected intra-vaginally [46] and in the spleen or liver in animals infected intravenously [3] , correlating with the major role of pDCs for local versus systemic IFN-I production in these experimental settings. Thus, pDC-derived IFN-I directly contributes to the control of HSV-2 replication in vivo in mice upon systemic or local infections. However, the picture appears much more complex for MCMV infection. In 129Sv mice, pDC-derived IFN-I likely contribute to MCMV control as anti-GR1 [12] or anti-BST2 (data not shown) antibody-mediated pDC depletion leads to a higher splenic viral load. In C57BL/6 mice, different approaches used to selectively affect pDC functions have led to different results. In vivo anti-GR1 [12] or anti-BST2 [39] antibody-mediated pDC depletion has no significant impact on viral replication in the spleen. Because this treatment is less efficient than in 129 mice (data not shown), it is not possible to rule out that residual pDCs still contribute sufficient IFN-I production to allow maximal antiviral effects. This interpretation could be consistent with the observation that complete abrogation of pDC IFN-I production in C57BL/6 mice leads to a significant increase in MCMV replication or in viral-induced mortality, as reported in the Ik L/L mice which lack mature pDCs [45] or in interferonregulatory factor 7 (IRF7) -/mice which have lost pDC ability to produce IFN-I [52]. However, because pDCs are not the only cell type affected in these two mutant mouse models, the interpretation of their phenotype is difficult. An alternative explanation for the differential impact of pDC depletion on MCMV replication in 129Sv versus C57BL/6 mice could be the specific capacity of the latter to mount other innate immune responses redundant for this function. Indeed, contrary to 129Sv mice, C57BL/6 mice have efficient anti-MCMV natural killer (NK) cell responses which are able to recognize and kill infected cells in vivo very rapidly after infection at the same time as pDCs are activated for IFN-I production [53] . This antiviral function of NK cells depends on their activation by IFN-I [22] . Thus, in C57BL/6 mice but not in 129Sv animals, low levels of residual IFN-I may be sufficient to promote efficient viral control by NK cells, even in the absence of strong direct antiviral effects of IFN-I, and irrespective of the cellular source of these cytokines.
In humans, several case reports have been published describing patients suffering from severe herpesvirus infections associated with decreased numbers of circulating pDCs as compared to healthy controls and with an impaired IFN-I production ability as assessed by in vitro restimulation of their PBMCs with the virus [54-56]. EBV infection of humanized NOD-SCID mice was reported to lead to a decrease in the numbers of circulating pDCs and in the ability of PBMCs to produce IFN-I upon in vitro restimulation [11] . This raises the question of whether the pDC deficiency reported in certain patients with severe herpesvirus infections could be a consequence rather than a cause of the disease. In any case, interestingly, reconstitution of NOD-SCID mice with pDC-enriched human PBMCs delayed their mortality upon consecutive infection with EBV [11] . It has been recently shown that chronic infection of mice with LCMV causes a long-lasting impairment of pDC ability to produce IFN-I upon activation with viral type stimuli and enhances sensitivity to a secondary unrelated infection with MCMV [57]. Thus, it has been proposed that enhanced sensitivity to opportunistic viruses in patients with human immunodeficiency type 1 (HIV-1) infection could in part result from decreased pDC responsiveness [57, 58] . A recent study indeed shows that while IFN-I can be detected in the spleens of patients with chronic HIV-1 infection, pDCs appear to make only a very low contribution to this function [59] In this regard, it is striking that herpesviruses are amongst the most common opportunistic agents causing life-threatening disease in HIV-1-infected infants [60] or in AIDS patients [61,62] who have a dramatic reduction in pDC numbers and an impairment of pDC functions [63] in addition to the deregulation of many other immune functions. Reactivation of latent infections with herpesviruses, in particular HCMV, is also a major cause of morbidity and mortality in bone marrow or solid organ transplantation recipients [64] , who undergo immunosuppressive treatments known to profoundly affect pDC numbers or functions [65] [66] [67] .
In summary, manipulation of pDC numbers or functions in mice and epidemiological analyses in humans strongly suggest that high level production of IFN-I by pDCs contributes to the control of infections by herpesviruses. However, its importance could vary significantly between individuals depending on their capacity to concomitantly mount other immune responses redundant for this function. There is currently no experimental system available to the scientific community that allows the disruption of pDC functions in vivo in an entirely specific and completely efficient way. The genetic engineering of mouse models designed to fill this need should be seen as a priority in this research field, because this will be the only rigorous way to evaluate the role of pDCs in vivo in the defense against herpesviruses and more generally in the orchestration of the immune responses to viral infections. The recent demonstration that the conditional knockout of the transcription factor TCF4 (also referred to as E2-2) in adult mice leads to a complete and selective loss of pDCs constitutes a significant advance towards this goal [68] .
Mouse splenic pDCs isolated around 1.5 days after MCMV infection of different mouse strains do not only secrete high levels of IFN-I but also produce significant amounts of IL-12p70, TNF-α, and CCL3, and induce mRNA expression for a number of other cytokines and chemokines including IL-1β, IL-6 and LTα [12, 13, 43] (Figure 1 ). Conversely, however, other cytokines that can be concomitantly detected in the spleens of infected animals seem to be mostly expressed in other specific innate immune cell types, for example IFN-γ in NK cells [13] or IL-15 in cDCs ( Figure 1 ). Like their murine counterparts, human pDCs activated with HCMV or HSV-1 are also induced to produce and secrete many cytokines and chemokines including TNF-α, IL-6, CXCL10 and CCL3 [8, 69] . However, unlike their murine counterparts, human pDCs do not express IL-12 or only at extremely low levels [31, 38] . pDCs can also produce anti-inflammatory cytokines such as IL-10 in response to EBV stimulation [11] .
The soluble factors produced by pDCs can act in a paracrine manner to recruit other cell types to the site of infection and induce them to produce a second wave of cytokines or chemokines. During MCMV infection, by producing IFN-I and CCL3, pDCs likely contribute to the induction of such a cytokine/chemokine cascade which has been demonstrated to be critical for NK cell recruitment to the sites of viral infection allowing the local delivery of their immunoregulatory and antiviral effector functions [70] . In addition, pDC-derived IL-12 directly induces IFN-γ production by NK cells [13, 22] while pDC-derived IFN-I induces NK cells to proliferate [22] and to acquire cytolytic granules containing perforin and granzyme B, at least in part indirectly by instructing other cell types, most likely cDCs, to produce IL-15 and transpresent it to NK cells in association with the IL-15Rα chain [21, 22, 71, 72] . Upon stimulation with HSV-1, pDCs have been reported to produce IL-18 and thus to most efficiently contribute to induce NK cell IFN-γ production [73] . Human pDCs stimulated with HCMV induce NK cells migration and IFN-γ secretion, but not cytotoxicity [5] which could be consistent with the poor expression of IL-15 by pDCs ( Figure 1 ). More generally, stimulation of pDCs with herpesviruses [69] as well as with other types of viruses including hepatitis C virus [74] or influenza virus [75] induces them to produce a variety of cytokines/chemokines which recruit and activate other innate immune cell types including cDCs, monocytes and NK cells. Finally, pDCs do not contribute to the activation of other innate immune effectors solely by the production of soluble factors but also through cell-cell contacts as illustrated by the demonstration that the ligand for the glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL) is preferentially expressed on human pDCs upon HSV-1 stimulation and is critical to promote NK cell IFN-γ production and cytotoxicity [76] .
In summary, although pDCs were initially characterized as professional IFN-I-producing cells upon in vitro stimulation with viruses or during viral infections in vivo, they also bear a significant contribution to the production of many other soluble factors under the same experimental settings. Thus, beyond their direct contribution to antiviral innate activities through IFN-I production, pDCs are very likely to play a critical role in the initiation and global orchestration of the recruitment and activation of other innate immune cells (Scheme 1).
Modulation of innate and adaptive immune responses by herpesvirus-activated pDCs. pDCs are able to sense infection with herpesviruses through TLR9 and/or TLR7. The triggering of these receptors in specialized endosomes, where they are preassembled with the MyD88 adapter molecule and the IRF7 transcription factor in multimolecular complexes, leads to the phosphorylation of IRF7 and its translocation to the nucleus where it associates with other partners to constitute a transcription initiation complex (TIC) able to induce the expression of IFN-I and other target genes. Largely due to their production of innate cytokines or chemokines, pDCs can exert a variety of stimulatory or inhibitory functions on other innate or adaptive immune cell types. The global effect of pDC responses on the overall immune response and on the promotion of health versus disease depends on the combination and levels of the cytokines that they produce as discussed in Scheme 2.
Before pDCs were discovered, IFN-I production during viral infections was believed to mainly occur in infected cells through the activation of intra-cytoplasmic sensors for viral RNA or DNA molecules such as the RNA-dependent protein kinase (PKR) or the more recently discovered RNA helicases RIG-I and MDA-5 [77] . However, pDCs are now known to be the major producers of IFN-I upon stimulation with herpesviruses as well as some other types of viruses in vitro, and also during in vivo infections with these same viruses [3, 12, 43, 46, 78] , while pDCs are most often not productively infected [4, 6, 13, 41, 79] . This apparent paradox was solved by the demonstration that pDCs express a unique set of endosomal receptors and associated signaling components allowing the detection of RNA or DNA sequences derived from engulfed viruses or infected cells. Indeed, pDCs express Toll-like receptors (TLR) 7 and 9 [80] which are localized in a specific endosomal compartment where they can respectively recognize single stranded RNA versus unmethylated CpG DNA sequences and consecutively initiate a signaling cascade ultimately resulting in high level IFN-I production. RNA or DNA molecules are not present in endosomes under steady state conditions in healthy individuals [81] . Thus, pDCs have the unique ability to detect viral infections without the requirement of endogenous viral replication, because they must be able to take up oligonucleotides from viruses or infected cells [82] into specialized endosomes for TLR7 or 9 triggering. The membrane receptors that must confer pDCs the ability to selectively recognize and engulf viral particles or apoptotic debris from infected cells remain to be identified.
MyD88 is a signaling adapter molecule used by all TLRs except TLR3 as well as by the receptors for IL-1 and IL-18. The study of MyD88 -/mice has allowed to demonstrate a crucial role of this molecule for pDC IFN-I production in response to in vitro stimulations by virus type stimuli as well as in vivo during viral infections [80] . TLR9 appears to be the only MyD88-associated receptor necessary for IFN-I production during HSV-2 infection [83] , which is consistent with the known presence of unmethylated CpG DNA sequences in the genome of herpesviruses. In contrast, during HSV-1 or MCMV infections, IFN-I production shows only a partial dependence on TLR9 [39,40, 84, 85] . This could be in part explained by the fact that other cells than pDCs contribute to IFN-I production, in a manner that is independent of MyD88 [39, 84, 86] but which may rely on intracellular detection of viral replication by cytoplasmic sensors for viral DNA [87] or RNA (as documented for EBV [88] ). However, we have also recently shown a partial redundancy between TLR9 and TLR7 in the MyD88dependent pDC activation for IFN-I production during MCMV infection in vivo [89] . Indeed, in TLR9 -/mice, TLR7 can promote sufficient residual pDC IFN-I production to allow a better control of viral infection and an enhanced resistance to viral-induced lethality as compared to MyD88 -/or TLR7 -/-TLR9 -/animals. The ability of pDCs to respond to a DNA virus through TLR7 is a strong argument in favor of their ability to engulf apoptotic bodies from dying infected cells for delivery of viral or host mRNA to TLR7-containing endosomes. Of note, TLR7 has recently been reported to be responsible for the activation of IFN-I production by cDCs upon infections with phagosomal bacteria [90] . Thus, TLR7 functions are clearly not restricted to the recognition of, and responses to, RNA viruses, but also extend to defense against herpesviruses and phagosomal bacteria. Interestingly, a cross-regulation between TLR9 and TLR7 has recently been demonstrated in mouse cDCs, whereby TLR9 outcompetes TLR7 for association with the scaffolding molecule UNC93B1 which is required for the trafficking of all endosomal TLRs from the endoplasmic reticulum to the endosomes. Thus, in wildtype animals, TLR responses in cDCs are biased towards DNA-but against RNA-sensing [91] . It will be interesting to determine whether this mechanism is also at play in pDCs, which have a particularly strong expression of TLR7 and an exquisite sensitivity to its specific ligands.
The unique ability of pDCs to produce very high levels of all subtypes of IFN-I in response to herpesvirus infections cannot be solely explained by their expression of TLR9 and by their ability to engulf viral particles or infected cells. Indeed, these two properties are shared with cDCs, especially the CD8α subset in the mouse, which still does not produce significant levels of IFN-I under the same experimental conditions. Indeed, additional properties have been demonstrated to contribute to the exquisite ability of pDCs to produce high levels of IFN-I upon viral stimulations (Scheme 1).
IRF7 is the master transcription factor controling IFN-I production in response to viral type stimuli, as IRF7 -/mice show dramatic decreases in IFN-I production and enhanced mortality in response to challenges with different viruses, including MCMV [52] or HSV-1 [92] . Human and mouse pDCs constitutively express very high levels of IRF7 [80] . In contrast, in cDCs and other cell types, IRF7 expression is induced only secondarily to a first, IRF3-dependent, wave of IFN-β/α4 production [93] but is critical to drive the consecutive production of the other IFN-α suptypes in a positive feedback loop. Loss of IRF7 expression abolishes pDC responses to MCMV [52], HSV-1 and all the other viral stimuli that have been tested [92] . Thus, high constitutive expression of IRF7 strongly contributes to the unique ability of pDCs to produce high level of all IFN-I subtypes very rapidly upon viral stimulation. In addition, as compared to cDCs, pDCs are characterized by a long retention time of CpG desoxyoligonucleotides in dedicated, transferrin receptor + , endosomes where preformed multimolecular complexes exist which bridge together TLR7/9 and their downstream signaling machinery including MyD88 and IRF7 [94, 95] .
The lack or low level of replication of certain herpesviruses and other viruses in pDCs may seem surprising considering the ability of many of these viruses to productively infect cDCs. The mechanisms that protect pDCs from productive viral infection are not entirely understood. However, autocrine IFN-I responses certainly play an important role as we initially reported by showing over a ten-fold increased proportion of infected pDCs in IFNAR -/versus WT mice challenged with an EGFPexpressing MCMV [13] . In these experimental settings, the ability to respond to IFN-I did not only protect pDCs from viral infection but also further enhanced their ability to produce IFN-I by about four-fold [12] . However, a significant production of IFN-I by pDCs still occurred in IFNAR -/animals.
In mice infected with vesicular stomatitis virus (VSV), pDCs have been demonstrated to produce IFN-I in a feedback loop independent manner [96] . The impact of pDC autocrine IFN-I responses has also been studied recently in the context of NDV infection. A crucial role of this positive feedback loop was shown both for sustained IFN-I production by pDCs and for protecting them from productive viral infection [97] . Indeed, in IFNAR -/-pDCs, IFN-I production is poorly sustained and entirely depends on viral replication in the pDCs and the detection of cytoplasmic viral RNA through mitochondrial antiviral signaling protein (MAV)-dependent pathways. Autocrine IFN-I activity has also been shown to protect pDCs from infection by mouse hepatitis virus [79] . Human pDCs are protected from HCMV infection due both to autocrine IFN-I effects and to other yet unidentified mechanisms [6] .
The selection during evolution of a cell type specialized for IFN-I production in response to viral infection may seem paradoxical with the fact that, when productively infected, any cell type is theoretically capable of producing high levels of these cytokines, including cDCs [98] . However, the major differences between pDCs and other cell types in the molecular mechanisms in place for the sensing of the viral infections and for the downstream induction of IFN-I production are revealing. Indeed, in most instances, pDC IFN-I production does not require endogenous viral replication in the cytoplasm but only detection in endosomes of engulfed oligonucleotides derived from other infected cells or viral particles. In fact, pDCs appear resistant to productive viral infection by several herpesviruses. In addition, IFN-I production by pDCs is not strongly dependent on a positive feedback loop, because pDCs constitutively express IRF7 at very high levels. In contrast, IFN-I production by most other cell types requires detection of viral RNA or DNA in the cytoplasm, and hence endogenous productive viral replication. Under these conditions, viruses have evolved a variety of strategies to inhibit both IFN-I production and IFN-I responses in infected cells very early on after initiation of their replication [87, [99] [100] [101] . Therefore, in infected cells, viruses do not only interfere with the intensity of the first wave of IFN-β/α4 but also greatly compromise the amplification loop for secondary induction of the other subtypes of IFN-α. It should be noted that HCMV has recently been reported to suppress IFN-I secretion by pDCs through its interleukin 10 homolog [102] . Nevertheless, pDCs may have a non redundant role in the rapid systemic production of IFN-I and the consecutive induction of a global innate antiviral state in the host under defined conditions of infection with herpesviruses, due to their general ability to escape viral interference with these functions.
Systemic innate cytokine responses can play protective antiviral functions but can also lead to severe immunopathology when too high. Thus, the benefits of activation of pDC antiviral defenses must be balanced against the risks of immunopathology. In this respect, it is likely that mechanisms are in place to finely tune the activation of pDCs depending of the nature and intensity of the environmental threats that they can perceive. The existence of such mechanisms is supported by the observation that certain autoimmune diseases including systemic lupus erythematosus (SLE) or psoriasis have been found to be associated with unbridled IFN-I production by pDCs [103] [104] [105] .
Several studies have recently demonstrated the existence of an ITAM-dependent signaling pathway, similar to that downstream of the B cell receptor, which is triggered on human pDCs by engagement of FcεRIγ-associated membrane receptors of the C-type lectin (CLEC4C/BDCA2) or immunoglobulin (LILRA4/ILT7) super-families, and which dampens pDC cytokine responses to TLR or viral stimulations in vitro [106] [107] [108] . This regulatory pathway must be evolutionarily conserved, because some of its most specific components including BLNK and CARD11 are selectively expressed in mouse pDCs as well [28] . Indeed, the mouse SIGLECH C-type lectin receptor which associates with the ITAM-bearing adaptor DAP12 has also been shown to inhibit pDC cytokine responses to TLR stimulations [109, 110] . DAP12 functions dampen pDC cytokine production [110] , including in vivo in a cell autonomous way during CpG challenge or MCMV infection [111] . Thus, it is likely that the mouse SIGLECH receptor modulates pDC function through the triggering of the same B cell receptorlike intracellular signaling pathway as the human CLEC4C receptor, although these receptors are not orthologous. The natural ligands of human CLEC4C and mouse SIGLECH still remain to be identified. However, BST2 has been very recently identified in humans as a ligand for LILRA4. BST2 is a membrane molecule induced on many cell types by IFN-I [112] . Thus, human pDCs are uniquely equipped to sense the paracrine response to their production of IFN-I, allowing for a negative feedback loop to prevent excessive production of the cytokines that could cause deleterious effects to the host. Future studies will likely show that this is also the case in the mouse.
Another negative feedback effect of IFN-I has been described in pDCs that is shared with cDCs. It relies on the IFN-I-dependent induction on DCs of the TAM receptor tyrosine kinases Axl, Tyro3, or Mer. These molecules associate with the α-chain of the IFN-I receptor and activate the suppressors of cytokine signaling-1 (SOCS-1) and SOCS-3 upon engagement by their ligands Gas6 or ProS which can themselves be produced by DCs [113] . Other cell-intrinsic mechanisms exist that also contribute to dampen pDC responses to TLR triggering, such as the engagement of the immunoreceptor tyrosinebased inhibition motif (ITIM)-bearing membrane receptor DCIR in human pDCs [114] . Interestingly, engagement of DCIR on pDCs increases their capacity to present antigen to T lymphocytes. Targeted delivery of vaccine antigens to mouse pDCs via coupling to an anti-SIGLECH antibody also promotes the induction of adaptive immune responses [115] . Thus, the mechanisms that terminate the production of innate cytokines by pDCs during responses to viral type stimuli may not only be in place to prevent the development of a cytokine shock but also to switch pDC functions towards direct activation of adaptive immunity once their initial role in the orchestration of innate immune defenses has been fulfilled.
In vivo during MCMV infection, pDC activation for innate cytokine production increases with the doses of viral inoculum used for the infection of a given mouse strain [116] . pDC cytokine responses to MCMV infection also increase dramatically in mice unable of efficient innate control of viral replication early after challenge, as a consequence of a selective impairment in antiviral NK cell activity, due either to antibody-dependent depletion of these cells or to the absence of the gene encoding the NK cell activation receptor specific for infected cells [116] . Reciprocally, early treatment of infected mice with an antiviral drug leads to a dramatic decrease of pDC cytokine production. Therefore, although innate cytokine production during MCMV infection is independent of endogenous viral replication in pDCs, it is tuned accordingly to the global level of viral replication in the host. Thus, rather than being the primary mechanism of defense against viral infections, high systemic production of IFN-I and other innate cytokines by spleen pDCs may represent a fail-safe mechanism. It may be turned on to maximal levels, at the risk of immunopathology, only in the case of high systemic viral replication associated to high levels of circulating ligands for TLR7 or TLR9, when other innate immune mechanisms have failed to efficiently control the virus. This hypothesis is supported by studies of airway infections of mice with viruses that do not belong to herpesviridae. For example, intranasal challenge with NDV leads to pDC production of IFN-I (in the spleen, specifically) only when the first line of defense made by alveolar macrophages is experimentally disrupted and the infection has spread systemically [47] .
In summary, pDCs are equipped with a unique set of sensors and associated signaling pathways to rapidly sense a variety of viruses, in particular herpesviruses, and to respond by high level production of IFN-I and other innate cytokines or chemokines, while protecting themselves from viral infection. In this respect, pDCs appear to play an important, non redundant role in vivo in innate immune defense against herpesviruses and other viruses such as mouse hepatitis virus [78] . However, pDC activation during viral infections appears to be tightly controlled in intensity, space, and time, by a variety of mechanisms, which may be in place to tune the risk of cytokine shock to the level of threat posed to a specific host by a given viral infection.
pDCs are not only professional producers of IFN-I and key players in innate immune defenses against viral infections. They have also demonstrated ability to contribute to the induction and regulation of adaptive immunity. Depending on the maturation signals they receive as well as on the nature and concentration of the antigen, pDCs can have contrasting effects on adaptive immunity (Scheme 1). They can promote anti-viral cellular adaptive immunity either directly through crosspresentation of exogenous antigens to CD8 T cells or indirectly through the promotion of cDC maturation. On the contrary, under different conditions, they can induce immune tolerance.
cDCs have been long known to be specialized in antigen processing and presentation to naïve T cells leading to the induction of specific adaptive cellular immune responses. By contrast, until recently, pDCs had often been reported to be poor activators of naïve T cells, owing to their purported lower efficiency to take up, process and present exogenous antigens (reviewed in [117] ). However, detailed and kinetic analyses of the expression of MHC class I and II in DC subsets upon activation have shown contrasting responses of pDCs as compared to cDCs, suggesting that these cell types function in a distinct and complementary fashion for the induction and maintenance of adaptive cellular immune responses.
Rapidly after antigen capture and receipt of maturation signals, cDCs very transiently upregulate and then drastically downmodulate antigen up-take as well as MHC class II neosynthesis and ubiquitination. This allows efficient, selective, and long-term presentation of the antigens that cDCs have encountered at the same time of receipt of the maturation signals, while the sampling and processing of the antigens encountered after maturation is shut off. Conversely, pDCs maintain MHC class II synthesis and ubiquitination after antigen capture, allowing continous sampling of the antigenic environment for processing and presentation to CD4 T cells [118, 119] . Under conditions of systemic immune activation as occurs during infections with blood-borne pathogens, this specialization may allow pDCs to present to CD4 T cells some microbial antigens synthesized late after the initial encounter with the infectious agent, much more efficiently than cDCs. In the case of herpesviruses which are opportunistic pathogens, one could expect that cDCs are already activated by the primary infectious agents and are then impaired in their ability to respond to the secondary infection by the herpesviruses, whereas pDCs may still be efficient.
Recently, both mouse and human pDCs have been shown able to cross-present exogenous antigens to efficiently activate naïve or memory CD8 T cells ( [120, 121] and reviewed in [117] ). Moreover, human pDCs have been demonstrated to harbor a unique, specialized endocytic compartment containing premade stores of MHC class I which allow rapid processing of exogenous antigens for cross-presentation to CD8 T cells [122] . Thus, it is quite possible that pDCs play a major role in the direct induction and maintenance of antiviral CD8 T cell responses during viral infections. However, very few studies have directly addressed the role of pDCs for T cell activation during infections with herpesviruses.
pDCs isolated from MCMV-infected animals up-regulate CD80, CD86, CD40 as well as MHC molecules and efficiently prime naive CD8 T cells in vitro for proliferation and IFN-γ production, when pulsed with exogenous antigens [13] . However, neither the ability of pDCs to naturally process and present MCMV antigens, nor the role of pDCs in the global shaping of antiviral CD8 T cells responses, have been addressed during the course of MCMV infection. During MCMV infection, pDC IFN-I production was required to promote cDC maturation and thus likely helped the induction of antiviral adaptive cellular immune response [13] . pDCs exposed in vitro to HSV-2 induced T cell proliferation, more strongly for CD8 T lymphocytes [4] . pDCs isolated from the draining lymph nodes of HSV-1-infected mice had only a poor ability to induce IFN-γ production by HSV-specific CD4 and CD8 T cells as compared to cDCs [123, 124] , suggesting that pDCs did not have a major role in the direct priming of T lymphocytes during HSV-1 infection. However, in these experimental settings, pDCs promoted optimal functions of cDCs via cell-to-cell contacts involving CD2 and CD40L, which contributed to enhance HSV-1-specific CD8 T cell responses [123] . Finally, in vitro stimulation of pDCs with HCMV or influenza promoted B cell activation and their differentiation into specific antibody-secreting plasma cells, through IFN-I-and IL-6-dependent effects [8, 125] .
In summary, in response to viral stimulations in vitro, or in vivo upon stimulation with synthetic TLR ligands and targeted delivery of antigens through antibody coupling, pDCs have been demonstrated to have a strong ability to cross-present exogenous antigens for the activation of naïve CD8 T cells. In addition, pDCs also play a key role in linking innate and adaptive immunity through their production of immunoregulatory cytokines or chemokines which can promote the recruitment and activation of cDCs and lymphocytes. However, the importance of these functions in vivo for the control of infections with herpesviruses still remains to be established.
To promote health over disease, antimicrobial immunity must be tightly regulated to allow the mounting of effector responses of sufficient strength and adequate quality for the control of the invading pathogen, but to prevent the development of immunopathology as could result from exacerbated inflammation. This delicate balance can be achieved in part through negative feedback regulatory loops acting at the level of innate immune responses as discussed earlier for pDC activation. Immunoregulatory mechanisms are also in place to finely tune adaptive immune responses, and pDCs have been demonstrated to be able to contribute to this function. In response to EBV stimulation, pDCs can produce anti-inflammatory cytokines such as IL-10 [11] . HSV-1-stimulated pDCs can downmodulate CD4 T cell activation directly through the production of IFN-I and IL-10, and indirectly through the induction of regulatory CD4 T cells, suggesting that pDCs may contribute to prevent excessive, detrimental, adaptive immune responses during viral infections [126] . The coculture of pDCs, but not cDCs, and CD4 T cells in the presence of HCMV leads to the induction of IL-10 producing T cells [127] . pDCs have also been shown able to express the enzyme indoleamine 2,3dioxygenase 1 (IDO), which can induce T cell tolerance through tryptophan catabolism and downstream production of pro-apoptotic metabolites [128, 129] . A role for pDC expression of IDO has been demonstrated in vivo in mice for the generation of an immunosuppressive environment in tumors [130] . In humans, the expression of IDO by pDCs has been proposed to contribute the immune deregulation during HIV-1 infection [131, 132] . However, to the best of our knowledge, there is currently no evidence that IDO can be expressed by pDCs in the course of infections by herpesviruses and that it can play a role in setting the balance between antiviral defense and immunopathology. The addition of an IDO inhibitor in cocultures of HSV-1-stimulated pDCs and CD4 T cells did not prevent the induction of regulatory T cells [126] .
To conclude, these data showed that, in response to certain herpesviruses, pDCs can secrete antiinflammatory cytokines and induce or activate regulatory T cells. However, these pDC functions have not yet been examined in vivo during herpesvirus infections. Regulatory T cells induced by pDCs could favor the development of highly specific adaptive immune responses targeted to the invading virus, by preventing bystander activation of T cells directed against other antigens, thereby contributing to promote antiviral defenses and to prevent immunopathology [133] . Interestingly, during intra-vaginal infections of mice with HSV-2, by controlling chemokine/cytokine production and overall immune activation in secondary lymphoid organs, regulatory T cells promote the trafficking of innate and adaptive antiviral effectors out of the lymph nodes to the site of infection for local control of viral replication [134] . Thus, taken together, these data suggest that the induction of regulatory T cells by pDCs may contribute to focus innate and adaptive antiviral immune responses to the appropriate antigens and anatomical sites to promote health over disease.
As discussed above, pDCs are potent antiviral cells which can produce high levels of a number of cytokines or chemokines and strongly stimulate both the innate and adaptive immune systems. Although pDC functions are regulated by a number of activating or inhibitory mechanisms, situations have been described where pDC responses are deleterious for the host. The role of pDCs in the development of autoimmune diseases, including SLE or psoriasis, have been discussed in detail elsewhere [135] . Recent data have also suggested a possible deleterious role of pDCs in the context of certain viral infections. This should not be a surprise considering that IFN-I have long been known to be a double-edged sword able to promote either health or disease depending on the physiopathological context (Table 2) .
For the infections with HIV-1 or simian immunodeficiency virus (SIV), there has recently been a striking paradigm shift regarding the role of pDCs in the natural history of the disease. HIV-infected individuals show decreased number of pDCs and their PBMCs are impaired for IFN-I production upon in vitro restimulation. Therefore, it was first hypothesized that a lack of pDC responses contributed to the failure to control viral replication early on after primary infection [63] . However, recently, evidences have been obtained of a massive IFN-I response during HIV-1 infection, starting early on during acute infection [136] and sustained thereafter [137, 138] . This chronic production of IFN-I has been proposed to contribute to CD8 T cell disarming and uninfected CD4 T cell killing [139] . The cellular source of the IFN-I produced during chronic infection is debated [59], but pDCs could contribute [140, 141] . More generally, pDCs have been reported to be hyperactivated during HIV infection and to likely contribute to immune deregulation and disease by a number of mechanisms [139, 141] . Strikingly, comparisons between pDC responses of non human primate models with contrasting susceptibilities to SIV-induced disease have demonstrated much higher pDC activation in the susceptible rhesus macaques as compared to the resistant African green monkeys [142] or sooty mangabeys [138] , associated with a polymorphism in the Irf7 gene between rhesus macaques and sooty mangabeys [138] . In summary, recent investigations strongly suggest that pDCs play a deleterious role for the host during infections with HIV-1 or SIV. This possibility has not yet been extensively examined during herpesvirus infections, but will be worth considering under specific conditions based on the data discussed below. Direct antiviral effects [15] Development of autoimmunity or immunopathology [15, 143] Promotion of cDC maturation [13, 23] Induction of DC apoptosis [144] promotion of cDC cross-presentation [24] Prevention of DC renewal [145] promotion of NK cell activation [19, 22] General inhibition of hematopoiesis [146, 147] Help to CD8 T lymphocytes [17, 18, 20] Anti-proliferative or pro-apoptotic effects on CD8 T cells [15, 148, 149] Help to B lymphocytes [150, 151] Susceptibility to bacterial surinfections [152, 153] 
Recently, the induction of IFN-β by TLR9 triggering on HCMV-infected fibroblasts has been reported to promote their survival and to increase their production of viral particles [154] . However, this proviral effect occurred only when fibroblasts were simulated with CpG shortly after infection. In contrast, a treatment concomitant to viral challenge almost completely abrogated fibroblast infection and virus production. Thus, it is unlikely that this mechanism plays a significant proviral role to the disadvantage of the host during infections with most herpesviruses in vivo, as systemic production of IFN-I by pDCs should lead to the induction of antiviral defenses in most cells before they encounter the virus. However, the possibility remains that this mechanism could have been exploited by herpesviruses which can productively infect pDCs such as HHV-6 and HHV-7 [9, 10] . Some data suggest that pDCs could participate to the local replication and to the dissemination of HHV-6 and HHV-7, as skin lesions induced by the infections with these viruses have been reported to be infiltrated by infected pDCs [10] , and infected pDCs can transmit the virus to stimulated T cells [9] .
As mentioned earlier, IFN-I can promote either health or disease depending on the physiopathological context (Table 2) . Therefore, high level production of IFN-I by pDCs such as occurs during certain herpesvirus infections could contribute to immunosuppression or even lead to immunopathology. Indeed, we have shown that high levels of pDC innate cytokine production during MCMV infection are associated with an ablation of cDCs and a significant delay in the induction of antiviral CD8 T cell responses for up to 48 hours [116] . This transient immunosuppression can be recapitulated by exogenous administration of IFN-I in mouse strains that mount low pDC responses to MCMV infection, pointing to a crucial role of pDC-derived IFN-I in this phenomenon [116] . This impact of pDC functions on the kinetics of induction of antiviral adaptive immunity is striking given the recent observation that better outcomes of early primary viral infection with LCMV in mice or SIV in macaques correlate with increased ratio between antigen-specific CD8 T cells and infected cells in situ very early after challenge [155] . TNF-α has been shown to contribute to the disease induced by MCMV infection, as it contributes to liver pathology independently of NK and T cell responses [156] . pDCs could also contribute to these effects since they are the major source of TNF-α early after infection in different mouse strains [43].
Because chronic IFN-I or TNF-α responses often occur in a variety of autoimmune diseases [157] , it will be worth examining whether pDC activation by herpesvirus infections may contribute to the development of autoimmunity. As mentioned earlier, a deleterious role of pDCs has been proposed for SLE or psoriasis [103, 104] . Although the etiology of these diseases is not entirely understood, it is possible that infections with certain herpesviruses could initiate the pathology in some patients. The possible implication of EBV infection in several autoimmune diseases including SLE is indeed debated [158, 159] .
Studies in mice have demonstrated a TLR9-and MyD88-dependent protective role of local CpG administration following intravaginal infection with HSV-2 [160] . pDCs were recruited in the vaginal mucosa 24 hours after CpG treatment. Anti-BST2 antibody-mediated pDC depletion in vivo abrogated the protection. In humans with genital infection by HSV, topical application on the affected regions of a cream formulation of a TLR7 agonist, imiquimod, allows remission of the lesions [161] , likely through the local recruitment and activation of pDCs for IFN-I production. Importantly, this treatment has proven efficient even in the case of recurrent lesions caused by viruses resistant to the classical antiviral drug acyclovir [161] . This was the case for an AIDS-patient whose pDCs were unable to respond to HSV-1 upon in vitro restimulation but produced high levels of IFN-I upon exposure to imiquimod [54] . Thus, boosting pDC responses on genital lesions caused by herpesviruses, through the local administration of TLR7 or 9 agonists, can lead to efficient control of the virus and cure of the disease through activation of antiviral innate immunity, in complement or alternatively to the use of classical antiviral drugs directly inhibiting the viral DNA polymerase (Scheme 2).
In mice, boosting pDC responses has also been shown to enhance resistance to systemic infection with MCMV or HSV-1. This has been achieved either through the systemic administration of the Thymosin-α1 peptide [41] or of FLT3L [162] . In both cases, enhanced protection against the viral infection resulted from increased IFN-I or IL-12 production. For the MCMV infection, this was shown to depend on the activation of the TLR9/MyD88/IRF7 pathway in pDCs and to lead to a higher NK cell activation. Since a deficiency in pDC responses has been proposed to be implicated in the susceptibility of bone marrow or solid organ transplant recipients to HCMV reactivation or primary infection [65, 67] , therapeutic strategies aimed at restoring normal pDC functions in this physiopathological context may bring clinical benefits (Scheme 2). However, as compared to the local activation of pDCs in skin or mucosa by topical application of TLR agonists, systemic activation of pDCs may bear increased risks of detrimental side effects. Thus, further studies will be required to determine the best strategy to harness pDCs for the promotion of efficient systemic innate antiviral defenses without triggering excessive inflammation and consecutive immunopathology. Scheme 2. Impact of the fine tuning of pDC activation on the promotion of health versus disease. During herpesvirus infections, disease can result either from immune failure to control viral replication early or from the development of immunopathology. Weak pDC activation could contribute to disease in the former case, and excessive pDC activation in the latter case. Immunopathology can cause immune-mediated damage to vital organs and/or compromise the ability of adaptive immunity to control viral replication later. Thus, a significant but controlled pDC activation is required to promote health over disease, by allowing early control of viral replication while not causing significant immunopathology. Therapeutic protocols aimed at boosting or dampening pDC responses could thus help to reach this balance and to fight disease under defined clinical conditions, as discussed in the body of this review.
As mentioned above, excessive pDC activation during infections with HIV-1/SIV or with MCMV can lead to an overwhelming production of IFN-I and other innate cytokines. This in turn can lead to immune deregulation, including delaying or disarming of adaptive immunity, or to the generation of a local inflammation which contributes to support viral replication and dissemination [163] . In addition, pDCs have been involved in autoimmune diseases for which certain herpesvirus infections have been suggested to be potential triggers [158, 159] . Thus, the dampening of pDC responses could be a reasonable component of immunotherapeutic strategies aimed at decreasing the immunopathology in these different conditions (Scheme 2). Interestingly, in a model of genital infection of macaques with SIV, the dampening of the pDC response locally in the vagina resulted in reduced local inflammation, including a decreased CD4 T cell infiltration, and led to a significant protection of the animals against repeated high dose challenge with SIV [163] . These effects were achieved by local application of glycerol monolaurate which dampened the activation of endocervical epithelial cells for CCL20 production and therefore prevented the downstream CCR6-dependent recruitment of pDCs in the vagina. Dampening systemic pDC responses in vivo could theoretically be achieved by a number of ways, including treatments with antagonist ligands of the TLR receptors [164] , or the administration of agonistic antibodies to the LILRA4 or CLEC4C receptors which are selectively expressed in pDCs and downmodulate their production of innate cytokines in response to TLR triggering [106] [107] [108] . However, it must be stressed that pDC responses should not be completely switched off, because this may lead to heightened viral replication, as recently demonstrated for inflammatory DCs in the case of a mouse model of intranasal influenza infection. Indeed, in this model, either excessive or absent inflammatory DC responses turned out to be deleterious for the host, due respectively to immunopathology versus loss of control over viral replication. In contrast, dampening of inflammatory DC responses through drug treatment allowed enhanced resistance to disease [165] . Thus, therapies aimed at dampening pDC responses to limit immunopathology in the course of viral infections should be combined with other treatments aimed at controling viral replication, such as antiviral drugs or immunotherapeutic restoration of the cytotoxic antiviral activity of NK cells or CD8 T lymphocytes.
Herpesviruses are well controlled by immunocompetent hosts, since most humans are infected by HCMV, EBV, or HSV, and yet do not develop any clinical symptoms. All three of these viruses appear particularly efficient at activating pDCs. Therefore, it seems reasonable to consider that pDCs may contribute to the induction of the strong, protective, adaptive immune responses observed to occur naturally in most infected individuals. Interestingly, the ability to trigger a variety of TLRs and to activate multiple DC subsets, including pDCs, is part of the explanation that has been proposed for the high efficacy of the attenuated yellow fever virus vaccine [166] . In the case of herpesviruses, other factors are also likely to contribute to the induction of long-lasting protective adaptive immunity. In particular, this is the case of the inflation over time of the memory CD8 T cells directed against the products of immediate early genes [167] . This inflation occurs due to the natural boosts provided by expression of these antigens during partial reactivation of virus from latently infected cells over the lifetime of the infected individuals [168] . Therefore, it would be worth considering the exploitation of herpesviruses as potent vectors for vaccination against other intracellular pathogens or tumors. Indeed, this has been already done rather successfully in mouse models of vaccination against influenza or LCMV, where the target antigens had been introduced under the control of the promoter of the gene encoding the immediate early antigen-2 [169] as well as very recently in rhesus macaques for vaccination against SIV although expression of the vaccine antigens had been driven by the promoter of a structural gene which should not induce CD8 T cell memory inflation [170] . The role of pDCs in the success of these vaccination protocols would be interesting to evaluate.
The various studies on pDCs in the context of infections by herpesviruses show a strong involvement of these cells in the antiviral immune response. Corresponding key issues are summarized in Table 3 . Both in vitro and in vivo, pDCs are the major source of IFN-I during stimulations or infections by herpesviruses. IFN-I responses have been shown to play a critical role in the control of these viral infections, in particular in the case of MCMV or HSV2. pDCs have a unique ability to rapidly produce high and systemic levels of IFN-I, without the requirement for endogenous viral replication, upon engulfment of viral products. This can be explained by the constitutive expression in pDCs of a specific set of viral sensors, including TLR7 and TLR9, and of downstream signaling molecules including MyD88 and IRF7, arranged as preassembled multimolecular complexes in special endosomes. Although the expression of some of these molecules can be shared with other immune cell subsets, this is not the case of the whole set altogether as a preassembled endosomal multimolecular complex. In addition, pDCs are also able to produce multiple other cytokines or chemokines upon activation by herpesviruses. Thus, pDCs are likely to play a non redundant role in the induction and regulation of innate immune responses to herpesviruses. It should be noted that humans with genetic deficiencies in signaling components known to be critical for pDC IFN-I production in response to viral stimulation do not appear to suffer from life threatening viral infections, except for herpes simplex encephalitis [171, 172] . This suggests that constitutive lack of pDC-derived IFN-I does not compromise the ultimate control of herpesvirus infections in humans in the absence of generalized immunodeficiency and in the context of the advanced health care system of developed countries. However, very recently, the sequencing and analysis of the ten human TLRs in over 150 individuals from various ethnicity demonstrated that the endosomal TLRs have evolved under strong purifying selection, including TLR7 and TLR9 which are selectively expressed to high levels in pDCs in humans. This suggests an essential non-redundant role of the endosomal TLRs in host survival "either via protective immunity against viral infections (present or past), or because of their additional involvement in other non immunity related processes of major biological relevance, or both" [173] . In any case, the possibility to harness pDC functions in the clinic to help treat infections with herpesviruses in immunocompromised individuals is promising. Topical treatments of genital lesions due to recurrent herpesvirus infections have indeed already given encouraging results. The role of pDCs in the regulation of adaptive immune responses to herpesvirus infections is less well understood. It is clear that pDCs can promote antiviral CD8 T cell responses through cDC instruction by soluble factors as well as cell-to-cell contacts. Whether pDCs bear a significant contribution to the direct priming of antiviral T lymphocytes in vivo during viral infections, as compared to cDCs, remains an open question on which future work will likely focus in the coming years. Moreover, pDCs are also able of inducing immunosuppression or tolerance under specific experimental conditions, which has not been yet studied in the context of infections with herpesviruses in vivo.
While attention has been mainly drawn on the goods of pDC responses to herpesviruses, one should not ignore the possibility that pDCs may also contribute to disease for given herpesviruses in specific physiopathological contexts. From this point of view, it is revealing that for infections with HIV-1 or SIV, a paradigm shift recently occurred regarding the role of pDCs in the disease. In contrast to the initial hypothesis that pDC responses should be protective, the current idea is that pDCs play an important deleterious role in the development of the disease. In the mouse model of MCMV infection, pDC hyperactivation can contribute to induce a transient suppression of antiviral adaptive immunity likely due in part to deleterious effects of high systemic levels of IFN-I on cDCs or CD8 T cells. pDCs have also been shown to be involved in a variety of autoimmune diseases for which infections with certain herpesviruses may constitute possible triggering agents. Under these physiopathological settings, therapeutic strategies aimed at dampening pDC activation may yield a clinical benefit.
Significant progress has been made in the last couple of years on the deciphering of the innate functions of pDCs and their regulation during viral infections in vivo. However, further studies are required to improve our knowledge of the overall role of pDCs in the physiopathology of the infections with herpesviruses as well as other viruses, and to deepen our understanding of how the positive and negative mechanisms which regulate pDC functions integrate in time and space to promote health over disease. To this aim, the generation of novel mouse models specifically devoid of pDCs, or selectively affected in their functions, will be instrumental. In addition, the comparison of the outcome of herpesvirus infections and of the antiviral immune responses between non human primate models naturally differing in their pDC responses to TLR stimulations, such as macaques and sooty mangabeys [138] , would be enlightening.  Herpesviruses can interfere with the induction of, or the responses to, IFN-I to escape immunity.
 pDCs are the main IFN-I producers in response to many viruses including all the herpesviruses tested.
 pDCs are able to sense herpesvirus infections through the TLR7/9 receptors in a MyD88 dependant manner.
 pDCs produce a large panel of cytokines/chemokines and thus must play a major role in the orchestration of early inflammation and downstream activation of innate and adaptive immune effectors.
 Mature pDCs can cross-present viral antigens for cognate CD8 T cell activation.  Communicable Diseases Prioritized for Surveillance and Epidemiological Research: Results of a Standardized Prioritization Procedure in Germany, 2011 INTRODUCTION: To establish strategic priorities for the German national public health institute (RKI) and guide the institute's mid-term strategic decisions, we prioritized infectious pathogens in accordance with their importance for national surveillance and epidemiological research. METHODS: We used the Delphi process with internal (RKI) and external experts and a metric-consensus approach to score pathogens according to ten three-tiered criteria. Additional experts were invited to weight each criterion, leading to the calculation of a median weight by which each score was multiplied. We ranked the pathogens according to the total weighted score and divided them into four priority groups. RESULTS: 127 pathogens were scored. Eighty-six experts participated in the weighting; “Case fatality rate” was rated as the most important criterion. Twenty-six pathogens were ranked in the highest priority group; among those were pathogens with internationally recognised importance (e.g., Human Immunodeficiency Virus, Mycobacterium tuberculosis, Influenza virus, Hepatitis C virus, Neisseria meningitides), pathogens frequently causing large outbreaks (e.g., Campylobacter spp.), and nosocomial pathogens associated with antimicrobial resistance. Other pathogens in the highest priority group included Helicobacter pylori, Respiratory Syncytial Virus, Varicella zoster virus and Hantavirus. DISCUSSION: While several pathogens from the highest priority group already have a high profile in national and international health policy documents, high scores for other pathogens (e.g., Helicobacter pylori, Respiratory syncytial virus or Hantavirus) indicate a possible under-recognised importance within the current German public health framework. A process to strengthen respective surveillance systems and research has been started. The prioritization methodology has worked well; its modular structure makes it potentially useful for other settings. The large number of infectious agents with different pathogenspecific, host-specific and socio-economic characteristics makes the allocation of the limited resources available within the area of prevention and control of communicable diseases both challenging and controversial. The amount of attention, efforts and funds spent on surveillance, control and research of infectious pathogens varies greatly between pathogens, settings and over time. This distribution often appears to be ambiguous, potentially guided by senior leaders' research interests, short-term political agenda or residuals of historic situations [1] . As many pathogens are potentially harmful for humans and may present serious public health threats, it is necessary to prioritize the resources dedicated for surveillance and epidemiological research of infectious diseases. This needs to be done sensibly and rationally bearing in mind different aspects of pathogens' characteristics, their impact on societies and long-term consequences of their presence or introduction into populations.
The rational and transparent setting of priorities for investment into health research is therefore becoming an essential part of research planning [2] [3] [4] . Usefulness of prioritization, irrespective of its methodology, has been demonstrated by several research groups [5] [6] [7] [8] [9] [10] [11] . Prioritization can provide directions for future resource allocation and strategic planning at different levels (institutional, regional, national or international) and act as a platform for inter-disciplinary debate involving decision-makers, researchers, clinicians and the general public [8, 12] .
Although today there are a number of published tools to guide the process of setting priorities, only a few publications openly describe the methodology in sufficient detail and transparency to allow reproducibility or adaptation in other settings [2, 3, 8, 13] . Furthermore, very little is published in terms of actual prioritization results.
The Department for Infectious Disease Epidemiology of the Robert Koch Institute (RKI), German national public health institute, is in charge of national surveillance, prevention, control strategies and epidemiological research in the field of infectious diseases. Together with external senior experts the Department initiated a prioritization process aiming to (1) develop a rational system for setting priorities in the area of infectious diseases using a metric-consensus approach, and (2) rank most common pathogens in accordance with their importance for national surveillance and epidemiological research to guide future work of the RKI.
In the absence of established standards we designed a methodology using elements of our previous work in 2004 [14, 15] and experiences of other groups [2, 3, 10, 11, 13] . Our multi-staged prioritization process included compilation of the list of pathogens to be prioritized, development of evaluation criteria, weighting of criteria and scoring of the pathogens. The methodology was based on the core domain for good practices in setting priorities for research in health, such as legitimacy and fairness [3, 8] , and represents the further development of the work from 2004.
The core team (YB, AG, GK) contacted relevant leading national public health institutions with the request to name a representative to take part in the Delphi consensus process that aimed at assessing the feasibility of the methodology and the relevance of the suggested criteria, as well as to discuss possible improvements and pathogens' scores. Ten external senior experts (BG, UG, JH, TJ, MK, MKr, TL, MP, NS, UU) were nominated. They represented the National Committee of Infectious Disease Epidemiology, the German Society of Hygiene and Microbiology, the German Society of Infectious Disease Specialists, the German Society of Epidemiologists, the National Reference Laboratories, the German Federal Ministry of Health, the RKI Scientific Committee, the German Federal Chamber of Physicians and the German Medical Association. Ten internal experts (GK, AG, YB, SB, RB, TE, OH, KS, OW, MM) represented units and departments of the RKI working in the field of infectious diseases. The participants shared expertise in bacteriology, virology, mycology, parasitology, general infectious diseases, tropical medicine, general medicine, epidemiology, public health, veterinary health and infection control.
To maintain a broader approach, we decided to evaluate pathogens rather than diseases for their importance. A list of pathogens was compiled according to the following selection criteria: (a) notifiable according to the German law for the control of infectious diseases [16] , (b) reportable within the European Union [17] , (c) reportable to the WHO under the International Health Regulations [18] , (d) agents with potential for deliberate release [19] , and (e) pathogens represented in dedicated chapters in an established infectious diseases manual [20] and occurring in Germany. Some pathogens were grouped together when biologically and clinically plausible. The list of pathogens was reviewed by the RKI experts and the Delphi process participants.
The twelve criteria used during our previous prioritization process in 2004 [14, 15] were further modified according to the feedback received from a broad group of different experts [21] . The newly suggested criteria and their three-tiered definitions were then reviewed by internal and external experts. The initial scoring of all pathogens according to each criterion was performed by the core team and internal experts from the RKI. The data supporting the scoring decisions and references to the data sources, or experts' own explanations were recorded in a structured format. The internal scoring was followed by a modified two-round Delphi consensus process (internal round with RKI and joint round with additional external experts) where scores were discussed.
Independent of the scoring, we invited a panel of external experts to assign a weight to each criterion. This invitation was sent to all 16 federal public health institutions, all 18 national reference centers, 49 consulting laboratories, 9 scientific and professional societies working in the area of control, prevention and research of infectious diseases and to all 72 participants of the 2006 online survey [21] . External experts were asked to assign a value ranging from 0 to 10 to each criterion, thus reflecting the criterion's contextual importance for surveillance and epidemiological research. The value 0 reflects the lowest and 10 the highest level of importance of a criterion. More than one criterion could be assigned the same weight, similarly to techniques used in other prioritization exercises [5] . The final criterion's weight was defined by the median value of all weights assigned by the participating experts.
Each score was multiplied by the weight for the respective criterion. The sum of these weighted scores reflects the total weighted score of a pathogen. The total weighted scores were finally re-scaled to a range from 0 to 100 in order to facilitate final interpretation.
Following the experience from Canada [11] , we did not focus on the exact numerical score assigned to a pathogen. The interpretation of the final weighted scores and their corresponding sequential ranks was done in priority groups reflecting four priority levels (the highest, high, medium and low priority). The cut-off limits for the groups were based on the equal ranges of 25 (0-25, 26-50, 51-75, 76-100). Distribution of the pathogens was later compared with their positions in the 2004 priority list.
In total 127 pathogens were selected for prioritization. Drugresistant strains were scored under the common pathogen group and were not assessed as a separate entity. During the Delphi rounds, changes in pathogens' scores were made based on the consensus approach.
The Delphi process also resulted in the recommendation to remove the criterion ''Emerging potential'' due to its ambiguous meaning (i.e. each infectious pathogen has an ability to emerge or re-emerge) and to rephrase the definitions of some criteria. The final criteria and their definitions are presented in Table 1 . The detailed scores are available in the Table S1 .
All criteria were weighted by a total of 86 experts (14 RKI and 72 external experts). All Delphi discussion participants took part in the weighting.
The opinions regarding importance of individual criteria varied greatly across the participants for some criteria, while it was similar for others. For example, eleven participants considered the criterion ''Public attention'' to be of a low importance (scoring it ''2'') and the same number considered it to be of a relatively high importance (scoring it ''6''). At the same time, other criteria such as ''Case fatality rate (CFR)'' and ''Trend'' were weighted in a similar way by the majority of the respondents. When looking at the median weights, the criterion ''Case fatality rate'' was considered the most important criterion (median weight of ''9'') while ''Trend'' and ''Public attention'' (both with a median weight of ''5'') were considered the least important ones ( Table 2) .
We analyzed weights assigned by the experts working in different areas of medicine: epidemiologists and public health specialists (n = 43), laboratory specialists (n = 35) and clinicians (n = 8) ( Table 2) . Several criteria were assessed similarly across the groups (for example, the criteria ''Prevention'', median weight of ''8'', or ''Trend'', median weight of ''5''). At the same time, the criterion ''Incidence'' was seen as one of the most important by Need for medical treatment is established but currently no effective treatment is available or AMR limits treatment options AMR = antimicrobial resistance. Note 1. All criteria apply to the geographical settings where the prioritization is conducted; the time-frame applicable to the requested epidemiological data should be defined prior to the process initiation and depend on a frequency with which pathogens are planned to be re-scored. The RKI conducted re-scoring relevant for Germany using a time-frame of 5 years. Indicated numerical thresholds apply to a country where the prioritization process is conducted; when the prioritization is conducted in other geographical settings, different thresholds may need to be considered. Note 2. Event is defined as the occurrence of a disease that is unusual with respect to a particular time, place or circumstances. For certain infectious diseases one case may be sufficient to constitute an event (e.g. polio virus). Public health actions are any kind of targeted actions aiming to identify the nature of the event and/or to apply control measures in response to the event occurrence. *assessed against the total burden of infectious diseases. **assessed for each particular pathogen in question, e.g., for the criterion ''Treatment possibilities and needs'' it therefore refers to availability and adequacy of treatment for each case of an illness caused by a particular pathogen and does not take into account the incidence of illnesses or the availability of preventive measures. doi:10.1371/journal.pone.0025691.t001
epidemiologists and public health experts (median weight of ''8'') and laboratory specialists (median weight of ''7'') but was seen as one of the least important by clinicians (median weight of ''5.5'').
Almost the reverse situation was observed with the criteria ''Work and school absenteeism'' and ''Health care utilization'' that were seen to be important by clinicians but perceived as of lower importance by the two other groups. Table 3 presents the allocation of the pathogens into four priority groups according to their weighted total score. The highest priority group contains 26 (20.5%) pathogens. Among those are the pathogens that already received the highest priority in our previous prioritization process, such as HIV, Mycobacterium tuberculosis, Staphylococcus aureus including methicillin resistant strains (MRSA), Influenza virus, Hepatitis C virus, Campylobacter spp., Neisseria meningitidis, Legionella pneumophila, Varicella zoster virus (VZV). It additionally contains a number of pathogens responsible for nosocomial infections (e.g., Klebsiella spp., Pseudomonas ssp., Enterococcus spp.) and Respiratory syncytial virus (RSV) that were not evaluated by us previously. Helicobacter pylori and Hantavirus now belong to this group, while in 2004 both pathogens held medium priority positions. Other pathogens that were scored highest in 2004 were now assessed to be in the medium priority group (e.g., Parvovirus B19).
The high priority group contains 39 (30.7%) and the medium priority group -45 (35.4%) pathogens. A number of pathogens that were ranked as the least important in 2004 (Vibrio cholera, Francisella tularensis, Bacillus anthracis, Bartonella quintana) were now assigned to the medium and high priority groups.
Out of 17 pathogens from the low priority group in 2011, 11 were newly added.
This prioritization approach allowed us to benefit from the cumulative knowledge of many experts. The results of our work seem convincing to us; they support our current activities as well as indicate new directions for the future work. For example, the ranking of the majority of the pathogens found in the highest priority group (e.g., HIV, M.tuberculosis, Influenza virus, Neisseria meningitides, Legionella pneumophila) is in line with strategic goals identified by a number of international agencies that focus both on resource-constrained health systems and on industrialized countries such as Germany [4, 9, [22] [23] [24] . The decision to evaluate a broad range of nosocomial pathogens in the current prioritization and their high ranks indicate a growing recognition of the problem of antimicrobial resistance and healthcare associated infections (HAI) and are in line with a number of new strategic international and national policies calling for enhancement of HAI surveillance activities and capacities [4, 25] .
The positioning of Helicobacter pylori, Hantaviruses, RSV and VZV among the pathogens in the highest priority group helped us to identify the under-recognized importance of the diseases with respect to surveillance and epidemiological research and call for actions in this respect.
Indeed, although there is a large amount of clinical and laboratory research dedicated to Helicobacter pylori and a number of clinical guidelines are available, very little is done in terms of public health surveillance, despite the growing rates of antimicrobial resistance seriously compromising treatment [26, 27] .
RSV remains the most common respiratory pathogen in infants and young children worldwide, often resulting in serious lower respiratory tract infections [28] , yet a routine surveillance system for RSV based on virological testing of sentinel respiratory samples has only been initiated recently, and the burden of disease at the population level in Germany is largely unknown. The placement of RSV in the highest priority group is particularly unusual as pathogens causing diseases in limited population groups, i.e. here in young children, are often believed to be of a lower overall importance.
The incidence of human disease caused by Hantavirus fluctuates significantly over time; its incidence has peaked in endemic areas in Germany in recent years, which may have contributed to the higher ranking of this pathogen in the 2011 prioritization and the call to enhance research activities. A population-based seroprevalence survey will indeed be initiated within the RKI-funded network of reference laboratories.
VZV, a virus that causes two frequent diseases in children and adults, was ranked as a pathogen with the highest importance both Table 2 . Median weight of each criteria defined by experts from different professional groups (criteria are ranked according to their priority positions among all participants).
All participants (n = 86) Area of professional activity [29] . The low priority group contains both pathogens with very low incidence in Germany (e.g., Mycobacterium leprae or helminths) and those much more common (e.g., Roseolovirus or Chlamydia pneumoniae), supporting our approach that the importance of a pathogen is defined by multiple factors rather than by its incidence or prevalence alone. Complex surveillance systems exist for some pathogens that were assigned to the medium priority group, for example, Francisella tularensis or Yersinia pestis. Although the amount of research that should be dedicated to those medium-and lowpriority pathogens must be revised, undoubtedly the need to investigate outbreaks associated with these pathogens, to maintain diagnostic capacity and to continue efficient surveillance to pick up increasing trends of these rare illnesses, remains.
Similarly to other research groups that initiated processes for setting priorities [2, 8, 13] , we found the compilation of the data necessary for the scoring process to be challenging due to a limited availability of reliable and published information. Participants did not find it easy to maintain sufficiently broad judgements despite the highly specialized expertise and focused research interest of some experts. Some degree of subjectivity can therefore never be avoided. Another limitation of our study was the difficulty to appropriately account for the decreasing trends of vaccinepreventable diseases that followed successful implementation of effective prevention programmes. Therefore, relatively low scoring of the respective pathogens for some of the criteria should not question the need to maintain adequate surveillance capacity for these illnesses.
In the applied methodology, we aimed at following the main principles of good practice in setting priorities in health research [3] and to reach maximum levels of objectivity, transparency, and reproducibility by integrating the following components: 1) a broad but systematic and reproducible selection of pathogens, rather than respective diseases to be included in the prioritization, 2) an explicit definition of each of the ten criteria using a threetiered grading approach, 3) a comprehensive individual pathogenspecific scoring according to the best available evidence reviewed by a multidisciplinary expert group, and 4) a separate weighting of the criteria based on the involvement of a broad range of internal and external experts. Although this approach required intensive preparation, we believe it assured a high level of objectivity and transparency as demanded by Nuyens et al [7] .
Our pathogen-specific approach allowed us to conduct prioritization not influenced by programmatic views and to compare pathogens' ranking within a group of diseases as well as across the groups (e.g., a pathogen being targeted by an antimicrobial resistance programme as well as by a zoonoses programme). This approach helped us uncover some differences in the importance of pathogens belonging to the same group, e.g., Neisseria gonorrhoeae and Trichomonias vaginalis.
Marked differences were observed in the weighting of the criteria between different professional groups. As the invitation to participate in the weighting was sent to various institutions and often forwarded further, it is not feasible to estimate the response rate. However, we received responses from at least one member of each contacted institution. The weighting of criteria is likely to correlate with societal values and reflect socio-economic, cultural and health system structure in a country. The fact that in Germany the criterion ''Case fatality rate'' was considered to be of the most importance, may reflect the high level of individualization and relative affluence of the German health care system. Comparisons to other countries are difficult since similar studies are lacking. One of the few other priority-setting initiatives with an explicit weighting procedure was conducted in Spain in which the criterion ''Burden and importance of illness'' was identified to be among the first three most relevant from a total of nine criteria, the other two being ''Potential to change health outcomes'' and ''Potential to translate new knowledge into clinical or health services practices'' [5] . We can conclude that weighting needs to be explicitly addressed in any prioritization exercise and it is particularly appropriate for a national public health institute in order to reflect expectations from a broad range of professionals as the respective stakeholders [30] . One way to even expand the societal perspective of the prioritization exercise could be to involve patients' representatives, similarly to how it has been done by Gooberman-Hill et al [31] , for example.
Conclusions: The prioritization methodology presented here is based on the systematic evaluation of evidence and the involvement of a broad range of external experts. We feel that the results provide internal consistency and are plausible in the public health perspective. Our comprehensive and transparent approach makes the results defensible and shall give guidance for current needs in surveillance and epidemiological research in Germany. The list of ranked pathogens established here will serve as a reference for our mid-term strategic decisions, which will include strengthening the existing or introduction of new surveillance systems for pathogens from the high priority group (e.g., RSV, VZV or Helicobacterpylori) and re-consideration of the research and surveillance needs for those from the lowest priority group. It has already influenced the decision process on the need for the installation of new and continuation of existing national reference centers in Germany and the internal planning for the respective allocation of resources (GK personal communication). We plan to conduct a re-assessment of priorities within a five-year time frame based on the same methodology. The prioritization tool or its components can be applied across different areas of infectious diseases (by re-weighting prioritization criteria by different professional groups for different purposes) and in different geographical areas (by re-scoring pathogens according to their characteristics relevant for particular countries or continents). We hope that the presentation of our methodology could be helpful to other institutions that choose to prioritize their resources based on a transparent and standardized process.  A Vesicular Stomatitis Virus Replicon-Based Bioassay for the Rapid and Sensitive Determination of Multi-Species Type I Interferon Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines. IFN-a and IFN-b are structurally related cytokines of the type I interferon family which mediate an early innate immune response to viral infections. There are 13 distinct IFN-a genes present in the human genome, and a single gene encoding for IFN-b. These genes are transcriptionally activated in cells sensing a virus infection through pattern recognition receptors such as the retinoic acid inducible gene I (RIG-I) helicase. Following secretion, IFN-a and IFN-b act similarly by binding to a common, ubiquitously expressed IFN-a/b receptor resulting in the activation of the JAK/STAT signal transduction pathway and transcription of ''IFN-induced genes'' [1, 2] . Several of these genes encode for proteins with strong antiviral activity, i.e. Mx protein, protein kinase R, and 29-59oligo(A) synthetase [3] . Due to their autocrine action, type I IFN may attenuate virus replication in infected cells. Probably more important is the paracrine action of type I IFN, which induces an antiviral state in previously uninfected cells, thereby blocking virus dissemination in the organism. In addition to this ''classical'' antiviral function, type I IFNs are known to affect cell proliferation and differentiation, to modulate the immune response, to inhibit angiogenesis, and to promote apoptosis [4, 5] . Genetically engineered type I IFNs are currently in clinical use for the treatment of multiple sclerosis [6] , chronic hepatitis C virus infection [7] , and certain types of cancer [8, 9] . An issue of increasing importance is the determination of neutralizing antibodies that are induced in some patients following recombinant IFN therapy [10] .
Apart from IFN-a/b, cytokines such as IFN-c (type II IFN) and IFN-l (type III IFN) exhibit antiviral activities, although they bind to distinct receptors. In particular, type III IFNs induce transcriptional activation of antiviral genes similar to those activated by type I IFN. Type III IFNs act primarily on epithelial cells [11] and probably play an important role in the innate immune response of epithelial tissues to virus infections [12, 13] .
The accurate determination of antiviral IFN activity is a cumbersome issue. In the ''classical'' bioassay, serial dilutions of both a test sample with unknown IFN activity and a type I IFN standard are incubated with an appropriate cell line prior to infection with a cytolytic virus such as vesicular stomatitis virus (VSV), encephalomyocarditis virus, or Sendai virus [14, 15] . The reciprocal value of the highest type I IFN dilution mediating protection of 50% of the cells from virus-induced cytopathic effects (CPE) is defined as one unit of type I IFN per volume. This classical IFN bioassay is time-consuming because the CPE normally needs 24 hours or more to develop. A faster readout can be achieved with recombinant viruses expressing reporter proteins such as green fluorescent protein (GFP) or firefly luciferase [16, 17, 18, 19] . However, any work with live virus requires appropriate biosafety containment. For example, a recently published IFN bioassay can only be performed in biosafety level 3 (BSL-3) facilities, because the test makes use of a recombinant Rift Valley Fever virus [19] . Viral replicon-based bioassays that take advantage of disabled propagation-incompetent viruses may provide an attractive alternative to live virusbased bioassays. A human hepatoma cell line harbouring a selectable hepatitis C virus replicon has been successfully employed for the measurement of type I IFN from patients with chronic hepatitis C virus infection [20] . However, as type I IFNs act in a species-dependent manner, this system may not be applicable to animal IFNs. Transgenic cell lines expressing a reporter gene under control of an IFN-responsive promoter may also be used to determine IFN activity under biosafe conditions [21, 22] . However, it is difficult to simply relate transcriptional reporter gene activation to antiviral activity.
In this report, a novel type I IFN bioassay is presented, which is based on BSL-1-classified VSV replicon particles. The assay is highly sensitive and quantifiable due to the expression of a firefly luciferase reporter gene. In addition, the assay can be rapidly performed within 6 to 7 hours and may be used to determine the antiviral activity of IFNs from humans as well as other species. Thus, this bioassay may be of general interest for all those who want to determine the antiviral activity of cytokines such as type I IFNs.
Cells BHK-21 cells were obtained from the German Cell Culture Collection (DSZM, Braunschweig, Germany) and grown in Earle's minimal essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS). BHK-G43, a transgenic BHK-21 cell clone expressing VSV G protein in a regulated manner, was maintained as described previously [23] . The porcine kidney cell line PK-15 (ATCC, Manassas, VA) was propagated in Dulbecco's modified Eagle medium supplemented with nonessential amino acids, 1 mM Na-pyruvate and 5% horse serum. D-17 canine osteosarcoma cells (ATCC), Calu-3 human lung adenocarcinoma cells (ATCC), and NHDF normal human dermal fibroblasts (Lonza, Cologne, Germany) were maintained in EMEM with 10% FBS. The UMNSAH/DF-1 (DF-1) chicken fibroblast cell line (ATCC) was maintained in Dulbecco's Modified Eagle's Medium and 10% FBS. All cell lines were cultured at 37uC in a humidified atmosphere containing 5% CO 2 , except DF-1 cells which were kept at 39uC. BALB 3T3 fibroblasts (subclone A31) were kindly provided by N. Pringle, University College, London, UK, and maintained in Dulbecco's Modified Eagle's Medium.
Human and murine IFN-b and human IFN-l (IL-29) were purchased from PBL InterferonSource (Piscataway, NJ). Recombinant porcine IFN-a1 [24] was kindly provided by Nicolas Ruggli (IVI, Mittelhä usern, Switzerland). Recombinant chicken IFN-a [25] was kindly provided by Peter Stä heli (University of Freiburg, Germany). Canine IFN-b [26] was kindly provided by Philippe Plattet (Department of Clinical Research and Veterinary Public Health, University of Bern).
A plasmid-based rescue system [27] was used to generate a Gdeleted VSV driving expression of firefly luciferase and eGFP. The previously described genomic plasmid pVSV*DG(HA) containing 6 distinct transcription units (N-P-M-HA-eGFP-L) [28] was modified by replacing the influenza virus HA gene in the fourth position with eGFP taking advantage of MluI and BstEII endonuclease restriction sites upstream and downstream of the HA ORF, respectively. The firefly luciferase gene was amplified from the pBI-L plasmid (Clontech) by Pfu PCR and inserted into the fifth transcrition unit using XhoI and NheI endonuclease restriction sites. The resulting plasmid was designated pVSV*DG(Luc) and contained 6 genes in the order N-P-M-eGFP-Luc-L (Fig. 1) .
For generation of VSV*DG(Luc) replicon particles, BHK-G43 helper cells were grown in 100-mm diameter culture dishes to 90% confluence and infected with recombinant modified vaccinia virus Ankara (5 pfu per cell) expressing T7 RNA polymerase (MVA-T7, a gift of Gerd Sutter, München, Germany). MVA-T7 has been classified by the German Central Committee for Biosafety as a BSL-1 organism (reference number 6790- [10] [11] [12] [13] [14] . Ninety minutes post infection, the medium was replaced with fresh EMEM containing 5% FBS and 10 29 M mifepristone (Sigma-Aldrich, Buchs, Switzerland) to induce VSV G expression [23] . Subsequently, the cells were transfected with 10 mg of pVSV*DG(Luc), 3 mg of pTM1-N, 5 mg of pTM1-P, and 2 mg of pTM1-L [28] using Lipofectamine TM 2000 transfection reagent (Invitrogen, Basel, Switzerland). The cells were trypsinized 24 hours post transfection and seeded into T75 flasks along with an equal number of fresh BHK-G43 cells. The cells were further incubated at 37uC for 24 hours in the presence of 10 29 M mifepristone. The cell culture supernatant was clarified by low-speed centrifugation and passed through a 0.20-mm-pore size filter to deplete vaccinia virus. VSV*DG(Luc) was further propagated on mifepristoneinduced BHK-G43 cells and stored frozen at 270uC. To determine infectious virus titers, confluent BHK-21 grown in 96well microtiter plates were inoculated in duplicate with 40 ml of serial tenfold virus dilutions for 1 h at 37uC. The wells additionally received 60 ml of EMEM and were incubated for 20 h at 37uC. The infectious titers were calculated according to the number of GFP-expressing cells/well and expressed as fluorescence-forming units per milliliter (ffu/ml).
MVA-T7 was titrated on DF-1 cells grown in 96-well microtiter plates. The cells were inoculated with tenfold serial virus dilutions for 90 min and overlayed with medium containing 0.9% methylcellulose. Following incubation for 48 h at 39uC, the cells were fixed with 3% paraformaldehyde, permeabilized with 0.25% Triton X-100, and subsequently incubated with the TW2.3 monoclonal antibody directed to the vaccinia E3L protein (kindly provided by Jonathan Yewdell, NIH, Bethesda, USA) and anti-mouse IgG conjugated to horseradish peroxidase (DAKO). Infected cell foci were visualized with the AEC peroxidase substrate and expressed as plaque-forming units per milliliter (pfu/ml). Using this assay, the final VSV replicon particle preparations proved to be free of MVA-T7.
Serial twofold or fourfold dilutions of type I IFN were prepared with cell culture medium containing 5% FBS. The IFN dilutions (100 ml) were added in quadruplicates to confluent cells grown in 96-wells (5610 4 cells/well) and incubated for either 1, 2, 4, or 20 hours at 39uC (DF-1 cells) or 37uC (all other cell lines). The cells were infected with VSV*DG(Luc) (m.o.i. of 5) and incubated for 5 hours at 37uC. The medium was aspirated and 30 ml of luciferase lysis buffer (Biotium Inc., Hayward, CA) was added to the cells. The cell lysates were stored at 220uC. Firefly luciferase activity was determined with a Centro LB 960 luminometer (Berthold Technologies). Luminescence was recorded for 1 s following injection of 30 ml of D-luciferin substrate (Biotium) to white 96-well plates containing 6-ml aliquots of cell lysate. The relative antiviral activity was calculated according to the following formula: Antiviral Activity (%) = 100 -[(RLU +IFN -Blank)6100/ (RLU 2IFN -Blank)]. Mock-infected cell lysates served as blanks and relative light units (RLU) detected with these samples were subtracted from the readings taken from VSV*DG(Luc)-infected cell lysates. RLU +IFN represents the RLU values from IFN-treated cells and RLU 2IFN the readings taken from reference cells, which had not received any type I IFN.
A conventional type I IFN bioassay was performed by incubating 96-well cell cultures for 20 hours with twofold dilutions of type I IFN as described above. The cells were infected with propagation-competent VSV (m.o.i. of 1 pfu/cell) and incubated until a cytopathic effect (CPE) was evident in mock-treated control cells. The cells were washed twice with PBS and stained for 1 h with 0.1% crystal violet in 10% formalin. The plates were washed with tap water to remove excess crystal violet and dried. The dye was dissolved by adding 100 ml of 70% ethanol to each well. The absorbance of crystal violet at 595 nm was determined with a microplate reader. The IFN titer was calculated as the reciprocal of the last IFN dilution causing 50% inhibition of virus-induced CPE (50 % reduction of absorbance at 595 nm compared to mock-infected cells) and was expressed as IFN units per volume. Alternatively, antiviral activity was calculated according to the following formula: Antiviral activity (%) = (OD595 +IFN -OD595 2IFN )6100/(OD595 Mock -OD595 2IFN ). OD595 +IFN and OD595 2IFN represent the absorbance of IFN-treated and non-treated cells following infection with VSV and staining with crystal violet, respectively. OD595 Mock denotes the absorbance of non-infected cells.
Mean values and standard deviation were calculated. Statistical analysis was performed using the paired Student's t-test. P,0.05 was considered significant.
Previously, a VSV replicon vector was generated by replacing the glycoprotein G gene of VSV with the hemagglutinin (HA) gene of an H7N1 influenza virus and inserting an extra transcription unit into the HA-L intergenic junction to drive expression of a modified GFP gene [28] . In the present work, this vector was modified by inserting the firefly luciferase gene into the transcription unit at position 5 (thereby replacing the GFP gene) and exchanging the HA gene with the GFP gene (Fig. 1a) . The resulting vector, VSV*DG(Luc), was propagated on BHK-G43 helper cells which express the VSV G protein in an inducible manner [23] . Up to 10 9 virus replicon particles (VRP) were released into the cell culture supernatant 24 h post infection (data not shown). Several mammalian and avian cells were found to be susceptible to infection with the VRPs in line with previous observations on the very broad cell tropism of VSV [29] . Infected cells were readily detected (5 to 6 h post infection) by means of GFP reporter expression (Fig. 1b) . However, non-helper cells were unable to complement the VSV G deletion and thus could not produce infectious progeny virus. Importantly, VSV*DG(Luc) did not induce type I IFN in the cell lines analysed (data not shown). This can be ascribed to the host shut-off activity of the VSV matrix protein [30] .
Infection of BHK-21 cells with VSV*DG(Luc) using a multiplicity of infection (m.o.i.) of 4 ffu/cell resulted in increasing firefly luciferase reporter activity with time (Fig. 1c) . In all subsequent experiments, luciferase activity was generally recorded at 5 h post infection as the signal-to-noise ratio was sufficiently high at this time. When BHK-21 cells were infected with different virus dose rates, luciferase reporter activity increased linearly between 0.004 and 40 ffu/cell (Fig. 1d) 
The luciferase reporter activity in VSV*DG(Luc)-infected cells depends on the replication/transcription levels of the RNA replicon. Thus, a reduction in viral genome replication as a consequence of type IFN action should lead to correspondingly lower luciferase levels. To test this hypothesis, normal human dermal fibroblasts (NHDF) were incubated for various time periods with serial dilutions of human IFN-b before infection with VSV*DG(Luc). A dose-dependent effect of IFN-b on firefly luciferase reporter activity was observed after 1 hour of incubation (Fig. 2a) . About 1 unit of IFN-b led to 50% suppression of luciferase activity. Extending the incubation time to 2 hours did not further improve the sensitivity of the test (p.0.05 for the 0.08 to 1.25 IFN units range). However, if the cells were treated for 20 hours, the dose response curve was significantly shifted to lower IFN units (p#0.001 for the 0.02 to 20 units range). Approximately 0.05 units of IFN-b were now sufficient to suppress reporter activity by 50%. These results indicate that an IFNb-induced antiviral state in NHDF cells can be detected as early as 1 hour after addition of IFN-b, although the sensitivity of the assay is higher if the cells were incubated with IFN-b for prolonged time.
VSV*DG(Luc) was also used to quantify the activities of porcine and chicken IFN-a. While the dose response curve of chicken IFNa on DF-1 chicken fibroblasts (Fig. 2b) was similar to the one of human IFN-b on NHDF (Fig. 2a) , porcine IFN-a showed a different kinetics on PK-15 porcine kidney cells. In these cells, a full antiviral state was accomplished only after 20 hours of treatment indicating that PK-15 cells respond rather slowly to the action of type I IFN (Fig. 2c) . Thus, the assay is applicable to type I IFN from different species but may perform differentially on distinct cell types. To further evaluate the bioassay, samples containing type I IFN of unknown activity were tested against a commercial IFN-b standard and the results were compared with those obtained with a conventional IFN bioassay based on VSV cytotoxicity. Human type I IFN was induced in NHDF human fibroblasts following infection with VSV*M q [30] . This propagation-competent virus expresses a mutant matrix protein that is unable to block the nucleocytoplasmic RNA transport of the cell. Before the samples were tested for antiviral activity, VSV*M q was inactivated for 30 min with 0.1 M HCl to avoid any interference with the bioassay (see also the section on thermal and pH stability of IFN). When the VSV*DG(Luc) replicon bioassay was used, about 0.8 pg/ml of a commercial IFN-b standard resulted in 50% antiviral activity. In consideration of the final dilution producing 50% antiviral activity, the type I IFN concentration of two test samples was defined as 4000 pg/ml and 1440 pg/ml, respectively (Fig. 3a) . When the samples were tested with the conventional bioassay (Fig. 3b) , the values were in the same range (5000 and 1500 pg/ml), indicating that both assays principally agree. Nevertheless, the replicon-based bioassay proved to be more sensitive than the conventional one as lower amounts of type I IFN were sufficient to reduce the luciferase reporter activity by 50% (compare curves shown in Fig. 3a with those in Fig. 3b ).
The species-dependent action of type I IFN was analysed by incubating human, canine, murine, and chicken cells for 20 hours with 5 units of type I IFN from either the homologous species or a different one. When cells were treated with the respective homologous IFN an antiviral state was induced as indicated by the lack of GFP expression 6 hours post infection with VSV*DG(Luc) (Fig. 4a) . In contrast, untreated cells or cells that had received type I IFN from a different species were not protected and showed GFP expression accordingly. To compare the effects of different concentrations of homologous and heterologous type I IFN on human cells, we treated NHDF for 20 hours with serial dilutions of human, porcine, and murine type I IFN prior to infection with VSV*DG(Luc). Firefly luciferase reporter activity in infected cell lysates indicated that porcine IFNa is active in NHDF albeit at reduced levels compared to human IFN-b (Fig. 4b; p#0 .0049 for the 0.008 to 2.0 units range). Murine IFN-b showed an even lower activity on NHDF (ED 50 of 500 units per 5610 4 NHDF cells; p#0.0003 for the 0.008 to 500 units range). These results demonstrate that type I IFNs act in a speciesdependent manner and accentuate the need for selecting appropriate cell lines to determine their activity.
We often encounter the problem that the activity of type I IFN has to be determined in a sample containing live virus. As virus infection may interfere with the bioassay, it has to be inactivated before the assay is performed, preferentially without touching the activity of type I IFN. As many viruses can simply be inactivated by treatment with heat, we first analysed the thermal stability of human IFN-b using the VSV*DG(Luc) bioassay. The antiviral activity of human IFN-b was fully maintained when the cytokine was incubated for 30 min at 50uC (Fig. 5a) . At 60uC, 99% of the activity was preserved (p = 0.021). At 70uC, the activity of human IFN-b dropped to 83% (p = 0.0017). Incubation at higher temperatures (80uC and 90uC) affected the activity more drastically, although some activity was still left at these temperatures. Only when human IFN-b was heated to 100uC for 30 min, activity was completely abolished. In contrast to human IFN-b, porcine IFN-a was completely inactivated at 70uC (p,0.0001), whereas human IFN-l (IL-29), a type III IFN, showed even higher residual activity at 80uC and 90uC (p,0.001), suggesting that these cytokines have different physicochemical properties.
To study the sensitivity of type I and type III IFNs to conditions of extreme pH, human IFN-b and human IFN-l were treated for 30 min with either 0.1 M HCl, 0.1 M NaOH or H 2 O, adjusted to neutral pH, and assayed on NHDF and Calu-3 cells, respectively. It turned out that the antiviral activity of human IFN-b was not significantly affected by acid (p.0.05) (Fig. 5b) , confirming the previously noted acid-stability of type I IFNs [31] . IFN-l showed similar properties as antiviral activity was maintained following treatment with 0.1 M HCl (Fig. 5c) . In contrast, alkaline treatment significantly reduced the activity of IFN-l (p,0.05 for the 250 2 1 ng/ml range), whereas IFN-b was not affected (p.0.05) (Fig. 5b) . Thus, treatment with acid may be employed to inactivate virus in a test sample without affecting the activity of type I and type III IFNs. On the other hand, alkaline may be used to differentiate between type I and type III IFNs.
Conventional bioassays take advantage of the antiviral activity of type I IFN to measure the inhibition of virus-induced cytopathic effects in cell culture [14] . In this study, we presented an improved bioassay by employing a recombinant VSV replicon equipped with two reporter proteins. This assay proved to be advantageous over the conventional assay with respect to biosafety, sensitivity, and time requirements.
The use of cytopathic viruses in conventional type I IFN bioassays makes appropriate biosafety measures necessary to reduce the risk of unwanted virus transmission and infection. In this regard, the VSV*DG(Luc) replicon particles can be regarded as biosafe. Since the modified VSV genome lacks the envelope glycoprotein (G) gene, VSV*DG(Luc) is propagated on helper cells providing the G glycoprotein in trans [23] . The replicon particles produced on these cells can run a single cycle of infection but cannot produce any infectious progeny [28] . Another aspect contributing to biosafety is that the RNA replicon replicates exclusively in the cytosol and does not produce cDNA intermediates. Thus, in the case of an accidental infection any risk of recombination with or integration into host chromosomal DNA can be excluded.
Virus preparations that are used for IFN bioassays must not contain any type I IFN which may distort the results. In addition, the viruses should not induce IFN in infected reporter cells to assure that the effects measured are solely due to the exogenous IFN added. Both criteria are fulfilled for VSV*DG(Luc). The helper cells used to propagate the replicon particles are derived from BHK-21 cells, which are defective in the synthesis of type I IFN [32] . In addition, the VSVDG replicon does not induce type I IFN in infected reporter cells, because the VSV matrix protein efficiently blocks the nuclear export of cellular mRNA including the IFN mRNA [30, 33] .
Conventional type IFN bioassays often take 24 hours or more to be completed as they rely on the inhibition of cytopathic effects. In contrast, the bioassay presented here takes advantage of the firefly luciferase reporter, which can be detected with high sensitivity long before a cytopathic effect is apparent. Although the luciferase reporter enabled us to unambiguously quantify the IFN-mediated reduction of virus replication, we frequently observed that the standard deviations for the antiviral activities increased when the cells were treated with low amounts of type I IFN. This likely reflects the high reporter gene expression at low levels of type I IFN and the inaccuracy associated with pipetting small volumes (6 ml).
The engagement of a common cellular receptor by type I IFNs leads to activation of the JAK/STAT pathway and transcriptional induction of several genes with antiviral activity [1, 2] . It is common practice that cells are incubated with type I IFN for several hours to induce an antiviral state [15, 19] . Our findings suggest that an antiviral state can be detected much earlier. For example, treatment of NHDF with 1 unit of human IFN-b for 1 hour and subsequent infection with VSV*DG(Luc) for 5 hours was sufficient to suppress firefly reporter expression by 50%, even though the effective dose was further lowered with longer incubation times. While DF-1 fibroblasts responded to chicken IFN-a with dose response kinetics similar to the one observed with human IFN-b on NHDF, PK-15 cells responded to porcine type I IFN in a much slower way. Thus, compared to conventional bioassays VSV*DG(Luc) may allow quantification of type I IFN in a considerably shorter time provided an appropriate cell line has been selected. The selection of a suitable cell line is also important with respect to the species it is derived from, as full activity of type I IFN was only observed with cells from the corresponding species. Since VSV is able to infect a broad spectrum of mammalian and avian cells, the new bioassay may be used to determine the bioactivity of type I IFNs from many different animals.
Although type I IFNs are the predominant cytokines with antiviral activity [34] , it cannot be excluded that test samples contain other cytokines that inhibit VSV replication. Indeed, using the VSV*DG(Luc) replicon assay, it was possible to quantify the antiviral activity of IFN-l (Il-29), a type III IFN. This raises the important question of how to distinguish between different antiviral cytokines? Certainly, neutralizing antibodies may be employed to specify the antiviral cytokine present in the sample. In addition, the extraordinary acid-stability of type I IFNs may be used to differentiate them from acid-labile cytokines such as type II IFNs [35] . However, acid may not be used to distinguish between type I and type III IFNs as IFN-l proved to be acid-stable as well. Since IFN-l but not IFN-b was sensitive to 0.1 M NaOH, treatment with alkaline may be used instead. However, the conditions of treatment still have to be optimized to guarantee the complete inactivation of type III IFNs while maintaining the activity of type I IFNs.
In addition to antiviral cytokines, test samples may also contain unknown viruses that potentially interfere with the bioassay. Treatment with heat or acid may be used to inactivate these viruses without affecting the bioactivity of type I and type III IFNs. However, as heat-inactivated influenza viruses still may be able to induce type I IFN [36] , virus inactivation by acid may be more convenient.
The correct and reliable determination of type I IFN biological activity is an important issue. The new bioassay presented in this study represents an attractive alternative to conventional type I IFN bioassays that work with cytotoxic live viruses. VSV*DG(Luc) is easily produced and handled under BSL-1 conditions. The activity of the replicon can be easily determined and standardized taking advantage of the two reporter proteins. It may be stored for prolonged time in lyophilized form without losing its activity. Finally, the proven sensitivity, rapidity, and accurateness of the assay recommends it for the determination of type I IFN from a number of mammalian and avian species. Finally, the test may be further developed to quantify the antiviral activity of other cytokines such as type II and type III IFNs. Characterization of Neutralizing Profiles in HIV-1 Infected Patients from whom the HJ16, HGN194 and HK20 mAbs were Obtained Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an ‘extended incubation phase’ PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development. Despite intense research efforts over nearly three decades, only minimal progress has been made in developing an HIV-1 vaccine. In retrospect, a number of reasons can be proposed for this failure such as the enormous genetic diversity of HIV, the camouflage of the neutralizing epitopes in the envelope spike by glycan shields, the presence of ''decoy'' immunodominant non-neutralizing antigenic determinants in non-conserved areas on the surface and the low gp120 trimer spike density on the virus membrane [1] . In addition, the most vulnerable regions may only be accessible for a short period. These short-lived structures include the so-called CD4 induced (CD4i) in gp120 and the pre-hairpin epitopes in gp41 that are only exposed following CD4 receptor binding and the subsequent conformational changes. Still, a few antibodies (Abs) are able to successfully interfere with the binding and fusion process, as seen in passive immunization studies in the macaque model. Such mAbs include 2G12 (binds to mannose residues on gp120); b12 and F105 (bind to the CD4 binding site, CD4bs); 17b and 65 (recognize conformational epitopes in the CD4i region); and 4E10 and 2F5 (bind to epitopes in the membrane proximal extracellular region or MPER of gp41). Last year, however, three new mAbs (HJ16, HGN194 and HK20) were reported from African patients from the ITM HIV-1 cohort. Taken together these mAbs target three different steps in viral entry: binding to CD4bs and thus preventing interaction of HIV-1 with CD4 by HJ16, binding to V3 and blocking the coreceptor binding by HGN194 and finally immobilizing the unfolding of the gp41 by the HK20 mAb [2] . Since HK20 targets HR1 instead of MPER or glycans in this region, it has the conceptual advantage over 4E10 and 2F5 of avoiding potential auto reactivity [2, 3] . Importantly, the HGN194 mAb has recently been found to confer protection in infant rhesus monkeys by the group of Ruprecht [4] .
In order to generate these mAbs, patient plasma were selected with a neutralization assay with an extended incubation time, using activated PBMC and a panel of clinically isolated replication competent HIV-1 strains. This assay differs from the classical 'short' PBMC neutralization assay by extending the incubation phase of plasma with virus from 1 to 24 hours. The importance of this format was shown in a SHIV challenge trial in rhesus macaques, where recombinant HIV envelope immunizations induced protection [5, 6] . Comparing various neutralization assays, we showed that the PBMC based assay with an extended incubation phase was able to discriminate between protected and non-protected animals after vaccination. Since we are attempting to develop a vaccine effective against a range of subtypes and because the subtype A, subtype C and circulating recombinant form (CRF) 02_AG are responsible for at least 75% of the current new infections worldwide, we identified patients, whose plasma could cross-neutralize mainly viruses from these three subtypes in the extended incubation PBMC assay. From the blood of selected patients, memory B cells were isolated and immortalized using an Epstein Barr Virus (EBV) based procedure [7] . Supernatants of B cell clones were tested in ELISA with recombinant gp41, trimeric gp120 and gp140 proteins from several subtypes as solid phase. Clones with binding activity to any of these antigens were expanded and supernatants were tested using a HOS based PV neutralization assay. This effort ultimately resulted in the selection of the new mAbs HJ16, HGN194 and HK20, which showed considerable breadth of neutralizing activity against a panel of HIV-1 primary isolates spanning both tier 1 and tier 2 viruses of different subtypes [2] .
Here, we present the characteristics of the patient's plasma and their respective mAbs in multiple neutralization assay formats. The results clearly demonstrate that patient selection was highly dependent on the neutralization assay. Although the crossneutralizing properties of the isolated Abs showed considerable variation with the neutralization assay format, all assays indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and also do not exclude the role that synergy between such Abs could play.
The study was approved by the Institutional Review Board of the Institute of Tropical Medicine and the Ethical Committee of the University Hospital of Antwerp. All participants understood and signed an informed consent.
Eligible patients visiting the ITM clinic in Antwerp had been infected for at least one year, were clinically asymptomatic and over 18 years old. Neither CD4 T cell counts nor viral loads were taken into consideration. Patients were preferentially selected from sub-Saharan regions where the subtypes A, C and/or CRF02_AG are prevalent. Plasma was subsequently screened for its ability to neutralize a panel of four subtype A, four subtype C and six CRF02_AG primary HIV-1 strains, in our extended incubation phase PBMC assay (see below).
HJ16, HK20 and HGN194 Abs were obtained as part of the Collaboration for AIDS Vaccine Discovery program from Dr. D. Corti (Institute for Research in Biomedicine, Bellinzona, Switzerland).
Buffy coats from healthy donors from the Red Cross Blood Transfusion Center at the University Hospital of Antwerp were used for isolation of PBMC by LymfoPrep (Axis-Shield, Oslo, Norway) centrifugation and adjusted to 1610 6 /ml in culture medium, consisting of RPMI 1640, 15% fetal calf serum (FCS), 0.03% L-glutamine and 50 mg/ml gentamycin (Lonza, Verviers, Belgium), 2 mg/ml polybrene (Sigma-Aldrich, Bornem, Belgium). Cells were stimulated with 0.5 mg/ml phytohemagglutinin (PHA, Oxoid, Hampshire, UK) for 2 days and 1 day with 200 U/ml interleukin-2 (IL-2; Gentaur, Brussels, Belgium) in a 7% CO 2 incubator at 37uC and then used for neutralization assays. The following cell lines were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZMbl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. and Ghost (3) . All isolates were classified by phylogenetic analysis of their envelope genes. All virus stocks were prepared and titrated on PHA/IL-2 stimulated PBMC. These strains have been extensively used for at least 10 years at ITM and are considered equivalent to neutralization resistant tier 2 viruses [8, 9] . Corresponding envelope PV constructs were obtained by DNA amplification of the complete env starting from PBMC co-cultures or by RT-PCR using plasma and subsequent cloning into an expression vector (pSV7d or pcDNA4/TO) [10] . These included the ITM strains VI 191 (A), VI 829 (C), VI 882 (C), VI 1358 (C), VI 824 (D), VI 1888 (CRF01), VI 1090 (CRF02), CI 20 (CRF02) and CA 18 (CRF02). The env expressing plasmids 92RW009 (A), SF162 (B) and 92BR025 (C) were provided by the EU Programme EVA Centre for AIDS Reagents, NIBSC, UK (AVIP Contract Number LSHP-CT-2004-503487). Sequencing of the PV constructs and phylogenetic analyses of the complete gp160 confirmed the identity of the PV and its corresponding virus. 
Since several parameters influence the observed neutralizing profile of a plasma or mAb, we included a comprehensive range of different neutralization assays with distinctive characteristics. Apart from the difference in target cell (primary cells vs. cell lines), incubation, absorption and culture phases were also investigated as determinants of neutralization outcome. Formats for the different assays are as shown in Table 1 .
PBMC based assays. All PBMC neutralization assays are described as a/b/c where 'a' is the incubation time in hours following mixing of mAb with virus, 'b' is the absorption time in hours during which the cells are exposed to the mAb/virus mixture. Cells are then washed and 'c' is the culture time in days (all at 37uC and 7% CO 2 ). In this study results were obtained in 24/1/14, 1/2/7 and 1/24/14 formats. These are named 'extended incubation', 'short incubation' and 'extended absorption' assays respectively. The extended incubation assay, which was originally used for patient selection has been described previously [6, 11] . Briefly, virus stock is diluted in a five fold series from 1/2 to 1/6250 in culture medium (RPMI-1640 medium supplemented with 15% FCS and 200 U/ml IL-2) to establish the titer (-log10 if the dilution at which 50% infection is achieved). A titer below 1 constitutes poor growth of the virus and the experiment is discarded. Ninety ml of each virus dilution are mixed with 5 ml of plasma or 50 mg mAb. In assays testing neutralization by plasma the mixture is complemented with 5 ml culture medium to give a final 1 in 20 dilution of plasma. When testing mAb, the mixture is complemented with 5 ml flow through (IgG was removed from HIV-1 negative plasma using a Protein G column [GE Healthcare Europe GmbH, Belgium]) to give 50 mg/ml of mAb. After the incubation phase 20 ml of each plasma or mAb/ virus mix are first dispensed in quadruplicate into flat bottom 96well microplates and 75,000 PBMC in 100 ml culture medium are added to each well. Plates are then left in a CO 2 incubator at 37uC during the absorption phase (b). Afterwards, cells are washed three times by centrifugation at 2000 rpm for 10 minutes, the supernatant is aspirated and 180 ml fresh culture medium are added to the cells. When cultured for 14 days, 125 ml of the medium is aspirated and replaced with 135 ml fresh culture medium. After c days, 200 ml of the supernatant are mixed with 50 ml Nonidet P40 (0.25% in PBS; Fluka, Sigma-Aldrich, Puurs, Belgium) to disrupt virions and this mixture is analyzed for the presence of HIV p24 antigen. As a control, pooled plasma of 100 HIV-1 negative donors are tested in parallel. For the short incubation and extended absorption phase assays, times of incubation are appropriately adjusted: 1/2/7 and 1/24/14 respectively. Neutralization activities are presented as the percentage reduction in infectious titer of a virus isolate following incubation with patient plasma or mAb relative to its titer following incubation with HIV-1 negative control plasma. Virus titers were calculated by the method of Reed and Muench [12] . An 80% reduction in titer was considered significant. By extending the usual one hour absorption phase of the extended and short incubation PBMC assay to 24 hours (1/24/14 format) we aimed to reproduce the conditions of the cell line based assays where the mAbs remain during the entire absorption and culture phase.
Pseudovirus based assays. Neutralization capacity of patient plasma and mAbs against PV on TZMbl and the HOS cell related GHOST.CD4-X4/R5 cells was determined as described [13, 14] . Luciferase reporter gene activity was quantified 48-72 h after infection upon cell lysis and addition of firefly luciferase substrate (Perkin-Elmer) as described. Emitted relative light units (RLUs) were quantified on a LB941 Berthold luminometer (Alabama, US). Infection of TZMbl cells was quantified using SteadyLite and infection of GHOST cells was quantified using BriteLite as a substrate (both Perkin-Elmer). In a preliminary experiment 1.10 4 TZMbl or GHOST cells were seeded in each well of 96-well, flat bottom plates and infected with a range of viral doses in a total volume of 200 ml to establish the dose, which resulted in a signal of 50,000 to 100,000 RLU in the presence of 10 mg/ml diethylaminoethyl-dextran (DEAE-dextran, Sigma, Belgium) to enhance virus infectivity in TZMbl cells, while no DEAE-Dextran was used in GHOST cells. In the actual neutralization experiments, mAbs or plasma were pre-incubated with PV for 1 h at 37uC. The mAb concentration or plasma dilution producing a 50% reduction in luciferase reporter gene production was determined by linear regression analysis in Microsoft Office Excel as described on http://www.hiv.lanl.gov/content/nab-reference-strains/ html/Protocol-for-Neutralizing-Antibody-Screening-Assay-for-HIV-1in-TZM-bl-Cells-November-2010.pdf. For IC50 of mAbs, the 50% inhibitory concentrations were determined via a linear interpolation method using the mean of duplicate or triplicate cultures. The assay readouts for the dilutions above and below the IC50 were joined with a straight line, plotted against the log concentration of mAb. The position where the line crossed the 50% assay readout was taken as the IC50 estimate. Where the IC value was outside the range of concentrations tested, it was recorded as either greater than the highest concentration used, or less than the lowest concentration, as appropriate. An ID50 for plasma and IC50 for Abs were calculated from a dilution series starting from 1:20 for plasma and starting from 50 or 150 mg for Abs depending on the Ab used.
The virus titer was calculated within each individual experiment using the method of Reed and Muench [12] . In the virus dilution series, doses ranged between those infecting all cultures (100%) to those infecting none (0%). Wells giving an OD.0.3, against a background of 0.03-0.05 in the ELISA, were considered to be infected. The infectious virus titer was calculated following virus incubation with mAb/plasma. The reduction in titer was calculated as a percentage of the virus titer following exposure to either IgG or plasma which was pooled from 100 HIV-1 negative donors. Purified IgG from this pool was used as the control for mAbs. Correlations were calculated using the Spearman Rank correlation test using Prism version 5.0. Differences or correlations between sets of data were considered significant if p#0.05 and r.0.5.
Over 1400 HIV-1 infected individuals regularly attend the clinic at ITM. Of these, 200 patients were identified whose origin was the sub-Saharan regions of Africa where subtype A, subtype C and/or CRF02_AG isolates are prevalent. Their plasma was evaluated when they were therapy naïve or at least 6 months therapy-free for the ability to neutralize primary HIV-1 strains from the A, C and/or CRF02_AG subtypes in the 24/1/14 extended incubation phase PBMC neutralization assay. About 25% of these patients had cross-neutralizing plasma i.e. plasma which neutralized at least 50% of strains belonging to one subtype plus at least 25% of strains from a second. We next classified the best responding plasma according to the HIV subtype they preferentially neutralized: e.g. when at least three out of four of the A or C strains (or five out of six CRF02 strains) gave greater than 80% neutralization. In Table 2 this neutralization profile is shown for 20 patients whose memory B cells were interrogated. According to these criteria, four patients' plasma preferentially neutralized subtype A strains (HGL-, HGD-, HQ-and HGN plasma) and two were more specific for subtype C (HVDA and HK plasma). We did not find any patients preferentially recognizing the CRF02 strains. Three of the tested patients neutralized subtypes A and C more than CRF02 (HMB-, HJ-and HGR plasma), one was more C and CRF02 subtype specific (HMQ plasma) and finally five patients displayed broad crossneutralizing activity over all three subtypes (HU-, HP/HM/ HGM-, HE-, HY-and HMV plasma). The remaining five interrogated patients did not display this subtype specific behavior (HL-, HZ-, HGP-, HR-and HMA plasma). There is no obvious association between the subtype infecting a patient and that neutralized by his or her plasma. The patients, from whom the newly isolated mAb were obtained, are underlined in the first column. Remarkably, plasma from these patients showed a rather subtype specific neutralization profile since plasma from patient 242315 (HJ patient) neutralized mainly A and C strains, plasma from patient 314994 (HGN patient) mainly A strains and plasma from patient 529552 (HK patient) mainly C strains. In Table 3 the clinical histories of these patients are summarized.
Patient 242315 from whom the CD4bs specific HJ16 mAb was obtained was a 45 year old Congolese woman who had been visiting our clinic since 1996. She received treatment intermittently and consequently had a varying CD4 count and viral load. Her neutralization profile had been obtained using plasma samples taken after stopping anti-retroviral therapy for 6 to 11 months but she was back on therapy at time of memory B cell interrogation for 7 months. Patients 314994-HGN and 529552-HK were not receiving antiretroviral treatment during this study. Patient 314994 from whom the V3 crown specific mAb HGN194 was obtained was a 41 year old woman from the Republic of Guinea who has been regularly attending our clinic since 1998. She has always maintained low viral loads and high CD4 T counts so far without treatment. Her viral loads varied between undetectable and 2,700 RNA copies/ml while her CD4 counts have fluctuated between 550 and 960 cells/ml. Patient 529552 whose HK20 mAb is specific for the HR1 region of gp41 was a 31 year old Ghanaian woman. Soon after arrival in Belgium in 2005 she tested positive for HIV. Her viral loads (1.500-40.000 RNA copies/ml) have Table 2 ). In view of the known neutralization resistance of these isolates [6, 8] they were considered to represent 'Tier 2 like'' strains. In the experiments represented in Table 4 , an additional panel of four subtype B, four subtype D and four CRF01_AE strains provided us with an overview of the neutralizing potential of the three selected patient plasma across six subtypes with a total panel of 26 ''tier 2 like'' strains.
As can be observed, the 242315-HJ patient plasma has a very broad neutralization spectrum with 21 of the 26 viruses neutralized, including all the C and CRF01 strains, 75% of the subtype A, B and D strains and 67% of CRF02 strains. The 314994-HGN plasma has a narrower range, neutralizing 13/26 viruses, including all of the B strains, 75% of the A strains, 67% of the CRF02 strains, 50% of C strains, but none of the D nor CRF01 strains. The 529552-HK plasma neutralized 12/26 viruses, including 75% of the C strains, 67% of the CRF02 strains, 50% of the A strains and 25% of the B, D and CRF01 strains.
Influence of neutralization assays on plasma neutralization profile of the three patients from whom the new antibodies were isolated In order to illustrate the influence of different assays on the neutralization spectra of these selected plasma, we compared results from all the assays shown in table 1 (except for the HOS-PV assay). The virus panel used in this comparison consisted of nine strains from our primary selection panel from which PV were also available. Three strains from the standardized ''NeutNet'' panel were added: A (92RW009, tier 2), B (SF162, tier 1A) and C (92Br025, tier 2) [15] and personal communication). Results are shown in Table 5 .
Comparing the neutralization breadth of the three patient plasma in three variants of the PBMC assay, indicates that the HJ and HK plasma neutralize much fewer viruses and the HGN plasma even loses all significant neutralization capacity in the classical short assay (1/2/7), implying that none of them would have been selected using results from this assay. Prolonging the absorption phase to 24 hours (1/24/14), to more closely resemble the cell line based assays (see table 1), only ''rescues'' some neutralization with the HGN plasma. No correlation was found between the results obtained in the different PBMC assays using the Spearman Rank correlation test. There was a correlation between results from the 24/1/14 extended incubation PBMC assay and those with the TZMbl assay using replication competent ''primary'' viruses for the 242315-HJ plasma (r = 0.62, p = 0.03) but not for the other 2 plasma samples. A stronger correlation (r.0.60 for all three plasma) was found for the 242315 and 314994 plasma between the 24/1/14 PBMC assay and the TZMbl_PV assay. The correlation was statistically significant (p,0.04). The strongest correlation (r.0.69) was observed between the two PV assays (TZMbl-PV and GHOST_PV). Correlations for all three plasma were significant (p,0.01).
Evaluation of plasma vs. antibodies in the 24/1/14 extended incubation PBMC assay. The neutralization profiles of the plasma are compared with those for their respective mAbs for the 24/1/14 extended incubation PBMC assay in Table 6 . The mAbs clearly neutralized a much more restricted range of isolates than the plasma. 242315-HJ plasma and HJ16 mAb both neutralized the subtype C isolate VI829, the subtype D CI 13 and two of the three CRF02_AG isolates, VI 1090 and CA18. 314994-HGN plasma and HGN194 mAb as well as 529552-HK plasma and HK20 mAb neutralized SF162 (B) and 92Br025 (C) while HGN194 mAb also neutralized VI 191 (A) and 89.6 (B). In Table 5 . Neutralization profile of patient plasma in different HIV-1 neutralization assays. addition, a number of qualitative discrepancies were observed in that growth of some viruses was strongly inhibited by the mAb, but enhanced by the plasma (e.g. subtype A 92RW009 with 242315-HJ plasma versus HJ16 mAb) or vice-versa (e.g. CRF02 strains with 314994-HGN plasma versus HGN194 mAb). This type of inconsistency was also observed by the group of Nussenzweig when they compared their new mAbs with results from the original plasma [16] . Some neutralization-sensitive isolates, CA1 (A), MN (B), BaL (B) and CI 13 (D), were added to the panel in table 6. However, there was only a limited increase in range with HJ16 mAb reaching 80% neutralization against CI 13 (D) and HGN194 mAb almost neutralizing BaL (B) to 80%. HK20 mAb had no activity against any of the extra isolates.
While the plasma demonstrated their broadest range of neutralization in the 24/1/14 assays they also showed activity in the other PBMC and cell-line assays. Since it is possible that the mAbs could share these activities we extended their evaluation to assays with the different formats ( Table 7) .
The range of HIV-1 isolates neutralized by both the plasma and mAbs is greatest in the extended incubation 24/1/14 PBMC assay while only three of the 36 mAb/isolate combinations show significant neutralizing activity in the extended absorption 1/24/14 PBMC assay. The HJ16 mAb neutralizes three (SF162, VI 1888 and VI 1090) of the six isolates (92RW009, 93Br025 and CA18) neutralized by the plasma in 1/2/7 PBMC assays (table 5). The HGN194 mAb neutralizes SF162, VI 1888 and 92RW009 while the corresponding plasma do not produce significant neutralization against any isolate in these assays. The HK20 mAb only neutralizes VI 1888. When the absorption and culture phases of the assay are extended to the 1/24/14 setup, HJ16 still neutralizes VI 1090, HGN194 neutralizes SF162 and VI 1888 while HK20 neutralizes VI 1090 for 80%.
With regard to cell line based assays, there is a good concordance for HGN194 mAb and plasma in the GHOST-PV assay. Four of these HGN194-PV combinations are also neutralizing when the target cells are TZMbl. However, when infectious virus is used 10/12 combinations are enhancing. HJ16 mAb neutralizes 92RW009 and VI 1090 in all three cell-line assays while the corresponding plasma failed to do so in these assays (see Table 5 ). Remarkably, however, 92RW009 was neutralized by 242315-HJ plasma in the 1/2/7 PBMC assay selectively and VI 1090 was neutralized by HJ plasma in both the 24/1/14 and the 1/2/7 PBMC assay. The HK20 mAb only neutralizes VI 882 in the GHOST_PV assay, SF162 in the TZMbl_PV assay and does not neutralize any infectious virus. There was no consistent statistically significant correlation between the levels of neutralization reached in the 24/1/14 PBMC assay and the others except where only a few isolates were actually neutralized or neutralization levels were low.
In order to link the present and previous studies, plasma were tested in TZMbl assays against a sub-panel of PV included in supplementary table 2 of reference 7.
Comparisons are presented in Table 8 . Again, there were anomalies with mAbs neutralizing isolates which were not neutralized by the corresponding plasma and vice versa. Similarly, the mAbs showed a reduced range of neutralization relative to their corresponding plasma. Plasma from the 242315-HJ patient is very effective against the three tier 1 strains and also against five out of 11 tier 2 strains. In contrast, the corresponding HJ16 mAb is not able to neutralize the tier 1 strains and although effective against six out of 11 tier 2 strains, these are not always the same isolates as neutralized by the plasma. Plasma from the 314994-HGN patient is able to neutralize all tier 1 strains as well as four tier 2 strains while the HGN194 mAb is also able to neutralize the tier 1 strains and three out of 11 tier 2 strains. However, again, these are not always the same strains that are neutralized by the plasma. Plasma from the HK patient is able to potently neutralize the three tier 1 strains but none of the tier 2
isolates while the HK20 mAb is effective against only one of the three tier 1 strains and one of the 11 tier 2 isolates. The proportion of isolates neutralized by an individual plasma was also markedly dependent on the panel of HIV isolates used. The patients' plasma were initially selected in 24/1/14 PBMC assays and the ITM panel of 14 primary infectious HIV-1 isolates (Table 4 ). In the smaller, modified panel of PV used in Antwerp (Table 5) 
The present study is based on an extensive program to employ naturally occurring broadly neutralizing Abs from HIV-infected patients as templates for immunogen design against A, C and CRF02 primary viruses. From the memory B cell interrogation of such patients many mAbs were generated, but only three of these (HJ16, HGN194 and HK20) showed interesting novel broad neutralizing capacity. Since the plasma and ensuing mAb were selected in different neutralization assays, we wanted to explore and understand the behaviour of these exceptional plasma and mAbs in various neutralization assays, based on PBMC or cell lines, using primary infectious viruses or non-replicating PV.
A first observation was that the three patients, from whom the neutralizing mAbs were generated, showed an intermediate breadth of neutralization, preferentially neutralizing subtype A (HGN patient) or C (HJ patient) or A and C (HK patient), which did not correspond with the subtypes of their infection. Another remarkable observation is that they all showed a low viral load without treatment at the time of sampling. Only patient HGN had the profile of a viraemic controller, whereas HJ was a chronic progressor and patient HK was probably still in an early phase.
In the last two years several groups have reported the discovery of new and promising Abs [17, 18, 19] . The HJ16, HGN194 and HK20 Abs obtained by our consortium were amongst those obtained by means of the interrogation of rather chronically HIV-1 infected patients. In the present study, the HGN194 patient was infected for at least 10 years and seemed to naturally control her HIV-1 infection, the HK20 patient may not have been infected for longer than a year and the HJ16 patient regularly required antiretroviral therapy to control her viral load. These data confirm that neutralizing Ab development does not protect against disease progression. Similarly, some of the broadest neutralizing plasma were obtained from patients who urgently required HAART (e.g. HY-plasma table 2, clinical history not shown).
A side-by-side comparison of the different neutralization assays used for characterization of these patients' plasma and the newly isolated Abs showed that the broadest spectrum of strains and subtypes was neutralized in the PV assays as well as in the 24/1/ 14 extended incubation PBMC assay with primary virus. In contrast, the classical short incubation phase assays as well as the extended absorption phase PBMC assays showed a reduction in the number of neutralized strains. The TZMbl assay using primary virus also showed this restricted profile despite the fact that it has an extended absorption phase in common with the cell line PV assays. Results for the three patient plasma that were selected for their cross-neutralizing capacity in the 24/1/14 PBMC assay correlated with those obtained in the TZMbl_PV assay for only two patients. It is unusual that these two substantially different techniques result in comparable neutralization profiles (own results and [20] ). Both PV based assays correlated strongly with each other.
Another observation was that all three isolated Abs have a narrower and partially different neutralization spectrum relative to the corresponding plasma in the extended incubation PBMC and TZMbl PV assays. Results with the HJ16 mAb from the PBMC, TZMbl and GHOST assays show good correspondence while for the HGN194 mAb the GHOST neutralization responses are broader. The HK20 mAb shows little to no neutralization in either the TZMbl or GHOST assays. Neutralization breadth across subtypes is unlikely to be due to endotoxin since plasma are negative in conventional assays where absorption phases (and therefore contact between plasma and cells) are longer [21] .
Several factors may be responsible for the reduction in the range of isolates neutralized by the mAbs. One reason could be the polyclonal character of the Abs in the plasma. Cross-neutralization may require interaction between Abs acting at several epitopes. In this scenario, reproducing the range of isolates neutralized by plasma would not be possible when an average of only one to two neutralizing mAb were isolated. This would be buttressed by methods to directly determine the number of individual neutralizing antibody clones in the patient's repertoire. We will also address this issue in new studies but this has also been examined by the group of Guan and Lewis [22] . Obviously, combining more isolated mAb might correspond better to the plasma results when additive and synergistic effects between Abs could be unveiled. Unfortunately, we did not obtain more mAbs for the 242315-HJ and 529552-HK patients. Although we did obtain more mAbs from the 314994-HGN patient, none of these, except for HGN194, were neutralizing in either the HOS or TZMbl assays. Nevertheless, there may be 'missing Abs' as has been previously suggested by the groups of Guan and Nussenzweig [16, 22] . However, the 'non-neutralizing' mAbs may still be relevant in the wider context since they could have other effector mechanisms such as Ab-dependent cell-mediated virus inhibition (ADCVI) or Ab-dependent cellular cytotoxicity (ADCC) through their FccRs [23, 24, 25 ]. An alternative explanation for why relatively few neutralizing mAb were obtained is that the primary screen was binding to monomeric or trimeric envelope protein in ELISA and this procedure might not be optimal. In the Walker study [17] , it was shown that the most potent Abs did not bind in an ELISA and even Abs that did bind had a low neutralizing profile. The inference was that specific quaternary protein structures should be used in a primary screening. This issue is being addressed with the most recent samples under interrogation at IRB within our consortium. It should also be noted that the Abs in the plasma probably originate in the plasma cells of the bone marrow while the mAbs are isolated from memory B cells in the circulation. These two cell populations may not produce the same range of Abs. It should be possible to culture individual plasma cells, clone their heavy and light chain variable regions and identify the IgG or IgA repertoires produced.
Selection of the patients from whom the mAbs were isolated was extremely assay dependent. The patients who gave the three interesting mAbs would not have been selected if any of the alternative assays had been used. The influence of target cells on neutralization has already been observed both by us and others [10, 20, 26, 27] . In particular, there is a three way interaction effect between the virus, antibody and target cells. Especially MPER specific Abs are more potent in PBMC based assays [4, 26, 27] . Since our data show that the HJ, HK and HGN patients are more potent in PBMC neutralization assays with an extended incubation phase it could be envisioned that these special patients could have a high proportion of gp41 specific Abs in their plasma.
In the past, naturally occurring cross subtype neutralizing Abs have already been used as templates for immunogen design but in most of these cases patients were selected using either the classical short PBMC assay (1/2/7 format) or PV assays and our results clearly show that a different group of patients is selected by the extended incubation PBMC assay (24/1/14 format). Testing the patient plasma against primary strains is also more stringent since the molecularly cloned PV seem to be more easily neutralized. Hence, we believe that our selection procedure against the primary ITM panel provided us with patients that had more potent responses.
A possible reason for any increased sensitivity of primary vs. pseudo viruses for identifying patients with potent neutralizing Abs could be the higher number of envelope glycoprotein spikes on the primary viruses relative to the PV [20, 28] . An alternative factor might be the density of (co)receptors on target cells, which has been implied by Corti et al who reported potent neutralization by HK20 in the HOS assays but almost no potency in TZMbl assays [2, 3] . Since HK20 recognizes an epitope in the gp41 region this could partially be explained by the high level of CCR5 expression on the TZMbl cells making it more difficult for anti-gp41 Abs to be effective [2, 29] . Also, the pathway employed by PV to enter TZMbl cells may be relevant so that HK20 could have been hindered by events following uptake into an endosome [30] .
Since the non-replicating PV constructs could not be assessed in the primary target cells the recent development of molecularly cloned constructs in a Renilla replication competent backbone is certainly a step forward in the development of a standardized PBMC based neutralization assay to assess neutralization in primary cells [31, 32] . It remains elusive whether the HJ16, HGN194 and HK20 mAbs would have been obtained from other patients. HK20 like Abs have been detected through ELISA and although the neutralizing capacity of this fraction was not shown it still provides proof that a significant number of HIV-1 infected patients have responded to the gp41-HR1 region which is only briefly exposed [2, 3] .
Even after almost 30 years of HIV research and the ongoing search for correlates of protection, there is still a critical need to determine how effective different types of antibody effector mechanisms can be in prevention of HIV-1 infection. Although many groups have tried to identify which neutralization assay can predict in vivo protection, this issue is still open to debate [33] . In several SIV and SHIV macaque studies neutralizing mAbs have correlated with protection [34, 35, 36, 37, 38, 39, 40, 41, 42, 43] , but there are also multiple counter examples [44, 45] . In this context, the most compelling demonstration that pre-existing Abs can be protective comes from passive immunization studies with either IgG or mAbs [28, 34, 38, 39, 40, 41, 42, 43, 46, 47, 48, 49] . The most recent study uses the HGN194 mAb against a SHIV strain containing an 'early' envelope and emphasizes the importance of potent neutralizing Abs that confer protection against a heterologous mucosal challenge [4] . The latter is highly significant since future vaccines will need to be effective against these relatively resistant early founder strains before infection is established in vivo [50] .
Taken together our observations show that a single neutralizing mAb from each of the three patients does not reflect the major neutralizing spectrum of the patients' plasma and there is no apparent correlation of the mAbs targeting HIV strains belonging to the subtype of virus infecting the patient. It is quite evident that different neutralization assays yield different results and it is still unclear which one is most predictive or suited to obtain neutralizing mAbs. Nevertheless, the strategy used for selection of plasma (in an extended incubation PBMC assay) and selection of mAb (based on ELISA binding and neutralizing capacity in a HOS_PV assay) yielded interesting new mAbs. A better understanding of in vitro neutralization characterizations of patient plasma and Abs and will hopefully lead to more effective ways of discovering new Abs that ultimately can be used for HIV-1 immunogen design and subsequent vaccine development. EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies. Epstein-Barr virus (EBV) is an almost ubiquitous human gamma herpes virus that infects resting human B-lymphocytes, including B-CLL cells, with high efficacy [1, 2] . EBV's B-cell tropism is mainly due to gp350, the viral envelope glycoprotein that interacts with the cellular complement receptor 2 (CR2, CD21) [3] on B cells. In EBV seropositive individuals, gp350 mainly elicits CD4+ T-cell responses [4] .
Exosomes are endosome-derived membrane vesicles, which are released by cells of diverse origin including dendritic cells, cancer cells [5] and EBV-infected B cells [6] . Exosomes bud from endosomal membranes and accumulate in multivesicular bodies, which eventually fuse with the cellular membrane and release the contained vesicles. Exosomes are rich in lipids and membrane proteins like MHC molecules, TNF-R and tetraspanins [5] but their specific composition depends on the cell of origin. Exosomes either fuse to the recipient cell membrane or are engulfed by phagocytic cells in such a way that exosome proteins are degraded and loaded onto MHC class II molecules [7] . Obviously, exosomes can deliver proteins as cargo in a very immunogenic manner so that they efficiently reactivate specific CD4+ T cell clones [8] . Hence, exosomes can induce strong and epitope-specific immune responses [9, 10] and can be used as an alternative to transfer strategies using gene vectors and as promising vaccines [11, 12] .
Chronic lymphocytic leukemia of B-cell origin (B-CLL) is the most common adult leukemia in the Western hemisphere. B-CLL is considered as a prototypic disease undergoing immune evasion as the malignant cells lack important accessory and co-stimulatory molecules. Thus, despite their expression of high levels of surface MHC class I and II molecules, which presumably present tumorassociated antigenic epitopes, the leukemic cells tend to induce tumor-specific T-cell anergy. Typically, activated T cells from patients show a significantly reduced expression of CD40 ligand (CD154) or are completely CD154-negative [13] . As a consequence, T cells from B-CLL patients cannot activate cells through the CD40 receptor. This interaction, however, is essential for CD40 signaling and subsequent induction of other immune accessory molecules like CD80 and CD86, which increase the antigen-presenting capacity of normal and B-CLL cells. On the other hand, the EBV-specific cellular immunity is relatively intact in these patients [2] . To overcome the dysfunction of potentially tumor-reactive T cells from patients with B-CLL, several approaches have been developed relying on the stimulation of B-CLL cells through the CD40 pathway, including the ectopic expression of CD154 on the leukemic cells, and aiming at the selfstimulation of these cells [14] [15] [16] [17] . In summary, immunotherapy of B-CLL is promising and CD154 is a potential candidate molecule to improve the patients' immune status and, eventually, the clinical outcome.
The robust cellular immunity in B-CLL patients against EBV [2] therefore prompted us to investigate the potential of tailored exosomes to redirect this immunity to malignant B cells. We present a novel approach for the targeted transfer of functional cellular proteins to B cells via tailored gp350+ exosomes. In this approach, gp350 has a dual function: (i) it confers B-cell tropism to exosomes so that they specifically co-transfer proteins of interest and (ii) it is a viral neo-antigen for these cells so that they efficiently reactive gp350-specific T cells. As a proof of concept, we show that tailored gp350+ exosomes can co-transfer functional CD154 as immune accessory molecule to B-CLL cells, which are subsequently stimulated to express surface molecules like CD54, CD80, CD86 and CD95 and stimulate autologous tumor-and EBVspecific T cells.
EBV gp350 is packaged into exosomes, confers B-cell tropism, and reactivates specific T cells EBV has a profound B-cell tropism that is mainly conveyed by gp350, which is the major EBV glycoprotein in the viral envelope and the ligand for cellular CD21 (CR2) on B cells. We knew from previous work that exosomes can transport ectopically expressed proteins such as green fluorescent protein (GFP), which is presumably present as a cargo in the exosomal lumen. In addition, several groups provided evidence that surface proteins are incorporated in exosome membranes [5] . We therefore asked whether gp350 could also become an integral part of exosomes and confer B-cell tropism to these vesicles. To answer this question, we cotransfected 293 cells with expression plasmids encoding BLLF1, the gene of gp350, and gfp. Three days later, we isolated vesicles from the supernatants of transfected HEK293 cells as described in Material and Methods and analyzed them by immunoblots for the presence of gp350 and exosome markers. Gp350 was detected in vesicles that floated at a density between 1.03 and 1.08 into an OptiPrep TM gradient, corresponding to a density between 1.13 to 1.18 in a sucrose gradient and thus in the density described for exosomes. The gradient also revealed the co-sedimentation of gp350 with the exosome markers hps70, tsg101 and CD63, indicating the nature of the gp350+ vesicles as exosomes ( Figure 1A ). Flow cytometry of exosomes coupled to latex beads revealed that gp350 is presumably located within the exosome membrane because it could be targeted with a specific antibody ( Figure 1B ). To demonstrate that gp350 confers B-cell tropism also to exosomes, we incubated gp350+/gfp+ exosomes with PBMCs from a healthy donor for one day and then quantified exosome binding by measuring GFP fluorescence by flow cytometry. This assay revealed that gp350+/gfp+ exosomes had an EBV-like tropism because they bound to CD19+ B cells but not to CD19negative cells ( Figure 1C ).
Phagocytic cells engulf exosomes, process their proteins in lysosomes and present epitops in association with MHC class II molecules to CD4+ T cells [10] . To further utilize the potential of gp350+ exosomes to specifically transfer exogenous proteins to B cells, which, in turn, may activate specific T cells, we generated exosomes that carried BNRF1, the major tegument protein of EBV, either alone (BNRF1+) or together with gp350 (BNRF1+/ gp350+). We then incubated purified CD19+ B cells with these exosomes overnight and used these PBMC as stimulators for an autologous BNRF1-specific CD4+ T-cell clone. As shown in Figure 1D , B cells incubated with BNRF1+/gp350+ exosomes activated the T-cell clone in a concentration-dependent manner whereas B cells incubated with BNRF1+ exosomes did not activate the T-cell clone. This result demonstrates the potential of gp350+ exosomes to transfer immunogenic foreign proteins to B cells that then can activate specific CD4+ T lymphocytes.
In a next series of experiments we wanted to elucidate whether 293/gp350+ exosomes can co-transfer functional membrane proteins to B cells. As a model system but also as a potential practical application, we chose B-CLL cells that express CD21 and become immunogenic upon ectopic expression of CD154 on malignant cells. We, therefore, transfected 293 cells with expression plasmids for gp350 and CD154 and isolated exosomes as described above. Again, an immunoblot with a CD154-specific antibody demonstrated the presence of CD154 in exosome preparations and an OptiPrep TM gradient revealed co-sedimentation with gp350 ( Figure 1A) indicating the presence of both proteins on exosomes ( Figure 2A ). In addition, flow cytometry revealed that gp350+/CD154+ exosomes specifically bound to CD19+ B cells from a B-CLL patient ( Figure 2B ).
To answer the question whether CD154 in gp350+/CD154+ exosomes was functional, HEK293 cells were transfected with a CD154 expression plasmid either alone or in combination with a gp350 expression plasmid. Transfer of exosomes to B-CLL cells was measured with an anti-CD154 antibody by flow cytometry. As shown in Figure 2C , exosomes that carry both proteins efficiently conveyed CD154 surface expression to B-CLL cells. CD154+/ gp350-exosomes were only transferred very inefficiently to B-CLL cells, probably due to an only weak interaction of CD154 with its receptor CD40 on the cell surface. In order to investigate the immune accessory function of CD154, we loaded B-CLL cells from an HLA II DR13+ donor either with 293 exosomes, with exosomes from 293 cells transfected with an expression plasmid for gp350 (gp350+ exo) or transfected with expression plasmids for gp350 and CD154 (CD154+/gp350+ exo). One day later, we added HLA-DR13-restricted gp350-specific CD4+ T cells and measured their activation with an IFN-c ELISA. This assay demonstrated the relevance of CD154 for T-cell recognition because B-CLL cells loaded with CD154+/gp350+ exosomes were significantly better stimulators than B-CLL cells loaded with gp350+ exosomes ( Figure 2D ). Induction of the accessory molecule ICAM-1 (CD54), the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), and the death receptor Apo1 (CD95) on B-CLL cells upon incubation with CD154+/gp350+ exosomes ( Figure 2E ) but not with exosomes from 293 cells provided an explanation for improved recognition by T lymphocytes.
We knew from previous experiments that PBMCs from healthy donors, which were incubated with gp350+ exosomes, efficiently re-stimulated an autologous gp350-specific CD4+ T-cell clone (data not shown). These experiments implicated that gp350, which is equally transferred to B-CLL cells by CD154+/gp350+ exosomes, can act as a neo-antigen in B-CLL cells, which are normally not infected with EBV [18] . This is a very interesting aspect because B-CLL patients usually maintain a robust and easily recruited CMV- [19] and EBV-specific T-cell response [2] although their T cells in general are functionally impaired. For instance, B-CLL cells infected with EBV or loaded with CMVpeptides are efficiently killed by autologous T lymphocytes from B-CLL patients [18, 20] . We thus aimed at investigating whether gp350 serves as a viral neo-antigen in exosome-treated leukemic cells and whether these cells become targets for gp350-specific autologous T lymphocytes. We stimulated PBMCs from B-CLL patients three times with CD154+/gp350+ exosomes as described above and elucidated the specificities and cytolytic potential of the activated T cells. To this end, we incubated autologous PBMCs for 24 hours with either CD154+/gp350+ exosomes, CD154+/gp350-exosomes or unmodified 293 exosomes or left them untreated. The next day, these cells were labeled with calcein and used as targets for autologous effector T cells. As shown in Figure 3 , T cells generated by stimulation with gp350+/ CD154+ exosomes efficiently killed autologous PBMCs that had been activated with CD154+/gp350-or CD154+/gp350+ exosomes as quantified by the release of calcein. Of interest, we did not observe notable activation of T cells against 293 proteins as PBMCs incubated with exosomes from non-transfected 293 cells were lysed to almost the same extent as non-activated autologous PBMCs.
These experiments also demonstrated that PBMCs loaded with CD154+/gp350+ exosomes were constantly better lysed than PBMCs loaded with CD154+/gp350-exosomes. This prompted us to investigate whether T cells specific for gp350 accounted for this improved lysis. Therefore, we tested the cytolytic activity of these T cells against EBV-infected lymphoblastoid cells (LCLs), which are known to be targets for gp350-specific T cells [21] , and against EBV-negative B blasts [22] from an HLA-DR13+ matched healthy donor. We found that the T cells efficiently lysed allogenic LCLs, whereas they completely ignored EBV-free B blasts, indicating the presence of EBV-specific T cells in the effector population ( Figure 3 ). These data corroborate that the stimulation of PBMCs with CD154+/gp350+ carrying exosomes is an efficient method for the activation of B-CLL cells and the reactivation and Immunoblots demonstrated that vesicles carry gp350 and that these vesicles co-sediment with vesicle that carry the exosome markers hsp70, tsg101, CD63 and ganglioside (GM)1 (www.exocarta.org). Calnexin is present in cell lysates (CL), its absence from exosomes demonstrated the purity of the preparation. (B) Latex beads were coated with gp350+ exosomes and stained with a gp350-specific antibody. gp350 is accessible and thus probably located in the exosome membrane (isotype control is shown as grey tinted histogram). (C) gp350 confers B-cell tropism to exosomes. PBMCs from a healthy donor were incubated for 18 h with gp350+/gfp+ exosomes and then analyzed for GFP fluorescence by flow cytometry. Only CD19+ B-cells stained positive, whereas an interaction of gp350+ exosomes with CD3+ T cells and CD19-cells (T and NK cells, monocytes) could not be observed. An immunoblot with a CD154-specific antibody revealed that CD154 is highly expressed in transfected cell and that the protein is incorporated into exosomes. Shown are immunoblots of cell lysates (CL) and lysates from purified exosomes (EL) of normal and transfected 293 cells, incubated with CD154-(upper panel) and tsg101-specific antibodies (middle panel). An OptiPrep TM gradient revealed co-sedimentation of CD154-and gp350-carrying vesicles (see Figure 1A ). (B) Exosomes carrying gp350+ specifically bind to CD19+ B cells from a B-CLL patient. Fresh B-CLL cells were incubated with purified exosomes from 293 cells transfected with a gp350 expression plasmid and binding of exosomes was measured by flow cytometry. (C) Exosomes carrying CD154 bind only weakly to B-CLL cells, probably through interaction with CD40, which is highly expressed on these cells. Binding of CD154-carrying exosomes is drastically enhanced by the co-expression of gp350. 293 cells were co-transfected with expression plasmids for CD154 and/or gp350. Fresh B-CLL cells were co-cultivated with CD154+/gp350-and CD154+/gp350+ exosomes for two days and binding of exosomes and thus transfer of CD154 was measured by flow cytometry. 
Having demonstrated that B-CLL cells activated by CD154+/ gp350+ exosomes achieve an activated phenotype, we next wanted to assess whether these B-CLL cells became immunogenic to autologous EBV-and CLL-specific T cells. Given the expected low number of these T cells in peripheral blood, we stimulated 1,5610 7 PBMCs from patients with B-CLL three times within 21 days with lethally irradiated autologous PBMCs that were incubated with different exosomes as described above. One typical experiment is shown in Figure 4 : before the first stimulation on day 0, PBMCs of this patient consisted mainly of malignant B cells with only 4% CD3+ T cells. After the third round of stimulation, cultures incubated with gp350+/CD154+ exosomes almost exclusively contained CD4+ and CD8+ T-cells ( Figure 4A ). In total, stimulation with CD154+/gp350+ exosomes yielded approximately 1610 7 vital cells on day 31, meaning that the CD4+ and CD8+ T cell counts had increased about 25-fold and 15-fold, respectively ( Figure 4B) , while the total cell number slightly decreased ( Figure 4C ). In contrast, no viable cells were detectable in those cultures that were treated with CD154-/gp350exosomes derived from non-transfected 293 cells, or that were left untreated. Thus, stimulation of T cells with B-CLL cells loaded with CD154+/gp350+ exosomes is a powe rful option to selectively expand specific T cells from B-CLL patients in vitro.
Next we wanted to elucidate the specificities and cytolytic activity of these T cells in more detail. As mentioned above, restimulated T cells form B-CLL patients efficiently lysed autologous PBMCs that were activated with CD154+/gp350-exosomes.
These exosomes do not contain gp350 and, therefore, the T cells must have recognized cellular antigens. To test whether T cells specific for B-CLL-associated antigens had been reactivated, we incubated them with irradiated EBV-negative B-blasts [22] from an HLA-A2-matched donor as a negative control or B-blasts loaded with HLA-A2 restricted peptides derived from B-CLLassociated antigens, namely MDM [23] , ETV5 [24] and PU.1 [25] . As shown in Figure 4D , peptide-loaded B-blasts were efficiently lysed by the T cells stimulated with CD154+/gp350+ exosomes whereas B blasts not loaded with peptides were completely ignored. Lysis of target cells was efficiently reduced upon addition of an MHC class I-specific antibody, W6/32, demonstrating the antigen specificity of the effector T cells.
CD154 is a known promising candidate molecule for the immunotherapy of B-CLL because it increases the immunogenicity of the tumor cells. In line, the ectopic expression of CD154 on B-CLL cells was shown to induce the expression of important costimulatory and adhesion molecules on the leukemic cells, turning them into efficient stimulators of autologous T cells. In principle, viral gene transfer of CD154 into B-CLL cells is a suitable approach to induce immunological reactions both in vitro and in vivo [14, [26] [27] [28] [29] but the low transduction efficiency and the resulting high dose of viral vectors may induce severe side effects. There also remain major concerns about the clinical use of viral vectors.
Exosomes are cellular microvesicles, which have already demonstrated their potential to induce specific immune responses. The majority of permanent cell lines spontaneously release exosomes into the supernatant, from where they can be easily purified and concentrated [30] . Transfecting HEK293 cells with expression plasmids coding for CD154 and gp350 resulted in the secretion of modified exosomes carrying both proteins. Upon interaction with their target B cells, an undetermined fraction of exosomes is presumably engulfed and degraded in lysosomes in such way that peptides of exosomal proteins are presented in association with MHC class II molecules. Exosomes that are not taken up by the target cells probably engage in protein-protein contacts with cellular surface molecules and lead to receptor activation and signaling. gp350 molecules on the surface of exosomes probably target these vesicles exclusively to human B cells which express relevant amounts of the receptor molecule for gp350, CD21. Exosomal co-transfer of both gp350 and CD154 is thought to lead to efficient CD154/CD40 ligand/receptor engagement on the B-cell surface activating the intrinsic CD40 signaling cascade.
Treatment of B-CLL cells with exosomes transferring functional CD154 protein causes leukemia cells to become efficient APCs. Activating the CD40 receptor leads to the induction of immune accessory molecules, making these leukemic cells potent stimulators of autologous T lymphocytes. Since almost all patients with CLL have a high prevalence of EBV (94%) [31] and thus possess EBV-specific memory cells with a high frequency of CD4+ gp350specific T cells [32] , the incorporation of gp350 into exosomes has a dual function: it confers B-cell tropism and serves as an immunodominant viral CD4 antigen. The incorporation of a viral protein as a tumor-specific antigen is straightforward, because the cellular immune system of cancer patients is usually impaired but virus-specific immune responses are detectable even in late-stage patients and T lymphocytes specific for herpes viruses are often present at high numbers [1, 33] . Given the fact that the vast majority of CLL patients are seropositive for EBV, gp350 and other EBV proteins have the potential as promising neo-antigens in B-CLL cells exploiting and redirecting the strong anti-viral cellular immune responses to leukemic cells.
Based on these results, we propose here a new immunotherapeutic approach for B-CLL, based on the simultaneous targeted transfer of functional CD154 and the EBV protein gp350 onto malignant cells using exosomes. gp350 is the major envelope protein of EBV and confers viral B-cell tropism by interacting with the complement receptor 2 (CD21), which is highly expressed on B lymphocytes. Here, we generated modified exosomes produced in 293 cells. As a result of gp350 incorporation, these particles have a profound B-cell tropism similar to wild-type EBV, so that gp350carrying vesicles specifically and efficiently bind to B cells. In addition, gp350 serves as a viral neo-antigen in B-CLL cells. We also found that CD154 on exosomes from HEK293 cells is functionally active as demonstrated by the induction of immune accessory molecules on B target cells probably through the CD40 pathway. Taken together, our experiments suggest that leukemia cells treated with CD154+/gp350+ exosomes are efficiently stimulated and subsequently killed by autologous B-CLL and gp350-specific cytolytic T lymphocytes.
In summary, our results demonstrate that modified exosomes carrying the EBV protein gp350 display a distinct tropism to normal and leukemic B cells and efficiently transfer CD154 as a functional protein onto these cells. Leukemic B cells treated with these particles acquire an activated phenotype and become potent stimulators of autologous T lymphocytes. Engineered exosomes can be easily generated and can readily be scaled up for clinical applications. In addition, they can be individually tailored to express additional accessory molecules like OX40L or the Fas ligand or alternative viral molecules to target other classes of cells like macrophages and DCs. The generation of modified exosomes is not limited to 293 cells, which we used in this proof-of-concept. Instead, other cell lines that are approved for human therapy, such as MRC-5 fibroblasts, should also be tested as an optional origin of exosomes to facilitate transition into clinical trials. Modified gp350-carrying exosomes can thus be regarded as powerful and promising tools for various immunotherapeutic approaches.
Peripheral blood samples were obtained from patients with diagnosis of B-CLL after informed consent approved by the Institutional Ethics Committee. Normal blood samples were taken from healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation on F/H. B cells were purified with CD19-specific MACS beads (Miltenyi, Bergisch Gladbach, Germany). Cells were cultured at 37uC in a 5% CO 2 atmosphere in standard medium with 10% fetal calf serum.
HEK293 is a human embryonic kidney cell line [34] , which spontaneously releases exosomes into the cell culture supernatant. For the generation of modified exosomes, 293 cells were cotransfected with expression plasmids for BLLF1 (gp350), CD154 and/or gfp. For the isolation of exosomes, 25 ml of conditioned supernatants were collected three days later, sterile filtrated and subjected to repeated centrifugations at increasing centrifugal force (10 min at 300 x g, 10 min at 5000 x g in a Heraeus 3SR+centrifuge, followed by 2 h at 100.000 x g in a Beckman LE-80K ultracentrifuge in a SW28 swing-out rotor. The pelleted particles were washed, resuspended in 500 ml volume PBS containing protease inhibitors (Complete Mini, Roche) and the protein content was analyzed in a Lowry microassay using reagents purchased from Bio-Rad (Munich, Germany). Exosomes were further purified by flotation into a 5-30% iodixanol gradient (OptiPrep TM , Sigma Aldrich, Deisenhofen, Germany).
B-CLL cells were cultivated with 100 mg of exosomes in a final volume of 2 ml for two days. Induction of surface accessory molecules was measured by flow cytometry using a FACS Calibur flow cytometer (Becton Dickinson). For bead-coupling assays 5 ml of surfactant-free sulfate/aldehyde latex beads were incubated with 10 mg of exosomes for 15 min at room temperature. Then 1 ml of PBS was added and the beads were incubated for another 2 hours. The beads were washed three times in PBS with 2% FCS and analyzed. Antibodies specific for tsg101 (sc-978) and CD154 (sc-7964) were purchased from Santa Cruz Biotechnology. E. Kremmer (Munich) provided the gp350-specific antibody 72A1. Fluorochrome-labeled secondary antibodies were obtained from Becton Dickinson (Heidelberg, Germany) or Immunotools (Friesoyte, Germany).
Vesicle preparations were spotted onto a PVDF membrane, incubated with with specific primary antibodies and an HRPcoupled secondary antibody and developed with the ECL system (GE Healthcare). Ganglioside M1 was detected with colera toxin (Sigma Aldrich).
In order to reactivate EBV-specific T-cells in B-CLL blood samples, 3610 7 cells were cultivated with 100 mg of exosomes in a final volume of 5 ml. Re-stimulations were performed on days 14 and 28 by adding 1610 7 lethally irradiated autologous PBMCs that have been loaded with 100 mg exosomes for 5h. Fresh medium was added once a week. After 31 days, cells were analyzed by flow cytometry for lineage markers. Interferon-cELISA assays were performed according to the manufacturers instructions (Mabtech, Uppsala, Sweden). The gp350-and BNRF1-specific CD4+ HLA-DR13-restricted T-cell clones used have been described elsewhere [8, 35] . All cells were tested in triplicates.
Calcein release assays were performed as described previously [36] . Briefly, target cells were labeled with Calcein-AM (1% solution in medium) for 30 min at 37uC. After intensive washing the cells were either incubated with 10 mg of exosomes for 4 h or loaded with CLL-specific, HLA-A2-restricted peptides (PU1.423: aa-sequence VLFYLGQYI, mdm2.53: YLAPENGYL and ETV5.45: ELFQDL-SQL) at 20 mg/ml for 1 h or left untreated. The cytotoxicity assays were performed over a time period of 6 h, using an effector-targetratio of 40:1, and the calcein released into the supernatant was quantified with excitation and emission wavelengths of 490 nm and 530 nm, respectively, in a plate-reader (Perkin Elmer, Waltham). All experiments were performed at least three times. The Ebola Virus Glycoprotein and HIV-1 Vpu Employ Different Strategies to Counteract the Antiviral Factor Tetherin The antiviral protein tetherin/BST2/CD317/HM1.24 restricts cellular egress of human immunodeficiency virus (HIV) and of particles mimicking the Ebola virus (EBOV), a hemorrhagic fever virus. The HIV-1 viral protein U (Vpu) and the EBOV-glycoprotein (EBOV-GP) both inhibit tetherin. Here, we compared tetherin counteraction by EBOV-GP and Vpu. We found that EBOV-GP but not Vpu counteracted tetherin from different primate species, indicating that EBOV-GP and Vpu target tetherin differentially. Tetherin interacted with the GP2 subunit of EBOV-GP, which might encode the determinants for tetherin counteraction. Vpu reduced cell surface expression of tetherin while EBOV-GP did not, suggesting that both proteins employ different mechanisms to counteract tetherin. Finally, Marburg virus (MARV)–GP also inhibited tetherin and downregulated tetherin in a cell type–dependent fashion, indicating that tetherin antagonism depends on the cellular source of tetherin. Collectively, our results indicate that EBOV-GP counteracts tetherin by a novel mechanism and that tetherin inhibition is conserved between EBOV-GP and MARV-GP.  Detection of a Fourth Orbivirus Non-Structural Protein The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4. The genus Orbivirus currently includes twenty two distinct virus species, with genomes composed of 10 segments of linear double stranded RNA (dsRNA), that are vectored by Culicoides midges, ticks, phlebotomine flies, anopheline or culicine mosquitoes. The three economically most important orbiviruses: Bluetongue virus (BTV) (the Orbivirus 'type-species') African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV) are all transmitted by Culicoides biting-midges [1] . Several tick-borne orbiviruses can infect humans, including members of the Changuinola virus, Corriparta virus, Lebombo virus, Orungo virus and Great island virus (GIV) species.
The coding assignments of the 10 BTV genome segments were initially determined in 1983 [2, 3, 4] . Seven distinct structural proteins (VP1 to VP7) and 3 distinct non-structural proteins (NS1, NS2 and NS3) were identified in orbivirus infected cells, or after in vitro translation of viral RNA. In most cases each genome segment encodes a single protein from a single open reading frame (ORF), expect segment 9 (Seg-9) and segment 10 (Seg-10), both of which encode two nearly identical proteins initiated from in-phase AUG codons close together near the upstream termini (VP6 and VP6a encoded by Seg-9, and NS3 and NS3a encoded by Seg-10) [2, 5] . However, in vitro translation of BTV RNA segments reproducibly generated a number of smaller translation products of unknown significance, that were usually dismissed as unimportant byproducts of translation [2] .
The icosahedral orbivirus core-particle is constructed as two concentric protein shells, the sub-core layer which contain 120 copies/particle of the T2 protein (VP3 of BTV), and the coresurface layer composed of 780 copies/particle of the T13 protein (VP7 of BTV). VP1, VP4 and VP6 are minor enzymatic proteins that are packaged along with the ten genome segments within the central space of the virus core [6, 7] . The orbivirus outer-capsid layer is composed of two additional structural proteins (VP2 and VP5 of BTV), which mediate cell-attachment and penetration during initiation of infection. These outer-capsid proteins are more variable than the core proteins and most of the non-structural proteins, and the specificity of their reactions with neutralising antibodies determines the virus serotype (as exemplified by VP2 of BTV [8] ). The relative number and locations of the BTV structural proteins have been determined in biochemical and structural studies using cryo-electron microscopy and X-ray crystallography [7, 9, 10, 11, 12] .
NS1 is the most abundant protein in BTV infected cells, forming tubules that may be involved in translocation of progeny virus particles to the cell membrane [13, 14] . BTV NS2 can be phosphorylated by ubiquitous cellular kinases and is an important matrix protein of the granular viral inclusion bodies (VIB) that form within the cytoplasm of infected cells. VIB represent the primary site of virus replication and assembly. The smallest of the BTV non-structural proteins that were previously identified, are membrane glycoproteins NS3 and NS3a, which are expressed in large amounts in insect cells, but not in mammalian cells. They are involved in the release of progeny virus particles from infected cells [15] . In some orbiviruses (e.g. AHSV) NS3/NS3a are highly variable and it has been suggested that they may be involved in determination of both vector competence and virulence [16] .
BTV-Seg-9 encodes the minor core protein VP6, which is a helicase. Recent bioinformatic analyses have identified a new overlapping ORF in Seg-9 of both insect-borne and tick-borne orbiviruses, although the putative protein (identified here as NS4) varies in size between 10 kDa and 22.5 kDa [17, 18] .
We report the synthesis and detection of NS4, in the cytoplasm and nuclei of cells infected with insect-borne and tick-borne orbiviruses (represented by BTV and GIV respectively).
All animal immunisation work was conducted according to the recommendations in the Animals (Scientific procedures) Act of the Home Office of the UK and the Directive on the protection of Animals used for Experimental and other scientific purposes of the EU. The protocol was approved by the Ethics Committee of animal experiments at the Institute for Animal Health in the UK (Project license number 70/7060). All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.
BHK-21 (American type cell culture collection) were grown at 37uC under 5% CO2 in Glasgow's minimum essential medium (GMEM), supplemented with 10% foetal bovine serum, 10% tryptose phosphate broth, penicillin G (100 IU/ml) and streptomycin (100 mg/ml). Culicoides sonorensis KC cells were grown at 28uC in Schneider's insect medium supplemented with 15% fetal bovine serum.
Confluent monolayers of BHK-21 cells were infected with either BTV-8 (isolate NET2006/04) or Great Island virus (GIV) (isolate CAN1971/01) at a multiplicity of infection (MOI) of 0.1 pfu/cell. Infected cell cultures were incubated at 37uC for 72 hours until cell lysis began. The cells were then scraped into the supernatant and centrifuged at 3,000 g for 10 minutes. RNA was extracted from cell pellets using guanidinium isothiocyanate (RNA NOW reagent: Biogentex, Tx, USA) as described earlier [19] . KC cells were infected at an MOI of 0.1 pfu/cell and then incubated at 28uC for 7 days. Both BHK-21 and KC cell pellets were used in western blot analyses as described below.
Viruses were purified from BHK-21 infected cells, as previously described using a discontinuous sucrose gradient [20] . Virus particles formed a blue opalescent band at the interface of the sucrose solutions. This was recovered and further purified by layering onto a continuous PercollH gradient as previously described [21] , using an SW41 rotor (100000 g, 1 hour, 4uC). The virus formed a blue band which was collected, diluted in 0.1 M Tris-HCl and pelleted at 10000 g for 1 hour.
Bioinformatic analyses of the overlapping ORF in Seg-9 of BTV and GIV
The hydrophobicity profile of different NS4 proteins was analysed using the Kyte and Doolittle hydrophobicity plot with a window size of 11 amino acids (aa) [22] . Sequence relatedness to proteins in public databases was assessed using the NCBI's BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)) and the pfam software (http://pfam.sanger.ac.uk/search/sequence). Amino acid alignments of NS4 of various orbiviruses were generated using the Clustal X program [23] and pairwise aa identities calculated using the MEGA 4 package [24] . The presence of 'coiled-coils' was indicated by analyses using the program 'COILS' (http://www.ch. embnet.org/cgi-bin/COILS_form_parser) and the PredictProtein server (http://www.predictprotein.org). The presence of nuclear localisation signals were analysed by PredictNSL, implemented in the PredictProtein server, and the cNLS Mapper (http://nlsmapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). Synonymous site conservation within the BTV VP6 coding sequence was analysed as described previously [25] . For this procedure, alignment columns in which the reference sequence (GenBank accession number: NC_006008) contained gap characters were removed so that the plots are in reference sequence coordinates.
The RNA of BTV-8 or GIV was separated by 1% agarose gel electrophoresis. Seg-9 was cut from the gel using a clean scalpel blade, purified using RNaid kit (MP Biomedicals) and cDNA was synthesised using a single primer amplification technique as previously described [19] ). The ORFs in Seg-9 from BTV-8 (between nucleotides 182 and 415, accession number: AM498059) and GIV (between nucleotides 176 and 748: accession number HM543473) were PCR amplified using specific primers tailed with restriction enzyme sites shown in table 1.
The pGEX-4T-2 vector and Seg-9 PCR products were doubledigested with EcoRI and NotI (BTV-8) or EcoRI and XhoI (GIV) enzymes (Invitrogen). The pCI-neo vector and Seg-9 PCR products were double-digested with EcoRI and NotI (BTV-8) or EcoRI and XbaI (GIV) enzymes. Digested products were gel purified using Genclean kit (Qbiogen). Corresponding vectors and PCR products were ligated overnight (O/N) at 16uC using T4 DNA ligase (Roche) to generate pGEX-BTVNS4, pGEX-GIVNS4, pCI-BTVNS4 or pCI-GIVNS4. These recombinant plasmids were used to transform XL1-Blue bacteria (Stratagene). Clones were recovered and grown in trypticase-soy-casein (TSC) medium containing 100 mg/ml ampicillin. The plamsids were subsequently purified using Qiaquick plasmid miniprep kit (Qiagen) and sequenced using the D-Rhodamine DNA sequencing kit and an ABI prism 377 sequence analyser (Perkin Elmer).
Confirmed pGEX-BTVNS4 or pGEX-GIVNS4 plasmids were used to transform BL21 or C41 bacteria. A single colony of each plasmid was grown overnight (ON) in TSC/ampicillin, then used to seed 200 ml of fresh TSC/ampicillin. The bacteria were grown until OD600 0.5, then 0.5 mM IPTG was added for induction, for 4 hours at 37uC, or for 8 hours at 28uC. The bacterial cells were pelleted and processed using Bugbuster protein purification (Novagen) as previously described [26] . The soluble fraction of the fusion protein was purified by glutathione affinity chromatography using glutathione sepharose, as directed by the manufacturer (GE Healthcare). Proteins were analysed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% polyacrylamide separating gel (Miniprotean III) with a 3% stacking gel and stained with Coomassie brilliant blue, as described Table 2 . Percentage amino acid identity values between NS4 of BTV, EHDV, AHSV, GIV, PHSV and YUOV. Amino acid identity ranged from 5% to50%. The highest identity exists between BTV and EHDV (50%) followed by PHSV and YUOV-1 (30%). Amino acid identity in NS4 between the tick-borne and insect-borne viruses ranged between 5% and 18%. doi:10.1371/journal.pone.0025697.t002 previously [21] . The purified fusion protein was used to immunize rabbits (Harlan) with an initial injection, followed by 4 boosts at 2 weeks interval in the presence of Montanide ISA50 (Seppic) as an adjuvant.
BTV-8 or GIV infected BHK-21 cells (5610 6 cells) and BTV-8 infected KC cells (5610 6 cells) were dissolved for 10 min at 100uC in 1 ml of sample denaturation buffer (160 mM Tris-HCl, 4 mM EDTA, 3.6% SDS, 60 mM DTT, 0.2% ß-mercaptoethanol, 0.8% methionine, 800 mM sucrose). A volume of 20 ml was analysed per well, by electrophoresis in a minigel (Miniprotean III tank -Bio-Rad). Purified and pelleted virus particles were also dissolved in sample buffer and analysed by SDS-PAGE, using a 4-20% gradient polyacrylamide gel.
Resolved proteins were electro-blotted on 0.2 mm nitrocellulose membrane (Bio-Rad) using 20 mM Tris, 0.05% SDS, 150 mM glycine and 20% V/V isopropanol transfer buffer. Membranes were blocked with 5% skimmed milk, in Tris buffered saline (TBS: 25 mM Tris/HCl, 150 mM NaCl, 2 mM KCl, pH 7.4) and incubated over night with a dilution of 1/300 rabbit antisera. Membranes were washed three times with TBS-Tween-20 (TBS containing 0.05% Tween-20) and further incubated with monoclonal, anti-rabbit, peroxydase conjugate (Sigma), diluted at 1/750 in 5% skimmed milk. After 2 hours the membrane was washed three times with TBS-Tween-20 and developed using 4-chloronaphthol (Sigma) in presence of hydrogen peroxide.
Logarithmically growing BHK-21 cells were infected with BTV-8 (1pfu/cell) for 24 hours then harvested and washed once with PBS. Nuclear extracts were prepared from 2.5610 7 cells using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce), as directed by the manufacturer. The nuclear extract was mixed volume to volume with sample denaturation buffer and analysed by SDS-PAGE using a 4-20% gradient polyacrylamide gel. Resolved proteins were electro-blotted on 0.2 mm nitrocellulose membrane as described above, blocked with 5% skimmed milk, in TBS-Tween-20 and incubated over night with a dilution of 1/300 anti-BTV-8 NS4 rabbit antiserum. The membranes were washed three times with TBS-Tween-20 and further incubated with monoclonal, anti-rabbit, peroxydase conjugate (Sigma), diluted at 1/750 in 5% skimmed milk. After 2 hours the membranes were washed three times with TBS-Tween-20 then incubated with Lumilight plus (Roche) chemiluminescent detection reagent, as described by the manufacturer. X-Omat radiographic films (Kodak) were exposed for 10 minutes to membranes then developed as described by the manufacturer.
BHK-21 cells were grown on coverslips placed at the bottom of a 24 well plates. 50% confluent cells were infected with 0.1 pfu/ cell of BTV-8 or GIV, incubated at 37uC for 4 hours or 24 to 72 hours, then fixed in 4% paraformaldehyde and processed for immuno-fluorescence. Briefly, rabbit antisera raised against NS4 of BTV-8 or GIV and a mouse anti-alpha tubulin antibody were both diluted 1/500 in PBS containing 0.5% bovine serum albumin (PBS-A) and applied to the fixed cell. After 1 hour incubation at room temperature (RT), slides were washed in PBS, then incubated with Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen) and Alexa Fluor 568 conjugated anti-mouse, both diluted 1/250 in PBS. After labelling with primary and secondary antibodies, the cells were stained with DAPI (1:10,000) for 15 
BHK-21 cells grown in 24 well plates (75% confluence), were transfected in triplicate, with pCI-BTVNS4 or pCI-GIVNS4 (4 mg/well) using Fugene-6 (Roche). At 48 hours post-transfection, the cells were fixed in 4% paraformaldehyde and processed for immuno-fluorescence, using anti-NS4 antibodies, as described above.
Identification of NS4 in cells transfected with pCI-BTVNS4, using anti-BTV-8 immune serum from infected mice BHK-21 cells were transfected with pCI-BTVNS4 using Fugene-6. At 48 hours post-transfection, the cells were dissolved in sample denaturation buffer as described above. Cell lysates were analysed by SDS-PAGE/Western blot, using an immune serum (diluted 1/50, in 5% skimmed milk) from mice infected with BTV-8. Non-transfected cells were used as control.
Nucleic acid protection assays dsRNA binding proteins can compete with Dicer (an endoribonuclease of the RNAse III family), reducing its ability to cleave long dsRNAs into 21 bp-long 'interfering' RNAs [27] . A dsRNA ladder (New England Biolabs) with sizes ranging from 500 to 21 bp was used as a template for Dicer cleavage. A competition assay with Dicer (Mobitech), was carried in the presence of 150 ng of expressed BTV-8 or GIV NS4, in a final volume of 20 ml containing 8 ml of the Dicer reaction, 2 mg of dsRNA, 1 mM ATP, 2.5 mM MgCl2. The reaction was incubated at room temperature for 20 minutes, followed by addition of a 1.5 U of Dicer, then incubated for a further 6 hours at 37uC. The reaction products were analysed by 3% agarose gel electrophoresis.
DNAse I is an endodeoxyribonuclease that can degrade dsDNA into 59 phosphorylated tetranucleotides [28] . A dsDNA ladder (Promega) with sizes ranging from 2645 to 36 bp provides a target for DNAse I cleavage. Competition assays between DNAse I (Roche) and BTV-8 or GIV NS4, were carried out in a final volume of 20 ml, containing 2 ml of 10X DNAse I buffer and 2 mg of dsDNA. The reaction was incubated at room temperature for 20 minutes, followed by addition of a 2 U of DNAse I, then incubated for a further 30 minutes at 37uC. The completed reaction was heated at 99uC for 1 minute to inactivate the DNase and the reaction products were analysed by 2% agarose gel electrophoresis.
The outer capsid protein VP9 of Banna virus (BAV, genus Sedaornavirus, family Reoviridae) expressed in E.coli [29] was used as a control in both RNAse and DNAse assays.
A colorimetric assay was developed to detect interactions between NS4 and dsRNA. Synthetic dsRNA was prepared with the 59-end of one strand linked to biotin via a 15-atom mixed polarity tetraethylene glycol spacer (59-Biotine TEG). This design allows the dsRNA to be captured at the bottom of a well of 96 well plate coated with streptavidin, while keeping the dsRNA free as a target for NS4 binding. The sequence of the +ve strand is: 59-Biotine-TEG-UGGAAGCGGCUGGCAAUUAAUUUUGGU-GUC-39 and that of the negative strand is 59-GACAC-CAAAAUUAAUUGCCAGCCGCUUCCA-39. Increasing concentrations (from 1 to 640 ng) of the dsRNA in PBS were added to separate wells of a streptavidin-coated 96 well plate (Pierce) and allowed to bind at room temperature for 2 hours. The wells were washed three times with TBS-Tween-20, then two hundred microlitres of a 5% solution of bovine serum albumin (BSA) in PBS, was added in each well, to block non-specific sites. After washing 3 times with TBS-Tween-20 a fixed amount (150 ng) of either BTV or GIV NS4 in binding buffer (20 mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM MgCl2, 2 mM MnCl2 and 5% glycerol) was added per well, prior to incubation for 30 minutes at 25uC. After the wells had been washed 3 times with TBS-Tween-20, rabbit anti-BTV or anti-GIV NS4 sera was diluted 1/250 in 5% BSA and 100 ml was added to each well, then the plates were incubated at 25uC for 2 hours. After washing three times, 100 ml of peroxydase conjugated antirabbit antibody was added (diluted 1/750 in 5% BSA) to each well. The plates were incubated at 25uC for 2 hours, then washed 3 times with TBS-Tween-20. One hundred microliters of SureBlue TMB 1-component microwell peroxidase substrate (tetramethyl benzidine from KPL) was added per well, then incubated for 30 minutes at 25uC. The reaction was stopped by adding 100 ml of 1 M HCl and the plate was read at OD 450 nm.
Wells not containing dsRNA/NS4, were included as negative controls. Wells from which the dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls.
The program MLOGD models and compares sequence evolution in single-coding and dual-coding sequences. It has previously been used to identify a second ORF, in a different but overlapping reading frame from that encoding the viral helicase (VP6 of BTV), within Seg-9 of the insect-borne orbiviruses [17, 18, 30] . This ORF was also identified in tick-borne orbiviruses [18] . The length of the putative translation product is highly variable, even between closely related Orbivirus species. In BTV and EHDV it is approximately 10 kDa, in Peruvian horsesickness virus (PHSV) and Yunnan orbivirus (YUOV) it is approximately 13.5 kDa, while in AHSV it is approximately 17 kDa, and in GIV it is approximately 22.5 kDa (twice as long as in BTV). These NS4 sequences contain a high proportion of charged residues, with basic R+K (arginine + lysine) content ranging from 13% to 22%, while acidic E+D (glutamic + aspartic acids) content ranges from 12% to 22%. Each NS4 protein contains 4-5 histidine residues, with the exception of the BTV protein, which contains none.
The levels of pairwise nucleotide conservation at synonymous sites within aligned sequences of the VP6 ORF, were used to assess the functional importance of the NS4-ORF. Complete or near-complete VP6-encoding sequences from BTV showed strikingly enhanced conservation in the region corresponding to the NS4 ORF (figure 1), supporting and extending previous computational analyses [17] . Enhanced conservation was also apparent at the 59 end of the VP6 coding sequence, indicating that this region (like the terminal noncoding regions of orbiviruses) is likely to contain functionally important elements.
Amino acid identity between NS4 of the different Orbivirus species compared ranged from 5% to 50%. Highest identity was detected between BTV and EHDV (50%), followed by PHSV and YUOV-1 (30%). Amino acid identity in NS4 between the tickborne and insect-borne viruses, ranged between 5% to 18% (table 2). Local blast analyses using BLAST-P or TBLAST-N identified significant matches (as defined by the E value in BLAST) between NS4 proteins encoded by other orbiviruses. Analysis of NS4 protein sequences using the pfam program, which uses the hidden Markov model (HMM) based profiles to identify or predict protein functionalities [31, 32] , revealed strong similarities to certain conserved functional motifs. AHSV NS4 exhibits strong relatedness over almost its entire length with DUF domains that have helical structures known to be involved in nucleic acid binding and/or modification [33] . Previous analysis of GIV NS4 identified a 72 amino acid fragment (aa 82 to 153) with 39% similarity to dsRNA-binding domains of similar length (approximately 68aa) in other reovirus proteins [ [18] or other dsRNA binding proteins [34] . BTV NS4 (77aa long) also exhibits relatedness (over aa 14-54) to a DUF domain, belonging to the MetJ/Arc repressor superfamily [35] , which has a ribbon-ribbonhelix-helix DNA-binding motif, with the beta-ribbon located in and recognising the major groove of operator DNA.
BTV NS4 shows strong relatedness to fzo-mitofusin protein, a putative transmembrane GTPase. The fzo protein has a coiled-coil structure and mediates mitochondrial fusion [36] . Another protein family with a coiled-coil structure, which also shows a strong match with BTV NS4, is EMP24_GP25L. Members of this family have been implicated in transporting 'cargo' from the endoplasmic reticulum (ER) and are related to the previously described GOLD domain [37] , which is always found combined with lipid-or membrane-association domains.
Sequence analyses indicate that PHSV NS4 (111 aa long) contains a coiled-coil domain between aa 75 and 111, YUOV NS4 (113 aa long) contains two coiled-coils domains between aa 5 to 45 and 75 to 105, and AHSV NS4 (143 aa long) contains coiled-coil domains between aa 5 to 85 and aa 110-140. The BTV NS4 (77 aa long) appears to contain only a single coiled coil structure, between aa 27 and 77.
Two overlapping potential nuclear localisation signals' (NLS) were identified in the aa sequence of PHSV NS4 ( . Although all of these NLS were monopartite, the GIV NS4 was found to contain a bipartite NLS (position 113-141: RKRGLEFLLLPLHEYVTHCAKEDIR-IYES). The prediction cut-off scores for all these NLS as defined by PredictNSL and cNLS ranged from 4 to 8, indicating dual nuclear/cytoplasmic localisations of a given protein [38] .
The aa region 55 to 129 of GIV NS4 showed 29% identity (55% similarity) to aa 1823 to 1890 of UTP20 (a component of the nucleolus).
NS4 of BTV and GIV were successfully cloned into pGEX-4T-2 and expressed in C41 at 28uC, as partially soluble proteins fused to GST (figure 2). The soluble fraction was used in competition assays with DNase I or endoribonucleases belonging to the RNAse III family and in binding assays with dsRNA. In contrast when these proteins were expressed in BL-21 they were totally insoluble and formed inclusion bodies. The inclusion bodies fraction was purified using bugbuster reagent, solubilised and used for immunization of rabbits.
Western blots analyses, using rabbit antisera raised against recombinant BTV-8 NS4, showed that NS4 is expressed in BTV-8 infected Culicoides KC cells and BHK-21 ( figure 3, figure 4) . The antiserum identified a single protein band with an apparent molecular weight of approximately 12 kDa in BTV-8 infected cells, which is close to the molecular weight calculated for NS4, from the sequence of Seg-9 (,10 kDa). The anti-BTV NS4 antiserum is therefore specific to NS4 and does not cross react with other viral proteins. Western blot analysis using non-infected BHK-21 cells, showed that anti-BTV NS4 rabbit antiserum does not cross react with cellular proteins. A similar analysis, using antisera raised against recombinant GIV NS4, identified a protein of approximately 20 kDa in GIV infected cells (figure 5), corresponding to the theoretical molecular weight of NS4 deduced from the sequence of GIV Seg-9. The anti-GIV NS4 antiserum is therefore specific to GIV NS4 and does not cross react with other viral proteins. Western blot analysis using non-infected BHK-21 cells, showed that anti-GIV NS4 rabbit antiserum does not cross react with cellular proteins. Western blot analyses of purified BTV virus particles showed no reaction with anti-BTV NS4 antibodies, indicating that NS4 is 'non-structural' ( figure 6A, 6B ). Figure 7 shows infected and non-infected BHK-21 cells probed with anti-BTV-8 VP2 antibodies raised in mice. Figure 8 shows infected and non-infected BHK-21 cells probed with anti-BTV8 immune serum from experimentally infected mice.
NS4 was identified in the nuclear fraction of BTV-infected BHK-21 cells harvested at 24 hours post-infection by western blot. Rabbit anti-BTV NS4 immune serum, identified the same band in the nuclear extract that was previously identified in infected cell lysates (figure 9). No band was identified in non-infected nuclear extracts.
NS4 was detected as early as 4 hours post-infection, mainly in the cytoplasm of BHK-21 cells infected with BTV-8 or GIV ( figure 10A, 10B) . At 24 hours post-infection, NS4 formed small aggregates throughout the cytoplasm and nucleus, suggesting that it makes specific interactions with itself and/or other infected cell components ( figure 11A, 11B) . This is consistent with bioinformatic analyses which identified nuclear localisation signals in NS4 of GIV and BTV (as well as YUOV, PHSV, EHDV, AHSV).
Although not all cells are morphologically intact at 72 hours postinfection, with GIV or BTV-8, at this stage NS4 was present in the cell membrane ( figure 12A, 12B) . This is consistent with bioinformatic analysis showing similarities between NS4 and membrane-associated proteins. Another set of cells, which were collected at 36 hours PI contained cells at different stages of infection. Those at an advanced stage of infection had depolymerised and depleted tubulin (figure 13A). No immuno-fluorescence signal was detected when non-infected cells were labelled using anti-NS4 antibodies (figure 13B, indicating that the anti-NS4 antiserum does not cross react with cellular proteins.
Further analyses with confocal microscopy identified nucleolar fluorescence using anti-NS4 antibodies in cells infected with either BTV or GIV. Localisation of the NS4 to the nucleoli was visible by confocal fluorescence as well as by overlaying the fluorescence signal onto cells imaged by differential interference contrast microscopy ( figure 14A, 14B) . Localisation of NS4 to the nucleoli was confirmed using anti-fibrillarin antibodies, giving a fluorescence signal that was super-imposable on that of NS4 in the nucleoli ( figure 15 ).
BHK-21 cells transfected with pCI-BTVNS4 or pCI-GIVNS4 resulted in expression of NS4 in both the cytoplasm and nucleus ( figure 16A, 16B) . NS4 was also detected in the nucleoli (figure 16B). Expressed NS4 was abundant in the cytoplasm where it formed aggregates similar to those found in infected cells. In many cells NS4 formed spherical bodies with 0.7 and 1 mm in diameter ( figure 16A and 16B) . Similar spherical bodies were occasionally also observed in cells infected with BTV-8 or GIV (figure 17A). Staining with the lipid stain oil-red-O, showed that these spherical bodies are associations between NS4 and lipid droplets ( figure 17B and figure 18A, 18B) . These bodies were identified in BTV-8 infected cells ( figure 17B and figure 18B) , where the oil-red-O stains lipids in the centre of the droplet while NS4 surrounds the lipid droplet. Figure 18A shows cells transfected with pCI-GIVNS4 stained with oil-red-O. Figure 19 shows non-infected cells stained with oil-red-O, where lipid droplets stain with red only. Similar data were recently reported for rotaviruses, where VP2, VP6 or NSP5 were found to associate with lipid droplets [39] .
The mice immune serum from an animal infected with BTV-8 identified a protein in cells transfected with pCI-BTVNS4 expressing BTV-8 NS4. The protein band had the same size as that identified by the anti-BTV NS4 rabbit immune serum in BTV-infected cells. No band was identified in non-transfected cells (figure 20).
Incubation of a dsRNA ladder (500-21 bp) with Dicer led to cleavage of long dsRNAs, generating 21 bp-long RNAs. Incubation of the dsRNA ladder with BTV NS4 or GIV NS4 alone did not alter dsRNA integrity. dsRNA preincubated with NS4 of GIV was protected against Dicer cleavage, consistent with previous findings regarding the presence of a dsRNA-binding domain. However, BTV NS4 did not protect dsRNA against Dicer and dsRNA was still processed into 21 bp long fragments, as analysed by agarose gel electrophoresis ( figure 21) . Incubation of dsRNA with BAV outer capsid protein VP9 (as a control) did not affect Incubation of a dsDNA ladder (2645-36 bp) with DNAse I led to degradation, while incubation with BTV or GIV NS4 only did not affect dsDNA integrity. However, dsDNA pre-incubated with either BTV or GIV NS4 was at least partially protected against 
In the colorimetric assay to detect NS4-dsRNA binding, the wells devoid of dsRNA were all negative, with a very low background (values close to zero). Negative control wells, containing only dsRNA, also had OD values close to zero (figure 23). Wells containing biotinilated dsRNA and BTV NS4 were also negative, indicating that BTV NS4 does not bind dsRNA. However, the wells containing dsRNA and GIV NS4, had increasing OD values with an almost linear relationship between the fixed NS4 concentration (150 ng/well) and the increasing dsRNA concentration, reaching a plateau at 320 ng of dsRNA/well (figure 23). This confirms the existence of a dsRNAbinding domain in GIV NS4, which is absent from BTV NS4.
Within the family Reoviridae, genome segments encoding more than one protein, from distinct, ORFs have been previously reported for the aquareoviruses, fijiviruses, orthoreoviruses, rotaviruses, phytoreoviruses and oryzaviruses [1] . Genome segments of the orthoreoviruses, phytoreoviruses, oryzaviruses and rotaviruses can be bi-or tri-cistronic with overlapping ORFs. Those in the phytoreoviruses (Seg-9 and Seg-12), orthoreoviruses (segment S1) and rotaviruses (Seg-11) were also found to be expressed in infected cell cultures [40, 41, 42, 43, 44] . Translation of overlapping ORFs from reovirus genome segments has usually been shown to be dependent on leaky scanning [41, 43, 44] although scanning-independent ribosome shunting has also been described [42, 45] .
An overlapping ORF in Seg-9, designated as ORFx, was recently identified by bioinformatic analysis in both insect-borne and tick-borne orbiviruses [17, 18] . ORFx appeared to encode a protein with the potential to bind dsRNA that was tentatively named as VP6db [18] . However, in line with previous orbivirus protein nomenclature, we have renamed these proteins, based on their theoretical size and absence from the virion, as non-structural protein 4 (NS4). GIV NS4 was previously shown to contain significant aa sequence matches with dsRNA-binding domains [18] . Further analyses of their amino acid sequences indicate that NS4 may be structured as 'coiled-coils' and that BTV NS4 exhibits significant relatedness (as identified by the pfam programme) with nucleic acid binding proteins that also have coiled-coils or helical structures and are associated with ER or cell membranes.
It was suggested that translation of ORFx may be initiated via 'leaky ribosome-scanning' [17] , although the presence of additional AUG codons between the VP6 initiation codon and the presumed NS4 initiation codon in some orbiviruses (including BTV) suggests that additional mechanisms for bypassing intervening AUG codons may be operating [17] . An A-rich polypurine tract is present upstream of the NS4 ORF in all of the sequenced Orbivirus species, except the tick borne St. Croix River virus (SCRV, [46] . The SCRV NS4-ORF (nt 101-379) is interrupted by a stop codon at position 217. Although hydrophobicity profiles of putative NS4 proteins from BTV, AHSV, PHSV, YUOV and GIV as analysed using the Kyte and Doolittle algorithm [22] are somewhat variable, overall they show broadly similar patterns of conserved domains, indicating that the NS4 proteins are generally hydrophilic ( figure 24 ).
BTV-8 infected KC or BHK-21 cells and GIV infected BHK-21 cells all contain NS4, as revealed by western blot analyses and confocal microscopy, confirming the existence of a new and previously un-described protein, encoded by ORFx of orbivirus Seg-9. NS4 has not previously been detected in purified BTV virus or core particles [20] . Western blot analyses of purified BTV-8 particles confirmed that the protein is 'non-structural'.
Bioinformatic analyses indicate that NS4 contains coiled-coils and is structurally related to other mammalian proteins, with helical or coiled-coil regions. These analyses also suggest that the NS4 may be functionally related to proteins involved in nucleic acid binding, or associated with lipids and membranes. Nuclear localisation signals were predicted in NS4 of PHSV, YUOV, EHDV, BTV, AHSV and GIV. All these proteins are rich in arginine and lysine residues that are essential for NLS [47] .
Double-stranded RNA-binding proteins (DRBPs) do not recognize specific nucleotide sequences but interact primarily with A-form double helix RNAs, which differ from the typical dsDNA B-form helix in that the minor groove is shallow and broad while the major groove is narrow and deep. This conformation allows DRBPs to bind non-specifically to dsRNAs. Indeed, the lack of nucleotide binding recognition suggests that target specificity may generally be governed through interactions with other proteins, since many DRBPs bind strongly but nonspecifically to any dsRNA structure in vitro. GIV NS4 can protect dsRNA from degradation by RNAse III endoribonucleases, confirming previous sequence analyses indicating the presence of a dsRNA-binding domains [18] . BTV NS4 which is half the theoretical size of its counterpart in GIV, lacks dsRNA binding domains and did not protect dsRNA from Dicer or RNAse III. NS4 of GIV and BTV both failed to protect ssRNA or ssDNA from degradation by RNAse A, or nuclease S1 respectively (data not shown). However, NS4 of both GIV and BTV did protect dsDNA from degradation by DNAse I, indicating an ability to bind dsDNA.
Fluorescent confocal microscopy confirmed that NS4 is expressed in both BTV and GIV infected cells, and starts to accumulate in the cytoplasm and nucleus (as fine aggregates) as early as 4 hours post-infection. However, at 72 hours postinfection NS4 was associated with the cell membrane. This is consistent with analyses suggesting similarities between NS4 and ER-lipid-or membrane-associated proteins [37] . Cells infected with BTV or GIV or transfected with plasmids expressing NS4, showed interaction of NS4 with lipid droplets within the cytoplasm. This is consistent with bioinformatic analysis that identified similarities between NS4 and lipid-associated domains. Similar data were recently reported for VP2, VP6 and NSP5 of rotavirus [39] . . Colorimetric assay to detect interactions of NS4 with dsRNA. The graph shows colorimetric OD readings plotted against concentrations of dsRNA. Increasing concentrations (from 1 to 640 ng) of a biotinylated dsRNA were bound to wells coated with streptavidin. BTV NS4 or GIV NS4 were added to the wells in triplicate. Wells not containing dsRNA/NS4 were included as negative controls. Wells from which dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls. Only wells containing the dsRNA to which GIV NS4 was added reacted with anti-GIV antibodies as indicated by increasing OD readings. The readings were almost linear (reaching a plateau at 320 ng of dsRNA) indicating that dsRNA acted as a target for binding of GIV NS4. doi:10.1371/journal.pone.0025697.g023
Viruses can interact with components of the nucleolus [48, 49] and viral proteins can co-localise with proteins such as nucleolin, B23 and fibrillarin (components of the nucleolus). The use of antifibrillarin antibodies identified NS4 in the nucleoli of cells harvested at 24 hours post-infection. Although NS4 was detected in the nucleoli late in infection, anti-fibrillarin antibodies failed to detect fibrillarin. This may reflect BTV induced apoptosis, leading to nuclear condensation and DNA fragmentation, blebbing of the plasma membrane and shrinkage [50, 51] , and/or host cell shut-off [4] . Similar findings were reported in rotavirus (another member of the family Reoviridae, genus Rotavirus) where NSP2 protein was found to cause depolymerisation of tubulin [52] .
As part of their replication strategy, viruses can use nucleolar components to favour viral transcription and translation, or alter the cell cycle [48, 49] . Western blot analysis indentified NS4 in the nuclear fraction of BTV infected cells, while immunofluorescence confocal microscopy co-localised NS4 to the nucleolus. GIV-NS4 showed sequence similarity to UTP20, a small subunit processome component and a component of the nucleolus. UTP20 is part of the U3 small nucleolar RNA (snoRNA) protein complex (U3 snoRNP) and is involved in 18S rRNA processing [53] . Whether NS4 interferes with the processing of the 18s rRNA remains to be clarified in future work.
The ability of GIV NS4 to protect dsRNA from cleavage by endoribonucleases of the RNAse III family and its ability to bind dsRNA agree with sequence analyses that indicated the presence of a dsRNA binding domain in GIV NS4 [18] . The inability of BTV NS4 to protect dsRNA from cleavage by endoribonucleases of the RNAse III family and its inability to bind dsRNA in a platebased colorimetric assay are in agreement with sequence analyses that failed to detect a dsRNA binding domain in its aa sequence [18] . Other reoviruses dsRNA-binding proteins include Sigma 3 of mammalian orthoreovirus (found in both the cytoplasm and nucleus), and pns10 of rice dwarf virus [54, 55] .
SCRV, which persistently infects tick cells but does not grow in mammalian cells, appears to have a non-functional NS4 ORF that is interrupted by a stop codon. These observations suggest that NS4 expression could play a role in productive infection of mammalian cells.
The data presented here show that the orbivirus genome encodes four distinct non-structural proteins (NS1-NS4). NS1 and NS3 play an important role in orbivirus exit mechanisms from infected cells [15] . BTV infects mammalian cells, usually resulting in a lytic infection, while infection of KC cells derived from the BTV vector Culicoides sonorensis, become persistently infected with little or no evidence of cell lysis [56, 57] . Previous work showed that intracellular expression of an NS1 specific antibody fragment (scFv) destabilised the formation of NS1 tubules in BTV infected cells [14] . As a consequence, cells became persistently infected and viruses exited by budding instead of via cell lysis. Although BTV NS3 is effectively expressed in insect cells [15] , it is much less abundant in mammalian cells [4] . It was suggested previously [14] that the relative levels of NS1 to NS3 synthesised during infection dictate the fate of cellular pathogenesis as of whether the virus exit occurs by lysis or budding.
The rapid accumulation of NS4 in the cytoplasm as early as 4 hours post-infection suggests that this protein plays an early role in the virus replication cycle. At 72 hours post-infection NS4 was absent from the nucleus which could be the consequence of changes affecting the nucleus and the integrity of the nuclear membrane. The presence of the NS4 in the plasma membrane late in infection suggests that it may play a role, alongside NS1 and NS3, in virus exit. Further co-localisation studies will be carried out to assess NS4 interactions with other viral or cellular protein components.  Geographic Distribution and Risk Factors of the Initial Adult Hospitalized Cases of 2009 Pandemic Influenza A (H1N1) Virus Infection in Mainland China BACKGROUND: As of 31(st) March 2010, more than 127,000 confirmed cases of 2009 pandemic influenza A (H1N1), including 800 deaths, were reported in mainland China. The distribution and characteristics of the confirmed cases in the initial phase of this pandemic in this country are largely unknown. The present study aimed to characterize the geographic distribution and patient characteristics of H1N1 infection in the 2009 pandemic as well as to identify potential risk factors associated with adverse patient outcome in China, through retrospective analyses of 885 hospitalized cases with confirmed H1N1 infection. METHODOLOGY/PRINCIPAL FINDINGS: The proportional hazards model was employed to detect risk factors for adverse outcome; the geo-statistical maps were used to characterize the distribution of all 2668 confirmed H1N1 patients throughout mainland China. The number of new cases increased slowly in May, 2009, but rapidly between June and August of the year. Confirmed cases were reported in 26 provinces; Beijing, Guangdong, Shanghai, Zhejiang and Fujian were the top five regions of the incidence of the virus infection. After being adjusted for gender, age, chronic pulmonary disease and other general symptoms, delay for more than two days before hospital admission (HR: 0.6; 95%CI: 0.5–0.7) and delayed onset of the H1N1-specific respiratory symptoms (HR: 0.3; 95%CI: 0.2–0.4) were associated with adverse patient outcome. CONCLUSIONS/SIGNIFICANCE: The 2009 pandemic influenza A affected east and southeast coastal provinces and most populous cities more severely than other regions in mainland China due to higher risk of high level traffic-, high population density-, and high population mobility-associated H1N1 transmission.The clinical symptoms were mild in the initial phase of infection. Delayed hospital admission and delayed appearance of respiratory symptoms were among the major risk factors for poor patient outcome. These findings may have significant implications in the future pandemic preparedness and response. The 2009 pandemic influenza A (H1N1) was first reported in Mexico and then rapidly spread around the world, unfortunately due to the 'convenience' of airplane-based modern transportation system. On 11 th June 2009, the World Health Organization (WHO) raised the warning level to the sixth phase, indicating that the episode of influenza had entered a pandemic stage [1] . As of 11 th April 2010, more than 214 countries and territories or communities had reported laboratory-confirmed cases of 2009 H1N1, including over 17798 deaths [2] . The first case in mainland china was reported on May 11 th , 2009. The Ministry of Health of the People's Republic of China released Guidelines for H1N1 Influenza Diagnosis and Treatment in the middle of July, 2009 [3] . By March 31 st 2010, more than 127,000 laboratorial confirmed cases including 800 deaths were reported in the country [4] .
Since the H1N1 pandemic in 2009, numerous field investigations and epidemiological studies have investigated the spatial-temporal dynamics, geographic distribution and patient characteristics of the 2009 H1N1 pandemic. Through analyses of a total of 377 H1N1associated deaths in the United States, Fowlkes et al. demonstrated that the H1N1-associated mortality rate varied substantially in different geographic regions (i.e. highest in Hawaii, New York and Utah) and in different age groups of the infected population (i.e. 76% in patients aged 18-65 years and 9% in patients aged $ 65 years) [5] . Similar geographic region-and patient age-dependent incidence of H1N1 infection and subsequent mortality have also been demonstrated in South America [6] , and Australia [7] . In addition, the susceptibility to H1N1 infection may vary in different races; Wenger et al. shown that Alaska Native people and Asian/ Pacific Islanders (A/PI) were 2-4 times more likely to be infected by H1N1 virus and hospitalized than white Caucasians [8] .
To date, little is known regarding the spatio-temporal characteristics of the 2009 pandemic H1N1 as well as factors affecting the recovery of the infected patients in mainland China, especially for the initial cases, who were younger and more mobile than general population. The present study was therefore conducted to investigate the geographic distribution of H1N1 infection in the 2009 pandemic as well as the risk factors for adverse patient outcome after initial H1N1 virus infection in mainland China. Our goal was to generate solid scientific bases for adequate preparedness for potential H1N1 pandemic in the future.
Basic characteristics/denigraphics of the patients Given the unique nature of 2009 pandemic H1N1, both suspected and laboratory confirmed cases had to be reported to the Chinese Centre for Disease Control and Prevention (CCDC). During May 7 th and August 12 th , 2009, the number of reported laboratory-confirmed H1N1 cases was 2668. In this study, we analyzed the data of 885 adult hospitalized patients, accounting for 68.6% of the total confirmed cases. The major demographic and clinical characteristics of these patients are summarized in Table 1 .
Of the 885 hospitalized adult cases, 590 (66.7%) experienced a delay#2 days in hospital admission, 815 (92.1%) completely recovered and were free of H1N1-associated symptoms on discharge whereas 70 (7.9%) were censored (not recovered when left hospitals). The median time from illness onset to hosptial discharge was 7 days (inter-quartile range 2-22).
The analyses using the proportional hazards model showed that gender, time from illness onset to hospital admission, general symptoms and respiratory symptoms were all related to the outcome of the H1N1 patients (Table 2) . Compared with male patients, females patients had a lower porpotion of recovery (HR 0.9, 95%CI: 0.7-1.0), but the differnence was not statistically significant. Delay in hospital admission (longer than 2 days) decreased the probability of recovery (HR 0.6, 95%CI: 0.5-0.7). General symptoms and respiratory symptoms were adversely associated with the rate of recovery in hospital with HR of 0.7 (95%CI: 0.5-1.0) and 0.3 (95%CI: 0.2-0.4), respectively. Showed in Figure 1 are the cumulative resolution rates. It is apparent that the cumulative resolution rate was lower in patients with delayed hospital admission than in those promptly admitted (x 2 = 60.978, P,0.001), and lower in patients with general symptoms (x 2 = 3.977, P = 0.046) or respiratory symptoms (x 2 = 100.261, P,0.001) than in those without corresponding symptoms. No significant difference was observed between genders (x 2 = 1.634, P = 0.201).
The weekly reported new cases of confirmed H1N1 infection in different geographic regions of mainland China during May 7 th to August 12 th , 2009 are presented in Figure 2 . A total of 26 provinces or cities had confirmed cases of infection, among which Beijing city had the highest number of infection (670 cases), followed by Guangdong province(628 cases), Shanghai city(318 cases), Zhejiang province (214 cases), and Fujian province(195 cases).
The number of new cases increased slowly in May 2009, but rapidly between June and August. In May, 87.3% of the cases were imported ones, while the domestic cases (67.3%) became dominant between June and August.
Most of the provinces had reported laboratory-confirmed cases by August. However, the cases were more likely to occur in the southeast coastal areas or areas that serve as major transport stations with relatively prosperous economy, convenient transport system and greater population.
In this study, patients with leukocyte and neutrophils abnormalities as well as patients undergoing antivirus treatment were excluded from the Cox regression model. In seasonal influenza, cough, mild leukopenia, and mild C-reactive protein elevation are relatively common clinical manifestations [9] . Pathologically, the H1N1 virus has features similar to those encountered in the infection with other highly virulent influenza A viruses such as the 1918 H1N1 and H5N1 viruses [10] . However, as the disease was generally a mild procedure, typical inflammation molecules might not be qualified to serve as diagnostic markers. For patients with complications, the secondary bacterial infection which causes elevation in leucopenia and C-reactive protein is most likely to occur. The widely used antivirus treatment like oseltamivir might increase the proportion of oseltamivir-resistant influenza A. Therefore, alternative treatment and vaccination (although, low coverage) are highly recommended.
It has been documented that the adverse outcome, particularly a severe form (i.e. entering ICU or death), after influenza infection is associated with numerous risk factors, including age and underlying medical conditions of the patients. Nolan et al. in a recent study on pandemic or seasonal influenza have observed that infants, the elderly and people suffering from chronic diseases are of high risks for adverse outcome [11] ; some other authors have demonstrated significant adverse clinical outcome in patients aged $20 years and patients delayed in hospital admission [12, 13] . Nevertheless, only adult hospitalized cases were included in the present study.
With the fast-growing public transportation and increasing socio-economic activities, population mobility has become a concerned problem in the prevention of infectious diseases. Ever since the outbreak of SARS, the central role that Geographic Information System (GIS) plays in the early detection and rapid response to infectious diseases has become evident to researchers as well as policy markers [14, 15] . However, how to integrate GIS into the traditional epidemiological model is a key challenge [16] . Use of GIS in our analyses in this study showed that the incidence of H1N1 infection in the 2009 pandemic in mainland China was higher in the coastal provinces in the southeast as well as in Beijing and Shanghai, two of the most populous cities in the country, where the infection occurred in clusters in the initial phase, followed by spread to adjacent provinces. Moreover, the analyses also demonstrated that within some of the provinces with confirmed cases, the number of H1N1 infection varied widely as well. In most cities and districts, there was still no H1N1 confirmed cases, the overall spread level of H1N1 virus was still regional. Nevertheless, our analyses on the H1N1 spread and the geographic distribution were qualitative only; further quantitative analyses such as correlation or Poisson regression are needed to test the hypothesis that the H1N1 virus spreads more quickly in the crowd area with more convenient travel system.
The limitation of this study is that it only enrolled the adult hospitalized patients. This study included no pediatric cases and therefore was unable to assess the differences in risk factors for adverse patient outcome between children and adults. In the initial phase of H1N1 infection in mainland China, hospital admission was required of every confirmed case. The information on the confirmed cases was from the Chinese Infectious Disease Surveillance System. Sub-clinical infection was common in H1N1 infection. People with mild symptoms might not be included in the Chinese Infectious Disease Surveillance System, possibly causing bias in our analyses. Further studies were needed in the mathematical model and simulation for the transmission characteristics.
Despite the limitations, this study through retrospectively analyses characterized clinical features, disease course, and hospitalization period of the 2009 pandemic H1N1 in mainland China. In adult H1N1 patients, age, chronic pulmonary disease and the appearance of general symptoms, delayed hospital admission and the appearance of respiratory symptoms but not gender were independent risk factors for adverse outcome. Our study also documented the spatial and temporal characteristics of the 2009 pandemic H1N1 at its early stage in mainland China.
The study was approved by ethic committees of both Capital Medical University in Beijing, China and the Chinese Centre for Disease Control and Prevention. 
Data on a total of 885 adult confirmed H1N1 cases from the CCDC. A confirmed case was defined as a person with influenzalike clinical manifestations and positive laboratory test for H1N1 virus, as assessed with the use of a reverse-transcriptasepolymerase-chain-reaction (RT-PCR) assay. Those patients who were still asymptomatic but positive for the RT-PCR assay were quarantined. The cases included in this study were those reported in the early phase of the epidemic between May 7 th and August 16 th , 2009 to the CCDC by sub-CCDC offices after face to face interviews with the individuals of suspected infection.
The case report form was used, which included the following information: gender, age, chronic pulmonary disease history, time from onset to hospital admission, general symptoms (at least one of the following: body temperature$37.5uC, dizziness, headache, chill, myalgia, arthralgia, weakness, anorexia, and/or conjunctivitis), respiratory symptoms (at least one of the following: runny nose, nasal obstruction, sneezing, dry cough, sputum, sore throat, pharyng itching, shortness of breath, and/or chest pain), pharyngeal abnormality, tonsil abnormality, white blood cells count, complication(s) (at least one of pneumonia, liver function impairment, and/or myocardic injury), antiviral medication and outcome.
Recovery was defined as resolution of symptoms as well as negative test results for H1N1 virus RNA for two consecutive days in throat swabs.
Survival analysis was used to evaluate adverse factors for recovery (days from illness onset to recovery) in the early phase of H1N1 epidemic. Potential risk factors assessed included gender, age, chronic pulmonary disease, time from onset to hospital admission, general symptoms, respiratory symptoms, pharyngeal abnormality, tonsil abnormality, white blood cells count, complication and antiviral medication.
The parameters of the recovery model were estimated by backward stepwise likelihood ratio test in proportional hazards model. Censored patient was considered as not recovered. Twosided P values and 95% confidence interval of the parameters were calculated. The cumulative resolution rate was analyzed using Kaplan-Meier analysis and log-rank test and compared between subgroups of patients. The subgroups were based on gender, time from onset to hospital admission, general symptoms or respiratory symptoms. Geo-statistical maps were used to characterized the spatial and temporal distribution of weekly confirmed cases during May 7 th and August 12 th . Longitude and latitude coordinates of each province were identified from a national GIS database. The procedure was fulfilled in a GIS (ArcInfo 9.2, ESRI, Redlands, CA) by overlaying a national vector map.
All statistical analyses were conducted with the SPSS 13.0 software for Windows (SPSS Inc, Chicago, IL, USA). Inferring viral quasispecies spectra from 454 pyrosequencing reads BACKGROUND: RNA viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. The genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences. RESULTS: In this paper, we introduce a new Viral Spectrum Assembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html. CONCLUSIONS: ViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations. Results: In this paper, we introduce a new Viral Spectrum Assembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html.
Conclusions: ViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations.
Many viruses (including SARS, influenza, HBV, HCV, and HIV) encode their genome in RNA rather than DNA. Unlike DNA viruses, RNA viruses lack the ability to detect and repair mistakes during replication [1] and, as a result, their mutation rate can be as high as 1 mutation per each 1,000-100,000 bases copied per replication cycle [2] . Many of the mutations are well tolerated and passed down to descendants, producing a family of co-existing related variants of the original viral genome referred to as quasispecies, a concept that originally described a mutation-selection balance [3] [4] [5] [6] [7] .
The diversity of viral sequences in an infected individual can cause the failure of vaccines and virus resistance to existing drug therapies [8] . Therefore, there is a great interest in reconstructing genomic diversity of viral quasispecies. Knowing sequences of the most virulent variants can help to design effective drugs [9, 10] and vaccines [11, 12] targeting particular viral variants in vivo.
Briefly, the 454 pyrosequencing system shears the source genetic material into fragments of approximately 300-800 bases. Millions of single-stranded fragments are sequenced by synthesizing their complementary strands. Repeatedly, nucleotide reagents are flown over the fragments, one nucleotide (A, C, T, or G) at a time. Light is emitted at a fragment location when the flown nucleotide base complements the first unpaired base of the fragment [13, 14] . Multiple identical nucleotides may be incorporated in a single cycle, in which case the light intensity corresponds to the number of incorporated bases. However, since the number of incorporated bases (referred to as a homopolymer length) cannot be estimated accurately for long homopolymers, it results in a relatively high percentage of insertion and deletion sequencing errors (which respectively represent 65%-75% and 20%-30% of all sequencing errors [15, 16] ).
The software provided by instrument manufacturers were originally designed to assemble all reads into a single genome sequence, and cannot be used for reconstructing quasispecies sequences. Thus, in this paper we address the following problem:
Given a collection of 454 pyrosequencing reads generated from a viral sample, reconstruct the quasispecies spectrum, i.e., the set of sequences and the relative frequency of each sequence in the sample population.
A major challenge in solving the QSR problem is that the quasispecies sequences are only slightly different from each other. The amount and distribution along the genome of differences between quasispecies varies significantly between virus species, as different species have different mutation rates and genomic architectures. In particular, due to the lower mutation rate and longer conserved regions, HCV quasispecies are harder to reconstruct than quasispecies of HBV and HIV. Additionally, the QSR problem is made difficult by the limited read length and relatively high error rate of high throughput sequencing data generated by current technologies.
The QSR problem is related to several well-studied problems: de novo genome assembly [17] [18] [19] , haplotype assembly [20, 21] , population phasing [22] and metagenomics [23] . As noted above, de novo assembly methods are designed to reconstruct a single genome sequence, and are not well-suited for reconstructing a large number of closely related quasispecies sequences. Haplotype assembly does seek to reconstruct two closely related haplotype sequences, but existing methods do not easily extend to the reconstruction of a large (and a priori unknown) number of sequences. Computational methods developed for population phasing deal with large numbers of haplotypes, but rely on the availability of genotype data that conflates information about pairs of haplotypes. Metagenomic samples do consist of sequencing reads generated from the genomes of a large number of species. However, differences between the genomes of these species are considerably larger than those between viral quasispecies. Furthermore, existing tools for metagenomic data analysis focus on species identification, as reconstruction of complete genomic sequences would require much higher sequencing depth than that typically provided by current metagenomic datasets.
In contrast, achieving high sequencing depth for viral samples is very inexpensive, owing to the short length of viral genomes. Mapping based approaches to QSR are naturally preferred to de novo assembly since reference genomes are available (or easy to obtain) for viruses of interest, and viral genomes do not contain repeats. Thus, it is not surprising that such approaches were adopted in the two pioneering works on the QSR problem [24, 25] . Eriksson et al. [24] proposed a multi-step approach consisting of sequencing error correction via clustering, haplotype reconstruction via chain decomposition, and haplotype frequency estimation via expectation-maximization, with validation on HIV data. In Westbrooks et al. [25] , the focus is on haplotype reconstruction via transitive reduction, overlap probability estimation and network flows, with application to simulated error-free HCV data. Recently, the QSR software tool ShoRAH was developed [26] and applied to HIV data [27] . Another combinatorial method for QSR was also developed and applied to HIV and HBV data in [28] , with results similar to those of ShoRAH. Our contributions in this paper are as follows:
• A novel QSR tool called Viral Spectrum Assembler (ViSpA) taking into account sequencing errors at multiple steps,
• Comparison of ViSpA with ShoRAH on HCV synthetic data both with and without sequencing errors, and
• Statistical and experimental validation of the two methods on real 454 pyrosequencing reads from HCV and HIV samples.
Our method for inferring the quasispecies spectrum of a virus sample from 454 pyrosequencing reads consists of the following steps (see Fig. 1 ):
• Constructing the consensus virus genome sequence for the given sample and aligning the reads onto this consensus,
• Preprocessing aligned reads to correct sequencing errors,
• Constructing a transitively reduced read graph with vertices representing reads and edges representing overlaps between them,
• Selecting paths in the read graph that correspond to the most probable quasispecies sequences, and assembling candidate sequences for selected paths by weighted consensus of reads, and
• Estimating candidate sequence frequencies by EM Below we describe each step separately.
We assume that a reference genome sequence of the particular virus strain is available (e.g., from NCBI [29] ). Since viral genomes do not have sizable repeats and the quasispecies sequences are usually close enough to the reference sequence, the majority of reads can typically be uniquely aligned onto the reference genome. However, a significant number of reads may remain unaligned due to differences between the reference genome and sequences in the viral sample.
In order to recover as many of these reads as possible, we iteratively construct a consensus genome sequence from aligned reads.
In particular, we first align 454 pyrosequencing reads to the reference sequence using the SEGEMEHL software [30] . Then we extend the reference sequence with a placeholder I for each nucleotide inserted by at least one uniquely aligned read. Similarly, we add a placeholder D to the read sequence for each reference nucleotide missing from the aligned read. Then we perform sequential multiple alignment of the previously aligned reads against this extended reference sequence. Finally, the consensus genome sequence is obtained by (1) replacing each nucleotide in the extended reference with the nucleotide or placeholder in the majority of the aligned reads and (2) removing all I and D placeholders, respectively corresponding to rare insertions and to deletions found in a majority of reads. Reads may contain a small portion of unidentified nucleotides denoted by N'swe treat N as a special allele value matching any of nucleotides A, C, T, G, as well as placeholders I, and D.
Iteratively, we replace the reference with the consensus and try to align the reads, for which we could not find any acceptable alignment previously. Our experiments on a dataset consisting of approximately 31,000 454 pyrosequencing reads generated from a 5.2kb-long HCV fragment (see data description in Results and Discussions) show that 85% of reads are uniquely aligned onto the reference sequence and an additional 9% of the reads are aligned onto the final consensus sequence. Reads that cannot be aligned onto the final consensus are removed from the further consideration. 
Since aligned reads contain insertions and deletions, we use placeholders I and D to simplify position referencing among the reads. All placeholders are treated as additional allele values but they are removed from the final assembled sequences. First, we substitute each deletion in the aligned reads with placeholder D. Deletion supported by a single read is replaced either with the allele value, which is present in all other reads overlapping this position, or with N, signifying an unknown value, otherwise. Next, we fill with placeholder I each gap in a read corresponding to the insertions in the other reads. All insertions supported by a single read are removed from consideration.
We begin with the definition of the read graph, introduced in [25] and independently in [24] , and then describe the adjustments that need to be made to read graph construction and edge weights to account for sequencing errors as well as the high mutation rate between quasispecies.
The read graph G = (V, E) is a directed graph with vertices corresponding to reads aligned with the consensus sequence. For a read u, we denote by b(u), respectively e(u), the genomic coordinate at which the first, respectively the last, base of u gets aligned. A directed edge (u, v) connects read u to read v if a suffix of u overlaps with a prefix of v and they coincide across the overlap. Two auxiliary vertices -a source s and a sink t are added such that s has edges into all reads with zero indegree and t has edges from all reads with zero outdegree. Then each st-path corresponds to a possible candidate quasispecies sequence. The read graph is transitively reduced, i.e., each edge e = (u, v) is removed if there is a uv-path not including edge e. Note that certain reads can be completely contained inside other reads. Let a superread refer to a read that is not contained in any other read and let the rest of the reads be called subreads. Subreads are not used in the construction of the read graph, but are taken into account in the final assembly of candidate sequences and frequency estimation.
Since the number of different st-paths is exponential, we wish to generate a set of paths that have high probability to correspond to real quasispecies sequences. In order to estimate path probability, we independently estimate for each edge e the probability p(e) that it connects two reads from the same quasispecies, and then multiply estimated probabilities for all edges on the path. Under the assumption of independence between edges, if we assign to each edge e a cost equal tolog (p(e)) = log(1/p(e)), then the minimum-cost st-path will have the maximum probability to represent a quasispecies sequence.
For reads without errors, [25] estimated the probability that two reads u and v connected by edge (u, v) belong to the same quasispecies as
is the overhang between reads u and v [25] , N = #reads, q = #quasispecies, and L = #starting positions. Thus, in this case the cost of an edge with overhang Δ can be approximated by Δ ∝ log(1/p Δ ).
To account for sequencing errors, we adjust the construction of the read graph to allow for mismatches. We use three parameters: (1) n = #mismatches allowed between a read and a superread, (2) m = #mismatches allowed in the overlap between two adjacent reads, and (3) t = #mismatches expected between a read and a random quasispecies. The probability that two reads u and v with j mismatches within an overlap of length o = e(u) b(v) belong to the same quasispecies can be estimated as:
where ε is the estimated 454 sequencing error rate. As in the case of error-free reads, defining the edge costs as ensures that stpaths with low cost correspond to most likely quasispecies sequences.
To generate a set of high-probability (low-cost) paths that are rich enough to explain observed reads, we compute for each vertex in the read graph the minimum cost st-path passing through it. Finding these paths is computationally fast. Indeed, we only need to compute two shortest-paths trees in G, one outgoing from s and one incoming into t; the shortest st-path passing through a vertex v is the concatenation of the shortest s v-and vt-paths.
Preliminary simulation experiments (see Additional File 1) show that better candidate sets are generated when edge costs c defined by (1) and (2) are replaced by e c . In fact, if we use even faster dependency on c then we obtain better candidate sets. The fastest growing cost effectively changes the shortest path into so called maxbandwidth path, i.e., paths that minimizes maximum edge cost for the entire path and for each subpath. So, ViSpA generates candidate paths using this strategy.
When no mismatches are allowed in the construction of the read graph, finding the candidate sequence corresponding to a st-path is trivial, since by definition adjacent superreads coincide across their overlap. When mismatches are allowed, we first assemble a consensus sequence from superreads used by the st-path. It may be not the best choice, especially when the coverage with superreads is low. Hence, we replace each initial candidate sequence with a weighted consensus sequence obtained using both superreads and subreads of the path, as described below.
For each read r, we compute the probability that it belongs to a particular initial candidate sequence s as:
where l and L denote the lengths of the read and initial candidate sequence, respectively, k is the number of mismatches between the read and the initial candidate sequence s, and t/L is the estimated mutation rate. Then final candidate sequence is computed as the weighted consensus over all reads, where the weight of a read is the probability that it belongs to the sequence. Note that, unlike the case without mismatches, the same candidate sequence can be obtained from different candidate st-paths, so we remove duplicates at the end of this step.
We assume that reads R with observed frequencies
where generated from a quasispecies population Q as follows. First, a quasispecies sequence q Q is randomly chosen accordingly to its unknown frequency f q . A read starting position is generated from the uniform distribution and then a read r is produced from quasispecies q with j sequencing errors. The probability of this event is calculated as h q r j l lj j , ( )
where l is the read length and ε is the sequencing error rate. 
In our simulation studies we use the following read data sets.
In order to perform cross-validation on the assembly method, we simulate reads data from 1739-bp long fragment from the E1E2 region of 44 HCV sequences [32] when sequence frequencies are generated according to some specific distribution. In our simulation experiments, we use geometric distribution (i-th sequence is constant factor more frequent than the (i + 1)-th sequence) to create sample quasispecies populations with different number of randomly selected above-mentioned quasispecies sequences. We first simulate reads without sequencing errors: the length of a read follows normal distribution with a particular mean value and variance 400, and a starting position follows the uniform distribution. This simplified model of reads generation has two parameters: number of the reads that varies from 20K up to 100K and the average read length that varies from 200bp up to 600bp.
Additionally, we simulate 454 pyrosequencing reads from 10 quasispecies sequences (following geometric distribution of frequencies) out of 44 HCV sequences [32] using FlowSim [33] . We generated 30K reads with average length 350bp.
The data set Data1 has been received from HCV Research Group in Institute of Biomedical Research, at University of Birmingham. Data1 contains 30,927 reads obtained from the 5.2kb-long fragment of HCV-1a genome (which is more than a half of the entire HCV genome). The average (aligned) read length average is 292bp but it significantly varies as well as the depth of position coverage (see Additional File 1 for details). The depth of reads coverage variability is due to a strong bias in the sequence start points, reflecting the secondary structure of the template DNA or RNA used to generate the initial PCR products. As a result, shorter reads are produced by GC-rich sequences. Data1 is available upon request from the authors.
The HIV dataset [27] contains 55,611 reads from mixture of 10 different 1.5kb-long region of HIV-1 quasispecies, including pol protease and part of the pol reverse transcriptase. The aligned reads length varies from 35bp to 584bp with average about 345bp (see Additional File 1 for details). In contrast to [27] , we do not filter out reads with low-quality scores.
In all our experimental validations, we compare the proposed algorithm ViSpA with the state-of-the-art tool ShoRAH as well as with ViSpA on ShoRAH-corrected reads (ShoRAHreads + ViSpA). We say the quasispecies sequence is captured if one of the candidate sequences exactly matches it. We measure the quality of assembling by portion of the real quasispecies sequences being captured by candidate sequences (sensitivity = +
) and its portion among candi- Here, we see advantage of ViSpA over ShoRAH.
Following [24] , we measure the prediction quality of frequency distribution with Kullback-Leibler divergence, or relative entropy. Given two probability distributions, relative entropy measures the "distance" between them, or, in the other words, the quality of approximation of one probability distribution by the other distribution. Formally, the relative entropy between true distribution P and approximation distribution Q is given by the formula:
where summation is over all reconstructed original sequences I = {i | P(i) > 0, Q(i) > 0} , i. e., over all original sequences that have a match (exact or with at most k mismatches) among assembled sequences. The relative entropy is decreasing with increasing of the average read length. It is expected since sensitivity is increasing with increasing of the average read length and EM predicts underlying distribution more accurately. ViSpA algorithm considerably outperforms ShoRAH (see Fig. 2 (right)).
However, ShoRAH has a significant advantage over ViSpA on a read data simulated by FlowSim both in prediction power and in robustness of results (see Table  1 ). Indeed, ShoRAH correctly infers 3 out of 10 real quasispecies sequences whereas ViSpA reconstructs only 1 sequence. Additionally, 10 most frequent assemblies inferred by ShoRAH are more robust with repeating up to 45% of times on 10%-reduced data versus 1% of times for ViSpA's assemblies. This advantage can be explained by superior read correction in ShoRAH. If ViSPA is used on ShoRAH-corrected reads, the results drastically improves: 5 quasispecies sequences are inferred and exactly 95% of times are repeated on reduced data, confirming that ViSpA is better in assembling sequences (see Table 1 ). Experimental validation on 454 pyrosequencing reads from HCV samples
We first discuss the choice of parameters of the read graph and candidate sequence assembly from stpaths. Then we give statistical validation for obtained 10 most frequent quasispecies sequences.
We infer quasispecies spectrum based on the read graphs constructed with various numbers n and m (numbers of mismatches allowed for superreads and overlaps corresponding to edges). We sort the estimated frequencies in descending order and count the number of sequences which cumulative frequency is 85%, 90%, and 95%. Fig. 3 reports these numbers as a percent of the total number of candidate sequences. There is an obvious drop in percentage for all three categories if we allow up to n = 6 mismatches to cluster reads and up to m = 15 mismatches to create edges. In this case, the constructed read graph has no isolated vertices.
To refine assembled candidate sequences, we use all reads and parameter t varying from 80bp till 350bp, or, in the other words, mutation rate varying from 1.75% up to 8% per sequence (which is in the range observed in [34] ). Out of 3207 max-bandwidth paths, we obtain as much as 938 distinct sequences (t = 80) and as low as 755 sequences (t = 350) for different values of t [80; 350].
The neighbor-joining tree for the most frequent 10 candidate sequences obtained by ViSpA and ShoRAH (see Fig. 4 ) reminds a neighbor-joining tree for HCV quasispecies evolution. Additionally, the most frequent candidate sequence found by ViSpA is 99% identical to one of the actual ORFs obtained by cloning the quasispecies. The quasispecies sequence is considered found if one of candidate sequences matches it exactly (k = 0) or with at most k (1 or 9) mismatches. All methods are run 100 times on 10% -reduced data. For the i-th (i = 1, .., 10) most frequent sequence assembled on the whole data, we record its reproducibility, i.e., percentage of runs when there is a match (exact or with at most k mismatches) among 10 most frequent sequences found on reduced data. "Reproducibility: Max" and "Reproducibility: Average" report respectively maximum and average of those percentages." Figure 3 Percentage of candidate sequences which cumulative frequency is 85%, 90%, and 95%. The values on x-axis corresponds to the number of allowed mismatches during read graph construction. n_m means that up to n mismatches are allowed in superreads and up to m mismatches are allowed in edges.
Viral sequences containing internal stop codons are not viable since the entire HCV genome consists of a single coding region for a large polyprotein. So the number of reconstructed viable sequences can serve as an accuracy measure for quasispecies assembly. Out of 10 most frequent sequences reconstructed by ViSpA, only 3 are not viable while ShoRAH is able to reconstruct only one viable sequence. This sequence has 99.94% similarity with the ViSpA's fourth most frequent assemblies. Both methods returned similar frequency estimations for this sequence: 0.017% (ShoRAH) and 0.019% (ViSpA).
Both ShoRAH and ViSpA (n = 6, m = 15) are run on eight 2.66GHz-CPUs with 8M cache. They take around 40 minutes to assemble sequences and estimate their frequencies. Smaller value of n increases ViSpA's runtime since its bottleneck (candidate sequences assembling) is proportional to the number of reads times number of paths. Indeed, smaller value of n results in larger number of superreads in built read graph, thus, in larger set of candidate paths. For example, ViSpA runs 90 minutes for n = 2, m = 2.
The plot on Fig. 5 shows validation results for 10 most frequent quasispecies sequences with respect to EM estimations assembled on Data1 by ShoRAH and ViSpA (n = 6, m = 15, and t = 120). Repeatedly, 100 times we have deleted randomly chosen 10% of reads and run both methods on each reduced read instance to reconstruct quasispecies spectrum.
The plot reports the percentage of runs when each of 10 most frequent sequences assembled on Data1 are reproduced among the 10 most frequent quasispecies Figure 4 The neighbor-joining phylogenetic tree for 10 most frequent HCV quasispecies variants on a 5,205bp-long fragment obtained by ViSpA and ShoRAH. Sequences are labeled with software name and its rank among 10 most frequent assembled sequences.
Percentage of runs when the i-th most frequent sequence is reproduced among 10 most frequent quasispecies assembled on the 10%-reduced set of reads. The i-th point at x-axis corresponds to the i-th most frequent sequence assembled on the 100% of reads. No data are shown for the sequences that are reproduced less than 5% of runs.
inferred on the reduced instances with no mismatches (k = 0), or with k = 1, 2, 5 mismatches. For example, for k = 0 ShoRAH repeatedly (35% of times) reconstructs only the third most frequent sequence while ViSpA reconstructs 7 sequences in at least 15% times, and the most frequent sequence is reconstructed 40% times. This plot shows that the found sequences are pretty much reproducible for ViSpa.
In order to compare ViSpA and ShoRAH, we run both of the methods on HIV dataset, used in the first experiment in [27] . As said above, we do not preprocess reads with respect to its 454 quality score, and it can explain poorer performance of ShoRAH. Indeed, ShoRAH correctly infers only 2 quasispecies sequences with at most 4 mismatches: one assembly has 3 mismatches with real quasispecies sequence, and the other has 4 mismatches.
ViSpA correctly reconstructs 5 quasispecies with at most 2 mismatches (3 of them among 10 most frequent assemblies): two sequences are inferred without any mismatches (one is among 10 most frequent assemblies), one assembly has 1 mismatch with real quasispecies sequence (and it is among 10 most frequent assemblies), and the rest sequences have 2 mismatches (one is among 10 most frequent assemblies). The assemblies correspond to a viable protein sequences.
If ViSpA is applied to ShoRAH-corrected reads, it can successfully infer three real quasispecies without any mismatches.
In this paper, we have proposed and implemented ViSpA, a novel software tool for quasispecies spectrum reconstruction from high-throughput sequencing reads. The ViSpA assembler takes into account sequencing errors at multiple steps, including mapping-based read preprocessing, path selection based on maximum bandwidth, and candidate sequence assembly using probability-weighted consensus techniques. Sequencing errors are also taken into account in ViSpA's EM-based estimation of quasispecies sequence frequencies.
We have validated our method on simulated error-free reads, FlowSim-simulated reads with sequencing errors, and real 454 pyrosequencing reads from HCV and HIV samples. We are currently exploring extensions of ViSpA to paired-end reads; the main difficulty is selection of pair-aware candidate paths. We also foresee application of ViSpA's techniques to the analysis of high-throughput sequencing data from microbial communities [23] and ecological samples of eukaryote populations [35] .
The ViSpA source code is available at http://alla.cs.gsu. edu/~software/VISPA/vispa.html.
Additional file 1: Supplementary Materials. The file contains derivation of edge cost formula (2) and EM algorithm, example of read graph construction and analysis of 454 pyrosequencing data. Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2)) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2) (VLP(VP40-GP)) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40) (particles lacking GP(1,2)) caused an aberrant response. This suggests that GP(1,2) binding to macrophages plays an important role in the immediate cellular response. Zaire ebolavirus (ZEBOV) is a member of the family Filoviridae within the order Mononegavirales [1] . It was discovered in 1976 in what is now the Democratic Republic of the Congo [2] as the etiological agent of a severe human viral hemorrhagic fever known as Ebola Hemorrhagic Fever (EHF). Infection with ZEBOV typically results in a rapidly fatal illness associated with high-level viremia, lack of an effective immune response, drastic lymphopenia, a severe coagulation disorder including disseminated intravascular coagulation and limited hemorrhages, widespread focal tissue necroses, systemic shock and multiorgan failure (reviewed in detail in [3] ). While the pathogenesis of ZEBOV infection has been relatively well described in experimental animals [4, 5] , only a few studies were reported that shed light on the molecular events following infection in humans. Unfortunately, these studies are partially contradictory. For instance, higher serum cytokine concentrations (IFN-a, IFN-c, IL-2, IL-6, and TNF-a) were measured in seven fatally infected patients compared to two survivors in one study, suggesting that a hyperactive immune responses may contribute to fatal outcome [6] . Other studies, describing the responses of eight fatally infected patients and four survivors, did not reveal significant concentration differences of IFN-a, IL-2, and TNF-a. On the other hand, that study suggested that fatal infections are due to generalized immunosuppression, including decreased IFN-c, IL-2, and IL-4 concentrations, lymphocyte apoptosis, and diminished IgG synthesis [7, 8, 9, 10] . The largest study to date included 42 fatally infected patients and 14 survivors. Hypersecretion of proinflammatory cytokines, chemokines and growth factors (IL-1b, IL-1RA, IL-6, IL-8, IL-15, IL-16, CXCL1 (GROa), CXCL10 (IP-10), eotaxin, M-CSF, MIP-1a, MIP-1b, MCP-1, MIF) and decreased concentrations of T lymphocyte-derived cytokines (IL-2, IL-3, IL-4, IL-5, IL-9, IL-13) concomitant to apoptotic loss of CD4 and CD8 T lymphocytes were typical for fatal cases [11] . Unfortunately, all these data reveal only the extent of homeostatic disarray in ZEBOV-infected individuals, but not its origin or genesis. It is therefore important to measure the responses of individual human cell types to infection, ideally in chronological order of their infection in vivo. Mononuclear phagocytes are very early, if not initial, targets of ZEBOV in humans and experimentally infected animals [12, 13, 14, 15] . In vitro, human and nonhuman primate macrophages are highly susceptible to ZEBOV infection with subsequent robust virus production [16, 17] , suggesting they may be the major source of the high viremia observed during the critical stages of infection. Studies performed in vitro are also strongly indicative that macrophages play a major role in inducing cytokine/chemokine dysregulation. For instance, human monocytes and macrophages infected with ZEBOV react with increased expression of MCP-1, CXCL1, IL-1b, IL-6, IL-8, MIP-1a, RANTES and TNF-a [16, 18] . Previous studies revealed that similar increased levels of expression of some of these cytokines were triggered by incubation of human macrophages with Ebola virus-like particles (VLPs) consisting of the ZEBOV matrix protein VP40 and the spike glycoprotein (GP 1,2 ) [19] or with UV-inactivated ZEBOV [16] . These findings indicated that virus replication might not be required for the activation of macrophages.
The aim of the current study was to determine the gene expression profiles of human macrophages exposed to infectious Ebola virions using DNA microarray technology (reviewed in [20, 21, 22, 23, 24] ) and therefore elucidate virus-host interactions. Here, we determine the initial responses of human macrophages to Ebola virion exposure. In a first set of experiments, DNA microarray analysis was performed to determine gene expression profiles of human macrophages 1 h and 6 h after in vitro exposure to Ebola virions in comparison to mock-exposed cells. In parallel, macrophages were treated with LPS to assess the responsiveness of the cells and to compare the response to different stimuli as genes responding to both virions and LPS might highlight possible response pathways. In a second set of experiments, we distinguished between responses induced by virion binding/entry and responses that require virus gene expression, cellular signals occurring after virion entry by exposing macrophages to Ebola VLPs. We found that Ebola virions exposure, as well as exposure to VLPs can trigger most of the detected changes after 1 h of exposure, and thus independent of virus replication.
Primary human macrophages were obtained from two sources. For the first set of experiments, fresh elutriated primary human monocytes from three donors (D1, D2, D3) were purchased from Advanced Biotechnologies, Columbia, MD. For the second set of experiments, primary human monocytes from three donors (Poietics CD14 + , untreated 2W-400 series) (D4, D5, D6) were purchased from Cambrex Bio Science Walkersville (Walkersville, MD). For differentiation of monocytes into macrophages, cells were cultivated in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 20% heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO), penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM). Human embryonic kidney (HEK) 293T epithelial cells and grivet (Chlorocebus aethiops) kidney epithelial Vero E6 cells (ATCC, Rockville, MD) were maintained in DMEM (Invitrogen, Carlsbad, CA) containing 10% heatinactivated fetal bovine serum (Invitrogen, Carlsbad, CA), penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM). All cells were incubated at 37uC in a humidified 5% CO 2 environment. The Mayinga strain of Zaire ebolavirus (ZEBOV), isolated in 1976 [2] , was used for all infections, which were performed under biosafety level 4 conditions at the National Microbiology Laboratory of the Public Health Agency of Canada in Winnipeg, Manitoba. Prior to use, virus stocks were propagated in Vero E6 cells and clarified by centrifugation at 3,000 g for 10 min at 4uC. Supernatants were then layered on TNE buffer (20 mM Tris [pH 7.5], 0.1 M NaCl, 0.1 mM EDTA) containing 20% sucrose and spun at 28,000 rpm at 4uC for 2 h by using an SW28 rotor with a Beckman Optima L-70K ultracentrifuge. The virion pellet was resuspended in RPMI 1640 and titers were determined by plaque assay as previously described [25] . As a control, supernatant from mock-infected Vero E6 cells was purified and quantified by the same procedures.
Generation and purification of virus-like particles (VLPs) were performed as described elsewhere [19] . In brief, ZEBOV VLP VP40-GP and VLP VP40 were generated by transient transfection of HEK 293T cells with plasmids encoding ZEBOV VP40 and/or ZEBOV GP 1,2 [19] and quantitated by electronmicroscopic particle counting [26] and a DC protein assay (BioRad, Mississauga, Ontario).
Electron-microscopic evaluation of VLPs was performed on a Phillips CM100 microscope with low dose software and Compustage attachments. Negative staining was performed on formvar carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA). Purified VLP solution (13 ml) was exposed to a freshly glow-discharged grid for 2 min, and the grid then transferred to a drop of 1% sodium silicotungstate (pH 7.5) for
Ebola virus causes a severe hemorrhagic fever syndrome in man with high case-fatality rates. Following infection, monocytes and macrophages are among the first cells targeted by the virus. These cells respond by increasing expression of inflammatory cytokines and chemokines that contribute towards pathogenesis. In order to more thoroughly characterize the host response to Ebola infection, primary human macrophages were infected with Zaire ebolavirus and samples harvested for transcriptional changes after 1 or 6 hours post infection. Whereas previous studies have analyzed a relatively small subset of host genes, this study examined the transcriptional profile of over 10,000 genes and employed rigorous pathway analyses to the datasets. Ebola virus was found to significantly regulate the expression of over 88 host genes. These changes occurred within the first hours of infection. Subsequent experiments demonstrated that virus replication was not necessary for activation. Indeed, noninfectious virus-like particles expressing the ebolavirus glycoprotein and matrix proteins were sufficient stimuli to induce activation. 
Prior to use, virion stocks, mock stocks, VLPs, and media were analyzed for endotoxin presence using the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD).
Six days after seeding, growth medium of human macrophages was replaced with fresh RPMI 1640 containing 2% human AB serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and Lglutamine (2 mM). Cells were then incubated for one day at 37uC in a humidified, 5% CO 2 environment. Cells from donors D1, D2, and D3 were infected with either mock virus preparation or ZEBOV at a multiplicity of infection (MOI) of 10. Cells of donors D4, D5, and D6 were infected with ZEBOV at an MOI of 100, mock virus preparation, ,100 particles/cell VLP VP40-GP , ,100 particles per cell VLP VP40 , mock VLP (plasmid only), ,100 latex particles/cell, or 10 ng/ml of lipopolysaccharide (LPS). Cell supernatants were removed from the cells 1 h or 6 h post infection, and RNA from cells was purified using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. The analysis of purified RNA from 10 MOI was analyzed by DNA microarrays as outlined below. The RNA obtained after infection with 100 MOI was subjected to quantitative real-time RT-PCR analysis only.
Genechip technology from Affymetrix (Santa Clara, CA) was used to study the transcriptional activity of human genes using the human GeneChipH array HG-U95Av2 with a total of 12,626 probes representing approximately 10,000 full-length genes (Affymetrix Technical Note to Human Genome U133 Genechip set). Normalized signal values were generated from image raw data and used to calculate p values indicating significance levels for signal strength (absent or present call) and log 2 ratio values in comparison files (change, increase, decrease calls) using the Affymetrix Microarray Suite 5.0 (MAS 5.0). Analysis was performed on these normalized data with a reduced set of genes after removing all genes that were absent on all arrays. The remaining data included 8,861 probe sets. Additional data reduction was achieved by excluding all genes that were identified as 'no change' (NC) in all six experiments when comparing Ebola virion-exposed with mock-exposed cells, respectively. The remaining dataset included 2,025 upregulated or downregulated genes. An analysis of variance (single factor ANOVA) and determination of the correlation coefficient (R 2 ) of changes in gene expression levels between ZEBOV-treated and VLP-treated cells was performed using Microsoft Office Excel. Array data were displayed on MA plots ( Figure S1 ), i.e. on a scatter plot showing the correlation between the average log 2 intensity versus log 2 ratio for a ZEBOV-treated versus mock-treated pair of cells. Log intensity was calculated as K (log(virion)+log(mock)). Genes unchanged between test and control have a log 2 value of zero; downregulated genes have negative values; and upregulated genes have positive values. Hierarchical clustering was performed using Stanford's GeneCluster and displayed with the TreeView program [27] . Clustering selection used average linkage clustering with the correlation uncentered.
InnateDb (www.innatedb.com) is a publically available resource which, based on levels of either differential gene expression, predicts biological pathways based on experiment fold change datasets [28] . Pathways are assigned a probability value (p) based on the number of genes present for a particular pathway as well as the degree to which they are differentially expressed or modified relative to a control condition. For our investigation input data was limited to the subset of 2,025 genes identified above. Additionally, functional networks were created using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA). Those genes with known gene symbols and their corresponding expression values were uploaded and mapped to their corresponding gene objects in the IPA Knowledge Base. Networks of these genes were algorithmically generated based on their connectivity and assigned a score. Genes are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). The intensity of the node color indicated the degree of up-or down-regulation. Genes in uncolored notes were not identified as differentially expressed in our experiment and were integrated into the computationally generated networks on the basis of the evidence stored in the IPA knowledge memory indicating a relevance to this network.
Reverse transcription and subsequent quantitative real-time polymerase chain reaction (qPCR) were performed as described previously [19] . Strand-specific qPCR was performed using strand-specific primers for the reverse transcriptase reaction. As controls for non-specific or self-priming events, control reverse transcriptase reactions lacking primer were performed in parallel. Relative amounts of different strands were determined by normalizing against the house-keeping gene GAPDH and by subtracting the amounts of PCR product resulting from selfpriming from strand-specific products. Relative quantification was performed using the comparative CT method (Applied Biosystems User Bulletin #2, Dec. 11, 1997).
DNA microarray technology was used to determine the initial response of human macrophages exposed to Ebola virions. Total RNA isolated from primary human macrophages of three donors (D1, D2, D3) at 1 h and 6 h after in vitro exposure to purified Ebola virions was compared to RNA from mock-exposed cells derived from the same donors. The time points were chosen as virus gene expression does not occur within one hour of cell-virion contact, whereas it will have commenced five hours later while ZEBOV replication is still absent (see below). A total of 12 HG-U95Av2 GeneChip microarrays were analyzed to determine differences in cellular gene expression levels between Ebola virion-exposed and mock-exposed macrophages from donors D1-D3 and also compared to the responses of LPS-treated cells. Genes detected as absent (p#0.05) on all 12 arrays were removed from a total of 12,606 probe sets prior to ratio analysis resulting in a reduced number of 8,861 probe sets. The log 2 ratio was plotted as a function of the average log 2 intensity of Ebola virion-exposed versus mock-exposed samples to test for hybridization quality and for the extent of variation among biological replicates (cells from the three donors). The MA plots for both the 1 h and 6 h time points demonstrate a low variability among donors and good overall reproducibility with the mean log 2 being zero over the EBOV-Induced Changes in Macrophage Gene Expression www.plosntds.org entire signal intensity range ( Figure S1 ). The low overall variability among donors was corroborated quantitatively by an analysis of variance of the signal strength. A single factor analysis of variance (ANOVA) indicated no significant difference among the 12 arrays (p = 0.83), confirming that the expression of the majority of cellular genes was not affected by exposure to Ebola virions. Genes responsive to Ebola virion exposure were identified by data reduction, i.e. by exclusion of all genes that were identified as 'no change' (NC, p#0.05) for all conditions tested. The resulting 2,025 probe sets (genes) were characterized by at least one significant change in one donor at either 1 h or 6 h post infection. ANOVA indicated that differences in expression patterns among these probe sets were statistically significant (p = 3610 26 ).
The 2,025 genes identified were then screened for patterns of cellular expression changes that could be biologically relevant. Two types of selection criteria were designed to determine whether expression of a cellular gene was significantly affected by Ebola virion exposure, threshold-based criteria and trend-based criteria. First, thresholds for fold-change, p-values, and signal strength were established. Specifically, a minimum of a 2-fold-difference in cellular gene expression levels in infected macrophages from at least two of the three donors in at least one of the two time points and p-values,0.05 were defined as pertinent ( Figure 1 ). Second, it was acknowledged that in the case of most genes the extent of changes in gene expression required for a biological impact are unknown. For instance, some genes might respond to virus infection with only minor changes in expression levels that may still be biologically relevant. Vice versa, rather dramatic changes may prove to be biologically irrelevant. Consequently, trend selection criteria were established to screen for a common tendency or direction of changes in cellular gene expression levels. Only changes with an associated p-value of #0.01 were considered, and no unique cutoff value for fold-change was specified. Accordingly, changes of less than 2-fold were accepted if the direction of change in macrophages from all donors was the same (all increased or all decreased). The two resulting gene sets were analyzed by hierarchical clustering (Stanford Cluster software) and the resulting clusters were visualized using TreeView (Figures 1 & 2) .
Application of the threshold-based selection criteria identified 205 cellular genes whose expression changed upon Ebola virion exposure ( Figure 1 ). Clustering of these 205 genes revealed two subclusters. Gene subcluster 1 was characterized by extensive expression variability across donors and time points. Inspection of transcript levels (signal strength) revealed very low signals, which may be a reason of the apparent variability among biological replicates. In contrast, many genes in subcluster 2 show consistent responses among all three donors in at least one time point. However, there are some genes that show similar responses in cluster 1. Since the overall variability in subcluster 1 could be due to low signal strength the dataset of 205 genes was subjected to the additional requirement that of the two compared signal levels the higher one (i.e. mock-signal for upregulated genes, Ebola-signal for downregulated genes) has a minimum signal value of 100 (signal threshold). Fifty-one genes remained when this added threshold criterion was applied. Most of them were located in subcluster 2 and are listed in Table S1 (threshold selection). The majority of the 51 genes were upregulated at 1 h post infection, at 6 h, or at both time points.
The second set of criteria, designed for selecting a trend of responses rather than assuming a threshold for fold-change, yielded a total of 66 probe sets (63 genes) (see Figure 2 and Table  S1 , trend selection). Approximately half of them were characterized by a fold-change of $2 and were also identified after the first set of selection criteria was applied. A total of 88 genes were identified after either criterion was applied. Among those 88 genes from threshold and trend analysis (Table S1 ), 26 fulfilled both selection criteria. Twenty-one of these genes were characterized by altered expression levels following exposure to Ebola virions in the same direction as after treatment with LPS. Interestingly, the expression levels of cellular genes identified by applying the trend analysis (Table S1 & Figure 2 ), all changed at the 1 h time point. Importantly, this set includes genes whose elevated expression was previously associated with human Ebola virus disease, such as the genes encoding CXCL1, IL-1b, IL-6, IL-8, and TNF-a [6, 11] , as well as genes previously unknown to play a role in Ebola virus disease, such as those encoding CCL-20, COX-2, IL-15 receptor a, phosphodiesterase 4B, and t-Pa. Expression levels of almost all identified genes were upregulated, with the exception of genes encoding GNA13, CREM, and LILRB2 at the 1 h time point and MRPS6, GADD45, and DUSP2 at the 6 h time point. Pathway over-representation analysis of gene expression data
Recently, the integration of bioinformatics to complex biological data sets has provided a network-based approach for delineating the host response. As cellular responses are mediated through the selective activation or repression of signaling pathways we sought to integrate our gene expression data into functional signaling networks. Functional networks were created from our biological data sets using Ingenuity Pathway Analysis (IPA) (Figure 3 ). Genes belonging to these functional networks were related to cell-to-cell signaling and interactions, hematological system development and function, immune cell trafficking, inflammatory response and cell movement. To expand on the biological significance of this analysis, and identify individual signaling pathways modulated following infection, we performed pathway over-representation analysis (ORA) with the online software tool InnateDB (www. innatedb.com) [28] and analyzed the 1 h and 6 h Ebola-infected vs. mock-infected comparative data sets. Integrated data was limited to the 2,025 genes identified above and fold-changes .1.5 and associated p-values.0.01 were chosen as parameters for pathway ORA. The resultant differentially regulated pathways with pvalues,0.1 are presented in Tables 1 and 2 .
Pathway ORA of the data set for the 1 h Ebola-infected vs. mockinfected treatments identified a number of pathways directly related to activation of the G-protein coupled receptor pathway ( Table 1) . As chemokines and inflammatory mediators activate GPCR signaling this correlates with previous investigations demonstrating increased secretion of cytokines and chemokines following Ebola virus insult [16, 18] . It was also demonstrated that many of the genes identified as central nodes in the IPA functional networks (IL-6, IL-10, IRF-7, etc.) also occupied central positions in the signaling pathways identified with InnateDB (heterotrimeric GPCR signaling pathways). Indeed, the upregulation of pathways related to interleukin (IL)-2, IL-12, IL-23 and IL-27 signaling pathways were also identified as being differentially upregulated in Ebola-infected cells as compared to the mock-infected treatment (Table S1 ). Further, GPCR-related signaling pathways (cytokine-cytokine receptor interaction pathway) and the Jak-Stat signaling pathway were also upregulated during the immediate response to infection. Interestingly, pathways related to fibrinolysis (dissolution of fibrin clot pathway; fibrinolysis pathway) were also upregulated by Ebola infection as compared to the mockinfected control (Table 1) . Previously, dysregulation of the fibrinolytic system was recognized in Ebola-infected macaques [13] . A limited number of downregulated pathways were identified in our analysis as being significantly downregulated at 1 h post-infection and were largely related to metabolic processes as well as inhibition of the negative regulation of GPCR signaling (Table 1) .
In contrast, differentially upregulated signaling responses at the 6 h time point were largely related to cell adhesion (syndecan-1-mediated signaling events), metabolism (prostaglandin and leukotriene metabolism; arachidonic acid metabolism; steroid hormone metabolism) and cytokine/chemokine signaling (cytokine-cytokine receptor interaction; chemokine signaling pathway; chemokine receptors bind chemokines) ( Table 2) . Interestingly, there was a large increase in the number of downregulated pathways at the 6 h time point as compared to the 1 h time point. Multiple pathways belonging to transcription and nucleotide/nucleoside metabolism were identified as being significantly downregulated in Ebola-infected cells as compared to the mock-infected controls 6 h post-infection ( Table 2) . Comparison of the differentially regulated pathway ORA data sets between the 6 h Ebola-infected vs. mock-infected and LPSstimulated vs. mock-stimulated treatments demonstrated minimal overlap of upregulated or downregulated pathways (data not shown). Whereas LPS stimulation resulted in the upregulation of pathways largely related to TNF-a, Toll-like receptor (TLR) or apoptosis signaling pathways these were not identified in the Ebola-infected samples. Thus, this likely indicates that the pro-inflammatory response to Ebola may be largely repressed by a yet unidentified mechanism at early time points following infection.
Real-time RT-PCR quantification of changes in cellular gene expression levels DNA microarrays are sensitive and specific in identifying regulated transcripts, but the technique commonly leads to underestimation of the degree of fold-change in gene expression. Comparison of the fold-changes in gene expression determined by DNA microarrays to a standardized quantitative real-time RT-PCR demonstrated that the underestimation error was larger when the fold-change differences were higher, corresponding to the finding that the quantitative range of real-time RT-PCR is larger than of DNA microarrays [29] . Consequently, real-time RT-PCR was performed for a subset of 16 selected genes that fulfilled at least one of the selection criteria to verify the results obtained by DNA microarray analysis. Some of the selected genes served as controls and are known to be influenced by ZEBOV infection, however, not necessarily at these early time points (TNF-a, IL-1b, IL-6, IL-8, CXCL1, RANTES, IL-10). Some genes were selected according to their potential relevance in infection due to their known functions and roles in modulation of immune response, viral infections or signaling pathways (hCOX-2, CCL20, 4-1BB, CSF-1, G-CSF, INDO, RSAD2, t-PA, DDX5).Verification was achieved when the direction of gene expression levels was identical and when the expression level changes determined by real-time PCR were equal or stronger than those determined by DNA microarray analysis. Figure 4 illustrates the comparison of the results of microarray analysis and real-time PCR quantifications over both time points. Real-time PCR confirmed the direction of changes in gene expression levels determined by microarray analysis and verified that most changes in cellular gene expression already occurred at the 1 h time point. In particular, upregulation of genes known to play an important role in Ebola hemorrhagic fever (CXCL-1, IL-1b, IL-6, IL-8, and TNF-a [6, 11] ) was verified, although some variation was seen among donors.
The observation that most cellular responses occurred within 1 h after exposure to Ebola virions suggested that virion binding/ Figure 5 ) confirmed that at 1 h cRNA amounts were below that of genomic RNA, whereas after 6 h cRNA was greatly increased. LPS served as a positive control for the responsiveness of the macrophages used in the virion exposure studies. A comparison of the effects of Ebola virion exposure versus LPS treatment revealed that the majority of genes responded similarly (Supplemental Table 1 ), further substantiating the hypothesis that the observed responses with Ebola virions were triggered by binding and/or entry. To further investigate which changes in expression levels in macrophages occur due to virion binding/entry, macrophage responses following exposure with infectious virions were directly compared to exposure to Ebola virus-like particles (VLPs) lacking viral genetic material and containing only the ZEBOV matrix protein VP40 and the ZEBOV spike glycoprotein GP 1,2 (VLP VP40-GP ) (see also [19] ). GP 1,2 is the only known surface-exposed structural component of Ebola virions and contains the cell-surface receptor-binding domain [30, 31] . We therefore hypothesized that if virion binding is responsible for the observed macrophage responses, then these effects would be mediated by GP 1,2 . To evaluate the role of GP 1,2 , VLPs consisting of VP40, but lacking GP 1,2 (VLP VP40 ), were tested in parallel with VLP VP40-GP . VLP VP40 particles were shown previously to be morphologically similar to the GP 1,2 -containing VLPs [32, 33] . LPS was again used as a control for macrophage responsiveness and also to directly compare the LPS-induced responses to those of Ebola virion and VLP exposures. Additionally, cells were exposed to latex particles, which were used in approximate equal quantity to VLPs. Ebola VLP preparations were tested by negative-stain electron microscopy for authentic appearance and presence (VLP VP40-GP ) or absence (VLP VP40 ) of GP 1,2 (data not shown). Particles resembled those previously described [32, 33] . In addition, Ebola virion, VLP, and latex preparations were tested for endotoxin-contamination before incubation with macrophages from donors D4, D5, and D6. The endotoxin concentrations of all samples used in this study were not above those of the tissue culture media (,0.5 U/ml). To determine whether the quantitative differences in gene expression levels between cells of donors D1-D3 observed in the first experiment were due to the MOI used, a higher MOI of Ebola virions (<100) was used for this experiment. Cells were exposed to VLPs and latex particles at a concentration of 100 particles per cell. LPS was used at the same concentration as in the first experiment (10 ng/ml), which allowed for a direct comparison of the responsiveness of macrophages used in both experiments. The results revealed that while a higher MOI did increase the overall intensity of responses compared to the first experiments (compare to Figure 4) , it did not eliminate or noticeably reduce the degree of variation of gene expression levels between donors ( Figure 6C &  6D ). This suggests that genetic variation among identical cell types of different donors may have an influence on the effect of ZEBOV infection and therefore may influence chances of survival.
Since all stimuli were given in parallel to macrophages from each donor, this experiment allowed for a direct qualitative comparison of the cellular responses to the different stimuli. Cellular gene expression levels were determined by real-time RT-PCR specific for 21 genes that were selected from the list of genes established using DNA microarray analysis in the first experiment. These 21 genes included the majority of genes previously analyzed by real time PCR (Figure 4 ), as well as additional genes that were suspected or known to play a role in ZEBOV infection in vivo.
The results of the real-time PCR quantification were analyzed by two-dimensional cluster analysis. The first clustering was performed to group genes according to similar behavior in expression changes (upregulation or downregulation) following a stimulus ( Figure 6A ). This first cluster was then used for clustering in a second dimension to group experimental stimuli according to similar effects they had on changes in gene expression ( Figure 6A , right cluster). Analysis revealed genes that were upregulated at the 1 h and then downregulated at the 6 h time point (GADD45, DUSP2, IL-10), genes that were first downregulated and then upregulated (IDO, ISG 45K), a large group of genes induced at both time points, and finally genes that were downregulated at both time points (TLR3, GNA13). The second clustering ordered the experimental groups according to similar responses in gene expression ( Figure 6B ) and revealed the following three major clusters: 1) 1 h exposure to Ebola virions, LPS, and VLP VP40-GP , 2) 6 h exposure to Ebola virions, LPS, and VLP VP40-GP , and 3) 1 h and 6 h exposure to VLP VP40 and latex particles. This result suggests that Ebola virions, LPS, and VLP VP40-GP caused strikingly similar responses in comparison to the responses to VLP VP40 and latex particles. This further supports the notion that binding/entry of virions mediated by GP 1,2 plays a significant role in the host response to infection. Thirteen of the 21 genes tested responded to Ebola virions, VLP VP40-GP , and LPS at both time points. TNF-a, IL-1b, IL-6 and IL-8 were also found in a separate study on the effects of VLPs on macrophages [19] and CXCL1 represents a gene known to be affected by ZEBOV infection. However, COX-2, GCH1, GM-CSF, MIP-3a, t-Pa, GADD45A, and IDO (6 h) are genes identified to play a role in Ebola hemorrhagic fever for the first time.
Within the first two clusters, various subclusters were identified. One revealed that out of the 13 genes that were upregulated at both time points, 9 responded, albeit weakly, to VLP VP40 at the 1 h time point. This supports the notion that the matrix protein VP40 might also be involved in cellular interactions upon binding/entry, which may participate in triggering part of the detected responses in macrophages, mainly involved in proinflammatory signaling. Exposure to latex particles did not induce a response, indicating that the signals mentioned above were not due to non-specific cellular uptake of particles. Another subcluster differentiated macrophages treated with VLP VP40-GP versus Ebola virions or LPS and included IFN-inducible genes such as RSAD2 and IFIT2, as well as G-CSF, TF, GADD45, DUSP2, IL-10, IDO, and CD 137lig. Another sub-cluster revealed 3 genes, DUDP2, IL-10, and TLR3, which were downregulated at the 6 h time point, and one gene, GNA13, which was downregulated at both time points by Ebola virions or VLP VP40-GP but not by LPS. 
The current study represents the first broad analysis of initial transcriptional responses to Ebola virions of human macrophages, the primary target cells of ZEBOV [12, 13, 14, 15] . To assure accurate identification of cellular genes that responded significantly to virion exposure, DNA microarray data were scrutinized and stringently filtered by two different sets of selection criteria and statistical evaluations. In addition, the expression levels of selected genes were experimentally verified by real-time PCR analysis. Using these methods for statistical verification and assay evaluation, our study permitted the identification of a large number of genes not previously implicated in early cellular responses to ZEBOV infection. The detected changes of gene expression levels occurred within the first hours after primary human macrophages were exposed to Ebola virions in vitro. In addition, we identified genes whose expression is known to be upregulated during acute Ebola hemorrhagic fever. This not only validated the experimental approach and data screening criteria, it also demonstrated that elevated levels of gene products, such as cytokines, are already induced in primary target cells within the first hours of virion binding and entry. Lending further credence to these assertions, pathway ORA of the significantly differentially regulated genes identified from our gene expression studies demonstrated a strong upregulation of specific host signaling pathways related to cytokine/chemokine signaling during the immediate response to Ebola infection. Indeed, the upregulation of signaling pathways related to cytokine/chemokine signaling events suggests that specific innate immune responses are mounted during the acute phase of Ebola viral insult. Through multiple pathway ORA analyses we identified broad cellular functional networks that are modulated during the early course of Ebola infection and, importantly, have correlated this with specific cell signaling pathways. The identities of the individual signaling pathways modulated by Ebola infection will provide critical information regarding disease pathogenesis and as well information for the development of novel antiviral therapeutics.
Some of the genes that had already been implicated in the pathogenesis of Ebola hemorrhagic fever such as IL-6 and TNF-a [6, 11] were induced after 1 h, but returned towards basal levels of expression after 6 h. Indeed, whereas LPS stimulation resulted in the activation of a large subset of TNF-a related signaling pathways at the 6 h time point there were no common pathways identified in the Ebola-infected pathway ORA. Similar results were obtained in another study, during which primary human macrophages, exposed to Ebola VLP VP40-GP for 24 h, were characterized by IL-1b, IL-6, IL-8, RANTES, and TNF-a expression that also peaked at 1 h or 6 h before receding to base levels [19] . These results contradict observations in vivo, which demonstrated upregulation of cytokines for extended periods of time [6, 7, 8, 9, 10, 11] . It is plausible that the early cytokine peak observed following high MOI infections in vitro is only achieved at a later time during in vivo infection. Another possible explanation for this difference is the duration of stimuli and thereby the duration of responses. For instance, in vivo, progeny virions are continuously produced by ZEBOV-infected cells and will therefore bind to and enter additional target cells, resulting in continuous stimuli that may maintain cytokine production and facilitate prolonged activation. Therefore, our in vitro approach most likely yielded results that are indicative of what continuously occurs in vivo. That increased cellular gene expression levels result in increased protein concentrations within 6 h was previously demonstrated in a study that used human macrophages exposed to VLP VP40-GP and ELISA measuring protein levels of IL-1b, IL-6, IL-8, TNF-a [19] .
The identified cellular genes whose expression levels were altered after exposure to Ebola virions belonged to different functional categories (Table S1 ). These genes include inflammatory cytokines, molecules that regulate blood coagulation (such as t-Pa, MMP-1, and serpine 1 and 2) genes involved in stressresponse, DNA repair, cell cycle arrest, and cell adhesion. In support of these functional categories, our pathway ORA also resulted in similar functional categorization of the differentially regulated signaling pathways following Ebola virus infection. The detection of a group of genes that responded to binding/entry of Ebola virions, VLP VP40-GP , as well as to LPS raises the question whether LPS and Ebola virions share receptors on the macrophage surface. Whereas receptors for Ebola virions on target cells remain elusive, it is known that LPS induces its effects by binding to TLR4 and MD2 and CD14 co-receptors. Recent studies demonstrated that Ebola VLP VP40-GP , but not VLP VP40 , induced cytokine and SOCS1 expression in a TLR4/MD2 dependent manner both in a human monocytic cell line (THP-1 cells) and in 293T cells expressing a functional TLR4/MD2 receptor [34] . The innate immune defense is achieved by activating NF-kB and type I IFN responses. It is already known that ZEBOV suppresses the host cell antiviral response by inhibiting interferon signaling via its VP35 and VP24 proteins [35, 36, 37, 38, 39, 40, 41] , and RNA silencing via VP35 [42] . Our studies revealed only limited IFN signaling in Ebola virion-exposed macrophages compared to those exposed to LPS, indicating that IFN signaling is inhibited early upon infection.
The host response to virulent pathogens is likely to fall into two categories [21] . First, there are common responses to unrelated pathogens, such as the type 1 IFN innate immune response that renders uninfected neighboring cells resistant to virus infection. Second, there are responses specific for individual pathogens. On the other hand, pathogens have evolved to counter these responses to ensure their own survival and transmission. Examples are how viruses of disparate families overcome the antiviral action of apolipoprotein B mRNA editing enzyme, catalytic polypeptidelike 3G (APOBEC3G) [43] , tripartite motif containing 5 (TRIM5a) [44] , bone marrow stromal cell antigen 2 (BST-2)/ tetherin [45, 46, 47] , or interferon induced transmembrane proteins (IFITM) [46, 48, 49] . It is important to remember that this study partially characterizes the response of humans to ZEBOV infection and that humans are not natural hosts of this virus. Therefore, host responses and virus counteractions are not in a state of equilibrium. It is therefore a fundamentally interesting question whether infected humans succumb to Ebola hemorrhagic fever because of direct effects exerted by the virus on the body or because of overbearing immune responses by the individual. Recently, various frugivorous bats have been implicated as potential filovirus reservoirs that seemingly remain unaffected by infection [50, 51] . It is therefore tempting to repeat our studies with cells from these animals to see whether their responses to Ebola virion exposure are fundamentally different.
Taking together, our data indicate that the immediate responses of early cellular ZEBOV targets in the human organism derive from virion binding/entry mediated by the ZEBOV spike glycoprotein and do not require virus gene expression. The fact that many surveyed genes responded similarly to VLP VP40-GP , but not to VLP VP40 , treatment clearly supports this notion. However, the fact that some genes, such as those encoding TF or skin collagenase, were triggered only by Ebola virions but not VLP VP40-GP or LPS indicate that these genes must be influenced by factors other than virus binding or entry, such as other components packaged within Ebola virions in addition to GP 1,2 and VP40. In this regard, it is important to remember that infectious Ebola virions not only consist of seven structural proteins (NP, VP35, VP40, GP 1,2 , VP30, VP24, and L) but also contain cellular transmembrane proteins that are usurped by budding virions [52] . Figure S1 MA plot depicting the overall distribution of gene expression changes as a function of average signal intensity for human macrophages obtained from three different donors. Each data set compares gene expression levels Figure 6 . Determination of relative changes in expression levels of 21 cellular genes in primary human macrophages. Depicted are fold-changes at the 1 h and 6 h time points after exposure to Ebola virions compared to mock-exposed cells, cells exposed to purified Ebola virionlike particles (VLPs) containing VP40 and GP 1,2 (VLP VP40-GP ) or VLPs containing VP40 only (VLP VP40 ), or cells treated with mock-VLP preparation. As controls, macrophages of each donor were treated in parallel with latex beads or with 10 ng of LPS. and changes in primary macrophages exposed to Ebola virions compared to mock-exposure at 1 h (A) and 6 h (B). (TIF)
Table S1 Summary of genes identified using threshold and trend analysis. 88 genes were identified from both threshold and trend analysis. Genes are grouped according to their known functions, determined by GeneOntology descriptors [53] . Many of these genes express proteins involved in apoptosis, inflammatory and acute immune responses, blood coagulation, and tissue remodeling. (DOCX) Case-based reported mortality associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection in the Netherlands: the 2009-2010 pandemic season versus the 2010-2011 influenza season BACKGROUND: In contrast to seasonal influenza epidemics, where the majority of deaths occur amongst elderly, a considerable part of the 2009 pandemic influenza related deaths concerned relatively young people. In the Netherlands, all deaths associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection had to be notified, both during the 2009-2010 pandemic season and the 2010-2011 influenza season. To assess whether and to what extent pandemic mortality patterns were reverting back to seasonal patterns, a retrospective analyses of all notified fatal cases associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection was performed. METHODS: The notification database, including detailed information about the clinical characteristics of all notified deaths, was used to perform a comprehensive analysis of all deceased patients with a laboratory-confirmed influenza A(H1N1) 2009 virus infection. Characteristics of the fatalities with respect to age and underlying medical conditions were analysed, comparing the 2009-2010 pandemic and the 2010-2011 influenza season. RESULTS: A total of 65 fatalities with a laboratory-confirmed influenza A(H1N1) 2009 virus infection were notified in 2009-2010 and 38 in 2010-2011. During the pandemic season, the population mortality rates peaked in persons aged 0-15 and 55-64 years. In the 2010-2011 influenza season, peaks in mortality were seen in persons aged 0-15 and 75-84 years. During the 2010-2011 influenza season, the height of first peak was lower compared to that during the pandemic season. Underlying immunological disorders were more common in the pandemic season compared to the 2010-2011 season (p = 0.02), and cardiovascular disorders were more common in the 2010-2011 season (p = 0.005). CONCLUSIONS: The mortality pattern in the 2010-2011 influenza season still resembled the 2009-2010 pandemic season with a peak in relatively young age groups, but concurrently a clear shift toward seasonal patterns was seen, with a peak in mortality in the elderly, i.e. ≥ 75 years of age. In 2009, the rapid spread of an emerging influenza virus, A(H1N1) of swine origin, resulted in the first pandemic of the 21 st century [1] . This pandemic influenza A (H1N1) 2009 virus has led to a limited outbreak in the Netherlands with, as in many other countries, generally mild illnesses in the majority of patients [2, 3] . The pandemic was considerably less lethal than was expected, with a low overall case fatality rate [4, 5] . Nevertheless, a considerable part of the pandemic influenza related deaths concerned relatively young persons (mainly young and middle aged adults) [3, [5] [6] [7] [8] . This is contrary to seasonal influenza epidemics, where deaths occur mainly amongst elderly aged 65 years or older [9] [10] [11] .
In the Netherlands, all deaths associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection had to be notified since 30 April 2009. This mandatory notification remained in place during the influenza season 2010-2011, which in Europe has been characterized predominantly by the influenza A(H1N1) 2009 virus, and to a lesser extent influenza virus type B [12, 13] . The national notification system provided detailed information of the clinical characteristics of all deaths associated with a laboratory-confirmed influenza A (H1N1) 2009 virus infection in the Netherlands. We performed a retrospective analysis of all fatalities, comparing the 2009-2010 pandemic season with the 2010-2011 influenza season, aiming to assess whether and to what extent pandemic mortality patterns concerning age distribution and underlying conditions were reverting to seasonal patterns.
In the Netherlands, laboratory investigation was indicated for all hospitalised and/or deceased patients with suspected influenza A(H1N1) 2009 virus during the 2009-2010 pandemic as well as the 2010-2011 influenza season. Following laboratory confirmation of influenza A (H1N1) 2009 virus infection, name and clinical characteristics of hospitalised and deceased patients had to be reported to the municipal health service (MHS) by both the attending medical doctor and the head of the involved microbiology laboratory. The MHS entered the notifications into a national anonymous and passwordprotected web-based database, including structured questions about patient demographics and information on underlying medical conditions, treatments, clinical presentation, and admission to an intensive care unit (ICU). In the pandemic season 2009-2010, additional information on underlying conditions for deceased patients was collected by the Centre for Infectious Disease Control (CIb) of the National Institute for Public Health and the Environment (RIVM) in consultation with the MHS and subsequently added to the notification database.
The notification database was used to perform a comprehensive analysis of all deceased patients with a laboratory-confirmed influenza A(H1N1) 2009 virus infection. Ethical approval was not required for this study as only anonymous data were used, and no (medical) interventions were made on human subjects.
Based on available clinical data, the underlying medical conditions were classified into nine groups: no underlying disorders, respiratory disorders, immunological disorders (including haematological malignancies), neurological disorders, intellectual disability (including Down syndrome), cardiovascular disorders, kidney and/ or liver pathology, other non-specified malignancies and metabolic disorders. Distinction was made between patients with single and multiple underlying disorders. Descriptive statistics were calculated for all available clinical and epidemiological characteristics. No statistical significant differences between the two seasons were found for these variables.
The mean age of the deceased patients in 2009/2010 was lower compared to that in 2010/2011, respectively 41 and 53 years (p = 0.02). Figure 1 shows the mortality rate per age group based on the total Dutch population in 2009 and 2010. During the pandemic season, the population mortality rates peaked in children aged between 0 and 15 years of age and in persons aged between 55 and 64 years. In the 2010-2011 influenza season, the first peak was considerably lower, while the second peak shifted to persons aged between 75 and 84 years.
Data on clinical presentation was available for 42 of the 65 fatalities (65%) during the pandemic seasons and for 28 of the 38 (74%) during the 2010-2011 season (table 1). In both seasons, fatal cases presented mainly with respiratory symptoms (41%), including acute respiratory distress syndrome (ARDS), followed by systemic symptoms (17%).
Immunological and respiratory disorders were the most commonly reported underlying medical conditions in the pandemic season, for respectively 23 (35%) and 22 (34%) of the 65 fatalities (table 2). In the 2010-2011 season, cardiovascular disorders and absence of medical underlying conditions were most common, respectively for 12 (32%) and 10 (26%) of the 38 deaths. Underlying immunological disorders were more common in the 2009-2010 compared to the 2010-2011 season (p = 0.02), while cardiovascular disorders were significantly more common in the 2010-2011 season (p = 0.005). Overall, multiple underlying conditions were reported for 27 of the 103 cases (26%). Particularly, intellectual disability (100%), cardiovascular (76%) and metabolic disorders (75%) were found in combination with other underlying conditions (data not shown).
The peak in mortality rates in persons aged between 55 and 64 years observed during the 2009-2010 pandemic, shifted to older age groups in the 2010-2011 influenza season. Furthermore, the peak in mortality rates in children younger than 15 years of age decreased considerably.
The decline of the peak in children might partly be explained by immunity in the youngest age groups, possibly related to high attack rates of influenza A(H1N1) 2009 virus in children during the pandemic season or to persisting vaccine-induced immunity [14] [15] [16] . Although the infection attacks rates during the pandemic season were very low in the older adults (≥ 40 years), the shift of the peak in mortality rates towards older age groups observed in our study might indicate increased circulation of the virus in the 2010-2011 influenza season in these age groups [14] . A shift of the age-specific mortality pattern similar to that observed in our study is also described for the post-pandemic seasons following the three pandemics in the 20 th century. During each of these earlier pandemics, persons younger then 65 years of age initially accounted for a high proportion of influenza-related deaths, followed by a declining proportion of deaths in the postpandemic seasons [17] . Simonsen et al. [17] hypothesised that younger persons may retain long-lasting immunity better than older persons after exposure to a new influenza virus subtype.
Recent studies on risk factors for influenza A(H1N1) 2009 deaths concluded that the majority of severe pandemic cases as well as fatalities had underlying medical conditions as previously also associated with severe seasonal influenza [4, 8, [18] [19] [20] [21] [22] . Our results are in line with previous studies in which respiratory disorders and immunosuppressive conditions were frequently reported as underlying diseases [4] [5] [6] 18, 21] . Furthermore, neurological disorders have been reported to be common underlying diseases in fatal pandemic influenza cases, especially in children and young adults [5, 19] . Patients with neurological and neuromuscular disease have also been recognized as high-risk group for severe disease from seasonal influenza [23] .
Our study showed a noticeable number of deceased patients (10%) with intellectual disability. Pérez-Padilla et al. [24] recently showed that Down syndrome was associated with adverse outcomes in cases of influenzalike illness (ILI) and severe acute respiratory illness (SARI) during the first months of the outbreak A (H1N1) 2009 influenza virus. All intellectual disabled patients in our study also had other chronic underlying conditions, making it impossible to assess the specific role of intellectual disability as a risk factor for fatal influenza.
Although it has been reported that the A(H1N1) 2009 influenza virus caused severe illness and death in pregnant and postpartum women [25] [26] [27] , as observed for seasonal influenza, no pregnancy-related pandemic influenza deaths were notified in the Netherlands. As we noted for intellectual disorders, also for pregnancy fatalities it is important to verify whether other chronic underlying conditions are present.
The relatively high number of fatalities with underlying cardiovascular disorders in the 2010-2011 influenza season might be associated with the shift of the mortality rates to elderly persons, since cardiovascular disorders are generally more common in elderly persons. Because of the relatively small numbers of fatalities, it is not possible to compare the differences in underlying conditions between the two seasons adjusted by age.
There remains a possibility that fatal case ascertainment is incomplete because of underreporting and -diagnosing. Especially in patients with severe underlying diseases and elderly, the generally non-specific symptoms may not have been recognized as being caused by influenza A(H1N1) 2009 virus infection. This is reflected by the fact that pandemic influenza was reported as contributing cause of death in some patients instead of the main cause of death. Moreover, this might also partly explain the relatively high mortality in patients with underlying immunological disorders in the 2009-2010 pandemic season compared to the 2010-2011 influenza season. It is plausible that testing for influenza was more common during the pandemic season because of heightened attention, particularly in patients with severe underlying diseases like immunological disorders.
To improve completeness of reporting in the hectic pandemic season, additional information on underlying conditions was actively collected where not available, which might have caused some information bias. Another limitation of this study is the lack of reliable historical records on deaths related to laboratory-confirmed influenza. Although deaths associated with laboratory-confirmed A(H1N1) 2009 virus infection were notifiable during the 2009-2010 and 2010-2011 seasons, clinical influenza diagnoses are generally not laboratory-confirmed during seasonal influenza epidemics. Nevertheless, estimates of the burden of seasonal influenza show that about 90% of influenzaassociated deaths occur in persons aged 65 years and older [9] . This is obviously different from the age specific mortality pattern seen during the 2009 and previous pandemics.
The maintenance of the mandatory notification of deaths associated with laboratory-confirmed influenza A (H1N1) 2009 made it possible to compare the fatal cases during the 2009-2010 pandemic season with that during the 2010-2011 influenza season. The mortality pattern in the 2010-2011 season still resembles the pandemic season with a peak in relatively young age groups, but concurrently shows a clear shift towards the seasonal pattern, as also described for previous pandemics in the 20 th century. A Chinese Herbal Formula to Improve General Psychological Status in Posttraumatic Stress Disorder: A Randomized Placebo-Controlled Trial on Sichuan Earthquake Survivors Introduction. Posttraumatic stress disorder (PTSD) is accompanied by poor general psychological status (GPS). In the present study, we investigated the effects of a Chinese herbal formula on GPS in earthquake survivors with PTSD. Methods. A randomized, double-blind, placebo-controlled trial compared a Chinese herbal formula, Xiao-Tan-Jie-Yu-Fang (XTJYF), to placebo in 2008 Sichuan earthquake survivors with PTSD. Patients were randomized into XTJYF (n = 123) and placebo (n = 122) groups. Baseline-to-end-point score changes in the three global indices of the Symptom Checklist-90-Revised (SCL-90-R) and rates of response in the SCL global severity index (GSI) were the primary endpoints. A subanalysis of the nine SCL factors and the sleep quality score were secondary endpoints. Results and Discussion. Compared to placebo, the XTJYF group was significantly improved in all three SCL global indices (P = 0.001~0.028). More patients in the XTJYF group reported “much improved” than the placebo group (P = 0.001). The XTJYF group performed significantly better than control in five out of nine SCL factors (somatization, obsessive-compulsive behavior, depression, anxiety, and hostility (P = 0.001~0.036)), and in sleep quality score (P < 0.001). XTJYF produced no serious adverse events. These findings suggest that XTJYF may be an effective and safe treatment option for improving GPS in patients with PTSD. On May 12, 2008 , an earthquake measuring 8.0 on the Richter scale hit Sichuan Province in southwestern China. According to the official data, more than 69,200 people were confirmed dead, more than 374,600 were seriously injured [1] , and at least 5 million were left homeless [2] . Recent literature shows that posttraumatic stress disorder (PTSD) and other psychological disorders such as anxiety and depression were fairly common and highly comorbid in 2008 Sichuan earthquake survivors [3] .
Posttraumatic stress disorder (PTSD) is a significant public health problem [4] . About 6.8% of adults develop PTSD in their lifetimes; 3.5% have the condition in any given year [5, 6] . Approximately 10%-50% of the survivors of traumatic events such as earthquakes and tsunamis will develop chronic PTSD [7] , which often persists for years if untreated [8] [9] [10] . The disorder is characterized by flashbacks and avoidance or numbness as well as hyperarousal after experiencing, witnessing, or confronting actual or potential death, serious phy sical injury, or a threat to physical integrity [11] . In addition to these symptoms, co-morbid psychiatric disorders are extremely common. In the National Comorbidity Survey (USA), approximately 80% of individuals with PTSD also met criteria for at least one other disorder listed in the diagnostic and statistical manual of mental disorders-III (DSM-2 Evidence-Based Complementary and Alternative Medicine III) [4] . Patients with PTSD often manifest other complications such as depression, anxiety, obsessive-compulsive behavior, hostility, and paranoid ideation disorders [3, [12] [13] [14] [15] [16] . Co-morbid psychiatric disorders and related subclinical symptoms combined with core PTSD symptoms result in poor general psychological status (GPS).
Selective serotonin reuptake inhibitors are the usual first level pharmacological treatment for PTSD [17] [18] [19] [20] [21] [22] . Other lines of drugs, such as benzodiazepines and monoamine oxidase inhibitors, are also commonly used [23] . However, the effects of these pharmaceuticals are not always satisfactory [23] [24] [25] , and undesirable side effects such as sleep disturbance, sexual dysfunction, and dizziness have been reported [23, [26] [27] [28] [29] .
For centuries, traditional Chinese medicine (TCM) has been widely used in China and some other Asian countries for psychological disorders, and many classic herbal formulas have been used to treat such maladies [30] [31] [32] [33] [34] [35] [36] [37] [38] . Xiao-Yao-San is one of the most popular [30] [31] [32] [33] [34] [35] [36] . We developed a modified, granulated form of Xiao-Yao-San, Xiao-Tan-Jie-Yu-Fang (XTJYF), by adding additional herbs, mainly from another classic TCM formula Er-Chen-Tang for treating depression, and we studied the safety and effects of this modification in cancer patients with depression (see Table 1 ) [39] . Because we found the formula effective and observed no serious side effects, we hypothesized that XTJYF would improve GPS in PTSD patients.
Patients were enrolled into this study five months after the 2008 Sichuan earthquake, between October 2008 and January 2009, through a community-based epidemiological survey of four settlements of a severely affected city, Dujiangyan. In the enrollment survey, the relationship between exposure to the earthquake and PTSD was assessed. Preliminary screening was performed in the communities by our researchers according to the DSM III for PTSD, Chinese version [40] . Eligible subjects were invited to participate in a diagnostic face-to-face or telephone interview with one of three experienced psychiatrists, each of which has at least eight years of clinical experience. Patients who met the inclusion and exclusion criteria were enrolled (see Patient Flow Chart, Figure 1 ), and our psychologists verified PTSD as the primary diagnosis of each enrollee. Inclusion criteria were age 16 or older, meeting DSM III criteria for PTSD with at least one of the nine Symptom Check-List-90-Revised (SCL-90-R) [41] subscores above the Chinese norm [42] , and being willing to be randomly assigned. Participants understood that those randomized into the placebo control group could receive XTJYF after completion of the whole trial if they wished. Exclusion criteria were past history of bipolarism, schizophrenia, or other psychotic disorders; current organic mental disorder, factitious disorder, or malingering; any past history of alcohol or substance dependence or abuse; evidence of clinically significant hepatic or renal disease or any other acute or unstable medical condition that might interfere with safe participation in the study; use of any medication with clinically significant psychotropic activity within two weeks of randomization; any cognitive-behavioral therapy during the trial; psychotherapy initiated or ending during the trial. For female patients of childbearing age, participation was contingent on a negative serum pregnancy test and a medically accepted method of contraception.
Written informed consent was obtained from all patients before participation. Patients were free to withdraw from the study at any time. Clinical diagnoses, physicals, and laboratory examinations were mainly conducted in the outpatient clinic at the Air Force Sanatorium in the city of Dujiangyan by our psychologist and other investigators. The research staff collected patients' weekly feedback on their medical conditions and delivered the XTJYF or placebo through inhouse visits. The trial protocol was approved by the Ethics Committee of Shanghai Changzheng Hospital and the Air Force Sanatorium in Dujiangyan.
A sociodemographic inventory and a medical history were taken, and a routine physical and laboratory examination (i.e., blood pressure, ECG, clinical chemistry and hematology tests, and urinalysis) was performed by the investigators as a baseline for future toxicology screening.
Eligible patients were randomized to either XTJYF treatment or placebo control. Random numbers were generated by computer software; treatment codes were held by the chief investigator, who was isolated from patients and outcome data. The chief investigator was also responsible for distributing the XTJYF and placebo with the assistance of our research staff. Patients, research staff, and data entry clerks were blinded to treatment group assignment. Treatment compliance was assessed by package count and observation by the research staff. Treatment codes were disclosed after the entire study was completed.
Interventions. All patients received 12 g packages of granulated XTJYF or placebo twice a day for eight weeks [39] and were instructed to drink the contents dissolved in warm, boiled water.
Each patient completed the SCL-90-R questionnaires twice, at baseline prior to randomization and in the eighth week after the randomization, that is, at the end of this clinical trial. The SCL-90-R is a questionnaire for self-reporting psychological distress. It is widely used in patients suffering from mental diseases and for psychological evaluation of healthy individuals. The instrument is well accepted for its good internal consistency, dimensional structure, reliability, and validity [43] [44] [45] . The Chinese SCL-90, translated and validated by Wang from the English version of the SCL-90-R, was used [46, 47] .
The SCL-90-R consists of 90 symptoms of distress. Patients were instructed to indicate the degree to which they had been troubled by each symptom during the preceding week by ranking the symptom from 0 to 4, with 0 being "not at all" and 4 being "extremely." The statements were classified into nine dimensions, or factors (F), that reflect various [41] . In addition, on the SCL-90-R, there are seven items not included in any of the nine factors, among which, three reflect sleep quality. Individual SCL-90-R factors have been used to evaluate the psychological condition of PTSD patients, and there is sufficient evidence to support the correlation of higher global SCL-90-R scores with the severity of a patient's core PTSD symptoms [12, [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] .
During the trial, patients were closely monitored for adverse events (AEs) and worsening of symptoms. The time of onset of any observed or spontaneously reported AE, its duration and severity, any action taken, and the outcome were recorded.
The original formula, Xiao-Yao-San, contains eight herbs:
Gan-Cao (Radix Glycyrrhizae preparata), and Sheng-Jiang (Rhizoma Zingiberis recens). Our modification, XTJYF, contains all the herbs of the original formula, except Sheng-Jiang, plus additional seven herbs, including Fa Ban-Xia (Rhizoma Pinelliae preparatae) and Chen-Pi (Pericarpium Citri reticulatae), that are commonly used for psychological disorders (see Table 1 ).
All herbal substances used in this trial are listed with the Pharmacopoeia Commission of China, 2005, and are accepted as suitable for human consumption when administered within standard dosage levels. None of these herbs is a controlled substance or an endangered species. Raw herbs were purchased from the Lei Yun Shang Pharmaceutical Company (Shanghai, China). The herbs were extracted with water, and the resulting granules were packaged by the Chinese Drug Preparation Department of Shanghai Changzheng Hospital. Levels of heavy metals and microbial and pesticide residues were carefully assessed, and all fell well within the normal range [58] .
The placebo granules, purchased from Jiangsu Tianjiang Pharmaceutical Company, Ltd., were designed to resemble the XTJYF granules in taste, smell, and appearance. The placebo was composed of dextrin, sunset yellow fcf, and a sweetener; the proportion was 1200 : 1 : 7. After being tested on five independent volunteers, the placebo was deemed indistinguishable from XTJYF. XTJYF and the placebo were dispensed in identical opaque packages.
2.6. Statistical Analysis. Quantitative data was summarized using mean, standard deviation (SD), or 95% confidence interval (95% CI). Qualitative data was described using proportion, as percentages. Baseline characteristics of the two groups were compared using the two-sided chi-square test or t-test at a significance level of 0.05.
Since this was a randomized, blind clinical trial, the statistical analyses for treatment effect evaluation of the primary and secondary outcomes are relatively straightforward. Baseline-to-end-point score changes in the three global SCL-90-R indices and rates of response in the GSI were computed as the primary endpoints. For defining rate of response, patients with a reduction of at least 30% from the baseline GSI score were classified as "much improved"; at least 50%, as "very much improved." Subanalyses of the baseline-toend-point score changes of the nine SCL factors and sleep quality score (the average of the scores of the three SCl-90-R items on sleep quality) were secondary endpoints. Statistical analysis on both primary and secondary outcomes was done using intention-to-treat analysis (ITT) with statistical software SPSS. Missing values in the SCL-90-R questionnaire for the patients who withdrew from the study before the eighth week were imputed using the last-observation-carried-forward method. For primary outcomes, effect sizes (for three global indices) and number needed to treat (NNT, for rate of response in the GSI), as well as the P values from two sample t-tests and chi-square tests, are reported in the treatment effect assessment. The same analytic approaches were applied to the secondary outcomes. Additionally, Fisher's exact test was used to compare the difference in dropout rate and AEs between the two treatment groups.
A total of 3478 individuals were screened, of whom 820 passed the preliminary screening and 245 were finally enrolled into the study; 575 were excluded. Of these, 372 were lost to follow-up or refused enrollment; 178 did not meet the inclusion criteria; 25 met the exclusion criteria. Enrolled patients were randomly assigned to XTJYF (n = 123) or placebo (n = 122) treatment. Of these, 102 (83%) of the XTJYF group and 99 (81%) of the control group completed the whole study. Reasons for withdrawal from the study are listed separately for each treatment group in Figure 1 , and a detailed discussion on treatment tolerability is provided in Section 3.4. Table 2 shows that randomization was effective and that there were no significant differences between the two groups in baseline demographics, core clinical PTSD symptoms, or baseline SCL-90-R global indices. Even though individual SCL-90-R factor scores and sleep quality scores at baseline are not shown here, we checked all of them and founded no significant differences between the two groups. Notice that women constituted 72% of XTJYF-treated and 71% of placebo-treated patients. Ages ranged from 16 to 85; 64% were over 45. Table 3 shows the urgency of the public health needs of these earthquake-affected PTSD patients and indicates Table 4 shows that patients in the XTJYF group experienced statistically significant improvement after treatment in all three supplementary global index scores compared to the placebo group. Based on the reported effect sizes, XTJYF treatment has a moderate effect on GSI and PSDI indices and a small effect on the PST index. Our findings on the rate of response, defin-ed based on GSI score improvement, are displayed in Figure 2 ; 50% of the XTJYF patients versus 28% of those in the placebo group were "much improved," providing statistically significant evidence supporting the advantage of XTJYF over placebo at the level of 0.05 (P value = 0.001). The NNT is 4.55. Additionally, as Figure 2 shows, 20% of the XTJYF patients versus 12% of those in the placebo group were "very much improved," but this result is not statistically significant (P value = 0.12).
Outcomes. The second part of Table 4 displays the treatment effects of XTJYF and placebo on the nine SCL factors and sleep quality score. The results indicate that, in comparison to placebo, the XTJYF group experienced statistically significant improvement after treatment in five of the nine SCL factors, somatization (P = 0.003), obsessive-compulsive behavior (P = 0.036), depression (P = 0.001), anxiety (P < 0.001), and hostility The original data was obtained from Jin et al. [42] . We recalculated the original data from "mean (sd)" to "mean (95% CI)" in order to make these data comparable. • The original data was obtained from Derogatis [41] . We recalculated the original data from "mean (sd)" to "mean (95% CI)" in order to make these data comparable. * Compared to the Chinese and American norms, P < 0.05. Compared to the American norms, P < 0.05. Table 4 : XTJYF treatment effect on primary and secondary outcomes. (1) Statistical analysis was done using intent-to-treat analysis (ITT) with SPSS. (2) Cohen's d effect size measure, in which an effect size of 0.2 to 0.3 is considered a "small" effect, around 0.5, a "medium" effect, and 0.8 to infinity, a "large" effect, is used here. (3) The P values come from the two sample t-tests.
(P = 0.019). Based on the reported effect sizes, XTJYF treatment has a moderate effect on somatization, depression, anxiety, and hostility, as well as a small effect on obsessivecompulsive behavior, interpersonal sensitivity, and phobic anxiety. Table 4 also shows that XTJYF treatment yielded statistically significant improvement in sleep quality at the end of the study, with a P value of less than 0.001 and a moderate effect size.
Overall, XTJYF was well tolerated. Compliance rate, 83% for the XTJYF and 81% for the placebo group, was reasonably high. Six in the XTJYF and five in the control group withdrew due to adverse effects, so reported AEs were similar in the two groups. The most frequently reported AEs were nausea (14.6% versus 9.0%; P = 0.24), diarrhea (10.6% versus 6.5%; P = 0.36), and malaise (10.6% versus 12.3%; P = 0.69). All AEs were minor and were determined to be unrelated to the ingestion of XTJYF.
In the XTJYF group, 21 subjects dropped out (17.1%); in the placebo group, 23 did (18.9%, P = 0.74). The primary reasons cited for dropout in the XTJYF and placebo groups, respectively, were AE (4.9% versus 4.1%; P = 1); lost to Patients with a score reduction of at least 30% from the baseline SCL-90-R GSI score were classified as "much improved," and 50%, as "very much improved." * XTJYF versus placebo, P < 0.05.
follow-up (1.6% versus 2.5%; P = 0.68); protocol violation (4.9% versus 3.3%; P = 0.75); lack of efficacy (3.3% versus 6.6%; P = 0.25); miscellaneous reasons, for example, disliked the taste of the herbs (2.4% versus 2.5%; P = 1). Subjects' laboratory values and vital signs were similar in the two groups. Changes in these values were minor, infrequent, and not considered clinically meaningful.
In the present study, we compared our data to the Chinese norm calculated by Jin et al. [42] and to the USA norm published by Derogatis [41] . (see Table 3 ). At baseline, the nine SCL factors and three global indices were higher than the norm in these earthquake-related PTSD subjects, suggesting that earthquake-related PTSD is accompanied by poor GPS. These findings are consistent with those reported by other investigators [3, [59] [60] [61] [62] [63] .
Hypothesizing that it would improve poor GPS in earthquake-related PTSD, we investigated a Chinese herbal formula, XTJYF, modified from a classic formula, Xiao-Yao-San, and found that, compared to placebo, XTJYF significantly improved all of the three global indices of SCl-90-R, and a significantly greater proportion of patients were "much improved" according to changes in GSI score. (See Table 4 and Figure 2) . A subanalysis provided a more detailed look at specific XTJYF effects on poor GPS, showing that five of the nine SCL factors and sleep quality score improved. (see Table 4 ).
These findings suggest that XTJYF may globally improve GPS in earthquake-related PTSD patients, specifically in somatization, obsessive-compulsive behavior, depression, anxiety, and hostility. In addition, the formula may improve the sleep quality of the patients and appears to be safe. Although a few subjects reported gastrointestinal complaints such as nausea and diarrhea during treatment, these were probably due to the poor diet available after the earthquake; these symptoms were also frequently reported in the placebo control group. Our findings are consistent with those of our previous study on XTJYF for cancer patients with depression [39] .
The results are meaningful because all five of the psychological disorders mentioned above are associated with high levels of functional and psychosocial disability in chronic PTSD patients [3, 4, 6, [12] [13] [14] [15] [16] [64] [65] [66] [67] [68] [69] , and most are reported to predict greater refractoriness to routine therapy in individuals diagnosed with PTSD [17, [70] [71] [72] [73] . For example, PTSD patients who report somatic symptoms also report higher overall PTSD symptoms [15, 64] and a higher frequency of depression [64, 74] . Patients with co-morbid PTSD and obsessive-compulsive behavior have been found to have a poorer response to cognitive behavioral therapy than those diagnosed with PTSD alone [73] . Co-morbid PTSD/depression appears to predict greater refractoriness to pharmacotherapy, greater symptom severity, lower levels of functioning and rates of recovery, and increased disability and potential of suicide [4, 6, [65] [66] [67] . Like depression, anxiety symptoms are associated with lower quality-of-life estimates and greater refractoriness to routine pharmacotherapy in PTSD patients [3, 68] . Hostility, which according to a meta-analysis of 39 studies is significantly elevated in individuals with PTSD [16] , is linked to adverse health outcomes, including cardiac death [69] . In the present study, XTJYF also appears to improve the sleep quality of these PTSD patients; sleep disturbances are among the most treatment-resistant symptoms of PTSD [75] . All of these symptoms are likely to contribute to alcohol and drug abuse [76, 77] as well as suicidal ideation [78] .
The psychological mechanisms of action of Xiao-Yao-San and its modifications have been investigated. It has been reported that the formula may act on psychological symptoms by upregulating central neurotransmitters such as serotonin. Bao et al. [79] reported that Xiao-Yao-San produced antidepressant effects in a mouse model of depression by ameliorating brain cortex 5-HT and 5-HIAA content. Other mechanisms of the formula have also been reported. Yue et al. [80] reported that Xiao-Yao-San suppressed chronic stress in a rat model by up-regulating GluR2/3, the AMPA receptor subunit 2/3, which mediates the postsynaptic depolarization that initiates neuronal firing [81] , and by downregulating PICK1, a protein that interacts with C-kinase 1, which may lead to AMPA receptor anchorage [82] in hippocampal regions CA1 and CA3. Similar findings, that Xiao-Yao-San upregulates AMPA receptor subunit mRNA expression in hippocampal region CA1 and the amygdala, were reported [83] . Furthermore, Xiao-Yao-San and its modifications were reported to suppress chronic stress by maintaining the stability of hippocampal neurons [84] , inhibiting hypothalamic-pituitary-adrenocortical axis negative feedback regulation [85] , and counteracting increase of Ca 2+ concentration in hippocampal synaptosomes [86] . Based on TCM theory, seven drugs were added in our modification, mainly from another classic TCM formula, Er-Chen-Tang. According to our previous preclinical study, this modification may 8 Evidence-Based Complementary and Alternative Medicine suppress depression by up-regulating the 5-HT 1A receptor in the hippocampus in a rat model of chronic stress [87] . However, because Xiao-Yao-San and its modifications contain multiple ingredients, specific active ingredients have not been identified, and the herbal interactions within the formula have not been systematically investigated. Further investigation to elucidate the mechanisms of action of this formula is warranted.
Several limitations to this study should be noted. First, our trial lacked a long follow-up assessment. This was largely due to the difficulties in following up this particular population, which consisted of earthquake survivors living in shelters with no specific address. In the patient recruitment stage, more than 45% (372 of 820) of those preliminarily screened for PTSD were lost to follow-up. Secondly, we did not include a questionnaire measuring specific PTSD core symptoms, mainly because of the low level of education in this mountain population. In our patient population, 43% had an elementary education or less and found it difficult to complete a single 90-question SCL-90-R questionnaire. However, although we did not include a specific questionnaire such as the Clinician-Administered PTSD Scale [88] or the Clinician-Rated Treatment Outcome PTSD Scale [89] to measure core PTSD symptoms, the widely used SCL-90-R captures a broader patient psychological profile than a specific PTSD questionnaire would do. Thirdly, only one dosage of XTJYF was used in this study, that used in our standard clinical practice. A higher dosage might benefit the nonresponders. Finally, more detailed information on types of trauma and the percentages of patients who suffered them should be gathered and analyzed.
Despite the limitations, our findings provide preliminary support for the use of TCM in treating GPS in earthquake survivors with PTSD. TCM has been used extensively in China to treat people suffering from various diseases after disasters, for it is readily available, reasonably cheap, effective, and safe. Because of their wide usage, the production of TCM herbal products is quick and cost effective in China. Traditional Chinese herbal medicine may provide an adjuvant therapy that is safe, effective, and timely for affected populations in natural disasters such as earthquakes. Quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species BACKGROUND: On the base of our previous study we were observed relevant studies on the hypothesis that the antiviral activity of quercetin 7-rhamnoside (Q7R), a flavonoid, won't relate ability of its antioxidant. METHODS: We were investigated the effects of Q7R on the cytopathic effects (CPE) by CPE reduction assay. Production of DNA fragment and reactive oxygen species (ROS) induced by PEDV infection were studied using DNA fragmentation assay and flow cytometry. RESULTS: In the course of this study it was discovered that Q7R is an extremely potent compound against PEDV. The addition of Q7R to PEDV-infected Vero cells directly reduced the formation of a visible cytopathic effect (CPE). Also, Q7R did not induce DNA fragmentation. Furthermore, ROS increased the infection of PEDV, which was strongly decreased by N-acetyl-L-cysteins (NAC). However, the increased ROS was not decreased by Q7R. Antiviral activity of antioxidants such as NAC, pyrrolidine dithiocarbamate (PDTC), and the vitamin E derivative, trolox, were hardly noticed. CONCLUSIONS: We concluded that the inhibition of PEDV production by Q7R is not simply due to a general action as an antioxidants and is highly specific, as several other antioxidants (NAC, PDTC, trolox) are inactive against PEDV infection. Many viruses are capable of inducing cell death, leading to lysis of the infected cells [1] [2] [3] [4] [5] [6] [7] . In late stages of virus infections, morphological changes, commonly known as cytopathic effect (CPE), can be microscopically observed. Virus-induced CPE is characterized by cell rounding, shinkage, deformation of nuclei and chomatin condensation. However, early death of infected cells may limit virus replication [8] . Also, apoptosis, or programmed cell death (PCD), during the late phase of viral infection has been suggested to play an important role in virus life cycle by facilitating viral progeny release and propagation [9, 10] . PCD is a process by which damaged, aged, or otherwise unwanted cells are eliminated though a series of steps that results in the destruction of their genome. The form of PCD known as apoptosis is characterized by a series of morphological changes, including nuclear condensation and fragmentation, cytoplasmic blebbing, and cell shinkage [4] .
Many viruses are capable of inducing reactive oxygen species (ROS) production. Results from many studies suggest that ROS are not directly involved in the induction of apoptosis in virus-infected cells [11, 12] . On the other hand, it has been demonstrated that virus infection increases the production of superoxide anion radicals from neutrophils and macrophages infiltrated into the lung of mice [13] , while transgenic mice carrying over-expressed extracellular superoxide dismutase exhibited less severe lung injury after influenza virus infection [14] . These studies, therefore, postulated that the pathogenesis of virus infection involves not only the virus proliferation mediated apoptotic cell death in the infected cells, but also the direct ROS-induced cellular injury by neutrophils and macrophages infiltrated into the virus-infected organs. But, despite many studies, the events leading to the generation of ROS during viral infections are still unclear.
In this paper, we was demonstrated the effects of quercetin 7-rhamnoside (Q7R) on production of CPE, ROS and DNA fragmentation inducted by PEDV infection and also studied the relationship of antiviral and antioxidant activity between Q7R and antioxidants.
Ribavirin and sulforhodamine B (SRB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were a reagent grade. Q7R was isolated from aerial parts of Houttuynia cordata using a previously described method [15] .
Vero (an african green monkey kidney cell line; ATCC CCR-81) was kindly provided by ATCC (American Type Culture Collection, Manassas, VA, USA). PEDV CV 777 (porcine epidemic diarrhea virus) was obtained from national veterinary research & quarantine service in Korea. Vero cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 0.01% antibiotic-antimycotic. Antibiotic-antimycotic, trypsin-EDTA, FBS and MEM were supplied by Gibco BRL (Grand Island, NY). The tissue culture plates were purchased from Falcon (BD Biosciences, NJ, USAs). Virus stock was stored at -70°C until use.
The antiviral activity and cytotoxicity of Q7R against viruses were determined by cytopathic effect (CPE) reduction method recently reported [15] . Also, the effect of Q7R on PEDV-induced CPE was observed by cytopathic effect (CPE) reduction method recently reported [15] . Ribavirin was used as positive, and was solublized in dimethylsulfoxide (DMSO) used as negative control.
The level of intracellular ROS was measured by the alteration of fluorescence resulting from oxidation of 2', 7'-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR). DCFH-DA was dissolved in DMSO to a final concentration of 20 mM before use. For the measurement of ROS, cells were treated with Q7R and other reagents for a time period indicated in the figure legends. After washing twice with cold PBS, they were incubated with 20 μM DCFH-DA at 37°C for 15 min. DCFH-DA is a stable compound that easily diffuses in to cells and is hydrolyzed by intracellular esterase to yield a reduced, non-fluorescent compound, DCFH, which is trapped within cells. The ROS produced by cells oxidized the DCFH to highly fluorescent 2', and 7'-dichlorodihydrofluorescein (DCF). The intensity of fluorescence was recorded using a flow cytometry (Becton Dickenson), with an excitation filter of 530 nm and an emission filter 575 nm. The ROS level was calculated as a ratio of: ROS = mean intensity of exposed cells: mean intensity of unexposed cells.
Vero cells were seeded onto a 6-well culture plate at a concentration of 2 × 10 4 cells per well. Next day, medium was removed and the cells were washed with PBS. Then, 0.09 ml of diluted virus suspension and 0.01 ml of medium supplemented with typsin-EDTA containing an appropriate concentration of the antiviral compound were added. It was a ten-fold dilution scheme for each compound. The culture plates were incubated at 37°C in 5% CO 2 for 2 days, the cells were lysed with lysis buffer (TE buffer;10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 0.5% SDS] and incubated for 10 min on ice, then centrifuged at 13,000 rpm for 30 min at 4°C. Cysolic DNA was extracted by phenol: chlororform (1:1) extraction of the supernatants. DNA was treated with 0.1 mg/ml Rnase A for 30 min at 37°C. The DNA was separated by agarose gel electrophoresis, and the DNA fragmentation was visualized from the digitized image of the gel as described [12] .
The effect of Q7R on PEDV-induced CPE After 2 day infections of Vero cells with PEDV, Mock cells ( Figure 1A ) or cells treated with 10 μg/ml Q7R ( Figure 1C ) or ribavirin ( Figure 1B) showed typical spread-out shapes and normal morphology. At this concentration, no signs of cytotoxicity of Q7R were observed. Infection with PEDV in the absence of Q7R resulted in a severe CPE ( Figure 1D ). Addition of Q7R on infected Vero cells inhibited the formation of a visible CPE ( Figure 1F ). However, the addition of ribavirin in PEDV-infected Vero cell was impossible to prevent CPE ( Figure 1E) . Thus, the CPE of the virus infection is prevented by the presence of Q7R.
To investigate the possible mechanisms of the observed antiviral effects of Q7R, we first examined whether the antioxidant property of Q7R contributed to its action.
To determine the influences of PEDV replication on intracellular ROS level, Vero cells were infected with PEDV for various periods of time, and a fluorescence probe, DCFH-DA, was added to the medium prior 15 min to use a flow cytometry. As shown in Figure 2A and 2B, exposure to PEDV resulted in increased ROS production which began at 2 h post-infection and peaked at 6 h (Figure 2A and 2B) .
To study the influence of antioxidant on ROS increased by PEDV infection, Q7R and NAC were used. As shown in Figure 3A and 3B, at 3 h post-infection, PEDV infection resulted in a drastic increase of intracellular ROS, which was strongly decreased by NAC but not by Q7R, according to increase of its concentration.
To study the influence of antioxidants on PEDV replication, the antioxidants, NAC, trolox and PDTC were used. As shown in Figure 4A and 4B, the four compounds did not exhibit cytotoxicity at different concentrations. Antiviral activity of NAC and PDTC was hardly present. However, trolox strongly exhibited antiviral activity at 100 μg/ ml concentration, but decreased rapidly according to a dose dependent manner compared with Q7R.
The effect of Q7R on the extent of DNA fragmentation resulting from PEDV infection was examined. The incubation with Q7R or ribavirin up to 100 μg/ml concentration for 48 h did not induce DNA fragmentation in mock-infected Vero cells ( Figure 5A or 5B, lanes 2-4). PEDV infection induced DNA fragmentation in Vero cells 48 h after infection in the absence of compounds ( Figure 5A or 5B, lanes 5). In the presence of Q7R, the DNA fragmentation was not induced in a dose-dependent manner ( Figure 5B, lanes 6-8) . But, DNA fragmentation was somewhat decreased when Vero cells infected with PEDV were treated with ribavirin at concentration of 1 or 10 μg/ml for 48 h. Incubation with ribavirin of 100 μg/ml did not induce DNA fragmentation.
Many viruses are capable of inducing cell death, leading to lysis of infected cells [1] [2] [3] . In late stages of PEDV infections, morphological changes commonly known as CPE, microscopically observed. The morphology of Vero cells after infection with PEDV was greatly decreased from that of PEDV by addition of Q7R. However, the addition of ribavirin to PEDV-infected Vero cell proved to be impossible in preventing CPE.
Viral infections such as rhinovirus, influenza virus, human immunodeficiency virus and bovine viral diarreha virus frequently result in the generation of oxidative stress in the infected cells [4] [5] [6] [7] . The events leading to the generation of ROS during viral infections are still unclear. Also, antioxidants have been shown to have Figure 1 The effects of Q7R on PEDV-induced CPE. Culture medium 6-well tissue culture plates were removed and the cells were washed with PBS. Then, 0.09 ml of diluted virus suspension and 0.01 ml of medium supplemented with typsin-EDTA containing Q7R of 10 μg/ml were added. After incubation at 37°C in 5% CO 2 for 2 days, the morphology of cells was investigated under microscope and a photograph taken.
antiviral activities against a variety of unrelated viruses by alleviating the oxidative stress generated by viruses [16] [17] [18] [19] [20] . Mechanistically, it is believed that these viruses induce apoptosis by oxidative stress mediated via ROS. Interference with this pathway by antioxidants is believed to inhibit virus-induced apoptosis and thus inhibit efficient virus multiplication. In contrast, there are also reports indicating that under certain conditions compounds act as a pro-apoptotic drug [21] [22] [23] . Depending on the viral system analyzed, antioxidative compounds differ in their ability to reduce virus growth [7, 24, 25] .
Flavonoids are a large class of polyphenolic compounds and Jung et al. (2003) reported that the relationship between flavonoid structure and antioxidant activity. They found that the inhibitory activities of flavonoids on total ROS are more strongly increases with the rising number of hydroxyl groups than the foavonoid glycosides on their structures [26] .
Our previous study showed that quercetin 7-rhamnoside (Q7R) didn't directly interact with PEDV particles and affect the initial stage of PEDV infection by interfering with its viral mRNA production [15] . In this report, we present evidence that Q7R, but not other commonly used antioxidants, are able to protect cells from PEDV induced death. Q7R are potent agents that have been shown to be involved in a number of processes, suggesting that their antiviral effects might not be due to its antioxidant functions alone. Nevertheless, further studies are needed to verify the underlying mechanism of Q7R action in inhibiting PEDV infection.
In conclusion, Q7R is an extremely potent anti-PEDV substance which reduces PEDV growth, inhibits Inhibition of PEDV replication by Q7R is independent of its antioxidant activity. Vero cells were infected with PEDV (MOI = 10) in the presence of Q7R. Relative ROS generation in Vero cells exposed to Q7R of 1, 10, 100 and 500 μg/ml for the indicated times. NAC exposed to 10 μg/ml for the indicated times. Redox-sensitive fluorescence probe DCFH-DA (20 μM) was added to the phosphate buffered-saline (pH 7.2) after harvest of Vero cells infected with PEDV at 6 h. Representative images of ROS-induced DCF fluorescence of infected cell at 6 h post-infection are shown at a flow cytometry histogram. All data represent mean values of six independent measurements ± S.D. Ctr2, control treated with DCFH-DA; NAC 10, treated with N-acetyl L-cysteine of 10 μg/ml; PEDV, infected with PEDV for 6 h; Q7R 500, treated with Q7R of 500 μg/ml. the CPE and DNA fragment of infected cells regardless of its antioxidant activity and then didn't directly interact with PEDV particles and affect the initial stage of PEDV infection by obstructing with its viral mRNA production. It will be interesting to further investigate the antiviral activity of the Q7R in preventing various PEDV-mediated injuries in in vivo pathological situations.  Use of Recombinant Adenovirus Vectored Consensus IFN-α to Avert Severe Arenavirus Infection Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens. The Arenaviridae family of viruses has several members that can cause viral hemorrhagic fever, an acute, often-fatal, viral syndrome characterized by intense fever, malaise, and less frequently, bleeding and neurologic manifestations. Case fatality rates of hospitalized patients suffering from arenaviral hemorrhagic fever (AHF) range from 15-30% [1, 2, 3, 4] . Arenaviruses known to cause AHF include Junín, Machupo, Guanarito, Sabiá, and Chapare in the South American continent, and Lassa and Lujo in west and southern Africa, respectively. Primary transmission of the arenaviruses from respective rodent reservoir hosts to humans occurs via exposure to contaminated excreta [5] . Person-to-person transmission can occur through contact with blood or other body fluids during the care and management of infected individuals [1, 6] . Notably, these viruses are considered a threat to national security and are classified as highest priority pathogens by the NIAID [7] .
At present, the treatment of AHF is limited to ribavirin and immune plasma [8, 9] . The latter has only been proven to be effective in treating cases of Argentine hemorrhagic fever (Junín virus infection) within 8 days of disease onset. Off-label usage of ribavirin has been shown to be effective in treating Lassa fever when therapy was initiated within 6 days of the development of clinical symptoms. However, there are toxicities associated with ribavirin therapy at dosages required for efficacious use, which may contribute to the observed poor patient compliance in completing prescribed treatment regimens [10, 11] . Very limited case data using ribavirin to treat other AHFs supports the use of emergency protocols [1, 12, 13] , however the utility of ribavirin therapy remains to be seen.
Interferon alpha (IFN-a) is an effective part of the host innate immune response, which can be manufactured as a recombinant human protein with broad clinical appeal [14] . Consensus (c)IFNa, also known as IFN alfacon-1 and Infergen, is a licensed, second generation IFN-a engineered to contain the most frequently occurring amino acids among the nonallelic IFN-a subtypes. Previously, we have demonstrated that cIFN-a can be used effectively alone, or in combination with ribavirin, to treat Pichindé virus (PICV) infection in hamsters [15, 16] , an experimental model of acute arenaviral disease [17] . However, while cIFN-a has clinical value, its usefulness is hindered by its short half-life and cost to manufacture. There is an initial distributive half-life of 7 minutes and a beta half-life of 2 to 5 hours [14] . The rapid systemic clearance requires frequent dosing to achieve desired therapeutic levels. Consequently, treatment can result in well-documented toxicities which include headache, depression, hair loss, fever, and malaise. In order to combat the rapid degradation, PEGylated forms of recombinant IFN-a have been introduced with half-lives that are on the order of days instead of hours, thus reducing the number of injections to once per week [18] . However, the cost to manufacture PEG-IFN-a is exceedingly high, and the PEGylation process has been shown to reduce the activity of IFN-a, thereby further increasing the production costs.
To circumvent the fast decay of cIFN-a, a replicationincompetent, recombinant adenovirus type 5 (rAd5) gene delivery platform was designed to drive constitutive expression of the cIFNa gene from transduced nasal epithelial target cells. This rAd5 cIFN-a virus, called DEF201, was first developed in mice and recently shown to be active against yellow fever virus (YFV) infection in hamsters [19, 20] . The intranasal (i.n.) inoculation used in the YFV study prevents the host immune system from recognizing the Ad5 vector, thereby bypassing any possible preexisting immunity [21] . In the present study, we evaluated the use of DEF201 administered i.n. for the prevention and treatment of PICV infection in hamsters.
All animal procedures complied with USDA guidelines and were conducted at the AAALAC-accredited Laboratory Animal Research Center at Utah State University under protocol 1229, approved by the Utah State University Institutional Animal Care and Use Committee.
Female golden Syrian hamsters were obtained from Charles River Laboratories (Wilmington, MA) and acclimated for a minimum of 6 days prior to experimentation. They were fed standard hamster chow and tap water ad libitum. Animals were approximately 7-9 weeks old at the time of virus challenge.
PICV, strain An 4763, was provided by Dr. David Gangemi (Clemson University, Clemson, South Carolina). The virus was passaged once through hamsters. Virus stocks were prepared from pooled livers harvested from infected hamsters. Virus dilutions were made in minimal essential medium (MEM), and infectious inoculum was given bilaterally in two intraperitoneal (i.p.) injections of 0.1 mL each. The recombinant adenovirus vectored cIFN-a (rAd5-huIFN-a; DEF201) and the rAd5 empty vector (rAd EV) control virus were provided by Defyrus, Inc. (Toronto, ON, Canada) at a concentration of 6610 9 and 2610 11 plaque-forming units (pfu)/ml, respectively. Both viruses were prepared in PBS for i.n. instillation in a 200 ml volume.
Virus titers were assayed using an infectious cell culture assay as previously described [22] . Briefly, a specific volume of liver or spleen homogenate or serum was serially diluted and added to triplicate wells of Vero (African green monkey kidney; American Type Culture Collection, Manassas, VA) cell monolayers in 96well microplates. The viral cytopathic effect (CPE) was determined 7 to 8 days post-virus inoculation, and the 50% endpoints were calculated as described [23] . The assay detection ranges were 2.8 to 9.5 log 10 50% cell culture infectious doses (CCID 50 )/g of liver or spleen and 1.8 to 8.5 log 10 CCID 50 /ml of serum. In samples presenting with undetectable liver or spleen virus, a value of ,2.8 was assigned (,1.8 for serum). Conversely, in cases wherein virus exceeded the detection range, a value of.9.5 (.8.5 for serum) was assigned. For statistical analysis, values of 2.8 or 9.5 log 10 (1.8 or 8.5 for serum) were assigned as needed for samples with undetectable or saturated virus levels, respectively.
Detection of ALT in serum is an indirect method for evaluating liver disease. Serum ALT levels were measured using the ALT (SGPT) Reagent Set purchased from Pointe Scientific, Inc. (Lincoln Park, MI) per the manufacturer's recommendations. The reagent volumes were adjusted for analysis on 96-well microplates.
Experimental design DEF201 dose range titration experiment. Hamsters were weighed on the morning prior to the day of infection and grouped (n = 15 for drug treatment groups, 26 for the placebo group) so that the average hamster weight per group across the entire experiment varied by less than 5 grams. Varying pfu amounts of DEF201, the rAd EV control virus, or saline placebo treatments were administered in a single i.n. dose 24 h prior to challenge with ,5 pfu of PICV. Five animals from each group were sacrificed on day 7 of infection. Serum was collected for assaying ALT activity, and virus titers were determined for liver, spleen, and serum samples as described above. The remaining 10 animals (21 for the placebo group) were observed 21 days for mortality and weighed individually every 3 days starting on day 0. Sham-infected normal controls (n = 3) were included for comparison.
Extended pre-exposure prophylaxis experiment. The design was similar to the DEF201 titration experiment with the following differences. Hamsters were weighed on the morning of initial pretreatment (day 214 relative to the infection) and grouped (n = 15 per group). Groups were treated once i.n. with 10 8 pfu of DEF201, rAd EV control virus, or saline placebo. Treatments were given 14 or 7 days prior to challenge with ,5 PFU of PICV. Animals were observed for 28 days post-challenge for mortality.
Post-exposure prophylaxis experiment. The design was similar to the DEF201 pre-exposure prophylaxis experiment with the following differences. Single dose i.n. treatments with 10 8 pfu of DEF201 or rAd EV were administered 24 h prior to, or 6, 24, or 48 h after challenge with ,5 pfu of PICV. On day 28 postinfection, the surviving animals (including 6 naïve sham-infected controls) were re-challenged. Morbidity and mortality were observed out to 58 days after the initial challenge.
Kaplan-Meier survival plots and all statistical evaluations were done using Prism (GraphPad Software, CA). The log-rank test was used for survival analysis. For analyzing differences in viral titers, ALT levels, and weight change, a one-way analysis of variance (ANOVA) with Newman-Keuls post test or the Kruskal-Wallis (two-tailed) test with the Dunn's post test was performed based on Gaussian distribution of the data.
In the initial trial, hamsters were treated with 10 6 to 10 8 pfu of DEF201 one day prior to challenge with a lethal dose of PICV.
Pretreatment with the highest dose of 10 8 pfu of DEF201 resulted in 100% survival, and 10 7 and 10 6 pfu doses also significantly protected 90% and 60% of hamsters, respectively, from mortality ( Figure 1A) . Moreover, the hamster that succumbed in the 10 7 group, survived 19 days. Importantly, only one out of ten hamsters treated with 10 8 pfu of the control rAd EV virus survived the infection; however, there did appear to be a slight delay in the time to death in the hamsters that received the control virus treatment.
The weights of the hamsters were measured every 3 days to assess weight gain over the course of the experiment as a marker of well being ( Figure 1B) . Notably, from day 3 to day 6, a time before weight loss due to illness from PICV infection would have been expected, hamster weights decreased as the dose of DEF201 increased. This would suggest that the higher treatment doses may have resulted in some loss of appetite, probably due to mild illness due to expression of consensus IFN since no overt effects were noticeable when handling the animals. The hamsters that received the 10 6 pfu dose of DEF201 gained weight through day 6 similarly to the animals treated with saline placebo and the normal controls (sham-infected, untreated) ( Figure 1B) . The high-dose of rAd EV control virus also resulted in a slight reduction in weight compared to the controls, suggesting that the immune response to the adenoviral vector alone may have caused some malaise in the animals.
There was no elevation in serum ALT levels on day 7 of infection in samples collected from parallel treated and infected hamsters receiving DEF201 (Figure 2A ). Eighty percent of the rAd EV group and 100% of the PBS placebo group had elevated levels of ALT, reflective of liver disease. Interestingly, the 10 7 and 10 8 pfu DEF201 groups presented with little to no day-7 virus burden in the serum, liver, or spleen, while the 10 6 group developed viral titers that were comparable to the rAd EV and placebo controls ( Figure 2B-D) . A delay in the development of liver disease in the 10 6 pfu DEF201treated animals may explain the reduced ALT levels. Alternatively, saturation of liver virus titers in the low-dose DEF201, rAd EV, and placebo groups may have masked a substantial difference between the former and the viral vector and vehicle control groups.
We next evaluated the prophylactic window of protection against PICV infection using the 10 8 pfu dose of DEF201. Animals were treated one or two weeks prior to challenge with a lethal dose of PICV. Consistent with the trend observed in initial dose titration study, hamsters treated with the 10 8 pfu dose of DEF201 had significantly reduced weights compared to those that received the rAd EV and placebo control treatments ( Figure 3A) . Nevertheless, the pretreatment with DEF201 seven days before infection was highly protective (90% survival rate; Figure 3B ). Notably, the single hamster that failed to survive the challenge succumbed on day 5, which was several days before the mean time to death measured in both the placebo and rAd EV groups. An autopsy to determine the cause of death was not performed.
In hamsters treated two-weeks prior to PICV challenge, DEF201 significantly reduced mortality (50% survival) and extended the time of death in the animals that succumbed ( Figure 3C ). In contrast, uniform lethality was seen with animals that received the rAd EV and placebo treatments. Of the 5 surviving animals pre-treated with DEF201, one was anorexic at the conclusion of the study on day 28 post-infection. This was reflected by a 27% weight loss compared to the animals starting weight. It is possible that this hamster, which appeared ill and lethargic, was not able to completely prevent the infection. It was unclear whether it would have ultimately recovered if the observation period had been extended.
On both the 7-day ( Figure 4A , C, E, G) and 14-day ( Figure 4B , D, F, H) pretreatments, DEF201 significantly reduced day-7 viral loads and liver disease (ALT) compared to the controls. The absence of elevated ALT levels in the DEF201-treated hamsters may be explained by the 2-3 log 10 reduction in liver virus burden ( Figure 4E , F) and a delay in the development of liver disease. Although tissue titers were slightly lower when DEF201 was given 7 days prior to challenge compared to the 14 day pretreatment, this was not evident with serum viral burden. Because most animals had measurable replicating PICV ( Figure 4C-H) , it is likely that survivors would have been immunized and protected from subsequent challenge. This may not be the case with hamsters treated with DEF201 24 h prior to challenge since most had no detectable virus titers in spleen, liver, or serum on day 7 of PICV infection ( Figure 2B-D) .
Having observed dramatic protection when administered up to 2 weeks prior to challenge, a final experiment was conducted to determine the therapeutic value of DEF201 in the hamster PICV infection model. When DEF201 was administered 6 or 24 h after challenge, highly significant protection was observed ( Figure 5 ). Efficacy waned when DEF201 treatment was delayed to 48 h postinfection. As anticipated, the treatment given 24 pre-challenge verified previous activity, with all animals surviving challenge.
Interestingly, there was higher than expected survival with the control rAd EV treatments initiated 24 and 48 h post-challenge, suggestive of a slight antiviral effect as the time of treatment was further delayed ( Figure 5) .
The surviving hamsters from this experiment were re-challenged with PICV to assess the ability of DEF201 to enhance longer-term protection via acquired immunity. With the exception of 4 animals in the 24 h DEF201 pretreatment group, and a single animal in the rAd EV 48 h group, all animals that were challenged with PICV on day 0 of the experiment survived a second challenge Figure 3 . DEF201 extended pre-exposure prophylaxis protects hamsters from lethal PICV challenge. Animals were treated i.n. with a single dose 10 8 pfu of DEF201, the rAd EV control virus, or PBS placebo 7 or 14 days prior to PICV infection. Animal weights were measured two weeks prior to, and at the time of, PICV challenge. The effect of 7-day and 14-day pretreatments on A) weight change over the two-week period prior to PICV challenge and the extended PICV prophylaxis efficacy data for the B) 7-day and C) 14-day pretreatments are shown. ***P,0.001 compared to respective placebo-treated animals. b P,0.01, c P,0.001 compared to respective rAd EV-treated animals. doi:10.1371/journal.pone.0026072.g003 on day 28 ( Figure 5 ). All six naïve animals that were initially shaminfected succumbed as expected.
In animals that were sacrificed on day 7 relative to the first infection, reductions in ALT and viral titers were most evident in the groups that received DEF201 within 24 h of infection ( Figure 6) . Notably, in the animals treated with the control rAd EV, there was an interesting trend that developed with the 6 h post-infection group having the greatest ALT levels and viral titers, followed by the 24, 48, and 224 h groups. This trend may suggest a low-level immune stimulation in the hamsters relative to the time at which the rAd EV was given. The resulting lack of measurable viral replication in the 224 h DEF201 group ( Figure 6B-D) is likely insufficient to elicit immunological memory. It is unclear as to why one of the first infection survivors from the 48 h rAd EV group ultimately succumbed to the second infection.
In the present study, our findings demonstrate that expression of cIFN-a following a single i.n. administration of DEF201 offers a strong protective effect in hamsters against challenge with PICV that included limiting liver disease and inducing an antiviral state that inhibited systemic and tissue viral replication. The lack of significant antiviral activity elicited by the rAd EV control virus suggests that the enhanced antiviral response produced by DEF201 is largely due to the expression of the cIFN-a gene. The weak stimulatory effect seen in 1 of the 3 experiments was not surprising considering the number of host systems that play a role in sensing the adenovirus vector [24] ; however, the effect was short-lived. In contrast, the enhancement of the host antiviral defenses by DEF201 was long-lasting with a 14-day pre-PICV challenge prophylactic window. Moreover, DEF201 was effective when given 1-2 days post-PICV infection. These data also suggest that sufficient viral replication may be necessary to elicit an adaptive immune response that confers lasting protective immunity, as, for the most part, only re-challenged animals from the 24 h DEF201 pretreatment group succumbed to a second challenge with a lethal PICV inoculum. Presumably, the robust innate immunity and antiviral state induced by the DEF201 pretreatment rapidly controlled the ,5 pfu challenge dose obviating the development of the adaptive immune response and immunological memory.
The pathogenic arenaviruses have evolved strategies to suppress and evade the host immune response [25, 26, 27, 28] , resulting in uncontrolled replication and broad dissemination. However, they appear to be unable to block the induction of IFN stimulated genes via exogenous type I IFN [29] , which may, in part, explain the success of DEF201 and cIFN-a treatments [15] . Also essential to the success of DEF201 was early intervention prior to significant viral replication and engagement of innate immune suppressive functions. Indeed, early induction of a strong type I IFN response is associated with favorable disease outcome in nonhuman primates challenged with Lassa virus [30] . Early post-exposure prophylaxis was also required with exogenous cIFN-a protein administered by the i.p. route [15, 16] . With the multiple strategies that arena and other pathogenic viruses have in place to subdue the IFN-mediated host antiviral response [31] , the utility of DEF201, recombinant IFN proteins, and IFN inducing agents will depend upon the nature of the IFN pathway blockade and require early administration to be effective post-exposure.
Notably, with daily cIFN-a protein injections of up to 40 mg/kg, significant protection was observed; however, survival rates did not exceed 80% in those studies employing the same PICV hamster model system and virus stock [15, 16] . In contrast, DEF201 consistently elicited greater protection (90-100%). The improved efficacy observed with DEF201 may be explained by a combination of factors that includes constitutive expression of fully glycosylated protein and reduced animal stress levels by avoiding daily injections for 7-10 days. We hypothesize that with the appropriate dose of DEF201, therapeutic levels of consensus IFN-a can be maintained, effectively eliminating the daily bolus effect produced by i.p. injections. In addition, because cIFN-a is produced in genetically engineered Escherichia coli, the native glycosylation pattern is lost. Conceivably, enhanced immunotherapeutic activity results from fully glycosylated cIFN-a expressed from cells transduced with DEF201.
Previous studies in mice with a related DEF201 virus expressing mouse IFN-a (mDEF201) have shown the utility of adenovirusbased system to counter viral infections [20, 32, 33] . More recently, in a different hamster model of viral hemorrhagic fever, several of us reported on efficacy of DEF201 in mitigating YFV infection and disease [19] . YFV infection appears to be more sensitive to the effects of DEF201, as a lower dose was able to provide complete protection. Taken together with the results of the present study, the experimental animal data support the broad use of DEF201 for extended pre-exposure and early post-exposure prophylaxis applications. Further investigations using advanced arenavirus models based on challenge of nonhuman primates with pathogenic arenaviruses [17] are needed to better evaluate the potential of DEF201 to prevent severe disease in humans. Nonhuman primate models should allow the full spectrum of cIFN-a activity not possible in hamsters or guinea pigs.
The familiarity of the FDA with adenovirus gene delivery technology and approved cIFN-a protein support the development of DEF201 for clinical use. An important step in the development process is the safety/toxicology testing in rodents, which is presently underway. In our studies, the highest dose of 10 8 pfu of DEF201 administered by the i.n. route appeared to be well-tolerated in hamsters despite evidence of weight loss. They did not appear visibly ill, but clearly the treatment was having some effect that possibly led to reduced food and water consumption consistent with mild toxicity seen with IFN-a therapy. The i.n. delivery route is designed to circumvent pre-existing immunity to adenovirus type 5 in humans [21, 34] , and may limit systemic inflammation that could occur by parenteral administration of large numbers of adenovirus particles. Ultimately, the production of a shelf-stable, powdered formulation of DEF201 for easy i.n. administration and long-term storage would be ideal for stock-piling in the event of the need for mass distribution due to intentional release or (re)emerging disease outbreaks of arena or other viral etiology. Protein Disulfide Isomerase and Host-Pathogen Interaction Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections. Host cells have the ability to cope with the progression and severity of infection in response to different types of pathogen. On the other hand, numerous mechanisms have evolved that support the use of the host cell machinery to facilitate pathogen survival and multiplication. Such co-evolutionary processes are directly affected by different physicochemical factors within different cell compartments, both in the host and in pathogen. For instance, pH critically affects antigen stability of the influenza virus which modulates endosome acidity that attenuates its own infection [1] . ROS (and Reactive Nitrogen Species) production and the redox state of different cell compartments are also critically involved in cellular hostparasite interaction. Among the many redox sensitive proteins that are altered during the course of different infections, protein disulfide isomerase (PDI-) mediated redox switches have been associated with pathogen attachment-internalization, antigen processing in the ER/phagosome, and the regulation of ROS production by Nox family enzymes. Thus, PDI emerges as a ubiquitous redox protein that regulates different steps of diverse infection processes. Several pathogens also have their own PDI that act as an important virulence factor (Table 1 ). Other redox modifications directly mediated by ROS and especially via nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS), which is abundant in phagocytic cells, have been reviewed elsewhere [2, 3] and are not considered in this article. Below, the main cellular redox aspects of host and pathogen PDI will be discussed.
The ancient PDI is a ubiquitous redox chaperone belonging to the thioredoxin oxireductase super family and can reduce (reaction 1), oxidize (reaction 2), and catalyse dithiol-disulfide exchange reactions (i.e., isomerase activities, reaction 3, Figure 1 ). Such broad range of activities overlaps with the chaperone role of PDI that overall performs a housekeeping function in helping to maintain proteins in a more stable conformation. There are around 20 PDI homologues, and the detailed structure and function of eukaryotic PDIs have been covered in recent excellent reviews [4, 5] . The classic mammalian PDI (55 kDa) has several domains ordered as a-b-b -a -c with 2 thioredoxin-like motifs (Trp-Cys-Gly-His-Cys) displayed in the a and a domain [4] [5] [6] (Figure 1 ). PDI is abundant in the ER (∼ 0.5 mM) where the relatively oxidizing conditions at basal level (i.e., GSH/GSSG ratios ∼ 2-3 : 1) favours PDI isomerase/oxidase activity, which is primarily involved in client protein redox folding (reaction 2-3, Figure 1 ). The oxidizing equivalents for this process are driven mainly by the ER thiol-containing oxidase, Ero1 (endoplasmic reticulum oxidase-1), which binds FAD and is in turn re-oxidized via electron transfer to oxygen, generating H 2 O 2 in the process [7] [8] [9] [10] . The H 2 O 2 destiny is elusive, but it can oxidize ER-located peroxiredoxin IV (PrxIV) that is further reduced by PDI that is oxidized in the process [11] . This redox circuit is thought to increase total protein folding and thiol oxidation via Ero1 [11] . However, even in the absence of Ero1, protein folding still occurs, and it is suggested that other oxidases may compensate for redox demand in the ER in some circumstances [12, 13] . Nevertheless, the PDI-Ero1-dependent oxidative activity is balanced to cytosolic glutathione levels suggesting a functional redox interplay between these compartments [12] . PDI reductase activity has been primarily associated to more reducing compartments (i.e., GSH/GSSG ratios ∼ 30-100 : 1), such as those in the vicinity of the plasma membrane [6, 10] . PDI redox versatility is mainly governed by the low pKa of the proximal cysteine on the active N-terminal a domain. Indeed, the lower pKa of 4.5 renders PDI a much better oxidase than thioredoxin, which has a pKa of 7.1 and is mainly a reductase in most neutral pH cell compartments. It should also be noted that PDI functions as a chaperone independently of its redox-active domains as especially required for its ATPase and Ca 2+ activity, although PDI redox motifs still stabilize binding interaction [4, 5, 10] . In the ER, PDI is tightly associated with prolyl-4 hydroxylase (the rate-limiting enzyme for collagen biosynthesis), the Sec61 translocon, and the MHC class I complex (see later). It can be also found as a heterodimer with microsomal triglyceride transfer protein [6, 10] . PDI is a soluble homodimer and does not have a transmembrane domain and, similarly to other ER chaperones, carries the KDEL C-terminal sequence which binds to respective receptors in the COP vesicles that circulate in the ER-Golgi vicinity, recycling proteins back to ER. PDI also undergoes intense intracellular trafficking and is found on the surface of diverse prokaryotic and eukaryotic cells [14] [15] [16] .
Despite limited knowledge about this traffic, it is possible that PDI exits the ER through the translocon Sec61 pore and/or via secretory vesicles [17] . PDI is thought to attach to lipids, glycans, and integral membrane proteins via electrostatic interactions at the cell plasma membrane [14, 15] , where its reductive activity mediates the infection of different pathogens ( Figure 2 , discussed later). PDI along with ERp57 have been found in the nuclei in association to DNA and affecting transcriptional activity of NF-kb, AP-1, and STAT3 [15] . These transcriptional regulators are key elements in many inflammatory processes, but their functional association to PDI and to different pathogen still elusive. In contrast to many other members of its family, such as thioredoxin itself and Erp57, PDI is not normally found in the cytosol, where it is likely cleaved by caspase-3 and -7 [18] . Protozoans and bacteria have their own PDIs ( Table 1 ). The function of these PDIs in protein folding is poorly understood; however, there are intriguing data correlating PDI expression and the pathogenicity of several parasites, especially obligatory intracellular protozoans. Leishmania that leads to distinct types [20] . If L. chagasi is a subgroup of L. infantum brought to America by European colonist or other specie in its own still a matter of controversy (see discussion in [21] ). The infection cycle in the vertebrate host and is initiated when Leishmania promastigote is injected into the skin by the insect vector. In the host, the promastigote is phagocytised especially by macrophages, and further it is converted into intracellular amastigote. Amastigote replicates inside the phagosome within the cell and is liberated after the cell lyses, subsequently infecting other cells resulting in the progression to disease [21, 22] . L. amazonensis has at least four PDIs, and the use of specific PDI inhibitors substantially affected parasite growth [23] . In L. major, the increased levels of Leishmania PDI (LmPDI) expression and secretion at the parasite surface reflects optimal protein folding balanced to parasite multiplication. Importantly, this is correlated to high virulence of the parasite strains [16] . More recently, the use of LmPDI antigens to generate a vaccine for L. major partially protected BALB/c animals and accelerated the cure of different strains of mice [24] . Similarly to Leishmania species, other parasites of the trypanosomatid group such as Trypanosoma contain several genes predicted to encode for PDIs, that can execute N-glycosylation and protein folding in the ER [25, 26] . Although PDI was considered essential for T. brucei survival, PDI activity was not essential for the growth of trypanosomes in vitro [26] . PDI is also expressed in different species of Plasmodium protozoans (Table 1) , the parasites that cause malaria [27, 28] . P. falciparum expresses at least nine different PDIs and the PfPDI-8 has great similarity to the prototype PDI and is expressed during all stages of parasite life cycle. This PDI facilitates the disulfide-dependent conformational folding of EBA-175 protein, an emerging candidate for the development of malaria vaccines [28] . This is intriguing given that malaria parasites express proteins with high content of cysteine, which are associated to parasite invasion and sequestration in the vertebrate host and transmission into mosquito host [28] . Finally, Toxoplasma gondii PDI was identified in host tears, suggesting an extracellular location and adhesion to host cells during the initial phase of infection [29] .
Antigen presentation occur through two distinct pathways. Antigen presenting cells (APCs; especially macrophages and dendritic cells; DCs) are long-lived cells that capture antigens and subsequently process and present them at the cell surface, where they are recognized by T-lymphocytes. This process provides a long-term adaptive immune response to fungi, bacteria, and parasite. After internalization by the APC, antigens pass through phagosome/lysosome vesicles, where they form complexes with MHC class II (Figure 2 ), which are recognized by helper CD4+ T lymphocytes (exogenous pathway). In contrast, self cell antigens and virus synthesized within cells (mostly non-APCs) are degraded by the proteasome in the cytosol and nucleus. In successive steps, the antigen is processed, folded, and incorporated into the MHC class I ( Figure 2 ) and the complex exposed on the cell surface, and recognized by cytotoxic CD8+ T lymphocytes (endogenous pathway). These two pathways overlap and some antigens are presented by both MHC class I and II, in a process called cross-presentation. This has been described in DCs responding to viral infection, transplant rejection, and some autoimmune diseases and cancer. Moreover, a wide range of pathogens passing or living in the phagosome such as Mycobacterium tuberculosis, Salmonella typhimurium, Toxoplasma gondii, and especially Leishmania spp and Trypanosoma cruzi are all crosspresented in association to high levels of CD8 + T cells [31] . PDI as part of the ER protein folding machinery directly regulates antigen processing of the MHC class I complex [32] [33] [34] [35] . Antigens that are degraded by peptidases and proteasome to shorter peptides in the cytosol and nucleus can be further transported to the ER through the TAP system, a transmembrane ER type of ATP-binding cassette (ABC) peptide transporter family [36] . ER-located PDI interacts with the peptide-loading complex (PCL) that efficiently promotes peptide assembly with MHC class I molecules and supporting the exit of the peptide-antigen complex from the ER [32] [33] [34] . Other PCL components include calreticulin, tapasin, ERp57 (another PDI family member), and the TAP transporter itself. Cells lacking PDI present much less peptide loading to MHC class I and the disulfide bridge between the peptide and MHC groove remains in a reduced redox state [32] . Normally, this interaction is affected by the redox exchange between PDI (predominantly oxidized) and ERp57 (predominantly reduced) [32] , a condition in which PDI favours the release of peptide-MHC class I from the PCL and the antigen-MHCI complex is exited from ER [34] . In fact, PDI-bound peptide facilitates the disassembly of the tapasin-ERp57 complex while the PDI unbound to the complex is unable to interact with tapasin-ERp57, retaining MHC I molecules in the ER [34] . Overall, PDI redox activity modulates the stability of the antigen peptide-MHC class I complex and further determines the transport of the complex to the plasma membrane [32] [33] [34] [35] . These redox effects may vary according to the type of antigen and some pathogens interfere with this pathway to escape antigen process and evading CD8 + T-cells recognition. This is the case for US3 protein from human cytomegalovirus, which enhances PDI degradation via the proteasome [32] . PDI participation in immune response, however, goes beyond its role in the ER protein folding machinery and it acts at other cellular steps of host-pathogen interaction. PDI in the ER is also thought to play a role in parasite phagocytosis, and the PDI displayed on the cell surface can mediate the entry of some viral, bacterial, and protozoan. PDI is also implicated in protein unfolding and trafficking of some pathogenic antigens across the endoplasmic reticulum and towards the cytosol by the endoplasmic reticulum-associated degradation system (ERAD). This is the main pathway where proteins are retrotranslocated from the ER to cytosol and further degradated by the proteasome. Next, we discuss some examples of the cellular association between host PDI and different pathogens.
Phagocytosis is the main gate for large microbes to enter into APCs. After binding and attaching to the pathogen, these cells can internalize organisms and large particles even bigger then their own size, which are then phagocytosed in an active process that involves intense membrane remodelling [37] . Proteomics studies accumulated over the last decade revealed the presence of ER chaperones in the isolated phagosome, uncovering a process called ER-mediated phagocytosis [38] [39] [40] [41] [42] [43] [44] [45] . ER chaperones were detected in phagosomes of macrophages exposed to different particulate material and pathogens, including latexbeads opsonised or not with immunoglobulin G (IgG) or mouse serum (to facilitate entry through the FcR or complement receptors), IgG-opsonized erythrocytes, promastigotes of Leishmania donovani derived from wild-type cells or cell-surface LPG knockout, among other parasites [38] . A mix of ER and endocytic vesicles in the formation of the parasitophorous vacuoles (PVs) during the uptake of different Leishmania spp. was recently shown in macrophages overexpressing ER-tagged green fluorescent protein [41] . The presence of the ER proteins Sec61, Bip/Grp78, and PDI in the phagosome of APCs [38, 41] support the idea that the ER provides the necessary machinery for antigen translocation from the phagosome to the cytoplasm and thus, possibly converges MHC class I and class II antigen cross-presentation [42] (Figure 2 ). There are several other complementary hypotheses on how peptides cross from phagosomes to cytoplasm during the cross-presentation process [43, 44] . Neutrophils are short-lived cells (half-life of 4-8 h in the human circulation) and very active in the phagocytosis of large microbes such as bacteria, parasites, and fungus. Contrary to APCs, neutrophils only contain restricted amounts of ER machinery and are thought to lack the ER-mediated phagocytosis process [38] .
Whether ER proteins functionally operate on phagocytosis-mediated infection has not been well characterised yet. An important work has shown that Dictyostelium lacking both ER calreticulin and calnexin present altered phagocytic cup formation and substantial decline in phagocytosis [45] . These two proteins utilise Ca, and their disruption per se affects actin filaments and plasma membrane remodelling during phagocytosis [45] . We recently showed that PDI is critically involved in Leishmania parasite infection in vitro [22] . We showed that phagocytosis of promastigotes (but not amastigotes) of Leishmania chagasi was significantly inhibited by macrophage incubation with the thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way [22] . The phenylarsine response is of particular interest, since this arsenic compound may act similarly to antimonials, widely used in leishmaniasis chemotherapy [46, 47] . PDI preferentially affects parasite internalization and the phagocytosis of the promastigote forms is increased when wild-type PDI is overexpressed in macrophages, an effect opposed by PDI knockdown. At later stages of infection (i.e., after 4 h), PDI from promastigote-infected J774 macrophages was immunoprecipitated and subsequently blotted with an anti-Leishmania antibody revealing a parasite band at ∼ 94 KDa [16, Figure 10 (b), lane 5]. Subsequent removal and analysis of this band by mass fingerprint spectrometry showed a 58% match with elongation factor 2 (EF2) of L. major (Q4Q259; data not shown). The incubation of purified bovine PDI (Sigma, P3818) and parasites did not yield any detectable protein complexes, suggesting that the macrophage milieu may be important to sustain PDI-EF2 association [22] . Interestingly, Leishmania EF2 has important virulent features and acts as a soluble antigen in lymphocyte stimulation in vitro [48] and in vivo [49] . Moreover, proteomics studies revealed that EF2 is secreted during promastigote differentiation into the amastigote stage with potential immunomodulatory proprieties in animal models [50] . Leishmania EF2 is therefore of particular interest for Leishmania therapeutic interventions such as vaccines. Although our studies did not address the role of the ER in mediating phagocytosis, these data provide compelling evidence for a functional role of ER-PDI in a host-parasite interaction. Other mechanisms underlining PDI-mediated L. chagasi promastigote phagocytosis involves its association to ROS production by phagocyte NADPH oxidase and this is discussed next.
The NADPH oxidase (Nox) family of enzymes uses NADPH as an electron donor to convert oxygen to superoxide anion (O 2
•− ), a precursor of H 2 O 2 and other powerful oxidants such as hydroxyl radical and peroxynitrite (in the presence of nitric oxide), collectively called ROS [3, 51, 52] . Each of the seven oxidase family members is characterized by a distinct catalytic subunit (i.e., Nox1-5 and Duox1-2), and has differing requirements for additional protein subunits [51, 52] . The prototypic member of the Nox family, Nox2 oxidase (or gp91 phox oxidase), is best known for its role in neutrophil and macrophage phagocytosis. Genetic defects in the enzyme are related to chronic granulomatous disease, a condition in which affected children suffer from recurrent severe fungal and bacterial infections due to defective phagocyte function [51] . Each Nox isoform forms heterodimers with a lower molecular weight p22 phox subunit and is predicted to be membrane-bound. Nox2 is normally quiescent and acutely activated by agonists such as PMA, LPG, and cytokines in a tightly regulated process in which cytosolic subunits (p47 phox , p67 phox , p40 phox , and Rac1 in the case of macrophages and dendritic cells, or Rac2 in neutrophil) associate with the Nox2-p22 phox heterodimer to initiate enzyme activity [51, 52] . Nox2 also has electrogenic features [53] and in APC cells is linked to the regulation of phagosome/lysosome pH and antigen processing [54, 55] . Usually, phagosome acidity is maintained by a vacuolar ATPase (V-ATPase) that transports protons from the cytosol into the phagosome lumen, therefore regulating the function of lysosome proteases in the fused phagolysosomes. Savina et al. [56] [57] [58] have shown that Nox2-derived superoxide in the phagosomal vesicle promptly consumes protons maintaining a higher pH ambient in dendritic cells during particle internalization, which favours antigen processing and presentation [56] [57] [58] . Opposite results were found in macrophages [56] [57] [58] . The selective role of Nox2 in different phagocytic cells remains to be defined. The jury is out on whether the results shown in macrophages (association of Nox2 to Rab27a; a member of Rab family of GTPases) are related to vesicle traffic molecule assembly and quality, or rather associated to degradation processes [59] .
Nox complex protein expression and function is greatly affected by redox compounds, and it is especially regulated by PDI with implications for cell signalling [60] . The association of PDI to p22 phox and other Nox isoforms in different cell types, especially in vascular cells, has been previously described [60] [61] [62] . A functional and spatial/physical interaction between PDI and the p22 phox oxidase subunit was shown in macrophages [22] and more recently between PDI and p47 phox in neutrophils [62] . In macrophages, PDI-Nox association was correlated to Leishmania infection in vitro [22] . It is well known that during phagocytosis of Leishmania, Nox2 is activated and parasite uptake is inhibited by antioxidants such as catalase [22] . Intriguingly, in the course of promastigote infection, some parasites evade that stressful condition and convert themselves into intracellular amastigotes, multiplying and resulting in progression to a disease process. Overall, our studies support the view that parasite phagocytosis/infection by macrophages is a redox process mediated by PDI in at least two ways. Initially, PDI-NADPH oxidase increases ROS production generating an oxidizing milieu, which seems to favour promastigote infection. The downstream role of ROS generated by PDI-NADPH oxidase remains unknown but can be related to the unfolded protein response signalling [63] or, similar to PDI-Ero1, to protein folding in the macrophage ER compartment with key implications for antigen processing. Nevertheless, at later stages of infection, macrophage PDI physically associates with Leishmania elongation factor-2 (as discussed earlier).
Some viruses envelop their genetic material within a protein-coated capsid in a further lipid membrane layout, for example, influenza virus, baculovirus, hepatitis-C, HIV, and Herpes virus. These enveloped particles require successive steps to successfully entry and infect host cells. They usually first attach onto host receptors (and attachment factors), and their membranes fuse to interact with endosome vesicles that traffic the virus toward the endoplasmic reticulum, where it is uncoated. The proteins are finally transported to the cytosol and nucleus [64] (Figure 2 ). There is convincing evidence showing that most viral infections are strongly influenced by changes in the redox environment and that host PDI mediates infection of enveloped viruses [65] [66] [67] [68] [69] [70] .
In the course of HIV infection, the virus first binds to attachment factors, for example, mannose binding C-type lectin receptor and intracellular adhesion molecule (ICAM-3) on the surface of host CD4 + T cells. The glycoprotein 120 (gp120) subunit of the virus envelope binds to immunoglobulin G of CD4 + and undergoes conformational changes, allowing the virus to interact with its coreceptors, CXCR4 or CCR5. These interactions favour downstream conversions of gp41 envelope subunit to a competent fusion conformation. Initial studies showed that membrane-impermeable PDI inhibitors and monoclonal antibodies against PDI prevent HIV-1 infection [65] . It was then revealed that the domain D2 of the CD4 has redox-active disulfide bonds and is regulated by thioredoxin [66] . Using membrane-impermeable reducing agents (especially arsenical-derived compounds) and labelling thiol reagents, it was demonstrated that CD4 + reactive thiols critically drive HIV entry into cells [66] . Work from another group also revealed that PDI, on the surface of HIV-1 target cells, reduces disulfide bonds of the recombinant envelope glycoprotein gp120 (reaction 1, Figure 1 ), a reaction prevented by the usual PDI inhibitors [67] . Intriguingly, PDI silencing in U373 and HeLa cells had little impact on HIV infection itself as compared to the effect mediated by general thiol inhibitors [68] . The reasons for this discrepancy remain to be elucidated and raise the question whether the reductive effect of PDI is coupled to other redox proteins (e.g., thioredoxin or Nox's) that could amplify virus-CD4 redox association in some cells. It is noteworthy that in these later studies, PDI knockdown on the cell surface was not evident as compared to massive loss of most PDIs within the ER; an observation that supports the idea that PDI in the ER has little impact in HIV-mediated infection [68] . Thiol inhibitors also affect viral fusion as that mediated by the fusion (F) protein from the Paramyxovirus Newcastle disease virus [69] . The overexpression of PDI family members PFDI and ERdj5 has also been shown to significantly catalyze the reduction of thiols in F protein, facilitating membrane fusion [70] . There is evidence suggesting a possible association between PDI and infection mediated by some members of the of Herpesviridae viruses family [71] .
PDI is also implicated in the attachment of some bacteria from different species of Chlamydia [72] [73] [74] . Chlamydia is an obligatory intracellular pathogen that causes diverse diseases in humans. The most common species are Chlamydia trachomatis, which is sexually transmitted and can cause blindness and infertility, and C. pneumoniae, which affects the respiratory tract. CHO cells have impaired endogenous PDI expression due to a defect in truncated mRNA processing, thus providing a valuable model to understand the effect of PDI-mediated cell-cell interaction and infection. These cells are very resistant to Chlamydia infection showing impaired attachment, an effect restored by ectopic expression of PDI [73] . Similar to HIV infection, the molecular mechanisms most likely include the reductive activity of PDI (reaction 1, Figure 1 ) on the surface of CHO cells [72] .
Crossing the endoplasmic reticulum (ER) membrane is an irreversible process for most proteins. In some cases, however, this flow is reversed and misfolded proteins retained in the ER are retrotranslocated to the cytosol via ERAD to be degraded by the proteasome. This pathway is also exploited by small pathogens, especially non-enveloped viruses and some bacterial toxins, to gain access to the cytosol. In these cases, antigenic particles that reach the ER by different means suffer molecular redox rearrangements and binding to PDI allowing them to be transported back to the cytosol or nucleus. Well-known examples are infections mediated by Polyomaviruses (Py) and Simian virus 40 (SV40) extensively studied in the field of carcinogenesis. After SV40 interaction with the GM1 receptor on the cell surface, the particle enters the host cell through endocytosis and traffics via the caveosome (a particular caveolin containing endosome with neutral pH) towards the ER compartment [75, 76] . SV40-coated pentamers are linked to each other by disulfide bonds between cysteine 104 (C104). Further isomerisation in the ER is crucial for the viral uncoating process. In vitro cell screening shows that among all ER-resident proteins, PDI and ERp57 more specifically regulate SV40 infection [75] . PDI silencing substantially decreases SV40 infection that is also dependent on some redox sensitive cysteines on the viral particle [75] . PDI cooperation with ERassociated ERAD proteins Derlin-1 and Sel1L is Ca dependent and facilitates SV40 traffic through ERAD [75] . A similar pathway is used by some nonobligatory intracellular bacteria that exert their effect through production of potent endotoxins, such as diphtheria toxin (DT) and cholera toxin (CT). These proteins function similarly to some plant toxins, such as ricin and abrin. Conversion into toxic proteins involves cleavage of their interchain disulfide bond, allowing them to traffic into the endocytic pathway within the host cell [77, 78] . In humans, CT is derived from the Bacterium Vibrio cholerae that causes cholera disease and has 2 subunits (A1 and A2). The protein first attaches to the host cell surface via GM1 and the subunit A2, which contains a KDEL sequence, and is transported back to the ER (see earlier discussion). There, PDI reduces and unfolds A2 and A1 that exit the ER via the Sec61 channel into the cytosol. PDI in the reduced state (reaction 1, Figure 1 ) binds to the toxin and subsequent oxidation of PDI, probably via Ero1α, enables the release of CT toxin [79, 80] . The active polypeptide A1 efficiently modifies a heterotrimeric G protein in the cytosol that leads to massive loss of chlorine and water secretion by intestinal epithelial cells in mammals, resulting in severe diarrhoea.
In this article we have reviewed the main cellular aspects of PDI-mediated host pathogen interactions and the pathways that are involved in viral, bacterial (including bacterial toxins), and parasitic infections. A number of cellular mechanisms through which PDI modulates some specific cellular pathways in immune cells have been described, such as redox-sensitive attachment, antigen presentation in the ER and exit from it, and association to phagosome and ROS production by NADPH oxidase (Figure 2 ). Many of these responses are antigen-specific and the precise mechanisms of action remain to be fully elucidated, especially in the context of redox changes in cross-presentation phenomena. Moreover, little is known about the role of PDI in infection per se, as well as how PDI signals to a more integrated cellular response to stress [63] . PDI global knockout mice are only viable until birth, but partial gene-modified mice and also modified pathogens will help to reveal the significant redox role of PDI and its redox partners. Overall, PDI is a key regulator that may propagate or limit the severity of the infection processes, depending on the infectious organism involved. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.
Endoplasmic reticulum MHC: Major histocompatibility complex H 2 O 2 : Hydrogen peroxide ERp57: A member of PDI family also know as glucose-regulated protein or 58-kD (GRP58) NF-kB: Factor nuclear kappa B AP-1: Activator protein 1 STAT-3: Signal transducer and activator of transcription 3. Improved Immunological Tolerance Following Combination Therapy with CTLA-4/Ig and AAV-Mediated PD-L1/2 Muscle Gene Transfer Initially thought as being non-immunogenic, recombinant AAVs have emerged as efficient vector candidates for treating monogenic diseases. It is now clear however that they induce potent immune responses against transgene products which can lead to destruction of transduced cells. Therefore, developing strategies to circumvent these immune responses and facilitate long-term expression of transgenic therapeutic proteins is a main challenge in gene therapy. We evaluated herein a strategy to inhibit the undesirable immune activation that follows muscle gene transfer by administration of CTLA-4/Ig to block the costimulatory signals required early during immune priming and by using gene transfer of PD-1 ligands to inhibit T cell functions at the tissue sites. We provide the proof of principle that this combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can improve persistence of transduced cells in experimental settings where cytotoxic T cells escape initial blockade. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward transgene products. Since the original reports describing the use of adeno-associated virus (AAV) vectors for transfer of β-galactosidase gene to muscle (Kessler et al., 1996; Xiao et al., 1996; Fisher et al., 1997) , recombinant AAVs (rAAV) have emerged as very efficient and potentially non-immunogenic vector candidates for delivering therapeutic genes to a variety of tissues and treating monogenic diseases. As they poorly activate innate immunity and weakly transduce dendritic cells, rAAV appear as far less immunogenic than adenoviral vectors (Zhang et al., 2000; Zaiss et al., 2002; McCaffrey et al., 2008) . Nevertheless, it has rapidly become clear that rAAV vectors carrying various transgenes can, under different conditions, induce potent immune responses that could ultimately lead to destruction of transduced cells in vivo and consecutive disappearance of transgene expression (Manning et al., 1997 (Manning et al., , 1998 Halbert et al., 1998; Brockstedt et al., 1999) . Not surprisingly therefore, their use has even been proposed in genetic vaccination protocols aimed at eliciting cellular and humoral immune responses against different microorganisms (Kuck et al., 2006; Du et al., 2008) . In primates and humans, rAAV administration has also been documented to elicit significant cytotoxic CD8 + T cell responses directed against the viral as well as the transgenic "exogenous" proteins, resulting in the destruction of transduced cells and complete loss of transgene expression (Manno et al., 2006; Mingozzi et al., 2007; Gao et al., 2009) . Additionally, on the side of the humoral immunity, production of neutralizing antibodies targeting capsid proteins may also prevent vector readministration and accelerate the loss of the therapeutic protein through the formation of immune complexes. Such immune complexes may further sensitize the cellular immune response by enhancing cross-presentation of the transgenic protein by the antigen-presenting cells (APC). Therefore, developing strategies to circumvent immune responses and facilitate long-term expression of transgenic therapeutic proteins has been identified as one of today's main challenges for the translation of rAAV vectors into the clinic (Mingozzi and High, 2011a,b; Nayak and Herzog, 2011) .
Depending on the experimental situation, rAAV-mediated gene transfer can either lead to durable transgene expression or, conversely, to the rapid formation of neutralizing antibodies and/or destruction of transduced cells by cytotoxic cells. Several factors influencing the immune response against transgenic proteins encoded by the rAAV vectors have now been identified including host species, route of administration, vector dose, immunogenicity of the transgenic protein, inflammatory status of the host and capsid serotype (Mays and Wilson, 2011) . These factors are thought to influence immunogenicity by triggering innate immunity, cytokine production, APC maturation, antigen presentation and, ultimately, priming of naïve T lymphocytes to functional effectors. Therefore, the idea to dampen immune activation by interfering with these very mechanisms has logically emerged with the aim to induce a short-term immunosuppression, avoid the early immune priming that follows vector administration and promote long-term tolerance (Zaiss and Muruve, 2008) .
Here, we evaluated two different strategies to inhibit the undesirable immune activation that follows muscle gene transfer by acting at two different checkpoints of the immune response, i.e., on T cell priming or on the functions of activated T cells that may escape such priming blockade. We used the administration of CTLA-4/Ig to inhibit the substantial immune priming that immediately follow vector injection. Indeed, CTLA-4/Ig represent a potent immunosuppressive fusion protein that reversibly prevents T cell activation (Wallace et al., 1995) and is now used in the clinic to treat inflammatory diseases such as rheumatoid arthritis (Bluestone et al., 2006) . Its immunomodulatory action depends on its competitive inhibitory effect on the CD28/B7 pathway thereby preventing the pivotal CD28-dependent costimulation required to fully activate T lymphocytes (Salomon and Bluestone, 2001) . As a second strategy, we turned to immunomodulatory molecules that could protect transduced muscle fibers from immune attacks by activated T cells. For that, we aimed at stimulating the inhibitory PD-1 molecule expressed on T cells upon activation, by the gene transfer of its ligands PD-L1 or PD-L2 to muscle cells (Freeman et al., 2000; Latchman et al., 2001; Ishida et al., 2002) . We show herein that acting on these two non-redundant mechanisms of tolerance provides synergistic effects that prolong transgene expression in muscle even in the presence of circulating cytotoxic T cells directed against the transgene product.
Female C57BL/6 (B6) mice were obtained from Centre d'Elevage Janvier (Le Genest Saint Isle, France). Mice were all between 7 and 10 weeks of age at beginning of experiments. For transduction with rAAV vectors, mice back legs were shaved under general anesthesia and titrated 1 × 10 11 vector genomes (vg) rAAV2/1-Ova or rAAV2/8-Ova were injected (50 μl in PBS) in the gastrocnemius muscles. Where indicated, 10 11 vg rAAV2/1-PD-L1 or rAAV2/1-PD-L2 were mixed with 10 11 vg rAAV-Ova and co-injected using the same procedure. For costimulation blockade experiments, mice were injected i.p. with 200 μg CTLA-4/Ig (Chimerigen laboratories, MF-110A4) diluted in 200 μl of PBS.
In some experiments, lymphocytes from tolerized mice were transferred to conditioned recipients to further evaluate the presence of anti-Ova lymphocytes and to study their functionality in vivo. For that, 50 × 10 6 splenocytes harvested from individual mice that have be treated 80 days before with AAV-Ova, with or without immunomodulatory regimens, were injected i.v. into 5 Gy-irradiated syngenic C57BL/6 recipient. One day after, each individual mouse was shaved and injected subcutaneously with 1 × 10 6 syngenic EG7 tumor cells expressing the Ova antigen and known to be sensitive in vivo to CD8 + T cell cytotoxicity in primed animals (Moore et al., 1988) . Tumor sizes were measured with a digital caliper three times a week during 26 days. Tumor volume was calculated as length × width × [(length + width)/2].
All animal experiments were approved by the local institutional ethic committee for animal experimentation (authorization #0211-22 "Comité Régional d'Éthique en Expérimentation Animale de Normandie").
The rAAV-Ova vector, a kind gift of Roland W. Herzog, was described previously (Wang et al., 2005) . The cDNA encoding mouse PD-L1 (CD274) or PD-L2 (CD273) were cloned using standard molecular biology procedures and introduced in the SSV9-CAG plasmidic backbone after digestion with EcoRI. The resulting expression cassette, flanked by AAV serotype 2 inverted terminal repeats (ITRs), contains the CAG promoter combining the cytomegalovirus early enhancer and the chicken β-actin promoter, a chicken β-actin intron, and a rabbit β-globin polyadenylation signal. rAAV2/1 and rAAV2/8 vectors were generated using a standard helper-virus free three-plasmid transient transfection method and pseudotyped with either AAV1 or AAV8 capsid proteins. Vectors were purified by two cesium chloride gradient centrifugations and dialyzed against PBS as described (Salvetti et al., 1998) . Genome titers of vector preparations were assayed by Dot-blot hybridization using a probe to detect the CAG or CMV promoter.
Quantification of soluble Ova (sOVA) concentration in serum was performed by Ova-specific ELISA. Microtiter plates were coated with polyclonal rabbit anti-Ova antibodies (1:3000 dilution, Ray-Biotech) and bound sOVA was detected using biotinylated rabbit polyclonal anti-Ova antibodies (1:5000 dilution, Abcam) and streptavidin-peroxidase (1:15000, Roche).
Detection of serum anti-Ova IgG antibodies was performed by ELISA using Ova-coated microtiter plates. Anti-Ova IgG antibodies were detected using biotinylated polyclonal goat anti-mouse IgG and revealed using the mouse ExtrAvidin kit (Sigma-Aldrich). IgG titers were defined as the dilution yielding the half maximum optical density obtained with control serums and was calculated using sigmoid curve fitting using GraphPad prism software.
To analyze the quantities of Ova DNA and Ova mRNA present in transduced muscles of treated mice at indicated time points, a real-time PCR assay was developed. Muscles collected from each mouse were kept at −80˚C before DNA and RNA extraction performed using the phenol/chloroform method and the RNeasy Fibrous Tissue Mini Kit (Qiagen) following the manufacturer's instructions.
For quantification of Ova DNA, the primers used were Ova-F (5 -AAG CAG GCA GAG AGG TGG TA-3 ), Ova-R (5 -GAA TGG ATG GTC AGC CCT AA-3 ), CD8a-F (5 -GGT GCA TTC TCA CTC TGA GTT CC-3 ), and CD8a-R (5 -GCA GAC AGA GCT GAT TTC CTA TGT G-3 ). For all reaction mixtures, 10 μl of FastStart Universal SYBR green master mix (Roche) was used in a final volume of 20 μl. Ova primers were used at 500 nM and CD8a primers at 400 nM. Approximately 10 ng of DNA was added in a 5-μl volume and always set up in duplicate. Each qPCR was performed under the following conditions: 10 min hot-start denaturation at 95˚C and 40 amplification cycles (10 s at 95˚C, Frontiers in Microbiology | Microbial Immunology 30 s at 60˚C). The melting temperatures of the final double-strand DNA products were determined by gradual heating from 60 to 95˚C over 20 min. All qPCRs were performed with a StepOne-Plus real-time PCR system (Applied Biosystems) and corresponding software. Absolute amounts of Ova and CD8a amplicons, in arbitrary units, were determined using serial dilutions of pAAV-CMV-OVA plasmid or pTOPO-CD8a plasmid as a standard. The data were expressed as Ova/CD8a ratios, fixed at 1 for PBS-injected control mice.
For quantification of Ova mRNA, 100 ng of total RNA were reverse transcribed using iScript DNA Synthesis Kit (Biorad). Then, 2 μl of cDNA were subjected to real-time PCR amplification using Ova primers, β-actin-F (5 -AAG ATC TGG CAC CAC ACC TTC T-3 ) and β-actin-R (5 -TTT TCA CGG TTG GCC TTA GG-3 ) primers. For all reaction mixtures, 10 μl of FastStart Universal SYBR green master mix (Roche) was used in a final volume of 20 μl, Ova primers were used at 500 nM and β-actin primers at 400 nM. The same qPCR program as above-described conditions were used. The absolute amount of Ova mRNA for each sample was then normalized against the β-actin mRNA amount (arbitrary units) and determined using serial dilutions of pAAV-CMV-OVA plasmid and β-actin purified PCR product.
Fluorescently labeled anti-CD4 (RM4-5), -CD8 (53-6.7), -CD44 (IM7), -CD62L (MEL-14), -PD-1 (J43), -PD-L1 (B7-H1), -PD-L2 (B7-DC) monoclonal antibodies (mAb), and unlabeled anti-CD16/CD32 antibodies were all purchased from eBioscience. PE-conjugated H-2K b /Ova 257-264 pentamers were used to detect the CD8 + cells that specifically recognize the immunodominant Ova-derived SIINFEKL peptide using the manufacturers' protocol (Proimmune). Flow cytometry measurements of single cell suspensions derived from lymph nodes, spleen, or blood samples were performed using standard procedures and acquired on a FACS-Canto (BD Biosciences) instrument. Flow cytometry analyses were performed using the FlowJo software (Tree Star).
Enzyme-linked immunospot (ELISpot) assays were performed following the instructions of the manufacturer (Diaclone). Briefly, PVDF membrane plates were coated with capture antibody against mouse IFNγ and blocked with a 2% skimmed milk solution prepared in PBS. 2 × 10 5 Splenocytes per well were cultured in vitro for 36 h in the presence of 10 μg/ml SIINFEKL peptide. Plates were revealed after incubation with anti-mouse IFNγ detection antibody coupled to biotin followed with a streptavidinalkaline phosphatase conjugate and exposure to a ready-to-use solution of nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3 -indolyphosphate (BCIP) for chromogenic development. Plates were analyzed with an ELISpot plate reader and a dedicated ImmunoSpots software (C.T.L.).
Results were expressed as mean ± SEM. Significance was assessed by non-parametric one-way ANOVA (Kruskal-Wallis tests) using the GraphPad Prism software. Results were considered statistically significant when the p value was inferior to 0.05 (*), to 0.01(**) or to 0.001 (***).
We used rAAV2/1-Ova and rAAV2/8-Ova administration to model a gene therapy setting where the transgene encodes for a highly immunogenic secreted protein. We first evaluated the capacity of these vectors to transduce muscle cells upon direct i.m. injection. Injection of 10 11 viral genome (vg) rAAV2/1-Ova or rAAV2/8-Ova similarly resulted in efficient transduction, as attested by the detection of Ova DNA and mRNA in the injected muscles quantified by qPCR and qRT-PCR respectively ( Figure 1A) . No significant difference was found when comparing the transduction efficiency of rAAV2/1-Ova and rAAV2/8-Ova in these conditions.
Transgene expression in muscle was found to be high early after transduction but rapidly declined over time, suggesting the occurrence of an immune response against transduced cells ( Figure 1A) . To investigate this point, we quantified the cellular and humoral immune responses directed against the xenogenic Ova transgene product by flow cytometry and ELISA. Staining with MHC-I pentamers presenting the immunodominant Ova-derived SIINFEKL peptide, designated hereafter as H-2K b /Ova pentamers, allowed to monitor the expansion of CD8 + lymphocytes in spleen and draining lymph nodes of rAAV-Ova challenged animals. This analysis revealed that both rAAV2/1-Ova and rAAV2/8-Ova injected in the gastrocnemius muscles induced a robust expansion of anti-Ova CD8 + lymphocytes (Figures 1B,C) . Interestingly, the kinetics and intensity of T cell priming was found to be different when comparing the cells harvested from the draining lymph nodes and from the spleen. In the lymph nodes draining the injected muscles, anti-Ova CD8 + T cells expanded early after AAV challenge to reach around 2% of the CD8 + T cell compartment at day 7 post-injection ( Figure 1C , left panel). In contrast, when analyzing splenocytes, anti-Ova CD8 + T cells were found to reach up to 10% of the total CD8 + subset but only at day 14 after i.m. AAV injection ( Figure 1C , right panel). These results are consistent with a model in which the local cellular immune response measured in the draining lymph nodes would be induced by an rAAV leakage from the injected muscle and the consequent transduction of non-muscle cells such as dendritic cells, as shown for AAV5 (Xin et al., 2006) or AAV1 (Lu and Song, 2009 ) for instance. The systemic response detected in the spleen would rather reflect systemic immunization directed against the circulating sOva protein after it has been produced by transduced muscle cells. This interpretation is supported by the kinetics of sOva apparition and antibodies production ( Figure 1D) . Indeed, in the serum of AAVtreated mice, sOva reached a maximum at day 7 post-injection and rapidly declined thereafter ( Figure 1D , green curves). Consistently, anti-Ova IgG appeared between day 7 and 12 after AAV i.m. injection, correlating with the disappearance of sOva in the serum after day 7 ( Figure 1D , blue curves). The latter probably reflects the in vivo clearance of sOva following the formation of antigenantibody immune complexes, although we cannot completely rule out that anti-Ova IgG antibodies may mask Ova epitopes in the immunoassay. Together, these data reveal that i.m. injection of www.frontiersin.org rAAV encoding sOva is strongly immunogenic and provides a mouse model that is suited to stringently evaluate the efficacy of tolerance induction protocols.
We investigated the possibility to block immune responses directed against the transgene product by two distinct immunomodulatory strategies, one blocking lymphocyte priming (i.e., CTLA-4/Ig) and the other inhibiting the function of lymphocytes targeting transduced muscle fibers (i.e., activation of the PD-1 pathway by muscle gene transfer of PD-1 ligands). Indeed, we have previously shown in transgenic mice that express Ova as a neo-autoantigen in muscle that tolerant transgenic anti-Ova CD8 + T cells up-regulated PD-1, suggesting that PD-1 plays a role in the induction of tolerance to skeletal muscle-expressed Ag (Calbo et al., 2008) . As expected, the anti-Ova CD8 + T cell response that followed an i.m. injection of rAAV2/1-Ova (Figures 1B,C) was accompanied by an up-regulation of PD-1 on CD8 + T cells that exhibit an activated CD44 hi phenotype (Figure 2A) . As activated T cells express PD-1 and PD-1 ligands inhibit their activation and function, we generated rAAV2/1-PD-L1 and rAAV2/1-PD-L2 vectors and first verified that they were able to efficiently transduce HEK cells in vitro ( Figure 2B) . The expected consequence of injecting these vectors i.m. is that they should not prevent T cell priming but rather inhibit the cytotoxic activity of T cells directed against muscle-expressed transgenic proteins.
To investigate the in vivo immunomodulatory potential of CTLA-4/Ig and rAAV2/1-PD-L1, we either administered 200 μg of CTLA-4/Ig i.p. or co-injected rAAV2/1-PD-L1 i.m. at the same time as rAAV2/1-Ova, and monitored the cellular and humoral immune response 14 day after. Results showed that even a single dose of CTLA-4/Ig was efficient to completely prevent the priming of anti-Ova CD8 + T cells (Figure 2C , left panel) and the apparition of anti-Ova IgG in the serum of treated animals ( Figure 2C , middle panel). Accordingly, this immunosuppressive treatment allowed a sustained sOva production that could be assayed in the serum at day 14 whereas it was undetectable in controls ( Figure 2C right panel). We concluded from these data that transient immunosuppression by a single dose of CTLA-4/Ig is very efficient to prevent the priming of the cellular and humoral response at early time points after AAV transduction.
In contrast, despite a slight but not statistically significant tendency to reduce immune responses, rAAV2/1-PD-L1 co-injected with rAAV2/1-Ova failed to provide the same effect on immune priming (Figure 2C , left and middle panels). Also, treatment with rAAV2/1-PD-L1 did not allow the persistence of sOva in the serum in agreement with the significant amount of anti-Ova IgG detected in the serum of these animals ( Figure 2C ). This expected result confirmed that local transduction of muscle cells with rAAV2/1-PD-L1 can not interfere with the systemic immune priming against sOva since activation of the PD-1/PD-L1 pathway was anticipated to regulate T cells functions rather than their priming.
We further analyzed the presence of Ova DNA and mRNA in muscles at day 40 in the different groups of treated mice by qPCR and qRT-PCR, respectively. This further confirmed that suppression of immune priming by CTLA-4/Ig promotes the persistence of transduced cells in vivo as measured by the detection of significantly higher levels of Ova DNA and mRNA in the muscles of treated animals ( Figure 2D ). By contrast, injection Frontiers in Microbiology | Microbial Immunology Figure 1 with 1 × 10 11 vg rAAV2.1-Ova in the gastrocnemius muscles at day 0. Splenic CD8 + T cells were analyzed at day 14 by flow cytometry for expression of CD44 and PD-1. (B) rAAV2/1-PD-L1 and rAAV2/1-PD-L2 vectors were designed, produced, and tested for their capacity to transduce HEK-293 cells in vitro. For this, cells were analyzed by flow cytometry 3-5 days after their transduction with 1/10th dilution of the concentrated virus stocks. Controls correspond to unmanipulated HEK-293 parental cells stained with the same antibodies (green histograms). (C) The immunosuppressive potential of rAAV2/1-PD-L1 and CTLA-4/Ig were evaluated in vivo. Mice were injected with 10 11 vg rAAV2/1-Ova in the gastrocnemius muscles and received or not a co-injection of 10 11 vg rAAV2.1-PD-L1, or 200 μg of CTLA-4/Ig injected contemporaneously by the i.p. route. Blood samples were then collected 14 days later to analyze the percentage of anti-Ova CD8 + T cells, the level of anti-Ova IgG and the presence of sOva in the serum. (D) Gastrocnemius muscles were then collected at day 40, and Ova DNA and mRNA were quantified by qPCR and qRT-PCR. of rAAV2/1-PD-L1 did not significantly improve transgene persistence in these settings, in line with the prominent systemic immune response detected at day 14 ( Figure 2D ).
Whereas a single dose of CTLA-4/Ig was efficient to suppress the systemic immune response at day 14 ( Figures 3A,B left panels), this was not the case when further monitoring the immune response at day 40. Indeed, anti-Ova CD8 + T cell expansion as well as anti-Ova IgG immune responses were readily detectable at day 40 in mice that had received rAAV2/1-Ova and CTLA-4/Ig at day 0 ( Figures 3A,B) . Therefore, early suppression of immune priming by a single dose of CTLA-4/Ig is not sufficient to permanently wipe out the immune responses against transgene product in these experimental conditions. Instead, continuous production of sOva by transduced muscle cells sensitizes the immune response at later time points when CTLA-4/Ig has been cleared from the circulation of treated mice.
We next tested whether rAAV2/1-PDL-L1 or rAAV2/1-PDL-L2 could synergize with CTLA-4/Ig to improve transgene tolerance at later time points when the initial CTLA-4/Ig monotherapy was evidently not efficient alone to completely block immune sensitization against sOva. This combined strategy may indeed target two non-redundant mechanisms of immunomodulation acting www.frontiersin.org either on the APC side for CTLA-4/Ig or on the target tissue side for rAAV2/1-PD-L1 and rAAV2/1-PDL-L2. To test this possibility, groups of mice were transduced i.m. with rAAV2/1-Ova and also received at the same time either rAAV2/1-PD-L1 or rAAV2/1-PD-L2 i.m. and CTLA-4/Ig i.p. combination therapies. We then evaluated the cellular and humoral immune responses at different time points and finally evaluated the persistence of transduced muscle cells by quantification of Ova DNA and mRNA at day 80 (Figure 3) .
Remarkably, whereas CTLA-4/Ig alone was inefficient, rAAV2/1-PD-L1 co-administered with CTLA-4/Ig at day 0 significantly improved transgene persistence, as measured by quantification of Ova DNA by qPCR (Figure 3C, left panel) . Accordingly, mRNA analysis by qRT-PCR at day 80 revealed sustained transcription of the transgene in muscle (Figure 3C, right panel) . Of note, rAAV2/1-PD-L2 appeared equally as effective as rAAV2/1-PD-L1 to provide this effect when combined with CTLA-4/Ig. Importantly, this protective effect occurred in a setting where neither CTLA-4/Ig alone nor the tested combination therapies could block immune response at days 40 and 80 (Figures 3A,B) . To further attest the presence of functional anti-Ova cytotoxic T cells at day 80 (a time point when pentamer staining is not sensitive enough to detect anti-Ova T cells, not shown), we analyzed by ELISpot the capacity of CD8 + T cells to secrete IFNγ when stimulated with the Ova-derived immunodominant SIINFEKL peptide. This assay revealed the presence of low numbers of functional anti-Ova CD8 + T cells in the three groups of animals that had received CTLA-4/Ig with or without rAAV-PD-L1 or rAAV-PD-L2, whereas the assay was negative for mice that did not receive rAAV-Ova and strongly positive for those that received rAAV-Ova in the absence of any immunoregulatory adjuvant therapy ( Figure 4A) . To definitively demonstrate that these IFNγ-secreting T cells were functional in vivo, we performed adoptive transfer experiments to non-lethally irradiated syngenic recipients that were inoculated with Ova-bearing tumor cells. Whereas tumors rapidly developed in recipients that had received T cells from control mice that only received PBS, recipient mice receiving lymphocytes from rAAV2/1-Ova injected donors rapidly developed an anti-tumor immune response that resulted in complete tumor rejection (Figure 4B ). In agreement with the lower numbers of anti-Ova CD8 + T cells detected by ELISpot, recipient mice that had received immune cells from donors treated with CTLA-4/Ig or the combined CTLA-4/Ig plus rAAV-PD-L1 or rAAV-PD-L2 therapy reacted with a slight delay but were also was also capable to efficiently control tumor growth ( Figure 4B) . Together, these results indicate that the cytotoxic activity of transgene product specific T cells that have escaped costimulation blockade can be functionally blocked by AAV-mediated gene transfer of PD-1 ligands in muscle tissues while they remain capable to reject tumor cells.
Gene therapy for muscular dystrophies and other monogenic diseases aims at achieving long-term expression of a functional form of an otherwise deficient gene in target tissues. In this context, the product of the therapeutic gene is structurally different from its abnormal or absent counterpart, and consequently viewed as nonself by the immune system. Additional signals from the viral vector may further strengthen adverse innate and adaptive immune reactions against the transgene product, while the vector itself can be targeted by antiviral pre-existing or acquired immunity. Although the presence of natural epitopes in the non-mutated regions of the protein or the occasional occurrence of mutation reversion in a minute population of patients' cells (Klein et al., 1992) may yield some level of immunological tolerance, it remains that acquired immunity to the transgene product still represents one of the major obstacles to the success of gene therapy. Here, we provide the proof of principle that a combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can promote persistence of transduced target cells and transgene transcription thereof even when some cytotoxic T cells have escaped initial control.
A first strategy for the blockade of adaptive immunity is obviously to target costimulation pathways and therefore provide an early control on lymphocyte priming. Whereas several costimulatory pathways have now been identified, CD28-mediated T cell costimulation by APC expressing CD80/CD86 molecules from the B7 family is clearly prominent. Indeed, CD28 signaling strongly enhances T cell proliferation and survival, cytokine production and prevents induction of anergy after TCR-mediated activation (Boise et al., 1995) . Consequently, anti-CD80 (B7.1) or anti-CD86 (B7.2) antibodies inhibit T cell priming and genetically engineered CD28-deficient T cells are strongly impaired in their capacity to expand (Green et al., 1994; Salomon and Bluestone, 2001) . T cells activation also promotes up-regulation of the negative regulator CTLA-4, another ligand of CD80 and CD86 molecules, which transmits inhibitory signals that regulate activated T cell and provides a key homeostatic mechanism of the immune response (Fife and Bluestone, 2008) . One of the early striking evidence of the key role of this molecule is that CTLA-4-deficient animal rapidly die FIGURE 4 | Detection of anti-Ova T cells in animals treated with combination therapies. Seven mice per group were injected with 1 × 10 11 vg rAAV2/1-Ova in the gastrocnemius muscles at day 0 and received at the same time combination therapies as in Figure 3 . Splenocytes were then harvested 80 days after and analyzed for the presence of lymphocytes capable to respond to Ova antigen. (A) Splenocytes were analyzed by ELISpot as described in the Section "Materials and Methods" for their capacity to secrete IFNγ after in vitro restimulation with the Ova-derived immunodominant SIINFEKL peptide. (B) 5 × 10 7 Splenocytes from the same mice were also adoptively transferred to 5 Gy-irradiated syngenic mice inoculated 1 day after with Ova-bearing EG7 tumor cells. The capacity of transferred lymphocytes to reject the tumors was monitored three times per week by measuring the tumor size with a digital caliper. www.frontiersin.org from massive lymphoproliferative syndrome and dysregulation of immunity (Waterhouse et al., 1995) .
One convenient way to pharmacologically block the CD28 costimulation pathway is to use the soluble CD80/CD86 ligand CTLA-4/Ig. This fusion protein prevents CD28 interaction with CD80 and CD86 molecules on naïve and activated T cells and therefore provides a strong and early inhibition on T cell priming (June et al., 1990; Lenschow et al., 1992; Larsen et al., 2005) . CTLA-4/Ig may also induce a negative signalization on the APC side through the induction of indoleamine2,3-dioxygenase (IDO) which catalyses the production of inhibitory kynurenine from tryptophan (Grohmann et al., 2002) . CTLA-4/Ig may also possibly provide some level of APC depletion in vivo, although this aspect has been poorly documented and should critically depend on the form of CTLA-4/Ig used. Importantly, CTLA-4/Ig is now clinically available for the treatment of diseases such as rheumatoid arthritis (Dumont, 2004) and it is thus reasonable to propose its evaluation in the field of gene therapy. Indeed, there has been experimental evidence that CTLA-4/Ig in combination with a monoclonal antibody to CD40L (MR1) in the context of gene therapy can block immune responses and allow vector readministration in the mouse (Halbert et al., 1998; Lorain et al., 2008) . It is difficult to weight the relative roles of CTLA-4/Ig and MR1 in these experiments but a plausible hypothesis is that CTLA-4/Ig provides the strongest immunoregulatory contribution since MR1 alone has been reported to be less efficient in similar experimental conditions (Manning et al., 1998) . The results reported herein are consistent with these earlier reports and provide definitive evidence that CTLA-4/Ig alone efficiently, but only transiently, blocks anti-transgene T cell priming.
One concern about using CTLA-4/Ig in gene therapy is that discontinuation of treatment exposes patients to immunization against the transgene product. The present results illustrate this point since initial CTLA-4/Ig efficiently blocks the anti-Ova cellular and humoral responses while sustained sOva production finally elicits this undesired response when CTLA-4/Ig is probably cleared from the circulation. Our experimental conditions have been purposely designed to model this very situation where the immune response escapes the initial immunoregulatory regimen in order to evaluate if a second strategy could provide additional protection of transduced cells, i.e., PD-L1/2 gene therapy.
The PD-1/PD-L1 pathway has recently been identified as a negative regulator of immunity . The PD-1 immunoreceptor is also a member of the CD28/CTLA-4 family which, upon interaction with either one of its two ligands PD-L1 or PD-L2, down-modulates TCR signaling, reduces cytokine production and affects T cell survival by recruiting the SHP-2 tyrosine phosphatase, thereby dampening the PI3K and Akt pathway (Francisco et al., 2010) . In BALB/c mice, PD-1 deficiency leads to the spontaneous development of a lethal autoimmune cardiomyopathy . This disease is mediated by autoantibodies directed against the cardiac muscle autoantigen troponin I (Okazaki et al., 2003) . Hence, PD-1 controls the physiological tolerance to muscle autoantigens. Also, PD-L1 was recently shown to regulate CD8 + T cell-mediated muscle injury in a mouse model of myocarditis (Grabie et al., 2007) . Several PD-1/PD-L1 dependant mechanisms may act synergistically to induce an effective effector T cell blockade and contribute to tolerance induction. As mentioned earlier, PD-1 engagement on the surface of T cell is known to significantly inhibit TCR signaling thereby preventing T cell activation and cytokine production. In addition to this mechanism, PD-L1 was recently shown to be a potent inducer of adaptive Tregs. Indeed, PD-L1 expressed on CD8 + dendritic cell subset plays an important role on the conversion of naive lymphocytes toward FoxP3 + regulatory T cells (Wang et al., 2008) . Thus, engagement of PD-1 on T cells could induce cell-intrinsic tolerance (by blocking TCR signaling) as well as a dominant form of immunological tolerance by promoting the emergence of FoxP3 + adaptive Tregs. Of note, manipulation of the PD-1/PD-L1 axis is also used by some tumors cells to tolerize surrounding lymphocytes. Indeed, expression of PD-L1 on the surface of malignant cells has been suggested to represent a subversive strategy used by tumors to escape to immuno-surveillance . Thus, we believe that up-regulation of the expression of PD-L1 on the surface of muscle cells by the means of gene transfer should be able to induce tolerization of the immune system by preventing cytotoxicity of PD-1-expressing activated lymphocytes. However, PD-L1 gene transfer alone was not sufficient in our model to prevent the immune response directed against transduced muscle fibers (Figure 2) , suggesting that muscle expression of PD-L1, i.e., at distance from lymphoid compartments, is probably not capable of inducing sufficient adaptive FoxP3 + Tregs. Another explanation would be that PD-L1 expressed in muscle is not effective to block fully activated T cells that have received normal costimulatory signals (i.e., in the absence of CTLA-4/Ig co-administration). This interpretation would be in line with the finding that PD-1 inhibitory pathway could be overcome by CD28 costimulatory ligation in the presence of IL-2 (Freeman et al., 2000; Latchman et al., 2001; Carter et al., 2002; Ishida et al., 2002) . Hence, the use of CTLA-4/Ig to block early CD28 costimulatory signaling may create favorable conditions where escaping T cells could become sensitive to PD-L1/2. This strengthens the notion that CTLA-4 and PD-1 signaling represent two distinct pathways acting synergistically to maintain tolerance (Fife and Bluestone, 2008) . As shown herein, neither CTLA-4/Ig alone nor combination therapies could block immune response at days 40 and 80 (Figures 3 and 4) . Our results are thus compatible with the notion that the use of rAAV2/1-PD-L1 or rAAV2/1-PD-L2 in combination with CTLA-4/Ig, does not completely inhibit the immune priming against sOva that most probably occurs at distance from the site of transduction (e.g., spleen and/or lymph nodes) but instead provides an additional protection by disarming lymphocytes within the target tissue.
It has been argued that the priming of T cells directed to the transgene product may be somehow defective after rAAVmediated gene transfer (Lin et al., 2007a,b) . However, in our experimental settings, transduced muscle fibers progressively disappear in the absence of immunoregulatory treatment ( Figure 2D) . Further, we show that even after CTLA-4/Ig alone or in combination therapies, an anti-Ova immune response is evidenced in ELISpot assays and in an in vivo model of tumor rejection upon adoptive transfer of T cells in recipient animals (Figure 4) . Hence, forced up-regulation of PD-1 ligands in muscle cells yielded a level of protection of transduced cells from the cytotoxic assault Frontiers in Microbiology | Microbial Immunology of circulating anti-Ova lymphocytes and provided in combination therapy a means to prolong transgene persistence and transcription. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward the transgene product and may deserve further investigation for non-secreted transgenic protein models and in larger animals.
Sahil Adriouch designed research, performed experiments, analyzed data, and co-wrote the manuscript; Anna Salvetti and Olivier Boyer designed research and co-wrote the manuscript; and Emilie Franck, Laurent Drouot, Carole Bonneau, and Nelly Jolinon performed experiments. Comparison of Humoral Immune Responses to Epstein-Barr Virus and Kaposi’s Sarcoma–Associated Herpesvirus Using a Viral Proteome Microarray Background. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi’s sarcoma–associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). Methods. To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi’s sarcoma or lymphoma. Results. In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1–positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. Conclusions. The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients. Background. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1).
Methods. To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi's sarcoma or lymphoma.
Results. In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1-positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile.
Conclusions. The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.
Epstein-Barr virus (EBV) and Kaposi's sarcomaassociated herpesvirus (KSHV) are human herpesviruses that differ dramatically in their geographic prevalence. EBV seropositivity in healthy populations is .90% worldwide. KSHV seropositivity is low (0-5%) in the United States, Canada, and Europe; intermediate (7-24%) in Italy and the Mediterranean; and high (23-70%) in sub-Saharan Africa [1] [2] [3] [4] . Seroprevalence for KSHV increases in individuals coinfected with human immunodeficiency virus 1 (HIV-1) and, in the United States and Europe, in men who have sex with men [5] [6] [7] . Primary infection with EBV can cause infectious mononucleosis, and both EBV and KSHV are associated with human cancers. EBV is associated with nasopharyngeal carcinoma, African Burkitt's lymphoma, a subset of Hodgkin's lymphoma, AIDS-related lymphoma, gastric carcinoma, peripheral T cell lymphoma, and posttransplant and other lymphoproliferative diseases in the immunodeficient [8] . KSHV is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Immunosuppression, such as that imposed by concurrent HIV-1 infection or transplantation regimens, increases the incidence of some of these virus-associated cancers [8, 9] .
The DNA genomes of EBV and KSHV encode an estimated 80 and 86 proteins, respectively. The degree to which these proteins are expressed differs depending on the stage of the viral life cycle. In vivo, the reservoir of EBV latent infection is resting memory B cells that have either no EBV protein expression or transient expression of the EBNA1 protein. Expression of the other latency proteins, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP-1, and LMP2A and LMP2B, has been detected in peripheral blood B cells shortly after primary infection and in B cells in tonsillar tissues [10, 11] . Lytic replication and expression of the EBV lytic proteins takes place in plasma cells and in epithelial cells [12] [13] [14] . Less is known about in vivo persistence of KSHV. B cells form a reservoir for latent KSHV infection, and differentiation of these cells also triggers lytic KSHV induction [15, 16] . KSHV endothelial and B cell tumors express the LANA, v-cyclin, and v-FLIP latency proteins, and vIRF3 is also detected as a latency protein in primary-effusion lymphoma cells. In addition, a small percentage of tumor cells express lytic cycle proteins such as the v-IL6 and the viral G-protein coupled receptor (v-GPCR), suggesting that KSHV lytic infection may contribute to the tumorigenic phenotype [17] .
Transcriptional profiling arrays have been developed to examine genome-wide expression of EBV and KSHV [18, 19] . However, humoral immune responses to EBV and KSHV proteins have predominantly been measured using virus-infected cell lines or enzyme-linked immunosorbent assays that incorporate just a few selected viral proteins [2] [3] [4] [20] [21] [22] . Although these assays have proven valuable for diagnostic and epidemiological studies, they do not provide a complete picture of the full repertoire of antibody responses generated by EBV and KSHV infection. High-throughput technologies allow cloning and expression of entire pathogen proteomes [23, 24] . We used this technology to generate proteomic microarrays for EBV and KSHV. These arrays allowed us to compare the global humoral immune responses to EBV vs KSHV in the same patient samples and within the same assay.
EBV and KSHV open reading frames (ORFs) were cloned and expressed as previously described [25] . Briefly, the open reading frames were polymerase chain reaction (PCR) amplified using primers based on the GenBank sequences V01555, AJ507799, and U756981. The bacterial artificial chromosome plasmids Akata BXI (EBV) and BAC36 (KSHV) [26] were used as templates (gifts from L. Hutt-Fletcher and S-J Gao). PCR products were cloned into the vector pDONR201. ORFs representing different virus strains were amplified directly from clinical samples or from cell lines. Proteins containing extensive repeats that limited expression in yeast (eg, EBNA1 and LANA) were amplified as N-terminal and C-terminal fragments. Escherichia coli bacteria were transformed with the reaction products and DNA prepared for sequence verification. Correct ORF-containing plasmid DNAs were then moved into a yeast destination vector, pEGH-A, a derivative of the yeast glutathione S-transferase (GST) vector, pEGH. Yeast cultures were induced with 2% galactose, and GST fusion proteins were isolated and purified on glutathione beads. One hundred and seventy-four appropriately sized EBV and KSHV proteins (Table S1 ; available online) were successfully purified based on immunoblot analysis using anti-GST antibody and were printed using a 48-pin contact printer (Bio-Rad) in duplicate on modified glass (Full Moon Biosystems) microscope slides along with controls. Each slide had 784 spots: 348 EBV and KSHV proteins, 112 control proteins (Table S1 ; available online), and 324 blank spots. The controls were used for orientation and to detect nonspecific interaction with proteins such as the GST fusion partner. Protein arrays were stored at 280°C.
Plasma from healthy blood donors and from patients with follicular B cell lymphoma (without HIV-1), AIDS-related lymphoma (ARL), and AIDS Kaposi's sarcoma was obtained with written informed consent in accordance with the Declaration of Helsinki after approval by the Johns Hopkins institutional review board.
Plasma was separated from peripheral blood collected in standard EDTA or acid citrate dextrose tubes and stored at 280°C.
Protein arrays were preincubated in 4 mL Superblock (Pierce) plus 2% bovine serum albumen (BSA) at 4°C overnight. Slides were assembled with 4-well modules and incubated with plasma (300 uL diluted in phosphate buffered saline [PBS] plus 2% BSA) for 1 hour at room temperature. Slides were washed with prewarmed (42°C) PBS Tween-20 (PBST) and incubated with secondary Cy3labeled antihuman IgG or IgA antibody (1:2000; Sigma and Jackson, respectively) diluted in PBS plus 2% BSA for 1 hour at room temperature. Slides were then washed 23 with prewarmed PBST, 13 with prewarmed distilled water, dried, scanned, and analyzed with GenePix Pro software (Molecular Devices).
The software GenePix Pro 7 was used to obtain the median foreground and background intensity for each spot. The raw intensity of the spot was defined as the ratio of the foreground to background median intensity. There are 324 ''blank'' spots on each protein chip, and the raw intensity distribution of these spots has a mean value of approximately 1. Assuming that the raw intensity distributions of ''blank'' spots are the same across all microarrays, we can standardize the protein signals such that
I2m r
where Z is Z-score of each spot, I is raw intensity of the spot, and m and r are mean value and standard deviation, respectively, of ''blank'' spots on the microarray. Each protein has 2 duplicated spots. The identified hits were defined as those proteins with Z-scores for both spots at or above the 5 SD cutoff. When comparing the appearance frequency for individual antigens for HIV/KS patients vs lymphoma or healthy normals, the enrichment score was reflected by ES 5 f ks =f 1 . We adopted binominal probability to calculate the significance between comparisons such that
where p is the probability that the protein was hit for at least k HIV/KS patients, n is the total number of HIV/KS patients, and f 1 is the frequency of the protein in the comparison group (either lymphoma or healthy normals). Proteins were selected as a significant biomarker for HIV 1 /KS patients only if they had at least 50% frequency in HIV 1 /KS patients, the enrichment score ES was larger than 1.6, and the P value was less than .005.
EBV and KSHV DNA in plasma were detected as previously described [27] . Viral DNA levels were determined by real-time, quantitative PCR using K8 primers for KSHV and BamHI-W primers for EBV. Controls were constructed by the addition of viral DNA to serum from healthy donors. Standard curves (with duplicate serial 10-fold dilutions of plasmid DNA that included a target sequence from 10 5 to 10 copies) were run in parallel with each analysis.
To systematically compare EBV and KSHV antibody responses, we utilized proteomic arrays displaying 174 virus proteins plus controls. The printing quality and the quantity of the immobilized proteins on the chip were monitored using anti-GST antibody followed by Cy3-labeled secondary antibody ( Figure 1A ). Preliminary analyses with arrays containing the 82 EBV and 92 KSHV ORFs revealed that plasma diluted up to 1:10 000 gave a signal on the arrays that was readily detected with Cy3-tagged antihuman IgG. A positive signal was set as one that was 5 SD or more above background. A section of the array illustrating positive and control signals is shown in Figure 1B . ORF38 is a tegument protein that has not been widely examined as an antigen. ORF73 encodes the latency LANA protein that is a gold standard for KSHV serology studies. The central region of LANA contains repeated sequences that limit effective expression of the protein in yeast. The N-terminus and C-terminus of LANA were therefore expressed separately for presentation on the array. The LANA C-terminus was recognized more frequently than the N-terminus (14 vs 4 positive plasma). This is consistent with a peptide mapping study that found more sera reacting to peptides mapping to the LANA C-terminus than to the N-terminus [28] . The median number of KSHV antigens recognized by the LANA-positive HIV 1 /KS plasma at 1:10 000 serum dilution was 6. Analysis of the EBV proteins recognized in the same assay by the same plasma samples revealed a different picture. EBV is a ubiquitous virus, and EBV proteins on the array were recognized using plasma from healthy donors, HIV 2 lymphoma patients, and HIV 1 /KS patients ( Figure 2B ). The median number of EBV proteins recognized at 1:100 serum dilution was 17, 18, and 27 for plasma from healthy donors, lymphoma patients, and HIV 1 /KS patients, respectively. Ninety-one percent of plasma specimens tested (32/35) reacted with the carboxy terminus of EBNA1 and 97% (34/35) reacted with the combination of EBNA3B and EBNA3C at 1:100 or 1:10 000 dilution. The single EBNA3-negative plasma specimen was from a lymphoma patient whose plasma did not recognize any EBV antigens. The difference between the normal and HIV 2 lymphoma population and the HIV 1 /KS population lay not in the individual antigens recognized, but rather in the heightened antigenic response to a range of EBV proteins. This is illustrated by the increased median number of antigens detected at 1:10 000 plasma dilution seen with HIV 1 /KS samples (15) vs the median number with plasma from healthy donors and lymphoma patients (7 and 5, respectively). Antigens with a statistically significant difference in appearance between healthy donors and HIV 1 /KS patients at a 1:10 000 dilution are shown in Figure 3 , which also includes the data for HIV-negative lymphoma patients for comparative purposes. The BFRF3 (p18) capsid antigen has been previously advocated as a serological marker for nasopharyngeal carcinoma diagnosis [29] .
Two subtypes of EBV, type 1 and type 2 (also called A and B), were originally defined on the basis of differences in the EBNA2 gene [30] . In this set, 10/35 sera showed discordant responses to the 2 EBNA2 proteins with either a different sensitivity of detection (1:100 vs 1:10 000) or recognition of only 1 of the 2 EBNA2 proteins. It is likely that these individuals are infected with only 1 of the 2 EBV subtypes. Note that 9/10 discordant sera had preferential recognition of type 1 EBNA2, which is consistent with the greater prevalence of type 1 EBV in clinical samples from the United States. Positivity to both proteins would represent recognition of common epitopes or reflect concurrent infection with both virus subtypes. In contrast to the antibody responses to KSHV proteins, the profiles of antibody responses to EBV proteins in these plasma samples were similar in the KS and lymphoma-carrying HIVpositive populations ( Figure 4B ). There was no significant difference in either the intensity of the positive signals or the number of antigens being recognized at a 1:10 000 serum dilution. Thus, the presence of KSHV-associated disease did not appear to have any impact on humoral responses to EBV antigens in the setting of concurrent HIV-1 infection. Again, the broader antigenic response to EBV compared with KSHV is striking. The W1 exon of EBV BamHI-W encodes 22 amino acids that form part of the EBNA-LP protein [33, 34] . The protein encoded by the entire EBV BamHI-W ORF (BWRF1) and the EBNA-LP protein were both printed on the array. We noted that 23/24 (95%) of the specimens that recognized EBNA-LP also recognized the protein encoded by BWRF1, implying that the common 22 amino acids contribute significantly to antibody recognition of EBNA-LP.
EBV and KSHV DNA are detected infrequently in serum or plasma from healthy individuals, but there is an increased prevalence in cancer patients and in HIV-1-infected individuals [35, 36] . A consistent correlation between EBV or KSHV DNA levels in serum or plasma and antibody titers to viral proteins has not been apparent [37] . To see if the more global antibody responses detected on the arrays would provide any additional insight, we examined KSHV and EBV DNA levels in the plasma from HIV-positive KS patients (see Figure 2A ). The plasma copy number for KSHV and EBV DNA was measured by real-time quantitative PCR using previously published primers [27] . A comparison of DNA copy number with antibody responses did not reveal any obvious correlation for either KSHV or EBV ( Figure 5) . However, the 2 samples with the highest KSHV DNA copy number (.180 000 copies) did not recognize either the K8.1 or ORF38 lytic proteins, whereas 61% (8/13) of the samples with ,50 000 copies recognized both of these antigens. Two samples are insufficient to allow any conclusions to be drawn, but the observation does raise the possibility that inadequate antibody production may have contributed to the robust KSHV replication detected in these individuals.
IgA responses to EBV EBNA1 and viral capsid antigens have long been used as a diagnostic tool for nasopharyngeal carcinoma [38] . In addition to nasopharyngeal carcinoma, IgA responses to VCA and to EBNA1 are frequently elevated in lymphoma patients and in individuals who are HIV-1 positive [39] . IgA responses to EBV proteins were examined at a 1:1000 dilution for 15 of the HIV-positive KS patients, 10 healthy individuals, and 10 HIV-positive lymphoma patients. Seven EBV antigens were consistently recognized by IgA across these patient groups, namely, EBNA1, the helicase BBLF4, the viral IL10 BCRF1, the uracil DNA glycoslyase BKRF3, the early protein BRRF1, the tegument protein BRRF2 [40] , and the latency membrane protein LMP2A ( Figure 6 ). The most intense IgA response detected was to EBNA1, which appeared to be recognized even more readily by IgA than IgG. All 25 normal and HIV-positive KS plasma tested recognized EBNA1 at a 1:1000 dilution, whereas in 4 of these samples there was no recognition of EBNA-1 by IgG even at a 1:100 dilution. However, the IgA response to the functionally equivalent protein in KSHV, LANA, was very different. Of 14 HIV-positive KS plasma samples that had IgG responses to LANA at a 1:10 000 dilution, only 2 had IgA responses at a 1:1000 dilution (data not shown). In 1 previous study, IgA antibody to KSHV LANA was detected in saliva but not in serum [41] .
A comparison of the IgG and IgA responses in the plasma from healthy donors also revealed differential responses to certain EBV proteins. All 10 normal sera had IgA responses at a 1:1000 dilution to the viral IL10 BCRF1, the tegument protein BRRF2, and the latency membrane protein LMP2A. In contrast, even at a 1:100 dilution, IgG responses to BCRF1, BRRF2, or LMP2A were not detected in these plasma samples. The IgA response to vIL10 is particularly interesting because vIL10 is an immune modulator that can have an impact on localized immune escape of EBV-infected cells.
Testing for EBV and KSHV has relied on assays such as immunofluorescence staining and Western blotting of infected cells. Assays using individual viral proteins have also been described. However, in the absence of a global comparison of protein immunogenicity, there has been inadequate information on which to base the choice of antigens to incorporate into these assays. The EBV latency proteins EBNA1, EBNA2, and EBNA3C and the KSHV LANA latency and K8.1 lytic proteins have been widely used as antigens, and these antigens were also frequently detected on the arrays. Our protein array screens also identified the EBV tegument proteins BBRF2, BGLF2, and BNRF1 (FGARAT); the small capsid protein BFRF3; the early protein BRRF1; and the KSHV ORF38 tegument protein as lytic viral proteins that are particularly well recognized by IgG. The screens also identified BBLF4, BCRF1, BKRF3, and BBRF1 as EBV lytic proteins that were well recognized by IgA. It should be noted that the EBV and KSHV proteins for our array were expressed in yeast which do not have the same machinery as mammalian cells for complex glycosylation. It is therefore likely that our assays underestimate the immunological response to glycosylated viral antigens such as the EBV envelope glycoprotein gp350/220 and KSHV K8.1.
Globally, the array analyses revealed a stark contrast between antibody responses to EBV and KSHV in the same patients. Multiple EBV proteins were consistently recognized by plasma IgG at the dilutions examined, whereas only the LANA (ORF73), ORF38, and K8.1 proteins of KSHV were detected with any consistency, even in patients with KS. This difference carried over to serum IgA responses where 2 latency EBV proteins and 5 lytic proteins were detected by the majority of the sera, while the only KSHV antigen detected by IgA was LANA, and that was seen only rarely. These observed differences may reflect the biology of in vivo persistence. Both viruses latently infect B cells, and both viruses are secreted into saliva [42, 43] . However, EBV can be routinely detected in circulating latently infected B cells of healthy seropositive individuals, whereas detection of KSHV in blood is rare [44] [45] [46] . The preferential ability to detect serum IgA responses against EBV vs KSHV on the protein arrays may also be related to in vivo infection of mucosal epithelial cells by EBV because the most frequently observed antigens were latency proteins or lytic replicative proteins known to be expressed in epithelial cells [47] [48] [49] [50] . KSHV infects endothelial cells as well as B cells, but the contribution of endothelial cells to in vivo viral persistence is not well understood.
The protein array described here provided a highly sensitive platform. Plasma with EBNA1 titers between 1:80 and 1:320 in conventional anticomplement immunofluorescence assays had titers of 1:10,000 or greater in the array assay (data not shown). The assay showed specificity. IgG responses to KSHV proteins were detected in sera from HIV-positive individuals with a diagnosis of KS or lymphoma but were not detected in a panel of HIV-negative lymphoma patients or healthy normals. This result, along with comparisons between EBV and KSHV responses in the HIV-positive samples, indicates that cross-reactivity between homologous proteins encoded by EBV and KSHV was not a confounding factor. This array provides a new platform for investigating the contribution of humoral immune responses in patients with EBV-and KSHV-associated pathologies.
Supplementary materials are available at The Journal of Infectious Diseases online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Figure 6 . IgA responses to EBV proteins. Plasma specimens from HIV-1-positive KS patients, AIDS-related lymphoma patients, and healthy individuals were incubated on the protein arrays at a 1:1000 dilution (upper panel). Heat map showing responses detected with Cy3-labeled anti-human IgA that were above the 5 SD cutoff (lower panel). Number of antigens recognized by each sample.
Financial support. This work was supported by Public Health Service grants from the National Cancer Institute of the National Institutes of Health (R37 CA42245 and R01 CA30356 to S. D. H, R21 CA138163 to S. D. H. and H. Z., RC2 CA148402 to G. S. H., and Cancer Center Core Grant P30CA006973 to William Nelson).
Potential conflicts of interest. All authors: No reported conflicts. Stem–loop structures can effectively substitute for an RNA pseudoknot in −1 ribosomal frameshifting −1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins. Ribosomal frameshifting is a translational recoding event in which a certain percentage of ribosomes are forced to shift to another reading frame in order to synthesize an alternative protein. This switch occurs at a specific position on the mRNA, called the slip site or slippery sequence, and can be either forwards (+1) or backwards (À1). The nature and efficiency of frameshifting depends on several factors, including tRNA availability and modifications, and mRNA primary and secondary structure (1, 2) .
The signals that are responsible for À1 frameshifting comprise two elements: a slippery sequence where the actual reading shift takes place, and a downstream located structural element which greatly stimulates the efficiency of frameshifting. Although the mechanism is still elusive, the present view is that the downstream structure forms a physical barrier that blocks EF-2 function and causes ribosomes to stall in their translocation step. This 'roadblock' puts tension on the mRNA-tRNA interaction. The tension can be relieved by the realigning of A-site and P-site tRNAs in the 5 0 -direction, whereafter EF-2 can do its work and the ribosome resumes translation in the À1 reading frame (3) .
In general, a pseudoknot is more efficient in stimulating frameshifting than a hairpin of the same sequence composition. This difference is likely related to a higher thermodynamic stability of the pseudoknot. Indeed, from thermodynamic analysis it appears that pseudoknots are more stable than their hairpin counterparts (4) (5) (6) . Recent studies employing mechanical 'pulling' of frameshifter pseudoknots have shown a correlation between the mechanical strength of a pseudoknot and its frameshifting capacity (7, 8) , and the influence of major groove and minor groove triplex structures (9) . The higher strength of a pseudoknot can be primarily attributed to the formation of base triples between the lower stem S1 and loop L2 ( Figure 1A ), making it more resistant against unwinding by an elongating ribosome (8, 10) . Base triples in several pseudoknots, such as Beet western yellows virus (BWYV) p1-p2 (11) , Pea enation mosaic virus type-1 (PEMV-1) p1-p2 (6) , Sugarcane yellow leaf virus (ScYLV) p1-p2 (12) and Simian retrovirus type-1 gagpro (SRV-1) (13, 14) have been shown to play an essential role in frameshifting. For pseudoknots with a longer stem S1 of 10-11 bp, like that of Infectious Bronchitis Virus (IBV), base triples do not appear to contribute to frameshifting (15) .
Although a hairpin is considered to be a less efficient frameshift-inducing secondary structure than a pseudoknot, some viruses like Human immunodeficiency virus (HIV) (16) , Human T-lymphotropic virus type-2 (HTLV-2) (17) and Cocksfoot mottle virus (CfMV) (18) make use of a simple hairpin to stimulate substantial levels of frameshifting. In addition, frameshifting in the prokaryotic dnaX gene requires, next to an upstream enhancer, the presence of a hairpin as well (19) . A few studies have investigated a correlation between hairpin stability and frameshift efficiency of natural shifty hairpins (19, 20) . Nonetheless, certain studies have shown that a hairpin composed of the same base pairs as a frameshifter pseudoknot is not very efficient in inducing frameshifting in mammalian cells and lysates (21) (22) (23) but is in other systems (24) .
Here, we have carried out a systematic analysis of the frameshift-inducing efficiency of hairpins derived from the SRV-1 gag-pro frameshifter pseudoknot. Investigation of about 30 different hairpin constructs revealed that next to thermodynamic stability, also loop size and composition, and stem irregularities can significantly influence frameshifting. Our data showed that there exists no base specific contacts between the hairpin and the ribosome during frameshifting and suggests that the hairpin primarily serves as a barrier to allow repositioning of tRNAs at the slippery site.
Mutations in the SRV-1 gag-pro frameshifting signal were made in an abridged version of plasmid SF2 (25) which is derivative of pSFCASS5 (26), a frameshift reporter construct. In this version, the entire BglII-NcoI fragment of pSF2 was replaced by a synthetic dsDNA fragment (5 0 -G ATCTTAATACGACTCACTATAGGGCTCATTTAA ACTAGTTGAGGGGCCATATTTCGC-3 0 , a SpeI restriction site is underlined). This yielded plasmid pSF208 in which the original GGGAAAC slippery sequence has been replaced by the more slippery UUUAAAC sequence (26) . pSF208 was digested with SpeI and NcoI, and sets of complementary oligonucleotides corresponding to the various mutants were inserted. A list of oligonucleotides is available upon request. All constructs were verified by automated dideoxy sequencing using chain terminator dyes (LGTC, Leiden).
DNA templates were linearized by BamHI digestion and purified by successive phenol/chloroform extraction and column filtration (Qiagen, Benelux). SP6 polymerase directed transcriptions were carried out in 50 ml reactions containing $2 mg linearized DNA, 10 mM NTPs, 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 10 mM DTT, 6 mM MgCl 2 , 2 mM spermidine, 6 U of RNase inhibitor (RNAsin, Promega, Benelux) and 15 U of SP6 polymerase (Promega, Benelux). After an incubation period of 2 h at 37 C, samples were taken and run on agarose gels to determine the quality and quantity of the transcripts. Appropriate dilutions of the reaction mix in water were directly used for in vitro translations. Alternatively, transcripts were purified by phenol/chloroform extraction and isopropanol precipitation and quantified by UV absorption as described previously (14) .
Experiments were carried out in duplicate using seriallyin water-diluted mRNAs with final concentrations of 5 nM. Reactions contained 4 ml of an RNA solution, 4.5 ml of rabbit reticulocyte lysate (RRL, Promega), 0.25-1 ml of 35 S methionine (Amersham, in vitro translation grade), 0.5 ml of 1 mM amino acids lacking methionine and were incubated for 60 min at 28 C. Samples were boiled for 3 min in 2Â Laemmli buffer and loaded onto 12% SDS polyacrylamide gels. Gels were dried and exposed to phosphoimager screens. Band intensity of 0-frame and À1 frameshift products was measured using a Molecular Imager FX and Quantity One software (Biorad). Frameshift percentages were calculated as the amount of À1 frameshift product divided by the sum of 0 and À1 frame products, corrected for the number of methionines (10 in the 0-frame product and 28 in the fusion product), multiplied by 100.
Candidates of interest were constructed in a dual luciferase vector, pDUAL-HIV(0), essentially as described previously (14, 27) . In short, pDUAL-HIV(0) was digested by KpnI and BamHI, followed by insertion of complementary oligonucleotides to clone the SRV-1 gag-pro pseudoknot, various hairpins as shown in Figures 2C and 5, and a negative control (NC) which formed no apparent secondary structure downstream of the slippery sequence. An in-frame control was constructed by inserting an A-residue upstream of the cytosine in the UUUAA AC slippery sequence of a 12 bp hairpin frameshift construct. HeLa cells were cultured in DMEM/high glucose/ stable glutamine (PAA Laboratories GmbH, Germany) and supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were kept in a humidified atmosphere containing 5% CO 2 at 37 C. Assay protocols were described previously (14) . Briefly, cells were transfected with 300 ng of plasmid using 1 ml of lipofectamine-2000 (Invitrogen) in a 24-well plate. Cells were lysed 24 h after transfection and luciferase activities were quantified by Glomaxmultidetector (Promega, Benelux) according to manufacturer's protocol. Frameshifting efficiency was calculated by dividing the ratio of Renilla luciferase (RL) over Firefly luciferase (FL) activity of the mutant by the RL/ FL ratio of the in-frame control, multiplied by 100.
In contrast to earlier reports involving the IBV frameshifting pseudoknot (21, 22) , we found that in the case of the SRV-1 gag-pro frameshift inducing pseudoknot a hairpin of similar composition as the pseudoknot did stimulate frameshifting in vitro ( Figure 1A and B). The 12 bp hairpin derivative of the SRV-1 pseudoknot (SRV-hp) showed 22% frameshifting efficiency, whereas the SRV-1 pseudoknot (SRV-pk) in this context yielded 31%. The pseudoknot in these experiments is a modified version of the wild-type SRV-1 pseudoknot previously used for NMR and functional analysis (14) . We note that the U UUAAAC slippery sequence was used to enhance the sensitivity of the in vitro frameshifting assay. This sequence is $1.5-fold more slippery than the wild-type GGGAAAC slippery sequence (28) . In the latter context, the hairpin was indeed less efficient (data not shown) while a non-slippery variant, GGGAAGC, was not effective at all (<0.2%, data not shown). Two other known efficient slip sites, AAAAAAC and UUUUUUA, caused 23 and 27%, respectively, of ribosomes to switch frame in the presence of the 12 bp hairpin (data not shown). These data showed that the 12 bp hairpin is a genuine stimulator of frameshifting. Since the hairpin construct also contained sequences resembling those of L2 of the pseudoknot construct, it was theoretically possible that these nucleotides could take part in the same base triples. To investigate this possibility, we replaced the downstream sequence in the hairpin construct (SRV-muthp). This did not affect the frameshift efficiency of the hairpin construct. In contrast, the same mutations in the pseudoknot context (SRVmutpk) reduced its activity about 6-fold ( Figure 1B) . Thus, it is unlikely that triple helix formation or other tertiary interactions contribute to hairpin-dependent frameshifting; the hairpin as such seems to be sufficient.
Next, we investigated the role of stem length on frameshifting efficiency. Increasing stem size from 12 to 15 or 21 bp did not significantly alter frameshifting (Figure 2A ). On the other hand, decreasing stem size led to a steady decrease in frameshifting efficiency which seemed to vanish around a stem size of 5 bp or ÁG 37 of À7.7 kcal/ mol ( Figure 2B ). Thermodynamic stabilities were calculated at the MFOLD website using version 2.3 parameters (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3), as these were previously shown to better fit in vivo hairpin stabilities (29) . These data support the notion that downstream structures serve as barriers to stall translating ribosomes to stimulate frameshifting, and demonstrate that there is a correlation between the thermodynamic stability of a hairpin and its frameshift inducing capacity.
A selection of above hairpins was cloned into a dualluciferase reporter plasmid and their frameshifting efficiency assayed in mammalian cells ( Figure 2C) . Although the absolute level of frameshifting was lower than in vitro, the trend was similar and showed maximal frameshifting of $8% around 12-15 bp. The pseudoknot in these assays was 1.6 times more efficient than the 12 and 15 bp hairpins, close to the in vitro ratio of 1.4 (see above). Thus, the hairpin derivative can effectively substitute for the SRV-1 pseudoknot in À1 ribosomal frameshifting.
Bulges and mismatches are known to change twisting and bending of a regular stem and are thus expected to influence the way in which a ribosome encounters a hairpin structure (11, 30) . To investigate a possible effect of helical twisting and bending on frameshifting, we introduced mismatches and bulges in the 12 bp stem at a position corresponding to the junction in the SRV pseudoknot ( Figure 3A) . Introduction of an A · A mismatch halfway through the stem (11 bp/AA) decreased frameshifting about 10 fold, although its predicted thermodynamic stability of À25.1 kcal/mol is comparable to that of a regular hairpin of 10 bp, yielding 13% frameshifting ( Figure 2B ). The frameshift inducing ability was recovered when the base pair was restored to A-U (12 bp/AU).
We also introduced a single or triple adenosine bulge at either side of the stem, to investigate potential bending effects on frameshifting. Figure 3A and B show that the single adenosine bulge mutant decreased frameshifting, depending on the location of the bulge, five to seven fold compared to the 12 bp hairpin construct. When the bulge was enlarged to three adenosines the frameshifting was almost abolished. Interestingly, the effect of bulges at the 3 0 side of the stem was less dramatic than those at the 5 0 side.
The loop composition plays a major role in hairpin stability, RNA/RNA and RNA/protein interaction. These factors may directly influence hairpin-induced ribosomal frameshifting efficiency. To explore the correlation between loop composition and frameshifting efficiency, a number of loop mutations were introduced in the context of a 9 bp stem (Figure 4 ). We note that the UUCG tetraloop with a CG closing base pair (cbp) has higher stability ($2 kcal/mol) than that with a GC cbp (31) . Therefore, we first tested if this different cbp affected frameshifting efficiency. Our results showed that there is no difference in frameshifting efficiency between UUCG and UUCG/cg constructs (Figure 4, bars 6 and 8) . Replacing the UUCG tetraloop by GGGC which, due to its high content of purines, is among the most disfavored tetraloops (32) had only a marginal effect on frameshifting (Figure 4 , compare bars 6 and 7 and Figure 5A, lanes 1 and 4) . Interestingly, increasing the loop size to 9 nt, which is predicted to lower the stability of stem did not affect frameshifting ( Figure 4 , bar 1; Figure 5A , lane 3).
Substituting UUCG by another stable tetraloop sequence (GAAA) resulted in a 2-fold decrease in frameshifting ( Figure 5A , lanes 1 and 2) either with GC ( Figure 4 , bar 10) or CG cbp (Figure 4 , bar 9). We designed another five loop mutants to try to explain the low efficiency of the GAAA tetraloop constructs. Constructs AAAA and CAAA induced 5.2% and 4.7% frameshifting, respectively (Figure 4, bars 2 and 3) , which is close to that of the GAAA constructs. The efficiency of two other A-rich loop mutants, ACAA and AAAU, was 7.5% and 7.1%, respectively (Figure 4 , bars 4 and 5), thereby closely matching that of the UUCG constructs. Finally, the GGGA tetraloop construct, belonging to the stable GNRA tetraloop family, induced 1.7-times more frameshifting than its GAAA sibling (Figure 4, bar 11) . These data suggest that the presence of 3 or 4 adenines at the 3 0 side of a tetraloop is unfavorable for frameshifting.
To further examine the role of the loop identity or size in ribosomal frameshifting, we cloned some of the above loop mutants into a dual-luciferase reporter plasmid and assayed their frameshifting efficiency in mammalian cells ( Figure 5B ). Our data show that the effects of loop nucleotides are comparable in vitro and in vivo. The stable GAAA tetraloop construct again had the lowest frameshifting efficiency ( Figure 5B , 2.9%), which was half that of the UUCG construct ( Figure 5B, 6 .1%).
Most RNA viruses that make use of ribosomal frameshifting employ pseudoknot structures instead of simple hairpins for this job. The reason for this may be the presence of a triple helix interaction between S1 and L2 in most frameshifter pseudoknots, which has been suggested to be a poor substrate for the ribosomal helicase (13, 33) and hence increases ribosomal pausing and the time window for slippage. Although pausing is critical, it is not sufficient for efficient frameshifting (34) . Previously, it was shown that a 17 bp hairpin with a calculated stability of À31.2 kcal/mol derived from the minimal IBV pseudoknot induced 5-to 10-fold less frameshifting in RRL (22) than its parent pseudoknot even though both the hairpin and the pseudoknot can pause ribosomes at the same position and to a similar extent (34) . In the present study, a 12 bp hairpin derivative of the SRV-1 gag-pro pseudoknot with a calculated stability of À26.9 kcal/mol was capable of inducing 22% of frameshifting, which is only 1.4-fold . Influence of loop sequence and closing base pair (cbp) on À1 ribosomal frameshifting efficiency. The composition of various loops capping a 9 bp stem is shown in bold, and CG-cbps are shown in lower case. The constructs are named after their loop sequence followed by the '/cg' extension when the cbp was changed from G-C to C-G. Slippery sequence and spacer are the same as in the construct shown in Figure 1A . Graph is similar to that of Figure 2B except that on the right y-axis ÁG starts from À18 kcal/mol. less than its pseudoknotted counterpart. This indicated that a non-natural hairpin can be an efficient frameshift stimulator, at least in the SRV-1 model. Furthermore, our results showed that the frameshifting efficiency increased upon elongation of the length of the hairpin up to 12-15 bp, which is consistent with our previous data using antisense oligonucleotides of 12-15 nt to induce ribosomal frameshifting (35) . More importantly, the frameshift inducing ability of these hairpin constructs with a perfect stem linearly correlated with the calculated thermodynamic stability, in agreement with two previous reports (19, 20) .
In the experiments of Bidou et al. (20) studying the HIV-1 gag-pol frameshift hairpin the stem-length was kept at 11 bp, while its stability was varied between À3.4 and À22.1 kcal/mol (recalculated using MFOLD 2.3) by changing the number of AU and GC base pairs in a small set of six hairpins. In the case of the dnaX gene of Escherichia coli 22 variants of the wild-type 11 bp hairpin were tested for their ability to stimulate À1 PRF at the AAAAAAG slippery sequence. Hairpin stabilities varied between À10.4 and À27 kcal/mol and a positive correlation between frameshifting efficiency and calculated stability was observed both in the presence (R 2 = 0.62) and absence (R 2 = 0.72) of upstream enhancer (19) . The dnaX gene with the highly efficient (prokaryotic) AAAAA AG slippery sequence is not directly comparable to our in vitro system; a 6 bp hairpin in the dnaX gene displayed 17% of frameshifting without upstream enhancer, whereas a 6 bp hairpin in our system induced only 3.5% of frameshifting.
In the HIV-1 gag-pol gene Bidou et al. (20) observed a 15-20% decrease in frameshifting in vivo with their most stable hairpin, similar to our results with the 21 bp hairpin. However, in our case, the stability at which this happened was À45 kcal/mol much higher than their most stable hairpin of À22.1 kcal/mol. It is possible that this difference is due to the different experimental systems. Although it has been suggested that too stable stems increase the time for tRNAs to shift back into the 0-frame again (20) we believe that our 21 bp hairpin is less efficient because it has more AU bps in the middle of the stem compared to the 12 and 15 bp hairpins (Figure 2A) . The experiments with hairpins harboring bulges or mismatches halfway through the stem demonstrated that this region is quite important for frameshifting ( Figure 3A and B) . Even though the overall stability of these constructs was comparable to that of a hairpin of 9 or 10 bp, their frameshift activity was equal or lower than that of a 6 bp hairpin of À13.1 kcal/mol: as if the mismatch or bulge after the 6th base pair disconnected the upper part of the stem. This observation is reminiscent of the overall destabilizing effect of mismatches in DNA hairpins. In a pioneer singlemolecule pulling study, it was shown that introducing a mismatch in a 20 bp DNA hairpin shifted its transition state close to the location of the mismatch (36) . Our data also comply with this mechanical study and suggest that mechanical stability may be a better parameter than thermodynamic stability to describe the frameshift efficiency of hairpins.
In addition to the mentioned dnaX and HIV-1 gag-pol hairpins, other examples of frameshifter hairpins are found in HTLV-2 and CfMV ( Figure 6 ). HTLV-2 gag-pro features a perfect 10 bp hairpin with CUA tri-loop which induces 9% frameshifting in RRL (16) . The CfMV 2a-2b frameshifting hairpin consists of 12 bp, one cytidine bulge close to the top, and a stable UACG tetraloop and is capable of inducing 11% of frameshifting in a wheat germ cell-free system (WGE) (17) . What these hairpins have in common is their length of 10-12 bp, their relatively low number of mismatches and bulges, their small loops and their high GC content, especially in the bottom 6 bp. These features are also applicable to the good frameshifters from our dataset. Interestingly, these features do not all apply to the minimal IBV hairpin ( Figure 6 ) that is derived from the so-called minimal IBV pseudoknot. Despite its large size of 17 bp, absence of mismatches and bulges, presence of a small loop, the stability of the middle part of the hairpin, i.e. bp 5-9, is not very high. This could be the reason why its activity in RRL is 5-10 fold lower (22) than of its parent pseudoknot, whose activity is 42% (25) . Surprisingly, in our assays the frameshift-inducing efficiency of the IBV hairpin was 26% (data not shown), which is in stark contrast to the 4-8% reported by Brierley et al. (22) . This discrepancy may be due to experimental conditions: in our experiments we used non-capped transcripts, a 7-nt spacer and RRL from Promega whereas the Brierley's lab used capped transcripts, a 6-nt spacer and in-house prepared RRL. On the other hand, the 26% we obtained for the IBV hairpin would be a factor of 1.6 lower than the 42% reported for the IBV pseudoknot (25) , and is similar to the ratio of 1.4 and 1.6 we obtained for SRV in vitro and in vivo, respectively.
Remarkably, in WGE the IBV hairpin has been reported to induce high levels (34%) of frameshifting versus 51% for the IBV pseudoknot (24) . In that study modified extracts were used that are somewhat more frameshift-prone than the standard wheat-germ extracts. Nevertheless, the ratio between pseudoknot and hairpin-induced frameshifting in this system is also 1.5. This number may reflect the additional interactions, like base triples, in a pseudoknot that make it a better frameshift stimulator than a hairpin.
In addition to stem size, loop composition is another determinant of hairpin stability. An important subgroup of hairpin loops is the tetraloop, which is the most common loop size in 16S and 23S ribosomal RNAs (37) . The tetraloops with consensus UNCG, GNRA, or CUUG loop sequence form stable loop conformations (38, 39) . As opposed to the mentioned stable tetraloops, purine-rich (32) and larger loops (40) are considered to be less favorable for hairpin formation. Our results showed that the GGGC loop is indeed less efficient in inducing frameshifting but the larger loop construct (9 bp/9 nt), although having a lower thermodynamic stability, showed comparable frameshifting efficiency to the stable UUCG tetraloop hairpin. This is consistent with previous studies that showed that increasing the size of the loop in a hairpin or pseudoknot can increase frameshift-inducing ability to a certain extent (21, 41) . Although larger loops seem efficient in inducing frameshifting, in known examples of frameshifter hairpins, there are no loop sizes of more than 5 nt. This could relate to hairpin folding kinetics (40) or to nuclease sensitivity.
Intriguingly, we found that a 9 bp stem capped with a GAAA tetraloop is 2-fold less efficient in inducing frameshifting than its UUCG counterpart in vitro and in vivo. It has been reported that GAAA tetraloops are frequently involved in RNA tertiary interactions (42) . We hypothesize that the GAAA tetraloop may be involved in an unknown RNA tertiary structure with ribosomal RNA, thereby interfering with frameshifting. The fact that in the known natural examples of frameshifter hairpins, the GAAA tetraloop, despite its high stability, is absent can be taken as support for this hypothesis (Olsthoorn, unpublished data) . Further investigation of this observation may lead to new insights in ribosomal frameshifting.
In conclusion, our data show that hairpins of various base composition in stem and loop can act as efficient frameshift stimulators. Combined with previous studies on antisense-induced frameshifting (43, 44) , these data support the notion that downstream structures primarily serve as barriers to stall translating ribosomes to stimulate frameshifting. Although there exists a linear relationship between calculated stability and frameshifting, local destabilizing elements like bulges or mismatches in a hairpin can greatly influence frameshift-inducing activity. Future experiments addressing the mechanical strength of these hairpins (7-9) may help to improve our understanding of the basics of ribosomal frameshifting. Usefulness of Published PCR Primers in Detecting Human Rhinovirus Infection We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)–specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology. H uman rhinoviruses (HRVs) cause more asthma exacerbations than any other known factor, in addition to causing most colds and infl uenza-like illnesses. The prevalence of HRV in published reports varies considerably. A novel HRV clade identifi ed in 2006, now known as HRV species C (HRV-C) (1), can be identifi ed only by PCR. Since 1988, seasonality and clinical outcomes and numerous different primer pairs have been used to identify HRV; how well these methods perform on new HRV types is uncertain. Given the likely variation in the preparation of RNA, the quality and formulations of commercial reverse transcription (RT)-PCR enzymes and reaction mix components and changes in thermal cyclers since 1988, not surprisingly many, perhaps most, of these assays are not being used in the manner they were originally described. For example, the fi rst HRV-specifi c primers reported (2) have subsequently been used with different RNA preparation methods, amounts of reverse transcriptase, cDNA priming strategies, dNTP concentrations, annealing temperatures (T M s), and cycling conditions (3, 4) .
We conducted a preliminary comparison of the relative sensitivity of a cross-section of published HRV-specifi c PCR primer pairs (most of which were fi rst published before HRV-C was reported), independent of most variables described above, by testing a panel of 57 clinical specimen nucleic acid extracts from combined nose and throat swabs from preschool children with colds and infl uenza-like illnesses in Melbourne, Australia. The study was approved by the Royal Children's Hospital Human Research Ethics Committee. The panel included representatives of the 3 HRV species (Figure) , human enteroviruses (HEVs), and extracts negative for picornaviruses. The HRVs had been previously detected by using a nested primer pair (online Appendix Table, www.cdc.gov/EID/content/17/2/294-appT.htm) (5). We used 10 different HRV primer pairs and also retested specimens by using the original primer pair with our standard reagents and equipment (5) . We applied the published T M when possible. The original descriptions of primer pairs 7 and 10 (online Appendix Table) lacked T M information, and after in-house calculations, we used T M s of 50°C and 58°C, respectively. We also deliberately standardized the reagents (OneStep RT-PCR kit, QIA-GEN, Doncaster, Victoria, Australia) and thermal cyclers used (Veriti, Applied Biosystems, Foster City, CA, USA) for conventional PCR and the RotorGene 3000 real-time cycler (QIAGEN). Because primer pair 1 had a published history of detecting types from all HRV species, we chose it to genotype HRV-positive samples by sequencing the amplifi ed products. Other pairs were used if pair 1 was unsuccessful.
We found that no primer pair detected the same HRVs and HEVs typed when the original pair (5) or pair 1 (online Appendix Table) was used. Five primer pairs, including real-time PCR (rtPCR) pair 5, did not amplify the HEVs, a positive feature for HRV-specifi c studies. Only 2 primer pairs amplifi ed anything from a specimen that was positive for both HRV and HEV, a problem for accurate estimation of the frequency of co-detections. The original primer pair screen detected 3 untypeable picornaviruses, which were not detected by any other pair or by repeat testing using the same pair. Only the second-round amplicon of the 3 nested sets of nested primer pairs (2, 3, and 9) was considered because the second round increased the total number of positive specimens over the fi rst round. The longest amplicon, produced by primer pair 7, was also a valuable genotyping target, but it detected only 14 of the original 27 HRV-positive specimens in this population.
We next selected 4 frequently published primer pairs (1, 5, 7, and 8) to examine 44 picornavirus-positive specimens (39 HRVs, 3 HEVs, and 2 untypeable picornaviruses) from nonhospitalized children with acute asthma exacerbation (6) . As before, primer pair 1 detected the greatest num- Most notably, primer pair 7 performed better than it had in the previous population, detecting only 1 fewer HRV than primer pair 1 and 9 more HRVs than pair 8. No speciesspecifi c bias was apparent, but generally, a specimen with a lower RNA concentration, as indicated by the cycle threshold from primer pair 5, was less likely to be detected or typed by using other primer pairs. Primer pairs 5 and 8 did not detect the 3 HEVs (HEV-68). We noted in both populations that primer pair 1 sometimes amplifi ed a region of human genomic DNA from chromosome 6 (GQ497714), for which amplicon size was indistinguishable from that expected due to HRV. It was not possible to use the precise conditions reported for the 10 compared assays; 1 was published >2 decades ago and used phenol chloroform extraction. Some of the original enzyme formulations or reagents are no longer available, and production processes have changed in the interim. Thermal cyclers have also changed. There was no consensus on enzymes and reaction mixes used. In addi-tion, the previously published primers were used in assays divided between those using 1-step RT-PCR and those using a separate RT cDNA synthesis step. A review of studies that detected HRVs with adequately described conditions during 2009-2010 found that fewer used a single-tube RT-PCR approach than a 2-step system. We conducted singletube RT-PCR to maintain the benefi ts of the so-called closed amplifi cation system of rtPCR. Thus, we chose to use a single common set of reagents as the fairest way to compare the primer pairs examined in this study. We believe the nature of this relative comparison best refl ects performance for the likely end users: clinical microbiology laboratories or researchers.
We compared primers rather than assay function using clinical material instead of cultured virus, plasmid or synthetic RNA standards, or screening contemporary or archived extracts, which are sometimes of low viral load. When picornavirus epidemiology is the primary research focus, we recommend using >2 primer pairs to maximize the detection of HRVs. Under our conditions, pairs 1-4 returned the highest number of positive results, and the rtP-CRs behaved similarly but with reduced sensitivity. The rtPCR that used pair 5 did not amplify known HEVs.
Many possible reasons could cause discrepant virus testing results between different sites, including changes to specimen integrity resulting from transport and variable amplifi cation resulting from low viral loads. The effects of viral load can be seen in this study: specimens in population 1 that were positive with multiple (>6 separate pairs) primer pairs had a mean cycle threshold of 33.3 (combining results from both rtPCRs), whereas those with <6 positive results had means of 39.3 cycles. Most (29/33) specimens with <3 positive primer pairs were negative by rtPCR. Amplifi cation variability can also be attributed to the substantial nucleotide sequence diversity between HRVs and the different temporal and clinical characteristics of the 2 specimen populations we used. Population diversity is a feature of HRV studies in the literature.
Our selection of published primer pairs includes those from studies that have informed our current understanding of HRV epidemiology. Finding such a high degree of variability in performance was thus noteworthy. Ineffi cient HRV detection by PCR may be a serious problem for research studies. Comparison of data between different HRV studies is confounded as are data from studies seeking to determine the effects of other respiratory viruses. The prevalence, seasonality, transmission, and clinical effects of HRV types and species require reexamination with tools that have been comparatively validated to ensure their sensitivity. Figure. Distribution of human rhinovirus (HRV) and human enterovirus (HEV) sequences used for primer pair studies. The HRV and HEV genotypes from the testing panel (indicated by fi lled circles) were aligned with the central 154 nt of the 5′ untranslated region (UTR) region of all complete HRV genomes and poliovirus-1. HRV-Ca and HRV-Cc refer to HRV-Cs with 5′ UTR sequences that have phylogenetic origins from either HRV-As or HRV-Cs, respectively. The tree was constructed by neighbor joining of maximum composite likelihood distance implemented in MEGA (www.megasoftware.net). NNAlign: A Web-Based Prediction Method Allowing Non-Expert End-User Discovery of Sequence Motifs in Quantitative Peptide Data Recent advances in high-throughput technologies have made it possible to generate both gene and protein sequence data at an unprecedented rate and scale thereby enabling entirely new “omics”-based approaches towards the analysis of complex biological processes. However, the amount and complexity of data that even a single experiment can produce seriously challenges researchers with limited bioinformatics expertise, who need to handle, analyze and interpret the data before it can be understood in a biological context. Thus, there is an unmet need for tools allowing non-bioinformatics users to interpret large data sets. We have recently developed a method, NNAlign, which is generally applicable to any biological problem where quantitative peptide data is available. This method efficiently identifies underlying sequence patterns by simultaneously aligning peptide sequences and identifying motifs associated with quantitative readouts. Here, we provide a web-based implementation of NNAlign allowing non-expert end-users to submit their data (optionally adjusting method parameters), and in return receive a trained method (including a visual representation of the identified motif) that subsequently can be used as prediction method and applied to unknown proteins/peptides. We have successfully applied this method to several different data sets including peptide microarray-derived sets containing more than 100,000 data points. NNAlign is available online at http://www.cbs.dtu.dk/services/NNAlign. Proteins are extremely variable, flexible and pliable building blocks of life that are crucially involved in almost all biological processes. Many diseases are caused by protein aberrations, and proteins are frequent targets of intervention. A plethora of highthroughput methods are currently being used to study genetic associations and protein interactions, and intense on-going international efforts aim at understanding the structures, functions and molecular interactions of all proteins of organisms of interest (e.g. the Human Proteome Project, HPP). In some cases, linear peptides can emulate functional and/or structural aspects of a target structure. Such peptides are currently identified using simple peptide libraries of a few hundreds to thousands peptides whose sequences have been systematically derived from the target structure at hand -that is, if this is known. Even when the native target structure is unknown, or too complex (e.g. discontinuous) to be represented by homologous peptides, the enormous diversity and plasticity of peptides may allow one or more peptides to mimic relevant aspects of a given target structure [1, 2] .
Peptides are therefore of considerable biological interest and so are methods aimed at identifying and understanding peptide sequence motifs associated with biological processes in health and disease. Indeed, recent developments in large-scale, high-density peptide microarray technologies allow the parallel detection of thousands of sequences in a single experiment, and have been used in a wide range of applications, including antibody-antigen interactions, peptide-MHC interactions, substrate profiling, identification of modification sites (e.g. phosphorylation sites), and other peptide-ligand interactions [3, 4, 5, 6, 7] . One of the major advances of peptide microarrays is the ease of generating large numbers of potential target structures and systematic variants hereof [8] .
Given the capability for large-scale data-generation already realized in current ''omics'' and peptide microarray-based approaches, experimentalists will increasingly be confronted with extraordinary large data sets and the consequent problem of identifying and characterizing features common to subsets of the data. These are by no means trivial problems. Up to a certain level of size and complexity, data can be presented in simple tabular forms or in charts, however, larger and/or more complex bodies of data (e.g. in proteome databases) will need to be fed into bioinformatics data mining systems that can be used for automated interpretation and validation of the results, and eventually for in silico mapping of peptide targets. Moreover, such systems can conveniently be used to design next-generation experiments aimed at extending the description of target structures identified in previous analyses [9] .
A wealth of methods has been developed to interpret quantitative peptide sequence data representing specific biological problems. By way of examples, SignalP, which identifies the presence of signal peptidase I cleavage sites, is a popular method for the prediction of signal peptides [10] ; LipoP, which identifies peptidase II cleavage sites, predicts lipoprotein signal peptides in Gram-negative bacteria [11] ; various prediction methods predict phosphorylation sites by identifying short amino acid sequence motifs surrounding a suitable acceptor residue [12, 13, 14, 15] etc. In general terms, these methods can be divided in two major groups depending on the structural properties of the biological receptor investigated, and of the nature of the peptides recognized. The simplest situation deals with interactions where a receptor binds peptides that are in register and of a known length. In this case, the peptide data is pre-aligned, and conventional fixed length, alignment-free pattern recognition methods like position specific weight matrices (PSSM), artificial neural networks (ANN), and support vector machines (SVM) can be used. Peptide-MHC class I binding is a prominent example of the successful use of such methods to characterize receptor-ligand interaction represented by pre-aligned data (reviewed in [16] ). Another more complex type of problems deals with interactions where either the motif lengths, and/or the binding registers, are unknown. In these cases, the peptide data must a priori be assumed to be unaligned and any bioinformatics method dealing with such data is faced with the challenge of simultaneously recognizing the binding register (i.e. performing an alignment) and identifying the binding motif (i.e. performing a specificity analysis). Peptide-MHC class II binding is a preeminent example of a receptor-ligand interaction represented by unaligned data. Several bioinformatics methods have been developed to identify binding motifs in such peptide data including Gibbs sampling [17] , hidden Markov models (HMM) [18] , stabilization matrix method (SMM) alignment [19] , and alignment using artificial neural networks [20] (for more references see [21] ). Another example of unaligned peptide data is that of antibodies interacting with linear peptide epitopes. Although B-cell epitopes frequently are conformational and three-dimensional in structure, some do contain linear components that can be represented by peptide interaction with the corresponding antibodies [22, 23, 24] .
Even though most of the methods described above are standard methods for data-driven pattern recognition, the development of a prediction method for any given biological problem is far from straightforward, and the non-expert user will rarely be able to develop their own state-of-the-art prediction methods. We have recently described a neural network-based data driven method, NN-align, which has been specifically designed to automatically capture motifs hidden in unaligned peptide data [20] . NN-align is implemented as a conventional feed-forward neural network and consists of a two-step procedure that simultaneously identifies the optimal peptide-binding core, and the optimal configuration of the network weights (i.e. the motif). This method is therefore inherently designed to deal with unaligned peptide data, and it identifies a core of consecutive amino acids within the peptide sequences that constitute an informative motif. Note that the method does not allow for gaps in the alignment. Although NNalign was originally developed with the unaligned nature of peptide-MHC class II interaction in mind -and independent validations have shown that NN-align indeed performs significantly better than any previously published methods for MHC class II motif recognition [25] -the unique ability of this method to capture subtle linear sequence motifs in quantitative peptide-based data and its adaptability makes it extremely attractive for other applications as well. Here, we have adapted and extended the NNalign method so that it can handle quantitative peptide-based data in general. Making this method generally available for the scientific community, we have embedded it into a public online web-interface that facilitates both handling of input data, optimization of essential training parameters, visual interpretation of the results, and the option of using the resulting method to predict on user-specified proteins/peptides. Through the server the user can easily set up a cross-validation experiment to estimate the predictive performance of the trained method, and automatically reduce redundancy in the data. The logo visualization is also improved with an algorithm that aligns individual neural networks to maximize the information content of the combined alignment. This web-based extension of the NN-align method empowers experimentalists of limited bioinformatics background with the ability to perform advanced bioinformatics-driven analysis of his/ her own sets of large-scale data.
Enabling any non-expert end-user to extract specific information from quantitative peptide data using an advanced bioinformatics approach, we have used our recently published NN-align method to generate a web-based extension with a reasonably simple, yet adaptable, web-interface and made this server publicly available at http://www.cbs.dtu.dk/services/NNAlign. Using this web server any user can submit quantitative peptide data (optimally based on actual discrete measurements, but even assigned classification, e.g. 0 and 1, can be used) and in return receive a trained method including training details and estimated predictive performance, a visual interpretation of the identified peptide pattern, and the trained model itself. The latter can be resubmitted to the web server at any later time and used to predict the occurrence of the learned motif in one or more concurrently submitted peptide sequences or FASTA format sequences.
The truly non-expert user has the option of using a set of default settings. Using these settings, the data is preprocessed using a linear transformation to make the data fall in the range from 0 to 1, and the NN-align method is trained using five-fold crossvalidation. For each cross validation partition five networks, each initiated from different initial configurations, are trained with 3 hidden neurons. The only critical parameter that the user is required to specify is the motif length. The value used for this parameter is specific to each problem and the user is recommended to define a motif length (or an interval of motif length) that is relevant to the biological problem investigated by the peptide data. The default settings will in most cases allow the user to obtain a first impression of the motif contained in the data, and achieve a prediction method that allows the user to make prospective studies on uncharacterized proteins/peptides. The more experienced user has several advanced options to customize the training. For details on these options refer to Materials and Methods section, or the help section of the web-server.
An example output from the NNAlign Server is shown in Figure 1 . Information about the training data is accompanied by a plot of the data distribution before and after the data processing needed to train the neural networks. An important feature is the possibility to download and save the trained model, and use it subsequently for predictions on new data. The results page also returns the performance of the method as estimated by crossvalidation, and provides links to a scatter-plot showing the correlation between measured and predicted values, as well as the complete alignment core on the training data. A sequence logo gives a visual representation of the identified sequence motif, which can also be viewed in a log-odds position-specific scoring matrix format. If any evaluation data has been provided at the time of method training, a section of the results will report the predictions of this evaluation set.
A few example applications illustrating the power of the NNAlign method are presented in the following sections. First, the method is applied to examples of pre-aligned peptide data using examples of MHC class I binding. Next, the alignment problem is included using MHC class II binding data, showing the ability of the method to identify at the same time the correct length of the motif, the binding register, and the sequence motif itself. An important output from the NNAlign method is a sequence logo representing the identified binding motif. Such sequence logos provide a highly intuitive representation of single-receptor specificities (as is the case Figure 1 . Example of output from the NNAlign server trained on MHC class II binding data for allele HLA-DRB1*0101. Links on the results page (in pink) redirect to additional files and figures relevant for the analysis. Run ID is a sequential identifier for the current job, and Run Name a user-defined prefix that is added to all files of the run. The ''view data distribution'' link shows the transformation applied to the data in preprocessing, which can be either a linear or logarithmic transformation. In this case the method was trained with a motif length of 9, including a PFR of size 3 to both ends of the peptide, and encoding in the network input layer peptide length and PFR length. The hidden layer was made of a fixed number of 20 neurons. Peptides were presented to the networks using a Blosum encoding to account for amino acid similarity, for 500 hundred iterations per peptide without stopping on the best test set performance. At each cross-validation step, 10 networks were trained starting from 10 different initial configurations. The subsets for cross-validation were constructed using a Hobohm1 method that groups in the same subset sequences that align with more than 80% identity (thr = 0.8). The model can be downloaded to disk using the dedicated link, and can be resubmitted to NNAlign to find occurrences of the learned pattern in new data. The estimated performance of the trained method is expressed in terms of Root Mean Square Error, Pearson and Spearman correlation. A visual representation of the correlation can be obtained from the scatterplot of predicted versus observed values. The ''complete alignment core'' link allows downloading the prediction values in cross-validation for each peptide, and where the core was placed within the peptides. Next follows a section on the sequence logo, showing a logo representation of the binding motif learned by the network ensemble. If the relative option is selected, links to logos for the individual networks in the final ensemble are also listed here. Finally, if an evaluation set is uploaded, an additional section shows performance measures and core alignment for these data. doi:10.1371/journal.pone.0026781.g001
for MHC class I and II binding data). Finally, to illustrate how the method is capable of handling and guide the semi-expert user in interpreting large-scale data sets, NNAlign is applied to data generated by a large-scale peptide microarray technology.
Binding of peptides to MHC class I molecules is highly specific, with only 1-5% of a set of random natural peptides binding to any given MHC molecule [26] . Moreover, in the vast majority of cases only peptides with length 8-10 amino acids can fit in the binding pocket of MHC class I molecules. The predictive performance of NNAlign on 12 human MHC class I alleles from data by Peters et al. [27] is shown in Table 1 (see the table footnote for the parameters used). The benchmark data sets contain quantitative binding data of a given length (9 amino acids) covering the whole spectrum from non-binding to strong-binding peptides, hence serving as a perfect illustration of the strength of the NNAlign method to handle pre-aligned peptide data. The overall performances of the three methods are comparable demonstrating that NNAlign competes with state-of-the-art methods designed specifically for MHC class I prediction.
As opposed to MHC class I binding, which is mostly limited to peptides of similar length, the MHC class II molecule interacts with peptides of a wide length distribution and high compositional diversity [28] . Binding of a peptide to an MHC class II molecule is primarily determined by a core of normally 9 amino acids, but the composition of the regions flanking the binding core (peptide flanking region, PFR) has been shown to also affect the binding strength of a peptide [29, 30] . Identifying the binding motif and binding register for MHC class II binding peptides is thus a problem that inherently requires simultaneous alignment and binding affinity identification. Here, an MHC class II benchmarking was obtained from the recent publication by Wang et al. [25] . The performance was estimated for each allele using a 5 fold cross validation, where at each step 4/5 of the data were used to train the neural networks, and 1/5 were left out for evaluation. For cross-validation, we preserved the same data partitioning as used in the original publication. In Table 1 , the performance of NNAlign on the Wang set is compared to other publicly available methods for MHC class II prediction. These include SMM-align [19] , ProPred/Tepitope [31, 32] , as well as the original version of the NN-align algorithm [20] . The NN-align-based methods outperform their competitors on all alleles, confirming the ability of the neural networks in dealing with alignment problems. The difference with the original NN-align method, which is due to differences in network architecture, is small and not significant (p.0.2, binomial test). For this example involving unaligned data, the NNAlign server competes with comparable state-of-the-art methods.
Choosing the optimal motif length Different positions in a binding motif can be more or less informative, and the ends of a motif can often not be clearly delineated. This prompts the question of how many positions are necessary and sufficient to represent a given motif and how the length of a motif is defined. NNAlign allows searching for the optimal motif length in a quantitative peptide data set. Here, the best motif length is the one that yields, in a cross-validation experiment, the lowest root mean square error (RMSE) between For MHC class I no significant difference is found in predicted performance between the NNAlign, SMM and ANN method (p.0.5, binomial test). The values for the SMM and ANN methods were taken from Peters et al. [27] . The method was trained using a fixed motif length of 9 corresponding to the peptide length, and constructing a network ensemble with multiple architectures using respectively 2,3,4,5 and 7 hidden neurons. Performance was measured in cross-validation, training each network for a fixed number of 500 iterations per sequence. The different MHC class II prediction methods are NN-align [20] , SMM-align [19] , and Propred [31, 32] . NNAlign server is the method described here. Performance values for first 4 methods are taken from [25] . NNAlign was trained with a motif length of 9, flanking regions of 3 amino acids, Blosum encoding including peptide length and flanking region length, and an ensemble of 2, 3, 5, 9 and 12 hidden neurons for each of 10 initial random configurations. In bold is highlighted the best performing method for each MHC allele. The column # gives the number of the peptides in the data set for the given allele. doi:10.1371/journal.pone.0026781.t001
observed and predicted values. By this token, a terminal position is included in the motif if it contributes with information at a level above what could be considered to be noise. In contrast, if the inclusion of a putative terminal position does not lead to a reduction in the RSME then it can be concluded that it does not add useful parameters to the model; rather, it lowers the predictive performance and should be omitted. This approach was used to suggest the motif length of the 14 MHC class II HLA-DR alleles, which were searched for optimal predictive performance by scanning through possible lengths from 6 to 11 amino acids. NNalign will report the length associated with the lowest RMSE value as the optimal motif length (see Figure 2 , left hand panel). Nonetheless, the user is advised to inspect the sequence logo as well as the performance plot of the RMSE as a function of the motif length to evaluate whether the dependence upon length appears significant. As defined here and illustrated in Figure 2 right panel, the 9-mer preference of HLA-DRB1*01:01 is significant, whereas the apparent 8-mer preference of HLA-DRB1*15:01 is not significant. In fact, for the 14 HLA-DR molecules included in the benchmark, only one was found to have a single consistent optimal motif length (DRB1*0101 with a motif length of 9 amino acids). For all other molecules the method did identify more than one possible optimal motif length. However, all motif lengths fell in the range of 7 to 10 amino acids, and in all cases a 9-mer motif was compatible with being the optimal motif length.
In order to enhance predictive performance, the NNAlign method exploits an ensemble of neural networks [20, 33] , which have been trained on different subsets of the data, and/or from alternative configurations of the network architecture (i.e. different number of hidden neurons and/or encoding schemes). As a consequence of different architectures and starting conditions, individual networks might disagree on the exact boundaries of the motif. This disagreement would complicate the visualization of the motif if this was represented as a simple overlay of the individual motifs as exemplified in Figure 3 , where sequence logos for four different networks from the ensemble trained on HLA*DRB1-04:01 binding data are shown in panels A through D. The individual networks agree on identifying the same strong primary anchor residues and positions, however, each single network identifies different ends (i.e. suggests different registers of the same motif; in casu starting at positions 1, 2, 2 and 3 of the predicted nonamer peptide). The weak C-terminal primary anchor residue of HLA*DRB1-04:01 probably explains why the boundaries are difficult to determine. A simple overlay of the predictions from individual networks would result in a muddled motif as depicted in Figure 3 , panel E. Implementing a Gibbs sampler approach, where matrix representations of the core motifs of different networks are aligned, we introduced an off-set correction for each network aiming at maximizing the information content of a combined logo representation of the motif. This approach led to a considerable improvement in the visual logo representation of the binding motif (Figure 3, panel F) . Offset correction is included as an integral part of the method to enhance motif visualization.
Characterizing the binding motif of HLA-DR molecules using the NNAlign method
To illustrate the power of the NNAlign method to capture the binding motifs within unaligned quantitative peptide data, we applied the method to derive sequence logo representations of the 14 MHC class II HLA-DR molecules included the Wang dataset. NNAlign was trained with a binding motif length of 9 amino acids, Blosum encoding, including peptide length and flanking region length, and PFRs of 3 amino acids, homology clustering at threshold 0.8 using all data points, 20 hidden neurons and a 5-fold cross-validation without stopping at the best test set performance. These parameters were found to be optimal in the original NNalign paper for MHC class II binding prediction [20] , with the only difference that here we choose a single value for hidden layer size for a matter of prediction speed. Individual networks are aligned to a common register using the offset correction strategy previously described. The sequence logos obtained are shown in Figure 4 . The sequence logos reflect the overall consensus of the binding motifs for HLA-DR molecules, namely a prominent P1 anchor with strong amino acids preference towards hydrophobic Figure 2 . Identification of optimal motif length using the NNAlign method. Left panel: Histogram of the optimal motifs lengths for the 14 HLA-DR molecules in the Wang dataset as identified by the NNAlign method. Right panel: Predictive performance measured in terms of the root mean square error (RMSE) between observed and predicted values as a function of the motif length for the two molecules DRB1*0101 and DRB1*1501. NNAlign was trained using the same parameters settings described in Figure 4 . At each motif length are shown the mean and standard error of the mean RMSE as estimated by bootstrap sampling. For DRB1*0101 a single consistent optimal motif length of 9 amino acids is found. For DRB1*1501 all motif length 8-11 had statistically indistinguishable performance (paired t-test). doi:10.1371/journal.pone.0026781.g002 amino acids in general, and aromatic amino acids as F and Y in particular, and the presence of two or more additional anchors at P4, P6 and/or P9 each with a unique amino acid preference. Even though most of these motifs exhibit a strong preference for hydrophobic and neutral amino acids at most anchor positions, some dramatic deviations from this general pattern exist. Examples of this are the motifs of DRB1*0301 and DRB1*1101 molecules that have strong preferences for charged amino acids at P4 and P6, respectively.
Handling large data sets exemplified by protease recognition of high-density peptide microarrays A peptide microarray containing a total of .100,000 peptides (49,838 of which were unique) was digested with the protease trypsin. The peptide sequences had been synthesized using the theme Ac-GAGAXXXXXGAGA, where Ac-is acetyl blocking the peptide alpha-amino group prior to digestion, and X represents amino acids chosen randomly from the 20 natural amino acids (except lysine, as this residue contains an epsilon-amino group, which even without digestion would be detectable (see Materials and Methods for details)). As a result, free amino groups can only be expressed by trypsin cleaved peptides, which then can then be labeled with Dylight549 and quantitated by fluorescence microscopy. A fluorescence microscopy picture of such a digested and stained peptide microarray (Figure 5a ) demonstrates both the resolution of the photolithographic peptide synthesis strategy and the dynamic range of the free amino group detection strategy. The resulting data was log-transformed and rescaled to obtain a data distribution covering the spectrum between 0 and 1, which -along with the corresponding peptide sequences encoded as Blosum scores without flanking regionswere used to train an NNAlign method. Training was done with a motif length of 5, a fixed number of 3 hidden neurons, 5-fold exhaustive validation, and stopping at the best test set performance. The prediction method yielded a Pearson correlation between measured values and predictions of r = 0.971, a Spearman correlation of r = 0.910, and receiver operating characteristics (ROC) area under the curve (AUC) of 0.997 (using The fundamental pattern appears in all these networks, but they place the anchors at different position of the core. e) shows the core of the 20 networks ensemble without offset correction; in f) offset correction was used to realign the logos to a common register. doi:10.1371/journal.pone.0026781.g003 a target threshold of t = 0.5). The very high performance measures of the resulting NNAlign method demonstrate both that the recorded peptide digestion data contains a consistent and intelligible signal, and that the NNAlign method is capable of deciphering and predicting this extraordinary large number of sequence-dependent peptide signals. The correlation scatterplot feature of the NNAlign web-server output, which compares predicted vs. observed values, further supports the validity of both the peptide microarray and of the NNAlign method. The correlation scatterplot for the trypsin digestion data reveals two major populations of peptides, one composed of non-degradable, non-predicted peptides and one containing weakly to strongly degradable, predicted peptides ( Figure 5b) . Few (0.7%) of the former peptides contained Arginine, whereas most (97.1%) of latter peptides contained Arginine. This is exactly what one would have expected from a peptide digestion with trypsin, which is known to cleave at the C-terminal side of amino acids Arginine (and Lysine, which has been excluded here, see above) [34] . For illustration purposes, Figure 5b includes a color-enhanced visualization of certain dipeptide sequences (note, this is not a standard feature of the NNAlign server) showing that RP sequences are resistant, RA sequences are quite susceptible, and RR sequences appear extremely susceptible to trypsin digestion. Thus, the known trypsin resistance of RP sequences is both demonstrated by the peptide microarray and subsequently captured by the NNAlign method. Note, that both the peptide microarray and the NNAlign generate a continuous set of measurements and predictions showing that trypsin cleavage involves a more complex interaction than a simple recognition solely of an Arginine residue (and by inference a Lysine residue), which would have resulted in a cleaved/non-cleaved classification [35] . It is also important to note that the detection strategy employed here does not reveal where the protease cleavage has occurred, but merely that the protease has recognized the peptide as a substrate and cleaved it somewhere.
A similar high-density peptide microarray driven approach was next used to address the specificity of the protease chymotrypsin, which is known to preferentially cleave at the C-terminal of tyrosine, phenylalanine and tryptophan (albeit not if followed by a proline). A high-density peptide microarray containing about 50,000 peptides (16,526 unique peptides) was generated according to the theme Ac-GAGAXXXXGAGA, treated with chymotrypsin, labeled with TAMRA and quantitated by fluorescence microscopy. The resulting data was used to train an NNAlign method (using the settings described in Figure 5 ). The correlation scatterplot of the measured versus predicted values exhibits a very strong linear correlation with a Pearson of r = 0.943 demonstrating that the peptide microarray data contains a consistent signal that reliably has been captured by the NNAlign method.
The amount of data deposited in genomic and proteomic databases has been growing exponentially for many years [36] . Due to recent technological advances that have enabled wholegenome sequencing and made whole-proteome analysis a realistic goal, sequence data will accumulate at an even faster pace in the future where single laboratories, even single experiments, can generate data at the ''omics'' level. This is amply illustrated here where a high-density peptide microarray technology allowed the parallel synthesis of more than 100,000 discrete peptide sequences per array, and the collection of a corresponding number of quantitative peptide-receptor interaction data -all within a single experiment.
The biggest hurdle of future ''omics'' research may easily become that of making sense of such large-scale biologic sequence data [37] . Presently, the ''omics'' experimentalist requires assistance from specialized and highly trained bioinformaticians capable of large-scale data handling and interpretation. Ideally, however, he or she should not only be armed with highthroughput data-generation technologies, but also with reasonably easy and robust bioinformatics methods allowing the experimentalist to analyze his or her own data. This would permit an immediate analysis of experimental results and assist in rational designs of next generation experiments aimed at extending the original analysis e.g. providing in silico tools for searches that potentially could encompass entire proteomes. Enabling the same person to do large-scale experiments and analysis should result in a better integration between design, experiment, and interpretation -and eventually support the development of new hypotheses. Unfortunately, suitable bioinformatics resources aimed at the nonexpert user are currently scarce, and rarely web-based. In our experience, open source software packages such as Weka [38] are not capable of performing concurrent alignment and motif identification, and are not suited for treating large-scale data sets. A widely used method for motif discovery, MEME [39] , can perform searches for un-gapped sequence patterns in DNA or protein sequences, and offers a user-friendly online server to the untrained user. However, this method is not designed for use in quantitative data, such as peptide-MHC binding or peptide microarray data.
To the best of our knowledge, NNAlign is the first web-based bioinformatics solution that allows non-expert users to discover short sequence motifs in quantitative peptide data. As shown here, NNAlign easily competes with state-of-the-art methods for identifying peptidebinding motifs of aligned (exemplified by MHC class I) as well as unaligned (exemplified by MHC class II) quantitative peptide sequence data. Further, demonstrating the general utility of NNAlign, we have used it to characterize the cleavage specificities of proteases from high-throughput peptide array data. If a sufficient number of training examples can be generated, including negative instances, we could envision applying the method also on data generated by phage display peptide libraries. Other instances of recognition of short specific peptide motifs occurs frequently in biology where they are involved in molecular interaction, recognition, signaling, internalization, modification etc (e.g. phosphorylation, dephosphorylation, trafficking motifs, SH2 and SH3 domains, glycosylation, lipidation, etc. In contrast to domain recognition, short linear peptide sequences are thought to be particularly difficult to identify due to their unordered structure [40] . NNAlign appears to be ideally suited to identify such short linear peptide targets. Due to its simple interface and robust performance, we believe the method to constitute a significant tool providing the non-bioinformatician end-user with the ability to perform advanced bioinformatics-driven analysis of largescale peptide data sets.
The data set of quantitative peptide-MHC class I binding affinity data published by Peters et al. [27] contains data from 48 different human, mouse, macaque and chimpanzee alleles. We selected 12 representative human alleles, and extracted binding data for 9-mer peptides maintaining the subsets of the original benchmark. This allows comparing the performance of NNAlign to the other methods presented in the paper by Peters et al. Figure 5 . Analysing high-density peptide array data with NNAlign. a) Fluorescence microscopy picture of a peptide microarray. The image is a magnified segment of the peptide chip used in the trypsin cleavage analysis. b) Trypsin peptide-chip data. The normalized observed (target) likelihood of cleavage as a function of the prediction score for the trypsin data set. Localizations of peptides containing the pairs of amino acids RP, RA or RR are highlighted in the plot. Proline (P) is known to prevent cleavage after arginine (R), whereas cleavage is observed with other amino acids such as R and A. c) Chymotrypsin peptide-chip data. Correlation plot between predicted and measured (target) data from the chymotrypsin data set. Values are binned by their x,y proximity, so that the scatterplot represents the density of data in each bin. NNAlign was trained with linear rescaling of the quantitative data, a motif length of 4 amino acids without inclusion of PFR encoding, Blosum encoding of peptide sequences, a combination of 3,7,15 hidden neurons, 10 initial seeds, 5-fold exhaustive cross-validation, training was stopped on the best test set performance. doi:10.1371/journal.pone.0026781.g005 MHC class II data set A large set of over 17,000 HLA-peptide binding affinities was published by Wang et al. [25] containing data from several different human alleles including HLA DR, DP and DQ alleles. For each allele, the predictive performance of various methods was estimated on the similarity reduced (SR) data set, where sequence similarity is minimized in order to avoid overlap between crossvalidation subsets. We preserved the same subsets for our crossvalidation, for easy comparison of the results and predictive performances.
Peptide arrays were synthesized by Schafer-N, Copenhagen, Denmark using a maskless photolithographic technique [41] in which 365 nm light is projected onto NPPOC-photoprotected [42, 43] amino groups on a glass surface in patterns corresponding to the synthesis fields. Details of the technique will be published elsewhere, but briefly, the patterns were generated using digital micromirrors and projected onto the synthesis surface using UVimaging optics. In each layer of amino acids, the relevant amino acids were coupled successively to predefined fields after UVinduced removal of the photoprotection groups in those fields. The couplings were made using standard Fmoc-amino acids activated with HBTU/DIEA in NMP. After coupling of the last Fmocamino acid in each layer, all Fmoc-groups were removed in 20% piperidine in NMP and replaced by NPPOC groups coupled as the chloroformate in DCM with 0.1 M DIEA. The procedure was then repeated until all amino acids had been added to the growing peptide chains. Final cleavage of side protection groups was performed in TFA:1,2-ethanedithiol:water 94:2:4 v/v/v for 2 h at room temperature.
Trypsin data. Peptide arrays were incubated for 30 min at room temperature with 0.1 g/L bovine Trypsin (Sigma T9201) dissolved in 0.1 M Tris/Acetate pH 8.0. After washing in the same buffer containing 0.1% SDS, the slides were washed with deionized water and air-dried. Staining of amino groups exposed by enzyme cleavage was made by incubation the slide for 30 min in 0.1 mg/mL Dylight549-NHS (Thermo Scientific) in 9:1 v/v nmethyl pyrrolidone:0.1M n-methyl morpholine/HCl pH 8 for 10 minutes.
Chymotrypsin. Peptide arrays were incubated for 30 min at room temperature with 0.1 g/L bovine Chymotrypsin (Sigma C4129) dissolved in 0.1 M Tris/Acetate pH 8.0. After washing in the same buffer containing 0.1% SDS, the slides were washed with deionized water and air-dried. Staining of amino groups exposed by enzyme cleavage was made by incubation of the slides for 10 min in 1 mM 5(6)-TAMRA (carboxytetramethylrhodamine, Fluka 21953) activated with 1 eq HBTU, 2 eq DIEA in nmethylpyrrolidone.
Recording of signals from peptide arrays. After incubation with activated fluorochromes, the peptide array slides were washed in the incubation buffer without fluorochrome followed by washings in n-methylpyrrolidone and dichloromethane and airdried. Images of the arrays were recorded using a MVX10 microscope equipped with a MT10_D fluorescence illumination system and a XM10 CCD camera (all from Olympus). The excitation wavelength was 530-550 nm and the emission filter was 575-625 nm. The images were analyzed using the PepArray analysis program (Schafer-N, Copenhagen Denmark).
Data pre-processing. The quantitative peptide data entered by the user is rescaled to be between 0 and 1 before being fed to the neural network. The user is also given the option to apply a logarithmic transformation to the raw data, if its distribution appears to be too squashed towards low values. Outliers deviating more than 3 standard deviations from the average, which after rescaling would produce sparse regions in the spectrum with no data, are set at a value of exactly 3 standard deviations. This procedure produces ideal data for artificial neural network (ANN) training, with all values in the range [0:1] and the bulk of the data in the central region of the spectrum. The parameters for the rescaling function are defined separately on each of the training sets used in cross-validation, and then also applied to rescale their relative test sets.
Subsets for cross-validation. In a n-fold cross-validation, n subsets are created from the complete dataset, and at each step n-1 subsets are used for training and 1 subset for testing. NNAlign offers three alternatives to create the subsets: i) random, splits the data into n subsets randomly; ii) homology clustering, uses a Hobohm 1 algorithm [44] to identify sequences that share an ungapped alignment with more than a specified fraction of matches; iii) common motif clustering, looks for stretches of identical amino acid between pairs of sequences as described by Nielsen et al. [19] . For both methods ii) and iii) similar sequences are grouped together in the same subset, but it is possible to choose to only include one representative for each group and disregard the other sequences from training. In this phase, if the input data contains repeated flanks (as might be the case in peptide array experiments, where linker sequences can be attached at the extremities of all peptides), these flanks are discarded, as they would affect the overlap estimation. If the user reckons that the repeated flanks might contain meaningful biological signal, an option allows retaining them in the training data. Note that in common motif clustering, the motif length is taken as the smallest in the interval of length given by the user. Thus, depending on the selected interval the subsets might be constructed in a different way and that could influence the cross-validated performance.
Neural network training. The neural network training is performed as described by Nielsen et al. [20] . Initially, all network weights are assigned random values. From the current network configuration, the method selects the optimal n-mer core (and potential peptide flanking residues) for each of the peptides within the training set. The network weights are next updated, to lower the sum of squared errors between the observed and predicted score, the cores are redefined based on the new network configuration, and the procedure is iterated.
An ensemble of ANNs is trained on the cross-validation subsets, with architecture parameters specified by the user. The motif length, encoding of flanks and peptide length determine the size of the input layer. If the motif length is given as an interval of values, multiple runs of ANN training are performed on the different lengths, and the length that produces the best cross-validated performance in terms of root mean square error (RMSE) is chosen for the final ensemble. The number of hidden neurons may be specified as a list of multiple values, so that an ensemble of networks is constructed with hidden layers of different sizes. Each architecture is trained multiple times, starting from different initial random configurations, to avoid as much as possible choosing suboptimal solutions producing local minima. Sequences can be presented to the network either with Sparse or Blosum encoding. In Sparse encoding, a vector of length N represents each amino acid, where all values are identical apart from the one representing the observed amino acid. Blosum encoding, on the other hand, takes into account amino acids similarity and partially allows substitutions of similar amino acids while penalizing very dissimilar ones [45] .
Performance measures. Cross-validation allows estimating a method performance without the need of external evaluation data. The subsets reserved as test-sets are run through the network trained in the same cross-validation step, and Pearson's correlation, RMSE and Spearman correlation are calculated between observed and predicted values.
It is possible to use the internal subsets to stop the training phase on the best test set performance in terms of RMSE. In this mode, performance can be estimated in an exhaustive or in a fast way. Exhaustive n-fold cross-validation (CV) consists of a nested CV procedure. At each step, 1 subset is left out as evaluation set, and the remaining subsets are used to generate a network ensemble in an n-1 CV training. In this CV training, the selected network configuration is the one that gives the minimum RMSE on the stopping set. Next the predictions for the evaluation data are estimated as a simple average of the prediction values for each network in the training ensemble. The exhaustive CV procedure adds one level to the cross-validation and increases greatly the running time. In alternative, the fast evaluation skips one nested level by using the same subset for stopping and evaluating performance, for a quicker but likely less accurate performance estimation.
Final network ensemble. With cross-validated ANN training, each network has been evaluated on data not included in the training. The networks can then be ranked by performance, and only the top N for each cross-validation step will be included in the final ensemble, with N specified by the user. The final network ensemble can be downloaded to local disk, and used for predictions on new data by loading it to the NNAlign server submission page.
Sequence motif logo. A list of 100,000 random naturally occurring peptides with length L = motif length+2 * flank length, generated from random UniProt [46] sequences, is presented to the individual networks in the ensemble. For each network, the 1% peptides that obtain the highest prediction scores are used to create a position specific scoring matrix (PSSM) that represents the motif captures by the neural network. Using a Gibbs sampler approach, all PSSMs are aligned to maximize the information content of the combined matrix. This ''offset correction'' step is obtained by repeatedly attempting to shift the starting position of randomly chosen PSSMs, and accepting/rejecting the move according to the conventional Metropolis Monte Carlo probability relation [47] :
Where DI is the change in information content between the new and old offset configuration and T is a scalar that is lowered during the calculation. The process assigns to each PSSM, and to its relative network, an offset value that quantifies the shift distance from other networks. The re-aligned cores from the 1% scoring of 100,000 peptides are finally used to generate a combined sequence logo with the WebLogo program [48] . The offset correction can be skipped if the user chooses to, and in this case the logo is simply created by presenting the list of random peptides to the ANN final ensemble and selecting the 1% peptides that obtain the overall best score. Evaluation data. Additional data not included in the training can be uploaded to the NNAlign Server as an evaluation set. Evaluation data must be a list of peptides, with or without associated values, or a file in FASTA format. In the first case, all peptides are run through the trained network ensemble, and scored accordingly to their best alignment core. If values are provided together with peptides, they are assumed to be target values for validation purposes, and statistical measures between these values and predictions are calculated. In the case a FASTA file is loaded as evaluation set, the sequences therein contained are cut into peptides of length L = motif length+2 * flank length, shifting the starting position of one amino acid at a time. The generated peptides are all fed to the network to identify those that most closely match the motif learned by the ANNs. The results are sorted by prediction value, so that the best candidates are displayed at the top of the list.
Sequence logos were introduced by Schneider et al. [49] as a way to represent graphically the pattern in a set of aligned sequences. The height R i of each column i in the logo is given as the information content in bits of the alignment at that particular position, and for a sufficiently large number of sequences and a 20letter alphabet it is calculated as:  Red blood cell transfusion in the critically ill patient Red blood cell (RBC) transfusion is a common intervention in intensive care unit (ICU) patients. Anemia is frequent in this population and is associated with poor outcomes, especially in patients with ischemic heart disease. Although blood transfusions are generally given to improve tissue oxygenation, they do not systematically increase oxygen consumption and effects on oxygen delivery are not always very impressive. Blood transfusion may be lifesaving in some circumstances, but many studies have reported increased morbidity and mortality in transfused patients. This review focuses on some important aspects of RBC transfusion in the ICU, including physiologic considerations, a brief description of serious infectious and noninfectious hazards of transfusion, and the effects of RBC storage lesions. Emphasis is placed on the importance of personalizing blood transfusion according to physiological endpoints rather than arbitrary thresholds. Red blood cell (RBC) transfusion is commonly required in critically ill patients. Several recent, observational, multicenter studies reported that approximately one third of critically ill patients received a blood transfusion at one time or another during their stay in the intensive care unit (ICU) ( Table 1) . Because of the frequent use of this intervention, it is important for the ICU physician to be aware of recent developments in this continuously evolving field of medicine. In this narrative review, we consider some key aspects of transfusion medicine in the ICU, focusing on aspects relevant to the critically ill patient, including prevalence and reasons for blood transfusion, epidemiology and etiology of anemia in these patients, pathophysiological considerations on tolerance to anemia, and efficacy of RBC transfusion. Safety concerns, including questions of RBC storage and leukoreduction, are then discussed, followed by a proposal for an integrated approach to transfusion decisions and a discussion on economic aspects and alternatives to blood transfusion.
Anemia is common in ICU patients and appears early in the ICU course [1] . In an observational, multicenter, cohort study in Scotland, 25% of patients admitted to the ICU had a hemoglobin level < 9 g/dl [2] . Similar results were reported in the ABC study [3] , in which 29% of patients had a hemoglobin concentration < 10 g/ dl on admission. Even in nonbleeding ICU patients, hemoglobin levels tend to decrease early [3] . This decrease is more pronounced in septic than in nonseptic patients [4] , at least in part because of their inflammatory response; more frequent blood sampling may also contribute.
Interestingly, anemia and the need to restore adequate oxygen delivery (DO 2 ) are the most common indications for transfusion, rather than acute bleeding [3, [5] [6] [7] [8] [9] [10] . Anemia in the critically ill patient is a multifactorial phenomenon that has been compared to the so-called "anemia of chronic illness" [11] . Apart from evident causes of anemia, such as primary blood losses (e.g., trauma, surgery, gastrointestinal bleeding), multiple other etiologies contribute to its pathophysiology and often coexist in the same patient [11] . These include blood losses related to minor procedures or phlebotomy, and hemodilution secondary to fluid resuscitation. Some studies have suggested that blood sampling may average as much as 40 ml/day [3, 4] , but the amount of blood required may decrease with technological developments in analytic methods. Other mechanisms for anemia include an inflammatory response with blunted erythropoietin (EPO) production, abnormalities in iron metabolism, and altered proliferation and differentiation of medullar erythroid precursors [11] . As a consequence, RBC deformability is decreased [12] , whereas RBC adherence to the endothelium is increased, especially in septic patients, potentially leading to microcirculatory impairment and tissue hypoxia [13] .
Tolerance to anemia in healthy subjects and in the critically ill patient Tolerance to anemia is highly dependent on the volume status of the patient, physiological reserve, and the dynamics of the anemia (for example, chronic, such as the anemia of sepsis, versus acute, such as hemorrhagic conditions). Normovolemic anemia is better tolerated than anemia in hypovolemic states (e.g., acute bleeding in trauma patients or surgery) in which cardiac output acutely decreases. In healthy subjects submitted to normovolemic hemodilution, cardiac output increases because of decreased blood viscosity (especially relevant in severe anemia) and increased adrenergic response, allowing tachycardia and increased myocardial contractility. Other phenomena include blood flow redistribution (to heart and brain) and an increased oxygen extraction ratio (reflected by a decrease in mixed venous saturation [SvO 2 ]). These mechanisms allow healthy humans to tolerate severe degrees of normovolemic anemia [14, 15] , although side effects, such as arrhythmias or ST changes, can be observed in extreme cases [16, 17] . The myocardium is the organ at risk in cases of acute anemia in which both tachycardia and increased ventricle contractility may increase myocardial oxygen demand. Because myocardial oxygen extraction is already almost maximal at rest, every increase in myocardial oxygen demand must be accompanied by increased coronary blood flow [18] . This can become problematic in patients with stenotic coronary arteries especially when tachycardia is present, which can decrease diastoledependent left ventricle perfusion. Therefore, in critically ill patients, especially those with heart failure or coronary artery disease (CAD), the myocardium may not tolerate such low hemoglobin levels [19] . In acute myocardial infarction, anemia may worsen myocardial ischemia, generate arrhythmias, and potentially increase infarct size [20] . In patients with acute coronary syndrome or heart failure, anemia increases morbidity and mortality [21, 22] . For these reasons, patients with cardiac problems should be managed with a more liberal approach to transfusion than other patients [23, 24] .
The primary purpose of blood transfusion is to increase DO 2 , which is determined by cardiac output and arterial content of oxygen, the latter being dependent on the hemoglobin level. Hence, blood transfusions can, theoretically at least, limit tissue hypoxia [13, 25, 26] . But does this really happen in clinical practice? It is obvious that RBC transfusions can be lifesaving in situations of acute severe anemia or in bleeding patients in whom RBC administration can increase both oxygen arterial content and cardiac output. However, in the absence of bleeding, the increase in hemoglobin concentration could very well be offset by a decrease in cardiac output because of the increase in blood viscosity associated with a decreased sympathetic response [27, 28] . DO 2 has been shown to increase following RBC transfusion in numerous studies [26] , but not in all [29] .
The effects of RBC transfusion on the relationship between DO 2 and oxygen uptake (VO 2 ) are even more difficult to predict. Some studies reported that VO 2 increased following RBC transfusion, whereas others did not [26] , and variable effects have been reported on tissue perfusion as assessed by gastric mucosal pH or near-infrared spectroscopy (NIRS) [30] . The reasons for these contradictory results lie primarily in the degree of severity of hypoxia preceding the RBC transfusion [31] , which influences the dependency of VO 2 on DO 2 . Methodological problems (imprecision in determination of VO 2 , assessment of global VO 2 instead of regional VO 2 , poor correlation between systemic oxygenation parameters, and oxygenation in the microcirculation [13] ) also may contribute to these discrepancies [31] .
Red blood cell transfusions have been associated with worse outcomes in several populations of patients, including critically ill patients. In a recent systematic review of 45 observational studies reporting the impact of transfusions on patient outcome (mortality, infections, acute respiratory distress syndrome [ARDS]) in populations of trauma, general surgery, orthopedic surgery, acute coronary syndrome, and ICU patients, Marik and Corwin [32] identified RBC transfusion as an independent predictor of death (pooled odds ratio (OR) from 12 studies, 1.7; 95% confidence interval (CI), 1.4-1.9), infectious complications (pooled OR from 9 studies, 1.8; 95% CI, 1.5-2.2), and ARDS (pooled OR from 6 studies, 2.5; 95% CI, 1.6-3.3). In ICU patients, the three studies included in the review (ABC study [3] , CRIT study [5] , and a study by Gong et al. [33] ) consistently showed a statistically significant association of RBC transfusion with mortality.
On the other hand, analysis of data from a multicenter, prospective, observational study of 3,147 patients in 198 European ICUs (the SOAP study) indicated that blood transfusions were not associated with increased mortality by multivariate analysis or propensity matching [34] . In contrast, an extended Cox proportional hazard analysis showed that patients who received a transfusion in fact had a better survival, all factors being otherwise equal. An increased rate of transfused leukoreduced RBCs reported in this study (in which 76% of centers routinely used leukoreduced RBCs) could perhaps account for the differences between the earlier ABC study [3] (in which 46% of centers used leukodepleted blood most of the time) and the SOAP study [34] . It also is possible that transfusion thresholds have become so low that the benefits of blood transfusion outweigh the risks.
In patients with acute coronary syndrome, several studies have shown poorer outcomes, including increased mortality, in transfused groups compared with nontransfused patients after adjustment for potential confounders [21, [35] [36] [37] ; similar findings have been reported in patients who undergo percutaneous coronary interventions (PCI) [38] . However, although still controversial, RBC transfusions may be useful in subgroups of elderly patients with acute myocardial infarction [39] or patients with ST elevation myocardial infarction (STEMI) [21] .
Patients who undergo cardiac surgery seem to have worse outcomes when transfused, including higher mortality [40, 41] , increased occurrence of postoperative infections [41, 42] , increased time on mechanical ventilation [40, 43] , and higher incidence of postoperative acute kidney injury [41, 44] .
Other studies have reported that trauma patients [45, 46] , including those with burns [47] , may have increased mortality rates associated with receiving blood transfusions. In contrast, RBC transfusion has been reported to be associated with improved outcomes in patients with traumatic brain injury or subarachnoid hemorrhage [48, 49] . In the early resuscitation of patients with severe sepsis, implementation of a therapeutic protocol that included RBC transfusion to obtain a hematocrit > 30% was associated with a significant reduction in hospital mortality [50] .
These results should be interpreted with caution, because most of these data come from observational, retrospective studies, which are subject to numerous biases and sometimes control poorly for confounders, despite the use of various statistical tools, such as logistic regression [51] . It is clear that analyses should not include only admission data. For example, in a welldefined patient population, such as after cardiac surgery, patients who develop gastrointestinal bleeding and require a blood transfusion have a worse prognosis, which is not necessarily the result of the blood transfusion. It is of paramount importance that all risks factors are taken into account. Ruttinger et al. [52] illustrated this point very well. In a series of more than 3,000 surgical patients, these authors showed by using a limited multivariable analysis that transfusions were associated with a worse outcome, but a more complete analysis cancelled out this statistical observation.
The reasons for the apparent worse outcome of transfused compared with nontransfused critically ill patients may be found in several detrimental effects of transfused blood, globally referred to under the acronym "Non-Infectious Serious Hazards Of Transfusion" or NISHOT ( Table 2 ) [53] . These include, among others, deleterious effects on the immune system (transfusion-related immunomodulation or "TRIM") or on the cardiopulmonary system, e.g., transfusion-related acute lung injury ("TRALI") [54] or transfusion-associated circulatory overload ("TACO"); the latter is currently the leading reported cause of transfusion-associated mortality [55] . These effects may be enhanced by pathologic conditions (e.g., sepsis) in which the microcirculation is impaired [56] and/or when the RBCs have been stored for some time.
During storage, RBCs undergo a series of biological and biochemical changes collectively referred to as "the storage lesion" [57] . This includes intracellular changes (progressive depletion of 2,3-diphosphoglycerate [2,3-DPG] with increased affinity of hemoglobin for oxygen, depletion of ATP), membrane changes (membrane vesiculation, morphological changes eventually leading to irreversibly deformed spheroechinocytes, lipid peroxidation and increased expression of phosphatidylserine, decreased deformability), and changes in the storage medium (decreased pH, increased potassium, release of proinflammatory cytokines). These stored RBCs also have an increased tendency to adhere to endothelium and could promote vasoconstriction; the stored RBCs act as a "sink" for nitric oxide [58] . Some animal studies [13] have shown deleterious effects of old RBCs on the microcirculation (potentially leading to tissue hypoxia and organ dysfunction). A human study found an inverse correlation between the age of transfused RBCs and maximal change in gastric mucosal pH, but these findings were challenged in subsequent studies [59] [60] [61] .
The clinical consequences of storage lesions are still not clear. A recent review of the literature [57] identified 24 studies that address the effects of RBC length of storage on clinical (mortality, infections, length of stay, length of mechanical ventilation) or physiological (microcirculation, gastric mucosal pH) endpoints. Some studies found associations between the age of transfused RBCs and poorer outcomes, whereas others did not. Overall, no clear detrimental effect of RBC age could be identified; however, definitive conclusions are difficult to obtain because of numerous statistical limitations and biases inherent to the study designs [51, 62] . Several, large, randomized, controlled trials in adult ICU and cardiac surgery patients are currently ongoing to address the clinical relevance of RBC storage. In the multicenter, double-blind prospective ABLE (Age of Blood Evaluation) study [63] , adult patients admitted to the ICU are randomly assigned to receive leukoreduced RBCs stored for less than 7 days or issued according to standard procedure (expected average storage time of 19 days). The primary endpoint of this study is 90-day all-cause mortality. The target number of patients is 2,510 (for an expected improvement in primary endpoint greater than 5%) with an anticipated completion date by April 2013. The Red Cell Storage Duration Study (RECESS) is a multicenter, randomized study in patients (age 12 years and older) who undergo complex cardiac surgery and are likely to require RBC transfusion [64] . Patients who need transfusion are randomized to receive RBCs stored for ≤ 10 days or ≥ 21 days. The primary endpoint of this study is the change in the Multiple Organ Dysfunction Score (MODS) from baseline to day 7, with secondary outcomes including all-cause 28-day mortality. The target number of patients is 1,832, and the anticipated completion date is September 2013.
The results of these trials, especially if older blood appears to be harmful, could have important logistic implications for blood banks [65, 66] .
Many of the adverse effects associated with the transfusion of allogeneic RBCs have been shown to be related to the infusion of white blood cells (WBCs) present in the blood product. Leukoreduction is a process in which WBCs are reduced in number through centrifugation or filtration [67] . This process allows removal of approximately 99.995% of WBCs, but several thousand leukocytes (0.005% of a 500 ml blood unit) may still be present in the processed blood [67] ; hence, the word "leukoreduction" is better than "deleukocytation." The beneficial effects of this process include decreased [67] , and possibly decreased lung injury, such as TRALI. Moreover, prestorage leukoreduction, in which WBC removal occurs before RBC storage, avoids the need for a leukodepletion filter during transfusion [67] (but a 170-200-μm filter still needs to be incorporated into the intravenous blood line).
In several studies, prestorage leukoreduction decreased RBC storage lesions, with fewer immunomodulating properties [68] and less adhesion of stored RBCs to the endothelium [69] . A clinical benefit of leukoreduction is still somewhat controversial, particularly in the critically ill patient where no randomized, controlled trial has been performed [70] . In a before-after study of 14,786 patients who underwent cardiac surgery, repair of hip fracture, or who required intensive care after surgery, there was a 1% decrease in mortality rate associated with the implementation of universal leukoreduction [71] . In a recent meta-analysis of nine RCTs involving 3,093 surgical patients, the use of leukoreduction significantly reduced the odds of postoperative infection (summary OR, 0.522; 95% CI, 0.332-0.821; p = 0.005) [72] . This observation had been suggested in a previous meta-analysis [73] but has been challenged by another recent meta-analysis [74] . Nevertheless, leukoreduction makes sense, and many countries have adopted it as routine, even though costs are elevated. In Europe, at the time of the SOAP study in 2002, 76% of centers reported using leukodepleted blood routinely [34] , whereas an earlier study performed in the same countries reported lower rates [3] .
Classically, the decision to transfuse is driven by arbitrary "triggers" (hemoglobin level) rather than clinical or physiologic findings. Data from the CRIT study [5] , in which there was little evidence that age or comorbidities significantly influenced transfusion practice, tend to support this view.
Current recommendations for RBC transfusion [75, 76] are mainly based on the famous "TRICC" (Transfusion Requirements In Critical Care) trial in which patients assigned to a restrictive transfusion strategy (transfusion if hemoglobin level < 7 g/dl) had similar 30-day mortality rates (and even lower mortality in subgroups with APACHE II < 20 and patients younger than age 55 years) than patients transfused according to a more liberal strategy (transfusion if hemoglobin level < 10 g/dl) [77] . In cardiac surgery patients, the recent randomized, monocenter "TRACS" (Transfusion Requirements after Cardiac Surgery) trial, which compared a restrictive to a liberal strategy (transfusion when hematocrit < 24% or < 30%, respectively), reported no difference in the primary endpoint (composite of 30-day mortality and morbidity [cardiogenic shock, ARDS, acute kidney injury]) between the groups [78] .
However, it is quite clear there is no "magic" hemoglobin or hematocrit trigger, and for the same level of hemoglobin, some patients will do well, whereas others will not. Thus, the decision to transfuse a patient should be individualized, taking into account several factors, including signs and symptoms of tissue hypoxia (angina pectoris, cognitive dysfunction diagnosed by neuropsychological tests, or increased P300 latencies [79] [80] [81] ), increased blood lactate levels [82] , or electrocardiographic changes suggestive of myocardial ischemia.
Indirect measures of oxygenation, such as a decreased SvO 2 or central venous oxygen saturation (ScvO 2 ), also may be considered [82] . For example, in a study of early goal-directed therapy in patients with severe sepsis or septic shock admitted to an emergency department, a decrease in ScvO 2 < 70% initiated a therapeutic intervention, including fluid resuscitation, inotropes, vasopressors, and RBC transfusion to increase hematocrit to > 30% [50] . Use of a decreased ratio of cardiac index to oxygen extraction (CI/EO 2 ratio) may be better, because this parameter also reflects the cardiac response to anemia [83] .
The costs of blood transfusion are particularly complex to assess because of the many factors that have to be taken into consideration (blood collection and screening for pathogens; blood component processing, including leukoreduction, storage, transport to the transfusion facility; administration of blood to the patient; management of potential short-and long-term transfusion-related side effects) [84] . The subtype of the blood unit also may play a role because some products, such as CMV-negative or autologous units, are costlier than classical allogeneic RBCs. Consequently, studies in this field have given extremely varied results, which are not easily comparable.
Evidence has shown increased costs of RBC transfusion over time [85] , related to various factors, including (but not limited to) use of leukoreduction and more sophisticated methods for pathogen detection, such as nucleic acid testing (NAT) [84] . For example, a study in Canada evaluated the mean societal cost of one allogeneic RBC unit at 264.81 US$, twice the cost estimated 7 years earlier [86] . Generally, these reported values are probably underestimated, and some have calculated that the cost of blood to society could in fact be twofold higher [84] .
Because of limited availability, costs and safety concerns related to blood transfusion, several strategies to reduce blood transfusions can be considered in addition to increasing transfusion trigger thresholds. These include approaches to reduce blood losses, for example use of antifibrinolytic agents, such as tranexamic acid or epsilon-aminocaproic acid (EACA) and techniques of cell salvage during surgery; also, the use of small volume sample tubes can limit the blood losses related to sampling for laboratory studies. In a meta-analysis of 9 randomized controlled trials [87] , subcutaneous administration of recombinant erythropoietin (EPO) in critically ill patients was shown to be associated with decreased transfusion rates, but this was not associated with improved mortality (except possibly in a subgroup of trauma patients [88] ). Concerns also have been raised about potentially increased rates of deep vein thrombosis [88] . The development of artificial oxygen carriers is under investigation, but these have their own problems [89] . Further research is needed to improve these alternative strategies.
RBC transfusion can be lifesaving. During the past two decades, however, safety concerns have emerged, with suggestions that morbidity and mortality may be increased in patients who receive blood transfusions. Therefore, the decision to transfuse should be individualized, based on a rational approach and taking into account physiologic variables in addition to the hemoglobin value. This strategy, along with the use of alternatives whenever possible to limit bleeding, should limit unnecessary exposure to RBCs.
Authors' contributions CL drafted the manuscript. The manuscript was revised for intellectual content by JLV. Both authors read and approved the final manuscript. Respiratory support by neurally adjusted ventilatory assist (NAVA) in severe RSV-related bronchiolitis: a case series report BACKGROUND: Neurally adjusted ventilatory assist (NAVA) is a new mode of mechanical ventilation controlled by diaphragmatic electrical signals. The electrical signals allow synchronization of ventilation to spontaneous breathing efforts of a child, as well as permitting pressure assistance proportional to the electrical signal. NAVA provides equally fine synchronization of respiratory support and pressure assistance varying with the needs of the child. NAVA has mainly been studied in children who underwent cardiac surgery during the period of weaning from a respirator. CASE PRESENTATION: We report here a series of 3 children (1 month, 3 years, and 28 days old) with severe respiratory distress due to RSV-related bronchiolitis requiring invasive mechanical ventilation with a high level of oxygen (FiO(2 )≥50%) for whom NAVA facilitated respiratory support. One of these children had diagnosis criteria for acute lung injury, another for acute respiratory distress syndrome. Establishment of NAVA provided synchronization of mechanical ventilatory support with the breathing efforts of the children. Respiratory rate and inspiratory pressure became extremely variable, varying at each cycle, while children were breathing easily and smoothly. All three children demonstrated less oxygen requirements after introducing NAVA (57 ± 6% to 42 ± 18%). This improvement was observed while peak airway pressure decreased (28 ± 3 to 15 ± 5 cm H(2)O). In one child, NAVA facilitated the management of acute respiratory distress syndrome with extensive subcutaneous emphysema. CONCLUSIONS: Our findings highlight the feasibility and benefit of NAVA in children with severe RSV-related bronchiolitis. NAVA provides a less aggressive ventilation requiring lower inspiratory pressures with good results for oxygenation and more comfort for the children. Neurally adjusted ventilatory assist (NAVA) is a new method of assisted ventilation that can be used for children regardless of weight and age. This ventilation mode is controlled by diaphragmatic electrical signals through a gastric tube with specific electrodes on its surface. The collected electrical signals allow synchronization of ventilation to spontaneous breathing efforts of a child, as well as providing pressure assistance proportional to the electrical signal and thus to the output of the child's respiratory centers.
NAVA has mainly been studied in children who underwent cardiac surgery [1] [2] [3] during the period of weaning from a respirator. We report here a series of 3 children with severe respiratory distress due to respiratory syncytial virus (RSV) bronchiolitis for whom NAVA facilitated respiratory support.
Our unit is a 12-bed tertiary care university hospital pediatric intensive care unit. Recruitment is both medical and surgical. NAVA has been used in our unit for weaning children from a respirator who were operated * Correspondence: jeanmichel.liet@chu-nantes.fr † Contributed equally 1 Unité de Réanimation Pédiatrique, Hôpital Mère-Enfant Faïencerie, CHU de Nantes, 38 Boulevard Jean-Monnet, 44093 Nantes, France Full list of author information is available at the end of the article on for congenital heart disease. The effectiveness of NAVA in these children led us to gradually expand the indications. We report a series of 3 children with severe respiratory distress due to RSV bronchiolitis for whom NAVA was used. The local ethics committee (groupe nantais d'éthique dans le domaine de la santé [GNEDS]) considered our report as non-interventional data research. The parents of all three children gave their written consent for publication. Starting NAVA requires the initial correct positioning of the "NAVA" gastric tube. This commercially available feeding tube equipped with sensors (Edi catheter, Maquet Critical Care, Solna, Sweden) permits the recording of electrical activity of the diaphragm (Edi) via a Servo-I Ventilator (Maquet Critical Care, Solna, Sweden) using a standardized method [4] . Settings are relatively simple and include positive end-expiratory pressure (PEEP), fraction of inspired oxygen (FiO 2 ), and level of NAVA assistance.
The Edi was multiplied was multiplied by the NAVA level to adjust the pressure assistance delivered to the child. The delivered pressure is equal to: NAVA level × (Edi max -Edi min) + PEEP. In clinical practice we usually started with a NAVA level of 1 cm H 2 O/μV that may have required adjustment if the Edi max signals deviated from a range between 5 and 20 μV. If the Edi signals turned out to be consistently greater than 20 μV, we increased the NAVA level until they are within this range. In the three reported cases, we did not need to do so.
During NAVA, the ventilator is triggered when the deflection in the Edi curve exceeds 0.5 μV. The assist is cycled-off when the Edi decreases to 70% of its peak value.
We assume that pressure support, which is pneumatically triggered, should remain a means of backup ventilation in case the Edi signal cannot be collected (e.g. if the child removes his/her Edi catheter). Therefore, we set the trigger of this pressure support high enough (typically 0 to -5 cm) so that this backup ventilation did not compete with NAVA ventilation.
We measured respiratory parameters (FiO 2 , tidal volume, mean airway pressure, peak inspiratory pressure, respiratory rate, and Edi max) directly from data exported from the respirator. Nurses recorded vital signs and SpO2. The oxygenation saturation index, OSI = (FiO 2 × mean airway pressure)/SpO 2 , was used to provide a non-invasive method of oxygenation assessment. This index can be used for the diagnosis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in children when SpO 2 values are ≤ 97% [5] . Diagnosis of ALI and ARDS required an acute onset of the process, bilateral infiltrates on a chest radiograph, no evidence of left atrial hypertension, and OSI > 6.5 (ALI) or > 7.8 (ARDS). Blood samples were also analyzed to provide blood pH and PCO 2 values.
Shemsy was one month old (3.8 kg) without any particular risk factors. Her parents referred her to the emergency room because of grunting and hypotonia. She had rhinitis for several days with a cough for 48 hours following a viral contamination 3 days prior. She presented with apnea, desaturation, bradycardia and altered consciousness. She was intubated and ventilated immediately, and then transferred to intensive care.
Ten hours later, respiratory parameters were as follows: synchronized intermittent mandatory ventilation (SIMV) with a tidal volume (VT) of 20 ml (5 ml/kg); rate, 30/min; PEEP, 5 cm H 2 O; and FiO 2 , 50%. Measured parameters (stable for 2 hours) included a SpO 2 of 91%, a mean airway pressure of 10 cm H 2 O, and a peak inspiratory pressure of 30 cm H 2 O (other parameters are also shown in Table 1 ). The OSI was 5.5. A chest X-ray showed poorly ventilated lungs with diffuse infiltrates. Since the child was agitated, the options for care were to increase sedation, or to attempt ventilation using NAVA. A brief test was undertaken to validate the use of NAVA, which proved successful. We chose to commence NAVA after a short period of decreased sedation (morphine was decreased to 8 μg/kg/h). Initial NAVA settings were PEEP, 5 cm H 2 O; NAVA level, 1 cm H 2 O/μV; and FiO 2 , initially 50% was then decreased by nurses to SpO 2 > 90%.
We observed a dramatic decrease in inspiratory pressure with a reduced requirement for oxygen (Figures 1  and 2 ). Within several minutes, the child's breathing became much more harmonious and smoother, while her respiratory parameters showed large variations from one cycle to another. SpO 2 was96%, mean airway pressure was 6 cm H 2 O, peak inspiratory pressure was 10 cm H 2 O, and minute volume was 0.6 l/min. Twelve hours later, FiO 2 was decreased to 21% with a mean airway pressure of 6 cm H 2 O. Detailed ventilatory parameters are reported in Table 1 . Since respiratory parameters were very low (Peak inspiratory pressure < 12 cm H 2 O with FiO2 < 25%) and blood gas values was normal, we extubated the child (10:30). She needed nasal continuous positive airway pressure for 3 days after which she was able to leave intensive care. Tracheal aspirate was positive for RSV and Streptococcus pneumoniae.
Matteo was 3 years old (14 kg), he was prematurely born (birth weight 1650 g) and he was mechanically ventilated in the neonatal period during 10 days for pulmonary hemorrhage.
He was recently hospitalized because of subcutaneous emphysema, with signs of acute respiratory failure from RSV infection. Because of an increased need for oxygen, he was intubated and ventilated with an FiO 2 of 100%, and PEEP was 3. A chest X-ray did not show pneumothorax that could be drained. Tracheal aspirate was positive for RSV and Hemophilus influenzae. A few days later, because of emergence of an alveolar syndrome associated with persistence of subcutaneous emphysema, he was transferred to our unit for possible extracorporeal membrane oxygenation (ECMO). Upon arrival, he had an oxygenation saturation index of 9.4 as well as with the other criteria for ARDS (bilateral infiltrates on a chest radiograph and no evidence of left atrial hypertension). Respiratory parameters were an SIMV with a VT of 85 ml (6 ml/kg), the rate was 25/ min, PEEP was 6 cm H 2 O, and FiO 2 was 70%. SpO2 was 89%, mean airway pressure was 12 cm H 2 O, peak inspiratory pressure was 28 cm H 2 O, and minute volume was 2 l/min. Venous blood gas showed a pH of 7.32, and a PvCO 2 of 53 Torr (7.1 kPa). Because of a slight improvement, the child was not treated by ECMO. He underwent fibroscopy to eliminate the diagnosis of foreign body inhalation, which would have explained the subcutaneous emphysema. Forty-eight hours later, because of an increase in cough, cutaneous emphysema worsened and became diffuse (cervico-thoraco-abdominal) despite the reduction in PEEP to 4 cm H 2 O (FiO 2 60%). We decided to use NAVA to improve the synchronization of mechanical ventilation with the child's spontaneous breathing. Sedation was reduced, midazolam was decreased to 80 μg/kg/h and sufentanil to 0.3 μg/kg/h. The child became alert. Initial NAVA settings were a PEEP of 5 cm H 2 O, NAVA level was 1.2 cm H 2 O/μV, and FiO 2 was 60%.
This change reduced the requirement for oxygen and normalized blood gases ( Table 2 ). The next day, the subcutaneous emphysema started to decline ( Figure 3 ). Peak inspiratory pressure was between 10 and 19 cm H 2 O with an FiO 2 of 50% for a SpO 2 of 90%. The child's level of distress was scored with the modified COMFORT scale [6] and it ranged between 11 and 14 (adequately sedated, as confirmed by the child). This clear improvement allowed us to reassign the child to the original hospital 48 hours after initiation of NAVA. NAVA was continued at the second facility, permitting extubation 3 days later.
Leane was 28 days old (3 kg) and was born at term. She had a 2-year-old brother with bronchiolitis. After an episode of rhinorrhea, she presented with feeding difficulties and was referred to the emergency room of a local hospital. Four hours later, she progressively presented with low oxygen saturation, tachypnea, and chest retraction. She was placed under nasal CPAP with 30% FiO 2 , and then transferred to our unit for severe RSV bronchiolitis. She was intubated on arrival because of clinical signs of respiratory distress and collapse. Although we suspected concomitant bacterial pneumonia because of a major inflammatory syndrome, we did not have bacteriological confirmation. Her respiratory status deteriorated rapidly. The OSI was 6.8 (with other diagnosis criteria for acute lung injury). Her need for oxygen increased rapidly with bilateral infiltrates on a chest radiograph. We chose to use NAVA after a short period of decreased sedation (morphine was decreased to 8 μg/kg/h). Initial NAVA settings were a PEEP of 5 cm H 2 O, NAVA level was 1 cm H 2 O/μV, and FiO 2 was initially 60% and was then adjusted by a nurse for a SpO 2 > 90% and < 98%. Six hours later, FiO 2 was 35%, while ventilatory pressures were lower than before starting NAVA ( Table 3) . As in the 2 other cases, ventilatory parameters were highly variable (Figure 4) , while chest movements of the child were smooth as if the child was not mechanically ventilated. Twenty-four hours after starting NAVA, FiO 2 was 21%. The modified COMFORT scale ranged between 7 and 13.
At 36 hours of NAVA ventilation, the child was accidentally extubated during coughing. She immediately presented marked signs of respiratory distress (Silverman score: 7/10). We then used noninvasive ventilation with the NAVA option. Edi max values were initially very high (80 μV) and gradually decreased over 1 hour after the establishment of noninvasive ventilation. Thereafter, nasal continuous airway pressure was applied and the child left the intensive care unit 3 days later.
As in many pediatric intensive care units, our rate of intubation of children hospitalized for bronchiolitis is tidal volume became much more variable from one cycle to another. Middle panels: One of the most remarkable changes observed with switching to the NAVA mode was the immediate reduction in the mean airway pressure (C) and in the peak airway pressure (D) which decreased from 30 to 10 cm H 2 O. Bottom panels: After starting NAVA, the respiratory rate became very variable over time (E). From a mandatory frequency set at 30 breaths per minute, respiratory rate increased to 40 and 60 breaths per minute. Clinically, the breathing became easier with harmonious chest movements. (F) Edi max that it is the sum of inspiratory Edi and Edi min corresponds to the peak of electrical activity of the diaphragm. In SIMV, this activity is depressed, and in NAVA, the inspiratory Edi (Edi max -Edi min) drives ventilation. Abbreviations: FiO 2 , fraction of inspired oxygen; Edi, electrical activity of the diaphragm; SIMV, synchronized intermittent mandatory ventilation. [5.7] Data in bold are prescribed settings. Other data are measured parameters that depend both on the ventilatory settings and the respiratory status of the child. * Data expressed in parentheses represent measurements that were very variable over time, and hence an estimate of the measured parameter is provided. Abbreviations: SIMV, synchronized intermittent mandatory ventilation; NAVA, neurally adjusted ventilatory assist; PEEP, positive end-expiratory pressure; FiO 2 , fraction of inspired oxygen; Edi, electrical activity of the diaphragm Figure 2 Screenshot with trends over 24 hours (case 1). In the window untitled "Courbes de tendances" (Trend curves), three panels report trends over 24 hours of peak inspiratory pressure (cm H 2 O), respiratory rate (resp/mn), and minute volume (l/min). On the right of these panels, the values of these ventilatory parameters were collected to the vertical bar (at 18:57 while the child was not receiving NAVA). The downward vertical arrow indicates the switch from SIMV to NAVA (at 20:30). Outside the window untitled "Courbes de tendances", on the right of the scrennshot, ventilatory parameters were collected the next day at 10:15 while the child was receiving NAVA. The upper pannel showed a decrease in peak inspiratory pressure after the switch of ventilation. The middle pannel showed the extreme variability of the respiratory ratein NAVA (the white area under the curve corresponds to the mandatory respiratory rate, while the black area corresponds to the spontaneous respiratory rate). The lower pannel showed minute volume that remained unchanged. When comparing values of ventilatory parameters in SIMV (at 18:57) with those in NAVA (next day at 10:15), peak inspiratory pressure decreased from 29 to 6 cm H 2 O, mean airway pressure decreased from 10 to 4 cm H 2 O, spontaneous respiratory rate varied from 0 to 29 breaths/min. Abbreviations: SIMV, synchronized intermittent mandatory ventilation; PEP, positive end-expiratory pressure; P crête, peak inspiratory pressure; P moyen, mean airway pressure; FR spont, spontaneous respiratory rate; F resp, respiratory rate; VM, minute volume; FiO 2 , fraction of inspired oxygen. low (< 20%) [7] . However, the severity of lung disease in some children still necessitates invasive mechanical ventilation. Our unit recruitment is both medical and surgical, and we therefore acquired expertise in NAVA through the weaning of children treated after cardiac surgery. We then expanded the indications of this mode of ventilation in children with severe respiratory disease. The positive results in the present study may be explained by the specific selection of children to whom we applied NAVA. First, there should be no specific contraindication to the placement of a nasogastric tube. Second, there should be no alkalosis or hypocapnia (in such cases there would not be sufficient diaphragmatic electrical activity). During alkalosis, the ventilatory brain centers no longer stimulate the diaphragm, and the respirator works on a backup mode, which is simply conventional ventilation. Finally, the level of sedation should not be too high, so that it does not depress brain centers that control breathing. If the sedation is too high, the Edi signal cannot be collected and the respirator works again in a backup mode. Likewise, neuromuscular connection from the respiratory center to the diaphragm must be intact. For example, NAVA cannot be used in case of post-surgical lesion of the two phrenic nerves [3] or diaphragmatic paralysis secondary to botulism [8] .
The main benefit observed in our cases was an improvement in oxygenation associated with a normalization of blood pH. This was achieved with marked decrease in peak airway pressure. This effect has been previously found in crossover studies reporting NAVA in weaning children from a respirator who were operated on for congenital heart disease and comparing NAVA with pressure support [1] [2] [3] . Clinically, breathing becomes easier with harmonious chest movements. In one of our cases, NAVA was very effective for ventilation in a child who had both ARDS and extensive cutaneous emphysema. The excellent synchronization of mechanical ventilation with the spontaneous breathing of the child improved oxygenation without aggravating the emphysema.
Several factors could explain the beneficial effects observed with NAVA in these three children who had severe respiratory distress. First, asynchrony is associated with increased morbidity, a longer duration of ventilation, and a longer hospital stay [9] [10] [11] . There are few pediatric data published regarding the adverse effects of long-term asynchrony between mandatory ventilation and the respiratory efforts of children, but it has been shown that infant-ventilator asynchrony (both inspiratory and expiratory asynchrony) may affect more than 50% of the total breath duration [12] . Second, NAVA provides assistance in synchronization, as well as in pressure assistance in Recording began with the establishment of NAVA. Upper panels: After starting NAVA, (A) FiO 2 gradually decreased to 35%, and (B) tidal volume became variable from one cycle to another ranging from 5 to 30 ml. Middle panels: Variability was also observed in measurements of pressure: (C) mean airway pressure, (D) peak inspiratory pressure. Bottom panels: (E) After starting NAVA, the respiratory rate became very variable over time. (F) Edi max corresponds to the peak of electrical activity of the diaphragm. The highest Edi values (recorded between 19:00 and 22:00) drove assistance with the highest pressures. A decrease in signal intensity was accompanied by a decrease in pressure, corresponding to an improvement in lung function. The requirement for oxygen decreased at the same time. Abbreviations: FiO 2 , fraction of inspired oxygen; Edi, electrical activity of the diaphragm; NAVA, neurally adjusted ventilatory assist.
proportion to the measured electrical activity of the diaphragm. This helps to limit the periods of insufficient assist delivery that could induce respiratory muscle fatigue with increased oxygen consumption, and periods of overassistance that can generate intrinsic PEEP with an inadequate increase in intrathoracic pressure [13] . NAVA can also prevent air swallowing and gastric distension by optimization of patient-ventilator synchrony [14] . Third, it is likely that NAVA can help clinician avoiding inappropriate ventilator settings that overload (or underload) respiratory muscles, preventing recovery. Finally, improvements in pulmonary gas exchange, systemic blood flow and oxygen supply to tissues which have been observed when spontaneous breathing has been maintained during mechanical ventilation with clinical improvement in the patient's condition [15] , are assumed to occur in NAVA, when breathing efforts by the patient and the initiated breaths are in synchrony.
Cost effectiveness studies are required, as NAVA requires probes that are single-patient use. It is possible that improving comfort provided by better synchronization between spontaneous breathing and mechanical ventilation could reduce the sedation and thus shorten duration of ventilation.
Based on three individual cases, NAVA appears to be a useful mode for weaning from a respirator and is an effective alternative treatment for children with severe respiratory distress. NAVA provides a respiratory support that is in harmony with the spontaneous efforts of breathing, allowing a decrease in inspiratory pressures and oxygen needs. Larger studies are required to compare NAVA with conventional respiratory support in children with various etiologies of respiratory distress. Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale BACKGROUND: Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products. PRINCIPAL FINDINGS: We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems. CONCLUSIONS: This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health. The development of high throughput techniques to identify the targets of cellular immunity on a proteome-wide scale will facilitate the development of vaccines against complex diseases. Almost all vaccines currently licensed for human use rely on antibody responses against the target pathogen. None are designed to induce protective T cell response, yet T cell responses are implicated as critical in protection against many pathogens, especially those with an intracellular stage such as the causative agents of malaria, leishmaniasis and Chagas disease [1, 2] . CD4 + T cells also play a key role in enhancing the pathogen-specific antibody responses [3] . The elucidation of the genome, proteome and transcriptome of important human pathogens, including the Plasmodium parasite, has provided a wealth of data that can potentially be mined to identify, on a proteome-wide scale, novel target antigens recognized by T cells and antibodies. However, how to effectively mine this data has proved challenging. In particular, technologies such as conventional protein expression methodologies which are well established on an individual antigen basis often cannot be translated directly to a whole proteome scale.
An important achievement of the scientific community, therefore, has been the development of technologies that allow the high throughput expression of recombinant proteins. These In Vitro Transcription and Translation systems (IVTT) or cell-free systems offer several advantages over traditional cell-based expression methods and are suitable for high throughput strategies due to reduced reaction volumes and process time [4, 5, 6] . Additional advantages include easy modification of reaction conditions for improving production of complex proteins, decreased sensitivity to product toxicity, and high yield. Importantly, cell-free systems have proved capable of generating proteins from complex parasites that have been difficult to produce in traditional cell-based systems, such as Plasmodium proteins [7, 8, 9, 10] . The most efficient expression to date has been achieved with an E. coli cell-free system which has yielded more than 93% efficiency of expression with a panel of 250 P. falciparum (Pf) proteins [11] . Eukaryotic based cell-free systems are also available with wheat germ, rabbit reticulocytes and insect cells. The eukaryotic lysate is considered by some to provide a better platform for production of complex proteins particularly with regard to post-translational modifications. Recently, up to 75% efficiency of production of Pf proteins in the wheat germ system has been reported [12, 13] but this is still less efficient than that observed with the E. coli system [11] .
The combination of these tools with large-scale cloning strategies, such as recombinatorial cloning, allow the generation of complete proteomes in vitro for multiple purposes. Several reports have shown the application of these tools for identifying antibody targets of complex diseases using protein arrays [11, 14, 15] . Such studies have established the feasibility of identifying, from a set of thousands of antigens, the most immunogenic targets of antibody responses which may correlate with protection as indicated by clinical disease stage classification [16] or virus neutralising activity [17] .
More challenging is the high throughput elucidation of T cell targets which is clearly of importance for those diseases where cell mediated immunity is implicated in protection, as well as for antibody mediated immunity where T cell help would be beneficial. Recently, the use of the E. coli based cell-free system (Rapid Translational system, RTS) for profiling of CD4 + T cell responses to vaccinia virus in humans was reported [18] . In that study, 180 predicted open reading frames of the vaccinia genome were expressed in the RTS system and unpurified RTS reaction products were tested for recognition by vaccinia virus-enriched T cell lines derived from 11 Dryvax smallpox vaccines, using 3 Hthymidine proliferation assays. Another study reported the use of IVTT products affinity purified on protein G-conjugated carboxylate microsphere beads to stimulate proliferative responses of polyclonal short-term T cell lines from cattle immunised with purified A. marginale outer membranes [19] . Of note, both studies used T cell lines, rather than unpurified splenocytes or bulk peripheral blood mononuclear cells, and neither reported a systematic evaluation of the T cell screening process.
Herein, we present a series of in vitro and in vivo experiments designed to demonstrate proof-of-concept for high throughput identification of antigens recognised by T cell responses in human or murine systems, using IVTT products unpurified, affinity purified through nickel resin or magnetic beads, or absorbed in beads to enhance the cell mediated immunogenicity by promoting dendritic cell uptake [20, 21] . IVTT produced antigens of FluM and FluHA from Influenza A virus [22] , CMVpp65 from Cytomegalovirus [23] and EBNA3A from Epstein-Barr virus [24] were assayed using bulk human PBMC for T cell recognition. Additionally, Plasmodium yoelii circumsporozoite protein (PyCSP) [25] IVTT products were assayed for antigenicity in vitro using splenocytes from PyCSP-immunized mice, and for immunogenicity in vivo as assessed by capacity to boost a PyCSP-specific immune response primed by plasmid DNA. In both human and murine systems, T cell responses were evaluated by IFN-c ELISpot and Cytometric Bead Array (CBA) cytokine assays. Robust IFN-c, TNF-a and IL-10 responses were detected, and IVTT products affinity purified through nickel resin or magnetic beads were highly effective. Considering the loss associated with purification, the nickel resin method proved the most optimal.
Coding sequences were expressed by coupled transcriptiontranslation in the E. coli cell-free IVTT system. An aliquot of each reaction mixture was analysed by SDS-PAGE and western blot, followed by chemiluminescence of the membrane to visualize the products formed. All four viral (FluHA, FluM, CMVpp65, EBNA3) and one parasite (PyCSP) recombinants could be produced using the manufacturer's recommended conditions. The average yield of each full-length recombinant, detected using an anti-HA antibody directed against the C-Terminal HA tag, was as follow: FluM, 1.15 mg/ml; FluHA, 0.73 mg/ml (two moles of HA tag per molecule of FLU-HA); CMVpp65, 0.7 mg/ml; EBNA3A, 4 ng/ml; and PyCSP, 0.6 mg/ml ( Figures 1A and B and Figure  S1A ). Western blot analysis using an antibody directed against the N-Terminal 6xHIS tag identified the presence of partial products for each viral recombinant (PyCSP not tested) ( Figure S1B ). Solubility analysis showed that a high proportion (at least 70%) of each recombinant was insoluble. A series of optimization studies including kinetics of expression (3 hr to 6 hr), reaction temperature (16uC, 25uC, 30uC), speed (stationary, 300 rpm, 600 rpm), addition of protease inhibitor cocktails, or addition of non-ionic detergents to promote protein solubilisation (Triton X-110 or Figure 1 . Recombinants produced using E. coli cell-free IVTT system. Western Blot and quantification of (A) viral antigens FluM, FluHA, CMVpp65 or EBNA3A and (B) parasite antigen PyCSP pIVEX HisHA IVTT products, probed with mAb against the C-terminal HA tag. Whole IVTT extracts (5 ml) of each antigen were run on a 12% NUPAGE gel, transferred to a PVDF membrane, and probed with anti-HA HRP antibody (1:500 dilution). Protein expression was quantitated against an IVTT-produced recombinant P. falciparum (PF14_0051) protein of known concentration expressing the same N-terminal 6xHis and C-terminal HA tags. Yields for each full length antigen were as follow: FluM, 1.15 mg/ ml; FluHA, 0.73 mg/ ml; CMVpp65, 0.7 mg/ ml; EBNA3A, 4 ng/ ml; and PyCSP, 0.6 mg/ ml. doi:10.1371/journal.pone.0027666.g001 Triton X-114) had no significant effect on the yield of full-length protein or partial products, or protein solubility (data not presented). Accordingly, a standard protocol of 4 hrs incubation at 30uC and 300 rpm was adopted for the IVTT reactions. IVTT products were used either unpurified (whole extract), associated to Polybeads or ProteinG beads, or purified using NI-NTA resin or MagneHis Ni-particles. The MagneHis purification method was associated with a very low recovery yield and this yield was much lower than with the NI-NTA system. For example, for PyCSP, the loss associated with purification using Ni-NTA or MagneHis was approximately 30% and 80% respectively.
We also evaluated production of the viral antigens in an insect cell based cell-free system (PyCSP not tested). In a limited number of attempts using the manufacturer's recommended conditions, only two of the four viral protein targets (FluM and CMVpp65) could be produced in this eukaryotic system. However, in contrast to results with the E. coli system, solubility analysis showed that at least 90% of the recombinants were soluble and partial polypeptides were not detected ( Figure 2 ).
In summary, all FluHA, FluM, CMVpp65, EBNA3 and PyCSP IVTT recombinants could be produced in the E. coli cell-free IVTT system, using the manufacturer's recommended conditions. However, at least 70% of each IVTT product was insoluble. In contrast, only two of the four viral antigens could be produced in the insect cell based system but in this system at least 90% of the product was soluble.
In vitro stimulation of PyCSP antigen-specific T cell response using IVTT products
The ability of IVTT products to stimulate an antigen-specific T cell response in vitro was evaluated in the P. yoelii CSP model. Splenocytes from mice (n = 5/group) immunized with VR2516 PyCSP plasmid DNA or VR1020 control plasmid were stimulated in vitro with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; rPyCSP IVTT purified using NI-NTA resin, MagneHis Ni-particles, or anti-HIS; or synthetic peptides representing defined T cell epitopes from PyCSP. Antigen-specific cytokine production was assayed using IFN-c ELISpot or CBA assays.
The number of IFN-c SFCs was significantly higher with splenocytes from VR2516 immunized mice as compared to VR1020 immunized mice when stimulated in vitro with PyCSP IVTT purified using NI-NTA resin (p = 0.013), MagneHis Ni-particles (p = 0.015) or PyCSP synthetic peptides (p = 0.003) ( Figure 3A ). There was no significant difference when splenocytes were stimulated with either unpurified IVTT or unpurified IVTT associated to Polybeads or Protein G beads ( Figure 3A ). Unexpectedly, the use of wells precoated with anti-HIS mAb to capture the tagged proteins was poorly effective with no difference noted between the VR2516 and VR1020 groups. Markedly higher background reactivity was noted with the unpurified or bound IVTT preparations as compared to the purified preparations, presumably due to the presence of high levels of LPS and proteins in the E. coli extract ( Figure 3A) .
Consistent with the ELISpot data, the amount of secreted IFN-c by CBA in cultures of splenocytes from VR2516 versus VR1020 immunized mice was significantly greater in cultures stimulated in vitro with PyCSP IVTT purified using NI-NTA resin (p = 0.005) or MagneHis Ni-particles (p = 0.003) or PyCSP synthetic peptides (p = 0.02) ( Figure 3B ). There was no significant difference when splenocytes were stimulated with either unpurified IVTT or unpurified IVTT associated to Polybeads or Protein G beads. No significant antigen-specific IL-10, TNF-a or IL-6 responses could be detected upon stimulation with purified or unpurified IVTT products; responses were detected upon stimulation with PyCSP peptides (Figures S2A and S2B ; data not presented for IL-6).
In summary, robust antigen-specific IFN-c responses from splenocytes of mice immunized with PyCSP plasmid DNA could be induced following in vitro stimulation with IVTT products purified by either NI-NTA resin or MagneHis Ni-particles, but not by unpurified IVTT reactions or IVTT products associated to Polybeads or Protein beads. There was no significant difference between responses induced by NI-NTA resin and MagneHis Niparticle purifications, but there was a much greater loss of recombinant associated with purification using the MagneHis Niparticles as compared to NI-NTA resin. In general, responses induced by stimulation with the purified PyCSP IVTT products were comparable to responses induced by stimulation with a pool of synthetic peptides representing defined T cell epitopes from PyCSP.
In vivo stimulation of PyCSP antigen-specific T cell response using IVTT products We next evaluated the capacity of unpurified and purified IVTT products to boost PyCSP primed immune responses in vivo. Mice (n = 5/group) were primed with VR2516 PyCSP plasmid DNA and then boosted in vivo with unpurified rPyCSP IVTT, PyCSP IVTT associated to Polybeads or ProteinG beads, or PyCSP IVTT purified using NI-NTA resin or MagneHis Niparticles, all adjuvanted with Alum. Splenocytes were stimulated in vitro with A20 target cells transfected with VR2516 PyCSP DNA or A20 cells pulsed with PyCSP synthetic peptides. Antigen-specific cytokine production was assayed using IFN-c ELISpot or CBA assays.
IFN-c responses were detected by both ELISpot and CBA following in vivo boosting with PyCSP DNA (p,0.001 for ELISpot and p,0.05 for CBA) or PyCSP IVTT purified using either NI-NTA resin or MagneHis Ni-particles (p,0.01 for ELISpot), as compared to Alum control ( Figures 4A and 4B ). Responses stimulated by purified IVTT products were almost as robust as those stimulated with PyCSP DNA. Low responses were noted when mice were boosted with unpurified whole extract or unpurified IVTT associated to either Polybeads or ProteinG beads.
Significant IL-10 responses were also detected following boosting with PyCSP IVTT purified using either NI-NTA resin (p,0.001) or MagneHis Ni-particles (p,0.001), as compared to Alum control, but not following boosting with unpurified whole extract or IVTT products associated to either Polybeads or ProteinG beads, or with PyCSP DNA ( Figure S2C ). TNF-a responses were not significantly different from the controls for any of the conditions ( Figure S2D ). IL-2 responses were induced only by purified IVTT products (data not presented). No significant responses were detected for other cytokines assayed using the CBA assay.
In summary, consistent with the results following in vitro stimulation with IVTT products, robust antigen-specific IFN-c responses could be induced by in vivo boosting with PyCSP IVTT products purified by either NI-NTA resin or MagneHis Niparticles, but not by unpurified IVTT reactions or IVTT products associated to Polybeads or ProteinG beads. There was no significant difference between purification with either NI-NTA resin or MagneHis Ni-particle, nor between purified IVTT products and plasmid DNA. The magnitude of Th1 (IFN-c and TNF-a) responses stimulated in vitro by purified IVTT PyCSP products were similar to those stimulated by PyCSP plasmid DNA.
In vivo stimulation of PyCSP antigen-specific antibody response using IVTT products
The ability of IVTT products to stimulate an antigen-specific antibody response was also determined. Sera from mice (n = 5/group) primed with VR2516 PyCSP plasmid DNA and boosted in vivo with rPyCSP IVTT purified using NI-NTA resin plus Alum were evaluated by ELISA against synthetic peptide representing the recombinant PyCSP protein. Data demonstrated that in vivo boosting with PyCSP IVTT products significant boosted antigen-specific antibody responses relative to responses induced by PyCSP plasmid DNA in the absence of boosting ( Figure 5 ).
The antigenicity of IVTT products in human PBMC cultures was next evaluated. The viral antigens FluM, FluHA, CMVpp65 and EBNA3A produced by IVTT were used unpurified, associated to Polybeads or ProteinG beads, or purified using NI-NTA resin or MagneHis Ni-particles to assay recall cytokine responses from PBMCs of 10 healthy humans by IFN-c ELISpot or CBA.
Consistent with the results in murine system, significant IFN-c ELISpot responses (p,0.05) were detected with all IVTT antigens purified using NI-NTA resin ( Figure 6A ) and for all antigens purified using MagneHis Ni-particles except FluM ( Figure 6B ) (as compared to PBS); the lack of responses with FluM was attributed to a low yield post MagneHis Ni purification (data not presented). Unexpectedly, significant IFN-c ELISpot responses were also noted with all IVTT viral antigens used unpurified (compared to empty pIVEX HisHA IVTT) ( Figure 6C ) or associated to ProteinG beads (compared to empty pIVEX HisHA IVTT associated with ProteinG beads) ( Figure 6D ), but only for only two or the four IVTT viral antigens associated with Polybeads (FluM and FluHA; compared to empty pIVEX HisHA IVTT associated with Polybeads) ( Figure 6E ).
Also consistent with the murine system, no significant responses were detected for any antigens when the ELISpot wells were precoated with anti-His mAb to capture the His-tagged IVTT proteins. The background responses with the unpurified or bound IVTT products were greater than for purified IVTT products, Figure 6 . Antigen-specific IFN-c ELIspot responses by human PBMC stimulated with IVTT-proteins. PBMCs were cultured with IVTTproduced FluM, FluHA, pp65 and EBNA3A purified using (A) NI-NTA nickel resin or (B) MagneHis Ni-particles; (C) unpurified; associated to (D) ProteinG beads or (E) Polybeads; or (F) added to wells precoated with Anti-His. Negative controls were medium only, unpurified whole extract, and whole extract associated to Polybeads or Protein G beads; positive control was CEF peptide pool. IFN-c ELIspot responses (spot forming cells, SFC) of cultured PBMCs were analysed after 36 hrs stimulation. Data are presented as mean +/-standard deviation of 10 volunteers. *P,0.05, **P,0.01 and ***P,0.001 compared to negative controls. doi:10.1371/journal.pone.0027666.g006
presumably as a result of prior exposure of the human subjects to E. coli bacteria or presence of LPS. Significant IFN-c responses to the CEF peptide pool p,0.0001, compared to PBS) were noted for all subjects (data not presented).
For all viral antigens, robust IFN-c responses were detected by CBA in cultures stimulated at 1:100 dilution with IVTT products purified using NI-NTA resin ( Figure 7A ) or MagneHis Ni-particles ( Figure 7B ) but not with purified products at 1:1,000 or 1:10,000 dilutions, presumably due to the low amount of antigen in culture ( Figures 7A and 7B ). With unpurified IVTT products or IVTT antigens associated to Polybeads or ProteinG beads, IFN-c responses were generally better at the higher dilution (1:10,000.1:1,000.1:100) where background E. coli responses were lower (Figures 7C-E) . However, none of these IFN-c responses were statistically different from the controls due to variations in the cytokine levels between individuals (p,0.05 only for EBNA3A-Protein G beads at 1:1000).
The profile of TNF-a responses by CBA was very similar to that of IFN-c ( Figure S3 ). For all viral antigens (except FluM-Ni-NTA), TNF-a responses were statistically significant compared to controls for cultures stimulated at 1:100 dilution with IVTT products purified using NI-NTA resin ( Figure S3A ) or MagneHis Niparticles ( Figure S3B ). Responses were also significant at 1:1,000 dilutions for CMVpp65 and EBNA3 all purified using NI-NTA resin or MagneHis Ni-particles; and FluHA purified with MagneHis Ni-particles. The TNF-a profile was also similar to that of IFN-c for unpurified and bead-associated IVTT products, with the highest responses almost always detected at 1:10,000 (except for EBNA3) (Figures S3C-E) . However, none of these TNF-a responses were statistically significant (except for unpurified CMVpp65 at 1:10,000), as noted above for IFN-c. The overall level of TNF-a was enhanced by association to Polybeads and Protein G beads, relative to unpurified IVTT products (up to 7-fold with EBNA3A-Polybeads at 1:100 dilution).
The profile of IL-10 responses by CBA was very similar to that of IFN-c and TNF-a ( Figure S4 ) for cultures stimulated with IVTT products purified using NI-NTA resin ( Figure S4A ) or MagneHis Ni-particles ( Figure S4B ) with the best responses detected at 1:100 ( Figure S4 ). However, for unpurified or beadassociated IVTT products, the IL-10 profile was inverse to that of IFN-c and TNF-a with the best responses at a 1:100 dilution ( Figures S4C-E) , consistent with potential immunosuppressive effects of IL-10. Significant response was found only in EBNA3A 1:100 purified with MagneHis Ni-particles. As noted for TNF-a, the overall level of IL-10 was enhanced by association to Polybeads and Protein G beads relative to unpurified IVTT products (up to 7-fold with EBNA3A-Polybeads at 1:100 dilution). Responses for other cytokines assayed by CBA were not significant (data not presented).
In summary, consistent with the murine data, robust antigenspecific IFN-c and TNF-a responses from human PBMCs could be induced following in vitro stimulation with IVTT products purified by either NI-NTA resin or MagneHis Ni-particles. In contrast to the murine system, positive IFN-c and TNF-a responses to some antigens could also be induced by unpurified IVTT reactions or IVTT products associated to Polybeads or Protein beads, indicating that the E. coli and LPS background of the whole extract was not sufficient to mask the immunogenicity generated by the IVTT recombinant products. For some but not all of the evaluated antigens, association of IVTT products to Polybeads or Protein G beads enhanced the antigenicity about 2fold to 4-fold.
The rapidly growing amount of genetic and proteomic information available in the post-genomic era precludes the use of traditional cell-based protein expression systems to screen proteins of interest for their potential as vaccine targets. The development of cell-free based systems, first described in 1960's [26] , allows the high throughput recombinant expression of thousands of proteins in reduced volume and time. The most popular cell-free systems are based in E. coli, wheat germ and rabbit reticulocytes extracts [4, 5, 27] . This platform has been applied in a number of proteomics-based studies including structural and functional proteomics [28, 29, 30] , protein evolution [31] , unnatural amino acids and protein labeling [32, 33] , protein interaction [34] , diagnostics and therapeutics [35, 36] and protein microarrays [15, 37, 38, 39] . The protein microarray platform, exploiting antigen-specific antibodies present in plasma or sera from exposed or immunized animals or humans, has been of particular interest to our laboratory to identify potential target antigens for malaria vaccine development [10, 11, 40] . Although Plasmodium proteins have proven particularly difficult to express using conventional cell-based methods, efficient expression of P. falciparum proteins ($ 93%) has been obtained using the E. coli cell-free system [11] . The wheat germ system has been also used for production of P. falciparum proteins, but with less efficiency (75%) [12] .
The success with cell-free protein expression suggest that this system could be also applied to cellular screening, to identify antigenic targets of T cell responses from genomic sequence data. However, this has not yet been adequately explored despite that T cells play a central role in orchestrating acquired immunity against infectious diseases [41, 42] . Both CD8 + and CD4 + T cells can mediate their effector function directly via cytotoxicity or indirectly via cytokines. CD4 + T cells can also provide help for CD8 + T cells or can recruit and activate B cells for antibody secretion. To date, the only reports have been restricted to measurement of proliferative T cell responses in PBMCs of smallpox vaccines or of cattle immunized with a purified Anaplasma marginale outer membrane preparation to a small number of IVTT produced vaccinia virus proteins or outer membrane proteins, respectively [18, 19, 43] . Neither of those studies comprehensively assessed and optimized the application of IVTT products for large-scale or proteome-wide cellular screening. Accordingly, herein, we report proof of concept for the potential of this approach a strategy for proteome-wide identification of antigens targeted by cell mediated immunity in both viral and parasite models, using IVTT products with specimens from human and mice.
Our human studies used well characterized antigens from EBV, CMV and Influenza A virus. which are known to be targeted by both CD4 + and CD8 + T cell responses and with defined T cell epitopes [44] . The murine studies used the well characterized sporozoite coat protein, the circumsporozoite protein (CSP), from the P. yoelii rodent malaria parasite. We tested each IVTT antigen individually and presented to T cells in different forms -either unpurified, purified using NI-NTA resin or MagneHis Niparticles, or associated to Polybeads or ProteinG beads. Synthetic peptides representing defined T cell epitopes from the respective antigens were assayed in parallel as positive controls. The primary immune readout was IFN-c production due to its crucial role in protection or pathogenesis of complex diseases [45] .
For all four viral and one parasite antigens tested in this study, partial products were common. The production of partial fragments in E. coli based IVTT reactions has been described previously and attributed to a phenomena called translational pausing but the presence of partial products did not adversely affect immunogenicity or antigenicity [46] . Since the antigens produced by E. coli based IVTT were at least partially insoluble, simple techniques for purification, such as antibody coated beads, were not effective. We therefore explored alternative purification options including Ni-NTA affinity resin and MagneHis Niparticles. Both purification systems tested using denaturating conditions proved efficient but a much higher product recovery was obtained with the Ni-NTA resin as compared to the MagneHis Ni-particles. Unexpectedly, with the eukaryotic based insect cell-free system, the solubility of the recombinants increased to almost 100%, but only two of the four proteins expressed in the E. coli cell-free system could be expressed in the insect cell based system, at least for the limited number of times expression was attempted. These data suggest that further studies with the insect cell-free system are warranted. Newer technologies of high throughput purification such as affinity ZipTips (Millipore, Ireland) and Ni-NTA plates (Qiagen, Valencia, CA) can facilitate the purification process, but do not effectively deal with solubility issues.
In both human/virus and murine/parasite models, IVTT products purified by either NI-NTA resin or MagneHis Niparticles were highly effective inducers of antigen-specific IFN-c and TNF-a responses in vitro. In the human/virus but not murine/ parasite model, antigen-specific IFN-c and TNF-a responses could also be induced by unpurified IVTT reactions or IVTT products associated to Polybeads or Protein beads, indicating that the E. coli and LPS background of the whole extract was not sufficient to mask the immunogenicity of the IVTT recombinant products. Addition of carboxylate beads to unpurified IVTT products increased the immunogenicity of the IVTT viral products by up 1.8-fold for IFN-c, 2.6-fold for IL-10 and 7-fold for TNF-a. The most robust and specific antigen-specific IFN-c and TNF-a responses (by CBA) were detected at the highest dilution of IVTT product, where background E. coli responses were lowest. This is a favorable scenario for proteomic-wide scale cellular screening, as the use of highly diluted IVTT products is more cost-effective. Unexpectedly, poor results were obtained with ELIspot wells precoated with anti-IFN-c mAb as well as anti-HIS mAb to bind the HIS tag on the IVTT product. In vivo studies with PyCSP IVTT products confirmed that the target protein was produced and that the IVTT produced proteins were immunogenic.
These data demonstrate the potential of IVTT products as a useful tool for the proteome-wide screening of cellular targets of viral, parasitic or bacterial immunity Overall, IVTT products affinity purified through nickel resin or magnetic beads proved the most efficient inducers of sensitive and specific antigen-specific cytokine responses, the nickel resin method was associated with the greater yield post-purification. Although not specifically evaluated herein, it is likely that such cell-free approaches may be suited to the identification of targets of CD4 + T cell responses, but not targets of CD8 + T cell responses due to a requirement for target antigen processing and presentation [43, 47, 48] . Rather, epitopebased approaches based on prediction of high affinity binding class I T cell epitopes using computerized algorithms, such as that reported by us previously [49] are probably more appropriate.
Overall, the work reported here provides proof of concept for the potential for high-throughput identification from genomic sequence data of antigenic targets of T cell responses from complex pathogens which threaten public health. Such antigens may represent promising candidates for the development of vaccines that have thus far proved elusive.
Ten healthy adult Caucasian volunteers (five male and five female; mean age 36.367.1 years old) were recruited with written informed consent under a protocol (P1111) approved by the Queensland Institute of Medical Research Human Research Ethics Committee. All studies with human specimens were approved by the Queensland Institute of Medical Research Human Research Ethics Committee (P1111) and conducted in compliance with all applicable regulations governing protection of human subjects. Although the history of vaccination in these subjects has not been documented, it is likely that all would have been vaccinated against or exposed to influenza, EBV, and CMV, given the documented prevalence of these viruses in human populations; and all were known to respond to the CEF peptide pool which comprises CD8 + T cell epitopes from FLU, EBV and CMV.
Female BALB/c (H-2 d ) mice aged 6-8 weeks were purchased from The Animal Resource Centre (Perth, WA) and maintained under standard conditions. All studies were approved by the Queensland Institute of Medical Research Animal Ethics Committee (protocol P1111).
The plasmid DNA vaccines encoding the P. yoelii circumsporozoite protein (CSP; VR2516) and the control plasmid (VR1020) have been previously described [50, 51] . The DNA vaccines were prepared using the EndoFree Plasmid Mega Kit (Qiagen, Valencia, CA) according to manufacturer's instructions and administered in sterile saline.
A custom vector was developed for protein expression in the E. coli cell-free transcription translation system. This vector, called pIVEX HisHA AmpR, was modified from the commercially available pIVEX 2.4d and pIVEX 2.5d vectors (Roche Applied Science, Mannheim, Germany) by incorporating both the N-terminal HIS tag and C-terminal HA tag into the one vector backbone. The HA tag was released from pIVEX 2.5d by digestion with BamHI and XmaI restriction enzymes (New England Biolabs, Ipswich, MA) for 4 hrs at 37uC (1 mg DNA/1 unit enzyme, 20 ml volume) and the fragment then extracted from a 3% agarose gel and purified using a commercially available QIAquick PCR purification kit (Qiagen, Germany) according to manufacturer's instructions. The purified HA-tag was ligated overnight at 16uC into BamHI and XmaI digested pIVEX 2.4d vector using 400 units T4 ligase (New England Biolabs), 25 ng of linearized pIVEX 2.4d vector and 70 ng of the HA-tag insert in a 20 ml reaction. The ligated product was purified from a 1% agarose gel as described above, transformed into TOP10 thermocompetent cells (Invitrogen), and grown on LB plates in the presence of 100 mg/ml ampicillin. Positive colonies were screened by colony PCR using 0.2 units/ml Expand Taq polymerase in Buffer 2 (Roche Diagnostics), 0.4 mM dNTPs and 0.4 mM each primer (forward: TAATACGACTCACTATAGGG; reverse TGCTAGT-TATTGCTCAGCGG) using the following conditions: initial denaturation at 95uC for 5 min; 35 cycles at 95uC for 30 sec, 55uC for 30 sec and 68uC for 1 min; and a final extension at 68uC for 10 min. #NC_007605) and P. yoelii circumsporozoite protein (PyCSP; strain 17XNL, accession #J02695) were amplified by PCR using gene-specific primers flanked with restriction enzyme sites ( Table 1 ). The 50 ml PCR reaction contained 0.2 units/ml Expand Taq polymerase in Buffer 2 (Roche diagnostics), 0.4 mM dNTPs and 0.4 mM each primer and 50 ng DNA template. PCR conditions were: initial denaturation at 95uC for 5 min; 45 cycles at 95uC for 30 sec, 55uC for 30 sec and 68uC for 3 min; and a final extension at 68uC for 10 min. The fragments corresponding to the expected size were excised from a 1% agarose gel, purified using the QIAquick PCR purification kit (Qiagen, Germany), and digested overnight at 37uC using the restrictions enzymes for which the cutting site was present in the flanking sequence. The pIVEX HisHA vector was also digested overnight at 37uC with the same set of restriction enzymes. After cleanup using the PCR purification kit, the gene fragments and the linearized vector were ligated overnight at 16uC at an insert:vector ratio of 1:20 using 30 ng of vector and 400 units T4 ligase (New England Biolabs) in a 20 ml reaction volume. The ligation product was transformed into TOP10 thermo-competent cells, grown overnight at 37uC on LB ampicillin plates (100 mg/ml), and colonies screened by PCR using the same protocol for the customized vector, except that the extension time of the colony PCR was increased to 3 min. One positive colony of each construct was grown overnight at 37uC in LB containing ampicillin (100 mg/ml) and the plasmid purified using the QIAprep Spin Miniprep kit (Qiagen) according to manufacturer's instructions. In a few instances where the IVTT production was poor, the DNA was further purified using phenol/chloroform extraction and ethanol precipitation which improved the yield, but this was not routinely done. All constructs were confirmed by DNA sequencing before use in the IVTT reactions.
Recombinant proteins were synthesized by cell-free in vitro transcription and translation using the Rapid Translation System 100 E. coli HY kit (Roche Diagnostics) or EasyXpress Insect Cell Protein kit (Qiagen) according to the manufacturer's instructions.
In some experiments, protease inhibitor cocktails (Cat P2714, Sigma-Aldrich, St Louis, MO; Cat 1836170, Roche Diagnostics), or addition of non-ionic detergents (Triton X-110 or Triton X-114) were added according to manufacturer's recommended protocol: 4 hr at 25uC. The recombinant antigens were stored at 220uC and used as immunogens for in vitro and in vivo up to one week after production. Protein expression of the full-length product (as evidenced by immunoblot using the anti-HA Cterminal tag mAb) was quantitated against an IVTT-produced recombinant P. falciparum (PF14_0051) protein of known concentration expressing the same N-terminal 6xHis and C-terminal HA tags. Immunoblot images were acquired in TIF format and analysed using the AnalySIS LS software (version 5.0; Soft Imaging Systems GmbH, Germany).
The E. coli IVTT products were purified using two different methods based on affinity to the N-terminal 6xHIS tag. One method used mini-spin columns with a cellulose acetate filter (Pierce, Rockford, IL) and NI-NTA resin (Qiagen). For that, 200 ml of NI-NTA resin was added to a spin column connected to a collection tube. The column was washed twice with 600 ml of milliQ water and twice with 600 ml of Binding Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM Imidazole and 8 M Urea pH 8) using centrifugation at 1500 rpm for 2 min. Then 50 ml of IVTT reaction was mixed with 700 ml of Binding Buffer and incubated at 4uC for 20 min in an orbital mixer, to solubilize the inclusion bodies. The solubilized sample was added to a NI-NTA resin column and incubated at 4uC for 30 min in an orbital mixer to maximize protein binding to the resin. The flow-through was removed via centrifugation at 1500 rpm for 2 mins and the column washed three times with 700 ml of Binding Buffer. The recombinant was eluted with 200 ml of Elution Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM Imidazole and 8 M Urea pH 8) incubated at 4uC for 20 min in an orbital mixer before collection via centrifugation.
The second method of purification used the commercially available MagneHis Protein Purification System (Promega, Madison, WI) according to the manufacturer's instructions. For that, 50 ml of IVTT reaction was mixed with 700 ml of Binding Buffer (100 mM HEPES, 20 mM Imidazole and 8 M Urea pH 7.5) and incubated at 4uC for 20 min in an orbital mixer, to solubilize the inclusion bodies. Then, 30 ml of MagneHis Niparticles was added to the sample and the mixture incubated at room temperature for 2 min. The sample tubes were connected to the MagneSphere Magnetic Separation Stand (Promega) and, after 30 sec of binding, the flow through was removed and discarded. The magnetic beads with bound protein were washed 3 times with 700 ml of Binding Buffer and the recombinant eluted in 200 ml of Elution Buffer (100 mM HEPES, 500 mM Imidazole and 8 M Urea pH 7.5) incubated at 4uC for 20 min in an orbital mixer before collection.
The eluted recombinants from both methods were dialyzed against PBS (desalting step) using Amicon ultra 0.5 ml 3 kDa cutoff centrifugal filters (Millipore, Ireland). For that, the centrifugal filters were wet with 300 ml of PBS pH 7.0 before adding 200 ml of the eluted protein. After 30 min of centrifugation at 14,000 rpm at 4uC, the volume of the protein was reduced to approximately 100 ml and 400 ml of PBS pH 7.0 were added. This process was repeated four times and the resultant sample collected and stored at 220uC.
Protein expression was confirmed by western blot. IVTT products were diluted 1:1 in 2x reducing sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% beta-mercaptoethanol) and denatured at 95uC for 5 mins. Samples (2.5 ml of E. coli IVTT whole extract or supernatant, and 5 ml of insect cell IVTT whole extract or supernatant) were run in parallel with molecular weight standard (BenchMark pre-stained protein marker, Invitrogen) on a 4-12% NuPage SDS Page gel (Invitrogen, Carlsbad, CA, USA) at 120 V for 70 min. Samples were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA) pre-wet with 100% methanol, using the BioRad Mini Trans-Blot system at 100 V for 1 hr, according to manufacturer's instructions. Membranes were blocked with PBS containing 5% skim milk powder overnight at 4uC with shaking, washed 3 times with PBS containing 0.05% Tween20, then probed with anti-HIS HRP (1:5000; Roche Diagnostics) or anti-HA HRP (1:500) (Roche Diagnostics) for 1 hr at room temperature. Membranes were Immunization of mice using the VR2516 PyCSP DNA vaccine and PyCSP IVTT product Mice (n = 5/group) were immunized intramuscularly in the tibialis anterior muscle with 50 mg of VR2516 PyCSP DNA vaccine or VR1020 negative control plasmid, two times at 3 week intervals. Splenocytes were harvested from DNA immunized mice or naïve control mice (n = 5) at 3 weeks post-boost for in vitro T cell assays using IVTT products. Serum was collected from select groups for antibody assays. The potential toxicity of IVTT products was assessed prior to use for in vitro stimulation of T cell responses by culturing splenocytes (5610 5 cells/well) of naïve BALB/c mice with IVTT-produced PyCSP either unpurified or purified using NI-NTA resin (Qiagen) or MagneHis particles (Promega) at dilutions of 1:100, 1:1000 and 1:10000, or media alone, and monitoring cell growth for 48 hours. No toxicity due to the unpurified or purified IVTT preparations was apparent, as indicated by cell death.
For the in vivo evaluation of IVTT products, mice (n = 5/group) were immunized intramuscularly with 50 mg/100 ml of VR2516 PyCSP DNA vaccine (split between two legs) and boosted 3 weeks later with PyCSP IVTT products formulated with 100 ml Aluminium Hydroxide gel adjuvant (Brenntag Biosector, Frederikssund, Denmark) as follows: A) 15 ml of IVTT reaction (9 mg PyCSP/mouse); B) 15 ml of IVTT reaction purified through NI-NTA resin (6.6 mg PyCSP/mouse); C) 15 ml of IVTT reaction purified through MagneHis Protein Purification System (3 mg PyCSP/mouse); D) 15 ml of IVTT reaction absorbed in 10 ml of PolybeadH Poly(methyl methacrylate) microspheres (PMMA microspheres, 0.08-0.09 mm diameter) (Cat #23570, Polysciences, Inc., Warrington, PA, USA) (9 mg PyCSP/mouse); and E) 15 ml of IVTT reaction associated to 10 ml of Protein G conjugated microspheres/carboxylate beads (Cat #21106, Polyscience) (9 mg PyCSP/mouse). The beads used in the immunizations were washed 3 times with PBS pH 7.0 before addition of the IVTT product, and used without further processing. Positive and negative controls groups (n = 5) were administered 50 mg of VR2516 PyCSP DNA vaccine intramuscularly or 100 ml of Alum mixed with 100 ml of PBS pH 7.0, respectively. Three weeks after immunization mice were sacrificed and the spleens harvested for use as effectors in T cell assays.
Spleens were macerated, washed in Dulbecco's solution containing 2% FCS, the red blood cells lysed with 0.09% NH 4 Cl for 5 min at 37uC, and then washed again. The splenocytes were resuspended in complete DMEM containing 10% FCS, 100 U/ml of Penicillin, 100 mg/ml of Streptomycin, 2 mM L-Glutamine and 0.05 mM ß-mercaptoethanol, and cultured in 96-wells flat-bottomed plates for ELIspot or CBA at a concentration of 5610 5 cells/well.
For in vitro evaluation of IVTT products, splenocytes were stimulated with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; or rPyCSP IVTT purified using NI-NTA resin or MagneHis Ni-particles. Unpurified IVTT products were used at a dilution of 1:200 or 1:1000 and purified IVTT products were used at a dilution of 1:100 or 1:200 for evaluation of cytokines in the supernatant using the Cytometric Bead Array assay or IFN-c secreting cells via ELIspot, respectively. Pilot experiments were carried out to find out the optimal dilutions of IVTT products for these assays (data not shown). An IVTT reaction using the empty pIVEX HisHA vector was used as negative control. A pool of synthetic peptides representing defined CD4 + and CD8 + T cell epitopes from PyCSP (residues 57-70, sequence KIYNRNIVNRLLGD [52] ; residues 280-288, sequence SYVPSAEQI [53] ; and resides 280-295, sequence SYVPSAEQILEFVKQI [54] ) at 10 mg/ml of each peptide was used as a positive control. ConA (Sigma-Aldrich, St. Louis, MO) at 5 mg/ml was used as a mitogen control. All stimuli were added in association with 1610 5 cells/well of A20/2 J cells (ATCC clone HB-98) irradiated at 1600 rads, as antigen presenting cells.
For in vivo evaluation of IVTT products, splenocytes were stimulated with 2610 4 cells/well of A20 cells transfected with VR2516 PyCSP DNA or VR1020 negative control using the AMAXA Nucleofector transfection kit (AMAXA, Cologne, Germany) according to manufacturer's manual, or with 1610 5 cells/well of A20 cells pulsed with the PyCSP peptide pool. A20 cells alone and medium were used as negative controls and ConA used as mitogen control. AMAXA transfections included parallel reactions with GFP plasmid as a positive control (data not presented); additionally, our laboratory routinely uses AMAXAtransfected A20 cells as APCs for in vitro T cell assays [55, 56] .
Peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll density gradient centrifugation and resupsended in complete RPMI containing 10% human AB serum, 100 units/ml of Penicillin, 100 mg/ml of Streptomycin, 2 mM L-Glutamine, 1 mM Sodium Pyruvate and 25 mM Hepes. Cells were cultured at a concentration of 1610 5 cells/well in 96-wells round-bottomed plates at 37uC in an atmosphere of 5% CO 2 . The cells were stimulated with IVTT-produced FluM, FluHA, CMVpp65 and EBNA3A antigens, either unpurified; associated to Polybeads or ProteinG beads; or purified using NI-NTA resin or MagneHis Niparticles. Unpurified and purified IVTT products were used at a dilution of 1:100, 1:1000 or 1:10000 for evaluation of cytokines in the supernatant using the Cytometric Bead Array assay, or at a dilution of 1:1000 (unpurified) or 1:100 (purified) for evaluation of IFN-c secreting cells via ELISpot. Negative controls included empty pIVEX HisHA IVTT reaction alone or associated to Polybeads or ProteinG beads, or medium only. A CEF peptide pool consisting of 32 synthetic peptides representing defined CD8 + T cell epitopes from human Cytomegalovirus, Epstein-Barr Virus and Influenza Virus (Anaspec, San Jose, CA) at 5 mg/ml total was used as a positive control [44] . Phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO) at 2 mg/ml was used as a mitogen control.
IFN-c secreting T cells were enumerated by ELISpot. Briefly, MultiScreen HTS IP 96 plates (Cat MSIPS4510, Millipore, Ireland) were pre-wet with 15 ml/well of 35% ethanol and washed twice with PBS (pH 7.4). For mouse ELIspot assays, wells were coated with 75 ml/well of sterile PBS (pH 7.4) containing 10 mg/ ml anti-mouse IFN-c (Cat 551216, Clone R4-6A2, BD Pharmingen, CA) with or without 1:500 anti-His mAb (Cat H1029, Sigma-Aldrich, St. Louis, MO) overnight at room temperature. For human ELIspot assays, wells were coated with 75 ml/well of carbonate buffer (pH 9.6) containing 10 mg/ml anti-human IFN-c (Cat 3420-3, Clone 1-D1K, MABTECH, Sweden) with or without 1:500 anti-His mAb (Cat H1029, Sigma-Aldrich, St. Louis, MO) overnight at 4uC. Plates were washed twice with 200 ml/well DMEM (splenocytes) or RPMI (PBMC) and blocked with 200 ml/ well of medium containing 10% FCS for 3 hrs at 37uC. After blocking, the wells coated with anti-His mAb were incubated with PyCSP, FluM, FluHA, CMVpp65, EBNA3A or empty pIVEX HisHA IVTT products diluted 1:1000 in DMEM or RPMI containing 2% FCS in a volume of 50 ml/well, overnight at 4uC (to absorb the His-tagged IVTT products to the anti-His mAb coated wells). Plates were washed 3 times with DMEM or RPMI, and the cells added to the IFN-c and His coated wells in triplicates. Alternatively, in the absence of anti-His pre-coating, IVTT products unpurified, associated to Polybeads or ProteinG beads, or purified using NI-NTA resin or MagneHis Ni-particles (see above), were added to the IFN-c coated wells together with the cells (100,000 PBMCs or 500,000 splenocytes), in triplicate. After 36 hrs incubation, plates were flicked to remove the cells and washed 6 times with PBS-Tween 0.05% pH 7.4 (PBS-T). Then 75 ml/well of biotinylated anti-mouse IFN-c (Cat 554410, Clone XMG1.2, BD Pharmingen) at 2 mg/ml in PBS or 75 ml of biotinylated anti-human IFN-c (Cat 3420-6, Clone 7-B6-1, MABTECH, Sweden) diluted 1:1000 in PBS was added to each well. Plates were incubated for 3 hrs at room temperature, washed 3 times with PBS-T, and 75 ml/well of Streptavidin-HRP for splenocytes cultures or Streptavidin-AP (Bio-Rad, Hercules, CA) for PBMC cultures diluted 1:1000 in PBS was added. After 1 hour incubation at room temperature, plates were washed 3 times with PBS-T followed by 3 times with PBS alone, and developed using the AEC substrate set (BD Pharmingen) for mouse splenocytes or SigmaFast BCIP/NBT (Sigma-Aldrich) for humans PBMC cultures, according to manufacturer's instructions. After 10 mins, the plates were rinsed extensively with dH2O to stop the enzymatic reaction, dried and stored in the dark. The number of IFN-c secreting cells was determined using the automated ELISpot reader (AID iSpot Reader, Autoimmun Diagnostika GmbH, Strassberg, Germany).
To determine the cytokines secreted by the stimulated splenocytes or PBMC, cells were cultured at a concentration of 5610 5 cells/well in 96-wells flat-bottomed plates (Corning Incorporated, Corning, NY, USA). The supernatant was collected after 48 hrs and 72 hrs stimulation and analyzed with Cytometric Beads Array flex kits (BD Biosciences, San Jose, CA, USA) containing IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IFN-c and TNF-a for mouse and IL-10, IFN-c and TNF-a for human samples. Data were acquired and analyzed using BD FACS Array Bioanalyser and FlowJo 7.6 software.
PyCSP-specific antibody responses in mouse sera were evaluated by peptide ELISA, as previously described [57] . Briefly, flatbottomed 96-well microtiter plates (Immulon 4; Dynex Technology Inc., Chantilly, VA, USA) were coated with 100 ml/well of recombinant PyCSP protein at a concentration of 1 mg/ml in carbonate-bicarbonate coating buffer pH 7.4, and incubated overnight at 4uC. Wells were blocked for 1 hr with 2% BSA in PBS containing 0.05% Tween 20 (Blocking Buffer) and washed three times with PBS containing 0.05% Tween 20 (PBS-T). Consecutive dilutions of individual sera diluted in PBS containing 0.01% Tween-20 were incubated for 2 hrs at room temperature. The plates were washed 3 times, and incubated with 100 ml/well Biotinylated anti-mouse IgG (Jackson ImmunoResearch) at a dilution of 1:20,000 for 1 hr. The plates were washed three times and incubated with Streptavidin HRP (BD Biosciences) at a dilution of 1:1000 for 1 hr. The plates were washed and developed for 10 mins with 50 ml/well TMB substrate (Sigma). Reactions were terminated by adding 50 ml of stopping buffer and the OD450 recorded using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results are expressed as mean OD readings of triplicate wells +/-SE.
Data from the in vitro and in vivo tests in mice were analyzed by the Student t-test and two-way ANOVA, respectively, where a p value,0.05 was considered significant. Data from human PBMC ELIspot and CBA were analyzed using two-way ANOVA and one-way ANOVA, respectively. A p value,0.05 was considered significant. Figure S1 Recombinants produced using E. coli cellfree IVTT system. Western Blot of (A) viral antigens FluM (32 kDa), FluHA (68 kDa), CMVpp65 (68 kDa) and EBNA3A (108 kDa), and parasite protein PyCSP (44 kDa), probed with anti-HA antibody, and (B) viral antigens FluM, FluHA, CMVpp65 and EBNA3A probed with anti-His antibody. Whole IVTT extracts (5 ml) of each antigen were run on a 12% NUPAGE gel, transferred to a PVDF membrane, and probed with anti-HA HRP antibody (1:500 dilution) or anti-His HRP antibody (1:5000 dilution). The western probed with anti-HA also detected a cross reactive band of 56 kDa in all expression extracts. The western probed with anti-His mAb showed the presence of partial products probably due to early termination of translation. (TIF) Figure S2 Antigen-specific TNF-a and IL-10 responses of mouse splenocytes stimulated in vivo or in vitro with IVTT-proteins. (A) and (B): splenocytes of mice immunized with VR2516 PyCSP plasmid DNA or VR1020 control DNA were cultured in vitro with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; rPyCSP IVTT purified using NI-NTA resin, MagneHis Ni-particles, or anti-HIS; or synthetic peptides representing defined T cell epitopes from PyCSP (positive control), as indicated. (C) and (D): splenocytes of mice immunized with VR2516 PyCSP plasmid DNA and boosted in vivo with unpurified rPyCSP IVTT; rPyCSP IVTT associated to Polybeads or ProteinG beads; or rPyCSP IVTT purified using NI-NTA resin or MagneHis Ni-particles; all formulated with Alum adjuvant. Parallel groups of mice were boosted with either Alum only or VR2516 as controls. Splenocytes were cultured in vitro with A20 cells transfected with VR2516 PyCSP plasmid DNA or A20 cells pulsed with synthetic peptides representing defined PyCSP T cell epitopes, as indicated. Secreted TNF-a (A and C) or IL-10 (B and D) in culture supernatant was measured by Cytometric Bead Array (CBA) after 48 hrs stimulation. *P,0.05, **P,0.01 and ***P,0.001 compared to negative controls. (TIF) Figure S3 Antigen-specific TNF-a responses by human PBMC stimulated with IVTT-proteins. PBMCs were cultured with IVTT-produced FluM, FluHA, CMVpp65 and EBNA3A purified using (A) NI-NTA nickel resin or (B) MagneHis Ni-particles; (C) unpurified; associated to (D) ProteinG beads or (E) Polybeads; or (F) added to wells precoated with Anti-His; IVTT products were diluted 1:100, 1:1000, or 1:10,000. Secreted TNF-a in the supernatant of cultured PBMCs was analyzed by Cytometric Bead Array after 48 hrs stimulation. * P,0.05 compared to negative controls. (TIF) Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities In all vertebrate animals, CD8(+) cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users. Major histocompatibility complex class I (MHC-I) molecules are found in all vertebrate animals where they play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They are highly polymorphic proteins that bind 8-11 amino acid long peptides derived from the intracellular protein metabolism. The resulting heterotrimeric complexes-consisting of the MHC-I heavy chain, the monomorphic light chain, beta-2 microglobulin (β 2 m), and specifically bound peptides-are translocated to the cell surface where they displayed as target structures for peptide-specific, MHC-Irestricted CTLs. If a peptide of foreign origin is detected, the T cells may become activated and kill the infected target cell.
MHC-I is extremely polymorphic. In humans, more than 3,400 different human leukocyte antigen class I (HLA-I) molecules have been registered (as of January 2011), and this number is currently growing rapidly as more efficient HLA typing techniques are employed worldwide. The polymorphism of the MHC-I molecule is concentrated in and around the peptide-binding groove, where it determines the peptide-binding specificity. Due to this polymorphism, it is highly unlikely that any two individuals will share the same set of HLA-I molecules thereby presenting the same peptides and generating T cell responses of the same specificities-something, that otherwise would give microorganisms a strong evolutionary chance of escape. Rather, this polymorphism can be seen as diversifying peptide presentation thereby individualizing T cell responses and reducing the risk that escape variants of microorganisms might evolve.
In 1999, we proposed that all human MHC specificities should be mapped ("the Human MHC Project") as a preamble for the application of MHC information and technologies in humans (Buus 1999) . Since then, we have developed large-scale tools that are generally applicable towards this goal: production, analysis, prediction and validation of peptide-MHC interactions (Ferre et al. 2003; Harndahl et al. 2009; Hoof et al. 2009; Larsen et al. 2005; Lundegaard et al. 2008; Nielsen et al. 2003 Nielsen et al. , 2007 Ostergaard et al. 2001; Pedersen et al. 1995; Stranzl et al. 2010; Stryhn et al. 1996) , and a "one-pot, read-and-mix" HLA-I tetramer technology for specific T cell analysis (Leisner et al. 2008) . Here, we demonstrate that many of these tools can be transferred to other vertebrate animals as exemplified by an important livestock animal, the pig. We have successfully generated a recombinant swine leukocyte antigen I (SLA-I) protein, SLA-1*0401, one of the most common SLA molecules of swine (Smith et al. 2005) . Using this protein, we have developed the accompanying biochemical peptide-binding assays and demonstrated that the immunoinformatics tools originally developed to cover all HLA-I molecules, despite the evolutionary distance, can be applied to SLA-I molecules. We suggest that the "human MHC project" can be extended to cover other species of interest.
All peptides were purchased from Schafer-N, Denmark (www.schafer-n.com). Briefly, they were synthesized by standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry, purified by reversed-phase high-performance liquid chromatography (to at least >80% purity, frequently 95-99% purity), validated by mass spectrometry, and quantitated by weight.
Positional scanning combinatorial peptide libraries (PSCPL) peptides were synthesized using standard solidphase Fmoc chemistry on 2-chlorotrityl chloride resins. Briefly, an equimolar mixture of 19 of the common Fmoc amino acids (excluding cysteine) was prepared for each synthesis and used for coupling in 8 positions, whereas a single type of Fmoc amino acid (including cysteine) was used in one position. This position was changed in each synthesis starting with the N-terminus and ending with the C-terminus. In one synthesis, the amino acid pool was used in all nine positions. X denotes the random incorporation of amino acids from the mixture, whereas the single letter amino acid abbreviation is used to denote identity of the fixed amino acid.
The peptides in each synthesis were cleaved from the resin in trifluoroacetic acid/1,2-ethanedithiol/triisopropylsilane/water 95:2:1:3 v/v/v/v, precipitated in cold diethylether, and extracted with water before desalting on C18 columns, freeze drying, and weighting.
Recombinant constructs encoding chimeric and SLA-1*0401 molecules A synthetic gene encoding a transmembrane-truncated fragment encompassing residues 1 to 275 of human HLA-A*11:01 alpha chain followed by a FXa-BSP-HAT tag (FXa = factor Xa cleavage site comprised of the amino acid sequence IEGR, BSP = biotinylation signal peptide, HAT = histidine affinity tag for purification purposes; see Online Resource 1) had previously been generated and inserted into the pET28 expression plasmid (Novagen) (Ferre et al. 2003) . Synthetic genes encoding the corresponding fragments of the SLA-1*0401 alpha chain (α 1 α 2 ) and α 3 , respectively, (Sullivan et al. 1997) were purchased from GenScript. To exchange domains and generate chimeric human/swine MHC-I gene constructs, a type II restriction endonuclease-based cloning strategy (SeamLess® Strategene; Cat#214400, Revision#021003a), with modifications, was used. All primers were purchased HPLC-purified from Eurofins MWG Operon (Ebersberg, Germany), and all PCR amplifications were performed in a DNA Engine Dyad PCR instrument (MJ Research, MN, USA). All constructs were validated by DNA sequencing. The following MHC-I heavy chain constructs were made HHH, HHP, HPP, PHP, and PPP, where the first, second, and third letter indicates domains α 1 (positions 1-90), α 2 (positions 91-181), and α 3 (positions 182-275), respectively, and H indicates that the domain is of HLA-A*11:01 origin, whereas P indicates that it is of SLA-1*0401 origin.
Constructs were transformed into DH5α cells, cloned, and sequenced (ABI Prism 3100Avant, Applied Biosystems) . Validated constructs of interest were transformed into an Escherichia coli production cell line, BL21(DE3), containing the pACYC184 expression plasmid (Avidity, Denver, USA) containing an isopropylβ-d-1-thiogalactopyranoside (IPTG)-inducible BirA gene to express biotin-ligase. This leads to almost complete in vivo biotinylation of the desired product (Leisner et al. 2008 ).
To maintain the pET28-derived plasmids, the media was supplemented with kanamycin (50 μg/ml) throughout the expression cultures. When appropriate, the media was further supplemented with chloroamphenicol (20 μg/ml) to maintain the BirA containing pACYC184 plasmid. E. coli BL21(DE3) cells transformed with appropriate plasmids were grown for 5 h at 30°C, and a 10-ml sample adjusted to OD (600) =1 was then transferred to a 2-l fedbatch fermentor (LabFors®). To induce protein expression, IPTG (1 mM) was added at OD (600) ∼25 and the culture was continued for an additional 3 h at 42°C (for in vivo biotinylation of the product, the induction media was further supplemented with biotin (Sigma #B4501, 125 μg/ ml)). Samples were analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before and after IPTG induction. At the end of the induction culture, protease inhibitor (PMSF, 80 μg/l) was added, and cells were lysed in a cell disrupter (Constant Cell Disruptor Systems set at 2,300 bar) and the released inclusion bodies were isolated by centrifugation (Sorval RC6, 20 min, 17,000×g). The inclusion bodies were washed twice in PBS, 0.5% NP-40 (Sigma), and 0.1% deoxycholic acid (Sigma) and extracted into urea-Tris buffer (8 M urea, 25 mM Tris, pH 8.0), and any contaminating DNA was precipitated with streptomycin sulfate (1% w/v).
The dissolved MHC-I proteins were purified by Ni 2 +/IDA metal chelating affinity column chromatography followed by Q-Sepharose ion exchange column chromatography, hydrophobic interaction chromatography, and eventually by Superdex-200 size exclusion chromatography. Fractions containing MHC-I heavy chain molecules were identified by A280 absorbance and SDS-PAGE and pooled. Throughout purification and storage, the MHC-I heavy chain proteins were dissolved in 8 M urea to keep them denatured. Note that the MHC-I heavy chain proteins at no time were exposed to reducing conditions. This allowed purification of highly active pre-oxidized moieties as previously described (Ostergaard et al. 2001) . Protein concentrations were determined by bicinchoninic acid assay. The degree of biotinylation (usually >95%) was determined by a gel-shift assay (Leisner et al. 2008 ). The pre-oxidized, denatured proteins were stored at −20°C in Tris-buffered 8 M urea.
Recombinant constructs encoding human and porcine beta-2 microglobulin Recombinant human β 2 m was expressed and purified as described elsewhere (Ostergaard et al. 2001) , (Ferre et al. 2003) . Using a previously reported E. coli codonoptimized gene encoding human β 2 m as template , a gene encoding porcine β 2 m was generated by multiple rounds of site-directed mutagenesis (QuikChange® Stratagene, according to the manufacturer's instructions) (Online Resource 2). Briefly, the genes encoding human or pig β 2 m were N-terminally fused to a histidine affinity encoding tag (HAT) followed by a restriction enzyme encoding tag (FXa), inserted into the pET28 vector and expressed in inclusion bodies in E. coli. The fusion proteins were extracted into 8 M urea, purified by immobilized metal affinity chromatography (IMAC), and refolded by dilution. The fusion tags were then removed by FXa restriction protease digestion. The liberated intact and native human or pig β 2 m were purified by IMAC and gel filtration chromatography, analyzed by SDS-PAGE analysis, concentrated, and stored at −20°C until use (Fig. 1 ).
Purification and refolding of recombinant porcine β 2 m proteins Porcine β 2 m was purified in the same way as human β 2 m (Ostergaard et al. 2001; Ferre et al. 2003) . Briefly, the ureadissolved β 2 m protein was purified by Ni 2 +/IDA metal chelating affinity column chromatography, refolded by drop-wise dilution into an excess refolding buffer under stirring (25 mM Tris, 300 mM urea, pH 8.00), and then concentrated (VivaFlow, 10 kDa). The refolded product was purified by Ni 2 +/IDA metal chelating affinity column chromatography again (this time in aqueous buffer, i.e., without urea). Fractions containing HAT-pβ 2 m were iden-tified by SDS-PAGE and pooled. Removal of the HAT tag was performed by cleavage with factor Xa restriction protease (FXa) followed by renewed purified by Ni 2 +/ IDA metal chelating affinity and Superdex200 gel filtration column chromatography, concentrated by spin ultrafiltration (10 kDa), mixed 1:1 with glycerol, and stored at −20°C.
Protein samples were mixed 1:1 in SDS sample buffer (4% SDS, 17.4% glycerol, 0.003% bromophenol blue, 0.125 M Tris, 8 mM IAA (iodoacetamide)) with or without reducing agent (2-mercaptoethanol) as indicated, boiled for 3 min, spun at 20,000×g for 1 min, and loaded onto a 12% or 15% running gel with a 5% stacking gel. Gels were run at 180 V, 40 mA for 50 min.
Peptide-MHC class I interaction measured by radioassay and spun column chromatography A HLA-A*11:01-binding peptide, KVFPYALINK (nonnatural consensus sequence A3CON1 ), was radiolabeled with iodine ( 125 I) using a chloramine-T procedure (Hunter and Greenwood 1962) . Dose titrations of MHC-I heavy chains (HHH or HHP) were diluted into refolding buffer (Tris-maleate-PBS) and mixed with β 2 m (human or porcine) and radiolabeled peptide, and incubated at 18°C overnight. Then binding of radiolabeled peptide to MHC-I was determined in duplicate by Sephadex™ G50 spun column gel chromatography as previously described . MHC bound peptide eluted in the excluded volume, whereas free peptide was retained on the microcolumn. Both fractions were counted by gamma spectroscopy, and the fraction peptide bound was calculated as excluded radioactivity divided by total radioactivity.
To examine the affinity of the interaction, increasing concentrations of unlabeled competitor peptide were added. When conducted under limiting concentrations of MHC-I molecule, the concentration of competitor peptide needed to effect 50% inhibition of the interaction, the IC 50 , is an approximation of the affinity of the interaction between MHC-I and the competitor peptide.
Peptide-MHC class I interaction measured by an enzyme-linked immunosorbent assay Peptide-MHC-I interaction was also measured in a modified version of a previously described enzyme-linked immunosorbent assay (ELISA) (Sylvester-Hvid et al. 2002) . Briefly, denatured biotinylated recombinant MHC-I heavy chains were diluted into a renaturation buffer containing β 2 m and graded concentrations of the peptide to be tested and incubated at 18°C for 48 h allowing equilibrium to be reached. We have previously demonstrated that denatured MHC molecules can de novo fold efficiently, however, only in the presence of appropriate peptide. The concentration of peptide-MHC complexes generated was measured in a quantitative sandwich ELISA (using streptavidin as capture layer and the monoclonal anti-β 2 m antibody, BBM1, as detection layer) and plotted against the concentration of peptide offered (Sylvester-Hvid et al. 2002) . A prefolded, biotinylated FLPSDYFPSV/ HLA-A*02:01 (Kast et al. 1994 ) complex was used as standard. Because the effective concentration of MHC (3-5 nM) used in these assays is below the equilibrium dissociation constant (K D ) of most high-affinity peptide-MHC interactions, the peptide concentration, ED 50 , leading to half-saturation of the MHC is a reasonable approximation of the affinity of the interaction.
The experimental strategy of PSCPL has previously been described (Stryhn et al. 1996) . The construction of the sublibraries and the ELISA-driven quantitative measurements of MHC interaction are as given above.
Briefly, the relative binding (RB) affinity of each PSCPL sublibrary was determined as RB (PSCPL)=ED 50 (X 9 )/ ED 50 (PSCPL) (where ED 50 is the concentration needed to half-saturate a low concentration of MHC-I molecules) and normalized so that the sum of the RB values of the 20 naturally occurring amino acids equals 20 (since peptides with a given amino acid in a given position are 20 times more frequent in the corresponding PSCPL sublibrary than in the completely random X 9 library). A RB value above 2 was considered as the corresponding position and amino acid being favored, whereas a RB value below 0.5 was considered as being unfavorable (these thresholds represent the 95% confidence intervals). An anchor position (AP) value was calculated by the equation ∑(RB−1) 2 . A primary anchor position is characterized by one or few amino acids being strongly preferred and many amino acids being unacceptable. We have arbitrarily defined anchor residues as having an AP value above 15 ). The peptide-SLA-I*0401 binding activity of each sublibrary was determined using previously published biochemical binding assay (ELISA) (Sylvester-Hvid et al. 2002 ) (with the modifications described above).
Sequences logos describing the predicted binding motif for each MHC molecule were calculated as described by Rapin et al. (2010) . In short, the binding affinity for a set of 1,000,000 random natural 9mer peptides was predicted using the NetMHCpan method, and the 1% strongest binding peptides were selected for construction of a position-specific scoring matrix (PSSM). The PSSM was constructed as previously described including pseudo-count correction for low counts. Next, sequence logos were generated from the amino acid frequencies identified in the PSSM construction. For each position, the frequency of all 20 amino acids is displayed as a stack of letters. The total height of the stack represents the sequence conservation (the information content), while the individual height of the symbols relates to the relative frequency of that particular symbol at that position. Letter shown upside-down are underrepresented compared to the background (for details see Rapin et al. (2010) ).
MHC distance trees were derived from correlations between predicted binding affinities. For each allelic MHC-I molecule, the binding affinity was predicted for a set of 200,000 random natural peptides using the NetMHCpan method. Next, the distance between any two alleles was defined, as D = 1− PCC, where PCC is the Pearson correlation between the subset of peptides within the superset of top 10% best binding peptides for each allele. In this measure, two molecules that share a similar binding specificity will have a distance close to 0 whereas two molecules with non-overlapping binding specificities would have a distance close to 2. Using bootstrap, 100 such distance trees were generated, and branch bootstrap values and the consensus tree were calculated.
We have previously generated highly active, recombinant human MHC-I (HLA-I) molecules and accompanying highthroughput assays and bioinformatics prediction resources. Here, we transfer the underlying approaches to an important domesticated livestock animal, the pig, and its MHC system, the SLAs. MHC-I molecules are composed of a unique and highly variable distal peptide-binding platform consisting of the alpha 1 (α 1 ) and alpha 2 (α 2 ) domains of the MHC-I heavy chain (HC) and a much more conserved proximal immunoglobulin-like membrane attaching stalk consisting of the alpha 3 (α 3 ) domain of the HC noncovalently associated with the soluble MHC-I light chain (β 2 m).
A priori, the establishment of recombinant SLA molecules is complicated by the lack of validated reagents. Any failure could therefore be caused either by real technical problems in generating SLA molecules, or merely by a lack of information about strong peptide binders to the SLA in question. To reduce this uncertainty, we decided to migrate from human to pig MHC-I in a step-wise manner and generate an intermediary chimeric MHC-I molecule composed of a well-known human peptide-binding platform attached to a SLA stalk, which might allow us to assess whether we could generate a functional SLA stalk consisting of SLA-1*0401 α 3 HC and pig β 2 m. To this end, we used the α 1 α 2 domains of the HLA-A*11:01 molecule, which we expected should be able to bind a known highaffinity HLA-A*11:01-binding peptide (KVFPYALINK). This peptide could be 125 I radiolabeled and used in a very robust peptide-binding assay testing whether the human stalk could be replaced with the corresponding SLA stalk. Once that had been successfully established, the entire SLA-1*0401 molecule would be constructed and tested.
We have previously expressed and purified the extracellular segment spanning positions 1-275 of the human HLA-A*11:01 in a denatured and pre-oxidized version that rapidly refold and bind appropriate target peptides (Ostergaard et al. 2001; Ferre et al. 2003) . Codon-optimized genes encoding the corresponding segments of SLA-1*0401 (α 1 α 2 ) and SLA-1*0401 (α 3 ) were constructed as described in the "Materials and methods" section and used to replace the HLA-A*11:01 gene segment in the above construct generating a new construct allowing for the expression of SLA-1*0401. For the generation of HLA-A*11:01/SLA-1*0401 chimeras, the genes encoding α 1 (spanning positions 1-90), α 2 (spanning positions 91-181), and/or α 3 (spanning positions 182-275) domains of HLA-A*11:01 and SLA-1*0401 were exchanged using Seam-Less and touch-down cloning strategies. Genes encoding the extracellular segments 1-275 of the above natural or chimeric MHC-I molecules were C-terminally fused to a biotinylation tag (as indicated for SLA-1*0401 in Online Resource 1), inserted into pET28, and expressed in inclusion bodies in E. coli (Fig. 2 shows SDS-PAGE of lysates of recombinant E. coli before and 3 h after IPTG induction). The fusion proteins were extracted into 8 M urea (without any reducing agents), purified by ion exchange, hydrophobic and gel filtration chromatography (all conducted in 8 M urea, without any reducing agents) (Fig. 3 shows SDS-PAGE of the purified SLA-1*0401 after gel filtration), concentrated, and stored in urea at −20°C.
Testing a chimeric molecule consisting of a SLA-1*0401 stalk and a HLA-A*11:01 peptide-binding platform-comparing human versus porcine β 2 m To test the proximal immunoglobulin-like membrane attaching SLA stalk, we generated recombinant porcine β 2 m and a chimeric human/porcine MHC-I heavy chain molecule where the α 1 α 2 were derived from the human HLA-A*11:01, and the α 3 was derived from the porcine SLA-1*0401. Since this construct contains the entire peptide-binding platform of HLA-A*11:01, we reasoned that the binding of the HLA-A*11:01 restricted peptide, KVFPYALINK, could be used as a functional readout of the refolding, activity, and assembly of the entire chimeric molecule including the porcine SLA stalk. For comparison, we tested the supportive capacity of human β 2 m and folding ability of the entirely human HLA-A*11:01. A total of four combinations could therefore be tested: porcine or human β 2 m in combination with either HHP or HHH (where the first letter indicates the origin of the α 1 domain (Human HLA-A*11:01 or Porcine SLA-1*0401), the second letter the origin of the α 2 domain, and the third letter the origin of the α 3 domain). A concentrationtitration of heavy chain was added to a fixed excess concentration (3 μM) of β 2 m and a fixed trace concentration (23 nM) of radiolabeled peptide. As shown in Figs. 4 and 5, the four combinations gave almost the same heavy chain dose titration with a half-saturation occurring around 1-2 nM heavy chain. Porcine β 2 m supported folding of the chimeric (HHP) α chain slightly better than it supported folding of the human (HHH) α chain. Human β 2 m supported folding of HHP and HHH equally well. Thus, a recombinant SLA stalk can fold and support peptide binding of the peptide-binding platform. These results also suggest that human β 2 m can support folding and peptide binding of porcine MHC-I heavy chain molecules.
Using a positional scanning combinatorial peptide library approach to perform an unbiased analysis of the specificity of SLA-1*0401 and human-pig chimeric MHC class I molecules Using human β 2 m to support folding, the recombinant SLA-1*0401 and human-pig chimeric MHC-I molecule were tested for peptide binding. We have previously described how PSCPL can be used to perform an unbiased analysis of MHC-I molecules (Stryhn et al. 1996) . A PSCPL consists of 20 sublibraries for each position where one of each of the 20 natural amino acids have been locked and all other positions contain random amino acids. Analyzing how much of each PSCPL sublibrary is needed to support MHC-I folding (see examples in Fig. 6 ) and comparing each sublibrary with a completely random library, the effect of any amino acid in any position can be examined and expressed as a RB value. Further, an AP value calculated as the sum of squared deviations of RB values for each position can be used to identify the most prominent anchor position (see "Materials and methods" for calculations). Thus, the specificity of a nonamer binding MHC-I molecule can be analyzed comprehensively with 9×20+1 completely random library=181 sublibraries (Stryhn et al. 1996) .
Here, this approach was used to perform a complete experimental analysis of SLA-1*0401 and a limited analysis of the chimeric HPP and PHP molecules. A nonamer PSCPL analysis of SLA-1*0401 can be seen in Table 1 . AP values identified positions 9, 3, and 2 (in that order of importance) as the anchor positions of SLA-1*0401. In position 9, the amino acid preferences were dominated by the large and bulky aromatic tyrosine (Y), tryptophane (W), and phenylalanine (F), all having RB values above 4 (Table 1 ). In the almost equally important position 3, preferences for negatively charged amino acids glutamic acid (E) and aspartic acid (D) were observed. In the lesser important position 2, the most preferred amino acids were the hydrophobic amino acids valine (V), isoleucine (I), and leucine (L), followed by the polar amino acids threonine (T) and serine (S).
Finally, a very limited PSCPL analysis was performed for the two chimeric human HLA-A*11:01/porcine SLA-1*0401 MHC-I molecules, HPP and PHP (Table 2) . For both chimeric molecules, it could be demonstrated that position 9 is a strong anchor position. The positively charged amino acids, arginine (R) and lysine (K), were preferred in position 9 of the chimeric HPP molecule, whereas the aromatic amino acid, tyrosine (Y), was exclusively preferred in position 9 of the chimeric PHP molecule. The positively charged amino acids, arginine (R) and lysine (K), were preferred in position 9 of the chimeric HPP molecule similar to the position 9 specificity of the HLA-A*11:01 molecule. In contrast, the aromatic amino acid tyrosine (Y) was preferred in position 9 of the chimeric PHP molecule similar to the position 9 specificity of the SLA-1*0401 molecule.
Using NetMHCpan to predict peptides that bind to SLA-1*0401 or to human-pig chimeric MHC class I molecules Our recently described neural network-driven bioinformatics predictor, NetMHCpan (version 2.0), has been trained on about 88,000 peptide-binding data points representing more than 80 different MHC-I molecules (primarily HLA-A and HLA-B molecules). We have previously shown that NetMHCpan is an efficient tool to identify peptides that bind to HLA molecules where no prior data exist (Nielsen et al. 2007 ) and demonstrated that NetMHCpan can be extended to MHC-I molecules of other species 1 (Hoof et al. 2009 ). We applied NetMHCpan to our peptide repository of about 10,000 peptides, which over the past decade have been selected to scan infectious agents (e.g., SARS and influenza, Sylvester-Hvid et al. 2004; Wang et al. 2010) , improve coverage of MHC-I specificities (e.g., Buus et al. 2003; Christensen et al. 2003) , etc. We extracted 29 peptides as predicted binders to either the SLA-1*0401, the HPP, or the PHP human/porcine chimeric class I molecules (some of the peptides were predicted to bind to two or even all three of these molecules). All these peptide-MHC-I combinations were tested for binding (Table 3) ; 13 of 14 peptides bound to the SLA-1*0401 molecule with an affinity (IC 50 value) better than 500 nM (6 with an affinity less than 50 nM); all 13 peptides tested on the PHP molecule were strong binders with IC 50 values below 50 nM; and 3 of 12 peptides tested on the HPP molecule bound with an affinity better than 500 nM. Of the 39 peptide-MHC-I combinations tested, 20 (51%) were found to be good binders, 9 (23%) were average binders, and 10 (26%) did not bind well (Table 3) . This is in stark contrast to the 0.5% frequency of binders among randomly selected peptides (Yewdell and Bennink 1999) .
Next, the NetMHCpan method was used to generate PSSMs and sequence logos from the corresponding amino acid frequencies as described by Nielsen et al. (2004) . For each position, the frequencies of all 20 amino acids were displayed as a stack of letters showing the sequence conservation/information content (the height of the entire 1 A preliminary report of SLA-1*0401 binding was given in Hoof et al. (2009) The normalized relative binding (RB) value indicates whether an amino acid is favored (RB>2, bold numbers) or disfavored (RB<0.5, italic numbers) in a given peptide position. The anchor position (AP) value is given by the equation ∑(RB−1) 2 . The important anchor positions 2, 3, and 9 for SLA-1*0401 are underlined stack) and the relative frequency of amino acids (the height of the individual amino acids). Figure 7 shows a specificity tree clustering of the SLA-1*0401 molecule compared to prevalent representatives of the 12 common HLA supertypes that NetMHCpan originally intended to cover . By this token, SLA-1*0401 most closely resembles that of HLA-A*01:01. The limited PSCPL analysis of the chimeric MHC-I molecules revealed strong P9 signals with specificities that seemed to be determined by the origin of the α 1 domain: the HPP chimera showed an HLA-A*11:01-like P9 specificity, whereas the PHP chimera showed a SLA-1*0401/HLA-A*01:01-like specificity. Since the NetMHCpan predictor successfully captured these chimeric specificities (see above), we reasoned that the predictor might also be used to perform in silico dissection of these specificities and used the P9 specificity as an example of such an in silico analysis. The NetMHCpan predictor considers a pseudo-sequence consisting of 34 polymorphic positions, which contain residues that are within 4.0 Å of the atoms of bound nonamer peptides (Nielsen et al. 2007 ). Of the 34 positions of the pseudosequence, 10 delineates the P9 binding pocket; however, only 3 of these, positions 74, 77, and 97, differ between SLA-1*0401 and HLA-A*11:01. To explore the effect of these three residues, we performed in silico experiments where we examined single substitutions Y74D, G77D, and S97I (the letter before the position number indicates the SLA-A*0401 single letter residue, whereas the letter after indicates the HLA-A*11:01 residue) as well as the corresponding triple substitution (YGS-DDI). As described above, PSSMs were generated for each of the in silico molecules followed by a specificity tree clustering (including SLA-A*0401, HLA-A*01:01, and HLA-A*11:01). Figure 8 shows this tree along with the sequence logo plots showing the predicted binding specificity of each in silico MHC-I molecule. Albeit the Y74D and G77D single substitutions showing some of the positively charged P9 peptide residue preference of HLA-A*11:01, they still clustered with HLA-A*01:01. In contrast, the in silico (YGS-DDI) triple substitution clustered with the HLA-A*11:01. This suggests that the NetMHCpan method is capable of defining the residues of the F pocket that determine the specificity of position 9.
We have previously suggested that the specificities of the entire human MHC-I system should be solved ("the human RB and AP values are defined as described in Table 1 MHC", Buus 1999; Lauemoller et al. 2000) . However, due to the extreme polymorphism of the MHCs, any attempt to address the specificity of the entire MHC system is a significant experimental undertaking. During the past decade, we have established a series of technologies to support a general solution of human MHC class I and II specificities. For MHC-I, this includes (1) a highly efficient E. coli expression system for production of recombinant human and mouse MHC-I molecules (both heavy chain and light chain (β 2 m) molecules) Ostergaard et al. 2001) , (2) a purification system for obtaining the highly active pre-oxidized MHC-I heavy chain species (Ferre et al. 2003) , (3) a high-throughput homogenous peptide-MHC-I binding assay for obtaining large data sets on peptide-MHC-I interactions (Harndahl et al. 2009 ), (4) a positional scanning combinatorial peptide library approach for a robust and unbiased analysis of the specificity of any MHC-I molecule (Stryhn et al. 1996) , (5) an immunobioinformatics approach to generate predictors of the peptide-MHC-I interaction, NetMHCpan, that allows predictions to be made for any human MHC-I molecules, HLA-I, even those that have not yet been covered by existing data set (Hoof et al. 2009; Nielsen et al. 2007) , and finally (6) we have demonstrated that pre-oxidized MHC-I molecules can be used to generate MHC-I tetramers in a simple "one-pot, mix-and-read" manner (Leisner et al. 2008) . Here, we propose that the next goal should be to extend the overall approach to MHC-I molecules of other species of interest. Mouse and rats have been extensively studied in the past, but much less reagents and information have accrued for the MHC-I molecules of other species. Here, we have used an important livestock animal, the pig, as a model system and demonstrated that it indeed is possible to transfer the original human approach to other species. Before attempting to generate a recombinant version of the entire porcine SLA-1*0401 molecule, we grafted the more conserved membrane-proximal "stalk" (the immunoglobulin-like class I heavy chain α 3 and β 2 m domains) of porcine SLA-1*0401 onto the peptide-binding domain of HLA-A*11:01 generating a chimeric human/ porcine MHC-I molecule. This chimeric molecule retained the peptide-binding specificity of the HLA-A*11:01 molecule, and it clearly demonstrated that the recombinant porcine stalk was functional and, by inference, also properly folded. It also suggests that the peptide-binding specificity of the distal domains do not crucially depend upon the identity of the proximal stalk. Further, comparing the ability of human and porcine β 2 m to support MHC-I complex formation using either a human or a porcine MHC-I stalk, we demonstrated that every combination (porcine β 2 m/human-α 3 , porcine β 2 m/porcine-α 3 , human β 2 m/human-α 3 , and human β 2 m/porcine-α 3 ) showed al- most the same heavy chain dose titration with identical half-saturations. These results illustrate the ability for porcine and human β 2 m to support complex formation of SLA molecules and vice versa and suggest evolutionary that the stalk is quite conserved. Next, we generated the entire SLA-1*0401 heavy chain and succeeded in generating complexes using human β 2 m as the light chain and PCSPL as peptide donors. The latter solved the a priori problem of not knowing which peptides would be needed to support proper folding of SLA-1*0401, and it did so in an unbiased manner. Furthermore, this approach is highly efficient since it readily establishes a complete matrix representing the amino acid preference for each amino acid and each position of a nonamer peptide. The specificity of SLA-1*0401 shows two primary anchors: one in positions 9 with a preference for aromatic amino acids and another in position 3 with a preference for negatively charged amino acids. In addition, the SLA-1*0401 features a secondary anchor in position 2 with hydrophobic or polar amino acid preferences.
An alternative approach to solve the problem of identifying peptides that support folding of MHC-I molecules of so far unknown specificity is to use our recently developed panspecific predictor, NetMHCpan. The successful use of this predictor to initiate peptide-binding studies was recently Fig. 7 Specificity tree clustering of the SLA-1*0401 molecule compared to prevalent representatives of the 12 common HLA supertypes ). The distance between any two MHC molecules and the consensus tree is calculated as described in "Materials and methods". All branch points in the tree have bootstrap values of 100%. Sequence logos of the predicted binding specificity are shown for each molecule. In the logo, acidic amino acids [DE] are shown in red, basic amino acids [HKR] in blue, hydrophobic amino acids [ACFILMPVW] in black, and neutral amino acids [GNQSTY] in green. The axis of the LOGOs indicates in all case positions one through nine of the motif, and the y-axis the information content (see Materials and methods) demonstrated for HLA-A*3001 . Although originally developed to cover all HLA-A and HLA-B molecules, it has also been shown to extend to MHC-I molecules of other species (Hoof et al. 2009 ). Here, we demonstrate that the NetMHCpan predictor is capable of extracting MHC-I sequence information across species and correctly relate this to peptide binding even in the absence of any available data for the specific query MHC-I molecule, i.e., the SLA-1*0401 as well as the chimeric HPP (hα 1 pα 2 pα 3 ) and PHP (pα 1 hα 2 pα 3 ) molecules. It is not clear why binding of the PHP chimera was more efficiently predicted than binding of the HPP chimera. One could speculate that NetMHCpan has not captured the effect of the different positions of the pseudo-sequence equally well and not all positions and pockets (and by inference-not all chimeric molecules) are therefore predicted equally well.
Using the NetMHCpan predictor to cluster SLA-1*0401 and representative molecules of 12 human HLA supertypes according to predicted peptide-binding specificities, the SLA-1*0401 specificity closely resembled that of HLA-A*01:01 (IEDB, http://www.immuneepitope.org/MHCalleleId/142, accessed March 9th 2011). This result was also obvious from an inspection of the PSCPL analysis of the SLA-1*0401. The PSCPL analysis of the P9 specificity of the SLA-1*0401 and the two chimeric molecules suggested that the P9 specificity primarily was determined by the α 1 domain. This contention was further strengthened by a NetMHCpan-driven in silico analysis of the residues delineating the F pocket, which interacts with P9. This suggests that NetMHCpan can be used to design and interpret detailed experiments addressing the structurefunction relationship of peptide-MHC-I interaction. In the case of SLA-1*0401, NetMHCpan suggests that Y74, G77, and S97 play a prominent role in defining the P9 F pocket. Whereas the NetMHCpan readily captured the P9 anchor residue of SLA-1*0401, it did not capture the P3 anchor (at least not in the 2.4 version). We surmise that this shortcoming is due to insufficient examples of the use of P3 anchors within the currently available peptide-MHC-I binding data. Inspecting the pseudo-sequence of SLA-1*0401 and HLA-A*01:01 vs. HLA-A*11:01 suggests that the presence of an arginine in position 156 might Fig. 8 Comparison of specific in silico mutations of the SLA-1*0401 molecule and comparison with the two HLA molecules: HLA-A*11:01 and HLA-A*01:01. The distance between any two MHC molecules and the consensus tree is calculated as described in "Materials and methods". All branch points in the tree have bootstrap values of 100%. The SLA-1*0401 mutations are indicated as Y74D, G77D, and S97I, where the letter before the position number indicates the SLA-1*0401 single letter residue and the letter after indicates the HLA-A*11:01 residue. YGS-DDI is the corresponding triple substitution. Sequence logos are calculated and visualized as described in Fig. 7 . The axis of the LOGOs indicates in all case positions one through nine of the motif, and the y-axis the information content (see Materials and methods) explain the preference for negatively charged amino acid residues in P3. Future NetMHCpan-guided experiments could pointedly address this question, and the resulting data could complement existing data and be used to update and improve the NetMHCpan predictor.
All in all the two complementary approaches, PSCPL and NetMHCpan, agreed on the specificity of the SLA-1*0401 molecule, as well as of the two chimeric MHC-I molecules. Thus, the specificity of SLA-1*0401 appear to be well established. This specificity has successfully been used to search for foot-and-mouth disease virus (FMDV)specific CTL epitopes in FMDV-vaccinated, SLA-1*0401positive pigs, and the recombinant SLA-1*0401 molecules have been used to generate corresponding tetramers and stain pig CTLs (Patch et al. 2011) . In conclusion, we here present a set of methods that can be used to generate functional recombinant MHC-I molecules, map their specificities and identify MHC-I-restricted epitopes, and eventually generate peptide-MHC-I tetramers for validation of CTL responses. This suite of methods is not only applicable to humans, but potentially to any species of interest. Immunogenetic Factors Associated with Severe Respiratory Illness Caused by Zoonotic H1N1 and H5N1 Influenza Viruses Following the 2009 H1N1 pandemic and ongoing sporadic avian-to-human transmission of H5N1 viruses, an emphasis has been placed on better understanding the determinants and pathogenesis of severe influenza infections. Much of the current literature has focused on viral genetics and its impact on host immunity as well as novel risk factors for severe infection (particularly within the H1N1 pandemic). An understanding of the host genetic determinants of susceptibility and severe respiratory illness, however, is currently lacking. By better defining the role of genetic variability in influenza infection and identifying key polymorphisms that impair the host immune response or correlate with protection, we will be able to better identify at-risk populations and new targets for therapeutic interventions and vaccines. This paper will summarize known immunogenetic factors associated with susceptibility or severity of both pH1N1 and H5N1 infections and will also identify genetic pathways and polymorphisms of high relevance for future study. Transmission of zoonotic influenza A viruses to humans is commonly the cause of new pandemics, which typically result in high disease burden and increased symptomatic severity and mortality. In order to predict which populations may be at highest risk of infection and to develop more effective therapeutic interventions and vaccines, a thorough understanding of both viral and host contribution to pathogenesis is required. In both the recent 2009 H1N1 (pH1N1) pandemic and the on-going rare avian-to-human transmission of H5N1, numerous studies have taken an indepth look at the impact of viral evolution and mutation on viral pathogenesis. Conversely, while both human and animal model studies of the host immune response to infection have identified correlates of severe disease, the contribution of host genetics to these correlates and to variability in susceptibility remains relatively unknown. Identification of host genetic polymorphisms contributing to altered susceptibility or disease severity has several benefits: identification of high-risk populations at greater need of prophylactic intervention, elucidation of host proteins important in virus-host interactions, and new targets for therapeutic interventions or vaccine development [1] . Studies of host genetics have provided important contributions to the study of other infectious diseases, including HIV, SARS, and HCV. This paper will describe what is currently known about the impact of host immunogenetics in both pH1N1 and H5N1 infections and will identify highly relevant polymorphisms and genetic pathways that could be investigated in future work.
H1N1 influenza viruses emerged as a result of a presumed or documented reassortment of segments from viruses of zoonotic origin with human-adapted influenza virus to cause pandemic spread in 1918 and again in 2009. The 2009 appearance of a swine-origin reassortant virus led to the first pandemic of the 21st century. During earlier pandemics, records indicate that certain individuals or populations appeared to be more susceptible to severe disease, but the 2 Clinical and Developmental Immunology ability to conduct studies in order to understand the immune mechanisms that underlay the increased propensity for complications was limited. The 2009 H1N1 (pH1N1) pandemic was accompanied by improved surveillance, thereby facilitating better estimation of disease severity and methods to examine the immune mechanisms behind complicated disease [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . This surveillance allowed for the identification of several novel risk factors among various populations, but with a limited understanding of the genetic variation that may contribute to those risk factors.
Infection. The 1918 H1N1 as well as the recent 2009 pandemics were both notable for the comparatively high rates of morbidity among healthy, young adults not typically observed with seasonal influenza [11] . During the recent pandemic, several studies of confirmed pH1N1 cases in Canada and the US reported the median age of severe infections to be 23-27 years old [8, 10] . In Canada, 30%-48% of infections also presented in persons with comorbidities; diabetes, heart disease, and immunosuppression were associated with the highest risk of severe infection, while lung diseases and obesity were among the most common underlying conditions [10, [12] [13] [14] . The role of pregnancy as a risk factor, regardless of the stage, was also supported by a myriad of reports; among hospital admissions, pregnancy accounted for roughly 30% of female cases aged 20-39 years old [9, 12, 15] .
Ethnicity was another major risk factor of pH1N1 susceptibility identified in several populations in North America and Australasia. The increased proportion of aboriginal individuals presenting with severe pH1N1 infection was not unique for this pandemic and was also seen in the 1918 H1N1 pandemic during which mortality in aboriginal communities in North America (3%-9%) was significantly higher than among nonaboriginal communities [16, 17] . In the 2009 pandemic, Pacific Islanders accounted for 2.5% of the Australian population but made up 9.7% of patients admitted to Australian ICUs with confirmed pH1N1. Maori individuals represent 13.6% of the New Zealand population, but accounted for 25% of ICU admissions in the ANZIC study [18] . Kumar et al. [12] also reported 25.6% of the individuals admitted to ICUs in Canada belonged to First Nations, Inuit, Metis, or aboriginal ethnicities; this is an overrepresentation compared to the 4.4% rate of selfreported aboriginal ethnicity according to the 2001 census (Statistics Canada). Similarly, pH1N1 mortality rates among American-Indian/Alaska Natives were four times higher than persons in all other ethnic populations combined in the United States [19] .
None of these studies examined the causal factors that lead to the higher influenza mortality in the high-risk groups described. It is clear that multiple converging risks account for the high rates of complications, including socioeconomic factors such as inability to access care, delayed seeking of care, higher rates of poverty, and greater numbers of household members. A few of the risk factors listed in the previous sections, however, share a degree of immune system impairment. One can, therefore, speculate that the partial protection afforded by the immune system, primarily by cross-reactive CD8+ T-cells recognizing viral epitopes, is decreased in some of the previously described groups (pregnancy is a good example). Additionally, genetic variation in immune-related genes leading to either gain-offunction or loss-of-function phenotypes could contribute to the variation observed in pH1N1 susceptibility and disease severity.
Associated with Severity of Pandemic H1N1 Infection. When a novel strain of influenza emerges, the pre-existing antibody response directed largely at the surface glycoproteins is rendered ineffective. In these cases, the mechanisms underlying heterosubtypic cross-protection assume a dominant role and it is, therefore, not surprising that immune dysfunction caused by underlying genetic polymorphisms may lead to impaired responses and would, therefore, be associated with adverse outcomes. During the 2009 H1N1 pandemic, several immunogenetic determinants of severe disease were identified.
When a novel strain of influenza emerges, the preexisting antibody response directed largely at the surface glycoproteins is rendered ineffective. In these cases, the mechanisms underlying heterosubtypic cross-protection assume a dominant role and it is, therefore, not surprising that immune dysfunction caused by underlying genetic polymorphisms may lead to impaired responses and would therefore be associated with adverse outcomes. During the 2009 H1N1 pandemic, several immunogenetic determinants of severe disease were identified.
Allele. The CCR5 protein is a chemokine receptor expressed primarily on T cells, macrophages, and dendritic cells. CCR5 plays a pivotal role in mediating leukocyte chemotaxis in response to chemokines (including RANTES, MIP-1α, and MIP-1β) and is believed to be important in the homing of many immune cell subsets, including regulatory T cells and Th17 cells, to mucosal surfaces. Until recently, the purported role of CCR5 in supporting the antiviral immune response was limited to appreciation of the effect of receptor deficiency in protecting from HIV infection and disease progression among individuals homozygous for the Δ32 allele. The understanding of the roles played by CCR5 was expanded when the Δ32 allele was found to be associated with an increased risk of symptomatic and fatal West Nile Virus (WNV) infection [20] [21] [22] , a severe adverse reaction to the live yellow fever virus vaccine, and with severe tick-borne encephalitis symptoms [23, 24] . Together, these data suggest that CCR5 may also play a critical role in the immune response to flavivirus infections. The spectrum of symptomatic severity observed during the 2009 H1N1 pandemic led our group to study CCR5 genotype among patients requiring intensive care admission and respiratory support for severe H1N1 symptoms. Among twenty samples of confirmed severe pH1N1 infection, the CCR5Δ32 allele was found in 5 out of 9 of the Caucasian individuals, giving a Caucasian allele frequency of 27.8% [25] (Table 1 ). This observed frequency is approximately 2.5 
Polymorphisms previously linked to IgG2 deficiency, but not corroborated in H1N1 patients [28] [29] [30] NLRP3
Association with dysregulation of inflammatory response (2107), alteration of NLRP3 mRNA stability and enhancer activity [33, 34] HLA Various alleles Influenza-specific CTL responses exhibit varying frequency and magnitude across various HLA alleles [35] times higher than that reported for local North American Caucasian populations [26, 27] . Given the small sample size available in this cohort, further studies will be required to conclusively determine the impact of CCR5 deficiency on pH1N1 susceptibility and severity.
Similarly to IgG1, IgG2a and IgG2b are able to bind to Fc receptors with high affinity and are thought to be important in protecting against influenza infection. A group of Australian investigators identified an index case of severe influenza in a pregnant woman with IgG2 subclass deficiency and subsequently measured total IgG and IgG subclasses in all patients with pH1N1 infection requiring ICU care (many of whom were pregnant) compared to less severe controls and asymptomatic pregnant women presenting to antenatal clinic. A low level of IgG2 was correlated with severe pH1N1 infection after multivariate analysis. Measurement of IgG2 after 90 days among 15 of the surviving IgG2-deficient patients showed that 11 remained IgG2 deficient despite albumin levels returning to baseline values [28] . Additionally, a case-control study from China enrolled 38 Asian patients with respiratory failure due to severe pandemic influenza and compared IgG2 levels with 36 mild cases. They did not find any cases of selective IgG2 deficiency, but did observe significantly lower levels of IgG2 among the severe cases (despite normal levels of the other IgG subclasses) [29] . The authors looked for the presence of FcγRIIa and IGHG2 genotypes ( Table 1 ) that were previously shown to be associated with IgG2 deficiency, but found similar rates among cases and controls. They did, however, corroborate the previously reported finding [30] of cytokine dysregulation among severe cases of infection and suggested that the mechanism responsible for the low IgG2 is the more robust Th1 response and a suppressed Th2 response. Given the lack of FcγRIIa and IGHG2 genotype data available from the Gordon et al. study [28] , the impact of these polymorphisms on IgG2 levels and severe pH1N1 infection remains to be determined.
Severe Influenza. Genetic polymorphisms associated with pH1N1 susceptibility and disease severity identified to date are limited, and much of the data is derived from small cohorts. An improved understanding of the sequence of immune responses to influenza as well as the application of newer technologies that employ high-throughput expression array or sequencing technologies can be used to guide a more focused approach to identify specific pathways that may be differentially activated by individuals with severe disease. Based on available data, we can identify several immune pathways and their genetic variants that warrant further investigation.
Recently, emphasis has been given to the role of the inflammasome in viral infections and, specifically, influenza. NLRP3 (NOD-like receptor family, pyrin domain-containing 3) inflammasomes are multiprotein complexes containing NLRP3, ASC (or Pycard), and caspase 1. Activation of cytokine/chemokine release through NLRP3 requires a signal derived from Toll-like receptor (TLR) stimulation, with resultant production of pro-IL-1β, -IL-18, and -IL-33. The prointerleukins are in turn cleaved to their respective active forms by caspase 1, which requires the input of an additional signal. The second signal in the context of influenza has been elegantly demonstrated by Ichinohe et al. [31] , who showed that golgi-localized H1N1 M2 is both necessary and sufficient to trigger inflammasome activation. Differential expression of the components of the two signaling cascades that are required for inflammasome activation may therefore explain differences in influenza disease severity. Indeed, mouse knockout studies have shown that intact inflammasomes are necessary for innate immune responses to influenza A, chemokine production, and late stage viral clearance (reviewed in [32] ). Evidence suggests that NLRP3-mediated signaling is also important in cellular recruitment and tissue repair during infection. A similar requirement for proper ASC function was observed in adaptive influenza immune responses. Multiple NLRP3 SNPs have been associated with dysregulated inflammation responses and NLRP3 mRNA stability in humans but have not been examined in the context of pH1N1 susceptibility or mortality [33, 34] (Table 1 ).
The CD8+ T-cell response is a strong predictor of vaccine-induced protection and is thought to be particularly valuable in the elderly. Because this response is focused on more conserved viral proteins, it has the additional benefit of providing some cross-reaction with new influenza strains [46, 47] . Undoubtedly, multiple factors underlie the differences in disease severity among ethnic groups, as previously discussed. From an immunogenetic perspective, however, HLA alleles are among the most variable human genes, and it is therefore conceivable that variable proportions of HLA class I alleles among ethnic groups may lead to qualitatively and quantitatively distinct CD8+ T-cell responses, as well as differences in immunodominant epitopes (Table 1) . Boon et al. [35] have demonstrated that the frequency of CTL responses specific for the HLA-B8restricted epitope NP 380-388 was lower in HLA-B27-positive donors than in HLA-B27-negative donors. They also showed that the HLA-A1-restricted epitope NP 44-52 responses were higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors. These observations suggest that the epitope specificity and magnitude of the CTL response is related to the HLA class I genetic background [35] .
The role of cell-mediated immunity in ameliorating infection caused by novel influenza strains has been the focus of intense study [48] and it is, therefore, the most compelling area to investigate in order to identify immunogenetic factors that predict severity of pandemic H1N1 influenza. A comprehensive investigation was undertaken by Bermejo-Martin et al. [49] in a study from Spain. They enrolled 19 critically ill patients with primary pH1N1 influenza pneumonia and used gene expression analysis in order to identify host immune responses associated with severe disease defined by illness requiring mechanical ventilation. They identified impaired expression of a number of MHC class II and MHC class I genes, T-cell receptor-associated genes, and also of a cluster of genes thought to be involved in dendritic cell maturation, indicating defective antigen presentation in the most severe group of patients. They found further evidence for the effect of altered antigen presentation on the development of an appropriate adaptive response against the virus in the impaired expression of a group of genes critical to the activation and function of both T and B cells. The group with severe illness also showed higher expression of genes involved in IL-6 and IL-10 pathways, and these results were in concordance with the high serum levels of IL-6 and IL-10 in the group dependant on mechanical ventilation. The authors concluded that severe disease is associated with an impaired transition from innate to adaptive immunity in response to the pH1N1 virus, similar to observations in the context of SARS and severe infections caused by H5N1. The impaired adaptive response was also associated with delayed viral clearance. This study did not, however, explore the role of genetic polymorphisms in this immune dysregulation.
Pathogenic avian influenza A/H5N1 viruses are endemic among poultry populations across Asia and Africa and present an ongoing risk for avian-to-human transmission. As of June 22, 2011, the WHO reports a total of 562 confirmed H5N1 cases and 329 deaths across 15 countries worldwide [50] . Although human-to-human transmission of H5N1 has so far been rare, the potential for viral evolution into a more transmissible strain raises the possibility that H5N1viruses could cause a pandemic. Given the high mortality rate currently associated with human H5N1 infection, a thorough understanding of the immune response and the underlying mechanisms of viral pathogenesis is crucial to improve treatment and to identify highly susceptible populations.
To date, much of the research into the immunobiology and pathogenesis of human H5N1 infection has focused on the H5 haemagglutinin protein and the impact of viral genetic polymorphisms on viral pathogenicity. Although studies have begun to characterize of the role of the host immune response in pathogenesis, the impact of genetic variability on susceptibility and disease severity remains an important gap in our current knowledge. By identifying host genetic polymorphisms that exacerbate immunopathology or provide protection, we will be able to improve treatment and future vaccines [1, 51] .
Severity. The precise impact of host genetic variability on H5N1 susceptibility remains somewhat controversial, given the limited case data available and the relatively low number of published studies. A case study in Indonesia [52] found evidence of clusters of H5N1 infection among blood relatives that may be indicative of shared genetic susceptibility, but the authors were unable to rule out a shared viral exposure or altered viral pathogenesis in any of the clusters. Similar observations in a number of additional studies have prompted several authors to suggest a potentially strong genetic basis for H5N1 susceptibility [53] [54] [55] [56] [57] . A compilation of confirmed H5N1 cases worldwide found that, on average, 22% of cases occurred in clusters, and only 6% of cases within the clusters were not genetically related to other cluster members [1] . While this data does not conclusively point to genetic variation as an important determinant of susceptibility, it is important to note that human-to-human transmission of H5N1 is very rare, and therefore does not likely explain the high degree of genetic relatedness among cases [1] . It would also be expected that clusters of nonrelated individuals working in the poultry industry would be more prominent than genetically related clusters, if people are at equal risk of infection [1] . Some studies, however, have suggested that the observed clustering of infections among families could occur due to chance alone at the low rates of infection that are observed in H5N1 and highlight the difficulty in drawing conclusions from the currently available data [58] . TLR3 908T/C Missense mutation identified in a patient with influenza-associated encephalopathy [45] host/virus interactions and the qualities of a protective immune response. To date, a number of candidate genes have been identified from both human and mouse immunobiology studies. Mouse models of influenza infection have the advantage of being able to dissect gene expression kinetics and the characteristics of the immune response at various stages of infection. Comparison of infection across inbred mouse lines demonstrates significant differences in viral titre and core temperature, as well as distinct patterns of immune gene upregulation, suggesting an important contribution of host genetic background [59] .
Interactions. Genetic variation affecting host proteins required for viral entry and pathogenesis may partially explain the sporadic and rare nature of avian-tohuman H5N1 transmission. Although humans do express the SAα-2,3Gal molecules that are efficiently bound by avian H5N1, their expression is usually limited to the lower respiratory tract and has only occasionally been detected in the nasal mucosa and upper respiratory tract [60, 61] . Additionally, the binding of several influenza strains to human erythrocytes is highly variable, with up to a 40fold difference between individuals tested in one study, suggesting a role for genetic polymorphism in regulating susceptibility [62] . Whether variation in the human ST3 beta-galactosamide alpha-2,3-sialyltranferase 1 (ST3GAL1) gene that produces the SAα-2,3Gal linkage affects H5N1 susceptibility is not known, but remains a possibility [63] . Interestingly, a population genetics study designed to detect human SNPs under virus-driven selective pressure found a significant enrichment of glycan biosynthesis gene SNPs associated with viral selection, including rs3758105 (intronic A/G SNP) in the ST3GAL1 gene [64] (Table 2) . Data demonstrating the infection of upper respiratory tract cells with H5N1 in vitro also suggests the presence of additional cellular receptors for the virus [61] . Alternately, prevention of viral attachment in the respiratory tract is accomplished by host proteins that can sterically hinder viral HA binding, or aggregate and opsonize the virus. These proteins include serum mannose-binding lectin 2 (MBL2) and surfactant, pulmonary-associated protein A1 and D (SFTPA1 and SFTPD, resp.). A SNP in MBL2 (230G/A), resulting in low serum MBL levels, is associated with SARS susceptibility [36, 37] , while polymorphisms in SFTPA1 and SFTPD are associated with other respiratory illnesses [65, 66] (Table 2 ). To date, none of these polymorphisms have been investigated with respect to H5N1 susceptibility.
Induction of an innate immune response following infection can occur as a result of the activation of pattern recognition receptors (PRRs), commonly known as Toll-like receptors (TLRs). TLRs recognize many elements of foreign pathogens, including LPS, flagellin, and dsRNA, and initiate signaling cascades that result in the production of type I interferons. Accumulating data suggests that genetic variation in TLRs and their associated signaling components modulates the response to TLR ligands and, consequently, the inflammatory immune response [67] . TLR3 is constitutively expressed on lung alveolar and bronchial epithelial cells and has been shown to contribute to the secretion of multiple cytokines following influenza A infection [68] . Given the data suggesting that H5N1 pathogenicity is due in part to alterations in innate immune responses and hypercytokinemia, it is plausible that polymorphisms altering TLR function could contribute to susceptibility or protection from infection. This hypothesis is supported by a genetic study of a case of influenza-associated encephalopathy, a condition associated with apoptosis and hypercytokinemia [45] . In this case, a missense mutation (908T/C) in the TLR3 gene was identified and was shown to be a loss-of-function mutation, suggesting a protective role for TLR3 signaling in severe influenza infection [45] ( Table 2) . Although these results are consistent with studies suggesting a protective effect of TLR3 in West Nile infection [69] , they are at odds with TLR3 null mouse studies, which have shown reduced proinflammatory cytokine production following cellular stimulation [70] [71] [72] . Consequently, the contribution of TLR genetic variants to H5N1 inflammatory responses remains to be resolved.
Induction of the type I interferon response during influenza infection appears to be important in both human and mouse models, as evidenced by the increased expression of genes including Irf1, Ifi202, Oas1, and Mx1 in mouse microarray studies [73] [74] [75] (reviewed in [76] ). This is consistent with the observation that viral evasion and attenuation of the IFN pathway contributes to H5N1 pathogenesis in humans and suggests potential targets for genetic studies [75, 77] . A strong target for analysis includes the myxovirus resistance (Mx) gene, which encodes interferon-induced antiviral proteins that inhibit viral RNA transcription and consequently confer influenza resistance in mouse lines with functional Mx1 alleles [78] . Polymorphisms in swine Mx genes have also been associated with influenza susceptibility [79] and multiple SNPs in the human MxA gene have been associated with variability in IFN responsiveness in Hepatitis C infection [80] and SARS susceptibility [38] [39] [40] (Table 2) . Specifically, the −123C/A promoter SNP associated with SARS protection correlates with increased basal MxA expression, leading the authors to speculate that −123 genotype may be an important determinant of H5N1 susceptibility [39] . Although human MxA protein has been shown to inhibit influenza replication [81] , no studies have looked for an association between MxA SNPs and H5N1 disease outcome or susceptibility. Because MxA is located on chromosome 21, studies have compared susceptibility to respiratory infections between wild-type and trisomy 21 patients (who exhibit increased MxA expression), but found greater susceptibility among the trisomy 21 group [82] . This group of patients is known to suffer from a multilevel T-cell dysfunction, however, making the role of MxA in respiratory immunity somewhat unclear.
Polymorphisms in OAS1 (2 ,5 -oligoadenylate synthetase 1; an interferon-induced antiviral protein) have also been associated with SARS susceptibility and progression [38, 40] and West Nile infection [41] . Consistent with the idea of increased H5N1 susceptibility and pathogenesis associated with poor IFN responses, the OAS1 SNP rs1077467 is correlated with reduced OAS-1 protein activity and associated with increased susceptibility to West Nile infection and in vitro viral replication [41] .
Comparison of severe H5N1 infections with uncomplicated seasonal influenza infections revealed a pattern of increased viral load and elevated cytokine production in the respiratory tract and serum [75, 83] . The robust cytokine/chemokine response often seen in H5N1 infected patients (hypercytokinemia) is believed to be at least partially responsible for the observed pathogenesis and high fatality of H5N1 infection. Elevated cytokines both in vivo and in vitro include IFNγ, sIL-2R, IL-6, IP-10, TNFα, and MCP-1 [75, [84] [85] [86] . Mouse models of H5N1 infection also demonstrate elevated levels of MCP-1, MIP-1α, IL-6, and IFNγ, even compared to 1918 H1N1 virus infection [87] . Knocking out IL-1R in mice exacerbates H5N1 pathology and suggests that IL-1β-mediated signalling may be important in protection [75] . In ferret models, IP-10 upregulation and signaling through CXCR3 was determined to be a major component of H5N1 disease severity and mortality [88] . Expression of many of these chemokines and cytokines in humans is modulated by SNPs in their promoter regions, including MCP-1 −2518 G/A [89] , IP-10 −201G/A [90] , and IL-6 −174G/C [91] . Genetic variants affecting expression and function of chemokine receptors may also modulate influenza pathogenesis, as CCR5 knock-out mice exhibit increased influenza mortality, whereas CCR2 knockout strains show increased survival ( Table 2) ; both of these effects appear to be related to the kinetics and strength of macrophage recruitment to the lung [42] . In vitro evidence further suggests upregulation of CCR5 on monocyte-derived macrophages that may enhance pathogenesis [92] .
Although relatively few studies have systematically evaluated the influence of genetic polymorphisms on susceptibility and disease severity in zoonotic H1N1 and H5N1 infections, the data available suggest that host immunogenetic variation could play an important role in determining the outcome of the immune response. With improvements in surveillance and case confirmation as well as new sequencing and gene expression platforms, we now have the capability to study host genetic variants among severe respiratory illness cases. Although several challenges to conducting such a study include ethical permission to carry out genetic polymorphism studies, the need for large numbers of wellcharacterised clinical specimens with relevant clinical data, difficulty to obtain sufficient number of samples from severe and fatal cases at a single institution, and difficulty in identifying mild controls. The extreme cases of human H5N1 disease are very rare, sporadic, with scattered cases in different countries, adding economic and political sensitivities associated with this disease. Overcoming these barriers and conducting collaborative research can lead to insights that will shed light on the varying degree of susceptibility observed between populations during the recent H1N1 pandemic and will provide greater insight into the hostpathogen interactions that determine disease course during severe H5N1 infection. In China, Students in Crowded Dormitories with a Low Ventilation Rate Have More Common Colds: Evidence for Airborne Transmission OBJECTIVE: To test whether the incidence of common colds among college students in China is associated with ventilation rates and crowdedness in dormitories. METHODS: In Phase I of the study, a cross-sectional study, 3712 students living in 1569 dorm rooms in 13 buildings responded to a questionnaire about incidence and duration of common colds in the previous 12 months. In Phase II, air temperature, relative humidity and CO(2) concentration were measured for 24 hours in 238 dorm rooms in 13 buildings, during both summer and winter. Out-to indoor air flow rates at night were calculated based on measured CO(2) concentrations. RESULTS: In Phase I, 10% of college students reported an incidence of more than 6 common colds in the previous 12 months, and 15% reported that each infection usually lasted for more than 2 weeks. Students in 6-person dorm rooms were about 2 times as likely to have an incidence of common colds ≥6 times per year and a duration ≥2 weeks, compared to students in 3-person rooms. In Phase II, 90% of the measured dorm rooms had an out-to indoor air flow rate less than the Chinese standard of 8.3 L/s per person during the heating season. There was a dose-response relationship between out-to indoor air flow rate per person in dorm rooms and the proportion of occupants with annual common cold infections ≥6 times. A mean ventilation rate of 5 L/(s•person) in dorm buildings was associated with 5% of self reported common cold ≥6 times, compared to 35% at 1 L/(s•person). CONCLUSION: Crowded dormitories with low out-to indoor airflow rates are associated with more respiratory infections among college students. ''Common cold'' is a conventional term for a mild upper respiratory illness, with symptoms such as nasal blockage and discharge, sneezing, sore throat and cough [1] . Adults typically have 2-5 common colds per year, and children 4-8 colds [2] . Although such infections are often regarded as trivial, the cost to society is large [3] . Rhinoviruses have been associated with 40-65% of ''common colds'' through the year [4] , and up to 80-92% of colds during outbreaks [5] .
Cross-infection from an infected person to a healthy person depends on a number of factors, including how many viral particles are shed by the infected person, and the viral particles' survivability, both over time and with respect to distance from source in a shared environment. Three main mechanisms have been proposed for transmission of viruses causing airways infections:
N contact with secretions that contain the virus, either directly (e.g. hand to hand) from an infected person or indirectly from surfaces (e.g. door knob), N ''large'' airborne droplets, which are produced by an infected person during talking, sneezing, or coughing, and can only spread in air for a distance of less than 1-2 m before falling down, N ''small'' droplet nuclei (dried droplets), that can stay airborne for an extended time and be transported long distances. Despite many years of study, the routes of spread of viral airways infections remain controversial. One opinion is that the virus is transferred through direct contact [6] , while the other is that the virus is transferred through airborne spread [7, 8] . During the SARS epidemic, early preventive messages to the public were to wash hands and, generally to avoid ''direct'' contact spread. Later, analysis of the temporal and spatial distributions of SARS cases in a large community outbreak in Hong Kong and the correlation of these data with the three-dimensional spread of a virus-laden aerosol plume indicated an important role for airborne spread of droplet nuclei [9] .
The influence of building characteristics including ventilation on the spread of viral respiratory infections has begun to receive increased attention from the public, government, media and scientists [10] . Brundage et al. [11] studied the risk of febrile acute respiratory diseases at four army training centers and found that disease rates were significantly higher among trainees in modern energy efficient barracks that had a low ventilation rate. Menzies et al. [12] suggested that there was a relationship between lower ventilation rates and more frequent tuberculosis infections among hospital workers. Milton et al. [13] reported an association between sick leave of employees and outdoor air supply rate. Myatt's [14] study showed that the probability of detecting airborne rhinoviruses was positively associated with weekly average CO 2 concentration in an office. Other factors found to be associated with rate of infectious diseases include occupancy level [15] , cleaning routines and ''damp'' buildings [16] . With respect to crowding, direct and surface contact as well as airborne transmission both appears to be factors in disease transmission. Hoge et al. found that severe overcrowding and inadequate ventilation contributed to an outbreak of pneumococcal disease in a large urban jail [17] .
In China, one 20 m 2 dormitory room is shared by 6-8 bachelor students or 4 master students or 3 PhD students. While such crowded spaces may be important sites for the propagation of respiratory infections, few studies have examined dorm room ventilation and its possible association with infection transmission.
The aim of this paper is to test whether the common cold is associated with how crowded a dorm room is and how well ventilated it is among college students in China.
Verbal consents were obtained from participants, since participants did not want to be tracked back by signature. Both the study and the consent procedure were approved by the ethics committee at Tianjin University.
This study is part of the ''Dorm Environment and Occupants' Health'' study, which was carried out from 2006 to 2007 at Tianjin University, China. Details of the recruitment process and questionnaire contents have been previously described [18] .
In brief, this study consisted of two phases. In Phase I, demographic information, the health status of 6500 students, and building and room characteristics of 2117 dorm rooms at Tianjin University were surveyed by questionnaires. The questionnaire survey was anonymous, but building number and room number were reported by participants. Project members visited dorm rooms, distributed questionnaires and explained to participants how to fill out questionnaires. The questionnaires were collected 2 days later. The questions on common cold infections were ''how many times have you had a common cold in the previous 12 months (options: ,6 times; 6-10 times; .10 times)'' and ''how long does a common cold usually last (options: ,2 weeks; 2-4 weeks; .4 weeks)''. Other questions were about frequencies of window opening, cleaning routines and environmental tobacco smoke (ETS) exposure.
In Phase II, air temperature, relative humidity and CO 2 concentration in dorm rooms were measured by indoor air quality monitor PS 31 (http://www.sensotron.pl) for 24 hours. Air quality monitors were calibrated at the International Center for Indoor Environment and Energy, Technical University of Denmark prior to measurements. Dorm occupants reported opening status of doors and windows at day and at night during measurement The out-to indoor air flow rate at night was calculated from an analysis of the build-up period of metabolic CO 2 produced by sleeping occupants (1:00 a.m.-8:00 a.m.) [19] . Calculation details are described in Information S1. CO 2 concentrations of dorm rooms were measured both in the summer (May-Jul., 2006) and in the winter (Dec., 2006-Apr., 2007) [20] . The average indoor air temperature and relative humidity at night were calculated (1:00 a.m.-8:00 a.m.). Outdoor CO 2 concentration and meteorological parameters were also measured on campus during the same time.
The associations among gender, age, whether family member ever had asthma and allergy, environmental tobacco smoke, cleaning routine, window opening frequency, occupancy levels, and self-reported common cold incidence and duration were analyzed by Chi-square tests. Adjusted odds ratios of crowdedness and air flow rate for common cold infections were evaluated in multiple logistic regression models. A carbon dioxide-based risk equation [21] was used to calculate the basic reproductive number of common colds which was compared to the self-reported infection rate.
A P value less than 0.05 indicates statistical significance. SPSS software 15.0 was used to perform the statistical analyses.
In Phase I, 3712 students living in 1569 dorm rooms in 13 buildings answered the questionnaire, giving a response rate of 57%. Surveys for 276 students were excluded from the analysis due to missing information. Forty eight percent (48%) of students were female. PhD students' mean age was 29 years, master students 25 years and bachelor students 22 years. Monday through Friday, 18% of participants spent less than 2 hours indoors watching TV/playing games per day, 36% spent 2-10 hours per day, and 46% spent more than 10 hours per day. Dorm buildings had 3-12 floors, with 26-43 dorm rooms per floor. All floors in each dorm building are homogeneous with regard to occupants' gender and education level. Dorm rooms consisted of one simple bedroom. Each floor provided two washing rooms and restrooms. Six bachelor students, 4 master students or 3 PhD students shared one dorm room with a volume of 50-70 m 3 . The average density was 5 m 2 per person. Based on the questionnaire data from Phase I, 238 dorm rooms with 473 students living in these dorms were evaluated for Phase II. The evaluated dorm rooms represented different building structures, construction periods, locations and occupancy levels. There were no significant differences in students' ages, gender, self-reported common cold incidence or duration between Phase I and Phase II.
In the questionnaire survey of Phase I, 249 out of 3436 (7.3%) students reported 6-10 common colds in the previous 12 months, while 94 (2.8%) reported more than 10 common colds. Four hundred and thirty six (12.8%) students had common colds lasting for 2-4 weeks, while 65 (1.9%) reported colds lasting more than 4 weeks. Demographic information and living habits of dormitory occupants, and their associations with common cold are summarized in Table 1 . Atopy was associated with increased incidence and longer duration of common cold. Male students were more susceptible than females, but had shorter duration colds. Females cleaned rooms more often than males, cleaning rooms at least twice per week 52% compared to 31% for males, and smoked less (1.4% vs. 15.9%). Passive smoking had a significant effect on the incidence of common cold (p = 0.029), but after adjustment for environmental tobacco smoke, males were still at greater risk for common colds (p = 0.010). Younger students lived in more crowded rooms and reported longer duration colds. Crowding, not age, was shown by stratification for occupancy level to be the significant association with common cold duration.
Self-reported common cold incidence and duration are compared for different occupancy levels in Figure 1 . With incrementally increasing occupancy in dorm rooms, the proportion of occupants with $6 common colds increased significantly (p = 0.002), as did the proportion of occupants with $2 weeks common cold duration (p = 0.000). The odds ratios of crowdedness for common cold incidence of $6 times and duration of $2 weeks, adjusted for gender, age, hours spent indoors, family members' asthma and allergy history, environmental tobacco smoke exposure are shown in Figure 2 . Students in 6-person rooms were about 2.0 times as likely to have a common cold incidence $6 times per year and a duration $2 weeks, as students in 3-person dorm rooms.
For Phase II, the evaluated dorm rooms were located in 13 buildings. Four were built between 1940 and 1960, two between 1977 and 1983, three between 1993 and 1999, and four after 2000. For newly constructed dorm buildings, concrete structure and PVC frame windows were used instead of the brick-stone structure and the wooden frame windows used in older buildings. Ventilation for all dorm rooms consisted solely of opening doors and windows. The out-to indoor air flow rates for rooms measured during summer varied significantly, from 0.8 to 110 L/s per person, with a median of 18 L/s per person. Air flow rates measured in the heating season (from Dec. 5, 2006 to Apr. 14, 2007) varied from 0.3 to 24 L/s per person, with a median of 3.0 L/s per person. Ninety percent of the dorm rooms had an outto indoor air flow rate less than 8.3 L/s per person.
The average indoor air temperature (mean 28.0uC, 95% confidence interval (CI) 27.8uC-28.3uC, range 22.0uC-32.1uC) and relative humidity (mean 54%, 95% CI 53%-55%, range 27%-78%) in summer were high and had large variations consequent to opening doors and windows as the sole mode of ventilation. During the winter season when the heating system was in use and doors and windows were closed, weather conditions had less influence on the indoor thermal environment (temperature: mean 21.0uC, 95% CI 20.7uC-21.3uC, range 15.4uC-26.5uC; relative humidity: mean 40%, 95% CI 38%-41%, range 18%-72%). Data for temperature and relative humidity in rooms with different occupancy levels and out-to indoor air flow rates are shown in Table 2 . In summer, relative humidity and temperature were not different in rooms with different air flow rates. An inverse association between occupancy level and relative humidity was caused by the measurement sequence (6-person dorms were measured at the driest time in May, whereas 3-person dorm rooms were measured in July when outdoor relative humidity was higher). Outdoor climate is the dominating factor in determining the indoor temperature and relative humidity in summer. In winter, rooms shared by 6 people had the highest relative humidity and temperature at night. A low out-to indoor air flow rate was related to a significantly higher relative humidity (p = 0.000).
However, common cold infections were not significantly associated with indoor air temperature (p = 0.806) and relative humidity (p = 0.642). Figure 3 shows that the lowest quartile of out-to indoor air flow rates per person in both summer and winter were associated with an increased proportion of occupants with $6 common colds in the previous 12 months. The adjusted odds ratios of ventilation rates for common cold infections increased slightly across the quartiles. The critical ventilation rate, below which common cold incidence increases, is identified. When ventilation rate is below 6 L/s per person, the common cold incidence in dorm rooms with average 4 occupants increased from 10% to 12%. When ventilation rate is below 1 L/s per person, the common cold incidence increased from 10% to 15%.
In our study, old buildings had more dampness problems, while new buildings using modern construction technologies had smaller ventilation rates [21] . Dampness problems have been reported to be associated with an increased incidence of common cold infections [18] . In order to eliminate the influence of indoor environmental factors other than poor ventilation, the mean ventilation rates in newly constructed dorm buildings were calculated and related to the percentage of occupants with common cold infections more than 6 times annually. The ventilation rates in winter are less than those in summer, and may help nail down the critical ventilation rate, below which common cold incidence increases. Figure 4 shows that the infection rate of common colds in the ''tight'' buildings constructed after 1993 is, in winter, associated with mean ventilation rate. There were 7 buildings constructed after 1993. One building was not included in the analysis because measurements were performed in only 9 dorm rooms. On average, there were 1140 occupants in each dorm building. A mean ventilation rate of 5 L/ (sNperson) was associated with $6 common colds per year in 5% of occupants , compared to a 35% for 1 L/(sNperson). There were 6 buildings constructed before 1993, among which 4 buildings had ,10 dorm rooms measured in winter and were excluded from the analysis. Of the remaining 2 buildings, one had mean ventilation rate of 5.7 L/(sNperson) and a common cold infection rate of 23.8%, while the other had a mean ventilation rate of 6.4 L/ (sNperson) and a common cold infection rate of 7.1%.
The Wells-Riley equation estimates the number of secondary infections that arise when a single infectious case is introduced into a population where everyone is susceptible [22] . This number is called the basic reproduction number. Rudnich and Milton [23] expanded the Wells-Riley equation to apply to situations with nonsteady state conditions and variable ventilation rates:
Where R A0 is the basic reproduction number; n is the number of occupants; f is the re-breathed fraction; and I is the number of infectors ( = 1). q is the quantum generation rate by an infected person (quanta/h), where a quantum is the amount of infectious material needed to produce infection in 63% of uniformly exposed animals, and is therefore 1.25 times the median infectious dose, 1.256ID 50 . t is the exposure time (h); f = (C-C 0 )/C a , where C a is the volume fraction of CO 2 added to exhaled breath, C is the volume fraction of CO 2 in indoor air, and C 0 is the volume fraction of CO 2 in outdoor air. The incidences (,6 times; 6-10 times; .10 times) and durations (,2 weeks; 2-4 weeks; .4 weeks) of common colds in the previous 12 months for different occupancy levels (6-people; 4people; 3-people per dorm) were self-reported by occupants. The mean duration of a common cold is 7-10 days [1] . For this study we assumed that the duration of a common cold was 9 days. Although many viruses can produce symptoms of common cold, rhinovirus is the most frequent cause of the common cold [24] . Riley and Nardell suggested that q for rhinovirus is in the range of 1-10/h [25] . Here we inferred q = 9/h. We assumed that the infector remained in the dorm room 8 hours per day. The average CO 2 concentrations in each dorm room from 1:00 a.m. to 8:00 a.m. were calculated. The estimated and self-reported number of common colds in each day in winter is compared (Table 3) . These two numbers fit very well indicating the validity of this CO 2 -based risk model in predicting infection rate of infectious disease like common cold.
If for a given population and infectious agent, the basic reproductive number .1 then that agent can spread in the population. The critical re-breathed fraction (f c ), corresponding to a basic reproduction number of 1, can be derived from Equation (1), 
In the present study, the critical re-breathed fraction in rooms with different occupancy levels and the associated critical indoor CO 2 concentrations above background (outdoor CO 2 concentration) were calculated from Equation (2), both as a function of exposure time ( Figure 5(a) ) and quantum generation rate ( Figure 5(b) ). Thus Figure 5 predicts the critical indoor CO 2 concentrations beyond which infectious disease will spread. The family of curves in Figure 5 (a) describes the trends of the critical indoor CO 2 concentrations above outdoor values (C-C 0 ) as a function of exposure times for risk of respiratory infections. The quantum generation rate used was 2/h. The critical CO 2 concentration above the background levels off if the common cold lasts more than 3 weeks (exposure time 8 hours/day, totally 168 hours) (Fig 5(a) ). This indicates that even for less infectious agents with quanta generation rate no more than 2/h, a full fresh outdoor air system without recirculation of indoor air needs to be used in environments where people spend extended time (for example bedrooms, dorms, schools, daycare centers) in order to prevent viral infections. In Figure 5 (b), the exposure time was assumed to be 56 hours (8 hours/day, i.e. 7 days). It shows that the current ASHRAE standard of 700 ppm above the background level [26] would not prevent the infection from being spread in a dorm room with 6 occupants unless the quantum generation rate of infectious agents is no more than 1 quantum/h (Fig. 5(b) ).
The campus living style and dormitory conditions of students at Tianjin University is typical of China. The sample size in our study is large, and the response rate was reasonably good (57%). No significant difference was found between respondents and nonrespondents in reporting wheeze and dorm room dampness [18] . Thus it is highly unlikely that selection bias impacted the findings of this study. Common cold is a conventional term for a mild upper respiratory illness. College students can be expected to understand what ''common cold'' refers to. There is no evidence to suggest that bachelor students have a different memory in reporting common cold infection, compared to PhD students. Compared to home environment, dorm buildings are perceived to be very crowded no matter whether 3 or 4 or 6 people share a 20 m 2 room. Even students in 3-people-shared dormitory think their space is crowded. Therefore, the significant association between occupancy level and incidence of common colds, and the dose-response relationship between ventilation rate and incidence of common colds cannot be explained by reporting bias.
The occupants' education level was not adjusted for when calculating the odds ratios of crowdedness for common cold infections since 3 PhD students or 4 master students or 6 bachelor students share one dorm room with similar volume. Education level itself should not be a confounding factor. Psychological stress, related to education status may have effect on common cold as indicated in a previous study [27] . However, our study found that less crowded dorm rooms occupied by PhD students were associated with less common cold infections. This cannot be explained by psychological stress since PhD students are supposed to be more stressed than master or bachelor students.
The summer measurement was from May to July and winter measurement from December to April. In summer measurements, 6-people-shared dormitories were measured first, followed by 4 or 3 people shared dormitories. In winter measurement, dorm Figure 3 . Associations between ventilation rate and common cold annual incidence $6 times. 1 Proportion of occupants with $6 common colds in the previous 12 months. 2 Odds ratios were adjusted for gender, age, family member allergy history, exposure to environmental tobacco smoke, building age and crowdedness. AOR: adjusted odds ratio; CI: confidence interval. doi:10.1371/journal.pone.0027140.g003 buildings were measured randomly. There could be a potential systematic bias for summer measurement, but not for winter measurements. During the measurements, outdoor CO 2 concentrations and meteorological parameters were monitored. In principle, air change rate in buildings with natural ventilation system is not influenced by air relative humidity. Outdoor air temperature itself and the consequent occupants' behavior (e.g. opening doors/windows) may influence the air change rate in dorm rooms. In our study, the opening of doors/windows was reported by occupants themselves. In winter, occupants tended to close doors and windows tightly, so that variations in winter outdoor temperature had little influence on ventilation rate in 
D i is the assumed number of common cold infections in winter under different self-reported incidence rate, times. i indicates common cold incidence. i = 1, 2, 3. 1common cold less than 6 times in the previous 12 months; 2-common cold 6-10 times; 3-comon cold more than 10 times. We assume D 1 = 3; D 2 = 6; D 3 = 8. O j is the occupancy level, person/room. j indicates occupancy level. j = 3, 4, 6. 3-three people per dorm room; 4-four people per dorm room; 6-six people per dorm room. D i,j is the proportion of students with different self-reported common cold incidences, %. M is the duration of a common cold, days. We assume M = 9 days [1] . T is days in winter season, 120 days. C j is the average CO 2 concentration from 1:00 a.m. to 8:00 a.m. in rooms with different occupancy levels, ppm. f j is the re-breathed fraction of indoor air in rooms with different occupancy levels. f j = (C j -C 0 )/C a . C a is the volume fraction of CO 2 added to exhaled breath, 37000 ppm. C 0 is the volume fraction of CO 2 in outdoor air, 300 ppm. q is the quantum generation rate by an infected person, quanta/h. We assume q = 9 quanta/h [25] . t is the time a infector remaining in the dorm room, hour/day. We assume t = 8 hours per day. doi:10.1371/journal.pone.0027140.t003 dorm buildings. In summer, the mean outdoor air temperature was 29.6uC, ranging from 22.5uC to 35.2uC. The median air change rate was 4.42 h 21 and 4.67 h 21 when outdoor air temperature is below and above 29.6uC. There was no significant difference of air change rate for different temperatures in summer (p = 0.319). Therefore, it is reasonable to assume that, the air change rates measured in summer and winter are representative for respective season, without influence from small climate changes within each period.
While it is possible that some of the self-reported common colds were influenza, the infection rate of flu among adults is approximately once per year in this part of China. Therefore, this possible error would not change our results. Moreover, common colds and influenza are spread in a similar way; the present study could have been titled ''airways infections''. In each dorm room, CO 2 concentrations were measured for 24 hours in both summer and winter. As measurements were made over a long period, i.e. summer measurements between May and July and winter measurements between December and April, and for 238 rooms, the mean values of ventilation rates should be valid for rooms with different occupancies and opening status of windows/ doors, and for changes in the outdoor climate.
There were imperfections in our data collection. In some rooms occupants may have had the window open during the night measurements in winter. Perhaps the incidence of common cold was influenced by an influenza epidemic. These sources of error would shift our findings towards the null hypothesis, that there was no association between common cold infections and dorm crowdedness or ventilation rate. Our findings are robust in spite of these possible problems. Thus, it is likely that more measurements and more accurate data on types of airways infections would show an even stronger association.
The out-to indoor air flow rate required by the Indoor Air Quality Standard of China is 8.3 L/s per person [28] . In the present study, 90% of the dorm rooms measured during winter had night-time ventilation rates less than this value. CO 2 concentration in corridors was not measured, so that the fresh out-to indoor air flow rate may have been even lower than the calculated value in cases when corridor windows were closed.
The suggested dose-response relationship between dorm ventilation rate and common cold infections among occupants can be extrapolated to other crowded public premises with substandard ventilation rate, meaning a possible important public health topic for e.g. schools, daycare centers.
Although it is widely held that people in crowded spaces have more airways infections [15, 29] , there are few studies on this. Our study is among the first published suggesting a relationship between occupancy levels, ventilation rates, and respiratory infections. With 6 occupants instead of 3 in a 20 m 2 dorm room, the proportion of occupants with incidence of more than 6 common colds in the previous 12 months doubled. When crowdedness is adjusted for, a lower ventilation rate is associated with an increased risk of common cold. This finding is consistent with Shendell's study in schools, which showed that a 1000 ppm increase in dCO 2 (difference between indoor and outdoor CO 2 levels) was associated with a 0.5%-0.9% decrease in annual average daily attendance [30] . For office buildings, Milton found that short-term sick leave was reduced by 35% at 24 L/s per person compared to 12 L/s per person outdoor air flow [13] .
A crucial question is whether the increased frequency of common colds in crowded places is due to direct contact (or via surfaces), via droplets or via droplet nuclei. The strong association with ventilation in this study indicates that airborne transmission is important and perhaps the main route.
Crowdedness and outdoor air ventilation per person are important for the spread of airborne infectious diseases in rooms such as dorms where people spend a lot of time. Respiratory viruses can be transmitted through air so that transmission is modulated by outdoor air supply rates. Further studies are warranted.
Information S1 Dormitory outdoor air flow rate calculation by using CO 2 method. (DOCX) Epidemiology and clinical characteristics of hospitalized patients with pandemic influenza A (H1N1) 2009 infections: the effects of bacterial coinfection BACKGROUND: Numerous reports have described the epidemiological and clinical characteristics of influenza A (H1N1) 2009 infected patients. However, data on the effects of bacterial coinfection on these patients are very scarce. Therefore, this study explores the impact of bacterial coinfection on the clinical and laboratory parameters amongst H1N1 hospitalized patients. FINDINGS: This retrospective study involved hospitalized patients with laboratory-confirmed H1N1 infections (September 2009 to May 2010). Relevant clinical data and the detection of bacterial coinfection from respiratory or sterile site samples were obtained. Multiplex PCR was used to determine the co-existence of other respiratory viruses. Comparison was made between patients with and without bacterial coinfection. The occurrence of coinfection was 34%; 14 (28%) bacterial and only 3 (6%) viral. Mycoplasma pneumoniae (n = 5) was the commonest bacteria followed by Staphylococcus aureus (n = 3). In univariate analysis, clinical factors associated with bacterial coinfection were age > 50 years (p = 0.02), presence of comorbidity (p = 0.04), liver impairment (p = 0.02), development of complications (p = 0.004) and supplemental oxygen requirement (p = 0.02). Leukocytosis (p = 0.02) and neutrophilia (p = 0.004) were higher in bacterial coinfected patients. Multivariate logistic regression analysis revealed that age > 50 years and combined complications were predictive of bacterial coinfection. CONCLUSIONS: Bacterial coinfection is not uncommon in H1N1 infected patients and is more frequently noted in the older aged patients and is associated with higher rates of complications. Also, as adjunct to clinical findings, clinicians need to have a higher index of suspicion if neutrophilia was identified at admission as it may denote bacterial coinfection. In April 2009, a novel influenza A (H1N1) virus emerged in Mexico and spread rapidly worldwide [1] . By June 11, 2009 nearly 30, 000 cases had been confirmed across 74 countries including Malaysia, prompting World Health Organization to raise its pandemic alert to phase 6 [2] . After the first reported H1N1 case in Malaysia in May 15, 2009, the numbers increased exponentially and as of May 31, 2010 they totaled 14, 821 with 87 deaths [3] . Thereafter, there have been numerous reports describing the epidemiological and clinical characteristics of H1N1 infections. However, studies focusing on the effects of respiratory pathogen coinfection on clinical and laboratory parameters in the H1N1 infected patients are scarce. Clinicians may assume that a single virus type is involved, as laboratory detection involves PCR specifically targeting H1N1. However, bacterial coinfection had been shown to contribute to morbidity and mortality in previous influenza pandemics [4] . Therefore, this study aims to explore the clinical and laboratory characteristics amongst patients hospitalized with laboratory-confirmed pandemic influenza A (H1N1) infection and the effects of bacterial coinfection on these parameters.
This retrospective study was conducted from September 2009 to May 2010 at Hospital Sultanah Aminah Johor Bahru (HSAJB). HSAJB is a 989-bedded tertiary referral centre and the government designated hospital for H1N1 testing in Johor State, Malaysia. As the main General Hospital of Johor, its' patient population is reflective of the larger community in Malaysia. During our study period, which coincided with the peak of H1N1 pandemic activity, all patients regardless of whether they were hospitalized or not, who presented with an influenza-like illness (ILI) were tested for H1N1.
Consecutive hospitalized patients with laboratory-confirmed H1N1 infections were identified from microbiology laboratory records. Laboratory diagnosis of H1N1 was made using the Centers for Disease Control and Prevention (CDC) real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol [5] . Relevant clinical data was retrieved from patients' medical records. The presence of bacterial coinfection from respiratory specimens (sputum, tracheal/nasopharyngeal aspirate, bronchoalveolar lavage) or sterile site samples (blood or pleural fluid) taken within 48 hours of admission was recorded. Mycoplasma pneumoniae infection was diagnosed by serology using particle agglutination test (Serodia-Myco II, Fujirebio Inc., Japan). A single titer of ≥ 160 was considered as diagnostic cut-off titer, based on population background study conducted in Malaysia [6, 7] . All samples confirmed H1N1 positive were stored at -80°C for further analysis using multiplex PCR (Seeplex RV Detection, USA) which detects adenovirus, influenza virus A and B, respiratory syncytial virus, parainfluenza types 1, 2 and 3 and human metapneumovirus. Hematological, liver and renal function parameters on admission were recorded.
Data was analyzed using SPSS version 17.0.1; comparing patients with and without bacterial coinfection with a P-value < 0.05 (two-tailed) taken as the level of significance. Variables associated with bacterial coinfection in the univariate analysis were then entered into multivariate logistic regression analysis.
After excluding 7 patients (5 incomplete data and 2 for presence of nosocomial pneumonia), data of 50 patients was available for analysis ( Table 1 ). The patients age ranged from 7 months to 82 years (median 20.3 years), with 90% patients (45/50) < 50 years. Excluding 6 * Not assessed in children < 3 years (n = 44) ** A patient may have more than one comorbidity or complications ¶ Includes asthma (n-= 9), Chronic obstructive airway disease (n = 1) and bronchiectasis (n = 1) Ψ Includes chronic myeloid leukemia (n = 1), acute myeloid leukemia (n = 1), meningioma of brain (n = 1) ¥ Includes autoimmune haemolytic anaemia (n = 1), idiopathic thrombocytopenia purpura (n = 1) £Includes cardiovascular disease (n = 2), immunosuppressives (n = 2), hypothyroidism (n = 1), stroke (n = 1) ® Includes liver impairment (n = 12), renal impairment (n = 4) septic shock (n = 2) and ARDS (n = 2). Ω Includes bacterial (n = 14), viral (n = 3). The sites for isolation of 9 non-Mycoplasma bacteria: (blood = 2, sputum = 3, nasopharyngeal aspirate = 3, bronchoalveolar lavage = 2) © Established values in our laboratory, Adults: leukocytes 4-11 × 10 9 /L; neutrophils 2-7.5 × 10 9 /L; lymphocyte 1.5-4 × 10 9 /L; Paediatrics: age-dependent ALF: abnormal liver function (n = 41) (raised alanine aminotransferase/ aspartate aminotransferase or both) ARF: abnormal renal function (n = 42) (raised creatinine) pregnancies, 24 patients (48%) had at least one preexisting comorbidity; lung disease being the commonest. The mean duration of symptoms before hospitalization was 4.4 ± 3.08 days (range 1-14 days). Cough (100%) and fever (98%) were the most common symptoms on admission. Twelve patients (24%) had oxygen saturation < 95% at presentation. Pneumonia was diagnosed in 25 patients (50%) based on clinical and radiological findings.
All patients received oseltamivir after admission. Twenty-two patients (44%) required oxygen supplementation. Nine cases (18%) were treated at the intensive care unit (ICU); 6 requiring mechanical ventilation. Thirteen patients (26%) developed complications (single or combination); liver impairment (n = 12), renal impairment (n = 4) septic shock (n = 2) and acute respiratory distress syndrome (ARDS) (n = 2). Two (4%) patients died, resulting from septicaemic shock and severe pneumonia respectively.
Forty-five patients (90%) had lower respiratory tract specimens sent for bacterial cultures. The 5 patients without these specimens were children who had difficulty in producing respiratory secretions, however, they appeared generally well with no evidence of pneumonia. Blood cultures were performed in 23 patients (46%) and Mycoplasma pneumoniae serology in 27 patients (54%). Of the 50 H1N1 patients, 17 (34%) were coinfected with a second respiratory pathogen; 14 (28%) bacterial and only 3 (6%) viral. Mycoplasma pneumoniae (n = 5) was the commonest bacterial coinfection followed by Staphylococcus aureus (n = 3), Klebsiella pneumoniae (n = 2), Streptococcus pneumoniae (n = 2), Moraxella catarrhalis (n = 1), Pseudomonas aeruginosa (n = 1), Streptococcus pyogenes (n = 1) and Streptococcus agalactiae (n = 1). Two patients had dual infection; M.pneumoniae/S.agalactiae and S.pneumoniae/M.catarrhalis respectively. The sites for isolation of 9 non-Mycoplasma bacteria were blood (2), sputum (3), nasopharyngeal aspirate (3) and bronchoalveolar lavage (2). The 3 virus detected were parainfluenza; these 3 patients presented with influenza-like illness with no deterioration of clinical findings.
A comparison between H1N1 patients with and without bacterial coinfection is shown in Table 2 . Although 90% of patients were < 50 years old, bacterial coinfection was more frequent in patients > 50 years (p = 0.02). The presence of underlying comorbidity provided a suitable niche for bacterial coinfection (p = 0.04). Although ICU admissions, mechanical ventilation, renal impairment, mortality and pneumonia were notably higher in patients with bacterial coinfection, they were not statistically significant. Other factors associated with bacterial coinfection in the univariate analysis were development of complications (p = 0.004), liver impairment (p = 0.02) and supplemental oxygen requirement (p = 0.02). Out of the 50 patients, 12 (24%) had leukocytosis and 13 (26%) neutrophilia. Bacterial coinfected patients demonstrated higher rates of leukocytosis (p = 0.02) and neutrophilia (p = 0.004). On the other hand, lymphopenia (n = 31) was notably higher in single viral H1N1 infection. Multivariate analysis revealed that age > 50 (OR 12.577; 95% CI 1-165.24; p = 0.05)) and development of complications (OR 9.01; 95% CI 1.70-47.67; p = 0.01) were predictive of bacterial coinfection.
Forty-one patients (82%) received antibiotics, either as empiric or definitive therapy upon admission and 16% prior to admission All patients with bacterial coinfection were treated with antibiotics; significantly higher rates compared to patients without bacterial coinfection (p = 0.05).
The bacterial coinfection rate of 28% amongst our H1N1 hospitalized patients was higher compared to other studies [8, 9] . A large laboratory-based study in the United States demonstrated comparable bacterial coinfection rates to our study with similarly very low frequency of viral copathogen detection [10] . Whilst our finding concurred with several studies [1, 8, 9, 11, 12] that showed H1N1 infections having a predilection for younger patients, patients > 50 years had higher risk of bacterial coinfection in our study.
Although concurrent bacterial infection was shown to have a major influence on mortality in previous influenza pandemics [4] , its' role in the current H1N1 pandemic is still evolving. Recent postmortem studies amongst fatal H1N1 cases established a link between bacterial lung infections and increased deaths [13] . Whilst an earlier study showed bacterial coinfection not to be a major contributor to severe disease [12] , a more recent study demonstrated otherwise [8] . In our study, patients with bacterial coinfection were found to have higher risk of developing complications. The presence of underlying comorbidity, liver impairment and supplemental oxygen requirement were significantly higher in bacterial coinfected patients in univariate analysis, although these factors were not predictive in multivariate analysis.
Unlike S.pneumoniae, S.aureus and S.pyogenes which are repeatedly reported as coinfecting agents [4, 8, 10, 13] , the high rates of M.pneumoniae coinfection was unique to our study. Although hematological parameters have been mentioned in few other studies [8, 9, 12] , to our best knowledge this is the first study that specifically explored the impact of bacterial coinfection on these parameters. CDC recognizes the importance of early empirical antibiotics in H1N1 infected patients who might have concurrent bacterial pneumonia [13] . Our study showed that leukocytosis and neutrophilia were notably higher in bacterial coinfected patients. This finding could alert physicians about the possibility of bacterial coinfection, as clinical diagnosis may be insufficient and bacterial cultures take time. Eighty-two percent of our patients received empiric or definitive antibiotics at some point during admission which was comparable to high rates in a China study [9] .
The limitation of our study includes its' retrospective design and a small sample size which was unavoidable, as we were limited by the actual number of cases during the study period and because it was a single centre study. As such, our study was inadequately powered to examine the influence of certain characteristics. Nasopharyngeal aspirates may have questionable pathogenic role, however the 3 patients with positive NPA were treated with appropriate antibiotics as they were felt to be clinically relevant. Mycoplasma serology was not performed in all patients and the request was based upon physicians' discretion, this may have underestimated the actual number of cases. The preadmission antibiotic therapy could underestimate the bacterial coinfection rates. Despite these limitations, we identified bacteria coinfection in 28% of our patients.
In conclusion, our study suggests that bacterial coinfection is not uncommon in H1N1 infected patients and laboratory investigations should go beyond establishing a viral cause alone. Bacterial coinfection was more frequently seen in the older age group and was associated with higher rates of complications. As adjunct to clinical findings, clinicians need to have a high index of suspicion if neutrophilia was identified on admission as it may denote bacterial coinfection. A larger scale study will be useful to further confirm our findings.  Clinical Review: Gene-based therapies for ALI/ARDS: where are we now? Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specific therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ARDS remains to be realized. Multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. Deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. Encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting. Gene-based therapy involves the insertion of genes or smaller nucleic acid sequences into cells and tissues to replace the function of a defective gene, or to alter the production of a specifi c gene product, in order to treat a disease. Gene therapy can be classifi ed into germline and somatic gene therapies. Germline approaches modify the sperm or egg prior to fertilization and confer a stable heritable genetic modifi cation. Somatic gene approaches use gene therapy to alter the function of mature cells. Commonly used somatic gene therapy strategies include the overexpression of an existing gene and/or the insertion of smaller nucleic acid sequences into cells to alter the production of an existing gene.
ALI/ARDS may be suitable for gene-based therapies as it is an acute but relatively transient process [8] , requiring short-lived gene expression, obviating the need for repeated therapies and reducing the risk of an adverse immunological response. Th e distal lung epithelium is selectively accessible via the tracheal route of administration, allowing targeting of the pulmonary epithelium [9] . Th e pulmonary vasculature is also relatively accessible, as the entire cardiac output must transit this circulation. Antibodies that bind antigens selectively expressed on the pulmonary endothelial surface can be complexed to gene vectors to facilitate selective targeting following intravenous administration [10] . It is also possible to use gene-based strategies to target other cells central to the pathogenesis of ALI/ARDS, such as leuko cytes and Abstract Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specifi c therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for genebased approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ ARDS remains to be realized. Multiple barriers to eff ective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection effi ciency of nonviral delivery methods. Defi cits remain in our knowledge regarding the optimal molecular targets for genebased approaches. Encouragingly, recent progress in overcoming these barriers off ers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting. fi bro blasts [11] . Furthermore, gene-therapy-based approaches off er the potential to selectively target diff erent phases of the injury and repair process. Th e potential to target specifi c aspects of the injury and repair processes such as epithelial-mesenchymal transition, fi brosis, fi brinolysis, coagulopathy and oxidative stress with these approaches is also clear.
Gene therapy requires the delivery of genes or smaller nucleic acid sequences into the cell nucleus using a carrier or vector. Th e vector enables the gene to overcome barriers to entry into the cell, and to make its way to the nucleus to be transcribed and translated itself or to modulate transcription and/or translation of other genes. Both viral and nonviral vector systems have been developed (Table 1) .
Viral vectors are the most eff ective and effi cient way of getting larger nucleic acid sequences, particularly genes, into cells (Table 1) . Th e viral genome is modifi ed to remove the parts necessary for viral replication. Th is segment is then replaced with the gene of interesttermed a transgene -coupled to a promoter that drives its expression. Th e modifi ed genome is then encapsulated with viral proteins. Following delivery to the target site, the virus binds to the host cell, enters the cytoplasm and releases its payload into the nucleus (Figure 1 ). Th e size of trans gene that can be used depends on the capsid size. A number of diff erent viral vectors have been used in preclinical lung injury studies to date.
Adenoviruses have double-stranded DNA genomes, have demonstrated promise in preclinical models [12, 13] and are well tolerated at low to intermediate doses in humans [14, 15] . Advantages include their ease of production, the high effi ciency at which they can infect the pulmonary epithelium [14, 16] and that they can deliver relatively large transgenes. A disadvantage of adenoviruses is their immunogenicity, particularly in repeated doses [14] . Newer adenoviral vectors, in which much of the immuno genicity has been removed, hold promise [17] . While adenovirus-mediated gene transfer in the absence of epithelial damage is relatively ineffi cient [18] , this may be less of a problem in ALI/ARDS that is characterized by widespread epithelial damage.
Adeno-associated viruses (AAVs) are single-stranded DNA parvoviruses that are replication defi cient [19] . A substantial proportion of the human population has been exposed to AAVs but the clinical eff ects are unknown.
AAV vectors have a good safety profi le, and are less immunogenic compared with other viruses, although anti bodies do develop against AAV capsid proteins that can compromise repeat administration. AAV vectors can insert genes at a specifi c site on chromosome 19 . Th e packaging capacity of the virus is limited to 4.7 kb, restricting the size of the transgene that can be used. AAVs are less effi cient in transducing cells than adenoviral vectors. Successful AAV vector gene transfer has been demon strated in multiple lung cell types including lung progenitor cells, in both normal and naphthaleneinduced ALI lungs [20] . AAV serotypes have specifi c tissue tropisms, due to diff erent capsid proteins that bind to specifi c cell membrane receptors. AAV-5 [21] and AVV-6 [22] exhibit enhanced tropism for the pulmonary epi thelium [21, 22] . AAVs can transduce nondividing cells and result in long-lived transgene expression. AAV vectors have been used in clinical trials in cystic fi brosis patients, underlining their safety profi le [23, 24] .
Th ese RNA viruses can transfect nondividing cells such as mature airway epithelial cells [25] . Th e virus stably but randomly integrates into the genome and expression is likely to last for the lifetime of the cell (~100 days). Th e transgene can be transmitted post mitosis, and there is also a risk of tumorigenesis if the transgene integrates near an oncogene. Th e development of leukemias in children following gene therapy for severe combined immunodefi ciency highlights this risk [26, 27] . While lentiviral vectors may be useful to correct a gene defi ciency associated with increased risk of ALI, the long-lived gene expression of lentiviral delivered genes may be more suitable for chronic diseases than for ALI/ARDS.
Nonviral delivery systems, while generally less effi cient than viral vectors in transfecting the lung epithelium, are increasingly used to deliver smaller DNA/RNA molecules (Table 1 ). Strategies include the use of DNA-lipid and DNA-polymer complexes and naked DNA/RNA oligonucleotides, such as siRNA [28] , decoy oligo nucleo tides [29] and plasmid DNA [30] . Nonviral delivery systems are less immunogenic than viral vector-based approaches, and can be generated in large amounts at relatively low cost.
Plasmid vectors are composed of closed circles of doublestranded DNA. As naked and plasmid DNA contain no proteins for attachment to cellular receptors, there is no specifi c targeting to diff erent cell types and thus it is essential that the DNA is placed in close contact with the desired cell type. Th ese limitations make this approach less relevant clinically.
Th e therapeutic DNA is held within a sphere of lipids, termed a lipoplex, or within a sphere of polymers, such as polyethyleneimine, termed a polyplex. Lipoplexes and polyplexes act to protect the DNA, facilitate binding to the target cell membrane and also trigger endocytosis of the complex into the cell, thereby enhancing gene expression. Th ese systems can be modifi ed to include a targeting peptide for a specifi c cell type, such as airway epithelial cells [31] . Th ese complexes effi ciently and safely transfect airway epithelial cells [31] , and they have demonstrated promise in human studies [32] .
siRNAs are dsRNA molecules of 20 to 25 nucleotides that can regulate the expression of specifi c genes. Specifi c siRNAs reduce infl ammation-associated lung injury in Table 1 .
Viral vector-delivered gene therapy
Relatively easily produced Immunogenic [14] Adenoviral transfer of genes for a surfactant (dsDNA genome)
Effi ciently transfect lung enzyme [49] , angiopoietin-1 [51] , HSP-70 [52] , epithelium [14, 16] apolipoprotein A-1 [53] , and Na + ,K + -ATPase pump Can deliver larger genes [55] genes attenuate experimental ALI Well tolerated in lower doses [1, 3] Adenoviral delivery of IL-10 gene attenuates zymosan ALI at low doses, but is harmful at high doses [58] Adeno-associated virus Good safety profi le; less Limited transgene size AAV vector gene transfer demonstrated in multiple vectors (ssDNA genome)
immunogenic Diffi cult to produce in large lung cell types including progenitor cells in both Inherently replication defi cient quantities normal lungs and following naphthalene-induced AAV-5 and AAV-6 lung epithelial ALI [20] tropism [10, 11] Transduce nondividing cells Long-lived gene expression Used in clinical trials for CF [12, 13] Lentivirus vectors Transduce nondividing cells [25] Oncogenesis risk due to Lentiviral transfer of shRNA to silence CD36 gene (RNA genome)
Integrate stably but randomly integration into genome expression suppresses silica-induced lung fi brosis into the genome [26, 27] in the rat [35] Nonviral gene-based strategies
Plasmid transfer (closed Easily produced at low cost No specifi c cell targeting Electroporation-mediated gene transfer of the dsDNA circles) Very ineffi cient Na + ,K + -ATPase rescues endotoxin-induced lung injury [60] Nonviral DNA complexes Complexes protect DNA Less effi cient than viral vectors Cationic lipid-mediated transfer of the Na + ,K + -(lipoplexes or polyplexes)
Complexes facilitate cellular ATPase gene ameliorated high-permeability targeting [31] pulmonary edema [59] Lipoplex-delivered IL-10 gene decreased CLP-induced ALI [61] Systemic cationic polyethylenimine polyplexes incorporating indoleamine-2,3-dioxygenase decreased ischemia-reperfusion ALI [62] DNA and RNA Easily produced at low cost No specifi c cell targeting Specifi c siRNAs reduce infl ammation-associated oligonucleotides (siRNA, Smaller molecules that can lung injury in humans [33] and in animal models shRNA, decoy easily enter cells [28, 34] oligonucleotides) Target regulation of specifi c genes shRNA-based approaches have reduced lung injury in animal models [35, 36] Cell-delivered gene therapy humans [33] and in animal models [28, 34] . shRNA is a single strand of RNA that, when introduced into the cell, is reverse transcribed and integrated into the genome, becoming heritable. During subsequent transcription, the sequence generates an oligonucleotide with a tight hairpin turn that is processed into siRNA. shRNAs have reduced lung injury in animal models [35, 36] . Decoy oligonucleotides are double-stranded DNA molecules of 20 to 28 nucleo tides, which bind to specifi c transcription factors to reduce expression of targeted genes, and have been successfully used in animal models [37, 38] .
An alternative approach is to use systemically delivered cells to deliver genes to the lung. Th is approach has been used to enhance the therapeutic potential of stem cellssuch as mesenchymal stem/stromal cells, which demon strate promise in preclinical ALI/ARDS models [39] . Fibroblasts have also been used to successfully deliver genes to the lung to attenuate ALI [40] . Preliminary data from a clinical trial in pulmonary hypertension show that endothelial progenitor cells overexpressing endothelial nitric oxide synthase (NOS3) decrease pulmonary vascular resistance [41] , highlighting the potential of cell-delivered gene therapy for ALI/ARDS.
Nebulization of genetic material into the lung is eff ective [42] , safe and well tolerated [32, 43, 44] . Th e integrity of AAV vectors [9, 43] and adenoviral virus vectors [44] are maintained post nebulization, as are cationic lipid vectors [32] and DNA and RNA oligonucleotides [45] . A number of gene therapy clinical trials have utilized nebulization to deliver the transgene to the lung [23, 43] , but without clear clinical benefi t to date [43, 44] .
Intravascular delivery approaches target the lung endothelium. Th ese approaches have been successfully used in preclinical studies of cell-based gene therapies [39, 40] , and also with vectors that incorporate components such as antibodies to target antigens on the lung endothelium [10] .
Successful gene-based therapies require the delivery of high quantities of the gene or oligonucleotide to the pulmonary epithelial or endothelial surface, require effi cient entry into the cytoplasm of these large and insoluble nucleic acids, which then have to move from the cytoplasm into the nucleus, and activate transcription of its product. Multiple barriers exist that hinder this process, not least the natural defense mechanisms of the lung, and additional diffi culties that exist in transducing the acutely injured lung (Table 2 ). Limitations regarding delivery technologies and defi ciencies in our knowledge regarding the optimal molecular targets also reduce the effi cacy of these approaches.
Th e lung has evolved eff ective barriers to prevent the uptake of any inhaled foreign particles [46] . While advantageous in minimizing the potential for uptake of external genetic material (for example, viral DNA), these barriers make it more diffi cult to use gene-based therapies in the lung. Barriers to entry of foreign genetic material into the lung include airway mucus and the epithelial lining fl uid, which traps and clears inhaled material. Th e glycocalyceal barrier hinders contact with the cell membrane, while the tight intercellular epithelial junctions and limited luminal endocytosis further restrict entry of foreign material into the epithelial cells.
Transducing the acutely injured lung may be diffi cult, due to the presence of pulmonary edema, consolidated or collapsed alveoli, and additional extracellular barriers such as mucus. Gene-based therapies targeted at the pulmonary epithelium may be less eff ective where there is extensive denudation of the pulmonary epithelium, as may occur in primary ARDS. Encouragingly, there is some evidence to suggest that ALI may not substantially impair viral gene transfer to the alveolar epithelium [47] .
Th e key limitation of nonviral vector approaches has been their lack of effi ciency in mediating gene transfer and transgene expression in the airway epithelium. Viral vectors are immunogenic, due to the protein coat of the viral vector, and the immune response is related to both vector dose and number of administrations. Th e potential to limit administration to a single dose in ALI/ARDS may reduce this risk. However, the development of an infl amma tory response resulting in death following administration of a fi rst-generation adenoviral vector highlights the risks involved [48] . Additional limitations of viral vectors include transgene size, which is limited by the size of the capsid that encloses the viral genes.
Th e therapeutic potential of gene therapy for ALI/ARDS is underlined by a growing body of literature demon strating effi cacy in relevant preclinical models. In considering the clinical implications of these studies, it is important to acknowledge that animal models of ARDS do not fully replicate the complex pathophysiological changes seen in the clinical setting. Th is is highlighted by the fact that many pharmacologic strategies demonstrating considerable promise in preclinical studies were later proven ineff ective in clinical trials. Nevertheless, these studies provide insights into the clinical potential of these strategies.
Adenovirus-mediated transfer of a gene that enhances surfactant production improves lung function and confers resistance to Pseudomonas aeruginosa infection ( Figure 2 ) [49] . Adenovirus-delivered superoxide dismutase and catalase genes protected against hyperoxic-induced, but not ischemia-reperfusion-induced, lung injury [50] . More recent studies have demonstrated the therapeutic potential of overexpression of a number of genes, including angio poietin-1 [51] , HSP-70 [52] , apolipo protein A-1 [53] , defensin β2 [54] and the Na + ,K + -ATPase pump [55] .
In contrast, overexpression of IL-1β can directly cause ALI [56] , while overexpression of suppressor of cytokine signal ing-3 worsens immune-complex-induced ALI [57] . Intriguingly, intra tracheal administration of adenoviral vector incor porating IL-10, prior to zymosan-induced lung injury, improved survival at a lower dose but was ineff ective and even harmful at higher doses [58] .
An early murine study demonstrated that cationic lipidmediated transfer of the Na + ,K + -ATPase gene ameliorated high-permeability pulmonary edema [59] . Electroporationassisted gene transfer of plasmids encoding for Na + ,K + -ATPase reverses endotoxin-induced lung injury [60] . Th e lipoplex-delivered IL-10 gene decreased lung and systemic organ injury induced by cecal ligation and puncture in mice [61] . Systemically administered cationic polyethyleni mine polyplexes incorporating indoleamine-2,3-dioxyge nase transduced pulmonary endo thelial cells and decreased lung ischemia-reper fusion injury [62] .
NF-κB decoy oligonucleotides, incorporated into viral vectors, attenuate systemic sepsis-induced lung injury when administered intravenously (Figure 3 ) [37] . In animal models, both intratracheal [34, 63] and intra venously [29, 64] administered siRNA successfully silence their target genes. shRNA-based approaches have been used to suppress silica-induced lung fi brosis [35] and to ameliorate lung ischemia-reperfusion-induced lung injury [36] .
More recently, aerosolization of siRNA that targets respiratory syncytial virus viral replication was safe and potentially eff ective in patients post lung transplant with respiratory syncytial virus infection [33] , clearly illustrating the therapeutic potential of these approaches for ALI/ARDS. 
Mei and colleagues enhanced the effi cacy of mesen chymal stem/stromal cells in endotoxin-induced ALI by transducing them to overexpress angiopoeitin-1 (Figure 4 ) [39] . Mesenchymal stem/stromal cells overexpressing IL-10 decreased alveolar infi ltration of CD4 and CD8 T cells following lung ischemia-reperfusion injury [65] . Bone marrow stem cells expressing keratinocyte growth factor attenuate bleomycin-induced lung injury [66] . Non stem cells can also be used to deliver genes to the injured lung [67] . Fibroblasts overexpressing angiopoeitin-1 attenuate endotoxin-induced lung injury [40] , while fi broblasts overexpressing vascular endothelial growth factor and endothelial nitric oxide synthase can attenuate or even reverse endotoxin-induced ALI [68] .
Advances in the identifi cation of therapeutic targets, improvements in viral and nonviral vector technologies, and regulation of gene-based therapies by temporal and spatial targeting off er the potential to translate the therapeutic promise of gene-based therapies for ALI/ ARDS to the clinical setting (Table 3) .
Viral vectors remain the focus of intensive research to optimize their effi ciency, to minimize their immuno genicity and to enhance their tissue specifi city [19, 31, 69, 70] . Strategies to develop less immunogenic vectors have focused on modifying the naturally occurring proteins in the viral coat [71] . Much research has been devoted to searching and characterizing both naturally occurring [71] and engineered capsid variants from mammalian species [72] . Capsid protein modification has also been used to enhance tissue specifi city [70] . Envelope protein pseudotyping involves encapsulating the modifi ed genome from one virus, such as simian immuno defi ci ency virus, with envelope proteins from another virus, such as vesicular stomatitic virus. Th is encapsu lation can enhance the therapeutic potential of viral vectors, by combining the advantages of one viral genome (for example, bigger payload or site-specifi c integration) with the tissue tropism of another virus.
Strategies to enhance the eff ectiveness of the lipoplexes used to deliver plasmids and other DNA/RNA oligonucleotides involve manipulation of the lipoplex lipid content and the use of targeting peptides. Th e choice of lipid infl uences expression effi ciency by enhancing release of the genetic material within the target cell [73, 74] . Targeting peptides increases transfection effi ciency by directing the lipid to a particular cell membrane or cell type [31] . Physical methods of plasmid delivery such as electroporation [60] and ultrasound can enhance gene transfer by bringing the plasmid DNA into closer proximity with the cell membrane and/or causing temporary disruption of the cell membrane. Other physical methods can also be used to increase in vivo gene transfer, including pressurized vascular delivery, laser, magnetic fi elds and gene gun delivery. Th ese systems enable plasmid-based gene delivery to reach effi ciencies close to that achieved with viral vectors.
Successful gene therapy relies upon being able to target the injury site, and to control the duration and levels of gene expression. Modifying the transgene DNA to exclude nonmethylated CpG motifs, typical of bacterial DNA, decreases the immune response and may increase transgene expression [75, 76] . High-effi ciency tissue-specifi c promoters may improve the effi ciency and specifi city of transgene expression. Lung-specifi c promoters include surfactant promoters [77] such as the surfactant protein C promoter [78] , a ciliated cell-specifi c promoter FOXJ1 [79] , the cytokeratin 18 promoter [80] , and the Clara cell 10-kDa protein [78] . Promoters can also be used to target a specifi c phase of illness, switching on when required to produce an eff ect at the optimal time point. A related approach is the development of promoters that allow for transfected genes to be turned on and off . Currently, the tetracycline-dependent gene expression vector [81] is the most widely used regulated system as it has a good safety profi le. Tetracycline is rapidly metabolized and cleared from the body, making it an ideal drug to control gene expression. However, the potential for an activator such as tetracycline to modulate the lung injury should be borne in mind. New-generation transactivators, with no basal activity and increased sensitivity, have now been developed [82] . In an ARDS context, conditional regulation of gene expression by the combined use of a lung-specifi c promoter and the tetracycline-dependent gene expression system may be a useful approach [83] . Capsid protein modifi cation to reduce immunogenicity [71] Capsid protein modifi cation to enhance tissue specifi city [70] Envelope protein pseudotyping
Manipulation of lipoplex lipid content to enhance cellular uptake [73, 74] Use of targeting peptides on lipoplexes and polyplexes [31] Strategies to enhance gene transfer; for example, electroporation, ultrasound, gene gun delivery
Modifying transgene DNA to eliminate bacterial motifs [75, 76] Development of high-effi ciency tissue-specifi c promoters [77] [78] [79] [80] Development of promoters that regulate gene expression [83] Enhanced therapeutic targeting Nebulization technologies [9] Strategies to target the pulmonary endothelium [10] Improved cellular uptake of vector Surface active agents to enhance vector spread [84] Reduce ubiquitination of viral capsid proteins [85] Better therapeutic targets Enhancement or restoration of lung epithelial and/or endothelial cell function [86] Strengthening lung defense mechanisms against injury [87] Speeding clearance of infl ammation and infection Enhancement of the repair process following ALI/ARDS [88] . 
An advantage of gene-based strategies is the ability to target specifi c cells within an organ; for example, the epithelial cells of the lung. Novel nebulization technologies, which facilitate the delivery of large quantities of undamaged vector to the distal lung, demonstrate considerable promise in this regard [9] . Alternative approaches to spatial targeting include targeting specifi c receptors that are plentiful on the target cell to increase transfection effi ciency. An interesting development in this regard is the targeting of systemically administered therapies to the pulmonary endothelium using antibodies to proteins expressed preferentially on these cells ( Figure 5 ) [10] . In these studies, the antioxidant enzyme catalase was conjugated with antibodies to the adhesion molecule PECAM, which is widely expressed on pulmonary endothelial cells, and to a nonspecifi c IgG antibody. Th e anti-PECAM/catalase conjugate, but not the IgG/catalase conjugate, bound specifi cally to the pulmonary endothelium and attenuated hydrogen peroxide injury.
Specifi c strategies have been developed to maximize uptake of vector into alveolar epithelial cells. It is possible to enhance lung transgene expression with the use of surface-active agents such as perfl urocarbon, which enhances the spread of vector and mixing within the epithelial lining fl uid [84] . Agents that reduce ubiquitination of AAV capsid proteins following endocytosis, such as tripeptide proteasome inhibitors, dramatically augment (>2,000-fold) AAV vector transduction in airway epithelia [85] .
Ultimately, the success or failure of gene-based therapies for ALI/ARDS is likely to rest on the identifi cation of better gene targets. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS continue to reveal novel therapeutic targets for gene-based approaches. Promising potential approaches include strate gies to enhance or restore lung epithelial and/or endothelial cell function [86] , to strengthen lung defense mechanisms against injury [87] , to speed clear ance of infl ammation and infection, and to enhance the repair process following ALI/ARDS [88] .
ALI/ARDS may be a particularly suitable disease process for gene-based therapies (Table 4 ). Th is is supported by increasing evidence from relevant preclinical ARDS models for the effi cacy of gene-based therapies that enhance or restore lung epithelial and/or endothelial cell function, strengthen lung defense mecha nisms against injury, speed resolution of infl ammation and infection, and enhance the repair process following ALI/ARDS. Despite this promising preclinical evidence, the potential for gene based approaches to ALI/ARDS in the clinical setting remains to be realized. Multiple barriers exist to the successful use of gene-based therapies in the lung, which limit the effi cacy of these approaches. Future research approaches should focus on overcoming these barriers, by developing more eff ective and less immunogenic vector delivery systems, developing strategies to focus gene expression on specifi c injury zones of the lung for defi ned time periods, and identifying better molecular targets that can take advantage of these potentially very powerful therapeutic approaches.
Abbreviations AAV, adeno-associated virus; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; IL, interleukin; NF, nuclear factor; shRNA, small hairpin RNA; siRNA, small interfering RNA.
The authors declare that they have no competing interests. Critical care services and the H1N1 (2009) influenza epidemic in Australia and New Zealand in 2010: the impact of the second winter epidemic INTRODUCTION: During the first winter of exposure, the H1N1 2009 influenza virus placed considerable strain on intensive care unit (ICU) services in Australia and New Zealand (ANZ). We assessed the impact of the H1N1 2009 influenza virus on ICU services during the second (2010) winter, following the implementation of vaccination. METHODS: A prospective, cohort study was conducted in all ANZ ICUs during the southern hemisphere winter of 2010. Data on demographic and clinical characteristics, including vaccination status and outcomes, were collected. The characteristics of patients admitted during the 2010 and 2009 seasons were compared. RESULTS: From 1 June to 15 October 2010, there were 315 patients with confirmed influenza A, of whom 283 patients (90%) had H1N1 2009 (10.6 cases per million inhabitants; 95% confidence interval (CI), 9.4 to 11.9) which was an observed incidence of 33% of that in 2009 (P < 0.001). The maximum daily ICU occupancy was 2.4 beds (95% CI, 1.8 to 3) per million inhabitants in 2010 compared with 7.5 (95% CI, 6.5 to 8.6) in 2009, (P < 0.001). The onset of the epidemic in 2010 was delayed by five weeks compared with 2009. The clinical characteristics were similar in 2010 and 2009 with no difference in the age distribution, proportion of patients treated with mechanical ventilation, duration of ICU admission, or hospital mortality. Unlike 2009 the incidence of critical illness was significantly greater in New Zealand (18.8 cases per million inhabitants compared with 9 in Australia, P < 0.001). Of 170 patients with known vaccination status, 26 (15.3%) had been vaccinated against H1N1 2009. CONCLUSIONS: During the 2010 ANZ winter, the impact of H1N1 2009 on ICU services was still appreciable in Australia and substantial in New Zealand. Vaccination failure occurred. Influenza A H1N1 2009 emerged in Mexico in early 2009 and spread rapidly causing a pandemic. The World Health Organization (WHO) declared a phase 6 influenza pandemic on 11 June 2009 and declared it to be over on 10 August 2010 [1] . The first wave of the H1N1 2009 outbreak was notable for the number of fatal cases among young people and atypical risk factors for developing severe diseases. Since 19 April 2009, WHO had reported over 491,766 laboratory-confirmed cases of H1N1 2009 and 18,449 related deaths [1] .
People in Australia and New Zealand (ANZ) were significantly affected by the virus, with a total of 43,700 confirmed cases in Australia as of October 2010, with 6,064 cases occurring between 1 January and 15 October 2010 [2] . We have reported previously the serious impact of the virus on the provision of critical care services in 2009 [3] . In order to rapidly inform health professionals in the Northern Hemisphere this study censored new incident cases before the end of the influenza season. In 2010, the deployment of vaccination and the acquisition of natural immunity against H1N1 2009 were expected to decrease the burden of disease due to influenza [4] . Australia and New Zealand were among the first countries to experience a second influenza season with H1N1 2009 following widespread deployment of vaccination.
In this report, we describe the incidence of intensive care unit (ICU) admissions, demographic and clinical characteristics (including vaccination status) and outcome of all patients with laboratory confirmed H1N1 2009 admitted to ICUs in ANZ during the second winter (2010) of this influenza epidemic. We compare these characteristics with those of patients admitted to ICU during the corresponding period of 2009.
We performed a multicentre study in 187 ICUs in ANZ comprising all adult, paediatric and combined adult and paediatric ICUs [5] . These ICUs had a total of 1,821 beds, of which 1,487 were equipped for mechanical ventilation. Each centre or region obtained Ethics Committee approval and the requirement for individual subject informed consent was waived at all sites. We report our findings according to STROBE guidelines for observational studies [6] .
Between 1 June and 15 October, in both 2009 and 2010, we screened for patients admitted to ICU with confirmed influenza A. Influenza A was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), antigen detection, or serology. H1N1 2009 and seasonal subtypes, H1N1 and H3N2, were determined by RT-PCR or specific serology. The laboratories were accredited by the National Association of Testing Authorities in Australia or by International Accreditation New Zealand. Population data for Australia and New Zealand were obtained from Australian Bureau of Statistics [7] and Statistics New Zealand [8] for 2009 and 2010.
We collected patient-specific data as described previously [3] , although vaccination status against H1N1 2009 virus was collected only during 2010. We divided patients into the age groups used in a previous report [9] . We calculated the duration of ICU and hospital stay and ICU occupancy rates for Australia and New Zealand. We recorded patient outcomes at ICU and hospital discharge status or as still in hospital or in ICU as of 15 October 2010 for patients admitted in 2010 and as of 23 November 2009 for patients admitted in 2009. Finally, we obtained data on vaccination in both countries.
We collected data using electronic case report forms. The study-coordinating centre was the Australian and New Zealand Intensive Care-Research Centre, Monash University, Melbourne, Australia [10] . H1N1 2009 infection is subject to mandatory reporting in both Australia and New Zealand and wherever possible diagnoses were confirmed with the relevant public health authorities. In addition, to confirm the completeness of case ascertainment, we contacted all ICUs that had no reported cases at the end of each study period. Cases transferred between ICUs were counted as a single ICU case. We made no assumptions for missing data and all proportions were calculated as percentages of available data.
We performed statistical analysis using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). We calculated descriptive statistics for all study variables. We report continuous variables as medians with interquartile range (IQR) and categorical variables as percentages with 95% confidence interval (95% CI) where appropriate. We estimated age-based population admission rates [7, 11] . We compared binomial variables of the second winter (2010) with those of the first winter (2009) using Chisquare tests for equal proportion or Fisher's Exact test where numbers were small. Comparisons between continuous variables were made using Wilcoxon rank sum test. A two-sided P-value of < 0.05 was considered to be statistically significant.
During the study period, from 1 June until 15 October 2010, 315 patients with confirmed influenza A were admitted to an ICU, compared with 1,113 patients during the same corresponding period in 2009. The predominant sub-type was H1N1 2009 in both years (90% in 2010 versus 83% in 2009, P = 0.002). The distribution of sub-types of influenza A is reported in Table 1 . In 2010 there were 283 patients with confirmed H1N1 2009 admitted to ICU, corresponding to a population incidence of admission to ICU of 10.6 (95% CI, 9.4 to 11.9) per million inhabitants [7, 8] . By comparison, during 2009 there were almost three times as many patients with confirmed H1N1 2009 (n = 921), corresponding to a population incidence of admission to ICU of 35.2 (95% CI, 32.9 to 37.5) per million inhabitants [7, 8] (P < 0.001). The geographical distribution of admissions was also different between 2010 and 2009 (Table 1 ). In 2010 the population incidence in Australia was 2-fold lower than in New Zealand (P < 0.001), whereas in 2009 this relationship was reversed with a 1.3-fold higher incidence in Australia (P = 0.009). The overall incidence, combining 2009 and 2010, was 45.3 (95% CI, 42.4 to 48.2) admission to ICU per million inhabitants in Australia and 47.1 (95% CI, 40.5 to 53.7) in New Zealand (P = 0.65).
The number of patients admitted to an ICU with H1N1 2009 according to study week is displayed in Figure 1 . In 2010 the onset of the epidemic was delayed (by five weeks) and the peak incidence was lower. The impact on ICU services was significantly lower in 2010 with peak daily ICU bed occupancy being 7.5 (95% CI, 6.5 to 8.6) per million inhabitants in 2009 compared with 2.4 (95% CI, 1.8 to 3.0) per million inhabitants in 2010 (P < 0.001) ( Table 1) .
The clinical characteristics, risk factors, and outcomes of patients admitted to ICUs with H1N1 2009 were broadly similar in 2010 to those admitted in 2009 ( Table 2 ). Comparing 2010 with 2009 there were no significant differences in the distribution of cases among different age groups. In both study periods the highest number of ICU admissions occurred among patients aged between 25 and 49. There was no difference in the proportion of patients with a body mass index greater than 35 kg/m 2 , who were pregnant or post-partum or who had no known predisposing factor between that observed in 2009 and 2010 ( Table 2) . The proportion of patients with asthma or chronic obstructive pulmonary disease, chronic heart failure, or an Acute Physiology Age Chronic Health Evaluation (APACHE) III or paediatric co-existing illness was lower in 2010, but these factors were still over-represented in comparison to the general population, as they were in 2009 [3] (Table 2) admissions due to confirmed influenza in both countries [3] . The second wave occurred later in the year but resulted in similar illness severity, affected similar groups at risk and caused similar in-hospital mortality. The incidence of H1N1 2009 influenza critical illness during 2010 was higher in NZ than in Australia, which was a reversal of the pattern in 2009. However, the combined incidence over both winters was similar in both countries. We observed vaccination failure in a substantial proportion of patients for whom vaccination status was known. Our observations do not support the view that the H1N1 2009 pandemic has come to an end [13] .
Although the incidence of critical illness was significantly lower in 2010 the pattern of illness among patients who were admitted to an ICU was similar to that observed in 2009 and in other reports of patients admitted to ICUs [14] [15] [16] . ARDS was present in more than 50% of patients, as previously described in ANZ [3] and worldwide [14, 15, [17] [18] [19] [20] [21] . The length of stay in ICU was unchanged [3] and similar to that reported elsewhere [14, 20] . The risk factors for admission to ICU were similar to those reported during the first wave [3, 22, 23] . In addition, the treatments administered and the mortality that occurred were similar to that observed in 2009 in ANZ as well as elsewhere [3, 13, 14, 18] .
The incidence of ICU admission per million inhabitants was higher in Australia than in New Zealand in 2009 (36.5 versus 28.3 admissions to ICU per million inhabitants) [3] . Conversely, in 2010 this incidence was higher in New Zealand. This difference is concordant with national data reporting 6,064 confirmed H1N1 cases in Australia (272 cases per million inhabitants) versus 1,810 in New Zealand (415 cases per million inhabitants in 2010) during the same period [2, 24] . The proportion of confirmed cases hospitalised (727 of 1,810 = 40.1%) [25] , and of hospitalised cases admitted to ICU (82 of 732 = 11.2%) in New Zealand was similar to 2009 [26] . Accordingly, the higher ICU admission rate per population observed in 2010 was due to a higher incidence of community infection, rather than a difference in severity of disease. The reason for the difference in infection rate between Australia and New Zealand in 2010 is unclear, as a similar proportion of the population was seropositive after the 2009 pandemic wave in Australia (22% (95% CI 19.1 to 24.9)) compared to New Zealand (26.7% (95% CI 22.6 to 29.4)) [27, 28] and a similar proportion were vaccinated (21.8% versus 24.1%, respectively) during the inter-wave period. Possible explanations include natural variations in community spread of influenza or an effect of the difference in vaccination deployment where Australia used both early monovalent and later polyvalent vaccines while New Zealand relied predominantly on delivery of the polyvalent vaccine alone, although overall coverage was similar.
We found that 15.3% of those patients for whom data were available had been previously vaccinated, which is consistent with another report of H1N1 2009 vaccination effectiveness [29] . In a critically ill patient with a severe pneumonia and a history of H1N1 vaccination the possibility of vaccination failure and H1N1 2009 infection should be considered.
Our data are subject to some limitations. To make this report available in a timely manner, we censored hospital outcome data. Ascertainment of cases of H1N1 2009 admitted to ICUs in 2010 as well as in 2009 may not have been complete, and we cannot exclude the possibility that a small number of cases were not reported to the registry, and false negative diagnostic tests may well have underestimated the true burden of H1N1 2009 in our patients. Among the patients with confirmed influenza A, there were 29 in whom the influenza was not sub-typed in 2010 and 132 in 2009. Thresholds for undertaking testing both in hospital and in the community were not standardised. It is not possible to reach a conclusion regarding the efficacy of vaccination as the vaccination status of many patients was unknown. Finally, while we report a similar severity of the illness to 2009, we do not have information about anti-viral treatment, potential viral mutation and resistance to anti-viral drugs. Finally, we did not evaluate the role played by a corticosteroid therapy on the outcome of patients with ARDS, and we were not able to cope with the controversy about the effect of this treatment [30] .
In conclusion, the impact of the second H1N1 2009 winter epidemic was still substantial although significantly less than in 2009. It had a lower peak occurring approximately five weeks later than in 2009, affected similar individuals, was similar in clinical severity, carried a similar mortality rate. In patients with H1N1 2009 infection requiring ICU admission a number of apparent vaccination failures were observed. Based on these data and despite the deployment of the vaccination, a second season of H1N1 2009 influenza may still have a substantial intensive care impact.
• H1N1 (2009) had a substantial impact on ICU resources during the winter of 2010 in Australia and New Zealand.
• Risk factors remain similar to those reported in 2009 and include obesity, pregnancy and presence of comorbidity.
• In 2010, the illness severity, reflected by treatment with mechanical ventilation, renal replacement therapy, vasopressor drug, and extracorporeal membrane oxygenation as well as by hospital mortality, was similar to that observed the previous year.  Early corticosteroid treatment for severe pneumonia caused by 2009 H1N1 influenza virus   Multinational, observational study of procalcitonin in ICU patients with pneumonia requiring mechanical ventilation: a multicenter observational study INTRODUCTION: The intent of this study was to determine whether serum procalcitonin (PCT) levels are associated with prognosis, measured as organ dysfunctions and 28-day mortality, in patients with severe pneumonia. METHODS: This was a multicenter, observational study of critically ill adult patients with pneumonia requiring mechanical ventilation conducted in 10 academic hospitals in Canada, the United States, and Central Europe. PCT was measured daily for 14 days using an immuno-luminometric assay. RESULTS: We included 175 patients, 57 with community acquired pneumonia (CAP), 61 with ventilator associated pneumonia (VAP) and 57 with hospital acquired pneumonia (HAP). Initial PCT levels were higher in CAP than VAP patients (median (interquartile range: IQR); 2.4 (0.95 to 15.8) vs. 0.7 (0.3 to 2.15), ng/ml, P < 0.001) but not significantly different to HAP (2.2 (0.4 to 8.0) ng/ml). The 28-day ICU mortality rate for all patients was 18.3% with a median ICU length of stay of 16 days (range 1 to 142 days). PCT levels were higher in non-survivors than in survivors. Initial and maximum PCT levels correlated with maximum Sequential Organ Failure Assessment (SOFA) score r(2 )= 0.50 (95% CI: 0.38 to 0.61) and r(2 )= 0.57 (0.46 to 0.66), respectively. Receiver operating curve (ROC) analysis on discrimination of 28-day mortality showed areas under the curve (AUC) of 0.74, 0.70, and 0.69 for maximum PCT, initial PCT, and Acute Physiology and Chronic Health Evaluation (APACHE) II score, respectively. The optimal cut-off to predict mortality for initial PCT was 1.1 ng/ml (odds ratio: OD 7.0 (95% CI 2.6 to 25.2)) and that for maximum PCT was 7.8 ng/ml (odds ratio 5.7 (95% CI 2.5 to 13.1)). CONCLUSIONS: PCT is associated with the severity of illness in patients with severe pneumonia and appears to be a prognostic marker of morbidity and mortality comparable to the APACHE II score. Respiratory tract infections requiring mechanical ventilation account for the majority of all infections treated in the intensive care unit (ICU) and are associated with prolonged hospital stay and high ICU mortality [1] [2] [3] . The Pneumonia Severity Index (PSI) is commonly used for risk stratification of patients with pneumonia. However, this parameter showed only moderate association with outcome prediction and was judged to be inadequate to guide clinical care [4] .
Numerous studies have evaluated the diagnostic performance of invasive procedures, or of biochemical and molecular markers in blood or bronchoalveolar lavage (BAL) in patients with ventilator-associated pneumonia (VAP), hospital acquired pneumonia (HAP) and community acquired pneumonia (CAP). These methods are difficult to apply to daily clinical practice and none has proved to be predictive of outcome [5] [6] [7] [8] . Furthermore, many aspects in the strategies for diagnosing HAP and VAP especially regarding the importance of invasive procedures are still controversial [9, 10] . Indeed, a recent study revealed that use of invasive procedures for etiological diagnosis of pneumonia varies considerably between European ICUs [11] . This uncertainty is most likely responsible for antibiotic overtreatment observed in this group of patients [12, 13] . Thus, measures to aid the early identification of patients with pneumonia are underdeveloped. Such measures are needed as patients with pneumonia are at high risk of death and would benefit from early adaption of therapy.
Procalcitonin (PCT), a relatively novel marker of infectious processes, has been shown to be associated with the severity of inflammation and prognosis during sepsis and septic shock [14] [15] [16] . In two large studies in the emergency department, low PCT-values were associated with a low risk of death in patients with CAP [17, 18] . Luyt and colleagues reported that PCT levels decreased during the clinical course of VAP but were significantly higher from Day 1 to Day 7 in patients with unfavorable outcomes [19] . The significance of PCT is emphasized by the observation that the course of PCT levels may safely guide antimicrobial therapy in patients with community acquired lower respiratory tract infections [20, 21] and ICU patients with suspected bacterial infections [22] . However, data about the significance of PCT in patients with hospital and ventilator acquired pneumonia requiring intensive care therapy are still limited.
The aim of this multicenter study was to test the hypothesis that serum PCT levels can assist in identifying patients with severe pneumonia who are at increased risk of poor outcome, measured as organ dysfunction and 28-day mortality.
In this multicenter, multi-national, observational study, patients admitted consecutively to the ICUs of 10 academic hospitals (8 in Canada and the United States and 2 in Europe) between 1 January 2003 and 20 November 2004 were screened for eligibility. The study protocol had been reviewed and approved by the Food and Drug Administration (protocol PCT-7; file number # I010023). Patients 18 years of age and older requiring mechanical ventilation with the new diagnosis of pneumonia within the last 48 hours were included. We excluded patients who were enrolled in a clinical study prior to baseline PCT sampling, had cardiogenic shock, had burns greater than 20% of total body surface, or were likely to die within 48 h, and postoperative patients following bone marrow transplant (within the last 6 months), coronary artery bypass grafts (within the last 7 days), and solid organ transplants (within the last 14 days). Patients were followed for 28 days after discharge from the ICU. The study was approved by local Institutional Review Boards/Ethics Committees of each participating institution and informed consent was obtained from the patients' next of kin.
Pneumonia was defined as the presence of new or progressive infiltrate(s), consolidation, cavitation, or pleural effusion on chest radiographs and the new onset of at least two of the following signs or symptoms: 1) cough; 2) production of purulent sputum or a change in the character of sputum; 3) auscultatory findings on pulmonary exam of crackles and/or evidence of pulmonary consolidation (dullness on percussion, bronchial breath sounds); and/or 4) the presence of acute or progressive dyspnea, tachypnea, or hypoxemia. In addition, at least one of the following criteria had to be fulfilled to establish the diagnosis of pneumonia: 1) fever, defined as body temperature > 38°C (100.4°F) taken orally; > 38.5°C (101.2°F) tympanically; or > 39°C (102.2°F
) rectally or via pulmonary artery (PA) catheter; and/or 2) elevated total white blood count (WBC) > 10,000/ mm 3 , or > 15% immature neutrophils (bands), regardless of total WBC, or leukopenia with total WBC < 4,500/ mm 3 . Microscopic examination of the Gram stained respiratory secretions had to show the presence of microorganisms, with ≥25 polymorphonuclear cells and ≤10 squamous epithelial cells per field at 100× magnification (low-power, 10× objective).
CAP [23] was defined as the occurrence of pneumonia in patients who had not resided in a long-term care facility for ≥14 days before the onset of symptoms and did not fulfill criteria of HAP, HAP [24] as pneumonia diagnosed in hospitalized patients or those residing in a long-term care facility (> 48 hours), such as a skilled nursing home facility or rehabilitation unit, or present < 7 days after a patient was discharged from the hospital with initial hospitalization of ≥3 days duration, and VAP [25] as pneumonia that developed more than 48 hours after intubation in mechanically ventilated patients who had no clinical evidence suggesting the presence or likely development of pneumonia at the time of intubation.
Within 48 hours of enrolment, we sought to establish a diagnosis of pneumonia through culture and susceptibility testing of respiratory secretions obtained by deep expectoration, nasotracheal aspiration, intubation with endotracheal suctioning, bronchoscopy with BAL or protected-brush sampling, or transtracheal aspiration. The diagnosis could also be supported by culture of samples obtained by percutaneous lung or pleural fluid aspiration, and/or single diagnostic antibody titer, (IgM), or a four-fold increase in paired serum samples (IgG) for the presumed pathogen. Patients with burns greater than 20% of total body surface, expected death within 48 h, post bone marrow transplant within the last 6 months, cardiogenic shock, cardiovascular bypass within the last 7 days, solid organ transplant within the last 14 days, or patients participating in other studies were excluded.
Key data were verified by source documents (hospital chart). Monitoring was conducted according to Good Clinical Practice (GCP) and standard operating procedures for compliance with applicable government regulations and was performed by an independent clinical research organization.
We recorded demographic data including date of birth, gender, ethnic origin, weight, and height, type of pneumonia, and admission Acute Physiology and Chronic Health Evaluation (APACHE) II score at study enrolment. Organ dysfunction status was assessed daily as described elsewhere [26] and worst values of each calendar day were reported. A modified Sequential Organ Failure Assessment (SOFA) score that excluded the Glasgow Coma Scale (GCS) was utilized.
PCT samples were collected for 14 days or until patients were discharged from the ICU and/or no longer required any mechanical ventilatory support. Blood samples not expected to be analyzed within 24 h of collection were frozen at -20°C for later analysis. PCT was measured using an immunoluminometric assay (LUMItest ® ; BRAHMS GmbH, Hennigsdorf, Germany). PCT levels were not available to the investigators until completion of the study and had no impact upon patient care during the course of the study.
The primary objective was to detect a correlation between maximum PCT and SOFA-score. A total of 180 subjects were required in order to significantly demonstrate that the correlation coefficient is above 0.2 with a power of 90%. Means ± standard deviations (SDs) or medians with interquartile ranges (IQR) are reported as appropriate. The three types of pneumonia were compared using tie-corrected exact Kruskal-Wallis tests. Pair-wise comparisons of HAP and VAP to CAP were added, based on tie-corrected exact Mann-Whitney U-tests. Odds ratios and receiver operating characteristic (ROC) curve methodology were used to judge the predictive power of PCT for outcome.
Of the 200 enrolled in this study, 25 patients were excluded from the analysis of the data. Of these, 21 patients had incomplete sampling and four patients met exclusion criteria. The characteristics on admission of the 175 patients included in our analysis study group are presented in Table 1 . Mean age was 62 years; roughly one-third had CAP, one-third had HAP, and one-third had VAP. The median hospital and ICU lengths of stay prior to enrolment were six days (range 0 to 368 days) and nine days (range 0 to 42 days), respectively.
Patients with CAP had higher APACHE II and SOFA scores at inclusion than patients with VAP. Such a difference was not observed between VAP and HAP patients. The incidence of cardiovascular co-morbid conditions on admission to the ICU was lower in patients with VAP than in the other groups (Table 1) . Positive cultures of the microbiological samples taken within 48 h were reported in 119 patients (67.4%). Gram-positive organisms were isolated in 75 patients (42.9%) and Gram-negative organisms in 63 patients (36.0%). The detected microorganisms are shown in Time course of PCT levels PCT levels were elevated at the time of enrolment in all groups (Table 3) . Initial PCT levels were higher in CAP than VAP patients. The maximum PCT levels were higher in patients with CAP than those with HAP or VAP. Maximum PCT occurred a median of one to two days after inclusion into the study. As shown in Figure  1 , PCT levels were persistently higher in patients with CAP than those with HAP during the first week following inclusion. There was no difference of initial PCT levels in culture positive and culture negative patients ( 
The overall 28-day mortality rate was 18.3% (n = 32) and the median ICU length of stay (LOS) was 16 (9 to 28.5) days (range 1 to 142 days). The 28-day mortality was higher in patients with severe CAP compared with those with HAP or VAP (36.8% vs. 10.5% and 8.2%, respectively, P < 0.01 each). Likewise, the maximum degree of organ dysfunction as assessed by the maximum SOFA score was higher in CAP compared with HAP and VAP patients. PCT levels were consistently higher in non-survivors than survivors throughout the observation period ( Figure 2 ). Initial PCT values of VAP patients were significantly higher in non-survivors than in survivors with a median PCT of 0.6 ng/ml in the latter group (Figure 3) . This difference between survivors and non-survivors was also observed in HAP but did not reach statistical significance. In the survivors, PCT values dropped to a median of 50.0% (27.3 to 100.0%) of the baseline value (P < 0.001) during the first five study days. A drop of similar magnitude with 53.7% (27.6 to 148.0%) was observed in the non-survivors without reaching statistical significance (P = 0.08). Initial and maximal PCT levels correlated with maximum SOFA score (r 2 = 0. 51 and r 2 = 0.57, respectively). The association between initial and maximum PCT levels and SOFA score was independent of the type of pneumonia (Figure 4) . In a ROC analysis on discrimination of 28-day mortality, the area under the curves (AUC) for maximum PCT, initial PCT, and admission-day APACHE II score were 0.74, 0.70, and 0.69, respectively ( Figure 5 ). The AUCs were not statistically different. The best cut-off of initial PCT to predict 28-day mortality was 1.1 ng/ml (odds ratio 7.0 (95% CI 2.6 to 25.2)) and that of the maximum PCT was 7.8 ng/ ml (odds ratio 5.7 (95% CI 2.5 to 13.1)). The highest AUC was observed in VAP patients with 0.71 (95% CI 0.92 to 1.01) compared to CAP with 0.41 (95% CI 0.24 to 0.92) and HAP with 0.56 (95% CI 0.58 to 0.96).
In this prospective multicenter study on a cohort of ICU-patients with severe pneumonia, median initial PCT levels were elevated above a normal value of 0.3 ng/ml in all groups. Those patients with ventilator associated pneumonia had the lowest initial PCT values. The maximum PCT levels were observed a median of one to two days after enrolment. Patients with severe CAP had highest initial median PCT values (2.4 ng/ml). These patients also showed greater disease severity, organ dysfunction, and mortality than HAP and VAP. This is in concordance with data from Valencia et al., who reported a mortality rate of 37% in CAP patients requiring ICU therapy [27] . Median admission PCTs of 3.4 ng/ml have been observed in patients presenting with CAP in the emergency department [17] .
PCT levels were higher, and remained persistently elevated, in non-survivors. Both, initial and maximum PCT values correlated with the maximum SOFA score and were a reasonable predictor of the risk of death within 28 days in these patients. In patients with severe pneumonia, initial PCT measurement allows a risk stratification similar to the APACHE II-score. The data agree with previous observations. In two studies in the emergency department with more than 1,600 patients each, PCT-values < 0.1 ng/ ml in CAP were associated with a low risk of death independent of the clinical risk assessment [17, 18] . PCT was also capable of identifying an unfavorable outcome in CAP patients staying at the ICU [28] . Impact of PCT-assessment is less well investigated in VAP and HAP compared to CAP. Patients with HAP not treated in an ICU have low median PCT values of 0.22 ng/ml [29] . In a single center study conducted in 44 patients with VAP, Duflo et al. found PCT to be significantly elevated in non-survivors: The best cut-off for serum PCT in the non-survivors in the VAP group was 2.6 ng/ml with a sensitivity of 74% and a specificity of 75% [7] . Likewise, Luyt et al. found high median PCT levels of about 3 ng/ml at Day 1 in patients with unfavorable outcomes during the clinical course of microbiologically proven VAP (n = 63) [19] . Interestingly, multivariate analyses further supported that serum PCT levels on days 1, 3, and 7 were strong predictors of unfavorable outcome [19] .
We found a significant association between PCT levels and organ dysfunction as assessed by the SOFA score. Similar observations were reported by Meisner et al. [30] and by Schroder et al. in surgical critically ill patients [31] . Hedlund et al. showed that the severity of disease measured by the APACHE II score was strongly associated with admission levels of PCT in 96 adult patients with CAP [32] . In 110 patients with CAP, Boussekey et al. found higher PCT levels in bacteremic patients and/or septic shock patients (4.9 ng/ml vs. 1.5 ng/ml) and in patients who developed infection-related complications (septic shock, multiorgan dysfunction, acute respiratory distress syndrome and disseminated intravascular coagulation) during their ICU stay [33] .
The association of PCT with morbidity and mortality may be of clinical importance not primarily for outcome prediction but to monitor success of therapy. Current data support the hypothesis that a drop in PCT levels represents an adequate antimicrobial therapy and may actually define a time point where antibiotic treatment can be safely withdrawn [20, 21] . Recently, this has been Continuous data are given as median (interquartile range) or mean ± standard deviation. CAP, community acquired pneumonia; HAP, hospital acquired pneumonia; ICU, intensive care unit; PCT, procalcitonin; n.s., not significant; SOFA, sequential organ failure assessment; VAP, ventilator associated pneumonia. demonstrated in ICU patients with suspected bacterial infection at admission or during their ICU stay [22] . More than 70% of these patients had pulmonary infections. Unsuccessful source control and poor outcome is associated with persistently elevated PCTs which should negatively affect outcome [14, 34] . Thus, increasing PCT or persistently elevated PCT values should trigger a change in antimicrobial therapy. In this study of severe pneumonia in mechanically ventilated patients, there was no difference in PCT levels between culture positive and culture negative pneumonia. In another study on patients with severe pneumonia as defined by a high Pneumonia Severity Index (PSI), PCT correlated with outcome but could not differentiate between bacterial and nonbacterial etiology of pneumonia [35] . In 72 children with CAP, Moulin et al. found PCT levels > 2 ng/ml in all 10 patients with blood culture positive for S. pneumoniae; PCT concentration was greater than 1 ng/ml in 86% of patients with bacterial infection, with the highest percentage being in those with positive blood culture [36] . This PCT-threshold was more sensitive and specific than CRP, IL-6, or white blood cell count for differentiating bacterial and viral causes of pneumonia. Likewise, Boussekey et al. found higher PCT levels in microbiologically documented CAP (median 4.9 ng/ml vs 1.5 ng/ml if no bacteria were found), but PCT levels could not discriminate between specific bacterial agents [33] . Duflo et al. identified VAP based on a positive quantitative culture of 10 3 colony-forming units/ml or more obtained via a mini-bronchoalveolar lavage. Median PCT values of VAP survivors at baseline were 0.6 ng/ml in this study. This low PCT value questions the validity of currently used VAP diagnostic criteria. Luyt et al. found a similar low PCT of about 0.5 ng/ml in VAP survivors and doubted the usefulness of this parameter for diagnosis of VAP [19, 37] . The 28-day mortality of 8.2% in patients with VAP in our study was very low. The Canadian Critical Care Trials group recorded an overall 28 days mortality rate of 18.7% in a large cohort of patients where VAP was diagnosed using similar criteria as in our study [5] . However, mortality rates between 9.8 and 93.3% have been observed depending on the presence of risk factors such as coexisting diseases, presence of bacteremia, arterial hypotension, or ARDS [38] . The low mortality rate of VAP patients and low PCT-values in the VAP survivors in this study may reflect the uncertainty in correctly diagnosing VAP despite the requirement for a positive Gram stain of respiratory secretion. Although VAP is the most frequent cause of death in hospital for patients with respiratory failure [39, 40] , the diagnosis of VAP is difficult. The optimal invasive procedure for diagnosing HAP or VAP remains poorly defined [9, 10] . Indeed, one study demonstrated that 29% of clinically suspected VAP cases were disproved by autopsy results [41] . In this study, microbiological proof of infection was possible in about 67% of the patients. This is in good agreement with findings in large sepsis trials where microbiological proof was possible in 41 to 51% of the patients with airway infections [42, 43] .
It should be noted that the immunoluminometric assay for PCT measurement applied in this study has been replaced today by more modern techniques with a higher accuracy especially in the low range of PCT levels. Such accuracy is a prerequisite when using PCT for antibiotic stewardship [20] . This study was focused on high PCT concentrations for their association with mortality and organ dysfunction. It is unlikely that such a relationship is affected by the type of assay.
Measurement of PCT levels in addition to the clinical judgement may offer a solution for this diagnostic dilemma since our data suggest that baseline PCT levels greater than 1.1 ng/ml identify a group of ICU patients with a high risk to develop multiorgan dysfunction followed by death. The quality of mortality prediction was similar to the APACHE II score. These data confirm the observation by Luyt et al., who found a PCT threshold of 1 ng/ml to predict unfavorable outcome [19] .
Furthermore, non-survivors showed no decrease in PCT suggesting that pneumonia remained uncontrolled. Assessing adequacy of antimicrobial therapy was not part of the study hypothesis and would have been beyond the scope of this trial. However, PCT measurement offers the possibility of being a marker for monitoring therapeutic success or failure, since successful therapy is associated with a decrease in PCT levels. A PCT guided algorithm has been shown to reduce duration of antibiotic therapy without affecting patients' safety [22, 44] .
In patients with severe pneumonia (CAP, VAP, HAP), PCT is associated with the severity of illness and is a good prognostic marker of morbidity and mortality in patients with pneumonia in demand of mechanical ventilation. The severity of illness as reflected by the degree of organ dysfunction may be a more important determinant of PCT levels than the type or cause of pneumonia.
• Procalcitonin (PCT) concentrations are associated with the severity of illness in patients with severe pneumonia in demand of mechanical ventilation.
• PCT is a good prognostic marker of morbidity and mortality in these patients.
• The severity of illness as reflected by the degree of organ dysfunction may be a more important determinant of PCT levels than the type or cause of pneumonia.
Abbreviations APACHE II: Acute Physiology and Chronic Health Evaluation II; AUC: area under the curve; BAL: bronchoalveolar lavage; CAP: community acquired pneumonia; CI: confidence interval; GCP: Good Clinical Practice; GCS: Glasgow Coma Scale; HAP: hospital acquired pneumonia; ICU: intensive care unit; IQR: interquartile range; PCT: procalcitonin; PSI: pneumonia severity index; SD: standard deviation; SOFA: Sequential Organ Failure Assessment; ROC: receiver operating characteristic; VAP: ventilator associated pneumonia; WBC: white blood cell count.
RB, PL, DA and KR helped to design the study, were responsible for the conduct of the trial, and helped to draft the manuscript. FMB conceived and designed the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Competing interests FB received a speaker fee from BRAHMS. ER receives research support from the National Institute of Allergy and Infectious Disease and the Aggennix Corporation and has served as one-time consultant for Aggennix Corporation, Eisai Pharmaceuticals, Idaho Technologies and Astra Zeneca. RB has received research support, consulting fees, and honoraria from BRAHMS and from bioMerieux. DA has received consultant fees from BRAHMS, performed PCT assays for the PCT-7 trial, and had access to equipment and assays by BRAHMS as part of NIH-funded studies. KR has received consultant fees from BRAHMS. FMB has received consultant and speaker fees and grant/research support from BRAHMS. JM, RD, JV, GG and PL declare that they have no competing interests. The role of receptor for advanced glycation endproducts (RAGE) in infection  During evolution, multicellular organisms have devel oped an impressive arsenal of defense and repair mechanisms to counteract threats such as infection and trauma. Such an infl ammatory response begins with the detection of the potential life-threatening event by recognizing so-called danger signals. Th ese signal molecules have been classically divided into: i) Exogenous, pathogen-associated molecular patterns (PAMPs) [1] , which are con served motifs on pathogens that are not found in higher eukaryocytes; and ii) endogenous innate danger mole cules, also named damage-associated molecular patterns (DAMPs) or alarmins, which are structurally diverse proteins rapidly released by the host itself during infec tion or (sterile) tissue damage [2] .
Known PAMPs include lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria, peptidoglycan (present in most bacteria), lipoteichoic acid (in many Gram-positive bacteria), bacterial DNA, viral DNA/RNA and mannans in the yeast cell wall. PAMPs are recognized by pattern recognition receptors (PRRs), in particular Toll-like receptors (TLRs) and Nod-like receptors (NLRs), leading to an infl ammatory response via several signaling pathways, including nuclear factorkappa B (NF-κB) activation and subsequent tumor necrosis factor (TNF)-α production.
Examples of putative DAMPs, the endogenous equivalents of PAMPs, are high-mobility group box 1 (HMGB1), some S100 proteins (S100A8/A9, S100A12), interleukins such as IL-1α, heat-shock proteins (HSPs), and nucleosomes [3] . DAMPs can be secreted either actively or passively following necrosis but are not released by apoptotic cells [4] and have activating eff ects on receptorexpressing cells engaged in host defense. DAMPs can also be detected by TLRs and NLRs and their engagement induces NF-κB activation as well, suggesting that DAMPs and PAMPs use, at least partially, the same receptors and signaling pathways. Liu et al. [5] however, propose that the immune system treats DAMPs and PAMPs diff erently; they suggest that DAMPs -but not PAMPs -bring CD24-Siglec G/10 into the proximity of TLRs/NLRs, resulting in repressed DAMP-induced TLR/NLR signaling.
When invaded by pathogens, host defense systems encounter PAMPs from microorganisms and DAMPs that are released from tissues, which are recognized by TLRs and NLRs to warn the host of imminent danger. In addition, the multiligand receptor for advanced glycation endproducts (RAGE) is regarded as a prototypic DAMP receptor that can bind several DAMPs, including HMGB1 and S100A12 [6] . Other known RAGE ligands include amyloid, β-sheet fi brils, S100B and S100P [7] ; furthermore, β2 integrins can interact with RAGE [8] . RAGE is expressed at high levels in the lungs and at low levels in normal adult tissues, including on cells involved in the innate immune system, e.g., neutrophils, T and B lymphocytes, monocytes, macrophages, dendritic cells, and endothelial cells [7] . Engagement of RAGE by its ligands leads to receptor-dependent signaling and activation of NF-κB and mitogen-activated protein kinase (MAPK) pathways [7] . Activation of RAGE plays a role in diverse experimentally-induced sterile infl ammatory and infectious diseases, including cecal ligation and puncture (CLP)-induced abdominal sepsis [9] , diabetic nephropathy, delayed type hypersensitivity, type II collagen induced arthritis, hepatic injury, and diabetic atherosclerosis [7, [10] [11] [12] . Th is review focuses on new insights into the pathogenesis of infectious diseases, including sepsis, peritonitis and pneumonia, off ered by studies conducted in the RAGE research fi eld. 
RAGE consists of three immunoglobulin-like regions, a transmembrane domain, and a highly charged short cytosolic tail that is essential for intracellular signaling [13] . Th e V domain in the extracellular part of RAGE is essential for binding of its ligands. Because of its ability to recognize three-dimensional structures rather than specifi c amino acid sequences, RAGE can interact with a wide range of ligands. RAGE was fi rst identifi ed as a receptor for advanced glycation endproducts (AGEs), explaining its name. AGEs are products of the nonenzymatic glycation and oxidation of lipids, proteins and other macromolecules that appear, in particular, under conditions of increased availability of reducing sugars and/or enhanced oxidative stress, especially when molecules turn over slowly and aldose levels are elevated. Further investigations showed that RAGE can recognize a diverse array of endogenous molecules that warn the immune system and induce a defensive immune response; the alarmins or DAMPs.
HMGB1 is a non-histone DNA-binding protein that serves as a structural component to facilitate the assembly of nucleoprotein complexes in the nucleus [14] . Extracellularly, HMGB1 functions as a cytokine. In response to infl ammatory stimuli, including PAMPs, HMGB1 can be actively released into the extracellular environment from a variety of cells including monocytes, macrophages, endothelial cells, enterocytes, pituicytes, dendritic cells, and natural killer cells [14] . HMGB1 can also be passively secreted into the extracellular milieu when cells die in a non-programmed way (necrosis), whereas apoptotic cells modify their chromatin so that HMGB1 binds irreversibly and consequently is not released [4] . During infectious diseases, increased HMGB1 concentrations may be due to active as well as passive release. Detection methods of HMGB1 that are currently used (and published) do not distinguish between these (and possible other) diff erent forms of HMGB1. More studies are necessary to: 1) Report the biological activity of (diff erent forms of ) HMGB1; and 2) develop HMGB1 ELISA assays that can distinguish between these (possibly also functionally) diff erent forms of HMGB1. Most investigations on HMGB1 and infection involve sepsis, the second leading cause of death in non-coronary intensive care units (ICUs) and the 10 th leading cause of death overall. Patients with severe sepsis display elevated circulating HMGB1 levels [15] [16] [17] and HMGB1 is predominantly released at the site of infection; patients with pneumonia and those with peritonitis showed increased concen trations in fl uid obtained from the bronchoalveolar space and abdomen, respectively [17] . In an animal model of CLP-induced sepsis, the kinetics of HMGB1 secretion in vivo was delayed and more sustained when compared with the release of pro-infl ammatory cytokines, like TNF-α, IL-1β and IL-6 [18, 19] . Similarly, various interventions that inhibit HMGB1 activity or production, such as anti-HMGB1 antibodies, the A-box segment of HMGB1, ethyl pyruvate, and nicotine, reduced CLPinduced sepsis and/or LPS lethality even if treatment was delayed for many hours, up to one day after the challenge [20, 21] . An implicated crucial event in sepsis pathophysio logy is apoptosis of immune cells, playing a major role in immunosuppression and lethality [22] . HMGB1 seems to be a downstream factor of apoptosis in the fi nal common pathway to organ damage in severe sepsis as indicated by observations that prevention of lymphocyte apoptosis improved survival after CLP [23] , whereas anti-HMGB1 treatment reduced lethality in the same model without infl uencing apoptosis [19] . Th is indicates that HMGB1 secretion is a relatively late event in sepsis that contributes signifi cantly to a worsened outcome. In addition, it has been reported that very pure HMGB1 does not have cytokine-inducing capacity itself, but activates cells indirectly by fi rst acquiring immune stimulating CpG DNA [24] , which is released in the bloodstream during bacterial sepsis. However, a recent study reported that HMGB1-mediated induction of macrophage cytokine production requires binding to TLR4, and that binding and signaling are dependent on a molecular mechanism that requires cysteine in position 106 within the B box [25] . Together these data indicate that HMGB1 may exert pro-infl ammatory eff ects in a direct TLR4-dependent way and an indirect way via binding of DAMPs and other agonistic molecules. S100A12 S100A12 is a calcium binding protein expressed in the cytoplasm of neutrophils, where it comprises 5% of the total protein content. Furthermore, S100A12 -also known as EN-RAGE (extracellular newly identifi ed ligand of RAGE) or myeloid-related protein (MRP)-6 -is found in monocytes and lymphocytes and provokes pro-infl ammatory responses in endothelial cells [26] . Although many RAGE ligands are promiscuous with regard to receptor use, S100A12 has only been shown to bind to RAGE. S100A12 expression is high in infl ammatory diseases such as atherosclerosis, rheumatoid arthritis, Crohn's disease, Kawaski disease, and cystic fi brosis. Within the lungs, S100A12 and RAGE are increased during acute lung injury (ALI) [26] . S100A12 expression may refl ect activation of neutrophils during pulmonary infl ammation and may contribute to endothelial activation via binding to RAGE [26] .
Recruitment of leukocytes to the site of infection is an essential step in host defense during infectious diseases against invading pathogens. RAGE plays a role in the regulation of cell migration in several ways. First of all, RAGE is a counter-receptor for integrins on leukocytes; in particular, RAGE has been identifi ed as a binding partner for the β2 integrins, Mac-1 and p150, 95 [8] . Second, by the interaction of RAGE with β2 integrinmediated leukocyte recruitment in vivo: RAGE -/mice displayed a diminished number of adherent infl ammatory cells on the peritoneum after CLP [9] and a reduction in neutrophil infl ux in the peritoneal cavity after thioglycollate peritonitis [8] . Interestingly, HMGB1 can activate lateral (in cis) RAGE-Mac-1 interactions on the leukocyte cell surface, enhancing Mac-1-intercellular adhesion molecule (ICAM)-1-dependent adhesion and migration [27] (Fig. 1 , indicated by the blue line and blue "+"). Furthermore, a recent report shows that endothelial expressed RAGE acts in concert with ICAM-1 in mediating β 2 integrin-dependent leukocyte adhesion during acute trauma-induced infl ammation [28] (Fig. 1 , indicated by the green line and green "+").
Th e signaling cascade(s) of RAGE -induced by engagement of its various ligands -that ultimately activates NF-κB is largely unknown. Th e predicted cytosolic portion of RAGE, consisting of 43 amino acids, is short compared to other PRRs, the TLRs and IL-1 receptors, and does not include a known signaling domain or motif. A RAGE mutant lacking this intracellular tail does not activate NF-κB and behaves like a dominant negative, preventing proinfl ammatory cytokine release from macro phages. Th ese data indicate a critical role of this cytosolic portion in transducing the signal from the cell surface to the nucleus. The damage-associated molecular patterns (DAMPs), high-mobility group box 1 (HMGB1) and S100A12, are released during infection ( [15, 17] , and unpublished data) and bind to and activate RAGE. It has to be determined whether other S100 proteins and other DAMPs are RAGE ligands (indicated as purple shapes) released during infection. It would be interesting to investigate whether RAGE can directly bind to, become activated and mount a fi rst immune reaction after ligation with specifi c PAMPs as well. Engagement of RAGE by its ligands results in receptor-dependent signaling and activation of NF-κB leading to a pro-infl ammatory response; the signaling pathway is largely unknown. In addition, RAGE interacts as an endothelial (and epithelial) adhesion receptor with the leukocyte integrin, CD11b/CD18 (Mac-1) (lower section) [8] . Furthermore, lateral (in cis) RAGE-Mac-1 interaction on the leukocyte surface is mediated by HMGB1 and activates Mac-1-intercellular adhesion molecule (ICAM)-1 dependent adhesion and migration and augments leukocyte recruitment [27] (indicated by the blue line and blue "+"). Moreover, a recent report shows that endothelially expressed RAGE acts in concert with ICAM-1 in mediating β 2 integrin-dependent leukocyte adhesion during acute trauma-induced infl ammation [28] (indicated by the green line and green "+"). One possibility is that RAGE uses as yet unknown adaptors framing a whole `new' signaling cascade to NF-κB. Another possibility is that the RAGE tail interacts with a Toll/IL-1 receptor (TIR)-containing protein which then recruits the downstream TIR-contain ing proteins in a way analogous to TLR-mediated signaling pathways. Finally, RAGE could transduce signals from the cell surface to the nucleus by bypassing the TIR-containing adaptor, directly interacting with member(s) of the signaling cascade.
In addition to triggering NF-κB activation, RAGE engage ment by its myriad ligands is linked to an array of signaling pathways, including MAPK family members, such as Jun-N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), PI3K/Akt, Rho GTPases, Jak/STAT and Src family kinases [7] .
Th is rather extraordinary variety of observed signals may be due to the broad expression of RAGE, its diversity of ligands, and possible contaminating elements in the preparations used in experiments.
Th e truncated form of full-length RAGE, soluble RAGE (sRAGE), consists of only the extracellular ligand-binding domain (V-C-C') lacking the cytosolic and trans membrane domains (i.e., the parts that transfer a signal into the cell) and circulates in the bloodstream. sRAGE has been indicated to be involved in infl ammatory processes in several ways. First, sRAGE blood concentrations are associated with various infl ammatory diseases in patients and in rats with experimentally induced ALI [29] . Further more, it is suggested that sRAGE can compete with full length cell-surface RAGE for ligand engagement, preventing these ligands from binding to RAGE or other receptors and/or exerting eff ects otherwise. Exogenous sRAGE treatment indeed attenuated infl ammatory responses in several animal models, including models of type II collagen-induced arthritis, hepatic injury, diabetic atherosclerosis, delayed type hypersensitivity, and experimental auto-immune encephalomyelitis [7] . Th e involvement of sRAGE during infection is not known. Based on experimental studies in rats and in patients with ALI, sRAGE has been described as a marker of lung injury [29] .
An increasing amount of research suggests a role for RAGE in the pathogenesis of pneumonia, peritonitis and sepsis, indicating that RAGE (ligand)-directed therapies might off er new treatment opportunities for human disease in the future.
Recent studies point to an important role of RAGE in the pulmonary compartment in physiological as well as in pathological circumstances. Physiologically, RAGE is expressed at high basal levels in the lungs relative to other tissues [26, [29] [30] [31] [32] [33] [34] , suggesting that RAGE may have lung-specifi c functions distinct from the role of RAGE in other adult tissues. In particular, although the kidney is dramatically aff ected by microangiopathy and fi brosiswhich is substantially attributed to RAGE -in patients with diabetes, the lungs, with a signifi cantly higher baseline RAGE expression than the kidney, remain unaff ected. In addition, RAGE has been found to be specifi cally localized near the basal cell membrane within alveolar pneumocytes [32, 35, 36] . Th ese two observa tions raise the question as to whether RAGE has a function in normal healthy lungs. Indeed, Englert et al. documented that aged RAGE -/mice develop pulmonary fi brosis-like alterations spontaneously; lungs from 19 to 24 month-old RAGE -/mice showed increased collagen staining and displayed increased levels of hydroxyproline relative to wild type mice [31] . RAGE knockdown in pulmonary fi broblasts increased their proliferation and migration in vitro, suggesting an important protective function of RAGE in the lungs and that loss of RAGE may be related to functional changes of pulmonary cell types resulting in fi brotic disease [34] . Another study demonstrated that RAGE on epithelial cells promoted their adherence to human collagen (a major component of the alveolar basal lamina) and a spreading morphology, which may facilitate gas exchange and alveolar stability in vivo [34, 35] . Together, these data suggest that RAGE plays a role in maintaining lung homeostasis in normal, healthy lungs. Further studies are needed to unravel the function(s) of pulmonary RAGE in physiology in more detail.
Th is putative functional role of RAGE in healthy lungs may be the explanation for the fi nding that the inhibition of RAGE signaling attenuates pathological sterile infl ammatory responses in diverse non-pulmonary experi mental studies [9] [10] [11] [12] , whereas in pulmonary non-infectious pathological infl ammatory conditions, somewhat confl ict ing results emerge. Lung injury induced by either bleomycin or hyperoxia is diminished in RAGE -/mice [37, 38] , suggesting a deteriorating attribution of RAGE. In contrast, Englert et al. showed that RAGE -/mice developed more severe lung fi brosis after asbestos adminis tration as measured by histological scoring and total lung hydroxyproline quantifi cation [31] . Of note, in all these studies, the mice were much younger at the time of sacrifi ce than the aged (19-24 month-old) RAGE -/mice that developed pulmonary fi brosis spontaneously in the experiment by Englert et al. [31] . Interestingly, lung homogenates and bronchoalveolar lavage (BAL) fl uid from patients suff ering from idiopathic pulmonary fi brosis reveal reduced membrane bound (and soluble) RAGE protein levels compared to healthy donor samples [31, 34] . 
Community-acquired pneumonia (CAP) is distinguished from hospital-acquired pneumonia (HAP) according to the time of acquisition of pneumonia and the pathogens involved. Th e Gram-positive bacterium, Streptococcus pneumoniae, is the single most frequent pathogen causing CAP, responsible for up to 60% of cases; Klebsiella pneumoniae, Haemophilus infl uenzae, Staphylococcus aureus, and viruses are isolated in about 10% of cases each and Mycobacterium tuberculosis is more prevalent in developing countries. Knowledge of the expression and role of RAGE in host defense during pneumonia is limited. Morbini et al. observed increased RAGE expression in patients with interstitial and postobstructive pneumonia [32] ; this report left unanswered whether patients with bacterial pneumonia were included in the analysis. Notably, two other studies showed that constitutively present RAGE was not upregulated during pulmonary infl ammation associated with ALI or acute respiratory distress syndrome (ARDS): First, rats with ALI induced by intratracheally administered LPS displayed no change in the distribution of RAGEexpressing cells [29] ; and second, patients with ARDS did not have increased pulmonary expression of RAGE [26] . sRAGE has been suggested to be a lung injury marker based on studies in patients with ALI and on experimental studies in rats [29] . sRAGE was increased in pulmonary edema fl uid and serum from patients with either ALI or ARDS and with hydrostatic pulmonary edema, and in BAL fl uid from rats with either LPS-or hydro chloric acid-induced ALI [29] .
Considering the ubiquitous expression of RAGE in the lungs, its putative involvement in the regulation of lung infl ammation and the somewhat inconsistent fi ndings which currently exist in the literature, we investigated its role during pneumonia, a major cause of morbidity and mortality world-wide. Recently, we reported that murine pneumonia induced by S. pneumoniae and by infl uenza A virus was associated with an upregulation of intraalveolar (membrane bound) RAGE expression [39, 40] . Furthermore, lung tissue of mice intranasally infected with the K. pneumoniae or with M. tuberculosis also showed increased RAGE expression (unpublished data). Th ese clinically very diff erent types of pulmonary infection and the involvement of RAGE therein will be discussed below.
Levels of the high-affi nity RAGE ligand, HMGB1, were higher in BAL fl uid from patients with pneumonia compared to BAL fl uid from healthy controls [17] . In experimentally induced pneumococcal pneumonia, the presence of RAGE was detrimental: Mice lacking RAGE had a better survival rate together with a lower pulmonary bacterial load and decreased dissemination of S. pneumoniae to blood and spleen compared to wildtype mice [39] . Th e diff erence was possibly partially due to an increased killing capacity of RAGE -/alveolar macrophages. Additionally, lung injury and neutrophil recruitment were reduced in the RAGE -/mice, which parallels fi ndings on RAGE as an endothelial counter receptor for the β2 integrin, Mac-1, [8] and the interplay between RAGE and Mac-1 on leukocytes, required for HMGB1-mediated infl ammatory cell recruitment [27] . In addition, blockade of the RAGE-HMGB1 interaction and prevention of the subsequent pro-infl ammatory stimulus might be an explanation for the less severe pulmonary damage in the RAGE -/mice during S. pneumoniae pneumonia.
Interestingly, in contrast to Gram-positive pneumonia, preliminary data from our laboratory reveal that RAGE plays a benefi cial role in mice during the host response to Gram-negative pneumonia (unpublished data). Indeed, RAGE defi ciency was associated with increased mortality and increased bacterial outgrowth and dissemination after K. pneumoniae inoculation (unpublished data). Relative to wild type mice, lung infl ammation was similar and cytokine and chemokine levels were slightly -if at allelevated. Moreover, RAGE -/mice showed an unaltered response to intranasally instilled Klebsiella LPS with respect to pulmonary cell recruitment and local release of cytokines and chemokines. Together, these fi ndings indicate that RAGE contributes to an eff ective antibacterial host response during K. pneumoniae pneumonia, whereas RAGE plays an insignifi cant part in the lung infl ammatory response to either intact Klebsiella or Klebsiella LPS.
It is unclear whether RAGE can also interact with ligands from pathogens. If so, this could be part of the explanation for our observation that RAGE involvement during Gram-positive and -negative pneumonia had such opposite eff ects on mortality. In addition, RAGEmediated eff ects on other fi rst-line defense mechanisms, such as chemotaxis, phagocytosis, killing (including respiratory burst), may depend on the pathogen and may contribute to the observed eff ects in Gram-positive and -negative pneumonia models. However, this remains speculative until investigations have been performed to analyze this interesting issue.
In addition to its potential to cause pandemics, seasonal infl uenza A virus infection causes over 200,000 hospitalizations and approximately 41,000 deaths in the United States annually, being the 7 th leading cause of mortality. We demonstrated that RAGE defi ciency resulted in a better outcome from pulmonary infl uenza A virus infection as indicated by a relative protection from infl uenza A virus-induced lethality in mice [40] . Th is was accompanied by improved viral clearance and enhanced cellular T cell response and activation of neutrophils, suggesting that endogenous RAGE impairs the cellular immunity against respiratory tract infection with infl uenza A virus. RAGE ligand, HMGB1, as well as sRAGE were upregulated in BAL fl uid during infl uenza A virus pneumonia. Hence, similar to pneumonia induced by the Gram-positive bacterium S. pneumoniae, RAGE is detrimental during pneumonia caused by infl uenza A virus. Th is is of particular interest, since it has been suggested that the greatest proportion of the mortality associated with infl uenza A virus infection is due to secondary bacterial pneumonia, with S. pneumoniae as the most frequent pathogen of the superinfection. Th erefore, RAGE is a potential treatment target in postinfl uenza pneumococcal pneumonia and further research is warranted to investigate this.
Th e role of RAGE in abdominal sepsis has been investigated in a limited number of studies so far. RAGEdefi cient mice showed decreased mortality after induction of polymicrobial sepsis induced by CLP in two reports [9, 41] . Moreover, anti-RAGE antibody yielded a better survival even when the anti-RAGE therapy was delayed up to 24 hours after CLP in mice receiving antibiotics [41] . Th e protective eff ect provided by the absence of RAGE was related to a fi rm inhibition of NF-κB activation, suggesting that the lack of excessive NF-κB activation in RAGE -/mice might have contributed to their reduced mortality [9] . In addition, RAGE defi ciency resulted in fewer infl ammatory cells in the peritoneum [9] , which parallels the results of an earlier investigation by the same group of authors identifying RAGE as a counter-receptor for the β2 integrin, Mac-1 (CD11b/CD18), and thereby as a mediator of leukocyte recruitment and adhesion [8] . Furthermore, the protective eff ect of RAGE inhibition in this CLP model could at least in part be the consequence of the inhibition of one of its ligands, HMGB1. Indeed, HMGB1 is secreted into the circulation after CLP and anti-HMGB1 antibody led to increased survival after CLP-induced peritonitis [18] .
In the same surgically (CLP)-induced model of sepsis, RAGE defi ciency and anti-RAGE therapy were reported not to aff ect bacterial outgrowth in the peritoneum, liver, or spleen [41] . Notwithstanding, a possible role of RAGE in antibacterial defense cannot be easily evaluated from this study because host defense against CLP depends, at least in part, on the extent of intestinal necrosis and the formation of a local abscess. Also, all mice in this experiment received broad spectrum antibiotics and bacterial outgrowth was only determined in mice that survived (i.e., not at predefi ned time points after CLP). For this reason, we used our model of abdominal sepsis induced by injection of the Gram-negative bacterium Escherichia coli into the peritoneum [42, 43] to study whether RAGE aff ects antibacterial defense. Th is model is a relevant tool to investigate the role of receptors/ mediators in limiting the growth and dissemination of bacteria after a primary intra-abdominal infection and to assess the contribution of these proteins to specifi c immune responses. RAGE expression was upregulated during E. coli induced sepsis [42] . RAGE defi ciency (either pharmacologically using anti-RAGE IgG antibodies or genetically using RAGE knock out mice) was related to a higher bacterial load and dissemination [42] . Th ese data indicate that RAGE signaling contributes to an eff ective antibacterial response during abdominal sepsis. RAGE exerted this eff ect probably indirectly and not via direct interaction with E. coli, considering the observation that leukocytes from RAGE -/mice had an unaltered capacity to phagocytose and kill E. coli in vitro. Furthermore, the fi nding that defi ciency of RAGE in general was associated with an exaggerated host response during E. coli sepsis [42] on the one hand, and with an attenuated infl ammatory response and better survival in (other) sterile models of intraperitoneal injection of LPS derived from E. coli [42, 44] on the other hand, suggests that although RAGE is involved in the immune reaction to E. coli, this function can be compensated for by other receptors in the presence of a growing bacterial load. Th e high-affi nity RAGE ligand, HMGB1, is secreted into the circulation systemically during clinical sepsis [16] [17] [18] as well as in our experimental sepsis model of E. coli [43] . Importantly, HMGB1 has been shown to transduce cellular signals in vitro and in vivo by interacting with at least three other receptors, i.e., TLR2, TLR4 and TLR9 when HMGB1 is complexed with CPG DNA [24, 44, 45] . One possible explanation for the increased response in the RAGE lacking mice during E. coli sepsis is, therefore, that the absence of RAGE could facilitate the interaction between HMGB1 and TLR2, TLR4 and/or TLR9.
Evidence of involvement of ligands of RAGE and HMGB1 in host defense in E. coli abdominal sepsis was recently published by our laboratory [43] . Inhibition of multiple RAGE ligands (by the administration of sRAGE) and inhibition of HMGB1 (by the administration of anti-HMGB1 antibodies) led to an enhanced bacterial dissemi nation of E. coli, denoting an advantageous role of RAGE ligands, including HMGB1, in the antibacterial response during Gram-negative sepsis.
Interestingly, we recently found that S100A12, another high-affi nity ligand of RAGE, is released systemically in patients during (abdominal) sepsis and also locally during peritonitis (unpublished data). Additionally, intravenous injection of LPS in healthy humans raised circulating S100A12 levels, implying that LPS might partially contribute to this upregulation during Gram-negative infection.
Payen et al. reported that, in patients with septic shock, mRNA S100A12 expression by circulating leukocytes was decreased during the recovery phase [46] . One possible function of S100A12 in host defense during infection and sepsis is its role as a DAMP. NF-κB mediated expression of pro-infl ammatory cytokines and upregulation of ICAM-1 and vascular cell adhesion molecule (VCAM)-1 on endothelium has been documen ted in vitro after S100A12 stimulation [47] . Furthermore, S100A12 could be of benefi t for the host during infection and sepsis due to its (more direct) antibacterial activity. Cole et al. determined that S100A12 has activity primarily against Gram-negative bacteria, including E. coli [48] . Because of the absence of S100A12 in rodents, a potential functional role of S100A12 during sepsis cannot be easily investigated by inhibiting/deleting S100A12 in animals. Altogether, the role of S100A12 during sepsis has yet to be evaluated using non-rodent models.
Bopp et al. documented that septic patients have elevated circulating sRAGE levels and that non-survivors show higher plasma sRAGE concentrations than survivors, suggesting that sRAGE is related to severity and clinical outcome in sepsis [49] . Knowledge on the role of endogenous sRAGE in sepsis is scarce. Hudson et al. demonstrated that sRAGE levels might represent an early marker of microvascular dysfunction, a pheno me non also present in sepsis [50] . Furthermore, increased sRAGE concentrations in sepsis might represent the acute infl ammatory status as splice variants of RAGE or as split off variants of the cell surface RAGE, the latter analogous to ICAM-1, another member of the immunoglobulin superfamily, which is a marker of cellular damage during sepsis. Another possibility is that systemic sRAGE levels might be elevated in parallel with HMGB1/ S100A12 levels as a counter-system against HMGB1/ S100A12 elicited tissue eff ects. More research is needed to clarify the functional role of sRAGE in sepsis and its putative role as a new sepsis marker.
Th e innate immune response is the fi rst line of defense against pathogens. Th e experimental studies described herein provide further insight into the role of RAGE and its ligands in host defense during clinically important infections, which eventually may contribute to better therapies against specifi c pathogens. While interpreting the results from preclinical investigations, one has to keep in mind that a careful balance between the infl amma tory and anti-infl ammatory response is vital in order to survive or recover from a severe infection.
Th e observation that lack of RAGE is of benefi t in one pneumonia model and detrimental in another, clearly adds to the notion that the way in which RAGE mediates host defense against diff erent pathogens relies on distinct mechanisms. It would be highly interesting to evaluate whether RAGE can directly bind to, become activated, and mount a fi rst immune reaction after ligation with specifi c PAMPs. Furthermore, RAGE-mediated eff ects on other fi rst-line defense mechanisms, such as chemotaxis, killing, phagocytosis and respiratory burst could depend on the pathogen. As such, targeting RAGE may be ineff ective or even harmful in some infectious conditions. Th erefore, more studies are necessary to justify clinical trials targeting RAGE in patients with severe infections. In this respect one could think of research on RAGE inhibition in pneumococcal and infl uenza A viral pneumonia. Additionally, experiments in which RAGE targeting is delayed until after bacterial/viral infection and combined with antibiotic/antiviral therapy should be considered. Moreover, more studies need to be conducted on the role of RAGE in critical organ derangements involved in the pathogenesis of severe infection, including activation of the coagulation system and the complement system. RAGE remains a potential yet promising therapeutic target that awaits further research. Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS. In May 2007, a county hospital in Xinyang City, Henan Province treated three patients with fever, abdominal pain, bloating, nausea, vomiting, gastrointestinal bleeding, and elevated aminotransferases. The local hospital diagnosed the disease as acute gastroenteritis. A family member of one patient reported the disease to the Henan Center for Disease Control and Prevention (CDC), which sent a team to investigate. The investigation revealed that the disease had the following characteristics: (1) acute onset with fever; (2) low white blood cell and platelet counts; (3) high levels of alanine and aspartate transaminases; (4) positive urine protein. On the basis of these features, the Henan CDC excluded the possibility of gastrointestinal disorders. In order to identify the disease etiology, the Henan CDC team used the above clinical characteristics as the case definition to search for similar cases in local hospitals in this and neighboring counties, while establishing a disease surveillance system that required all medical institutions to report cases that met the above case definition. Altogether, 79 cases were found in 2007 in Henan, with 10 fatalities (case fatality rate, 12.7%). All patients were farmers and resided in mountainous or hilly villages, and many had reported tick bites 7-9 days before illness, further suggesting an infectious etiology. In recent years, patients with similar clinical symptoms were reported with human granulocytic anaplasmosis (HGA; Anaplasma phagocytophilum) in neighboring Anhui province [1] . In 2005, there was an epidemic of Tsutsugamushi (scrub typhus/ Orientia tsutsugamushi) in this area [2] . Clinical investigations, epidemiological analyses, and laboratory testing prompted consideration of rickettsial diseases as possible causes, including HGA, human monocytic ehrlichiosis (HME; Ehrlichia chaffeensis), and Tsutsugamushi disease. Specific methods such as polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) for these pathogens were then used to determine if these cases were attributable to HGA or HME [3, 4] . However, only 18 of 79 (22.7%) patients were positive for A. phagocytophilum based on serology and DNA testing. Thus, the disease was initially considered at least partly caused by A. phagocytophilum, and cases were provisionally diagnosed as suspected HGA based on clinical and epidemiological data [1] [2] [3] [5] [6] [7] . In the 3 years since 2007, 206 suspected cases have been discovered in Henan, but there was only a very low positive rate of A. phagocytophilum confirmation (6 of 206 patients) and no pathogen was isolated. Similar cases were also reported in the mountainous and hilly areas of nearby Shandong, Jiangsu, Hubei and Anhui provinces, indicating that the disease already existed for some time and was widely distributed [7] . We decided to address the possible causative pathogen underlying this infection.
On the basis of epidemiological and clinical characteristics, we considered two types of diseases to be possible: rickettsial and arthropod-borne viral disease. Because of the low rates of A. phagocytophilum (rickettsial disease) detection, the research team intensified its virus search to take into account arthropod-borne viruses, including Flaviviridae (Dengue viruses [DENV] , Japanese encephalitis virus [JEV]), Togaviridae (Chikungunya virus, Eastern equine encephalitis virus [EEEV] , Western equine encephalitis virus), and Bunyaviridae (Crimean-Congo hemorrhagic fever virus, Hantaan virus, and Rift valley fever virus) [8] . Specific PCR assays for these viruses were used [9] [10] [11] [12] [13] [14] [15] [16] . However, none of the patients from 2007 to 2010 was positive for these viruses, suggesting a new infectious agent, possibly a virus, remained to be discovered. Thus, the syndrome was considered an emerging infectious disease and was named fever, thrombocytopenia and leukopenia syndrome (FTLS). To identify the etiology, the research team adopted the following strategy: 1) sequencing of randomly amplified cDNA/ DNA from FTLS patient samples using high-throughput Illumina sequencing to specifically explore viral communities present in patients suffering from FTLS, 2) PCR detection of target DNA directly from clinical specimens, 3) viral culture, 4) immunodetec-tion methods, and 5) electron microscopic study of the morphology of the cultured virus.
Culture followed by serological and molecular tests is a standard approach for identifying an unknown virus. However, culture of an unknown virus is time-consuming, even taking several years to confirm a novel infection like HIV [17] . Otherwise, virus culture often fails because of the lack of cell lines capable of supporting propagation of viruses (e.g., hepatitis B and C virus). Methods for cloning nucleic acids of microbial pathogens directly from clinical samples offer opportunities for pathogen discovery, thereby laying the foundation for future studies aimed at assessing whether novel or unexpected viruses play a role in disease etiology. Random PCR and subtractive cloning sequencing have identified previously unknown pathogens as etiological agents of several acute and chronic infectious diseases [18, 19] . Recently, high-throughput sequencing approaches have been used for pathogen detection and discovery in clinical samples [20] [21] [22] . We also developed a method for exploring viruses, both known and novel, using highthroughput Illumina sequencing. In this study, high-throughput Illumina sequencing was applied to specifically explore the viral communities in patients with FTLS, using healthy subjects as controls. Here, we provide evidence for the discovery of a novel bunyavirus associated with FTLS through high-throughput sequencing. Subsequent culture of the virus and PCR detection of the specific virus in patient specimens confirmed these findings. 
Given the serious nature of FTLS, it was decided to handle all clinical specimens and perform all experiments involving live virus in a biosafety level-3 (BSL-3) facility.
We studied 285 Henan province patients with FTLS whose samples were submitted to the Henan Province CDC between May 2007 and July 2010. Acute-phase serum samples from all patients were collected. Paired convalescent sera were available from 95 patients.
Diagnostic testing of sera for microbial agents possibly related to FTLS Sera were tested by reverse transcription (RT)-PCR, PCR, and/ or indirect IFA serological assays for a number of microbial agents, including A. phagocytophilum, Ehrlichia chaffeensis, Dengue fever virus, Japanese encephalitis virus, Chikungunya virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Sandfly fever Naples Sabin virus, and Hantavirus [3] [4] [5] [6] [9] [10] [11] [12] [13] [14] [15] [16] 23] . Antigen slides for diagnosis of HGA (A. phagocytophilum) were purchased from Focus Diagnostics (IF1450G, CA, USA). Antigen slides for diagnosis of other pathogens were prepared by our laboratories. Fluorescein isothiocyanate (FITC)-conjugated goat anti-human
Initially in 2007, and again between 2008 and 2010, cases of a life-threatening disease with sudden fever, thrombocytopenia, and leukopenia were reported in Henan Province, China. Patient reports of tick bites suggested that this disease could be infectious or tick-transmitted. Many patients were provisionally diagnosed with human granulocytic anaplasmosis (HGA). However, only 24 of 285 (8%) had objective evidence of HGA, suggesting that other pathogens likely contributed to fever, thrombocytopenia and leukopenia syndrome (FTLS). Illumina sequencing was used for direct detection in clinical samples of pathogens possibly associated with FTLS. A novel bunyavirus was found only in samples from FTLS patients. Further epidemiologic and laboratory investigation confirmed that the novel bunyavirus was associated with FTLS. The results illustrate that metagenomic analysis is a powerful method for the discovery of novel pathogenic agents. Combined with epidemiologic investigation, it could assist in rapid diagnosis of unknown diseases and distinguish them from other diseases with similar symptoms caused by known pathogens.
IgG (Fc) was purchased from Sihuan Sci-Technics Company (Beijing, China).
Equal quantities (100 mL) of acute-phase sera from 10 FTLS patients who had a history of tick bite were pooled and centrifuged at 1000 x g for 10 minutes. The supernatant was collected for DNA and RNA extraction. The same was done for 10 sera from healthy subjects (control).
DNA was extracted from 140 mL of each sample using the QIAamp DNA mini Kit (Qiagen, 51304) according to the manufacturer's instructions. DNA was eluted from the columns with 50 mL water containing 20 mg/mL RNaseA. After incubation at 37uC for 15 minutes to eliminate RNA, DNA was used immediately or stored at 280uC.
Total RNA was extracted from 140 mL of each sample using the QIAamp viral RNA mini Kit (Qiagen, 52904) according to the manufacturer's instructions. RNA was eluted from the columns with 50 mL of diethyl pyrocarbonate (DEPC)-treated water containing 1 U DNaseI. Samples were incubated at 37uC for 15 minutes to eliminate human DNA, followed by DNase inactivation at 95uC for 10 minutes. RNA was used immediately or stored at 280uC. 
Random hexamer PCR was carried out in a 25-mL mixture containing 4 mL of cDNA or DNA, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl 2 , 100 mM dNTPs, 1 U Taq DNA Polymerase (Promega, M1661) and 100 ng of random hexamer primers containing a linker (5'-GCCGGAGCTCTGCAGAA-TTCNNNNNN-3'). After denaturing at 95uC for 5 minutes, targets were amplified by 45 cycles of 95uC for 30 seconds, 40uC for 30 seconds, 50uC for 30 seconds, and 72uC for 90 seconds. The amplified products were detected by agarose gel electrophoresis. Pure water was used as a negative control.
FTLS patient and control genomic DNA/cDNA libraries were constructed according to the manufacturer's instructions (Illumina). In brief, RT-PCR and PCR products were roughly quantified by UV absorption and equal amounts of each sample were mixed. Nucleic acids within the mixtures were sheared by sonication, and fragments in the 150-180-bp range were collected by cutting bands from an agarose gel after electrophoresis. The sheared DNA and cDNA ends were repaired using Klenow DNA polymerase, after which 5' termini were phosphorylated and 3' termini were polyadenylated. The adaptors were added, PCR enrichment was performed, and 150-180-bp fragments were collected for sequencing by the Illumina method.
The sequencing procedure was performed according to the manufacturer's instructions (Illumina). In this process, template library DNA was hybridized to the surface of the flow cells and multiple copies of DNA were made to form clusters using the Illumina cluster station. Workflow steps included template hybridization, isothermal amplification, linearization, and final denaturation and hybridization of sequencing primers. Paired-end sequencing (100 cycles) was performed using a four-color DNA Sequencing-By-Synthesis (SBS) technology following the manufacturer's instructions.
Short Oligonucleotide Analysis Package (SOAP) was used to handle the large amounts of short reads generated by parallel sequencing [24] . Briefly, after filtering out highly repetitive sequences and adaptor sequences, the overlapping datasets between FTLS and healthy subjects were analyzed by subtracting fragments that mapped to both host genomic-plus-transcript and bacteria databases. The non-redundant reads were mapped onto a virus database downloaded from NCBI (ftp://ftp.ncbi.nih.gov/ genbank/). The resulting alignments were filtered to identify unique sequences by examining alignment (identity $80%) and Evalue scores (e#10 22 ). Filtered unique alignments were examined in the taxonomy database (NCBI) using a custom software application written in Perl (BioPerl version 5.8.5). Unmapped reads were examined in GenBank nucleic acid and protein databases using BLASTn and BLASTx, respectively [25] [26] [27] . Unique alignments were examined in the taxonomy database (NCBI). Sequences without hits were placed in the ''unassigned'' category. Sequences were phylotyped as human, bacterial, phage, viral, or other based on the identity of the best BLAST hit. Considering misannotation and low-complexity for Illumina short reads, sequences assigned to the same virus family were further assembled into contigs with Velvet 1.1.04 (K-mer length = 21; coverage cutoff: default 0; Insert length: PE only; minor contig length: 42) for direct comparison with GenBank nucleic acid databases using BLASTn [28] . Contigs were also blasted with GenBank protein databases using BLASTx [28] . An E-value cutoff of 1610 25 was applied to both BLASTn and BLASTx analyses. Sequences phylotyped as viral were placed in the ''viral'' category.
Our mass sequencing data revealed that some sequences showed possible infection with human bocavirus, which belongs to the Parvoviridae family. PCR performed to detect human bocavirus tentatively identified in sera from FTLS patient samples amplified a 291-bp fragment of the NS1 gene, as described previously [29] . Amplified products were detected by agarose gel electrophoresis and sequenced using an ABI 3730 DNA Sequencer.
Our mass sequencing data revealed a 168-bp sequence (C361, Accession: HQ412604) indicating the possible presence of a novel virus with closest identity (i.e., lowest E-value) to Tehran virus which belongs to the Phlebovirus genus of the Bunyaviridae family. We developed a PCR strategy based on the 168-bp sequence identified in sera from FTLS patient samples to detect the novel bunyavirus using forward (PF: 5'-GAC ACG CTC CTC AAG GCT CT-3') and reverse (PR: 5'-GCC CAG TAG CCC TGA GTT TC-3') primers designed with Primer3 (Supplementary Figure S1) . PCR was carried out in a 25-mL mixture containing 4 mL of cDNA, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl 2 , 100 mM dNTPs, 1 U Taq Pol (Promega, M1661), 0.25 mM forward primer, and 0.25 mM reverse primer. Thermocycling conditions were as follows: 95uC for 4 minutes (denaturation), followed by 35 cycles of 94uC for 30 seconds, 54uC for 30 seconds, and 72uC for 30 seconds. Amplified products were detected by agarose gel electrophoresis and sequenced using an ABI 3730 DNA Sequencer.
The Vero E6 cell line (African green monkey kidney cell) was selected for isolation of the novel bunyavirus associated with FTLS because it supports the growth of many bunyaviruses [30, 31] . Vero E6 cell lines were inoculated with six serum samples that contained novel bunyavirus RNA. Each sample underwent at least three cell culture passages in Vero E6 cell line before being considered negative. Medium was replenished on day 7, and cultures were terminated 14 days after inoculation. All cultures were observed daily for cytopathic effect (CPE). Virus-infected cells and uninfected cells were also examined for the novel bunyavirus by RT-PCR at each passage. Vero cell cultures with obvious CPE and containing novel bunyavirus RNA were further analyzed by morphology, genome sequencing, and serology.
Cells showing CPE and containing novel bunyavirus RNA were collected for thin-section electron microscopy. After discarding the culture supernatant, virus-infected cells (50 mL) were mixed 1:1 with 4% glutaraldehyde (paraformaldehyde), placed onto Formvar-carbon-coated grids, and stained with 1% methylamine tungstate. Specimens for thin-section electron microscopy were prepared by dehydrating washed cell pellets with serial dilutions of acetone and embedding in epoxy resin. Ultrathin sections were cut on an Ultracut LKBV ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEM-1400).
The medium from 20 mL of novel bunyavirus-infected Vero E6 cells was centrifuged at 1,000 x g for 10 minutes and then at 4,000 x g for 10 minutes, after which the supernatant was collected. PEG8000 was added to the supernatant at a final concentration of 10% (w/v) followed by centrifugation at 20,000 x g for 2 hours. The pellet was resuspended in 2 mL 16phosphate-buffered saline (PBS) for RNA extraction. Random RT-PCR was performed, and the products (500-1500 bps) were collected and ligated into the pGEM-T vector (Promega, A3600) by incubating overnight at 16uC. Escherichia coli JM109 were transformed with the ligation mixture and cultured on LB agar containing X-gal. White clones were sequenced using an ABI 3730 DNA Sequencer. Contaminating human and extraneous sequences were eliminated using Cross-Match, and the complete sequence was assembled using Phred-Phrap-Consed [32] . Bridge RT-PCR was employed for gap-closure.
Phylogenetic analyses were performed using the neighbor-joining method in the MEGA software package, version 4.0.2 [33] . Available nucleotide or protein sequences from known viruses were obtained from GenBank for inclusion in the phylogenetic trees. Selected sequences from GenBank included those with the greatest similarity to the sequence read in question based on BLAST alignments as well as representative sequences from all major taxa within the relevant Bunyaviridae family. To further establish the relationships between the new virus and the members of the Phleboviruses genus, we included all sequences for phleboviruses available in GenBank. Branching orders of the phylograms were verified statistically by resampling the data 1,000 times in a bootstrap analysis with the branch-and-bound algorithm, as applied in MEGA.
After successful isolation of the novel bunyavirus, we developed an indirect IFA to detect specific antibodies in patient serum specimens, as previously described [4] . In brief, monolayers of virus-infected Vero E6 cells showing CPE and containing novel bunyavirus RNA were harvested, and one volume of infected cells was mixed with 0.5 volumes of non-infected cells. The mixture was centrifuged at 1,000 x g for 10 minutes, after which cells were resuspended in 16PBS, spotted onto 12-well glass slides, and fixed with acetone for 10 minutes. Sera from patients with FTLS (including 285 acute-phase samples and 95 paired sera), patients with respiratory diseases (80 serum samples), and healthy subjects (50 serum samples) were applied to the cells. Samples (diluted 1:20 in PBS) were screened by first spotting 50 mL of each serum sample per well and incubating for 30 minutes at 37uC. After washing for 10 minutes in PBS, 20 mL of FITC-conjugated goat anti-human IgG (Sihuan Sci-Technics Company, Beijing, China) diluted 1:40 in buffer containing Evans blue was added to each well and incubated for 30 minutes. After washing, slides were mounted in glycerin and examined by immunofluorescence microscopy. A titer of 1:20 was considered positive. 
The median age of patients was 57.2 years (range, 23-88) and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5-14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.
The main clinical features in confirmed patients included sudden onset of fever (.37.5uC 240uC) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia (Table 1) . A small number of cases suffered more severe complications, including hypotension, mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary hemorrhage, respiratory failure, disseminated intravascular coagulation, multiple organ failure, and/or death. Most patients had a good outcome, but elderly patients and those with underlying diseases, neurological manifestations, coagulopathy, or hyponatremia tended to have a poorer outcome.
Laboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels ( Table 2 ). Biochemical tests revealed generally higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes, especially AST.
One lane for each of the two sample pools (FTLS and healthy controls), each consisting of 10 samples, was sequenced. The proportions of high-quality sequences in FTLS and control pools were 98.08% (10, 198 ,407/10,397,161) and 97.88% (10,250,809/ 10,472,315), respectively. Unique, high-quality sequence reads were then classified into broad taxonomic groups based on the taxonomy of the most frequent top-scoring BLAST matches for each sequence. Virus sequences constituted 0.0065% (67,969) and 0.0048% (50,677) of the reads in FTLS and control libraries, respectively (Table 3) . After contig assembly, the number of sequences phylotyped as ''viral'' decreased to 3,163 and 2,412, respectively, in the two pools (Table 4) . To screen for possible viruses, we focused exclusively on viruses that were present in patient sera.
For detection of known viruses, non-redundant reads were directly aligned with the GenBank database of nucleic acids using BLASTn software. Some sequences from Adenoviridae (human adenovirus), Herpesviridae (herpesvirus), Papillomaviridae (papillomavirus) and Retroviridae (human endogenous retrovirus) were detected in both FTLS patients and healthy subject samples. Some hepatitis B virus (HBV) and human bocavirus sequences were detected only in patient sera, indicating the presence of HBV and human bocavirus infections among these patients. To detect novel viruses, we examined sequence data against the GenBank protein database using BLASTx. An analysis of the deduced protein sequences revealed four different virus families in sera from FTLS patients (Table 4 ). Among these were viruses from Hepadnaviridae, which are not known to cause FTLS; Torque teno virus (TTV) from Anelloviridae, which has been reported to be associated with certain inflammatory states [34] , but is not known to be transmitted by arthropods; and viruses from the Parvoviridae family, including human bocavirus, which could cause febrile illness and signs of FTLS. Although human bocaviruses are not known to be transmitted by arthropods, feline panleukopenia virus, a parvovirus, is strongly suspected to be transmitted by arthropods [35] . All family Parvoviridae sequences detected in FTLS samples were also assembled and their protein sequences deduced. Included among these samples were four fragments, all of which were found to be highly homologous to human bocavirus; one fragment showed the greatest similarity to human bocavirus 2 isolate 53044 (identity = 86%) with the lowest E-value (8610 217 ). Human bocavirus was further detected by PCR in the pool of 10 serum samples, but only one individual sample within the pool tested positive for bocavirus. The final virus family detected in sera from FTLS patients was the Bunyaviridae family, which contains viruses known to cause FTLS after tick bites [8, 30] . All family Bunyaviridae sequences detected in FTLS samples, including 11 fragments (1 S-segment, 2 M-segments, and 8 L-segment fragments), were assembled and their protein sequences were deduced. Among these 11 novel virus fragments was a 168-bp fragment (C361, Accession: HQ412604) of the polymerase gene that showed the greatest similarity to Tehran virus (identity = 36%) with the lowest E-value (3610 27 ). This suggested the presence of a novel virus or a known virus whose genome had not yet been sequenced.
To more accurately assess the genetic relationships to known viruses, we constructed a phylogenetic tree using a neighbor-joining method [33] . The result showed that the potentially novel virus clustered with Toscana virus, Uukuniemi virus, and Rift Valley fever virus of the Phlebovirus genus ( Figure 1A ). The pools of 10 serum samples from patients and 10 serum samples from healthy subjects were also screened by PCR for the presence of the novel bunyavirus. All 10 samples from patients tested positive for the novel bunyavirus; however, all 10 samples from healthy subjects tested negative. Thus, we further focused on the virus from the family Bunyaviridae.
Using specifically designed RT-PCR primers, we detected viral RNA in 223 of the 285 acute serum samples tested ( Table 5 ). The specificity of the RT-PCR was confirmed by sequencing selected PCR products. None of the 80 sera from patients with respiratory diseases or the 50 sera from healthy subjects was positive using the novel virus-specific RT-PCR.
Six acute serum samples that tested positive for the novel bunyavirus by specific RT-PCR were inoculated onto Vero E6 cells, and four virus strains were isolated. The initial CPE analysis showed rounded refractile cells 2-4 days after inoculation. CPE did not progress in the initial cultures, but appeared slightly at 24 hours in subsequent passages (Figure 2A) . RT-PCR revealed the presence of RNA for the novel bunyavirus in all four virus strains, and all isolates reacted with the serum of a convalescent patient in IFA ( Figure 2C ). In addition, electron microscopy showed the presence of virus particles approximately 80-90 nm in diameter-a size compatible with a bunyavirus (Figure 3 ). Virus particles were presumably localized to the Golgi apparatus ( Figure 3 ).
Genome sequencing of one isolate (HN01) revealed three segments of negative polarity, single-stranded RNA, including a large segment (L; GenBank HQ642766), a medium-sized segment (M; GenBank HQ642767), and a small segment (S; GenBank HQ642768). The deduced amino acid sequence of the L segment had the highest homology (34%, E value = 3610 2163 ) to RNA polymerase of the Uukuniemi virus of the Phlebovirus genus, whereas the M segment had the highest homology (26%, E value = 8610 255 ) to glycoprotein genes of the Punta Toro virus in the Phlebovirus genus. Of the two proteins encoded by the ambisense S segment, one had the highest homology to nucleocapsid protein (39%, E value = 3610 240 ) of the Rift Valley Fever virus, and the other had the highest homology to nonstructural protein genes (24%, E value = 0.049) of the Punique Virus, both of which belong to the Phlebovirus genus. Collectively, these findings confirm that this virus belongs to the Phlebovirus genus of Bunyaviridae.
During the revision of this manuscript, some new sequences of SFTSV (severe fever with thrombocytopenia syndrome) were released. A comparison of these new SFTSV sequences with the sequence of this novel virus showed that they were highly homologous (.99% identity). Using sequences of Phlebovirus available in GenBank, a phylogenetic analysis showed that, although most closely related to the Uukuniemi virus of the Phlebovirus genus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage), the three genomic segments of the novel virus, along with the SFTSV sequences, were highly divergent ( Figure 1B-1E ).
IgG antibodies to the novel bunyavirus were detected in 80 of 285 acute-phase serum samples from patients with FTLS (Table 5) . Of 95 patients from whom paired acute-and convalescent-phase sera were available, 52 had seroconversions and 21 had greater than 4-fold increases in antibody titer to the virus. Six had less than a 4-fold increase in antibody titer to the virus, but all paired sera tested positive. Sixteen patients tested negative to the virus, suggesting that some non-FTLS patients with similar symptoms were included in this study, a situation that is not surprising given that FTLS is a newly emerging disease. The acute-phase sera of four patients from whom the virus was isolated tested negative for IgG antibody to the virus. All convalescent sera obtained 2 months later from the same four patients contained IgG antibody to the virus. None of the 130 sera from patients with respiratory diseases or healthy subjects had detectable antibody.
Since 2007, there has been an increase in reported cases of FTLS in Xinyang City, Henan Province. These patients were tentatively diagnosed as having A. phagocytophilum infection. However, only a few (8.4%, 24/285) such patients had evidence for A. phagocytophilum infection, and none of the 285 patients tested positive for the many other pathogens capable of causing similar clinical and laboratory manifestations that were also investigated. These findings suggested novel infectious agents, including viruses.
Traditionally, virus culture is very important for identifying an unknown viral infection. Before performing the Illumina sequencing strategy, we attempted viral and rickettsial culture with DH82 and BHK cell lines, but the lack of an obvious CPE led us to initially abandon this approach. Here, mass sequence data obtained by Illumina sequencing revealed four virus families that appeared only in FTLS patient sera. Among these four virus families, viruses from the Parvoviridae and Bunyaviridae families reportedly can cause signs of FTLS and be transmitted by arthropods. However, only one sample from a pool of ten samples tested positive for bocavirus by PCR, suggesting that bocavirus from the Parvoviridae is not likely involved in FTLS. For viruses in the Bunyaviridae family, the incidence of infection is closely linked to vector activity. For example, tick-borne viruses are more common in the late spring and late summer when tick activity peaks. Human infections with certain Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus, are associated with high levels of morbidity and mortality [30] . Considering the tick-bite history of many FTLS patients, we focused on Bunyaviridae family viruses.
The entire Bunyaviridae family contains more than 300 members arranged in four genera of arthropod-borne viruses (Orthobunyavirus, Nairovirus, Phlebovirus and Tospovirus) and one genus (Hantavirus) of rodent-borne viruses [30, 36] . The Phlebovirus genus currently comprises 68 antigenically distinct serotypes, only a few of which have been studied. The 68 known serotypes are divided into two groups: the Phlebotomus fever group (the sandfly group, transmitted by Phlebotominae sandflies) comprises 55 members, and the Uukuniemi group (transmitted by ticks) comprises the remaining 13 members. Of these 68 serotypes, eight are linked to disease in humans, including the Alenquer, Candiru, Chagres, Naples, Punta Toro, Rift Valley fever, Sicilian, and Toscana viruses [30] . Phleboviruses have tripartite genomes consisting of a large (L), medium (M), and small (S) RNA segment.
In screening for unknown viruses, species hits alone likely carry little weight. Thus, we used all sequences in the family Bunyaviridae for our analysis. A 168-bp fragment of the polymerase gene with the lowest E-value and high sequence identity was used as the sequence of the unknown virus. This virus sequence was detected in all 10 pooled samples, indicating that the virus is involved in FTLS. After detecting a possible novel bunyavirus through highthroughput Illumina sequencing, we inoculated Vero cell lines, which are known to be sensitive to phleboviruses, with sera from six positive patients and were subsequently able to detect the virus by RT-PCR [30, 31] . Although the CPE was modest, RT-PCR confirmed the infection. Genome sequencing was performed and a phylogenetic analysis of the genome sequence showed that this virus clustered into the Phlebovirus branch, but was divergent from other known phleboviruses. These results confirm the novelty of this virus within the Phlebovirus genus of the family Bunyaviridae [36] . Furthermore, virus size and propagation in cells were similar to that of the bunyaviruses.
PCR and serological tests were performed to further test the causal link between the new virus and FTLS. Although we have not completely fulfilled Koch's postulates, evidence implicating this new bunyavirus in the outbreak of the disease among patients with FTLS is compelling.
In view of the fact that the disease is caused by a novel bunyavirus, and taking into account that the disease was first discovered in Henan (HN), we propose the name "Henan Fever" for the FTLS disease cause by the novel virus (proposed name ''Henan Fever Virus'' [HNF virus]). Since the submission of this manuscript, a bunyavirus was identified as the cause of FTLS in Chinese patients from other regions of China, and the authors have named this virus ''SFTSV'' to indicate that it is the cause of severe fever with thrombocytopenia syndrome [37] . After release of the GenBank sequences referred to in the Yu paper, we compared the sequences of SFTSV with those of FTLSV and found that they were nearly identical (.99% identity). As we first identified the syndrome in 2007 and described the presence of the virus in patients between 2007 and 2010, we suggest that the name ''HNF virus'' should take precedence. The most distinctive feature of the current work includes the use of an unbiased metagenomic approach for viral pathogen discovery that facilitated the rapid creation and implementation of standard culture, serological, and molecular diagnostic approaches. However, there are other differences between the results described here and those reported by Yu et al; notably, we observed slight, but distinctive, CPE in Vero cells. The reason for the failure to observe CPE in Vero cells infected with the ''SFTSV'' bunyavirus [37] , whose genome is nearly identical to that of bunyavirus isolated from our FTLS patients, is unclear. Perhaps this reflects the fact that the ensuing CPE is not dramatic. Alternately, this could indicate the existence of distinct viral strains that vary in pathogenicity, virulence, and possibly even disease manifestations. This is an area of active study in our laboratories.
The discovery of this new virus will assist in the rapid diagnosis of this disease and help to distinguish it from other diseases caused by pathogens such as A. phagocytophilum, E. chaffeensis, Crimean-Congo hemorrhagic fever virus, Hantavirus, dengue virus, Japanese encephalitis virus, and Chikungunya virus. Furthermore, the availability of the new virus will facilitate the future development of new therapeutic interventions, such as vaccines and drugs. Figure S1 Sensitivity and dynamic range of real-time PCR in the detection of bunyavirus RNA. To evaluate sensitivity of our RT-PCR, a real-time PCR was performed. Serial dilutions of in vitrobtranscribed bunyavirus RNA sequences were tested. A wide linear range (from 5 copies to 5610 7 copies of control RNA per reaction) was detected in this assay. (TIF) Clinical aspects and cytokine response in severe H1N1 influenza A virus infection INTRODUCTION: The immune responses in patients with novel A(H1N1) virus infection (nvA(H1N1)) are incompletely characterized. We investigated the profile of Th1 and Th17 mediators and interferon-inducible protein-10 (IP-10) in groups with severe and mild nvA(H1N1) disease and correlated them with clinical aspects. METHODS: Thirty-two patients hospitalized with confirmed nvA(H1N1) infection were enrolled in the study: 21 patients with nvA(H1N1)-acute respiratory distress syndrome (ARDS) and 11 patients with mild disease. One group of 20 patients with bacterial sepsis-ARDS and another group of 15 healthy volunteers were added to compare their cytokine levels with pandemic influenza groups. In the nvA(H1N1)-ARDS group, the serum cytokine samples were obtained on admission and 3 days later. The clinical aspects were recorded prospectively. RESULTS: In the nvA(H1N1)-ARDS group, obesity and lymphocytopenia were more common and IP-10, interleukin (IL)-12, IL-15, tumor necrosis factor (TNF)α, IL-6, IL-8 and IL-9 were significantly increased versus control. When comparing mild with severe nvA(H1N1) groups, IL-6, IL-8, IL-15 and TNFα were significantly higher in the severe group. In nonsurvivors versus survivors, IL-6 and IL-15 were increased on admission and remained higher 3 days later. A positive correlation of IL-6, IL-8 and IL-15 levels with C-reactive protein and with > 5-day interval between symptom onset and admission, and a negative correlation with the PaO(2):FiO(2 )ratio, were found in nvA(H1N1) groups. In obese patients with influenza disease, a significant increased level of IL-8 was found. When comparing viral ARDS with bacterial ARDS, the level of IL-8, IL-17 and TNFα was significantly higher in bacterial ARDS and IL-12 was increased only in viral ARDS. CONCLUSIONS: In our critically ill patients with novel influenza A(H1N1) virus infection, the hallmarks of the severity of disease were IL-6, IL-15, IL-8 and TNFα. These cytokines, except TNFα, had a positive correlation with the admission delay and C-reactive protein, and a negative correlation with the PaO(2):FiO(2 )ratio. Obese patients with nvA(H1N1) disease have a significant level of IL-8. There are significant differences in the level of cytokines when comparing viral ARDS with bacterial ARDS. Originating from Mexico and spreading initially in the United States and Canada, a novel influenza A(H1N1) virus infection (nvA(H1N1)) of swine origin spread globally during spring 2009 to mid-February 2010. Rates of hospitalization and death have varied widely according to country [1] . Among hospitalized patients 9 to 31% have been admitted to intensive care units (ICUs) where the rate of death was 14 to 46% [2] [3] [4] [5] [6] .
In Romania the pandemic wave lasted from September 2009 to February 2010, reaching a peak in December. The Romanian Ministry of Health reported 7,008 confirmed cases of nvA(H1N1) influenza, the death rate being 1.9%.
Primary influenza pneumonia had a high mortality rate during pandemics not only in immune-compromised individuals and patients with underlying co-morbid conditions, but also in young healthy adults [7] .
During nvA(H1N1) virus infection, experimental and clinical studies have identified dysregulated systemic inflammation as an important pathogenetic mechanism correlating with severity and progression of the disease [8, 9] . The role of most immune responses in controlling and clearance of H1N1 influenza A or its contribution to severe respiratory compromise is not well known.
To and colleagues found higher plasma levels of proinflammatory cytokines and chemokine in the group of patients with acute respiratory distress syndrome (ARDS) caused by viral A(H1N1) influenza, throughout the initial 10 days after symptom onset [8] . Bermejo-Martin and colleagues found that mediators involved in the development of Th17 cells (IL-6, IL-8, IL-9, IL-17), Th1 cells (TNFα, IL-15, IL-12p70) and type II interferon (IFNγ) had high systemic levels in hospitalized patients with nvA(H1N1) influenza [9] . The detrimental or beneficial role of these cytokines in severe illness is not known.
The aim of our study was to further investigate the profile of Th1 and Th17 mediators and interferoninductible protein-10 (IP-10), an innate-immunity mediator, as early host response in a group of critical and noncritical hospitalized patients with nvA(H1N1) from Cluj-Napoca, Romania, and to correlate them with the clinical aspects.
The study was performed between October 2009 and February 2010 in the ICUs of the Emergency County Clinical Hospital and of the Teaching Hospital of Infectious Diseases, Cluj-Napoca, Romania.
Thirty-two patients hospitalized with nvA(H1N1) infection were enrolled in the study: 21 patients with nvA(H1N1)-ARDS, and 11 patients with nvA(H1N1)mild disease. Additionally, 20 patients with bacterial sepsis-ARDS were included and served to compare the cytokine levels between the nvA(H1N1)-ARDS group and the bacterial sepsis-ARDS group.
The study protocol was approved by the Ethics Committee for Clinical Research of the University of Medicine and Pharmacy 'Iuliu Hatieganu' Cluj Napoca and the hospital authority. Informed consent was obtained from each patient or their legal representative.
The inclusion criteria were age > 16 years, symptoms compatible with influenza and confirmed nvA(H1N1) virus, bacterial severe sepsis with ARDS, and informed consent. The exclusion criteria were age < 16 years, known infection by human immunodeficiency virus, patients with other respiratory viral infections, bacterial sepsis without ARDS-syndrome, and refusal to consent.
The control group included 15 healthy volunteers without chronic or acute disease. Data were recorded prospectively by investigators at each hospital. The following data were recorded: age, sex, pregnancy, underlying diseases (chronic obstructive pulmonary disease, asthma, diabetes, chronic heart failure, chronic renal failure, cirrhosis, immunosuppression), obesity defined as body mass index > 30, and the time in days from symptom onset to hospital admission. Hematological, biochemical and microbiological results were included in the database. The extension of lung infiltrates on chest X-ray scan was registered as the number of quadrants involved. The severity and prognosis of the illness was assessed in adults using the Acute Physiology and Chronic Health Evaluation II (APACHE II) score and the Sepsis-related Organ Failure Assessment (SOFA) score. ARDS was defined using the 1994 American-European Consensus Conference definitions [10] . The pulmonary dysfunction score was based on the PaO 2 :FiO 2 ratio, ranging from 0 to 3 where grade 0 represented a ratio less or equal to 250; grade 1, a ratio ranging from 250 to 175; grade 2, a ratio ranging from 100 to 175; and grade 3, a ratio less or equal to 100 [11] .
A(H1N1) influenza virus presence was confirmed by testing nasopharyngeal swabs or bronchoalveolar lavage specimens with real-time PCR (commercial kits: Full Velocity SYBR Green QRT-PCR/SuperScript III Platinum One-Step Quantitative RT-PCR Taqman; Invitrogen Corporation, Carlsbad, California, USA) at The National Influenza Centre of Cantacuzino Institute, Bucharest, Romania.
In patients with nvA(H1N1)-mild disease, the serum samples were taken on hospital admission. In patients with nvA(H1N1)-ARDS infection, the serum samples were taken on admission to the ICU and 3 days later to determine cytokine kinetics. The installation of ARDS, either viral or bacterial, in the course of the disease determined the time of admission to the ICU. In patients with bacterial sepsis-related ARDS, the serum samples were taken on admission to the ICU.
The enrolled patients and the healthy volunteers gave whole blood, which was clotted for 30 minutes at 37°C and stored at -70°C until use. The resulting serum was used for cytokine determination.
Seven different serum cytokines (IL-6, IL-8, IL-12p70, IL-15, IL-17, TNFα and IFN-γ) were measured with Luminex 200 (Luminex Corporation, Austin, TX, USA) using a multiplex cytokine kit along with the assay performed in accordance with the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Additionally, we used ELISA kits for quantitative determination of the two cytokines IL-9 and IP-10 (Quantikine; R&D Systems).
Subjects were stratified into three groups: 11 patients with nvA(H1N1)-mild disease, 21 patients with nvA (H1N1)-ARDS, and 20 patients with bacterial sepsis-ARDS.
Descriptive statistics included means and standard deviations or medians and interquartile ranges for continuous variables of normal and non-normal distributions. Clinical and biochemical characteristics and cytokine levels were compared. The Fisher exact test and the chi-square test were used for categorical variables. The Mann-Whitney U test was used for nonparametric variables. The Wilcoxon test (nonparametric test) was used to compare two paired groups. The association between nonparametric variables was determined by the Spearman correlation coefficient (r). Any value of P < 0.05 was considered statistically significant. GraphPad Prism version 5.03 Software for Windows (GraphPad Software, La Jolla, California, USA) was used.
A total of 32 patients with confirmed nvA(H1N1) infection and 20 patients with bacterial sepsis-ARDS were enrolled over the study period. Their demographic, co-morbidities and clinical characteristics are presented in Table 1 . Patients in the nvA(H1N1)-ARDS group were significantly older than those in the nvA(H1N1)-mild disease group (median age 42 years vs. 33 years, P = 0.009). Obesity was more common in the nvA(H1N1)-ARDS group. The median interval between onset of illness and admission was 6 days (interquartile range 3.5 to 8.5) in the nvA(H1N1)-ARDS group and 2 days (interquartile range 2 to 3) in the mild disease group (P < 0.001) ( Table 1 ). All the patients with nvA(H1N1) virus infection presented symptoms of acute respiratory viral infection on admission. The median length of hospital stay was higher in the nvA(H1N1)-ARDS group compared with the mild disease group (11 days vs. 6 days, P < 0.001). All patients with nvA(H1N1) virus infection received oseltamivir on admission: the standard dose (150 mg/day) was administered for patients with mild disease, and a higher dose (300 mg/day) was used for nvA(H1N1)-ARDS patients. During the ICU hospitalization, critical patients with influenza virus infection (ARDS) received corticosteroid therapy (hydrocortisone or methylprednisolone). In agreement with our protocol, empirical antibiotics were started on admission.
Among 21 patients with nvA(H1N1)-ARDS, four developed acute renal failure requiring renal replacement therapy, two developed secondary bacterial pneumonia and three developed pneumothorax (Table 1 ). Ten patients from the nvA(H1N1)-ARDS group received non-invasive ventilation and 11 patients received mechanical ventilation.
Pregnancy was another risk factor for nvA(H1N1)-ARDS infection and ICU admission (3/21 cases; Table  1 ). Two pregnant women were in the third trimester and one was in the second trimester. No underlying disease was noted. The range interval after symptom onset and ICU admission was 3 to 7 days. Caesarean delivery was necessary in two cases. All pregnant women required respiratory support (two invasive and one noninvasive) during hospitalization and all survived.
Seven patients died in the nvH1N1-ARDS group. Histopathological changes were similar in all cases: tracheitis, bronchitis with focal squamous metaplasia, necrotizing bronchiolitis, emphysema, extensive diffuse alveolar damage associated with alveolar hemorrhage and marked hyaline membrane formation, fibrosis and granulocyte pulmonary infiltrates. Pulmonary thromboemboli with focal infarcts were observed in three cases.
The lymphocyte count was significantly lower in the nvA(H1N1)-ARDS group than in the mild disease group (P = 0.011) ( Table 2 ). Comparing laboratory abnormalities on hospital admission we found that patients with nvA(H1N1)-ARDS were more likely to have elevated levels of serum lactate dehydrogenase, alanine and aspartate aminotransferase (P < 0.001, P = 0.049 and P < 0.001, respectively) than patients with nvA(H1N1)mild disease ( Table 2) .
Twenty patients with bacterial sepsis-ARDS were included to compare the cytokine levels in viral and bacterial ARDS. Immune suppression (six patients with cancer) was more common in the bacterial sepsis-ARDS group (P = 0.044). The mean (standard deviation) APACHE II score, SOFA score and PaO 2 :FiO 2 ratio were similar in both groups ( Table 1 ). The leukocyte count, C-reactive protein and procalcitonin levels were higher in the bacterial ARDS group than in the nvA (H1N1)-ARDS group (P = 0.047, P = 0.05 and P < 0.001, respectively) ( Table 2) .
The results of the cytokine profile are shown in Figure  1 . At admission, only IL-6, IL-12, IP-10 and TNFα were significantly higher in the mild disease group than in the control group. Except for IL-17 and IFNγ, all cytokine levels were higher in critical patients with nvA (H1N1)-ARDS than in the control group. Compared with the mild disease group, significantly higher levels of IL-6, IL-8, IL-15 and TNFα were found in the nvA (H1N1)-ARDS group (P < 0.001, P < 0.001, P < 0.001 and P < 0.05, respectively). Compared with controls, the levels of IL-6, IL-8, IL-9, IL-15, IL-17, IP-10 and TNFα were significantly elevated in the bacterial sepsis-ARDS group. Levels of IL-8, IL-17 and TNFα were significantly higher in the bacterial-ARDS group versus the nvA (H1N1)-ARDS group (P = 0.05, P = 0.004 and P = 0.011, respectively; Figure 1 ).
Patients with pandemic influenza virus (severe ARDS and mild disease) were stratified according to the interval between symptom onset and admission. Levels of IL-6, IL-8, IL-15 and IFNγ were significantly higher in patients with delayed admission, > 5 days after symptom onset (P = 0.006, P = 0.037, P = 0.013 and P = 0.027, respectively) ( Table 3 ).
Serum cytokine levels over time (3 days after admission and antiviral treatment) showed a decrease of IL-6, IP-10, TNFα, IFNγ and IL-17 in critical patients with nvA (H1N1)-ARDS (Table 4 ). Serum cytokine levels over time in nvA(H1N1)-ARDS survivors showed a significant decrease of IL-6, IP-10 and TNFα (Table 5 ). In nonsurvivors versus survivors from the nvA(H1N1)-ARDS group, the levels of IL-6 and IL-15 on admission and 3 days after were significantly higher ( Table 6 ). IL-17 was higher in nonsurvivors 3 days after admission (Table 6) . Correlation between cytokine levels and clinical or laboratory characteristics in patients with confirmed nvA(H1N1) infection was determined by Spearman correlation coefficient. We found significant correlation of IL-6, IL-8 and IL-15 levels with C-reactive protein (r = 0.67, P < 0.001; r = 0.5, P = 0.003; and r = 0.48, P = 0.005, respectively), with PaO 2 :FiO 2 ratio (r = -0.556, P = 0.001; r = -0.574, P < 0.001; and r = -0.614, P < 0.001, respectively) and with interval between symptom onset and hospital admission (r = 0.51, P = 0.002; r = 0.41, P = 0.019; and r = 0.48, P = 0.004, respectively).
IL-8 was significantly higher (P = 0.013) in obese versus nonobese patients with nvA(H1N1) infection.
In this study we presented the cytokine profiles following nvA(H1N1) infection in 32 hospitalized patients (11 mild and 21 severe disease) and the cytokine profiles found in 20 cases of bacterial sepsis.
The patients with severe nvA(H1N1) disease were younger than the patients with bacterial sepsis (no statistical significance). Similarly to other study groups, we found that obesity was more common in the nvA (H1N1) ARDS group, suggesting it may be a risk factor for complications and admission to the ICU [2, 5, 6] . Laboratory findings in the same group of patients include lymphocytopenia and elevation in levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatinine -as in other patient groups with novel influenza virus infection [4, 6] . In contrast, the bacterial-ARDS group presented no lymphocytopenia, lower elevation in serum liver enzymes and higher levels of C-reactive protein and procalcitonin. No significant differences were found between bacterial and viral ARDS groups in SOFA and APACHE II scores at admission. The pulmonary histopathological findings in nvA(H1N1)-ARDS nonsurvivors were similar to other fatal cases of nvA(H1N1) virus infection [12, 13] .
Installation of ARDS in the course of the disease was the moment of blood sampling for cytokine measurements. There was a difference regarding the time of symptom onset and hospital admission between the severe and mild groups of nvA(H1N1) disease that could affect the comparison of cytokine levels between the two groups. For this reason we not only compared the cytokine levels between mild and severe disease, but also mixed the patients with nvA(H1N1)-mild and severe disease and compared the level of cytokines according to the interval between symptom onset and admission (first interval 1 to 5 days, second interval 6 to 14 days). We found that not all cytokines had the same behavior against the time of symptom onset and admission.
The pattern of immune response in patients with nvA (H1N1) virus infection is incompletely characterized. CD4 + T cells are known to play an important role in the initiation of immune responses by providing help to other cells. T-helper cells could be divided into subsets: Th1, Th2 and Th17.
Th1 cells mainly develop following infections by intracellular bacteria and some viruses [14] . The mediators involved in the development of Th1 are IL-12, IFNγ, IL-15, IL-18 and TNFα.
IL-12 bridges the early nonspecific innate immunity and the subsequent antigen-specific adaptative immunity [15] . IL-12 was shown to inhibit apoptosis of T cells [16] and of dendritic cells [17] . Alveolar macrophages have a functional IL-12 receptor, and virus-infected macrophages in the presence of IL-12 might be protected from apoptosis limiting viral clearance [18] . Apoptosis of virus-infected cells was shown to be an effective mechanism for viral clearance [19] . Bermejo-Martin and colleagues reported more significant IL-12 results in the critical A(H1N1) group of patients [9] . In our study, IL-12 is significantly higher in the nvA (H1N1)-mild disease group and in the nvA(H1N1)-ARDS group versus the control group and is not significantly higher in the bacterial ARDS group. IL-15 plays a critical role in protecting CD8 + T cells from apoptosis during the contraction phase following microbial infection [20, 21] . The CD8 + T cells surviving in the presence of IL-15 might be pathogenic in lung injury following highly pathogenic influenza A virus infection [22] . IL-15 activates the effector function of memory phenotype CD8 + cells [23] . In our study, IL-15 is significantly higher in the nvA(H1N1)-ARDS group versus the nvA(H1N1)-mild disease group, but without significant difference in the nvA(H1N1)-ARDS versus bacterial-ARDS groups. Similar to our results, IL-15 was a hallmark of critical illness in the Hong Kong and Spanish nvA(H1N1) cytokine studies [8, 9] . IL-15 is significantly higher at admission (P1) and 3 days later (P2) in the nvA(H1N1)-ARDS group for nonsurvivors versus survivors, so it might be pathogenic in lung injury influenza A virus infection. Similarly, To and colleagues found IL-15 significantly higher in critical A(H1N1) patients and very significant in the A(H1N1)-ARDS death group [8] .
IFNγ is a cytokine of innate and adaptative immunity. Its major functions are activation of macrophages, differentiation of Th1 from T cells, inhibition of the Th17 pathway and control of intracellular pathogens [24] . Bermejo-Martin and colleagues found high systemic levels of IFNγ in hospitalized patients with nvA(H1N1) [9] . In contrast, in the present study there were no differences between the control and study groups. The IFNγ level over time in the nvA(H1N1) ARDS group was higher at admission than 3 days later, without significant difference between survivors versus nonsurvivors.
TNFα is a cytokine of innate immunity. The principal cellular targets and biologic effects include activation of endothelial cells, neutrophil activation, fever, liver synthesis of acute phase proteins, muscle and fat catabolism, and apoptosis of many cell types. In our study, we found highly increased TNFα levels in the nvA(H1N1)mild disease, nvA(H1N1)-ARDS and bacterial ARDS groups compared to the control group. TNFα is significantly higher in nvA(H1N1)-ARDS versus nvA(H1N1)mild disease, with similar results being found by To and colleagues and Bermejo-Martin and colleagues [8, 9] . This cytokine is also significantly increased in bacterial-ARDS versus nvA(H1N1)-ARDS.
For the groups of patients with nvA(H1N1), according to the time interval between symptom onset and hospital admission, there were no significant differences found for IL-12 and TNFα levels, but there were significant differences for IL-15 and IFNγ, levels being higher when the time interval was between 6 and 14 days. None of our patients were on oseltamivir medication between symptom onset and admission.
Th17 cells are effective in host defense against certain pathogens and tissue inflammation. Th17 mediators for the development of Th17 cells are IL-6, transforming growth factor beta, IL-8, IL-9, IL-17, IL-1 and IL-23.
IL-6 is a cytokine of innate immunity, its principal targets being the liver cells, the β cells and the naïve T cells [25] . Despite the apparently beneficial role that macrophages play in controlling early viral replication, several reports have demonstrated a more deleterious effect of these cells in influenza A viral infections by excessive inflammation in the lung attributed to IL-6 and TNFα [26] . In our study, IL-6 is increased in nvA(H1N1)-ARDS versus nvA(H1N1)-mild disease. Similarly, IL-6 and IL-15 constituted a hallmark of critical illness in the Hong Kong and Spanish nvA(H1N1) cytokine studies [8, 9] . In the nvA(H1N1)-ARDS group, the IL-6 serum level is significantly higher at admission than 3 days later. In the same group, IL-6 is significantly higher in nonsurvivors versus survivors at admission and 3 days later, which seems to further contribute to pulmonary damage and death. We found positive correlations between IL-6, IL-15 and IL-8 levels and a longer than 5 days interval between symptom onset and admission, as well as with C-reactive protein, but a negative correlation with the PaO 2 :FiO 2 ratio, indicating the severity of the disease. IL-8 is a chemokine of innate immunity. The chemokine's principal biologic effect is chemotaxis, being a major chemokine for neutrophil activation, and migration into tissues [24] . In our study, IL-8 is highly significant in the nvA(H1N1)-ARDS and ARDS bacterial groups versus the control group, but is not significant in mild disease. In contrast, IL-8 was increased in both critical and noncritical nvA(H1N1) hospitalized patients in the Spanish and Hong Kong studies. In our study, IL-8 is higher in nvA(H1N1)-ARDS versus nvA(H1N1)-mild disease and in bacterial ARDS versus nvA(H1N1)-ARDS. The obese patients with nvA(H1N1) disease had a significant level of IL-8. Plasma IL-8 levels are increased in normoglycemic obese subjects, related to fat mass and the TNFα system [27] .
IP-10 is a chemokine of innate immunity, and macrophages and dendritic cells are the principal cell source. We found a higher level of IP-10 in nvA(H1N1)-mild disease, nvA(H1N1)-ARDS and bacterial-ARDS groups versus the control group, and no other differences between groups. In the nvA(H1N1)-ARDS group, the IP-10 level is higher at admission than 3 days after admission because of the survivors' cytokine profile. An increased level of IP-10 was found in the Spanish group as early response to nvA(H1N1) infection in both hospitalized and mild patient disease, as in the present study, while in the Hong Kong group IP-10 was significantly higher in critical patients only. In our study, IP-10 levels in nvA(H1N1)-ARDS nonsurvivors remained higher at admission and 3 days later, being not significantly correlated with the clinical outcome. Emphysema was one of our hystopathological findings and thus it might be speculated that a high level of IP-10 in nonsurvivors could be correlated with emphysema. IP-10 released by lung CD41 and CD81 T cells stimulates alveolar macrophage production of matrix metalloproteinase-12, which digests lung elastin [28, 29] .
IL-17 is a cytokine of adaptative immunity. Principal cellular targets include endothelial cells with increased chemokine production and macrophages with increased chemokine and cytokine production. This cytokine's principal biologic effect is proinflammatory [24, 25] . In the present study IL-17 is significantly higher in the bacterial ARDS group versus the control group and is higher in the bacterial ARDS group versus the nvA(H1N1)-ARDS group. No significant differences between nvA(H1N1)-mild disease versus controls and between nvA(H1N1)-ARDS versus controls were found. In the nvA(H1N1)-ARDS group, IL-17 was higher at admission and lower 3 days later. In the Spanish study the IL-17 level was increased in hospitalized noncritical patients, and in the Hong Kong study no differences between groups were found, similar to the present study.
IL-9, like IL-6, is a Th2 cytokine that induces differentiation of Th17 cells and has anti-inflammatory properties. IL-9 is a cytokine of current interest associated with allergic Th2 responses and is a key modulator of antiviral immunity [30] . In our study IL-9 is significantly higher in the H1N1-ARDS group versus the control group, and is not significantly increased in mild disease -in contrast to the Spanish study, where IL-9 was increased in both critical and noncritical hospitalized patients.
Regarding the behavior of Th17 mediators in nvA (H1N1) groups of patients according to the time interval between symptom onset and admission, there were no differences for IL-9, IL-17 and IP-10 and there were significant differences for IL-6 and IL-8, the levels being higher when the interval was between 6 and 14 days. All our patients with ARDS disease were on corticosteroid treatment, because deficient corticosteroid-mediated downregulation of inflammatory cytokine transcription in ARDS patients is associated with disease progression and mortality. Many studies reported that prolonged corticosteroid treatment was associated with a significant reduction in markers of systemic inflammation [31, 32] . In the present study the blood samples for cytokine measurements were taken at admission for the bacterial-ARDS group of patients, and at admission and 3 days later for the nvA(H1N1) group of patients -for this reason, corticosteroid could not significantly affect cytokine levels.
The small number of patients enrolled in the mild disease group is one of our study limitations. Among hospitalized patients with mild flu-like syndrome, only those with risk of severe complications and of secondary outbreaks in the exposed population were sampled for real-time PCR. On the contrary, the laboratory of the National Influenza Centre of Cantacuzino Institute, Bucharest was overwhelmed, being the only centre for influenza PCR diagnosis. Another limitation is the exclusion of children, an important group with nvA (H1N1) virus infection.
In our critically ill patients with nvA(H1N1) virus infection we found increased levels of some cytokines: IP-10, TNFα, IL-15, IL-12, IL-6, IL-8 and IL-9. The hallmarks for the severity of the disease were IL-6, IL-15, IL-8 and TNFα. We found a positive correlation of IL-6, IL-15 and IL-8 with the admission delay and C-reactive protein and a negative correlation with the PaO 2 :FiO 2 ratio. The obese patients with nvA(H1N1) disease had a significant level of IL-8. There were significant differences in the level of cytokines when comparing viral ARDS with bacterial ARDS.
• In the influenza-related ARDS group, the levels of IL-6, IL-8, IL-9, IL-12, IL-15, IP-10 and TNFα are significantly increased versus the control group. In the bacterial sepsis-ARDS group, levels of IL-6, IL-8, IL-9, IL-15, IL-17, IP-10 and TNFα are also increased versus the control group. When comparing these two groups, the levels of IL-8, IL-17 and TNFα are significantly higher in bacterial ARDS versus viral ARDS, and IL-12 is increased only in viral ARDS whereas IL-17 is increased only in bacterial ARDS. When comparing the mild nvA (H1N1) and critical ARDS influenza A groups, IL-6, IL-8, IL-15 and TNFα are significantly higher in critical ARDS patients being hallmarks of disease severity.
• The serum levels of IL-15, IL-6, IL-8 and IFNγ according to the interval between symptom onset and admission in hospitalized nvA(H1N1) patients are significantly higher when this interval is longer than 5 days.
• In nonsurvivors versus survivors from the nvA (H1N1)-ARDS group, IL-6 and IL-15 are increased at admission and stay higher 3 days later -which seems to further contribute to pulmonary damage and death.
• There is a positive correlation of IL-6, IL-8 and IL-15 levels with C-reactive protein and with > 5-day interval between symptom onset and hospital admission, and a negative correlation with the PaO 2 :FiO 2 ratio.
• The obese patients versus nonobese patients with nvA(H1N1) infection have a significant level of IL-8.  Clinical review: Idiopathic pulmonary fibrosis acute exacerbations - unravelling Ariadne's thread Idiopathic pulmonary fibrosis (IPF) is a dreadful, chronic, and irreversibly progressive fibrosing disease leading to death in all patients affected, and IPF acute exacerbations constitute the most devastating complication during its clinical course. IPF exacerbations are subacute/acute, clinically significant deteriorations of unidentifiable cause that usually transform the slow and more or less steady disease decline to the unexpected appearance of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) ending in death. The histological picture is that of diffuse alveolar damage (DAD), which is the tissue counterpart of ARDS, upon usual interstitial pneumonia, which is the tissue equivalent of IPF. ALI/ARDS and acute interstitial pneumonia share with IPF exacerbations the tissue damage pattern of DAD. 'Treatment' with high-dose corticosteroids with or without an immunosuppressant proved ineffective and represents the coup de grace for these patients. Provision of excellent supportive care and the search for and treatment of the 'underlying cause' remain the only options. IPF exacerbations require rapid decisions about when and whether to initiate mechanical support. Admission to an intensive care unit (ICU) is a particular clinical and ethical challenge because of the extremely poor outcome. Transplantation in the ICU setting often presents insurmountable difficulties. death in all patients aff ected, and IPF exacerbations constitute the most devastating complication during its course [1] [2] [3] [4] [5] [6] . IPF exacerbations appear more frequently than previously thought and represent a common terminal event [7, 8] . IPF lacks eff ective treatment, and survival is approximately 3 years [2, 6, 9, 10] . Best supportive care constitutes the only attainable therapeutic strategy and includes a more or less eff ective attempt to alleviate symptoms and prevent complications and a far more effi cacious interventional approach consisting of the withdrawal of corticosteroids and immunosuppressants (commonly administered by clinicians) that are ineff ective and harmful [2, 9, 11] . Transplantation is the only thera peutic option [12] .
IPF exacerbations represent acute and clinically signifi cant deteriorations of unidentifi able cause, transform ing the slow and more or less steady disease decline [13] to the unexpected appearance of acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) ending in death [6, 14] . Occasionally, IPF exacerbations may present in a previously apparently healthy or minimally symptomatic individual and might represent acute progression of an unsuspected or undiagnosed early IPF [3, 15] . Defi nition criteria include IPF diagnosis, unexplained worsening or development of dyspnea within 30 days, new lung infi ltrates (mainly ground glass upon honeycomb), and exclusion of any identifi able or treatable cause of lung injury [6] . Surgical lung biopsy per se constitutes a risk factor for their development [16] but, when performed for the investigation of the etiology of exacerbations or in autopsies, discloses a histological picture of diff use alveolar damage (DAD), which is the ARDS tissue counterpart, upon usual interstitial pneumonia (UIP), which is the IPF tissue equivalent [4, 8, [17] [18] [19] .
In IPF, anachronic and reiterative epithelial injury and loss of the alveolar-capillary integrity constitute the initial event and 'the point of no return' that trigger aberrant repair pathways leading to inappropriate, progressive, and heterogeneous lung scarring (UIP) [20] [21] [22] . DAD upon UIP might represent massive epithelial and endothelial injury of the lung areas yet preserved from Abstract Idiopathic pulmonary fi brosis (IPF) is a dreadful, chronic, and irreversibly progressive fi brosing disease leading to death in all patients aff ected, and IPF acute exacerbations constitute the most devastating complication during its clinical course. IPF exacerbations are subacute/acute, clinically signifi cant deteriorations of unidentifi able cause that usually transform the slow and more or less steady disease decline to the unexpected appearance of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) ending in death. The histological picture is that of diff use alveolar damage (DAD), which is the tissue counterpart of ARDS, upon usual interstitial pneumonia, which is the tissue equivalent of IPF. ALI/ ARDS and acute interstitial pneumonia share with IPF exacerbations the tissue damage pattern of DAD. 'Treatment' with high-dose corticosteroids with or without an immunosuppressant proved ineff ective and represents the coup de grace for these patients. Provision of excellent supportive care and the search for and treatment of the 'underlying cause' remain the only options. IPF exacerbations require rapid decisions about when and whether to initiate mechanical support. Admission to an intensive care unit (ICU) is a particular clinical and ethical challenge because of the extremely poor outcome. Transplantation in the ICU setting often presents insurmountable diffi culties.
scarring [9, 23] . Putative initiators of IPF include viruses, cigarette smoke, gastroesophageal refl ux, and occupational exposure to wood and metals [24, 25] . Aging, by reducing effi ciency in repairing damage, represents a cofactor [26] . Th e development of DAD upon UIP may relate to a clinically occult infection [14, 27] , aspiration, or a distinct pathobiological manifestation of IPF [6] . 'Treatment' with high-dose corticosteroids with or without an immunosuppressant proved ineff ective and represents the coup de grace for these patients [8] . IPF exacerbations require rapid decisions about when and whether to initiate mechanical support. However, the con sideration of admission to an intensive care unit (ICU) is a particular clinical and ethical challenge because of poor outcome [28] [29] [30] [31] [32] . Transplantation in this setting presents insurmountable diffi culties.
Th e incidence of IPF exacerbations varies greatly between studies (from 8.5% to 60%) mainly because of diff erences in their design [3] [4] [5] [6] 8, 14, 16, 19, 28, 29, [31] [32] [33] [34] [35] : (a) case series and retrospective cohorts [4, [17] [18] [19] , (b) randomized controlled trials of specifi c treatments for IPF [3] , (c) autopsy reviews [8, 16, 33] , and (d) retrospective reviews of ICU admissions [28, 29, 31, 32] . Discrepancies in reported frequen cies should be attributed to the diffi culty in strictly respecting the defi nition criteria especially concerning symptom duration (less than 4 weeks) and the defi nite exclusion of infection [3, 6, 36] . IPF exacerbations do not appear to be linked to disease duration, functional derange ment, age, gender, or smoking history [4, 29] , although further studies are necessary to confi rm early development as well as lack of association with immunosuppression [37] . Exacerbation mortality approaches 100%, questioning the need for ICU admission [2] [3] [4] [5] [6] 8, 14, 16, 19, 28, 29, [31] [32] [33] [34] .
Th e defi nition of IPF exacerbations 'after excluding identi fi able causes of lung injury' implies that in 'idiopathic' pulmonary fi brosis, 'idiopathic' exacerbations occur [3, 4, 6] . However, in clinical practice, when such a patient is referred to the emergency department (ED), the attending clinician has to face one of three clinical scenarios [38] (Figure 1 ). Th e fi rst scenario is the case in which the physical evolution of IPF comes to the fi nal end in which spontaneous breathing becomes unsup portable [39] (Figures 1a and 2) . In this scenario, the exclusion of 'identifi able-treatable causes of lung deterior ation' is demanding, but the only option attainable is palliation. Th e second scenario refers to 'true' IPF exacerbation that brings the patient to the ED (Figures 1b and  3 ). In this case, after admission to the hospital ward, the patient usually becomes unable to maintain spontaneous breathing within hours or very few days, often not enough time for the extensive work-up required to identify treatable factors of deterioration, and needs ventilatory support and ICU transfer [5, 7, 8] . Th e third scenario refers to the admission to the hospital ward of an IPF-deteriorated patient because of reversible causes either aff ecting the lung or not; in this case, early identifi cation of the precipitating factor(s) and their prompt treatment are imperative (Figures 1c and 4) . Nevertheless, borders between the above scenarios are unclear in routine clinical practice since exacerbations occur as a spectrum rather than a clearly defi nable event.
However, even after the exclusion of any identifi able and treatable factor(s) inducing IPF exacerbations, the most important etiologic hypothesis remains that of a clinically occult infection that precipitates an already UIP-scarred lung into DAD [6] . For several reasons, viruses are the best etiologic candidates: (a) Epstein-Barr, cytomegalovirus, hepatitis C, herpes simplex, parvovirus B19, torque teno, and especially herpes viruses 7 and 8 have been implicated in IPF pathogenesis [40] [41] [42] ; (b) fl ulike illness heralds the onset of exacerbations, and IPF mortality seems to peak in winter time and coincides with the peak of viral respira tory infections [43] ; (c) in the mice pulmonary fi brosis experimental model, gamma herpesvirus induces exacer ba tions [44] as well as other viruses in vivo [45] ; and (d) latent lung viral infections may reactivate under immuno suppression commonly used by clinicians [41] . Th erefore, in IPF, viruses may act as both initiators and exacerbators because of their formidable ability to induce ARDS [46] . Besides viruses, microbials in traction bronchiectases/ bronchiolectases are equally strong candidates. Bronchiec tases are among the most common of the whole spectrum of lesions that characterize the architectural distortion in IPF. Interleukin-8, neutrophils, and alphadefensins are increased or activated in stable or exacerbated patients with IPF [47, 48] and possibly play a role in triggering ARDS. In addition, immunosuppressive treatment certainly increases suscep ti bility to microbials.
Accordingly, further considerations have to be made. ALI/ARDS, acute interstitial pneumonia (AIP), and IPF exacerbations have common clinical, physiological, imaging, and histopathology features, and it is incon ceivable that they do not also have common etiopathogenetic mechanisms ( Figure 5 ). ALI/ARDS develops by diff erent insults to the lung, and the mainstay of its treatment is provision of excellent supportive care and etiologic manage ment of the underlying cause [46] . AIP is precisely an ARDS of 'unknown cause' , and no specifi c clinical clues to diff erentiate between 'known and unknown cause' ARDS exist [49] . Criteria for the diagnosis of AIP are the same as in IPF exacerbations with the exception of the 'incubation' time (2 months instead of 4 weeks) and the prerequisite of normal chest roent genogram. AIP, incomprehensibly, is included among the idiopathic interstitial pneumonias (IIPs) and probably should be added to the list of unknown cause ALI/ARDS, although some believe that AIP may represent a fulminant presentation of IIP secondary to imprecise autoimmune factors [1, 2] . Although there are no controlled trials of specifi c treatment, intensive immunosuppression has been the mainstay of treatment (usually under the coverage of several broad-spectrum antimicrobials, although this is not always stated) because of the inclusion of AIP among the IIPs [50] . However, AIP mortality approaches that of IPF exacerbations, and the provision of excellent suppor tive care and further search of underlying causative factors and adequate treatment seem more logical. In stable IPF (in contrast to other pneumonias), lung damage is not resolved by restitutio ad integrum. IPF exacerbations characterized by DAD upon UIP may represent the acute response of scarred and irreparably damaged lung. Epithelial cell apoptosis involves and is considered to be among the main pathogenetic mechanisms in the development of any DAD [51, 52] . Th erefore, it seems incoherent that DAD, which is the common denominator of all ALI/ARDS, AIP, and IPF exacerbations and which develops upon diff erent histology substrates (UIP in IPF exacerbations, normal lungs in AIP, and normal or diseased lungs in ARDS), presents at diff erent time intervals (7 days for ARDS [46, 53] , 4 weeks for IPF exacerbations [7] , and 2 months for AIP [1] ) and requires diff erent pharmacologic approaches, which proved certainly fatal in AIP and in IPF 'true' exacerbations.
Early, accurate, and secure diagnosis is critical in IPFexacer bated patients with reversible precipitating [7] . For details about laboratory tests and blood/sputum/bronchoalveolar lavage (BAL) tests, see the 'Clinical and laboratory assessment' section. Cardiac echo, cardiac echocardiography; CTPA, computed tomography pulmonary angiography; HRCT, high-resolution computed tomography; ICU, intensive care unit; PE, pulmonary embolism; PH, pulmonary hypertension; PNX, pneumothorax; proBNP, pro-brain natriuretic peptide. factor(s) (Figure 1 ) [2] . Investigation into medical history should focus on smoking habits, toxic exposures, prescribed medications, immunosuppression, and signs and symptoms of potentially undiagnosed autoimmune rheumatic disease [2, 54, 55] . Physical examination frequently reveals tachypnea, cyanosis, digital clubbing, bilateral inspiratory crackles, and lower extremity edema. In the most severe cases, the patient may be obtund or comatose because of severe hypoxemic and potentially hypercapnic respiratory failure. Th e presence of arrhyth mias, chest pain, hemoptysis, or hemodynamic instability should guide the physician to an overlapping or alternative diagnosis such as acute coronary syndrome or pulmonary embolism.
Chest roentgenograms, including past imaging data, may help to orientate the clinician toward the identifi cation of the causative agents of the exacerbation. Computed tomo graphy pulmonary angiography is mandatory to exclude pulmonary embolism, and high-resolution computed tomography (HRCT) may document extension High-resolution computed tomography shows mild reticulation. (d) Roentgenogram of the patient 24 months after diagnosis demonstrates worsening of the reticular pattern superimposed on a ground-glass pattern. The patient was admitted with severe breathlessness and productive cough. Her symptoms were severely aggravated in the last 9 months and she was hospitalized many times. She had received corticosteroids and mycophenolate mofetil, which were discontinued months prior to this roentgenogram because of lower respiratory tract infections. At the time of the roentgenogram, she was receiving only proton pump inhibitors. She deteriorated further despite best supportive care and died while on palliation treatment. Our putative diagnosis was IPF progressing to the fi nal end.
of honey combing or other lung comorbidities (Figure 1 ). Echo cardio graphy may also be useful. When early undiagnosed IPF presents with fulminant respiratory insufficiency and ARDS [3, 29] , honeycombing with bibasilar and subpleural distribution on HRCT [1] can establish the diagnosis of IPF exacerbation and diff eren tiate defi nitely from AIP [49] . In IPF exacerbations, HRCT reveals new bilateral ground-glass abnormalities or consolidations (or both) upon UIP pattern [6] . A ground-glass pattern, especially if extensive, is not a feature of stable IPF, and its rapid development away from areas of fi brosis heralds DAD. Akira and colleagues [18, 56] have proposed a classifi cation of acute exacer bations of IPF on the basis of three ground-glass and consolidation computed tomography patterns that appear to have prognostic implications: (a) peripheral, (b) multifocal, and (c) diff use, though others did not confi rm a similar assumption [57] .
Since no laboratory test is specifi c to IPF exacerbations, most tests are performed to exclude treatable causes of deterioration and to document the severity of the exacerbations. Th e standard laboratory work-up should include all necessary tests for the investigation of a critically ill patient with impeding ALI/ARDS of unknown etiology. ALI and ARDS criteria (arterial partial pressure of oxygen/fraction of inspired oxygen [PaO 2 /FiO 2 ] of less than 300 and less than 200, respectively) should prepare the clinician for the possibility of mechanical support. Accurate diagnosis in IPF exacerbations requires bronchoalveolar lavage (BAL) to exclude infection or alternative diagnoses; BAL is best performed before mechanical support or immediately afterwards [2] . Perform ing lung biopsy could be justifi able when facing a disease with grave prognosis but bears an increased risk for postsurgical complications and should be individual ized to each patient [2] .
IPF exacerbations lack an eff ective treatment. Intensive immunosuppression proved harmful and fatal [2] . Patients presenting with IPF exacerbations must be managed in centers specializing in interstitial lung diseases (ILDs) with the availability of various specialties and departments such as a respiratory ward with a respiratory ICU/highdependency unit (RICU/HDU), an ICU, and possibly a cardiothoracic transplantation center on a 24-hour basis. Lung transplantation constitutes a treat ment option for IPF 'true' exacerbations [2, 38] but faces insurmountable diffi culties, even in specialized centers.
Management depends on the clinical scenario ( Figure 1 ). In case of progression to the fi nal end (Figure 1a) , palliation is more appropriate [2] . Noninvasive ventilation (NIV), by decreasing breathing work, is considered a major palliative option that, together with best supportive care, may help to reduce patient discomfort and permits management in an RICU [58, 59] . Patients with 'true' IPF exacerbations (Figure 1b) , in which the diagnostic approach fails to identify a possible infective etiology, must continue to receive empirical antimicrobial therapy that takes into consideration factors such as immunosuppression, previous colonization, BAL timing, onset of mechanical support, and results of obtained cultures [2] . 'Specifi c' therapies for 'true' IPF exacerbations until now have consisted of highdose intravenous corticosteroids plus an immunosuppressant [2] . However, Cochrane reviews for the effi cacy of these therapies concluded that there is no evidence for any benefi t of both cortico steroids and immunosuppressants in IPF [60, 61] . Besides, both progression of fi brosis on native lung in single-lung transplant patients and IPF 'true' exacerbations have been described in the heavily immunodepressed trans planted patient [10] . NIV may also help to wean the very few survivors from the IPF exacerbations and also constitutes the bridge to transplantation [62] . To promptly recognize and treat reversible precipitating factors implicated in IPF exacerbations (Figure 1c) , recovery in the RICU/HDU or (in case of multiorgan failure) in the ICU is mandatory [63] .
An IPF patient is referred to the ICU for severe acute respiratory failure as a consequence of the clinical scenarios (mentioned above) that may lead to ventilatory support (Figure 1 ). Progression to the fi nal end reaches a point at which spontaneous ventilation in no longer possible (Figure 1a ). ICU admission of these patients, because of the poor outcome, should be avoided [2] ( Figure 2 ). In 'true' IPF exacerbations (Figure 1b) , ventilatory support and ICU transfer buy time and could have some infl uence on fi nal outcome in specifi c patients. Unfortunately, in the vast majority, this does not happen, and the mortality of this patient population is high, higher even than that predicted by the usual clinical score HRCT shows, at the lung bases, ground-glass opacities upon extensive peripheral thickening of intralobular septa. The patient was a 65-year-old male with IPF and initiated treatment with high doses of corticosteroids. (c) Four months later, HRCT denotes diff use ground-glass with irregular reticulation. Note the extensive lipomatosis of the mediastinum due to chronic steroid use. Owing to deterioration of dyspnea, he was admitted to another hospital, where bronchoalveolar lavage (BAL) was performed and the immunosuppressive treatment was intensifi ed. A few weeks later, he was admitted to our department with respiratory failure, severe corticosteroid-related myopathy, diabetes mellitus, severe dyspnea, and purulent sputum. Clinical examination disclosed herpes simplex virus keratitis in the left eye, and BAL cultures grew positive for Pseudomonas aeruginosa. Corticosteroids were tapered, and antimicrobial and antiviral treatment was initiated. Both eye and lower respiratory tract infections subsided, and the patient was discharged home a few weeks later. (d) Eighteen months after the exacerbation, the groundglass opacities completely resolved as did the lipomatosis of the mediastinum. The patient is still alive and at home. [2, 31] (Figure 3 ). Admission of an IPF patient to the ICU because of reversible causes either aff ecting the lung or not (Figures 1c and 4 ) bears better prognosis, but special attention should be paid to avoid further complications. Th e complexity of the above scenarios underscores the importance of good communication between referring and ICU physicians.
So far, the studies of IPF patients in the ICU have had many limitations (Table 1) [4, 28, 29, 31, 32, 34, 59, 64] . Th ese studies are usually retrospective and single-centered and include limited numbers of patients. In addition, most of these studies include all IPF patients admitted to the ICU for respiratory failure regardless of etiology, the proportion of patients with confi rmed diagnosis is variable, the ventilator parameters are usually not reported, and the pharmacologic therapy demonstrates a signifi cant diversity. Th e only common parameter is the conclusion: the prognosis of ventilated IPF patients is disappointing [2] . Given these results, what may be the goals of ICU support for a patient with an IPF exacerbation? Although defi nite conclusions cannot be drawn, there is a general feeling that mechanical ventilation and intensive support do not have a signifi cant eff ect on outcome [2] . Could this be due to the disease itself, ventilator-induced lung injury, complications of intensive support (sepsis, critical care myoneuropathy, or ventilator-associated pneumonia), or a combination of the above? Only assumptions can be made, and patients (at an earlier stage) and relatives as well as physicians outside of the ICU before or at admission should become aware of the poor prognosis. Th is does not mean that IPF patients with acute respiratory failure should be denied admis sion; in many hospitals, the ICU is the right place to perform in a safe and timely fashion the necessary extended investigation to exclude reversible causes of deterioration in these patients.
Ventilating a patient with an IPF exacerbation is a diffi cult and demanding task, and no 'cookbook' prescriptions can make the work easier for the intensivist. Th e evidence for the best ventilator strategy applying to an IPF exacerbation is extremely scarce, and the eff ect of ventilatory management on outcome has not been system atically assessed; therefore, every suggestion is based on theoretical principles and pathologic data that are characterized mainly by extended DAD [7] . Recently, Bates and colleagues [65] introduced the concept of percolation, according to which the progression of parenchymal lung disease can suddenly reach a threshold that dramatically alters the mechanical properties of the lung. IPF exacerbations that require ventilator support could be an example of crossing this percolation threshold. Under these circumstances, mechanical ventila tion could represent a second hit for the lung parenchyma, further deteriorating the mechanical properties of lung parenchyma and introducing a vicious cycle that ends in death. Mechanical ventilation with conventional volumes (8 mL/kg) in patients without lung injury can induce severe surfactant impairment, and sustained plasma cytokine production has been demonstrated in patients without ALI ventilated with conventional tidal This non-proportional fi gure denotes the incoherence of the clinical signifi cance of acute respiratory distress syndrome (ARDS), acute interstitial pneumonia (AIP), and idiopathic pulmonary fi brosis (IPF) exacerbations in which DAD, despite being the common denominator, develops upon diff erent histology substrates (UIP in IPF exacerbations, normal lungs in AIP, and normal or diseased lungs in ARDS) and, according to current defi nitions, presents at diff erent time intervals: 7 days for ARDS, 4 weeks for IPF exacerbations, and 2 months for AIP. This incoherence led also to a diff erent pharmacologic approach, which proved to be unsuccessful at least in AIP and in IPF true exacerbations. ALI, acute lung injury.
volumes (10 mL/kg) compared with those ventilated with low tidal volumes (6 mL/kg) [66, 67] . So it should not be surprising, although it may be very diffi cult to prove, that the employment of traditional tidal volumes in patients with IPF exacerbations would be detrimental given that their lungs are characterized by extended parenchymal alterations, severe inhomogeneity, and decreased compliance even prior to initiation of mechanical ventilation. Especially the inhomogeneity of the lung parenchyma could cause severe overinfl ation of the 'healthy' lung units with higher compliance and jeopardize the 'healthy' parenchyma left. A ventilation strategy employing low tidal volumes (4 to 6 mL/kg ideal body weight), such as that used for patients with ARDS, seems prudent and is advised by many experts [68] . Positive end-expiratory pressure (PEEP) should be used moderately because of the aforementioned risk of overinfl ation of intact lung units. Fernández-Pérez and colleagues [64] demonstrated that high PEEP was independently associated with increased mortality in chronic ILD [64] . In the same context, there is no place for recruitment or prone position [69] . Given that intubated patients with IPF exacerbations require high-minute volume because of increased dead space, the respiratory frequency should be increased to the maximum acceptable rate and the target of a normal PaCO 2 (arterial partial pressure of carbon dioxide) should be abandoned. Th is high respiratory rate might require the use of heavy sedation and quite often paralysis, and care should be given to avoid auto-PEEP [70] . Th e eff ect of prolonged sedation and paralysis on the neuromuscular function of these patients, who have often been administered steroids for a long time, is an unavoidable cost. Th e earliest possible interruption of sedation will facilitate weaning provided that gas exchange and lung mechanics have improved.
NIV has some theoretical advantages in IPF patients and has been used extensively in cases of acute respiratory failure to avoid intubation. Unfortunately, the studies about its use have the same methodological problems as those for the invasive ventilation studies mentioned previously, and no fi rm conclusions can be drawn. Th ere are two things that make NIV more 'attractive' in this setting: the almost absolute mortality that invasive mechanical ventilation carries and the avoidance of intubation and ventilation risks (aspiration, ventilator-associated pneumonia, and ventilatorassociated injury). Th e problem is that in most cases the excessive work of breathing associated with IPF exacerbation cannot be managed eff ectively by NIV. Extracorporeal membrane oxygenation could represent a valuable adjunct to conventional treatment for selected cases of IPF. Limited availability, high cost, complicated technology, and increased rates of complications have been the most important factors limiting its use so far [71] [72] [73] . In the setting of IPF therapeutics, it has been used mainly as a bridge to transplantation [74] . Transplantation represents the fi nal line of defense for the IPF patient and is the only therapy with a proven survival benefi t. Early referral (even at the time of diagnosis) to a lung transplant center is mandatory [75] because of the prolonged waiting-list time, which sometimes exceeds the patient's life expectancy.
IPF exacerbations constitute the most devastating compli cation of IPF. Diff erent and hard-to-diff erentiate clinical scenarios may reproduce the hallmark of their defi nition: subacute/acute deterioration of dyspnea and bilateral chest infi ltrates, corresponding in 'true' IPF exacerbations, to a tissue pattern of DAD upon UIP. Also, ALI/ARDS and AIP present DAD. Intensive immunosuppression proved ineff ective and represents the coup de grace for these patients. Provision of excel lent supportive care and the search for and treatment of the 'underlying cause' remain the only options. Th e unravelling of Ariadne's thread continues.
Abbreviations AIP, acute interstitial pneumonia; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; DAD, diff use alveolar damage; ED, emergency department; HDU, high-dependency unit; HRCT, high-resolution computed tomography; ICU, intensive care unit; IIP, idiopathic interstitial pneumonia; ILD, interstitial lung disease; IPF, idiopathic pulmonary fi brosis; NIV, non-invasive ventilation; PEEP, positive end-expiratory pressure; RICU, respiratory intensive care unit; UIP, usual interstitial pneumonia.
The authors declare that they have no competing interests. Influence of genetic variability at the surfactant proteins A and D in community-acquired pneumonia: a prospective, observational, genetic study INTRODUCTION: Genetic variability of the pulmonary surfactant proteins A and D may affect clearance of microorganisms and the extent of the inflammatory response. The genes of these collectins (SFTPA1, SFTPA2 and SFTPD) are located in a cluster at 10q21-24. The objective of this study was to evaluate the existence of linkage disequilibrium (LD) among these genes, and the association of variability at these genes with susceptibility and outcome of community-acquired pneumonia (CAP). We also studied the effect of genetic variability on SP-D serum levels. METHODS: Seven non-synonymous polymorphisms of SFTPA1, SFTPA2 and SFTPD were analyzed. For susceptibility, 682 CAP patients and 769 controls were studied in a case-control study. Severity and outcome were evaluated in a prospective study. Haplotypes were inferred and LD was characterized. SP-D serum levels were measured in healthy controls. RESULTS: The SFTPD aa11-C allele was significantly associated with lower SP-D serum levels, in a dose-dependent manner. We observed the existence of LD among the studied genes. Haplotypes SFTPA1 6A(2 )(P = 0.0009, odds ration (OR) = 0.78), SFTPA2 1A(0 )(P = 0.002, OR = 0.79), SFTPA1-SFTPA2 6A(2)-1A(0 )(P = 0.0005, OR = 0.77), and SFTPD-SFTPA1-SFTPA2 C-6A(2)-1A(0 )(P = 0.00001, OR = 0.62) were underrepresented in patients, whereas haplotypes SFTPA2 1A(10 )(P = 0.00007, OR = 6.58) and SFTPA1-SFTPA2 6A(3)-1A (P = 0.0007, OR = 3.92) were overrepresented. Similar results were observed in CAP due to pneumococcus, though no significant differences were now observed after Bonferroni corrections. 1A(10 )and 6A-1A were associated with higher 28-day and 90-day mortality, and with multi-organ dysfunction syndrome (MODS) and acute respiratory distress syndrome (ARDS) respectively. SFTPD aa11-C allele was associated with development of MODS and ARDS. CONCLUSIONS: Our study indicates that missense single nucleotide polymorphisms and haplotypes of SFTPA1, SFTPA2 and SFTPD are associated with susceptibility to CAP, and that several haplotypes also influence severity and outcome of CAP. Community-acquired pneumonia (CAP) is the most common infectious disease requiring hospitalization in developed countries. Several microorganisms may be causative agents of CAP, and Streptococcus pneumoniae is the most common cause [1] . Inherited genetic variants of components of the human immune system influence the susceptibility to and the severity of infectious diseases. In humans, primary immunodeficiencies (PID) affecting opsonization of bacteria and NF-Bmediated activation have been shown to predispose to invasive infections by respiratory bacteria, particularly S. pneumoniae [2] . Conventional PID are mendelian disorders, but genetic variants at other genes involved in opsonophagocytosis, with a lower penetrance, may also influence susceptibility and severity of these infectious diseases with a complex pattern of inheritance [3] .
In the lung, under normal conditions, microorganisms at first encounter components of the innate immune response, particularly alveolar macrophages, dendritic cells and the lung collectins, the surfactant protein (SP)-A1, -A2 and -D. SP-A1, -A2 and -D belong to the collectin subgroup of the C-type lectin superfamily, and contain both collagen-like and carbohydrate-binding recognition domains (CRDs) [4] . Upon binding to pathogen-associated molecular patterns (PAMPs), SP-A and SP-D enhance the opsonophagocytosis of common respiratory pathogens by macrophages [5, 6] . Mice rendered SP-A or SP-D deficient exhibit increased susceptibility to several bacteria and viruses after intratracheal challenge [7] [8] [9] . SP-A1, -A2 and -D also play a pivotal role in the regulation of inflammatory responses [4, 10, 11] and clearance of apoptotic cells [4, 12, 13] . In mice, SP-A and SP-D have been shown to be nonredundant in the immune defense in vivo [9] .
The human SP-A locus consists of two similar genes, SFTPA1 and SFTPA2, located on chromosome 10q21-24, within a cluster that includes the SP-D gene (SFTPD) [11] . The nucleotide sequences of human SFTPA1 and SFTPA2 differ little (96.0 to 99.6%) [14] . Single nucleotide polymorphisms (SNP) at the SFTPA1 codons 19, 50, 62, 133 and 219, and at the SFTPA2 codons 9, 91, 140 and 223 have been used to define the SP-A haplotypes, which are conventionally denoted as 6A n for the SFTPA1 gene and 1A n for the SFTPA2 gene (see Table E1 in Additional File 1) [15] . Variability at the SFTPD gene has been also reported. Particularly, the presence of the variant amino acid (aa)-11 (M11T) has been shown to lead to low SP-D levels [16] .
In the present study, we assessed the potential association of missense polymorphisms of the SFTPA1, SFTPA2 and SFTPD genes as well as the resulting haplotypes, with the susceptibility to and the severity and outcome of CAP in adults. In addition, we evaluated the existence of linkage disequilibrium (LD) among these genes, and the effect of genetic variability on SP-D serum levels.
We studied 682 patients and 769 controls, all of them Caucasoid Spanish adult individuals from five hospitals in Spain. Foreigners and individuals with ancestors other than Spanish were previously excluded in the selection process. The diagnosis of CAP was assumed in the presence of acute onset of signs and symptoms suggesting lower respiratory tract infection and radiographic evidence of a new pulmonary infiltrate that had no other known cause. A detailed description of the exclusion criteria and clinical definitions are shown in Methods in Additional File 1 [17] [18] [19] . The control group was composed of healthy unrelated blood donors from the same hospitals as patients.
For susceptibility, a case-control study was performed. Severity and outcome were evaluated in a prospective study of CAP patients. Demographic and clinical characteristics of CAP patients included in the study are shown in Table E2 in Additional File 1.
In order to analyze the effect of the SFTPD aa11 on SP-D levels in our population, protein levels were measured in serum samples from individuals in the control group by means of a Surfactant Protein D ELISA kit (Antibodyshop ® , Gentofte, Denmark).
Four haplotypes of SP-A1 (6A, 6A 2 , 6A 3 and 6A 4 ) and six of SP-A2 (1A, 1A 0 , 1A 1 , 1A 2 , 1A 3 and 1A 5 ) are found frequently (>1%) in the general population [15] . On the basis of the differences in non-synonymous SNPs (SFTPA1-aa19, -aa50, -aa219, SFTPA2-aa9, -aa91, -aa223) the most frequent conventional haplotypes of these genes, except 1A and 1A 5 , can be unambiguously identified (see Table E1 in Additional File 1). However, this method does not allow for the differentiation of some of these haplotypes from those rare haplotypes (frequency equal or lower than 1%) identified with the SNPs indicated in Table E1 in Additional File 1. For comparative purposes, in our study each haplotype was denoted by the name of the most frequent haplotype for a given combination of non-synonymous SNPs. Genomic DNA was isolated from whole blood according to standard phenol-chloroform procedure or with the Magnapure DNA Isolation Kit (Roche Molecular Diagnostics, Pleasanton, CA, USA). Genotyping of polymorphisms in SFTPA1 (aa19, aa50, aa219), SFTPA2 (aa9, aa91, aa223) and SFTPD (aa11) genes was carried out using minor modifications of previously reported procedures [15, 20] . The accuracy of genotyping was confirmed by direct sequencing in an ABI Prism 310 (Applied Biosystems, Foster City, CA, USA) sequencer.
Haplotypes for each individual were inferred using PHASE statistical software (version 2.1) [21] . The haplotype of SFTPA1, SFTPA2 or the haplotype encompassing SFTPA1, SFTPA2 and SFTPD was ambiguous or could not be assigned in 12 individuals, who were excluded from the study. The order used for the haplotypes nomenclature is SFTPD-SFTPA1-SFTPA2. Linkage disequilibrium (LD) was measured by means of Arlequin (version 3.11) [22] and Haploview [23] softwares in the control group. In addition, pairwise LD between haplotypes of SFTPA1 and SFTPA2 as well as with the SFTPD SNP was characterized using Arlequin 3.11. The existence of LD was considered if D' >0.4.
Informed consent was obtained from the patients or their relatives. The protocol was approved by the local ethics committee of the five hospitals. All steps were performed in complete accordance to the Helsinki declaration.
Bivariate and multivariate statistical analyses were performed using SPSS (version 15.0) (SPSS, Inc, Chicago, Ill, USA) and R package [24] . A detailed description of the statistical methods is shown in Methods in Additional File 1.
Susceptibility to CAP related to SFTPA1, SFTPA2 and SFTPD gene variants Seven non-synonymous SNPs were genotyped across the region containing the SFTPD, SFTPA1 and SFTPA2 genes ( Table 1) . None of the SNPs showed a significant deviation from Hardy-Weinberg equilibrium in controls. Several major alleles were overrepresented in controls compared with patients, but only SFTPA1 aa50-G, SFTPA2 aa9-A and aa91-G remained significant after Bonferroni correction for multiple comparisons. A dominant effect of SFTPA2 aa9-A, and a recessive effect of SFTPA1 aa50-G and aa219-C as well as SFTPA2 aa223-C were associated with a lower risk of CAP (see Table 1 ). When haplotypes were inferred, seven different haplotypes were found for SFTPA1 and eight for SFTPA2 (see Table 2 ). All haplotypes except 6A 5 , 6A 15 , 1A 10 and 1A 13 had frequencies higher than 1% in our population. The most frequent haplotype for SFTPA1 and SFTPA2 were respectively TGC and AGC, which correspond mainly with the 6A 2 and 1A 0 haplotypes respectively. The frequencies of both haplotypes were significantly lower in patients compared to controls (P = 0.0009, OR = 0.78; 95% confidence interval (CI) 0.67 to 0.91, for SFTPA1 6A 2 . P = 0.002, OR = 0.79; 95% CI 0.68 to 0.92, for SFTPA2 1A 0 ), even when Bonferroni correction was applied. Several haplotypes were overrepresented in patients compared with controls, but only 1A 10 (P = 0.00007, OR = 6.58; 95% CI 2.24 to 26.22) remained significant after Bonferroni correction. For the observed odd-ratios, the power of the tests with a significance level of 1% were 84.16%, 79.09% and 94.04% for the haplotypes 6A 2 , 1A 0 and 1A 10 respectively. In addition, dominant and recessive models showed a significant dominant effect on CAP susceptibility for haplotypes 6A 3 , 1A 0 , 1A 7 and 1A 10 and a recessive effect for haplotype 6A 2 (see Table 2 ).
Linkage disequilibrium of SFTPA1, SFTPA2 and SFTPD genes
Pairwise LD (D') measured by means of Arlequin confirmed the existence of LD among several SNPs at SFTPA1 and SFTPA2, whereas SFTPD aa11 was only observed in LD with SFTPA1 aa19 (see Figure 1) . A similar pattern of LD was observed when D' was measured by means of the Haploview software (data not shown). SFTPA1 and SFTPA2 were previously found to be in LD [25, 26] . The value of LD measured as r 2 was very low for every pair of SNPs (data not shown), and none of the studied SNPs could be used as haplotypetagging SNP to infer the observed haplotypes. When pairwise LD was measured among haplotypes instead among SNPs, SFTPA1 was found to be in LD with SFTPD aa11, but only a marginal LD was found between SFTPA2 1A and SFTPD aa11 (see Table E3 in Additional File 1).
Susceptibility to CAP related to haplotypes encompassing SFTPA1, SFTPA2 and SFTPD When haplotypes encompassing both SFTPA genes were studied, we observed 39 of the 64 expected haplotypes, and only 14 haplotypes had frequencies higher than 1% (data not shown). The most common SFTPA1-SFTPA2 haplotype, 6A 2 -1A 0 , was underrepresented in patients (P = 0.0005, OR = 0.77; 95% CI 0.66 to 0.90), whereas 6A 3 -1A was overrepresented (P = 0.0007, OR = 3.92; 95% CI 1.63 to 10.80) (see Table 3 ). Both differences remained significant after Bonferroni correction. For the observed odd-ratios, the powers of the tests with a significance level of 1% were 87.76% and 84.04% for the haplotypes 6A 2 -1A 0 and 6A 3 -1A respectively. On the other hand, dominant and recessive logistic regression models showed a significant dominant effect on CAP susceptibility for haplotypes 6A 3 -1A and 6A-1A 1 and a recessive effect for haplotype 6A 2 -1A 0 (see Table 3 ). We also intended to analyze whether phased variants encompassing the three genes were involved in susceptibility to CAP. Only 68 of the 128 expected haplotypes were observed, and 16 of them had a frequency over 1%. Chromosomes containing C-6A 2 -1A 0 were decreased in patients when compared with controls (P = 0.00001, OR = 0.62; 95% CI 0.50 to 0.77), a difference that remained significant after Bonferroni correction. C-6A 2 -1A 0 was also significantly associated with protection against CAP in a dominant model (see Table 3 ).
A similar pattern of haplotype distribution was observed when individual as well as two-and three-gene based haplotypes were compared between pneumococcal CAP patients and healthy controls (see Table E4 in Additional File 1), though no significant differences were now observed after Bonferroni corrections.
Outcome and severity of CAP patients related to genetic variants at SFTPA1, SFTPA2 and SFTPD genes When fatal outcome was analyzed, patients who died within the first 28 days showed a higher frequency of haplotypes 6A 12 , 1A 10 and 6A-1A, and a lower frequency of the major SFTPA1aa19-T and aa219-C alleles and of haplotypes 6A 3 and 6A 3 -1A 1 (see Table 4 ). Similar results were observed when 90-day mortality was analyzed (see Table 4 ). For the observed odd-ratios, the power of the tests with a significance level of 5% was 82.64% when the protective effect of 6A 3 -1A 1 on 28-day mortality was evaluated, and 81.45% and 80.79% concerning the effect of 6A 3 and 6A 3 -1A 1 on 90-day mortality respectively. Kaplan-Meier analysis ( Figure 2 ) and log-rank test (Table 4 ) also showed significantly different survival for the above mentioned alleles and haplotypes. Cox Regression for 28-day survival, adjusted for age, gender, hospital of origin and co-morbidities, was significant for haplotypes 6A 12 and 6A-1A, and it remained significant for haplotypes 6A 3 and 6A-1A when 90-day survival analysis was performed (see Table 4 ). We also analyzed Cox Regression adjusted for hospital of origin, PSI and pathogen causative of the pneumonia, and we found similar results: for 28-day Figure 1 Genomic organization, location of SNPs, and linkage disequilibrium (D') map for SFTPD, SFTPA1 and SFTPA2 genes. SNPs: Single-nucleotide polymorphisms. All the D' values higher than 0.3 were statistically significant (P < 0.05). Linkage disequilibrium was measured in the control group.
survival it remained significant for haplotype 6A-1A (P = 0.029, OR = 2.45; 95% CI 1.10 to 5.46), although for 6A 12 haplotype it was not significant (P = 0.072); for 90-day survival it was significant for both 6A 3 (P = 0.038, OR = 0.52; 95% CI 0.28 to 0.96) and 6A-1A (P = 0.045, OR = 2.12; 95% CI 1.02 to 4.44) haplotypes. No effect of the SFTPD aa11 SNP was observed. Due to the high number of observed haplotypes, and because of the limited sample size in the patient groups when they were stratified on the basis of severity and outcome, the haplotypes including SFTPA1, A2 and D were not studied.
The relevance of these genetic variants in the severity of CAP was also evaluated by analyzing predisposition to acute respiratory distress syndrome (ARDS) and to multiorgan dysfunction syndrome (MODS) (see Tables 5 and  6 ). The SFTPD aa11-C allele was significantly overrepresented in patients with MODS or ARDS. Haplotypes 6A and 6A-1A, were also associated with the development of ARDS, and SFTPA2 1A and 1A 10 were associated with the development of MODS. For the observed odd-ratios, the power of the association of 1A with predisposition to MODS was 89.29%. However, the number of individuals included in the analysis of outcome was relatively small and the power of the tests with a significance level of 1% was lower than 80%. These associations remained significant in multivariate analysis adjusted for age, gender, hospital of origin and co-morbidities, as well as for hospital of origin, PSI and causative microorganism (see Tables 5 and  6 ). By contrast, 6A 3 -1A 1 was associated with protection against MODS, although this difference was not significant in the multivariate analysis.
In order to study whether variants at the pulmonary collectins were associated with differences of serum levels of SP-D, this protein was measured in serum from healthy controls with known genotypes. The SFTPD aa11-C SNP associated with lower SP-D serum levels (905.10 ± 68.38 ng/ml for T/T genotype, 711.04 ± 52.02 ng/ml for T/C, and 577.91 ± 96.14 ng/ml for C/C; ANOVA P = 0.017) (see Figure 3 ). 
This study is unique in reporting a genetic association between non-synonymous SNPs at SFTPD, SFTPA1 and SFTPA2, as well as of haplotypes encompassing these genes, with the susceptibility, severity and outcome of CAP. The major alleles of SFTPA1 aa50-G, aa219-C as well as SFTPA2 aa9-A and aa91-G or genotypes carrying these alleles were associated with protection against CAP. The frequencies of the different SNPs and haplotypes of SFTPA1, SFTPA2 and SFTPD observed in our study were similar to those previously reported in European populations [25] . SFTPA1 and SFTPA2 were reported to be in strong LD [26, 27] , and several haplotypes of these loci tend to segregate together, being 6A 2 -1A 0 the major haplotype [27] . A protective role against CAP was associated with 6A 2 , 1A 0 and 6A 2 -1A 0 in our survey but only the rare 1A 10 and 6A 3 -1A haplotypes were significantly associated with susceptibility to CAP. Similar results were observed in susceptibility to pneumococcal CAP. Several SNPs and Table 4 . [15] . † P-value for the bivariate comparison. ‡ P-value for multivariate analysis, including the variables age, gender, hospital of origin and co-morbidities. For those bivariate comparisons that resulted in nonsignificant differences, multivariate analysis were not calculated. § P-value for multivariate analysis, including the variables hospital of origin, PSI (Pneumonia Severity Index) and pathogen. haplotypes were also associated with a higher severity and poor outcome; MODS, ARDS, and mortality were selected because they represent the more severe clinical phenotypes. Particularly, 1A 10 and 6A-1A were overrepresented among patients who died at 28 or 90 days, and they also predisposed to MODS and ARDS respectively. Likewise, 6A was associated with ARDS, and 1A was associated with MODS. By contrast, 6A 3 and 6A 3 -1A 1 were underrepresented in patients who died. The SFTPD aa11-C allele was associated with the development of MODS and ARDS, but no significant effects on mortality were observed. In spite that the power of the test for some associations with outcome and severity were higher than 80% for the observed OR with a significance level of 5%, the number of individuals included in the analysis of outcome was relatively small. Consequently, associations with outcome should be interpreted with caution.
Only a few studies have addressed the role of the genetic variability at SFTPA1, and SFTPA2 in infectious diseases [28] [29] [30] [31] . In bacterial infections, homozygosity for the 1A 1 haplotype was reported to be associated with meningococcal disease [30] . Noteworthy, 6A 2 -1A 0 was protective against acute otitis media (AOM) in children [32] . Haplotypes 6A 2 and 1A 0 may also be involved in protection against respiratory syncytial virus (RSV) disease [29, 33] . Considering the high difference in the frequencies with the corresponding alternative alleles and haplotypes, it is tempting to speculate that 6A 2 , 1A 0 and 6A 2 -1A 0 could have been maintained at high frequencies partly by their protective effect against respiratory infections. The 6A and 6A-1A haplotypes were found to be associated with an increased risk of wheeze and persistent cough, presumably triggered by respiratory infections or environmental contaminants, among infants at risk for asthma [27] . Regarding SP-D, the SFTPD aa11-T allele was associated with severe RSV bronchiolitis [34] , whereas the SFTPD aa11-C variant was associated with tuberculosis [30] .
In sharp contrast to the potentially proinflammatory effects after PAMP recognition by collectins, mice deficient in SP-A or SP-D develop enhanced inflammatory pulmonary responses [35] [36] [37] . SP-A and SP-D play a dual role in the inflammatory response. They interact with pathogens via their CRD, and are recognized by calreticulin/CD91 on phagocytes through the N-terminal collagen domain, promoting phagocytosis and proinflammatory responses [10, 13] . By contrast, binding of the CRD to signal inhibitory regulatory protein α (SIRPα) on alveolar macrophages suppresses NF-B activation and inflammation, allowing the lung to remain in a quiescent state during periods of health [10] . A similar dual effect is observed in the promotion or inhibition of apoptosis [12] . SP-A and SP-D can also inhibit inflammation by blocking, through the CRD, Toll-like receptors 2 and 4 [38, 39] . In agreement with previous results [16] , we have observed that the SFTPD aa11-C allele associates with significantly lower SP-D serum levels than the aa11-T allele, and this effect was dose-dependent. The aa11-C/T SNP, located in the Nterminal domain, influences oligomerization of SP-D and explains a significant part of the heritability of serum SP-D levels [16, 40] . Serum from aa11-C homozygotes lack the highest molecular weight (m.w.) forms of the protein, which binds preferentially to complex microorganisms whereas the low m.w. SP-D preferentially binds LPS [16] .
As a consequence of intracellular oligomerization, monomeric SP-A subunits fold into trimers, and supratrimeric assembly leads to high-order oligomers [41, 42] . The degree of supratrimeric oligomerization is important for the host defense function [14, 41, [43] [44] [45] . SP-A1 and SP-A2 differ in only four amino acids (residues 66, 73, 81 and 85) located in the collagen domain [46] . In most functions examined, recombinant human (rh) SP-A2 shows higher biological activity than SP-A1 [14, 41, [47] [48] [49] [50] .
The significance and the nature of functional differences between variants at SP-A1 and SP-A2 are poorly understood [14, 49, 50] . Variants aa50 (SP-A1) and aa91 (SP-A2) are located in the collagen region. These changes may affect the oligomerization pattern and binding to receptors such as calreticulin/CD91 or the functional activity of the protein. Likewise, the variants aa219 (SP-A1) and aa223 (SP-A2) are located in the CRD, and might directly influence the binding properties to microorganisms or to surface receptors such as SIRPα or TLR4. Residue 9, and frequently residue 19, is located in the signal peptide, and it is not know whether these variants may affect the function of the protein [14, 44] . Alternatively all the missense variants could be in LD with SNPs in regulatory regions that might affect translation and RNA stability [51, 52] .
Native SP-A is thought to consist of hetero-oligomers of SP-A1 and SP-A2, and properties of co-expressed SP-A1/SP-A2 are between those of SP-A1 and SP-A2 [41, 46] . However, the extent of oligomerization of SP-A, as well as the SP-A1/SP-A2 ratio, may be altered in various diseases and can vary among individuals [53, 54] . The combination of both gene products may be important for reaching a fully native conformation [41] . In fact, it was recently shown that both SP-A1 and SP-A2 are necessary for the formation of pulmonar tubular myelin [55] . Therefore, the effect of a given haplotype may be largely influenced by haplotypes at the other gene. Our results suggest that the 6A 2 to1A 0 haplotype is more protective against CAP than both 6A 2 and 1A 0 .
It was previously reported that the SFTPD aa11 SNP is in LD with SFTPA1 and SFTPA2 [25] . A protective effect of the 6A 2 to 1A 0 haplotype was even higher when this haplotype co-segregates with the SFTPD aa11-C allele. Likewise, one haplotype containing 6A 2 -1A 0 and the G allele of the SFTPD aa160 SNP could be protective against severe RSV disease [29] . Haplotypes at SFTPA1 are in LD with SFTPD aa11 in our population, but only a marginal LD between SFTPA2 and SFTPD aa11 was observed. In addition, no LD between 6A 2 to A 0 and SFTPD aa11 was found in controls (D' = 0.09) or CAP patients (D' = 0.024) in our study. These findings suggest that the protective effect of the co-segregation of SFTPD aa11-C with 6A 2 to 1A 0 on CAP susceptibility may rather reflect genetic interactions. Alternatively, the SFTPD aa11 SNP may be a marker of other SNPs in LD with SFTPA1 and SFTPA2. The gene of another collecting, the mannose-binding lectin (MBL), is located at 10q11.2-q21. We have previously observed that MBL deficiency predisposes to higher severity and poor outcome in CAP [56] , and LD of the SP genes with MBL2 cannot be ruled out.
Despite modern antibiotics, CAP remains a common cause of death, and the search for new therapeutic approaches has been redirected into non-antibiotic therapies [57] . SP-A levels are reduced in several pulmonary diseases [58] [59] [60] . SP-D may also be reduced in some patients with ARDS [59] . In Sftpa -/and Sftpd -/mice, intratracheally administered SP-A or SP-D can restore microbial clearance and inflammation [8, 35] . Exogenous surfactant preparation containing the hydrophobic SP-B and -C are nowadays widely used for replacement therapies in infantile RDS. In addition, intratracheal instillation of recombinant SP-C reduced mortality in patients with severe ARDS due to pneumonia or aspiration [61] . Some of the genetic variants analyzed in our survey, such as 1A 10 , although rare, may have a high impact on susceptibility, severity and outcome of CAP. Validation of our results in other populations, and a better knowledge of the functional and clinical significance of the genetic variability at SFTPA1, SFTPA2 and SFTPD could be relevant for future investigations in the use of these collectins in the treatment of respiratory infectious diseases.
The surfactant proteins A1, A2 and D are key components of innate immune response and the antiinflammatory status in the lung. Genetic variability at the genes of these collectins influences susceptibility and outcome of community-acquired pneumonia. These results could be relevant for future investigations in the use of these collectins in the treatment of respiratory infectious diseases.
• The SFTPA1 and SFTPA2 haplotypes 6A 2 , 1A 0 and 6A 2 to 1A 0 , and the SFTPD-SFTPA1-SFTPA2 haplotype C-6A 2 to 1A 0 are associated with a protective role against the development of Communityacquired pneumonia (CAP).
• 1A 10 and 6A 3 to 1A haplotypes are associated with increased susceptibility to CAP.
• Haplotypes 6A and 6A to 1A are associated with development of ARDS, while 1A and 1A 10 are associated with MODS in patients with CAP.
• The variant SFTPD aa11-C leads to decreased SP-D serum levels, and predisposes to development of MODS and ARDS in patients with CAP.
• Haplotypes 6A 12 , 1A 10 and 6A to 1A are overrepresented among patients who died at 28 or 90 days. By contrast, 6A 3 and 6A 3 to 1A 1 are protective against 28-day and 90-day mortality.
Additional file 1: Further description of methods, definitions and statistical analysis, and Tables E1-E4. The file contains additional information on exclusion criteria and definitions of PSI, ARDS and MODS. The statistical tests used are described. The additional file also includes four tables. Table E1 defines the resulting haplotypes from SNPs combination in SFTPA1 and SFTPA2 genes. Table E2 presents demographic and clinical characteristics of CAP patients. Table E3 shows the pairwise linkage disequilibrium measure for surfactant proteins A1, A2 and D alleles. Table E4 compares haplotypes of SFTPA1, SFTPA2 and SFTPD between patients with pneumococcal CAP and controls.  Survival of Influenza A(H1N1) on Materials Found in Households: Implications for Infection Control BACKGROUND: The majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. However the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. We therefore measured the viability over time of two H1N1 influenza strains applied to a variety of materials commonly found in households and workplaces. METHODOLOGY AND PRINCIPAL FINDINGS: Influenza A/PuertoRico/8/34 (PR8) or A/Cambridge/AHO4/2009 (pandemic H1N1) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. Virus genome was measured by RT-PCR; plaque assay (for PR8) or fluorescent focus formation (for pandemic H1N1) was used to assess the survival of viable virus. CONCLUSIONS/SIGNIFICANCE: The genome of either virus could be detected on most surfaces 24 h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. In contrast, virus viability dropped much more rapidly. Live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at 24 h. We conclude that influenza A transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). In situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals. Influenza transmission is well documented in households and other residential settings [1] [2] [3] [4] . Yet the underlying mechanisms of transmission remain poorly understood and hotly debated [5, 6] . Although transmission by aerosols (particles typically ,5 mm in diameter), larger droplets and contact transmission (direct and via fomites) probably all play some role, the relative importance of each is uncertain, which has led to difficulties regarding the provision of evidence-based infection control advice for both pandemic and seasonal influenza [7] . If virus can survive for meaningful periods on surfaces and objects, or alternatively, if surfaces are frequently re-inoculated (e.g. by toddlers), then it is feasible that transmission via fomites might occur.
The potential for transmission of influenza by indirect contact (i.e. via fomites) is linked to the ability of virus to survive in transmissible titres on commonly touched surfaces; however few data exist on this subject. Parker et al (1944) demonstrated improved survival of influenza viruses in the presence of human mucus [8] ; and in 1962, Buckland demonstrated experimentally that influenza virus was inactivated relatively quickly on glass, probably through desiccation [9] . In 1982, widely cited work by Bean et al showed that both influenza A and B, directly applied to stainless steel surfaces or hard plastic, could survive for 24-48 hours, and be transferred, from there to hands, for 24 hours; survival was much shorter on porous materials such as paper and cotton (8-12 hours) , with transferability to hands for only 15 minutes [10] . In contrast, Thomas et al, recently demonstrated survival of human seasonal A (H1N1) and A (H3N2) on Swiss banknotes for up to three days, increasing to up to eight days when applied with nasopharyngeal secretions from children (17 days if applied at very high concentration). Although viable virus was recovered at each of these time points, it was noted that virus load declined sharply after the first few days; no other materials were tested [11] . Other studies have detected influenza virus on fomites in homes and health and childcare facilities, using RT-PCR to establish the presence of the viral genome [12] [13] [14] . However, data obtained using this technique (even quantitatively) do not distinguish adequately between viable and non-viable virus and are therefore problematic to interpret in the context of practical infection control guidance. In another recent study, virus was detected by PCR on commonly touched household surfaces, but only one sample proved culture positive [15] . However, the time from deposition to recovery was not known, nor the extent of any cleaning undertaken.
We evaluate the survival of influenza A (H1N1) viruses deliberately applied to a range of commonly touched household and workplace surfaces, using RT-PCR for genome detection and culture methods to determine viability. We conclude that RT-PCR is only useful to demonstrate the absence of virus and that on most surfaces, virus viability drops rapidly. Nevertheless, on certain non-porous surfaces, viable virus persists for several hours, rendering fomite transmission possible without re-inoculation.
To test the surface survival of influenza virus, we used a variety of materials commonly encountered in the home and workplace, including a hospital setting ( Table 1) ; choice of surfaces to be tested was discussed with the Department of Health, England to ensure relevance to public health policy. These included fibrous materials such as the ubiquitous J-clothH (Associated Brands) widely used for cleaning, a silver impregnated fabric with known bacteriostatic properties (Toray Textiles Europe Ltd.) of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, as well as fabric from a child's soft toy. The latter fabric was made of non-absorbent polyester and, although a porous item overall, individual fibres might perform as a nonporous surface. A variety of non-porous plastic surfaces representing objects highly likely to be touched by multiple individuals such as light switch, telephone and keyboard plastics were also tested, as well as porous and non-porous 'background' materials such as various wood surfaces, glass, Perspex/plexiglass (poly (methyl methacrylate) -a thermoplastic often used as a light or shatterresistant alternative to glass) and metals. As a control surface, we used standard laboratory polystyrene culture dishes. As viruses, we used two human H1N1 strains: the laboratory adapted A/Puerto Rico/8/34 (PR8) strain because of ready availability and robust, convenient assay systems with a wide dynamic range, and an isolate of the current 2009 pandemic virus A/Cambridge/AH04/ 2009 (AH04), as a low passage history representative of a virus likely to be encountered in the current environment. The source and disinfection method used to clean the various surfaces before testing are listed in Table 1 .
Human influenza A virus PR8 (Cambridge lineage) was grown in embryonated hens' eggs and harvested at a titre of 9610 8 pfu/ ml. For inoculation of the surfaces, the virus was diluted 1:10 in 1% BSA and serum free media (Dulbecco Modified Eagles Medium, DMEM, Gibco, UK). This represented a viral titre approximating 1.5610 8 TCID50/ml, just above the upper end of titres reported for human shedding [10, 11] . Preliminary experiments established that virus survival was improved by the addition of extra protein to the suspension. We tested 0.5% or 1% BSA as well as four preparations of artificial mucus produced from pig stomach mucosa (NBS Biologicals), pig stomach mucin types II or III or bovine sub maxillary glands mucin, type I-S (all from Sigma Aldrich). 1% BSA had the largest effect on titre and duration of survival, followed by the bovine mucin (data not shown). In the interests of simplicity and reproducibility, 1% BSA was therefore used in all subsequent experiments.
To test a 2009 pandemic influenza A (H1N1) virus strain on selected surfaces, a clinical isolate designated influenza A/Cambridge/AHO4/2009 (AH04) was passaged once in MDCK cells and then grown in Caco-2 cells (colorectal adenocarcinoma cells, ATCC HTB-37 TM ). The virus is a recent isolate from an Received sterile in packaging from manufacturer.
Fumigation in a CLIII room using a Laycock Fumigator (Tolbest Ltd). doi:10.1371/journal.pone.0027932.t001 immunocompetent patient who was hospitalised briefly at the start of their illness, but recovered. This virus does not form discrete plaques in MDCK cells and could therefore not be titred by this method. Instead, virus stocks were quantified by qPCR for segment 7 [12] . Although this method scores viable and non-viable virus particles alike, preparations of wild type influenza A viruses generally have similar particle:PFU ratios and quantitative comparison of RT-PCR and other titration methods have shown good agreement [13, 14] . The AH04 stock contained 6.5610 8 genome copies/ml and was used at a 1:10 dilution as for PR8. For comparison, the PR8 stock had a genome titre of 1.6610 11 genome copies/ml.
Mouse monoclonal AA5H (Abcam) was used to detect influenza NP by immunofluorescence.
Surfaces were cut into 2 cm 2 pieces and sterilised by a variety of means depending on the surface to be tested (e.g. autoclaving, fumigation etc). Sterile surfaces were glued into sterile 6-well tissue culture dishes using cyanoacrylate adhesive (Henkel, UK). Preliminary experiments (data not shown) demonstrated that dried adhesive alone was non-inhibitory to influenza virus. Under the same conditions of temperature and humidity (ranges 17-21uC and 23-24% respectively), 10 ml volumes of virus were applied to six samples of each surface at the same time. Sampling was conducted immediately -time zero -to demonstrate recoverability. A cotton swab was moistened by dipping in 3 ml of virus transport medium (VTM, Remel, UK) and then wiped carefully in 6 different directions for 1 minute across the top of the surface. Keeping everything on ice, the swab was placed into the tube containing the residual (3 ml) volume of VTM and vortexed for 1 minute. After this, the sample was split directly into 6 eppendorf tubes and stored on dry ice prior to freezing at 270uC. The remaining samples in the plate were kept in a plastic, lidded box at constant temperature and humidity. At 4, 9, 24, 48 and 72 hrs, further samples were taken and stored. After initial experiments it was clear that the virus did not survive in detectable amounts for more than 24 hrs, therefore for the majority of the experiments only the first 4 time points (0, 4, 9, and 24 hrs) were taken. Initial experiments with PR8 virus also showed that loss of virus on the swab was not a major factor, with recovery of virus at time zero from polypropylene surfaces approaching 50% of initial titre (data not shown).
The qRT-PCR assay used has been described previously [12] . In brief, primers and probes to the Matrix gene of influenza A were used to detect the presence of the virus on the surfaces. Samples from all time points were stored and then extracted. Virus genome was amplified to check that the quantity of virus deposited on the different surfaces was consistent and to determine whether any of the surfaces affected the genome over time.
Plaque assays were performed as previously described in MDCK cells using Avicell overlays [15, 16] , in duplicate or where possible in triplicate.
To detect AH04 virus by fluorescent focus assay, infectious material from swabs was first allowed to amplify by inoculation into 1610 6 MDCK cells and incubation for 48 h. Supernatant virus was then diluted 1:10 in serum free DMEM and 250 ml used to inoculate 1.5610 5 MDCK cells in a 24 well tissue culture plate. After virus absorption, the cells were overlaid with 1 ml serum free DMEM media containing 1 mg/ml Worthington's trypsin and 0.14% BSA and incubated overnight at 37uC. The following day they were fixed with 4% formaldehyde in PBS, permeabilised by the addition of 0.2% Triton 6100 in PBS for 5 minutes at RT and fluorescently stained with anti-NP monoclonal antibody and counterstained for DNA with 4,6-diamino-2-phenylindole (DAPI) as previously described [17] . Cells were examined blind by two people and scored semi quantitatively for the presence of infected cells using a standardised schema (2 no fluorescence seen; +/2 some fluorescence seen (,5% cells infected); +5-10% cells infected; ++ .10% cells infected). The literature indicates immunofluorescence to be at least as sensitive in general as plaque assay [13, 18, 19] . Confirming this, tests using serial dilutions of known quantities of PR8 virus, our method reliably detected 20 PFU of virus in the original sample prior to amplification and 50% of the time detected 2 PFU (data not shown).
To test the surface survival of the virus genome, replicate samples of the various materials were inoculated with 10 ml samples containing 1610 6 PFU of virus and incubated for defined periods of time before sample recovery was attempted by swabbing. It was noted that the liquid was absorbed by the wooden surfaces within 5 minutes whereas a droplet could be seen on non-porous surfaces for considerably longer, although in all cases, surfaces had dried by 7 hours. Material eluted from the swabs was then titred for virus genome by quantitative RT-PCR. For both PR8 (Fig. 1A , Table 2 ) and AH04 (Table 3) viruses the results were unambiguous. On most surfaces, the viral genome persisted well, with only around a 10-100 fold drop from the initially recoverable titre after 24 h. The exceptions were unsealed wood surfaces, where both viruses lost genome titre rapidly and on pine surfaces in particular, became undetectable after a few hours. Thus in general, viral RNA survives well for at least 24 h and few surfaces had any significant 'contact effect' in immediately reducing genome titre.
When PR8 surface viability was assessed by plaque assay, virus inoculated onto a control surface of a tissue culture dish could be recovered efficiently at t0, but thereafter infectivity fell away rapidly with no live virus recovered at 24 h (Table 4 ). Fitting the data to a one-phase exponential decay model (Fig. 1B ) estimated the t 1/2 of the virus under these conditions to be around 1.5 h. A similar pattern of rapid loss of infectivity was seen when the household surface samples were tested, with the difference that greater initial losses of infectivity ranging between 20-fold (telephone handset) to nearly 4000-fold (unsealed pine) were seen (Table 4 ). Nevertheless, viable virus was recovered at 4 h (but not later) from the silver-impregnated cloth, soft toy fabric and in trace quantities, from light switch material. The only material (other than the control tissue culture dish) for which even low amounts of viable virus could be detected at 9 h was stainless steel. Thus despite the persistence of the viral genome on a wide variety of household surfaces, PR8 infectivity decayed sharply, with evidence of significant contact effects from some materials; most notably unsealed pine, but also a wide variety of other porous and nonporous surfaces.
To test whether these findings could be extrapolated to a currently circulating virus, we next tested the survival of AH04 virus, a 2009 pandemic isolate, on a subset of the materials. Unlike PR8, as a recent clinical isolate this virus does not grow to high titres in the laboratory and nor was a workable plaque assay available. We therefore used a fluorescent focus assay in which live virus is detected by immunofluorescent detection of the viral nucleoprotein in infected cells. To boost the sensitivity with which viable virus could be detected, infectious virus present in the swabs was first amplified by growth in MDCK cells before subsequent assay. The assay therefore provides a highly sensitive but semi quantitative measure of virus infectivity, ideally suited to working with low titre samples [13, 19] . By this measure, the AH04 virus persisted for at least 24 h on the control tissue culture dish material, although titres were evidently lower at 9 and 24 h ( Table 5) . Consistent with the results obtained with PR8 virus, all household surfaces tested showed lower persistence of infectious virus, with none providing recoverable titre at 24 h and the majority failing to produce live material at 9 h. Once again the pine surface showed very rapid inactivation of viability, with no infectivity recovered at 4 h. Thus both an historic virus isolate and an example of the recent pandemic strain fail to survive in high titres for long periods of time on a variety of household surfaces, but with significant survival over shorter time spans on certain materials.
Prior to the influenza A(H1N1) pandemic of 2009-10, few data were available with regard to virus survival on different household surfaces. With a few notable exceptions [10, 11] , the majority of studies had been carried out based on RT-PCR to detect the presence of the genome [20] [21] [22] ; these shed no light on the presence or absence of viable virus. In this study we sought to provide contemporary data about virus survival on a wider range of materials found in or on household surfaces than previously described in the literature; these exemplars were chosen after discussion with UK pandemic policy makers. However, one limitation is that our study was confined to H1N1 influenza A viruses (PR8 and the 2009 pandemic virus) due to resource issues. However, we know of no evidence to suggest there are substantial differences in survival between human influenza viruses. Moreover, when we compared the survival of PR8 virus with two seasonal isolates of influenza A (A/Solomon Islands/12/5/08 (H1N1) and influenza A/Brisbane/12/5/08 (H3N2), obtained from Professor Alan Hay of the National Institute of Medical Research, Mill Hill), we saw no significant differences, with all three viruses losing plaque titre on a plastic surface with a t1/2 of around 90 minutes (data not shown). We therefore think it is reasonable to generalise from the findings here to other human strains of influenza A. Further studies, especially of influenza B are warranted however. We applied concentrations of virus (,1610 6 TCID 50 ), which were within the range of those reported in the respiratory secretions of naturally infected individuals [5, 10] . In addition, we suspended virus in 1% bovine serum albumin (BSA), reflecting our (unpublished) finding that BSA improved virus survival, and similar findings from Thomas et al [11] using mucus obtained from children. Our experiments were conducted within a narrow range of humidity and temperature conditions consistent with normal human indoor living conditions in temperate zones, and all survival assays were performed in duplicate and where possible triplicate. We used plaque assay techniques and immunofluorescence techniques for PR8 and pandemic viruses, respectively. The differing methodologies used to detect the two strains of H1N1 virus (lower titre inoculum of the pandemic AH04 virus but higher sensitivity detection method) make it difficult to directly compare the survival of the two strains, but we see little to suggest any major difference.
Our data on the survival of the laboratory adapted PR8 virus indicated that viable virus was no longer recoverable in detectable amounts from 9 of 14 (64%) surfaces four hours after deposition; however, contrary to the findings of Bean et al., non-porous surfaces were not consistently more conducive to virus survival than porous ones [10] . Nevertheless, no test surfaces supported detectable virus survival beyond nine hours. Broadly similar outcomes in which infectivity tended to be lost after 4-9 hours were obtained with the recent pandemic isolate AH04. Overall, our results indicate that influenza virus does not remain viable in large quantities on most surfaces in indoor domestic conditions for more than a few hours. Our data are consistent with recent findings from a study of environmental deposition of pandemic H1N1 virus in the homes of infected patients, involving our laboratory, when almost 10% of tested surfaces yielded viable virus [15] . However, in this and similar studies in community settings where environmental samples are taken relatively infrequently and the infectious source remains present, it is not possible to establish the time elapsed since virus deposition [15, 23] .
With regard to the testing of specific materials, we examined survival on a range of porous items: a children's soft toy, a silver impregnated fabric with known bacteriostatic properties of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, and a branded cleaning cloth (J clothH, Associated Brands). We hypothesised that the inclusion of an antimicrobial agent, MicrobanH (Microban International Ltd) in the J cloth might inhibit viral growth. MicrobanH is based on triclosan and has been demonstrated to have anti-bacterial and anti-fungal activity; it has not however, been demonstrated or claimed to be anti-viral. Notwithstanding, in our laboratory setting, some constituent or quality of the J clothH appeared to limit virus survival to under 4 hours. The result for the silver impregnated fabric also deserves further comment. Whilst silver has been demonstrated to have bacteriostatic properties, it has not been documented to show antiviral activity. Our data would tend to suggest that it is not significantly inhibitory to influenza A. Surfaces that allowed PR8 virus to survive longest (between four and nine hours) included light switch material (polyvinyl chloride) and a computer keyboard. Interestingly these are likely to be the materials from which the most frequently touched communal household objects are made. Both PR8 and pandemic viruses survived less than four hours on all of the wood surfaces tested. This may have been due to a number of factors including porosity of the surface, oils in the wood or a potentially virucidal 'contact effect' of varnish finishes. Pine oil in particular has been demonstrated to have virucidal activity against respiratory viruses [24] . Our findings suggest they are not hospitable environments for enveloped viruses.
As observed in other studies, we found that stainless steel supported the viability of influenza viruses longer than other tested metals. Metals have been demonstrated to have low levels of anti viral activity [25] [26] [27] ; and stainless steel has previously been demonstrated to support influenza virus viability for longer than that of copper [28] . Confirmation of these results raises questions about the use of stainless steel in healthcare and daycare settings in particular.
In conclusion, testing two H1N1 strains of influenza A (one of which was a 2009 pandemic virus) demonstrates that in an environment that is consistent with indoor domestic settings in temperate zones, virus deposited onto the touched environment is likely to survive up to a few hours, though rarely more than nine hours, on the vast majority of surfaces. Metallic and non-metallic non-porous materials pose the greatest risk and should be targeted for frequent cleaning if situated in close proximity to patients infected with influenza virus; fortunately the latter are also more conducive to surface cleaning with a wide variety of simple cleaning agents [12] . Whilst our data suggest that the risk of virus transmission might last several hours after deposition, we generated very little data suggesting that appreciable amounts of virus survived much beyond nine hours. This probably means that frequently touched environments such as classrooms, offices and living rooms, which are then left unoccupied overnight, will not contain much viable virus on surfaces by the next morning. Nevertheless, the data still support frequent cleaning of commonly touched items and surfaces throughout the working day, particularly when symptomatic persons are present, for example in physician waiting rooms. In terms of cleaning regimens, one critically important consideration is that survival of virus in high titres for prolonged periods is not necessary for fomite transmission if surfaces are frequently re-inoculated (e.g. by toddlers). However the contribution of such indirect transmission relative to respiratory droplets directly from one person to another or relative to aerosol transmission remains unknown. Respiratory failure presenting in H1N1 influenza with Legionnaires disease: two case reports INTRODUCTION: Media sensationalism on the H1N1 outbreak may have influenced decisional processes and clinical diagnosis. CASE PRESENTATION: We report two cases of patients who presented in 2009 with coexisting H1N1 virus and Legionella infections: a 69-year-old Caucasian man and a 71-year-old Caucasian woman. In our cases all the signs and symptoms, including vomiting, progressive respiratory disease leading to respiratory failure, refractory hypoxemia, leukopenia, lymphopenia, thrombocytopenia, and elevated levels of creatine kinase and hepatic aminotransferases, were consistent with critical illness due to 2009 H1N1 virus infection. Other infectious disorders may mimic H1N1 viral infection especially Legionnaires' disease. Because the swine flu H1N1 pandemic occurred in Autumn in Italy, Legionnaires disease was to be highly suspected since the peak incidence usually occurs in early fall. We do think that our immediate suspicion of Legionella infection based on clinical history and X-ray abnormalities was fundamental for a successful resolution. CONCLUSION: Our two case reports suggest that patients with H1N1 should be screened for Legionella, which is not currently common practice. This is particularly important since the signs and symptoms of both infections are similar. Media sensationalism with respect to the swine flu outbreak may have influenced decisional processes and clinical diagnosis. We report two cases of patients who present during 2009 in whom H1N1 and Legionella infection coexisted. Secondary bacterial pneumonia is recognized as one of the most common causes of death in influenza cases. Coinfection has been found in 30% of all influenza cases in persons with seasonal influenza. The pathogens most often involved are Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenza [1, 2] . From July 2009 through February 2010 in Italy, 2500 confirmed cases of pandemic influenza and four and a half million cases of influenza-like illnesses were reported to the sentinel surveillance system.
A total of 1278 (50%) confirmed cases of H1N1 were hospitalized. Of these, 271 (21%) cases presented with pneumonia, which was attributed to bacterial coinfection in 33 cases. Of the 33 cases with pneumonia due to a bacterial coinfection, six (18%) were due to the Legionella pneumophila serogroup 1 [3] .
Our first case is a 69-year-old Caucasian man with a past medical history of coronary artery disease, chronic renal insufficiency, hypertension and type 1 diabetes. Two weeks earlier, he had been exposed to a child with an upper respiratory infection. He lived in a rural area. He had no history of insect bites, but was exposed to farm animals and pond water.
He had been well until nine days earlier, when dry cough, myalgias, fever (39.4°C), malaise, sore throat and nasal congestion presented. On physical examination in the Emergency Room (ER), the patient had a temperature of 39.2°C, a heart rate of 50 beats per minute and a respiratory rate of 35 breaths per minute. A buccal swab was negative for influenza A and B antigens, and no parasites were seen on a peripheral-blood smear.
Acetaminophen, ketorolac, levofloxacin and normal saline were administered.
After 24 hours he presented with persistent fever (39.0°C), dry cough and respiratory failure and was admitted to the intensive care unit (ICU). Vital signs were as follows: blood pressure 135/70 mm Hg; heart rate 50 beats per minute; respiratory rate 34 breaths per minute; oxygen saturation 88%, on 50% inspired oxygen. On physical examination rhonchi were detected in the lower lung fields.
Repeated tests of nasopharyngeal secretions for influenza viruses, parainfluenza virus, respiratory syncytial virus, and adenovirus were negative. Testing for antibodies to toxoplasma was suggestive of past infection. Cultures of specimens of blood, urine, and sputum were sterile. Polymerase chain reaction determination of buccal swab for H1N1 influenza A virus was positive. Urinary tests for Legionella antigens were positive.
On admission main laboratory examations were as follows: white blood cell count (WBC) was 9.8 K/mL (87% neutrophils, 4% lymphocytes); C-reactive protein 205 mg/L; serum sodium 132 mEq/L; serum phosphorus 2.3 mg/dL; serum glutamate pyruvate transaminase (SGPT) 175 IU/L; serum oxaloacetate transaminase (SGOT) 184 IU/L; serum ferritin 4100 ng/mL; creatinine phosphokinase (CPK) 241 IU/L. Winthrop scale score was > 15.
Chest radiograph showed low lung volumes, with patchy air-space disease consistent with multifocal pneumonia (Figure 1 ). Intravenous azithromycin (500 mg twice daily), levofloxacin (500 mg twice daily) and oral oseltamivir (150 mg twice daily) were administered. Within 18 hours after arrival, tachypnea and hypoxemia (PaO 2 : 58 mm Hg, while breathing 50% oxygen) increased further requiring intubation and mechanical ventilation. Hypotension and renal failure developed; methylprednisolone and vasopressors were administered.
On the third day reverse transcriptasepolymerase chain reaction (RT-PCR) on a broncoalveolar lavage specimen was still positive for H1N1 influenza infection. Legionella antigens were also confirmed positive. On the fifth day he was extubated and non-invasive ventilation was started. He was discharged from the ICU on day 21.
Our second case is a 71-year-old Caucasian woman with a past medical history significant for hypertension, type 1 diabetes and chronic hepatitis C.
She reported an eight day history of dry cough and fever (39.2°C) associated with sore throat and nasal congestion. She lived in a peripheral urban area. On emergency room examination, her Glasgow Coma Score (GCS) was 12 (E = 3 V = 4 M = 5), temperature was 39.6°C, blood pressure was 80/40 mm Hg, heart rate was 54 beats per minute, respiratory rate was 40 breaths per minute and oxygen saturation was 88% on 50% inspired oxygen. Her chest radiograph was consistent with multifocal pneumonia (Figure 1 ). There were rhonchi in the left and right lung fields. A rapid test of a specimen from a buccal swab was negative for influenza A and B antigens, and no parasites were seen on a peripheral-blood smear. Acetaminophen, ketorolac, levofloxacin and normal saline were administered.
After one hour she was admitted to the ICU and due to worsening hypoxemia (PaO2 48 mm Hg, while breathing 50% inspired oxygen), neurological impairment with a GCS of 10 (E = 2 V = 3 M = 5) and hemodynamic instability (blood pressure 80/40 mmHg, heart rate 46 beats per minute), she was intubated and mechanically ventilated. RT-PCR on a broncoalveolar lavage specimen was positive for H1N1 influenza infection.
On admission main laboratory examination results were as follows: white blood cell (WBC) count was 5.8 Iannuzzi et al. Journal of Medical Case Reports 2011, 5:520 http://www.jmedicalcasereports.com/content/5/1/520 K/Ml (77% neutrophils; 5% lymphocites); C-reactive protein was 312 mg/L; serum sodium was 129 mEq/L; serum phosphorus was 2.5 mg/dL; serum SGPT were 215 UI/L; serum SGOT were 220 UI/L; serum ferritin was 5280 ng/mL; CPK was 445 IU/L. Her Winthrop scale score was > 15.
Intravenous levofloxacin (500 mg twice daily) and oral oseltamivir (150 mg twice daily) were administered. On the second day, hypoxemia, hypotension and renal failure developed; norepinephrine was administered after fluid challenge. For persistent hypoxemia she was ventilated in the prone-supine position for 12 hour intervals daily. Tests of the urine for legionella antigens were positive. Azithromycin (500 mg twice daily) was added her treatment.
On the ninth day she underwent percutaneous tracheostomy. She was discharged from the ICU on day 35.
During the Spring of 2009, a novel influenza A (H1N1) virus of swine origin emerged to cause infections in humans in North America [4] .
The pandemic was carefully followed by the media but with a touch of sensationalism that caused a widespread a sense of fear in the population. Health care systems and physicians were suddenly in the spotlight. In our cases all the signs and symptoms (including respiratory failure, refractory hypoxemia, leukopenia, lymphopenia, thrombocytopenia, and elevated levels of creatine kinase and hepatic aminotransferases) were consistent with critical illness due to infection with the 2009 H1N1 virus [1, 4, 5] .
Other infectious disorders may mimic H1N1 viral infection especially Legionnaires' disease. Because the swine flu H1N1 pandemic occurred in Autumn in Italy, Legionnaires' disease was to be highly suspected since its peak incidence usually occurs in early fall.
Initial attempts to diagnose H1N1 infection using immunochromatography relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1. Hence, tests with monoclonal antibodies that react with H1N1 but not seasonal influenza A (H1N1 and H3N2) or B viruses were developed.
Recognizing viral hemagglutinin and nucleoprotein, specifically allows the detection of H1N1 virus in nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses.
Early and rapid diagnosis of H1N1-related respiratory insufficiency needs rapid screening during a pandemic but clinicians cannot rely only on the buccal swab test and need to rule out false positive and negative cases by RT-PCR on oral/nasal fluids or bronchoalveolar lavage specimens.
In case one the visit by a child with an upper respiratory infection five days before the onset of illness in the patient represents also a plausible exposure to the 2009 H1N1 virus; in case two the lack of positive anamnesis for other suspicions together with the finding of positive specimens for H1N1 influenza A infection could have caused us arrive at a fashionable diagnosis and stopped us from further investigations. We do think that the immediate suspicion of Legionella infection based on clinical history, X-ray abnormalities and Winthrop University Hospital Infectious Disease Division's diagnostic weighted point system scale (Table 1) were fundamental for a successful resolution [6, 7] .
Almost all patients affected by pandemic H1N1 infections admitted to an ICU because of lung involvement receive empiric antibiotic therapy. However, preliminary clinical data have failed to demonstrate a consistent role of bacterial co-infection suggesting that severe pulmonary damage occurs as a result of viral pneumonia [1, 11] . A recent autopsy study revealed evidence of concurrent bacterial infection in 29% of cases [8] . In 45% of these the pathogen was S. pneumoniae. These findings confirm the results of previous studies of autopsy specimens showing that most deaths attributed to influenza A virus occurred concurrently with bacterial pneumonia [9] . On the other hand, they highlight the importance of treating influenza patients with both empiric antibacterial therapy and antiviral medications. According to our experience we believe that zoonotic infections had to be ruled out. The lack of known contact with animals in an immunocompetent host appears to rule out zoonotic infections, such as Coxiella burnetii. One of the patients had worked near water ponds, which could have been contaminated by animal urine, but he did not have pulmonary hemorrhage so leptospirosis seems unlikely. Without exposure to birds, Chlamydia psittaci is unlikely. Community-acquired pneumonia (Streptococcus pneumoniae, Haemophilus influenzae, S. pyogenes, or Staphylococcus aureus) can cause severe pulmonary disease, especially in patients with antecedent influenza. These pathogens should have responded to the broadspectrum antimicrobial therapy; therefore, they are unlikely to have been the sole cause of illness. Atypical bacterial pathogens such as Legionella pneumophila cause multifocal pneumonia but usually do not cause upper respiratory tract symptoms [1, 2] .
Anaplasmosis could result in a lower respiratory tract disease but fulminant disease is rare and clinical improvement should have occurred with levofloxacin treatment [10] . Radiographic abnormalities also needed to be ruled out. Many critically ill patients have radiographic findings of viral pneumonitis, with bilateral interstitial and alveolar infiltrates. Multifocal and patchy abnormalities as seen in these patients have been reported in cases of 2009 H1N1 influenza A virus infection but do not completely rule out invasive bacterial infection [2] . Ground glass opacity and cavitary lobar opacity should focus attention on Legionnaire's disease [11] .
Another potential contributory factor that needed to be ruled out was viral infection of the respiratory tract. Infection with adenovirus or influenza virus must be considered. Adenovirus type 14 is the most likely cause of severe viral pneumonia in adults. Radiographic findings may include lobar infiltrates, although these are more characteristic of bacterial pneumonia [12, 13] . The fact that bacterial infections should have responded to levofloxacin argues against the fact that a secondary bacterial pneumonia superimposed with influenza A or B causing severe pulmonary disease. The lack of recent travel in H5N1 (bird flu) endemic areas or exposure to sick or dead poultry argue against H5N1 influenza (bird flu) [14] .
Our two case reports suggest that patients with H1N1 should be screened for Legionella, which is not currently common practice. This is particularly important since the signs and symptoms of both infections are similar. Doctors should never be dazzled by contingency and media sensationalism in decision making. With prompt identification of the bacterial etiology of pneumonia, appropriate treatment can be started with both antibacterial therapy and antiviral medications. The length of hospital stay and the mortality of both pandemic and seasonal influenza can be reduced.
Written informed consent was obtained from both patients for publication of this case report and any ccompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Authors' contributions MI collected patient data regarding the ER and ICU and was a major contributor in writing the manuscript. OP was responsible for buccal swab and bronchoalveolar lavage specimens and for RT-PCR processing. FR interpreted radiological findings and provided the radiological differential diagnosis. GS provided a major contribution in data analysis and interpretation. RT provided a major contribution in data analysis and interpretation. EDR collected data regarding ER and ICU, contributed to the analysis and interpretation, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. Agricultural intensification, priming for persistence and the emergence of Nipah virus: a lethal bat-borne zoonosis Emerging zoonoses threaten global health, yet the processes by which they emerge are complex and poorly understood. Nipah virus (NiV) is an important threat owing to its broad host and geographical range, high case fatality, potential for human-to-human transmission and lack of effective prevention or therapies. Here, we investigate the origin of the first identified outbreak of NiV encephalitis in Malaysia and Singapore. We analyse data on livestock production from the index site (a commercial pig farm in Malaysia) prior to and during the outbreak, on Malaysian agricultural production, and from surveys of NiV's wildlife reservoir (flying foxes). Our analyses suggest that repeated introduction of NiV from wildlife changed infection dynamics in pigs. Initial viral introduction produced an explosive epizootic that drove itself to extinction but primed the population for enzootic persistence upon reintroduction of the virus. The resultant within-farm persistence permitted regional spread and increased the number of human infections. This study refutes an earlier hypothesis that anomalous El Niño Southern Oscillation-related climatic conditions drove emergence and suggests that priming for persistence drove the emergence of a novel zoonotic pathogen. Thus, we provide empirical evidence for a causative mechanism previously proposed as a precursor to widespread infection with H5N1 avian influenza and other emerging pathogens. Preventing and controlling emerging zoonoses require identification of the processes that drive cross-species pathogen transmission [1] . Agricultural intensification has been proposed as a major underlying cause of pathogen emergence from wildlife and domestic animal populations into human populations [2, 3] ; however, the precise mechanisms by which this occurs have rarely been demonstrated. Specific agricultural practices may increase frequency of cross-species pathogen transmission, setting the stage for persistence of new pathogens to occur. Pathogen introduction into a partially immune population-such as a population where some individuals are vaccinated or were previously infected-can result in longer and potentially larger epidemics than introduction into a naive population [4] . We refer to this phenomenon as immunity-based, population-level 'priming' for persistence. The potential of such priming to drive zoonotic emergence has been demonstrated theoretically in the general case [4] , as a possible precursor to measles emergence [5] and as a mechanism that could facilitate widespread emergence of H5N1 avian influenza in poultry [6] . Here, we present evidence that this process is not only theoretically possible but is likely to have played a key role in the first known outbreak of Nipah virus (NiV) encephalitis, and therefore for the emergence of a lethal zoonosis.
NiV is a paramyxovirus that emerged in people in Malaysia in 1998 [7 -9] . Serology, virus isolation and polymerase chain reaction detection indicate that NiV is maintained in Pteropus spp. fruit bats (flying foxes), including P. vampyrus and P. hypomelanus in peninsular Malaysia [10] [11] [12] . Transmission from flying foxes to pigs is thought to occur via saliva on fomites (discarded fruit pulp) or via faecal or urine contamination of pigsties [11] . Pigs act as amplifier hosts, enabling infection of humans via droplet transmission during respiratory infections [13] . The risk of direct transmission from bats to humans in Malaysia is believed to be low [14] .
Between September 1998 and April 1999, NiV caused 246 reported cases of febrile encephalitis in humans in peninsular Malaysia and Singapore [7, 8] and an epidemic of respiratory and neurological disease in commercially farmed pigs [9, 15] . Initial human cases were seen in Tambun village and surrounding areas in Perak State, followed by a large epidemic in the southern states of Negeri Sembilan and Selangor and several cases in Singapore (figure 1a). The majority of these cases occurred in pig farmers and abattoir workers [17] . During the course of the outbreak investigation, nine cases of NiV encephalitis were retrospectively diagnosed in the Tambun area with onset dates between January 1997 and May 1998, five of which were associated with the index farm of the 1998 -1999 outbreak in Perak (figure 1a). Six of the nine cases are considered confirmed cases on the basis of serological tests performed in 1999 [16] ; the remaining three are probable cases.
Several mechanisms have been previously proposed for the appearance of NiV in pig and human populations in 1998. One frequently cited explanation is that flying foxes harbouring NiV were new to the region where transmission to pigs occurred, driven there by climatic anomalies. In late 1997, an El Niñ o Southern Oscillation-related drought and burning of forested land in Sumatra and Kalimantan (Indonesia) created an atmospheric haze across Sumatra and peninsular Malaysia [18] . One of us (K.B.C., see group author list) hypothesized that this atmospheric haze caused atypical flying fox immigration from Sumatra across the Straits of Malacca into peninsular Malaysia and northward towards Perak [18] . This hypothesized movement of flying foxes into an area with large pig farms potentially outside the species' normal range was proposed to have precipitated transmission of the virus from bats to pigs. However, a retrospective diagnosis of human NiV encephalitis cases on the index farm in early 1997 indicates that bat-to-pig transmission did not result from these specific environmental conditions. In particular, seven of these cases occurred prior to the rise in airborne particulate matter that is diagnostic of the haze event, which peaked in September 1997 [18] . The recognition of cases prior to the haze event refutes the hypothesis that this event drove initial cross-species transmission (table 1) . Furthermore, at the time of the major outbreak in pigs and humans, NiV antibodies were found to be widespread in flying fox colonies within peninsular Malaysia [12] , suggesting that the outbreak was not the result of a (recent) point source introduction of the virus into the bat population, as previously suggested [18] .
Since its discovery in 1999, NiV has been identified as the cause of human neurological and respiratory disease in India and Bangladesh [23, 24] , and molecular evidence suggests a wide distribution of henipaviruses in the reservoir hosts [25] [26] [27] [28] [29] . Despite the continued threat that NiV and related viruses represent to global health, no detailed studies have combined data on pig populations, bat colonies and human cases to examine the mechanisms that drove the first known and largest outbreak. This paper describes an interdisciplinary approach that synthesizes all available data to improve and update our understanding of the process of NiV emergence in Malaysia. The study has three main branches, all of which build on earlier work.
First, Chua et al. [18] identified the presence of fruit trees on the index farm as a plausible link between flying foxes and pigs that could have precipitated introduction of the virus. We place this finding in a broader context by examining spatial and temporal patterns of agricultural production in peninsular Malaysia prior to and at the time of the outbreak. We also investigate, in detail, the history and status of fruit crop production on the index farm.
Second, our preliminary models suggested that repeated introduction could plausibly lead to persistence of the virus in a generic large pig population [30] . In this study, we use detailed information on management practices and production patterns at the index farm to parametrize models of this population. We use these models to examine whether repeated introduction was necessary and sufficient to allow the virus to persist on the farm. We then compare model predictions of piglet mortality patterns with data from production records to assess whether repeated introduction led to persistence of the virus in this population.
Third, Johara et al. [12] identified Pteropus spp. as likely reservoirs of NiV infection. Here we examine the distribution of Pteropus bats in peninsular Malaysia, present data on NiV serology from all known roosts and examine the opportunities for transmission of the virus from bats to pigs by placing these findings in the context of large-scale agricultural production patterns.
Finally, we bring together the three branches of the study to describe the events that led to NiV emergence in Malaysia and Singapore and discuss the causal nature of different contributing factors.
2.1.1. Large-scale patterns. Both pig and mango (Mangifera indica) production tripled in peninsular Malaysia between the early 1970s and the late 1990s (figure 1c). The intensification of production of pigs and mangoes was loosely correlated during this time period. The marked decline in both end-of-year standing pig population (SPP) and annual mango production between 1998 and 1999 indicates that this correlation may reflect widespread dual use of agricultural land to produce both pigs and mangoes, with the decline in commercial mango harvest reflecting the widespread abandonment and culling of infected pig farms that occurred in the first half of 1999 (following the discovery of NiV). Table 1 . Proposed drivers of emergence. Hill outlined nine criteria to be used to assess causality for epidemiological outcomes with complex origins [19] . A useful discussion of the implementation of these criteria in disease ecology is provided by Plowright et al. [20] . This figure 1a ; [18] a Temporality is the only criterion that is logically necessary (in bold).
Nipah virus emergence J. R. C. Pulliam et al. 93
30 000 (approx. the same size as at the time the farm was culled in 1999). Mangoes, jackfruit (Artocarpus heteropyllus) and durian (Durio sp.) were grown on the index farm, while other farms in the Tambun area grew primarily pomelos (Citrus maxima), which are not eaten by flying foxes. As Chua et al. [18] identified, several large mango trees were planted directly adjacent to pig enclosures. Approximately 400 mango trees were planted on the farm in 1983, and the trees began producing fruit in 1987. The location of these trees, in relation to the areas where pigs were kept, is shown in the electronic supplementary material, which also includes additional details of the timeline of pig and fruit production on the index farm.
2.2.1. Dynamic models. We analysed pig production data from the index farm and developed an agestructured model of NiV dynamics within the farm population. Our model shows that multiple transmission events from bats to pigs are the best explanation for the observed pattern of human cases and pig mortality. As piglets aged in the index farm, they were moved from the breeding sections into the growing section, and finally into the finishing section for the last six weeks before they were sent to slaughter. When the virus was initially introduced into the pig population, individuals entering the growing section were immunologically naive. As the virus spread throughout the farm, the proportion of pigs entering this section with active immunity increased to the point where too few susceptibles remained for the chain of transmission to continue, and the virus could not be maintained within the farm population (figure 2a,c). The first, rapid epizootic of NiV in the pig population on the index farm thus would have produced highly localized human infections (figure 1a) and the virus most probably went extinct in the pig population at this time (figures 2a,c and 3). Our models suggest that, when NiV was reintroduced to the farm, it circulated enzootically until the farm was depopulated in March 1999 (figures 2 and 3). This finding is consistent with the timing of human cases associated with the index farm and with the high antibody prevalence detected in sows and piglets in Breeding section 2 upon depopulation of the index farm [31] . The change in dynamics between initial and subsequent introductions of the virus on the index farm results from the presence of acquired immunity in the sow population and conferred immunity in the young pig population. The age structure within the growing section, along with its rapid turnover and large population size, allowed the virus to circulate enzootically. On reintroduction of the virus into the growing section, the population had a different immunological profile from the time of initial introduction. Many young pigs moving from the breeding sections into the growing section now carried maternally derived passive immunity rather than active immunity obtained from exposure to the virus (figure 2b). We estimate that maternal antibodies are lost at approximately 14 weeks of age (electronic supplementary material). Pigs born with maternal antibodies would therefore become susceptible to the virus roughly four weeks after entering the growing section, where they would remain for another six weeks. These dynamics produce a steady inflow of susceptible individuals, which is sufficient to maintain the virus over a period of 2 years or more ( figure 2b,d) , a period similar to that observed between the index case for NiV in Malaysia in 1997 and the onset of the large-scale outbreak. The population size and turnover rate of the finishing section suggest that it may also have permitted enzootic circulation of the virus (electronic supplementary material). Stochastic simulations demonstrate that priming for persistence on the index farm is the most likely scenario consistent with pig and human epidemiology for R 0 values .2.5 (up to R 0 ¼ 30, the highest value examined), and simulations with an R 0 value in this range best reproduce the very high seroprevalence (.95% for sows and . 90% for piglets) detected in Breeding section 2 at the time of depopulation [31] (electronic supplementary material).
Although no direct prevalence data exist for NiV in the pig population on the index farm, the presence of the virus in the breeding sections can be inferred from production records kept by the farm's managers (electronic supplementary material) and from reported serological findings [31] . These data are consistent with the scenario predicted by the model and with the timing of human cases: there was a period of approximately five to six months between the first introduction of the virus into the pig population and subsequent, smaller peaks in piglet mortality (figure 1b), when the virus was probably reintroduced into the farm-probably from the flying fox reservoir, or possibly from other pig farms in the area. The virus appears to have spread into each of the three breeding sections between December 1996 and February 1997, with strong evidence of infection (unusually high piglet mortality) in at least two of the breeding sections (Breeding Nucleus and Breeding section 2) during January 1997, and significant mortality on a number of subsequent occasions, following an interim period in which piglet mortality returned to baseline levels (figure 1b and electronic supplementary material).
2.3.1. Bat distribution. We conducted a countrywide survey of the distribution and infection status of the two Pteropus species in peninsular Malaysia. We located 14 active P. vampyrus roost sites across peninsular Malaysia, four P. hypomenlanus roosts on islands surrounding the peninsula and one mixed roost near Langkawi Island off the northwest coast. We estimated the current distribution of Pteropus spp. bats in peninsular Malaysia using combined results of wildlife surveys and analysis of hunter licensing data (figure 4 and electronic supplementary material). The overall distribution of flying foxes in peninsular Malaysia is similar to that observed in a large-scale survey of flying fox populations conducted in 1999 [32] , although there are fewer permanent roost sites. Satellite telemetry studies show that the bats are highly mobile, moving between Indonesia, Malaysia, Singapore and Thailand [33] .
We found that flying foxes are consistently present throughout Perak State, including near the index farm, where we located two seasonal P. vampyrus roosts: one in Lenggong, Perak, ,50 km from the index farm, where we observed a maximum population of approximately 2500 bats, and another near Tambun, Perak, within 2 km of the index farm, which had been previously reported in 1999 [32] and where we observed bats in 2003 -2005 [33] . The distance between these roosts and the index farm is within the bats' nightly foraging range. Flying fox roosts were not found in the more densely populated pig-farming regions of southern Selangor and Negeri Sembilan [32, 33] (figure 4). 
NiV emergence in Malaysia occurred in two phases, and our current understanding of the processes leading to its emergence is outlined in figure 5 . Phase I of emergence, the occurrence of human cases, required that only two criteria be met: circulation of the virus in a local bat population and the existence of a pathway for transmission from Pteropus bats to pigs. In phase I, early human cases were directly linked to the transmission of virus from bats to pigs, with all identified cases occurring in the immediate vicinity of the index farm where bat-to-pig transmission ignited rapid pig-to-pig transmission and produced a tight cluster of human cases on the index farm and an adjacent property. Phase II of emergence, incident human cases outside the area of transmission from flying foxes, required that the criteria for phase I be met but also mandated the existence of a pathway for transmission to humans that had become unlinked from the wildlife reservoir. In principle, several scenarios could have produced such a pathway, as illustrated in figure 5 ; however, the evidence amassed here indicates that the causal pathway realized (highlighted in dark blue). Alternative pathways that would have been sufficient to produce both phases of emergence-had they occurred-are indicated by dashed arrows. Specifically, introduction of Nipah virus into an area with extremely dense pigfarming activity (such as Port Dickson in Negeri Sembilan or Sabang Perai Utara in Pulau Penang) probably could have resulted in viral persistence without priming and reintroduction. However, these areas lacked factors further up the causal pathway: the absence of Pteropus bats in these areas prevented viral introduction prior to the establishment of an alternative source of infection (e.g. persistent viral circulation on the index farm in Kinta, Perak). Similarly, if Nipah virus in Malaysia had resulted in human-to-human transmission, an alternative causal pathway could have produced incident human cases outside the area of transmission from flying foxes, as was seen in the Faridpur outbreak in Bangladesh in 2004 [34] , but there is no evidence that such human-to-human transmission occurred in Malaysia [35] . The causal pathway that was realized in Malaysia most probably involved (i) the creation of a pathway for transmission from bats to pigs via agricultural intensification (i.e. dual-use agriculture, or the practice of planting fruit trees on land used for livestock production, and increased fruit production through time) and (ii) repeated introduction of the virus into a high-turnover commercial pig population in Perak that led to viral persistence and set the stage for phase II of the emergence process. FF, flying fox. some years after the maturation of the mango trees owing to chance events within the system, e.g. fruit bat population dynamics, migratory behaviour or the dynamics of NiV in bat populations. This is supported by our own work on Hendra virus which suggests that, when the virus is actively transmitted within a bat colony, there is a heightened chance of repeated spillover, but circulation of the virus in bats is unlikely at any given place and time [36] . On the other hand, we cannot rule out the possibility that specific local conditions in late 1996 and early to mid-1997 caused increased viral shedding among bats in the area surrounding the index farm, thereby increasing the risk of cross-species transmission to pigs. Given the high mobility of flying foxes and the seasonal fluctuation in colony sizes in peninsular Malaysia [33] , NiV is probably transmitted regularly between roost sites and throughout the region. Such dynamics explain the ubiquity of NiV antibodies, and our data suggest that flying foxes and possibly NiV were regularly present near the index farm in Perak prior to the 1998 -1999 outbreak. Thus, we propose that it is plausible that flying foxes repeatedly introduced the virus onto the index farm in 1997.
Our analyses have shown that the initial introduction on the index farm was most probably insufficient to induce persistent circulation of the virus. Furthermore, the lack of human cases on other farms between the time that this epizootic would have burned out and the reappearance of human cases associated with the index farm strongly suggests that the virus was not circulating on farms in the surrounding area in the interim. The short duration of this initial epizootic appears to have provided an insufficient window of time for transportation of pigs between farms to spread the infection. On the other hand, reintroduction of the virus into the 'primed' pig population on the index farm provided a substantially longer window of opportunity for spread. Our model's results indicate that reintroduction of the virus at the time of the reappearance of human cases on the index farm would have permitted enzootic circulation of the virus until the farm was culled in 1999.
During this time, the infection spread to other farms in the Tambun area, probably via movement of infected pigs from the index farm, which supplied gilts and piglets to smaller operations in the vicinity. Subsequent pig movement (including 'fire sales' of pigs in reaction to the cluster of human cases in September-November 1998) allowed the virus to spread throughout Perak and eventually south to Negeri Sembilan and Selangor [15] . Because there was no evidence of transmission between humans [35] , the pattern of human infection necessarily followed the spread of the virus in pigs, explaining why human cases were much more widespread in 1998 -1999 than in 1997. In addition, the high density of pig farms in many parts of the south (electronic supplementary material) may have allowed respiratory transmission of the virus between adjacent farms without physical movement of infected pigs, contributing to the rapid spread of infection and high number of human cases in the south. On the other hand, these areas were buffered from phase I emergence via bat-to-pig transmission by the absence of flying foxes in the area [33] ( figure 4) and, possibly, a lesser degree of overlap between fruit and pig production (electronic supplementary material).
We have identified two synergistic component causes [37] that precipitated each phase of emergence ( figure 5) . The existence of a pathway for transmission from flying foxes to pigs was a necessary complement to viral circulation in the bat population to produce phase I of emergence, and viral persistence in the pig population on the index farm created an infection source outside the flying fox reservoir that, combined with transportation of pigs, was sufficient to produce phase II of emergence. Each of these component causes was brought about by a phenomenon that-while not strictly necessary-did ultimately permit emergence in this context. We refer to these factors as 'drivers' of emergence, and table 1 outlines the evidence for interpreting each of these drivers as a causal factor. Agricultural intensification (namely, dual use of agricultural land and increases in production) resulted in the direct overlap of mango production and livestock rearing and therefore produced a pathway for a virus circulating in flying foxes to infect an intensively managed commercial pig population, driving phase I of emergence. Because NiV had a very low probability of persisting on the index farm without reintroduction (figure 3), priming for persistence was necessary to bring about enzootic circulation in this context and appears to have been sufficient to do so, driving phase II of emergence.
This study illustrates how a broad, interdisciplinary approach to the study of emerging zoonoses focused on data from specific emergence events can illuminate emergence processes. We have demonstrated the specific role of agricultural expansion, particularly dual use of agricultural land and intensified pig production practices, in NiV emergence. A role for long-term changes in species interactions has previously been hypothesized for numerous host-jumping viral species including various primate retroviruses [38, 39] , SARS [40] and avian influenza viruses, and our evidence suggests that cross-species transmission of NiV most probably resulted from the expanded interface between wild animal reservoirs and human or domestic animal populations, although it is possible that short-term ecological conditions were also a contributing factor.
Our findings have important implications for control of NiV in commercial pig farms, prevention of widespread epidemics in livestock and our general understanding of zoonotic disease emergence. First, although restrictions on planting fruit trees near pigsties appear to have prevented introduction of the virus into pig populations in Malaysia since 1999, there is a possibility of reintroduction both there and in other countries where flying foxes are found. Regular surveillance of pigs sent to markets and abattoirs in areas where pig farming overlaps flying fox distributions, along with hospital-based surveillance of encephalitis cases, may permit identification of initial NiV introductions and allow for early intervention and prevention of subsequent spread. Second, widespread prophylactic vaccination of pig populations is likely to be cost-prohibitive because of the rapid turnover of commercial pig populations. In addition, if vaccine coverage were not kept at a sufficient level, the dynamics of the virus when introduced into the population would resemble those following reintroduction on the index farm, inducing long-term persistence and increasing the risk of phase II emergence (electronic supplementary material).
Finally, an important feature of the NiV outbreak in Malaysia and Singapore was the two-phase process by which emergence occurred, which implies that there was a missed opportunity for earlier recognition of this novel aetiological agent and potential intervention. Zoonotic pathogens often go unnoticed during the initial stages of 'viral chatter'-that is, repeated introductions that cause only a small number of cases [41] -because they are not yet easily transmissible in humans (e.g. simian retroviruses), are initially asymptomatic (e.g. HIV), are clinically similar to other diseases (e.g. NiV in Malaysia, which was originally misdiagnosed as Japanese encephalitis despite its epidemiological distinctiveness) and/or occur in areas with poor surveillance and diagnostic testing (e.g. NiV in Bangladesh and India). Other pathogens are noticed relatively quickly because they cause dramatic disease (e.g. Ebola haemorrhagic fever); however, even in these situations, we cannot rule out the possibility of previous undocumented cases, and the discovery of historical cases after the identification of an emerging pathogen is common (NiV [16] , SARS [42] , HIV [38] and H5N1 influenza [43] ).
These patterns support previous calls for increasing targeted global surveillance and pathogen discovery in atypical disease outbreaks [44, 45] . As the Malaysian NiV outbreak highlights, surveillance of livestock and livestock workers in regions of high wildlife biodiversity would improve the chances that viral chatter of wildlife origin is detected prior to a widespread epidemic in livestock or people. This approach may be critical for identifying new outbreaks of NiV, which has recently demonstrated a potential to produce propagated outbreaks in Bangladesh, where short chains of human-to-human transmission have occurred [34] . It may also provide a strategy for the identification of unknown henipaviruses, and other novel agents, prior to epidemic or pandemic emergence. 
In order to better understand the history, layout and daily operation of the index farm, J.R.C.P. conducted field interviews of farm workers, including two private veterinarians who oversaw the health of the animals, and the orchard manager, as well as government veterinarians who oversaw and participated in the depopulation campaign.
4.2.1. Dynamic models. Population dynamic models of production dynamics on the index farm were developed and parametrized from descriptions of farm-management practices provided during interviews with farm workers and detailed production records from 1 January 1995 to 31 October 1996. Models of infection dynamics were then constructed by building on these baseline population models. Dynamics were explored using a deterministic ordinary differential equation (ODE) model with a cutoff value of one infected individual (calculated as the sum of individuals in all exposed and infectious classes), below which the infection was considered to have gone extinct. A stochastic individual-based model (IBM) was then used to confirm that the qualitative dynamics observed in the ODE model were robust to the incorporation of empirically derived waiting time distributions and other factors that could not be represented in the ODE framework. The stochastic model was run for a wide range of parameter combinations to assess the probability of extinction following initial and subsequent introductions under different scenarios and to assess the range of transmission parameters consistent with observed seroprevalence levels in Breeding section 2. Detailed descriptions of the ODE model and the IBM are given in the electronic supplementary material.
Statistical analyses of production records were developed to detect evidence of the virus in the breeding sections. A baseline model for pre-weaning piglet mortality was formulated for each breeding section on the basis of production records from 1 January 1995 through 31 October 1996, and candidate models were compared by Akaike information criterion. For all litters born after this period, a piglet mortality index (PMI) was calculated that indicates the extent to which the observed mortality deviates from the expected mortality for a litter of the same size during the baseline period. Litter-level data are shown in the electronic supplementary material.
For the sake of interpreting large-scale patterns over time, litters were binned according to farrowing date. We established a baseline for variation across bins in the average PMI based on the period from 1 January 1995 to 31 October 1996. In figure 1b, the binned PMI values are scaled in terms of the binned PMI percentile (relative to variation within the parametrization period). Absolute values and further details of the analysis are shown in the electronic supplementary material. We collaborated with the federal wildlife department and enlisted a network of sport hunters as informants to locate and monitor flying fox roost locations throughout peninsular Malaysia. A full description of census techniques (and additional findings) has been published elsewhere [33] . We also developed two indices of flying fox density based on different expectations for how hunting practices reflect bat distribution. These indices and their underlying assumptions are described in the electronic supplementary material.
Flying foxes were non-randomly sampled using mist nets and anaesthetized. Three millilitres of blood was collected from the brachial vein and stored for 24 h at 48C to allow for serum separation. The separated serum was then stored at 2208C until use. All bats were released at the site of capture after recovery from anaesthesia. Serum neutralization tests were conducted on all serum samples at the Australian Animal Health Laboratory, Geelong, Australia, under Biosafety Level 4 conditions. Details on anaesthesia and testing protocols are given in the electronic supplementary material. P.D. conceived and directed the study; P.D. and J.R.C.P. cowrote the paper with assistance from J.H.E.; J.R.C.P. designed and conducted the modelling and statistical analyses with J.D. and A.P.D., and conducted all fieldwork related to livestock; J.H.E. and S.A.R. led the bat survey work, and S.A.R. conducted some of the sample testing. M.B. collected field data on pig production during the NiV outbreak investigation. A.A.J. facilitated data acquisition and all field activities in Malaysia. A.D.H. directed sample testing. H.E.F. assisted in study design, helped direct field activities and provided veterinary input. All authors were involved in the design of the study and the interpretation of the results and commented on the manuscript. All other Do expert assessments converge? An exploratory case study of evaluating and managing a blood supply risk BACKGROUND: Examining professional assessments of a blood product recall/withdrawal and its implications for risk and public health, the paper introduces ideas about perceptions of minimal risk and its management. It also describes the context of publicly funded blood transfusion in Canada and the withdrawal event that is the basis of this study. METHODS: Interviews with 45 experts from administration, medicine, blood supply, laboratory services and risk assessment took place using a multi-level sampling framework in the aftermath of the recall. These experts either directly dealt with the withdrawal or were involved in the management of the blood supply at the national level. Data from these interviews were coded in NVivo for analysis and interpretation. Analytically, data were interpreted to derive typifications to relate interview responses to risk management heuristics. RESULTS: While all those interviewed agreed on the importance of patient safety, differences in the ways in which the risk was contextualized and explicated were discerned. Risk was seen in terms of patient safety, liability or precaution. These different risk logics are illustrated by selected quotations. CONCLUSIONS: Expert assessments did not fully converge and it is possible that these different risk logics and discourses may affect the risk management process more generally, although not necessarily in a negative way. Patient safety is not to be compromised but management of blood risk in publicly funded systems may vary. We suggest ways of managing blood risk using formal and safety case approaches. Blood is a special product, being integral to life and inside our bodies. As Chan [1] notes (following Titmuss [2] ), there is much symbolism associated with blood. As a medical solution to a health problem it depends on the gift of others. Its gift or donation is altruistic and seen as sharing what is essential to life. In Christian societies, blood is seen as having the ability to wash away sins [3] . It represents purity, intensified by its gift to others. Blood donation is a very visible connection to others, even in societies where payments are made to donors [4] . These payments remain controversial and may lead to compromises in the safety of blood products. Blood donation is indeed a two-edged sword: a lifesaver or a transmitter of disease. The struggle over successful compatibility of blood types and to combat transmissible diseases has been a long but largely successful story [5] . The hazards in receiving transfused blood were heightened by HIV/AIDS, which as Chan [1] argues, led to the blood risk being intensified by this stigmatising illness. But since the mid-1980s, screening tests for sexually transmitted infections, including HIV and, later, other transmissible diseases have been implemented in many countries worldwide. In fact, scientific risk assessments, the vigilance of existing or new federal agencies, enhanced public information and disclosure have greatly improved both the calculated and perceived risk of 'bad blood'. As we shall see, Canada is no exception but it is a jurisdiction where there is particular salience.
Blood transfusions are overwhelmingly safe, with about 0.5 to 3 percent of all transfusions resulting in adverse consequences [6] . A proportion of these adverse events result from error in preparation and administration. Other adverse events are classified as infectious (e. g. HIV/AIDS, hepatitis, human T-Cell lymphotropic virus (HTLV), West Nile virus) or non-infectious (these are commoner and include acute haemolytic reaction, transfusion associated acute lung injury, allergic reactions, graft-host disease). While severe non-infectious complications are rare, case fatality is high. Infectious disease risks associated with blood transfusion in Canada are currently estimated at 1 per 7.8 million units transfused for HIV, 1 per 2.3 million for hepatitis C, and 1 per 153,000 for hepatitis B [7] . For manufactured plasma-derived products, the risk is estimated at less than 1 in 10 million or theoretical [8] . Bacterial infection from contaminated blood products is also possible but routine bacterial screening of high risk products (platelets) and changes in blood collection techniques have reduced this risk significantly. Methods to inactivate pathogens in blood products (those currently known and future threats), have been developed for plasma and platelets and methods applicable to red cells are under development. Canadian blood suppliers are currently exploring the feasibility of implementing these new technologies once they are available [9] . So despite public concern (see Lee [10] ), experts would surely see risk as minimal and theoretical. And they do. But this does not prevent every 'unsafe' blood incident from being rigorously pursued. But this assumes that all experts may have the same goals or different pathways to similar goals which may shape the treatment of the hazard differently. In part they do -the safety of the product and public health. But do different groups of experts have other goals or interpretations which may shape the treatment of hazard in specific ways? This is the broad question explored in this paper, the major contribution of which is to examine the divergence of expert opinion even where science is consistent and the goal universal.
To answer our question, a constructivist approach is adopted. Assessing and managing all kinds of risk is a challenging task but this is especially the case in those risks pertaining to human health which are largely dealt with in the public domain. Because these concerns do invoke public discourse, much attention has been paid to the differences, if any, between expert and public perceptions and assessments [11] . For twenty years or more, constructivist analyses have brought to the fore the importance of prior social, cultural, institutional and political factors that shape and are embedded in both lay and expert risk assessments (see [12] ). Lay people and experts use similar cognitive processes with both falling prey to errors from anchoring, overconfidence and the gambler's fallacy (see [13] ) Attention has also turned to the perceptions and assessments of experts themselves. Van Zwanenberg and Millstone [12] comment that the construction of scientific claims can inform risk assessments. Furthermore, a "coherent realm of expertise" may not exist, as Haggerty [14:201] points out with respect to crime risk. He adds that expert opinion about crime risk is incredibly fractured with there being differences in professional opinion with respect to dimensions of high-profile risks as well as the nature and efficiency of crime prevention. Sjoberg [15] also points out that there is likely to be a whole range of expertise and this range is not well-articulated in many risk perception studies. If, therefore, there is this range of expertise, why should we expect expert opinions to converge, although overconfidence in technical and medical assessments may exist (but see [16] )? In fact, we shall note certain biases in expert opinion.
Shanteau [17] questions the convergence of expert opinion which is based on the widespread use of statistics and economics in assessments and on the widespread search for generalizability in how people think about risks. Shanteau argues then that the bases of convergence are in themselves flawed (see also [18] [19] [20] ). Of course, it is recognised that different risk logics or discourses -i.e. different conceptions of an activity or event as risk -exist, but these have seldom been applied to expert opinion (see [21, 22] ). Silva et al. [23] show, in an experimental setting, that despite differences in scientific background and political beliefs, scientists tend towards a precautionary stance over the setting of safety standards. Yet scientists are not the only type of expert involved in such matters. McMahan et al., [24] point to the differences in how scientists and risk assessors regard electric and magnetic fields. Furthermore, Chalmers et al. [25] show how the opinions of dentists and nursing directors vary over dental care in nursing homes. Chociolko [26] provides an example of expert disagreement often found in sometimes adversarial settings in which experts 'take sides' representing, say, industry or community.
How might we assess expert opinion and convergence on risky matters? Shanteau [17] points to the importance of the level of decision, made by experts using a medical analogy of diagnosis (what is it?), prognosis (what is the likely outcome?) and treatment (what to do about it?), with a different logic applied on different levels. Furthermore, experts and professionals possess similar cognitive properties as non-experts. "Experts are not immune to the cognitive illusions that affect other people" [27] . And it is increasingly noted that cognition and emotion are intertwined in decision-making (see [28] ). As Cross [29:28] comments: "when an attitude or an activity is of considerable importance to a person, the individual is loathe to believe that it is hazardous." Furthermore, biases or heuristics found in studies of perceived risk may be found among experts and affect their assessments and opinions. In this paper, we address some of these issues about expert opinions, utilising the ideas of non-convergence (especially at different levels of an assessment), and of the heuristics made by different professional groups (commitment to a particular practice, reliance on rational models and thinking), to understand different concepts of risk relating to the blood supply arising from a case when supply of 'safe blood' was potentially disrupted in one Canadian city. So all may want 'safe blood' but do the management and decision-making styles and discourses of all actors converge or vary?
The Context and the Case 'Safe blood' has been a major policy issue in Canada. This is also a sensitive issue, which likely arises from the 'tainted blood tragedy' of the 1970s and 1980s. At that time, the blood supply was organised and managed by the Canadian Red Cross Society (CRCS) as this body had been responsible for blood supply during the Second World War. CRCS is a not-for-profit, humanitarian society, with many diverse activities extending beyond the responsibility for the blood supply which are largely carried out by volunteers. A chronic lack of funding for the CRCS held back technical developments to collect and supply blood in Canada. Government funding only began to increase in the mid-1970s as procedures for blood collection became modernised. But its principles as part of the international Red Cross (non-discrimination against individuals and independence from government) meant its screening and management procedures became fatally flawed in the Canadian context, with often strained relations between volunteer oversight and professional activities. These limitations set the stage for tragedy with the discovery of blood-borne diseases as recruitment of donors was still in volunteer hands and carried out with self-answered questionnaires. The recipe for disaster was set, and the situation was worsened by the failure to track those who had received tainted blood and a failure to apologise or compensate affected individuals on the parts of the provincial and federal governments (see [30, 31] ). As Picard [30] has noted, 'tainted blood' was arguably the largest public health catastrophe in Canada. "About 1,000 individuals who received blood transfusions in Canada between the late 1970s and 1980s were infected with HIV and another 30,000 were infected with hepatitis C" [32] . Eventually, this led to the establishment of a public inquiry in 1993 with the final report of the Krever Commission being delivered in 1997 [31] . Krever recommended the creation of a new blood system and the Commission's recommendations led to the creation of Canadian Blood Services to operate the system in all provinces, except Quebec which formed its own agency, Héma-Québec. With the creation of these agencies, the Canadian blood system joined the U.S. and West European countries in having an expert-based and scientifically-based system and CRCS went on to different humanitarian tasks.
For the blood system and perhaps other aspects of the Canadian healthcare system, Krever highlighted two key elements -the institution of precautionary measures and the creation of a governance system emphasising safety (see [32] ). Risk assessment became based on scientific tests and evidence and the management system highly coupled and structured to protect human health. The Canadian blood system is currently seen as a high reliability organisation, emphasising safety and responsiveness to problems [33] . In fact, these elements were introduced so the Canadian blood system could respond to threats in a timely and effective way. This was the case for threats from infectious agents with product recall or donor deferral being rapidly instituted for variant Creutzfeldt-Jacob disease (See [33, 34] ), West Nile virus (see [35] ) and SARS. It is also the case for 'organisational threats' in which practice errors over labelling, documentation, or other deviations from procedures are dealt with through product withdrawal. For such threats, it may be said that the Krever report is one dimension in the increasing use of quality assurance for among other things patient safety in health care. But we know from other settings that challenges occur and 'accidents' (often resulting from operator error or technical malfunction) are normal in complex systems [36] . It seems therefore worth discovering if in this highly coupled, apparently well-managed system, all experts agree about the nature of arising hazards.
Recalls involving small numbers of blood products occur frequently (daily), for example, when donors provide post-donation information that affects their eligibility as a blood donor. Whereas withdrawals, which occur less frequently, are often due to operational deviations where the risk is minimal or unknown and may involve a large number of blood products. Withdrawal involving a large number of blood products can jeopardize the availability of an adequate blood supply.
When there is a real or perceived threat to the safety of the blood supply, withdrawals occur without reference to cost or potential short-term shortages. A major withdrawal of blood products in Ontario in 2005 involved over 3500 blood product components. The reason for this withdrawal was clerical in nature associated with records of donation which are completed at the time each donor gives blood. The risk to the blood supply was likely minimal or nonexistent. Individuals in the appropriate organizations joined forces to deal with the matter. But it brought to light many of the challenges faced by both hospitals and the blood suppliers around dealing with error and emphasized gaps in the system related to timeliness; communication; risk perception; and recipient notification. It provides an example of what we call organizational 'threat' or, more positively, a learning opportunity. We suggest that this approach to identifying error as a potential for hazard and risk is useful in that it focuses attention on possible differences in organizational responses and public notification.
So given the context, it would appear that the case could have been dealt with procedurally with all parties -regulators, blood product supplier, hospitals and treatment facilities and agents -being on the same page. The risk from the blood product should have been seen in a universal way by all parties involved in this particular case. But was it? If there are different levels of decisionmaking and risk assumptions do expert assessments necessarily converge? The answer, to anticipate, is yes and no.
This paper is an outgrowth of a study undertaken to determine the current procedures and protocols for handling the recall/withdrawal of blood products in Ontario and beyond [37, 38] . In this we adopt a quasifoundational position, with the experiences and views of respondents being seen in the answers to our questions (see Additional File 1: Appendix 1) from which exemplars in the form of quotations are drawn (see [39] ). We undertook a number of one-on-one interviews with key stakeholders in the blood supply and management arena, both within and beyond Ontario. Interviews were conducted with individuals from Canadian Blood Services, Héma-Québec, Ministry of Health and Long Term Care (Ontario), relevant staff at hospitals and hospital laboratories, as well as blood product recipients. These recipients were not professional or credentialized experts but it was felt that their experience of the blood transfusion system might add a different perspective to our investigation. Within hospitals, we interviewed physicians (both transfusion medicine physicians and physicians who do not work in the transfusion service but who use blood products), hospital administrators, CEOs, risk managers and public relations personnel. Within hospital laboratories, we interviewed managers, technologists and transfusion safety officers. In other words, we interviewed in total 45 professionals involved in blood supply -regulators, suppliers, treatment individuals and agencies, administrators, laboratory personnel and transfusion recipients. The project's steering committee (composed of individuals from the blood supplier, hospitals and laboratories) helped to identify individuals from the blood supply and management arena who had experience of blood product recalls and withdrawals and who could therefore be anticipated to be able to provide rich data. This purposeful sampling was augmented by asking those individuals interviewed to suggest others whom they thought it would be helpful to interview. We employed a multilevel sampling framework in that we wished to compare the perceptions of those with potentially different stances on blood risk management (see [40] ). We recognise that the sample size in our study limits the generalisability of our findings and interpretations, and it may indeed be exploratory with our practice suggestions being essentially that-suggestive. Table 1 shows the number of participants interviewed within each category. As well as interviews, copies of written relevant rules and procedures pertaining to recalls/withdrawals were obtained. And we may askdoes opinion converge on the type and nature of the risk?
We also planned to conduct interviews with individuals from the Ontario Hospital Association and from Health Canada (the regulator of the Canadian blood system). It became clear while talking to individuals from the Ontario Hospital Association that the association does not currently play a role in blood product recalls/withdrawals within the province. Health Canada, despite being asked on several occasions, declined to participate in the project, having been advised by their legal department not to do so in order to avoid the possibility of liability issues. Recipients of blood products 2
Note: Numbers in the text after job title refer to individuals in that category interviewed.
In order to obtain as comprehensive an understanding as possible of the recall/withdrawal process across the province of Ontario, we made sure that the different types of hospitals were represented (large urban teaching hospitals, smaller urban hospitals and rural hospitals). We also interviewed individuals from Héma-Québec, to get a sense of how the process compares between blood suppliers and key informants from other provinces (Alberta, British Columbia, Nova Scotia, and Saskatchewan).
The protocol was approved by the Research Ethics Board of McMaster University. The interview guide included questions on understanding the terminology involved in recall/withdrawal situations; individuals' experiences of these situations and the actions taken by different individuals and or stakeholders at different stages in the process; existing policies and procedures; questions related to disclosure of information and notification of recipients, including who should be involved in this process and how it should be done. Respondents were also asked for any suggestions they might have on improving the process (see Additional File 1: Appendix 1).
Interviews were conducted over a four month period from May to August 2006, either in person or by telephone. The interviews were conducted by two of the research team members (EA and BMcC). To maximise consistency of questioning, a semi-structured interview guide was followed in each of the interviews. The questions were open-ended and interviews lasted between 45 minutes to 1 hour. All interviews were audio-taped and then fully transcribed. The transcriptions were checked for accuracy against the taperecordings and then imported into NVivo 7, a qualitative data management software program commonly used in qualitative research [41] .
A team analysis approach was employed whereby as transcripts became available, they were read independently by several members of the research team (EA, BMcC, and JE). The team then met at regular intervals to discuss the content of interviews and to identify the themes and issues emerging from the data. A schematic for coding the data (identifying discrete passages of text that contain the same idea) was developed by EA and BMcC and then this coding scheme was applied to a new batch of interviews by EA, BMcC and JE. Interrater agreement was then calculated and was found to be very high (100% for major codes and 94.3% for minor codes) thus indicating that the coding scheme was working well. This schematic was then used to code the entire data set by BMcC, so that all interviews were coded using the same coding themes. This schematic acted as a taxonomy, classifying and organizing the complexity of the 45 responses and there is a close parallel between the taxonomy (reported in [38] ) and the interview guide. Further, deeper coding of the data generated other propositions, which may be seen as second order constructs (see [42] [43] [44] ). The analytical development of these constructs allowed relationships to be identified between the codes in the taxonomy (see [45] ). These also emerged because of our interest in blood risk management. Thus the issues of notification, response to minimal risk, responsibility and legal requirement are transformed into those appearing in the results section, guided by themes and theories of risk management and expert judgement outlined above, i.e. the heuristics and logics used to deal with uncertainty and to manage risk for protective, liability and precautionary reasons. In presenting the findings we have selected quotes that illustrate these ideas, allowing as many respondents as possible to speak. We suggest, therefore, that our paper makes a methodological contribution by using the typifications of social phenomenology to transform respondents' concerns into specific risk discourses.
As a foreshadowing to outline different conceptions of how blood product risk should be managed, we note confusion among respondents over the meaning of the terms 'recall' and 'withdrawal'. There is meant to be clarity in:
"With respect to a health product, other than a medical device, means a responsible party's removal from further distribution or use, or correction, of a distributed product that presents a risk to the health of customers or violates legislation administered by Health Products and Food Branch Inspectorate (HPFBI)" [46] .
Withdrawal "The removal from further distribution or use, or correction of a distributed product where there is no health and safety risk and no contravention of the legislation administered by the HPFBI. It is not considered to be a recall" [46] .
Yet only 14/45 (30%) of individuals interviewed indicated that they knew the difference between the two terms. The difference between the terms is confusing for both hospital and blood supplier personnel:
"I think they are confusing. The only reason, I'll be honest; the only reason that I'm familiar with it is because of the incident that we went through ..." (Laboratory Manager-01) "Well ... it's very confusing. We are not exactly sure when they recall something or withdraw something. It's, you never know why they are asking you to return something. And they don't give you any extra information, and it is somewhat frustrating, because you know why are they doing this?" (Lab Technologist-06) "Um... gosh... there probably is a very important difference and I ... I would be lying to you if I said with confidence that I could tell you the difference" (Physician using blood products-01)
The confusion is mainly associated with diagnosis (what is it?) and does not appear to influence the action (what is to be done?) taken to deal with the recall or withdrawal notification at the hospital level. Regardless of whether a recall or withdrawal is issued, the initial action taken by the hospital Transfusion Service is the same: implicated products are removed from useable inventory. So would uncertainty be removed if one term was applied?
"I don't think they should be handled differently. You know if for whatever reason a product is being taken off the market, it would, it's unfortunate that you have two terms where again they have different connotations. I think one term should suffice for all and... they should be handled in the same way." (Physician Blood Bank-03) "Well I guess the fact that we have so much trouble remembering which is which... could be problematic. I think for the regulator they do require. I mean it's important to have two different terms... with two different definitions because they, they are two different matters. In practice that we get the two terms mixed up, I'm not sure it really matters..." (Blood Supplier-07)
While there were differences in labelling what was happening, there were none in terms of prognosis (likely outcome), seen universally as the removal of unsafe product. In other words, expert opinion converges with respect to the goal -safe blood -but on how and why this might be done there is divergence, thus revealing differences in management perspectives. We identify three approaches to managing the risk from this organisational threat: risk as hazard, risk as liability and risk and precaution.
Risk as hazard emphasises the potential adverse consequences to patient well-being. Such an approach demands immediate removal and the full disclosure of what is happening to patients. Furthermore, it suggests that those notifying patients should be as close to the patients as possible, usually the treating or transfusing physician. This risk is almost a given and most comments refer to the importance of physician notification to allay any or all fears about hazard, the physician being seen as trusted, knowledgeable and close to the patient. There is agreement on who is central in this immediate task of ensuring blood safety, the blood supplier, but different stakeholders hold the risk as hazard view for different reasons. The blood supplier itself sees a distinctive chain of responsibility and action if blood may be unsafe -from themselves to the physician to the patient.
"... you know we are in this era of informing the patient, but I think to some extent the pendulum can go too far ... and that we need to be careful about not giving patients information that's no of value... I think we can overdo some of the informing of patients, it's almost like we're passing the buck and not kind of letting the responsibility stop somewhere along the way with a physician..." (Blood Supplier-07)
This discussion on the need to notify seems supported by all stakeholders. Hospitals see that any notification from the blood supplier highlights a concern and the need for action.
"Now, I would anticipate that if the blood... supplier... was concerned enough to notify us as an organization to recall a product, then the degree of risk is always such that it would be important for us to notify the patient. You know what I mean. Like, the risk assessment has already taken place at the blood supplier" (Hospital-Risk Management-02) "I guess from my perspective there is either risk or there is no risk, and if there's no risk they're not notifying us. If they're notifying us it's because there is risk" (Hospital-PR-01) Physicians tend to agree but see themselves and are seen as those best positioned to make patients aware of potential problems as 'they know' their patients best.
"In my opinion, it's the role of the clinician who's caring for the patient to manage those things" (Physician using blood products-05) "It's useful to have a well-informed recommendation from the Blood Supplier but the hospital always has to use its discretion and the difference there is that we know our patient population" (Physician using blood products-07) "I think ultimately the physician always has that, you know, discretion. And that's clear in case law" (Blood Supplier-04).
Others feel that the physician may not have all the skills necessary to manage risk as hazard, especially if their style is based on a confident, medical approach to the issue.
"So I would be perfectly supportive, of a hospital system or provincial system or even a nation-wide system that developed advisory guidelines. But I don't think it's a situation where if it is ... that a physician doesn't want to notify her patients that, that somehow some other agency needs to get involved and compel that notification" (Physician-Blood Bank-03) "I don't know whether there would be a place for a third party to come in, where a third party would contact the recipient and say 'I know that your physician spoke with you, or that you received a letter, this is just a reminder that you might need to go for further testing'" (Laboratory Technician-04) "Well I don't know if everybody has them but a patient representative would be one option. I think somebody with those sets of skills. Like not just the technical skills but the people with counselling skills and discussion. I mean they'd have to have some kind of knowledge about blood and risk" (Provincial Health Ministry-02) Risk as hazard was the best articulated of the discourses on how to manage. Yet risk as liability, where the potential consequences for system integrity of specific practices were central, received greater expression from respondents. Patient well-being is of course still vital but now disclosure to patients of events that might affect their wellbeing protects the blood supply institution as well. Such management is seen particularly at the supplier and hospital levels. The identification of the risk and its disclosure were closely related. But under many comments lies an often implicitly stated concern over liability, i.e. what is our liability if we do not act (disclose) and something happens? In other words, their responses are anchored around current operational characteristics of the healthcare system and their legally demarcated roles within that system.
Liability is often framed in terms of outside perceptions, specifically and not unusually, on how public perceptions of expertise and expert response may be framed. In this discourse, the role of the blood supplier is central but responses to their notices may vary depending on how others see the issue and their liability. The blood supplier must always give an opinion.
"We [Blood Supplier] would always give our opinion as to follow up." (Blood Supplier-01)
The importance of local circumstances and their possible impact on liability are recognised by the blood supplier. It must balance its obligations with that of the independence (and discretionary action) of institutions such as hospitals and doctors.
"What we do in our centre is... we take a look at the reason for the recall, we may provide that information. The hospital doesn't do much with it, and they know they'll get a supplementary letter from me if I think recipient notification is required so that decision whether recipient notification is required is made here. I think that ... works better in our environment because I have more experience in this than the regional lab tech ... and we don't have experienced blood bank directors in most of the blood banks." (Blood Supplier-02) Yet circumstances at the local level may affect response to this opinion.
"But the Blood Supplier has their own ideas about what, in what situations do recipients need to be notified so they advise us. In our opinion, you do or do not have to notify the recipient if this product has been transfused already and we sometimes do what the Blood Supplier asks, although we're a little more aggressive about notifying patients than what the Blood Supplier does." (Physician using blood products-01). "I think you know, some hospitals are in a position where they absolutely must rely on the expertise of the Blood Supplier. I mean, they, you know, primary care hospitals that I assume are somewhat more comfortable just saying, 'Look, just tell us what you want to do. Tell us what you want to say and we're not gonna do any independent analysis. Just, you know, provide us with what your recommendation is.' Other hospitals are saying, 'okay Blood Supplier, thanks for the information. We're gonna consider this independently. We have the expertise to do so, you know. The only thing we want you to do is provide as much information as you can on the risk and we're all consider it at our Transfusion Committee and we'll all decide ultimately what we think our physicians should do in terms of patient notification.' ... that kind of thing. You know, so there's quite a difference. So that is a part that we're struggling with a little bit in terms of how do we fulfill our obligations? We never want to be, we never want to fetter anyone's discretion in terms of notification. That's for sure. Though part of us is saying, you know, part of the time we think, okay, we can provide all of the, all of the information, all of the risk information as clearly as we can, and that's it and then the hospital can sort of make their decisions. But there's another component then because we don't want to be in a position of... we don't want to abdicate our responsibility and thrust the decision making on hospitals, you know, so there's sort of a tension there between those two" (Blood Supplier-04).
The hospital is perhaps in a position of having to respond to local pressures before the full facts are known because they are local institutions and have explicit liability concerns.
"I believe in full disclosure even if all you can say is here's what we think happened, here's why it happened, here's what we know and don't know, and here's a mechanism for either monitoring or what have you down the road. So in the absence of that, in the event that there's some new novel research finding in the not so distant future, I've never been told about this, I didn't know I had exposure, some marvellous new technology or technique comes along that would perhaps allow me to be more definitive. Well I don't even know about it to pursue that or include it in my own medical history" (Hospital Adminstrator-02)
While some groups which are closest to the patient that is being transfused with blood see most recalls as leading to minimal risk, perhaps displaying an overconfidence in the role of scientific assessment, others are concerned about outside perceptions and what these might do to the situation.
" ... we couldn't identify what the risks were and the risks were minimal, therefore we should not disclose, and [the Chief of Governance] was adamant that we needed to ... I was really surprised that the recommendation by the Transfusion Committee was totally disregarded" (Transfusion Safety Officer-01)
Waiting for an assessment can lead to much time thinking through the issue. This may result in an affect response, especially with respect to the media. This may be exacerbated by public attention being roused before a system-based announcement can be made.
"And sometimes we've got no, we don't have an assessment yet... so we're sitting ... waiting, you know and there's a time delay there so... my biggest fear is in the meantime it hits the media, now we've got an issue" (Laboratory Manager-03) "But the problem is, is that you have media that's watching, and so then, then there's a different spin put on this when it hits the newspaper. You know... another bloody scandal. So are you more concerned about your public relations or are you more concerned about the effect you're going to have on patient care?" (Laboratory Manager-01)
Much of the liability discourse takes into account the perceptions and roles of other stakeholders, both inside and outside the blood supply system. The third discourse -risk as precaution -does that too but also seems to treat problems on a case-by-case basis as uncertainty cannot be fully removed or explained. It is a view of risk often held and articulated by system administrators. A precautionary approach has been based on a changed perspective toward patients and institutional/ professional partners. And precaution -taking care -is necessary as interests and ways to achieve them may be dissimilar. The blood supplier sees precaution as necessary because of these dissimilarities.
"I don't know how much a hospital's disclosure policy would vary from one hospital to another. I mean one would hope that there's some uniformity in that or else disclosure of things is going to be a problem for very much more than recalls and withdrawals because I think there's all kind of things in a hospital that one might decided you disclose or you don't disclose" (Blood Supplier-07) "I'm not looking at it in the capacity of disclosure and our disclosure policy and for us it's not as simple as just telling people something went wrong. We have to ... weigh out what the risk of telling someone versus the benefit of telling someone ... we very much are very strong proponents of disclosure and do it unless the risk of advising is significantly worse than not we certainly learn towards advising patients" (Hospital Administrator-03) Furthermore, the blood supplier argues for precaution because of the need to respect the different needs and sensibilities of different types of patient.
"Because right now, the decision whether to inform a patient is based upon the doctors and the doctors in hospitals alone. The patient has no input whatsoever.... now the CJD thing was a perfect example of some people probably didn't want to know all the recalls, because what good could this have done. But some people did. So that there has to be a choice made by the patients... and my view and most patients' view on that is that they have no business making that decision for patients. Now, it's just the whole attitude of the health system, it's not anyone's particular fault, but it's just the attitude of you know, it's your responsibility and we'll make the decision. I think patients feel you know a little ticked with that" (Transfusion Recipient-02) Often, agencies lack information about these patients: the recipients of the transfused products.
"We only have part of the story when we do these recalls and withdrawals which is the information from our end about the donor or about the component but we don't know anything about the recipient at the other end. And the importance of the information and about what should be done has to be determined in the context of the recipient" (Blood Supplier-03) "I know our medical staff has a problem with the word recommendation because, I think it centres around the fact that they are dealing with facts about the unit and have no facts about the patient. Certainly, if you go to the level of individual patients, you can, I think justify different actions because of the different situations of patients" (Blood Supplier-01)
Precaution on the part of local system administrators is weighed not only with respect to hazard but also institutional and patient autonomy.
"For us it's not as simple as just telling people that something went wrong. We have to, we like to, weigh out what the risk of telling someone versus the benefit of telling someone ... so I think there needs to be a way to bring hospitals together and recognising the hospitals are independent and are free to make their own choices to the extent that that can be coordinated goes a long way because ... we have to have a plan B that while we didn't think disclosure was appropriate, in the event that another hospital chose to disclose, we had to be prepared for how we would respond to that" (Hospital Adminstrator-03) "It's a very paternalistic approach to say 'oh well we know it's bad for you and so you know we want to spare you the pain of having to you now think about things like this so just let us do it.' That has not worked well in the past and we are a sort of newer generation of people where the attitude has changed... So, give the sort of transition in the social more that is out there.... I think trying to take that paternalistic approach that we will hide things from you for your own best interest it just won't sell" (Transfusion Recipient-01)
In managing the possible consequences of this error, a quite minor, almost theoretical, risk to the safety of the blood supply, there was uncertainty about what was being managed -a recall or withdrawal. This has now been resolved in part through the authors' report (see [38] ). The term recall is now used. Clarity in identifying responses to a possible hazard is necessary. But the then uncertainty around terminology did not stop the problem from being tackled but it caused pause for thought. We note too in this intensely regulated, safetyfirst environment that conventional risk discourse -as hazard -and management as its removal or mitigation did not loom large. It is a given in this tightly knit and collegial community that patient safety is paramount. There appears to be significant levels of trust between the different groups of professionals, something not always found in hazard management settings (see [47] ). This trust and respect provide an excellent basis for existing and enhanced communication about blood hazards in these groups. Communication between parties with respect to the issue, what it means, and what responses are possible is key. It remains important to remember that there may be differences in approach to, say, discourse, or treatment and some adjustments may be required. And while the discourses are ones of engagement in this case, that does not necessarily mean that there will not be adversarial interactions over preferred management strategies and rationales, especially in the importance given to patient notification. (See [48] for a discussion of expertise and collaboration). This need for disclosure may in fact lead to a further risk management challenge. In our case, there was discussion among risk managers about risk amplification through disclosure. Research [49] has certainly shown the importance of the physician communicating risk issues to the public.
Risk as liability views system integrity and maintenance as an important goal. Management entails not only the use of knowledge about patient safety and system practice but also a cognitive and emotional commitment to the aims and goals of the organisation. The supplier agency has created that commitment and loyalty within a changing Canadian health care system (see [37] ). Yet uncertainty remains and this may be due to the centrality of precautionary principles in the blood supply system. Thus, it is not surprising that risk and precaution are seen simultaneously as two dimensions of hazard management. Good record keeping and monitoring of transfused blood (and its recipients) enables precaution to be central in risk management. Kaplan et al [50] advocate a medical event reporting system for transfusion and we concur with their suggestions. Such a system (Transfusion Error Surveillance System -TESS) is currently being developed and piloted in Canada [9] . Furthermore, the difference discourses in expert groups within the same decision-making system (but likely at different levels of decision-making) may be beneficial, if recognised as such. 'Hazard' is a safety-first approach protective of health; 'liability' ensures that the challenges of risk amplification and perceived injury may be dealt with if an unknown risk is disclosed and 'precaution' ensures due diligence and quality assurance before action is taken.
With respect to the three risk discourses, some are more likely to be articulated by some professional groups -hazard by physicians, liability by administrators (hospital and blood supplier), precaution by administrators (especially the blood supplier). Those groups are largely responsible for managing the risk from these respective vantage points. There is largely a coherence of expertise within those realms, although it is in part challenged by laboratory technicians (hazard and liability) and transfusion recipients (liability and precaution). A vital strategy in managing any risk or consequences of error is a coherent response. This coherence may break down as different dimensions of risk management are required (patient safety, system maintenance) or different professional and lay groups engaged. In fact, it is possible for different discourses to be used by one or more different groups. In all discourses, it is possible for different heuristics to guide management response, overconfidence with respect to medical expertise and the scientific assessment of risk (physicians and blood supplier), anchoring to the legal and formal positions of institutions (hospitals), and affect with respect to feelings about uncoordinated public announcement of a risk and perceived media response (laboratory technicians and transfusion staff). The blood supplier uses all discourses because of its variable role in supplying safe products.
In adopting a quasi-foundational approach, there are ways to ensure rigour and the trustworthiness of interpretations. The social phenomenology of Schutz, used to derive themes and as a basis for theory development from respondent perceptions, requires meeting three postulates, namely logical consistency, subjective interpretation and adequacy. For the first, we have highlighted how the research problem and methods were derived from a real world problem on which was based the questionnaire, sampling strategy and the need to interview; for the second, by using respondents' views to develop interpretation (different than most studies as the first order or naturalistic constructs are expert ones); and for the third, by linking second order constructs to activities and phenomena in blood risk management. Furthermore, trustworthiness can be demonstrated by credibility and confirmability (see [51, 52] for practice-based examples). We have provided a trail from problem identification, questionnaire development, coding taxonomies to themes and theoretical development. We have also provided the rationale of respondent selection and sampling design as well as an outline of procedures followed to arrive at constructs or themes. We have derived through these themes risk discourses and logics relevant to blood risk management. Before finalization, the study results and recommendations were presented at a consensus conference of study participants and stakeholders, to ensure validity of data interpretation. All suggestions made were incorporated into the final results. Although we recognize that all researchers will not necessarily agree to the ways we have sought validity for these findings (see [53] ), first author conversations with risk managers point to the utility of the interpretations. Thus this qualitative investigation has contributed a nuanced risk characterization.
In fact, all discourses are necessary to manage this low-level risk. This coherence and differentiation of expertise around managing blood risk has practical consequences. As Hunt [54] notes, governance of risk is characterised by risk avoidance rather than risk management and is in turn dominated by a preoccupation with safety. The further corollary is the expanding panoply of regulation and guidelines. This may be noted in blood supply. And when 'something happens'-a threat to safety-the virtuous cycle of risk, regulation, and prescription is interrupted. The relentless pressure for the systemisation and integration of risk management practices continues as a watchword for corporate social responsibility (see [55] ). This virtuous cycle is reinforced by the use of a precautionary logic. As Haggerty [14] , notes, precaution emphasises the worst eventualities. It is not so much about risk. It "invites one to anticipate what one does not yet know, to take into account data, hypothesis and simple suspicions" [56:288] . With a product as vital and special as transfused blood, precaution is necessary. But societally it may feed anxieties and increase risk aversion. Practically, we point to consideration being given to enterprise risk management (ERM), recently developed as ISO 31000 (see [57] ) and accepted by the Canadian Standards Association in 2010.. ERM considers any risks or uncertainties affecting objectives, requires a flexible organization to tailor risk management, formalising monitoring review and consultation, and demands accountability from all those dealing with the risk. All senior managers must be committed to the process which must be used by all decision-makers in a flexible organizational structure. In its stages, it considers risk in careful ways. In setting the context and identifying risk, it suggests consideration of risk appetite and triggers. In analyzing and treating risk, ERM points towards acceptance, control and mitigation. It suggests ensuring that residual risk and its potential impacts are not ignored as it is not possible to remove all uncertainty. ISO 31000 may be complemented by using a safety-case approach which requires the incorporation of all evidence to ensure the system is safe to operate (see [58] , potentially modified to permit inputs from all appropriate stakeholders to organize heterogeneous information and concerns to ensure the safety and dependability of a larger network (i.e. the blood system).
Elements of the blood management system have been drawn to ISO 31000. Furthermore, processes such as recording, inter-professional communication, notification and disclosure have been applied in dealing with blood risk. Risk triggers and residual risk point to the relevance of hazard and liability discourses, especially as in other domains, use of the precautionary principle has been shown to trigger concerns and lower trust in governance structures (see [59] ). So for the institution of precaution we must be clear on what we are managing, why and in what ways. For the Canadian blood system, a new procedure of providing reasons for recall will assist hospitals and other donation agents in managing perceived risks. Yet the role of expert biases and domain interests are likely to continue to exist, and must be understood and incorporated to ensure blood safety and continued public trust and to provide timely responsiveness in such a high reliability system. We suggest the framework of ISO 31000 emphasising context, risk identification and assessment, risk treatment, communications and consultation is useful, along with a safety-case approach. Communication about ways to protect public safety is always necessary and this must include clarity on definitions, responsibilities, and public perceptions and what the consequences of even minor errors mean in a complex system. Furthermore, error as a risk state needs careful theorizing and application in systematic risk management approaches.
Additional file 1: Appendix 1: Interview guide for understanding the management of blood products in Ontario. Appendix provides the questionnaire used to explore risk management of blood products under conditions of uncertainty. Noninvasive positive pressure ventilation for acute respiratory failure in children: a concise review Noninvasive positive pressure ventilation (NPPV) refers to the delivery of mechanical respiratory support without the use of endotracheal intubation (ETI). The present review focused on the effectiveness of NPPV in children > 1 month of age with acute respiratory failure (ARF) due to different conditions. ARF is the most common cause of cardiac arrest in children. Therefore, prompt recognition and treatment of pediatric patients with pending respiratory failure can be lifesaving. Mechanical respiratory support is a critical intervention in many cases of ARF. In recent years, NPPV has been proposed as a valuable alternative to invasive mechanical ventilation (IMV) in this acute setting. Recent physiological studies have demonstrated beneficial effects of NPPV in children with ARF. Several pediatric clinical studies, the majority of which were noncontrolled or case series and of small size, have suggested the effectiveness of NPPV in the treatment of ARF due to acute airway (upper or lower) obstruction or certain primary parenchymal lung disease, and in specific circumstances, such as postoperative or postextubation ARF, immunocompromised patients with ARF, or as a means to facilitate extubation. NPPV was well tolerated with rare major complications and was associated with improved gas exchange, decreased work of breathing, and ETI avoidance in 22-100% of patients. High FiO(2 )needs or high PaCO(2 )level on admission or within the first hours after starting NPPV appeared to be the best independent predictive factors for the NPPV failure in children with ARF. However, many important issues, such as the identification of the patient, the right time for NPPV application, and the appropriate setting, are still lacking. Further randomized, controlled trials that address these issues in children with ARF are recommended. Breathing difficulties are common symptoms in children and common reason for visits to the emergency department [1] . In United Kingdom, respiratory illnesses (both acute and chronic) accounted for 20% of weekly general practitioner consultations, 15% of hospital admissions, and 8% of deaths in childhood in 2001 [2] . Although the great majority of cases are benign and self-limited, requiring no intervention, some patients will require a higher level of respiratory support. Invasive mechanical ventilation (IMV) is a critical intervention in many cases of acute respiratory failure (ARF), but there are definite risks associated with endotracheal intubation (ETI) [3] . By providing respiratory support without ETI, non-invasive positive pressure ventilation (NPPV) may be, in appropriately selected patients, an extremely valuable alternative to IMV. It is generally much safer than IMV and has been shown to decrease resource utilization and to avoid the myriad of complications associated with ETI, including upper airway trauma, laryngeal swelling, postextubation vocal cord dysfunction, and nosocomial infections [3] . NPPV usually refers to continuous positive airway pressure (CPAP) or bilevel respiratory support, including expiratory positive airway pressure (EPAP) and inspiratory positive airway pressure (IPAP), i.e., biphasic positive airway pressure (BIPAP) and bilevel positive airway pressure (BiPAP), delivered through nasal prongs, facemasks, or helmets. Although there is high-level evidence in the literature to support the use of NPPV for the treatment of ARF due to different causes, such as exacerbation of chronic obstructive pulmonary disease [4] and acute cardiogenic pulmonary edema [5] in adults, there are few reports about its use in this acute setting in children. So far, case series constitute the vast majority of the available knowledge in this age group. However, there is an increasing interest in the use of NPPV as a therapeutic tool for children with respiratory distress that is clear from the increasing number of published studies over time ( Figure 1) ; a research of studies on the use of NPPV in children > 1 month of age, published before December 30, 2010 (database: MEDLINE via PubMed; keywords: noninvasive ventilation, non-invasive ventilation, noninvasive positive pressure ventilation, non-invasive positive pressure ventilation, bipap, continuous positive airway pressure; age limits: children from 1 month to 18 years old) identified 332 relevant articles, of which 48% were published during the past 5 years. This concise review is designed to focus on the effectiveness of NPPV in children > 1 month of age with ARF (excluding patients with neurologic or chronic lung disease).
The frequency of ARF is higher in infants and young children than in adults. This difference can be explained by defining anatomic compartments and their developmental differences in pediatric patients that influence susceptibility to ARF [6] . In addition, respiratory failure often precedes cardiopulmonary arrest in children, unlike in adults where primary cardiac disease often is responsible. Therefore, prompt recognition and treatment of pediatric patients with pending respiratory failure can be lifesaving [6] .
Respiratory failure is a syndrome in which the respiratory system fails in one or both of its gas exchange functions: oxygenation and carbon dioxide elimination. In general, patients with respiratory failure may be classified into two groups, depending on the component of the respiratory system that is involved: hypoxemic respiratory failure and hypercapnic respiratory failure [7] .
Hypoxemic respiratory failure (known as type I) Hypoxemic respiratory failure (type I) can be associated with virtually all acute diseases of the lung, such as status asthmaticus, bronchiolitis, pneumonia, and pulmonary edema, which interfere with the normal function of the lung and airway. The predominant mechanism in type I failure is uneven or mismatched ventilation and perfusion (intrapulmonary shunt) in regional lung units. This is the most common form of respiratory failure, characterized by a PaO 2 < 60 mmHg with a normal or low PaCO 2 . The primary treatment of type I respiratory failure in children is to administer supplemental oxygen at a level sufficient to increase the arterial oxygen saturation (SaO 2 ) to greater than 94%. In situations when a fraction of oxygen in inspired gas (FiO 2 ) of greater than 0.5 is necessary to achieve this goal, this often is referred to as "acute hypoxemic respiratory failure" [7] . In this setting, NPPV may be considered.
Hypercapnic respiratory failure (known as type II)
Hypercapnic respiratory failure (type II) is a consequence of ventilatory failure and can occur in conditions that affect the respiratory pump, such as depressed 1993 1993-1995 1996-1998 1999-2001 2002-2004 2005-2007 2008-2010 Time years
References (n) neural ventilatory drive, acute or chronic upper airway obstruction, neuromuscular weakness, marked obesity, and rib-cage abnormalities. Alveolar hypoventilation is characterized by a PaCO 2 > 50 mmHg [7] . The onset of type II failure may be insidious and may develop when respiratory muscle fatigue complicates preexisting disorders, such as pneumonia or status asthmaticus, which present initially with hypoxemia without hypoventilation. Aministration of oxygen alone is not an appropriate treatment for hypercapnic respiratory failure and can result in the patient retaining even more carbon dioxide, especially in situations where the child has adapted to chronic hypercapnia and is relatively dependent on oxygen-sensitive peripheral chemoreceptors to maintain ventilatory drive. In addition to supplemental oxygen, therapies to reduce the load on the respiratory muscles and increase the level of alveolar ventilation should be instituted in children with type II respiratory failure.
When to use NPPV for acute respiratory failure?
When the cause of ARF is reversible, medical treatment works to maximize lung function and reverse the precipitating cause, whereas the goal of ventilatory support is to "gain time" by unloading respiratory muscles, increasing ventilation, and thus reducing dyspnea and respiratory rate and improving gas exchange. Two recent physiological studies have demonstrated these beneficial effects of NPPV in children with ARF [8, 9] . NPPV is increasingly used for treatment of ARF in children. Tables 1 and 2 summarize the studies reporting the effectiveness of NPPV in children with ARF of various etiologies [8, . However, the determinants of success of NPPV relate more prominently to the primary diagnosis as discussed below.
Lower airway disease is a common cause of ARF. Asthma accounts for the largest percentage of this group, but infections, such as viral bronchiolitis, also are common and predominantly impact the small airways. Physicians caring for acutely ill children are regularly faced with this condition. Both non-invasive and invasive ventilation may be options when medical treatment fails to prevent respiratory failure. ETI and positive pressure ventilation in children with lower airway obstruction may increase bronchoconstriction, increase the risk of airway leakage, and has disadvantageous effects on circulation and cardiac output. Therefore, ETI should be avoided unless respiratory failure is imminent despite adequate institution of all available treatment measures. NPPV can be an attractive alternative to IMV for these patients. Clinical trials in children with acute lower respiratory airway obstruction have suggested that NPPV may improve symptoms and ventilation without significant adverse events and reduce the need for IMV [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . NPPV theoretically improves the respiratory status of patients with lower respiratory airway obstruction by several mechanisms [37] . During acute bronchospastic episodes, patients have an increase in airway resistance and expiratory time constant. The combination of prolonged expiratory time constant and premature closure of inflamed airways during exhalation results in dynamic hyperinflation, which causes increased positive pressure in the alveoli at end-expiration (auto-PEEP). Because the alveolar pressure must be reduced to subatmospheric levels to initiate the next breath, this auto-PEEP increases the inspiratory load and induces respiratory muscle fatigue. The EPAP delivered by NPPV may help to decrease dynamic hyperinflation by maintaining small airway patency and may reduce the patient's work of breathing by decreasing the drop in alveolar pressure needed to initiate a breath. In addition, inspiratory support, i.e., IPAP delivered by NPPV, helps to support fatigued respiratory muscles, thereby improving dyspnea and gas exchange. Needleman et al., in a physiological study, found that the NPPV use in children with status asthmaticus was associated with a decrease in respiratory rate and fractional inspired time and an improvement of thoracoabdominal synchrony in 80% of patients [12] . A few clinical studies of small size (3-73 patients) reported the use of NPPV for treatment of status asthmaticus in children (Table 1 ) [10, 11, 13, 14] . NPPV was well tolerated with no major complications and was associated with an improvement of gas exchange and respiratory effort (Table 1) . Viral bronchiolitis, mainly due to respiratory syncytial virus, represents the largest cohort of children treated with NPPV [15] [16] [17] [18] [19] [20] . Use of NPPV in infant with severe bronchiolitis was associated with improved respiratory rate [15, 19] and PaCO 2 [16, 19, 20] , decreased work of breathing [17] , and ETI avoidance in 67-100% of patients (Table 1 ) [17, 18] .
In children, dynamic upper airway obstruction can present as an acute life-threatening condition and leads to severe alveolar hypoventilation. In 2006, a survey of French PICU group found that 67% of pediatric intensivists applied frequently or systematically NPPV in the management of dynamic upper airway obstruction in children [38] . However, there is a paucity of literature on the use of NPPV in the acute setting of upper airway obstruction in children. NPPV was associated with a significant decrease in respiratory effort [21] and a sustained improvement in gas exchange [22] in children with dynamic upper airway obstruction (Table 1) . 
The main goals of NPPV in patients with parenchymal lung disease, such as pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS), are to improve oxygenation, to unload the respiratory muscles, and to relieve dyspnea. The first goal can usually be achieved by using EPAP to recruit and stabilize previously collapsed lung tissue [39] . Unloading of the respiratory muscles during NPPV with IPAP has been reported by L'Her et al. in adult patients with ALI [39] . The authors concluded that adding IPAP to EPAP may be indispensable in patients with ALI treated with NPPV [39] . Indeed, IPAP allows a better respiratory system muscle unloading, alveolar recruitment, oxygenation, and CO 2 washout improvement. Although NPPV seems disappointing in ARF owing to pneumonia in adult patients, with failure rates of up to 66% [40] , several noncontrolled trials have suggested that NPPV could improve symptoms and ventilation without significant adverse events and reduce the need for IMV in children with ARF due to pneumonia [22] [23] [24] [25] [26] [27] . Use of NPPV in this acute setting in children was associated with reduction in ETI rates ranging from 50-100% (Table 1) [23, 24] .
The most challenging application of NPPV may be in patients with ARDS. Studies of NPPV for the treatment of ARDS in adult population have reported failure rates of 50-80% [40] . A meta-analysis of the topic in adult population concluded that NPPV was unlikely to have any significant benefit [41] . In children, the use of NPPV for the treatment of ARDS was associated with a failure rate of 78%, and 22% of them died (Table 1 ) [27] . Therefore, NPPV use in such a patient group is rarely justified. However, if a trial of NPPV is initiated, patients should be closely monitored and promptly intubated if their conditions deteriorate, so that inordinate delays in needed interventions are avoided.
Acute chest syndrome (ACS) is one of the leading causes of death and hospitalization among patients with sickle cell disease [42] . Approximately 70% of patients (adults or children) with ACS are hypoxic [43] . Indeed, patients with sickle cell disease are prone to infarctive crises. Thoracic bone infarction (usually in the ribs) in such patients leads to pain, splinting, hypoventilation, and the clinical signs of ACS. In situ red blood cell sickling in the lung vasculature is possibly a consequence of hypoventilation with subsequent infarction of lung parenchyma. NPPV has been proposed as a therapeutic option for patients with ACS. By improving patient oxygenation, NPPV could prevent progression from painful crisis to ACS, and ultimately to ARDS. Three retrospective studies reported favorable outcomes in children with ACS treated with NPPV (Table 1) [22, 27, 28] .
Postoperative pulmonary complications are a major cause of morbidity, mortality, prolonged hospital stay, and increased cost of care [44] . It has been reported that 5-10% of all surgical adult patients experience postoperative pulmonary complications [45] . Atelectasis, postoperative pneumonia, ARDS, and postoperative respiratory failure have all been classified as postoperative pulmonary complications. Postoperative respiratory failure is most commonly defined as the inability to be extubated 48 hours after surgery [46] , although some investigators have used 5 days [47] . NPPV has been successfully used to treat postoperative respiratory failure in both pediatric and adult patients. Compared with standard treatment, NPPV used after major abdominal surgery improved hypoxemia and reduced the need for ETI in adult population [48] . NPPV application in children with postoperative respiratory failure was associated with improved respiratory effort, gas exchange, oxygen saturation, and reduced the need for ETI (Table  1 ) [8, 24, 26, 27, 29, 30] .
The need for reintubation after failed extubation is associated with increased morbidity and high mortality [49] . NPPV has been proposed as a means of "facilitating" weaning from IMV, and as a "curative" treatment for postextubation respiratory failure. Although several studies have shown the efficacy of NPPV in weaning from IMV in adult population [50] , its application for postextubation respiratory failure is not supported by randomized, controlled trials [51] . In children, two noncontrolled trials assessed the efficacy of NPPV in these settings: the application of NPPV as a means of "facilitating" ventilation weaning, and as "curative" treatment for postextubation respiratory failure was associated with success rates of 81-86% and 50-75%, respectively [31, 32] .
ARF in immunocompromised patients most often results from infections, pulmonary localization of the primary disease, or even postchemotherapy cardiogenic pulmonary edema. Treatment of such patients often requires intubation and mechanical ventilation. Avoidance of the infectious complications associated with IMV is particularly attractive in these high-risk patients, in whom this could be devastating, if not fatal. Results of randomized, controlled trials have proven the beneficial effects of NPPV in immunocompromised adult patients [52, 53] . Some case series reported the use of NPPV in the treatment of respiratory failure in immunocompromised children (Table 2 ) [23, 27, [33] [34] [35] [36] . The likelihood of NPPV success in immunocompromised children seems to be related rather to the type of pulmonary disease: the ETI avoidance rates varied from 40% for ARDS to 100% for pneumonia ( Table 2) .
Are there predictive factors of NPPV failure in children with ARF?
It is not always apparent which patients will initially benefit from NPPV; some patients do not obtain adequate ventilation with NPPV. The NPPV failure rate may be fairly consistent for certain diseases, and NPPV failure eventually requires intubation. Inability to early identify patients who will fail NPPV can cause inappropriate delay of intubation, which can cause clinical deterioration and increase morbidity and mortality.
Knowing the predictors of NPPV failure in patient with ARF is therefore crucial in deciding if and when to apply this ventilatory technique. Several authors have identified different predictive factors of NPPV failure in children with ARF: the results of studies are given in Table 3 [20, 24, 26, 27, 31, 54, 55] . The best predictive factors for the NPPV failure in ARF appear to be the level of FiO 2 and PaCO 2 on admission or within the first hours after starting NPPV (Table 3) .
During recent years, there has been an increasing interest in the use of NPPV for children with ARF. There are some promising studies supporting its use in this acute setting. NPPV was well tolerated with rare major complications and was associated with improved gas exchange, decreased work of breathing, and decreased need for ETI. Both critical care ventilators and portable ventilators have been used for NPPV. However, the vast majority of the available knowledge in this acute setting results from noncontrolled trials and case series of small size. As such, many important issues, such as the identification of the patient, the right time for NPPV application, and the appropriate setting, are still lacking. Further randomized, controlled trials addressing these issues in children with ARF are needed to define better the patients who are likely to benefit from this alternative method of respiratory support. Also, the respective place of NPPV and high flow oxygen therapy in children with ARF due to different conditions has to be determined [56] . Najaf-Zadeh and Leclerc Annals of Intensive Care 2011, 1:15 http://www.annalsofintensivecare.com/content/1/1/15 Pandemic A/H1N1v influenza 2009 in hospitalized children: a multicenter Belgian survey BACKGROUND: During the 2009 influenza A/H1N1v pandemic, children were identified as a specific "at risk" group. We conducted a multicentric study to describe pattern of influenza A/H1N1v infection among hospitalized children in Brussels, Belgium. METHODS: From July 1, 2009, to January 31, 2010, we collected epidemiological and clinical data of all proven (positive H1N1v PCR) and probable (positive influenza A antigen or culture) pediatric cases of influenza A/H1N1v infections, hospitalized in four tertiary centers. RESULTS: During the epidemic period, an excess of 18% of pediatric outpatients and emergency department visits was registered. 215 children were hospitalized with proven/probable influenza A/H1N1v infection. Median age was 31 months. 47% had ≥ 1 comorbid conditions. Febrile respiratory illness was the most common presentation. 36% presented with initial gastrointestinal symptoms and 10% with neurological manifestations. 34% had pneumonia. Only 24% of the patients received oseltamivir but 57% received antibiotics. 10% of children were admitted to PICU, seven of whom with ARDS. Case fatality-rate was 5/215 (2%), concerning only children suffering from chronic neurological disorders. Children over 2 years of age showed a higher propensity to be admitted to PICU (16% vs 1%, p = 0.002) and a higher mortality rate (4% vs 0%, p = 0.06). Infants less than 3 months old showed a milder course of infection, with few respiratory and neurological complications. CONCLUSION: Although influenza A/H1N1v infections were generally self-limited, pediatric burden of disease was significant. Compared to other countries experiencing different health care systems, our Belgian cohort was younger and received less frequently antiviral therapy; disease course and mortality were however similar. On March 2009, in Mexico, a novel recombinant influenza strain (A/H1N1v) of swine origin was discovered as an infective agent in humans [1] . This new virus spread rapidly, first to USA and Canada, then all over the world, causing the "new 2009 influenza A/H1N1v pandemic" [2] . Worldwide, the burden of disease was significant and subsequent efforts from health care systems were required to face an overload of patient's consultations as well as to implement vaccination and surveillance programs. Although consequences of the pandemic were less dramatic than initially feared, the World Health Organization (WHO) estimated that the virus was responsible of at least 17700 deaths worldwide and the Centers for Disease Control and Prevention (CDC) reported 59 millions infected people in the USA [3] [4] [5] ).
During this A/H1N1v flu wave, children and young adults were identified as a particular risk group. They presented a higher attack rate than older adults [6] and a greater mortality rate than previously observed with classical seasonal flu [7, 8] . Several reports on influenza A/H1N1v in pediatric settings have now been published [9] [10] [11] [12] , but information on clinical presentation and severity of infection in European children remains limited [13] [14] [15] . However, these data could be of the great interest to guide future recommendations for vaccination and antiviral therapy during forecoming flu seasons.
Belgium experienced the influenza A/H1N1v epidemic from July 2009 to January 2010. Pandemic vaccine (adjuvanted Pandemrix ® ) was only available after the peak occurred in October and was given with priority to risk groups (defined as health care workers, pregnant women and chronically ill patients) [16] . According to our national surveillance system, around 214531 people were infected, 733000 could benefit from vaccination and 19 deaths were attributable to the virus [17] .
In this context, we conducted a multicenter study analyzing influenza A/H1N1v pediatric cases hospitalized in four tertiary medical centers of Brussels, Belgium. Our study aimed to offer a comprehensive description of influenza A/H1N1v infection in children, in the light of other recently published data from countries experiencing different health care systems [9] [10] [11] [12] .
In collaboration with infection control units and microbiology laboratories, we prospectively registered all proven and probable pediatric cases of influenza A/H1N1v infections hospitalized in four tertiary facilities of Brussels (Hôpital Universitaire des Enfants Reine Fabiola, Universitair Ziekenhuis of Brussels, Cliniques Universitaires Saint-Luc and Hôpital Saint-Pierre). These facilities totalize 406 pediatric beds, representing 80% of the total pediatric beds available in Brussels (about 1 million inhabitants in 2009). Moreover, three of the hospitals have a Pediatric Intensive Care Unit (PICU) where critically-ill children from other hospitals of Brussels and the surrounding areas are referred to (in total 32 PICU beds available).
The study period extended from July 1, 2009, to January 31, 2010. Children were included if they were aged from 0 to 18 years, presented with clinical symptoms compatible with influenza (fever and/or respiratory signs/symptoms) and had either positive PCR results for influenza A/H1N1v (proven cases), or an antigen and/or a positive culture for influenza A (probable cases). The latter cases were included because virtually no other seasonal influenza A viruses were circulating in Belgium during the epidemic period (less than 0.4%, data from the Belgian National Institute of Public Health). Moreover, specific H1N1v PCR confirmation was no longer carried out routinely at the end of the epidemic, due to the high cost of this testing and the limited number of cases after December 2009.
Data were collected retrospectively from patients' medical files using a standardized questionnaire.
A pre-existing co-morbidity was defined as a chronic condition requiring long term medication or medical follow up. Co-morbidities were listed based upon CDC H1N1 flu guidelines http://www.CDC.gov/h1n1flu.htm and other recent publications [8] . Co-morbidities were not mutually exclusive, so that a child could participate in several categories.
Fever was defined as a central temperature above 38°C elsius. Nosocomial infection was defined as a proven or probable case occurring after more than 48 hours of hospitalization.
Respiratory samples collection included nasopharyngeal aspirates, nasopharyngeal flocked swabs (Copan Diagnostics, Corona, CA), throat flocked swabs (Copan Diagnostics, Corona, CA) and sputum.
Antigen testing was assessed by immunochromatographic rapid antigen detection (RAT) in three of the four centers or also by direct immunofluorescence (DIF) technique (Argene SA, Verniolle, France) in one of them. Both DIF and immunochromatography use highly sensitive monoclonal antibodies directed against either influenza A or B nucleoprotein antigens [18] . RAT was performed using two different testing: the Coris Influ-A&B Respi-Strip (Coris Bioconcept, Gembloux, Belgium) and the Binax Now influenza A & B (Binax Inc., Inverness medical, Maine, USA). Direct antigen testing was unavailable in the fourth participating hospital, representing 26% of our cohort of patients.
Viral culture was performed on the three following cell lines: Vero, MRC-5 and LLC-MK2 in two centers; and MDCK, Hep-2, MRC-5 and LLC-MK2 in a third hospital, as described elsewhere [19] . In the fourth center which used DIF and RAT for antigen detection, respiratory samples were not cultured as antigen detection was followed directly by real-time PCR. (This center represented 22% of the cohort).
Biomolecular testing consisted, for three of the four centers, firstly in detection of influenza A virus by a home-made real-time RT-PCR (RT-PCR InflA) targeting the matrixprotein-coding gene and consequently by specific detection of the circulating pandemic variant using two monoplex real-time RT-PCR assays as described in the Centers of Disease Control (CDC) protocol: the SW InfA PCR (SWINE) and the SW H1 PCR (RT-PCR A/ H1N1) [20] . In the fourth center (26% of samples), a commercial available PCR kit was used directly for detecting the pandemic strain: "Swine influenza virus (sw H1N1) Real-time PCR" (Diagenode Diagnostics, Liège, Belgium).
Statistical analyses were performed using Graph Pad Prism Software, Inc, 2003, San Diego, USA. Chi square or Fisher's exact tests were used to compare non continuous variables and Mann Whitney u-test was used to compare continuous variables. A two-tailed p-value less than 0.05 was considered as statistically significant.
Approval of the Medical Ethics Committees of the four hospitals was obtained before starting the study. A code number was attributed to each child so that data collected remained strictly confidential.
During the epidemic period, an excess of 18% (+10486) of pediatric outpatients and emergency department visits was registered, as compared with the mean measured over the 3 previous years during the same months. Figure 1 represents the evolution of H1N1v 2009 pediatric hospitalized cases over time in the four hospitals, with peak of the epidemic observed between the end of October and the beginning of November 2009. 215 children were hospitalized with proven or probable influenza A/ H1N1v infection; representing 2% of the total hospitals' admissions registered during the whole study period but 6% (191/3144) of those during the four weeks of the peak of the epidemic. Additionally, the PICU occupation rate by influenza A/H1N1v infected children was 3.5% over the whole study period and reached 8% during the peak of the epidemic.
Among our cohort of 215 children, 57% were male. The median age of the patients was 31 months (range: < 1 to 208 months), with 19% of the children having less than 3 months of age ( Figure 2 ). As shown in Table 1 , 101/215 (47%) children presented with one or more underlying co-morbid condition, principally chronic lung diseases and neurological disorders. The median age of patients presenting co-morbidities was significantly higher than of those without (50 versus 14 months, p < 0.0001).
The major clinical features, reasons for hospitalization and blood diagnostic results are summarized in Table 2 . The median duration of symptoms before admission was 2 days (IQR: 1-4 d). Presentation on admission consisted mainly in febrile respiratory illness, with a high prevalence of gastrointestinal symptoms, independently of age groups (Table 3) . Moreover, 21 children (10%) presented initially with neurological manifestations. Based upon initial clinical presentation, diagnosis of influenza A/H1N1v infection was suggested by clinicians in 56% of children (Table 2) . 74/215 (34%) children had chest X-ray confirmed pneumonia, associated in 6% with pleural effusion. Three patients had confirmed bacterial superinfection with Streptococcus pneumoniae (positive blood cultures). For five patients (2%), the influenza infection was hospital acquired.
As shown in Table 3 , children aged less than 3 months old had a significantly lower rate of pneumonia and tended to have less neurological manifestations than older ones. For this age group, the main reason for hospitalization was surveillance of acute fever without focus. Initial clinical presentation was globally similar between patients with and without chronic co-morbidities, except for hypoxemia (Table 3) .
Blood inflammatory profile on admission was variable, with 13% of the children presenting with leukopenia and 6% with thrombocytopenia ( Table 2) . Respiratory samples collected to diagnose influenza A/ H1N1v infection were nasopharyngeal aspirates and nasopharyngeal swabs in 58% and 39% of the patients, respectively. Compared to PCR (considered as the gold standard), the sensitivity of antigen by RAT was low, being only 29% (10/34) and 57% (47/83) using Coris RespiStrip and Binax Now testing, respectively (data not calculated for DIF method, as only performed on 11 samples). The sensitivity of culture was quite better at 76% (107/141).
Among the 215 children, only 51 (24%) received oseltamivir (doses according to weight and age [21] ). For a large majority of them (37/47, data unavailable for three children), antiviral therapy was started directly on admission and was continued for five days (35/41, data unavailable for 9 patients). Rate of oseltamivir prescription reached 42% and 71% for children having pneumonia and for those requiring PICU admission, respectively. No significant related adverse events were reported. Oseltamivir was significantly more frequently prescribed among children older than 2 years compared to younger ones (Table 4 ) and among children with underlying disease (39/101 [39%]) versus 12/114 [11%], p < 0.0001). Additionally, 57% (123/215) of the patients were treated with antibiotics, for a median duration of seven days. In only 17 of them (14%), antibiotic therapy was discontinued after obtaining confirmation of influenza A/H1N1v infection (data unavailable for seven cases). Among children treated by antibiotics, 54% had a diagnosis of pneumonia. Finally, antibiotics were similarly used in all patients' age groups (Table 4) .
Intensive Care 21/215 patients (10%) had to be admitted to PICU, mainly within 24 hours of admission. Among them, the prevalence of co-morbidity (62%) tended to be higher than observed among ward patients, with a predominance of neurological disorders ( Table 1 ). The median age of PICU children was 75 months (IQR 47-130). PICU admissions were significantly more frequent in children above two years of age (Table 4 ) and no infant less than three months old required intensive care.
The major reason for PICU admission was respiratory failure subsequent to pneumonia (Table 3) . Seven patients (3% of the global cohort) presented an Acute Respiratory Distress Syndrome (ARDS) and three had pleural effusion. Two patients were admitted for surveillance because of severe underlying disease. None of the 21 children presented seizures or signs of viral encephalitis ( Table 3 ). The median duration of PICU stay was four days and ranged from 1 to 90 days. 13/21 (59%) children received respiratory support with non invasive ventilation (NIV); eight (38%) required mechanical invasive ventilation for a median duration of six days (range 1 to 45). One previously healthy child presenting with severe ARDS followed by cardiac-respiratory arrest had to undergo Extra Corporeal Membrane Oxygenation (ECMO) support during 12 days, but survived with mild respiratory sequelae.
The median duration of hospitalization was three days (IQR: 2-6 d). This result was unaffected by the patients' age (Table 4) . Parental request/non compliance 2 (1)
Legend: *IQR = interquartile range; **PMN = polymorphonuclear leukocytes; ***CRP = C-reactive protein; 1 201 children with blood results available on admission; 2 defined as Hb oxygen saturation less than 94%; 3 transcutaneous saturation The case-fatality rate among the global cohort was 2% (5/215). The five deaths were directly attributable to influenza A/H1N1v with or without bacterial superinfection and occurred in children with co-morbidities who would otherwise have died from their underlying disease. These underlying diseases consisted in neurological disorders from various etiologies (extensive central nervous system glioma, polymalformative syndrome, Hurler syndrome, cerebral palsy and severe encephalopathy with pontocerebellar hypoplasy). For all of them, severe pneumonia was notified, associated for three children with ARDS. All deaths concerned children aged more than two years old ( Table 4 ). Four of the five children had received oseltamivir within 48 hours of clinical symptoms.
Three additional patients (1%) were cured from influenza A/H1N1v infection but still suffered from sequelae at the end of the study (two had bronchiectasis with emphysema and one a pulmonary restrictive syndrome needing tracheotomy and NIV support at home).
Even though consequences were less dramatic than initially feared, the 2009 influenza A/H1N1v pandemic has caused a significant burden of disease worldwide, especially in the pediatric population [6] [7] [8] . Higher attack rate was observed among children, causing important overload in outpatient and emergency departments [11] as well as in PICU [22] . Unfortunately, our study was not designed to assess the epidemiological impact of influenza A/H1N1v infection over the whole pediatric population of Brussels. Moreover, by selecting only laboratory confirmed infections, we underestimated the number of hospitalized cases, especially since diagnosis confirmation by PCR was no more routinely performed at the end of the epidemic. However, we were able to notice an important pediatric burden of disease in Brussels, as illustrated by an increased rate of outpatients visits of 18% during the epidemic period compared to the three previous years and a high rate of PICU occupation by influenza A/H1N1v infected patients (8% during the peak of epidemic). It would have been of interest to compare the rate of hospitalization related to H1N1 with those registered for seasonal flu during the 3 previous years but these data were unfortunately not available.
This study offers a comprehensive description of influenza A/H1N1v pattern of infection among Belgian hospitalized children, in the light of recent publications from other continents. Consistently with these reports [9] [10] [11] [12] 23, 24] , co-morbidities were highly prevalent among influenza A/H1N1v infected hospitalized children (47%). The co-morbidities were not different from those observed during seasonal flu. As described by others [7, 9, 22] , the presence of at least one co-morbidity was significantly more frequent in children of more than two years of age and constituted a risk factor for severity of disease, in terms of PICU admissions and casefatality rate. Furthermore, influenza A/H1N1v illness course differed according to patients' age groups. Indeed, children less than two years of age (46% of the cohort), and especially those less than three months, presented milder patterns of infection and were often hospitalized only for observation of fever without focus. 86% of PICU admissions and all deaths occurred in children over two years of age (with 80% of deaths among children > five years old). Although this issue is conflicting in the medical literature [7, 10] , similar findings have been reported in a large series by investigators from the CDC [25] . This observation differs from what is seen during seasonal flu, where young children and especially infants presented a higher mortality-rate compared to older ones [26, 27] . Nevertheless, during this pandemic wave as well as during previous flu seasons, the highest rate of hospitalization was generally reported among young age groups [8, 9, 11, 24] . This was particularly true in our series, as reflected by a median of age of 31 months, which was even younger than among American and Israeli hospitalized children (median age ranging from four to six years) [9, 10] . If unexplained by the severity of infection, this finding probably illustrates differences in clinical practices and hospitalization policies. In Belgium, the National Healthcare System renders the access to inpatients pediatric facilities easy, so that hospitalization of infants presenting with fever without focus, especially those younger than 3 months of age, is largely recommended and not restricted to the most severe cases as in other countries [28, 29] .
Although we focused on hospitalized cases, influenza A/H1N1v episodes were mainly self-limited, consisting of febrile respiratory illness and requiring short duration of hospitalization (median 3 days) with or without oxygen supplementation. Initial clinical features did not differ from seasonal flu [30] , except for the higher proportion of children presenting with gastrointestinal manifestations, as described in previous studies [12, 23, 24] . This involvement of the gastrointestinal tract could be subsequent to a high rate of influenza A/ H1N1v virus replication [31] . Neurological manifestations were also frequent (10% of children) but in contrast with other reports [9, 22] were not correlated with PICU admission or fatality. Finally, more than one third of the whole cohort and 71% of PICU patients had pneumonia confirmed on chest X-rays. Even though only 3 (1%) children had evidence of bacteremia (all due to S. pneumoniae), it seems very likely that a significant proportion of pneumonia, especially those with lobar infiltrates, were associated with bacterial super-infections. According to some series, bacterial super-infections after influenza A/H1N1v episodes were found in about 4% of hospitalized children [11, 12, 23] but reached 20 to 38% among fatal cases [7, 25, 32] . Considering the low rate of positive blood cultures (BC) in pediatric bacterial community-acquired pneumonia (2.5 to 5%) [33, 34] , these published rates as well as our data likely constitute an under-estimation, as bacterial pneumonia diagnosis relied on positive cultures from sterile sites or autopsies and as part of children had received antibiotics prior to microbiological documentation. Finally, among our whole cohort, no necrotizing pneumonia, empyema or sepsis due to S. aureus or group A Streptococcus were reported, even though those pathogens are frequently involved in other series [9, 25] . Surprisingly, a majority of children were treated by antibiotics, even after the diagnosis of influenza A/ H1N1v infection was obtained. As mentioned above, confirming bacterial pulmonary super-infection after influenza illness is challenging and diagnostic relies more on clinical presumption and unspecific blood results [35] . However, this could only partly explain the high rate of antibiotics use, as only 54% of those children treated by antibiotics presented pulmonary infiltrates. Rate of antibiotics prescriptions was uniform among all age groups and was also high in other pediatric studies [10] [11] [12] . The exact reasons sustaining this practice remain unknown but should be worth to investigate in further prospective studies. Contrastingly, our study showed a particularly low percentage of oseltamivir prescriptions. Indeed, only 24% of the children were treated compared with 45 to 84% in other similar studies [8, 9, 11, 12] . In our four centers, the use of oseltamivir was significantly higher in children above 2 years of age and/or suffering from comorbidities. This more "watch and wait" practice was in line with the restrictive national recommendations issued for oseltamivir pediatric use during the 2009 pandemic wave, which suggested cautious prescription under one year of age, regarding the absence of safety data among infants [16] . Moreover, in Belgium, prescription of antiviral drugs during seasonal flu is very limited and kept for management of severe diseases or immuno-compromised patients [36] . Obviously, the limitations associated with the retrospective design do not allow us to conclude on treatment efficacy. It is however interesting to note that, although oseltamivir was scarcely used, fatality rate and PICU admissions were comparable to the other above-mentioned reports [9, 10, 12] .
In Argentina [8] , the case-fatality rate of influenza A/ H1N1v infected children was 5%, with a global pediatric mortality rate 10 times greater compared to previous flu seasons. National surveys in the United Kingdom [7] and U.S [23] reported also a higher influenza related mortality rate during the pandemic influenza A/H1N1v than observed with seasonal flu. However, consequences in the Northern hemisphere were less dramatic than anticipated. Studies from these countries reported case-fatality rate among hospitalized children ranging from 0.6 to 3% [9] [10] [11] [12] , similar to our findings (2%). As previously hypothesized [10] , these North/South differences in patients' outcome could partly be explained by an easier access to the health care system in Europe, Israel and North America. In Israel [10] , as well as in our series, the median duration of symptoms before hospitalization was only 2 days compared to 4 days in Argentina [8] . On another hand, 2 patterns of influenza A/H1N1v related deaths have been described [7] : those occurring after several days of hospitalization in chronically ill patients (80%), in contrast to those observed after acute evolution of viral infection in previously healthy children (20%). Despite a small number of cases, a similar profile seemed to happen in our series, as the five children who died suffered from chronic neurological disorders and one previously healthy child presented a fulminant viral infection causing cardio-respiratory arrest and requiring nine days of ECMO support to be cured.
Although influenza A/H1N1v infections were globally self-limited, pediatric burden of disease was significant. Children of more than 2 years old and/or suffering from chronic co-morbidities were shown at higher risk of severe infection. Compared to other countries experiencing different health care systems, our Belgian cohort was younger and received less frequently antiviral therapy; disease course and mortality were however similar. A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders. Proteases of the caspase family are known as important mediators of apoptosis and have been commonly subdivided based on their roles in apoptosis or inflammation (apoptotic initiator, apoptotic executioner or inflammatory caspases). This definition however has become somewhat inaccurate as an increasing number of non-apoptotic roles for both initiator and executioner caspases have been identified that mediate cell differentiation, maturation and signaling events [1] .
Caspases can further be distinguished based on their inherent differences in caspase substrate preference that are defined by the shape and electrostatic potential of the active site cleft [2] . Using positional scanning of peptide libraries, consensus recognition sequences have been proposed for each caspase and have led to the development of peptide substrates as well as inhibitors that typically consist of 4 amino acids (i.e. DEVD for caspase-3), followed by a fluorescent tag such as Afc (7-amino-4-trifluoro methylcoumarin) for a substrate or a 'warhead' such as fmk (fluoromethylketone) that covalently binds the enzyme for an inhibitor. These reagents are useful to investigate caspases that constitute the majority of caspase-like activity in a sample, as it may be assumed for active caspase-3 in highly apoptotic extracts [3] . However, with Km/kcat ratio differences of less than 10 fold for many widely used peptide substrates [4] , these reagents are not particularly useful for investigating the activity of a caspase present at lower concentrations in cell culture and tissue samples. In particular in developmental or signalling processes that do not involve cell death, intracellular caspase activity is likely under tight control by endogenous caspase inhibitors or the proteasome [5, 6] and the resulting low levels of activity are difficult to detect with peptide substrates.
In biologic protease substrates, additional factors outside the 4 amino acid recognition site can influence the selectivity and efficiency of proteolytic cleavage. For caspases, it has been shown that the amino acid residue directly after the scissile bond (P19) is an important determinant of cleavage, since charged or bulky residues are not well tolerated [7] . Furthermore, domains far away from the cleavage site can mediate the interaction between substrate and protease (exosites), and although such interactions have not yet been shown for proteases of the caspase family, the high variability of cleavage site motifs in natural caspase substrates argues in favour of the presence of exosites. Known substrates for caspase-6 show a particularly high variability in their recognition sequences [8] , with cleavage sites other than I/D/E/L/T/V, E/ D/Q, X, D found in substrates such as the presenilins (ENDD, [9] ), huntingtin (IVLD, [10] ), DNA Topoisomerase I (PEDD, [11] ), AP-2 alpha (DRHD, [12] ), Periplakin (TVAD, [13] ), FAK (VSWD, [14] ) and TGEV (VVPD, [15] ).
Caspase-6 has garnered much attention recently since it has been shown that it is involved in the developmental pruning of axons [16, 17] , and it has been suggested that similar pathways might erroneously be activated in neurodegenerative disorders such as Alzheimer's (AD) and Huntington disease (HD) [16, 18] . The presence of activated caspase-6 and cleavage of caspase-6 substrates is indeed a hallmark of AD, HD and cerebral ischemia, and has been shown in a number of different animal models and patient brain tissue [18, 19, 20, 21, 22] . To assess caspase-6 activity in cell and tissue samples, peptide substrates or inhibitors need to be titrated accurately to yield meaningful results, since the peptide substrate commonly used to assess caspase-6 activity, VEID, can be cleaved by other caspases as well as the proteasome when used at too high concentrations [23, 24] . In addition, even low concentrations of a VEID substrate can lead to inaccurate results if the relative amount of other proteolytic activity in the sample is significantly higher than that of caspase-6 due to small differences in kcat/Km [4] . A recently developed method using the biotinylated caspase inhibitor zVAD is significantly more specific, but to achieve this specificity, immunoprecipitation and subsequent detection of the active caspase by Western blotting is required [23] . For a more quantitative way of assessing caspase-6-specific activity, we have developed a novel method based on the cleavage of lamin A [24] . Here we show that this method is sensitive, specific and allows for the detection of caspase-6 activity in complex samples. The assay quantitatively measures caspase-6 activity and can be useful for investigating caspase-6 biology as well as the screening and intracellular efficacy assessment of caspase-6 inhibitors.
In order to develop an activity assay that is more specific for caspase-6 than available methods relying on the cleavage of the VEID peptide, we decided to investigate the cleavage of known caspase-6 substrates. To be useful for the measurement of caspase-6 activity, a substrate would ideally be as specific as possible. In particular it should not be cleaved by other proteases activated during apoptosis and be cut by caspase-6 in detectable quantities. The protein substrate should be easy to purify and thus available in larger amounts, and existing neo-epitope antibodies allowing for the specific detection of the cleavage fragment would be an advantage. The nuclear lamin proteins were among the first caspase-6 substrates characterized [25] , and knockdown studies strongly suggest that their cleavage during apoptosis is highly dependent on the presence of caspase-6 [24] . Furthermore, purified lamin A as well as a variety of neo-epitope antibodies against the cleavage fragments are readily available, and high stability of the cleavage fragments without further degradation during the apoptotic process has been described [26] , which should facilitate their detection.
The VEID peptide sequence commonly used in caspase-6 substrates and inhibitors is derived from the lamin cleavage site, but since protein context is known to change the kinetics and specificity of proteolytic events, we decided to compare the cleavage of the VEID peptide to that of the full-length lamin A protein.
The lamin A protein is a more specific substrate than its VEID peptide
We therefore decided to investigate whether lamin A is a specific substrate for caspase-6. In particular, we were interested in the ability of other executioner caspases to cleave lamin A, since these are likely to show high activity in apoptotic extracts where accurate quantification of caspase-6 activity will be of interest. We first subjected the Ac-VEID-AFC substrate to digestion by caspases -3, -6 and -7, respectively. The amount of active enzyme used in each reaction was normalized to the concentration of active sites in the sample as determined by titration against the irreversible inhibitor zVAD-fmk (Fig. S1 ). As expected, the VEID peptide substrate, although it is best processed by caspase-6, still shows significant cleavage by caspase-3 and -7 (Fig. 1A ) at high substrate concentrations (100 mM). Lower substrate concentrations can be used to achieve greater specificity, however, the difference in kcat/Km for VEID is less than 3 fold between caspase -3 and -6 [4] . This results in a small concentration window that can be exploited to achieve selectivity for caspase-6, if the same amounts of active caspase-3 and -6 are present. However, this window will be lost if the sample contains a higher concentration of active caspase-3 than caspase-6. As shown in Figure 1B , an 8 fold molar excess of caspase-3 over caspase-6 is enough to lead to a significantly higher signal from the non-specific caspase-3, making VEID-based assays problematic for the use in apoptotic samples or other cell and tissue lysates with high levels of active caspase-3. Lamin A, on the other hand, only showed proteolytic processing when incubated with recombinant active caspase-6, not with the corresponding amounts of caspases -3 and -7 at concentrations up to 300 mM (Fig. 1C) . We therefore focussed on lamin A as a substrate for the development of a specific caspase-6 activity assay.
To confirm the selectivity of lamin cleavage by caspase-6, we made use of mouse embryonic fibroblast (MEF) cells generated from wild-type and caspase-62/2 (C6wt and C6ko) mice [27] . These cells only differ in their expression of caspase-6 ( Fig. 2A) , making them an ideal system to study the specificity of caspase-6 substrates. Apoptosis was induced by the addition of 50 nM staurosporine to the culture medium, and the activation of caspase-6 was monitored over time by assessing the cleavage of endogenous lamin A via Western blotting (Fig. 2B ) or with the Ac-VEID-AFC peptide substrate (Fig. 2F ). The antibody used to detect full length lamin A at 70 kDa cross-reacts with the closely related lamin C protein (60 kDa, [28] ), and the N-terminal fragments of both cleaved lamin A and C are detected at the same size of 28 kDa (Fig. 2B+E) . Furthermore, using a cell line that does not express caspase-3 but expresses caspase-6 ( Fig. 2C ), we found that the lamin proteins are still processed after the induction of apoptosis with camptothecin ( Fig. 2D ), confirming the requirement of caspase-6 but not caspase-3 for this process.
An increase in VEID cleavage over time was observed for both C6wt and C6ko cell lines (Fig. 2F ). For C6wt cells, significant increases over baseline were observed at all time points, whereas the C6ko cells show lower VEID processing initially with a nonsignificant increase in the first 2 h of treatment. However, at later timepoints C6ko cells exhibit similar levels of VEID cleavage as C6wt with a highly significant increase over non-treated controls (Fig. 2F) , suggesting that other proteases make up for a large proportion of the VEID cleavage activity in apoptotic extracts. These results indicate that the lamin protein substrates are much more specific for cleavage by caspase-6 than the VEID peptide.
Development of an electrochemiluminescence-based ELISA method for the quantitative assessment of cleaved lamin A In order to accurately quantitate the amount of cleaved lamin A generated by caspase-6, we turned to an ELISA-based assay format using the MesoscaleH platform, which allows for a fast and sensitive detection with minimal background using electrochemi-luminescence [29] . To determine whether cleavage of lamin A is more sensitive than cleavage of the VEID peptide substrate in detecting low levels of active caspase-6, we incubated different concentrations of the enzyme in parallel either with Ac-VEID-AFC or purified lamin A protein and determined the amount of cleavage after 30 min by measuring the fluorescence in the sample (for Ac-VEID-AFC) or by subjecting the sample to an ELISA using the lamin A neo-epitope antibody (for lamin A cleavage). Comparison of the results showed that both assays perform at least equally well in detecting active caspase-6 concentrations down to 10 nM, with the lamin cleavage assay showing a linear concentration-response relationship down to the lowest caspase concentrations tested (Fig. 3A) . Signal-to-noise ratios were at or above 3 for the ELISA assay for as low as 10 nM caspase-6, whereas the signal-to-noise dropped below 3 for the VEID-based assay at this concentration (Fig. 3B ). Using a peptide inhibitor for caspase-6, VEID-CHO, both assays furthermore arrived at a similar IC 50 value (lamin-based ELISA: 64620 nM, VEID-based assay: 5666 nM), indicating that the ELISA method is suitable to assess the inhibition of caspase-6 by small molecules or peptides. Overall, our method shows a slight increase in sensitivity over commonly used VEID cleavage methods and is able to reliably detect caspase-6 concentrations down to 10 nM.
Next, we wanted to assess whether the lamin cleavage assay shows higher specificity than the VEID-based system for caspase-6 over other proteases that are activated after induction of apoptosis.
To this end, we tested lysates derived from staurosporine-stressed C6wt and C6ko MEFs with our newly developed ELISA and found a linear increase in signal over time in wt samples, whereas the signal from C6ko samples remained stable at background levels that were similar to the background seen in untreated C6wt (Fig. 3D ). This indicates that the amount of intracellularly cleaved, Figure 1 . The lamin A protein is a more specific substrate than its VEID peptide. A: 100 mM VEID-Afc was incubated with different amounts of caspase -3, -6 or -7 for 1 h at 37uC. Fluorescence generated by cleavage was monitored over time, and the initial, linear portion of the curve was used to calculate the reaction velocity. VEID is preferentially cleaved by caspase-6, but also cross-reacts with caspases -3 and -7 at higher concentrations. Error bars are the SEM of N$3 of 3 independent experiments. B: 5 mM VEID-Afc was incubated with 0.5 mM caspase-6 or different amounts of caspase-3 for 1 h at 37uC. The reaction velocity was calculated as in A. Even at this low concentration of VEID-Afc, the peptide substrate can be cleaved by caspase-3, and an 8 fold higher molar concentration of caspase-3 than caspase-6 results in a higher signal for VEID cleavage by caspase-3 than caspase-6. Error bars are the SEM of N = 3 independent experiments, statistical significance was assessed by 1-way ANOVA and posthoc Dunnett comparisons: *** p,0.0001. C: Pure lamin A protein was incubated with different amounts of caspase -3, -6 or -7 for 30 min at 37uC. Samples were separated by SDS-PAGE and both fragments of cleaved lamin A was detected by Western blotting with antibodies #2031 (full-length lamin A and N-terminal fragment) and #2032 (C-terminal fragment). No cleavage was observed with caspases -3 or -7, while caspase-6 generated lamin A fragments in a dose-dependent manner. A representative image of 3 independent experiments is shown. doi:10.1371/journal.pone.0027680.g001 endogenous lamin A protein is a highly specific readout for the quantification of caspase-6 activated during staurosporine-induced apoptosis.
Kinetic measurements of either Ac-VEID-AFC or lamin A cleavage by fluorescence or our newly developed ELISA allowed comparison of the kinetic parameters of caspase-6 for the two substrates. We find that the full-length lamin A protein has a more than 1000fold lower Km than its cleavage site peptide VEID (Table 1) . Although the kcat value is also decreased, the kcat/Km is still more than 10fold higher for lamin A (Table 1 ). This indicates that the binding between lamin A and caspase-6 is tighter, which might be mediated by domains outside the cleavage site. Such binding sites could also be responsible for the observed specificity of caspase-6 for the full-length protein substrate. The Km and kcat values for VEID are furthermore in good agreement with previously reported data [4] .
Caspase-6 is postulated to be involved in neuronal degeneration and apoptosis [16, 17, 18, 20, 22, 30] , and detection of its activity in neuronal cultures is therefore of paramount interest. Previous studies have frequently used TUNEL assays as a measure of apoptosis, looked at VEIDase activity or the presence of the active caspase-6 fragment by Western blotting or immunostaining and assessed the presence of cleaved caspase-6 substrates [8, 18, 20, 21, 22] . The most specific methods to assess the effect of drug treatments on caspase-6 activity developed so far use antibodies against the active caspase-6 fragment, levels of which Figure 2 . VEID, but not lamin A+C, is cleaved in the absence of caspase-6. A: Caspase-6 protein (full-length, 32 kDa) is detected in MEFs generated from C6wt, but not C6ko mice. B: Endogenous lamin A protein (70 kDa) is cleaved in wt, but not C6ko MEFs after staurosporine stress for 4 h or longer. The antibody cross-reacts with full-length lamin C (60 kDa), and the cleaved band at 28 kDa has the same size for both lamin A+C (lower panel). C: MCF-7 cells express caspase-6, but not caspase-3 protein, whereas both C6wt and C6ko MEFs contain both caspases. hu: human, m: mouse, ns: non-specific band. D: MCF-7 cells were stressed with 5 mM camptothecin for different amounts of time and the cleavage of endogenous lamin A and C proteins was monitored by Western blotting with antibodies antibodies #2031 (full-length lamin A+C) and #2032 (C-terminal fragments). E: Schematic representation of lamin A and C and the caspase-6 cleavage site at AA 230. The N-terminal fragments generated by caspase-6 cleavage (red) have the same size (28 kDa) for both lamin A+C. F: C6wt or C6ko MEFs were stressed with 50 nM staurosporine for different amounts of time, lysates were generated and analyzed for cleavage of VEID-Afc. C6wt cells show a significant increase in fluorescence at each timepoint, the fluorescence signal obtained from C6ko lysates only reach a statistically significant difference from baseline after 4 h. would not necessarily change upon inhibitor treatment, or immunoprecipitation of active caspases with biotinylated zVADfmk, which depends on efficient precipitation and Western blotting for quantification [23] .
Our newly developed ELISA method, however, could not be directly applied to neuronal cultures, since lamin A is not expressed in embryonic mouse brain [31] . Neurons in early stages of development up until postnatal day 5 express lamins of the B1 and B2 subtypes, which differ from the lamin A and C sequence at the caspase cleavage site (VEVD in B-type lamins and VEID in lamins A and C (Fig. 4A) [24, 31] ). Furthermore, the cleavage of lamins B1 and B2 at this site is not specific for caspase-6, as has been shown in a caspase-6-deficient cell line [24] . In agreement with these findings, we observed cleavage of lamin B1 in primary cortical neurons derived from both C6wt and C6ko mice at embryonic day 16.5 ( Fig. 4B) , while lamins A and C were not detected (data not shown). To overcome this obstacle, we decided to spike C6wt and C6ko neuronal lysates with purified lamin A protein, and after incubation at 37uC to allow for its cleavage by endogenous caspase-6 activity, we subjected the samples to our ELISA assay. We detect a significant increase in cleavage of lamin A protein when the incubation was performed in the presence of extracts derived from camptothecin-stressed C6wt neurons, whereas no increase in cleaved lamin A signal was observed in the presence of camptothecin-stressed C6ko neuronal extracts (Fig. 4C) . The ELISA method is therefore also suitable to detect caspase-6 activity in samples that do not contain endogenous lamin A or C protein.
Detection of caspase-6 activity in the absence of endogenous lamin cleavage Caspase-6 activity can be localized to the nucleus, which is commonly associated with cell death and the cleavage of nuclear substrates such as lamin A, whereas a cytoplasmic localization of active caspase-6 as it is the case in neurodegeneration does not result in immediate apoptosis [5, 21, 32] . We therefore decided to test whether spiking of cell extracts with pure lamin A protein can detect active caspase-6 at early timepoints, before its translocation to the nucleus and cleavage of endogenous lamin A. To this end we transiently transfected COS7 cells with full-length human caspase-6, a system in which the enzyme auto-activates in the cytosol before translocating to the nucleus [32] . After transfection, the active form of caspase-6 becomes detectable by Western blotting at the 9 h timepoint (Fig. 5A) . After 24 h of incubation, low levels of endogenous lamin A cleavage were observed with the ELISA method, indicating that at this time point active caspase-6 is present in the nucleus (Fig. 5B) . However, when cell lysates were supplemented with purified lamin A protein and endogenous, active caspase-6 was allowed to cleave the spiked protein, activity could already be detected after 9 h and increased dramatically at later time points (Fig. 5B ), in agreement with the Western blot data (Fig. 5A ). This indicates that the spiking method can detect caspase-6 activity before endogenous lamin is cleaved and is thus suitable to detect non-nuclear caspase-6 activity.
Although our Mesoscale ELISA method shows significant advantages over commonly used, VEID-based assay systems to measure caspase-6 activity, it still requires manual cell lysis and protein quantification steps that are not easily amenable to highthroughput screening campaigns. We therefore turned to a highcontent imaging platform that allows automated liquid handling as well as immunofluorescence imaging and quantification. Using a primary antibody specific for caspase-6 cleaved lamin A, we found increased perinuclear staining in C6wt cells after induction of cell death by camptothecin (Fig. 6A) . The observed blebbing of the nuclear membrane is consistent with the breakdown of the nuclear lamina during apoptosis. No staining for cleaved lamin protein was observed in non-stressed C6wt, non-stressed C6ko or camptothecin-stressed C6ko cells (Fig. 6A ). Using nuclear DAPI staining as a reference point, we quantified the perinuclear immunofluorescence in a ring around the nucleus (Fig. 6A, insets) , and found a 2fold increase in staining intensity in camptothecin-stressed versus non-stressed wt MEFs (Fig. 6B) . Only background signal was observed in C6ko MEFs even after camptothecin treatment, indicating that the detection of cleaved lamin A by immunofluorescence is also specific to caspase-6 (Fig. 6B) . This method is ideally suited for the high-throughput intracellular testing of modulators of caspase-6 activity in in the presence of confounding proteolytic activity, i.e. that of caspase-3.
The study of the role of caspases during apoptosis, but also during non-apoptotic developmental and signalling processes, is hampered by the lack of specific assays to measure the activity of single members of the caspase family. Neo-epitope antibodies against the active forms of caspases are available and can be used in immunohistochemical and Western blotting applications. However, the presence of active caspase fragments does not necessarily correlate with proteolytic activity, since the proteases could be bound to either endogenous or exogenous inhibitors, which is especially problematic in high-throughput screening campaigns aiming to identify caspase inhibitors. Another method using immunoprecipitation with biotinylated zVAD, on the other hand, is specific to active caspases, but quantitation relies on the efficacy of immunoprecipitation and Western blotting [23] .
To be able to accurately quantify the activity of caspase-6 in cell culture, we therefore developed novel activity assays based on the cleavage of the caspase-6 specific substrate protein lamin A [24] . Here, we confirm the specificity of lamin A cleavage by caspase-6 using C6wt and C6ko MEFs that were stressed with staurosporine to undergo apoptosis. Although the antibodies we used in this study cross-react with cleaved lamin C, specificity for caspase-6 cleavage is maintained, since both proteins arise from the same gene by alternative splicing, contain the same caspase-6 cleavage site and only differ in their C-termini with lamin A being 10 kDa longer (Fig. 2D) [28, 33] . The cleaved fragment was detected in C6wt, but not in C6ko MEFs, and we developed an electrochemiluminescence-based ELISA method to accurately quantify cleaved lamin using the Mesoscale platform. The new method has an improved detection limit and signal-noise ratio over commercially available caspase-6 activity assays using the VEID peptide substrate. Furthermore, our assay is specific to caspase-6 and thus superior for the detection of caspase-6 activity in complex samples with a variety of different proteolytic activities such as cell lysates. Through spiking with purified lamin A protein, the assay can be applied to samples lacking endogenous lamin A and C such as embryonic primary neurons. Furthermore, the method is amenable to samples where caspase-6 activity does not lead to the cleavage of the endogenous nuclear lamins and is therefore not necessarily associated with apoptosis.
The specificity is due to the use of a protein instead of a peptide substrate, and even though the VEID sequence is derived from the caspase-6 cleavage site in lamin A (aa227-230), we show that the Kcat/Km value for lamin A is more than 10fold higher than for VEID, indicating that lamin A is a better substrate for caspase-6. A similar effect has been shown for proteases involved in blood Figure 6 . Caspase-6 activity can be quantified by immunofluorescent staining for cleaved lamin A. A: C6wt and C6ko MEFs were stressed with camptothecin, stained with an antibody against cleaved lamin A and analyzed on an automated imaging platform. Nuclei were counterstained with DAPI, and identified by the software (blue circles). Debris (red circles) was not analyzed. The signal intensity in the cleaved lamin A channel was quantified in a ring around the nucleus (green circles). B: Quantitation of the perinuclear staining from cleaved lamin A is graphed. Untreated C6wt MEFs were normalized to 100% and the fold change in stressed C6wt, stressed and non-stressed C6ko MEFs are compared. Error bars are the SEM of N$3 of 3 independent experiments. Statistical significance was assessed by 2way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001. doi:10.1371/journal.pone.0027680.g006 coagulation [34, 35] , where substrates are bound outside the active site of the protease (exosite). There has been speculation about the presence of exosites in caspases [36] , but their existence has not been proven conclusively yet. The example of exploiting protein substrate specificity for the development of an activity assay could be used for the development of more specific assays for other members of the caspase family, such as p23 for caspase-7 [3] .
Caspase-6 has recently emerged as an important player in neuronal dysfunction and degeneration and its activation has been linked to several neurodegenerative conditions such as AD, HD and stroke [16, 17, 18, 19, 20, 21, 22, 30, 37] . Direct inhibition of the enzyme as well as targeting of other players in the activation pathway might therefore be beneficial in these disorders. We show here that the cleavage of lamin A can be assessed in a highthroughput setting using a high-content imaging platform, making it an ideal intracellular readout to test interventions that decrease caspase-6 activity.
All experiments were carried out in accordance with protocols (Animal protocol A07-0106) approved by the UBC Committee on Animal Care and the Canadian Council on Animal Care.
Ac-VEID-Afc, Ac-DEVD-Afc, Ac-VEID-CHO, zVAD-fmk and active caspase-3, -6 and -7 enzymes were purchased from Enzo Biosciences. Lamin A antibodies were from Cell Signaling Technology: cleaved lamin A and total lamin A/C for Western blotting (cat. nos. 2031 and 2032), cleaved lamin A for Mesoscale ELISA (cat. no. 2036) and immunofluorescence staining (cat. no. 2036). Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against fulllength caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R). Camptothecin and staurosporine were from Sigma, cell culture reagents were from Gibco.
The amounts of enzyme indicated in the figures (5-500 nM) were incubated with 100 mM Ac-VEID-AFC at 37uC in caspase cleavage buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT) for 1 h in a black 96well plate (Nunc). Fluorescence was measured every 5 min with excitation at 400 nm and emission at 505 nm. The initial linear part of the curve was analyzed.
For the calculation of active site concentrations, Ac-DEVD-AFC was used for caspases-3 and -7 according to the same protocol, and different amounts of zVAD-fmk were added to all reactions. The ratio between reaction velocity with inhibitor (Vi) over reaction velocity without inhibitor (V0) was plotted against the inhibitor concentration, and the concentration for y = 0 was determined as the active site concentration of the caspase.
The specific activity of the caspase enzymes used was determined with a standard curve using free AFC and the Ac-VEID-AFC or Ac-DEVD-AFC substrate for caspase-6 and caspases -3 and -7, respectively. The resulting specific activities were: 11.7 nmol Ac-VEID-AFC/min/mmol caspase-6, 13.7 nmol Ac-DEVD-AFC/min/mmol caspase-3 and 9.9 nmol Ac-DEVD-AFC/min/mmol caspase-7.
For the assessment of VEID cleavage in cell lysates, lysates corresponding to 100 mg protein were mixed with an equal volume 200 mM Ac-VEID-AFC in 26 caspase cleavage buffer. Fluorescence was measured as described above.
Different concentrations of Ac-VEID-Afc substrate (1-150 mM) were digested with 10 nM caspase-6 in caspase cleavage buffer for 1 h in a black 96well plate (Nunc). Fluorescence was measured every 5 min with excitation at 400 nm and emission at 505 nm. The initial linear part of the curve was analyzed.
Different concentrations of lamin A protein (3-400 nM) were digested with 20 nM caspase-6 in caspase cleavage buffer for 15, 30, 45, and 60 minutes at 37uC. The reactions were stopped by shock-freezing, and thawed samples were analysed with the Mesoscale ELISA system. The cleavage rate was calculated at each lamin concentration and plotted against the concentration of Lamin A protein to calculate the Km and Kcat (Fig. S2 ).
Caspase-6 activity assays using the Mesoscale ELISA system 100 ng pure lamin A protein was subjected to digestion by different concentrations of caspase enzymes as indicated in the figures (0.05-1000 nM) in caspase cleavage buffer for 30 min at 37uC. The reactions were stopped by shock-freezing, and thawed samples were analysed with the Mesoscale ELISA system: 5 ml of each reaction, corresponding to 25 ng lamin A protein, were spotted onto Mesoscale ELISA plates. Samples were incubated at room temperature for 1 h, then 150 ml 5% BSA in PBS was added per well and the plate was again incubated for 1 h at room temperature. All wells were briefly washed 3 times with 150 ml PBS containing 0.05% Tween-20, and 25 ml antibody mix was added per well (cleaved lamin A antibody, Cell Signaling 2036, 1:100, goat anti-mouse sulfo-tag secondary antibody, MSD technology, 1:500 in 1% BSA/PBS). After 1 h incubation at room temperature, all wells were briefly washed 3 times with 150 ml PBS containing 0.05% Tween-20, and 150 ml 26 reading reagent (MSD technology) was added per well. The plates were then read on a Mesoscale platform electrochemiluminescence reader (MSD technology) according to manufacturer's instructions.
For assays using MEF or MCF-7 cell lysates, lysates were diluted in PBS to 0.2 mg/ml, 5 ml were spotted in each well of a Mesoscale ELISA plate and the plate was developed as described above.
For assays using neuronal or transfected COS7 cell samples, lysates corresponding to 20 mg total protein were mixed with 100 ng lamin A protein in 16 caspase cleavage buffer. Samples were incubated for 3 h at 37uC, 5 ml were spotted in each well of a Mesoscale ELISA plate and the plate was developed as described above.
Mouse embryonic fibroblasts (MEFs) from a C6ko mouse and its C6wt littermate were generated from day 12.5 embryos resulting from timed-pregnant heterozygous breedings and tissues not used for culture were genotyped. The generation and characterization of C6ko mice is described elsewhere [27] . Single pups were dissected in ice cold PBS, the body without head, limbs and liver, lung and heart was minced, the centrifuged pellet was digested with 0.25% Trypsin-EDTA at 37uC for 15 min and neutralized with MEF medium (Dulbecco's modified Eagle medium with high glucose, 10% fetal calf serum, 2 mM L-Glutamine, 100 mM non-essential amino acids, 1 mM sodium pyruvate, 1 mM b-mercaptoethanol). Cells were passaged through a pipette tip, centrifuged and suspended in MEF medium containing DNase I. After centrifugation, cells were suspended in fresh medium, incubated for 2 min at room temperature to let debris and cell clumps settle and cells in the supernatant were seeded into cell culture flasks. At passage 2, cells were immortalized by transfection with pSV3-neo SV40 large T antigen (ATCC) with the Fugene reagent (Roche) according to manufacturer's instructions. Immortalized cells were selected and propagated through the addition of 600 mg/ml G418 to the medium.
For the activation of caspase-6, cells were stressed with 50 nM staurosporine for 0-6 h and harvested by trypsinization. Cell pellets were lysed in 50 mM Tris pH 8, 150 mM NaCl and 1% Igepal with 4.2 mM Pefabloc and 'Complete' protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation.
Cortical neuronal cultures were prepared as described previously [38] from E16.5 littermate embryos obtained from timedpregnant heterozygous breedings and tissues not used for culture were genotyped. Cultures were maintained at 37uC under 5% CO 2 and half of the culture media was exchanged every 4-5 days. At day 10 in vitro, 5 mM camptothecin was added to the media to induce caspase activation [39] , and cells were harvested after 30 h of treatment by scraping in ice-cold PBS supplemented with protease inhibitors (4.2 mM Pefabloc and 'Complete' protease inhibitor cocktail (Roche)). Cells were pelleted by centrifugation and stored at -80uC until lysis. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and 'Complete' protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation.
Culture, transfection and lysis of COS7 cells COS7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 2 mM L-Glutamine. Cells were transfected with human caspase-6 cDNA with a C-terminal DDK tag using the Fugene reagent (Roche) according to manufacturer's instructions. Cells were harvested 0-24 h after transfection by trypsinization. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and 'Complete' protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation.
Culture and lysis of MCF-7 cells MCF-7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 2 mM L-Glutamine. Cells were stressed with 5 uM camptothecin for different amounts of time and harvested by trypsinization. Cell pellets were lysed in 50 mM HEPES pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA and 10% glycerol with 4.2 mM Pefabloc and 'Complete' protease inhibitor cocktail (Roche) on ice, and protein concentrations were determined in the cleared lysates after centrifugation.
Western blotting 20 ng of pure lamin A protein was subjected to digestion by different concentrations of caspase-3, -6, and -7 (0.75-6 mM) in caspase cleavage buffer for 30 min at 37uC. The reactions were stopped by shock-freezing, and thawed samples were run on 4-12% Bis-Tris gels (Nupage, Invitrogen), transferred to PVDF membranes by electroblotting and membranes were developed with primary antibodies in 5% BSA/PBS. Fluorescently labelled secondary antibodies and the LiCor Odyssey Infrared Imaging system were used for detection.
For Western blotting of cell lysates, 50 mg total protein were run on 4-12% Bis-Tris gels (Nupage, Invitrogen) and processed as described above.
C6wt and C6ko MEFs were seeded at 2500 cells per well into 96well plates. N = 3 for each treatment. The following day, cells were treated with camptothecin (5 mM final) for 16 hours. Post stress, cells were washed in PBS, fixed in 4% formaldehyde (EMS)/PBS for 60 minutes at 4C, washed with PBS, and permeabilized with 0.3% Triton-X 100/PBS. Primary antibody to mouse cleaved lamin A (Cell Signaling Technology) was added at 1:100, in normal goat serum, and incubated at 4C overnight. Plates were washed twice in PBS before adding secondary antibodies, AlexaFluor 488 anti-mouse IgG (Invitrogen), each at 1:800 in normal goat serum, and DNA staining dye, Hoechst 33342 (Invitrogen), at 1:10000, for 90 minutes at room temperature. Plates were washed again twice and left in PBS.
Labeled cells were analysed using the ThermoFisher Cellomics ArrayScan VTI, a high content scanning (vHCS) microscope, using version 6.6.2.0 software. The ArrayScan VTI captures images using an ORCA-ER camera in two channels applicable to the assay. XF-53 filters for 405 and 488 nm were used in automated image analysis to quantify nuclei number, and cleaved lamin A, respectively. The Cellomics toolbox, Compartmental Analysis, used an algorithm to encircle perinuclear staining of cleaved lamin A. The primary object in the Hoechst channel was identified as the nucleus of the cell of which 500 total were counted per well. In channels 2, the perinucleus was identified as a ''circ'', which was 3 pixels outside the primary object, where the algorithm measured pixel intensity above background staining. The ''MEAN_AvgCirIntensity'' for each channel was used for analysis, under the vHCS analysis tool, and exported to Microsoft Excel spreadsheets and GraphPad Prism for further analysis. Figure S1 Active site titrations for caspases -3, -6, 7. The exact active site concentration of each caspase used was determined by titrating the enzymes against the pan-caspase inhibitor zVAD-fmk [27] . (TIF) Figure S2 Km determination for lamin A. A: The indicated concentrations of lamin A protein were digested with 20 nM caspase-6 and samples were analysed with the Mesoscale ELISA system. B: Cleavage rates were determined as the slope of the curves in (A) and plotted against the lamin A concentration to obtain values for Km and kcat through curve fitting using the built-in function of the GraphPad Prism 5.0 software package. (TIF) Quantifying social distancing arising from pandemic influenza Local epidemic curves during the 1918–1919 influenza pandemic were often characterized by multiple epidemic waves. Identifying the underlying cause(s) of such waves may help manage future pandemics. We investigate the hypothesis that these waves were caused by people avoiding potentially infectious contacts—a behaviour termed ‘social distancing’. We estimate the effective disease reproduction number and from it infer the maximum degree of social distancing that occurred during the course of the multiple-wave epidemic in Sydney, Australia. We estimate that, on average across the city, people reduced their infectious contact rate by as much as 38%, and that this was sufficient to explain the multiple waves of this epidemic. The basic reproduction number, R(0), was estimated to be in the range of 1.6–2.0 with a preferred estimate of 1.8, in line with other recent estimates for the 1918–1919 influenza pandemic. The data are also consistent with a high proportion (more than 90%) of the population being initially susceptible to clinical infection, and the proportion of infections that were asymptomatic (if this occurs) being no higher than approximately 9%. The observed clinical attack rate of 36.6% was substantially lower than the 59% expected based on the estimated value of R(0), implying that approximately 22% of the population were spared from clinical infection. This reduction in the clinical attack rate translates to an estimated 260 per 100 000 lives having been saved, and suggests that social distancing interventions could play a major role in mitigating the public health impact of future influenza pandemics. Infectious diseases are commonly controlled by minimizing contact between infectious and susceptible individuals. Personal measures to reduce potentially infectious contacts are sometimes referred to as 'social distancing'. It has been suggested that policies encouraging social distancing may be effective against pandemic influenza (Bell et al. 2006; Glass et al. 2006) . It is unclear, however, whether individuals can reduce their infectious contact rate to a level low enough to return a worthwhile public health outcome. An examination of levels of social distancing actually achieved during previous epidemics can provide useful guidance as to the effectiveness of social distancing interventions during future influenza pandemics.
The infectiousness of a disease is characterized by the basic reproduction number (R 0 ), which for our purposes is the expected number of infectious contacts per infective when there are no pharmaceutical or behavioural interventions in place and every individual is equally susceptible. More sophisticated definitions are required where individuals have substantially different risks of infection; the methods described by Diekmann & Heesterbeek (2000) are useful in defining and calculating R 0 when contact structures and other kinds of heterogeneity are important. In practice, when an epidemic occurs, the effective reproduction number (R) differs from R 0 due to the deployment of interventions, the build-up of herd immunity and possibly pre-existing immunity.
The benefit arising from interventions that additionally decrease R beyond that expected based on herd immunity alone may differ depending on the magnitude of the decrease and its timing (Bootsma & Ferguson 2007; Hatchett et al. 2007 ). If a reduction in the infectious contact rate can be introduced early and sustained, the overall attack rate can be reduced. For a given decrease in the contact rate, the relative reduction in the attack rate is smaller for larger R 0 (figure 1). For example, halving the infectious contact rate may lead to a major epidemic being averted (i.e. a 100% reduction in the attack rate) when R 0 Z2, but, at most, approximately only a 20% reduction in the attack rate if R 0 Z4 (figure 1).
It is more realistic to assume that interventions to reduce R cannot be sustained indefinitely. If interventions are introduced, and subsequently removed before herd immunity has increased sufficiently to reduce R to approximately 1, this will postpone and diminish the peak incidence of the epidemic (though not necessarily the eventual attack rate), thus reducing the peak load on health services. Finally, we will argue that if the introduction of timelimited interventions (e.g. social distancing) is timed in such a way as to minimize the number of active infective cases as R approaches unity, then the minimum achievable attack rate can be obtained.
Through a combination of geographical isolation and public health measures, the city of Sydney, Australia, delayed the introduction of the Spanish flu by several months until early 1919, at which point public health officials responded almost immediately (McCracken & Curson 2003) . As with many populations affected during the 1918-1919 pandemic (e.g. Geneva, Switzerland; Chowell et al. 2006) , Sydney experienced multiple epidemic waves. There are several theories explaining the multiple waves, including transient post-infection immunity, viral antigenic drift and the involvement of multiple viral strains; substantial counterarguments exist for all these theories and the issue remains unresolved (Taubenberger & Morens 2006) . In the case of Sydney, the beginning of a second wave coincided with the lifting of public infection control measures, suggesting that transient adoption of social distancing measures could underlie the observed dynamics (McCracken & Curson 2003) . More broadly, Hatchett et al. (2007) observed that the quality and timing of non-pharmaceutical public health interventions aimed at decreasing disease transmission by reducing social contact rates appeared to influence the course of influenza epidemics in 17 large US cities during 1918, with second waves occurring only after the relaxation of interventions. We hypothesize that the public of Sydney in 1919 initially responded to the public health measures and subsequently rising and/or high incidence of cases and, particularly, case fatalities by reducing their exposure to potentially infectious contacts. Bootsma & Ferguson (2007) have documented a similar reactive reduction in contact rates in response to high mortality rates arising from pandemic influenza. As the perceived risk decreased, the public subsequently relaxed, returning to normal behaviour. There is a delayed negative feedback between the contact rate and the incidence, and, as with many dynamical systems that experience time lags, oscillations develop. We assume that R 0 is constant over the duration of the epidemic. This is in contrast to Chowell et al. (2006) for example, who assumed that R 0 differed between waves-we consider this to be a phenomenological rather than explanatory assumption.
In this paper, we seek to estimate the degree of social distancing that occurred in Sydney in 1919. To do this, we use the epidemic curve and other historical data to estimate (i) the disease reproduction number over the course of the 1919 Sydney influenza epidemic, (ii) bounds on the fraction of people who were asymptomatic seroconverters (whether infectious or not) in that epidemic and (iii) bounds on the fraction of people who were resistant before the epidemic began (e.g. owing to heterotypic immunity).
The methods used in this paper are described in three sections. Section 2 establishes the relevant aspects of the historical background, including why we argue for attributing the epidemic waves to the effect of social distancing. Section 3 measures the reproduction number on each day of the Sydney epidemic by applying the method of Wallinga & Teunis (2004) . Section 4 presents methods for using the observed reproduction numbers and the cumulative number of cases to derive relationships between the serological attack rate and the initial fraction of the population that are susceptible. Each of these quantities has direct policy implications for an epidemic. They are often incorporated into models (e.g. Ferguson et al. 2005; Longini et al. 2005) , despite considerable uncertainty about which values are appropriate for pandemic influenza.
In this section, we describe the history of the epidemic in Sydney and what is known about the population's behaviour at each stage. The method we subsequently present in §5 relies on using the historical record to identify periods during the epidemic when the population behaved normally with regard to the transmission of disease. We assume that the public's willingness to reduce transmission relies on their perception of the risk associated with the epidemic. We argue that the historical record, as described by McCracken & Curson (2003) , shows periods during which the perceived risk would be high (owing to high infection incidence or the imposition of control measures), and periods when the risk would be perceived as low. Three periods (labelled A, C and E) are associated with a high perceived risk and three others (B, D and F) are associated with a low perceived risk, and consequently normal transmission. Figure 2a shows a summary of these periods and a detailed explanation follows. If the intervention is not introduced immediately and sustained indefinitely, a lower reduction will be achieved.
We define period A as beginning from the time when the first cases were identified (27 January 1919). During this period, extensive infection control measures were imposed, including: closing theatres and public places of entertainment; compulsory wearing of masks on all public transport and in public places; closure of schools; prohibition of race meetings and church services; and removal of patients to hospital and strict quarantine of contact (see McCracken & Curson (2003) for a complete list). As the incidence remained low in comparison with severe epidemics reported from elsewhere around the world, authorities deemed that the threat had passed and most measures were lifted on 1 March. From 1 March until the reimposition of control measures on 24 March (period B), the incidence rose exponentially. Even so, the daily death rate was low in absolute terms (figure 2a) because initial incidence was low, and the mean delay between symptom onset and death was 8.5 days (Armstrong 1920) . During this period, we assume that the population approached normal behaviour.
Things changed on the weekend of 22-23 March, when 20 people died of influenza; infection control measures were reimposed around the end of March. We assume that, from 25 March, the perceived severity of the disease was high enough to reduce transmission. These measures were continued throughout the first wave (period C).
We assume that the decreasing incidence led to a decreased perceived risk and that the public started to resume normal behaviour as the authorities lifted infection control measures in the middle of May. We assume that behaviour approached normal during the period D, 25 March to mid-June.
A second wave began shortly after the infection control measures were lifted (i.e. during period D), and was clearly apparent by mid-June. Even though infection control measures were not reimposed, we assume that the high incidence was a sufficient threat to alter people's behaviour. We define this period of altered behaviour (period E) as running from mid-June to 12 August.
We assume that people resumed normal behaviour by 12 August (thereafter period F), as by then the incidence of hospitalizations and deaths had dropped substantially, and the number of hospitalizations ceased to be reported in daily papers.
We argue that social distancing is an appropriate explanation for the waves for several reasons. Seasonal changes in virus transmissibility, while possible, cannot be of sufficient size to cause multiple waves-particularly over such a short time period. Indeed, seasonal influenza epidemics on an annual basis cannot occur if the difference attributable to seasons is more than approximately 10% of R. Multiple circulating viruses may have contributed to the waves in Europe, where repeat infection was documented (Ministry of Health 1920). However, this could not have occurred in Sydney, where reinfection was extremely rare, and when it did occur the symptoms were mild (Armstrong 1920 ). Armstrong reports that 814 out of 1488 (55%) health care workers were attacked once, yet only four of these (0.5%) were recorded as being attacked twice.
It might be argued that the first and second waves in Sydney were caused by different strains which provided cross-protection. For this to produce two comparable waves would require that the second strain be substantially more infectious (higher R 0 ) than the first to overcome the effects of herd immunity. We will show that the reproduction numbers during both waves in Sydney were remarkably similar. Finally, if applied in a transient manner (i.e. applied then lifted too early), there is an underlying mechanistic explanation of resulting waves (Bootsma & Ferguson 2007) .
Daily hospital admissions attributable to influenza were collated from the Sydney Morning Herald, which published a daily report except that data for weekends were not broken into separate days. Daily data on deaths attributable to influenza came from the New South Wales Statistical Register 1919-1920 (table 105) . These data have already been given in figure 2. Land and sea border control/quarantine surrounding Sydney meant that the overwhelming majority of cases were not imported.
At the height of the epidemic, the Sydney hospitals were overloaded and turned away patients who would have otherwise been admitted (McCracken & Curson 2003) . During the period where the hospitals were not overloaded, the epidemic curve and time-dependent effective reproduction number (see §3.2) can be inferred from either the hospitalization or death data.
We estimated the effective reproduction number R(t) for each day of the epidemic using the method of Wallinga & Teunis (2004) . The method assumes that the infectiousness function, which describes the rate at which an infected individual transmits infection over the course of their infection (Becker 1989) , is known. We derived an average infectivity profile from Ferguson et al. (2005) and defined b(a) to be the average relative infectivity of a person on day a of their infection; transmission was assumed not to occur after 10 days. The mean serial interval arising from the resulting infectivity profile was 2.6 days. The method was applied separately to both the death and hospitalization data. Strictly speaking, the method of Wallinga & Teunis (2004) should be applied to incident infections. As infectious events are rarely observed, we (and previous authors) must use symptom onset, death or some other measure as a surrogate marker for infection. Two issues arise, which are as follows. First, notifications of markers (e.g. deaths) may be substantially thinned versions of incident cases. Second, there is a delay, most likely of variable duration, between the infection and the chosen marker. Wallinga & Teunis (2004) showed that a small degree of thinning (e.g. resulting from under-reporting of cases) would not bias estimates of R(t), but did not investigate the effect of using only a small fraction of cases (as when using deaths as a surrogate when the case-fatality rate is low) to estimate R(t). In the Sydney 1919 epidemic, the probability of hospitalization and death for a given clinical infection was 4.8 and 1.2%, respectively. We used repeated stochastic simulations of an epidemic with R 0 in the range of 1.5-2.5 in a population of 800 000, with the number of daily cases thinned to 5 and 1% to confirm that thinning per se results in no discernible bias in the resulting estimates of R over the course of an epidemic. If the delay from the infection to the chosen marker (e.g. death) is fixed, there is no bias in the resulting estimates of R(t). Conversely, if there is variability in the delay, then there is a potential for bias, particularly if the distribution of the delay is right-skewed. The effect of the time-to-marker delay distribution is to widen the epidemic curve of the marker, relative to the true incidence curve. The wider the distribution from the infection time to the marker, the greater the potential bias. On theoretical grounds, it is easy to show that during the early and late exponential phases of an epidemic (i.e. its leading or trailing edge), every marker gives an unbiased estimate of R, provided that the exponential phase is itself long in duration compared with the width of the distribution for the marked event. During the peaks of the epidemic, the epidemic curve is not exponential and the above result does not apply. In our case, Armstrong (1920) provided data on the distribution of time from the onset of influenza symptoms to death (mean 8.86 days, s.d. 6.0 days), which is well described by a gamma (kZ2.74, qZ3.23) distribution. We repeated our epidemic simulations; this time, modelling the time from infection to death using this gamma distribution shifted 1.5 days to the right to account for the disease incubation period (assumed fixed). Applying the method of Wallinga & Teunis (2004) to the death data confirmed that the resulting daily estimate of R has little discernible bias during the early exponential growth period and again during the final days of the epidemic. Our application of the method to death data does underestimate R during the middle of the epidemic, and overestimate it at the start of the declining phase; the extent of the bias increases with increasing R 0 , though it is less than 10% for a freely evolving epidemic with R 0 Z1.5. During periods when R is close to 1, the bias is also small-this is reflected in the similarity of the hospitalization and death results.
Given that we are predominantly interested in the reproduction numbers during the periods of early exponential growth and during the final cases of the epidemic ( §4.5), we consider that the method of Wallinga & Teunis (2004) produces estimates of R that are adequate for our purposes. To remove dayto-day variation in estimates of R(t) for the purpose of making inference, we fitted a smooth curve to the daily estimates of R(t) using cubic splines with knots every 7 days. Figure 2b shows the daily estimates of the effective reproduction number R(t) based on both the hospitalization and death data. The estimates are noisier during the periods when case numbers are small (e.g. before day 75 and after day 200). As expected,RðtÞ begins above 1 and drops below 1 as the first wave peaks, though not by much ðR min ðC ÞZ 0:85G0:01ðGs:e:ÞÞ. It returns to greater than 1 at approximately day 130 (figure 2b), which is approximately when the second wave of the epidemic began to grow, and remained above 1 until day 165, the peak of the second wave. It is apparent that RðtÞ based on the hospital admissions underestimates R(t) during both waves due to hospitals being overloaded (figure 2b). At times other than early in the epidemic when the number of deaths is very small, the estimates of R(t) based on either hospitalizations or deaths are very similar (allowing for deaths to lag hospitalized cases; figure 2b). We henceforth use deaths only to make inference on R(t). Indeed, we expect there to be less bias in the estimates of R(t) arising from deaths compared with hospitalizations. This is because being admitted to hospital is dependent on many factors unrelated to the epidemiology of disease that may vary over time (e.g. perceived need for hospital care based on the case-fatality rate). The maximum value of the smoothed curve during period B gave an estimate ofRðBÞZ 1:59G0:02ðGs:e:Þ (figure 2b). The mean of the daily reproduction number in period F waŝ RðFÞZ 0:95G0:04ðGs:e:Þ.
In this section, we present a method for inferring the degree of social distancing during different periods of the epidemic. Our method relies on knowing the reproduction number operating at each time (established in §3). We attribute part of the variation in this reproduction number to herd immunity and the remainder to social distancing.
The total population size of Sydney was NZ810 700, of which at least 14 130 (1.74%) were admitted to hospital and approximately 3500 (0.43%) died as a result of influenza infection (McCracken & Curson 2003) . Based on a survey of 600 establishments covering 106 923 employees, the proportion of workers that were absent from duty as a result of influenza was 36.6% (Armstrong 1920, p. 144 ). This was considered as an unbiased estimate of the clinical attack rate, although we argue that the serological attack rate (proportion of workers who developed resistance) may have differed.
We denote the proportion of the population that were recorded as being hospitalized or as having died on day t as h(t) and d(t), respectively; these are known from the data. We denote the proportion susceptible as s(t), and the per capita incidence on day t as i(t). We do not assume that infectives were necessarily symptomatic, but they are all assumed to have become immune. Our model assumes that mixing within the population can be approximated as homogeneous. We assume a form for the effective reproduction number that incorporates the build-up of immunity in the population and social distancing,
where s(t) is a scalar, which describes the extent to which behaviours resulting in disease transmission are maintained. A reduction in s(t) indicates that disease transmission has decreased for some reason other than the depletion of susceptibles. For example, when the population is behaving normally (i.e. no social distancing), s(t)Z1, and when potentially infectious contacts are reduced by half, s(t)Z0.5. We consider that the population closely approached normal behaviour during periods B and F, and possibly during period D, i.e. s(B)Zs(D)Zs(F)Z1 (table 1) . Our aim is to use this model to estimate s(t) by estimating R(t) and s(t). More specifically, we seek to estimate R A ðtÞ Z R 0 sðtÞ Z RðtÞ sðtÞ ; ð4:2Þ
which we refer to as the 'adjusted reproduction number'the adjustment referring to the correction of the effective reproduction number for the proportion of the population that are susceptible. When there is no social distancing, R A ðtÞZ R 0 . Our goal is to estimate how much of the variation in the reproduction number exceeds that which can be attributed to the build-up of immunity, and to attribute that to social distancing. We define s min to be the lowest value of s(t) obtained from the analysis, corresponding to the point of greatest social distancing.
The serological attack rate (final proportion infected and developing solid immunity) is aZs(0)Ks(N). The fraction of the population remaining susceptible at time t is equal to the initial proportion susceptibleKthe cumulative proportion infected by t, sðtÞ Z sð0ÞK ð t 0 iðt 0 Þdt 0 : ð4:3Þ
We do not observe i(t) and must infer it from the daily death and/or hospitalization data. In the case of deaths (which in §3.3 we show yields the best estimate of R(t)), we must account for the time delay (t) between infection and death. The time from symptom onset to death was remarkably similar across all age groups with a mode of 7 days (Armstrong 1920; figure 3 ). We add 1.5 days for the incubation period (Ferguson et al. 2005 ) and round to the nearest integer, so that tZ9.
Hence, re-expressing equation ( are compatible with the observed reproduction number over the course of the epidemic.
In this section, we discuss the possible range of values of the serological attack rate. During the preparation of this paper, a similar theory has been presented (Bootsma & Ferguson 2007 ), which we present in more detail. If social distancing is sufficiently effective (s!1/R 0 ) and can be maintained, then an epidemic will go extinct by the epidemic threshold theorem (Becker 1989) . In a large population, the fraction who become infected in this case is negligible. This may have contributed to the extinction of SARS virus (Riley et al. 2003) .
If an epidemic cannot be contained by social distancing, and goes on to infect a sizeable fraction, the serological attack rate a must lie between a minimum value a min and a maximum value a max . Consider two hypothetical major epidemics, the first without social distancing and the second with what we will argue is optimum effective social distancing. For an epidemic in a reasonably well-mixed population, unimpeded by social distancing, a max is obtained from R 0 and s(0) by the final size equation (Diekmann & Heesterbeek 2000) a max Z sð0Þð1Ke Ka max R 0 Þ: ð4:5Þ
Given estimates of R 0 and s(0), we use equation (4.5) to obtain an estimate of this maximum serological attack rate ðâ max Þ, noting that this estimate is quite robust to a range of underlying spatial contact structures and variation in infective potential among individuals (Ma & Earn 2006) . In the second scenario, we assume that the eventual extinction of the epidemic is a result of the development of resistance in the wider community. The optimum attack rate is obtained by applying social distancing such that as the proportion of susceptibles in the community falls below 1/R 0 , the number of infectives is so small that the epidemic fades out without infecting a significant fraction of the remaining susceptible population. Here, the ultimate proportion of the population remaining susceptible is s(N)Z1/R 0 (if s(N)O1/R 0 , reintroduction of the infection could lead to another epidemic wave). This condition allows us to define a min Zs(0)K1/R 0 and, given estimates of R 0 and s(0), we can obtain an estimate of this minimum attack rate ðâ min Þ. One would think that achieving this limit in practice should be rather difficult due to its extreme nature.
The difference between the scenarios arises due to the following reasons. Once the proportion of susceptibles in the community falls below 1/R 0 , the effective reproduction number drops below unity regardless of the degree of social distancing, and the epidemic is doomed to extinction. A freely flowing epidemic, however, overshoots a min because at this stage the largest number of infectives is active. In the optimal case, social distancing is used to minimize the number of infectives at this stage, so that there is no overshoot. The ultimate attack rate therefore depends on how many individuals are infected as R(t) crosses 1.
In Sydney 1919, the attack rate must have lain between these extremes: a min % a% a max . Based on the difference between our estimates of a and a max , we scale up the number of lives actually lost to estimate how many lives might have been lost if the epidemic had been entirely unimpeded by social distancing. By this measure, the number of deaths per 100 000 of the population that were prevented by social distancing (D) was DZ ða max =a K1Þp !10 5 .
Whether or not social distancing has occurred during an epidemic, if it is relaxed (i.e. s(N)Z1) during the final cases, it follows from equation (4.1) that RðNÞ Z R 0 ð1KaÞ: ð4:6Þ
Under optimal social distancing with a minimum possible attack rate ðaZ sð0Þ K 1=R 0 Þ, we expect R(N) to be unity. In epidemics where transmission is unimpeded (s(t)Z1 throughout), epidemic decline is much more rapid. During the final phase, there are sufficient infectious cases in that many susceptibles are infected, even though the reproduction number is well below 1.
To estimate the reproduction number during periods B and D (RðBÞ andRðDÞ, respectively), we took the maximum of the smoothed estimate of R(t). Our estimate of the final reproduction number (RðFÞ) was the mean of the daily estimates during period F, weighted by the number of deaths on that day. By substituting equation (4.4) into equation (4.1) and solving for s(0) after setting s(B)Zs(F)Z1, we obtain a relationship between s(0) and a along with the reproduction numbers during periods B and F and the associated cumulative number of per capita deaths,
Here, t B refers to the time until the peak in the effective reproduction number during period B, and t F is the time to the middle of period F. We could have additionally usedRðDÞ; however, a priori we were less confident that the population was behaving normally during period D. All analyses were undertaken using the computing environment R v. 2.5.0 (R Development Core Team 2007).
Having estimated the values ofRðBÞ andRðFÞ, equation (4.7) establishes a unique relationship between a and s(0). The reported clinical attack rate is the obvious first choice as an estimate of a, but may be biased for several possible reasons: (i) it is conceivable that clinical cases may not have conferred solid immunity, (ii) cases that seroconvert may be asymptomatic, and (iii) illness may have been mistakenly attributed to influenza when it was in fact caused by another influenza-like illness (e.g. respiratory syncytial virus). Hence, we explore the values of a to be an arbitrary 10% below (0.329) and 10% above (0.403) the reported clinical attack rate. The upper value turns out to be just above the maximum possible under our final approach to estimating a; that is, to use equation (4.7) under the assumption that everyone was initially susceptible to infection (i.e. s(0)Z1). We therefore explore three estimates of a. For each estimate, we compute the corresponding values of s(0), a max , a min , R A (B), R A (D), R A (F) and s min . We estimate R 0 as the average of R A (B), R A (D) and R A (F).
Setting the serological attack rate to the observed clinical attack rate of 0.366 estimates the initial susceptible proportion to be s(0)Z0.912 andR 0 Z 1:76 (table 2) .
Setting the serological attack rate to aZ32.9% (i.e. 10% lower than the clinical attack rate) corresponds to an initial susceptible proportion of s(0)Z0.821. This scenario requires that 10% of those who developed clinical symptoms were not solidly protected against future severe attack, contradicting contemporary observations of influenza-dedicated hospital staff (Armstrong 1920) .
If the population was initially fully susceptible (s(0)Z1.0), a serological attack rate of aZ0.401 is required to explain the epidemic dynamics. Again, if we assume that 0.366 is an accurate measure of the clinical attack rate, then it follows that 8.7% of those infected developed immunity without having developed clinical symptoms to the extent that they did not attend work. Although we do not give credence to a scenario that assumes s(0)Z1.0, as it is probable that there was at least some heterotypic immunity from seasonal influenza, we note that it creates an upper bound of 8.7% for the fraction of infectives who could have been asymptomatic transmitters.
We suggest that, of these three estimates, the survey-based estimate of the clinical attack rate (0.366) is probably closest to the true value of the serological attack rate (i.e. aZ0.366) and hence our preferred estimate of R 0 is 1.76 (table 2; figure 4) .
Each of the three scenarios returns the same value of s min (this is a mathematical consequence of our methods), corresponding to a reduction in the infectious contact rate of 38% during the first wave (table 2) . During the second wave, the maximum estimated reduction in the infectious contact rate was less (24%). Interestingly, the second wave was perceived as being more severe than the first, so the difference between these values may be attributable to the public health policy of encouraging social distancing during the first wave. Alternatively, the difference could be explained by the exceptionally heavy rain that fell nearly throughout the month of May (following the first wave), thus discouraging people from getting out and circulating in the wider population (McCracken & Curson 2003) .
Assuming homogeneous mixing, no social distancing, s(0)Z31.2% and R 0 Z1.76, using equation (4.5), we would expect an attack rate of 58.8%-much greater than the 36.6% observed. Assuming that the number of deaths is directly proportional to the attack rate, the reduction indicates that DZ260 per 100 000 lives were possibly saved as a result of social distancing.
The estimated value of a min was approximately 6% less than the modelled serological attack rate for the three parameter combinations examined. This suggests that few additional lives could have been saved by increasing the degree of social distancing, unless it was able to eliminate the epidemic. The observation that R(t) reduces to near 1 for a prolonged period during the last days of the epidemic further supports the conclusion that a was close to a min . Substituting aZ0.588 into equation (4.6), the expected reproduction number during the final stages of the epidemic is 0.725substantially less than the 0.95 observed. Table 2 . Values of the attack rate a and the corresponding values of the initial susceptible proportion (s(0)), the basic reproduction number (R 0 ), the minimum and maximum fractions that could have been infected (a min , a max ), the adjusted reproduction numbers during periods when we expect that social distancing is at a minimum (R A (B), R A (D) and R A (F)), the social distancing coefficient when social distancing was at its greatest (s min ), and the estimated number of deaths avoided per 100 000 (D). (Values in the first row are computed by assuming that a was 10% less than the reported clinical attack rate with s(0) allowed to vary freely. The second row is computed using the clinical attack rate as an estimator for a. The third row is computed by adjusting a to obtain s(0)Z1.0.) The relationship between the adjusted reproduction number and the number of daily deaths for the first and second waves shows a negative trend-more deaths mean greater social distancing (figure 5a,b). For figure 5c,d, we wish to plot the reproduction number against the number of infections on the same day. Since the number of infections is unknown, we use the number of deaths 9 days later as a proxy. The clockwise cycles reveal the delay between the infection and the subsequent decline in R A (t)-and hence the degree of social distancing.
We have assumed an 'all or nothing' model of prior immunity, meaning that a fraction of individuals were totally protected from infection during the pandemic period. The main alternative model of prior immunity is that a fraction of the population is partially immune, having a lower (but non-zero) risk of infection. Under some circumstances, there will be material differences between the behaviour of these prior immunity models: if R 0 is very large, all susceptibles, whether fully or partially immune, will inevitably be infected; alternatively, if there is assortative mixing between classes of susceptibles, fully susceptibles will be overrepresented during the early stages of the epidemic and underrepresented in later stages. These circumstances do not apply to the Sydney 1919 epidemic-there was a reasonably low attack rate (less than 50%) and little evidence to support strongly assortative mixing. While our model result is that 10% of the population were fully immune, for these data we cannot easily distinguish this from alternatives, such as where 20% of the population had 50% of the normal risk of infection.
While the infectivity profile we use has empirical support, it is interesting to consider the effect of changing the infectivity profile. Had we used an infectivity profile with a shorter mean serial interval, we would have obtained reproduction numbers closer to 1, meaning that smaller changes in the degree of social distancing would explain the epidemic waves. However, the reproduction number cannot be reduced much below 1.6 before it becomes impossible to achieve an attack rate of 36.6%, in an epidemic with two waves of similar magnitude. On the other hand, a longer serial interval would have produced higher estimates of R 0 . In this case, we have underestimated the social distancing achieved during the 1919 epidemic.
It is possible that other interventions, such as closing schools and quarantining infectives, played a role in containing the epidemic. We argue that most of these can be broadly categorized as social distancing. Measures such as quarantine are likely to have been practised more or less constantly throughout the epidemic and probably did not contribute to the changes in R(t).
We conclude that the variable application of social distancing, whereby individuals reduced their infectious contact rate in response to the perceived risk, is a plausible explanation for the multiple waves of pandemic strain influenza seen during 1919 in Sydney, Australia. Indeed, while the waxing and waning of the multiple waves appears dramatic, the degree of social distancing required to explain this (in this case, at most, halving one's infectious contact rate) seems quite possible. More generally, Bootsma & Ferguson (2007) and Hatchett et al. (2007) have demonstrated that variation in the timing of introduction and lifting of non-pharmaceutical interventions aimed at reducing contact rates can explain why cities experienced different inter-wave periods, ranging from being so short as to be undetectable through to several months (Taubenberger & Morens 2006) . We note, however, that transient social distancing certainly does not explain why the case-fatality rate of the 1918-1919 pandemic typically was higher during the second wave, as indeed was the case for Sydney (McCracken & Curson 2003) . However, note that the very similar reproduction numbers observed during both waves of the epidemic support our initial assumption that R 0 did not differ over the course of the epidemic. Subject to the assumption that infection at any time conferred protection against a subsequent severe attack, we conclude that approximately 9% of the population were resistant to the epidemic strain prior to the epidemic, and that, during the epidemic, not more than approximately 9% of infections that conferred resistance to the epidemic strain were subclinical to the extent that people were able to continue working. Using our best estimate that 91.2% of individuals were initially susceptible, the R 0 of the 1919 influenza epidemic in Sydney was 1.8, consistent with recent estimates that have used a similar mean serial interval (Ferguson et al. 2005; Sertsou et al. 2006) . The observed attack rate, however, was substantially less than would be expected for this basic reproduction number, and we argue that social distancing is a plausible reason for this. This result underlines the effective role that social distancing could possibly play in mitigating the effects of a future pandemic of influenza. Novel Inhibitor Design for Hemagglutinin against H1N1 Influenza Virus by Core Hopping Method The worldwide spread of H1N1 avian influenza and the increasing reports about its resistance to the current drugs have made a high priority for developing new anti-influenza drugs. Owing to its unique function in assisting viruses to bind the cellular surface, a key step for them to subsequently penetrate into the infected cell, hemagglutinin (HA) has become one of the main targets for drug design against influenza virus. To develop potent HA inhibitors, the ZINC fragment database was searched for finding the optimal compound with the core hopping technique. As a result, the Neo6 compound was obtained. It has been shown through the subsequent molecular docking studies and molecular dynamic simulations that Neo6 not only assumes more favorable conformation at the binding pocket of HA but also has stronger binding interaction with its receptor. Accordingly, Neo6 may become a promising candidate for developing new and more powerful drugs for treating influenza. Or at the very least, the findings reported here may provide useful insights to stimulate new strategy in this area. In recent years, severe flu-like human cases were reported around the world and subsequently the causative virus was identified as the influenza A virus [1, 2] . The virus was spreading rapidly around the world and had been identified as a new reassortant with three genetic lineages, mainly with a swine origin. Therefore, it was called swine-origin influenza virus (S-OIV). Owing to its extremely rapid human-to-human transmission rate, within only two months the 2009 S-OIV had been detected throughout the entire world. On June 11th, 2009 the World Health Organization (WHO) declared an official pandemic, the first pandemic in the 21st century [3] .
Influenza A virus that belongs to the Orthomyxoviridae family is a negative-strand segmented RNA virus, in which the surface membrane proteins are constituted by three important components: M2 proton channel, hemagglutinin (HA), and neuraminidase (NA). The M2 proton channel is responsible for proton conductance vitally important to viral replication. HA is responsible for binding to the surface of the infected cell as a trimer leading to the attachment and subsequent penetration by viruses into the target cell. NA is responsible for cleaving the terminal sialic acid moieties from the receptors to facilitate the elution of the progeny virions from the infected cell [4] . Therefore, any of the three components can be the target for drug design against influenza virus. Recently, stimulated by the successful determination of its high-resolution three-dimensional structure [5] , many discussions about the M2 channel have been made in this regard [5, 6, 7, 8, 9] . The two existing M2 drugs, amantadine (Symmetrel) [10] and rimantadine (Flumadine) [10] approved by FDA, are no longer effective because of their inefficacies to influenza virus.
Sialic acid (SA) as a natural ligand combines with both of the glycoproteins (HA and NA) and located at the membrane of host cell, which is the basis of heme-agglutination when viruses are mixed with blood cells and entry of the virus into cells of the upper respiratory tract [11, 12] . According to the mutagenic analysis the residues of both HA1 and NA binding sites are quite conserved for most influenza A strains [13, 14] .
Owing to its deep active site cleft, the NA has been an attractive target for drug design. Both zanamivir and oseltamivir were designed by modifying the sialic acid (SA) structure. The two FDA-approved clinical drugs were once successfully used to inhibit the spread of influenza viral progeny [15] by binding to viral surface glycoprotein of neuraminidase (NA) [15] . However, it has also been found from several clinical cases [16, 17, 18] that oseltamivir failed to treat avian influenza virus. It is both antigenic drift (sequence base mutations) and antigenic shift (genetic recombination) of segmented RNA genome of influenza viruses that have caused the NA inhibitor being resistant [19, 20] .
HA facilitates viral entry through binding to the host surface sialic acid residues [21] . Accordingly, if HA is blocked at its sialic acid binding site by a small molecule, the viral entry process will be stopped and the penetration of viruses into host cell prevented. In comparison with NA inhibitors, the HA inhibitors were usually more effective in inhibiting influenza virus. For all the HA subtypes (H1-H16) so far identified [22] , the HA1 subtype from the recent pandemic H1N1/09 virus was taken as the target for constituent screening and drug design [23] .
Despite of many year scientific research efforts, so far there is no clinical available inhibitor against HA1. On the other hand, many studies have indicated that computational approaches, such as structural bioinformatics [24, 25] , molecular docking [26, 27] , pharmacophore modeling [28] , identification of proteases and their types [29] , and HIV protease cleavage site prediction [30, 31] , can timely provide very useful information and insights for drug development. Encouraged by the aforementioned studies, the present study was initiated in an attempt to find a new antiinfluenza compound by screening the fragment database for the optimal constituent inhibitor. Meanwhile, the techniques of the core hopping with glide docking and molecular dynamic simulation were also utilized to analyze the binding interactions between the inhibitor and HA1, in hopes that the findings thus obtained will be useful for developing new and powerful drugs against H1N1 influenza virus.
The crystal structure for the HA1 subtype from the recent pandemic H1N1/09 virus was downloaded from the PDB Bank [32] . Its PDB ID is 3AL4. The antigenicity of the HA1 from the swine-origin A (H1N1)-2009 influenza A virus is quite similar to that of the HA from the 1918 pandemic virus [23] . The bindingsite was identified by the SiteMap tool in Schrodinger Suite 2009 (www.schrodinger.com) as described in [34, 35, 36] . The bind-site encompassed the ligand N-Acetyl-D-Glucosamine (NAG), which is observed in complex with HA1 of the crystal structure (PDB: 3AL4). Shown in Fig. 1 is a close-up view for the binding site of protein HA1 rendered by the molecular surface. The binding pocket is formed by those residues that have at least one heavy atom (i.e., an atom other than hydrogen) with a distance ƒ5 Å away from any heavy atom of NAG ligand when it is bound to the receptor at the binding site, as elaborated in [37] . The segments of loop1, loop2, loop3, and loop4, which play an important role in the interactions with the ligand, are shown by four different colors with their respective key residues: Ser92, Glu72, Pro143, and Arg227 ( Fig. 1) . The motions of such four residues were monitored during the molecular dynamics simulations.
The drug-like database and the fragment database derived from ZINC [38] were used for virtual screening and core hopping searching, respectively.
The Glide5 docking program [39] interfaced with Schrodinger Suite 2009 [33] was used to screen the drug-like database from ZINC [38] based on the 3D structure of 3AL4. The preparation and refinement protocols for protein receptor and all compound structures were performed on the Protein Preparation Wizard and LigPrep modules embedded in Schrodinger 2009 [33] , respectively. For protein preparation, the process included assigning bond orders, adding hydrogen, treating metals, treating disulfides, deleting waters and alleviating potential steric clashes, adjusting bond order and formal charges by protein minimization with the OPLS2005 force field [40] , the constrained refinement value of RMSD for the protein was limited to 0.3 Å . Meanwhile, for the compounds, the preparation consisted of the generating possible states by ionization at target pH 7.062.0, desalting, retaining chiralities from 3D structure and geometry minimization with the OPLS2005 force field [40] . When the above steps were accomplished, all investigated compounds were docked into the receptor pocket through the rigid docking model with the Standprecision (SP) scoring function [41, 42] to estimate the binding affinities.
Many useful clues for drug design can be achieved through molecular docking studies (see, e.g., [24, 27, 43, 44, 45, 46] ). In order to gain even more useful information in this regard, the novel drug design algorithm called ''Core Hopping'' [33] was used in this study that has the function to perform both the fragment-based replacing and molecular docking. Such method is particularly useful for de novel drug design because it can improve the activity of the template, which was ZINC01602230 compound in this study. As a lead compound screened out from the drug-like database, the template was taken as an initial structure to subject to optimization via the core hopping method by finding the optimal cores that are attached to the scaffold part of the template in binding with the protein receptor.
During the process of core hopping, the first step was to define the points at which the cores were attached to the scaffold. It was performed in the Define Combinations Step from the Combinatorial Screening panel [33] . The second step was to define receptor grid file, which was done in the Receptor Preparation panel [33] . The third step was to prepare the cores attached to the scaffold for the fragment database derived from ZINC [38] . Finally, the cores thus obtained were sorted and filtered by goodness of alignment and then re-docked into the receptor after attaching the scaffold, followed by using the docking scores to sort the final molecules.
Many marvelous biological functions in proteins and DNA and their profound dynamic mechanisms, such as switch between active and inactive states [47, 48] , cooperative effects [49] , allosteric transition [50, 51] , intercalation of drugs into DNA [52] , and assembly of microtubules [53] , can be revealed by . The binding pocket is defined by those residues that have at least one heavy atom with a distance 5 Å from the NAG ligand [37] . The four loops (loop1, loop2, loop3, and loop4) that play an important role in interacting with the ligand are represented by round ribbons of four different colors as well as their key residues Ser92, Glu72, Pro143, and Arg227, respectively. The motions of such four residues were monitored during the molecular dynamic simulation. The docked poses for ZINC01602230, Neo and Neo6 are shown with the stick model colored in purple, yellow and dark green, respectively. doi:10.1371/journal.pone.0028111.g001 studying their internal motions [54] . Likewise, to really understand the action mechanism of a receptor with its ligand, we should consider not only the static structures concerned but also the dynamical information obtained by simulating their internal motions or dynamic process.
In order to examine whether the designed inhibitor remains bound in the presence of explicit solvent from a dynamic point of view, the molecular dynamic simulation was performed with GROMACS 96-53a6 force fields [55] with the periodic boundary conditions (PBC) by using GROMACS 4.0 package for Linux. The topology files and charges for the ligand atoms were generated by the Dundee PRODRG2.5 Server (beta) [56] . Before starting the simulations, all the models were solvated with the explicit simple point charge (SPC) water in a cubic box. The models were covered with a water shell of 1.0 nm from the surface of the protein. The system was neutralized with six chlorine ions to replace the six SPC water molecules. Subsequently, the energy minimization was performed for the system concerned by using the steepest descent until touching a tolerance of 100kJ/mol. And then, the 10 ns MD simulations were carried out with a time step of 1 fs; the corresponding coordinates were stored every 100 fs. The PME algorithm was used to calculate the electrostatic interactions. All simulations were run under the periodic boundary condition with NVT ensemble by using Berensen's coupling algorithm for keeping the temperature at 310 K and pressure at 1atm. All bonds were constrained by using the LINCS algorithm. The GROMACS 4.0 package was utilized to analyze the results.
The drug-like database from ZINC [38] was screened by using Glide5 for its near-optimal performance aimed on targeting the HA1 receptor (PDB ID:3AL4). The top hit (ZINC01602230) or (2amino-N-(7H-purin-6-yl) acetamide) (Fig. 2) , a compound condensation product of Glycine and Adenine, which was considered as the most potential lead compound for further modification. Subsequently, the core hopping method was employed to search the fragment database for replacing the adenine part. Finally, the new structure Neo was discovered that has more strong affinity than ZINC01602230.
The flowchart to show the process of finding the desired inhibitor is given in Fig. 2 , from which we can see that after the ZINC01602230 was screened out from the Zinc drug-like database, the core hopping method was used to optimize the core1 to core2 by means of searching the ZINC fragment database. The binding affinity between the NAG and the receptor was used as the filtering set. As a result, the compound with the top hits, Neo, was selected for further optimization.
As shown in Fig. 1 , the rigid core2 fragment in Neo sticking out of the active pocket might not well adhere to the active pocket surface. To improve its binding affinity to the target protein, the bond C-N was cut off at the site shown in Fig. 2 . As a consequence of doing so, only the pyridine remained and the whole structural flexibility was enhanced so as to have the ability to stretch out to complement the surface of HA1 binding site.
The new scaffold as a building block was further optimized through the second core hopping process by replacing the core3 with various fragments by searching the fragment database. Interestingly, the best substitute core4 also contains the same glycine as the terminal fragment on the other side of Neo or ZINC01602230. Subsequently, a series of compound candidates modified from the Neo structure were generated, and then the top ten compounds with the best binding affinity computed by Glide5 program [39, 42] were listed in Fig. 3 .
As can be seen from Fig. 4 , the result obtained from the docking simulation has proved that the compound binding interactions with residues ARG227 and ASP92 were fully consistent with the previous report [57] . The structure of Neo6 complemented the shallow pocket of HA1 with the optimal conformation. The side chains of the key residues, such as Arg227, Pro143, Glu72 and Asp92 in protein, made a major contribution to the receptor-ligand binding affinity by forming H-bonds with the different heavy atoms (e.g. O, N) of the Neo6 (Fig. 4) . Besides the common H-bonds formed between the three residues (Arg227, Pro143, and Glu72) and the compound Neo6 as in [58] , the other two H-bonds were formed between the two nitrogen atoms of the new extensible fragment core4 and the oxygen atom of Asp92 residue. Consequently, compared with ZINC01602230, the binding affinity of Neo6 with the receptor was strengthened from 25.83 kcal/mol to 28.38 kcal/mol (Fig. 3 ).
Furthermore, molecular dynamics simulations were performed for the inhibitor-complexed system HA1-Neo6 and the inhibitoruncomplexed system HA1, respectively. The root mean square deviation (RMSD) from initial conformation is a central criterion used to evaluate the difference of the protein system. The stability of a simulation system was evaluated based on its RMSD. The RMSD values for both Neo6-HA1 (green curve) and HA1 (red curve) versus the simulation time were illustrated in Fig. 5A , in which the RMSD for Neo6-HA1 system is a little smaller than that of HA1 system, indicating that the flexibility of HA1 was decreased after the Neo6 binding to HA1. In order to investigate the motions about the important residues interacted with the inhibitor in the binding site defined as loops (Loop1-Loop4) in Fig. 1 , the root mean square fluctuations (RMSF) for all the sidechain atoms of protein were calculated, as shown in Fig. 5B . The curves of RMSF associated with Loop1, Loop2, Loop3, and Loop4 are colored orange, light blue, dark blue, and maroon, respectively. It can be clearly seen from Fig. 5 that the fluctuating magnitudes of the four loops in HA1 are much larger than those in Neo6-HA1, clearly indicating that the receptor HA1 is more stable after binding with the ligand Neo6. The RMSD for all backbone atoms of the Neo6-HA1 system (green) and the HA1 system (red). (B) The RMSF for side-chain atoms of the Neo6-HA1 system (green) and the HA1 system (red). The curves associated with Loop1, Loop2, Loop3, and Loop4 are colored orange, light blue, dark blue, and maroon, respectively. doi:10.1371/journal.pone.0028111.g005 Accordingly, among the series of Neo compound candidates, Neo6 is anticipated to be a promising drug candidate for further experimental investigation to develop new and effective drug against influenza viruses. Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to “right next door”: one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors. It is generally accepted that viruses, released as cell-free virions from an infected cell, transmit to distant cells and tissues. This spreading pathway contributes to wide-ranged diffusion of cell-free viruses. However, in this spreading pathway, viruses are exposed to host anti-virus defense systems. In contrast, direct infection to a neighboring cell is considered to be beneficial for the virus in terms of evasion from the host anti-virus defense. There are two typical manners in infection to ''right next door'': one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission [1, 2] .
The cell-cell fusion infection pathway is characteristic for a variety of virus such as paramyxoviruses, herpesviruses, some retroviruses, and so on. For example in the case of measles virus belonging to Paramyxoviridae, infection is initiated by the interaction of the viral hemagglutinin glycoprotein with host cell surface receptors. The virus penetrates into the cell through membrane fusion mediated by the interaction of the fusion glycoprotein. In later stages of infection, newly synthesized glycoproteins accumulate at the cell membrane resulting in fusion of the infected cell with neighboring cells by producing syncytia. Thus, viruses can spread from cell to cell without producing cell-free virus particles.
The examples of the cell-to-cell transmission are diverse, and these mechanisms are dependent on pairs of viruses and host cells. Vaccinia virus particles bound on the filopodium of an infected cell are repelled toward neighboring uninfected cells by the formation of filopodia using actin filament [3] . The filopodia direct viruses to uninfected cells. Immunotropic viruses including retroviruses utilize an immunological synapse, designed as virological synapses for the cell-to-cell transmission [4] [5] [6] [7] . Claudin-1 and occludin, components of tight junction, are involved in hepatitis C virus (HCV) entry through the cell-to-cell transmission [8, 9] . The cell-to-cell transmission through tight junction is also observed in other viruses which infect epithelial layers [10, 11] . These retroviruses and HCV remain on the surface of an infected cell even after budding. The uninfected cells adjacent to these infected cells can accept or take over viruses from the infected cell. Thus, the cell-to-cell transmission can be categorized into two manners based on the state of infecting viruses, either cell-free or cell-associated virions.
Influenza virus, belonging to the family of Orthomyxoviridae, is one of the most serious zoonotic pathogens and causes seasonal epidemics or periodic pandemics among human beings around the world. The viral envelope consists of a lipid bilayer derived from cells that anchors three of viral transmembrane proteins, hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Influenza virus infection is initiated by the attachment of HA on virus particles to cell surface receptors containing sialic acids [12] . It has been known that the specific interaction between HA and sialic acid species is one of the determinants of the host range of influenza viruses [13] . Beside its role in the viral attachment, HA is also involved in intracellular fusion between viral envelope and host cell endosome membrane in the endocytotic pathway, by which the virus content is released inside the host cell [14] . The functional maturation of HA is mediated by the cleavage of HA into two disulfide-linked glycopolypeptides, HA1 and HA2 [15] , accomplished by trypsin or trypsin-like proteases derived from host cells [16] [17] [18] [19] . The membrane fusion is induced by a conformational change in the mature HA, which is triggered at low pH in the endosome, allowing viral ribonucleoprotein complexes to release into the cytoplasm [20, 21] . Thus, HA plays a critical role in initiation and progression of influenza virus infection. Influenza virus NA possesses the enzymatic activity that cleaves a-ketosidic linkages between terminal sialic acids and adjacent sugar residues of cellular glycoconjugates [22] . The sialidase activity of NA removes terminal sialic acid residues from HA and NA proteins as well as host cell surface glycoproteins. Since the terminal sialic acid of sialyloligosaccharides is critical for HA binding, the receptordestroying activity of NA serves to counter the receptor-binding activity of HA. It is quite likely that this activity contributes to prevention of successive superinfection of an infected cell [23] . In the absence of the functional sialidase activity, progeny virions aggregate on the cell surface due to the HA receptor-binding activity and can not be released [24, 25] . Thus, NA cleaves sialic acids from the cell surface and facilitates virus release from infected cells. However, it is not clear whether every progeny virion is released as cell-free virion to infect the uninfected cells after diffusion into the extracellular environment. Influenza viruses are generally transmitted as cell-free viruses from infected to uninfected cell but they may also infect through the cell-to-cell transmission, in particular during local lesion formation.
Here, we examined whether influenza virus transmits from an infected cell to adjacent uninfected cells without virus release. Live cell imaging techniques showed that a recombinant influenza virus, in which the NA gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. Furthermore, progeny virions remain associated on the surface of infected cell even after budding, and then progeny virions could be passed to adjacent uninfected cells.
To examine the transmission pathway of influenza virus, we performed immunofluorescence analyses by using anti-nucleoprotein (NP) polyclonal antibody. Influenza virus can form an infection center even in the presence of oseltamivir, a potent NA inhibitor (commercially known as Tamiflu) [26] [27] [28] . Oseltamivir at the concentration of 50 mg/ml completely prevented the release of progeny influenza viruses ( Figure 1A ). Noted that a large number of single fluorescent foci caused by initial infection markedly expanded and formed cell clusters consisting of 5-10 infected cells in an MDCK cell monolayer ( Figures 1B and S1 ), suggesting influenza virus can spread to some extent in the presence of oseltamivir. To verify that NA is not involved in this spreading, we generated an NA-deficient influenza virus by a reverse genetics method as described previously [29, 30] . The NAdeficient influenza virus contains a mutated NA segment, in which the NA coding region including a sialidase catalytic domain was replaced with the enhanced green fluorescent protein (EGFP) gene [29] . By this replacement, the NA activity is eliminated from the recombinant influenza virus, and EGFP can be utilized as a marker for viral infections. Immunofluorescence analyses demonstrated that the NA-deficient influenza virus also forms infected cell , culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate standard deviation (s.d.) from 3 independent experiments. (B) Confluent MDCK cells were infected by wild-type influenza virus A/WSN/33 or NAdeficient influenza virus at MOI of 0.0001 in the presence or absence of 50 mg/ml oseltamivir phosphate. NA-deficient influenza virus was generated by reverse genetics as previously described [29] . After incubation at 37uC for 36 hours, immunofluorescence analyses were performed using anti-nucleoprotein (NP) polyclonal antibody and antirabbit IgG antibody conjugated to Alexa Fluor 568 (Invitrogen). Scale bar, 100 mm. doi:10.1371/journal.pone.0028178.g001 clusters similarly to those formed by wild-type influenza virus in the presence of oseltamivir ( Figure 1B) . The fluorescence pattern of NP overlapped with the localization of GFP derived from the EGFP gene of the NA-deficient influenza virus ( Figure S2 ). Thus, NA-deficient influenza virus can be used to investigate the NAindependent infection pathway of influenza virus.
Next, we performed live cell imaging analyses to directly observe the infection time course of the NA-deficient influenza virus. The GFP fluorescence derived from the NA-deficient influenza virus first appeared in a single cell on an MDCK cell monolayer at 24 hours post infection. The virus started to spread from an infected cell to adjacent cells in 5-6 hours after the first appearance of a GFP-positive cell ( Figure 2 and Video S1). The spreading rate was clearly faster than the rate of cell divisions. The mean doubling time of uninfected MDCK cells was 20-24 hours under the condition employed here, and it is expected that the proliferation speed would be much slowly because infected MDCK cells were maintained in the serum-free medium and formed cell monolayer at the high cell density. These suggest that NA-deficient influenza viruses may infect adjacent cells through the cell-to-cell transmission mechanism without apparent production of cell-free virions.
The cell-to-cell virus transmission pathway could be interpreted as one of viral evolving strategies to avoid neutralizing antibody responses [2, 31, 32] . Therefore, we examined the effect of neutralizing antibody on NA-deficient influenza virus. A polyclonal antibody with the neutralizing activity against influenza virus particles inhibited infection of cell-free viruses to less than 50% at the concentration of 0.03%, although the cell cluster formation was observed at the concentration less than 0.01%. On the other hand, the NA-independent transmission of the NA-deficient influenza virus was blocked only when neutralizing antibody was present at the concentration of 0.3% ( Figure 3 ). These results indicated that the NA-independent transmission of influenza viruses is less sensitive to the neutralizing antibody.
Next, to investigate the mechanism of NA-independent transmission of influenza virus, we examined whether HA is involved in this transmission. In the absence of the NA activity, virus spreading from an infected cell to adjacent cells was dramatically suppressed by omission of trypsin, essential for maturation of HA, from the experimental condition ( Figure 4A ). The GFP fluorescence derived from NA-deficient influenza virus appeared in a single cell at 24 hours post infection. However, this virus did not spread, but rather disappeared during subsequent 24 hours (Video S2). These observations indicate that the NAindependent cell-to-cell transmission of influenza virus is dependent on HA maturation mediated by trypsin, as is the case for the general cell-free transmission of this virus.
To clarify whether virus particles or viral RNP complexes are transmitted to adjacent cells, we examined the effect of amantadine on the cell-to-cell transmission of influenza virus. Amantadine inhibits the early step of uncoating of influenza virus RNP from virion in endosomes [33, 34] . For this study, other influenza virus strain, influenza virus A/Udorn/72, was used instead of influenza virus A/WSN/33 because influenza virus A/ WSN/33 is highly resistant to amantadine [35] . We confirmed that influenza virus A/Udorn/72 is sensitive to oseltamivir ( Figure  S3 ) and could also spread via cell-to-cell transmission independent of the NA activity as did for influenza virus A/WSN/33 ( Figures 1B and 4B ). In the case of a single administration of amantadine, fluorescent foci derived from infected cells scattered, and the number of single foci was greatly decreased compared with that in the absence of the drugs. In contrast, a single administration of oseltamivir, fluorescent foci formed some clusters and expanded in a time-dependent manner ( Figure 4B ). This dissimilarity of inhibitory manner was caused by the difference of the sites of action between amantadine and oseltamivir. Amantadine inhibits the replication of influenza A virus by preventing the translocation of vRNP complexes from endosomes to the cytoplasm, whereas oseltamivir has no effects on viral replication itself but inhibits the release of cell-free virions from infected host cells. We investigated the inhibitory effect of amantadine on the cell-to-cell transmission of influenza viruses. The formation of infected cell clusters was observed with co-administration of amantadine and oseltamivir, as well as with a single administration of oseltamivir ( Figure 4B ). However, the quantitative analysis revealed that the size of infected cell clusters with the coadministration were decreased as compared to that with oseltamivir alone ( Figure 4C ). These observations indicated that the NA activity-independent cell-to-cell transmission of influenza virus was susceptible to the inhibitory effect of amantadine, suggesting that the cell-to-cell transmission undergoes through endocytosis but vRNP complex itself is not incorporated in the infected cells by adjacent cells.
The virus transmission undergoes from infected to uninfected cells through either basolateral [36] [37] [38] or apical [39] [40] [41] [42] sides. In the case of influenza virus, cell-free progeny virions are released only from the apical surface of polarized epithelial cells [43] . This releasing polarity is achieved by directed transport of viral membrane proteins to the apical plasma membrane [44] . Indeed, that HA and NA glycoproteins are associated with lipid rafts, and the raft association has been implicated in apical transport [45, 46] .
To determine whether or not the cell-to-cell transmission of the NA-deficient influenza virus occurs on the apical surface, we performed transwell assays in the presence of the neutralizing antibody to influenza A viruses. The neutralizing antibody was added to infected MDCK cell monolayer from apical or Immunofluorescence analyses were performed with cells infected with wild-type influenza virus at 18 hpi using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). GFP fluorescence derived from the recombinant virus was observed at 36 hpi. Scale bar, 100 mm. (C) The level of viral spreading was indicated in the graph by measuring NP and GFP derived from wild-type and NA-deficient virus, respectively. Five different microscope fields were taken randomly, and then the intensity of green color was analyzed with ImageJ NIH image processing software. Each result was represented by a value relative to that in the absence of neutralizing antibodies. Error bars indicate s.d. from 3 independent experiments. doi:10.1371/journal.pone.0028178.g003 basolateral side, and the inhibitory effect on the spread of GFP fluorescence derived from the recombinant virus was examined. Addition of high concentrations of the neutralizing antibody from the apical side blocked the cell-to-cell transmission of the NAdeficient influenza virus, whereas the addition from the basolateral side had no effect ( Figure 5 ). These observations indicated that the polarity in the influenza virus budding in the cell-to-cell transmission pathway is apical.
Previous report showed that influenza viruses were refractory to superinfection with a second cell-free virus [23] . In the case of the cell-to-cell transmission of influenza virus in the presence of oseltamivir, it is possible that a progeny virion is temporarily bridged by HA between an infected cell and adjacent uninfected cells, since viruses can not be released from infected cell surface due to the inhibition of the NA activity by oseltamivir. The cellassociated progeny virion may have an opportunity to re-infect the previously infected cell, compared to a cell-free progeny virion in the general spreading. Thus, we examined whether influenza viruses can infect the cell which had already been infected, using ts53 mutant and wild-type influenza virus A/WSN/33. ts53 virus has a substitution mutation from U to C at the nucleotide position of 701 in the PA gene. This substitution introduces an amino acid change from wild-type Leu 226 to Pro 226 and gives a defect in the viral genome replication process [47, 48] . At first, cells were infected with ts53 virus at moi of 10, and after incubation for 0, 2, 4, 6, and 8 hours, cells were superinfected with wild-type virus at moi of 10. The amount of segment 3 viral RNA (vRNA) encoding PA was determined quantitatively by RT-PCR. Then, using a mutated primer for PCR, we could introduce a Stu I site only in the PCR products derived from the wild-type sequence ( Figure 6A ). Thus, DNA fragments amplified from the wild-type and ts53 could be distinguished by Stu I digestion. The digested DNA fragments containing 220 and 199 base pairs derived from ts53 and wildtype, respectively, were separated through PAGE. After 6 hours or later post infection, re-infection with the second challenging virus hardly occurs in the absence of oseltamivir. However, in the presence of oseltamivir, appearance of wild-type fragment suggests that the re-infection had occurred ( Figure 6B ). The result indicates that progeny virus particles remain on the surface of infected cell even after budding, and can infect the cell previously infected, as well as uninfected cells adjacent to the infected cell, when oseltamivir is present.
With the except for the virus which spreads through the cell-cell fusion transmission, virus infection is initiated by the binding of cell-free virions to their host cells. Recently, the virus transmission mechanism from an infected cell to adjacent cells without virus diffusion into the extracellular environment is highlighted from the aspect of its significance in virus spreading in the presence of antibodies [1, 2] . This antibody-insensitive pathway is often called cell-to-cell transmission [2] . The cell-to-cell transmission may be categorized into two pathways, i.e., transmission of cell-free virions to adjacent uninfected cells, and transmission of progeny virions associated on the surface of an infected cell even after budding through narrow synaptic space between an infected cell and adjacent uninfected cells. As an example of the former mechanism, cell-free vaccinia virus particles associated with the filopodium of an infected cell are repelled toward neighboring uninfected cells by inducing the formation of actin filament [3] . Several cases have been reported for the latter mechanism: Immunotropic viruses including retroviruses utilize the immunological synapses [4] [5] [6] [7] . Immune cells are not constitutively polarized, but contain the machinery that directs their secretory apparatus towards a cell that is involved in an immunological synapse. This machinery can be subverted by retroviruses containing human immunodeficiency virus (HIV). An HIV-infected cell can polarize viral budding towards a target cell expressing receptor through a structure called a virological synapse. Virions bud from an infected cell into a synaptic cleft, from which they fuse with the target-cell plasma membrane [49] [50] [51] [52] . The progeny virions of HCV are trapped between infected and uninfected cell membranes at the tight junction. Using Claudin-1 known as a component of the tight junction and one of the entry factors of HCV [8] , virions fuse with and penetrate uninfected target cells [31] . Therefore, HCV may acquire the ability to spread within polarized liver epithelium. Thus, the cell-to-cell transmission certainly plays significant roles for the dissemination of several enveloped viruses. However, the cell-to-cell transmission of influenza virus has not been discussed well. Here, we have shown that influenza virus spreads by forming infected cell clusters even in the presence of an NA inhibitor. Live cell imaging clearly showed that influenza virus lacking the NA activity spreads from an infected cell to adjacent cells through the cell-to-cell transmission mechanism (Figure 2 ). This was also the case for wild-type influenza virus during early phases of infection ( Figure 4B ). In the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, and then these progeny virions can be passed on to adjacent uninfected cells.
We showed that the cell-to-cell transmission of the NA-deficient influenza virus depends on functional HA. The viral spreading was dramatically suppressed without HA activation by trypsin treatment ( Figure 4A) . Moreover, the cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes [33, 34] . These findings indicate that functional HA and endosome acidification by M2 ion channel are required for the cell-to-cell influenza virus transmission, thereby allowing viruses to enter the adjacent cells through the endocytotic pathway ( Figure 4) .
Our findings showed that the NA-deficient influenza virus is not diffused into the extracellular environment. The viral spreading in the absence of oseltamivir appears to be much faster compared to the viral spreading in the presence of the drug, suggesting that NA could be involved in determination of spreading speed ( Figure 4B ). The NA activity prevented progeny virions from entering cells which virus came from (Figure 6 ), implying that progeny virus particles should be transmitted to adjacent uninfected cells. The cell-to-cell transmission started in early phase of infection, and the virus spread through diffusion of cell-free viruses ( Figure 4B ). Indeed, it was reported that the cell-to-cell transmission is a rapid spreading pathway in the case of vaccinia virus [3] . Vaccinia virus induces a blocking mechanism of superinfection and thereby infects to adjacent uninfected cells efficiently. In early phases of vaccinia virus infection, viral proteins A33 and A36 are expressed at the infected cell surface. Once cell-free virus particles contact the filopodium, the A33/A36 complex induces the formation of actin filament, which causes this superinfected virion to be repelled toward uninfected cells [3] . Influenza viruses can re-infect the cells previously infected in the presence of oseltamivir (Figure 6 ), suggesting that a progeny virion may be bridged by HA between infected and adjacent uninfected cells temporarily. Thus, in the case of the cell-to-cell transmission of influenza virus, we propose that progeny virions associated with the surface of infected cells even after budding are directed to adjacent uninfected cells. The cell-to-cell transmission mechanism of influenza virus is distinctly different from that of vaccinia virus in the infecting virus status: Infected cell-associated virions and cell-free virions are involved in the cell-to-cell transmission of influenza virus and vaccinia virus, respectively. The strategy for influenza virus appears to be similar to that for HCV. HCV progeny virions budded from an infected cell are trapped between infected and uninfected adjacent cell membranes at the tight junction. HCV virions then, enter into adjacent cells through endocytosis and low pH-dependent membrane fusion using Claudin-1 [8] . The cell-to-cell transmission of influenza virus also required functional HA and endosome acidification by M2 ion channel. However, it has not been reported that HCV has a gene encoding a receptor destroying enzyme similar to NA of influenza virus. We speculated that HCV progeny particles are bridged between infected and adjacent uninfected cells temporarily like influenza virus in the presence of oseltamivir. Progeny influenza virus particles could be transmitted to adjacent uninfected cells efficiently in the presence of the NA activity, suggesting that the cell-to-cell transmission of influenza virus is more strategic than that of HCV.
Our findings raise an interesting question as to what is the biological significance of cell-to-cell transmission for influenza virus infection in vivo. Until now, it had been believed that influenza virus was released from infected cells as cell-free virions and then spread from cell to cell as well as from organism to organism. The transmission mode by cell-free virions undergoes the extremely highspeed of its diffusion and causes epidemic or pandemic infection.
The tropism in an infected animal body is generally restricted to respiratory tract or lung and its periphery, and the requirement of a trypsin-like protease has been generally described for the reason of the restriction. It is possible that the cell-to-cell transmission mode may play a significant role for the virus spreading inside of organism, although cell-free influenza virions are causative of highspeed spreading. At the least, the limited but distinct level of infection followed by replication could provide some opportunity to generate influenza virus variants. It is an open question whether the cell-to-cell transmission mode is involved in the pathogenesis caused by influenza virus infection in vivo.
The existence of cell-to-cell transmission pathway gives a caution when NA inhibitors are used, because NA inhibitors may not be sufficient to completely block the spread of influenza Figure 6 . Influenza viruses can not re-infect previously infected cells. (A) A method for determination of the amount of segment 3 genome derived from ts53 and wild-type. Total RNA was reverse-transcribed with the primer PA-895-rev, which is complementary to the segment 3 positivesense RNA. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut partially corresponding to segment 3 positive sense RNA between the nucleotide sequence positions 678 to 700 except for 696 and 697, which are shown in red letters. Since segment 3 of ts53 has a substitution mutation from U to C at the nucleotide position of 701, the PCR product derived from wild-type could be digested by Stu I but not that from ts53. Then, PCR products were digested with Stu I and separated through 8% PAGE. (B) Detection of the genome of the segment 3 derived from ts53 or wild-type. At 3 hours post superinfection of wild-type virus, total RNA was extracted, and semi-quantitative RT-PCR was performed. Subsequently, the amplified DNA products were digested with Stu I and separated through 8% PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively. The relative amount of wild-type segment 3 to that at 0 hour in the absence of oseltamivir phosphate was shown in the graph. Error bars indicate S.D. from 3 independent experiments. White bar, in the absence of oseltamivir phosphate; black bar, in the presence of oseltamivir phosphate. doi:10.1371/journal.pone.0028178.g006 virus in local microenvironments. Since this cell-to-cell transmission pathway exists, development of antiviral therapeutic strategies in addition to NA inhibitors is highly recommended.
Madin-Darby canine kidney (MDCK) cells were kindly gifted by A. Ishihama (Hosei University), and maintained in minimal essential medium (MEM) (Nissui) containing 10% fetal bovine serum. Human embryonic kidney 293T cells were kindly gifted by Y. Kawaoka (University of Tokyo), and maintained in Dulbecco modified Eagle medium (DMEM) (Nissui) supplemented with 10% fetal bovine serum. Influenza virus A/Udorn/72 was grown in allantoic sacs of 11 day-old embryonated eggs (MIYAKE HATCHERY). Wild-type influenza virus A/WSN/33 and ts53 mutant were used after single-plaque isolation. MDCK cells were infected with influenza virus A/WSN/33 or ts53 at a multiplicity of infection (MOI) of 0.1 PFU/cell, and incubated at 37uC and 34uC, respectively. After incubation for 24 h, the culture fluid was harvested and centrifuged at 1,7006 g for 10 min. The virus suspension was stored at 280uC until use.
The production of rabbit polyclonal anti-NP antibody was described previously [53] , and this antibody was used as a primary antibody for indirect immunofluorescence assay. A goat antirabbit IgG antibody conjugated to Alexa Fluor 488 or Alexa Fluor 568 was purchased from Invitrogen and used as a secondary antibody for indirect immunofluorescence assay. A polyclonal antibody against influenza A virus was obtained from 2-month-old female rabbit immunized with 250 mg of purified virions of influenza virus strain A/Puerto Rico/8/34 [54] . The generation of antibodies was boosted three times and used as neutralizing antibodies to block the influenza virus infection.
MDCK cells were infected with influenza virus A/WSN/33 at a multiplicity of infection (MOI) of 0.001 PFU per cell. After virus adsorption at 37uC for 1 hour, the cells were washed with serumfree MEM and incubated at 37uC with maintenance medium (MEM containing vitamins and 0.1% BSA) containing oseltamivir. At 48 hours post infection (hpi), culture supernatant was collected, and then its viral titer was determined by plaque assays.
An NA-deficient influenza virus possessing the terminal sequences of NA segment but lacking the NA coding region, which was replaced with enhanced green fluorescent protein (EGFP) gene, was generated by reverse genetics as described previously [29, 30] . For reverse genetics, we used plasmids containing cDNAs of the influenza virus A/WSN/33 viral genome under the control of the human RNA polymerase I promoter (referred to as Pol I plasmids). Briefly, 293T cells were transfected with seven Pol I plasmids for production of all vRNA segments of influenza virus A/WSN/33 and one for the mutant NA vRNA segment containing EGFP ORF, together with protein expression vectors for PB2, PB1, PA, and NP controlled by the chicken b-actin promoter (pCAGGS). TransIT-293 (Mirus) was used for transfection. At 24 hours post transfection, recombinant viruses were harvested from the cell surface using bacterial NA derived from Clostridium perfringens (sigma). MDCK cells were infected with harvested recombinant viruses treated with N-tosyl-L-phenyl-alanine chloromethyl ketone (TPCK)-trypsin (1 mg/ml). After confirmation of GFP fluorescence derived from amplified recombinant virus genomes at 48 hours after infection, the recombinant viruses on the cell surface were collected using bacterial NA. The viral titer of recombinant viruses was determined by counting the number of infected foci using a fluorescence microscopy (Carl Zeiss).
Cells on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% NP-40 in PBS. The coverslips were soaked in 1% bovine serum albumin in PBS, and then incubated at room temperature for 1 hour with a primary antibody. After being washed twice with PBS, the coverslips were incubated at room temperature for 1 hour with a secondary antibody. The coverslips were then incubated at room temperature for 5 min with 3 mM 49,69-diamidino-2phenylindole (DAPI) and finally mounted on glass plates, and cells were observed under the fluorescence microscope.
Living cells were analyzed using BioStation ID system (GE Healthcare). Confluent MDCK cells were infected with the NAdeficient influenza virus at the multiplicity of infection (MOI) of 0.0001 in the presence or absence of 1 mg/ml TPCK-trypsin. At 24 hours post infection, culture dishes containing infected cells were set into the chamber of BioStaion ID system, which was maintained at 37uC under 5% CO 2 and 95% humidity. Then, images were acquired during next 24 hours at interval with 1 hour. The excitation wavelength was controlled by a manual filter wheel equipped with filters suitable for enhanced green fluorescence protein (EGFP).
Confluent MDCK cell monolayer was prepared on transwell inserts (BD Falcon, pore size 0.4 mm) and infected with the NAdeficient influenza virus at MOI of 0.0001. After virus adsorption at 37uC for 1 hour, the cell monolayer was washed with serum-free MEM, and maintenance medium was added into both sides within the transwells. The neutralizing antibody to influenza A virus was added into the inside or the outside of transwell inserts with the maintenance medium. Subsequently, cells were incubated at 37uC for 36 hours followed by analyses using the fluorescence microscopy.
ts53 virus has a substitution mutation from U to C at the nucleotide position of 701 in the PA gene. This substitution introduces an amino acid change from wild-type Leu 226 to Pro 226 and gives a defect in the viral genome replication process [48] . However, under the permissive temperature, the level of viral genome replication is no difference between wild-type and ts53 [47] . To discriminate the genome of wild-type and that of ts53, total RNA was reverse-transcribed by reverse transcriptase (TOYOBO) with PA-895-rev (59-TTAATTTTAAGGCATC-CATCAGCAGG-39), which is complementary to the segment 3 positive sense RNA. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut (59-TCTCCCGCCA-AACTTCTCAGGCC-39) partially corresponding to segment 3 positive sense RNA between nucleotide sequence positions 678 to 700 except for nucleotide positions 696 and 697. Since segment 3 of ts53 has a substitution mutation from U to C at the nucleotide position of 701, the PCR product derived from wild-type was digested by Stu I but not that from ts53. After PCR reactions, PCR products were digested with Stu I and separated through PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively. DNA was stained with GelRed (BIOTIUM) and visualized by UV illumination.
Figure S1 Formation of cell cluster caused by initial infection. MDCK cells were infected with influenza virus A/ WSN/33 at moi of 0.0003 in the presence or absence of 50 mg/ml oseltamivir phosphate. After incubation for 8 and 24 h, immunofluorescence analyses were performed using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). Nuclear DAPI and viral NP staining patterns are shown in blue and green, respectively. Enlarged views are shown in red borders. Scale bar, 100 mm. (TIF) Figure S2 The expression of GFP derived from NAdeficient influenza virus overlapped with the localization of NP. MDCK cells were infected with NA-deficient influenza viruses at MOI of 0.0001. After incubation at 37uC for 48 hours, immunofluorescence analyses were performed using anti-NP antibody. Scale bar, 100 mm. (TIF) Figure S3 Influenza virus A/Udorn/72 was sensitive to oseltamivir. MDCK cells were infected with influenza virus A/ Udorn/72 at a MOI of 0.001 PFU per cell. At 36 hpi, the culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate s.d. from 3 independent experiments. (TIF) Video S1 NA-deficient influenza virus spreads through cell-to-cell transmission. Confluent MDCK cells were infected with NA-deficient influenza virus at MOI of 0.0001 in the presence of trypsin. After incubation at 37uC for 24 hours, a single GFP-positive cell and its vicinity were traced it during the period from 24 hpi to 48 hpi at interval of 1 hour. Live cell imaging data analyses was performed by Biostation ID (GE healthcare). Scale bar, 50 mm.
Video S2 NA-deficient influenza virus does not spread in the absence of trypsin. Confluent MDCK cells were infected with the NA-deficient influenza virus at MOI of 0.0001 in the absence of trypsin. After incubation at 37uC for 24 hours, a single GFP-positive cell was detected, and then this cell and neighborhood cells was traced during the period from 24 hpi to 48 hpi at interval of 1 hour. Live cell imaging data analyses were performed by Biostation ID (GE healthcare). Scale bar, 50 mm. (MOV) Persistent Expression of Hepatitis C Virus Non-Structural Proteins Leads to Increased Autophagy and Mitochondrial Injury in Human Hepatoma Cells HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol. Hepatitis C virus (HCV) is an enveloped, positive, singlestranded RNA virus in the family of Flaviviridae [1] . The linear, non-segmented HCV genome of 9.6 kb encodes a polyprotein that undergoes post-translational cleavage by cellular and viral proteases to yield at least 10 mature proteins [2] [3] [4] . HCV infection is a major cause of chronic liver disease and is the major cause of liver cancer in the United States. HCV produces a chronic infection in 50-80% of infected patients; among them, roughly 20% will eventually develop liver cirrhosis. It is widely accepted that insufficient host immune response in eliminating HCV leads to persistent infection and the eventual development of liver diseases [4] [5] [6] . Interferon-a and ribavirin treatments have been prescribed either to stimulate immune response for clearance of viruses or to disrupt viral replication. However, high toxicity and low efficacy toward the two most prevalent HCV subtypes, 1a and 1b, in the US has been a barrier to effective eradication of persistent HCV infections [7] .
To address the pathogenesis caused by HCV infection, recent studies have begun to focus on direct cytopathic effects. HCV proteins associate with different subcellular structures, including mitochondria, endoplasmic reticulum (ER), and lipid droplets, to facilitate replication and assembly of viral particles [2] . These associations lead to alterations of the integrity and functions of organelles. HCV-mediated oxidative stress is commonly observed and is achieved by increasing reactive oxygen and nitrogen species (ROS and RNS) or by altering cellular antioxidant capacities [8] [9] [10] [11] . In particular, HCV core proteins are shown to be closely associated with the mitochondria and cause increases in ROS and RNS production and lipid peroxidation [11] [12] [13] [14] , reduction in GSH and NADPH concentrations, reduction in mitochondrial complex I activities, and increase in mitochondrial Ca +2 uptake, which ultimately disrupts mitochondrial membrane permeability and leads to mitochondrial dysfunction [14, 15] . HCV nonstructural proteins have also been implicated in disturbing the redox balance and altering antioxidant enzyme levels [16, 17] . Specifically, NS5A is shown to up-regulate Mn superoxide dismutase (MnSOD) through AP1 transcription factor in the p38 MAPK and JNK signaling pathways [18, 19] . Additional studies showed the involvement of NS5A in ER stress and disturbance of intracellular Ca +2 homeostasis, which leads to increased mitochondrial ROS production and altered mitochondrial function [18, 20] . Because of the relationship between chronic HCV infection and the development of hepatocellular carcinoma, studies have also been carried out to identify HCV proteins that may be responsible for the hepatocarcinogenesis. For example, the HCV core protein has been shown to promote immortalization of primary human hepatocytes [21] , whereas the non-structural proteins NS3 and NS4B have been shown to transform NIH 3T3 cells either individually or in combination with Ha-ras [22, 23] .
Most studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. We hypothesize that different components of HCV polyprotein, depending on their subcellular distributions, cause different types of cytopathic effects and elicit different types of responses in different subcellular compartments. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used hepatoma cells constitutively expressing the HCV genomelength replicon, the subgenomic replicon, or the core protein to model the state of persistent HCV infection and took a comparative approach to dissecting the role of various HCV proteins in cellular pathogenesis in this study.
Based on the SEAP activities measured from multiple sampling, the protein expression levels of HCV genome-length and subgenomic replicons were comparable ( Figure S1A ). Core protein expression levels, on the other hand, suggested that Core-on cells produced higher levels of core protein than the genome-length replicon cells ( Figure S1B ). To identify the subcellular location of HCV proteins in genome-length replicon, subgenomic replicon, and Core-on cells, immunogold EM was carried out with antibodies against core, NS5A, and NS5B proteins. The majority of core, NS5A, and NS5B proteins were located in the mitochondria and the ER with minor differences in their distribution within these organelles ( Figure 1A and Table S1 ). Positive signals were also observed in the nucleus, lipid droplets, and the Golgi. NS5A signals were observed in autophagocytic vacuoles in 25% of the electron micrographs examined, with an average signal intensity of 3-6 gold particles per autophagocytic vacuole (Table S1 ).
To identify ultrastructural changes caused by the presence of HCV proteins, electron microscopic analyses were carried out. The study results revealed mitochondrial injury as a common defect among genome-length replicon, subgenomic replicon, and Core-on cells with enlarged mitochondria and focal loss of cristae ( Figure 1B ). Consistent with this observation, measurement of the cross sectional area of mitochondria directly from electron micrographs showed a significant increase in mitochondrial sizes ( Figure 2A ). The data indicates mitochondrial degeneration and the possible loss of mitochondria. Consequently, the number of mitochondria per cell was significantly reduced ( Figure 2B ). In addition to mitochondrial defects, accumulation of lipid droplets was common among all three HCV protein-expressing cell lines ( Figure 1B and Figure S2 ). Genome-length and subgenomic replicon cells also showed focally dilated rough endoplasmic reticulum (RER) and prominent presence of autophagocytic vacuoles ( Figures 1B and 3A ).
Increased oxidative damage is the likely culprit for the observed mitochondrial degeneration and reduced mitochondrial number in HCV protein-expressing cells. To determine the extent of ROS generation in the mitochondria, MitoSOX was used for live cell staining. The results showed a significant increase in ROS production in the mitochondria across all three cell lines ( Figures 2C and 2D ). After adjusting to the average number of mitochondria per cell, the level of MitoSOX intensity was similar among genome-length replicon, subgenomic replicon, and Coreon cells. Cytosolic and mitochondrial aconitases (ACO1 and ACO2) are very sensitive to inactivation by reactive oxygen and nitrogen species, and reduction in aconitase protein levels and enzyme activities have been used as indicators for increased oxidative stress in their respective subcellular compartments [24, 25] . Our studies showed that ACO1 existed at low levels, whereas ACO2 was barely detectable by western blot analysis, in Huh7 and HCV protein-expressing cells. ACO1 protein level was reduced by 18% in cells with the genome-length replicon ( Figure  S3A ), and total aconitase activity was reduced by 5-23% ( Figure  S3B ) in genome-length and subgenomic replicon cells. The reduction in total aconitase activity most likely reflected increased oxidative stress in the cytosol because the majority of aconitase in Huh7 cells was in cytosolic form.
From ultrastructure analyses, we consistently observed the presence of autophagocytic vacuoles and primary autophagocytic vesicles ( Figures 1B, 3A , and 3B) in genome-length replicon and subgenomic replicon cells and, to a lesser extent, in Core-on cells.
To confirm the initial observation, immunocytochemical and western blot analyses were carried out to determine the status of LC3, which is an integral part of the autophagosome membrane. LC3-positive punctuate structures in the cytoplasm were prominent in genome-length replicon and subgenomic replicon cells ( Figure 3C ), but they were only present at a very low level in Coreon cells. Consistent with this result, western blot analyses showed a marked increase of LC3-I and LC3-II in genome-length replicon and subgenomic replicon cells ( Figure 3D ). However, the ratios between LC3-I and II were not changed. In contrast to the prominent accumulation of autophagocytic vacuoles in genomelength and subgenomic replicon cells, autophagosomes were not detected in HCV transgenic mice expressing the full-length HCV polyprotein (data not shown).
To determine if oxidative stress played a role in HCV-mediated activation of autophagy, cellular redox state was altered by either enhancing the cellular antioxidant capacity through dual overex-pression of superoxide dismutase (SOD) and catalase (CAT), or increasing cellular oxidative stress by xanthine/xanthine oxidase (X/XO) treatment. Changes in total LC3 and LC3-II levels were used as indicators of autophagy. Significant reduction of total LC3 and LC3-II levels was observed in subgenomic replicon, Core-on, and Core-off cells with overexpression of CuZnSOD/cytosolic catalase (SOD1/cCAT) or MnSOD/mitochondrial catalase (SOD2/mCAT) ( Figure 4A ). Although similar trends were observed in Huh7 cells, the extent of reduction did not reach a significant level. Despite comparable levels of CuZnSOD, MnSOD, and catalase expression ( Figure S4 ), LC3 levels were not altered in genome-length replicon cells. In contrast, X/XO treatment significantly increased total LC3 levels in genomic replicon, subgenomic replicon, and Core-off cells ( Figure 4B , left panel). However, LC3-II levels were only significantly increased in genomic replicon and Core-off cells ( Figure 4B , right panel). No significant change in LC3-II level was observed in Huh7, subgenomic replicon, and Core-on cells. Furthermore, addition of CAT to the X/XO treatment diminished the increase of total LC3 in genome-length and subgenomic cells ( Figure 4B ). The data suggest that the enhanced autophagy from X/XO treatment is mediated through H 2 O 2 in genome-length and subgenomic replicon cells.
To find out whether increased mitochondrial stress and ER stress led to changes in antioxidant profiles, western blot analyses were carried out to determine the protein levels of CuZnSOD, MnSOD, peroxiredoxin 1 (PRDX1), peroxiredoxin 3 (PRDX3), thioredoxin 1 (TRX1), and thioredoxin 2 (TRX2). Among them, CuZnSOD, PRDX1, and TRX1 are cytosolic proteins, and MnSOD, PRDX3, and TRX2 are mitochondrial proteins. The sulfonylated peroxiredoxins (PRDX-SO3), which are the end products of irreversible oxidation of the active site cysteine in peroxiredoxins, were also monitored and served as indicators for the redox state in HCV protein-expressing cells ( Figure S5C ). Significant increases in the protein levels of CuZnSOD, MnSOD, PRDX1, PRDX3, TRX1, and TRX2 were consistently observed in the subgenomic replicon cells, and the extent of increase ranges from 1.5-to 4.9-fold ( Figure 5 ). The genome-length replicon cells showed a modest (1.8-fold) but significant increase in TRX2, and a marginal decrease in CuZnSOD (p = 0.0734) and PRDX3 (p = 0.0739) ( Figure 5 ). No remarkable changes were observed between Core-on and Core-off cells. The ratios of PRDX1-SO3/ PRDX1 and PRDX3-SO3/PRDX3 were also used to gauge the redox state in the cytosol and the mitochondria, respectively, and no significant changes were observed across all cell lines analyzed ( Figure S5A and S5B).
To determine if ultrastructural changes and increased mitochondrial ROS production affect cell proliferation and survival, population doubling time was determined during the exponential phase of cell growth. Genome-length replicon and Core-on cells showed a 41% and 11% increase in population doubling time, respectively, without a significant increase in cell death during the 72-hr period when cell number increase was monitored ( Figure 6A ). On the other hand, subgenomic replicon cells had a comparable population doubling time to that of Huh7 and Coreoff cells. To determine if cell cycle regulation is affected in HCV protein-expressing cells, in-cell westerns were carried out to determine total cyclin D1 levels. Consistent with increased population doubling time in genome-length replicon cells, cyclin D1 level was decreased ( Figure 6B ). There was also a trend in decreased cyclin D1 levels in Core-on cells.
In this study, we showed that the most dominant subcellular location for HCV core, NS5A, and NS5B proteins was in the mitochondria, followed by the ER and the Golgi. Consequently, expression of HCV core or non-structural proteins led to increased ROS generation in the mitochondria, which most likely contributed to mitochondrial injury and degeneration. Expression of non-structural proteins also led to membrane blebbing in the ER and accumulation of autophagocytic vacuoles. Despite these changes, only cells with the subgenomic replicon (i.e. cells expressing the non-structural proteins) showed consistent upregulation of mitochondrial and cytosolic antioxidant enzymes. Cells with the genome-length replicon, on the other hand, did not have a robust antioxidant response and showed prolonged population doubling time and a decrease in cyclin D1 expression. Apart from the shared mitochondrial phenotype, increased lipid accumulation was also common among all three HCV proteinexpressing cell lines. Subcellular locations of HCV proteins have provided important clues to the site of viral genome replication and assembly, as well as the cytopathic effects caused by HCV infection. The ultrastructure study with immunogold labeling showed a distinct localization of core, NS5A, and NS5B proteins in the mitochondria and the ER. Significant core, NS5A, and NS5B distributions were observed in other subcellular compartments, including the Golgi apparatus, the nucleus, and lipid droplets. The study results are in general agreement with previously published data [26] [27] [28] [29] [30] [31] . However, the inner membrane location of core protein was unexpected because previous studies using proteinase K digestion suggested association of core protein with the mitochondrial outer membrane and the mitochondrial-associated membrane compartment [15, 28] . The discrepancy could be due to the compromised mitochondrial membrane integrity in genome-length replicon and Core-on cells, or due to artifacts from sample preparations for EM analysis. The association of core protein with lipid droplets also appeared to be lower than previous study results, and the discrepancy could be due to differences in the antibodies used.
A significant number of studies have focused on the role of HCV core protein in mitochondrial dysfunction [11, 14, 15, 20, 26, 32] . In this study, cells expressing only the nonstructural proteins were also shown to have suffered from mitochondrial damage, and the extent of damage, in terms of mitochondrial size and number, was comparable to that of cells expressing the full-length polyprotein or core protein (Figures 2A  and 2B ). In addition, the level of ROS production in the mitochondria, as detected by MitoSOX, was also comparable among the three HCV protein-expressing cell lines after adjusting for total mitochondria (Figures 2C and 2D) . The data suggest that multiple HCV proteins are capable of entering the mitochondria and cause increased oxidative stress, although the underlying mechanism may not be the same for each protein. The core protein-expressing cells had the lowest number of mitochondria per cell ( Figure 2B ). The data implies that Core-on cells may suffer from the most severe mitochondrial loss among the three HCV protein-expressing cell lines. Whether this is due to the higher level of core protein expression in Core-on cells ( Figure S1A ) will need to be examined with other approaches.
Besides increased ROS production in the mitochondria of all three cell lines analyzed, our data on aconitase protein levels and enzyme activities also suggested an increased ROS production in the cytosol in genome-length and subgenomic replicon cells. The reduction in ACO1 protein level in genome-length replicon cells is reminiscent to that observed in CuZnSOD deficient mice [25] , in which heightened state of oxidative stress in the cytosol may lead to increased degradation of irreversibly inactivated aconitase. Subgenomic replicon cells had reduced aconitase activity without a significant reduction in the protein level ( Figures S3A and S3B) . The data suggests that ACO1 may be reversibly inactivated due to increased oxidative stress in the cytosol. Taken together, our study results suggest that HCV non-structural proteins are capable of causing increased oxidative stress in both the mitochondrial and the cytosolic compartments, whereas the effects of core protein is mainly limited to the mitochondria.
Cells respond to a variety of internal stress or cellular damages by forming autophagosomes to degrade damaged components and to recycle usable elements for future biosynthesis [33] . However, some RNA viruses, most notably, poliovirus, dengue virus, mouse hepatitis virus, and foot-and-mouth disease virus develop the abilities to overcome such cellular defense by hijacking the autophagocytic pathway to facilitate viral replication [34] [35] [36] [37] . Multiple studies in recent years have shown that HCV infection of the human hepatoma cell line Huh7 and immortalized human hepatocytes induce autophagocytic vacuoles [38, 39] and that autophagy machinery is required for the initiation of HCV replication [40] and the production of infectious particles [41] . HCV induces the accumulation of autophagosomes via activation of the unfolded protein response pathway without enhancing autophagocytic protein degradation [38, 42] . Consequently, inhibition of autophagy inhibits HCV replication [41] . Although most studies are carried out with HCV genotype 2a, it is now well accepted that autophagy probably also occurs with infection of other HCV genotypes. What is not clear from these studies is whether enhanced autophagy persists during the chronic phase of HCV infection, and if and to what extent oxidative stress contributes to HCV-mediated activation of autophagy. In this study, we observed the accumulation of autophagocytic vacuoles and up-regulation of the autophagosome marker, LC3, in the genome-length and subgenomic replicon cells (Figures 3B-3D) . The data suggests that HCV non-structural proteins play a role in inducing autophagy, possibly through ER stress ( Figure 1B) , mitochondrial damage ( Figures 1B and 2A) , and induction of oxidative stress (Figures 2D and 4B ). Core-on cells, on the other hand, had a slight increase in the number of autophagocytic vacuoles without a significant increase in LC3-II or total LC3 levels ( Figures 3B-3D) .
HCV-mediated oxidative stress was likely a contributor in the activation of autophagy because enhanced antioxidant capacity significantly reduced total LC3 and LC3-II levels ( Figure 4A ), whereas increased oxidative stress led to a further increase in total LC3 and LC3-II ( Figure 4B ). ROS generated in the cytosol and mitochondria was equally capable of enhancing autophagy because increased antioxidant capacity in either compartment achieved comparable levels of reduction. In addition, H 2 O 2 appeared to be the main culprit in ROS-mediated activation of autophagy within this experimental system since addition of catalase effectively suppressed X/XO-mediated activation of autophagy ( Figure 4B ). Despite up-regulation of multiple antioxidant enzymes in subgenomic replicon cells ( Figure 5 ), these cells responded to further increase in antioxidant enzymes with reduced autophagy. It is important to note that although the antioxidant enzymes clearly can modulate the effects of viral proteins, they only serve to reduce the LC3 increase by ,26% in subgenomic replicon cells. The data suggest either that other mechanisms, such as ER stress, are important as well or that the antioxidant effect is incomplete. Overexpression of antioxidant enzymes also led to a reduction of autophagy in Core-on and Core-off cells. Antioxidant enzyme overexpression in cells with genome-length replicon, on the other hand, failed to reduce the level of autophagy even though the enhanced antioxidant capacity from SOD/CAT expression vectors was comparable to that of other cells ( Figure  S4 ). In contrast, increased oxidative stress effectively increased autophagy in genome-length replicon cells ( Figure 4B ). The data suggest that oxidative stress may not play a significant role in the basal level of autophagy in genome-length replicon cells; however, the cells are sensitive to additional oxidative stress and are capable of accumulating more autophagosomes under conditions of increased oxidative stress. Although total LC3 levels were increased in X/XO-treated subgenomic replicon cells, LC3-II levels were not changed with the added oxidative stress ( Figure 4B ). It is possible that the conversion of LC3-I to LC3-II was already at the maximum level in subgenomic replicon cells, and additional oxidative stress was not able to enhance the reaction further.
NS5A and, to a lesser extent, NS5B and the core protein have been detected in autophagocytic vacuoles (Table S1 ). Whether the NS5A localization in the autophagosome observed in our study is a cellular defense mechanism elicited to degrade foreign antigens or a viral mediated self-preserving mechanism to enable viral replication remains to be determined. HCV transgenic mice expressing the full-length HCV polyprotein were also examined for the accumulation of autophagocytic vacuoles in the liver. However, no evidence of enhanced autophagy was detected. It is possible that additional factors are needed to cause the accumulation of autophagosomes in vivo or that very low viral protein expression levels in the transgenic mice were not sufficient to induce these changes. A previous study using the same line of transgenic mice [43] showed that long-term iron overload was necessary to induce the accumulation of autophagosomes. Since iron overload increases oxidative stress in the liver, the result is consistent with our finding in HCV protein-expressing cells in the relationship between oxidative stress and autophagy.
Huh7 cells with the subgenomic replicon showed significant upregulation of multiple antioxidant enzymes that belonged to the cytosolic and mitochondrial compartments ( Figure 5 ). Despite the up-regulation of multiple antioxidant enzymes, the ratios of oxidized (Sulfonic form) to total peroxiredoxin 1 and 3 in the subgenomic replicon cells remained the same as that in Huh7 cells ( Figure S5 ), suggesting that the overall redox environment was still more oxidizing. This conclusion was also supported by the increased MitoSOX staining and reduced aconitase activities ( Figures 2C and 2D and Figure S3B ). Whether the up-regulation of multiple antioxidant enzymes in the subgenomic replicon cells is a direct response to the presence of HCV non-structural proteins or is merely a clonal variation will need to be deciphered with additional studies in the future. However, it is worth noting that previous studies with different HCV subgenomic repliconexpressing cells also showed a similar up-regulation in various antioxidant enzymes [17, 44] . Huh7 cells with the genome-length replicon had a 1.8-fold increase in the mitochondrial form of thioredoxin (TRX2), but a 15% reduction of its upstream enzyme PRDX3 ( Figure 5 ). The net result was a slightly higher ratio of PRDX3-SO3 to total PRDX3; however, the increase was not significantly higher than that of Huh7 cells ( Figure S5B ). The expression level of non-structural proteins should be comparable between the genome-length and subgenomic replicon cells based on the SEAP reporter assay ( Figure S1A ) and published results [45] . Therefore, the lack of antioxidant response in cells with the genome-length replicon was probably not due to a difference in the expression of non-structural proteins; but rather, it could be due to the additional cytopathic effects from core, E1, E2, and p7 proteins that counteract the antioxidant enzyme response induced by non-structural proteins. Consequently, the genome-length replicon cells had a prolonged population doubling time and reduced cyclin D1 expression (Figure 6) , which suggested delayed cell cycle progression. In contrast, Core-on cells showed no changes in any of the antioxidant enzymes examined.
In summary, our studies using Huh7 cells expressing different parts of the HCV polyprotein suggest that the presence of the fulllength HCV polyprotein leads to the most severe cellular damage, including generation of ROS, accumulation of lipids, ER stress, mitochondrial injury and degeneration, accumulation of autophagosomes, and prolonged population doubling time. Despite the elevated levels of ROS, these cells failed to mount a robust antioxidant response. Huh7 cells with only the non-structural proteins, on the other hand, have the same cytopathic changes as that observed in the genome-length replicon cells, but the cells showed a robust antioxidant response even though the response was not sufficient to suppress HCV-mediated ROS production. Our data suggest that HCV-mediated ER stress, mitochondrial injury, and oxidative stress form the three arms of mediators for the activation of autophagy (Figure 7) . Their effects are additive and inter-related. Without a concomitant increase in downstream protein degradation, autophagosomes accumulate and provide a sanctuary for HCV replication and protection from host immune surveillance [42] . The process likely helps to sustain a low level of viral production, which perpetuates chronic HCV infection and chronic liver injury (Figure 7) . In addition to activation of autophagy, HCV-mediated ER stress can lead to imbalance in Ca homeostasis, which further contributes to cellular oxidative stress [14, 15, 46, 47] . Mitochondrial injury can lead to metabolic deficits Figure 7 . Diagrammatic presentation of the cytopathic effects caused by various HCV proteins. Replication of HCV RNA and production of HCV structural (core, E1, E2, and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) in the rough ER and the subsequent partition of HCV proteins to different subcellular compartments lead to ER stress, mitochondrial injury, and the production of ROS. These cytopathic effects lead to activation of autophagy without a concomitant increase in protein degradation. Consequently, autophagosomes accumulate in HCV infected cells. EM photo with normal mitochondria (M) and rough ER (RER) was taken from healthy Huh7 cells; EM photos with enlarged mitochondria, ER blebbing, lipid droplet (L), and autophagosomes (A) were taken from genome-length and subgenomic replicon cells. The HCV genetic materials contained in genome-length replicon, subgenomic replicon, and Core-on cells are depicted at the top of the diagram. doi:10.1371/journal.pone.0028551.g007 and further exacerbates cell injury and dysfunction. Oxidative stress can cause increased DNA damage and mutation, inactivation of redox-sensitive proteins, and activation of redox sensitive signaling pathways such as MAPK and AP-1 [48] [49] [50] . These cytopathic effects work in concert in the course of chronic HCV infection to promote cell death, hepatocyte turnover, alteration of the liver microenvironment, and ultimately cell transformation and tumorigenesis.
All animal procedures were reviewed and approved under the protocol number HUT100602MOU by the VA IACUC committee (Subcommittee on Animal Studies, NIH assurance number A3088-01) at the VA Palo Alto Health Care System and in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals.
Cell culture and mouse model Control Huh7 cells and Huh7 cells with genome-length replicon, subgenomic replicon, or the tet-inducible core expression construct were used for this study. All three HCV proteinexpressing cell lines have been described previously [20, 26] . Genome-length replicon and subgenomic replicon were derived from a genotype 1a H77c infectious molecular clone [20] . The genome-length replicon encodes the full-length HCV polyprotein (core, E1, E2, p7, and NS2-NS5), whereas the subgenomic replicon encodes only the non-structural proteins NS2-NS5B [20] . The tet-inducible core-expressing cells express core protein in the absence of doxycycline. These cells are referred to as Core-on cells when core expression is turned on and as Core-off cells when core expression is turned off by the addition of doxycycline to the culture medium. All cells were maintained in DMEM with 10% tetracycline-free fetal calf serum (FCS, Clontech, Mountain View, CA, USA), Pen/Strep (50 IU/ml penicillin and 50 mg/ml streptomycin), and G418 (200 mg/ml) plus the following antibiotics: genome-length and subgenomic replicon cells, 5 mg/ml blasticidin; Core-off cells, 20 mg/ml doxycycline. When cells were plated for an experiment, only DMEM with 10% tetracycline-free FCS and Pen/Strep was used. All cells were incubated at 37uC with 5% CO 2 . Expression of HCV proteins in genome-length and subgenomic replicon cells were monitored by following the specific activities of the reporter gene, secreted alkaline phosphatase (SEAP), in the culture medium at 48 to 72 hrs after the initial plating [51] ; HCV core protein expression in genome-length replicon and Core-on cells were monitored by western blot analysis.
HCV transgenic mice expressing the full-length HCV polyprotein [12] were used at 3 months of age. Only male mice were used in this study. All mice were kept in a barrier facility with a 12hr dark-light cycle, given food and water ad libitum, and maintained in microisolators with a constant temperature between 20uC and 26uC. All animal procedures were reviewed and approved by the IACUC committee at the VA Palo Alto Health Care System and in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals.
Control Huh7 cells and genome-length replicon, subgenomic replicon, Core-on, and Core-off cells were fixed with 2.5% glutaraldehyde in 0.1 M Na Cacodylate, pH 7.4 for 2 hrs, postfixed with 2% aqueous osmium tetroxide for 2.5 hrs, and subsequently stained en bloc in 2.5% uranyl acetate (in water) overnight before dehydration and embedding in Eponate 12 resin (Ted Pella, Inc., Redding, CA, USA). Thick (1 mm) sections of the embedded cells were examined at the light microscopic level. The cell blocks were further trimmed to obtain thin sections (80 nm), stained with saturated solution of uranyl acetate (15 min) followed by Reynolds' lead citrate (8 min) , and examined with a JEOL JEM 100CX II transmission electron microscope (JEOL Ltd., Tokyo, Japan).
HCV transgenic and non-transgenic mice were perfused through the left ventricle of the heart, first with 10 U/ml heparin in saline until the liver was cleared of blood (about 5 min), then with fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1M Na Cacodylate, pH 7.4) until the liver was completely fixed (about 6-8 min). Heparin and fixative solutions were delivered by a peristaltic pump (VWR, Westchester, PA, USA) with the flow rate set at 4.5 ml/min. One mm cubes were prepared from the fixed liver and were left to continue fixation at room temperature (RT) overnight. These specimens were processed and embedded in Eponate 12 resin as described above.
For mitochondrial size determination, electron micrographs taken at 7.2K magnification were used, and mitochondrial sizes from 3-4 cells from each cell line were measured. Image J was used to determine the area occupied as pixel number, which was then converted to mm 2 . For determination of the number of mitochondria and autophagocytic vacuoles/vesicles, electron micrographs taken at low magnifications (1.9-3.6K), with the requirement of being able to fit an entire cell into the view, were used and 10-26 cells from each cell line were analyzed.
For live MitoSOX and MitoTracker staining and imaging, 2610 4 cells were seeded onto each chamber of tissue culture treated 8-chamber slides (Millicell EZ slides, Millipore, Billerica, MA, USA) and incubated overnight. For MitoSOX staining to detect superoxide generation in the mitochondria, culture media was replaced with 200 mL of staining solution containing 5 mM MitoSOX Red (Invitrogen, Carlsbad, CA, USA) and 5 mg Hoechst 33342 (for nuclear staining, Invitrogen) in HBSS, and cells were incubated at 37uC for 10 minutes. For equilibration, staining solution was replaced with 500 mL pre-warmed HBSS and returned to 37uC for another 10 minutes before imaging. For MitoTracker staining to visualize the mitochondrial network, culture media was replaced with 100 mL of staining solution containing 200 nM MitoTracker Green FM (Invitrogen) and 5 mg Hoechst 33342 in HBSS, and cells were incubated at 37uC for 30 minutes. The staining solution was then replaced with 500 mL pre-warmed HBSS before imaging. Cells were imaged directly in HBSS with a 40x (NA = 0.6) objective on an Olympus IX71 inverted fluorescence microscope equipped with a Coolsnap HQ monochrome camera (Photometrics, Tucson, AZ, USA). For direct comparison of staining intensity, exposure time was kept constant at 500 msec for MitoSOX and at 200 msec for MitoTracker. Staining intensities of MitoSOX and MitoTracker were determined with Image J as pixel intensities, and a minimum of 40 cells each were analyzed.
Expression level of the reporter gene, secreted alkaline phosphatase (SEAP), was used to monitor the expression of the HCV genome-length and subgenomic replicons. Huh7 cells with the genome-length or subgenomic replicon were cultured to ,80% confluency and cell culture medium from each cell line was removed for SEAP assays. The SEAP reporter assay was performed using the Great EscAPe SEAP Fluorescence Detection Kit (Clontech), and SEAP activities were normalized to total cellular proteins. To determine total aconitase activities, Huh7 and HCV protein-expressing cells were cultured to 90% confluency in 60 mm plates. Cell pellets were resuspended in aconitase buffer (50 mM Tris-Cl, pH 8, 2 mM citric acid, and 0.6 mM MnCl 2 ) and passed through two rounds of freeze-thaw cycle between liquid nitrogen and room temperature water to break open membranes. Due to the low level of aconitases in Huh7 cells, total aconitase activities were determined with a kinetic assay [24] coupled to the PMS/MTT color reaction to enhance the sensitivity [25] . Following a 3-minute preincubation at 37uC in the dark, the reaction was continued at 37uC and the OD change was monitored every 5 minutes at 590 nm for up to 60 minutes using a plate reader (SpectraMax M3, Molecular Devices, Mountain View, CA, USA). The reaction appeared to be most linear in the first 30 minutes and consequently, OD change per minute, as a function of enzyme activities, was calculated for that time period. The final results were normalized to the amount of total proteins in the cell lysates.
To enhance cellular antioxidant capacity, expression constructs for dual expression of CuZnSOD/cytosolic catalase (SOD1/ cCAT) or MnSOD/mitochondrial catalase (SOD2/mCAT) were used (constructed by SKZ, unpublished data). Expression of SOD1 and SOD2 are controlled by elongation factor-1 alpha (EF-1á) promoter, and expression of catalase is controlled by CMV promoter. Cells were grown to 50% confluency in 6-well plates and were transfected with 2.5 mg of each expression construct using TransITH-LT1 Transfection Reagent (Mirus Bio LLC, Madison, WI, USA). Cell lysates were prepared 36 hrs after the transfection for western blot analysis of LC3. CuZnSOD, MnSOD, and catalase levels were also determined to ensure comparable expression of each protein among different cell lines. To increase oxidative stress, cells were grown to 50% confluency in complete culture medium in 6-well plates and were then switched to 2% FCS-containing medium with 0.25 mM xanthine (X) and 20 mU/ml xanthine oxidase (XO). Since X/XO generates a combination of superoxide and H 2 O 2 , catalase (CAT, 40 mU/ml) was added to a subset of cultures to eliminate H 2 O 2 . Cells were incubated with X/XO or X/XO/CAT for 72 hrs; culture medium was changed every 24 hrs to maintain a steady level of X, XO, and CAT.
Several immunochemical procedures are described below; antibodies used in this study are listed in Table 1 .
Immunogold labeling. For immunogold EM localization of HCV proteins, cells were fixed (0.1% glutaraldehyde, 4% paraformaldehyde in phosphate buffered saline) and embedded in LR Gold resin (Ted Pella) using techniques described by Berryman and Rodewald [52] . Antibodies were pre-absorbed with Huh7 cell homogenate for 1-24 hrs. Thin sections on grids were blocked with 3% bovine serum albumin in TBS (Tris buffered saline) for 1 hr at RT, incubated with primary antibodies for 1 hr at RT, followed by gold-labeled goat anti-mouse IgG or goat antirabbit IgG for 1 hr at RT. The antibody complexes were stabilized with 2% glutaraldehyde in water, and the sections were stained with osmium vapor and lead citrate and examined as described above [53, 54] . Huh7 and Core-off cells were used as negative controls for antibody bindings. Multiple non-overlapping areas were scored for the frequency of HCV antigen localization and for the abundance of the antigen in each area. A positive signal is defined as the presence of at least two gold particles in a given organelle.
Immunocytochemistry staining for LC3. To determine the status of LC3 protein, cells were seeded on 12 mm Fisherbrand Coverglass for Growth (Fisher Scientific, Pittsburgh, PA, USA) and cultured in a 24-well plate overnight. Huh7 cells treated overnight with Bafilomycin A1 (200 nM, Wako Chemicals, Richmond, VA, USA) were used as positive controls for autophagy. Cells were fixed in 4% paraformaldehyde (in PBS, pH 8), quenched in 50 mM NH 4 Cl for 10 min, permeabilized in 0.1% Triton X-100/PBS for 10 min, and blocked in 1% BSA for 10 min. Cells were then incubated with 15 ml rabbit anti-LC3 polyclonal antibody for 60 min, followed by goat anti-rabbit IgG conjugated with Alexa Fluor 488 for 30 min. Cell nuclei were stained with 10 mg/ml Hoechst 33258 (Invitrogen). All steps were carried out at RT. Cover slips were washed in water, mounted in a drop of ProLong Gold antifade reagent (Invitrogen), and air-dried in the dark overnight. Cells were imaged with a 40x (NA = 0.75) objective on a Zeiss AxioVision microscope equipped with a Hamamatsu monochrome camera. For direct comparison of staining intensity, exposure time was kept constant at 89 msec.
Western blot analyses. To determine the protein level of HCV core protein, CuZn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD), Peroxiredoxin 1 and 3 (PRDX1 and 3), sulfonylated peroxiredoxin (PRDX-SO3), and cytosolic and mitochondrial aconitase (ACO1 and ACO2), cell pellets were incubated on ice with Tissue Protein Extraction reagent (T-PER, Fisher Scientific) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA), passed through 26 gauge needles several times, and centrifuged at 13,000 g, 4uC, for 2 min to remove cell debris. Total protein concentration was determined with a NanoVue Spectrophotometer (GE Healthcare, Piscataway, NJ, USA), and 50 ìg total proteins per lane were used for western blot analyses. Proteins were separated by 4-20% Mini-PROTEANH TGX gels (Bio-Rad, Hercules, CA, USA) (core, CuZnSOD, MnSOD, ACO1, and ACO2) or 16% Novex Tris-Glycine gels (Invitrogen) (PRDX1, PRDX3, and PRDX-SO3) and transferred to 0.2 mm nitrocellulose membranes (Bio-Rad). PRDX-SO3 antibody only detects sulfonylated PRDX and is not specific to PRDX1-SO3 or PRDX3-SO3. Consequently, sulfonylated PRDX1 and PRDX3 were distinguished based on their size difference ( Figure S5C ). To determine LC3 protein levels, cells cultured in 60 mm dishes were washed once with PBS and then scraped directly in the cell lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 2% SDS, 1 mM EDTA, 1x protease inhibitor cocktail) on ice. To break open the membranes, cell lysates were freeze-thawed two times and passed through a 26 gauge needle five times. Fifty mg of each cell lysate was separated with Any KD Mini-PROTEANH TGX gels (Bio-Rad) and transferred to 0.2 mm nitrocellulose membranes. The same membranes were used for thioredoxin 1 (TRX1) and thioredoxin 2 (TRX2) analyses. Alternatively, LC3-I and II were separated by 12% NuPAGE Bis-Tris gels (Invitrogen) in a subset of the studies. All membranes were incubated with primary antibodies overnight at 4uC followed by IR-labeled secondary antibodies for 1 hr at RT on a shaker. Membranes were washed three times each between primary and secondary antibody incubation and after secondary antibodies with PBST (PBS with 0.1% Tween 20). Membranes were then analyzed with the Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE, USA). Signal intensity of each protein was normalized to that of âactin. Three to four independent experiments were carried out for each protein; all results are plotted as mean 6 SEM.
The statistical analysis program GraphPad Prism (version 4.03, GraphPad Software, Inc., San Diego, CA, USA) was used for data analyses. One-way ANOVA analyses with Dunnett's post-hoc test were carried out initially to see if there was an effect of HCV proteins or treatments on the cellular indices being measured. Two-tailed Student's t tests were then used for pair-wise comparison between Huh7 and Genome-length replicon, subgenomic replicon, or Core-on cells. Student's t tests were also carried out for comparisons between Core-on and Core-off cells. Figure S1 Genome-length replicon, subgenomic replicon, and core protein expression. A, SEAP activities are used to monitor the expression of genome-length and subgenomic replicons in Huh7 cells. B, western blot analysis is used to determine the expression of core protein in genome-length replicon cells and Core-on cells.
(TIF) Figure S2 Oil-red-O staining for lipid deposit in HCV proteinexpressing Huh7 cells. Nuclei are stained with hematoxylin. Pictures were taken using a 20x objective. (TIF) Figure S3 Cytosolic aconitase (ACO1) protein levels and total aconitase activities. ACO1 protein levels (A) were determined by western blot analyses, and total aconitase activities (B) were determined with a kinetic assay coupled to the PMS/MTT color reaction. Mitochondrial aconitase (ACO2) protein levels were too low to be reliably quantified. Data are presented as mean 6 SEM of three independent experiments. (TIF) Figure S4 Overexpression of superoxide dismutase (SOD) and catalase (CAT) in HCV protein-expressing Huh7 cells. Cells were transfected with expression vectors designed for dual expression of CuZnSOD/cytosolic catalase (SOD1/cCAT) or MnSOD/mitochondrial catalase (SOD2/mCAT) to increase antioxidant capacity in the cytosol or mitochondria, respectively. For each set of transfection, the order of cells loaded (from left to right) is Huh7, genome-length, subgenomic, Core-on, and Core-off cells. SOD1 and SOD2 are tagged with V5 and CAT with Myc epitope and are detected with antibodies against these epitopes. Endogenous SOD1, SOD2, and CAT are not detectible by V5 or Myc antibody and are therefore, not visible in this blot. (TIF) Figure S5 The redox state of peroxiredoxin 1 (PRDX1) and 3 (PRDX3). The ratios of PRDX1-SO3 to PRDX1 (A) and PRDX3-SO3 to PRDX3 (B) were determined by sequential western blot analyses (see C below). C, representative western blots showing the separation of PRDX1 and PRDX3 in 16% polyacrylamide gels and the sequential binding of specific antibodies to PRDX-SO3, PRDX1, and PRDX3. Data are presented as mean 6 SEM of four independent experiments. (TIF)
Table S1 Immunogold EM localization of HCV proteins. The subcellular location of HCV core, NS5A, and NS5B proteins and the frequency at which they are observed in each organelle by immunogold electron microscopy is presented.
(DOCX) Neighborhood Properties Are Important Determinants of Temperature Sensitive Mutations Temperature-sensitive (TS) mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method–logistic regression–to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations. 0.38 -0.03 0.48 1.11 0.00 Turn breaker: turn is defined by DSSP [2] 0.58 -0.03 0.48 0.32 0.00 * ACC = accuracy, MCC = Matthews correlation coefficient, AUC = area under the curve, KL = Kullback-Leibler divergence, DD = distribution distance. These values were calculated from a ten-fold cross-validation of each feature. Missing and accounted for: gaps and areas of wealth in the public health review literature BACKGROUND: High-quality review evidence is useful for informing and influencing public health policy and practice decisions. However, certain topic areas lack representation in terms of the quantity and quality of review literature available. The objectives of this paper are to identify the quantity, as well as quality, of review-level evidence available on the effectiveness of public health interventions for public health decision makers. METHODS: Searches conducted on http://www.health-evidence.ca produced an inventory of public health review literature in 21 topic areas. Gaps and areas of wealth in the review literature, as well as the proportion of reviews rated methodologically strong, moderate, or weak were identified. The top 10 topic areas of interest for registered users and visitors of http://www.health-evidence.ca were extracted from user profile data and Google Analytics. RESULTS: Registered users' top three interests included: 1) healthy communities, 2) chronic diseases, and 3) nutrition. The top three preferences for visitors included: 1) chronic diseases, 2) physical activity, and 3) addiction/substance use. All of the topic areas with many (301+) available reviews were of interest to registered users and/or visitors (mental health, physical activity, addiction/substance use, adolescent health, child health, nutrition, adult health, and chronic diseases). Conversely, the majority of registered users and/or visitors did not have preference for topic areas with few (≤ 150) available reviews (food safety and inspection, dental health, environmental health) with the exception of social determinants of health and healthy communities. Across registered users' and visitors' topic areas of preference, 80.2% of the reviews were of well-done methodological quality, with 43.5% of reviews having a strong quality rating and 36.7% a moderate review quality rating. CONCLUSIONS: In topic areas in which many reviews are available, higher level syntheses are needed to guide policy and practice. For other topic areas with few reviews, it is necessary to determine whether primary study evidence exists, or is needed, so that reviews can be conducted in the future. Considering that less than half of the reviews available on http://www.health-evidence.ca are of strong methodological quality, the quality of the review-level evidence needs to improve across the range of public health topic areas. Using Systematic Reviews A systematic review consists of an examination of all of the primary studies on a topic, which includes searching for, collating, and assessing the studies, to establish conclusive evidence about a topic [1] . The Cochrane Collaboration is an international body that produces systematic reviews of primary research at the highest standard, and as such, this is a commonly accepted definition of systematic reviews. Evidence-informed public health advocates the incorporation of the best available scientific evidence into decision making [2] . Review level evidence is an important part of evidence-informed public health decision making, since reviews synthesize the results of individual studies, providing a more accurate estimate of the effects of an intervention [3] . Rigorous synthesis of primary research minimizes bias [4] [5] [6] , explains differences among studies relating to the same research question [7, 8] , and presents more precise and consistent summary statistics than the effect sizes found in individual studies [5, [8] [9] [10] . Well-conducted reviews provide high-quality, accurate evidence [4] [5] [6] , increasing decision-makers' confidence in the strength of the review evidence and in applying the findings in practice [4] . Public health decision makers prefer using systematic reviews to assist in decision-making given that review level evidence saves time and is more efficient compared to using primary studies [6, 11] . Systematic review findings can be generalized to a larger sample, providing a great evidence base for users; such external validity is essential to ensure adaptability and applicability of evidence-based interventions into the local context [12] . Consequently, systematic reviews are useful for informing and influencing public health policy and practice decisions [7, 13, 14] .
While the value of review level evidence is acknowledged and well documented, public health decision makers encounter a number of challenges in incorporating systematic reviews in their decision making.
Review level evidence is available in journals and obtainable through bibliographic database searches [15] , yet barriers still exist in accessing the information. Public health decision-makers often have difficulties locating systematic reviews in the published literature due to database indexing limitations, limited availability of relevant public health reviews, lack of primary study evidence and thus a lack of reviews in some topic areas, and a lack of interest in certain topic areas by researchers conducting reviews [3, 15, 16] .
Even when systematic reviews are identified, only a small proportion of those are relevant to public health. For example, one search strategy captured 41, 871 abstract titles across all research topics in public health but once screened, only 1, 356 were identified as being potentially relevant, of which only 207 reviews were actually deemed relevant to public health [16] . The majority of published systematic reviews pertain to clinical topics rather than public health [16] . Consequently, there are gaps at the systematic review level across the spectrum of public health practice [6, 17] .
When relevant reviews are located, users still need to be critical of that evidence. There are a number of search engines that provide evidence from various databases, such as the Cochrane Database of Systematic Reviews, PubMed, and the Campbell Collaboration, but the evidence is not critically appraised [17, 18] . While some of these databases which include public health relevant evidence assess the quality of the evidence, many do not [19] . To reduce bias in evidence-informed practice, public health decision makers need to be able to assess the methodological quality of systematic reviews [4] . However, critical appraisal skills have been identified as a significant barrier to using research evidence in decision making [20] . Development of individual capacity is important in addressing appraisal challenges as well as providing support [21] .
Health Evidence is a research and service organization aimed at supporting Canada's public health decision makers in accessing and interpreting research evidence. The target audience for Health Evidence includes medical officers of health, policy makers, program managers, and frontline workers in public health. Given the audience, decision making may take place at the local level (such as public health units/regional health authorities), provincial level (such as ministries), or federal level (such as government). Our most widely accessible resource is the http://www.health-evidence.ca online registry of systematic reviews; a free, user-friendly, searchable database of public health relevant, qualityappraised systematic reviews published since 1985 evaluating the effectiveness of public health interventions. Given that unpublished literature, such as conference abstracts, provide little added value [22] , Health Evidence mainly focuses on published review literature. In order to identify the scope of interventions to include in the health-evidence.ca registry, qualitative interviews were conducted, as well as seeking organizational charts and information from every province and territory in Canada on the services public health units provide. Systematic reviews are considered relevant if: 1) the article is a review, which includes the synthesis of more than one primary study; 2) the intervention is relevant to public health practice; 3) the effectiveness of an intervention is evaluated; 4) the evidence on health outcomes is reported; and 5) the search strategy is described [19] . To assess the methodological quality, the following ten criteria are used: 1) a clearly focused question was stated; 2) inclusion criteria were explicitly stated; 3) a comprehensive search strategy was described; 4) an adequate number of years were covered in the search; 5) a description of the level of evidence was provided; 6) the methodological rigor of primary studies was conducted and results were described; 7) the methodological quality of primary studies was assessed by two reviewers and the level of agreement was provided; 8) tests of homogeneity or assessment of similarity of results across studies was conducted and reported; 9) appropriate weighting of primary studies was conducted; and 10) the author's interpretation of the results were supported by the data [19] . Each criterion is equally weighted and a final methodological score is tallied out of 10. Reviews with an overall rating of eight or more are considered strong, five to seven, moderate, and below four are considered to be weak in methodological quality.
Due to competing demands, it is necessary for decision makers to quickly find, assess and use evidence to inform their decision making. The health-evidence.ca registry eliminates the need for users to search individual databases, identify relevant reviews, and conduct critical appraisal on the effectiveness of public health interventions. The tools used by Health Evidence to assess relevance and conduct critical appraisal are available online http://health-evidence.ca/html/HowJudgefor-Yourself, accessed 6 May 2011), and users can view completed critical appraisal tools for each review in the registry.
In order to reach public health decision makers, Health Evidence is promoted at conferences, workshops, and site-visits, through outreach and engagement, networking, and listservs via website posts and e-newsletters, and through social media, such as Twitter and YouTube. Health Evidence also connects with public health decision makers through various partnerships and collaborations with the National Collaboration Centres for Public Health, public health units, the Canadian Best Practices Portal, and the Public Health Agency of Canada. The registry is also listed as a resource on several public health organization and university websites, such as Research into Action, Pan American Health Organization, KT+ Knowledge Translation, Canadian Institute for Health Research (CIHR) Knowledge Translation and Commercialization, Nova Southeastern University, Dalhousie University, and more.
Health-evidence.ca has nearly 5, 000 registered users, and sees over 40, 000 visitors annually representing more than 150 countries. In the development of the health-evidence.ca registry, 21 topic areas of interest to public health decision makers were identified through focus groups and consultations with key informants within the public health setting. The purpose of the registry is to facilitate access to review-level evidence for decision makers working in program planning and policymaking in public health and health promotion [19] . All reviews in the registry are indexed according to these 21 Focus of Review topic areas allowing site visitors to search the registry using common public health terms. In addition, each registered user completes a profile when signing up to the site checking off as many of the 21 topic areas relevant to them. This enables each registered user to receive a list of reviews related to their areas of interest, along with a rating of the methodological quality of each review, each quarter when the registry is updated.
Unfortunately, for some public health topics, there are limited or no high quality reviews available and for others the reviews that are available are not of good methodological quality, meaning that use of these findings in decision making requires careful consideration.
A thorough search of http://www.health-evidence.ca allowed us to indentify the top areas of interest to public health decision makers, and provide an overview of the availability of review-level evidence within these areas. In this paper we will not only identify topic areas of high interest to public health decision makers, we will also highlight existing gaps as well as identify topic areas with an abundance of high-quality evidence. One objective of this paper is to identify the quantity of systematic reviews available on the effectiveness of public health interventions, so as to encourage researchers and research funders to conduct/fund systematic reviews where gaps exist. A second objective is to identify the quality of systematic review evidence on the effectiveness of public health interventions in order to encourage higher quality methodological reviews and higher level synthesis of topics areas rich in high-quality reviews (e. g., review of reviews).
Populating the health-evidence.ca registry of systematic reviews
The health-evidence.ca registry of systematic reviews is populated through an extensive ongoing search (1985present) of seven electronic databases (MEDLINE, EMBASE, CINAHL, PsycINFO, Sociological Abstracts, BIOSIS, SportDiscus), handsearching of 46 journals, and screening the reference lists of all relevant reviews [19] . Reviews are assessed for relevance, and then relevant reviews are indexed by commonly-used public health terms and quality assessed by two independent reviewers who come to agreement on the final rating of each review (strong, moderate, weak). More detail on http://www.health-evidence.ca has previously been published [19] .
Assessing health-evidence.ca user and visitor areas of interest Registered user areas of interest were assessed by querying the health-evidence.ca registered user database and looking at the areas of interest identified by all users who registered up to December 31, 2010. Data were aggregated by topic area. Registered user data is provided voluntarily by users and aggregation ensures individual data remain anonymous. Topic areas of interest were ranked from highest to lowest rates of user interest. The top 10 areas of interest were summed to generate the denominator: total user interest in the top 10 topic areas.
Visitor areas of interest were assessed by summing frequency of visitor searches of the 21 Focus of Review topic areas and visitor use of the topic area browse menu for the period January 1, 2010 to December 31, 2010. Visitor site usage is tracked via Google Analytics, a web analytics tool that collects and aggregates nonpersonal data to report on visitor interaction with the health-evidence.ca website. Total search and browse access by unique visitors were ranked from highest to lowest pageviews. The top 10 areas of interest were summed to generate the denominator: total visitor interest in the top 10 topic areas.
The health-evidence.ca registry was used to identify gaps and areas of wealth in the public health review literature. Each of the 21 Focus of Review topic areas were searched, and the quantity and proportion of reviews rated methodologically strong, moderate, and weak were identified. Three categories were used to define availability of reviews within each topic area: (+) few, representing 1-150 reviews; (++) moderate, representing 151-300 reviews; and, (+++) many, representing topic areas possessing greater than 301 reviews. Reviews that addressed multiple topics were accounted for within each topic area that they addressed (e.g., a review on the effectiveness of exercise in preventing chronic disease would be categorized as both physical activity and chronic disease).
As of December 31, 2010, there were 4, 842 health-evidence.ca registered users, with each user identifying an average of 6.3 areas of interest, resulting in a total of 30, 363 identified topic areas. Upon registration, each user is asked to indicate as many areas of interest as they find relevant, which results in more identified areas of interest than total users. For the purpose of accurately representing the data showing all interest, we have included all indications in interest in each topic area, knowing that the denominator used represents total expressions of interest as opposed to total users. The top 10 registered users' topic areas are represented in Figure 1 . Registered users' top three topic areas out of the top 10 include by order of interest: 1) healthy communities, 2) chronic diseases, and 3) nutrition. order of interest include: 1) chronic diseases, 2) physical activity, and 3) addiction/substance use.
The top 10 topic areas of interest of registered users and the top 10 topic areas of interest of visitors of http:// www.health-evidence.ca, as well as the availability of review evidence by methodological quality, are identified in Table 1 . The top areas of interest and the total number of reviews available included: addiction/substance use (355), adolescent health (367), adult health (552), child health (409), chronic diseases (702), communicable disease/infection (241), healthy communities (134), injury prevention/safety (296), mental health (336), nutrition (426), parenting (287), physical activity (353), reproductive health (240), and social determinants of health (66).
While there was overlap between six of the registered users' and visitors' top areas of interest, the topic areas healthy communities, adult health, adolescent health, and, communicable disease/infection were preferred by registered users alone, and visitors had preferences for addiction/substance use, parenting, injury prevention/ safety, and, reproductive health.
For the six areas of interest similar to both registered users and visitors, differences exist in the order of expressed interest. Three topic areas ranked similarly among the top 10 for both registered users and visitors with a difference of only one rank apart: chronic disease ranked high on both top 10 lists, ranking first for visitors and second for registered users; nutrition ranked third for registered users and fourth for visitors; and social determinants of health ranked sixth for visitors and seventh for registered users. The remaining three topic areas that both visitors and registered users were interested in included: mental health, ranking seventh for visitors and ninth for registered users; physical activity, ranking second for visitors and fifth for registered users, and, child health, ranking fourth for registered users and ninth for visitors. In the top five highest ranking common topic areas, both groups had interest in chronic diseases, nutrition, and, physical activity Characteristics of registered users of and visitors to the Health Evidence registry are provided in Table 2 . Based on the sample, 82.7% of registered users and 65.0% of visitors to health-evidence.ca are Canadian, and English is the language of preference for 98.1% of registered users and 83.3% of visitors. Upon registering to health-evidence.ca, users are asked to provide their organizational affiliation; 54.6% of users work in the field of public health or health services. As of December 31, 2010, 94.8% of registered users were subscribers to the quarterly Health Evidence tailored e-newsletter.
As of April 1, 2011 there were 2, 175 systematic reviews evaluating the effectiveness of public health and health promotion interventions indexed in the health-evidence. ca registry. Table 3 provides an overview of the availability of reviews within each of the 21 Focus of Review topic areas. Figure 3 depicts the relationship between registered users' interests, visitor searches, and available reviews within each of the 21 topic areas.
Topic areas with fewer than 150 reviews included: food safety and inspection (13) , dental health (62), social determinants of health (66), environmental health (69), and healthy communities (134). Of the areas with fewer than 150 reviews, healthy communities ranked first as an area of interest for registered users with 2, 548 registered users indicating interest in this topic. Social determinants of health ranked sixth and seventh for visitors * Visitor data for organization type is shown for organizations with five or more visits in 2010. † Visitor data was collected by internet service provider and many organizations could only be identified by internet service provider (e.g. Rogers, Shaw, Bell, etc.) and registered users respectively, with 1, 528 visitor searches submitted in 2010 and 1, 734 registered users indicating interest in the topic. The majority of healthevidence.ca registered users did not have preference for the other three identified areas with fewer than 150 reviews.
Topic areas with a moderate number of reviews (151-300 reviews) included: infant health (153), senior health (152), sexual health (195), sexually transmitted infections (208), communicable disease/infection (229), reproductive health (232), parenting (282), and, injury prevention/safety (241). Four of these eight topic areas have expressed interest by visitors or registered users. For visitors, ranking at fifth, eighth and tenth place respectively, searches submitted in 2010 for parenting totalled 1, 813 pageviews, for injury prevention/safety 1, 360, and, for reproductive health 1, 273 pageviews. Ranking tenth place, 1, 422 registered users indicated interest in the topic communicable disease/infection.
Topic areas with a large quantity of systematic reviews (301 or more reviews) include: mental health (336), physical activity (353), addiction/substance use (355), adolescent health (367), child health (409), nutrition (426), adult health (552), and chronic diseases (702). All of the topic areas with many available reviews (301+) were of interest to registered users and/or visitors. Chronic diseases, nutrition, and, physical activity were the three highest-ranking areas of interest common across both groups. The number one visitor area of interest and number two registered user area of interest was chronic diseases with 3, 115 visitor searches submitted in 2010, and 2, 153 registered users expressing interest in the topic. Nutrition ranked third for registered users and fourth for visitors with 1, 877 user interests, and 2, 010 visitor searches respectively. Physical activity ranked second for visitors with 2, 350 searches, and fifth for registered users with 1, 824 interested in the topic. Child health and mental health were two additional topics with many available reviews that rank in the top ten for both registered users and visitors. In the area of child health, 1, 846 registered users expressed interest and 1, 293 visitors submitted searches, ranking it fourth and ninth respectively, and the area of mental health ranked seventh for visitors with 1, 502 searches submitted, and ninth for registered users with 1, 459 interested in the topic. The remaining three topic areas with many available reviews were preferred by either visitors or registered users (i.e., not common to both groups). Addiction/substance use ranked third for visitors with 2, 283 search page views in 2010. Adult health and adolescent health ranked sixth and eighth respectively for registered users with 1, 778 and 1, 636 registered users indicating interest in each of these topics.
A master list categorizing all of the 21 topic areas and the corresponding number of reviews available on the effectiveness of public health interventions, as well as the methodological quality, are listed in the additional file 1.
The 21 Focus of Review topic areas were further broken down into 291 sub-topic categories. There were 34 sub-topics with no reviews available including: hormone replacement therapy, infertility, Norwalk virus, autism, and elder abuse, among others ( Table 4 ). The 21 Focus of Review topic areas that had sub-topics with no review included adult health, communicable disease/infection, dental health, environmental health, food safety and inspection, parenting, and senior health. The largest proportion of sub-topic with no review was observed within communicable disease/infection (n = 12). Adult health was ranked sixth and communicable disease/ infection ranked tenth by registered users. Parenting was ranked as a fifth preference for visitors. The remaining sub-topic with no reviews within dental health, environmental health, food safety and inspection, and senior health were not a preference for either registered users or visitors. (+)few reviews as 1-150, (++)moderate reviews as 151-300, (+++)many reviews as 301 and greater
In addition there were 68 sub-topics with fewer than five reviews available, such as lung cancer, testicular cancer, food service inspection, fetal alcohol syndrome, sexual assault, and social justice. The full list of subtopics with fewer than five reviews is included in Table  5 . Most of the sub-topics with fewer than five reviews were within the registered users and visitors' topic areas of interest. Topic areas which were of interest to both registered user and visitors only had a small proportion of sub-topics with less than five reviews available (child health, chronic diseases, mental health, nutrition, and social determinants of health). Whereas the communicable disease/infection topic area, which ranked tenth among registered users, had the largest proportion of sub-topics with fewer than five reviews (n = 16). While environmental health and food safety and inspection were not preferred topic areas for registered users and visitors, a large portion of the subtopics in these two categories had fewer than 5 reviews. Although there is currently a lack of review literature in these areas, these topics have been mentioned by public health professionals as relevant to public-health practice.
Also, there were numerous sub-topics with a great number of reviews available. Table 6 identifies 71 subtopics with more than 25 reviews available. Such subtopics included, but were not limited to, alcohol abuse/ use, smoking cessation, women's health, cancer, cardiovascular disease, lifestyle behaviours, disease transmission, depression, diet, healthy weight, exercise, and HIV. As well, most of these sub-topics were within the registered users and visitors' topic areas of interest, with the exception of dental health, senior health, sexual health, and sexually transmitted infections.
The Health Evidence methodological quality rating is based on the ten criteria used to assess the strength of the methods. The proportion of reviews rated as having strong, moderate, or weak methodological quality was constant across all topic areas. 80.2% of the reviews on health-evidence.ca were of strong (43.5%) or moderate (36.7%) methodological quality. These well-done reviews included slightly more strong review quality ratings compared to the moderate review ratings. The remaining 19.8% of reviews in the top areas of interest were of weak methodological quality. These weak quality reviews met four or fewer methodological quality criteria, and as such, scored poorly on six or more of the ten criteria. Based on the weak reviews, 10.5% did not have a clearly focused question, 46.5% did not use appropriate inclusion criteria, 87.7% did not have a comprehensive search strategy, 34% did not cover an adequate number of years, 48% did not describe the level of evidence in the primary studies, 96.6% did not assess the methodological 
A number of sub-topic areas within public health featured no reviews or a very small number of reviews including those within the main topic areas for women's health (sub-topics include female genital mutilation, hormone replacement therapy, and infertility as examples); communicable disease/infection (sub-topics include food-borne diseases, hantavirus, and Norwalk, among others); food safety and inspection (sub-topics include botulism, food processing and inspection, and Hepatitis A as examples); dental health (sub-topic: dental implants); environmental health (sub-topics include asbestos, carbon monoxide poisoning, environmental epidemiology, extreme temperature, swimming pools, and flood, as examples). In these areas, review literature is needed to add to the existing body of literature from which decision makers can draw. In topic areas lacking in review literature, realist reviews, which provide explanatory analyses [23] , are a relatively new type of review that may provide further insight into the topic area. While these areas show the deficits in review literature, in 25 other sub-topic areas there was a wealth of systematic review literature of moderate or strong quality, including but not limited to, alcohol abuse/use, smoking cessation, women's health, cancer, cardiovascular disease, lifestyle behaviours, disease transmission, depression, diet, healthy weight, exercise, and HIV. In these areas offering many reviews, most had 15 or more which were of strong methodological quality. While review groups can identify and fill gaps in areas where evidence is lacking, there is also an opportunity to produce higher level syntheses where good-quality review evidence is available. Based on this analysis of the published, public health review literature catalogued in http://www.health-evidence.ca, all of the topic areas having many available reviews were also preferred areas of interest for users of the site. It is unclear how the availability of evidence is linked to interest in a topic, but it may be that demand can generate reviews in a particular topic area and that the public spurs research to fill gaps [24] . Alternately, preference for a topic may be a reflection of there being available evidence that drives interest in the topic and web site updates regarding that topic. Interestingly, the 15 priority topic areas indicated by a 2005 Cochrane global priority setting exercise [24] still closely match the top 10 topic areas of interest indicated by health-evidence.ca users and visitors.
While resources such as http://www.health-evidence. ca, provide synthesized, high quality research evidence relevant to public health practice, coverage of health topics is not equal [25] . Compared to clinical review literature, there is far less population health systematic review literature [6] and within public health, a number of topic areas are without a solid base of review evidence evaluating the effectiveness of interventions (e.g., environmental health, social determinants of health) [18, 26, 27] . Public health studies are difficult to design and results are drawn from natural experiments (e.g. one health unit adopting a program compared to another health unit), thus fewer studies have been developed on the effectiveness of public health interventions compared to randomized controlled trials on medical treatments [28, 29] . However this lack of review-level evidence doesn't necessarily indicate a lack of evidence [30] . There may be primary research or other forms of evidence that can inform decisions but which may not yet have been synthesized. In cases where there are no eligible studies available to be reviewed for a particular topic, the result can be an "empty review", meaning that no studies were located which met the inclusion criteria to answer the question for review. Empty reviews can go unreported but may in fact be useful since empty reviews indicate interest in an area, highlight gaps, and offer a snapshot of the state of research evidence at the time of publication [31] . Even in light of a lack of evidence, or poor reporting of the evidence, decision makers can and should take an informed approach to having insufficient evidence [30] . An informed approach may be needed in topic areas that demonstrated a lack of review-level evidence, such as dental health, environmental health, food safety and inspection, and seniors' health, and particularly for public health priority areas such as healthy communities and social determinants of health. Consequently, the best literature may be sparse or of low quality in these particular topic areas. In these areas, new systematic review literature is needed to inform practice and policy decision making. It is unclear at this time whether gaps pertain only to reviews lacking in these topic areas, or whether there is a corresponding lack of primary studies as well, hindering the production of reviews. In some instances it may be necessary to first develop the primary study base in order for studies to be available for synthesis in a systematic review. Future reviews should be conducted on these broad topic areas for which review-level evidence on the effectiveness of interventions is lacking. Funding organizations should generate calls for syntheses to address these gap areas, while non-government and public health organizations should provide feedback on the lack of evidence in areas of interest to them to potential funders. Funding priorities for syntheses should reflect those areas which are priorities for public health decision makers both in Canada and internationally.
There are promising indicators of demand for reviews, including actions being taken to promote the use of reviews [6, 11, 19, 30, [32] [33] [34] [35] [36] , an awareness of sites providing access to review-level evidence [19] and an increasing number of groups generating summaries of reviews [33] . Despite this heightened activity, given the gaps, a greater investment is needed to provide an evidence base that can meet demand and determine how to apply existing good quality systematic reviews in different contexts [25] . Organizations involved in the conduct of systematic reviews should direct synthesis funding to areas lacking in review content, or should consider higher-level reviews of reviews (where appropriate), where large bodies of review evidence already exist. Examples of organizations that conduct systematic reviews and reviews of reviews include: The Cochrane Database of Systematic Reviews http://www.cochrane.org/reviews/ index.htm, The Campbell Collaboration http://www. campbellcollaboration.org, The Centre for Reviews and Dissemination (CRD) http://www.york.ac.uk/inst/crd/ index_databases.htm, Health Technology Assessment international (HTAi) http://www.htai.org/, Effective Public Health Practice Project (EPHPP) http://www.ephpp. ca, CDC Guide to Community Preventative Services http://www.thecommunityguide.org, Canadian Agency for Drugs and Technology in Health (CADTH) http:// www.cadth.ca/, Agency of Healthcare Research and Quality (AHRQ) http://www.ahrq.gov/ and the National Institute for Health and Clinical Excellence (NICE) http://www.nice.org.uk. Some groups have a prioritization process to identify and meet needs for systematic reviews; for example, the Agency of Healthcare Research and Quality has a topic prioritization group to determine the relative importance of their effectiveness reviews against a standard set of criteria to prioritize unmet needs [37] . The Cochrane Collaboration aims to increase the quantity and quality of public health systematic reviews specifically [32] , with the new Health Promotion and Public Health Review Group announced in 2006 to prioritize and produce public health relevant reviews [38] . Authors have suggested that a global registry of anticipated public health studies could help to fill the gaps by making it easier to identify relevant, but potentially unpublished, primary studies available for review [6] .
While well-done reviews in a large number of areas are available, it is important to continue to improve the quality of the overall body of public health review literature. Considering that the majority of weak reviews scored poorly on assessing the methodological quality of the primary studies, transparency, methods for combining or comparing results, conducting a comprehensive search strategy, and data supporting the author's interpretations, review authors should be cognisant of these criteria when conducting systematic reviews. In improving the quality of systematic reviews, the overall goals should ensure there are high quality reviews in all public health topic areas and a higher standard across the board.
Although this paper provides information on the quantity and quality of systematic reviews on the effectiveness of public health interventions, there are a few limitations, including the lack of information regarding visitors to the Health Evidence registry. While the majority of visitors access the registry from various internet service providers, we are unable to determine their organization and the relevance of the registry content to a particular organization. Additionally, it is unclear at this time whether visitors and registered users are using reviews housed in the registry to inform their practice decisions. Studies are ongoing to evaluate the registry's usefulness and effectiveness. Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU. Nairobi sheep disease virus (NSDV) is a member of the genus Nairovirus within the family Bunyaviridae and causes acute hemorrhagic gastroenteritis in sheep and goats, with very high morbidity and mortality rates in susceptible animals [1] . It was originally isolated in Nairobi, Kenya in 1910 by inoculation of sheep with the blood of sheep suffering from acute gastroenteritis. NSDV was originally thought to be endemic only in East Africa; recent sequence data showed that the same virus can also be found in many places in India and Sri Lanka where it is called Ganjam virus (GV) [2] . Daubney and Hudson showed that NSDV is primarily transmitted in East Africa by the hard tick Rhipicephalus appendiculatus, and that animals that were bred in areas where this tick was prevalent were immune, but animals that were moved into such areas died in large numbers [3, 4] . The virus is therefore only of limited effect on stable populations, but can be a severe limitation on trade or attempts to improve stocks through introduction of new animals. There is no current vaccine. In India, the virus is found in a number of tick species, primarily Hemaphysalis intermedia [5] . Sheep and goats are the only known vertebrate hosts of NSDV/GV [6, 7] , although one or two cases of human infection through needle-stick injury have been reported as leading to mild febrile illness [8, 9] .
Nairoviruses are small, enveloped RNA viruses in which the genome consists of three segments of single stranded, negative sense RNA, designated Large (L), Medium (M) and Small (S) [10] . The S, M and L segments encode, respectively, the nucleocapsid protein (N), at least two envelope glyoproteins (Gn and Gc) and the viral RNA-dependent RNA-polymerase (L). The L segment is unusual among bunyaviruses, being extremely long (.12 kb), encoding a single protein of .450 kDa. The carboxyterminal half of this protein contains most of the polymerase motifs, while the amino-terminal part is largely of unknown function. The genus contains more than 30 different virus isolates, loosely grouped based on serum cross-reactivity and hemagglutination inhibition [11] , since sequence data on all but a few of these viruses has been limited or missing until recently. The most important serogroups are the NSDV serogroup, which also includes Dugbe virus (DUGV) and Kupe virus, and the Crimean Congo hemorrhagic fever virus (CCHFV) serogroup, which contains CCHFV and Hazara virus, both human pathogens. CCHFV causes a severe disease in human beings, with a reported mortality rate of 3-30% [12] . The disease is very similar to that caused in sheep by NSDV infection and is characterised by haemorrhage, myalgia, and fever.
The first line of defence against virus infections is innate immunity. The key players are interferons (IFNs) and other cytokines that are rapidly produced in virus infected cells (Reviewed in [13] ). Three major classes of IFNs are known. Type I IFNs comprise the largest group, with multiple distinct IFNa genes, one to three IFNb genes and other genes (IFNv, -e, -d, -k). The first two are induced directly in response to viral infection whereas the others play less defined roles. Type II IFN has a single member, namely IFNc, which is secreted by activated T cells and natural killer cells rather than in direct response to virus infections. A third class of IFNs has been described recently that shares the same pathway to sense viral infection as type I IFNs and is also induced directly in response to viral infection [14, 15, 16] . After an infected cell senses a viral infection, IFNs are produced and released from the cell to induce an antiviral state in both itself and neighbouring cells. Both type I and type II IFNs bind to their cognate cell-surface receptors, thereby activating a signal-transduction pathway that triggers the transcription of several hundreds of genes [17] . These IFN-stimulated genes (ISGs) have either IFNstimulated response elements (ISRE) or GAS (Gamma-activated sequence) elements in their promoter region. Type I IFNs such as IFNa can lead to transcription of ISGs with ISRE or GAS elements, whereas IFNc can only induce ISGs with GAS elements (reviewed in [18] ).
It seems that most viruses studied so far have developed a strategy to counteract the host innate immune system [13] . Two Nairoviruses (CCHFV, DUGV) have been shown to be inhibited by MxA, a protein induced by type I IFNs [19, 20] . Since the viruses are inhibited by IFN-induced proteins, viral virulence will depend at least in part on the ability of the virus to avoid or block the type I IFN response, and it is therefore very likely that Nairoviruses have developed tactics against this host defence mechanism. The current knowledge on how Nairoviruses manipulate the host innate immune system comes mostly from studies with CCHFV. Andersson et al. have shown that CCHFV showed a markedly delayed type I IFN response in cell culture, up to 48 hours after infection, possibly by interfering with the pathway that leads to activation of interferon regulatory factor 3 (IRF3) [21] . In addition they found that CCHFV is insensitive to IFNa treatment applied six hours post-transfection. One possible explanation for the delay in the IFN response is given by another study showing that CCHFV has developed a mechanism to remove the 59-terminal triphosphate group from its genome segments, thereby avoiding retinoic acid inducible gene I protein (RIG-I)-dependant IFN induction [22] . Another possible explanation may be the activity of the ovarian tumour-like (OTU) domain found in the amino-terminus of the viral L protein. Sequence analysis of the L proteins of Nairoviruses identified this domain, which represents a unique class of deubiquitinating enzymes (DUBs) [23] . Recent studies with the CCHFV OTUcontaining L protein showed that it decreases the coupling of ubiquitin (Ub) and the ubiquitin-like protein (Ubl) encoded by interferon stimulated gene 15 (ISG15) to cellular proteins [24] . The post-translational modification of proteins by Ub and Ubls regulates essential processes in the type I IFN response to viral pathogens [25, 26] . Cellular DUBs have been found which appear to act as part of negative feedback control systems for IFN induction pathways [27, 28] . Removal of Ub and/or ISG15 from their protein conjugates will disrupt a number of elements of the IFN induction pathway, and targeting the ISG15 and/or the ubiquitin system is a common strategy used by different viruses to inhibit innate immune responses [29, 30, 31, 32] .
Up till now there is no information available on if and how NSDV/GV manipulates the host innate immune system. Because of the difficulties in handling CCHFV, there is only limited correlation of studies on virus and on viral proteins. We provide here the evidence that NSDV/GV is able to inhibit IFN induction as well as IFN action. We could identify the viral L protein as being responsible for these inhibitory effects. Furthermore NSDV is able to reduce total protein ubiquitination and ISG15ylation in infected cells. This deconjugating activity, as with CCHFV, is located in the N-terminal part of the NSDV L protein containing the OTU domain. Inactivation of the OTU enzymatic activity resulted in loss of its ability to antagonise IFN responses.
Two isolates of NSDV/GV were available to us for these studies, one a highly tissue-culture passaged isolate of the virus from Uganda (and therefore notionally NSDV), the other an isolate from India (and therefore notionally GV) which had been passaged a limited number of times in mouse brain or BHK 21 cells. Although these two isolates have been shown phylogenetically to be the same virus [2] , we will refer to them as GV and NSDV in this paper to indicate the two isolates. The GV isolate proved to be still pathogenic in sheep, while the NSDV isolate was nearly completely attenuated, and the GV isolate is therefore probably nearer to wild type virus.
As described in the Introduction, studies with CCHFV have shown it has a delayed IFN response in infected cells [21] . We wanted to know if NSDV/GV is able to interfere with the early induction of type I IFNs. To address this question we transfected Vero cells with a reporter gene construct containing the firefly luciferase under the control of the IFNb promoter. Luciferase expression is taken as a measure of protein production from this specific promoter after activation of transcription, which in turn is taken as a measure of IFNb induction. Although this system is commonly used to study the control of IFN induction, we recognise that failure to express luciferase could reflect viral effects at a number of different points in the induction, transcription and protein synthesis pathway. As a positive control for the induction of IFNb we used Newcastle disease virus (NDV), a paramyxovirus which has been reported as being unable to block IFN induction in mammalian cells [33] and therefore is frequently used as a model stimulator of cytoplasmic PRRs. Vero cells lack a functional IFN-b gene [34, 35] which made them a useful tool for our experiment because they allowed us to measure direct activation of the IFNb promoter in infected cells excluding any indirect effect of IFN synthesis in neighbouring uninfected cells. The transfected Vero cells were infected with the NSDV or GV isolate or with NDV and the amount of synthesised luciferase was determined at the indicated time points ( fig. 1a&b ). Infection with NDV induced a rapid activation of the IFNb promoter ( fig. 1a ); after 4.5 and 6.5 hours of NDV infection very high amounts of luciferase are already detected when compared to mock-infected cells, with levels of luciferase already decreasing at 9hpi. In contrast, at 4.5hpi there was no increase in promoter activity detectable in GV/ NSDV-infected cells when compared to mock-infected cells ( fig. 1b) . A very slight increase in luciferase was seen at 10hpi in NSDV-infected cells, and at 14 hpi there is a clear significant increase in the reporter gene activity induced by NSDV or GV infection with further increases up to 24 hours of infection. Recent evidence suggests that many negative-strand RNA viruses do not generate dsRNA during infection [36] and only induce IFN rapidly if they contain defective interfering particles (DIs) [37] . It is likely therefore that our NDV preparation is acting through a significant content of such DIs. Neither NSDV/GV isolate, however, induced interferon until later stages in the infection cycle. There was a significant difference between the two isolates, NSDV and GV, regarding the kinetics of activation of the IFNb promoter. Activation by the GV isolate was later than that induced by the NSDV isolate and also the transcriptional activation observed was lower when compared to the NSDV isolate ( fig. 1b ). This might be due to the fact that the NSDV isolate was previously passaged over 60 times in cell culture, in contrast to the GV isolate which has primarily been maintained in mouse brain culture and has been passed twice in BHK-21 culture in our hands; this difference may have resulted in the NSDV isolate having a reduced efficiency to evade IFN induction, or a more rapid growth in the cultured cells, leading to a more rapid production of one or more PAMPs recognised by the host cell. This second possible explanation is supported by the growth kinetics of GV and NSDV in Vero cells, which showed that NSDV replication rates were faster compared to GV, although both isolates grew to the same final titer (Lidia Lasecka, pers. communication).
NSDV/GV is able to block transcription from the IFNb promoter at early stages of infection
Next we wanted to examine if the absence of IFNb promoter activation in early stages of NSDV infection upon NSDV/GV infection is due to an active block or if NSDV/GV is rather avoiding the activation of the IFNb promoter by a similar mechanism as already described for CCHFV [21] . On that account we transfected Vero cells with the IFNb reporter gene plasmid. One day later cells were infected with the GV or NSDV isolate, and subsequently super-infected with NDV. Immunofluorescence experiments showed that approx 85% of cells were infected with NSDV/GV, and that prior infection with NSDV/ GV did not block NDV infection in these cells (data not shown). The reporter gene activity was determined as described in material and methods ( fig. 2 ). In cells infected with NDV alone the expected increase in the activity of the IFNb promoter was observed when compared to uninfected cells. Interestingly, when the NDV infection was preceded by infection with GV or NSDV there was a clear reduction in the promoter activation detectable compared to cells infected solely with NDV. The reduction in the effects of NDV in these assays was only 40% even though about 85% of the cells were infected with NSDV/GV. This may have been because there was insufficient NSDV/GV protein at the time of the NDV superinfection to provide a complete block of the IFN induction pathway inside each infected cell; the reduction in reporter gene activity was indeed less pronounced if NDV was applied after shorter periods of GV or NSDV infection such as four hours post infection (data not shown), showing that it takes some time for the active block of the IFN induction pathway to take effect. This discrepancy may also reflect the limited nature of the block provided by NSDV/GV, which is unable to completely prevent activation of the IFN-beta promoter later in its own infection (FIg 1b) , as well as the very strong stimulus provided by the NDV superinfection in these experiments. Nevertheless, these results indicate that NSDV and GV are able to actively suppress activation of the IFNb promoter, and are not simply avoiding detection by cellular PRRs.
We wanted to know if NSDV is able to interfere with type I and type II IFN signalling. Vero cells were transfected with plasmids having the luciferase ORF under the control of the mouse Mx1 promoter (a promoter strongly activated by type I IFN) or one containing multiple copies of a GAS element (responds to type II IFN). Those cells were subsequently infected with either the GV or the NSDV isolate. After eighteen hours of infection cells were treated with IFNa or IFNc or left untreated. Finally the luciferase activity was determined as described in material and methods. Treatment of cells with IFNa induced high levels of luciferase activity in cells transfected with the Mx-1 reporter plasmid ( fig. 3a ) which is in accordance with the literature [38] . Infection with either GV or NSDV resulted in a significant reduction in IFNinduced Mx-1 promoter activity when compared to uninfected cells. The same effect could be observed when cells were treated with IFNc to induce the GAS element ( fig. 3b ). GV and NSDV were similarly effective in reducing the promoter activity in the presence of IFNc to less than forty percent of the activity in IFNcstimulated uninfected cells. These data strongly suggest that NSDV/GV is able to counteract the type I and II IFN induced transcription of genes that have an ISRE or GAS element in their promoters.
The binding of IFNa/b to the type I IFN receptor results in the autophosphorylation and activation of JAK (Janus activated kinase) 1 and tyrosine kinase 2, which are both members of the JAK family and associated with the receptor (reviewed in [18] ). The activated JAKs phosphorylate specific tyrosines of STAT1 (signal transducer and activator of transcription 1) and STAT2. Upon phosphorylation, STAT1 and STAT2 form heterodimers and, in association with other factors, translocate to the nucleus to bind to ISREs to initiate transcription of ISGs. Binding of IFNc to its receptor similarly induces phosphorylation of STAT1 which then forms homodimers that translocate to the nucleus to activate the transcription of genes with GAS elements. Targeting the activation of STAT proteins or their transfer to the nucleus are efficient ways to block innate immunity that are used by several different viruses (reviewed in [13] ). To investigate whether NSDV/GV are inhibiting IFN action through an effect on STAT phosphorylation, we infected Vero cells with NSDV or GV for 16 hours and then stimulated the infected cells with IFNa or IFNc. Samples from those cells were subjected to immunoblot analysis to check the phosphorylation status of STAT1 and STAT2 ( fig. 4 ). In uninfected cells IFNa treatment induced phosphorylation of STAT1 and 2 ( fig. 4a ). An almost complete block of STAT1 phosphorylation was observed in NSDV infected cells, especially at later time points. The GV isolate also inhibited STAT1 phosphorylation, though was clearly less effective. This reduced effectiveness of the GV isolate corresponds to a reduced ability to block IFN action in the reporter gene assay ( fig. 3 ), and may be due to the slightly slower growth of this isolate in cell culture; at later time points of infection (18hpi) the GV isolate was The GV L protein inhibits transcription from the IFNb promoter So far the viral RNA-dependent RNA polymerase (RdRP) from CCHFV and DUGV are the only nairoviral proteins known to interfere with the host innate immune response [24] . We wanted to identify the NSDV protein(s) that is/are responsible for the inhibitory effects on IFN induction and action in infected cells. For that reason we made viral protein expression plasmids that were derived from the GV isolate as it is far less tissue culture adapted than the NSDV isolate. We cloned the open reading frame (ORF) of the S and M segments of GV into a mammalian expression vector under the control of a CMV promoter and with a V5-tag in frame at the C-terminus of each protein. For the M segment, which is translated into a polyprotein that produces at least two glycoproteins, this produces a V5 tag at the C terminaus of Gc, the most distal of the glycoproteins produced from this segment. We were unable to clone the complete ORF of GV (or NSDV) L into a plasmid due to recombination events that took place in Escherichia coli, even in strains such as STBL2, SURE2, ABLE K, XL-10 and MDS42 which are engineered to support unstable DNA. We mapped the toxic/unstable sequence to a region of approximately 1 kb found roughly in the middle of the L ORF. We were able to construct plasmids containing either half of this sequence but not the whole piece, and were therefore able to prepare expression constructs encoding the amino-and carboxy-terminal parts of GV L protein (aa L1-1757 and aa L1749-3391 respectively), thereby covering the whole protein with a short overlap between the constructs. In addition we cloned two shorter fragments of the amino terminal part of the viral polymerase that contained the OTU domain (aa L1-169) or the OTU domain and the following zinc-finger domain (aa L1-667). We transfected different amounts of these expression plasmids to approximately equalize the expression levels of the individual viral proteins in the reporter gene assays. In fig. 5a typical protein expression levels are shown that were used in our studies. Only the expression of the glycoprotein Gc which had the carboxy-terminal V5 tag could be analysed. The level of Gn could not be checked due to the lack of glycoprotein-specific antibodies for NSDV. However we assumed that the expression levels of the two glycoproteins are similar as they are expressed as a single polyprotein.
We wanted to know if one of these viral proteins is able to block the transcription from the IFNb promoter in Vero cells infected with NDV. To address this question we co-transfected these viral protein expression constructs together with our reporter plasmids pIFNb-luc and pJATLacZ into Vero cells. Then the transfected cells were infected with NDV and luciferase activity in cell extracts was analysed ( fig. 5b ). Neither the nucleoprotein nor the glycoproteins could impede NDV-induced transcriptional upregulation of the IFNb promoter. All three amino-terminal expression constructs containing the OTU domain of the GV L protein (L1-169, L1-667 and L1-1757) were able to significantly decrease luciferase expression, whereas the C-terminal part of L (L1749-3391), which contains parts of the polymerase but no OTU domain, showed no effects on NDV-induced reporter gene expression. The protein L1-169 was more effective than L1-667 and L1-1757 in blocking NDV-induced IFNb-promoter activity. These data strongly suggest that the GV OTU domain is responsible for the inhibitory effects on type I interferon induction observed in infected cells (fig. 2 ).
Our previous experiments have shown that NSDV is able to inhibit the action of type I and type II IFNs in infected cells ( fig. 3) . To identify the viral protein that is responsible for this effect we transfected Vero cells with reporter plasmids carrying a luciferase gene under the control of type I and II IFN-responsive promoters (pGL3-Mx-1-luc and GAS-luc) together with the viral protein expression plasmids. The cells were treated with IFNa or IFNc to induce transcription from the IFN-responsive promoters and the luciferase induced was measured ( fig. 6 ). The reporter gene activity in cells treated with IFNa or IFNc was significantly reduced in the presence of the GV L protein constructs containing the OTU domain (L1-169, L1-667 and L1-1757) compared to cells transfected with empty plasmid only, whereas in cells expressing the nucleoprotein, the glycoproteins or the carboxy-terminal part of the L protein, treatment with IFNa or IFNc induced comparable levels of reporter gene activity to that found in cells expressing no viral protein. As in the studies of IFN induction, the two shortest versions of the GV L protein were the most efficient in blocking the action of IFNa and IFNc.
Data from studies with CCHFV L protein showed that the OTU domain is enough to inhibit total protein ubiquitination and ISG15ylation in 293T cells [24] . We wanted to know if NSDV/GV has any effects on total protein ubiquitination and/ or ISG15ylation in cells. We investigated this using Vero cells transfected with expression constructs for tagged forms of Ub or ISG15, with appropriate supporting plasmids as required for the ISG15 system [24, 39] . Cells were transfected with a plasmid expressing HA-tagged ubiquitin and subsequently infected with the GV or NSDV isolates. Twelve hours post-infection cell extracts were prepared and the expression of ubiquitin conjugated-proteins determined by immunoblotting ( fig. 7a) , showing that infection with either isolate caused a decrease in total protein ubiquitination compared to uninfected cells. In a similar way we studied the effects of NSDV/GV on ISG15ylation of proteins in infected host cells. ISG15 is a Ubl that is very rapidly induced in type I IFN-treated cells [40] and is thought to play important roles in anti-viral responses, either as a monomer or by conjugation to host cell proteins (reviewed in [41] ). ISG15 conjugation to host cell proteins exerts antiviral activity against influenza virus [42] and Sindbis virus [43] . For our studies on NSDV and its effects on host ISGylation during infection we made use of the fact that ISG15ylation can also be generated by transfecting expression constructs for ISG15 along with plasmids encoding the core components of the ISG15 conjugation system, the E1 activating protein (mUBE1L), E2 conjugating protein (UbcM8) and E3 ligase (mHerc6) into cells in the absence of IFN [39] . After transfecting these four plasmids into Vero cells, the cells were subsequently infected with the GV or NSDV isolates or left uninfected. Twelve hours post-infection cells were lysed and the level of ISG15-conjugates were examined by immunoblotting ( fig. 7b) . Infection of NSDV/ GV resulted in a drastic decrease in ISG15-conjugates found in Vero cells when compared to uninfected cells. Importantly, the infection with NSDV itself did not cause a reduction in the total amount of mono-ISG15 expressed from the transfected plasmid, as shown by tracks 5-7 of fig. 7b , where the helper plasmids were omitted, so one is simply comparing ISG15 expression in uninfected and infected cells. This excludes any indirect effect of infection on ISGylation through an effect on ISG15 expression or stability.
Since the virus itself reduces Ub and ISG15 coupling to cell proteins, we wanted to confirm that the OTU domain is responsible for these decreased levels of conjugates in NSDV/ GV as it is for CCHFV. For these studies we used 293 cells, as those were the cells used in the studies on CCHFV proteins. We determined the level of ubiquitination in the presence of the GV L1-169 protein in 293FT cells and compared it to the level found in cells transfected with empty plasmid or with the CCHFV L1-354 protein (containing the OTU domain of CCHFV) (fig. 7c) . The CCHFV OTU-containing protein contains an HA tag and so appears in the same blots as the HA-Ub and HA-Ub conjugates. The CCHFV OTU completely abolished ubiquitination as previously shown [24] . The GV OTU was also effective in decreasing the levels of cellular Ub-conjugates, but was less effective compared to the CCHFV OTU domain. In the same way we investigated whether the OTU domain of NSDV/GV was responsible for the reduction in ISG15ylation. For this purpose we transfected 293FT cells with the core components of the ISG15 . 7d) . Expression of the GV OTU domain efficiently blocked conjugation of ISG15 to cellular proteins. The CCHFV OTU also blocked ISG15ylation in 293FT cells as already published [24] . Again the CCHFV OTU was more efficient than the GV OTU in reducing the amounts of ISG15conjugated proteins in the cell. Both virus isolates of NSDV are therefore able to decrease total cellular ubiquitination and ISG15ylation levels during infection. The GV OTU domain, when expressed alone, could reproduce these effects exerted by the virus during infection.
To determine the role of the catalytic activity of the OTU domain in IFN antagonism, we changed the cysteine at position 40 and the histidine at position 151, two components of the catalytic triad, to alanine (C40A and H151A respectively). A third mutant was created where glutamine 16 was replaced by arginine (Q16R); Q16 has been described as important for the binding of the CCHFV OTU to ubiquitin but with little importance for the binding of ISG15 [44, 45] , so this mutation was designed to allow us to study solely the effects of deISG15ylation on the innate immune system. The mutations were introduced individually into the GV L1-169 construct, and transfected into 293FT cells to examine their effect on ubiquitination and ISGylation ( fig. 8a, b) . Mutation of C40 or H151 resulted in a complete loss of deubiquitinating ( fig. 8a) and deISG15ylating (fig. 8b) activity. The Q16R mutant performed like the wildtype regarding its ability to remove ISG15 from cellular substrates ( fig. 8b ) while its deubiquitinating activity was only slightly reduced when compared to the wildtype OTU ( fig. 8a) . In previous studies on CCHFV a similar mutant was described as being unable to hydrolyze a fluorogenic model DUB substrate (Ub-AMC) [44] . The difference might be explained by the fact that we are using the OTU domain from a different virus and also a different assay for DUB activity.
We tested these mutants regarding their ability to interfere with IFN induction. For this purpose, cells were transfected with L1-169 or the catalytic mutants C40A, H151A or Q16R, along with the reporter plasmids pIFNb-luc and pJATLacZ ( fig. 8c) . The IFNb promoter was activated by infection of cells with Sendai virus (SV) for eight hours. Interestingly all three mutants lost their ability to block SV-induced IFNb promoter activity when compared to the wildtype. These results showed that the catalytic activity of the OTU is necessary to counteract IFN induction. The results with the Q16R mutant were interesting, as it retained its deubiquitinating and deISG15ylation activity but, despite that, was not able to block IFN induction.
We also examined the ability of L1-169 C40A, H151A, and Q16R to block type I ( fig. 8d) and II (fig. 8e ) IFN action. We used the same experimental setup as described before when we tested the different viral proteins for their ability to block IFN action ( fig. 6 ). The catalytic site mutants of L1-169 no longer blocked type I IFN-induced gene expression (fig. 8c) . The Q16R mutant did block the induced transcription from the Mx-1 promoter to some extent, but not as efficiently as the wildtype. All mutants lost their ability to block type II IFN-induced gene expression (fig. 8d) . These data show that the enzymatic activity of the OTU domain in the nairovirus L protein plays a pivotal role in antagonizing IFN induction and action. In addition, the data from the Q16R mutant strongly suggests that specific, yet different, targets are involved in antagonising these three activities, since this protein is still able to remove ubiquitin and ISG15 from the dominant cellular substrates, but lost its ability to antagonize IFN induction and type II IFN action while retaining some ability to block type I IFN action.
NSDV is regarded as one of the most pathogenic diseases in sheep and goats with mortality rates ranging from 40% in Merino sheep to 90% in Masai sheep [1] . Judging by its high pathogenesis, NSDV has most likely developed efficient mechanisms to circumvent or inhibit innate immunity. The first response of the immune system against virus infections is the production and secretion of type I IFNs. We could show that the induction of transcription from the IFNb promoter in infected cells by NSDV/ GV is delayed and reduced when compared to another negative strand RNA virus ( fig. 1) . We do not observe the extensive delay described previously in CCHFV infected cells [21] ; however, that observed in our studies would give the NSDV enough time to establish its infection and produce progeny virions (which takes approx 12 hours in Vero or BHK21 cells).
A possible reason for the delayed induction could be a reduced production of PAMPs by the virus, such as the removal of the 59 triphosphate from progeny viral genome transcripts, as observed for CCHFV [21] . Interestingly our results showed that NSDV is able to actively suppress the induction of IFNb in infected cells, as both isolates were clearly able to reduce the NDV-induced transcription from the IFNb promoter by approximately 40% (fig. 2 ) when assayed at 8-12hpi, before either NSDV/GV isolate showed strong induction of IFN.
Expression of the N-terminal part of the RNA-dependent RNA polymerase (L), which contains the OTU domain, was sufficient to reproduce the antagonistic effect on IFN induction in a reporter gene-based assay (fig. 5) , and we could show that mutations that affected the catalytic site of the OTU protease were no longer antagonists ( fig. 8 ). Ubiquitination and modification of cellular proteins with ubiquitin-like molecules such as ISG15 play important roles in regulating IFN induction through both the Toll-like receptor (TLR) and RIG-I-like receptor (RLR) pathways [46, 47] . Lys-63-linked polyubiquitination of RIG-I has been shown to be crucial for its ability to induce type I IFNs [48] . Arimoto et al. [49] showed that virus-induced IRF3 and NF-kB activation is dependent on the polyubiquitination of the protein NF-kB essential modulator (NEMO). Furthermore NF-kB activation is known to depend on ubiquitination of the inhibitor protein I-kB, targeting it for degradation [50] , while ISG15ylation enhances NF-kB activity by conjugating to and suppressing protein phosphatase 2Cb, which suppresses dephosphorylation of I-kB [51] . ISG15ylation positively regulates IRF-3 activation by preventing its interaction with PinI [52] . Wholesale removal of conjugated ubiquitin and ubiquitin-like modifiers would therefore greatly inhibit the IFN induction pathway. Indeed we observed a significant reduction in the levels of cellular ISG15-and ubiquitinconjugates during NSDV/GV infection ( fig. 7a, b) which could be attributed to the OTU domain of the L protein ( fig. 7c, d) . The enzymatic activity proved to be essential for the observed inhibition of IFN induction ( fig. 8e) .
Several other viruses have exploited this mechanism of negatively regulating the IFN induction by encoding proteases of the OTU family [24, 30, 53, 54] . All these proteases exert deconjugating activities towards ubiquitin or the ubiquitin-like (ubl) molecule ISG15 or both. Interestingly, cells themselves make use of DUBs to regulate the IFN pathway but, in contrast to nairoviral OTUs, cellular DUBs tend to have highly specific targets [27, 55, 56, 57] . For example, deubiquitinating enzyme A (DUBA) selectively cleaved polyubiquitin chains on tumor necrosis factor receptorassociated factor 3 (TRAF3) and was identified in a small interfering RNA-based screen as a negative regulator of type I IFN production [27] . The crystal structure of the CCHFV OTU domain revealed a unique structure allowing it to bind ISG15 as well as ubiquitin, and this ability to interact with both conjugating proteins is one of the underlying reasons for the promiscuous activity of the nairovirus OTUs [44, 45, 58] .
We also found that NSDV is able to inhibit the action of type I and II IFNs, and that this activity involves the inhibition of phosphorylation of both STAT1 and STAT2. The mechanism(s) of this inhibition remains to be determined. There is no reduction in the levels of STAT1/2, and an inhibition of their phosphorylation suggests either binding/sequestration of one or both STATs or inhibition of the IFN receptor-associated JAKs. We observed that IFNc-induced STAT1 phosphorylation was blocked similarly by both isolates at all times post infection, whereas IFNainduced STAT1/2 phosphorylation was dependent the degree of growth of the virus, the block developing more slowly in cells infected with the slower-growing GV isolate. This suggests that the mechanisms by which the virus exerts the blockade of type I and 2 IFNs are not the same. Further evidence for this came from the GV L1-169 Q16R mutant, which retained most of its DUB activity and its full deISG15ylating activity and still shows significant blockade of IFNa-induced gene expression but no longer blocks IFNc-induced gene expression ( fig. 8c, d) . It could be that this part of the N-terminus contains a specific-substrate binding site in addition to the catalytic core of the OTU domain, contributing to the effect of the OTU domain on specific cellular substrates; in this case the Q16R mutation exerts a steric inhibition on binding to the target involved in blocking type II IFN action, rather than an inhibition which is dependent on the OTU enzymatic activity. For the herpesvirus-associated ubiquitinspecific protease (HAUSP or USP7), an additional binding site with affinity for its target protein has been described in addition to its catalytic protease domain [59] .
The virus had a stronger effect on IFNa-induced STAT1 phosphorylation than on STAT2 phosphorylation at all times, suggesting that STAT1 is the primary target. As with the block of IFN induction, the blockade of both type I and type II IFN actions mapped to the OTU domain of the L protein and required a functional OTU catalytic site. So far no definitive role for ubiquitination or ISG15ylation in type I or type II IFN signalling has been shown. JAK1 and STAT1 have been shown to be conjugated by ISG15 [60, 61] ; however, STAT1 phosphorylation in response to IFN is normal in cells from ISG15 knock-out mice [62] , suggesting that ISG15 plays no role in the immediate cell response to IFN.
Differences were seen in the effectiveness of the different OTU-containing fragments of the GV L to block IFNa-or IFNcinduced gene transcription when expressed in our reporter genebased studies. GV L1-169 and L1-667 reduced the transcriptional activity induced by IFNa or IFNc to 30% and 40% respectively of the positive control, whereas the protein L1-1757 was less effective in reducing the transcriptional activity of the Mx-1 promoter or the GAS promoter (55% and 65% of the positive control respectively). These differences might reflect slight differences in the amounts of protein expressed in the transfected cells that cannot be detected by immunoblotting. Alternatively, the catalytic domain of the NSDV L protein might be autoinhibited by folding or oligomerisation in the longer construct L1-1757, in contrast to the shorter versions containing the OTU domain. A similar observation was made with the OTU domain containing nonstructural protein 2 (nsp2) from porcine reproductive and respiratory syndrome virus where a longer fragment was less effective in blocking NFkB promoter activity than a smaller version of this protein [30] . In addition it has been shown that CCHFV full length L displayed significantly less DUB activity than shorter versions of the protein [24] . Further crystal structures to extend that of the basic OTU domain [44, 45, 58] will clarify these points.
One important factor that needs to be examined is the relationship of the reactivity of these OTU domains to species specificity of the viruses. Influenza B was found to inhibit the human but not the mouse ISG15ylation system [39] . The nairovirus OTUs appear to act by cleaving Ub and ISG15 from their respective conjugates, since mutations in the active site abolish activity [24] , and the OTUs are therefore active against both human and murine systems. However, there was a clear difference between the activity of the CCHFV and NSDV/GV OTUs in the murine ISG15ylation system used in this study. We know little of the ruminant equivalents of the Ubls, and it is possible that the OTUs of different viruses may be adapted to species differences in these modifying proteins which could lead in turn to differences in the species specificity of pathogenesis. It will be important to examine the actual level of modification of host cell proteins when these viruses are grown in cells from different hosts to establish whether there is any degree of correlation between OTU cleavage activity and host cell species.
Within this study we could demonstrate that NSDV/GV is able to block the innate immune system at three different stages, type I IFN induction, type I IFN action and type II IFN action. NSDV/ GV seems to be another example of a pathogen that exploits the host cell ubiquitin pathways for its own good by encoding an enzyme with deubiquitinating and deISG15ylating activity. However, to fully clarify the role of the OTU activity, an OTU knock-out virus is needed to evaluate the importance of the OTU domain in vivo, and we are working towards such a system. An advantage of NSDV/GV will be the ability to carry out such experiments in the natural host.
The Vero cells (African green monkey kidney cells) used in these studies were a modified line that expresses CD150 (aka Signaling Lymphocyte Activation Molecule (SLAM), as these were the Vero 
Except where indicated, all DNA manipulation was done following standard methods. Plasmids were cloned and grown in Escherichia coli DH5a or SURE2 (Stratagene). Routinely plasmid DNA was purified on CsCl gradients. The plasmids pJAT-lacZ, pGAS-luc, and pIFNb-luc were the kind gifts of Prof Steve Goodbourn, St. George's Hospital Medical School, London, United Kingdom. The pGL3-Mx1P-luc was kindly provided by Prof Georg Kochs, Department of Virology, University of Freiburg, Germany. The following plasmids were used for the ISG15ylation and ubiquitination experiments: pCAGGS.MCS-6HismISG15, and plasmids expressing mHerc6, Ubcm8 and mUbE1L were provided by Prof Deborah J. Lenschow, Washington University School of Medicine, St. Louis, Missouri. Plasmid pHA-CCHFV-L1-354 is in pCAGGS-MCSII and was the gift of Dr Natalia Frias-Staheli, Mount Sinai School of Medicine, New York.
Construction of viral protein expression plasmids. Total RNA from GV-infected BHK21 cells was extracted by using RNeasy Mini Kit (Quiagen) which served as a template for cDNA synthesis using random priming oligos and SuperscriptII Reverse Transcriptase (Invitrogen). The viral genome was amplified by PCR using NSDV/GV genome-specific oligos and subsequently blunt-end cloned into pT7Blue (Novagen). All PCRs were performed using proofreading polymerase (KOD; Novagen). The genome of both strains was completely sequenced. Plasmids pcDNA-GV-N and pcDNA-GV-M were made by cloning the complete ORFs of GV S and GV M segments into pcDNA6/V5-His (Invitrogen), expressing C-terminal V5-tagged N and the glyoproteins (only Gc has a V5 tag at its C-terminus). GV L1-169 and L1-1757 were cloned in pcDNA6 with a C-terminal V5 tag. GV L1-667 and L1749-3391 were cloned into pTriEX (Novagen) such that the expressed proteins have a 6xHis tag at the C-terminus. To generate catalytically inactive variants of L1-169 in pcDNA6/V5-His, single amino acid mutations were introduced by overlap PCR mutagenesis. All mutations were confirmed by sequencing the entire open reading frame.
Mouse monoclonal antibody against phosphotyrosine 701-STAT1 were purchased from BD Biosciences. Polyclonal antibodies against STAT1, STAT2, and phosphotyrosine 689-STAT2 were obtained from Upstate. Mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA) was obtained from Santa Cruz Biotechnology. Mouse monoclonal anti-His antibody was purchased from Sigma Aldrich and HRP-tagged anti-HA antibody from Roche. The rabbit anti-N antiserum recognizing the amino terminus of the NSDV and GV N protein were previously made in our laboratory. Mouse monoclonal antibody against the V5 epitope tag was purchased from AbD Serotech. Mouse monoclonal antibody U85 recognising Newcastle disease virus was the kind gift of Ruth Manvell, AHVLA, Weybridge, UK.
All transfections were carried out with TransIT LT1 (Mirus) according to the manufacturer's instructions. A ratio of 2 or 3 ml LT1 per mg DNA was used. Cells were plated at 10 5 per well in 12well plates 1 day before use. Usually, 24 hours post transfection the medium containing the transfection mix was removed and replaced with fresh medium. The cells were then lysed in 200 ml lysis buffer (120 mM NaCl, 50 mM TrisCl pH 7.5, 0.05% Nonidet P-40). The samples were centrifuged for 1 min at full speed in a table top centrifuge. 50 ml of the cleared cell extract was taken and the luciferase activity was measured after adding 50 ml of luciferase assay reagent (Promega) to each sample. The following settings were used to measure the relative light units: integration time: 10 sec, sensitivity: 200 or 230 and Filter set 1. To determine the b-galactosidase activity 150 ml of assay buffer (48 mM Na 2 HPO 4 , 32 mM NaH 2 PO 4 , 8 mM KCl, 0.8 mM MgSO 4 , 3.2 mg/ml o-Nitrophenyl b-D-Galactopyranoside) were added to the samples after the luminescence was measured. The samples were incubated at 37uC for 30 min to 60 min before absorbance at 420 nm was measured. The luminescence and absorbance measurements were done in a Synergy 2, Multi Detection Microplate Reader (BioTek Instruments) using Gen5 software. The values were normalised and statistically analysed as previously described [63] .
Vero cells were plated at an initial seeding density of 1610 5 / well in 12-well plates. One day later cells were infected with GV or NSDV at a multiplicity of infection (MOI) of 1. The virus inoculum was removed one hour after infection, cells were washed once with PBS and fresh medium was added. The infected cells were further incubated for 14 h before being treated with 1000 IU/ml recombinant human aA-Interferon (IFN) or 1000 IU/ml recombinant human IFN-c. IFN-aA was purchased from Calbiochem and IFN-c was obtained from Millipore. Cells were treated for 30 min with or without IFN before being harvested and lysed with 100 ml of 1x SDS sample buffer (New England Biolabs). SDS-PAGE and Western blots were carried out as previously described [64] . A Human Monoclonal Antibody with Neutralizing Activity against Highly Divergent Influenza Subtypes The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of “universal” prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies. Influenza, one of the diseases that has shaped human history [1, 2] , still has an evident clinical and socio-economical impact [3, 4] . The 2009 pandemic has raised several major concerns related to the few prophylactic and therapeutic measures available. Antiviral compounds have drawbacks caused by the rapid emergence of drug-resistant isolates [5, 6] , require prompt administration to be effective [7] , and have several associated side-effects especially in high-risk categories, including children and pregnant women [8, 9] . Additionally, the vaccinal strategy is exposed to the annual risk of being ineffective due to possible mismatches between the predicted strains included in the vaccine and those actually in circulation; moreover, it would not engender a prompt response in pandemic settings [10] . In this scenario, new broadrange ''universal'' anti-influenza strategies are required [11, 12] . In particular, it would be important to identify and eventually elicit what has recently been described as an unusually extreme broadrange immunity directed against broadly conserved viral regions, differing from the more common and restricted immunity directed against highly variable regions [12] . A number of approaches have already been proposed in literature [10, 12, 13, 14, 15, 16] , but a pivotal role, both in the prophylactic and therapeutic field, may be played by the availability of broad-range neutralizing human monoclonal antibodies (mAbs) allowing the identification of human B epitopes widely shared among different influenza subtypes [11, 12] . Indeed, it is accepted that antibodies are key players in natural protection against influenza viruses, and that hemagglutinin (HA) is the main target for the virus-neutralizing antibody response [17] . However, although a single influenza infection provides lifelong immunity against the infecting virus and a limited number of antigenically correlated strains, the host can remain susceptible to infection with an antigenically drifted variant due to HA variability [18] .
HA is the major glycoprotein of the influenza virus; it binds sialic acid on the surface of the cells through its globular head (HA1 domain) and makes possible the fusion of the viral envelope with the endosomal membranes through its stalk region (mainly formed by the HA2 domain) [17] . The sixteen known subtypes of HA, sharing between 40% and 60% amino acid sequence identity, have been clustered in two distinct phylogenetic groups: group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16) and group 2 (H3, H4, H7, H10, H14, and H15) [12, 14] . The subtypes, H1, H2, H5 and H9 in group 1, and H3 and H7 in group 2 have been isolated in humans and in particular H1, H2 and H3 subtypes have been responsible of the reported influenza pandemic outbreaks. In this study, we describe a human mAb, named PN-SIA28, that is capable of neutralizing all tested group 1 isolates, as well as isolates belonging to H3N2, the only group 2 subtype capable, so far, of causing a pandemic.
The binding and neutralizing features of PN-SIA28 were initially studied using the Fab fragment molecule produced in E. coli, demonstrating that Fab PN-SIA28 recognizes an epitope on the stem region of HA and is able to strongly neutralize all tested H1N1 strains [19, 20] .
It is well documented in the literature that bivalency of a whole IgG molecule may be an essential features for the biological activity of a mAb [21, 22, 23, 24] . For this reason, in this work, to evaluate PN-SIA28 features as whole IgG molecule, IgG PN-SIA28 was generated and tested in different neutralization assays against human, swine and avian influenza A viruses belonging to both HA based phylogenetic groups and encompassing all subtypes responsible of described pandemic events. The results obtained showed that IgG PN-SIA28 strongly neutralizes viruses belonging to the group 1 as well as those of the group 2. More in details, IgG PN-SIA28 neutralized all the H1N1 tested viruses with an half maximal inhibitory concentration (IC 50 ) ranging between 0.4-3.7 mg/ml, the H5N1 viruses with IC 50 ranging between 0.9-2.8 mg/ml, the H2N2 subtype isolate with an IC 50 of 0.8 mg/ml and the H9N2 subtype strain with an IC 50 of 0.9 mg/ml (Table 1, Figure S1 ). More interestingly, IgG PN-SIA28, also potently neutralizes H3N2 viruses circulating in the years from 1968 to 1975 with IC 50 ranging between 0.8-2.6 mg/ml (Table 1, Figure S2 ) despite the great phylogenetic distance of these viruses compared to those belonging to group 1 ( Figure 1 ). No detectable neutralizing activity was observed against more recent H3N2 tested viruses (Table 1, Figure 1 and S3) as well as for the H7N2 tested isolate.
Screening of a random 12-mer peptide phage displayed library. In order to select peptides able to bind antibody PN-SIA28 that could possibly indicate the region that the antibody recognizes on the HA, a phagemidic random 12-mer peptide library was screened. After three rounds of panning, PN-SIA28 binding phages were eluted and used to infect E. coli XL1-Blue for an ELISA screening on PN-SIA28. Twenty-five positive clones were sequenced. An in silico analysis was performed, using Mimox and Pepitope servers, comparing the selected peptides to the available crystal structure of H1N1 A/South Carolina/1918 (A/ SC/1918) (ID code 1RD8) and A/Puerto Rico/8/34 (A/PR/8/ 34) hemagglutinins (ID code 1RU7). This study allowed the identification of several possible residues located on the stem region of HA potentially involved in the binding of PN-SIA28: Asn336, Ile337, Pro338, Trp357 and Thr358 (sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709) ( Figure 2 ).
In vitro selection of escape mutants. To further define the region bound by PN-SIA28, an assay aiming at the evaluation of PN-SIA28 capability to induce escape mutant was performed. A/ PR/8/34 H1N1 virus was cultured under constant selective pressure of PN-SIA28, and after several rounds of cell infections, two escape mutants were generated. Sequencing analysis of the generated neutralization escape variants revealed two different mutants carrying each a single amino acidic mutation in position 361 (Ile361Thr) and in position 362 (Asp362Gly) in the HA2 subunit compared to wild type.
Alanine scanning mutagenesis study. On the basis of these results, HA mutants carrying an alanine substitution in position 361 (Ile361Ala) or in position 362 (Asp362Ala) were generated and the PN-SIA28 binding to the mutants was evaluated by FACS analysis evidencing that PN-SIA28 was not longer able to bind to the mutated HAs. In order to identify other amino acidic residues involved in the binding of PN-SIA28 to HA, a large panel of HAalanine mutants was generated (Table S1 ). FACS analysis showed that binding of PN-SIA28 to HA was decreased by His25Ala, His45Ala mutants on HA1 and Thr358Ala, Met360Ala, Ile361Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala and Val395Ala mutants on HA2. (Figure 2 ). All other mutations listed in the Table S1 did not have any effect on PN-SIA28 binding. An extra HA-mutant in position 361 (Ile361Val) was generated. Indeed, in position 361 either an Isoleucine or a Valine residue can be present in different isolates belonging to either to group 1 either to group 2 ( Figure 2 ). No binding reduction of PN-SIA28 to this mutant was observed.
Based on the results obtained with the HA mutants, an in silico analysis on the HA crystal structure of A/SC/1918 and A/PR/8/ 34 was carried out for the amino acid residues that the alanine scanning study showed to influence the binding of PN-SIA28 to HA. The analysis confirmed that the residues identified lie on the stem region of HA, that they belong to the HA1 and HA2 subunits and that they are exposed on the surface of the HA molecule ( Figure 3) .
A similar approach was followed also for the H3 subtype. Based on the results obtained for the H1N1 study, four H3 alanine mutants were initially generated on the A/Aichi/2/68 (H3N2) HA: His34Ala, Asn54Ala on HA1 and Ile363Ala and Asp364Ala on HA2 (corresponding to His25, His45, Ile361 and Asp362 in H1N1 sequencing numbering) ( Figure 2 , Figure 4 and Table S1 ). None of these alanine mutated HAs was bound by PN-SIA28. Furthermore, the Asn54His mutant (corresponding to position 45 in H1N1 numbering) on A/Aichi/2/1968 HA was also generated due to the presence of a Histidine in the corresponding position in H1N1 viruses. An increased binding of PN-SIA28 to this mutant was observed compared to wild type H3 HA. In order to deeper investigate on the absence of neutralizing activity of PN-SIA28 against most recent H3N2 strains, three more alanine-mutants (Asp18, Lys57 and Ile70) (see Table S1 ) were generated considering the differences in the stem region between H3N2 A/Victoria/3/1975 (the most recent neutralized isolate) and H3N2 A/Philippines/2/1982 (the earliest non neutralized strain).
A 30% binding decrease was observed for Lys57Ala HA mutant, while the Asp18Ala and Ile70Ala did not have any effect on PN-SIA28 binding to H3N2 HA ( Figure 5 and Table S1 ).
In the present study we describe the binding features and the neutralizing activity of a human monoclonal antibody named PN-SIA28 previously described as Fab fragment [19, 20] . IgG PN-SIA28 was tested against a large panel of influenza A viruses belonging to group 1 (H1N1, H2N2, H5N1, H9N2) and group 2 (H3N2, H7N2) subtypes showing a broader neutralizing activity compared to its monovalent molecule, possibly due to the bivalency feature of the IgG. Indeed, IgG PN-SIA28 showed a Figure 1 . PN-SIA28 Neutralizing activity. Influenza hemagglutinin unrooted phylogenetic tree of the different viral strains tested in neutralization assays with PN-SIA28. Viral isolates belonging to group 1 and group 2 are presented in the box B and box A, respectively. A green '+' indicates positive neutralizing activity, a red '2' indicates negative neutralizing activity. As reported in the text, PN-SIA28 is able to neutralize all of the group 1 strains and is also able to neutralize all of the H3N2 isolates spanning 1968 and 1975. *Recombinant HA from (H1N1) A/South Carolina/1/ 1918 pandemic strain was previously shown to be bound by PN-SIA28 [19, 20] . Analogously, recombinant HA from H5N1 A/Cygnus Olor/Italy/742/ 2005 was recognized by PN-SIA28 (data not shown). # H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA28 as Fab fragment [19, 20] . doi:10.1371/journal.pone.0028001.g001 robust neutralizing activity (IC 50 = 0.4-3.7 mg/ml) against all tested strains belonging to group 1, thus demonstrating that its epitope is broadly shared among group 1 viruses, including the highly divergent H9 subtype. More importantly, PN-SIA28 showed a potent neutralizing activity also against group 2 viruses demonstrating that its epitope is also present in the highly Figure 2 . Sequence conservation in hemagglutinin groups and subtypes. Boxes indicate mutated residues which decrease PN-SIA28 binding to mutated HAs. Circles on the top indicate PN-SIA28 percent binding to each HA alanine mutants compared to binding to wild-type HA: red 25% binding, yellow 50-75% binding. Sequence numbering is based on H1N1 A/PR/8/34 coding region (GenBank accession number ABO21709). Neutralizing activity of PN-SIA28 against each strain is highlighted by a green '+' or a red '2' on the left, indicating neutralizing activity and no neutralizing activity, respectively. *Recombinant HA from H1N1 A/South Carolina/1/1918 pandemic strain was previously shown to be bound by PN-SIA28 [19, 20] ; analogously, recombinant HA from H5N1 A/Cygnus Olor/Italy/742/2005 was recognized by PN-SIA28 (data not shown). # H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA28 as Fab fragment [19, 20] . doi:10.1371/journal.pone.0028001.g002 divergent H3N2 subtype. Indeed, IgG PN-SIA28 was able to potently neutralize H3N2 strains circulating from 1968 to 1975 (IC 50 = 0.8-2.6 mg/ml), despite the important phylogenetic distance between these viruses and those belonging to group 1 ( Figure 1 ). No neutralizing activity was observed against the dramatically divergent H7N2 isolate tested in this study, nor against the more recent H3N2 isolates.
The broad neutralizing activity of PN-SIA28 against highly divergent influenza viruses, suggests that the epitope recognized is extremely conserved. The lack of hemagglutination inhibitory activity and the competition with a mouse mAb (C179) directed against the stem region of HA, had already suggested that PN-SIA28 epitope is not localized on the globular head and that it is shared between HA1 and HA2 domains [19, 20] . Several approaches have been used in this study to better define the region recognized by PN-SIA28. The screening of a phagemidic random peptide library suggested the binding of PN-SIA28 to the stem region in the proximity of the viral membrane. Interestingly, the sequence analysis of A/PR/8/34 (H1N1) escape mutants, generated under PN-SIA28 selective pressure, identified two residues (Ile361 and Asp362) localized in close proximity to the residues identified with the peptide library approach. It is worth noting that none of these two specific mutations is present in any of the more than 6,000 sequences available in public influenza databases (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), suggesting that the immune pressure on these residues is poor and that the region is functionally highly conserved in all subtypes (rate of non-conservative substitutions: 0.17% for residue 361, and 8.7% for residue 362). Based on these data, A/PR/8/34 (H1N1) HA mutants carrying an alanine substitution in position 361 (Ile361Ala) or in position 362 (Asp362Ala) were generated and the FACS analysis showed that PN-SIA28 lost most of its capability to bind the mutated HA, in concordance with the data obtained from the escape mutants. The residue in position 361 was also mutated in Valine (Ile361Val), an aminoacid largely present in most of the neutralized isolates (Figure 2 ), indeed confirming that the presence of this residue did not affect binding.
With the help of available HA crystal structures, a larger panel of HA-A/PR/8/34 (H1N1) alanine mutants was analogously generated and several residues (His25Ala, His45Ala on HA1; Thr358Ala, Met360Ala, Ile361Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala and Val395Ala on HA2), close to those identified with the escape mutants approach, influenced the interaction of PN-SIA28 with HA ( Figure 2 and 3) . The more restricted panel of alanine mutants (His25Ala, Asn45Ala, Ile361Ala and Asp362Ala) generated for A/Aichi/2/68 (H3N2) HA, confirmed the key role of some of the residues already identified as implicated in the interaction of PN-SIA28 with HA (H1) (Figure 4 ). Three more mutants were generated considering the differences among solvent exposed residues close to PN-SIA28 epitope-core on HA stem region between A/Victoria/3/1975 (the most recent neutralized isolate) and A/Philippines/2/1982 (the earliest non neutralized strain) isolates. The only mutant partially affecting PN-SIA28 binding was the one on Lys57, a residue present among all H3N2 neutralized strains and substituted by several different residues among H3N2 not neutralized strains. This could partially explaining the lack of neutralizing activity against most recent strains ( Figure 5 and Table S1 ).
Overall, these results indicate that the region bound by PN-SIA28 encompasses residues on the HA stem, and that it is partially overlapping from the binding region of other human neutralizing antibodies with heterosubtipic neutralizing activity limited to influenza A group 1 viruses [25, 26, 27, 28, 29] or group 2 viruses [30, 31] . In addition, the region bound is also partially shared with FI6v3, an optimized human mAb able to crossneutralize influenza A group 1 and group 2 viruses [32] . Importantly, both the heavy chains of PN-SIA28 and FI6v3 derive from rearrangement of the same VH gene germline (VH3-30). As already hypothesized for the VH1-69 subfamily, widely shared among human mAbs with heterosubtypic neutralizing activity limited to group 1 viruses, the VH3-30 gene structure may be important in conferring the broader heterosubtypic activity observed for PN-SIA28 and FI6v3. The future definition of HA/ PN-SIA28 crystal structure and its comparison with HA/FI6v3 complex may help confirm the possible role of conserved VH3-30 residues in conferring such unusual neutralization properties.
Although the resolution of the crystal structure will be necessary for the fine definition of the PN-SIA28 epitope, several important considerations may already be made. Firstly, it was previously speculated that the presence of an additional glycosilation site (Asn54 corresponding to His45 present on group 1 viruses) on the HA1 portion of the stem region in H3N2 viruses is the reason for the lack of anti-H3N2 activity of other mAbs with heterosubtypic activity limited to group 1 subtypes [25, 26, 30] . The present study demonstrates that this extra glycosilation site on H3N2 does not preclude PN-SIA28 binding and neutralizing activity against viruses belonging to this subtype ( Figure 2 ). The testing of HA alanine mutants for position His45 on H1N1 and Asn54 on H3N2, and the increased binding of PN-SIA28 to Asn54His mutant on H3N2, confirm that this key residue is particularly important for the interaction of PN-SIA28 with HA. However, other residues, including Lys57 and others not yet identified, certainly play an important role in the PN-SIA28 binding to HA, as shown by the lack of activity observed against the recent H3N2 isolates tested, as well as against the H7N2 isolate, despite a substantial identity in the studied region ( Figure 2 ).
In conclusion, the molecule described in this paper is a singular human mAb featuring a broad heterosubtypic neutralizing activity encompassing group 1 and group 2 influenza A subtypes, and therefore recognizing a broad-range neutralizing human B epitope in the stem region of HA. Indeed, PN-SIA28 neutralizes influenza viruses belonging to all subtypes that have caused pandemics in humans, as well as other subtypes with pandemic potential.
The data presented may therefore be crucial not only for the improvement of classical passive anti-influenza prophylactic and therapeutic strategies [33, 34] , but also for the correct understanding of mechanisms leading to in vivo protective anti-influenza immunity and therefore for the design of more effective vaccinal strategies. Indeed, several pharmacokinetic studies have shown that the extremely low IC 50 featured by PN-SIA28 against most of the isolates tested can easily be reached in humans after systemic administration [35, 36] . Given the frequent correlation between a low IC 50 value and in vivo efficacy observed for other anti-influenza mAbs [25, 26, 27, 28, 29, 30, 31, 37, 38, 39] , PN-SIA28 could be a really promising compound to be used in passive immunization strategies.
More importantly, PN-SIA28 represents the molecular evidence that an extremely wide unusual neutralizing immunity although, uncommon, may be elicited during the course of a natural infection and, potentially, even after a new-generation vaccinal approach focused on its epitope [40] . As a consequence, PN-SIA28 could be crucial in the identification of promising HAbased vaccinal strategies aimed at the elicitation of a broadly heterosubtypic protective immunity similar to the one represented by this human mAb. As described for a few other mAbs [25, 26, 27, 28, 29, 30, 31, 32, 41] , PN-SIA28 is mainly directed against the highly conserved HA2 domain whose use in novel anti-influenza vaccinal approaches is being widely investigated. However, PN-SIA28 demonstrates that not all antibodies directed against the stem region have the same characteristics, and that their neutralizing activity is modulated by the specifically recognized epitope. Indeed, the data presented in this paper show that in the design of such innovative approaches (i.e. the so-called ''headless approach'' [16] ) it is important to identify also residues belonging to HA1 domain to be preserved to elicit the widest immunity. Several immunogens are usually obtained on the basis of in silico analysis, but their real protective potential can only be ascertained after time-consuming and expensive in vivo studies. Under this perspective, PN-SIA28 could therefore be an extremely important reagent to identify immunogens more likely to stimulate antibodies with a similar broad heterosubtypic neutralizing activity that will constitute the best candidates for further in vivo studies.
The isolation of PN-SIA28 has been previously described [19, 20] . In brief, PN-SIA28 was isolated from a 55 year old patient with a negative clinical history of infection from influenza viruses during the past ten years, and with a detectable serum neutralizing titre (half maximal inhibitory dilution 2 ID 50 . = 1:20) against two reference strains belonging to two distinct HA-based phylogenetic groups: the H1N1 strain A/Puerto Rico/8/1934 for group 1 and the H3N2 strain A/Port Chalmers/1/1973 for group 2. It was then expressed in E. coli as Fab fragment to define its features.
To obtain the whole IgG described in this study, the BD BaculoGold System (BD Biosciences Pharmingen, San Diego, CA, USA) was used. Briefly, nucleotide sequences codifying heavy and light chains of PN-SIA28 Fab fragment were sub-cloned into the baculovirus expression vector pAc-k-Fc (PROGEN Biotechnik GmbH, Heidelberg, Germany). Sf solution containing the antibody was dialyzed against PBS and then concentrated using Amicon Ultra-15 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Antibody concentration was determined by SDS-PAGE gel and by spectrophotometric measurement at 280 nm.
An anti-influenza A antibody directed against the HA (H1N1 subtype), named RB62, and an anti-HCV E2 glycoprotein antibody, named e137, produced and purified with an identical procedure were used as controls in all experiments.
The following reference strains were tested in the BLS3 laboratory of the Vita-Salute San Raffaele University: (H1N1 . The A/swine/Parma/1/97 isolate was analogously grown on NSK (Newborn Swine Kidney) cells, kindly provided by the Zooprophylactic Institute of Brescia, Italy. At 80% confluence, cells in MEM supplemented with 2 mg/ml serum-free TPCKtrypsin (Roche Applied Science), were infected with each strain at a MOI of 0.001. After 1 hour of infection, cells were washed with PBS (Phosphate buffered saline); MEM supplemented with 2 mg/ ml trypsin was then added and cells were incubated at 37uC in 5% CO 2 atmosphere. Cells were observed daily to monitor the cytopathic effect and, usually after 72-96 hours, the supernatant was collected, centrifuged at 1000 rcf for 10 minutes to eliminate cells debris and filtered with 0.22 mm filters (Millipore, Billerica, MA, USA). The supernatant was then aliquoted and stored at 280uC as cell-free virus stock.
Fluorescence inhibition assay. Each viral isolate was titrated by the limiting dilution method and the viral titer calculated by the Reed-Muench formula. Neutralizing assays were carried out in 96 wells plate using MDCK cells (46104cells/ well). Serial dilutions, 30 mg/ml-0.03 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with 100 TCID50 of each H1N1 or H3N2 virus. Following incubation, 100 ml of the mix antibody-virus were added to the cells and incubated for another hour at 37uC in 5% CO2. At the end of this incubation, cells were washed with PBS and 100 ml of MEM TPCK-Trypsin (2 mg/ml) were added in each well. Cells were incubated for 7 hours at 37uC in 5% CO2 and then washed with PBS, fixed and permeabilized with ice-cold ethanol. Cells were incubated with anti-influenza A mouse antibody (Argene, Shirley, NY, USA) for 30 minutes at 37uC in a humid chamber. The cells were then washed and incubated for 30 minutes at 37uC in a dark humid chamber with a FITC-conjugated secondary antibody (Argene, Shirley, NY, USA). Nuclei staining was obtained with Hoechst 33342 (Sigma Aldrich). An infection control without antibody was included, as well as a negative control with an anti-HCV/E2 antibody (e137). Each neutralization assay was performed in triplicate and repeated in two different sessions.
The neutralization activity for each antibody concentration was expressed as the percentage reduction of fluorescent nuclei compared with the nuclei count in the infection control. Nuclei counting was performed by using the GE Healthcare's IN Cell Analyzer 1000, an automated epifluorescence based microscope system. The neutralization curves were then fit by non-linear regression with the GraphPad Prism software, allowing IC 50 calculation.
Colorimetric assay. Each viral isolate was titrated to establish working dilution that produces 15-30 foci forming units per well in 96 tissue culture plates. Neutralizing assays were carried out in 96 wells plate using MDCK/SIAT-1 cells. Serial dilutions, 30 mg/ml-0.37 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with the subset of viruses. Following this incubation, 100 ml of the antibody-virus mix was added to the cells and incubated for another hour at 37uC in 5% CO 2 . At the end of this incubation, the cells were washed twice in PBS and 100 ml of virus growth media containing 2 mg/ml of TPCK treated trypsin was added. Cells were incubated for 12-16 hours at 37uC in 5% CO 2 and then washed with PBS, fixed and permeabilized with ice cold methanol/acetic acid (95:5) for 30 min at 220uC. Cells were incubated with anti-NP antibodies (Millipore, Billerica, MA, USA) for 30 minutes at 37u. The cells were then washed and incubated for 30 additional minutes at 37uC with a mouse HRP-conjugated secondary antibody. True Blue chromogenic substrate (KPL) was used to count the number of foci.
Plaque reduction assay. Each viral isolate was titrated by the limiting dilution method and the viral titre calculated by the Reed-Muench formula. The viral titre was also calculated as plaque forming units (PFU) on six-well flat-bottomed plates (Corning, Corning, NY, USA). Neutralizing assays were carried out in 6 wells plates using MDCK cells (5610 5 cells/well). Two dilutions, 1-0.1 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with 100 TCID 50 each of H1N1 or H3N2 virus. Following this incubation, 1 ml each of virus-antibody mix was added on MDCK monolayer and the plate was incubated 1 hour at 37uC in 5% CO 2 . After this incubation, the medium was removed and the monolayer washed twice with PBS. Two ml of MEM-agarose 0.8% supplemented with penicillin (50 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), streptomycin (100 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM) (Gibco Invitrogen, Carlsbad, CA, USA) and trypsin (2 mg/ml) (Roche Applied Sciences) were gently added to each well and the plates were incubated 48 hours at 37uC in 5% CO2. After this incubation the agarose medium was removed from each well and 1 ml of 70% methanol-crystal violet 1% (w/v) was added to each well at room temperature. Finally, the wells were washed with tap water and dried. An infection control without antibody was added as well as a negative control with anti-HCV/E2 e137 mAb. The neutralization was determined counting the PFU reduction in presence of antibodies in comparison with the infection control.
A phagemidic 12-mer peptide library (Ph.D.12 TM Phage Display Peptide Library, New England Biolabs Inc., Boston, MA, USA) was screened against PN-SIA 28 Fab fragment. Briefly, 300 ng of PN-SIA 28 were coated on four wells (96-wells plate, COSTAR), and the peptide library was amplified transforming electrocompetent E. coli XL1 Blue. 70 ml of the obtained phage preparation was incubated with PN-SIA 28 for 1 hour at 37uC and subsequently the unbound phages were removed by washing with PBS 1X-Tween20 using progressive Tween20 concentration starting from 0.1% to 0.5%. The phage that bound PN-SIA 28 was then eluted using low pH and, once neutralized, used to infect E. coli XL1 Blue for a subsequent ELISA screening on 100 ng/well coated PN-SIA 28. Positive clones were then sequenced using the kit-provided primers (New England Biolabs Inc., Boston, MA, USA).
Positive clones were then sequenced. An in silico analysis was performed, using Mimox and Pepitope servers, comparing the selected peptides to the available crystal structure of A/PR/8/34 and A/South Carolina/1918 haemagglutinins (1RU7.pdb and 1RD8.pdb).
The experiment was performed on 90% confluent MDCK cells growing in T25 flasks (Nunc, Rochester, NY, USA) in MEM supplemented with 10% FBS. PN-SIA28 as well as e137 mock control, were diluted in 1.5 mL MEM supplemented with TPCK trypsin (2 mg/ml) to obtain final concentrations of 2 mg/ml, 10 mg/ml and 20 mg/ml of antibody. 100 TCID 50 of A/PR/8/34 were prepared in 1.5 mL of the same medium. The two solutions, containing antibodies and virus, were mixed in order to obtain a final concentration of 1 mg/ml, 5 mg/ml and 10 mg/ml for the mAbs in a final volume of 3 mL. The mixes were then incubated 1 h at 37uC. An infection positive control (virus without PN-SIA28) was included as well as non-infected cells. After two washes with sterile PBS 16 (Gibco Invitrogen, Carlsbad, CA, USA), 1 ml of each neutralization mix was added to the flasks containing MDCK cells and the infection was performed for 1 h at 34uC in 5% CO 2 . After absorption, the medium was removed and the monolayer washed twice with sterile PBS. 3 mL of MEM supplemented with trypsin (2 mg/ml) was added to the infection positive control and to non-infected cells. Antibody PN-SIA28 and e137 were added to the previously treated infected cells, maintaining the concentrations used during the infection step. Cells were incubated at 34uC in 5% CO 2 and regularly checked for 48 hours for the presence of cytopathic effect (CPE) by comparing the positive infection control to the treated infected cells. The supernatant was then centrifuged (2000 rcf for 10 minutes), collected and stored at 280uC. All the viral stocks were titrated and used to infect new cell preparations, increasing, when it was possible, the concentration of PN-SIA28. After ten passages, the cells of the infection positive control and mock control were compared with infected cells under selective pressure of PN-SIA28. Once a strong cytopathic effect was evident in the positive and mock controls, the presence or absence of CPE in the PN-SIA28 treated infected cells was evaluated. All the supernatants were collected, centrifuged, stored and used to perform a full length DNA sequence of influenza genomic fragment 4 coding for viral HA.
A/PR/8/34 (H1N1) HA was amplified as previously described [19, 20] using the following PCR-oligonucleotides: APR834_s: 59-CACCATGAAGGCAAACCTACTGGTCCTGTTATGTG-39; APR834_as: 59-TCAGATGCATATTCTGCACTGCAAAGAT-CCATTAGA-39. A/Aichi/2/1968 (H3N2) HA was amplified using the following PCR-oligonucleotides: Aichi/2/68H3N2f: 59-CACCATGAAGACCATCATTGCTTTG-39; Aichi/2/68H3-N2r: 59-TCAAATGCAAATGTTGCACCTAATG-39. The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, alanine mutants for H1N1 and H3N2 HA were generated using Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 HA mutants were generated for H1N1 and 7 for H3N2 (Table S1 ).
The binding activity of PN-SIA28 was assayed using full-length wild type and mutants HAs cloned as described above. In brief, 1610 6 human epithelial kidney (HEK) 293T cells (ATCC CRL-1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) with 4 mg of pcDNA 3.1D/V5-His-TOPO vector containing the HA nucleotide sequences. After centrifugation and fixation with paraformaldehyde 4%, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA28 or a conformational control for H1N1 (RB62) or a control for H3N2 (12D1, kindly provided by P.Palese) at 1 mg/ml and 10 mg/ml. Additionally, the isotype control, e137 (1 and 10 mg/ml) was introduced as well as untransfected cells and a mouse anti-H1 subtype monoclonal antibody directed against a linear epitope (anti-influenza A hemagglutinin [12D1102] GeneTex Inc., Irvine, CA, USA) to evaluate the transfection efficiency for each HA. The cells were then washed and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) monoclonal antibodies. Afterwards, the cells were washed and analysed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA28 to the different HA-mutants was expressed as a binding percentage compared to wild-type.
For sequences analysis the following software packages were used: SeqScape (Applied Biosystems), ClustalX (Toby Gibson), Bio Edit (Tom Hall, Ibis Therapeutics), and Treeview (GubuSoft).  Tissue Tropism and Target Cells of NSs-Deleted Rift Valley Fever Virus in Live Immunodeficient Mice BACKGROUND: Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time. CONCLUSIONS/SIGNIFICANCE: These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection. Rift Valley fever virus (RVFV) is an arthropod-borne member of the Bunyaviridae family, genus Phlebovirus that causes recurrent outbreaks affecting humans and animals. The virus can be transmitted by Aedes and Culex mosquitoes [1] , although it can also be transmitted by inhalation or physical contact with the body fluids from infected animals [2, 3] . Identified in the 1930s in Kenya, RVFV has spread during recent years to most sub-Saharan African countries, in Egypt and in the Arabian Peninsula, and in the Indian Ocean islands of Grande Comore and Mayotte [4, 5, 6] . In humans, RVFV infections are generally either asymptomatic or characterized by a feverish syndrome without any severe sequelae. However, a small percentage of patients exhibit complications, characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis [7, 8, 9, 10] . A relationship has been demonstrated between high viral load in blood and death of the patient [11, 12] . RVFV infects domestic ruminants, including sheep, cattle, goats, and camels. It is responsible for massive abortion events in pregnant ruminants and high mortality in lambs and calves. High viremia associated with hepatic necrosis and increase of liver enzymes are hallmarks of severe acute lethal infection in ruminants [13, 14] . Encephalomyelitis has been described in calves [15] . Laboratory rodents such as mice are also highly susceptible to RVFV infection. In outbred Swiss mice, the survival time was inversely proportional to the logarithm of the viral dose inoculated via the intravenous route [16] . Depending on their genotype, males from various inbred strains of mice inoculated by the peritoneal route with 10 2 PFU of the virulent Egyptian ZH548 strain die between 4 to 10 days after inoculation, illustrating natural variation in susceptibility of the host to RVF [17] . The main damages of mouse infection with RVFV can be observed early in the liver, with extensive apoptosis of hepatocytes, accompanied in the blood by a peak in liver enzymes, along with increased bilirubin levels [18, 19, 20] . It has been recently shown that mice that survive hepatitis develop later infection of the brain, and eventually die from meningoencephalitis [21] . Interestingly, a diverse set of cell types from a number of tissues was found to contain RVFV antigens, including mononuclear phagocytes, but also cardiac myofibers, pancreatic islet cells, and adrenal medullary cells [21] . These data showed that RVFV exhibits a large tropism for a variety of tissues and individual cell types. Quantitative real-time PCR have also been used to study the kinetics of RVFV infection in the blood and organs of infected mice [22] . High amounts of RVFV RNA were found in blood, liver and brain samples shortly after infection with the highest viral RNA levels in the liver. We hypothesized that in vivo imaging might be an alternative method to assess viral replication using a recombinant RVFV carrying a reporter gene that allows the monitoring of viral expression in live animal.
RVFV has a tripartite negative-sense, single-stranded RNA genome with large (L), medium (M) and small (S) segments. The L segment encodes the viral RNA-dependent RNA polymerase, the M segment the two virion glycoproteins (G N and G C ) and the NSm nonstructural proteins, and the S segment the N nucleoprotein and the NSs nonstructural protein. Reverse genetic systems have been successfully developed for the recovery of recombinant RVFV (reviewed in [23] ). These rescue systems rely on transfection with plasmids expressing the three viral RNAs, and the N nucleoprotein and L RNA-dependent RNA polymerase, which are required for the packaging and replication of the viral RNAs. The RVFV RNA genome segments are expressed under the control of either the cellular DNA-dependent RNA polymerase I promoter [24, 25, 26] or the bacteriophage T7 promoter in cells that constitutively express T7 RNA polymerase [24] . Such rescue systems have been used to produce recombinant RVFV, including various mutants that lack the NSs, NSm genes, or carry specific mutations [26, 27, 28, 29, 30] . Viral strains that express reporter genes were also generated [24, 31, 32] . Importantly, in these recombinant viruses, the reporter gene activity directly reflects the extent of both viral transcription and replication.
In this study, we aimed to detect and quantify viral replication in living animals using two recombinant RVFV strains expressing either humanized version of the luciferase gene of Renilla reniformis (hRLuc) or enhanced green fluorescent protein (GFP) gene of Aequora victoria. Both RVFV viruses lack a functional NSs gene, which is a main factor of virulence in mice [33] , and are therefore avirulent in immunocompetent mice. However, in mice that are nonresponsive to type I IFN, the virus expressing either hRLuc or GFP caused lethality within 3 days, in agreement with previous data for the NSs-deficient Clone 13 [24] . In these mice, virus infection could be tracked by luciferase imaging in live animals and by the detection of GFP-positive cells from infected animals, by use of flow cytometry. We observed qualitative and quantitative differences in the in vivo tropism of RVFV in mice and show previously unsuspected sites of virus replication and modes of virus spread.
Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture to perform experiments on live mice, in appliance of the French and European regulations on care and protection of the Laboratory Animals (accreditation number B 75 15-01 and B 75 15-07). The veterinary staff of the Institut Pasteur animal facility approved protocols. Protocols were performed in compliance with the NIH Animal Welfare Insurance #A5476-01 issued on 02/07/2007. Inbred 129S2/SvPas mice with knockout at the interferon a and b receptor 1 locus (Ifnar1 2/2 ) and control mice (Ifnar1 +/+ ) were bred at the Institut Pasteur [34] . Vero E6 cells were grown in DMEM supplemented with 10% FCS. BHK21/T7 cells [35] were grown in MEM supplemented with 5% FCS and tryptose phosphate broth powder (Sigma-Aldrich, Gillingham, UK). The cell culture media were supplemented with 10 IU/ml of penicillin and 10 mg/ ml of streptomycin. Stocks of the virulent RVFV Egyptian ZH548 strain were produced under biosafety level 3 (BSL3) conditions.
Plasmids pPol I-LZH, pPol I-MZH, and pPol I-SZH carrying the L, M and S segments of ZH548, respectively, were cloned in the plasmid pRF108, [24, 36] . The plasmid pPol I-SZHDNSs, derived from pPol I-SZH, carries two BbsI cloning sites in place of NSs [24] . The humanized Renilla reniformis luciferase sequence from phRL-SV40 (Promega, Charbonnières-les-Bains, France) was inserted in pPol I-SZHDNSs to give pPol I-SZHDNSs-hRLuc. The structure of pPol I-SZHDNSs-hRLuc plasmid was confirmed by sequence analysis. Recombinant rZHDNSs-GFP [24] and rZHDNSs-hRLuc RVFV stocks were produced under BSL3 conditions. Approximately 5610 5 BHK21/T7 cells were seeded in triplicate in 35 mm culture dishes. The following day, they were combined with FuGENEH6 transfection reagent (Roche Applied Science, Indianapolis, IN) and 0.5 mg each of pTM1-L and pTM1-N [25] , and 1 mg each of pPol I-LZH, pPol I-MZH, and either pPol I-SZHDNSs-GFP or pPol I-SZHDNSs-hRLuc in OptiMEM (Gibco, invitrogen, Carslbad, CA). One day later, the medium was renewed. Five days later, the supernatant containing the rescued virus was collected and stored at 280uC. To produce
Rift Valley fever, caused by a member of the Bunyaviridae family, has spread during recent years to most sub-Saharan African countries, in Egypt and in the Arabian peninsula. The virus can be transmitted by insect vectors or by direct contacts with infectious tissues. The analysis of virus replication and dissemination in laboratory animals has been hampered by the need to euthanize sufficient numbers of animals and to assay appropriate organs at various time points after infection to evaluate the viral replication. By following the bioluminescence and fluorescence of Rift Valley fever viruses expressing light reporters, we were able to track the real-time dissemination of the viruses in live immunodeficient mice. We showed that the first infected organs were the thymus, spleen and liver, but the liver rapidly became the main location of viral replication. Phagocytes also appeared as important targets, and their systemic depletion by use of clodronate liposomes decreased the number of viruses in the blood, delayed the viral dissemination and prolonged the survival of the infected mice. viral stocks of rZHDNSs-GFP and rZHDNSs-hRLuc, Vero E6 cells were infected with the rescued virus at a MOI of 0.001 and 0.01, respectively. At 72 h post-infection, the supernatant was collected and the viral suspension was titered.
Vero E6 cells, infected with serial dilutions of viral suspension, were incubated under an overlay of DMEM supplemented with 2% FCS, antibiotics and 1% agarose. Four days later, the plates were stained with 0.2% crystal violet in 10% formaldehyde, 20% ethanol and the lytic plaques were counted.
To test the luciferase expression within cells after infection with rZHDNSs-hRLuc, Vero E6 cells were infected using a MOI of 0.3 and 3. Next, every 2 h, for 12 h, luciferase activity was measured in triplicates by Renilla Luciferase Assay System (Promega, Madison, WI). To check the stability of luciferase expression through passages, Vero E6 cells were infected using an MOI of 3 and the luciferase activity was measured in triplicates at 8 h postinfection.
To test the GFP expression, Vero E6 cells were infected with rZHDNSs-GFP using a MOI of 1. At 15 h post-infection, the cells were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS, permeabilized for 10 min with 0.5% Triton X100 in PBS and incubated for 30 min at room temperature in 5% bovine serum albumin (BSA) in PBS. The cells were next incubated for 30 min at 37uC with a mouse anti-N antibody diluted in 5% BSA in PBS (dilution 1:800), washed with 5% BSA in PBS and incubated for 25 min at 37uC with the secondary antibody Alexa Fluor 555 goat anti-mouse (Invitrogen, Paisley, UK) diluted in 5% BSA in PBS (dilution 1:1200). Finally, the cells were washed with 5% BSA in PBS and then in water. The slides were mounted with Fluoromont-G (SouthernBiotech, Birmingham, AL). The cells were observed under an Axioplan 2 Imaging microscope (Zeiss, Le Pecq, France) using excitation and emission filter allowing simultaneous detection of GFP and Alexa Fluor 555.
One week prior to infection, five to six week-old mice were transferred in BSL3 isolators to allow acclimatization. After this period, they were inoculated intraperitoneally (i.p.), intradermally (i.d.) or intranasally with 10 4 PFU ZH548, rZHDNSs-hRLuc or rZHDNSs-GFP RVFV in DMEM supplemented with 2% FCS, and antibiotics. For i.p. infection, mice were inoculated with 100 mL of viral suspension. For i.d. and intranasal infections, mice were first anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg) administered i.p., then inoculated with either 15 to 30 mL or 15 mL of viral suspension into the ear (i.d.) or intranasally, respectively. Mortality was recorded at least twice a day from day 1 to 4 post-infection and once a day after day 5 postinfection until the end of the observation period. Animals were observed for a maximum of 14 days.
To improve bioluminescence imaging, hairs were removed [37] . We observed the mice to detect clinical signs due to the infection prior to imaging. Mice that exhibited no severe clinical signs were anesthetized and injected i.p. with 100 mL h-coelenterazine (1 mg/ml). The h-coelenterazine stock solution provided by Nanolight Technology (Pinetop, AZ) was solubilized in ethanol-propylene glycol solution (1:1) at 10 mg/ml. This solution was diluted in PBS (1:9) just before imaging. The h-coelenterazinetreated mice were immediately placed in a hermetically sealed light-tight-transparent chamber (TEM Sega, Lormont, France) equipped with two HEPA filters. One HEPA filter was connected to an air pump, thus allowing air renewal during the imaging. The bio containment chamber allowed simultaneous imaging of 6 mice. The mice were imaged 15 and 20 min after h-coelenterazine injection for the whole body and the thorax, respectively [38] . Imaging was performed with a Xenogen's IVIS 100 system, including a cooled charge-coupled device (CCD) camera [39, 40] . Integration periods ranged from 0.5 to 120 s depending on the amounts of light emitted at various infection sites. Images were obtained using Living ImageH 3.1 software (Xenogen, Alameda, CA). Specific regions of interest (ROI) on the images were defined without overlay, using the anatomic location of the different organs and their visual observation through the skin when possible. For each imaging session, a mock-infected mouse was used as a negative control. A signal was considered significant if its intensity in infected mice was at least twofold higher than the background luminescence in the mock-infected mouse. After the last imaging time point, mice were euthanized.
For ex vivo imaging, selected mice were euthanized at different times after infection for harvest of the following organs: liver, spleen, thymus, lung, kidney, stomach, small and large intestine, heart, ovary and uterus, testis, epididymis, seminal vesicles and preputial glands. The organs were placed in 6 (intestine) or 2 ml (all other organs) of PBS. Before imaging, 1 mL/ml h-coelenterazine 5 mM in ethanol and propylene glycol (1:1) was added [41] . Imaging was performed in a hermetically sealed chamber to avoid light. Images were acquired 10 min after the addition of hcoelenterazine. Integration period ranged from 0.5 to 60 s depending on the amounts of light emitted from various organs.
RNA was extracted using Trizol LS reagent (Invitrogen, Carslbad, CA) and suspended in RNase free water. RNA was quantified using Nanodrop 3300 (Thermo Scientific, Courtaboeuf, France). The M segment of RVFV was amplified with primers 59-CATGGATTGGTTGTCCGATCA-39 and 59-TGAGTGTAA-TCTCGGTGGAAGGA-39. Quantitative RT-PCR assays were performed using StepOne Plus Real-Time PCR System (Applied Biosystem, Courtaboeuf, France) in 96-well plates. Reverse transcription using MultiScribe Reverse Transcriptase (Applied Biosystem) at 48uC for 30 min was performed followed by a standard amplification program. The size of the amplification product was 108 pb. A standard curve was generated using duplicates of 10-fold serial dilutions of RNA of the M segment ranging from 10 9 to 10 2 copies. Quantification of viral RNA was done by comparison of the threshold cycle (Ct) values of the samples to the standards.
Histopathological and immunohistochemical analysis of wildtype mice infected with 10 4 PFU ZH548 RVFV was performed 3 to 5 days after i.p. inoculation. rZHDNSs-hRLuc-infected Ifnar1 2/2 mice were euthanized at 8, 16 and 34 h after i.p. inoculation. For each time point, a complete post-mortem examination was carried out. The lung, brain, kidneys, spleen, liver, pancreas, thymus, testis, uterus and ovaries were removed and immediately fixed for one week in 10% neutral buffered formalin. Samples from each organ were embedded in paraffin and five-micrometer sections were then cut and stained with hematoxylin and eosin (HE). The histological characterization of lesions was completed by an immunohistochemical detection of the RVFV using mouse antibodies against the RVFV (dilution 1:100) visualized with the Histofine Simple Stain MAX-PO kit (Histofine Biosciences inc, Cambridge, UK).
Eleven Ifnar1-deficient 129S2/SvPas mice were either infected i.p. with 10 4 PFU rZHDNSs-GFP RVFV (N = 6) or mock-infected (N = 5). Twenty-four hours later, the spleen was harvested. Erythrocytes were lysed using NH 4 Cl (9 g/L) buffer. The rat anti-mouse CD16/CD32, clone 2.4G2 antibody (BD Pharmingen, San José, CA) was used to block non-antigen-specific binding of immunoglobulins to Fc-receptors. Cells were stained using a combination of the following antibodies: (i) PE-conjugated rat anti-mouse NKp46/CD335 (BD Pharmingen). (ii) PerCP-Cy5.5-conjugated hamster antimouse CD3 (BD Pharmingen). (iii) APC-conjugated rat anti-CD19 (BD Pharmingen). (iv) Pacific Blue-conjugated rat antimouse CD11b/Mac-1 (eBioscience, San Diego, CA). (v) APCconjugated hamster anti-mouse CD11c/Itgax (BD Pharmingen). (vi) Alexa Fluor 700-conjugated rat anti-mouse MHC Class II (I-A/I-E) (eBioscience). (vii) PE-conjugated rat antimouse Ly6G/Gr-1 (BD Pharmingen). (viii) Biotin-conjugated anti-mouse CD115/c-Fms (eBioscience) with Streptavidin-PerCP-Cy5.5 (BD Pharmingen) as second-step reagent. All staining procedures were conducted on ice. Then, the cells were fixed with 4% formaldehyde. Fluorescence was measured using a FACSAria II flow cytometer (BD Biosciences, San Jose, CA), and data analysis was performed using CellQuest (BD Biosciences) and FlowJo (Ashland, OR) softwares. Dead cells were visualized using the Fixable Aqua Dead Cell Stain kit (Invitrogen, Carlsbad, CA). Fluorescence compensation settings for multicolor flow cytometric analysis were optimized based on single-stained polystyrene microparticles (Comp-Beads, BD Pharmingen).
Clodronate (Cl2MBP; dichloromethylene-biphosphonate)-loaded liposomes (CLL) were used to deplete phagocytic cells [42, 43] . Clodronate was a gift of Roche Diagnostics GmbH, (Mannheim, Germany). It was encapsulated in liposomes as described earlier [42, 43] . Mice were injected i.p. with 300 mL and i.v. with 200 mL CLL. Control mice were treated i.p. and i.v. with PBS-loaded liposomes. Twenty-four hours later, single-cell suspensions were prepared from blood and spleen. FcR blocking reagent mouse (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to block non-antigen-specific binding of immunoglobulins to Fc-receptors. Cells were stained using combination of the following antibodies: FITC-conjugated rat anti-mouse CD11b (BD Pharmingen), PEconjugated rat anti-mouse CD115 (eBioscience.com), APCconjugated rat anti-mouse F4/80 (eBioscience.com), PE-conjugated hamster anti-mouse CD11c (BD Pharmingen) and Pacific Blueconjugated rat anti-mouse Ly6G/Gr-1 (eBioscience.com). All staining procedures were conducted on ice. Fluorescence data were obtained and analyzed using MACSQuant Analyzer and MACSQuantify software (Miltenyi Biotec). Challenge with 10 4 PFU rZHDNS-hRLuc was performed by injection into the ear, 24 h after liposome treatment.
The survival curves were compared using the logrank test. The bioluminescence signals and blood plasma viral loads were analyzed with the nonparametric Mann-Whitney test. All tests were performed using the StatView 5.0 software (SAS Institute Inc, Cary, NC).
The generation of a recombinant RVFV expressing a green fluorescent protein (GFP), rZHDNSs-GFP, has previously been described [24] . Our previous attempts to generate a recombinant RVFV expressing a humanized firefly luciferase (hFLuc) have been confounded by genetic instability and the rapid emergence of mutants with deletions [24] . Therefore, we generated rZHDNSs-hRLuc RVFV that carries a humanized Renilla luciferase (hRLuc) gene using Pol I based plasmids as previously described [24] . The rescued rZHDNSs-hRLuc was amplified in Vero E6 cells and stocks produced. The titer reached 8610 7 PFU/ml. The plaques formed by rZHDNSs-hRLuc were fuzzy with a faint staining inside, as those obtained with the rZHDNSs virus that carries a deletion of the NSs gene [24] . To test the luciferase expression within the infected cells, Vero E6 cells were infected with rZHDNSs-hRLuc at a MOI of either 0.3 or 3, and lysed every 2 hours for 12 h. Luciferase activity was measured using coelenterazine, a specific substrate of Renilla luciferase, and found to be expressed at significant levels from 2 h post-infection onwards, while uninfected Vero E6 cells showed no luciferase activity. The luciferase activity increased with time and was dependent on the MOI (data not shown). This is consistent with previous reports [31, 32] . To check the stability of the recombinant virus in vitro, rZHDNSs-hRLuc was passaged on Vero E6 cells and, at each passage, we measured the viral titer in the supernatant at 72 h post-infection and the luciferase activity within the infected cells at 8 h post-infection with a MOI of 3. Both the viral titer and the luciferase activity remained stable over at least 8 passages, varying from 10 7 to 10 8 PFU/ml and from 10 7 to 10 8 raw light units (RLU)/s per 3610 5 cells, respectively. To test the virulence of the recombinant virus, wild-type 129S2/SvPas mice (N = 5) and 129S2/SvPas mice deficient for IFN-a/b receptor subunit 1 (Ifnar1 2/2 ) (N = 10) were infected i.p. with 10 4 PFU of rZHDNSs-hRLuc. All wild-type mice survived the infection for 13 days with no signs of disease, as seen following infection with rZHDNSs in which the NSs gene is totally deleted [24] or Clone 13, a natural isolate that lacks 69% of the NSs open reading frame [33] . In contrast, all rZHDNSs-hRLuc infected Ifnar1 2/2 mice died within 45 h (Figure 1 ) from severe hepatitis with no signs of neurological disorder. Infections of Ifnar1 2/2 mice with rZHDNSs or with Clone 13 gave similar results ( [24] and data not shown).
To evaluate the stability of the recombinant viruses in live animals, total RNAs were extracted from the liver of rZHDNSs-hRLuc-or rZHDNSs-GFP-infected Ifnar1 2/2 mice at 34 h postinfection and RT-PCR assays were performed using primer pairs flanking the hRLuc or GFP reporter gene. The amplification product sizes were those expected from the structure of pPolI-SZHDNSs-hRLuc and pPolI-SZHDNSs-GFP plasmids (data not shown). No amplification products with smaller sizes were observed, suggesting that the recombinant viruses maintained their own genomic stability not only in cultured cells, but also during in vivo infection. Furthermore, to examine the reporter expression stability after in vivo infection, the recombinant rZHDNSs-GFP was harvested from the liver of an infected Ifnar1 2/2 mouse at 34 h post-infection and then used to infect Vero E6 cells at a MOI of 1. The percentage of cells positive for the N viral protein that were also GFP-positive was measured. The percentage of N-positive, GFP-positive cells was almost identical to that of the initial viral stock (84%61.90% vs. 85%67.34%). Altogether, these results suggest that the recombinant viruses were stable for the time of infection in live mice.
To visualize the spread of the virus, Ifnar1 2/2 mice were infected i.p. with 10 4 PFU of rZHDNSs-hRLuc. At 8, 16 and 34 h postinfection, h-coelenterazine was injected i.p. This route of hcoelenterazine administration was preferred to tail-vein injection due to slower kinetics of light production, as previously reported [39] . Mice were observed with real-time in vivo imaging 15 and 20 min after the injection of h-coelenterazine for the whole body and the thorax, respectively. At 8 h post-infection, luminescence was readily detected. Short integration periods (15 s) were sufficient to acquire a significant signal. We observed strong signals between the forelegs in the thoracic cavity, and below the xiphoid cartilage in the abdominal cavity, respectively (Figure 2A) . Imaging of the left profile showed an additional signal in the spleen ( Figure 2B ). In some experiments, animals were euthanized for ex vivo imaging, the organs of the thorax and abdomen were harvested, and the individual organs were imaged ( Figure 2D ). In the thorax, the greatest signal originated from the thymus, whereas the signal from the lungs was only slightly above background. In the abdomen, the pancreas was the most luminescent organ. The spleen and the liver also emitted significant luminescence. On average, a ten-fold higher luminescence signal was observed in the harvested pancreas compared to the liver. This suggests that the liver, the critical target organ of the disease, was not among the main replication sites for RVFV at this early stage of infection. At 16 h post-infection, the whole body luminescence was higher than at 8 h post-infection (Figure 2A) . The signal spread out the abdominal cavity. The high intensity of luminescence in the abdominal cavity precluded detection in other locations unless integration was limited to the thorax ( Figure 2C ). Dissection and ex vivo imaging showed a gradual increase of luminescence in the pancreas and in the liver. Additional sources of luminescence were the intestine mesentery, kidneys, ovaries and uterus in females, the seminal vesicles, preputial glands, epididymis and testis in males ( Figure 2D , and data not shown). The intensity of these signals was quite similar to that measured in the spleen and in the liver (data not shown). At 34 h post-infection, the intensity of the signal led to saturation of the camera using a 0.5 s integration period, thus preventing identification of individual organs (Figure 2A) . Ex vivo imaging revealed that the highest signal was in the liver. Other organs with intense luminescence were the spleen, intestine mesentery and pancreas (data not shown). Quantification of the luciferase expression in living mice during the time course of infection showed that the luminescence signal originated from the thymus remained constant from 8 h postinfection onwards, whereas luminescence profiles were increased in the liver and pancreas, suggesting a progressive increase of viral replication in these organs (Figure 3 ).
To determine whether there was a correlation between the luminescence detected by the camera and the amount of virus genomes in the tissues, rZHDNSs-hRLuc-infected Ifnar1 2/2 living mice were subjected to imaging. Next, the animals were euthanized and the organs harvested and imaged. Total RNAs were extracted from the organs and RVFV RNA copy numbers were measured by quantitative real time RT-PCR. We observed a highly significant correlation between the luminescence emitted by the pancreas in living mice and RVFV RNA copy number ( Figure 4A ). Similarly, luminescence intensity significantly correlated with the RVFV RNA copy number in the harvested pancreas, spleen and liver ( Figure 4B-D) .
The ability of h-coelenterazine to cross the blood-brain barrier is unknown. To determine whether rZHDNSs-hRLuc can infect the brain, we dissected and soaked the brain in an hcoelenterazine solution and imaged ( Figure 2D ). At 8 h postinfection, the luminescence intensity was ten-fold higher in the brain from infected mice compared to control (10 4 photons/ second/cm 2 /steradian [p/sec/cm 2 /sr] vs. 10 3 p/sec/cm 2 /sr), showing that the RVFV replicated in the brain at an early stage. Light emission increased through 16 h and 34 h post-infection to reach 7610 5 and 7610 6 p/sec/cm 2 /sr, respectively. Importantly, the intensity of luminescence in the brain was ten-to hundred-fold lower compared to the intensities in the thymus, pancreas, spleen, liver, and intestine mesentery, suggesting that the viral load was lower in the brain than in the thoracic and abdominal organs.
RVFV can be transmitted through injection of infectious saliva from mosquito into the dermis or direct inhalation from body fluids, such as blood of infected animals [2, 3] . To approximate these two natural routes of infection and to monitor their effects, we compared the light production after intraperitoneal, intradermal or intranasal inoculation of 10 4 PFU rZHDNSs-hRLuc into Ifnar1 2/2 mice. Following intradermal inoculation of the ear pinna (N = 5), luminescence was first visible in the neck on the side of the injected ear, and in the abdominal cavity at 24 h postinfection ( Figure 5A ). Histologic analysis established the source of the light in the neck; the neck signal came from the lymph nodes draining the injected ear (data not shown). At 40 h post-infection, organs in the abdominal cavity, including the pancreas and the liver were highly luminescent. All mice succumbed to infection by 69 h post-infection, a survival time significantly longer than after i.p. inoculation (P,0.025). Luminescent virus inoculated intranasally was already detected 24 h post-infection in the abdomen. This mode of inoculation caused an interstitial pneumonia that initiated as a distinctive luminescence signal in the lungs from 48 h post-infection onwards ( Figure 5B ). Mice infected intranasally (N = 5) survived significantly longer than after i.p. inoculation (P,0.0047); all were dead by 69 h post-infection.
To clarify the identity of the RVFV target cells, we carried out histopathological analysis in RVFV-hRLuc-infected Ifnar1 2/2 No histological lesions were detected in the other organs. In the liver, lung and spleen, the lesions were similar to those previously reported in RVFV-infected wild-type mice [18, 19, 20, 21] . Diffuse apoptosis of lymphoid cells have been previously reported in areas with or without RVFV antigen in the thymus of infected BALB/c [21] . Accordingly, we identified the thymus as one of the major targets of RVFV by bioluminescence. However, the pancreas and reproductive organs were also luminescent although none of these organs are known as tissue targets of RVFV. Therefore, to identify cell types that support RVFV replication in these organs, we studied tissue samples by histology and immunohistochemistry with antibodies against the RVFV. In the pancreas, no histological lesions were found in the exocrine or endocrine components ( Figure 6A ). However, a multifocal inflammatory lesion was observed in the mesentery around pancreatic acini (peritonitis), characterized by necrosis of adipocytes associated with infiltration of macrophages and neutrophils ( Figure 6A-C) . Viral antigens were present only in the cytoplasm of macrophages ( Figure 6D ) and, more rarely, in neutrophils ( Figure 6E ), confirming that the virus did not target the pancreatic exocrine or endocrine cells but macrophages. Similarly, in the ovaries, viral nucleocapsid-positive macrophages were seen in the stroma ( Figure 6F ). Thus macrophages appeared as important cell targets for the replication of RVFV-hRLuc in Ifnar1-deficient mice. To examine whether macrophages are also cell targets for the replication of virulent RVFV in wild-type mice, histopathological and immunohistochemical analysis was performed in wildtype 129S2/SvPas mice (N = 3) infected i.p. with 10 4 PFU ZH548. Post-mortem analyses were carried out once mice displayed clinical signs, i.e. three to five days after the inoculation. Histopathological analysis of the pancreas and its mesentery revealed no peritonitis. However, numerous macrophages containing intracytoplasmic viral antigens were observed in the sinus of the pancreaticoduodenal lymph node (Figure 6, G and H) . These macrophages occasionally displayed a hyperbasophilic and condensed nucleus, a morphological change that is characteristic for irreversible cell injury (Figure 6 , I). Collectively, these results confirmed that macrophages are important cell targets of the RVFV in the mouse.
To further dissect target cells of RVFV replication in Ifnar1deficient mice, we used the recombinant virus rZHDNSs-GFP that carries GFP in place of the NSs gene. We have shown previously that cells infected in vitro with rZHDNSs-GFP are fluorescent upon excitation at 488 nm [24] . Ifnar1-deficient mice were either infected i.p. with 10 4 PFU rZHDNSs-GFP (N = 6) or mocktreated (N = 5). At 24 h post-infection, the spleen was dissected and single-cell suspensions were analyzed by flow cytometry for GFP expression. At this time point, 0.54% (range 0.14-1.53%) of the total hematopoietic cell population of the spleen from rZHDNSs-GFP-infected mice expressed GFP whereas no GFP-positive cells were found in splenocytes after mock infection ( Figure 7A ). We examined the expression of GFP in various subsets of antigen presenting cells based on the surface expression patterns of CD45.2, CD11b, CD11c, Ly6G, CD19, CD3, NKp46, CD115 and MHCII class II by multicolor flow cytometric analysis. Among the CD11b + CD115 + Ly6G 2 (macrophages), CD11c + CD11b + MHC II + (dendritic cells) [44] and CD11b + CD11c 2 Ly6G + (granulocytes), on average 5.58% (range 2.15-8.71%), 4.5% (range 0.82-8.49%) and 1.96% (range 0.05-6.07%) cells expressed GFP, respectively ( Figure 7C [left panels], B [right panels], and C [right panels], respectively). The percentage of GFP-expressing cells within the total cell population of spleen varied from one infected mice to another, indicating that the dynamics of RVFV infection progression was not identical in all individuals. However, each of the three subsets of immune cells was infected with the same efficiency in the different mice. This is shown by the fact that the ratio of GFP-expressing macrophages, dendritic cells or granulocytes was highly correlated with the ratio of GFP-expressing cells in the total cell population from the spleen (Pearson correlation coefficient 0.97, 0.85 and 0.79, respectively) . These findings suggest a distinct pattern of susceptibility to infection by the RVFV-GFP for different immune cells in the following order: CD11b + CD115 + Ly6G 2 (macrophages).CD11c + CD11b + MHC II + (dendritic cells).CD11b + CD11c 2 Ly6G + (granulocytes). On average, less than 0.4% (range 0-1.19%) of NKp46 + CD3 2 natural killer (NK) cells were positive for GFP ( Figure 7E, left panel) . Finally, GFP fluorescence was seen on average in only 0.25% (range 0.07-0.57%) of CD19 + CD3 2 (B lymphocytes) cells and 0.20% (range 0.05-0.43%) NKp46 2 CD3 + (T lymphocytes) cells ( Figure 7D and E, right panels). Thus, at 24 h post-infection, RVFV replicated in cells of the myeloid lineage, primarily in mononuclear phagocytic cells, such as macrophages, dendritic cells and granulocytes.
To study the significance of virus replication in phagocytic cells in vivo, we injected intraperitoneally (i.p.) and intravenously (i.v.) clodronate-loaded liposomes (CLL) to Ifnar1-deficient mice prior to infection with RVFV. These liposomes are widely used to deliver clodronate to phagocytic cells, especially macrophages, and the accumulation of clodronate leads to irreversible metabolic damages, which will eventually result in apoptosis [45] . As reported previously [42, 43] , the i.p. administration of CLL kills macrophages in the peritoneum and spleen of wild-type mice whereas i.v. administration affects mainly macrophages in the spleen and liver. We first analyzed the effect of CLL treatment on the phagocytic cell population of the blood and spleen of Ifnar1deficient mice 24 h after i.p. and i.v. administration. Flow cytometric analysis was performed to compare the percentage of macrophages/monocytes, dendritic cells and granulocytes in samples from mice treated with CLL (N = 3) and PBS liposomes (PBSL) (N = 3). The CLL treatment resulted in a 23-fold reduction of CD11b + CD115 + cells (monocytes) and a 6-fold reduction of CD11b + F4/80 + -expressing macrophages in the blood and spleen, respectively. In addition, CD11b + CD11c + F4/80 2 cells (dendritic cells) were decreased 9-fold in the blood. By contrast, CD11b + CD11c 2 Ly6G + cells (granulocytes) were not depleted in the blood and spleen, as previously reported [46] . Altogether this analysis showed that, 24 h after CLL treatment, blood monocytes and dendritic cells and spleen macrophages were efficiently depleted in Ifnar1-deficient mice whereas granulocytes were not affected. Next, CLL-or PBSL-treated mice were infected intradermally with 10 4 PFU rZHDNSs-hRLuc at 24 h after liposome administration. To investigate whether the clodronate treatment affected viral replication in vivo, we first observed PBSL-and CLL-administered infected mice using the imaging of whole bodies at 24 and 40 h post-infection. The profile of bioluminescence signals was similar in PBSL-and CLL-administered mice (N = 5 in each group) (data not shown). However, we observed weaker signals in CLLadministered mice. Indeed, the signals in the liver region were on average fifteen and four-fold lower at 24 and 40 h post-infection respectively in CLL-administered mice compared to PBSLtreated mice (P,0.05). We then measured the viraemia at 24 
In vivo imaging studies using reporters, such as hRLuc and GFP, may provide a more complete picture of the spatiotemporal progression of a viral disease [47, 48] . In this study, we report the use of recombinant RVFV-hRLuc and RVFV-GFP strains to investigate the in vivo dynamics of RVFV infection progression in living mice and identify the virus-expressing cells. The recombinant viruses were generated by replacing the NSs gene with the reporter gene. Hence, these viruses were avirulent in immunocompetent mice when compared with wild-type virus but they were highly pathogenic in mice lacking interferon-a/b receptor, enabling to use them for pathogenesis studies in this mouse model. We were able to detect luciferase reporter expression at early stages of infection in the main known sites of viral replication, the liver, the spleen, the thymus and the brain [18, 21, 49, 50, 51] . The pancreas appeared as an unexpected site of virus replication. Using ex vivo imaging and histological examination, we primarily identified macrophages infiltrating the adipose tissue surrounding the pancreas as primarily virus-expressing cells. Similarly, RVFVexpressing macrophages were identified in the stroma of the ovary. The RVFV-GFP confirmed the importance of macrophages as specific host cells for the virus in Ifnar1 2/2 mice. It further allowed the identification of dendritic cells and granulocytes as target cells for RVFV replication. Interestingly, only a low number of B-, T-and NK-cells expressed the GFP reporter. Viral antigens have been previously detected in mononuclear phagocytic cells and dendritic cells in the lymph nodes, spleen and thymus from infected wild-type mice [21] and in macrophages in the lymph nodes from infected rats [51] . Interestingly, Smith and colleagues [21] also noticed that lymphocytes did not appear stained with the RVFV antibody in agreement with our observations. Although RVFV replication in the human macrophage-like cell line U937 [52] and in cultured peritoneal macrophages from susceptible rats [53] have been previously documented, this is, to our knowledge, the first study to evaluate the infection rates of various subsets of cells of the myeloid lineage in vivo. Because the level of fluorescent GFP directly reflects the extent of transcription and replication of the recombinant virus, we assume that the virus is highly expressed in GFP-positive cells. However, since macrophages, dendritic cells and granulocytes are able to uptake cell debris, it is possible that some of these cells are GFP-positive following phagocytosis of debris of RVFV-GFPinfected cells in vivo.
Phagocytic cells function as pathogen sensors. Macrophages and neutrophils provide the first line of defense following infections. Macrophages and dendritic cells are antigen presenting cells and play a crucial role in the establishment of the adaptive immune response. Infection with RVFV-GFP showed that phagocytic cells are also target cells for RVFV. We investigated the importance of the in vivo interaction between phagocytic cells and RVFV. We treated Ifnar1-deficient 129S2/SvPas mice with CLL to deplete the population of phagocytic cells, and showed that, following intradermal infection with RVFV, the depleted mice allowed reduced RVFV replication compared to control mice, as assessed both by in vivo imaging and viral titration from blood samples. Accordingly, CLL-treated mice displayed enhanced survival time compared with control mice, indicating that phagocytic cells are involved in the pathogenesis of RVF. Altogether, our data suggest that during the initial stages of infection of Ifnar1 2/2 mice, the virus replicates inside macrophages and dendritic cells. On the other hand, since the RVFV replicates in diverse cell types in peripheral tissues, the infection may progress rapidly and lead to acute hepatitis and death.
RVFV is thought to be transmitted primarily by bites of infected mosquitoes, by direct contact with infected body fluids or through airborne transmission. It has been confirmed that exposure of mice to aerosols containing RVFV is able to induce infection [54] . Following inoculation into a dermal site, RVFV-hRLuc expression was seen in the draining lymph node which became the main site of replication early after infection while the virus was still weakly detected into the abdominal cavity. Later, virus spread and caused severe hepatitis within 69 h post-infection. Following intranasal inoculation, virus replicated in the lung where it caused pneumonia within 48 h post-infection. Its dissemination to the abdominal cavity was rapid and mice succumbed at 69 h postinfection. Thus, typical routes of exposure were associated with clear differences in the spatial and temporal progression of RVFV and caused delayed death compared with i.p. inoculation.
Type I interferons (IFNs) are essential elements during host antiviral defense [55] . Both recombinant RVFV strains inoculated i.p. were able to kill Ifnar1-deficient 129S2/SvPas mice within 2 days whereas wild-type 129S2/SvPas mice survived infection, indicating that a functional IFN-a/b pathway is critical for the protection of mice from fatal infection with these attenuated viruses. We showed that the recombinant viruses could replicate in known target tissues and cells of RVFV. It is not clear whether in the absence of the IFN-a/b receptor, the reporter RVFV can replicate in tissues and cells that are not normally susceptible to infection with a fully virulent RVFV in wild-type mice. Hence, we infected wild-type 129S2/SvPas with the virulent RVFV ZH548 strain and observed infected macrophages in the spleen and pancreaticoduodenal lymph node. However, we failed to identify the peritonitis seen repeatedly in recombinant RVFV-infected Ifnar1 2/2 mice. This suggests that, in Ifnar1 2/2 mice, cells of the macrophage lineage displayed an increase susceptibility to RVFV compared to wild-type mice. The high susceptibility of cells of the macrophage/dendritic lineage to viral infection in the absence of a functional type I IFN system has been previously observed. An increased infection of cells of the macrophage/dendritic lineage was observed in Ifnar1 2/2 mice infected with either the Sindbis virus [56] , or the mouse hepatitis virus [57] . Similarly, macrophages showed the greatest increase in susceptibility among the different splenocyte populations in West Nile virus-infected Ifnar1 2/2 mice compared to wild-type mice [58] . More generally, an increase in the replication of viruses in tissues and cells normally susceptible to virus infection has been previously observed in Ifnar1 2/2 mice. Coxsackievirus replicated dramatically in the liver of Ifnar1-deficient compared with wild-type mice [59] . Finally, previous investigation of poliovirus replication sites in infected Ifnar1 2/2 mice expressing the human poliovirus receptor showed that nontarget tissues became potentially permissive for virus infection when IFNa/b signaling was disrupted [60] . Therefore, the fact that Ifnar1 2/2 mice inoculated with NSsdeficient RVFV strains develop acute hepatitis and eventually die, as wild-type mice infected with a virulent RVFV strain, does not mean that the exact mechanisms of the cellular pathogenesis are the same in Ifnar1 2/2 and wild-type mice. Thus, although Ifnar1 2/2 mice have proven to be a tractable system in which to study the progression of RVFV infection in vivo, the immunocompromised nature of this mutant strain remains a limitation in translating these results directly to wild-type mice. Additional work needs to be done to develop similar whole-organ imaging and flow cytometry analysis in immune-competent mice. Further studies involving the use of fully virulent RVFV -i.e. carrying the NSs gene -which express a reporter gene, might allow us to give a comprehensive picture of the dynamics of natural infection in mammals. Our work provides the basis for the use of bioluminescent and fluorescent RVFV to study the effects of specific mutations in the viral genome and of host genetic factors on the tissue tropism and replication kinetics in living mice.  Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. To date, quantitative real-time PCR (qRT-PCR) is the most reliable and easy to perform technique to measure the expression level of a selected gene of interest (GOI) by quantifying mRNA transcripts. qRT-PCR is fast and the sensitivity of the method allows precise quantification of minimal differences in expression across a wide dynamic range even when working with limited amounts of starting material. However, several variables associated with the different steps of qRT-PCR experimental procedures can lead to considerable inter-sample variation and possibly to erroneous results: the different amount and quality of starting material; RNA integrity; efficiency in cDNA synthesis and PCR amplification; and differences between tissues or cells in overall transcriptional activity [1] . Among the strategies proposed to control for technical and sample variation in qRT-PCR experiments [2] , the use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors generated in the quantification of gene expression. In this normalization strategy, reference genes are used as internal controls and are submitted to the same experimental protocol of the GOI. The expression level measured for the target gene is then normalized according to the values of the internal controls. It is clear, therefore, that an ideal reference gene should be stably expressed within the samples to be compared irrespective of experimental conditions or external factors, otherwise the detection of small changes become unfeasible and unreliable. A number of studies have well assessed that genes classically thought to be stable for their ubiquitous expression and involvement in cell homeostasis (e.g., GAPDH, ACTB, 18S rRNA) are not always the best reference genes, as they show different behaviour across various cell types and tissues [3, 4] . Accordingly, a proper evaluation of several candidate genes should be performed before any gene expression study [2] .
Studies aimed at identifying the most suitable reference genes in the ovine species have been performed in nervous tissues, spleen, mesenteric lymph node, ileum, lung and pulmonary artery [5] [6] [7] [8] . However, no specific information on whole blood is currently available. A reference gene for use in peripheral blood mononuclear cells was selected [9] , but this study was based on the analysis of the standard deviation of cycle threshold (C t ) and not on specifically designed algorithms. Nevertheless, blood is a readily accessible source of material for analysis and some attempts to identify gene expression markers by qRT-PCR in order to develop blood tests in sheep have been reported for prion diseases. For example, in 2001, Miele et al. discovered a novel erythroid-associated factor (ERAF) and demonstrated a dramatic decrease in expression of the specific transcript within rodent models of prion diseases, providing the first easily detectable molecular marker in a readily accessible tissue [10] . More recently, analysis of blood by qRT-PCR from sheep experimentally infected with scrapie revealed that the extent of differential expression of ERAF in peripheral ovine blood may be insufficient to provide a discriminatory diagnostic test [11] . However, the lack of a set of validated reference genes for sheep whole blood did not allow for the proper normalization of gene expression data and, in this study, glycophorin C (GYPC) was arbitrarily chosen as normalizer based on its higher expression in the human erythroid lineage. Moreover, the use of a single gene to normalise expression is no longer considered sufficient [12] [13] [14] [15] . Vandesompele et al. (2002) demonstrated that errors of up to 20-fold in expression data can be generated by the use of only a single reference gene [1] .
The aim of the present study was to identify a set of reference genes to be used for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression level in whole blood samples from control and disease-stressed sheep, both separately and combined, in order to select genes whose stability was unaffected under stress conditions. The geNorm and NormFinder applets [1, 16] were used for validating the reference genes; sample processing and experiments were carried out according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [17] .
Preliminary qRT-PCR experiments carried out to set up optimal reaction conditions showed that all candidate reference genes were expressed in ovine whole blood. qRT-PCR optimisation was performed using pooled cDNA samples in parallel with sheep genomic DNA. All primer pairs spanned two exons, generating melt-curve profiles specific to cDNA and genomic DNA amplification. While this strategy entailed much more effort in primer design and reaction optimisation, it assured specific amplification of mRNA transcripts by avoiding/recognizing interference of genomic DNA in quantification. Actually, in most cases DNAse treatment does not completely eliminate genomic DNA contamination, especially when RNA extraction is performed using reagents based on mono-phasic solution of phenol and guanidine isothiocyanate (personal observation). Gene-specific amplification was confirmed for all selected genes by a single-peak in melt-curve analysis and subsequent sequencing of amplicons. The determined reference gene sequences have been submitted to the GenBank database under the accession numbers JN811677-JN811687.
The highest expression was obtained with ACTB, B2M and RPL19 with C t averages of 14.64, 15.82 and 15.95, respectively, whereas the lowest expressed gene was GYPC (mean C t , 27.50). For all analysed genes, the relative standard curve gave correlation coefficients greater than 0.985 and efficiencies between 90 and 110%.
To select the optimal set of reference genes, expression values of the candidate genes were submitted to analysis by the geNorm and NormFinder applications. Table 1 reports the expression stability values (M) of the candidate reference genes in control, disease-stressed and combined sheep groups as calculated by the geNorm applet. The M values are used to rank genes on the basis of their stability: high M values indicate increased gene expression variability, whereas the most stable genes should exhibit M values <1.5 [1] . All studied genes reached acceptable stable expression with low M values, less than 1.5. Based on M value ranking, SDHA appeared to be the most stably expressed gene in control sheep with an average M value of 0.316, followed by YWHAZ and HPRT. In disease-stressed sheep, the overall stability of the candidate genes was lower and YWHAZ was the most stably expressed gene with an average M value of 0.624, followed by GAPDH and SDHA. When data from the two groups were combined, SDHA and YWHAZ To determine the optimal number of reference genes needed to calculate a normalization factor (NF), geNorm measures the pairwise variation between two sequential NFs with an increasing number of reference genes. A cut-off value of 0.15 is usually considered acceptable; it indicates that the control gene combination ensures satisfactory stability and that an additional gene need not be included. In the panel of candidate genes studied here, the use of two genes as references proved to be sufficient for accurate normalization in all sheep groups (Figures 1B, 2B and 3B). 
NormFinder ranks a set of candidate genes according to their expression stability measure (ρ) based on the similarity of their expression profiles. Lower values are assigned to the most stable genes. Table 2 reports the results of the NormFinder analyses. The ranking appears to be consistent to the one previously determined using geNorm. SDHA, YWHAZ and HPRT still occupy the highest positions in control animals, with stability values of 0.068, 0.125 and 0.132, respectively; while YWHAZ, GAPDH and SDHA shows the highest stability values in disease-stressed sheep (ρ values = 0.046, 0.064 and 0.188, respectively). When expression data from control and disease-stressed animals were combined, the resulting ranking confirmed SDHA and YWHAZ in the top positions with stability values of 0.093 and 0.096, respectively, followed by ACTB (ρ value = 0.099). TFRC and PGK1 are equally defined as the least reliable controls by both software and in all sheep groups. 
We examined the expression of 11 genes in ovine whole blood by using two commonly accepted softwares (geNorm and NormFinder). Both software algorithms are frequently used and freely available but have a different working rationale. NormFinder selects out of a set of potential reference genes one single best-performing reference gene that shows the least variation within the analysed group. GeNorm focuses on pairwise comparisons of reference gene expression in the experimental samples and so is less appropriate for identifying co-regulated genes [18] . To avoid possible bias, we therefore selected the candidate reference genes on the basis of differences in their physiological functions.
To investigate the influence of the animal health status on the stability of the candidate reference genes, the analyses were performed in whole blood of control sheep and of sheep showing disease symptoms after clinical evaluation. Moreover, disease-stressed animals were sampled and analysed twice in order to monitor gene stability in disease-stressed sheep not only at different time points, but also under heat stress conditions, which, in association to disease status, really represent an extreme situation (see the Experimental Section for details on animal selection and sampling procedure). In all sheep groups, the results obtained with geNorm and Normfinder were consistent although not identical, as similarly reported elsewhere [19] [20] [21] [22] . SDHA and YWHAZ can be considered the most stably expressed genes in ovine whole blood ranking at the top positions in the control and disease-stressed sheep, both when they were analysed separately and when they were combined. SDHA and YWHAZ stability appears to be reliable as it was affected neither by disease status alone nor in association with heat stress. Moreover, geNorm indicated SDHA/YWHAZ as the best reference gene combination in the control and disease-stressed sheep joined datasets, a situation likely to fit most experimental contexts involving case and control animals; however, the SDHA/YWHAZ combination would be suitable for normalization of gene expression data also in studies carried out under physiological conditions, as these two genes demonstrated high stability in the control sheep group as well. Although data in sheep are still limited, SDHA appears to have good stability in this species as it was included in the reference genes required for reliable normalisation in several tissues (cerebrum, spleen, mesenteric lymph node and ileum). Similarly, YWHAZ was included in the optimal panel of reference genes to be used in the cerebellum, obex and ileum [5] . HPRT expression stability was evaluated only in the lung and pulmonary artery of brainstem death and control sheep, but it performed poorly as reference in both tissues on separate and combined analyses [7] . Actually, HPRT ranked as the third most stable gene of the control group in sheep whole blood, but its stability strongly decreased under disease conditions, emphasizing that proper validation of reference genes in a cell type or tissue of interest and under different experimental settings is mandatory before reporting qRT-PCR results. B2M showed stable expression in one study on human leukocytes from 13 healthy donors [1] . B2M also had stable expression in a large study in which 526 human whole blood samples represented healthy individuals and six disease groups [23] . In sheep, however, B2M is outperformed by other genes and demonstrates suboptimal suitability as reference gene in whole blood. In humans, GYPC expression is considerably higher in erythroid lineage cells than in non-erythroid cells [24] . GYPC was therefore used by Brown et al. (2007) to normalize qRT-PCR analyses of erythroid markers in sheep whole blood [11] . In our study, however, both geNorm and NormFinder classified GYPC in the bottom half of the stability ranking under both control and disease conditions, showing that ubiquitously expressed genes would provide a more relevant comparison for measuring erythroid gene expression. In control sheep, GAPDH resulted in being the gene with the highest degree of individual variation in expression level. This finding was not surprising, as GAPDH can be regulated under a large number of physiological states and is generally not considered a good reference gene [25, 26] . Nevertheless, GAPDH ranked as the second most stable gene in the disease-stressed and the combined sheep groups. Taylor et al. (2008) found that the levels of GAPDH transcription were the most stable of the genes tested in ovine peripheral blood mononuclear cells during infection with Mycobacterium avium subsp. paratuberculosis [9] . This finding is consistent with our results indicating stability of GAPDH in whole blood under disease condition. However, it must be taken into account that Taylor's results could also be attributable to a characteristic of the specific cell type or to the fact that the study based its observations on analysis of the standard deviation of C t values and not on specifically designed algorithms.
A distinctive point of our study is the major effort put into primer design with the aim to validate only oligos spanning at least one intron. This aspect has been neglected in the previous works on reference gene validation in sheep, probably because of the lack of ovine genomic DNA sequences available in the public databases. Indeed, we were able to retrieve intron-spanning primers from previous publications for only three genes (PGK1, SDHA and G6PD) among those included in our study. Nevertheless, this approach is highly recommended in combination with DNAse I treatment to avoid/recognize co-amplification of contaminating genomic DNA [1] , since spurious PCR signals could affect the selection of reliable references by mimicking individual variation with lower stability scores. Also, when searching RTPrimerDB, a reference database for qRT-PCR primers [27] [28] [29] [30] , we noted that among 8329 real-time PCR primer sets for 5758 genes of 26 organisms available at the time of writing, only 16 SYBR Green assays were deposited under Ovis aries. Importantly, therefore, an additional outcome of our study is a set of validated primer sequences suitable for gene expression experiments based on SYBR Green chemistry to be carried out with other ovine tissues and cells.
Fresh whole blood samples were collected into EDTA tubes from 28 Biellese sheep belonging to three different farms. The animals included in the study were unrelated. Sheep were submitted to clinical evaluation by a veterinarian and categorized as control animals (n = 18), not showing any clinical sign, and disease-stressed animals (n = 10). Specifically, they had chronic diarrhoea (n = 5), lameness (n = 2), abscesses (n = 2) and respiratory syndrome (n = 1). The blood samples from disease-stressed sheep were collected twice: the first sampling was carried out in August, when the environmental temperature was of 35 °C, and the second in September with an environmental temperature of 26 °C. Every sampling was preceded by clinical evaluation of sheep to confirm disease status. The blood samples were immediately transferred to the lab and submitted to nucleic acid isolation.
Total RNA was extracted using the QIAamp RNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions. Contaminating genomic DNA was removed by on-column treatment of each sample with DNase I (Qiagen). Purity, concentration and integrity of total RNA were assessed using two independent techniques. RNA purity and concentration were evaluated by absorbance readings using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). RNA quality was determined with an RNA 6000 nano LabChip Kit in the Agilent Bioanalyzer 2100 system. Quality was evaluated using the RNA Integrity Number (RIN) [31] .
The mean total RNA concentration was 96 ng/µL while A260/A280 and A260/230 ratios ranged from 1.99 to 2.04 and 2.02 to 2.16, respectively. Therefore all samples were pure, free from protein and organic pollutants derived from RNA extraction. The RIN obtained for all samples ranged from 7.2 to 8.2 with a mean value of 7.6.
Total RNA (500 ng) was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol in a final volume of 20 µL. The cDNA was subsequently stored at −20 °C. Pooled cDNAs were then used in preliminary experiments to evaluate primer performance and specificity and for PCR protocol optimization. Subsequently, the expression profile of the selected genes was analysed in each cDNA sample separately.
We selected 11 genes belonging to various functional classes and frequently used as references in qRT-PCR gene expression experiments: β-actin (ACTB); tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); hypoxanthine phosphoribosyl-transferase I (HPRT); transferrin receptor (TFRC); succinate dehydrogenase complex, subunit A (SDHA); β-2-microglobulin (β2M); phosphoglycerate kinase I (PGK1); glyceraldehyde-3phosphate dehydrogenase (GAPDH); glucose-6-phosphate dehydrogenase (G6PD); ribosomal protein L19 (RPL19); and glycophorin C (GYPC). Specifically, GYPC was included into the panel because of its expression in the erythroid lineage and because it was used in a previous study as normalizer to quantify the expression of erythroid genes in sheep blood [11] .
Primers for PGK1, SDHA and G6PD were based on previous publications [5, 7] . The other primers were designed using Primer3 software [32] by aligning ovine sequences available in GenBank with bovine and human homologous genes. Primers were selected to produce amplicons spanning two exons and their specificity was tested using ovine pooled cDNA and genomic DNA in preliminary PCR assays. The PCR products were subsequently run on 2% agarose gel to check for size specificity and, eventually, sequenced. Table 3 summarises primers information including sequences, product size, putative exon position, estimated size of the amplicon, efficiency of RT-PCR (E) and correlation coefficients (R 2 ). 
All PCR reactions were performed in a 25-µL final volume containing 2× Brilliant II SYBR Green Master Mix (Stratagene), 300-900 nM of each specific primer and 1 µL of cDNA. PCR amplification was run on a Mx 3005P QPCR System (Stratagene) using 96-well optical plates under the following conditions: 10 min at 95 °C for polymerase activation, and 40 cycles of 3-segment amplification with 30 s at 95 °C (for denaturation), 30 s at 56-60 °C, and 40 s at 72 °C for elongation. Primer concentration and annealing temperatures were optimised to individual genes; specifications are available from the Authors upon request. A dissociation step was added after elongation to ensure that the desired amplicon was detected. The dissociation step eliminates non-specific fluorescence signal and ensures accurate quantification of the desired product. Finally, a melting curve was produced to confirm single gene-specific peaks and to detect primer/dimer formation by heating samples from 60 to 95 °C. PCR efficiencies were calculated using a relative standard curve derived from a pooled cDNA mixture (a 10-fold dilution series with five measuring points). All experiments were replicated twice for each gene with triplicate sample runs within each replication and a no-template control was included using water instead of cDNA.
qRT-PCR data were analysed for reference genes expression stability using two different statistical algorithms: geNorm version 3.5 [1] and NormFinder version 0.953 [16] according to the developers' recommendations. Raw quantification cycle (C t ) values were converted to relative quantities using the comparative C t method as input data for the two applets. Preliminary analyses performed separately on qRT-PCR data from disease-stressed animal sampled at different time points and under different environmental temperatures (August and September) retrieved consistent results. Therefore C t values were averaged after inter-run calibration according to Hellemans et al. [33] and submitted to subsequent data analysis. The combined analysis was performed processing samples from ten randomly chosen control sheep together with the disease-stressed sheep and then joining expression results in a combined dataset.
A number of studies have been carried out to identify reliable reference genes in specific tissues in various species [34] [35] [36] [37] [38] . In sheep, analyses of expression stability of candidate reference genes are limited to a restricted range of tissues [5] [6] [7] [8] and data on ovine whole blood were still lacking. However, peripheral whole blood is attractive because of its accessibility and usefulness in monitoring several physiological and pathological conditions. As regards disease status, blood certainly represents the best tissue for in vivo test development since collection is non-invasive and easy to perform. For example, the identification of differentially expressed genes acting as indirect in vivo markers in blood would represent a major breakthrough for the diagnostics of non-conventional agents (like prions) which currently cannot be detected by standard methods as the diagnosis still rely on post mortem investigations. This study provides a panel of optimal control genes for use in qRT-PCR studies in sheep whole blood. The two softwares tested, based on different algorithms and analytical procedures, produced highly comparable results. SDHA and YWHAZ represent good reference genes for gene expression studies in sheep peripheral whole blood, unaffected by disease status and heat stress conditions, and the geometric mean of these two stable genes is an accurate normalization factor [1] . Our results may be useful for the identification of genes differentially expressed in a readily accessible tissue, with the potential of discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci (eQTLs) [39, 40] . Normal variation in thermal radiated temperature in cattle: implications for foot-and-mouth disease detection BACKGROUND: Thermal imagers have been used in a number of disciplines to record animal surface temperatures and as a result detect temperature distributions and abnormalities requiring a particular course of action. Some work, with animals infected with foot-and-mouth disease virus, has suggested that the technique might be used to identify animals in the early stages of disease. In this study, images of 19 healthy cattle have been taken over an extended period to determine hoof and especially coronary band temperatures (a common site for the development of FMD lesions) and eye temperatures (as a surrogate for core body temperature) and to examine how these vary with time and ambient conditions. RESULTS: The results showed that under UK conditions an animal's hoof temperature varied from 10°C to 36°C and was primarily influenced by the ambient temperature and the animal's activity immediately prior to measurement. Eye temperatures were not affected by ambient temperature and are a useful indicator of core body temperature. CONCLUSIONS: Given the variation in temperature of the hooves of normal animals under various environmental conditions the use of a single threshold hoof temperature will be at best a modest predictive indicator of early FMD, even if ambient temperature is factored into the evaluation. Foot-and-mouth disease (FMD) is a highly infectious viral disease of cloven-hoofed animals, both domestic and wild. The disease is caused by a small RNA virus, which is 28 nm in diameter and exists as seven serotypes. The disease is characterised by fever, and blisters in the mouth, on the feet and on the teats and these rupture and are associated with slobbering and lameness. Adult animals may suffer weight loss and milk production can decline significantly. Though most animals eventually recover from FMD, the disease can lead to myocarditis and death, especially in newborn animals [1] . FMD is found regularly in parts of South America, Africa, the Middle East and other parts of Asia and periodically spreads to affect normally disease free countries. It is a significant impediment to trade in livestock and their products as countries with the disease face restrictions for exporting to disease free regions. Moreover, the disease is difficult and costly to control and eradicate. The Royal Society [2] estimated that during the 2001 epidemic in the UK, in which some six million animals were culled, the losses to agriculture and the food chain were £3.1 billion and some £2.5 billion was paid by the UK Government in compensation for slaughtered animals and clean-up costs. Losses were also experienced in tourism and business directly affected by tourism; it has been estimated these were between £2.7 and £3.2 billion [3] . Two other epidemics highlight the global impact of the disease; the first a major epidemic in Argentina in 2001 and the second in Japan during 2010; in the first two thousand five hundred and nineteen herds were infected [4] and in the second two hundred and fifty (Office international des épizooties-World Organisation for Animal Health, 2010. Follow-up report Early identification of animals infected with FMD virus is vital if disease outbreaks are to be rapidly diagnosed and controlled. Thorough screening to identify signs of FMD is time consuming and labour intensive since it requires the capture and restraint of suspect animals for clinical examination. This can be particularly difficult in some situations, for example where animals are at pasture, are difficult to handle or are present in very large numbers. Animals with FMD often develop a fever with temperatures in excess of 40°C and vesicular lesions around the coronary band, in and around the mouth and on the mammary gland. The vesicular lesions are associated with local inflammation giving rise to an increase in skin temperature which can be detected by palpation [1] . On their own, these temperature changes are not pathognomonic for FMD but can be used to select animals that warrant closer examination to detect more definitive signs and/or enable sampling for confirmatory testing.
Infrared thermography (IRT) can be used to measure the heat emitted from a surface and to display and store an image and associated data. The technique has been used by the medical profession over recent years across a range of human conditions, to identify local inflammations or pyrexia [5] and in the detection of fever associated with SARS and avian influenza [6] . IRT has also been used by those involved with animal disease [7] [8] [9] . Workers at the Pirbright Laboratory of the Institute for Animal Health (IAH-Pirbright) and at the Plum Island Animal Disease Center (PIADC), USA have reported that IRT can be used to measure the temperatures of animals that need to be checked for possible onset of FMD [10] [11] [12] . These workers studied groups of animals with experimentally-induced FMD and measured temperatures (primarily around the coronary band) as disease progressed. It was found that increases in temperature associated with FMD could be detected, sometimes prior to the development of visible lesions. Unpublished work by the current authors involving five cattle, five sheep and five pigs infected with the Asia 1 strain of FMDV discovered that it was easy to measure the feet temperatures of the animals and established that there was potential for using the technique in the field. Cattle feet temperatures ranged from 18.7°C to 31.7°C, with the highest value being recorded the day before foot lesions were visible, but at the same time as the first lesion on the tongue. Prior to the first appearance of lesions temperatures were below 27°C.
To optimise interpretation of temperature measurements and to demonstrate the reliability of the technique to differentiate between infected and healthy livestock requires further IRT data from uninfected animals, kept at different ambient temperatures and under different husbandry conditions. This shortcoming is addressed here by IRT measurements and analysis from healthy cattle.
The experimental period was divided into two phases. The first phase was designed to make observations under different IRT/animal configurations and environmental conditions, the second to examine the changes in an animal's hoof temperature over a daily cycle of activity.
In the first phase, five separate sets of temperature data were taken over a period of five months using a TIR1 imager manufactured by Fluke (temperature range -20°C to 100°C, accuracy +/-2°C, operated at a distance of 1 to 2 m, emissivity 0.95). Two groups of nine and ten cattle initially aged 12 and 3 months old respectively, were housed in small groups in pens in an open barn at the Institute for Animal Health farm at Compton, Newbury; one half of each pen had a concrete floor and the other a slightly raised straw filled area.
Each animal in turn was restrained either by hand or in an animal crush and four IRT measurements were taken of each of the animal's feet from different aspects (front, back, lateral and medial) and a measurement was also taken of the left eye (see Figure 1 for an example). These two sites were selected because, as mentioned above, researchers working on FMD had detected an increase in temperature around the coronary band and it is hypothesised that temperatures around the eye provide a non-invasive indicator of an animal's core temperature. Other sites commonly affected by FMD lesions such as the mouth and udder are less accessible and/or only applicable to lactating animals. To investigate the link between eye temperature and body temperature each animal's rectal temperature was taken at the same time as the thermal images using a digital thermometer. The IRT images were taken twice within ten minutes from each animal to evaluate repeatability of the measurements and correct for minor variations in the angle of the imager to the animal. Ambient temperatures were measured with a Fisher Scientific model FB70357 digital thermometer.
Care was taken throughout the experiment when handling the cattle, as it was appreciated that even the simple act of gathering animals can cause an increase in stress which in turn may result in an increase in the animal's temperature.
As it was not practical to measure changes in an animal's hoof temperature over an extended period of activity using an IRT imager, a second temperature measuring device was used for this purpose (IButton data loggers, type DS1921G, temperature range -40°C to 70°C, accuracy +/-1°C, data recording rate every second or every two seconds, manufactured by Embedded Data Systems). The IButtons were strapped to the animals' hooves as shown in Figure 1 . To compare the results of IRT and IButtons, hoof temperatures were measured for two cattle. TIR1 images were taken either immediately prior to IButton attachment, simultaneously with attachment, but for another foot, or immediately after the IButton was removed. Identical readings from the two instruments were not expected as both devices measure temperature in different ways (TIR1-radiative and IButton-thermal contact). However, it was anticipated that similar trends could be detected using both sensors.
The final phase of the work was to investigate changes of hoof temperature as a function of activity. These were established using IButtons and a video security camera (Solidex Day Night DomeCam Varifocal Lens combined with a Solidex 4 channel DVR) placed above the pen holding two of the cattle (chosen for ease of visual recognition). IButtons were attached to the two hind feet of the cattle and data recorded at a frequency of once or twice per minute for a period of approximately twenty hours. Air temperatures were recorded using an IButton suspended in free air close to the animal pen. The experiment was done twice. TIR1 images were processed using Smart View Software (V2.1.0.10), supplied by Fluke. For each image, an area of approximately 2 cm 2 above and below the coronary band was selected and the maximum temperature within this area (see Figure 1 ) was recorded and transcribed to an Excel spreadsheet for subsequent statistical analysis. Additionally, an area at least 10 cm above the hoof was selected to determine whether a ratio between hoof and more proximal leg surface temperature could help compensate for hoof temperature changes caused by ambient temperature changes. An area of approximately 2 cm 2 around the eye was selected for analysis and the hottest temperature within this area, including the eye itself, was recorded (see Figure 1 ).
For comparing IButtons and IRT, the area covered by the attached IButton was selected and the average temperature within this area recorded and further analysed in an Excel spreadsheet. IButton data was analysed with TempIT software supplied by Signatrol (Version 4.1.8) and the data transferred to the master Excel spreadsheet. To determine the animal's movements the security camera images were replayed and activity allocated into one of four categories (lying down, standing on deep straw, standing on concrete and outside of the holding pen). The date and time for each change in activity category was recorded for comparison with the IButton data.
Two separate analyses of the data were carried out to assess: (i) the repeatability of thermal image measurements taken sequentially within a ten minute interval; and (ii) the potential for defining a threshold temperature above which cattle would be considered abnormal based on IRT.
The repeatability of thermography was assessed by computing the difference in temperature as measured by corresponding images (i.e. for the same hoof with the same aspect on the same day) for each animal and determining whether the median differed significantly (P < 0.05) from zero using a Wilcoxon signed rank test.
The potential for defining a threshold temperature to identify unhealthy cattle based on IRT was examined using a Bayesian hierarchical model, which incorporates between-animal variation and facilitates predictions outside the data which allow for parameter uncertainty. In this approach, the observed hoof temperature (T jk ) for the jth observation on animal k was described by,
jk is the expected hoof temperature, σ 2 e is the error variance, the b (k) i s are parameters and X ijk is the value of the ith factor (e.g. hoof, aspect or ambient temperature) for the jth observation on animal k. Betweenanimal variation was modelled by assuming that the parameters for each animal are drawn from higherorder distributions, such that,
Non-informative priors were used for the higher-order parameters: diffuse Normal distributions for the μ b is and diffuse gamma distributions for the σ b is. Parameters in the model were estimated using Markov chain-Monte Carlo methods implemented in WinBUGS [13] . Two chains of 50,000 iterations were run for each model, with the first 10,000 iterations discarded to allow for burn-in of the chain. Each chain was then thinned by sampling every tenth iteration to reduce autocorrelation amongst the samples.
The fits of different models were compared using the deviance information criterion (DIC) [14] . Posterior predictions for the expected hoof temperature as a function of ambient temperature were generated by sampling from the joint posterior density for the higher-order parameters. A range of percentiles of the resulting distribution were used to define thresholds for identifying abnormal animals and the specificity of a classification scheme based on these thresholds (essentially the proportion of animals below the threshold) was assessed.
In the first phase of the experiment, between July and November 2009, around two thousand three hundred thermal images of cattle hooves were taken. During these experiments, ambient temperatures ranged from 10°C to 24.8°C and general weather conditions from a warm summer's day through to cold and damp winter conditions. Hoof temperatures measured by IRT ranged from approximately 10°C to 36°C (Figure 2a ) and depended markedly on ambient temperature (Figures 2d &3) . Furthermore, the variability in hoof temperatures was greatest at lower ambient temperatures (Figure 2d ). The median range in hoof temperatures for individual animals on a given day was approximately 6°C, but in some cases it was > 12°C (Figure 3 ). This range often reflected one hoof or side being markedly warmer than the other (for example, animals 362, 762, 766 and 769), but sometimes there was no clear explanation for the difference (for example, animals 763 and 777).
Differences in hoof temperature between corresponding images recorded on the same day were typically small ( Figure 2b ) and did not differ significantly (P > 0.05) from zero for 13 (out of 19) animals. For six animals (animals 379, 762, 766, 768, 769 and 774), the median difference in repeated observations was significantly (P < 0.05) different from zero, though the median difference in each case was only a fraction of a degree (range: -0.3°C to 0.2°C). Eye temperature measured by IRT provided a reasonable proxy measure for body temperature, with eye temperatures approximately 2°C lower than rectal temperature ( Figure 2c) and not significantly affected by ambient temperature (P > 0.05).
An adequate model to describe the hoof temperature data included ambient temperature (°C), hoof (coded as: front-left, front-right, hind-left and hind-right) and camera aspect (coded as: front, lateral, medial and rear) ( Table 1) ; removing any of these terms from the model significantly worsened model fit (full model: DIC = 17270.6; removing ambient temperature: DIC = 18846.8; removing hoof: DIC = 17309.8; removing aspect: DIC = 17297.2). Adding extra terms to the model improved the model fit, often markedly so; for example, a quadratic term for ambient temperature (DIC = 14730.3) or eye temperature as a normalising factor (DIC = 16648.1). However, this was at the expense of the resulting model being poor as a predictive tool, because the variance for the higher-order parameters needed to be so large to incorporate the observed differences amongst animals. Accordingly, the adequate model was used in subsequent analyses.
The analysis indicated that there was variation in temperature amongst hooves on the same animal, but these differences were not systematic between animals, as evidenced by means for the hoof parameters which do not differ significantly from zero, but which have a high standard deviation (Table 1) . Camera aspect did influence hoof temperature measurement, with images taken from the lateral, medial or rear aspect being around 1°C lower than those taken from a front aspect (Table 1) . However, ambient temperature had the greatest impact on hoof temperature (Figures 2d &3; Table 1 ).
By sampling from the joint posterior density for the higher-order model parameters (and integrating out the effects of hoof and camera aspect) it was possible to generate predictions for hoof temperature as a function of ambient temperature. The 75th, 90th and 95th percentiles for these predictions were then used to define thresholds by which to identify healthy cattle, with a further refinement that the maximum threshold temperature was set equal to the mean rectal temperature for the animals (38.3°C) (Figure 4a ; Table 2 ). The specificity of a classification scheme based on these thresholds was investigated. For a threshold based on the 75th percentile, the predicted specificity was low, especially at ambient temperatures below 20°C (< 80% specificity, Figure 4b ). The specificity was improved by setting a threshold based on the 90th or 95th percentile with specificity > 90% predicted above temperatures of 15°C and 10°C respectively (Figure 4c, d) .
A simple comparison between the TIR1 and three IButtons, on a shaded uniform temperature carpet tiled floor revealed that both instruments recorded similar temperatures with the TIR1 being warmer than the IButton by 0.1 to 1.4°C (IButton no./TIR1/IButton: 1/24.3/23.0; 2/24.3/ 22.9; 2/23.6/22.9; 3/24.3/23.5; 23.6/23.5°C). It was also established that the IButtons, given a sudden temperature change of 15°C took fifteen minutes to reach equilibrium. Table 3 presents the results from the comparison between the IButton and TIR1 for two animals on two separate days. The data show that temperature measured by the IButton approximately fifteen minutes after attachment and just before removal relates well to the average temperatures measured by the TIR1. The IButton temperatures were consistently warmer than the average temperature measured by TIR1 (average temperature differences 5.3, 5.8, 4.4 and 1.9°C). This trend was observed for all data collected during the comparison of the IButton and TIR1. The IButton as well as the TIR1 record sudden temperature changes equally well as seen on one occasion where the average temperatures for all feet for one animal measured by the TIR1 were 10.2/11.2/12.3 and 9.6°C before IButton attachment; whereas after the removal of the IButton average temperatures for all legs measured by the TIR1 were only very slightly raised (0.1-1.3°C) apart for one foot where the temperature was raised by 14.7°C. The IButton recorded 14°C and 15°C for three out of the four legs at fifteen minutes after attachment and before removal, but recorded temperatures of 18°C after fifteen minutes and 29.5°C before removal for the leg with the raised temperature (data not shown).
The extended measurement period using both the IButton and security surveillance camera showed that the animals' hoof temperatures varied by as much as 20°C depending upon a combination of activity and ambient air temperature. A typical analysis is given at Figure  5 where it can be seen that when the animal was standing on the concrete temperatures were much lower than when it was lying down in the straw with its feet tucked under its body. This effect was consistent in each of the animals whose temperatures were measured. Maximum temperatures of 38°C were recorded and this was very close to the animal's rectal temperature.
To detect inflammatory conditions such as FMD affecting cattle feet, IRT needs to be able to identify abnormal surface temperature elevations. This raises the challenge of being able to distinguish such elevations from the spectrum of variability found in uninfected animals. Similar challenges affect the use of the technique in screening human subjects, for instance for pyrexia at airports [6] . Two approaches can be envisaged for FMD. First, IRT could prove very useful if a threshold temperature were to be established above which a foot temperature triggers a suspicion of an inflammatory condition. This approach has been suggested by Rainwater-Lovett [12] . However, the current study shows that this technique may be too simplistic in its approach as an animal's hoof temperature is significantly affected by ambient temperature and posture/activity. Thermal image data reported by Bashiruddin [11] from FMDV-infected cattle were compared with the thresholds shown in Table 2 to determine if these thresholds could provide a basis for early stages of FMD infection to be detected. At the ambient isolation facility temperature of~16°C, Table 2 suggests that hoof temperatures of 30.5°C (75 th percentile), 34.9°C (90 th percentile) and 37.6°C (95 th percentile) would indicate an elevated temperature indicative of infection. However, of the five animals which became infected, only one showed a temperature above 30°C (two hooves) and this was when vesicular lesions were visible. Although definitive conclusions will require study of greater numbers of infected animals, these results suggest that the threshold temperatures determined in the present study will result in a low sensitivity, unless specificity is reduced. An alternative approach is for the operator to use IRT to identify hot-spots. These are identified as either part or all of a hoof that is hotter than the surrounding skin or hotter than other feet. In this approach, it is relative rather than absolute temperatures that matter. Previous studies [11, 12] have demonstrated that areas of raised temperature on an animal's hoof can be detected. To investigate this approach, a "blind test" was conducted using forty four thermal images from six cattle either before infection or in the early stages of FMD [11] . One of the authors was invited to categorise the images as "not a concern", "unlikely to be infected", "possibly infected", "suspicious" or "highly suspicious". Once an animal displayed clinical signs evident upon close physical examination, it was considered infected and it was excluded from further analysis the day afterwards, since temperatures of the feet often decline within a day or two of the formation of vesicles even if ruptured lesions remain evident. The results from this pilot revealed a 70% sensitivity (7 out of 10 images) and 79% specificity (scoring possibly infected and above as positive) (27out of 34 images) or 30% sensitivity (3 out of 10 images) and 94% specificity (scoring suspicious and above as positive) (32 out of 34 images). Whilst these results are encouraging, further work using images collected from a larger number of infected animals is needed before a conclusion can be reached concerning the merits of this approach.
This study has been completed under ideal field conditions. The situation in the field is likely to be less favourable. For example the animal's feet may be wet, covered in grass, muddy or covered in faeces. These variables need to be studied in more detail before IRT can be used with confidence to detect FMD in the field. Other parts of the body affected by inflammation in FMD, such as the mouth are not readily visualised by an infrared camera, whilst changes in the udder are limited in application to female dairy breeds. The use of IRT eye measurements seems a promising method to measure body temperature and therefore merits further evaluation in animals affected with FMD and other pyrexic conditions. If IRT technology is to be useful in the field it has to be both technically capable of distinguishing between infected and non infected animals and be a cost effective diagnostic tool. In the field two scenarios are likely; the first where animals are housed or can be easily corralled and are readily accessible at close range and the second where they are at pasture and less easy to gather. In the first instance the current cost of an IRT camera will be in the range £2-10 k but in the second, where the equipment is required to operate at longer ranges, it is likely that a more powerful telephoto lens would be required. The cost of this significantly increases the price of the equipment possibly up to £20 k.
The study has identified that an animal's hoof temperature is influenced by its activity prior to the point at which thermal screening is performed. Consequently, a period of acclimatisation is required prior to an image being taken. This is particularly the case if the animal has been lying down with its feet tucked under its body.
The work has shown that IRT images of an animal's eye temperature may be a useful proxy for core temperature and could be used to detect pyrexia as an indicator for selecting animals for closer examination. This conclusion supports the observation by Dunbar [10] who compared high quality thermograms of the eye (n = 16) to body temperature and found them not to be different (p = 0.19). However, further work is required with animals infected with FMDV to confirm this. Cochrane Systematic Reviews of Chinese Herbal Medicines: An Overview OBJECTIVES: Our study had two objectives: a) to systematically identify all existing systematic reviews of Chinese herbal medicines (CHM) published in Cochrane Library; b) to assess the methodological quality of included reviews. METHODOLOGY/PRINCIPAL FINDINGS: We performed a systematic search of the Cochrane Database of Systematic Reviews (CDSR, Issue 5, 2010) to identify all reviews of CHM. A total of fifty-eight reviews were eligible for our study. Twenty-one of the included reviews had at least one Traditional Chinese Medicine (TCM) practitioner as its co-author. 7 reviews didn't include any primary study, the remaining reviews (n = 51) included a median of 9 studies and 936 participants. 50% of reviews were last assessed as up-to-date prior to 2008. The questions addressed by 39 reviews were broad in scope, in which 9 reviews combined studies with different herbal medicines. For OQAQ, the mean of overall quality score (item 10) was 5.05 (95% CI; 4.58-5.52). All reviews assessed the methodological quality of primary studies, 16% of included primary studies used adequate sequence generation and 7% used adequate allocation concealment. Of the 51 nonempty reviews, 23 reviews were reported as being inconclusive, while 27 concluded that there might be benefit of CHM, which was limited by the poor quality or inadequate quantity of included studies. 58 reviews reported searching a median of seven electronic databases, while 10 reviews did not search any Chinese database. CONCLUSIONS: Now CDSR has included large numbers of CHM reviews, our study identified some areas which could be improved, such as almost half of included reviews did not have the participation of TCM practitioners and were not up-to-date according to Cochrane criteria, some reviews pooled the results of different herbal medicines and ignored the searching of Chinese databases. Traditional Chinese Medicine (TCM) is an essential part of the healthcare system in several Asian countries, and is considered a complementary or alternative medical system in most Western countries [1] . Chinese herbal medicines (CHM) are an essential part of TCM [2] . The 2002 National Health Interview Survey showed that 18.6% of adults used CHM in the United States, while it was 12.1% in 1997 [3] . With the increased use of CHM, questions arise from clinicians, patients, and policymakers as to the effectiveness of these interventions [4] . In an era of evidence-based healthcare, systematic reviews of randomized controlled trials (RCTs) are becoming increasingly important as a source of evidence for decision-making. As the number of systematic reviews of CHM increase, the quality of which has been highlighted and called into question. Some studies have assessed the quality of CHM reviews published in Chinese journals, in general, they have been criticized for lacking a comprehensive search for clinical trials, ignoring the characteristics of TCM, using inappropriate criteria to assess the methodological quality of included studies, and addressing too broadly defined questions [5] [6] [7] . All [8] these aspects could have contributed to a poor quality review.
The Cochrane Collaboration is an international organization that aims to prepare and maintain rigorous systematic reviews in order to help people make well-informed decisions about health care [8] . Compared with reviews published in paper-based journals, Cochrane reviews are noted to have greater methodological quality [9] . Ever since 1999 when the first Cochrane review of CHM was published, a sharp increase has been observed in the number of similar reviews. However, no previous studies have systematically assessed the methodological quality of Cochrane reviews of CHM. Therefore, we did this overview of systematic reviews. Our study had two objectives: a) to systematically identify all existing Cochrane reviews of CHM; b) to assess the methodological quality of included reviews.
Data for this study was acquired through previously published work, no patient or hospital data was accessed. Therefore, written consent and institutional ethical review was not required for this research. The PRISMA checklist and flow diagram are available as supporting information; see PRISMA Checklist S1 and PRISMA Flow Diagram S1.
In order to identify reviews focusing on CHM, we searched the titles and abstracts of all reviews contained within the Cochrane Database of Systematic Reviews (CDSR) (Issue 5, 2010) using the following terms: Chinese or herb* or traditional or plant or medic*.
We included all Cochrane reviews of CHM. Protocols and reviews which have been withdrawn from publication were excluded. We defined CHM as preparations derived from plants or parts of plants (e.g. leaves, stems, buds, flowers, roots or tubers) that grow in China and have been widely used for medical purpose. CHM include single herbs (or extracts from single herbs) and compound formulas of several herbs in all forms of preparation formulation (e.g. oral liquid, tablet, capsule, pill, powder, plaster or injection liquid). It should be noted that our definition of CHM does not include plant-derived chemicals or synthetic chemicals which contain constituents of plants. For example, although Huperzia serrata has its origin in China, according to our definition, Huperzine A does not belong to CHM because it is a kind of alkaloid extracted from Huperzia serrata. In addition, we only included reviews discussing herbs which originated from China, reviews on herbs such as Passiflora and Echinacea, both of American origin, were invariably excluded.
Oxman-Guyatt Overview Quality Assessment Questionnaire (OQAQ) [10] The OQAQ instrument was selected as the quality appraisal tool, which was designed to evaluate whether the authors of a systematic review conducted a comprehensive search, minimized bias in the selection of primary studies, evaluated the primary literature, and pooled the results appropriately. It consists of 10 questions, the first 9 questions are designed to assess different aspects of methodological quality and have set answers of ''yes'', ''partially/can't tell'', or ''no'', question 10 is an assessment of the overall scientific quality of the systematic review on a scale of 1 to 7, it is answered based on how well the review scored on the first 9 questions.
We established a database (using Microsoft Excel 2007) to extract data. The database had two components: 1) general characteristics, including country of first author and number of authors, whether the review had the participation of TCM practitioners, number of trials and participants included, disease, the year of review last assessed as up-to-date, conclusions drawn by the reviewers (by assessing the reviewers' abstract conclusions statements), interventions in experimental groups, number of herbs included, and whether the results of different herbal medicines were pooled; 2) methodological quality of included reviews, including OQAQ scale, the approach to assessment of methodological quality of primary studies, the number of trials with adequate sequence generation and allocation concealment, and type and number of English and Chinese databases searched. Two reviewers (Jing Hu and Wei Zhao) independently extracted the information of each review, disagreements between the two reviewers were resolved by discussion.
The questions addressed by a review may be broad or narrow in scope, each review was assigned into one of the following two categories: 1) narrowly focused reviews, intervention in each review was single herb or herbal preparation, as an example of ''Chinese herbal medicine suxiao jiuxin wan for angina pectoris''; 2) broadly focused reviews, including reviews concerned multiple Chinese herbs or a family of herbal medicines sharing similar efficacy, such as ''Chinese herbal medicine for premenstrual syndrome'' and ''Chinese herbal medicine Huangqi type formulations for nephrotic syndrome''. For broadly focused reviews, we listed the number of herbs included and assessed whether the results of different herbal medicines were pooled.
A review was believed to have the participation of TCM practitioners if at least one author works in TCM department, university or hospital, or it stated that it had got suggestion from TCM practitioners.
When we assessed the type and number of English and Chinese databases searched, we only listed the databases which at least 4 reviews searched. In addition, the Cochrane Specialized Register and databases/websites for ongoing trials were also searched in some reviews, we did not list them in our study.
278 potentially relevant reviews were obtained, after selection (according to inclusion and exclusion criteria), a total of 58 Cochrane reviews were eligible for our study, a full list of reviews is included in Table S1 . Of the 58 reviews, one review [49] included herbs originated in China, India and Japan; interventions in another review [55] concerned both herbal and chemical medicines. In these two cases, we extracted and analyzed the information relating to CHM.
The number of authors in the 58 reviews ranged from 1 to 10, the first authors were most often from China (46 [79%]), followed by UK (n = 8), and Netherlands, Canada, USA and Australia each have one first-authored review. Twenty-one (36%) of the included reviews had at least one TCM practitioner as its co-author. 7 (12%) reviews didn't include any primary study, of the remaining reviews (n = 51), a total of 671 studies and 75,609 participants were included, the median number of studies and participants included were 9 (Quartile: 3, 15) and 936 (Quartile: 492, 1567) respectively. 50% of the reviews were last assessed as up-to-date prior to 2008, of reviews considered out-of-date, one was last updated in 2000. In total, 44 diseases were investigated in the included reviews, 18 (31%) reviews addressed cerebral vascular and cardiovascular diseases (9 reviews focused on stroke), followed by reviews focused on respiratory diseases (n = 6) and gynecological/pregnancy diseases (n = 6).
Of the 51 nonempty reviews, only one review concluded positively, 27 (53%) concluded that there might be benefit of CHM for treating specific health conditions, which was limited by the poor quality or inadequate quantity of studies, 23 (45%) reviews concluded that the currently available data do not allow any conclusion to be drawn, generally because of low methodological quality of studies, small number of studies and participants included or publication bias.
Nineteen reviews focused on 13 single herbs or herbal preparations, while the remainder (39 [67%]) addressed broad questions, in which 34 reviews concerned multiple Chinese herbs or multiple formulations of Chinese herbs, 5 reviews involved a family of herbal medicines sharing similar efficacy, including Huangqi type formulations (including Huangqi injection and Huangqi-Danggui mixture), Chuanxiong preparations (including Nao-an capsule, Xifeng wan and Apoplexy Preventing Dry Ointment Powder), Dan Shen agents (including Compound Danshen Dripping Pill, Compound Danshen injection, Danshen injection, Yiqi huoxue injection, and Quyu huatan xiezhuo fang) and Sanchi (including Xinnaotai, Sanchitongshu capsule, Naoming injection, Xuesaitong soft capsule, Sanqitongshu capsule, Xuesaitong and Xueshuantong injection). Of the 39 reviews, 4 didn't include any primary study, of the remaining reviews (n = 35), the median number of herbal medicines involved was 6 within a range of 1 to 71, results of different herbal medicines were pooled in 9 reviews, in which 7 reviews pooled the results of all Chinese herbal medicines, one review pooled all Danshen agents, and one pooled Sanchi. Table 1 presents a summary of OQAQ items of the included reviews, the mean score (item 10) was 5.05, 95% CI (4.58, 5.52). 41 of 58 reviews attempted to minimize bias during the selection of studies by at least two reviewers independently select eligible studies.
All reviews reported assessing the methodological quality of included primary studies, 21 (36%) reviews used the Cochrane Collaboration's 'Risk of Bias' tool, the Jadad scale was used in 6 reviews, 12 (21%) reviews used unnamed checklist. Among 671 included studies, 108 (16%) used adequate sequence generation, allocation concealment was adequate in 50 (7%) studies.
The median number of databases searched in 58 reviews was 7 within a range of 4 to 15. Regarding to the English language databases, the most searched was MEDLINE (98%), followed by EMBASE (97%) and CENTRAL (97%). CBM was the most searched Chinese database (78%), the second most used was CNKI (45%) and the third was VIP (24%) ( Table 2 ). All reviews searched at least 2 English databases, while 10 reviews did not search any Chinese database. 41 (71%) reviews searched at least 3 English databases, while only 6 (10%) reviews searched at least 3 Chinese databases (Table 3 ).
Evidence-based health care involves the systematic collection, synthesis and application of scientific evidence to guide clinical practice and policy-making. Systematic reviews are a key component of evidence-based health care. Currently, CDSR has included 58 systematic reviews of CHM. Almost seventy percent of included reviews' topics were too broad, the percentage was much higher than that of similar reviews published in Chinese journals, which was 38 percent (41 among 107 reviews) [69] . It is difficult to develop a comprehensive search strategy for broadly focused reviews, for instance, one hundred and sixty herbal medicines are now available for coronary heart diseases treatment, a systematic review of CHM for coronary heart disease will have to include clinical trials of all these herbal medicines, it is easily to cause the incomplete identification of relevant studies. Choosing broad topics for reviews will require more resources in data collection and analysis, the results may also be too complicated to interpret. So topic selection of CHM reviews should focus on specific clinical problems, the extensive titles are not recommended.
As broad questions of reviews may be addressed by large sets of heterogeneous studies, the data synthesis may be particularly challenging. In our study, 9 reviews (among the 39 broadly focused reviews) pooled the results of different herbs, which did not identify potentially important differences in effects across different interventions. Systematic reviews can, but do not have to use meta-analysis when combining data from primary studies, prior to conducting a meta-analysis, reviewers should examine the consistency of the interventions. It is recommended that the data of each intervention should be analyzed and presented separately if several different interventions for the same condition were tested in one review.
The goal of a systematic review is to identify relevant studies completely and unbiasedly [70] . It has been demonstrated [71, 72] that significant amounts of evidence would potentially be missed if the search is limited to English-only sources. Because a considerable number of clinical trials on CHM were published only in the Chinese language journals, so a comprehensive search of Chinese databases is essential for a systematic review of CHM. However, we were disappointed to find that almost twenty percent of reviews did not search any Chinese database in our study. One study [73] compared four Chinese databases and concluded that CBM is the preferred database for systematic reviewers to retrieve relevant Chinese studies, while CNKI is recommended for non-Chinese-speaking researchers due to its free searched English version website (www.global.cnki.net) and ''Cross-Language Search'' functions. CBM has no English website and a fee is charged for searching, now many Chinese medical universities have got the permission to search CBM, so maybe the most costand time-efficient way to search it is to enhance collaboration with Chinese researchers.
In doing a review of CHM, professional advice from TCM practitioners is of great value. It is generally assumed that the characteristics of TCM would be well taken into account in a review if one or some of its reviewers majored in TCM. In our study, we found that more than sixty percent of reviews did not have one TCM practitioner in the authors list, which might lead to insufficient consideration of the characteristics of TCM (e.g. determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs) and incorrect results. We suggest that future reviews should be authored by a group of individuals with both clinical expertise and methodological expertise.
Of the 671 primary studies included, less than twenty percent of studies used adequate sequence generation, and only seven percent used adequate allocation concealment. Because of the poor methodological quality of primary studies, nearly half of reviews were reported as being inconclusive, while 27 reviews provided preliminary evidence of CHM's benefits to certain conditions, which should be considered tentative and need to be confirmed with rigorous RCTs. The Chinese government has been aware of the importance of conducting scientifically sound RCTs and has made substantial investments into funding clinical researches of CHM, now many well-designed RCTs of CHM with rigorous methodology are in progress or have been completed in China [74] , we believe future updates of currently inconclusive Cochrane reviews of CHM may reach more definitive conclusions.
Although we believed a review has a greater chance of considering the characteristics of TCM if at least one author works in TCM department, university or hospital, or it stated that it had got suggestion from TCM practitioners, it is quite possible that some reviewers had consulted TCM experts while designing and doing the review, but did not report it in articles. As cases of this kind could not be ruled out, we therefore might have underestimated the proportion of reviews getting support from TCM practitioners. In addition, we restricted our search to Cochrane reviews because they are generally less prone to bias than systematic reviews published in paper-based journals [9, 75, 76] . However, this might cause the results of this study to be only applicable to review articles in the Cochrane database. Further evaluation is needed in order to know whether the systematic reviews of CHM published in English paper-based journals even in the leading journals have the same problem.
Table S1 List of all included Cochrane reviews of CHM. (DOC) PRISMA Checklist S1
(DOC) PRISMA Flow Diagram S1 (DOC)  Current Status of the Immunomodulation and Immunomediated Therapeutic Strategies for Multiple Sclerosis Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, and CD4(+) T cells form the core immunopathogenic cascade leading to chronic inflammation. Traditionally, Th1 cells (interferon-γ-producing CD4(+) T cells) driven by interleukin 12 (IL12) were considered to be the encephalitogenic T cells in MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Currently, Th17 cells (Il17-producing CD4(+) T cells) are considered to play a fundamental role in the immunopathogenesis of EAE. This paper highlights the growing evidence that Th17 cells play the core role in the complex adaptive immunity of EAE/MS and discusses the roles of the associated immune cells and cytokines. These constitute the modern immunological basis for the development of novel clinical and preclinical immunomodulatory therapies for MS discussed in this paper. Multiple sclerosis (MS) was initially identified in 1868 by Charcot. This disease often begins in young adulthood with intermittent episodes of neurological dysfunction, including visual impairment, ataxia, motor and sensory deficits, and bowel and bladder incontinence. These are attributable to recurrent inflammatory attacks on the white matter of the brain and spinal cord, which lead to the accumulation of perivascularly distributed inflammatory cells within the brain and spinal cord white matter [1] . Beeton et al. first established an animal model of MS in the 1930s, when they immunized monkeys with a central nervous system (CNS) homogenate to induce what is now known as experimental autoimmune encephalomyelitis (EAE) [2] . Since this pilot animal study, EAE has become the most accepted animal model of MS. In recent decades, pathogenic hypotheses have been investigated and novel therapeutic agents tested in this model in the fields of CNS inflammation and demyelination. Therefore, EAE provides a valuable tool for the investigation of the T-cell-dependent pathogenesis of autoimmune inflammation in the CNS and the orchestration of the autoimmune demyelinating inflammation in the CNS of MS patients. Mice and/or genetically modified mice have also been of fundamental value in the exploration of the complex pathogenesis of MS [3, 4] . EAE is undoubtedly the best animal model in which to study autoimmune diseases and particularly the demyelinating diseases of the CNS, such as MS [5] .
Myelin basic protein-(MBP)-specific T cells isolated from the peripheral lymphocytes of human individuals with MS and encephalitogenic T cells recovered from circulating 2 Clinical and Developmental Immunology autoreactive T cells of either immunized or naïve animals have shown that autoreactive T-cell lines that recognize the encephalitogenic part of MBP in vitro can be distinguished from an unprimed rat T-cell population. This confirms that autoreactive T cells play a central role in the pathology of MS [6] [7] [8] . EAE can also be induced by adoptively transferring an expanded population of myelin-reactive encephalitogenic CD4 + (T helper [Th] ) cells, which allows the further dissection of the immunopathogenic potency of different encephalitogenic CD4 + cell populations [9] . In the 1990s, Mosmann and Coffman postulated that Th cells can be classified into two distinct subsets, Th1 and Th2. Th1 cells produce large quantities of interferon γ (IFNγ), driven by interleukin 12 (IL12), which promotes cellular immunity directed against intracellular pathogens. Alternatively, Th2 cells, which secrete IL4, IL5, IL13, and IL25, are essential in the destruction of extracellular parasites and the mediation of humoral immunity [10, 11] . Selfreactive Th1 clones derived in vitro are capable of adoptively transferring EAE to naïve recipients [12] . Increased levels of Th1 cytokines are particularly evident during EAE/MS relapse, whereas increased Th2 cytokines are found during remission in MS patients when compared with control levels [13] . Clinical and hematological symptoms are exacerbated in relapsing/remitting MS patients following the administration of IFNγ, and this is also observed in other Th1type diseases, whereas it is less apparent in Th2 diseases [14, 15] . Th1 cells were earlier thought to be pathogenic T cells, whereas Th2 cells were thought to confer an antiinflammatory potential, constituting protective T cells in both MS and EAE [16] [17] [18] [19] .
However, this clear-cut immunodysregulation of the Th1/Th2 balance in EAE and MS may be part of a hidden complex of interactions underlying EAE and MS [20] . The Th1-driven nature of the EAE/MS disease was challenged by the finding that IFNγ-and IFNγ-receptor-deficient mice, as well as mice that lack other molecules involved in Th1 differentiation, such as IL12p35, IL12 receptor β2 (IL12Rβ2), and IL18, were not protected from EAE, but instead were more susceptible to the disease [21] [22] [23] [24] [25] . Unexpectedly, mice deficient in IL12α (IL12p35), a component of the Th1 paradigm, are vulnerable to EAE. Similarly, IL12Rβ2deficient mice develop more severe clinical manifestations of EAE, whereas IL12p40-deficient mice are resistant to EAE [23, 24, 26] . These discrepancies and conflicting data indicate that an imbalance in the Th1/Th2 milieu cannot explain the overall immunopathogenic mechanisms underlying EAE and MS.
p19, a novel cytokine heavy-chain homologue of the IL6 subfamily, was discovered as a computational sequence [27] . When the p19 chain is linked to the p40 chain, a subunit of IL12 (another subunit of the IL12 heterodimers is the p35 chain), it forms a novel cytokine designated IL23. Therefore, the deletion of IL12p40 will affect the functions of both IL12 and IL23. Cua and colleagues verified that Il23 but not Il12 is essential for the induction of EAE by generating Il23p19 knockout (KO) mice and comparing them with IL12p35 KO mice [28] . Furthermore, an IL17-producing Tcell subset, driven and expanded by IL23, can pathogenically induce EAE when adoptively transferred into naïve wildtype mice [29, 30] . These IL17-producing T cells were dramatically reduced in the CNS of IL23p19-deficient mice. Based on these studies, researchers confidently suggested that IL17-producing CD4 + T cells are a distinct and novel Th subset that exacerbates autoimmunity, and designated them Th17 cells [31, 32] . Th17 cells are a Th-cell subset distinct from Th1 and Th2 cells in terms of their differentiation, expansion, and effector functions [33, 34] . The discovery of Th17 cells further clarifies the cytokine profile of MS [35] .
Recently, the levels of IL17 produced by MBP-stimulated peripheral blood cells obtained from MS patients or controls were shown to correlate with the active lesions in MS patients observed with magnetic resonance imaging (MRI) [36] . Like other Th subsets, the Th17 lineage is activated by a specific cytokine milieu. However, IL23 cannot produce Th 17 cells de novo from naïve T cells, and the IL23 receptor (IL23R) is not expressed on naïve T cells [37] . Transforming growth factor β (TGFβ) upregulates IL23R expression, thereby conferring responsiveness to IL23, which confirms that TGFβ is a critical cytokine in the commitment to Th17 expansion in vitro and in vivo [38] . In mice, TGFβ together with IL6 can activate antigen-responsive naïve CD4 + T cells to develop into Th17 cells [39] . In humans, naïve CD4 + cells exposed to IL6, TGFβ, and IL21 can develop into Th17 cells, and the production of IL23 plays a role in maintaining these Th17 cells [40, 41] . Altogether, Th17 cells require IL23, TGFβ, IL6, and IL1 for their generation. Th17 cells produce IL17A and IL17F, which are upregulated in chronic lesions [42] , and IL22, which is also involved in the pathogenesis of MS. Thus, Th17 cells are a recently discovered, unique Th lineage that produces a repertoire of signature cytokines, including IL17A, IL17F, IL21, and IL22, that are essential for the development of autoimmune diseases such as MS [43] .
The discovery of transcription factors that are key regulators of the cytokine expression required to launch lineagespecific transcriptional programs has greatly extended our understanding of Th-cell lineage commitment [44] . It has been shown that T-bet and STAT4 program the commitment of the Th1 lineage and Th1 cytokine production [45] , whereas GATA-binding protein 3 (GATA3) and STAT6 drive Th2 population expansion and Th2 cytokine production [46, 47] . The T-bet and STAT4 (necessary for Th1 differentiation) transcription factors are important in the differentiation of autoimmune T cells in the EAE model [48] , and T-betand STAT4-deficient mice are resistant to EAE. However, these transcription factors do not mediate the induction of Th17 cells. Instead, in a unique inductive milieu, Th17 differentiation is driven by distinct transcription factors: retinoic acid receptor-related orphan receptor-γt (Rorγt) and Rorα [33, 34] . Stat3 deletion in T cells also prevents autoimmune uveitis and EAE and increases the expression of IL10 and forkhead box P3 (FoxP3) [49] , and the expression of FoxP3 programs the development and functions of Treg cells [50] . In humans, IL23 and IL1β also induce the development of Th17 cells expressing IL17A, IL17F, IL22, IL26, IFNγ, the chemokine CCL20, and the transcription factor RORγ [51] [52] [53] , as illustrated in Figure 1 (adapted from Hirota et al. [54] ).
Recent microarray studies of lesions in MS patients demonstrated an increased expression of IL17, confirming that Th17 cells play an important role in the development of inflammation and demyelination and in the eventual damage of the CNS. IL17 is a recently described cytokine produced in humans almost exclusively by activated memory T cells and can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. Patients with MS have greater numbers of IL17-mRNAexpressing mononuclear cells in the cerebrospinal fluid (CSF) than in the blood. Previously, no increase in the numbers and expression of IL17 mRNA by mononuclear cells isolated from the CSF was observed in patients with MS, but higher levels of IL17 mRNA were observed in the CSF than in the blood, with the highest levels in the blood detected during clinical exacerbations [56] . These data confirm the pivotal role of IL17 in MS both peripherally and centrally.
Myelin is expressed in the circulation, and other CNS antigens are thought to be expressed in the cervical lymph nodes, which can trigger the conversion of autoaggressive myelinreactive T cells to pathogenic T cells. Adhesion molecules, the integrins, allow these myelin-reactive T cells to penetrate the blood-brain barrier (BBB) under inflammatory conditions, and in this way, activated and memory T cells can enter the CNS [57] . Autoaggressive myelin-reactive T cells migrate into the CNS, where they recognize their cognate target antigens, and the movement of antigen-presenting cells (APCs) into the CNS is essential for lymphocyte reactivation within the CNS compartment and the initiation of the inflammatory cascade in the development of EAE [58] . Subsequently, inflammatory and immune cells, such as granulocytes and macrophages, are attracted into the CNS parenchyma, where they mediate tissue inflammation, leading to demyelination and tissue damage [59] . The brain was formerly considered an immunoprivileged organ, but this perspective has been revised in the last two decades [60] . Today, we understand that any damage to the CNS can activate immune cells in situ in the CNS, particularly microglial cells. Deshpande et al. demonstrated the transient inactivation of microglial cells via a cell-specific deficiency of CD40 expression, indicating that microglial cells are crucial for maintaining the autoimmune responses in the CNS [61] . The major histocompatibility complex (MHC, also known as "human leukocyte antigens" in humans) class II molecules are only displayed on specialized APCs (e.g., dendritic cells [DCs], B cells, and macrophages), whereas MHC class I molecules are expressed by all cells in the inflammatory milieu of the CNS [62] . Microglial cells upregulate the expression of MHC and costimulatory molecules to initiate the generation and maintenance of the inflammatory milieu. DCs seem to play a critical role in antigen presentation to invading T cells and in the release of cytokines and chemokines, thereby guiding the entry of monocytes, lymphocytes, and cells with a phenotype similar to that of DCs into the lesion [63] .
Th cells recruit macrophages, which release proinflammatory cytokines and destructive molecules (such as nitric oxide [NO], IL1, IL6, tumor necrosis factor α [TNFα], and matrix metalloproteinases (MMPs]), and CD8 + T cells also directly attack MHC class I-expressing cells, such as oligodendrocytes and neurons [64, 65] . The secretion of destructive molecules, such as NO and TNFα, and the degradation of myelin are consequences of this cascade. TNF receptor 1 (TNFR1) but not TNFR2 signaling is critical for demyelination and the limitation of T-cell responses during immune-mediated CNS disease [66] . This complicated process triggers the recruitment of innate immune cells, generally consisting of T cells, macrophages, and microglia, which in turn mediate demyelination, axonal damage, and lesions.
In autopsy samples from MS patients, the expression of IL17 is evident in perivascular lymphocytes and in astrocytes and oligodendrocytes located in the active areas of CNS lesions. IL17R is also identifiable in acute and chronic MS plaques of patients with MS, suggesting the enrichment of Th17 and CD8 + T cells in active MS lesions, and confirming an important role for IL17 in the pathogenesis of MS [67] . Th17 cells are identified by their expression of IL23R and the memory T-cell marker CD45RO in situ. Other markers that have been investigated including the chemokine receptor, CCR6, and RORC variant 2, which is a central transcription factor for Th17-cell development [42, 68] . Microarray analysis of MS lesions has also demonstrated increased transcripts of genes encoding inflammatory cytokines, particularly IL6, IL17, and IFNγ and associated downstream pathways [56] . A significant increase in IL23 mRNA and protein expression is found in lesion tissues compared with nonlesion tissues. Activated macrophages/microglia have been shown to be important sources of IL23p19 in active and chronically active MS lesions. IL23p19-expressing mature DCs are preferentially located in the perivascular cuffs of active lesions. This data on the expression of IL23p19 in MS lesions improves our understanding of the pathogenesis of MS [69] .
There is also evidence that MS endothelial cells express high levels of IL17R and are more permeable to IL17 than are non-MS endothelial cells. Perivascular DCs also express high levels of granzyme B in inflammatory lesions, polarizing naïve CD4 + T cells into Th17 cells. These Th17 cells transmigrate efficiently across BBB endothelial cells (BBB-ECs), leading to the destruction of human neurons and initiating CNS inflammation through Th-cell recruitment [70] . Similarly, the expression of IL17R and IL22R on BBB-ECs has been examined in MS lesions, and IL17 and IL22 have been shown to disrupt BBB tight junctions in vitro and When naïve CD4 + TCRαβ + T lymphocytes, classified by their low expression of CD44, absence of CD25, and high levels of CD62L, encounter their cognate antigens, they can differentiate into several previously identified effector subsets. It is likely that several "master" transcription factors, individually required for T-cell differentiation towards one of the end effector stages, are initially expressed upon engagement of the TCR with costimulatory receptors. Each transcription factor drives a specific set of genes required for lineage commitment and the expression of signature cytokines and negatively affects alternative pathways. However, the local microenvironment is the driving force that determines the outcome of the differentiation course. Th1 cells are established in the presence of IFNγ and IL12 and signaling via STAT1 and STAT4, resulting in the expression of the master transcription factor T bet. Th2 cells depend on IL4 and STAT6 for the increased expression of GATA3, whereas the simultaneous presence of TGFβ results in the development of Th9 cells, utilizing an undefined master transcription factor. The presence of TGFβ, with IL2 signaling via STAT5, is known to generate, at least in vitro, inducible Treg, which utilize FOXP3 like those Treg generated in the thymus. Again, it is TGFβ in combination with IL6 signaling via STAT3 that drives the expression of RORγt, resulting in the differentiation of Th17 cells. However, the initiation of the developmental program of these T helper subsets may not be completed in the presence of only these driving cytokines. Several additional factors may be required for their subsequent functional maturation or may be responsible for the fine tuning of their effector phases. Several of these factors are indicated, together with the characteristic cytokine profiles of each subset (adapted from [54] ).
in vivo. IL6 transsignaling may also play a role in the autoimmune inflammation of the CNS, mainly by regulating the early expression of adhesion molecules, possibly via cellular networks at the BBB [71] . Ifergan et al. demonstrated that a subset of CD14 + monocytes migrate across the inflamed human BBB and differentiate into CD83 + CD209 + DCs under the influence of BBB-secreted TGFβ and granulocytemacrophage colony-stimulating factor (GM-CSF). These DCs can produce IL12p70, TGFβ, and IL6 and promote the proliferation and expansion of distinct populations of Th1 and Th17 cells. The abundance of such DCs in situ is strongly associated with microvascular BBB-ECs within acute MS lesions and with a significant number of Th17 cells in the perivascular infiltrate [72] .
Astrocytes play significant physiological roles in CNS homeostasis and act as a bridge between the CNS and the immune system. Astrocytes also contribute to the complex interactions during CNS inflammation. IL17 functions in a synergistic manner with IL6 to induce IL6 expression in astrocytes. Astrocytes upregulate the expression of IL17 and IFNγ genes and proteins in T cells, which is consistent with the astrocytes' capacity to express IL23 subunit p19 and the common IL12/IL23 subunit p40, but not IL12 subunit p35 when these two cell types are cocultured [73] . Das Sarma et al. demonstrated increased IL17RA expression in the CNS of mice with EAE and the constitutive expression of functional IL17RA in mouse CNS tissues. They also identified the expression of IL17RA in both astrocytes and microglia in vitro. In that study, the secretion of the chemokines Mcp1, Mcp5, Mip2, and CxcL1 was upregulated in these cells, suggesting that the upregulation of chemokines by glial cells is the result of IL17A signaling through constitutively expressed IL17RA [74] .
Ma et al. demonstrated that the suppressor of cytokine signaling 3 (Socs3) participates in IL17 functions in the CNS as a negative feedback regulator, using mouse models of Socs3 small interfering RNA (siRNA) knockdown and Socs3 deletion. These mice with loss of Socs3 function showed enhanced IL17 and IL6 signaling in astrocytes via the activation of the NF-κB and Mapk pathways, indicating that astrocytes can act as a target of Th17 cells and IL17 in the CNS [75] . Similarly, Kang et al. constructed specific deletion mutants of Act1, a critical component required for IL17 signaling, in mice with EAE to examine CNS inflammation in endothelial cells, macrophages, microglia, and the neuroectoderm (neurons, astrocytes, and oligodendrocytes). In these Act1-deficient mice, Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils, and macrophages. Therefore, astrocytes are critical in IL17-Act1-mediated leukocyte recruitment during EAE [76] .
Interestingly, Merkler et al. demonstrated that macrophages respond to the Th1 milieu and neutrophils respond to Th17 cytokines in a marmoset monkey model of EAE. They also showed dense accumulations of T and B lymphocytes, MHC-II-expressing macrophages/microglia, and early activated macrophages at the sites of perivascular and parenchymal lesions in the neocortex and subcortical white matter, indicating that the inflammatory response, especially macrophage and microglia activation, may be regulated differently in the gray matter areas of the primate brain [77] .
In summary, DCs in the peripheral tissues and microglia in the CNS are responsible for cytokine polarization and the expansion of Th17 cells. The complex interactions of Th17 cells with different DCs, such as microglia, astrocytes, and peripheral DCs (including neutrophils and macrophages), all contribute to the immunopathogenesis of EAE and MS.
IL1R KO mice have impaired Th17 cells and are protected from EAE [78] , and IL1β increases the susceptibility to and progression of relapse onset in MS [79] , implying a role for IL1β in the development of EAE and MS. EAE was abolished by a virus-expressing IL4 but not by a virus-expressing IL10 in chronic relapsing EAE. Therefore, the cytokine environment was converted from a disease-promoting IL23producing condition to a disease-limiting IL4-producing condition by the local expression of IL4 from a Herpes simplex virus vector delivered to the brain [80] . Moreover, the increased expression of IL4 in glial cells was associated with the reduced severity of EAE [81] , suggesting that the upregulation of Th2 cytokines inhibits the propagation of the inflammation of EAE/MS by encephalitogenic Th17 cells. CD4 + CD25 + Foxp3 + T cells, well-known regulatory T cells (Tregs), retain the potential to inhibit the autoimmune response, and protect against inflammatory injury. TGFβ is a key cytokine in the generation of Tregs. Tregs are not only primarily involved in the regulation of Th17 cells but can also regulate the functions of Th1/Th2 cells [82] . A distinction has been drawn between the generation of pathogenic Th17 cells that induce autoimmunity and the generation of Tregs that inhibit autoimmune tissue injury [39] . Although EAE was once considered a classical Th1 disease, it has been proposed that it is predominantly Th17 driven. Recently, Singh et al. demonstrated that the overexpression of IL17 in T cells did not exacerbate EAE. Moreover, genetic and antibody studies have indicated that the absence of IL17A or IL17F does not reduce the incidence or severity of EAE. The collective findings of IL17 and IFNγ studies indicate that their roles may depend on the nature of the immune response and that the IL17 that occurs in the brain may overcome the inhibitory effect of IFNγ, which generally prevents inflammation at that site [83] . When pure Th17 cells from myelin oligodendrocyte glycoprotein-(MOG-) immunized mice, polarized with TGFβ to deplete any IFNγ production, are adoptively transferred to mice, they do not induce EAE, suggesting that the reciprocal interactions among Th17-related cytokines enrol and activate the involvement of associated immune cells. Interestingly, when Th17 cells are combined with Th1 cells, they can fully induce EAE disease [84] . Liu et al. also demonstrated that the loss of STAT3 by Th cells results in an intrinsic developmental defect that renders STAT3 −/− mice resistant to CNS inflammatory diseases. STAT3 is required for the production of IL17 by Th17 cells, the generation of double positive T cells expressing IL17 and IFNγ, and T cell trafficking into CNS tissues. This suggests that STAT3 may be a therapeutic target for modulating CNS autoimmune diseases, and that Th1 cells can facilitate the entrance of Th17 cells into the CNS during EAE [85] .
An encephalitogenic Th1 cell line that induces the recruitment of host Th17 cells to the CNS during the initiation of EAE has been reported [49] . Stromnes et al. showed significant differences in the regulation of inflammation in the brain and spinal cord, depending on different Th17/Th1 ratios, by demonstrating that specific T-cell populations targeting different myelin epitopes are characterized by different Th17/Th1 ratios in EAE [86] . Therefore, Th1 cells have the potential to reciprocally regulate Th17 cells during EAE.
IL21 is a type I four-α-helix bundle cytokine that belongs to the IL2 family and functions as a "growth hormone"like cytokine. After the antigen-responsive differentiation phase, Th17 cells enter the amplification stage, and IL21 plays a pivotal role in the expansion and differentiation of the Th17 lineage, providing an autocrine and paracrine stimulus for Th17 cells [41, 87] . During clonal expansion, IL21 also promotes IL23R expression in differentiated Th17 cells, which plays an important role in the stabilization of the Th17 lineage in the presence of IL23 [88] . Although no effects were observed when Il21 was administered after EAE progression, the administration of IL21 boosted natural killer (NK) cell functions before the induction of EAE, including the secretion of Ifnγ. Therefore, IL21, by affecting NK cells, has various effects during the initiation and progression of EAE [89] .
Alternatively, IL27, an IL12/IL23 family member, is a negative regulator of Th17 cell differentiation and can prevent inflammatory demyelination in the EAE model [44] . IL27 drives the expansion and differentiation of IL10producing Tr1 cells by inducing the expression of three key molecules: the transcription factor c-MAF, the cytokine IL21, and ICOS. Moreover, IL27-driven c-MAF expression transactivates the production of IL21, which acts as an autocrine growth factor for the expansion and/or maintenance of IL27-induced Tr1 cells. ICOS also promotes IL27-driven Tr1 cells. Each of these elements is essential, because the loss of c-MAF, IL21 signaling, or ICOS reduces the frequency of IL27-induced differentiation of Tr1 cells ( Figure 1 ) [90] . Exacerbation of EAE was demonstrated in IL27-deficient mice, and interestingly, Il27-treated mice had markedly reduced CNS inflammatory infiltration, indicating the downregulation of Th17 phenomena [91] .
Recently, a novel effector T-cell subset, Th9 cells, has been identified, and the ability of this T-cell subset to induce EAE is currently being investigated. Jäger et al. generated Mog-specific Th17, Th1, Th2, and Th9 cells in vitro to directly characterize their encephalitogenic potency after their adoptive transfer. They found that Mog-specific Th1, Th17, and Th9 cells, but not Th2 cells, induce EAE. Interestingly, each T-cell subset induced disease in a distinct pathological manner, suggesting that the different effector Th subsets that induce EAE do so differently and implying that the pathological heterogeneity in MS lesions might be partly attributable to various characteristics of myelinreactive effector T cells [92] . The authors also suggested that MS might be a disease caused by multiple distinct myelinreactive effector cells. The disease induced by Th17 cells in some animals exhibited symptoms atypical of EAE, including ataxia, severe imbalance, and weight loss associated with high mortality. Some animals had a mixture of atypical and typical EAE symptoms. When cells were recovered from the CNS, it appeared that the transferred Th9 cells produced IFNγ. The identities of the other cell populations did not seem to drift after their in vivo transfer [93] .
Nowak et al. recently demonstrated that like other T cells cultured in the presence of TGFβ, Th17 cells produce IL9. Th17 cells generated in vitro with IL6 and TGFβ and ex vivo-purified Th17 cells both produced IL9. Data show that IL9 neutralization and IL9R deficiency attenuate the disease, and this correlated with reductions in Th17 cells and IL6-producing macrophages in the CNS. These authors also confirmed the role of IL9 in the development and progression of EAE and implicated Il9 as a Th17derived cytokine that contributes to inflammatory disease [94] .
Together, Th2 cells, Tr1 cells, and Tregs exert repressive effects on Th17 cells, and Th9 cells have a stimulatory effect on Th17 cells, suppressing EAE and MS. However, Th1 cells play dual roles in EAE.
Our understanding of the pathophysiology and neurodegenerative processes of MS has led to the development of novel therapeutic strategies. Since the early 1990s, diseasemodifying drugs have been introduced for the selective management of MS, including IFNβ and glatiramer acetate (GA), which have become the standard treatment for relapsing/remitting MS [95] . Most recommendations previously made by the Multiple Sclerosis Therapy Consensus Group (MSTCG) on the use of disease-modifying drug therapies remain valid [96, 97] . Hermmer and Hartung have published an apparent review of the development of rational therapies in MS [98] . Therefore, we will discuss four domains of novel immunomediated therapeutics used for MS and their current status. The first domain includes immunosuppressive agents, such as mitoxantrone, laquinimod (ABR-215062), cladribine (Mylinax ), and teriflunomide (probably via the suppression of TNFα and IL2 production). The second domain includes immunomodulatory agents: (1) cytokine inhibitors such as IFNβ; (2) agents that deplete specific immune cell subsets, such as alemtuzumab (a human monoclonal antibody [mAb] that targets CD52 expressed by T and B cells, producing long-term T-cell depletion) [99, 100] and rituximab (which targets CD20 to deplete human B cells) [99, 101] ; (3) agents that selectively block coreceptors and costimulators, such as daclizumab (an anti-CD25 mAb that inhibits activated T cells and induces regulatory immune cells) [102] . The third domain involves the development of migration-modifying therapies: (1) agents that affect adhesion molecules, such as natalizumab (an mAb that blocks very late antigen 4 [VLA-4]) and (2) sphingosine 1phosphate receptor (S1PR) agonists: fingolimod (FTY720). The fourth domain includes neuroprotective agents associated with immunomodulation, including broad-spectrum immunomodulators such as statins, PPAR agonists (e.g., pioglitazone, gemfibrozil), the sex hormone estriol (E3), fumarate, minocycline, and erythropoietin (EPO), all of which have been effective in the treatment of both EAE, and MS. IFNβ has been clinically introduced to treat patients with MS based on its ability to shift a Th1-mediated response to a Th2-mediated response [92] . However, microarray studies have indicated that a number of genes in patients with MS are upregulated by the cytokines associated with the differentiation of cells into Th1 lymphocytes rather than into Th2 lymphocytes, suggesting that this shift may not be the only therapeutic mechanism of IFNβ in MS [103] . IFNβ therapy also reduces IL23 mRNA levels [104] . IFNβ inhibits human Th17 cell differentiation, so the Th17 axis could be another target of IFNβ therapy [105] . IFNβ-mediated IL27 production by innate immune cells has been shown to play a critical role in the immunoregulatory role of IFNβ in EAE by inhibiting Th17 cells in EAE mice and MS patients [91, 106, 107] . Besides, Galligan et al. evidence further that IFNβ(−/−) mice exhibited an earlier disease onset and a more rapid progression of EAE compared to IFNβ(+/+) mice of EAE and IFNβ(−/−) mice of EAE had increased 7 numbers of CD11b(+) leukocytes infiltrating affected brains and an increased percentage of Th17 cells in the CNS with augmentation of autoreactive T cells,suggesting that IFNβ acts to suppress the production of autoimmune-inducing Th17 cells during the development of disease as well as modulating proinflammatory [108] . In addition, the therapeutic effect of IFNβ is probably attributable to the induction of the regulatory cytokine IL10 [104] . Furthermore, Axtell et al. design a delicate study to further clarify the role of IFNβ in MS/EAE [109] . Likewise, They demonstrate that IFNβ was effective in reducing EAE symptoms transferred by Th1 cells transfer but exacerbated disease by Th17 cells transfer and effective treatment of IFNβ in Th1-induced EAE correlated with augmented IL10 production; differently, in Th17-induced EAE, the amount of IL10 was unaffected by treatment of IFNβ. Likewise, a high IL17F level in the serum of people with RRMS is associated with fail of IFNβ therapy. This characteristic of IFNβ might contribute to explore some logical biomarkers for predictive assessment of the response to a popular therapy for MS [109, 110] . Although, B cells may have a dual role in the pathogenesis of MS that they contribute to the induction of the autoimmune response but also mediate the resolution of the CNS inflammatory infiltrate [111, 112] . However, Ramgolam et al. demonstrate further that supernatants transferred from IFNβ-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the RORC and IL-17A and secretion of IL-17A. Likewise, IFNβ-1b also induces B cells' IL-10 secretion which may mediate their regulatory potent [113] . Thus, IFNβ-1b exerts its therapeutic effects at least in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS, which may uncover extra mechanisms of the B-cell contribution to the autoimmune effects and provide novel targets for future selective treatment of MS [113] .
Glatiramer acetate (GA; Copaxone; copolymer 1) exerts a clinical response in MS patients via its modulation of IFNγ and IL4 by reducing the expression of IFNγ and ensuring the stable expression of IL4 in anti-CD3/CD28stimulated peripheral blood mononuclear cells (PBMCs) [114] . Moreover, GA enhances the suppressive effects of Tregs in both EAE and MS [115, 116] . Studies of human DCs have shown that GA modulates the production of inflammatory mediators without affecting DC maturation or immunostimulatory potential. DCs exposed to GA secrete low levels of the Th1-polarizing factor IL12p70 in response to lipopolysaccharide and triggering of the CD40 ligand [117] . Human DCs exposed to GA also induce IL4-secreting effector Th2 cells and increase their expression of IL10 [118] . These results show that APCs, including DCs, are essential for the GA-mediated shift in Th-cell phenotypes and indicate that DCs are an important target of the immunomodulatory effects of GA.
Patients with MS show a threefold to fourfold increase in the expression of the α4 subunit of the integrin VLA-4, which is normally expressed on activated lymphocytes, monocytes, and other cell types in the CSF and circulation [119] . Elovaara et al. confirmed that methylprednisolone reduces the adhesion molecules in the blood and CSF in patients with MS [120] , implying that targeting leukocyte trafficking may be a possible therapeutic strategy for MS [121] . Therefore, natalizumab, a humanized mAb directed against the VLA-4 adhesion complex, has been introduced into the treatment of MS and reduces the risk of sustained progression of disability and the rate of clinical relapse in patients with relapsing MS [122] . However, during clinical trials, two natalizumab-treated MS patients developed progressive multifocal leukoencephalopathy (PML), which resulted in the voluntary removal of the drug from the market in February 2005 [123, 124] . A retrospective safety evaluation was subsequently conducted, and natalizumab was consequently returned to the market as a monotherapy in July 2006 for the treatment of relapsing MS; however, there were 111 cases of PML reported subsequently in natalizumab-treated MS patients as of April 2011 [125] . More evidently, the risk of developing PML for a MS patient on natalizumab (Tysabri) is almost 100 times higher if the patient (1) has been taking the drug for more than two years, (2) has a prior history of immunosuppressant use, and (3) tests positive for antibodies to the JC virus [126] , compared to a patient with none of these three risk factors [127] . Instead, there is currently no convincing evidence that natalizumab-associated PML is restricted to combination therapy with other disease-modifying or immunosuppressive agents [128] . Nevertheless, natalizumab use must be restricted to the indicated patients.
Mitoxantrone, a cytotoxic drug with immunomodulatory properties, is used to treat progressive forms of MS [129] . Mitoxantrone increases the ex vivo production of the Th2 cytokines IL4 and IL5, but with no significant changes in IFNγ, TNFα, IL10, or IL17 expression by PBMCs or CD4 + T cells, indicating that the immunomodulation afforded by mitoxantrone treatment in MS acts through the enhancement of Th2-type cytokines [130] .
Currently, a head-to-head race for approval had initially developed between two under spotlight oral immunomodulatory agents-fingolimod and cladribine ( Figure 2 ) [131] . Fingolimod (FTY720/Gilenya, Novartis), an S1PR modulator [132] , is under the spotlight because it has completed phase III trials [133] and has been approved by the US Food and Drug Administration as the first oral, first-line treatment for relapsing MS [134, 135] . S1PR is mainly expressed by immune cells, neuronal cells, endothelial cells, and smooth muscle cells [136] [137] [138] [139] . The key roles of S1PR in angiogenesis, neurogenesis, and the regulation of immune cell trafficking, endothelial barrier function, and vascular tone were demonstrated with the genetic deletion of S1pr in a murine model [140] [141] [142] . The immunomodulatory effect of fingolimod acts in two pathways. In one pathway, it inhibits the function of S1PR, which facilitates the CC-chemokine receptor 7-(CCR7-) mediated retention of lymphocytes in the lymph nodes, including naïve T cells and central memory T cells, but not effective memory T cells. This significantly reduces the infiltration of inflammatory cells into the CNS [143, 144] and reduces the numbers of autoreactive Th17 cells that are recirculating via the lymph and blood to the CNS [145] [146] [147] . The second pathway prohibits neuroinflammation via the modulation of the . This activation results in the increased production of proinflammatory cytokines, which lead to the aberrant activation of Th1 and Th17 proinflammatory responses. Activated encephalitogenic adaptive immune effectors (such as Th1 cells, Th17 cells, CD8 + cells, and B cells) express surface molecules that allow them to penetrate the blood-brain barrier and to enter the central nervous system (CNS). The presence of autoreactive immune effectors, together with abnormally activated CNS astrocytes and microglia, leads to the increased production of reactive oxygen species, excitotoxicity, autoantibody production, and direct cytotoxicity, which are all involved in the demyelination and axonal and neuronal damage that is present in patients with MS. Potential therapeutic interventions at different levels of the immunopathological cascade are shown in the filled yellow boxes (cytotoxic T lymphocytes [ [55] .). S1PR1 expressed on oligodendrocytes, neurons, astrocytes, and microglia [76, 148, 149] . Another oral immunomodulatory drug Cladribine (2-chlorodeoxyadenosine) is a synthetic chlorinated deoxyadenosine analog [150] that is activated by intracellular phosphorylation in specific cell types, resulting in preferential and sustained reduction of peripheral T and B lymphocytes, mimicking the immunedeficient status of hereditary adenosine deaminase deficiency [151] . Orally administered cladribine shows significantly efficacy in patients with RR-MS [152] . Relative to placebo, oral cladribine reduces relapses by 55-58% and has an impact on disability progression and all MRI outcome markers in patients with RR-MS [152] [153] [154] . Nevertheless, to exactly weight the benefits of both novel immunomodultory agents against the potential risks is necessary and must be monitored continually.
These advances in identifying unique therapeutic targets for MS have instigated numerous phase II and phase III clinical trials, for example, trials of various mAbs, including those directed against CD52 (alemtuzumab), CD25 (daclizumab), and CD20 (rituximab), and trials of disease-modifying therapies, such as teriflunomide, laquinimod, and fumarate [135, 155] . For example, alemtuzumab, a humanized mAb, targets the surface molecule CD52 on all T-cell populations and other cellular components of the immune system, such as thymocytes, B cells, and monocytes [156] .
Offner reported that estrogen and its derivatives exert neuroimmunoprotective effects against EAE and that E2 upregulates the expression of Foxp3 and Ctla4, which contribute to the activity of Tregs, suggesting the therapeutic application of estrogen to MS [157] . Papenfuss et al. also demonstrated that estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE and they confirmed that E3 protects mice against EAE by inducing DCs to increase their expression of inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3) and deviate towards a Th2 phenotype [158] .
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily, which includes receptors for steroids, retinoids, and thyroid hormones, all of which are involved in the immune response [159] . Natarajan et al. demonstrated that PPARγ agonists inhibit EAE by blocking IL12 production, IL12 signaling, and Th1 cell differentiation [160] . Kanakasabai et al. further demonstrated that the PPARδ agonists ameliorate EAE by blocking IFNγ and IL17 production by Th1 and Th17 cells. The inhibition of EAE by PPARδ agonists is also associated with reductions in IL12 and IL23 and increases in IL4 and IL10 expression in the CNS and lymphoid organs. This indicates that PPARδ agonists modulate the Th1 and Th17 responses in EAE, and suggests their use in the treatment of MS and other autoimmune diseases [161] .
Minocycline, an oral semisynthetic tetracycline antibiotic, can penetrate the CNS and has interesting pleiotropic biological functions and neuroprotective effects, including in demyelinating diseases such as MS [55] . Nikodemova et al. have shown that minocycline attenuates EAE in rats by reducing T-cell infiltration into the spinal cord and downregulating LFA-1 on T cells, but without modifying the production of dominant cytokines [162] . Zabad et al. demonstrated in a cohort study the impact of oral minocycline on clinical and MRI outcomes and serum immune molecules during the 24 months of open-label minocycline treatment. No relapses occurred between months 6 and 24, and the levels of the p40 subunit of IL12 were elevated during the 18 months of treatment, which might have counteracted the proinflammatory effects of IL12R. The downregulation of MMP9 activity was reduced by minocycline treatment [163] .
Brines et al. have demonstrated that EPO mediates neuroprotection against experimental ischemic brain injury [164] . Agnello et al. have shown that EPO exerts an antiinflammatory effect that ameliorates EAE [165] . Yuan et al. also demonstrated that EPO retains its immunomodulatory capacity in both the periphery and the inflamed spinal cord by promoting a massive expansion of Treg cells, inhibiting Th17 polarization and abrogating the proliferation of antigen-presenting DCs [166] . We observed significantly reduced levels of both Th1 and Th17 cells in the CNS and a significantly increased proportion of splenic Tregs in EPOtreated Mog-EAE mice. We also demonstrated that MOGspecific T-cell proliferation was suppressed in the EPOtreated group [167] .
The immunomodulatory mechanisms of immunomediated therapeutic agents are not fully understood. Here, we report our current understanding of the immunomodulatory effects of clinically proven and clinically tried agents, and of potential candidate agents, such as decoy receptor 3 (DcR3). We have selectively reviewed their immunomodulation in EAE and MS. Demjen et al. showed that the neutralization of CD95L (FasL) promoted axonal regeneration and functional improvement in an injured animal model, suggesting that this therapeutic strategy may constitute a potent future treatment for human spinal injury [168] . DcR3 is a recognized member of the TNFR superfamily and is predominantly expressed in tumor cells, allowing them to evade immune attack [169] . DcR3 is a soluble receptor that binds to members of the TNF family and can competitively inhibit the binding of TNF to TNFRs [170] . FasL, LIGHT, and TNF-like molecule 1A (TL1A) are all confirmed ligands of DcR3 [171, 172] . When DcR3 binds to FasL, it inhibits FasL-induced apoptosis [169] . It has also recently been shown that DcR3 counteracts the effects of Th17 cells by interfering with FasL-Fas interactions [173] . We have demonstrated that DcR3 ameliorates EAE by directly counteracting inflammation and downregulating Th17 cells in situ [174] , implying that DcR3 downregulates the Th17 response and inhibits the inflammation of the CNS in situ during EAE by blocking ligand-receptor interactions, such as Fas-FasL, DR2-LIGHT, and/or DR3-TL1A. Therefore, we introduce DcR3, another immunomodulatory molecule, as a potential candidate for consideration in the clinical treatment of MS.
In summary (Figure 2 ), these immunomodulatory agents and neuroprotective therapies for MS have great value as clinical agents, to be tested in clinical trials or preclinical studies, and in the development of novel therapeutic strategies for MS [55] .
MS is the most common disabling CNS disease in young adults. It is characterized by recurrent relapses and/or progression, which are attributable to multifocal inflammation, demyelination, and axonal pathology within the brain and/or spinal cord [175] . The effector Th cells play a well-recognized role in the initiation of autoimmune tissue inflammation, and these autoreactive effector CD4 + T cells have an established association with the pathogenesis of this disorder [17] . However, in models thought to be driven by Th1 cells, mice lacking the hallmark Th1 cytokine IFNγ were not protected from EAE but tended to display enhanced susceptibility to this disease [26] . The identification of Th17 cells has shed light on this apparent discrepancy. Like Th1 cells, polarized Th17 cells have the capacity to cause inflammation and autoimmune disease. A deficiency of the Th17-related cytokine IL23, but not of the Th1-related cytokine IL12, induces resistance to EAE, implying that Th17 cells are the chief contributors to EAE/MS [28] , whereas Th1 cells can consistently transfer EAE disease [16, 17] . Komiyama et al. demonstrated that EAE was significantly suppressed in Il17 −/− mice, manifested as delayed onset, reduced maximum severity, ameliorated histological changes, and early recovery [176] . However, the outcomes have varied when the differentiation and/or functions of Th17 cells have been blocked in clinical trials of human autoimmune diseases, with notable success only in psoriasis and Crohn's disease, but negative results in relapsing/remitting MS. The strategy of inhibiting the Th17 response has had even less support in preclinical studies in animal models [177] .
These data raise the questions of whether MS is mediated solely by Th1 cells or solely by Th17 cells, whether it is mediated by both pathways, or whether perhaps it is mediated by neither pathway [175] . There is growing evidence that autoreactive T cells (particularly Th1 and Th17 cells) participate in the pathophysiology of MS. Although the exact roles of Th1 and Th17 cells in the development of MS lesions are not well understood, it appears that both these effector T-cell populations can cause CNS inflammation and demyelinating lesions in MS and EAE [50, 178] .
Our increasing understanding of the immunopathogenic roles of Th1, Th2, and Th17 cells and Tregs in MS/EAE should facilitate the development of novel immunomodulatory therapeutic approaches to MS [179, 180] . The treatment of MS has always been hampered by the untoward adverse effects caused by immunosuppression with agents such as natalizumab [128] . Currently approved disease-modifying treatments achieve their effects primarily by blocking the proinflammatory response in a nonspecific manner. Their limited clinical efficacy calls for a more differentiated and specific therapeutic approach. We can confidently say that IFNβ, GA, and mitoxantrone are fairly clinically effective for MS patients. The addition of estrogen(s) or minocycline has also shown benefits in the treatment of MS. We have established the protective effects of DcR3 and EPO against EAE [174, 181] , but further evidence is required before they can be used clinically for the treatment of MS. More immunomodulatory therapeutic agents are currently in clinical trials, including fingolimod (FTY720), alemtuzumab, and rituximab add-on therapies [182] . The extensive clinical application of these potential novel immunomodulatory therapeutic agents will be under close scrutiny in the near future. Towards cross-lingual alerting for bursty epidemic events BACKGROUND: Online news reports are increasingly becoming a source for event-based early warning systems that detect natural disasters. Harnessing the massive volume of information available from multilingual newswire presents as many challanges as opportunities due to the patterns of reporting complex spatio-temporal events. RESULTS: In this article we study the problem of utilising correlated event reports across languages. We track the evolution of 16 disease outbreaks using 5 temporal aberration detection algorithms on text-mined events classified according to disease and outbreak country. Using ProMED reports as a silver standard, comparative analysis of news data for 13 languages over a 129 day trial period showed improved sensitivity, F1 and timeliness across most models using cross-lingual events. We report a detailed case study analysis for Cholera in Angola 2010 which highlights the challenges faced in correlating news events with the silver standard. CONCLUSIONS: The results show that automated health surveillance using multilingual text mining has the potential to turn low value news into high value alerts if informed choices are used to govern the selection of models and data sources. An implementation of the C2 alerting algorithm using multilingual news is available at the BioCaster portal http://born.nii.ac.jp/?page=globalroundup. As electronic data expands, online reports are coming to represent a new modality in early warning surveillance for natural disasters such as epidemics [1] , typhoons and earthquakes [2, 3] . Recent studies in disease surveillance such as [4] have shown that significant challenges still exist for fine-grained automated understanding of event dynamics.
Since 2006, BioCaster [5] has been performing gathering, semantic analysis and mapping of global news reports to provide a near-real time summary of human epidemics. The system is used regularly by both national and international health agencies as well as a growing base of individual users. Recent advances include expanding the number of diseases to include animal and crop pathogens as well as extending the number of languages from 4 to 13. With the increase in data came an understanding that public health analysts needed more help finding novel trends in the event stream.
In order to support the task of detecting the unusual, we compare five widely used temporal aberration detection algorithms to look for spikes in the news event stream. This paper builds on our previously reported study for monolingual news alerting [4] by seeking to explore the hypothesis that cross-lingual events from text mining can provide improved detection rates. Although we focus here on newswire as a source we believe the results should have applicability for other unverified reports such as email lists and the rapidly developing space of user generated content.
The 2009 H1N1 pandemic illustrated how dependent each country is on the surveillance capacity in other states. Reducing public health risk depends on an overall strengthening of global health event monitoring as well as locally available sources such as clinical data and over-the-counter sales data. The Web provides a low cost surveillance infrastructure that has been shown to offer a timely means of detecting epidemics such as SARS [6] that is often several days ahead of the official reporting curve. In addition to work on BioCaster, there is a small but growing body of work looking at the issues of online public health monitoring such as GPHIN [6] and MedISys/PULS [7] . However, studies providing details of recall/precision/timeliness for end user tasks in media-based health surveillance are still surprisingly limited. To the best of our knowledge no previous study has explored the multilingual effects in this area. Several characteristics of early epidemic detection make the problem particularly challenging. Firstly, we want to catch epidemics as early as possible before they develop into humanitarian crises; Secondly, not every epidemic is of equal importance -those that are of most concern to the international community are described by the International Health Regulations [8] ; Thirdly, patterns of media coverage are complex [9] , at times focussing on dramatic and emotive imagery, at others prioritizing the reader's security and economic interests. In many ways the connection between media interest and the population at risk is often blurred.
How is this work different to various research in topic detection and tracking (TDT) [10] that has been undertaken for the last 14 years? Whilst both tasks look for events that are highly localized in time and space, the task we undertake begins with a predefined event semantics and a desire to distinguish the unexpected from the typical. Put another way, bursts in media interest do not always correspond to public health significance. The stream of work here seeks to uncover underlying trends and factors. Neither is this task entirely the same as TDT's first topic detection since we measure performance partly by the number of days before the silver standard that we can capture an event.
In general it is extremely difficult to determine ground truth for the actual numbers and durations of disease outbreaks. As a silver standard we have chosen the best publicly available human network of reporters which is ProMED-mail [11] . ProMED-mail is a program of the International Society for Infectious Diseases with many expert volunteer reporters globally and a sophisticated staged editorial process. Outbreak reports are distributed to 40,000 subscribers by email, RSS feed and Web portal -precisely the audience we target in our automated system.
In this study we have used quite coarse-grained granularity by choosing countries and days as the units. This is due to the current limits of reliable location detection in the system and also the frequency of news that we observe. The recorded time for each event was normalized to system download time which takes place every hour of each day.
Evaluation uses the standard classification test measures of sensitivity (recall), specificity, positive predictive value (PPV or precision), negative predictive value (NPV) and timeliness. We also measured the average number of system alarms per 100 days and compared this to the silver standard. The F-measure (F1) is calculated in the usual way as the harmonic mean of sensitivity and PPV.
As in our previous study, the standard for a true positive was to obtain a system alert on a country-disease event on or before the silver standard alert. To allow for compatibility and comparison we kept the period for a qualifying system alert as up to 7 days prior to and including a qualifying ProMED report on the same topic. Other history period lengths might be more or less effective but were not the target of the investigation in this study. True positives were increased by 1 if there was any system alert that fell within the 7 day period. Multiple system alerts did not count twice. False positives were increased by 1 for each system alert that fell outside of the 7 day window. False negatives were counted as the number of qualifying alert periods when there were no system alerts. True negatives were counted as the number of days outside of any qualifying alert period when no system alert was given. In testing we tried to maximize F1 together with timeliness. Figure 1 shows the 16 event streams that we explored. The events chosen for this study were determined based on diversity of geographical and media coverage rather than random selection. The 16 event streams contain 2064 surveillance days with 153 events (7.4% of alerting days) (Note that system data from the study will be made publicly available online for re-use via the GENI database interface on BioCaster). Since we wanted to explore the hypothesis that linguistic coverage in multiple languages could strengthen detection rates and timeliness we compared English news coverage against all languages including English for each of 16 disease outbreaks. English was chosen as the baseline because of its overall geographic representativeness. An alternative and perhaps more realistic approach might have been to use the native language for each outbreak country as the baseline which we will consider in future investigations. Because cross-lingual events on the 13 languages were only available in our system from December 2009, the trial period was from January to May 2010.
ProMED reports used in the silver standard excluded those that fell outside our case definition, based on the International Health Regulations [8] decision tree instrument. For example, requests for information, reports primarily focussed on control measures and aggregated summary reports not arising from specific events.
The text mining system we explored involves a semantic pipeline of modules running on a high throughput cluster computer with 48 Xeon cores. Throughput is approximately 9000 articles per day. System news was gathered from multiple news sources through Google News and MeltWater News as well as specialized sources such as the European Media Monitor, IRIN and ReliefWeb. (Note that no ProMEDmail messages were included in the system data for this study using a block on the Internet domain and message title). In total this gives us access to over 80,000 news sources globally. The languages used in the study (in ISO-639-1) are: ar,zh,nl,en,fr,de,it, ko,pt,ru,es,vi and th.
Underlying the system is a publicly available multilingual application ontology [12] which is used within the rule books to make basic inferences such as countries from names of provinces, or diseases from causal pathogens. The BioCaster ontology (BCO) rules also allow us to unify variant forms of terms such as the 11 forms of A(H1N1).
After data sourcing, translation takes place from the twelve non-English languages used in this study using Google's online translation system. As a quality reference point we refer to a recent large-scale evaluation of machine translation for European language pairs [13] . In this study on news texts it was found that across a wide variety of metrics Google's online system consistently performed among the highest quality systems for Spanish-English, French-English and German-English language pairs. Following machine translation, text classification using Naive Bayes (F1 0.93) removes non-disease outbreak news before text mining is applied. Rules are based on a regular expression matching toolkit called the Simple Rule Language [14] and divided between 18 entity types and template rules. The final structured event frames in XML includes slot values normalized to BCO root terms for disease, pathogen (virus or bacterium), time period, country and province. Additionally we also identify 15 aspects of public health events critical to risk assessment. For the purpose of this study we only made use of disease and country slots. Events in the 13 languages are treated in this study as being part of a univariate model for comparison purposes against English events.
Latitude and longitude of events down to the province level are found automatically using Google's API up to a limit of 15000 lookups per day, and then using lookup on 5000 country and province names harvested from Wikipedia.
We experimented with a range of popular models for early alerting used in the public health community: the Early Aberration and Reporting System (EARS) quality control chart models C3, C2 and W2 as well as the F-statistic and the Exponential Weighted Moving Average (EWMA). All were implemented in Excel for the purpose of this study. The models are what might be termed 'snapshot' models because they all use short 7 day baselines that assume a relatively stationary background, i.e. ignoring medium to long term periodic variations such as seasonal cycles. The baselines are used to predict future trends against which the current day values are compared. All models also use a 2 day 'guard period' just before the target day t to prevent the current day's data from being included in the baseline. All models use a minimally supervised method by setting a threshold parameter which we determined using the same 5 held out data sets used by [4] . These were 0.2 (C2 and W2), 0.3 (C3), 0.6 (F-statistic) and 2.0 (EWMA). A minimum standard deviation was set at 0.2 and a frequency purge was applied to remove singleton events, i.e. those with counts of 1 per day.
The EARS algorithms [15] are based on cumulative sum calculations commonly used in quality control. C2 triggers an alert when a test statistic S t exceeds a number k of standard deviations above the baseline mean:
where C t is the event count on the target day, µ t and s t are the mean and standard deviation of the counts during the baseline period. We set k to 1 for all experiments.
C3 is a modified version of C2 so that the previous 2 observations (within the guard period) are added to the test statistic if the counts on those days does not exceed a threshold of 3 standard deviations plus the mean on those days. The rationale here is to extend the sensitivity of C2.
W2 [16] is a stratified version of C2 which compensates for weekend data outages by removing Saturday and Sunday data counts from the baseline. Alerting though can take place on any day.
The calculation for the F-statistic [17] is:
Collier Journal of Biomedical Semantics 2011, 2(Suppl 5):S10 http://www.jbiomedsem.com/content/2/S5/S10
where s t 2 approximates the variance during the testing window and s b 2 approximates the variance during the baseline window.
Calculation is as follows:
EWMA Unlike other models in our test, the EWMA provides for a non-uniformly weighted baseline by down-weighting counts that are on days further from the target day:
where 1 >l > 0 is a parameter that controls the degree of smoothing. The optimal level found from held out data was found to be 0.2. The test statistic is calculated as:
. m s l l 2 0 5
As above, µ t and s t are the mean and standard deviation on the baseline window.
Interestingly we found that approximately 80% of news reports covered only about half the ProMED-mail alert disease-country topics, implying that the remaining 20% of news has to provide coverage for almost half the topics. Surprisingly, the trend was broadly similar for both English and all language news. Although the sample size is relatively small, given that the events we chose were from all regions of the world, this implies that having news in more languages may have a deepening effect rather than a broadening effect on event coverage. The three notable exceptions were in the cases of FMD in China (e4 in Figure 1 ), Dengue in Brazil (e12) and Dengue in Bolivia (e13).
Results for global events on English (Table 1) show an advantage for the F-statistic if we are primarily concerned with sensitivity (recall) and alerting rates (shown in column B ). However the F-statistic has a clear disadvantage with PPV (precision) which impacts heavily on the number of false alarms. This can be seen clearly by comparing the alarm rate per 100 days of 16.2 in column A with the ProMED average of 7.4. Both advantages and disadvantages are amplified when we add cross-lingual events.
Whilst the F-statistic has the highest overall F1, its high rate of false alarms reflected in the PPV makes it potentially an undesirable choice. If we seek for the best balance of F1 and timeliness with a minimum of false alarms then C3 looks like a more desirable alternative.
Cross-lingual event capture seemed to extend sensitivity in all models, improving F1 and timeliness. To see if we could harden our intuitions about these effects we looked specifically at South East Asia -a region where we would expect the representation of Chinese to be proportionately greater than English. Table 2 shows results which largely mirror those for the world as a whole. The noticable exception though is that EWMA shows a large drop in performance.
Although the sample size is limited, the data suggests trends in model performance. C3 seems to perform best when we consider that the high false alarm rate for the F-statistic could desensitize users. Cross-language events generally seem to improve F1 performance by several points across most models except for EWMA. The benefits come from an extension in sensitivity but could be focussed on topics where we already have large coverage of English news. This is not to say that multilingual news is not useful, as we comment below, it could be that it has a greater role to play in extending detection rates of novel events at lower levels of geographic granularity than the country.
Beyond the cross-lingual effects, drill down analysis revealed that bag-of-words topic classification and event extraction using intra-sentential regular expressions were still letting through a proportion of non-events. We sampled 274 English news articles by hand from the BioCaster portal's implementation of the C2 algorithm using a 7 day baseline window and found that approximately 30% of positively classified news articles fell just below the borderline of our case definition. Commonly misclassified topics included: vaccination campaigns, factual advice on avoidance and treatment of infectious diseases, improvements in surveillance facilities and surveillance exercises. Often the articles mentioned an infectious disease and cited facts about case numbers such as "90% of cholera cases reported annually…" or an analysis of historical events. This points to a need to strengthen discourse analysis, such as inter-sentential causality and inclusion relations between events which the current intra-sentential template driven approach does not handle well. The study also raises several questions about factors in the imbalance of reporting: why did Dengue in Brazil (e12) or FMD in China (e4) receive such massive local coverage but disproportionately less in the English media? Why did cholera in Angola (e9) or influenza in Romania (e8) receive comparatively low coverage overall? We also observed that the USA epidemics (e15 and e16) were widely reported in English but not so greatly in other languages.
In order to illustrate the potential complexity of the task we provide a detailed drilldown analysis of one of the outbreaks in our data set, i.e. cholera in Angola. Just to put the reporting of this outbreak into context: Angola itself is a former Portuguese colony which has suffered major outbreaks (e.g. 2006 to 2008) of cholera due to poor sanitation, drinking water infrastructure and environmental conditions. Although UNI-CEF has commented on recent advances, the country remains at risk, especially during the rainy season from January to mid-May. In this case BioCaster was more successful for English than for the multilingual system because a false spike of reports occluded subsequent true positives. In the case of the silver standard report on 19/3/2010, the cited English source was not detected but its Spanish translation was found a few days later -still much earlier than the ProMED-mail report.
The example is a relatively special case that illustrates an event that was not widely re-reported. The reports were made in English, Portuguese, French and Spanish from Angop. Externally, the 4/3/2010 article from Angop was republished in http://allafrica. com and http://africanseer.com on the 4th March. It was also referenced in a blog by the Namibia online community.
Automated health surveillance using text mining is not intended as a substitute for skilled human analysts but as these results show, it does have the potential to reduce their information burden if informed choices are used to govern the selection of models. In order to help guide users in the significance of news events we implemented the C2 algorithm for multilingual news alerting in the 'Global Roundup' section of the Bio-Caster Web site at http://biocaster.nii.ac.jp. The results, updated each hour, show the test statistic value, the disease, country, province, focus species daily news frequency and the baseline mean and standard deviation. Additionally, citation links are provided to the news articles with a list up of all languages that contributed to the alert. The output is available as both RSS and a Twitter feed. Obvious improvements to the techniques described here could take place by modeling lower geographic granularity and reducing size differences between geo-units. More sophisticated approaches might incorporate proximity information between events or model how events propagate through news space. A more subtle effect of the granularity restriction is that the models we presented do not allow us to follow what might be called 'late warning' signals. i.e. follow on events within the country's borders. For this reason detecting events below the country level is desirable. Future work will need to concentrate on maximizing system sensitivity to overcome the fragmentation of the event distribution that occurs when we bucket events into smaller geographic units.  Reducing mortality in sepsis: new directions Considerable progress has been made in the past few years in the development of therapeutic interventions that can reduce mortality in sepsis. However, encouraging physicians to put the results of new studies into practice is not always simple. A roundtable was thus convened to provide guidance for clinicians on the integration and implementation of new interventions into the intensive care unit (ICU). Five topics were selected that have been shown in randomized, controlled trials to reduce mortality: limiting the tidal volume in acute lung injury or acute respiratory distress syndrome, early goal-directed therapy, use of drotrecogin alfa (activated), use of moderate doses of steroids, and tight control of blood sugar. One of the principal investigators for each study was invited to participate in the roundtable. The discussions and questions that followed the presentation of data by each panel member enabled a consensus recommendation to be derived regarding when each intervention should be used. Each new intervention has a place in the management of patients with sepsis. Furthermore, and importantly, the therapies are not mutually exclusive; many patients will need a combination of several approaches – an 'ICU package'. The present article provides guidelines from experts in the field on optimal patient selection and timing for each intervention, and provides advice on how to integrate new therapies into ICU practice, including protocol development, so that mortality rates from this disease process can be reduced. Sepsis is the tenth most common cause of death in the US [1] . A recent US study reported that severe sepsis accounts for in excess of 215,000 deaths annually from a total population of approximately 750,000 patients-a mortality rate of approximately 29% (with published studies quoting a range of 28-50%) [2] . This persistent, high mortality rate is clearly unacceptable, given that it ranks sepsis above some of the higher profile causes of in-hospital death, including stroke (12-19% risk of death in the first 30 days) and acute myocardial infarction (AMI) (8% risk of death in the first 30 days) [3] . Moreover, the actual number of deaths associated with the condition may be even higher than current estimates suggest. Many sepsis patients have at least one comorbidity and deaths are often attributed to these conditions rather than to sepsis [4] [5] [6] . Unfamiliarity with the signs and symptoms of sepsis may further hinder accurate diagnosis.
There are many possible reasons for this high mortality. Sepsis is certainly a complex disease state; the pathophysiology is only now beginning to be unraveled, and it is complicated by heterogeneous presentation (possible signs of sepsis are presented in Table 1 ). While none of these signs alone is specific for sepsis, the otherwise unexplained presence of these signs should signal the possibility of a septic response.
Many cases of sepsis are recognized late, and patients are often inappropriately treated before entering the intensive care unit (ICU) by physicians unfamiliar with the signs and symptoms of the condition. Furthermore, treatment may be initiated by any of a number of physicians (anesthetists, hematologists, intensivists, infectious disease specialists, pulmonologists, and emergency physicians). There are presently various defined supportive strategies for treating patients with sepsis, but improvements are needed to reduce the unacceptably high mortality rate.
Moreover, as with other areas of medicine, the application and integration of new but proven strategies for reducing morbidity and mortality into clinical practice has been slow.
Encouraging new data have recently been presented on new approaches to the management of patients with sepsis. Many of these approaches attempt to modulate or interrupt the sepsis cascade and to address the cause of multiorgan dysfunction. Although many of these approaches are in early phases of development (e.g. antibodies to tumor necrosis factor [TNF] alpha, bactericidal permeability increasing protein, high-flow hemofiltration to remove circulating inflam-matory mediators, platelet-activating factor acetyl hydrolase, and antielastases), other approaches are more advanced and are already beginning to impact on outcomes in the ICU.
At a roundtable discussion in London in June 2002, Professor Jean-Louis Vincent brought together five experts to discuss more effective implementation of five exciting new interventions in the ICU setting to decrease the unacceptable burden of mortality in patients with severe sepsis.
Each of the roundtable panelists is a highly respected physician in the world of sepsis and critical care medicine. The interventions discussed encompassed low tidal volume in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) (Edward Abraham), early goal-directed therapy (EGDT) (Emanuel Rivers), drotrecogin alfa (activated) (Gordon Bernard), moderate-dose corticosteroids (Djillali Annane), and tight control of blood sugar (Greet Van den Berghe).
The purpose of the roundtable discussion was to provide guidance for clinicians on the integration of new interventions into the ICU to reduce the mortality in sepsis, on appropriate patient selection for these interventions, and on appropriate timing of these interventions.
The present review reports the discussions and recommendations of the panel.
The overall 30-day mortality in the ICU is typically ~20% [8] . The 30-day mortality in the population with severe sepsis, defined as sepsis with organ dysfunction, is 30-50%. It is clear from this figure that severe sepsis contributes disproportionately to the overall 30-day mortality in the ICU and compares unfavorably with some of the higher profile acute killers in hospital (e.g. stroke and AMI) [3] . Despite the general improvements in medicine overall, this mortality rate has remained essentially unchanged for the past 25 years. This has contributed to a feeling of pessimism among Table 1 Possible signs of sepsis (adapted from [ intensivists and other medical professionals regarding treatment prospects for severe sepsis, and a reluctance to rapidly incorporate new interventions into clinical practice [9] .
Although the sepsis mortality rates are unacceptable, they camouflage some significant developments that are and have been occurring for hospital patients, for the general ICU population and, particularly, for those with severe sepsis. Direct comparison of mortality rates among patients with identical Acute Pysiology and Chronic Health Evaluation (APACHE) scores in the placebo arm of anti-TNF or anti-endotoxin studies published 10-15 years ago [10] [11] [12] with more recent studies [13, 14] , demonstrates that the mortality rate is much lower in more recent studies. Interestingly, this decrease was apparent even before the five interventions discussed in the present article were published, reflecting improvements in the general supportive care of sepsis patients.
Indeed, the panel contends that mortality from septic shock has already been reduced. Some patients who in the recent past would have died from severe sepsis or septic shock do not reach the ICU now because they are well managed on the wards, in the emergency department, and even during preoperative and postoperative care. For example, those sepsis patients that receive prompt antibiotic therapy have a 10-15% lower mortality rate than those who receive antibiotic therapy later in their care [15] .
Progress is also being made in diagnosing sepsis: more patients are being tested to identify the source of infection and the pathogens involved, supportive care measures have been improved (e.g. hemodynamic support), and other measures have been put in place to reduce the incidence of nosocomial infections (e.g. reducing the need for pulmonary artery catheters by using echo techniques to assess cardiac function). There has also been a realization of the importance of specially trained intensive care physicians in the ICU. It has been internationally recognized that changing the ICU from an 'open format', whereby patients are cared for by their admitting physician, to a 'closed format', whereby patients are managed by appointed intensivists, reduces mortality rates [16] .
Although the mortality rate is beginning to decline, it still remains unacceptably high. Furthermore, the number of patients with severe sepsis and septic shock is increasing; people are living longer, and there has been a rise in the number of immunocompromised patients due to aggressive cancer therapy and the increased prevalence of HIV.
In-hospital AMI-associated mortality rates averaged approximately 25-30% in the 1960s [3] . This clearly unacceptable mortality rate was addressed by the development of a number of new pharmacological and mechanical interventions together with improvements in supportive care.
In the landmark Second International Study of Infarct Survival  trial, published in 1988, 17,187 suspected AMI patients were  treated with either streptokinase or aspirin, with both drugs, or with neither. The mortality rate in the combination group of this trial was 8%, compared with 13.2% in those patients given neither streptokinase nor aspirin [17] .
Cardiologists have effectively implemented multiple pharmacologic and supportive care interventions to reduce mortality in AMI from 25-30% to 8% and lower. Not satisfied with this already remarkable figure, they are trying to reduce it further.
Physicians treating patients with sepsis are clearly faced with a very different situation to those treating patients with AMI, and so direct comparisons are not possible. However, several factors have contributed to the success of AMI therapy and possibly to the lack of such success in sepsis (Table 2) .
Sepsis is undoubtedly complicated. However, many of the lessons that have been learned through effective application of therapies in other disease states can be applied to severe sepsis. Furthermore, the encouraging data that are beginning to appear in the literature indicate that sepsis may not be as intractable to treat as once thought.
The following sections provide salient information on five interventions that have shown a significant positive impact on mortality rates in sepsis, severe sepsis, septic shock, or sepsis-related diseases in recent clinical trials. The interventions were presented at the roundtable by one of the principal investigators of the key trial of the intervention. Each section concludes with recommendations for the integration of the particular intervention into clinical practice.
The traditional approach in patients with ALI/ARDS is to ventilate using tidal volumes between 10 and 15 ml/kg body weight, almost twice the average tidal volume at rest (7-8 ml/kg body weight), and to maintain a low positive endexpiratory pressure (PEEP). The purpose of this approach is to achieve normal values for the pH and partial pressure of arterial carbon dioxide. However, this method leads to high inspiratory airway pressures and to excessive stretch of the aerated lung.
In 1997, Tremblay et al. examined the effect of ventilation strategy on lung inflammatory mediators in the presence and absence of a pre-existing inflammatory stimulus in Sprague-Dawley rats [18] . In both stimulated and nonstimulated groups, the presence of inflammatory mediators (TNF-α, IL-1β, IL-6, IL-10, macrophage inflammatory protein 2, and IFN-γ) was highest in those rats ventilated with a large tidal volume and zero PEEP. Furthermore, in a study by Ranieri et al. in 1999 [19] , the concentration of inflammatory mediators 36 hours after randomization of the groups was significantly lower in the lung-protective strategy group (tidal volume, 7.6 ± 1.1 ml/kg) than in the control group (tidal volume, 11.1 ± 1.3 ml/kg) (P < 0.05).
Following on from the positive results in the Tremblay et al. trial [18] , a small study (53 patients) was carried out by Amato et al. in Brazil [20] . The mortality rate was 38% in patients given 'protective' ventilation (PEEP above the lower inflection point on the static pressure-volume curve, tidal volume < 6 ml/kg ideal body weight, driving pressures < 20 cmH 2 O above the PEEP value, permissive hypercapnia, and preferential use of pressurelimited ventilatory modes) compared with 71% in patients on conventional ventilation (P < 0.001). This impressive reduction in mortality was tempered by the higher than normal mortality level in the control group, prompting the National Institutes of Health-funded Acute Respiratory Distress Syndrome Network to set up a similar, larger (861 patients), prospective, multicenter, randomized trial in the US [21] .
For a summary of the protocol used in this study, see Appendix 1.
The trial was stopped after the fourth interim analysis because the use of lower tidal volumes was found to be associated with a significantly reduced mortality (P = 0.005 for the difference in mortality between groups).
The primary endpoints were mortality prior to hospital discharge with unassisted breathing and ventilator-free days (days alive, off mechanical ventilation, between enrollment and day 28). Both of these endpoints were achieved (Figs 1-3 ).
In addition, patients receiving a tidal volume of 6 ml/kg ideal body weight had increased organ failure free days and lower IL-6 levels.
ALI is seen in 25-42% of patients with sepsis [22] . Although the approach has only been tested in patients with ALI/ARDS, a tidal volume of 6 ml/kg ideal body weight is at the lower end of the range of physiologic ventilation. Hence, this approach should be suitable for most patients in the ICU setting.
Furthermore, as many patients with severe sepsis or septic shock progress to frank ALI/ARDS, the panel believes that low tidal volume therapy is a valid option in these patients, and an option that may indeed prevent the development of ALI/ARDS.
Although patient selection in the clinical trial specified both blood gas and lung infiltrate criteria, at least 90% of patients in the general ICU setting meet the criteria for blood gas but Table 2 A comparison of acute myocardial infarction (AMI) and sepsis
Market issues Significant publicity surrounding and general awareness Lack of understanding among physicians and the of the condition; large trials general public Diagnosis A relatively straightforward and relatively common Complicated by a long list of signs and symptoms diagnosis (electrocardiogram, enzymes, troponin), and and few objective tools for validation one that can be made by generalists, not just cardiology specialists
Generally single organ disease (notable exception when Often chronic or acute comorbidities complicated by cardiogenic shock)
Generalists have been taught to recognize the signs Sepsis patients often come 'second hand' from a and symptoms of AMI; initial treatment is usually specialist who may not be appropriately trained to provided by emergency physicians, who are trained diagnose, manage, and refer patients with sepsis to treat these patients Mortality prior to hospital discharge in patients receiving a tidal volume of 6 and 12 ml/kg ideal body weight. 
Acidosis is more likely to develop in patients with severe lung problems rather than in those exhibiting milder disease when tidal volumes are kept low. However, acidosis is seldom a clinical problem and rarely requires administration of bicarbonates.
One of the issues with low tidal volume therapy is that the patients are often more uncomfortable, at least initially, when they are being ventilated with a tidal volume of 6 ml/kg ideal body weight. The patients tend to exhibit tachypnea and may become more agitated. Sedation is generally required, but the ventilator setting can be maintained. Of more concern is that ICU staff may consider a respiration rate of 40/min to be a sign of something more serious and may attempt to terminate the intervention. Education of staff is clearly essential.
The strategy assessed in this trial not only includes ventilation with a low tidal volume, but also the provision of extrinsic PEEP. There may be some concern that an increased respiratory rate may result in intrinsic PEEP and hemodynamic problems (e.g. decreased cardiac filling, decreased cardiac output, and diminished blood pressure). The panel believes that auto PEEP was not an issue in the Acute Respiratory Distress Syndrome Network study. In addition, in the groups with low tidal volume, at least 10% more oxygen was required to maintain the fraction of inspired oxygen (FiO 2 ), suggesting that there was very little auto PEEP occurring.
When mechanical ventilation is indicated for treatment of patients with ALI/ARDS, the tidal volume should be limited tõ 6 ml/kg ideal body weight.
Goal-directed therapy represents an attempt to adjust the cardiac preload, afterload, and contractility to balance systemic oxygen delivery with oxygen demand. In patients with severe sepsis and septic shock, such an approach would seem eminently reasonable as part of general supportive measures to restore and maintain adequate cellular perfusion and to prevent organ dysfunction. In the setting of the ICU, however, supranormal and normal approaches have met with little or no success [23, 24] . It is possible that, by the time these therapies are applied in the ICU, any such intervention may have been too late. Hence, the focus has shifted towards hemodynamic optimization in the early presentation of disease, such as in the emergency department.
A prospective, randomized, predominantly blinded study was initiated by the Early Goal-Directed Therapy Collaborative Group to examine the results of hemodynamic interventions in the emergency department [25] . In this study, patients were randomly assigned to either 6 hours of EGDT or to standard therapy prior to admission to the ICU.
Baseline characteristics (including the adequacy and duration of antibiotic therapy) in the EGDT and standard therapy groups were not significantly different. The vital signs, resuscitation endpoints, organ dysfunction scores, and coagulation-related variables were similar in these groups at baseline [25] . However, there were some important differences between the treatment groups (see Table 3 ).
Available online http://ccforum.com/content/6/S3/S1
Proportion of patients alive and off the ventilator having been ventilated with a tidal volume of 6 and 12 ml/kg ideal body weight. Median number of ventilator-free days in patients receiving a tidal volume of 6 and 12 ml/kg ideal body weight. Patients randomized to EGDT received the same therapy but, in addition, were monitored for the endpoint of central venous oxygen saturation (ScvO 2 ) > 70%. EGDT patients were given more intravenous fluids (including blood transfusions) and more inotropic support (mostly dobutamine).
For more information on the protocol used in this study, see Appendix 2.
Key data are presented in Table 4 . The in-hospital mortality was 30.5% in the group assigned to EGDT and was 46.5% in the group assigned to standard therapy (P = 0.009), indicating that EGDT provides significant benefits in improving outcomes in patients with severe sepsis and septic shock.
During the interval from 7 to 72 hours, patients assigned to EGDT exhibited a more significant improvement in mean ScvO 2 (70.4 ± 10.7% versus 65.3 ± 11.4%), in lactate concentration (3.0 ± 4.4 mmol/l versus 3.9 ± 4.4 mmol/), in base deficit (2.0 ± 6.6 mmol/l versus 5.1 ± 6.7 mmol/l), and in pH (7.40 ± 0.12 versus 7.36 ± 0.12) than patients assigned to standard therapy (P ≤ 0.02 for all comparisons). During the same period, the mean APACHE II scores were significantly lower, indicating less severe organ dysfunction, in the patients assigned to EGDT than in those patients assigned to standard therapy (13.0 ± 6.3 versus 15.9 ± 6.4, P < 0.001).
The protocol was based predominantly on guidelines published in 1999 by the Society of Critical Care Medicine [26] . However, these guidelines have not been universally followed in clinical practice since their publication. An increasing number of critically ill patients are presenting to, and being treated in, emergency departments [27, 28] . This is present-ing significant resource challenges in the emergency department environment. The inability to institute EGDT may thus not be a conscious decision by the clinician not to follow the Society of Critical Care Medicine guidelines. Emergency medicine in general may have to develop and formulate the cost-benefit analysis to support or implement such care in this environment in order to improve outcomes.
There are sufficient evidence-based data to recommend that all patients with severe sepsis or septic shock should receive early and aggressive resuscitation based on this EGDT protocol (see Appendix 2) . It is important that the interventions are individualized to each patient. A negative or positive value indicates how the control group therapy compares with the treatment group. a P < 0.001, b P = 0.01, c P = 0.02, d P = 0.03, e P = 0.04. EGDT, early goal-directed therapy. it is possible to identify patients with profound global myocardial dysfunction who are hence at risk of impaired perfusion. These patients, almost 15% of those in the EGDT group, received dobutamine during the first 6 hours because myocardial suppression was diagnosed. Once myocardial dysfunction is corrected (and compliance improved), these patients become more suitable for volume loading, so this group received almost 3.5 liters more fluids in the first 6 hours than the control patients. Therefore, although vasopressor use was similar in the first 6 hours, patients in the EGDT group were more aggressively weaned off these agents during this period, resulting in fewer patients in this group entering the ICU on vasopressors than in the control group. The lack of aggressive volume loading in the control group led to greater use of vasopressors in patients over the subsequent 72 hours. In spite of more volume loading, the EGDT group received less mechanical ventilation over the subsequent 72 hours than in the standard treatment group.
Why was cardiovascular collapse a significant cause of death in the control group?
Cryptic shock (shock with normal vital signs) is a frequent occurrence in early severe sepsis and septic shock. Despite resuscitation to the goals for mean arterial blood pressure and CVP, almost 40% of control patients continued to exhibit global tissue hypoxia (decreased ScvO 2 and increased lactate levels); in these patients, there was a twofold increase in hemodynamic deterioration, requiring more mechanical ventilation, pulmonary artery catheterization, and vasopressor use in the subsequent 72 hours.
How do severe sepsis and septic shock differ hemodynamically in the early stages compared with that classically described in the ICU?
Patients presenting with early sepsis and septic shock are characterized by hypovolemia (low CVP), normal to increased blood pressures, and decreased cardiac output (decreased central venous oxygen saturation and low cardiac index). This is in contrast to ICU patients who are euvolemic, have high ScvO 2 , and have elevated cardiac indices [29] .
What are the most important ways in which EGDT can improve outcomes?
The key factors are early detection of high-risk patients in cryptic shock, early reversal of hemodynamic perturbations and global tissue hypoxia, prevention of acute cardiovascular collapse, and the possibility of preventing the inflammatory aspects of global tissue hypoxia that accompany the inflammation or infection.
Severe sepsis and septic shock patients should receive early aggressive therapy to restore and maintain oxygen availability to the cells. There should also be generous use of fluids and inotropic agents titrated by appropriate hemodynamic monitoring.
Background A large number of observational studies have shown that patients with sepsis have severe depletion of protein C [30, 31] . A number of studies have also shown the association of protein C depletion with high mortality in sepsis [32] [33] [34] . Furthermore, baboon studies have demonstrated that treatment with activated protein C prevents death from live Escherichia coli infusions [35, 36] .
Activated protein C exerts a number of actions. Anticoagulant action includes the inactivation of coagulation factors Va and VIIIa, and the inhibition of the formation of thrombin. Profibrinolytic action allows the activity of tissue plasminogen activator (endogenous tissue plasminogen activator), by inactivating plasminogen activator inhibitor 1 and thrombin activatable fibrinolysis inhibitor. Finally, anti-inflammatory action reduces IL-6 (in vivo) and proinflammatory cytokines (in vitro).
The specific mechanisms by which drotrecogin alfa (activated) exerts its effect on survival in patients with severe sepsis are not completely understood.
The efficacy of drotrecogin alfa (activated) (recombinant human activated protein C) in reducing mortality in patients with severe sepsis was investigated in a large multicenter, blinded, placebo-controlled, randomized, phase III clinical trial, the Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial [14] . All patients in the PROWESS trial received standard supportive care in addition to either drotrecogin alfa (activated) or placebo.
For a summary of the protocol used in the PROWESS study, see Appendix 3.
The overall mortality in patients treated with drotrecogin alfa (activated) was 24.7% compared with 30.8% in patients receiving placebo, an absolute risk reduction of 6.1% (P = 0.006) (see Fig. 4 ). The absolute risk reduction in patients with high risk of death defined by an APACHE II score ≥ 25 was 12.8% (P < 0.001). The absolute risk reduction in patients with high risk of death defined by multiple organ failure was 7.4% (P = 0.006).
No substantial differences in drotrecogin alfa (activated) treatment effects were observed in subgroups defined by gender, ethnic origin, or infectious agent.
Can drotrecogin alfa (activated) be used in patients on dialysis for pre-existing renal failure, a category that was specifically excluded in the PROWESS trial?
No pharmacokinetic data were available on drotrecogin alfa (activated) in patients on chronic dialysis when the PROWESS trial began, so such patients were excluded from the trial. Subsequent research has shown that the pharmacokinetics of drotrecogin alfa (activated) are not substantially changed in patients on chronic dialysis.
The design of the PROWESS trial allowed a maximum of 48 hours between the onset of first organ dysfunction and the receipt of drotrecogin alfa (activated) (a 24-hour window was allowed for receipt of the drug following the first confirmation of first organ dysfunction, which in turn had to have been present for no more than 24 hours). The treatment effect of drotrecogin alfa (activated) was consistent across all time intervals from meeting the entry criteria to the receipt of the study drug. Treatment with drotrecogin alfa (activated) thus does not appear to be as time critical as interventions such as tissue plasminogen activator in stroke or myocardial infarction. Because most of the experience with drotrecogin alfa (activated) was based on organ failure times less than 48 hours, treatment should not be delayed when an appropriate candidate is identified. The time window employed in the PROWESS trial should allow a full history to be taken and other tests to be performed to determine the bleeding risk.
As with all anticoagulants, drotrecogin alfa (activated) is associated with a risk of severe bleeding. During the infusion period in the PROWESS trial, the bleeding rates were 2.4% in the drotrecogin alfa (activated) group versus 1.0% in the placebo group (P = 0.024). The risk of bleeding was fairly constant across most subgroups. However, severe thrombocytopenia (< 30,000/mm 3 ) was commonly associated with serious bleeding and intracerebral hemorrhage.
Patients at high risk of death in the PROWESS trial were most likely to benefit from drotrecogin alfa (activated). In the PROWESS trial, the APACHE II score was the most effective predictor of risk of death and likelihood of benefit from drotrecogin alfa (activated), particularly in those patients with an APACHE II score ≥ 25.
In the PROWESS trial, the number of organ dysfunctions was also an important indicator that supported an association between likelihood of benefit from drotrecogin alfa (activated) and risk of death. Two or more organ dysfunctions identify a population that responds well to therapy, and is a practical measurement.
The panel believes that acute respiratory failure or hypotension unresponsive to fluid challenge should suggest the use of drotrecogin alfa (activated). However, coagulopathy, a platelet count < 80,000/mm 3 , acidosis, or low urine output alone should not suggest its use.
A very large international study of 11,500 patients will be started in late 2002 to investigate the efficacy of drotrecogin alfa (activated) in patients with a single organ failure and/or APACHE scores < 25.
The decision on whether to administer the drug should ultimately depend on whether the patient meets the selection criteria. A patient presenting in the emergency room with acute respiratory failure or acute cardiovascular decompensation should receive appropriate treatment there.
The drawback to treatment in the emergency room is that there may not be sufficient time in which to evaluate the patient's bleeding risks. Delaying treatment for a few hours will enable more tests to be performed and a fuller history to be taken, both of which will provide a better indication of whether drotrecogin alfa (activated) is appropriate.
The dose is always the same (24 µg/kg/hour), regardless of the type of organ failure or the degree of sepsis severity. In addition, the 96-hour window of treatment is always the same so that interruptions of treatment are made up at the end to maintain a total of 96 hours of treatment. Twenty-eight-day survival in patients treated with drotrecogin alfa (activated) or placebo: all-cause mortality. 
Do patients require any laboratory testing before they receive drotrecogin alfa (activated)?
No laboratory testing was carried out in the PROWESS trial, and subgroup analysis identified no biochemical marker that conclusively indicates treatment. For example, treatment-associated reductions in mortality were observed in patients with normal protein C levels and in those with low protein C levels. Clinical criteria are recommended for the initiation of therapy.
Aspirin (650 mg/day) was allowed in the PROWESS trial. Patients on glycoprotein IIb/IIIa inhibitors were excluded because no data were available regarding drug interactions and pharmacokinetics. Use of these types of agents is likely to increase the risk of bleeding with drotrecogin alfa (activated) therapy. The anticipated benefits must therefore be weighed against the potential risks. In the PROWESS trial, efforts were made to correct the international normalized ratio towards normal if it was greater than 3 at any time during infusion of drotrecogin alfa (activated).
Approximately one-third of patients in the PROWESS trial received steroids at the same time as drotrecogin alfa (activated). There was no interaction with steroid use, presumably because the mechanism of action of steroids is so different from that of activated protein C. Hence, steroids should be used if they are needed, and if the patient qualifies for drotrecogin alfa (activated) the two should be used together.
Drotrecogin alfa (activated) should be considered for use in all adult patients with recent onset severe sepsis or septic shock, and a high risk of death.
The value of steroids in the treatment of patients with severe sepsis and septic shock has been fiercely debated for some time. Although a number of well-designed, randomized, controlled trials failed to show any benefits of steroid therapy in terms of improved survival in patients with severe sepsis (reviewed in [37, 38] ), with mortality increased in many as a result of an increased incidence of nosocomial infections, these trials were primarily investigating the efficacy of short courses of high-dose steroids. The question of whether lower doses of steroids may provide benefit in these patients has only recently been addressed.
There is a relatively strong rationale for considering the use of steroids in patients with refractory septic shock. Relative adrenal insufficiency is common in patients with refractory septic shock (50-75% of patients) [39] . In addition to such relative adrenal insufficiency and the blunted response to corticotrophin, a large body of evidence indicates that sepsis and refractory septic shock are characterized by peripheral tissue resistance to corticosteroids [40, 41] . In septic patients, this can be evidenced in a variety of ways. First, global cortisol binding, which carries cortisol from the adrenal glands to the tissues, decreases in patients with severe sepsis [42] . Second, the number and binding affinity of glucocorticosteroid receptors may be reduced in patients with sepsis and severe sepsis [43] , leading to a decrease in the conversion of cortisone to its active form, cortisol, particularly by IL-2 levels in the tissues. Finally, data have been published demonstrating that moderate doses of steroids may restore cell sensitivity to vasopressors [44] . This may reduce the intensity of the inflammatory response and decrease organ dysfunction. Low-dose steroid treatment is also well tolerated [40] .
This body of evidence prompted the initiation of a phase III randomized, controlled trial performed in 19 centers in France with 300 patients [45] . The aim of the trial was to determine whether moderate-dose corticosteroid therapy affected survival in patients with refractory septic shock and adrenal insufficiency.
All patients had to be treated with vasopressor agents and mechanical ventilation. For a summary of the protocol used in this study, see Appendix 4.
Patients were stratified according to their response to the adrenocorticotrophic hormone (ACTH) test. Nonresponders were defined by an increment in cortisol levels < 9 µg/dl or < 250 nM/l after challenge with 250 µg cosyntropin. Of the 300 patients included, there were 229 nonresponders to the corticotropin test (placebo, 115 patients; steroids, 114 patients). A significant survival benefit was demonstrated among nonresponders receiving moderate-dose corticosteroids. There were 73 deaths in the placebo group (63%) and 60 deaths in the steroid group (53%) (hazard ratio, 0.67; 95% confidence interval, 0.47-0.95; P = 0.023).
No beneficial effects were observed in the subset of patients who were classified as responders. Hence, in this paradigm, the ACTH test serves as a useful prognostic measure. Since a beneficial effect was observed in the total population, however, the need for an ACTH test can be challenged and further studies are required.
If an ACTH test is performed, corticosteroid administration can be started before results are received.
Moderate-dose corticosteroids should be administered to patients with established refractory septic shock.
What is the optimal dose for this intervention?
Hydrocortisone should be given daily at a dose of 200-300 mg. Fludrocortisone should be given daily at a dose of 50 µg.
What is the optimal duration for this intervention?
Moderate doses of steroids should be given for 7 days.
Hydrocortisone can be administered as serial boluses or as a continuous infusion. It may be that rebound phenomena at treatment discontinuation are more frequent when hydrocortisone is given as a continuous infusion. In addition, in the phase III randomized trial, hydrocortisone was given as serial boluses.
The phase III randomized trial has shown that the combination of hydrocortisone and fludrocortisone increased survival. In addition, sepsis is more frequently associated with a mineralocorticoid deficiency than a glucocorticoid deficiency. Hence, fludrocortisone should be added to hydrocortisone.
Administration of moderate-dose corticosteroids should be considered in cases of refractory septic shock, particularly in those with relative adrenal insufficiency. It is recommended that an ACTH test be carried out before starting the intervention.
Hyperglycemia, caused by insulin resistance in the liver and muscle, is a common finding in ICU patients. It can be considered an adaptive response, providing glucose for the brain, red cells, and wound healing, and is generally only treated when blood glucose increases to > 215 mg/dl (> 12 mmol/l).
Previous studies have shown that high levels of insulin-like growth factor binding protein 1 (a very good marker of lack of hepatic insulin effect) predict mortality [46, 47] . Patients with high insulin-like growth factor binding protein 1 also tend to have the lowest insulin levels, indicating that beta cell function is impaired and, therefore, not enough insulin is being produced. These results indicate that hyperglycemia may not always be adaptive and that it should be treated to avoid the onset of specific complications.
Nevertheless, conventional wisdom in the ICU has been that hyperglycemia is beneficial and that hypoglycemia should be avoided.
The hypothesis that hyperglycemia (> 110 mg/dl, > 6.1 mmol/l) predisposes to specific ICU complications, prolonged intensive care dependency and death was tested in a prospective, randomized, controlled trial [48] .
For a summary of the protocol used in this study, see Appendix 5.
Thirty-five of the 765 patients (4.6%) in the intensive insulin group died in the ICU, compared with 63 patients (8.0%) in the conventional therapy group. For further mortality data on both the length of hospital stay and the cause of death, see Tables 5 and 6 . For morbidity data, see Figure 5 .
Tight control of blood sugar, as outlined in Appendix 5, requires a strict protocol for insulin administration and repeated determination of blood sugar.
This is yet to be proven, and is the subject of an ongoing study. Because medical patients tend to stay in the ICU longer than surgical patients, the results from this study indicate that this intervention would be even more favorable to medical ICU patients. However, one needs to be careful with application of the algorithm in certain disease states, especially severe hepatic dysfunction and renal failure.
No, all carbohydrates are included. See Appendix 5 for guidelines on feeding.
The level was chosen because it is in the physiologic range for healthy people.
As well as its effect on glycemia, insulin has been shown to inhibit TNF-α and macrophage inhibitory factor (when infused concomitantly with glucose). This has led to some doubts as to whether the effect in this study was due to normalization of blood glucose levels. However, multivariate analysis of all the risk factors for mortality, including severity of illness on admission, indicated that blood glucose determines the outcome; there was a 75% increase in risk of death per 50 mg/dl increase in blood glucose.
It is not yet possible to determine this. Although it was blood glucose levels that were measured, the effects of insulin may in fact be on free fatty acids, as they change in parallel with S11 blood glucose. One of the key mechanisms may be prevention of hypertriglyceridemia and high concentrations of free fatty acids.
It is strongly advisable to tightly control blood sugar close to physiologic levels, especially in surgical patients. Implemen-tation of this recommendation requires a well-defined ICU protocol.
The interventions discussed in the present article have been applied in different patient populations and at different times in the course of the disease (see Table 7 ).
It is essential for physicians to understand that these therapies are not mutually exclusive. Optimal patient management may require a combination of approaches: mechanical ventilation to preserve lung function, hemodynamic support to maintain adequate ScvO 2 , intensive insulin therapy to normalize blood sugar, steroids to provide adequate immunosuppression, and drotrecogin alfa (activated) to prevent the systemic coagulopathy characteristic of severe sepsis and, hence, to preserve organ function. A sound understanding of the indications and contraindications of these interventions will guide appropriate intervention.
Similarly, the timing of therapy needs to be closely monitored. Education in the signs and symptoms of sepsis and severe sepsis should prompt early initiation of therapy. Many of the interventions discussed in this article were tested at specific Available online http://ccforum.com/content/6/S3/S1 Multiple organ failure, no sepsis focus 18 14 Multiple organ failure, with sepsis focus 33 8
Most important effects on morbidity [46] . CVVH, continuous venovenous hemofiltration; ICU, intensive care unit; NNT, number needed to treat; RRR, relative risk reduction. 
Despite the wealth of data to support the approaches discussed, it is clear that uptake of these interventions into clinical practice has been slow. Although there may be practical reasons for this, it would appear in many cases to involve either unfamiliarity with the data or a reluctance, or at least inertia, to change established practices (witness the necessity of proving that hypoglycemia is beneficial in ICU patients despite no good evidence to the contrary).
The ICU has changed in the past 30 years; there are more tools to use and more interventions to implement. Despite application of new methods, however, outcomes have changed very little and certainly not in proportion to the changes that were expected based on the results from clinical trials. Efficient integration of new interventions into the wider ICU population is clearly essential.
The panel believes that optimal use of existing therapies and the integration of proven new therapies will reduce mortality rates. Further positive results from new trials with improved trial designs should encourage intensivists to incorporate new interventions into their practice.
Protocols are essential to ensure efficient integration of new therapies and to improve outcomes on the wards. Morris predicted in a recent paper that an increase in compliance with evidence-based recommendations through the use of protocols would decrease error and would enhance patient safety [49] . However, a complete treatment protocol is only effective when each ward (inside and outside of the ICU) has the trained staff to implement it, and when a skilled intensive care physician is available to lead the team. Training and education of staff is essential. 
All five of the interventions discussed in this article have generated convincing evidence for their use, and they hold out hope for reducing mortality in patients with sepsis, severe sepsis and septic shock. Yet, despite compelling data, the application of these interventions has yet to become routine practice in most ICUs.
It is our hope that this article will enable physicians to understand how best to apply these therapies in clinical practice; from appropriate patient selection and timing of therapy, to combining different approaches for optimal patient management.
A willingness to embrace new interventions, coupled with the development and implementation of rigorous protocols to ensure appropriate use, will improve outcomes and lead to a substantial reduction in mortality in these patients.
• A respiratory rate ≥ 20 breaths/min or a partial pressure of arterial carbon dioxide ≤ 32 mmHg, or the use of mechanical ventilation for an acute respiratory process. • A white cell count ≥ 12,000/mm 3 or ≤ 4000/mm 3 , or a differential count showing >10% immature neutrophils.
Patients should meet at least one of the following five criteria: 
• Pregnancy or breastfeeding.
• Aged younger than 18 years or weight >135 kg.
• Platelet count < 30,000/mm 3 .
• Conditions that increase the risk of bleeding: • surgery requiring general or spinal anesthesia within 12 hours before the infusion, the potential need for such surgery during the infusion, or evidence of active bleeding postoperatively; • a history of severe head trauma requiring hospitalization, intracranial surgery, or stroke within 3 months before the study, or any history of intracerebral arteriovenous malformation, cerebral aneurysm, or mass lesions of the central nervous system; • a history of congenital bleeding diatheses; gastrointestinal bleeding within 6 weeks before the study unless corrective surgery had been performed; or • trauma considered to increase the risk of bleeding. • A known hypercoagualable condition including:
• resistance to activated protein C; • hereditary deficiency of protein C, protein S, or antithrombin III; • presence of anticardiolipin antibody, antiphospholipid antibody, lupus anticoagulant, or homocysteinemia; or • recently documented (within 3 months) or highly suspected deep-vein thrombosis or pulmonary embolism. • Patient's family or physician, or both, not in favor of aggressive treatment of the patient, or the presence of an advanced directive to withhold life-sustaining treatment.
• Patient not expected to survive 28 days because of an uncorrectable medical condition, such as poorly controlled neoplasm or other end-stage disease. • Moribund state in which death is perceived to be imminent. • Human immunodeficiency virus infection in association with a last known CD4 cell count ≤ 50/mm 3 . • History of bone marrow, lung, liver, pancreas, or smallbowel transplantation. • Chronic renal failure requiring hemodialysis or peritoneal dialysis (acute renal failure was not an exclusion criterion). • Known or suspected portosystemic hypertension, chronic jaundice, cirrhosis, or chronic ascites. • Acute pancreatitis with no established source of infection.
• Participation in an investigational study within 30 days before treatment. • Use of any of the following medications or treatment regimens:
• unfractionated heparin to treat an active thrombotic event within 8 hours before the infusion (prophylactic treatment with a dose of unfractionated heparin of up to 15,000 U/day was permitted); • low molecular weight heparin at a higher dose than recommended for prophylactic use (as specified in the package insert) within 12 hours before the infusion; • warfarin (if used within 7 days before study entry and if the prothrombin time exceeded the upper limit of the normal range for the institution); • acetylsalicylic acid at a dose of more than 650 mg/day within 3 days before the study; • thrombolytic therapy within 3 days before the study (thrombolytic agents permitted for the treatment of thromboses within a catheter); • glycoprotein IIb/IIIa antagonists within 7 days before study entry; • antithrombin III at a dose of more than 10,000 U within 12 hours before the study; • protein C within 24 hours before the study.
Drotrecogin alfa (activated) should be given at a dose of 24 µg/kg/hour for 96 hours.
Infusion should be interrupted 1 hour prior to any percutaneous procedure or major surgery, and should be resumed 1 and 12 hours later, respectively, in the absence of bleeding complications. 
There was an 8-hour time window from shock onset to check for eligibility and to perform a short ACTH test (blood samples before and 30 and 60 min after a 250 µg intravenous bolus of tetracosactrin). Patients were then randomly assigned to receive 50 mg hydrocortisone as an intravenous bolus every 6 hours and one 50 µg tablet of fludrocortisone through a nasogastric tube once a day, or their respective placebos. Treatments were given for 7 days, and patients were followed up for 1 year.
On admission, patients should receive continuous intravenous glucose (200-300 g over 24 hours). After 24 hours, total parenteral, combined parenteral and enteral, or total enteral feeding should be instituted: 20-30 nonprotein kcal/kg/day with a balanced composition (0.13-0.26 g nitrogen/kg/day and 20-40% nonprotein calories in the form of lipids). Total enteral feeding should be attempted as early as possible. Synergistic Roles of Eukaryotic Translation Elongation Factors 1Bγ and 1A in Stimulation of Tombusvirus Minus-Strand Synthesis Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3′ end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation. Plus-stranded (+)RNA viruses recruit numerous host proteins to facilitate their replication and spread [1, 2] . Among the identified host proteins are RNA-binding proteins (RBPs), such as ribosomal proteins, translation factors and RNA-modifying enzymes [1] [2] [3] [4] [5] . The subverted host proteins likely affect several steps in viral RNA replication, including the assembly of the replicase complex and initiation of RNA synthesis. However, the detailed functions of recruited host RBPs in (+)RNA virus replication are known only for a small number of host factors [2, [6] [7] [8] .
Tomato bushy stunt virus (TBSV) is model plant RNA virus coding for two replication proteins, p33 and p92 pol , which are sufficient to support TBSV replicon (rep)RNA replication in a yeast (Saccharomyces cerevisiae) model host [9, 10] . p33 and p92 pol are components of the membrane-bound viral replicase complex, which also contains the tombusviral repRNA serving not only as a template for replication, but also as a platform for the assembly of the viral replicase complex [11] [12] [13] . Recent genome-wide screens and global proteomics approaches with TBSV and a yeast host revealed a large number of host factors interacting with viral components or affecting TBSV replication. The identified host proteins are involved in various cellular processes, such as translation, RNA metabolism, protein modifications and intracellular transport or membrane modifications [14] [15] [16] [17] .
Various proteomics analyses of the highly purified tombusvirus replicase has revealed at least five permanent resident host proteins in the complex, including the heat shock protein 70 chaperones (Hsp70) [18] [19] [20] [21] , glyceraldehyde-3-phosphate dehydrogenase [4] , pyruvate decarboxylase [21] , Cdc34p E2 ubiquitin conjugating enzyme [4, 21, 22] , eukaryotic translation elongation factor 1A (eEF1A) [23, 24] and two temporary resident proteins, Pex19p shuttle protein [25] and the Vps23p adaptor ESCRT protein [24, 26, 27] . The functions of several of these proteins have been studied in some detail [4, 17, 18, 19, 20] .
The emerging picture from systems biology approaches is that eukaroyotic translation elongation factors (eEFs), such as eEF1A, play several roles during TBSV replication. Accordingly, eEF1A has been shown to facilitate the assembly of the viral replicase complex and stimulate the initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) [23, 24] . Another translation elongation factor identified in our genomewide screens with TBSV is eukaryotic elongation factor 1Bgamma (eEF1Bc) [15] . eEF1Bc is an abundant, but not essential cellular protein, which is part of the eukaryotic translation elongation factor 1B complex also containing the eEF1Ba subunit in yeast and the eEF1Ba and eEF1Bd subunits in metazoans [28] .The eEF1B complex is the guanine nucleotide exchange factor for eEF1A, which binds and delivers aminoacyl-tRNA in the GTPbound form to the elongating ribosome. Additional roles have been ascribed to eEF1Bc in vesicle-mediated intracellular protein transport, RNA-binding, vacuolar protein degradation, oxidative stress, intermediate filament interactions and calcium-dependent membrane-binding [29, 30, 31] .
In this paper, we characterize the function of eEF1Bc in TBSV replication. Our approaches based on yeast and in vitro replication assays reveal that eEF1Bc is a component of the tombusvirus replicase and binds to the 39-end of the viral RNA. Using a cellfree replication assay, we define that eEF1Bc plays a role by enhancing minus-strand synthesis by the viral replicase. The obtained data support the model that eEF1Bc opens up a 'closed' structure at the 39-end of the TBSV (+)RNA, rendering the RNA compatible for initiation of (-)-strand synthesis. Moreover, we find that eEF1Bc and eEF1A play nonoverlapping functions to enhance (-)-strand synthesis. Altogether, the two translation factors regulate TBSV replication synergistically by interacting with different portions of the viral (+)RNA and the replication proteins.
Deletion of eEF1Bc inhibits TBSV RNA accumulation in yeast model host eEF1Bc is coded by TEF3 and TEF4 nonessential genes in yeast [32, 33] . Single deletion of TEF3(CAM1) or TEF4 reduced TBSV repRNA accumulation to ,25% ( Figure 1A , lanes 3-8), while deletion of both genes resulted in even more inhibition, supporting TBSV repRNA accumulation only at 15% level (lanes 9-11). Expression of eEF1Bc (Tef4p) in tef4D yeast increased TBSV replication to ,80%, demonstrating that the defect in TBSV repRNA replication in tef4D yeast can be complemented.Altogether, these data established that eEF1Bc plays an important stimulatory role in TBSV replication.
To obtain direct evidence on the involvement of eEF1Bc in TBSV replication, we prepared cell-free extracts (CFE) from a yeast strain lacking the TEF4 gene or from wt yeast. These yeast extracts contained comparable amount of total proteins ( Figure 1C , right panel). The CFE extracts were programmed with the TBSV (+)repRNA and purified recombinant p33 and p92 pol obtained from E. coli. Under these conditions, the CFE supports the in vitro assembly of the viral replicase, followed by a single cycle of complete TBSV replication, resulting in both (-)-stranded repRNA and excess amount of (+)-stranded progeny [20, 34] . Importantly in the case of a translation factor, this assay uncouples the translation of the viral proteins from viral replication, which are interdependent during (+)RNA virus infections. CFE obtained from tef4D yeast supported only 29% of TBSV repRNA replication when compared with the extract obtained from wt yeast ( Figure 1C , lane 2 versus 4). These data demonstrate that Tef4p plays an important role in the activity of the viral replicase complex.
To test if the decrease in TBSV repRNA replication in vitro was due to reduced (+) or (-)-strand synthesis, we measured the replication products under non-denaturing versus denaturing conditions ( Figure 1C ). We found that the amount of dsRNA [representing the newly-synthesized 32 P-labeled (-)RNA product hybridized with the input (+)RNA; lane 1, Figure 1C , see also ref. [23] ] and the newly-synthesized (+)RNA both decreased by ,3fold in CFE obtained from tef4D yeast in comparison with those products in the wt CFE (lane 3). Since the ratio of dsRNA and ssRNA did not change much in the CFEs ( Figure 1C ), the obtained data are consistent with the model that Tef4p (eEF1Bc) affects the level of (-)RNA production, which then leads to proportionately lower level of (+)RNA progeny.
Adding purified recombinant eEF1Bc to CFE from tef4D yeast supported TBSV repRNA replication to similar extent as the CFE from wt yeast (i.e., containing wt eEF1Bc, Figure 1D , lanes 3-6 versus 1-2), indicating that the recombinant eEF1Bc can complement the missing Tef4p in vitro, when the same amount of p33 and p92 pol was provided. Using large amount of eEF1Bc in the CFE-based assay did not further increase TBSV repRNA replication ( Figure 1D , lanes 3-4), suggesting that eEF1Bc should be present in optimal amount during TBSV replication.
To obtain additional evidence if eEF1Bc could stimulate RNA synthesis by the viral RdRp, we used the E. coli-expressed recombinant p88C pol RdRp protein of Turnip crinkle virus (TCV). The TCV RdRp, unlike the E. coli-expressed TBSV p92 pol or the closely-related Cucumber necrosis virus (CNV) p92 pol RdRps, does not need the yeast CFE to be functional in vitro [35, 36] . Importantly, the template specificity of the recombinant TCV RdRp with TBSV RNAs is similar to the closely-related tombusvirus replicase purified from yeast or infected plants [10, 36, 37, 38] . The recombinant TCV RdRp preparation lacks co-purified eEF1Bc (E. coli does not have a homolog), unlike the yeast or plant-derived tombusvirus replicase preparations, facilitating studies on the role of eEF1Bc on the template activity of a viral RdRp. When we added various amounts of the highly purified recombinant eEF1Bc to the TCV RdRp assay programmed with TBSVderived SL3-2-1(+) RNA template, which is used by the TCV RdRp in vitro to produce the complementary (-)RNA product [37] , we observed a ,2-to-4-fold increase in (-)RNA synthesis by the TCV RdRp (Figure 2A , lanes [3] [4] [5] . eEF1Bc in the absence of the TCV RdRp did not give a 32 P-labeled RNA product, excluding that our eEF1Bc preparation contained RdRp activity (not shown). Altogether, our data suggest that eEF1Bc can stimulate in vitro activity of TCV RdRp on a TBSV (+)RNA template, confirming a direct role for eEF1Bc in viral (-)RNA synthesis by a viral RdRp.
To test if the stimulating activity of eEF1Bc on the in vitro RdRp activity was due to binding of eEF1Bc to the (+)RNA template and/or to the TCV RdRp protein, we performed assays, in which the recombinant eEF1Bc was pre-incubated with the TCV RdRp or the (+)RNA template prior to the RdRp assay. These experiments revealed that pre-incubation of the purified eEF1Bc with the TBSV-derived SL3-2-1(+) RNA template prior to the RdRp assay led to a ,4.5-fold increase in (-)RNA products ( Figure 2B, lanes 1-2) . In contrast, pre-incubation of the TCV Author Summary RNA viruses recruit numerous host proteins to facilitate their replication and spread. Among the identified host proteins are RNA-binding proteins (RBPs), such as ribosomal proteins, translation factors and RNA-modifying enzymes. In this paper, the authors show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bc) reduces Tomato bushy stunt virus (TBSV) replication in a yeast model host. Knock down of eEF1Bc level in plant host also decreases TBSV accumulation. Moreover, the authors demonstrate that eEF1Bc binds to the viral RNA and is present in the tombusvirus replicase complex. Functional studies revealed that eEF1Bc promotes minusstrand synthesis by serving as an RNA chaperone. The authors also show that eEF1Bc and eukaryotic translation elongation factor 1A, another host factor, function together to promote tombusvirus replication. Cell-free TBSV replicase assay supports a role for eEF1Bc in minus-strand synthesis. Purified recombinant TBSV p33 (12 pmol) and p92 pol (1 pmol) replication proteins in combination with DI-72 (+)repRNA (4 pmol)were added to the whole cell extract prepared from tef4D (lanes 1-2) or WT yeast strains. Left panel: The nondenaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. The full-length single-stranded repRNA is pointed at by an arrow. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heattreated replicase products (ssRNA is present). The amount of ssRNA and the ratio of ssRNA/dsRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (-)RNA and the input (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right panel shows the coomassie-blue stained SDS-PAGE gel to visualize total protein levels in the whole cell extracts. (D) eEF1Bc stimulates TBSV repRNA synthesis in whole cell extract prepared from tef4D. Increasing amounts of purified recombinant eEF1Bc (lanes 3-4, 26 pmol; lanes 5-6, 13 pmol) were added to tef4D CFE and the in vitro synthesized 32 P-labeled TBSV repRNA was measured on denaturing PAGE. See further details in panel C. Note that the recombinant eEF1Bc added to the tef4D CFE is about 10-fold less than the total eEF1Bc present in the WT CFE. doi:10.1371/journal.ppat.1002438.g001
RdRp with the (+)RNA template ( Figure 2B , lanes 3-4) or eEF1Bc with the TCV RdRp ( Figure 2B , lanes 7-8) prior to the RdRp assay did not result in increase in (-)RNA synthesis. Overall, data shown in Figure 2B imply that eEF1Bc can stimulate (-)RNA synthesis only when eEF1Bc binds to the (+)RNA template before the RdRp binding to the template.
To further test the stimulatory effect of eEF1Bc, we also tested the RdRp activity in the presence of eEF1Bc using a mutated (+)RNA template. The mutation [SL3-2-1m(+)] opens up the closed structure in the promoter region that leads to increased template activity [39] . The mutated template showed only ,2-fold increased RNA products in the RdRp assay with eEF1Bc ( Figure 2C , lanes 3-4 versus 1-2). In contrast, eEF1Bc did not stimulate RNA products when the negative-stranded RI-III(-) RNA was used as a template in the TCV RdRp assay ( Figure 2C , lanes 9-10 versus 7-8). Thus, these data support the model that eEF1Bc can mainly stimulate (-)-strand synthesis by the RdRp on the wt 39 TBSV sequence, while it is not effective on the (-)RNA template.
To test if eEF1Bc directly binds to a particular region within the TBSV repRNA, we performed electrophoretic mobility shift (EMSA) experiments with purified eEF1Bc and 32 P-labeled regions of (+)repRNA that included known cis-acting elements involved in (-)RNA synthesis [39, 40, 41] . These experiments revealed that eEF1Bc bound efficiently to the 39-end of the TBSV (+)repRNA (construct SL3-2-1, carrying the terminal 3 stem-loop structures, Figure S1 ). Template competition experiments confirmed that SL3-2-1 RNA bound competitively to eEF1Bc in vitro( Figure S1B ).
To further define what sequence within SL3-2-1 is bound by eEF1Bc, we used complementary DNA oligos to partially convert portions of SL3-2-1 into duplexes (RNA/DNA hybrids) as shown The TCV RdRp assay had two steps: first, the shown components were incubated at room temperature to facilitate their interaction, followed 5 min later the addition of the shown component and the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 5-6 and 9-10). The RNA transcript (20 pmol), eEF1Bc (20 pmol) and purified TCV RdRp (2 pmol) were used in these assays. (C) The effect of eEF1Bc on the TCV RdRp activity with additional templates. One of the templates was SL3-2-1 m(+) with a point mutation within the promoter sequence (carrying SL1m mutation), which is being used more efficiently than the wt SL3-2-1(+) by the TCV RdRp in vitro. The second template was RI-III ( in Figure 3A . EMSA assay with purified recombinant eEF1Bc revealed that the very 39-terminal SL1 region had to be ''free'' (not part of the duplex) for eEF1Bc to bind efficiently to the SL3-2-1 RNA (compare lane 1 with lane 5 in Figure 3A ).
Since eEF1Bc is known to bind to A-rich single-stranded sequences [32] , we mutagenized the tetraloop (GAAA) sequence to either CUUG or GUUU tetraloop sequences ( Figure 3B ) that are expected to maintain the stability of the double-stranded stem. EMSA analysis showed that neither RNAs with the new tetraloop sequences bound efficiently to eEF1Bc ( Figure 3B , lanes 5-7 and 11-13). Based on the EMSA data, we conclude that the GAAA tetraloop region of SL1 is an efficient binding site for eEF1Bc in vitro. However, we cannot exclude that eEF1Bc binding may be dependent on stabilizing effects of the GNRA tetraloop on the stem structure. The loop nucleotides may or may not be involved in protein-RNA contacts.
Binding of eEF1Bc to the 39 end of the TBSV RNA is required for stimulation of (-)-strand RNA synthesis in vitro
To examine if binding of eEF1Bc to SL1 is important for stimulation of (-)-strand RNA synthesis by the viral RdRp, we performed an in vitro RNA synthesis assay using a mutated SL3-2-1 carrying the 'CUUG' tetraloop instead of the wt 'GAAA' tetraloop sequence ( Figure 4A ). Unlike for the wt SL3-2-1 RNA, eEF1Bc could not stimulate complementary RNA synthesis by the viral RdRp on the SL3-2-1cuug(+) template ( Figure 4A , lanes 7-10 versus 1-4). These data suggest that binding of eEF1Bc to the 'GAAA' tetraloop sequence of SL1 is important to stimulate (-)strand synthesis by the viral RdRp in vitro.
Since the TBSV (+)RNA, including the minimal SL3-2-1 sequence, forms a secondary structure where the replication silencer sequence (RSE) in SL3 base-pairs with the 39-terminal 5 nts within the genomic promoter (gPR) (both sequences are highlighted with gray boxes in Figure 4A ), it is possible that eEF1Bc helps (-)-strand synthesis by opening up the gPR. The single-stranded gPR sequence would be more accessible for (-)strand synthesis as shown based on RNA mutagenesis [39] . To test this model, we obtained a complementary RNA that formed a duplex with SL1 and neighboring sequences, but leaving SL1 including the 'GAAA' loop-sequence nonbase-paired to facilitate binding to eEF1Bc ( Figure 4B ). Interestingly, eEF1Bc was able to stimulate (-)-strand synthesis by 70%, suggesting that eEF1Bc might indeed facilitate opening up the 39-terminal structure when it is part of a duplex. eEF1Bc co-purifies with the viral replicase complex and it binds to TBSV repRNA in yeast To test if eEF1Bc is a component of the tombusvirus replicase, we purified the His 6 -Flag-tagged p33 (HF-p33) replication protein via Flag-affinity purification from the detergent-solubilized membrane fraction of yeast [10] . We detected both p33 and eEF1Bc in the purified preparation ( Figure 5A , lane 1), suggesting that eEF1Bc is likely part of the replicase complex [21] . Importantly, eEF1Bc was not found in the control samples containing the His 6 -tagged p33 (H-p33) that were also purified via the Flag-affinity procedure ( Figure 5A , lane 2). Since eEF1Bc does not seem to bind to p33 or p92 replication proteins (data not shown), it is likely that eEF1Bc was co-purified with p33 via the viral RNA template in the viral replicase complex.
To demonstrate that eEF1Bc can indeed bind to the TBSV (+)repRNA in cells, we Flag-affinity-purified His 6 -Flag-tagged eEF1Bc from the detergent-solubilized membrane fraction and also from the soluble (cytosolic) fraction of yeast. Interestingly, the viral RNA was co-purified with eEF1Bc from both fractions ( Figure 5B, lanes 3 and 7) . These data confirmed that eEF1Bc binds to the viral RNA in yeast.
Since eEF1Bc was found in association with the TBSV repRNA in the cytosolic fraction of yeast, it is possible that eEF1Bc might affect the viral RNA recruitment from the cytosol into replication that takes place on the peroxisomal or ER membrane surfaces [42, 43] . Therefore, we tested the recruitment of the TBSV (+)repRNA to the membrane fraction in our CFE assay [23] . We found that eEF1Bc did not facilitate the association of the TBSV (+)repRNA with the membrane when applied in the absence of p33/p92 replication proteins ( Figure S2 ). Moreover, eEF1Bc did not further increase the amount of TBSV (+)repRNA bound to the membrane in the presence of p33/p92 replication proteins, which are needed for RNA recruitment ( Figure S2 , lanes 3-4 and 8-10) [24] . Therefore, we conclude that eEF1Bc is unlikely to promote the recruitment of the TBSV (+)repRNA to the membrane.
Since both eEF1Bc and eEF1A bind to the 39-terminal region of the TBSV (+)RNA ( Figure 3 ) and ref: [23, 24] , it is possible that they could affect each other's functions during replication. To test the mutual effect of eEF1Bc and eEF1A on the (-)-strand RNA production of the viral RdRp, we performed in vitro RdRp assays with purified eEF1A and recombinant eEF1Bc as shown in Figure 6 . Based on previous experiments, eEF1Bc was known to stimulate (-)-strand synthesis the most when pre-incubated with the template (+)RNA ( Figure 2B ). In contrast, pre-incubation of eEF1A with the viral RdRp was more effective than preincubation of eEF1A with the template RNA [23] . Therefore, we performed the pre-incubation experiments prior to the RdRp assay as shown in Figure 6 . We found the largest stimulation of (-)-strand synthesis by the viral RdRp in a dual pre-incubation assay, when eEF1Bc was pre-incubated with the viral RNA template, while eEF1A was separately pre-incubated with the viral RdRp ( Figure 6, lanes 3-4) . Pre-incubation of eEF1Bc with the viral RNA template (lanes 5-6) or pre-incubation of eEF1A with the viral RdRp (lanes 7-8) were about half as efficient in stimulation of (-)-strand synthesis than the dual pre-incubation assay (lanes [3] [4] . Therefore, these data support the model that eEF1Bc and eEF1A both promote (-)-strand synthesis and their effect is synergistic, likely involving separate mechanisms (see Discussion). in vitro binding assay with purified eEF1Bc using an ssDNA oligo/ssRNA template duplex. The annealed ssDNA (purple)/ssRNA (black) duplexes representing the 39 end of the TBSV RNA are shown schematically. The assay contained the annealed ssDNA/ssRNA plus 0.6 and 0.4 pmol purified recombinant eEF1Bc, respectively. The 32 P-labeled free ssDNA and ssDNA/ssRNA duplex were separated on nondenaturing 5% acrylamide gels. Quantification of the ssDNA/ssRNA duplex was done with ImageQuant. (B) RNA gel shift analysis shows the role of the SL1 tetraloop in binding to eEF1Bc. The RNA templates representing the 39 end of the TBSV RNA and the mutations (circled nucleotides) are shown schematically. The eEF1Bc -32 P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. The RNA transcript (0.2 pmol), and eEF1Bc (0.4, 0.5 and 0.6 pmol) were used in these assays. doi:10.1371/journal.ppat.1002438.g003
To obtain evidence on the importance of eEF1Bc in TBSV replication in the natural plant hosts, we knocked down the expression of the eEF1Bc gene in Nicotiana bethamiana leaves via VIGS (virus-induced gene silencing). Efficient knocking down of eEF1Bc mRNA level in N. benthamiana ( Figure 7B ) only resulted in slightly reduced growth of the plants without other phenotypic effects ( Figure 7A ). The accumulation of TBSV genomic RNA, however, was dramatically reduced in both inoculated ( Figure 7B , lanes 1-5) and the systemically-infected young leaves ( Figure 7C , lanes 1-4) when compared with the control plants infected with the 'empty' Tobacco rattle virus (TRV) vector. The lethal necrotic symptoms caused by TBSV in N. benthamiana were also greatly attenuated in the eEF1Bc knock-down plants ( Figure 7A ). Therefore, we conclude that eEF1Bc is essential for TBSV genomic RNA accumulation in N. bethamiana.
To test if eEF1Bc is also needed for the replication of other plant RNA viruses, we infected eEF1Bc-silenced N. benthamiana leaves with the unrelated Tobacco mosaic virus (TMV) RNA ( Figure 8A ). We found that the severe symptoms caused by TMV were greatly ameliorated in eEF1Bc knock-down plants ( Figure 8A ). Accumulation of TMV genomic RNA was also dramatically reduced in both inoculated ( Figure 8B ) and systemically-infected ( Figure 8C ) leaves of the eEF1Bc knock-down plants. Based on these data, eEF1Bc seems to be needed for TMV replication and/or spread in plants. Thus, our data have revealed new functions for eEF1Bc in plant RNA virus replication and spread.
Tombusviruses, similar to other (+)RNA viruses, subvert a yet unknown number of host-coded proteins to facilitate robust virus replication in infected cells. The co-opted host proteins could be part of the viral replicase complexes and provide many yet undefined functions. Translation factors, such as eEF1Bc and eEF1A, are among the most common host factors recruited for (+)RNA virus replication [23, 24] . While eEF1A is an integral component of the tombusvirus replicase complex [23, 24] and several other viral replicases [44, 45, 46] , the function of eEF1Bc in tombusvirus replication is studied in this paper. Co-purification experiments with the p33 replication protein, which is the most abundant protein component in the tombusvirus replicase complex [21, 22] , revealed that eEF1Bc is a permanent member of the replicase ( Figure 5A ). eEF1Bc is likely recruited into the viral replicase via the viral (+)RNA, which is bound to eEF1Bc in both cytosolic and membranous fractions ( Figure 5B ). The possible role of host proteins or membrane lipids in assisting the recruitment of eEF1Bc for TBSV replication cannot be excluded. Accordingly, eEF1Bc has been shown to bind to a large number of host proteins (www.yeastgenome.org). For example, eEF1A, which is also a permanent member of the tombusvirus replicase, is known to interact with eEF1Bc [47, 48, 49] and eEF1A might facilitate the recruitment of eEF1Bc and possibly other translation factors. The binding of eEF1Bc to intracellular membranes has also been shown before [32] . Altogether, our model predicts that the viral (+)RNA could be involved in recruitment of eEF1Bc into viral replication ( Figure 5) . However, the opposite model that eEF1Bc facilitates the recruitment of the TBSV (+)RNA into replication is not supported by our in vitro data ( Figure S2) . Indeed, addition of eEF1Bc to the CFE assay did not increase the membrane-bound fraction of TBSV (+)repRNA in the absence or presence of the viral replication proteins ( Figure S2 ). eEF1Bc selectively enhances minus-strand synthesis by opening the closed 39-terminus during TBSV RNA replication
We confirmed a direct role for eEF1Bc in RNA synthesis in vitro by using a cell-free extract prepared from tef4D yeast that supported (-)-strand RNA synthesis ,3-fold less efficiently than CFE from wt yeast (Figure 1 ). Moreover, in vitro assays with highly purified eEF1Bc and the recombinant TCV RdRp, which is closely homologous with the TBSV p92 pol , also revealed that eEF1Bc stimulates (-)-strand synthesis by binding to the viral (+)RNA template ( Figure 3) . Accordingly, pre-incubation of eEF1Bc and the TBSV-derived template RNA prior to the RdRp assay led to the highest level of stimulation of (-)RNA synthesis ( Figure 2 ). On the other hand, eEF1Bc does not stimulate the RdRp activity directly, since pre-incubation of eEF1Bc with the RdRp did not lead to more efficient (-)-strand RNA synthesis in vitro ( Figure 2 ). We propose that eEF1Bc modifies the structure of the (+)-strand template prior to initiation of (-)-strand synthesis that leads to more efficient RNA synthesis as described below.
In vitro initiation of (-)-strand synthesis by the viral RdRp requires the gPR promoter consisting of a short 39-terminal singlestranded tail and a stem-loop (SL1) sequence [39, 50] . However, Figure 6 . Synergistic effect of eEF1Bc and eEF1A on stimulation of minus-strand synthesis by the closely-related TCV RdRp. Purified eEF1Bc (20 pmol) and eEF1A (20 pmol) were added to the TCV RdRp (2 pmol) assay as shown. The RdRp assay had two steps: first, the shown components on the top and bottom were incubated in separate tubes at room temperature to facilitate their interaction, followed 5 min later by mixing the components from the two tubes and addition of the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 1-2 and 9-10). The gel image shows the results of RNA synthesis in the presence of equal amounts of purified eEF1Bc and eEF1A as shown in a TCV RdRp assay. doi:10.1371/journal.ppat.1002438.g006 The FLAG/His 6 -tagged HF-p33 was purified from yeast extracts using a FLAG-affinity column. The purified HF-p33 and the co-purified His 6 -tagged eEF1Bc were detected with anti-His antibody. Bottom panel: Western blot of HF-p33 and the His 6 -tagged eEF1Bc in the total yeast extract using anti-His antibody. (B) RT-PCR analysis to detect the co-purified TBSV (+)RNA in the affinity-purified His 6 -tagged eEF1Bc preparation from yeast replicating TBSV repRNA. Both the membrane and soluble yeast fractions were used for eEF1Bc purification and subsequent RT-PCR analysis to detect (+)repRNA. ''+'' and ''-'' mean that His 6 -tagged eEF1Bc was expressed from a plasmid or not in yeast. Samples were used for RT-PCR (lanes 3-4 and 7-8) or for PCR (without RT reaction, lanes 1-2 and 5-6). doi:10.1371/journal.ppat.1002438.g005 the gPR region is present in a 'closed' structure in the TBSV (+)RNA due to base-pairing of a portion of the gPR with the RSE present in SL3 as shown in Figure 9 . This interaction makes the TBSV (+)RNA poor template in the in vitro assay due to the difficulty for the viral RdRp to recognize and/or open the 'closed' structure [39] . Our current work with eEF1Bc, however, suggests that eEF1Bc can bind to the tetraloop region of SL1 (and to an Arich sequence in SL2) that leads to melting of the base-paired structure and opening the stem of SL1 and the RSE-gPR basepairing as shown schematically in Figure 9B . We propose that the open structure can be recognized efficiently by the viral replicase leading to efficient initiation of (-)-strand synthesis ( Figure 9B ). This model is supported by several pieces of evidence presented in this paper, including (i) stimulation of (-)-strand synthesis by eEF1Bc when the wt SL1 is present in the template; (ii) lack of stimulation of(-)-strand synthesis by eEF1Bc when a mutated SL1 (tetraloop mutant), which does not bind efficiently to eEF1Bc, was used as a template in the in vitro assay; (iii) stimulation of (-)-strand synthesis when eEF1Bc was pre-incubated with the (+)-strand template, but not when eEF1Bc was pre-incubated with the viral RdRp ( Figure 2) ; and (iv) the lack of stimulation of (+)-strand synthesis on a (-)-strand template by eEF1Bc (Figure 2 ). In addition, eEF1Bc stimulated (-)-strand synthesis by the viral RdRp when a partially complementary RNA oligo was hybridized with the SL1 region ( Figure 4B ). However, eEF1Bc could not efficiently bind to the 39-end of the TBSV RNA when it formed a hybrid (duplex) with a perfectly complementary DNA oligo ( Figure 3A) , suggesting that eEF1Bc can melt only the local secondary structure, but cannot unwind more extended duplex regions. An alternative possibility is that eEF1Bc protein stabilizes the unpaired structure (when the SL1 structure is kinetically pairing/unpairing), rather than implying that it actively "opens" the structure.
An intriguing aspect of our model is the possible regulation of the ''open'' and ''closed'' structure of the 39 UTR by eEF1Bc. Displacement of eEF1Bc bound to the 39-end by the viral replicase during (-)-strand synthesis could make the 39-terminus of the (+)strand RNA fold back into a 'closed' structure. This could prevent efficient re-utilization of the original (+)-strand template during TBSV replication, and the switch to efficient (+)-strand synthesis on the (-)RNA intermediate ( Figure 9B ). This model can also explain why the newly made (+)-strand RNA progeny will not enter the replication cycle in the absence of bound eEF1Bc within the originally-formed replicase complexes as observed previously in the CFE assay [20] . We propose that the new (+)RNA progeny need to leave the replicase complex, then bind to eEF1Bc in the cytosol and assemble new replicase complexes, followed by a new round of viral RNA replication. Thus, this model suggests that eEF1Bc plays a key role in regulation of the use of (+)-strand RNAs in TBSV replication ( Figure 9B ).
Our finding of TBSV RNA binding by eEF1Bc adds to the growing list of RNAs bound by eEF1Bc. For example, the 39 UTR of vimentin mRNA is bound by eEF1Bc [51] , which led the authors to suggest that eEF1Bc plays a role in vimentin mRNA subcellular localization by also binding to cytoskeleton or membranes. eEF1Bc also binds to the tRNA-like structure at the 39 UTR of BMV, albeit the relevance of this binding is currently unclear [51] . Also, the actual role of eEF1Bc in the VSV replicase is currently not defined [31] .
Translation elongation factors seem to be important for replication of many RNA viruses. For example, EF-Tu and EF-Ts play a role in replication of bacteriophage Qbeta [52, 53] . The eukaryotic homolog of EF-Tu, eEF1A was found to bind to viral RNAs, such as TBSV, Turnip yellow mosaic virus (TYMV) [54] , West Nile virus (WNV), Dengue virus, hepatitis delta virus, TMV, Brome mosaic virus, and Turnip mosaic virus [55, 56, 57, 58, 59, 60] and to viroid RNAs [61] . Therefore, it is highly probable that many (+)strand RNA viruses recruit translation elongation factors to facilitate and regulate their replication in infected cells.
Nonoverlapping roles of eEF1Bc and eEF1A in stimulation of (-)-strand synthesis
The emerging picture on the functions of eEF1Bc and eEF1A is that these translation elongation factors play different, yet complementary roles in TBSV replication as suggested in Figure 9B . While eEF1Bc binds to SL1, eEF1A has been shown to bind to both p92 pol RdRp and the SL3 region of TBSV (+)repRNA [23, 24] . The binding of the RNA by eEF1Bc promotes the opening of the closed 39-terminal structure, whereas eEF1A facilitates the proper and efficient binding of the RdRp to the 39 terminal RSE sequence of the viral RNA, which is required for the assembly of the viral replicase complex [11, 39] , prior to initiation of (-)-strand synthesis (Figure 9 ) [23, 24] . The binding of eEF1A-RdRp complex to the RSE might lead to proper positioning of the RdRp over the 39-terminal gPR promoter sequence opened up by eEF1Bc, thus facilitating the initiation of (-)RNA synthesis starting from the 39-terminal cytosine ( Figure 9B) . Altogether, the two translation factors facilitate the efficient initiation of (-)-strand synthesis in addition to reducing the possibility of re-utilization of the (+)-strand template for additional rounds of (-)-strand synthesis. This regulation of RNA synthesis by the co-opted host factors shows the specialized use of host components to serve the need of viral replication.
The current work also provides evidence that eEF1Bc is a key factor in TBSV replication in yeast ( Figure 1 ) and in N. benthamiana (Figure 7) . Since eEF1Bc is a highly conserved protein in all eukaryotes [32] , it is not surprising that yeast eEF1Bc, similar to the plant eEF1Bc, can be co-opted for TBSV replication. Interestingly, deletion of either TEF3 or TEF4 genes reduced TBSV repRNA accumulation in yeast, suggesting that eEF1Bc is present in limiting amount or eEF1Bc is present in not easily accessible forms (in protein complexes) and/or locations in yeast cells. Silencing of eEF1Bc in N. bethamiana showed even more inhibition of TBSV RNA accumulation than deletion of eEF1Bc genes in yeast. This is likely due to the robust antiviral response (i.e., induced gene silencing) of the plant host, which could result in degradation of the small amount of viral RNA produced by the less efficient viral RNA replication in the presence of limited eEF1Bc in the knock-down plants.
Silencing of eEF1Bc expression in N. benthamiana also reduced the accumulation of the unrelated TMV (Figure 8 ), which belongs to the alphavirus-like supergroup. These data suggest that eEF1Bc is likely involved in TMV replication, which also contains a highly structured 39-end [54] . Therefore, it is possible that eEF1Bc is co-opted by different plant RNA viruses, and possibly other RNA viruses as well.
Overall, the current work suggests three major functions for eEF1Bc in TBSV replication ( Figure 9 ): (i) enhancement of the minus-strand synthesis by opening the 'closed' 39-end of the template RNA; (ii) reducing the possibility of re-utilization of (+)strand templates for repeated (-)-strand synthesis; and (iii) in coordination with eEF1A, stimulation of the proper positioning of the viral RdRp over the promoter region in the viral RNA template. These roles for eEF1Bc and eEF1A are separate from their canonical roles in host and viral protein translation.
Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) and the single-gene deletion strain of the TEF4-encoded form of eEF1Bc (tef4D) were obtained from Open Biosystems (Huntville, AL). TKY680 strain in which both yeast encoded eEF1Bc, TEF4 and TEF3 were deleted (MATa ura3-52 leu2D1 his3D200 trp1D101 lys2-801 tef3::LEU2 tef4::TRP1) and its isogenic wild type TKY677 (MATa ura3-52 leu2D1 his3D200 trp1D101 lys2-801) as well as the isogenic single deletion mutant strains, TKY678 (MATa ura3-52 leu2D1 his3D200 trp1D101 lys2-801 tef3::LEU2) and TKY 679 (MATa ura3-52 leu2D1 his3D200 trp1D101 lys2-801 tef4::TRP1) were published previously [30] . The following plasmids pESC-GAL1-Hisp33/GAL10-DI-72, pGAD-CUP1-p92 pYES-GAL1-p92, pCM189-TET-His92 were described earlier [21, 22] . URA3 based pGBK-ADH-Hisp33/ GAL1-DI72, pGBK-CUP1-HisFLAGp33/GAL1-DI-72, and pGBK-CUP1-Hisp33/GAL1-DI-72 plasmids were constructed by Daniel Barajas (unpublished result). The URA3 based, low copy-number plasmid, pYC-GAL1-Tef4 expressing non-tagged full-length Tef4 protein was constructed as follows: pYC/NT-C plasmid was digested with BamHI and XhoI restriction enzymes and then PCR product of the TEF4 gene was generated with primers #2089 (ccgcGGATCCATGTCCCAAGGTACTTTA-TAC) and #2320 (CGCCTCGAGTTATTTCAAAACCT-TACCGTCAACAATTTCC) and digested with the same restriction enzymes, followed by ligation. The plasmid pYES-NTC2-GAL1-HisTef4 expressing His 6 -tagged Tef4p protein was created with the same restriction enzymes using pYES-NT-C2.
HIS3-based pEsc-His/Cup-FLAG plasmid [20] was digested with BamHI and XhoI restriction enzymes and then PCR product of the TEF4 gene was generated with primers #2089 and #2320 and digested with the same restriction enzymes, followed by ligationto obtain pEsc-His/Cup-FLAG-TEF4.
HIS3 based pESC-GAL1-His33/GAL10-DI-72 and LEU2 based pGAD-CUP1-Hisp92 plasmids were transformed into tef4D strain. In the in vivo complementation assay, non-tagged Tef4p protein was expressed from URA3 plasmid pYC-GAL1-Tef4 and TEF4 mRNA was detected with a specific probe generated by the T7 transcription of the PCR product obtained with primers #2089 and #3788 (TAATACGACTCACTATAGGATTATT-TCAAAACCTTACCGTCAACAATTTCC).
TKY680 (tef3D/tef4D), the isogenic TKY679 (tef4D), TKY678 (tef3D) and wild type TKY677 yeast were transformed with plasmids pESC-GAL1-His33/GAL10-DI-72 and pCM189-TET-His92. Yeast was pre-grown at 23uC overnight in 3 ml synthetic complete dropout medium lacking the relevant amino acids containing 2% glucose and 1 mg/ml doxycyclin to suppress p92 expression by the inhibition of TET promoter and then TBSV replication was launched by replacing the media with 2% galactose without doxycycline. Cells were harvested at 48 h time point. Total RNA extraction from yeast cells and Northern blotting and Western blotting were done as previously described [15, 24] .
Expression and purification of recombinant eEF1Bc protein pEsc-His/Cup-FLAG-TEF4 plasmid was transformed into tef4D strain. Yeast was pre-grown overnight at 29uC in 2 ml synthetic complete dropout medium lacking histidine (SC-Hmedium) containing 2% glucose. The volume of the media was increased up to 100 ml 16 h later and copper sulfate was added to a final concentration of 50 mM for induction of protein expression. Yeast was grown to 0.8 OD 600 (,4-6 h). Then, yeast cells were harvested and broken by glass beads in a FastPrep cell disruptor followed by Flag-affinity purification of FLAG-Tef4p protein [34] . The bacterial heterologous expression and purification of His 6tagged Tef3 protein from plasmid pTKB523 was performed as described in ref: [62] using only the Ni affinity column step.
Yeast extract capable of supporting TBSV replication in vitro was prepared as described [20] . The newly synthesized 32 P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.5x Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85uC for 5 min in the presence of 50% formamide [20] .
To test the in vitro activity of Tef4p, different concentrations (26 and 13 pmol) of purified FLAG/His 6 -Tef4p was added to 0.25 mg (4 pmol) DI-72 (+)repRNA transcript and incubated in the presence of yeast cell-free extract and reaction buffer for 10 minutes at RT followed by the addition of MBP-p33 and MBP-p92 along with the rest of the reaction components. The reaction was performed at 25uC for 3 h and analyzed as above.
The TCV RdRp reactions were carried out as previously described for 2 h at 25uC [36] , except using 7 pmol template RNA and 2 pmol affinity-purified MBP-p88C. Different concentrations of eEF1Bc (6xHis-affinity purified recombinant Tef3p obtained from E. coli or Flag-affinity purified HF-Tef4p obtained from yeast) were added to the reaction at the beginning or as indicated in the text and Figure 2 . legend. The 32 P-labeled RNA products were analyzed by electrophoresis in a 5% PAGE/8 M urea gel [63] . The 86-nt 39 noncoding region of TBSV genomic RNA and its mutants were used as the template in the RdRp assay [24, 36] . RNA templates were generated with T7 transcription using PCR products obtained with the following primers: #1662 (TAATACGACTCACTATAG GACACG GTTG ATC TC ACC-CTTC) and #1190 (GGGCTGCATTTCTGCAATG) for SL3-2-1(+), #1662 and #4390 (GGGCTGCACAAGTGCAAT-GTTCCGGTTGTCCGGT) for SL3-2-1cuug(+). SL3-2-1m(+) RNA was generated with T7 transcription on PCR products amplified with primers #1662 and #1190, on a plasmid template harboring GGGCU nucleotide-deletion in SL3 region as described [39] . A duplex RNA was generated by hybridizing SL3-2-1(+) and SL3-2-ds1(-) made by T7 transcription of the PCR product using primers #4361 (GTAATACGACTCACTA-TAGGGCTACTTCCGGTTGTCCGGTAGTGCTTCC) and 
For EMSA, 6xHis-Flag tagged Tef4p was purified from a yeast tef4D strain with anti-FLAG M2-agarose affinity resin. Different concentrations (0.6, 0.5 and 0.4 pmol) of HF-Tef4p protein was used for incubation with 0.2 pmol of 32 P-labeled SL3/2/1(+) RNA or mutated RNAs at 25uC in a binding buffer [50 mM Tris-HCl (pH 8.2), 10 mM MgCl 2 , 10 mM DTT, 10% glycerol, 2 U of RNase inhibitor (Ambion)]. Samples were incubated at 25uC for 15 min, then resolved in 4% nondenaturing polyacrylamide gel [23] . Similar experiments were also performed with 6xHis-affinity purified recombinant Tef3p obtained from E. coli (not shown).
For the co-purification of TBSV DI-72 repRNA and eEF1Bc protein, the yeast tef4D strain was co-transformed with pGBK-ADH-Hisp33/GAL1-DI72, pGAD-CUP1-Hisp92 and pESC-CUP1-HisFLAG-Tef4. The pESC-CUP1-FLAGHis-Tef4 plasmid was replaced with the pESC plasmid in the control experiment. Yeast was pre-grown overnight at 29uC in 2 ml SC ULHmedium containing 2% glucose and 5 mM copper sulfate. The volume of the media was increased to 20 ml after 16 h for an additional 10 h (OD 600 of ,0.8), then the cultures were transferred to 20 ml SC ULHmedium containing 2% galactose to induce TBSV DI-72 RNA transcription at 23uC. The transcription of DI-72 RNA was stopped by changing to the media containing 2% glucose after 8 h. The cultures were diluted to 200 ml and copper sulfate was added to a final concentration of 50 mM to induce the expression of Flagtagged Tef4 protein. After incubation at 23uC for 24 h, the samples were centrifuged at 3000 rpm for 4 min. Cells (,1 g) were re-suspended in 2 ml TG Buffer (50 mM Tris-HCl [pH 7.5], 10% glycerol, 15 mM MgCl 2 , and 10 mM KCl) supplemented with 0.5 M NaCl and 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma) and RNase inhibitor (Ambion). Yeast cells were broken by glass beads in a FastPrep cell disruptor (MP Biomedicals) 4 times for 20 sec each at speed 5.5. Samples were removed and incubated 1 min in an ice-water bath after each treatment. The samples were centrifuged at 500 6g for 5 min at 4uC to remove glass beads, unbroken cells and debris then supernatant was moved into fresh pre-chilled tubes. After being centrifuged again at 500 6g for 5 min at 4uC supernatant transferred into fresh pre-chilled tubes and soluble (SU) and membrane (ME) fractions containing the viral replicase complex were separated with centrifugation at 35,000 6g for 15 min at 4uC. The SU fraction was applied on 0.1 ml anti-FLAG M2agarose affinity resin (Sigma) and Tef4 protein tagged with 6xHisand FLAG affinity tags was purified. Before applying ME fraction on the anti-FLAG M2 resin, solubilization of the membranebound replicase was performed in 1 ml TG buffer with 0.5 M NaCl, 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma), and 2% Triton X-100 via rotation for 2 hours at 4 uC. The solubilized membrane fraction was centrifuged at 35,000 6g at 4uC for 15 min and the supernatant was added to the resin preequilibrated with TG buffer supplemented with 0.5 M NaCl and 0.5% Triton X-100, followed by gentle rotation for 2 h at 4uC. The unbound proteins were removed by gravity flow, and the resin was washed two times with 1 ml TG buffer supplemented with 0.5 M NaCl, 0.5% Triton X-100 and once with 1 ml TG buffer, 0.5% Triton without NaCl. The bound proteins were eluted with 150 ml TG buffer without NaCl, 0.5% Triton X-100, supplemented with 150 mg/ml flag peptide and 1% yeast protease inhibitor cocktail via gentle tapping the column occasionally for 2 h at 4uC. After centrifugation at 600 6g 2 min at 4uC, semiquantitative RT-PCR was performed to detect TBSV repRNA copurified with eEF1Bc using primers, #359 (GTAATACGACT-CACTATAGGAAATTCTCCAGGATTTC) and #1190, amplifying full length (+)repRNA.
To test if eEF1Bc is present in the viral replicase, yeast tef4D strain was transformed with pGBK-CUP1-HisFLAGp33/GAL1-DI-72, pGAD-CUP1-Hisp92 and pYES-GAL1-HisTef4. In the control experiment, 6xHisp33was expressed from pGBK-CUP1-Hisp33/GAL1-DI-72. Yeast cultures were grown in SC-ULHmedia containing 1% raffinose and 1% galactose with 5 mM copper-sulfate for 4 days with increasing the volume of the culture from 2 ml to 100 ml to a final OD 600 of, 1.0. After harvesting of cells, co-purification of 6xHis-tagged Tef4p with HF-p33 (part of the viral replicase) was conducted by using anti-FLAG M2-agarose affinity resin as described above (in the section: FLAG-affinity purification of eEF1Bc-TBSV repRNA complex), with the exception that only solubilized ME fraction was loaded on the column. Proteins bound to affinity resin were eluted by incubation with 150 ml buffer containing FLAG peptide and precipitated with Trichloroacetic acid (TCA) [64] . Samples were analyzed by SDS-PAGE and Western blotting.
Virus-induced gene silencing (VIGS) in N. benthamiana was done as described [65, 66] . To generate the VIGS vector (pTRV2-eEF1BcNt), a 314-bp cDNA fragment of NteEF1Bc was RT-PCR amplified from a total RNA extract of N. benthamiana using the following pair of primers: #2993 (CGCGGATCCAAAG-GTTTCTGGGACATGTATGA) and #2994 (CGCCTCGA-GACACGCTCCTTCTGTGATTCATC) and inserted into the corresponding (BamHI/XhoI) restriction sites of pTRV2 plasmid.
The sequence of the N. tabacum eEF1Bc gene (GenBank: ACB72462.1) was derived via a BLASTP search based on the Cterminal (translation elongation factor) domain (aa 252-412) of the Saccharomyces cerevisie Tef4 protein. The selected sequence (TC64920) from the Solanaceae Genomics Resource (www.tigr. org) gave 98% identity with N. tabacum EF1Bc -like gene (GB#: EU580435.1).
To confirm the silencing of the EF1Bc gene in N. benthamiana, we performed RT-PCR amplification with primer pairs: #2952 (CGCGGATCCGGAAAGGTTCCTGTGCTTGA) and #2992 (C G C CT C G A G GTCCAGAAGTATCTCTCTACA TGTGG) on total RNA extract of pTRV2-EF1BcNt and pTRV2 empty agroinfiltrated N benthamiana plants. PCR conditions were as follows: 27 cycles of 94uC 20sec, 60uC 30sec, 68uC 30 sec with HiFi Taq polymerase. Tubulin mRNA control from the same total RNA samples was detected by RT-PCR using primers #2859 (TAATACGACTCACTATAGgaACCA AA TC AT T CATGTT-GCTCTC) and #2860 (TAGTGTATGTGATATCCCACCAA) [65] . The leaves of VIGS-treated plants were sap inoculated with TBSV, or TMV on the 9 th day after silencing [65] . Total RNA was extracted 3 or 5 days post inoculation [65] . For Northern blot analysis of the viral RNA level, we prepared 32 P-labeled complementary RNA probes specific for the 39-ends of the viral genomic RNAs based on T7 transcription. To obtain the PCR templates for the probes, we used the following primers for TBSV: #1165 (AGCGAGTAAGACAGACTCTTCA) and #22; for TMV: #2890 (TCTGGTTTGGTTTGGACCTC) and #2889 (GTAATACGACTCACTATAGGGATTCGAACCCC-TCGCTTTAT).
The TBSV viral RNA is recruited to the membrane from the soluble fraction with the help of TBSV replication proteins and host factors present in the yeast CFE. The in vitro RNA recruitment reaction was performed according to [20, 23] , except that 32 Plabeled DI-72 (+)repRNA were used and rCTP, rUTP, 32 P-labeled UTP, and Actinomycin D were omitted from the assay. As a negative control, p33 and p92 were omitted from the reaction to detect DI-72 binding nonselectively to host proteins present in the membrane. In vitro binding assay with purified recombinant eEF1Bc (Tef3). The TBSV (+)RNA templates were the four noncontiguous segments of the TBSV (+)RNA that are present in defective interfering RNAs, including DI-72 repRNA used in this study. RI(+) represents the 59-UTR, RII(+)-SL is an internal highly conserved sequence that binds to p33 replication protein, RIII(+) is ashort conserved sequence closed to the 39 end, andSL3-2-1(+), which contains the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The assay contained 32 P-labeled free ssRNA (as shown), plus 0.6 pmol purified recombinant eEF1Bc, respectively. The bound RNA-protein complexes were separated on nondenaturing 5% acrylamide gels. Quantification of the free (unshifted) RNA was done with ImageQuant. (B) RNA gel shift analysis shows SL3-2-1(+) RNA binds competitively to eEF1Bc. The RNA templates representing the 39 end of the TBSV RNA and the deleted nucleotides are shown schematically. The cold competitor was SL3-2-1(+) RNA, which represents a large portion of the 39-UTR ( Figure 4A ). The eEF1Bc -32 P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. (EPS) Figure S2 eEF1Bcdoes not affect the template recruitment step in vitro. Purified recombinant p33/p92 and 32 P-labeled DI-72 (+)repRNA and eEF1Bc (affinity purified recombinant Tef3) were added to a whole cell extract (CFE), followed by centrifugation/ washing to remove the 32 P-labeled repRNA that is not bound to the membrane. Then the membrane-bound RNA was analyzed in a denaturing PAGE gel. Note that the repRNA binds to the cellular membrane fraction nonspecifically (,20% level) in the absence of the viral replication proteins. (EPS) Mannose-Binding Lectin Contributes to Deleterious Inflammatory Response in Pandemic H1N1 and Avian H9N2 Infection Background. Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. Methods. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Results. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Conclusions. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response. The pandemic H1N1 2009 (pdmH1N1) influenza A virus has spread globally since the outbreak first started in Mexico in 2009. As of August 2010, the virus had already caused more than 18 449 deaths in at least 214 countries worldwide [1] . The World Health Organization officially announced the step-down of pdmH1N1 to postpandemic phase on 10 August 2010, and the virus was expected to circulate as a seasonal virus in the human population thereafter [2] . Gene segments of the pdmH1N1 virus are derived from multiple lineages, including the Eurasian swine, the classical swine, and the triple reassortant swine lineages. Multiple genetic reassortment events of viral components have taken place and thus gave rise to this novel pandemic virus [3] . Clinical symptoms of pdmH1N1 infection are usually mild, possibly due to the cross-protection offered by memory cytotoxic T lymphocytes established from previous exposure to seasonal influenza [4] .
H9N2 avian influenza virus (H9N2/G1) is widely prevalent among poultry in various Eurasian regions, including mainland China and Hong Kong. In 1999 and 2003, H9N2 influenza was reported in 3 children in Hong Kong. All 3 of them developed relatively mild symptoms and recovered within a week [5, 6] . The 6 internal genes of H9N2/G1 virus were found to be related to the highly pathogenic avian influenza virus A/Hong Kong/483/97 (H5N1). Although the H9N2/G1 virus was found mainly in poultry, it has the ability to transmit across species, and with its genetic similarity to the highly pathogenic H5N1, it also poses a threat of becoming pandemic [7] .
Mannose-binding lectin (MBL) is a serum protein primarily produced by the liver. It belongs to the collectin family that comprises the collagen-like domain and the carbohydrate recognition domain (CRD). MBL functions as a key patternrecognition molecule recognizing a wide range of pathogens [8, 9] . Lectin pathway activation [10] and opsonophagocytosis are triggered upon MBL binding to pathogens [11] . While the MBL gene is highly polymorphic in humans, clinical association studies have demonstrated that MBL deficiency was associated with increased susceptibility to certain infections [12, 13] .
The antiviral role of MBL in influenza virus infection remains controversial. Previous studies suggested that MBL demonstrates in vitro anti-influenza virus function, including inhibition of viral hemagglutination and direct neutralization of the virus either in a complement dependent or independent manner [14] [15] [16] . However, other studies have shown that the antiviral function of MBL may vary among different strains of influenza viruses, depending on the number of potential glycosylation sites on the viral hemagglutinin (HA) globular domain [17, 18] . Influenza virus-infected epithelial cells and macrophages can initiate a cellspecific response that includes the transcription and release of proinflammatory cytokines and chemokines [19, 20] . Although some studies have indicated that MBL may regulate proinflammatory cytokine and chemokine release from phagocytes in response to bacterial stimulation [21, 22] , little is known about its immunomodulatory role in influenza [23] .
In the present study, we investigated whether MBL could display any in vitro or in vivo antiviral function toward pdmH1N1 and H9N2/G1 viruses, as well as whether it could modulate the inflammatory response upon infection by these two strains of influenza virus.
Influenza virus A/California/04/2009 (pdmH1N1) were propagated in embryonated chicken eggs and purified by ultracentrifugation with minor modification of our previous work [4, 24] . Influenza virus A/Quail/Hong Kong/G1/97 (H9N2/G1) was grown in Madin-Darby canine kidney (MDCK) cells with modified Eagle's medium (Invitrogen) containing 2 lg/mL N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)treated trypsin (Sigma-Aldrich). Virus stocks were purified by adsorption to and elution from turkey red blood cells and stored at 280°C until use as previously described [25] . The determination of virus titer was performed by titrating virus in MDCK cells, with daily observation of cytopathic effect and confirmation by hemagglutination assay. The tissue culture infective dose affecting 50% of the cultures (TCID 50 ) was calculated by the Reed-Muench formula. Ultraviolet (UV)-irradiated virus was prepared by irradiation with energy of 0.2 J in a UV crosslinker as described previously [26] .
The binding assay was performed as described previously [12] . In brief, 96-well flat-bottom polystyrene plates (Corning-Costar) were precoated with 100 lL/well of 10 2 , 10 3 ,10 4 , and 10 5 TCID 50 UV-irradiated influenza viruses or phosphate-buffered saline (PBS). After incubation at room temperature overnight, wells were blocked for 2 hours at room temperature with 1% bovine serum albumin (BSA) in PBS with 0.05% sodium azide. Different concentrations of recombinant human MBL (rhMBL) (0, 0.5, 2, 6, or 8 lg/mL), which was kindly provided by Dr K. Takahashi (Laboratory of Developmental Immunology, Harvard Department of Pediatrics, Massachusetts General Hospital, Boston), were added and incubated overnight at 4°C. Then 100 lL of 0.2 lg/mL biotinylated monoclonal anti-MBL antibody (HYB131-01, Antibody Shop) diluted in PBS with 1% BSA was added into each well. Bound antibody was detected by using horseradish peroxidase-conjugated streptavidin and tetramethybenzidine substrate solution (R&D Systems). The binding of MBL to influenza virus was evaluated by the absolute absorbance values measured at 450 nm (A 450 ).
Breeding pairs of MBL wild-type (WT) and MBL knockout (KO) mice on C57B6/J were provided by Dr Takahashi [27] . They were maintained under specific pathogen-free conditions in the animal facilities of the Laboratory Animal Unit, The University of Hong Kong. Female mice were used at 6-10 weeks of age. They were anesthetized and inoculated intranasally with 30 lL of 10 3 TCID 50 pdmH1N1 virus, 10 5 TCID 50 H9N2/G1 virus, or PBS at day 0. Virus-infected or mocktreated mice were weighed daily. All animal care and experiments were conducted in accordance with the Committee on the Use of Live Animals in Teaching and Research guidelines of the University of Hong Kong.
Virus-infected or mock-treated mice were sacrificed at days 3, 7, and 14 after infection. The lungs were harvested and homogenized by a tissue homogenizer (Omni International). The homogenates were centrifuged at 2500 rpm for 10 minutes at 4°C. Supernatants were used for virus titer determination and cytokine detection.
Expression levels of interleukin (IL) 1a, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF) a, interferon (IFN) c, macrophage inflammatory proteins (MIP)-1a, MIP-1b, monocyte chemotactic protein (MCP)-1, MCP-3, and Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) in the lung homogenates were quantitatively determined by flow cytometrybased immunoassay (Mouse Th1/Th2 cytokine 10plex and Mouse Chemokines 6plex Flowcytomix Multiplex, Bender MedSystems) according to the manufacturer's protocol. Interleukin 1b and keratinocyte chemoattractant (KC) simplex were purchased separately (Bender MedSystems). In brief, lung homogenates were prepared and processed accordingly. The samples were acquired on a BD LSRII (BD Bioscience), and the amount of cytokine (ng/mL) was calculated by FlowCytomix Pro 2.3 software (Bender MedSystems).
Virus-infected or mock-treated mice were sacrificed at the indicated time point for histopathologic analysis. The lung tissues were fixed in 10% formalin and embedded in paraffin. Fivemicrometer-thick, paraffin-embedded sections were cut and stained with hematoxylin and eosin (H&E) to analyze histological lesions. Histopathologic score of lung tissues was examined by a board-certified pathologist blinded to the exposure status. Lung inflammatory changes were graded using a semiquantitative scoring system based on the following parameters: peribronchiolar and bronchial infiltrates, bronchiolar and bronchial luminal exudates, perivascular infiltrates, parenchymal pneumonia, and edema, as previously described [28] . Each parameter was graded on a scale of 0-4 with 0 as absent, 1 as slight, 2 as mild, 3 as moderate, and 4 as severe. The total lung inflammation score was expressed as the sum of the scores for each parameter. The degree of cell infiltration was independently scored on an increasing scale of 0-3 with 0 as no cells, 1 as few cells, 2 as moderate influx of cells, and 3 as extensive influx of cells [29] .
Data were expressed as mean (standard error of the mean). Unpaired Student t test in GraphPad Prism 5.0 software (GraphPad) was used for statistical analysis. A P value ,.05 was considered significant. 
rhMBL Binds Both pdmH1N1 and H9N2/GI Viruses A microtiter capture assay demonstrated that MBL could bind to pdmH1N1 and H9N2/G1 in vitro ( Figure 1 ). The MBL-virus binding occurred in a dose-dependent manner ( Figure 1A and 1D). Similarly, increased amount of virus could also result in increased binding by MBL ( Figure 1B and 1E) . MBL utilizes the CRD to recognize pathogens in a calcium-dependent manner [30] . Further addition of ethylenediaminetetraacetic acid (EDTA) in the assay inhibited the binding of MBL to both strains of influenza virus ( Figure 1C and 1F) , suggesting that the binding occurred through the CRD of MBL.
Wild-type and MBL KO mice, 6-10 weeks of age, were infected intranasally with 30 lL of 10 3 TCID 50 pdmH1N1 virus or 10 5 TCID 50 H9N2/G1 virus. The viral dosage chosen for the experiment was previously demonstrated to be sublethal (data not shown). Mice were inoculated with 30 lL of PBS as the mock treatment. No mice died throughout the 14-day experiment. The body weight of mice, which was a physiological value indicating infection progress and the health of the animals, was recorded daily. Mice receiving mock treatment did not lose body weight, demonstrating the absence of potential harmful effects due to anesthetics and intranasal inoculation (Figure 2A ).
Both strains of influenza virus could successfully infect MBL WT and MBL KO mice as evidenced by the significant weight loss in these mice after virus infection. Compared to MBL KO mice, MBL WT mice had more weight loss upon virus infection. For pdmH1N1 virus infection, MBL WT mice showed a significantly greater body weight drop on days 3-12 compared with MBL KO mice ( Figure 2B ). The mean peak body weight loss observed in the MBL WT mice and MBL KO mice were -23.51% and -17.38%, respectively. For H9N2/G1 virus infection, MBL WT mice only showed a significantly greater weight loss on day 8 when compared with MBL KO mice ( Figure 2C ). Although similar mean peak weight loss was observed between the MBL WT mice and MBL KO mice after H9N2/G1 virus infection, MBL WT mice recovered more slowly than the KO mice. Collectively, these data suggested that the presence of MBL caused a more severe infection by the pdmH1N1 and H9N2/G1 viruses.
Wild-type and MBL KO mice infected with pdmH1N1 or H9N2/G1 virus were sacrificed on days 3, 7, and 14 after infection. Virus titers in lung homogenates were determined by TCID 50 . As shown in Figure 3 , these 2 strains of influenza virus were detectable in the lung homogenates collected from MBL WT and KO mice on day 3 and day 7, confirming viral lung infection. On day 14, titers for both strains of virus were undetectable, which was consistent with the regain of body weight by MBL WT and MBL KO mice and suggested recovery from the infection. For pdmH1N1 virus infection, there was no significant difference in the lung virus titer between MBL KO and MBL WT mice on days 3 and 7 after infection ( Figure 3A ). In contrast, significantly less virus titer was detected in MBL KO mice on day 7 but not on day 3 after H9N2/G1 virus infection compared to that in MBL WT mice ( Figure 3B ). 
To further investigate whether MBL would modify the inflammatory response upon pdmH1N1 and H9N2/G1 virus infection, a panel of 14 proinflammatory cytokines and chemokines were examined in the lung homogenates collected from the infected MBL WT and MBL KO mice. The simultaneous profiling of the cytokines and chemokines was examined by using bead-based suspension array, which could allow the sensitive and specific detection of these proteins in the available amount of lung homogenates.
As shown in Figures 4 and 5 , upon pdmH1N1 and H9N2/G1 virus infection, inflammatory response assayed by cytokines and chemokines production was triggered in both MBL WT and MBL KO mice. Except for IL-2, of which the level remained constantly low during the course of experiment, the kinetics of individual proteins were similar in that they were readily detectable on day 3, reached the highest level on day 7, and declined on day 14. Strikingly, MBL KO mice had reduced inflammatory responses during infection. Among the 14 cytokines examined, the majority of them showed significantly lower amounts in MBL KO mice lung homogenates than in MBL WT mice, including IL-1a, IL-1b, IL-6, IL-10, TNF-a, IFN-c ( Figures 4A and 5A) , KC, MIP-1a, MIP-1b, MCP-1, MCP-3 and RANTES (Figures 4B and 5B) . These results suggested that MBL upregulates the inflammatory response to influenza virus infection, resulting in elevated production of proinflammatory cytokines and chemokines in the MBL WT mice as compared with the MBL KO mice.
To further confirm the severe inflammatory response in the MBL WT mice compared with MBL KO mice, lung sections were stained with H&E for histological analysis to evaluate inflammation-associated lung damage caused by pdmH1N1 and H9N2/G1 influenza virus infection. In the histological sections of MBL WT mice, more severe lung inflammation and more cell infiltration were observed when compared to that of MBL KO mice on day 7 ( Figure 6 ). Consistent with our cytokine and chemokine data, the pulmonary histological analysis suggested that the MBL WT mice had a more severe inflammatory response upon pdmH1N1 and H9N2/G1 virus infection.
Mannose-binding lectin is a pattern-recognition molecule, which provides first line of host defense. Accumulating evidence has suggested that MBL exhibits in vitro anti-influenza virus properties by direct neutralization, inhibiting influenza virus hemagglutination, binding to the influenza virus as an opsonin, and activating the complement system through the lectin pathway [14, 16, 23] . However, these properties vary among different virus strains and subtypes [17, 18] . In this study, we focused on the pandemic influenza A H1N1 virus and avian influenza A H9N2/G1 virus, which are of potential threat to the global community.
We demonstrated that despite these 2 strains of viruses being bound by rhMBL via the CRD of MBL at the physiological level, they infected both MBL WT and MBL KO mice effectively. Our results are consistent with a recent in vitro study by Job et al [18] , in which MBL was found to bind to pdmH1N1 fairly in vitro but the virus was resistant to the antiviral activity of MBL. The number and position of potential glycosylation sites on the viral HA globular domain determine the binding affinity between MBL and the virus. Even though MBL can physically bind to the virus, the binding may be insufficient for executing any antiviral function. Arguably, Chang et al [23] recently reported that MBL deficiency increases susceptibility to infection with influenza A virus Philippine 82 H3N2 (Phil82), which is a human strain. We reconcile with the suggestion that MBL effects would differ depending on strains of influenza A virus and thus MBL causes variable antiviral activities and host responses. The degree of glycosylation on the globular head of the HA molecule is believed to be essential for MBL to exhibit its antiviral properties. For Phil82 virus, the high-mannose oligosaccharide at residue 165 of the HA molecule has already been shown to be crucial for the neutralization by MBL [31] . Although pdmH1N1 virus contains a single potential glycosylation site at the base of the HA globular head (Asn 104 ), it lacks potential glycosylation sites on the globular head region of HA (Asn 142 , Asn 144 , Asn 172 , Asn 177 , and Asn 179 ) [18] . To our knowledge, binding of MBL on H9N2/G1 virus is not well documented in the literature. Therefore, we analyzed the potential glycosylation sites on HA of H9N2/G1 virus based on an in silico approach as suggested by Job et al [18] . The HA sequence data was retrieved from GenBank (AAF00706.1) and we used NetNGlyc 1.0 server to predict the number of potential glycosylation sites. We found that there was no potential glycosylation sites near residue 165 of the HA molecule of H9N2/G1 virus. We speculate that as a result of the absence of potential glycosylation sites near the receptor-binding domain of the HA globular head of both pdmH1N1 virus and H9N2/G1 virus, MBL fails to interfere with the viral binding to target cells despite its ability to bind the virus. This can adequately explain the discrepancy between the present in vivo data and Chang's study [23] .
In this study, MBL WT mice were found to have a more severe disease in terms of greater weight loss and worse lung pathology than MBL KO mice during either pdmH1N1 or H9N2/G1 virus infection. This suggests that MBL may contribute to the disease severity seen in the MBL WT mice. To elucidate the mechanisms, we investigated the immune response, such as production of cytokines and chemokines at various time points during the infection. Most cytokines and chemokines were detected with similar kinetics, with the peak on day 7 following influenza virus infection in both MBL WT and MBL KO mice. These data suggest that the most critical phase of influenza infection occurs around day 7 after infection, and this is Interestingly, we found that MBL contributed to a more severe proinflammatory response by increasing the production of several proinflammatory cytokines, such as IL-1a, IL-1b, IL-6, TNF-a, and IFN-c. Interleukin 1a and IL-1b are multifunctional proinflammatory cytokines produced readily by influenzainfected leukocytes. They are capable of inducing fever, anorexia, and weight loss [32] . Enhanced production of these cytokines can contribute to the acute lung immunopathology after influenza virus infection in mice [33] and induce gene expression of other cytokines like IL-6 and TNF-a [34, 35] . Despite the abundance of IL-6 following the influenza virus infection, in vivo studies showed that it does not contribute significantly to the pathogenesis of influenza virus infection because the mortality and morbidity observed in mice infected with H5N1 are comparable in both MBL WT and IL-6 deficient mice [36, 37] . Tumor necrosis factor a is readily produced by influenza virusinfected leukocytes and can activate macrophages, stimulate dendritic cell maturation and neutrophils, further enhance the inflammatory response, and activate efficient antigen presentation system in the infected site [20, 38] . Excessive production of TNF-a causes tissue injury, hemorrhagic shock, and death in mice [39, 40] . Interferon c is also an important proinflammatory cytokine that has different functions, including the activation of macrophages, differentiation of Th1 from T cells, enhancement of antigen presentation, and expression of the chemokine gene [41, 42] . These proinflammatory cytokines are commonly found in the acute-phase response to influenza virus infection and may induce immunity but also cause damage to the host tissue [43] . These cytokines were also increased in our infected murine lung, with significantly higher levels in MBL WT mice than in MBL KO mice on day 7, coinciding with body weight loss and lung histological findings. Interleukin 10, an antiinflammatory cytokine, was also significantly higher in MBL WT mice than in MBL KO mice on day 7. Interleukin 10 deficiency was reportedly protective in high-dose influenza virus infection [44] , implying that increased IL-10 may be deleterious to the host. As a consequence of such an overwhelming ''cytokine storm'' [45, 46] , the MBL WT mice were found to have a worse disease course than in the MBL KO mice, including greater body weight loss and more severe lung inflammation.
In addition, we also found that most chemokines, including KC, MIP-1a, MIP-1b, MCP-1, and MCP-3, were elevated in MBL WT mice compared with the MBL KO mice in both pdmH1N1 and G1/97 virus infection. Influenza virus-infected macrophages produced large amounts of these chemokines in vitro [25, 47, 48] . Functionally, these chemokines are important mediators for immune cell activation and chemotactic factors, which recruit leukocytes to the infected sites [49] . This may help account for our histological observation that more inflammatory cell infiltration was observed in MBL WT mice than in MBL KO mice.
The role of MBL in modulating immune responses has also been observed in Staphylococcus aureus infection. It was shown that MBL amplifies the host immune response during S. aureus infection by cooperating with Toll-like receptors 2 and 6 and augments the production of proinflammatory cytokines and chemokines [50] . The observation from our present study prompted us to further investigate whether MBL may also cooperate with other pattern-recognition receptors and thus further amplify the host response during influenza virus infection.
In conclusion, we have shown for the first time that MBL is a risk factor leading to a more severe pdmH1N1 and H9N2/G1 virus infection by upregulating proinflammatory responses.
Financial support. This work was supported in part by the National Essential epidemiological mechanisms underpinning the transmission dynamics of seasonal influenza Seasonal influenza has considerable impact around the world, both economically and in mortality among risk groups, but there is considerable uncertainty as to the essential mechanisms and their parametrization. In this paper, we identify a number of characteristic features of influenza incidence time series in temperate regions, including ranges of annual attack rates and outbreak durations. By constraining the output of simple models to match these characteristic features, we investigate the role played by population heterogeneity, multiple strains, cross-immunity and the rate of strain evolution in the generation of incidence time series. Results indicate that an age-structured model with non-random mixing and co-circulating strains are both required to match observed time-series data. Our work gives estimates of the seasonal peak basic reproduction number, R(0), in the range 1.6–3. Estimates of R(0) are strongly correlated with the timescale for waning of immunity to current circulating seasonal influenza strain, which we estimate is between 3 and 8 years. Seasonal variation in transmissibility is largely confined to 15–30% of its mean value. While population heterogeneity and cross-immunity are required mechanisms, the degree of heterogeneity and cross-immunity is not tightly constrained. We discuss our findings in the context of other work fitting to seasonal influenza data. Seasonal influenza causes significant levels of morbidity and mortality around the world each year, yet its dynamics and the annual sequence of pathogen subtypes are hard to predict [1] . Understanding the mechanisms underlying the annual behaviour of influenza and their sensitivity to parameters is important for the forecasting and control of seasonal epidemics and also in assessing the effect of the introduction of novel antigenic strains. The 2009 H1N1 pandemic is a recent example [2] . The initial outbreak of this novel H1N1 strain occurred in spring and summer, 'out of season' in the Northern Hemisphere. Uncertainty with regard to intensity of transmission during summer substantially complicated forecasting of the likely trajectory of the epidemic over the following months.
Many respiratory transmissible diseases exhibit seasonal epidemics. Annual periodic forcing causes a wide range of oscillatory epidemic behaviour, as illustrated by incidence rates for measles and pertussis [3] [4] [5] . The source of seasonal forcing in those cases is largely attributed to annual variations in the intensity of contact between children, but a range of other mechanisms have been proposed. In the case of influenza, two recent hypotheses have centred on variation in vitamin D levels and air humidity [6, 7] .
Influenza dynamics are additionally complicated by considerable antigenic diversity in human influenza viruses. Within each of the two influenza A subtypes (H3N2 and H1N1) co-circulating prior to 2009, continual evolution selecting for antigenic novelty was seen [8] . The dynamics of intra-subtype evolution is characterized by relatively stable strains periodically replaced by antigenically distinct types in punctuated evolution events [8] [9] [10] . In addition, there is evidence of immune-mediated competition between subtypes [10] . These mechanisms combine to generate complex seasonal behaviour, featuring a range of annual attack rates (AARs) and epidemic durations and alternating sequences of dominant annual subtypes and strains [11] . Detailed and computationally intensive simulations are required to fully integrate the epidemiological and genetic aspects of long-term behaviour [10, 12] . However, strain-specific data with sufficient temporal resolution to fit such a model are not available. We therefore adopt a simplified description here and model two weakly interacting subtypes, with evolution within each subtype being represented by a gradual loss of immunity of the previously exposed host population, a so-called SIRS (susceptible-infected-recoveredsusceptible) model. While use of SIRS models to represent intra-subtype evolution is not uncommon [13] [14] [15] , previous models of influenza time series have not accounted for dynamics generated by multiple interacting subtypes.
In this paper, we identify a set of key features characterizing seasonal influenza-like illness (ILI) incidence time series and use these to define information measures for the distance between a transmission model and empirical observations. Use of summary statistics (rather than attempting to directly fit models to timeseries data) adds robustness to variations in reporting (much ILI is not even caused by influenza) and shortterm fluctuation in the incidence data. Our aim is not to precisely match the incidence time series, but to find broad regions of parameter space which are consistent with the characteristics of seasonal flu epidemics and hence identify which epidemiological mechanisms are essential and acceptable ranges for their parameters. For this purpose, an elaborate model and detailed data are not necessary.
We use the following features of the time series to characterize seasonal influenza incidence in temperate countries of the Northern Hemisphere:
-Epidemic duration. The vast majority of seasonal influenza outbreaks are observed between the extremes of mid-November and the end of April [11] . Since a background rate of ILI incidence is present throughout the year, duration is difficult to define precisely. The range of values quoted for the duration of an annual epidemic (described with the acronym ADE in this paper) is typically between three and 16 weeks [1, 16] . -Attack rate. The proportion of the population infected in a year, which we term the AAR, is very difficult to determine, as a significant proportion of infections are asymptomatic and only a proportion of symptomatic cases seek healthcare. Hence sentinel data on ILI can only give relative information, such as the fractional variation in incidence over time. In addition, measures such as ILI are non-specific for influenza and can be caused by a variety of respiratory pathogens. That said, ILI data from France and the UK indicate a standard deviation for AAR across successive years of approximately 40 per cent of the mean attack rate [17, 18] . Results from serological and virus isolation data from closely monitored populations indicate an AAR range of approximately 10-20% for seasonal influenza, rising to 30 per cent or more for influenza pandemics [1, 17] . Information on consultation rates for known cases also suggests a mean AAR of around 15 per cent [13, 18, 19 ]. -Periodic behaviour. Seasonal influenza is characterized by annual outbreaks in temperate countries in the sense that there is almost invariably a marked seasonal increase in case rate during the winter months. However, it cannot be said to be periodic in the sense that successive outbreaks are comparable in magnitude and form with each other. In this sense, seasonal influenza has no clear periodicity (annual, biennial and triennial) and contrasts with the behaviour of diseases such as measles, which has a pronounced biennial structure in the pre-immunization period [5] . The metrics that we use to compare model and time-series behaviour are able to pick up this characteristic aperiodicity. -Strain variation. Virus isolation studies show that individual seasonal epidemics are usually dominated by a single type and/or subtype, although others may be present at low levels [13] . Successive seasons are usually dominated by different types and subtypes, although often the same strain is present for several seasons (figure 1).
Seasonal influenza is characterized by annual outbreaks in temperate countries in the sense that there is almost invariably a marked seasonal increase in case rate during the winter months. However, it cannot be said to be strictly periodic in the sense that successive outbreaks are comparable with each other in magnitude and duration. In this sense, seasonal influenza has no clear periodicity and contrasts with the behaviour of diseases such as measles, which has a pronounced biennial structure in pre-immunization period [5] .
We use a deterministic SIRS compartmental model, to which we have added a number of refinements to capture critical aspects of influenza infection and immunity. The basic dynamics of infection and immunity in the model are
where l j is the force of infection of strain j and S n is the part of the population that is immune to strain n. Infected individuals recovering from strain n enter a class entirely immune to n (e.g. S 0 ! S 1 , S 1 ! S 12 ). Immunity to a given strain is lost at a rate s. Figure 2 illustrates the flows of individuals between the various immunity classes. The model is further complicated by stratification by age (children and adults) with heterogeneous mixing, differential infectiousness and susceptibility by age and a realistic infectiousness profile. The full details of the model are described in the electronic supplementary material. The effect of seasonal forcing is included through a time-varying contact parameter,
Mechanisms of seasonal influenza J. Truscott et al. 305
We express this variation in terms of peak R 0 and relative amplitude of variation throughout. The sinusoidal form is a good match for variation in infectiousness as a function of absolute humidity in temperate regions [14] , but we also examine the effect of using school terms as a seasonal driver using step-function forcing (see the electronic supplementary material). In determining feasible ranges for these parameters, we reviewed the range of estimates that exist for R 0 . The majority of these are calculated for the major global epidemics (1918, 1957, 1968) , since the antigenic novelty of pandemic viruses means an assumption of a serologically naive population can be made, making analysis simpler. For seasonal influenza, knowledge of population susceptibility is necessary to estimate R 0 (as opposed to the effective reproduction number, R). Estimated values for the 1918 pandemic range from 1.3 to 2.8 [20] . Similar values are found for the 1957 and 1968 epidemics [21] . These values also correspond well with those from studies of seasonal influenza, giving winter R 0 of 1.7 and school holiday R 0 of 1.4 [16] .
The periodic forcing of systems of ordinary differential equations leads to a rich variety of behaviour, characterized by solutions with periods that are multiples of the forcing period (see [22] for SEIR example). Typically, smooth variation of the parameters can lead to sudden changes in the periodicity of the stable behaviour of the system. As will be seen in §3, the goodness of fit of the model is strongly dependent on the periodicity of the model's solution.
We represent the generation time for the pathogen by an Erlang distribution with shape parameter k ¼ 4 and mean 1/a, where a ¼ 2.7 days [20, 23] . Our model incorporates two strains of influenza, for instance, representing H1N1 and H3N2, to try and capture aspects of the co-circulation of multiple influenza types and subtypes [11] . There are four immune states for individuals in the model; entirely susceptible, immune to either strain 1 or 2 and immune to both strains. The formulation allows for the inclusion of a basic cross-immunity mechanism, whereby an individual infected with either strain has a probability, f, of becoming immune to the other as well (assuming that this was not already the case) The flow between different immune states is illustrated in the electronic supplementary material. We note that this cross-immunity response is different from the shortterm non-specific response with regard to influenza strains considered elsewhere [10] , though cross-immunity is assumed to wane at the same rate as strain-specific immunity.
Our model also includes a mechanism for loss of immunity, returning individuals to a susceptible state. Surveillance data show that human influenza strains can be grouped into clusters within each of which there is a high level of cross-immunity, but between which cross-immunity is much lower [8] . The appearance of a new cluster therefore corresponds to a step change in the susceptibility of the population to the current strain. Our model caricatures this process with a timescale, D, for resistant individuals to become susceptible to the current strain again. As antigenically distinct clusters appear every 2 -8 years [8, 24] , this is our expected range for values of D. There is evidence from contact studies and from modelling of influenza epidemics that infectious contact between individuals is highly assortative and agedependent, with the highest rates among school-age children [16, 25, 26] . We include these effects by stratifying the population into children (less than or equal to 14 years) and adults (more than 14 years) and employing a mixing matrix to describe contact between the two groups. The degree of assortativity is controlled by the parameter, u, and can be varied between random mixing (u ¼ 0), where groups contact each other proportional to the fraction of the population they represent, and wholly assortative (u ¼ 1) where each group mixes only with itself. Differences in intensity of contact are captured by relative susceptibility and infectiousness parameters, r and c (see the electronic supplementary material for details).
To assess the quality of fit of the model behaviour to the data, we compare the distribution of key features in the time-series data with those generated by the epidemic model using the Kullback-Leibler (KL) information distance. We use normal distributions to characterize the empirical distributions of AAR and epidemic duration across a number of years. As discussed above, ignorance of the reporting rate makes it hard to know the underlying 'real' infection rate and also makes it difficult to compare reported incidence collected under different surveillance systems. In order to compare the data from the UK and France, we assume constant reporting rates for the UK and French surveillance systems, respectively, and scale the reported values linearly such that each has a mean AAR of 15 per cent (see the electronic supplementary material). Both dataset yield standard deviations of around 35 per cent of mean value for AAR and 11 + 2 weeks for epidemic duration.
We calculate the KL information distance between model and data, I, for each of the key features as follows:
where f is the distribution taken from the data, g is the approximate distribution of the same feature recovered from the model over many simulated years and p is a vector of model parameters. (See the electronic supplementary material for implementation.) The overall measure of goodness of fit used is the unweighted sum of the information distances for AAR and duration. We explore parameter space to identify regions where model behaviour most closely resembles empirical patterns. Although a simplified description of the epidemiological and evolutionary mechanisms of human influenza, our model nevertheless incorporates a substantial number of parameters. We focus on the following groupings:
-the seasonal peak value of R 0 , here termed R p , and its relative amplitude d (R p (1 -d) being the seasonal minimum value of R 0 ). Strictly, these parameters control overall transmissibility and the magnitude of seasonal forcing of transmission; -the timescale for the generation of antigenically new strains, represented by mean duration of immunity to the current influenza strain, D, and the crossimmunity between strains, f ( §2); -the degree of assortativity in the contact patterns between children and adults, u; -external force of infection, e, representing effect of contact between members of the modelled population and infected individuals outside the modelled population.
The values of other parameters are listed in table 1 and discussed in the electronic supplementary material. In discussing the behaviour of the model, periodicity refers to the periodicity of the overall case rate with time, rather than for an individual strain. Simulations were run using a population of 60 million, approximating the population of the UK. Owing to the large population, a deterministic model was used. Tests using the corresponding stochastic models showed no qualitative variation from the deterministic dynamics and the presence of a continuous low level external force of infection precluded the possibility of extinction. Figure 3 illustrates the behaviour of the model and aspects of the information distance between model and data as a function of R p and D. The region of best fits is located in a narrow diagonal band spanning 1.6 , R p , 2.5 and 3 , D , 8 (figure 3d ). Along this band, increasing reproduction number is compensated for by a longer period of effective immunity that decreases the susceptible proportion of the population, giving a constant mean attack rate. The acceptable region is bounded in part by the duration of the model epidemics. The lowest and highest values of R p generate epidemics that are too broad and too narrow, compared with the target distribution. The fit of the model is strongly constrained by the dynamics of the model, which exhibits a wide range of periodic behaviour across quite small changes in parameter values (figure 3a). While AAR changes smoothly with the parameters, abrupt changes in periodicity lead to qualitative changes in the distributions of AAR and epidemic duration and hence the KL distance. Best-fit behaviour is associated with long-period behaviour of the model (4þ years). Here, the model generates a range of AARs clustered around the mean and matching the target distribution. The qualitatively different forms of behaviour are well characterized by the total KL distance measure (figure 4). KL distances less than 200 correspond to realistic behaviour with appropriate mean attack rate distributions (figure 4a). KL distance between 200 and 300 match either with realistic behaviour interspersed with large-scale epidemic episodes or with realistic behaviour but with a mean attack rate displaced from the target value ( figure 4b) . Larger values represent dynamics and mean attack rates greatly different from the observed time series (figure 4c). Figure 5 illustrates a strong sensitivity to the amplitude of variation of the contact parameter, d, with the best-fit lying in the range 0.15-0.3. Although the mean AAR is not strongly dependent on d (figure 5a), the bifurcation behaviour of the model means realistic solutions (resembling figure 4a,b) can be found for higher amplitudes of seasonal variation but not in a contiguous region (figure 5b). Solutions with smaller seasonal variations are rejected on the epidemic duration component of the information distance. Low amplitudes generate broad epidemics which do not match the target distribution.
The behaviour of our model is quite sensitive to the assumed external force of infection, e. The level of external forcing assumed for most of this work ( §2) is negligible compared with the average force of infection generated by the indigenous population. However, long-period and chaotic solutions for seasonally forced SIR models generate very low infection prevalence during epidemic troughs, even low levels of importation of infectives strongly encourages annual and biennial behaviours and removes highly chaotic solutions from the optimal region. The effect of importation rate can be seen in figure 6 . For external forces of infection above about 10 25 yr -1 , only annual and biennial solutions are found. Optimal behaviour is found for an external force of infection of approximately 6 Â 10 27 yr -1 . Figure 7 explores the sensitivity of the model to the degree of heterogeneity and cross-immunity in the population. The choice of R p and D lies in the wellfitting band in figure 3d. It is clear that a wide range of values for these parameters allow the model to fit the behaviour of the time series quite well. The model's qualitative behaviour (in terms of its periodicity) is more stable with respect to these mechanisms and variation within the closest fit region mainly affects the mean attack rate. There is a broadly inverse relationship between the well-fitting values of the parameters u and f. Increasing the assortativity of mixing concentrates infections more strongly in age groups, decreasing the available susceptibles and hence the attack rate. Increasing cross-immunity has an equivalent effect by increasing the effective loss of susceptibles caused by any single infection event and hence reducing the attack rate. As can be seen from figure 7a, values of the cross-immunity parameter f outside the range 0.3-0.6 drive the model into unfavourable periodicities, giving very poor fits. This suggests that a model with two strains interacting via cross-immunity is necessary to reproduce the dynamics seen in influenza time series and that it is insufficient to have two independent strains (f ¼ 0) or two antigenically identical strains (i.e. f ¼ 12equivalent to a single strain model). Similarly, extreme values of u also lead to poorly fitting model behaviour, suggesting that a uniformly mixing population (u ¼ 0) would also not generate matching behaviour.
In this work, we have identified a minimal set of mechanisms necessary to match the long-term temporal behaviour seen in ILI time series. We find that an age-structured population and multiple strains with cross-immunity are necessary to recreate the distributions of AAR and epidemic duration seen in time-series data. Nonlinear models of this type with temporal forcing are well known for having complex bifurcation structures affecting their periodic behaviour. On the timescale of a single disease season or single epidemic, these would have little effect on parameter estimation. Over many seasons, however, the periodicity of the underlying model is crucially important. We would argue that capturing the long-term trends in behaviour is at least as important as the detail of individual seasonal outbreaks and our fitting approach focuses on these aspects. The complex bifurcation structure of the model means that the information distance is not necessarily a smooth function of the parameters. This makes finding a unique set of parameters giving an overall minimum distance impossible. Although mean attack rate and duration generally vary smoothly, the period of the model solution changes discontinuously (inevitably, as it only takes values that are multiples of the annual forcing period). Solutions with longer periods generate a wider range of AAR and epidemic durations over an extended period of time and are therefore capable of fitting the target data distributions better. As a result, the best fits are strongly associated with longer periods in model solutions and the quality of fit of the model can change abruptly over small changes in parameter values.
Peak R 0 , the amplitude of variation of R 0 and the duration of immunity are all strongly constrained. Figure 3 illustrates that increasing R 0 is offset by a longer duration of immunity reducing the susceptible population. The narrowness of the well-fitting parameter region is a result of the sudden changes of behaviour generated by changes in these parameters. Figure 5 also illustrates this feature. The closest fitting region is found for d between 0.15 and 0.3, but patches of well-fitting solutions are scattered a range of values of R p and d owing to the sensitivity to the system to temporal forcing. We note that a change in the mode of forcing from sinusoidal to school-term leads to generally broader ranges of acceptable parameter values (see the electronic supplementary material), perhaps indicating that the presence of this mechanism is a strong contributor to the variable annual behaviour observed in the ILI dataset. Because of the difficulties in knowing the 'true' incidence rate, the mean AAR is not precisely known and a range of 10-20% is often quoted. To allow for this uncertainty, we investigated allowing the information distance calculation to be based on the best-fit mean AAR from the range 10-20%, rather than precisely 15 per cent per year. Resulting best-fit parameter regions for R 0 against D and u against f were not significantly changed, owing almost certainly to the dominance of qualitative model behaviour as described above.
Incidence periodicity of the model is much less sensitive to cross-immunity and age structure, resulting in a wide region of close fitting behaviour for values of u between about 0.2 and 0.6 and f between 0.3 and 0.5. As discussed in §3, this strongly suggests that an agestructured population and, in particular, a pathogen population with more than one strain and crossimmunity, are necessary to reproduce the patterns of behaviour found in the ILI time series. Models based on single strains and well-mixed populations generate annual and biennial behaviour for the same parameter values. The necessity of multiple strains has been noted in other work modelling seasonal influenza [14] , although in that work the two strains did not interact.
Within the best-fitting parameter regimes, incidence periodicity for each modelled strain is basically biennial with strains dominating alternate years. While real strain dynamics are clearly more complex than this (figure 1), observed patterns do show a tendency for a particular strain not to dominate in successive years. Within the model, cross-immunity generates a negative correlation between strains, causing them to alternate in successive years. For strong cross-immunity, both strains become antigenically similar and goodness of fit falls off rapidly.
It is instructive to compare our results with those of previous papers fitting simple models to influenza incidence data. Work by Xia et al. [15] used a simple single strain SIRS, but with a more detailed description of temporal variation in contact rate and loss of immunity post-recovery. In addition, the infection rate was described by the phenomenological term b I a g(S). Exponents of this type are well known to facilitate fitting [5] , but are hard to interpret. We note that both the form of this term and the non-exponentially distributed duration of immunity in that study affect epidemic attack rates as a function of transmissibility and the periodicity of epidemics, and hence may play an equivalent role to age structure and the incorporation of two subtypes in our model in allowing a good fit to the data.
Shaman et al. [14] use a similar SIRS model to investigate the possibility that seasonal changes in absolute humidity can generate recorded patterns in pneumonia and influenza mortality data. The model was stochastic, has no age structure, and effectively uses only one strain. Best-fit parameter values are similar to those found in this work, although the relative amplitude of variation in R 0 is in the range 0.4-0.5, significantly higher than our findings. Best-fit parameter sets showed considerable lack of correlation with each other, which the authors attribute to the stochastic nature of their model. Our work suggests that this scatter may be the result of the complex bifurcational structure of such models. As already discussed, our results indicate that a two strain model without cross-immunity or age structure is unlikely to fit patterns of seasonal influenza from a temperate region. However, there are several significant differences between the two systems. Shaman et al. employ a significantly higher background force of infection than ours (approx. 7.3 Â 10 24 yr -1 ), which would place our model in a strongly annual or biennial regime. Hence that model may reproduce the mean attack rate well, but not its variability.
Our assessment of goodness of fit is currently focused primarily on distribution of AARs and duration of epidemics, although we also take account of the sequence of strains and the age-distribution of cases (see the electronic supplementary material). Future work will test our conclusions against a full description of the incidence data as well as against different choices of key features in the data, such as time of epidemic onset. Amiodarone Exposure During Modest Inflammation Induces Idiosyncrasy-like Liver Injury in Rats: Role of Tumor Necrosis Factor-alpha Amiodarone [2-butyl-3-(3′,5′-diiodo-4’α-diethylaminoethoxybenzoyl)-benzofuran] (AMD), a class III antiarrhythmic drug, is known to cause idiosyncratic hepatotoxic reactions in human patients. One hypothesis for the etiology of idiosyncratic adverse drug reactions is that a concurrent inflammatory stress results in decreased threshold for drug toxicity. To explore this hypothesis in an animal model, male Sprague-Dawley rats were treated with nonhepatotoxic doses of AMD or its vehicle and with saline vehicle or lipopolysaccharide (LPS) to induce low-level inflammation. Elevated alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase activities as well as increased total bile acid concentrations in serum and midzonal hepatocellular necrosis were observed only in AMD/LPS-cotreated rats. The time interval between AMD and LPS administration was critical: AMD injected 16 h before LPS led to liver injury, whereas AMD injected 2–12 h before LPS failed to cause this response. The increase in ALT activity in AMD/LPS cotreatment showed a clear dose-response relationship with AMD as well as LPS. The metabolism and hepatic accumulation of AMD were not affected by LPS coexposure. Serum concentration of tumor necrosis factor-alpha (TNF) was significantly increased by LPS and was slightly prolonged by AMD. In Hepac1c7 cells, addition of TNF potentiated the cytotoxicity of both AMD and its primary metabolite, mono-N-desethylamiodarone. In vivo inhibition of TNF signaling by etanercept attenuated the AMD/LPS-induced liver injury in rats. In summary, AMD treatment during modest inflammation induced severe hepatotoxicity in rats, and TNF contributed to the induction of liver injury in this animal model of idiosyncratic AMD-induced liver injury. Amiodarone [2-butyl-3-(3#,5#-diiodo-4'a-diethylaminoethoxybenzoyl)-benzofuran] (AMD), a class III antiarrhythmic drug, is known to cause idiosyncratic hepatotoxic reactions in human patients. One hypothesis for the etiology of idiosyncratic adverse drug reactions is that a concurrent inflammatory stress results in decreased threshold for drug toxicity. To explore this hypothesis in an animal model, male Sprague-Dawley rats were treated with nonhepatotoxic doses of AMD or its vehicle and with saline vehicle or lipopolysaccharide (LPS) to induce low-level inflammation. Elevated alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase activities as well as increased total bile acid concentrations in serum and midzonal hepatocellular necrosis were observed only in AMD/LPS-cotreated rats. The time interval between AMD and LPS administration was critical: AMD injected 16 h before LPS led to liver injury, whereas AMD injected 2-12 h before LPS failed to cause this response. The increase in ALT activity in AMD/LPS cotreatment showed a clear doseresponse relationship with AMD as well as LPS. The metabolism and hepatic accumulation of AMD were not affected by LPS coexposure. Serum concentration of tumor necrosis factor-alpha (TNF) was significantly increased by LPS and was slightly prolonged by AMD. In Hepac1c7 cells, addition of TNF potentiated the cytotoxicity of both AMD and its primary metabolite, mono-N-desethylamiodarone. In vivo inhibition of TNF signaling by etanercept attenuated the AMD/LPS-induced liver injury in rats. In summary, AMD treatment during modest inflammation induced severe hepatotoxicity in rats, and TNF contributed to the induction of liver injury in this animal model of idiosyncratic AMD-induced liver injury.
Key Words: amiodarone hepatotoxicity; inflammation; lipopolysaccharide; idiosyncratic adverse drug reactions; drug metabolism; tumor necrosis factor-alpha.
Idiosyncratic adverse drug reactions (IADRs) typically occur only in a small fraction of patients who are treated with certain drugs at therapeutic doses. IADRs are usually unrelated to the pharmacological target of the drug. They present a serious human health problem and are usually not predicted by current preclinical safety evaluation during drug development. During the period 1975-2000, 10% of newly approved drugs were withdrawn from the U.S. market or received black box warnings due to these adverse reactions Uetrecht, 2007) .
The mechanisms by which IADRs occur are not clear. Evidence from experimental animals indicates that mild inflammation can decrease the threshold for toxicity and thereby render an individual susceptible to an adverse drug reaction that would not otherwise occur . Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, is widely used as an inflammagen in these animal studies. A nonhepatotoxic dose of LPS can interact with nontoxic doses of several IADR-associated drugs from different pharmacologic classes to induce liver damage in rodents (Deng et al., 2006; Luyendyk et al., 2003; Waring et al., 2006; Zou et al., 2009b) .
Amiodarone [2-butyl-3-(3#,5#-diiodo-4'a-diethylaminoethoxybenzoyl)-benzofuran] (AMD), a class III antiarrhythmic drug, is effective in increasing the survival of patients after myocardial infarction or congestive heart failure (Singh, 1996) . Since the approval of AMD by the U.S. Food and Drug Administration in 1985, the use of this drug has been associated with a variety of adverse effects, including liver dysfunction, pulmonary complications, thyroid dysfunctions, and ocular disturbance (Rotmensch et al., 1984) . The reported frequency of liver abnormalities in patients receiving AMD varies from 14 to 82% (Lewis et al., 1989) . Most of these reactions are mild, with serum transaminase elevation within threefold of the upper limit of normal (ULN), but some are more severe (Babatin et al., 2008) . Cases of liver reactions after intravenous administration of AMD are rare, but damage can be acute and marked (Rätz Bravo et al., 2005) . Fulminant hepatic failure or death associated with AMD hepatotoxicity has also been reported (Babatin et al., 2008) .
There is evidence that the interaction between LPS-induced cytokines and drugs or their metabolites plays an important role in the LPS-drug interaction (Zou et al., 2009a) . Tumor necrosis factor-alpha (TNF) is a proximal mediator of the inflammatory cascade induced by LPS (Beutler and Kruys, 1995) and is critically involved in many models of liver injury, such as ischemia/reperfusion (Teoh et al., 2004) , alcoholic liver disease (Yin et al., 1999) and some drug/LPS-induced liver injury models (Shaw et al., 2009b; Tukov et al., 2007; Zou et al., 2009a) . As an example, TNF selectively augmented the cytotoxicity of sulindac sulfide, which is the major toxic metabolite of sulindac (Zou et al., 2009a) . In the case of amiodarone, the major metabolite of AMD is mono-N-desethylamiodarone (DEA), which shares similar pharmacological (Talajic et al., 1987) and pharmacokinetic (Shayeganpour et al., 2008) characteristics with AMD. DEA has antiarrhythmic properties, a very long half-life, and accumulates in the liver and many other tissues. In primary hepatocytes, HepG2 cells and other cell types, DEA is much more cytotoxic than AMD (Waldhauser et al., 2006) . The plasma concentration of DEA is greater in cases of AMD-associated IADRs (O'Sullivan et al., 1995) , suggesting a possible role for this metabolite in AMD toxicity.
The purpose of this study was to test the hypothesis that inflammatory stress induced by LPS potentiates amiodaroneinduced hepatotoxicity in rats. When the results demonstrated a hepatotoxic interaction between inflammatory stress and amiodarone, the roles of metabolism and TNF were explored.
Materials. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St Louis, MO). The activity of LPS (Lot 075K4038, derived from Escherichia coli serotype O55:B5) was 3.3 3 10 6 endotoxin units (EU)/ mg, which was determined by a Limulus Amebocyte Lysate Kinetic-QCL kit from Cambrex Corp. (Kit 50-650U; East Rutherford, NJ). The reagents for the measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT) activities were purchased from Thermo Electron Corp. (Waltham, MA). The kit for total bile acids measurement was purchased from Diazyme Laboratories (Poway, CA).
Animals. Male Sprague-Dawley rats (Crl:CD(SD)IGS BR; Charles River, Portage, MI) weighing 250-370 g were used for in vivo studies. They were fed standard chow (Rodent Chow/Tek 8640; Harlan Teklad, Madison, WI) and allowed access to water ad libitum. Animals were allowed to acclimate for 1 week in a 12-h light/dark cycle prior to experiments. They received humane care according to the criteria in the Guide for the Care and Use of Laboratory Animals.
Experimental protocol. In all the experiments, rats were fasted for 12 h before administration of LPS and food was returned thereafter. A 20 mg/ml solution of AMD was made in its vehicle (0.18% Tween 80), and 4.1 3 10 5 EU/ml solution of LPS was made in sterile saline. To determine the optimal time interval between AMD and LPS treatments, rats were treated with AMD (300 mg/kg, ip) 2 h, 8 h, 12 h, 16 h, or 20 h before LPS (1.6 3 10 6 EU/kg, iv). For the evaluation of the dose-response relationship for AMD, rats were treated with AMD (0-400 mg/kg, ip) and 16 h later with LPS (1.6 3 10 6 EU/kg, iv) or saline. For the evaluation of the dose-response relationship for LPS, rats were treated with AMD (400 mg/kg, ip) or vehicle and 16 h later with LPS (0-1.6 3 10 6 EU/kg, iv). In subsequent studies, rats were treated with AMD (400 mg/kg, ip) or vehicle and 16 h later with LPS (1.6 3 10 6 EU/kg, iv) or saline. In the etanercept treatment study, rats were treated with etanercept (8 mg/kg) or sterile water by sc injection 1 h before LPS.
Rats were anesthetized with isoflurane, and blood and liver samples were taken. Serum was prepared from blood, and plasma was prepared from blood collected into a syringe containing 3.2% sodium citrate (BD Biosciences, San Diego, CA). The right medial lobe of the liver was rapidly frozen for immunohistochemistry, and the left lateral lobe of liver was fixed in 10% neutral-buffered formalin and stored in 70% ethanol for histopathology.
Evaluation of liver injury. Liver injury was estimated from the serum activities of ALT, AST, ALP, and GGT and from the serum concentration of total bile acids.
Formalin-fixed liver samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) staining. The stained liver sections were examined using light microscopy.
Drug and metabolite analysis. Serum samples and liver homogenates were mixed with acetonitrile containing ethopropazine as internal standard (IS). After vortexing and centrifugation, protein was removed, and the supernatant was diluted and transferred to autosampler vials for liquid chromatographytandem mass spectrometry (LC/MS/MS) analysis. LC/MS/MS analysis was performed by use of a Shimadzu LC-20 high performance liquid chromatography (HPLC) system coupled to a QTRAP 3200 tandem quadrupole mass spectrometer (AB SCIEX, Foster City, CA) operated under control of Analyst v. 1.4.2 software. The Ascentis Express C18 HPLC Column (5 cm 3 2.1 mm, 2.7 lm) was maintained at 50°C. A volume of 2 ll was injected into the HPLC system and eluted with a gradient based on 10mM ammonium acetate in H 2 O (solvent A) and methanol (solvent B): 0-0.5 min, 10% solvent B; 0.5 -1 min, 10-98% solvent B; 1-4 min, 98% solvent B; 4-6 min, 10% solvent B; flow rate, 0.3 ml/min. Positive mode electrospray ionization was used for all analyses. Mass spectrometry parameters, including declustering potential and collision energy, were optimized independently for each analyte and IS. Multiple reactions monitoring the m/z transitions were used for the quantitative analysis of AMD (m/z 646.1/201.1), DEA (m/z 618.1/547.0), and ethopropazine (m/z 313.1/114.1). The LC/MS/MS method achieved lower limits of quantification: 50 ng/ml for AMD and 5 ng/ml for DEA. Analytical reproducibility was judged to be ± 10.2% in the middle of the calibrated range of concentrations. The Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL) was used to determine protein concentration in the liver homogenates.
Assessment of cytotoxicity in vitro. The murine hepatoma cell line Hepa1c1c7 purchased from American Type Culture Collection (Manassas, VA) was used to assess cytotoxicity in vitro. Hepa1c1c7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 1% antibiotic-antimycotic (Invitrogen) and 10% heat-inactivated fetal bovine serum (SAFC Biosciences, Lenexa, KS) in 75-cm 2 tissue culture flasks at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Cells were plated in 96well plates at 15,000 cells per well and allowed to attach for 8 h before medium was replaced. Various concentrations of AMD, DEA, and/or TNF were added to designated wells, and cells were incubated under maintenance conditions. Twenty-four hours later, lactate dehydrogenase (LDH) activity released into the culture medium was measured using the Cytotox-One Homogeneous Membrane Integrity Assay (Promega, Madison, WI). The percent LDH release was calculated as LDH in supernatant/(LDH in supernatant þ LDH in cell lysate). Lysate LDH was determined after addition of Triton to lyse the cells.
Statistical analysis. The results are expressed as means ± SEM. One-way or two-way ANOVA was applied as appropriate; Tukey's method was employed as a post hoc test. Grubb's test was used to detect outliers. At least three biological repetitions were performed for each experiment. The p value < 0.05 was set as the criterion for statistical significance.
Serum ALT activity did not increase from treatment with either LPS or AMD alone (Fig. 1) . In the AMD/LPS group, administration of AMD at 16 or 20 h before LPS resulted in significant serum ALT activity increase, whereas AMD injected 2-12 h before LPS failed to cause this response. The 16-h interval between AMD and LPS treatments was selected for future studies.
Neither AMD alone nor LPS alone affected ALT activity at any of the doses tested. In rats cotreated with AMD and LPS, serum ALT activity was dependent on both AMD ( Fig. 2A) and LPS (Fig. 2B) doses. Significant increases in ALT activity were observed with AMD doses 300 mg/kg (p < 0.05) and with LPS doses 1.2 3 10 6 EU/kg (p < 0.05). 400 mg/kg and 1.6 3 10 6 EU/kg were selected as AMD and LPS doses for subsequent studies, respectively.
In the time course study, the serum activities of both ALT and AST were measured as markers for hepatocellular injury (Figs. 3A and B). Neither AMD nor LPS alone affected serum ALT activities at any time examined. For the AST activity, LPS alone had no effect at any time examined, and AMD alone caused a slight increase at 2, 4, and 10 h. Significant elevation of serum ALT and AST activities were only observed in AMD/ LPS cotreatment, and the increases started between 4 and 6 h after LPS administration and continued to increase through 10 h.
At 10 h after LPS administration, serum activities of ALP and GGT and concentration of bile acids were measured as indicators of cholestatic injury (Figs. 4A-C). Only AMD/LPS cotreatment caused significant increases in these serum markers, whereas AMD or LPS treatment alone had no effect.
All the saline-treated control rats were free of liver lesions (Fig.  5A) . No microscopic evidence of hepatic pathology was found in four of the six LPS-treated rats (Fig. 5B ). Liver sections from two of the LPS-treated rats had a few small foci of midzonal hepatocellular necrosis with an associated neutrophilic influx. In contrast, a widespread, mild-to-marked fibrinopurulent capsulitis was present in the liver sections from all the AMD-treated rats (Fig. 5C ). This was characterized by a thickening of the hepatic capsule due to edema and a conspicuous inflammatory exudate comprising mainly neutrophils, lesser numbers of mononuculear cells, and various amounts of amorphorous proteinaceous material. This fibrinopurulent exudate was also often scattered along the outer peritoneal surface of the capsule. Focal areas of subcapular hepatocellular necrosis were occasionally associated with the capsulitis.
The most profound hepatic histopathology was found in animals treated with both AMD and LPS (Fig. 5D ). All these rats had a mild-to-marked fibrinopurulent capsulitis with occasional subcapsular necrosis similar to that found in the AMD-treated rats, but in addition, all these animals had conspicuous areas of midzonal hepatocellular necrosis. The latter lesion ranged from widely scattered focal areas of necrosis in midzonal regions to widespread hepatocellular necrosis with coalescence of affected midzonal and occasionally centriacinar regions (bridging necrosis). Accumulations of neutrophils (inset, Fig. 5D ) were present in all these necrotic regions. Periportal regions were spared of AMD/LPS treatment-related injury. Rats were treated with AMD (0-400 mg/kg, ip) and 16 h later with saline or LPS (1.6 3 10 6 EU/kg, iv). (B) Rats were treated with AMD (400 mg/kg, ip) or its vehicle and 16 h later with LPS (0-1.6 3 10 6 EU/kg, iv). Serum ALT activity was measured at 10 h after LPS administration for both data sets. # indicates significantly different from respective groups not given LPS; * indicates significantly different from respective group not given AMD. p < 0.05, n ¼ 3-14.
Accumulation of AMD AMD and DEA concentrations in rat serum and liver homogenates were determined at various times after LPS administration (Figs. 6A-D). From 2 to 10 h after LPS or saline administration (i.e., 18-28 h after AMD administration), the serum and tissue concentrations of AMD and DEA were unaffected by LPS cotreatment. The average serum concentrations of AMD and DEA were 1100 and 128 ng/ml, respectively; and the average liver concentrations of AMD and DEA were 157 and 53 ng/mg protein, respectively.
Serum TNF concentration was measured at 2 and 4 h after LPS administration (Fig. 7) . AMD by itself had no effect on serum TNF concentration. At 2 h after LPS, the serum TNF concentrations in rats treated with AMD/LPS or with vehicle/ LPS were similar. However, by 4 h after LPS, the concentration of TNF in serum of AMD/LPS-treated rats was significantly greater than that in vehicle/LPS-treated rats.
Hepa1c1c7 cells were exposed to AMD or DEA, and 24 h later, cytotoxicity was assessed by measuring LDH activity released into the culture medium (Figs. 8A and B). Both AMD and DEA caused concentration-dependent LDH release. Significant cytotoxicity was observed with AMD concentrations greater than 20 ug/ml and DEA concentrations greater than 7 ug/ml. Addition of TNF (3 ng/ml) did not cause cytotoxicity alone but significantly potentiated the cytotoxicity of AMD and DEA.
Etanercept is a soluble TNF receptor construct that inactivates TNF. Etanercept (8 mg/kg, sc) injected 1 h before LPS inhibited the biological activity of TNF in rats and was not hepatotoxic by itself (Tukov et al., 2007; Zou et al., 2009a) . The same treatment was used in this study. AMD/LPS cotreatment increased serum ALT activity, and etanercept significantly attenuated this increase (Fig. 9A) . Changes in serum ALT activity were supported by histological Rats were treated with AMD (400 mg/kg, ip) or vehicle and 16 h later with LPS (1.6 3 10 6 EU/kg, iv) or saline. They were examined 2, 4, 6, or 10 h after LPS injection. Activities of (A) ALT and (B) AST in serum were measured. # indicates significantly different from respective groups not given LPS; * indicates significantly different from respective group not given AMD. p < 0.05, n ¼ 4-9.
AMD HEPATOTOXICITY DURING MODEST INFLAMMATION examination of H&E-stained liver sections: the severity and frequency of necrotic foci were markedly reduced in rats cotreated with etanercept (Fig. 9B) .
Since its introduction in Europe in 1962, amiodarone has been associated with idiosyncratic hepatotoxicity (Lewis et al., 1989) . A linear correlation between serum AMD concentration and serum ALT activities has been established (Pollak and You, 2003) ; however, in that study, the ALT values did not exceed 3 3 ULN and were not considered to be clinically significant. For the cases of severe liver injury caused by intravenous amiodarone loading, patients' serum ALT activities were up to 10-206 3 ULN. There was usually a 24-72 h delay between the initial loading of AMD and the onset of elevation in ALT activities; and in many cases, the ALT activity returned to normal after a few days of continuation of maintenance dosing (Rätz Bravo et al., 2005) . Accordingly, it is hard to draw a simple linear relationship between the magnitude or frequency of severe hepatotoxicity and serum AMD concentration. An effort to establish a model for AMD-induced liver injury in healthy rodents was unsuccessful. Neither short-term, large dose nor long-term, small dose administration of AMD led to observable liver damage (Young and Mehendale, 1989) . The cause of severe AMD hepatotoxicity is more likely to be a combination of AMD and other factors, e.g., inflammatory Various concentrations of (A) AMD or (B) DEA were added to cultures of Hepa1c1c7 cells together with TNF (3 ng/ml) or saline. Twenty-four hours after treatment, LDH activity released into the culture medium was measured. The percent LDH release was calculated as described in ''Materials and Methods'' section. * indicates significantly different from respective groups not given TNF; # indicates significantly different from respective groups not given AMD or DEA. p < 0.05, n ¼ 3. episodes. In the present study, regardless of cotreatment with LPS, the serum concentration of AMD was about 1100 ng/ml, which is very close to the steady-state serum concentration of AMD in human patients (1500 ng/ml) under long-term oral amiodarone therapy (Pollak et al., 2000) . Our findings support that at this clinically relevant concentration of AMD in serum, hepatotoxicity can be induced by a concurrent inflammatory episode related to LPS exposure.
Previous studies in rodents have suggested a possible association between inflammation and liver injury for several drugs associated with human IADRs, including chlorpromazine (Buchweitz et al., 2002) , ranitidine , diclofenac (Deng et al., 2006) , trovafloxacin (Shaw et al., 2007) , sulindac (Zou et al., 2009b) , and halothane (Dugan et al., 2010) . The results of the present study expand these findings and demonstrate that a nonhepatotoxic dose of AMD is rendered hepatotoxic when acute inflammation is triggered by LPS administration. Acute increases in serum markers for hepatocellular and cholestatic injury were found in rats cotreated with AMD/LPS. These resemble the clinical hepatic chemistry changes in human idiosyncrasy during AMD therapy (Rätz Bravo et al., 2005) . The midzonal and bridging necrosis and infiltration of inflammatory cells in AMD/LPS are also consistent with some of the histological changes found in human patients (Babatin et al., 2008; Rätz Bravo et al., 2005) . However, the histological characteristics in people with AMD-induced liver injury were variable. Different patterns of hepatocellular necrosis, such as midzonal, centrilobular, bridging, and panlobular, have been reported (Lewis et al., 1989) . Genetic differences, concurrent medications, and even different origins of inflammation might account for these varied responses; nevertheless, the AMD/LPS interaction model in rats mimics important aspects of AMD-induced IADRs in human patients.
The timing of AMD and LPS dosing in this model was important for the development of severe liver damage. A minimal interval of 16 h was required for AMD and LPS to interact to induce liver injury. When LPS was injected within 16 h after AMD, no liver injury was observed. Absorption, distribution, metabolism, and clearance as well as toxicological actions could contribute to this timing requirement. The elimination of AMD is primarily through hepatic metabolism and biliary excretion, and its half-life in plasma is very long (55 days in humans) (Pollak et al., 2000) . Accordingly, the loss of AMD due to elimination within 16 h is minimal. AMD has a dose-dependent effect on the respiratory chain and b-oxidation in the mitochondria (Fromenty et al., 1990a, b) , and it can also affect the function of lysosomes and other acidic organelles (Stadler et al., 2008) . The 16-h interval may be required for AMD to distribute into the liver, accumulate in organelles, and sensitize hepatocytes to interact with LPS or its downstream cytokines.
In other drug/LPS models, there is also a dependence on the temporal relationship between administrations of drug and LPS, and the time interval needed for a maximal hepatotoxic response is different for different drugs (Shaw et al., 2007; Zou et al., 2009b) . This time interval requirement might help to explain the low frequency of IADRs in human patients: i.e., only when the inflammatory episode happens at a certain time during drug therapy would idiosyncratic hepatotoxicity occur.
In rats, AMD is deethylated by cytochromes P450 (CYPs) 3A4, 1A1, 2D1, and 2C11 in the liver (Elsherbiny et al., 2008) . DEA, the major metabolite, is three-to fivefold more toxic than AMD to cultured HepG2 cells (Waldhauser et al., 2006) and to primary rat hepatocytes (Gross et al., 1989) . In our treatment of Hepa1c1c7 cells, a similar trend was observed (Fig. 8) . The administration of LPS affects the expression and activities of CYPs in rats (Sewer et al., 1997) . This raised the possibility that LPS might potentiate the toxicity of AMD by increasing its metabolism to DEA. To evaluate this, serum and liver concentrations of AMD and DEA were measured with LC/ MS/MS. The average serum concentration of DEA in AMDtreated rats was 128 ng/ml, which is about 1/10 of the serum AMD concentration. This ratio is commonly seen in the serum after an intravenous loading dose of AMD, both in people (Ha et al., 2005) and rats (Shayeganpour et al., 2008) . Neither the serum nor the liver concentration of AMD or DEA was affected by LPS cotreatment, suggesting that neither AMD accumulation nor DEA generation was affected by LPS.
Intratracheal instillation of AMD in vivo or exposure of alveolar macrophages to AMD in vitro led to TNF production (Futamura, 1996; Reinhart and Gairola, 1997) . AMD treatment also increased TNF production by alveolar macrophages on LPS stimulation (Punithavathi et al., 2003) . In the present study, treatment of rats with AMD alone did not cause serum TNF elevation, but it did increase the concentration of TNF in serum of LPS-treated rats. These results suggest that the increased appearance of TNF in AMD/LPS-cotreated rats was probably not an additive effect; rather, AMD appeared to potentiate the production or diminish the clearance of TNF caused by LPS. The concentration of TNF in serum increases rapidly in LPS-treated rats, peaks at around 1.5-2 h, and then returns to basal levels at around 6 h (Tukov et al., 2007) . In the present study, the concentration of TNF around the peak time (i.e., 2 h) in LPS-cotreated rats was not affected by AMD, but the TNF concentration was greater in AMD-cotreated rats at a later time (4 h). These data suggest that AMD prolonged the elevation in TNF caused by LPS administration. This seemingly small difference in TNF concentration was shown to be critical to liver pathogenesis in another drug/LPS model of liver injury involving trovafloxacin (Shaw et al., 2009b) . Accordingly, it is possible that prolongation of the LPS-induced TNF response is a critical event across models of LPS-drug interaction.
The importance of TNF in the AMD model was explored by preventing its binding to cellular receptors with etanercept. Etanercept pretreatment reduced hepatotoxicity, indicating that TNF has an important role in AMD/LPS-induced liver injury. Because no liver injury was observed after treatment with LPS AMD HEPATOTOXICITY DURING MODEST INFLAMMATION alone, the large TNF peak caused by LPS was not hepatotoxic by itself; however, this amount of TNF could be critical for the induction of hepatocellular injury by potentiating the toxic effect of AMD and/or DEA. Signaling from an activated TNF receptor can lead to lysosomal leakage, mitochondrial damage, and caspase activation (Wullaert et al., 2007) . All three of these events were also found in AMD and DEA cytotoxicity in vitro (Agoston et al., 2003; Spaniol et al., 2001) . Further support for a critical role for TNF came from our in vitro study in Hepa1c1c7 cells in which TNF increased the cytotoxicity of both AMD and DEA. As a proximal proinflammatory cytokine, TNF can also contribute to liver damage by inducing downstream inflammatory events, such as coagulation activation and neutrophil activation (Shaw et al., 2009b; Tukov et al., 2007) . The etanercept treatment herein reduced the ALT activity to half of the level seen in the absence of this inhibitor, whereas the same dose of etanercept reduced the ALT activity almost to control level in trovafloxacin/LPS and sulindac/LPS models (Shaw et al., 2007; Zou et al., 2009a) . This suggests that other factors induced by LPS might act in parallel with TNF in the AMD/LPS model. In other models of potentiation of xenobiotic toxicity by LPS, factors such as neutrophils (Luyendyk et al., 2005) , the coagulation system (Shaw et al., 2009a) , and prostanoids (Ganey et al., 2001) play important roles, and these factors might be relevant in the model presented here.
In summary, AMD was rendered hepatotoxic in rats in the presence of a coexisting inflammatory stress induced by LPS. AMD/LPS-cotreated rats developed liver pathology and blood chemistry changes that resemble AMD-induced idiosyncratic hepatotoxicity in human patients. LPS did not interact with AMD by changing the metabolism or distribution of AMD. AMD enhanced the increase in plasma TNF concentration caused by LPS, and neutralizing TNF reduced liver injury from AMD/LPS coexposure. Moreover, TNF potentiated the cytotoxicity of both AMD and DEA in vitro. These findings add support to the idea that inflammatory stress can interact with IADR-associated drugs to cause liver injury by a mechanism involving TNF and suggest that a similar mode of action might apply to several drugs that cause idiosyncratic hepatotoxicity in humans.
National Institutes of Health (R01DK061315). Polyvalent DNA Vaccines Expressing HA Antigens of H5N1 Influenza Viruses with an Optimized Leader Sequence Elicit Cross-Protective Antibody Responses Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic. The continuous spread of highly pathogenic avian influenza Type A (HPAI) H5N1 viruses in avian species across multiple continents and frequent reports of human H5N1 infection in China and Southeast Asia highlight the threat of a potential flu pandemic in the human population. At the same time, H5N1 viruses have grown into genetically and antigentically diversified viruses. Based on phylogenetic analysis of hemagglutinin (HA) protein gene sequences, at least 10 clades of H5N1 viruses (clades 0-9) have been identified [1, 2, 3, 4, 5] . Recent studies have further assigned these viruses into four major antigenic groups (A-D) [3] . HPAI H5N1 viruses from more than one clade have caused human infection since 1997.
A key component in the global strategy to prepare for and control any pending influenza pandemic is the development of an effective vaccine. Several versions of inactivated as well as live attenuated H5N1 vaccines have been tested in humans and showed an overall good safety and immunogenicity profile mainly by using a clade 1 H5N1 virus (A/Vietnam/1203/04) as the vaccine strain per recommendations by the World Health Organization (WHO) [6, 7, 8] . Given that the majority of the world's human population is naïve to H5N1 influenza, two immunizations are needed to achieve desired levels of protective immune responses against H5N1 in contrast to the annual seasonal flu vaccine which requires only one immunization, presumably due to the priming effects by either exposure to circulating H1, H3 or Type B influenza viruses in humans or history of prior seasonal flu vaccination. The likely requirement of two immunizations in conjunction with the genetic complexity of H5N1 viruses, as evidenced by their separation into multiple subgroups, makes it difficult to prepare for the timely production of a sufficient number of doses of H5N1 vaccines in the event of an H5N1 pandemic; therefore, supplemental strategies are needed. As shown by our previously published report [9] and confirmed by other recent studies [10] , a DNA prime-inactivated vaccine boost is highly effective in eliciting higher protective immune responses than using either DNA or inactivated flu vaccine alone. Therefore, it may be possible to use DNA vaccines as the first dose of immunization that can be given either long before the pandemic (pre-pandemic vaccination) or shortly after the outbreak, to reduce the burden on the production of inactivated vaccines at the time of the outbreak. Furthermore, DNA vaccines can be stockpiled for a long period of time, which makes this method even more attractive.
One key issue that needs to be analyzed for the above strategy is the cross reactivity between DNA vaccines expressing H5 HA antigens from different clades. It is critical to first optimize the immunogenicity of H5 HA DNA vaccines and then to test how much cross protection can be achieved with optimized H5 HA DNA vaccines. In the current report, we constructed DNA vaccines to express wild type HA antigens without mutations at the HA1 and HA2 cleavage site from four key H5N1 strains that have caused major human infection: HK/156/97 (clade 0), VN/1203/ 04 (clade 1), Ind/5/05 (clade 2.1), and Anhui/1/05 (clade 2.3). Rabbit sera immunized with these HA antigens were examined for their protective antibody responses against either homologous or heterologous H5N1 viruses. Our results demonstrated an imperfect cross-reactivity profile for the protective antibody responses among these four viruses. A polyvalent formulation including three different H5 HA DNA vaccines was able to produce broad protective antibody responses with high titers against these key H5N1 isolates. Information learned from this study should facilitate the selection of candidate H5N1 vaccines to form polyvalent H5N1 DNA vaccines as part of the global strategy to prevent and control a potential avian flu pandemic.
One of the key findings from our previous study was that HA antigens from H1 and H3 serotypes had different structure preferences in order to elicit optimal protective antibodies [11] . Two of the HA antigen designs used in that study were also included in the current study to identify the optimal design for the H5 serotype HA antigens: one used the wild type HA antigen insert (H5.wt), which has the exact same amino acid sequences found in the natural viral isolate, and the other used a truncated HA antigen insert (H5.dTM), which removed the transmembrane (TM) and intracellular segments of the HA2 domain ( Fig. 1) . In addition, a third HA antigen insert was created (H5.tPA), in which a human tissue plasminogen activator (tPA) sequence replaced the original wild type leader sequence from the HA antigen (Fig. 1) . The third HA antigen design was adopted because the H5.dTM design also used a tPA leader sequence and the H5.tPA insert served as a control for the H5.dTM insert to understand the role of the tPA leader when it is incorporated as the only change in the design from the original wild type HA antigen insert.
In order to maximize the immunogenicity of HA DNA vaccines as shown in previous studies [9, 11] , HA genes used in the current study were also codon optimized and chemically synthesized. In addition, the HA gene sequences used in the current study express intact HA amino acid sequences at the cleavage site between HA1 and HA2 (PQREXRRKKRQG) of HA proteins in highly pathogenic H5N1 viruses [12, 13, 14] . This cleavage was shown to be important for the pathogenesis of H5N1 viruses [12, 15] . For inactivated H5 serotype flu vaccines, these residues were removed to improve the safety profile of such vaccines for both manufacturing and mass immunization purposes [7] [16, 17, 18] .
Expression and immunogenicity of different forms of DNA vaccines coding for the HA antigen of a 1997 H5N1 influenza Hong Kong isolate
The first set of H5 HA DNA vaccines was produced by cloning codon optimized HA genes based on the amino acid sequences of HA antigen from an H5N1 influenza isolate A/HongKong/156/ 97, which was responsible for the first outbreak of H5N1 avian influenza in humans in 1997, into a DNA vaccine vector [19] . The expression of HA antigens from three different H5-HK DNA vaccines was examined in transiently transfected 293T cells ( Fig. 2A) . Regardless of whether the natural HA leader or tPA leader was used, HA proteins expressed from two full length H5-HK DNA vaccine constructs (HA-HK.wt and HA-HK.tPA) were detected in cell lysate but not in the supernatant, suggesting they are mainly cell-associated. In contrast, truncation of the Cterminal segment, including the removal of the TM domain in the H5-HK.dTM DNA vaccine construct, was able to significantly increase the secretion of HA protein (detected in supernatant) ( Fig. 2A) . H5-HK HA proteins expressed by all of three HA DNA vaccine designs were able to be cleaved into HA1 and HA2 subunits ( Fig. 2A) .
New Zealand White (NZW) rabbits were used in the current study to produce large quantities of sera for both binding antibody and functional antibody analyses. Animals were immunized with one of the three H5-HK HA DNA vaccines (individually) via gene gun. Positive antibody responses were elicited in immunized rabbit sera against the HK HA antigen and levels of such responses increased with repeated immunizations while the negative control rabbit group that received empty DNA vector did not have HAspecific antibody responses (Fig. 2B ). As measured by both temporal and peak-level antibody responses, there was no difference in the ability of the three forms of H5-HK HA DNA vaccines to elicit H5 HA-specific antibody responses ( Fig. 2B and 2C) .
However, functional antibody analyses with the rabbit anti-HA immune sera showed a very different picture when these sera were further analyzed by either the hemagglutination inhibition (HI) or microneutralization (MN) assays. All three forms of H5-HK HA DNA vaccines induced protective antibody responses against the autologous wild type virus A/HongKong/483/97, but levels of protective antibodies were different among sera induced by different designs of H5-HK HA DNA vaccines. The DNA vaccine with the full length HA insert under the tPA leader sequence (H5-HK.tPA) elicited consistently higher HI and MN antibody titers when compared to the other two forms of HK-HA inserts, the full length HA with a natural leader sequence (H5-HK.wt), and the transmembrane region (TM) truncated HA (H5-HK.dTM). The difference was statistically significant (p,0.05) between H5-HK.tPA and H5-HK.dTM sera based on the HI assay (Fig. 2D ) and between H5-HK.tPA and H5.HK.wt sera based on the MN assay (Fig. 2E) . Expression and immunogenicity of different forms of DNA vaccines coding for the HA antigen of an H5N1 isolate A/VietNam/1203/04
In order to rule out that the above finding was not only unique to this H5N1 virus isolate from Hong Kong in 1997, similar designs of HA inserts were produced by using a codon optimized HA DNA gene from the H5N1 strain A/VietNam/1203/04, a well-studied representative isolate for the H5N1 viruses [20, 21] . The pattern of H5-VN HA expression was similar to that of H5-HK HA antigens. Only cell-associated HA antigens were detected with H5-VN.wt and H5-VN.tPA constructs in contrast to that identified with the H5-VN.dTM, which had HA antigen expression in both cell lysate and supernatant fractions (Fig. 3A) . Rabbits were immunized with the electroporation method as previously reported [22] . Similar to H5-HK DNA plasmids, binding antibody responses, as measured by ELISA, showed similar levels among sera elicited by the three H5-VN DNA vaccines with different HA gene insert designs (Fig. 3B) . However, functional antibodies, as measured by HI and MN antibody analyses, revealed again that the H5-VN.tPA design induced the highest levels of functional antibody responses ( Fig. 3C and 3D ). In the case of functional antibody responses against the wild type virus A/VietNam/1203/04, the differences between H5-VN.tPA and the other two forms were statistically significant by both HI and MN assays (p,0.05 or p,0.01).
In order to ensure that the difference in protective antibody responses between sera elicited by H5-VN.wt and H5-VN.tPA was not the result of repeated immunizations, sera collected after one or three immunizations were also measured (Fig. 4) . Pseudotyped viruses expressing VN HA antigen were used to measure the neutralizing antibody activities in rabbit sera with less immunizations. The strength of protective antibodies was measured at two levels: inhibition concentrations that can block either 50% (IC50) or 90% (IC90) of virus infection to target cells. Both measurements showed that H5-VN.tPA-elicited rabbit sera had significantly higher titers of neutralizing antibody activities than the wild type H5 HA design, especially when using the more stringent IC90 as a cut-off (p,0.05 or p,0.01) (Fig. 4) . Sensitivity to deglycosylation treatment for HA antigens expressed by different forms of H5-VN HA DNA vaccines Additional studies were conducted to ask if glycosylation of HA has been affected with the use of a different leader sequence which may influence the immune responses. Our previous study with a hepatitis B surface antigen suggested that post-translational modifications including glycosylation may affect the immunogenicity of DNA vaccine delivered antigens [23] . The Asn (N)-linked glycosylation of influenza HA proteins are essential for virus infectivity and vaccine immunogenicity [24, 25] . Based on sequence analysis, there are 8 to 9 N-linked glycosylation sites (Asn-X-Ser/ Thr) in H5N1 HA proteins. We next investigated whether the HA antigens expressed by different designs of H5 HA inserts in the above DNA vaccinations have similar levels of N-linked glycosylation. The HA antigens expressed in 293T cell lysate transfected with the H5-VN.wt, H5-VN.tPA or H5-VN.dTM DNA vaccines and HA antigen expressed in the 293T cell supernatant transfected with the H5-VN.dTM DNA vaccine were analyzed for their susceptibility to PNGase treatment, which can cleave any type of Nlinked sugar including complex oligosaccharide structures resulting from the maturation of high mannose moieties during transport of the glycoprotein through the Golgi.
Treatment with PNGaseF allowed for the removal of N-linked glycosylations, as shown by the reduction of apparent molecular weight of HA0, HA1, and HA2 species in Western blot analysis for HA proteins expressed in cells transfected by these H5-VN HA DNA vaccines ( Figure S1 ). The molecular weight reduction patterns for HA0, HA1, and HA2 antigens were the same between cells transfected by H5-VN.wt and H5-VN.tPA DNA vaccines, suggesting that HA antigens expressed by these two HA DNA vaccines were similarly glycosylated. A smaller molecular weight HA2 antigen was observed in cells transfected with the H5-VN.dTM DNA vaccine, presumably due to the truncated size of HA2 domain in this particular HA insert design; however, PNGaseF treatment also led to a proportional reduction of molecular weight for truncated HA2 protein in both supernatant and cell lysate preparations. The only unique finding is that the cell-associated HA antigens in H5-VN.dTM transfected cells showed a high level of heterogeneity and a small portion of the HA1 proteins was not fully deglycosylated by PNGaseF treatment, reflecting the continued presence of different forms of glycosylated HA proteins in H5-VN.dTM transfected cells. Otherwise, the overall glycosylation pattern, as probed by deglycosylation treatment, was very similar among HA antigens produced by three different types of H5 HA DNA vaccines.
Cross-protective antibody responses induced by individual DNA vaccines expressing HA antigens from key H5N1 viral isolates Based on the above results, additional HA DNA vaccines with the HA.tPA insert design were produced by using codon optimized HA genes that encode the HA proteins from H5N1 viral strains A/Anhui/1/2005 and A/Indonesia/5/2005, both have caused human infection in recent years [26, 27] . Given the circumstance that H5N1 influenza antigen drifts have occurred since the first human outbreak in Hong Kong in 1997, it would be important to determine if H5 HA vaccines developed based on H5N1 viruses isolated at different epidemic time points can induce cross antibody responses against other H5N1 viruses.
One set of experiments was conducted to understand the cross protection between paired H5N1 HA antigens. Rabbits were immunized with individual H5-HK.tPA, H5-VN.tPA, and H5-AH.tPA DNA vaccines and rabbit immune sera were examined for HA antigen-specific antibody responses. H5 HA-specific antibody responses against H5 HA antigens from different viruses were analyzed by ELISA and the potential cross-protective antibody responses against different H5N1 viruses were evaluated by HI and MN assays.
H5-HK (A/HK/156/97), a clade 0 H5N1 isolate, and H5-VN (A/VN/1203/04), a clade 1 H5N1 isolate, represent H5N1 viruses isolated from the first human outbreak in Hong Kong in 1997 and a subsequent outbreak in Vietnam in 2004, respectively. Results shown in Fig. 5 indicate that H5-HK.tPA DNA vaccine-immunized rabbit sera showed high antibody responses recognizing both the autologous H5-HK HA antigen and the heterologous H5-VN HA antigen. However, the HA-specific IgG titers against the autologous H5-HK antigen were higher than those observed against the heterologous H5-VN antigen (p,0.05). Conversely, the H5-VN.tPA DNA vaccine elicited high level HA-specific IgG responses against autologous H5-VN and heterologous H5-HK HA antigens although the overall titers against its autologous H5-VN HA antigen may be higher (not statistically significant).
Protective HI and MN antibody responses induced by H5-HK.tPA and H5-VN.tPA DNA vaccines were further compared against either A/HK/483/97 or A/VN/1203/04 wild type viruses (Fig. 5 ). HI and MN titers were present in both HK.tPA and H5-VN.tPA DNA vaccine-immunized rabbit sera against Similar analysis was conducted with rabbit immune sera elicited by H5-VN and H5-AH HA DNA vaccines (Fig. 6 ). H5-AH (A/ Anhui/1/05), a clade 2.3 H5N1 isolate, represents the H5N1 virus isolated from a human outbreak in China in 2005 [26] . The cross reactivity between H5-VN and H5-AH immune sera and viruses was very similar to that observed above between H5-HK and H5-VN immune sera and viruses. For binding antibody responses, H5-VN rabbit immune sera had significantly higher recognition to its autologous H5-VN HA antigen than the heterologous H5-AH HA antigen (p,0.05). H5-AH rabbit immune sera elicited higher antibody responses recognizing the autologous H5-AH HA antigen than the heterologous H5-VN HA antigen (not statistically significant) (Fig. 6) . For functional antibodies, both HI and MN analyses showed preference for H5-VN and H5-AH rabbit immune sera against their respective autologous wild type viruses, A/ VN/1203/04 and A/Anhui/1/05 (p,0.05 or p,0.01) (Fig. 6 ).
Cross-protection by a polyvalent DNA vaccine formulation expressing HA antigens from three representative H5N1 viral isolates
The above results indicate that while antibody responses against one H5N1 HA antigen or virus may well cross-react with another H5N1 HA antigen or virus, the levels of antibody responses against the autologous antigen or virus were always higher. Given the uncertainty regarding which H5N1 virus may ultimately cause a pandemic H5N1 outbreak, it is important to develop a vaccine strategy that can maximize the protection efficacy against a wide spectrum of H5N1 viruses from different clades (or subtypes). It is possible that a consensus HA antigen, or a structurally-optimized HA antigen design, can cover different H5N1 viruses at various levels of protective efficacy but there is no actual data showing that such HA antigen can achieve the maximum protective antibody responses against several H5N1 viral isolates from different clades.
One alternative approach is to produce a polyvalent HA formulation by including multiple H5N1 HA DNA vaccines in one injection to produce an immune sera that can induce the highest antibody response against a wide range of H5N1 viral isolates. The pseudotyped virus system, developed in recent years and widely used in leading influenza studies, provides high sensitivity in detecting functional antibody responses against influenza HA antigens, and at the same time, eliminates the influence of other influenza viral gene products since a common viral backbone is used for different HA pseudotyped viruses [28, 29] . It is an ideal system as a high-throughput assay for multiple serum samples against a wide range of viruses. As shown in Fig. 6 , rabbit sera elicited by either the 3-valent HA DNA vaccine formulation (H5-VN+H5-AH+H5-IN) or the monovalent H5 HA DNA vaccines (HK, VN, AH, and IN) were tested for their antibody responses against three pseudotyped viruses expressing H5-VN, H5-AH, or H5-IN HA antigens. The matched monovalent rabbit sera consistently showed the highest functional antibody responses against autologous pseudotyped viruses (Fig. 7A-7C) . However, the 3-valent serum was the only one that showed high level antibody responses against all three pseudotyped viruses while one non-matched monovalent rabbit serum could neutralize one or two viruses but not all three, further confirming the hypothesis that a polyvalent HA formulation is capable of protect against multiple H5N1 viruses.
According to phylogenetic analysis, H5N1 viruses can be divided into 10 clades (0-9). Since 1997, humans have mainly been infected by H5N1 viruses from clades 0, 1, and 2, although there have also been reports of infection by clade 7 virus [5] . Clade 2 is the most complicated in its genetic evolution and has been further divided into five subclades (2.1 to 2.5). In the current study, While progress has been made in reducing the number of required immunizations during vaccination with inactivated H5N1 vaccines by incorporating various adjuvants into the vaccine formulations, a major next-step for H5N1 vaccine research is to determine to what degree the immunity elicited by one H5 avian influenza vaccine (currently, many candidate H5N1 vaccines were developed based on a clade 1 virus (A/VietNam/1203/2004)) can cross-protect against H5N1 viruses from other clades. Unlike human Type A influenza viruses (H1 or H3 serotypes), any potential pandemic caused by an H5N1 virus will be of avian origin and, in theory, any of the current known H5N1 avian viruses may jump to the human population leading to the next pandemic. Therefore, a systemic examination on the cross-protection among HA antigens from different clades is needed for strategic planning to determine whether more than one H5N1 vaccine is needed based on the analysis of protection profiles, and if so, what particular viral strains should be selected to provide the maximum breadth of protection.
DNA vaccination is an attractive strategy to provide relatively quick and straightforward production of vaccines against an influenza pandemic when the demand for such vaccines suddenly increases. However, a key issue surrounding the use of DNA vaccines is their low immunogenicity in humans. In recent years, the success of the prime-boost strategy has greatly enhanced the utility of DNA vaccination for future human applications [10, 30] . At the same time, optimization of the design of antigen inserts based on the uniqueness of each antigen (a process of ''antigen engineering'') [31, 32] can also play a key role.
Results included in the current report indicated that the tPA leader sequence and the C-terminal transmembrane domain/ cytoplasmic region of H5 HA both contribute to better functional antibody responses in H5.tPA DNA vaccines when compared to H5.wt and H5.dTM DNA vaccines. The above findings were different from our previous results on protective antibody responses induced by differently designed flu H1 and H3 HA DNA vaccines [11] . In this previous study, only the full length H1.wt but not transmembrane truncated H1.dTM induced high level HI and MN responses against H1 virus while both the full length H3.wt and truncated H3.dTM induced similar HI and MN responses against H3 virus [11] . These results provide a strong indication that the HA antigens from different influenza A subtypes (H1, H3 and H5) may have different preferences for antigen structure designs in order to generate optimal protective antibody responses.
Studies were conducted in this report to identify the mechanism responsible for better protective antibodies in rabbit immune sera elicited by the H5.tPA HA insert design but the exact mechanism is currently unclear. First, we tested whether a higher level of HA antigen expression was produced with the tPA-leader design. As shown in Fig. 2A and 3A, antigen expression levels between WTleader design and tPA-leader design were similar, thus excluding this possibility. Next, we asked whether there is increased secretion of the HA antigen due to the use of the tPA leader. However, as shown in Fig. 2A and 3A , there is no major detectable level of secreted HA antigens in supernatant for either the WT-design or tPA-design. Furthermore, the dTM design did have a higher level of secretion due to the deletion of transmembrane and intracellular portion of HA protein but did not elicit better protective antibody responses. Finally, a study was conducted to examine the possible role of post-translational processing, such as a change in glycosylation, of HA the antigen, which may affect the antigen processing pathway, as we previously reported with a hepatitis B surface antigen DNA vaccine [33, 34, 35] . However, Figure S1 showed that there is no major difference between WTdesign and tPA-design after de-glycosylation treatment.
Therefore, it is very likely that HA antigen expressed with the tPA leader may be more effective in eliciting conformational antibodies. This hypothesis is supported by two pieces of evidence. First, there was no difference in the levels of binding antibodies as measured by ELISA, which indicated that there was no difference in the general immunogenicity between WT and tPA leader designs; only a difference in the functional antibody was observed. Second, the HA.dTM DNA vaccine design also used the tPA leader but did not have better functional antibodies, proving that the proper folding of the HA antigen in the presence of a tPA leader is important and is dependent on the presence of an intact HA2 domain. It is possible that such an HA antigen conformation is part of a trimer structure of HA since the HA2 domain is involved in the formation of HA trimers. Only antibodies against the trimer structure are more functionally relevant to block the trimer form of HA spikes on viral particles. By using the optimal H5.tPA HA insert design, studies in this report further demonstrated that there are good levels of cross protection by one H5 HA DNA vaccine against multiple H5N1 viruses from different clades. It is well documented that cross protection among H5N1 viruses can be detected [33, 34, 35] . Clade 1 H5N1 vaccines (VN) cross protect against both clade 1 and clade 2 (Indonesia) viruses in ferrets with the use of a strong adjuvant [36] . Using live attenuated cold adapted (ca) viruses expressing HA and NA from 1997, cross protection was observed against the late H5 virus from 1997 to 2005 [37] and ferrets [37] and ferrets [38] and mice [39, 40] and mice [39, 40] .
However, as shown in the current study, not all H5 HA vaccines can elicit the same levels of cross protective antibodies, and more significantly, maximum levels of protective antibodies were usually detected against the autologous viral isolates. Given the knowledge that protective anti-flu antibody responses in humans are much There were 3 rabbits/group in Vector, HK, VN, AH, and Ind groups, and 4 rabbits/group in 3-valent groups. Rabbit sera tested by the pseudotyped NAb assays were collected at 2 weeks after the 4 th DNA immunization. '','' denotes below detection level. The arrow denotes neutralization against the autologous H5 pseudotyped virus. The statistical differences between the testing group and the autologous neutralization group or 3-valent group are indicated by ''*'' when the p value was less than 0.05. doi:10.1371/journal.pone.0028757.g007 lower than in experimental animals, cross protection may not be very high in humans with one randomly selected H5 HA vaccine. In the current set of studies, it was encouraging to observe that the polyvalent H5 HA DNA vaccine was able to elicit high level protective antibody responses against multiple key H5N1 viruses. Such a polyvalent flu DNA vaccine can be used for stockpiling against a potential H5N1 pandemic even before information is available on which viral isolates may cause a human outbreak. At the same time, there are alternative approaches including the use of consensus HA antigen designs to achieve a broad coverage of various viral strains (personal communication with David Weiner). It will be interesting to compare the relative efficacy between polyvalent and consensus HA DNA vaccines in their abilities to elicit protective antibody responses. One unique advantage of the polyvalent formulation is its flexibility; one alternative HA antigen can replace or be added to the earlier polyvalent formulation in the event that a new strain of virus becomes a threat while it will be necessary to re-design the whole consensus HA insert in order to allow for broader coverage.
While HI titers against heterologous virus (cross-clade) were significantly reduced compared to HI titers against homologous virus in the current study, the heterologous virus titers were generally above 1:100, and in humans, a HI titer of 1:40 has been associated with protection [41] , and so all of the constructs might be protective after multiple immunizations. At the same time, the titers observed in mice may not be the same as in humans. While HI titers have been associated with protection, H5 HA DNA vaccines described in the current report were not tested for protection against challenge and so there is the possibility that only some of these constructs may not protect against clinical disease or lethal infection.
No matter the design of DNA vaccines that may be used, recent studies have indicated that DNA priming immunization is effective as part of the prime -boost strategy for flu vaccine applications. In addition to DNA prime-inactivated flu vaccine boost [9, 10] , a study published in 2011 further demonstrated that DNA primelive attenuated flu vaccine boost was equal to or more effective than twice immunization with the live attenuated flu vaccine against the H5N1 viruses based on antibody responses and viral clearance in immunized ferrets [42] . Since live attenuated vaccines are considered the most immunogenic form of vaccines, it is impressive to observe that one time DNA prime was able to achieve the same priming effect as a live attenuated flu vaccine. In this particular study, the H5-VN.tPA DNA insert was used as part of the collaboration with the manufacturer of live attenuated H5N1 flu vaccine.
Based on the results published in the current report and other recent similar studies, H5N1 HA DNA vaccines evaluated in the current study should be included in the design of human studies to understand whether results reported here can be reproduced in humans when they are used as part of DNA prime, either individually or as part of the polyvalent HA DNA formulation. The finding from such studies will be very useful in the identification of simple yet powerful approaches to develop vaccines against major influenza pandemics. preference of Homo sapiens. The less optimal codons in HA genes were changed to the preferred codons in mammalian systems to promote higher expression of the HA proteins, as previously described [11] . These codon optimized HA genes were chemically synthesized by Geneart (Regensburg, Germany) with added restriction enzyme sites of PstI and BamHI for subcloning purpose immediately upstream of the start codon and downstream of the stop codon, respectively.
For either H5-HK or H5-VN HA DNA vaccines, three versions of codon optimized HA gene inserts were cloned into DNA vaccine vector pSW3891 [43] . For the first version, the full length H5-HK or H5-VN HA gene insert (568 aa,) with their natural HA leader sequences subcloned, individually, into the pSW3891 vector at the PstI and BamHI sites, designated as H5-HK.wt or H5-VN.wt DNA vaccine constructs. For the second version, the HA natural leader sequence (the first 15 aa at N-terminus for both H5-HK and H5-VN) was replaced by a human tissue plasminogen activator (tPA) leader sequence. The H5-HK and H5-VN HA gene inserts coding for aa 16-568 were PCR amplified from the full length codon optimized H5 HA genes using the following primers: H5-HA-opt-1 (gtcgctccgctagc GACCAGATCTGCATC-GGCTAC) and H5-HA-opt-2 (agtcacggatcc TCAGATGCA-GATCCGGCACTG). The individual H5-HK or H5-VN HA gene was cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.tPA or H5-VN.tPA DNA vaccine constructs. For the third version, the HA natural leader sequence was replaced by a tPA leader sequence and the transmembrane (TM) and cytoplasmic region (37 aa at the C-terminus) of H5 and HA was deleted for both H5-HK and H5-VN. The truncated H5-HK or H5-VN HA genes were PCR amplified from the full length codon optimized H5-HK or H5-VN HA gene using primer pairs: H5-HA-opt-1 and H5-HA-opt-4 (agtcac ggatccTCACTGGTAG-GTGCCCATGCTCTC), or H5-HA-opt-1 and H5-HA-opt-8 (agtcacggatccTCACTGGTAGATGCCGATGCTTTC), respectively. The truncated H5-HK or H5-VN gene insert was individually cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.dTM or H5-VN.dTM.
For the H5-AH HA DNA vaccine, the construct with the full length HA under tPA-leader sequence was made as described above. Each individual DNA vaccine plasmid was prepared from Escherichia coli (HB101 strain) with a Mega purification kit (Qiagen, Valencia, CA) for both in vitro transfection and in vivo animal immunization studies.
NZW rabbits (,2 kg body weight) were purchased from Millbrook Breeding Labs (Amherst, MA) for immunogenicity studies, and housed in the Department of Animal Medicine at the University of Massachusetts Medical School in accordance with IACUC approved protocol. The rabbits (3 rabbits/group) were immunized with a Helios gene gun (Bio-Rad) at the shaved abdominal skin as previously reported [44] with a total of 36 mg H5 HA DNA vaccine plasmid or vector control plasmid at each immunization. DNA immunizations were given at weeks 0, 2, 4, 8. Serum samples were taken prior to the first immunization and 2 weeks after each immunization for study of H5 HA-specific antibody responses.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Massachusetts Medical School's Institutional Animal Use and Care Committee (IACUC) (Protocol: A-1674). All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.
Transient expression of the HA antigens from various HA DNA vaccine constructs were verified by Western blot analysis. HA DNA vaccine constructs were first transfected into the human embryonic kidney 293T cells using the calcium phosphate precipitation method. Briefly, 2610 6 293T cells at 50% confluence in a 60 mm dish were transfected with 10 mg of plasmid DNA, and a total of 3 ml supernatant and 100 ml of cell lysate were harvested 72 hours later. Equal amounts of each transiently expressed HA antigen (10 ng of protein in 10 ml) were loaded for the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under denatured conditions, then transferred onto PVDF membranes (Bio-Rad, Hercules, CA). After being blocked overnight at 4uC in blocking buffer (0.2% I-block, 0.1% Tween-20 in 16PBS), the membranes were incubated with a 1:500 dilution of rabbit sera immunized with HA DNA vaccines for 30 min followed by washes. Then, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG at 1:5000 dilution for 30 min. Following washes, the signals were detected using a chemiluminescence-based Western-Light Kit (Tropix, Bedford, MA).
To analyze the N-linked glycosylation of H5 HA antigens expressed by various forms of H5 HA DNA vaccines, the HA antigens expressed from 293T cells [45, 46] were treated with PNGaseF (New England BioLab, Beverly, MA) [47, 48, 49] . Briefly, the HA proteins were first denatured at 100uC for 10 min in glycoprotein denaturing buffer and then chilled on ice. Following addition of G7 reaction buffer, the deglycosylation enzyme cocktail was added and incubated reaction at 37uC for 4 hours. Either mock-treated or deglycosylated HA samples were subjected to SDS-PAGE and Western blot analysis was performed as described above.
ELISA was conducted to measure HA-specific antibody (IgG) responses in immunized rabbits and mice. The 96-well flat-bottom plates were coated with 100 ml of ConA (50 mg/ml) for 1 hour at room temperature, and washed 5 times with PBS containing 0.1% Triton X-100. Subsequently, the plates were incubated overnight at 4uC with 100 ml of transiently expressed HA antigen at 1 mg/ ml. After being washed 5 times as above, the plates were then blocked with 200 ml/well of blocking buffer (5% non-fat dry milk, 4% Whey, 0.5% Tween-20 in PBS at pH7.2) for 1 hour. After five washes, 100 ml of serially diluted rabbit or mouse serum was added in duplicate wells and incubated for 1 hour. After another set of washes, the plates were incubated for 1 hour at 37uC with 100 ml of biotinylated anti-rabbit or anti-mouse IgG (Vector Laboratories, Burlingame, CA) diluted at 1:1000 in Whey dilution buffer (4% Whey, 0.5% Tween-20 in PBS). Then, 100 ml of horseradish peroxidase-conjugated streptavidin (Vector Laboratories) diluted at 1:2000 in Whey buffer was added to each well and incubated for 1 hour. After the final washing, the plates were developed with 3,39,5,59 Tetramethybenzidine (TMB) solution at 100 ml per well (Sigma, St. Louis, MO) for 3.5 minutes. The reactions were stopped by adding 25 ml of 2 M H 2 SO 4 , and the plates were read at OD 450 nm. The end titration titer was determined as the highest serum dilution that has an OD reading above twice of that from the negative control serum.
Influenza A viruses of the H5N1 A/HongKong/483/97 (H5N1), A/Viet Nam/1203/04 (H5N1), A/Anhui/1/2005 (H5N1), and A/ Indonesia/5/2005 (H5N1) were grown in the allantoic cavity of 10day-old embryonated hen eggs at 37uC for 26 to 40 h. Allantoic fluid pooled from multiple eggs was clarified by centrifugation and frozen in aliquots at 270uC. The 50% egg infectious dose (EID50) for each virus stock was calculated by the method of Reed and Muench following serial titration in eggs. All experiments with HPAI viruses were conducted under Biosafety Level 3 containment.
Hemagglutination-inhibition (HI) assay HI assays were performed by standard methods [50] . Briefly, the assay was performed using, 0.5% v/v fowl or horse [30] red blood cells, 4 HA unit of reference H5N1 virus (A/HongKong/ 483/97, A/VietNam/1203/04, A/Anhui/1/2005, A/Indonesia/ 5/2005) and specific sera treated with receptor destroying enzyme. The HI titer was defined as the highest dilution of the serum able to inhibit hemagglutination.
MN assays was performed as described previously [51] . In brief, influenza virus containing 100 TCID 50 was incubated with equal volume of two-fold dilutions of the specific heat-inactivated serum overnight at 37uC in a 5% CO2 humidified atmosphere for 1 hr. After the incubation, 100 ml virus-serum samples were added to a 96-well plate containing Madin Darby Canine Kidney (MDCK) cell monolayer and incubated for 5 days at 37uC and 5% CO2. The microneutralization titer was defined as the highest dilution of serum that neutralized 100 TCID 50 of virus in MDCK cell [52, 53] cultures (as detected by the absence of cytopathic effects). The MN assays were conducted in two different labs: 1) National Microbiology Laboratory, Public Health Agency of Canada, and 2) Beijing Institute of Microbiology and Epidemiology, according to the viruses available.
The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [28, 54, 55] . To produce H5N1 pseudotyped viruses, 293T cells [45, 46] (5610 6 cells plated the day before) were transfected with 13.43 mg of pNL 4-3.Luc.R-E-(NIH AIDS reference and reagent program), 1.2 mg of H5-HA-wt DNA vaccine plasmid and 0.3 mg of N1-NA plasmid using 75 mg of polyetheleneimine transfection reagent. Supernatants were harvested 48 hours later, frozen at 280uC and then standardized by infectivity in 293A cells using a luciferase-based TCID50 measurement. For neutralization assays, serum samples (5 ml) were heat inactivated at 56uC for 30 minutes, then threefold serially diluted in culture medium in flat-bottomed microtiter plates. Pseudotyped H5N1 virions were then added to the plates at 200 TCID50/well and incubated for 1 hour at 37uC. After incubation, 293A cells were trypsinized and added to each plate at a dilution of 1610 4 cells per well. Following a 48-hour incubation, plates were developed using a luciferase assay system (Promega). Values averaged from triplicate wells were then used to determine IC50 based on wells that displayed 50% reduction in infection as compared to control wells containing virus plus pre-immune sera.  ELM—the database of eukaryotic linear motifs Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instances. Short linear motifs (SLiMs, LMs or MiniMotifs) are regulatory protein modules characterized by their compact interaction interfaces (the affinity and specificity determining residues are usually encoded between 3 and 11 contiguous amino acids (1)) and their enrichment in natively unstructured, or disordered, regions of proteins (2) . As a result of limited intermolecular contacts with their interaction partners, SLiMs bind with relatively *To whom correspondence should be addressed. Tel: +49 (0) 6221 3878398; Fax: +49 (0) 6221 387517; Email: gibson@embl-heidelberg.de low affinity (in the low-micromolar range), an advantageous attribute for use as transient, conditional and tunable interactions necessary for many regulatory processes. Due to the limited number of mutations necessary for the genesis of a novel motif, SLiMs are amenable to convergent evolution, functioning as a driver of network evolution by adding novel interaction interfaces, and thereby new functionality, to proteins. This evolutionary plasticity facilitates the rapid proliferation within a proteome, and as a result, motif use is ubiquitous in higher eukaryotes.
SLiMs play an important role for many regulatory processes such as signal transduction, protein trafficking and post-translational modification (3, 4) . Their importance to the correct functionality of the cell is also reflected by the outcome of motif deregulation. For example, point mutations in SLiMs have been shown to lead severe pathologies such as 'Noonan-like syndrome' (5) , 'Liddle's syndrome' (6) or 'Retinitis pigmentosa' (7) . Furthermore, mimicry of linear motifs by viruses to hijack their hosts' existing cellular machinery plays an important role in many viral life cycles (8) . However, despite their obvious importance to eukaryotic cell regulation, our understanding of SLiM biology is relatively limited, and it has been suggested that, to date, we have only discovered a small portion of the human motifs (9) .
Several resources are devoted to the annotation and/or detection of SLiMs [Prosite (10), MiniMotifMiner (11) and Scansite (12) ]. Here, we report on the 2012 status of the Eukaryotic Linear Motif database.
The ELM initiative (http://elm.eu.org) has focused on gathering, storing and providing information about short linear motifs since 2003. It was established as the first manually annotated collection of SLiM classes and as a tool for discovering linear motif instances in proteins (13) . As it was mainly focused on the eukaryotic sequences, it was termed the Eukaryotic Linear Motif resource, usually shortened to ELM. The ELM resource consists of two applications: the ELM database of curated motif classes and instances, and the motif detection pipeline to detect putative SLiM instances in query sequences. In the ELM database, SLiMs are annotated as 'ELM classes', divided into four 'types': cleavage sites (CLV), ligand binding sites (LIG), sites of posttranslational modification (MOD) and subcellular targeting sites (TRG) ( Table 1) . Currently, the ELM database contains 170 linear motif classes with more than 1800 motif instances linked to more than 1500 literature references (Table 1 ). Each class is described by a regular expression capturing the key specificity and affinity determining amino acid residues. A regular expression is a computer-readable term for sequence annotation and is used by the ELM motif detection pipeline to scan proteins for putative instances of annotated ELM classes. The search form for sequence input is shown in Figure 1 , while the results page showing the putative and annotated instances is illustrated in Figure 2 .
The ELM resource is powered by a PostgreSQL relational database for data storage and a PYTHON web framework for data retrieval/visualization. The main tables within the database contain information about ELM classes, ELM instances, sequences, references, taxonomy and links to other databases [the database structure is described in greater detail in (14) ].
Since the last release (14) , 24 new ELM classes have been added to the ELM database (Table 1 ) and several more have been updated. One of the newly annotated motif classes is the AGC kinase docking motif (LIG_AGCK_PIF), consisting of three distinct classes. It is present in the non-catalytic C-terminal tail of AGC kinases that constitute a family of serine/threonine kinases consisting of 60 members that regulate critical processes, including cell growth and survival. Deregulation of these enzymes is a causative factor in different diseases such as cancer and diabetes. The motif interacts with the PDK1 Interacting Fragment (PIF) pocket in the kinase domain of AGC kinases. It mediates intramolecular binding to the PIF pocket, serving as a cis-activating module together with other regulatory sequences in the C-tail. Interestingly, in some kinases the motif also acts as a PDK1 docking site that trans-activates PDK1, which itself lacks the regulatory C-tail, by interacting with the PDK1 PIF pocket. PDK1 in turn will phosphorylate and activate the docked kinase. Other novel classes (Table 2) include phosphodegrons, which are important mediators of phosphorylation-dependent protein destruction, and the LYPxL motif, which is involved in endosomal sorting of membrane proteins but is also implicated in retrovirus budding.
Annotated ELM instances serve as representative examples of the respective ELM class. They are also invaluable for the computational analysis and classification of motifs (15) . Therefore, special emphasis has been put on the curation of more than 500 novel ELM instances (in 40 different classes) by scanning and annotating more than 400 articles. The number of protein databank (PDB) entries annotated have been increased to 195 (Table 1 ), meaning that for 10% of all instances there is a 3D Figure 1 . ELM start page. The user can submit a query sequence to the motif detection pipeline either as UniProt accession number or in FASTA format. Filtering criteria such as taxonomic range or cellular compartment should be activated to limit the resulting list of SLiM instances.
protein structure annotated, giving more detailed information about the biological context of the respective motif.
The ELM website at http://elm.eu.org can be used in two ways: first, as a front-end to explore the ELM database of curated ELM classes and instances, and second, to run the motif detection pipeline to detect putative SLiM instances in query sequences. Both interfaces have been improved with the most notable changes listed below.
The database user interface, having been stable for many years, has been overhauled and replaced by a novel interface introducing several new features ( Figure 1 ). Up-to-date web technologies have been used to improve the general user experience: the PYTHON framework DJANGO (http://www.djangoproject.com) dynamically creates and serves all HTML pages, while JavaScript was used to make the whole site more interactive and thus improve the user experience. In particular, the ELM detail pages (Figure 3) , which hold the most (18) . The lower part contains the annotated and putative ELM instances for the given protein sequence (Epsin1, UniProt accession Q9Y6I3). The background is colored according to the structural information available. Each box represents one ELM instance, the color of which indicates the likelihood that this instance is functional: grey instances are buried within structured regions, while shades of blue represent instances outside of structured regions and hint on sequence conservation, with pale blue representing weak sequence conservation and dark blue indicating strong sequence conservation. Red ellipses or boxes mark instances that are annotated in the query sequence or a homologous sequence, respectively.
important information about each ELM class including references, regular expression, taxonomic distribution and gene ontology terms (Table 3) , have been updated by annotating the protein domain interacting with the respective motif. Where available, a 3D model of representative protein databank structures of linear motif interactions was added to the ELM detail page ( Figure  3 , top right).
To cope with the increasing amount of annotated classes as well as instances, a novel query interface was introduced to assist the user in finding information of interest. The ELM browser (Figure 4 ) now features a search interface for free text search. In addition, the search results can also be filtered and reordered using buttons (Figure 4 , left side) and table headers, respectively, and be downloaded as tab-separated values (TSV).
Further, improvements to the ELM database include revising the experimental methods used for annotation by using a standardized methods vocabulary [in sync with PSI-MI ontology (16, 17) ].
A candidate page has been introduced to display novel ELM classes that have not yet been annotated in detail or are currently undergoing annotation. We invite researchers to send us their feedback and expert opinion on these classes and to contribute novel motif classes that will be added to the candidate page and ultimately be turned into full ELM classes ( Figure 5 ). Minimum requirements are at least one literature reference as well as a short description. In addition, a draft regular expression or a 3D structure showing the relevant interaction would also be helpful. Currently, the number of possible ELM classes on this candidate list (awaiting further annotation) exceeds the number of completely annotated classes, indicating the great demand for further annotation.
The ELM motif detection pipeline scans protein sequences for matches to the regular expressions of annotated ELM classes ( Figure 2 ). The query output combines these putative instances with information from the database (annotated ELM instances) as well as predictions from different algorithms/filters. The ELM resource employs a structural filter (18) to highlight and mask secondary structure elements, as well as SMART (19) to detect protein domains. Furthermore, an additional disorder prediction algorithm (IUPred) (20) has been included to predict ordered/disordered regions within the protein.
IUPred uses a cutoff of 0.5 to classify a sequence region as either structured or disordered, with values above this threshold corresponding to disorder, highlighted in green background and lower values indicating structured regions, displayed in red background in the output graph. Disorder and domain information is combined by Motifs, present in proteins in several repeats, which mediate binding to the hydrophobic cleft created by subdomains 1 and 3 of G-actin LIG_Actin_WH2_2 LIG_Actin_RPEL_3
The AGCK docking motif mediates intramolecular interactions to the PDK1 Interacting Fragment (PIF) pocket, serving as a cis-activating module LIG_AGCK_PIF_2 LIG_AGCK_PIF_3
IAP-binding motifs are found in pro-apoptotic proteins and function in the abrogation of caspase inhibition by inhibitor of apoptosis proteins in apoptotic cells LIG_BIR_III_1 LIG_BIR_III_2 LIG_BIR_III_3 LIG_BIR_III_4
Motif binding to the dorsal surface of eIF4E LIG_eIF4E_2
A proline-rich motif binding to EVH1/WH1 domains of WASP and N-WASP proteins LIG_HCF-1_HBM_1
The background coloring to highlight structured regions within the protein, which allows inspection of SLiMs that reside at domain boundaries and emphasizes motifs in disordered regions.
The conservation of linear motifs can help in assessing the functional relevance of putative instances, with functional instances showing higher overall sequence conservation than non-functional ones (21) . Therefore, sequence conservation of the query protein is calculated using a tree-based conservation scoring method (22) and highlighted in the graphical output. Here, lighter shades of blue represent low conservation while dark blue shading corresponds to high-sequence conservation. The actual conservation score can be inspected by moving the mouse over the respective ELM instance (Figure 2) .
The functionality of linear motifs can be modulated by modifications such as phosphorylation (23, 24) . To enable the user to investigate phosphorylation data in the context of putative linear motif instances, phosphorylation annotations from the Phospho.ELM resource (25) have been added to the graphical output (Figure 2, top row) . The phosphorylated residues are highlighted in different colors (serine: green, threonine: blue, tyrosine: red); each phosphorylation site is linked to a page showing detailed information about the respective modification site from the manually curated data set of the Phospho.ELM resource.
The importance of the short linear motifs in virus-host interactions makes the ELM resource an important tool for the viral research community. For example, Cruz et al. (26) analyzed a protein phosphatase 1 (PP1) docking motif in 'protein 7' of transmissible gastroenteritis virus using the ELM class LIG_PP1. This conserved sequence motif mediates binding to the PP1 catalytic subunit, a key regulator of the cellular antiviral defense mechanisms, and is also found in other viral proteomes, suggesting that it might be a recurring strategy to counteract the hosts' defense against RNA viruses by dephosphorylating eukaryotic translation initiation factor 2a and ultimately ribonuclease L. To reflect our increasing awareness of viral motifs (8), special focus has been attributed to the annotation of viral instances in the ELM database: in the latest release, more than 200 novel ELM instances found in 84 different viral taxons have been added. The notion of viruses abusing existing SLiMs in their hosts is demonstrated by viral instances being annotated alongside instances in their hosts' proteins. For example, the ELM class LIG_PDZ_Class_1 contains 12 instances in human proteins but has recently been expanded with 5 instances from 5 different human pathogenic virus proteins. . ELM instances browse page. A full-text search (here, search term used was 'AP2', filtering for 'true positive' instances in taxon 'Homo sapiens', yielding 58 instances) assists in finding annotated instances. A search can be restricted to a particular taxonomy or instance logic (top) or ELM class type (buttons on the left). The list can also be exported to TSV or FASTA format for further processing.
The importance of SLiMs is further corroborated by the occurrence of pathologies that are caused by mutations that either mutate existing linear motifs or create novel linear motifs (of undesired function) (27) . Examples include 'Usher's syndrome' (28) , 'Liddle's Syndrome' (6) or 'Golabi-Ito-Hall Syndrome' (29) . The developmental disorder 'Noonan Syndrome' can be caused by mutations in Raf-1 that abrogate the interaction with 14-3-3 proteins mediated by corresponding SLiMs and thereby deregulate the Raf-1 kinase activity (30) (the Raf-1 protein sequence features two LIG_14-3-3_1 binding sites that are annotated at 256-261 and 618-623 in the ELM resource). A related disease, 'Noonan-like Syndrome', is caused by an S to G mutation at position 2 of the SHOC2 protein, creating a novel myristoylation site (annotated as ELM class MOD_NMyristoyl). This irreversible modification results in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation (5) . More information about the implication of short linear motifs on diseases is collected at http://elm.eu.org/infos/diseases.html.
By providing a high-quality, manually curated data set of linear motif classes with experimentally validated SLiM instances, the ELM database has proven to be invaluable to the community: small-scale (single protein) analyzes benefit from the detailed annotation of each ELM class in attributing novel features to proteins of interest. By using in vitro and in vivo studies, von Nandelstadh et al. (31) could validate a PDZ class III motif, detected by ELM at the carboxy terminus of myotilin and the FATZ (calsarcin/myozenin) families. This evolutionarily conserved carboxy-terminal motif mediates binding to PDZ domains of ZASP/Cypher and other Enigma family members (ALP, CLP-36 and RIL) and disruption of these interactions results in myofibrillar myopathies (32) . Additionally, ELM annotations can contribute to high-throughput screenings (33) as well as development of novel algorithms (34) (35) (36) , methods (37) and databases (38) . Furthermore, the highly curated data of the ELM resource are used as a benchmarking data set to evaluate the accuracy of prediction algorithms (21, 39, 40) .
For any such analysis, the user should be aware that many matches to ELM regular expressions are false positives. Before conducting experiments based on ELM results, it is strongly advisable to check if a motif match is conserved, exposed in a cell compartment in which the motif is known to be functional. The ELM resource applies several filters to provide the user with such information that should ideally also be supported by the experimental evidence.
The importance of SLiMs is highlighted by the growing number of instances with relevance to diseases or viruses. Yet, despite their importance and abundance, our understanding of linear motifs is still limited. This is mainly owing to the fact that they are still quite difficult to predict computationally and to investigate experimentally (3, 41, 42) . By better understanding the biology of linear motifs, we hope to increase our insight into diseases and viruses (and vice versa). The ELM resource tries to aid the researcher in the search for putative SLiM instances by providing a feature-rich toolset for sequence analysis. Consequently, with the aforementioned additions and changes, we hope that the ELM resource continues to be a valuable asset to the community. Epithelial Cells Derived from Swine Bone Marrow Express Stem Cell Markers and Support Influenza Virus Replication In Vitro The bone marrow contains heterogeneous population of cells that are involved in the regeneration and repair of diseased organs, including the lungs. In this study, we isolated and characterized progenitor epithelial cells from the bone marrow of 4- to 5-week old germ-free pigs. Microscopically, the cultured cells showed epithelial-like morphology. Phenotypically, these cells expressed the stem cell markers octamer-binding transcription factor (Oct4) and stage-specific embryonic antigen-1 (SSEA-1), the alveolar stem cell marker Clara cell secretory protein (Ccsp), and the epithelial cell markers pan-cytokeratin (Pan-K), cytokeratin-18 (K-18), and occludin. When cultured in epithelial cell growth medium, the progenitor epithelial cells expressed type I and type II pneumocyte markers. Next, we examined the susceptibility of these cells to influenza virus. Progenitor epithelial cells expressed sialic acid receptors utilized by avian and mammalian influenza viruses and were targets for influenza virus replication. Additionally, differentiated type II but not type I pneumocytes supported the replication of influenza virus. Our data indicate that we have identified a unique population of progenitor epithelial cells in the bone marrow that might have airway reconstitution potential and may be a useful model for cell-based therapies for infectious and non-infectious lung diseases. Bone marrow contains a variety of stem cells that include hematopoietic stem cells, mesenchymal stem cells or stromal cells (MSC), and multipotent adult progenitor cells [1] . Many reports using a variety of animal models have demonstrated that bone marrow cells (BMCs) may have a role in the repair and regeneration of injured lung, infarcted myocardium, and damaged bone, tendon and cartilage [2, 3, 4, 5, 6, 7, 8] . BMCs cultured in vitro can differentiate into type I, II, and basal and airway epithelial cells and express the cystic fibrosis transmembrane conductance regulator (CFTR) protein [9] . BMCs have been shown to improve survival and attenuate lung inflammation in bleomycin-and endotoxin-induced lung injury [8, 10, 11, 12] . Following infusion of BMCs in animal models, these cells have been identified as type I and II alveolar epithelial cells, endothelial cells, fibroblasts, and bronchial epithelial cells [12] . However, precise identity of specific subpopulation of BMCs that engraft in the lung parenchyma and have regenerative potential is still not clear.
Recently, Wong and colleagues [13] reported the isolation of progenitor epithelial cells from mouse and human bone marrow. These cells expressed Clara cell secretory protein (Ccsp), a marker of airway progenitor cells [14] , CD45 and mesenchymal markers CD73, CD90, CD105. These cells differentiated into multiple epithelial cell lineages, including type I and II pneumocytes in vitro. Furthermore, these progenitor epithelial cells preferentially homed to naphthalene-injured lung following intratracheal or systemic inoculation.
Influenza viruses belong to the family Orthomyxoviridae and cause highly contagious respiratory infections in humans and animals. These viruses cause seasonal epidemics and infrequent pandemics in humans. Seasonal influenza epidemics are responsible for between 200,000 and 500,000 influenza-related deaths each year [15] . Avian influenza viruses caused three human pandemics during the last century. The 2009 pandemic, the first pandemic of 21 st century, was caused by a triple reassortant H1N1 influenza virus of swine lineage [16] . In addition to seasonal and pandemic viruses, highly pathogenic avian influenza (HPAI) H5N1 virus has crossed species barrier to infect humans. As of August 9, 2011, more than 500 human cases with over 300 deaths have been reported worldwide [17] . H5N1 viruses replicate to higher titers in lungs and extra-pulmonary tissues leading to acute respiratory distress syndrome, multiple-organ dysfunction, lymphopenia, and hemophagocytosis [18, 19, 20] . Influenza viruses, therefore, pose a constant public health threat, and it is important to understand its pathogenesis to devise effective control measures.
Swine are gaining popularity as a useful large animal model for stem cell therapy for important human diseases or conditions such as myocardial infarction, diabetes, atherosclerosis, traumatic brain injury, retinal damage, and tooth regeneration [21, 22, 23, 24, 25] . Like humans, pigs are an outbred species. As well, they are similar to humans in anatomy, physiology, and immune responses [26, 27, 28, 29] . Additionally, swine can serve as an excellent animal model for influenza virus pathogenesis studies. The clinical manifestations and pathogenesis of influenza in pigs closely resemble to what is observed in humans. Furthermore, the cytokine responses in branchoalveolar lavage (BAL) fluid from swine influenza virus-infected pigs are also identical to that observed in nasal lavage fluids of experimentally infected humans [30] . These observations support that pigs serve as an excellent animal model to study the pathogenesis of influenza virus [31] .
In this study, we report the isolation of previously undocumented progenitor epithelial cells in pig bone marrow that expressed Clara cell secretory protein (Ccsp), a marker for lung progenitor cells, and the stem cell markers octamer-binding transcription factor (Oct4) and stage-specific embryonic antigen-1 (SSEA-1). These progenitor cells showed increased self-renewal capacity and expressed epithelial cell markers such as pancytokeratin (Pan-K), cytokeratin 18 (K-18), and occludin. Importantly, these cells expressed receptors for both mammalian and avian influenza viruses and were permissive to infection with these viruses. The progenitor cells differentiated into type I and II pneumocytes and type II pneumocytes also supported replication of influenza virus. These data provide new insights into the pathogenesis of influenza virus. Further, porcine progenitor epithelial cells described here may serve as a useful model in cellular therapy strategies for epithelial diseases.
During the culture of BMCs for the isolation of mesenchymal stromal cells (MSC), we observed colonies of epithelial cells surrounded by mesenchymal cells. The colony cells exhibited cuboidal morphology typical of epithelial cells (Fig. 1A) .
To characterize epithelial colony cells, we expanded individual colonies in vitro and performed immunocytochemistry by using a panel of stem cell and epithelial cell-specific antibodies. We found that the epithelial colony cells expressed Ccsp, Oct4, and SSEA-1 (Fig. 1B) . We detected the expression of Oct4 in the nuclei, whereas SSEA-1 was mainly found on the cell surface and in the cytoplasm of the progenitor epithelial colony cells (Fig. 1B) . The colony cells expressed epithelial specific markers Pan-K, K-18, and occludin ( Fig. 1C) but not the mesenchymal markers CD29, CD44, and CD90 (data not shown). Differentiation potential of progenitor epithelial cells
To address the self-renewal and differentiation potential of these progenitor epithelial cells, individual colonies were isolated from primary cultures and seeded in tissue culture plates precoated with collagen I in 50% epithelial growth medium and 50% MSC conditioned medium (MSC-CM); referred to as epithelial differentiation medium in the text. In epithelial differentiation medium, the cells continued to grow, became flattened, and appeared as thinly spread cell clusters ( Fig. 2A) . By day 5, the expression of pro surfactant protein C (SPC) was detected in the cytoplasm of these flattened cells (Fig. 2B ). These features are consistent with the type II pneumocytes. Also, expression of aquaporin 5 (Aqua5) protein, a marker for type I pneumocytes was detected on expanded cells which were significantly larger than that of the parental primary epithelial colony cells (Fig. 2B ). The expression of SPC or Aqua5 was not detected on primary undifferentiated cells. These results suggest that bone marrow progenitor epithelial cells have the potential to differentiate into type I and II like pneumocytes.
Detection of a-2,3and a-2,6-linked sialic acid receptors on progenitor epithelial cells Influenza virus infects cells through binding to cell surface sialic acid receptors. To examine the susceptibility of progenitor epithelial cells to influenza virus, we first evaluated by flowcytometry these cells for the presence of sialic acid receptors. The a-2,3-and a-2,6-linked sialic acid receptors were detected on the surface of progenitor epithelial cells by lectin staining. A majority of progenitor cells (.95%) expressed both a-2,3and a-2,6-linked sialic acid receptors, indicating that viruses of both avian and mammalian lineages might replicate in these cells (Fig. 3A ).
After confirming the expression of sialic acid receptors on progenitor epithelial cells, we next examined the susceptibility of progenitor epithelial cells to mammalian and avian influenza viruses. Cells were inoculated with SwIV, AvIV or HuIV at a MOI of 1 for 1 hour (h). As shown in Fig. 3 , infectious viruses were observed in culture supernatants after 24 h after infection and viral titers slightly increased at 36 h. Among the viruses tested, SwIV produced highest cellular lysis (Fig. 3B ) and replicated to highest titers followed by AvIV and HuIV (Fig. 3C) . The production of cytopathic effects required live, infectious virus because cell cytotoxicity was not observed in heat-inactivated virus-infected cultures (data not shown).
In addressing whether in vitro differentiated type I and II pneumocytes are susceptible to influenza virus infection, progenitor cells were differentiated to type I and II pneumocytes for 5 days in epithelial differential medium. The differentiated cultures were then exposed to SwIV. Co-immunostaining revealed SwIV replication in differentiated type II pneumocytes as indicated by the presence of viral proteins whereas no viral proteins were detected in differentiated type I pneumocytes (Fig. 4 ).
In this study, we report the isolation and identification of previously undocumented stem/progenitor epithelial cells from bone marrow of pigs. These cells expressed the stem/progenitor cell markers Oct4, SSEA-1, and Ccsp, and the epithelial markers Pan-K, K-18, and occludin. Upon culture in epithelial differentiation media, these cells differentiated into type I and II pneumocytes. Most importantly, progenitor cells were targets for mammalian and avian influenza virus replication.
We detected the expression of Oct4 in progenitor epithelial cells. Oct4, a member of the POU family of transcription factors, is expressed in pluripotent stem cells such as embryonic stem cells, induced pluripotent stem cells, and lung stem cells where it regulates self-renewal and pluripotency [32, 33] . These Oct4 + colony cells also expressed other stem cell markers; SSEA-1, Ccsp and epithelial markers; pan-K, K-18, and occludin. Clara cells, airways progenitor cells in the lungs which have been involved in lung regeneration and repair, also express Ccsp and cytokeratins [14, 32, 34] . Importantly, these Oct4 + Ccsp + colony cells differentiated into cells possessing Aqua5 and SPC which are markers for type I and II like pneumocytes respectively. Expression of stem cell markers in progenitor epithelial cells may, therefore, be associated with differentiation potential of these cells.
The porcine progenitor epithelial cells reported in this study share similarities with recently reported human and mouse cells that both cell types express Ccsp, but unlike human and mouse cells, porcine progenitor epithelial cells did not express mesenchymal markers such as CD29, CD44 and CD90 [13] . Porcine progenitor epithelial cells also expressed stem cell markers; Oct4 and SSEA-1; however, mouse and human cells were not tested for expression of these markers [13] . Similar to human and mouse bone marrow cells, porcine bone marrow progenitor cells expressed both Ccsp and SPC. Ccsp and SPC expressing broncho-alveolar stem cells (BACS) were recently identified in adult mouse lung [35] . Mouse BACS were found to express hematopoietic markers, whereas our porcine bone marrow progenitor epithelial cells lacked the expression of these markers. Importantly, we were able to passage progenitor epithelial cells up to passage 8 suggesting that these cells have extensive self-renewal and proliferation properties.
The role of bone marrow stem cells in tissue regeneration including lungs is well established [36, 37, 38, 39] . However, the precise identity of specific subpopulation of BMCs that has tissue regeneration capacity is not known. As reported for human progenitor epithelial cells, porcine Oct4 + Ccsp + colony cells were detected in the plastic-adherent fraction surrounded by mesenchymal cells [40] . Therefore, it is likely that the progenitor epithelial cells were derived from mesenchymal precursors. Previously, bone marrow and even cord blood MSC were shown to have epithelial differentiation potential. Cord blood MSC when cultured in vitro expressed lung-specific markers such as Ccsp, SPC, and CFTR [41] . Intravenous administration of cord blood-MSC into NOD-SCID mice resulted in a low level of airway engraftment of the cord blood-MSC that expressed cytokeratin and CFTR. Similarly, bone marrow MSC differentiated to type I and II pneumocytes in vitro and following intravenous or intratracheal administration, engrafted in recipient lung paren-chyma as type I and II pneumocytes [3, 42] . These observations suggest that bone marrow MSC could serve as precursors of differentiated epithelial cells. Moreover, in this study we observed that progenitor epithelial cells when cultured in 50% MSC-CM were able to differentiate into type I and II pneumocytes further providing evidence that mesenchymal stroma is important for the pluripotency of epithelial colony cells. Future work will be directed to identify the molecular signals provided by the mesenchymal precursors that regulate the differentiation potential of epithelial progenitor cells.
Bone marrow cell therapy may be effective in conditions that currently lack effective treatment. Stem cells isolated from different anatomic niches of lungs [34, 35, 43, 44, 45] have been shown to differentiate into multiple epithelial cell types following lung injury. Our lab and Wong and colleagues [13] demonstrated the existence of progenitor epithelial cells in the bone marrow of pigs and humans respectively which are capable of differentiating into type I and II pneumocytes suggesting that these cells will be valuable for lung therapies. Functionally, type I pneumocytes are important for gas exchange, water permeability and the regulation of alveolar fluid homeostasis whereas type II pneumocytes produce lung surfactants that reduce the alveolar surface tension [46] . The isolation and expansion of lung-derived autologous cells from humans is difficult, whereas bone marrow progenitor cells can be easily isolated and expanded. In other preliminary studies, we have isolated progenitor epithelial cells from the lung of pigs. Future in vivo experiments in pigs will be designed to compare the lung repair potential of bone marrow-and lung-derived progenitor epithelial cells following infection and/or LPS-induced lung injury.
Excessive virus replication, multi-organ failure, and hyperimmune activation have been detected in humans and laboratory animals infected with HPAI H5N1 and with recreated 1918 pandemic influenza virus [47, 48, 49, 50, 51] . The mechanisms responsible for deterioration of lung function and the loss of capacity for lung repair after influenza virus infection are not well understood. Although influenza virus primarily replicates in lung, virus has been detected in extra pulmonary tissues including bone marrow [52, 53] . Our in vitro findings show that progenitor epithelial cells in bone marrow are permissive to influenza virus suggesting that in the event of influenza virus infection, bone marrow progenitor cells may not be available to home to the injured lung to participate in tissue repair. Additional experiments are needed to confirm the infection of progenitor epithelial cells in vivo and to delineate further their roles in local and lung repair following infection with influenza virus.
In conclusion, we have isolated Oct4 + Ccsp + SSEA-1 + expressing progenitor epithelial cells in the bone marrow of swine which are capable of differentiating into type I and II pneumocytes. Additionally, we have demonstrated that these cells serve as targets for influenza virus infection. Further characterization of these progenitor cells may help in understanding the pathogenesis of infectious and non-infectious epithelial diseases and the mechanisms of lung injury repair.
Femur bones were obtained from 4-to 6-week old germ-free pigs. BMCs were isolated by previously described methods [54, 55, 56] . Briefly, the tip of each bone was removed and the marrow was harvested by inserting a syringe needle into one end of the bone and flushing with Dulbecco's Modified Eagle's Medium (DMEM; Gibco). The bone marrow cells were filtered through a 70-mm nylon mesh filter (BD, Falcon, USA) and mononuclear cells were obtained by density gradient centrifugation over Ficoll-Hypaque (gradient density 1.077). Cells (1-5610 5 /cm 2 ) were plated in 25 cm 2 cell culture flasks in DMEM containing 10% fetal bovine serum, 2 mm L-glutamine, 1% antibiotic solution (Gibco, USA). Cultures were incubated at 37uC in a humidified atmosphere containing 95% air and 5% CO 2 . The non-adherent cells were removed after 72 h of culture. Individual epithelial colonies surrounded by mesenchymal cells were plucked and were expanded in vitro by culturing in DMEM media.
Stem cell and epithelial cell markers on progenitor epithelial cells were detected by immunofluorescence assay (IFA). Epithelial colony cells were fixed in methanol: acetone (1:1) for 3 min at room temperature and then blocked with 3% bovine serum albumin (BSA) for 30 min. Cells were incubated at 4uC with following primary antibodies: rabbit anti-human Oct4 (Santa Cruz Biotechnology), mouse anti-human SSEA-1(Millipore), goat antimouse cc10 (Santa Cruz Biotechnology), mouse anti-human pan cytokeratin, mouse anti-human cytokeratin 18 (Sigma), rabbit anti-human occludin (Invitrogen), mouse anti-human CD90, rat anti-mouse CD44 and mouse anti-pig CD29 (BD Pharmingen). After overnight incubation at 4uC, cells were washed and incubated for 1 h at room temperature with the following respective FITC-labeled secondary antibodies: donkey anti-goat IgG, goat anti-rabbit IgG, and goat anti-mouse IgG (Sigma). Cell nuclei were then counterstained with 49,6-diamidino-2-phenylindole (DAPI).
Porcine bone marrow MSC were grown and expanded as described above. When MSC reached at about 80% confluence, medium was aspirated and cells were washed three times with phosphate-buffered saline. Cells were cultured in serum-free medium for 24 h. CM was removed and centrifuged to remove cellular debris and used in epithelial differentiation assays.
To analyze differentiation potential of porcine progenitor epithelial cells, cells were seeded in culture dishes pre-coated with collagen I. The cells were cultured in epithelial cell differentiation medium containing 50% epithelial growth media supplemented with bovine pituitary extract (70 mg/ml), human epidermal growth factor (5 ng/ml), insulin (5 mg/ml), and hydrocortisone (0.5 mg/ ml) (MEGM, Lonza, Walkersville, MD) and 50% MSC-CM medium. After 5 days of incubation at 37uC, cells were observed for morphology and cells grown on coverslips were examined for the expression of Aqua5 (marker for type I pneumocytes) and SPC (marker for type II pneumocytes) by using goat anti-human Aqua5 (Santa Cruz Biotechnology) and rabbit anti-human SPC (Millipore) primary antibodies by IFA as described above.
Sialic acid receptors on progenitor epithelial cells were detected by flowcytometry [57] . Madin-Darby canine kidney (MDCK) cells have been shown to express both a-2,3-linked sialic acid receptors and a-2,6-linked sialic acid receptors; avian and mammalian influenza virus receptors, respectively. Therefore, MDCK cells were included as positive controls. The cells were incubated with FITC-labeled Maackia amurensis lectin II (MAA) or Sambucus niagra agglutinin (SNA) (EY Laboratories) at 4uC for 30 min in dark and acquired by Accuri C6 flow cytometer (BD Accuri) and analyzed using CFlow plus software (Accuri).
Influenza virus strains; swine influenza virus (SwIV; Sw/OH/ 07, H1N1), avian influenza virus (AvIV; Ch/NY/95, H7N2) and human influenza virus (HuIV; Hu/OH/06, H1N1) were propagated in 10-day-old embryonated chicken eggs (AvIV was kindly provided by Dr. S.M. Goyal, Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN). Progenitor epithelial cells were infected with SwIV, AvIV and HuIV at a multiplicity of infection (MOI) of 1. After adsorption for 1 h, the cells were washed and fresh medium was added to the cells. At intervals after infection, released viruses in culture supernatants were titrated in MDCK cells [58] .
To examine whether differentiated progenitor epithelial cells support influenza virus infection, epithelial colony cells were cultured in collagen I coated dishes for 5 days in epithelial cell differentiation medium. On day 5, cells were infected with SwIV and influenza viral nucleoprotein (NP) was detected at 24 h after infection by IFA using mouse anti influenza A NP (Millipore) as primary antibody and rhodamine-labelled goat anti-mouse (Molecular Probes) as secondary antibody. Two Birds with One Stone? Possible Dual-Targeting H1N1 Inhibitors from Traditional Chinese Medicine The H1N1 influenza pandemic of 2009 has claimed over 18,000 lives. During this pandemic, development of drug resistance further complicated efforts to control and treat the widespread illness. This research utilizes traditional Chinese medicine Database@Taiwan (TCM Database@Taiwan) to screen for compounds that simultaneously target H1 and N1 to overcome current difficulties with virus mutations. The top three candidates were de novo derivatives of xylopine and rosmaricine. Bioactivity of the de novo derivatives against N1 were validated by multiple machine learning prediction models. Ability of the de novo compounds to maintain CoMFA/CoMSIA contour and form key interactions implied bioactivity within H1 as well. Addition of a pyridinium fragment was critical to form stable interactions in H1 and N1 as supported by molecular dynamics (MD) simulation. Results from MD, hydrophobic interactions, and torsion angles are consistent and support the findings of docking. Multiple anchors and lack of binding to residues prone to mutation suggest that the TCM de novo derivatives may be resistant to drug resistance and are advantageous over conventional H1N1 treatments such as oseltamivir. These results suggest that the TCM de novo derivatives may be suitable candidates of dual-targeting drugs for influenza. The first global pandemic of the 21st century was announced by the World Health Organization (WHO) in 2009 due to the worldwide spread of influenza A subtype H1N1 (H1N1/09) [1] . More than 214 countries have reported laboratory confirmed cases, and more than 18,449 deaths have been recorded [2] . Currently, the neuraminidase inhibitor TamifluH (oseltamivir) remains the primary drug prescribed to patients infected with H1N1/09 [3] . However, the emergence of drug resistant viral strains [4] and limited drug administration window [5] exemplifies the need for additional therapies.
Important constituents of influenza surface membrane proteins include hemagglutinin, neuraminidase, and the matrix protein 2 (M2) proton channel [6, 7] . Hemagglutinin mediates the binding of viral particles to host cell surface sialic acid and the invasion of viruses into host cell [8] [9] [10] . Neuraminidase is responsible for the cleavage of sialic acid residues to promote the release of progeny viruses [11, 12] . M2 proton channels are critical for viral mRNA incorporation into the virion and virus budding [13] . Over one hundred serological subtypes [14] have been identified through different combinations of the 16 hemagglutinin (H1-H16) and nine neuraminidase groups (N1-N9) currently known. The 3Dstructure of M2 proton channels have recently been solved in both influenza A and B [15, 16] , allowing more in depth studies regarding its biological function and action mechanism [17] [18] [19] . These proteins have been used as targets for rational attempts to design drugs for influenza [20] [21] [22] [23] [24] [25] [26] [27] .
The H1N1/09 virus strain is a triple reassortant that contains gene segments from avian, swine and human influenza viruses [28] . In addition to antigenic shift that can lead to fundamental changes in influenza surface antigens, antigenic drift could reduce binding affinity of host antibodies to antigens [29, 30] . A major challenge in influenza vaccine development is the rapid evolution of influenza viruses, causing vaccines to be easily outdated and reformulation necessary each year [31] [32] [33] . Although the H1N1/ 09 virus is susceptible to neuraminidase inhibitors, cases regarding oseltamivir-resistant viruses with neuraminidase mutation (such as H275Y) have been reported [34, 35] . Given that influenza viruses have RNA genomes that are prone to changes, it is imperative to devise new therapies. Much effort has been made to investigate the mechanism and devise alternative drugs against the drugresistance issue of H1N1 [36] [37] [38] [39] [40] . Developing inhibitors that target both H1 and N1 antigens can reduce resistance issues resulting from the mutation of a single target antigen.
Computational approaches have been widely applied to molecular biology and medicine [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] . Structure-based methods, including docking and MD simulation, are invaluable tools in
The influenza A subtype H1N1 (H1N1/09) pandemic raised public concerns due to drug resistance strains. Drug resistance occurs from conformational changes causing the original drug to lose binding ability and exhibit biological effects. The world's largest TCM Database@Taiwan was employed to screen for potential leads that simultaneously bind to H1 and N1. Three de novo compounds derived from Rosemarinus officinalis and Guatteria amplifolia were identified as having dual binding properties to H1 and N1. Structural analysis indicated that the candidates bind to multiple residues in both H1 and N1. In addition, the de novo derivatives were predicted as bioactive using four different computational models. The compounds are validated as potent dual targeting influenza drug candidates through multiple validations. Key advantages of the candidates include (1) binding to H1 and N1 through multiple amino acids, and (2) not binding to known mutation residues in H1 or N1. Such advantages can reduce drug resistance caused by single point mutations. On a broader context, features important for successful H1N1 drug development are discussed in hopes of providing starting templates for drug development and improvements. drug discovery and design. Computational docking is important for investigating ligand-protein interactions and elucidating binding mechanisms [51] [52] [53] [54] [55] [56] [57] . Since publication of the pioneer paper in 1977 [58] , it has been established that low-frequency motions existing in proteins and DNA can help reveal dynamic mechanisms underlying fundamental biological functions [59] [60] [61] [62] [63] . NMR observation later confirmed such inferences and the findings were applied to medical treatments [64] [65] [66] [67] . In recent years, application of molecular dynamics to investigate internal motions and biological functions of biomacromolecules has opened new frontiers. Vast amounts of information on molecular recognition and binding [68] [69] [70] [71] , conformations or conformational changes [72] [73] [74] [75] , molecular mechanisms of bioactivity and stability [76] [77] [78] [79] , and drug discovery [80] [81] [82] [83] [84] have been found. To understand interaction of drugs with proteins or DNA, consideration should be given not only to the static structures but dynamical information obtained by simulation through a dynamic process. In this regard, both docking and MD simulation were utilized in this study to provide comprehensive analysis protein-ligand interactions under static and dynamic conditions. Much effort has been placed on developing new, effective influenza treatments, but most have focused on neuraminidase or M2 as the target protein [37, 38, [85] [86] [87] . To date, no hemagglutinin inhibitor is available. Traditional Chinese medicine (TCM) has been used extensively for finding effective drugs [88] , and we have successfully designed novel medicinal compounds and identified potential drug leads through traditional Chinese Medicine Database@Taiwan (TCM@Taiwan) [89] . Preliminary studies conducted in this lab show potential for TCM compounds to serve as neuramindase and hemagglutinin inhibitors individually [90] [91] [92] [93] [94] [95] . In view of the current needs for drugs effective against native and mutant H1N1/09 and our promising preliminary results, the present study integrates the concept of ''dual targeting'' with the aforementioned computational tools and TCM in the attempt to identify dual-targeting inhibitors of H1N1 that may be useful for drug development.
The experimental procedures and screening results after each filtering step are summarized in Figure 1 . Among the 829 native TCM compounds, 81 docked into both H1 and N1 and were used for de novo evolution (Table S1 ). De novo compounds with dual binding capacities to H1 and N1 were ranked by combined DockScore and the top ten derivatives are listed in Table 1 . Nine of the ten top ranking de novo compounds were derived from Rosmaricine, a natural compound isolated from Rosemarinus officinalis [96] . The remaining de novo compound was based on Xylopine, which is naturally found in Guatteria amplifolia [97] .
The top three derivatives, Xylopine_2, Rosmaricine_14 and Rosmaricine_15, have in common a pyridinium addition to their native structure ( Figure 2) . The pyridinium addition could be the main explanation for higher DockScores of these three derivatives compared to their native compounds and the other derivatives. Rosmaricine_14 and Rosmaricine_15 differed by the number of fused rings, but the slight difference in DockScore suggests that addition of an acyclic ring has little influence on binding affinity.
Docking of the de novo compounds back to the receptor provides insights to modifications that can be made to modulate or enhance molecular properties and also highlights important protein-ligand interactions. When docked into the N1 protein binding site, Xylopine_2 interacts with Asp151 via a protonated amino group and has pi and hydrogen bond (H-bond) interactions with Trp179 and Glu228, respectively ( Figure 3A ). Rosmaricine_14 ( Figure 3B ) and Rosmaricine_15 ( Figure 3C ), have interactions with Asp151 and Arg293 via the carbonyl group and Glu228 through the 2-aminopyridinium group.
TamifluH forms H-bond interactions with Arg156, Arg293 and Arg368, but not with Asp151 or Glu228 ( Figure 3D ). Both Asp151 and Glu228 have been reported as one of the major residues in the N1 ligand binding site [98, 99] . The ability of the de novo derivatives to form interactions with both Asp151 and Glu228 may account for the higher DockScores.
Binding of the top three de novo derivatives to H1 site is detailed elsewhere [92] . The ability to bind with important H1 residues Asp103 and Arg238 [100] indicates the dual targeting possibility of the candidates. Figure 4A . All values were within the 95% prediction bands and the r 2 value = 0.8043. The SVM model was constructed using identical molecular descriptors and ligands as the MLR model. The r 2 value of the SVM model was 0.8605 and the correlation between observed and predicted activities of 27 ligands are illustrated in Figure 4B . Table 2 summarizes the pIC 50 values of TamifluH and the top three candidates as predicted by the generated MLR and SVM models. The predicted activity of TamifluH using the generated MLR model (pIC 50 = 7.613) is similar to observed bioactivity values reported in the literature (pIC 50 = 7.823) [101] . This indicates that the generated MLR model is a good prediction model. Predicted activity values using the SVM model indicate a lower pIC 50 with regard to TamifluH. Nonetheless, both models indicate that all TCM de novo derivatives are good candidates with neuraminidase inhibitory activity. MLR and SVM models for predicting hemagglutinin inhibitory activity were not established due to the lack of available hemagglutinin inhibitor structures in the literature.
Stability profile analysis. Root mean square deviation (RMSD) and total energy results from MD are summarized in Figure 5 and provide information on N1-ligand complex and ligand stability. During the 20 ns simulation process, the RMSDs of the four complexes ranged between 1.4-1.7 Å . Xylopine_2 stabilized after 15 ns, and the total energy of the complex equilibrated at 219,000 kcal/mol. The ligand RMSD of Rosmaricine_14 remained stable throughout the simulation, and no evident changes in total energy were observed after 17 ns. The RMSD and total energy of Rosmaricine_15 stabilized after 13 ns. Fluctuations in ligand RMSD was observed in TamifluH for the first 5 ns, but no changes were observed in ligand RMSD and total energy from 5 ns until the end of MD. The larger ligand RMSD fluctuations and higher total energy observed for Xylopine_2 may be attributed to its spatial structure and docking characteristics. Xylopine_2 consists of a bulky xylopine and a 2-aminopyridinium residue linked through 1 in a way similar to that of cis conformations ( Figure 6 ). It is well established that cisconformations are less stable than their transcounterparts, and thus may explain the higher total energy levels for Xylopine_2. In addition, Xylopine_2 binds to N1 though the aminopyridinium residue, allowing the xylopine structure more freedom to rotate and thereby increasing ligand RMSDs and total energy levels.
H-Bond network during MD simulation. The H-bond occupancy of each compound in N1 is summarized in Table 3 Figure 7 . The distance between Xylopine_2 and Glu228 was maintained between 2-3 Å ( Figure 7A ). The distance of Rosmaricine_14 and Glu119 and Glu 228 also remained between 2-3 Å ( Figure 7B ). From Table 3 , Rosmarcine_15 and TamifluH did not form high occupancy H-bonds with Tyr402. Intriguingly, bond distance profiles indicate that Tyr402 was one of the key amino acids for H-bond formation ( Figure 7C , 7D). Tyr402 bond distance generally exceeded 2.5 Å in Rosmaricine_15, thus explaining the low occupancy rate in Table 3 . For TamifluH, the distance in the first ns was between 3-4 Å and then decreased to 2-3 Å from 1-20 ns, thus accounting for the low occupancy rate as well. Despite the low occupancy rates, the bond distance profiles suggest that Tyr402 is an important N1 binding site for Rosmaricine_15 and TamifluH.
Possible mechanism for protein-ligand interaction. Insights to how ligand stabilization occurs within the protein binding site can be discerned from MD simulation. The H-bond formations with Leu224 at 2 and Glu228 at 3 and 4 ''sandwich'' the aminopyridinium and anchors Xylopine_2 ( Figure 6A ). However, the xylopine moiety remained unattached, causing strain to the compound and possibly contributing to large H-bond distance fluctuations ( Figure 7A ). At approximately 15 ns, attraction between Glu228 and 3 causes the terminal amine residue to torque towards Glu228 increasing the distance from Leu224 ( Figure 6A ). As a result, the stable H-bond with Leu224 was lost, and an additional H-bond with Glu228 was formed from 2. At the end of MD simulation, the primary binding force for Xylopine_2 was H-bonds formed with Glu228. In contrast to Xylopine_2, multiple binding sites secured Rosmaricine_14 and 15 within the binding site as reflected by the small H-bond distance fluctuations compared to Xylopine_2 ( Figure 7B , 7C). Rosmaricine_14 bound to Glu119, Glu228, and Arg293 through 6, 7, and 8 respectively ( Figure 6B ). The multiple attachment points anchor both the aminopyridine moiety and the rosmaricine moiety, reducing strain on the molecular structure as reflected by the low total energy and bond distance fluctuations. The increase in bond distance from Arg293 in Figure 7B starting at 3 ns was due to Arg293 intermolecular Hbond formation between the O atom and NH 2 residue. Nonetheless, all H-bonds were maintained throughout the MD simulation, suggesting good stability of Rosmaricine_14. The mechanism for stability of Rosmaricine_15 is similar to that of Rosmaricine_14. In addition to H-bonds at 9 with Glu228 and 10 with Arg293 which are identical to Rosmaricine_14, H-bonds are formed at 11 with Glu228, 12 with Thr226, 13 with Asn262, and 14 with Tyr402 ( Figure 6C ). These additional anchor points stabilized residues that were available for rotation in Rosmaricine_14, further lowering the total energy of the compound ( Figure 5C ). Within the anchored ligand, twisting of 15 contributes to H-bond fluctuations at Glu228 such as Figure 6D ). The stability of Tamiflu as a result of these binding anchors is reflected in the low total energy profile ( Figure 5 ) and small H-bond distance fluctuations ( Figure 7D ). The ability of the de novo derivatives to form stable H1-ligand complexes has also been assessed [92] . All de novo derivatives were capable of forming H-bonds at Glu83 and Asp103, the key binding sites on H1.
The torsion angles of flexible bonds in each candidate when in complex with H1 and N1 are summarized in Figure 8 . In Xylopine_2, all monitored bonds were stable in H1 except for e ( Figure 8A ). The fluctuations could be attributed to the attraction between the amine group H atoms and Asp 103. When bound to N1, b was the primary location for torque changes in Xylopine_2. The recorded torsion angle changes at b support our previous speculation that the unattached xylopine moiety is a key source of instability for Xylopine_2. Torsion angle fluctuations of Rosmar-icine_14 in both H1 and N1 were mainly due to rotations at g and j ( Figure 8B ). Such changes are expected as the H atoms on the amine group continuously rotate to form H-bonds with key amino acids. Bonds in Rosmaricine_15 ( Figure 8C ) exhibited similar characteristics to those in Rosmaricine_14. Rapid rotations at the amine groups l and o are visualized by the recorded angle trajectories. NAG ( Figure 8D ) and Tamiflu ( Figure 8E ) both have relatively stable intermolecular torsion changes. This indicates that the lower stability of NAG and Tamiflu in H1 and N1, respectively, are not due to instability of their ligand structures, but may be attributed to weaker or unstable ligand-protein affinities.
Hydrophobic interactions. Hydrophobic interactions also played a role in stabilizing ligands within H1 ( Figure 9 ) and N1 ( Figure 10 ) binding sites during MD. Due to differences in ligand structure and binding conformation, amino acids with which hydrophobic interactions were formed differed. In H1, amino acids involved in hydrophobic interactions included Pro82, Asp103, Asn104, Cys107, Cys153, and Pro154. More stabilizing interactions including H-bonds and hydrophobic interactions were observed in the N1 binding site (Figure 10 ). For the TCM candidates and Tamiflu, the spatial distribution of H-bonds coupled and hydrophobic interactions limits the free movement of ligands within N1, thus increasing stability of the N1-ligand complex.
To further investigate docking features, CoMFA and CoMSIA models were built and validated using 27 neuraminidase inhibitors listed in Table S2 . The PLS analyses results for CoMFA and CoMSIA models are shown in Table 4 . The CoMFA model was generated using both steric and electrostatic fields and yielded a (Table 5) .
The validated CoMFA and CoMSIA maps were used to assess ligand bioactivity. Contour of the de novo compounds at 20 ns MD simulation to the relative spatial positions of CoMFA and CoMSIA feature maps are shown in Figure 11 . In Xylopine_2, Rosmaricine_14 and Rosmaricine_15, the H-bond between the 2-aminopyridinium group and Glu228 matched the electropositive group feature of the CoMFA model ( Figure 11A,11C,11E ) and the H-bond donor feature in CoMSIA model ( Figure 11B,11D,11F) . The hydrophobic benzene structures of Xylopine_2 matched the steric favoring region of the CoMFA map and the hydrophobic feature of the CoMSIA map. The carbonyl groups in Rosmaricine_14 and Rosmaricine_15 which formed H-bonds with Tyr402 satisfied the H-bond acceptor feature in the CoMSIA model. TamifluH also contours to both CoMFA and CoMSIA models. The 3-methoxypentane group close to Arg293 and Asn344 matched the steric favoring region of CoMFA ( Figure 11G ) and the hydrophobic feature of CoMSIA ( Figure 11H ). This residue has similar characteristics to the 2aminopyridinium group in the de novo derivatives. In addition, the N-methylacetamide group in TamifluH, which forms H-bond with Tyr402, is located near the H-bond donor feature in CoMSIA. Though all compounds contoured to the N1 inhibitor features identified by CoMFA and CoMSIA, a critical difference was observed between TamifluH and the TCM de novo derivatives. All compounds except TamifluH formed H-bonds at Glu228. As Glu228 is a primary binding site of N1 [99] , ability of the TCM de novo derivatives to maintain stable binding with Glu228 during MD simulation supports the potential of these compounds as drug alternatives to TamifluH.
Due to the lack of reported H1 ligand bioactivities in the literature, direct assessment of bioactivity through construction of CoMFA and CoMSIA models was not possible. Alternatively, indirect support was provided by assessing the ability of de novo derivatives to maintain contour to the N1 CoMFA/CoMSIA maps while forming interactions at key residues in H1, Glu83 and Asp103 [92] . As illustrated in Figure 12 the TCM de novo derivatives docked into the H1 binding site and formed critical interactions at Glu83 and Asp103 without losing contour to the CoMFA and CoMSIA maps. These results suggest that not only were the TCM de novo derivatives capable of docking into both H1 and N1, but that biological activity was also predicted in both binding sites, thus it is possible to develop dual-targeting drugs from the selected de novo derivatives.
Important features for potential H1 and N1 inhibitors are summarized in Figure 13 . For H1, a salt bridge with Glu83 and H-bond donor and/or electrostatic interactions with Asp103 are important characteristics that should be met. Potential inhibitors for N1 should have salt bridge and/or H-bond formation at Glu228 and interactions with Asp293. These features can be used to identify or design novel drugs for H1 and/or N1. In the case of the TCM de novo derivatives from this study, each compound could structurally fulfill the requirements of both H1 ( Figure 13A,13B,13C ) and N1 ( Figure 13D,13E,13F) binding sites, thus supporting their potential as dual-targeting compounds.
In this research, we identified Xylopine_2, Rosmaricine_14, and Rosmaricine_15 as the top three de novo derivatives exhibiting binding affinity to H1 and N1. Addition of a pyridinum residue to the native structures of xylopine and rosmaricine contributes to bond formation at key residues in both H1 (Glu83, Asp103) and N1 (Glu228, Arg292). The de novo derivatives were predicted as active by the SVM and MLR models, and contoured well to the 3D-QSAR models. The TCM de novo derivatives were able to maintain contour while forming key binding interactions in H1, thus providing indirect support for bioactivity in H1. The results of this study indicate that the TCM de novo derivatives not only can bind to, but can also exhibit biological activities in both H1 and N1.
Key binding locations of the de novo derivatives include Glu83 and Asp103 for H1, and Glu228 and Arg292 for N1. Mutations currently attributed to oseltamivir resistance are located at H275 and N295S of the NA [103] . Since the key binding locations of the TCM derivatives do not overlap with those causing oseltamivir resistance, derivatives will be able to bind to viruses that are currently resistant to TamifluH. In addition, the de novo derivatives do not bind to amino acids in H1 or N1 that are prone to mutation ( Table 6, Table 7 ) [40, 104] , thus would likely be able to exert activity across a range of mutant H1N1 viruses. Last but not the least, multiple bond formations observed in MD provide additional insurance against possible mutations at key binding residues. In the case of a single point mutation, the de novo compounds will remain bound to the H1 and N1 sites through another key residue, therefore resisting the development of drug resistance in the virus. Based on the results and observations of this study, the TCM de novo derivatives may be attractive compounds for designing novel dual-target inhibitors for H1 and N1. 
Virtual screening, de novo derivative generation, and molecular dynamics (MD) simulation were performed using Discovery Studio Client v2.5.0.9164 (DS2.5; Accelrys Inc., San Diego, CA). The two-dimensional and three-dimensional structures of TCM compounds were generated using ChemBioOffice 2008 (Perki-nElmer Inc., Cambridge, MA). Comparative molecular field analysis (CoMFA) and comparative molecular similarities indices analysis (CoMSIA) models were constructed using SYBYLß 8.3 package (Tripos Inc., St. Louis, MO).
Compounds from the TCM Database@Taiwan were docked to H1 and N1 protein active sites reported in our previous study [91] . All procedures were completed under the forcefield of Chemistry at HARvard Molecular Mechanics (CHARMm) [105] . The virtual screening process was performed using LigandFit. The conformational search method was based on the Monte Carlo algorithm. Rigid body minimization following initial ligand placement was completed using Smart Minimizer. Scoring functions used by LigandFit were DockScore.
TCM compounds that docked into both H1 and N1 proteins were selected and then ranked by the sum of their H1 and N1 DockScore.
TamifluH was used as the control for N1, and its N1 docking score was set as the minimum requirement. The top TCM compounds that passed the filtering were selected for de novo evolution.
In de novo evolution, TCM compounds were placed into the H1 and N1 protein binding sites described previously, and Ludifragments were attached to the native structure. The new derivatives were generated in full evolution mode. Derivatives from de novo evolution were subjected to additional screening through Lipinski's rule [106] to rule out orally unstable or pharmacologically inapplicable compounds. As de novo products generated for H1 and N1 proteins differed, all de novo products were re-docked to H1 and N1 proteins to assess binding affinity. De novo products that docked into both H1 and N1 proteins were selected and ranked by the sum of their respective H1 and N1 DockScore. The top ten compounds with the highest DockScore were selected for further structure-based analysis.
The 27 neuraminidase inhibitors used, including 24 training set compounds and 3 test set compounds, were adapted from Zhang's study [102] . Compounds were drawn using ChemBioOffice 2008 (PerkinElmer Inc., Cambridge, MA) and modified to physiological ionization using the Prepare Ligand function in DS 2.5. Bioactivity values (IC 50 ) were also obtained from Zhang's study though the original sources were not clarified, and converted to pIC 50 (log(1/ IC 50 )). Molecular descriptors of the compounds were calculated using Calculate Molecular Properties in DS 2.5 and the GFA was used to select the best representative molecular descriptors [107] . Utilizing the best representative molecular descriptors identified through GFA, MLR and SVM models were constructed using MATLAB (The Mathworks Inc., Natick, MA) and LibSVM [108] , respectively, and used to predict the bioactivity of TCM de novo compounds.
The MD simulation was performed using the Molecular Dynamics package of DS 2.5. The complexes were created with a 10 Å solvation shell of TIP3 water around the protein. Sodium cations were added to each system for neutralization. Minimization using Steepest Descent and Conjugate Gradient were performed at 500 cycles each. Each protein-ligand complex was gradually heated from 0K to 310K over 50 ps, followed by a 200 ps equilibration phase. The production stage was performed for 20 ns using NVT canonical ensemble and trajectory frames were saved every 20 ps. SHAKE algorithm was applied to immobilize all bonds involving hydrogen atoms throughout the MD simulation. Long-range electrostatics were treated with PME method. Time step was set to 2 fs for all MD stages. The temperature coupling decay time for the Berendsen thermal coupling method was 0.4 ps. Post processing of the trajectory was performed using Analyze Trajectory module. Torsion angles of each bond were also monitored through DS 2.5. LIGPLOT [109] was used to generate schematic diagrams of protein-ligand interactions for each candidate and control in H1 and N1.
CoMFA and CoMSIA models were constructed through the partial least square (PLS) analysis using previously described neuraminidase inhibitors [102] . The optimal number of components was obtained from leave-one-out method to yield the highest r 2 and q 2 values in non-cross validation and cross-validation, respectively. Biological activities of the TCM de novo compounds were evaluated based on contour to the generated 3D-QSAR map. Table 6 . HA mutation points between the 1918 H1N1 and H1N1/09 viruses. Table 7 . NA mutation points between 1918 H1N1 and H1N1/09 viruses. Supporting Information  Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst BACKGROUND: Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material. RESULTS: In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy. CONCLUSIONS: This study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts. NeuNAc is the most prevalent exponent of sialic acids [1] . In mammals, sialic acids are usually found as terminal residues of glycol conjugates on the outermost cell surface. As a result of their location and their negative carboxylate functionality, sialic acids play important roles in mediating cellular recognition and adhesion processes [2] and in the infection cycles of severe viral diseases, such as influenza viruses A and B [3] . In these cases, de novo-synthesized viral particles attach to their respective sialic acids at the cell surface. Neuraminidase (sialidase) activity is needed for the propagation of the virus in the host. Consequently, sialic acid derivatives are successfully applied in the therapy of such virus-related diseases. One well-known product that functions as a neuraminidase inhibitor is Relenza. Its active pharmaceutical ingredient is Zanamivir, which is a direct derivative of the NeuNAc precursor [4] .
Traditionally, NeuNAc is prepared through extraction from natural sources, such as bird nests, milk, or eggs [5] , through the hydrolysis of colominic acid (a homopolymer of NeuNAc) in a culture broth of Escherichia coli K1 [6] , or through chemical synthesis [7] . Methods for NeuNAc production have included a chemo-enzymatic process [8, 9] , a two-enzyme reaction process [10, 11] , a biotransformation process using E. coli [12] , and an E. coli whole-cell system [13] . However, the requirement for ATP or an excess of pyruvate and the subsequent expensive downstream processing has kept the costs of NeuNAc production considerably high (current market price is $100/g).
Chitin is considered the second most abundant biomass available on earth [14] . The estimated annual biosynthesis of chitin is more than 10 11 tons in marine waters alone [15] . Unlike cellulose, the other dominant biopolymer, chitin can serve as a source for both carbon and nitrogen (C:N = 8:1) [16] . This property suggests that chitin is an optimal resource for the production of NeuNAc (C:N = 11:1) because no additional nitrogen would need to be applied as it would be if glucose or cellulose were used as raw material. Chitin is found in the exoskeletons of arthropods, such as crustaceans (including crab, lobster, and shrimp) and insects (including ants and beetles), the cell walls of fungi, the radula of mollusks, and the beaks of cephalopods (including squid and octopi). This polymer is composed of β-(1,4)-linked units of the amino sugar N-acetylglucosamine (GlcNAc) that is currently produced using hydrolysis of deproteinized and demineralized crustacean shells [17] . Chitinolytic enzymes from fungi of the genus Hypocrea have been extensively studied for decades [18] . More recently, the chitinolytic enzyme system of H. jecorina has been studied using genome-wide analysis [19, 20] . Unlike their bacterial counterparts (e.g., Serratia marcescens [21] ), Hypocrea chitinolytic preparations have a high ratio of exochitinase to endochitinase activity and almost exclusively release monomeric GlcNAc from chitin [22] , which is another advantageous aspect of chitin compared to cellulose. Nevertheless, this raw material has not been adequately used. Therefore, the basic premise of this study was to exploit the potential of a saprophytic fungus to degrade the cheap biowaste chitin to its monomer GlcNAc and to further metabolize this product to NeuNAc.
Engineering a NeuNAc synthesis pathway into Hypocrea
The biosynthesis of NeuNAc begins with the formation of N-acetylmannosamine (ManNAc) from GlcNAc or UDP-N-acetylglucosamine (UDP-GlcNAc). In mammals, ManNAc is then phosphorylated to give ManNAc-6phosphate (ManNAc-6P). The second step involves the condensation of either ManNAc (in bacteria) or Mac-NAc-6P (in mammals) with phosphoenolpyruvate (PEP) to give NeuNAc or NeuNAc-9P, respectively. In mammals, NeuNAc-9P is then dephosphorylated to generate NeuNAc (see Figure 1 ). Hypocrea naturally degrades chitin almost exclusively to GlcNAc [22] . Therefore, the challenge was to engineer a pathway to convert GlcNAc to NeuNAc via ManNAc, which would enable the use of Hypocrea as a whole-cell catalyst.
Lee and coworkers found that whole-cell extracts of several photobacteria could convert GlcNAc to ManNAc [13] . Among them, Anabaena sp. CH1 exhibited the highest GlcNAc 2-epimerase activity; consequently, they cloned and characterized a gene encoding GlcNAc 2-epimerase from Anabaena sp. CH1 (E.C. 5.1.3.8), which was used in the present study as a Hypocrea codon-optimized gene. For the second step (the condensation of ManNAc to NeuNAc), the currently used enzyme-catalyzed processes use a lyase, which requires an excess of pyruvate. Use of this incurs high downstream processing costs. Therefore, we used the NeuNAc synthase (EC 2.5.1.56) from Campylobacter jejuni [23] in the Hyprocrea process. This enzymatic step entails the use of PEP instead of pyruvate, which in the intended in vivo process is supplied by the fungus, thereby leading to an irreversible and more efficient reaction towards NeuNAc [24] . Moreover, the need for an excess of pyruvate becomes obsolete, and the resulting downstream process is significantly simplified. Similar to the GlcNAc 2-epimerase, the coding sequence for the NeuNAc synthase was codon-optimized for the usage in Hypocrea. The synthetic pathway is presented in Figure 1 . The complete nucleotide sequences for both genes encoding the recombinant enzymes, tbage and tneub, are shown in additional file 1.
As the ability of the fungus to metabolize NeuNAc is an important issue, a possible uptake of NeuNAc by H. jecorina was investigated. Therefore, the fungus was pre-grown on glycerol in liquid culture, and half of the mycelium was autoclaved and half of it was harvested. The dead and living mycelia were transferred to glycerol-containing medium to study growth conditions or to medium without a carbon source to study resting cell conditions. NeuNAc was added to both media, and cultures were incubated for 8 h. Supernatants from all conditions were analyzed after incubation for 0 and 8 h by HPLC after derivatization using 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB). Similar amounts of NeuNAc were present under all conditions regardless of whether the fungus was alive or dead ( Figure 2a ). This result indicates that NeuNAc uptake does not occur in H. jecorina. As a positive control experiment we did a similar experiment but instead of NeuNAc, GlcNAc was added to the media. As can be inferred from Figure 2b GlcNAc was completely consumed after eight hours under both growth and resting cell conditions when the mycelium was viable.
Recombinant Hypocrea strains were generated using protoplast transformation of H. jecorina QM9414. In the derived strains, the two Hypocrea codon-optimized genes (without GST-tag) were placed under the control of either the H. jecorina pyruvate kinase (pki) promoter, which is a strong constitutive promoter, or the H. jecorina xylanase 1 (xyn1) promoter, which is a strict shut-off system if an inducer (e.g. D-xylose) is missing. Such a system was used to avoid interference of the introduced recombinant pathway with cell wall biosynthesis and consequently, biomass formation. However, when comparing both promoter systems the strong pki promoter did not lead to decreased growth, diminished cell integrity or other adverse effects (data not shown). Therefore, we used strains in which both genes were under the control of the pki promoter for further studies as we observed a remarkably higher Neu-NAc formation.
Transcriptional analysis of the recombinant H. jecorina strains was done by RT-qPCR to compare expression of both inserted genes using sar1 (SAR/ARF-type small GTPase) as a stable reference gene [25] . Furthermore, the copy numbers of both genes was measured by qPCR of genomic DNA using pki as a reference, which in the native H. jecorina genome is present as a single copy gene. Based on these analyses a strain (termed PEC/PSC1) was chosen for further investigations because it showed the highest equal expression of both inserted genes. This was confirmed by the finding that this strain bears two copies of each recombinant gene in the genome. These data were also supported using Southern blot analysis (data not shown).
Both recombinant enzymes were heterologously expressed as glutathione S-transferase (GST) fusion proteins in E. coli; the affinity chromatography purified proteins were used to confirm that their enzymatic capability was not altered by the codon usage adaptation and to provide a positive control for the enzymatic assays later on.
To determine if the recombinant enzymes were functional, both GST fusion proteins were used in an enzymatic assay. The presence of GlcNAc and the formation of the intermediate product ManNAc and the final product NeuNAc were monitored using HPLC-MS analysis and results are presented in Figure 3 . Application of the GST-fusion proteins of both enzymes in the in vitro assay led to the formation of ManNAc and NeuNAc demonstrating that the synthetic genes are expressed as functional proteins (Figure 3a1 and 3b1) .
According to the GST-fusion proteins, cell-free extracts of the recombinant H. jecorina strain PEC/PSC1 were applied in the enzymatic assay. The formation of ManNAc ( Figure 3a2 ) and NeuNAc could be detected (Figure 3b2 ). This demonstrates that both enzymes are also fully functionally expressed in the recombinant H. jecorina strain PEC/PSC1. Neither ManNAc nor NeuNAc was detected using cell-free extracts from the parental strain in the assay (Figure 3a3 and 3b3) , indicating that these pathways are normally not active in Hypocrea.
To investigate the stability of NeuNAc in cell-free extracts of the recombinant strain, according cell-free extracts obtained from the cultivation in a bioreactor on chitin (vide infra) were spiked with NeuNAc and incubated for 24 h. As a control, a heat-inactivated cell-free extract was similarly treated. Using HPLC analysis after derivatization with DMB, similar amounts of NeuNAc were detected in both extract preparations (Figure 3c) , suggesting that components of the cell-free extract do not actively degrade NeuNAc. In addition, a similar amount of NeuNAc was measured in a NeuNAc-spiked cell-free extract of the recombinant strain that was not incubated, assuming that the 24-h incubation period at 30°C did not decrease the NeuNAc levels. As a final control, a cell-free extract without NeuNAc was also analyzed after a 24-h incubation period and, as expected, showed a lower amount of NeuNAc, which could only have resulted from its formation during the cultivation on chitin. In summary, we did not observe degradation of NeuNAc by H. jecorina. These data suggest that NeuNAc is not metabolized by the recombinant Hypocrea strain.
We next addressed whether the recombinant H. jecorina strain had the ability to produce NeuNAc in vivo. To test this, the strain was grown on GlcNAc in shake flasks and cultivated on colloidal chitin in a bioreactor. Data on the corresponding cultivation monitoring are provided in additional file 2. As a positive control, an enzyme assay using the GST fusion proteins was again performed and resulted in the detection of ManNAc using HPLC-MS analysis as shown in Figure 4a1 . Notably, the intermediate ManNAc was detected in the recombinant strain, regardless of the carbon source (Figure 4a2 und Figure  4a4 ), whereas the parental strain did not form ManNAc (Figure 4a3 ). In the parental strain, only the first metabolite, GlcNAc, was detected, and it was present because it was either directly used as a carbon source or formed by degradation of the biopolymer chitin due to the native chitinolytic activity of the fungus. The synthesis of the product NeuNAc was analyzed using HPLC-MS/MS analysis (Figure 4b) . As a positive control, the reaction products (ManNAc, NeuNAc) generated by the use of the GST fusion proteins in an enzymatic assay are shown (Figure 4b1) . Importantly, the recombinant H. jecorina strain formed NeuNAc using either carbon source, Figure 2 The analysis of possible metabolization of NeuNAc in H. jecorina. The parental strain was pre-cultured on glycerol, and half of the mycelia were autoclaved. Living (light grey) and dead (dark grey) mycelia were transferred to MA media containing glycerol or MA media that lacked a carbon source, and NeuNAc (a) or GlcNAc (b) (as positive control) was added to both media. Samples were collected at 0 and 8 h. For NeuNAc analysis supernatants were derivatized using DMB befor analyses using HPLC. The presented values are the means of two biological duplicates that were derivatized in duplicate. Error bars indicate the standard deviations. GlcNAc or chitin (Figure 4b2 and 4b4) , whereas in the parental strain no formation of NeuNAc was detected (Figure 4b3 ). This analysis allowed us to estimate that 13 μg NeuNAc per g mycelium (dry weight) was formed in the recombinant strain. Thus, on its own, this would not be a competitive production process, but it does demonstrate the possibility for engineering a saprophyte and using it as a whole-cell catalyst that expresses a bacterial enzyme cascade. This method has enormous potential considering its use of a cheap starting material and the relatively simple, inexpensive cultivation of a fungus.
Taken together, we successfully engineered Hypocrea in a way that this fungus now produces NeuNAc from the biopolymer chitin by employing its natural saprophytic activity in combination with the introduction of a bacterial enzyme cascade. Because human society will face severe bottlenecks in the supply of energy and in obtaining certain raw materials in the upcoming years, we hope that this study will highlight the potential advantages of biopolymers, such as chitin, and stimulate their efficient usage. Furthermore, we anticipate that such strategies will support efforts to create sustainable production processes.
The parental strain H. jecorina (T. reesei [26] ) QM9414 (ATCC 26921) was maintained on malt extract (MEX) agar.
Mycelia for the enzymatic assay were cultivated in 3% (w/v) MEX medium using 10 8 conidia/L at 30°C.
Cultivation of H. jecorina on colloidal chitin was performed in a bench top bioreactor (Bioengineering, Wald, Switzerland) as previously described [27] . Briefly, 500 mL Mandels-Andreotti (MA) [28] medium containing 1% (w/v) colloidal chitin [29] , 0.5% GlcNAc, and 0.1% (w/v) bacto peptone (Difco, Detroit, US) was inoculated with 10 8 conidia/L. Some drops glanapon (Becker, Wien, Austria) were added to the medium to avoid excessive foam formation. Cultivation was performed at 30°C temperature, pH 5, 0.3 vvm aeration rate, and 500 rpm agitation rate for 96 h. Each sample drawing was followed by a microscopic analysis for infection control. Culture supernatant and mycelia were separated by filtration through GF/F glass microfiber filters (Whatman, Brentford, UK). All strains (parental, recombinant) showed similar growth on rich media as well as MA medium.
The synthetic gene tbage (for sequence see additional file 1) is based on the protein sequence of Anabaena sp. CH1 GlcNAc-2-epimerase (GenBank: ABG57042) and was reverse translated into a nucleotide sequence using the GeneOptimizer ® software (Geneart, Regensburg, Germany). The codon usage was optimized for H. jecorina (http://www.kazusa.or.jp/codon). The synthetic gene tneub (for sequence see additional file 1) was similarly obtained based on the protein sequence from Campylobacter jejuni NCTC11168 NeuNAc synthase (http://old.genedb.org/ genedb/cjejuni/index.jsp, Cj1141).
The synthetic genes tbage and tneub were excised from the production plasmid using XbaI/NsiI digestion and inserted into pRLM ex 30 [30] to generate the plasmids pMS-PEC and pMS-PSC.
For the construction of pGEX-epi and pGEX-syn, the oligonucleotides GEXfw and GEXrev (Table 1) were used to introduce an XbaI and NsiI site into plasmid pGEX4T-2 (GE Healthcare, Chalfont St Giles, UK), yielding pGEX-MS. tbage and tneub were inserted into pGEX-MS via XbaI/NsiI digestion to yield the plasmids pGEX-epi and pGEX-syn.
The protoplast transformation of H. jecorina was performed as described previously [31] . The plasmid pHylox2 (2 μg) [32] , which confers hygromycin B resistance [30] , and 4 μg of each plasmid pMS-PEC and pMS-PSC were co-transformed into the fungal genome.
Fungal genomic DNA was isolated as described previously [31] . Southern hybridization and detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit II following the manufacturer's instructions (Roche, Basel, Switzerland).
RNA extraction, cDNA synthesis and qPCR analysis were performed as described elsewhere [25] . Primer sequences are given in Table 1 .
GST fusion proteins of GlcNAc-2-epimerase and Neu-NAc synthase were generated using plasmids pGEX-epi and pGEX-syn in E. coli BL21 (DE3). Purification of the proteins was performed using GSTrap™FF (GE Healthcare) according to standard procedures. 
Harvested mycelia were ground into fine powder and resuspended in 0.1 M Bicine buffer (pH 8) containing protease inhibitors (2 μM leupeptin, 1 μM pepstatin A, and 10 μM PMSF) (0.3 g mycelia/mL). The suspension was sonicated using a Sonifier ® 250 Cell Disruptor (Branson, Danbury, US) (power 40%, duty cycle 50%, power 20 sec, 40 sec pause, 10 cycles). Insoluble compounds were separated using centrifugation (10 min, 13000 g, 4°C). Enzymatic analysis was performed according to a previously described modified protocol [33] . The assay was performed in a total volume of 100 μL containing 10 mM GlcNAc, 10 mM PEP, 12.5 mM MnCl 2 , 100 mM Bicine buffer (pH 8) and 40 μL cell-free extract. Reactions were incubated for 60 min at 37°C, terminated at 85°C for 10 min and analyzed using HPLC. As a positive control, 5 μL of both GST fusion proteins were applied in place of the cell-free extracts.
The stability of NeuNAc in the cell-free extract was determined by adding NeuNAc (150 μM) and incubating for 24 h at 30°C. After derivatization with DMB [34] , the NeuNAc quantity was measured using HPLC.
Harvested H. jecorina mycelia were ground into fine powder and resuspended in water (0.3 g mycelia/mL). The suspension was sonicated using a Sonifier ® 250 Cell Disruptor (Branson) (power 70%, duty cycle 50%, power for 1 min, 1 min pause, 3 cycles). Insoluble compounds were separated using centrifugation (10 min, 13000 g, 4°C), and the supernatant was analyzed using HPLC-MS/MS.
H. jecorina mycelia were pre-grown on MA containing 1% glycerol, transferred to MA medium containing 1% glycerol or no carbon source, spiked with 30 μM Neu-NAc or GlcNAc, respectively, and incubated for 8 h at 30°C. Autoclaved mycelia served as a negative control. After derivatization with DMB [34] , the NeuNAc quantity was measured using HPLC.
NeuNAc, ManNAc and GlcNAc formation was measured using LC-MS (IT-TOF-MS) (Shimadzu, Kyoto, Japan) with a Rezex™ RHM-Monosaccharide H + -column (8%, 300 × 7.8 mm) (Phenomenex, Torrance, USA). The mobile phase consisted of water with 0.1% (v/v) trifluoroacetic acid, the flow was 0.6 mL/min, the column temperature was 80°C, and the injected volume was 10 μL. MS detection was performed in ESI+ mode, covering a scan range of 60-600 amu. The retention times were determined using pure standard substances. The identity of NeuNAc was confirmed by both, chromatographic retention time and mass spectral signal, which are very well matched by authentic standards of NeuNAc. The better the mass accuracy obtained from exact mass determination by HR-MS, the lower is the number of possible isobaric candidates (e.g. [35] ). In this case the mass accuracy is better than 2 ppm, leading to the number of candidates reduced to less than 10, with an even further reduction in the number of potential candidates because the isotopic pattern is also taken into account (what the software of the used IT-TOF-MS instrument does automatically).
DMB derivatives of NeuNAc were separated on a Kinetex RP C18 (Phenomenex) at 0.75 mL/min with a 40°C column temperature and a mobile phase of water: methanol:trifluoroacetic acid (74.25:25:0.75). A Shimadzu RF-20AXS fluorescence detector (excitation 373 nm, emission 448 nm) was used for detection.
Additional file 1: Coding sequences of the synthetic genes tbage and tneub. Coding sequences of the synthetic genes tbage and tneub. The sequences are provided in FASTA format. The XbaI site is underlined, and the NsiI site is double-underlined. The start codon ATG and the stop codon TAA are presented in bold letters.  mRNA pseudoknot structures can act as ribosomal roadblocks Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses. The reading frame of the vast majority of mRNAs is determined by the start codon after which the downstream cistron is translated in the same frame. Maintenance of the reading frame occurs without further signals to the ribosome. However, examples of genes containing information for programmed frameshifts can be found in most organisms, or in some of their IS sequences, transposable elements, retroelement-derived sequences or viruses. The sequence-information needed for programmed ribosomal frameshift varies and both +1 and À1 frameshifts can be induced (1) (2) (3) .
Here, we focus on the frameshifting signal found in several viruses (1) , including infectious bronchitis virus (IBV) and SARS-CoV. The signal leads to programmed ribosomal À1 frameshift, whereby multiple proteins are produced from a single polycistronic messenger RNA (mRNA) (4, 5) . The frameshift efficiency, i.e. the fraction of ribosomes, which change reading frame, is important to ensure a correct stoichiometric relationship between the different products of translation. It has been shown that altered frameshift efficiency has detrimental effects on the proliferation of HIV-I and the yeast L-A viruses (6, 7) . In order to induce À1 frameshift, these viruses rely on three physical features on the mRNA: a heptanucleotide sequence, a spacer and a downstream structure (8) . The heptanucleotide sequence, called the slippery sequence, is where the À1 frameshift occurs and typically has the following sequence: X XXY YYZ, where X, Y and Z denote nucleotide species and spaces indicate initial reading frame. The spacer is a stretch of 6-9 nt positioning the ribosome correctly at the slippery site when encountering the downstream structure. The downstream structure is most often found to be a pseudoknot. The pseudoknot structure probably functions as a physical barrier deforming upon approach of the translating ribosome (9) , thereby assisting the frameshifting process; however, geometry and surface charge of the structure may also play a role for the frameshifting (10) .
In bacteria and yeast, programmed frameshift signals can have rather different elements, as, e.g. the upstream Shine-Dalgarno binding element in the autoregulatory RF2 gene frameshift site first described in Escherichia coli (11) or the different pattern of the +1 frameshift stimulating heptanucleotide sequences present in Saccharomyces Ty elements (2) . However, many frameshift signals deviate little from those described for the virus-derived system used here and many signals are of such general character that ribosomes from different kingdoms of life will respond to them by shifting frame (12) . This happens not always with the same efficiency as in the original organism (12, 13) and there are even examples found where a frameshift element can direct the ribosomes into À2 or +1 frameshift depending on the test organism (14) . Here, we challenged E. coli ribosomes by constructing artificial frameshifting signals containing pseudoknot-like structures with strong stems. Using a refined frameshift assay, involving two-dimensional (2D) gel electrophoresis of pulse labelled proteins, we show that a significant amount of frameshifted ribosomes permanently stall within the strongest pseudoknots which therefore efficiently act as roadblocks.
The small ribosomal subunits have been shown to be sensitive towards mRNA secondary structure in the process of translation initiation and mRNA structures can exclude initiation both in eukaryotes during the scanning process (15) and in prokaryotes for binding between the mRNA and the 3 0 -end of 16S RNA (16) . The fully assembled and translating 70S or 80S ribosomes seem to be more robust. It is, however, broadly accepted that mRNA secondary structures can function as obstacles to translating ribosomes (17, 18) although examples exists of large secondary structures in mRNA that are translated without any ribosomal delay (19) . Nevertheless, there is compelling evidence from in vitro experiments showing that ribosomes may pause upstream to such structures, most pronounced if the structures form pseudoknots (20) (21) (22) . Possibly the lack of rotational freedom in the helix of stem 1, due to the pairing in stem 2, makes pseudoknot structures harder to 'unzip' by the ribosome than simple stem-loop structures (23) . This may explain why pseudoknots can pause ribosomes. Examples from nature show the existence of diverse peptide sequences, often present in regulatory circuits, which will stall ribosomes (24), but to our knowledge, a permanent halt of ribosomes caused by mRNA structures has not been shown previously.
Recent single molecule investigations suggest that the mechanical strength of pseudoknots correlate with the ability of the pseudoknot to stimulate frameshift (25) (26) (27) , at least in a certain interval. However, the calculated Gibbs free energy does not always correlate with frameshift efficiency. Not only the strength of the stems, but also the interaction between the loop and the stems might be of importance for the ability to induce frameshift and for the overall mechanical strength and brittleness of the structure. If the pseudoknot becomes too strong the ribosome, frameshifted or not, might not be able to open it and continue translation, whereby the pseudoknot acts as a roadblock. Often in literature (25) (26) (27) (28) (29) (30) (31) frameshifting assays were performed on constructs exhibiting the common feature that the stop codon for the normal reading frame was located at the entrance of the pseudoknot (or inside the pseudoknot) and the stop codon for the successful À1 frameshift was located downstream of the pseudoknot. In most frameshifting assays, the amount of frameshifting is determined by quantifying the amount of full-length frameshifted versus non-frameshifted products. However, for this to be a correct measure, the frameshifted ribosome must continue translation through the pseudoknot and beyond to the À1 frameshifted stop codon. If the À1 frameshifted ribosome permanently stalls inside the pseudoknot, it would falsely be interpreted as if the ribosome did not frameshift. Therefore, there is a serious pitfall in the classical methods which renders the amount of frameshifted ribosomes to be non-correctly determined, i.e. be underestimated, potentially leading to false hypotheses regarding the physical mechanism of frameshifting.
The observation that strong pseudoknot-like structures can stop translation lead to the hypothesis that the largest amount of frameshifted product will be produced if the pseudoknot is mechanically strong but without a significant roadblocking effect. Most likely, this is exactly the balance exhibited by naturally occurring viral pseudoknots.
Escherichia coli strain MAS90 [E. coli K-12, recA1 D(pro-lac) thi ara F 0 : lacI q1 lacZ::Tn5 proAB + ]. Liquid cultures were grown in minimal MOPS media (32) using glycerol as carbon source. Cultures were incubated with shaking at 37 C for at least 10 generations in the log phase prior to being used in frameshift assays.
Pseudoknots were designed using custom-made software, which ensued that the codon usage was appropriate for expression in E. coli and that the sequences were likely to fold into the correct structure as determined by pknotsRG (33) . Hence, the resulting sequences are artificial pseudoknot-like structures and there is always a risk that the structure does not fold as anticipated. The selected sequences were synthesized by GeneScript and were subsequently inserted into plasmid OFX302 [containing slippery sequence, spacer and pseudoknot (25) ] between HindIII and ApaI restriction sites.
The in vivo frameshift assays were performed as described previously (25) . Briefly, 1 ml of an exponentially growing culture was induced with Isopropyl b-D-Thiogalactopyranoside (IPTG) to a final concentration of 1 mM at an optical density of 0.4-0.7 measured at 436 nm (OD 436 ). After induction for 15 min, the culture was pulse-labelled with $10 mCi L-[ 35 S]-methionine for 20 s and chased with 100 mg L-methionine for 2 min before being transferred to 25 ml of chloramphenicol (100 mg/ml) on ice. The cells were harvested by centrifugation and proteins were boiled in SDS buffer and separated by 9% SDS-PAGE. The gel was dried and placed on a phosphor imager screen (Molecular Dynamics) and left to expose for 1-3 days. Relative amount of protein of the relevant polypeptides was quantified using ImageQuant software and the frameshift efficiency (e) was determined as follows:
where V FS is the relative radioactivity in the frameshift product, n met,FS is the number of methionines in the frameshift product, V STOP is the relative radioactivity in the in-frame stop product and n met,STOP is the number of methionines in the in-frame stop product.
Two-dimensional SDS-gels were performed as described (34) with a few modifications (35) using samples from the frameshift assay described above. The frameshift efficiency was determined as described for the frameshift assay above, although polygonal shapes were used to encircle the polypeptides of interest and quantify the relative amount of radioactivity in them.
Polypeptides originating from stalled ribosomes were found as radioactive polypeptides with appropriate isoelectric point and molecular weight appearing on gels when the translated transcript contained a pseudoknot. These polypeptides were absent when a transcript without a pseudoknot was translated. The weakest stalled protein spots were difficult to distinguish from spots originating from endogenous gene expression on these gels (compare to the 0 construct in Supplementary Figure S5 ) and their determination is connected with some uncertainty. The statistical analysis used to compare the stalling efficiency between pseudoknot 22/6a and 22/6b was an unpaired one-tailed Student's t-test with a significance level of 0.05.
Total RNA was extracted from 1.5 ml culture samples by the 'Hot-phenol' extraction method and separated according to size by electrophoresis on 1.2% agarose, 6% formaldehyde gels in recirculating 1xMOPS buffer. Capillary blots were performed onto Hybond-N + (Perkin Elmer) membranes, and the RNA was crosslinked to the membrane by 0.12 J/cm2 UV light in a Stratalinker 1800. Riboprobes covering mRNA sequences as described in Figure 4 were made by T7 RNA polymerase transcripts from the pMAS39 'downstream' template (19) or from templates made by PCR where one primer included 'hanging out' T7 promoter sequences (gene10 and lacZ 5 0 probes). The riboprobes were synthesized in the presence of 32-P-UTP and the final specific activity was about 40 Ci/mmol of nucleotide. Hybridization and stripping of membranes were performed following standard protocols (Amersham, Hybond-N+ booklet, 2006). The membranes were wrapped in Saran wrap and placed on a phosphor imager screen (Molecular Dynamics) and left to expose over night. Signals were visualized using ImageQuant software.
We created a series of plasmids containing different pseudoknots and where the in-frame stop codon was placed either immediately upstream ('Upstream stop') or $150 nt downstream ('Downstream stop') from the pseudoknot ( Figure 1A ). The 'Upstream stop' constructs had an in-frame stop codon in the spacer between the slippery sequence and the pseudoknot. This caused non-frameshifted ribosomes to produce a 28 kDa polypeptide (gene10 from phage T7) while ribosomes undergoing a À1 frameshift continued through the pseudoknot and into lacZ producing a 148 kDa fusion protein of the T7 gene10 and lacZ sequences. In the 'Downstream Stop' constructs we replaced the UAA stop codon immediately upstream from the pseudoknot with a lysine codon (AAA). This change caused non-frameshifting ribosomes to continue through the pseudoknot and terminate at a downstream UGA codon producing a 37 kDa polypeptide. The pseudoknot constructs based on the plasmid OFX302 (25) are detailed in Figure 1B . We systematically increased the length of stem 1 and in pseudoknot 22/6a through 22/6c, we exchanged GC with UA base pairs, thus, gradually decreasing the stability of stem 1.
Often, the number of ribosomes which undergo À1 frameshift has been determined from constructs such as our 'Upstream stop' constructs, by separating radioactively labelled proteins by SDS-PAGE and quantifying the relative amount of protein in each of the two polypeptides (28 versus 148 kDa). Given the limited resolution of SDS-PAGE, it is, however, impossible to clearly differentiate between polypeptides produced by ribosomes that terminate at the in-frame UAA stop codon and ribosomes that undergo À1 frameshift but stall within the pseudoknot. In order to overcome this problem, we invoked 2D SDS-PAGE (34) whereby polypeptides were separated not only by molecular weight but also by their isoelectric point (pI).
While polypeptides originating from ribosomes stalled in the pseudoknot varied only slightly in molecular weight, they varied significantly in their pI. Based on the 'Downstream Stop' construct, we calculated a theoretical 2D SDS-PAGE assay of a growing polypeptide as consecutive codons are translated (shown in Figure 2A ). At around 28 kDa, the trace splits into two, the triangles denote the non-frameshifted product and the circles denote the À1 frameshifted product. Red symbols denote codons inside the pseudoknot. Experimental data originating from the 'Downstream Stop' construct is shown in Figure 2B , the theoretically expected features are indeed present, e.g. both the non-frameshifted (DS-stop) and the À1 frameshifted (FS) products are visible. The heat shock proteins GroEL and DnaK serve as landmarks on the gel. Interestingly, a series of polypeptides originating from ribosomes stalled inside the pseudoknot appeared (inside dashed red line). For comparison, a standard SDS-PAGE of the same sample is shown in Figure 2C , here, the second level of information (isoelectric point) is lost and the relative blurry bands are difficult to interpret.
The results shown in Figure 2 revealed that a 1D SDS-PAGE assay could not firmly identify polypeptides originating from a À1 frameshifted ribosome stalled in the pseudoknot from the non-frameshifted product in a 'Downstream Stop' construct. In order to quantify the amount of À1 frameshifted ribosomes stalled inside the pseudoknot, we performed a 2D SDS-PAGE separation of the radioactively labelled proteins originating from the 'Upstream Stop' construct (Supplementary Figures S4 and  S5) , which is the type of construct most commonly used throughout literature. The advantage of a 2D-gel analysis is that all the unfinished protein chains with different lengths concentrate in a common spot when they have the same pI. This made it possible to identify randomly stalled translation products inside the pseudoknot sequence and we quantified the amount of radioactivity in all identified additional spots. This produced a conservative estimate of the amount of stalled translations.
The result of quantifying the fraction of in vivo À1 frameshifted ribosomes, both those which made it all the way to the lacZ stop codon (gene10/lacZ fusion) and those which stalled inside the pseudoknot, is shown in Figure 3A . The hatched bars denote the À1 frameshift efficiency taking into account only the end product of À1 frameshift (148 kDa gene10/lacZ fusion). This frameshift efficiency was calculated as (intensity of FS product)/ (intensity of non-FS product+intensity of FS product). The filled bars denote the À1 frameshift efficiency when both the end product (148 kDa gene10/lacZ fusion) and the products originating from stalled ribosomes are taken into account. This frameshift efficiency was calculated as (intensity of FS product+intensity of stalled product)/(intensity of non-FS product+intensity of FS product+intensity of stalled product).
In addition to the six artificial pseudoknot-like structures, we also analysed two earlier investigated pseudoknots PK400 and PK401 (25), with over-all A B Figure 1 . Frameshift assay and pseudoknot structures. (A) All plasmid constructs contain an IPTG inducible promoter in front of T7 gene10 (light grey), a complete frameshift signal, and lacZ (dark grey). The frame shift stimulating pseudoknot-like structure is inserted downstream of gene10. Immediately, downstream from the pseudoknot lacZ is inserted in the À1 reading frame relative to gene10. In the 'Upstream Stop' construct the non-frameshifting ribosomes will translate gene10 and terminate at a UAA stop codon in the spacer sequence and produce a 28 kDa polypeptide. Ribosomes undergoing À1 frameshift at the slippery sequence translate lacZ thus producing $148 kDa polypeptide. In the 'Downstream Stop' construct the UAA stop codon is replaced by an AAA lysine codon thus resulting in $37 kDa polypeptide being produced by non-frameshifting ribosomes which terminate at an UGA stop codon downstream from the pseudoknot. (B) Sequence and structure of the inserted pseudoknots, the slippery sequence and the spacer. In pseudoknot, 10/6, 22/6a, 22/6b and 22/6c the first base in loop 2 has been removed in order to maintain the downstream reading frame (underlined). The boxed insert in panel B shows the structure and sequence of previously described constructs (25) .
structures more similar to naturally occurring pseudoknots (Figure 1 insert), inspired from structures in the infectious bronchitis virus (22, 28, 30) . The pseudoknot structures in this type of virus are selected for their effects on vertebrate ribosomes, but the stem1 length variations were found to yield approximately the same relative stimulatory effect in E. coli (25) and suggest that stem1 strength is equally important for stimulating bacterial ribosomes to frameshift.
All pseudoknots investigated stalled some fraction of the frameshifted ribosomes, however, significantly more ribosomes stalled in the artificial pseudoknots than in those resembling naturally occurring pseudoknots (PK400 and PK401).
To quantify the amount of ribosomes stalling within a pseudoknot in vivo we calculated the ratio of (stalled+non-stalled frameshifted ribosomes) to (non-stalled frameshifted ribosomes), the result is shown in Figure 3B . For the IBV inspired pseudoknots, this ratio was close to 1 signifying that essentially no ribosomes stalled. However, the ratio was significantly larger than 1 for the more artificial pseudoknots which acted as roadblocks for a large amount of frameshifted ribosomes. The length of stem 1 did not significantly influence on the amount of frameshifted or stalled ribosomes. Interestingly, within pseudoknots with the same overall structure (22/6a-c) 22/6a stalls a significantly higher fraction of frameshifted ribosomes than 22/6b (verified by Student's t-test, n = 4, a = 0.05, P = 0.012), which again stalls more than 22/6c. Hence, the ability to stall a ribosome correlated with the strength of the pseudoknot base pairs, the stronger the base pairs the more frameshifted ribosomes were stalled.
Earlier studies have shown that the insertion of sequences able to form mRNA secondary structures into a gene may cause the RNA polymerase to stall or invoke a target for endonucleolytic attacks (19) . Therefore, in our analysis of mRNA pseudoknot-stalled ribosomes, it was important to verify that there was no significant population of mRNAs that ended within the pseudoknot structure. If such truncated transcripts were abundant, it would be difficult to distinguish between protein products from ribosomes stalled within the pseudoknot and protein products originating from ribosomes ending translation at 'non-stop' mRNAs having their 3 0 -ends within the pseudoknot sequence. In the latter case translation would be terminated by tmRNA trans-translation thus rendering the protein products unstable due to the tmRNA-encoded tag (36) . In the following subsections 'Identification of transcripts from the T7gene10-PK-lacZ gene fusions', 'Messenger RNA stability' and 'Coupling between translation and transcription is required for full-length transcripts', we will show that the observed proteins did indeed originate from stalled ribosomes and that they were not caused by other effects.
Identification of transcripts from the T7gene10-PK-lacZ gene fusions. To identify the major class of transcripts from our pseudoknot containing constructs, we made a northern blot with RNA from all strains used to measure frameshift frequencies, which are those containing the upstream stop. We used three different probes hybridizing either upstream of the pseudoknot, immediately downstream of the pseudoknot or in the very end of the lacZ reading frame ( Figure 4A ). Figure 4B -D, there was an unspecific hybridization from all three probes to the 23S and 16S ribosomal RNAs. In E. coli, ribosomal RNA constitutes between 80% and 90% of total RNA depending on growth conditions and some cross-hybridization to these species is often seen in northern blots. Here, the uninduced culture in Figure 4B -D, lane '0 no IPTG', made it possible to estimate the unspecific probing to rRNA and the two bands were used as size markers on the blots.
Following induction with IPTG, all strains showed increased hybridization above the 23S RNA band compared to the uninduced control with all three probes. The so-called 0 construct was described in reference (25) , and contains a slippery sequence and the UAA stop codon but no pseudoknot-like structure. In all strains, except the one with the 0 construct, there were a distinct band (Fl) representing the expected full-length transcript. The full-length transcript reached from transcription start to the stem-loop structure downstream of the 3 0 -end of the lacZ open reading frame ('hp' in Figure 4A ). This mRNA stem-loop structure has been shown to stabilize the lacZ transcript by reducing 3 0 -end exonucleolytic attacks (37) . The core plasmid contained no distinct transcription termination signal after the lacZ gene, and accordingly we found transcripts that exceeded far beyond the full-length Fl band ( Figure 4B-D) .
In the beginning of lacZ, $200 nt into the open reading frame, there is a site, called 'pt' in Figure 4A , where the RNA polymerase is caused to terminate if there is inefficient translation initiation of the lacZ gene (38) . In the 0 construct there is no pseudoknot to stimulate frameshift at the slippery site. Therefore, virtually no ribosomes were expected to follow the RNA polymerase from gene 10 into the lacZ part of our gene fusion. As expected, Figure 4B and C, lane '0' shows a prominent band ('SP' for stop polymerase) corresponding in size and probe-ability to this premature termination product. Also, corresponding low amounts of high molecular weight transcripts are detected for this construct. All the other constructs shown in Figure 4 contained frameshift stimulating pseudoknots and a inspection of the northern blot showed that the 'SP' bands probed with both gene10 and lacZ5 0 sequences were present in sizes which correspond to the sizes of the pseudoknots inserted.
Messenger RNA stability. The wild type lacZ mRNA half-life is close to the average mRNA half-life in E. coli (120 s) and transcription takes close to 80 s due to the length of the lacZ gene (three times longer than the average gene). Therefore, a northern blot of wild type lacZ mRNA under steady state transcription will always include a lot of unfinished native transcripts, as well as mRNAs under degradation. Here, our gene10-lacZ fusion was even longer and transcription should take $120 s. Accordingly, all induced strains included in Figure 4 show a distinct smear of mRNA fragments recognized by all three probes. In order to examine the half-life of our artificial transcripts, we made experiments where transcription from the P tac promotor was stopped due to removal of the inducer ( Figure 5 ). Two minutes after IPTG removal, any remaining smear should originate from mRNA degradation because most of the RNA polymerase should have reached the end of the gene fusion at this time.
From the experiment, shown in Figure 5 , it is evident that both the 'Fl' and the 'SP' mRNA fragments had a half-life close to the average 2 min E. coli mRNA half-life. In addition, both the pseudoknot containing constructs (10/6 and 22/6a) revealed the existence of a short mRNA fragment that was recognized only by the gene10 probe but not the lacZ5 0 and 3 0 probes (indicated by 'asterisks' in Figure 5 ). This fragment includes the transcription start in the 5 0 -end and the pseudoknot in the 3 0 -end. We suggest that the pseudoknot acts as an exonuclease barrier like the natural stem-loop structure in the 3 0 -end of the wild type lacZ transcript (37) and thereby induces a degradation intermediate of a distinct length with increased half life compared to unstructured mRNA sequences like those from construct 0. Alternatively, but not mutually exclusive, a pseudoknot acts like a rho-independent termination signal to the RNA polymerase. However, the sequences were not followed by a row of uridine residues, which would be necessary to make a stem-loop structure into a functional transcription terminator.
Coupling between translation and transcription is required for full-length transcripts. The final test of our model for the transcription pattern in our artificial gene fusion was to establish translational coupling beyond the slippery sequence and into the polar termination site (SP) in lacZ. By changing the upstream stop codon between the slippery site and the pseudoknot region into a sense codon ribosomes should, frameshifted or not, follow the RNA polymerase into the beginning of the lacZ sequence.
The 22/6a and the 0 constructs are the two constructs with the lowest frequency of frameshifting. Therefore, they have the least ribosome traffic into the lacZ sequence. Alteration of the UAA stop codon into a lysine AAA codon in the spacer between the slippery sequence and the pseudoknot changed the pattern of transcripts immensely. These two downstream stop variants ('DS. stop' in Figure 6 ), which did not contain a stop codon upstream from the structure, expressed significantly more full-length ('Fl') transcript and only insignificant amounts of premature transcription stop fragment ('SP') compared to their sister constructs containing the UAA stop codon upstream from the structure ( Figure 6 ). Our control construct, PK401, which stimulated 14% frameshift, showed no premature transcription stop fragment ('SP') and therefore no change in transcription pattern was observed as a consequence of removing the upstream UAA stop codon ( Figure 6 ) thus confirming that the major effect causing the 'SP' fragment is polarity in the lacZ gene and not transcription termination caused by the pseudoknot sequences. Also, the very short band marked by 'asterisks' that appeared from the 22/6a construct was not present in the 'DS. stop' variant ( Figure 6 ). This exclude this mRNA fragment to be causal for the appearance of stalled protein products, because 22/6a ('DS. stop') is the construct that caused the highest frequency of stalling (compare Figure 2 and Supplementary Figure S5 ).
Our conclusion is that the stable proteins observed from within the pseudoknot structures (Figure 2 , Supplementary Figure S1 , S2, S4 and S5) were products from stalled ribosomes. The stalling of the ribosomes was directly caused by the tertiary structure and not by some secondary effect, as, e.g. stop codon-less mRNA fragments ending within the structure sequences.
The structures analysed in this study are artificial and were designed to fold into pseudoknot-like structures with a gradually increasing mechanical strength. The mechanical strength was adjusted by changing the base pairs of the two stems, which seems to be a reasonable way of crudely varying the mechanical strength, as the energy involved in base pairing is higher than the energies involved in, e.g. the electrostatic interaction of the loop with the stems. It is, however, likely that the loop-stem interaction, surface charges or other players than just mechanical strength influence frameshift stimulating effect of mRNA structures. As there is a consensus in recent literature that pseudoknot mechanical strength correlates with frameshifting efficiency (23) (24) (25) , it was intriguing that the amount of frameshifted product was reduced by the stronger pseudoknot 22/6a compared to the weaker 22/ 6b or c ( Figure 3A ). This proved to be caused by stalling of a significant amount of frameshifted ribosomes by the strong pseudoknots ( Figure 3B ). Future studies will show whether significant stalling can also be caused by naturally occurring pseudoknots.
Quantitative northern blot analysis was used to examine whether the observed translation products ending within the pseudoknot structure arose from fragments of mRNA produced either by low RNA-polymerase processivity or specific endonucleolytic attacks by RNases at the pseudoknot sequences. No evidence was found of a specific population of transcripts that could explain the amounts of protein products attributed to originate from pseudoknot-stalled ribosomes. Also, our protein-stability assay showed that the translational products from the stalled ribosomes were stable for at least 80 min (Supplementary Figure S1) , thus indicating that the stalled ribosomes are not rescued by tmRNA and that the stalled proteins do not originate from truncated mRNA.
We also checked whether the protein products from within the pseudoknot structure could arise from very slow rather than permanently stalled ribosomes. A pulse chase experiment (Supplementary Figure S2 ) revealed that within 16 min there was no sign of a redistribution of label between the stalled spots and the stop codon-terminated downstream stop product, thus proving the possibility of very slow ribosomes to be unlikely. It is possible that the newly discovered ribosome rescue factor, ArfA (39) could be active at pseudoknot-stalled ribosomes and that nascent proteins would be more stable than if saved by tmRNA. However, as can be seen in Supplementary Figure S3 , the growth of strains expressing pseudoknot 22/6a was severely affected by induction and showed a decrease in growth rate correlating to the amount of stall product observed. Because ribosomes are limiting in growing cells (40) , the sequestration of ribosomes by engagement in induced overexpression of a gene from a plasmid will often cause a strain to grow slower than the uninduced counterpart. The enhanced reduction in growth rate upon induction of 22/6a compared to the 0 construct (Supplementary Figure S3 ) could indicate that stalled ribosomes were not rescued at a sufficiently high rate and we suggest that either the ribosomal rescue systems were titrated by the large amount of mRNA induced from the plasmid alleles, or alternatively, that no rescue is possible for pseudoknot-stalled ribosomes.
Our results are in agreement with the observation that the amount of protein produced from an mRNA can be reduced when a pseudoknot is located upstream (29) . Also, they provide a possible explanation for the reduction in frameshift efficiency observed by, e.g. Napthine et al. (30) when increasing the thermodynamic stability of stem 1 above a certain threshold. This apparent reduction in frameshift efficiency (observed by 1D SDS-PAGE) could be caused by the fact that a significant fraction of the 'frameshifted' ribosomes permanently stalled within the pseudoknot.
We propose that pseudoknot induced frameshifting efficiency can be viewed as a balance between two competing effects (as visualized in Figure 7) , the mechanically stronger the pseudoknot, the larger the frameshifting efficiency (25) (26) (27) , however, the stronger the pseudoknot the larger the likelihood of stalling the frameshifted ribosome, thus preventing the translation of full-length frameshift product. Possibly, evolution optimized viral pseudoknots to balance these two effects. Hence, in measurements of frameshifting efficiency it is important to take into account the roadblocking effect of mRNA pseudoknots. Figure 7 . Model of frameshifting efficiency. Increasing the strength of a pseudoknot causes the pseudoknot to induce frameshifting at a higher frequency. However, the stronger the pseudoknot the larger the likelihood that it will act as a roadblock for the ribosome, reducing the amount of frameshifted product produced. The optimal frameshifting efficiency is achieved by balancing the two contributions. Secretory phospholipase A2 pathway in various types of lung injury in neonates and infants: a multicentre translational study BACKGROUND: Secretory phospholipase A2 (sPLA2) is a group of enzymes involved in lung tissue inflammation and surfactant catabolism. sPLA2 plays a role in adults affected by acute lung injury and seems a promising therapeutic target. Preliminary data allow foreseeing the importance of such enzyme in some critical respiratory diseases in neonates and infants, as well. Our study aim is to clarify the role of sPLA2 and its modulators in the pathogenesis and clinical severity of hyaline membrane disease, infection related respiratory failure, meconium aspiration syndrome and acute respiratory distress syndrome. sPLA2 genes will also be sequenced and possible genetic involvement will be analysed. METHODS/DESIGN: Multicentre, international, translational study, including several paediatric and neonatal intensive care units and one coordinating laboratory. Babies affected by the above mentioned conditions will be enrolled: broncho-alveolar lavage fluid, serum and whole blood will be obtained at definite time-points during the disease course. Several clinical, respiratory and outcome data will be recorded. Laboratory researchers who perform the bench part of the study will be blinded to the clinical data. DISCUSSION: This study, thanks to its multicenter design, will clarify the role(s) of sPLA2 and its pathway in these diseases: sPLA2 might be the crossroad between inflammation and surfactant dysfunction. This may represent a crucial target for new anti-inflammatory therapies but also a novel approach to protect surfactant or spare it, improving alveolar stability, lung mechanics and gas exchange. Phospholipase A2 biology Phospholipases A2 are a widely distributed group of enzymes primarily implicated in the turnover of membrane phospholipids and lipid digestion. They are also crucial for the inflammation pathways, as they are the first step for the production of eicosanoids and other inflammatory mediators [1, 2] . Secretory phospholipase A2 (sPLA2) is the low molecular, well conserved and secreted form of the enzyme. It is excreted into the alveoli mainly by macrophages and mast cells [1] [2] [3] . sPLA2 has a dual role, as it contributes to the inflammation pathway and it is also the main enzyme involved in the catabolism of surfactant [1, 2, 4] This complex proteo-lipid mixture is essential for the alveolar opening and the maintenance of an adequate gas exchange.
sPLA2 is well known to be involved in lung inflammation and surfactant degradation based on animal and human studies in adults [4] . Therefore, it is conceivable that sPLA2 through either its pro-inflammatory role or the surfactant catabolism, might be involved in the pathogenesis of several critical respiratory diseases. Basic data have shown that both sPLA2 activity and expression are regulated by many factors including steroids, Clara Cell Secretory Protein (CCSP), Tumor Necrosis Factor-α (TNFα), Surfactant protein A (SP-A) and certain surfactant phospholipids, Interleukine-1 (IL-1) and some other cytokines [4] . Imbalance in the sPLA2 pathway due to different production of its modulators may account for increased surfactant degradation or lung tissue inflammation. Schematic representation of sPLA2 pathway is presented in Figure 1 : the possible roles of the enzyme in the pathogenesis of acute respiratory distress syndrome (ARDS), infant respiratory distress syndrome (iRDS), broncho-pulmonary dysplasia (BPD), infection related respiratory failure (IRRF) and meconium aspiration syndrome (MAS) are also illustrated. Data about the sPLA2 Figure 1 Possible involvements of sPLA2 pathway in the pathophysiology of critical respiratory diseases in infants. Full lines with arrows and hatched lines with squares indicate stimulatory and inhibitory actions on the enzyme activity and expression, respectively. Bold arrows show the direct consequences of the enzymatic activity in different diseases. ARDS: acute respiratory distress syndrome; BPD: bronchopulmonary dysplasia; iRDS: infants' respiratory distress syndrome; IRRF: infection related respiratory failure; MAS: meconium aspiration syndrome; sPLA2: secretory phospholipase A2; CCSP: clara cell secretory protein; IL-1β: interleukine-1 β; SP-A: surfactant protein-A; TNFα: tumor necrosis factor-α; MV: mechanical ventilation. role in each of the above-mentioned diseases are described in the following paragraphs.
A wide body of literature suggests a role for sPLA2 in the development of ARDS and acute lung injury (ALI), its milder form. sPLA2 interferes with the surfactant activity and so reduces compliance [4] [5] [6] . sPLA2 starts a vicious cycle in which it damages the surfactant; since some surfactant components have their own inhibitory effect on sPLA2 activity and expression [7, 8] , the sPLA2-induced surfactant damage reduces this inhibition and thus the enzyme is able to further catabolize the surfactant phospholipids [4] . Thus, sPLA2 facilitates the action of other injurious agents against the lung epithelium, leading to further surfactant damage, alveolar collapse and respiratory impairment [4] . This process is also linked to the lung tissue inflammation, since TNFα and some other pro-inflammatory cytokines are strong sPLA2 inductors throughout the regulation of NFkB nuclear transcription factor [4, 7, 8] . Moreover, sPLA2 itself starts the inflammatory cascade, since it is the first step in the biochemical pathway leading to the production of arachidonic acid derivatives [9] . Inflammation may further inactivate surfactant, contributing to the above-described vicious cycle [4] [5] [6] . sPLA2 activity is raised in broncho-alveolar lavage fluid (BALF) in animal models of ARDS and in adult patients and this correlates with the clinical severity and mortality [4, 5, 8, 10] . We recently found raised enzyme levels in post-neonatal ARDS, similarly to the adult findings [11] . Moreover, respiratory syncytial virus (RSV) infection seems to cause a more severe ARDS because of the sPLA2 overexpression, triggered by the RSV itself [12] . Consistently, transgenic animals defective for the CCSP gene, experience higher inflammation and mucous production, when infected by RSV [13] .
In animal models, the administration of sPLA2 inhibitors reduced lung inflammation and improved both compliance and oxygenation, especially if the inhibitor is administered early during the injury development [8, 14] . Similarly, the inhibitor was able to reduce sPLA2 activity in BALF of patients with iRDS, IRRF, MAS and postneonatal ARDS [15] .
The importance of surfactant is well known in neonatal critical care. An inadequate surfactant production is the pivotal cause of hyaline membrane disease, also called infant respiratory distress syndrome (iRDS), the most frequent respiratory disease of preterm infants [16] . Although exogenous surfactant administration is curative in many preterm infants, long-term respiratory sequels are still a significant problem in this population, with 20% of the surviving preterm babies affected by BPD [17] . Moreover, the tiniest babies born at the limit of viability often require multiple surfactant administrations. Many of these very preterm deliveries are associated with infections and chorioamnionitis [18] . In these cases, inflammation lengthens the lung injury, decreasing the usefulness of exogenous surfactant and damaging the lung tissue [18] . In such situation, sPLA2 is likely to play a crucial role: we found increased sPLA2 levels in babies with iRDS comparing to normal term neonates [19] . sPLA2 was found to increase foetal neutrophil migration and so to enhance lung tissue inflammation [20] . Babies with higher sPLA2 activity are likely to be the ones needing repeated surfactant administrations and they are at higher risk for chronic lung disease.
Some authors recently tried to administer CCSP to preterm neonates. CCSP is a natural inhibitor of sPLA2 in the lung [21, 22] . This drug has been given endotracheally together with surfactant [21] achieving a significant reduction in lung tissue inflammation. Similar results in terms of inflammatory markers and lung function have been obtained in animal models of iRDS and MAS [23] [24] [25] .
Other authors have proposed the same approach with endotracheally administered budesonide, vehicled by surfactant [26] . Budesonide inhibits sPLA2 and has been associated with a decrease in the release of sPLA2induced pro-inflammatory cytokines [27] . Surfactant is the cornerstone of iRDS therapy and the sPLA2 inhibition could theoretically protect it, reducing the need for repeated doses and improving the long-term respiratory outcome.
During sepsis or pneumonia surfactant may be inadequately produced and recycled or it may be inactivated by lung tissue inflammation [28, 29] . In these cases surfactant therapy is often less useful and does not achieve the clinical improvement usually seen in iRDS [28, 29] . Mortality rate for such condition is still remarkable in term infants and even higher in preterm babies, who often experience sepsis or pneumonia as nosocomial infections acquired in the intensive care units [16] . sPLA2 is raised in BALF of neonates with IRRF [19] . This is consistent with animal and cellular studies showing that bacterial membrane lipopolysaccharide is a potent inductor of sPLA2 [4] . Nonetheless, no definite data are available about the role of sPLA2 and its pathway during IRRF.
Pancreatic sPLA2 has been indicated as a main etiological agent of MAS, one of the worst form of neonatal lung injury, characterized by massive surfactant inactivation, lung tissue inflammation and airway obstruction [30] [31] [32] . Meconium carries high amounts of sPLA2 and bile acids that are likely to contribute to lung injury, increasing sPLA2 activity and causing further surfactant inactivation [33] . Moreover, not only the pancreatic sPLA2 but also the pulmonary isoforms of the enzyme may be involved in the syndrome, [34] as lung sPLA2 production may be boosted by the meconium-induced release of pro-inflammatory cytokines [35] [36] [37] and through a specific cross-talk between different enzyme isoforms [38] . Consistently, we have recently found raised levels of pulmonary sPLA2 in BALF of patients affected by MAS when compared to their own meconium and to control babies [39] . MAS still has a mortality rate of about 50% and sometimes requires invasive treatments as broncho-alveolar lavage using saline/surfactant solutions or extra-corporeal life support [40, 41] .
sPLA2 is also involved in neonatal bile acids pneumonia, a more rare form of lung injury, [42] in which neonatal lungs are challenged with the bile acids coming from the maternal circulation when the mother is affected by obstetric cholestasis [43, 44] . In this condition, the neonatal lung may experience a sPLA2 overactivation [33] due to the bile acids coming from maternal circulation. This may lead to severe respiratory failure, since bile acids increase the sPLA2 activity enabling the presentation of the phospholipid substrate to the catalytic site of the enzyme [45] .
It is known that some sPLA2 gene polymorphisms are associated with chronic obstructive pulmonary or coronary artery disease [46, 47] . Given the wide role of sPLA2 in many critical respiratory conditions, an individual predisposition due to different polymorphisms is likely to exist. Nevertheless, data about sPLA2 genetics, its association with respiratory failure and its clinical severity have not been published.
Available data allow hypothesizing a role for sPLA2 or its modulators in the pathogenesis, in the clinical severity and in the development of complications of the above mentioned types of lung injury. Our aim is to clarify such role, in order to better understand whether or not sPLA2 therapeutic inhibition might be a helpful strategy. To do that, we are planning to:
1. Identify the exact subtype(s) of sPLA2 produced and secreted into the alveoli during post-neonatal ARDS-ALI, MAS, iRDS and IRRF. This is important to know because sPLA2 inhibitors may have a different specificity for the various enzyme subtypes. In animal models, distinct sPLA2 subtypes have been associated to lung dysfunction [1, [48] [49] [50] . 2. Study the main modulators of sPLA2 expression and activity (TNFα, CCSP, SP-A and IL1). This will allow identification of possible pathway imbalances and eventually new therapeutic targets. 3 . Clarify what happen to sPLA2 and its pathway when exogenous surfactant is administered, as it usually occurs to preterm neonates. This will allow to understand if there is a link between sPLA2 activity/overexpression, the repeated need for surfactant and BPD occurrence. 4. Clarify if there is a genetic predisposition due to different sPLA2 genes polymorphisms which could lead to more severe clinical pictures in iRDS, ARDS, MAS or IRRF or to a long term negative outcome.
This is the first study aimed at investigating the whole sPLA2 pathway in the above-described types of lung injury. To date, no study has addressed the functioning of the whole sPLA2 pathway, including the role of genetics, pathway modulators and related exogenous therapies that may affect it. This is essential because the diseases in which sPLA2 is thought to be important are basically different and the enzyme could play a different role through different subtypes, with different influence of its modulators and various response to the exogenous surfactant administration. Moreover, gene polymorphisms may play a role affecting the enzyme activity and so the clinical picture. Furthermore, many respiratory diseases potentially caused or influenced by sPLA2 are typical of newborn infants (e. g.: MAS, iRDS) or are present both in adults and in children, but with different causes and characteristics (e.g.: ARDS) [51] . Thus, data coming from animal studies or from adult experience cannot be directly applied to children and a specific study is warranted. The data coming from the present project will be crucial for future studies targeted at developing an anti-sPLA2 therapeutics.
A multicenter design has been previewed and the project will be coordinated at the Laboratory of Clinical Molecular Biology of the University Hospital "A. Gemelli", Catholic University of the Sacred Heart in Rome. A Study group on Secretory Phospholipase in Paediatrics (SSPP) has been arranged and project coordinators will be a clinical pathologist/biochemist (Prof. E. Capoluongo, Head of the Lab) and a paediatric intensivist/neonatologist (Dr. D. De Luca). SSPP will consist of two working groups for this project:
a. Laboratory group. This consists of biochemists and biologists experts in several molecular biology techniques applied to BALF specimens. These investigators will remain blinded to the clinical data, which will be known only to the project coordinators. b. Clinical group. This will consists of all clinicians -neonatologists and/or pediatric intensivists -working in intensive care units (at the University Hospital "A.Gemelli" or in so called "Collaborating centers") where patients' enrolment, samples and data collection will be performed.
Clinical investigators will meet a project coordinator regularly before the beginning of the study. This will happen by tele-conference or by visiting the collaborating centre. All investigators will remain in contact during the entire project by e-mail and/or tele-conference. One of the project coordinator will also give a 24h/7d availability by phone in case of urgent matters.
The project is still open and other intensive care units are welcomed to participate as collaborating centres. Interested colleagues should contact the corresponding author to discuss the study feasibility (please see at the end of manuscript).
To accomplish the study purposes, the work will be subdivided in two phases: 1) clinical phase; 2) bench phase.
Enrolment The following group of patients will be identified: iRDS, IRRF, MAS, ARDS-ALI. To be enrolled in a group babies must fulfil all the following inclusion criteria:
A. Preterm neonates (gestational age ≤ 37 sett) with iRDS C-Reactive protein (CRP) < 10 mg/L or procalcitonin (PCT) < 0.6 ng/mL in the first 72 hours of life; Chest-Xrays typical for iRDS; no clinical signs of sepsis; need for mechanical ventilation.
B. Infants and neonates with IRRF (regardless of the age) B1. Early IRRF. Neonates from mother with vaginal or urine positive cultures. Respiratory distress signs and CRP > 10 mg/L [52] or PCT > 0.6 ng/mL [53] in the first 72 hours of life; clinical signs of sepsis or blood/BALF positive culture; need for mechanical ventilation.
B2. Late IRRF. Neonates with respiratory distress signs beyond the first 72 hours of life or infants, irrespectively of the age and CRP > 10 mg/L [52] or PCT > 0.6 ng/mL [53] ; clinical signs of sepsis or blood/BALF positive culture; need for mechanical ventilation.
C. Neonates with MAS Neonates with meconium stained and thick amniotic fluid who required broncho-aspiration following Neonatal Resuscitation Program guidelines [54] . Continuous need for mechanical ventilation at 15 minutes of life. Chest-X rays typical for MAS.
D. Infants with ARDS-ALI [55] Infants beyond neonatal age (> 30 days of life) and < 1 year of age under mechanical ventilation and having PaO 2 /FiO 2 ratio < 200 (ARDS) or < 300 (ALI), chest-X rays typical for ARDS-ALI, acute onset of the respiratory distress and no cardiogenic oedema/increase in left atrial pressure.
A control group has also been previewed, as follows:
Patients ventilated for non-pulmonary reasons (e.g.: anaesthesia, central nervous system diseases), PaO 2 /FiO 2 ratio > 300 or FiO 2 = 0.21, negative CRP and PCT, normal chest-X rays and chest clinical examination.
A careful revision of the clinical characteristics will be done for each patient at the moment of discharge (or death). This will be done in each centre to ensure the appropriateness of diagnosis and internal validity. Procedures to be performed in the intensive care units 1. Broncho-alveolar lavage.
This procedure will be performed as soon as possible from the fulfilling of the enrolment criteria. In case of neonates, broncho-alveolar lavage will be performed in the following schedule:
• PRE-SURFACTANT • POST-SURFACTANT (after at least 12 hours from the surfactant administration) • PRE-2 nd SURFACTANT (only for babies needing a second dose) • POST-SURFACTANT (after at least 12 hours from the 2 nd surfactant administration)
Obviously, for infants receiving just a single surfactant dose or no surfactant at all, only one or two bronchoalveolar lavages will be carried out.
This procedure is to be intended a non-bronchoscopic lavage: it will be performed according to our previously described and well standardized technique [15] and following the advices of the European Respiratory Society guidelines [56] . All BALF specimens will be added with 0.9% saline up to 2 mL and a small aliquot of the fluids will be sent for microbiological culture.
2. 1.5 mL blood drawing in a vial with no anti-coagulant to be centrifuged (see below).
If a baby undergoes repeated broncho-alveolar lavages, the blood drawing will be repeated each time. Every BALF and blood specimens must be obtained within 1 hour from each other.
3. 0.5 mL blood drawing into an EDTA vial to be immediately stored at 4°C. This blood will be used for DNA extraction to analyse sPLA2 genes polymorphisms and will be drawn only once for each baby.
In general, blood drawings will be performed from an indwelling arterial line or from a central venous line to avoid haemolysis. Blood without anti-coagulant and BALF samples will be immediately centrifuged at 3000 rpm for 10 minutes to separate the serum or the supernatants which will be immediately stored at -80°C. Data to be registered in the intensive care units The following data will be recorded either from the vital parameters monitors, from the ventilator screen or the clinical files. • Type of mechanical ventilation provided • Peak inspiratory pressure, positive end-expiratory pressure, mean airway pressure (Pāw), Expired tidal volume (pro Kg) • Total respiratory rate and spontaneous respiratory rate (if any) • Dynamic compliance over ten mechanical breaths or static compliance using end-expiratory occlusion (depending on the ventilator) § • Total respiratory system resistances over ten mechanical breaths (If patients are ventilated with high frequency oscillatory ventilation, instead of the above-mentioned parameters, Pāw, amplitude and frequency will be recorded. If a specific flow-sensor [57] is available the tidal volume delivered during oscillations will also be registered and used for further calculation [see below]).
• Cumulative dose of exogenous surfactant (for neonates) • FiO 2 • Oxygen saturation at the right hand These data must be recorded as close as possible to the broncho-alveolar lavage/blood drawing (max within 1 hour from such procedures). These data will be recorded in real time in an appropriate electronic database provided by the coordinating centre to each intensive care unit participating in the study.
Moreover, using the above-mentioned data, the following indexes will be calculated:
• Oxygenation index (FiO 2 × Pāw/PaO 2 ) • PaO 2 /FiO 2 ratio • Ventilatory index (Peak -end expiratory pressure) × respiratory rate × PaCO 2 /1000 (if conventional ventilation is provided) • Alveolar ventilation estimate during high frequency oscillatory ventilation (DCO 2 = frequency * (tidal volume) 2 ], if a specific flow sensor is available) [58] Moreover, the following data will be recorded:
• Mortality • Intensive care unit length of stay • Duration of invasive ventilation • Duration of oxygen therapy • Any neurological sequel at the discharge • Oxygen requirement after discharge • Diagnosis of chronic lung disease, for preterm neonates, according to the NICHD definition of BPD [59] .
Exclusion criteria Patients with one of the following characteristics will not be enrolled in any group:
1. Congenital lung malformations of any type 2. Lung or thoracic surgery 3. Lung cancer of any type 4. Congenital complex malformations 5. Patients undergoing extracorporeal life support.
Storage and transfer of data and samples All data will be anonymously stored in the above described electronic database and will remain property of the enrolling centre. At the end of the clinical phase they will be checked for validity in each centres and then sent in a secured way to the Coordinating centre. At that time all specimens will be also sent under dry ice to the Coordinating centre.
2) Bench phase sPLA2 pathway In serum and BALF supernatants the following analyses will be performed:
• Western blotting for sPLA2-IIA, -V, -X. For this procedure external (actinin) and internal (recombinant human sPLA2 subtypes, -IIA, -V, -X) controls will be used and total protein measurements will also be performed with Bradford's method [5] .
• TNFα assay • SP-A assay • IL1 assay These assays will be performed using specific ELISA/ EIA kits already used to analyse BALF. These methods have been proven to do not cross-react with other cytokines and with sPLA2; in previous studies, coefficients of variation of the standard curve resulted always ≤ 9% [15, 39, [60] [61] [62] .
• sPLA2 global activity assay To do this assay, all samples will be centrifuged (for 10' at 12000 rpm and then for 3' at 3500 rpm) through a membrane-filter with a molecular weight cut-off of 30 kDa (Amicon Ultra centrifugal filter; Millipore, Billera, MA-USA), to separate the secretory and cytosolic phospholipases (which weight ≈14 kDa and ≈80 kDa, respectively) [39, 62, 63] .
• High sensitivity urea nitrogen assay in the BALF supernatant.
All measurements in BALF will be corrected for the serum-to-BALF urea ratio, as previously described [64] . sPLA2 genetics sPLA2-IIA [HGNC:9031], -V [HGNC:9038] and -X [HGNC:9029] genes polymorphisms will be studied in the patients' leukocytes. We found 15 single nucleotide polymorphisms (SNP) for these genes. These polymorphisms were searched in the dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), JSNP (http://snp.ims.u-tokyo.ac.jp), GenBank at the NCBI (http://www.genbank.com) and Applied Biosystems genotyping databases (http://www.appliedbiosystems.com), as well as in a previous study linking them to coronary artery disease [47] . Analysis of genetic polymorphisms SNP genotyping will be performed by TaqMan allelic discrimination assay [65] . Polymerase chain reaction will be performed with specific primers at concentrations of 900 nM. Fluorescence data files from each plate were analyzed by a specific software. In order to verify the correct genotype assignment, we will randomly analyse some of the above screened samples by means of genetic sequencing (BigDye terminator technique).
All laboratory procedures will be carried out respecting safety regulations and bench investigators will be blinded to the patients' group of origin and to their clinical data. To accomplish this blindness, before starting the bench phase all vials will be re-labelled with a new code and only the project coordinators will be aware of the new code. Statistics and sample size Data will be tested for normality and then analyzed with parametric or non-parametric procedures, as appropriate. Accordingly, univariate analysis using Student and analysis of variance or Mann-Whitney, Wilcoxon, Kruskal-Wallis and Friedman tests will be performed. Some laboratory data will be subjected to correlation analysis with clinical findings, using Pearson's, Spearman's or Kendall's technique, according to data characteristics.
Subsequently, if needed, significant results will be subjected to multiple curve estimation procedure [66] and/ or multivariate analysis according to data characteristics and the results of the univariate analyses. The genetic data will be analyzed using χ 2 test for the Hardy-Weinberg equilibrium of alleles at the individual loci. The association between genotypes and clinical data will be tested with χ 2 -or Fisher test and then with logistic regression or analysis of co-variance, as appropriate [67] . All statistical analyses will be performed by the project coordinators, who have a long experience and formal training in biostatistics.
Despite a formal sample size calculation is not warranted, based on the available data [5, 11, 19] , we previewed a convenience sample size, as follows: 50-60 preterm infants affected by iRDS, with at least 20 receiving a second surfactant dose; for ARDS and IRRF groups 20 patients will be also convenient, while 10 control neonates or infants will be considered. These sample size have been checked for power regarding the correlation between sPLA2 activity and two selected clinical variables (using Power and Precision demo rel. 3.2 [68] ). Given α-error of 0.05 and a correlation coefficient (r) ≥ 0.6 and ≥ 0.85 [5, 11, 19] for the respiratory compliance and the PaO 2 /FiO 2 ratio, respectively, the power resulted > 80% in both cases.
The study is supposed to last 12-18 months for the enrolment phase in each collaborating centre, and 3-6 months for the laboratory phase at the coordinating centre.
The protocol and consent form have been approved by the Ethical Committee of the University Hospital "A. Gemelli" at the Catholic University of the Sacred Heart (Rome, Italy) as coordinating centre. Local ethical boards in each collaborating centre have also approved the protocol.
The participation to the study will not change in any way the routine clinical assistance previewed for every patient. Furthermore, the participation to the study will respect all the local Regulations about safety procedures and the privacy. Informed consent will be given by parents or tutors of each baby, before the enrolment in the study.
Study results will be presented to each investigator by teleconference and/or e-mail. If possible a meeting in occasion of one of the major congresses in the field of Paediatrics or Critical Care (like the European Society for Paediatric Research or European Society for Paediatric and Neonatal Intensive Care Congresses) will be organised. Data will be also presented at these meetings and the subsequent manuscripts will be circulated between all investigators for revision. All resulting manuscripts will be authored by the project coordinators and by the group authorship (SSPP: Study group on Secretory Phospholipase in Paediatrics).
This study, thanks to its multicenter design, will clarify the role(s) of sPLA2 and its pathway modulators in several paediatric and neonatal forms of lung injury. In fact, enough evidence is available to indicate sPLA2, or at least some of its subtypes, as a key point in the pathophysiology of certain critical respiratory diseases. Inflammation is a complex process and is essential in many of these conditions: sPLA2 might be the main crossroad between inflammation and surfactant catabolism. Since this latter is surely an important component of some clinical situations, sPLA2 is worth to be studied in its metabolic and genetic issues, trying to correlate them to the clinical pictures.
Given the peculiarity of such diseases and the relative rarity of some of them, only a multicentre design will be able to clarify this field. The purpose is not free from practical consequences, as several sPLA2 inhibitors are now available or under advanced development [69] . Many drugs are already used in respiratory critical care [70] but sPLA2 blockade might represent a new antiinflammatory therapy and a novel approach to protect surfactant or spare it, improving alveolar stability, lung mechanics and gas exchange.
The establishment of a net of centres involved in clinical research, along with the support of bench investigators, will help in understanding this field and in building future randomised interventional studies.
Footnote § Compliance measurement depends on the type of ventilator. Those with the end-expiratory occlusion will provide a static measure, otherwise a dynamic measurement will be done using the hot-wire anemometer flow sensor. In that case, to increase the measurement accuracy, the spontaneous breathing must be temporarily avoided, gas leaks must be < 5% and stable respiratory conditions with minimal airway secretions must be achieved. Conditions and technique for this measurement have been described in details elsewhere [15] . The study has received funds by Catholic University of the Sacred Heart (part of Research fundings D1-2011, to E.Capoluongo) and by a charity program of a private engineering company (QProgetti srl, Rome, Italy, funds 2011), which has nothing to do with this research field and will have no role at all in the project. Transmissibility and temporal changes of 2009 pH1N1 pandemic during summer and fall/winter waves BACKGROUND: In order to compare the transmissibility of the 2009 pH1N1 pandemic during successive waves of infections in summer and fall/winter in the Northern Hemisphere, and to assess the temporal changes during the course of the outbreak in relation to the intervention measures implemented, we analyze the epidemiological patterns of the epidemic in Taiwan during July 2009-March 2010. METHODS: We utilize the multi-phase Richards model to fit the weekly cumulative pH1N1 epidemiological data (numbers of confirmed cases and hospitalizations) as well as the daily number of classes suspended under a unique "325" partial school closing policy in Taiwan, in order to pinpoint the turning points of the summer and fall/winter waves, and to estimate the reproduction numbers R for each wave. RESULTS: Our analysis indicates that the summer wave had slowed down by early September when schools reopened for fall. However, a second fall/winter wave began in late September, approximately 4 weeks after the school reopened, peaking at about 2-3 weeks after the start of the mass immunization campaign in November. R is estimated to be in the range of 1.04-1.27 for the first wave, and between 1.01-1.05 for the second wave. CONCLUSIONS: Transmissibility of the summer wave in Taiwan during July-early September, as measured by R, was lower than that of the earlier spring outbreak in North America and Europe, as well as that of the winter outbreak in Southern Hemisphere. Furthermore, transmissibility during fall/winter in Taiwan was noticeably lower than that of the summer, which is attributable to population-level immunity acquired from the earlier summer wave and also to the intervention measures that were implemented prior to and during the fall/winter wave. Although the first known imported case of 2009 pandemic influenza (pH1N1) arrived in Taiwan on May 18 from the U.S. via Hong Kong, Serological evidence has indicated that the pH1N1 virus had spread to central Taiwan by April-June [1] . Local infections and laboratory-confirmed pH1N1 cases in Taiwan started to mount in significant numbers in July-August when the schools were in summer recess. By the time the schools reopened in September, multiple intervention measures had been implemented by the government, which include strict border temperature screening starting in May, a "325" class suspension policy [2, 3] implemented in September, and later a mass immunization program [3] [4] [5] starting in November. The number of cases began to decline by the end of the year, and continued to do so into early next year, until the government announced on February 23 the end of the fall/winter outbreak [6] with over 3000 laboratory-confirmed cases reported, 910 hospitalizations, and 41 deaths [7] .
Although school closing was a widely used method of intervention around the world during the pH1N1 outbreak (see, e.g., [8] [9] [10] [11] [12] [13] ), its suitability, timing, and the manner of implementation remains controversial. When K-12 schools (kindergarten through high schools) reopened on August 31 in Taiwan, the government implemented a unique partial school closing policy called the "325" class suspension policy aimed toward kindergarten through secondary schools (K-9), cram schools, and after-school institutions. Under this policy, if within any three (3) consecutive school days, two (2) or more students in the same class are diagnosed with influenza, then that class will be suspended for the next five (5) days including weekends and holidays [2, 3] . The policy was designed to minimize the potential social impact of full-scale school closings in the event of a major influenza outbreak in the community; to detect cluster infections in school settings early and swiftly; and to contain the infections locally without disruption for the other students in the school. At the height of the class suspensions in late November, more than 1800 classes with more than 50,000 students from almost 800 schools in Taiwan were suspended on a single school day (Figure 1 ), yet without any visible disruption in the normal functioning of the society.
Moreover, starting November 1, a mass immunization program was initiated in Taiwan sequentially, according to a priority list of 12 target groups [4] , with healthcare and public health personnel having the highest priority [5] . Subsequently, preschool children were immunized starting on November 9; and followed by pregnant women, K-6 schoolchildren, and people with major illness/injury being vaccinated starting on November 16; 7-12 year-olds on November 23; and the general population on December 12. By March 16, a total of 5.66 million doses of AdimFlu-S (unadjuvanted H1N1v from Adimmune) or Focetria ® (MF59 ® adjuvanted H1N1v from Novartis) were administered, and more than 5 million of the 23 million Taiwanese had been immunized [14] . Children 12 and under were advised to receive two doses of vaccine, although many of them eventually received only one dose due to various reasons.
A simple mathematical model, the Richards model, is utilized to fit publicly accessible cumulative epidemic data in order to obtain estimates for the turning points (the peaks and volleys of the incidence curve) and the reproduction number R of a particular wave of infections. Examples of applications of the Richards model to infectious diseases include those of SARS [15, 16] , dengue [17, 18] , and the 2009 pH1N1 epidemic [19, 20] . In this study, we will make use of the Richards model to pinpoint the turning points of each wave of the epidemic, in order to ascertain the temporal changes of the epidemic in Taiwan in the summer months and during the fall and winter days. The transmissibility of the pH1N1 virus during the outbreak is determined through its reproduction number.
The data was accessed from the Central Epidemic Command Center website of the Taiwan Centers for Disease Control (TCDC). Samples were collected from hospitals and clinics participating in the Taiwan Influenza surveillance system under the Taiwan National Influenza Center (Taiwan NIC), which was established in 2006 to integrate all existing efforts of influenza surveillance and notification with laboratory analysis systems throughout Taiwan in order to enhance the epidemic data collection capacity in Taiwan [21] . The weekly laboratory confirmed pH1N1 case data (by the week when the samples were collected and sent to the TCDC-contracted laboratories) and the weekly hospitalization data (by the week the lab-confirmed cases were hospitalized) from June 28, 2009 (epidemiological week or e-week 27 of 2009) to March 27, 2010 (e-week 12 of 2010) was accessed from the weekly Influenza Express made publicly available on the internet by the TCDC [22] during the epidemic. The surveillance protocols in Taiwan remained essentially the same throughout the data period since, by the time the data were collected, clinical characteristics of the pH1N1 infection had already been well understood from the spring outbreaks around the world.
We also accessed the daily record of numbers of classes suspended and number of schools with at least one class suspended during the fall school semester (September 9, 2009 to January 20, 2010) from the TCDC daily pH1N1 updates [23] during the epidemic. The time series of class suspension data is given in Figure 1. Since this data is for school days only, the days are specified in the horizontal axis of Figure 1 in weekly increments of 5 school days, except for weeks with less than 5 school days at the beginning and the end of the school semester as well as the week containing the New Year holiday (January 1).
The Richards model [24] is of the form:
where the prime symbol "'"
denotes the rate of change over time which is in eweeks. C(t) is the cumulative number of cases at time t (in weeks), K is the cumulative case number over a single wave or phase of outbreak, r is the per capita growth rate of the infected population, and a is the exponent of deviation. The explicit solution of the equation is
Here the parameter t m is related to the turning point t i of a wave (or the inflection point of the cumulative case curve) by the simple formula t m = t i + lna/(ra), where ln denotes the natural logarithm function. Moreover, R 0 = exp(rT) where T is the generation interval of the disease, or the average time interval from the onset of one infected person to the time when the onset of his or her contacts occurs. It has been shown mathematically [25] that, given the growth rate r, the expression R 0 = exp(rT) provides an upper bound of the basic reproduction number regardless of the distribution of the generation interval that is being used. In this work, we will use the term effective reproduction number R instead, due to the community-level immunity likely achieved by July and the interventions implemented during the two waves.
The Richards model is a phenomenological model which can be used to describe the phenomenon of a biological growth (of cumulative number in this case) without requiring detailed information on the actual process of disease transmission. The basic premise of the Richards model is that the incidence curve of a single wave of infections contains a single peak of high incidence, resulting in an S-shaped cumulative epidemic curve and a single turning point (or peak incidence) of the outbreak. The turning point, defined as the point in time at which the rate of accumulation changes from increasing to decreasing, or vice versa in the event of a multi-wave outbreak, can be easily pinpointed by locating the inflection point of the cumulative case curve, i. e., the moment at which the trajectory begins to decline, as demonstrated in previous applications (see, e.g., [15] [16] [17] [18] [19] [20] . This quantity has important epidemiologic implications, indicating either the valley (i.e., moment of acceleration after deceleration) or peak (i.e., moment of deceleration after acceleration) of a disease incidence curve. Multi-wave outbreaks also can be modeled by using the multi-phase Richards model [16, 18] . Simultaneous estimates of the model parameters r, a, t i , and K, based on fitting the explicit solution of the Richards model for C(t) to the epidemic data used in the study, can be obtained easily and efficiently using any standard software with a nonlinear least-squares approximation tool, such as SAS or Matlab. The procedure for locating multiple turning points for multi-wave outbreak, which required the use of the multistage Richards model, is detailed in [16] and hence is omitted here.
We first fit the weekly laboratory confirmed pH1N1 case data by sample receiving week in Taiwan Table 1 with the model fit shown in Figure 2 .
The turning points for the two waves are estimated at 8.50 weeks after e-week 29 and 7.96 weeks e-week 39, respectively. Subsequently, the weeks in which the turning points for temporal changes in the weekly confirmed pH1N1 case number took place on e-week 36 (8/30-9/5) for the first wave with a 95% CI range of (36.62, 37.38), and on e-week 47 (11/15-11/21) with a 95% CI range of (46.42, 47.50) for the second wave. We note that the above results were obtained by rounding off the estimates to the next largest integer, e.g., e-week 27+8.50 = 35.50 and hence e-week 36 is the week during which the turning point for the first wave occurred, and similarly for the second wave.
To compute the effective reproduction number R, we use the generation time T = 1.91 days (95% CI: 1.30-2.71) for the 2009 pH1N1 in Mexico estimated by Fraser et al. [26] . We note that the given CI's for R 0 reflect the uncertainty in the generation time T as well as in the uncertainty in the least-squared estimates for r, and does not reflect the error due to the model itself, which is always difficult to measure.
We also fit the weekly confirmed pH1N1 hospitalization data by hospitalization week in Taiwan from eweek 29 (7/12-7/18) of 2009 to e-week 12 (3/21-3/27) of 2010 to the Richards model. The results are given in Table 2 . The data also fit a two-phase Richards model with the first wave spanning e-weeks 27-39 (7/12/09-9/ 26/09) of 2009 and the second wave from e-week 39 (9/ 20-9/26) of 2009 to e-week-12 (9/27/09-3/27/10) of 2010 ( Figure 3) .
The turning points for the weekly confirmed pH1N1 hospitalizations occurred on e-week 37 (9/6-9/12) for the first wave with a 95% CI range of (36.12, 36.82) and on e-week 46 (11/8-11/14) with a 95% CI range of (44.64, 46.62) for the second wave, which were the same weeks as the case number data turning points. The estimate for R using an estimated generation time T for pH1N1 in Mexico [26] is again provided.
To further analyze and compare our previous results, we also make use of the daily class suspension data in Taiwan from September 9, 2009 to January 20, 2010, which allows us to ascertain the temporal changes in this intervention measure during the time period. Since this dataset started near the end of the first wave, according to our previous results, only one wave was modeled via the Richards model. The estimation results for model fit using the daily class suspension number data as well as the daily number of schools with at least one class suspended are given in Table 3 The actual confirmed case number (approximated by K in our model) is 1742 during the first wave and 3238 for the two waves. point. A graphical illustration of the temporal timelines of the epidemic, as illustrated by the three model fits, is given in Figure 6 . Moreover, an illustrative comparison of the estimates for R as obtained by the model fits is also provided in Figure 7 . In both Figures 6 and 7 , the results from fitting the number of schools with class suspended are omitted for brevity, since they are similar to that of the fitting with class suspension data. Figure 2 Model fit for the 2-wave Richards model using weekly confirmed pH1N1 case data by sample receiving week in Taiwan. The dots are the real cumulative data, the blue curve denotes the first wave, and the red curve denotes the second wave. The arrows indicate the weeks in which turning points had occurred. The actual number of confirmed hospitalizations is 297 for the first wave and 910 for the two waves.
The estimates for effective reproduction number R obtained from the confirmed case and hospitalization data are in good agreement, with R in the range of 1.04-1.27 for the first summer wave during July-September, and 1.01-1.05 for the second wave in fall/winter, using the generation time estimated by [26] for the spring outbreak in Mexico. Serological evidence has indicated that approximately one in every ten persons was infected with the 2009 pH1N1 virus in central Taiwan by April- June [1, 27] ; hence the estimates using data after July does not yield, and can reasonably be expected to be lower than, the more commonly known basic reproduction number R 0 .
A recent modeling study [28] of the 2009 pH1N1 epidemic by geographic region in Mexico reveals a threewave pandemic, with an initial wave in April-May (Mexico City area), a second wave in June-July (southeastern states), and a geographically widespread third wave in August-December. The estimates for the regional reproduction numbers R were 1.8-2.1, 1.6-1.9, and 1.2-1.3 for the spring, summer, and fall waves, respectively. The second and third waves in Mexico occurred, respectively, one month earlier than the summer (July-early September) and fall/winter (late September-March 2010) waves in Taiwan under study here and exhibit similar decreasing trend, although with higher R.
Transmissibility of the fist pH1N1 wave in Taiwan during the summer in July-September, as measured by R, was lower than that of the earlier spring outbreak in North America [20, 26, 29, 30] and Europe [31] , most likely, at least in part, due to decreased social contacts among the population triggered by public awareness of the earlier, well-publicized outbreaks in Mexico and North America as well as the subsequent preemptive government campaign to reduce transmissions. It was also lower than that of the winter outbreak in the Southern Hemisphere around the same time [19, 32, 33] , perhaps attributable to the fact that it was the winter influenza season in the Southern Hemisphere. Moreover, It is lower than the final size estimate of R 0 (1.87; 95% CI: 1.68-2.06) obtained from serological study of a cohort household population in central Taiwan during the same period of time [1] . However, we note that this disparity is reasonable since the serologic data used for this estimate accounts for the asymptomatic cases among the cohort group. The decreased transmissibility (smaller R) during fall/winter can be reasonably attributed to increased community-wide immunity from the first wave, and perhaps to the 325 class suspension policy initiated in early September before the start of the fall/winter wave.
Significantly higher estimate of R (focused on schoolchildren) in the range of 2.0-2.6 was found for the initial pandemic wave in Japan [34] . Using updated epidemic data and an age-structured model, the same authors also estimated R for the subsequent community-wide wave in Japan in early summer to be much lower (1.21-1.35) [35] , although different population and modeling methodology also may have played a role in the decrease in R in subsequent waves. Similar decreases in estimates of reproduction number of 2009 H1N1 when more than one pandemic wave had occurred have been reported in many countries, including Mexico [28] , Argentina and Brazil [19] , Canada [20] , and Japan [34, 35] . Furthermore, these studies show that it is not uncommon for multiwave outbreaks to be more transmissible in a first wave but less widespread with a smaller number of infections (or perhaps limited to a small subpopulation as was in the case of pH1N1 in Japan), when compared to subsequent waves. Moreover, the second wave in Taiwan started shortly after the school opened in September, when additional infections occurring in school settings (as demonstrated by substantial number of class suspensions) contributed to a large number of cases, but perhaps with relatively less per contact transmissibility when compared to household contacts, as it has been reported that sitting next to a case or being the playmate of a case did not significantly increase the risk of H1N1 infection [36] .
The estimates for R using laboratory-confirmed case data by sample receiving weeks are slightly lower than those obtained by using confirmed hospitalization data. Although both the confirmed case and hospitalization datasets identify week 39 as the cutoff week for the two waves, the estimates of turning points for each wave differ by about one week when using the two datasets. Since only the more severe confirmed cases were hospitalized, the individuals in the resulting hospitalization time series is a selected subset of those in the confirmed case time series. Subsequently, the temporal trends of the two time series might not be closely comparable. However, the cumulative curves in Figures 2, 3 , 4, 5 indicate some similarity in the temporal trends of the cumulative data, mainly in the form of the turning points. The reproduction numbers of the two datasets, on the other hand, are indeed comparable since they mostly are generated from the initial growth rates and hence less affected by any selection bias.
The confirmed case data is generated by sampling week, which could be different from the week of symptom onset and hence pose a potential source of some bias in data. However, samples were typically taken when the physicians diagnosed and reported H1N1 cases. We refer to 2003 SARS outbreak in Taiwan, when it was estimated that the onset-to-diagnosis interval is 1.20 days for previously quarantined persons and 2.89 days for non-quarantined persons [37] . Given the similarity in symptoms of SARS and influenza as well as the heightened public awareness due to the world-wide alarm over the seriousness of the pH1N1 pandemic by September, it is more than likely that the time delay from symptom onset to diagnosis (and sample collection) of pH1N1 cases in Taiwan would be no more, if not less, than that of 2003 SARS. Moreover, one would expect that the lesson of SARS and the subsequent efforts by the government to educate has taught the general public in Taiwan to avoid delays in seeking medical care. Subsequently, this delay of one or two days in the weekly data can be expected to be most likely not significant. The use of hospitalization data is mainly for the purpose of estimation of reproduction number and comparison with the resulting estimates using the confirmed case data, which is not affected by this delay that might be present in both data.
Estimates of R obtained by using other (larger) estimated generation time in literature result in larger values for R, but generally are well within the ranges of the other studies (see, e.g., [19, 20, 26, [29] [30] [31] [32] and Table 2 [33]) and hence is omitted for brevity. Note also that the formula for R used here yields an upper bound over all possible distributions for T given the growth rate r, and hence might result in an overestimate of its true value.
In Taiwan, the fall session for kindergarten to high school started on August 31, while the universities started the fall semester two weeks later, around mid-September. Our analysis using the weekly confirmed case and confirmed hospitalization data shows that the initial summer wave of pH1N1 epidemic in Taiwan had peaked by e-week 36-37 (8/30-9/12), around the time schools from kindergarten to grade 12 reopened on August 31. However, a second fall/winter wave of cases started to emerge near the end of September around eweek 39 (9/27-10/3), approximately 4 weeks after the schools reopened, which did not reach its peak until mid-November (e-week 46-47 or 11/8-11/21) and lasted until the turn of the year. It is interesting to note that the state-specific fall pandemic waves in Mexico began 2-5 weeks after school reopened [28] , which is consistent with our results on the start of the fall wave in Taiwan. Note that both turning points of the two waves in Taiwan fell on neighboring week using either the lab-confirmed case or hospitalization data. This is reasonable since the hospitalization of confirmed cases and the time that the samples were received by laboratories are closely related, although not necessarily in any particular order.
The class suspension data started on September 9 near the end of the first wave when the earliest class suspension occurred, according to our 2-wave fitting in Tables 1 and  2 , hence only one wave was modeled via the Richards model (Table 3) . Moreover, November 19 (95% CI: November 18-20) was determined to be the turning point for the daily class suspension data, while November 17 (95% CI: November 16-18) is the turning point for the daily number of schools with class suspended. Both days fall on e-week 47, which coincides with the week where the turning point had occurred as pinpointed by using the confirmed case data and one week after the turning point obtained by using the hospitalization data. It is reasonable to expect the class suspension to take place following the occurrence of case reporting and hospitalization. Moreover, the use of daily data allows a more precise estimation of the turning point.
Also of interest is the possible impact of major intervention measures implemented by the Taiwan government during this time period, which including the aforementioned "325 class suspension" policy and the mass immunization program. The daily number of class suspensions started to increase in early September and continued until late November after the implementation of mass immunization campaign (Figure 1 ). In particular, the 325 policy, which was designed to minimize the potential social impact of full-scale school closings in the event of a major influenza outbreak in the community; deserve special attention to ascertain its actual effectiveness. In fact, the lower estimates of R for the second wave and for the school closings data might indeed be attributable to the possible effects of school closings after September. However, more detailed class suspension data as well as age-specific epidemic data is needed to further quantify the actual impact or effectiveness of this very unique approach of partial school closure and localized class suspensions on the infections in the school and in the community in a qualitative modeling analysis (see, e.g., [12, 13, 38] ).
Using routine influenza surveillance data, we modeled the temporal changes of the two waves of pH1N1 epidemic in Taiwan in summer and in fall/winter. The mass H1N1 vaccination program was first initiated sequentially on November 1, where a typical delay of at least two weeks from immunization is needed for protection from the vaccine to take effect in human bodies. Our results suggest that the turning point for the second wave of infections in the fall had occurred around mid-November (e-week 46-47 or 11/8-11/21). Moreover, the class suspension data indicate that the number of class suspensions had peaked by November 20, less than three weeks after the start of mass immunization and most likely before the impact of mass immunizations started to become significant. However, the mass immunization, and perhaps the voluntarily decreased social contacts by the general public in response to the well-publicized mass immunization campaign by the government, could have contributed to the overall mitigation of the disease in the community, as indicated by the early saturation of the winter epidemic by early February. However, this cannot be modeled without detailed vaccination data.
The Richards model considers only the cumulative infected population size with saturation in growth as the outbreak progresses, which can be caused by other factors such as implementation of control measures. Although data by reporting date is often and typically scrambled by artificial factors such as health system alertness, public response, and government responsiveness, the Richards model is able to capture the turning points of outbreaks because they are often results of these artificial factors. We note, however, that the skewness in an epidemic curve, as quantified by the exponent of deviation "a" in the Richards model which describes the curvature of a given cumulative case data, also could conceivably arise from various other intrinsic factors such as spatial heterogeneity and individual heterogeneity in contact (see [39] , pp. 281 for example) which is not captured by this simple model. This type of modeling, although somewhat simplistic and subsequently limited in its quantification of complex factors, nevertheless enables us to ascertain the impact of these artificial factors through the temporal changes of an outbreak, especially in the events when detailed epidemic data describing disease transmissions and other relevant data (such as that of intervention measures in this case) are not readily available for the construction of a complete disease transmission model and the reliable estimation of model parameters, as in this study Moreover, the use of cumulative numbers could often, or at least partially, smooth out stochastic variations that typically occur in epidemic data, and hence the Richards model could be a valuable tool in providing clues to the challenging task of public health policy evaluation and planning. Molecular mechanisms of inflammation and tissue injury after major trauma-is complement the "bad guy"? Trauma represents the leading cause of death among young people in industrialized countries. Recent clinical and experimental studies have brought increasing evidence for activation of the innate immune system in contributing to the pathogenesis of trauma-induced sequelae and adverse outcome. As the "first line of defense", the complement system represents a potent effector arm of innate immunity, and has been implicated in mediating the early posttraumatic inflammatory response. Despite its generic beneficial functions, including pathogen elimination and immediate response to danger signals, complement activation may exert detrimental effects after trauma, in terms of mounting an "innocent bystander" attack on host tissue. Posttraumatic ischemia/reperfusion injuries represent the classic entity of complement-mediated tissue damage, adding to the "antigenic load" by exacerbation of local and systemic inflammation and release of toxic mediators. These pathophysiological sequelae have been shown to sustain the systemic inflammatory response syndrome after major trauma, and can ultimately contribute to remote organ injury and death. Numerous experimental models have been designed in recent years with the aim of mimicking the inflammatory reaction after trauma and to allow the testing of new pharmacological approaches, including the emergent concept of site-targeted complement inhibition. The present review provides an overview on the current understanding of the cellular and molecular mechanisms of complement activation after major trauma, with an emphasis of emerging therapeutic concepts which may provide the rationale for a "bench-to-bedside" approach in the design of future pharmacological strategies. Despite significant advances in injury prevention, prehospital resuscitation strategies, and modern intensive care, trauma remains the main cause of death in young people in the United States, resulting in more years of potential life lost before the age of 75 years than any other disease [1] [2] [3] [4] . Until present, the pathophysiology of major trauma remains poorly understood [5, 6] . In principle, the pathophysiological sequelae of major injuries are characterized by the initial traumatic impact (so-called "first hit"), followed by a cascade of subsequent immunological reactions, which render the patient susceptible to a potentially detrimental "second hit" insult [7] . The activation of innate immune response mechanisms has been characterized as a crucial event initiating the early phase of hyperinflammation within hours to days after major trauma [6] [7] [8] . While innate immunity is classically considered to be the immediate "first line of defense" against non-self antigens (e.g. infectious pathogens), a traumatic insult can induce a similarly potent acute inflammatory response [9] [10] [11] [12] [13] . The trauma-induced immune response may be limited locally, as in isolated injuries, or result in a massive systemic immune activation, as in patients with multiple injuries [1] . The endogenous triggers of trauma-associated inflammation have been thoroughly investigated and characterized in recent years [7, 14] . The so-called "first hit" induced by a traumatic impact leads to the appearance of an arsenal of "damage-associated molecular patterns" (DAMPs) that are recognized by receptors of immune cells [15] . DAMPs represent a recently characterized large superfamily of danger signals which can activate innate immune responses after trauma or trauma-induced complications, such as infection and sepsis [7, 16] . The DAMP family of danger signals includes the so-called "pathogenassociated molecular patterns" (PAMPs) and molecules termed "alarmins" [17] . The list of molecules belonging to the DAMP family has been increasing dramatically in recent years, and their pathophysiological function in mediating trauma-induced inflammation is far from being fully understood [18] . PAMPs represent a heterogenic entity of recently described inflammatory molecules related to the innate immune system [17, 19] . These microbial molecules are recognized by the immune system as foreign due to their characteristic molecular patterns. In contrast, the so-called "alarmins" represent the correlate of PAMPs for all non-pathogen-derived danger signals which originate from tissue injury [17] . This heterogeneic group of danger molecules is capable of activating innate immune responses in response to tissue damage and cell injury. The alarmins comprise the "heat-shock proteins" (HSPs), annexins, defensins, as well as "classical" markers of tissue injury, such as the S100 protein and the high mobility group box 1 (HMGB1) protein [17, 20] .
Immunologically competent cells recognize both PAMPs and DAMPs through multiligand receptors expressed on their surfaces, such as Toll-like receptors (TLRs) [21, 22] .
The very early stage after tissue trauma is characterized by activation of cellular and molecular effectors of the innate immune system, including complement activation and recruitment and activation of neutrophils (polymorphonuclear leukocytes; PMNL) [6, 7] . The complement system appears to represent the crucial effector of innate immune responses in the early phase after major trauma [23] [24] [25] . Once the cascade is activated through one of three (five) established pathways (Figure 1 ), complement plays a critical role in the elimination of invading pathogens by opsonization for phagocytosis (C3b, C4b), chemotaxis of leukocytes (C3a, C5a), and by direct lysis of pathogens through the membrane attack complex (MAC, C5b-9) [23, 26, 27] . The generation of anaphylatoxins C3a and C5a provides potent chemoattractants for phagocytes and neutrophils, and recruit these immune cells to the site of injury [24, 28, 29] . The anaphylatoxins further induce degranulation of mast cells, basophils and eosinophils and mediate the hepatic acute-phase response [30, 31] . Finally, the generation of C5b by cleavage of C5 initiates the terminal complement pathway with MAC formation. The MAC forms through the self-association of C5b along with C6 through C9 and leads to the formation of a large membranolytic complex capable of lysing prokaryotic and eukaryotic cells [32] . Multiple previous studies have unequivocally shown that trauma activates complement, both locally at the site of injury, and systemically. Early studies in the 1980s revealed that the complement cascade is activated at the level of C3 in serum of trauma patients, and the extent of activation correlates with the severity of injury [33, 34] .
The neutrophil (or PMNL) has been established as the cellular counterpart to the humoral immune response mediated by complement activation, and represents a "key effector" cell of the early posttraumatic immune response. Within minutes, and up to several days after injury, neutrophils play an important role in mounting the immunological defense and the debridement of injured tissue. Primed neutrophils are capable of mediating an inflammatory response, characterized by release of cytokines, chemokines, reactive oxygen species, and tissue-toxic enzymes, such as myeloperoxidase and elastase [20, 35] . Aside from the beneficial role of neutrophils in host-defense and clearance of damaged tissue after trauma, excessive priming and cellular PMNL activation may lead to an overwhelming inflammatory response and "innocent bystander" injury to host tissue [35, 36] . Uninjured tissue may become damaged by the local release of toxic metabolites and enzymes, thus contributing to remote organ injury (e.g. to brain and lungs), by contributing to tissue edema and secondary tissue damage [12, 35, [37] [38] [39] .
Based on the delicate balance between protection and harm, the posttraumatic inflammatory response has been rightfully termed a "double-edged sword" [40] [41] [42] . The present review will outline the current understanding of complement activation and regulation after major trauma, with a focus on specific injury patterns, including musculoskeletal trauma, ischemia/reperfusion, chest and brain injuries. We will furthermore discuss potential new pharmacological strategies related to the targeted inhibition of complement, which may shed some hope into the design of new immunomodulatory treatment modalities for severely injured patients in the future.
The complement system represents one of the phylogenetically oldest cascade systems of the body, consisting of a proteolytic cascade of more than 30 soluble and surface-bound proteins that can be activated by the classical, the lectin and the alternative pathway [32, 43, 44] .
Recently, two additional complement activation pathways have been described, i.e. the properdin and the thrombin pathways, both of which will be discussed in more detail below. Figure 1 depicts a rough schematic of the so far known complement activation pathways and of the biological functions of activated complement components. In brief, the three main activation pathways converge in the formation of enzymatic complexes termed the C3 convertases and C5 convertases, which cleave the two main components of the complement system, C3 and C5. The two proteolytic fragments generated by the action of the convertases are the anaphylatoxins C3a and C5a. Both can trigger proinflammatory signaling through binding to their corresponding receptors, the C3a receptor (C3aR) and C5a receptor (C5aR and C5L2), on various myeloid and non-myeloid cells [28, 29, 45, 46] . C5a is a powerful chemoattractant for neutrophils that recruits immune cells to the site of injury and activates cellular attack mechanisms like oxidative burst and lysosomal enzyme release [47, 48] . Furthermore, the anaphylatoxins contribute to the degranulation of mast cells and basophils, induce the expression of adhesion molecules on endothelial cells, cause smooth-muscle contraction and enhance the acute phase response of the liver [48] . The cleavage of C3 by C3 convertases leads to the generation of a second major fragment, C3b, which acts as an opsonin facilitating the removal of bacteria and cell detritus by phagocytic cells [49] . Finally, the formation of C5b by cleavage of C5 initiates the assembly of a multimolecular complex, the MAC (C5b-9), that perforates membranes of bacteria and nucleated cells and causes rapid cell lysis and death [45, 50, 51] .
Recently, a second initiation mechanism of the alternative activation pathway was described, termed the properdin pathway [52] . Properdin is capable of recognizing several DAMPs and PAMPs on foreign and apoptotic cells, thus allowing C3 convertase assembly on the target surface [32, 52] . Properdin also functions as a stabilizer for C3 convertase complexes of the alternative pathway. In addition to properdin, a fifth complement activation pathway has been described, which identified the clotting factor thrombin as a C5 convertase. This notion was supported by the observation that thrombin is capable of generating C5a in the absence of C3, thus providing a direct link between the complement and coagulation system [53, 54] .
Traumatic brain injury (TBI) induces a profound inflammatory response that contributes to brain edema, neuronal cell death, and adverse outcome [55] [56] [57] . Posttraumatic activation of the complement cascade has been shown to play a pivotal role in the development of secondary brain injury (Table 1) [10, 12, 23, 24, 58, 59] . Multiple experimental Table 1 Insights from experimental complement inhibition based on genetically engineered mice and pharmacological approaches in models of traumatic brain injury (TBI). and clinical studies have revealed elevated levels of complement components and complement activation fragments in serum, cerebrospinal fluid (CSF), and brain parenchyma after head injury [12, 23, 60, 61] . Intracerebral complement deposition after TBI derives either from an altered permeability of a dysfunctional blood-brain barrier (BBB), or from posttraumatic biosynthesis of complement components by resident and infiltrating cells of the central nervous system (CNS) [12, [62] [63] [64] . Most studies have focused on the central complement component C3, and on the potential neuroprotective effects of inhibiting C3 convertases, the level at which the three main activation pathways merge, thus inhibiting downstream complement activation. Clinical studies revealed elevated C3 levels in the CSF of patients with severe TBI [65] . Experimental brain injury models described intracerebral PMNL infiltration and concomitant accumulation of complement C3 in cortical and hippocampal brain sections after experimental TBI in rats [66] . In those studies, C3 accumulation was significantly related to places of intracerebral cell death and to increased intracerebral myeloperoxidase activity [66] . In accordance with these findings, C3-deficient mice were found to have lower neutrophil extravasation and cerebral lesion volumes in a freeze model of brain injury [67] . In light of the central role of C3 and downstream complement activation fragments in the pathophysiology of TBI, much emphasis has been recently devoted to elucidating therapeutic aspects of C3 convertase inhibition, in various experimental model systems [68] [69] [70] [71] [72] . Genetically engineered mice, either deficient in the C3 gene, or with transgenic CNS-restricted overexpression of Crry -a soluble inhibitor of C3 convertases in mice-showed a significant extent of neuroprotection after brain injury, compared to wild-type animals [67, 70] . The GFAP-sCrry transgenic mice showed a significantly improved neurological outcome and an attenuated extent of posttraumatic BBB dysfunction in a model of closed head injury [70] . Based on these insights, the concept of Crry-mediated neuroprotection was extrapolated to a pharmacological approach, by posttraumatic injection of a recombinant chimeric Crry-Ig molecule in the same model of closed head injury [71] . The systemic injection of Crry-Ig during an early therapeutic "window of opportunity" within one hour to 24 hours after trauma resulted in a significant neurological improvement and reduced extent of neuronal cell death, compared to vehicle-injected control mice [71] .
A similar therapeutic approach was tested in a fluid percussion model of brain injury, using recombinant Vaccinia virus complement control protein (VCP), a potent inhibitor of alternative and classical pathway C3 convertases [69, 72] . In these studies, the intracranial administration of VCP mediated neuroprotective effects related to posttraumatic preservation of spatial memory, as compared to vehicle-injected controls [69, 72] .
Further therapeutic approaches were designed to more specifically target "key" effector components of complement activation, such as the anaphylatoxin C5a and its receptor (C5aR, CD88) [29, 67, 73, 74] . In addition, more attention was recently devoted to target specific pathways of complement activation exclusively, in order to overcome the potentially deleterious effects of a complete "shut-down" of complement activation at the central C3 level. This notion is based on the fact that complement also mediates neuroprotective effects in the injured brain, as e.g. shown by a dose-dependent protection of glutamate-induced excitotoxicity against neurons by the C3derived proteolytic fragment, anaphylatoxin C3a [75] , and by C3a-mediated induction of nerve growth factor (NGF) by microglia [76] . Based on the recent concept of a "dual role" for complement in the pathophysiology of brain injury, by promoting both early neurotoxic and late neuroreparative mechanisms after TBI [12, 77, 78] , the exclusive targeting of selected complement pathways was given more consideration, as opposed to the "pan" inhibition at the C3 convertase level [79] [80] [81] [82] . Among these, the targeted inhibition of the alternative pathway has drawn particular attention in recent years [79, 80, 83] . Factor B, the "key" component of the alternative pathway, was previously reported to be significantly elevated in the intrathecal compartment of patients with severe TBI [65] . Experimental studies on factor B-deficient mice (fB-/-), which are devoid of a functional alternative pathway, revealed significant neuroprotection after closed head injury, in conjunction with a decreased extent of posttraumatic complement activation [79] . These positive findings derived from studies in gene knockout mice were extrapolated into a pharmacological approach, using a neutralizing monoclonal anti-factor B antibody (mAb1379) in the same model system [80] . The postinjury injection of mAb1379 led to significantly attenuated extent of complement activation and anaphylatoxin C5a generation, and was associated with an improved neurological recovery and reduced neuronal cell death after experimental closed head injury [80] . These data imply an important role of the alternative complement pathway in contributing to the delayed neuropathology after TBI, and provide strategic opportunities for therapeutic targeting of alternative pathway molecules as a potential future pharmacological strategy.
An additional avenue of research has been focusing on the terminal complement pathway, or "membrane attack" pathway, which results in cellular lysis by the MAC/C5b-9 [51, 84, 85] . In clinical studies, elevated levels of activated soluble MAC/C5b-9 were detected in the CSF of severely head-injured patients [62] . Moreover, the extent of intrathecal complement activation was associated with secondary cerebral insults in TBI patients, including post-injury BBB dysfunction [10, 62, 64] . Experimental studies have revealed that the intracerebroventricular injection of MAC induced a marked upregulation of adhesion molecule expression and leukocyte infiltration in the subarachnoid space and cerebral parenchyma [84] . In addition, MAC injection into hippocampus evoked seizures and neurocytoxic effects in rats [85] . Local MAC deposition in the injured brain was demonstrated in experimental models [86] and in injured human brains [87] . The complement regulatory molecule CD59 represents the main controlling molecule of MAC formation and an essential protector from neuronal cell injury after complement activation [51, 88] . Neurons express CD59 constitutively, as a protective mechanism from autologous "innocent bystander" cell lysis after complement activation in the brain [51, 89] . However, the posttraumatic activation of phosphatidyl-inositol-specific phospholipase C (PI-PLC) after traumatic brain injury renders neurons vulnerable to MAC-mediated lysis by shedding of the glycosyl-phosphatidyl-inositol (GPI)-anchored glycoprotein CD59 from neuronal membranes [88, 90] . A recent experimental study on closed head injury in mice lacking the gene for Cd59a (CD59a -/-) revealed increased susceptibility to brain injury in CD59a -/mice, compared to wild-type littermates [88] . In fact, head-injured CD59a -/mice showed increased neuronal cell death in tissue sections assessed by TUNEL histochemistry, in conjunction with elevated serum levels of neuron specific enolase (NSE), an indirect marker of neuronal injury [88] . These data corroborate the crucial role of the complement regulatory molecule CD59 in protecting neurons from complement-mediated lysis, and emphasize the impact of the terminal complement pathway in contributing to the pathophysiology of delayed neuronal cell death after TBI.
Until present, there is a lack of specific pharmacological therapy designed to avoid induction of secondary brain injuries and delayed neuronal cell death [91] . There have been some significant advances in the field of therapeutic complement inhibitor development, in recent years [43, 74, [92] [93] [94] . While some of these inhibitors have been successfully tested in experimental head injury models (Table 1) [67, 68, 71, 80] , the "bench-tobedside" extrapolation to clinical applications in headinjured patients has yet to be accomplished [91] .
Severe blunt chest trauma with associated pulmonary contusions is characterized by a robust inflammatory reaction which can result in exacerbated lung injury, acute respiratory distress syndrome (ARDS), multiple organ failure, and death [95] [96] [97] [98] [99] . Activation of alveolar macrophages and recruitment of neutrophils into the interstitial and alveolar compartments are followed by the release of an arsenal of proteinases and oxidants causing leakage of the pulmonary microvasculature and destruction of the alveolar epithelium [100] [101] [102] [103] . Various experimental models of lung injury could yield important insights into the critical role of complement activation products, particularly anaphylatoxin C5a, in the pathophysiology of trauma-induced lung inflammation and progressive alveolar injury [28, [104] [105] [106] . Elevated levels of C5a have been described in broncheoalveolar fluid samples from patients with acute lung injury [28, 107, 108] . When C5a was applied intratracheally in rats exposed to an IgG immune complex model, increased intrapulmonary generation of chemokines, accumulation of neutrophils and changes in vascular permeability could be detected [106] . The protective effects of anti-C5a were further corroborated by the observation that the antibody also suppressed release of tumor necrosis factor (TNF) into bronchoalveolar lavage [109] . Furthermore, C5a was shown to be required for TNF-dependent upregulation of intercellular adhesion molecule-1 (ICAM-1), an essential endothelial adhesion molecule required for neutrophil migration [109] . Czermak and colleagues demonstrated that both the in vitro and in vivo blockade of C5a led to significantly reduced production of CXC and CC chemokines [110, 111] .
A proposed model for the current understanding of C5a-mediated inflammatory pathophysiology of acute lung injury is depicted in Figure 2 . Anaphylatoxin C5a has been shown to induce the early release of proinflammatory cytokines by alveolar macrophages, such as TNF and interleukin (IL)-1β [104] . Interaction of endothelial adhesion molecules (e.g. ICAM-1) with their corresponding receptors on neutrophils (e.g. CD11b/ CD18) leads to adhesion and transmigration of neutrophils into the alveoli [104] . Furthermore, release of TNF and IL-1β can also function in an autocrine way and activate alveolar macrophages to generate chemokines [112] . Among these, the different chemokines have been shown to further mediate neutrophil infiltration [113] . Activated neutrophils, alveolar macrophages and epithelial cells release reactive oxygen species and proteinases that cause diffuse alveolar and microvascular damage, thus exacerbating acute lung injury [111] .
The interaction of C5a with its receptors, C5aR (CD88) and C5L2, is crucial for mediating the pulmonary inflammatory response. Bronchial and alveolar epithelial cells have been shown to express the C5aR [114, 115] . Mice lacking the C5aR gene showed a decreased extent of pulmonary inflammation, as characterized by attenuated myeloperoxidase production by neutrophils and decreased vascular leakage [116] .
Furthermore, the use of a specific C5aR antagonist led to similar attenuation of inflammation signs in immune complex-induced lung injury, indicating the C5aR as a predominant effector of the C5a-mediated inflammation in the lung [117] . A recent study could point out that the cellular responses induced by C5a/C5aR interaction are potentiated by a tight connection between complement and Fcγ receptors [118] . Both C5aR and FcγR are known to be expressed on alveolar macrophages [111] . Shushakova et al. found that C5a causes induction of the activating FcγRIII and suppression of the inhibitory FcγRII during lung injury resulting in a pro-inflammatory reaction. Genetic ablation of C5aR expression in mutant mice completely abolished C5a/C5aR-induced regulation of FcγRs and led to decreased intrapulmonary generation of TNF and neutrophil accumulation [118] . Taken together, C5a seems to have a broader critical function through FcγR regulation, thus augmenting inflammation in the lung. In contrast to the C5aR, the effects of C5a are limited by C5L2 that is co-expressed with the C5aR on many cells including neutrophils [119] . Besides of C5a, C5L2 can also bind C5a desArg and potentially additional complement fragments [120] . Gerard et al. could demonstrate a greater influx of inflammatory cells and an enhanced release of IL-6 and TNF in C5L2-deficient mice in the model of immune complex-induced lung injury [121] . This observation proposes an anti-inflammatory role of C5L2 in the lung that seems to counteract C5a/C5aR-mediated inflammation.
The complement-induced pulmonary response after chest trauma has been suggested to depend on a delicate balance between pro-and anti-inflammatory transcription factors [111] . Alveolar macrophage activation is characterized by increased nuclear translocation of nuclear factor(NF)-B and activator protein-1 (AP-1) representing an initial event in the genesis of the inflammatory cascade [112, 122] . In contrast to NF-B and AP-1, the transcription factor STAT3 has emerged as a negative regulator of the inflammatory response [28] . Interestingly, C5a has been shown to be responsible for STAT3 activation in lungs and alveolar macrophages after immune complex-induced lung injury whereas no complement-dependence could be found for activation of AP-1 [122, 123] . STAT3 has been hypothesized to act as a transcriptional mediator for the anti-inflammatory cytokine IL-10, and might contribute to a negative feedback system in acute lung injury [28, 111, 124] . In addition to the above described "classic" lung injury models, a recent study has paid more attention to the immune response after experimental blunt chest trauma induced by a blast wave [104] . Flierl and colleagues reported complement activation after trauma-induced bilateral lung contusion in rats with C5a-dependent perturbations in neutrophil functions. Treatment with anti-C5a antibody abolished functional deficits in neutrophils and reduced intrapulmonary levels of leukocytes and of cytokines [104] .
Taken together, there is evidence from various animal models that support a predominant role of C5a in initiating a cascade of inflammatory events during acute lung injury. If lung trauma is severe, activation of the innate immune system can lead to a dysregulated inflammatory response resulting in ARDS [125] . Elevated levels of C3a and C5a were measured in plasma of patients with ARDS [126] . In addition, experimental complement inhibition led to attenuated pathology in an animal model of lung injury [126] [127] [128] . Thus, it is tempting to speculate that C5a might act as a potential target for immunomodulation after chest trauma [74] , to avoid the deleterious effects of posttraumatic inflammation, which lead to ARDS, multiorgan failure, and death [97, 129] .
Experimental models of musculoskeletal trauma demonstrated that the early posttraumatic inflammatory response is often accompanied by robust generation of complement activation products [66, 104, 105] . However, up to now, the involvement of the complement cascade in bone and cartilage trauma has only been marginally investigated [130] . In recent years, increased attention has been devoted to the investigation of the role of complement in bone biology and fracture healing [131] . Mesenchymal stem cells as progenitor cells of osteoblasts were shown to express the complement receptors C3aR and C5aR, and the complement regulator molecules, CD55 and CD59 [132] [133] [134] . Moreover, osteoblastic differentiation as a key aspect of bone formation and remodeling induces upregulation of a number of complement-related genes, like C1q, C4, C3aR, properdin, C1-inhibitor (C1-INH) and complement factor H [135] . Pobanz and colleagues reported the expression of a functional C5aR by a human osteoblast-like cell line and detected increased osteoblast IL-6 production after stimulation of these cells with C5a [136] . Furthermore, vitamin D3 has been described to regulate C3 production by murine osteoblastic cells both in vitro and in vivo [137] [138] [139] . Complement C3 was postulated to exhibit a modulating influence on the differentiation of bone marrow cells into osteoclasts [139, 140] . Additional studies pointed out that complement appears to be involved in the transformation of chondral precursors to bone tissue during the enchondral ossification process, involving both the classical and alternative pathway complement activation [141, 142] . Consequently, complement components were hypothesized to be also involved in the inflammatory response after musculoskeletal trauma, and in mediating induction of fracture repair processes [131] . A recent study revealed that the C5aR is expressed in fracture callus by differentiated osteoblast, chondroblast-like cells, and osteoclasts [143] . Since fracture healing is known to be delayed in case of additional trauma-induced injuries, it furthermore remains to be examined if systemic complement generation might be the initiator of this delayed recovery after musculoskeletal trauma [144] .
In addition to the role in fracture healing, the effect of complement activation on cartilage destruction after joint injuries has been discussed in recent years [130] . Gene expression analyses demonstrated that chondrocytes express a broad range of complement components and complement regulatory proteins [145] [146] [147] . The origin of complement components in the synovial fluid remains a topic of debate [130, 148] . Aside from chondrocyte-induced biosynthesis, it appears that multiple other non-cartilaginous sources contribute to complement release in the inflamed joint, including synovial cells and infiltrating leukocytes [130] . We recently hypothesized that chondrocytes may release pro-inflammatory cytokines, express neoantigens and undergo enhanced apoptosis after cartilage injury [130] . However, until present, the involvement of the complement system in posttraumatic joint inflammation and the development of posttraumatic osteoarthritis remains poorly understood, and requires further research.
The pathophysiology of musculoskeletal trauma and of skeletal muscle ischemia/reperfusion is summarized in Figure 3 . The oxygen deficit in major trauma, in conjunction with subsequent reperfusion of ischemic tissues has been recognized as a trigger of an intense inflammatory response that may cause damage both locally in the affected muscle and also in remote organs primary not involved in the ischemic insult [149] [150] [151] [152] . Complement activation and consumption represents a critical event in the early phase of limb ischemia/reperfusion (I/R) injury resulting in the release of potent complement fragments like C3a and C5a [150, 153, 154] . It has been suggested that binding of preexisting natural IgM antibodies to neoantigen expressed by hypoxic cells after interruption of the blood flow is responsible for the activation of the classical complement pathway that importantly contributes to skeletal muscle I/R injury [155] [156] [157] . This hypothesis is strengthened by the fact that mice genetically deficient of mature B and T cells and natural antibodies (Rag1 -/mice) show significant reductions of tissue damage in a model of hindlimb ischemia and reperfusion [155, 158] . Furthermore, muscle edema and secondary neutrophil accumulation in the lung, as signs of reperfusion injury, were attenuated in C1q -/and C4 -/mice deficient in central components of the classical complement pathway [159, 160] . Aside from the classical pathway, recent data indicate important involvement of the classical and the lectin pathway in skeletal muscle I/R injury [159, 161] . A protective effect was attributed to the complement regulatory molecules decay-accelerating factor (DAF/CD55), C1-INH, and soluble complement receptor type 1 (sCR1) after skeletal muscle reperfusion injury [162] [163] [164] . Moreover, a pivotal role of C5a in causing lung damage after hindlimb I/R was shown in an experimental study in rats [165] . In accordance with this observation, multiple markers of local and remote organ injury were markedly reduced in C5-deficient mice, and in mice treated with a neutralizing C5aR antagonist [74, [166] [167] [168] .
In summary, complement activation appears to play a significant role in contributing to post-injury inflammation in musculoskeletal trauma, including fractures, cartilage injury, and skeletal muscle I/R injury.
Polytrauma is characterized as a syndrome of multiple injuries with defined severity which leads to a massive systemic immune activation and to secondary dysfunction and failure of remote, initially uninjured, organs [1, [5] [6] [7] . Clinical studies have demonstrated that complement activation occurs in plasma of patients after major trauma, as early as at the time of presentation in the emergency department [169] [170] [171] . The extent of complement-mediated inflammation was correlated with injury severity, tissue hypoperfusion, and posttraumatic [171, 172] . Serum levels of C3 and C3a were identified as markers of injury severity and outcome in multiply injured patients [173, 174] . Moreover, expression profiles of complement regulatory molecules and of the anaphylatoxin C5a receptor (C5aR/CD88) appeared to be significantly altered in leukocytes of multiply injured patients during the early phase of polytrauma, compared to blood samples from healthy volunteers [175] . The expression profiles of CD46 (membrane cofactor protein; MCP), CD59, and C5aR (CD88) on neutrophils correlated inversely with the severity of injury, an observation which was attributed to an intriguing trauma-induced "complementopathy" in multiply injured patients [175] .
Sepsis represents a lethal complication of major trauma, characterized by an uncontrolled complement activation, as determined by significantly elevated plasma levels of C3a, C4a and C5a [176] [177] [178] . The anaphylatoxin C5a appears to represent the central molecule in the development of the overwhelming inflammatory response in sepsis, and has been coherently described as "too much of a good thing" [179] [180] [181] (Figure 4 ). Blockade of C5a was linked to improved survival in different experimental models of sepsis [182] [183] [184] [185] . Persistent elevation of C5a during progressive sepsis was related to a posttraumatic immunparalysis with "shutdown" of crucial neutrophil functions, including a loss of chemotactic and phagocytotic activity, impairment of the oxidative burst, and disturbances in intracellular signaling pathways [48, 186, 187] . Recent studies corroborated an important contribution of C5a in modulating apoptosis in different cell types during sepsis. While apoptosis rates in neutrophils were shown to be significantly attenuated during sepsis, lymphocytes, thymocytes and adrenal medullary cells exhibited increased C5a-dependent susceptibility to programmed cell death [188] [189] [190] [191] [192] . The latter phenomenon was hypothesized to be responsible for impaired adreno-medullary catecholamine release predisposing the development of septic shock [191] . Excessive C5a levels during sepsis were furthermore associated with reduced myocardial contractility and cardiac output, a phenomenon described as "cardiomyopathy of sepsis" [193] . In general, multiple organs seem to be put at increased risk for C5a-mediated damage induced by an abrupt upregulation of the C5aR in a variety of tissues (heart, lung, kidney, liver, thymus) in early phases of sepsis [194, 195] . A recent study implied that C5a-mediated signaling through the two C5a receptors (CD88 and C5L2) contributes to adverse outcome from sepsis [196, 197] . In experimental models of sepsis, the blockade of C5a and its receptors has been shown to protect end-organ function and to improve outcomes, thus providing a future new avenue for pharmacological treatment of this detrimental complication of major trauma [198] [199] [200] [201] . Future studies will have to be designed to validate this promising notion in a clinical setting.
In recent years, multiple experimental and clinical studies have substantiated the notion of "key" role of complement activation after major trauma in contributing to the deleterious pathophysiological sequelae in the injured brain, lungs, and musculoskeletal system. Complement activation furthermore significantly contributes to the mechanisms of systemic post-injury complications, such as I/R injury, sepsis, and multiple organ failure. Therapeutic options aimed at attenuating the inflammatory complications of major trauma are currently unsatisfactory, and research strategies have largely failed in extrapolation from "bench to bedside". Experimental data from recent animal studies highlight the potential for complement inhibitors aimed at targeting central complement components and specific complement activation products, as promising future pharmacological agents in patients with major trauma. In this regard, site-targeted complement inhibition by new generation chimeric molecules which link pharmacological inhibitors to the local site of complement activation and tissue deposition may represent the future pharmacological "golden bullet". These chimeric molecules act locally at the site of injury and inflammation, and thus avoid the unwanted negative and adverse effects of a systemic complement blockade. Clearly, there is a tremendous need for well-designed experimental studies to shed some further light into our understanding of the complement-mediated pathology of major trauma, with the hope of designing and implementing new clinical treatment strategies for severely injured patients in the near future. Predicting Biological Functions of Compounds Based on Chemical-Chemical Interactions Given a compound, how can we effectively predict its biological function? It is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. In this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) Carbohydrate Metabolism, (2) Energy Metabolism, (3) Lipid Metabolism, (4) Nucleotide Metabolism, (5) Amino Acid Metabolism, (6) Metabolism of Other Amino Acids, (7) Glycan Biosynthesis and Metabolism, (8) Metabolism of Cofactors and Vitamins, (9) Metabolism of Terpenoids and Polyketides, (10) Biosynthesis of Other Secondary Metabolites, (11) Xenobiotics Biodegradation and Metabolism. It was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. Besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. Furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. Interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. It is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions. Metabolism refers to a collection of chemical reactions in vivo, which keep an unceasing supply of matter and energy for living organisms to maintain life (e.g., growth and reproduction) [1] . These energy-using and energy-releasing chemical reactions catalyzed by enzymes are organized into many metabolic pathways. Some compounds/small molecules play major roles in these pathways and are vital for many activities essential for life. For example, during the digestion, the energy rich molecules (i.e. carbohydrate) are broken apart to provide energy, which is then used by cells to build up complex molecules from simple molecules, such as utilizing amino acids to synthesize new proteins that the body needs. Identifying the biological functions of compounds is an effective way to study the mechanisms of many basic biological processes [2] . On the other hand, small molecules are the cause, and the cure, for many diseases. For example, diabetes mellitus is a metabolic disease caused by insufficient or inefficient insulin secretary response and elevated blood glucose level [3] . Compounds such as sulfonylureas [4] , acarbose [5] , biguanides, thiazolidinediones [5] , and sitagliptin [3] have been used as effective drugs for diabetic therapy. Therefore, it is essential to annotate the bioactivities of compounds, which will benefit drug design and disease treatment.
Besides the conventional biochemical experiments, computational methods are alternative ways to annotate the biological functions of compounds. In recent years, various bioinformatics and structural bioinformatics [6] tools were developed to address this issue, such as Quantitative Structure Activity Relationship (QSAR) [7, 8] , pharmacophore modeling [9] , molecular docking [10] , and Monte Carlo simulated annealing approach [11, 12] . Different from these methods, Lu et al. [1] and Cai et al. [2] analyzed the biological functions of compounds by mapping them to the corresponding metabolic pathway classes, which are strongly associated with the biological functions of compounds. The functional group composition was used to represent the compounds, and the Nearest Neighbor Algorithm and AdaBoost learner [13] were used to construct the prediction models by Cai et al. [2] and Lu et al. [1] , respectively. Both the two prediction methods achieved quite promising results on their own datasets.
However, none of their datasets contained the ''multi-function'' compounds that belong to two or more metabolic pathway classes. Since these authors were only focused on addressing the singlelabel classification problem, their methods could not be used to deal with the ''multi-function'' compounds. Actually, according to KEGG [14] , among all the compounds with functional annotations, the ''multi-function'' compounds occupy about 8%. Particularly, these multi-function compounds may play some unique role intriguing to both basic research and drug development and hence are worthy of our special attention.
Recently, the systems biology methods based on protein-protein interactions have been widely applied for predicting protein attributes [15, 16, 17, 18, 19] . These algorithms suggest that interactive proteins are likely to share the common biological functions [16, 17, 18, 19] , also more likely tending to have the same biological function than non-interactive ones [20, 21] . Likewise, we can assume that the interactive compounds may tend to share the common biological functions. In this study, the chemical-chemical interactions were retrieved from STITCH [22] (Search tool for interactions of chemicals), where the interaction unit consists of two chemicals and their interaction weight. The interaction weight (confidence score) represents the probability that the interaction occurs between the two chemicals concerned. The interactive compounds can be classified into the following three categories: (I) ones that participate in the same reactions; (II) ones that share the similar structures or activities; (III) ones with the literature associations [22] . In a metabolism system, chemical reactions are organized into many metabolic pathways, thus the compounds involved in the same reactions are in the same metabolic pathways. Similar structures or activity means that they share the similar functions, and hence they are likely to be in the same metabolic pathways. The co-occurrence of two compounds in many literatures suggests some kinds of direct or indirect relationships, indicating they have the potential to be in the same metabolic pathways. Accordingly, it is rational to suppose that the interactive compounds tend to participate in the same metabolic pathways.
In this study, we proposed a multi-target model based on chemical-chemical interactions for predicting the metabolic pathways where compounds participate in. Our method sorts the possible metabolic pathways that are associated with the query chemical, providing a more comprehensive view of the biological effects of the compound.
According to a recent comprehensive review [23] , to establish a really useful statistical predictor for a biological system, we need to consider the following procedures: (1) construct or select a valid benchmark dataset to train and test the predictor; (2) formulate the statistical samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the attribute to be predicted; (3) introduce or develop a powerful algorithm (or engine) to operate the prediction; (4) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor. Below, let us describe how to deal with these steps.
The compounds were retrieved from public available database KEGG [14] (Kyoto Encyclopedia of Genes and Genomes) compound
[ftp://ftp.genome.jp/pub/kegg/release/archive/ kegg/42/ligand.tar.gz] (release 42.0). Subsequently, these compounds were mapped to the following 11 major metabolic pathway classes that are strongly associated with the biological functions of compounds (http://www.genome.jp/kegg/pathway. Table 1 under the title of Group-I). From the 4,366 compounds of Group-I, 3,137 compounds were retrieved that can interact with any of the others as annotated by STITCH database [22] (see Table 1 under the title of Group-II).
Of the 4,366 compounds of Group-I, 4,027 are involved in only one metabolic pathway class, 246 in two metabolic pathway classes, 54 in three metabolic pathway classes, 24 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes. Of the 3,137 compounds of Group-II, 2,820 are involved in only one metabolic pathway class, 226 in two metabolic pathway classes, 53 in three metabolic pathway classes, 23 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes.
Note that since one compound may occur in more than one pathway class, the sum of the compounds over the 11 pathway classes in Group-I turns out to be 4,860, which is greater than 4,366. Likewise, the sum of the compounds over the 11 pathway classes in Group-II is 3,606, which is greater than 3,137. This is quite similar to the case of proteins with multiple location sites, as elaborated in [24, 25] .
The chemicals interactions were retrieved from STITCH [22] , a large database of known and predicted interactions of chemicals and proteins derived from experiments, literature, databases, and so on. As mentioned in Introduction, there are three types of associations between two compounds in STITCH: (I) cooccurrence in reactions, (II) similar structures or activities, and (III) literature associations. In the downloaded STITCH chemicals interactions file: chemical_chemical.links.detailed.v2.0.tsv from http://stitch.embl.de/cgi/show_download_page.pl, there are 337,482 pairs of interactive compounds belonging solely to type I, 73,598 pairs solely in type II, 2,152,508 pairs solely in type III, 384 pairs in both type I and II, 120,936 pairs in both type I and III, 10,372 pairs in both type II and III, and 1,990 pairs in the three types, in total of 2,697,270 interactions. Each of the interaction is quantified by the interaction confidence score, which represents the likelihood that the interaction occurs. In this study, the interactions with both interactive compounds occurring in the 4,366 compounds of Group-I were extracted. As a result, 3,137 compounds with 75,949 interactions were collected to constitute the benchmark dataset of the current study (see Table 1 under the title of Group-II).
Besides the 4,366 compounds (cf. Table 1 under the title of Group-I) with known metabolic pathway classes, there are 11,661 compounds without known metabolic pathway classes in KEGG. Among these compounds, 5,549 compounds that have annotated interactions with the compounds of the 4,366 compounds in STITCH were collected. Such 5,549 compounds are to form an independent dataset, being used to test our prediction method in hopes to acquire useful information for further investigation.
As mentioned in Introduction, the interactive compounds tend to participate in the same metabolic pathways. Accordingly, for a query compound, the higher interaction confidence score with its interactive compound, the more likely they are to participate in the same metabolic pathway. The more its interactive compounds involving in a certain metabolic pathway, the more likely it is to participate in such metabolic pathway. Based on these points, we should count not only the number of compounds interacting with the query compound, but also the corresponding interaction scores. Thus, the desired predictor can be formulated via the following procedures.
Suppose the training dataset contains n compounds, which are denoted as fC 1 ,C 2 ,:::,C n g. The 11 metabolic pathway classes (cf. Table 1 ) are expressed as fP 1 ,P 2 ,:::,P 11 g, where P 1 represents the 1 st metabolic pathway class (''Carbohydrate Metabolism''), P 2 the 2 nd metabolic pathway class (''Energy Metabolism''), P 3 the 3 rd metabolic pathway class (''Lipid Metabolism''), and so forth. Thus, the descriptor of metabolic pathway classes to which the compound C i belongs to can be formulated as P(C i )~½p i,1 ,p i,2 ,:::,p i,j ,:::,p i,11 T (i~1,2,:::,n; j~1,2,::
where
Given a query compound C q , its interaction with the compounds in the training dataset can be defined as W (C q )~½w q,1 ,w q,2 ,:::,w q,i ,::
where w q,i represents the interaction confidence score between C q and C i . T is the transpose operator, and w q,i~0 if no interaction exists between them. Here, we did not consider the selfinteraction, therefore w q,i~0 when q~i. Accordingly, the likelihood that the query compound C q is involved in the j-th metabolic pathway class can be formulated by the following score
which is the sum of the interaction confidence scores of C q with its interactive compounds in the training dataset by counting both the number of interactive compounds and the interaction confidence scores. Obviously, the higher the score of Eq. 4, the more likely C q is to be involved in the j-th metabolic pathway C j . Thus, for a given query compound C q , we can use Eq. 4 to calculate its 11 scores, with each associated with one of the 11 metabolic pathway classes. The class to which the compound C q most likely belongs should be the one with the highest score. In other words, the query compound C q will be predicted to belong to the mth metabolic pathway class if m~arg max j S(C q [j)jj~1,2,:::,11
where m is the argument of j that maximize the value of S(C q [j).
Since the problem in this study is of multi-label classification, we intend to provide flexible information by predicting some candidate metabolic pathway classes for the query compounds, rather than just the most likely metabolic pathway class. Therefore, instead of Eq. 5, let us consider the following equation containing 11 scores in a one-column vector: where D ; is a descending operator that sorts the 11 scores of Eq. 4 for S(C q [j) according to the descending order (S 1 §S 2 § Á Á Á §S j § Á Á Á §S 11 ). If there is a tie among these scores, a random order will be made among those with a tie. Consequently, the predicted metabolic pathway classes for the query compound can be derived according to the descending order of Eq. 6; i.e., if S 1~S (P k [6), S 2~S (P k [1), S 3~S (P k [10) , then it follows that the query compound C q is involved in the 6 th metabolic pathway class (''Metabolism of Other Amino Acids'') will be ranked as the highest in the likelihood, that C q in the 1 st metabolic pathway class (''Carbohydrate Metabolism'') as the 2 nd , and that C q in the 10 th metabolic pathway class (''Biosynthesis of Other Secondary Metabolites'') as the 3 rd . The corresponding results thus obtained are, respectively, called the 1 st -order, 2 nd -order, and 3 rd -order predicted metabolic pathway classes. And so forth.
In statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (such as 5-fold, 7-fold, or 10-fold cross-validation) test, and jackknife test [26] . In this study, the 5-fold cross-validation was employed to examine the performance of our method. The concrete procedures were that the training dataset were divided into five groups by splitting each of its subsets into five approximately equal-sized subgroups. Each of these five groups was in turn used as a testing dataset and the rest used as training dataset, thereby generating five different success rates, with their average representing the success rate by the 5-fold cross-validation.
For the j-th order prediction, the accuracy W j was calculated by 
where M j is the number of the compounds whose j-th order predicted metabolic pathway class is one of the true pathway classes that the compounds are involved with, and N is the total number of compounds in the dataset. Such 11-order accuracies were used to evaluate our prediction method. It is obvious according to the definition of Eq. 7 that, the higher the value of W j with a smaller value of j, or the lower the value of W j with a larger value of j, the better the prediction quality will be by our method.
In the dataset, the average number of metabolic pathway class that each compound is involved in is calculated as
where E i is the number of metabolic pathway classes that the compound C i is involved with. Hence, another measurement -the likelihood that the first k order predicted metabolic pathway classes cover all the true metabolic pathway classes that the compound is involved in -can be formulated as
Usually, k is the smallest integer equal or greater than the average number of metabolic pathway classes (H). It is obvious from Eq. 9
that the larger the value of L k , the better the prediction quality will be by our method.
Given a query compound, according to the information of its interactions with the 4,366 compounds in Group-I ( Table 1 ) whose metabolic pathway classes are known, the likelihood of its belonging to each of the 11 metabolic pathway classes can be easily calculated according to Eq. 4. And the scores thus obtained were sorted according to a descending order (Eq. 6) to yield the predicted metabolic pathway classes according to their different ranks or orders.
In this study, our method was evaluated by the 5-fold crossvalidation on the benchmark dataset that contains 3,137 compounds in Group-II of Table 1 . The 11-order prediction accuracies are shown in Figure 1 . The first order (most likely) prediction accuracy is 77.97%, and the last order (least likely) prediction accuracy is 0.38%, which indicates a quite good performance of our method.
The average number of metabolic pathway classes with which each compound is involved is 1.15 (cf. Eq. 8), meaning that the average success rate by a random guess would be 1.15/ 11 = 10.45%, which is much lower than that by our method.
Accordingly, the parameter k in Eq. 9 was set to (1.15+1) = 2; i.e., we may select the results of the first two orders of the predicted metabolic pathway classes for the query compounds. As we can see from Figure 1 , the accuracies of both the 1 st and 2 nd order predictions are higher than that of the random guess. According to Eq. 9 the metabolic pathway classes predicted by the 1 st and 2 nd orders have actually covered more than 80% of all the true metabolic pathway classes, suggesting that, of the results predicted by the 11 orders, more attention should be paid to those by the first two orders.
Listed in Table 2 are the accuracies by each of the 11 prediction orders for the 3,137 compounds about their involvement in the 11 metabolic pathway classes using the 5-fold crossvalidation test. The highest accuracy achieved by the 1 st -order prediction was 80.96% for the 1 st metabolic pathway class (''Carbohydrate Metabolism''). And the results obtained by the 1 st and 2 nd prediction orders have covered 89.00% of the true metabolic pathway classes. The second highest accuracy by the 1 storder prediction was 78.77% for the 11 th metabolic pathway class (Xenobiotics Biodegradation and Metabolism), while the results obtained by the 1 st and 2 nd prediction orders have covered 87.00% of the true metabolic pathway classes. Both the two 1 st -order accuracies are higher than the overall 1 st -order prediction accuracy of 77.97%, and each of their combinations with the 2 nd -order predictions is also higher than the overall likelihood of 80.00%. As for the metabolic pathway classes with less compounds, such as ''Glycan Biosynthesis and Metabolism'' class that contains only 68 compounds in Group-I and 43 in Group-II (cf . Table 1) , the predicted accuracies were relatively not as good as the others. It is anticipated that with more experimental data are available in future for the compounds in these classes, the corresponding prediction success rates will be improved. Overall speaking, the aforementioned results are quite encouraging, indicating that our approach may become a useful tool to deal with this kind of very complicated systems.
As stated in the Method section, the interactive compounds derived from STITCH tend to participate in the same metabolic pathways. For example, Table 3 lists the interactions of dihydrouracil with other compounds. Among the 32 interactive compounds, most of them appear in ''metabolism of cofactors and vitamins'' or ''metabolism of other amino acids'' or ''nucleotide metabolism'' pathway class (cf. Table 1 ) just like dihydrouracil. Dihydrouracil and uracil participate in pyrimidine metabolism pathway (belong to ''nucleotide metabolism''), where 5,6-dihydrouracil and NADP+ are catalyzed by dihydropyrimidine dehydrogenase (DPD) to form uracil and NADPH+H+ [14, 27] . They are also co-mentioned in many PubMed Abstracts such as [28, 29, 30, 31, 32, 33, 34, 35, 36, 37] . Another two interactive compounds -dihydrouracil and dihydrothymine share a very similar structure, the only difference is that dihydrothymine has a methyl at the 5th position of the hexatomic ring while dihydrouracil has not [38] . According to the prediction criteria, when dihydrouracil was treated as a query compound, the first three order predicted metabolic pathways that it participates in are ''nucleotide metabolism'', ''metabolism of cofactors and vitamins'' and ''metabolism of other amino acids'', respectively, which are consistent with the true metabolic pathways that it is involved in.
Predicted results for the compounds with unknown metabolic pathway Encouraged by the quite promising results obtained by the 5fold cross-validation test on the benchmark dataset of the 3,137 compounds, we applied the method to the 5,549 compounds whose metabolic pathways are unknown as mentioned in the Materials and Methods section. The predicted results thus obtained are given in Table S1 . As discussed above, we selected the metabolic pathway classes obtained by the 1 st and 2 nd order predictions for these compounds, in hoping that the information thus obtained may provide useful clues for further investigations. Actually, it is interesting to see that many of our predicted results have proved to be reasonable according to the reports from other investigators. For example, N-acetylgalactosamine 4-sulfate and its interactive compounds with pathway information are shown in Table 4 . N-acetylgalactosamine 4-sulfate can bind to sulfate, glucuronic acid, galactose, xylose, fucose, Na(+), glycerol, and phosphate to form complex to perform the biological function [39] . In PubMed Abstracts, N-acetylgalactosamine 4-sulfate is comentioned with sulfate [40] , glucuronic acid [41] , galactose [42] , 39-phospho.pho. [43] , sugar-1-phosph. [44] , UDP-GlcNAc [45] , indole-3-glyce. [46] , N-acetyl-D-glucosamine [47] , and GDPmannose [44] . Besides, N-acetylgalactosamine 4-sulfate and Nacetyl-D-glucosamine share a similar structure and the difference is that N-acetylgalactosamine 4-sulfate has a sulfate at the position 4 of the ring while N-acetyl-D-glucosamine has not [38] . From these evidences, N-acetylgalactosamine 4-sulfate is supposed to participate in the same metabolic pathways as its interactive compounds. It can be seen from Table 4 that most of the interactive compounds of N-acetylgalactosamine 4-sulfate belong to the 1 st and 2 nd metabolic pathway classes. By considering all the interactions and the interaction confidence scores, it was predicted that Carbohydrate Metabolism (the 1 st class) and Energy Metabolism (the 2 nd class) would be the possible metabolic pathway classes that N-acetylgalactosamine 4-sulfate belongs to. Actually, as a carbohydrate, N-acetylgalactosamine 4-sulfate reacts with Chondroitin 4-sulfate to form hydrogen oxide and G12336 (i.e. (GalNAc) 2 (GlcA) 1 (S) 2 ), one kind of glycan which can participate in Carbohydrate and Energy Metabolism. Therefore, N-acetylgalactosamine 4-sulfate may also participate in Carbohydrate and Energy Metabolism. Another example is that cyclopropylamine in Table 4 has 23 interactive compounds with known pathway information. Cyclopropylamine, cyanuric acid, ammonia, N-cyclopropylammelide, c0761, hydroxyl radicals are in the same pathway -N-cyclopropylmelamine degradation [48, 49] , where N-cyclopropylmelamine first reacts with hydrogen oxide to form N-cyclopropylammeline and ammonia, and then N-cyclopropylammeline also reacts with hydrogen oxide to form Ncyclopropylammelide and ammonia. After that, N-cyclopropylammelide reacts with hydrogen oxide to form cyanuric acid, cyclopropylamine and hydroxyl radicals. Finally, cyanuric acid is transformed into hydrogen oxide and ammonia through cyanurate degradation. Cyanuric acid, N-cyclopropylammelide, and c0761 are all in the 11 th pathway class. Therefore, cyclopropylamine may also belong to the 11 th pathway class (Xenobiotics Biodegradation and Metabolism). For other interactive compounds, they are comentioned with cyclopropylamine in PubMed Abstracts, such as polyethylene [50] , 1-aminocyclopropane-1-carboxylic acid [51] , cyclopropanecarboxylic acid [52] , 3-hydroxyphenylacetic acid [53] , and acetophenone [54] . In Table 4 , most of the interactive compounds of cyclopropylamine belong to the 11 th metabolic pathway classes. According to above analysis, cyclopropylamine is suggested to participate in the Xenobiotics Biodegradation Metabolism, which was the 1 st -order predicted class for cyclopropylamine by our method. Accordingly, it is quite reasonable to expect that our method may provide useful information for further investigating into biological functions of compounds from the viewpoint of system biology.
As indicated by the above discussion and analysis, the results derived from the 1 st and 2 nd order predictions should be considered as the candidates for the metabolic pathway classes with which the query compound may be involved. In view of this, biochemical experiments should be conducted by mainly focusing on the targets predicted by the 1 st and 2 nd order predictions. The results obtained by the last five order predictions can be ignored due to their very low likelihood (,2%). Consequently, the current prediction method can provide useful clues for further validation by experiments and expedite the research progress by prioritizing the targets concerned.
It is instructive to note that for the 4,366 compounds in Group-I of Table 1 , there are still 1,229 compounds that can not be processed by the current method due to lack of the interaction information with other compounds within the dataset. It is expected that the problem can be solved by collecting as much chemical-chemical interaction information as possible from STITCH, which is a large-scale and well-maintained resource in chemical biology, including the interactions information for over 2.5 million proteins and over 74,000 small molecules in 630 organisms. With the continuous increase of the interactions information, the performance of our method will be further improved.
Based on the chemical-chemical interactions information, a multi-target model was proposed for identifying the metabolic pathway classes with which a query compound is involved. Since some compounds may be involved with more than one metabolic pathway class, our method is featured by the capacity able to provide a series of potential metabolic pathway classes for each of the query compounds investigated, instead of only one metabolic pathway class. It is anticipated that our method may become a useful tool in helping annotate the compound for their biological functions.
Table S1 Each order predicted metabolic pathway class for the collected 5,549 compounds without known metabolic pathway classes. The predicted metabolic pathway class code corresponds to the code in Table 1 . Among the 11 predicted pathway classes, the first 2 order predicted metabolic pathway classes should be paid more attention to. (PDF) Identification and Characterization of a Novel Non-Structural Protein of Bluetongue Virus Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell. Bluetongue is a major infectious disease of ruminants caused by an arbovirus (Bluetongue virus, BTV) transmitted by biting midges (Culicoides spp.) [1] [2] [3] . Historically, bluetongue has been endemic almost exclusively in temperate and tropical areas of the world where the climatic conditions favour both the spread of the susceptible insect vector population and the virus replication cycle within the vector [4] . However, in the last decade BTV has spread extensively in several geographical areas including Southern Europe and also, unexpectedly, in Northern Europe causing a serious burden to both animal health and the economy [5, 6] .
From a molecular and structural virology perspective BTV is one of the best understood animal viruses. BTV is a member of the Orbivirus genus, within the Reoviridae family, and possesses a doublestranded RNA genome formed by 10 segments (Seg-1 to Seg-10) of approximately 19200 base pairs in total [1, 3] . Until now, the BTV genome has been shown to encode for 7 structural and 3 non-structural proteins. The BTV genome is packaged within a triple layered icosahedral protein capsid of approximately 90 nm in diameter [1, [7] [8] [9] [10] . The outer capsid of the virion is composed by 60 trimers of VP2 and 120 trimers of VP5 [11] and differences within this outer capsid define the 26 BTV serotypes which have been described so far [12, 13] . The outer capsid proteins, and VP2 in particular, stimulate virus neutralizing antibodies which in general protect only against the homologous serotype [14] . The internal core is formed by two layers, constituted by VP3 (subcore) and the immunodominant VP7 (intermediate layer) [7] . Three minor enzymatic proteins, VP1 (RNA dependent RNA polymerase), VP4 (capping enzyme and transmethylase) and VP6 (RNA dependent ATPase and helicase) are contained within the core that is transcriptionally active in infected cells [15] [16] [17] [18] [19] [20] [21] .
The BTV genome encodes also 3 non-structural proteins: NS1, NS2 and NS3/NS3a. NS1 and NS2 are highly expressed viral proteins and their multimers are morphological features of BTVinfected cells. Multimers of the NS1 protein form tubules (approximately 50 nm in diameter and up to 1000 nm in length) that appear to be linked to cellular cytopathogenicity [22] , while NS2 is the major component of the viral inclusion bodies. NS2 plays a key role in viral replication and assembly as it has a high affinity for single stranded RNA and possesses phosphohydrolase activity [23] . NS3/NS3a are glycosylated proteins involved in BTV exit. There are two isoforms of NS3: NS3 and NS3a with the latter lacking the N-terminal 13 amino acid residues [24] [25] [26] .
Therefore, the segmented genome of BTV has been thought to be monocistronic (i.e. ten genome segments encoding for 10 proteins) for almost three decades [27, 28] . Segment 9 however, contains the open reading frame (ORF) encoding VP6 but also a smaller coding sequence in the position +1 reading frame that is present in BTV and some related Orbiviruses such as African horse sickness virus and others [29] . Bioinformatic analysis predicts that the BTV ''ORFX'' encodes for a protein of 77-79 amino acid residues. This putative ORFX is subject to functional constraints at the amino acid level and its level of conservation is higher compared to that of the overlapping VP6. In addition, the ORFX putative AUG initiation codon has a strong Kozak context suggesting that this protein might be translated by leaky scanning [29] . Alternative reading frames are expressed in a variety of RNA viruses and they can play fundamental roles in viral replication and virus-host interaction. In this study, we identified a previously unknown non-structural protein and characterized its biological properties.
All experimental procedures carried out in this study are included in protocol number 5182/2011 of the Istituto G. Caporale 
Initially, the open reading frame expressing ORFX (NS4) was amplified by PCR from BTV-10 (GenBank accession number D00509) and cloned into the pCI Mammalian Expression Vector (Promega) resulting into pCI-NS4. The BTV-8 NS4 was cloned into the peGFP-N1 vector (Clontech), resulting in plasmid pNS4-GFP. pNS4 7-77 -GFP, pNS4 13-77 -GFP and pNS4 19-77 -GFP are mutants derived from pNS4-GFP expressing NS4 truncated of the amino terminal 6, 12 and 18 amino acid residues, respectively. pNS4 7-77 -GFP, pNS4 13-77 -GFP and pNS4 19-77 -GFP maintain the methionine and valine residues in position 1 and 2 of NS4. Note that BTV-10 and BTV-1 NS4 are 100% identical at the amino acid level. While BTV-8 and BTV-1 NS4 differ for a single amino acid residue in position 6. The set of BTV-1 and BTV-8 plasmids necessary to rescue these viruses in vitro by reverse genetics were obtained following the method recently published by Boyce and colleagues [26] . Briefly, total RNA was extracted from infected cells using Trizol (Invitrogen) according to the manufacturer's instructions. Each BTV genome segment was amplified by RT-PCR using the AccuScript PfuUltra II RT-PCR Kit (Agilent) from either BTV-1 or BTV-8 dsRNA preparations and the resulting PCR products were gel-purified (Qiagen) and cloned into either pUC57 (Fermentas) or pCI. Each BTV segment was cloned downstream of a T7 promoter and upstream of a BsaI or SapI restriction site. All of the mutants described in this study were obtained using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. All plasmids used in this study were completely sequenced before use. Sequences of PCR primers used in this study are available upon request. Antisera used in this study included polyclonal rabbit antisera raised against BTV VP7, NS1, NS2, NS3 and ORFX (NS4) expressed in bacteria as Glutathione S-transferase (GST)tagged recombinant proteins (Proteintech Group, Inc.). Antiserum against BTV-1 NS4 was raised against a recombinant GST fusion protein including the entire NS4 protein expressed in bacteria. Polyclonal rabbit antiserum against BTV VP6 was kindly provided by Polly Roy as previously described [32] . Antibodies against B23 and c-tubulin were obtained commercially (Sigma Aldrich).
BTV-8 (IAH reference collection number NET2006/04) was originally isolated from a naturally infected sheep during the 2006 outbreak in Northern Europe [33] . The virus was passaged once in KC cells and once in BHK 21 cells. The reference strain of BTV-1 was originally isolated at the ARC -Onderstepoort Veterinary Institute (IAH reference collection number RSArrrr/01) and was adapted to cell culture by passaging it twice in embryonated eggs and 9 times in BHK 21 cells. Both viruses were kindly provided by Peter Mertens. Virus stocks were prepared by infecting BSR cells at a multiplicity of infection (MOI) of 0.01 and collecting the supernatant when obvious cytpopathic effect (CPE) was observed. The supernatants were clarified by centrifugation at 500 g for 5 min and the resulting virus suspensions aliquoted and stored at 4uC for short term usage and at 270uC for long term storage. Virus titres were determined by standard plaque assays using BSR or CPT-Tert cells [34] .
Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an ''arbovirus'' transmitted from infected to susceptible hosts by biting midges. Historically, bluetongue has been endemic almost exclusively in temperate and tropical areas of the world. However, in the last decade BTV has spread extensively in several geographical areas causing a serious burden to both animal health and the economy. BTV possesses a double-stranded RNA segmented genome. For over two decades, it has been widely accepted that the 10 segments of BTV genome encode for 7 structural and 3 nonstructural proteins. In this study we discovered that BTV expresses a previously uncharacterized non-structural protein that we designated NS4. Although BTV replicates exclusively in the cytoplasm, we found NS4 to localize in the nucleoli of the infected cells. Our study shows that NS4 is not needed for viral replication both in mammalian and insect cells, and in mice. However, NS4 confers a replication advantage to BTV in cells in an antiviral state induced by interferon. In conclusion, we have elucidated a possible route by which BTV can counteract the defences of the host. Sequence analysis 65 full-length segment 9 sequences representing 24 BTV serotypes were obtained from GenBank. Amino acid conservation plots and secondary structure predictions were obtained using the CLC Genomics Workbench (CLC, Aarhus, Denmark) software and bioinformatics tools available online (the PSIPRED server [http:// bioinf.cs.ucl.ac.uk/psipred], and the Network Protein Sequence Analysis (nps@) server, [http://npsa-pbil.ibcp.fr/]).
Recombinant BTVs were rescued by reverse genetics as previously described [26] . Briefly, plasmids containing the genomic segments of BTV-1 or BTV-8 or resulting mutants were linearized with the appropriate restriction enzymes and then purified by phenol-chloroform extraction. Digested plasmids were used as a template for in vitro transcription using the mMESSAGE mMA-CHINE T7 Ultra Kit (Ambion), according to the manufacturer's instructions. ssRNAs were purified sequentially by phenol/chloroform extraction and through Illustra Microspin G25 columns (GE Healthcare Life Sciences), following the manufacturer's protocol. Monolayers of 95% confluent BSR cells grown in 12 well plates were transfected twice with BTV RNAs using Lipofectamine 2000 (Invitrogen). Firstly, 0.5610 11 (BTV-1) or 1610 11 (BTV-8) molecules of each of the BTV segments encoding VP1, VP3, VP4, NS1, VP6 and NS2 were diluted in Opti-MEM I Reduced Serum Medium containing 0.5 U/mL of RNAsin plus (Promega) and then mixed with Lipofectamine 2000 diluted in Opti-MEM I Reduced Serum Medium. After 25 min of incubation at room temperature, the mixture was added to the cells. 16 to 18 h after the first transfection, the cells were transfected as before but with all 10 BTV segments. 3 to 4 h after the second transfection the cells were overlaid with 2 ml of minimal essential media containing 1.5% agarose type VII and 2% FBS, and monitored for development of plaques. Finally, individual BTV rescued clones were picked through the agarose overlay and used to infect fresh BSR cells in order to obtain a virus stock. Where necessary, BTV dsRNA was extracted from infected cells using Trizol (Invitrogen). The ssRNA fraction was precipitated using lithium chloride, and the harvested dsRNA fraction was precipitated using isopropanol in the presence of sodium acetate.
Growth curves of BTV recombinant viruses used in this study were derived in cells infected at a MOI of 0.05 and testing for the presence of infectious virus in supernatants collected at 8, 24, 48, 72 and 96 h post-infection. Virus growth was also assessed in cells in the presence of 1000 antiviral units/ml (AVU/ml) of interferon Tau (IFNT) or universal type I interferon (UIFN). Recombinant ovine IFNT was kindly provided by Tom Spencer. IFNT was produced in Pichia pastoris and purified as described previously [35] . Universal type I Interferon (UIFN) was obtained from PBL InterferonSource. BFAE cells and CPT-Tert cells were treated with 1000 AVU of IFN 20 h prior infection with BTV recombinants at a MOI of 0.1 (BFAE and CPT-Tert), 0.01 or 0.001 (CPT-Tert). Two hours after infection, the medium was replaced and the cells maintained in the presence of either IFNT or UIFN at the original concentration. Cell supernatants were collected at 24, 48 and 72 h post-infection, centrifuged for 5 min at 500 g in order to pellet cell debris and virus infectivity was subsequently titrated by endpoint dilution analysis on BSR cells. Viral titers were calculated by the method of Reed & Muench and expressed as log 10 TCID 50 /ml [36] . Each experiment was performed two to three times, each time in duplicate, using different stocks for each virus.
CPT-Tert cells were plated in 24-well plates and treated for 20 h with 1000 AVU/ml of IFNT or UIFN and then infected with either BTV-1 or BTV-8 at different MOIs (0.1, 0.01 and 0.001). The medium was replaced 2 h after infection and the cells maintained in the presence of either IFNT or UIFN at the original concentration. At 72 h post-infection, the cells were washed once with phosphate buffered saline (PBS; pH 7.4) and stained for 16 h using a 0.5% crystal violet/10% formaldehyde solution. We used Image-Pro Plus (MediaCybernetics), in order to quantify in each well the percentage of the monolayer that was disrupted after BTV replication. Results were expressed as the percentage of destroyed monolayer by calculating for each well the following formula: (number of pixels above background: total number of pixels times) X 100.
BSR cells were transfected with 0.6-1.8 mg of either pCI-NS4, pNS4-GFP or derived deletion mutants, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
For western blot analyses of intracellular proteins, cells were lysed by standard techniques as described previously [37] . For viral pellet analysis, cell supernatants were collected and viral particles concentrated 200 times by ultracentrifugation as previously described [38] . Protein expression was assessed by sodiumdodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using the various antisera as indicated above. Membranes were incubated with a horse radish peroxidaseconjugated secondary antibody (GE Healthcare Life Sciences) and developed by chemiluminescence using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences).
Experiments were performed using BSR, BFAE, CPT-Tert or C6/36 cells cultured in two-well glass chamber slides (Lab-Tek, Nalge Nunc International). Cells were either transfected with appropriate plasmids or infected with various BTV strains at a MOI between 0.01 and 1.5. Cells were washed with PBS and fixed with 5% formaldehyde for 15 minutes. The fixed cells were then processed as described previously [39] and incubated with the appropriate antisera. Secondary antibodies were conjugated with Alexa Fluor 488 (Invitrogen, Molecular Probes) or Alexa Fluor 594 (Invitrogen, Molecular Probes). Slides were mounted using VECTASHIELD Mounting Medium with DAPI (49,6-diamidino-2-phenylindole, Vector Laboratories). Slides were analysed and images collected using a Leica TCS SP2 confocal microscope.
BSR cells were infected with BTV-1, BTV-8 or the corresponding deletion mutants at a MOI of 0.05 in 35 mm dishes. At 24 h post-infection, cells were fixed using cold 2.5% gluteraldehyde and 1% osmium tetroxide. Cells were subsequently pelleted through 1% SeaPlaque agarose (Flowgen), dehydrated using a graded alcohol series and embedded in Epon 812 resin, followed by cutting and analysis in a Joel 1200 EX II electron microscope.
Animal experiments were carried out at the ''Istituto G. Caporale'' (Teramo, Italy) following local and national approved protocols regulating animal experimental use. Study 1. Litters of 3-day old NIH-Swiss mice (n = 8-12), were inoculated intra-cerebrally with 10 3 TCID 50 of either BTV-1, (2/2) 129/Sv], were inoculated intraperitoneally with 100 PFU of either BTV-1, BTV-8, BTV-1DNS4 or BTV-8DNS4. For each virus, two groups (n = 5) of mice were inoculated using two different virus preparations. Survival plots were constructed using data collected from two experimental groups (n = 10) with the exception of a mock infected group that was constituted by a single group of 6 mice. Formalin-fixed and paraffin-embedded brains tissue sections from inoculated (and mock inoculated) mice were used in immunohistochemistry. Sections (4-6 mm) were examined for the presence of BTV NS4 using a polyclonal NS4 antiserum and the EnVision (DAKO) detection system.
The BTV genome is formed by 10 segments. Segment 9 contains an open reading frame (ORF) between nucleotides 182 and 418 ( Figure 1A ) in position +1 with respect to the major ORF expressing VP6 [29] . In silico analysis showed that this extra ORF is highly conserved and encodes a putative protein of 77-79 amino acid residues. A stretch of 11 basic amino acid residues is present in the N terminal portion of the protein (residues 3 to 20). In Figure 1A) .
We generated a polyclonal antiserum towards ORFX, in order to assess whether BTV expressed this previously uncharacterized protein. We detected ORFX in BSR cells infected with either BTV-8 or BTV-1 by western blotting ( Figure 1B) . Controls included BSR cells transfected with plasmids expressing ORFX either in its native form, or with eGFP fused to its C terminus. These data confirm that BTV expresses a protein encoded by an alternative reading frame located in segment 9.
We subsequently investigated whether ORFX was a structural or non-structural protein. We infected BFAE cells with BTV-1 and analysed supernatants (containing viral particles) and total cellular protein extracts. Unusually for mammalian cell lines, BFAE cells show very little BTV induced cytopathic effect (CPE), thus facilitating the efficient discrimination between all BTV proteins present in the cellular fraction and the structural proteins present in purified and concentrated viral particles released from infected cells. By western blotting, we detected NS1 and ORFX in the cellular fraction, while VP7 was abundantly present in the viral fraction (concentrated by ultracentrifugation) and barely visible in the cellular fraction ( Figure 1C) . We obtained the same results by infecting C6/36 mosquito cells (data not shown). We detected VP6 in both the cellular and the viral fraction ( Figure 1C) . Interestingly, unlike VP7, VP6 appeared to be relatively more abundant in cell lysates compared to the viral pellets, suggesting that there is an intracellular pool of this protein that is not incorporated in the BTV virions.
The absence of ORFX in the viral pellet strongly suggested that this is a non-structural protein expressed by BTV. In light of these data, we designated this protein NS4. NS4 was also expressed in vivo, as shown by immunohistochemistry of brain sections of mice inoculated intracerebrally with BTV ( Figure 1D ).
By confocal microscopy of cells transiently transfected with pCI-NS4, we observed that NS4 localized mainly in the nucleus (Figure 2A) where it showed a strong co-localization with the nucleolar marker B23 [40] . Importantly, cells infected with either BTV-1 or BTV-8 also showed a strong nuclear co-localisation between NS4 and B23 [40] (Figure 2B ). We also observed NS4 to localise in the nucleus of the C6/36 insect cells ( Figure 2C ).
NS4 does not have a canonical nuclear localization signal (NLS) but possesses a stretch of basic amino acid residues, at the amino terminus portion of the protein, that could drive nuclear localization [41] (Figure 2D ). We constructed an NS4 expression plasmid (pNS4-GFP) and a series of deletion mutants (pNS4 7-77 -eGFP, pNS4 13-77 -eGFP and pNS4 19-77 -eGFP) lacking the 6, 12 and 18 amino terminal residues, respectively. pNS4-GFP and pNS4 7-77 -eGFP transfected cells showed a strong nuclear localization of NS4. On the other hand, NS4 showed a predominantly cytoplasmic localization in cells transfected with either pNS4 13-77 -eGFP or pNS4 19-77 -eGFP. These data suggest that the amino terminal basic domain of NS4 may play an important role in the nuclear localization of this protein.
Interestingly, BFAE cells infected with BTV-1 revealed that NS4 expression was evident as early as 2 hours post infection, similar to that observed for other BTV structural and nonstructural proteins (Figure 3 ).
The data above clearly show that a previously uncharacterized BTV protein, here referred to as NS4, is a non-structural protein that localises to the nucleolus of infected cells. Next, we generated by reverse genetics BTV NS4 deletion mutants in order to assess the requirement of this protein for viral replication. We generated a set of plasmids necessary for the rescue of BTV-1 and BTV-8 and engineered three mutations in the plasmids containing segment 9 of BTV-1 and BTV-8 such that the NS4 initiation codon was removed along with the introduction of two stop codons in the NS4 coding sequence. All the mutations introduced were designed in order to leave the VP6 amino acid sequence unaltered ( Figure 4A ). As a negative control for BTV rescue, we designed a VP6 deletion mutant with a premature stop codon incorporated into the VP6 coding sequence (position 79). As shown in Figure 4B , viable BTV1-DNS4 and BTV8-DNS4 were rescued with similar efficiency to the respective wild-type (wt) viruses, upon transfection of RNA transcribed in vitro from the appropriate plasmids representing the genomic segments of wt or mutated BTV-1 and BTV-8. As expected, BTV-1DVP6 and BTV8-DVP6 could not be rescued.
We did not detect any variation in the migration pattern of dsRNA genomic segments extracted from all the wt or the NS4 deletion mutant viruses ( Figure 4C ). The RNA profiles of both the wt and DNS4 rescued viruses were identical to the corresponding profile of the stock viruses from which the segments were originally cloned. For each virus, segment 9 was completely sequenced in order to confirm the presence of the introduced mutations.
We confirmed, by western blotting and confocal microscopy, that the DNS4 mutants do not express NS4 but express levels of VP7 and NS2 comparable to the parental wild type viruses. It was also evident that in BSR cells BTV-8 expresses lower amounts of NS4 relative to BTV-1 ( Figure 4D and not shown). However, BTV-1 replicates better than BTV-8 in these cells and differences in the steady-state levels of VP7 between these two viruses were also observed ( Figure 4D ). In cells infected by BTV1-DNS4 or BTV8-DNS4, we found by electron microscopy all the ultrastructural features of BTV-infected cells (e.g. viral inclusion bodies, NS1 tubules, viral particles) ( Figure 4E ).
We next assessed the replication kinetics of the rescued viruses in a variety of mammalian and insect cell lines, including those corresponding to the natural hosts (sheep and cattle) and vector (midges). All subsequent experiments were performed using the rescued versions of the wt viruses as they represent a more homogenous population and are therefore more directly comparable to the rescued DNS4 viruses. Cells were infected with a MOI of 0.05 and supernatants were collected at various times postinfection ( Figure 5 ). No obvious difference was obtained in the replication of wt and DNS4 viruses, regardless of the cell lines used in the assay ( Figure 5) . Interestingly, the cell adapted BTV-1 viruses consistently grew more efficiently in vitro than the BTV-8 BTV-8 at a MOI of 0.05. At 48 h post-infection, cells were fixed and analyzed by immunofluorescence as for expression of NS4 as indicated in panel A. Scale bars correspond to 11 mm. (D) Confocal microscopy of CPT-Tert cells transfected with pNS4-GFP or the truncated mutants indicated above each panel. The red box corresponds to the first two amino terminal amino acid residues of NS4 that were maintained in all mutants. At 24 h posttransfection, cells were fixed and analyzed by immunofluorescence. Scale bars correspond to 18 mm. doi:10.1371/journal.ppat.1002477.g002 BTV NS4 confers a replication advantage to BTV-8, but not BTV-1, in mammalian cells treated with interferon BTV, like most RNA viruses, is a strong inducer of interferon, both in vivo in its natural hosts and in vitro [42] [43] [44] . Given that other RNA viruses express proteins that counteract the innate immunity of the host, we hypothesised that NS4 might aid BTV replication in the presence of interferon (IFN). We treated cells with two type I IFNs: IFN tau (IFNT) and universal IFN (UIFN). IFNT is secreted by the ruminant conceptus and it is intimately linked to pregnancy recognition signalling and possesses antiviral activity [45] while UIFN is an alpha interferon hybrid constructed from recombinant Human IFNs alpha A and alpha D, and is known to stimulate an antiviral response in a wide variety of mammalian cells.
CPT-Tert cells were pre-treated with IFNT or UIFN for 20 h prior to infection with BTV-1 or BTV-8 (or mock infection) with MOIs ranging from 0.001 to 0.1. Both wt and the DNS4 mutants, destroyed 80 to 100% (depending on the MOI used) of the monolayer of infected cells in absence of IFN treatment ( Figure 6 ). On the other hand, pre-treatment with both types of IFN significantly reduced BTV-induced CPE. Interestingly, in the presence of IFN, BTV-8 wt consistently induced a more pronounced CPE than BTV8-DNS4. Conversely, only minor differences were observed in the CPE induced by both wt BTV-1 and BTV-1DNS4 in the presence of IFN ( Figure 6 ).
Subsequently, we performed multi-step virus growth curves in order to further assess the replication of BTV wt and DNS4 in the presence or absence of IFN. CPT-Tert cells were treated with interferon, as described above, and infected at a MOI of 0.01 with wt and mutant viruses. At 24, 48 and 72 h post infection the cell supernatants were collected and the virus titrated in susceptible cells.
BTV-8DNS4 consistently reached lower titres (approximately 10 to 25 fold) than wt BTV-8 in cells treated with 1000 AVU/ml of either IFNT or UIFN (Figure 7) . Similar to what was observed in the IFN protection assays, there was no discernable difference in the replication growth of BTV-1 and BTV-1DNS4 after treatment with either IFNT or UIFN. Similar patterns with both BTV-1 and BTV-8 wt and the DNS4 mutant viruses where observed when the input viruses were used at a MOI of 0.1 and 0.001 in CPT-Tert (data not shown), or in BFAE cells treated with UIFN and infected at a MOI of 0.1 (data not shown).
We next ruled out that the mutations inserted in segment 9 of BTV-8DNS4 had a negative effect on VP6 expression (the other protein expressed by segment 9). As shown in Figure 8A , BTV-8 wt and BTV-8DNS4 express similar amounts of VP6, reinforcing the notion that the biological differences observed between these two viruses were indeed due to the expression of NS4.
Therefore, the data presented so far suggested that either the BTV-1 NS4 was somewhat defective or that the influence of this protein on viral replication in the presence of IFN varies from strain to strain. In order to discern between these two possibilities, N in position 6) in order to render this protein identical to the homologous BTV-1 protein ( Figure 8B ). Both cytopathic protection assays and multistep growth assays clearly showed that BTV-8/1S9 replicated more efficiently than BTV-8/ 1S9DNS4 in the presence of IFN ( Figure 8C, D) . Similar results were obtained with BTV-8/1NS4, which replicated more efficiently than BTV-8DNS4 in cells pre-treated with IFN, while no major differences were observed between BTV-1/8S9 and BTV-1/8S9DNS4. Collectively, these data strongly indicate that the NS4 of BTV-1 is not defective and can function within the context of BTV-8.
DNS4 BTV mutants are pathogenic in mice models of disease Next, we assessed the virulence of DNS4 BTV mutants in two murine models of bluetongue infection [46, 47] . 129sv IFNAR (2/2) mice, which are deficient in the type I IFN receptor, are susceptible to infection and disease induced by BTV inoculated by various routes [46, 48] . Newborn NIH-Swiss mice inoculated intracerebrally are also susceptible to BTV infection [47] . These models have been previously used to assess BTV virulence [47, 49] .
In this study, we infected 129sv IFNAR (2/2) mice with either BTV-1, BTV-8 or the corresponding DNS4 mutants. No major differences were observed in the virulence of wild type and DNS4 viruses; all viruses employed in this study killed 100% of the inoculated mice by day 8 post-infection ( Figure 9 ). We also inoculated 3-day old NIH-Swiss mice intracerebrally with the same viruses as above. Once again, both wild type and DNS4 viruses were able to kill 100% of the inoculated mice with no major differences in the virulence observed ( Figure 9 ).
In this study we have shown that BTV expresses a previously uncharacterised non-structural protein that favours viral replication in cells in an antiviral state. By constructing deletion mutants by reverse genetics, we showed that NS4 is dispensable for viral replication in vitro, both in mammalian and insect cells, and in vivo in murine experimental models. However, the coding sequence in the NS4 reading frame of segment 9 is highly conserved in BTV and in related Orbiviruses [29, 50] , suggesting that it must be essential for the maintenance of BTV in nature. Indeed, we have found that NS4 confers a replication advantage to BTV-8 in cells pre-treated with type I IFN.
We found NS4 to have strong nucleolar localization, although it may shuttle between the nucleolus and cytoplasm and possibly carry out its biological functions in the latter. The nucleolus is a dynamic sub-nuclear structure that plays crucial roles in ribosome subunit biogenesis, the response to cellular stress and cell growth [51, 52] . Several examples of viral proteins targeting the nucleolus have been discovered in recent years [53] . The retroviral Rev and Rev-like proteins for example, shuttle between the nucleolus and cytoplasm, and function as post-transcriptional regulators of viral gene expression [54] [55] [56] [57] . One of the main functions of these proteins is to facilitate the export of unspliced viral mRNA (transcribed from the proviral DNA copy of the retroviral genome stably integrated in the cell genome) by simultaneously binding an RNA structure in the viral RNA and the karyopherin export factor Crm1 (chromosome region maintenance 1) [58] . Other RNA viruses (including those that replicate exclusively in the cytoplasm) have also been found to possess proteins that target the nucleoli. Examples include, among others, avian infectious bronchitis virus [59] , porcine reproductive and respiratory syndrome virus [60] , Newcastle disease virus [61] , Semliki forest virus [62] , dengue virus [63] , West Nile virus [64] , influenza virus [65] , avian reovirus [66] and encephalomyocarditis virus [67, 68] The reasons for the nucleolar targeting of many of these proteins have not always been entirely clear. 10 (TCID 50 /ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed two times, each time in duplicate. doi:10.1371/journal.ppat.1002477.g008
The avian reovirus sA protein is a structural protein and is a major component of the inner capsid shell. Although the sA protein localises mainly in viral factories in the cytoplasm of infected cells, it also localizes in the nucleoli [66] . sA has a strong affinity for dsRNA and it may provide protection against the IFNinduced and dsRNA dependent PKR response. Interestingly, sA mutants that do not bind dsRNA are also unable to reach the nucleoli, suggesting that dsRNA binding and nucleolar targeting may be strictly linked [69] .
BTV NS4 may also bind nucleic acids but, unlike the reovirus sA, we show strong evidence that NS4 is not a structural protein.
Indeed, by western blotting we did not detect NS4 in viral particles but only in lysates of BTV infected cells. In addition, by confocal microscopy we did not detect NS4 in viral inclusion bodies but predominantly in the nucleoli of viral infected cells. We cannot exclude completely that small amounts of NS4, below the limits of detection of our western blotting analysis, are present in viral particles.
The predicted structural features of NS4 resemble those of a transcription factor of the bZip family with a basic domain followed by a leucine zipper motif [70] . Thus, NS4 may function as a nucleic acid binding protein and either repress or enhance transcription of genes linked directly or indirectly to the IFN response of the cell. However, a BTV-8 recombinant virus (BTV-8DLZNS4) expressing an NS4 with all the 4 leucine residues forming the putative leucine zipper mutated (into either glutamine or serine) replicated as efficiently as BTV-8 wt in cells pre-treated with IFN (data not shown). Thus, more studies will be necessary to explore this possibility.
The organization of VP6/NS4 ORFs in segment 9 of BTV mirrors that of NSP5/NSP6 in the rotavirus segment 11 [71] . The rotavirus NSP6 is not essential for virus replication but unlike the BTV NS4, does not localize in the nucleus of infected cells [72] .
To date, limited information is available on the interplay between BTV and the host innate immune system. BTV has been recognized as a potent inducer of type I IFN in sheep [42] , cattle [43] and mice [73] . However, limited data have been available on how BTV induces the IFN response of the cell and, more importantly, what counteracting measures the virus utilises to overcome this response. Our data suggest that BTV may use NS4 to defend itself from the innate immune response of the host given that replication of BTV8-DNS4 in cells treated with IFN is 10 to 25 fold less efficient compared to wild type BTV-8. In addition, the cytopathic effect in cells treated with IFN is more pronounced when cells are infected by wild type BTV-8 compared to cells infected by BTV8-DNS4.
Viruses have evolved a variety of strategies to evade the host innate immunity [74] . Other dsRNA viruses such as rotaviruses, use different mechanisms (which vary between strains and the type of infected cells) to modulate the type I IFN response. For example, rotaviruses use NSP1 protein to promote the proteasome-dependent degradation of IRF proteins [75] [76] [77] and mediate repression of NF-kB, resulting in a reduction of IFN induction [78] . Rotaviruses also induce shut off of cellular protein synthesis resulting from the detection of dsRNA by PKR which, in turn is responsible for phosphorylation and consequent inhibition of the eukaryotic translation initiation factor eIF2a [79] . The blocking of host cell protein synthesis is another likely strategy used by some RNA viruses to counteract the IFN response [67, 68] . BTV also blocks host cell protein synthesis early after infection, although the mechanisms underlying this phenomenon are not clear [27] .
Interestingly, we found that BTV1-DNS4 replicated as efficiently as wild type BTV-1, even in cells treated with IFN. However, the NS4 of BTV-1 appears to possess the same biological properties of the NS4 of BTV-8. Indeed, a BTV-8 reassortant containing the entire segment 9 of BTV-1 (BTV-8/ 1S9) or a recombinant BTV-8 expressing an NS4 100% identical to the homologous BTV-1 protein (BTV-8/1NS4), maintained the phenotype of wt BTV-8. Thus, it is possible that the role played by NS4 in counteracting the IFN response of the host could vary between different virus strains. It is important to stress that the strain of BTV-8 that we used in this study has been passaged only a few times in culture (once in KC cells and three times in BHK 21 cells) after isolation from blood of an infected animal. On the other hand, BTV-1 was derived from the ''reference'' South African strain passaged twice in embryonated eggs and 9 times in BHK 21 . BTV-1 appears to grow slightly faster than BTV-8 in culture, especially at the early time points post infection. Thus, faster replication may help BTV1-DNS4 to escape the IFN response of the cell more efficiently, as already suggested for some strains of influenza, and this may render NS4 less critical in these in vitro assays [80] .
More in vivo experiments will be needed in order to determine the role of NS4 in the interplay with the natural host of BTV infection. We observed no differences between wild type BTV-8 and BTV8-DNS4 in experimental mouse models, although it remains possible that differences could be identified in sheep. It is possible that NS4 is required for viral replication in insects, although we have established in this study that no differences are observed on the replication of the DNS4 mutants in insect cells in vitro.
In conclusion, in the present study we have identified a previously uncharacterized non-structural protein of BTV. The identification of this highly conserved protein opens the way to understand finer details of virus-host interaction and pathogenesis. In addition, the distinct nucleolar localization, in a virus that replicates exclusively in the cytoplasm will offer new avenues to understand the various roles played by these organelles in the biology of the cell. Interference of H-bonding and substituent effects in nitro- and hydroxy-substituted salicylaldehydes Two intramolecular interactions, i.e., (1) hydrogen bond and (2) substituent effect, were analyzed and compared. For this purpose, the geometry of 4- and 5-X-substituted salicylaldehyde derivatives (X = NO(2), H or OH) was optimized by means of B3LYP/6-311 + G(d,p) and MP2/aug-cc-pVDZ methods. The results obtained allowed us to show that substituents (NO(2) or OH) in the para or meta position with respect to either OH or CHO in H-bonded systems interact more strongly than in the case of di-substituted species: 4- and 3-nitrophenol or 4- and 3-hydroxybenzaldehyde by ∼31%. The substituent effect due to the intramolecular charge transfer from the para-counter substituent (NO(2)) to the proton-donating group (OH) is ∼35% greater than for the interaction of para-OH with the proton-accepting group (CHO). The total energy of H-bonding for salicylaldehyde, and its derivatives, is composed of two contributions: ∼80% from the energy of H-bond formation and ∼20% from the energy associated with reorganization of the electron structure of the systems in question. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00894-011-1044-1) contains supplementary material, which is available to authorized users. and (3) of the 4-hydroxy-salicylaldehyde (in red, see Table 2 ); in parenthesis SESE for para and meta di-substituted benzene derivatives (in green, Tables 3 and 4 ). (2) and (3) of the 5-nitro-salicylaldehyde (in red, see Table 2 ); in parenthesis SESE for para and meta di-substituted benzene derivatives (in green, Tables 3 and 4 ). (2) and (3) of the 4-nitrosalicylaldehyde (in red, see Table 2 ); in parenthesis SESE for para and meta di-substituted benzene derivatives (in green, Tables 3 and 4 ).  Acute Respiratory Distress Syndrome Induced by a Swine 2009 H1N1 Variant in Mice BACKGROUND: Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus. METHODOLOGY PRINCIPAL FINDINGS: Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8–10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. CONCLUSIONS/SIGNIFICANCE: These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus. A novel influenza A (H1N1) virus of swine origin emerged among humans in Mexico during the spring of 2009 and rapidly spread worldwide [1] . The pandemic prompted the World Health Organization (WHO) to raise the alert level to the highest rating of six, the pandemic phase, within 2 months [2] . In August 2010, WHO officially declared that the disease was in the post-pandemic period [3] ; however, it is still circulating among humans, together with seasonal viruses. Although most influenza cases caused by 2009 H1N1 virus infection typically display mild upper respiratory tract syndrome, some cases progress to severe pneumonia and acute respiratory distress syndrome (ARDS) [4, 5] . Many studies have shown that ARDS caused by 2009 H1N1 virus results in 17.3-56% mortality [4, 6, 7, 8] , which was regarded as the major cause of death by 2009 H1N1 virus infection [9] . ARDS is the result of acute injury to lung tissue, commonly resulting from sepsis, trauma, and severe pulmonary infections [10] . Infectious factors, most of which are viruses, have become one of the most important causes of ARDS in humans [11, 12, 13] . Clinical cases and established animal models have revealed that the pathogenesis and pathological features of ARDS induced by different viral pathogens are distinct [14, 15] . However, knowledge of the pathogenesis of 2009 H1N1 virus, especially ARDS induced by 2009 H1N1 virus, is still limited and hinders therapeutic strategies. Therefore, it is necessary to evaluate the pathogenesis of ARDS caused by 2009 H1N1 virus infection in an appropriate animal model to assess potential therapies.
Mice are a good model for evaluating the pathogenesis and antiviral therapy of influenza pneumonia, due to the general fidelity of the illness in mice to the human disease [16] . Moreover, a mouse model of ARDS caused by highly pathogenic H5N1 avian influenza virus infection has been well established [13] . The typical 2009 H1N1 virus, such as A/California/04/2009 (CA/04), can efficiently replicate in mouse lungs without prior host adaptation. However, it only causes moderate lung lesions and no mortality, even when inoculated at a high dose of 10 6 pfu [17, 18] . Thus, such typical 2009 H1N1 viruses may not be able to induce ARDS in a mouse model.
In the present study, we used a virulent variant 2009 H1N1 virus, which was isolated from a pig and possessed a virulenceassociated HA-D222G mutation, to establish an ARDS mouse model. The model established here provides a useful tool to explore the mechanism of ARDS, as well as screening and therapeutic options.
Six-week-old female mice were infected intranasally (i.n.) with 10 2.5 pfu SD/09 virus. Some of the infected mice showed signs of illness, such as altered gait, inactivity, ruffled fur, and anorexia on day 2 post-infection (p.i.). From day 2 p.i., the body weight of most mice significantly decreased ( Figure S1 ). By day 6 p.i., most mice presented with more severe clinical signs of respiratory disease, including labored respiration and respiratory distress, and most mice lost almost 20% of their initial body weight. On day 8 p.i., most mice were nearly unable to respond to exterior stimuli, and acute respiratory rates and labored respiration were observed (Video S1, and Video S2 for control). Approximately 60% of mice died between days 8 and 10 p.i. Gross observation of infected mice showed that the lungs were highly edematous, with profuse areas of hemorrhage and consolidation. No obvious gross lesions were observed in the kidneys, liver, spleen or brain of infected mice.
Mice were infected i.n. with 10 2.5 pfu SD/09 virus, and three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the virus titers in viscera were determined. As shown in Figure 1A , the virus titer in the lung gradually increased between days 2 and 6 p.i., and reached a peak on day 6 p.i. The virus titers in the lung gradually decreased from day 6 p.i., and only one of three mice possessed detectable virus in the lungs on day 10 p.i. No viruses were detected in other organs, including heart, spleen, liver, kidneys, blood and brain, at the indicated time. These results indicate that SD/09 virus could replicate efficiently in mouse lung but did not cause systemic infection.
As shown in Figure 1B , the effect of SD/09 viral infection on lung wet:dry weight ratio did not change significantly within 2 days p.i. However, a dramatic increase was observed from day 4 p.i., and reached a peak on day 8 p.i., which was nearly twice that observed in control group lungs (p,0.01). The change in lung wet weight:body weight ratio was similar to the change in lung wet:dry weight ratio ( Figure 1C ). The results indicated that the SD/09 virus could induce acute lung edema in mice.
Kinetic observation of lung lesions of SD/09-virus-infected mice is shown in Figure 2 . On day 4 p.i., lung lesions were characterized by dropout of mucous epithelium and inflammatory cells adhering to the bronchiolar surface ( Figure 2C , D). On day 6 p.i., severe edema could be seen around blood vessels ( Figure 2E ); interstitial pneumonia was also observed that showed interstitial edema and thickening of the alveolar walls; and the alveolar lumen was flooded with detached alveolar cells, erythrocytes, and inflammatory cells ( Figure 2E , F). On day 8 p.i., the virus caused more severe interstitial pneumonia and peribronchiolitis, characterized by edema and extensive of lymphocytes, neutrophils and plasma cells around the area of bronchiolitis ( Figure 2G , H). Lesions in the lungs of infected mice were still severe on day 14 p.i., with extensive alveolar collapse, and remaining alveoli were filled with fibrin, desquamated alveolar cells, and inflammatory cells. Lymphocytes and alveolar macrophages were the predominant inflammatory cells observed at high magnification ( Figure 2J ). Masson's stain revealed that alveolar walls and spaces were filled with collagen fibers ( Figure 3A , B), indicating that proliferative fibroblastic lesions may develop. In comparison, lungs from mockinfected control mice had no apparent histological changes ( Mice were inoculated i.n. with 10 2.5 pfu SD/09 viruses; tissues were collected at indicated times p.i. and viruses were titrated in MDCK cells. Body weight, lung wet and dry weight were determined and recorded. The lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema. *p,0.05, **p,0.01, ***p,0.001, comparison between ratios obtained from the virusinfected and control groups. Bars represent means 6 SD of data from three mice. doi:10.1371/journal.pone.0029347.g001 Immunohistochemistry revealed viral antigens in the epithelial cells of the bronchioles ( Figure 3D ), terminal bronchioles, and alveolar epithelial cells ( Figure 3E ). These data indicated that SD/ 09 virus could infect the epithelia of the lower airway and cause viral pneumonia in mice.
As shown in Table 1 , virus-infected mice showed a slightly decreased partial pressure of arterial oxygen (Pa O2 ), saturation of arterial oxygen (Sa O2 ), and slightly increased partial pressure of arterial carbon dioxide (Pa CO2 ) from days 4 to 6 p.i. Most infected mice presented with severe clinical signs of respiratory distress on day 8 p.i., and blood gas analysis also showed that Pa O2 and Sa O2 dramatically decreased compared with the controls (p,0.05). These results suggested acute respiratory dysfunction and severe hypoxemia in virus-infected mice.
The number of leukocytes in BALF from SD/09-infected mice showed an increase from day 4 p.i. ( Table 2 ). The BALF of virusinfected mice on day 6 p.i contained 1.1610 6 cells/ml and was significantly different from the 1.6610 5 cells/ml observed for PBSinoculated mice (p,0.001). These data indicate a dramatic increase in inflammatory cells in the lungs of SD/09-infected mice. To quantify the immune cell subpopulations responding to viral infection, we next determined cell differential counts in the infected lungs by Wright staining. Compared with PBS-inoculated animals, mice infected with SD/09 virus exhibited an increase of neutrophils from 4 days p.i., and the peak was 18-fold greater than that of the control group on day 8 p.i. Leukopenia was detected on day 4 p.i., and was statistically significant on day 6 p.i. (p,0.05); the lowest value appeared on day 8 p.i. (p,0.001). Furthermore, differential blood counts revealed that the number of lymphocytes sharply decreased in infected mice. The lowest number of lymphocytes observed occurred on day 8 p.i. (Figure 4B ), which dropped to ,20% of the control group number (p,0.001).
To determine the cytokine responses that occur after SD/09 virus infection, we measured the levels of five cytokines and chemokines in lungs of infected mice on days 2, 4, 6, 8 and 10 p.i. As shown in Figure 5 , all five were significantly different between virus-infected and control mice. Interleukin (IL)-6 and IL-10 in the virus-infected mice reached peak levels as early as day 2 p.i. and were significantly higher than those of the control group (p,0.001). Interferon (IFN)-c, monocyte chemotactic protein (MCP)-1, and tumor necrosis factor (TNF) dramatically increased in mouse lungs on days 6-8 p.i. (p,0.001), consistent with the appearance of pulmonary lesions. These results showed that infection with SD/09 viruses resulted in elevated amounts of proinflammatory chemokines and cytokines in the lungs of mice.
In the spring of 2009, a novel influenza A(H1N1) virus rapidly spread worldwide, resulting in the first influenza pandemic of the 21st century [1] . Critically ill cases caused by 2009 H1N1 virus retrospectively showed that most had progressed or died due to ARDS [4, 6] . However, the pathogenesis and therapeutic intervention of ARDS caused by 2009 H1N1 infection have still not been elucidated. Animal models of disease are important for characterizing pathogenesis and developing the preclinical evidence for revised approaches to ventilating patients with ARDS [19] . Here, we present a mouse model for the study of ARDS induced by SD/09 virus, a virulent 2009 H1N1 variant.
Previous studies have indicated that typical 2009 H1N1 viruses such as CA/04 bind only to a-2,6-linked sialic acid (SA) receptor [17] , but only the a-2,3-linked SA receptor is found in the mouse respiratory tract [20] . Therefore, such a typical 2009 H1N1 virus may not be able to induce ARDS in mice. In fact, we used CA/04 virus to induce ARDS in mice; however, animals inoculated with a high dose of CA/04 virus (10 6.5 pfu) only showed moderate respiratory symptoms, and no lethality was observed (unpublished data). It has been shown that 2009 H1N1 virus possessing a D222G mutation in hemagglutinin (HA) could increase the pathogenicity in mice [21, 22] and binding to the a-2,3 SA receptor [23] . Moreover, clinical data indicate that such variants are only associated with severe H1N1 human infection [24] . Therefore, we suggest that the variant possessing the D222G mutation in HA can induce ARDS in a mouse model. The virus used in the present study was isolated from swine in 2009, and sequence analysis revealed that all the eight genes of the isolate had a close relationship with the 2009 H1N1 influenza virus circulating in humans. Notably, the swine isolate, SD/09, had a D222G mutation in HA. Compared with CA/04 virus (LD 50 .10 6 pfu), SD/09 showed significantly increased virulence in mice, with an LD 50 of 10 2.25 pfu, which was nearly identical to that of the mouse-adapted strain A/Hong Kong/415742Md/09 (LD 50 = 10 2.2 pfu) [17, 21] . Mice infected i.n. with 10 2.5 pfu SD/ 09 virus showed obvious respiratory symptoms, including visually prominent signs of respiratory distress and abdominal respiration, with approximately 60% mortality between days 8 and 10 p.i. The lungs of virus-infected mice were highly edematous, which was also demonstrated by dramatically increased lung wet:dry weight ratio. Pathological changes presented a progressive pattern, typically diffuse alveolar damage, interstitial and alveolar edema, neutrophil and macrophage-dominant inflammatory cellular infiltration, and areas of hemorrhage and necrotizing bronchiolitis. Arterial blood gas saturation is a key parameter of ARDS in humans [25] . In the present mouse model, Pao 2 and Sao 2 of infected mice were significantly lower than in the control group from day 2 p.i., especially on day 8 p.i., where these parameters sharply decreased, and most virus-infected mice began to die. These changes in arterial blood gas demonstrated that most infected mice developed severe hypoxemia consistent with of the appearance of clinical signs and lung lesions of ARDS. Previous studies showed that mice infected with typical 2009 H1N1 virus only exhibited mild interstitial inflammatory infiltration and limited alveolitis [18, 21] , whereas severe lung damage was found in the SD/09-infected mice, including severe edema around the blood vessels and bronchiolitis, and extensive inflammatory accumulation from 4 to 8 days p.i. At 10 days p.i., the surviving mice developed an irreversible fibrosis involving collagen deposition in alveolar walls and spaces, which was similar to that observed in human ARDS patients with 2009 H1N1 infection [26] . Our histopathological results were consistent with ARDS induced by other influenza viruses. Mice infected with mouse-adapted virus of the A/Puerto Rico/8/34 (H1N1), or high pathogenic H5N1 virus also showed a progressive series of pathological changes from interstitial pneumonia to diffuse alveolar damage [13, 27] . However, in contrast to highly pathogenic H5N1 virus, mice infected with SD/09 virus did not show viral spread to extrapulmonary organs.
Immunohistochemical examination revealed the presence of viral antigens in the bronchioles, terminal bronchiolar epithelium, and alveolar epithelial cells. Perhaps SD/09 virus infection of the alveoli, particularly type II pneumocytes, rather than bronchioles, is a key to the development of ARDS. Type II pneumocytes are responsible for the production and secretion of surfactant to lower the surface tension of water and allow membrane separation, and insufficient pulmonary surfactant in the alveoli may result in alveolar collapse [28, 29] . The 1918 pandemic H1N1 and high pathogenic H5N1 viruses preferentially infect type II pneumocytes and alveolar macrophage in mice [15, 30] . Alveolar macrophages may play a critical role in disease pathogenesis, not through production of infectious virus but rather through the upregulation of proinflammatory cytokines that may further damage alveolar pneumocytes [31] . These phenomena suggest that viral cell tropism may determine the processes of ARDS.
Pulmonary aberrant immune response is considered a significant feature of ARDS induced by 2009 H1N1 virus [19, 32] . In the present mouse model, the number of leukocytes observed in the BALF of virus-infected mice significantly increased compared with the control mice on day 8 p.i. Different counts in BALF showed that the proportion of neutrophils dramatically increased. These innate immune cells were capable of reducing the virus load in the lung [33] ; however, they could cause lung injury through direct or indirect mechanisms. Neutrophil oxidants and proteases can cause direct injury of cells in the alveolar-capillary membrane [34] . Neutrophils and macrophages can secrete copious amounts of chemokines and cytokines that can recruit more immune cells into lung tissues, and produce a ''cytokine storm'', one of the most important factors in the production of ARDS [35] .
A retrospective cohort study of 74 2009 H1N1 patients found that higher levels of proinflammatory cytokines and chemokines in plasma were observed in the ARDS-death group compared with the survived-without-ARDS or the mild-disease groups [5] . Another study in critically ill patients with ARDS caused by 2009 H1N1 virus infection has shown that the hallmarks of disease severity were elevated levels of IL-6, IL-15, IL-8 and TNF-a [36] . We examined the levels of five cytokines and chemokines in infected mouse lungs and found significant differences between the virus-infected and mock groups. It has proved that high levels of IL-6 were able to mediate acute lung injury [37] , and had a negative correlation with the Pa O2 :Fi O2 ratio in severely affected patients with 2009 H1N1 virus infection [36] . Our data showed SD/09 viral infection induced high levels of IL-6 in mouse lung, which may also play an important role in the course of ARDS. Hagau etc. found the levels of TNF-a increased significantly in the 2009 H1N1-related ARDS patients [36] . In present study, TNF levels also dramatically increased in the lungs of virus-infected mice, and were consistent with the clinical symptoms and reached peak levels when mice began to die. In addition, high levels of IL-10, IFN-c and MCP-1 were also present in the virus-infected mouse lungs, similar to observations found in severely affected humans with 2009 H1N1 infection [38] .
In summary, we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant, which demonstrated key human ARDS clinical and pathological features, such as respiratory distress, low Pa O2 , exudative, proliferative and fibrotic lung, and high levels of inflammatory cells and cytokines. The mouse model may contribute to the study of the pathogenesis and therapy of ARDS induced by 2009 H1N1 virus. To determine LD 50 of SD/09 virus, eight 6-week-old female BALB/c mice per group were inoculated i.n. with 10 1 -10 6 pfu (50 ml) viruses and monitored for 14 days. The value of MLD50 was calculated using the Spearman-Karber method and expressed by pfu per MLD 50 [39] .
We evaluated the pathogenicity of the virus in mice and found that it could efficiently replicate in the lungs of mice with high lethality (10 2.25 pfu per MLD 50 ). To determine the optimal dose of inoculation, 10 mice in each group were infected i.n. with 10 1.5 , 10 2.5 or 10 3.5 pfu viruses, and the signs, body weight, and mortality were monitored daily for each group for 14 days. Pilot experiments indicated that a dose of 10 2.5 pfu was optimal, because the course of the disease was prolonged and the mice presented with obvious signs of respiratory illness.
BALB/c mice were lightly anesthetized and inoculated i.n. with 50 ml 10 2.5 pfu SD/09 virus in PBS. Mock-infected animals were inoculated i.n. with 50 ml PBS. At the indicated time, infected mice were sacrificed, and the parameters that present the course of the disease were determined. Twenty mice (10 infected with SD/09 virus and 10 inoculated with PBS) were used to investigate clinical signs and mortality for 14 days.
Three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i. and their organs were collected. The collected tissues were weighed, and 10% homogenates were prepared in cold PBS. The homogenates were centrifuged at 3000 rpm for 10 min to remove cell debris, and then the supernatants were 10-fold serially diluted for viral titer determination by plaque assay in MDCK cells. Virus titers were expressed as mean log pfu/g 6 standard deviation (SD).
Three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the lungs were removed and weighed and then desiccated in an oven at 60uC for 72 h. The lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema, as previously described [40] .
Three mice were euthanized on days 4, 6, 8 and 14 p.i. The lungs were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Lungs on day 14 p.i. were also stained with Masson's trichrome.
Lung tissue sections taken on day 6 p.i. were stained for influenza A virus antigens. An anti-influenza nucleoprotein monoclonal antibody (AA5H; Abcam, Hong Kong) was used to identify influenza A virus nucleoprotein in sections. Secondary antibody (Millipore, Billerica, MA, USA) against the primary antibody was labeled with horseradish peroxidase, and the color reaction was developed with a horseradish peroxidase reaction kit (diaminobenzidine-tetrahydrochloride; Sigma, St. Louis, MO, USA).
Blood gas analysis was performed as previously described [13, 41] . Three mice were anesthetized with Zoletil (tiletamine-zolazepam; Virbac; 20 mg/g) on days 4, 6, 8, 10 and 14 p.i. Arterial blood samples were withdrawn into a heparinized syringe by percutaneous left ventricular sampling of lightly anesthetized mice that were spontaneously breathing room air. Blood gas analysis was immediately performed using a Vetstat Electrolyte and Blood Gas Analyzer (Idexx laboratories, Westbrook, MA, USA).
Leukocyte counts in BALF were performed as previously described [42, 43] . Briefly, three mice were euthanized on days 4, 6, 8 and 10 p.i., and the lungs were lavaged twice in situ with the chest cavity opened by midline incision with a total volume of 1.0 ml saline (4uC) inserted through an endotracheal tube. The rate of recovery of BALF was not less than 90% for all animals tested. After the amount of fluid recovered was recorded, an aliquot of BALF was diluted 1:1 with 0.01% crystal violet and 2.7% acetic acid for leukocyte staining and erythrocyte hemolysis. The number of leukocytes in the BALF was counted with a hemacytometer under a microscope. For differential counts, the BALF samples from each mouse were stained with Wright stain, and the numbers of monocytes, neutrophil and lymphocytes were determined, on the basis of morphologic criteria, under a light microscope, with evaluation of at least 200 cells per slide. All slides were counted twice by different observers blinded to the status of the animal.
Heparinized blood samples were collected on days 4, 6, 8 and 10 p.i. The total numbers of leukocytes and differential blood counts for three individual mice were analyzed using an automated hematology analyzer.
IL-6, IL-10, TNF, IFN-c and MCP-1 levels were determined in lung homogenates using a cytometric bead array technique (BD Cytometric BEAD Array Mouse Inflammation Kit; BD Bioscience, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 50 ml mouse inflammation capture bead suspension and 50 ml PE detection reagent were added to an equal amount of sample standard dilution and incubated for 2 h at room temperature in the dark. Subsequently, samples were washed by adding 1 ml wash buffer and centrifugation at 2006 g at room temperature for 5 min. Supernatants were discarded and 300 ml wash buffer was added. Samples were analyzed on a BD FACSArray bioanalyzer (BD Bioscience) according to the manufacturer's instructions. Standard curves were prepared similar to the method above. Data were analyzed using BD CBA Software (BD Bioscience). Finally, the chemokine or cytokine levels were recorded as pg/ml homogenate.
Data were analyzed by two-way analysis of variance using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). When a significant effect was observed, pairwise comparisons were performed using the Bonferroni post-hoc test. All data are reported as mean 6 SD.  True versus False Parasite Interactions: A Robust Method to Take Risk Factors into Account and Its Application to Feline Viruses BACKGROUND: Multiple infections are common in natural host populations and interspecific parasite interactions are therefore likely within a host individual. As they may seriously impact the circulation of certain parasites and the emergence and management of infectious diseases, their study is essential. In the field, detecting parasite interactions is rendered difficult by the fact that a large number of co-infected individuals may also be observed when two parasites share common risk factors. To correct for these “false interactions”, methods accounting for parasite risk factors must be used. METHODOLOGY/PRINCIPAL FINDINGS: In the present paper we propose such a method for presence-absence data (i.e., serology). Our method enables the calculation of the expected frequencies of single and double infected individuals under the independence hypothesis, before comparing them to the observed ones using the chi-square statistic. The method is termed “the corrected chi-square.” Its robustness was compared to a pre-existing method based on logistic regression and the corrected chi-square proved to be much more robust for small sample sizes. Since the logistic regression approach is easier to implement, we propose as a rule of thumb to use the latter when the ratio between the sample size and the number of parameters is above ten. Applied to serological data for four viruses infecting cats, the approach revealed pairwise interactions between the Feline Herpesvirus, Parvovirus and Calicivirus, whereas the infection by FIV, the feline equivalent of HIV, did not modify the risk of infection by any of these viruses. CONCLUSIONS/SIGNIFICANCE: This work therefore points out possible interactions that can be further investigated in experimental conditions and, by providing a user-friendly R program and a tutorial example, offers new opportunities for animal and human epidemiologists to detect interactions of interest in the field, a crucial step in the challenge of multiple infections. Numerous parasites species circulate simultaneously in natural populations. Many of them are able to infect a same host species and a host individual can therefore be infected by several parasites at the same time. These multiple infections are not only common in nature but usually more frequently encountered than infections by a single parasite [1] . Within a host individual, parasites can thus interact, either in a synergistic manner (parasite A favours infection by parasite B or worsens the symptoms caused by B) or in an antagonistic manner (parasite A decreases the infection risk by parasite B or reduces the symptoms caused by B) [2] . As these interactions can have important epidemiological, biological and clinical consequences (e.g., [3] [4] [5] [6] [7] ), detecting, understanding and evaluating them is essential to understand the phenomena and to control and manage infectious diseases.
In recent years, the question of polyparasitism has attracted considerable attention [4, 8, 9] , although in reality the subject has a long history of experimental investigation under laboratory conditions [10, 11] . Many epidemiological studies have also been conducted on the main human pathogens, motivation for the study of polyparasitism being in particular driven by the urgency to understand the epidemiological and clinical consequences of infection by parasites potentially interacting with HIV and other emerging diseases [12] and the mechanisms of their interactions. A large amount of work indeed revealed interactions between HIV and tuberculosis, malaria, sexually transmitted diseases, and helminths (e.g., [6, [13] [14] [15] [16] [17] [18] [19] [20] ); as well as interactions between plasmodia parasites and helminths (e.g., [21] [22] [23] [24] ). Studies on animal hosts also revealed interactions between their parasites, with many studies on helminth communities (mammals: [4, 25, 26] , birds: [27] , fish: [28] ), and fewer on protozoan species (e.g., [29] ) or viruses (e.g., [30] ). Many diseases have been revealed to be affected by the presence of other disease-causing agents, altering the rates of species co-occurrence, levels of infection and disease severity. Parasite interactions have also been shown to affect the success of parasite vaccination strategies [31] and could be involved in disease (re)emergence [32] , reinforcing the interest of these studies.
If laboratory experiments have clearly demonstrated that interspecific parasite interactions occur, often mediated by host immune responses [1, [33] [34] [35] , attempts to detect such effects in natural populations have generally been less successful. Indeed, detecting their existence on the field is not easy, due to complex networks of indirect effects making it difficult to infer underlying processes. Field studies are however essential as experimental systems are oversimplified and require an existing suspicion of interaction between the studied parasites. In addition, only studies in natural populations can give access to infection and co-infection probabilities. In other words, before studying their mechanisms in the lab, interactions of interest must be identified in the field. Main difficulties encountered in field studies are methodological. Many confounding factors can create statistical associations between parasites even if there is no true biological interaction between them, which may alter conclusions about the importance of interspecific interactions [36] [37] [38] [39] [40] . A similar transmission mode, for example, can alone increase the risk of co-infection. The excess of positive associations found in strongylid communities in domestic horses, ruminants and macropod marsupials is in particular likely to be due to the common habit of these hosts feeding on pastures contaminated with the larvae of a number of nematode species [41] [42] [43] . In addition, environmental, behavioural or host-specific factors can be associated with both types of infection and influence epidemiological and geographic patterns of infection and disease. Among such common risk factors, some have long been recognised, such as sexual behaviours for sexually transmitted diseases (e.g., [44] ), socio-economic status for infections particularly prevalent in poor regions such as helminth infection and malaria [45] , or age for many diseases (e.g., [46] ). As apparent associations between two infections may be due to common risk factors, they are crucial to identify and to take into account in the analysis. However, such confounding factors are difficult to control and few methods enable to take them into account.
A variety of analytical approaches have been suggested to detect associations in parasite communities, primarily focusing on macroparasite (parasitic helminth) communities (e.g., [4, 39, 47, 48] ). However, they implicitly assume that the direction and strength of an observed association between parasite species reflects an underlying biological interaction, and their reliability to detect interactions has been recently questioned [49] . The adoption of a generalized linear mixed modelling (GLMM)-based approach has been rather suggested by Fenton et al. [49] (see also [50] ). Apparently more robust to detect interactions between macroparasites, this method has the advantage of offering the opportunity of taking into account the variance caused by other factors.
Nevertheless, field data, particularly relating to microparasites, are most of the time serological (i.e. presence-absence data). Indeed, viral excretion is usually too short to make antigen detection an efficient tool to follow microparasites in natural populations, as host capture and sampling would have to be done exactly during the excretion period, especially during nonepidemic phases. Most field data are thus limited to observed frequencies of seronegative, seropositive and doubly seropositive individuals. In this context, the search for potential interactions between pairs of microparasites is traditionally done by calculating odds ratios in stratified data or by a Pearson's chi-square test of independence (e.g., [29, 51, 52] ). The latter compares the observed frequencies to the frequencies expected if parasites are independent, under the null hypothesis that the joint distribution of the cell counts in a 2-dimensional contingency table is the product of the row and column marginals. However, such methods ignore confounding factors and/or the possible simultaneous action or interaction of several of them. Significant associations detected in this manner can therefore be either true biological interactions or statistical associations, with no means of distinguishing the two. Alternative methods have been therefore proposed to determine the expected frequencies in a modified chi-square analysis. Some are based on the estimation of ''pre-interactive'' species prevalences [53] , which requires previous knowledge of dominance relationships between parasites species. Some others are based on log-linear models [e.g., [54] [55] [56] . In addition, another way to take risk factors into account is to include them in a logistic regression analysis and to determine whether parasite B status is still a predictor of parasite A status [52, 57] . However, the main drawback of methods based on log-linear or logistic regression models is that they are based on an asymptotic approximation of the deviance, which might not be relevant for small sample size data.
In the present paper, we propose another method (termed the ''corrected chi-square'') to detect microparasite interactions from serological data, based on an adaptation of the Pearson's chisquare test. By combining logistic regressions and chi-square tests, we are able to calculate the expected frequencies of co-infected individuals if parasites are independent considering their risk factors, and to compare them to the observed ones. In a first step, we perform a theoretical comparison of the robustness of the corrected chi-square and the logistic regression approaches. In a second step, both approaches are applied to serological data obtained in natural populations of domestic cats to search for potential interactions between four feline viruses. The domestic cat is indeed an appropriate model to investigate such questions as its main viruses are well known and rather easy to survey on the field, and its natural populations, although very flexible in their social and spatial organisation, have been extensively studied [32, [58] [59] [60] [61] [62] .
1.1. Logistic regression analysis. A first way to test the interaction between two pathogens is to test the effect of the serological status to one virus on the probability of being seropositive to the other. A logistic regression was used for that purpose. The approach allows correcting for common risk factors by adding known or suspected risk factors as correction variables. The logistic regression model reads:
Where F k denotes the k-th risk factor, p 1 is the probability of seropositivity to pathogen 1 and S 2 the serological status to pathogen 2. The coefficients a k (k = 0…K) and b are the coefficients of the logistic regression. The interaction between the two pathogens was tested using a likelihood-ratio test (LRT) testing H0: b = 0 vs H1: b=0. The asymptotic chi-square approximation was used to derive the Pvalue of the test of independence between the two viruses [63] .
1.2. Corrected Pearson's chi-square tests. The corrected chi-square approach is based on the idea that the coefficients of the logistic regression of the two viruses can be used to estimate the number of seronegative, single-and double-seropositive individuals expected if the two pathogens are independent. As the classical chi-square, the corrected chi-square compares the observed (O i,j ) and theoretical (E i,j ) numbers of individuals with different combinations of status (seropositive or seronegative) for the two pathogens using the chi-square statistic:
where i is the status to pathogen 1 (0 for seronegative and 1 for seropositive) and j is the status to pathogen 2. To calculate the E i,j , for each pathogen taken separately, a logistic regression including K risk factors (see previous section) is run to estimate the probability of being seropositive for each individual (termedp p p,x for individual x and pathogen p, p[ 1,2 f g):
Whereâ a p,k denotes the estimation of the regression coefficients for pathogen p and F k,x the value of the k-th risk factor in individual x.
The theoretical contingency table is then deduced from these probabilities:
For each pair of viruses, the distribution of the corrected chisquare was determined by a parametric bootstrap run as follows:
Step 1: Estimated seropositivity probabilities (p p p,x ) are used to generate in silico serological data for both pathogens independently.
Step 2: The corrected chi-square is calculated for this in silico dataset.
Steps 1 and 2 were repeated 1000 times, leading to 1000 independent realisations of the corrected chi-square statistic under the null hypothesis of independence between the two pathogens.
Two ways of calculating the P-value were derived from this procedure. P-value1 was estimated assuming that the corrected chi-square is proportional to a chi-square with one degree of freedom, the coefficient of over-(or under-) dispersion (ĉ) being defined by the mean of the bootstrapped corrected chi-square. P-value2 was given by the proportion of bootstrapped corrected chisquares which were smaller than the observed value. In principle, P-value2 is better (no assumption on the distribution of the Likelihood Ratio Test, LRT, is made), but requires running enough simulations, which may be long in some cases. P-value1 allows working with smaller numbers of simulations when simulation times are too long.
The R program is available as supplementary file (File S2) and can be applied to any presence-absence data to calculate the corrected chi-square and the associated P-values. A tutorial example (File S3) illustrates its use step-by-step using an example dataset (File S4).
The main criticism that could be made to the logistic regression approach is that it is based on the asymptotic distribution of the LRT. In practice, the chi-square approximation is true only for large datasets. In the present paper we investigated the robustness of the logistic regression to different sample sizes and numbers of correction risk factors. We also aimed to compare how robustness is affected by the type of risk factors considered (qualitative or quantitative). The same investigations were performed with the corrected chi-square test to compare the robustness of the two approaches.
For that purpose, random seroprevalence datasets were generated, assuming independent viruses. Random data were always generated assuming that all individuals had an independent 0.5 probability of being seropositive for each pathogen. N F randomly generated risk factors were considered in the logistic regression for the two pathogens. By construction these factors have no effect (they are chosen independently of the serological status of the individuals) but from a theoretical point of view it is interesting to measure how their inclusion in the model can introduce biases depending on the approach.
Randomly generated factors could be either qualitative or quantitative. For simplicity, qualitative factors had only two modalities, individuals having a 0.5 probability of being in each one. Quantitative factors were chosen for each individual randomly according to a standard normal distribution. To investigate how the nature of risk factors affects robustness, three scenarios were tested: i) all factors are qualitative; ii) all factors are quantitative and iii) half of the factors are quantitative while the other half are qualitative factors (mixed scenario).
Our objective now was to understand how data characteristics (the number of individuals, n, the number of factors, N F and their type, scenario i, ii or iii) would affect the probability of wrongly concluding that there is an interaction between the two pathogens (type I error). For a given combination of these characteristics, a thousand random seroprevalence datasets were generated and we estimated the type I error associated to each approach as the proportion of random datasets for which the P-value was below 5%.
3. Application to cat data 3.1. Ethics Statement. The field work has been made by qualified people according to the French legislation. Accreditation has been granted to the UMR-CNRS 5558 (accreditation number 692660703) for the program.
3.2. The feline viruses. The Feline Immunodeficiency Virus (FIV), is a major non-traumatic cause of death in adult cats, and is associated with immunosuppression causing secondary infections [64] . This retrovirus can infect other felids, most of which are threatened or endangered species e.g., the European wildcat (F. s. silvestris) [64] [65] [66] . It is mainly transmitted by bites, through a direct horizontal mode [67] , principally during aggressive or sexual contacts [64, 68] . The Feline Herpesvirus (FHV) and the Feline Calicivirus (FCV) are responsible of upper respiratory tract disease, of concern in veterinary medicine [69, 70] . Both viruses are transmitted through 'amicable' contacts, by oral, nasal and ocular secretions during close interactions [71, 72] . FHV infected cats become asymptomatic carriers, but the latent infection can be reactivated by a stress (i.e., change of habitat, lactation or fights between males; [73] ). The Feline Parvovirus (FPV) infects all felids, as well as other carnivores [74] , and FPV infection may be fatal especially in kittens [75] . The virus is transiently excreted in feces, urine, saliva and vomiting and its high resistance in the environment (still infectious after 13 months at 4-25uC; [76] ) makes indirect transmission through feces and contaminated areas largely predominant [77, 78] .
3.3. Serological data. The serological statuses for FIV, FHV, FCV and FPV were obtained in 2007 in 15 natural rural populations of domestic cats in North-Eastern France [62, 79] . Cats were captured using baited traps or directly caught by the owner, anaesthetized, measured, and blood samples were taken from the jugular vein. FIV-antibodies were immediately searched for with a commercial kit using the ELISA method (SNAP Combo +, Idexx), whereas specific antibodies against FHV, FCV or FPV were measured by a specific blocking ELISA [80] . None of the cats was vaccinated. All six pairs of viruses were tested for potential association. Between 467 and 474 cats were tested for each virus and 465 to 469 were double-tested (depending on the virus pair).
Previous analyses using logistic regression models with the same dataset revealed the combination of risk factors that were supported by our data [62] . Five factors were initially investigated: age (AGE), sex (SEX), way of life (owned or unowned, WOL), orange phenotype (orange or non orange, PHENO) and body mass (MASS) and one correction factor (the population of origin, POP) was considered. For each virus, the most appropriate model was selected using the Akaike Information Criterion adjusted for small sample size (AICc, [81] ). Ideally, all factors potentially creating apparent associations should be included in the model. But to limit the number of correction risk factors, the minimal model containing the identified risk factors for the two viruses was retained as a compromise for each pair (Table 1) .
The corrected chi-square was robust for all tested sample sizes and numbers of parameters, whatever the nature of the factors (scenarios i, ii, iii) and the method used to calculate the P-value (P-value1, P-value2, see File S1 and Fig. S3 for more details). The type I error of this method remained indeed very close to 5% (Fig. 1) . On the contrary, the robustness of the logistic regression approach decreased with the N F /n ratio (number of factors/sample size). In scenarios i (only qualitative factors) and ii (only quantitative factors), the type I error was around 5% for a ratio of 0.005, around 8% for a ratio of 0.15 and around 20% for a ratio of 0.35. It became significantly different from 5% for ratios larger than 0.12 (type I error = 6.7%, z = 2.47, p = 0.019) and 0.08 (type I error = 7.9%, z = 4.21, p = 5.7610 25 ) for scenarios i and ii, respectively. In the mixed scenario (iii), the type I error became significantly different to 5% for all N F /n ratio larger than 0.075 (type I error = 7.1%, z = 3.047, p = 0.0038). More details are available in File S1 and Fig. S2 . Taken together, these results show that, as a rule of thumb, the logistic regression approach is robust for N F /n ratios below 0.1 for all types of factors.
The two approaches (corrected chi-square and logistic regression) were used for the analysis of the interactions between four cat viruses ( Table 2) .
Results showed that the interaction was not significant for pairs involving FIV. All other pairs (FHV-FCV, FHV-FPV and FCV-FPV) were found to interact, i.e., the number of individuals coinfected by two viruses could not be explained by shared risk factors. The three significant associations were all positive, meaning that there were always more co-infected individuals than expected considering shared risk factors (Table 3) .
Pairwise interactions between FHV, FCV and FPV could have come from the fact that one virus was a common risk factor for the two others. This possibility was tested (see the three last lines of Table 2 ) by adding the serological status to one virus as a common risk factor for the two others. Results led to reject this hypothesis, meaning that the observed associations cannot be solely explained by the fact that one virus interacts with the two others.
The two P-values obtained for the corrected chi-squares are coherent. As for the P-values obtained for the logistic regression approach, they are usually slightly lower than those of the corrected chi-squares, probably because of the over-predictive trend of logistic regressions.
In addition, as with simulated data, the logistic regression approach was less robust to small sample sizes than the corrected chi-square (Table S1 ). This was tested by randomly sampling smaller subsets of the cat data in order to increase the N F /n ratio.
Finally, to emphasise the need to consider risk factors in the analysis of interactions, we also calculated the classical independence Pearson's chi-square. This approach, which does not integrate risk factors, predicted an association between five of the six tested pairs. In the case of the FIV-FCV and FIV-FHV pairs, it would lead to wrongly conclude on the existence of an interaction, whereas the two approaches have shown that these apparent interactions were in fact explicable by shared factors.
Common risk factors can create statistical associations. This work confirmed that ignoring them would lead to wrong conclusions. Ignoring them would indeed result in an over-estimation of the number of interactions as any association, biological or statistical, would be put in one basket. The loss of significance after controlling for other factors was illustrated in this paper with feline viruses data, and was previously found by Behnke et al. [39] for helminth parasites of the wood mice. The next step was to identify an appropriate way to take those risk factors into account.
Two approaches to take risk factors into account with serological data (i.e., presence-absence) were proposed and examined. Those are the use of logistic regression models as Table 1 . Risk factors models used to test for potential association between pairs of feline viruses. previously done by some authors [52, 57] , or an adaptation of the chi-square test for independence presented for the first time in this paper.
To determine which method should be used under which circumstances, we need to make the following considerations.
First, the corrected chi-square involves 2n+2 estimations of the logistic regression coefficients, n being the number of bootstraps. In comparison, only two models must be parameterized in the logistic regression. As a consequence, the logistic regression approach is much faster to run (less than a second versus 2.5 minutes for the corrected chi-square for a model with 6 factors in full interaction and 300 individuals, for 1000 bootstraps, using a desktop computer with an Intel(R) core(TM)2 Quad CPU Q6600 processor). Second, the corrected chi-square is more robust than the logistic regression, especially for small sample size. A first solution would be to use the corrected chi-square as soon as simulation times are acceptable. For a 5% rejection threshold, a more straightforward alternative is to use the corrected chi-square by default as soon as the ratio between the sample size and the number of parameters is below 10 and the logistic regression in the opposite case. However, we did not test all potential situations and further analyses are needed to determine the limit of robustness of the logistic regression approach (in particular in situations where the probability of infection is not 50% and can be affected by risk factors).
Two P-values have been proposed for the corrected chi-square. The first one relies on the assumption that the corrected chi-square is proportional to a chi-square with one degree of freedom; the second one simply counts the proportion of in silico datasets for which the value of the corrected chi-square is above the observed value. Both P-values led to consistent results using a 5% rejection threshold, consistently with the fact that for all tested pairs the corrected chi-square fitted well with an under-dispersed chi-square with one degree of freedom (Fig. S1, Fig. S2 ). Which one should be used in practice actually depends on the simulation time. If simulations are fast enough and if running 1000 bootstrap is acceptable, P-value2 should be preferred. In the opposite case, a good option is to run much less bootstraps (typically 30) and to use P-value1.
Even if other alternative methods allow taking covariates into account, we only compared the corrected chi-square to the logistic regression approach. We could have compared it as well to loglinear models, which model the probability of infection with single and multiple parasite species from contingency tables and allow including known risk factors. However, in this approach the independence between parasites is tested using likelihood ratio tests, which are based on an asymptotic approximation of the deviance as in the logistic regression approach. They should therefore have the same limitations than logistic regressions and their robustness should be similarly influenced by the N F /n ratio. In addition, continuous variables are usually discretized in loglinear models, whereas the corrected chi-square allows working with continuous data.
After correction by the known risk factors of the viruses, three pairs of feline viruses out of six appeared to be significantly associated. The N F /n ratio being 0.04 to 0.06, the logistic regression approach can be considered robust, at least for a 5% rejection threshold.
First, it is worth noting that age is a crucial covariate. The infection probability of all viruses increases with host' age [62] , thus age must strongly participate in the generation of false interactions. This age-dependence is due to both a biological effect (i.e., behaviors and immune defenses may evolve with age, [82, 83] ) and a mechanical effect (i.e., older individuals are more likely to be seropositive because of a longer exposure time). Disentangling both effects would require the use of Susceptible-Infected-Recovered (SIR) models, but was not necessary here. Indeed, to The type I error of the corrected chi-square tests represented here is based on P-value2 but similar results were observed with P-value1 (Fig. S3) . Note that for the logistic regression approach, points resulting from a given sample size were linked to see the effect of the N F /n ratio for different sample sizes (solid line: n = 100, dashed line: n = 200, dotted line: n = 300). The dashed horizontal line represents a type I error of 5%. doi:10.1371/journal.pone.0029618.g001 correct for age in the study of interactions, the important is to model the evolution of the probability of infection with age.
Correcting for all risk factors, no pair of viruses involving the Feline Immunodeficiency Virus (FIV-FHV, FIV-FCV, FIV-FPV) was significantly associated. This result is at first surprising because, as in humans infected by HIV, feline AIDS is characterised by a chronic immunodeficiency, allowing subsequent opportunistic infections (review in [84] ). Indeed, although FIV positive cats can mount immune responses to administered antigens other than during the terminal phase of infection, their primary immune responses may be delayed or diminished [85, 86] . Experimental studies also revealed that cats co-infected by FIV and FCV or FHV had more severe disease signs than non-FIV infected cats [87, 88] . In addition, the presence of FHV was shown to accelerate FIV transcription through the activation of the FIV long terminal repeat [89] , a phenomenon that was also shown in vitro for the human versions of the viruses, HSV2 and HIV [90] [91] [92] [93] . Those laboratory experiments show that FIV infection may increase the severity of FHV or FCV-induced clinical signs but do not address the question of the effect of FIV on the sensitivity to FHV or FCV infection. Furthermore, the few epidemiological studies interested in the question did not demonstrate any epidemiological association between FIV and FHV [94] . In other words, if experimental investigations suggest a synergy between FIV and FHV and between FIV and FCV towards a more severe disease, our sero-epidemiological study suggests that the identified risk factors explain by themselves the apparent increase of double sero-positive individuals.
As for the FIV-FPV pair, this study is to our knowledge the first to search for a potential association. Whether risk factors were taken into account or not, we did not find any significant association between the two viruses. Again, this could be at first surprising as both viruses are supposed to be immunosuppressive [84, 95, 96] . In experimental conditions, FPV infection is more severe in FIV-infected cats [97] . Consequently, a positive association could have been expected if infections had facilitated each other (leading to numerous co-infections) or a negative association if the co-infection had led to a strong host mortality (leading to few co-infections). However, the FPV-induced decrease in the immune response is transient and more likely to occur in young kittens, whereas FIV infection is more frequent in adult cats. The persistence of FPV-antibodies can be longer than 7 years [98] , and consequently, double seropositivity against FPV and FIV is not synonymous of co-infection. It is likely that co-infections by the two viruses are not frequent and mainly occur in adult animals which are less sensitive to FPV. As no association was evidenced for these three pairs of viruses, the FIV infection does not seem to modify the risk of infection by another virus. However, our results do not exclude the occurrence of an interaction once both parasites are in contact within the host (e.g., directly through competition or indirectly via the host immune system), as suggested by several experimental co-infection studies. In addition, the FIV seropositivity status may encompass different stages of the infection with various degrees of immunodeficiency. The results of this study do not exclude the possibility that late stage FIV infection may increase the sensitivity to the other feline viruses.
On the contrary, the three other pairs (FHV-FCV, FHV-FPV and FCV-FPV) were significantly associated after correction by their known risk factors. It is to our knowledge the first evidence of a possible interaction between those viruses. As more double seropositive cats than expected under the independence hypothesis were observed, possible synergies are suggested. After an acute infection, FHV is known to persist life-long in a latent form, which can be reactivated in stressful conditions [73] . Infection with FPV or FCV could thus be responsible for the reactivation of FHV in latently infected animals, resulting in seroconversion against both FHV and the new infecting virus. This could explain the FHV-FCV and FHV-FPV associations. In addition, since FPV is more immunosuppressive than FCV, the interaction between FPV and FHV is expected to be stronger than that between FCV and FHV, which is consistent with our results. The immunosuppressive effect of FPV could also explain the association with FCV. In that case however, contrary to FHV, it would require that the FCVinfection occurs at the time of the immunosuppression occuring within the two weeks post-FPV infection. Interestingly, a similar association between FPV and FCV antibodies was described in free-ranging lions in East Africa [99] .
This work pointed out new probable synergies between feline viruses that can now be further investigated in laboratory conditions. However, the associations could also result from the existence of an unknown confounding factor common to FHV, FCV and FPV. The feline parvovirus is immunosuppressive, as a result of the strong leukopenia occurring within the two weeks post-infection [95, 96] . This virus could therefore be a confounding factor to the FHV-FCV pair if FPV-seropositive cats are more susceptible to FHV and FCV at the same time. However, as shown in this paper, the FHV-FCV interaction remained significant after correction by FPV (Table 2) .
If FPV is not a confounding factor, we cannot exclude the existence of another one, such as a greater susceptibility of certain individuals to infections whatever the parasite involved. Numerous studies have shown that an extensive inter-individual variability exists in response to certain pathogens, such as HIV (review in [100] ), trypanosomiasis (review in [101] ), or human and bovine tuberculosis (reviews in [102, 103] ), including variations in susceptibility to the parasite, its transmission, and/or the course of disease progression. It has been attributed to host determinants and variability in multiple genes that regulate virus cell entry, acquired and innate immunity (e.g., macrophages, molecular and cellular actors of the inflammatory reaction), and others that influence the outcome of the infection. Hosts with a diminished or delayed innate immune response may in fact be more susceptible to any infection, with physiological parameters, such as hormonal profiles (e.g., [104] ), possibly playing a role in the modulation of transmission efficiency and/or in the immune response intensity. A weaker physical condition could also lead to a higher sensitivity to infectious agents (lower dose-effect, different intra-host dynamic) (e.g., [105] ). More generally, individuals' personality may as well be involved [61, 106] . A better understanding of genetic, physiological and immunological basis of such inter-individual variability would therefore be of particular interest in the context of polyparasitism. Another perspective of this work is the development of new methods able to distinguish pairwise interactions from those due to common confounding factors shared by the three viruses. Such methods could use the proportion of infected individuals that are in reality triply infected.
While the study of macroparasites usually uses quantitative data (i.e., parasite load per individual host), the study of microparasites on the field is most of the time limited to presence-absence data (i.e., serology), making the detection of associations between parasites more complicated from a methodological point of view. The corrected chi-square proposed in this study is, with the logistic regression approach, currently one of the rare ways to search for interaction between parasites from presence-absence data. This work provides evidence of the efficiency of such methods to reduce the bias introduced by common risk factors and encourages their use. However it also points out the low robustness of the likelihood ratio test for certain data characteristics. The corrected chi-square test must indeed be preferred for small sample size.
Those methods can be applied to any epidemiological study based on serology, within human or animal host populations. Applied here to feline viruses, they revealed significant associations between three pairs of feline viruses. If they still do not allow us to decide whether such associations are really true interactions or whether they reveal the existence of ''over-susceptible'' hosts, we believe it is an important step forward as it offers the possibility to point out parasites associations that should be further investigated in experimental conditions. The understanding of parasites interactions and of their consequences on diseases evolution, emergence and management is indeed a crucial challenge for human and animal epidemiologists of our time. Figure S2 Issue of the conformity tests of the type I error to 5% according to the N F /n ratio for the logistic regression approach. The issue was coded 1 when the test was significant, 0 when not and the resulting logistic regression was drawn (dark line). Three scenarios are considered: i) all factors are qualitative (A); ii) all factors are quantitative (B) and iii) half of the factors are quantitative and the other half are qualitative (mixed scenario, C). (EPS) Figure S3 Type I error (%) of the corrected chi-square tests according to the N F /n ratio and the type of P-value used for the corrected chi-square: P-value1 (blue empty points) or P-value2 (red full points). Three scenarios are considered: i) all factors are qualitative (A); ii) all factors are quantitative (B) and iii) a half of the factors is quantitative and the other half is qualitative (mixed scenario, C). The dashed horizontal line represents a type I error of 5%.
Table S1 Corrected chi-square tests and logistic regressions to search for feline viruses' interactions using subsets randomly sampled in cat data such that the N F / n ratio takes various values.
File S1 Robustness of the logistic regression approach and of the corrected chi-square test. (1) Conformity tests of the type I error to 5%, (2) Influence of the way to calculate the Pvalue of the corrected chi-square test on the robustness of the study. (DOC) File S2 ''Chi2corr'', an R program for the application of the corrected chi-square test to any presence-absence data: test statistic, observed and expected frequencies, estimated dispersion coefficient (parametric bootstrap), P-values and distribution of the bootstrapped corrected chi-square.
File S3 A step-by-step example of application of the corrected chi-square test to search for interaction between two parasites, using a provided dataset (''da-ta_example.txt'', File S4) and the provided R program (''Chi2corr.R'', File S2).  The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues. CEACAM1 is a member of the carcinoembryonic antigen (CEA) gene family of Ig-like cell-cell adhesion molecules [1, 2] . Like other members of this family, CEACAM1 is a type I transmembrane protein with a heavily glycosylated extracellular region composed of four Ig-like domains, a transmembrane domain and a cytoplasmic tail [3] . In the rat liver there are two allelic variants of CEACAM1 which differ by 16 amino acids in their N-terminal domains [2, 4] and two major splice variants, designated 4L and 4S, that are distinguished by differences in the length of their cytoplasmic tails of 70-72 amino acids and 10-12 amino acids, respectively [2, 4, 5] .
Both isoforms of CEACAM1 are down-regulated in epithelial cancers arising in the liver, prostate, bladder and colon [6, 7, 8, 9, 10, 11, 12] , a finding that prompted re-expression analysis aimed at defining structure and function relationships. Restoration of expression by infecting rat cell lines derived from primary hepatocellular carcinomas (r-HCC) with a CEACAM1-4L retrovirus resulted in potent growth suppression in vitro and tumor suppression in vivo [13] . Further analysis showed that the 4L cytoplasmic domain was necessary and sufficient for tumor suppression [14] , an activity that required phosphorylation of serine 503 and in colon carcinoma cells, concurrent phosphorylation of tyrosine 488 [15, 16] .
In contrast to CEACAM1-4L, CEACAM1-4S failed to generate a tumor suppressor phenotype when re-expressed in r-HCC or mouse colon carcinoma cell lines [13, 17, 18] . However when expressed in MCF7 mouse mammary carcinoma cells, CEA-CAM1-4S induced glandular morphogenesis, an activity requiring phosphorylation at one or more sites in the 4S cytoplasmic domain [19, 20, 21] . Site directed mutagenesis further showed that mutation of phenylalanine 445 at the C-terminus of the CEACAM1-4S cytoplasmic domain not only compromised interactions with the actin cytoskeleton but also inhibited lumen formation, suggesting interactions of CEACAM1-4S with the cytoskeleton were an important determinant of glandular morphogenesis. Interestingly, when mouse mammary carcinoma cells were grown in humanized NOD/SCID mouse mammary fat pads, only the 4L isoform initiated morphogenesis, the opposite of what was observed in vitro [21] , raising questions about the equivalence of in vitro and in vivo models of morphogenesis.
Because of its role in cell adhesion, the CEACAM1 N-terminal Ig domain [22, 23, 24] , like the cytoplasmic domain, has been the focus of numerous investigations. The adhesive epitope within the N-terminal Ig-domain has been defined for rat [24] , mouse and human CEACAM1 [22, 23] , the evolutionary relationships between CEACAM1 from different species has been determined [25, 26] and the three dimensional structure has been established by X-ray crystallography [27] . In comparison, the CEACAM1 transmembrane domain has received relatively little attention, perhaps because transmembrane domains have often been viewed as passive anchor sequences that span the lipid bilayer. Over the last 10 years, this simplistic viewpoint has fallen by the wayside in the face of accumulating evidence implicating transmembrane domains in helix-helix interactions leading to dimerization, oligomerization and signal transduction [28, 29, 30] . The possible involvement of transmembrane-transmembrane domain interactions in the functionality of CEACAM1 was suggested by the presence of repeating GXXXG motifs (where X represents any amino acid), sequences known to control protein dimerization and signaling [30, 31] , and the presence of transmembrane C-terminal tyrosine residues shown in other proteins to be mediators of molecular recognition, self assembly and signal transduction [32] . In the present investigation, we have examined the effect of transmembrane domain mutations on the ability of CEACAM1-4S to confer an anchorage independent phenotype when expressed in a clonal line of CEACAM1 negative, anchorage dependent rat hepatocellular carcinoma cells, designated 253-NT. Our results show that transmembrane mutations in both GXXXG and tyrosine residues have both positive and negative effects on the anchorage independent phenotype produced by wild type CEACAM1-4S.
The origin and characteristics of MAb 5.4 specific for CEACAM1 and MAb 188.A2 specific for rat transferrin receptor have been described previously [33, 34] . Monoclonal antibody 9.2 (MAb 9.2) was provided by Drs. Werner Reutter and Oliver Baum at the Free University, Berlin, Germany [35] . Mouse anti-human HLA antibody was purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO). The preparation of polyclonal rabbit antipeptide antibodies specific for the CEACAM1-4L and CEA-CAM1-4S has been previously described [36] . The secondary antibodies used for indirect immunofluorescence labeling were Alexa-488 conjugated goat anti-mouse and goat anti-mouse-HRP conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA).
The parental cell line 253T was established from a 2acetylaminofluorene induced rat hepatocellular carcinoma, as described previously [35] . The anchorage dependent 253T-NT cell line was isolated from 253T by limiting dilution cloning. 253T and 253-NT cells were grown in Waymouth medium (Sigma, St. Louis, MO, USA) supplemented with 15% FBS, 1% glutamine (Invitrogen), 0.1% Gentamycin (Invitrogen), and 0.2% Normocin (Invivogen, San Diego, CA, USA). For cell proliferation assays, 1.5610 4 cells were plated in a 24-well plate. At 24, 48, 72, and 96 hours after plating, cells were trypsinized (Invitrogen), stained with trypan blue and counted using a hemocytometer.
RNA was isolated from a normal Fisher rat liver using RNAzol B according to the manufacturer's instructions (Tel-Test, Friendswood, TX). cDNA was synthesized from the purified RNA according to the manufacturer's instructions using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen). CEACAM1-4S cDNA was amplified by PCR from the total cDNA product using primers: CEACAM1-4S Forward 59CAGGAATT-CATGGAGCTAGCC-TCGGCT-39 and CEACAM1-4S Reverse 59-CGAGTCGACT-CGTCAGAAGGAC CCAGATCC-39. The primers contained EcoRI and SalI restriction sites.
A restriction digest was performed on both the CEACAM1-4S PCR product and the pCl-neo plasmid (Promega, Madison, WI, USA) using EcoRI and SalI (New England Biolabs, Ipswich, MA, USA). Following digestion, the plasmid and the PCR product were dephosphorylated using Antarctic phosphatase (New England Biolabs), heat-treated to inactivate the phosphatase, and run on a 1% agarose gel. Bands corresponding to the plasmid DNA and CEACAM1-4S PCR product were purified using the Geneclean spin kit (Qbiogene, Morgan Irvine, CA, USA). The CEACAM1-4S PCR product was ligated into the pCI-neo plasmid using T4 DNA ligase (New England Biolabs). Ligated plasmid was transformed into One Shot OmniMax 2 T1 Phage-Resistant Cells (Invitrogen) according to the manufacturer's protocol. Transformed cells were plated onto LB/ CARB plates and resulting colonies were screened by PCR using forward and reverse primers for CEACAM1-4S (see above). Plasmid DNA from four CEACAM1-4S positive clones was isolated using the Qiagen Endofree maxiprep kit (Valencia, CA, USA) and submitted for DNA sequencing to the W.M. Keck Biotechnology Laboratory at Yale University (New Haven, CT).
Mutation sites were chosen based on the consensus location of the transmembrane domain predicted by the different membrane topology algorithms shown in Table 1 . The results from this analysis showed that the various transmembrane prediction algorithms delineated transmembrane domains with variable N (G424 to I431) and C (Y445 to S449) termini and different numbers of potential GXXXG motifs. All of the predicted transmembrane domains lacked the terminal GLSE predicted by Kyte Doolittle and Hopp-Wood (Table 1 ) and thus did not include the G420-LSE-G424 motif. Ten of the 12 predicted transmembrane domains included G428, strongly suggesting the presence of the two motifs centered on G432 (G428-IVI-G432 and G432-SVA-G436). Although G424 was present in only 3 of 12 predicted TM, G424's location at the N-terminus of a tandem array of three classic GXXXG motifs caused us to consider that the G424-AIA-G432 sequence might function as a dimerization motif when all the other GXXXG motifs were disrupted by G428L and G436L mutations. With regard to the C terminus, all of the predicted transmembrane domains contained Y445 and 7 of 12 contained Y448, leading us to conclude with reasonable certainty that Y448 was within the transmembrane domain or at the very least, at the interface between the transmembrane and cytoplasmic domains. Taking all of these considerations into account, we arrived at the transmembrane sequence proposed in Table 2 . The brackets denote the uncertainty with regard to G424 and Y448. In recognition of the variability in the predicted N-terminus and the uncertainty regarding the functionality of the G424-AIA-G428 motif, we introduced a G424L mutation to disrupt this motif and a G432L mutation to knock out both the G428-IVI-G432 and the G432-SVA-G436 motifs. Disruption of all three of the possible GXXXG motifs was accomplished by introducing both G424L and G432L mutations. To disrupt tyrosine mediated interactions, Y445 and Y448 were replaced with V, the choice of a tyrosine to valine mutation being based on the following considerations: 1) valine lacks a hydroxy group; 2) valine does not have an aromatic side chain; 3) valine is non-polar and thus should not disrupt the alpha helix or alter transport to the plasma membrane.
Site-directed mutagenesis was performed on the pCI-neo plasmid containing the CEACAM1-4S gene using the Quik-Change II XL Site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. The oligonucleotide primers used for mutagenesis were as follows: 
For transfection of 253-NT cells, 1610 6 cells at 75% confluence were transfected with 7.5 mg of plasmid DNA in Lipofectamine LTX reagent (Invitrogen) following the manufacturer's recommended protocol. After incubation for 24 hours in complete medium, stable transfectants were selected by maintaining cells in complete medium containing geneticin (Invitrogen) at 600 mg/ml with medium changes every 48 hours. For all subsequent experiments, geneticin resistant cells were maintained in complete selection medium.
Cells were seeded in permanox chamber slides (Nalge Nunc International, Rochester, NY) at a density of 1610 5 cells/ml and incubated in a 5% CO 2 humidified chamber for 48-72 hours. Cells were washed three times in ice-cold PBS and fixed for 10 min in ice-cold acetone. Normal liver sections and frozen tumor sections were prepared as previously described [37] . Cells were blocked for 15 min with 1% normal goat serum (Sigma-Aldrich) in PBS and incubated for 30 min at room temperature with a 1:200 dilution of primary MAb 9.2 specific for CEACAM1-4S. After three washes in PBS, cultures were fixed with 4% paraformaldehyde for 1 min, quenched in 0.1 M glycine in PBS, and incubated at 4uC for 30 min in Alexa 488 conjugated goat anti-mouse IgG (1:400 dilution). Cells were examined by fluorescence microscopy using a Nikon Eclipse E800 microscope (Nikon Instruments, Inc., Melville, NY).
Confocal images were acquired with a Nikon PCM 2000 (Nikon Inc. Mellville, NY, USA) using the Argon (488) and the green Helium-Neon (543) lasers. Serial optical sectioning was performed with Simple 32, C-imaging computer software (Compix Inc, Cranberry Township, PA, USA). Z series sections were collected at 0.5 mm using a 606 PlanApo objective and a scan zoom of 26. Images were processed in NIH Image J (National Institutes of Health, Springfield, VA, USA).
Cells were harvested at 75% confluence using non-enzymatic cell dissociation solution (Sigma-Aldrich) and labeled in suspension with anti-CEACAM1-4S monoclonal antibody MAb 9.2 as described above. Primary and secondary antibodies were diluted in 1% normal goat serum in PBS and incubated sequentially with cells at 4uC for 20 min. Cells were washed with sterile ice cold sort buffer (Ca/Mg++ free, pH 7.0 PBS, 5 mM EDTA, 2 mM HEPES and 1% FBS) and resuspended at a final concentration of 8-10610 6 cells/ml. CEACAM1-4S positive cells were isolated by FACS as previously described by Comegys et al. [38] .
Cells were harvested using non-enzymatic cell dissociation solution (Sigma-Aldrich), suspended in 0.76% low melting point agar diluted 1:1 with 26 complete Waymouth medium (Sigma-Aldrich) and seeded in 6 well plates (10 4 cells/well) coated with 2 mls of 1.25% low melting point agar (Sigma-Aldrich) diluted 1:1 with 26 complete Waymouth medium. Plates were incubated at 37uC for three weeks and analyzed microscopically to identify anchorage independent clones. Colony size was determined from digital micrographs using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Bethesda, MD) to determine the average area calculated from measurements made on a total of 15-30 colonies.
All animal studies were performed using protocols approved by the Rhode Island Hospital Institutional Animal Care and Use Committee (cmtt# 0268-98). Athymic nude mice were purchased from Harlan (Indianapolis, IN). Twenty-four hours prior to transplantation of tumor cells, animals were injected intraperitoneally with anti-LY2.2 antibodies containing anti-asialo GM1 to suppress T-cell and NK cytotoxic activity, respectively [39, 40] . Cultured cells were harvested as described previously [13] . Subcutaneous injections with 5610 6 cells per site were performed under aseptic conditions into both of the upper flanks. For intraperitoneal injections, mice were injected with 2610 6 cells suspended in HBSS. Tumors were excised at three weeks postinjection, weighed, frozen in a hexane/acetone bath and stored at 280uC. Tumors were analyzed by indirect immunofluorescence as described above.
Immunoblot Analysis Following Blue-Native (BN) PAGE BN-PAGE analysis was performed on crude membrane isolates prepared from 253T-NT cells stably transfected with empty vector, wild type CEACAM1-4S and each of the single and double glycine mutants described above. Cell pellets were thawed on ice and lysed following 10 passages through a 20-gauge needle. Lysates were centrifuged at 3,500 rpm for 15 min at 4uC and MgCl 2 and benzonase (Sigma Aldrich) were added to the supernatant to give final concentrations of 2 mM and 1 unit/ml, respectively. After incubation at room temperature for 30 min, membranes were centrifuged at 17,000 rpm for 15 min at 4uC. Blue-native (BN) PAGE electrophoresis was performed using the Native Page Novex Bis-Tris gel system (Invitrogen). Membrane pellets were solubilized on ice for 30 min in 16 Native Page sample buffer (Invitrogen) containing 1% dodecyl-ß-D-maltoside (Invitrogen) and protease inhibitors. After a 30 min incubation, the samples were centrifuged at 20,0006 g for 20 min at 4uC to remove insoluble material. A Bradford assay (Bio-Rad, Hercules, CA, USA) was performed to determine protein concentration. One ml of 5% NativePage G-250 sample additive (Invitrogen) was added to each sample, and the samples and a Native Mark protein ladder (Invitrogen) were loaded into the wells of a 4-16% Bis-Tris gel (Invitrogen). After running at 150 V for approximately 30 min, the cathode buffer was changed from 0.02% to 0.002% G-250 and electrophoresis at 150 V was continued for an additional 120 min. BN-PAGE gels were immunoblotted onto PVDF membranes (Bio-Rad). Transfer was performed using Bio-Rad's semi-dry transfer apparatus for 1 hour at 25 V. After transfer, membranes were incubated in 8% acetic acid for 15 min, rinsed with water and air-dried. PVDF membranes were re-hydrated with methanol, rinsed with water and blocked in 5% milk overnight at 4uC. Blots were labeled with antibodies as previously described [24] .
Immunoblots were prepared with extracts from 80% confluent cell cultures lysed in RIPA buffer (Pierce, Rockford, IL, USA) containing protease and phosphates inhibitors (Calbiochem). Cell lysates were centrifuged at 14,000 rpm for 15 min and resolved by SDS-PAGE. Immunoblots were prepared and visualized as previously described, using MAb 9.2 to detect CEACAM1-4S [13] . The density of the MAb 9.2 reactive bands was determined by image analysis of digital images of immunoblots captured using a Versadoc Imaging System (Bio-Rad) and Quantity One software (Bio-Rad), as previously described [13] .
A paired t-test was performed to determine statistical significance using GraphPad QuickCalcs software (GraphPad Software Inc., La Jolla, CA).
In previous investigations, we focused on the structural and functional determinants of tumor suppression mediated by CEACAM1-4L, one of two major splice variants expressed in rat liver [13, 16] . The primary goal in these studies was to gain insight into the role of the cytoplasmic and extracellular domains in cell adhesion and tumor suppression. In the present report, we have focused on the CEACAM1 transmembrane domain, a relatively uncharacterized region shared by both the 4L and 4S isoforms. Our cell model for these studies was an anchorage dependent, CEACAM1 negative subclone designated 253-NT that was derived clonally from the parental rat 253T cell line [6] . When suspended in soft agar, 253T cells continued to grow and by three weeks had formed well-defined anchorage independent colonies ( Figure 1A ). In comparison, soft agar cultures of 253-NT cells showed no evidence of significant growth and after three weeks were composed almost entirely of single cells ( Figure 1B) . In contrast, 253-NT cells stably transfected with a wild type CEACAM1-4S expression plasmid and enriched by FACS to yield cultures at least 70% positive for CEACAM1-4S expression regained the anchorage independent phenotype of the parental 253T cells ( Figure 1C ) while cells stably transfected with empty vector ( Figure 1D ) had not grown significantly after 3 weeks in soft agar.
Wild Type and Transmembrane Domain Mutants were Expressed at Similar Levels on the Surface of Stably Transfected 253T-NT Cells Sequence analysis showed that the CEACAM1-4S transmembrane domain contained multiple GXXXG dimerization motifs and two C-terminal tyrosine residues which in other transmembrane receptors were involved in transmembrane domain dependent signaling events [30, 31, 32] . To determine if these transmembrane domain elements played a part in the anchorage independent phenotype induced by CEACAM1-4S, nine expression vectors encoding CEACAM1-4S with transmembrane domain mutations in the GXXXG motifs and/or tyrosine residues were constructed as described under Methods and shown in Table 1 . These vectors were used to produce stably transfected sublines of 253-NT. Single G424L or G432L mutations or double G424L/G432L mutations were introduced to disrupt two or more GXXXG motifs. Substituting leucine for glycine introduced a large hydrophobic amino acid that maintained the hydrophobic character of the transmembrane domain but disrupted the conformation of the transmembrane domain helix [28] . Transmembrane domain tyrosine residues were mutated to valine, a substitution that replaced tyrosine with a non-polar hydrophobic amino acid lacking a hydroxyl group in its side chain. In other systems, proper spacing of the tyrosine hydroxyl group had been shown to be necessary for signaling events dependent upon protein-protein interactions [41] .
Examination by confocal fluorescence microscopy of cultures established from CEACAM1-4S positive cells isolated by FACS showed intense membrane fluorescence when labeled by indirect immunofluorescence with CEACAM1 specific MAb 9.2, confirming that both wild type and mutant forms of CEACAM1-4S were properly transported to the plasma membrane ( Figure 2 , A-L). Empty vector transfected and untransfected 253-NT cells showed no detectable reactivity with MAb 9.2 ( Figure 2 , E and J, respectively). Immunoblot analysis indicated that the wild type and mutant proteins all had an apparent molecular mass of 105 kDa, the expected size for CEACAM1-4S isoform [2] , suggesting that post-translational processing has not been altered ( Figure 3 ). Quantitative analysis by flow cytometry of FACS selected, stably transfected cells labeled by IIF with MAb 9.2 indicated that 72-83% of the cells were positive for either wild type or mutated forms of CEACAM1-4S (Figure 4 ).
The effect of transmembrane domain mutations on anchorage independent growth was determined from changes in the average areas of soft agar colonies ( Figure 5 and 6) . When 253T-NT cells expressing wild type ( Figure 1C) , single or double tyrosine mutants of CEACAM1-4S were grown in soft agar, sublines expressing tyrosine mutants (Figure 5 D, E, F and Figure 6 ) showed a 3.5-fold increase in colony size relative to cells transfected with the wild type protein ( Figure 1C and Figure 6 ). In contrast, 253T-NT cells expressing CEACAM1-4S with GXXXG motifs disrupted by G to L mutations ( Figure 5A , B, C and Figure 6 ) either did not grow or formed colonies that for the double glycine mutant ( Figure 5C and 6), were less than half the size of those formed by cells expressing the wild type protein ( Figure 1C and Figure 6 ). Moreover, the rapid growth phenotype conferred by tyrosine to valine mutants appeared to be dominant over the growth suppressed phenotype displayed by glycine to leucine mutants since all of the sublines with both glycine to leucine and tyrosine to valine mutations displayed enhanced anchorage independent growth ( Figure 5G , H, I and Figure 6 ).
Proliferation assays were carried out to ascertain whether the size of soft agar colonies ( Figure 5 and 6 ) was proportional to the rate of proliferation in vitro calculated from changes in cell number as a function of time. As shown in Figure 7 , 253T-NT cells expressing wild type CEACAM1-4S proliferated 2.2 times faster than 253T-NT cells carrying the empty vector and from 1.4-2.2 times faster than cells transfected with glycine mutants. Consistent with their rapid growth in soft agar, 253T-NT cells expressing CEACAM1-4S with single or double tyrosine mutants proliferated at rates that were 1.4-1.6-fold higher than cells with wild type CEACAM1-4S. Taken together, these data suggested that the CEACAM1-4S transmembrane domain was controlling interactions involved in growth under anchorage independent conditions, interactions that were altered by GXXXG or tyrosine mutations.
To determine if changes in anchorage independent growth induced by transmembrane domain mutations were mirrored by altered tumorigenicity, nude mice were injected subcutaneously in the upper flanks with 253T-NT cell lines expressing wild type or mutant CEACAM1-4S. Subcutaneous tumors were harvested from animals at three weeks after injection, a time point chosen by necessity because of the large tumor burden in animals injected with 253T-NT cells expressing tyrosine mutants. On average, tumors produced by cells expressing wild type CEACAM1-4S were 5.5-and 6.5-fold larger by weight than those generated by cells transfected, respectively, with the empty vector or the single G424L mutant (Figure 8 ). While cells expressing the wild type protein formed tumors comparable in weight to those formed by cells expressing the double G to L mutant, 253T-NT cells expressing CEACAM1-4S with single Y448V, double Y445V/ Y448V or quadruple G424L/G432L/Y445V/Y448V mutations produced tumors that were 1.9-, 1.25-, and 2.14-fold larger than those produced by cells transfected with the wild type protein (Figure 8 ). Indirect immunofluorescence analysis of frozen tumor sections, confirmed that tumor nodules formed by 253-NT cells transfected with either wild type or transmembrane domain mutants remained strongly positive for CEACAM1-4S (Figure 9 ).
Previous reports have demonstrated that dimerization via transmembrane domain helix-helix interactions are often mediated by GXXXG or GXXXA motifs within the transmembrane domain [30, 42] . To determine if the GXXXG motifs within the transmembrane domain of CEACAM1-4S played a role in the dimerization of CEACAM1-4S [36, 43] , the effect of G to L mutations on CEACAM1-4S interactions was analyzed by Blue-Native polyacrylamide gel electrophoresis (BN-PAGE). In BN-PAGE, Coomassie G-250 is used in place of SDS to coat proteins with a uniform negative charge without causing denaturation or disruption of protein-protein interactions [44] . As shown in Figure 10 , wild type and single G-to-L mutants of CEACAM1 demonstrated an apparent molecular mass by BN-PAGE that was approximately 100 kDa higher than the double G to L mutant, a difference approximately the size of CEACAM1-4S resolved by reducing SDS-PAGE. These data suggested that a single GXXXG motif was sufficient to mediate dimerization of CEACAM1-4S and/or interaction with another yet-to-be identified transmembrane protein, interactions that appeared to require helix-helix interactions mediated by GXXXG motifs.
The ability of CEACAM1-4S expression in the context of the proteome of 253-NT cells to restore the tumorigenic and anchorage independent growth characteristics of the parental 253T cell line [6] provided a quantifiable, reproducible endpoint for examining the functionality of the CEACAM1-4S transmembrane domain. The idea that CEACAM1 phenotypes could be context specific came from our previous studies showing that CEACAM1-4L expression dramatically suppressed the tumorigenicity of CEACAM1 negative PC-3 human prostate carcinoma cells [13] . With continued passage, however, cells eventually reacquired a tumorigenic phenotype without losing expression of CEACAM1-4L, suggesting a strong selection for cells with proteomes that were unable to support CEACAM1 mediated tumor suppression. Context dependent effects on CEACAM1-4S phenotypes were also suggested by the ability of CEACAM1-4S to induce morphogenesis of MCF7 cells in vitro but not in vivo [21] .
Analysis of the amino acid sequence of the CEACAM1-4S transmembrane domain revealed the presence of four GXXXG sequences (Table 1 and 2), a motif known to drive high affinity helix-helix interactions that stabilize the dimerization/oligomerization of many well characterized proteins such as glycophorin A, epidermal growth factor receptor and the G protein-coupled afactor receptor of budding yeast [30, 42, 45] . For the latter protein, disruption of the GXXXG motif not only impaired oligomerization but also disrupted signaling [30, 46] . Previous investigations have shown that both the long and short isoforms of CEACAM1 form cis-dimers [36, 43] , an interaction that we hypothesized should be stabilized by GXXXG mediated helix-helix associations. To test this idea, we introduced glycine-to-leucine mutations that disrupted one or more transmembrane domain GXXXG motifs. FACS, fluorescent confocal microscopic and immunoblot analysis of cells stably transfected with the glycine-to-leucine mutants confirmed that the mutant proteins were expressed on the cell surface at the same level and the same size as wild type CEACAM1-4S. However, after 3 weeks in soft agar, cells expressing single glycine mutants showed significantly lower rates of proliferation and significantly smaller colonies when compared to cells transfected with wild type CEACAM1-4S, suggesting that a single glycine-to-leucine mutation had compromised the ability of CEACAM1-4S to induce anchorage independent growth. From these results, we concluded that the smaller size of colonies produced by glycine mutants resulted at least in part from a decrease in the rate of proliferation.
Since the double glycine mutation disrupted all of the GXXXG motifs, a corresponding decrease in either homo-or hetero-cisdimerization would be expected if these motifs were the sole mediators of this interaction. However, impaired dimerization could also occur if a reduction in GXXXG motifs destabilized cisdimers formed by single glycine mutants with a subsequent reduction in their steady state levels. Blue native-PAGE analysis showed that the apparent size of the double but not the single glycine mutants showed approximately a 100 kDA decrease in size, a shift consistent with impaired dimerization. Moreover, since single glycine to leucine mutations produced the growth suppressed phenotype but only double mutations hampered dimerization, it was concluded that even without an apparent effect on dimerization, glycine to leucine point mutations in the transmembrane domain had a profound effect on the growth phenotype produced by CEACAM1-4S.
With one exception, the effects of transmembrane domain mutations on growth in vivo were similar to those observed in vitro.
In keeping with their limited capacity for anchorage independent growth, cells transfected with empty vector were poorly tumorigenic relative to cells expressing the wild type CEACAM1-4S. As predicted from their growth in soft agar, cells expressing the G424L mutant formed tumors considerably smaller than those derived from cells expressing wild type CEACAM1-4S. However, the similarity in the size of tumors generated by cells expressing the wild type, double glycine and double tyrosine mutants was at odds with in vitro assays where cells expressing wild type CEACAM1-4S showed a significantly greater or lesser capacity, respectively, for anchorage independent growth than cells expressing double glycine or tyrosine mutants. This discrepancy is reminiscent of the differential in glandular morphogenesis exhibited in vitro and in vivo by MCF7 mammary carcinoma cells [21] and thus could reflect differential effects of the subcutaneous microenvironment.
In general, tumors formed by cells expressing the single and double tyrosine mutants were much larger than those generated by cells transfected with wild type CEACAM1-4S or empty vector, a trend consistent with the greater rate of proliferation shown in vitro by tyrosine mutants. When viewed as a whole, these findings show that the phenotypes manifested in vitro by cells expressing transmembrane domain mutants are recapitulated in vivo, suggesting these changes are intrinsic to the transmembrane domain and are not the result of extrinsic factors e.g., microenvironment, that change the phenotypic effects of CEA-CAM1-4S by altering its proteomic context.
The decrease in soft agar growth produced by glycine mutations and the increase by tyrosine mutations relative to the wild type CEACAM1-4S would classify these, respectively, as loss-of-function and gain-of-function mutations [47, 48, 49] . Gain-of-function mutations are usually dominant, a characteristic apparent for the glycine/tyrosine mutants of CEACAM1-4S where the growth enhanced phenotype of tyrosine mutants was dominant over the growth inhibited phenotype resulting from glycine mutations. Based on the known functions of GXXXG motifs, it seems likely that the loss-of-function caused by glycine mutations is related to changes in helix-helix interactions that destabilize rather than prevent the formation of cis-dimers [28, 50, 51] , a possibility consistent with the fact that single G424L or G432L mutations failed to disrupt dimerization ( Figure 10 ) but did cause a decrease in anchorage independent growth. Also noteworthy is the marked decrease in cisdimerization ( Figure 10 ) when mutations were introduced at both G424 and G432, a result that suggested G424-AIA-G428 was a functional GXXXG motif capable of directing cis-dimerization in the absence of the two motifs centered on G432 and necessary to sustain the level of growth produced by wild type CEACAM1-4S. Although this conclusion is seemingly at odds with the 9 prediction algorithms that placed G424 outside the transmembrane domain (Table 1) , we suggest that a combination of structural and functional data provides a more accurate location of the transmembrane domain, one that incorporates the GAIAG motif at the N-terminus.
Similar reasoning can be applied to the C-terminus where 5 of 12 transmembrane prediction algorithms placed Y448 outside the transmembrane domain. However, mutation of either Y445 or Y448 led to the soft agar growth enhanced phenotype, indicating first, that Y448 was functionally equivalent to Y445 and thus likely to be located within the transmembrane domain and second, that both tyrosines were required to sustain anchorage independent growth at the level produced by wild type CEACAM1-4S. However, whether Y448 was or was not in the transmembrane was moot since single Y445V or Y448V mutations produced the growth-enhanced phenotype in soft agar. Put another way, the presence of both tyrosines appeared to be necessary to suppress anchorage independent growth, a suppressive effect that apparently involved more than interactions between aromatic side chains since in both single tyrosine to valine mutants (VFLY or YFLV) the remaining tyrosine was paired with F446. Whether the two tyrosines without phenylalanine would be able maintain the wild type CEACAM1-4S phenotype is an open question that is beyond the scope of the present investigation.
While the mechanism behind the gain-of-function produced by the Y mutations is less clear, there is increasing evidence supporting the functional importance of interactions between transmembrane aromatic amino acids [41] . Although aromatic residues make up only a small percentage of the amino acids in any given protein, they are generally the most highly conserved residues. Interactions between the aromatic rings of phenylalanine, tryptophan (W) or tyrosine are thought to involve pi-stacking, a process that creates an attractive force when aromatic rings assume energetically favored stacking geometries. Accumulating evidence suggests that pi-stacking plays an important role in molecular recognition and self assembly either by contributing energy for driving self assembly or by providing directionality and orientation through stacking geometries [52, 53] . When a bacterial transmembrane database was statistically analyzed by Sal-Man et al, these investigators found that aromatic pairs (WXXW or YXXY) were significantly over-represented compared to their predicted frequency, suggesting a functional role for these sequences [52] . The stabilization of transmembrane domain selfassociation following substitution of tyrosine for glutamine (Q) and serine (S) in the known dimerization motif, QXXS, provided support for this idea and led these authors to conclude that stabilization of transmembrane associations by aromatic residues may be a general mechanism for generating specificity in transmembrane-transmembrane interactions. In considering which amino acid to substitute for the transmembrane tyrosine residues, phenylalanine would intuitively seem to be the logical When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant. doi:10.1371/journal.pone.0029606.g010 choice. However, based on recent reports, tyrosine to phenylalanine mutations may or may not disrupt function. Stevens et al [54] reported that a YS/FA mutation of the IgM transmembrane domain had no effect on anti-Ig induced signaling as measured by the activation of tyrosine phosphorylation and did not disrupt the association between IgM and its signaling partners, Ig-alpha/Igbeta. In contrast, a YS/VV mutation diminished both signaling and association with Ig-alpha/Ig-beta. In cases where phenylalanine substitutions fail to mimic tyrosine, the lack of a properly spaced hydroxy group may be the reason [41] . Based on these considerations, it was decided to introduce tyrosine to valine mutations.
GXXXG motifs and aromatic amino acids are common features of the transmembrane domains of many different plasma membrane proteins with GXXXG motifs most often located at the N-terminus [55, 56, 57] and Y/F/W aromatic amino acids at the C-terminus of the transmembrane domain [58, 59] . Many of the proteins also have Sternberg-Gullick dimerization motifs, a family of sequences discovered in the transmembrane domain of tyrosine kinase growth factor receptors [60] . Members of this family, which includes a subset of GXXXG/AXXXG/SXXXG dimerization motifs, are composed of 5 amino acids arranged with G, A, S, T or P in the first position (N-terminus), an A, V, L or I in the fourth position and G or A in the fifth position. Our findings demonstrate that a single amino acid substitution in the transmembrane domain of CEACAM1-4S can produce dramatic effects on cell proliferation, anchorage independent growth and in vivo tumorigenicity. It seems clear that the transmembrane domain and more specifically GXXXG motifs and tyrosine residues make a significant contribution to the functionality of CEACAM1-4S and by extension, to other transmembrane proteins with similar characteristics. Further studies are needed to define the downstream signaling events impacted by re-expression of CEACAM1-4S or by transmembrane domain mutations, studies that should provide valuable insights into events controlled by transmembrane domain mediated interactions. Of particular interest will be the identity of pathways inactivated/activated by tyrosine mutations that lead to the positive growth effects of tyrosine mutations and the ability of these pathways to counteract the tumor suppressor activity of CEACAM1-4L, the larger splice variant with the same transmembrane domain as the 4S isoform. Human Subtilase SKI-1/S1P Is a Master Regulator of the HCV Lifecycle and a Potential Host Cell Target for Developing Indirect-Acting Antiviral Agents HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1) – or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care. Hijacking of host lipids and their biosynthetic pathways is a common strategy for microbial infection. Human enveloped viruses including hepatitis C virus (HCV) and human immunodeficiency virus (HIV)-1 use cholesterol-rich lipid rafts for entry [1, 2] , assembly [3] , and/or replication [2, 4] . Lipid droplets (LDs), once considered static storage vesicles for host lipids, are now appreciated as dynamic organelles [5] that are also utilized in the lifecycles of pathogenic human viruses including rotavirus (RV) [6] , dengue virus (DV) [7] , and HCV [8] . HCV in particular requires host LDs for assembly of nascent viral particles [9] [10] [11] .
HCV is a globally important human pathogen afflicting more than 170 million people worldwide [12, 13] . HCV, a hepacivirus member of the Flaviviridae family and an enveloped virus, is encoded by a single-stranded positive-sense RNA genome [14] . Viral RNA is directly translated by the host machinery into a single polyprotein, which is cleaved by host and virus-encoded proteases to release the individual structural (core, E1, and E2) and non-structural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [15] . During infection, HCV-encoded proteins promote reorganization and accumulation of LDs in the perinuclear region of the cell [16] . The HCV core protein is targeted to LDs [17] and orchestrates the assembly and release of infectious viral particles during the late stages of infection [18] . Hence, disrupting the interaction of the HCV core protein with LDs compromises this essential stage within the HCV lifecycle [8, 10, 11] .
Several host metabolic pathways tightly control cellular lipid synthesis. Targeted disruption of these pathways [19] [20] [21] by HCVencoded proteins has been linked with liver steatosis [22, 23] in HCV-infected individuals. Importantly, there is a correlation between the degree of steatosis and both the severity of chronic HCV infection [13, 24] and the response to treatment with pegylated-interferon-a and ribavirin [25, 26] .
Overstimulation of host lipid metabolism by HCV during infection is achieved through a variety of molecular mechanisms (reviewed in [27] , [28] , and [29] ). For example, HCV employs multiple strategies to activate the sterol regulatory element binding protein (SREBP) pathway, which is important for regulation of host lipid homeostasis [19, [30] [31] [32] . SREBPs are endoplasmic reticulum (ER), membrane-anchored transcription factors that respond to changes in intracellular sterol levels through interactions with sterol-sensing proteins (reviewed in [33] ). When sterol levels are high, SREBPs are retained as inactive precursors in the ER [34, 35] . Under low sterol conditions, SREBPs are escorted to the Golgi, where two resident endoproteases (subtilisin kexin isozyme/site-1 protease (SKI-1/S1P) and SREBP Site-2 protease (S2P); reviewed in [36] and see below) cleave the precursor polypeptide SREBP, allowing the release of transcriptionally active SREBP molecules from the ER [37, 38] . The released SREBP fragment migrates to the nucleus and binds to sterol response elements in the promoters of cholesterol and fatty acid biosynthetic target genes, and it activates their transcription [39] [40] [41] . Activation of the SREBP pathway by HCV aids the virus lifecycle and may ultimately promote the development of steatosis and liver disease in chronically infected individuals [42, 43] .
Human site-1 protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin-isozyme-1 (SKI-1), is a membranebound subtilisin-related serine endoprotease that belongs to a group of nine mammalian proprotein convertases (PCs) in family S08 [44] [45] [46] . SKI-1/S1P displays unique substrate specificity among the PC members by showing preferred cleavage after non-basic amino acids [47] . SKI-1/S1P cleaves at the carboxyterminus of the peptidyl sequence Arg/Lys-Xaa 3 -Xaa 2 -Leu/ Ser/Thr [48] , where Xaa 3 is any amino acid except Cys at the P3 position of the scissile peptide bond and Xaa 2 is a hydrophobic amino acid containing an alkyl side chain at the P2 position [49] . In addition to the proteolytic processing of transcription factors [36] , SKI-1/S1P participates in the proteolytic activation of viral-envelope glycoproteins of the Lassa virus [50] , the lymphocytic choriomeningitis virus [51] , and the Crimean Congo hemorrhagic fever virus [52] . Importantly, endoproteolytic processing of these viral glycoproteins by SKI-1/S1P is a critical step for the production of infectious progeny viruses, suggesting that SKI-1/S1P may represent an attractive target for therapeutic intervention against human pathogenic arenaviruses [47, 48, [50] [51] [52] .
Given that SKI-1/S1P-dependent proteolytic cleavage of SREBPs is a master molecular switch for controlling host cell cholesterol homeostasis [41, 53] , we hypothesized that inhibiting SKI-1/S1P endoproteolytic activity in the secretory pathway would prevent HCV hijacking of host lipid metabolic pathways and thus compromise the virus lifecycle. Because of our previous success with engineering serine protease inhibitors (serpins) to develop effective and selective PC inhibitors [54, 55] , we hypothesized that engineering a naturally occurring serpin scaffold could also provide a powerful approach for developing selective SKI-1/S1P inhibitors. Serpins differ from non-serpin inhibitors in that they require a large inhibitor conformational change in order to trap proteases in an irreversible complex [56] . The conformational change is initiated by reaction of the active serine of the protease with the reactive center loop (RCL) of the serpin, which results in a covalent species involving an acyl ester linkage to the cO of the protease active site serine [57] . This cleaves the RCL, which then moves 71 Å to the opposite pole of the serpin, taking the tethered protease with it [57] .
We selected a Drosophila serpin, Spn4A, as a prototype macromolecular inhibitor scaffold. The identification of Spn4A by our group as the most potent natural serpin inhibitor of the human PC furin (K i ,13 pM [58] ) provides us with a novel and unique molecular tool for dissecting the contribution of SKI-1/ S1P-dependent proteolytic activity in the secretory pathway to viral infection of eukaryotic cells. We report that engineering of the RCL of Spn4A to mimic the consensus sequence Arg/Lys P4 -Xaa 3 -Xaa 2 -Leu/Ser/Thr P1Q for cleavage by SKI-1/S1P (Spn4A RCL: Arg P4 -Arg-Lys-Arg P1Q -. Arg P4 -Arg-Leu-Leu P1Q ) resulted in the development of Spn4A-RRLL, a novel, selective, and effective serpin-based inhibitor of SKI-1/S1P. We demonstrated the antiproteolytic and anti-HCV activities of our new recombinant adenovirus-expressing Spn4A-RRLL ''secreted'' (s) variant directed at the secretory pathway SKI-1/S1P. Expression of Spn4A.RRLL(s) in Huh-7.5.1 cells results in a strong inhibition of the SKI-1/S1P-mediated activation of SREBP-1 and downregulation of SREBP target gene products. As hypothesized, inhibiting SKI-1/S1P activity robustly blocked HCV infection of Huh-7.5.1 cells in a dose-dependent manner. We found that specific inhibition of SKI-1/S1P activity by Spn4A.RRLL(s) dramatically reduced the abundance of LDs in hepatoma cells. Use of the specific active site-directed small-molecule inhibitor of SKI-1/S1P, PF-429242, confirmed the results of our studies with the protein-based therapeutic Spn4A.RRLL(s), with a robust inhibition of HCV infection.
The results of our studies contribute to our understanding of the HCV lifecycle and HCV-associated steatogenesis and to efforts in developing novel host-directed antiviral therapeutic agents against HCV. In addition, with the finding that an increasing number of human enveloped viruses employ host LDs for infection [6, 7] , our results suggest that SKI-1/S1P-directed inhibitors may allow the development of novel broad-spectrum antiviral agents.
Protein engineering of the Spn4A scaffold and drug delivery strategy to target secretory pathway SKI-1/S1P
Our previous studies have demonstrated that Spn4A architecture can inhibit two evolutionary divergent members of the PC family (furin and PC2) [58] . We selected this novel Drosophila melanogaster serpin scaffold to engineer a novel protein-based inhibitor directed at the PC SKI-1/S1P. First, we cloned our pre-His/FLAG-tagged
Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver cancer and liver transplantation worldwide. No vaccine is available for preventing the spread of HCV, and the current therapeutic regimen is only moderately effective and causes serious side effects. New antiviral agents are required to treat HCV infection, but the high mutation rate of HCV hinders the effectiveness of virus-specific inhibitors. Targeting the host enzymes required for HCV to replicate offers a promising new direction for antiviral therapy. During infection, HCV promotes excessive fat accumulation in the liver, which benefits the virus as this promotes formation of lipid droplets, a cellular organelle essential for assembly of new HCV infectious viral particles. Here, we report the development of a specific inhibitor targeting SKI-1/S1P, a host enzyme required for lipid production in human cells. We show that inhibiting SKI-1/S1P activity in human liver cells effectively blocks lipid droplet formation and HCV infection. Many prevalent human viruses, such as dengue, rotavirus, and hepatitis B virus, hijack host lipid metabolic pathways similar to those targeted by HCV to complete their lifecycle. Thus, we propose that cellular SKI-1/S1P is a potential target for developing desperately needed novel broad-spectrum antiviral drugs.
Spn4A construct [58] into an adenoviral shuttle vector to generate Spn4A.RRKR(r) ( Figure 1A ). Spn4A is a unique ''retained'' (r) serpin that presents, at its C-terminus, an HDEL sequence ( Figure 1A) , a functional variant of the C-terminal KDEL retention signal that directs secretory protein retention in the ER [59] . Because SKI-1/S1P cleavage of SREBP substrates occurs in the Golgi apparatus [35] , we next needed to generate a ''secreted'' (s) variant of Spn4A, Spn4A.RRKR(s) ( Figure 1A ). We hypothesized that only an Spn4A (s) variant, which traffics through the late secretory pathway prior to secretion in the extracellular space, would encounter active SKI-1/S1P molecules in the early Golgi compartment. This was accomplished by inserting a stop codon before the C-terminal ER-retention signal, HDEL. Next, we employed site-directed mutagenesis to optimize the interactions between the RCL of Spn4A and the substrate binding sites of SKI-1/S1P. Residues at positions P2 and P1 of the Spn4A RCL ''bait'' region Arg P4 -Arg P3 -Lys P2 -Arg P1Q were substituted to generate Arg P4 -Arg P3 -Leu P2 -Leu P1Q , thereby mimicking the Lassa virus glycoprotein precursor GP-C cleavage site [50] ( Figure 1A and 1B: Spn4A.RRLL(r) and Spn4A.RRLL(s), respectively).
To test the serpin-like properties and antiviral activities of our Spn4A variants in cellulo, recombinant adenoviruses (Ad) expressing the Spn4A constructs Ad-Spn4A.RRLL(r) and Ad-Spn4A.RRLL(s) were produced as described previously [55] . As adenoviruses display strong tropism for the liver [60] , the major site of HCV infection [13] , these recombinant adenoviruses are especially useful molecular tools for HCV research, including the use of human hepatoma Huh-7.5.1 cells, which support robust HCV infection in cellulo [61] .
Robust intracellular expression and differential secretion of adenovirus-expressed Spn4A.RRLL(r) and Spn4A.RRLL(s) in human hepatoma Huh-7.5.1 cells
The level of expression of Spn4A variants was first optimized by infecting human hepatoma Huh-7.5.1 cells (highly permissive for HCV JFH-1 infection) with different multiplicity of infection (moi) of recombinant adenovirus expressing intracellularly retained Spn4A.RRLL(r). Cellomics HCS analysis revealed that over 90% of these Huh-7.5.1 cells expressed Spn4A.RRLL(r) at a moi of 50 ( Figure S1 ). Cellomics HCS was also used to determine if cell death occurs following 2 days of pre-treatment with Spn4A.RRLL(r) or Spn4A.RRLL(s) compared to the control (Ad-Empty) followed by 72 hours of HCV infection as employed in the experiments below ( Figure S2 ). We observed no significant reductions in total cell numbers under these experimental conditions. The high-content analysis of cell death was confirmed using an MTS-based cell viability assay (data not shown). The lack of detectable toxicity induced by Ad-Spn4A.RRLL(r) and Ad-Spn4A.RRLL(s) up to a moi of 50 showed that these variants can be tested over a very wide dynamic range in hepatoma cells.
The serpin-secretion profile in Huh-7.5.1 cells was examined by Western blotting of cell lysates and extracellular media. As expected, a prominent 45-kDa band was detected in Ad-Spn4A.RRLL(r)-infected and Ad-Spn4A.RRLL(s)-infected cell lysates using anti-FLAG antibody (Figure 2A, lanes 2 and 3) . Furthermore, as hypothesized, the 45-kDa band of Spn4A.RRLL(r) was found only in lysed cell extracts and was not found secreted into extracellular media (Figure 2A Serpin-like properties and intrinsic specificity of Spn4A.RRLL(r) and Spn4A.RRLL(s) A stable, acyl-enzyme complex is formed between a protease and a functional inhibitory serpin following RCL cleavage. This allows for detection of the high molecular weight, heat-stable, and SDS-stable enzyme-inhibitor (EI) complex by standard SDS- Spn4A.RRKR(r) encodes for the naturally occurring serpin Spn4A, isolated from Drosophila melanogaster, with potent inhibitory activity against the human proprotein convertase furin. Spn4A.RRKR(r) contains the alpha-1 antitrypsin signal peptide (SP) at the N-terminus followed by a tandem His-tag (HHHHHH) and FLAG-tag (DYKDDDDK) sequence (HF). The P4 -P1 furin cleavage sequence in the RCL is Arg-Arg-Lys-Arg. Spn4A.RRKR(r) also contains the His-Asp-Glu-Leu (HDEL) ER retention motif (r) at the C-terminus. The secreted (s) serpin, Spn4A.RRKR(s), contains a stop codon before the HDEL signal. The RCL of Spn4A-RRKR(r) and (s) was modified to mimic the predicted SKI-1/S1P target cleavage site present in the Lassa virus glycoprotein pre-GP-C, which is Arg-Arg-Leu-Leu. Thus, Spn4A.RRLL(r), which is also retained in the ER, encodes the P4 -P1 Arg-Arg-Leu-Leu cleavage recognition sequence in the RCL. Spn4A.RRLL(s) contains a stop codon before HDEL, allowing the serpin to be secreted. (B) In silico homology model of the Spn4A.RRLL(r) variant was generated as described in the Materials and Methods. Ribbon diagram of the molecular model was generated using Pymol. The side chains of the RRLL residues within the flexible ''bait region'' of the RCL are shown as sticks in wheat colour. Sheet A is shown in yellow, sheet B is in blue, and sheet C is in cyan. Alpha-helices are red and loops are green. doi:10.1371/journal.ppat.1002468.g001 PAGE and Western blot [54, 55, 58, 62] . To determine if Spn4A.RRLL(s) is a functional and selective inhibitor of SKI-1/ S1P, recombinant Spn4A variants (furin-and SKI-1/S1P-directed inhibitors) were expressed in Huh-7.5.1 cells for 72 hours. Cell media and extracts containing recombinant serpins were then harvested and incubated with purified recombinant His-tagged human SKI-1/S1P or furin ( Figure 2B ) as described previously [47, 54, 58] . Reaction mixtures were analyzed by Western blot and probed for EI complex formation with anti-His antibody ( Figure 2B ) and anti-FLAG antibody ( Figure S3 ). The results shown in Figure 2B clearly demonstrate EI complex formation between recombinant SKI-1/S1P and Spn4A.RRLL(s) in cell media and in cell extracts (lane 5, upper and lower panels). As expected, Spn4A.RRLL(s) did not form a complex with furin ( Figure 2B , lane 10, upper and lower panels), whereas the furindirected serpin Spn4A.RRKR(s) formed an EI complex with furin but not with SKI-1/S1P in cell media and extracts ( Figure 2B , lanes 9 and 7, respectively, upper and lower panels). Lysed cellular extracts expressing Spn4A.RRLL(r) also demonstrated EI complex formation with SKI-1/S1P ( Figure 2B , lane 12, bottom panel). The results of our biochemical analysis confirmed the serpin-like properties of recombinant Spn4A.RRLL(r) and (s) biosynthesized in human hepatoma cells and the selectivity of Spn4A.RRLL(s) against SKI-1/S1P. Importantly, Spn4A.RRLL(s) inhibits SKI-1/ S1P by a suicide substrate mechanism and forms a kinetically trapped heat-and SDS-stable complex with SKI-1/S1P as is characteristic of other physiological serpin-protease pairs [58, 63] .
Spn4A.RRLL(s) is a potent inhibitor of SKI-1/S1P-mediated endoproteolytic cleavage of SREBPs, of their downstream effector gene expression, and of intracellular cholesterol-ester accumulation
To confirm that expression of Spn4A.RRLL(s) in Huh-7.5.1 cells inhibits endogenous SKI-1/S1P-mediated cleavage of SREBP molecules, we examined nuclear SREBP-1 protein levels in cells infected with adenovirus-expressed Spn4A variants. As a positive control, we also treated cells with the selective, reversible, and competitive small-molecule inhibitor of SKI-1/S1P: PF-429242 [64, 65] . This compound was recently synthesized and characterized both in vitro and in vivo for its anti-lipidemic properties including efficient inhibition of nuclear SREBP accumulation. As previously described [64, 66] , the calpain inhibitor, alpha-N-acetyl-Leu-Leu-Nle-CHO (ALLN), was employed to facilitate the detection and accumulation of the Nterminal fragment of SREBP-1 in the nucleus. An anti-fibrillarin antibody was used to positively identify the nuclear fractions ( Figure 3A ) [67] . As expected, Western blotting of nuclear extracts from cells treated with 10 mM of PF-429242 (PF-429242 + ALLN), using an antibody against the N-terminal fragment of SREBP-1, revealed a complete block of SREBP-1 accumulation in the nucleus ( Figure 3A ). Nuclear extracts from cells infected with Ad-Spn4A.RRLL(s) (RRLL(s) + ALLN) also exhibited a dramatic decrease in nuclear SREBP-1 accumulation when compared to Ad-Empty (control + ALLN) and Ad-Spn4A.RRLL(r) (RRLL(r) + ALLN)-infected cells. These results confirm that expression of recombinant Spn4A.RRLL(s) in the secretory pathway of Huh-7.5.1 cells inhibits SREBP-1 processing by SKI-1/S1P.
Next, to determine whether the Spn4A.RRLL(s)-mediated reduction in nuclear SREBPs was associated with a concomitant decrease in the protein levels in SREBP-target genes, we examined the fate of three SREBP-dependent gene products, SREBP-2, LDLR, and PCSK9. We investigated these host cell proteins because of their proposed contribution to HCV entry (LDLR and PCSK9) and propagation (SREBP-2) [19, 68, 69] . A time course analysis of cells expressing Spn4A.RRLL(s) in complete media showed the most significant block in SREBP-regulated LDLR expression after 72 hours ( Figure S4 ). As HCV is known to induce SREBP activation [19, 31, 32] , we then analyzed the expression of SREBP-regulated proteins under SREBP-activated conditions ( Figure 3B ). Huh-7.5.1 cells were depleted of exogenous sterols for 24 hours to induce SREBP transport from ER-to-Golgi prior to infection with Ad-Empty, Ad-Spn4A.RRLL(r), or Ad-Spn4A. RRLL(s). The levels of LDLR, PCSK9, and SREBP-2 (all regulated by nuclear SREBPs [70] [71] [72] [73] ) were then measured using Western blot analysis of lysed cell extracts ( Figure 3B ). After 72 hours of Spn4A.RRLL(s) expression, mature LDLR (160 kDa) levels were reduced by 74% compared to Ad-Empty-treated cells. Similarly, an 85% block in mature PCSK9 (60 kDa) expression and a 79% reduction in full-length SREBP-2 expression were observed. No significant reductions in LDLR or SREBP-2 levels following Spn4A.RRLL(r) treatment were observed. Interestingly, a significant reduction in PCSK9 expression was detected in Spn4A.RRLL(r)-expressing cells ( Figure 3B ). The expression of btubulin and of the Golgi marker GM130 were not affected by Spn4A.RRLL(r) or Spn4A.RRLL(s) expression ( Figure 3B ). These results confirm that expression of Spn4A.RRLL(s) in Huh-7.5.1 cells specifically inhibits the SREBP pathway including target genes identified as cellular cofactors affecting HCV infection.
A critical function of the SREBP pathway and the genes that it regulates is to control lipid homeostasis [36] . We investigated the impact of inhibiting SKI-1/S1P using both Spn4A.RRLL(s) and PF-429242 on total intracellular lipid levels, specifically cholesterol, cholesterol-esters, triglycerides, and phospholipids ( Figure 3C and 3D). Among the cell lipids examined, Spn4A.RRLL(s) and PF-429242 had the most dramatic impact on cholesterol-ester levels, a major constituent of cellular LDs [5] ; these were reduced by 74% in Spn4A.RRLL(s)-treated cells compared to control (Ad-Empty)-treated cells ( Figure 3C ). Similarly, PF-429242 reduced cholesterol-ester levels by , 63% compared to control cells treated with DMSO ( Figure 3D ). A 14% reduction in triglycerides was also induced by Spn4A.RRLL(s), although this reduction did not reach significance, whereas PF-429242 caused a significant 51% reduction in total intracellular triglycerides. A significant 5% reduction in free cholesterol levels was also observed in Spn4A.RRLL(s)-treated cells and a 25% reduction was observed in PF-429242-treated cells compared to respective controls. No significant reductions in phospholipid levels were detected ( Figure 3C and 3D). These results suggest that sustained inhibition of SKI-1/S1P-mediated cleavage and activation of SREBP causes increased cellular utilization of lipid stores.
Expression of secretory pathway Spn4A.RRLL(s) in Huh-7.5.1 cells dramatically reduces the abundance of cellular lipid storage droplets LDs are dynamic intracellular lipid storage compartments made up of triglyceride and cholesterol esters, surrounded by a phospholipid membrane and associated with specific marker proteins including adipose differentiation-related protein (ADRP), also known as perlipin 2 [74] . Because SREBP activation controls the expression of genes directly involved in intracellular fatty acid and cholesterol biosynthesis (reviewed in [36] ) and because cholesterol-ester levels were reduced by Spn4A.RRLL(s), we investigated the effect of serpin-mediated SKI-1/S1P inhibition on cellular LD abundance. Fluorescence microscopy was used to determine the relative abundance of LDs stained with BODIPY 493/503 in Huh-7.5.1 cells infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), and Ad-Spn4A.RRLL(s). After 72 hours, the level of BODIPY-stained LDs in Spn4A.RRLL(s)-expressing cells was dramatically reduced compared to empty vector-treated cells ( Figure 4A ). By contrast, Spn4A.RRLL(r)-expressing cells had no apparent reduction in LD size or abundance compared to controltreated cells ( Figure 4A ). Quantification of confocal microscopy images demonstrated that, on average, LD abundance was reduced by 80% in Spn4A.RRLL(s)-expressing cells compared to controls ( Figure 4B ). The effect of Spn4A.RRLL(s) expression on cytosolic LD abundance was confirmed by visualizing the LD marker ADRP/perilipin 2 using confocal microscopy ( Figure 4C ) and by using quantitative Western blot ( Figure 4D ). Spn4A.RRLL(s) was observed to reduce ADRP/perilipin 2 protein expression by 50% compared with control cells ( Figure 4D ), whereas there was no reduction in ADRP/perlipin 2 expression following Spn4A.RRLL(r) treatment. We subsequently confirmed that reduced cellular LD levels were due to inhibition of SKI-1/S1P using 10 mM PF-429242 whereupon ADRP/perilipin 2 levels were reduced by 62% compared to control DMSO-treated cells ( Figure 4E ). These results confirm that Spn4A.RRLL(s)-and PF-429242-mediated inhibition of SKI-1/S1P enzymatic activity dramatically reduces intracellular LD abundance in Huh-7.5.1 cells.
Expression of secretory pathway Spn4A.RRLL(s) in Huh-7. 5 
Since the SREBP signaling pathway is induced by HCVencoded proteins during infection [19, [30] [31] [32] , we tested the effect of inhibiting this pathway on the HCV lifecycle in human hepatoma cells. Huh-7.5.1 cells were treated with increasing moi (1-50) of Ad-Spn4A.RRLL(r), Ad-Spn4A.RRLL(s), or Ad-Empty (control) for 48 hours in complete media with or without exogenously added sterols followed by 72 hours of infection with HCV. The number of HCV-infected cells, as evidenced by positive core protein expression, was measured using Cellomics HCS ( Figure 5A ). It was determined that Spn4A.RRLL(s) expression inhibited HCV infection in a dose-dependent manner compared to control-treated cells. HCV infection was not significantly reduced in cells infected with Ad-Spn4A.RRLL(s) at a moi of 1. A moi of 12.5, however, caused a 40% reduction, a moi of 25 caused a 60% reduction, and a moi of 50 caused a 75% reduction in the number of HCV-infected cells compared to controls. Spn4A.RRLL(r) had no significant impact on HCV infection up to adenovirus moi 50 when compared to the control ( Figure 5A ). Supplementing sterol and lipid metabolites significantly restored infectivity when cells were treated with moi 50 of Ad-Spn4A.RRLL(s), where a 2-fold increase in HCV infection compared to non-supplemented cells was observed ( Figure 5A ). These results show that the anti-HCV activity of Spn4A.RRLL(s) is, at least in part, associated with its capacity to decrease intracellular lipid stores within the host cells.
The anti-HCV properties of Spn4A.RRLL(s) were confirmed and extended by further virological studies on cells pre-treated with our serpin-based inhibitors for 48 hours prior to 72 hours of HCV infection ( Figure 5B and 5C). First, quantitative Western blot analysis revealed a 64% reduction in the expression of intracellular HCV core protein in Spn4A.RRLL(s)-expressing cells compared to the control ( Figure 5B ). Similarly, Spn4A.RRLL(s) treatment was found to reduce extracellular infectious HCV titers by 76% ( Figure 5B) . A 65% reduction in intracellular HCV RNA levels was also observed using quantitative PCR (QPCR analysis ( Figure 5C ). Spn4A.RRLL(r) expression did not significantly impact any aspect of the HCV lifecycle examined ( Figure 5A , 5B and 5C). These results demonstrate that inhibition of SKI-1/ S1P-mediated proteolytic activation of SREBP molecules using secretory pathway protein-based inhibitors is an effective antiviral strategy to robustly block HCV infection in Huh-7.5.1 cells.
To determine if a decrease in intracellular HCV RNA in Spn4A.RRLL(s)-treated cells ( Figure 5C ) is due to reduced viral replication or alternatively due to reduced HCV entry, we examined cells transfected directly with total genomic HCV RNA for 3 days following 48 hours of adenovirus-mediated serpin expression. We found that under these experimental conditions, when receptor-mediated HCV entry was bypassed, no significant . Acquisition and analysis were performed using the same intensity and threshold settings across all images. (C) The LD marker ADRP was detected in cells treated with Spn4A.RRLL(r), Spn4A.RRLL(s), and Ad-Empty (control) using rabbit anti-ADRP antibody (green), and images were obtained using an Olympus Fluoview FV1000 laser scanning confocal changes in intracellular HCV RNA levels were detected by QPCR ( Figure 5C ). Examination of total cell extracts by Western blot revealed that HCV core levels are not reduced following HCV RNA transfection in serpin-treated cells ( Figure S5) , confirming that Spn4A.RRLL(s) does not interfere with HCV replication when HCV entry is bypassed.
The impact of Spn4A.RRLL(s) treatment on HCV replication was further investigated using HCV subgenomic replicons [75] . Human hepatoma cells harbouring stable HCV replicons encoding wild-type NS5A (Huh.8 cells) or NS5A with an adaptive mutation (Huh.2 cells) were treated with recombinant adenoviruses for 5 days. Total RNA levels were then harvested and the level of HCV RNA was quantified using QPCR analysis. No significant differences were observed between HCV replicon levels treated with Spn4A.RRLL(s), Spn4A.RRLL(r), and the control (Ad-Empty) ( Figure 5D ). These results confirm that Spn4A. RRLL(s) does not inhibit HCV replication. These findings, in addition to the measured decrease in LDLR expression presented in Figure 3B , strongly suggest that the robust reduction in intracellular HCV RNA levels observed in Spn4A.RRLL(s) pretreated cells prior to HCV infection is, at least in part, due to reduced viral attachment or entry.
Next, we wanted to test whether extracellularly applied PF-429242 would effectively inhibit the endoproteolytic activity of secretory pathway SKI-1/S1P and reduce HCV infection in Huh-7.5.1 cells. First, using an MTS-based cell viability assay, we confirmed that no major cytotoxic effects occur in Huh-7.5.1 cells treated with up to 50 mM of PF-429242 ( Figure S6 ). Next, cells were treated with increasing concentrations (0.05 mM to 50 mM) of PF-429242 for 24 hours before the cell media was replaced and cells were infected for 48 hours with HCV. The number of HCVinfected cells, indicated by positive core protein expression, was measured using Cellomics HCS ( Figure 6A Because PF-429242 and Spn4A.RRLL(s) decrease the abundance of LD components, we hypothesized that HCV assembly, rather than HCV replication, can also blocked by SKI-1/S1P inhibition. To support this hypothesis, Huh.8 and Huh.2 cells were treated with 10 mM PF-429242 for 72 hours. No significant changes in HCV subgenomic RNA levels in either cell line ( Figure 6C ) treated with PF-429242 were observed (compared to DMSO-treated control). In summary, with the lack of effect of PF-429242 on HCV replicon levels and comparing the two sets of data presented in Figure 6B , which demonstrate a strong antiviral effect of PF-429242 when added either pre-or post-HCV inoculation, we can propose that pharmacological inhibition of SKI-1/S1P endoproteolytic activity by PF-429242 impacts late assembly stages of the HCV lifecycle.
It is now well established that hijacking of host-cell biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle [76] . The next frontier is identifying common, critical, host cell pathways that are hijacked by pathogenic human viruses, in order to develop broad-spectrum, host-directed antivirals with novel mechanisms of action. In this study, we hypothesized that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of antiviral agents against infection associated with human enveloped viruses such as HCV, whose replication and pathogenesis depend on the interaction with lipid droplets (LDs) [8] . In the case of HCV, overstimulation of host lipid metabolism in the liver during viral infection promotes cholesterol intracellular storage in host LDs, a critical cellular event for the HCV lifecycle that leads to steatosis of the liver in HCV-infected patients [8, 18, 77] . One such master regulator of cholesterol metabolic pathways is the host proprotein convertase SKI-1/S1P [36, 78] . SKI-1/S1P plays a critical role in the proteolytic activation of SREBPs, which control expression of key enzymes of cholesterol and fatty-acid biosynthesis [41, 53] . Here, we report that strategic manipulation of cellular SKI-1/S1P activity levels by protein-based or small-molecule protease inhibitors provides a means of effectively microscope. (D) Huh-7.5.1 cells infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 72 hours were harvested and subjected to SDS-PAGE and Western blot analysis. Mouse anti-ADRP antibody was used to detect protein expression levels in serpin-treated cells compared to control-treated cells. Relative protein expression was quantified by normalizing to b-tubulin expression. The inset shows a representative Western blot. (E) Huh-7.5.1 cells were treated with DMSO (control) or with 10 mM PF-429242 for 24 hours, the compound was removed, and the cell lysates were harvested after an additional 48 hours. Relative ADRP expression (normalized to b-tubulin) in inhibitor-treated cells compared to control cells was quantified by subjecting total cell lysates to Western blot analysis. Values are plotted relative to protein expression in control cells, which are set to 1. Results (mean 6 SEM) from 3 independent experiments are shown. *p,0.05. doi:10.1371/journal.ppat.1002468.g004 inhibiting HCV infection (JFH-1 strain) of Huh-7.5.1 cells in a dosedependent manner. Furthermore, we reveal the common molecular and cellular mechanisms of action of the SKI-1/S1P inhibitors and demonstrate that they act as negative modulators of cytoplasmic LD abundance (Figure 7) , an organelle central to HCV assembly [8] and liver steatosis.
To investigate the biological consequences of inhibiting SKI-1/ S1P endoprotease activity on biochemical pathways of lipid homeostasis hijacked by HCV, we first employed a protein-based inhibitor strategy. We engineered and developed a novel, recombinant adenovirus expressing an effective and specific secretory pathway, SKI-1/S1P-directed serpin, Spn4A.RRLL(s). We showed that Spn4A.RRLL(s) forms a kinetically trapped heatand SDS-stable complex with SKI-1/S1P molecules characteristic of other physiological serpin-protease pairs. We then demonstrated that it blocks the SKI-1/S1P-mediated cleavage of endogenous SREBP-1 and the expression of SREBP downstream effector gene products (e.g., SREBP-2, LDLR, and PCSK9). The SREBP target gene products identified in Spn4A.RRLL(s)-treated cells are involved in lipid homeostasis and reported to participate in HCV-host interactions. SREBP-2, along with the other SREBP isoforms SREBP-1a and -1c, are activated by HCV-encoded proteins or during HCV infection [19, 31] . PCSK9 has been implicated in HCV infection through its regulation of two HCV entry factors: CD81 and LDLR [68] . Although the specific role of LDLR in HCV infection is unclear, increasing evidence indicates that LDLR promotes attachment and uptake of lipoproteinassociated HCV particles into hepatocytes [29, 69, 79, 80] .
In addition to blocking SREBP-mediated up-regulation of hepatic genes during HCV infection, we hypothesized that blocking SKI-1/S1P endoprotease activity would also compromise cellular lipid storage. Analysis of intracellular lipid content and cytoplasmic LD abundance in Spn4A.RRLL(s)-expressing cells confirmed our hypothesis. In addition, we observed a decrease in ADRP/perilipin 2 abundance in Spn4A.RRLL(s)-expressing cells compared to control-treated cells. The physiological role of ADRP has yet to be fully established but it plays an important role in LD structure and formation. Interestingly, ADRP is degraded through the proteasome-dependent pathway during regression of lipidstoring cells, indicating that when ADRP is not bound to LDs, such as in SKI-1/S1P-inhibited cells, it will be susceptible to rapid proteasomal degradation [81] . Clinical studies have demonstrated that there is a correlation between the level of ADRP and the degree of hepatocyte steatogenesis in humans [82] . In addition, ADRP is found to be up-regulated in fatty liver in humans and in mice with liver steatosis [83] . Collectively, these observations suggest that inhibition of SKI-1/S1P offers an attractive therapeutic target for reducing HCV-induced liver steatosis.
As hypothesized, inhibiting SKI-1/S1P-mediated SREBP endoproteolytic cleavage events using Spn4A.RRLL(s) resulted in a dose-dependent decrease in HCV infection. We demonstrated that HCV core expression and HCV RNA levels are reduced in Spn4A.RRLL(s)-expressing cells following HCV infection, leading to a robust reduction in extracellular infectious HCV particle release. Western blotting also confirmed that HCV core protein post-translational processing was unaltered by serpin expression because the size of the protein was unaltered. Supplementing 1) The inactive SKI-1/S1P zymogen is biosynthesized in the ER and traffics to the Golgi apparatus following intramolecular autocatalytic maturation of the proenzyme [35, 46, 99, 100] . (2) During HCV infection, the SREBP pathway is activated by a variety of molecular mechanisms [19, [30] [31] [32] . (3) For SREBP to activate genes involved in lipid biosynthesis, its N-terminal domain must be released through sequential endoproteolytic cleavage first by SKI-1/S1P and then by S2P [37, 38] . (4) The released N-terminal domain translocates to the nucleus and activates various aspects of lipid metabolism [36] . (5) Activation of lipid biosynthesis increases LD formation where the HCV core protein localizes to orchestrate HCV assembly and subsequent secretion [8, 10, 16] . (6) Biosynthesis of LDLR, a proposed receptor for HCV entry, is also activated by SREBP signaling [69, 79, 101] . (7) Spn4A.RRLL(s) is a secretory pathwayexpressed serpin (Figure 1 ). (8) Spn4A.RRLL(s) interacts and forms a covalent complex with enzymatically active SKI-1/S1P molecules (Figure 2 and S3) in the Golgi apparatus preventing SKI-1/S1P-mediated endoproteolytic cleavage of SREBP protein ( Figure 3A ). (9) A small-molecule inhibitor PF-429242 also efficiently inhibits SKI-1/S1P endoproteolytic activity ( Figure 3A ). (10) SKI-1/S1P inhibition blocks expression of the putative HCV receptor, LDLR ( Figure 3B and S4) , and reduces HCV entry ( Figure 5) . (11) The expression of other SREBP-regulated genes, such as PCSK9 and SREBP-2, are also blocked ( Figure 3B ). (12) Downstream lipid synthesis is interrupted resulting in overall reduced intracellular cholesterol-ester and triglyceride abundance ( Figure 3C and 3D) . (12) This is then detected as a decrease in LD abundance (Figure 4) , which impedes assembly and secretion of infectious HCV particles. doi:10.1371/journal.ppat.1002468.g007
Spn4A.RRLL(s)-expressing cells with compounds such as mevalonate, oleate, and cholesterol resulted in an incomplete rescue of HCV infection, suggesting that the antiviral activity of our proteinbased inhibitor cannot be explained solely by the decreased availability of lipids in these cells. This also supports our hypothesis that reduced LDLR levels may compromise HCV entry into Spn4A.RRLL(s)-treated hepatocytes. LDLR, a well-established SREBP-regulated gene, has been repeatedly shown to support HCV entry into hepatocytes [69, 80] . Also, no significant reductions in HCV RNA levels were observed in Spn4A. RRLL(s)-treated cells harbouring subgenomic HCV replicons or following full-length genomic HCV RNA transfection in Huh-7.5.1 cells. Altogether, these studies indicate that the observed decline in HCV RNA levels following HCV infection may result from compromised HCV entry.
To gain further insight into the different stages of the viral lifecycle targeted by our SKI-1/S1P inhibitor, we used an activesite-directed small-molecule inhibitor of SKI-1/S1P, PF-429242. This pharmacologic inhibitor of SKI-1/S1P has recently been characterized for its effectiveness in blocking cleavage of SREBP-2, for blocking expression of SREBP-activated genes, and also for inhibiting arenavirus glycoprotein processing [64, 84] . In contrast to our recombinant adenovirus-expressed serpins, PF-429242 can be added extracellularly to rapidly inhibit SKI-1/S1P. This allows us to study the biological impact of blocking SKI-1/S1Pdependent pathways during both early and late stages of HCV infection.
We first confirmed a reduction in abundance of neutral lipids and ADRP/perilipin 2 expression in PF-429242-treated cells. Then, we confirmed that inhibition of SKI-1/S1P using PF-429242 blocks HCV infection and extracellular infectious virus production in a dose-dependent manner. The anti-HCV activity of PF-429242 is very robust and particularly striking. A single 24hour pre-treatment with the compound was sufficient to block HCV infection, and the antiviral effect of PF-429242 was still apparent 72 hours post-treatment. Importantly, pharmacological treatment of already infected HCV cells resulted in a 90% reduction of HCV virus production. Similar to Spn4A.RRLL(s) treatment, PF-429242 did not reduce HCV RNA levels in the two stable subgenomic replicon cell lines that were examined. This, in combination with the observed reduction in abundance of a central organelle (LD) involved in HCV assembly, and the reduction in HCV particle secretion in cells treated 24 hours after HCV inoculation, supports PF-429242 as an inhibitor of late stages of the HCV lifecycle, i.e., during assembly or egress. Taken all together, these results indicate that inhibiting SKI-1/S1P can interrupt the HCV lifecycle at multiple stages of viral infection both preventing naïve cells from becoming infected and preventing virus release from already infected cell populations. Thus, developing more effective active-site-directed SKI-1/S1P small-molecule inhibitors (, nM range) with better pharmacokinetic properties [64] could lead to novel indirect-acting antiviral treatment options for HCV-infected patients [42, 76, 85, 86] . Importantly, inhibiting the SREBP pathway in HCV-infected cells, which have exacerbated lipid production and which are steatotic, may relieve symptoms caused by chronic HCV infection in addition to blocking viral infection [87] .
In conclusion, pharmacologic inhibition of SKI-1/S1P offers a very promising avenue for the development of novel anti-HCV therapeutics (Figure 7) . On one hand, targeting a host cell master molecular switch such as SKI-1/S1P with a novel class of drugs compromising multiple stages of the virus lifecycle would have the main advantage of making it more difficult for the virus to develop escape mutations [86, 88] . On the other hand, the toxicity issues associated with the inhibition of host cell proteases such as SKI-1/ S1P [89] could be addressed by using adjunctive therapy, combining our novel class of lipid-modulating agents with the current standard of care or with the appropriate synergistic directacting antivirals [85, 86] .
Finally, our results reveal that targeting host LD biogenesis by inhibiting SKI-1/S1P endoproteolytic activity may have farreaching applications in the therapeutic treatment of other important human Flaviviridae viruses such as dengue virus, whose replication and pathogenesis also depend on the interaction with lipid droplets [7] . 
A plasmid containing the cDNA of an HCV consensus clone isolated from a Japanese patient with fulminant hepatitis (JFH-1) (GenBank accession number AB047639) [91] cloned behind a T7 promoter (pJFH-1; a generous gift from Dr. Takaji Wakita, National Institute of Infectious Diseases, Tokyo, Japan) was used to generate genomic HCV RNA and infectious HCV stocks as previously described in [61] .
Purified HCV RNA was used to transfect Huh-7.5.1 cells as a means of studying HCV infection independently of receptormediated entry. Five micrograms of purified RNA was incubated with 10 ml of lipofectamine 2000 (Invitrogen) in minimal essential media (MEM) for 30 minutes. The RNA-lipid complexes were added to cells in MEM for 16 hours; then cells were washed with phosphate-buffered saline (PBS) and complete media was added for the remainder of the experiment.
The amount of infectious HCV particles generated for viral stocks or in the described experiments was determined using a modified, previously described protocol [61] . Briefly, 1610 4 Huh-7.5.1 cells were plated in each well of a 96-well plate and infected with 10-fold serial dilutions of HCV-infected cell media. At 72 hours post-infection, cells were fixed and probed as described in the ArrayScan Quantification methods section. An ArrayScan VTI High Content Screening (HCS) Reader (Thermo Scientific) was used to acquire images of the entire group of infected wells. Titers were determined by manually counting foci (fluorescence forming units (FFU)) in the lowest dilutions with positive signal.
In silico homology model of Spn4A.RRLL variant
The Drosophila melanogaster Spn4B sequence (GeneBank Accession number gi|24586105|ref|NP_524955.2) exhibits 34% sequence homology with the human neuroserpin (hNS), for which a crystal structure is available [94] in the Protein Data Bank (PDB ID: 3F5N). Of the five chains in this pentameric structure of hNS, chain B is most well resolved with the fewest missing residues, and it was used as the template for the homology model presented in Figure 1B . The model was built and refined using the SwissPDB Viewer. The C-alpha residues in this model structure align to 1.9 Å RMSD with reference to the hNS structure.
The first 997 amino acids of human SKI-1/S1P lacking the Cterminal transmembrane domain but containing a C-terminal 8-His-tag (PGDDDDKHHHHHHHHSGS) were expressed in Sf9 insect cells as previously described [47] . Two liters of cell culture supernatant were used for purification. Two hundred milliliters of 200 mM Tris/HCl pH 8.0, 500 mM NaCl was added, and then the pH was adjusted to pH 8.0 by further addition of 2 M NaOH. The resulting precipitate was removed by centrifugation at 10000 x g for 30 minutes and subsequently filtered through a glass filter. The cleared supernatant was then applied to a small (0.9 ml column volume) IMAC column (Ni-Sepharose, GE Healthcare, Freiburg, Germany) by continuous flow (1.0 ml/minutes). The column had previously been equilibrated in 50 mM Tris/HCl pH 8.0, 500 mM NaCl (buffer A), and bound recombinant SKI-1/S1P was eluted with a continuous gradient over 30 column volumes to buffer A plus 300 mM imidazole. Collected fractions were assayed for SKI-1/S1P enzymatic activity as previously described [47] using the paranitroanilide (p-NA) acetylated (Ac) tetrapeptidyl substrate Ac-RRLL-pNA [custom synthesized by Peptides International (Louisville, Kentucky, USA)]. The most active fractions were pooled. Concentration and buffer exchange to buffer A was then done using spin concentrators (Millipore, Billerica, MA, USA) with a molecular weight cutoff of 30 kDa. The final preparation was, after addition of 30% v/v glycerol, stored at -80uC and had a specific activity of 0.018 U/mg (measured as above). Recombinant His-tagged furin (0.432 mg/ ml) was purchased from R & D Systems (Minneapolis, MN, USA), and reactions with adenovirus recombinant serpin were performed under the buffer conditions for furin assays as previously described [54, 58] .
Cultured cells were washed with ice-cold PBS and re-suspended in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% octylphenyl-polyethylene glycol [IGEPAL], 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] containing 1 X Complete, EDTA-free, protease inhibitor cocktail [Roche, Laval, QC, Canada]). Whole cell extracts were vortexed and then clarified by centrifugation at 12000 x g for 15 minutes. Soluble extracts mixed with 2 X sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, and 5% beta-mercaptoethanol) were electrophoresed on 8-15% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) for one hour, and proteins of interest were detected by probing with the appropriate primary and secondary antibodies diluted in Odyssey blocking buffer containing 0.1% Tween 20. Protein bands were detected and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). All immunoblots were scanned at a wavelength of 700 nm for detecting IRDye 680 labeled antibodies and at a wavelength of 800 nm for IRDye 800CW conjugated antibodies [95, 96] . Signal intensities were quantified by means of the Odyssey software version 3.0. Beta-tubulin was always used as a loading control and for normalizing protein expression. Media samples analyzed for secreted Spn4A variants were taken directly from cultured cells, mixed with 2 X sample loading buffer, and subjected to the described Western blot analysis. [47] ; furin buffer contains 100 mM HEPES, pH 7.5, 1 mM CaCl 2 , 0.5% Triton X-100 [54, 58] , 1 X Complete EDTA-free protease inhibitor cocktail), and 11.6 ng/ml SKI-1/ S1P or 2.4 ng/ml furin. The enzyme mixture was incubated at 30uC for 30 minutes, and the reaction was stopped with 12.5 mM EDTA (final concentration). After completion, products were resolved on a 10% SDS-gel. The high-molecular weight band (EI) was visualized as described above for Western blotting.
In black flat-bottom 96-well plates (BD Biosciences), cells were plated (.10,000 cells/well) and infected as described in the methods below. Following infection, cells were fixed in 4% formaldehyde v/v diluted in PBS and blocked in PBS containing 3% BSA, 0.3% Triton X-100, and 10% FBS. Cells were first probed with HCV anti-core antibody (1:500) in PBS containing 3% BSA and 0.3% Triton X-100 (Binding Buffer), then incubated with Alexa Fluor-568-conjugated donkey anti-mouse secondary antibody (1:1000) and 10 mg/ml Hoechst dye. Cells were analyzed by a quantitative, high-throughput, fluorescence microscope system called the Cellomics ArrayScan VTI High Content Screening (HCS) Reader (Thermo Scientific) using the software Target Activation BioApplication (TABA). TABA was used to count the total number of cells (Hoechst-stained nuclei) and the percentage of those cells that were expressing HCV core (positive signal at 568 nm wavelength). . Nuclear fractionation of cell lysates was performed at 4uC as described previously with modifications [64, 66] . Cells were harvested in 400 ml buffer C (10 mM HEPES/KOH, pH 7.6, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose) containing 1 X complete protease inhibitor cocktail (Roche). To shear DNA, the cells were passed 20 times through a 23-gauge needle. The lysate was centrifuged at 1100 x g for 7 minutes and the resulting supernatant was centrifuged again at 25000 x g for 60 minutes to obtain the membrane pellet. The pellet containing membranebound SREBP-1 was re-suspended in 75 ml of SDS-lysis buffer (10 mM Tris HCl, 100 mM NaCl, 1% SDS, 1 mM EDTA, 1 mM EGTA, pH 6.8). The pellet from the 1100 x g spin was resuspended in 100 ml buffer D (20 mM HEPES/KOH, 420 mM NaCl, 1.5 mM MgCl 2 , 2.5% glycerol, 1 mM EDTA, 1 mM EGTA, pH 7.6) containing 1 X complete protease inhibitor cocktail. Nuclear pellets were rocked for 1 hour, after which the samples were centrifuged at 25000 x g for 60 minutes to obtain the clarified supernatant containing the nuclear fraction.
After Huh-7.5.1 cells were seeded onto coverslips for 24 hours, they were infected with adenovirus (moi 50) for 72 hours. Cells were fixed in 4% v/v formaldehyde in PBS, then permeabilized and blocked in PBS containing 0.05% saponin (wash buffer) and 1% BSA (binding buffer). Blocking of cells stained with BODIPY 493/503 was done in the presence of 0.2 M glycine to reduce background fluorescence. Cells were probed with primary antibodies in binding buffer, then incubated with a secondary antibody, Hoechst dye (10.0 mg/ml), and BODIPY 493/503 (1.0 mg/ml; when indicated) diluted in PBS. Cells were mounted onto slides with an anti-fade solution and sealed with clear nail polish. The slides were then imaged using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) or an Olympus Fluoview FV1000 laser scanning confocal microscope (Olympus Corporation, Tokyo, Japan) [95] [96] [97] . Leica MM AF Software (Leica Microsystems) was used to count the number of LDs (green channel) in cells expressing Spn4A.RRLL(r) (n = 21) or Spn4A.RRLL(s) (n = 15) (cells positive in the red channel). LDs in cells treated with Ad-Empty (n = 23) were also enumerated. All quantified images were acquired using the same laser intensity and gain settings, and LDs were enumerated by applying the same threshold setting to each image.
Huh-7.5.1 cells were infected with recombinant adenovirus at different moi in complete media with or without exogenous sterols (50 mM sodium mevalonate, 20 mM sodium oleate, 5.0 mg/ml cholesterol). After 48 hours, the cells were infected with HCV moi 0.1 or transfected with purified HCV genomic RNA for 72 hours, and then cells were analyzed by Cellomics ArrayScan HCS, or total RNA was isolated from cell extracts using the RNeasy plus kit (Qiagen, Mississauga, ON, Canada) including on-column DNase digestion. Media from treated and infected cells were harvested for HCV titer determination as described above. To examine the Spn4A.RRLL(s) mediated block in PCSK9, LDLR, and SREBP-2 expression, Huh-7.5.1 cells were grown in media supplemented with LPDS for 24 hours, infected with adenovirus variants, and harvested 72 hours later.
Purified total RNA was reverse transcribed to cDNA using TaqMan reverse transcription reagents (random hexamers; Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR was carried out using Brilliant II Fast QPCR reagents (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions on an Mx3005P QPCR system (Stratagene). Online ProbeFinder software (Roche Applied Science) was used to find primers that would allow amplification of the HCV RNA 59 end in combination with the Human Universal Probe Library from Roche (Roche Applied Science). For amplification of the HCV RNA 59 region, 400 nM of both forward primer (59-CAT-GGCGTTAGTATGAGTGTCG-39) and reverse primer (59-GG-TTCCGCAGACCACTAT-39) were used in combination with 200 nM of probe #75 from the Human Probe Library (Roche). HCV RNA levels were relatively quantified across samples and normalized to beta-actin RNA levels using 500 nM primers (forward: 59-GCC CTG AGG CAC TCT TCC and reverse: 59 GGA TGT CCA CGT CAC ACT TC-39) and 250 nM probe (59AC TCC ATG CCC AGG AAG GAA GGC-39 with a 59 Cy5 fluorophore and 39 black hole quencher).
Cell viability was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). This assay employs a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS], which is bio-reduced by cells into a colored formazan product that can be detected in tissue culture media at 490 nm wavelength [98] .
PF-429242, an active site-directed small-molecule inhibitor of SKI-1/S1P [64, 84] , was synthesized by Dr. Peter Chua at the Center for Drug Research and Development (CDRD) at the University of British Columbia (Vancouver, BC, Canada) according to previously described protocols [65] . To investigate the antiviral activity of the small molecule, Huh-7.5.1 cells were treated with PF-429242 for 24 hours. After 24 hours of treatment, the media was removed and then cells were infected with HCV (moi 0.1) for 48 or 72 hours. Alternatively, cells were first infected with HCV for 24 hours; then, the media was removed and replaced with media containing various concentrations of PF-429242 for a further 48 hours. Intracellular HCV infection levels were determined using Cellomics HCS ArrayScan. Infectious extracellular titers were determined in media of 72-hour HCVinfected cells.
To measure the level of intracellular lipids following 72-hour recombinant adenovirus expression or 48-hour PF-429242 treatment, cellular extracts were harvested in 1% triton-X 100 in PBS (for phospholipid, cholesterol, and protein assay) or 5% triton-X 100 in H 2 O (for triglyceride assay). To extract triglycerides, samples were slowly heated to 90uC and brought to room temperature, twice. Total cholesterol, cholesterol esters (Amplex Red Cholesterol Assay kit, Invitrogen), phospholipids, and triglycerides (EnzChrom, BioAssay Systems, Hayward, CA, USA) were quantified using commercially available kits. Lipid levels were normalized to cellular protein content (DC Protein Assay, Bio-Rad, Hercules, CA, USA).
The sigmoidal fit function in Igor Pro software (WaveMetrics, Inc., Portland, OR, USA) was used for fitting HCV and PF-429242 inhibition curves and for determining EC 50 values. The reported EC 50 values are the average of the values calculated from three independent experiments plus or minus the standard deviation. The student's t-test (unpaired) was used to calculate significance, which is represented in the figures by the following notation: * denotes p,0.05, ** denotes p,0.01, and *** denotes p,0.005. Figure S1 Optimization of adenovirus-expressed Spn4A.RRLL(r) expression in Huh-7.5.1 cells. Huh-7.5.1 cells were infected with moi 1, 12.5, 25, and 50 of the intracellularly retained serpin Ad-Spn4A.RRLL(r). Treated cells were fixed 48 hours post-infection and probed for serpin expression using mouse anti-FLAG antibody. Cell nuclei were stained with Hoechst dye to determine the total cell number. The percentage of Spn4A.RRLL(r)-expressing cells was quantified using Cellomics HCS. Results (mean 6 SEM) from 2 independent experiments are shown. (TIF) Figure S2 Effect of Spn4A variant treatment and HCV infection on Huh-7.5.1 cell growth. Huh-7.5.1 cells were infected with various moi (1 -50) of Ad-Empty, Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 48 hours in complete media. Treated cells were infected with HCV (moi 0.1) and fixed 72 hours post-infection. Fixed cells were probed with Hoechst dye to stain for cell nuclei, which were then quantified using Cellomics HCS to determine the relative number of cells in each well under the varying conditions. All values are expressed as relative cell number in serpin-treated cells compared to cells infected with Ad-Empty, which is set to 1. Results (mean 6 SEM) from 3 independent experiments are shown. (TIF) Figure S3 Serpin-like properties of recombinant adenovirus-expressed Spn4A variants expressed in Huh-7.5.1 cells. Huh-7.5.1 cells were infected with recombinant adenovirus expressing the His-and FLAG-tagged Spn4A variants indicated or the Ad-Empty control for 72 hours. Media alone (upper panels) or cell extracts (lower panels) lysed in RIPA buffer were combined with recombinant His-tagged SKI-1/S1P [47] or His-tagged furin for 30 minutes at 30uC. Samples were prepared for Western blot analysis and probed with rabbit anti-FLAG antibody to detect SDS-and heat-stable protease-serpin complex formation and also to distinguish serpin bands from protease bands on the Western blots. All Western blots shown are representative of at least 2 independent experiments. (TIF) Figure S4 Time course analysis of LDLR expression in Spn4A.RRLL(s)-treated cells. Huh-7.5.1 cells were grown in complete media for 24 hours and then infected with Ad-Empty (control) or Ad-Spn4A.RRLL(s). Cell extracts were harvested for Western blot 24, 48, and 72 hours post-infection, and lysates were then subjected to Western blot. Anti-LDLR antibody was used to detect protein-expression levels in control-and Ad-Spn4A. RRLL(s)-treated cells, and b-tubulin was probed for normalizing LDLR expression. Values are plotted relative to LDLR expression in control (Ad-Empty)-treated cells, which was set to 1. The representative results of 2 independent experiments are shown. (TIF) Figure S5 Spn4A.RRLL(s) does not block HCV core production post-transfection of HCV RNA. Huh-7.5.1 cells were infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) (moi 50) for 48 hours in complete media and then transfected with genomic HCV RNA for 72 hours. Relative HCV-core expression (normalized to b-tubulin) in serpin-treated cells compared to control-treated cells was quantified by examining total cell lysates using Western blot analysis. Results (mean 6 SEM) from 2 independent experiments are shown. A representative Western blot is shown to the right of the graph. (TIF) Figure S6 The effect of PF-429242 on cell viability. Huh-7.5.1 cells were treated with DMSO (control) or various concentrations of PF-429242 for 24 hours before the inhibitor was removed, and fresh complete media was added to the cells for an additional 48 hours. The relative cytotoxicity of the compound was then determined using an MTS-based cell viability assay. The absorbance measured at 490 nm is proportional to the number of living cultured cells. Results (mean 6 SEM) from 3 independent experiments are shown. Statistical significance was calculated for PF-429242-treated cells compared to DMSO-treated cells.
(TIF) HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells Dendritic cells (DCs) initiate immune responses by transporting antigens and migrating to lymphoid tissues to initiate T-cell responses. DCs are located in the mucosal surfaces that are involved in human immunodeficiency virus (HIV) transmission and they are probably among the earliest targets of HIV-1 infection. DCs have an important role in viral transmission and dissemination, and HIV-1 has evolved different strategies to evade DC antiviral activity. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can act as an alarmin, a danger signal to alert the innate immune system for the initiation of host defense. It is the prototypic damage-associated molecular pattern molecule, and it can be secreted by innate cells, including DCs and natural killer (NK) cells. The fate of DCs is dependent on a cognate interaction with NK cells, which involves HMGB1 expressed at NK–DC synapse. HMGB1 is essential for DC maturation, migration to lymphoid tissues and functional type-1 polarization of naïve T cells. This review highlights the latest advances in our understanding of the impact of HIV on the interactions between HMGB1 and DCs, focusing on the mechanisms of HMGB1-dependent viral dissemination and persistence in DCs, and discussing the consequences on antiviral innate immunity, immune activation and HIV pathogenesis. High mobility group box 1 (HMGB1) is a nuclear DNAbinding protein actively released by innate immune cells in response to exogenous pathogen-derived molecules, acting as a danger signal and triggering inflammation. HMGB1 is a proinflammatory cytokine that is essential for maturation of dendritic cells (DCs), their migration to lymphoid tissues and Th1 polarization of naïve T cells. HMGB1 signals by binding to Toll-like receptor 4 (TLR4) to activate MyD88-dependent nuclear translocation of NF-kB, which upregulates the expression and release of cytokines and other inflammatory mediators. HMGB1 is expressed at the synapse between NK cells and DCs, it is pivotal during NK-DC cross talk, promoting DC maturation and protecting them from lysis. HMGB1 triggers human immunodeficiency virus (HIV) replication in latently infected primary myeloid cells.
What are the molecular mechanisms involved in vivo in the disruption of NK cell-DC cross talk during chronic HIV-1 infection, and what are the consequences on both NK cell killing activity and DC-dependent promotion of adaptive immune responses? Does HMGB1 has a role in the trans-infection of T lymphocytes with HIV-1 through the exosome-dissemination pathway? Given that HMGB1 can combine with LPS to trigger TLRs, and TLR-mediated immune activation results in the production of proinflammatory cytokines, to what extent does HMGB1 contribute to generalized immune activation and disease progression in HIV-1-infected individuals? What is the contribution of HMGB1 to HIV dissemination and the establishment of HIV reservoirs in DCs? Would the specific targeting of c-FLIP or c-IAPs in DCs contribute to the depletion of HIV-1 reservoirs? Given the expression of HMGB1 and its receptor RAGE in active neurological diseases, including multiple sclerosis and Alzheimer's disease, does it has a role in HIV-associated neurological disorder?
High-mobility group box 1 protein (HMGB1) (also known as amphoterin or HMG1) was originally defined as a non-histone nucleosomal protein that is important for the regulation of transcription. It is a 215 amino-acid protein, encoded on chromosome 13q12, which is highly conserved between species (99% species homology between rodents and humans). HMGB1 contains two internal repeats of positively charged domains, the A-and B-Box, in the N terminus, and a negatively charged COOH terminus ( Figure 1 ). The two boxes bind to the minor groove of chromatin, thus modifying DNA architecture. 1 This facilitates the binding of regulatory protein complexes to DNA such as V(D)J recombinases 2 and p53-p73 transcriptional complexes. [3] [4] [5] In its resting state, the acidic tail of HMGB1 interacts with specific residues in the A-Box and B-Box, forming an extended and flexible segment, shielding them from other interactions that might occur before HMGB1 binds DNA. 6 HMGB1 likely has a role in DNA repair and replication. HMGB1 overexpression, which is observed in many tumors, accelerates cell cycle progression, and recent data suggest that endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival 7 and that HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells. 8 The discovery by Kevin J Tracey et al. (1999) in a mouse model of endotoxaemia that lipopolysaccharide (LPS)activated macrophages release HMGB1, but later than secretion of the pro-inflammatory cytokines TNF-a and interleukin 1 (IL-1), and that protection against endotoxin lethality could be obtained by administration of anti-HMGB1 antibodies 9 has revealed that HMGB1 is a proinflammatory mediator able to alert the immune system to tissue damage and to trigger an immediate response. The term 'alarmin' has been proposed to differentiate the endogenous molecules that are very rapidly released or produced in response to microbial infection or tissue injury, and act as potent effectors of innate defense. 10 Alarmins have antimicrobial, enzymatic or chromatin-binding activities and they share common features, including their rapid passive release from necrotic cells or secretion from cells of the innate immune system (macrophages, natural killer (NK) cells) in response to infection, they bind to TLRs and receptors of antigen-presenting cells such as DCs, thus promoting adaptive immunity, and they are involved in the reconstruction of tissues destroyed secondary to inflammation. 11 Based on these criteria, a list of putative alarmins has been proposed, including the defensins, eosinophil-derived neurotoxin, thymosins, annexins, HSPs, or IL-1a. 12 HMGB1 remarkably fulfills these criteria and it is probably the best-characterized alarmin. The crucial role of HMGB1 not only in response to infection, injury and inflammation, but also its pathological effects in many diseases have recently challenged important questions regarding its biological activities and pathological effects.
Recent studies have established the involvement of HMGB1 in not only acute and chronic inflammatory conditions, including sepsis, 13 rheumatic diseases, 14 or SLE, 15 but also viral infectious diseases, such as that induced by SARS, hepatitis viruses, influenza viruses 16 or HIV. 17 It is currently unclear whether HMGB1-mediated inflammatory response contributes to the pathogenesis of various viral diseases, but it has been suggested to be involved in SARS-associated injurious pulmonary inflammatory response, persistent liver injury in hepatitis patients, or pathogenesis of West Nile encephalitis. 18 Regarding HIV infection, elevated plasma levels of HMGB1 were detected during progressive HIV-1 infection, positively associated with viral replication. 17 In vitro, HMGB1 may trigger or inhibit HIV-1 replication, depending on the target cell and the microenvironment. 19, 20 Recent studies analyzed the impact of HMGB1 on the fate of HIV-1-infected DCs, and the data suggested not only a possible contribution of this protein to the functional impairment of DCs but also to HIV dissemination and persistence. 21, 22 This review will discuss the mechanisms whereby HMGB1 contributes to innate immunity by regulating maturation and functions of DCs but also how it may contribute to viral latency and HIV disease pathogenesis.
HMGB1, a DAMP that Likes DCs HMGB1, a sentinel for nucleic-acid-mediated response in DCs. During microbial infection, the activation of innate immune responses by DNA and RNA is essential to protective immune responses and is mediated by the NH2  COOH  A box  B box  Acidic tail   1  79  89  163  186  215  DNA binding domain  DNA binding domain   150  183  RAGE-binding domain   28  44  NLS1   179  185  NLS2  C106   TLR4 binding site   Proinflammatory  cytokine-domain   89  108   100  189  202 transmembrane TLRs and cytosolic receptors. 23 TLRs belong to a family of pattern recognition receptors (PRRs) that have essential roles in innate immunity. They are a class of single-membrane-spanning receptors that have the ability to recognize structurally conserved molecules from bacteria. Engagement of TLRs activates the immune response.
HMGB proteins bind to all immunogenic nucleic acids and have recently been identified to serve as universal sentinels for nucleic-acid-mediated innate immune responses. 24 Activation of macrophages or DCs with microbial cytosinephosphate-guanosine (CpG)-DNA, resulting in secretion of proinflammatory cytokines, involves TLR9 initially localized in the endoplasmic reticulum (ER). The mechanism of TLR activation by CpG-DNA was recently discovered and HMGB1 was found to have a crucial role. HMGB1 binds to CpG-DNA and receptor for advanced glycated endproducts (RAGE), the first identified receptor for HMGB1, and this DNA-protein complex preassociates with TLR9 in the ER-Golgi intermediate compartment, thus accelerating the delivery of microbial DNA to TLR9 and leading to the recruitment of the TLR adaptor molecule MyD88. 25 Thus, HMGB1 and RAGE are pivotal for TLR9-dependent induction of genes encoding type I interferon after stimulation of DCs with DNA-containing complexes. Ablation or depletion of HMGB1 impairs redistribution of TLR9 to early endosomes in response to CpG-oligodeoxynucleotides (ODN), leading to a decreased response to CpG-ODN, but which could be complemented by extracellular HMGB1. 25 In addition, HMGB1 was found to be a key factor in immune complex-triggered activation of autoreactive B cells and the induction of type I interferon by plasmacytoid DCs, thus possibly contributing to SLE. 25 HMGB1, a cytokine that initiates host defense. HMGB1 is a nuclear protein and, to function as a cytokine, it must be released in the extracellular milieu. This occurs either via passive release from necrotic cells, 26, 27 or by active secretion by cells of the innate immune system. Wang et al. 9 first reported that HMGB1 was liberated from macrophages stimulated with LPS, and that HMGB1 had an important role in experimental sepsis. HMGB1 secretion from LPS-primed macrophages was shown to require the inflammasome components apoptotic speck protein containing a caspase recruitment domain (ASC), caspase 1 and NALP3. 28 Monocytes, macrophages and immature DCs (mDCs) secrete HMGB1 in response to LPS, TNF-a, or IL-1b stimulation. 29 IFN-g can induce HMGB1 release from macrophages that, at least in part, requires induction and signaling through TNF-a. 30 Secretion of a nuclear protein requires a tightly controlled relocation program. Several forms of post-translational modifications, such as acetylation, phosphorylation and oxidation result in the accumulation of HMGB1 in the cytosol. [31] [32] [33] [34] Upon activation with LPS, monocytes and macrophages acetylate HMGB1 extensively, allowing its relocalization from the nucleus to the cytosol, and further concentration into secretory lysosomes. 35 Thus, because it does not contain a leader sequence, HMGB1 is secreted via a non-classical vesiclemediated secretory pathway.
A second pathway of HMGB1 nuclear/cytoplasmic shuttling has been reported involving phosphorylation, 33 mediated by protein kinase C. 36 In neutrophils, HMGB1 is post-translationally methylated, which alters its conformation and weakens its DNA-binding activity, causing its cytoplasmic localization. 34 Recently, HMGB1 has been shown to undergo oxidation that may have the potential to modulate various aspects of its function, including subcellular localization, interaction with DNA, cytokine activity and proinflammatory activity. 37 Indeed, induction of immunological tolerance by apoptotic cells was shown to require caspase-dependent ROS production by mitochondria, which oxidized the danger signal HMGB1, and thereby neutralized its damage-associated molecular pattern molecule (DAMP) function, including the ability to activate DCs. 38 From the reverse perspective, in necrotic cells that do not generate ROS and provide fully active HMGB1, treatment with H 2 O 2 inactivates the DAMP function of the protein. Thus, DAMP is released and can work, but briefly and in a short range, until it gets oxidized. This restricts its activity temporally and spatially, preventing a prolonged stimulation of its targets and thereby limiting inflammation. 39 HMGB1 is essential for DC maturation, migration and Th1 polarization. Inflammatory signals activate antigenpresenting DCs, which undergo a differentiation process referred to as maturation and migrate to secondary lymphoid organs. HMGB1 has a crucial role in this process, acting as a chemoattractant for immature DCs (iDCs) that involves RAGE, and further inducing DC maturation, as shown by the upregulation of the surface markers CD80, CD83, CD86 and human leukocyte antigen (HLA)-A, B, C, and DC production of cytokines, including IL-6, CXCL8, IL-12p70 and TNF-a. 11, 40 The mobilization of DCs from peripheral tissues is critical for the establishment of T-cell-dependent immune responses or tolerance, because the physical interaction of DCs with naive T cells takes place in the T-cell areas of lymph nodes. Importantly, in vivo homing of DCs to draining lymph nodes depends on RAGE, 41 a finding consistent with the in vitro upregulation of the CCR7 and CXCR4 receptors upon autocrine secretion of HMGB1 by mature myeloid DCs. 40 Thus, the autocrine/paracrine release of HMGB1 and the integrity of HMGB1/RAGE pathway are required for the migratory function of HMGB1. Moreover, HMGB1 secreted by DCs is required for clonal expansion, survival and functional Th1 polarization of naïve T cells, which occurs through the secretion of proinflammatory cytokines, including IL-12, IL-18 and IFN-g 42, 43, 21 In the presence of inhibitors of HMGB1 or of RAGE, DC activated with pathogen-associated molecular patterns (PAMPs) fail to mature 43 ( Figure 2 ).
The inflammatory properties of HMGB1 depend on the ability to complex with soluble moieties, including nucleic acids, microbial products (LPS), cytokines (IL-1b) and chemokines. Campana et al. 44 reported that HMGB1 secretion is required for CXCL12 (SDF-1)-dependent migration of DCs. In addition, HMGB1 protects the conformation of CXCL12 in a reducing environment, a state existing in the draining lymph node. However, only a partial inhibition of DC migration was observed in the presence of a CXCL12 inhibitor or HMGB1 A box, suggesting that multiple receptors are responsible for mediating the DC response to the HMGB1/ CXCL12 complex. An interesting study performed in CD24deficient mice revealed how the host distinguishes between danger versus pathogen PAMPs. 45 The proinflammatory activity of HMGB1 is regulated by CD24, which associates with HMGB1, thus inhibiting NF-kappa B activation occurring through CD24 association with Siglec-10. Thus, this CD24-Siglec-10 pathway protects the host against a lethal response to pathological cell death, but it would allow an appropriate response to invading pathogens. 45 
DCs, pivotal to adaptive immunity, are early targets of HIV. All lentiviruses can infect macrophage lineage cells in which they generate a persistent infection. HIV-1 has developed a broader tropism leading to preferential infection of CD4 þ T cells, which are progressively destroyed both as a direct viral cytopathic effect and a bystander induction of apoptosis in uninfected cells. 46 Studies in animal models have revealed that critical events to establishing systemic infection take place very quickly at the mucosal portal of entry. Following mucosal exposure to high doses of simian immunodeficiency virus (SIV), the virus can cross the mucosal barrier and it establishes within 3-4 h a small founder population of productively infected cells. 47, 48 Infection then expands locally before virus detection in the draining lymph node, and systemically throughout the secondary lymphoid organs. 49 In vivo, resting CD4 T cells are the initially infected cells in lymphoid tissues. 48 However, the first cells to be infected at the mucosa are the intraepithelial DCs, as shown in vivo in the genital tract of rhesus macaques 1 h after intravaginal inoculation of SIV, and infected DCs reach the draining lymph nodes 18-24 h after SIV exposure, much earlier than CD4 þ T cells. 50 As sentinels, DCs are crucial for the generation of antiviral immunity. They are the most potent antigen-presenting cell in the immune system owing to their superior capacity for acquiring and processing antigens for presentation to T cells and their potential to express high levels of the co-stimulatory molecules that drive T-cell activation and polarization. 51 Thus, they effectively link the innate recognition of viruses to the generation of the appropriate type of adaptive immune response. DCs are a heterogeneous family, including langherans cells in the epidermis, interstitial DCs found in all peripheral tissues, myeloid DCs and plasmacytoid DCs found in the blood. Their heterogeneity resides at several levels, including anatomical location, phenotype and function. 52 DCs express a large repertoire of PRRs and, in response to signals from these receptors, they are activated and migrate to the T-cell area of regional lymph nodes where mDCs present virus-derived epitopes to CD4 þ or CD8 þ T cells. 53 DCs have a prominent role in promoting viral dissemination. Viruses, including HIV, have evolved Figure 2 HMGB1-mediated cross talk between DCs and NK cells is pivotal to DC maturation and further induction of adaptive immunity. The disruption of an epithelial barrier allows invasion of microbial pathogens, which elicit an innate response at the site of infection (1). Neutrophils and macrophages infiltrate the site of tissue infection and release alarmins, including HMGB1 (2). HMGB1 recruits iDCs, resulting in an increase in local mobilization of iDCs (3). Functional DC maturation requires a cross talk with NK cells, which involves HMGB1 expressed at NK-DC synapse (4). Immature to mDC conversion allows DCs to migrate to secondary lymphoid organs (5) and contributes to the enhanced uptake, processing and presentation of microbial antigens to naïve T cells, thus polarizing a Th1 response (6) . This T-cell response involves IL-12 and IL-18 released by mDCs. Cognate interaction between NK cells and DCs may also lead to the selective killing of DCs that are not appropriate for antigen presentation to T cells (7). This editing process, which involves TRAIL, allows NK cells to control the quality of DCs, thus regulating adaptive immunity different strategies to evade DC antiviral activity and to propagate and persist in DCs. HIV-1 replication in DCs is usually weakly productive, and the frequency of HIV-1infected DCs in vivo is much lower than that of HIV-1-infected CD4 þ T cells. 54 However, DCs do not need to be productively infected to transmit the virus to CD4 T cells and to spread it in an infectious form. Analysis of conjugates between DCs and T cells revealed the recruitment of HIV and its receptors CD4, CCR5 and CXCR4 to DC-T cell junction, thus facilitating transmission of HIV during the formation of an infectious synapse in the absence of antigen-specific signaling. 55 This process, known as 'trans-infection', takes place when part of the virus evades classical degradation pathways, being maintained in endosomal acidic compartments, thus retaining viral infectivity for long periods and promoting efficient HIV transfer to CD4 T cells. 56, 57 Trans-infection ability may be restricted to mDCs that display a greater ability to capture incoming virions, retain them in an infectious form in large vesicles within the cells, and transmit them to target CD4 þ T cells, 58, 59 thus augmenting viral dissemination in the lymphoid tissues and significantly contributing to HIV disease progression. Therefore, the viral dissemination that mDCs potentially mediates in vivo is powerful, as viral transmission through trans-infection does not rely on antigen presentation, many CD4 þ T cells being exposed to mDCs-exposing virus.
Recently, it has been suggested that HIV can exploit a preexisting exosome trans-dissemination pathway intrinsic to mDCs, thus allowing trans-infection of CD4 þ T cells. 60, 61 Exosomes are membrane vesicles of 30-100 nm in diameter and of endocytic origin, which are produced and secreted in vitro by living cells of diverse origin. They are involved in the stimulation of a specific immune response and they can transfer antigens from infected, tumoral, or antigen-presenting cells to mDCs, increasing the number of DCs bearing a particular antigen, thus amplifying the initiation of primary adaptive immune response. 62 Exosomes from DCs loaded with tumor-derived epitopes on MHC-I molecules are able to stimulate in vivo cytotoxic T lymphocyte-mediated anti-tumor responses, 63 and to indirectly activate in vivo naïve CD4 þ T cells through the exchange of functional peptide-MHC complexes between DCs through a trans-dissemination mechanism. 64 A recent study has shown that upon maturation, DCs are able to capture large amounts of HIV, HIV-gag-VLP, or exosomes, resulting in localization within a CD81 þ compartment, and efficient transmission of captured particles to target T cells in an envelope glycoprotein-independent manner. 65 This 'Trojan exosome pathway' 60 used by mDCs that allows HIV to move between cells in the absence of fusion events could have a prominent role in promoting viral dissemination.
DC-dependent activation of NK cells. NK cells are involved in early viral control and by interacting with DCs they have a crucial role of producing pro-inflammatory cytokines and lysing infected cells. In addition, they can interact with T cells and DCs to shape the magnitude and quality of adaptive immune responses. [66] [67] [68] Following tissue invasion by pathogens and subsequent initiation of inflammatory responses, NK cells migrate to lymphoid tissues in response to the chemokines IL-8 and fractalkine (CX3CL1). In a mouse model, Lucas et al. 69 showed that, in vivo, naive NK cells do not acquire effector function unless a priming step has occurred by contact with DCs in draining lymph nodes. The requirements for NK priming was independent of IL-12 and it included secretion of IFN-a by TLR þ cells, which induced upregulation of IL-15Ra and IL-15 expression by DCs, and trans-presentation of IL-15 by DCs to NK cells in the lymph node. NK cells are not fully activated during priming by DCs, as they do not spontaneously produce IFN-g or mediate cytotoxicity. Full activation of NK cells requires an additional contact with mDCs. This results in mutual activation of previously primed NK cells and DC, 70 and in the release of IFN-g by NK cells, thus contributing to antigen-driven T-cell activation. 71 Blocking of IL-12 abolishes DC-induced IFN-g secretion by NK cells, whereas membrane-bound IL-15 on DCs is essential for NK cell proliferation and survival 72 ( Figure 2 ).
NK-dependent maturation of DCs. NK/DC cross talk has an important role in the process of DC maturation. NK cells are involved in the positive selection of mature myeloid DCs that, after migration to secondary lymphoid compartments, induce priming of Th cells. 73, 74 Primed NK cells upregulate their cytotolytic function and they release cytokines such as TNF-a and IFN-g, which in turn promote the maturation program of DCs that have captured the antigen. [75] [76] [77] At this stage, NK cells acquire the capability to kill autologous iDC, an event that is dependent upon a process of NK cell activation involving the NKp30 receptor 78 and the TNFrelated apoptosis-inducing ligand (TRAIL)-DR4 pathway. 22 NK cells would spare those DCs that after antigen uptake express high levels of HLA class I molecules, whereas they would kill those DCs (recruited in inflamed tissues) that failed to undergo a full maturation. This process involves inhibitory CD94/NKG2A receptors specific for the non-classical HLA-E molecules. 74 mDCs are resistant to NK killing due to the upregulation of HLA class I expression including HLA-E. 79 mDCs also upregulate an array of additional surface molecules including CCR7, CD80, CD86 and HLA class II, which allow their migration to lymph nodes and their optimal interaction with T cells during their priming phase. Thus, during the early phases of inflammation, DC maturation is under the control of NK cells that have a major role in keeping in check the quality of DC undergoing maturation (Figure 2 ). HMGB1 at the crossroad between innate and adaptive immunity. HMGB1 is expressed at the synapse between NK cells and DCs (Figure 3) , and recent studies highlighted the pivotal role of this cytokine during NK-DC cross talk. Semino et al. 80 showed that NK cells trigger immature DCs to polarize and secrete IL-18 at NK-DC synaptic cleft, thus instructing NK cells to release HMGB1, which promotes DC maturation and protects DCs from lysis. Interestingly, the ability of different NK cell subsets to induce DC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from the nucleus to the cytoplasm, which is strongly enhanced by engagement of the surface molecule NKp30. 81 Moreover, in the presence of DC-derived cytokines, such as IL-12, a cooperation between NKp30 and DNAM-1 to induce NK cells to kill DCs, release TNF-a and promote DC maturation were evidenced. 82 HMGB1 shows a nuclear localization in primary resting NK cells sorted from the blood, and the NK cell activation induces HMGB1 relocalization from the nucleus to the cytosol followed by extracellular release. 21 During the cross talk between activated NK (aNK) cells and iDC, both NK cells and DCs express HMGB1 21 that appears essential for the upregulation of CD80, CD83 and CD86 maturation markers on DCs and for IL-12 production. The HMGB1 secreted during NK-DC cross talk is also essential for Th1 polarization of naïve CD4 T cells, and RAGE is required for HMGB1 effects on DCs. 43, 21 Moreover, the autocrine/paracrine release of HMGB1 is required for the upregulation on DCs of the CCR7 and CXCR4 chemokine receptors and their migration in response to the chemokines receptor ligands CCL19 and CXCL12, respectively, 40 and RAGE has a nonredundant role in DC homing to lymph nodes, as shown in mice by noninvasive imaging by magnetic resonance. 41 Overall, HMGB1-RAGE pathway is activated in DCs that are committed to maturation in peripheral tissues, and it controls the expression of chemokine receptors in DCs that acquire the ability to reach secondary lymphoid organs where they initiate the clonal expansion of Ag-specific T cells. The disruption of HMGB1-RAGE pathway by specific inhibitors 43, 21 or the genetic deletion of RAGE 41 interrupts this circuit, possibly limiting the initiation of T-cell adaptive response. . NK cells and DCs were stained with red and green Cell Trackers, respectively. During the coculture, one NK cell interacted several times with the DC (pointed out with a star), leading to the killing of the DC. The DC died by apoptosis, as shown by the blebs (indicated with the yellow arrows). This editing process occurred very rapidly, within less than 1 min following the kiss of death by NK cells. 22 (e) Mitochondria rearrangement at NK-DC synapse, detected with a green MitoTracker cells. This inhibitory effect of HMGB1 was caused by repression of LTR-mediated transcription. 83 Extracellular HMGB1 also showed a dichotomic effect in different cell types. Addition of HMGB1 to primary monocytes with active HIV-1 infection was reported to suppress viral replication, associated 19 or not associated 20 to HMGB1-mediated increased release of b-chemokines (RANTES, MIP-1a and MIP-1b), strong inhibitors of HIV entry. In contrast, extracellular HMGB1 increased HIV-1 replication in the chronically infected monocytic cell line U1, 19 a process that did not require de novo protein synthesis. 84 HIV-1 induction relied on HMGB1-RAGE interaction, involved p38, ERK and NF-kB pathway, and stimulated the release of TNF-a. 84 Interestingly, HMGB1 could reactivate ex-vivo quiescent HIV-1 from latently infected PBMC collected in aviremic HIV-infected patients. 84 Thus, HMGB1 may reduce viral replication in acute infection by inducing inhibitors of viral entry, but it may trigger viral replication in latently infected cells, including in cells from HIV-infected patients.
NK-DC cross talk contributes to HIV replication through HMGB1. The mechanisms involved in NK-DC interaction during viral infections are poorly understood. It was recently reported in murine CMV (MCMV) infection that MCMVinfected DCs were capable of activating syngeneic NK cells in vitro and also capable of enhancing NK-dependent clearance in vivo, 85 demonstrating the crucial role of NK-DC cross talk in controlling viral replication. In HIV infection, NK-DC interaction was found defective in viremic HIV-1-infected patients, characterized by abnormalities in the process of reciprocal NK-DC activation and maturation. 86 Recently, we investigated the impact of HIV-1 on NKdependent maturation and function of iDCs in an ex-vivo model of cross talk between purified primary NK cells and monocytes-derived iDCs. We discovered that maturation of HIV-1-infected DCs required aNK to occur and this process involved HMGB1. Blocking HMGB1 with specific antibodies or glycyrrhizin, a specific inhibitor of HMGB1, impaired maturation of infected DCs. However, the cross talk between HIV-1-infected DCs and aNK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs, known to trigger the adaptive response. 21 Moreover, the interaction between aNK and HIV-1-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. This process was mainly triggered by HMGB1, released both by NK cells and DCs, and blocking HMGB1 strongly inhibited HIV replication in both isolated infected DCs and DCs cocultured with aNK cells 21 (Figure 4) . Thus, these findings provide evidence for the crucial role of NK-DC cross talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS. NK-DC cross talk and HMGB-dependent HIV persistence in DCs. The NK cell-mediated editing process of DCs, which is required to keep in check the quality of DCs prone to mature and further present the antigen to T cells, is compromised during HIV infection. Indeed, NK cells from viremic patients show a decreased ability to kill immature DCs. 87 The defect is associated with an increase in the proportion of CD56 À NK cells with impaired NKp30 function. 86 In addition, increased production of IL-10 during HIV-1 infection can protect immature DCs from NK cellmediated lysis, resulting in accumulation of partially mature, poorly immunogenic DCs in the lymph nodes of infected individuals. 88 In an ex-vivo model of NK-DC cross talk, we showed that HIV-1-infected DCs become resistant to NK cellmediated lysis due to an upregulation in DCs of two apoptosis inhibitors, cellular FLICE-inhibitory protein (cFLIP) and cellular inhibitor of apoptosis protein 2 (c-IAP2). 22 The expression of these inhibitors was upregulated by HMGB1, released by aNK cells at NK-DC synapse, and they protected HIV-1-infected DCs from TRAIL-dependent apoptosis. 22 Blocking HMGB1 with specific antibodies restored the susceptibility of infected DCs to NK killing, and similar effect was observed knocking down c-FLIP or c-IAP2 by siRNA 22 (Figure 5 ). Overall, these findings suggest that impaired NK-DC cross talk during HIV-1 infection is a consequence of bidirectional alteration of both DC and NK cell functions, and they reveal the pivotal role of HMGB1 in HIV-1 persistence in DCs.
Extracellular HMGB1 is an important component contributing to tissue injury in acute and chronic inflammatory conditions. HMGB1 and its receptors RAGE, TLR2 and TLR4 have been implicated in mechanisms of many diseases, including cancer, sepsis, atherosclerosis, stroke, rheumatoid arthritis and many other inflammatory conditions. 89 Plasma levels of HMGB1 are elevated during the course of HIV-1 infection 17 and positively associated with high viral load. 90 HMGB1 can be passively released by virus-infected cells including primary CD4 T cells infected with HIV-1, and this was associated with both necrotic and apoptotic cell death. 91 HMGB1 can also be released by non-infected apoptotic CD4 T cells that die through a bystander killing process, which is mainly induced by extracellular HIV-1-encoded proteins and by HIV-1associated chronic immune activation. 46 Increased circulating HMGB1 levels detected in progressive HIV-1 infection, combined with microbial products and TLR ligands, may contribute to gut inflammation and subsequent microbial translocation, suggested to have an important role in HIV pathogenesis. 92 Microbial translocation is the leaking of normally friendly commensal bacteria from the gut -where they are usually contained -into the systemic circulation. Brenchley et al. 92 proposed that this phenomenon contributes to immune activation in patients with HIV, and thus has a causative role in the progression of the disease. Markers of microbial translocation found in the bloodstream include LPS and bacterial DNA, and the level of circulating LPS in the first year of chronic HIV infection is a strong predictor of disease progression independent of CD4 T-cell count and HIV viraemia. 93 The possible link between circulating LPS and HMGB1 levels in inflammatory conditions is suggested by following recent observations: (1) priming of macrophages with LPS induces the processing and releasing of HMGB1 in addition to IL-1b, IL-18 and TNF-a, which requires the inflammasome components ASC, caspase 1 and NALP3; 28 (2) conversely, NALP3 silencing has a protective effect in a murine model of liver ischemia-reperfusion injury, associated with decreased production of HMGB1, IL-1b, IL-18, TNF-a and IL-6; 94 (3) the resistance of caspase-1-deficient mice to LPS correlates with reduced serum HMGB1 levels. 28 (4) HMGB1 forms highly inflammatory complexes with LPS, and signals through the TLR4; 95 (5) an association of elevated circulating levels of LPS and high viral load was reported in HIV-infected patients. 90 Thus, HMGB1-LPS complexes may be important in perpetuating inflammatory amplification loops in HIV disease. Thus, HMGB1 by itself, or combined to LPS or other TLR ligand or cytokines, may induce a self-perpetuating cycle by contributing to immune activation that creates new T-cell targets for viral infection and subsequent increased rate of cell death and release of HMGB1, but also by stimulating HIV replication and viral persistence in DCs. This vicious circle may facilitate HIV disease progression and contribute to AIDS pathogenesis ( Figure 6 ).
HMGB1 is an endogenous danger signal that can be released into the extracellular milieu during states of cellular stress or damage, and that is also actively produced by innate effectors such as NK cells subsequently involved in promoting adaptive immunity. HMGB1 has a pivotal role during NK-DC cross talk but, in the context of a chronic viral infection such as that induced by HIV-1, it triggers viral replication in DCs and blocks NK-mediated killing of infected DCs, thus contributing to viral persistence. Increased circulating HMGB1 levels are detected in progressive HIV-1-infected individuals and, in combination with microbial products and TLR ligands, it may also contribute to gut inflammation and subsequently increase microbial translocation and chronic immune activation, a hallmark of HIV-1 disease progression. In order to get a better understanding on the role of HMGB1 in HIV disease and to develop interventions aimed at enhancing immunity to HIV-1, several major questions need to be addressed.
The authors declare no conflict of interest. Figure 6 Proposed contribution of HMGB1 to HIV persistence and dissemination. HIV disease progression is characterized by gut inflammation and consequently microbial translocation leading to the release of LPS in the bloodstream (1). Circulating LPS may induce active release of HMGB1 by innate cells, including macrophages and DCs (2) . Necrotic and apoptotic cells that accumulate during chronic HIV infection may also be a constant source of HMGB1 (3). HMGB1 activates DCs by signaling through the receptor RAGE or TLR4 if cooperate with TLR4 ligand LPS, thus triggering NF-kB activation (4) . This results in the release of HMGB1 (5) and proinflammatory cytokines (6) , and the triggering of HIV replication in mDCs (7). Trans-infection of HIV from mDCs to CD4 T cells involves HIV capture by DC-SIGN followed by its recruitment at the site of T-cell interaction (8) . This infectious synapse will lead to the productive infection of CD4 T cells (9) . Trans-infection of HIV can also be mediated by exocytosis of the HIV-1 particles captured by DCs. After endocytosis, the captured HIV-1 particles are targeted to a multi-vesicular endosomal body (MVB) in DCs (10) . Although some of the MVB-localized virus fraction is targeted to the lysosome and degraded to be further presented to TCR in the context of MHC molecules, fusion of MVB with the plasma membrane results in the release of virus particles along with exosomes (11) . Virus produced by infected CD4 T cells, DCs and macrophages spread the infection to the draining lymph nodes and other lymphoid tissue Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis BACKGROUND: Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. METHODS: Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. RESULTS: The lower detection limits ranged from 10(1) copies/µL to 5×10(1) copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. CONCLUSIONS: The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens. Bacterial meningitis is an infectious disease with high rates of mortality and severe sequelae. Early identification of causative bacterial and viral pathogens is important to enable prompt and appropriate treatment and thereby prevent life-threatening clinical outcomes. Patients with suspected meningitis are generally prescribed antibiotics before hospital admission or cerebrospinal fluid (CSF) collection for examination. However, the admin-istration of antibiotics may render it difficult to culture the causative bacteria, thereby impairing microbiological diagnosis [1, 2] . To counter this problem, alternative methods of molecular identification, including 16S/23S rRNA gene amplification followed by sequencing and real-time PCR, have been developed. However, the clinical application of such techniques is limited because of the presence of interfering bacterial or viral DNA, timeconsuming nature of these assays, and small number of detection channels available on the real-time PCR platform [3] [4] [5] [6] [7] .
http://dx.doi.org/10.3343/alm.2012.32. 1.44 www.annlabmed.org These limitations can be overcome by the use of multiplex PCR methods, which enable the rapid and accurate identification of up to 9 bacterial and viral pathogens in clinical samples [8] [9] [10] . We evaluated the diagnostic value of the newly developed Seeplex Meningitis ACE Detection kit (Seegene Inc., Seoul, Korea), a multiplex PCR kit that uses dual priming oligonucleotide (DPO) methods. This kit detects the 12 most common bacterial and viral pathogens of acute meningitis, namely, Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), Neisseria meningitidis (NM), Group B streptococci (GBS), Listeria monocytogenes (LM), herpes simplex virus (HSV)-1 and HSV-2, Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV-6), and human enterovirus (HEV).
The Seeplex Meningitis ACE Detection kit has 3 components: Seeplex Meningitis-B, which detects 5 bacteria (SP, HI type b, NM, GBS, and LM); Seeplex Meningitis-V1, which detects 6 viruses (HSV-1, HSV-2, VZV, EBV, CMV, and HHV-6); and Seeplex Meningitis-V2, which detects HEV. The Seeplex Meningitis-B and V1 assays amplify DNA, whereas the Seeplex Meningitis-V2 assay amplifies cDNA reverse-transcribed from viral RNA. The target genes amplified from the 12 strains are shown in Table 1 .
Each PCR amplification was performed using 5 μL of isolated nucleic acid solution, 2 μL of 10 × primer mixture, and 10 μL of 2 × Multiplex Master Mix (Seegene Inc.) in a total volume of 20 μL.
The amplification protocol was as follows: initial denaturation at 94°C for 15 min, 40 cycles of denaturation at 94°C for 30 sec, annealing at 63°C for 90 sec, and extension at 72°C for 90 sec. The amplified PCR products were electrophoresed in 2% (w/v) agarose gels and stained with ethidium bromide.
PCR products were cloned into the vector pUC19 (size, 2,686 bp). The cloned plasmids were harvested, DNA concentrations were measured, and the numbers of target copies were calculated (1 µg of a 1,000-bp DNA unit equals 9.1 × 10 11 copies). The sensitivity of the system was evaluated by amplifying 10-fold serial dilutions of each plasmid (concentrations, 10 4 to 10 -1 copies/20 μL), with distilled water as the negative control. Samples were PCR-amplified and electrophoresed in 2% (w/v) agarose gels. Analytical sensitivity was defined as the lowest template copy number for which amplified products were consistently [11, 12] . Bacterial meningitis was differentiated from viral or aseptic meningitis by a WBC count of more than 100 cells/mm 3 with neutrophil dominance and a ratio of CSF glucose/serum glucose level of less than 0.4. Cases with insignificant WBC count or discrepant results in different parameters were classified into the undetermined group. The median patient age was 27 yr (range, 1 month to 72 yr). For the verification study, we tested only the 78 samples collected at the time of diagnosis, i.e., before antibiotic therapy was initiated. Another set of 118 samples was used to assess discrepancies in positive results obtained from the same patients. Samples that were blood-colored, showed leakage, were aged over 12 hr, and strongly suspected of being contaminated (with discrepancies in the pathogens detected in consecutive tests), were excluded. All samples were assessed by conventional CSF microscopic analysis, staining, and culture. Control CSF samples were collected from 27 patients; of these 
Positive results were confirmed by conventional PCR amplification by using single primer pairs and subsequent sequencing. Amplicons were purified by using a PCR purification kit (Sol-Gent, Daejeon, Korea) and sequenced by using the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3730XL DNA analyzer (PE Applied Biosystems). Sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) provided by The National Centre for Biotechnology Information (http://www.ncbi.nlm.nih. gov/BLAST).
The detection sensitivity was the highest for GBS [lower limit of detection (LoD) of 5 genomes/mL] and the lowest for HI (LoD of 57 genomes/mL). The LoDs for HEV, EBV, VZV, HSV-2, HHV-6, NM, CMV, SP, HSV-1, and LM were 10, 11, 11, 12, 12, 13, 16, 19, 38, and 51 genomes/mL, respectively. The analytical sensi-tivities for all the 12 target pathogens were higher than the 100 copies per reaction stated by the manufacturer. No cross-reaction was evident with human genomic DNA and other reference strains. The Seeplex Meningitis-V2 ACE Detection kit successfully differentiated all the 19 HEV species from the other strains. CSF samples were obtained from 78 patients at the time of diagnosis of active meningitis and before administration of antibiotic treatment. Among these samples, 44 (56.4%) yielded positive results with the Seeplex kit (Table 3 ). Five cases (6.4%) showed positive results for HEV (4) and HSV-1 (1), which were consistent with the results of conventional studies. However, in 2 cases the results were not consistent with the results of conventional studies. One was positive for both LM and EBV in Seeplex Meningitis ACE detection kit, but positive only for LM in the conventional study; the other tested positive for HEV in Seeplex Meningitis ACE detection kit but positive for VZV in the conventional study. Each case presented a bacterial and a viral features, respectively in CSF analysis.
The Seeplex Meningitis ACE detection kit detected causative pathogens for 37 cases (47.4%) that tested negative in the conventional studies. Two patients had single bacterial infection with GBS and LM, and CSF analysis revealed bacterial and undetermined features, respectively. Single viral infections were evident in 16 patients with HEV, 9 with VZV, 3 with HSV-1, and 1 each with HSV-2 and EBV. All the cases with positive results for viruses showed viral or undetermined features in CSF analysis. Five patients showed simultaneous infection with 2 bacterial or viral pathogens: 2 tested positive for both SP and HEV and 1 each tested positive for SP and HSV-1, SP and VZV, and EBV and HSV-1. One of the cases positive for both SP and HEV had CSF findings compatible with bacterial meningitis, while the others had features consistent of viral meningitis, with lymphocyte dominance and high glucose level. Sequencing of amplicons showed no evidence of false-positive cross-reactivity; the rates of BLAST match were 98-100%. CSF samples obtained from the remaining 34 patients did not test positive with the Seeplex kit. Sequential work-up with conventional studies showed that 1 patient each was infected with EBV, VZV, SP, HEV, and HSV-1 and had consistent CSF findings. The pathogenic agents in the other 29 patients could not be identified, although CSF analysis indicated that 6 had bacterial infection, 13 had viral infection, and the remaining 10 had indeterminate findings. None of the control cases showed false-positive results.
Acute meningitis requires prompt treatment to avoid life-threatening clinical outcomes and severe sequelae. Since various viral and bacterial pathogens can cause acute meningitis, the causative agent must be identified before treatment is initiated. Conventional diagnostic tools, including CSF staining, culture, and antigen analysis, have limited diagnostic sensitivity and specificity. This has prompted the development of several molecular methods employing conventional, real-time, or multiplex PCR. Although previously developed multiplex PCR methods can rapidly identify up to 9 bacterial and viral agents, the Seeplex Meningitis ACE detection kit, which employs a DPO method, can identify the 12 most common bacterial and viral agents that cause meningitis: SP, HI, NM, GBS, LM, CMV, HEV, EBV, HSV-1, HSV-2, HHV-6, and VZV. DPOs, which consist of 2 separate priming regions joined by a polydeoxyinosine linker, yield 2 primer segments with distinct annealing properties. The long 5′-segment initiates stable priming, whereas the short 3′-segment controls target-specific extension; the use of these 2 primer segments effectively eliminates non-specific priming and yields consistently high PCR specificity even under less-than-optimal PCR conditions [13] . This method is highly appropriate for the identification and differentiation of viral and bacterial pathogens with very variable genetic characteristics and low availability of primer sites. Several multiplex PCR kits using the DPO technology have been developed to identify common viral and bacterial pathogens causing respiratory infections and diarrhea, and such kits are currently being used in clinical laboratories [14] [15] [16] .
We found that the Seeplex Meningitis ACE Detection kit offered high sensitivity and specificity for identifying bacterial and viral pathogens in patients diagnosed with acute meningitis. The LoDs for the 12 pathogens ranged from 10-50 copies/µL, and no cross-reaction with human genomic DNA or reference (clinically identified) bacterial and viral pathogens was evident, indicating the high specificity of the kit.
Our validation study revealed that the kit successfully identified pathogens in the CSF samples collected from 44 of the 78 (56.4%) patients who were clinically diagnosed with acute meningitis. Only 6 of the 44 pathogens identified using the Seeplex Meningitis ACE Detection kit were also identified by other laboratory methods, with the pathogen being HEV in 4 cases and HSV-1 and LM in the remaining 2 cases. Six CSF samples were simultaneously positive for 2 pathogens, suggesting co-infection or contamination rather than false-positivity, because subsequent sequencing of PCR products yielded 98-100% matches with sequences in the BLAST database. Of the 12 pathogens assayed, HEV had the highest prevalence and was detected in 23 samples by the Seeplex Meningitis ACE Detection kit. Of the bacterial strains, SP was the most frequently detected; SP was detected in 5 samples, although it was detected along with viruses, including HEV, HSV-1, and VZV, in 4 samples.
Five of the 34 patients, whose samples were negative at the time of diagnosis were later found to have EBV, VZV, SP, HEV, and HSV-1 infection when investigated by conventional studies. These discrepancies were perhaps attributable to inter-specimen differences at varying stages of infection. Of the other 29 patients who tested negative for all pathogens, 10 had indeterminate CSF parameters, whereas 19 showed findings consistent with bacterial or viral infection. The presence of clinical features of meningitis and indeterminate CSF findings in the 10 patients may be attributed to conditions other than acute viral or bacterial meningitis. In contrast, meningitis in the 19 patients with CSF findings consistent with bacterial or viral infections may have been caused by pathogens other than the 12 assayed by the Seeplex Meningitis ACE Detection kit. Alternatively, the concentration of pathogens in the CSF may have been too low to permit detection.
In conclusion, the Seeplex Meningitis ACE Detection kit showed high sensitivity and specificity for the 12 most common bacterial and viral agents causing acute meningitis. A high detection rate was observed in the CSF samples obtained from patients clinically diagnosed with acute meningitis. This kit may enable the rapid identification of pathogens in patients with acute meningitis. ColorPhylo: A Color Code to Accurately Display Taxonomic Classifications Color may be very useful to visualise complex data. As far as taxonomy is concerned, color may help observing various species’ characteristics in correlation with classification. However, choosing the number of subclasses to display is often a complex task: on the one hand, assigning a limited number of colors to taxa of interest hides the structure imbedded in the subtrees of the taxonomy; on the other hand, differentiating a high number of taxa by giving them specific colors, without considering the underlying taxonomy, may lead to unreadable results since relationships between displayed taxa would not be supported by the color code. In the present paper, an automatic color coding scheme is proposed to visualise the levels of taxonomic relationships displayed as overlay on any kind of data plot. To achieve this goal, a dimensionality reduction method allows displaying taxonomic “distances” onto a Euclidean two-dimensional space. The resulting map is projected onto a 2D color space (the Hue, Saturation, Brightness colorimetric space with brightness set to 1). Proximity in the taxonomic classification corresponds to proximity on the map and is therefore materialised by color proximity. As a result, each species is related to a color code showing its position in the taxonomic tree. The so called ColorPhylo displays taxonomic relationships intuitively and can be combined with any biological result. A Matlab version of ColorPhylo is available at http://sy.lespi.free.fr/ColorPhylo-homepage.html. Meanwhile, an ad-hoc distance in case of taxonomy with unknown edge lengths is proposed. Many datasets are "naturally" structured as hierarchical classifications. In particular, the Darwin's evolution theory ensures that the relationships between species can be expressed within a tree (named "phylogenic tree"/"taxonomic tree"). However, the exploration of large trees (and graphs) is not easy. 1 Visualising taxonomy together with other pieces of information (obtained from various biological analyses for example) is often extremely instructive; some examples can be found in. [2] [3] [4] In those cases, a level of granularity of the tree is usually chosen and a color is assigned to each subclass thus defined. 4 Several drawbacks can be easily identified. The granularity level is obviously subject to arbitrariness. Moreover, proximity relationships between subclasses are ignored as well as their subdivisions.
In the following, we set up an automatic coloring method in order to address these problems. Indeed, we describe a simple method that automatically generates an intuitive color code showing proximity relationships between data in any hierarchical classification. The presented algorithm, named ColorPhylo, associates a specific color to each item so that the taxonomic relationships are shown by color proximity (the closer two items in the tree, the more similar their colors). Colors can thereafter be used in any user's analyses and figures so as to display taxonomy.
The above research field is yet relatively unexplored. An automatic coloring tool for tree exploration has been proposed by Fua and co-authors: 5, 6 in order to explore a given taxonomy, leaves are considered as an ordered list of items (the order can result for example from the reading direction when the tree is displayed on a phylogram). Each node can then be related to a color according to a chosen LUT (Look-Up Table) . The LUT may be locally stretched or compressed in order to focus on a given area. Such an interactive control of the LUT allows exploring the taxonomy while considering various depths. This approach suffers however from a major shortcoming: the leaf ordering is not unique (trees are invariant with respect to permutation of linked branches). Very different colorings can then result from various choices. Moreover, close colors can correspond to items that belong to different branches and somewhat different colors can rely to close species. These drawbacks can be observed in Figures 6 and 8 .
In the biological field, several tools may be used to color species in taxonomic tree. 4, 7, 8 In particular, PhyloView 4 has been specially designed to display taxonomy and other analyses (phylogenetic trees) together. Strictly speaking, however, these tools are not automatic coloring tools: the user has to define the colors for taxonomic groups that are relevant from his point of view.
Node coloring is of main concern in Self-Organising Map (SOM) framework. 9,10 SOM is a non-linear dimensionality reduction method. It is designed to map items (generally from a high dimensional Euclidean space) onto a discrete grid: data are aggregated on each neuron (vector quantisation). Because the grid is discrete, the visualisation of gaps between clusters-if they exist-is uneasy. Various coloring methods have been proposed to account for this drawback, including. [11] [12] [13] [14] [15] [16] [17] In particular, Kaski et al 14, 15 display neurons in a CIELab colorimetric space; 16 Johan Himberg 17 uses a hierarchical classification in order to find clusters and use a cut-off function in the resulting tree so as to linking neurons to a LUT (thanks to a method close to the Fuas' one). 5, 6 However, the SOM context differs from the one of our present objective, despite approaches that are connected. Indeed, we focus here on taxonomies and SOM deals with multidimensional data. Moreover, if hierarchical classifications are often involved in automatic coloration of SOM, the tree is not the input of the algorithm, but a step in the method (especially in the context of cluster discovering). Such methods can be considered, however, as sources of inspiration for the present paper.
One of the examples used in the present paper (see section 3.2) is based on the Genbank taxonomy (http://www. ncbi.nlm.nih.gov/sites/entrez?db=taxonomy). Please note that the Genbank taxonomy can not be considered as a conclusive phylogeny, as clearly stated by Genbank owners (http://www.ncbi.nlm.nih.gov/Taxonomy/ taxonomyhome.html/index.cgi?chapter=howcite). We are only looking here for a way to display any given hierarchical classification, whether it is considered as a phylogeny, a taxonomy or so.
ColorPhylo aims at assigning a unique color to each species of a given set so that the color differences reflect the taxonomic "distances" between species (see Fig. 1 ):
Step 1: If not available, taxonomic distances are calculated from the taxonomic tree. Two procedures are considered depending on whether the edge lengths are known or not (see section 2.2). Thus, a distance matrix between all species is obtained.
Step 2: Species are mapped onto a 2D space while preserving the distance matrix as much as possible (section 2.3).
Step 3: The map is rescaled (and possibly rotated) (section 2.4) in order to fit a 2D colorimetric subspace (section 2.5).
Step 4: Each species located in the colorimetric subspace is consequently given a unique color. The 
Relation to the colorimetric space each species gets its own color … where the color-code is used. species coloring can therefore be used in subsequent analyses to visualise the taxonomic relationships (as done in section 3).
The generation of a color-code for hierarchicallyorganised data.
The presented methodology relies on the availability of a distance based on a taxonomic tree.
Two cases are possible:
I. Edge lengths are known. Distances between species can be immediately deduced: the distance between two species corresponds to the sum of the length of edges connecting them. The "bananaquit bird" dataset offers a good illustration of that case (section 3.1). II. Edge lengths are unknown (the GenBank taxonomy dataset: section 3.2). We then have to (arbitrary) choose the length of edges. Many solutions can be considered. One may assign the same length for all edges. However, such a choice is inappropriate when the tree depth depends on the level of refinement along branches. Such a situation is rather common in taxonomy: the Eukaryote classification is actually more refined than that of Bacteria and Achea (Fig. 2 ). If the length of edges is set constant along the tree, humans appear closer to some bacteria (which are microscopic prokaryotes) than to octopus (which is also an animal). The toy example presented in Figure 3 , left insert (a) provides a good illustration of this bias.
In fact, as far as the tree of life is concerned, the distance between two species belonging to the same subclass (branch) must always be smaller than the distance between one of these species and any species that does not belong to the subclass. In order to ensure that this property is always true, we propose to attribute a variable length for edges: an important length is given to edges close to the tree root, and the length is successively reduced when the edge gets away from the root. A geometric progression can be implemented here. The length of edges at the tree root is 1. The length of any other edge equals half the length of the parent edge (Fig. 3 , right insert (B)).
This arbitrary choice has a critical property: Proposition: The distance between two species belonging to the same subclass is always smaller than the distance between one of these species and any species that does not belong to the subclass.
Proof: Let us consider three species A, B and C where A and B belong to a same subclass, contrary to C: species A and B are linked by node N 1 , and species The DD-HDS projection in the two-dimensional output space is computed (code available at http://sy.lespi. free.fr/DD-HDS-homepage.html). A DD-HDS parameter (λ) can be adjusted in order to more or less emphasize smallest distances: 0 (1 respectively) for minimum (maximum respectively) consideration of longer distances. In the following examples, λ is set to 0.5. The output of DD-HDS is the set of coordinates in a 2D space (coordinates
It is worth noting that additive distances (distances in a tree) cannot be perfectly preserved onto a 2D space. However, this fact is not a drawback in the present case because we are less concerned by distances preservation than subclasses segmentation. Indeed, even if the distance mapping is not perfect, it still emphasizes the various levels of the classification thanks to DD-HDS that avoids both "false neighbourhoods" (when a small distance in the output space becomes a large distance in the data space) and "tears" (when a large distance in the output space becomes a small distance in the data space). 19 
After being mapped, data are translated and rescaled so as to occupy a circle having a radius equal to 1. This step is required in order to ensure that the map can be related to the circle-shaped HSB colorimetric space (see section 2.5). Coordinates are subsequently cen-
, ,
″ ″ + and its angle is ϕ ρ
0  and ϕ ρ otherwise. Rotation around the centre and symmetry can take place now. Rotation and symmetry does not impact MDS solutions but allow modifying the color map at will while preserving relative color differences between species (for example, it may be useful to make colors close to the ones the user is familiar with).
Each position in the mapping can now be related to a color according the HSB (Hue, Saturation, Brightness) colorimetric conic-shaped space (The HSB space is also called HSV space (V for Value) or HSL space C is linked to node N 1 by node N 2 (similarly to the example in Fig. 3) . d(x, y) corresponds to the distance between elements x and y in the tree. According to the properties of geometric progression with a common ratio equal to 1/2 (basically,
Thus, the contribution of edges far from the tree root is reduced, which makes it possible to emphasize most important classes.
It must be pointed out that such a procedure should not be considered as a way to assess "true" edge lengths but rather as a heuristic method to account for classes and subclasses within the actual automatic coloring procedure.
Whatever the case, the next steps starts from the resulting matrix of taxonomic distances (noted d thereafter).
Non-Linear Multi-Dimensional Scaling (MDS) is a set of methods designed to show relationships between items. The explored dataset is displayed as points on a low-dimensional output space. Most of the time, the output space is a Euclidean 2D space, which is proposed as a "map" of data. These techniques are particularly used to explore high dimensional data. Indeed, MDS techniques are expected to retrieve the spatial organisation of high dimensional dataset. To achieve this goal, linear, as well as non-linear Multi-Dimensional Scaling methods preserve the distances between data "as much as possible", according to criteria depending on the method. 18 Usually, small distances are emphasized.
As far as our data are concerned, MDS methods are consequently expected to display taxonomic relationships (a distance matrix) onto small dimensional spaces. To achieve this goal, we have chosen the DD-HDS algorithm (Data-Driven High Dimensional Scaling), 19 for its efficiency whatever the distribution of input distances.
Evolutionary Bioinformatics 2011:7 (L for Lightness)). The HSB space relates each position in a cone to a color:
• Hue corresponds to the angle on a circle (the base of the cone) where 0° is pure red. • Saturation corresponds to the distance to the centre of the circle: 0 for 0% of saturation (pure white), 1 for 100% (pure color). • Brightness corresponds to position on the axe of the cone. No brightness (black) at the cone tip to full brightness at the base.
Brightness is not used in the present framework (Brightness is set to 1).
A position in a circle is characterised by hue and saturation values (Fig. 4) . Species can subsequently be given a color, norm (ρ i ) being related to saturation and angle (ϕ i ) being related to hue. The continuity of color in the HSB space ensures that color proximity is related to taxonomic proximity Figure 7 (section 3.2).
Angle corresponds to hue while distance to centre corresponds to saturation.
In the automatic coloring of SOM framework, Kaski et al 14, 15 propose to embed data in a CIELab colorimetric space. 16 Properties of this colorimetric space also fit very well with our context: indeed distance between positions in the CIELab colorimetric space is supposed to be linked with the human perception of color difference. However, significant constraints are placed on the mapping due to the particular shape of the CIELab space. In contrast, the mapping on circleshaped HSB sub-space is easier. Most of the times, data can be expected to fill a large part of the space after a simple rescaling procedure. Moreover, the mapping can be easily rotated and returned in the space in order to find the most convenient color code.
It is worth noting that a three-dimensional mapping (such as RGB space) may offer in some instances a better fit with the matrix of distance. However, every color would be eligible, and a species color could be close (or similar) to the background color. With the two-dimensional mapping related to HSB colorimetric space, no species can correspond to grey, which can be chosen as background color.
ColorPhylo is tested on two biological applications: the first one refers to a published ornithological study, 20 the second one is related to the analysis of genomic signatures (DNA sequences characterised by oligonucleotide frequencies).
It has to be remembered that, on the one hand we study specified knowledge (or analysis results) related to a given dataset (here, birds' geographical positionfirst example-and oligonucleotide counts-second example-) and on the other hand, a taxonomy (the tree) of these data is available. In the present paper, we aim to display the taxonomy and the knowledge together. The procedure described in section 2 provides a taxonomic color-code used here to color geographic position of birds or genomic signatures.
Application to the work of E. Bellemain and co-authors: linking geographical distribution and phylogenetic data for bananaquit birds Bellemain and co-authors performed a phylogenetic analysis of bananaquit birds (Coereba flaveola) caught in various places in Latin America and Caribbean islands. 20 Geographical positions of catching sites are displayed (in their Fig. 1 ) and the genetic analyses led to two phylogenetic trees (their Figs. 2 and 3) . Relationships between geographical origin of birds and taxonomy are discussed, and conclusions about the evolutionary history are made. Despite that this work has been successfully achieved without the help of any visualisation tool, a coloring based on the taxonomic tree would have considerably helped these authors.
In the following, ColorPhylo is used to assign a color to each bird according to its position in the taxonomic Saturation Hue Figure 4 . hSB colorimetric 2D sub-space (Brightness is set to 1). tree (based on combined mitochondrial data, Figure 2 in) . 20 Here, edge lengths (provided by the phylogenetic analysis) are known and used (see section 2.2, subsection I). Birds are displayed as colored dots, positioned on the map on their catching site (Fig. 5) .
Dot coloring allows easy observation of the genetic groups: Lesser Antilles and Puerto Rico (green dots), Bahamas and Quintana Roo (orange dots), continent (purple dots), and Greater Antilles except Puerto Rico (red dots) as well as smaller relationships within groups.
We performed the analysis on the same dataset, according to the Fua et al 5,6 coloring method (Fig. 6) .
It must be pointed out that the interactive local stretching and compression of the LUT-an essential feature of the Fua et al method-cannot be implemented here (obviously impracticable on a printed document!). For that reason, a comparison between ColorPhylo and the Fua et al algorithm from the present figures exclusively would be highly unfair. However, there are noticeable differences between Figures 5 and 6 . A high diversity in the population of birds from Lesser Antilles and Puerto Rico could be inferred from figure 6 right insert, whereas it is not supported in fact by the evolutionary tree (Fig. 6 , left insert). Moreover, the high difference between birds 
Confrontation of geographical positions of birds and phylogenetic analysis Figure 6 . Analysis of bananaquit birds data, similar as the one presented in Figure 1 and Figure 5 , except that the Fua et al 5, 6 coloring method is used rather than colorPhylo. from Lesser Antilles and birds from Bahamas and Quintana Roo is clearly underestimated.
Dot coloring allows easy observation of the genetic groups: Lesser Antilles and Puerto Rico (green dots), Bahamas and Quintana Roo (orange dots), continent (purple dots), and Greater Antilles except Puerto Rico (red dots) as well as smaller relationships within groups.
Coloring according to the Genbank tree: Species paths are from Genbank so that "root, cellular organism, Eukaryota, Fungi/Metazoa group, Metazoa; Eumetazoa, Bilateria, Coelomata, Deuterostomia, Chordata, Craniata, Vertebrata, Gnathostomata, Teleostomi, Euteleostomi, Sarcopterygii, Tetrapoda, Amniota, Mammalia, Theria, Eutheria, Euarchontoglires, Primates, Simiiformes, Catarrhini, Hominoidea, Hominidae, Homo/Pan/Gorilla group, and Homo Sapiens" qualifies human for example. Paths are derived from a rooted tree where edge lengths are unknown. Taxonomic distances are calculated according to the procedure described in section 2.2, subsection II. Colors are then selected according to section 2.3, 2.4 and 2.5. Data embedded in the colorimetric space are displayed in Figure 7 .
Species are embedded in a two-dimensional space generated from the taxonomic distance matrix. Colors are assigned from the position in the mapping (by construction, colors are smoothly Contrasting with figure 7 , the three domains of life are not clearly segmented by color in Figure 8 .
An example of color code use: Study of the link between taxonomic proximity and genomic signature proximity: The whole set of short oligonucleotide frequencies observed in a DNA (Deoxyri-boNucleic Acid) sequence is species-specific and is thus considered as a "genomic signature". 2,21 Moreover, a DNA segment as short as 1 Kb (kilobase) is sufficient to characterise the genomic signature of the species. As a consequence, genomic signature appears qualifying the "writing style" of the species. The genomic signature is species specific:
it allows finding the species of origin of a DNA fragment with a fairly good efficiency. 2, 22 Note that because the genomic signature is stable along the genome, non-homolog fragments of species can be compared (homology is required for most other methods devoted to comparative genomics). Lastly, proximity between species in terms of genomic signature is known to be linked to evolutionary proximity. Indeed many phylogenies based on hierarchical classifications of signatures have been proposed. [23] [24] [25] [26] [27] [28] Another powerful approach to describe the taxonomic organisation of genomic signatures uses dimensionality reductions: data are embedded on a two-dimensional (or sometimes three-dimensional) space. These representations have been achieved by Principal Components Analysis (PCA), 2,3 by Self Organising Map (SOM) 29 or by Data-Driven High Dimensional Scaling (DD-HDS). 19 However, details of the spatial organisation of genomic signatures cannot be easily observed on a large dataset because of the limitation in term of discrete colors. In the following example we propose to visualise the taxonomic tree of species on the DD-HDS mapping of genomic signatures by means of the color code provided by ColorPhylo and Figure 7 .
Prior to mapping, signatures are corrected using 1-order Markov model 30 as recommended in. 25, 27 A distance matrix between signatures is then obtained, based on the Pearson's correlation coefficient as recommended by. 26 The mapping is subsequently generated: spatial proximity between items expresses proximity between species from the genomic signature point of view. Colors are provided by the ColorPhylo procedure (the map of species in the related taxonomic color-space is shown in Fig. 7) : color proximity between items relies on taxonomic proximity between species. As a result, the various levels of taxonomy are simultaneously observable on a single figure (Fig. 9) . The taxonomic organisation of signatures is clearly demonstrated. In particular, the patches of homogeneous colors support concluding that the similarity between genomic signatures accurately matches the taxonomic tree of the species, the signatures come from.
Using ColorPhylo is straightforward. Hierarchical classifications can be easily displayed together with their relationships with any other organisation of the data. We have observed on real life examples that the interpretation of the resulting color code is fully intuitive. In the first example, geographical origins of bananaquit birds are related to phylogenetic data in order to analyse the evolutionary history. However, because the number of items and the complexity of the dataset was somewhat low, Bellemain and co-authors succeeded in describe the relationships between taxonomy and geographical distribution in their publication (but at the price of more irksome work). When the size of the dataset and/or the complexity of their relationships increase, ColorPhylo can provide a critical benefit, as it can be observed for the second application. In that example, (genomic signature) colors express the membership of items to one of the three domains of life, with subtle shades showing subclasses. In both cases, we have instantaneously access to the structure of classification through an attractive visualisation plot.
Although variations of color are theoretically unlimited, we rely on the perceptual discriminative power of the human eye. Surprisingly, the method gives access to a remarkable degree of detail (well above what is expected with a "manually" defined color code). In addition, a focus on a small region of the data is always possible by an ad hoc local color reallocation. Similarly, Colorphylo may be adapted to fit color-impaired users' requirements. The 2-dimensional color-space can be modified at will according to any desired effect, including of course satisfaction of the user's color perception.
In this paper, we have proposed a method to study the relationship between a given knowledge on a set of data (here, the birds' geographical position and the oligonucleotide frequencies in DNA sequences of species) and a specific organisation of these data, expressed by a taxonomic tree. Our approach can easily be adapted to other contexts. For example, if the organisation of the data results from an analysis based on a distance matrix (such as the ones performed by Neighbor joining, 31 Fitch-Margoliash, 32 …), the original distance matrix may be preferred to the taxonomic distance (such an approach may have been implemented for the bananaquit birds dataset). In fact, the procedure may be extended to the analysis of any kind of organisation of the data, given it is expressible as a distance matrix for which a 2D mapping makes sense.
A matlab version of ColorPhylo is available at http://sy.lespi.free.fr/ColorPhylo-homepage.html.
SL and BF conceived the method together and wrote the manuscript. Both authors read and approved the final manuscript.
We thank Eva Bellemain and collaborators, as well as Biomed Central, for the permission to reproducing their figure. We also thank Mikael Cugnet for its useful comments.
Author(s) have provided signed confirmations to the publisher of their compliance with all applicable legal and ethical obligations in respect to declaration of conflicts of interest, funding, authorship and contributorship, and compliance with ethical requirements in respect to treatment of human and animal test subjects. If this article contains identifiable human subject(s) author(s) were required to supply signed patient consent prior to publication. Author(s) have confirmed that the published article is unique and not under consideration nor published by any other publication and that they have consent to reproduce any copyrighted material. The peer reviewers declared no conflicts of interest. 
In the Bananaquit bird example, a very similar color has been given to birds from all great Antilles Islands. We subsequently run ColorPhylo again while focusing on the Antilles Islands subclass. The result is displayed on Annexe Figure 1 . The tight matching between phylogeny and geographical data is demonstrated in details by the color code. publish with Libertas Academica and every scientist working in your field can read your article "I would like to say that this is the most author-friendly editing process I have experienced in over 150 publications. Thank you most sincerely."
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"LA is different, and hopefully represents a kind of scientific publication machinery that removes the hurdles from free flow of scientific thought." Your paper will be: • Available to your entire community free of charge • Fairly and quickly peer reviewed • Yours! You retain copyright http://www.la-press.com Deconstructing host-pathogen interactions in Drosophila Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. As a result, Drosophila can be used to deconstruct many of the key events in host-pathogen interactions by using a wealth of well-developed molecular and genetic tools. In this review, we aim to emphasize the great leverage provided by the suite of genomic and classical genetic approaches available in flies for decoding details of host-pathogen interactions; these findings can then be applied to studies in higher organisms. We first briefly summarize the general strategies by which Drosophila resists and responds to pathogens. We then focus on how recently developed genome-wide RNA interference (RNAi) screens conducted in cells and flies, combined with classical genetic methods, have provided molecular insight into host-pathogen interactions, covering examples of bacteria, fungi and viruses. Finally, we discuss novel strategies for how flies can be used as a tool to examine how specific isolated virulence factors act on an intact host. Drosophila has emerged as an important model for examining the function of genes that are relevant to diverse human diseases affecting a broad range of cell types (for reviews, see Bier, 2005; Bier and McGinnis, 2008) . Additionally, this model organism can serve as a host for a surprising variety of bacterial and viral pathogens. Seminal discoveries in the field of host-pathogen interactions have been made in Drosophila. For example, the Toll signaling pathway, which plays a central role in innate immunity, was first identified in Drosophila, and studies using this model organism have helped to identify and delineate fundamental conserved host genetic pathways involved in barrier formation and maintenance (Mace et al., 2005; Martin and Parkhurst, 2004; Pearson et al., 2009; Ting et al., 2005) , innate immune signaling (Agaisse and Perrimon, 2004; Dionne and Schneider, 2008; Ferrandon et al., 2007; Igaki et al., 2010; Ryu et al., 2010) , the RNA interference (RNAi) response (Sabin et al., 2010) , pathogen engulfment (Meister, 2004) , and the evolution of intracellular pathogens (Haselkorn, 2010; Serbus et al., 2008) . As discussed in detail below, Drosophila has also been used to identify and analyze the function of pathogen-derived virulence factors (Avet-Rochex et al., 2005; Avet-Rochex et al., 2007; Botham et al., 2008; Guichard et al., 2010; Guichard et al., 2006; Shelly et al., 2009; Guichard et al., 2011) .
Many genetic tools are available to address the mechanisms of pathogen action in Drosophila. These include comprehensive genetic screens, or genome-wide RNAi screens, in cell lines and intact flies that can identify host pathways required to defend against pathogens. Reciprocally, it is also possible to search for pathogen-encoded factors that are required for virulence in flies. In vivo studies in flies are greatly facilitated by the ability to direct expression of transgenes encoding host or pathogen proteins in specific cell types using the GAL4-UAS transactivation system (Brand and Perrimon, 1993) or the new independently acting LexA system, which allows for combinatorial expression of genes in distinct or overlapping patterns (Yagi et al., 2010; Pfeiffer et al., 2010) . Moreover, it is possible to perform epistasis experiments using combinations of dominant and recessive mutations (or mutants with opposing phenotypes) in a given pathway to determine the sequence in which genes act in that pathway. These versatile tools, combined with the rapid Drosophila life cycle, allow detailed genetic analysis of virulence factors that act on tissues or organs; such experiments would be much more difficult to conduct in vivo in mammalian systems.
In this review, we first outline the basic host defense mechanisms used by Drosophila to resist and respond to invading pathogens to provide context for our discussion of how flies can be used to deconstruct key mechanisms of host-pathogen interactions. We then focus on three related topics: (1) genome-wide RNAi screens in Drosophila cell lines infected with pathogens to identify host pathways for defense or that are exploited by pathogens (e.g. bacteria, fungi, viruses); (2) classical genetic and RNAi screens conducted in intact flies to delineate host defense pathways that are active in specific tissues (e.g. the gut) or to identify important virulence factors produced by the pathogen; and (3) analysis of the function of specific pathogen virulence factors in an intact organism. The studies reviewed here highlight the speed and power of Drosophila genetics for uncovering new pathways and factors in host-pathogen interactions, as well as for characterizing unknown activities of specific virulence factors. Identification of such elements in the host-pathogen relationship should help to guide studies in vertebrate systems and contribute to defining new targets for potential therapeutic intervention.
Host-pathogen interactions in Drosophila PERSPECTIVE and an internal component comprising the gut (or endoderm). The formation of both the outer epithelial barrier and the inner intestinal barrier depends on the formation and maintenance of intercellular junctions, and many basic discoveries in this field have been made in Drosophila (Banerjee et al., 2006; Furuse and Tsukita, 2006; Wirtz-Peitz and Zallen, 2009 ). Such studies have delineated key mechanisms involved in establishing apical-basal polarity, including the assembly of distinct protein complexes at adherens junctions and septate junctions (claudin-dependent junctions that share important similarities with vertebrate tight junctions). One highly conserved feature of this process is the role of the exocyst protein complex in trafficking proteins such as cadherins and cell signaling components to adherens junctions (Andrews et al., 2002; Beronja et al., 2005; Blankenship et al., 2007; Jafar-Nejad et al., 2005; Langevin et al., 2005; Mehta et al., 2005; Murthy et al., 2003; Murthy et al., 2005; Murthy and Schwarz, 2004; Murthy et al., 2010) .
At first glance, the mammalian epidermis seems very different from that of flies ( Fig. 1A ), but there are striking parallels with respect to the formation and maintenance of epithelial barriers in the two species, illustrating a probable common ancestral origin. For example, claudin-family proteins forming the tight junctions between epithelial cells seem to have similar functions in both flies (Behr et al., 2003; Nelson et al., 2010; Wu et al., 2004) and mice (Furuse et al., 2002) [see the 2009 article by Furuse for a review on the role of claudins and other tight junction proteins in mammalian epithelia (Furuse, 2009) ]. Similarly, the transcription factor Grainyhead (Grh) plays an important role in regulating the expression of genes that are required to form the cross-linked outer epidermal surface both in flies (Bray and Kafatos, 1991) and mice (Matsuki et al., 1998; Ting et al., 2005) (although the set of Grh target genes seems to be different in each species). Grh also regulates genes that are involved in wound repair both in flies (Mace et al., 2005) and mice (Ting et al., 2005) .
It is noteworthy that Drosophila and vertebrate intestinal epithelia are also similar in several respects. These parallels include: the fact that stem cells play an important role in replacing cells that have undergone pathogen-dependent apoptosis; the sequential deployment of Wnt and Hedgehog (Hh) signaling during the differentiation of intestinal epithelial cells (Pitsouli and Perrimon, 2008; Takashima et al., 2008) ; and the formation of the morphologically specialized brush border microvilli and the underlying cytoskeletal terminal web (Li et al., 2007; Morgan et al., 1995; Phillips and Thomas, 2006) .
A challenge faced by intestinal cells is that they must tolerate commensal bacteria, with which they have a mutualistic relationship (Backhed et al., 2005; Dale and Moran, 2006; Sansonetti and Medzhitov, 2009) , while also mounting a vigorous response to pathogens (for a review, see Ryu et al., 2010) . One important pathway involved in this distinction controls the production of reactive oxygen species (ROS) by the dual-oxidase (Duox) transmembrane protein (Ha et al., 2009; Ha et al., 2005a; Ha et al., 2005b) , which also plays a key role in the human gut (for a review, see Ryu et al., 2010) . Genetic analysis in Drosophila has revealed bi-stable control of Duox activity in the gut. In the presence of commensal bacteria and absence of pathogenic species, low-level activation of the immune deficiency (IMD) pathway of the innate immune system (see later) induces negative feedback of the Duox pathway (at both the level of expression and activity), resulting in low basal levels of ROS production. By contrast, when invading pathogens are detected by host immune signaling, expression and activity of Duox components is greatly increased, leading to destruction of the pathogenic bacteria (Ha et al., 2009) .
The inducible ROS-producing Duox system works in parallel with other immune pathways, such as the Jun N-terminal kinases (JNK) pathway. JNK signaling is activated in intestinal epithelial cells of adult flies following ingestion of pathogenic Pseudomonas aeruginosa , which leads to proliferation of intestinal stem cells to compensate for apoptotic loss of mature infected cells (for a review, see Pitsouli et al., 2009 ). An interesting aspect of this pathogen in Drosophila is that, in combination with an activated oncogenic form of RAS, it can lead to overproliferation of stem cells to form tumors . Whether the elevated incidence of human cancers of the intestinal tract as a result of associated bacterial infection (Bornschein et al., 2009; Selgrad et al., 2008) is similarly influenced by RAS activation remains to be determined. Another interesting emerging theme is the elucidation of host pathways involved in detecting cell damage in the intestine, which then regulate stem cell mediated repair of the damaged epithelium. These studies have revealed important contributions of the insulin (Amcheslavsky et al., 2009 ) and TSC-TORC1 (Amcheslavsky et al., 2011) pathways, as well as of Hippo (Hpo)-mediated activation of the JAK-STAT and Epidermal growth factor receptor (EGFR) pathways (Ren et al., 2010) . Finally, experiments involving oral infection of flies with Erwinia carotovora, a natural Drosophila pathogen, suggest that gut homeostasis is maintained by active tissue repair of cell damage caused by bacteria (Buchon et al., 2009) . The observation that ROS can trigger apoptosis followed by repair in the larval gut (Gupta et al., 2010) suggests that the Duox pathway provides compensatory feedback to pathways controlling apoptosis and stem cells to ensure that host cells damaged by ROS exposure are duly replaced. Overall, these studies provide an excellent foundation for further analysis of how the gut responds to pathogens by repairing damage and differentially responding to commensal versus pathogenic bacteria.
Broadly speaking, the innate immune response consists of three parts:
(1) pathogen immobilization (Fig. 1B) , (2) core immune signaling pathways (Toll, IMD and JAK-STAT) (Fig. 1C) and (3) the RNAi pathway (Fig. 1D ). Because there have been several excellent reviews describing these pathways, we only summarize here their key elements, as depicted in Fig. 1 , and refer the reader to other sources for more in depth descriptions (Agaisse and Perrimon, 2004; Akira et al., 2006; Bhavsar et al., 2007; Brodsky and Medzhitov, 2009; Diacovich and Gorvel, 2010; Dionne and Schneider, 2008; Ferrandon et al., 2007; Folsch et al., 2003; Sansonetti, 2008) .
The most basic innate response to bacterial or fungal infection is a cellular response (Jiravanichpaisal et al., 2006) that immobilizes the invading microbe by phagocytosis, engulfment or a Host-pathogen interactions in Drosophila PERSPECTIVE melanization reaction that traps it (Fig. 1B) . Pathogens can also be immobilized in flies and other insects by a clotting reaction (Dushay, 2009) . Once immobilized, the pathogen can then be either destroyed extracellularly by antimicrobial peptides (AMPs) or eliminated intracellularly. Three basic types of Drosophila blood cell (known as hemocytes) perform these functions: plasmatocytes, which are professional phagocytic cells akin to mammalian macrophages; lamellocytes, which wrap themselves around invading microorganisms to form an enveloping capsule; and crystal cells, which contain the enzymes that catalyze melanization (Meister, 2004) (Fig. 1B) . As discussed later in more detail, many host genes that are required for phagocytosis have been identified using Drosophila in a series of genome-scale cell-based screens. Similar studies in the future might shed light on genes that are essential for lamellocyte and crystal cell function. Autophagy is another general mechanism important for clearing bacteria (Yano et al., 2008) and viruses (Cherry, 2009; Shelly et al., 2009) . It should be pointed out, however, that autophagy can also be hijacked for the benefit of the pathogen, as in the case of poliovirus, which derives its envelope membranes from autophagic vesicles (Suhy et al., 2000) .
The second part of the Drosophila innate immune response comprises a set of core signaling pathways (Fig. 1C) : the Toll pathway, the IMD pathway and the JAK-STAT pathway. The activities of these pathways are modulated by other pathways, such as that mediated by target of rapamycin (TOR) or Eiger-Wengen [Drosophila homologs of human tumor necrosis factor (TNF) and TNF receptor]. When induced following pathogen infection, innate immune pathways result in the production of AMPs such as Drosomycin and Diptericin (Dionne and Schneider, 2008; Ferrandon et al., 2007; Agaisse and Perrimon, 2004; Dionne and Schneider, 2008; Ferrandon et al., 2007; Folsch et al., 2003) .
The third part of the Drosophila innate immune response is the double-stranded RNAi pathway that is involved in defending against many types of viral infections, and which also protects against viral infection in plants and animals (for a review, see Sabin et al., 2010) (Fig. 1D ). The RNAi pathway is activated by viral nucleic acids and can be broken down into two main steps: (1) biogenesis of 21-base-pair double-stranded viral small interfering RNAs (siRNAs), which is accomplished by the Dicer protein complex, and (2) the silencing of viral RNAs by the host-induced viral siRNAs, which is accomplished by the RNA-induced silencing complex (RISC). This innate protective system has been highly amenable to analysis using genome-wide screening in Drosophila cells (see below).
One of the great recent technical advances in the field of Drosophila cell biology has been the development of efficient whole genome RNAi screens to identify genes required for specific cellular processes (Mohr et al., 2010; Perrimon and Mathey-Prevot, 2007; Perrimon et al., 2010) . In such assays, Drosophila cell lines such as hemocyte-derived S2 cells or Kc cells (which can be induced by hormone treatment to differentiate into neurons) are grown in 384well plates and treated with a library of double-stranded RNAs that have been designed for highly selective RNAi-mediated knockdown of each of the predicted Drosophila coding messenger RNAs (mRNAs). These cells are then assayed for performance of a cellular process such as cell viability, cell shape changes or bacterial uptake by phagocytosis. By screening such libraries in replicate and then re-screening RNAi candidates that test positive for a specific effect, it is possible to approximate genome-wide coverage of all genes required in these cells for a given process [for an excellent, comprehensive review of such RNAi screens, see Cherry (Cherry, 2008) ]. Such screens have been used to identify many host response factors that are crucial during infection by bacteria, fungi and viruses.
Several straightforward RNAi screens have been conducted to identify genes that are required for phagocytosis of various species of bacteria by S2 cells. For these experiments, ingestion of bacteria expressing green fluorescent protein (GFP) is monitored and host genes involved in phagocytosis are revealed on the basis of the identity of specific RNAi molecules that inhibit uptake of fluorescence. These screens have revealed that distinct sets of host genes are essential during infection by various pathogens. For example, different pathogens are recognized by distinct cell surface receptors, such as peptidoglycan recognition proteins (PGRPs) (Ramet et al., 2002) , SR-C1 (Ramet et al., 2001) , Eater (Kocks et al., 2005) , Nimrod (Kurucz et al., 2007) or DSCAM (Watson et al., 2005) . However, these screens also defined a core set of intracellular uptake components that are regulated in all types of bacterial infection tested: these included genes required for actin remodeling (e.g. genes encoding proteins of the Arp2/3 complex) and endocytosis (e.g. COPI and COPII), as well as genes encoding factors that are required to recycle endosomes to the cell surface, such as proteins in the exocyst complex (Agaisse et al., 2005; Cheng et al., 2005; Philips et al., 2005; Ramet et al., 2002; Stroschein-Stevenson et al., 2006; Stuart et al., 2007) .
Other genes involved in the response to bacterial infection that have been identified in RNAi screens are required for host cells to clear ingested bacteria. Again, these screens defined a set of generally required genes that limit bacterial survival or replication, such as genes encoding endosomal sorting complex required for transport (ESCRT) proteins (Philips et al., 2008) , as well as genes preventing the growth of specific pathogens, such as lysosomal hexosaminidase, which restricts growth of Mycobacterium marinum but not Listeria monocytogenes or Salmonella typhimurium (Koo et al., 2008) . In other standard genetic studies, intracellular microorganisms such as Wolbachia were found to also engage in mutualistic symbiotic relationships with the host, such as protecting the host against viral infection (Hedges et al., 2008; Teixeira et al., 2008) and nutritional supplementation (Brownlie et al., 2009) , which presumably arose during co-evolution of the endosymbiont and host.
RNAi technology can also be used in a combinatorial fashion to knock down the activity of two or more genes at a time, which permits detection of genes acting in parallel in a given process or pathogenic infection. In one study, Dorer and colleagues performed Host-pathogen interactions in Drosophila PERSPECTIVE a series of single and double gene knockdown experiments of 73 genes in Kc cells to test the hypothesis that Legionella pneumophila, the agent of Legionnaires' disease, recruits membrane material from endoplasmic reticulum (ER)-to-Golgi trafficking (Dorer et al., 2006) . Although few single knockdowns had much of an effect, the authors found evidence supporting their hypothesis in several double knockdown experiments. For example, double knockdown of the intermediate compartment and Golgi-tethering factor transport protein particle (TRAPP) together with the ER SNARE protein Sec22 resulted in reduced pathogen replication efficiency. They also showed a requirement in bacterial replication for the Cdc48-p97 complex that is involved in ER-associated degradation, and demonstrated that this complex is also important for Legionella pneumophila replication in mouse bone-marrow-derived macrophages. These studies underscore the role of endocytosis in phagocytic host cells and, owing to the combinatorial power of the system used, revealed a role for endocytic steps carried out by parallel mechanisms.
Fungi generally activate the Toll signaling pathway of the Drosophila innate immune system via a specific set of PGRP detection peptides (Fig. 1C ). RNAi screens similar to those performed to identify host genes required for phagocytosis of bacteria have also been carried out to identify host factors involved in response to fungi such as Candida albicans (Stroschein-Stevenson et al., 2006; Stroschein-Stevenson et al., 2009) . Beyond identifying genes with broad expected functions, such as regulators of the actin cytoskeleton and vesicular trafficking, these studies also identified genes required for the uptake of specific fungal pathogens. One of these proteins, Macroglobulin complement related (Mcr), is a secreted protein that binds directly to C. albicans and promotes its internalization. Interestingly, Mcr is related to four other Drosophila thioester proteins (Teps), two of which are selectively required for phagocytosis of specific bacterial species (TepII for Escherichia coli and TepIII for Staphylococcus aureus), but not for phagocytosis of C. albicans (Stroschein-Stevenson et al., 2006) .
In addition to being susceptible to infection by bacterial and fungal pathogens, Drosophila is also a natural host for viruses such as Drosophila C virus (DCV), Drosophila X virus (DCX) and Flock House virus, and, perhaps surprisingly, by a broad variety of viruses causing disease in humans such as Sindbis virus, vesicular stomatitis virus (VSV; a virus of the Rhabdoviridae family, which includes the well-known rabies virus), Rift Valley fever virus, dengue virus and West Nile virus (Cherry et al., 2005; Cherry et al., 2006; Cherry and Perrimon, 2004; Galiana-Arnoux et al., 2006; van Rij et al., 2006; Wang et al., 2006) . Genome-wide RNAi screens have identified several important host factors that are exploited by viruses, such as factors required selectively for replication of influenza virus (Hao et al., 2008) or propagation of dengue virus (Sessions et al., 2009) . Similarly, viruses such as DCV that have transcripts with internal ribosome-binding sites depend on several host translation factors that are not required for other types of viruses lacking these sites (Cherry et al., 2005) . DCV also requires the host factor COPI to generate a vesicular compartment, which is necessary for viral replication, and COPI is also required for the replication of the related poliovirus in human cells (Cherry et al., 2006) . As another example, infection by vaccinia virus (the prototypical poxvirus) was found to depend on the AMP-activated kinase (AMPK) complex, the master energy sensor of the cell, for endocytic entry and actin remodeling (Moser et al., 2010) . The authors found a similar requirement for AMPK in facilitating vaccinia infection of mouse embryonic fibroblasts and showed that this kinase was also involved in viral entry via the process of macropinocytosis.
As mentioned above, the RNAi pathway plays a key role in defending against viral infection. Genome-wide and targeted RNAi screens have contributed to the elucidation of this pathway (Galiana-Arnoux et al., 2006; Nayak et al., 2010; Otsuka et al., 2007; Sabin et al., 2009; van Rij et al., 2006; Wang et al., 2006) (for a review, see Sabin et al., 2010) and the importance of the systemic spread of an RNAi activating signal (probably some large viral doublestranded RNA) for stimulating RNAi-dependent immunity throughout the organism (Saleh et al., 2009) . Interestingly, siRNAs do not spread from cell to cell in Drosophila (Roignant et al., 2003) , in contrast to the mechanism by which RNAi molecules are directly distributed in plants (Palauqui et al., 1997; Winston et al., 2002) and nematodes (Fire et al., 1998; Voinnet et al., 1998) to mediate systemic immunity.
As a complement to cell-based screening methods, it is also possible to screen for host genes that are required to combat pathogen infection using intact flies. Although these screens are more laborious than screens in Drosophila cell lines, or wholegenome RNAi screens in worms (i.e. Caenorhabditis elegans screens can be done on plates), screens using intact flies can be accomplished either by classic mutagenesis or by screening high quality collections of stable UAS-RNAi stocks. A great advantage of the latter approach is that one can use the GAL4-UAS expression system (Brand and Perrimon, 1993) to drive expression of UAS-RNAi constructs throughout the organism or in specific subsets of cells or stages of development (Fig. 2B ). For such experiments, a strain of flies carrying a transgene under the control of the yeast upstream activating sequence (UAS) is crossed to a strain of flies expressing the GAL4 transcription factor (which binds to the UAS sequence and activates transcription in a particular pattern, e.g. in the gut). The progeny then express the UAS transgene of interest in the pattern determined by the GAL4 'driver' stock, permitting expression of genes in specific cell types at specific stages of development. This level of control permits investigators to identify the cells or organs in which gene functions are required [e.g. epidermis, fat body (the main source of systemic AMPs, and an approximate model of the mammalian liver), hemocytes or gut].
In one screen using adult flies, host defense factors that are required to protect against intestinal infection with the opportunistic broad-host-spectrum pathogen Serratia marcescens were first identified by using a large collection of fly lines in which 13,000 individual RNAi molecules were used to knock down target gene expression throughout the organism (Cronin et al., 2009) . RNAi molecules that caused increased lethality following infection were then tested further for their role in defending Host-pathogen interactions in Drosophila PERSPECTIVE against S. marcescens infection of the gut by expressing the relevant UAS-RNAi constructs with gut-specific and hemocytespecific drivers. These studies first confirmed the dependence on the IMD (but not Toll) innate immune pathway for responding to infection by S. marcescens (as would be expected for a Gramnegative bacterium), and also revealed an important role for the JAK-STAT pathway in responding to infection in the gut (Fig. 2C ). Further analysis of JAK-STAT signaling showed that this pathway regulates stem cell proliferation and thereby intestinal epithelial homeostasis during infection. These results obtained in intact flies provide an important complement to screens performed in C. elegans, which also identified several signaling systems important for innate immunity (Irazoqui et al., 2010) . Studies of damage and repair by gut pathogens can be conducted in Drosophila because flies, but not worms, have intestinal stem cells that replenish epithelial cells after they undergo programmed cell death during infection (see above).
Systematic screens such as that mentioned above (Cronin et al., 2009) can also be used to identify host factors co-opted by a pathogen that, when mutated, render the host resistant to the pathogen. An example using a traditional genetic approach is the case of Vibrio cholerae, in which investigators showed that feeding V. cholerae bacteria to flies caused rapid death (i.e. in 2-3 days) that required the function of the primary virulence factor cholera toxin (CTX) (Blow et al., 2005) . CTX is an ADP ribosyl transferase that specifically ribosylates the Gs subunit of a host trimeric Gs protein, resulting in constitutive activation of adenylate cyclase (Middlebrook and Dorland, 1984) . The dependence on CTX was Host-pathogen interactions in Drosophila PERSPECTIVE unexpected because flies lack the enzymes required to synthesize the GM1 ganglioside that serves as the CTX receptor and that is present in most vertebrates and a few invertebrates. Accordingly, feeding flies purified CTX holotoxin had no effect (Blow et al., 2005) . Paradoxically, however, full virulence of ctx-mutant bacteria could be restored by feeding infected flies purified CTX, suggesting that, in the presence of the bacteria, a novel alternative route of CTX delivery to host cells in the gut might be employed. Further analysis showed that several host target factors known from mammalian studies were required by V. cholera to infect flies, such as proteins mediating the dehydrating effects of CTXdependent cAMP production -including a Gs subunit, adenylate cyclase and an SK-type potassium ion channel (Blow et al., 2005) . Having established flies as a model for V. cholerae infection, the authors then screened a large collection of stocks with mapped transposon insertions into the fly genome and identified mutations that either enhanced or reduced severity of infection. This strategy identified several host genes important for the response to V. cholerae infection, including those conferring resistance when mutated and that presumably are exploited by bacteria (e.g. components of the TNF and IMD pathways) as well as those used in host defense (e.g. the apoptotic pathway) (Berkey et al., 2009 ).
Adult flies and cells have also been used to screen for pathogenencoded factors that contribute to virulence. One particularly elegant screen for bacterial virulence factors was carried out for P. aeruginosa, an opportunistic human pathogen that can cause serious disease. Over 4000 transposon insertion mutants of the bacterium were screened by injecting them into the adult fly hemolymph and measuring percent lethality. This resulted in the identification of 15 different bacterial loci that contributed significantly to virulence (Kim et al., 2008) . The authors examined the basis of virulence for one of these genes, hudR. hudR encodes a transcription factor that represses expression of the neighboring gene hudA, which is involved in ubiquinone biosynthesis. On the basis of their genetic analysis, the authors hypothesized that the decreased virulence of hudR mutants resulted from overexpression of hudA. They confirmed this hypothesis by showing that overexpression of hudA in a hudR-mutant background resulted in attenuated virulence of P. aeruginosa in flies and that hudA hudR double mutants had normal virulence.
Flies have also been used to differentiate virulence of P. aeruginosa strains, such as those isolated from the sputum of cystic fibrosis patients (who are particularly sensitive to infection by this pathogen) (Lutter et al., 2008; Salunkhe et al., 2005; Sibley et al., 2008) . For these assays, flies are either fed different strains of bacteria obtained from burn wounds or from cystic fibrosis patients, or bacteria are inoculated by wounding flies. Similar infection experiments can be performed to identify interactions between P. aeruginosa and other microbes present in sputum that could contribute to the virulence of this pathogen (Sibley et al., 2008) .
Virulence factors of human fungal pathogens can also be identified in Drosophila (Ben-Ami et al., 2010; Chamilos et al., 2010; Chamilos et al., 2008; Lamaris et al., 2007) . For example, gliotoxin produced by the filamentous fungus Aspergillus fumigatus is required for virulence of this pathogen in both flies and mice (Spikes et al., 2008) , and Cas5 has been shown to be a transcription factor regulating a set of genes required for integrity of the cell wall of C. albicans . Adult flies have also been used as an intact organism to screen for drugs that block fungal infection (Chamilos et al., 2006a; Chamilos et al., 2006b; Lamaris et al., 2009; Lamaris et al., 2008; Lionakis et al., 2005) .
In the previous section, we discussed strategies by which flies can be used to screen for pathogen virulence factors. In this final section, we consider the advantages of Drosophila as a model for analyzing virulence factor function, and for identifying the host proteins and pathways that they target (e.g. Fig. 2C ). Although cellbased expression systems and biochemical experiments performed with purified virulence factors can be invaluable for establishing mechanism of action, they do not necessarily predict how such factors will act in an infected organism -either systemically or in selected tissues, in which cell-autonomous and non-cellautonomous processes might be important. Model systems such as flies and worms are ideal for this level of analysis owing to the great variety of genetic tools available to tease apart the effects that such factors might have on specific host pathways and biological processes. Although flies and worms are only distantly related to humans, many virulence factors target host proteins and pathways that are among the most conserved in eukaryotes -thus, studying the effect of pathogens in these organisms is often highly relevant to human disease. In addition, as discussed below, studies in model organisms also enable examination of the combinatorial effect of two or more virulence factors, which is more challenging in intact mammals. Finally, we highlight in Box 1 how studying the effect of toxins can shed light on basic cellular processes.
Drosophila is an excellent in vivo genetic system for analyzing toxin activities in a multicellular and organ context given the highly conserved nature of many host targets of these virulence factors. For example, flies have been used to study the activity of the virulence factor ExoS from P. aeruginosa, which encodes a factor
One of the first uses of toxins in flies was to genetically ablate specific cells with cell-lethal toxins such as diphtheria toxin (Kunes and Steller, 1991) or ricin (Moffat et al., 1992) . It is also possible to block the neuronal activity of cells without killing them, as with tetanus toxin (TTX), which was used to block synaptic transmission in the nervous system (Allen et al., 1999; Baines et al., 1999; Reddy et al., 1997; Sweeney et al., 1995) and activity-dependent regulation of synaptic size and function (Nakayama et al., 2006) . TTX has been used in a myriad of Drosophila studies to inhibit neurotransmission in various processes, including learning and memory, locomotion and courtship (for a review, see Martin et al., 2002) , circadian rhythms (Johard et al., 2009; Kaneko et al., 2000) , and the serotonin-dependent response to light (Rodriguez Moncalvo and Campos, 2009) . Similarly, application of cholera toxin (CTX), an ADP-ribosylation factor, was used to study the function of the G protein Concertina, which is involved in initiating embryonic gastrulation (Morize et al., 1998) . Similarly, transgenic expression of a UAS-CTX-A construct helped to distinguish which heteromeric G proteins contribute to wing maturation (Katanayeva et al., 2010) . Indeed, these and other toxins, which neutralize or alter the activities of multiple host proteins, can be used to perform a variety of in vivo pharmacological studies to complement classical genetic loss-offunction studies.
containing a domain with Rho-GAP activity (which can inactivate host small GTPases of the Rho/Rac subfamily). During P. aeruginosa infection, ExoS is injected into host cells by a type-II secretion system (TTSS), and infection of flies with P. aeruginosa leads to rapid death that depends on TTSS function (Fauvarque et al., 2002) . When the GAP domain of ExoS (ExoSGAP) is expressed in fly hemocytes, phagocytosis is inhibited (Avet-Rochex et al., 2005) . In addition, expression of ExoSGAP in flies increases their sensitivity to infection by P. aeruginosa (Avet-Rochex et al., 2005) , and this effect can be rescued by co-expressing host Rac2 with ExoSGAP (Avet-Rochex et al., 2007) . These studies provide evidence that host Rac2 is inhibited by bacterial ExoSGAP during infection.
A second example illustrating the utility of Drosophila for investigating toxin activities in vivo is provided by studies of Helicobacter pylori (Fig. 2C) , which is associated with the development of gastric ulcers and cancer in humans. Under normal circumstances, ligand-initiated receptor tyrosine kinase (RTK) signaling in both fly and mammalian cells is mediated by a receptorassociated protein complex including Grb2 (Drk), Gab (Dos) and Shp-2 [Corkscrew (Csw)] (Drosophila protein names are shown in parentheses) that then activates signaling via the downstream components of the Ras-MAPK pathway. Drosophila played a prominent role in discovering key components of this pathway and in establishing the order of molecular events that take place during signaling (Simon, 2000) . In mammalian cells, the H. pylori virulence factor CagA activates RTK signaling at the level of SHP-2, a tyrosine phosphatase that is homologous to Drosophila Csw (Hatakeyama, 2008; Hatakeyama, 2009) , which acts downstream of Gab (Dos in flies) (Herbst et al., 1996; Raabe et al., 1996) . Studies in Drosophila confirmed the hypothesis that CagA can bypass the need for signaldependent activation of Dos in an intact organism, because CagA expression in Drosophila embryos or in the adult eye was capable of rescuing dos-mutant phenotypes (Botham et al., 2008) . Furthermore, the ability to activate effectors of the Sevenless RTK pathway in the eye was shown to be dependent on the downstream effector Csw, validating the hypothesized role of CagA in the RTK signaling pathway acting between Gab (Dos) and SHP-2 (Csw).
Beyond providing a multicellular model for assigning known biochemical activities to virulence factors, Drosophila can also be used as a tool to discover completely new activities of virulence factors. For example, Bacillus anthracis, the etiological agent of anthrax, produces two toxic factors required for systemic virulence (Lacy and Stevens, 1999; Mourez, 2004; Tournier et al., 2007; Guichard et al., 2011) : lethal factor (LF), a zinc metalloprotease that cleaves MAPKKs (Duesbery et al., 1998; Vitale et al., 1998) , and edema factor (EF), a highly active calmodulin-dependent adenylate cyclase (Leppla, 1982) . Both LF and EF are essential for the lethal effects of anthrax (Pezard et al., 1991) , which culminates in vascular failure and septic-shock-like death. An important unanswered question is, how do LF and EF, with such seemingly disparate enzymatic activities, collaborate during infection (particularly within vascular endothelial cells, which become leaky at advanced stages of disease, leading to death)?
In initial studies, we showed that anthrax toxins act on Drosophila homologs of their known targets in mammalian cells (Guichard et al., 2006) . In addition to these known effects of LF and EF, we observed that both toxins also caused adult wing and bristle phenotypes similar to those caused by inhibition of the Notch signaling pathway, and blocked expression of Notch target genes in developing wing imaginal discs (Guichard et al., 2010) . Moreover, these toxins interacted in a synergistic fashion to block Notch signaling (Fig. 3A) . Further analysis of this Notch-like phenotype revealed that it resulted from failure to recycle the Notch ligand Delta to the cell surface (Guichard et al., 2010) . EF was found to reduce the levels and activity of the small GTPase Rab11, whereas LF reduced cell surface levels of the Rab11 binding partner, Sec15 (Fig. 3B ). Sec15 is part of an octameric protein complex known as the exocyst, which targets proteins, including Delta and the cell adhesion molecule DEcadherin, to adherens junctions (accordingly, DE-cadherin trafficking to adherens junctions was also reduced by EF and LF). These results from flies were validated in human vascular and lung endothelial cells by our collaborators in Victor Nizet's laboratory (Fig. 3C) , who also showed that EF reduced epithelial barrier integrity in a cell culture assay and in vivo in mice (Guichard et al., 2010) .
Maintenance of vascular integrity depends on cell-cell adhesion (Dejana et al., 2009) , and cell-cell communication mediated by Notch signaling plays a role in promoting the formation of primary (or patent) vessels over more permeable microvessels (Hellstrom et al., 2007; Leslie et al., 2007; Lobov et al., 2007; Roca and Adams, 2007; Siekmann and Lawson, 2007; Suchting et al., 2007) . By inhibiting these two interrelated processes, and possibly interactions between endothelial cells and other vascular cell types such as mural cells, anthrax toxins might contribute to the latestage effects of anthrax infection when disruption of endothelial barrier function leads to lethal vascular collapse. Once sufficient levels of anthrax toxins are produced, they can be fatal even if the bacterial infection is eliminated with antibiotic treatment. Thus, these studies of anthrax toxins initiated in flies and validated in mammalian models might ultimately have therapeutic implication for treating humans infected with anthrax or for other conditions compromising vascular integrity.
Given the compact sizes of viral genomes, only few viral proteins fall into the category of bona fide virulence factors, similar to the potent bacterial toxins discussed above. By contrast, most viral proteins are dedicated to basic processes essential to the virus life cycle, such as entry or exit, replication, or manipulation of host processes such as transcription or translation. Model organisms are useful for examining specific interactions between viral and host proteins to gain insights into their mechanisms of action.
An excellent example of using the full complement of Drosophila tools to study a viral pathogen was carried out by Cherry and colleagues, who showed that fly cells can be infected with VSV. VSV can replicate in these cells to generate mature viral particles that can infect mammalian cells. They showed that infection of adult flies with VSV induces autophagy (Shelly et al., 2009) and that autophagy was mediated by VSV-G, a pathogen surface protein that is recognized by Drosophila cells. The authors found that induction of autophagy plays an important role in protecting against VSV infection and then asked what host pathways might mediate the autophagy response to VSV. A variety of elegant genetic epistasis experiments demonstrated that the PI3K-Akt pathway was attenuated by VSV infection, thereby relieving its constitutive (Guichard et al., 2010) . WT, wild type. (B)Analyze mechanisms of toxin action. The Notch-like phenotypes caused by expression of LF or EF in the wing both result from inhibition of endocytic recycling of membrane cargo to the AJ by the exocyst complex. EF acts by reducing the levels and activity of the Rab11 GTPase, which indirectly results in a loss of large vesicles containing its binding partner Sec15-GFP, a component of the exocyst complex. LF does not seem to alter Rab11 levels or function, but inhibits the formation of large Sec15 vesicles (Guichard et al., 2010) . (C)Validate toxin mechanism in vertebrates. Human brain microvascular endothelial cells were treated with purified EF toxin or LF toxin. As in fly cells, both toxins greatly reduce the number of Sec15-GFP vesicles in these cells and reduce cadherin expression (Guichard et al., 2010) . (D)Examine interactions between toxins. Cooperative interactions between toxins or other virulence factors can be assessed by co-expressing them in specific cells and comparing the effects of both toxins to that of the action of either toxin alone. In the example shown, anthrax toxins were expressed alone or in combination using a weak GAL4 driver to express low levels of the toxins. Each panel consists of an adult wing (top) and a larval wing imaginal disc showing expression of the Notch target gene wingless (wg) along the future edge of wing in third instar larvae (bottom). Expression of LF or EF alone (+LF or +EF, respectively) has little or no effect on formation of the wing margin (compared with WT). When LF and EF are co-expressed, the wing margin virtually disappears, as does expression of wg along the primordium of the wing margin. (E)In vivo structure-function analysis of toxins. The systemic activities of mutant forms of toxins or other virulence factors can be assessed in Drosophila. Such activities include cell-non-autonomous effects mediated by intercellular signaling systems, which are difficult to screen for in cell culture. In the simple case shown in this panel, high levels of LF expression lead to reduced wing size (middle panel) and a single point mutation in the LF catalytic domain renders it inactive (right panel). Panels A-D adapted from Guichard et al. (Guichard et al., 2010) with permission. Panel E adapted from Guichard et al. (Guichard et al., 2006) , with permission.
Host-pathogen interactions in Drosophila PERSPECTIVE inhibition of autophagy ( Fig. 2A) . Akt activation is also attenuated by expression of the SARS-Coronovirus Membrane protein in flies, which in this case results in increased apoptosis (Chan et al., 2007) . In another study, host factors required for the HIV accessory protein Nef to downregulate expression of the human CD4 protein were identified by RNAi screening in Drosophila S2 cells expressing human CD4. These factors included components of the clathrinassociated AP2 complex, which was then validated as an essential cellular component mediating a similar Nef-CD4 interaction in human cells (Chaudhuri et al., 2007) .
Classic genetic approaches in Drosophila can also be applied to probing structure-function relationships of toxins or other virulence factors. One straightforward approach to define domains of a toxin that are important for producing the phenotype of interest is to mutagenize flies carrying a UAS-toxin construct and to screen for loss of the phenotype that results from expression of the wild-type toxin (Fig. 3E ). One can then PCR amplify the mutated UAStransgenes and sequence the putative mutant allele to determine the molecular nature of the loss-of-function mutation. It is also possible to screen for mutations in the transgene that results in altered phenotypes caused by dominant gain-of-function mutations (Guichard et al., 2002) .
An important goal of this review has been to convince readers from other fields that flies provide a broad range of advantages for studying host-pathogen interactions at the level of the cell, tissue, organ and intact organism. As discussed, genome-wide RNAi screens in Drosophila cell culture have generated a wealth of new information regarding the genes involved in mediating basic host cellular responses to pathogens, such as those involved in innate immunity, phagocytosis and restriction of intracellular pathogen survival. These cellular studies can be complemented by studies that aim to identify host resistance factors and pathogen virulence factors using intact flies as infection models. Studies in flies also provide the potential to explore mutualistic interactions with intracellular endosymbionts, and to conduct mechanistic analysis of specific virulence factors, and combinations of these factors, using the state-of-the-art genetic tools available in Drosophila.
A particular advantage of model systems such as yeast, C. elegans and Drosophila is the potential to examine arrays of genetic combinations to identify factors produced by the host or pathogen that act redundantly (host) or that genetically interact (host or pathogen). These types of studies are inherently number intensive (known as 'the n problem'), because many combinations must be analyzed in a comprehensive fashion. However, there are excellent examples in which combinatorial genetic analysis has been used to investigate cellular processes involved in other types of human disease. For example, the interacting components of the DNA mismatch repair machinery were first identified in yeast, and the same components were found to interact in humans in a dominant manner and to contribute to cancer (Kolodner, 1995) . Drosophila cells and intact fly mis-expression systems (e.g. combined RNAi expression) are also well suited for such analyses, which would be prohibitively expensive and labor intensive in vertebrate models. It is of course important to validate results obtained in single cells or invertebrate model systems in vertebrates, a process previously referred to as 'closing-the-loop' (Bier, 2005) . It is possible to envision a tiered system of analysis in which initial discoveries that are made using powerful model genetic systems, including yeast, worms and flies (in cells and in whole organisms), are then validated in vertebrate models, including zebrafish, mice and human cells, and finally are linked via human genetics to specific disease processes. For example, in a recent study of genes on human chromosome 21 causing congenital heart defects when overexpressed in individuals with Down syndrome, a combined genetic analysis in flies, mice and humans pointed to two interacting genes, DSCAM and COL6A2, as contributing to formation of atrial septal defects (Grossman et al., 2011) .
In addition to assessing combinatorial contributions of host factors, Drosophila is well suited for examining cooperative interactions between pathogen virulence factors, as presented in the examples above. Many pathogens produce a complex cocktail of virulence factors, subsets of which are often co-expressed from neighboring genes in so-called pathogenicity islands. These coregulated virulence factors are typically delivered by a dedicated injection system and often act by unknown means in various combinations in different host cell types. Such virulence factors from a given pathogenicity island can be expressed in various combinations in specific cell types to identify specific cellular contexts in which they interact. Given the great success of the fly for analyzing the activities of single host or pathogenic factors in disease processes, it will interesting to see whether it also serves as a robust system to study more complex networks of interactions between host pathways or pathogen virulence factors. With the advent of whole genome RNAi tools and comprehensive mutant collections, flies should also provide an important intact model system for identifying unknown activities of virulence factors that act in a multicellular context to inhibit specific signaling systems or to alter contact between neighboring cells in structured tissues and organs. Combined use of Drosophila cells and intact flies in moderate-to high-throughput drug screens is also emerging as an effective strategy to identify compounds, or combinations of existing compounds, that alter the activity of host pathways to counter the effect of pathogens. Clearly, flies have a bright future as tools for further deconstructing human host-pathogen interactions. The Organisation of Ebola Virus Reveals a Capacity for Extensive, Modular Polyploidy BACKGROUND: Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. CONCLUSIONS: The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope. Viruses have evolved as genome packaging machines to efficiently transfer nucleic acids between susceptible host cells, ensuring replication. The majority of viruses have hollow, quasispherical shells rather than tubular structures, perhaps because this gives the most efficient packaging of nucleic acid with a fixed copy number of coat protein subunits. In non-enveloped viruses, the volume enclosed by the (usually) icosahedral structure is a constraint on the size of the genome, giving a limited capacity to encode capsid proteins, and usually restricts the genome copy number, or ploidy of the virion, to one [1] . Most membraneenveloped viruses are also quasi-spherical, but their symmetry is frequently less well-ordered, which is usually described as pleomorphic. This feature allows some flexibility in volume, which could accommodate variation in the size of the genome or its copy number. Nevertheless, most viruses, irrespective of their architecture, appear to have evolved to encapsidate only a single copy of their genome within the protein or protein/lipid shell, or a dimeric copy in retroviruses. Notable exceptions are the Paramyxoviridae and the Birnaviridae where particles may contain up to four copies of the RNA genomes [2, 3] . Although some strains of influenza can produce elongated virions, there is a mechanism that selectively encapsidates only one set of genome segments in each virion [4] .
The Filoviridae family, including the Ebolavirus and Marburgvirus genera, cause haemorrhagic fevers with high mortality in humans, and no effective treatments are currently approved [5] , although candidate vaccines are promising [6] . The 18.9 kb single-stranded negative-sense non-segmented RNA genome of Ebola virus (EBOV) codes for at least eight proteins. The ribonucleoprotein complex is composed of the nucleoprotein (NP), polymerase protein (L), VP24, VP30, and VP35. The trimeric transmembrane glycoprotein (GP) forms surface spikes on the virion envelope and also has a soluble form, while the matrix protein, VP40, is associated with the inner surface. [5, [7] [8] [9] . The GP spike, a class I fusion protein, mediates cellular attachment and entry and is extensively glycosylated, especially in the glycan-rich mucin-like domain [10] [11] [12] [13] . Three proteins, VP24, VP35 and NP are essential for nucleocapsid formation [14] . Although some of the major protein interactions that occur during EBOV morphogenesis have been characterised [14, 15] , the three-dimensional (3D) structure and molecular arrangements have not been previously determined. Structural details are essential to understand how protection of the genome, cell binding, entry, and immune evasion are achieved in a filamentous animal virus, and to determine how this unique morphology plays a role in pathogenesis.
Research on filoviruses has been hampered by their status as biosafety level 4 pathogens. Previous investigations of filovirus structures within embedded, sectioned and metal-stained cells by electron tomography revealed few details of the high resolution oligomeric structure [16, 17] . It has been demonstrated that aldehyde-fixation alone, and subsequent cryo-electron microscopic imaging in the frozen-hydrated state preserves structures, at least up to 12 angstroms resolution [18] , and in some cases, fixation improves the resolution achievable [19] . In addition, it has also been shown that high-resolution X-ray structures can also be obtained in the presence of aldehyde fixatives [20] . Therefore, we analyzed purified and isolated EBOV and Ebola virus-like structures using cryo-electron microscopy (cryo-EM), and cryoelectron tomography (cryo-ET). In the current study, the Zaire strain of EBOV was purified and inactivated by paraformaldehyde fixation: excess fixative was then removed by dialysis to reduce beam damage for imaging in the frozen-hydrated state. The flashfreezing at liquid ethane temperatures used in cryo-electron microscopy preserves the structural and molecular detail, avoiding artifacts associated with conventional EM methods, such as dehydration and/or sectioning or staining, that usually prevent detailed structural analysis. Digital image processing reveals the 3D organization of EBOV, including the structural arrangement of component molecules at resolutions of 14-19 Å .
We identified filamentous EBOV particles 20 microns or longer, with a well ordered internal structure, and a helical nucleocapsid giving an internal ''herring-bone'' appearance using cryo-EM and cryo-ET (Figures 1, 2 , S1, S5). The nucleocapsid, as observed within intact viral particles, has a uniform helical structure (Figures 1, 2, 3, 4) and is enveloped by a membrane coated by an external layer of GP spikes. From the same image data set, we combined extracted volumes from tomograms with 2-D single particle processing to determine the structure of the GP spikes ( Figure 5 ) to a resolution of 14 Å as measured by the Fourier Shell Correlation (FSC) 0.5 criterion. Virions are rarely straight. Variation in the overall length of virions is non-random, and they fit into ordered size classes ( Figure 1 ). Analysis of 2090 distinct intact virions with a nucleocapsid from cryo-electron micrographs shows that the most common class length (53%) of virus particles is 982679 nm ( Figure 1A , Table S1 ). The other size classes are multiples of this length. Enveloped filovirus particles have several Empty and linked EBOV structures were excluded from the histogram data. A single G1-single/comma shaped EBOV is shown (inset on the right, G1 = 1 copy of genome). (B) Low magnification cryo-images showing: G1-single/comma shape, G1-single/ linear, G5-continuous (G5 = 5 copies of genome). (C) High magnification of a G1-(single genome) virion with a region filtered to emphasize the nucleocapsid. (D) Low magnification image of a G4-linked EBOV, each genome copy is indicated and numbered, the red arrows show the transition points between nucleocapsids. The circular holes (filled with vitreous ice) appear as lighter regions and the support film (''quantifoil'') appears dark grey. A ''linker'' region is shown at higher magnification (inset). doi:10.1371/journal.pone.0029608.g001 different morphologies (shown in Figures 6, S1 , S5). These configurations are ''single'' particles, containing a nucleocapsid of uniform length, (which we postulate to contain one copy of the genome), ''continuous'' particles, with nucleocapsids of a length of the single virion multiplied by an integer of 2 or greater; and ''linked'' virions composed of a series single-genome nucleocapsids connected by short sections of empty envelope. Negative staining can cause drying and staining artefacts which hamper accurate measurements and preclude 3D analysis. Nevertheless, in previous studies using this technique, the average length of Marburg virus Figure 2 . Image processing of Ebola virus. Linear 2D averaging of EBOV: the envelope and nucleocapsid are prominent features (A). The line trace is colour-coded as follows: red, spike; beige, lipid envelope; green, membrane-associated proteins; white, membrane-nucleocapsid gap; blue and purple, outer and inner nucleocapsid. (B) 2D class averages of envelope plus inner face. (C) VP40 VLPs, showing 2D averages from the from side regions (first two) and end-on/central regions (last three). In (A-C) representative individual repeats have been highlighted in color using the same scheme as in (A). (D) Schematic model of the nucleocapsid and envelope, highlighting the relative distribution of NP to VP40. (E,F) 3D reconstruction of the nucleocapsid with the same colour scheme as in (A). The location of the inner nucleocapsid, and the bridge are indicated. The reconstruction is presented at a volume threshold that would encompass a single copy of each of these proteins, and the viral RNA. In (E) the vertical (protein-protein) and horizontal (protein-RNA) contacts are indicated by yellow and white arrows, respectively. (G) Various recombinant nucleocapsid-like structures, and authentic EBOV, which have been studied by electron microscopy [14, 15, 21, 28] . 3D schematics of these structures highlighting the RNA and protein composition and the diameter of these structures, at the same scale for comparison to (E). doi:10.1371/journal.pone.0029608.g002 was measured as 790 nm, and EBOV as 970 nm [21, 22] : the latter is very similar to the mean length of 982 nm measured in the current study by cryo-TEM of frozen hydrated specimens. In addition, discrete sub-populations of virions of double or triple the average length were also previously reported in centrifuged virus preparations [21] .
We also observed ''empty'' filaments that lack nucleocapsids, with a random length and a smaller diameter ( Figure S1C ). These empty nucleocapsids are also visible in previously published thinsection micrographs of human EBOV infected pathology specimens, e.g. [23] , and are thus probably not an artifact of cell culture. It is also unlikely that the polyploidy we have observed in EBOV is a peculiarity of the particular viral isolate or of the Vero cell line that we used for this investigation. Previous studies using negative stain EM have shown that both Marburg virus and several different EBOV species can produce filamentous virions up to 14 mm long when grown in several different cell lines [21, 22] . Blood specimens from Guinea-pigs and monkeys that were inoculated with primary isolates of the Marburg agent, when centrifuged and observed by negative stain, also showed filamentous virions of variable length, with the average reported as about 1 mm in length but with a smaller proportion being over 2 mm in length [24] . In addition, viral filaments of lengths from 1.4 to 1.6 mm long, and occasionally as long as 2.6 mm, can also be seen in published ultrathin sections of EBOV infected human and monkey tissues [23, 25] . Thin-section EM always underestimates the lengths of virions, since filament profiles are frequently truncated by the section plane: in addition, tissue shrinkage of 10 to 20% during dehydration and resin embedding is common. Thus, it is probable that polyploid EBOV with multiple nucleocapsids are also produced in naturally infected humans and animals, though it is not possible to make a direct comparison with cell cultured virus: it would be very difficult to obtain large enough quantities of concentrated virus from animals to carry out detailed cryo-TEM analysis and measurements.
The Ebola nucleocapsid structure was solved to 19 Å resolution (FSC 0.5 criteria) using linear regions of 34,605 images (taken at 3-4 mm defocus, Figure 2 ). Since virus particles were not straight enough for conventional helical image processing, a combination of tomography, sub-tomogram averaging, single particle averaging, and the iterative helical real-space construction method were used [26, 27] . The EBOV nucleocapsid is a right-handed doublelayered helix with an outer diameter of 41 nm and a hollow inner channel 16 nm in diameter as determined by image analysis and tomography (Figures 2, 3 , 4, S2, S4, movie S1). The pitch is 6.96 nm, with 10.81 repeats per helical turn ( Figure 2 ). The inner nucleocapsid, composed of large subunits, is linked by vertical and horizontal contacts between the large subunits ( Figure 2E ,F). The horizontal contacts occur between the large subunits at a diameter of 22.3 nm. The vertical contacts linking the coils have a higher density than the horizontal ones, so we interpret the horizontal contacts as involving viral RNA (white arrow, Figures 2E, 4E ; movie S1) and the vertical contacts as protein-protein interactions (yellow arrow, Figure 2E ). The NP subunits are also linked by an outer horizontal layer at a diameter of 37 nm. This layer consists of a ring of bridges between adjacent large subunits ( Figure 2 ). The bridges joining the NP subunits are composed of two lobes, one of which of which is slightly bigger than the other. Previous studies that produced recombinant nucleocapsid-like structures showed that expressed VP24 and VP35 both independently associate with NP, but that all three proteins together are necessary to produce ,50 nm diameter helical nucleocapsid-like structures. When VP35, VP30, VP24, and NP were transfected together, approximately 50 nm diameter helical nucleocapsid-like structure was also generated, whereas NP alone generated helical NP-RNA complexes ,20-25 nm in diameter, which were nuclease sensitive [14, 28, 29] . Taken together these results suggest that VP24 and VP35 are the structural components of the bridge located on the periphery of the nucleocapsid ( Figure 2G ). It is not possible to accurately delineate VP24 and VP35 within the bridge at this resolution, however the density of the bridge is consistent with a predicted total mass of 60 kD, thus each bridge is composed of one molecule of VP24 and one molecule of VP35. It is likely that the larger lobe is VP35 and that VP24 therefore resides within the smaller lobe ( Figure 2F ). Thus, each bridge is composed of a VP24-VP35 heterodimer that holds adjacent NP molecules together horizontally. This structure explains how VP24 and VP35 are able to independently interact with NP, and why all three are required for the formation of a double-layered nucleocapsid. In addition, it implies that VP24 and VP35 can interact with each other as well as each interacting with a different site on the NP molecule in order to make an oligomeric structure.
The recombinant nucleocapsid data indicates that both VP35-VP30-VP24-NP and VP35-VP24-NP produce approximately 50 nm diameter helical structures that are indistinguishable from nucleocapsids produced by EBOV. This indicates that VP30 does not increase the diameter of the nucleocapsid. We propose that VP30 lies in the interior of the nucleocapsid and is not part of the bridge on the periphery of the nucleocapsid. This localization is consistent with previous work showing that VP30 is a component of the nucleocapsid, and associates with NP, but is non-essential for nucleocapsid formation [14, 30, 31] . Our model, in which the inner layer at 22.3 nm diameter is RNA-NP, and the outer bridge centered at 37 nm is composed of VP24-VP35 heterodimers, with VP30 bound to NP, is thus consistent with these previous observations [14, 28, 29] , and suggests that the outer VP24-VP35 heterodimer bridge functions in the stabilization and/or protection of the nucleocapsid.
We have modeled the arrangement of the RNA within the EBOV nucleocapsid (Table S2 ). Since no atomic resolution data on the ribonucleoprotein structure of filoviruses have yet been reported, the ribonucleoprotein ring-structures of other members of the mononegovirales are useful for comparison. Although the mass of the nucleoproteins, and the number of nucleotides per subunit differs amongst these virus families, a model of the 18.9 kb EBOV genome with the RNA following a circular fixed radius as in respiratory syncytial virus (RSV) [32] would make a nucleocapsid containing one copy of the EBOV genome about 914 nm long (Table S2 ). This fits closely with the 982 nm length class (53% of the particles observed in this study) containing a single copy of the EBOV genome threaded through the NP at a fixed radius. Allowing 33 nm of space for the membrane envelope to curve around at each end of the virus particle gives an estimated length for a single-genome virus particle of 980 nm, which is very close to that observed (982679 nm). The majority of the virus particles fall into size categories that are a multiple of the putative single genome length (G1), giving size classes of 1.960.15 mm (G2: 18.7% of the particles having 2 genomes); 2.960.2 mm (G3: 12% of particles having 3 genomes) and so on ( Figure 1A and Table S1 ) . The length of the longest particle measured was consistent with having 22 genome copies. Our model predicts a nucleotide to NP ratio of 13.0 (Table S2) , which is within the range of 12 to 15 nucleotides per NP molecule as measured by biochemical studies of Marburg virus [33] . In partially full virions, the membrane envelope is constricted at the transition point where the nucleocapsid ends and the empty membrane tube begins (Figures 1, S1C ). Empty virions ( Figure  S1C ) have a similar structure to VP40-GP virus-like particles ( Figure 3A ). Both full and empty virions have a continuous layer of spikes projecting from the surface (Figures S1, S2, S3, movie S2), giving an overall diameter of 120 nm for full particles. The majority of virions are linear (Figures 1, S1A, S5 ), others have a ''comma-shaped'' appearance, with a globular head containing portions of the nucleocapsid that are curled-up or bent at one end ( Figures 1A,B and S1B). It is clear that ''toroidal'' virions previously identified by negative staining [22, 34] are a variation of comma-shaped virions. Internal vesicles of 20-40 nm in size are also sometimes observed at the ends of virions ( Figure S1D ). These vesicles appear to be formed during the process of envelopment of the nucleocapsid, since they were not observed in preparations of VP40 or VP40-GP VLPs. VP40 VLPs had wavy envelopes with an irregular diameter ranging between 48 nm and 142 nm (N = 49) compared to VP40-GP VLPs which were more ordered with a diameter between 50 nm and 91 nm (N = 26), (Figure 3 ). Thus the presence of GP, and certain contacts between the GP and VP40, play a part in stabilizing the tubular membrane envelope structure, and our observed structure agrees with previous reports that GP enhances VP40 VLP budding [35, 36] . The previously reported ''branched'', filamentous forms [34] were rarely observed: these consist of empty tubes (data not shown). It is possible that centrifugal virus purification disrupted most branched structures that were seen previously with negative staining of cell culture supernatant.
The VP40 matrix protein shows a regular 5 nm lattice spacing ( Figure 2B , C). Both the nucleocapsid and the VP40 layers are ordered, however the contacts between them appear to be nonsymmetrical ( Figure 2D ), implying some flexibility in their intermolecular contacts. It has been shown that VP35 interacts with the VP40 and can be packaged into VP40 VLPs [37] . The localization of the VP24-VP35 bridges on the periphery of the nucleocapsid, may allow interactions of one or more of the nucleocapsid proteins with VP40, possibly through projecting low-density protein loops. There is a 6-7 nm gap of low density between the nucleocapsid and the VP40 layer, but tomography also shows discrete areas of connectivity between the nucleocapsid and matrix protein layers, which may be connections between the envelope and the nucleocapsid ( Figure 3I ). These results are substantiated by the analysis of sub-tomograms where helical symmetry is clearly evident but was not imposed (Figures 4, S4) . The use of sub-tomogram analysis improves the resolution of the data by averaging sub-tomograms together which are at different angular orientations. The helical nucleocapsid is clearly identified in the structure, including the gap between the envelope-VP40, and the nucleocapsid-VP40. The right-handed pitch of the helix is clearly discernable (Figure 4E ), as well as the putative location of several VP40 proteins, although the resolution of the tomographic data by itself is slightly lower than with single particle analysis. While showing the overall organization of EBOV, tomography also allows estimation of the stoichiometry of the major structural proteins (Figure 3 , movie S2). Both EBOV and VP40-GP VLPs have an irregular distribution of the GP spikes on the surface (Figures 3, S3) demonstrating the lack of any ordered lattice-like arrangement with the matrix protein VP40. The spikes are clustered, and the average centre-to-centre spacing is 15.2 nm ( Figure S3, and movie S3) . We calculate that a virion of 982 nm in length would have about 1888 copies of the GP spike, and 8391 copies of VP40. The wide spacing of the EBOV GP in the viral envelope allows plenty of space for free access and binding of any neutralizing antibodies directed at both the club-shaped head and stem region without stearic hindrance (Figures 3J, S3) .
The nucleocapsid structure implies equimolar ratios of NP, VP24, VP30, and VP35. A genome of 18.9 kbp with 13 bases per NP with a single genome copy gives 1454 molecules of NP, VP24, VP30, and VP35 per virion. Previous analyses of Coomassie blue stained gels of purified EBOV predicted 625, 1208, 833, and 2686 protein molecules of NP, VP24, VP30, and VP35, per virion respectively [38] , which is in the same stoichiometric range as predicted by our model for the nucleocapsid, taking into account the variability of individual protein band staining by Coomassie blue, which is affected by factors such as distance of migration [39] , basic amino acid content [40] , and the extent of glycosylation [41] . Since NP is glycosylated, we anticipate underestimation of the NP content of virions [14, 41] .
The GP spike is necessary for cellular attachment and fusion of EBOV. A definitive cell surface ligand for the receptor binding domain has not been identified, and a number of different cell surface proteins are able to enhance infection [42, 43] . Cleavage of GP results in two domains: GP1 containing the receptor binding domain and GP2 that contains the fusion and transmembrane domains. The structure of a smaller engineered fragment of GP1-GP2 has recently been determined by x-ray crystallography (3CSY pdb [42] ). We determined the structure of the entire EBOV GP trimeric spike at a resolution of 14 Å (FSC 0.5 criteria), by combining sub-tomograms from the spikes of VP40-GP VLPs (234 images) with EBOV images for the side perspective data (8084 images) using projection matching as previously described [44] ( Figure 5 and movie S4). In our reconstruction, the spike is in situ in the viral membrane, thus the transmembrane region and base of the spike, adjacent to the membrane, are less well defined than the distal region of the spike, due to the smaller differences in contrast between lipid and protein versus water and protein, as well as Fresnel fringes at the edge of the viral membrane. The spike extends 10 nm from the surface of the envelope, with a clubshaped head 6.5 nm in height, and a 3.5 nm long stalk. Docking of the previously determined X-ray structure into our cryo-EM map shows a good fit, with a correlation coefficient of 0.75 using the docking and correlation program SITUS [45] . The difference map calculated between our cryo-EM map and the GP1-GP2 structure identified volumes corresponding to the domains deleted to generate the 3CSY GP1-GP2 structure ( Figure 5 ). We show that the mucin-like domains (connected at V310 and E502 -shown in Green in Figure 5 ) completely fill the previously described bowllike chalice which contains the putative receptor binding sites [42] . The proximity of glycosylation sites in the GP1-GP2 structure suggests that the distal density of the spike contains the glycans that were deleted in order to construct the GP1-GP2 structure. In addition, each mucin-like domain has an ''arm-like'' projection, which extends radially at the distal end of the spike, to a maximum diameter of 13 nm. The localization of the mucin-like domain is consistent with previous studies showing that endosomal proteolysis plays a role in enhancing infectivity as well as binding of the Ebola GP to the plasma membrane [46, 47] .
The other two major deletions in the 3CSY structure (N278-R299 and A189-Y214) are situated at the midpoint of the structure just above the stalk (shown in pink in Figure 5 ). Inclusion of the KZ52 Fab in the docked structure demonstrates that this neutralizing epitope (from a human survivor) is localized on the side of the stem region of EBOV GP trimer at the base of the clubshaped head, and that the Fab domain lies close to the lipid envelope when bound, and approximately tangential to the viral envelope. The densities putatively corresponding to N278-R299 and A189-Y214 are close to the KZ52 neutralizing site, but do not obstruct antibody binding. The mucin-like domains are out of the way and cannot interfere stearically with KZ52 Fab binding. This is consistent with previous results indicating that KZ52 binding does not require cathepsin cleavage [48] . We have thus delineated the low resolution structure of the glycocalyx or ''glycan cap'' that covers the distal end of the uncleaved EBOV GP spike, which is consistent with a proposed role in immune evasion [42] . Our data will enable docking of future structures to investigate receptor binding, antigenicity, and fusion mechanisms.
We have shown that EBOV particles are capable of a high degree of polyploidy, made possible by the extreme length polymorphism of budding virus particles. Polyploidy in filoviruses may be more extensive than in any other virus family, with 46% of virions having more than one genome copy, and some having up to 22 copies. Polyploidy has been shown to increase infectivity rates in paramyxovirus and birnavirus [2, 3] . Attempts to investigate infectivity rates of EBOV by centrifugal fractionation of the different sized particles are stymied by the extreme filamentous morphology, as well as that fact that EBOV of different lengths have the same buoyant density (personal observations). A complex double layered helical nucleocapsid appears to be unique to filoviruses. In the case of rhabdoviruses, the bullet-shaped nucleocapsid precludes them from being linked sequentially. Although some influenza strains can produce filamentous virions, this morphology appears to be driven by the matrix protein only [49] . A filamentous morphology may have evolutionary implications by allowing genome length flexibility. It could also enhance the ability for viral dissemination in infected tissues, for example by diapedesis of budding filamentous virions through epithelial layers.
Zaire Ebolavirus was propagated in Vero E6 cells and purified as previously described [50] . Ebola enriched samples were checked by SDS-Page and Western blotting, and rendered non-infectious by fixation with 4% paraformaldehyde. Excess fixative was removed by placing the fixed samples in a Slide-A-Lyzer G2 cassette with a 0.5 ml capacity, and a 10,000 MWCO (Thermo Scientific Pierce Protein Research Products, Rockford, Illinois, USA), followed by dialysis against PBS. Virus-like particles were produced as previously described [51] . All work with infectious Ebola virus (virus culture and purification) was performed in the biosafety level 4 laboratories at the National Microbiology Laboratory of the Public Health Agency of Canada, Winnipeg, Manitoba.
Samples for cryo-electron microscopy (cryo-EM), and cryoelectron tomography (cryo-ET) were mixed with BSA coated Accessories, Wageningen, The Netherlands) at a ratio of 2:1 (virus:gold) for cryo-ET, and (9:1) for cryo-EM. Specimens (4 ml) were then applied to glow-discharged quantifoil grids with 2 mm holes spaced at 1 mm intervals (Quantifoil MicroTools GmbH, Jena, Germany). Grids were subsequently plunge cooled in liquid ethane using a Vitrobot Mark IV (FEI Company, Hillsboro, Oregon, USA). Specimens were transferred to a Tecnai 20 G2 transmission electron microscope (FEI) operated at 200 kV, equipped with a Gatan CT3500TR single tilt rotation lowtemperature specimen holder. For cryo-EM imaging was conducted at temperatures of ,2185uC. Images were recorded using an Eagle 4K CCD camera (FEI Company, Hillsboro, Oregon, USA). For single particle image analysis, images were taken at 50,0006 or 80,0006 magnification at 2-4 mm defocus, with a dose of 10 electrons/Å 2 . This corresponded to a pixel size at the CCD detector of 2.147 Å /pixel and 1.353 Å /pixel, respectively. For virus length measurements low magnification cryo-EM images were taken at 5,0006, 3,5006 and 2,5006. For cryo-ET single axis tomograms were taken at 25,0006, 29,0006 or 50,0006 magnification, at 28 m or 26 m defocus, with angle steps of 2u24u. Data were collected within tilt ranges of 660u, or 652u, with a total dose/tomographic data set of 47-60 electrons/Å 2 . For cryo-EM, data collection was done using the low-dose unit and software coupled with the TEM Imaging & Analysis (TIA) software (FEI Company, Hillsboro, Oregon, USA). Automated eucentricity determination, and focusing were performed using the Xplore3D data acquisition software (FEI Company, Hillsboro, Oregon, USA). For cryo-ET, data collection was done using the Xplore3D data acquisition software, the low-dose unit, and the TIA software (FEI Company, Hillsboro, Oregon, USA).
The exact magnification in the microscope at the CCD detector was determined using a calibration grid (Pelco International, Redding, CA). Ebola virus length measurements (n = 2090) were made using the Image J software package [52] using the free hand line tool, and the analyse/measure function. For this analysis only viruses containing a continuous nucleocapsid were measured. Viruses with linked nucleocapsids and empty viruses were omitted. The measurements that were made in image J were then collated, analysed, and plotted, using Microsoft Excel.
Tomographic image analysis of cryo-ET data was carried out with the Inspect3D Xpress software package (FEI Company, Hillsboro, Oregon, USA). The tomographic images were aligned to each other by a two-step process. The first step involved alignment of adjacent images by cross correlation. This process was repeated several times until the shift between adjacent images was below one pixel in either the X or Y plane. The second step involved the alignment of the entire image stack using 10 nm colloidal gold particles as fiducial markers. In this instance the term ''image stack'' refers to all the images collected in a single tomographic tilt. This procedure involves the selection of ten to twenty of the 10 nm gold particles and the subsequent identification and tracking of these particles in all of the images in the image stack. The locations of these particles are then used in conjecture with the tilt angles of each image to globally align all of the images to each other. The last step in this process was to calculate the three dimensional reconstruction of the tomogram from the aligned images. In this study we used the simultaneous iterative reconstruction technique (SIRT) algorithm with 10 iterations to calculate the final three-dimensional reconstruction (tomogram).
Sub-tomogram image analysis of cryo-ET data was carried out with the Automated Recognition of Geometries, Objects, and Segmentations (ARGOS) software package (FEI Company, Hillsboro, Oregon, USA). For this analysis an 80 3 pixel subtomogram was extracted from a tomogram using the Chimera [53] software package. In this analysis the tomogram used contained a linear region of the Ebola virus ( Figure S4 ), and the 80 3 pixel sub-tomogram contained a single linear segment. This template was then used by the ARGOS software to conduct an exhaustive search of the original tomogram for similar structures. This analysis involved a six dimensional search matrix (three positional variants, and three rotational variants). The entire search process was sped up by the ARGOS software by utilizing parallel processing on the computer's graphics processing unit (GPU). Once individual sub-tomograms were selected based on correlation, they were inspected and compared to the initial template sub-tomogram. The extracted and aligned sub-tomograms were subsequently averaged with a filter that minimized the missing wedge artifact. This average structure then was used as the reference and the entire procedure was repeated several times.
Single particle image analysis: software and hardware
Single particle cryo-EM image processing was carried out using the EMAN/EMAN2 and SPIDER/WEB image processing program packages [54, 55] . Particle selection (EMAN) and contrast transfer function correction (EMAN2) were conducted on an Apple Inc. Mac Pro computer (12-core, Intel Xeon Nehalem processors 2.93 GHz, 32 GB Ram, Mac OS X 10.6.7). All subsequent calculations were performed on a Dell PowerEdge R900 4-way 64-bit Xeon X7460 processors, Six Core 2.67 GHz CPUs with 256 GB Ram running Linux (CentOS 5.2). Images were corrected for contrast transfer function (ctf) using the ''e2ctf.py'' function in the EMAN2 software package 5 , which estimates defocus and corrects for ctf by phase-flipping. Images of the spike (n = 8084 side perspective; n = 234 end-on perspective) and nucleocapsid (n = 34,605) were selected for image analysis. The resolution of the cryo-EM reconstruction was estimated by Fourier shell correlation using the FSC 0.5 criteria. In all subsequent sections image analysis procedures were conducted using the SPIDER software package unless otherwise stated.
Analysis of initial images of the ''straight'' linear segments of the Ebola virus using Fourier transformation indicated that there was sufficient bending of the helical nucleocapsid to make standard helical analysis problematic. Therefore, an initial reference free single particle 2D analysis was conducted in EMAN using the ''startnrclasses'' program to identify any potentially recurring motifs within linear regions of the Ebola virus.
In order to further investigate the nucleocapsid repeat identified in the 2D analysis the iterative helical real space reconstruction method (IHRSR) was implemented [26, 27] . This procedure requires an initial 3D helical reference structure which is used for image alignment. In this investigation a linear region of the Ebola virus which was extracted from a tomogram was used to generate this helical reference structure. This initial 3D structure was first pre-treated with a Gaussian mask to select only the nucleocapsid-containing region of the virus tomogram. An auto correlation function was then performed in which the volume was rotated around the helical axis, and translated along the axis of the nucleocapsid. At each rotational and translational position and autocorrelation value was calculated (between the shifted and unshifted volume). The net result of this process was the determination of the helical symmetry present in the tomogram. In order to analyse the data generated by this procedure the Microsoft excel spread sheet program was used. The correlation plots generated by this process solved the handedness, pitch, and number of repeats per turn for the nucleocapsid. These symmetry parameters were then applied to the tomogram to generate the initial 3D model. The IHRSR method was then applied to the 34,605 single particle images of the nucleocapsid as previously described [26, 27] .
For the spike dataset two image populations were combined composed of side, and end-on perspectives. For the side view perspective images were subfield directly off of the Ebola virus cryo-EM images. For the end-on perspectives sub-tomographic volumes were extracted from the tomographic reconstructions of the Ebola VLP. The 3D volumes were then added in the ''Z'' plane to generate 2D projection averages which were then used for the subsequent single particle image analysis. The data were the processed using EMAN to generate an initial 3D reconstruction, which was then refined in SPIDER using the projection matching technique as previously described [44, 56] . The docking of the 3CSY.pdb [42] structure to the cryo-EM structure of the spike was accomplished using the SITUS [57] software package with the exception that only the GP1 and GP2 components of the 3CSY structure were used for the docking process. The ''floodfill'' program in SITUS was used to segment the spike component of the 3D cryo-EM reconstruction from the envelope component of the reconstruction. The segmented volume was then used for the docking procedure using the 'colores' function in SITUS. Once docked the entire 3CSY.pdb structure which included the Fab of the KZ52 neutralizing antibody was superposed over the docked GP1/GP2 component of the structure.
The 3D cryo-EM reconstructions, cryo-ET reconstructions, 3D models of the Ebola virus, and the atomic resolution structure 3CSY.pdb were visualized using UCSF Chimera software package (Computer Graphics Laboratory, University of California, San Francisco, supported by NIH P41 RR-01081) [53] . The 3D images and movies presented in this manuscript were generated directly by UCSF Chimera software package. , Region three is shown, with the locations of correlation maxima shown with an X. The angular distance between each maximum was calculated from several plots. A total of 71 measurements gave an average angular distance of 33.6u+/ 28.5u between helical repeats, resulting in 10.7 repeats per turn. Using the 6.96 nm pitch (Fig. S8 ) the step in Z per helical repeat was calculated as 0.65 nm. These helical symmetry values were then imposed on the nucleocapsid tomogram and this structure was used as the initial reference volume for refinement using the iterative helical real space reconstruction method. (TIF) Figure S3 Surface spike distribution in the Ebola VLP. Longitudinal Z-slices through the top and middle of the particle are shown, as well as the end-on view (A). The tomogram is shown as a shaded surface at a density threshold that indicates the spikes (B). The volume from one side of the tomogram has been extracted, and a red-blue color scheme shows the depth at which the spikes are located. This region of the envelope has a surface area of 15,651 nm 2 . Selected spikes have been identified by red circles, the single particle reconstruction of the spike is shown at the same scale to the right in a red square for comparison. The same region in (B) is shown in (C) with a solid orange cylinder to provide a visual cue for the viral envelope. Eighty-six individual spikes were counted (white spheres) and have a patchy distribution (D), each spike would occupy an average area of 182 nm 2 , giving an average spacing between spikes of 15.2 nm. The reconstruction of the spike (blue) with the docked KZ52 Fab (purple) has been included to show that there is ample room for antibody attachment. (TIF) Figure S4 Extraction of Ebola nucleocapsid structure for sub-tomographic analysis. The tomogram of a linear region of the Ebola virus was used as the first reference for subtomogram analysis (A). When viewed along the helical axis (Y) or from the end perspectives (X,Z) the basic components are visible. The tomographic volume was also cylindrically masked along the X-axis, selecting only the density containing the nucleocapsid, to highlight the components of the nucleocapsid in the tomogram (B). Two-dimensional single particle image analysis was carried out with cryo-images (C) (not tomographic data sets), for comparison to the 3D tomographic data. The average shown in this panel was generated by reference free classification, using the ''startnrclasses'' program in EMAN [54] . The 6.96 nm helical pitch can be easily seen in the 2D average, but is also visible in the projections of the tomographic volume in (A, B) . (TIF) Figure S5 Representative low-magnification images of Ebola virus. Frozen hydrated virus is clearly visible with sections of the filamentous virus over both the support film and across the holes in the quantifoil film. Individual G1 (single genome copy) virus is circled in red, several sections containing a nucleocapsid are indicated by a blue arrowhead, and regions without a nucleocapsid are indicated by a magenta arrowhead. Globular heads are identified by yellow arrowheads. In this image the circles (light grey, 2 m diameter) are filled with frozen hydrated virus in a thin aqueous layer, and the quantifoil support film appears as darker grey.
Table S1 Length analysis of ''continuous'' Ebola virus particles. The length of 2090 EBOV particles were measured using ImageJ [52] . The values in the ''model length'' column are based on multiples of the G1 mean length. The values in the in the ''mean length'' column were calculated directly from the data. Only full particles containing a continuously packaged nucleocapsid were measured, all others (linked-nucleocapsid and empty particles) were omitted from this analysis. The terms G1-G22 indicate the number of genomes/viral particle (i.e. G22 = 22 genomes). All measurements are in mm.
Modeling of RNA in the Ebola nucleocapsid. Two previously determined atomic resolution structures of negative stranded RNA viruses (VSV (2GIC.pdb) [58] , and RSV (2WJ8.pdb) [32] ) were used to estimate the EBOV nucleocapsid length and number of nucleotides per nucleoprotein. Images of VSV (A) and RSV (B) are shown as a molecular surface with the protein in orange and the RNA as a green ribbon. From left to right, they show a surface view from the side, a side-on cross section, an end-on view, the RNA density alone in projection, and a rotational average of the projection. The VSV-based estimate, with a saw-tooth pattern of RNA in the helix, gave a nucleocapsid which 614.37 nm long, too short for the measured length of the G1 EBOV (982 nm). The RSV-based model, with a relatively straight/circular pattern of RNA in the helix predicted a nucleocapsid 914.55 nm long, which closely fits the measured length of G1 virions, after allowing ,34 nm space at each end to accommodate the curve of the envelope containing GP spikes and matrix proteins. The RSV-like model gives 13 nucleotides per nucleocapsid protein which is similar to previous biochemical estimates of 12-15 for Marburg virus [33] , suggesting that the RNA in the EBOV nucleocapsid is arranged in a smooth helical pattern at a diameter of ,22 nm. (TIF)
Movie S1 3-D reconstruction of Ebola virus nucleocapsid. This movie shows the three-dimensional structure of the of the Ebola virus nucleocapsid as shaded surface representation. The surface is set at a density threshold which would include one copy of NP, VP24,VP30, VP35, and the RNA. The nucleocapsid rotates, and then is sliced through the Z-axis to show the internal components of the structure. Movie S3 Ebola virus spike distribution. This movie shows one surface of a cryo-electron tomogram of an Ebola viruslike particle, generated by expressing the VP40 and GP proteins. The structure rotates showing the distribution of spikes. The locations of individual spikes are identified by white spheres, and the reconstruction is replaced by a cylinder to show the patchy distribution of spikes on the surface of the virus-like particle.
Movie S4 3-D reconstruction of Ebola virus spike. This movie shows the three-dimensional structure of the of the Ebola virus GP spike. The structure rotates showing views from different angles, and indicates the location of the docked 3CSY.pdb [42] structure with the GP1 and GP2 domains and KZ52 antibody (purple). (MOV) 3D QSAR Pharmacophore Modeling, in Silico Screening, and Density Functional Theory (DFT) Approaches for Identification of Human Chymase Inhibitors Human chymase is a very important target for the treatment of cardiovascular diseases. Using a series of theoretical methods like pharmacophore modeling, database screening, molecular docking and Density Functional Theory (DFT) calculations, an investigation for identification of novel chymase inhibitors, and to specify the key factors crucial for the binding and interaction between chymase and inhibitors is performed. A highly correlating (r = 0.942) pharmacophore model (Hypo1) with two hydrogen bond acceptors, and three hydrophobic aromatic features is generated. After successfully validating “Hypo1”, it is further applied in database screening. Hit compounds are subjected to various drug-like filtrations and molecular docking studies. Finally, three structurally diverse compounds with high GOLD fitness scores and interactions with key active site amino acids are identified as potent chymase hits. Moreover, DFT study is performed which confirms very clear trends between electronic properties and inhibitory activity (IC(50)) data thus successfully validating “Hypo1” by DFT method. Therefore, this research exertion can be helpful in the development of new potent hits for chymase. In addition, the combinational use of docking, orbital energies and molecular electrostatic potential analysis is also demonstrated as a good endeavor to gain an insight into the interaction between chymase and inhibitors. Raised blood pressure, especially systolic pressure (hypertension), is one of the striking factors inducing various diseases like heart failure, stroke, myocardial infarction and arterial aneurysm, and is a leading cause of chronic kidney failure [1] . A treatment of hypertension is to decrease the circulating volume and/or to slack the blood vessels [2] . Angiotensin II has important roles not only in the regulation of blood pressure but also in the development of vascular wall remodeling [3] . Conversion of angiotensin I (Ang I) to angiotensin II (Ang II) is catalyzed by well-known angiotensin-converting enzyme (ACE), which is a metallo-proteinase with dipeptidyl-carboxypeptidase activity. However, chymase (EC 3.4.21.39) which is a chymotrypsin-like enzyme expressed in the secretory granule of mast cells, also catalyzes the production of angiotensin II in vascular tissues even when ACE is blocked ( Figure 1 ).
Chymase-dependent conversion of angiotensin I to angiotensin II and precursors of TGF-β and MMP-9 to their active forms.
Chymase converts Ang I to Ang II with greater efficiency and selectivity than ACE [4] . The rate of this conversion by chymase is approximately four fold higher than ACE. Chymase shows enzymatic activity immediately after its release into the interstitial tissues at pH 7.4 following various stimuli in tissues. Since chymase has no enzymatic activity in normal tissues, chymase inhibitors are expected to have high safety because chymase inhibitors may not have an effect on any other targets in normal tissues [5] . In order to generate Ang II, human, monkey, dog and hamster chymases cleave the angiotensin I at Phe8-His9 peptide bond. Chymase also converts precursors of transforming growth factor-β (TGF-β) and matrix metalloproteinase (MMP)-9 to their active forms thus contributing to vascular response to injury. Both TGF-β and MMP-9 are involved in tissue inflammation and fibrosis, resulting in organ damage [6] . Previous studies have demonstrated the involvement of chymase in the escalation of dermatitis and chronic inflammation pursuing cardiac and pulmonary fibrosis [7] . Therefore, inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, allergic inflammation, and fibrotic disorders. Chymase inhibition may also be useful for preventing the progression of type 2 diabetes, along with the prevention of diabetic retinopathy [8] . Moreover, the role of chymase in inflammation has prompted its restorative value in diseases such as chronic obstructive pulmonary disease (COPD) and asthma [9] .
Chymase inhibitors are imperative for elucidation of the physiological functions of chymase and potentially useful therapeutic agents. Several chymase inhibitors such as sulfonyl fluoride derivatives [10] , Boc-Val-Pro-Phe-CO 2 Me [11] , Z-Ile-Glu-Pro-Phe-CO 2 Me, (F)-Phe-COGlu-Asp-ArgOMe [12] , N-(2-Naphthyl) carboxamido derivatives [13] , N- (2,2-dimethyl-3-(N-(4-cyanobenzoyl) amino) nonanoyl)-L-phenylalanine ethyl ester [14] , 3-benzylazetidine-2-one derivatives [15] , 1,3-diazetidine-2,4-dione derivatives [16] , methyllinderone derivatives [17] , chloromethyl ketone derivatives [18] , 1-oxacephem derivatives [19] , and 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives [20] have been reported previously. In general, chymase inhibitors readily decompose in plasma, thus the stability of the chymase inhibitors in human plasma has always been a matter of great concern. For a drug candidate, it is essential to enhance the stability of the active compound in human plasma. So, there is always a dire need to search for more stable inhibitors with high activity against human chymase.
Many studies have indicated that computational approaches, such as predicting drug-target interaction networks [21] , prediction of body fluids [22] , predicting HIV cleavage sites in proteins [23, 24] , predicting protein metabolic stability [25] , predicting signal peptides [26] , identification of DNA Binding Proteins [27] , predicting the network of substrate-enzyme-product triads [28] , predicting protein subcellular locations [29, 30] , predicting proteases and their types [31] , predicting antimicrobial peptides [32] , predicting membrane proteins and their types [33] , predicting GPCRs and their types [34] , identifying nuclear receptor subfamilies [35] , predicting gram-negative bacterial protein cellular locations [36] , and predicting transcriptional activity of multiple site p53 mutants [37] , can provide many useful insights and data for which it would be time-consuming and costly to obtain by experiments alone. Actually, these data, combined with the information derived from the structural bioinformatics tools (see, e.g., [38] ), can timely provide very useful insights for both basic research and drug development. In view of this, the present study attempts to develop a new computational modeling method in the hopes it may become a useful tool for the drug development.
A quantitative structure-activity relationship (QSAR) study is a helpful approach to quantitatively understand the relationships between molecular structures of inhibitors and their biological activities [39] [40] [41] [42] [43] [44] [45] [46] . Pharmacophore modeling and 3D-QSAR studies have been successfully applied previously for various drug discovery research, including glycoprotein (GP) IIb/IIIa antagonists, H 3 -antihistaminics, and dihydrofolate reductase inhibitors [47] [48] [49] [50] [51] . Electronic molecular features such as electron density, frontier molecular orbital density fields such as lowest unoccupied molecular orbital (LUMO), highest occupied molecular orbital (HOMO) and molecular electrostatic map have also been revealed to be significant in other QSAR studies to explain biological activity and molecular properties [52] . The HOMO density field was useful in a study of ACE inhibitors, and the LUMO density field was found to be important for explaining the TA100 mutagenicity [53, 54] . Thus, determining molecular electronic properties responsible for the potent activity of selected chymase inhibitors should illuminate the fundamental molecular level forces responsible for their potency.
Various QSAR studies for chymase inhibitors have also been performed. The QSAR analysis of anhydride-type chymase inhibitors showed that aromatic substituents played an important role in determining the inhibitory potency of the compounds [55] . While, Hayashi and coworkers showed that introduction of various substituents in chloromethyl ketone derivatives resulted in a variation in their activity against human chymase [18] . A 3D QSAR model for the identification of stable chymase inhibitors has also been developed by Yuuki et al. 2003 [56] .
The subject of the present study is to develop QSAR models and explore the key molecular features of chymase inhibitors influencing the protein-ligand binding and interaction, by exploring the dependence of inhibitory activities upon various physiochemical properties of these compounds. In order to accomplish these tasks, an exclusive computational strategy is applied by using various QSAR model building techniques such as pharmacophore modeling, molecular docking, and Density Functional Theory (DFT) (Figure 2 ). In the first phase of calculations, a pharmacophore model (Hypo1) comprising key chemical features for the identification of novel and diverse chymase inhibitors has been generated. After validation, this pharmacophore model is used as a 3D structural search query to find new classes of compounds with similar chemical features from chemical databases. The obtained hits are scrutinized based on their estimated activity and calculated drug-like properties. Molecular docking is also performed for the evaluation of compounds for important binding site interactions and affinity. Finally, we have carried out DFT-based QSAR studies on a set of chymase inhibitors retaining structural diversity and a wide biological activity range, along with potent hits retrieved by newly developed pharmacophore model (Hypo1). The objective of this DFT study is two-fold. One purpose is to derive the QSAR model itself and the other is to scrutinize the usefulness of conceptual DFT quantities. Moreover, it also served as a validation technique for the generated pharmacophore model. Various electronic properties such as LUMO, HOMO, and locations of molecular electrostatic potentials, are computed. The results of this study are expected to explore the crucial molecular features contributing to binding specificity and be useful for understanding the molecular mechanism by which these compounds act and can be further utilized to get compounds with better activity by rational modification.
One of the main objectives of the present study is to generate a pharmacophore model for the identification of novel chymase inhibitors. To accomplish this, ten hypotheses with imperative statistical parameters were generated by HypoGen module of DS using a training set of 20 compounds (Figure 3 ).
The hypotheses are generated with cost functions and correlation values by which they are estimated. The fixed cost, total cost and null cost values are calculated by HypoGen module during the hypotheses generation. The fixed cost is the lowest possible cost representing a hypothetically simplest model that fits all data perfectly, whereas the null cost value is equal to the maximum occurring error cost. For a more statistically significant hypothesis, there should be greater difference between these two cost values. The possibility of correlating the experimental and estimated activity data enhances to 75-90% with a cost difference of 40-60 bits between the total and null cost values [57, 58] . In the present work, the null cost value of the top 10 hypotheses is 182.366 and the fixed cost value is 75.791. Thus, a difference of 106.575 bits between fixed cost and null cost consigns to a meaningful pharmacophore model. Moreover, the total cost of the generated hypothesis should be closer to the fixed cost. All ten generated hypotheses scored a total cost closer to the fixed cost which leads to a good model. Statistically significant factors which include cost values, correlation coefficients (r), pharmacophore features, and root mean square deviations (RMSDs) of all 10 hypotheses are listed in Table 1 . The configuration cost enumerates the entropy of the hypothetical space and its value should not exceed a maximum value of 17 for a significant pharmacophore model [59, 60] . The configuration cost value of 16.601 was obtained for this pharmacophore generation calculation.
Seven of the 10 hypotheses were made of five pharmacophoric features while another three had shown four features. The HY-AR was the common feature among all hypotheses. Nine of the 10 hypotheses had Hydrogen-bond acceptor (HBA), three hypotheses had ring aromatic (RA) while only one hypothesis was made of hydrogen bond donor (HBD). Hypo1 consists of two HBA and three HY-AR features and scored the better correlation and cost difference values. The RMSD value indicates the quality of "prediction" for the training set. The RMSD of all ten hypotheses ranged from 1.176 to 1.421 Å while the Hypo1 showed the lowest RMSD value of 1.176 Å. The correlation coefficient for the Hypo1, 0.942, represents a good correlation by linear regression of the geometric fit index. All these results construe that Hypo1 is the best ranking pharmacophore model among other hypotheses ( Figure 4 ). On the basis of the activity, compounds belonging to training and test sets were categorized into activity scales: most active (++++, IC 50 (inhibitory concentration) < 20 nM); moderately active (+++, ≥20 IC 50 < 200 nM); less active (++, ≥200 IC 50 < 2000 nM); inactive (+, IC 50 ≥ 2000 nM). Activities of all compounds were estimated based on the best ranking pharmacophore model, Hypo1. The experimental and estimated activity values for the 20 training set compounds based on Hypo1 are listed in Table 2 . Analysis of the activity prediction of training set compounds revealed that all the most active compounds were predicted in the same scale, whereas only one moderately active compound was estimated as less active and three inactive compounds were estimated as less active compounds among the 20 compounds of training set. The estimated activity values of most and least active compounds of the training set based on Hypo1 were 0.27 and 4800 nM, respectively, which are very close to that of their experimental activity values (0.46 and 5900 nM). This result revealed that the structural characteristics which can explain the difference in their biological activities are present in Hypo1 ( Figure 5a ). The most active compound 1 could map all the features of the best pharmacophore model, Hypo1, with a fit value of 9.04. The carbonyl oxygen atoms attached with the piperazine ring and azetidinone moiety were mapped onto the two HBA features. All three phenyl rings present in this most active compound mapped over three HY-AR features. The least active compound 20 in the training set maps Hypo1 with a fit value of 4.79 missed two HY-AR features as compared to compound 1. Carbonyl group of imidazolidine-dione and the only carboxyl group of this least active compound mapped both the HBA features whereas the phenyl ring attached to the imidazolidine-dione mapped over one of the HY-AR features ( Figure 5b ).
The validation of suggested pharmacohore model, Hypo1, was performed by two different validation methods, namely, test set prediction and Fischer randomization methods. A test set containing 97 compounds, representing diverse activity classes and different functional groups, is used in this validation process. These test compounds were imported into the DS and diverse conformers were built in the same manner as for training set compounds. The estimated activities of these test set compounds were calculated based on the geometric fit of these compounds over Hypo1. Analyses of the estimated activities of test set compounds demonstrated remarkable results. From the 97 test set compounds, 94 compounds showed error values less than 5 which is hardly different from the experimental and estimated activity values (Table 3) . Eight out of nine of the most active compounds were estimated in the same activity scale, whereas the ninth compound was predicted as moderately active. Seventeen out of 26 moderately active compounds were estimated in the same scale, whereas the remaining nine were estimated as less active compounds. All the 40 less active compounds were estimated in the less active scale. Furthermore, only three of the 22 inactive compounds were predicted as less active compounds. Thus, the ability of Hypo1 to forecast the activity of test set compounds was very impressive and outstanding. A correlation value of 0.928 was achieved between experimental and estimated activities of test set compounds. A correlation plot showing the correlation between the experimental and estimated activity values of training and test set compounds was generated and displayed in Figure 6 . 
Another validation method based on Fischer randomization was also performed on the training set compounds to verify the quality of Hypo1. In this validation process, a confidence level of 95% was selected and thus 19 spreadsheets (Table 4 ) were generated. The data obtained from this validation method did not produce any better statistical values compared with that of Hypo1. Out of the 19 runs, only three had a correlation value between 0.90 and 0.92 which was comparatively less than the correlation value of Hypo1. The total cost values of all randomized models and RMSD values were higher than Hypo1, which is not appropriate for a good pharmacophore model. Therefore, this validation test also endows the Hypo1 with a high level of assurance.
The suggested pharmacophore model Hypo1 developed so far divulges a fairly accurate idea of the required molecular features for a new lead. Therefore, Hypo1 was applied as a search query to retrieve molecules with novel and desired attributes from chemical databases (Maybridge and Chembridge). A total of 2202 hit compounds, 1478 compounds from Maybridge and 724 compounds from Chembridge, respectively, were obtained. Molecular properties were calculated for all hit compounds retrieved from databases. The 181 hit compounds (124 from Maybridge and 57 from Chembridge database, respectively) with an estimated activity value closer to the most active compound in the training set were selected for further evaluation. These hits were further filtered by using Lipinsiki's rule of five which evaluates drug-likeness, or determines if a chemical compound with a certain pharmacological or biological activity has properties that would make it feasible to be an orally active drug in humans. The 49 compounds of Chembridge database and 23 compounds from Maybridge database have satisfied the requirements of Lipinsiki's rule of five for a drug-like compound. Thus, these 72 hit compounds that satisfied the Lipinsiki's rule of five from a total of 181 hits were subjected to molecular docking.
All of the 20 training set compounds along with the 72 database hits retrieved from the database screening process were docked into the protein active site using the GOLD (Genetic Optimization for Ligand Docking) docking program. GOLD fitness score which differentiates molecules on account of their interacting pattern is calculated for all molecules. The most active compound of training set (compound 1) scored a docking score of 66.6 and exhibited various hydrogen-bonding interactions with the key active site residues (Figure 7a ). Moreover, two of the carbonyl oxygen atoms near the middle ring that mapped on the HBA features of "Hypo1" showed hydrogen-bonding interactions with Gly193 and Ser195 residues of the active site. Previous studies of chymase have also divulged the importance of Gly193 and Ser195 as key amino acids in active site region of the enzyme [9, 13] . Along with diverse hydrogen-bonding contacts, the phenyl group of compound 1, which was mapped on the HY-AR feature of "Hypo1" showed π···σ interactions with the aromatic ring of residue F191. Moreover, compound 1 also showed hydrophobic interactions with Y215 and L99 amino acids. Several hit compounds obtained from database screening process also showed high GOLD fitness scores and formed interactions with the active site residues. The hit compounds that showed a fitness score of more than 66 were selected as final hits for further evaluation process. Intriguingly, all the final three compounds were obtained from Maybridge database and none from the Chembridge database. Compound HTS12673 which showed an estimated activity value of 6.716 nM has scored a GOLD fitness score of 78.73. It has also exhibited key interactions with the important amino acids like Gly193, Ser195, Y215, and H57 at the active site of the enzyme (Figure 7b ). The phenyl part of anisole ring and pyridine ring that mapped over the HY-AR features of "Hypo1" instigated the improved binding of this compound through better hydrophobic interactions. Compound BTB02076, which was also retrieved from the Maybridge database, with an estimated activity value of 8.605 nM has shown a GOLD fitness score of 72.40. This compound has formed various close contacts that lead to the important ligand-enzyme interaction such as hydrogen bonding interactions with Gly193, Ser195 and hydrophobic interactions with Phe191 amino acid in the active site of the enzyme (Figure 7c) . Moreover, important π···π interactions between the fused ring system of BTB02076 and the side chain imidazole ring of His57 amino acid were also revealed. Furthermore, it also showed hydrophobic interactions with Y125 and L99 amino acid residues of protein through the hydrophobic groups mapped over HY-AR features of Hypo1. Third hit, JFD00311, with the estimated activity value of 4.661 nM and GOLD fitness score of 74.51 has formed hydrogen bond network with the active site residues Gly193, and Ser195 (Figure 7d ). The benzene rings and oxygen atoms of the benzenesulfonic acid moieties in this hit compound that overlaid the HY-AR and HBA features of "Hypo1", respectively, enabled considerable hydrophobic and polar interactions with the important amino acids in the active site. The mapping of these top three database final hits on Hypo1 and their 2D molecular structures are depicted in Figures 8 and 9 , respectively. All three hit compounds have mapped the entire features of the best pharmacophore model, Hypo1. Thus, in the design of potent inhibitors of chymase, compounds HTS12673, BTB02076, and JFD00311 which showed important results with respect to all properties such as estimated activity, calculated drug-like properties and better GOLD fitness scores can be proposed as potential leads. Novelty search using SciFinder Scholar and PubChem compound search has also ascertained that these hits were not reported earlier for chymase inhibition.
The electrostatic features impacting the inhibitory effect of chymase inhibitors have been investigated aiming at providing useful information for understanding the structure inhibition relationships of chymase inhibitors. Structures of the most and least active compounds of the training set are optimized along with the three final database hit compounds at B3LYP/6-31G* level. Statistically significant factors such as HOMO, LUMO, and MESP, for all compounds are calculated. According to Fukui's frontier orbital approximation, the frontier orbitals HOMO and LUMO of a chemical species are very important in defining its reactivity. Fukui first recognized the importance of frontier orbitals as principal factors governing the ease of chemical reactions and the stereoselective path while Parr and Yang demonstrated that most frontier theories can be rationalized from DFT.
When the whole dataset of molecules was taken into account, an apparent trend of inhibitory activity (IC 50 ) data with an increase in HOMO energy was observed ( Figure 10 ). For all compounds, HOMO energy ranges between −5.619 and −6.415 eV. High value of E HOMO is likely to indicate a tendency of the molecule to donate electrons to appropriate acceptor molecule of low empty molecular orbital energy. The correlation of HOMO energies with IC 50 data indicates that the HOMO of the inhibitor may transfer its electrons to less energy, LUMO, of some amino residues in the active site of chymase. The calculations show that compounds 1 and 20 have shown the highest (−5.873 eV) and lowest (−6.415 eV) HOMO level energies respectively. This trend is in good agreement with the experimental observations suggesting that compounds 1 and 20 have exhibited the highest (0.46 nM) and lowest (5900 nM) inhibitory profile, respectively, in all investigated chymase inhibitors. While BTB (BTB02076) has shown highest (−5.619 eV) HOMO level energy among hit compounds even higher than HOMO energy level of compound 1, the other two hit compounds also showed higher E HOMO than the least active compound of the data set. In a previous study, a high HOMO energy level also played an important part in activity of the most active dual and selective LOX inhibitors [61] . Moreover, a clear trend between the inhibitory activity (IC 50 ) data and LUMO energy of all compounds was also revealed. For all compounds, LUMO energy ranged between −0.631 and −2.275 eV. Compound 1 and BTB showed highest LUMO level energies; and least active compounds 19 and 20 demonstrated LUMO with lowest energies.
HOMO and LUMO sites are plotted onto the molecular surface of most active (1) and least active (20) compounds of the data set along with the two hit (BTB, HTS) compounds ( Figure 11 ). Most often, the heteroaromatic rings, which contain the heteroatoms such as nitrogen and oxygen, are the regions in all these compounds that can act as electron donors or acceptors to the active site of the chymase. Experimental study also deduced that introduction of heteroatoms to the inhibitor compound enhanced its stability in human plasma (20) . For instance, the placement of an ethoxy group in compound 2 instigated its stability. Electron donor rings can be identified as those with the greatest electron density from the HOMO. In the case of compound 1, HOMO is scattered over the 4-methylpiperazine moiety together with the carbonyl group and LUMO is spread over the region 2-hydroxyl-4-oxoazetidine containing heteroatoms like oxygen and nitrogen. Docking results also showed that this region of compound 1 is involved in important interactions with the key residues of protein. For compound 20, HOMO is composed of aniline ring and LUMO spreads over sulfonyl and benzoic acid groups. LUMO plot over methylbenzenesulfonamide group in hit compounds BTB showed hydrogen bonding interactions with important amino acids Gly193 and Ser195 at the active site of the enzyme. Whereas the HOMO plot is scattered on 2-methoxyphenol group and dihydroquinazolin moiety, the six membered ring part of dihydroquinazolin group is involved in important π···π interactions with the side chain imidazole ring of His57 amino acid. For HTS hit compound, HOMO and LUMO are composed of methoxybenzene, benzoindazole moieties, and oxadiazole substituted pyridine moiety, respectively. Overlay of HTS on "Hypo1" and its docking with the protein also speculated the involvement of these groups in key interactions with the active site of protein. The effect of the orbital energies on the inhibition activities can be associated with the charge transfer, π···π, or π···σ stacking between inhibitors and aromatic amino acid residues in the binding site of chymase. The result of molecular docking studies on chymase inhibitors also proved the presence of such kind of interactions.
Electrostatic potential is widely used in characterizing molecules, especially for biomolecules, and takes special effect in the biomolecular recognition and in the prediction of the functional sites [62] . Nam et al. reported their discovery that electrostatic interactions accounted for the majority of the rate acceleration in the mechanism of RNA transphosphorylation in solutions catalyzed by the hairpin ribozyme [63] . Daga and Doerksen have stated the binding mode and the role of stereoelectronic properties in binding of spiroquinazolinones showing phosphodiesterase 7 (PDE7) inhibitory activities [64] . Recently, the electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential was reported by Dehez et al., driving the diphosphate nucleotide rapidly toward the bottom of the internal cavity of membrane-protein mitochondrial ADP/ATP carrier by forming a privileged passageway [65] . Considering these discoveries comprehensively, we supposed that the electrostatic potential of the inhibitor also played a significant role in the binding and interaction with chymase together with orbital energy and consequently influenced the inhibition effect. The 3D isosurface maps of MESP were interpolated on the electron density surfaces of constant electron charge density (0.0004 e/au 3 ). As is well known, the electrostatic potential is defined as the interacted energy of a positively remote charge point with the nuclei and the electrons of a molecule. The 3D plots of electron density (ED) and the MESP for compounds 1, 20, BTB and HTS are shown in Figure 12 . The red and the blue color represent the electronegative and electropositive potentials whereas the green represents a potential halfway between the two extremes.
The coloring area of the surface represents the overall molecular charge distribution with the electrostatic potential. As for the compounds in this study, the electronegative potential (MESP min ) was coded with red on the MESP maps in a range from 202.16 to 152.27 kcal/mol indicating a strongest attraction while the interpolated blue map represents the electropositive potential (MESP max ) of a strongest repulsion varying from 15.68 to 42.67 kcal/mol. The predominance of green region in the MESP surfaces corresponds to a potential halfway between the two extremes that are indicated in red and blue colors, respectively. MESP plotted onto constant electron density surface for most active compound 1 showed the most electronegative potential region (red color) over the oxygen atom of the carbonyl group near the piperazine moiety. However, in the case of the least active compound 20, most negative potentials due to sulfonyl and carbonyl oxygen atoms are missing. For hit compounds, appearance of localized negative potential regions located at the oxygen atoms of the carbonyl and sulfonyl groups and nitrogen of the pyridine ring are consistent with the docking results which recognized this region as hydrogen bond acceptor. Moreover, one more prominent localized negative charged region protruding over the oxadiazole group was oriented adjacent to Gly193, to be recognized as a hydrogen bond acceptor. The strong electrostatic interaction of the negative potential with key residues Gly193 and Ser195, namely the formation of the hydrogen bond, will enhance the inhibition effect substantially together with the orbital interaction through the exchange of energy. The blue electropositive maps of these compounds were mainly distributed over the methyl group. The hydrogen atoms attached to the six-membered rings also bear the maximum brunt of positive charge (blue region). Due to the accumulation of positive potential, these moieties exhibited π···π and π···σ interactions with the aromatic residues of active site. These molecular electrostatic potential features are also in concert with the key chemical features (HBA and HY-AR) of pharmacophore model (Hypo1) which was successfully employed as a 3D structural query for virtual screening of databases for the identification of new potent chymase inhibitors. Thus electrostatic potential of the inhibitors can play a significant role in the binding and interaction with chymase together with orbital energies, and consequently influence the inhibition effect.
A set of 117 structurally distinct compounds reported as chymase inhibitors with their diverse experimentally known inhibitory activity (IC 50 ) data was compiled from the literature such as life science journals [14] [15] [16] [17] [18] [19] [20] 55, [66] [67] [68] . All of the inhibitory activities were obtained using the same biological assay method [14] . To form a training set, 20 compounds with distinctive structural motif and wide activity range (0.46 to 5900 nM) were selected. For all compounds in the training set, energy minimization process was performed with CHARMM forcefield. Poling algorithm was applied to generate a maximum of 255 diverse conformations with the energy threshold of 20 kcal·mol −1 above the calculated energy minimum for every compound in the dataset. These conformers were generated using Diverse Conformer Generation protocol running with Best/Flexible conformer generation option as available in Accelrys Discovery Studio v2.5 (DS), Accelrys, San Diego, CA, USA. This method ensures the best coverage of conformational space by performing a more rigorous energy minimization in both torsional and cartesian space by using poling algorithm.
All the 20 training set compounds associated with their conformations were submitted to the HypoGen module of DS. The HypoGen algorithm implemented for the pharmacophore hypothesis generation process is executed in three phases, namely, constructive, subtractive, and optimization phases. In constructive phase, identification of features common to the most active compounds takes place whereas all pharmacophoric features that are also present in the least active compounds are removed in subtractive phase. Finally, in the optimization phase, the hypothesis score is improved by regression parameters which are used for the estimation of the activity value of each training set compound. The relationship between the geometric fit value and activity value is utilized for this computation. Pharmacophore hypotheses showing best correlation in the 3D arrangement of features in a given training set compounds with the corresponding pharmacological activities are formed and ranked. Several structure activity relationship (SAR) pharmacophore models were derived from training set compounds using HypoGen module of DS. In this study, the top 10 hypotheses which were returned by the hypotheses generation process with significant statistical parameters were selected for further calculations.
The generated quantitative pharmacophore model was validated to find out whether it is competent enough to identify the active structures and estimate their activity values precisely. This validation process was performed based on test set prediction and Fischer randomization methods. In developing statistical models, the following three cross-validation methods are often used to examine a model or predictor for its effectiveness in practical application: independent dataset test, subsampling test, and jackknife test [69] . However, of the three test methods, the jackknife test is deemed the most objective [29] . The reasons are as follows. (i) For the independent dataset test, although all the samples used to test the model or predictor are outside the training dataset used to train it so as to exclude the "memory" effect or bias, the way of how to select the independent samples to test the model or predictor could be quite arbitrary unless the number of independent proteins is sufficiently large. This kind of arbitrariness might result in completely different conclusions. For instance, a model or predictor achieving a higher success rate than the other model or predictor for a given independent testing dataset might fail to keep so when tested by another independent testing dataset [69] ; (ii) For the subsampling test, the concrete procedure usually used in literatures is the 5-fold, 7-fold or 10-fold cross-validation. The problem with this kind of subsampling test is that the number of possible selections in dividing a benchmark dataset is an astronomical figure even for a very simple dataset, as elucidated demonstrated by Equations 28-30 in [70] . Therefore, in any actual subsampling cross-validation tests, only an extremely small fraction of the possible selections are taken into account. Since different selections will always lead to different results even for a same benchmark dataset and a same model or predictor, the subsampling test cannot avoid the arbitrariness either. A test method unable to yield a unique outcome cannot be deemed as a good one; (iii) In the jackknife test, all the samples in the benchmark dataset will be singled out one-by-one and tested by the model or predictor trained by the remaining samples. During the process of jackknifing, both the training dataset and testing dataset are actually open, and each sample will be in turn moved between the two. The jackknife test can exclude the "memory" effect. Also, the arbitrariness problem as mentioned above for the independent dataset test and subsampling test can be avoided because the outcome obtained by the jackknife cross-validation is always unique for a given benchmark dataset. Accordingly, the jackknife test has been increasingly and widely used by those investigators with strong math background to examine the quality of various predictors (see e.g., [30, [71] [72] [73] [74] [75] ). However, to reduce the computational time, we adopted the independent testing dataset cross-validation in this study as done by many investigators with SVM as the prediction engine. A test set comprising 97 compounds with experimentally known chymase inhibitory activity values was used in test set prediction method. Ligand Pharmacophore Mapping protocol running with BEST/Flexible conformation generation option was used to map the test set compounds. Fischer randomization method as available in DS was applied on training set compounds to prove that the generated pharmacophore model was not obtained by chance. A pharmacophore is only useful as a predictive model in finding novel, potential leads suitable for further development only if it is able to detect the compounds with known inhibitory activity. In order to identify new potential lead compounds, the selected pharmacophore model was subsequently used as 3D structural search query to screen the Maybridge and Chembridge chemical databases consisting of 60,000 and 50,000 of structurally assorted compounds, respectively. All queries were performed using Ligand Pharmacophore Mapping protocol running with Best/Flexible search method in DS. To be retrieved as a hit, a molecule must fit all the features of the pharmacophore hypothesis. The hits obtained through database screening were further filtered using Lipinsiki's rule of five in order to carry only drug-like compounds in further studies.
Computational docking operation is a useful vehicle for investigating the interaction of a protein receptor with its ligand and revealing their binding mechanism as demonstrated by a series of studies [18, 46, [76] [77] [78] [79] [80] [81] [82] [83] [84] . Docking plays a significant role in predicting binding orientation and affinity of small molecule drug candidates to their protein targets with known 3D structures [85, 86] . Hence, docking serves as an important tool in the rational computer-assisted drug design [87, 88] . GOLD 4.1 (Genetic Optimization for Ligand Docking) from Cambridge Crystallographic Data center, UK uses a genetic algorithm for docking ligands into protein binding sites to explore the full range of ligand conformational flexibility with partial flexibility of protein [89] . In this study, it has been utilized for the docking of training set compounds along with the new hits retrieved from chemical databases. Protein coordinates from the crystal structure of chymase co-crystallized with β-ketophosphonate (PDB ID: 1T31), determined at a resolution of 1.9 Å were used to define the active site [9] . All the water molecules present in the protein were removed and hydrogen atoms were added. The active site was defined with a 10 Å radius around the ligand present in the crystal structure. At the end of the computation, the 10 top-scoring conformations of every ligand were saved. Early termination option was applied to pass over the genetic optimization calculation when any five conformations of a particular compound were envisaged within an RMS deviation value of 1.5 Å. The GOLD fitness score is calculated from the contributions of hydrogen bond and van der Waals interactions between the protein and ligand, intramolecular hydrogen bonds and strains of the ligand. The protein-ligand interactions were scrutinized by DS.
As far as computational technique is considered, many practices have ascertained that DFT, which takes into account the exchange and correlation effects effectively, is most likely one of the best methods to study medium-size or larger molecular systems and appropriate for QSAR study, with exhibiting excellent performance than semiempirical method or some other ab initio methods. Complete geometry optimization for data set compounds was carried out using DFT with Becke's three-parameter exchange potential and the Lee-Yang-Parr correlation functional (B3LYP), using basis set 6-31G* level [90] . A useful kind of net atomic charges, called electrostatic potential (ESP)-fitting charges, were derived from the DFT calculated molecular electrostatic potential distribution using CHelpG method, which produces charges fit to the electrostatic potential at points selected. Vibrational frequencies were computed at the same B3LYP/6-31G* level to characterize the stationary points on the corresponding potential energy surfaces. All calculations were performed using the Gaussian 03 suite of programs.
Based on structural diversity and wide biological activity range, four chymase inhibitors including most and least active compounds, were selected from the training set. While, three final hits BTB02076, HTS12673, JFD00311 retrieved from Maybridge database by the selected pharmacophore model, which showed important results with respect to all properties like molecular interactions with the active site components, estimated activity, calculated drug-like properties, and high GOLD fitness score, were also selected. Thus, data set employed for DFT study consisted of seven compounds. Various quantum-chemical descriptors such as LUMO, HOMO, and locations of molecular electrostatic potentials (MESP), were computed.
The mapping of the electrostatic potential is an established technique for investigation of biologically active compounds because it plays a key role in the initial steps of ligand-receptor interactions. The formatted checkpoint files of the compounds generated by the geometric optimization computation were used as input for CUBEGEN program interfaced with Gaussian 03 program to compute the MESP. The MESP isopotential surfaces was produced and superimposed onto the total electron density surface (0.0004 e/au 3 ). The electrostatic potential of the whole molecule is finally obtained by superimposing the electrostatic potentials upon the total electron density surface of the compound.
Since user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful models or predictors [91] , we shall make efforts in our future work to provide a web-server for the method presented in this study.
Combining various theoretical methods like pharmacophore modeling, database screening, molecular docking and DFT calculations, an investigation for identification of novel chymase inhibitors and to specify the key factors crucial for the binding and interaction between chymase and inhibitors was performed. The highly correlating (r = 0.942) pharmacophore model (Hypo1) with two hydrogen bond acceptors, and three hydrophobic aromatic features was generated. After successfully validating "Hypo1" using test set and Fischer randomization methods, it was further used in database screening. Hit compounds were subjected to various drug-like filtrations and molecular docking studies. Finally, three structurally diverse compounds with high estimated activity and strong molecular interactions with key active site amino acids were identified. Furthermore, a DFT study, which articulated the influence of the electrostatic features of compounds on their inhibitory activity well, was performed. Analysis of orbital energies and plots of MESP has shown very clear trends between electronic properties and inhibitory activity (IC 50 ) data. An increasing trend was observed between IC 50 and HOMO energy values. The molecular electrostatic potential features were also consistent with the key chemical features of "Hypo1" thus successfully validating "Hypo1" by the DFT method. Therefore, the results of this study will be helpful, not only in the development of new potent hits for chymase, but also in providing a better understanding of the interaction between the chymase and inhibitors. This will in turn assist in the rational design of novel potent enzyme inhibitors. Perspectives on Immunoglobulins in Colostrum and Milk Immunoglobulins form an important component of the immunological activity found in milk and colostrum. They are central to the immunological link that occurs when the mother transfers passive immunity to the offspring. The mechanism of transfer varies among mammalian species. Cattle provide a readily available immune rich colostrum and milk in large quantities, making those secretions important potential sources of immune products that may benefit humans. Immune milk is a term used to describe a range of products of the bovine mammary gland that have been tested against several human diseases. The use of colostrum or milk as a source of immunoglobulins, whether intended for the neonate of the species producing the secretion or for a different species, can be viewed in the context of the types of immunoglobulins in the secretion, the mechanisms by which the immunoglobulins are secreted, and the mechanisms by which the neonate or adult consuming the milk then gains immunological benefit. The stability of immunoglobulins as they undergo processing in the milk, or undergo digestion in the intestine, is an additional consideration for evaluating the value of milk immunoglobulins. This review summarizes the fundamental knowledge of immunoglobulins found in colostrum, milk, and immune milk. The topic of immunoglobulins in milk immediately brings to mind the relationship between mother's milk, transfer of passive immunity from mother to neonate, and the immature immune system of the neonate. Research in this field dates back to the late nineteenth century, however for many centuries herdsmen have capitalized on the linkage between maternal immune status and the immunological protection and development of the neonate [1, 2] . Immunoglobulins in mammary secretions come from several sources and represent a history of the antigen exposure of the mother and the response of her immune system. Immunoglobulins are transported through the mammary epithelial cells by receptor-mediated mechanisms and transferred out of the mammary gland by milk ejection during suckling. The immunoglobulins then enter the environment of the gastrointestinal tract of the neonate. Although that environment is primarily geared toward digestion to gain nutritional benefit, the immunoglobulins remain sufficiently stable to provide protective benefits for the neonate, either through uptake into the vascular system in the newborn of some species or through immunological function in the gastrointestinal tract. The immunoglobulins found in milk and the transfer of passive immunity from mother to neonate have been reviewed by many authors, with a partial listing referenced here [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] .
In addition to the importance of homologous transfer of passive immunity between mother and neonate, there is considerable interest in the potential for heterologous transfer of passive immunity, such as immunoglobulins obtained from one species and utilized for passive immunity in another species. The ability to manipulate the immunological status of animals through vaccination against diseases that affect humans and the opportunity to harvest those immunoglobulins in the form of colostrum or milk has long been recognized [19, 20] , and continues to be a topic of interest in both animal science and human medicine [13, 16, 17, [21] [22] [23] .
This review begins with a summary of some of the research on what has been termed -immune milk‖ and then discusses various aspects of immunoglobulins in mammary secretions (structure, function, concentration, sources, transport, species differences, and roles of immunoglobulins). Finally, traits related to stability and processing methods for collecting milk immunoglobulins are reviewed.
One intriguing application of our knowledge about bovine colostral and milk immunoglobulins comes through the opportunity to provide passive immunity against diseases in other species, especially in humans. The ability to direct the cow's immune system to produce antigen-specific antibodies that are secreted in colostrum and milk and may be used to provide protection against a specific disease continues to be an area of interest. For example, the widespread consumption of immune milk from cows inoculated against diseases such as avian influenza, SARS, and other human respiratory diseases, has been suggested as a potential means of slowing outbreaks of the disease before they reach epidemic levels [24] . A number of reviews have summarized and evaluated early attempts to develop and test the use of immune milk products to provide passive immune protection [21] [22] [23] [25] [26] [27] [28] [29] [30] [31] . Several immune milk products are available commercially [13, 17, 22, 23, 32] . Safety issues associated with use of bovine immune milk products for human use have been discussed by others [23, [33] [34] [35] . The discussion below provides some examples of immune milk products and their use against some animal and human diseases (sections 2.2-2.7).
Secretion of antibodies in breast milk from naturally immunized mothers can provide protection against enteric and other diseases in children [11] . For example, elevated concentrations of antibodies specific for enteric pathogens, such as Vibrio cholerae, in the mother's breast milk do not prevent colonization with the bacterium in the nursing child, but do seem to protect the infected child from developing diarrhea [36] . Breast feeding is associated with a reduced incidence of Campylobacter diarrhea in young children compared with children that do not breast feed [37] . In those children that are breast fed and do develop diarrhea, the human milk consumed may not contain IgA antibodies specific for the common antigen of Campylobacter [37] , suggesting a degree of antigen specificity contained in the breast milk.
The idea of immunizing the pregnant animal with the intent of controlling neonatal morbidity and mortality is well established [38] . Vaccination or natural immunization of the pregnant cow, ewe or sow against enterotoxigenic Escherichia coli [38] [39] [40] or intestinal viruses [41, 42] , can provide a degree of protection for the newborn. As an example, while only limited protection against viral challenge occurred in calves fed once shortly after birth with a pooled colostrum from cows immunized against bovine rotavirus, a shorter duration of diarrhea was observed [43] . On the other hand, calves fed milk supplements with low levels of a similar immune colostrum at each feeding for two weeks did have reduced virus sheading and reduced incidence of diarrhea [44] .
In primates, immunization of pregnant baboons with a rhesus rotavirus vaccine increased milk immunoglobulin and virus neutralizing titre [45] . Prenatal immunization of pregnant women with a single dose of meningococcal vaccine not only increased antigen-specific IgG antibody in the infant's serum during the initial 2-3 months after birth, but antigen-specific IgA in milk continued to be elevated at least up to 6 months [46]. As discussed in section 4, IgG transfer to the offspring in humans occurs during late pregnancy and provides the initial systemic source of that immunoglobulin. Infants consuming breast milk will primarily be consuming secretory IgA (section 4), which has significant protective activity in the intestine, as discussed in section 5.2.
The above examples of homologous transfer of passive immunity set the stage for considering the opportunities for heterologous passive transfer. Immune milk products generally are some form of protein product derived from the colostrum and/or milk of dairy cattle. The cows typically are hyperimmunized against one or more antigens representing pathogens of bacterial or viral origin. Crude preparations of the immunoglobulin from colostrum or milk may range from essentially no alteration of the immunoglobulin concentration in the product to partial immunoglobulin isolation or concentration in a whey protein concentrate.
The primary immunoglobulin in cow colostrum and milk is IgG, whereas the primary immunoglobulin in human milk is IgA [1] . Nevertheless, bovine IgG from colostrum or milk can be effective as a means of providing passive immunity to protect animals and humans from disease. The use of bovine colostral immunoglobulin preparations from immunized cows for disease protection of the neonate of other species has been demonstrated in swine [47] , and experimental animal models such as mice [48, 49] . There also are a number of examples of the use of bovine immune milk products in the treatment or prevention of human disease, especially in cases where the pathogen acts by way of the gastrointestinal tract. When considering these studies, it should always be kept in mind that the colostrum or milk preparations potentially contain other immune modulating substances than immunoglobulins, as discussed briefly below (section 6.3).
The concept of using immune milk derived from hyperimmunized cows for treatment of human disease can be traced back to the 1950s and earlier [19, 20] . Some of the early efforts in this field involved using immune milk products for treatment of rheumatoid arthritis and hay fever [19] . Immune milk preparations produced from milk from cows immunized with a heat-killed, lyophilized mixture of bacteria found to reside in the human gastrointestinal tract has been studied for the prevention and treatment of rheumatoid arthritis, high blood cholesterol, high blood pressure, and oral submucous fibrosis [50] [51] [52] [53] . On the other hand, most studies on the use of immune milk have examined the potential of immune milk for prevention and treatment of infectious diseases, particularly gastrointestinal disease.
Even milk that does not come form hyperimmunized cows may in some sense be regarded as immune milk. Bovine anti-human rotavirus IgG1 antibodies have been found in raw and pasteurized milk from cows that had not been specifically immunized against that virus [54] . Milk from non-immunized cows also has been found to contain measurable antigen-binding activity against several human pathogenic bacteria [55] .
Several authors have tested the efficacy of immunoglobulin preparations with antibody activity against human rotavirus as a means of providing passive immunity to children. For example, children consuming a defatted colostrum preparation from cows immunized against a strain of human rotavirus had no improvement of symptoms when the infection was established (patients admitted to a hospital with rotavirus infection), however the preparation was effective in limiting diarrhea in children when consumed prior to the infection [56, 57] . In another study, cessation of excretion of rotavirus in the stool of infants with acute rotavirus gastroenteritis was correlated with the presence of neutralizing activity in the stool after ingestion of a bovine whey protein concentrate from rotavirus-hyperimmunized cows [58] , although there was not a significant decrease in duration of diarrhea in that study. Other studies have found that treatment of children with hyperimmune bovine colostrum from cows immunized with human rotavirus serotypes reduces the duration and severity of diarrhea due to rotavirus [59] , and can provide significant protection from rotavirus infection [60] .
Enteropathogenic bacteria have also been the target for development of immune milk. Over 80% of childrens' stools became negative for the E. coli strains used to hyperimmunize the cows that provided the source of immunoglobulin in a bovine colostrum/milk immunoglobulin concentrate consumed by children for 10 days [61] . Interestingly, only one in nine children treated with the immunoglobulin concentrate, and having diarrhea that was associated with E. coli strains which were not used in the immunization of the cows, developed negative stools, underscoring the importance of the bacterial strain-specificity of the immune product. Consumption of a hyperimmune immunoglobulin concentrate with a high antibody titer against a lipopolysaccharide isolated from Shigella flexneri 2a also has been shown to provide protection against a challenge with the same strain [62] . However, no difference in diarrhea or other symptoms in children with stools positive for S. dysenteriae was found whether treated with bovine colostrum from cows immunized against S. dysenteriae or with colostrum from cows not hyperimmunized [63] .
Enterotoxigenic E. coli also is commonly associated with traveler's diarrhea. Prophylaxis against this infection may be achieved by providing passive immunity with immune milk. A bovine whey protein concentrate from cows immunized with enterotoxigenic E. coli serotypes and consumed 3-times daily for seven days protected all of the adult volunteers from developing diarrhea after being challenged with an enterotoxigenic E. coli strain [64] . In contrast, 90% of the volunteers who received control immunoglobulin concentrate prior to challenge developed diarrhea after the E. coli challenge. Subsequent studies using IgG isolated from bovine colostrum from cows hyperimmunized against specific E. coli colonization factor antigens also have shown protective effects in volunteers challenged with colonization factor antigen-bearing enterotoxigenic E. coli [65] , however other studies by the same group did not demonstrate significant effects of similar milk immunoglobulin products [66] .
Bovine colostrum concentrate preparations derived from cows that have not been hyperimmunized against specific antigens also may provide some benefit via passive immunization for some diseases. For example, a commercial product which is made from large standardized pools of colostrum collected from over 100 cows has been used to treat a number of diseases [22, 23] , including diarrhea caused by diarrheagenic E. coli [67] . Similar preparations from non-immunized cows may provide protection against bacterial toxins that are the cause of diarrhea in AIDS patients [68] . These studies, along with the above mentioned study comparing colostrum preparations from cows immunized against S. dysenteriae or non-immunized cows [63] , demonstrate that bovine colostrum contains significant antimicrobial properties as a result of natural exposure of the cows to antigens of pathogens that may afflict humans.
Another example of a potential use for bovine immunoglobulin preparations to control bacterial populations comes from studies on dental caries formation [69] . The concept of prenatal immunization of the pregnant mother to protect the neonate against dental caries was demonstrated in rats [70] . In applications to humans, bovine whey preparations of colostrum from cows immunized with caries-inducing bacterial strains (Streptococcus mutans and Streptococcus sobrinus), and containing over 60% immunoglobulin of which 80% was IgG1, has been used in several studies evaluating its effect on caries-producing bacteria. The colostral whey preparation reduced adherence of Streptococcus mutans in vitro and caused aggregation of suspended bacteria [71] , as well as inhibited glucose uptake by the test organism [72, 73] . The whey preparation from hyperimmunized cows opsonized bacteria and enhanced in vitro phagocytosis of bacteria by human leukocytes [74] .
Antibodies in the whey preparation remained functional when added to milk that had been treated via ultra-high temperature pasteurization or milk that was fermented to extend shelf-life [75] .
Immune milk from cows hyperimmunized against seven Streptococcus mutans strains reduced the recoverable bacterium in plaque samples from volunteers within seven days of initiation of mouth rinsing with the whey concentrate product [76] . Mouth rinsing with immune milk collected from cows immunized with a fusion protein representing two of the major factors implicated in oral colonization by Streptococcus mutans inhibited recolonization of saliva and plaque by that organism [77, 78] .
Immunodeficiency disorders often are associated with cryptosporidiosis, which can lead to chronic malabsorption and weight loss. In a case study of a child with congenital hypogammaglobulinemia, severe vomiting and diarrhea due to cryptosporidiosis, gastric infusion with hyperimmune bovine colostrum from cows immunized with cryptosporidium oocytes resolved the symptoms within a few days and oocyts were no longer found in stool samples after about eight days [79] . Similarly, in a child with AIDS who had severe diarrhea caused by cryptosporidiosis, administration of a commercial hyperimmune bovine colostrum preparation with anticryptosporidial activity improved the diarrhea and eliminated the parasite [80] .
In the cases where immune milk is collected from cows immunized against one or more pathogens, the immunization regimen occurs during the prepartum period of the cow. To put this in perspective relative to the lactation cycle of a cow, a brief reminder of that cycle may be helpful. Depending on the management system used by a farm, most dairy cattle will have their first calf early in their third year, marking the start of their first lactation. The cow will be re-bred about two to three months into lactation. Pregnancy is approximately 280 days. At about 2 months before expected calving date, or approximately 10 months into lactation, milk removal is halted and the cow is given what is called a -dry‖ period. The mammary gland undergoes a process of involution during the early dry period where most residual milk components are broken down and resorbed [81] . The mammary gland begins a redevelopment phase several weeks prior to calving. Colostrum formation occurs in the days leading up to calving, coinciding with the early phase of lactogenesis (initiation of lactation). In the cow, lactogenesis begins shortly prior to calving and extends into the first few days postpartum. Colostrum collected at the first milking of the cow after calving represents the accumulation of colostral products during the days leading up to parturition, including immunoglobulins which are at their highest concentration in the first milking. Concentrations of immunoglobulins then decline rapidly in the subsequent several milkings [82] .
One application for immunization of pregnant or lactating animals comes from the arena of mastitis control in cattle. Mastitis is the major disease in dairy cattle and most often is caused by intramammary infection [83, 84] . Vaccination of cattle against mastitis-causing pathogens has been an area of study for many years [85, 86] . Optimization of immunization schedules continues to be investigated [87] . Effective vaccines against mastitis-causing pathogens can increase antigen-specific immunoglobulins in the serum, which in turn can be increased in the mammary secretions. In the case of the J5 E. coli bacterin vaccine, the immunization also may be causing the mammary gland to become hyper-responsive to bacterial challenge [88] , reminding us that enhancement of antigen-specific antibodies in the milk is not the only mechanism by which the vaccine may be having its effect.
Because the peripartum and early lactation periods are times of high susceptibility of the mammary gland to mastitis, many immunization schedules include prepartum immunizations during the dry period when milk is not removed and the mammary gland undergoes involution. It is also important to remember that cattle are generally immunosuppressed during the peripartum period [88, 89] , potentially compromising the impact of immunizations administered just before or just after calving.
Coliform mastitis is one of the major types of mastitis in cattle [90] . The more successful vaccination protocols for mastitis control have been with the J5 E. coli bacterin vaccine which is administered initially either just before or at the time of drying off [87, [91] [92] [93] [94] [95] [96] . These typically are followed by additional vaccine doses approximately mid-dry period. Some protocols include an additional immunization within several days after calving [87, 91, 92, 94] , while others also continue immunizations into the first three months of lactation [87, 94] . Attempts to vaccinate against other mastitis-causing pathogens have been met with more limited success. Such vaccination protocols range from immunizations during the dry period [97] , to peak lactation [98] , and even late lactation [99] . Although most of the immunization protocols used in mastitis control administer the vaccine either intramuscularly or subcutaneously, intramammary immunization also can result in an increase in antigen-specific immunoglobulin in milk, as well as in the serum [100] [101] [102] [103] .
A look across the immunization protocols used in studies to produce many immune milk products shows considerable variation, especially in the number and timing of immunizations. In those specifically collecting colostrum shortly after calving, multiple immunizations are administered during late pregnancy when the cow would be in the dry period [56] [57] [58] 60, 61, 64, 71, 72, 77, 79, [104] [105] [106] . Mammary secretions then are collected either only at first milking [79] , pooled from the first 4 to 6 milkings [56, 57, 72, 104] , pooled from the first 6 to 10 days after calving [58, 61, 64, 105] , or collected for longer periods into lactation [77] . Other studies have initiated immunizations during the late dry period and then continued vaccinating throughout lactation [50,51,76], or only vaccinating during lactation [107] . Many of these studies used intramuscular or subcutaneous immunization, although some also have incorporated intramammary [58, 79, 105] , or intravenous infusion [61] .
Newer technologies for vaccine development and delivery may further enhance the production of immune milk products. Immunization protocols that expose animals to specific antigens may enhance humoral immune responses in the mammary gland, including peptide-based vaccines [108] , and DNA-based vaccines [109, 110] . Delivery of antigen to the animal can also be achieved with antigen encapsulated in biodegradable microspheres [111] , and with antigen-release devices [112] . Transgenic animals also have been used to produce antigens that then may be used to vaccinate animals against viral disease [113] .
The immunoglobulins, or antibodies, found in colostrum or milk are the same as those found in the blood or mucosal secretions. They are a family of proteins with a range of protective bioactivities. Immunoglobulins are divided into several classes including IgM, IgA, IgG, IgE, and IgD [114] , and IgG, IgA and IgM are the major immunoglobulin classes in mammary secretions. IgM is the class that appears initially when an organism is exposed to an antigen for the first time (primary infection). IgM has a low specificity and hence a lower potency in defeating the infection. IgA is the major immunoglobulin class found in mucosal secretions and prevents mucosal infections by agglutinating microbes, whereas IgG is the primary immunoglobulin class found in bovine colostrum and milk. Several subclasses of IgG exist, with IgG1 and IgG2 being the major immunoglobulins in serum. All monomeric immunoglobulins have the same basic molecular structure, being composed of two identical heavy chains and two identical light chains, with a total molecular mass of approximately 160 kilodaltons (for details on immunoglobulin structure see [5, 14, 16] ). Both the heavy and light chains have constant regions and variable regions. Heavy and light chains are linked together by disulfide bonds, resulting in the classic Y-shape of the immunoglobulin molecule [114] . The number and location of the disulfide bonds is dependent on the class of immunoglobulin. Each immunoglobulin molecule has two antigen binding sites which comprise the antigen-binding fragment (Fab). The Fab includes the variable amino acid domain. At the other end of the molecule is the constant fragment (Fc) which has a constant amino acid sequence among molecules of the same subclass and which confers the identity of an immunoglobulin as a particular subclass. The Fc region of the molecule is the region that binds to Fc receptors on various cell types.
In the case of polymeric immunoglobulins, including the polymeric forms of IgA and IgM that are found in milk, the monomeric forms of the immunoglobulins are linked together through the covalent interaction with a joining (J) chain [114, 115] . The result is a dimeric form of IgA and a pentameric form of IgM. Binding of these immunoglobulins to the J chain also results in them having several special features, including: a high valency of antigen-binding sites, allowing them to agglutinate bacteria; limited complement-activating activity, which allows them to act in a noninflammatory manner; and a high affinity for the polymeric immunoglobulin receptor (pIgR) that is responsible for transepithelial transport of IgA and IgM into mucosal secretions such as milk [116] . The pIgR and its relationship to the secretory component (SC) associated with secretory IgA and secretory IgM is discussed further below (section 5.2).
The content of immunoglobulins in colostrum and milk is highly dependent on the animal species [1, 14] . The same holds for the relative proportion of the immunoglobulin classes. These species differences are adaptations to the reproductive strategies of the animals and the degree of maturation of the offspring at birth. Animal species may be divided into three classes [1] : (1) species where immunoglobulins are transferred mainly to the fetus via the placenta (humans and rabbits);
(2) species where offspring are born agammaglobulinemic and immunoglobulin transmission occurs via mammary secretions (ungulates such as horses, pigs, cows, and goats); and (3) species where immunoglobulins are transferred both via placenta and mammary secretions (rats, mice and dogs).
These adaptations have several consequences both for the composition of immunoglobulins in colostrum and milk, and for the role of colostrum. Indeed, for animals like rats, mice, dogs and ungulates, uptake of colostrum of adequate quality and sufficient quantity is important for the offspring to boost the systemic immune function in the short term, whereas colostrum consumption in the human infant provides more protection for the gastrointestinal tract (see section 6.3). This is reflected in a lower total immunoglobulin content in human colostrum as compared to colostrum from the other species ( Figure 1 ) [1, 3, 117] . Human colostrum has a low content of IgG (2%), and the IgG required to provide systemic immunity is transferred across the placenta before birth. In contrast, colostral IgG content in many other species is typically greater than 75% of the total immunoglobulin content (Figure 1 ). An additional consequence of different routes of immunoglobulin transmission relates to the changes in relative contents of immunoglobulins that occur in the transition from colostrum to milk within certain species (Figure 1 ). For example, the profile of immunoglobulins in human colostrum is similar to that found in milk, where the IgA level is high in both colostrum and milk (88-90% of total immunoglobulin). This is in contrast to the bovine mammary secretions where the high concentration of IgG in colostrum declines rapidly with successive milkings. For animals like rats, mice, dogs and ungulates, the role of colostrum and milk immunoglobulins is to provide immune protection both systemically and for the gastrointestinal tract, which is reflected in large changes in the profile of immunoglobulins during the transition from colostrum to mature milk ( Figure 1 ). Thus, for many species the proportion of IgA increases between colostrum and milk. [1] ; human and pig [3] ; and horse [117] .
Immunoglobulins found in mammary secretions arise from systemic and local sources. In the case of IgG in milk, the major portion comes from the serum [14] . While IgG producing plasma cells may occur within the mammary tissue, their contribution to the IgG in colostrum is minor compared with the IgG derived from the serum.
Although limited paracellular passage of immunoglobulins may occur during inflammation (mastitis), uptake and transport of immunoglobulin across the mammary epithelial barrier is thought to occur primarily through an Fc-receptor-mediated process [1, 7, [118] [119] [120] . Immunoglobulins are thought to bind to receptors at the basolateral surfaces of the mammary epithelial cell. These receptors are specific for the Fc portion of the immunoglobulin molecule. The receptor-bound immunoglobulin is internalized via an endocytic mechanism [121] , transported to the apical end of the cell and released into the alveolar lumen. Recent studies have shed additional light on the details of this process [122] .
In the case of IgG, the receptor responsible for transcytosis of IgG into colostrum is referred to as FcRn, or the neonatal Fc receptor, because it was initially identified in the neonatal rodent intestine as the receptor responsible for the specific uptake of maternal IgG [123, 124] . The FcRn also has been implicated in the trans-placental transport of IgG in humans and other species [125] [126] [127] , which may involve an endocytic and transcytotic process [128] . Since its initial discovery, FcRn has been described in many tissues [122] . The receptor is a heterodimer composed of a membrane-bound α-chain similar to MHC class-1 molecules and a smaller MHC class-1 protein, β2-microglobulin [129] . Binding of IgG to FcRn is pH-dependent, with high affinity binding occurring at acidic pH, but only weak binding at neutral or basic pH [122] . This observation suggests that IgG taken up by the epithelial cells may bind to FcRn within an acidic environment in the endosomes. The precise mechanism of transport across the epithelial cell and release into the colostrum or milk remains to be demonstrated.
The half life of IgG in serum is typically longer (1-3 weeks) than that for IgA or IgM (1-2 days), and the half-life of IgG2 is slightly longer than for IgG1 [122] . Evidence suggests that IgG2 has a higher affinity for FcRn than IgG1 [122] . In bovine colostrum, IgG1 is many fold greater in concentration than IgG2 [82] , although they are of approximately equal concentrations in serum. It may be that the majority of the IgG2 taken up by the mammary epithelial cell during colostrum formation is not passed on to the alveolar lumen, but rather is recycled back to the extracellular fluid. The FcRn is thought to have a major role in the recycling of IgG in various tissues in the body [130] [131] [132] . That is, IgG that potentially may be lost through various tissues is recycled by the respective cells by binding to FcRn and recycled back to the blood or lymph. This is supported by studies of overexpression of FcRn in transgenic mice where there is an extension of the half-life of serum IgG [133, 134] , as well as a boosting of the overall humoral immune response of the mice [135] .
Localization of FcRn in bovine, sheep and water buffalo mammary tissue indicates that the receptor is homogeneously distributed throughout the epithelial cells prior to parturition, but primarily localized at the apical surface of the mammary epithelial cells after parturition [136] [137] [138] [139] . While this type of observation corroborates the conclusion that FcRn plays an important role in IgG transport during colostrum formation, at least in ruminant species, the precise meaning of this redistribution of FcRn staining in mammary cells remains to be determined. It is also interesting to note that the initial report of this distribution pattern in sheep mammary epithelium included the observation that the staining pattern became diffuse within the cells during mammary involution [136, 137] . Transport of IgG also may increase transiently in mammary secretions during involution in cattle [140] . Hormonal and local factors have been implicated in the control of immunoglobulin transport during colostrum formation [32] .
Haplotypes of the FCGRT gene, coding for the MHC Class I α-chain of FcRn, are associated with serum concentrations of IgG in neonatal beef calves [141] and associated with IgG concentrations in colostrum of dairy cows [142] . Haplotypes of the β2-microglobulin gene (β2M) also are associated with serum IgG concentrations in newborn calves [143] . In estimating mass transfer of IgG1 into colostrum in dairy cattle, 10% of cows had mass transfer greater than one standard deviation above the mean, perhaps indicating a genetic or hormonal regulation of the variance of transport [144] . Clearly there is opportunity for genetic manipulation of IgG transport in the mammary gland to enhance the concentrations of immunoglobulins in colostrum and milk. However, it should be remembered that serum IgG concentrations in the periparturient cow are already decreased as a result of the extensive IgG transport into the colostrum [145] , and as indicated above, the cow is in an immunosuppressed state during the peripartum period [88, 89] .
The other major classes of immunoglobulins transported into colostrum and milk are IgA and IgM. Immunoglobulin A is the major immunoglobulin in human colostrum and milk (Figure 1 ), however it is also present in milk of most other species. Colostrum and milk IgA and IgM are found in the form of secretory IgA, or sIgA, and sIgM. Much of these are produced by plasma cells in the mammary tissue. The plasma cells are part of the gut-associated lymphoid tissue (GALT), the largest immune organ of an organism, which includes the Peyer's patches, lymphoid and myeloid cells in the lamina propria and intraepithelial lymphocytes [146, 147] . Lymphocytes from the GALT system migrate to the mammary gland and provide a direct link between the antigen exposure response in the mother's mucosal immune system, especially via the enteric mucosal immune system, and the secretory immunoglobulin repertoire of the mammary gland [18] . This means that maternal colostrum and milk will contain antibodies specific for pathogens that may be encountered by the neonate's intestine and other mucosal tissues [10, 18, 148] , providing a rationale for the observations summarized above that bovine colostrum from nonimmunized cows also may afford passive immune protection against human pathogens [54, 55] .
The immune connection between the GALT and the mammary gland is of particular interest with respect to human milk where the major immunoglobulin is sIgA, which accounts for one of the key factors underlying the importance of breast feeding [10] . The immune activation of GALT in the human infant is delayed, and the milk sIgA and sIgM provide the neonatal intestine a level of protection through their immune exclusion actions and their anti-inflammatory effects [10, 18] .
Transepithelial transport of IgA and IgM across the mammary epithelial cells occurs via the polymeric immunoglobulin receptor (pIgR) which is responsible for binding dimeric IgA and pentameric IgM in mucosal tissues [149, 150] . The polymeric nature of IgA and IgM arises from their binding with the J-chain peptide [116] . Only IgA or IgM that contain the J chain have a high affinity for pIgR [116, 151, 152] . In fact, the J chain has been evolutionarily conserved within tetrapods to the point where human polymeric IgA can bind to the pIgR from the amphibian Xenopus laevis [152] . Polymeric IgA or IgM bound to pIgR is internalized and transported to the apical end of the mammary epithelial cell by an endocytic process. The pIgR molecule is cleaved to release a receptor fragment, called secretory component (SC), which remains bound to the immunoglobulin molecule [119, 149] . In the case of pIgR receptor sites that are not occupied by immunoglobulin, the secretory component is still cleaved from the membrane-bound portion of pIgR, resulting in release of free secretory component. The secretory component has protective effects of its own, potentially blocking epithelial adhesion of enterotoxigenic E. coli and neutralizing the effects of other pathogens [148] .
Expression of pIgR in the mammary gland is under control of hormones responsible for initiation of lactation [153] . Elevated transport of IgA also may occur during mammary gland involution in cattle and persist longer into the involution process [140] .
Part of the transfer of passive immunity story in mammals involves the timing and location of transfer of immunoglobulins from the mother to the offspring, while another part encompasses the fate and function of the immunoglobulins once in the neonate [1, 7, 127] . In humans, intestinal transfer of maternal IgG from colostrum is sparse in the neonate and their immune competency is assured by transfer via the placenta. In rats and mice, there is FcRn-mediated uptake of IgG from the colostrum and milk in the neonate intestine. In ungulate species such as cattle, sheep, goats and pigs, the young are born essentially agammaglobulinemic and rely entirely on uptake of colostral immunoglobulins, especially IgG, for systemic immune protection.
The consumption of colostrum by the neonatal calf has significant effects on the gastrointestinal tract [154] . The intestinal uptake in the immediate period after birth is transient and nonselective in species such as cattle, sheep, goats, swine and others. The intestinal cells become unable to absorb macromolecules within 24-36 h after birth probably as a result of developmental processes occurring in the enterocytes [155] . The process whereby the intestinal cells gradually stop absorbing macromolecules is termed -closure‖. Before closure, the enterocytes will nonselectively absorb large molecular weight proteins and other molecules [155] . Macromolecules so transported are released into the lamina propria and then are absorbed into the lymphatic or portal circulation. Failure of passive transfer of immunity in these species is defined as occurring when a threshold concentration of IgG is not reached before closure occurs, which in the calf corresponds to serum IgG levels less than 10 mg/mL [156] . The maternal IgG in the calf's blood gradually declines over the initial month after birth, and has a half-life of approximately 16 days [157] .
Milk sIgA is not taken up by the infant's intestinal mucosa [148, 158] . In fact, gut closure in humans occurs before birth and little immunoglobulin is absorbed intact in the intestine after birth [148, 158] . However, the presence of sIgA in the intestinal lumen is part of the protective function of the epithelial barrier in the intestine [159] . Milk sIgA in the intestine will bind bacteria, toxins and other macromolecules, limiting their ability to bind to intestinal cells and thereby be transported through the mucosa to the lamina propria to cause a systemic immune response [160] . In adults of a pIgR-deficient strain of mice, which do not transport sIgA into the intestinal lumen, there is an increased serum IgA and IgG that react with commensal organisms and food antigens [161] . This may be occurring because sIgA is not being secreted into the intestinal lumen to participate in its role in immune exclusion (see section 6.3), and resulting in an increased uptake of food antigens and microbial antigens from the intestinal lumen which pass to the lamina propria and stimulate specific antibody responses [161] . Development of the GALT system is dependent on microbial stimulation [148, 158] . The microbe binding function of sIgA then modulates the early microbial colonization of the gastrointestinal tract and the interaction of those microbes with the developing neonatal immune system [148, 158, 160] .
From the discussion of immune milk products above it was clear that these products have protective effects on neonatal health, as well as infant and adult human health. The exact mechanisms by which immune milk products have their effects are less clear and deserve further investigation. Below are summarized several perspectives to consider when evaluating the effects of immune milk products and the role of immunoglobulins in achieving those effects.
It should be remembered that colostrum and milk not only contain immunoglobulins, but also contain a range of antimicrobial factors and factors that may impact the immune system [10, 154, 160, [162] [163] [164] [165] [166] [167] [168] . These include the iron-binding antimicrobial protein lactoferrin, antibacterial enzyme lactoperoxidase, antibacterial and lytic enzyme lysozyme, oligosaccharides that function as analogues of microbial ligands on mucosal surfaces, antimicrobial heat stable peptides (defensins), and soluble CD14. In addition, colostrum and milk contain leukocytes, including activated neutrophils, macrophages and lymphocytes. Colostrum also contains cytokines and growth factors that may affect neonatal intestinal development, as well as intestinal immune responses to disease in adults [166, 169] . The relative concentrations of these factors vary considerably among species. Furthermore, colostrum provides a source of energy which may impact IgG absorption in the neonate [170] , and provide additional energy for an effective immune response.
Another point to consider is that, while most macromolecules are degraded by digestive enzymes, some portion of macromolecules is transported across the intestine intact, including proteins [171, 172] . Much of the immunoglobulin consumed in an immune milk can be expected to be partially or completely digested (discussed in section 7.3), however some portion of the immunoglobulin will remain intact or at least partially intact and capable of binding to an antigen.
All colostrum and milk will contain some sIgA, even those collected from cattle. The sIgA present in these secretions may contribute to the protective effects of immune milk products. Secretory IgA is considered to be the primary immunoglobulin responsible for immune protection of mucosal surfaces such as the intestine [158] . Secretory IgA and sIgM, as polymeric forms of the respective immunoglobulins, are stabilized by their binding to SC. They have antimicrobial properties such as agglutination of microbes and neutralization of viruses, and noninflammatory extracellular and intracellular immune exclusion by inhibiting adherence and invasion of mucosal epithelia [158] . The intracellular immune exclusion occurs when sIgA is being transcytosed by the enterocytes and comes into contact with viral particles within the endosomic system [15] . Secretory IgA also neutralizes pathogens in the intestinal lumen [173] . Bacterial enterotoxins may be neutralized by binding sIgA and internalization into intestinal epithelial cells [174] .
In addition, IgA has a major role in the immunosuppressive mechanisms in the intestine that inhibit proinflammatory responses to oral antigens, which is part of the oral tolerance mechanisms in the intestine [158] . This suppression of the proinflammatory mechanisms is counterbalanced by systemic immune factors, including systemic IgG, which may result in inflammation and tissue damage once an antigen crosses epithelia barrier to the lamina propria [158] .
After closure, any IgG localized in the lamina propria, whether from systemic sources or from uptake from the intestinal lumen, could contribute to proinflammatory responses in the intestine [158] . Indeed, post-closure uptake of IgG can occur via the FcRn receptor. FcRn has been identified in the human adult intestine [175, 176] , consistent with the hypothesis that FcRn is involved in IgG recycling (discussed in section 5.1). However, the transport of IgG across the enterocyte seems to be bidirectional, lending support to the concept that IgG in the intestine is involved in immune surveillance and defense of the mucosal lining [176] [177] [178] . Intestinal FcRn may deliver IgG-antigen immune complexes to the lamina propria for immune processing [158, 177] , thereby enhancing local mucosal immune response.
On the other hand, functionally intact IgG that remains in the intestinal lumen might be expected to bind antigens and participate in protection of the tissue through immune exclusion. The intestinal mucus layer does provide an important protective barrier in the interactions of the intestinal tissue with microbes [179] . Interestingly, an IgG Fc binding site has been identified in association with the intestinal mucus [180] [181] [182] . This IgG Fc binding protein is distinct from the FcRn receptor. The Fc binding protein may block passage of IgG-antigen complexes to the enterocyte surface, thereby blocking their uptake and transport to the lamina propria, and perhaps allowing the complexes to be degraded in the intestinal lumen and excreted [169, 182] .
Consumed colostrum also may impact immunological development of the neonate [1] . These maternal antibodies may then inhibit infant responses to vaccine administration and impact development of the infant's immunity [183] .
In the case of dairy animals producing colostrum or milk immunoglobulins for human consumption, immunoglobulins are harvested at milking and undergo various types of processing whether it is to prolong the shelf-life of the milk, to concentrate or isolate the immunoglobulins from the mammary secretion, or to digest the milk in the intestine. Through such processing, immunoglobulins are exposed to a number of conditions that may alter the structure and function of the protein. Some of methods used to concentrate or isolate the immunoglobulins include steps that involve exposing the protein to heat, acid or pressure which may affect the conformation of the protein, and ultimately the immunological activity of the antibody.
A range of methods have been used for isolation of immunoglobulins from colostrum or milk. These include traditional methods of ammonium sulfate precipitation and column chromatography [3, 5, 145, 184] . Affinity chromatographic methods used to isolate IgG include lectins [185] ; protein A or G chromatography [186, 187] , and more recently, isolation with protein A/G immobilized electrospun polyethersulfone membranes [188] ; metal chelate chromatography [189, 190] ; and adsorption with polyanhydride microparticles [191] . The range of detection and quantification methods for IgG, most often analyzed by radial-immunodiffusion [192] or enzyme-linked immunosorbant methods [193] , are now expanding to include methods that detect multiple proteins, such as thermally addressed immunosorbant assays [194] , and rapid methods that may be integrated into milking systems, such as surface plasmon resonance-based immunosensors [195] .
Pepsin is a major proteolytic enzyme produced by the stomach. Pepsin digestion of IgG yields an F(ab') 2 fragment that includes the two antigen-binding (Fab) sites of the IgG molecule [5, 114, 196, 197] . Intact immunoglobulin, F(ab') 2 and other antibody formats are being exploited in development of antibody therapeutics [198] .
In the small intestine, immunoglobulins are further digested by pancreatic enzymes. One of them, trypsin, preferentially digests bovine IgG1 over IgM, whereas another enzyme, chymotrypsin, preferentially hydrolyzes IgM over IgG [199] . Bovine IgG1 is more susceptible to hydrolysis by pepsin than IgG2, while IgG2 is more susceptible to trypsin [200] .
Immunoglobulins are relatively more resistant to gastrointestinal digestion than other milk or colostral proteins. Upon ingestion and entry into the stomach, the caseins form a curd under the influence of the acidic environment and proteolytic activity. As a consequence, casein is retained in the stomach of the neonate longer than the whey proteins, including IgG [201] . In the intestine, the fate for the other major whey proteins is rapid digestion for α-lactalbumin, while β-lactoglobulin is more slowly digested. Intestinal digestion of IgG is among the slowest of the whey proteins and IgG provides the smallest proportion of amino acids to the neonate relative to the other major whey proteins [201] . In vitro incubations of IgA and IgG with small intestinal content of young lambs have shown that IgA is more resistant towards digestion than is IgG [17] .
In adult humans consuming a bovine whey protein concentrate, approximately 59% of IgG and IgM was detected by radial immunodiffusion from effluents from the jejunum, while 19% was detected in the ileum [202] . These estimates of digestion of immunoglobulin compare with estimates of digestion of milk proteins in adult humans which are approximately 42% complete at the end of the jejunum and 93% complete by the end of the ileum [203] , again underscoring the relative resistance of immunoglobulins to digestion in the gastrointestinal tract. Detectable immunoglobulin in stool samples of infants fed the same immune product accounted for 10% of the ingested immunoglobulin [58] . In adults fed a bovine immunoglobulin concentrate, fecal IgG was typically less than 4% of ingested dose [204] . Detectable IgG in the stool [204] , or ileal effluent samples of adults [205] , is not significantly increased by prior treatment with a proton pump inhibitor to reduce stomach acid production. However, encapsulation of the immunoglobulin product can significantly increase the IgG detectable in the stool [204] , although only low levels of IgG are detectable in the ileum of adults ingesting encapsulated immunoglobulin [205] . These studies suggest that degradation of immunoglobulins is occurring throughout the intestinal tract [202] .
The primary structure of the immunoglobulin found in intestinal effluents most likely is the immunoglobulin Fab or F(ab') 2 fragments found in their stool [58, 202] , which nevertheless maintains its antigen-binding activity, as indicated by the correlation between appearance of the immunoglobulin and the virus-neutralizing activity observed in stool samples [58] . In adults ingesting bovine anti-Clostridium difficile immunoglobulins, toxin-neutralizing activity paralleled the bovine IgG content in ileal effluent [205] , and in stool samples [204] . A pepsin-resistant form of bovine IgG representing approximately 10% of colostral immunoglobulin has been isolated with a lectin that binds O-linked oligosaccharides [185] , indicating that some proportion of IgG in the gastrointestinal tract may remain intact.
The pH of bovine mammary secretions transiently drops at calving (to approximately pH 6.4), then increases over several days to pH 6.6 to 6.9 [206] , which is the pH characteristic of mature milk. Therefore, bovine colostrum is slightly more acidic than mature milk. Studies of isolated immunoglobulin stability over a pH range indicate that bovine IgG isolated from milk is stable for several hours at 37 °C when in pH 6-7, however stability is significantly reduced at pH ≤ 3 and ≥10 [207, 208] . The negative effect of pH on IgG stability, even in the range of 4.5-6.5, is enhanced under elevated temperature conditions [209, 210] . The use of a multiple emulsion to encapsulate milk IgG may increase stability of the protein against extreme acidic or alkali conditions, as well as against proteolytic degradation [211] . However, emulsification by homogenization may reduce the residual IgG content of the emulsion product [211] , probably as a result of high shear forces [208] . Ultrasonic treatment of isolated IgG also decreases residual IgG content [208] .
Immunoglobulins are thermolabile. Exposure to temperatures of 75 °C can reduce detectable isolated bovine IgG by 40% in 5 min, and by 100% at 95 °C for 15 s [208] . Heat exposure causes conformational changes in the IgG molecule [212] . Antigen-binding activity of bovine IgG also is reduced after heat treatment [209, 213] . This is consistent with studies that suggest that the antigen-binding region of the immunoglobulin molecule is more thermolabile than the other regions of the molecule [209, 214] . Detectable IgG in colostrum or colostral whey also are reduced by heat treatment, however at a slower rate than for isolated IgG. Thermal protectants such as sugars or glycerol can increase the stability of isolated IgG to heat treatment [208, 215] .
Many milk processing protocols include heat treatment of the colostrum, milk or whey. Of the major immunoglobulin classes in bovine milk, IgG is the most thermostable and IgM is the least thermostable [214] . Commercial milk samples that have undergone a typical pasteurization process, including skim milk powder, can retain 25-75% of the IgG concentration compared with raw milk, while milk undergoing ultra-high temperature (UHT) pasteurization contains little detectable IgG [192, 216] . Nevertheless, antigen-specific IgG in milk is relatively stable under typical conditions of pasteurization when compared with that in UHT milk or cow milk-based infant formulas that undergo high-temperature processing [54, 217] .
Flash-heat treatment of human breast milk, a method recommended by WHO to reduce vertical transmission of HIV in resource-poor regions, has minimal effects on milk IgA and antimicrobial activity of the milk [218, 219] . This method involves placing a jar of milk into a water bath, the water bath is heated to boiling, and then the jar of milk is removed and allowed to cool. The milk reaches a maximum temperature of 72-73 °C and is above 56 °C for over 6 min [218, 219] .
Alternative methods of achieving microbial inactivation may offer a means of avoiding the impact of heat treatment on IgG solutions. For example, high voltage pulsed electric fields have been used as a nonthermal processing method for pasteurization in various foods [220] [221] [222] . Pulsed electric field processing also generates heat, however temperature exposure of the fluid is less than 50 °C, and total treatment time exposure is in milliseconds [223] . That compares with more typical pasteurization process at about 72 °C for 2 min. Microbial inactivation in bovine IgG solutions as a result of pulsed electric fields did not change the secondary structure or the thermal stability of the secondary structure of the IgG [224] , and antigen-binding activity was unchanged [223] . Another emerging technology that may provide a nonthermal microbial inactivation treatment for milk uses exposure to pulsed ultraviolet light [225] .
High-pressure processing is another non-thermal method with the potential for inactivation of microbial and certain enzymes in food products, thereby extending shelf-life of the product [226] . While the high-pressure process also generates heat during the treatment of the sample, lowering the initial temperature of the sample allows for control of the maximum temperature reached to be maintained within a desired range [227] . To be effective in inactivating bacterial spores, high-pressure processing needs to be combined with moderate temperature treatment [228] . Moderate to extensive loss of immunoactivity of IgG may occur depending on the conditions used for high-pressure processing of colostrum or other IgG-containing fluids [227, 229] . High-pressure processing also has been used for human breast milk with minimal effect on the milk IgA [230] .
The issue of heating effects on immunoglobulin and colostrum also is important for control of various diseases that occur in cattle. Collection and storage of colostrum from dairy cows shortly after calving has long been a common procedure. The stored colostrum then is fed to newborn calves to assure adequate uptake of IgG for protection of the calf. Several pathogens can be transmitted from cow to calf via colostrum or milk [231] . Colostrum may contain these pathogens as a result of shedding from the mammary gland, contamination of the colostrum after harvesting or improper storage of colostrum prior to feeding calves [231] . One approach to allowing the neonate the benefits of colostrum from infected cows is collecting colostrum and batch pasteurization of pooled colostrum prior to feeding to the calves [232] . Volume of the batch of pooled colostrum that is pasteurized affects measurable IgG concentrations in the colostrum, as well as IgG serum concentrations attained in calves after feeding the colostrum [232] . Heat treatment of colostrum at 60 °C for one to two hours does not alter measurable IgG concentrations or viscosity of the colostrum, nor does that treatment affect antibody activity [231, 233] . In addition, bacteria inoculated into colostrum prior to a heat treatment of 60 °C for one hour are not detectable after the heat treatment [234] . On-farm heat treatment of colostrum (60 °C for one hour) results in higher concentrations of serum IgG and greater apparent absorption efficiency of IgG in new born calves consuming the treated colostrum than consumption of raw colostrum [235] [236] [237] .
Colostrum and milk are rich sources of immunoglobulins. These secretions have developed through evolution to ensure homologous transfer of passive immunity from mother to offspring. The immunoglobulins that are passed from mother to her offspring, whether by transplacental transfer or by ingestion of colostrum and milk, can form an important link between the immunological experience of the mother and the immune capacity of the newborn. This immunological link also includes many immune factors that may be present in mammary secretions other than the immunoglobulins. The immunoglobulins in colostrum and milk also provide opportunities to harness their immunological function for the benefit of other animals, including humans. Research has demonstrated that bovine colostrum and milk, whether or not they are from cows immunized against specific pathogens, provide a medium for the heterologous transfer of passive immunity, and may offer disease protection in a range of species. New technologies for enhancing efficacy of vaccination, enhancing stability and extending shelf-life of the immunoglobulin preparation while minimizing the impact of the processing, and extending the effectiveness of the immunoglobulin in the intestine, may enhance future use of colostrum and milk based on their potent immunological activity. While the mechanisms by which immunoglobulins are transferred from mother to neonate and their role in the neonate have become well documented, additional research is needed to clarify the mechanisms of action of the immunoglobulins derived from milk or colostrum when used in animals that are developmentally more mature.  The crazy-paving pattern: a radiological-pathological correlation The crazy-paving pattern is a linear pattern superimposed on a background of ground-glass opacity, resembling irregularly shaped paving stones. The crazy-paving pattern is initially described as the pathognomonic sign of alveolar proteinosis. Nowadays this pattern is a common finding on high-resolution CT imaging, and can be seen in a number of acute and chronic diseases. The purpose of this paper is to illustrate different diseases that cause this crazy-paving pattern and to correlate the radiological findings from computed tomography with the histopathological findings. The superimposition of a linear pattern on ground-glass opacity on computed tomography images results in a pattern that is termed crazy-paving pattern, resembling the structure of irregularly shaped paving stones [1, 2] . The crazy-paving pattern is a common finding on thin-section computed tomography (HRCT), but also on multidetector computed tomography (MDCT). Ground-glass opacity is defined as a hazy increase in lung density with preservation of airway and vessel margins [3] . Ground-glass opacity occurs when there is a mild decrease in the amount of air in the airspaces and a filling of the airspaces with fluid, cells or other material, thickening of the alveolar walls or thickening of the interstitium. The linear component of this pattern can be caused by a thickening of the interlobular septa (septal lines), a thickening of the intralobular septa and the intralobular interstitium (intralobular reticular pattern and intralobular branching lines) or a linear deposition of material within the airspaces at the borders of the acini (periacinar pattern) ( Fig. 1 ) [4] . The crazypaving pattern was initially described as a pathognomonic sign of alveolar proteinosis; however, nowadays, this pattern has been reported in a variety of acute and chronic diseases as summarised in Table 1 [2, [5] [6] [7] [8] . The purpose of this paper is to illustrate different diseases showing a crazypaving pattern. The diagnosis is made based on clinical or on histological findings. If histopathological proof is available, a radiological-histopathological correlation is made.
A retrospective review of the medical records of our radiological computed tomography database was performed, from 1 January 2008 until 31 December 2008, searching for patients reported to have a "crazy-paving" pattern on a CT of the chest. In total, 98 patients with a crazy-paving pattern were retained and reviewed. To rule out acute pulmonary embolism, most of the patients underwent interstitial pathological features or underwent their chest CT in an oncological setting. All these patients underwent a dedicated MDCT of the chest with 100 mAs, Fig. 1 a Anatomy of the secondary pulmonary lobule. b-e The reticular pattern: b thickening of the interlobular septa; c thickening of the intralobular interstitium; d irregular areas of fibrosis; e periacinar pattern 120 kV, slice thickness of 1 and 3 or 5 mm, and table feed of 12 mm per rotation, with or without intravenous contrast administration, according to the indication of chest CT. Only seven patients with a crazy-paving pattern on chest CT also underwent an open lung biopsy to make the definitive diagnosis. In 59 patients, the definitive diagnosis was made on a clinical basis. In the remaining 32 patients, the cause of the crazy-paving pattern remained undecided, because patients were not followed further in our institution.
Ninety-eight patients with a crazy-paving pattern were retained and reviewed. Table 2 summarises the different causes of the crazy-paving pattern as found on open lung biopsy or based on clinical decision. Only seven patients underwent open lung biopsy to establish the diagnosis.
A 46-year-old man presented with a 1-week history of progressive dyspnoea. He also complained of a cough and the production of white mucus in the morning. He reported a smoking habit of one pack of cigarettes per day with no further information regarding his past smoking history.
Chest radiograph and CT were undertaken. Chest radiograph (Fig. 2a) showed a reticular pattern more pronounced in the central parts of the lungs. There was also an increase in lung density centrally in both lungs. No pleural fluid was noted, and the heart and central vascular structures were normal. On CT, there was a patchy distribution of areas with increased lung attenuation throughout both lungs. Superimposed on this increased lung attenuation a linear pattern was seen. There were multiple small regular and irregular lines. Some of them were thickened interlobular septa. More lines were visible in the centre of the secondary pulmonary lobule in a very irregular pattern suggesting thickening of the intralobular interstitium.
Histopathological evaluation of a specimen from open lung biopsy out of the right lung showed amorphous eosinophilic material in the alveoli, positive on periodic acid Schiff (PAS) staining. This eosinophilic material corresponded with deficient surfactant (Fig. 2c) . The lines visible on CT corresponded to deposition of material within the airspaces at the borders of the acini in the secondary pulmonary lobules (periacinar pattern; Fig. 2b ). The diagnosis of alveolar proteinosis was made.
A 62-year-old woman with progressive shortness of breath on exercise. Chest radiograph and CT were undertaken. Chest radiograph showed a patchy distribution of areas with increased lung density (Fig. 3a) . There was also an increase in linear markings in both lungs. On CT, a crazy-paving pattern was seen with a geographic distribution. Some of the lines were thickened interlobular septa. Centrally in the secondary pulmonary lobule we could also see a spider of lines: thickening of the intralobular septa. These findings were seen predominantly in the upper lung areas (Fig. 3b) .
Although the patient had no history of bird exposure, serum precipitins against pigeons were elevated. To resolve this paradox, an open long biopsy was performed. Histology demonstrated interstitial pneumonia with lymphocytes, plasma cells and foamy macrophages in the interstitium. Epithelioid granulomas without caseation were also seen. There was no fibrosis (Fig. 3c) .
The diagnosis of hypersensitivity pneumonitis was made.
An 80-year-old man with rapidly progressive dyspnoea. A chest radiograph and CT were undertaken. The chest radiograph showed a patchy distribution of areas with consolidation. There was also a fine reticular pattern, most pronounced in the periphery of both lungs (Fig. 4a ). Chest CT, performed to rule out acute pulmonary embolism, was negative for the presence of lung emboli. A crazy-paving pattern with a scattered distribution of ground-glass opacities and a linear pattern superimposed, with multiple small irregular lines, was visible. Traction bronchiectasis was seen in the periphery of both lungs (Fig. 4b) . Chest radiograph showed a reticular pattern that was most pronounced in the central parts of the lungs. There was also a decrease in the lung translucency centrally in both lungs. Heart and central vessels were normal. There was no pleural effusion. b On CT, a patchy distribution of a crazy-paving pattern was visible. The lines corresponded to a deposition of material within the airspaces at the borders of the acini (1) in the secondary pulmonary lobules, but also along the interlobular (2) and intralobular septa (3): the periacinar pattern. c Radiological-histopathological correlation. Histopathological evaluation of a specimen out of the right lung showed amorphous eosinophilic material in the alveoli (*) positive on periodic acid Schiff (PAS) staining. This material corresponded to deficient surfactant. Filling of the alveoli (*) was responsible for the ground-glass appearance on CT. When the airspaces adjacent to the inter-and intralobular septa (black arrow) and to the alveolar walls filled, the periacinar pattern became visible On histology, thickening of the interstitium with variable degrees of severity was seen, leaving some alveolar septa almost completely normal, whereas others were thickened. Fibrinous exudates, honeycombing and mild inflammatory alveolitis were also present (Fig. 4c) .
The diagnosis of usual interstitial pneumonia (UIP) was made.
A 56-year-old woman with increasing dyspnoea.
A chest radiograph and CT were undertaken. The chest radiograph showed a reticulation of the lung parenchyma, diffusely spread in both lungs, centrally and peripherally (Fig. 5a) . Chest CT showed a crazy-paving pattern especially in the periphery of both lungs. There was an increase in lung Fig. 3 Hypersensitivity pneumonitis. a Chest radiograph showed patchy distribution of areas with increased lung density. There was also an increase in the linear pattern in both lungs. b On CT, a crazy-paving pattern was seen with a geographic distribution of ground-glass opacities with the superimposition of thickened inter-(1) and intralobular (2) septa. The findings were seen predominantly in the upper lung areas. c Radiological-histopathological correlation. Histology demonstrated interstitial pneumonia with lymphocytes, plasma cells and foamy macrophages in the interstitium. Epithelioid granulomas without caseation were also seen. There was no fibrosis. The alterations in the walls of the alveoli and the inflammation in the interstitium were visible as thickening of the inter-and intralobular lines Fig. 4 Usual interstitial pneumonia. a Chest radiograph showed patchy distribution of areas with consolidation and a fine reticular pattern, most pronounced in the periphery of both lungs. b A crazy-paving pattern was visible with scattered distribution. Superimposed on the ground-glass opacities a linear pattern with multiple small irregular lines was visible (intralobular fibrosis) (1). Traction bronchiectasis was seen in the periphery of both lungs (white arrow). c Radiologicalhistopathological correlation. On histology, thickening of the interstitium (arrow) with variable severity was seen, leaving some alveolar septa almost completely normal, whereas others were thickened. Fibrinous exudates, honeycombing (*) and mild inflammatory alveolitis were also present Fig. 5 Non-specific interstitial pneumonia. a Chest radiograph showed reticulation in the lung parenchyma, diffusely spread in both lungs, centrally and peripherally. b Chest CT showed a crazy-paving pattern especially at the periphery of both lungs. There was an increase in lung attenuation (ground-glass opacification) with a superimposition of a reticular pattern with thickening of the inter-(1) and intralobular (2) septa. c Radiologicalhistopathological correlation. Histological evaluation showed a homogeneous fibrotic thickening of the interstitium with inflammation. Macrophages were visible within the alveolar septa. Homogeneous interstitial inflammation was seen, corresponding to the diffuse ground-glass opacities, whereas fibrosis in the interstitium and alveolar septa (black arrow) was related to the superimposed linear pattern Fig. 6 Radiation pneumonitis. a Chest radiograph showed an area of consolidation in the right lung with an air bronchogram. There was also loss of volume of the right lung. b CT showed the therapy response of the tumour. There was patchy distribution of a crazy-paving pattern with increased lung attenuation (ground-glass opacity) and thickening of the interlobular septa in the right lung (1) . c Radiologicalhistopathological correlation. Histological examination after autopsy showed airspace filling with an exudate in combination with thickening of the interlobular septa (arrow), thickening of the interstitium surrounding the airspaces and also the presence of irregular fibrosis (dotted arrow). Alveolar spaces filled with an exudate of proteinaceous material were responsible for the ground-glass opacities on CT. The reticular pattern was due to congestion of capillaries and oedema of the interstitium Fig. 7 Exogenous lipid pneumonia. a Chest radiograph showed a decrease in lung translucency in the caudal region of the right lung with an air bronchogram. b Chest CT showed a crazy-paving pattern with areas of increased lung attenuation and with thickening of interlobular septa (1), even thickening of the intralobular interstitium (2) . c Radiologicalhistopathological correlation. Histological examination showed alveoli filled with lipid particles (*), some ingested in macrophages (+) with the formation of lipid granulomas Fig. 8 Lymphangitic carcinomatosis. a Chest radiograph showed a pleural effusion in the right haemothorax. An increased linear pattern was seen in the left and right upper lung. b CT showed a diffuse crazy-paving pattern with areas of groundglass attenuation and thickening of the interlobular septa (1). There were also some small nodular lesions visible mostly in the left upper lobe suggestive of pulmonary metastases (2) . c Radiological-histopathological correlation. Histological examination of the autopsy specimen demonstrated thickening of the interlobular septa (*) due to fibrosis and the presence of tumour cells. There was also perivascular (arrow) thickening due to an expansion of lymphatic spaces by tumour cells. The histological reaction was that of diffuse alveolar damage and consisted of hyaline membranes in the alveolar ducts and respiratory bronchioles while the alveolar spaces fill with an exudate of proteinaceous material. This corresponded to the ground-glass opacities on CT. The reticular pattern was due to congestion of capillaries and oedema of the interstitium attenuation (ground-glass opacification) with a superimposition of thickened inter-and intralobular septa (Fig. 5b) .
Histological evaluation showed a homogeneous fibrotic thickening of the interstitium with inflammation. Macrophages were visible within the alveolar septa (Fig. 5c) .
The diagnosis of non-specific interstitial pneumonia (NSIP) was made.
A 71-year-old man with a limited small cell lung cancer developed fever and a cough after radiation therapy.
A chest radiograph and CT were undertaken. The chest radiograph showed an area of consolidation in the right lung with an air bronchogram. There was also loss of volume of the right lung (Fig. 6a) . CT showed a decrease in the size of the tumour consistent with response to therapy. There was a patchy distribution of a crazy-paving pattern with ground-glass opacities and thickening of the interlobular and intralobular septa (Fig. 6b) .
Histological examination after autopsy showed airspace filling with an exudate in combination with thickening of the interlobular septa, thickening of the interstitium sur-rounding the airspaces and also the presence of irregular fibrosis (Fig. 6c) .
The diagnosis of radiation pneumonitis was made.
A 54-year-old man with progressive dyspnoea. A chest radiograph and CT were undertaken. The chest radiograph showed decreased translucency with an air bronchogram in the right lower lobe. There were no signs of interstitial lung disease (Fig 7a) . Chest CT showed a crazypaving pattern with areas of increased lung attenuation and with thickening of the interlobular septa, even thickening of the intralobular interstitium in the right middle and lower lobe (Fig. 7b) . Histological examination showed alveoli filled with lipid particles, some of them ingested in macrophages with the formation of lipid granulomas (Fig.7c) .
The diagnosis of exogenous lipid pneumonia was made.
A 73-year-old woman with an insidious onset of unexplained and progressive dyspnoea. Acute respiratory distress syndrome. CT revealed bilateral areas with ground-glass attenuation superimposed with a reticular pattern. These lines corresponded to thickening of the interlobular septa, but also thickening of the intralobular interstitium A chest radiograph and CT were undertaken. The chest radiograph showed a pleural effusion in the right hemothorax. An increased reticular pattern was seen in the left upper lung field and to a lesser degree also in the right upper lung field (Fig. 8a ). CT showed a diffuse crazypaving pattern with areas of ground-glass attenuation and thickening of the interlobular septa. There were also some small nodular lesions visible, mostly in the left upper lobe, suggestive of pulmonary metastases (Fig. 8b) .
Histological examination of the autopsy specimen demonstrated heterogeneous thickening of the interlobular septa due to fibrosis and the presence of tumour cells. There was also perivascular thickening due to an expansion of lymphatic spaces by tumour cells.
The diagnosis of lymphangitic carcinomatosis was made.
A 34-year-old woman with thrombotic thrombocytopaenic purpura and severe myasthaenia gravis developed progressive respiratory insufficiency. A chest CT was undertaken. CT showed a patchy distribution of areas with ground-glass opacification in both lungs, more pronounced in the central parts of both lungs (Fig. 9) . There was also a superimpo-sition of a linear pattern. Most of the lines were thickened interlobular septa. The diagnosis of pneumocystis jirovecii pneumonia was made based on clinical and laboratory findings.
A 67-year-old woman who received a total knee prosthesis developed septic shock with ARDS in the postoperative period. A chest CT was undertaken (Fig. 10) . This CT revealed bilateral areas with ground-glass attenuation superimposed with thickened interlobular septa but also thickening of the intralobular interstitium.
An 83-year-old man with the diagnosis of acute lymphatic leukaemia developed cardiac decompensation with oedema of the lower limbs. CT of the chest (Fig. 11) showed a patchy distribution of areas with ground-glass opacification. A superimposed linear pattern was also present. Most of the lines were thickened interlobular septa. Within the secondary pulmonary lobule, enlarged vascular structures with a spider configuration were seen. There were also some other intralobular lines. Fig. 11 Pulmonary oedema. CT showed patchy distribution of areas with ground-glass opacification and a linear pattern. Most of the lines were thickened interlobular septa. Within the secondary pulmonary lobule, enlarged vascular structures with a spider configuration were seen. There were also some other intralobular lines Fig. 12 Sarcoidosis. CT showed a diffuse increase in lung attenuation (ground-glass attenuation) with the superimposition of an irregular reticular pattern: thickening of the interstitium and thickening of the peribronchovascular interstitium
A 44-year-old man with sarcoidosis underwent a control CT of the chest. There was diffuse increased lung attenuation with the superimposition of multiple irregular lines and also irregular thickening of the bronchovascular bundles: the crazy-paving pattern (Fig. 12) . Interstitial fibrosis was the cause of the irregular thickening of the interstitium.
A 40-year-old man with haematopoietic stem cell transplantation. He developed dyspnoea, and CT was undertaken. CT revealed multiple areas of ground-glass attenuation and consolidations. There was also a superimposition of multiple lines: thickened inter-and intralobular septa in intralobular lines caused by fibrosis (Fig. 13) .
The diagnosis of graft-versus-host disease was made.
A 24-year-old woman with bilateral lung transplantation. A control CT was performed and showed patchy distribution of areas of ground-glass opacification with the superimpo-sition of thickened interlobular septa: the crazy-paving pattern (Fig. 14) . The diagnosis of organising pneumonia was made on a clinical basis.
A 75-year-old man known to have bronchioloalveolar carcinoma. Chest CT showed a patchy distribution of areas with increased density, areas of ground-glass opacification and areas with consolidation. Superimposed on these areas there was a reticular pattern (Fig. 15 ). These lines correspond to a thickening of the interstitium. The diagnosis was made based on biopsy, which was not performed in our institution.
The crazy-paving pattern is a non-specific pattern. Initially, this pattern was considered to be highly suggestive of alveolar proteinosis. Nowadays, we can find this pattern in different lung diseases: airspace diseases and interstitial diseases [8] . The crazy-paving pattern consists of scattered or diffuse ground-glass attenuation with superimposition of Fig. 13 Graft-versus-host disease. CT revealed multiple areas of ground-glass attenuation and consolidations. There was also a superimposition of multiple lines: thickened inter-and intralobular septa and intralobular fibrosis Fig. 14 Organising pneumonia. CT showed patchy distribution of areas of ground-glass opacification with the superimposition of thickened interlobular septa a linear pattern. These lines can be: thickened interlobular septa (septal lines), thickened intralobular septa and thickening of the intralobular interstitium (intralobular reticular pattern and intralobular branching lines), or it can be a linear deposition of material within the airspaces at the borders of the acini and the secondary pulmonary lobules (periacinar pattern) [4] .
Alveolar proteinosis and exogenous lipid pneumonia are airspace diseases. In alveolar proteinosis, airspaces are filled with a phospholipoproteinaceous material. On CT, the filling of the alveoli is responsible for the ground-glass appearance. When the airspaces adjacent to the inter-and intralobular septa and to the alveolar walls fill, the periacinar pattern becomes visible (Fig. 2c) [9] [10] [11] .
Exogenous lipid pneumonia is the result of chronic inhalation of oily substances and is primarily a disease that affects the alveolar spaces. On CT, diffuse ground-glass opacities and consolidations, sometimes with fat attenuation caused by large lipid particles and numerous lipid-laden macrophages distending the alveolar spaces, can be seen, especially in the lower lung areas (Fig. 7c) [9, 12, 13] .
Pneumocystis jirovecii pneumonia is a common pulmonary infection in severely immunocompromised patients. Our patient was receiving treatment with Neoral, Imuran, Medrol and Mestinon. Chest radiographs can be normal in up to 18% of patients. Typical radiographic manifestations on CT are bilateral, perihilar reticular and poorly defined ground-glass opacities with superimposition of lines, which can be associated with interlobular septal thickening [14] . As described by Rossi et al. histological features contributing to the ground-glass attenuation include the foamy nature of the alveolar exudates and thickening of the alveolar walls by oedema and cellular infiltrates [2] .
Hypersensitivity pneumonitis, UIP , NSIP, radiation pneumonitis and lymphangitic spread of carcinoma are interstitial diseases. In hypersensitivity pneumonitis, antigen-antibody complexes around the microvasculature cause a neutrophil-rich inflammatory response and subsequent tissue injury. Biopsy in the subacute phase shows heavy infiltrates of lymphocytes and plasma cells in the walls of the alveoli in combination with poorly formed granulomas containing foreign body giant cells. In chronic phases, the interstitial inflammation remains, but fibrosis becomes more apparent and honeycombing can occur. On CT, the alterations in the walls of the alveoli and the inflammation in the interstitium are visible as thickening of the inter-and intralobular lines and thickening of the intralobular interstitium (Fig. 3c) [15] .
The cardinal features of UIP on CT include subpleural reticular opacities (intralobular and interlobular septal lines) and honeycombing, increasing from the apex to the base. Ground-glass opacities are inconspicuous or absent in UIP, but focal areas of GGO may be present [16] . On histology, the hallmark is a geographically and temporally heterogeneous parenchymal fibrosis against a background of continuing mild inflammation (Fig. 4c) [17] .
In NSIP the predominant finding on HRCT is subpleural, patchy, ground-glass opacification [18] . Traction bronchiectasis, subpleural microcystic honeycombing and irregular linear opacities can be seen in more advanced cases. On histology, homogeneous interstitial inflammation is seen, corresponding to the diffuse ground-glass opacities, whereas fibrosis in the interstitium is related to the superimposed linear pattern (Fig. 5c) [19] .
The inflammation of lung tissue, secondary to radiation therapy, is localised in the tissue within the radiation field and depends on the interval since completion of treatment.
In the acute phase (4 to 12 weeks after completion of radiation therapy), the histological reaction is that of diffuse alveolar damage and consists of hyaline membranes in the alveolar ducts and respiratory bronchioles while the alveolar spaces fill with an exudate of proteinaceous material. This corresponds to the ground-glass opacities typically manifesting on CT. The reticular pattern that can be seen in this phase is due to congestion of capillaries and oedema of the interstitium (Fig. 6c) [20] .
Pulmonary lymphangitic carcinomatosis is a metastatic lung disease characterised by diffuse spread of tumour to the pulmonary lymphatic system. When tumoral cells spread to the pulmonary lymphatic system and peri- Fig. 15 Bronchioloalveolar carcinoma. CT showed patchy distribution of areas with ground-glass opacification and areas with consolidation. Superimposed on these areas there is a reticular pattern corresponding to the thickening of the interstitium lymphatic interstitial tissue, interstitial thickening is seen on CT. The proliferation of these cells in combination with lymphatic dilatation contributes to this interstitial thickening (Fig. 8c) [21] .
Sarcoidosis is a systemic entity characterised by the development of non-caseating granulomatous inflammation [22] . The most common parenchymal findings include irregular thickening of the bronchovascular bundles and small nodules in a perilymphatic distribution. Ground-glass attenuation and crazy-paving pattern are also described in sarcoidosis [1] . The linear pattern is caused by interstitial fibrosis.
Adult respiratory distress syndrome (ARDS) is a form of pulmonary oedema. Diagnosis is based on impaired diffusion capacity, reduced compliance of the lung and typical radiological findings. Chest CT features are bilateral consolidation and ground-glass attenuation [23] . Other findings such as reticular and linear opacities can also be seen. Histological features include oedema of the alveoli and perivascular spaces with filling of the alveoli by a protein-rich fluid [22, 24, 25] . The progress to architectural distortion and honeycombing with thickening of the inter-and intralobular septa is responsible for the linear accentuation.
The CT findings in patients with leukaemia consist mainly of ground-glass attenuation, centrilobular nodules and thickening of the bronchovascular bundles in the peripheral lung. The combination of ground-glass opacities and the thickening of the bronchovascular bundles can produce the crazy-paving pattern [26] . Accumulation of fluid in the alveolae causes the ground-glass opacification. Accumulation of fluid along the interlobular septa and along the walls of the alveolae can cause the periacinar pattern.
More than half of allogeneic haematopoietic stem cell transplant (HSCT) recipients develop graft-versus-host disease (GVHD), which remains a major cause of morbidity and mortality. HRCT findings in patients with GVHD are non-specific: diffuse interstitial and alveolar infiltrates are the most prominent features [27] . Depending the interstitial and alveolar component, a crazy-paving pattern can also be seen. On biopsy multiple hyaline membranes and fibroproliferative alterations can be seen, caused by the interstitial fibrosis and responsible for the linear pattern on CT. The multiple exudates into the alveolae are responsible for the ground-glass attenuation [28] .
Organising pneumonia is a chronic inflammatory process characterised by plugs of granulation tissue in the lumen of distal small airways, often extending into the alveolar spaces, associated with an interstitial cellular response [29] . Typical CT features are scattered and asymmetric bilateral subpleural as well as peribronchovascular consolidation. A crazypaving pattern can be seen but is an uncommon finding [30] .
Bronchioloalveolar carcinoma (BAC) has been classified into mucinous and non-mucinous subgroups and is characterised by a lepidic growth pattern through the airways and air spaces with preservation of the lung architecture. BAC may present with a variety of CT appearances. Features of BAC are the CT angiogram sign or air bronchograms in solitary nodules and in the periphery of larger consolidations, unifocal or multifocal ground-glass opacities, the crazy-paving pattern, and lobar or multilobar consolidation and cavitating nodules [31] . In patients with a crazy-paving pattern, the ground-glass attenuation reflects the lowdensity intra-alveolar material (glycoprotein), whereas the superimposed lines are due to infiltration of the interstitium by inflammatory or tumour cells [32] .
The crazy-paving pattern on CT is a non-specific finding. It is characterised by scattered or diffuse areas of groundglass attenuation with superimposition of a linear pattern. This linear network can be caused by thickening of interlobular or intralobular septa or the presence of intralobular fibrosis, or it can be caused by a linear deposition of material within the airspaces. Most diseases can be diagnosed based on clinical and radiological findings. In a minority of cases a biopsy with histopathological examination is needed to establish the diagnosis. Comparison of percutaneous radiofrequency thermal ablation and surgical resection for small hepatocellular carcinoma BACKGROUND: The purpose of this investigation was to compare the outcome of percutaneous radiofrequency thermal ablation therapy (PRFA) with surgical resection (SR) in the treatment of single and small hepatocellular carcinoma (HCC). METHODS: We conducted a retrospective cohort study on 231 treatment naive patients with a single HCC ≤ 3 cm who had received either curative PRFA (162 patients) or curative SR (69 patients). All patients were regularly followed up after treatment at our department with blood and radiologic tests. RESULTS: The 1-, 3- and 5-year overall survival rates after PRFA and SR were 95.4%, 79.6% and 63.1%, respectively in the PRFA group and 100%, 81.4% and 74.6%, respectively in the SR group. The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 82.0%, 38.3% and 18.0%, respectively in the PRFA group and 86.0%, 47.2% and 26.0%, respectively in the SR group. In terms of overall survival and recurrence free survival, there were no significant differences between these two groups. In comparison of PRFA group patients with liver cirrhosis (LC) (n = 127) and SR group patients with LC (n = 50) and in comparison of PRFA group patients without LC (n = 35) and SR group patients without LC (n = 19), there were also no significant differences between two groups in terms of overall survival and recurrence free survival. In the multivariate analysis of the risk factors contributing to overall survival, serum albumin level was the sole significant factor. In the multivariate analysis of the risk factors contributing to recurrence free survival, presence of LC was the sole significant factor. The rate of serious adverse events in the SR group was significantly higher than that in the PRFA group (P = 0.023). Hospitalization length in the SR group was significantly longer than in the PRFA group (P = 0.013). CONCLUSIONS: PRFA is as effective as SR in the treatment of single and small HCC, and is less invasive than SR. Therefore, PRFA could be a first choice for the treatment of single and small HCC. Hepatocellular carcinoma is a major health problem worldwide, with an estimated incidence ranging between 500,000 and 1,000,000 new cases annually. It is the fifth most common cancer in the world and the third most common cause of cancer-related death [1] . The prognosis of HCC is generally poor. Surgical resection (SR) remains the best hope for a cure but is suitable for only 9 to 27% of patients [2, 3] . The presence of significant background liver cirrhosis (LC) often precludes hepatic resection in patients with HCC. Recurrence in the liver remnant is also common in patients who have undergone radical hepatic resection.
Currently, local ablative therapy competes with surgical resection and liver transplantation as primary treatment for small HCC. Various locoregional therapies are used to treat patients who are not candidates for surgery because of the severity of the underlying liver disease. Percutaneous radiofrequency thermal ablation (PRFA), a recently developed local ablative technique, has attracted the greatest interest and popularity because of its efficacy and safety [4] . Previous studies have shown PRFA to give good results from the perspective of tumor control, with complete tumor ablation rates of 90 to 95%, and low local tumor progression rates of 5 to 10% [5] [6] [7] [8] . Prospective randomized trials have shown PRFA to be better than percutaneous ethanol injection (PEI) in producing a higher rate of complete ablation with fewer numbers of treatment sessions [9] . However, there is still debate with regard to whether PRFA or SR is the most suitable therapy of small HCC.
In the present study, we conducted a retrospective cohort study to compare the results of PRFA and SR in the treatment of small HCC.
Between January 2004 and January 2010, 231 patients with single HCC ≤ 3 cm in diameter received curative treatment using PRFA or SR in our department. Before performing PRFA or SR, a full discussion was made between physician and surgeon. After giving enough information including contents of the discussion between physician and surgeon to patients, patients themselves made decisions whether they received PRFA or SR. In patients with the tumor sites extremely difficult to perform PRFA such as the site directly under the hepatic dome or the heart or with poor visibility of the tumor under ultrasonography owing to extreme obesity or impossibility of breath hold when performing PRFA, SR was performed. And in patients whom high rates of complications were expected as when tumors at the site of hepatic hilar lesion were treated by PRFA, SR was performed. In patients whom informed consent could not be obtained upon SR for the reason such as physical burden, PRFA was performed. Even in patients with poor liver function such as Child-Pugh C, if they wished to treat HCC and there were no ascites, treatment for HCC was performed after fully explaining the risk for treatment. PRFA was administered to 162 patients and 69 patients underwent SR. Written informed consent was obtained from all patients. The ethics committee of our department approved the protocols for PRFA and SR. The present study comprised a retrospective analysis of patient records and all treatments were conducted in an openlabel manner. The primary end point was overall survival and the secondary end point was recurrence free survival.
HCC was diagnosed using abdominal ultrasound and dynamic computed tomography (CT) scans (hyperattenuation during the arterial phase in all or some part of the tumor and hypoattenuation in the portal-venous phase) and/or magnetic resonance imaging (MRI), mainly based on the recommendations of the American Association for the Study of Liver Diseases [10] . Arterial and portal phase dynamic CT images were obtained at approximately 30 s and 120 s, respectively, after the injection of the contrast material. Abdominal angiography combined with CT (angio-CT) assistance was performed on all patients before PRFA and SR. This was due to the fact that Yamasaki et al. reported that this technique was useful for detecting small satellite nodules [11] . Then, we confirmed the presence of single HCC ≤ 3 cm in diameter with no vascular invasion using CT during hepatic arteriography (CTHA) and arterial-portography (CTAP). With regard to the diagnosis of liver cirrhosis, resected specimen at surgery was used in the SR group, and biopsy specimen was used in the PRFA group, respectively.
We routinely used a cool-tip needle (Radionics Corp., Burlington, MA, USA) while performing PRFA. Using the intercostal or subcostal approach, a 17-gauge, 2 or 3 cm cooled-tip electrode was inserted under real-time ultrasound guidance. The initial treatment was planned with one ablation for tumors of < 2 cm in diameter, and two or more ablations with the overlapping technique for tumors of ≥ 2 cm in diameter. After insertion of the electrode into the tumor, we started ablation at 60 W for the 3-cm exposed tip and 40 W for the 2-cm exposed tip. The power was increased to 120 W at a rate of 10 W/min. The duration of a single ablation was 12 min for the 3-cm electrode and 6 min for the 2-cm electrode. After PRFA exposure, the pump was stopped and the temperature of the needle tip was measured. When the temperature reached > 60°C, additional ablation was not performed. When tumor ablation was complete, thermal ablation was performed along the needle track. All patients were carefully observed for treatment-related complications. All procedures were performed under ultrasound guidance by one of five operators who had at least 3 years of experience of performing PRFA. We used the artificial ascites technique to prevent collateral thermal injury when the anticipated PRFA zone was in contact with a critical organ, such as the hepatic flexure of the colon. We also used this technique to improve visibility when the index tumor was located in the hepatic dome area. In the present study, for all patients who had received PRFA, we confirmed that the ablative margin surrounded the entire circumference of the tumor by using dynamic 16-column multi-detector CT (MDCT) using 3-mm slice scans within 1 week after PRFA and 1 month after PRFA.
All procedures were performed by one of four surgeons who had at least 10 years of experience of surgical resection. Surgical resection was carried out under general anesthesia using a right subcostal incision with a midline extension. We performed anatomic partial hepatectomy with a resection margin of at least 1 cm over the tumor, based on intraoperative ultrasonography (IOUS) guidance. IOUS was routinely performed to estimate the location, size, number and feeding vessels of the tumor, as well as to give an exact vascular map of liver anatomy. The Cavitron ultrasonic aspiration (CUSA, Valley Lab Corp, USA) was used to dissect the liver tissue. Hemostasis was achieved with dipolar electric coagulation and suturing. The Pringle maneuver was usually used in case of cirrhotic liver, with a clamp/unclamp time of 15 min/5 min policy. When liver function approached normal and adverse events had disappeared after surgical resection, we permitted patient discharge.
Follow-up consisted of monthly blood tests and monitoring of tumor markers, including des-γ-carboxy prothrombin, which was measured using a chemiluminescent enzyme immunoassay (Lumipulse PIVKAII Eisai, Eisai, Tokyo, Japan). Dynamic CT scans were obtained every 3-4 months after PRFA and SR. No patients were lost to follow-up.
Differences between the two groups were analyzed using the unpaired t-test for continuous variables, and the categorical variables were analyzed using the χ 2 test or continuity correction method. The overall survival curves and the recurrence-free survival curves were generated using the Kaplan-Meier method and compared using the log-rank test. The relative prognostic significance of the variables in predicting overall survival were assessed using univariate and multivariate Cox proportional hazards regression models. All variables with a P value < 0.05 evaluated using univariate analysis were subjected to multivariate analysis. Results of the multivariate analysis were presented as the hazard ratio (HR) with a corresponding 95% confidence interval (CI). All statistical tests were two-sided. All data were analyzed using SPSS software, version 9.0 (SPSS Inc., Chicago, IL, USA) for Microsoft Windows. Data are expressed as means ± standard deviation (SD). Values of P < 0.05 were considered to be statistically significant.
The baseline characteristics of the two groups are shown in Table 1 . Between the two groups, there were significant differences in tumor size (P = 0.001), platelet count (P = 0.004) and PIVKAII value (P = 0.037).
Fifty-four of 69 patients were treated with segmentectomy; 12/69 patients received bisegmentectomy; 3/69 patients underwent hemihepatectomy. The histological diagnoses of 69 patients were as follows: well-differentiated hepatocellular carcinoma (3/69), moderately differentiated hepatocellular carcinoma (36/69) and poorly differentiated hepatocellular carcinoma (30/69). Using dynamic CT performed within 1 month after SR, we confirmed no residual HCC in the liver remnant of all patients.
The mean number of treatment sessions for the 162 PRFA treated patients was 1.80 ± 0.37. Target biopsy prior to PRFA was not performed on any of the patients because of the specific complication of tumor seeding. We confirmed that all of the PRFA treated patients achieved complete ablation (ablated zone totally enveloped the tumor without enhancement) using dynamic CT prior to patient discharge and 1 month after PRFA.
In the present study, there were 3 patients with Child-Pugh C who underwent PRFA. Their Child-Pugh scores were all 10 points and PRFA was performed safely in these 3 patients.
The median follow-up period was 3.1 years (0.2-7 years) in the PRFA group and 3.3 years (0.7-7 years) in the SR group, respectively. Thirty-three patients (20.4%) in the PRFA group died during the follow-up period. The causes of death were HCC recurrence (24 patients), liver failure (6 patients) and miscellaneous (3 patients).
Twelve patients (17.4%) in the SR group died during the follow-up period. The causes of death were HCC recurrence (9 patients), liver failure (2 patients) and miscellaneous (1 patient). The 1-, 3-and 5-year overall survival rates after PRFA and SR were 95.4%, 79.6% and 63.1%, respectively in the PRFA group and 100%, 81.4% and 74.6%, respectively in the SR group (Figure 1) . The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 82.0%, 38.3% and 18.0%, respectively in the PRFA group and 86.0%, 47.2% and 26.0%, respectively in the SR group (Figure 2) .
In terms of overall survival (P = 0.259) and recurrence free survival (P = 0.324), there were no significant differences between these two groups. Figure 1 Cumulative overall survival rate. The 1-, 3-and 5-year overall survival rates after percutaneous radiofrequency thermal ablation (PRFA) and surgical resection (SR) were 95.4%, 79.6% and 63.1%, respectively in the PRFA group and 100%, 81.4% and 74.6%, respectively in the SR group. There was no significant difference between these two groups as determined using the log-rank test (P = 0.259).
We defined local tumor progression as the presence of a hypervascular nodule adjacent to the ablated area of PRFA or the resected area of SR using dynamic CT scan. 20 patients in the PRFA group and 10 patients in the SR group had local tumor progression during the observation period. The 1-, 3-and 5-year local tumor progression rates after PRFA and SR were 2.0%, 14.3% and 28.3%, respectively in the PRFA group and 2.8%, 14.3% and 22.8%, respectively in the SR group. (Figure 3 ) In terms of local tumor progression, there was no significant difference between these two groups (P = 0.746).
There were 127 patients in PRFA group patients with LC and 50 patients in SR group patients with LC, respectively. The 1-, 3-and 5-year overall survival rates after PRFA and SR were 94.2%, 75.8% and 56.4%, respectively in the PRFA group with LC and 100%, 78.0% and 67.8%, respectively in the SR group with LC. (Figure 4 ) The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 86.0%, 35.0% and 14.8%, respectively in the PRFA group with LC and 79.5%, 39.3% and 23.8%, respectively in the SR group with LC. (Figure 5 ) In terms of overall survival (P = 0.521) and recurrence free survival (P = 0.669), there were no significant differences between these two groups.
There were 35 patients in PRFA group patients without LC and 19 patients in SR group patients without LC, respectively. The 1-, 3-and 5-year overall survival rates Figure 2 Cumulative recurrence free survival rate. The 1-, 3-and 5-year recurrence free survival rates after percutaneous radiofrequency thermal ablation (PRFA) and surgical resection (SR) were 82.0%, 38.3% and 18.0%, respectively in the PRFA group and 86.0%, 47.2% and 26.0%, respectively in the SR group There was no significant differences between these two groups as determined using the log-rank test (P = 0.324). after PRFA and SR were 96.6%, 87.2% and 74.4%, respectively in the PRFA group without LC and 100%, 95.6% and 95.6%, respectively in the SR group without LC. (Figure 6 ) The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 93.0%, 52.5% and 22.2%, respectively in the PRFA group with LC and 100%, 75.7% and 30.4%, respectively in the SR group with LC. (Figure 7) In terms of overall survival (P = 0.276) and recurrence free survival (P = 0.258), there were no significant differences between these two groups.
Serious adverse events were significantly more frequent in the SR group than in the PRFA group (6/69 versus 3/ 162; P = 0.023). Serious adverse events in the SR group were as follows: bile leakage (2 patients); refractory ascites (2 patients); acute respiratory distress syndrome (ARDS) (1 patient); and massive gastrointestinal bleeding (1 patient). Serious adverse events in the PRFA group were as follows: biloma (1 patient); refractory ascites (1 patient); and intra-abdominal bleeding (1 patient).
The hospitalization length was significantly longer in the SR group (18.1 ± 10.4 days) than in the PRFA group (14.7 ± 5.7 days) (P = 0.013). In addition, there was no patient who died within the same hospitalization, making the mortality rate 0% in two groups.
In the univariate analysis of factors contributing to overall survival, hepatitis C virus (HCV) versus non HCV (P = 0.042), serum albumin (g/dL) (> 3.5 versus ≤ 3.5) (P = Figure 3 Cumulative local tumor progression rate. The 1-, 3-and 5-year local tumor progression rates after percutaneous radiofrequency thermal ablation (PRFA) and surgical resection (SR) were 2.0%, 14.3% and 28.3%, respectively in the PRFA group and 2.8%, 14.3% and 22.8%, respectively in the SR group. There was no significant differences between these two groups as determined using the log-rank test (P = 0.746). 0.003), and platelet count (× 10 4 /mm 3 ) (> 10 versus ≤ 10) (P = 0.045) were found to be significant factors (Table 2) . However, in the multivariate analyses involving these three factors, serum albumin (g/dL) (> 3.5 versus ≤ 3.5) was the sole significant factor contributing to overall survival.
Similarly, in the univariate analysis of factors contributing to recurrence free survival, HCV versus non HCV (P = 0.022), LC versus non LC (P = 0.002) and platelet count (× 10 4 /mm 3 ) (> 10 versus ≤ 10) (P = 0.005) were found to be significant factors (Table 3) . However, in the multivariate analyses involving these three factors, the presence of LC was the sole significant factor contributing to recurrence free survival.
Partial hepatectomy in patients with resectable HCC, who have normal liver function and are in good general condition is still considered the gold standard therapy with the aim of delivering curability [12] . In recent years, it has been possible to reduce perioperative mortality to less than 5% depending on the extent of resection and hepatic reserve [13] . The improved outcome is primarily as a result of advances in surgical and radiologic techniques, perioperative care and more cautious patient selection [14] .
Patients not eligible for resection because of their medical condition might be candidates for local ablative therapy, such as percutaneous ethanol injection (PEI) and PRFA. Many clinical trials comparing PRFA and PEI have demonstrated the clear superiority of PRFA over PEI [9, [15] [16] [17] . However, a major limitation of PRFA is the small volume of tumor that can be treated. The rate of complete ablative necrosis decreases with the size of the tumor, particularly in the case of tumors Figure 4 Cumulative overall survival rate between percutaneous radiofrequency thermal ablation (PRFA) group patients with liver cirrhosis (LC) (n = 127) and surgical resection (SR) group patients with liver cirrhosis (LC) (n = 50). The 1-, 3-and 5-year overall survival rates after PRFA and SR were 94.2%, 75.8% and 56.4%, respectively in the PRFA group with LC and 100%, 78.0% and 67.8%, respectively in the SR group with LC. There was no significant differences between these two groups as determined using the log-rank test (P = 0.521).
larger than 3 cm. There is general consensus that complete response to PRFA therapy in patients is associated with improved outcome [18] [19] [20] . Therefore, in the present study, objectives were limited to patients with HCC ≤ 3 cm in size.
HCC mainly disseminates through the portal and hepatic veins. The micro-dissemination can invade the tributaries of the portal branches and shed tumor emboli in the neighboring branches of the same liver segment [21] [22] [23] [24] . However, in the present study, with regard to recurrence free survival, there was no significant difference between the two treatment groups. One possible reason for this is that a sufficient ablative margin around the tumor when PRFA is administered may suppress the invasion of the micro-dissemination. Previous studies have reported that the initial treatment contributes to the survival of HCC patients treated using PRFA [19, 25] . In PRFA therapy, obtaining sufficient ablative margin around the tumor seems to be essential.
The findings of the present study indicated that the overall and recurrence free survivals were the same for patients with a single HCC ≤ 3 cm in diameter treated with either PRFA or SR. In addition, PRFA was demonstrated to have an advantage over SR in causing less serious adverse events and a shorter hospitalization length. Figure 5 Cumulative recurrence free survival rate between percutaneous radiofrequency thermal ablation (PRFA) group patients with liver cirrhosis (LC) (n = 127) and surgical resection (SR) group patients with liver cirrhosis (LC) (n = 50). The 1-, 3-and 5-year recurrence free survival rates after PRFA and SR were 86.0%, 35.0% and 14.8%, respectively in the PRFA group with LC and 79.5%, 39.3% and 23.8%, respectively in the SR group with LC. There was no significant differences between these two groups as determined using the log-rank test (P = 0.669).
Chen et al conducted a randomized control trial (RCT) on 180 patients with a single HCC ≤ 5 cm to receive either PRFA or surgical resection [12] , and Lu et al carried out another RCT on 105 patients with early HCC [26] . These two RCTs presented similar findings to those of our study. Additionally, four non-randomized controlled studies also reported similar findings of ours [27] [28] [29] [30] . And our study suggests that PRFA is less invasive than SR. It seems that PRFA can be a first choice for the treatment of small HCC. On the other hand, a recent study indicated that surgical resection provided better survival and lower recurrence rates than RFA for patients with HCC that conformed to the Milan criteria for a RCT [31] . However, in comparing their results with ours, the mean age of their patient population was more than 10 years younger than ours. In the etiology of liver disease in their study, patients with chronic hepatitis B were in the majority [31] . However, in our study, patients with chronic hepatitis C were in the majority. Therefore, their study results did not reflect the actual situation in Japan where Japanese HCC patients consist of many elderly patients, and the etiology of background liver disease involves chronic hepatitis C which accounts for about 80% of Japanese HCC patients. Hence, we should interpret their study results with caution.
Our study had several limitations. First, it was a retrospective cohort study. Patients who had a good hepatic reserve tended to receive surgical resection, and this could have possibly led to bias. Second, we did not assess the histopathologic diagnosis of HCC in the PRFA group. Tateishi et al reported that patients with Figure 6 Cumulative overall survival rate between percutaneous radiofrequency thermal ablation (PRFA) group patients without liver cirrhosis (LC) (n = 35) and surgical resection (SR) group patients without liver cirrhosis (LC) (n = 19). The 1-, 3-and 5-year overall survival rates after PRFA and SR were 96.6%, 87.2% and 74.4%, respectively in the PRFA group without LC and 100%, 95.6% and 95.6%, respectively in the SR group without LC. There was no significant differences between these two groups as determined using the log-rank test (P = 0.276). Cumulative recurrence free survival rate between percutaneous radiofrequency thermal ablation (PRFA) group patients without liver cirrhosis (LC) (n = 35) and surgical resection (SR) group patients without liver cirrhosis (LC) (n = 19). The 1-, 3-and 5-year recurrence free survival rates after PRFA and SR were 93.0%, 52.5% and 22.2%, respectively in the PRFA group without LC and 100%, 75.7% and 30.4%, respectively in the SR group without LC. There was no significant differences between these two groups as determined using the logrank test (P = 0.258). poorly differentiated HCC had a poorer outcome than patients with well to moderately differentiated HCC after PRFA [32] . Third, our study patients were limited to patients who have undergone curative treatment. These problems should be resolved in a future prospective study.
In conclusion, we demonstrated that PRFA is as effective as SR in the treatment of single and small HCC patients who have undergone curative treatment, and that PRFA is less invasive than SR. Therefore, PRFA can be a first choice for the treatment of single and small HCC.  Making sense of perceptions of risk of diseases and vaccinations: a qualitative study combining models of health beliefs, decision-making and risk perception BACKGROUND: Maintaining high levels of childhood vaccinations is important for public health. Success requires better understanding of parents' perceptions of diseases and consequent decisions about vaccinations, however few studies have considered this from the theoretical perspectives of risk perception and decision-making under uncertainty. The aim of this study was to examine the utility of subjective risk perception and decision-making theories to provide a better understanding of the differences between immunisers' and non-immunisers' health beliefs and behaviours. METHODS: In a qualitative study we conducted semi-structured in-depth interviews with 45 Australian parents exploring their experiences and perceptions of disease severity and susceptibility. Using scenarios about 'a new strain of flu' we explored how risk information was interpreted. RESULTS: We found that concepts of dread, unfamiliarity, and uncontrollability from the subjective perception of risk and ambiguity, optimistic control and omission bias from explanatory theories of decision-making under uncertainty were useful in understanding why immunisers, incomplete immunisers and non-immunisers interpreted severity and susceptibility to diseases and vaccine risk differently. Immunisers dreaded unfamiliar diseases whilst non-immunisers dreaded unknown, long term side effects of vaccines. Participants believed that the risks of diseases and complications from diseases are not equally spread throughout the community, therefore, when listening to reports of epidemics, it is not the number of people who are affected but the familiarity or unfamiliarity of the disease and the characteristics of those who have had the disease that prompts them to take preventive action. Almost all believed they themselves would not be at serious risk of the 'new strain of flu' but were less willing to take risks with their children's health. CONCLUSION: This study has found that health messages about the risks of disease which are communicated as though there is equality of risk in the population may be unproductive as these messages are perceived as unbelievable or irrelevant. The findings from this study have implications beyond the issue of childhood vaccinations as we grapple with communicating risks of new epidemics, and indeed may usefully contribute to the current debate especially in the UK of how these theories of risk and decision-making can be used to 'nudge' other health behaviours. Few would argue against the success of mass vaccination programmes in reducing and, in the case of smallpox, eliminating infectious diseases. Continued success however, requires adequate coverage which in turn requires parents to be committed to vaccination as an effective method of preventing their children from contracting diseases. Such commitment may be adversely affected by an increasingly complicated immunisation schedule for an increasing number of diseases and scares of vaccine safety (e.g. MMR debate that continues in the UK). It has also been argued that the very success of mass vaccination programmes has limited parents' experience of vaccine preventable diseases and thus affected their assessment of the severity of diseases and importance of prevention [1, 2] . There is therefore, a continued need to better understand parents' perceptions of what is serious, what is risky and what is best for their children's health. Indeed with SARS, bird and swine flu, this extends to what people think about how best to protect their own health. Theories of health beliefs, decisionmaking and subjective risk perception have all been used in attempts to explain parents' decisions with respect to immunisation [3] [4] [5] , but with limited success in explaining why parents differ in their perceptions of risk. With the exception of Hawe's et al research, [6] public health and health promotion campaigns have not drawn explicitly on or tested these theories. A recent review discussed how decision-making theories might help to explain parent attitudes and behaviour with respect to MMR vaccine uptake, but provided no primary evidence [7] . We argue in this paper that our understanding of immunisation choices and how we may best influence those choices may be increased through a synthesis of health beliefs, decision-making and subjective risk perception theories. This paper describes the findings from a qualitative study examining the utility of the risk perception and decision making theories to provide a better understanding of the differences between immunisers' and non-immunisers' health beliefs and behaviours, when considering the risks of a 'new strain of flu'.
Theories of health protective behaviour offer an appealing framework in which to interpret differences in compliant and non-compliant parents with respect to childhood vaccinations [8] . While several models have been developed to account for people's adoption of health protective behaviour such as the Theory of Reasoned Action [9] , the Triandis Model [10] , Multi-Attribute Utility (MAU) Theory [11] and the Subjective Expected Utility Theory [8] , the Health Belief Model is possibly the simplest and the most widely used and tested [12, 13] . The four elements of the Health Belief Model are: perceived susceptibility (likelihood of getting the disease), perceived severity (perception of how serious an outcome or consequence is from the disease), perceived benefits (efficacy of preventive action undertaken) and perceived barriers (time, effort, money, inconvenience, pain, side effects of preventive action) [12, 13] .
Attempts to assess the association of these elements and childhood immunisation uptake have been inconclusive with some studies reporting expected relationships [14] [15] [16] and others contrary to what would be expected [17, 18] . These contradictory findings have led to the conclusion that the health beliefs of mothers are not important contributors to immunisation uptake or completion and are less important than socio-demographic factors. That is, incomplete immunisation (fall behind the immunisation schedule or fail to complete) is associated with being poor [17] , and being a single parent [19, 20] . Low maternal education has usually been found to be a risk factor for not completing immunisation [20] . Being anti immunisation on the other hand, is associated with high education [21] .
While in some instances theories of health protective behaviours have been shown to differentiate between complete, incomplete and non-immunisers, none of them provides an explanation of how people perceive risks or how their perceptions might influence behaviour. Indeed these models assume a rational basis for these decisions: a simple weighing up of information regarding severity, susceptibility, benefits and barriers. There is not, however, a simple relationship between mortality or morbidity figures and the perception of risk [22] . People do not perceive, interpret or act on risk information in the way expected by risk experts in general [22] nor specifically when considering vaccines and diseases [4, 23] .
Two domains which have addressed lay rather than expert perceptions of risk and what influences decisions encompassing risk are studies of the subjective perception of risk [24] [25] [26] and the study of decision-making under uncertainty (also referred to as the psychology of choice) [27, 28] . Both approaches focus on risk as a subjective rather than an objective concept, and both involve social and psychological aspects that impact on the individual cognitive structure of risk perception.
Research into the subjective perception of risk generally involves asking study participants to rate a heterogeneous set of environmental or health risks including risks from individual activities, residential or work conditions, hazards from technologies, substances or products, and natural hazards. Respondents are asked to rate these activities or hazards in terms of perceived magnitude of risk, the acceptability of the risk and other aspects such as likelihood of death, catastrophic potential, avoidability, fear, familiarity, imposed or personal choice, time scale of impact, benefits of risk source, degree of concern, personal exposure etc.
Studies including vaccination (otherwise unspecified) as a hazard have reported vaccination as low risk [24, 25] . Vaccination is generally considered to be a risk that is not 'dreaded' (controllable, not fatal, individual, low risk to future generations) but is somewhat 'unknown' (not observable, effect delayed, new risk, risk unknown to science). Slovic [25] , in a study of risk perception of prescription drugs including vaccines, found vaccines were generally considered beneficial by the sample. However, people associating negative meanings to drugs tended to judge drugs and vaccines as having higher risks and lower benefits than people who associated positive meanings to drugs.
This research has consistently found that risks are perceived more negatively if exposure to the hazard is involuntary, people perceive they have little personal control over outcomes and there is uncertainty about the consequences of the outcome(s), the hazard is unfamiliar, the effects of the hazard are delayed, the hazard has catastrophic potential, the benefits are not immediately apparent and the hazard is caused by human rather than natural causes [24] . These have been summarised by two factors labelled 'Dread' (uncontrollable, feared, involuntary exposure, inequitable distribution of risk, not easily reduced, catastrophic, risk increasing, fatal consequences, risk to future generations) and the 'Unknown' (not observable, risk unknown to science, delayed effect, new risk) [29] . People make judgements about the persuasiveness and trustworthiness of experts involved in communicating risk and find risks less acceptable if they believe that the communication of the risks between experts and the community is poor [30] .
Research into decision-making under uncertainty showed that the rational subjective expected utility models which presumes that a good or 'rational' decision maker will sum the utilities and choose the action with the greatest total utility, does not explain how people make decisions [27, 28] . Kahneman and Tversky's research confirmed findings from the subjective perception of risk and further contributed to an understanding of how information about risks or uncertainties of outcomes influences decisions [27, 28] . Using hypothetical scenarios, studies have shown that people consistently underestimate risks of familiar and frequent events and overestimate the occurrence of low probability, but high consequence risks. Thus, rare events are perceived as more likely to occur than they do and common events are thought to occur less often than they do. The classic problems devised by Kahneman and Tversky involved asking subjects to choose between the certainty of saving 200 out of 600 lives or taking a 1 in 3 chance of saving 600 lives. They found that people's responses to possible negative outcomes are more extreme than their responses to possible positive outcomes. People also demonstrate a tendency to believe that their own risks are less than others, particularly if they believe that their exposure to risk is in some way under their control [31] . The impact of this tendency, described as unrealistic optimism and/or the illusion of control, is to reduce the perceived need to take protective measures [32] .
Of particular importance to the decision to immunise are studies describing the operation of omission bias and choices involving ambiguous situations [33] . Omission bias describes a preference for taking no action if the action might cause harm, even if there is a greater risk of harm by 'doing nothing'. Ambiguity describes the decision-maker's feeling that there is missing information relevant to outcome or choice [33] . The effect of ambiguity on choice is to reduce people's willingness to act or to postpone the action until the missing information can be obtained [33] . These studies used hypothetical scenarios, making decisions about others, and participants were generally tertiary students and sometimes, parents.
As stated above the Health Belief Model by itself has been found wanting in terms of being able to explain parents' behaviour with respect to immunising their children. Conceptual pieces have been written describing how subjective risk perception and risky decision-making theories may be useful in understanding parents' behaviour and choices although these have not drawn on primary data [7] . Using a qualitative design, the aim of this study was to: explore the salience of the decision-making and risk perception findings to parents' choices to immunise their young children; examine the utility of the risk perception and decision-making theories to provide a more detailed explanation of the differences between immunisers' and nonimmunisers' perceptions of severity, susceptibility to disease and benefits of vaccines; and understand how these theories might explain perceptions of risk and reactions to a 'new strain of flu' for themselves and their children.
Sampling A stratified purposeful sampling strategy [34] , was used to identify first time and experienced mothers of infants who were completely immunised (for age), incompletely immunised (behind the recommended immunisation schedule), partially immunised (parents chose or advised not to have a specific immunisation) or who had no immunisations. Initially, mothers of children between 14-16 months were approached. This age allowed a range of immunisation experiences to be discussed with participants while minimising the time since the first immunisation. To obtain sufficient numbers of nonimmunisers and partial immunisers this age range was broadened to include 3 to 30 months. It was initially proposed that interviewing approximately 8 mothers from each immunisation category would be sufficient to discern patterns of similarity and difference between these categories. If preferred by the mother, both parents could participate in the interview.
Possible participants were identified by Maternal and Child Health (M&CH) nurses in five metropolitan local government areas in Melbourne, Australia. Nurses were asked to approach mothers fitting the immunisation categories and to include, to the best of their knowledge, mothers of high and low education (< Year 11) and high and low income (held a Health Care Card). Parents were identified as fitting these categories from informal information available to the nurses. Parents from non-English speaking backgrounds whose English was poor were not interviewed. The Nursing Mothers Association group for the area also advertised the study.
Ethics approval was granted by the Royal Children's Hospital Ethics in Human Research Committee. Participation was voluntary, with written consent required. Participants provided informed consent to be interviewed, and for the interview to be taped. Participants were assured that they could stop the interview at any time, could choose not to answer any question if they didn't want to and that their responses would be confidential and transcripts anonymised. The interviewer did not have a dual relationship with the participants (i.e. she was neither a clinician nor provider of health services/care).
Recruiting and interviewing continued until 'saturation' occurred [34] (i.e. no new information was obtained from the interviews) for complete, incomplete and non-immunisers or until no more new parents fitting the categories could be identified, as was the case for partial immunisers.
Over the period of data collection, 94 families were identified as possible participants. Forty-eight interviews were arranged and 45 completed. One participant withdrew consent prior to the interview (she did not believe she had anything to say). Two participants were not at home at the time scheduled for the interview and neither returned follow-up phone calls. Of those not interviewed, 17 fitted categories for which a sufficient number of interviews had been conducted (complete immunisers); 11 were not interviewed due to language difficulties; 12 could not be contacted by the nurses and 6 refused. Interviews were undertaken in 1995-96. For six interviews both mother and father participated in the interviews (three of these were non-immunising families). All interviews were conducted in the participants' homes.
Semi-structured, one-on-one interviews were used to collect information from parents about their children's health, the experience of illness in the family, their understanding and interpretation of risk and how all of these related to their decision to immunise. The method of one-on-one interviews rather than focus groups, was chosen as the aim was to explore the parents' experiences and path to choosing to immunise or not, rather than a group discussion of the pros and cons of immunisation. To understand the context of parents' decisions to immunise, the interviews covered four themes: (1) how mothers keep their children healthy; (2) experience, familiarity and concerns regarding both vaccine preventable diseases and other diseases; (3) concepts and influences on risk perception and (4) the decisions, experience and outcomes regarding immunisation. The interview began with questions about health as a nonthreatening introduction and to place the consequent discussions about disease and disease prevention in the framework or context of health.
Questions about diseases and the family's experience of them were included to explore the relationship between common illnesses experienced by the family and vaccine preventable diseases. What was of interest here was which diseases were familiar, which were unfamiliar, which were to be avoided if possible and which were 'just' childhood illnesses.
To aid the investigation of how parents understand and interpret risk information the following two hypothetical news items about an influenza outbreak were read to the participants.
Health authorities issued a warning today about a new strain of flu expected this winter. The flu affects the airways, making breathing difficult and causing repeated bouts of coughing. Long term effects of pneumonia and brain inflammation have been reported in some cases. This strain appears to affect adults between the ages of 20-50 years. Several deaths occurred last year from the A-strain of this virus.
Doctors have recommended that all adults should be vaccinated, especially those who are overworked, stressed and tired.
Health authorities issued a warning today about a new strain of flu expected this winter. The flu affects the airways, making breathing difficult and causing repeated bouts of coughing. Long term effects of pneumonia and brain inflammation have been reported in some cases. Several deaths occurred last year from the A-strain of this virus. Many of those who died were children under 5 years. Doctors have recommended all young children should be vaccinated.
The description of symptoms and complications was taken from a description of the complications for pertussis [35] . The doctors' recommendations were written so that the parents could consider themselves 'at risk' in the 1st instance, parents of young children often feeling overworked and tired, and in the 2nd scenario their child/children fitted the 'at risk' group.
Omission bias was examined in this study by asking parents to respond to the following statement:
STATEMENT 1 Some people say they won't vaccinate because they would feel worse if their child died because of the injection than if the child was not immunised and died from the disease.
Participants were asked their opinion and were then read a second statement: STATEMENT 2 Some people say they would vaccinate because they would feel worse if their child got the disease and died or was brain damaged when they could have had an injection to prevent it.
These statements were used rather than the more complicated scenarios developed by others (e.g. [33] ) because it was believed they captured the essential element of omission bias in circumstances with which the parent could identify.
The interview concluded with discussions of the process of deciding to immunise or not and included a discussion of structural and non-structural barriers.
All interviews were conducted in the participants' homes at times convenient to them by the first author. Interviews lasted between 45 to 90 minutes. Socio-demographic information including family size and type (two or one parent family), mother's age, parental occupations and education levels was collected at the end of the interview. For those children who were immunised, immunisation status was determined from the immunisation records held by the parent. All interviews were audio-taped and fully transcribed. The interview focussed on the sole or youngest child in the family. Previous experience of disease and immunisations for older children was discussed in terms of its effect on decisions for the youngest child.
Interviews were thematically coded after all interviews had been collected. This analysis focussed on determining whether parents' descriptions of their experiences and beliefs were congruent or incongruent with theories of health behaviour, decision-making and risk perception. The coding was undertaken by the first author. No formal testing of the reliability of the coding was undertaken although discussions with colleagues about the analysis and the meanings and patterns derived from this were extensively undertaken.
Interviews were completed with 16 mothers whose children had completed immunisations appropriate for their age, 12 whose children were incompletely immunised, seven whose children were partially immunised (chose or advised not to have at least one component), and ten whose children had no immunisations. All families with incomplete immunisations had two or more children. (See Table 1 .)
The following section presents a brief summary of similarities between immunisers and non-immunisers in terms of the concepts in the Health Belief Model. This is followed by a critical interpretation of the data linking this model with the theories of subjective perception of risk and decision-making under uncertainty. Finally the differences found between complete, incomplete, partial and nonimmunisers in terms of these theories are summarised. Table 2 summarises the differences and similarities between complete, incomplete and non-immunisers in terms of the core concepts of the Health Belief Model from these interviews (see [16] for further details). Partial immunisers formed two groups; those whose child had had a severe reaction to DTP (Diphtheria, Tetanus, Pertussis vaccine) (n = 3) where the parents had been advised not to continue with vaccination and those who chose to only undertake some vaccinations or changed their mind about vaccinations after the first DTP vaccine (n = 4). The former of these expressed views similar to complete immunisers and the latter to nonimmunisers. To better understand these differences in perceptions, the interviews were analysed firstly for themes from the studies of risk perception-dread, familiarity and controllability.
Dread was an important determinant of what the participants in this study perceived as high risk. What was dreaded, however, differed between the immunisers and the non-immunisers. Immunisers dreaded the outcomes of the diseases, especially those with which they were unfamiliar. This fear motivated them to take the risk of immunising.
"The life threatening ones really concerned me, like ones I didn't know anything about...Polio really scared me and the thought of whooping cough... those are quite scary sort of concepts. Meningitis was frightening." (Complete immuniser, #11) "I'm not sure about whooping cough, it just has horrible connotations in my mind but I'm not quite sure why, what can happen... Yeah, it's interesting isn't it, you know, people think it's the ones that you don't know about that you're likely to dismiss but it doesn't seem to me that way." (Complete immuniser, #13)
Polio, diphtheria, tetanus and meningitis were unfamiliar to these mothers but they conjured vivid images of severe outcomes. Of this group (immunisers), parents considered their children to be at greatest risk from meningitis. Even though most considered it unlikely that their children would contract these diseases, it was easy to imagine that if contracted, the worst was likely to happen.
LB: "If M hadn't been immunised, how likely do you think she would get these diseases?" The risks associated with vaccination were also perceived as being rare but rather than imagining the worst in this instance, they believed that one would be unlucky to have severe reactions.
Thus, on balance, the risk of not immunising was not worth taking and a 'common sense' approach was necessary. They likened vaccination to taking other safety precautions. In terms of the theories of risk, respondents were perceiving the diseases as less familiar therefore dreaded and unknown, and therefore possibly overestimating their risk, and perceiving vaccines as more familiar, and possibly underestimating their risk.
In contrast, non-immunisers dreaded the unknown or uncertain outcomes of the vaccines with major fears being for invisible/undetectable/distant problems such as the vaccines causing leukaemia, SIDS, AIDS and brain damage. For these parents, vaccines were not only ineffective but they were actively dangerous to children's health.
"Brain damage, affecting limbs. I've read there are long term effects which we really don't know about. There are new diseases coming up. Polio is no longer life threatening but there are cancers and AIDS, long term effects on the immune system. And this is because we are interfering, causing genetic changes." (Non-immuniser #26) "They don't work and they do harm. Putting these things into their bodies-germs and all the other products-mercury aluminium etc cannot be good. It suppresses/disrupts the child's immune system. It doesn't work and it is harmful. It's not just the risk of the side effects but the long term effects that we don't know about now. Basically so many things which we did in the past we now know better and think were barbaric. I would rather not do something to my child when we don't know what the long term effects might be. There have been studies which have related these to leukaemia and other cancers, asthma eczema and all sorts of things. I don't want to do that to my child." (Non-immuniser, #19)
On the other hand, severe outcomes of the diseases were believed to be rare or only a problem for children with poor nutrition, poor sanitation, and compromised immune systems. Non-immunisers believed it was unlikely that their children would suffer serious complications if they contracted these diseases because they had healthy immune systems.
"If D did get one of these diseases it wouldn't necessarily be catastrophic. Some people do get very sick or die but what we don't know, what they don't tell us is that those children were probably not well to start with. Fairly sick children are more likely to get serious long term effects. Their health before the illness is crucial to how their bodies cope with the disease." (Non-immuniser,# 19) "...a rejection of the notion that children have to be immunised against these diseases because the disease itself will automatically be worse than the immunisation and a concern that the you know the vaccination itself can have problems." (Non-immuniser,# 21)
For one non-immuniser, who was not 'against' using conventional medicine' she believed her children were protected from disastrous consequences because they had easy access to modern medical intervention.
"Well I just can't see the need. If your child catches measles and... that develops into anything else... we're not stuck in the middle of the country without good doctors or hospitals..." (Non-immuniser, #29)
As reported previously, both pro-and anti-immunisers were concerned about vaccines overloading even healthy but immature immune systems [16] "I do sort of worry that we are vaccinating too much. I just worry about what it does to your immune system, to all our immune systems" (Complete immuniser, #14) "He's still very thin but he's past the point of where I sort of see him [as] very vulnerable...like now I'm happy to give them to him." (Incomplete immuniser, #24) "I mean I know she is a strong as a horse and I know she could have every shot under the sun and she'd be fine I just don't think it's a proper thing to do ... in only two-month olds." (Non-immuniser, #29)
As would be expected from risk perception theory, diseases that were familiar to parents were not dreaded. Measles, mumps and rubella were not considered serious or life threatening by most parents irrespective of immunisation status. Most mothers were familiar with these diseases. They had had personal experience of these and remembered them as mild.
"[If] she happened to get measles, well I'm not that worried...because I had it and it was fine." (Complete immuniser, # 11)
The motivation to immunise against these diseases was, therefore, less than for diseases that were unfamiliar.
"...If it's a disease like measles, mumps, chicken pox, things like that you can let them get through fine, then if you got into meningitis, polio, well yeah, you'd have to think again." (Complete immuniser, #31) "See, measles and German measles-I know that they brought the immunisation in because there are complications and there have been kids with complications. But see, like I remember from my generation a lot of us that was just the normal. Kids had the measles. So I am not so sure about those two whether it is important." (Incomplete immuniser, #15)
Measles, mumps and rubella were perceived as diseases that...'every child's got to get' and rather than avoid these diseases it was best to 'get them out of the way' as early as possible, especially as it was believed that these diseases were more serious in adults. There was no urgency in having their children vaccinated for these diseases.
The idea of control was also used by parents to explain their choice to vaccinate. One explanation for immunising given by complete immunisers was that they could not control their children's exposure to diseases and hence, it was safer to vaccinate. By doing so they could control, to some extent, the diseases that their children were at risk of contracting.
"I think the world of her and I thought if [I] can prevent her getting any of these diseases I will. (Complete immuniser, #7)
Incomplete immunisers believed vaccination would contain or reduce the effects of disease rather than prevent it completely.
"...kids still get measles and mumps so that's the silly thing isn't it really? It's only to prevent it, it can't cure, do you know what I mean? I've heard of kids still getting measles." (Incomplete immuniser #30) "But it doesn't prevent the flu, you still get the flu but not a strong dosage." (Incomplete immuniser, #44)
In contrast, non-immunisers talked about being able to control their children's environment and therefore their exposure to disease. This non-immunising mother spoke of her reasons for vaccinating the family dogs: 
The explanatory power of ambiguity, outrage, omission bias and optimistic control was also examined in these interviews.
Perceived lack of information or insufficiency of information should either provoke outrage [30] or hesitation from acting [33] . Participants provided examples of both. Lack of information about susceptibility to vaccine side effects caused mothers in some instances to refrain from vaccinating their child or to hesitate about immunisation. One mother had hesitated to immunise her second child until she could be reassured that he would not have severe side effects from the vaccine. She had reason to believe he would be particularly susceptible to such side effects because he was 'not robust', he had many food allergies and his father had collapsed after immunisation as an infant. She expressed an equivalent concern about the child's susceptibility to disease especially as he had a school-aged sibling who could expose him to disease. The major reason for her hesitation was that no one had seriously considered her questions or considered her son's case on an individual basis.
"I want someone to look at him as an individual and I don't feel that they are the medical community....I don't want people making the decisions for me. ...I want that information available so that I can make an informed choice" (Non-immuniser, #18)
Being aware that children could react to the MMR vaccine but not being told what that reaction was or what to expect also caused some hesitation with some mothers. Some mothers hesitated about immunising against Hepatitis B which was at the time of the study recommended for 'at risk' groups. It was unclear to these mothers what this phrase meant and if it applied to their children.
[I] believe he should have the hepatitis one 'cause if he comes in contact with another child that's got it, but they say he's not at risk... but what makes him not at risk to get it? So I've been umming and aahing whether to get that one."(Complete Immuniser, #10)
The reverse of hesitating to act because of insufficient information was shown by others who figuratively 'shut their eyes' to the information about vaccine risk because it was unsettling.
"I think honestly speaking, this sounds stupid, but I think well, I don't want to hear it [about side effects], because it scares me. I know it might be stupid because you think, well you know they're s'posed to have it but if you start thinking well, what if you know if this happens and that happens well, then you wont immunise your children, so, there's a risk I s'pose."(Incomplete immuniser, #42)
Another response to a perception of insufficient information was anger or outrage. During the interview some mothers apologised for not being better informed about diseases.
Others were angry at their lack of knowledge about diseases and vaccines. This anger was not directed at themselves but at unspecified others. Anger was more often expressed by non-immunisers who believed that drug companies and doctors knew vaccines were not safe but kept the information from the public.
Whether parents choose not to act, when action may cause harm, was explored. This was done with the use of two statements-describing whether (1) it would be worse to have your child die due to your action (immunise) or (2) it would be worse due to inaction (die from disease) (see Methods section for statements). Both statements presented uncomfortable possibilities to parents.
"You can't ... I mean as far as I'm concerned you lose a child you lose it and it's painful either way I like to think that I've done the best I can to protect him from it um and if you know it's because of the injection well to some degree I'm fatalistic. I mean if it's meant to be it's meant to be. There's not much you can do about it but I would rather know that I've taken every precaution I can instead of you know leaving him open and susceptible to these things." LB: Some people say they would vaccinate because they would feel worse if their child died from an illness which they could have prevented. "Possibly I would sit in that category." (Complete immuniser, #1)
Most parents, irrespective of the immunisation status of their children identified more with the second statement, with only a few parents identifying with the first:
"I'd have to agree with that. I think if you've given birth to a perfect healthy child and then you've introduced foreign substances into their body which has then damaged them in some way, ah, yeah, I don't know, I don't think I could live with myself. Whereas if they've caught the disease that's kind of c'est la vie you know. I mean it's still awful. It's still a great tragedy, especially if you do lose them. But I think that's that. If you talk about metaphysics, I believe in metaphysics and all the rest of it, so I'd sort of say well, they're meant to be here, they're meant to experience it, they're meant to deal with it or not deal with it depending on what they're here for. So I have to take a philosophical approach. It'd be devastating." (Non-immuniser, #32)
Opposite to what would be predicted, many of the non-immunisers disagreed with the first statement and were adamant that this did not form part of their reason not to immunise.
FATHER: "I think you would be foolish to reach [that conclusion] I mean we're not foolish. I couldn't possibly say that I would be more comfortable with the you know..." MOTHER: "The child dying or at least you know that he died from the disease." FATHER: "Yeah a natural thing rather than induced. Yeah that's where the natural therapy philosophy goes too far...That would never be reason to [not immunise]." MOTHER: "No, for not immunising him. Yes. I can't even relate to it as a distinction." (Non-immunisers #27)
The main reasons parents gave for not agreeing with the first statement was the perception that this scenario was unlikely to occur and, irrespective of immunisation status, most parents believed they had done everything they could to prevent disease. Thus, parents used their perceptions of the risks of the outcome of death from vaccine or the risk of getting the disease to explain their choice.
"No, well I'd feel, well I think that that's part of the risk, that there is a small risk that your child will have a reaction to the immunisation that's that's minimal compared to the risk of them getting the disease if you don't immunise so I'd always opt to immunise. (Complete immuniser, #5) "I think you've got more, to me I think she's got more of a chance getting something not being vaccinated than, she's a healthy little girl isn't she?" (Complete immuniser,#7)
For those who agreed with Statement 1, they based this on their belief that there was a greater risk from vaccines so the first statement was the more likely scenario.
Participants' responses to the two hypothetical radio news items, was concordant with the theories that perception of risk may be influenced by an unrealistic optimism about one's own risks or unrealistic perception of control over one's life. The participants generally did not believe that they would be at risk from the flu. They believed themselves to be healthy, not susceptible to flu and that they were strong enough to fight it off:
"I tend to think that couldn't happen to me 'cause I'm young and healthy and couldn't possibly die of flu...Its a few cases [dying] and that happens." (Complete immuniser, #13)
Although the scenarios were written specifically so that those being interviewed fitted the 'at risk group', the participants did not identify with this group. They believed that the people who suffered serious consequences of flu were different to themselves. When they heard similar items on the news they assumed the people who were badly affected were old, frail, sick, had not been eating well, had poor immune systems, low resistance or were people who did not look after their health.
"It says doctors recommend all should be vaccinated especially those who are overworked, stressed and tired. Well of course they would be the ones whose immune systems would not cope." (Non-immuniser, #19)
All non-immunisers believed the risk of this hypothetical flu vaccine was higher than the risk of the disease but some immunisers also perceived high risk and little benefit from the vaccine. They related incidents of relatives who had bad experiences after receiving a flu injection. They believed that there were other ways of reducing the risk of flu such as taking supplements, improving lifestyle and avoiding people with the disease. Those who did believe their family to be at risk of this flu already received annual flu injections, with the occurrence of serious illness in these families prompting them to have flu vaccines.
Participants were also asked whether knowing someone who had the illness and had been very ill would affect their decision to immunise against this hypothetical flu. Again the response was that it would depend on the state of the friend's health and their habits: whether they were unhealthy or stressed, or careless of their health. While it would be more concerning to hear of a friend who was ill, most did not think it would mean they themselves were more at risk.
In the second news item the 'at risk' group was children under five. All parents stated this item of news would be of greater concern to them. Their first action however, would be to seek more information from their health advisers before immunising. The risk of their children becoming ill was more important than the risk to themselves and all believed that their responsibility as parents was to do everything they could to protect their children. This responsibility made it stressful to make decisions for children because 'you can't afford to make the wrong choice', and one can't take risks for one's children where one might take risks for oneself.
"...[I'm] not prepared to risk them, I can control my risks." (Non-immuniser, #29) "You've got to take the risk to prevent them getting sick." (Incomplete immuniser, #20) "Take all the risk factors out of it and make sure they have a good life." (Incomplete immuniser, #34) "Because I am so much more protective of their health than mine. I am concerned that they don't have the option of making choices as easily as I do and that is why I would like to make informed choices. And I feel like if I make the wrong choice for yourself and that is something that I wear, I am responsible for it. If I make the wrong choice for them it is more serious." (Non-immuniser, #21)
There was however, a reluctance to immunise. Many believed there was an over-reliance on immunisations and antibiotics and that they would only immunise if the disease was widespread or local (Statewide). If it were not widespread it was not worth the risk of preventative medicine. Table 3 summarises the factors found to influence the decision to immunise and corresponding aspects of the explanatory theories used as a theoretical framework for this study. In this table we aim to show how the information from risk perception and decision making under uncertainty allow for a greater explanation or point to more nuanced action. For example, consideration of unfamiliarity with diseases would be part of the Health Belief Models framework of considering perceived severity. However the Health Belief Model does not explain or allow us to understand what people perceive as familiar or unfamiliar. One might think that if parents are not familiar with a disease they may not think it is serious, whereas subjective perception of risk would indicate that the reverse may be operating: unfamiliarity increases people's perception of risk. Similarly, where parents don't think their child is susceptible to the disease (a Health Belief Model construct), the idea of having 'optimistic control' helps to explain why they might think this.
In considering the news reports respondents were asked how many would 'several deaths' be to cause them to worry about the risks of the disease. It was difficult for the participants to respond to this question meaningfully and the production of these numbers was somewhat arbitrary; most were not comfortable giving their response, and many could not say. For instance, one couple said they could give an answer if it was needed to meet the research requirements but it would be meaningless. The numbers given varied from only one or two deaths, five in the State, or between one and ten percent. Others gave figures of more than fifty percent of those who got the disease would have to die for them to be concerned.
The question was useful, however, because it provoked participants to define the type of information they wanted in order to make sense of reports such as those they had just heard. For instance, they said their response to the number of deaths would depend on how similar to their own circumstances were those who had died. Did they live in Australia, Victoria or developed countries? Were those who were dying, previously Table 3 Factors influencing the decision to immunise drawing on the Health Belief Model, subjective perception risk and risky decision-making theories healthy people or those who were sick and thus, more susceptible? Was it a familiar disease (flu) or rare (Ebola virus)? If it was unfamiliar it was frightening. If it was familiar (flu) they wanted to know the details of who it was who had died or suffered complications. Thus, it was not the statistics that were important for deciding on risk, but the characteristics of those who had the disease and the familiarity or unfamiliarity of the disease.
The decision to immunise or not is complex with perceptions of risks of vaccines, diseases and robustness of the child's health to be considered. In this study we have identified and clarified differences in perception between complete, incomplete and non-immunisers and also identified similarities between all mothers with respect to the decision to immunise their young children.
While the decision to immunise young children can be understood to some extent in the context of perceptions of severity and susceptibility to disease and benefits and barriers to immunisations, the theories of risk perception and decision-making add a depth of understanding to the differences found between these parents in terms of their perceptions and interpretations of what is risky and what is not. Two aspects in particular appear to be important: the familiarity or unfamiliarity with the disease and perceived control over risks or outcomes. Thus, perceptions of severity of disease are influenced by the unfamiliarity of the disease and/or the perception that these diseases will have uncontrollable outcomes. That is, diseases with which parents are least familiar are perceived as more severe than those with which parents are more familiar and therefore worth taking preventive action. Being a familiar disease contributed to delays in immunisation, or not immunising as these familiar diseases were not considered to be severe.
This finding is congruent with other primary studies. For example, Hilton et al [36] reported 'of all the diseases... measles was the one that parents most commonly reported having as a child. ...Indeed their experience of measles often rendered it a less threatening disease.... While parents with no experience of measles entertained the long-term damage it could inflict, those with experiences of it tended to minimise the risks' (p 174, authors' italics). From this it would appear that it is too simplistic to attribute reduction in immunisation uptake to a growing lack of familiarity with diseases because of the success of the immunisation programmes.
Understanding people's perceptions of what can and cannot be controlled is important to understanding their behaviour. There is both an aspect of fearing uncontrollable or unknowable outcomes and therefore taking preventive action and an optimistic belief or an illusion that the environment or risks can be controlled. Unlike the non-immunisers in this study and others [21] , immunisers choose immunisation because they believe they have limited control over their children's environment and contacts. Many believed they could control risks to their own health or were willing to 'take the risk' with their own health.
Parents were less willing to take risks with their children's health than with their own. This was partly because children were perceived as more susceptible than healthy adults and partly because their children were dependent on them making good decisions. This unwillingness to take risks included being cautious of preventive action as well as cautious about diseases. An important barrier to action was the tension between what is 'natural' and medical intervention. For many mothers there was something 'unnatural' about medical intervention. They held a belief that medical intervention was necessary for important diseases but that it was not safe or necessary to use for all problems. Again, this perception that what is unnatural is more risky is congruent with the studies of subjective risk perception, but could not be predicted from the Health Belief Model.
Importantly, the participants believed that the risks of diseases and complications from disease were not equally spread throughout the community. When listening to reports of epidemics, it is not the number of people who are affected but the familiarity or unfamiliarity of the disease and the characteristics of those who had the disease that caused parents to worry about taking preventive action. Poor information or communication creates barriers to immunisation completion which can be understood in terms of the concepts of outrage and ambiguity. Lack of trust and poor communication between providers and parents exacerbated the belief that information was being kept from them. Because they all believed that parents were ultimately responsible for their children, this feeling that information was denied them frustrated and angered them.
This study has used qualitative methods to determine if aspects of theories of risk and decision-making can help to explain parents' decisions about immunising their children. Traditionally qualitative research has been associated with the generation, rather than the testing, of hypotheses, however, this denies a major strength of qualitative research which is to examine theories in the light of data. As such it is a method suited to producing understanding and to generating solutions to problems [34] . We believe this method, therefore, is appropriate to provide a greater understanding of complexities of decision making and perceptions of disease and vaccines and a depth of information not available from large scale quantitatively based surveys. The benefits of using the qualitative method described in this study is the large quantity of detailed information it provides, however, using a small non-randomly selected sample can present problems in determining the generalisabilty of the ensuing information. The results of this study confirm, complement and extend the findings of other studies in this area [4, 5, 36, 37] .
The data that this paper draws on were collected in the late 1990s and some may wonder at their currency. We believe that this might be a problem if the focus of the paper was about which diseases or vaccines are an issue; with scares and controversies these can change over time. The point of this paper however, has been to examine the utility of synthesizing theories of health protective behaviours, risk perception and decision-making. While we recognise that different socio-temporal contexts may create different issues (e.g. the impact of the MMR controversy was substantially greater in the UK than Australia), we argue that the approach we have taken in this paper provides a framework for us to make sense of people's reactions to and perceptions of old (and new) diseases and vaccinations at any time. We therefore think this work can contribute to, and be of particular importance in informing the public health approaches to new flu epidemics. It provides data supporting commentary and critique of the current public health approach to the issues of vaccine risk and immunisation uptake, being that continued provision of better risk information is not the answer [7] .
We have found and would argue that the theories of risk perception and aspects of decision-making under uncertainty have been useful for understanding the differences and similarities between pro-immunisers and non-immunisers, except for the issue of omission bias. Parents in this study, whether immunisers or not, generally did not agree that a negative outcome was preferable or more acceptable from inaction. This finding raises some doubts about the methods and/or generalisability of findings from studies of this phenomenon, which usually involve multiple, similar, hypothetical situations with limited contextual information, presented in mathematical and probabilistic language. Participants in this study responded to these omission bias statements by generally denying that either contributed to their decision to immunise their child.
Similar findings to this study have been reported by others [3] [4] [5] 37] and have important implications for how public health addresses the issues of trust and communicates risk information. As others have noted there is more to risk communication than providing more facts about risks [3, 4, 7, 23] . To paraphrase Hobson-West, from these studies and critiques, it is clear that education is not the main policy tool and ignorance is not the main enemy for maintaining immunisation uptake (p 279 [23] ).
Drawing on the aspects of subjective perceptions of risk and decision-making under uncertainty, we believe the following needs to be considered in communicating risk information and health messages:
Facts and figures are not interpreted or acted upon rationally: dread, catastrophic potential and familiarity with the risk influences interpretation and action People act as lay epidemiologists. Thus providing information about risks as though everyone has the same risk makes the advice unbelievable and can be discounted
Parents are more willing to take risks about their own health than with their children's health but this greater caution about their children's health does not automatically mean they will accept medical intervention From the theory of subjective perception of risk people may well be wary of novel vaccines or therapies (manmade versus natural risks) hence there may be some hesitation in the uptake of such vaccines People will discount their risks-'the people affected are not like me'. This may have implications for how people will assess their risk of being badly affected by any outbreak of new strains of influenza such as H1N1.
Clear communication which involves listening to, and not dismissing people's concerns, is valued.
This study has shown that there is a need to understand and take into account how people subjectively perceive risks and how that influences their decision-making, in order to understand their choices and behaviours. Theories of risk perception and decision-making can help to explain differences in perceptions of severity and susceptibility of diseases and vaccines. Importantly, this study has found that health messages about the risks of disease which are communicated as though there is equality of risk in the population may be unproductive as these messages are perceived as unbelievable or irrelevant. The findings from this study have implications beyond the issue of childhood vaccinations as we grapple with communicating risks of new epidemics. Using and developing a more complex theoretical approach to public health issues may increase the likelihood we can understand the barriers to action and develop effective methods of communicating risk and delivering acceptable public health interventions. And indeed may usefully contribute to the current debates, especially in the UK, of how these theories of risk and decision-making can be used to 'nudge' other health behaviours [38, 39] . Development and Applications of VSV Vectors Based on Cell Tropism Viral vectors have been available in various fields such as medical and biological research or gene therapy applications. Targeting vectors pseudotyped with distinct viral envelope proteins that influence cell tropism and transfection efficiency are useful tools not only for examining entry mechanisms or cell tropisms but also for vaccine vector development. Vesicular stomatitis virus (VSV) is an excellent candidate for development as a pseudotype vector. A recombinant VSV lacking its own envelope (G) gene has been used to produce a pseudotype or recombinant VSV possessing the envelope proteins of heterologous viruses. These viruses possess a reporter gene instead of a VSV G gene in their genome, and therefore it is easy to evaluate their infectivity in the study of viral entry, including identification of viral receptors. Furthermore, advantage can be taken of a property of the pseudotype VSV, which is competence for single-round infection, in handling many different viruses that are either difficult to amplify in cultured cells or animals or that require specialized containment facilities. Here we describe procedures for producing pseudotype or recombinant VSVs and a few of the more prominent examples from envelope viruses, such as hepatitis C virus, Japanese encephalitis virus, baculovirus, and hemorrhagic fever viruses. Viruses are obligate parasites of living organisms, and their replication is absolutely dependent on the host cell's machinery. The entry of enveloped viruses requires host cell binding and membrane fusion that is mediated by envelope proteins located on the surface of the virion.
Some viruses utilize a single molecule as a receptor for entry into the host cell, while many viruses require co-receptor(s) localized near the receptor for complete entry. Identification of viral entry receptors that are composed of membrane proteins, lipids, or carbohydrates is important for examining the life cycle of a virus and for further developing entry inhibitors. However, receptors or co-receptors of several viruses have been difficult to identify because of the lack of reliable cell culture systems, an insufficient amount of native viral particles, or difficulty in handling because of the requirement for biosafety level (BSL)-3 or -4 containment. Therefore, several surrogate systems have been developed to study the initial step of infection. One of the most primitive assays is a binding assay. Purified soluble envelope proteins, viral-like particles which are produced in insect cells by baculoviral vectors, and authentic viral particles obtained from patients have been used to study the mechanisms of viral attachment and to identify binding receptor molecules. However, these binding assays cannot be used to analyze further steps of infection such as fusion and penetration. A cell fusion assay was established to examine the membrane fusion activity of viral envelope proteins. This assay is sensitive and can easily determine cell fusion using reporter genes. Pseudotype virus systems based on vesicular stomatitis virus (VSV), influenza virus, retroviruses, and lentiviruses have also been established to examine entry mechanisms and to identify putative entry receptors for targeted viruses ( Table 1) . Pseudotype viruses have also been applied in neutralization tests for antibodies and vaccine development ( Table 1) . As for the application of a pseudotype virus system for VSV, a recombinant virus system with a heterologous viral envelope gene together with a reporter gene encoded into its own genome instead of the G gene has also been developed.
In this paper, we describe the properties of pseudotype or recombinant VSVs and their application to some enveloped viruses we have studied, such as the hepatitis C virus (HCV), Japanese encephalitis virus (JEV), baculovirus, and hemorrhagic fever viruses.
Vesicular stomatitis virus is a non-segmented, negative-stranded RNA virus that belongs to the family Rhabdoviridae, genus Vesiculovirus. VSV infects a broad range of animals, including cattle, horses, and swine. The genome of the virus codes for five major proteins, glycoprotein (G), matrix protein (M), nucleoprotein (N), large protein (L), and phosphoprotein (P). The G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis. The L and P proteins are subunits of the viral RNA-dependent RNA polymerase.
The simple structure and rapid high-titer growth of VSV in mammalian and many other cells has made it a useful tool in the fields of cellular and molecular biology and virology, and this was further strengthened with the establishment of the reverse www.frontiersin.org genetics system for VSV. Recombinant VSV in which native envelope G protein is replaced with a foreign reporter gene such as a fluorescent reporter protein, luciferase, or secreted alkaline phosphatase (SEAP) can normally bud from producing cells even in the absence of G protein, and heterologous viral envelope proteins are incorporated into the virion. Previous studies demonstrated that VSV forms a "pseudotype" when a cell is co-infected with VSV and other enveloped viruses (Huang et al., 1974; Witte and Baltimore, 1977) . A pseudotype virus is defined as a viral particle harboring other types of viral envelopes or host cellular proteins with or without its own envelope. By virtue of these characteristics of VSV, pseudotype virus systems, in which VSV G proteins are completely replaced with other types of viral envelope proteins, have been established (Figure 1) . Up to the present, numerous types of pseudotype viruses have been constructed with heterologous viral envelope proteins and used in studies examining the entry of viruses, for identification of novel viral receptors, for development of neutralization tests, and as vaccine vectors ( Table 1 ). In particular, availability of pseudotype viruses has been useful in the study of several high-risk viruses that require high-level containment facilities, i.e., in the handling of BSL-3 or -4 viruses. Infectivity of these pseudotype viruses can be easily and quantitatively evaluated by measurement of the reporter gene activities. A recombinant virus system, which encodes a heterologous viral envelope gene instead of an envelope gene in its own genome, has also been made available by establishment of reverse genetics (Figure 2 ). This recombinant virus is replication-competent both in vitro and in vivo and can contribute to the study of targeted viruses that inefficiently replicate in experimental systems. Although the pseudotype virus is limited to single-step infection and therefore provides a poor model for actual infection processes, the recombinant virus is a far more authentic and powerful tool for investigating targeted viral infection. Currently, this system is applicable to the VSV or other FIGURE 1 | Schematic representation of the production of pseudotype VSV. Producer cells were transfected with an expression plasmid encoding foreign envelope genes and then infected with a VSV G-complemented pseudotype virus (*G-VSVΔG). The pseudotype virus released from the producer cells infected target cells but was not able to produce infectious progeny viruses. several viruses and not to retroviruses or lentiviruses. Recombinant VSV can be produced in various cells without regard to transfection efficiency; on the other hand, recovery of pseudotype VSV as well as pseudotype retroviruses or lentiviruses is restricted to 293T or some other type of cells that exhibit a high competency of transfection. Recombinant VSV could also lead to the induction of cellular and humoral host immunity (Schnell et al., 1996) .
Seeded or recombinant VSVs in which the G gene is replaced by a foreign reporter gene such as a fluorescent reporter protein (green fluorescent protein, GFP; red fluorescent protein, RFP; and so on), luciferase, or SEAP or each viral envelope gene were generated as described below. Either 293T or BHK cells were grown to 90% confluence on 35-mm tissue culture plates. The cells were infected with a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase (vTF7-3) at a multiplicity of infection (MOI) of 5. After incubation at room temperature for 1 h, the cells were transfected with helper plasmids, pBS-N, pBS-P, pBS-L, and pBS-G, and template plasmids, pVSVΔG-GFP (RFP), pVSVΔG-Luci, pVSVΔG-SEAP, or pVSVΔG-Env using a cationic liposome reagent. After 4 h, the supernatants were Table 1 | Application studies of pseudotype and recombinant VSV. Takada replaced with 10% FBS DMEM, and cells were incubated at 37˚C for 48 h. The supernatants were then filtered through a 0.22-μmpore-size filter to remove vaccinia virus and were applied to 293T or BHK cells that had been transfected with pCAGVSVG 24 h previously. If BHK cells constitutively expressing the bacteriophage T7 RNA polymerase (BHKT7) were utilized, the cells were only transfected with helper plasmids, pIRES-N, pIRES-P, pIRES-L, pIRES-G, and template plasmids using a cationic liposome reagent without the vaccinia virus infection. Recovery of the virus was assessed by examining the cells for the cytopathic effects that are typical of a VSV infection after 24 h. Stock of * G-complemented viruses, i.e.,VSVΔG virus or recombinant viruses transiently bearing VSV G protein on the virion surface, were grown from the single plaque on BHK cells transfected with pCAGVSVG and then stored at −80˚C. The infectious titers of the recovered viruses were determined by a plaque assay. To generate pseudotype virus, 293T, BHK, or some other type of cells that exhibit a high competency of transfection were transfected with a plasmid expressing the envelope protein using a cationic liposome reagent. After 24 h of incubation at 37˚C, cells were infected at an MOI of 0.5 with the * G-VSVΔG-Luci and * G-VSVΔG-SEAP, or 5 with * G-VSVΔG-GFP (RFP). The virus was adsorbed for 2 h at 37˚C and then extensively washed four times or more with serum-free DMEM.
After 24 h of incubation at 37˚C, the culture supernatants were collected, centrifuged to remove cell debris, and stored at −80˚C.
To generate recombinant virus in various mammalian cells, cells were infected with the * G-complemented VSVΔG-Env at an MOI of 5 for 2 h at 37˚C and then extensively washed four times or more with serum-free DMEM. After 24 h of incubation at 37˚C, the culture supernatants were collected and stored at −80˚C. The infectious titers of the viruses were determined by evaluation of each reporter assay or a focus-forming assay. Further details of the protocol can be found in a recent paper (Whitt, 2010) .
Hepatitis C virus has already infected more than 3% of the worldwide population and 80% of those infected develop persistent HCV infection (Cerny and Chisari, 1999; Theodore and Fried, 2000) . Persistent HCV infection often leads to chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. Currently, there are still 1.5 million or more HCV carriers in Japan.
In past years, anti-hepatitis C therapy has modestly improved; however, a currently available combination therapy, consisting of interferon and the nucleoside analog, ribavirin, shows a sustained response in only less than half of the treated patients. The development of innovative treatment alternatives for patients infected with HCV is urgently required, and a better understanding of the life cycle of HCV should allow us to improve HCV therapies. However, due to the lack of an in vitro cell culture system for the isolation of virus directly from patient sera at present, various surrogate systems such as replicon cells (Lohmann et al., 1999) , pseudotype viruses (Lagging et al., 1998; Matsuura et al., 2001; Bartosch et al., 2003; Hsu et al., 2003; Tani et al., 2007) , or trans-complement particles (Ishii et al., 2008; Steinmann et al., 2008) have been developed to study each step of HCV infection. Although in vitro binding assays using soluble purified envelope proteins or HCV-LPs have identified several candidate receptors for HCV, the final determination of a true entry receptor or coreceptor capable of internalizing HCV may be made using an infection assay. Toward this end, pseudotype virus systems based on VSV and retrovirus or lentivirus have been established and applied to identify entry receptors for HCV. Although it is still unknown how HCV envelope proteins retained in the endoplasmic reticulum (ER) are incorporated into both VSV and retroviruses, which naturally bud from the plasma membrane, significant infectivity of these pseudotype viruses has been exhibited in several human hepatoma cell lines. These infections could be inhibited by treatment with antibodies or soluble proteins against putative receptors or HCV envelope proteins, or by a knockdown of receptor molecules by small interfering RNAs (siRNAs), suggesting that innate HCV infection had occurred. We also successfully generated infectious pseudotype and recombinant VSVs incorporating unmodified HCV envelope proteins in hepatic and non-hepatic human cell lines. These viruses exhibited high infectivity in a human hepatoma cell line, Huh7, which is highly susceptible to infection by cell-cultured HCV (HCVcc). The recombinant virus, but not the pseudotype virus, was able to propagate and form foci only in Huh7 cells. The infection of Huh7 cells with pseudotype and recombinant viruses was inhibited by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. These viruses, as well as pseudotype retroviruses (HCVpp) or HCVcc, were sensitive to the inhibitors of vacuolar acidification, such as ammonium chloride, concanamycin A, or bafilomycin A 1 , or formation of clathrin-coated pits, chlorpromazine, suggesting that these viruses enter via pH-dependent and clathrin-mediated endocytosis into target cells (Blanchard et al., 2006; Tani et al., 2007) . The infectivity of the recombinant virus was inhibited by an ER αglucosidase inhibitor, N -(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin www.frontiersin.org (Tani et al., 2007) . Focus formation of the recombinant virus was also impaired by Nn-DNJ treatment. It was obvious that the appearance of infectious or non-infectious viruses was dependent on the cell type as a result of the infectivity of the recombinant viruses generated from various cell lines. Although the precise mechanisms of HCV assembly or budding that cause the differences in infectivity of viruses generated from different cell lines is still unclear, host cellular factors might be involved in the assembly or budding steps in the generation of infectious particles.
Japanese encephalitis virus, a mosquito-borne zoonotic pathogen, is the leading cause of viral encephalitis in humans, with ∼50,000 cases reported annually worldwide. JEV is an enveloped virus belonging to the family Flaviviridae and the genus Flavivirus, which also includes Dengue virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus (Gubler et al., 2007) . The genome consists of a single-stranded positive-sense RNA of approximately 11 kb, encoding a single large polyprotein, which is cleaved by host-and virus-encoded proteases into three structural (C, PrM, and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The envelope protein (E) is a 53-kDa glycoprotein, which is a major component of the virion surface and has been found to be associated with all the biological properties of the virus, such as attachment to cellular receptors, penetration, fusion with the endosomal membrane, host cell range and cell tropism, and neutralization to antibodies. Although a number of cellular components that interacted with E protein, such as heat shock cognate protein 70 (Ren et al., 2007) , heat shock protein 70 (Das et al., 2009) , vimentin (Das et al., 2011) , glycosaminoglycans (Su et al., 2001; Lee and Lobigs, 2002) , and laminin (Boonsanay and Smith, 2007) , have been shown to participate in JEV binding or penetration, the precise mechanisms remain largely unknown. The pseudotype and recombinant VSV systems have offered us useful tools to focus on the study of entry mechanisms of JEV E proteins by using control viruses harboring an appropriate protein on identical particles. Both pseudotype (JEVpv) and recombinant (JEVrv) VSV bearing the JEV E protein exhibited high infectivity for the target cells, and JEVrv, but not JEVpv, was able to propagate and form foci, as did authentic JEV . Both JEVpv and JEVrv were neutralized by anti-JEV E antibodies. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH-and clathrin-dependent endocytic pathways. Treatment of the JEVpv and JEVrv with cholesterol drastically reduced the infectivity, as previously reported on authentic JEV (Lee et al., 2008) . In contrast, depletion of cholesterol from the viruses by treatment with methyl β-cyclodextrin enhanced the infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and JEVrv . These enhancements were inhibited by treatment with an SMase inhibitor or C 6 -ceramide. Involvement of ceramide in the entry of JEV was confirmed by co-precipitation of the JEV E protein with labeled-ceramide . In our study, it was demonstrated that cellular lipid components such as cholesterol and ceramide play crucial roles in the entry of JEV. Modification of sphingolipids on the plasma membrane of the cells might be a novel target for the development of antivirals against JEV infection.
Baculovirus vectors have been shown to exhibit not only a highlevel of gene expression in insect cells but also efficient gene transduction into a wide variety of mammalian cells with lower cytotoxicity (Hofmann et al., 1995; Boyce and Bucher, 1996; Shoji et al., 1997) . In contrast, the complement systems of animals have been defined to represent a potent primary barrier to in vivo application of baculovirus vectors produced in insect cells (Hofmann and Strauss, 1998; Tani et al., 2001 Tani et al., , 2003 . However, pseudotype viruses based on retroviruses or lentiviruses bearing baculovirus envelope protein GP64 have recently been shown to exhibit efficient gene transduction into mouse organs for long periods compared with those bearing the VSV G protein, which is commonly used for pseudotyping (Kumar et al., 2003; Schauber et al., 2004; Kang et al., 2005; Sinn et al., 2005 Sinn et al., , 2008 . It was considered that serum resistance of the pseudotype viruses bearing GP64 protein is caused by differences between insect and mammalian cells, because pseudotype retroviruses or lentiviruses were generated from mammalian cells, and in contrast, baculovirus was generated from insect cells. Therefore, we generated recombinant VSV bearing GP64 protein in both insect and mammalian cells and examined the role of the GP64 on resistance to inactivation by human or guinea-pig sera . Recombinant VSVs generated in human cell lines exhibited the incorporation of human decay accelerating factor (DAF) in virions and were resistant to serum inactivation, whereas those generated in insect cell lines exhibited no incorporation of human DAF and were sensitive to complement inactivation. Recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation, suggesting that acquisition of resistance to human complement by the incorporation of DAF with baculovirus GP64 represents a step in the development of novel viral vectors for improved gene therapy. in the cells treated with various entry inhibitors depended on the species of pseudotype virus, suggesting that several entry mechanisms were involved in the infection of arenaviruses (Tani et al., unpublished data) . In studies on serological diagnosis of arenaviruses, cross reaction occurred among species of arenaviruses in enzyme-linked immunosorbent assay or immunofluorescence assay, whereas neutralization tests using pseudotype viruses exhibited a specific reaction with each species of virus (Nakauchi et al., 2009; Iha et al., unpublished data) . Although Old or New World arenaviruses have been shown to utilize α-dystroglycan or human transferrin receptor 1, respectively, as one of the cellular receptors, infectivities of the pseudotype viruses have not been consistent with the expression levels of the receptor molecules in our preliminary studies. The infection of pseudotype viruses was not completely inhibited by soluble protein or antibodies of receptor molecules, suggesting that another receptor molecule(s) might be involved in the entry of these viruses. Although further characterization of the pseudotype viruses bearing GPC envelope proteins of arenaviruses will be needed, these viruses are thought to mimic the functional properties of wild type arenaviruses and are suitable for the study of entry mechanisms, including investigation of novel cellular receptor(s), neutralization tests, or vaccine development.
Up to the present, various viral vectors aimed at gene transfer or therapy have been developed and applied in biological and medical research fields. Pseudotype or recombinant VSV are useful tools as alternative viruses to study entry mechanisms, identification of novel cellular receptors, screening antiviral libraries, or development of serological diagnosis for various kinds of viruses, especially unmanageable BSL-3 or -4 viruses. These viruses have also been applied in targeting vectors to specific cells. VSV vectors with monoclonal antibodies against specific oncogenic proteins or viral receptor molecule(s) incorporated on virion surface have been targeted specifically to cells expressing oncogenic proteins or infected cells expressing the viral envelope proteins, respectively, without any influences on normal or uninfected cells. This raises the possibility of the elimination of cancer cells or chronic viral infections by using acute VSV infection. Genetically engineered VSVs encoding suicide cassettes or immune response genes have also been generated as more specific, safer, and effective agents for cancer therapies. Further studies and applications of VSV vectors will provide us not only with useful tools for virological studies but also various benefits for biological sciences and medical research.
This work was supported in part by grants-in-aid from the Ministry of Health, Labour and Welfare; the Ministry of Education, Culture, Sports, Science, and Technology; the 21st Century Center of Excellence Program of Japan; the Global Center of Excellence Program; and the Foundation for Biomedical Research and Innovation, Japan. Distinct Regulation of Host Responses by ERK and JNK MAP Kinases in Swine Macrophages Infected with Pandemic (H1N1) 2009 Influenza Virus Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2′5′-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics. Swine influenza is an acute respiratory disease caused by swine influenza viruses (SIV). The symptoms and signs generally include fever, sneezing, nasal rattles, and respiratory distress in pigs. Pigs recover within a few days, but severe signs can develop and mortality can reach up to 10% when highly virulent strains are involved [1] or pigs are infected at young ages [2, 3] . Pigs have long been considered to be the intermediate host of various subtype viruses and ''mixing vessels'' for the evolution and genesis of influenza viruses with pandemic potential because of their susceptibility to swine, avian, and human influenza viruses [4, 5, 6] . This broad susceptibility is due to the presence of both sialic acid (SA)2,3 Gal-and SA2,6-Gal receptors present in the respiratory epithelium.
Three major SIV subtypes are prevalent: H1N1 (classical swine H1N1 and avian-like H1N1), H3N2 (triple reassortant H3N2 and human-like H3N2), and H1N2 [2, 7, 8, 9, 10, 11] . Pigs are also susceptible to and show clinical signs when infected with pandemic (H1N1) 2009 virus (referred to hereafter as H1N1pdm) [12] , which emerged in April 2009 in North America [13] , arising at least in part from contemporaneous SIV. To date H1N1pdm has been found in a few swine farms [12, 14, 15] , which further demonstrates a two-way process of both gene and virus trafficking between humans and pigs. Though H1N1pdm has remained antigenically and genetically stable in humans since its emergence, a novel reassortant SIV containing a H1N1pdm-like NA and seven other genes from triple-reassortant H1N2 and European ''avian-like'' H1N1 viruses was identified in early 2010 [16] , and that same year H1N1pdm was shown to be evolving genetically at a faster pace in pigs than it was in humans [12, 15, 17] . Effective control of circulating influenza viruses in swine populations is key to reducing consequent genesis of novel pandemic strains that threaten the health of both humans and animals.
Studies have been conducted to identify proinflammatory cytokines including TNF-a, IL-6, IL-12, and IFN-a or IFN-c, which are upregulated in lung or bronchoalveolar secretions in SIV-infected pigs [18, 19, 20, 21] and may be correlated with clinical manifestations. In an alveoli macrophage-depleted pig model, macrophages appeared to be indispensible to effective clearance of SIV from lungs. A higher frequency of cytotoxic T, cd T, and Treg cells were also detected in infected pig lungs [18] , which together with the induction of cytokines, contribute to pathogenesis of influenza infection in pigs. Exploring the mechanism of regulation of host responses is crucial for understanding the pathogenesis of SIV and for controlling swine influenza in pigs. Macrophages residing beneath the respiratory epithelium and surrounding alveoli are part of the first line defenses against influenza viruses. During influenza viral replication in bronchial epithelial cells, macrophages are one of the earliest targets to be infected. Together with dendritic cells, macrophages coordinate innate immune responses, which subsequently lead to adaptive immunity by initiating antigen presentation and lymphocyte activation. Macrophages are indispensable in alveolar host defense and controlling influenza virus in pulmonary organs in pigs [22] . While protective in launching host antiviral responses and restricting virus spread, induced proinflammatory cytokines and chemokines are also the cause of pathogenicity for the host and may lead to acute respiratory failure (ARF), a major cause of death in highly pathogenic H5N1 or H1N1pdm-infected humans [23] . Needless to say, the roles of macrophages are critical to pathogenicity as well as host protection in SIV-infected pigs. However, little is known about the mechanisms of how host responses are regulated in pigs or their macrophages.
Considering the critical role macrophages play in SIV infections, and the threat that H1N1pdm could further evolve higher virulence in pigs and subsequently infect humans, we were interested in profiling host responses of swine macrophages to H1N1pdm, and more importantly, in exploring the underlying mechanism of host response regulation including antiviral, proinflammatory responses, and apoptosis in pigs. In this report, we will demonstrate that swine macrophages are susceptible to infection by H1N1pdm. We will show a unique pattern of proinflammatory cytokine responses to the infection, which are distinctly regulated by swine mitogen-activated protein (MAP) kinases. We have also observed cross-talk between MAP kinase and NFkB pathways, and our data indicate that MAP kinase ERK1/2 and JNK1/2 may impact the activation of NFkB through the induction of RIG-1, leading to IFN-b induction in H1N1pdm-infected swine macrophages.
The 3D/4 cells used in our study are a spontaneouslytransformed line of swine macrophages purchased from ATCC (Manassas, VA) and grown in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS). Mouse anti-ERK and anti-JNK antibodies as well as rabbit anti-phospho ERK and antiphospho JNK antibodies (Cell Signaling), anti-cytochrome c, anti-influenza NS1, and alkaline phosphatase (AP)-conjugated anti-rabbit and anti-mouse IgG antibodies (Santa Cruz Biotechnology) were obtained from their respective providers. Anticleaved caspase antibody was obtained from Cell Signaling Technology, and anti-Bak antibody was obtained from EMD Chemicals. The chemicals purchased from EMD Chemicals also included inhibitors for MAP kinases, U0126 (ERK1/2), SB203580 (p38), and InSolution JNK Inhibitor II (JNK1/2), and the inhibitors for NFkB and IKK (6-Amino-4-(4-phenoxyphenylethylamino) quinazoline (Cat. 481406) and Wedelolactone (Cat. 401474), respectively).
A/Nanjing/108/2009 (H1N1), a pandemic (H1N1) 2009 virus, was isolated from a swab sample of an outpatient febrile child at the Nanjing Children's Hospital during the pandemic in 2009, Nanjing, China. The sampling procedure was performed in accordance with the rules set by the Institutional Review Board at the Hospital. The eight genomic segments of this virus have been fully sequenced and the raw data are deposited at Genbank under accession numbers JQ173100 through JQ173107. The virus was grown in 9-day-old embryonating chicken eggs; virus allantoic fluid (VAF) was harvested 48 hrs after inoculation, then titrated with standard haemagglutination tests (HA) and plaque assays in MDCK cells for HA and infectious viral titers, respectively [24] . For viral infection, the 3D/4 cells were trypsinized, resuspended in RPMI 1640 medium containing 10% FBS, and plated on 6-cm tissue culture plates at 5610 6 cells per plate 12 hrs before infection. The cells were infected with H1N1pdm inocula in VAF at a multiplicity of infection (MOI) of 1. After 1 hr of adsorption, the virus inocula were discarded and 3 ml of serum-free RPMI 1640 medium containing TPCK-trypsin (1 mg/ml, Sigma) was added. The cells were incubated at 37uC and 5% CO 2 for various time points before cell lysates or total RNA extraction were prepared.
mRNA transcript levels of IFN-b, IL-1b, IL-6, IL-8, CCR5, IP-10, TNF-a, FasL, TRAIL, Mx, 2959-OAS, retinoic acid-inducible gene I (RIG-1), melanoma differentiation-associated antigen 5 (MDA-5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were analyzed by a two-step real-time RT-PCR assay as described previously [25] . 1 mg of total RNA, prepared from the cells using the RNeasy kit (Qiagen), was reverse transcribed with the QuantiTect reverse transcription kit (Qiagen) following the manufacturer's instructions. The sequences of primers used in the study are listed in Table 1 . The RT reaction was carried out with the RNA after treatment with DNase I at 42uC for 2 min. Real-time PCR was conducted with 1 ml of cDNA in a total volume of 25 ml with the iQ SYBR Green Supermix (Bio-Rad) following the manufacturer's instructions. Relative expression values were normalized using an internal GAPDH control. The fold change of relative gene expression levels was calculated following the formula: 2 (DCt of gene2DCt of GAPDH) [25, 26] . For each reaction, melting curves were analyzed to determine the specificity of each amplicon. To determine the viral RNA level, the total RNA from infected cells was reverse transcribed and cDNA used for Taqman-based real-time PCR (Applied Biosystems) to measure viral M gene transcripts in the infected cells [27] .
Cell lysates were prepared by lysing uninfected and infected 3D/4 cells in 1% NP-40 lysis buffer containing 1 mM PMSF, 1% aprotinin, 20 mg leupeptin ul 21 and 1 mM sodium vanadate (Sigma) as described previously [10] . Cell lystes were clarified by low speed centrifugation (1000 g, 5 min at 4uC) and subjected to SDS-PAGE (10 to 12%). Proteins were transferred to the Immuno-Blot PVDF membrane (Bio-Rad), and western blot analysis was performed following standard protocols [28] using rabbit or mouse anti-MAP kinase or phosphor-MAP kinase antibodies (1:500) in TBST containing 5% fat-free milk powder for 90 mins incubation at RT. After washes, incubation with APconjugated anti-rabbit or anti-mouse IgG antibody for another 90 mins followed. After incubation and thorough washes, BCIP/ NBT reagents (Sigma) were used for the development of colorimetric signals on the membrane. The membrane was also blotted with a monoclonal anti-actin antibody (Santa Cruz Biotechnology) for input control.
For statistical analysis, a two-tailed Student's t-test was used to evaluate realtime RT-PCR data. An x 2 analysis was used to evaluate significant differences of the data in two and more groups. The 0.05 level of probability (p,0.05) was considered statistically significant.
To examine the susceptibility of pig macrophages to H1N1pdm originating from a human host, we infected 3D/4 cells with the A/ Nanjing/108/2009 (H1N1). Typical cytopathic effect (CPE) appeared 16 hrs post infection and the cell monolayer was destroyed 32 hrs post infection (Fig. 1A) . This result demonstrated that H1N1pdm retains the ability to infect and replicate in swine macrophages, and can reach 1.8610 4 PFU/ml as shown in a replicative curve (Fig. 1B) . Apoptosis occurred and proceeded through the course of the infection, as we observed cleaved/ activated caspase-9 as well as the emergence of downstream executioner caspases-6, -7, and -3, which eventually destroyed the infected swine macrophages (Fig. 1C) . Clearly, cytochrome c was released into the cytosol (Fig. 1E) , which activated mitochondriamediated intrinsic apoptosis as early as 3 hrs post infection. Bak, a pro-apoptotic Bcl-2 family member, was upregulated as detected in the infected cells (Fig. 1D) , and may be involved in the release of cytochrome c from mitochondria in swine macrophages.
To elucidate the pathogenesis of H1N1pdm in pigs, we examined the pattern of cytokine responses in pH1N1-infected swine macrophages. Total RNA from infected and uninfected 3D/ 4 cells collected at different time points post infection (p.i.) were prepared and used for realtime RT-PCR analyses with specific primers to swine cytokines. We found that the levels of proinflammatory cytokines IL-6 and IL-8 were upregulated up to 51-and 38-fold at 16 hrs, respectively, and the level of IL-8 continued to rise up to 142-fold at 36 hrs p.i.. However, the level of IL-1b remained unchanged throughout the infection ( Fig. 2A) , indicating that IL-6 and IL-8, as well as TNF-a (Fig. 2B ) as described later, were the main proinflammatory cytokines upregulated.
We observed a robust induction of antiviral IFN-b, which rose up to 620-and 5,100-fold at 16 and 36 hrs p.i., respectively (Fig. 2C ). IFN-inducible antiviral proteins Mx and 295.-OAS were induced accordingly up to 910-and 12,510-fold, respectively, at 36 hrs p.i. (Fig. 2C) .
TNF family members were also induced in response to H1N1pdm infection, which may be attributable to cell death. We found that in pig macrophages the levels of FasL and TNF-a remained undetectable, while TNF-related apoptosis-inducing ligand (TRAIL) seemed to be most abundant before infection, based on Ct values from realtime RT-PCR (data not shown). FasL and TNF-a were induced most robustly, but TRAIL was only mildly induced in response to infection (Fig. 2B) . Among the induced, the level of TNF-a, critical in both cell death and inflammation, was sharply upregulated up to 14-and 162-fold, and FasL up to 43-and 22-fold at 16 and 36 hrs p.i., respectively. FasL and TNF-a may play a major role in H1N1pdm-triggered extrinsic apoptosis.
To understand the mechanism of proinflammatory cytokine and TNF family ligand induction in H1N1pdm-infected swine macrophages, we investigated how MAP kinases were activated and whether their signaling pathways were involved in the regulation of various cytokines and TNF family ligands in pig immune cells.
3D/4 cells were infected with H1N1pdm, and cell lysates were prepared at various time points for SDS-PAGE and western blot analyses with specific anti-ERK1/2 and anti-JNK1/2 antibodies. Activated forms of ERK and JNK (phospho-ERK1/2 and phosphor-JNK1/2) were detected by anti-phospho-ERK1/2 and anti-phospho-JNK antibodies. As shown in Figure 3A , ERK1/2 was basally phosphorylated at a low level before infection, but further phosphorylated between 9 and 18 hrs and thereafter p.i.. Phosphorylation and activation of JNK1/2 appeared at 9 hrs and increased to the peak around 18 hrs p.i. (Fig. 3B) . Although both ERK1/2 and JNK1/2 were activated in response to H1N1pdm infection in swine macrophages, ERK1/2 remained active at basal level even before infection, so did JNK1/2 as shown in some of our experiments (Fig. 4B) . However, our data showed that basal level phosphorylation of both ERK1/2 and JNK1/2 remained unchanged in uninfected 3D/4 cells through the period of our infection. In addition to ERK1/2 and JNK1/2, we have also observed the phosphorylation and activation of p38 MAP kinase in H1N1pdm-infected cells (data not shown). 
To evaluate the role of MAP kinases in the regulation of proinflammatory cytokine responses in H1N1pdm-infected swine macrophages, we pre-treated 3D/4 cells with specific inhibitors for ERK1/2, p38, and JNK1/2 1 hr prior to infection. We then infected the cells with the virus and observed how infectioninduced activation of MAP kinases was affected by inhibition of the respective MAP kinases.
As shown in Figure 4A , 3D/4 cells were pre-treated with inhibitors of ERK1/2 (U0126), p38 (SB230058), and JNK1/2 (JNK InSolution), at concentrations of 10 mM, 5 mM, and 50 mM, respectively. While the phosphorylation of ERK1/2 was unaffected by treatment with the p38 and JNK inhibitors, it was completely abolished at both 18 and 30 hrs p.i. (lines 4-5) by the ERK1/2 inhibitor U0126 (Fig. 4A) . We noted that the basal level phosphorylation of ERK1/2 diminished in the presence of U0126. On the other hand, in light of the p38 and JNK inhibition with their specific inhibitors, the phosphorylation of ERK1/2 appeared to be enhanced (Fig. 4A, lines 6-9 ), indicating that a compensatory mechanism may exist among MAP kinases.
We observed a similar response in which a complete suppression of JNK1/2 phosphorylation was observed (lines 8-9) when the cells were pre-treated with the JNK1/2 inhibitor (Fig. 4B) . However, the phosphorylation of JNK1/2 was not suppressed at all by the inhibitors of ERK1/2 and p38. We noted that there were double bands for JNK1, and a lower band of JNK1 usually appeared at a later stage of infection (30 hrs p.i.). This band was detected mainly by anti-JNK1/2, but not by anti-phospho-JNK1/ 2, indicating that JNK activation was transient and dephosphorylation of JNK occurred at later stages of infection, probably by an uncharacterized MAP kinase phosphatase (MKP) present in pigs.
A basal level phosphorylation of NFkB was also observed in 3D/4 pig macrophages, and was further enhanced upon H1N1pdm infection, indicating that the NFkB pathway was activated as well in infected pig macrophages (Fig. 4C) . When the cells were pre-treated with specific inhibitors of NFkB (10 nM) or IKK (10 mM), the phosphorylation/activation of NFkB was effectively decreased or diminished.
MAP kinases and NFkB pathways were activated in H1N1pdm infected pig macrophages, which could be reversed or inhibited by their specific inhibitors. We used these inhibitors to study the regulation of host responses, which may be controlled by these pathways. 
To determine how cytokine responses are regulated by individual MAP kinases, we pre-treated the cells with ERK1/2 and JNK1/2 inhibitors, respectively, and measured the induction of the cytokines after infection with realtime RT-PCR. We observed that IL-1b was barely detected and not induced during H1N1pdm infection. Interestingly, we noticed that IL-1b was upregulated in the presence of the JNK inhibitor, although no change was observed after the treatment by the ERK inhibitor, indicating that IL-1b could have been induced in swine macrophages infected with H1N1pdm, but was virtually suppressed by JNK1/2 (Fig. 5A) .
We observed that the induction of IL-6, IL-8, and IL-10 was completely suppressed in the presence of the ERK1/2 inhibitor, which indicates that IL-6, IL-8, and IL-10 inductions are all dependent on the ERK signaling pathway (Fig. 5B-D) . It is interesting to note that JNK1/2 may play different roles in the induction of IL-6, IL-8, and IL-10 based on their responses in the presence of the JNK inhibitor. JNK1/2 may have moderate effects in the induction of IL-6 ( Fig. 5B ), but may be not relevant at all to the induction of either IL-8 or IL-10 ( Fig. 5C-D) .
We also noted that CCL5 (RANTES) was strongly regulated by ERK1/2 and JNK1/2 in swine immune cells. As shown in Figure 5E , induction of CCL5 was efficiently blocked in the presence of either ERK1/2 or JNK1/2 inhibitors, indicating that CCL5 is induced by H1N1pdm infection through ERK and JNK signaling pathways.
As for antiviral IFN-b, which was robustly induced with H1N1pdm infection in swine macrophages, ERK1/2 appeared to be essential since the induction of its mRNA transcripts was virtually abolished in the presence of the ERK inhibitor (Fig. 6A ). JNK1/2 may also play a role in IFN-b induction because of its significant decrease at the earlier stage of infection (16 hrs p.i.) when 3D/4 cells were pre-treated with the JNK inhibitor. However, ERK1/2 seemed to be the primary pathway in the IFN-b induction in swine macrophages. The distinct contributions to the induction of IFN-b by ERK1/2 and JNK1/2 were also reflected in the decreased mRNA transcript levels of IFN-inducible antiviral proteins, Mx and 2959-OAS, in the presence of ERK and JNK inhibitors, respectively (Fig. 6B-C) , which is in accordance with the suppression of the IFN-b induction by these same compounds in the infected cells. Both Mx and 2959-OAS were suppressed significantly by the ERK inhibitor, but only the 
In contrast to the abundance of TRAIL transcripts, mRNA levels of FasL and TNF-a were barely detectable by realtime RT-PCR in swine macrophages (data not shown). However, both FasL and TNF-a were induced profoundly in response to pH1N1 infection ( Fig. 2B and 7A-C) , while the change of TRAIL was mild. By using inhibitors, we concluded that the induction of FasL and TNF-a are mainly controlled by the ERK1/2 and JNK1/2 pathways in pig macrophages.
The NFkB pathway could also be critical in host responses, as has been shown in humans and mice infected with influenza A virus. NFkB can be phosphorylated and activated in swine macrophages in response to H1N1pdm infection ( Fig. 8A and 4C ), albeit at a later stage. Interestingly, when the cells were pre-treated with ERK1/2 or JNK1/2 inhibitors, the phosphorylation of NFkB was also suppressed. However, when the cells were pre-treated with the p38 inhibitor, NFkB phosphorylation decreased much less than with ERK1/2 or JNK1/2 inhibitors (Fig. 8A) . This result suggests that a cross-talk may exist between MAP kinase and NFkB pathways, and that among the MAP kinases, ERK1/2 and JNK1/2 are mainly involved. Figure 5 . Regulation of swine proinflammatory cytokine gene transcripts by MAP kinases. 3D/4 cells were pretreated with U0126 and InSolution JNK inhibitor, which are inhibitors of ERK1/2 and JNK1/2, respectively, 1 hr before H1N1pdm infection. Total RNA was prepared at 24 and 36 hrs post infection for reverse transcription. cDNA was used for realtime PCR with specific primers to measure fold changes of cytokine transcripts at different time points. Each assay was repeated at least twice. A-E. Regulation of IL-1b, IL-6, IL-8, IL-10, and CCL5, respectively, by ERK1/2 and JNK1/ 2 inhibitors. Data show mean fold changes plus standard deviation of two or three independent assays. *p,0.05, Student's t-test. doi:10.1371/journal.pone.0030328.g005
We next examined the expression levels of RIG-1 and MDA-5, the RLR family members and cytosolic sensors for RNA viruses. We found that RIG-1 in particular was significantly induced up to 1280-fold, while MDA-5 was also upregulated up to 42-fold in infected pig macrophages (Fig. 8B) . We further examined the induction of RIG-1 and MDA-5 and their relevance to MAP kinases. To do this, we pre-treated the cells with inhibitors of MAP kinases. As shown in Figure 8C , the induction of RIG-1 was completely abolished by the inhibition of ERK1/2 or JNK1/2 inhibitors, and to a much lesser extent, by the p38 inhibitor, suggesting that the induction of RIG-1 was dependent on ERK1/ 2 and JNK1/2, but not as much on p38. This differentially regulated pattern of RIG-1 induction by ERK1/2, p38, and JNK1/2 was similar to the suppression of NFkB phosphorylation/activation by MAP kinases (Fig. 8A) , suggesting that the induction of RIG-1 was associated with ERK1/2 or JNK1/2 activation, but to a much lesser extent with p38. Since NFkB could be downstream activated by RIG-1/IPS-1 [29, 30] , we postulate that ERK1/2 or JNK1/2 may activate NFkB through the activation of RIG-1/IPS-1 during H1N1pdm infection in pig macrophages.
A similar, albeit less dramatic, induction and suppression of MDA-5 expression was also observed (Fig. 8D) , which indicated that MDA-5 might also be an intermediate adaptor bridging the MAP kinases ERK1/2 and JNK1/2 to the NFkB pathway activation. The cells were pretreated with U0126, SB230058, and InSolution JNK inhibitor, 1 hr before H1N1pdm infection. Total RNA was prepared from infected cells for reverse transcription. cDNA was used for realtime PCR with RIG-1 and MDA-5 primers to measure fold changes of RIG-1 and MDA-5 transcripts in treated swine macrophages. Each assay was repeated at least twice. Data show mean fold change plus standard deviation of two or three independent assays. *p,0.05, Student's t-test. doi:10.1371/journal.pone.0030328.g008
In the present study, we have demonstrated a pattern of host responses in swine macrophages to H1N1pdm infection. Strong proinflammtory and antiviral cytokine responses including IL-6, IL-8, TNF-a, as well as IFN-b, were observed. In contrast, IL-1b was not induced, and was barely detectable in pig macrophages. This pattern differs from that in bronchoalveolar secretions of SIV-infected pigs in which IL-1b was induced but IL-8 was not [19, 20, 21, 22, 25] . The different cell types involved (macrophages and epithelial cells) may account for the difference. It has previously been reported that in human immune cells and patients a weak innate immune response, evidenced by a poor induction of proinflammatory and antiviral cytokines including IFN-b and TNF-a, has been observed in human monocyte-derived DCs and macrophages infected with H1N1pdm, compared to seasonal H1N1 infection [31] . Highly pathogenic H5N1 viral infection in human macrophages induced higher expression of IL-6 and CCL5 (RANTES) than pH1N1 [32] , which may explain generally mild clinical disease among H1N1pdm-infected patients. In human macrophages, similar to our findings, IL-1b was not detected.
MAP kinase signaling pathways and their roles in the regulation of cytokines and viral replications have not been characterized in influenza-infected pig immune cells. In this study, we found that ERK1/2 and JNK1/2 could both be activated in swine macrophages. We noted that ERK1/2 was phosphorylated and active at a low level constitutively, which may be important for the rapid physiological responses required upon infection.
To elucidate the mechanism that regulates swine host responses, we used specific inhibitors of MAP kinases to pre-treat macrophages before infection. We determined that the induction of IFN-b, IL-6, IL-8, and IL-10 were regulated by ERK1/2, while JNK1/2 may only play a minor or no role in the regulation of these cytokines. As described earlier, IL-1b was not induced in response to the pH1N1 infection, which could be explained by our data indicating that its induction was in fact efficiently suppressed by JNK1/2 in swine macrophages. This may be the first time that JNK1/2 inhibitory effects on the induction of proinflammatory cytokines have been demonstrated. Previous studies found that IFN induction was dependent on the JNK1/2 signaling pathway in epithelial cells infected with influenza virus infection [33] . However, our data clearly demonstrate that ERK1/2 plays a major role in the regulation of IFN-b in pig macrophages, which may indicate that the regulation of IFN differs in different cell types. We noted that basal level activities of both ERK1/2 and JNK1/2 were constitutively present in non-infected 3D/4 cells, which may be important in the induction of proinflammatory and antiviral cytokines at the early stages of infection. Our data indicate that the induction of IL-6, IL-8, IL-10, CCL-5, as well as IFN-b, were apparent at the earliest stages of viral infection even before ERK1/2 was further activated.
We realized that a transformed monocytic cell line, instead of primary cells, was used in the study, which may compromise the significance of our data. Basal level phosphorylation of both ERK1/ 2 and JNK1/2, which may affect certain cytokine production, would be minimal in primary monocytes. However, specific inhibitors used in the study completely wiped out phosphorylation of both ERK1/2 and JNK1/2 (Fig. 4) . The effect of MAP kinase phosphorylation and activation on the regulation of affected cytokines as observed in our study with the inhibitors is, therefore, valid, even though the cells were not primary cultures.
Macrophages appear to die inevitably of apoptosis when infected with influenza virus [26] . The Fas-mediated extrinsic apoptotic pathway is apparently triggered by TNF family ligands. While both FasL and TNF-a were induced vigorously upon the viral infection, induction of TRAIL was rather mild in H1N1pdminfected swine macrophages. We knew previously that FasL and TNF-a were barely detectable, while the level of TRAIL remained high prior to the infection based on our realtime RT-PCR data (Ct) (Xing et al., unpublished data). We can therefore presume that H1N1pdm-induced apoptosis may be mainly attributed to FasL and TNF-a, while pig macrophages could be resistant to TRAIL, since the cells remained intact despite the presence of a high level of TRAIL before infection. Furthermore, we were also able to determine that both ERK1/2 and JNK1/2 were involved in the induction of FasL, TNF-a, and TRAIL. FasL is also regulated by ERK1 in chicken macrophages infected with an H9N2 avian influenza virus [34] .
Both toll-like receptors (TLR) and RNA helicases, such as RIG-1 and MDA-5, are critical to antiviral innate immunity [35, 36] . As a cytosolic sensor, RIG-1 binds to dsRNA and viral ssRNA that contain a 59-triphosphate not present in host RNA, and then is recruited to mitochondrial protein IPS via the CARD domain, leading to activation of NFkB, IRF-3/-7, and induction of IFN [37, 38, 39] . RIG-1 can be induced by viral infection [40] . In this study, we observed a robust induction of RIG-1 and MDA-5 in H1N1pdm-infected swine macrophages, which appeared to be suppressed completely by inhibitors of ERK1/2 or JNK1/2, but to be a much lesser extent, by the inhibitor of p38. This indicates that the induction of RIG-1 or MDA-5 depends on the activation of ERK1/2 and JNK1/2 in pig macrophages. We postulate a mechanism, therefore, that the cross-talk between MAP kinase and NFkB pathways is through the regulation of RIG-1 and maybe MDA-5, and that ERK1/2 controls the activation of NFkB, leading to the induction of IFN in swine macrophages. International Society for Disease Surveillance Conference 2011: Building the Future of Public Health Surveillance: Building the Future of Public Health Surveillance  Public health surveillance (also called field epidemiology) as defined by Centres for Disease Control and Prevention (CDC) is the ongoing systematic, collection, analysis and interpretation of outcome-specific data essential to the planning, implementation and evaluation of public health practises closely integrated with the timely dissemination of these data to those who need to know (1) . The IDSR is a strategy of the WHO Afro region adopted by the member states in 1998 as a regional strategy for strengthening weak national surveillance systems in the African region (2, 3) . The DSNOs under the supervision of the Medical Officers of Health (MOHs) are responsible for surveillance activities within their Local Government catchment area. Therefore, their role is very crucial to the success of the IDSR strategy.
The study was conducted in Lagos State, South Western Nigeria, in June 2011. A quassi experimental, before and after study was done. Participants were DSNOs and assistant DSNOs of the 20 Local Governments in Lagos State. Training materials were received from the Lagos State Ministry of Health,World Health Organisation (WHO) and the Central Public Health Laboratory, Lagos. Pre-and posttests were conducted using questions developed for the purpose. Participants scores were categorised as fail (B50%) and pass (50% and above). The impact of the training was assessed by statistical evaluation of the pre-and posttests results.
Participants were 25 DSNOs and assistant DSNOs including 11 (44%) males and 14 (56%) females. Their mean age was 39.7 years (SD, 7.8 years) with ages ranging from 25 to 57 years.
They had been employed for an average of 3.6 years (SD, 2.4 years). Most (88%) of the participants were married. Only about a third has had relevant working experience prior to this job. The mean pretest score was 34.0% (SD, 2.1), which increased to 56.3% (SD, 2.3) at posttest. The mean paired difference in score between post-and pretest of 22.3% (SD, 10.4) was statistically significant p 00.000. There was also a statistically significant difference between the proportion of participants who passed the posttest but failed the pretest [16 (69.6%)] and the proportion who passed the pretest but failed the posttest [0 (0%)]; p 00.000. The mean score of male participants was higher at the pretest (35.6% vs. 32.0%) while the females performed better at the posttest (57.3% vs. 55.6%), although this difference was not statistically significant. The ages of participants and the number of years of employment were also not statistically associated with their performance in the pre-and posttests. 
In the last decade, the scope of public health (PH) surveillance has grown, and biosurveillance capacity has expanded in Duval County. In 2004, the Duval County Health Department (DCHD) implemented a standalone syndromic surveillance (SS) system, which required the manual classification and entry of emergency department (ED) chief complaints by hospital staff. At that time, this system, in conjunction with other external systems (e.g., CDC ILInet, FluStar and NRDM) were used to conduct surveillance for health events. Recommendations from a 2007 ISDS panel were used to strengthen surveillance within Duval County. Later that year, the Florida DOH moved to a statewide SS system and implemented ESSENCE, which has been expanded to include (1) ED record data from 176 hospitals (8 within Duval County); (2) reportable disease case records from Merlin; (3) Florida Poison Information Network consultations; and, (4) Florida Office of Vital Statistics death records (1) . ESSENCE has subsequently become a platform for rapid data analysis, mapping and visualization across several data sources (1) . As a result, ESSENCE has improved business processes within DCHD well beyond the initial scope of event detection. These improvements have included (1) expansion of the ability to create visualizations (e.g., epi-curves, charts and maps); (2) reduction in the time required to produce reports (e.g., newsletters and media responses); (3) reduction in staff training needs; and (4) augmentation of epidemiology processes (e.g., active case finding, emergency response and quality improvement [QI] ) and closing the PH surveillance loop.
To quantify the impact of ESSENCE on PH activities, an evaluation of epidemiology business processes pre-and post-ESSENCE was conducted. Staff time, computer programs/ systems utilized and computational steps were compared for tasks within three quantifiable areas: visualization creation, report production and activities during PH responses. Visualizations included production of a map, chart, table and epidemic curve for the same data. Report production tested the ability to create nontextual documents with multiple graphics. Conducting ad hoc ED surveillance was compared between ESSENCE and e-mail/telephone methods. QI reviews were compared between ESSENCE and Merlin Analysis Tools. The number of software or systems requiring training, which were replaced by ESSENCE, was reviewed. Computational steps included page clicks, exporting/importing data between systems and data management. Testing was conducted by a single reviewer proficient in current systems performing the task a single time to completion. Time and computational steps were measured from the initiation of a task to the completion of a usable product and then averaged across the three quantifiable areas.
ESSENCE created visualizations on average 89% faster than previous methods, requiring 43% fewer computational steps. For the production of reports ESSENCE was 86% faster, requiring only 4.55 minutes compared to 27.83. For tasks relating to PH responses, initiating ad hoc ED surveillance was 96% faster through ESSENCE without comparing resource savings during follow-up monitoring. However, conducting QI reviews was faster using previous systems with modules designed for such tasks. ESSENCE also reduced the need for staff training, as one system was used for most tasks instead of five (Excel, ArcGIS, Access, SAS/SPSS and Merlin).
Using ESSENCE had advantages compared to previous methods that extended beyond basic processes. The analytic and computational abilities of the system are more advanced, more accessible and more user friendly than those of previous systems. The ability to save and share queries and visualization dashboards as well as the added efficiency of navigation reduce redundancy and improve user functionality. Ultimately, the user experience is enhanced, and resources are optimized. In the future, ESSENCE will continue to expand available data sources as well as increase analytical capacity based on user feedback and identified system needs.
Biosurveillance; syndromic; epidemiology; business processes Reference 1. Kite-Powell A, Hamilton J, Wojcik R, Loschen W, Hopkins R. Florida's ESSENCE system*from syndromic surveillance to routine epidemiologic analysis across syndromic and nonsyndromic data sources. Emerg Health Threats J 2011;4(Suppl 1).
*Taj Azarian E-mail: tajazari@gmail.com
The Public Health*Seattle & King County (PHSKC) syndromic surveillance system has been collecting emergency department (ED) data since 1999. These data include hospital name, age, sex, zip code, chief complaint, diagnoses (when available), disposition and a patient and visit key. Data are collected for 19 of 20 King County EDs, for visits that occurred the previous day. Over time, various problems with data quality have been encountered, including data drop-offs, missing data elements, incorrect values of fields, duplication of data, data delays and unexpected changes in files received from hospitals. In spite of close monitoring of the data as part of our routine syndromic surveillance activities, there have occasionally been delays in identifying these problems. Since the validity of syndromic surveillance is dependent on data quality, we sought to develop a visualization to help monitor data quality over time, in order to improve the timeliness of addressing data quality problems.
Methods SAS version 9.2 (Carey, NC) was used to create two groups of visualizations: (1) a separate heatmap for each hospital, showing how each individual ED performs on each of 13 data quality measures and (2) a separate heatmap for each data quality measure, showing how data quality varies by ED. The heatmaps summarize data by month and year, though other visualizations (e.g., daily or weekly) are also possible. For each row on the heatmap, a color change indicates that data quality has shifted over time. Blocks with stable color over time suggests that there has not been a change in data quality. White space on the heatmap highlights periods of time where data were not recorded by the system and can provide a visual cue for newly added EDs, hospital closures or data drop-offs. The heatmaps are generated monthly for each of 13 data quality measures. SAS code for generating the heatmaps will be provided at the session.
Two heatmaps are provided as examples of our visualization approach (see Fig. 1 ). Since applying this visualization to our syndromic data, PHSKC has identified several data quality errors that are likely to have gone undetected or been slow to detect otherwise, including out of range ages and sudden data drop-offs. Consequently, we have adopted the methodology to other nonsyndromic data sources, including notifiable condition reporting to the health department.
Syndromic surveillance systems commonly encounter problems with data quality. These problems can result in imprecise counts and can adversely affect detection of trends, outbreaks and situational awareness. The heatmap visualizations have been a useful tool for PHSKC to identify problems with data quality in a timely manner. The code can be easily adapted to display other data quality measures, stratifications and data sources beyond the ED setting. 
The Washington Comprehensive Hospital Abstract Reporting System (CHARS) has collected discharge data from billing systems for every inpatient admitted to every hospital in the state since 1987 (1) . The purpose of the system is to provide data for making informed decisions on health care. The system collects age, sex, zip code and billed charges of the patient, as well as hospital names and discharge diagnoses and procedure codes. The data have potential value for monitoring the severity of outbreaks such as influenza but not for prospective surveillance: Reporting to CHARS is manual, not real-time, and there is roughly a 9-month lag in release of information by the state. In 2005, Public Health*Seattle & King County (PHSKC) requested that hospitals report pneumonia and influenza admissions (based on both admission and discharge codes) directly to the PHSKC biosurveillance system; data elements included hospital name, date/time of admission, age, sex, home zip code, chief complaint, disposition and diagnoses. In 2008, reporting was revised to collect separate admission and discharge diagnoses, whether the patient was intubated or was in the ICU and a patient/visit key. Hospitals transmit data daily for visits that occurred up to 1 month earlier.
Previously, we identified a strong concordance between the volume of influenza diagnoses recorded across the PHSKC and CHARS systems over time (2) . However, discrepancies were observed, particularly when stratified by hospital. We undertook an evaluation to identify the causes of these discrepancies.
We included patients with a diagnosis of influenza (ICD9 codes 487.0, 487.1, 487.8, 488.0 or a textual variant of 'influenza', excluding 'H. influenza'). We also focused on 2008 data exclusively, since at the time of the analysis, more recent CHARS data were unavailable. Of the 20 hospitals in King County, 10 provided admissions data in 2008, but data from only 9 hospitals were available in CHARS for comparison. For each of the 180 influenza hospitalizations identified by the PHSKC system, we manually attempted to find a matching record in CHARS according to hospital name, discharge month/ year, age, sex and zip code. We flagged all influenza admissions in the PHSKC system that did not have a matching record in CHARS. Next, we asked hospitals with unmatched records to reverse-identify patients and retrieve their medical charts for PHSKC review.
In 2008, the PHSKC system (which searches through admission and discharge diagnoses) identified 180 patients hospitalized with influenza, compared with 161 patients identified by CHARS (which is based on discharge diagnoses exclusively). Thus far, PHSKC has reviewed 46 charts from 8 hospitals to validate system accuracy; review of data from the remaining hospital is pending. We identified 3 hospitals that were transmitting incorrect data to PHSKC and requested correction and resubmission of historical data from these hospitals. Preliminary analysis revealed that 35 of the 180 influenza hospitalizations captured by the PHSKC system (19%) were missed by CHARS; however, 15 of these patients (43%) were admitted with presumptive diagnoses of influenza but were determined not to have influenza by the time of discharge. Also, 28 of 161 influenza hospitalizations (17%) captured by CHARS were missed by the PHSKC system; however, we had no means of reverse-identifying CHARS records and, therefore, could not evaluate the validity of these data by chart review.
This evaluation identified several problems with data quality, which were substantial though not universal across hospitals. We plan to continue the analysis using 2009 data, to ensure that data quality issues have been resolved. A key limitation of this analysis is that CHARS is an imperfect gold standard for identifying King County influenza admissions; we could not independently identify admissions based on laboratory data to determine which system performed better.
With the proliferation of social networks, the web has become a warehouse of patient discussions and reports, estimated at 10 billion records and growing at a rate of 40 percent per year. First Life Research, Ltd. (FLR), has searched and mapped thousands of these discussions and indexed hundreds of millions of reports (currently 960M) and is engaged in building web-based solutions that enable the public and public health practitioners to access massive health-related information and knowledge generated from the crowd.
FLR's competency is the ability to identify, analyze, index and aggregate user-generated content by collecting billions of testimonials from social networks. It utilizes cutting edge technologies for massive data aggregation and applies advanced natural language processing (NLP) techniques for continuous analyses, in order to convert this unstructured data into refined information. The insights gained can be used to support and enable better informed decision making processes, both for patients and healthcare providers.
A platform of data investigations utilizing the 'Wisdom of the Crowd' focusing on biosurveillances aspects as follows:
1.
PharmacovigilanceÁbrand monitoring and safety alerts: Crowd trial provides a dashboard of parameters on medications, their side effect profile, interactions and drug's comparative advantage. 2. Social Health at a glance:
Temporal overview of prevalence and statistics of the most engaging health issue discussed across the social web, represented by aggregation of the reports (citations) generated by the e-patients (Table 1 ). 3. Health trends detected by harnessing the social web:
This public feedback exists in real-time, large scale and enables ongoing observational studies by tapping into the health reports involving a massive sample size (Fig. 1) .
The value of crowd trial to public health is new and complementary to what the existing monitoring processes provide. Moreover, user-generated content contains valuable feedback on medication usage and health information.
Thus the emerging wisdom of the crowd analytics potentially represents a new phase and eventually new tools using data evaluation based on large scale population inputs, and it will benefit greatly all public health environment. Introduction People usually celebrate holidays by inviting family and friends to have food at home or by gathering and eating at restaurants or in other public venues. This increased exposure to food with a common source can create conditions for outbreaks of gastrointestinal illnesses. Holidays can also be targeted by bioterrorists who seek to maximize physical damage, psychological impact and publicity around dates of patriotic or religious significance. They might aim at contaminating food and water supplies, especially with CDC-defined category B agents that can cause diseases such as salmonellosis, shigellosis, cholera, crytosporidiosisand infections with Escherichia coli O157:H7 and the Epsilon toxin of Clostridium perfringens. Hence, there is a need to quantify whether gastrointestinal illnesses increase around holidays. This can also help determine a baseline of the incidence to which future holiday periods should be compared to. This research does not focus on specific reportable diseases. That will be the purpose of forthcoming research. Instead, ED visits with gastrointestinal symptoms are used to leverage the capability of syndromic surveillance for early detection.
A query with the stringˆvomitˆ, or,ˆdiarrheaˆ, or,ˆgastroenter-itisˆ(VDG) was performed in the Electronic Surveillance System for Early Notification of Community-based Epidemics (ES-SENCE) during a 7-day period surrounding the 10 Federal Holidays of each year of the quadrennial 2007Á2010. The count of ED patients during the 7-day period was compared to the count of a 28-day background by calculating a ratio between the 2 periods for both, the 4-year average and year-specific. The analysis was broken down by age groups (0Á4, 5Á17, 18Á64, 65 and plus and all-age). Database analysis was conducted using SAS 9.2.
President's Day and Labor Day were associated with the highest 4-year average increases (12% each). Decreases in the 4-year average only occurred around two of the holidays, Independence Day ((7%) and Memorial Day ( (5%). Age groups 0Á4 and 5Á 17 had their largest 4-year average spikes around Labor Day (' 24% among 0Á4 and '30% among 5Á17), right after the beginning of classes as well as around President's Day (12% and 13%). The 18Á64 age group had its largest 4-year average increases during Christmas (19%) and Thanksgivings Days (15%). As for the 65' age group, Christmas (15%) and President's Day (14%) showed the largest increases. The span was much wider when analyzing year-specific holidays, from ' 40% after Martin Luther King Day in 2010 to (17% after Independence Day on the same year. Factors other than holidays could have also influenced the increases in ED visitors, such as the beginning of classes in August of each year and the H1N1 influenza epidemic in 2009. This research hinges on the comparison of the holiday period to a 28-day background. Future tracking of VDG should also be based on comparing the current holiday period to its mean of previous years to control for seasonal or day-specific effects. The availability of only 4 years of data prevented us from removing the seasonal effect in this research.
ESSENCE can help to track the incidence of gastrointestinal symptoms in the community during holiday periods. The incidence of reportable gastrointestinal diseases during holiday periods should also be ascertained in a future research.
Syndromic; surveillance; holidays; gastrointestinal; illness 
INDICATOR is a multistream open source platform for biosurveillance and outbreak detection, currently focused on Champaign County in Illinois. It has been in production since 2008 and is currently receiving data from emergency department (ED), patient advisory nurse (PAN), outpatient convenient care clinic (CC), school absenteeism, animal control and weather sources. (Table 1) .
We performed simple pairwise correlation between signals using the longest period of mutual data availability, e.g., between convenient care data and patient advisory nurse data, we compared the period between April 2007 and August 2011. We also offset signals by up to 14 days in each direction to investigate whether there were lag relationships between them.
ILI surveillance source: Analysis of the relationships between the signals for PAN, ED and CC are heavily influenced by a strong day of the week effect in the PAN data. Considering all of the data, including periods of high and low ILI activity, the correlation between ED and PAN shows a stronger relationship when the ED signal lags the PAN by 1 day (r 00.653 vs. r 0 0.617). There is a less clear relationship between ED and CC with the strongest correlation occurring when CC lags ED by 2 days (r 00.669 vs. r 00.659). The relationship between CC and PAN is unclear. These relationships are also all valid when just considering the period of the 2009 H1N1 pandemic, but interestingly not the last strong seasonal influenza in 2008. For the 2008 season, for which we do not have ED data, the signal for CC clearly lags the signal for PAN with a peak correlation at a lag of 6 days (r00.561 vs. r00.413). ILI and climate: There are significant negative correlations between all three signals and daily temperature, e.g., CC and Tmin (r0(0.265, df 02004, p B 0.0001), but not precipitation, e.g., CC (r 0(0.011) when considering all data. There are no clear relationships between temperature or precipitation and CC or PAN during the 2008 seasonal outbreak, but there is a clear negative correlation between temperature and CC, PAN and ED during the 2009 H1N1 pandemic, e.g., ED and Tmin (r0(0.366, df 0 119, pB 0.001). There was no clear correlation with precipitation.
Zoonotic reports: There are some very strong correlations between the signals, such as the correlation between daily minimum temperature and patients seeking treatment for insect bites (r 00.602, df0821, pB0.0001), with a less strong correlation between precipitation and insect bites peaking with a bite lag of 24 days (r00.163, pB0.0001). Not as expected is the correlation between animal bites and Tmin (r 00.178, pB0.001).
There are clear indications of temporal relationships between different surveillance signals, with PAN consistently giving a 1-day lead over ED for ILI. The strong relationship between temperature and ILI cases during the 2009 H1N1 pandemic is most likely coincidental since the onset in Champaign County coincided with the change of season in September/October. The lack of a relationship between ILI and weather during the 2008 seasonal outbreak remains to be understood.
ILI; zoonoses; correlation; multistream; weather Introduction Syndromic surveillance of ED and PCC data has been widely used for the detection, tracking and monitoring of health events (e.g., bioterrorism, disease outbreaks and environmental exposures) over the past decade (1) . In recent years, these data have been found to be useful for public health programs not normally associated with syndromic surveillance (e.g., injury prevention, drug abuse and environmental health (1)). In 2010, the first calls referencing exposure to products marketed as 'legal highs' and 'bath salts' were received by PCCs in the United States (2) . Synthetic drugs, such as those commonly known as bath salts, often are labeled as 'not for human consumption' and, thereby, circumvent normal legal control procedures that control the sale and distribution of recreational drugs (3) . The purpose of this study was to evaluate the emerging trends for the use of bath salts in Ohio.
Syndromic surveillance data from ED chief complaints were collected and analyzed from Ohio's syndromic surveillance application, EpiCenter for 2010Á2011. Because the term bath salts refers to a grouping of drugs, and the effects of ingestion or inhalation of these drugs can vary widely, a specific classifier was created to define ED visits related to bath salts. This classifier included many variations of the common street names for bath salts. Human exposure calls to the PCCs in Ohio related to use of bath salts were also collected and analyzed from the National Poison Data System (NPDS) during the same time period. These data were combined and a correlation analysis was performed, using SAS v 9.2 to evaluate the relationship between the two data types and to illustrate the trends in designer drug use for bath salts. Due to small daily counts of both ED visits and PCC exposure calls, the data were totaled by month for all time-series and correlation analyses.
In 2010, there were very small numbers (nB5) of both ED visits and PCC calls for bath salts. In 2011, ED visits and PCC calls related to bath salts increased dramatically. ED visits for bath salts totaled 166 and PCC calls totaled 480 through July 2011. A time-series chart of these data, analyzed by month, as shown in figure 1 . Pearson correlation analysis showed a strong relationship between ED visits and PCC calls for bath salts (r 00.83, p 00.02).
These results suggest an emerging, upward trend in the use of bath salts in Ohio beginning in early 2011. Syndromic surveillance provides a useful and inexpensive way to track trends in designer drug use. In order to identify these types of trends, knowledge of the subject matter and common name of the substance being tracked is essential. Although this type of analysis significantly underestimates the number of people using drugs, it can be used to identify the arrival and pace of adoption of a new drug. This information can be used by prevention programs and lawmakers to reduce the likelihood of widespread adoption. In mid-July 2011, Ohio passed legislation banning the sale of products containing the chemicals found in bath salts. The law took effect on October 15, 2011. The Ohio Department of Health will continue to monitor ED and PCC data for the next designer drug.
Syndromic surveillance; designer drugs; 'bath salts' 
With an estimated 500 million people infected each year, dengue ranks as one of the most significant mosquito-borne viral human diseases and one of the most rapidly emerging vectorborne diseases (1) . A variety of obstacles including bureaucracy and lack of resources have interfered with timely detection and reporting of dengue cases in many endemic countries (2) . Surveillance efforts have turned to modern data sources, such as Internet search queries, which have been shown to be effective for monitoring influenza-like illnesses (3, 4) . However, few have evaluated the utility of web search query data for other diseases, especially those of high morbidity and mortality or where a vaccine may not exist.
Bolivia, Brazil, India, Indonesia and Singapore were chosen for analysis based on data availability and adequate search volume. For each country, a univariate linear model was built by fitting a time series of the fraction of Google search query volume for specific dengue-related queries from that country against a 'gold standard' time series of dengue case counts for a time-frame within 2003Á2010. The specific combination of queries used was chosen to maximize model fit. Spurious spikes in the data were also removed prior to model fitting. The final models, fit using a training subset of the data, were cross-validated against both the overall dataset and a holdout subset of the data. All search queries were fully anonymized. This methodology is similar to the approach used to develop Google flu trends (3) .
Dengue generated over a million Google search queries every month. Some queries showed that the user was looking for more information about the disease, while others were looking for symptoms or treatments. Model-fitted 'expected' epidemic curves matched official case counts 'observed' epidemic curves quite well for all 5 countries in most countries (Fig. 1) , with validation correlations ranging from 0.82 to 0.99. Dengue queries were not as influenced by mass panic-induced searching.
Web search query data were found to be capable of tracking dengue activity in Bolivia, Brazil, India, Indonesia and Singapore. Whereas traditional dengue data from official sources are often not available until after some substantial delay, web search query data are available in near real-time and could serve as a useful low-cost complement to traditional surveillance. Even if peaks are no earlier, there is value in 'nowcasting'*predicting the present where there are delays in gaining access to current official data (5) . More broadly, these results also contribute to a growing pool of evidence demonstrating the capability of relatively novel sources such as webbased data to assist with public health goals. The product of this work is freely available at www.google.org/denguetrends. 
Dengue fever is endemic in over 100 countries and there are an estimated 50Á100 million cases annually (1) . There is no vaccine for dengue fever yet, and the mortality rate of the severe form of the disease, dengue hemorrhagic fever, ranges from 10-20% but may be greater than 40% if dengue shock occurs (2) . A predictive method for dengue fever would forecast when and where an outbreak will occur before its emergence. This is a challenging task, and truly predictive models for emerging infectious diseases are still in their infancy.
Predictive disease modeling attempts to exploit the complicated relationship between disease outbreaks and measurable environmental, biological, ecological and sociopolitical variables. Previous studies (3Á5) identified factors associated with dengue outbreaks such as: past cases, ambient temperature, precipitation, Normalized Difference Vegetation Index, Enhanced Vegetation Index, Southern Oscillation Index, sea surface temperature anomalies and socioeconomic factors. We obtained and preprocessed these variables to get one value per district per week. The epidemiological dengue fever case data used span 2001Á2009 and cover several districts in Loreto, Peru. We computed incidence rate per 1000 residents, enabling us to deal with significantly different population sizes in the different districts. The method predicts incidence rates 4 weeks in advance. We developed logistic regression (LR) models using part of the data set available. The second part, not previously used for model development, was used for testing. The ROC curve and positive predictive value (PPV) for the test set are shown in Fig. 1 . High specificity is easier to obtain than high sensitivity. The preliminary results are encouraging: when sensitivity is 0.375, we obtain a specificity of 0.987 and a PPV of 0.6.
Effective methodologies to predict outbreaks of dengue fever may facilitate public health interventions to mitigate the impact of the disease. For best results, the researchers must have access to data streams with timely, detailed and accurate values of predictor variables. High PPV is of principal importance as health officials may be unlikely to spend resources on mitigation efforts based on model predictions without evidence of accuracy on past outbreaks.
Dengue; disease prediction; logistic regression 
Block 3 of the U.S. Military ESSENCE system affords routine access to multiple sources of data. These include administrative clinical encounter records in the Comprehensive Ambulatory Patient Encounter Record (CAPER), records of filled prescription orders in the Pharmacy Data Transaction Service (PDTS), developed at the DoD Pharmacoeconomic Center, Laboratory test orders and results in HL7 format and others. CAPER records include a free-text Reason for Visit field, analogous to chief complaint text in civilian records, and entered by screening personnel rather than the treating healthcare provider. Other CAPER data fields are related to case severity. DoD ESSENCE treats the multiple, recently available data sources separately, requiring users to integrate algorithm results from the various evidence types themselves. This project used a Bayesian network (BN) approach to create an ESSENCE module for analytic integration, combining medical expertise with analysis of 4 years of data using documented outbreaks.
The strategy was to emulate a domain expert's use of ESSENCE by means of a BN whose inputs were outputs of alerting algorithms (1) applied to data streams chosen for specificity to acute illness in outpatient encounters, laboratory tests and prescribed medications in the chosen syndromes. Efforts were restricted to 5 syndrome groups seen as amenable to fusion of the ESSENCE sources: influenza-like illness, gastrointestinal, fever, rash and neurological.
Major subtasks included modifying the ESSENCE chief complaint processor (2) for CAPER syndrome classification, selection and judicious use of fields in chemistry and microbiology test data, selection of generic code number (GCN) groups of prescribed medications, development and implementation of an algorithm testbed for the various streams to be fused and elicitation of domain expertise to design BNs for practical decision support.
Initial findings from fusion using severity concepts in CAPER data yielded sharp alerting reduction from pure algorithmic methods, with a timeliness loss of 1 day in 2 known outbreaks, no days in 4 others. The alert reduction was dramatic in datasets from small facilities, typically reducing the alert rate from 20 per year to below 5. In larger facilities, the reduction was less dramatic but often over 50%. Time series chosen from the laboratory test and GCN groupings were tested using additional known outbreaks. Results of fusing all algorithmic output streams will be presented.
The VA has employed ESSENCE for health monitoring since 2006 (1) . Epidemiologists at the Office of Public Health (OPH) monitor the VA population at the national level. The system is also intended for facility-level monitoring to cover 152 medical centers, nearly 800 community-based outpatient clinics (CBOC), and other facilities serving all 50 states, the District of Columbia and U.S. territories. For the entire set of facilities and current syndrome groupings, investigation of the full set of algorithmic alerts is impractical for the group of monitors using ESSENCE. Signals of interest may be masked by the nationwide alert burden. Customized querying features have been added to ESSENCE, but standardization and IP training are required to assure appropriate use.
We derived and refined default alerting filters relevant to the monitor's purview, beginning with three jurisdictional levels: (1) facility; (2) facility group or station, including all clinics and divisions associated with a parent VA medical center; and (3) superuser, referring to routine system-wide monitors. The filters were based on the number of patients, statistical significance of alerts and a composite severity measure. This measure was derived from case-based criteria developed for the Department of Defense ESSENCE (2) and adapted by OPH epidemiologists for the VA population. These criteria are based on separate monitoring of evaluation\management codes for complex cases, OPH-selected procedure codes, 'bounce-backs' to an emergency setting, patient age distributions anomalous for a VA facility and extreme spikes; case disposition was not yet available. We tested candidate filters by tabulating alert rates and sensitivity to known outbreaks using 13 months of all-VA historical outpatient data. For the superuser, the filter required at least one severity factor among records composing an alert, at least 3 cases (5 for the common syndromes) and only p-values B 0.01. These restrictions were reduced in localized user filters.
The full set of unfiltered ESSENCE alerts at levels of high and moderate significance, applied for 8 syndrome groupings to all medical centers, CBOCs and other facilities sending outpatient and emergency department data, was on average 410 per day (539 per week day). Table 1 illustrates the sharp drop in alerting using filters developed to present only sets of records selected for investigation at each level. Additional results will show the sensitivity of the resultant hierarchical system to documented outbreaks at the various levels.
Hierarchical filtering can furnish practical, canonical alert criteria applied to algorithm alerts. As circumstances change, users may reconfigure for increased sensitivity or altered coverage with ESSENCE customization tools. The planned addition of new data elements (laboratory/micro, pharmacy and radiology) will further refine alerting. These refinements can increase detection/response capability by focusing attention on signals of interest with a reasonable alert burden. Novel approach to statewide biosurveillance using emergency medical services (EMS) information
The purpose of the National Collaborative for Bio-preparedness (NCB-P) is to enhance biosurveillance and situational awareness to better inform decision-making using a statewide approach. EMS represents a unique potential data source because it intersects with patients at the point of insult or injury, thus providing information on the timing and location of care. North Carolina uses a standardized EMS data collection system, the Prehospital Medical Information System (PreMIS), to collect information on EMS encounters across the state using the National EMS Information System (NEMSIS) template (1) . Since NEMSIS is planned to be incorporated by EMS agencies in every state, an EMS-based approach to biosurveillance is extensible nationally.
Carolina from 2009 to 2010 were utilized in the project. Based upon a previous analysis of emergency department (ED) presentations, an interdisciplinary team (EMS, emergency department, epidemiology and public health) then developed an approach to assign EMS records to 1 of the 20 symptom-based illness categories (gastrointestinal illness, respiratory, etc). EMS encounter records were characterized into these illness categories using a novel text analytic program (SAS Institute, Cary, NC). Baseline patterns of EMS encounters were developed for each illness category across the state, local regions (3-digit zip code) over time. Event alerts were identified across the state and by regions in illness categories using either change detection with cumulative sum (CUSUM) analysis (3 standard deviations) or a novel text-proportion (TAP) analysis approach (SAS Institute).
year period were analyzed. The initial analysis focused upon gastrointestinal illness given the potential relationship of gastrointestinal distress to infectious outbreaks, food contamination and intentional poisonings (ricin). After accounting for seasonality, a significant gastrointestinal event was detected in February 2010 (see red circle on graph in Fig. 1 ). This event coincided with the announcement of a norovirus outbreak (2) . The use of CUSUM approach (yellow circle on graph) detected the alert event as early as January 24, 2010. Using the novel TAP approach on a regional basis detected the alert as early as December 6, 2009 .
Advantages of EMS data include being an early point of contact with patients and providing information on the location of insult or injury. Surveillance based on EMS information system data can detect outbreaks of illness of interest to public health.
A novel text proportion technique shows promise as a useful early event detection method.
Biosurveillance; analytics; detection; emergency; preparedness References (2) . College-aged drinkers tend to binge drink at a higher frequency than the general population, putting them at greater risk for unintentional injuries and unsafe sex practices (3) . Identifying collegespecific patterns for alcohol-associated morbidity have important policy implications to reduce excessive drinking and associated harms on and around college campuses.
An ''alcohol'' syndrome was developed based on alcohol-related chief complaint keywords sensitive and specific to acute or chronic alcohol ED visits, and validated by an ICD-9 field. The data were aggregated by day from 2008 to 2010, by age and age group. These data were analyzed using general linear modeling (PROC GENMOD), a time trend analysis, and a temporal SaTScan using age groups. Potential time periods of interest were major holidays, days of week, and college start and end periods.
Alcohol-related ED visits for college-age patients have increased since 2003. When analyzed with respect to holidays and days of week, college-age specific trends begin to emerge: college-aged ED visits differ from the general population by day of week (Fig. 1) . Additionally, college age groups can be distinguished from other ages on certain days of the year. Most notably, early summer and early fall show these age-specific increases, and this age group drives some holiday spikes, such as New Year's Day/ Eve.
Further analyses need to be performed to refine a college drinker age group from syndromic data, as well as potentially identify this population spatially, such as in college-dense areas.
In addition, a larger collaboration with the DiSTRIBuTE network as an extrapolation of this work (4). This would ideally help to improve the definition of an alcohol syndrome and expand the identification of these problem drinkers in other jurisdictions. 
The spatial scan statistic proposed by Kulldorff (2) has been widely used in spatial disease surveillance and other spatial cluster detection applications. In one of its versions, such scan statistic was developed for inhomogeneous Poisson process. However, the underlying Poisson process may not be suitable to properly model the data. Particularly, for diseases with very low prevalence, the number of cases may be very low and zero excess may cause bias in the inferences. Lambert (3) introduced the zero-inflated Poisson (ZIP) regression model to account for excess zeros in counts of manufacturing defects. The use of such model has been applied to innumerous situations. Count data, like contingency tables, often contain cells having zero counts. If a given cell has a positive probability associated to it, a zero count is called a sampling zero. However, a zero for a cell in which it is theoretically impossible to have observations is called structural zero.
We assume that the case-counts in the regions follow independent ZIP random variables with the same probability p of a structural zero. The ZIP model allows for additional flexibility when compared to the Poisson. When structural zeros occur, the ZIP model accounts, in average, for a reduction in the casecounts. The ZIP model allows for superdispersion or extra-Poisson variation, while the Poisson model often understimates the observed dispersion. Regarding the likelihood ratio test formulation for the ZIP model we describe our Scan-ZIP statistic considering (a) we know when a zero count is a structural one and (b) we do not know, for sure, whether or not a zero count is a structural one. For the latter case, the Scan-ZIP statistic is obtained through an EM procedure.
Our methodology was evaluated by means of a numerical case study. We constructed artificial clusters using a map consisting of 203 hexagonal cells arranged in a regular grid, 15 of which are structural zeros. An example can be seen in Fig. 1 . Gray regions indicate the ''true'' cluster while the )'s indicate structural zeros. We compare the Poisson, ZIP and ZIP'EM scans in terms of power, sensitivity and positive predictive value (PPV). The Scan-ZIP and Scan-ZIP'EM methods presented systematically and significantly better results when compared to the Scan-Poisson, as shown in Table 1 for the given cluster of Fig. 1 . More examples and a real data application will also be presented.
The Scan-ZIP statistic has shown to be more suitable for the detection and inference of spatial clusters for data with zero excess as it outperforms the Scan-Poisson statistic in terms of power of detection, sensitivity and PPV.
Spatial clusters; spatial scan statistic; zero-inflated Poisson (1). However, this system may not represent the true epidemic situation of infectious disease in community, particularly those who do not seek medical care (2) . Moreover, the epidemiological settings, sources of the infection and social network all together may still facilitate the transmissions. These rooted problems cannot be rapidly solved.
We present our web-based technical framework designed with social network theory. Using cloud computing technology, user only needs internet to access our system and webpage, and database was built by Joomla Framework, HTML, CSS, PHP and MySQL. National Health Insurance Database (NHID), which has over 98% Taiwan citizen coverage rate in 2009, and National Notifiable Reporting System (NNRS) were used to evaluate our system; data of syndrome groups by ICD-9CM codes from these two systems during 2009, with pandemic influenza, were first analyzed.
Daily symptoms can report into database, with time-spatial information. Statistic methods (e.g., CUSUM) were built in server (Fig. 1) . The real-time data, with cloud-computing, can be calculated online. Also the system can gain a better feedback and sharing timely information among decision makers, health workers and citizens. User-interface (UI) of system, including main home page with Map-API, reporting entrance and latest news, was user-friendly.
Using the 2009 pandemic influenza, results of evaluation are shown (Fig. 2) . Except the pattern of 'ILI' (Fig. 2C ), other curves, using our easily understood definitions, show similar increase trend in week 34 with the gold standard (NNRS) ( Fig. 2A) , the first outbreak signal NNRS had detected. With CUSUM, case numbers did increase in week 34Á35 and fell out thresholds in week 35Á38, except 'Fever'Cough'.
In conclusion, easily defined syndrome groups for public surveillance is feasible and can complement with traditional passive surveillance systems. More potential case can be detected earlier, particularly those who do not seek medical care. Certainly, this newly developed and user-friendly surveillance system can be applied to study transmission of infectious disease within socialnetwork and also to allow public's participating surveillance leading to public health efforts in disease prevention will be no longer limited to healthcare system and thus become more effective. 
Most outbreaks are small and localized in nature, although it is larger outbreaks that result in the most public attention. So, a solution to manage an outbreak has to be able to accommodate a response to small outbreaks in a single jurisdiction scalable up to outbreaks that involve thousands of cases across multiple jurisdictions and to handle different types of situations with different questions and response required. To make this happen, information and resources need to be shared more consistently and efficiently to help facilitate the communication that occurs at all levels and to support day-to-day operations in order to ensure consistent use.
During the period from early 2008 through mid-2009, representatives from many of the operational public health groups across the state worked together to identify the requirements necessary to support the improvement of outbreak management in New York. These requirements were prioritized by the project team and the highest level requirements were approved to begin the design and development of the Outbreak Management System (OMS). The system included the following features: creating a central outbreak incident to record incident level information, generating a unique identifier that can be shared across integrated applications to facilitate aggregated query and reporting capabilities with a common data set shared across the jurisdictions and program areas involved in an outbreak investigation and providing forms that are customizable for an outbreak but use standard sets of questions and a common vocabulary where possible. The OMS user guide and training were provided to those who will be using it to manage outbreak incidents.
A project charter including project mission, proposed solution, guiding principles, project scope, critical risk factors, communication plan and project team was approved in 2008. Regular project team meetings were conducted, and functional requirements and software specification documents were completed in 2009. A diagram of data flow is shown in Fig. 1. An incident screen with create, update and search utilities was designed and completed in May 2011. The incident screen collects incident, disease-and event/facility-specific data, case definition and coordinator/investigator information. Web services were applied to create basic reports (Fig. 2) , including case counts by case status, county, age group and a de-identified line list extracting from the Communicable Disease Electronic Surveillance System (CDESS). User acceptance testing was completed in July and webinar trainings to all users were completed in September 2011. The content and structure of food and waterborne outbreak investigation forms have been developed. The prototype of user interface for entering forms currently is under development.
The Outbreak Management Solution consists of a combination of systems development, training and technical communications enhancements. The OMS will increase ability to provide timely and consistent information to the public and healthcare practitioners, to improve ability to coordinate response activities. The system will be able to answer general inquiries and generate reports and to calculate performance measures. 
Although development of computerized medical record systems in the United States is a high priority, there are relatively few instances of such systems supporting disease surveillance systems. The Indian Health Service (IHS) has had an electronic record database for over 30 years; however, implementation of point of care electronic health records (EHR) and use of these data for public health surveillance have begun only over the past 4 years.
The IHS health database is distributed across the United States with most data maintained at 465 local care facilities. Among these 465 facilities, there are over 235 EHR deployments, using similar but separately maintained configurations of the IHS EHR. Data are entered into the EHR system as part of daily clinical care or transcribed from paper for results of clinical referrals or outside tests. We developed a surveillance system that identifies reportable cases, notifies providers and provides data to a dedicated national surveillance database. Cases are found using a locally deployed extension to the local data system that searches for a combination of ICD-9 codes, clinical data and laboratory data, based on Council of State and Territorial Epidemiologists (CSTE) case definitions, on a nightly basis. Reports for situational awareness and response are made locally, regionally and nationally using an a priori established priority ranking of the public health importance of a case or outbreak.
Pandemic influenza (pH1N1) was the first health condition targeted for surveillance in 2009. Through an iterative process, a combination of ICD-9 codes and measured fever at the time of visit yielded the highest sensitivity and specificity using the case definition for influenza-like illness (ILI) found in the Centers for Disease Control and Prevention's (CDC) ILInet surveillance system. Formal evaluation of ILI surveillance using review of local medical records (electronic and paper) in one region of the country found that the system had a sensitivity of 96.4% and specificity of 97.8%. IHS is expanding EHR surveillance to capture cases of chlamydia, syphilis, HIV, invasive pneumococcal disease, measles, Haemophilus influenza type b (Hib), meningococcal disease, hepatitis B, hepatitis C and tuberculosis.
PyConTextKit is a web-based platform that extracts entities from clinical text and provides relevant metadata*for example, whether the entity is negated or hypothetical*using simple lexical clues occurring in the window of text surrounding the entity. The system provides a flexible framework for clinical text mining, which in turn expedites the development of new resources and simplifies the resulting analysis process. PyCon-TextKit is an extension of an existing Python implementation of the ConText algorithm (1), which has been used successfully to identify patients with an acute pulmonary embolism and to identify patients with findings consistent with seven syndromes (2) . Public health practitioners are beginning to have access to clinical symptoms, findings and diagnoses from the EMR. Making use of these data is difficult, because much of it is in the form of free text. Natural language processing techniques can be leveraged to make sense of this text, but such techniques often require technical expertise. PyConTextKit provides a web-based interface that makes it easier for the user to perform concept identification for surveillance.
PyConTextKit's annotation lexicon can be derived from existing lexicons or ontologies and then used to extract concepts relevant to a particular domain or syndrome. In this case, the symptoms from the Syndromic Surveillance Ontology (3) and the Extended Syndromic Surveillance Ontology (ESSO) (4) have been imported into PyConTextKit. Users can create their own text classifier by porting concepts from ESSO and by adding new concepts. Concepts are ultimately mapped to standardized vocabularies like the Unified Medical Language System. PyConTextKit currently supports the following six features: view of documents to be annotated,management of a lexicon, document annotation using the lexicon,view of annotation results, document classification based on the annotations and summary statistics generation.
PyConTextKit allows the user to manage a lexicon for extraction targets, such as symptoms. It also allows the user to manage a lexicon for modifiers, such as negation cues (e.g., 'no' and 'absence of') and temporality cues (e.g., 'history of'). The modifiers are applied to the targets by pyConTextKit during the annotation phase, and the user can determine the criteria for extraction of a target from a report based on the modifiers. For example, the user may only want to extract symptoms that occurred recently and not historically. The document classification feature identifies documents containing the targets and modifiers specified by the user. For instance, the user may want to identify documents with recent and nonnegated instances of respiratory symptoms and diagnoses.
Finally, PyConTextKit also enables the user to view summary statistics such as the number of documents in the dataset meeting the specified criteria. If the application were run on a dataset involving patients from a particular population, for example, the user could view the number of patients meeting the criteria in that population.
PyConTextKit is aimed at a clinical audience attempting to apply NLP to clinical reports. The strength of PyConText-Kit lies in its flexibility in incorporating new knowledge, its hopefully intuitive interface and the sophistication of its document-level analysis. 
Influenza is a serious disease that seasonality causes substantial but varying morbidity and mortality. In Taiwan, estimates of the influenza mortality burden were based on post-hocanalyses of national mortality statistics and not available until at least six months after the corresponding epidemic. Timely monitoring and early detection of influenza-associated excess mortality can guide antiviral or vaccine interventions and help healthcare capacity planning. Beginning April 2009, Taiwan Centers for Disease Control (TCDC) has been collaborating with the Department of Health (DOH) Office of Statistics to develop an automated system for real-time P&I mortality surveillance (1).
Taiwan's Mortality Information Regulations require medical institutions to report any mortality to DOH through the National Death Certificate System (NDCS) within 7 days after a death certification is issued. Automated data from the NDCS were daily submitted to TCDC by secure electronic transmission and processed and analyzed using SAS Enterprise Guide 4.3 (SAS Institute Inc, Cary, NC). For each report, the underlying cause of death was determined by applying the World Health Organization classification principles (2) and searched for freetext traditional Chinese 'pneumonia', 'influenza', or 'flu' to identify P&I deaths. Reporting timeliness and completeness of this surveillance system was assessed by comparing reporting data with post-hoc mortality statistics for the year of 2008. We used an R-package 'surveillance' to detect aberrations in the P&I mortality weekly data (3) .
In 2008, the number of deaths for which P&I was listed as the underlying cause in the national mortality statistics was 8,665; of these, 6,795 (78%) were reported through the NDCS. The weekly surveillance-based P&I mortality estimates had a consistently strong correlation with those obtained from mortality statistics data (correlation coefficient 0.85, p B0.0001). Eighty seven percent of the reports were received within 7 days after death (median 2 days). During the 2010Á11 influenza season, an increase in mortality was observed in January 2011, with the highest weekly number of P&I deaths to be 421 (week 5 of 2011) (Fig. 1) . From 2010 through 2011, consecutive alarms were generated for week 26Á27, 31Á32 and 36Á39 of 2010, and week 2Á15 of 2011 ( Fig. 1) .
Taiwan has established an early warning system for P&I mortality to assist with characterization of influenza severity. 
Previous studies in developed countries showed school absenteeism data can serve as a proxy for monitoring infectious disease activities and facilitate early community outbreak detection. However, absenteeism patterns may differ in developing settings and affect the utility of the surveillance system. Despite the nonspecific nature of absenteeism data, other practical challenges will need to overcome for system set up and maintenance in remote area.
Weekly electronic school attendance reports were received from the participating schools by short message service (SMS) or direct communication by phone to the central office of The Cambodian Children's Advocacy Foundation, Cambodia, a local nongovernmental organization for initial data processing. Absenteeism data were anonymized. Overall absenteeism data were weekly aggregated and sent to Hong Kong, via email for further analysis.
Implementation of the electronic surveillance system was feasible after initial staff training, purchasing necessary equipments for communication and standardizing data formats. The protocol for data sending, receiving and analyzing were stable. Data transfer procedures were simple and acceptable according to the school and CCAF staff's feedback. Data quality was monitored by occasional onsite school visits by the investigators.
A total of 430 students (47.4% female) from 17 preschools have absenteeism data recorded since November 27, 2010. From March 1, 2011 onward, a total of 1437 students (47.6% female) from 47 preschools (including 30 public preschools) have absenteeism data recorded. The mean weekly overall absenteeism rate from November 27, 2010, to July 12, 2011, was 23.2% (maximum 32.4%, minimum 13.6%, standard deviation 4.5%), whereas the mean weekly absenteeism rate due to sickness was 2.3% (maximum 4.0%, minimum 0.7%, standard deviation 1.0%). We are currently seeking reference disease surveillance data to evaluate the accuracy of this system and negotiating with the village chiefs to set up disease surveillance data dissemination points for risk communication with the villagers.
While school absenteeism data are preexisting, easily accessible and require minimum time and resource for data collection and database maintenance after initial development, it can potentially serve as a convenient syndromic data source for disease surveillance targeting school age children in the population. The system will be particularly useful in resource limited settings where health care and laboratory capacity are insufficient for disease surveillance purposes.
School absenteeism; rural area; disease surveillance; electronic data; short message service 
A devastating cholera outbreak began in Haiti in 2010. Sequencing of Vibrio cholerae isolates showed that the epidemic was likely the result of the introduction of cholera from a distant geographic source. The same strain of cholera was detected in other countries within 100 days. The unique instigation and geographic spread of this epidemic highlight the need for improvements in timely global outbreak surveillance. Novel information sources have been shown to provide early information about public health events and disease epidemiology. Particularly, volume of Internet metrics such as web searches or microblogs have been shown to be a good corollary for public health events (1) . In this study, we evaluate geographic trends in online social media following an infectious disease outbreak to determine whether this may enable prediction of secondary outbreak locations.
We examined Twitter postings from the first 100 days of the Haitian cholera outbreak. Twitter is a microblogging service in which users can give information in 140 character length posts, 'Tweets'. We selected Tweets containing the word 'cholera' including those with the Twitter hashtag identifier ('#cholera'). Our search captured English, French and Spanish mentions of the word cholera. We define an outbreak as cholera incidence beyond an isolated case. Six countries in which cholera did or was suspected to have spread, and without endemic cholera, were examined: Canada, Dominican Republic, Mexico, Spain, USA and Venezuela. We first collected 'Twitter Updates' for each country, Tweets that came from users in a particular country, normalized by the number of Twitter users in the country. Second, we filtered Tweets in which the keyword cholera as well as a country's name was mentioned, 'Twitter Mentions'. Logistic models were constructed to analyze the relationship between volume of updates and mentions and the occurrence of a secondary cholera outbreak in the chosen countries. We evaluated our models through the Hosmer-Lemeshow (HL) test and also by cross-validation with data from Puerto Rico, in which there was concern of a potential outbreak.
Global Tweets regarding a disease outbreak include concern from family or friends, local happenings and reiteration of news reports. Fig. 1 illustrates example Tweets and distribution of Twitter Updates in this study. The HL test yielded p-values of  1 and 0.185 (updates and mentions models). Large p-values indicate that the null hypothesis cannot be rejected and the model fits the expected distribution of values well, in this case, better for the updates model. Both models output a low probability (mentions: 0.04, updates: 6e-11) of an outbreak in Puerto Rico within 100 days, and there was no actual outbreak.
Global discussion of disease outbreaks may indicate where an outbreak will spread. This is the first study to examine how these discussions, via social media, can be used to understand and predict geographic spread of disease. Both the models demonstrated good fit to expected distributions through the HL test and correctly predicted no outbreak in Puerto Rico. We are working on incorporating data from more countries into the model, as well as other covariates such as environmental factors that would contribute to a country's tendency toward an outbreak. Although the global microblogging community is currently limited in demographics, penetrat\ion of consumer technology is increasing worldwide and could be a useful complementary tool for timely and cost-effective disease outbreak surveillance. 
For the 2010Á2011 influenza season, Spokane Regional Health District required hospitals to report any admissions with laboratory-confirmed influenza using traditional NC surveillance methods. Simultaneously, HIE records from four Spokane facilities were monitored for flu diagnoses (i.e., records with ICD9 487Á488 listed in the working or final diagnoses) and positive flu laboratory test results (including rapid antigen, DFA, culture or PCR). Records from the NC system and the HIE were matched using facility name, age, gender, county and admission date. The medical records of cases detected by the HIE but not reported through the NC system were evaluated to determine true case status. Sensitivity and PPV were calculated for each surveillance system. Timeliness, completeness and representativeness of records received through the HIE were evaluated against NC reporting.
One hundred forty-six true laboratory-confirmed influenza cases were identified (Fig. 1 ). By including records with a flu diagnosis or a positive flu lab result (excluding records with a negative flu lab), the sensitivity of the HIE was 90% and the PPV was 94%. In comparison, the sensitivity of NC reporting was 91%. HIE cases were detected a median of 5 days after admission versus 2 days through the NC system. Data for influenza hospitalizations from the HIE did not differ signifi-cantly from data collected through the NC system with regard to sex, age, pregnancy status and mortality. The time series of influenza-related hospital admissions from the HIE and NC system were highly correlated (r 00.99).
HIE data are a useful resource for influenza hospitalization surveillance. They are sensitive, specific and representative of the true population of laboratory-confirmed influenza patients admitted to the hospital. It also provided data that were adequately timely and complete. Microbiology laboratory data improved the sensitivity and PPV of the Public Health Surveillance HIE feed to levels near that of NC reporting when used in combination with discharge diagnoses. Thus, for influenza, this enhanced syndromic data feed is comparable to traditional clinical surveillance.
Health information exchange; influenza; surveillance (3), a resource developed by a working group of 18 researchers representing 10 syndromic surveillance systems in North America. ESSO encodes almost three times as many clinical concepts as the Syndromic Surveillance Ontology and incorporates eight syndrome categories, in contrast to the Syndromic Surveillance Ontology's four (influenza-like illness, constitutional, respiratory and gastrointestinal). The new clinical concepts and syndrome groupings in ESSO were developed by a board-certified infectious disease physician (author JD) in conjunction with an informaticist (author MC). In order to evaluate and audit these new syndrome definitions, we initiated a survey of syndromic surveillance practitioners.
We designed an online survey that presented respondents with all the clinical concepts associated with each syndrome definition, and the question 'To what extent do you agree that the following concepts are potentially indicative of SYNDROME?' For each clinical concept, the respondent then indicated their agreement from 'Strongly disagree' to 'Strongly agree'. We publicized our survey through the ISDS Newsletter.
As September 5, 2011, 24 people have participated in the survey, with 14 completing all the questions. Although providing personal information was optional, half the respondents supplied biographical details. Most of the respondents were based in North America, typically from state or county public health departments, although three were based outside North America (one from Taiwan and two from the UK NHS). Apart from one assistant professor, all the respondents had either 'public health', 'epidemiologist' or 'syndromic surveillance' in their job titles. Strong disagreement was expressed by a minority of respondents on 7 of the 279 ESSO concepts (see Table 1 ). Only 2 concepts*'hoarseness' (respiratory syndrome) and 'concussion' (neurological syndrome)*elicited disagreement (or strong disagreement) with ESSO syndrome definitions among a majority of respondents. We are currently developing a strategy to 'flag' concepts with high levels of disagreement in order to better inform ESSO users.
Ontology; terminology; informatics 
The ability to rapidly detect any substantial change in disease incidence is of critical importance to facilitate timely public health response and, consequently, to reduce undue morbidity and mortality. Unlike testing methods (1, 2), modeling for spatiotemporal disease surveillance is relatively recent, and this is a very active area of statistical research (3) . Models describing the behavior of diseases in space and time allow covariate effects to be estimated and provide better insight into etiology, spread, prediction and control. Most spatiotemporal models have been developed for retrospective analyses of complete data sets (4). However, data in public health registries accumulate over time and sequential analyses of all the data collected so far is a key concept to early detection of disease outbreaks. When the analysis of spatially aggregated data on multiple diseases is of interest, the use of multivariate models accounting for correlations across both diseases and locations may provide a better description of the data and enhance the comprehension of disease dynamics.
When small area disease data in the form of counts are available, Bayesian hierarchical Poisson models are commonly used to describe the behavior of disease (5) . In this study, we use the convolution model (6) to describe the behavior of disease under endemic conditions. Each time new observations become available, we show how the conditional predictive ordinate (CPO, 7), which is a Bayesian diagnostic tool that detects unusual observations, can be adapted in a surveillance context to detect small areas of unusual disease aggregation (8) .
For the joint analysis of two or more diseases, we introduce a generalization of the shared component model (9) where the underlying risk surface for each disease is separated into shared and disease-specific components. We then propose a multivariate extension of the surveillance CPO that incorporates information from the different diseases and, consequently, facilitates the outbreak detection work. The multivariate surveillance technique has the ability to detect outbreaks of disease in either one or in a combination of diseases.
We analyze weekly emergency room discharges for acute upper respiratory infections, influenza, acute bronchitis, asthma and pneumonia in 2009. The data are available by county for the 46 counties of South Carolina. The use of a shared component model accounting for correlation across diseases provides a better overall fit. In addition, the use of the multivariate SCPO increases the statistical power for detection of outbreaks.
Public health surveillance; spatial data; Bayesian hierarchical models; joint disease mapping; conditional predictive ordinate
An expanded ambulatory health record, the Comprehensive Ambulatory Patient Encounter Record (CAPER) will provide multiple types of data for use in DoD ESSENCE. A new type of data not previously available is the reason for visit (ROV), a freetext field analogous to the CC. Intake personnel ask patients why they have come to the clinic and record their responses. Traditionally, the text should reflect the patient's actual statement. In reality, the staff often 'translates' the statement and adds jargon. Text parsing maps keywords or phrases to specific syndromes. Challenges exist given the vagaries of the English language and local idiomatic usage. Still, CC analysis by text parsing has been successful in civilian settings (1). However, it was necessary to modify the parsing to reflect the characteristics of CAPER data and of the covered population. For example, consider the shock/coma syndrome. Loss of consciousness is relatively common in military settings due to prolonged standing, exertion in hot weather with dehydration, etc., whereas the main concern is shock/coma due to infectious causes. To reduce false positive mappings, the parser now excludes terms such as syncope, fainting, electric shock, road march, parade formation, immunization, blood draw, diabetes, hypoglycemic, etc.
First, a set of syndromes in an existing JHU-APL CC parser used in civilian versions of ESSENCE were selected for evaluation. The CC parser was then used to categorize the ROV from 3 months of records from all DoD facilities (about 12 million records total). From the records matched to each syndrome, 2000 were selected; the 1000 most common strings and an additional 1000 strings at random. Two analysts evaluated the sample strings independently and identified key decisions they each used to decide whether the match between the text string and the syndrome was accurate. They then attempted to reconcile those cases where they disagreed. Unresolved differences were reviewed by a DoD consultant who offered revised rules based on clinical experience and known practice patterns among DoD providers. The modified rules were incorporated into the CC parser and tested on miniature data samples for accuracy, and then the CC parser was rerun on the full record set. The cycle of independent analysis and review was repeated with additional modifications to correct any remaining errors followed by a final full run. Before and after contingency tables were used to compare CCbased versus diagnosis-based classifications. The final product was a modified CC parser input file tailored for use with DoD ambulatory healthcare records.
The following before/after contingency Table 1 illustrates the results for two syndromes: one common, influenza-like illness (ILI), and one rarer syndrome, neurological (Neuro). Additional results will show effects of the CC noise removal on alerting using datasets with documented outbreaks.
This iterative method produced a CC parser with substantially improved performance, eliminating many obvious incorrect classifications and resulting in a smaller number of more meaningful alerts for public health investigation. The process could be used to tune parsers to meet the unique medical terminology used in different communities. Moreover, daily CC and diagnostic counts should not be crudely pooled, but do provide complementary views of the population health status. 
To review observations and conclusions from a recent global biosurveillance conference, provide an assessment of the scientific and technical capabilities and gaps to achieve an effective and sustainable integrated global biosurveillance (InGBSV) system, and recommend research and development priorities enabling InGBSV.
Life science and biotechnology advances have provided transforming capabilities that could be leveraged for InGBSV. Global infectious disease surveillance holds great promise as a tool to mitigate the endemic and pandemic infectious disease impacts and remains an area of broad international interest. All nations have significant needs for addressing infectious diseases that impact human health and agriculture, and concerns for bioenergy research and environmental protection. In January 2011, Los Alamos National Laboratory, Department of State and the Defense Threat Reduction Agency co-hosted the 'Global Biosurveillance Enabling Science and Technology' conference. Guided by the National Strategy for Countering Biological Threats and joined by major government stakeholders, the primary objective was to bring together the international technical community to discuss the scientific basis and technical approaches to an effective and sustainable InGBSV system and develop a research agenda enabling a long-term, sustainable capability. The overall objective of the conference was to develop a technology road map for InGBSV, with three underlying components: (1) identify opportunities for integrating existing biosurveillance systems, the near-term technological advancements that can support such integration and the priority of future research and development areas; (2) identify the required technical infrastructure to support InGBSV, such as methodologies and standards for technology evaluation, validation and transition; and (3) identify opportunities, and the challenges that must be overcome, for partnerships and collaborations.
To achieve the objectives, the conference was structured to review the current state of biosurveillance, identify core components for a comprehensive capability and scientific and technical bases to support this capability and explore the critical improvements needed to enhance the existing regional and global disease outbreak prediction capabilities. Open discussion time was planned in order to engage broad participation during the conference to recommend approaches to establishing an effective international network, propose implementation strategies and the measures of effectiveness and identify the challenges that must be overcome in the next 3Á5 years in order to establish an initial biosurveillance capability that will have significant positive impact on biothreat nonproliferation, economy and public health.
We will report the principle observations from the conference. All participants were keenly aware of the complexity of developing an InGBSV, passionate about and committed to pursuing biosurveillance and supportive of the initial focus on application of existing information technology tools. It was largely agreed that:
Á Scientific understanding of pathogen and pathogen-humanenvironment interaction is the foundation for integrated InGBSV; Á Emerging technology being developed by the R&D community for basic and applied life science advancement provides tremendous support for InGBSV; Á InGBSV can be 'jump started' with initial focus on information science and technology applications and integration; Á Challenges were recognized as multidimensional, but opportunities for developing a GBSV exist and are invigorated by international health security policies; Á Formulating unconventional partnerships and establishing an advanced concept demonstration is an effective near-term path forward.
An effective integration of existing technologies can provide great potentials to pursuing the opportunities afforded by establishing and operating an integrated global biosurveillance system (Fig. 1) . A common appreciation is also evident for the challenges associated with planning, obtaining necessary resources and establishing the desired functional biosurveillance capability. 
It is admitted that real time surveillance system permits to reduce delay of outbreak detection and preventive measures implementation (1) . It is usually based on prediagnostic numeric data collection and transmission (2) . ASTER (Alerte et surveillance en temps réel) is a real time surveillance system for French Armed Forces deployed in French Guiana and Djibouti ( Fig. 1) , constituted by 2 kinds of networks: several declaration networks and one analysis network (3). On June 2011, an outbreak occurred among a French Army Regiment in Djibouti, which has permitted to evaluate ASTER in real conditions.
Declaration network: at the end of medical consultation, each medical staff member declares clinic signs of his patient using a numeric standardized form on computers (specific declaration software). They transmit this anonymous form to a data base located in a Military Surveillance Disease Centre in France. Analysis network: observed data are automatically compared with historical data every 10 minutes, using current past graph method (specific analysis software), to produce alarm signals. These signals have to be analysed by epidemiologists to confirm or not the real alert about outbreak occurrence.
Data base already contained administrative data about all the soldiers present in Djibouti; and in case of illness symptoms in cause with date of onset and rapid antigenic tests results. Fiftyone cases of tonsillitis were declared during 4 days on 646 soldiers (attack rate 08%), with 18 positive streptotests on 25 performed (72%) (Fig. 2) . Epidemic curve had only one peak as if it was one source of contamination. A retrospective cohort study found one meal at risk (RR 012.8, IC95% 0[7.9Á20.6]), prepared by a local food provider.
ASTER produced an early warning signal, 7 days before the classic surveillance system, and only 1 day after the beginning of symptoms. It is based on clinic signs surveillance, which is more sensitive than disease surveillance. It permitted to perform immediately the description of the outbreak, using the realtime database without disturbing physicians and, therefore, to change the local food provider. The quality of data was good although physicians were busy because of the number of patients.
Real time surveillance; outbreak; early warning; sensitivity 
Data obtained through public health surveillance systems are used to detect and locate clusters of cases of diseases in spacetime, which may indicate the occurrence of an outbreak or an epidemic (1Á5). We present a methodology based on ALRs to compare the null hypothesis (no outbreaks) against the alternative hypothesis (presence of an emerging disease cluster).
The ALR preserves the martingale structure of the regular likelihood ratio, which allows the determination of an upper limit for the false alarm rate, depending only on the quantity of evaluated cluster candidates.
A fast computational algorithm incorporates this important property, determining the cutting point to control the false alarm rate, thus making a viable tool for the detection of emerging clusters in geographical maps, where the baseline of the number of cases has nonconstant average. The greater flexibility of the candidate clusters' shape produces a better estimation of the most likely cluster.
However, the large cardinality of the set of candidate clusters is an obstacle for the application of the ALR procedures, generating function values so small that the alarm may not ring, even if an emerging cluster exists. To solve this problem, we propose the use of an adaptive approach also for the clusters' configuration space.
Performance is evaluated by the following criteria: average detection delay and probability of correct detection in space,
given that an outbreak really exists. We present simulations with artificial data and applications for thyroid cancer in New Mexico and hanseniasis in children in the Brazilian Amazon.
An empirical analysis based on simulations was obtained, with very satisfactory results. Those performance results suggest that the ALR strategies, working in both adaptivity levels of parametric space and clusters' configuration space, are very effective in the surveillance of space-time disease clusters. Objective To assess the effectiveness of a public health automated phone campaign to increase vaccination uptake in targeted neighborhoods. To identify alternative predictors of variation in vaccination uptake, specifically to assess the association between vaccination uptake and weather conditions and day-of-week.
Work on vaccination timing and promotion largely precedes the 2009 pandemic. Postpandemic studies examining the wide range of local vaccination efforts mostly have been limited to surveys assessing the role of administrative strategies, logistical challenges and perceived deterrents of vaccination (1).
We used a quasi-Poisson logistic regression model to analyze daily vaccination counts at Montréal's mass vaccination centers (MVC; n 018) before and after an automated phone campaign promoting pandemic vaccination in 13 of the city's 29 health districts. We then used a similar model to test a more mundane explanation for the considerable variation in daily use of MVC: that inclement weather and weekends deterred vaccination.
We found a nonsignificant increase in vaccinations following the phone campaign, with fewer than 1000 estimated additional vaccinations (results not shown). The association between weather conditions and vaccination was strong and significant when controlling for variation between MVC (Table 1) . We found no evidence of day-of-week effect.
Only 50% of Montréal Island was vaccinated, which was well short of the public health goal for 'herd immunity'. Uptake was below 30% in some census tracts. Vaccination capacity was not the limiting factor.
Despite targeting neighborhoods with the lowest uptake, the Health Department's campaign did not appear to increase vaccination, reflecting ineffectual communication or a more troubling lack of trust in health authorities (2) . Launching the campaign earlier might have been more effective.
The strong association between vaccination and weather ( Fig. 1) , suggests that many individuals either were easily deterred from vaccination or delayed their trip to MVC. For wait-and-see individuals, even a short postponement may well have become nonvaccination (3) .
These findings suggest that vaccination uptake could be improved by allocating more resources at the start of the vaccination campaign.
Vaccination campaign; pandemic; influenza; weather 
In 2004, the Marion County Public Health Department (MCPHD), which serves a county population over 890,000, began using a real-time syndromic surveillance system, ES-SENCE (Electronic Surveillance System for the Early Notification of Community-based Epidemics) to assist in detecting possible disease outbreaks. Today, about 1600 emergency department visits occur daily in Marion County's 14 emergency departments. Epidemiologists from MCPHD have contributed to the city's extreme temperature plans for the last few years. While most of the previous increases in heat-related illnesses in Marion County have been attributed to prolonged heat exposure in connection with local auto races, the county had not activated the county wide emergency response plan in several years. From Tuesday, July 19 through Friday, July 22, 2011, the Marion County Extreme Temperature Plan was put into action in response to several days of a high heat index.
As the written plan indicated, a MCPHD epidemiologist checked ESSENCE every 3 hours and sent updated numbers to the Emergency Operations Center three times a day via e-mail. The query used the following terms: 'ˆheatˆ, or,ˆdehydˆ,or,ˆhotˆ,andnot,ˆgunshotˆ'. This is one of several pieces of information used to guide decision making when considering opening additional cooling centers and creating press releases for the public.
The MCPHD sister's agency is the area hospital that accepts medically underserved patients. With this access to the patient system, the electronic medical records of 21 people meeting the search criteria and seeking care at this emergency department between June 1, 2011, and July 29, 2011 were reviewed. An extended time period was reviewed to see if there were obvious differences in the counts of patients or terminology used in the chief complaint once the heat wave was upon the city. Five (24%) sought care in June 2011, 16 (76%) in July. Fifty percent of those seeking care for heat-related issues were seen in a 2-day period in July. Six people (29%) developed symptoms while at work. Work-related tasks included roofing, painting, working in metal tanks and driving trucks without air conditioning. Two homeless persons (10%) sought care during the 2-day time frame when half of the cases were identified as well as four of the six who developed symptoms while at work. Alcohol and drugs may have been a contributing factor for three (14%) of those seeking care. Nine individuals (43%) were treated for medical conditions, such as chronic obstructive pulmonary disease, diabetes, urinary tract infections, gastrointestinal infections or pneumonia.
Although the current query did produce a few 'false positives', the MCPHD staff has decided to continue the use of the same terminology since a significant amount of the cases detected were indeed heat related. The counts from the days of the heat wave were significantly higher than in previous summer months. The use of syndromic surveillance during a heat event can provide meaningful information for decision makers in emergency preparedness. Heat-Related Chief Complaints 
The CDC's BioSense Program receives near real-time health care utilization data from a number of sources, including Department of Defense (DoD) healthcare facilities from around the globe and nonfederal hospital emergency departments (EDs) in the United States, to support all-hazards surveillance and situation awareness. Following the tsunami in Japan on March 11, 2011 , the BioSense Program modified its surveillance protocols to monitor: (1) injuries and possible radiationassociated health effects in Japan-based DoD facilities and (2) potential adverse health effects associated with the consumption of potassium iodide (KI), a salt used to prevent injury to the thyroid gland in the event of radiation exposure, among persons attending participating EDs in the US. We present the findings from that enhanced surveillance.
The BioSense Program monitored healthcare activity in 20 DoD facilities located in Japan from March 17 through April 11, 2011. In Japan-based outpatient DoD facilities, we monitored 10 health conditions, which are associated with injuries, and possible syndromic presentations of radiation exposure, which included nausea/vomiting, diarrhea, headache, hypotension, rash, convulsion, dyspnea, dizziness and anemia. We also searched for radiation exposure-specific International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) 990, 508.0, 558.1, E926 and E926.9. There was a 2-to 5-day lag time between the time of patient visit and time when ICD-9-CM-coded final diagnoses were available. To monitor healthcare utilization for potential adverse effects associated with KI exposure in the United States, we searched ED chief complaint (CC) data from all 635 nonfederal BioSense hospitals for the following keywords indicating a KI-related visit: (1) potas*, pottas*; (2) iodine, iodide; (3) KI; (4) radiation, radeation; (5) nuclea*; (6) Japan. A given ED visit was considered a match if the content of the CC met the following keyword inclusion criteria: 1 and 2, 3, 4 and 5 or 6. Perl Regular Expressions were used to take into account upper-and lowercase letters and word boundaries. CC data were updated within 0Á2 days following the visit date.
To identify clusters of patient visits of interest, we used a modified version of the early aberration reporting system (EARS) C2 statistic (1) . For both surveillance efforts, this signal detection method was run upon the data for individual facilities.
Additionally, visit data from all 20 DoD facilities were analyzed as a group.
From March 17 through April 11, 2011, the BioSense Team created daily reports for the CDC's Emergency Operation Center and DoD counterparts. Reports included time series graphs for each of the 10 health conditions. Fig. 1 shows an example of a time series for diarrhea and total visits in the 20 DoD facilities. During this surveillance period, no clusters or radiation exposure coded visits were detected in Japan-based DoD facilities. In the United States, no ED-visit clusters associated with KI intoxication were found in nonfederal US EDs.
BioSense is an adaptable electronic all-hazards public health surveillance system that can provide near real-time health situational awareness during large-scale natural disasters. 
This study examines healthcare records, for the time period January 1, 2010ÁJune 30, 2011, from a North Carolina-based hospital system composed of three different hospitals for clusters of visits related to exposures. Visits were identified based upon the inclusion of terms in chief complaint data related to chemical, carbon monoxide, meningitis, food poisoning and other types of exposures. For each hospital, time series of visit counts from 1 hour cells, based on patient time-of-visit, were monitored using the past 60 days' cell counts as a baseline. Either a Poisson or a negative binomial distribution was assumed, depending on the baseline mean and variance for each hospital/hour cell. A p-value was calculated for the probability of at least as many visits as observed, and an alert was issued if this value was below 0.01, a threshold chosen to minimize the burden on the PHEs to investigate the alert.
For comparison with traditional NC DETECT methods, we also applied Biosense's modified C2 algorithm (2) to the daily exposure-related visit counts for this study. Table 1 shows how C2 and TOA alerting are complementary at the 3 study hospitals. Line lists provide relevant information for each patient cluster. Table 2 gives an example of a TOA alert from a late morning cluster at a single hospital.
TOA monitoring of exposure-related ED visits efficiently complements daily syndromic surveillance, finding additional clusters of potential interest. This method can be adapted to distributed as well as centralized alerting systems. 
To examine the completeness of data submitted from clinical information systems to public health agencies as notifiable disease reports.
Electronic laboratory reporting (ELR) was demonstrated just over a decade ago to be an effective method to improve the timeliness of reporting as well as the number of reports submitted to public health agencies (1) . The quality of data (including completeness) in information systems across all industries and organizations is often poor (2) , and anecdotal reports in the surveillance literature suggest that ELR may not improve the completeness of the data in the submitted reports (3).
The scope of our research included the following aims: (1) the development of a method for evaluating the completeness of laboratory data in the context of public health reporting; (2) measuring the completeness of laboratory data received from clinical information systems and an HIE using the method; and (3) comparing the completeness of the 'raw' data from clinical information systems (e.g., unaltered and unedited ELR messages) with the completeness of 'enhanced' data from the HIE (e.g., ELR messages having syntax corrected and concepts mapped to standard vocabularies).
A comparison of 7,592,039 raw messages and 16,365 enhanced messages revealed a number of differences with respect to data completeness. Data field completeness within the ELR messages varied from 0.01% to 84.6% across the two samples. Completeness was generally higher in the enhanced message sample as shown in Table 1 .
To effectively perform surveillance, public health agencies require access to 'timely, accurate, and complete data' (4) . Unfortunately, data quality is an issue for many clinical information systems that capture data utilized in public health surveillance processes. This study assessed the completeness of real-world ELR data from multiple provider organizations using a variety of laboratory information systems, documenting evidence that ELR data are heterogeneous in their completeness across and within information systems. In many cases, data important to public health surveillance processes are missing, indicating suboptimal ELR data quality. The study further documented evidence that a statewide or regional HIE can employ methods to mitigate ELR data deficiencies, leading to improvements in the completeness of ELR data prior to transmission to public health agencies.
Electronic laboratory reporting; health information exchange; data quality; completeness 
No off the shelf software was available that met local health department system requirements and budget constraints. Thus, a collaborative team of public health professionals and database programmers convened to establish a project charter that outlined the system requirements, personnel responsible, timeline and budget. A qualitative analysis of current county level systems and data helped to establish the requirements of the FITS system, as well as direct the reporting capabilities and features. The fixed budget expenditure and early deadline led to an expedited timeframe; the system was completed within 4 months.
A FITS was created in accordance with the project charter, timeline, user specifications and budget and included but was not limited to the following technologies: CentOS, Ruby and Excel. An innovative feature of the FITS system is the live lookup of national drug codes (NDC) hosted on the FDA's website, which autopopulates the FITS vaccination database with up-to-date information (2) . FITS allows flu immunization records to be accurately captured at the county level, resolving the dilemma created by the PREP amendment. County health professionals were invited to a system users training to orient them with the system, record management and downloading/ uploading of data. After an initial rollout of FITS at the county level, it is expected to be promoted statewide to increase accurate reporting of immunization administration.
The field of public health informatics calls for the conceptualization, design, development, deployment, refinement, maintenance and evaluation of surveillance systems (3) . This project exemplifies the positive outcomes that can result from collaborative efforts within the informatics framework. It is anticipated that this system will serve as a model for other counties across the state and will lead to improved reporting of flu vaccination records across the State of Ohio and perhaps beyond. Introduction Spatial cluster analysis is considered an important technique for the elucidation of disease causes and epidemiological surveillance. Kulldorff's spatial scan statistic, defined as a likelihood ratio, is the usual measure of the strength of geographic clusters (1). The circular scan (2), a particular case of the spatial scan statistic, is currently the most used tool for the detection and inference of spatial clusters of disease. Kulldorff's spatial scan statistic for aggregated area maps searches for clusters of cases without specifying their size (number of areas) or geographic location in advance. Their statistical significance is tested while adjusting for the multiple testing inherent in such a procedure. However, as is shown in this work, this adjustment is not done in an even manner for all possible cluster sizes (3).
We pose a modified inference question: what is the probability that the null hypothesis is rejected for the original observed cases map with a most likely cluster of size k, taking into account only those most likely clusters of size k found under null hypothesis for comparison? This question is especially important when the p-value computed by the usual inference process is near the alpha significance level, regarding the correctness of the decision based in this inference.
Numerical experiments are made showing that the proportions of rejections of the null hypothesis differ noticeably, by employing the usual critical value, compared with using the data-driven critical values. It is also shown that the computational cost of estimating the data-driven critical value may be reduced through the use of a simple interpolation.
A practical procedure is provided to make more accurate inferences about the most likely cluster found by the spatial scan statistic. The proposed method is more useful when the computed p-value using the classical inference is close to the significance level; otherwise, there will be no change in the decision process. In this situation, it is recommended that the data-driven inference should be performed, especially when the observed most likely cluster has relatively large size.
Spatial scan statistic; inference; data-driven Significant multiple high-and low-risk regions in event data maps Emerson Bodevan 1 , Luiz Duczmal 2 *, Gladston Prates Moreira 3 , Anderson Duarte 3 and Flá via Oliveira Magalhã es 4
The Voronoi Based Scan (VBScan) (1) is a fast method for the detection and inference of point data set space-time disease clusters. A Voronoi diagram is built for points representing population individuals (cases and controls). The number of Voronoi cells boundaries intercepted by the line segment joining two cases' points defines the Voronoi distance between those points. That distance is used to approximate the density of the heterogeneous population and build the Voronoi distance Minimum Spanning Tree (MST) linking the cases. The successive removal of its edges generates subtrees, which are the potential space-time clusters, which are evaluated through the scan statistic. Monte Carlo replications of the original data are used to evaluate cluster significance. In the present work, we modify VBScan to find the best partition dividing the map into multiple low-and high-risk regions.
In our novel approach, we use the previous VBScan recursively on the map with case-control point event data. At each recursive step, we compute two functions: (i) the likelihood ratio of the multiple components and (ii) the likelihood ratio increase since the previous step. As the first function always increases monotonically with every added component to the partition, the last function is used as a measure of the cost-benefit of adding a further region to the partition. This is done employing a multicriteria decision process, determining the nondominated partition solutions. Through Monte Carlo replications under null hypothesis, we compute the significance of the nondominated solutions and choose the best partition.
Our method was tested on several different simulated maps partitioned into different numbers of components (ranging from 2 to 4). The relative risks for each component were chosen as 3 sigma, (3 sigma and 0 sigma, corresponding respectively to high-, low-and neutral-risk spots. We evaluate the power of detection and matching (a measure of overlap between the real and detected partitions) for each set of 1000 Monte Carlo replications. The average power varies from 0.686 to 0.803, and matching varies from 0.566 to 0.850 for the several sets of simulations.
We also applied the method on a case study of dengue fever in a small Brazilian town in 2010 (1). Fig. 1 shows the MST linking the 57 cases (small circles) distributed among 3929 controls. The optimal partition consists of three components: two high-risk regions (red and blue) and a low-risk region (white).
The proposed method is fast, with good partitions' accuracy determination. The dengue fever application's result shows that our method is in very good agreement with the previous analysis with VBScan, which indicates two significant high-risk clusters (the red region is the primary cluster with p-value 0.004, and the blue region is the secondary cluster with p-value 0.016).
Case-control; disease cluster; spatial scan statistic; space partition; dengue fever 1 
The spatial scan statistic (1) is the most used measure for cluster strenght. The evaluation of all possible subsets of regions in a large dataset is computationally infeasible. Many heuristics have appeared recently to compute approximate values that maximizes the logarithm of the likelihood ratio. The Fast Subset Scan (2) finds exactly the optimal irregularly spatial cluster; however, the solution may not be connected.
The spatial cluster detection problem was formulated as the classic knapsack problem (3) and modeled as a biobjective unconstrained combinatorial optimization problem.
Dynamic programming relies on the principle that, in an optimal sequence of decisions or choices, each subsequence must also be optimal. During the search for a solution, it avoids full enumeration by pruning early partial decision solutions that cannot possibly lead to optimal solutions.
We propose a novel method, the Geographical Dynamic Scan (GDScan) to find optimal connected clusters. It employs an adaptation of the NemhauserÁUllman algorithm for the 0Á1 knapsack problem (4). We minimize a biobjective vector function F(z)0((C(z),N(z)), where C(z) and N(z) are the number of cases and the population of the candidate cluster z, respectively. Then, we show that the solution which maximizes the spatial scan statistic is included in the set of nondominated solutions of F (the Pareto set), showing that the dynamic programming algorithm allows to solve the unconstrained maximization of the scan statistic for any given spatial dataset. However, this is typically not sufficient to solve practical spatial detection problems.
The dynamic programming algorithm is thus modified to consider (i) a geographical proximity constraint and (ii) a connectivity constraint, for each region j. Assuming that the geographical proximity of a region j contains k regions, the geographical dynamic scan guarantees the optimal solution within the collection of 2ˆk subsets, searching for only a small number of subsets, which depends almost linearly on k, on average.
We conducted numerical simulations showing that GDScan has good power of detection, sensitivity and positive predicted value.
An application is shown for the dataset of Chagas' disease cases in the population at risk of puerperal women in Minas Gerais state, Brazil, in 2006 (5). Fig. 1 shows the nondominated sets of solutions obtained by GDScan for neighborhood sizes of 5, 20, 40 and 90.
GDScan is a fast and an efficient method to detect connected arbitrarily shaped disease clusters in aggregated area maps.
Disease cluster; dynamic programming; spatial scan statistic 
Ordering-based approaches (1,2) and quadtrees (3) have been introduced recently to detect multiple spatial clusters in point event datasets. The ALG (4) is an efficient graph-based data structure to handle the communication of cells in discrete domains. This adaptive data structure was favorably compared to common tree-based data structures (quadtrees). An additional feature of the ALG data structure is the total ordering of the component cells through a modified adaptive Hilbert curve, which links sequentially the cells (the orange curve in the example of Fig. 1 ).
We combine ordering-based approaches with the ALG structure to identify multiple clusters in case-control datasets, in a fivestep procedure. In the first step, we subdivide adaptively the domain into square cells (blue squares in Fig. 1 ), with controls (pink points) and cases (little dotted black squares). In the second step, the cell's ordering given by the Hilbert curve is used to sequentially join cells with the highest proportion of cases over controls. This produces loose groups of higher than average rates of disease. The adjacent groups thus formed are themselves united, according to certain criteria, into larger groups in the third step, forming the cluster candidates. In the fourth step, Kulldorff's spatial scan statistic is computed for each group, and the clusters are ranked. Finally the cases are randomized, and steps 1Á4 are repeated. That is done hundreds of times to compute the significance of each cluster. Only significant clusters are reported.
As an application, we find clusters of dengue fever for Lassance City, in southeast Brazil, 2010 (5). Fig. 1 shows the three significant clusters found, displayed as the three green patches.
The previous adaptive subdivision of the domain is essential to define more homogeneous regions within the study area.
Also, instead of just applying some ordering-based approach, our method introduces an intermediate step (step 3) to combine the separated regions of high incidence. Those two features produce more reliable clusters, compared with the usual ordering-based methods.
Spatial cluster; autonomous leaves graph; ordering-based; spatial scan statistic; quadtree 
In April 2009, a novel strain of influenza A was detected in Mexico, which quickly spread to the United States and the rest of the world. In response to the pandemic, the New Hampshire Department of Health and Human Services (NH DHHS) developed a web-based school absenteeism reporting system to track and record overall absenteeism and influenza-like illness (ILI)-related absenteeism in New Hampshire schools.
An absenteeism reporting form was developed and placed on a NH DHHS website. Access to the reporting form website was through a secure NH Department of Education (DOE) web portal, and school nurses were asked to voluntarily report data into the system. The questionnaire asked for the number of students absent, the number of students absent for ILI, the number of staff absent and the number of staff absent for ILI in addition to the name of the reporting school ( Fig. 1 ). Data were exported and analyzed daily by NH DHHS staff using Microsoft Excel. School enrollment data for each school were provided by DOE so that rates of absenteeism could be calculated. Rates for overall absenteeism and absenteeism due to ILI were aggregated by school administrative unit (SAU) and posted on the NH DHHS website twice weekly in the form of a map. Schools reporting absenteeism greater than 10% for any given day were contacted to determine whether an ILI outbreak was occurring and to recommend control measures.
Between September 7 and December 23, 2009, statewide overall school absenteeism ranged from 0.0% to 29.3% and statewide ILI-related absenteeism ranged from 0.0% to 14.2%, both of which peaked the week of November 1Á7, 2009. The observed peak of school absenteeism was consistent with data observed in other ILI surveillance systems such as over-the-counter sales of cough and cold medications and visits to emergency departments ( Fig. 2) . At the peek of absenteeism, 346 of 479 (72%) of all elementary, middle and high schools in New Hampshire were reporting into the system. NH DHHS identified 103 outbreaks using the school absenteeism reporting system; a state public health nurse investigated each outbreak. Timeliness of outbreak detections were within 24 hours due to daily reporting.
The newly developed school absenteeism reporting system provided important public health surveillance data to evaluate potential outbreaks or clusters of disease in communities and resulted in the detection of and response to 103 outbreaks of ILI that would not have been detected otherwise. Enhanced interaction with school nurses through the development of the surveillance system resulted in increased awareness in school populations about the influenza A/H1N1 (2009) virus. Furthermore, the rapid detection and response to potential outbreaks identified using the system may have minimized the effect of ILI in the school system and the community, through heightened awareness and implementation of control measures.
Although the reporting system started as a way to monitor the impact of the 2009 influenza A/H1N1 pandemic, NH DHHS has continued to routinely monitor student absence ever since. School absenteeism surveillance has the potential to aid in early detection, and mitigation, of other communicable diseases in the school system, an important indicator of illness in the communit. 
EDs supply critical infrastructure to provide medical care in the event of a disaster or disease outbreak, including seasonal and pandemic influenza (1). Already overcrowded and stretched to near-capacity, influenza activity augments patient volumes and increases ED crowding (2, 3); high ED patient volumes expected during a true influenza pandemic represents a significant threat to the nation's healthcare infrastructure (4) . EDs ability to manage both seasonal and pandemic influenza surges is dependent on coupling early detection with graded rapid response. While practical use of traditional surveillance systems has been limited due to the several week lag associated with reporting, new internet-based surveillance tools, such as GFT, report surveillance data in nearreal time, thus allowing rapid integration into healthcare response planning (5) .
October 2010) at an urban academic hospital with physically and administratively separate adult and pediatric EDs. We collected weekly data from GFT for the city of Baltimore, ED CDC reported standardized influenza-like illness (ILI) data, laboratory-confirmed influenza data and ED crowding indices (including patient volume, number of elopements, waiting room time and length of stay for admitted and discharged patients). Pediatric and adult data were analyzed separately using crosscorrelation with GFT.
GFT correlated with both number of positive influenza tests as seen in Figure 1 (adult ED r 00.876, pediatric ED r 00.718) and number of ED patients presenting with ILI (adult ED r00.885, pediatric ED r 00.652). Pediatric but not adult crowding measures such as total ED volume (r 00.649) and left without being seen (r 00.641) also had good correlation with GFT. Adult crowding measures for low acuity patients such as waiting room time (r00.421) and length of stay in discharged patients (r00.548) had moderate correlation with GFT.
City-level GFT shows strong correlation with local influenza cases and ED ILI visits, providing first time evidence of its utility for local ED surveillance and, potentially, response planning. Importantly, GFT correlated with several pediatric ED crowding measures as well as those for low acuity adult patients.
Influenza, Google flu trends, emergency department, crowding 
To describe a real-time reportable disease and surveillance solution focused on local public health department needs and compatible with state health departments, regardless of meaningful use certification status of health care providers.
Multiple options (1, 2) are available for health care provider organizations to receive assistance in demonstrating compliance with meaningful use requirements for public health reporting (3) . A certified EHR solution is a requirement for participation in these programs; vast majority of health care providers do not yet have such a solution. No funding programs are currently available to assist public health agencies, especially local public health departments (4) . As a result, most providers and local public health agencies are seemingly left without viable options except spending significantly in a tight budget environment.
Prior to ARRA 2009/Meaningful Use, KHA, KY CHFS and ETI created an electronic disease-reporting solution for hospitals and associated local public health. The Community Surveillance project operates in 3 population centers within Kentucky and provides real-time surveillance and disease reporting for up to 18 prevalent disease conditions across more than 20 provider locations and 6 provider organizations within the Northern Kentucky, Lexington/Fayette County and Louisville Metro communities. The software solution, ETI's HealthSIS, is able to accept meaningful use certified messaging, uncertified messaging formats and custom data streams for both reportable disease and syndromic surveillance information from a single stream within each provider location. The messaging stream used for identifying reportable or syndromic conditions is a copy of the content generated by normal hospital operations; specialized or additional data input is not required from health care provider staff. HealthSIS is configured to submit only data relevant to surveillance goals of the community from source systems; this configurable filtering capability allows for reduced resource requirements, reduced data management resources, and a manageable data set for analysis.
Since mid-2008, NKIDHD receives electronic disease reports and surveillance support for what is now the 18 most prevalent disease conditions in the community. SEMC (6 facilities) has realized 75%Á90% reduction in public health reporting effort as a result of the capabilities provided by HealthSIS. Since mid-2009, LFCHD receives syndromic surveillance data from ambulance runs, initially to support preparations for the 2010 Alltech-FEI World Equestrian Games; LFCHD began receiving electronic disease reports for 9 prevalent disease conditions in mid-2010 from CBH and UKMC. Since late 2009, LMPHW receives electronic disease reports for 5 prevalent disease conditions from BHE and notifications of same from JHSMH; in 2011, NHC began participating in the disease-reporting process.
Installing and maintaining electronic disease and syndromic surveillance to support public health agencies is possible without large budgets and without massive systems upgrades within health care provider organizations; benefits are clearly measurable Á independent study in Washington state in 2010 confirms benefit of the concept (5). Emergint's HealthSIS software and the Community Surveillance solution implemented in Kentucky presents a systems approach that can be replicated across the country, whether fed by providers or HIEs and regardless of meaningful use certification status. 
The electronic surveillance system for the early notification of community-based epidemics (ESSENCE) is the web-based syndromic surveillance system utilized by DHMH. ESSENCE utilizes a secure, automated process for the transfer of data to the ESSENCE system. Data sources in the Maryland ESSENCE system include emergency department (ED) chief complaints, poison control center calls, over-the-counter (OTC) medication sales and pharmaceutical transaction data (for certain classes of antibacterial and antiviral medication). All data sources have statewide coverage and are captured daily in near real-time fashion. OIT developed a web-based application in conjunction with OP&R to allow the epidemiologists involved in the ESSENCE program to monitor and audit the transfer of this data. The application allows the user to indicate whether or not each data file has been consumed into ESSENCE for any date of the year. The user can edit these daily entries at any time to update the status of the data that have been received. The user may also query the database by data source, date and date range to generate a report. The database also contains contact information for technical and infection control staff at the hospitals that participate in the ESSENCE program. Finally, the application can also generate reports that detail which users have logged into ESSENCE, when the log-in occurred, and which pages within ESSENCE were visited.
Forty-six EDs, two major pharmacy chains, two poison control centers and the Centers for Disease Control and Prevention (CDC; through a pilot partnership), all contribute data to ESSENCE on a daily basis. Thirty-six separate SSH File Transfer Protocol (sFTP) data feeds are required to transfer and incorporate these data into ESSENCE. Beginning January 1, 2011, the web-based application developed by OIT was utilized on a daily basis to ensure that the transfer of data was monitored and recorded regularly. Each morning, an epidemiologist from OP&R logs into ESSENCE and verifies which data points have been consumed into the system. The presence or absence of each data point is then recorded in the data tracking application. An automated e-mail is generated that details which data sources are absent. This e-mail is sent to the OIT employees involved in the ESSENCE program, who then check the server to see if the data were transferred or if there was an error during the transfer and consumption process. The epidemiologists then contact each data source that failed to send the data on that particular day.
Data are presented for January 1, 2011, through July 31, 2011 (212 total days). Between the ED chief complaint data, the poison control data, the OTC medication sales data and the pharmaceutical transaction data, there are a total of 50 data points on any given day in the Maryland ESSENCE system. This amounts to a total of 10,600 possible data points that can be in the ESSENCE system during this time frame. Using the data tracking application, OP&R has managed to acquire and consume 10,527 data points or 99.31% of all possible data points for this time period. The poison control data are complete for this time frame, as is the pharmaceutical transaction data, and one of the major pharmacy chain's data. The other major pharmacy chain whose data are not complete is only missing 2 data points (99.10% complete). Twenty-five of the 46 EDs have transferred 100% of the possible data points to the ESSENCE system. All other EDs contributing data to the ESSENCE system are at least 95.75% complete.
The data tracking application developed by OIT and utilized by OP&R to monitor and audit the transfer of syndromic surveillance data has thus far been successful in ensuring data completeness. Those involved with the program have been able to monitor and document that over 99% of all possible data are incorporated into the surveillance system. The data source with the lowest completion percentage has over 95% of its data incorporated into the system. DHMH will continue to use this system moving forward to ensure that the syndromic surveillance data transfers continue to be successful.
Data; transfer; audit; ESSENCE *Zachary Faigen E-mail: zfaigen@dhmh.state.md.us Introduction ILI data are collected via an Influenza Sentinel Provider Surveillance Network at the state level. Because participation is voluntary, locations of the sentinel providers may not reflect optimal geographic placement. This study analyzes two different geographic placement schemes*a maximal coverage model (MCM) and a K-median model, two location-allocation models commonly used in geographic information systems (GIS) (1). The MCM chooses sites in areas with the densest population. The K-median model chooses sites, which minimize the average distance traveled by individuals to their nearest site. We have previously shown how a placement model can be used to improve population coverage for ILI surveillance in Iowa when considering the sites recruited by the Iowa Department of Public Health (IDPH) (2) . We extend this work by evaluating different surveillance placement algorithms with respect to outbreak intensity and timing (i.e., being able to capture the start, peak and end of the influenza season).
We developed a web-based site placement calculator to aid public health officials in designing their surveillance system. We then compare the two algorithmic site placement schemes against each other by simulating the spread of influenza across the state of Iowa. Our simulations are based on an Iowa Medicaid dataset comprised two million cases classified with 30 different ILI ICD-9 codes and their corresponding geocodes from 2000 to 2008. We use the Huff Model (3) to determine whether or not a case might have been detected by a particular network of sites. Using this scheme, we compare surveillance networks based on outbreak intensity (i.e., which networks detect the highest percentage of cases) and outbreak timing (i.e., which networks detect cases temporally in sync with the true start, peak and end of the disease season). To compare network outbreak timing, we generate the noise parameters of a state space time series model using the expectationmaximization (EM) algorithm implementation provided by Shumway and Stoffer (4). We then perform an analysis on which ICD-9 codes a network might consider.
Considering outbreak intensity, we show that sites chosen by our approach outperform the sites used by the IDPH. In other words, we can provide a more accurate representation of disease burden during an influenza season. However, our approach does not provide a substantial difference in the detection of the start, peak and end of the influenza season. In addition, when using the noise values generated by the EM algorithm to analyze the minimal number of sites needed to estimate the timing of the influenza season, our results were highly influenced by both the number of different ICD-9 codes considered and the number of cases considered.
We built a web-based tool to assist public health officials in designing their sentinel surveillance site network. Through simulation, we show that the sites our tool selects allow for better representation of disease burden. We also show that selecting the correct ICD-9 codes that the surveillance network should consider may be as important as selecting the locations of the sites themselves. Using our methods, we can help public health officials design surveillance systems, which are smaller and more easily managed but still detect cases that reflect reliable estimates of both disease burden and timing. 
Noroviruses are the single most common cause of epidemic, nonbacterial gastroenteritis worldwide. NoVs cause an estimated 68Á80% of gastroenteritis outbreaks in industrialized countries and possibly more in developing countries.
Data were analyzed from 900 RT-PCR-confirmed NoVoutbreaks extracted from a systematic review of articles published from 1993 to 2011, indexed under the terms 'norovirus' and 'outbreak'. SeventyÁfour variables were included in the database.
Of the 894 outbreaks documenting year of occurrence, 72% occurred between 2000 and 2010. More than 90% of outbreaks occurred in the northern hemisphere and 45% took place during the winter. In general, we found the number of primary cases and persons at risk was significantly lower in outbreaks related to food and waterborne transmission as well as foodservice and healthcare settings. The attack rates were significantly higher in outbreaks related to food, water and those that occurred in the winter. Attack rates were also lower in healthcare-related outbreaks, perhaps on account of proper infection control practices and active surveillance by healthcare facilities to limit the spread of disease.
Multivariate regression analyses demonstrated that higher attack rates were significantly associated with foodservice (b 0 14.70, p00.02) and winter outbreaks (b09.81, p0B0.01). A combination of strains was most common among food and waterborne outbreaks. Waterborne outbreaks were also significantly associated with GI strains (odds ratio [OR] 00.10, 95% confidence interval [CI] 00.03Á0.41 where the odds of an outbreak being caused by a GII strain are less than the odds of the outbreak being caused by a GI strain), while healthcarerelated (OR 040.42, 95% CI 02.09Á783.15 where the odds of the outbreak being caused by a GII strain are greater than the odds of the outbreak being caused by a GI strain) and winter outbreaks (OR 05.56, 95% CI 01.97Á15.69) were associated with GII strains (Table 1) .
Food and waterborne outbreaks may have greater attack rates due to: (1) efficient viral transmission, especially within smaller confines, via drinking water or food items, and (2) more accurate identification of persons at risk.
Decreased mobility of infected persons in healthcare settings may also limit transmission of NoV to healthy individuals. As mentioned previously, the clustering of people indoors during seasonal cold weather may facilitate person-to-person NoV transmission. These results identify important trends for epidemic NoV detection, prevention and control. 
The intrinsic variability that exists in the cases counting data for aggregated-area maps amounts to a corresponding uncertainty in the delineation of the most likely cluster found by methods based on the spatial scan statistics (3) . If this cluster turns out to be statistically significant, it allows the characterization of a possible localized anomaly, dividing the areas in the map in two classes: those inside and outside the cluster. But, what about the areas that are outside the cluster but adjacent to it, sometimes sharing a physical border with an area inside the cluster? Should we simply discard them in a disease prevention program? Do all the areas inside the detected cluster have the same priority concerning public health actions? The intensity function (2), a recently introduced visualization method, answers those questions assigning a plausibility to each area of the study map to belong to the most likely cluster detected by the scan statistics. We use the intensity function to study cases of diabetes in Minas Gerais state, Brazil.
We use the intensity function to visualize the plausibility of each area of some study map to belong to a possible cluster in the map. Fig. 1 presents the most likely cluster found by circular scan. Fig. 2 shows the intensity function. The intensity function map (Fig. 2) shows clearly that areas with the highest quantiles correspond ( Fig. 1 ) to areas belonging to the primary cluster detected by circular scan. But, the intensity function map also shows a significative number of areas (red color) with a high plausibility to belong to a possible real cluster and some areas (orange color) with intermediate to high intensity function values.
Given a study map with an observed number of cases distributed among its areas, the intensity function value for each area represents the importance of that particular area in delineating a possible cluster of anomalies in the map. This is shown clearly in the results obtained for diabetes cases in Minas Gerais.
Intensity function; spatial scan statistics; diabetes; delineation of spatial clusters 
Cardiovascular event prediction has long been of interest in the practice of intensive care. It has been approached using signalprocessing of vital signs (1Á4), including the use of graphical models (3, 4) .
Our approach is novel in making data segmentation as well as hidden state segmentation an unsupervised process and in simultaneously tracking evolution of multiple vital signs. The proposed models are adaptable to the individual patient's vitals online and in real time, without requiring patient-specific training data if the patient-specific feedback signal is available. Additionally, they can incorporate expert interventions, produce explanations for alarm predictions and consider effects of medication on state changes to reduce false alert probability.
The proposed model represents distributions of patient data (vitals, state and treatment) as a dynamic Bayesian network. The state of the patient is observed only when an alarm is triggered. The arity of the state variable is estimated from data via E-M optimization. The state and another discrete observable, treatment (a vector of administered medications), influence the continuous output variables that represent the vital signs. The vitals are segmented adaptively using a Kalman filter to reflect a potentially nonstationary periodicity of signals. The segmented vitals are then represented with a continuous Semihidden Markov Model. The trained system is capable of predicting the patient's state on-the-fly from currently observed vitals. It can also learn on-the-fly whenever user feedback is available in the form of correct labels of the predicted states.
We conducted evaluations using the MIMIC II data (5). We used ECG and respiratory rate as input vitals in an attempt to predict heart failure alarms. The results shown in Table 1 were obtained per-patient, by subsampling, using data from the patients held out of the training set. The proposed approach brings the AUC metric (area under the receiver operating characteristic diagram) to 0.66 on average, the patient-specific model offers an improvement, and the inclusion of treatment information provides further benefits.
We have outlined a probabilistic modeling system that is capable of predicting heart failure alarms using time series of vital signs. It is able to learn the key parameters from data (state and temporal resolution) and allows fast adaptation to personalized features of a specific patient. Tests involving a limited set of vital signs indicate improved predictability of heart failure events when compared to a model relying only on prior probabilities. The next steps involve adding more vital signs to the input space to realize improvements of predictive accuracy. 
Influenza is a major cause of mortality. In developed countries, mortality is at its highest during winter months, not only as a result of deaths from influenza and pneumonia but also as a result of deaths attributed to other diseases (e.g., cardiovascular disease). Understandably, much of the surveillance of influenza follows predefined geographic regions (e.g., census regions or state boundaries). However, the spread of influenza and its resulting mortality does not respect such boundaries.
Data on influenza and pneumonia mortality were collected from 97 cities over 11 years (1996 through 2007), as reported in the MMWR (1). We used a novel method of computing the pairwise distance between two time series based on the Mahalanobis Distance derived from the time-series state-space-modeling framework. Mahalanobis Distance is a scale invariant form of Euclidean Distance that also takes correlations of the data set into account. This is an extension of a previously devised Kullback-Leibler Information-based time-series-clustering discrepancy measure (2) . All pairwise distances between cities were then used in a clustering procedure known as QT_Clust (3). This procedure was initially developed for the clustering of high dimensional genomic data. However, QT_Clust may be applied to many time-series-data sets where the trajectory rather than the process of a time series is of interest. A measure of cluster size and within-cluster distance is used to compare how geographically based influenza surveillance performs as opposed to nongeographically based surveillance.
The average within-cluster distance for the nine census regions is 5205 units. Ignoring geography, we found that our nine largest clusters held 85 of the cities (87.6% of the total cities observed) and maintained an average within-cluster distance of 4918 units. This amounted to a 5.5% reduction in the within-cluster distance. The largest of these clusters held 33 cities from all but one census region and had a within-cluster distance of 4295 units, meaning that its within-cluster distance was 17.5% smaller than that of the mean within-cluster distance of the nine census regions, the largest of which only held 17 cities.
It is natural to think of geographic proximity as an indicator of how likely a city or region's pattern of influenza mortality mirrors that of another region. However, we hypothesize that the relatively high level of travel within the country will affect the pattern of mortality such that cities across the nation may resemble one another more closely than cities within a predefined geographic region. Our approach involved the creation of a discrepancy measure specifically designed for time series data and the application of a clustering routine that seeks to create high quality clusters (rather than high-inclusion clusters). The largest cluster held roughly a third of the observed cities and yet still had a low within-cluster distance when compared to the geographic census regions. This result suggests that many cities observe similar influenza and pneumonia mortality patterns despite varying geographical locations. There are several limitations to this study. First, while our discrepancy works well in the presence of missing data, a preponderance of consecutive missing time points can negatively affect performance. We determined that 25 cities out of the original 122 cities reported too sporadically for analysis. Furthermore, our clustering technique depends upon the arbitrary selection of a 'quality criterion' that is very data driven. High quality clusters can be obtained, but this often leads to a large number of clusters. Conversely, a small number of clusters can be obtained by lowering the quality criterion. Future work will determine if we can use this time-seriesclustering approach to find repeatable clusters that may or may not suggest changes to current geographic boundaries in an effort to coordinate future influenza surveillance activities. 
Based on the definition of the U.S. Centers for Disease Control and Prevention (2), an updated definition of syndromic surveillance was developed, taking into account the evolution of syndromic surveillance in the past decade. Further, an inventory of syndromic surveillance systems in Europe has been started. To identify human syndromic surveillance systems, a literature review was first performed using Pubmed and Google, identifying 40 relevant publications. A brief questionnaire was sent to Triple-S partners, the European Center for Disease Prevention and Control (ECDC) and other contact persons to identify existing, past, pilot and planned systems in the different countries and the reference person for each system. The reference persons were then asked to complete a long online questionnaire for collecting detailed information (e.g., objectives, data sources, timeliness, statistical methods for outbreak detection, reporting tools and response measures).
For the inventory of veterinary syndromic surveillance systems, a similar method was used. The brief questionnaire was sent to the European Food and Safety Agency (EFSA) focal points and chief veterinary officers of each Member State and to the members of the European College of Veterinary Public Health. Differently from the inventory of human systems, the veterinary inventory included mortality surveillance systems.
Eight site-visits to existing systems are scheduled between June 2011 and May 2012, e.g., United Kingdom, France and Denmark/Sweden. Open to 6Á10 project partners and participants from all European countries, the visits offer an in-depth understanding of a variety of systems and facilitate knowledge transfer, through discussions on practical experiences with national and regional stakeholders (e.g., strengths and weaknesses of the systems, lessons learned from the operators and users and expectations of decision makers).
The first output of the project was the adoption of the definition of human and animal syndromic surveillance.
The initial results from the inventory of syndromic surveillance systems and the geographical distribution of identified systems will be presented. As a result of the literature review and the responses to the brief questionnaire for human systems, 19 active systems, 11 pilot or planned systems and 4 systems for past mass gathering events since 2000 (e.g., Olympic games) have been identified to date. For veterinary systems, only 5 systems were identified by the literature review, whereas 16 active, 7 pilot and 2 expired systems have been identified by the brief questionnaires.
The first 4-day site-visit took place in the United Kingdom (Birmingham and Glasgow) in June 2011, where there are several systems based on different data sources: emergency departments, general practitioners (Q-surveillance, Piper and SISRS), phone calls to help lines (NHS Direct, NHS24), out-ofhours primary care and pharmacy prescription data.
By December 2011, four site visits have been conducted. The synthesis of the first visited systems, including their main characteristics and the experiences and lessons learned through those visits, will be presented.
Results from the inventory and the site visits will constitute the foundation for the development of guidelines for improving syndromic surveillance in Europe. Objective To present the Triple-S project, which aims to increase the European capacity for real-time surveillance and monitoring of the health burden of expected and unexpected health-related events.
A European project to develop guidelines to strengthen public health surveillance and rapid response was launched in September 2010 for 3 years, under the name Triple-S (Syndromic Surveillance Survey, Assessment toward Guidelines for Europe). The project, co-financed by the European commission through the Executive Agency for Health and Consumers, involves 24 organizations from 13 countries (Fig. 1) . The DG Sanco, ECDC, WHO/Europe and ISDS are members of the advisory board of the project, to ensure an exchange of practices and expertise at both the European and the global level ( Fig. 1 ).
The Triple-S project coordinated by the French Institute for Public Health Surveillance (InVS) is divided into six work packages (WP). Three are horizontal managerial WP for coordination, dissemination and evaluation. The other three concern an inventory of existing systems (WP4), country visits (WP5) for knowledge exchange and a deeper understanding of a selected number of systems. WP4 and 5 will lead to the development of guidelines for implementing syndromic surveillance in Europe (WP6).
The inventory will identify competent organizations and reference persons for animal and human syndromic systems in the European Union. A questionnaire will allow the collection of detailed characteristics of established, pilot and planned systems.
The Triple-S consortium has adopted a proactive approach to stimulate knowledge exchange through eight country visits.
The projects's final guidelines will provide scientific and technical guidance and tools for the development and implementation of syndromic surveillance systems for both human and animal health, according to the needs and expectations of the different EU member states taking into consideration the different data sources available and the different aims of syndromic surveillance systems. An additional outcome of the project is to build a sustainable network of organizations, which can provide support and advice on tasks related to syndromic surveillance: management, partnership with data providers and users, statistical methods, definitions of syndromes and dissemination.
For this, the triple S project will develop links with different projects in the USA and Canada. For organizations planning to start or reestablish a syndromic surveillance system in their own country, this network could help raise awareness of opportunities and of pros and cons of the anticipated syndromic surveillance system.
The objective of the project is not to create one single European syndromic surveillance system but to review and analyze syndromic surveillance activities across Europe to produce guidelines, while respecting the diversity of health systems and potential data sources for syndromic surveillance across Europe. 
Based on the descriptive analysis, a linear yearly trend, the days of the week, the period of school holidays, a seasonal effect and the unusual days (day-off) have a marked effect on the daily fluctuations of ED visits. The pattern of the daily visits during the summer periods has been modeled using a quadratic function. The day-of-week effect has been differentiated by seasonal period of the year. The final model based on the combination of variables explains 78% of the daily variations of the ED attendances and shows a good predictive performance on 2010.
This study is a first approach to improve the knowledge of the factors that influence the ED visits. In previous work, we proposed a process by which only posts that are based on specific 'important' topics are read, thus drastically reducing the amount of posts that need to be read. The process works by finding a set of 'bellwether' users that act as indicators for 'important' topics and only posts relating to these topics are then read. This approach does not consider the text of messages, only the patterns of user participation.
Our text analysis approach follows that of Cataldi et al. (1), using the idea of semantic 'energy' to identify emerging topics within Twitter posts. Authority is calculated via PageRank and used to weight each author's contribution to the semantic energy of all terms occurring in within some interval ti. A decay parameter d defines the impact of prior time steps on the current interval.
We considered 3 models of authority: (1) (emerging topic detection) ETD uniform (i.e., equal weight); (2) ETD PageRank; and (3) bellwether. For 1 and 2, we built a directed graph of user activity, using thread coparticipation to define edges. Each EIN post was text tokenized, POS-tagged, and had stop words removed. We use a time window t 0 5 days to aggregate messages terms and a decay parameter d0 60 days. We identified emerging terms using an energy threshold approach, where emerging is defined as any term where energy! k*m energy over the interval t, where k is some constant; we used k 0 1.3. Any term identified as emerging that occurs in the postsubject line is flagged as important. For the bellwether model, we algorithmically selected 80 and 90 authors from the year prior to the one under analysis and flagged threads as important when one of those bellwethers participated in it. We also conducted 1000 random trials of selecting subsets of 80 users to follow.
For evaluation, we used an annotated set of EIN threads identified as clinically important. We measured the total number of threads each authority model flagged for reading versus the number of actual important messages.
The performance of each authority method, measured as threads recommended by each model and evaluated over all messages from January 2003 to March 2009 (Table 1) .
The bellwether model performs best overall, requiring the least messages read while detecting more important threads at less cost of reading unimportant threads. The differences between 80 and 90 bellwethers reflect how parameters influence the tradeoff between these 3 measures. There was no significant benefit gained from viewing the EIN network in terms of a PageRank over equal authority, which aligns with previous work identifying PageRank's limitations in identifying experts (2) . Moreover, compared to randomly selecting 80 authors to follow, the performance is worse with our chosen parameters. Future work will examine incorporating bellwether authority into a textual analysis framework. 
We chose the following aspects of patient care to be included in the database form: presurgery patient condition and medications, anesthesia information, perfusion information, surgery information, recovery information, status of the patient at discharge and30 days and 365 days postsurgery follow-up information. Information was collected through structured questionnaire by trained data abstractor and entered into Microsoft Access software. On the basis of research hypotheses, specific data chunk was extracted and analyzed in SPSS (Statistical Package of Social Sciences) software. Impact in clinical practice Before this database, there was no way to monitor mortality and morbidity. Fortunately, with the development of database, postsurgery mortality and morbidity rates could easily be generated. It helped in development of strict enforcement of protocol to reduce the mortality and morbidity rates. It also helped in controlling preventable postsurgery complications. It also helps in identification of a gap inpatient knowledge regarding the use of warfarin after heart valve surgery and deficiencies in laboratory capabilities, both causing catastrophic complications. As a result, we modified our practice in an effort to address these issues and reduce the complication rates after heart valve surgery. Furthermore, identification of the need to quantify the midterm functional status of in-person and telephonic interview, resulting in the development of a questionnaire that has been added to our protocol 1-year postsurgery.
More meticulous record keeping, including long-term follow-up for 5 years will be collected. In addition to this, the development of a separate congenital/pediatrics cardiac surgery database will also be developed.
Updated and stringently maintained database helps to identify deficiencies in practice and provides a direction for future improvement.
Database; coronary artery bypass grafting; warfarin; mortality; quality of care; evaluation
The Ohio Department of Health (ODH) has created a biosurveillance network, which was originally based on the Real-time Outbreak and Disease Surveillance (RODS TM created by the University of Pittsburgh and is now developed as EpiCenter TM and managed by Health Monitoring Systems, Inc. (HMS) (1Á3). The web-based system uses advanced statistical algorithms to generate alerts from thresholds that are tuned at the state scale of geography for surveillance of chief complaints during emergency department admissions. Interpretation of output and the use of the system is geared toward trained epidemiologists and infection control practitioners.
The need to evaluate biosurveillance data within a local, realworld context is necessary to maximize situational awareness at the community level given that: emergencies begin and end as local events, hospital emergency department demographic catchments may vary significantly within a county and some emergency department capabilities may be unique to particular hospitals. With the addition of data analysis methods, which produce intuitive summary visualizations of large data sets, it is hoped that individuals whose training is not as specialized as the EpiCenter TM users will gain a greater understanding of the biosurveillance data and its relevance to the local population.
Reusable procedures were created for MySQL TM (4) to load and transform standard EpiCenter TM data exports. Structured Query Language (SQL) statements process export file data elements to create: more granular temporal elements, category groupings to mirror daily activities and generalized lifestyles, categories based on spatial calculations and scores derived from the frequency of symptom and syndrome categories.
Additional blocks of code were created in R TM (5) for automated analysis, visualization and reporting. The information graphics produced by R with enhanced biosurveillance data includes pivot tables, thematic maps, heatmaps, pictograms and charts. Edits of the code may be done with a text editor, and the local geographic coordinates of zip codes and hospital facilities can be obtained on the Internet.
The information graphics are to be assembled and annotated as an annual reference resource, Cuyahoga County Hospital Emergency Department Service Utilization Profile (HEDSUP). This output of localized biosurveillance data analysis summarizes the facility admissions, catchment demographics and symptoms using various visualizations, which aim to be understandable by an audience of nonstatistical experts.
Facility specific sections will be made available to hospital infection control practitioners and emergency department managers to evaluate patterns of service utilization and to improve data quality. Local health departments are considered to be the primary users through daily epidemiology and surveillance requiring decision support during disease outbreak investigation and response activities across all hospital emergency departments within a health jurisdiction.
The high availability of open source components cited above, the ease of integration and minimal development cost may provide low-resource environments, such as local health departments, increased functionality through a sustainable and customizable framework of modular components. The utilization of more intuitive data visualizations will shift the burden of advanced data analysis to a less technical interpretation of information by a broader user base for a greater understanding of local public health dynamics.
Visualization; open source; localized; situational awareness; biosurveillance Introduction An increase in tuberculosis (TB) among homeless men residing in Marion County, Indiana, was noticed in the summer of 2008. The Marion County Public Health Department (MCPHD) hosted screening events at homeless shelters in hopes of finding unidentified cases. To locate men who had a presumptive positive screen, the MCPHD partnered with researchers at Regenstrief Institute (RI) to create an alert for healthcare providers who use the Gopher patient management system in one of the city's busiest emergency departments. A similar process was used at this facility to impact prescription behavior (1). A similar method was also used at the New York City Department of Health and Mental Hygiene (2).
MCPHD and RI created a legal memorandum of understanding so that MCPHD could share the names and date of births of suspect cases to the programmers at RI. The alert went into effect in July of 2010. When a healthcare provider's search is also one of the suspected names, an alert appears on the screen informing the provider that this person should have a chest x-ray as part of a follow-up to a TB outbreak investigation. A phone number of a MCPHD nurse on call is provided. The suspect list is periodically updated to remove names of patients who have been located in other medical settings. The novel aspect of this system is that normal methods of locating these individual such as phone or address was not available. Additionally, other traditional public health methods to contact these patients had not proved successful.
Fifty-three different patients have been on the alert list since activation. Only one notification has occurred in the 13 months of activation. On December 1, 2010, the TB program reported that a provider had seen one of the suspect cases and the alert prompted the provider to order a chest x-ray and notify the MCPHD staff.
A review of a hospital patient management system revealed that 12 other patients were seen in the emergency departments while on active alert lists. Some patients were seen more than once while on the list. Some cases showed that the chest x-rays were performed as requested but the patient records did not indicate if the procedure was prompted due to the alert or because the patient presented with symptoms of TB. A review of the process is underway to better understand why these encounters did not provide a notification to the MCPHD.
Several MCPHD staff work in local homeless shelters daily looking for the suspect patients. If a staff member prompts an individual to go to the ED, that encounter is not recorded in the MCPHD Patient Management System. Therefore, the provider may ignore the TB alert on these patients since the patient was already suspected for TB.
In some instances, the patients were seen at the MCPHD TB clinic within a couple of days of being seen at the hospital ED. Again, the records do not indicate if the patient was prompted to go to the TB clinic. Interestingly, no new TB cases among this population were reported for the month of August.
Future attempts at locating patients: Since many of the identified cases have known psychiatric issues, outreach workers in the TB program are hoping to partner with the mental health community to reach some of the suspect cases. MCPHD is working with a local mental health clinic to create a similar alert in their patient management system. Also, RI may help develop another alert that includes contacts of cases, rather than suspect patients in hopes of completing the first initial screening for that segment of the population.
The most important outcome is getting the patients tested and treated if infected. Assessing the effectiveness of the alert is difficult due to a lack of encounter data. Only one-fourth of the patients (13 of the 53) visited the emergency department during the year of the alert. This is another reminder that multiple strategies must be used to reach this population for care. Health informatics can be an aid to public health in such endeavors. 
The VHA is the VA organization responsible for providing healthcare to over 5 million patients annually at 153 medical centers and over 900 outpatient clinics across the United States and U.S. territories. The VA Subject Matter Expertise Center for Biological Events (SMEC-bio) aims to leverage data in the extensive VHA electronic health records system and other sources to provide decision support to leadership for emerging infectious disease threats. Initial SMEC-bio work to examine this capability suggested that the increased incidence of dengue disease in the VHA patient population in PR in 2010 may be related to increased rainfall (1). This present work analyzes dengue incidence in the PR VHA patient population over time to understand disease trends and contribute to a framework for predictive analysis.
VHA administrative inpatient and outpatient datasets were queried separately for monthly occurrence of dengue and dengue-like diagnosis codes (ICD-9-CM 061, 065.4, 066.3 and V73.5) in PR for Federal Fiscal Year (FY) 1997 to FY 2011 (up to August). Patients are unique within each FY and are counted at the first occurrence of the noted code(s). Wavelet time series analysis using the Morlet wavelet was performed to identify the presence and scale of periodic components in the inpatient and outpatient time series. Wavelet coherence analysis was subsequently carried out to determine the statistical association R(a,t) between the two time series at multiple periodic scales 'a' and time scales 't' (see equation).
Finally, the phase lag between the two time series was determined by computing their phase difference as the ratio of the imaginary and real parts of the cross-spectrum of the two time series.
Wavelet power spectral analysis of the inpatient and outpatient time series identified statistically significant oscillatory modes across different years and at all periodic scales examined* indication of the endemic nature of dengue in PR. Crossspectral analysis of the two time series revealed three distinct areas of significant coherence as seen in Fig. 1 . At the 1-year period, the two time series have strong association during 2004Ápresent, with waves of outpatient cases lagging behind those of inpatient cases by an average lag time of 7 days (not shown). At the 1.5-year period, the two time series cohere during 1999Á2003, indicating that dengue outbreaks did not occur yearly in the VHA population prior to 2004. At the 3.5Á4-year period, they cohere across all years in the data, an indication of a multiyear interepidemic period.
Endemic and epidemic signatures of dengue were shown to be present in the PR VHA patient population. Furthermore, an average lag time of 7 days between the epidemic waves of inpatient and outpatient dengue cases was observed at the annual scale, not reported previously. In light of these observations, future studies will examine whether the dengue dynamics in the VHA patient population in PR, coupled with other data such as weather parameters, can be used to forecast dengue epidemics in VHA patients and perhaps inform dengue dynamics in the general population in the region. 
Increasingly, epidemiologists have been tasked with interpreting multiple streams of heterogeneous data arising from varied surveillance systems. However, public health personnel have experienced an overload of plots and charts, as information visualization techniques have not kept pace.
Elucidating design objectives based on analysis of current system functionality and gaps in currently available systems; 2.
Developing a conceptual model that captures and represents the mental model of the prototypic public health epidemiologist; 3.
Developing novel visualization paradigms for the public health domain; 4.
Developing sample data set to support development and testing of the prototype.
The prototype was populated with real world data and used to visualize a gastrointestinal disease outbreak caused by cryptosporidium and respiratory virus outbreaks including influenza and RSV. The prototype was well received by regional epidemiologist practicing in the state and local health departments of Utah.
The EpiCanvas display provides a novel visualization that facilitates situational awareness. Based on heuristic mental models of end users, this display encourages visual correlation, data interrogation, exploration and discovery (Fig. 1) . The prototype also provides the first iteration of an integrated infectious disease weather map for use by public health professionals.
Visualization; analytics; surveillance 
In this project, we present a robust methodology for evaluating the fitness of the Semi-Naïve Bayesian Network (SNBN) disease model structure used by Geographic Utilization of Artificial Intelligence in Real-Time for Disease Identification and Alert Notification (GUARDIAN)*a real-time, scalable, extensible, automated, knowledge-based infectious disease detection and diagnosis system*for determining the appropriate thresholds, which separate between different 'goodness of fit' categories, and for tuning the model parameters to optimize its applicability within a particular healthcare facility.
Each of the infectious diseases that GUARDIAN monitors is modeled in GUARDIAN's knowledge base as a SNBN (1) of diagnostic features. Each model is developed through extensive literature review by infectious disease experts and encoded as a standardized probabilistic SNBN (2) . GUARDIAN uses these networks to rapidly assess each patient whose data are presented to the system. General model fitness is evaluated using 10-fold cross-validation. The overall, threshold-independent, model quality is determined through analysis of the receiver operating characteristic (ROC) curve (3) of each model using de-identified emergency department patients for the true negatives. To find the thresholds for each relative risk category, we use Gaussian decomposition (4) over the probability distribution of known negatives; known positives of other diseases; and known positives of confirmed, suspected and probable cases of the disease of interest.
To tune the SNBN model parameters for a particular healthcare facility, we use a hill-climbing algorithm (5) over known positives from literature and a sample of patients (negative and, if available, positive) from that healthcare facility. The hillclimbing algorithm uses the 10-fold cross-validation area under the ROC curve as the metric to optimize.
We performed the process outlined above for severe acute respiratory syndrome (SARS), because its similarity to influenza presents the largest challenge of all of the diseases in GUAR-DIAN's current knowledge base. Even with this challenge, the area under the ROC curve was 0.929 and was optimized to 0.962 through hill-climbing. Gaussian decomposition showed good fidelity using 5 risk categories (Fig. 1) .
As we have demonstrated for SARS, GUARDIAN's infectiousdisease knowledge base uses robust models, which accurately discriminate among related diseases with a high level of accuracy, confidently place each diagnosis within its appropriate relative risk category and optimize its performance for a particular healthcare facility. These results illustrate the improvement that disease-specific expert systems, such as GUARDIAN, represent over traditional trend-based anomaly detection systems. The robust process presented here is currently being used to validate GUARDIAN's existing and ever-growing knowledge base containing a number of diseases of interest for public health surveillance. 
The Centers for Disease Control and Prevention (CDC) case definition of influenza-like illness (ILI) as fever with cough and/ or sore throat (1) casts a wide net resulting in lower sensitivity, which can have major implications on public health surveillance and response.
This is a retrospective cross-sectional study conducted August 1, 2009, to July 31, 2011, in the emergency department of an academic medical center. The sample consisted of 2661 patients who received a nasopharyngeal swab followed by polymerase chain reaction (PCR) testing for respiratory viruses. Geographic Utilization of Artificial Intelligence in Real-Time for Disease Identification and Alert Notification (GUARDIAN)*a syndrome surveillance program*was utilized to review patients' records and detect the presence or absence of 37 influenza-associated symptoms (e.g., rhinorrhea, myalgias, headache and nausea among others) including reported and measured fever, cough and sore throat. Demographic factors such as age groups and gender were included in the analysis. Descriptive and x 2 test were used to determine a subset of significant signs and symptoms. A binary logistic regression with backward selection option was employed to further narrow down significant symptoms.
Females represented 53.4% of the sample. The percent of positive influenza cases based on PCR results was 9.2% (i.e., 245 cases). The majority of positive influenza cases (55.9%) occurred below 50 years of age. Positive influenza patients with fever, cough or sore throat were 207 (84.5%), 218 (89%) and 56 (22.9%), respectively. Based on x 2 test, fever, sore throat, cough, myalgias or body aches, chills or rigors, rhinorrhea or nasal congestion or sinusitis or nasal symptoms, dyspnea, upper respiratory infection symptoms or viral illness, rash and age groups were statistically significant. Apart from fever and cough, myalgias and rhinorrhea were significant associated symptoms of influenza based on multivariate analysis (Table 1) .
Based on these results, some of the recommended ILI case definitions could be (1) fever with cough and/or myalgias and/or rhinorrhea (i.e., based on only positive odds ratios among symptoms); (2) fever with cough (i.e., based on highest positive odds ratios among symptoms 
Detection of BTAs is critical to the rapid initiation of treatment, infection control measures and public health emergency response plans. Due to the rarity of BTAs, standard methodology for developing syndrome definitions and measuring their validity is lacking.
BTA profile development consisted of the following steps.
Step 1: Literature scans for BTAs: articles found in a literature review on BTAs that met predefined criteria were reviewed by multiple researchers to independently extract BTA-related data including physical and clinical symptoms, epidemiology, incubation period, laboratory findings, radiological findings and diagnosis (confirmed, probable or suspected).
Step 2: Data analysis and transformation: articles were randomly divided, taking into account reported diagnosis and sample size, to generate detection (75% of articles), and testing (25% of articles) profiles. Statistical approaches such as combining frequencies, weighted mean, pooled variance, min of min and max of max were utilized for combining the data from articles to generate the profiles.
Step 3: Missing data analysis: based on generated statistical and clinical judgment, specific reasonable assumptions about the missing values for each element (i.e., always reported, never reported, representative and conditionally independent) were applied to the profile. Imputed case analysis (ICA) strategies (1) used these data assumptions to fill in missing data in each metaanalysis of the summary data.
Step 4: Translation: the generated profiles and synthetic positive BTA cases were reviewed (via clinical filters and physician reviews) and programmed into GUARDIAN.
Step 5: Prior probability determination: using archived, historical patient data, the probabilities associated with each element of the profile were determined for the general (non-BTA) patient population.
Step 6: Validation and testing: multiple mutually exclusive samples of ED cases along with synthetic/real positive BTA cases were utilized to perform 10-fold cross-validation as well as testing to generate statistical measures such as positive predicted value, negative predicted value, sensitivity, specificity, accuracy and receiver operating characteristic curve (ROC) for each BTA.
To demonstrate the applicability and usability of BTA methodology, severe acute respiratory syndrome (SARS) was chosen since differentiating SARS symptoms from regular influenza is difficult and presents challenges for even robust surveillance systems.
Literature scan yielded 34 articles with 4265 cases and 90 unique signs, symptoms and confirmatory features (frequency data068 and continuous data022) for SARS. After combining the data and assigning the assumptions, there were 18 representative, 63 conditionally independent and 9 confirmatory features. Applying the BTA methodology for SARS, the positive predicted value, negative predicted value, sensitivity, specificity and accuracy based on 10-fold cross-validation were 55.7%, 94.6%, 74.8%, 88.1% and 85.8%, respectively. An ROC curve analysis revealed an area under the curve of 0.929. The main features contributing toward identifying the positive SARS cases were fever, chills/rigors, nonproductive cough, fatigue/ malaise/lethargy and myalgias. The identified features were in agreement with clinicians' judgment.
The GUARDIAN BTA profile development methodology provides a sound approach for creating disease profiles and a robust validation process even in a BTA (e.g., SARS) that may closely resemble regularly occurring diseases (e.g., influenza). The BTA profile development methodology has been successfully applied to other BTAs such as botulism, brucellosis and West Nile virus, with high sensitivity and specificity.
Biological threat agents; real-time surveillance; surveillance methodology 
In the summer of 2001, New Jersey (NJ) was in the process of developing surveillance activities for bioterrorism. On September 11, 2001, the United States suffered a major terrorist attack. Approximately a month later, anthrax-laced letters were processed through a NJ Postal Distribution Center (PDC). As a result of these events, the state instituted simplistic surveillance activities in emergency departments (EDs). Over time, this initial system has developed into a broader, more streamlined approach to surveillance that now includes syndromic data, e.g., Influenza-like illness (ILI), as well as the use of technology (automated surveys, real-time data connections and alert analysis), to achieve surveillance goals and provide daily information to public health partners in local health departments and DHSS response colleagues.
Daily response rates over time were analyzed to determine whether enhancements to surveillance produced any improvement in participation by EDs. During the timeframe used for the study, the total number of EDs varied due to facility closures and reorganizations and, therefore, daily response was measured by using the percentage of facilities responding each day versus the actual number. 
With each implementation of a new form of data collection and more advanced analysis, the response rate increased (see Fig. 1 ). In addition, the time involved for surveillance activities decreased for DHSS staff since increased automation led to fewer errors and a reduced need for follow up.
As automation in surveillance activities has increased, participation rates of facilities improved as well. Hospital staff became more engaged when there was a more defined purpose to reporting ED visits and admissions (e.g., The Republican National Convention and the H1N1 Novel Influenza A outbreak). Based on the improvements observed, the state is undertaking a project to move all NJ EDs into a real-time, syndromic surveillance system. This implementation is expected to further enhance data reporting and increase response rates beyond the current 86.4%. 
We used Amazon's EC2 (Elastic Compute Cloud) services to experiment with cloud computing for syndromic surveillance. We applied two cloud service models: Infrastructure as a Service (IaaS) and Software as a Service (SaaS). Our first goal was to apply cloud computing technologies in order to reduce computational time needed for syndromic analyses. Scan statistics, due to their reliance on Monte Carlo simulation to find confidence levels, are particularly well suited to being improved by parallel computation. We used the R package DCluster to calculate scan statistics and combined that with the SNOW (Simple Network of Workstations) package. We also experimented with using cloud computing to parallelize the AMOEBA approach to cluster detection.
Our second goal was to determine the practicality of easing the barrier of software complexity. To that end, we created software packages that include data import, analysis and visual presentation of results and released them as freely available virtual machines, or images, for the public to use. The GeoViz Toolkit was one of the software packages delivered in this manner (Fig. 1) .
We found that both Infrastructure as a Service and Software as a Service cloud computing service models can help reduce barriers to effective use of sydromic surveillance methods. Easy provision of many computers allowed us to speed up the computational times by an order of magnitude. The creation of integrated software services to perform disease surveillance is the easiest way to deliver complex functionality.
We conclude that, in the future, cloud computing can and should play a more prominent role in disease surveillance.
Cloud computing; scan statistics; informatics Introduction Emergency management during a disaster entails innumerable challenges. Each disaster uniquely shapes the types and timing of information needed both to manage the disaster and to measure the impact on available resources, the environment and community systems. Traditional public health surveillance methods typically preclude providing a real-time, comprehensive estimate of public health impacts related to the disaster while the disaster is unfolding. Traditional methods can also be resource intensive and costly, require active cooperation of medical systems involved in a disaster response and are often conducted postdisaster.
Syndromic surveillance of emergency department (ED) chief complaints and over-the-counter (OTC) medication sales was reinstituted in the Austin area in the fall of 2010. In 2011, the Austin area was hit with three natural disasters: a winter ice storm; a summer of extreme heat/extended drought; and a week of significant wildfires. Each disaster varied greatly in type, size, intensity and duration. The Austin/Travis County Health and Human Services Department (A/TCHHSD), in partnership with Austin/Travis County EMS (ATCEMS), was able for the first time to provide near-real time data to emergency managers on the potential health impact during each of the 2011 disasters using the syndromic and EMS electronic data systems. The data were used to provide situational awareness and guide selected response actions during the course of the disaster, as well as document potential areas for future mitigation efforts.
A/TCHHSD uses two syndromic surveillance systems: (1) Realtime Outbreak and Disease Surveillance (RODS) system* utilizes chief complaint data from emergency department visits in 14 Austin Metro area hospitals; and (2) National Retail Data Monitor (NRDM)* utilizes OTC medications sales data. ATCEMS has an automated system to track the types of calls to EMS and transport to area hospitals. All three systems also provide data on patient age, sex, home zip code and receiving hospital. Information on the use of syndromic surveillance and EMS systems for each natural disaster (ice, extreme heat and fire) will be presented. Each case study will provide information on: (1) salient features of the natural disaster; (2) rationale for the type(s) of surveillance resources employed; (3) data analysis; (4) results; (5) data dissemination; (6) advantages and limitations; (7) lessons learned; and (8) process improvements.
Ice storm: Piloted the use of 'keyword' surveillance in our jurisdiction. Local hospitals were asked to include the word 'weather' in the chief complaint of patients presenting to the ED. The major trauma hospital in the Austin area implemented keyword surveillance within 4 hours of the request. Keyword surveillance provided insight into the impact of injuries during the ice storm. This approach was essentially resource neutral, both for the health department and the hospitals. The RODS system was also used to track chief complaints of hyperthermia and exposure. Data were reported twice a day during the ice event.
Drought/heat: This is an ongoing surveillance effort. We will present RODS data and EMS data from May through September 2011 which describe the pattern of heat-related illness over time. The pattern of heat-related illness diverged over time from the heat index. These data were reported to emergency management daily during the most extreme heat index days and weekly for the rest of the summer.
Wildfires/smoke incident: We were asked to provide an estimate of the impact of air quality from the wildfires. We examined ED chief complaint data, OTC medication sales and EMS data. These data are still being analyzed.
Syndromic surveillance/EMS data systems are extremely valuable in providing situational awareness during an emergency incident. Use of electronic data systems are essentially resource neutral and can provide near real-time data. These systems do not replace the need for traditional disease/injury surveillance but can help fill a need during a crisis. Response partners must be educated as to the limitations of the systems. 
To demonstrate how event-based biosurveillance, using direct and indirect indications and warning (I&W) of disease, provides early warning and situational awareness of the emergence of infectious diseases that have the potential to cause social disruption and negatively impact public health infrastructure, trade, and the economy (1). Specifically, tracking of I&W during the 2011 enterohemorrhagic Escherichia coli (EHEC) O104:H4 outbreak in Germany and Europe was selected to illustrate this methodology.
Argus is an event-based, multilingual surveillance system, which captures and analyzes information from publicly available Internet media. Argus produces reports that summarize and contextualize I&W of emerging threats and makes these reports available to the system's users (1) . The significance of the EHEC outbreak analyzed here lies primarily in the fact that it raised epidemiological questions and public health infrastructure concerns that have yet to be resolved, and required the development of new resources for detecting and responding to newly emerging epidemics (2) .
Argus reports meeting the following inclusion criteria were reviewed: (1) entities: E. coli and food/crop contamination, (2) location: Germany and the European Union (EU), (3) time period: MayÁJuly 2011. The reports were reviewed for relevant I&W with the primary goal of identifying factors that inhibited effective control of the outbreak and resulted in public health infrastructure strain. Geospatial visualizations of the Argus outbreak reports were created as the event unfolded.
On May 23, a surge in EHEC infections was reported at hospitals mainly in northern Germany; the outbreak was unusual in that it caused atypically severe symptoms in adult females. By May 26, state health authorities had identified over 600 EHEC cases, including 214 severe cases with hemolytic uremic syndrome (HUS), and confirmed the causative agent as a highly virulent HUS-associated EHEC 41 strain belonging to serotype O104:H4. Faced with a rapidly growing number of cases, health authorities notified the EU of a potential public health emergency of international concern and implemented new surveillance systems (2) . Media reports suggested that the public health infrastructure was strained to a breaking point, as hospitals in northern Germany issued appeals for blood donations and transferred cases to hospitals in neighboring states. These problems were compounded by the lack of an effective HUS treatment, causing health officials to resort to an emergency experimental treatment instead. As the outbreak continued to spread, up to 130 cases primarily associated with travel were detected in 13 other European countries (3). The EU responded by implementing a new case definition twice over the course of 1 month, to allow for effective surveillance and treatment of cases (3, 4) . By June 29, an investigation launched by the European Food Safety Agency (EFSA) had determined that contaminated fenugreek seeds imported from Egypt were the most probable source of the outbreak (3, 5) . Previous efforts to locate the source of infection had failed, resulting in strained trade relations and major economic losses among EU member states (3). On July 26, Germany's Robert Koch Institute (RKI) declared the outbreak over and reported a cumulative total of 4321 EHEC cases, including 852 HUS cases and 52 fatalities (6) .
This study highlights the challenges faced in providing a timely response to a rapidly spreading infectious disease outbreak and the role that event-based biosurveillance can play in quickly identifying areas for public health intervention. Argus reporting identified that the EHEC outbreak fundamentally challenged the public health system in Germany, by exposing deficiencies in infectious disease surveillance. More importantly, it evidenced that even a strong public health system must be able to adapt rapidly to challenges posed by the changing epidemiology of infectious diseases (2) . To that end, an interdisciplinary approach to event-based biosurveillance that allows for the timely detection of outbreaks and astute analysis of pertinent I&W is of paramount importance.
Event-based biosurveillance; infectious disease; social disruption; E. coli O104:H4; food contamination
In 2010, as rules for the Centers for Medicaid and Medicare Electronic Health Record (EHR) Incentive Programs (Meaningful Use) (1) were finalized, ISDS became aware of a trend toward new EHR systems capturing or sending emergency department (ED) chief complaint (CC) data as structured variables without including the free-text. This perceived shift in technology was occurring in the absence of consensus-based technical requirements for syndromic surveillance and survey data on the value of free-text CC to public health practice.
On January 31, 2011, ISDS, in collaboration with the CDC BioSense Program, recommended a core set of data for public health syndromic surveillance (PHSS) to support public health's participation in Meaningful Use. This study was conducted to better support a requirement for ED CC as free-text, by investigating the relationship between the unstructured, freetext form of CC data and its usefulness in public health practice.
PHSS analysts from 40 public health agencies that contribute syndromic data to the ISDS Distribute project were asked to take an online survey.
The survey, developed in consultation with state-and locallevel syndromic surveillance experts and implemented using SurveyMonkey † , consisted of 15 questions, which were crafted to obtain data in four areas: (1) basic system design and coverage;
(2) CC data formatting and classification practices; (3) CC data use; and (4) impact of codifying CC on PHSS capabilities.
Participants had 2 weeks to complete the survey. ISDS staff contacted nonrespondents to encourage participation 7 and 3 days before the end of the survey period. Qualitative survey data from open-ended questions were reviewed and grouped into themes or categories.
PHSS epidemiologists or analysts from 87.5% (35 out of 40) of the Distribute-contributing health authorities completed the survey. Within the respondent group, 9 cover local jurisdictions, 25 state jurisdictions, and one was from CDC BioSense. Combined, the 35 agencies captured EHR data from 1344 ED.
Survey results revealed that 97% of participants receive ED patient CC data in free-text (Fig. 1) . ED triage staff presumably capture these data in an EHR, based on a patient's presenting condition as an open-ended, unstructured memo. Some survey participants also reported receiving ED CC in coded formats, either as ICD-9 codes (34%) or as text from a drop-down menu (20%).
A majority of survey respondents (74%) reported having used free-text CC to monitor public health in over 17 different emergencies over the past 2-3 years. Most frequently, free-text CC was used to monitor the impact of H1N1, heat waves, infectious disease outbreaks, and winter storms.
Through a national survey of PHSS epidemiologists, ISDS identified that public health agencies benefit from free-text CC data, and this format needs to be maintained. ISDS also learned that as newly certified EHR systems are switching CC from freetext to a structured format, the advantages for making this transition are not fully known to public health practitioners.
Syndromic surveillance; Meaningful Use; free-text; EHR Introduction NC DETECT provides near-real-time statewide surveillance capacity to local, regional and state-level users across NC with twice daily data feeds from 117 (99%) emergency departments (EDs), hourly updates from the statewide poison center and daily feeds from statewide EMS runs and select urgent care centers. The NC DETECT Web application provides access to aggregate and line listing analyses customized to users' respective jurisdictions. The most active users are state-level epidemiologists (DPH) and hospital-based public health epidemiologists (PHEs). The use of NC DETECT is included in PHE job descriptions, and functionality has been developed specifically to meet their surveillance needs, including data entry of aggregated laboratory results for flu and respiratory panels. Interviews of local health department (LHD) users completed as part of an evaluation project have suggested that functionality specifically tailored to LHDs may increase their use of the NC DETECT Web application (1). As of June 2011, there were 139 LHD users with active NC DETECT accounts (out of 384 total users with active accounts). Methods Initial information-gathering sessions were held with DPH stakeholders on April 7 and 12, 2011. Mock-ups based on these meetings were discussed with LHD focus groups on April 13 and 14 via Web conference. A later version of the prototype was shown in person at a health department epidemiology team meeting on May 13, and feedback from that meeting was incorporated into the initial release of the dashboards, which were made available to LHD users on June 14, 2011. On June 21, 2011, drill down functionality was added to the dashboards, and on June 30, 2011, the dashboards were made available to DPH users. The dashboards were developed in Java to integrate with our existing Web application using Java and jQuery.
The dashboards are organized by tabs; current tabs include Overview (Fig. 1) , Hot Topics, Heat, Animals/Vectors, Hurricane, Foodborne, PHE Weekly Report summary and users comments on signals and events investigations. The tabs will change in subject in the fall and winter months, e.g., including a Flu tab. The average number of LHD logins into the NC DETECT Web application has not increased significantly since the release of the dashboards (Fig. 2) .
Average LHD logins per week for June and July 2011 (n 015) are significantly lower than for PHEs (45 per week on average for 12 total PHE level users). Dashboard interfaces may be particularly beneficial and used more during large scale events of public health significance monitoring, e.g., the 2012 Democratic National Convention in Charlotte, NC. We will continue to work with LHD users to design easy-to-use reports to meet their surveillance needs.
Dashboards; all-hazards surveillance; user interface design 
To explore the possibility of using statistical methods to detect Shigella outbreaks, assess the effectiveness of the methods to signal real outbreaks, provide manageable information for follow-up activities and avoid unnecessary surveillance work.
Shigella remains highly infectious in the United States, and rapid detection of Shigella outbreaks is crucial for disease control and timely public health actions. The New York State Department of Health (NYSDOH) implemented a Communicable Disease Electronic Surveillance System (CDESS) for local health departments (LHDs) to collect clinical and laboratory testing information and supplement epidemiologic information for the patients from New York State, excluding New York City, with infectious diseases. The CDESS includes reported cases that are involved in outbreaks and which constituted the base for identifying any outbreak. The selection of a fitted outbreak detection method would play a critical role in enhancing disease surveillance.
Weekly case numbers were obtained from CDESS and counted patients with Shigella who had diagnosis or specimen collection dated between January 1, 2006, and December 31, 2010. Six statistical models were applied to the weekly case numbers in generating signals to identify outbreaks, and signals were compared to the actual outbreak to evaluate their detection powers. Outbreak-related cases from CDESS were removed for the modeling purpose except for the cumulative sum-related methods, which used all cases. The sensitivity (SE), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) were calculated to evaluate the performance of each method.
General Linear method (GL) Yt0a'abi ct; i; i01 . . . 52; where Yt is the expected number of cases in week t, ct,i is the dummy value which equals 1 if the week of the year for Yt is the same as i, else it equals 0.
It applies the same statistical procedure as GL except for the assumption that the case numbers follow Poisson distribution.
Yt0a'bt'c1sin(2pt=52)'c2sin(4pt=52)'c3sin(6pt=52)' d1cos(2pt=52)'d2cos(4pt=52)'d3cos(6pt=52)'at; where Yt is the expected number of cases in week t, and at is the random error.
A signal was generated when the case number exceeded the 95% confidence limit for the prediction value from the above three methods.
A signal was created when the case number exceeded the baseline mean, i.e., mean of previous two weeks, plus three standard deviations.
Historical Limit method (HL) Similar procedures applied as CuSum except that data for the prior 8 weeks of the last year were used as the baseline.
Negative Binomial CuSum method (NBC) Prior 8 weeks of data excluding current week were used to calculate the baseline mean and variance, which derived the NBC parameter. A signal occurred when the parameter exceeded the threshold value.
For the purpose of evaluations, an outbreak week was defined as any week that had over two outbreak-related cases during the study period.
Fourteen outbreak weeks were identified to evaluate the detection ability of the six methods. The The SPs did not vary much across six methods while the SE of the PO method was higher than the rest. The PPV ranged from 11% to 63%, and the NPV did not vary greatly. The total numbers of signals generated from the PO and NBC methods were higher than the rest.
Among the above six methods, the PO method had the ability to detect a high percentage of true outbreaks. However, the high number of signals and the relatively low PPV indicated the limitations of the PO method. Other information such as geographical clusters should be considered in determining further public health investigations as needed.
There is national recognition of the need for cross-programmatic data as well as system coordination and integration for surveillance, prevention, response and control implementation. To accomplish this, public health must develop an informatics competency and create an achievable roadmap, supported by performance measures, for the future. Within the New York State Department of Health, Office of Public Health (OPH), a cross-organizational and cross-functional Public Health Information Management Workgroup (PHIM-WG) was formed to align public health information and technology goals, objectives, strategies and resources across OPH. In June 2011, the OPH Performance Management Initiative, funded by the Centers for Disease Control and Prevention's National Public Health Improvement Initiative, offered strategic planning workshops, funded by the Association for State and Territorial Health Officials (ASTHO), to PHIM-WG.
Senior management of the major programmatic areas within OPH including, Communicable and Chronic Disease, Family Health, HIV/AIDS, Environmental Health and Wadsworth Center Laboratory, identified representatives to participate in PHIM-WG. Informatics, information technology (IT) and information management (IM) literature was reviewed to determine a framework upon which to build the strategy (1). Words and concepts with multiple interpretations were identified and agreed-upon definitions were used for planning discussion. An assessment of the as-is and desired state formed the basis of the strategic objectives and destinations. A community-balanced scorecard (CBSC) approach (2), grounded in the Public Health Accreditation Board Essential Services (PHAB-ES), is being used to guide the development of a strategic plan, to include performance metrics.
PHIM-WG includes physician, epidemiology, program management, policy and planning, IT, quality improvement and project management representatives. IM, composed of the integration of program, processes, policy and technology, was the selected framework. An initial informatics lexicon was developed. Using CBSC, identified strategic destinations were aligned with PHAB-ES objectives, which were then adapted and aligned with the IM framework. An IM vision and strategy map, including strategic objectives and destinations, were produced. Public health IM desired state, objectives, and activities were linked to the PHAB-ES within four major community perspectives; health status, implementation, process and learning, and assets. PHIM-WG is working to produce a more-fully developed strategy and implementation plan, including engaging internal and external partners, defining associated performance metrics to measure progress to the desired state and aligning with NYSDOH strategic planning efforts.
As a cornerstone of public health, IT/IM should be and can be aligned with or directly linked to the public health essential services. The development and promotion of a common informatics lexicon and workforce engagement and training are critical to public health, especially for advancing data analysis, use, and dissemination capabilities. PHAB-ES-based IM strategic planning can be an essential first step for community collaborators to define the vision, objectives and measurable activities to advance the technology, research and practice of public health surveillance.
Informatics; information management; strategic planning; public health; cross program
Beginning on March 13, 2011, ACDC experienced an unusual increase in reported bacterial meningitis cases in Los Angeles (LA) County. Early in the investigation, there were few epidemiological links between the cases. Three cases were homeless; two resided at the same Skid Row shelter in downtown LA. ACDC assessed its syndromic surveillance databases to help gauge the scope of the outbreak and detect potentially overlooked cases.
Electronic ED chief complaints (CC) from January 1, 2011, to April 10, 2011. were queried from eight EDs within an 11-mile radius of Skid Row. Only visitors with resident zip codes that corresponded to Skid Row or that were blank to account for homelessness were included. Visits were reviewed if CC included keywords based on common meningitis symptoms and also those of confirmed cases.
Coroner deaths from the same time period were reviewed for location of death and homeless status. Real-time LA City emergency dispatch calls were also reviewed if they were made from the same homeless shelter in which the two confirmed cases resided.
Two hundred and thirty-eight ED visits met the meningitis syndrome definition; however, there was no substantial increase ( Fig. 1 ). Within the same zip code catchment area, there were no ED visitors with mention of homelessness or shelter residence in their CC.
There was no overall increase in the total number of homeless coroner deaths. Two of 45 deaths took place in shelters*one death in January from 'cardiomyopathy' that occurred at the homeless shelter of interest, and another nonspecific shelter death in March from 'strep pneumonia'.
Forty-one 911 ambulance calls were made from the homeless shelter associated with the confirmed meningitis cases. While there was no overall increase in call volume, one call matched a confirmed case fatality.
One limitation of ED data in this investigation is that they do not contain patient resident addresses, making restriction to the homeless or shelter residents impossible. While no additional cases were found, the absence of an increase provides validation that a large countywide outbreak had not occurred.
Both coroner and 911 call databases were more flexible than ED data, containing fields facilitating focused queries on the key epidemiological links of homelessness and shelter residence. Coroner data are limited, however, in that there is a 2-day reporting lag. While many homeless deaths were found, few had precisely reported death locations.
Many 911 calls were reported from the shelter of interest. While medical information was vague, additional details enabled ACDC to match one call to a confirmed case. Follow-up for diagnosis information is possible when ED transportation information is present. Precise caller locations make 911 calls particularly useful for investigations with a strong emphasis on location such as point source outbreaks. Querying preestablished ED visit, coroner death, and 911 call feeds can provide a relatively quick supplement to traditional outbreak investigations.
Coroner; 911 call; dispatch; emergency department; outbreak 
Chronic diseases are the leading causes of mortality and morbidity for Americans but public health surveillance for these conditions is limited. Health departments currently use telephone interviews, medical surveys and death certificates to gather information on chronic diseases but these sources are limited by cost, timeliness, limited clinical detail and/or poor population coverage. Continual and automated extraction, analysis and summarization of EHR data could advance surveillance in each of these domains.
We leveraged the Electronic medical record Support for Public Health (ESP) surveillance platform to create a chronic disease surveillance module. ESP is an open source software (esphealth.org) that reads structured EHR data, analyzes them for events of public health interest and communicates findings to public health agencies. We created algorithms to identify diabetes types using a combination of diagnosis codes, laboratory tests and medication prescriptions. We then applied these algorithms to the ESP installation in Atrius Health, a multisite, ambulatory practice with over 700,000 patients. We programmed ESP to create patient level linelists each night that detail patients' demographics (age, sex, race/ethnicity and zip code), vitals (body mass index, blood pressure and pregnancy status), key laboratories (hemoglobin A1C and cholesterol levels), diabetes type and care (medications and medical nutrition counseling). De-identified linelists are transmitted nightly to a secure website called the 'RiskScape' that automatically maps selected health indicators and stratifies results by age group, race/ethnicity, year of diagnosis and body mass index. Users can customize indicators and stratifications displayed by RiskScape.
The RiskScape presents a timely, clinically rich picture of the health of large populations using EHR data that is refreshed nightly. Examples of RiskScape views and report options are shown in Fig. 1 and 2. Fig. 1 maps the rate of nutrition referrals by zip code amongst women with gestational diabetes. Fig. 2 stratifies these results by age and race/ethnicity within the greater Boston area and compares them to statewide averages.
Automated analysis and presentation of EHR data can provide a rich, timely picture of chronic disease prevalence, care and complications for large populations. This technology has a great potential to advance public health practice by highlighting specific populations with gaps in care that merit targeted interventions. 
Much progress has been made on the development of novel systems for influenza surveillance (1, 2) or explored the choices of algorithms for detecting the start of a peak season. The use of multiple streams of surveillance data has been shown to improve performance (3) but few studies have explored its use in situational awareness to quantify level or trend of disease activity. In this study, we propose a multivariate statistical approach, which describes overall influenza activity and handles interrupted or drop-in surveillance systems.
A multivariate dynamic linear time series model was fitted to data on influenza-like illness (ILI) rates among networks of public and private general practitioners and school absenteeism rates, plus drop-in fever count data from designated flu clinics (DFC) that were created during the pandemic. The data streams were assumed to follow an underlying latent process with local linear trend. The estimated level and trend of the latent process reflect the magnitude and direction of influenza activity, which are then combined to infer an overall influenza activity index. Correlations between the estimated influenza level from the model and laboratory isolation rate were calculated to assess its performance before and during the 2009 pandemic.
ILI rates from public outpatient clinics and the estimated influenza level from the multivariate model had the highest correlations with laboratory isolation data before the 2009 pandemic (r00.57 and 0.58, respectively) but the former was interrupted during the pandemic period due to activation of the DFC. The estimated influenza level from the multivariate model captured the influenza level well during the pandemic period (r00.76), significantly better than the best surveillance data in the same period (p-value 00.03). The inferred influenza activity index is able to reflect the influenza activity (Fig. 1) .
The use of a multivariate method to integrate information from multiple sources of influenza surveillance data can improve situational awareness of influenza activity, with the advantage of maintaining performance when data streams are interrupted or supplemented by additional systems during certain critical periods such as the 2009 influenza pandemic.
Keywords Sentinel surveillance; influenza; multivariate analysis; pandemic Introduction NPDS is a national database of detailed information collected from each call, uploaded in near real-time, from the 57 participating regional poison centers (PCs) located across the United States. NPDS is owned and operated by the American Association of Poison Control Centers (AAPCC). Since 2001, scientists from the Centers for Disease Control and Prevention collaborated with AAPCC to use NPDS for surveillance of chemical, poison and radiological exposures. In March 2011, a 9.0 magnitude earthquake and tsunami damaged the reactors at the Fukushima Daiichi nuclear power plant in Japan, causing a radiological incident classified as a 'major accident' according to the International Nuclear Event Scale. The incident resulted in the release of radioactive iodine (IÁ131) into the global environment, which was detected in precipitation in parts of the United States. While no adverse health effects were expected, concerned citizens contacted public health officials at the local, state and federal levels. Many started to acquire and use potassium iodide (KI) and other iodide-containing products intended for thyroid protection from IÁ131, even though this was not a public health recommendation by state and federal public health agencies. Shortly after international media coverage began, regional PCs began receiving calls regarding the Japan radiological incident. State and federal health officials were interested in identifying health communication needs and targeting risk communication messages to address radiation concerns and KI usage recommendations as part of the public health response. This was done in part through NPDS-based surveillance.
A new, unique event code was created for staff of all 57 regional PCs to use for coding calls related to this incident. This enabled CDC and AAPCC to track incident-related information requests and exposure calls using NPDS. Calls involving either information requests or reported exposures to radiation, potassium iodide and other iodide-containing products were identified, reviewed and tabulated daily. For each exposure call, individual PCs were then contacted by AAPCC officials to obtain additional data not uploaded to NPDS. CDC epidemiologists and toxicologists reviewed these data daily using set criteria to determine if a true exposure had occurred. Aggregate NPDS data were reported daily to CDC's Emergency Operations Center leadership to enhance situational awareness.
During the time period that the CDC Emergency Operations Center (EOC) was activated for this response (March 11 to April 18), there were 404 calls nationally regarding the Japan radiological incident. Three hundred and forty (84%) were calls requesting information about KI, iodide/iodine containing products or radiation associated with the Japan radiological incident. The remaining 64 calls (16%) were potential incidentrelated exposure calls. Of these, KI (n020), other iodidecontaining products (n017) and radiation (n 015) were reported most frequently. The number of information calls peaked on March 16 (n054), and the number of exposure calls peaked on March 17 (n09). Thirty-four (53%) of exposure calls were confirmed KI and iodide/iodine containing product exposures, 23 (36%) were calls regarding incident-related exposures, which were unable to be confirmed, and 7 (11%) were determined to be nonexposures.
Collaboration between CDC, AAPCC and PC staff were crucial to surveillance efforts during the Japan radiological incident response. National surveillance using NPDS demonstrated utility for conducting near real-time human health effects and exposure surveillance associated with a known public health emergency. Surveillance efforts identified confirmed exposures to KI and iodide-containing products. The CDC used this information, along with other media sources, to identify health communication needs and implement appropriate health messaging. 
We assessed destination information from the EDN system for immigrants and refugees arriving during 2009 and 2010 with TB disease (Class A TB with waiver) or a radiographic TB without positive smear or culture for TB (Class B1), or LTBI (Class B2), or contact with a TB case. The destination information was mapped with ArcGIS software to the county level and aggregated at the national level. Data were categorized by region according to the 10 Agency for Toxic Substances and Disease Registry (ATSDR) regions (5) . Since the change of location after arrival can be entered into the EDN system by the health department, this information was assessed for the system's ability to provide secondary migration information.
The 
Over one third of immigrant and refugee arrivals with TB notifications were in the region comprising Arizona, California, Hawaii and Nevada. The increase in TB notifications in this region was attributed to those born in Vietnam. Secondary migration data were available, but the relatively early change in locations may indicate corrections to initial destination data rather than true secondary migration.
Tuberculosis; immigrants; refugees; electronic disease notification; EDN
Modern public health surveillance systems have great potential for improving public health. However, evaluating the performance of surveillance systems is challenging because examples of baseline disease distribution in the population are limited to a few years of data collection. Agent-based simulations of infectious disease transmission in highly detailed synthetic populations can provide unlimited realistic baseline data.
Dynamic social networks for the Boston area (4.1 million individuals) were constructed based on data for individuals, locations and activity patterns collected from the real world. We modeled a full season of endemic influenza-like illnesses (ILI), healthcare seeking behavior and a surveillance system for outpatient visits. The resulting in silico surveillance data contain the demographics and complete history of disease progression for all individuals in the population; those who are in a specified surveillance system create a data stream of ILI visits. Outbreaks of influenza are artificially inserted into this surveillance data. Outbreak detection using space-and-time scan statistics was used to analyze the background with and without the inserted outbreaks. The performance of the algorithm was assessed under different levels of coverage and catchment distributions. One hundred unique baseline data sets were generated. Twelve artificial outbreaks were inserted in each. Six different surveillance system designs were assessed.
We present a robust framework for using highly detailed simulations to provide the foundation for evaluating and designing a surveillance system's ability to detect outbreaks. A small demonstration study shows that detection rates varied from 17% to 80% across the different surveillance systems. Increased coverage did not linearly improve detection probability for all surveillance systems. Surveillance systems with uniform coverage of the population did not perform better than one based on a real-world system with nonuniform coverage. Higher coverage improved the timeliness of detection but, for most cases, by only 1 or 2 days on average. Additional results can be found online (http://ndssl.vbi.vt.edu/insilicoSurveillance/).
Highly detailed simulations of infectious disease transmission can be configured to represent nearly infinite scenarios, making them a powerful tool for evaluating the performance of surveillance systems and the methods used for outbreak detection. 
The 'wisdom of the crowd' or the 'crowd trial' is a process of taking into account the collective intelligence of a large population sharing experiences regarding health issues and treatments online via social media platforms [Health 2.0], generating novel data sets comprising massive unstructured user-generated content of health reports.
Unlike regulated formal postmarketing reports, the crowd trial takes place spontaneously, continuously and on a very large scale. This crowd trial provides a snapshot of health trends and has become a proxy of postmarket clinical trials of medications and other therapies.
The purpose of this case report is to demonstrate how applying an additional data source originated from e-patient reports helps support drug surveillance and pharmacovigilance processes.
Singulair (Montelukast Sodium) is a leukotriene receptor antagonist, indicated to prevent asthma attacks in adults and children. It is also used to relieve allergies in adults and children.
Singulair was approved by the FDA in February 20, 1998. In March 2008, the FDA informed healthcare professionals of investigating the possible association between Singulair usage and behavior/mood changes, suicidality and suicide.
First Life Research (FLR) identifies, analyzes, indexes and aggregates user-generated content by collecting billions of testimonials from social networks. It utilizes cutting edge technologies for massive data aggregation and applies advanced natural language processing (NLP) techniques for continuous analyses, in order to convert this unstructured data into refined information.
With the proliferation of social networks, the web has become a warehouse of patient discussions and reports, estimated at 10 billion records and growing at a rate of 40 percent per year. These reports are spread across more than 150,000 (and growing) English-language sites, forums and blogs. FLR has searched and mapped thousands of these sites and indexed hundreds of millions of posts (currently 800M) and is engaged in refining statistical methods of signal detection that enables investigation of health trends. FLR can look at large samples and discover small changes, such as drug side effects, which may not be discovered by other means for years.
In this case, FLR detected the mentioned FDA alerts and related clinical manifestations prior to the official alert by 'listening' to the 'crowd trial', in that case, the Singulair users. 
This report shows that by 'listening' into the social web, unforeseen phenomena may be revealed. Specifically, it is evident that advanced technological solutions and signal detection algorithm were able to detect neuropsychiatric events (side effects) in the case of Singulair, more than 2 years prior to any official warning by the regulator or the manufacturer.
'Crowd trial' provides a dashboard of health trends and grants feedback on medications, drug safety, side effects, interactions and drug comparisons.
The insights gained and demonstrated as aforementioned can be used to support and enable better informed decision making processes, both for patients and healthcare providers.
Drug surveillance; user generated content; crowd trial; adverse drug reaction
The emerging 'wisdom of the crowd' analytics potentially represents a new phase and eventually new tools using data evaluations based on large scale population inputs, and it will benefit greatly all public health environment. 
The American Recovery and Reinvestment Act (ARRA) initiated a broad range of national implementation activities. In order to support the critical activities of meaningful use (MU), ONC established the S&I Framework. In the beginning of 2011, the Laboratory Reporting Interface (LRI) Public Health (PH) Work Group (WG) was formed as a subworking group of the S&I Framework LRI activity. This LRI PH WG, besides providing PH required data elements to the LRI, assessed a need for documentation of the broad landscape of public health data exchange transactions. As a result, this WG recommended to participants and leadership of the ONC S&I that a new initiative, the ONC S&I PH-R activity should be established. In July 2011, a team of PH practitioners (co-authors of this presentation) started working on a charter and proposed deliverables for the group.
Findings by ONC S&I LRI PH WG demonstrated that there are significant gaps in development of (a) functional requirements for PH-R and (b) interoperable standards-based specifications that support PH-R electronic data exchange from clinical care to public health and within public health. In order to strengthen PH-R, the S&I PH-R activity defined the following priorities: (1) Compile the full picture of all aspects of PH reporting; (2) Review and define public health and patient safety business processes and functional requirements and develop Á HIT interoperability specifications; (3) Align public health objectives in MU Stage 1 with the needs of other public health domains and programs that were not explicitly mentioned in MU Stages 1; (4) Develop a roadmap for aligning public health, patient safety and clinical objectives in MU with regards to HIT standards, development, harmonization, testing and certification; (5) Develop a roadmap for deploying standard-based certified HIT applications in PH agencies and for patient safety reporting.
Even though practical tasks of the S&I PH-R initiative are at the initial stage, this activity will evolve into an important national forum that embraces stakeholders critical for improvement of population health tasks including system vendors, clinical care and public health professionals. Addressing gaps in interoperability of data exchange between clinical and population care should foster progress in situational awareness, PH emergency response and quality of population care.
Public health reporting; ONC S&I; informatics *Nikolay Lipskiy E-mail: dgz1@cdc.gov Introduction Using an EHR system, we tracked an outpatient population from a series of primary care providers to identify ILI as part of a multistate effort directed by the Centers for Disease Control and Prevention. From these patients, we also collected deidentified project-specific information and symptoms using an electronic template to evaluate possible differences among patient groupings as well as longitudinal population patterns.
We selected a series of providers using NYC DOHMH's EHR network, from which we could obtain practice characteristics (i.e., number of patient visits, type of practice and age distribution) and evaluation score developed to rate a practice's ability to use EHRs. We then set up an electronic template at each practice and scheduled the transmission of a report with de-identified patient characteristics and patient counts. Nasopharyngeal samples were collected from each patient presenting with ILI to test for influenza subtypes including influenza A (H1, H3 and H1N1) and influenza B by RT-PCR. Samples negative for influenza were tested for other respiratory viruses including rhinovirus, metapneumovirus (MPV), respiratory syncytial virus (RSV), parainfluenza virus (PIV) and adenovirus by RT-PCR by Luminex. We analyzed the data for completeness to evaluate the success of electronic surveillance. We also compared the data by gender, age group, symptoms as well as evaluated virus frequency over time.
Compared to paper-based records, EHR-based tracking reduced time and manpower requirements by the automation of data acquisition from each practice and improved capabilities for determining ILI incidence by reporting a patient denominator along with the number of ILI cases. Proper training and selecting the right practice played a large role in that success. Some initial challenges included providers overlooking the symptomology associated with ILI in the CDC guidelines, which led to failing to identify ILI cases and unfamiliarity with the electronic template. This was especially an issue in a larger practice that had a large number of rotating staff.
The results of PCR testing for influenza subtypes evolved from almost exclusively H1N1 in 2009Á2010 to the cocirculation of H1N1, seasonal H3 and influenza B in 2010Á2011. Luminex testing was only performed in 2010Á2011, and we found that rhinovirus and MPV were most common and were present over most of the season. Other viruses showed peaks at certain times of the year.
This project demonstrates that EHRs can improve surveillance capabilities by streamlining and standardizing reporting. This can help to establish a more sophisticated reporting tool using gold standard methods on a larger scale, which will in turn improve public health by providing information on the most common circulating virus at the time of diagnosis, and especially in the event of outbreaks such as pandemic H1N1. In addition, longer term longitudinal use of EHRs for this type of surveillance can determine whether the pattern observed one season is repeated the next.
Keywords Electronic health record system; influenza-like illness; surveillance; influenza
The status of each ICU patient is routinely monitored, and a number of vital signs are recorded at subsecond frequencies (1), which results in large amounts of data. We propose an approach to transform this stream of raw vital measurements into a sparse sequence of discrete events. Each such event represents significant departure of an observed vital sequence from the null distribution learned from reference data. Any substantial departure may be indicative of an upcoming adverse health episode. Our method searches the space of such events for correlations with near-future changes in health status. Automatically extracted events with significant correlations can be used to predict impending undesirable changes in the patient's health. The ultimate goal is to equip ICU physicians with a surveillance tool that will issue probabilistic alerts of upcoming patient status escalations in sufficient advance to take preventative actions before undesirable conditions actually set in.
To generate potentially informative events from vital signs, we first segment each data channel into sequences of k consecutive measurements. We then perform Fourier transformation to obtain spectral profiles of each segment of raw signal. Multiple spectral profiles, extracted from periods of observation that are considered medically benign, are then assembled to form a kdimensional flat table. We apply principal component analysis to this, and the top p components are considered further. These p components form a null space model of the expected normal behavior of the given vital sign. We build one null space model for each channel separately; this concludes the learning stage of the process.
Each newly observed set of k consecutive measurements is then processed through Fourier transform and projected onto the p principal components of the corresponding null space models. Over time of observation, these projections produce p time series per measurement channel. We apply a cumulative sum (CuSum) control chart to each of these time series and mark the time stamps at which CuSum alerts are raised. These moments correspond to circumstances in which the observed spectral decomposition of a vital sign does not match what is expected. We consider each such event as potentially informative of near-future deteriorations in the patient's health status. We quantify the predictive utility of each type of these automatically extracted events using training data, which contain actual health alerts, in addition to the vital signs data. To accomplish the task, we perform an exhaustive search across all pairs of CuSum event types (inputs) and alert types (outputs) and identify pairs with high values of the lift statistic (2). Input-output pairs with lifts significantly greater than 1.0 can be expected to enable prediction of health status alerts. Fig. 1 depicts an example result obtained with the presented method. The CuSum Events (green spikes) obtained for the 9th principal component of Modified Chest Lead 1 (MCL1) signal, and the alerts (red spikes) are critical apnea conditions. We can see that, for this patient, the CuSum events most of the time precede apnea alerts, and they can potentially be used to predict an upcoming apneas.
We have outlined a method of processing vitals collected routinely at the bed side of ICU patients. It identifies signals that can be predictive of upcoming adverse health events.
Critical care; event detection; data mining 
Influenza is a recurrent viral disease that requires timely and accurate detection. The use of Twitter as a source for biosurveillance has been shown useful (1). However, these efforts target messages in English, omitting from surveillance the part of users that speaks other languages, such as Spanish.
We implemented a system that builds upon existing technologies and services. The open source platform Ushahidi (2) was used to automatically search for content. An initial query report was generated from Twitter, includes username, content and timestamp. The city of each user was extracted from their profile and a query to GoogleMaps gave us the coordinates. At the end, this new information was uploaded to Ushahidi. We used the keyword 'gripa' (Spanish for flu) and scheduled hourly updates of the search.
The prototype website operated for a pilot period of 1 month starting April 11h, 2011. A total of 473 unique occurrences worldwide were captured, of which 29% are located in Mexico (138/478) and 52% in Colombia (244/473).
We observed a higher number of incidences in Colombia relative to Mexico (Fig. 1a) . When comparing these findings with the data on the reported cases of influenza (Fig. 1b) from the World Health Organization Flunet Biosurveillance (3), the results were consistent.
Our approach has promising potential for timely detection of ILI-related incidences in the areas previously underrepresented. Future work is to include different linguistic and contextual representations.
Biosurveillance; influenza; twitter; open source; spanish A demonstration of meaningfully using the ISDSrecommended data elements Introduction National Health IT Initiatives are helping to advance the state of automated disease surveillance through incentives to healthcare facilities to implement electronic medical records and provide data to health departments and use collaborative systems to enhance quality of care and patient safety. While the emergence of a standard for the transfer of surveillance data is urgently needed, migrating from the current practice to a future standard can be a source of frustration.
This project will investigate tools that can be used to support ingestion and translation of public health meaningful use data in the HL7 formats. Open source tools, such as Mirth, have been identified as early candidates to support this function. After the necessary translations have been made, this project will investigate transfer methods to move the meaningful use data from a public health department to a cloud environment. With data available in the cloud, the project will then investigate methods for putting the ESSENCE system in a cloud environment as well. This will provide the collaborative team a platform to evaluate the utility of both the meaningful use data and potentially the value of having regional and national data sharing aspects available to the public health users. Finally, the team will determine the scalability and performance of a cloud environment for disseminating these tools to other jurisdictions across the country.
Early research for this project has already shown the need to redesign aspects of the ESSENCE system to support the additional meaningful use data fields. These changes involved modifications to the database design and the utilization of a more flexible configuration system. We fully expect additional modifications to be made to better support the cloud environment. These findings and the results of the public health evaluation of the system will be presented.
Public health departments will soon be flooded with mountains of new data. Having tools that can translate, transfer and utilize these new data sets effectively will be necessary. This collaborative team will research and put into practice solutions that can be used throughout the country.
Electronic medical records for public health; meaningful use; interoperability; cloud Introduction Domains go through phases of existence, and the electronic disease surveillance domain is no different. This domain has gone from an experimental phase, where initial prototyping and research tried to define what was possible, to a utility phase where the focus was on determining what tools and data were solving problems for users, to an integration phase where disparate systems that solve individual problems are tied together to solve larger, more complex problems or solve existing problems more efficiently. With the integration phase comes the desire to standardize on many aspects of the problem across these tools, data sets and organizations. This desire to standardize is based on the assumption that if all parties are using similar language or technology, then it will be easier for users and developers to move them from one place to another.
Normally the challenge to the domain is deciding on a vocabulary or technology that allows seamless transitions between all involved. The disease surveillance domain has accomplished this by trying to use some existing standards, such as Health Level 7 (HL7), and trying to develop some of their own, such as chief complaint-based syndrome definitions. However, the standards that are commonly discussed in this domain are easily misunderstood. These misunderstandings are predominantly a communication and/or educational issue, but they do cause problems in the disease surveillance domain. With the increased use of these standards due to meaningful use initiatives, these problems will continue to grow and be repeated without improved understanding and better communication about standards.
After reviewing presentations and participating in many discussions at conferences and with public health officials, a number of topics were identified that many believe use or are standards. These topics included HL7, syndrome definitions, analytical algorithms definitions and the definition of what is or is not a disease surveillance system. Next, the common understandings of each were compiled and compared with actual definitions and real world experiences from users of the standards. From this, a list of misunderstandings or poorly communicated aspects of each topic was derived.
The results of this process have pointed out a number of inconsistencies with general assumed knowledge and actual truth related to many standards. The HL7 standard is just one example of a standard that is misunderstood in many aspects. Many believe that HL7 is a transport protocol, others believe that is a file format, others believe that it defines specific locations for data elements and still others believe that HL7 'set the language, structure and data types required for seamless integration from one system to another' (1). Each of these beliefs has nuggets of truth in them but do not explain the full story of HL7. Those that believe an HL7 message from one hospital can be fully read and understood in the exact same way as a second hospital may also be mistaken. Even though this is the hope of a standard, to have a standards-based tool that can be used over and over in different situations, real world experience tells us a different story about this so-called standard. Similarly, each topic has beliefs that are partially true, but by not understanding the whole truth, the standards can lead to complications.
Standards are highly beneficial to a domain. They provide efficiency in tool development and promote interoperability between organizations. Sadly, fully understanding a standard can sometimes be difficult, and misunderstandings can allow decisions to be made on untrue assumptions about a standard. The word standard has a meaning attached to it that can easily confuse someone into believing a capability exists that actually does not. Through improved education and communication, we can benefit from these standards without getting caught in their traps.
Standards; HL7; syndrome definition; detector interfaces; system definition
In development for over 14 years, ESSENCE is a disease surveillance system utilized by public health stakeholders at city, county, state, regional, national and global levels. The system was developed by a team from the Johns Hopkins University Applied Physics Laboratory (JHU/APL) with substantial collaborations with the U.S. Department of Defense Global Emerging Infections Surveillance and Response System (DoD GEIS), U.S. Department of Veterans Affairs (VA) and numerous public health departments. This team encompassed a broad range of individuals with backgrounds in epidemiology, mathematics, computer science, statistics, engineering and medicine with significant and constant influence from many public health collaborators.
We created a timeline of events, such as a particular partner's need (Florida Department of Health's desire to detect outbreaks based on patient time of arrival) or a public health outbreak (SARS) and correlated each one with design and architecture decisions that influenced ESSENCE. We used these events to describe the epidemiology, technology, analytical, administrative, political, legal and monetary factors that were considered at each point. Looking historically and critically at each decision point, we analyzed the benefits and costs of each decision. These benefits and costs were described from many different points of view, including those of the developer, user, administrator and others.
After walking through the historical timeline, we described the current architecture and feature set of ESSENCE. We also were able to point out the unique features between different instances of ESSENCE.
Based on user feedback, understanding outside influences and internal research, the ESSENCE team is always looking to improve the system. Part of this presentation will be to describe the future plans for the ESSENCE system from both architecture and feature stand points.
Public health user needs and preferences have strongly influenced and prioritized the growth of ESSENCE, sometimes in unforeseen directions. Conversely, the evolving domain of syndromic and disease surveillance has broadened the situational awareness, perspectives and sometimes the responsibilities of public health monitors. The ESSENCE system has provided those monitors with the tools to help detect and investigate public health situations in their communities.
The utility of the ESSENCE system can be traced back directly to the influence of public health users and to the design decisions of the ESSENCE team. Understanding the history of disease surveillance in this context can help clarify current situations faced by today's public health practitioners as well as prepare them for tomorrow.
Keywords ESSENCE, disease surveillance, system architecture Introduction VA ESSENCE analyzes ICD-9 diagnosis codes and demographic data from outpatient and emergency department (ED) visits using complex aberrancy-detection algorithms (1). In 2010, a new instance was stood up (VA Inpatient ESSENCE), which receives weekly feeds of inpatient data from all VA acute care hospitals starting October 1, 2009. Data include demographics, admission/ discharge data (including ICD-9 diagnosis codes), diagnosisrelated group (DRG), bedsection, procedure and surgery data.
For this demonstration, we selected one disease for which we currently perform routine outpatient/ED ESSENCE surveillance (influenza) and one HAI of interest [C. difficile infection (CDI)]. First, we queried VA Inpatient ESSENCE for hospitalizations with an influenza diagnosis code (ICD-9: 487, 488). These data were compared to CDC's AHDRA hospitalizations, a voluntarily reporting system for laboratory-confirmed influenza-associated hospitalizations. Second, we queried VA Inpatient ESSENCE for hospitalizations with the CDI diagnosis code (ICD-9: 008.45) as well as total monthly discharges. Monthly rates for CDI were then calculated per 1000 total discharges. CDI rate per 100,000 population for FY 2010 was calculated using the total enrollees in VA Health Care in FY 2010 (8.343 million) as the denominator. Previous analysis from a non-VA hospital demonstrated good correlation between the CDI code and positive toxin assay (2) .
Alerts for influenza were observed on multiple consecutive days during the fall wave of the H1N1 pandemic as well as during the peak of the 2010Á2011 influenza season. Peaks in weekly influenza hospitalizations appeared to correlate well temporally between the VA and CDC's AHDRA data (Fig. 1) . From October 1, 2009 to July 31, 2011 more than 12,500 CDI codes were identified among nearly 1.13 million hospitalizations with a calculated mean CDI rate of 11.1 per 1000 discharges (Fig. 2) . The CDI rate for FY10 was 78/100,000 population.
Inpatient data provide robust and valuable information for VA beyond what was previously available in outpatient ESSENCE data or through manual methods. Inpatient data can be monitored year-round, which provides more complete situational awareness for planning and response. Future plans include (1) developing inpatient-specific alerting algorithms, (2) establishing a single VA ESSENCE application that combines both outpatient and inpatient data and (3) imsproving timeliness of inpatient data receipt and adding additional data elements to improve system specificity. 
Of the 13 million people in Malawi (1) 85% are rural and the country has high burden of under-five morbidity and mortality due to preventable infectious diseases. Respiratory, febrile and diarrhea diseases are the top 3 morbidity and mortality illnesses in most developing countries (2) . Acute medical care has greatly improved these conditions, but widespread and uncontrolled use of antibiotics threatens to reverse gains achieved so far. Drug sensitivity tests are a prerequisite to guide prescription practices.
An evaluative study on all 28 district hospital laboratories in all regions of Malawi. The data are routine quarterly assessments covering from October 2009 to April 2011. The main focus was on performance of culture procedures, drug sensitivity testing practice, documentation and demand and use of drug sensitivity results by clinicians.
Malawi has 29 district hospital laboratories of which only 12 (41%) are currently able to perform culture procedures. Only four (14%) of the laboratories performing culture procedures are able to perform drug sensitivity cultures, which should inform prescription practices. There is lack of demand and reliance on drug sensitivity tests by the prescribing clinician. Clinicians sited the lack of laboratory capacity and also the delays that go with culturing procedures.
Inadequate laboratory performance of drug sensitivity tests coupled with syndromic clinical diagnosis are the culprits of antimicrobial resistance and treatment access in Malawi.
There is no laboratory-based data forming sensitivity profiles of most antibiotics used to treat common infectious diseases.
Malawi is one of the many low income countries that can claim no substantive laboratory-based data on antimicrobial susceptibility. Laboratory surveillance of antimicrobial resistance is a prerequisite to guide informed selection and purchase of drugs for local use based on scientific proof. This is more cost effective and may lead to modification of treatment procedures as necessary.
Drug sensitivity tests; prescription practices; clinicians; laboratory -based data.
Seasonality has a major effect on the spatial and temporal (i.e., spatiotemporal) dynamics of natural systems and their populations (1) . Although the seasonality of influenza in temperate countries is widely recognized, interregional spread of influenza in the United States has not been well characterized.
Cities Mortality Reporting System (1996Á2008) to construct weekly time series of P&I mortality for each year and Census Bureau Division. The timing of each seasonal wave was determined by identifying a significant increase and subsequent decrease in P&I mortality plus a lead-in and a lead-out week. Average time to death ([S[(t)(nt)]]/N; where N 0total P&I deaths for all weekly periods in the season, t0week of season (e.g., 1, 2, etc.), and nt 0total P&I deaths for week t) was used to determine the timing and velocity of each seasonal influenza wave. Ordinary least squares regression was used to develop trend lines and spread vectors for annual influenza epidemics in order to determine the directionality of annual influenza waves. Average time to national spread, average time to national peak P&I mortality and average P&I mortality were also determined and compared between influenza subtypes.
For the years 1972Á1988 and 1996Á2008, annual influenza epidemics needed an average of 7.9 weeks to spread across the country and lasted an average of 22 weeks. Seasons where H3N2 was the dominant influenza subtype (N013) were, on average, significantly shorter (20.3 vs. 26.7 weeks p00.0049) and spread quicker (time to death: 10.3 weeks vs. 13.8 weeks, p 00.0053) than seasons with H1N1 as the dominant subtype (N03). There was also a significant difference in the average time to national spread between H3N2-dominant seasons and H1N1-dominant seasons (6.1 vs. 13 weeks, p 00.0253) ( Table 1) . Moreover, an average seasonal traveling wave of influenza began in the East North Central region then took two routes: (1) eastward then southward along the Atlantic coast and (2) westward to the Pacific coast.
Preliminary results of this analysis suggest that certain temporal patterns of influenza seasons vary by influenza subtype. Future analyses will focus on determining the temporal characteristics for influenza seasons between 1989 and 1996 (and for seasons between 1996 and 2007, using complete NVSS mortality sets) and assessing the intercounty spread of epidemic influenza. Accurately identifying spatiotemporal patterns could improve epidemic prediction and prevention as well as aid the creation of efficient containment policies for pandemic influenza (2) . This analysis will aid public health in developing more effective and efficient strategies to decrease morbidity and mortality associated with seasonal influenza in the United States.
Pandemic preparedness; spatial dynamics; geographic synchrony 
To outline the mechanism of a pilot educational brucellosis prevention program among selected high-risk groups in an endemic region of Uzbekistan.
One goal of the Biological Threat Reduction Program (BTRP) of the US Defense Threat Reduction Agency (DTRA) is the enhancement of surveillance of especially dangerous pathogens of both humans and animals within countries of the former Soviet Union. One of the diseases of interest to the program is brucellosis, which is a life-threatening condition and constitutes a major health and economic challenge around the world. This is also true for Uzbekistan (UZ), where brucellosis is endemic in a number of regions. In the Samarqand region of UZ, for example, studies have reported a 9.3%, and 3.6% seroprevalence for humans and farm animals, respectively (1).
The lack of awareness about brucellosis in at-risk populations, shepherds, veterinarians and people who handle raw milk is believed to significantly contribute to the spread of disease from animals to humans. Here, we suggest mechanisms to evaluate awareness about the disease and the impact of an educational intervention in at-risk groups.
The intervention and two control groups will include subjects from the at-risk groups in the Samarqand region. In all three groups, the selection of study subjects will be done from nonbrucella-related visits to primary care centers by at-risk patients with no previous history of brucella. At-risk subjects within the intervention and first control group will be asked to complete a questionnaire to assess their awareness about brucellosis, specifically about its clinical presentation and risk for exposure in people. At-risk subjects in the second control group will not receive any questionnaire. The educational intervention procedures will consist of briefings to a group of healthcare professionals, delivered through BTRP regular training events, together with printed materials to be explained by the physicians to patients in the intervention group. The briefings and materials will show practical ways of preventing the spread of brucellosis targeted at common practices within the atrisk groups. The seasonality of the disease in endemic regions like Samarqand dictates that the best timing for the intervention program is in the fall (SeptÁDec), before lambing season (FebÁJun). Our measurable outcome is the number of newly acquired human brucellosis cases among the three study groups registered after the intervention. Registration of brucellosis cases will follow existing protocols within the Uzbek healthcare system. Additionally, the questionnaire administered to the intervention and one control group will provide an insight of the baseline awareness about the disease. Adequate sample size and analysis of the data will allow comparisons between the three study groups and between strata within the groups, e.g., veterinarians and farmers. The control group not exposed to the questionnaire will allow an assessment of the impact of possible increased awareness as a result of our interventions.
Disease awareness questionnaires, educational materials and further details of our study design will be presented at the conference. The anticipated increase in knowledge about risk practices associated with the transmission of brucellosis from animals in at-risk populations should lead to a reduction in human cases of brucellosis in the intervention group, compared to control groups.
The epidemiology of brucellosis among humans and animals is well-characterized. Preventive measures for the diseases are well known; yet, applying this knowledge in resource-poor countries remains a constant challenge. Having effective health education programs is a vital component in efforts to reduce the disease burden by reducing the animal-to-human transmission rate. (3) an isolated case, uninvolved in recent transmission (i.e., neither source nor recipient). Source and secondary cases require more intense intervention due to their involvement in a chain of transmission; thus, accurate and rapid classification of new patients should help public health personnel to effectively prioritize control activities. However, the currently accepted method for classification, DNA fingerprint analysis, takes many weeks to produce the results (1); therefore, public health personnel often solely rely on their intuition to identify the case who is most likely to be involved in transmission.
Various clinical and sociodemographic features are known to be associated with TB transmission (2). By using these readily available data at the time of diagnosis, it is possible to rapidly estimate the probabilities of the case being source, secondary and isolated. 
Performance of the prediction model was promising as it was significantly better than random prediction (i.e., the AUCs were higher than 0.5). Small proportions of source and secondary cases in the available data may have limited performance. However, the model can be an effective decision support tool if its ability to identify a case likely to be involved in transmission is superior to the intuition of public health officials. Thus, further evaluation of the model in the context of TB control program should be conducted. If effective, the model would be particularly useful when incidence of TB increases in a resource limited setting, in which efficient prioritization of investigation is desired. Overall, the current study has important implications in promoting the approach of evidence-based practice in control of TB.
Tuberculosis; transmission; prediction model; public health; decision support 
Information on established syndromic surveillance systems was collected from peer-reviewed articles (found in MEDLINE, Scopus and Google Scholar), proceedings from all ISDS Conferences and other conferences and searches through reference lists of papers. In addition, web pages of international health organizations, surveillance networks and Ministries of Health were explored. Identified syndromic surveillance systems were categorized by country, resource level and surveillance methodology, among other features. Eight systems were selected and examined in detail to extract transferable information.
The literature demonstrates the many diverse, yet successful, syndromic surveillance efforts being implemented at the national and regional levels. Existing systems utilize a variety of data sources, data transmission techniques and analysis methodologies, ranging from low-tech, highly manual systems to automated, electronic systems. Frequently, syndromic surveillance systems are a coordinated effort among several partners, supplement existing systems, incorporate both specific and nonspecific disease detection and are used in conjunction with laboratory-based surveillance.
Though not without challenges, syndromic surveillance has the potential to serve as a valuable disease detection tool in resource-limited settings. Further examination and evaluation of these systems will benefit global disease surveillance capacity.
Biosurveillance; syndrome; developing countries Introduction Alcohol abuse is one of the major leading causes of preventable mortality in the United States (1). Binge drinking or excessive alcohol consumption, categorized as a pattern of drinking that brings a person's blood alcohol concentration (BAC) to 0.08 (2), has become a major cause for concern, especially in the 18-to 20-year-old population. Iowa City is home to the University of Iowa, a large public university of 30,000 students. On June 1, 2010, the city council enacted a new ordinance prohibiting persons under 21 from entering or remaining in bars (establishments after 10:00 PM whose primary purpose is the sale of alcoholic beverages) after 10:00 PM (3). Prior to the ordinance, Iowa City was the only municipality in the region where underage patrons were allowed on premises. The new ordinance was enacted largely in response to public safety concerns, including perceptions of increased violence and sexual assaults, especially at bar closing time.
Our hypothesis is that the under 21 ordinance also resulted in changes to travel behavior, where prior to the ordinance, the campus bar culture constituted an 'attractive nuisance', attracting a volatile mix of college students and nonlocals of all ages.
Arrest records were obtained from the University of Iowa Police Department containing all alcohol-related citations from January 1, 2004 to June 26, 2011. As the University of Iowa Police Department is one of the 4 local law enforcement agencies (Iowa City Police, Coralville Police and Johnson County Sheriff), these 7002 records represent a sample of alcoholrelated arrests, albeit one focused on the downtown bar area frequented by college students. Each record contains the date of the arrest as well as the age and home address of the offender, allowing us to compare 'in town' offenders (i.e., from within Iowa City, Coralville and transients) with 'out of town' offenders. Records corresponding to football Saturdays, where some 50,000 people come to Iowa City to tailgate and attend the Big10 football game, were excluded from the analysis as not representative of the usual bar culture. A total of 1490 alcoholrelated arrest records remained in the analysis.
A Fisher's exact test was used to test the hypothesis of whether the proportion of arrests of out of town patrons versus in town patrons is independent of the under 21 ordinance.
Data analysis confirms that, following the ordinance, the proportion of arrests involving out of town patrons to in town patrons was significantly reduced (Fisher's exact test, p 5 0.0001). Similar results were obtained for only under 21 arrests (Fisher's exact test, p0 0.0095) and over 21 arrests (Fisher's exact test, p 0 0.0058), suggesting that the campus bars were equally attractive to all age groups prior to the ordinance.
Immediately following the ordinance, the average weekly number of alcohol-related arrests increased from 9.3 to 16.3. Since over 21 arrests also increased, the change cannot be attributed solely to the new ordinance; indeed, additional police resources were deployed in a deliberate attempt to change the drinking culture. Of course, since the arresting officer cannot generally detect residency prior to arrest, arrest data still represent a geographically unbiased sample of bar patrons and can be used to explore changes in the mix of patrons.
We hypothesize that the changes detected in the proportion of arrests of in town and out of town patrons reflect a more homogeneous student clientele, where town-gown tensions are less likely to arise. Of course, any reduction in out of town patrons also corresponds to a reduction in the risk of DUIrelated fatalities, since students walk to the bars.
There are several shortcomings to this study. First, our data are incomplete as data from other enforcement agencies was not available. Second, we were unable to directly confirm the link with violence or sexual assault, as additional data would be required to do so: these are our next steps.
Alcohol; binge drinking; college binge drinking
The Border Infectious Disease Surveillance (BIDS) program was established in 1999 by the Centers for Disease Control and Prevention and Mexico Secretariat of Health, following mandates from the Council of State and Territorial Epidemiologists (CSTE) and the United StatesÁMexico border health association to improve border surveillance. The BIDS program is a binational public health collaboration to create an active sentinel-site surveillance of infectious disease among the United StatesÁMexico border. It is a collaborative effort between local, state, federal and international public health agencies throughout both countries in the border region. This project is aimed at using the best aspects of both countries surveillance system.
We established a network of sentinel clinic and hospital sites along the geographical United StatesÁMexico border region. We utilized a shared syndromic case definition that is compatible between both countries. Standardized data collection instruments allows for exchange of surveillance data. We increased the laboratory capacity for to test for diseases of public health importance.
This effort has been successful at building a regional surveillance system. In the 2010, three pilot hospital sites were enrolled to conduct severe acute respiratory infection (SARI) surveillance. These patients were tested for viral, bacterial and important fungal infections that cause respiratory disease. Fig. 1 includes results of the 74 hospitalized SARI patients who were enrolled in the 2010Á2011 influenza season. The SARI patients were 54% (n040) male and had a median age of was 62.5 years (range, 0Á 87 years). The expansion of this surveillance system requires additional sentinel hospital-sites and additional syndromes. A syndrome of acute diarrheal illness will be the focus of surveillance at one new pilot sentinel site, with potential to expand in the future.
A surveillance system using syndromic and CSTE case definitions allows for comparison of morbidity in the United States/ Mexico border region, increased communication and bidirectional sharing of information across the border. Creating and expanding a regional surveillance system that crosses an international boundary requires coordination and collaboration from all agencies involved. These surveillance data allow for examination of the border region as one epidemiologic unit. Consistent communication with clinicians and hospital staff helps to build credibility and interest. Simplicity in surveillance procedures encourages compliance. These surveillance efforts can guide vaccine allocation planning and efficiency in evaluating illnesses that maybe vaccine preventable. Systems to share information between various states in the United StatesÁMexico border region are important to develop binational control strategies.
Surveillance; syndromic; border; binational; Mexico Novel conceptual framework and toolset for countrywide assessments of opportunities and challenges for public health interventions Introduction Imbalances in wealth, education, infrastructure, sociopolitical leadership, healthcare and demographics create opportunities and challenges when implementing public health interventions. Understanding these, while embracing 'smart power', one can objectively assess a country's receptivity for support. Therefore, we developed a novel conceptual framework and toolset that objectively measured opportunities and challenges to inform decision-making, specifically about future implementation of the Electronic Integrated Disease Surveillance System (EIDSS)*a computer-based system for national reporting and monitoring of reportable human and veterinary infectious diseases*in East Africa and the Middle East.
After conceptualizing and designing the toolset architecture, we gathered objective data to calculate indicators using a systematic approach from published reports; articles from peer-reviewed journals; and websites of international organizations and national Ministries in each country. We also interviewed stakeholders. Indicators were weighted to reflect the level of impact on elements and domains, and standardized baselines were established to uniformly measure outcomes. Outcomes for each element and domain were then calculated based on the weighted, indicator data.
One hundred twenty-four indicators were identified that measured 16 elements that defined 7 domains of country-specific opportunities and challenges: political will, stakeholder involvement, culture, public health functionality, healthcare, laboratory and communication infrastructure. Thirty (24%) of the 124 indicators were chosen from the reporting requirements of the 2005 International Health Regulations. In the pilot, we found various positive and negative implementation characteristics in Uganda, Kenya, Tanzania, Afghanistan, Iraq and Pakistan.
We conceived a new and useful approach to objectively analyze opportunities and challenges for public health interventions within a country. With respect to introducing EIDSS, we piloted the toolset and described a balanced view of the opportunities and challenges. Application of this novel framework should be useful for other public health interventions, and validation and further testing of the toolset should be performed.
Public health evaluation; assessment; SWOT; smart power 
To present the prevailing global public health informatics landscape in developing countries highlighting current mobile system requirements and usage for disease surveillance and revealing gaps in the technology.
Mobile technology provides opportunities to monitor and improve health in areas of the world where resources are scarce. Poor infrastructure and the lack of access to medical services for millions have led to increased usage of mobile technology for health-related purposes in recent years. As adoption has increased, so has its acceptance as a viable technology for health data collection. The ability to provide timely, accurate and informed responses to emerging outbreaks of disease and other health threats makes mobile technology highly suitable for use in surveillance data collection activities and within the arena of global health informatics overall. The American Public Health Association (APHA) defines global health informatics as the application of information and communication technologies to improve health in low-resource settings, which include the following:
. linking disparate sources of data together through natural language processing; . use of mobile health technologies for disease surveillance;
. use of telemedicine to manage chronic disease;
. use of digital libraries to increase knowledge and awareness of public health events.
Based on donor-funded global health projects, systems requirements were gathered and existing mobile systems were evaluated for use in surveillance in low-resource settings. In advance of the tools evaluation, literature reviews were performed, and informatics experts at the Centers for Disease Control and Prevention (CDC), the World Health Organization (WHO) and various global nongovernmental organizations (NGOs) and associations were consulted and then recommendations were formulated. Systems were evaluated based on minimum requirements, which included maturity, usage, scalability, interoperability, functional features related to data collection and attributes that enable country ownership and generate high data quality.
In our evaluation, no single system was found to meet the needs of all the surveillance requirements. Mobile technology standards and guidelines were searched for, with none being found. An open-source, end-to-end software solution that is readily available and able to meet the needs of health surveillance was not identified, although several systems were deemed promising and have garnered significant use. Key features of an end-to-end mobile surveillance system would include the following:
. easily adoptable;
. open source or public domain;
. able to support multiple mobile platforms;
. form design environment;
. enumeration, case selection and case management;
. multilingual and Unicode functionality;
. client-server deployment (local and cloud based);
. SMS enabled; . rational database system data storage;
. data extraction to statistical file formats;
. embedded analysis and report capability;
. GIS/GPS enabled, with global mapping capability;
. geospatial analytic capability;
. data visualization.
Mobile technology has emerged as a key component of global health informatics. With the expansion of this technology, a plethora of tools and systems have materialized. With so many systems, it is difficult to know which tools to apply. To add to the confusion, no standards or guidelines currently exist. Additionally, there is a clear need for an end-to-end, opensource, scalable mobile system that incorporates functionality for questionnaire design, data management, analysis and reporting. These gaps must be addressed in order for mobile surveillance technologies adoption to advance adequately.
Introduction Disease screening facilitates the reduction of disease prevalence in two ways: (1) by preventing transmission and (2) allowing for treatment of infected individuals. Hospitals choosing an optimal screening level must weigh the benefits of decreased prevalence against the costs of screening and subsequent treatment. If screening decisions are made by multiple decision units (DU; e.g., hospital wards), then they must consider the disease prevalence among admissions to their unit. Thus, the screening decisions made by one DU directly affect the disease prevalence of the other units when patients are shared.
Because of this interdependent relationship, one DU may have an incentive to ''free-ride'' off the screening decisions of others as the disease prevalence declines. On the other hand, DUs may find it futile to invest in screening if they admit a large number of infected patients from neighbors who fail to screen properly. This problem is important in determining the optimal level of unit autonomy, since increasing a unit's level of autonomy in screening effectively increases the total number of DUs.
We develop a theoretical model that incorporates the two channels through which screening may reduce prevalence. The model is based on a hospital composed of N treatment units (e.g., ICU and ER) divided into n DUs, that transfer patients between one another and an outside population. Disease prevalence in each DU is determined by an SIS model based on the multi-institutional framework of Smith, et al. (1, 2) . A DU's prevalence is a function of its own screening level (s) and that of their neighbors (š).
We develop a cost structure similar to Armbruster and Brandeau that incorporates the various costs to screen for and treat a disease. (3) Given these costs, a single DU chooses the screening level that minimizes its net present value of discounted future costs. We solve for the symmetric, pure-strategy Nash equilibrium.
As the rate of recovery following treatment (t) increases relative to screening and treatment costs, the DU's best response curve transitions from an inverted-U pattern to one that is monotonically decreasing (Fig. 1) . Additionally, the equilibrium screening value is monotonically decreasing in the number of DUs (Fig. 2) . Here the best response curves intersect the line of equal screening values.
When treatment is less effective, free-riding is less severe and a DU's optimal screening may actually increase with its opponents level. However, as treatment becomes more effective, optimal screening levels are strictly decreasing in the other DU's allocation: free-riding takes full effect. As the number of DUs increases, so does the opportunity to free-ride. This means optimal screening will decrease and disease prevalence will increase as the number of DUs increases. Therefore, in a purely symmetric environment increasing unit autonomy may adversely affect disease prevalence: authority for screening should be centralized. 
The time series of syphilis cases has been studied at the country and state level at the yearly basis (1, 2) , and it has been found that syphilis has a periodicity of approximately 10 years (2). However, to inform prevention efforts, it is important to understand the short-term dynamics of disease activity.
We used data from the MMWR. It contains weekly syphilis counts per state. We consider the time period from 1995 to 2009. We removed week 53 when present, due to inconsistencies in reporting. We considered 53 locations: the 50 states plus Puerto Rico, and the cities of New York City and Washington DC. To predict disease activity in each state, we constructed a series of linear lagged regression models that used several states as covariates. To benchmark our models, we constructed a basic ARIMA model with one autocorrelation term. All the models were constructed to forecast 4 weeks in advance. Prediction at week t was performed by fitting the models using all past data prior to week tÁ4. To identify bellwether states, we proceeded as follows. First, we repeatedly fitted 2-covariate models to forecast each state and obtained the top 5 most frequent bellwether states for each state. Then, we obtained the most frequent bellwether states from the above lists.
We found that forecasting states using less than 10 states as covariates is better than using more or the state itself as covariate (ARIMA), as shown in Fig. 1 . An example of out of sample prediction is shown in Fig. 2 , for New York City.
We also found that the 10 most frequent states in models with two covariates are California, Virginia, Florida, New York City (treated as state), Alabama, Ohio, Tennessee, North Carolina, New Hampshire and New Mexico. The first 5 are covariates of 40 states, and the amount increases to 50 when adding the later.
Using several states as covariates in models seem to improve their forecasting power. This suggests that these models 'learn' the dynamics of syphilis between different states. In addition, we have identified the existence of specific bellwether states. By using these bellwether states, it is possible to forecast syphilis cases in almost all the states in the country.
Several limitations undermine the quality of the predictions. First, cases are counted at reporting time instead of acquisition time (3) . Second, the MMWR file supposedly contains cumulative numbers within a year, but this is not always true. Third, some states exhibit strong yearly periodicity, which seems to be due to patterns in disease reporting.
Syphilis time series; forecasting; disease surveillance 
Argus is an event-based surveillance system, which captures information from publicly available Internet media in multiple languages. The information is contextualized, and indications and warning (I&W) of disease are identified. Reports are generated by regional experts and are made available to the system's users (1) . In this study a small-scale disease event, plague emergence, was tracked in a rural setting, despite media suppression and a low availability of epidemiological information.
Argus reports meeting the following inclusion criteria were selected retrospectively: (1) disease: plague, (2) location: Peru, (3) time period: AprilÁOctober 2010. The reports were reviewed for relevant I&W of plague infection, with the goal of identifying factors that contributed to disease spread and ineffective public health response.
From the time period specified, media reported on a human plague outbreak in northern Peru where all 3 clinical forms of plague were identified (septicemic, pneumonic and bubonic); in one area, bubonic plague was registered for the first time in over a decade while pneumonic plague was reported for the first time ever in the country, according to an official (2) .
The first human case of bubonic plague was reported in April, followed by a 2-month reporting lull from May to July. Subsequently, new media information revealed ongoing human plague cases, including nosocomial pneumonic infections which had spread from one patient to medical staff and one relative, as well as a severe lack of biosafety personal protective and laboratory equipment (3) .
Retrospective review of Argus reports later identified 3 key factors that limited the effectiveness of disease management in the region: (1) a lack of government leadership and accountability, (2) poor sanitation leading to an inability to decrease the vector population and (3) an inadequate regional healthcare infrastructure (4) . Media sources recognized discrepancies in medical information provided by health officials and the medical community, and as the outbreak continued, protests erupted over poor sanitary conditions and insufficient medical resources as observed by healthcare workers. In August, the Minister of Health (MOH) declared that the outbreak had been 'controlled'; however, the media continued to report human plague cases and noted concern regarding the potential danger of plague spreading to urban markets. Travel restrictions were applied and reports later speculated that the World Health Organization (WHO) would close ports and issue a national quarantine if plague extended into coastal export areas (5, 6) . Further, officials declared a latent risk of disease transmission to bordering countries. At the end of the study reporting timeframe, media continued to identify the confirmation of new human bubonic plague cases, the implementation of vector control efforts, and the ongoing risk to residents despite attempted disease management efforts.
The use of an event-based methodology provided detailed insight into a localized, small-scale disease situation where limited medical and epidemiological information was available. Argus documentation of this event allowed for a retrospective review, which identified deficiencies in the current disease management system in Peru and drew attention to the potential negative impact of social and political context on public health efforts.
Surveillance; plague; emergence; intervention; isolated
Heat waves have serious health impacts such as heat exhaustion, heat stroke, dehydration and death. Heat illness morbidity and mortality can be reduced with the identification of vulnerable populations and targeted public health interventions. In June and July of 2011, a heat wave occurred in Nebraska in which 28 days reached 90 F or higher. Syndromic surveillance data were used to describe heat-related illness emergency department (ED) visits during this time.
Eight hospitals currently submit syndromic surveillance ED data to Nebraska Department of Health and Human Services (NeDHHS), representing approximately 18% of all ED visits for the state. Five hospitals reported complete data for the selected study period, June 1, 2010ÁAugust 10, 2011. The three hospitals not reporting complete data for the study period were excluded. These records represent approximately 15% of all ED visits in the state for JuneÁAugust. Cases of heat-related illness were identifiedusing ICD9CM diagnostic and external cause of injury codes: 992, 705.1, 708.2 and E900. Additional cases were identified from the chief complaint field using the SAS INDEX function to locate the following words within the text field: 'HEAT', 'HEATED', 'DEHYDRATED' and 'HYPERTHERMIA'. Each record returned from these searches was examined to confirm the presence of heat illness. Chief complaint fields containing keywords but not involving heat-related illness, i.e., 'applied heat to swollen ankle', were eliminated. with heat-related ED vists were 55% male (n 0273) with a median age of 34 years. Further analyses will assess correlation between heat index and heat illness in Nebraska.
The rate of heat-related illness ED visits was slightly higher in the summer of 2011 than in 2010. This system provides an effective method to identify and track heat illness. Timely identification of patients with heat illness using this system can facilitate rapid and focused public health response and reduce heat*related morbidity and mortality.
Syndromic surveillance; heat illness; heat wave Introduction Commonly used syndromic surveillance methods based on the spatial scan statistic (1) first classify disease cases into broad, preexisting symptom categories (prodromes) such as respiratory or fever, then detect spatial clusters where the recent case count of some prodrome is unexpectedly high. Novel emerging infections may have very specific and anomalous symptoms, which should be easy to detect even if the number of cases is small. However, typical spatial scan approaches may fail to detect a novel outbreak if the resulting cases are not classified to any known prodrome. Alternatively, detection may be delayed because cases are lumped into an overly broad prodrome, diluting the outbreak signal.
We propose a new approach to detect emerging patterns of keywords in the chief complaint data. Our semantic scan statistic has three steps: automatically inferring a set of topics (probability distributions over words) from the data using Latent Dirichlet Allocation (2), classifying each chief complaint to the most likely topic, and then performing a spatial scan using the case counts for each topic. We compare three variants of the semantic scan: static (topics are learned from historical data and do not change from day to day), dynamic (topics are recalculated each day using the most recent two weeks of data) and incremental (not only using the static topics but also learning additional 'emerging' topics that differ substantially from the static topics).
We compared the three semantic scan methods to the standard, prodrome-based spatial scan using synthetic disease outbreaks injected into real-world emergency department data from Allegheny County, PA. We first considered 55 different outbreak types, corresponding to all distinct ICD-9 codes with at least 10 cases, which were mapped to one of the existing prodromes. For each outbreak type, we generated spatially localized injects with chief complaints sampled from the cases with that ICD-9 code (Fig. 1) . The static, dynamic and incremental methods required an average of 7.7, 7.1 and 6.9 days, respectively, to detect and were able to precisely characterize the outbreak based on the detected topic (e.g., top keywords for ICD-9 code 569.3 were 'rectal', 'bleed', and 'bleeding'). The prodrome method achieved more timely detection (5.0 days to detect) but with much less precise characterization (e.g., 'hemorrhagic' for ICD-9 code 569.3). Next, we considered both randomly selected, unmapped ICD-9 codes and synthetically generated unprecedented events, such as an outbreak that makes the patient's nose turn green. The prodrome method required 10.9 days to detect these outbreaks, while the semantic scan was able to achieve much faster detection. For example, for the green nose outbreak, the static, dynamic and incremental methods detected in 6.4, 5.3 and 5.6 days, respectively. The dynamic and incremental methods correctly identified the emerging topic (keywords 'green', 'nose', 'nasal', etc.), while the static method did not, since the outbreak did not correspond to any of the topics learned from historical data.
The semantic scan statistic can successfully capture emerging spatial patterns in free-text chief complaint data, enabling more timely detection of novel emerging outbreaks with previously unseen patterns of symptoms. Other advantages include more accurate characterization of outbreaks (identifying a set of keywords that precisely describe the disease symptoms) and the ability to detect outbreaks without preexisting syndrome definitions. Additionally, our methods have the potential to achieve more timely detection by incorporating free-text data sources, such as Twitter and other social media tools, into the surveillance process.
Text mining; event detection; semantic scan
Optimal sequential management of disease outbreaks has been shown to dramatically improve the realized outbreak costs when the number of newly infected and recovered individuals is assumed to be known (1, 2) . This assumption has been relaxed so that infected and recovered individuals are sampled, and therefore the rate of information gain about the infectiousness and morbidity of a particular outbreak is proportional to the sampling rate (3). We study the effect of no recovered sampling and signal delay, features common to surveillance systems, on the costs associated with an outbreak.
We develop a stochastic compartment model for disease populations consisting of susceptible (S), infected (I), recovered (R) and deceased (D) individuals. This model contains four parameters determining the rates of these transitions: S 0I, I0R, I0D and S 0R (vaccination). While all vaccination and death transitions are observed completely, the infected and recovered transitions are observed through sampling possibly with a delay between the transition and when the information can be used in a decision.
Sequential inference of parameters is performed using Bayesian updating, which is available in closed form when independent gamma priors are assumed, and the current system state is known. For the two sampled transitions, the associated parameters are updated in a manner that is consistent with how information is gained during sampling so that the rate of information gain is proportional to the sampling rate.
A cost structure is developed to weigh the outbreak morbidity and mortality versus the cost of active outbreak control (isolation, vaccination and increased sampling). The morbidity cost is quadratic to account for increased costs that occur when many individuals are sick simultaneously. Control costs include fixed and running costs, which are a function of the current number of infected individuals (3) .
The effect of recovered sampling and delay is primarily assessed by running separate scenarios that have combinations of sampling and delay and calculating the average outbreak cost under these scenarios. In addition, allowing recovered sampling in a control allowed analysis of how often and when the optimal outbreak management utilized this sampling.
As a case study, we use a recent measles outbreak in Harare, Zimbabwe, as our basis. At outbreak onset, we assume 20,000 susceptible individuals ( Â1% of total population in accordance with vaccination coverage rates) and 20 infected individuals. Priors for outbreak parameters are vague but informative, e.g., a 95% interval for infectiousness is 4 to 11 days.
Relative to the base-case scenario where immediate sampling is performed on both newly infected and recovered individuals, the following results are observed. Eliminating recovered sampling increases average costs by 5%, a one-period delay between transitions and control action increases costs 6%, a two-period delay increases costs 14% and eliminating all sampling increases costs by 34%.
When allowing increased sampling as a possible outbreak control measure, the optimal decision was to utilize sampling of infected and recovered individuals about 20% of the time.
Typical syndromic surveillance systems have taken the first step, which is to provide a measure of the number of newly infected individuals. Costs being equal, this research suggests this was the best investment for surveillance. We hope future research with different diseases and surveillance possibilities will elucidate where money should be spent in improving surveillance practices. 
Data were extracted from the weekly Zimbabwe cholera epidemiological bulletins available in the World Health Organization's Zimbabwe cholera epidemiological bulletin archive (2) . The focus of the data collection was on the tables titled 'Distribution of Measles IgM Positive by Age group and District of residence', which typically contained both cumulative and new cases of IgM-confirmed measles cases by district and age categories. Although not entirely consistent, the age categories were younger than 9 months, 9Á11 months, 1Á4 years, 5Á14 years, and 14 years and older.
The statistical software R (3) was used for data cleaning (an extensive process) and exploratory analysis. The maptools package (4) was used to generate maps of the geographical disease progression. Fig. 1A provides an example time series for the cumulativeconfirmed measles cases in Harare, the capital of Zimbabwe, where all age categories have been combined. Indicated in green is the mass vaccination campaign that took place between May 24 and June 2. Fig. 1B provides an example map displaying the geographical distribution of confirmed measles cases upon extinction of the outbreak. The darker color indicates a higher attack rate (number of confirmed cases divided by total population); the darkest red area is Harare.
This exploratory analysis questions the utility of the mass vaccination campaign since the campaign came after the peak of the outbreak in the hardest hit district in Zimbabwe. But since Harare was one of the earliest districts affected, perhaps the campaign prevented further spread to other districts. In addition, it is possible that suspected cases in Harare were more likely to become confirmed cases due to geographical proximity of testing laboratories, thereby inflating the relative attack rate.
Measles; Zimbabwe; exploratory analysis; geographical; R 
In March 11, 2011, the big earthquake attacked eastern Japan followed by huge tsunami and nuclear plant accident. Consequently, a lot of people could not help living in evacuation sites. Since those evaluation sites have high density of population and were not necessarily good in sanitary condition, outbreaks of influenza, norovirus or other infectious diseases were concerned.
We developed a web-based evacuation site surveillance system with 8 syndromes including acute gastroenteric symptoms; influenza or influenza-like-illness; acute respiratory symptoms other than influenza; rash and fever; neurologic symptoms including tetanus, meningitis and encephalitis; cutaneous symptoms; wound-related infectious diseases; icterus and death. Age of the patients was classified into three categories: younger than 5 years, 5 to 64 years and 65 years old or older. Analysis by evacuation site was performed automatically, and if some aberrations were found, the system showed an alert sign on the screen of a computer. The information on patients was shared with the public health center and the local government office simultaneously.
Evacuation site surveillance started in Fukushima prefecture on March 25, 2011, and in Miyagi prefecture on May 8, 2011.
About 400 sites in Miyagi prefecture were covered until the end of May. When the surveillance found an aberration, the public health center investigated the site and started taking an action for control.
This system raised awareness of infectious diseases and provided good information for risk assessment. Before the earthquake, the pharmacy surveillance and the school surveillance (only in Miyagi prefecture), which are nationwide syndromic surveillance in Japan, were operating, and these played a complementary role for evacuation site surveillance and the official surveillance. Our experience showed that it would be too late to start to develop the system from the scratch after a disaster occurred. Thus, it is essential to make a plan on activation of the system in advance in case a severe disaster occurs and to prepare and stockpile the hardware that is necessary for an early activation of evacuation site surveillance. The necessary hardware, for example, includes battery and communication tool even if electronic power, internet and (mobile) phone network are shut down. This is the next challenge. 
Syndromic surveillance systems were designed for early outbreak and bioterrorism event detection. As practical experience shaped development and implementation, these systems became more broadly used for general surveillance and situational awareness, notably ILI monitoring. Beginning in 2006, ISDS engaged partners from state and local health departments to build Distribute, a distributed surveillance network for sharing de-identified aggregate emergency department (ED) syndromic surveillance data through existing state and local public health systems (1). To provide more meaningful cross-jurisdictional comparisons and to allow valid aggregation of syndromic data at the national level, a pilot study was conducted to assess implementation of a common ILI syndrome definition across Distribute.
Six jurisdictions provided 4 years of baseline ED data using a common ILI definition comprising 3 subsyndrome components defined by a formal code-set (Fig. 1) . Distribute sites were invited to participate in the assessment based on geography, jurisdiction size and ED coverage. Invited sites were asked to provide historical data consisting of total and ILI-related daily visit counts by age group ( B2, 2Á4, 5Á17, 18Á44, 45Á64 and 65' years). The common ILI syndrome and subsyndrome case definitions for the pilot were defined from coded or free text ED patient electronic chief complaint data as 'fever and cough', 'fever and sore throat', and 'flu'. Evaluation included comparison of syndrome time-series, subsyndrome and age-specific distribution of visits and signal-to-noise measures.
We found less variation between jurisdictions in weekly ratios using the common ILI definition (mean 2%; range 1.5Á3.1%) than locally preferred syndromes (mean 4.9%; range 1.8-8.4%), and influenza epidemic signal-to-noise ratios were comparable for most jurisdictions during the study period. The findings suggest that the common syndrome improves comparability without an overall cost in terms of epidemic signal discrimination.
The results of this common ILI assessment suggest that disparate local systems can adopt a harmonized syndrome definition allowing for meaningful comparisons and national aggregation while maintaining the ability to use local systems and definitions. The common ILI syndrome provided more directly comparable time-series, both during baseline periods and epidemics. Use of the common syndrome did not have an overall or systematic cost in terms of epidemic signal discrimination. Where the signal-to-noise ratio was not improved, differences were usually minimal. Also, the use of the common syndrome did not restrict the use of the locally defined syndromes for local detection. This collaborative pilot was useful in synthesizing local experience in the creation of a nationally harmonized ILI syndrome definition.
Influenza; surveillance; epidemiology; syndrome standard; emergency department 
We describe the initial phase of the ISDS Distribute pilot for monitoring acute gastroenteritis (AGE) syndromic emergency department (ED) visits and present preliminary analysis of agespecific trends documenting a dramatic shift in AGE consistent with US rotavirus vaccine policy and use.
Epidemic AGE is a major contributor to the global burden of morbidity and mortality. Rotavirus and norovirus epidemics present a significant burden annually, with their predominant impact in temperate climates occurring during winter periods. Annually, epidemic rotavirus causes an estimated 600,000 deaths worldwide and 70,000 hospitalizations in the United States, primarily among children younger than 5 years (1). The U.S. burden from norovirus is estimated at 71,000 hospitalizations annually, with the impact more generally across age groups (2) . Changes in rotavirus vaccine use have significantly reduced the impact of epidemic rotavirus (3).
The Distribute project began in 2006 as a distributed, syndromic surveillance effort networking state and local health departments to share aggregate ED based influenza-like illness (ILI) syndrome data (4) . The AGE pilot was conducted to assess the feasibility of generalizing the Distribute model from ILI trends to monitoring other syndromes. Distribute participating jurisdictions were asked to submit diarrheal and vomiting AGE syndrome ED data, following a commonly used syndrome definition. Of the 10 Distribute participating jurisdictions that submitted AGE data, 6 provided historical baseline data going back to January 2006 or earlier. Of these, 3 were state, 3 large city or county jurisdictions, located in Northeastern, Mid-Atlantic, Midwestern and Western U.S. surveillance regions. Syndrome time-series ratios [(weekly AGE syndrome count)/ (total ED visit count)] were assessed by jurisdiction and age group. To aid comparison of seasonal trends across jurisdictions, time-series were normalized around their baseline as a measure of relative increase [(weekly AGE ratio)/(weekly lowerquartile)]. Rotavirus vaccine 2006 pre-and postlicensure periods were compared.
All jurisdictions submitting AGE data to Distribute presented seasonal trends with predominant winter peaks. Across the pilot jurisdictions, seasonal peaks from 2003/04 to 2005/06 occurred during MarÁApr, while 2006/07 to 2009/10 seasonal trends peaked predominantly in DecÁFeb. Overall, epidemic timing was similar across age groups; however, the shifting pattern in impact after the 2006/07 season presented a greater drop among young children. (Fig. 1) .
The results of the pilot suggest the Distribute model can be successfully generalized to monitoring AGE trends, specifically the age-specific timing and impact of winter-seasonal epidemic rotavirus and norovirus. The case study of 2006 rotavirus vaccine implementation and subsequent shift in timing and impact of AGE trends suggest that syndromic ED data can potentially provide a useful surveillance indicator of populationlevel vaccine effect.
Gastroenteritis; norovirus; rotavirus; epidemiology; emergency department 
Cost-effective, flexible and innovative tools that integrate disparate data sets and allow sharing of information between geographically dispersed collaborators are needed to improve public health surveillance practice. Gossamer Health (Good Open Standards System for Aggregating, Monitoring and Electronic Reporting of Health), http://gossamerhealth.org, is an open source system, suitable for server or 'cloud' deployment, which is designed for the collection, analysis, interpretation and visualization of syndromic surveillance data and other indicators to monitor population health. The Gossamer Health system combines applied public health informatics research conducted at the University of Washington (UW) Center for Public Health Informatics and Washington State Department of Health, in collaboration with other state and local health jurisdictions, the International Society for Disease Surveillance and the Centers for Disease Control and Prevention.
Gossamer combines work on (1) methods for automated surveillance based on summarized clinical data, such as the influenza and visit counts used in the Distribute project (1), (2) methods developed for the modularization of surveillance processes developed for the Shoki project (2), (3) methods developed for the automated processing of Health Information Exchange data (HIE) as part of the CDC HIE initiative (3) and (4) standard industry server virtualization and deployment techniques (4).
Gossamer uses code developed at UW and additional open source components. Most components are distributed under the '3-clause BSD license', permitting free use, modification and redistribution. Automated modules include (1) HL7 message receipt, processing and storage, (2) compilation of line listing data from HL7 Minimum Biosurveillance Data Set (MBDS) and Meaningful use (under development) messages, (3) classification of cases into syndromes and compilation of syndrome data into indicators, (4) receipt, storage, aggregation and management of indicator data, and (5) analysis, visualization and reporting (AVR) of indicator data. Modules may be deployed locally or in the EC2 cloud and communicated using standard protocols to let deployment strategies be mixed across the system to support both sharing and shared use of components, as well as load balancing and optimization. This presentation will talk about the goals of the open source system and give underlying details of the technical implementation using virtual machines. As an example, we will discuss an application of the Gossamer system instance developed to let a state public health agency disseminate summarized laboratory test results for multiple (14) respiratory viruses (see Fig. 1 ).
To support existing and emergent surveillance needs, the UW has worked with local and state health jurisdictions to identify features that allow for user-defined indicators of chronic and infectious disease surveillance. An important aspect of the Gossamer Health vision is its support for public health agencies to collaborate in cross-jurisdictional surveillance efforts through both on-demand and automated sharing of standards-based data feeds.
Gossamer is a work in progress, but it is a community work. All are welcome to participate in its development. 
Cross-jurisdictional sharing of public health syndrome data is useful for many reasons, among them to provide a larger regional or national view of activity and to determine if unusual activity observed in one jurisdiction is atypical. Considerable barriers to sharing of public health data exist, including maintaining control of potentially sensitive data and having informatics systems available to take and view data.
The Distribute project (1,2) has successfully enabled crossjurisdictional sharing of ILI syndrome data through a community of practice approach to facilitate control and trust and a distributed informatics solution.
The Gossamer system (3) incorporates methods used in several UW projects including Distribute. Gossamer has been designed in a modular fashion to be hosted using virtual or physical machines, including inside cloud environments. Two modules of the Gossamer system are designed for aggregate data sharing and provide a subset of the Distribute functionality.
The Distribute and Gossamer systems have been used for ad hoc sharing in three different contexts: sharing of common ILI data for research into syndrome standardization, sharing syndromic data for specific events (2010 Olympics) and for pilot regional sharing of respiratory laboratory results. Two additional projects are underway to share specific syndromes of recent interest: alcohol-related and heat-related ED visits.
The Distribute system was initially designed to share 4 syndromes (broad and narrow ILI, and GI syndromes). To reduce barriers to entry, the Distribute project does not impose strict syndrome definitions. This lack of standardization introduces variability between jurisdictions and a pilot has been undertaken to compare sites with preferred definitions and to develop a common ILI definition. To enable the addition of a common syndrome considerable modifications to the structure of the Distribute system were required. The approach taken allowed for the use of arbitrary indicators and stratification ranges.
The Gossamer system uses a similar data storage architecture to that of the current version of Distribute, though Gossamer is more modular and better able to use external services. These features make it useful for moving beyond specific political structures or disease content areas.
The expanded data model has now been used to support the ILI standardization effort through comparison of newly contributed 'ILI-S' syndrome data. Distribute was also used to develop a site to allow Washington State DoH to share specific syndromic data with British Columbia during the 2010 Olympics. An instance of Gossamer demonstrated sharing laboratory results for 14 viral isolates between two states. In addition to community-driven comparisons of ILI and GI syndromes, the data model has been applied at the design level to two additional syndrome types for ad hoc data sharing: alcohol intoxicationrelated visits and heat exposure-related ED visits.
While built around similar data models, each system has strengths and weaknesses for ad hoc sharing of data. Advantages of the Distribute system for sharing additional data include making use of the existing trust and community that is based around the system, which reduces many barriers to sharing data and facilitates adding more community members. In addition, data feeds and administrative details are already in place.
Disadvantages of using Distribute include limitations in the common data transmission format, limitations in stratifiers and limitations in compartmentalization.
The implementation of the very similar data model in Gossamer is able to address some of these issues by various strategies including virtualization and modular architecture, while extending the flexibility which supports new applications of the data collection, quality and analysis methods developed for use with influenza syndromes in Disribute.
The 5 examples illustrate the strengths of the community of practice approach to sharing data. The Distribute and Gossamer systems illustrate how lightweight systems can be designed to easily facilitate ad hoc sharing between jurisdictions.
Informatics; surviellance; architecture; data sharing; public health practice
Distribute is a national emergency department syndromic surveillance project developed by the International Society for Disease Surveillance (ISDS) for influenza-like illness (ILI) that integrates data from existing state and local public health department surveillance systems. The Distribute project provides graphic comparisons of both ILI-related clinical visits across jurisdictions and a national picture of ILI.
Unlike other surveillance systems, Distribute is designed to work solely with summarized (aggregated) data, which cannot be traced back to the unaggregated 'raw' data. This and the distributed, voluntary nature of the project create some unique data quality issues, with considerable site to site variability. Together with the ISDS, the University of Washington has developed processes and tools to address these challenges, mirroring work done by others in the Distribute community.
University of Washington together with the ISDS has undertaken a comprehensive analysis of the quality of the data being received by Distribute, primarily using visual methods, examining data quality characteristics within and between sites. This process included basic exploratory analysis of data quality problems and analytical analysis of specific aspects of data quality, including the relationship between timeliness, completion and accuracy.
Considerable variability was seen between sites in terms of timeliness and completion, and completion rates did not necessarily correlate with accuracy. In our talk, we will present results comparing the quality of data between sites (sites will be unidentified), in particular comparisons between timeliness, completion and accuracy. We will also examine the types of observed relationships between timeliness, completeness and accuracy exhibited across the sites.
The purpose of this talk is to facilitate discussion between Distribute participants around data quality and the role that the ISDS can play in ensuring data quality. We will show prototypes of two features that could be hosted on the Distribute restricted site. The first feature would allow each site to compare the quality of their data (identified only to them, with site linked to the id of the user) with the remaining sites (each unidentified). The second feature would allow each site to see time series of their data together with prediction intervals for the accuracy of the ILI ratio for recent dates where the data are incomplete (see Fig. 1 ).
Our goal is to spark discussion on data quality with respect to syndromic surveillance data and, in particular, how the Distribute project can be leveraged to improve the quality of aggregate data produced by participating sites.
Keywords Data quality; surveillance; public health practice; data quality *Ian Painter E-mail: ipainter@uw.edu (page number not for citation purpose) 
Distribute is a national emergency department syndromic surveillance project developed by the International Society for Disease Surveillance for influenza-like illness (ILI) that integrates data from existing state and local public health department surveillance systems. The Distribute project provides graphic comparisons of both ILI-related clinical visits across jurisdictions and a national picture of ILI.
Unlike other surveillance systems, Distribute is designed to work solely with summarized (aggregated) data, which cannot be traced back to the unaggregated 'raw' data. This and the distributed, voluntary nature of the project creates some unique data quality issues, with considerable site to site variability. Together with the ISDS, the University of Washington has developed processes and tools to address these challenges, mirroring work done by others in the Distribute community.
The University of Washington together with the ISDS has undertaken a comprehensive analysis of the quality of the data being received by Distribute, primarily using visual methods, examining data quality characteristics within and between sites. Several visualization tools were developed to assist in analyzing and characterizing data quality patterns for each site: upload pattern graphs (Fig. 1) , stacked lag histograms and arrays of lagged time series graphs. Upload pattern graphs are heat maps comparing upload dates with encounter dates (an example figure is given below for three sites). Stacked lag histograms provide a succinct view of the complete distribution of data timeliness for a particular site. Arrays of lagged time series graphs provide an in-depth look at how timeliness patterns manifest in time series graphs. Implementation of the latter two visualizations required implementing a specific database architecture to enable reconstruction of the data at any prior upload date.
In our talk, we will present these visualizations and demonstrate how they can be used to discover several common and some unusual data quality patterns and issues. We will also discuss the underlying architecture that allows us to reconstruct prior views and discuss the importance of examining data quality in terms of prior data views.
Visualizations; data quality; surveillance 
During responses, an electronic medical record (EMR) allows federal emergency response staff to view and evaluate near realtime clinical encounter data. Analysis of EMR patient data can enhance situational awareness and provide decision advantage for headquarters' staff during both domestic and international events. The EMR was utilized by field medical personnel during the response to the Haiti earthquake.
During the U.S. response to the Haiti Earthquake in January 2010, patient demographic and clinical treatment data were collected by ESF-8 responders through the EMR. Data were collected throughout the patient experience during registration, triage, treatment and discharge. Inclusion criteria for encounter records in the main analysis were entered into the EMR between January 18, 2010, and February 22, 2010, encounter occurred at one of the HHS sites in Haiti and data downloaded no later than February 23, 2010. Data were then analyzed in order to identify potential emerging conditions and operational medical needs during the entire response.
We analyzed 8925 patient encounter records entered into the EMR between January 18 and February 22, 2010. Of those records, 4612 (51.8%) were coded as female, 3995 (44.8%) as male and 303 (3.4%) were not specified. Additionally, 1444 (16.2%) of the encounters were coded as less than 6 years old, 1638 (18.3%) were coded as 6Á18 years old, 4352 (48.8%) were coded as 19Á49 years old, 1004 (11.2%) were coded as 50Á65 years old, 283 (3.2%) were coded as more than 65 years old and 204 (2.3%) were not specified. Mean age was 27.1 (SD 019.1) years with a minimum of 1 day and a maximum of 100 years. Additionally, 6575 (75.1%) records were coded as nonurgent, 1889 (21.6%) as urgent and 295 (3.3%) as emergent. Daily surveillance of the records resulted in the identification many of suspected or confirmed symptom and disease occurrences. They included 8 cases of chicken pox/herpes zoster, 46 cases of conjunctivitis, 1 case of hemorrhagic fever, 23 guns shots wounds, 15 cases of malaria, 1 case of measles, 3 cases of meningitis, 2 cases of mumps, 53 cases of acariasis (including scabies), 1 case of typhus, 3 cases of tetanus, 3 cases of tuberculosis and 7 cases of pneumonia. We also detected 714 instances of fever and 550 instances of diarrhea.
During the 2010 earthquake response in Haiti, knowledge of the medical encounters through EMR data in the field provided indications of need for patient care. The surveillance of suspected and confirmed condition and diseases of concern allowed for timely decisions on adjustments to the response. Event burden could be quickly assessed through electronic reporting. EMR data can enhance and inform emergency response decision-making during domestic and international events and may be a useful tool for field public health and medical surveillance and situational awareness during future disaster responses. 
Epidemiological data suggests that there have been disproportionate numbers of non-White persons hospitalized due to 2009 pandemic influenza (H1N1) in MA. Population-based statewide descriptions of H1N1-related hospitalizations according to race/ ethnic group and SES have not been described.
We identified those discharged from any MA hospital during the H1N1 pandemic in the Hospital Discharge Database (HDD) with ICD-9 diagnosis codes correlating highly with positive viral specimens (1). Using five-digit zip codes as an identifier, we linked census data to the HDD population to provide a measure of SES indicator through aggregate levels of affluence. We used random-effects multivariate logistic regression models to explore the above objectives.
Results 9737 individuals met inclusion criteria, and 1529 individuals (16%) were admitted to the ICU. Hispanics had the lowest rates of ICU stay (11% Hispanics in the ICU had the highest length of stay (8.1 days), the youngest mean age (26 years), nearly a third (32%) were B18 years, 52% were from the lowest SES group, and 58% were female (Table 1) . Differences between race/ethnic groups and SES exist (Table 1) . Results from multivariate regressions indicate that Hispanics are at 27% lower risk for ICU stay compared to Whites (OR 00.73, p B0.001*data not shown).
Hispanics were particularly vulnerable to exposure and susceptibility to H1N1 (2). However, Hispanics had the lowest rates of H1N1-related ICU admission and significantly lower risk of having H1N1-related ICU visits. Logistic regression models indicate that these differences are not explained by the large differences in SES. This is contrary to other reports and could be related to the low mean age of this group. Future work should address how lower age among Hispanics influences H1N1-related ICU stay Á especially in young Hispanic women.
Pandemic H1N1 influenza; epidemiology; disparity research; race/ethnicity Objective
To investigate the utility of spatial analysis in the tracking of the stages of the HIV epidemic at an administrative territory level, using the Odessa region, Ukraine, as an example.
Detection of the signs of HIV epidemic transition from concentrated to generalized stage is an important issue for many countries including Ukraine. Objective and timely detection of the generalization of HIV epidemic is a significant factor for the development and implementation of appropriate preventive programs. As an additional method for estimating HIV epidemic stage, the spatial analysis of the reported new HIV cases among injection drug users (IDU) and other populations (due to sexual way of transmission) has been recommended. For studying new HIV cases in small societies, relative risk (RR) rates are preferred over incidence indicators. Spatial clustering based on the calculation of RR rates allows us to locate the high-risk areas of HIV infection with greater accuracy.
In our opinion, in the process of epidemic generalization, the spatial divergence of epidemic will be observed as well. In particular, clusters with high RR of sexual HIV transmission independent from the clusters with high RR of injection HIV transmission may appear.
We used spatial clustering based on reported HIV cases acquired through IDU and sexual transmission from 1994 to 2009 in the smallest administrative units (called Radas) in the rural territory of the Odessa region, Ukraine. For the formal spatial clustering, we used Kulldolf Spatial Statistics, realized in the SatScan program. Clustering was conducted by the Poisson model. We used the circle window and set the cluster size limit empirically at 15% of the at-risk population. The study was done in clusters with high RR.
Visualization was carried out on QuantumGIS.
With clustering, the HIV incidence due to IDU and sexual intercourse were mostly identical in the 1994Á1999 and 2000Á 2004 periods. However, three spatial clusters of sexually acquired HIV emerged in the 2005Á2009 period (RR 0 3.44, p 0 0.0005; RR 0 10.60, p0 0.011; RR 0 2.18, p 0 0.0265), which did not correspond to an increased RR of IDU-acquired HIV (see Fig. 1 ). Proportion of Radas, simultaneously included in the clusters of both types of HIV transmission, decreased from 64.58% in 2000Á2004 to 48.33% in 2005Á2009.
To test the effectiveness of the method, we compared the number of Radas where HIV cases were registered due to sexual transmission only and were not detected due to IDU. In the 2005Á2009 period, we observed an increase in the number of Radas reporting sexually acquired HIV cases but not IDUacquired HIV cases.
The spatial clustering of the HIV epidemic in the rural areas of the Odessa region showed a divergence in the spatial distribution between IDU and sexually transmitted HIV. We believe this finding may indicate the generalization tendencies of HIV epidemic. Our hypothesis has been supported by other epidemiological characteristics, such as: increase in number of sexual HIV cases and their proportion in the total number of HIV cases; increase in proportion of Radas reporting sexually acquired HIV cases but not IDU-acquired HIV cases; increase in the proportion of sexual HIV cases reported in these Radas; HIV seroprevalence among pregnant women in the region accounted in average to 0.9Á1.1%.
To estimate HIV epidemic stage, additional methods of epidemiological analysis like spatial analysis of morbidity can be used. 
OHA, in collaboration with the Johns Hopkins University Applied Physics Laboratory, implemented a syndromic surveillance system, Oregon ESSENCE. A critical component to developing and growing this statewide system is obtaining buy-in and voluntary participation from hospital EDs. This process involves approval at multiple levels within a hospital facility from administration to information technology (IT) staff responsible for sending electronic ED data to the Oregon ESSENCE system. Therefore, developing marketing materials that appeal to a wide range of recruitment audiences is a key step in obtaining stakeholder buy-in. OHA adopted the ISDS and CDC syndromic surveillance standards for the public health objective of the Center for Medicaid and Medicare Services (CMS) Meaningful Use Programs. However, Oregon hospitals will not receive financial incentive to participate in Oregon ESSENCE from CMS until 2014 during stage two of Meaningful Use. Consequently, this project's early years will focus on obtaining voluntary participation from hospitals.
OHA developed a recruitment packet to provide information to hospital Chief Executive Officers, Chief Information Officers, Infection Preventionists, Meaningful Use coordinators and IT staff. The packets will be distributed in a number of ways: primarily, during face-to-face meetings with hospital and public health stakeholders, and also during other forums such as meetings of the Oregon Association of Hospitals and Health Systems as well as broader Meaningful Use seminars. Recruitment folders include a brief overview of syndromic surveillance and the ESSENCE system (Welcome to Oregon ESSENCE); a description of utility (Oregon ESSENCE: Real-time Data for Public Health Action); a list of the requested variables (Oregon ESSENCE Data Fact Sheet); examples of effective uses of ESSENCE (ESSENCE success stories); a visual diagram of the data flow process (Oregon ESSENCE data flow); and a list of action steps to begin participation (Let's Roll).
We developed an informative packet of materials for a variety of audiences that is both appealing and concise. Oregon's hospitals come in all shapes and sizes, each with unique approval processes for engaging in data sharing, prioritization of voluntary public health projects, coordination of Meaningful Use efforts and IT support. Therefore, we expect that the breadth and depth of the marketing materials will be a critical component to successful recruitment of hospitals.
We developed appealing and concise information packets for a variety of audiences. While each individual may not need the full breadth of the information we are providing, depending on their role at the hospital, we anticipate that the recruitment packet provides a useful overview of Oregon ESSENCE and syndromic surveillance to a variety of hospital and public health stakeholders.
Emergency departments; data exchange; marketing *Melissa Powell E-mail: melissa.e.powell@state.or.us
Oregon Health Authority (OHA), in collaboration with the Johns Hopkins University Applied Physics Laboratory, recently implemented Oregon ESSENCE, an automated, electronic syndromic surveillance system. One way to strengthen syndromic surveillance is to include data from multiple sources. We are integrating data from emergency departments, state notifiable conditions and vital statistics and the Oregon Poison Center (OPC). Implementing ESSENCE in Oregon provided the opportunity to automate poison center surveillance, which was previously done manually. In order to achieve this, OHA needed a daily data feed of OPC data to upload into Oregon ESSENCE servers. For OPC to do this directly, they would have incurred significant costs to develop the necessary electronic infrastructure to query and send the data; furthermore, OPC does not employ IT staff. OHA does not currently have funding available to support IT system interoperability with Oregon ESSENCE; so, we sought a low-cost solution that would build upon existing systems that utilized the National Poison Data System (NPDS) web service.
OPC facilitated OHA access to the NPDS web service, which OHA could use free of charge. Access to the web service consisted of requesting approval from the local poison center and adhering to an NPDS web service data use agreement between OPC and OHA. We use FileMaker, a commercial off the shelf database application, to automatically query the NPDS web service on a daily basis. The queried data are then automatically sent from a local database temporarily storing the information to the ESSENCE servers. OHA already uses FileMaker for managing notifiable conditions data (i.e., communicable disease reporting); so, there were no new licensing costs associated with this method.
OPC data are available within the ESSENCE application to OHA syndromic surveillance staff. Sending OPC data into ESSENCE allows OHA staff to monitor timely OPC data in an automated, routine manner. When alerts are generated within the ESSENCE system, they are first assessed by syndromic surveillance staff. Those that require follow-up trigger a call between OHA and OPC. Oregon is the first state to use the NPDS web service to upload poison center data into ESSENCE.
OHA previously monitored OPC data using two methods: (1) through the NPDS system using a web-based interface; and (2) through ToxiTrack, poison center database software. ToxiTrack software is a companion software to Toxicall † , the data collection software system utilized by OPC. Data from Toxicall were transferred via VPN to OHA, where ToxiTrack software was used to view data. Although both of these systems provide unique capabilities for viewing summarized case data, there are limitations in their functionality for situational awareness. ESSENCE offers OHA the ability to easily analyze and report on these data and geospatial graphing capabilities without having to use additional statistical and GIS software.
Integration of poison center data into Oregon ESSENCE supports the initiative to develop a statewide syndromic surveillance system that includes a variety of data sources. It also addresses the need for improved, timely communication between OPC and OHA that was identified following Oregon's response to the 2011 Japanese Earthquake and Radiation event. Because OPC data are integrated into ESSENCE, OHA staff members are able to develop an understanding of expected call volumes and types during day-to-day operations. This is an important component of ongoing situational awareness as we learn what to expect and also how to interpret data from OPC. This resource-effective solution can be applied to jurisdictions that use a variety of applications to monitor their poison center data.
Poison center; web service; integration across data sources; resource-limited settings *Melissa Powell E-mail: melissa.e.powell@state.or.us Introduction Syndromic surveillance of health care data such as the International Classification of Diseases, Ninth Revision (ICD-9), codes related to ILI, was used to track the progression of the 2009 Fall Novel H1N1 outbreak in the Madison area (1) . Early studies focused on prediction of an outbreak; however, further investigation of patient resource utilization would be helpful in developing an action plan for addressing community and patient needs during future outbreaks. There is a paucity of research comparing ED and urgent care utilization rates during the 2009 Novel H1N1 pandemic, though there are regional data suggesting that urgent care centers bore a larger portion of the burden of H1N1 influenza than EDs (2) . Furthermore, one group found that ILI-related phone calls to urgent care centers predicted influenza outbreak at least 1 week ahead of peaks in the ILI hospital care consultation rates (3) . ED data on its own have proven useful for public health disease surveillance (4, 5) , and many studies group urgent care and ED care together. The literature is lacking subgroup analysis of these two very different care environments. Understanding the correlation between urgent care and ED utilization rates will provide a more in depth understanding of the stress that the 2009 Fall Novel H1N1 placed on community resources in our geographic region.
This study is a cross-sectional retrospective analysis of ED vs. urgent care utilization rates for ILI in the greater Madison area from October 2009 to December 2009. The proportion of ILI encounters was calculated for two university-based urgent care centers (grouped) and compared with the ED data from the same university-based system. Proportions were calculated from ICD-9 and total daily encounter volume data.
The average proportion of encounters for ILI at urgent care centers was 0.298 in comparison with 0.125 for ED visits during the 2009 Fall Novel H1N1 influenza outbreak. Graphical trends in illness were comparable.
Patients in our geographic region were 2.4 times more likely to seek care at urgent care centers for ILI during the Fall wave of the H1N1 influenza pandemic. Neither care site predicted the outbreak more effectively than the other. 
The critical need for population-level interventions to support the health needs of the growing population of older adults is widely recognized (1). In addition, there is a need for novel indicators to monitor wellness as a resource for living and a means for prediction and prevention of changes in community health status (2) . Smart homes, defined as residential infrastructure equipped with technology features that enable passive monitoring of residents to proactively support wellness, have the potential to support older adults for independence at the residence of their choice. However, a characterization of the current state of smart homes research as a population health intervention is lacking. In addition, there is a knowledge translation gap between the smart homes research and public health practice communities.
The EBPH movement identifies three types of evidence along a continuum to inform population health interventions: Type 1 (something should be done), Type 2 (this should be done) and Type 3 (how it should be done) (3). Type 2 evidence consists of a classification scheme for interventions (emerging, promising, effective and evidence based) (3). To illustrate typology use with an example: the need for population health interventions for aging populations is well known (Type 1 evidence), many studies show that smart home technologies can support aging in place (Type 2 evidence), but there are few, if any, examples of smart homes as population health interventions to support aging in place (Type 3 evidence).
Our research questions for this systematic review are as follows:
1) What categories of Type 2 evidence from the scientific literature uphold smart homes as an EBPH intervention? 2)
What are the novel health indicators identified from smart home studies to inform design of a community health registry that supports prediction and prevention of negative changes in health status? 3)
What stakeholders are reported in studies that contribute Type 2 evidence for smart homes as an EBPH intervention? 4)
What gaps exist between Type 2 and Type 3 evidence for smart homes as an EBPH intervention?
Our search methodology includes searches of MEDLINE, CINAHL and IEEE conference proceedings databases to provide coverage across a literature that is found in many disciplines and is not well-indexed. As the term 'smart home' is not well-defined, our search terms also include 'telemedicine', 'telehealth', 'e-health', 'health monitoring', 'gerontechnology' and 'gerotechnology' in combination with 'older adult', 'elderly', 'aging', 'ageing', 'community-dwelling' and 'senior'. Our inclusion criteria include any study that describes a technology designed for an older adult audience to support wellness management through social, spiritual, physical or cognitive means (4). Our exclusion criteria include smart homes designed for efficiency and nonhealth-related surveillance technologies.
Initial search results indicate many studies that can be classified as Type 2 evidence along the continuum of emerging, promising, effective and evidence-based smart home interventions. Initial findings are that Type 3 evidence is lacking and public health policy makers are underrepresented.
Early analysis of complete search results will be presented for (1) categorizations of evidence according to the evidence-based public health typology, (2) enumeration of stakeholders reported in included studies and (3) identification of novel indicators of health to inform design of a standards-based community health registry for older adults.
Smart homes; population health; aging in place; older adults; informatics
In May 2001, Boston released a strategic transportation plan to improve bicycle access and safety (1) . According to the Boston Transportation Department, ridership has increased 122% between 2007 and 2009 (2). A collaborative public health and public safety task force was initiated in 2010 to foster a safe and healthy bicycling environment.
The Boston Public Health Commission (BPHC) syndromic surveillance system receives information from ED visits from all 10 acute care hospitals in Boston every 24 hours. Data received include visit date, demographics, ZIP code of residence, chief complaints and ICD-9 CM-coded final diagnosis. Disposition information was reported from 9 of these hospitals in 2010. BPHC collaborated with CDC's BioSense Program to specify a BRI syndromic case definition that combined chief complaint and ICD-9 CM-coded information and excluded motor cycle only related events. Subsyndromes were used to assess the type of injury and severity based upon 47 standard BioSense subsyndromes and 21 subsyndromes developed for this study. The data sample used for this study included over 2 million visits between 2007 and 2010. Injury visits were categorized at the neighborhood level using a standard ZIP code of residenceto-neighborhood mapping. Results were stratified by age, patient neighborhood of residence, race/ethnicity, gender and disposition (2010 data only).
Over the study period, a total of 4510 ED visits were classified as BRIs (0.22%). The percentage of BRI visits increased from 0.18% in 2007 to 0.27% in 2010. The majority of injuries (69%) occurred between May and September (Fig. 1) and likely corresponds to increased bicycling activity during those months.
Seventy-five percent of persons presenting with BRIs were male and 60% reported race/ethnicity as white. Persons aged 18Á 25 years represented 28% of visits and those aged 6Á17 years accounted for 17%. Boston residents accounted for 52% of BRI visits; 15% were from bordering communities. One Boston neighborhood with the highest BRI rate by patient residence also has a large college student population.
Throughout the entire study period (2007Á2010), nearly one quarter (1082) of BRI visits were associated with fractures and dislocations; whereas less than 10% of visits were for sprains or strain injuries. Head injuries were associated with 84 (1.9%) of BRI visits.
In 2010, 149 (11%) of the 1411 BRI visits resulted in admission, most commonly for fractures and dislocations. Twenty-two percent were among individuals aged between 50 and 59 years; 21% were among persons aged 18Á24 years. Fiftyfour percent of all BRI admissions were associated with fractures and dislocations. Thirty-one (2.2%) BRI visits in 2010 were associated with head injuries; of which 11 (35%) were admitted for care. For BRI visits involving falls, 8% were admitted versus 17% for BRI visits associated with a motor vehicle.
Syndromic surveillance can be used to monitor and track BRI and to inform targeted prevention activities such as education and outreach to select at-risk populations (e.g., college students). Presently, information on the environmental context of injuries, such as the precise location of the accident, is limited. As bicycle use increases, improved methods to combine syndromic surveillance, emergency medical services and public safety information are needed to identify accident 'hot spots' to guide implementation of preventive measures.
Injury; prevention; emergency; bicycle; syndromic surveillance 
Animal bites may have potentially devastating consequences, including physical and emotional trauma, infection, rabies exposure, hospitalization and, rarely, death (1). NC law requires animal bites be reported to local health directors (2) . However, methods for recording and storing bite data vary among municipalities. NC does not have a statewide system for reporting and surveillance of animal bites. Additionally, many animal bites are likely not reported to the appropriate agencies (3) .
NC DETECT provides near-real-time statewide surveillance capacity to local, regional and state-level users with twice daily data feeds from NC EDs. Between 2008 and 2010, 110 to 113 EDs were submitting visit data to NC DETECT. Several animal bite-related online reports are available and provide aggregate and visit-level analyses customized to users' respective jurisdictions. The NC DETECT ED visit database currently provides the most comprehensive and cost-effective source of animal bite data in NC.
Several NC DETECT animal bite-related reports were developed based on chief complaint and triage note keyword searches and ICD-9-CM codes. Using the Animal Bite Keyword Report, statewide ED visit data were extracted for 2008Á2010. ED visit records in NC DETECT were examined manually to assess the performance of case definition keywords. Using the Animal Bite ICD-9-CM Code Report, statewide ED visit data were extracted for 2008Á2010. The following ICD-9-CM injury codes are included in this report: E906.0 (dog bite), E906.1 (rat bite), E906.3 (bite of other animal except arthropod) and E906.5 (bite by unspecified animal). The burden of ICD-9-CMÁcoded animal bite visits to total ED visits was examined by age group and gender.
Review of Animal Bite Keyword Report data revealed several additional case definition inclusion and exclusion keywords. This knowledge has led to continued development of keyword reports. The Animal Bite ICD-9-CM Code Report indicated a total of 33,294 ED visits for animal bite from 2008 to 2010. For each year, the highest proportion of ICD-9-CMÁcoded animal bite ED visits to total ED visits were for 5Á9 year olds (Fig. 1) . Across all 3 years, males had a slightly higher proportion of animal bite-coded ED visits to total ED visits (0.28%) compared to females (0.23%).
Case definition development for the Animal Bite Keyword Report is an iterative process. Sensitivity and specificity of keyword reports must be considered, and case definitions should depend on the report's intended use. Evaluation of the Animal Bite ICD-9-CM Code Report showed 5Á9 year olds and males have the highest proportion of animal bite-coded ED visits in NC. A snake bite report and animal bite incidence rate reports are under development. NC DETECT is a valuable source for animal bite surveillance in NC.
Animal bite; surveillance; emergency department 
In MarchÁApril 2011, Salt Lake Valley Health Department (SLVHD) investigated an outbreak of measles (N 09) resulting from a single imported case from Europe. Syndromic surveillance was used to identify measles-like illness (MLI) and enhance early case detection, which is crucial for proper public health intervention (1).
Daily text-based chief complaint data, March 23ÁMay 5, 2011, from 15 syndromic sites were obtained from EpiCenter (2) (funds provided by Utah Department of Health), mapped to 5 MLI syndromes (Table 1 ) and summarized using the Early Aberration Reporting System (EARS) (3) . Events of interest included all 'rash' events that contributed to an alert or had a concerning chief complaint (e.g., eye pain), all 'febrile rash' events that had a concerning chief complaint, all 'prodrome' events that had a concerning chief complaint, all 'case definition' events April 7, 2011 onward (date after which public health intervention was still possible) and all 'measles/testing' events. Visit notes, laboratory tests and results were obtained daily for each event of interest and reviewed for MLI. Summary findings, including diagnoses, laboratory results, rash descriptions and suspect exposures, were documented and non-MLI events were ruled out. Events of high suspicion for measles were further investigated via patient interview by phone and/or home visit.
Ninety-seven events of interest (of 2365 events captured in MLI syndromes) were identified: 32 rash, 58 febrile rash, 1 prodrome, 12 case definition and 8 measles/testing (14 were categorized in ! 1 syndrome). Eighty-four events of interest were ruled out based on chart findings. Thirteen events of high suspicion for measles required further investigation. Twelve events were ruled out based on negative measles IgM results, evidence indicating other diseases (fifth disease and Kawasaki syndrome), vaccine reaction or inaccurate documentation of clinical symptoms. One event was found to be confirmed by positive measles IgM (Fig. 1) .
Early identification of a measles case using syndromic surveillance during an outbreak was crucial in reducing contact exposures, preventing additional cases and reducing the cost associated with proper public health intervention. We estimate that early detection of the remaining 8 confirmed cases by syndromic surveillance could have reduced the direct cost of the outbreak by 82%. Syndromic surveillance played a significant role in curtailing the outbreak as a valuable tool to supplement active surveillance.
Measles; syndromic surveillance; early detection; outbreak; cost Introduction Syndromic surveillance systems use electronic health-related data to support near-real time disease surveillance. Over the last 10 years, the use of ILI syndromes defined from emergency department (ED) data has become an increasingly accepted strategy for public health influenza surveillance at the local and national levels. However, various ILI definitions exist and few studies have used patient-level data to describe validity for influenza specifically.
A retrospective design was used to evaluate clinical records for a predictive model of lab-confirmed influenza. Children who presented to the ED at Seattle Children's Hospital between January 1, 2001 and May 31, 2005 were eligible for inclusion in the study. The accuracy of four syndrome definitions were compared for identifying lab-confirmed influenza: (1) ILI from chief complaint (CC) or diagnoses codes (''ILI''); (2) ILI from CC alone; (3) febrile illness from CC or diagnoses (''Febrile''); and (4) febrile illness from CC alone. We evaluated syndrome validity over two distinct time periods: (1) the ''discrete'' 2003Á04 influenza season, which had relatively less co-circulation of influenza and respiratory syncytial virus (RSV) compared to most years, and (2) the ''concomitant'' 2000Á05 influenza seasons (excluding 2003Á04), when influenza and RSV co-circulation was high. Analyses during the concomitant years were further stratified by age B5 years and ]5 years. Multiple imputation was used to address missing viral lab results. The imputation model was based on testing guidelines in place at the hospital during the time of study.
We studied approximately 14,000 visits during the discrete year and 32,000 visits during concomitant years. Viral results were unavailable for approximately 75% of respiratory visits and multiple imputation was used to impute values. During the discrete year, sensitivity and specificity were 0.49 (95% Confidence Interval [CI]: 0.30, 0.68) and 0.72 (CI: 0.70, 0.74) respectively, for the ''Febrile'' definition and 0.29 (CI: 0.13, 0.54) and 0.89 (CI: 0.87, 0.90) for the ''ILI'' definition. ILI sensitivity was 2.05 (CI: 1.08, 3.91) times greater and its false positive fraction 44% (CI: 37%, 49%) lower in concomitant years compared to the discrete year. Greater sensitivity and false positive fractions (1-specificity) tended to be produced by the febrile definitions than by the ILI definitions; and by definitions derived from CC or diagnoses as compared to those from CC alone. The false positive fraction of all syndrome definitions was higher in younger children compared to older children.
Although the sensitivity of syndromic ILI definitions was not high by clinical standards, our interest was to understand the proportion of influenza cases in the community being captured by the system. ED ILI may provide a more robust estimate of the burden of disease than laboratory surveillance, which captures only a subset of patients seen by a healthcare provider and who were tested. The higher specificity of the ILI definitions suggests it is best used for situational awareness during influenza outbreaks and for distinguishing influenza from other viral agents. The use of several definitions throughout the season may be most appropriate in some settings. Public health practitioners should bear in mind that syndrome performance may vary by season and year. Higher syndrome specificity among older children suggests specificity in adults should be higher than that observed for younger populations. However, the generalizability of these results to adult populations and other hospitals is uncertain and should be further studied.
The electronic surveillance system for the early notification of community-based epidemics (ESSENCE) is the web-based syndromic surveillance system utilized by the Maryland Department of Health and Mental Hygiene (DHMH). ESSENCE utilizes a secure, automated process for the transfer of data to the ESSENCE system that is consistent with federal standards for electronic disease surveillance. Data sources in the Maryland ESSENCE system include ED chief complaints, poison control center calls, over-the-counter (OTC) medication sales and pharmaceutical transaction data (specifically for antibacterial and antiviral medications). All data sources have statewide coverage and are captured daily in near real-time fashion.
Forty-six EDs, two major pharmacy chains, two poison control centers and the Centers for Disease Control and Prevention (through a pilot partnership), all contribute data to ESSENCE on a daily basis. Data reported from June 1, 2009, through January 1, 2011, were used to examine the relationships between ED visits for ILI and antiviral (M2 inhibitors and neuraminidase inhibitors) prescription medication transactions in the state of Maryland. ArcGIS 9.2 was used to spatially evaluate these relationships. Data for the total population of Maryland by jurisdiction were obtained from the U.S. Census Bureau, Census 2010 PL94-171 release and prepared by the Maryland Department of Planning, Projections and Data Analysis/State Data Center, April 2011.
Generally, jurisdictions with the highest populations also had the highest number of ILI ED visits and the highest numbers of antiviral prescription medication transactions. These results did not vary based on type of antiviral medication. There was one exception to the general trend: County 14 had the lowest percent of ILI ED visits (0.45%) but the highest percentage of antiviral prescription medication transactions (1.26%). Spatial analysis showed that the highest number of ILI ED visits were in the National Capital Region (NCR) and Central Maryland while the highest numbers of antiviral prescription medications were in the NCR Region.
The trends seen in this analysis follow what is to be expected; the counties with the larger populations had higher numbers of ILI ED visits and higher antiviral prescriptions. These larger counties have more hospitals, which allows for greater access to EDs. County 14 has only one hospital that contributes data to the ESSENCE system; thus, residents may have traveled to an ED in another county but filled a prescription in their home county. This could account for why County 14 had the lowest number of ILI visits and the highest number of antiviral prescriptions. This county also has a very high median income; thus, it is possible that ED visits were lower because more individuals sought medical attention from primary care physicians. Other counties may follow these same trends.
The ESSENCE system has been a useful tool in the tracking and monitoring of diseases such as influenza. It is also used as an indicator to local health departments to begin preparation for flu season. DHMH will continue to use syndromic surveillance on a daily basis for early detection of seasonal and pandemic influenza. 
Evaluation was conducted through electronic telecommunication with the disease control staff of the D/M HOs and checking of websites and S-R electronic bulletins.
Together with the staff of the Sardjito Hospital (teaching hospital of the GMU SM), local D/MHOs and the GMU MS Department of Public Health, the CHSM developed Standard Operating Procedures for S-R core and support functions of MNCH priority diseases (e.g., postpartum bleeding, preeclampsia/eclampsia, LBW and pneumonia) as well as of other major diseases (e.g., malaria, TB, DM and hypertension). The training sessions and consultations were effectively executed in the target districts and municipalities. Based on the performance of the electronic bulletins, however, only one DHO has a functioning S-R support unit. Efficacy of the S-R Systems strengthening approach used by the CHSM could not be evaluated by way of on-the-spot interviews, observation and review of S-R records/ reports due to financial constraints.
A more intensive strengthening method is required to ensure sustainable operation of core and support functions of S-R Systems. The MoH is considering to post at least one Field Epidemiology Training Program (FETP) graduate at the D/M HOs. These FETP graduates, and students, could be used to build and enhance S-R systems for priority diseases and to conduct valid monitoring-evaluation.
Surveillance-response; MNCH; strengthening; Indonesia
The Government of Indonesia (GoI) aims to eliminate malaria by 2030 in 4 stages (1). To reach the elimination phase, High Case Incidence (HCI) areas go through a preelimination phase. The aim of the proposed project is to support one of the Stage 3 provinces in reaching the preelimination phase by 2015 and to assist its HCI districts and municipalities in reorienting their programs to malaria elimination. The preelimination phase can be attained by following these evidence-based technical strategies: (1) prompt and accurate diagnosis of cases; (2) prompt treatment with effective medicines, including intermittent preventive treatment in pregnancy (IPTp); (3) selective, targeted and integrated vector control; and (4) emergency and epidemic preparedness (2) .
These strategies can only be properly carried out if the District/Municipal Health Offices (D/M HOs) have a timely, useful and reliable malaria surveillance-response (S-R) system. The use of computers and electronic telecommunication networks has sped up the flow of institution-based case surveillance data to the D/M HO.
To increase its usefulness, however, the S-R system must include data of cases detected in the community along with data of the disease agent and environment. The proposed project will include the collection of all these surveillance data in order to be useful for the implementation of malaria control strategies. Furthermore, to increase timeliness and reliability, the project will support the malaria S-R systems of the Bangka Belitung Province, Sumatera, by means of cell phone (CP) applications through the following activities:
To develop a CP software for a malaria S-R system and to set up a malaria S-R central data bank (CDB) that will be placed at a commercial hosting server.
To set up a village CP network in each HCI village for demographic data and home malaria management (HMM) data reporting by households.
To train village midwifes or village malaria workers (VM/ MW) to provide HMM, to administer IPTp to pregnant members, to send diagnosis, treatment and IPTp data to the CDB and to obtain blood films for microscopic examination by the HC parasitology microscopist.
To train HC parasitology microscopists to perform microscopic examinations, to use a microscopy-enabled CP for sending microscopy images to the Provincial Lab for reliability testing and to send data to the CDB.
To train HC coassistant entomologist, D/M HO assistant entomologist and Provincial HO entomologist to collect vector and environment data, to use a CP for sending vector control targets data to the CDB and to use a microscopy-enabled CP for sending microscopy images to the Provincial Lab for reliability testing.
To provide consultations, training and resources for the D/ MHOs to facilitate the utilization of CP applications for reporting demographic and HMM data and drug and insecticide resistance/efficacy sentinel surveillance and to facilitate sector and intersector surveillance-based rapid and planned response decision making.
To ensure the attainment of project objectives, the GMU CHSM and collaborating institutions will (1) obtain endorsements from the heads of the District/Municipal and Provincial Governments; (2) engage MoH Directorate of Malaria and GMU Medical School staff in workshops and technical guidance; (3) engage malaria program managers of the District/Municipal and Provincial HOs as project field coordinators; and, (4) recruit experienced technicians as on-the-job trainers in parasitological microscopy, entomology and information technology and experienced experts as trainers and consultants in public health surveillance and management.
Poster presentation at the ISDS 10th Annual Conference 2011.
Description: CDC works to save lives and protect people during major public health events. In an effort to support these processes, CDC established CTS, which is maintained within the Division of Informatics Solutions and Operations (DISO), in the Public Health Informatics and Technical Program Office (PHITPO). CTS consists of four system components, which interoperate to improve communications and event response efficiency while still functioning independently, recognizing the unique requirements and use cases for each system. Collectively, the data consolidated from these systems can show population coverage, numbers of untreated individuals, drug and equipment shortages, need for resupply and more. The web-based applications are deployed centrally at CDC and use the CDC's secure data access method for security.
The first of these components is the Inventory Management and Tracking System (IMATS), currently under development. IMATS provides state and local public health providers with a tool to track medical and nonmedical countermeasure inventory and supplies during daily operations or an event. The solution tracks quantities of inventory, monitors reorder thresholds and facilitates warehouse operations including receiving, staging and storing of inventory.
The Communications Portal is a web-based content management system in development, which consolidates important event response details into one place and will provide timely and adequate information to states and other jurisdictions. This system is complementary to the IMATS as it manages communications related to, but not limited to, Emergency Use Authorization (EUA), Investigational New Drug (IND) and recall notices.
Preparing for the future of public health surveillance also requires innovative and appropriate informatics systems that provide timely and accurate response to all-hazards events. CTS and its components are developed to assist in all types of surveillance needs.
Countermeasure; all hazards; public health; inventory; tracking
For public health surveillance to achieve its desired purpose of reducing morbidity and mortality, surveillance data must be linked to public health response. While there is evidence of the growing popularity of syndromic surveillance (1, 2) , the impact or value added with its application to public health responses is not well described (3) .
Ontario's 36 public health units, the provincial ministry of health and federal public health agency completed a web survey in 2010 to identify surveillance systems used routinely and during the pandemic and to describe the perceived utility of systems for monitoring pandemic activity and informing decision making. Follow-up semi-structured interviews were conducted with key informants to elucidate drivers for specific public health actions taken during the pandemic and, specifically, to understand the role syndromic data played in influencing decisions among those who had access.
The web survey identified 20/38 (53%) organizations which use at least one syndromic surveillance system; key informant interviews identified another 2 organizations, for a total of 22/38 (58%) syndromic surveillance 'users'. Mirroring routine surveillance, traditional surveillance systems, specifically laboratory and reportable disease data (iPHIS) and school absenteeism data were the most frequently used sources during the pandemic (Fig. 1) . Laboratory data were considered the most useful data source for monitoring the epidemiology (71% of organizations perceived it as 'essential') and informing decision making (76%), while emergency department screening data were considered the most useful syndromic surveillance source (52% and 70%). Syndromic data were found to support two specific public health actions taken during the pandemic: influenza assessment centers and communications, including recommendations to the public. Informants felt that syndromic data provided confidence to decide whether and when to open/close influenza assessment centers and when/what to communicate to the public. Nonsyndromic users utilized stakeholder consultations and traditional surveillance data to support these decisions. Syndromic surveillance did not appear to have a role in supporting decisions around immunization clinics, school closures or recommendations to health care professionals; rather vaccine availability and ministerial guidance acted as drivers for these decisions.
While traditional surveillance systems were considered most essential for monitoring the pandemic locally and informing decisions, syndromic surveillance was found to support several public health actions taken during the pandemic. Understanding how syndromic surveillance systems are valued, utilized and linked to public health action is necessary to inform investments to build surveillance capacity.
Public health surveillance; syndromic surveillance; pandemic influenza; evaluation 
Informal surveillance systems like HealthMap (HM) are effective at the early detection of outbreaks (1, 2) . However, reliance on informal sources such as news media makes the efficiency of these systems vulnerable to newsroom constraints, namely highprofile disease events drawing reporting resources at the expense of other potential outbreaks and diminished staff over weekends and holidays. To our knowledge, this effect on informal or syndromic surveillance systems has yet to be studied.
Using HM's English-language global infectious disease database events (3), we identified expected periods of decreased reporting of infectious disease due to newsroom constraints between July 1, 2008, and August 31, 2011. Crowdout events were defined as averaging greater than five events per day for at least 2 weeks, plus making up at least 50% of daily events. Meeting these criteria were H1N1/swine flu (two instances), Haiti's cholera epidemic, 2010, and the E.coli outbreak in Germany, 2011. The December holiday period, when most of the newsroom is off duty, was also tested. We examined whether the average number of noncrowdout events differed significantly from the average daily HM events at baseline, defined as similarly structured periods without holidays or high-profile epidemic events. Baselines were measured before and after the crowdout period, plus during the same time period in other applicable years. Means were compared using paired t tests with unequal variances.
The two instances where H1N1 met inclusion criteria both resulted in average numbers of daily events significantly lower than similar periods before, after and parallel to the time period in question (Fig. 1) . See Table 1 for more results. On average, the greatest number of daily events occurs on Thursdays, least on Sundays. The outbreaks of cholera in Haiti and E. coli in Germany showed no significant crowdout effect at both global and regional levels. A reduction in the average events per day during the December holiday period was not significant.
Informal surveillance has limitations that are exacerbated by newsroom constraints. During the global H1N1 pandemic, significantly fewer infectious disease events were recorded by HM's informal surveillance system. Crowdout poses a risk for epidemiological surveillance since decreased relative surveillance may postpone reporting of outbreaks. Moreover, crowdout during H1N1 showed that this phenomenon can endure for long time periods. However, regional outbreaks like cholera in Haiti or E. coli in Germany do not appear to affect informal surveillance on a global or regional scale.
Informal surveillance; syndromic surveillance; infectious disease; epidemics; media 
Epi-X is an internet-based secure website for the exchange of information regarding developing public health events. Reports are exchanged with state epidemiologists, state health officers and other key public health officials. Provisional and secure information is regularly posted on Epi-X. The Epi-X user base is restricted to public health officials at the local, state, federal and international levels. Private healthcare practitioners who do not otherwise hold a government position are not given access to Epi-X. As of August 2011, Epi-X has approximately 6000 users, of which approximately 1600 are authorized to directly contribute reports regarding developing public health events. Epi-X is frequently used to seek reports of cases of illness related to an outbreak, cluster or increased occurrence of a specific infectious disease. The usability and usefulness of Epi-X in this capacity have not previously been assessed.
A total of 52 case-seeking reports were posted on Epi-X during calendar year 2010, all of which were used to seek cases of infectious disease. Epi-X staff were successful in eliciting contributor feedback in regards to 30 of these reports. Four questions were asked that assessed the motivation behind posting a case-seeking report on Epi-X, the practicality of posting a case-seeking report on Epi-X, the successfulness of finding related cases by posting a case-seeking report on Epi-X and if the contributor intends to use Epi-X for this purpose in the future.
Of the 52 case-seeking reports posted on Epi-X during calendar year 2010, all were posted with the intent of seeking cases of illness caused by infectious disease. One report was broad based and also sought cases of illness caused by injury. These reports were categorized by type of infectious agent, depending upon commonality of symptoms and routes of transmission. Epi-X contributors posted case-seeking reports for 19 individual confirmed or suspected infectious agents in 2010. The top four infectious agents for which case-seeking reports were posted on Epi-X in 2010 were Salmonella (10 reports), Legionella (9), hepatitis A virus (4) and measles virus (4) . Other infectious agents included Influenza, Bordetella, Cryptosporidium, Escherichia coli and Listeria. Three reports were posted for which the infectious agent was unknown.
The 52 reports were contributed by 44 contributors. Epi-X staff were able to elicit feedback from contributors for 30 reports. In regards to usability, the system was considered practical for 28 of the 30 reports for which feedback was elicited. In regards to case-seeking usefulness, 2 of the 30 case-seeking reports for which feedback was elicited were considered not successful; eight were considered moderately successful, and 15 were considered fully successful. For five reports, the contributor was unable to rank the success. Of the 30 respondents, 28 stated their intent to use Epi-X for this purpose in the future.
Epi-X case-seeking reports were considered at least moderately successful in 23 of 30 reports for which feedback was elicited. In some instances, investigators expected to find no other related cases but posted their reports to make sure they had been thorough. Some investigators regarded their report(s) as successful, despite not finding any additional cases. Investigators may become more confident that all related cases have already been identified if they do not find additional cases as a result of posting on Epi-X.
Epi-X has become a standard method of identifying related cases. For investigators seeking additional cases in other statelevel jurisdictions, posting case-seeking reports on Epi-X is a practical method. Increased use would likely strengthen the public health response to emerging infectious-disease events.
The Epidemic Information Exchange; Epi-X; surveillance; secure; case finding *James Schwendinger E-mail: bnz2@cdc.gov 
Hepatitis Avirus (HAV) infection is usually mild in childhood but more severe in adolescents and adults. An estimated 1.4 million cases of HAV infection occur annually in the world. The casefatality rate among patients of all ages is approximately 0.3% but tends to be higher among older persons (approximately 2% for 40 years or older). HAV is a notifiable disease on weekly basis where health centers and hospitals report cases to the health directorates, which in turn report electronically to the Communicable Diseases Directorate, with subsequent paper reporting of detailed epidemiological description. The due time is Tuesday next week. Diagnosis is clinically based and depends on case definition. A previous study in Jordan revealed that reporting rate increased from 6.4 in 2004 to 7.9 in 2008/100,000, the highest reporting rate was in the North region, mainly Mafraq.
Ten health centers and one hospital were randomly selected; 13 weeks were also selected randomly from the year 2009. The reporting process was reviewed in the three levels for the number of reported cases of HAV in the selected weeks: the peripheral level by reviewing the reporting forms, notifiable logbooks of the reporting sites; the intermediate level in the health directorate by reviewing the specific notification forms(SNF) from each reporting site, and the comprehensive forms from all reporting sites; and the central level by reviewing the electronic and paper reporting to the communicable diseases directorate.
The SNF were found for only 15% of reported HAV from Health Directorate in 2009. All the selected reporting sites had commitment in reporting. The sensitivity of reporting from reporting sites to health directorate was 96%; nevertheless, 38% of the reporting sites reported zero cases. HAV surveillance in Mafraq was evaluated upon application of CDC criteria for evaluation of surveillance system (as demonstrated in Table 1 ).
The increased number of HAV-reported cases in Mafraq is not related to a public health hazard; it is probably a result of relatively reasonable surveillance system. The reporting protocol is not well implemented, it is mostly phone based, and this will weaken the sensitivity of surveillance system; therefore, paper-based reporting should be enhanced.
Hepatitis A; surveillance; evaluation; Mafraq, Jordan The diagnosis is clinically based and does not depend on laboratory test. Surveillance does not require complex training, equipments or fulltime working personnel. Reporting procedure is telephone based and 'regular mail' based, which is affordable to all reporting centers
The sureveillance system for HAV is clinically based, it includes also suspect and probable cases, and the reporting is according to available facilities. Case definition could be easily modified to cope with any addition Acceptability Timeliness Almost 38% of the reporting sites did not report any case in 2009; also, 23% of the reporting sites reported three cases or less, this could reflect that the surveillance for hepatitis A is not well accepted Almost all reporting centers reported to the health directorate in exact time by telephone, this is followed by paper reporting. Only 42% of the reports from Mafraq health directorate to the Communicable Diseases Directorate were done electronically; about 90% of the electronic reporting was done on time, 'Tuesday' the next week
Data quality HAV surveillance is considered representative as monthly reports give detailed epidemiological information
The SNFs were found for only 15% of reported HAV cases from Health Directorate; none of HAV cases were investigated. Discrepancies were observed in the reported numbers, as 7% in excess was found in the reporting centers registry in comparison with the original reporting center SNFs; while 6% less was observed between the numbers of reported HAV cases in health directorate registry in comparison with reporting center registry 
The spread of infectious diseases is facilitated by human travel. Disease is often introduced by travelers and then spread among susceptible individuals. Likewise, uninfected susceptible travelers can move into populations sustaining the spread of an infectious disease.
Several disease-modeling efforts have incorporated travel and census data in an effort to better understand the spread of disease. Unfortunately, most travel data are not fine grained enough to capture individual movements over long periods and large spaces. Alternative methods (e.g., tracking currency movements or cell phone signals) have been suggested to measure how people move with higher resolution but these are often sparse, expensive and not readily available to researchers.
FourSquare is a social media application that permits users to 'check-in' (i.e., record their currentlocation at stores, restaurants, etc.) via their mobile telephones in exchange for incentives (e.g., location-specific coupons). FourSquare and similar applications (Gowalla, Yelp, etc.) generally broadcast each check-in via Twitter or Facebook; in addition, some GPS-enabled mobile Twitter clients add explicit geocodes to individual tweets.
Here, we propose the use of geocoded social media data as a real-time fine-grained proxy for human travel.
Sixty-eight million geocoded entries (tweets and check-ins) from 3.2 million users were collected from the Twitter streaming API for the period from September 11, 2010 through January 28, 2011. The Twitter API provides a random sample of tweets; nongeocoded tweets or tweets originating from outside the United States were discarded. In addition, users with fewer than 6 records, or those who check in too frequently (more than once in 5 seconds) or travel too quickly (faster than 1800 km/hr) were removed to exclude automated bots or other location spam.
We analyzed a 5-week subset of the data (September 11, 2010 through October 26, 2010) consisting of 3 million record intervals from 165,000 users.
We display intrastate travel by aggregating each user's consecutive records within each state and plotting only transitions between states (Fig. 1) . The denser edges represent more frequent transitions, illustrating the pattern of travel on a national scale. We also constructed a heat map representation of Manhattan (Fig. 2) by aggregating users' check-ins with 500 m resolution. A larger bubble represents a denser set of records in that geographic area.
By linking each individual users' consecutive location records together, we computed the statistical distribution of time interval and distance traveled between records. About half of the checkins are less than 6 hr and no more than 1km apart from each other.
We show that social media location data can be used as multiscale proxy for travel at the national, state and urban level. These data are inexpensive and easily obtained. Furthermore, they can be used not only to understand historical travel but also to monitor in realtime changes in travel behavior to help inform disease surveillance.
Future work, currently underway, will validate this source of information against other sources of travel data and will investigate its value to better understand the spread of infectious diseases for disease monitoring and surveillance purposes. 
Adverse drug events (ADEs) are a major cause of morbidity and mortality (1, 2) . However, postmarketing surveillance systems are passive, and reporting is generally not mandated (2) . Thus, many ADEs go unreported, and it is difficult to estimate and/or anticipate side effects that are unknown at the time of approval. ADEs that are reported to the FDA tend to be severe, and potentially common, but less serious side effects are more difficult to characterize and document. Drugs with a high risk of harm outweighing the therapeutic value have recently been subjected to a greater level of interest with the Food and Drug Administration's Risk Evaluation and Mitigation Strategies (REMS) (3). However, no rapid method to detect if the REMS produce the desired effect and assessment of the impact is conducted by the drug manufacturer.
Increasingly, Americans have been turning to the internet for health-related information, largely by the use of search engines such as Google. The volume of searches for drugs and ADEs provides a unique insight about the interest in various medications and side effects as well as longitudinal changes.
We generated a list of the 179 most commonly used drugs in the United States in 2008 based on the Agency for Healthcare Research and Quality's Medical Expenditure Panel Survey (MEPS). Using this list of drugs, we consulted MicroMedex, a drug database, for information regarding possible ADEs for each drug. Next, we then obtained search volume data from Google Insight for all possible pairs of drugs and ADEs.
Using a set of searches restricted to only the known ADEs for a given drug, we coded each ADE as either common or other as listed by MicroMedex. Based on this categorization, we conducted a Wilcoxon two-sample signed rank test. Finally, we constructed a negative binomial model to explain the number of ADEs found by Google Insights. The total number of detected ADEs was modeled using the number of common ADEs in MicroMedex, the number of other ADEs in Micro-Medex and the number of prescriptions for the drug based on 2008 data from MEPS as covariates.
A second list of 149 drugs with REMS was obtained from the FDA and search volume as collected for each of the drugs. We fit a generalized linear model to the data starting 1 year before and ending 1 year after the initial REMS approval date. The model included a dummy variable indicating if the month occurred before or after the initial approval of the REMS. The interaction between this variable and the time covariate was used to determine if the REMS had any impact on interest as measured by search volume.
Both the Wilcoxon signed rank test and the negative binomial model indicate that Google Insight more readily detected common ADEs compared to the other ADEs. The Wilcoxon rank sum test indicated a shift toward more complete detection for the common ADEs compared to other ADEs (pB 0.001).
The negative binomial had similar results. The marginal increase in the number of ADEs detected by Google at the median for both the common and other ADEs was similar at 1.27 and 1.29, respectively. However, the median values were 7 and 39, respectively.
Only 40% (59/149) of drugs with a REMS approval demonstrated a change in slope with 90% confidence. The remaining 60% (90/149) indicated no significant change in interest over the time frame.
Our data help validate the use of Google Insights and search volume as a means to estimate the relative incidence of ADEs. In addition, internet search volume can be used as a rapid means for detecting new or changing ADEs after approval. Finally, the severity and frequency of ADEs may vary within a particular drug class, and search volume may provide additional information for guiding clinicians to select a given drug within a class.
The release of the REMS failed to create a change in search volume for the majority of the drugs. This may be due to prior elevated interest as the result of previous safety alerts or may be an indication that the REMS fails to create increased awareness of the risks of the drug. Further analysis of FDA safety alerts or change point analysis may provide a greater understanding of the effect of various risk management methods.
Keywords adverse drug events, Google Insights, post-marketing surveillance
To assess the impact of use of mobile phones use on the efficiency and effectiveness of the Integrated Disease Surveillance Project (IDSP) in the state of Andhra Pradesh (AP).
Public health surveillance systems are constantly facing challenges of epidemics and shortage in the healthcare workforce. These challenges are more pronounced in developing countries, which bear the greatest burden of disease and where new pathogens are more likely to emerge, old ones to reemerge and drug-resistant strains to propagate. In August 2008, a mobile phone-based surveillance system was piloted in 6 of the 23 districts in the state of AP in India. Health workers in 3832 hospitals and health centers used mobile phones to send reports to and receive information from the nationwide Integrated Disease Surveillance Project (IDSP). Like in many other states, the IDSP in AP is facing many operational constraints like lack of human resource, irregular supply of logistics, hard to reach health facilities, poor coordination with various health programs and poor linkages with nonstate stakeholders. The mobile phone-based surveillance system was an attempt to tackle some of the barriers to improving the IDSP by capitalizing on the exponential growth in numbers as well as reach of mobile phones in the state. Promising results from the pilot of the system led AP state to extend it to about 16,000 reporting units in all 23 districts. This study evaluates how the system has affected the efficiency and effectiveness of IDSP in the state.
Key informant interviews, focus group discussions, record reviews and surveillance data analysis were conducted at the District Surveillance Units (DSUs), Primary Health Centers(PHCs)and Health Subcenters (HSCs). Five out of the 23 districts were selected for the evaluation using a probability proportion to size sampling strategy. Six PHCs were selected randomly from each of these 5 districts and 1 HSC was selected randomly from each of the PHCs. A total of 30 PHCs and HSCs were visited for evaluation.
The mobile phone-based system was being used only by 20 to 60% of the reporting units. Since the start of the system, there was an increase of 10 to 25% in completeness of IDSP reports. There were significant gains (12Á30%) in timeliness of reports. The system was saving time and money on logistics when compared to paper-based reporting. Public health workers in the field were enthusiastic about the system but were not using it as widely or extensively as was possible, because of lack of clear directives for implementation; lack of guidelines for usage and lack of systematic training of workforce for using the system.
Use of mobile phone technology has the potential to enhance the overall efficiency and effectiveness of the IDSP but will require clear policy directives and guidelines for deployment and usage, systematic training plans and adequate resources for the technology to be accepted and used universally in the state. Our evaluation findigs suggest that to maximize the potential benefits of mobile technology in health systems, its use should be based on evidence from operational, technical and technological feasibility studies. This study will prove useful for scaling up such strategies toward disease surveillance systems in countries with similar operational challenges and ready access to mobile phones.
Integrated disease surveillance; mobile technology; surveillance quality; feasibility; policy
The use of syndromic surveillance systems by state and local health departments for the detection of bioterrorist events and emerging infections has greatly increased since 2001. While these systems have proven useful for tracking influenza and identifying large outbreaks, the value of these systems in the early detection of bioterrorism events has been under constant evaluation (3, 4) .
Several U.S. anthrax infections have been identified since the 2001 Amerithrax attacks. These cases were investigated by a number of local, state and federal agencies, and most were subsequently associated with exposure to imported animal hides contaminated with anthrax spores of natural origin (5Á7). Each incident presented a unique diagnostic challenge since all three forms of the disease (inhalation, cutaneous and gastrointestinal) were identified. All of the cases were reviewed to determine which laboratory and surveillance systems were used to first identify possible cases and the number of days required to confirm the diagnosis of anthrax. The role of syndromic surveillance and other advanced surveillance systems in identifying these cases and searching for additional cases was evaluated. Efforts to coordinate surveillance and communication efforts among the various jurisdictions involved in the investigation of these cases were also noted.
A review of these post-Amerithrax incidents revealed that all the cases were identified by astute clinicians using improved laboratory techniques. The time required to suspect and confirm the diagnosis of anthrax decreased with each subsequent incident, with increased awareness of animal sources of anthrax combined with improved compliance with laboratory reporting protocols. While syndromic surveillance systems did not identify the initial patients, these systems were used to search for additional cases. These efforts were enhanced when they were well coordinated among all jurisdictions.
The single local sources of exposure in most of these cases limited the value of these incidents to test the ability of syndromic surveillance systems to detect potential bioterrorist attacks. However, each incident provided valuable experience in the use of advanced laboratory and syndromic surveillance systems in the identification of anthrax cases. Although 10 years of surveillance system development has enhanced our nation's preparedness, use of outbreak modeling exercises in conjunction with regional and national multijurisdictional public health working groups, such as the Distribute Community of Practice, can further test and develop our ability to respond to bioterrorist attacks and emerging disease.
Anthrax; syndromic surveillance systems; disease detection; modeling
Parallel surveillance, separate monitoring of each continuous series, has been widely used for multivariate surveillance; however, it has severe limitations. First, it faces the problem of multiplicity from multiple testing. Also, the ignorance of CBS reduces the performance of outbreak detection if data are truly correlated. Finally, since health data are normally dependent over time, CWS is another issue that should be taken into account. Sufficient reduction methods are used to reduce the dimensionality of a simple multivariate series to a univariate series, which has been proved to be sufficient for monitoring a mean shift in multivariate surveillance (1, 2) . Having considered the sufficiency property and the nature of health data, we propose a sufficient reduction method for detecting a mean shift in multivariate series where CWS and CBS are taken into account. (2) proposed a sufficient reduction method used for monitoring a mean shift in multivariate series where observations are assumed independent. Also, the former allows for CBS while the latter does not. In this study, we further develop sufficient reduction methods by taking CWS and CBS into account. At each time point, data from p-dimensional multivariate series are used to calculate sufficient statistics derived from the likelihood ratio between out of control and in control states. The evaluation of this method is by simulation study, where bivariate series are generated daily from different sets of parameters (whether or not CWS and/or CBS are present) (3). Detection of a mean shift, which is 2, 3 or 4 times standard deviation of background data, is investigated. A EWMA chart is used to monitor the resultant series of sufficient statistics, and the conditional expected delays (CED) and false alarm rates (FAR) (4) from four methods are compared.
Three examples from our simulation study are shown in Table 1 . Data are generated from three different sets of parameters (CWS (8) and CBS (r)), and the aim is to detect a shift in mean of 2s.d. Dataset 1 has no CWS and CBS (800 and r 00). Dataset 2 includes CWS (8 00.4), while dataset 3 presents both CWS and CBS (8 00.4 and r00.3). For all datasets, the parallel method gives longer delays compared with other methods. In the case of dataset 1 (no CWS and CBS), the last three methods perform similarly. When CWS is present (dataset 2), the proposed method performs better than the others with slightly lower delay and much lower FAR. This pattern is repeated when CBS is incorporated (dataset 3).
When CWS is present, CWS should be taken into account to the sufficient reduction method as ignoring CWS delays detection and gives more false alarms. Sufficient reduction methods derived for independent observations (1, 2) do not take CWS into account; therefore, the effect of CWS is still present in their derived series of sufficient statistics. This effect violates the assumptions of EWMA chart, for which data are assumed to be independent and then produces a high FAR. Although the sufficient reduction method proposed by Wessman (1) allows for CBS, it does not allow CWS. Incorporating both CWS and CBS in our proposed sufficient reduction method substantially improves the performance in detecting a mean shift in multivariate surveillance data.
Introduction Disease surveillance data often have an underlying network structure (e.g., for outbreaks that spread by person-to-person contact). If the underlying graph structure is known, detection methods such as GraphScan (1) can be used to identify an anomalous subgraph, indicative of an emerging event. Typically, however, the network structure is unknown and must be learned from unlabeled data, given only the time series of observed counts (e.g., daily hospital visits for each zip code).
Our solution builds on the GraphScan (1) and Linear Time Subset Scan (LTSS) (2) approaches, comparing the most anomalous subsets detected with and without the graph constraints. We consider a large set of potential graph structures and efficiently compute the highest-scoring connected subgraph for each graph structure and each training example using GraphScan. We normalize each score by dividing by the maximum unconstrained subset score for that training example (computed efficiently using LTSS). We then compute the mean normalized score averaged over all training examples. If a given graph is close to the true underlying structure, then its maximum constrained score will be close to the maximum unconstrained score for many training examples, while if the graph is missing essential connections, then the maximum constrained score given that structure will be much lower than the maximum unconstrained score. Any graph with a large number of edges will also score close to the maximum unconstrained score. Thus, we compare the mean normalized score of a given graph structure to the distribution of mean normalized scores for random graphs with the same number of edges and choose the graph structure with the most significant score given this distribution.
We generated simulated disease outbreaks that spread based on the zip code adjacency graph with additional edges added to simulate travel patterns and injected these outbreaks into real-world hospital data. We evaluated detection time and spatial accuracy using the learned graphs for these simulated injects (Fig. 1 ). This figure also shows the detection performance given the true (adjacency plus travel) graph, the adjacency graph without travel patterns and the average performance given randomly generated graphs. We observe that the learned graph achieves comparable spatial accuracy to the true graph, while the adjacency graph has lower accuracy. Additionally, the learned graph is able to detect outbreaks over a day earlier than the true graph and 1.5 days earlier than the adjacency graph. Thus, our method can successfully learn the additional edges due to travel patterns, substantially improving detection performance.
We proposed a novel framework to learn graph structure from unlabeled data. This approach can accurately learn a graph structure, which can then be used by graph-based event detection methods such as GraphScan, enabling more timely and accurate detection of outbreaks, which spread based on that latent structure. Our results show that the learned graph structure is similar to the true underlying graph structure. The resulting graph often has better detection power than the true graph, enabling more timely detection of outbreaks, while achieving similar spatial accuracy to the true graph.
Keywords Event detection; biosurveillance; graph learning 
The illegal wildlife trade is a multifaceted, clandestine industry that has led to the disruption of fragile ecosystems, facilitated the spread of pathogens and led to the emergence of novel infectious diseases in humans, domestic animals and native wildlife (1, 2) . The trade is as diverse as it is large, with live and dead wildlife, representing multiple species sold to satisfy human demands for food, medicine, pets and trophies. Wildlife are harvested at astonishing numbers and used for such things as exotic pets, ornamental jewelry and clothing and traditional Chinese medicine (3) . An estimated 75% of recently emerging infectious diseases originated from animals (4), which can include both live animals and animal products.
Freely available RSS feeds from official sources, such as organizations dedicated to ending the illegal wildlife trade to include TRAFFIC, WildAid and the Coalition Against Wildlife Trafficking (CAWT), were used to obtain information on illegal wildlife and wildlife product confiscations. In addition, information was obtained from freely available, disparate Internet sources (including discussion forums, mailing lists, news media outlets and blogs) by utilizing specific keyword search strings. For a 1-year period beginning August 1, 2010, English-language reports were collected on the illegal wildlife trade and interception points were analyzed (Fig. 1 ). When available, the origin and intended destinations of illegal wildlife products were also collected to aid in the development of proposed wildlife trade routes and hot-spot regions. Lastly, a comprehensive list of commonly traded species was compiled along with the potential zoonotic diseases that could be spread from traded animals to humans.
From 858 reports collected, elephants (n0107, 12.5%), rhinoceros (n0103, 12.0%), tigers (n068, 7.9%), leopards (n054, 6.3%), and pangolins (n045, 5.2%) were among the most commonly intercepted species (to include live animals and wildlife products). Zoonotic diseases associated with these species include rabies, cowpox, echinococcosis, anthrax, and tuberculosis. Countries with the most illegal wildlife product interceptions included India (n 0146, 15.6%), the United States (n0143, 15.3%), South Africa (n075, 8.0%), China (n041, 4.4%), and Vietnam (n037, 4.0%).
Available at http://www.healthmap.org/wildlifetrade, the digital wildlife surveillance tool is freely available to both wildlife conservation officials as well as members of the general public and shows real-time reports of illegal wildlife trade activity worldwide as an interactive visualization. The system combines official and unofficial reports with an overall goal of providing a greater understanding of the global wildlife trade network in addition to providing a jumping-off point for the identification of hot spot regions where enhanced surveillance should be implemented for emerging zoonoses. 
The HEDSS system was implemented in 2004 to monitor disease activity (1). Twenty of 32 emergency departments (ED) and 1 urgent care clinic provide data. Chief complaints are routinely categorized into 8 syndromes.
Although previous studies have shown that ED syndomic surveillance is not useful for early detection of GI outbreaks (2) , it has demonstrated utility in monitoring trends in seasonal norovirus activity (3) . An evaluation to assess the utility of HEDSS to characterize endemic and outbreak levels of GI illness has not been previously conducted in Connecticut.
In Connecticut, Campylobacter, Cryptosporidium, Cyclospora, shiga toxin-producing Escherichia coli (STEC), Giardia, Listeria, Salmonella, Shigella, Vibrio and Yersinia are laboratory reportable findings. Aggregate hospital admissions data are reported daily by all hospitals. Facility and community GI outbreaks are also reportable events. Weekly percentage of HEDSS GI syndrome visits (combined GI, vomiting, diarrhea and bloody diarrhea) were compared to the number of GI hospital admissions, number of facility and community GI outbreaks and reportable enteric diseases using correlation coefficients. GI syndrome ED visits were also examined by geographical region and age.
Vomiting and diarrhea were each highly correlated with the combined GI syndrome (r 00.99, p B0.0001; r 00.93, p B0.001, respectively), although vomiting has a greater magnitude than diarrhea. ED GI visits were correlated with GI hospital admissions (r 00.73, p B0.0001). Similar results were also seen when comparing HEDSS GI data to the number of total reported outbreaks (r00.76, pB0.0001) and facility outbreaks (r00.71, p B0.0001) but not community outbreaks alone (r 0 0.09, p00.23). The combined GI syndrome was inversely correlated with laboratory confirmed cases of Giardia (r0( 0.18, p 00.02), Campylobacter (r 0(0.45, p B0.0001), STEC (r0(0.32, p B0.0001), Listeria (r0(0.23, p 00.004), Salmonella (r0(0.41, p B0.0001), Shigella (r0(0.19, p 00.01), Vibrio (r 0(0.36, p B0.0001). No significant positive correlations were detected when controlling for seasonality or using a narrower syndrome definition. There was no significant geographic variation in GI illness by region. Children younger than 5 years had a proportion of ED visits for GI illness that was consistently higher than all other age groups.
There is a strong and consistent association between ED visits for GI illness and facility outbreaks, the majority of which are suspected to be caused by norovirus (4, 5) . The strength of observed associations was similar when using a vomiting, diarrhea or combined GI syndromes; no significant correlations were observed when using the narrow bloody diarrhea syndrome. HEDSS GI syndromes were inversely correlated with illness caused by bacterial enteric pathogens, even when using the bloody diarrhea syndrome to identify more severe illness or controlling for seasonality. The HEDSS system is a critical tool for situational awareness of community gastrointestinal illness, particularly that which is caused by norovirus. Since norovirus is not a reportable condition in Connecticut, this system is used as the primary source of monitoring community GI illness.
Keywords Syndromic surveillance; gastrointestinal; public health practice; evaluation
Asthma is a chronic condition of public health concern associated with morbidity, mortality and healthcare utilisation. It disproportionately affects certain ethnic and demographic groups.
Asthma admission records in London (2001Á2006) were used. Negative binomial regression was used to model the effect of demographic (sex, age & ethnic group), diagnostic (primary & secondary diagnosis, method of admission) and temporal (day of the week, meteorological season & year of admission) factors on the LOS, accounting for the random effects of each patient's attendance, as model 'I' and again for area of residence, model 'A'. Akaike information criterion (AIC) was used to compare the two models.
The median and mean asthma LOS over the period of study were 2 and 3 days, respectively. Admissions increased over the years from 8308 (2001) to 10,554 (2006) , but LOS declined within the same period. Fewer males (48%) than females (52%) were admitted and, the latter had longer LOS compared to males. Only 5% were primarily diagnosed as predominantly allergic, whilst >94% were classified as 'asthma, unspecified'. Younger people were more likely to be admitted than elderly, but the latter had higher LOS (p B0.001). The secondary diagnosis and method of admission were important diagnostic determinants of length of stay, with very marginal differences between the two statistical models ('I' & 'A'). Again, all the temporal factors were significant determinants of LOS. Overall the patient cluster model (AIC=239394.8) outperformed the area model (AIC=247899.9).
Asthma LOS is best predicted by demographic, diagnostic and temporal factors with individual patients as a random effect.
Asthma; length of stay; spell duration; risk factors; hospital admission *I. Soyiri E-mail: soyiriin@yahoo.com 
The spatial scan statistic (1) detects significant spatial clusters of disease by maximizing a likelihood ratio statistic F(S) over a large set of spatial regions, typically constrained by shape. The fast localized scan (2) enables scalable detection of irregular clusters by searching over proximity-constrained subsets of locations, using the linear-time subset scanning (LTSS) property to efficiently search over all subsets of each location and its k(1 nearest neighbors. However, for a fixed neighborhood size k, each of the 2 k subsets are considered equally likely, and thus the fast localized scan does not take into account the spatial attributes of a subset. Hence, we wish to extend the fast localized scan by incorporating soft constraints, which give preference to spatially compact clusters while still considering all subsets within a given neighborhood.
For a given local neighborhood with center location s c and size k, we place a bonus or penalty^i0h(1 Á 2d i /r) on each location s i , where d i is that location's distance from the center, r is the neighborhood radius and h is a constant representing the strength of the compactness constraint. Each^i can be interpreted as the prior log-odds that s i will be affected, and thus the center location (d i 00,^i 0h) is e h times as likely as its (k ( 1)th nearest neighbor (d i 0r,^i0(h). We demonstrate that the penalized score function F'(S) 0F(S)'Ss i # S^i can be efficiently maximized over all subsets S for each neighborhood.
To do so, we show that F(S) can be written as an additive function (sum over locations) conditioned on the relative risk in region S, and therefore F'(S) is additive given the risk as well. We then jointly maximize F?(S) over all subsets S and all values of the risk.
The penalized subset scan was evaluated using emergency department (ED) data from 97 Allegheny County zip codes. We compared detection power and spatial accuracy, with and without compactness constraints, on synthetic, spatially localized outbreaks injected into the ED data. Our results show that including compactness constraints allows the penalized subset scan methods to detect outbreaks earlier and improve spatial accuracy, as compared to the unpenalized fast localized scan and circular scan, over a wide range of neighborhood sizes (Fig.  1) . Without compactness constraints, the method averaged 7.6 days to detect and an overlap coefficient of 54.9% for neighborhood size of k 010, but detection performance degraded rapidly for smaller or larger values of k. Performance of the compactness-constrained methods was less dependent on neighborhood size, requiring 7.4 days to detect and achieving an overlap coefficient of 59.3% for well-chosen parameter values.
Our results demonstrate that the incorporation of soft compactness constraints substantially improves the timeliness and accuracy of outbreak detection. Our new approach based on additive linear-time subset scanning enables efficient maximization of penalized scan statistics over subsets of the data, thus improving detection of irregular clusters.
Outbreak detection; spatial scan; penalized subset scan 
Although the advent of the ONCs 'meaningful use' criteria has added significant new incentives for healthcare organizations to provide the necessary data for implementing syndromic surveillance, incentives alone are not sufficient to sustain a robust community of practice that engages public health and healthcare practitioners working together to fully achieve meaningful use objectives. The process for building a successful community of practice around syndromic surveillance is primarily applicationagnostic. The business process has many of the same characteristics regardless of application features and can be incrementally customized for each community based on the unique needs and opportunities and the functional characteristics of the application. This presentation will explore lessons-learned in the north central Texas region with BioSense 1 and ESSENCE over the past 6 years and will describe the multiphase process currently underway for BioSense 2.0. Key program process steps and success criteria for public health and healthcare practitioners will be described. This road map will enable other local health department jurisdictions to replicate proven methodologies in their own communities. The presentation will also highlight what it takes for an existing community of practice with a home-grown system to move processes and protocols to the cloud.
The NACCHO Advanced Practice Centers (APC) Program is a network of local health departments whose mission is to promote innovative and practical solutions that enhance the capabilities of all local health departments and the public health system to prepare for, respond to, and recover from public health emergencies. Real world practice situations are supported and evaluated, resulting in the creation of tools designed to export and scale roll outs of lessons learned to other jurisdictions.
Several products or tools specific to biosurveillance, disease detection and investigation were created through the APC Program methodology. Highlighted in this talk will be the Building a Public Health Community of Practice*A Biosurveillance Resource Compendium is a CD toolkit intended to help public health agencies implement an effective, comprehensive biosurveillance program. Providing approximately 40 resources, the CD includes a series of articles on implementing biosurveillance initiatives, materials defining and discussing the development of a public health community of practice, specific examples of real-world tools and resources that have proven beneficial in North Texas (including system response protocols) and a research report on biosurveillance system efficacy. The CD can help public health agencies strengthen partnerships with stakeholders at the federal, state and local levels and with the medical community, law enforcement, first responders and schools; it details how Tarrant County Public Health accomplished those goals and shares tools that were instrumental to the agency's success.
Lessons learned from a systematic approach to building and sustaining a regional and local biosurveillance community of practice have been documented in a meaningful way. These lessons can and should be leveraged as more of the country engages in syndromic surveillance through meaningful use incentives and the BioSense 2.0 infrastructure. The NACCHO sponsored north central Texas APC and tools derived from their work is a proven method to provide such assistance to local health departments across the country.
Informatics; advanced practice centers; sustained relationships *Michael Coletta E-mail: mcoletta@naccho.org
The ability to estimate and characterize the burden of disease on a population is important for all public health events, including extreme heat events. Preparing for such events is critical to minimize the associated morbidity and mortality (1, 2) . Since there are delays in obtaining hospital discharge or death records, monitoring of ED visits is the timeliest and an inexpensive method for surveillance of HRI (1). Aside from air temperature, other environmental variables are used to issue heat advisories based on the heat index, including humidity and wind (3) . The purpose of this study was to evaluate the relationship between HRI ED visits and weather variables as predictors, in Ohio.
Syndromic surveillance data from ED visits were collected and analyzed from Ohio's syndromic surveillance application, Epi-Center, for July 2011. Since the physical effects of HRI can vary greatly and affect multiple body systems, a specific classifier was created to query ED visits that were likely related to HRI and was defined as chief complaints referencing heat 'exhaustion or exposure', dehydration or hyperthermia. Measurements for weather variables included temperature, dew point, humidity, pressure and wind speed. The average daily values of these variables were calculated from seven geographically representative cities in Ohio and used as a surrogate for statewide data. These data were obtained from Weather Underground, which collects data from Automated Surface Observations System (ASOS) stations located at airports throughout the United States. These data were analyzed via time-series analyses and stratified by age group and gender. Correlation and linear regression analyses were performed, using SAS v 9.2 to determine which weather variables were the best predictors of HRI, as defined by ED chief complaint data.
During the third week of July 2011, Ohio experienced a heat wave with multiple heat advisories throughout its various cities. The total ED visits related to HRI peaked on July 21 (n0170, 107 males, 63 females), which was also the day with the highest maximum temperature (97.4 F). A time-series chart of these ED visits by age group is shown below. The data show that the most sensitive populations (ages 0-5 and 65 and older) were the least affected and likely were adhering to the heat advisories. The 18Á 39-and 40Á64-year-old age groups were most affected by the heat. Pearson correlation showed a strong relationship between HRI visits and mean temperature and dew point (r00.76 and r00.66), p B0.0001. Multiple linear regression analyses were completed to determine which weather variables were the best predictors with HRI. The best model showed that for every 1 unit increase in ED visits, there was a 3.88 unit increase in mean temperature, independent of mean humidity and wind speed, p B0.0001. The addition of mean dew point caused the model to have a high colinearity and was removed from the model.
These results suggest the advisories provided to the public during the heat wave in Ohio were most adhered to by the sensitive populations (very young and elderly). Middle-aged males were most susceptible to HRI during the peak of the heat wave. Temperature and dew point showed a strong relationship with HRI and were modeled as significant predictors of HRI. Additional analyses should be completed to further evaluate this relationship. Finally, obtaining patient diagnosis records from the hospital EDs would provide strength in validating the observed results.
Heat-related illness; weather; predictor; classifier; correlation This not only represents a huge opportunity for public health to collect more data to enhance disease detection and control, improve safety, and reduce health disparities, but also presents an integration challenge.
In 2011, NH DPHS initiated a project with Orion Health to build a Rhapsody integration engine (1) portal to receive the three types of Public Health data. A Syndromic surveillance pilot was chosen since 25 of 26 hospitals were already sending real-time data in HL7 format to the statewide syndromic surveillance system. NH DPHS collaborated with the NH Regional Extension Center (REC) to host MU guidance and brokered with Orion Health, the Office of the National Coordinator (ONC), and CMS to offer hospitals the option to use a modular certification for MU public health measures by selecting the Orion Health module (2) . Selecting this module allows hospitals to send data to the NH DPHS Rhapsody portal in whatever format they choose; then, the NH DPHS Rhapsody system converts these messages to the approved ONC standards for public health reporting. Orion Health contractors set up the Rhapsody server, configured data routes and built validation, filtering, and mapping logic. Mapping to HL7 2.5.1 was performed, but additional mapping to 2.3.1 was done before sending data to the syndromic surveillance application. Hospitals were directed to reroute data transmissions to the new Rhapsody VPN IP address and port, and Rhapsody was configured to pass traffic to the original surveillance application address and port. Additionally, data was sent through the normal VPN connection to compare the accuracy and performance of the new path.
This MU project generated more hospital participation than was realized prior to initiating the Rhapsody integration.
Negligible syndromic surveillance processing time degradation was realized with the added Rhapsody processing. This processing allowed NH DPHS to implement its last acute care hospital into the existing syndromic surveillance application (using Rhapsody mapping), filter existing hospital syndromic surveillance transmissions on specific patient types (preventing unwanted types), receive MU ELR and immunization data prior to expected timelines, increase hospital MU certification reimbursement without additional MU expenditure and decrease the hospital laboratory staff reporting burden, which previously was manual.
Conclusions NH DPHS was able to take advantage of opportunities and resources beyond the State of NH. The brokered Orion Health Rhapsody MU certification solution provided a lower cost certification solution to hospitals (as compared to purchasing Rhapsody or certifying their EHR). NH DPHS was able to build an expandable public health MU infrastructure easily integrated with the NH HIE. The MU REC website provided guidance, FAQs, state rules and allowed NH DPHS to communicate effectively with hospital partners and the NH REC, to take advantage of REC expertise, and keep all partners informed.
In response to the terrorist attack of September 11, 2001 , the NH Department of Health and Human Services (NH DHHS) engaged state and external partners in the design of an early warning surveillance system to support bioterrorism and emergency preparedness. Initially, NH DHHS began collecting four syndrome counts from 13 hospital emergency departments (EDs) by fax. Automation began in 2002, when an over-thecounter (OTC) syndromic surveillance pilot system was implemented by Scientific Technologies Corporation (STC). In 2003Á2004 this system, the Syndromic Tracking and Encounter Management System (STEMS), was expanded to include school absentee and occupational health reports. Over time, an internal death data application was automated to query vital record deaths, and in 2005, a real-time ED surveillance pilot, the Automated Hospital ED Data System (AHEDD), was developed by STC to replace manual ED surveillance. Over the past decade, NH continued to expand the original concept with innovative approaches to identify undetected or underreported disease outbreaks.
NH's surveillance consists of assessing individual but compatible surveillance systems for (1) rapid detection of a covert bioterrorism attack and (2) early detection of naturally occurring outbreaks (i.e., influenza). The OTC pharmaceutical system was implemented with automated data processing and alerting within an enterprise architecture. Modified Shewhart charting was developed with dynamic system modeling using a knowledge base technique. Community health status was charted with a set of state syndrome variables and dynamic processes, where baselines, thresholds, trend analysis and alerts from historic data were automatically charted (1). This technical framework was implemented in STEMS with OTC data, school absentee data and occupational health data, then later in AHEDD. The AHEDD system also used the Real-Time Outbreak and Disease Surveillance CoCo chief complaint classifier with electronic data feeds from four hospitals. AHEDD was later expanded to include drill down custom querying for all 26 acute care hospitals (allowing NH to realize statewide ED surveillance).
Over time, custom querying included data mining techniques adapted from the death data application, (2) to detect narrowly defined chief complaint health conditions and cluster activity.
This together with a 'Google'-like query tool allow NH surveillance staff to quickly assess any situation. Recently, a single portal infrastructure, based on AHEDD, was created to receive all external syndromic surveillance, Electronic Lab Reporting and immunization transmissions, helping hospital partners meet Meaningful Use (MU), which paves the way for integration with a statewide Health Information Exchange.
Over the past 10 years, the usefulness of NH's surveillance systems has been demonstrated repeatedly. STEMS detected influenza and school norovirus outbreaks (3), and AHEDD tracked H1N1 and acute respiratory illness during the flu season, detected anthrax exposures during a gastrointestinal anthrax investigation and identified reportable disease occurrences (i.e., Lyme disease) and nonreportable clusters (i.e., carbon monoxide). These narrowly defined chief complaint queries have been found to be more useful than broad-based queries in detecting daily illness and heath risks. Results of individual surveillance systems assessed together validate individual system detections (i.e., increased sales of OTC antiviral medication and increased school ILI absenteeism validate ED flu spikes).
Ten years of NH syndromic surveillance tool development has established a critical biosurveillance infrastructure with emergency preparedness response capability during disease outbreaks and natural disasters. These syndromic surveillance tools are now integral to the daily efforts of epidemiologists and public health professionals.
The association of influenza vaccination with influenzalike illness among adults aged 65 years and older in the United States Mayuko Takayama 1,2 *, Catherine Wetmore 1 and Ali Mokdad 1 Objective To explore the association of influenza vaccination with influenza-like illness (ILI) among adults aged 65 years and older
After the 2009 H1N1 influenza pandemic, CDC initiated community-based surveillance of self-reported influenza-like illness (ILI) (1), defined as the presence of fever with cough or sore throat. Although ILI is frequently attributed to other pathogens, including rhinovirus, routine surveillance of ILI at the population level does aid in the detection of nascent influenza outbreaks. In the United States, approximately 90% of influenza-related deaths occur among adults aged 65 years and older (2) . We explored the association of influenza vaccination with ILI, among this vulnerable age group.
Self-reported survey data from the 2010 Behavioral Risk Factor Surveillance System (BRFSS) was analyzed. Because the relationship between ILI and influenza infection is strongest during the influenza season, we limited the study sample to adults aged 65 years and older who participated between January and March 2010 (N035,628). We adjusted for three categories of individual-level factors: sociodemographics, health behaviors, and history of chronic disease diagnoses. We used stratified, weighted multivariable logistic regression to estimate the association between receipt of the influenza vaccine in the past year and report of ILI in the past month via adjusted odds ratios (aOR) and 95% confidence intervals (95% CI).
Recent ILI was reported by 3.37% (95% CI: 3.02Á3.73%) of responders. 67.7% (95% CI: 66.8Á68.6%) reported receiving the influenza vaccine in the past year. After adjusting for sociodemographics, health behaviors, and chronic disease diagnoses, receipt of influenza vaccination was significantly associated with recent ILI, with vaccine recipients being more likely to report ILI (aOR 01.50, 95% CI: 1.01Á2.24). Persons who are underweight (BMIB18.5, compared with normal weight) (aOR 03.21, 95% CI: 1.19Á8.65), and those diagnosed with asthma (aOR 02.45, 95% CI: 1.65Á3.62), coronary heart disease (aOR 01.77, 95% CI: 1.17Á2.65), and stroke (aOR 0 1.75, 95% CI: 1.07Á2.87) were also more likely to report ILI.
Our study showed an association between influenza vaccination and influenza-like illness among persons aged 65 years and older. This is a counterintuitive finding as vaccines are known to reduce the burden of influenza. Although our study is crosssectional and we cannot determine a causal pathway, it is possible that individuals with greater susceptibility to influenza infection (e.g., persons with chronic diseases) were more likely to get vaccinated. Indeed, these findings suggest the success of targeted public health messaging regarding the importance of vaccination among high risk individuals.
Influenza; vaccination; influenza-like illness; surveillance
Public health officials and epidemiologists have been attempting to eradicate syphilis for decades, but national incidence rates are again on the rise. It has been suggested that the syphilis epidemic in the United States is a 'rare example of unforced, endogenous oscillations in disease incidence, with an 8Á11-year period that is predicted by the natural dynamics of syphilis infection, to which there is partially protective immunity' (1). While the time series of aggregate case counts seems to support this claim, between 1990 and 2010, there seems to have been a significant change in the spatial distribution of the syphilis epidemic. It is unclear if this change can also be attributed to 'endogenous' factors or whether it is due to exogenous factors such as behavioral changes (e.g., the widespread use of the internet for anonymous sexual encounters). For example, it is pointed out that levels of syphilis in 1989 were abnormally high in counties in North Carolina (NC) immediately adjacent to highways (2) . The hypothesis was that this may be due to truck drivers and prostitution and/or the emerging cocaine market (1). Our results indicate that syphilis distribution in NC has changed since 1989, diffusing away from highway counties (see Fig. 1 ).
Using CDC data for syphilis, we construct county-level syphilis distribution maps for NC and Florida and time series (1990-2010) of spatial distributions of syphilis for Florida. Additionally, for comparison, we construct county-level (from 2004 to 2010) and state-level (from 1995 to 2010) syphilis distribution time series.
Maps of cases (per 100,000) in NC show that the disease has spread into rural counties and is no longer concentrated along the highway (see Fig. 1 ). In Florida, along with the overall decrease in syphilis incidence, the distribution of cases becomes more concentrated from 1990 to 1998. When, in 1999, syphilis incidence rates begin to increase again, the distribution again widens and spreads to more rural communities (see Fig. 2 ).
The time series of national state-level syphilis distribution indicates an increase in the number of states at the extremes of the distribution (i.e., with very high or very low case counts). However, at the same time, the national county-level distribution remains stationary. This indicates that counties with high case counts are clustering in states with high case counts and similarly counties with low case counts are clustering in states with low case counts.
The county-level spatial distribution of syphilis has changed significantly since 1990 and in ways that may depend on exogenous factors. Higher prevalence of syphilis in states seems more due to an increase in syphilis in counties that earlier had a low incidence of the disease. County-level syphilis data present a rather nuanced picture of how syphilis incidence has changed over the years and may form the basis for effective interventions.
Keywords Spatial distributions; time-series analysis; syphilis 
Event-based biosurveillance monitors diverse information sources for the detection of events pertaining to human, plant and animal health using online documents, such as news articles, newsletters and blogs (1) . Machine learning techniques have been successfully used for automated document classification, an important step in filtering source information (2Á15).
We review studies on document classification using machine learning for event-based biosurveillance and comparatively summarize them for close examination. Table 1 lists relevant studies we identified. These studies differ in target regions, languages, event types and surveillance criteria, as well as classification methods. This diversity illustrates the complementarity of all the approaches.
Common challenges shared by these methods include detection of rare events and practical evaluation of the employed methods. The comparative advantages of each method remain unclear because of the lack of benchmark data. A community effort is necessary to develop an event ontology and benchmark corpora.
Keywords biosurveillance; text classification; machine learning 
Tracking ED asthma visits is an important part of asthma surveillance, as ED visits can be preventable and may represent asthma control failure (1). When using limited clinical ED datasets for secondary purposes such as public health surveillance, it is important to employ a standard approach to operationally defining ED visits attributable to asthma. The prevailing approach uses only the primary ICDÁ9ÁCM diagnosis (Dx) for the ED visit (2); however, doing so may underestimate the public health impact of asthma. We conducted this pilot study to determine the value of including ED visits with asthma-related Dx in secondary or tertiary positions. For example, for an ED visit with a primary Dx of upper respiratory infection and secondary Dx of asthma, it is possible that the infection triggered the asthma exacerbation and the visit could be attributed to both infection and asthma.
We utilized all ED visit data for 2008Á2009 from the state public health surveillance system (3), accounting for 99.5% of the visits to North Carolina EDs. Included were visits with an ICD-9-CM diagnosis code for asthma (493.xx) in any Dx position (1Á11). We then grouped asthma visits into 11 strata based on the Dx position containing the asthma code. We identified the most frequent chief complaint and primary Dx categories for each of the 11 asthma Dx positions. We also grouped procedure codes (ICDÁ9ÁCM and CPT) for potential asthma (e.g., nebulized medications) and cardiac (e.g., electrocardiogram) conditions for each Dx position.
Results 350,341 (4.0%) of the 8.7 million ED visits had a diagnosis of asthma in 1 of the 11 Dx positions. The most common chief complaints for visits with asthma were: Dx positions 1 and 2-dyspnea and asthma, and Dx positions 3Á5-injury. 69,877 (19.9%) of the asthma visits had at least 1 procedure code assigned, those with asthma or cardiac procedure code are shown in Fig. 1 .
Restricting the definition of an asthma-related ED visit to the first diagnosis position may miss a substantial proportion of the asthma-related public health burden. Further analysis is in progress to evaluate the validity of these preliminary findings.
Public health surveillance; ED data; asthma Data elements include disposition, initial vital signs, up to 11 ICD-9-CM final diagnosis codes, up to five external causes of injury codes (E-codes), as well as the arrival date and time, patient sex and age, patient zip and county and chief complaint. As of January 2008, NC DETECT emergency department data covered 99% of the NC population and captures approximately 4.5 million ED visits each year. As a result, requests for data from researchers continue to increase. Use of the data for public health purposes is covered by the mandate requiring hospitals to submit their emergency department data to NC DPH.
Data requesters must use the ED data in NC DETECT for public health-focused studies. Data requests from commercial entities are not approved. The data request process occurs primarily via paper and e-mail, although we have implemented a centralized tracking system to store all documentation for data requests and track changes to them over time. To initiate the process, requesters view a presentation on https://www.ncdetect. org/ReportsPortal/public/dataRequest.do and then enter information on the study purpose, the researchers involved, any grants covering the research and the specific data requested* specifically the data elements, time frame and file format. Researchers must get approval from their home institution's Institutional Review Board and sign a Data Use Agreement (DUA) with NC DPH. The DUA outlines data use requirements such as securing the data during the study, presenting data in aggregate form only (in a manner in which an individual cannot be identified), sharing materials with NC DPH prior to presentation or publication and destroying data upon completion of the study. Data requests within NC DPH typically do not require a DUA and are exempt from this process. In addition, researchers who need to determine the feasibility of a study before submitting a full data request can go through an exploratory process that requires a DUA but no IRB. While data requests are ongoing, a small Data Oversight Committee (DOC) meets once monthly to review these requests and to discuss status and outstanding issues. The NC DETECT DOC includes representatives from the NCDPH, CCHI, NC Hospital Association (NCHA) and DPH legal personnel (as needed).
We currently have 31 data requests in our online tracking system. Each data request represents multiple e-mails and phone calls, iterative revisions to the data request and multiple data pulls from the NC DETECT database. Requests can be delayed when researchers request data elements that are not collected by NC DETECT, submit unclear requirements, change requirements, do not understand the challenges of processing free text data and/or add/change researchers who will be accessing the data. Requesters have used NC DETECT data to publish manuscripts on topics including the health effects of wildfires, ED visits for cancer patients, tick-borne illness and asthma, and comparison of NC ED visit data to national data, among others. Because the ED visit data are collected under a state mandate and in collaboration with the NCHA, their release and use for research is thoroughly evaluated by the DOC. The complexity of the data requests over time has resulted in changing of data use restrictions, as well as revisions of the DUA wording. We do not have enough resources to closely monitor the use of the data once they are provided to researchers. However, researchers are expected to abide by all provisions detailed in their DUA and by signing acknowledge the potential penalties for violation of the terms the agreement.
Data requests can take a considerable amount of time and iterative discussions with the requester, even with a welldefined process and clear documentation. Understanding the administrative and technical time commitments involved is important when considering making syndromic surveillance data available to external users for research.
Data sharing; data use agreements; data requests
Recent events have focused on the role of emerging and reemerging diseases not only as a significant public health threat but also as a serious threat to the economy and security of nations. The lead time to detect and contain a novel emerging disease or events with public health importance has become much shorter, making developing countries particularly vulnerable to both natural and manmade threats. There is a need to develop disease surveillance systems flexible enough to adapt to the local existing infrastructure of developing countries but which will still be able to provide valid alerts and early detection of significant public health threats.
In collaboration with the Philippine National Epidemiology Center (NEC) and the Philippines-AFRIMS Virology Research Unit, Armed Forces Research Institute of Medical Sciences, the EDE program, which was developed by Johns Hopkins UniversityÁApplied Physics Lab, was introduced in the Philippines to augment the data analysis capability of the Philippine Integrated Disease Surveillance and Response (PIDSR) System. Reported significant increases in the number of suspect dengue cases/outbreaks at the municipality, provincial and regional level, which were reported to and investigated by NEC from July 1, 2011, to August 31, 2011, were used as a reference point. A defined period, 30 days prior to the date when the event was officially reported, was retrospectively analyzed. The day when an EDE alert was first triggered and the number of EDE alerts detected during this period were described. Since NEC analyzes data by morbidity week, municipalities that were detected to be above the NEC alert or epidemic threshold during a randomly selected morbidity week (week 31; reporting date of August 6, 2011) were compared with the number of EDE alerts triggered during the past 7 days before the report date of morbidity week 31.
Retrospective analysis done during the past 30 days before the event was officially reported showed that EDE Alerts were already triggered as early as 30 days (median of 27.5 and range 14Á30 days) prior to the date of the NEC report. The number of days associated with EDE 'alerts' out of the 30 days prior to the NEC official report date had a median of 9.5 days (range of 3Á14 days). A total of 17 municipalities had reported dengue cases above the alert or epidemic threshold with 8/17 of these municipalities having at least 1 day in the previous week with a case count of more than 5 while 9/17 had case counts of 5 or less for all 7 days in the past week. For municipalities with at least 1 day with a case count of 5 or more for the previous 7 days, the median of the number of days with associated EDE alerts was 5 (range 0Á7 days). For municipalities with case counts of 5 or less for all 7 prior days, no alerts were usually generated (median 0 and range 0Á2 days).
A surveillance system's usefulness for outbreak detection should be correlated with its ability to increase the lead time in detecting outbreaks of public health significance, which should subsequently lead to a more timely intervention. Analysis of currently available data seem to show promising applications of EDE in early warning alert capability of impending increases in dengue cases in the Philippines though when case counts are 5 or less, alert results may not be very reliable. Validity of alerts generated by EDE for early detection of outbreaks should be further investigated using other diseases, prediagnostic/ nonclinical/nontraditional data and syndromes, taking into consideration effect of seasonality, weekly trends or holidays.
Surveillance, outbreak, dengue, predictive, validity
In Réunion Island, the nonspecific surveillance was mainly developed during A(H1N1) influenza pandemic in 2009 (1, 2). In March 2010, a new surveillance system was implemented from National Health Insurance data. This monitoring was based on the weekly consultation number and home visits by general practitioners.
The data based on the activity of general practitioners were transmitted on week W and covered the consultations and home visits carried out by the general practitioners in the week W (1.
These data were updated week by week according to the flow of repayments. The data received were aggregated, and no personal information was communicated.
The thresholds corresponding to the statistical alarms for the weekly numbers of all consultations were based on a calculation using adapted versions of two historical methods (log-linear regression model of Farrington and historical limit method) and CUSUM methods.
The surveillance period was spread over 134 weeks from week 1 of 2009 to week 29 of 2011. For the two historical methods, expected numbers of all consultations were calculated during these 134 weeks, using a training period of at least 3 years. A 95% confidence interval was calculated for each weekly expected number. A weekly count observed was considered significantly greater than the expected value if it was above the 95% upper confidence limit.
For the CUSUM methods, only few weeks were necessary to calculate a one-sided positive cumulative sum. An alarm was obtained if this cumulative sum was greater than a fixed decision value.
The data covered 72% of the population of Réunion Island. Over the surveillance period, 11,048,739 consultations were recorded with an average of 82,453 consultations per week (min: 56,682; max: 120,432). An illustration of the results obtained with the historical limit method is presented in Fig. 1 . The first alarms that occurred on week 34 to week 36 of 2009 corresponded to the influenza A(H1N1) epidemic with a peak in week 35. Statistical alarms observed on week 8 of 2010, and week 7 to 9 of 2011 were related to the season circulation of respiratory syncytial virus. These results were confirmed by the laboratory data. During the austral winter of 2010, one alarm was obtained in week 39 corresponding to the influenza epidemic.
This surveillance system based on the data of the National Health Insurance is a complementary tool to nonspecific monitoring in Reunion Island. Not only does it ensure the detection of unusual health events but it also allows to quantify a public health impact for major events. It brings information about the recourse to the so-called 'non emergency' cares that will allow public health authorities to implement adapted control measures. The major advantage of this system is its exhaustive data use that ensures a global view on all consultations carried out in the island and to have a denominator to calculate other indicators. 
Mining text for real-time syndromic surveillance usually requires a comprehensive knowledge base (KB), which contains detailed information about concepts relevant to the domain, such as disease names, symptoms, drugs and radiology findings. Two such resources are the Biocaster Ontology (1) and the Extended Syndromic Surveillance Ontology (ESSO) (2) . However, both these resources are difficult to manipulate, customize, reuse and extend without knowledge of ontology development environments (like Protégé) and Semantic Web standards (like RDF and OWL). The cKASS software tool provides an easy-touse, adaptable environment for extending and modifying existing syndrome definitions via a web-based Graphical User Interface, which does not require knowledge of complex, ontology-editing environments or semantic web standards. Further, cKASS allows for*indeed encourages*the sharing of user-defined syndrome definitions, with collaborative features that will enhance the ability of the surveillance community to quickly generate new definitions in response to emerging threats.
We have developed a web-based prototype of the cKASS system that allows individual users or collaborative communities to access the service anytime and anywhere, without a complex technical configuration process (Fig. 1) . Two types of databases are used to support cKASS. First, a relational database is used to store user information and KB descriptors(e.g., KB domain and status). Second, KBs are stored as RDF triples using triple store and queried using SPARQL, an RDF query language, with the Jena SDB (SPARQL database,) providing robust and scalable storage. Existing resources stored in standard RDF and OWL formats can be easily loaded into the triple store and used as a basis for constructing new syndrome definitions.
The web interface is designed to support both individual and collaborative KB development. cKASS consists of two zones:
User workspace, where registered users can create, browse, modify and publish customized syndrome definitions constructed from either publicly available or user-created resources.
Community space, where anyone can browse and search shared KBs. Users who choose to share their KBs can make them available to the general community in this space.
cKASS also provides search/query capabilities at different levels.
For example, the user can search within a specified domain or within a named KB for terms or concepts. Queries can either be simple strings or can consist of arbitrarily complex SPARQL and SQL queries. Further, users can import queried results into their syndrome definitions (for example, concept only, concept and its attributes or concept and all its subclasses). Finally, once created, KBs can be exported in standard formats, such as XML, CSV or RDF, for use with other tools.
Currently, two existing syndromic surveillance oriented ontologies* Biocaster and ESSO*have been loaded into the cKASS triple store and can be used as a basis to construct new syndrome definitions. Both KBs can be queried using SPARQL and SQL. Conclusions cKASS offers public health professionals and clinicians an environment to support the extension and modification of existing KBs, without the need to use complex ontology editing environments and formalisms, allowing the user to rapidly develop or augment existing syndrome definitions and react quickly to the changing surveillance landscape. Keywords Syndrome surveillance; knowledge authoring; ontology (1). Producers were informed of the problem by their swine processing facility. Tissue samples from affected producers were culture-positive for Mycobacterium avium. In the spring of 2010, USDA Veterinary Services (VS) began monitoring weekly ADRS STB carcass condemn data after a VS Staff Officer was made aware of unusual increases in STB condemns in another region. By June 2010, STB condemn rates in both of the affected areas decreased to typical seasonal levels; however, beginning January 2011, rates again rose beyond baseline seasonal highs, exceeding those seen in the 2010 outbreak.
The ADRS provides weekly condemn data to VS along with information on species and total number of animals slaughtered. June 2007ÁMay 2011 ADRS market swine data were grouped by three major swine production areas (basins) to (1) identify preoutbreak condemn baselines, (2) quantify differences in the 2010 and 2011 STB outbreaks and (3) ascertain the geographical extent of the outbreaks. In addition, a fourth basin was created representing the remainder of U.S. market swine slaughter plants.
To identify critical weeks of anomalous condemns, a modification of the 'C3' version of the Early Aberration Reporting System was applied to the STB condemn series (2) . In addition to examining alerts by basin, the alerting algorithm was applied to individual plant data. Data processing was performed using SAS version 9.1 and an Excel-based 'Alerting Algorithms Tool' developed and published by Dr. Howard Burkom (3) .
Mean weekly swine TB condemn rates, which seasonally ranged from 5.7 to 21.4 per 100,000 between 2007 and 2009 (mean 010.7, SD03.0), increased above typical levels beginning January 2010 and rose even higher beginning January 2011 (Fig. 1) . Most of the increase was due to condemns in Basin 3, which experienced 670/ 100,000 weekly STB condemns at the height of the 2011 outbreak. Summary data for Basins 1, 2 and 4 indicate that condemn profiles remained at nonoutbreak levels over the 4-year data series. Retrospective analysis by individual facility suggests that while condemns in Basin 1 appeared to coincide with typical seasonal STB rates, the pattern of alerts for one facility may have signaled STB outbreaks in both outbreak periods.
By combining 'on-the-ground' practitioner-based information with geographically broader analysis of ADRS data, we identified an emerging disease situation involving TB lesions in swine. A preliminary epidemiological investigation suggests that likely principal risk sources are related to feed and ground water (1). Analysis of ADRS condemn data suggests that several lower Midwest slaughter facilities in addition to the affected plant used by producers involved in the investigation may have been affected by STB.
Abattoir; surveillance; syndromic
Hypoglycemia is a serious sequel of diabetes treatment that is not tracked by current health surveillance efforts despite substantial related morbidity and mortality (1). We take a novel approach to hypoglycemia surveillance, engaging members of an international online diabetes social network (SN) in reporting about this issue as members of a consented, distributed public health research cohort.
We collected structured self-reported data about hypoglycemia and related harms using a software application called TuAnalyze that supports SN-mediated health research (2) . Odds for harms were estimated controlling for demographics, diabetes type and health insurance.
Of 2538 TuAnalyze users, 608 (24% response rate) completed two complementary surveys on hypoglycemia and diabetes care. Of these, 169 (27.8%) reported ]1 severe low in the past 12 months.
Harms were high; one in seven reported an accident or serious injury; over 40% reported high daily worry, and the frequency of reported withdrawal behaviors ranged from 20 to 50%. Experience of ]1 past 12-month severe low was associated with added risk for each of the six harms, and for experiencing multiple harms. (Tables 1 and 2) .
Hypoglycemia prevalence is high and exerts a considerable toll in terms of physical and social harms in this sample of predominantly type 1 or insulin-treated patients. Hypoglycemia surveillance is feasible using a novel approach that affords opportunity for bidirectional communication and tracking* capabilities important to ameliorating this problem.
Diabetes; hypoglycemia; surveillance; social networking 
The Veterans Health Administration (VHA) uses the Electronic Surveillance System for the Early Notification of Communitybased Epidemics (ESSENCE) to detect disease outbreaks and other health-related events earlier than other forms of surveillance (1). Although Veterans may use any VHA facility in the world, the strongest predictor of which healthcare facility is accessed is geographic proximity to the patient's residence. A number of outbreaks have occurred in the Veteran population when geographically separate groups convened in a single location for professional or social events. One classic example was the initial Legionnaire's disease outbreak, identified among participants at the Legionnaire's convention in Philadelphia in the late 1970s (2) . Numerous events involving travel by large Veteran (and employee) populations are scheduled each year.
An H1N1 influenza outbreak was identified at a Veteran Benefits Administration (VBA)-sponsored conference in Baltimore, MD, in July 2009 in which affected VBA employees (both local and from out-of-town) sought healthcare at the VA Maryland Health Care System*Baltimore Medical Center. Using ESSENCE, daily counts of ICD-9 codes related to influenza diagnoses (as defined by VA ESSENCE influenzalike illness [ILI] syndrome group) were collected from the VHA Baltimore Medical Center from March 01, 2009, to September 12, 2009 . Data included case status (as defined by ICD-9 code and chart review), date and location of visit and patients' zip code of residence. We also accessed data from the VA Planning System and Support Group to determine whether the patients' residential ZIP code fell within the Baltimore VA Medical Center's catchment area. Using SAS, a p-chart (where the denominator was the daily number of patient ILI encounters) was run to determine days during which an aberrant proportion of patients from out-of-catchment zip codes were encountered.
An aberrant proportion of out-of-catchment zip code ILI encounters signaled an out-of-control process (or alert) on July 23, 2009, 2 days later than the beginning of the influenza outbreak at the facility (Fig. 1 ) on the date when the majority of affected participants were evaluated for flu symptoms. (The alert on July 26, 2009, was a part of this same outbreak.) Using this algorithm, there were two other days in the 7-month period during which the chart signaled that the process was out-of-control: March 21, 2009, and September 6, 2009 . Investigations are being conducted to determine the nature of these other signals.
Using p-charts to detect unusual clusters of patients' residential zip codes that fall outside of facilities catchment area is likely a method of detecting disease outbreaks previously not utilized. Future work includes running this algorithm in all VA Medical Centers to prospectively identify disease outbreaks involving increased proportions of patients residing outside of the medical center's catchment area.
Surveillance; p-chart; algorithm; signal detection; outbreak Objective To present the development and implementation of the SIPS project, a statewide, hospital-based surveillance system for severe community-acquired pneumonia (sCAP) in Kentucky.
The threat of epidemics due to nonhuman strains of influenza A viruses is ever present (1). Surveillance is a critical aspect of pandemic preparedness for early case detection (2) . Identification of the index cases of a pandemic virus can trigger public health mitigation efforts (3) . To develop an appropriate surveillance process, it is important to understand the two possibilities of pandemic evolution. A new pandemic may begin with mild cases, during which surveillance should be concentrated on work/school absenteeism and in physician offices. The other possibility begins with severe cases, characterized by sCAP, respiratory failure and ICU admission. As the syndrome of pneumonia is not reportable to health agencies for public health surveillance, a year-round, hospital-based surveillance mechanism may be an important tool for early case detection in the event of an epidemic of sCAP. To fill these gaps, we developed a statewide, hospital-based surveillance network for sCAP surveillance in Kentucky.
All acute care hospitals in Kentucky were invited to participate in the project. A case of sCAP was defined as a patient admitted to an ICU with the physician diagnosis of CAP. Upon patient identification, demographic and clinical characteristics were entered into an Internet-based data collection form. All patients had a nasopharyngeal swab sent to the University of Louisville Infectious Diseases Reference Laboratory for identification of viral pathogens. The Luminex xTAG respiratory viral panel multiplex PCR was used for viral identification. Clinical cultures were utilized to identify bacterial and fungal causes of sCAP. Statistical Process Control (SPC) charts were used to identify outbreaks. Chloropleth maps were used for spatial analysis. Each analytical mechanism was provided in real-time via the study website.
Surveillance for sCAP began in December 2008, prior to the 2009 H1N1 influenza A pandemic. Six facilities representing all areas of the state, both rural and metropolitan were included. The website, www.kyflu.net was developed for study coordination. From December 1, 2008, through August 2011, 458 cases of sCAP were identified. There were multiple areas of specialcause variance on the SPC charts, though there were no unusual clusters upon spatial evaluation of the maps. The most common virus identified in patients with sCAP was rhinovirus (n039, 20%), followed by 2009 H1N1 influenza A virus (n034, 18%). These viruses were cultured in chicken eggs, genetically analyzed and further studied in mouse and ferret models to determine viral evolution and virulence mechanisms. One influenza virus was found to be hypervirulent compared to other strains.
The SIPS project is an ongoing effort that has thus far successfully identified patients with sCAP of viral etiology. Surveillance for sCAP is important not only for the early detection of cases in the event of a pandemic of influenza but for other etiologies as well. Furthermore, through translational research activities, we were able to identify novel strains of influenza and are working to further characterize the evolution of these viruses in our state.
Influenza; respiratory virus; outbreak; pandemic; epidemic
Adverse drug events (ADEs) are a significant source of morbidity and mortality. The majority of postmarketing surveillance for ADEs is passive. Information regarding ADEs is reported to the medical community in peer-reviewed journals. However, in most cases, there is significant lag in the publication of peer-reviewed articles concerning ADEs. Within medical journals, our intuition is that letters to the editor may provide the earliest reports of ADEs. They often report single case reports or a collection of cases and usually precede more formal investigations and reports. Although these letters may contain useful and timely information, the challenge is that letters to the editor may be 'buried' inside print journals. Furthermore, they may be more difficult to find and access even when using electronic searches because unlike other published reports, there is no corresponding abstract to view. Due to the lack of an abstract, detection depends almost exclusively upon words in a title or manually applied Medical Subject Headings (MeSH). We propose that searching the full text of letters to the editor can provide a faster and perhaps more complete detection of ADEs compared to searches based on MeSH terms or titles alone.
We first identified a list of the most commonly used 179 drugs in 2008 based on the Agency for Healthcare Research and Quality compiled Medical Expenditures Survey. We then used Micro-Medex, a commercial drug information service, to find a list of key publications describing ADEs for these drugs. Next, we obtained the text for the majority of letters to the editor published in The Lancet (6558 from 1967 to date; 82% of total) and The New England Journal of Medicine (3524 from 1966 to date; 75% of total). We restricted the letters to those that had the MeSH term 'adverse effect' in the indexing data. We also eliminated the letters with label 'Comment' to avoid searching letters specifically referencing a previously published paper in each journal, respectively. The resulting dataset contained 2166 letters for The Lancet and 1449 for The New England Journal of Medicine.
We then compared the results from two different search strategies. In the first, an emulation of a PubMed search, we only examined the MeSH terms and the title for the letter. Our second approach included a search of the full text of the letters in addition to the title. Using these two strategies, we were able to determine the 'earliest' letter in these two journals for a given drug/ADE pair. We compared this date against the date of the citation referenced by MicroMedex to determine which search method provided the earliest detection.
Both search strategies, with and without full text, were able to find the particular drug/ADE pair mentioned in letters before the corresponding Micromedex reference. However, using fulltext search outperformed title/MeSH-based search, not only based on the number of drug/ADE pairs found but also on the time of detection. The percentage of letters in the dataset that are not related to specific articles is 0.6% using title/MeSH, 2.1% using title/full-text. Furthermore, we found that MeSH terms are not always reliable. For example, some of the letters had MeSH terms like ''Adverse Effect'' but no mention of adverse effects in the letter. Not surprisingly, since MeSH is a controlled vocabulary, some of these terms do not appear in the full text of the letters. These findings are shown in Table 1 .
Our results suggest that the full texts of the letters to the editor provide a potential stream of information regarding early warnings for ADEs. Future work will need to expand the number of journals considered and, furthermore, consider the potential for 'false positive' warnings.
Adverse drug events; letters to the editor; signal detection Objective To examine the incidence and characteristics of heat illness during sports and recreation.
Although heat illness is preventable, it is a leading cause of death among U.S. high school and college athletes (1) . Despite this, the total burden of heat illness during sports and recreation is unknown. With over 250 million U.S. residents reporting occasional participation in sports or recreational activities (2), there is a large population at risk.
We used two national injury surveillance systems to examine heat illnesses in two different U.S. population subsets. We used the National Electronic Injury Surveillance System-All Injury Program (NEISS-AIP) to examine heat illness incidence and characteristics among sports and recreation participants of all ages from 2001 to 2009, and we used the National High School Sports-Related Injury Surveillance Study (High School RIO TM ) to examine heat illness incidence and characteristics among high school-aged athletes from 2005 to 2009 (Table 1) . NEISS-AIP, operated by the U.S. Consumer Product Safety Commission, monitors consumer product-related injuries treated in a nationally representative sample of 66 U.S. hospital emergency departments (EDs) (3). Trained coders enter demographics, a brief narrative and consumer product information for each injury presenting to their ED. High School RIO TM , operated by the Center for Injury Research and Policy at Nationwide Children's Hospital (Columbus, OH), monitors sports injuries in a nationally representative sample of 100 high schools (4). Certified athletic trainers at participating schools report exposure and injury data electronically.
Using NEISS-AIP, we calculated an estimated 5946 (95% confidence interval [CI]04194Á7698) ED visits for sports-and recreation-related heat illnesses occurred annually from 2001 to 2009. Incidence was highest among males (72.5%) and among persons aged 15Á19 years (35.6%) and occurred most commonly during football (24.7%) and exercise (20.4%). Using RIO TM , we calculated an estimated 9237 (95% CI 08357Á10,116) heat illnesses resulting in time lost from participation occurred during high school sports annually from 2005 to 2009, most commonly during football (70.7%).
National injury surveillance systems provide a unique opportunity to examine heat illness in sports and recreational settings. NEISS-AIP and High School RIO TM demonstrate different approaches to studying this problem. Results from both analyses indicate that heat illness causes substantial morbidity among sports and recreation participants. We need to find new ways to target effective heat illness prevention messages to those at greatest risk to reduce morbidity and prevent mortality. Continued surveillance is also warranted to monitor trends and evaluate interventional activities.
Injury; surveillance; heat illness; NEISS; RIO TM Introduction Syndromic surveillance uses syndrome (a specific collection of clinical symptoms) data that are monitored as indicators of a potential disease outbreak. Advanced surveillance systems have been implemented globally for early detection of infectious disease outbreaks and bioterrorist attacks. However, such systems are often confronted with the challenges such as (i) incorporate situation specific characteristics such as covariate information for certain diseases; (ii) accommodate the spatial and temporal dynamics of the disease; and (iii) provide analysis and visualization tools to help detect unexpected patterns. New methods that improve the overall detection capabilities of these systems while also minimizing the number of false positives can have a broad social impact.
In this paper, we propose an inference model for determining the location of outbreaks of epidemics in a network of nodes. In our setting, the network is the NC counties where the basic model incorporates spatial geographical relationships between the counties. The model is epidemiological, by choice, to process daily flu counts from the counties in order to infer when an outbreak of flu is present in a county that is distinguishable from background counts. The methodology incorporates Gaussian Markov random field (GMRF) and spatio-temporal conditional autoregressive (CAR) modeling.
The methodology has some nice features including timely detection of outbreaks, robust inference to model misspecification, reasonable prediction performance as well as attractive analytical and visualization tool to assist public health authorities in risk assessment. Based on extensive simulation studies and synthetic data generated from a dynamic SIR model, we demonstrated that the model is capable of capturing outbreaks rapidly, while still limiting false positives.
In this paper, we have presented a new methodology that adapts the existing GMRF class of models to deal with spatio-temporal surveillance data. When the data are mainly spatial and coarsely discretized in time, simple models such as the CAR model will continue to be valuable for descriptive analysis. However, when data have a fine resolution in both the spatial and temporal dimensions, our model, which explicitly incorporates the directional nature of time by conditioning future events on past outcomes, is likely to be more insightful.
Syndromic surveillance; spatio-temporal; Markov random field; conditional autoregressive Introduction Livestock owners normally pay the full cost of disease testing. As a result the number of laboratory submissions is dependent on the owner's perception that testing is beneficial. This decreases the likelihood of an accurate diagnosis and biases the number and type of samples received by a laboratory. Despite these limitations, laboratory data are commonly used for passive disease surveillance.
The Ontario Farm-call Surveillance Project (OFSP) analyzed disease-related farm call data supplied by livestock veterinarians. Project goals were to provide a new data source for livestock disease monitoring and to improve the quality of laboratory data. As an incentive for participation, veterinarians were not charged when diagnostic samples were sent to the Animal Health Laboratory (AHL), University of Guelph.
The OFSP veterinary clinics were a convenience sample of foodproduction and equine clinics in Ontario. Clinics participating in OFSP were offered two incentives: (1) free diagnostic testing at the AHL and (2) $175.00 per farm call if postmortems (PMs) were performed and farm call data were received within 10 days of the call. The first incentive was offered for the duration of the project; the second was available from October 2010 to June 15, 2011.
The average number of days from farm call completion to data submission was compared pre-and post-PM incentive.
The rate at which a veterinarian submitted samples for diagnostic testing to the AHL was calculated (total number of submissions/total number of farm calls). Only 20/28 OFSP clinics were enrolled in the study pre-PM incentive. A comparison of the number of submissions to the AHL for those clinics pre-and post-PM incentive was performed. Submissions of animals for necropsy or tissue for histology were classified as 'pathology' submissions. The proportion of livestock pathology submissions that were from the OFSP were compared to the total livestock pathology submissions pre-and postcommencement of the PM incentive. AHL reporting rates of livestock zoonotic diseases were compared pre-and post-commencement of the OFSP (total number of positive livestock zoonotic disease laboratory submissions/total number of livestock laboratory submissions).
One hundred and eight veterinarians from 28 livestock clinics contributed data to the surveillance project between April 2009 and June 2011. No clinics withdrew from the study. Fig. 1 illustrates the timeliness of reporting before and after the PM incentive.
Veterinarians participating in OFSP submitted a sample to the AHL 11% of the time they completed a disease-related farm call. A comparison of 20 OFSP clinics revealed that 458 more cases were submitted to the AHL while those clinics were participating in the OFSP than the year prior to participation. OFSP clinics represented 19% (28/147) of the clinics submitting pathology samples during the time period the PM incentive was offered. OFSP pathology submissions represented 36% (712/ 1984) of the total pathology livestock submissions for the same time period. For the same period, the previous year (pre-PM incentive) OFSP pathology submissions accounted for 7.7% (141/1822) of the total pathology submissions.
The proportion of laboratory submissions from OFSP clinics positive for a zoonotic disease increased from 4.3% prior to participation in the project to 7.7% while part of the OFSP.
Incentives are needed to ensure adequate compliance with a surveillance program. The OFSP incentives were considered a key factor in the number of veterinarians participating in the study as well as the 0% drop out rate.
Receiving data quickly is critical when monitoring for new or emerging diseases. Animals found dead or moribund are an important group to monitor for livestock disease surveillance but producers often do not want to pay the cost of a PM. The ability to provide better client service made the incentives offered by OFSP appealing to veterinarians.
The OFSP incentives increased submissions to the laboratory, improved the laboratory data for passive surveillance and, specifically, increased zoonotic disease reporting.
Thanks to the OFSP veterinarians and clinic staff for their effort and support with this project.  RNA-Seq Based Transcriptional Map of Bovine Respiratory Disease Pathogen “Histophilus somni 2336” Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify “novel” genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method. The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations. Systems biology approaches are designed to facilitate the study of complex interactions among genes, proteins, and other genomic elements [1, 2, 3] . In the context of infectious disease, systems biology has the potential to complement reductionist approaches to resolve the complex interactions between host and pathogen that determine disease outcome. However, a prerequisite for systems biology is the description of the system's components. Therefore, genome structural annotation or the identification and demarcation of boundaries of functional elements in a genome (e.g., genes, non-coding RNAs, proteins, and regulatory elements) are critical elements in infectious disease systems biology.
Bovine Respiratory Disease (BRD) costs the cattle industry in the United States as much as $3 billion annually [4, 5] . BRD is the outcome of complex interactions among host, environment, bacterial, and viral pathogens [6] . Histophilus somni, a gramnegative, pleomorphic species, is one of the important causative agents of BRD [6] . H. somni causes bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis [7] . H. somni strain 2336, the serotype used in this study and isolated from pneumonic calf lung, has a 2.2 Mbp genome and 2044 predicted open reading frames (ORFs), of which 1569 (76%) have an assigned biological function.
Genome structural annotation is a multi-level process that includes prediction of coding genes, pseudogenes, promoter regions, repeat elements, regulatory elements in intergenic regions such as small non-coding RNAs (sRNA), and other genomic features of biological significance. Computational gene prediction methods such as Glimmer [8] or GenMark [9] use Hidden Markov models which are based on a training set of well annotated genes. Although these methods are quite efficient, they often miss genes with anomalous nucleotide composition and have several well-described shortcomings: because bacterial genomes do not have introns, detecting gene boundaries is comparatively difficult; due to the usage of more than one start codon, computational genome annotation methods may predict overlapping ORFs [10] ; prediction programs use arbitrary minimum cutoff lengths to filter short ORFs, which may lead to under-representation of small genes. In case of sRNA (small non-coding RNA) prediction, the lack of DNA sequence conservation, lack of a protein coding frame, and the limited accuracy of transcriptional signal prediction programs (promoter/Rho terminator prediction) confound computational prediction [11, 12] .
Computational prediction methods are a ''first pass'' genome structural annotation. Whole genome transcriptome studies (such as whole genome tiling arrays [13, 14, 15] and high throughput sequencing [16, 17] ) are complementary experimental approaches for bacterial genome annotation and can identify ''novel'' genes, gene boundaries, regulatory regions, intergenic regions, and operon structures. For example, a transcriptomic analysis of Mycoplasma pneumoniae identified 117 previously unknown transcripts, many of which were non-coding RNAs, and two novel genes [18] . Transcriptome analyses identified novel, non-coding regions in other species, including 27 sRNAs in Caulobacter crescentus [15] , 64 sRNAs in Salmonella Typhimurium [17] , and a large number of putative sRNAs in Vibrio cholerae [16] . sRNAs found in pathogen genomes are known to be involved in various housekeeping activities and virulence [19] .
In this study we used RNA-Seq for the experimental annotation of the H. somni strain 2336 genome and to construct a single nucleotide resolution transcriptome map. Novel expressed elements were identified, and where appropriate, computational predictions of previously described gene boundaries were corrected.
In 2008 the complete genome sequence of the H. somni strain 2336 became available (GenBank CP000947). The 2,263,857 bp circular genome has a GC content of 37.4%, and 87% of the sequence is annotated to coding regions. The genome has 2065 computationally predicted genes, of which 1980 are protein coding. We sequenced the transcriptome of H. somni using Illumina RNA-Seq methodology, and obtained 9,015,318 reads, with an average read length of approximately 76 bp. We mapped approximately 9.4% reads onto the reference DNA sequence of H. somni strain 2336 using the alignment program Bowtie [20] . To determine expressed regions in the genome, we estimated the average coverage depth of reads mapped per nucleotide/base. We used pileup format, which represents the signal map file for the whole genome in which alignment results (coverage depth) are represented in per-base format. Regions where coverage depth was greater than the lower tenth percentile of expressed genes were considered significantly expressed [21] ; in the current study, this corresponded to a coverage depth of 7 reads/bp in pileup format.
As another measure for estimating background expression level, we analyzed the coverage in the intergenic regions of the genome. We assumed that at least half of the intergenic region is not expressed (considering the presence of known expressed regions, such as 39 and 59 UTR of genes, intergenic region of the operons, and sRNAs) and calculated the coverage, which corresponded to #6 reads per base, lower than our first cutoff estimate. We retained the most conservative cutoff for expression, i.e., 7 reads per base for describing the expression map of H. somni. Nucleotides in the genome sequence with coverage depth above our threshold value were considered to be expressed. This resulted in the generation of a whole genome transcriptome profile of H. somni 2336 at a single nucleotide resolution. Figure 1 show the steps involved in the analysis of expressed intergenic regions.
We compared the RNA-Seq based transcriptome map with the available genome annotation to identify expressed, novel, and intergenic regions in the genome. Promoters and terminators were predicted across the genome to add confidence to the identified novel elements. For the first time, we report the identification of 94 sRNAs (Table 1) in the H. somni genome. The start and end for sRNA in Table 1 refer to the boundaries of transcriptionally active regions (TAR, putative sRNAs). Of these, twelve were similar to wellcharacterized sRNA families that are described in many bacterial species, such as tmRNA, 6S, and FMN ( Figure 2 ). The total of 82 novel sRNAs reported in this study has not been reported earlier.
The majority of the identified sRNAs (.75%) were shorter than 200 nucleotides (length range 70-695 nucleotides). The average GC content of sRNA at 39.3% was slightly higher compared with the 37.4% GC content of the genome. Promoters within 50 nt upstream/downstream of the TAR boundaries were predicted for 68 sRNA. Similarly, Rho-independent transcription terminators were predicted within 50 bp upstream/downstream of 40 sRNA. Figure 3 shows the depth of coverage for one of the identified novel sRNA ''HS46'' viewed in the Artemis genome browser [22] .
BLAST analysis of the sRNA sequences against the nonredundant, nucleotide database at NCBI revealed that 31 of the sRNA sequences were unique to the H. somni 2336 genome. Another 41 were highly conserved (.95% identity with .95% coverage) only in H. somni strain 129PT, which is a commensal, preputial isolate. A set of 11 sRNAs were conserved in the related Pasteurellaceae family, which includes genomes such as P. multocida, H. influenzae, H. parainfluenzae, and H. ovis. Only 11 sRNAs were conserved in distant bacterial genomes from genera Streptococcus, Clostrodium, Actinobacillus, Vibrio, and others. This lack of sRNA sequence conservation beyond the species could indicate that sRNA sequences are under strong selection pressure, and that they could be responsible for the adaptation of many species to different environmental niches.
We searched all H. somni sRNA sequences against the Rfam database [23] to determine their putative functions. We found that 12 sRNAs were homologs to well characterized sRNAs in other genomes. The identified functional categories included FMN riboswitches, gcvB, glycine, intron_gpII, lysine, alpha_RBS, LR-PK1, isrK, MOCORNA, RNaseP_bact_a, tmRNA, and 6S. sRNAs for which no Rfam function could be predicted represent a completely novel set of non-coding sRNAs. Functions of these novel sRNA need to be determined by further experiments.
We evaluated the coding potential of all expressed intergenic regions, by conducting BLASTX based sequence searches against the non-redundant protein database at NCBI followed by manual analysis and interpretation. We identified 38 novel protein coding regions ( Table 2 ). The average length of the identified novel proteins was around 60 amino acids (ranged from 19 to 135 amino acids). The majority of the novel proteins (30) were conserved hypothetical proteins present in related species such as H. somni 129PT, M. haemolytica, and H. influenzae. Some of the novel proteins had predicted functions, such as DnaK suppressor protein, toxic membrane protein TnaC, and predicted toxic peptide ibsB3 ( Table 2 ). Figure 4 shows an example of a novel protein ''HSP7'' that is similar (74% similarity and 100% coverage) to a putative, phage-related DNA-binding protein of Neisseria polysaccharea.
The single nucleotide resolution map described in this study enabled us to correct the start site for five genes based on the current genome annotation (Table 3) . These genes were annotated as phospholipid synthesis protein, ribosomal protein S2, aconitate hydratase 2, peptide chain release factor 2, and DUF411, a protein of unknown function. Based on evidence from RNA-Seq data, we performed a BLAST comparison with other phylogenetically similar proteins to confirm the new gene boundaries (Table 3) .
The comparison of the transcriptome map of the H. somni genome with predicted proteins revealed the presence of frameshift mutations. Four genes have non-functional start codons, resulting in a predicted protein, truncated at the amino terminus (based on BLAST comparison with homologous proteins in other species), although full length mRNA was present. An example is presented for the gene ''HSM_0748'', annotated as ''Alpha-Lfucosidase'' ( Figure S1 ). The other three genes, HSM_0603, HSM_1666 and HSM_1668, encode a hypothetical protein, type III restriction protein res subunit, and CTP synthase, respectively. Two genes with frameshifts causing protein truncations (based on BLAST comparison with homologous proteins) are HSM_1385 (beta-hydroxyacyl dehydratase, FabA) and HSM_1744 (alcohol dehydrogenase zinc-binding domain protein). The transcriptome map revealed a full length mRNA for these two genes that code for truncated proteins.
Our transcriptome map of H. somni identified expression from 1636 (approximately 80%) of the predicted genes. The expressed genes were distributed evenly across all TIGRFAM functional categories (Table S1 ). The transcriptome map allowed identification of operon structures at a genome scale, critical for identifying co-expressed genes and for understanding coordinated regulation of the bacterial transcriptome. We identified co-expression for 452 pairs (total 730 genes) of H. somni genes ( Table S2 ) that were transcribed together and constituted a minimal operon. By joining consecutive overlapping pairs of co-expressed genes, we identified 278 distinct transcription units (Table S3) .
We compared our experimentally identified co-expressed genes with computationally predicted operons. The overlap between computational prediction of co-expressed genes using DOOR [24] and this study was 86% (394 gene pairs) (Table S4) . Thus, our dataset validates expression of 394 computational gene-pair predictions. We identified 59 new gene pairs that are co-expressed and were not predicted by DOOR, which could be part of unidentified, new operon structures. For example, further in-depth analysis indicated a new operon consisting of three genes: HSM1354, HSM1355 and HSM1356, annotated as ribosomal protein L20, ribosomal protein L35, and translation initiation factor IF-3 respectively, which were not predicted computationally ( Figure 5 ). The orthologs of these genes are well known to form a functional operon of ribosomal proteins (IF3-L35-L20) in Escherichia coli [25] .
In this study using RNA-Seq we describe the whole genome transcriptome profile of H. somni 2336, a bovine respiratory disease pathogen. The single nucleotide resolution map helped uncover the structure and complexity of this pathogen's transcriptome and led to the identification of novel, small RNAs and protein coding genes as well as gene co-expression. Prokaryotic genome annotation is performed often using computational gene prediction programs [8, 9] . However, these prediction algorithms are not able to identify the non-coding sRNAs, antisense transcripts, and other small proteins. To overcome the shortcomings of computational genome structural annotation, various experimental methods are used for identification of novel expressed elements [13, 14, 15, 16, 17, 18, 26, 27, 28] . Deep transcriptome sequencing (RNA-Seq) has emerged recently as a method that enables the study of RNA-based structural and regulatory regions at the genome scale. RNA-Seq technology has many advantages compared with existing array based methods for transcriptome analysis. In particular, RNA-Seq does not require probes, so the process is free from probe design issues or bias from hybridization issues. Also, the transcriptome coverage from RNA-Seq is very high [29, 30] . RNA-Seq was demonstrated to be effective for the discovery of bacterial non-coding RNAs, accurate operon definition, and correction of gene annotation [27, 31, 32] . Therefore, in the current study, we used RNA-Seq for profiling H. somni 2336 transcriptome.
Mapping of RNA-Seq reads onto the H. somni genome sequence resulted in more than 94% coverage with at least one read per base. This observation is consistent with the reported 94% genome expression in Bacillus anthracis, 89.5% in Sulfolobus solfataricus, and 95% in Burkholderia cenocepacia, studied under one or more experimental growth conditions using RNA-Seq [32, 33, 34] . These results indicate that most of the bacterial genome sequence is expressed at some basal level. To identify significantly expressed regions above this baseline, we used two alternative methods (discussed in Results section) to estimate the background expression. Both methods yielded similar results (6-7 reads per base). We selected the higher stringency cutoff of 7 reads per base to minimize the number of false positives.
We identified a total of 95 sRNAs in the H. somni genome. Twelve of these were predicted by Rfam [23] and are similar to conserved sRNA (e.g., 6S, tmRNA, FMN) in other bacterial species, which helps validate our approach. The 83 novel H. somni sRNAs may have housekeeping function, regulatory activity, or participate in virulence as described in other pathogenic bacteria [19, 35, 36] . The identified sRNAs did not show any location specific bias across the genome. Similarly, genes known to be associated with virulence are known to be scattered across bacterial genomes [37, 38] . However, the tendency to form clusters was observed with sRNAs, which could indicate that functionally related sRNAs tend to be located in close proximity. The RNA-Seq based transcriptome map of H. somni identified 38 novel protein coding genes that were missed by the initial annotation. The average length of the proteins coded by these genes exceeds 60 amino acids, suggesting that length based cutoff was not the main reason that these genes were missed by computational gene prediction programs. The novel protein coding genes identified in the current study could serve as a training set to improve gene prediction algorithms.
The transcriptome map helped to identify incorrect annotation of start codons in the genome. Transcriptional mapping does not provide direct evidence of translational start sites. However, location of identified transcriptional start sites suggest that the annotated start codons are incorrect, an observation that is confirmed by BLAST comparisons against homologous genes in other bacterial species. Transcriptional mapping revealed genes where the 59 untranslated sequence extended well beyond the translational start. BLAST comparisons indicated that these genes have either nonsense or missense base changes relative to homologous genes in other bacterial species, causing apparent ''truncated'' proteins compared with those in other species. Further work is needed to determine whether these 59 untranslated regions serve regulatory functions or they are vestigial.
RNA-Seq data enabled us to determine operon structures at a genome scale, and it allowed identification of some operons not predicted by the computational operon prediction method. Operon structures that include genes not expressed under the experimental growth condition used in the current study, could not be identified. Our results support the notion that using a combination of experimental operon identification by RNA-Seq and computational prediction can improve operon identification in bacterial genomes [39] .
For the first time, we report the RNA-Seq based transcriptome map of H. somni 2336 and describe novel expressed regions in the genome. Whereas the results are interesting, we are aware of the limitations of the study. Because the RNA-Seq protocol was not strand specific, we could not determine the strand specificity of expressed novel transcripts. Therefore, Table 1 lacks information about sRNA orientation in the genome. Because strand specific information was missing, we could not describe antisense expression in the genome. For protein coding genes, we derived strand specificity based on alignment of the BLAST hit. Despite this shortcoming, we identified novel expressed regions and transcriptional patterns across the whole genome at a high coverage, which is not possible by other transcriptome analysis methods.
Overall, this study describes RNA-Seq based transcriptome map of H. somni for identification of functional elements in a pathogen of importance to agriculture. Our genome-wide survey predicts numerous, novel, expressed regions that need biological characterization for understanding disease pathogenesis. Description of all functional elements in the H. somni system is a prerequisite for conducting holistic systems approaches to understand the complex pathogenesis of bovine respiratory disease.
We propagated H. somni 2336 on three TSA-blood plates (with 5% sheep red blood cells) for 16 hr or until a fresh lawn of cells was visible. IBC approval was not required for acquiring the plates as they were purchased through a commercial vendor: Fisher Scientific (Pittsburgh, PA), and manufactured by Becton Dickinson Diagnostic Systems, (Franklin Lakes, NJ). We washed the plates with brain heart infusion (BHI) broth, adjusted the culture to an OD620 nm = 0.8, and supplemented with RNAprotect reagent. The cells were harvested by centrifugation and stored at 280uC. We extracted total RNA using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Total RNA was treated with RNase-free DNAse (Invitrogen, Carlsbad, CA). Using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), we determined the RNA integrity number (RIN) of total RNA to be greater than 8. MICROBExpress TM Kit (Ambion, TX, USA), which specifically removes rRNAs, was used for mRNA enrichment. Small RNAs (i.e., tRNA and 5S rRNA) are not removed with this enrichment step (confirmed by Bioanalyzer).
We used 100 ng enriched mRNA with Illumina mRNA-Seq sample preparation kit (Illumina, San Diego, CA) for library construction following the manufacturer's protocols. Briefly, mRNA was fragmented chemically by divalent zinc cations and randomly primed for cDNA synthesis. After ligating paired-end sequence adaptors to cDNA, we isolated fragments of approximately 200 bp by gel electrophoresis and amplified. We 
We checked all Illumina reads for quality, and removed sequence reads containing ''Ns''. Custom perl script was written to convert Illumina reads into fastq format. The script ''fq_all2std.pl'' from MAQ [40] converted fastq format to Sanger fastq format. Reads in sanger fastq format, were mapped onto the Histophilus somni 2336 genome sequence (GenBank Accession number. CP000947) using the alignment tool Bowtie [41] , allowing for a maximum of two mismatches. The reads that mapped to more than one location were discarded. We used Samtools [42] to convert data into SAM/BAM format, and to generate alignment results in a pileup format. Pileup format provides the signal map file and has per-base format coverage. Custom perl scripts were written to calculate the background expression. Processed data was deposited in GEO with the accession number GSE29578.
We used in-house perl scripts to extract novel expressed intergenic regions to identify novel small RNAs, riboswitches, and putative novel proteins. sRNA ,70 bp in length were discarded to minimize the number of false positives. For each novel expressed region, BLAST sequence searches were performed against the non-redundant protein database at NCBI to identify potential protein coding regions. Intergenic regions within predicted operons [24] represent expressed regions and can be mis-classified as sRNAs. Therefore, these regions were excluded.
We analyzed BLAST results manually, to identify novel protein coding regions and start codon corrections. If no protein coding region was found in the intergenic expressed regions, the presence of a promoter or a rho-independent terminator allowed us to classify the regions as sRNA. Bacterial promoter sequences were predicted by Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html) [43] . Rho-independent transcription terminators were identified using the program TransTermHP [44] . For functional annotation, all identified identified sRNA sequences were searched against the Rfam database [23] . sRNA sequence conservation among other genomes was determined by blastn searches against nonredundant nucleotide database at NCBI. We mapped sRNAs, along with additional features, onto genome browsers like IGV [45] and Artemis [46] for further visualization, manual analysis, and interpretation.
Gene expression: expressed reads with coverage above background were mapped onto the annotated genes of H. somni 2336. Genes that had a significantly higher proportion of their length (.60%) covered by expressed reads were considered to be expressed.
Operons: RNA-Seq can identify and predict operon structures in bacteria. We considered two or more consecutive genes to be part of an operon, if they fulfilled the following criteria: (a) they are expressed; (b) they are transcribed in the same direction; and (c) the intergenic region between the genes is expressed. Overlapping pairs of such genes were joined together to identify large operon structures. We used in-house perl scripts for the analyses. Figure S1 Mutated start codon. The Figure shows that the predicted protein coding frame (MH_748) is shorter at the 59 end than the corresponding transcript level shown by the RNA-Seq coverage. Although the transcript is longer near 59 end, no start codon is found in that region which might be a result of the mutation in that region of the start codon. This was further validated using homology searches of the full length transcript which shows high homology (95% Identity and .95% coverage) to a alpha-L-fucosidase protein from M. haemolytica PHL213. (TIF)  A review on chemical and biological properties of Cayratia trifolia Linn. (Vitaceae) Cayratia trifolia Linn. Domin Syn. Vitis trifolia (Family: Vitaceae) is commonly known as Fox grape in English; Amlabel, Ramchana in Hindi and Amlavetash in Sanskrit. It is native to India, Asia and Australia. It is a perennial climber having trifoliated leaves with 2-3 cm long petioles and ovate to oblong-ovate leaflets. Flowers are small greenish white and brown in color. Fruits are fleshy, juicy, dark purple or black, nearly spherical, about 1 cm in diameter. It is found throughout the hills in India. This perennial climber is also found in the hotter part of India from Jammu and Rajasthan to Assam extending into the peninusular India upto 600 m height. Whole plant of Cayratia trifolia has been reported to contain yellow waxy oil, steroids/terpenoids, flavonoids, tannins upon preliminary phytochemical screening. Leaves contain stilbenes (piceid, reveratrol, viniferin, ampelopsin). Stem, leaves, roots are reported to possess hydrocyanic acid, delphinidin and several flavonoids such as cyanidin is reported in the leaves. This plant also contains kaempferol, myricetin, quercetin, triterpenes and epifriedelanol. Infusion of seeds along with extract of tubers is traditionally given orally to diabetic patients to check sugar level of blood. Paste of tuberous is applied on the affected part in the treatment of snake bite. Whole plant is used as diuretic, in tumors, neuralgia and splenopathy. Its climbers wrapped around the neck of frantic bullock and poultice of leaves are used to yoke sores of bullock. The bark extract shows the antiviral, antibacterial, antiprotozoal, hypoglycemic, anticancer and diuretic activity. This article focuses on the upgraded review on chemical and biological properties of Cayratia trifolia Linn. and triggers further investigation on this plant.  Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/ncomms1466) contains supplementary material, which is available to authorized users. T he precise assembly of many ion channels relies on a multimerization domain that assembles compatible subunits and excludes incompatible subunits. In the voltage-gated family of channels, these multimerization domains take the form of either an amino-terminal tetramerization domain (T1 domain) 1,2 or a carboxy-terminal four helix coiled-coil domain [3] [4] [5] . Although channel assembly is generally permitted without these multimerization domains, such domains confer subunit-specific assembly, often restricting the channel composition to like-subunits.
Cyclic nucleotide-gated (CNG) channels, however, have a more complex subunit composition. Native CNG channels are composed of a stereotyped number of CNGA subunits and CNGB subunits. CNGA and CNGB subunits are structurally related and both are thought to contribute to the formation of the pore. The CNG channel from rod photoreceptors is composed of three CNGA1 subunits and one CNGB1 subunit [6] [7] [8] , whereas the CNG channel from olfactory receptors is composed of two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit 9 . The cone CNG channels comprise CNGA3 subunits and CNGB3 subunits, though the subunit stoichiometry is still unknown 8, 10 . The presence of the CNGB subunits confers enhanced Ca 2 + permeation, modulation by Ca 2 +calmodulin, and native-like cyclic-nucleotide specificity compared with homomeric CNGA channels [11] [12] [13] [14] [15] [16] [17] . The precisely controlled subunit compositions, therefore, tune the CNG channels for their specialized roles in phototransduction and olfactory transduction.
CNG channels are activated by the direct binding of intracellular cyclic nucleotides 18 . They are members of the voltage-gated ion channel superfamily, consisting of four subunits around a centrally located pore. The intracellular carboxy-terminal region of each subunit consists of a C-linker region, a cyclic nucleotide-binding domain (CNBD), and a post-CNBD region (Fig. 1a) . The Xray crystal structure of the carboxy-terminal region of the related hyperpolarization-activated cyclic nucleotide-gated channel reveals a four fold symmetric tetramer with non-interacting CNBDs and extensive intersubunit interactions between C-linker regions 19 .
How does the complex subunit composition of CNG channels come about? The CNGA subunits contain a carboxy-terminal leucine zipper (CLZ) domain in the post-CNBD region (Fig. 1a,b) 8 . On the basis of the finding that the CLZ domain from CNGA3 forms homotypic trimers in solution, it was proposed that trimerization of this domain constrains the channel to contain three CNGA subunits 8 . Previously, it has been shown that point mutations in this domain decreased the proportion of heteromeric channels (relative to CNGA homomers) but did not alter the 3:1 stoichiometry of the heteromeric channels 20 .
Here we test the role of the CLZ domain by measuring the subunit composition of CNGA1 + CNGB1 channels when the CLZ domain in the CNGA1 subunit was deleted. Using electrophysiology and fluorescence measurements, we found that deletion of the CLZ domain relaxed the constrained subunit stoichiometry. This allowed more than one CNGB1 subunit in the channel and conferred novel pharmacological properties and cyclic nucleotide specificity. Using X-ray crystallography, we found that the CLZ domains of both CNGA1 and CNGA3 formed homotypic parallel 3-helix coiled-coil domains, consistent with their proposed role in regulating subunit assembly.
To determine the role of the CLZ domain in controlling heteromeric assembly of CNGA1 + CNGB1 channels, we first measured the functional effects of deleting the CLZ domain. CNG channels were expressed in Xenopus oocytes and studied using the inside-out configuration of the patch-clamp technique. Homomeric channels of CNGA1 subunits are efficiently activated by saturating cGMP, but poorly activated by saturating cAMP (I cAMP /I cGMP = 0.05 ± 0.01, n = 6) (Fig. 2a , black is with 2.5 mM cGMP, red is with 16 mM cAMP). However, incorporation of a single CNGB1 subunit per tetramer confers a significant elevation in cAMP efficacy (I cAMP / I cGMP = 0.22 ± 0.03, n = 7) (Fig. 2a) . The mechanism of the increased cAMP efficacy by the CNGB1 subunit is unknown but could involve either cAMP binding to CNGB1 or simply a more favourable opening transition with CNGB1 (or both). This increased efficacy is constant over a wide range of CNGB1 expression levels, indicating that the CNGB1 subunit incorporates into the channel with a fixed and tightly-regulated stoichiometry 15 .
Previously, it has been shown that the amino-terminal region of the CNGB1 subunit interacts with the CLZ domain of the CNGA1 subunit and regulates trafficking of heteromeric channels 21 . To test for a possible role of this interaction in subunit assembly, we deleted the amino-terminal region (amino acids 2-765) of CNGB1 (CNGB1-∆N). Co-expressing CNGA1 and CNGB1-∆N produced robust currents with amplitude similar to that of CNGA1 + CNGB1 (ref. 21 ). In addition, as shown in Figure 2a , deletion of the aminoterminal region of CNGB1 had no effect on the cAMP efficacy of the heteromeric channels (I cAMP /I cGMP = 0.21 ± 0.03, n = 7). These results suggest that the amino-terminal domain of CNGB1 is not required for the proper assembly of functional heteromeric channels.
Next, we tested the effects of deletion of the CLZ domain (amino acids 609-693) of the CNGA1 subunit (CNGA1-∆CLZ). Deletion of the CLZ domain had no effect on the expression or measured functional properties of CNGA1-∆CLZ homomeric channels (Fig. 2a) 21 . However, it had a dramatic effect on expression and functional properties of CNGA1-∆CLZ + CNGB1 heteromeric channels. As shown previously, co-expression of CNGA1-∆CLZ with CNGB1 at a typical 1:4 RNA ratio virtually eliminated all functional channel expression (Fig. 2a) 21 . This dominant negative effect may underlie one form of retinitis pigmentosa resulting from deletion of a portion of the CLZ domain in CNGA1 (refs 21,22) and suggests that the CNGA1-∆CLZ channels are still interacting with CNGB1, perhaps in an uncontrolled manner. To recover functional expression of heteromeric channels, we co-injected CNGA1-∆CLZ with CNGB1-∆N containing a deletion of the trafficking signal in the amino-terminal region of CNGB1 (ref. 21) . Even at an RNA ratio of 1:2 CNGA1-∆CLZ: CNGB1-∆N, the effect of the CLZ domain deletion on the heteromeric channels was striking. Co-expression of CNGA1-∆CLZ and CNGB1-∆N yielded channels with a highly elevated cAMP efficacy (I cAMP /I cGMP = 0.38 ± 0.02, n = 10) (Fig. 2a) , significantly higher than CNGA1 homomeric channels and CNGA1 + CNGB1 heteromeric channels (Fig. 2b) . The elevated cAMP efficacy seen with the CLZ domain deletion in heteromeric channels, but not homomeric channels, suggests the possibility that CNGA1-∆CLZ and CNGB1-∆N form heteromeric channels with a novel stoichiometry, perhaps with more than one CNGB1 subunit per channel.
To further test for incorporation of multiple CNGB1 subunits on deletion of the CLZ domain, we measured the pharmacological properties of heteromeric channels. Although 100 µM L-cisdiltiazem is ineffective at blocking CNGA1 homomers, it potently blocks CNGA1 + CNGB1 heteromers (Fig. 3a , black is control, blue is 100 µM L-cis-diltiazem) 17 . The block is voltage dependent with a Hill slope of one, suggesting a pore-blocking mechanism (Fig. 3a,b) . The CNGA1-∆CLZ mutation had no effect on block of homomeric channels, and the CNGB1-∆N mutation had no effect on block of heteromeric channels. Surprisingly, the block of CNGA1-∆CLZ + CNGB1-∆N heteromers was intermediate between CNGA1 (or CNGA1-∆CLZ) homomers and , and CnGA1-∆CLZ + CnGB1-∆n (n = 10). The expected subunit stoichiometry for each case is shown at the bottom. Data are plotted as mean ± s.e.m. Asterisks denote statistically significant differences with P < 0.01. CNGA1 + CNGB1 (or CNGA1 + CNGB1-∆N) heteromers (Fig. 3a) . Previously, it was shown that heteromers formed with point mutations in the CLZ domain of CNGA3 exhibited intermediate block by L-cis-diltiazem because they formed mixtures of CNGA3 homomers and CNGA3 + CNGB1 heteromers 20 . However, that was not the case here. As shown above, CNGA1-∆CLZ + CNGB1-∆N heteromers actually exhibit greater cAMP efficacy than either CNGA1 homomers or CNGA1 + CNGB1 heteromers (Fig. 2) . Together, these results indicate the formation of a previously uncharacterized population of heteromeric CNG channel complexes.
To unravel the cause of this intermediate block, we measured the dose-response relations for L-cis-diltiazem block at + 60 mV. The dose-response relations for channels containing CLZ domains were well fit with a single Langmuir isotherm, suggesting a single population of channels (Fig. 3b) . The block of CNGA1 and CNGA1-∆CLZ homomers were similar and low affinity (K 1/2 = 174 ± 7.2 µM, n = 5 and K 1/2 = 164 ± 8.2 µM, n = 6, respectively), and the block of CNGA1 + CNGB1 and CNGA1 + CNGB1-∆N heteromers were similar and high affinity (K 1/2 = 2.45 ± 0.28 µM, n = 5 and K 1/2 = 1.40 ± 0.21 µM, n = 5, respectively) (Fig. 3b) . The dose-response relation for block of CNGA1-∆CLZ + CNGB1-∆N heteromers, however, was not fit well by either a single Langmuir isotherm, nor by the sum of two Langmuir isotherms with affinities constrained to those of CNGA1 homomers and CNGA1 + CNGB1 heteromers. Instead, fits to the sum of two Langmuir isotherms revealed a small high affinity component similar to that of CNGA1 + CNGB1 channels (K 1/2 = 0.56 ± 0.09 µM, n = 4), and a larger component with an affinity intermediate between that of CNGA1 homomers and CNGA1 + CNGB1 heteromers (K 1/2 = 50 ± 5 µM, n = 4) (Fig. 3b) . These results suggest that the heteromers formed from CNGA1-∆CLZ and CNGB1-∆N are not a mixture of CNGA1 homomers and normal CNGA1 + CNGB1 heteromers, but, instead, a mixture of normal 3:1 CNGA1 + CNGB1 heteromers and novel CNGA1 + CNGB1 heteromers.
One intriguing possibility is that the novel heteromers formed without a CLZ domain contain multiple CNGB1 subunits. This would also suggest that whereas one CNGB1 subunit confers much greater L-cis-diltiazem affinity, extra CNGB1 subunits lower the L-cis-diltiazem affinity (Fig. 3d) . This model predicts that lowering the relative amount of expressed CNGB1 subunits should actually increase the fraction of the high affinity component (due presumably to channels with the normal 3:1 CNGA1:CNGB1 stoichiometry), because a lower amount of CNGB1 should favour a subunit stoichiometry with fewer CNGB1 subunits. The results of such an experiment are shown in Figure 3c . Lowering the RNA co-injection ratio of CNGA1-∆CLZ and CNGB1-∆N from 1:2 to 1:1 significantly increased the fraction of the high affinity component from 0.21 ± 0.01, n = 3 to 0.40 ± 0.01, n = 4. In addition, the dose-response relation is now poorly fit by a sum of two Langmuir isotherms and is suggestive of a third component with an affinity similar to CNGA1 homomers. Together, these results suggest that deletion of the CLZ domain removes the constraint allowing only one CNGB1 subunit in the channel tetramer. This allows multiple CNGB1 subunits that confer higher efficacy for cAMP and (surprisingly) lower affinity for L-cis-diltiazem block (Fig. 3d ). If channels containing three CNGB1 subunits were not functional or not trafficked properly to the plasma membrane, then this loss of controlled assembly could also explain the large dominant negative effect of CNGB1 subunits, when the CLZ domain is deleted from the CNGA1 subunit.
To directly measure the presence of multiple CNGB1 subunits in the channel, we turned to an approach based on fluorescence resonance energy transfer (FRET) 23 . FRET reports the proximity of two fluorophores based on the efficiency of energy transfer between a donor fluorophore and an acceptor fluorophore. FRET can be detected by an enhanced emission of the acceptor fluorescence on excitation of the donor. Cyan fluorescent protein (eCFP) and yellow fluorescent protein (eYFP) are genetically-encoded donor and acceptor fluorophores, respectively, that can be fused to proteins of interest 24 . They exhibit 50% energy transfer at a distance of about 50 Å and measurable transfer up to 80 Å (ref. 25) , making them ideally suited for measurements of subunit composition 7 . Previously it was shown that if CNGA1 subunits are fused to eCFP and eYFP (individually) and co-expressed with CNGB1 subunits in Xenopus oocytes, there was significant FRET for channels in the plasma membrane 7 . However, if CNGB1 subunits were fused to eCFP and eYFP and co-expressed with CNGA1 subunits, there was little or no FRET (Fig. 4a) . These results revealed that heteromeric rod CNG channels in the plasma membrane contain multiple CNGA1 subunits but only a single CNGB1 subunit (3:1 stoichiometry).
We performed a similar experiment to determine if deletion of the CLZ domain permitted incorporation of multiple CNGB1 subunits into the heteromeric channels. If there are multiple CNGB1 subunits in the heteromeric channels, then we should observe FRET between CNGB1 subunits when fused to eCFP and eYFP and co-expressed with CNGA1-∆CLZ subunits (Fig. 4a) .
For these experiments, we fused eCFP and eYFP (individually) to the amino-terminal end of CNGB1-∆N (eCFP-CNGB1-∆N and eYFP-CNGB1-∆N respectively). The eCFP and eYFP fusion constructs exhibited functional properties indistinguishable from their non-fusion counterparts (data not shown). As the amino-terminal region is deleted in this subunit, the fluorescent proteins should be near the inner leaflet of the membrane, close to the S1 transmembrane segment. Based on the structure of the related Kv1.2 channel 26 , two fluorescent proteins in the same channel should be about 56 Å apart (assuming they are in adjacent subunits) or 79 Å apart (assuming they are in nonadjacent subunits).
Representative results from co-expression of CNGA1-∆CLZ with eCFP-CNGB1-∆N and eYFP-CNGB1-∆N are shown in Figure 4b . FRET was measured from the sensitized emission of the acceptor using fluorescence spectra 23 . The fluorescence emission spectra from channels in or near the plasma membrane of live Xenopus oocytes were measured with confocal microscopy. Excitation of eCFP with a 458 nm laser produced a complex emission spectrum (red trace) with both eCFP and eYFP emission components. The eYFP component (orange trace) was extracted by subtracting off a scaled emission spectrum from eCFP alone (blue trace). This extracted spectrum contains eYFP emission from both FRET and direct excitation of eYFP by 458 nm light. To isolate the component due to FRET, the fractional excitation of eYFP by 458 nm light relative to 488 nm light (Ratio Ao) (Fig. 4c, Methods) was subtracted off the corresponding fractional excitation for eCFP and eYFP-containing channels (Ratio A). Ratio A-Ratio Ao is directly proportional to the FRET efficiency 23 . As shown in Figure 4d , Ratio A-Ratio Ao was significantly larger with CNGA1-∆CLZ than with CNGA1, when the CNGA1 subunit was coexpressed with fluorescent CNGB1. These results directly demonstrate that deletion of the CLZ domain caused multiple CNGB1 subunits to assemble.
A possible alternative explanation for the FRET seen between CNGB1 subunits with deletion of the CNGA1 CLZ domain is that the CNGB1 subunits are forming homomeric channels at low levels. Indeed, the fluorescence is considerably dimmer with co-expression of CNGA1-∆CLZ than with CNGA1. Previously, however, it has been shown that, in the absence of CNGA1, CNGB1 subunits do not form functional homomeric channels and are only very weakly expressed in the plasma membrane 9, 17 . Similarly, we found that, in the absence of a CNGA1 subunit, both CNGB1 and CNGB1-∆N did not produce detectable currents and are poorly expressed in the plasma membrane ( Supplementary Fig. S1 ). In addition, it was shown previously that the CNGB1 subunits expressed alone do not exhibit appreciable FRET 9 . This makes it unlikely that the increased FRET seen between CNGB1 subunits with deletion of the CNGA1 CLZ domain is from CNGB1 subunits forming homomeric channels.
Nevertheless, to prove that multiple CNGB1 subunits are coassembled with CNGA1-∆CLZ subunits, we examined the FRET between CNGA and CNGB subunits. For acceptor sensitization, the apparent FRET efficiency increases as the donor-to-acceptor ratio increases 27 . Therefore, if the donor fluorophore (eCFP) was fused to the CNGB subunit and the acceptor fluorophore (eYFP) was fused to the CNGA subunit, then an increase in apparent FRET should be seen if multiple CNGB1 subunits assembled in the channel complex ( Supplementary Fig. S2) . Indeed, the results show a significant increase in FRET between eCFP-CNGB1-∆N and eYFP-CNGA1-∆CLZ compared with eYFP-CNGA1 (Fig. 4e) . These results indicate that multiple CNGB1 subunits are co-assembling with CNGA1-∆CLZ subunits. Overall, it is clear that deletion of the CLZ domain relaxes the 3:1 constraint on subunit assembly.
The above results suggest that the CLZ domain has an important role in constraining the stoichiometry of rod CNG channels to 3:1 CNGA1:CNGB1 subunits. Previously, it has been shown that a protein fragment containing the CLZ domain from cone CNGA3 forms homotypic trimers in solution 8 . To determine whether the CLZ domain from CNGA1 would also form trimers, and to localize the region involved, we screened a number of carboxy-terminal fragments of both CNGA1 and CNGA3 using fluorescence-detection size-exclusion chromatography (FSEC) (Supplementary Fig. S3) 28 . The protein fragments were expressed in bacteria as N-terminal (NGFP) or C-terminal (CGFP) fusions to GFP. After cell lysis, the crude supernatant was then loaded on a size-exclusion chromatography system with an inline fluorescence detector. All the expressing fragments that contained the CLZ domain showed an appreciable component that eluted at 13 mL, consistent with the formation of a trimer (Fig. 5a) . To verify the composition of the high molecular weight component, samples of the purified protein, following cleavage of GFP, were analysed by light scattering size-exclusion chromatography (LS-SEC) (Fig. 5b) 29, 30 . For both CNGA1 and CNGA3, the molecular weights determined from LS-SEC were within about 1% of the predicted molecular weights for a trimer (Table 1) . Similar results with smaller fragments revealed a minimal fragment, corresponding to the CLZ domain, that formed trimers in both CNGA1 (amino acids 621-667) and CNGA3 (626-672) subunits.
The sequence of the CLZ domain exhibits periodic heptad repeats (a-b-c-d-e-f-g) n in which the 'a' and 'd' positions are hydrophobic residues (Fig. 1b) 8 . This motif is characteristic of coiled-coil domains 31, 32 . There are two noncontiguous heptad repeat regions in each sequence; the first has three repeats and the second has two repeats. The two regions are out of register, with the 'a' position of the first region becoming the 'd' position of the second region.
While the presence of heptad repeats is fairly indicative of coiledcoil domains, it is difficult to determine from the sequence alone the oligomeric state, parallel versus antiparallel arrangement, and details of the coiled-coil packing and surface architecture 32 . These properties have direct bearing on the mechanism of assembly, the interpretation of mutations, and the interaction with other proteins or domains. For these reasons, we were interested in obtaining the high-resolution structure of the CLZ domains.
We have solved the high-resolution X-ray crystal structures of the CLZ domains of both CNGA1 and CNGA3. The proteins were expressed and purified from bacterial cell lysates and crystallized under vapour diffusion. Crystals of CNGA1#621-690 grew in space group P2 1 2 1 2 1 with three molecules in the asymmetric unit and diffracted to 2.14 Å. The structure of CNGA1#621-690 was solved using molecular replacement with the structure of HIV gp41 N-trimer pocket region 33 (3L36) as a search probe (Supplementary Table S1 ). The structure of CNGA3#626-672 in space group P12 1 1 was solved to 1.9 Å using molecular replacement with the structure of SARS virus S2 protein 34 (1ZVB) as a search probe (Supplementary Table S1 ).
The structures of both CNGA1#621-690 and CNGA3#626-672 consisted of long (65 Å) parallel three-helix coiled-coil domains (Fig. 6) . The sequence beyond the CLZ domain in CNGA1 (amino acids 665-675 for chain A; 665-676 for chain B; and 665-670 for chain C) flared outward and was partially involved in crystal packing interactions (Supplementary Fig. S4 ). In addition, the CNGA1#621-690 structure contained two bound heavy atoms per trimer with an electron density greater than 10 sigma ( Fig. 6 ; Supplementary Fig. S5 ). These heavy atoms were coordinated by acidic residues (E629 and D633 for the proximal site, and E671 and E623 for the distal ion) at the interface between different trimers in the crystal and, therefore, are presumed to be non-physiological ( Supplementary Fig. S5 ). They are likely to be Zn 2 + ions because of their coordination and the requirement for Zn 2 + in the crystallization solution to produce crystals.
The packing of the coiled-coil domains followed the characteristic 'knobs into holes' arrangement with the 'a' and 'd' positions forming alternating layers of the hydrophobic core of the coiled coil ( Fig. 7) 32, 35 . The two noncontiguous heptad repeat regions assembled into a single continuous coiled coil. Interestingly, the CNGA1 and CNGA3 structures differed in the length of the two heptad repeat regions. For CNGA1, the first region contained 3 heptad repeats and the second contained 3.5 repeats (Fig. 7b) . For CNGA3 the first region contained 4 heptad repeats and the second contained 2.5 repeats (Fig. 7c) . The difference appears to be the substitution of an isoleucine for a leucine after the third heptad repeat in CNGA1 (Fig. 1b) .
Although the structures of CNGA1 and CNGA3 were generally similar, they were not identical. As stated above, different residue positions were used in the helical packing between the two heptad repeat regions in the sequence. In addition, the supercoiling was different between CNGA1 and CNGA3, with a somewhat longer pitch for CNGA1 (181 Å) than for CNGA3 (149 Å) (Supplementary Table S2 ). These differences were also reflected in the structural alignments of the individual subunits of CNGA1 and CNGA3 (rmsd of Cα ranging from 0.87 Å to 2.0 Å). These differences in structure, however, do not have any known functional consequences.
The results from the above experiments, and those of others, suggest a molecular mechanism for the controlled assembly of rod CNG channels with a 3:1 stoichiometry of CNGA1:CNGB1 subunits (Fig. 8a) [6] [7] [8] [9] 20 . In this mechanism, the CNGA1 subunits initially assemble into trimers through homotypic association of their carboxy-terminal CLZ domains into a parallel three helix coiled coil. Subsequently, a CNGB1 subunit, if present, is preferentially incorporated into the remaining slot of the channel tetramer because of a high affinity association with CNGA1 subunits. This mechanism incorporates aspects of two previous models for heteromeric assembly of CNG channels: initial assembly of CNGA1 trimers 8 , and high affinity association between CNGA1 and CNGB1 subunits 9,21 . The trimerization of the CLZ domain imposes the constraint that the channels contain at least three CNGA1 subunits, whereas the high affinity association between CNGA1 and CNGB1 assures that the fourth subunit is CNGB1. In support of this mechanism, we showed with electrophysiology and fluorescence that deletion of the CLZ domain in CNGA1 causes multiple CNGB1 subunits to incorporate into the heteromeric CNG channel. Furthermore, with X-ray crystallography we showed that there is a trimerization of the CLZ domain for both CNGA1 and CNGA3 (Fig. 5) that results from the formation of a parallel three helix coiled coil (Fig. 6) .
In an elegant series of experiments, Yau and co-workers have proposed a mechanism for 3:1 CNGA:CNGB assembly that involves initial assembly of CNGA trimers 8 . They show that other three helix coiled coils, but not two helix or four helix coiled coils, could substitute for the CLZ domain of CNGA channels to reproduce normal heteromeric channel function and expression 20 . However, whereas we found that deletion of the CLZ domain causes an increase in CNGB1 incorporation in the channels, they showed that mutations in the CLZ domain decreased the incorporation of CNGB1 subunits 20 . Their results were explained by an assembly model where excess CNGA1 monomers outcompete CNGB1 monomers for channel incorporation. Instead, we propose that CNGB1 monomers outcompete CNGA1 monomers for channel incorporation owing to a high affinity association of CNGA1 and CNGB1 subunits. A high affinity association between CNGA and CNGB subunits has been previously proposed to explain the dependence of the CNGA:CNGB ratio in the surface membrane as a function of the ratio of their RNAs injected into the oocyte 9 . A high affinity association of CNGA1 and CNGB1 subunits also explains the large dominant negative effect of CNGB1 on deletion of the CLZ domain in CNGA1 (Fig. 2a) 21 . When the CLZ domain is deleted, the CNGA1 subunits preferentially interact with CNGB1 subunits, resulting in channels with multiple CNGB1 subunits that are not as efficiently expressed in the plasma membrane (Fig. 8b) . This defect has been proposed to underlie one form of inherited retinitis pigmentosa resulting from deletion of the last two heptad repeats of the CLZ domain of CNGA1 (refs 21,22) .
Previously, it has been shown that deletion of the aminoterminal region of CNGB1 could partially restore expression of these CNGA1-∆CLZ-containing channels 21 , suggesting that the amino-terminal region of CNGB1 might be involved in the highaffinity association between CNGA1 and CNGB1 subunits. Alternatively, the amino-terminal region of CNGB1 could contain a trafficking signal for incorrectly assembled heteromeric CNG channels 21 . This trafficking signal could provide a mechanism for error-correction where channels with more than one CNGB1 subunit are either poorly trafficked to the plasma membrane or rapidly removed from the membrane.
Beyond channel assembly, the CLZ coiled-coil domain may also serve as a scaffold for interactions with other proteins. Previously, it has been shown that the calmodulin binding site in the aminoterminal region of CNGB1 directly interacts with the CLZ domain of CNGA1 (refs 36,37) . Furthermore, Ca 2 + -calmodulin disrupts this interaction resulting in separation of the amino-terminal region of CNGB1 from the carboxy-terminal region of CNGA1 (ref. 38) . Also, the CLZ coiled-coil domain may be an important site of interaction for protein complexes that regulate channel activity, as has been proposed for Kv7 channels [39] [40] [41] .
Although the subunit stoichiometry of the rod CNG channel has been well established, the stoichiometry of the cone CNG channel is still uncertain. Whereas some studies have suggested it also has a 3:1 stoichiometry of CNGA3:CNGB3, others have suggested a 2:2 stoichiometry 8, 10 . Here we find that the CLZ domain of CNGA3 forms a parallel three helix coiled coil that is similar to that of CNGA1. This suggests that the stoichiometry and mechanism of assembly for the cone CNG channel will be similar to that of the rod CNG channel. In addition, it has been shown that other three helix coiled-coil domains can substitute for the CLZ domain in cone CNG channels, but two and four helix coiled-coil domains cannot 20 . Interestingly, the olfactory CNG channel has a stoichiometry of 2:1:1 CNGA2:CNGA4:CNGB1b 9 . Both the CNGA2 and CNGA4 subunits have a CLZ domain (Fig. 1b) . It remains to be determined whether the 2:1:1 stoichiometry comes about from a heterotypic three helix coiled coil of the CNGA2 and CNGA4 subunits, or from other intersubunit interactions such as seen at the level of the C-linker regions in the related hyperpolarization-activated cyclic nucleotide-gated channels 19 . In addition, the 3:1 stoichiometry of the TRPP2/PKD1 complex has recently been proposed to involve a trimeric coiled-coil domain in the carboxy-terminal region of TRPP2 (ref. 42) , suggesting that the assembly mechanism of CNG channels may extend to other distantly related proteins.
Molecular biology. The bovine CNGA1 complementary DNA used was described previously 43, 44 and contains a C-terminal FLAG epitope (DYKDDYK). The bovine CNGB1 cDNA was a kind gift from Dr. R. Molday 14 and the human CNGA3 cDNA was a kind gift from Dr. K.W. Yau 45 . CNGA1-∆CLZ (CNGA1-∆609-693) and the CNGB1-∆N (CNGB1-∆2-765) were made as described 21 . CNGB1-∆N had a further manipulation, where its internal NcoI restriction site was removed via silent mutation and re-inserted at the initial methionine codon using a PCRbased method. Improved monomeric versions of eCFP and eYFP (mCerulean and mCitrine) were employed in the generation of all fluorescent protein fusions with CNG subunits used for FRET experiments. cDNAs for mCerulean and mCitrine were kindly provided by Dr D Piston 46 and Dr RY Tsien 47 , respectively. All constructs were confirmed with fluorescence-based automated sequencing. cDNAs were subcloned into the high-expression pGEMHE vector 48 for expression in Xenopus oocytes. Vectors were linearized and complementary RNA (cRNA) was transcribed using the mMessage mMachine kit (Ambion). For co-injection experiments, relative amounts of RNA were quantified on agarose gels with a densitometric method. CNGA1 and CNGB1 were injected at an RNA ratio of 1:4 that has previously been The CnGA1 subunits (red) initially assemble into trimers through homotypic association of their carboxy-terminal CLZ domains into a parallel three helix coiled coil. subsequently, a CnGB1 subunit (green), if present, is preferentially incorporated into the remaining slot of the channel tetramer due to a high affinity association with CnGA1 subunits. (b) Deletion of the CLZ domain in CnGA1 allows CnGA1 subunits to assemble with CnGB1 subunits. This produces channels with multiple CnGB1 subunits and a large dominant negative effect of CnGB1.
or the sum of two Langmuir isotherms:
-diltiazem cis where K 1/2 is the apparent affinity and X is the fraction of the high affinity component (K a 1/2 ) relative to the low affinity component (K b 1/2 ).
For FRET experiments, fluorescence images were collected using a X20 objective from the animal pole of oocytes with a confocal microscope (Leica SP1) using laser excitation at 458 nm and 488 nm. Regions of interest were drawn by hand in MetaMorph (Molecular Devices), and background fluorescence was quantified from the blank area inside the cell and subtracted ( Supplementary Fig. S6 ). The pinhole was adjusted so that the brightest cells were within the linear range of the detector with a maximum pinhole setting of 1 Airy unit. Data from cells with very dim fluorescent signals were discarded. Emission spectra were constructed over a range of wavelengths with an emission window of 2 nm. Spectra closely resembled the published spectra 46, 47 , suggesting that the fluorescent properties of these fluorescent protein variants are retained despite their incorporation into fusion proteins. FRET calculations were made using the spectral FRET method, as previously described 7, 9, 23 .
Protein biochemistry. Four different CLZ containing constructs of varying lengths were generated for both CNGA1 and CNGA3 ( Supplementary Fig. 3) . Each of these eight constructs was subcloned into two bacterial expression vectors, pNGFP_BC and pCGFP_BC (kindly provided by Dr. E. Gouaux), creating aminoterminal and carboxy-terminal GFP fusion proteins respectively. Each of these 16 constructs was screened by FSEC as previously described 28 . Briefly, 5 ml bacterial cultures were induced, spun down, and resuspended in 800 µl of 150 mM KCl, 30 mM HEPES, pH 7.5 containing 2.5 µg ml − 1 DNAse and cOmplete protease inhibitor tablets (Roche). The cells were lysed with a probe sonicator; the lysate was cleared by centrifugation (1,700 × g for 30 min); and 100 µl of the supernatant was loaded on a Superdex 200 10/300 GL column (GE Healthcare) mounted on an FPLC system with a fluorescence detector set for detection of GFP fluorescence. Four candidates from the FSEC screen (CNGA1#621-690, CNGA1#621-667, CNGA3#626-694, and CNGA3#626-672) were subjected to further analysis. For large scale protein preparations, the CNGA1 and CNGA3 channel fragments were subcloned into the pETGQ and pETM11 vectors, respectively, creating amino-terminal poly-His fusion proteins. For the CNGA3#626-672 construct, the NcoI site at the amino terminus was deleted using PCR-based methods. Two litre bacterial cultures were grown to mid-log phase and induced with 1 mM IPTG overnight at 18 °C. The cultures were spun down and resuspended in 150 mM KCl, 30 mM HEPES, pH 7.5 containing 1 mM AEBSF, 2.5 µg ml − 1 DNAse and cOmplete protease inhibitor tablets (Roche). Cells were lysed with an EmulsiFlex C-5 homogenizer (Avestin), and the lysate was cleared by centrifugation at 131,000 × g for 45 min at 4 °C. The proteins were then purified on a Ni 2 + affinity resin column (HisTrap HP, GE Healthcare). The octahistidine tag was removed by thrombin (1) (1) (2) (2) or TEV cleavage, and the proteins were further purified on an anion or cation exchange column (HiTrap Q HP or HiTrap SP FF, GE Healthcare) and concentrated.
Two of the samples mentioned above (CNGA1#621-690, CNGA3#626-694) were submitted to the Biophysics Resource of the W.M. Keck Biotechnology Facility at Yale University (New Haven, CT) for LS-SEC analysis. The samples were run on a Superdex 200 10/300 GL column (GE Healthcare) in 150 mM NaCl, 20 µM EDTA, 50 mM Tris-HCL, pH 8.0 (CNGA1#621-690) or 150 mM KCl, 30 mM HEPES, pH 7.5 (CNGA3#626-694). The molecular weight was determined by solving the equation that relates the excess scattered light, measured at several angles, to the concentration of solute and the weight-average molar mass using the ASTRA program 30 .
X-ray crystallography. Crystals were grown by the sitting drop, vapour diffusion method at 20°C. Crystals of CNGA1#621-690 grew using a 1:1 mixture of protein and reservoir solution containing 18.2% (w/v) PEG 3350, 45 mM Zn 2 + acetate, 9 mM CoCl 2 . These conditions produced crystals within 8 days with space group P2 1 2 1 2 1 that diffracted to a resolution of 2.14 Å. Crystals of CNGA3#626-672 grew using a 1:1 mixture of protein and reservoir solution containing 20% (w/v) PEG 3350, 200 mM K + acetate. These conditions produced crystals within 1 day with space group P12 1 1 that diffracted to a resolution of 1.9 Å.
For diffraction data collection, crystals were immersed in liquid nitrogen after cryoprotection in 20% glycerol. Data were collected at 110 °K on beamline 8.2.1 at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley). Integration, scaling and merging of the diffraction data were done with the Mosflm program 52 . The structures were solved by molecular replacement using the programs Phaser 53 and Phenix 54 . For CNGA1, the structure of HIV gp41 N-trimer pocket region 33 (3L36) was used as a search probe. For CNGA3, the structure of SARS virus S2 protein 34 (1ZVB) was used as a search probe. Molecular models were rebuilt using ARP/wARP (ref. 55) (CNGA1) or the Phenix software suite 54 (CNGA3). Structure refinement and validation were performed using the Phenix software suite 54 and the program Coot 56 using Fo -Fc ′omit′ maps. Based on the Ramachandran plot, the percent of amino acids in favoured/allowed/ disallowed conformations were 99.37/0.63/0.0 for CNGA1 and 100.00/0.0/0.0 for CNGA3. The coiled-coil parameters were analysed by the program TWISTER 57 .
Statistical analysis. Data parameters were plotted as mean ± s.e.m. Student's test was used to determine significance at P < 0.05. The 2011 Retrovirology Prize winner Masao Matsuoka: forward looking and antisense Masao Matsuoka wins the 2011 Retrovirology Prize. LTR DNA sequence of the HTLV-1 provirus is preferentially methylated while the 3' LTR of the provirus is not. His work in this area is independent of and contemporaneous with similar observations made in human immunodeficiency virus type 1 (HIV-1) research and has added to our insights on how retroviral latency is achieved and how it might be thwarted by employing targeted therapeutic agents.
Because Matsuoka found that the 3' LTR of the HTLV-1 provirus is surprisingly hypomethylated, this led him to consider whether such a state would favor an antisense transcript originating from this end of the proviral genome. HIV-1 researchers have long ago discounted the importance of a 3' LTR driven antisense transcript. Yet, in the last decade, independent results from Mesnard (France) and Matsuoka (Japan) have strongly established the existence and biological importance of an HTLV-1 antisense transcript called HBZ. The HBZ RNA encoded by the minus strand of the provirus is capable of encoding a bZIP protein, and Matsuoka and his colleagues have shown that this RNA and its protein are expressed in all Adult T-cell leukemias (ATLs). Importantly, Matsuoka is the first to demonstrate that the expression of HBZ promotes the proliferation of T cells, and he has reported that HBZ expressing transgenic mice develop T cell lymphomas.
Professor Matsuoka has been a founding member of Retrovirology's editorial board. To understand better Masao's views, I asked for his answers to several questions.
KTJ: Tell me what you find the most rewarding about being a retrovirologist?
MM: Human retroviruses are big threats to humans. They evolve to replicate elaborately in human cells using their small genomes. Whenever I find clever mechanisms of retroviruses, they never failed to surprise Correspondence: kjeang@niaid.nih.gov Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, the National Institutes of Health, 9000 Rockville Pike, Bethesda 20892, MD, USA me. At the same time, I feel the challenge to eliminate them from humans and to prevent retroviral diseases. As a retrovirologist, I am fascinated to study these small, but immensely important genomes with their ingenious mechanisms.
KTJ: Did you ever have second thoughts about being a scientist? What would you have done if you did not become a scientist?
MM: When I was a boy, I was fascinated with living fish and insects. I was very much interested in living animals. It is a reason that I love fishing. If I did not become a scientist, I would have liked to be a fisherman. However, for Japanese fishermen, it is very competitive to catch good fish for "Sushi". I am skeptical that I can succeed as a fisherman. I guess that being a scientist is better for me.
KTJ: The 21 st century is said by many to be the "Pacific century"how do you see science being different in the 21 st century compared to the 20 th century?
MM: I am confident in the 21 st century that people in the Pacific countries will be influential in science. A striking feature of this region is diversity in many aspects, including race, culture, politics, and so on. I hope that this huge diversity generates new science, and contributes to human society to solve many challenging puzzles like climate changes, and ever changing diseases (diabetes mellitus, cancer, infectious diseases, etc.), and the exhaustion of natural resources. Of course, none of these issues are easy to be solved. I really hope that diverse people in Pacific countries contribute to these tough problems.
KTJ: Young scientists face many obstacles today in developing their careerswhat do you see as some obstacles facing young Japanese scientists and how would you advise them?
MM: In most Japanese universities, associate and assistant professors work under the supervision of full professors. Full professors get grant money from the government or funding agencies, and they usually determine the research theme for young scientists. It is not an actual independent position. I would like to recommend young scientists to be independent as possible as they can or to choose a good boss who permits them to do independent research.
KTJ: Someone told me once that every 10 years he reinvents himself. What do you see yourself doing 10 years from now that might be different from what you are doing today?
MM: I agree that reinventing oneself is a nice and cool idea. However, I think that people (except rare persons) tend to behave as they always are. Before I moved to this Institute, I was a clinician, and took care of many patients in the hospital. I spent small efforts for my research at that time (half clinician, and half scientist). However, now I spend much more time for my research in Institute for Virus Research, Kyoto University. My past background is good experience for me since I can appreciate different points of view for retrovirology. Actually, I think understanding how the virus induces various clinical conditions (inflammation, hypercalcemia, immunodeficiency, etc.) from virological and clinical points of view provide informed and synergistic perspectives. Sometimes, these different views lead to new ideas.
KTJ: You have published 6 papers [4] [5] [6] [7] [8] [9] in Retrovirologytell me what do you think about Open Accessshould this principle be important to Japanese scientists, why or why not?
MM: I strongly agree with Open Access. Access to knowledge should be open to anyone who wants it. Recently, Japanese government and funding agencies do not request open access to scientists. This policy should be changed. Kyoto University has its own repository (Kyoto University Research Information Repository), Kurenai (that means "truly red" in Japanese), to access publications from Kyoto University. The world is sometimes unfair. But, science should be open and fair for anyone who wants to know new findings and knowledge. It should be the concrete basis for science.
KTJ: When the time comes and you retire from doing science, how and for what would you like to be remembered?
MM: When I retire from science, I would like to be remembered as a researcher who connected clinical science and retrovirology. Of course, I also study drug development for HIV-1 and resistance mechanisms. I am confident that there are many other important contributors to this field of HIV.
KTJ: Finally, if you have an opportunity to address a gathering of all the world leaders at the United Nation, in 250 words or lesswhat would you say to them?
MM: Now, the world has become closely connected more than at any other time of human history in many aspects. Economies are tightly linked, and an economical crisis like the Greek crisis in one country quickly affects many other world economies. Climate changes also influence the world. In the latter, a limitless desire of human exploitation of our environment has exacerbated these situations. A new viral infection quickly spreads within weeks like SARS. The HIV pandemic is a big tragedy to human, and avian influenza will be a new threat. To respond to these infections, we need a new platform of collaboration and friendship. Thus, we must appreciate that people and countries are indivisibly united. However, the sad thing is that people of the world are stuck in old thinking. We need to understand that in today's world it is not possible for one country to pursue mainly its own benefit.
The "chyu-you (derived from the famous Chinese book)" means the balanced state or person with harmonized character, which is appreciated by people for a long time in Japan. I hope that people and countries should keep this word in mind, and exercise control over themselve. The host and the pathogen co-evolve to adapt to each other. It is time for humans to harmonize the world and society. Science is an important arena which gives us many opportunities to achieve this. Effect modification of environmental factors on influenza-associated mortality: a time-series study in two Chinese cities BACKGROUND: Environmental factors have been associated with transmission and survival of influenza viruses but no studies have ever explored the role of environmental factors on severity of influenza infection. METHODS: We applied a Poisson regression model to the mortality data of two Chinese metropolitan cities located within the subtropical zone, to calculate the influenza associated excess mortality risks during the periods with different levels of temperature and humidity. RESULTS: The results showed that high absolute humidity (measured by vapor pressure) was significantly (p < 0.05) associated with increased risks of all-cause and cardiorespiratory deaths, but not with increased risks of pneumonia and influenza deaths. The association between absolute humidity and mortality risks was found consistent among the two cities. An increasing pattern of influenza associated mortality risks was also found across the strata of low to high relative humidity, but the results were less consistent for temperature. CONCLUSIONS: These findings highlight the need for people with chronic cardiovascular and respiratory diseases to take extra caution against influenza during hot and humid days in the subtropics and tropics. Influenza used to be considered as a "cold" disease as it usually returns every cold winter in temperate countries. However, recent studies have shown that influenza can be active throughout the year in the warm tropics and subtropics and the disease burden of influenza there can be as heavy as that in temperate climates [1] [2] [3] [4] . It has been proposed that influenza seasonality is driven by complicated interactions between antigenic drifts of virus strains, environmental factors, host susceptibility and behavior changes [5] [6] [7] . Among these potential factors, environmental factors including temperature and relative humidity have been most thoroughly explored by laboratory and observational studies [8, 9] . Recent studies raised a hypothesis that absolute humidity is one of drivers for influenza seasonality in temperate regions [10, 11] , but the mechanism behind various seasonal patterns of influenza outbreaks under different climates remains unclear.
Given the association of environmental factors with both influenza virus activity and mortality, these factors have been adjusted for as confounders in the statistical models for influenza associated mortality burden [2, 12] . However, few of previous laboratory or epidemiological studies tackled the potential modifying role of environmental factors on severity of infection, or mortality risks associated with influenza. Such effect modification is plausible because extreme weather conditions could have a synergistic effect with influenza on mortality burden. Both cold and hot temperatures have been associated with increased mortality risks of respiratory diseases in numerous studies [13] [14] [15] . Our previous study in Hong Kong also found a two-peak seasonal variation in mortality burden of influenza, which is similar to the pattern of influenza seasonality, suggesting that environmental factors might also affect the severity of seasonal influenza infection [16] . In this study we applied a Poisson regression model to the data of two subtropical Chinese cities: Guangzhou and Hong Kong, to examine the possible effect modification of environmental factors on the severity of influenza infection measured by the mortality attributable to influenza. These two cities are geographically close, with Guangzhou located at latitude 23°N and Hong Kong at 21°N ( Figure  1 ). Both cities have a typical subtropical climate, but on average Hong Kong has a higher temperature and humidity. Hong Kong also has a larger population than Guangzhou (6.8 million vs. 3.7 million).We examined three environmental factors which have been documented to regulate virus survival and transmission: temperature, relative humidity and absolute humidity. According to Hong Kong Law all deaths from natural causes are required to be registered, so the mortality data in Hong Kong was fairly complete. The registered death data collected by the Guangzhou Department of Health covered the residents from the eight urban districts, but did not include the immigrant population and residents living in the two suburban districts. Therefore, the population denominator of Guangzhou did not change much during the study period. Given the fact that most immigrant workers are young adults and few died in Guangzhou, we can assume that our mortality data was complete and representative of the general population of Guangzhou. We aggregated the weekly numbers of deaths with underlying cause of cardiorespiratory disease, pneumonia and influenza, and allcause mortality. The corresponding International Classification of Diseases, Tenth Revision (ICD10) codes adopted by Guangzhou and Hong Kong are I00-I99, J00-J99 for cardiorespiratory, J10-J18 for pneumonia and influenza, and A00-R99 for all-cause deaths. We used the accidental deaths (ICD10 codes S00-T989) in Hong Kong as the control disease. Weekly mean temperature and relative humidity were separately derived from the National Meteorological Information Centre of China and Hong Kong Observatory for Guangzhou and Hong Kong respectively. Weekly mean vapor pressure was calculated as a metric for absolute humidity following an equation provided by Basu et al. [18] , vaporpressure = 0.06112*relativehumidity% * 10 7.5*temperature( • C)/(237.7+temperature( • C)) (1) Vapor pressure will be used for absolute humidity hereafter in this paper.
A generalized additive model (GAM) with a log link function and Poisson error was fitted to the weekly numbers of deaths. The Poisson model has been widely applied to the estimation of influenza associated disease burden and recently been validated by an empirical dataset of laboratory confirmed influenza cases [19] . First, long-term trends and seasonal patterns of causespecific mortality counts, as well as environmental factors including temperature and relative humidity, were adjusted for as confounders by building a core model:
Here y t denotes the number of deaths at week t. ns(t), ns(temp t ), and ns(humd t ) denote the natural cubic spline smoothing functions of time, weekly average temperature and relative humidity. The natural spline smoothing function was used to remove the small variation while maintaining the major trend of each variable, i.e. to make them smoother. The aim of smoothing was to increase the efficiency in estimating the model coefficients [20] . Given the high correlation between temperature and vapor pressure, the smoothing function of vapor pressure was not added in order to avoid collinearity between the variables. The adequacy of this core model was evaluated by the absence of any obvious pattern in partial autocorrelation functions of its residuals (Additional file 1: Figure S1 ). Second, the weekly proportions of specimens positive for influenza A or B were then added into the core model as a variable for influenza virus activity to obtain a main effect model.
The main effects of influenza have been presented elsewhere [21] . To explore the effect modification of environmental factors on influenza associated mortality, we added into the core model the product terms of influenza proportion variable and dummy variables for periods of normal and extreme (high or low) weather conditions as interaction terms between virus activity and environmental factors. For example, the interaction model for temperature and influenza mortality was:
where Lowtemp i = 1 for the periods within low temperature ranges and 0 for otherwise, and Midtemp t and Hightemp t are similarly defined as the dummy variables for the middle and high temperature periods. The smoothing function of temperature ns(temp t ) was kept in the model in order to adjust for the association of temperature and mortality. It is reasonable to assume that the cutoff point of extreme weather may differ across cities as people may adapt to prevailing climates. We used the first (25%) and third quartiles (75%) of weekly average temperatures (or humidity) as the cutoff points to define the low, middle and high temperature (or humidity) periods [22] . The presence of effect modification by environmental factors was evaluated using likelihood ratio tests between the interaction and main effect models. To measure the effects of influenza on mortality, we computed the percentage change of mortality counts associated with 1% increases of influenza virus activity for the low, middle and high periods. The formula for the low temperature period is
Here β 1 was obtained from the above interaction model. To test whether our results were robust to various definitions of periods, we chose two sets of extra cutoff points: 20th and 80th, 30th and 70th percentiles of weekly average data in each city. Although we removed the autocorrelation and seasonal trends within mortality data, there are still concerns that such adjustment was inadequate and the remaining uncontrolled seasonal factors may cause interaction terms of environmental factors and virus activity to appear significant in our models. To rule out this possibility, we used accidental deaths that were expected to be unrelated to influenza infection as a control mortality group to show that our findings are unlikely the spurious results of under-adjustment of seasonal confounding factors in modeling. Some previous studies used the anomalies to assess the effects of meteorology factors on influenza [11] .We therefore conducted a sensitivity analysis by defining the strata by anomalies, instead of absolute values of meteorology factors. The anomalies were defined as the deviations of observed metrological data from a seasonal curve with a constant and a sinusoidal pair fitted to these observed data. All the analyses were performed using the mgcv package of R software (version 2.5.1.) [23] .
Hong Kong has a larger population than Guangzhou (6.8 million versus 3.7 million) during our study period. On average, Hong Kong has a slightly higher temperature, relative humidity and vapor pressure, and smaller annual variations than Guangzhou ( Table 1 ). The mean of the weekly proportions of specimens positive for influenza A or B was higher in Hong Kong than in Guangzhou. In 2004 and 2006, there were two peaks of virus activity in Hong Kong (one in February/March and another in June/July), but only one broad peak in Guangzhou ( Figure 2 ). The influenza seasonality was similar between these two cities in 2005.
More deaths with underlying causes of cardiorespiratory, pneumonia and influenza or all-cause were recorded in the low temperature (or low vapor pressure) periods for both Guangzhou and Hong Kong ( Table 2) . For both cities, the mortality counts did not show any obvious difference across levels of relative humidity. High influenza virus activity coincided with high levels of temperature, relative humidity or vapor pressure, with the only exception of Guangzhou which had higher mean proportions when temperature is within the middle range.
Significant interaction between temperature and influenza on the mortality risks was only found in cardiorespiratory mortality in Hong Kong (p < 0.05), and the interaction between relative humidity and influenza was found significant for all-cause mortality in both Guangzhou and Hong Kong, and for cardiorespiratory mortality only in Guangzhou ( Table 3 ). The patterns of influenza impact across the low to high temperature periods were not consistent among the two cities. In Guangzhou, the highest risk of mortality tended to be observed in the middle-temperature period, whereas for Hong Kong the largest changes in risk were found in the periods with high temperature for the three mortality categories ( Table 3 ). An increasing pattern of influenza associated mortality risks could be observed along low-, mid-and high-relative humidity periods in Guangzhou and Hong Kong, but most of estimates for low-and high-periods were not statistically significant ( Table 3) .
The interaction between vapor pressure levels and virus activity were found to be significant (p < 0.05) for allcause and cardiorespiratory mortality, but not for pneumonia and influenza mortality (Table 3) . Consistently higher mortality risks were found at the high levels of vapor pressure, with only exception of P&I mortality in Hong Kong. The influenza effects on mortality of cardiorespiratory, pneumonia and influenza, and all causes were found significant during the middle-and highvapor pressure periods (with the only exception of allcause mortality in Hong Kong), but not significant when vapor pressures remained at the relatively low levels. For the high vapor pressure periods, all-cause excess mortality counts attributable to influenza would increase by 0.35% and 0.26% for per 1% increase of virus activity, and the corresponding increases for cardiorespiratory mortality were 0.54% and 0.49% for Guangzhou and Hong Kong, respectively (Table 3) . We also assessed the effect modification of environmental factors on influenza effects (i.e. interaction of each factor and influenza) for the age group younger than 65 years (< 65) and the elderly aged 65 years or older (≥65). The results of the ≥65 age group were consistent with those for the all-ages group, with an increasing trend over vapor pressure levels observed for all-cause and cardiorespiratory mortality in the two cities (Figure 3 ). For the < 65 age group, this trend could also be observed in most city-specific disease categories, with the only exception of all-cause mortality in Hong Kong. But the interaction terms were statistically significant (p < 0.05) only in the ≥65 group. The modifying effects of temperature and relative humidity on influenza effects were found quite similar between the all-ages and ≥65 age groups, but not between the allages and < 65 age groups (data not shown). The models with indicators for different cutoff points returned similar estimates for temperature, relative humidity and vapor pressure (Additional file 1: Figure S2 ). We did not observe any significant interaction between influenza and environmental factors in terms of their effects on the control mortality category of accidental mortality in Hong Kong. Influenza associated accidental mortality risks were also not statistically significant.
To ensure the standardized comparison between Guangzhou and Hong Kong, we decided to apply the same modeling approach to the data of same study period, because the core model would have been slightly different if we used the longer time series of Hong Kong. To check the robustness of our conclusions, we repeated the above analysis using a longer time series data of Hong Kong during 1998-2006 and the estimates were shown in Additional file 1: Table S1 . The statistical significance of interaction terms was close to those from the study period of 2004-2006. The estimates for the low temperature periods became larger and statistically significant; those for the high relative humidity periods were smaller and comparable to the middle relative humidity; and for the low vapor pressure periods, the estimates were similar but became significant. The increasing trend across the low, middle and high levels of temperature and relative humidity was less evident.
The results of stratification analysis by anomalies are shown in Additional file 1: Table S2 . The estimates were similar to those for the periods defined by absolute values of meteorological factors, in terms of magnitude and changing patterns. But the likelihood ratio tests showed more significant interaction for temperature or vapor pressure, and less for relative humidity.
In this study we quantified influenza associated mortality risks at the various ranges of temperature and humidity, and compared the results between two large subtropical cities. An increasing pattern of influenza-associated mortality risks on all-cause and cardiorespiratory along the low to high periods was found for temperature, relative and absolute humidity in both cities during the period of 2004-2006. The interaction between vapor pressure indicators and virus activity were also consistently significant for relative and absolute humidity. Although lower vapor pressure (or temperature) has been found to facilitate virus transmission and survival in the guinea pig model [10] , our results suggested that higher vapor pressure (or temperature) was associated with a higher mortality risk attributable to influenza. Severity of seasonal influenza epidemics were not only determined by virus transmission efficiency and outdoor weather conditions, but also largely affected by host resistance, indoor living environment and social behavior [5] . The high vapor pressure periods coincided with the summer peaks of influenza in both Guangzhou and Hong Kong, indicating that influenza could pose a higher risk when reaching its peak in seasons with high vapor pressure in subtropical cities. Our results may help to interpret our previous findings that the effects of influenza were significantly higher in the humid and warm spring/summer period than in the dry and cold winter period in Hong Kong [16] . The extreme low vapor pressure was usually recorded during December-January and the highest appeared during June-July, which coincided with the trough and peak periods of excess risks associated with influenza viruses. Effect modification of environmental factors was only detected in all-cause and cardiorespiratory mortality, but not in pneumonia and influenza, suggesting that the synergistic interaction between high humidity (or temperature) and virus activity may mainly lie in their similar regulation pathways in cardiovascular systems. These results are also in agreement with our previous findings that pneumonia and influenza mortality risks attributable to influenza did not exhibit a seasonal variation [16] . Extreme heat has been documented to increase blood viscosity through evaporation of body fluid and trigger intravascular coagulation through damaging endothelial cells [24] . Influenza infection has a similar pro-thrombotic effect by inducing inflammation around blood vessels and rupturing atherosclerotic plaques [25] . As a result, mortality risks would be dramatically raised by the stress of both extreme weather and influenza infections. The results suggested that we need extra precautionary measures to reduce influenza infections in people with cardiovascular diseases, especially under the frequent hot and humid weather conditions experienced in the tropical and subtropical areas. Although the experiments of influenza virus transmission between guinea pig hosts found the higher transmission rates occurred under dry air (vapor pressure below 10hPA) [10] , our results indicated that the mortality risks associated with influenza under the low vapor pressure environment were lower than the rest of study period. The reason could be the short time of exposure to the very low level of vapor pressure. During our study period there were only 2 and 13 weeks with an average vapor pressure below 10hPA in Hong Kong and Guangzhou, respectively. Most weekly average vapor Percentage change (%) of mortality counts for all-cause and cardiorespiratory (CRD) mortality. The risks associated with 1% increase in influenza virus activity during the low-, middle-and high-vapor pressure periods were plotted for the age groups younger than 65 years (< 65) and equal or over 65 years (≥65). The 95% confidence intervals were shown in vertical bars. An asterisk is added above the bars if the interaction between vapor pressure and influenza is shown statistically significant by the likelihood ratio test between main effects and interaction models. pressures during the low vapor pressure period were within the range of 10-20hPA, in which the guinea pig experiments showed dramatically reduced transmission rates [10] . In future, we may examine the seasonal variation in influenza effects in other cities to assess whether such a seasonal variation, if common in subtropical and tropical cities, is consistently determined by environmental factors, or by other factors such as host immunity and virus virulence. The results for the different age groups suggested that the modification effects of environmental factors may mainly lie in the elderly aged over 65 years, as the consistent increasing trend over the low to high vapor pressure periods was only observed in this age group. However, since over 65% of deaths occurred in this age group for both cities (Table 1 ), the small numbers of weekly death counts in the younger age group (< 65 years) may not have had enough power to allow assessment of effect modification based on the data over the 3 years. A future study with a long study period or a large population may help answer whether young people maybe also expose to higher mortality risks during the hot and humid days.
In this study, we used the quartiles of weekly data in each city, to separately define the periods with normal (middle) and extreme (low and high) weather. Given the difference in weather conditions between these cities, we think that it is not appropriate to use the same cutoff points for temperature or humidity to compare their modification effects on influenza associated mortality, as people living in hot subtropical and tropical regions may adapt well to the year-round hot and humid climate and have a higher threshold for adverse effects of weather. For example, although it is widely accepted that the temperature effects on mortality exhibited a U-or V-shape curve in both temperate and tropical/subtropical areas, the turning point of this curve varied across different cities. A study conducted in 11 cities of the US found that the turning point of temperature for its effects on mortality could range from 18.4°C to 32.4°C [26] . To our best knowledge, so far there are no studies that have ever assessed the effect modification of temperature on influenza effects. Therefore, the commonly adopted cutoff points of city-specific quartiles seem appropriate at this stage [21, 27] .
There are several limitations in our study. Firstly, our study is based on 3 years of surveillance data which may not have enough power to allow assessment of exposureresponse curves for the effects of environmental factors. Nevertheless, our findings did suggest an increasing trend of influenza associated mortality risks across the periods of low, middle and high vapor pressure, although such findings may be applicable only to the warm climates. Secondly, we only investigated the effect modification of environmental factors through a simple interaction model, but there were other unadjusted factors, including host susceptibility and virulence of influenza strains. These factors are unlikely to work independently with environmental factors. Other environmental factors such as ultraviolet radiation [28] , rainfall [29] have been proposed to play a role in the regulation of influenza seasonality, although evidence is rather limited compared with the three factors we chose to investigate [30] . Lastly, we did not adjust for the vaccination rate in our model. In 2003, vaccination rate was 191 doses per 1,000 total population in Hong Kong [31] , slightly higher than the rate of 129 doses/1,000 total population in Guangzhou [21] . However, it is not clear when people received vaccination; therefore we were unable to assess the role of vaccination in our study.
This study provides a piece of key evidence to the effect of environmental factors on severity of seasonal influenza under warm climates and helps reveal the mechanism behind global influenza seasonality. It also highlights the need for people with chronic cardiovascular and respiratory conditions to take extra caution against influenza during the hot and humid days in the subtropics.
Additional file 1: Tables S1 and S2; Figures S1 and S2. Influenza A/H1N1 septic shock in a patient with systemic lupus erythematosus. A case report BACKGROUND: Immunocompromised patients, such as systemic lupus erythematosus (SLE) sufferers have an increased risk of mortality, following influenza infection. In the recent pandemic, influenza A H1NI virus caused 18449 deaths, mainly because of adult respiratory distress syndrome or bacterial co-infections. CASE PRESENTATION: In this case report, an SLE patient with viral-induced septic shock, without overt pulmonary involvement, is discussed. The patient was administered oseltamivir and supportive treatment, including wide-spectrum antibiotics, vasopressors and steroids, according to the guidelines proposed for bacterial sepsis and septic shock. She finally survived and experienced a lupus flare soon after intensive care unit (ICU) discharge. CONCLUSIONS: To our knowledge, this is the first case to report severe septic shock from influenza A/H1N1 virus, without overt pulmonary involvement. Infections are among the most important causes of morbidity and mortality in systemic lupus erythematosus (SLE). However, viruses are not considered to cause serious infections in these patients; they, usually, represent reactivation of herpes viruses, such as herpes simplex virus and varicella-zoster virus [1] . Nevertheless, it is reported that immunocompromised patients have an increased risk of mortality, following influenza infection [2] .
In the recent pandemic, influenza A H1N1 virus has been estimated to cause approximately 18.449 deaths in 214 different countries until August 1 st 2010 [3] . Adult respiratory distress syndrome (ARDS), along with bacterial co-infections were the direct causes of death in most cases [4] . However, no cases of viral-induced septic shock without severe pulmonary involvement have been reported. Nevertheless, little is known about this infection in SLE patients [5] . Herein, we report a case of an SLE patient, who developed septic shock due to influenza A H1N1 infection, without acute lung injury.
A 46-year old female was admitted to the hospital because of low-grade fever, sore throat and fatigue for four days; she was on clarithromycin 500 mg twice daily, as she was considered to suffer from upper respiratory tract infection by her general practitioner.
The patient had a history of SLE for 24 years, antiphospholipid syndrome and autoimmune hypothyroidism. SLE was diagnosed in the background of immune thrombocytopenic purpura (ITP), starting at the age of 8. At the age of 12, splenectomy was performed to control refractory thrombocytopenia. Currently, SLE was adequately controlled (Systemic Lupus Erythematosus Disease Activity Index, SLEDAI = 0, anti-dsDNA antibodies negative, C3 and C4 levels normal); medication included methylprednisolone 8 mg/day, azathioprine 50 mg/day, aspirin 50 mg/day and levothyroxine 150 μg/day. The patient had been vaccinated against seasonal influenza and Streptococcus pneumoniae a month before, but not against A/H1N1 virus.
On admission, she was in severe cardiovascular instability with hypotension (BP = 60/40 mmHg), tachycardia (HR = 130/min), tachypnea (RR = 30/min), hypothermia (< 35.5°C), along with oliguria and altered mental status. Oxygenation fraction PaO 2 /FiO 2 was over 350. No obvious site of infection could be identified. The initial chest X-ray was normal, as well as computed tomography of the thorax ( Figure 1 ). Heart ultrasound revealed mild diastolic dysfunction with preserved ejection fraction and no valvular disease. Concerning other organ involvement, there was mild prerenal azotemia (urea 94 mg/dl, creatinine 1.5 mg/dl) and severe liver impairment (alanine aminotransferase 2837 U/L, aspartate aminotransferase 3165 U/L).
The patient was considered to suffer from systemic inflammatory response syndrome (SIRS) and was treated with vigorous fluid resuscitation (crystalloids and colloids), oseltamivir 150 mg/day and moxifloxacin (400 mg/day, after appropriate cultures were obtained). A few hours later, she was intubated and carried to the ICU, because of refractory shock, where vasopressor therapy (noradrenaline up to 2 μg/kg/min) was administered to maintain mean arterial pressure ≥65 mmHg.
Arterial pressure wave form analysis (FloTrac/Vigileo System), continuous ScVO 2 and CVP monitoring were used to estimate patient's hemodynamics. After initial fluid resuscitation, cardiac index, ScVO 2 and CVP measurements were 2.7 L/min/m 2 , 75% and 13 mmHg, respectively (mean values). Blood lactate levels were 4.8 mmol/L. The ventilatory support consisted of controlled MV with tidal volume 7 ml/kg and respiratory rate 15/min. Initial plateau pressure was 16 cmH 2 O with respiratory system compliance 58 ml/cmH 2 O.
The patient responded after 24 hours; noradrenaline was tapered to 0.25 μg/kg/min and hydrocortisone (300 mg/ day) was added as adjunctive therapy. Additional antibiotics included piperacillin/tazobactam (18 g/day) and linezolide (1.2 g/day). Blood, urine and bronco-alveolar lavage (BAL) cultures were negative. Real-time PCR for influenza A/H1N1 virus (BAL specimen) was positive and oseltamivir was administered at 300 mg/day.
In ICU, the clinical course was complicated by bacterial co-infections 10 days after admission; septicaemia due to carbapenem-resistant Klebsiella pneumoniae and ventilator-associated pneumonia due to multi-drug resistant Acinetobacter baumanii. Gentamicin and colistin were administered, according to strain sensitivity, leading to complete resolution of the lesions. She was extubated after 19 days and transferred to the ward.
Concerning H1N1 virus, BAL rt-PCR was persistently positive for 21 days. Oseltamivir was administered in high doses (300 mg/day) for 28 days and was discontinued after negative PCR.
The patient experienced a lupus flare (SLEDAI = 4, C3 = 65 mg/dl, C4 = 7.4 mg/dl), soon after extubation with thrombocytopenia and severe haemolytic anemia. The flare was successfully managed with steroids and intravenous immunoglobulins (IVIGs). Her clinical condition was complicated by severe critical illness polyneuropathy/myopathy (CIP/CIM), managed with long term physiotherapy. In four months, previous therapeutic regimen was re-established and satisfactory performance status was maintained.
Influenza A/H1N1 pandemic represents a global health issue, as it was estimated to be the direct cause of over 600 million respiratory infections and over 50 million hospitalizations, until August 10 th , when WHO announced that H1N1 was in post-pandemic period [6]. Immunosupression is a well-defined risk factor for worse outcome in H1N1 disease. ARDS and secondary bacterial super-infections, leading to septic shock and multiple organ failure are considered to be the primary causes of death [4, 7] . However, primary viral septic shock, especially by influenza, is rarely reported in the literature [8] [9] [10] [11] .
The mortality pattern in SLE is reported to be biphasic; major infections play an important role during the first years of disease, while cardiovascular and other disease complications account for most deaths in long-lasting disease [12] . Viral infections, however, are not reported to affect mortality in SLE [1, 13] .
Severe cardiovascular instability, as indicated by the hemodynamic monitoring of the patient, with refractory shock in H1N1 infection, has not been reported so far in the literature. Acute lung injury and/or ARDS due to H1N1 virus could not be identified, as initial chest Xray and thorax CT were normal and oxygenation was not impaired (PaO 2 /FiO 2 ≥350) throughout the disease course. Bacterial infections, as potential causes of septic shock, were not diagnosed; serial blood, urine and BAL cultures were negative. The therapeutic approach was that of conventional septic shock, according to 2008 guidelines and resulted in patient recovery [14] .
Long lasting viral persistence, despite recommended oseltamivir therapy, probably reflects a poor immune status and defective natural and acquired antibacterial immunity, due to several reasons. Patients with SLE are considered to be immunocompromised either because of the disease itself or due to the immunomodulating agents used for disease management [15] . On the other hand, splenectomy is not considered to represent a major risk factor for viral infections.
Vaccination is suggested to be less effective in SLE, especially in the background of azathioprine treatment, although measurement of antibody titers can only indirectly assess the efficacy of vaccination [2] . Latest studies supported that seroprotection after a single vaccination in SLE patients was significantly reduced compared to healthy controls [16] . The presented patient was vaccinated against seasonal influenza but not against H1N1 virus. Although recent reports suggest that CD8+ T cells are able to cross react with H1N1 virus, they are functionally impaired [17] .
The major complication in this patient, after ICU discharge, was a disease flare (thrombocytopenia and hemolytic anemia) along with CIP/CIM. The latter complicates approximately 30-50% of ICU patients, particularly in cases of multiple organ failure and septic shock [18] . The pathophysiologic process behind CIP/CIM is not fully elucidated; reactive oxygen intermediates, drug toxicity, steroid therapy and poorly controlled hyperglycemia are considered to be critical predisposing factors [18] . Treatment options are not well validated, although intravenous immunoglobulins seem to have a beneficial effect [19] .
IVIGs were administered to this patient for managing severe autoimmune haemolytic anemia and thrombocytopenia; their benefit in recovery from CIP/CIM can not be assessed directly. However, IVIGs allowed quick steroid tapering and, possibly, prevention of further nosocomial infections.
To our knowledge, severe septic shock from influenza A/H1N1 virus, without overt pulmonary involvement, has not been reported in the literature. The authors of this case report of special interest from many points of view, feel it would be a positive step to share their experience with experts in the field. Physicians' awareness and prompt and aggressive supportive treatment are expected to optimize patient outcomes.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent (in Greek) is available for review by the Editor-in-Chief of this Journal. Timeliness of contact tracing among flight passengers for influenza A/H1N1 2009 BACKGROUND: During the initial containment phase of influenza A/H1N1 2009, close contacts of cases were traced to provide antiviral prophylaxis within 48 h after exposure and to alert them on signs of disease for early diagnosis and treatment. Passengers seated on the same row, two rows in front or behind a patient infectious for influenza, during a flight of ≥ 4 h were considered close contacts. This study evaluates the timeliness of flight-contact tracing (CT) as performed following national and international CT requests addressed to the Center of Infectious Disease Control (CIb/RIVM), and implemented by the Municipal Health Services of Schiphol Airport. METHODS: Elapsed days between date of flight arrival and the date passenger lists became available (contact details identified - CI) was used as proxy for timeliness of CT. In a retrospective study, dates of flight arrival, onset of illness, laboratory diagnosis, CT request and identification of contacts details through passenger lists, following CT requests to the RIVM for flights landed at Schiphol Airport were collected and analyzed. RESULTS: 24 requests for CT were identified. Three of these were declined as over 4 days had elapsed since flight arrival. In 17 out of 21 requests, contact details were obtained within 7 days after arrival (81%). The average delay between arrival and CI was 3,9 days (range 2-7), mainly caused by delay in diagnosis of the index patient after arrival (2,6 days). In four flights (19%), contacts were not identified or only after > 7 days. CI involving Dutch airlines was faster than non-Dutch airlines (P < 0,05). Passenger locator cards did not improve timeliness of CI. In only three flights contact details were identified within 2 days after arrival. CONCLUSION: CT for influenza A/H1N1 2009 among flight passengers was not successful for timely provision of prophylaxis. CT had little additional value for alerting passengers for disease symptoms, as this information already was provided during and after the flight. Public health authorities should take into account patient delays in seeking medical advise and laboratory confirmation in relation to maximum time to provide postexposure prophylaxis when deciding to install contact tracing measures. International standardization of CT guidelines is recommended. Aircrafts can function as transport vehicle for patients infected with influenza, leading to introduction of a new virus strain to non-endemic areas [1, 2] . Although the risk is small, passengers might be infected by a contagious patient during the flight [3] [4] [5] [6] , as well as during public transport [7] . Transmission during the flight increases the possibility of further transmission in the area of destination. For these reasons, during the initial phase of the influenza A/H1N1 2009 pandemic, many countries initiated contact tracing among flight passengers of flights where contagious patients with laboratory confirmed influenza A/H1N1 2009 were notified.
A risk assessment guideline for infectious diseases transmitted on aircrafts has been developed by the European Centre for Disease Prevention and Control (ECDC) [8] , which includes influenza. Literature study revealed on-board transmission in flights with a duration of less than 8 h. The majority of infected contacts during these flights were seated on the same row, or one or two rows in front of behind the index [9] [10] [11] [12] . Contacts up to 8 and 10 rows distance from the index were infected in one study [10] . As these contacts also had personal contact with the index during the flight, transmission across a distance of so many rows is not proven. The guideline concludes that it is difficult to design a single contact tracing algorithm for influenza. Due to the short incubation period of influenza, it is almost impossible to provide contacts with postexposure prophylaxis (PEP) within the time that it is most effective, which is 48 h after exposure [13] . Therefore, the main aim of contact tracing might be to interrupt the chain of transmission by alerting contacts for early diagnosis and treatment. Although the World Health Organization (WHO) developed technical advice for case management of influenza A/H1N1 2009 in air transport during the pandemic [14] , no international standardized protocol for contact tracing for this pathogen was available.
In line with the ECDC guideline [8] and the Dutch guideline for 'Incidental introduction of a new influenza strain' [15] , in the Netherlands close contacts of a patient with laboratory confirmed pandemic influenza were identified. In case the index had been contagious during a flight with a duration of ≥ 4 h, passengers and cabin crew were to be informed on signs and symptoms of the disease and to seek medical care in case they would occur. In addition, close contacts, defined as passengers seated on the same row, two rows in front and two rows behind the index case, as well as the cabin crew working in this compartment, were traced by public health authorities to provide a 10 day prophylactic course of oseltamivir as soon as possible (preferably within 48 h after exposure).
Schiphol Airport is the only airport in the Netherlands where trans-Atlantic flights arrive. Its Municipal Health Services (MHS, GGD Kennemerland) and the Center for Infectious disease Control (CIb-RIVM) frequently experienced that, despite all efforts, the time period elapsing from exposure to administration of the first oseltamivir dose exceeded the required 48 h. Acquiring contact details from airlines was time consuming, and contact details on passenger lists were often minimal, so that contacts were difficult to trace. In this study, we assess the time delay in contact tracing of flight passengers for influenza A/H1N1 2009 as performed in the Netherlands during the initial phase of the pandemic. Our data show that despite all efforts the effectiveness of this control measure in daily practice is minimal.
From April 29th until June 22nd 2009, contact tracing among flight passengers in the Netherlands was indicated for laboratory confirmed influenza A/H1N1 2009 cases, who traveled on a flight for 4 h or longer while being contagious, defined as 1 day before, until 7 days after disease onset. These criteria were installed by the CIb, which also functions as National focal point (NFP).
The procedure for contact tracing is complex, see Figure 1 . Requests for contact tracing to the CIb for Dutch index patients originate from any Dutch MHS which identifies a patient who traveled by plane while being contagious for an infectious disease which requires contact tracing. Other nation's health authorities will make a request to the CIb in case they diagnosed a patient which arrived at Schiphol airport for transit while being infectious. Requests for CT in the last group are submitted to the National Focal Point (NFP) or through the Early Warning and Response system of the EU (EWRS). The CIb verifies laboratory confirmation, and the indication for contact tracing regarding flight duration. The MHS of the airport where the specific flight landed coordinates contact tracing for flight passengers. In case of Schiphol, MHS Kennemerland approaches the involved airline company requesting the passenger list. The airline provides passenger lists with at least passenger names, seat numbers and booking or contact details. MHS Kennemerland then completes contact details through booking offices or using other search methods. Close contacts living in the Netherlands are traced by the respective Dutch MHS's. For tracing foreign contacts, the CIb sends a notification with contact details to the NFP of the country of final destination, or through the EWRS system for EU countries.
During the pandemic, CT requests were turned down if more than 4 days had elapsed after flight arrival, as contact tracing was not considered to have additional value. During the study period, passenger locator cards (PLC) only were used on direct flights from Mexico during the initial phase of the pandemic. These flights were all run by Dutch airlines.
For each contact investigation performed in the period April 29th until June 22nd 2009, the following data were collected: flight arrival date, first day of illness of index patient, date of laboratory diagnosis, date of contact tracing request and the date passenger lists were obtained and contact details were completed ('contacts details identified'). From these data, time intervals (in days) between flight arrival and date of diagnosis (interval I), between diagnosis and request dates (interval II) and between request and contact details identified dates (interval III) were calculated, see Figure 2 . Date of actual contact tracing and oseltamivir administration was not available in this study, but is inherently always hours if not days later. As the airline company traces contacts amongst crewmembers, these are not included in this study.
Data were analyzed using SPSS software (version 18, USA). The influence of availability of PLC's on timeliness and the origin of the airline company (Dutch or non-Dutch) were statistically analyzed.
In the period April 29th until June 22nd 2009, 24 indications for CT were identified. Three international requests concerning CT for influenza patients diagnosed outside the Netherlands were declined as already more than 4 days had elapsed since flight arrival. In 17 out of the 21 remaining contact investigations, passenger lists with contact details were obtained within 7 days after arrival (81%), see Table 1 . In total contact details of 451 close contacts were identified, of which 199 contacts lived in the Netherlands, and 252 contacts abroad. The average number of close contacts per flight was 27 (range: 8-44). In four contact investigations (19%), contact details were not obtained, or provided later than 7 days after flight arrival and CT was stopped. These CT were all related to non-Dutch airlines, and total delay *:. In the beginning of the pandemic one request for contact tracing was accepted after 7 days **: these late CT requests were accepted as the passenger lists of the concerned flights already were available from earlier contact investigations ***: date of diagnosis not known ª: Passenger Locator Card was stated 8 days for further data processing. Of the 21 requests, the total delay between request and contact detail identification was longer for non-Dutch airlines (mean 6,3 SD 2,7) compared with Dutch airlines (mean 4.1 days, SD 1,5)(1-sided Mann-Whitney test, p = 0,033).
For the 17 completed contact investigations, interval I was the largest interval in the contact tracing procedure (mean 2,6 days, range 1-6, 95% CI 1,6-3,6, n = 13). The other intervals II and III were shorter, with a mean of 0,8 days and 0,6 days respectively, see Table 1 . Figure 3 shows the medians of the described intervals. Since 15/ 17 index cases were already ill before, or during the day of arrival of the flight, the delay in interval I is mainly caused by delay in seeking medical advice and diagnostic procedure itself. After acceptance of the request for CT by the CIb, GGD Kennemerland needed on average 0,6 days (range 0-2, 95% CI 0,3-0,9 days) to collect the passenger list from the airlines and complete contact details (interval III). The total delay between flight arrival and identification of contact details was on average 3,9 days (range 2-7 days, 95% confidence interval 3,2-4,7 days), see Table 1 . In only 3 out of 17 contact investigations (18%), contacts were identified within 2 days after arrival. In 2 out of these 3 contact investigations, PLC's were available. Interval III of the 5 CT with PLC's available was shorter (0,4 days, SD 0,5) than for 12 CT's without PLC (0,7 days, SD 0,7), this was not significant however (p: 0,25). Overall delay in CT with PLC's also was shorter (mean 3,6, SD 1,8), but not significant, when compared to CT without PLC's (mean 4,1, SD 1,4) (p:0,25).
In this study we evaluated the timeliness of contact tracing (CT) of flight contacts in daily practice. We conclude that the prevailing policy to provide close contacts antiviral PEP during the early phase of the influenza pandemic is very difficult to implement effectively and therefore has little effect to control disease spread. Active case finding through contact tracing of exposed persons is an important procedure during the containment phase of an emerging communicable disease. However, our data show that, even in a small-industrialized country with modern communication tools, tracing of flight contacts exceeds the required maximum of 48 h after exposure.
For influenza, close contacts of contagious index cases are entitled to receive antiviral PEP within 48 h after exposure to prevent them from becoming ill and further spreading of the disease. Starting oseltamivir within 48 h does not prevent disease but shortens the disease period, mitigates symptoms and might decrease further transmission. Awareness among contacts to seek medical evaluation when influenza-like (ILI) symptoms occur, for both proper antiviral treatment and (home-) isolation advice, reduces further spreading. As influenza has a relative short latent period, for influenza A(H1N1)/2009 varying between 0,7-3,1 days [16, 17] , contacts ideally should be informed within 1 day. Oseltamivir postexposure prophylaxis for this pandemic strain is reported to be effective even when administrated more than 48 h after exposure in household settings [18] , however, delays in administration are not specified. We cannot exclude the possibility that in our study, even delayed administration of oseltamivir prophylaxis may have prevented some people from becoming ill, although we anticipate the effectiveness of the intervention overall to be less in this setting than in households.
Our study among 17 contact investigations showed an average total delay of 3,9 days between flight arrival and identification of contacts by passenger list, which is too late for effective PEP, and late for alerting on first symptoms of disease. Only in three contact investigations (18%), contact details were obtained within 48 h. However, after identification of passenger details, health authorities need time to actually trace the contact and administer PEP. It is highly unlikely that this was achieved within the same 48 h. We therefore conclude that contact investigation for provision of PEP as conducted here was ineffective.
Regarding the awareness of ILI symptoms, Schiphol Airport handed all passengers on flights arriving from Mexico information leaflets on influenza A/H1N1 2009 with information on early symptoms and requesting them to seek medical advice in case of fever and respiratory symptoms such as coughing. Posters with this information were placed in passenger halls, to inform passengers arriving indirectly from Mexico via transit through other airports, or arriving from non-endemic areas with higher transmission (e.g. USA). As contact details were identified on average 3.9 days after exposure, however not contacted yet, we conclude that CT did not have additional value for timely achievement of increased awareness.
It is not a new finding that contact tracing of flight passengers is a time-consuming procedure [8] . In one study among flight passengers during the pandemic in 2009, 52% (53/95) of the contacts were reached within 72 h [5] . In a measles contact investigation, 75% (202/ 275) of responding passengers were contacted within 72 h. In this study however, the diagnosis of measles was already suspected during the flight, and laboratory confirmation was initiated immediately after landing [19] . It also helped that many contacts were tourists staying at the same hotels, which facilitated tracing them.
Our study shows that the longest delay before identification of contact details for an influenza index case is caused by the time between arrival and laboratory diagnosis (interval I, 2,6 days). This delay is a result of patients delay in seeking medical care, and doctor's delay, including laboratory confirmation. For influenza, the indicated laboratory test was Polymerase Chain Reaction, which takes several hours to obtain the result and in the beginning of the pandemic, the PCR test was not yet available in many laboratories.
Patients delay was considerable however. It even took the seven passengers with date of onset before the flight, and therefore symptomatic during the flight, 1 to 2 days after arrival before laboratory confirmation was made. Also, none of the airline reported that these patients already were identified during the flight, nor that infection control measures were taken. For the indexes that became ill on the day of arrival, delay until laboratory confirmation still lasted 3 days (range 1-6 days). A prepandemic study by Sharangpani et al. among flight passengers showed that they are more willing to seek physicians care in case they developed flu-like symptoms when the perceived the pandemic as serious [20] . Leggat et al. demonstrated during the pandemic that only a minority (35,5%) of Australian citizens would cancel their air travel in case of cough and fever lasting more than 1 day. This was higher among persons who were more concerned about the pandemic [21] . In the Netherlands, the perceived severity of the disease decreased significant during this study period [22] . We expect that the delay until laboratory diagnoses in this study considerably is affected by patients delay seeking medical care, which might be better in diseases experienced as more threatening.
Collecting passenger details from foreign airlines also caused considerable delay because of differences in time zones and the need to convince the concerned airline companies about the urgency to collect and hand-over passenger lists with contact details. Sometimes official request letters were necessary for legal reasons to release personal contact details. Dutch companies were easier to convince by Dutch health authorities to hand over passenger details. Our data show that contact details that were identified too late or not at all, indeed more often originated from non-Dutch than from Dutch airline companies. An internationally standardized contact tracing protocol, communicated with the International Civil Aviation Organization (ICAO) and International Air Transport Association (IATA), would facilitate the timeliness, and therefore effectiveness of contact tracing.
Although one might expect differently, timeliness of CT for flights where PLC's were available, was not better than CT for flights without PLC. However, PLC's reduces the effort, in terms of staff support for airline companies and the municipal health service to collect useful passenger information considerably. PLC's were only used by Dutch airlines, who already were able to provide passenger lists relatively quickly. This also explains the limited attributed shortening in timeliness. Contact details on PLC's might be more accurate to trace the passenger than details provided by the passenger list or booking station. This is further investigated.
This study has several limitations. As available data were recorded in days, and not in hours, it was not possible to determine the time intervals more precisely. As this was both with first and last date of the intervals, we expect no negative or positive bias. Secondly, the arrival date was used for date of exposure, while the actual exposure might have already taken place the day before at departure of the flight. This would imply an increase in delay and decrease the effectiveness of contact tracing. Also, we have no data if, and when contacts were actually reached and oseltamivir was administered. Since several steps were still required to reach the contacts after they were identified through passenger lists, this only would have lead to further delay in administrating prophylaxis. Further investigation into the timeliness of administration of prophylaxis among these contacts is initiated, to have insight in the delay of this last interval to facilitate future decisions on the effectiveness and necessity of contact tracing among flight passengers. Lastly, this study includes CT initiated at only one airport. CT procedures might be different at airports in other countries, which influences interval III. As this is not causing the main delay, we do not expect that in other countries CT would be much faster.
We conclude that tracing close contacts among flight passengers during the initial phase of pandemic A/ H1N1 2009 was not effective, as timely provision of PEP could not be achieved in most cases. Most contacts came from an endemic area (Mexico) or areas with well known increased transmission during the first 2 months of the pandemic. The additional risk for those travelers of being a close contact during a long haul flight is small (3,5%) [5] . Furthermore, airline companies and/or Schiphol airport already provided contacts with information on the disease and its symptoms by. The benefit to inform them of the fact that they were contacts of a laboratory confirmed case did not justify the extra effort health authorities invested in contact tracing, especially during a period where public health officials, airports and airline companies were absorbed by efforts of other pandemic related control measures.
In hindsight, the limited burden of disease of influenza A/H1N1 2009 did not justify contact tracing efforts. The main reason for flight contact tracing is raising alertness for possible exposure to uncommon infectious diseases, enabling early recognition and treatment of the disease and timely installation of control measures (e.g. SARS and viral hemorrhagic fevers). For some diseases, PEP is indicated as well. The risk assessment upon which the decision to install contact tracing is based should incorporate -apart from an evaluation of the severity and rarity of disease -an assessment of the required timeliness of effective control measures [23] . The expected time for laboratory confirmation of index cases and identification and tracing of contacts should be related to the maximum period during which quarantine, PEP or other control measures are effective in order to decide on the benefit of this time consuming procedure. Lastly, also cabin crew should be aware of their role of signaling infectious patients. In consultation with medical professionals, direct control measures can be installed, as well as medical evaluation after landing. Validation of Self-swab for Virologic Confirmation of Influenza Virus Infections in a Community Setting Few studies have investigated the validity of self-collected nose and throat swabs for influenza confirmation in community settings. We followed outpatients with confirmed influenza with sequential measurement of viral loads and applied log-linear regression models to the viral shedding patterns. Among 176 outpatients with confirmed influenza, the detection of virus and quantitative viral loads obtained from self-swabs was consistent with statistical predictions based on earlier and later measurements, suggesting that self-collected nose and throat swabs can be a valid alternative for virologic confirmation of influenza A or B infection in a community setting. Few studies have investigated the validity of self-collected nose and throat swabs for influenza confirmation in community settings. We followed outpatients with confirmed influenza with sequential measurement of viral loads and applied loglinear regression models to the viral shedding patterns. Among 176 outpatients with confirmed influenza, the detection of virus and quantitative viral loads obtained from self-swabs was consistent with statistical predictions based on earlier and later measurements, suggesting that selfcollected nose and throat swabs can be a valid alternative for virologic confirmation of influenza A or B infection in a community setting.
In community-based studies of acute respiratory illnesses, clinical specimens from the upper respiratory tract may be collected from patients at different stages of disease for virological testing. Although those clinical specimens are typically collected by trained healthcare professionals (HCPs) in a clinic setting, selfcollection by the patient at home may be a more acceptable, economical, and logistically feasible alternative. We investigated whether self-collected nose and throat swabs (NTSs) from patients in a community setting could provide a valid alternative for virologic confirmation of influenza A or B virus infection. Based on 2 similarly designed community-based studies, we modeled the viral shedding patterns from illness onset, adjusting for delays between clinical symptom onset and specimen collection, and compared the quantitative viral load measurements in self-swabs with model-based predictions.
We conducted 2 separate community-based studies of influenza virus infection in Hong Kong with broadly similar protocols for recruitment and follow-up. In both studies, outpatients with recent-onset acute respiratory illness who presented within 48 hours of symptom onset were recruited; of individuals who provided informed consent, those with a positive result on a QuickVue Influenza A1B rapid diagnostic test (Quidel Corp) were invited to continue with follow-up. In one prospective, multicenter study (Influenza Resistance Information Study [IRIS] ), patients recruited between 20 January 2010 and 24 November 2010 were followed up to examine natural prevalence and/or emergence of resistance to antivirals among circulating influenza virus strains. In a separate household transmission study (HTS), eligible patients were recruited between 9 January 2008 and 29 September 2008 and followed up as part of a study investigating the effectiveness of nonpharmacological interventions [1] .
In the IRIS, NTSs were collected by a trained HCP on days 1 and 6 after recruitment in the outpatient clinic and self-collected by subjects at home on day 3 after receiving detailed instruction on swab technique from the HCP at baseline. During the clinic visits, the nasal swab was collected by inserting and rotating a separate flocked sterile swab (Copan) through each nostril into the posterior nares, and throat swabs were collected by swabbing a sterile flocked swab on both the tonsillar fossae and posterior pharynx. The flocked end of the 2 nasal swabs and the throat swab were then transferred to a vial containing Copan Universal Transport Medium by breaking the prescored breaking point of the plastic swab shaft. Specimens collected in the clinic on day 1 and day 6 were stored directly in a clinic refrigerator at 4°C-8°C after collection.
Face-to-face instruction on how to perform a nasal and throat swab on oneself or one's child was given to the patient or to the parents of children aged ,8 years by the HCP during the baseline visit on day 1. Patients also received a patient instruction leaflet and a kit containing the 3 swabs, an individually wrapped tongue depressor, a transport medium vial, and a sealable plastic bag. Patients kept the day 3 swabs in a refrigerator at home after collection and returned them to the clinic on the day 6 visit. All specimens were sent by courier in insulated transport container to the central laboratory at Erasmus Medical Centre within 7 days of collection.
In the HTS, all NTSs were collected by a trained HCP at home visits on days 1, 4, and 7 after recruitment. Nasal swabs were collected by inserting and rotating a sterile plain swab (viscosetipped collection swab with a snappable plastic stick; EURO-TUBO) into the anterior nares, and throat swabs were collected by rubbing a second sterile swab against the tonsillar fossa. Both swabs were then snapped off into a tube containing viral transport medium (0.5% bovine serum albumin in Earle's balanced salt solution with antibiotic). Specimens were stored in an insulated transport container with at least 2 ice packs immediately after collection. Specimens were then either delivered directly or first stored overnight in a study outpatient clinic in a 2°C-8°C refrigerator and then delivered the next day to the central testing laboratory at Queen Mary Hospital by courier in ice boxes.
Slightly different laboratory procedures were used in the 2 studies. For the IRIS, influenza A and influenza B matrix gene-specific reverse-transcription polymerase chain reactions (RT-PCRs) were performed as described elsewhere [2] . Dilutions of an electron microscopic-counted influenza virus A/PR/8/34 stock (Advanced Biosciences) and B/Lee/40 (Advanced Biotechnologies) were run in parallel for conversion of RT-PCR threshold cycle (Ct) values into a quantitative measurement of viral particles per milliliter (vp/mL) [2] .
For the HTS, samples were eluted and cryopreserved at 270°C immediately after receipt in the laboratory. Specimens were then tested by a quantitative RT-PCR assay to detect the presence of influenza A or B virus and determine molecular viral loads in RNA copies per milliliter (copies/mL) using standard methods as described elsewhere [1, [3] [4] [5] .
Previous studies have suggested that following influenza virus infection, viral load rises to a peak around the time of illness onset and then, for influenza A, declines approximately log-linearly over the subsequent 5210 days to undetectable levels and, for influenza B, plateaus with a more gradual decline [6, 7] . We specified multivariable linear regression models for the log viral load on the first and third measurement (typically 0 and 7 days, respectively, after recruitment), with the same slope but separate intercepts for each individual to allow for between-person variability in peak viral loads. We fitted separate models for each study and for influenza A and B and adjusted for age and oseltamivir treatment. Interaction terms with time were included to allow the slope of the regression line to vary by age and oseltamivir treatment. Viral loads for specimens with measured load below the lower limit of quantification (LLOQ) were imputed as half the LLOQ. This random-effects regression model constructed using the first and third measurement was used to predict viral loads expected on the second measurement (typically 3 days after recruitment), which were then compared with the observed viral loads on the second swabs, which were collected by the patients in IRIS and by an HCP in the HTS. We calculated mean differences with 95% confidence intervals (CIs) based on the t distribution.
One hundred thirty-eight subjects with confirmed influenza A and 58 with confirmed influenza B were recruited in the IRIS in 2010, including 43% aged ,15 years (range, 2-85 years); 53% were female, and 53% were prescribed oseltamivir treatment. One hundred eighty-eight subjects with confirmed influenza A and 118 with confirmed influenza B were recruited into the HTS in 2008, of whom 73% were aged ,15 years (range, 0-79 years); 54% were female, and 25% were prescribed oseltamivir treatment. The demographic characteristics of subjects with influenza A versus B were similar. Oseltamivir treatment was more common during the period of peak pandemic A (H1N1) activity (data not shown).
Among subjects with a positive RT-PCR result for influenza A at the first measurement and a self-swab available, 109 of 121 (90%) subjects in the IRIS had detectable virus in the selfcollected swab 2-5 days after illness onset. In the HTS with an HCP-collected swab, 132 of 183 (72%) subjects had detectable virus in the swab 2-7 days after illness onset. For influenza B, the corresponding statistics were 49 of 55 (89%) for the IRIS (2-5 days after onset) and 74 of 117 (63%) for the HTS (2-7 days after onset).
Trends in influenza A viral load are shown in Figure 1A for 138 subjects from the IRIS and in Figure 1B for 188 subjects from the HTS. In the IRIS, the influenza A viral loads determined from swabs taken at the second measurement were slightly lower on average than the expected values based on the random-effects regression model ( Figure 1E ). The mean difference between observed and predicted viral load on the second measurement was 20.50 (95% CI, 2.69 to 2.31) log 10 vp/mL. In the HTS, viral loads determined from swabs taken at the second measurement were slightly higher on average than the expected values based on the random-effects regression model ( Figure 1F) , with a mean difference of 0.31 (95% CI, .08-.54) log 10 copies/mL. Trends in influenza B viral loads are shown in Figure 1C for 58 subjects from the IRIS and in Figure 1D for 118 subjects from the HTS. In both studies, the differences between observed and predicted viral loads on the second measurement were small and statistically insignificant ( Figure 1G and 1H) . In the IRIS, the mean difference was 0.16 (95% CI, 2.18-.51) log 10 vp/mL, and in the HTS, it was 0.14 (95% CI, 2.16-.43) log 10 copies/mL.
Results from the HTS, in which all 3 swabs were collected by trained HCPs, showed that the viral load on the second measurement could accurately and reliably be predicted from a loglinear model based on the first and third measurements. Applying the same approach to the IRIS data, we found that viral loads from self-swabs on the second measurement were very similar to the viral loads that we would have expected if the second swab had been collected by a trained HCP. Our results therefore support the feasibility and validity of using self-swabs as an alternative approach to permit laboratory confirmation of influenza-associated illnesses in a community setting.
Previous studies have demonstrated the feasibility of using parent-collected NTSs from children, in either a hospital setting or community setting, for laboratory confirmation of influenza and other respiratory virus infections without any significant loss in sensitivity [8] [9] [10] . Our results further extend this to selfcollected swabs by patients in the community setting, both for qualitative disease confirmation and quantitative viral load estimation. In the IRIS, 90% of self-collected swabs contained detectable influenza virus approximately 4-6 days after illness onset, indicating no substantial loss in sensitivity for qualitative virus detection through this approach. In the HTS, the lower proportion of specimens with detectable virus in the second swab can be attributed to the slightly longer average delay from illness onset.
Our results also suggest that self-swabs work generally well for quantitative measurement of viral loads. For influenza B, there was no significant difference between those obtained from selfswabs and the values predicted from the other 2 swabs by HCPs ( Figure 1G and 1H) . For influenza A, overall trends in viral loads between the 2 studies also appear similar ( Figure 1A and 1B) . Although self-swabs from the IRIS were associated with a lower viral load than the predicted value ( Figure 1E and 1F), these results should be interpreted with some caution as differences in the type of swabs and transport media used, collection site and technique, delays between collection and transport to the laboratory, and laboratory procedures between studies may have led to artifactual differences. We therefore only compared the predicted and observed viral loads within each study because results from the 2 studies were not directly comparable. Although the log-linear model fit the data well and provided reasonable predictions of viral loads in the HTS (Figure 1 ), more complex models might better represent the decline in viral loads over time. Although we did not explicitly model the shedding patterns of different influenza A subtypes, we have not previously identified substantial differences [2, 5] .
No reports in the literature exist on the validity of self-swab for longitudinal studies of influenza virus infection and illness in Figure 1 . A-D, Molecular viral loads on first and third measurements (circles ) and second measurement (crosses ) for influenza A from the Influenza Resistance Information Study (IRIS) (A ) and the household transmission study (HTS) (B ) and for influenza B from the IRIS (C ) and the HTS (D ). E-H, Difference between observed and expected molecular viral load (VL) at the second measurement, with a histogram summarizing the differences, for influenza A from the IRIS (E ) and the HTS (F ) and for influenza B from the IRIS (G ) and the HTS (H ). The second measurement (crosses ) was collected by self-swab in the IRIS (A, C, E, G ) and by a healthcare professional in the HTS (B, D, F, H ). For the IRIS, the lower limit of detection (LLOD) of the influenza A assay was 54 viral particles per milliliter (vp/mL), and the lower limit of quantification (LLOQ) was 131 vp/mL; the LLOD of the influenza B assay was 168 vp/mL, and the LLOQ was 194 vp/mL. For the HTS, the LLOD of the influenza A and B assays was 550 copies/mL, and the LLOQ was 900 copies/mL. a community setting. Because of the need for multiple sequential respiratory specimens over the course of illness, such studies typically require multiple clinic visits by the patients or multiple home visits by the HCPs and are thus costly and complicated, which may also affect study compliance. Self-swab would thus be an attractive alternative, and further validation of this approach would benefit the design of future community-based studies. Further studies employing collection of NTSs by both the patients and HCPs in a parallel or randomized fashion could help to enable finer calibration of the measurements obtained by self-swabs. Molecular Mimicry as a Mechanism of Autoimmune Disease A variety of mechanisms have been suggested as the means by which infections can initiate and/or exacerbate autoimmune diseases. One mechanism is molecular mimicry, where a foreign antigen shares sequence or structural similarities with self-antigens. Molecular mimicry has typically been characterized on an antibody or T cell level. However, structural relatedness between pathogen and self does not account for T cell activation in a number of autoimmune diseases. A proposed mechanism that could have been misinterpreted for molecular mimicry is the expression of dual T cell receptors (TCR) on a single T cell. These T cells have dual reactivity to both foreign and self-antigens leaving the host vulnerable to foreign insults capable of triggering an autoimmune response. In this review, we briefly discuss what is known about molecular mimicry followed by a discussion of the current understanding of dual TCRs. Finally, we discuss three mechanisms, including molecular mimicry, dual TCRs, and chimeric TCRs, by which dual reactivity of the T cell may play a role in autoimmune diseases. Chronic autoimmune diseases are the by-product of the immune system recognizing self-antigens as foreign, which can lead to inflammation and destruction of specific tissues and organs (immunopathology) [1] . The impact of these diseases is global and heterogeneous with over 100 million people afflicted with more than 80 different autoimmune diseases [2] . While the etiology of autoimmune diseases is not fully elucidated, the causes are likely based on a combination of hereditary and environmental factors [3] . Although host genetic background contributes to the induction of an immune response to self, epidemiological and molecular evidence implicates infectious agents (viral and bacterial) as the principal environmental insults responsible for the induction of autoimmune diseases (reviewed in [4] [5] [6] ). Prolonged proinflammatory responses to infections have been associated with the initiation and exacerbation of autoimmune diseases (reviewed in [4, 7, 8] ). Inflammation is facilitated by proinflammatory cytokines such as type I interferon (IFN), interleukin (IL)-1β, IL-12, IFN-γ, IL-17, and tumor necrosis factor (TNF)-α (reviewed in [7, 9, 10] ). However, these proinflammatory cytokines are critical for clearance of pathogens, suggesting that environmental factors are able to divert the immune response towards immunopathogenesis. Although a number of immune cells are responsible for secreting proinflammatory cytokines, the primary cell types implicated in a vast majority of autoimmune disorders are autoreactive B and T cells, or antibody recognition of self [11] . Although a number of viruses and bacteria have been linked to the initiation of certain autoimmune diseases, identifying a particular virus or bacteria that is solely responsible for the induction of an autoimmune response is rare. This occurrence is due to the potential for multiple infections being involved in priming the immune system and other infections triggering disease, which could explain why no one viral infection has been conclusively linked to the development of immune-mediated autoimmune diseases [7] . However, there are a variety of examples of bacterial infections initiating and exacerbating autoimmune diseases. Streptococcus pyogenes is a gram-positive bacterium which causes group A streptococcal infection that is responsible for a number of diseases. The complications associated with S. pyogenes are rheumatic fever and glomerulonephritis. The infection causes the production of cross-reactive antibodies in response to the bacteria. Antibodies recognize the M protein (virulence factor) and the N-acetyl-β-Dglucosamine (GLcNAc) of S. pyogenes and cross-react with myosin leading to heart damage (reviewed in [8, 12, 13] ). Further evidence of molecular mimicry due to the production of cross-reactive antibody includes infection with gram-negative bacteria, such as Klebsiella pneumoniae and Campylobacter jejuni. Infection with K. pneumonia or C. jejuni leads to the production of cross-reactive antibodies able to recognize the self-antigens histocompatibility leukocyte antigen (HLA)-B27 and gangliosides, which induce ankylosing spondylitis and Guillain-Barré syndrome, respectively (reviewed in [8, 14] ). Examples of human autoimmune diseases with possible links with molecular mimicry are presented in Table 1 .
The immune system has a number of mechanisms that are able to detect foreign pathogens by utilizing the major histocompatibility complex (MHC). This locus encodes the HLA genes and a variety of immune response (Ir) genes, thereby shaping the immune system that protects against pathogens. There are two main types of HLA antigens, HLA class I and class II. The function of HLA class I molecules is to present viral peptides at the surface of an infected cell to a T cell receptor (TCR) on a CD8 + T cell. The activation of these CD8 + T cells leads to the killing of the virally infected cell. This role of HLA class I, the identification of cells that are infected, explains why all nucleated cells have the capacity to express these MHC molecules. HLA class II molecules, in comparison, are expressed almost exclusively on the surface of dendritic cells, B lymphocytes, macrophages, endothelial cells, and activated T cells. Functionally, the HLA class II molecules present peptides to the TCR on CD4 + helper T cells. The engagement of the TCR by the peptide-MHC complex is necessary for the activation of CD4 + and CD8 + T cells, thereby leading to an effective adaptive immune response against an invading pathogen [15]. CD4 + T cells are central mediators of the adaptive immune response including cytokine secretion and cellular and humoral defenses against a pathogen. The HLA locus is extremely polymorphic leading to a heterogeneous population ensuring propagation of a species against novel pathogens. Unfortunately, this genetic heterogeneity adds to the complexity of identifying HLA genes implicated in autoimmune diseases.
In addition to its role in protection against pathogens, a second critical role of the MHC and Ir genes is to safeguard against self-reactivity by restriction of the immune response to self. In this regard, the immune system has developmental checkpoints for the maturation of a T cell. As a naïve T cell expressing a pre-TCR migrates from the bone marrow to the thymus, rearrangement of α and β TCR genes occurs and T cells that have either too high avidity or lack of recognition of self-antigens are selected against and subsequently programmed for cell death. This selection mechanism for generating mature αβ TCRs is named central tolerance. Further, peripheral mechanisms of tolerance are able to suppress autoreactive T cells through certain subsets of cells including regulatory T cells (Tregs) that are able to inhibit self-reactive immune cells in the periphery.
Unfortunately, there are a variety of mechanisms including molecular mimicry, bystander activation, exposure of cryptic antigens, and superantigens by which pathogens can aid in the expression of an autoimmune disease [16] [17] [18] [19] [20] [21] . Inflammation induced by exposure to a foreign antigen can lead to autoimmune diseases from cross-reactive epitopes (molecular mimicry). These epitopes are segments of foreign antigens which, when presented to either T or B cells in the context of the MHC, can activate CD4 + or CD8 + T cells. The induction of the immune response and subsequent proinflammatory cytokine release is critical for clearance of a virus or bacteria. However, a sustained proinflammatory response against specific host tissues can occur when there is sequence or structural homology between foreign antigens and selfantigens, termed molecular mimicry [18] . Although this concept has been associated with autoimmunity, there are instances where mimicry (cross-reactivity) provides protection for the host, termed heterologous immunity [22] . Cross-reactivity or mimicry between various strains of viruses or bacteria could help explain how protective immunity arises in certain individuals even in the absence of prior exposure to an emerging pathogen. This example of sequence homology in which molecular mimicry between viruses leads to protective immunity is in contrast to a pathogen mimicking host epitopes (reviewed in [11] ).
Over 30 years ago, molecular mimicry by either a virus [18] or bacteria [23] was hypothesized to initiate and exacerbate an autoimmune response through sequence or structural similarities with self-antigens. Currently, molecular mimicry is the prevailing hypothesis as to how viral antigens initiate and maintain autoimmune responses which lead to specific tissue damage [18] . Initial work by Fujinami, Oldstone, and colleagues identified mouse antibodies to measles virus and herpes simplex virus (HSV-1) obtained from antibody-secreting B cell clones [18] . These antibodies were reactive to both intermediate filaments of normal cells and the proteins of measles virus and HSV-1, [114, 115] , reviewed in [116] thereby demonstrating a relatedness between host and viral antigens [18] . Further work by Fujinami and Oldstone used myelin basic protein (MBP), a nerve sheath protein containing an encephalitogenic T cell epitope in rabbits. The hepatitis B virus polymerase (HBVP) protein was found through computer analysis to share six consecutive amino acids with the encephalitogenic MBP epitope [16] , and when rabbits were sensitized with either MBP or HBV peptides, the rabbit's tissue serum reacted against MBP. Further, rabbits sensitized with the HBVP peptide developed central nervous system (CNS) pathology similar to rabbits sensitized with whole MBP protein or the MBP peptide [16] . Importantly, the rabbits sensitized with HBVP did not contract hepatitis but still developed encephalomyelitis and presented with a similar pathology as MBPsensitized mice. These experiments were the first experimental demonstration of molecular mimicry, whereby a microbial peptide with similar amino acid sequences to the self-peptide was able to activate autoreactive T cells and subsequently cause specific tissue damage.
Immune cells of the adaptive immune response are specifically activated, but the hallmark of autoimmunity is the dysregulation of the immune system, especially T and B cells recognizing self-antigens as foreign. Activation of an autoimmune response could be enhanced by a variety of other, albeit, non-mutually exclusive non-specific mechanisms including bystander activation and superantigens. The difference between other non-specific mechanisms that initiate autoimmunity and molecular mimicry is that microbial mimics specifically direct the immune response towards a tissue and/or organ. Originally, T cell recognition was postulated to be highly specific and cross-reactivity was thought to be a rare phenomenon. However, the structural requirements for peptide binding by MHC class II molecules that are presented to T cells were found to be based on amino Linear sequence matches in amino acid motifs is not the only criteria for mimicry [32] . It has been hypothesized that self-reactive immune cells are primed by molecular mimicry and bystander activation, thereby sensitizing the immune cells and leading to a "fertile field" but no apparent disease. Subsequent environmental insults could induce these sensitized autoreactive cells to cause an autoimmune disease. Work from our laboratory demonstrated that recombinant viruses having molecular mimicry with self-CNS antigens were unable to initiate an autoimmune disease individually [38] . However, infected mice that were subsequently challenged, after viral clearance, with a non-specific immunologic insult developed disease [38] . Further, subsequent experiments showed that conventional inflammatory responses to specific pathogens were able to induce disease in animals primed with a molecular mimic to a CNS antigen [39]. Therefore, not only is the priming of the immune system necessary for an autoimmune disease but the milieu to which the primed immune cells are exposed is an important factor in initiating an autoimmune disease. Animal models of various autoimmune diseases have explored the role of molecular mimicry as a contributing factor ( Table 2) .
The use of transgenic (tg) mice expressing virus proteins as transgenes in specific organs has been an important model for providing evidence for molecular mimicry. The expression of lymphocytic choriomeningitis virus (LCMV) viral antigens in pancreatic islet cells and the subsequent cross of this tg mouse with a TCR-tg mouse specific for LCMV glycoprotein resulted in an animal that only developed autoimmune disease if virally infected [40, 41] . These results demonstrated that "self"-reactive T cells are present in the periphery and the immune cells appear to remain quiescent until an appropriate signal (viral infection) triggers the T cells to respond.
There are a variety of non-mutually exclusive factors that lead to a fully activated T cell, such as the quantity of peptide-MHC presented on the surface of antigenpresenting cells and TCR avidity. The interaction between the peptide-MHC and TCR is critical for the initiation of an adaptive immune response and clearance of a pathogen [15] . In order for T cells to reach maturity, the T cell goes through a number of developmental checkpoints leading to somatic recombination of various gene segments. The TCR αand β-chains are generated by V-D-J recombination, which leads to αβ TCRs expressed on the surface of T cells [42, 43] . Although it was believed that T cell signaling was mediated by a single antigen receptor, recent evidence demonstrates that T cells are capable of expressing functional dual Vα TCRs at a frequency of approximately 30% in humans and 15% in mice; however, an accurate number of dual specific TCRs is lacking due to the limited availability of anti-Vα monoclonal antibodies (mAbs) [44] [45] [46] . Interestingly, in contrast to the high frequency of dual expressing Vα T cells, only 1% of humans and 5-7% of mice express two β-chains due to allelic exclusion mechanisms, but the frequencies of dual Vβ TCRs have been found to be higher with age and in TCR-tg mice [47] [48] [49] . Expression of multiple TCR Vαs on the surface of a T cell is the result of simultaneous rearrangement of both TCRα loci during thymocyte development [50-52]. Further, TCR Vβ-chains preferentially bind to certain Vαchains leading to differential expression of chimeric TCRs on the surface of T cells [51, 53, 54].
Due to the heterogeneity of TCRs normally expressed in the periphery of humans and mice, TCR-tg mice have been used to track and determine the fate of T cells expressing dual TCRs. The use of TCR-tg mice has led to the identification of a potential role for dual TCRs in a variety of conditions including graft-versus-host disease, human immunodeficiency virus infection, inflammatory bowel disease, T cell leukemia, T cell lymphoma, and MS [55-61].
The expression of dual TCRs by the same T cell has been proposed to be a potential mechanism for autoimmune disease. Normally, high avidity self-reactive T cells are thymically depleted, but it has been hypothesized that the expression of a self-TCR on a T cell is lower when presented in the context of a second TCR, thereby providing a cover for high avidity self-TCRs from both central and peripheral tolerance. Blichfeldt et al. [62] demonstrated that dual tg-TCRs, which have lower expression of each TCR on the surface of a T cell, needed higher concentrations of peptide, presented by MHC, to induce a similar T cell proliferative response compared to a single receptor T cell.
A potential role of dual TCRs in autoimmunity is in the rescue of autoreactive T cells from thymic selection. For example, the double tg mouse for autoimmune diabetes, in which the mice express a TCR specific for peptide 111-119 of hemagglutinin (HA) (TCR-HA) under the control of the rat insulin promoter and develop spontaneous diabetes and insulitis [63], were used to determine how T cells could escape tolerance mechanisms even if the antigen was ubiquitously expressed [64]. Low expressing TCR-HA coexpressing T cells were more effective at transferring diabetes than TCR-HA high dual TCRs, suggesting that the surface level expression of a dual TCR can be modulated by a second TCR expressed on the same T cell, thus "escape" of autoreactive T cells could be the first step in an autoimmune disease.
The "trigger" of an autoimmune disease could be linked to environmental insults, such as viruses. A T cell co-expressing TCRs specific for a self-antigen and a foreign antigen could potentially allow for autoreactive T cells to be activated if the host is exposed to that foreign antigen. The activation of a subset of T cells could than lead to tolerance being broken and the initiation of an autoimmune disease if these T cells experienced a particular organ or tissue that expressed the self-antigen for the other TCR expressed at the surface of the T cell. In support of a role for dual TCRs in autoimmune diseases, work performed in our laboratory characterized autoreactive CD8 + T cells isolated from the spleens of Theiler's murine encephalomyelitis virus (TMEV)infected SJL/J mice [65] . In vitro assays testing CD8 + T cell killing activity found a population of CD8 + T cells that killed uninfected syngeneic cells [65] . Adoptively transferring these TMEV-specific autoreactive CD8 + T cells into non-infected SJL/J mice caused CNS pathology [65] . Further support for the importance of the mechanism by which viral infection could induce an autoimmune disease through dual TCR-expressing T cells was performed by Ji et al.
[61] using MBP(79-87) TCR-tg mice [66] . Cytometric phenotyping, in vitro CD8 + T cell killing assays, and adoptive transfer experiments were used to track the expansion and killing capacity of Vα8Vβ8 MBP (79-87)-specific TCR and Vα8Vβ6-vaccinia virusspecific TCR. Infection of these tg mice with vaccinia virus induced autoimmune disease, thus demonstrating a virus triggering an autoimmune disease through dual TCR expressing T cells [61] . Although several tg TCR β-chains have been described on peripheral T cell [61, 67-70], there is no evidence that co-expression of dual TCRs leads to autoimmunity without the use of TCR-tg mice. As described above, current work in our laboratory has characterized TMEV-specific autoreactive CD8 + T cell clones derived from a wild-type animal, and these autoreactive TMEV-specific T cell clones express dual TCRs (manuscript in preparation). Importantly, we were able to induce CNS pathology in naïve SJL/J mice by adoptively transferring the TMEV-specific clones. Although further work is needed in order to identify the self-antigen that activates these CD8 + T cells, to our knowledge these results are the first demonstration of an autoimmune disease initiated by a dual expressing TCR characterized in the virus' natural host.
Taken together, three possible mechanisms could explain how the dual reactivity of the TCR may play a role in autoimmune diseases (manuscript in preparation). The first mechanism is molecular mimicry, whereby the induction of an autoimmune response to self is due to a single TCR recognizing both a virus and a self-antigen. The second mechanism is the expression of dual TCRs on a single T cell, where one TCR is able to recognize a microbial antigen and the other TCR recognizes self. The third mechanism involves a T cell expressing chimeric TCRs generated from either a single Vα combining with two different Vβs or a single Vβ combining with two different Vαs, resulting in a T cell with the potential of expressing two different chimeric TCRs specific for a self-antigen and a foreign antigen.  Low usage of government healthcare facilities for acute respiratory infections in guatemala: implications for influenza surveillance BACKGROUND: Sentinel surveillance for severe acute respiratory infections in hospitals and influenza-like illness in ambulatory clinics is recommended to assist in global pandemic influenza preparedness. Healthcare utilization patterns will affect the generalizability of data from sentinel sites and the potential to use them to estimate burden of disease. The objective of this study was to measure healthcare utilization patterns in Guatemala to inform the establishment of a sentinel surveillance system for influenza and other respiratory infections, and allow estimation of disease burden. METHODS: We used a stratified, two-stage cluster survey sample to select 1200 households from the Department of Santa Rosa. Trained interviewers screened household residents for self-reported pneumonia in the last year and influenza-like illness (ILI) in the last month and asked about healthcare utilization for each illness episode. RESULTS: We surveyed 1131 (94%) households and 5449 residents between October and December 2006 and identified 323 (6%) cases of pneumonia and 628 (13%) cases of ILI. Treatment for pneumonia outside the home was sought by 92% of the children <5 years old and 73% of the persons aged five years and older. For both children <5 years old (53%) and persons aged five years and older (31%) who reported pneumonia, private clinics were the most frequently reported source of care. For ILI, treatment was sought outside the home by 81% of children <5 years old and 65% of persons aged five years and older. Government ambulatory clinics were the most frequently sought source of care for ILI both for children <5 years old (41%) and persons aged five years and older (36%). CONCLUSIONS: Sentinel surveillance for influenza and other respiratory infections based in government health facilities in Guatemala will significantly underestimate the burden of disease. Adjustment for healthcare utilization practices will permit more accurate estimation of the incidence of influenza and other respiratory pathogens in the community. As the 2009 influenza A (H1N1) pandemic highlighted, surveillance for influenza is now a worldwide priority. [1, 2] At the 58 th World Assembly in 2005, The World Health Organization adopted a resolution calling for Member States to fortify and coordinate national strategies to prepare for an influenza pandemic, including establishment of surveillance systems for human influenza. [3] To assist with the development of standardized influenza surveillance systems in the Americas, the Pan American Health Organization (PAHO) and the United States Centers for Disease Control and Prevention (CDC) developed a generic protocol for influenza surveillance incorporating two sentinel surveillance systems, one hospital-based system for severe acute respiratory infections (SARI) and SARI-related mortality and another for influenza-like illness (ILI) based in ambulatory clinics. [4] Sentinel surveillance for influenza can provide information on trends in viral circulation patterns and seasonality, along with virus characteristics to help guide decisions on vaccine composition. However, healthcare seeking behaviors can affect who accesses care at the sentinel site, limiting the ability to gather information to guide public health policies. Without understanding patterns of healthcare seeking behavior, it is not possible to calculate the burden of disease, generalize findings to a larger population or identify risk groups.
Healthcare utilization surveys (HUS), one method of determining the healthcare utilization practices for specific diseases in defined populations, have been conducted in several countries. [5] [6] [7] [8] [9] [10] [11] [12] In HUS, random samples of the catchment population are interviewed with respect to their healthcare seeking and treatment behaviors during recent episodes of disease. These data can be used in a number of ways to support the interpretation of information from sentinel surveillance sites: first, to establish correction factors for estimates of incidence in the community based on numbers of cases presenting at the sentinel surveillance site, including the incidence in particular population sub-groups; second, to describe the actual catchment population accessing healthcare at the sentinel site to determine generalizability; and third, to identify other healthcare providers who may be recruited to participate in the surveillance system.
To inform the establishment of a surveillance system for influenza and other respiratory infections, and the implementation of the PAHO/CDC standard protocol for influenza surveillance in Guatemala, we conducted a HUS among residents of the Department of Santa Rosa, Guatemala to describe the healthcare seeking behavior for acute respiratory illnesses.
Guatemala, with a population of 12,755,366 in 2008, had a gross national income per capita of $2680 and is considered a middle-income country by the World Bank (http://data.worldbank.org/indicator/NY.GNP.PCAP.CD, accessed on 1 September 2010). Guatemala is divided into 22 departments, which are further subdivided into 10-29 municipios (similar to counties), made up of multiple communities. The Guatemalan Ministry of Public Health and Social Welfare (MSPAS) provides free healthcare in several different settings, including hospitals, health centers, health posts, and outreach centers. Hospitals and health centers are staffed by physicians and nurses, whereas health posts are staffed by nurses. Outreach centers provide preventive and primary healthcare but are only visited by trained medical staff a few days each month. In addition to the MSPAS facilities, formally-employed workers who contribute to the Guatemalan Institute of Social Security (IGSS) can receive healthcare from IGSS hospitals and health centers, which are concentrated in Guatemala City. Other non-governmental locations where people may seek healthcare are private hospitals and clinics, pharmacies, drug shops, and traditional healers and midwives. Communities in Guatemala vary in their access to healthcare depending on their size and location (e.g., urban vs. rural).
Santa Rosa is a mostly agrarian department located in the southeastern part of the country approximately 80 km from Guatemala City. The population in 2006 was 308,522 residing in 14 municipios with an estimated 768 communities. Cuilapa is the department's capital city. In contrast to the country as a whole, which is almost half Amerindian indigenous, only 3% of Santa Rosa's residents are Mayan or Xinca, and Spanish is spoken by approximately 91% of the inhabitants. The mortality rate of children <5 years of age for Santa Rosa is 58 per 1000 live births, significantly higher than the average for the country (45 per 1000 live births). [13] Government-run healthcare facilities within the department include one hospital (the National Hospital of Cuilapa, 176 beds), 14 health centers (one in each municipio) and 56 health posts in the outlying communities. There is one IGGS facility that treats only patients involved in motor vehicle accidents. Two small private hospitals as well as approximately 133 private ambulatory clinics are available for those who choose to pay for healthcare. Additionally, healthcare services can be sought from more than 114 pharmacies or drug shops, and an unknown number of traditional healers, midwives and community healthcare workers. Healthcare may also be accessed in Guatemala City and neighboring departments.
We conducted a cross-sectional survey to determine healthcare utilization patterns for acute respiratory, diarrhea, neurologic and febrile illnesses: we report here only the results for acute respiratory infections. We used a stratified, two-stage cluster sampling procedure. Communities in the 2002 Guatemalan census were stratified as to whether or not they had a hospital or health center located in their community. As the first stage of sampling, 30 communities were selected within each stratum, using probability proportional to the population of each community, for a total of 60 clusters. Maps detailing household locations were obtained from the Guatemalan Census Bureau for these communities and 20 houses were randomly selected for a total of 1200 houses.
Interviews were conducted in person from October 1 through December 13, 2006. All persons who had lived in the house for at least six of the preceding 12 months were considered members of the household and eligible for inclusion, including persons deceased at the time of the survey if they had been resident during the reference period. Infants <6 months of age were included if they had lived in the household since birth. Households were excluded if a head of household or consenting adult was unavailable after visiting the house on three separate occasions over at least two days, or if the household head declined to participate. Excluded households were not replaced. If a house was abandoned or no longer existed, another house was randomly chosen for inclusion.
The adult respondents from each household were read a consent statement and asked to give verbal consent for their household's participation. The protocol for this study was reviewed and approved by the institutional review boards of the Centers for Disease Control and Prevention (Atlanta, GA) and the Universidad del Valle de Guatemala (Guatemala City, Guatemala) and approved by the MSPAS (Guatemala City, Guatemala).
The sample size of 600 households per strata (1200 total) was based on calculations for diarrhea, a more common syndrome, rather than for pneumonia as information was not available on the expected incidence of pneumonia. However, assuming a 10% household nonresponse rate, and 4.8 persons per household, a sample of 600 households per stratum should yield between 39 and 156 persons with pneumonia, assuming an annual incidence of between 1.5% and 6.0%, respectively. Given a design effect of two due to the clustering of pneumonia cases by community and household, this sample would be large enough to estimate the proportion of the population seeking healthcare outside of the house for pneumonia with a precision of 10%, assuming 70% of persons with pneumonia seek care for their illness.
A structured survey was completed for each participating household with information on both household-and individual-level characteristics. All household members were enumerated and an adult proxy was interviewed for children <15 years old or older residents not present at the time of the interview to determine whether any household member met any of the case definitions (mild or severe respiratory, diarrhea, acute febrile and acute neurologic illness) during the prescribed time period. A clinical history was obtained for each illness episode (if more than two episodes of the same illness were reported, the most recent illness episode was used as the reference) along with a history of healthcare treatment seeking. Proxies of household residents who died but met the case definition for one of the illnesses in the relevant time period before death were also administered the illness-specific forms.
A case of severe respiratory illness, referred to as pneumonia, was defined as self-reported cough and difficulty breathing for two or more days, or a physician-diagnosis of pneumonia; the reference period was the 12 months prior to the interview. This case definition has been used in several studies of self-reported pneumonia in the community [12, 14] and is based on questions that were moderately sensitive and specific for pneumonia from a World Health Organization verbal autopsy questionnaire. [15] Additionally, severe pneumonia was defined for children <3 years old who met the pneumonia case definition as any of the following: blue lips and/ or nails, inability to breastfeed or drink, convulsions, unconsciousness or decreased activity. For those ≥3 years old who met the pneumonia case definition, severe pneumonia was defined as fast breathing with confusion.
A case of mild respiratory illness, referred to as ILI, was defined as subjective fever with either cough or sore throat in the 30 days prior to the interview. If a respondent reported both an ILI and pneumonia for the same month, the ILI data were excluded.
Outpatient providers were defined as all sources of healthcare that did not admit patients for overnight stays, and included government and private ambulatory clinics, pharmacies, drug shops, the IGSS and traditional healers. Inpatient providers included government and private hospitals.
Survey forms were received at the offices of the CDC-UVG Collaboration at the Universidad del Valle de Guatemala for optical scanning into a database using the Cardiff Teleform system (Vista, CA). Each Teleform entry was checked manually with the original forms to ensure accurate scanning and coding. Socioeconomic status (SES) was estimated using a wealth index generated using the factor effects derived from the first principle component of a principle component analysis of household goods, house construction material, source of water supply, source of cooking fuel and sanitation facility; the wealth index was categorized into quintiles with households weighted by number of residents and sample weights. [16, 17] To account for the complex survey design, sample weights were applied in all analyses. We used the Wald Chi-square statistic to test for differences between proportions and logistic regression to test for trends related to age and SES. Analyses were conducted with SAS version 9.1 (SAS Institute, Cary, NC) using PROC SURVEYFREQ or PROC SURVEYLOGISTIC.
We approached 1200 households but residents could not be reached at 33 (3%) locations after three visits, and in 36 (3%), the household head declined to participate. We interviewed residents from 1131 (94%) households and gathered information on a total of 5449 persons of which 2806 (52%) were female and 586 (12%) were children <5 years old (Table 1) .
We found 323 persons (6%, 95% confidence interval [CI] 6-7%) who met the pneumonia case definition in the previous year. Almost all (87%) met the case definition with self-reported cough and difficulty breathing for at least two days; 2% reported only a physician's diagnosis of pneumonia; and 12% reported both. There were 60 cases (11%, 95% CI 9-13%) of pneumonia reported among children <5 years old, and 263 cases (6%, 95% CI 5-6%) among persons aged five years or older. Among the children <5 years old, 31 (6%, 95% CI 5-7%) met the case definition for severe pneumonia.
The proportion of pneumonia cases reported by month increased from October 2005 through September 2006 (Figure 1 .) More than half of the pneumonia cases were reported from the last five months prior to the survey.
There were 628 (13%, 95% CI 12-14%) persons who reported ILI in the previous month. A case of ILI was reported by 106 (19%, 95% CI 17-20%) children <5 years old and 522 (12%, 95 CI 11-13%) persons aged five years or older.
The most common symptoms reported by persons with pneumonia were difficult breathing (100%), cough (99%), and feverishness (88%) ( Table 2 ). The mean duration of illness of all persons with pneumonia was 13 days (range 2-120); more than one-quarter had symptoms for seven days or more.
Among persons who reported ILI, the most common symptom besides feverishness (100%) was sore throat (97%), headache (89%) and cough (88%). Signs of lower respiratory tract infection, such as difficult or fast breathing and wheezing, were less common among person who reported ILI than those with pneumonia. The mean duration of illness among persons with ILI was seven days (range 1-90); more than half of person who reported an ILI had symptoms for seven days or more.
The age distribution of persons with pneumonia was significantly different from the surveyed population without pneumonia (P<0.0001), with more children <5 years and adults ≥60 years old among the persons reporting pneumonia than among the surveyed population without pneumonia (Table 1) . Similarly, the age distribution of those with ILI was younger than the surveyed population without ILI (P = 0.001). The distribution of the person who reported ILI by household wealth index was significantly different from those without ILI (P<0.0001) with more cases among persons in the lowest wealth category and fewer in the wealthiest category. There was no difference in household wealth between persons with and without pneumonia (P = 0.14). 
Among the 60 children <5 years old reporting pneumonia in the last year, 55 (92%) sought care outside the home. All subsequent analyses of healthcare-seeking behavior are based on those who sought care outside the home. Sixteen (27%) children sought care from more than one source. Hospitals were consulted by 17 (25%) children <5 years old with pneumonia, and most were government hospitals (Table 3) . Nine (12%) children <5 years old with pneumonia were admitted for at least one night in a hospital. Outpatient care providers were visited by 38 (75%) children <5 years old with reported pneumonia. Overall, the most frequently reported source of healthcare for children <5 years old with pneumonia were private ambulatory clinics, which attended to more than half the reported cases. More than half (55%) of the children <5 years old with reported pneumonia received care at least once during their illness from government facilities, either government hospitals or ambulatory clinics. Among the 263 persons five years or older who reported pneumonia in the last year, 199 (73%) sought healthcare outside their home, with 21 (8%) seeking care from more than one source. Among persons in this age group who sought care outside the home, 28 (12%) sought care at hospitals (Table 3) , and 8 (4%) were admitted for at least one night. Government hospitals provided most of the hospitalized care. Among outpatient care providers, the most frequently sought source of care were private clinics, which provided care to 65 (31%) persons with pneumonia aged five years or older, along with government ambulatory clinics (46, 27%). A considerable proportion (16%) of persons with pneumonia aged five years or older sought care at pharmacies. Care for ILI was sought outside the home by 87 (81%) children <5 years old, and 6 (6%) sought care from multiple sources (Table 3) . Government clinics were the source of healthcare most often consulted by children <5 years old for ILI; 34 (41%) children <5 years old reported seeking care at a government clinic, whereas 20 (20%) reported consulting a private clinic. Hospitals were consulted by 6 (5%) children <5 years old for ILI, and 4 (5%) were hospitalized for one night or more. Care was sought at pharmacies and drug shops for nearly one-third of children <5 years old with ILI.
Care for ILI was sought outside the home by 337 (65%) persons aged five years or older, and 13 (3%) consulted multiple sources. Government clinics were consulted for ILI by 111 (36%) persons aged five years or older. Pharmacies were consulted for ILI by 110 (29%) persons aged five years or older. One (0.1%) ILI patient five years or older was hospitalized for more than one night.
Among the respondents with pneumonia who did not seek healthcare for their illness, the perception that their illness was not severe enough to warrant treatment (28/69, 42%) and the cost of treatment (13/69, 20%) were the major reasons cited for not seeking care. Among persons with ILI who did not seek healthcare for their illness, insufficient severity of illness (68/204, 31%), cost of treatment (37, 18%), lack of medical services (13, 9%) and spontaneous improvement (19, 7%) were the major reasons cited.
Sociodemographic and illness characteristics associated with seeking treatment at government facilities There was no significant association between sex of the respondent and whether care was sought for pneumonia (P = 0.34) or ILI (P = 0.15) at a government hospital or clinic (Table 4 ). Children <5 years old were more likely to receive healthcare for pneumonia and ILI at government facilities than persons aged five years or older, but this difference was statistically significant only for Numbers will not necessarily add up to 100% because more than one healthcare provider can be consulted in the course of an illness. Percentages are calculated using sample weights.
pneumonia (P = 0.03). There was a significant inverse trend across SES status with persons of higher socioeconomic status less likely to seek care for pneumonia and ILI at government facilities (P = 0.005 and P = 0.001, respectively). The duration of illness was not associated with consultation at a government facility for either pneumonia (P = 0.37) or ILI (P = 0.25). Severity of pneumonia was not associated with seeking care from a government facility (P = 0.13).
We conducted a HUS to help inform establishment of a sentinel surveillance system for pneumonia and influenza-like illness in Santa Rosa, Guatemala, and found that private clinics are the single most important source of healthcare for pneumonia both in children <5 years old and older persons. For more mild ILI, both children <5 years old and persons aged five years or older are more likely to consult government clinics than other sources of health care. These findings suggest that in Santa Rosa, sentinel surveillance for pneumonia in government hospitals will significantly underestimate the burden of disease, by up to 75% for children <5 years old and 88% for persons aged five years and older. Government healthcare clinics will underestimate the number of cases of ILI by about 59% for children <5 years old and 64% for persons five years and older.
Our study is consistent with other studies of healthcare-seeking behavior in Guatemala and Central America that have found private clinics to be common sources of healthcare. Van der Stufyt et al. reported that more than 40% of Guatemalan families sought healthcare for their children <5 years old from private physicians, compared with 26% consulting a governmental health center.
[18] Focus group interviews from three countries in Central America found that persons considered the healthcare obtained through private physicians and clinics preferable to public options because of prompt attention and a perception that healthcare is better. [19] Another study evaluating healthcare utilization among children in rural Guatemala who reported a diarrheal or respiratory illness found private physicians were more likely to be consulted by households with higher income. [20] The population that uses government health clinics and hospitals for respiratory illnesses in Santa Rosa is younger and poorer than the general population. We found a trend for decreasing use of government facilities with increasing age and household wealth. These findings should be taken into account to improve the generalizability of burden of disease estimates made from sentinel surveillance data.
The results of this study are subject to several important limitations. We used a case definition that required self-report or report by a proxy of an illness that occurred up to one year prior to interview. It is well known that such self-reports are limited by recall decay, and recent episodes are more likely to be recalled than earlier episodes. [21] This could be noted in our data that demonstrated a decreasing report of pneumonia with increasing time before the survey. As long as recent illness episodes do not differ from prior episodes with regard to patterns of healthcare-seeking behaviors, there is no reason to believe that recall decay causes bias with regard to these variables. Because our case definition is based on self-report, there is substantial potential for misclassification, especially between mild and severe acute respiratory illness, and this can be seen in the report of some lower respiratory tract symptoms among respondents with ILI. However, the behaviors associated with episodes of ILI compared to pneumonia (lower probability of seeking care outside the home, less likely to seek treatment at a hospital) are suggestive of a more mild illness and we are reasonably confident that we have described the healthcare seeking behaviors that are broadly associated with both pneumonia and ILI. An additional limitation is our sample size, which is too small to permit age to be stratified into more than two groups, restricting our ability to model healthcare-seeking behaviors more precisely by smaller age groups. Finally, it is not clear whether results from one area of Guatemala with a significantly lower indigenous population than the rest of the country can be generalized to the nation as a whole.
Despite the limitations of this and similar surveys, our findings indicate that Guatemala and other countries in the region can improve the estimation of the burden of influenza and other respiratory pathogens from sentinel surveillance by taking healthcare utilization into account. As a large proportion of the population with respiratory disease in Guatemala does not attend government health facilities for treatment, this approach could help correct government surveillance data for missing cases and facilitate comparison of the burden of influenza with other countries. Understanding the clinical spectrum of complicated Plasmodium vivax malaria: a systematic review on the contributions of the Brazilian literature The resurgence of the malaria eradication agenda and the increasing number of severe manifestation reports has contributed to a renewed interested in the Plasmodium vivax infection. It is the most geographically widespread parasite causing human malaria, with around 2.85 billion people living under risk of infection. The Brazilian Amazon region reports more than 50% of the malaria cases in Latin America and since 1990 there is a marked predominance of this species, responsible for 85% of cases in 2009. However, only a few complicated cases of P. vivax have been reported from this region. A systematic review of the Brazilian indexed and non-indexed literature on complicated cases of vivax malaria was performed including published articles, masters' dissertations, doctoral theses and national congresses' abstracts. The following information was retrieved: patient characteristics (demographic, presence of co-morbidities and, whenever possible, associated genetic disorders); description of each major clinical manifestation. As a result, 27 articles, 28 abstracts from scientific events' annals and 13 theses/dissertations were found, only after 1987. Most of the reported information was described in small case series and case reports of patients from all the Amazonian states, and also in travellers from Brazilian non-endemic areas. The more relevant clinical complications were anaemia, thrombocytopaenia, jaundice and acute respiratory distress syndrome, present in all age groups, in addition to other more rare clinical pictures. Complications in pregnant women were also reported. Acute and chronic co-morbidities were frequent, however death was occasional. Clinical atypical cases of malaria are more frequent than published in the indexed literature, probably due to a publication bias. In the Brazilian Amazon (considered to be a low to moderate intensity area of transmission), clinical data are in accordance with the recent findings of severity described in diverse P. vivax endemic areas (especially anaemia in Southeast Asia), however in this region both children and adults are affected. Finally, gaps of knowledge and areas for future research are opportunely pointed out. Plasmodium vivax is the most geographically widespread species of Plasmodium causing human disease, with most cases reported in Central and Southeast Asia, in the horn of Africa and in Latin America [1] . It is considered to be a potential cause of morbidity and mortality amongst the 2.85 billion people living at risk of infection, excluding the large African populations who are mostly Duffy negative and, therefore, naturally less susceptible to this infection. However recent data suggest that the parasite is evolving and may use alternative receptors other than Duffy (DARC) for erythrocyte invasion [2] . It is estimated that 5.5% of the population under risk live in the Americas [3] . The major biological characteristic of this parasite is the presence of liver hypnozoites responsible for the frequent relapses, which add a substantial number of cases to the general burden of the disease, what is being faced as one of the most challenging bottlenecks for vivax malaria eradication [4] .
Although often regarded as causing a benign infection, there is recent increasing evidence that the overall burden, economic impact, and severity of P. vivax have been underestimated, in part due to a bias in the scientific literature which traditionally devoted most of its attention to the more lethal parasite Plasmodium falciparum, probably as a reflection of a more substantial funding [5] . Until 16 October 2011, the search in MED-LINE using P. vivax as keyword retrieved 5,026 indexed abstracts; using P. falciparum, on the other hand, retrieved almost five times more abstracts: 25, 807 . Even in places where P. vivax represents the major local problem to be tackled, clinical research is still focused on P. falciparum [6] . There is robust evidence in the past decade from hospital-based studies in India and Indonesia that P. vivax is able to cause severe disease [7, 8] . Some authors argue that this clinical severity may only now be properly recognized and announced by researchers in the field, but these complications apparently are not new from a historical perspective [9] . Actually, the case fatality rate (CFR) related to malarial infections in the English marshes during the 16th and 17th centuries, corresponding to the Little Ice Age, suggest that P. vivax (a parasite more prone to persist in vectors even under low temperatures) may have killed part of this population already victimized by famine [10] . During the first half of the 20th century, malariotherapy in patients with neurosyphilis, using essentially the 'nonsevere' P. vivax parasite, led to diverse complications, CFR ranging from 3.3 to 30.3% [11] . The major related complications in these co-infected patients were liver damage, ruptured spleen, jaundice, delirium, uncontrolled vomiting and persistent headaches [11] . That reinforces the concept that P. vivax infection may synergize with other co-morbidities resulting in more complicated disease. Added to local geographical and social determinants, wide Annual Parasite Incidence (API) and CFR variations due to this species are seen around the world.
In summary, P. vivax, which has long been neglected and mistakenly considered 'benign' [12] , is receiving an increasing amount of importance in the debates taking place on malaria epidemiology and control, drug resistance, pathogenesis and vaccines [13] . As reviewed elsewhere, the good clinical characterization of severe disease in vivax infection is the first step to understand how the inflammatory response to this parasite contributes to pathogenesis [14] .
Traditionally, Brazil has been responsible for almost half of all cases of malaria in Latin America. In 2009, 308,498 cases of malaria were reported in this country (257,571 caused by P. vivax), representing 54.9% of all the malaria reported in the Americas [15] . Cases are virtually restricted to the Amazon Basin (constituted by the states of Amazonas, Acre, Roraima, Amapá, Pará, Tocantins, Rondônia, and parts of Mato Grosso and Maranhão). Amazonian urban agglomerations under continuous economical development trigger intense migration flows, such as in the city of Manaus (in the Western Brazilian Amazon), helping to maintain the disease under endemic levels [16, 17] . Malaria in Brazil is mostly related to P. vivax since the 1990s, when the available tools for control at the moment were put together and intensified, such as the fast diagnosis through thick blood smear (TBS) in all febrile patients, and free access to anti-malarials, integrated through a decentralized primary care-centred public health system [18] . Allied to that, an active community of local malariologists has been persistently identifying the profile of anti-malarial resistance with permanent counseling to the Brazilian Ministry of Health, which responds promptly to these evidences, changing the first line regimens [19] . As the sexual forms (which are infective for the vector) of P. falciparum generally appear later in the course of infection, opportune diagnosis and treatment tend to have a high impact on reducing the transmission intensity of this species but the same is not true for P. vivax, whose gametocytes are present in the very first days of the infection, before efficacious treatment is usually started. In 2008, 59% of all malaria cases registered in the Brazilian Amazon were treated in the first 48 h after appearance of symptoms (SIVEP-Malaria, 2009). These public health measures allied to a regularly updated online information system also impacted the number of deaths related to P. falciparum, which were not more than 58 in 2009 (Brazilian Ministry of Health, 2010). As a consequence, even in the non-indexed literature, severity due to P. falciparum is not frequently reported anymore in Brazil.
Brazil has reported 85% of its cases related to P. vivax in 2010, which puts this country in a peculiar epidemiological situation, as one of the few countries around the world with P. vivax predominance. The impact of P. vivax/P. falciparum co-infections or simultaneous circulation of both species with similar frequencies in a given population, upon the immunological status and clinical presentation of malaria is still unclear [20, 21] , but most probably clinical data from population from certain areas should not be extrapolated to other areas in distinct epidemiological conditions. Actually, the lack of data on clinical presentation of P. vivax infection allied to the several particularities of this region, including the diverse genetic background of its population, implicate that the generalization of the findings from Southeast Asia may be inappropriate.
In Brazil, in 1903, the young physician Carlos Chagas (most known for the discovery of American trypanosomiasis afterwards) wrote his MD thesis on the haematological complications of malaria, which, at that moment, also occurred in the non-Amazon area. His major findings in studying P. vivax patients were severe anaemia, splenomegaly, leukopenia, cachexia and jaundice associated to concomitant staphylococcal disease [22] . Bone marrows were also analysed in these patients with no conclusive findings. Later on, during the 1940s, Djalma Batista in Manaus described a series of malarial cases from his outpatient clinics in whom large splenomegaly, cachexia and minor bleeding were frequent among those with the 'benign' tertian malaria [23] . More recently, from 1998 to 2008, 234 deaths related to vivax disease were officially reported to the Brazilian Ministry of Health [18] , and an increase in the hospitalization trends for vivax patients was published in a tertiary care hospital from Manaus [24] . To complicate matters, these facts parallel a lack of robust biomarkers and specific criteria for severe disease for this species in the literature. A sine qua non requisite in the analysis of clinical severity related to P. vivax infection is the exclusion of mixed infection with P. falciparum through a more sensitive technique such as PCR and the exclusion of other co-morbidities which may be responsible for the clinical presentation per se. In the literature, in general, reports of 'complicated/severe' cases lack more precise and uniform definition criteria, in part due to the rare application of more robust endpoints such as death and admission to the intensive care unit (ICU), and therefore end up suffering bias through individual judgment of authors, editors and reviewers. As in most of the data published there were no systematic exclusion of co-morbidities and/or mono-infection confirmation using PCR, performing a meta-analysis of severe manifestations of P. vivax becomes virtually impossible. The other bias in the case of Brazil is that many relevant data are confined in abstracts from national scientific meetings and graduate students' dissertations and theses. The systematic review of these unpublished data therefore could contribute to the understanding of the clinical spectrum of vivax infection in this country and ultimately as a representative sample from Latin American vivax malaria.
The sources for published data on clinical aspects of vivax infection in Brazil were MEDLINE (1948 to February 2011) and LILACS (1982 to February 2011). The following search strategy was devised for both databases: (Plasmodium vivax).mp. AND (Brazil).mp. All types of study designs with primary data were included (cross-sectionals, case-controls, cohorts, case series and case reports). The abstracts were analysed in details by two independent reviewers and publications were selected if they mentioned any type of clinical complication (no specific criterion was used) in at least one patient with the diagnosis of vivax infection. Disagreement between the two reviewers was solved through consensus. Articles were excluded if they were reviews and also if they did not contain primary data on clinical aspects. For included studies, there were extracted data on date of publication, location, number of patients, and characteristics of participants (age range, pregnancy status, presence of co-morbidities), if molecular diagnosis through PCR was used to assess vivax malaria mono-infection and fatality. Exclusion criteria for analysis were participants with mixed infections (P. falciparum/ P. vivax); studies in where patients with P. falciparum and P. vivax were both presented but the clinical data reported was not individualized for each species; and studies reporting the same patients from previous studies from the same authors. Through abstract analysis, 297 articles were retrieved and after application of the inclusion and exclusion criteria, 27 articles (from 1987 to 2011) were selected, which are presented in Table 1 .
Unpublished studies were searched manually in the Annals of the Congress of the Brazilian Tropical Medicine Society (published in supplements of the indexed journal Revista da Sociedade Brasileira de Medicina Tropical [Journal of the Brazilian Society of Tropical Medicine]), from 1964 to 2011. This is the most traditional scientific event for tropical medicine clinicians in Brazil. Similar inclusion and exclusion criteria were used in this search. However if the same abstract data were published afterwards as a full paper, the published paper information was presented here. If the abstract referred to a dissertation or thesis, this more detailed information was presented instead. Forty-five abstracts were retrieved from 1995 to 2011. Of these, 17 fulfilled any of the exclusion criteria and therefore, 28 abstracts are presented in Table 2 .
Masters' dissertations and doctoral theses abstracts since 1987 were searched in the online database http:// capesdw.capes.gov.br/capesdw/Teses.do maintained by the Coordination for the Improvement of Higher Education Personnel (CAPES), the institution which coordinates and supervises all the Brazilian Graduate Programmes in all areas of knowledge. The full original electronic documents were downloaded from the website when available or obtained through contact with the respective graduate students. Ten dissertations and three theses are presented in Table 3 .
Classical malaria paroxysms are typically short and sharply delineated within a period of less than eight hours. Fever is one feature that is almost invariably present during a paroxysm. Any of other common symptoms of the febrile syndrome, such as chills, rigours and sweating, are also described. These symptoms of a paroxysm could be accompanied by others, including headache, nausea and vomiting, and moderate to severe muscle, joint and back pain [93] . Indeed high fever tends to be more evident in vivax disease even with lower parasitaemia, due to its recognized lower fever-threshold (around 100 infected RBCs/microlitre) [94] . Therefore, any description of these classical symptoms, together or isolated, should be regarded by any experienced clinical as non-severe malaria, regardless of their intensity, because they are not associated to increased rates of hospitalization or fatality.
In the Brazilian literature reviewed, a wide spectrum of clinical complications aside from the classical symptoms of vivax malaria was found throughout the 68 indexed and non-indexed publications, despite the low number of deaths attributed to this species in this literature sample. The major complications are addressed as follows:
World Health Organization (WHO) criterion for severe anaemia is haemoglobin below 5 g/dL in children and under 7 g/dL in adults. However the clinical manifestations due to anaemia per se are not known and to what extent it contributes to the respiratory distress associated with the hyperdynamic status of the febrile syndrome. There is scarce literature on malarial anaemia in population-based studies in Latin America, as reviewed elsewhere [95] . On top of that, major differences in Latin America are seen when the same methodology is applied. That is probably related to distinct genetic background and environmental factors, e.g. in the Amazon Basin (intense racial mixture) and in the Colombian Pacific Coast (nonmixed black population) [96] . It is not known if anaemia is as frequent among patients from Brazil as in Southeast Asian patients, where P. vivax is considered to be a disease of children because the acquisition of immunity against this species occurs much faster than for P. falciparum, in highly endemic areas [97] . In Brazil only 25% of vivax disease affects children 0-14 years of age, however severe anaemia was reported in hospitalized children and adults, needing red blood cell (RBC) transfusions [45] . A key description of anaemia in vivax malaria children in Latin America was published in Venezuela in 2006 [98] . The 'congenital malaria' in newborns from the present series of reports with severe anaemia confirms previous findings that vivax malaria has an important clinical impact in children under 3 months [99] . Non-severe anaemia, however, seems to be as frequent as 25.8% among the population of a recent occupation area in Rondônia, where hydroelectric power plants are being built [100] . The cut-off of haemoglobin under 12 g/dL as a criterion of anaemia however should be seen with scepticism because of age ranges and the lack of baseline levels of haemoglobin validated to specific populations, which makes meta-analyses susceptible to misclassification. Major confounding factors in the global analysis of anaemia are the local contributors to this haematological complication such as iron-deficiency anaemia, which was found to occur in 5.6% of a rural Amazonian population, mostly among school children and women [101] . Another important associated condition, which may interfere in the comparison between distinct populations, is the prevalence of intestinal helminthic infection. In a study performed with anaemic children, the presence of hookworms and malnutrition was cited [30] . However some controversy exists regarding this influence since in a cohort study, children with any intestinal helminth were protected from anaemia triggered by acute vivax infection [47] . In fact anti-helminthic treatment and iron supplementation reduced the haematological indexes in the population from an endemic area for malaria [102] .
No Brazilian study has addressed the concomitant diagnosis of parvovirus B19 as a contributing factor to anaemia in malaria, considering that recent evidence supports that the use of chloroquine (CQ) may stimulate viral replication in the bone marrow, worsening anaemia [103] .
Apparently pregnant women develop anaemia as a major complication in vivax infection [32, 78] , and the impact upon the concept needs further investigation. Chronic comorbidities affecting erythrocyte physiology, such as sickle cell anaemia (SCA), may be related to more severe haemolysis and severe anaemia as well [40] .
Thrombocytopaenia as defined by platelet counts under 150,000/μL seems to be very frequent among patients with vivax malaria and apparently more frequent in vivax than in falciparum patients [104] , despite not being a consensus [105] . The increase in the report of thrombocytopaenia in several reference centres could also be a reflection of a better laboratorial infrastructure. Only in recent decades in developing countries automated full blood counts included platelet count as a routine. Many studies in Brazil confirm that platelet counts are directly correlated to peripheral parasitaemia [89, 90] , but the meaning of this finding is still unknown. However, only mild bleeding is usually associated with this haematological complication in studies where detailed and systematic clinical description of the patients was made, even for severe thrombocytopaenia, which means in general platelet count under 50,000/μL [81, 89, 90] . In fact, there is no report in the whole literature of a fatal case of patient presenting exclusively with severe thrombocytopaenia, even for P. falciparum. That is probably why thrombocytopaenia, regardless of being described as a complication by WHO, is not strictly-speaking considered a severity criterion by itself [106] . What happens most of the time is that thrombocytopaenia is usually taken as a surrogate marker for DIC in settings where no specific examinations to confirm this severe complication are available, such as prothrombin activation time, D-dimers and fibrin degradation products. However, there is a disproportionate difference in the proportions of thrombocytopaenia, which is considered relatively frequent in large studies for frequency estimation [90] and of DIC, which is a rare complication, very scarcely reported in the literature associated to P. vivax infection [107, 108] . Actually, there is some coagulation cascade activation, but usually with minor impact on coagulation tests and platelet counts [83] . It is important to consider however that in areas where dengue is also endemic, as is the case of Brazil, thrombocytopaenia studies should obligatorily rule out this viral infection, which also presents a substantial percentage of thrombocytopaenia as part of its non-severe presentation [109] . In fact there are cases of co-infection already reported in the Brazilian Amazon recently [110] , but the literature poorly describes the clinical aspects of this coincidental infection [111] .
Respiratory distress is defined by oxygen saturation less than 94%, or deep breathing (acidotic breathing), or an age-stratified increased rapid respiratory rate (> 32/min in adults, > 40 in children 5-14 y, > 50 in children aged 2 mo to 5 y, and > 60 in babies less than 2 mo) [112] . However this syndromic approach does not translate any mechanism of disease and may be associated to the clinical presentation of febrile syndrome during the malarial paroxysm, severe anaemia, metabolic acidosis, lung oedema, pneumonia or acute respiratory distress syndrome (ARDS). In most of the cited Brazilian studies, there are no described criteria on how respiratory distress was defined, which makes comparisons with the general literature impossible. Sometimes imprecise clinical presentation is simply defined as pulmonary manifestations. In only one, ARDS is well characterized, comprising detailed radiological characterization and arterial gas analysis (FiO 2 /PaO 2 ) [37] . Lung oedema is usually based on clinical and radiological parameters and the effect of fluid overload is not clear for vivax infection, since only a few cases were reported so far, Brazilian cases included [39, 64, 65, 113, 114] . The impairment of respiratory symptoms after the beginning of treatment with CQ referred elsewhere [115] was not mentioned in any of the present reports, which could be due to inappropriate study design. Ruling out pneumonia is not easy because of the low frequency of positive blood cultures and due to the fact that in most of these patients with pulmonary complications empirical antibiotics are initiated as a rule. Data from the Papuan Indonesia indicate that many infants who die with P. vivax have radiological evidence of pneumonia [116] , but the specificity of radiological findings to differentiate vivaxinduced pulmonary abnormalities from pneumonia is questionable. In Mozambique, pyogenic bronchopneumonia was a common cause of respiratory distress in autopsied pregnant women with falciparum malaria, in both HIV positive and negative [117] . In the Amazon, HIV prevalence is estimated to be~1% (unpublished data), which makes opportunistic diseases less prone to impact on severe clinical complications of vivax malaria, as is the case for falciparum malaria in Africa.
This is classically the most lethal clinical complication of severe falciparum malaria and the definition is also very imprecise with a wide spectrum of possible presentations, such as: impaired consciousness or unrousable coma (Glasgow coma score ≤10 or Blantyre coma scale ≤2); prostration, i.e. generalized weakness so that the patient is unable walk or sit up without assistance; failure to feed; or multiple convulsions (more than two episodes in 24 h). Despite being infrequent in our studies, the phenomenon was also reported but not only in children [36, 86] . These reports must be very cautious in terms of ruling out other malarial complications as the cause of the neurological manifestations, such as hypoglycaemia and metabolic acidosis, but also associated infections as bacterial or viral meningoencephalitis. In India, acute intermittent porphyria was an unexpected co-morbidity associated to the neurological manifestations of patients with vivax malaria [118] . In Papua, P. vivax-associated coma was rare, occurring 23 times less frequently than that seen with falciparum malaria, and was associated with a high proportion of non-malarial causes and mixed infections detected using PCR [119] .
This complication is suspected in cases of oliguria and confirmed if serum creatinine is higher than 3.0 mg/dL. Bacterial sepsis, dehydration, shock and past history of chronic renal failure should be routinely searched in the differential diagnosis. It was also reported in the Brazilian literature [38, 48] , but in one study one case was found in a patient with arterial hypertension, what could be a triggering condition [45] . Despite not being frequent in Brazil, Plasmodium malariae is found in some scattered areas [120] , and as a potential cause of glomerulonephritis [121] , this parasite should be ruled out by molecular biology tools whenever acute renal failure is detected in a malarial patient with vivax infection, due to similarities of these two species at routine optical microscopy.
New WHO guidelines already point to hyperbilirubinaemia (total bilirubin > 3.0 mg/dL) as being a weak marker of severity, unless it is followed by any other vital organ dysfunction [106] . This finding seems to be the most frequent among children and adults with vivax disease considered as 'severe' [122, 123] . Since haemolysis is not usually as severe as to cause significant clinical jaundice, most of these patients actually have some hepatocyte necrosis as evidenced by the mild to moderate liver enzymes (AST/ALT) increase with subsequent cholestasis [124] . It was shown that icteric syndrome was a common cause of hospitalization in pregnant women with vivax malaria in Manaus [87] . It was also detected in newborns [59, 74] , which makes vivax malaria an obligatory differential diagnosis of neonatal sepsis. Jaundice in the presence of vomiting and upper abdominal pain should raise suspicion on acalculous cholecystitis, a poorly described complication apparently with good prognosis [125] . Other diseases that may evolve to an icteric syndrome may be ruled out, especially because they are also more frequent in the tropics, such as leptospirosis [34] and typhoid fever [126, 127] . Hepatitis A virus (HAV) and vivax co-infection has already been reported as cause of jaundice and high elevation of transaminases [45] . Hepatitis B virus (HBV) is also highly prevalent in Brazil, especially in the Amazon [128] and there is some evidence that P. vivax/HBV co-infection may be related to more frequent jaundice [80] and higher transaminase levels.
Algid malaria refers to the shock syndrome usually defined as circulatory collapse (systolic pressure under 70 mmHg in adults or under 50 mmHg in children) non-responsive to fluids. In the present vivax malaria reports, it was more reported most frequently among patients who died, suggesting that, as expected for this severe clinical complication, it could be regarded as a good marker of severity. However, the aetiology of this complication is still unclear even for P. falciparum. Apparently it is multifactorial and the complication should be regarded as a syndrome where cardiac dysfunction, dehydration, bleeding, adrenal insufficiency, and bacterial sepsis could all play a role [129] . A review of all malaria deaths in the USA found that 5% were due to P. vivax associated with cardiac disease [130] , which suggests cardiac dysfunction as a contributing factor to algid malaria. There is robust evidence that bacteraemia in Africa is associated with higher fatality in falciparum malaria in children [131] . Less frequently shock occurs isolated, but usually as part of multi-organ dysfunction syndrome (MODS), leading to a clinical picture suggestive of 'malaria-induced toxic shock' [132] .
Metabolic acidosis (plasma bicarbonate < 15 mmol/L) and hyperlactataemia (lactate > 5 mmol/L), which are common in severe falciparum malaria and are good predictors of fatal outcome, have never been described in vivax severe disease. In a series of children with vivax infection admitted to the ICU, metabolic acidosis is mentioned [92] , however concomitant sepsis is described in this series and specificity for malaria cannot be assumed. If one admits lactic acidosis as a consequence of hypoxia triggered by microvasculature obstruction in falciparum disease, the scarcity of data on the frequency of this phenomenon in vivax disease may simply reflect the less severe obstruction due to less cytoadhesion, as already suggested elsewhere [133, 134] . In the case of hypoglycaemia (blood glucose < 40 mg/dL), the complication has been rarely described elsewhere [123, 135] , and in only two studies in Brazil this finding was reported among children and pregnant women [32, 92] .
The impact of vivax infection upon pregnancy and the concept is less clear in Brazil and Latin America as a whole, despite robust evidence that vivax malaria causes low birth weight and maternal anaemia exists in Thailand [136] and Indonesia [137] . The burden of the infection due to this species in Brazilian pregnant women from a highly endemic area in the Amazon seems to be high [138] . Malaria anaemia in pregnant women with vivax is already known [137] and data from Brazil confirm that this is the most common complication among these women [78] . Additionally the few reports in the present series also point to low birth weight, vaginal bleeding, amniorrhexis, abortion, premature delivery, hypoglycaemia, hepatitis and jaundice as complications [31, 32, 49, 70, 87] . Hyperemesis gravidarum may superimpose to the febrile syndrome and to the gastrointestinal side effects associated to CQ in pregnant women, contributing to uncontrolled vomiting and consequent metabolic disorders. Apparently in the case of pregnancy, co-morbidities do not seem to be frequent among patients with clinical complications. Despite the need of more pathogenesis studies with the infected placenta, ultrasound studies in order to search for prognostic markers are urgently needed.
Some atypical complications are not frequently described for malaria and likewise are not classically referred as severe malaria. Rhabdomyolysis has been reported for vivax in 1993 in a patient with myoadenylate deaminase deficiency [139] ; only one case was reported in Brazil in a patient without co-morbidities [46] . Rarely, patients with vivax malaria could evolve with immune thrombocytopenic purpura (ITP) as a complication of the acute infection [33] . To confirm this diagnosis, the patient has to be followed up with persistent thrombocytopaenia for many weeks after the efficacious anti-malarial treatment and diseases, in which ITP is more frequently seen, such as HIV, should be discarded. The mechanisms involved are poorly understood. Splenomegaly is considered a typical finding in the physical examination of a patient with vivax disease, but the occurrence of spleen haematomas evolving with rupture and fatal outcome is relatively rare [41, 52, 91] despite being more frequent among this species as compared to falciparum [140] . In any case, patients with vivax malaria referring abdominal pain should be investigated for this complication as some patients may evolve with a bad prognosis if not properly managed by a surgeon.
Ocular manifestations in vivax disease apparently have no relation to cerebral malaria or bad prognosis, as is the case for falciparum [141] . Few reports have been published on vivax patients with non-severe disease and retinal haemorrhage [142] and in Brazil this fundoscopical finding was associated with hypovitaminosis A [35] . Another atypical complication, which may be more frequent than expected for vivax infection, and with outstanding impact upon the development of some emerging economies in the globe, is poor school performance which should be a surrogate marker for the intellectual impairment related to malaria [143] . Acute malnutrition has been shown to be a complication of vivax malaria in highly endemic areas [144] . In Brazil a few evidences show that malnourishment and vivax co-exist but the impact of this association is still unknown [145] . In only two studies was malnutrition referred to as a possible cause of the reported clinical complication [92, 146] . Vasculitis [66] , leukemoid reaction [76] and pleural effusion [62] as a marker of severity seems to be speculative and details of these reports do not support any in-depth analysis.
High parasite density as a marker of severity for P. vivax, as it is for P. falciparum, still needs additional studies, considering this parasite infects preferably reticulocytes. The same occurs with the presence of schizonts in peripheral blood, which is usually associated with high sequestered biomass and severity for falciparum [147] , but is still an unexplored aspect for vivax. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP) and the gradation of disease severity [48] . Super-oxide dismutase-1 (SOD-1) seems to be a powerful predictor of disease severity in individuals with different clinical presentations of vivax malaria [148] .
As soon as precise markers of severity are available, it would be possible to design studies powered to analyse the influence of the host genetics in the development of severe vivax disease. Some association between pulmonary manifestations and TNF and IL-12 polymorphisms has been attempted [79] . It has been proposed for the first time in Manaus that G6PD deficiency could protect against vivax malaria, in a cross-sectional study, based on past history of the enrolled population [149] . This protection was later confirmed in Pakistan [150] . Male hemizygotes for this deficiency also showed to be protected against severe falciparum malaria [151] . No data exist on the protection against severe vivax disease. Likewise, people with the FYA/FYB genotype presented higher susceptibility to clinical vivax malaria [152] . Since the discovery in Brazil that Duffy-negative individuals could be infected by P. vivax [153] , some speculation on the other possible invasion receptors has emerged. However, cohort studies are needed to investigate the real impact of the distinct Duffy genotypes on clinical malaria incidence, submicroscopic asymptomatic infection, malaria-triggered anaemia and lower parasitaemia, as already suggested for FYB/FYX and FYA/FYX genotypes in the Brazilian Amazon [154] .
The major advance in the study of the pathogenesis of severe vivax disease was the demonstration of P. vivaxinfected RBCs cythoadhesion on human lung endothelial cells (HLEC) and placental tissue ex vivo [134] . This cythoadhesion was obviously lower than P. falciparuminfected RBCs adhesion, but with similar stability. However the next challenge is to try to link this finding to the in vivo phenomena [155] . The increased adhesion with the addition of LPS in the P. vivax ex vivo model suggests that endothelial activation may be an enhancing event. The role of augmented platelet-derived microparticles [156] and CD4 + CD25 + FoxP3 + regulatory T cells (Tregs) cells found in vivax disease should also be investigated in severe disease. Plasma levels of TNF, IFN-γ and also IFN-γ/IL-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity [48] . Patients with severe disease also presented higher haemolysis and higher plasma concentrations of Cu/Zn SOD-1 and lower concentrations of PGE-2 and TGF-β than those with mild disease [157] . Oxidative stress was also proposed as a mechanism for thrombocytopaenia found in vivax disease [158, 159] , as well as its association with TNF [85] . Circulating immune complexes were not associated to vivax thrombocytopaenia [89] , but polymorphisms of the highly immunogenic AMA-1 were associated to platelet count in these patients [160] , suggesting that immunological mechanisms are involved in platelet destruction. In the case of anaemia, there is no correlation between the presence of anti-erythrocyte and anti-cardiolipin antibodies and the presence or intensity of this haematological finding [161] . Auto-immunity induced by secondary cryoagglutinins should be explored [54] . Erythropoiesis seems to be affected [162] , and the finding of parasites inside the bone marrow [43] stimulate the search for mechanisms of diserythropoiesis in this milieu, despite technical limitations to analyse this tissue in humans. The role of the spleen in severe disease is still unknown, as well as the role of the variant subtelomeric multigene vir family, which may influence the sequestration of infected RBCs in this organ [163] . Parasite genetics, such as MSP-1 and CSP polymorphisms, has not been shown to be associated with clinical severity [164] .
In summary, the immune response in patients with severe vivax disease has not been fully addressed in the general literature, and further approaches are needed in order to unveil immune mechanisms related to these complications.
In terms of therapy, CQ and primaquine (PQ) are still the drugs of choice for the treatment of vivax malaria in many endemic areas, Brazil included. It is important however to keep in mind that side effects of these drugs could be erroneously taken as clinical severity associated to the parasite infection. In the case of CQ, it is considered a safe drug, despite the occurrence of pruritus, which most of the time is considered to be a minor effect and rarely requires the drug withdrawal [165] . Psychosis on the other hand is a more severe complication [75, 166] , as well as cardiac arrhythmia [29] . Atypical complications of its use such as severe gastric bleeding were associated with haemophilia A [50] . In the case of PQ, tranquillity is not the same as with CQ, because PQ is able to induce metahaemoglobinaemia [42, 51] and severe haemolysis [44] in patients with G6PD deficiency. The burden of the deficiency in Brazil is poorly measured but the few data available in endemic areas for malaria has shown it to be between 3.0% [149] and 5.8% [167] among men, since the deficiency is linked to the X-chromosome. In the case of Brazil, the prescription of PQ in the abbreviated regimen (0.5 mg/kg/day for 7 days) without any routine G6PD screening may contribute to increase the frequency and severity of the side effects triggered by this drug, as confirmed by the reports of patients with blackwater fever after PQ use, including one fatal case [63, 71, 91, 92, 168] . To complicate matters, for the radical cure, the new drug under late stage clinical investigation, tafenoquine, shows no evidence that it is safer than PQ in G6PD deficient [169] .
The simultaneous occurrence of severe vivax disease and CQ-resistance in some countries has raised the question of a possible association between severity and resistance, especially for anaemia [170] . CQ resistance actually has been reported in Brazil almost at the same time as clinical severity [171, 172] , but some studies argue against that, showing that severe patients responded to CQ [45] . Added to that, reliable genetic markers of resistance are lacking [173] . Increased levels of pvmdr-1 and pvcrt-o RNA in a single severe patient with vivax malaria however paved the way to the study of gene expression in association to resistance [174] .
As suggested by the present data, 11 cases were reported in Brazilian travellers who live in the non-endemic area and occasionally go to the Amazon. Regarding the possibility of severe disease triggered by P. vivax, Travel Outpatient Clinics should emphasize to their clients the possible complications of this disease, still considered 'benign' in most of the educational folders and travellers' guides, especially because no good chemoprophylaxis against relapses related to this species is available to date. On top of that, retarded diagnosis and treatment outside the Amazon area contributes to the higher fatality rate of P. falciparum patients [18] . A similar situation could be observed for P. vivax, being this disease misdiagnosed as other febrile diseases.
Despite the increasing evidence of CQ-resistance worldwide, the Brazilian Ministry of Health still recommends CQ as the first line therapy for vivax treatment, considering that only one single study has properly shown~10% of resistance in the area of Manaus [172] . The few available efficacy studies on ACT for the treatment of vivax were reviewed recently [175] , and give good evidence for their use in vivax malaria, however, more studies are needed. Only recently the Brazilian Ministry of Health followed the WHO recommendations to manage vivax severe patients with parenteral artemisinin derivatives as if they had severe falciparum infection, considering that a submicroscopic mixed infection could be misdiagnosed in the routine TBS [106] . This recommendation was already stated by the famous Brazilian parasitologist Samuel Pessôa in his Medical Parasitology textbook, from 1967 [176] . Supportive therapy is even more neglected and there is virtually no study focusing in the clinical management of patients with severe vivax disease.
There are actually many priorities in clinical research related to vivax disease. The major ones were discussed previously. Considering that asymptomatic infections due to P. vivax are even more common in endemic areas for both species [177] , the likelihood of an asymptomatic patient becoming ill due to another microorganism is not improbable, which requests a good epidemiological characterization of the endemic area where the severe cases are being reported and systematic exclusion of mixed infections through PCR, due to the possibility of submicroscopic infection with P. falciparum. Another major priority in vivax research is the investigation of concurrent infections through systematic laboratory exclusion of the most prevalent infectious diseases in severe patients. In Figure 1 , the major research questions are addressed.
In the present systematic review, the major limitation was the fact that most of the information was retrieved from non-peer reviewed sources. However, it seems clear that vivax patients in Brazil are calling the attention of their physicians only recently. Like other infectious diseases, defining severity criteria is a major challenge. As an example, dengue fever specialists have defined 'warning signs' for dengue haemorrhagic fever, the most lethal complication of the infection due to dengue virus, which are early signs that should raise the suspicion of severe dengue but are not applied themselves to the final classification [178] as proper intervention can avoid the patient evolving to more severe stages. Sometimes in the literature potential 'warning signs' for severe vivax malaria are mistaken for severity criteria, which are those ultimately related to increased fatality. WHO severity criteria formerly developed for falciparum disease seem to apply reasonably to vivax disease as well, but there are clearly 'warning signs' that should motivate clinicians from the tropics to observe patients more closely, such as isolated thrombocytopaenia, isolated jaundice or the presence of chronic or acute co-morbidities. For example, during influenza outbreaks, the virus does not necessarily kill per se, but compromises the most vulnerable population and facilitates fatal secondary bacterial infections. The most common complications observed in the field are not necessarily the most frequently reported in the literature, sometimes biased by the uniqueness or exoticness of the cases reported. It is only after 1987 that these cases started to be reported in Brazil in indexed and nonindexed publications, which may simply parallel the increase in the absolute numbers of vivax cases in Brazil, culminating in the more frequent observation of rare clinical events triggered by this parasite. Publication bias may also impact the chronology of these complicated case reports, especially when research group leaderships based in the endemic areas start to look for clinical aspects more closely. It is noteworthy however that studies on pathogenesis must be careful when dealing with severe vivax disease as a single entity. The best approach is to study groups of patients with specific complications (e.g., severe anaemia or ARDS) in order to minimize the risk of heterogeneous groups with probable multifactor causality, including the diversity of host genetics. The amount of complications related to anti-malarial drug use is not negligible, especially primaquine. Multicentric studies using standard protocols, with the proper care of confirming mono-infection by more specific tools (e.g. PCR) and ruling out co-morbidities, are urgently needed to characterize the real spectrum of vivax disease worldwide. Tissues from deceased patients are also waited, in order to support more robust analyses of the mechanisms of death. Without that information, vaccine clinical trials against P. vivax will not be able to include among their endpoints the protection against the severe disease (essentially severe anaemia), which parallels the frequency of severe falciparum anaemia in some endemic areas. The recent discussion on malaria eradication will only succeed if the two parasites which most affect humans begin to be treated as distinct and not causing a single disease. Clinical characterization is the first step to estimate its burden and ultimately to plan any control strategy in the near future.  Protective Role of the ACE2/Ang-(1–9) Axis in Cardiovascular Remodeling Despite reduction in cardiovascular (CV) events and end-organ damage with the current pharmacologic strategies, CV disease remains the primary cause of death in the world. Pharmacological therapies based on the renin angiotensin system (RAS) blockade are used extensively for the treatment of hypertension, heart failure, and CV remodeling but in spite of their success the prevalence of end-organ damage and residual risk remain still high. Novel approaches must be discovered for a more effective treatment of residual CV remodeling and risk. The ACE2/Ang-(1–9) axis is a new and important target to counterbalance the vasoconstrictive/proliferative RAS axis. Ang-(1–9) is hydrolyzed slower than Ang-(1–7) and is able to bind the Ang II type 2 receptor. We review here the current experimental evidence suggesting that activation of the ACE2/Ang-(1–9) axis protects the heart and vessels (and possibly the kidney) from adverse cardiovascular remodeling in hypertension as well as in heart failure. All epidemiological studies show that the risk of adverse cardiovascular (CV) outcomes, such as stroke, myocardial infarction (MI), heart failure (HF), and kidney disease [1] , increase progressively with increasing blood pressure (BP). On the other hand, clinical trials demonstrate that lowering BP reduces such risks [1] . All antihypertensive medications lower BP, but specific drug classes display effects beyond BP reduction (pleiotropic effects) that might contribute to cardiovascular risk reduction.
Remodeling of the cardiovascular structure occurs in response, not only to changes in BP and flow, but also to modifications in the neurohormonal environment, in which the rennin-angiotensin-aldosterone system (RAAS) exerts a most predominant influence [2] .
The RAAS is a major regulator of BP [3, 4] . In addition, the RAAS has a role in the vascular response to injury and inflammation [4] . Chronic RAAS activation, through both angiotensin (Ang) II and aldosterone, leads to hypertension and perpetuates a cascade of proinflammatory, prothrombotic, and atherogenic effects associated with endorgan damage [3, 4] . Based on these facts, several drugs have been developed that work by (a) reduction of Ang II levels, (b) inhibition of the Ang II type 1 receptor (AT1R), (c) blockade of the aldosterone receptor, and (d) renin receptor blockade [5, 6] . During the last 25 years several clinical trials have shown the benefits with these drugs that inhibit the RAAS with regard to BP reduction, regression of cardiac hypertrophy, prevention of kidney damage and reduction of cardiovascular morbidity reduction in hypertensive patients. Besides, with most of these RAAS blockers, quality of life as well as survival has been significantly improved in patients with heart failure. Consequently, the RAAS is currently a main therapeutic target in hypertension treatment [3, 4] . Aggressive BP control improves outcomes in patients with CV disease, stroke, and nephropathy and might have beneficial effects beyond BP lowering [7] .
Despite the reduction of CV events and end-organ damage with the current pharmacologic strategies, CV disease remains the primary cause of death in the world, and more than 94,000 Americans annually experience progression to end-stage renal disease (ESRD). As population ages, the proportion affected by end-organ damage is expected to grow [8] . Thus, it is most relevant to find new molecules 2 International Journal of Hypertension in order to prevent and reduce hypertension as well as pathologic CV and kidney remodeling and dysfunction. In this regard, activation of the new ACE2/Ang-(1-9) pathway seems to counterbalance the damage due to the RAAS system activation.
We review here the current experimental evidence suggesting that activation of the ACE2/Ang-(1-9) pathway protects the heart and vessels (and possibly the kidney) from adverse cardiovascular remodeling in hypertension as well as in heart failure.
The discovery of angiotensin-converting enzyme homologue, ACE2, added further complexity to the main axis of the RAAS, in which Ang II and its forming enzyme ACE play major roles [9, 10] . A growing body of evidence points to a possible promising role for this new member of the RAAS by opposing to the effects of the main axis [11, 12] . ACE2 has dramatically changed the direction of cardiovascular and renal research in view of the pivotal role of this enzyme in the regulation of the RAAS [12, 13] .
ACE2 is the newest member of the RAAS and shares approximately 40% similarity with the somatic form of ACE [9, 10] . ACE2 is a membrane-bound carboxypeptidase and its cellular and tissue distribution is different from that of ACE. While ACE is expressed in the endothelium throughout the vasculature, ACE2 is distributed in tissues with the most abundant expression in heart, kidney, lung, small intestine, and testis [14] . ACE2 can be released into the circulation and urine by shedding [15] . Tumor necrosis factoralpha-converting enzyme (TACE/ADAM17) is the sheddase responsible for the ectodomain cleavage and shedding of ACE2 [16] .
However, normal ACE2 enzymatic activity in plasma is very low, probably due to the presence of an endogenous inhibitor [17] [18] [19] . ACE2 is different from ACE in both substrate specificity and functions [9, 20, 21] . ACE2 can form (a) Ang-(1-7) through hydrolysis of Ang II and (b) Ang-(1-9) through hydrolysis of Ang I. This last reaction is negligibly slow and is several hundred times slower than Ang II hydrolysis by ACE2 to form Ang(1-7)-a vasodepressor peptide counterbalancing the vasopressor effect of Ang II [20, 21] . Ang-(1-7) can be subsequently converted to Ang-(1-5) by ACE [9, 20] or by neutral endopeptidases [9] , while Ang-(1-9) may be converted to Ang-(1-7) by ACE [9] . There is little evidence proving the existence of alternative hydrolysis of Ang-(1-9) to Ang II in some tissues. Drummer et al. [22] proved that homogenates of rat kidney, and in a lesser extent of lung, convert Ang-(1-9) to Ang II due to an ACEindependent aminopeptidase and N-like carboxypeptidase. Singh et al. [23] confirmed that the pathway Ang I-Ang-(1-9)-Ang II really exists in glomeruli of streptozotocininduced diabetes mellitus rats. Moreover, in human heart tissue the main products of Ang I degradation are both Ang-(1-9) and Ang II generated by heart chymase, ACE and a poorly identified carboxypeptidase A [24] . Although the data proving the existence of alternative pathways of Ang II production, in clinical practice we can still block only ACE or AT1R.
ACE2 does not act on bradykinin metabolism and its activity is not inhibited by classic ACE inhibitors (ACEIs) [9] . Thus it has been proposed that ACE2 activity may counterbalance the effects of ACE by preventing the accumulation of Ang II in tissues where both ACE2 and ACE are expressed [25, 26] . ACE2 has several biological substrates and it is considered a multifunctional enzyme. Acting as a monocarboxypeptidase, it cleaves several other non-RAAS peptides which have roles in maintaining cardiovascular homeostasis such as (des-Arg9)-bradykinin, a member of the kininogen-kinin system [13] . (des-Arg9)-Bradykinin is formed from bradkinin by the action of carboxypeptidases and is an agonist of the B1 receptor, which is induced after tissue injury [27] . Bradykinin, a vasodilator which acts through the B2 receptor, is produced from its precursor kininogen by kallikrein and is degraded by ACE [13] . While, degradation of bradykinin by ACE is known to be an important aspect of BP regulation, the significance of the degradation of (des-Arg9)-bradykinin by ACE2 remains to be established. In addition to (des-Arg9)-bradykinin, ACE2 is also able to degrade apelin-13, a peptide proposed to cause vasoconstriction and known to regulate fluid homeostasis, and other non-RAAS peptides such as kinetensin, dynorphin A and neurotensin [20] .
For a long time, Ang-(1-7) was thought to be devoid of biological activity, in spite of early reports on biological effects [28] . The importance of Ang-(1-7) was emphasized by the discovery of ACE2. Ang-(1-7) has been shown to release vasopressin as effectively as Ang II from neurohypophyseal explants [28] and to have actions opposing those of Ang II, namely vasodilation, antitrophic effects and implications of vasodilation caused by bradykinin [29, 30] .
Several experiments suggest an important interaction between Ang-(1-7) and prostaglandin-bradykinin-nitric oxide (NO) systems. Ang-(1-7) binds to the Mas receptor (G protein-coupled receptor) which mediates vasodilating and antiproliferative actions of this peptide [31] . The Mas receptor can hetero-oligomerize with the AT1 receptor and acts as a physiological antagonist of Ang II [32] . Studies revealed that Ang-(1-7) activated endothelial nitric oxide synthase and NO production via Akt-dependent pathways [33] . Furthermore, Tallant et al. [34] showed that the presence of an antisense probe directed against Mas abolished the Ang-(1-7)-induced inhibition of protein synthesis in cardiomyocytes. This study also revealed that Ang-(1-7) decreased serum-stimulated ERK1/ERK2 mitogen-activated protein kinase activity, a response that was blocked by D-Ala 7-Ang-(1-7), an antagonist of Mas receptor.
Ang II binds with high affinity to two different receptor subtypes-AT1R and AT2R-which are members of the seven-transmembrane-domain G-protein-coupled receptors (GPCR) superfamily, through Gq and Gi, respectively [35] . Whereas the AT1R mediates most of the recognized actions of Ang II, it appears that the AT2R opposes, in part, to the effects mediated by the AT1R. As the AT2R is expressed in adult tissues in smaller amounts than the AT1R, the actions and cell signaling of AT2R have been less well characterized International Journal of Hypertension 3 than those of AT1R [36] [37] [38] . Current knowledge suggests that AT2R stimulation mediates vasodilation, antigrowth, proapoptotic and antiinflammatory effects [39, 40] . Hence, the AT2R can modulate cardiovascular remodeling as well as progression of atherosclerosis.
AT2R stimulation activates the NO-cGMP-dependent pathway [41] . This occurs either directly or indirectly through bradykinin or by increased endothelial NOS activity or expression. AT2R activation is associated with phosphorylation of JNK, PTPs, IκBα (inhibitor of NF-κB), and the transcription factor ATF2, and dephosphorylation of p38MAPK, ERK1/2, and STAT3, which are linked to antiproliferative and antiinflammatory effects and apoptosis [38, [42] [43] [44] . AT2R may induce relaxation by opening large-conductance Ca 2+ -activated K + channels (BKCa) [45] and by negative regulation of the vascular Rho A/Rho kinase pathway. The AT2R also enhances the activity of tyrosine phosphatases and vanadate-sensitive phosphatases MKP1 (DUSP1), SHP1 (PTPN6) and PP2A [46, 47] .
There is little information in the literature with respect to Ang-(1-9) probably because this peptide was initially thought to be active only after conversion to Ang-(1-7). Ang-(1-9) can be generated by several carboxypeptidase-type enzymes including ACE2 or cathepsin A [48, 49] . Ang-(1-9) is present in healthy volunteers, in patients or in animals treated with ACE inhibitors (ACEIs) or AT1 receptor blockers (ARBs) [50] [51] [52] , and its circulating levels are increased by pathological conditions (i.e., early after MI) [51] . However, very little is currently known about Ang-(1-9) biological effects [50, 53] .
Initial studies showed that incubation of Chinese hamster ovary cells (CHO) with Ang-(1-9) potentiated the release of arachidonic acid by [Hyp 3 Tyr(Me) 8 ]BK, elevated [Ca 2 ]i and also resensitized the B2 receptor desensitized by BK [48] . At the same time, Jackman et al. [54] showed in CHO cells and in human pulmonary endothelial cells that Ang-(1-9) was significantly more active than Ang-(1-7) enhancing the effect of an ACE-resistant bradykinin analogue on the B2 receptor and that Ang-(1-9) also augmented arachidonic acid and NO release by kinin [54] .
Some studies have suggested that Ang-(1-9) may be an endogenous inhibitor of ACE. Donoghue et al. [9] proposed that Ang-(1-9) is a competitive inhibitor of ACE because it is by itselfan ACE substrate. Under conditions of ACE inhibition, such as after long-term administration of an ACEI in rats, Ang-(1-9) levels increased in plasma and kidney [50, 53] . This increase in Ang-(1-9) steady-state levels could be due to decreased catabolism of Ang-(1-9) by ACE. Conversely, the increased levels of Ang-(1-9) could be due to increased production by ACE2 as a result of increased availability of Ang I substrate. These results indicate that an alternate pathway of Ang I metabolism by ACE2 exists and that this pathway may be amplified in the presence of ACE inhibitors.
To determine whether Ang-(1-9) is active per se or it becomes active only after conversion to Ang-(1-7), Chen et al. [55] examined the metabolism of Ang I, Ang-(1-9) and Ang-(1-7) in stably transfected CHO cells that express human ACE and human bradykinin B2 receptors coupled to green fluorescent protein (B2GFP). They found that Ang-(1-9) was hydrolyzed 18 times slower than Ang I and 30% slower than Ang-(1-7). Ang-(1-9) inhibited ACE and it resensitized the desensitized B2GFP receptors, independently of ACE inhibition [55] . This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B2 receptors. They concluded that Ang-(1-9) enhanced bradykinin activity, probably by acting as an endogenous allosteric modifier of the ACE and B2 receptor complex. Therefore, when ACE inhibitors block conversion of Ang I, other enzymes like ACE2 can still release Ang I metabolites like Ang-(1-9) and enhance the efficacy of ACEIs.
Recently, Flores-Muñoz et al. [56] using radioligand binding assays observed that Ang-(1-9) is able to bind the Ang II type 2 receptor (AT2R) (pKi = 6.28 ± 0.1). They demonstrated that Ang-(1-9) and not Ang II, affected hypertrophy through the AT2R, as PD123319 (an AT2 receptor blocker) did not alter Ang II-mediated growth but did block the effects of Ang-(1-9). Despite having ∼100fold lower affinity than Ang II for the AT2R [57] , the selective AT2R activity of Ang-(1-9) is not inconsistent with current pharmacological models of G protein-coupled receptor signalling and activation. Indeed, the concept of functional selectivity, where individual receptor ligands have the capacity to selectively stabilize conformations which lead to distinct signalling outcomes [57] [58] [59] , is supported by a previous study in which the critical amino acids and the mode of binding of ligands at the AT1R and AT2R were investigated [60] . While agonist activation of the AT1R was particularly sensitive to peptide modifications that disrupted contact points between Ang II and its receptor, substitutions within Ang II were far better tolerated by the AT2R [60] . The AT2R exists in a relaxed conformation and Ang II therefore binds to multiple indistinct contact points [60] . Since Ang-(1-9) contains the entire Ang II sequence plus a C-terminal histidine, these observations indicate that this difference may stabilize the AT2R in a conformation able to counteract hypertrophic signalling in cardiomyocytes. Flores-Muñoz et al. [56] did not observe functional competition between Ang II and Ang-(1-9) at the AT2R and they concluded that that Ang-(1-9) is able to antagonize Ang II signalling in cardiomyocytes selectively via the AT2R, highlighting that Ang-(1-9), along with Ang-(1-7), makes up part of the counter-regulatory arm of the RAS. What remains to be determined is the downstream signalling effects from Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) . Preliminary studies indicate that the classical pathways via PKC translocation and ERK1/2 activation [61] [62] [63] are not different between Ang II-, Ang-(1-7)-and Ang-(1-9) stimulated cells. Since the downstream signalling from the AT2R is unclear at present, future studies will be required to establish these mechanisms.
Crackower et al. [64] were the first to test ACE2 as the gene underlying the blood pressure locus on the X chromosome. They showed reduced expression of renal ACE2 in the salt-sensitive Sabra hypertensive rat compared with the normotensive rat. Both spontaneously hypertensive rats (SHR) and spontaneously hypertensive stroke-prone rats (SHRSP) rats showed reduced renal ACE2 protein levels compared with the normotensive Sabra and Wistar Kyoto (WKY) strains. Two other groups confirmed some of these findings showing lower renal ACE2 mRNA, protein, and activity in the SHR compared to WKY rats [65, 66] . However, other investigators were unable to detect any difference in renal ACE2 mRNA, protein, and activity between adult hypertensive rats and their normotensive controls [67] . Rentzsch et al. [68] , assessed in SHRSP (that display reduced ACE2 mRNA and protein expression compared with control animals in the kidney) the role of ACE2 in the pathogenesis of hypertension. They generated transgenic rats on a SHRSP genetic background expressing the human ACE2 in vascular smooth muscle cells by the use of the SM22 promoter, called SHRSP-ACE2. In these transgenic rats, vascular smooth muscle cells (VSMC) expression of human ACE2 was confirmed by RNase protection, real-time RT-PCR, and ACE2 activity assays. Transgene ACE2 expression leads to significantly increased circulating levels of Ang- (1-7) , a prominent product of ACE2. Mean arterial blood pressure was reduced in SHRSP-ACE2 compared to SHRSP rats, and the vasoconstrictive response to intraarterial administration of Ang II was attenuated. The latter effect was abolished by previous administration of an ACE2 inhibitor. To evaluate the endothelial function in vivo, endotheliumdependent and endothelium-independent agents such as acetylcholine and sodium nitroprusside, respectively, were applied to the descending thoracic aorta and blood pressure was monitored. Endothelial function turned out to be significantly improved in SHRSP-ACE2 rats compared to SHRSP. These data indicate that vascular ACE2 overexpression in SHRSP reduces hypertension probably by local Ang II degradation and by improving endothelial function [68] .
A target gene therapy strategy holds significant potential to translate the available fundamental research of ACE2 into therapeutics. In fact, initial animal experiments have been extremely encouraging. For example, in SHR, viralmediated ACE2 overexpression in the heart decreased high BP [69] . This strategy also preserved cardiac function, as well as left ventricular wall motion and contractility, and attenuated left ventricular wall thinning induced by myocardial infarction [70] . ACE2 overexpression in the rostral ventrolateral medulla causes significant decreases in BP and heart rate (HR) [71] .
Compared with ACEIs and ARBs, the targeting of ACE2 has the following potential therapeutic advantages, first, it degradates both Ang I to generate Ang-(1-9) and Ang II to generate Ang- (1-7) . Thus, targeting ACE2 would not only produce the antihypertrophic peptide Ang-(1-9) [52] and the vasoprotective/antiproliferative peptide Ang-(1-7) [72] [73] [74] , but would also influence the vasoconstrictive/proliferative effects of the ACE/Ang II/AT1R axis [75] . Second, it is a multifunctional enzyme with many biologically active substrates [9, 20] . Third, unlike ARB/ACEI therapy, ACE2 is an endogenous regulator of the RAS [75] . Fourth, it is a part of the vasodilatory/antiproliferative axis of the RAS [20] and fifth, although treatment with ACEIs or ARBs indirectly increases ACE2 expression, direct activation of this enzyme could result in a better outcome in cardiovascular diseases [68, 75] . Thus, the activation of the ACE2 axis may be a novel therapeutic strategy in hypertension.
So far, all attention has been focused on Ang- (1-7) , that opposes the pressor, proliferative, profibrotic, and prothrombotic actions mediated by Ang II [76] . Experimental and clinical studies have demonstrated a role for the Ang-(1-7)/ACE2/Mas axis in the evolution of hypertension, the regulation of cardiovascular and renal function, and the progression of cardiovascular and renal disease including diabetic nephropathy [77] . Additional evidence suggests that a reduction in the expression and activity of this vasodepressor component may be a critical factor in mediating the progression of cardiovascular and renal disease. These findings support a role for the Ang-(1-7)/ACE2/Mas axis and, in particular, on its putative role as an ACE-Ang II-AT1 receptor counter-regulatory axis within the RAS [76, 77] .
Recently, the alternative angiotensin peptide, Ang-(1-9) has shown relevant biological functions. Ocaranza et al. [51] have observed increased ACE2 activity and Ang-(1-9) plasma levels in MI and sham rats treated with enalapril for 8 weeks while circulating Ang-(1-7) levels did not change in any phase after MI [51] (Figure 1 ). These findings support the hypothesis that, in this second arm of the RAS, ACE2 through Ang-(1-9) instead of Ang-(1-7), could act as a counterregulator of the first arm, where ACE catalyzes the formation of Ang II.
Besides, in experimental hypertension (DOCA salt model) and in normotensive sham animals, RhoA/Rho-kinase inhibition (a signaling pathway that participates in pathological cardiovascular and renal remodeling and also in blood pressure regulation) by fasudil reduced BP and increased vascular and plasma ACE2 enzymatic activity. At the same time, fasudil reduced Ang II and increased Ang-(1-9) plasma levels ( Figure 2 ) [78] . No modifications were observed here in Ang-(1-7) levels despite increased ACE2 levels with RhoA/Rho-kinase inhibition [78] . Thus, RhoA/Rho-kinase inhibition, by increasing eNOS and/or by reducing both ACE and Ang II, does not activate the Ang-(1-7) pathway. This novel effect of RhoA/Rho-kinase inhibition on both ACE2 expression and Ang-(1-9) levels might additionally contribute to the antihypertensive effects of RhoA/Rhokinase inhibitors. Besides, these results strongly suggest that in this experimental model, hypertension is more dependent on ACE2 and Ang-(1-9) levels than on ACE and Ang II levels. Therefore, this second RAAS axis through ACE2 and Ang-(1-9) could be an important target for the treatment of hypertension.
International Journal of Hypertension 5 Figure 1 : Plasma levels of Ang-(1-7), Ang-(1-9) and Ang II in rats with myocardial infarction treated with the ACE inhibitor enalapril (8 weeks). Increased plasma levels of Ang-(1-9) were observed in rats with myocardial infarction treated with the ACE inhibitor enalapril. Myocardial infarction was induced by coronary artery ligation. Data are presented as mean ± SEM (n = 12/group). AMI: acute myocardial infarction, E: enalapril. * P < 0.05 compared to both Sham and untreated myocardial infarction groups; * * P < 0.05 compared to both Sham and enalapril-treated myocardial infarction groups. (adapted with permission from [51] ).
The vascular wall is continuously exposed to hemodynamic forces such as the luminal pressure and shear stress. Changes in these forces, either physiological or pathological, lead to functional and/or structural alterations of the vascular wall [79] . Acute changes in hemodynamic forces can modify vessel diameter. Chronic changes in hemodynamic forces result in structural alterations of the vessel wall, indicated by changes in wall diameter and thickness. In addition, changes in vascular structure are not solely determined by hemodynamic forces [80] , but also by inflammatory responses and changes in extracellular matrix components [81] . Structural changes of the medial layer of the vascular wall during hypertension are termed "eutrophic remodeling" [82] and subsequently translate to other vascular pathologies. This involves an inward encroachment of the arterial wall thereby, reducing the diameter of the lumen [83] . Several RAAS components are involved in neointimal formation after vascular endothelial damage [84] . In particular, Rakugi et al. [85] observed that vascular endothelial damage results in the induction of vascular ACE. Their results suggested that inhibition of vascular ACE might be critical in the prevention of restenosis after balloon injury. Patients with previously untreated essential hypertension and eutrophic inward remodeling appears to respond to antihypertensive medication. Reduction in BP with drugs that block the RAAS such as ACEIs [86] [87] [88] or ARBs [86, 87, 89] and calcium channel antagonists [90] are able to reverse the eutrophic inward remodeling [88] .
The protein and mRNA of ACE2 are expressed in human coronary arteries and arterioles and the vasa vasorum of most organs [9, 91] . Recently, ACE2 expression has also been (1-7) , Ang-(1-9) and Ang II in DOCA salt hypertensive rats treated with the Rho kinase inhibitor fasudil. Increased plasma levels of Ang-(1-9) were observed in DOCA salt hypertensive rats treated with the Rho kinase inhibitor fasudil. Fasudil (100 mh/kg/day) by gavage was administered during 3 weeks, starting on the third week after DOCA administration. Data are presented as mean ± SEM (n = 8-11/group). DOCA: deoxycorticosterone, F: fasudil. * P < 0.05 compared to both Sham and untreated DOCA groups (adapted with permission from [78] ). observed in the large conduit arteries (aorta and carotid) in the HR [92] . ACE2 localizes preferentially in endothelial cells and arterial smooth muscle cells (SMCs) [9, 91] . As for the role of ACE2 in vascular remodeling, the effect of ACE2 on neointima formation has not yet been studied, but Ang-(1-7) infusion after balloon-catheter injury of the rat carotid artery reduced neointima formation [93] . This effect was probably mediated by its inhibition of vascular SMC proliferation [94] . In hypertensive animal models, ACE2 mRNA and protein were associated with immunoreactive Ang-(1-7) in the large conduit arteries of SHRs. Treatment with an ARB induced a fivefold increase in ACE2 mRNA and was associated with a significant increase in aortic Ang-(1-7) protein expression. This effect was associated with a decrease in aortic medial thickness, suggesting that this may be a protective mechanism in the prevention of cardiovascular events during hypertension [94] . Igase et al. [95] showed that ACE2 protein is expressed not only in the media of the carotid artery but also in the neointima of the ballooninjured carotid artery in SHR. The increase in ACE2 protein expression in the neointima following exposure of the rats to an ARB compared to vehicle was associated with a reduction in neointima thickness. These results lead to the hypothesis that there is a strong correlation between the increase in ACE2 protein in the injured carotid artery of SHR and vascular remodeling during blockade of Ang II receptors [95] .
There is known the prothrombotic effect of Ang II [96, 97] and the antithrombotic action of Ang-(1-7) [98] in renovascular hypertensive rats. Thus, in this context, the question arises whether Ang-(1-9) effects are similar to Ang II or to Ang-(1-7) in in vivo conditions. Kramkowski et al. [99] described that Ang-(1-9) enhances electrically stimulated thrombosis in rats and that this effect was abolished by losartan-an antagonist of the AT1 receptor. The prothrombotic activity of Ang-(1-9) was accompanied by the enhancement of ex vivo platelet aggregation and in vitro Ang-(1-9) increased platelet aggregation. However, there are some points in this paper that should be clarified. First, thrombus formation was initiated by electrical stimulation producing arterial injury that is unrelated to a clinical situation. Second, the prothrombotic effect of Ang-(1-9) was much weaker, to the prothrombotic action of Ang II [96, 97] . Third, Ang-(1-9) slightly increased platelet aggregation in in vitro conditions.
On the contrary Ocaranza et al. [78] showed that by inhibiting the RhoA/Rho-kinase pathway with fasudil, gene expression and enzymatic ACE activity and plasma levels of Ang II were reduced ( Figure 2 ) and whereas aortic gene expression and ACE2 activity were importantly increased. Simultaneously, plasma levels of Ang-(1-9) (Figure 2 ), mRNA eNOS levels increased and the aortic overexpression of the remodeling promotion proteins TGF-β1, PAI-1, and MCP-1 as well as the increased aortic NADPH oxidase activity and O 2− production were reduced, as a consequence of direct RhoA/Rho-kinase inhibition [100] . This novel effect of RhoA/Rho-kinase inhibition on ACE2 gene expression, enzymatic activity, and Ang-(1-9) levels might additionally contribute to its benefits in hypertension, atherosclerosis, and in cardiovascular and renal pathologic remodeling. This is the first observation concerning a pharmacologic ACE2 and Ang-(1-9) levels activator, both in normotensive and in hypertensive animals, one of the most interesting findings of that study (Figure 2 ). Additionally, in experimental hypertension, direct RhoA/Rho-kinase inhibition also normalizes overexpression of genes that promote vascular remodeling. Interestingly, the observed changes in ACE/ACE2 and in Ang-(1-9) levels were present only during fasudil treatment both in sham and in the DOCA hypertensive rats [78] . Thus, vascular remodeling could be more dependent on the tissue ACE2/Ang-(1-9) axis than on Ang-(1-7) levels in normotensive as well as in hypertensive rats.
In vessels, new members of the RAS have been detected, including ACE2, Ang-(1-7) and Mas. Vascular ACE2 is functionally active and generates Ang-(1-7) from Ang II. Ang-(1-7) is found in the endothelium and vascular wall [101] [102] [103] and immunohistochemical staining shows abundant presence in aortic perivascular adventitial tissue [104, 105] . Ang- (1-7) , by binding to receptor Mas on endothelial cells, opposes Ang II actions by mediating vasodilation, growthinhibition, antiinflammatory responses, antiarrhythmogenic and antithrombotic effects [33, 68] through NOS-derived NO production, activation of protein tyrosine phosphatases, reduced MAPK activation and inhibition of NADPH oxidase-derived generation of reactive oxygen species (ROS) [106, 107] . Overexpression of ACE2 in the vascular wall of SHR is associated with improved endothelial function and attenuated development of hypertension [68] . Ang-(1-7)-Mas can hetero-oligomerize with AT1R, thereby inhibiting Ang II actions. The ACE2/Ang-(1-7)-Mas axis is now considered as a counter-regulatory system to the ACE-Ang II-AT1R axis in the vasculature [107] , although some evidence indicates that Ang-(1-7) may also promote fibrosis and inflammation in certain conditions [108, 109] .
After myocardial injury or in response to chronically increased hemodynamic load, cardiac mass increases as a result of cardiomyocyte hypertrophy and ventricular wall thickening. Initially these changes are compensatory mechanisms which help to maintain ejection performance and heart function. With continued hemodynamic overload the heart becomes dilated and its walls thinner, resulting in a geometry that contributes to systolic dysfunction by increasing wall stress [110] . At the cellular level, cardiac myocytes increase in size (hypertrophy), rearrange within the myocardial matrix (cell slippage), and die, to be replaced by fibrous tissue, which include fibroblasts and collagen. These changes are collectively referred to as "remodeling" [111] . Cardiac remodeling has been consistently associated with an impaired prognosis in patients with hypertension, MI and chronic heart failure (CHF) [112] . Despite recent advances in our understanding of the ACE2/Ang-(1-7)/axis, the functional role of ACE2 in the heart is somewhat controversial. Crackower et al. [64] originally reported a progressive reduction in LV contractile function in ACE2-null mice without significant changes in fibrosis, left ventricular and cardiac myocyte hypertrophy, or in mean arterial pressure [64] . Interestingly, whereas plasma and tissue levels of Ang II were increased, a decrease in blood pressure was only observed in 6-month-old male ACE−/− homozygote mice but not in age-matched females or 3month-old males. Conversely, Gurley et al. [113] reported that ACE2 deletion enhanced the susceptibility to Ang IIinduced hypertension but had no effect on cardiac structure or function [113] . Huentelman et al. [114] showed that the ACE2 overexpression protects the heart from Ang II-induced hypertrophy and fibrosis. More recently, in SHR hypertensive rats Díez-Freire et al. by using lentiviral-based ACE2 gene transfer, attenuated cardiac fibrosis and hypertrophy [70] and also improved LV and remodeling after experimental MI [115] . Finally, Yamamoto et al. [116] reported that ACE2 deletion exacerbated pressure overload-induced cardiac dysfunction and remodeling that was associated with increased intracardiac Ang II levels and AT1R activation. The reasons for these discrepancies seem to be: (a) the genetic background of the mice used for ACE2 gene deletion [113] , (b) global versus tissue-specific ACE2 manipulation, or (c) the cardiac responses were monitored under basal or pathophysiological conditions.
In MI Ocaranza et al. [51] observed that (a) circulating and LV enzymatic activities of ACE2 were downregulated in the long-term phase of LV dysfunction in rats, (b) these effects were prevented by the conventional ACE inhibitor enalapril, (c) plasma Ang-(1-9) levels were significantly increased when MI rats or sham-operated rats were treated with enalapril for 8 weeks but circulating Ang-(1-7) levels did not change at that time ( Figure 1) Ang-(1-9) Figure 3 : Signaling events and cellular effects induced by Ang II via AT1R and opposing effects of Ang-(1-9) acting through AT2R. Proposed Ang-(1-9)-dependent mechanisms that antagonize the cardiovascular remodeling effects of Ang II. ACE2 can directly cleave Ang I to form Ang- (1) (2) (3) (4) (5) (6) (7) (8) (9) . This peptide activates the AT2R to initiate signaling pathways that antagonize AT1R-mediated tyrosine kinase cascades. In this simplified scenario, Ang-(1-9) increases SHP-1 tyrosine phosphatase activity to inactivate src-dependent signaling. AT2R activation also acts other pathways such as NO-AKT. AT1R: Ang II type 1 receptor; AT2R: Ang II type 2 receptor; ERK1/2: extracellular signal-regulated kinase 1/2; JAK: Janus-activated kinase; MAPK: mitogen-activated protein kinase; p38: p38 MAPK; PKC: protein kinase C; STAT: signal transducer and activator of transcription; NO: nitric oxide; SHP-1: protein tyrosine phosphatase SH2 domain-containing phosphatase 1; MEK: mitogen/ERK kinase. Solid arrows indicates activation broken arrows indicates inactivation. findings, it was proposed in this model of HF, that Ang-(1-9) rather than Ang-(1-7) acts as a counterregulator of Ang II [51] .
Recently, in MI rats randomized to receive either vehicle, the ACEI enalapril, or the ARB candesartan for 8 weeks, Ocaranza et al. [52] observed that both drugs prevented LVH and increased plasma Ang-(1-9) levels by several folds. Ang-(1-9) levels correlated negatively with different LVH markers with or without adjustment for BP reduction. This effect was specific as neither Ang-(1-7), Ang II nor bradykinins were correlated with LVH. Chronic administration of Ang-(1-9) to MI rats by osmotic minipumps versus vehicle for two weeks decreased plasma Ang II levels, inhibited ACE activity and also prevented cardiac myocyte hypertrophy. Because there are in vitro evidences that the incubation of Ang-(1-9) with ACE generates Ang-(1-7) [9] , and Ang-(1-7) negatively regulates hypertrophy [34, 117] , the authors used the Ang-(1-7) receptor blocker A779 to investigate whether Ang-(1-7) could mediate the effects of Ang-(1-9). Even though A779 was bioactive, with significant increase in circulating Ang-(1-7) levels by 2.7 fold, this compound did not modify the Ang-(1-9)-dependent suppression of cardiac myocytes hypertrophy induced by MI [52] . In in vitro experiments with cardiac myocytes incubated with norepinephrine (10 μM) or with IGF-1 (10 nM), Ang-(1-9) also prevented hypertrophy and this effect was not modified by the coincubation with Ang-(1-9) and A779 [52] .
To further understand the role of Ang-(1-9) compared to Ang-(1-7) in cardiomyocyte hypertrophy, Flores-Muñoz et al. [56] studied Ang-(1-9) effects in rat neonatal H9c2 and in rabbit left ventricular cardiomyocytes. Cardiomyocyte hypertrophy was stimulated with Ang II or vasopressin, significantly increasing cell size by approximately 1.2-fold as well as stimulating expression of the hypertrophy gene markers atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain and myosin light chain (2-to 5fold). Both Ang-(1-9) and Ang-(1-7) were able to block hypertrophy induced by either agonist. The effects of Ang-1-9) were not inhibited by captopril, supporting previous evidence that Ang-(1-9) acts independently of Ang-(1-7). The authors investigated receptor signalling via angiotensin type 1 and type 2 receptors (AT1R, AT2R) and Mas. The AT1R antagonist losartan blocked Ang II-induced, but not vasopressin-induced, hypertrophy. Losartan did not block the antihypertrophic effects of Ang-(1-9), or Ang-(1-7) on vasopressin-stimulated cardiomyocytes. The Mas antagonist A779 efficiently blocked the antihypertrophic effects of Ang-(1-7), without affecting Ang-(1-9). Furthermore, Ang-(1-7) activity was also inhibited in the presence of the bradykinin type 2 receptor antagonist HOE140, without affecting Ang-(1-9). Moreover, Flores-Muñoz et al. [56] observed that the AT2R antagonist PD123,319 abolished the antihypertrophic effects of Ang-(1-9), without affecting Ang-(1-7), suggesting Ang-(1-9) signals via the AT2R. Radioligand binding assays 8 International Journal of Hypertension demonstrated that Ang-(1-9) was able to bind the AT2R (pKi = 6.28 ± 0.1). The data indicate that ACE2/Ang-(1-9) axis, acting as a counterregulator of Ang II, is an effective, and possibly direct novel anticardiac hypertrophy axis.
Pharmacological treatments based on the RAS blockade are used extensively for the treatment of hypertension and CV remodeling. However, in spite of their success in pharmacological blockade of the RAS, the prevalence of end-organ damage has risen steadily in the last several decades. These observations indicate that novel and innovative approaches must be used in an attempt to promote a more effective treatment for the residual CV remodeling. In this environment, the ACE2/Ang-(1-9) axis is an important target, that is critical in tipping the balance of vasoconstrictive/proliferative to vasodilatory/antiproliferative axis of the RAS. Conceptually, the ACE2/Ang-(1-9)/AT2 axis balances the adverse effects of the ACE-Ang II-AT1 receptor axis ( Figure 3 ). Accumulating evidence suggests that ACE2 expression and Ang-(1-9) levels are altered in diastolic and systolic dysfunction and remodeling and the activation of the ACE2/Ang-(1-9) axis protects the heart and vessels from cardiovascular remodeling. In conclusion, the noncanonical RAS arm has new biological effector Ang-(1-9) to counterregulate the classical RAS. Viral Proteins Acquired from a Host Converge to Simplified Domain Architectures The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for manipulation of viral infection. Many studies, mainly from bacteria and unicellular eukaryotes, focus on the exchange of genetic material between viruses and cellular hosts. Sequences are best studied through their structural and functional domains [1, 2, 3, 4, 5] . The evolution of domains is a significant force for shaping the proteins along the tree of life. Sequence exchange between genomes within and between superkingdoms is evident from the appearance of a domain in a particular phylogenetic branch [6] . The contribution of horizontal gene transfer is not limited to bacteria but has occurred across distant species [3] . For example, some signaling domains in bacteria are the consequence of a horizontal gene transfer [7] .
The viruses are parasitic agents that maintain an intimacy with their host cells. Consequently, an extensive horizontal evolution [8] is associated with the viral life cycle. The lack of similarity of viral proteins (e.g., capsid proteins) with any cellular organisms is in accord with their early and unique origin [8, 9] . Most likely, the modern viruses originated at the early RNA world of the primordial genetic pool.
With the increasing numbers of sequenced viruses, similarity among seemingly unrelated viruses was reported. A role of the hosts as vehicles for such cases is proposed. For example, the structural similarities observed between bacterial viruses (PRD1, Bam35), Chlorella virus (PBCV-1) and adenovirus in the coat proteins, led to the proposal that all viruses are old, probably preceding the cellular life. Furthermore, it is compatible with polyphyletic virus origins, as opposed to the monophyletic origin of cellular life [10] . Still, assignment of viruses to the phylogenetic tree of life remains unresolved [11] .
Notably, viruses as vectors (mainly RNA viruses) have the potential to rearrange the genomic material, and thus, to change the domain architecture [12, 13, 14] . Studies on horizontal gene transfer focused primarily on viruses infecting bacteria and archaea (e.g., bacteriophages) [15, 16] . The co-evolution of viruses toward their hosts indicates an active crosstalk on an evolutionary time scale [17, 18, 19] .
Several studies reported on a handful of cases of functional mimicry by viral proteins [20] . In few cases, evidence for gene transfer from the host to the virus is obvious. For example, the photosynthetic efficiency in cyanobacteria (Synechococcus and Prochlorococcus) relies on components of the photosystem II. These critical components express in the respective phages [21] . In the case of the phytoplankton-virus system, the DNA virus EhV that infects the microalge (Emiliania huxleyi), contains a complete metabolic pathway as a result of a horizontal gene transfer [22] . A similar case is demonstrated for the dUTPase genes (Dut) that are necessary for regulating the cellular levels of dUTP. Phylogenetic analysis revealed the origin of the viral Dut sequence in a monophyletic cluster of DNA viruses with eukaryotic hosts [23] .
The Acanthamoeba polyphaga Mimivirus and the family Phycodnaviridae [24] , contain many genes that are found in cellular organisms. For example, the giant virus Cafeteria roenbergensis virus (CroV) includes numerous eukaryotic-like genes for translation factors, ubiquitin pathway components, intein elements, histone acetyltransferase and more [25] . These are extremely large viruses of aqueous environments that infect bacteria, animals and protists [26] .
A search for similarities between viral and host proteins has largely been focused on herpesviruses [27] , Hepadnaviridae [28] and others. However, the high mutation rate of RNA viruses [29] and the coexistence between viruses and their hosts for millions of years has most likely blurred the sequence similarity. Recently, several studies challenged the origin of ancient viral segments in metazoan genomes. These sequences that are called EVE (for endogenous viral element) encompass all virus-derived genomic loci [30] .
In this paper, we present a coherent survey on protein sequences that are shared between viruses and their hosts. We assess the scale of the phenomenon by focusing on the viral-related protein sequences that appear in metazoa. We have used the current archive of all proteins [31] as the basis for identifying sequences with a potentially common origin. Presumably, their appearance in the virus reflects virus-acquired sequences. Of about 190 instances of highly similar viral-eukaryotes sequences, we recognize that only a small number originated from a host origin. We extended the collection of viral proteins that have a host origin by investigating the eukaryotes-viruses Pfam families [32] .
We focused on the 670 Pfam cross-taxa families that contain viruses and metazoa. A careful examination reveals that these instances reflect either missed annotations or the remnants of sequence exchange by virus infection. To distinguish these possibilities, we constructed sequence alignment trees for all 670 Pfam families. From the properties of the trees, we focused on 335 families that most likely contain viruses that hijacked sequences from their host. We found that most of the viral proteins in the orthologous families are much shorter and composed of simpler domain architectures. In almost all cases, the number of domains and the sequence of the tails and the inter-domain linkers are considerably shorter in the viral proteins relative to their counterpart host proteins. We discuss the potential of such short viral proteins to interfere with critical cellular functions and thus are candidates for manipulation strategies in defeating viral infection.
Genetic material exchange between viruses and their hosts Figure 1A shows two over-simplified scenarios in support of a genetic exchange from the virus to the host genome and in the reverse direction, from the host to the viral genome. In the first scenario, a viral sequence is detected in the host (e.g., human) but not in the rest of the phylogenetic branch. The following scenario accounts for viral sequences acquired from the host ( Figure 1A , right). Under this scenario, the viral gene sequence is identified in a broad group of organisms that belong to a phylogenetic tree that includes the host (human). Therefore, the sequence in the virus is most likely a reflection of a hijacking event, according to an argument of maximum parsimony.
Supporting evidence for the directionality of the genetic exchange of viral and cellular organisms relies on a detailed phylogenetic analysis. The topology of the reconstructed tree is used to support the most parsimonious scenario (see Materials and Methods). The simplified illustrations in Figure 1A do not address the more complicated, realistic instances in which different viruses carry sequences that resemble various organisms. An additional criterion used in supporting the occurrence of sequence acquisition by viruses is the presence of a sequence resemblance in the known host. The origin of viruses is probably preceding the cellular life [8, 10] . Thus, the ancient events in which viral sequences were incorporated into an ancestor eukaryote cannot be traced by their sequence similarity. Still, a conserved functional or structural similarity could expose such early events [33] . In this study, we have not attempted to date the horizontal transfer event. Furthermore, we will not discuss the events of genetic material exchange (see discussion in [34] ), but limit our study to the acquisition of coding sequences in viruses and metazoa.
There are about one million viral proteins in the UniProt database (990,049, August 2010) that represent about 66,000 viral strains. This is a highly redundant resource and about half of it composed of medically relevant strains including Hepatitis B viruses (HBV) and Human immunodeficiency virus (HIV). We took advantage of a reliable source of UniRef [35] that unifies sequences according to their identity level along the sequence length. We used UniRef90 classification (see Materials and Methods). There are .165,000 UniRef90 clusters that contain at least one viral protein ( Figure 1B) . However, from this set, we only considered 262 instances that contain at least two proteins, where one of them must be a eukaryote ( Figure 1B) . Of the 5,482 cross-taxa clusters that contain sequences from viruses and cellular organisms, 95% are sequences of bacteriophages and plasmids confined to the bacteria [36] . We will not further discuss the events that are confined to bacteria and archaea.
A taxonomical view shows the diversity of the organisms that share the UniRef90 clusters with viral proteins ( Figure 1C ). It shows that the eukaryotes are the most diverse group with 106 species that share their homologues with viral proteins. This result
Many studies focused on the exchange of genetic material between viruses and cellular hosts. The diversity of viruses argues that, along the evolutionary history, viruses have shaped the host genomes. While most viruses have many opportunities to exchange genetic material with their hosts, tracing such events is challenging as the origin of the sequences is masked by the high mutation rate of many viruses. On the other end, for completing a successful infection cycle the viruses must cope with the cell machinery for entry, replication and translation while hiding from the host immune system. We collected evidence for instances of viral protein sequences that were most probably ''stolen'' from the hosts. Additionally, a shared ancestry with metazoa is associated with 670 Pfam domain families. For half of these families, the origin of the viral proteins from its host is supported. For about 75% of the cross virus-metazoa families, the viral proteins are significantly shorter than their counterpart host proteins. Most of these cross-taxa viral proteins are single domain proteins and proteins with a simple domain composition relative to the proteins of their hosts. These viral proteins provide insights on the overlooked intimacy of viruses and their multicellular hosts. suggests that the phenomenon of shared sequences is quite broad, and many eukaryotes have been subjected to a genetic material exchange. Among the UniRef90 clusters that contain viruses and eukaryotes ( Figure 1B) , ,70% are from tetrapoda, 13% plants, 13% arthropoda, 4% fungi and only a smaller percentage of other taxa. We focused on the cross-taxa clusters of viruses and mammals (118 clusters include ,2,200 proteins, Figure 1D ). Viruses from Class I (dsDNA viruses with no RNA stage) and class VI (Retro-transcribing ssRNA, Plus strand) are prevalent among those that infect mammals [17] . The dominating Class VI viruses are characterized by their ability to integrate sequences into the host ( Figure 1A , left). Table 1 lists the Class I viral proteins that share sequences with mammals (23 clusters, Figure 1D ). In Class I viruses, the virus enters the nucleus before its replication (with the exception of Poxvirus family) and its infectivity is strongly dependent on the host cell division.
Discriminating whether a sequence has originated from the virus or the host is not straightforward. We generated for each cluster a phylogenetic dendogram and analyzed the connectivity of the viral protein in view of its neighboring sequences. Often the analyzed cluster is too small. In such cases, we expanded the cluster to the relaxed UniRef50 classification. We applied additional criteria in support of a virus having acquired protein sequences from the host: (i) The tested sequence appears in several organisms ($2) on the same evolutionary branch (as in Figure 1A , right); (ii) The tested sequence is not associated with viral contamination. Most analyzed cross-taxa clusters derive from the contamination by viral proteins following integration of the virus to a mammalian genome. Several instances were contaminated by the extensive use of viral vectors as vehicles in variety of molecular manipulations (e.g., Adenoviruses, Table 1 ).
Another source of contamination is from cancerous cells infected by viruses (e.g., human papilloma virus). In such instances, some sequences that are assigned as 'human' are incorrectly annotated. In these instances that reflect the incorporation of the virus to the host, different protein sequences from the same virus are identified which are best explained as a result of infection or an integration event. For example, the proteins in UniRef90 clusters P06426, P06463, P21735, P06788, P36741 and P21736 belong to Human papilloma virus (Table 1) .
Studies on the viral sequences that were integrated into the vertebrate germ line and hence shaped the vertebrate genetic heritage were reported [37, 38, 39] . Herein, we only consider the protein sequences that are shared by viruses and their metazoan hosts. The principal virus families that infect multicellular eukaryotes are listed in Supportive data Table S1 .
For few instances, a support exists for viruses that hijacked sequences from the host. Among Class I viruses (Table 1 ) the shared functions include interlukin-10 (IL-10) (Figure 2 ), beta-1,6-Nacetylglucosaminyltransferase (b1,6GnT) ( Figure S1 ) and Ubiquitin. The b1,6GnT and IL-10 are found exclusively in metazoa and the multicellular eukaryotic branch. The key features and the functional amino acids are conserved in the viral and the corresponding mammalian proteins (Figure 2A , Figure S1 ). Indeed, in human cells lacking b1,6GnT gene, the Bovine herpesvirus 4 (BoHV-4) sequence fully recovered the missing enzymatic activity [40] .
Resolving the evolution of the Ubiquitin in the genome of Pestivirus suggested that the virus hijacked Ubiquitin-related sequences in two consecutive events [41] . A browsable table is available at www.protonet.cs.huji.ac.il/ virost/tables/UniRef90-Class1.html. Figure 2 shows a prototypic case of viral proteins that resemble the host protein. Interleukin 10 (IL-10) inhibits the induction of pro-inflammatory cytokines. IL-10 was found in many viruses including Epstein-Barr virus (EBV), equine herpesvirus (EHV) and cytomegalovirus (CMV) [42] . Presumably, the gene product protects the infected cells from the host defense mechanism. An extended cluster of IL-10 (Table 1) covers 20 viruses and 96 cellular organisms (UniRef50_P22301). Representatives of viral and metazoan proteins are shown by the multiple sequence alignment (MSA) ( Figure 2B ). Most of the variations in the viral and metazoan protein reside in the sequence of the N-terminal that covers the signal peptide ( Figure 2B ). Traces of a genomic organization of the host in the viral genome were reported. For example, IL-10 like sequence from the gammaherpesvirus ovine herpesvirus 2 includes 5 exons and 4 introns [43] .
Inspecting the UniRef90 clusters that contain proteins from viruses and metazoa (187 clusters) shows a wide variation in the distribution of protein lengths (Supportive data Table S2 ). Viruses tend to reduce their production load by deleting and reducing the unessential genetic material [44] . While this length reduction is an absolute necessity for most viruses, some giant viruses (e.g., Mimivirus, Chlorovirus, and Cafeteria roenbergensis virus) include ,1000 proteins [24, 25] . The evolution origin of proteins from the Giant viruses remains unknown [45] . Still, 12% of the Acanthamoeba polyphaga mimivirus (APMV) proteins constitute a large number of host related sequences. The average length of this subset of the Mimivirus proteins (523 amino acids) is similar to the length of their homologous sequences.
The small numbers of cases of viral acquired sequences (Table 1,  Supportive data Table S2 ) may indicate the sequence divergence that had occurred throughout evolution. We therefore expanded the analysis for remote homologous. We questioned whether the viral protein sequences that were already substantially diverged due to a rapid evolution rate, or a long evolutionary history still maintain the host protein's functional domain.
The Pfam provides a comprehensive resource of functional and structural families and domains. Each Pfam entry represents a statistical model with an average sequence identity of 30-40% among the members of the family. Currently, Pfam covers 11,912 families, where 1,165 families include at least a viral protein and a eukaryotic protein representative. Some Pfam families are extremely large. Among families that contain metazoa and viral proteins are 'Helix-loop-helix DNA-binding domain' (,6000 proteins) and 'Sugar transporter' (,12,000 proteins). Contamination of viral proteins in metazoan proteomes (e.g., Capsid, Env, Tat) occurs mainly as a result of viral vector manipulations in cell lines, leading to incorrect assignment as a viral-eukaryotic crosstaxa family. An example is the GFP family (PF01353) that we have manually removed from the analysis. To reduce such sporadic instances, we considered Pfam families having at least two metazoan proteins, resulting in a list of 667 Pfam families.
Supportive data Table S3 lists the species, composition of the domains and the proteins' length.
The relatively small numbers of cases of viral acquired sequences (Table 1, Supportive data Table S2 ) may indicate the sequence divergence that had occurred throughout evolution. Therefore, we expanded the analysis for remote homologous. We questioned whether the viral protein sequences that were already substantially diverged due to a rapid evolution rate, or a long evolutionary history still maintain the host protein's functional domain.
Over 300 cross-taxa Pfam families (virus-metazoa) are best explained by a viral acquisition of host sequences. Instances of lateral gene transfer between bacteria and their bacteriophages dominate many of the cross-taxa Pfam families. Other families contain genuine viral proteins contaminated by metazoan proteins.
In order to justify the directionality of sequences from the hosts to the virus, we constructed for each of the 667 Pfam families a sequence-based tree (MSA based on the domain and not the full length sequence). We considered Pfam families in which only 1-2 viral proteins are included in the family, and families in which the percentage of the virus proteins in the family is small (,5%, Figure 3A ). The vast majority (547 families, 82%) of the analyzed Pfam families fulfilled these criteria ( Figure 3A , blue). We also requested that the viral proteins are clustered in sub-trees within the family tree. We counted the number of viral proteins spreading within the sequence alignment tree. We suggest that viral proteins that are clustered in a defined sub-tree (called Viral Cluster, VC) are likely to represent a single episode of acquired sequence from the host. Consequently, only a limited diversity among the closely related viruses is expected in view of the rest of the tree. 64% of the 547 Pfam families from the previous selection fulfill the requirement for clustered viral proteins. These are the Pfam families that contain #2 viral clusters (60%), and other families (4%) that are specified by a high degree of condensation (i.e. the ratio of the viral proteins to the number of VCs is $3). These filtration steps further reduced the list of relevant Pfam families to 335 ( Figure 3B ).
We show a tree constructed for one of the 335 families. The IL-6 (PF00489) family contains 10 viral proteins ( Figure 3C , blue) that are split to two sub-trees of viral clusters (VC) and other 136 Metazoan proteins (marked as collapsed sub-trees). The maximal depth in this tree is 19 (included in the collapsed sub-tree, red triangle). The deepest viral protein in the tree is of depth = 9, and its normalized depth is 9/19 = 0.474. The depth of the viral cluster (VC, the maximal sub-tree which contains all viral proteins) is therefore, 4/19 = 0.211.
The normalized depth for all the proteins in the 335 Pfam families ( Figure 3D, top) is analyzed in view of the distribution of the normalized depth of the viral proteins within these families ( Figure 3D, bottom) . It seems that the two distributions are remarkably different ( Figure 3D ) which is in accord with the notion that the viral proteins are relatively isolated subsets among the proteins from the cellular organisms in the relevant Pfam families. Table 2 shows a sample of these families along with the cellular process and the protein function in the viral life cycle. A full list of the 667 Pfam families with the analyzed properties of their alignment trees is provided in Supportive data Table S3 .
One of the families that exemplified the trend found in virusmetazoa Pfam families is the PAAD/DAPIN/Pyrin family (PAAD_DAPIN, PF02758). This domain family is a diverse family (26% average sequence identity) that includes 34 cellular species and 5 dsDNA viruses that belong to the Poxviridae. The PAAD domain is at the N-terminal regions of proteins. This domain occurs in several multicellular organisms, in the context of inflammation, signaling and apoptosis ( Figure 4) .
Several observations could be extracted for the PAAD domain: (i) Based on a multiple sequence alignment (MSA) of the PAAD domain sequences it is evident that the 5 viral proteins were diverged significantly ( Figure 4B ). All 5 viral proteins reside in one cluster, in the phylogenetic tree, together with other mammals as their sibling in the tree ( Figure 4A, blue font) . The domain architecture within the protein of the family is best explained by an initial extensive duplication of the PAAD domain ( Figure 4A Figure 4A , red font). Note that these proteins spread throughout the sequence-based tree. Presumably, it is a reflection of a domain loss event. Some of these proteins are fragments (e.g., Q5T3V8_HUMAN), and others include less characterized PfamB domains [32] (e.g., IFI4L_MOUSE, Q3UPZ5_MOUSE).
The initial tests on UniRef90 covered 14,000 proteins in relatively small clusters (,90 proteins on average, Supportive data Table S2 ). In contrast, the collection of the cross-taxa Pfam families (Table S3) covers 161,000 viral proteins and 400,000 metazoan proteins. Therefore, focusing on the cross-taxa Pfam families provides an opportunity to increase the statistical power of the tests.
Several statistical observations regarding the sequences among the cross-taxa families of viruses and multicellular organisms can be made: (i) The average length of the metazoan proteins is 507 amino acids, while the average length for the viral proteins in these families is only 396 amino acids (P-value of ,1.0e-17 by the KS-test, Figure 5A ). (ii) For 73% of all families, the viral proteins are shorter than the length of the average metazoan proteins in the family (P-value,1.0e-13 by the Hypergeometric test). (iii) In 67% of the families, the number of Pfam domain appearances (including several repeats of the same domain or different ones, Figure 5B ) is smaller in the viral proteins relative to the metazoan proteins in the family (P-value,1.0e-40 by the KS test). (iv) In 62% of the families, the number of different Pfam domains is higher in the metazoan proteins relative to the viral proteins. (v) For the discussed families, the median number of Pfam domains is 1.06 while, for the metazoan proteins, this value is 1.7 (Pvalue,1.0e-32 by KS test).
Many metazoan proteins are multi-domain (colored rectangle, Figure 5B ). We tested whether the viral acquired sequences that belong to multi-domain proteins displayed a stronger tendency for a size reduction (see scheme, Figure 5B ). A reduction in length of viral proteins may be a reflection of reducing the number of domains ( Figure 5B, b-c) , shortening the length of the linker sequences ( Figure 5B , a) or even the trimming of the length of the domain itself.
Among the 667 analyzed Pfam families, in 103 of them, the metazoan proteins contain at least 3 Pfam domains. In 85% of this set (88 families), the viral proteins are shorter ( Figure 5B, virus) . Remarkably, the average length of these 103 metazoan proteins families is 912 amino acids relative to 503 amino acids for the viral proteins that belong to these families. Similarly, in this set of multidomain proteins the viral proteins have an average of 2.9 domains, while the metazoan proteins have 4.6 domains on average (paired t-test, p-value of 1.0e-11). This shows that the tendency to reduce the protein length and the number of domains is stronger when the number of Pfam occurrence in the original host protein is higher.
In order to reduce the risk of misclassification, we further restricted the analysis to Pfam families of viruses-metazoa (with $3 Pfam domains) that contain at least 2 viral proteins (total of 50 families). The length of the viral proteins is significantly reduced. For 90% of these families (above the reference line, Figure 5C ), the viral proteins are shorter than their matched metazoan proteins. In order to determine whether the reduction in length is due to a reduction in the number or the properties of the domains, we repeated the analysis for the ratio of the number of distinct domains (depicted by the different colored rectangles, Figure 5B) in the viral and their relevant metazoan sequences ( Figure 5D ). For 80% of the families (families above the reference line), number of different Pfam domains that are associated with viral proteins is reduced. Note that by this measure, a short viral protein ( Figure 5B a-b) still has a ratio of 1.0. We show that the viral proteins are not only significantly shortened, but have also converged to a simpler domain composition.
The length of the individual domains between the viral and the metazoan host proteins is identical (Supportive data Figure S2 ). Recall, that this observation may be mainly due to the definition of belonging to a Pfam domain family.
The high statistical significance of these trends is consistent with a possibility that the short viral proteins have resulted from the acquisition of fragments from the host protein. Alternatively, it can be the result of a refinement of the acquired sequences during viral evolution. We separated each protein into three segments: (i) The Pfam domain(s); (ii) The tail linker (TAIL) that combines the amino acid extension towards the N-and the C-termini of the protein, beyond the boundary of the domain(s); (iii) The internal domain linker (IDOL) that comprises the sum of the amino acid spacers between domains. Clearly a single domain protein lacks IDOL.
We performed a separate analysis for the TAIL and the IDOL sequences ( Figures 6A-6D) . The study was performed on all the families that have at least 2 Pfam domains (unique or repeated). The average TAIL in viral proteins is 14 amino acids while the metazoan protein TAIL length is 85 amino acids (p-value,1.0e-150, Figures 6A-6B ). Trimming of protein tails at both termini often leads to a loss of cellular localization signals (e.g., KDEL, PDZ binding sites are found at C-termini) [17] . Importantly, the average IDOL length of the viral proteins in the Pfam families is 30 amino acids, while, for the metazoan equivalent proteins, the length is 67 amino acids (p-value,1.0e-150, Figures 6C-6D) . While a short TAIL may be explained by the viruses having acquired a fragmented sequence from their hosts, the same trend was found for the IDOL. Figure 6C shows that while only 54% of the metazoa IDOL have a length of ,40 amino acids, in the viral proteins from the same Pfam families, 96% of the proteins have IDOL that are shorter than 40 amino acids. These results are consistent with an active trimming and refinement process throughout viral evolution. Short IDOL length is advantageous in suppressing protein misfolding, and hence, improving translation effectiveness [46] . Similarly to the finding of short IDOL sequences in viral proteins, we identified instances of an internal domain which is missing in the viral protein while the flanking domains are maintained in the same order in the eukaryotic homologous protein ( Figure 6E) . The viral putative phosphatidylinositol kinase L615 (UniProt: Q5UR69) is a 701 amino acid protein from the Acanthamoeba polyphaga Mimivirus (APMV) that infects Amoeba. It has two Pfam domains: FYVE (PF01363) followed by PI3_PI4_kinase (PF00454). There are no other known proteins with identical domain architecture in the Amoebozoa kingdom (there are 7 such proteins in other kingdom, e.g., Stramenopiles and Excavata). However, there are 3 proteins from the genus Dictyosteliida (slime molds) that do belong to the Amoebozoa kingdom (UniProt D3BQ22, Q54UU9 and EGC34678). In all 3 of these proteins, the architecture is composed of FYVE domain followed by PI3Ka and PI3_PI4_kinase. The missing domain of PI3Ka in the Mimivirus (APMV) provides an evidence for an active elimination of an internal domain based on parsimonious argument. The findings of shorter IDOL (Figures 6C-6D) or absence of internal domains ( Figure 6E ) are probably the result of the trimming and shortening of the sequences after their acquisition by the virus. The possibility of a domain insertion in eukaryotes cannot be excluded.
The exhaustive search for sequences that were hijacked by viruses from their host allowed us to speculate on the underlying modes of mimicry. It was shown that once a mimicry function by a virus is established, the corresponding functional partner protein of the host undergoes a fast positive selection to overcome the deleterious effect of the viral mimicry [20] .
According to these findings, the viral proteins that originated from the hosts are short versions of the full-length host proteins ( Figures 5-6, Supplemental Figures S2, S4) . Furthermore, these proteins are characterized by a substantial reduction in the architectures of the domains ( Figure 5 ) and the protein linkers ( Figure 6 ). We classified these proteins into distinct (yet not exclusive) modes of action. For simplicity, we unified the viral acquired sequences from the cross-taxa families to 5 strategy modes (Figure 7) .
Mode A depicts a competition on a receptor binding by a viral ligand that replaces the natural one. Examples for this mode are the expression of the secreted IL-10 ( Figure 2 ), IL-8 (UniProt: Q98158, Q98314, D2E2Z5) and PDGF (UniProtKB Q80GE8, Q2F842 and D0VXD7). These secreted mitogens are identified in class I and class VI viruses (Table 2) . Viral proteins participate in a rich protein-protein interaction (PPI) network [47] . Mode B illustrates PPI, where the virus uses an acquired sequence for replacing a host partner protein or for interacting with a preexisting protein complex. The result is an alteration of the cells' function. Examples for viral proteins that interfere with the host PPI are the anti-apoptotic Bcl-2 sequences and Profilin (Table 2, for example UniProt: Q5IXM3, P33828,  P68695) . Mammalian Semaphorins (Sema7) and the Smallpox virus A39R protein ( Table 2 , UniProt: Q775N9, B7SV99, Q0N658, A0ES13) share identical binding modes with a crossreactivity towards common receptors [48] .
Mode C depicts the role of protein modifications (e.g., phosphorylation). A viral protein can either mimic the host modifications (Figure 7, marked C1) . Alternatively, a modification occurs by a viral enzyme (Figure 7 , marked C3). Such mimicry can lead to a modification of the original site or at an entirely new site (Figure 7, marked C2) . Apparently, there are instances in which both the modifying enzyme and the target proteins are both sequences that were acquired from the host (Figure 7, marked C4) . This mode is dependent on the presence of active kinases (or phosphatases). For example, human cytomegalovirus (HCMV) kinase introduces phosphorylation sites that perfectly mimic the function of the cellular CDK2 (cyclin dependent kinase) [49] . An evolutionary tree alignment for viral B1R protein kinase (Supplemental Figure S3 ) supports the functional overlap and mimicry with the closely related cellular kinases.
Mode D depicts the importance of nucleic acid regulation of transcription. In this mode, a viral protein mimics the host regulation by either competing for an existing transcription factor (Figure 7, marked D1) , or by modifying the transcription program following a DNA/RNA binding (Figure 7 , marked D2). For example, the Epstein-Barr virus (EBV) encodes an activator protein that is similar to Fos/Jun family (bZIP_1, PF00170. For example, UniProt: Q80GR6, Q8QQX9, Q6USE5, D2Y5S7). The difference in specificity and the dimerization properties of the EBV activator allows the activation of an alternative transcription program [50] .
Mode E collectively points to the generic strategies for damaging and deactivating the host proteins. It could be achieved by protein tagging (i.e., SUMO, ubiquitin), or the activation of viral proteases. Among the cross-taxa Pfam families, some families are associated with specialized proteases (Table S3) . Mode E shows the various routes by which acquired sequences alter key cellular processes. Molecular mimicry in trafficking and the subcellular localization is common to many viruses. For example, Soluble N-ethylmaleimide sensitive factor Attachment Protein (a-SNAP) is a conserved protein among all eukaryotes. It was also found in Canarypox and Fowlpox viruses [51] . These proteins may alter the balance of the vesicular trafficking, docking and the membrane fusion machinery. In autophagy, viral proteins exploit processes such as membrane fusion and protein folding for the benefit of their replication [52] .
We limit the discussion to the modes by which the shorter versions of the viral acquired proteins exhibit their impact on some cellular functions. The described modes (A-E) are effective in additional instances of molecular and functional mimicry [53, 54] .
Inspecting the viral proteome is challenging, as the majority of viral sequences are redundant and poorly annotated. Importantly, the rapid evolution and the high mutation rate in some viral classes often leads to the loss of a detectable sequence similarity and, therefore, additional cases of virus hijacking events cannot be detected based on sequence similarity search methods. Despite these drawbacks, we have traced hundreds of viral proteins with respect to their hosts. Only a small fraction of them shows high sequence similarity with corresponding host proteins. For the majority of the cases, the origin of the viral sequences and possible derivations from the host call for applying powerful models for remote homologues.
We provided analysis for 670 homologous families (according to the Pfam definition). For half of these families we provided support for sequence acquisition by the viruses from their hosts. The candidate sequences for a host to viral acquisition are useful in exploring the mechanisms by which viruses hijack and refine sequences.
We found that most of the viral proteins that potentially originated from host sequences are significantly shorter and contain fewer domains. Furthermore, we propose that the sequence refinement by the virus is a dynamic process. The inter-domain linkers (e.g., sequences connecting domains, but excluding the amino-and carboxyl tails) are significantly short, relative to other related proteins ( Figure 6 ). The viral proteins act in the cell according to a finite number of strategies. The simpler domain composition of these viral proteins is sufficient for the utilization of functional mimicry. Currently, we are expanding the analysis by identifying short peptides in viral proteomes that serve as competition agents for neutralizing critical cellular functions.
The collections of 187 UniRef90 clusters and the 667 Pfam cross-taxa families are available as interactive tables. These tables are available at:
www.protonet.cs.huji.ac.il/virost/tables/UniRef90.html www.protonet.cs.huji.ac.il/virost/tables/Pfam.html 
UniProKB includes 990,049 sequences (taxonomy-viruses). The viral proteins include ,15,000 reviewed proteins (UniProt/ SwissProt). The rest of the proteins are from UniProt/TrEMBL. There are 430.6 K sequences after removal of HIV and HBV sequences. Only 241.8 K are full-length (56.1%), while the rest are denoted as 'fragments'. The percentage of full-length proteins in metazoa is 54% (1.191 M/2.2051 M). The pre-calculated classifications of UniRef90 (i.e., identity of .90% at the amino acid level) reduce the UniProKB set to 175,236 clusters. Additional steps of filtrations are: (i) Considering only clusters with a minimal size of 2 proteins (62,129 clusters); (ii) Clusters that also include the metazoan proteins (187 clusters).
ViralZone is a database that manually assigns host-virus pairs (http://www.expasy.ch/viralzone, coordinated by UniProt/ SwissProt). ViralZone holds reference strains viruses that belong to 83 families and 330 genera. This is a high quality collection of 'complete proteome'. All viruses are classified into Table S1 .
Pfam 24.0 (11,912 families) [32] is a high quality resource for domains and families. A valid cross-taxa list was generated. Eukaryotes and viruses cross-taxa resulted in 1,165 Pfam entries. The following filtration steps were applied: (i) Pfam families with at least one viral protein and at least one metazoan protein (taxid: 33208), total of 859 Pfam families. (ii) Restricting the Pfam to families that have at least one metazoan protein and at least one metazoan-infecting virus resulted in 796 Pfam families. (iii) Pfam families with .95% viral proteins for structural element of the virus (e.g., Env, Coat, Capsid). (iv) Enzymes of the replication system were excluded, as these genes are the outcome of several events of genetic exchange [55] . Specifically, we excluded families of RNA/DNA polymerases (39 families), Exo/ Endonuclease (16 families), Helicase (15 families), tRNA synthetase (8 families) and Primase (8 families). We also manually eliminated the cluster represented by the GFP (PF01353) that reflects the inevitable contamination from the extensive use of GFP as vectors in many molecular biology techniques. The filtered list includes 667 protein Pfam families (Supplemental data Table S3 ).
We define linker sequences as TAILs (Tail Linkers) and IDOLs (Inter Domain Linkers). The TAILs are all sequences at the two terminals external to the first and last domain in the protein. Each protein provides two entries. The IDOL is a collection of all interdomain sequences (excluding TAIL). Protein TAIL's length was defined as the mean of the two tail segments. In the same way, IDOL length was defined as the mean of the lengths of the inter domains linkers.
We collected the Pfam data for all proteins having at least 2 domains (i.e., having at least one IDOL) and one of the domains belong to the 667 Pfam domain families (Table S3 ). There are ,57,000 such viral proteins and ,98,000 metazoan proteins.
Statistical tests were applied for the set of viral proteins in view of the host cellular protein for each cluster (or Pfam family collection). We applied statistical confidence tests (P-values) based on the non-parametric Kolmogorov-Smirnov (KS), Student t-test and the hypergeometric distribution tests. The KS test is based on the maximum distance between the two cumulative curves based on the separated viral and host proteins and viral and metazoan for the TAILs and IDOLs.
Multiple sequence alignments (MSA) by ClustalW were used for constructing the Phylogenetic trees. Local alignment searches are from NCBI-BLAST. BLAST was activated with a 'gap costs' for Existence: 10 and for Extension: 1. The resetting of the BLAST parameters was needed for systematic identification of missing domains detection scheme. The phylogenetic trees were built using the iTol [56] . Table S3 ). The graphs show the distribution of averages proteins length (two distributions per each Pfam family: one for the metazoan proteins and one for the viral proteins. A statistical KS test was performed on the domains length. No significant difference between the metazoan domains and the counterpart viral domains is detected. The same results were observed when using other statistical tests (e.g., t-test, not shown). The average and median proteins length and the average and median domain length is shown, next to the results of the statistical significant tests. (PPT) Figure S3 Phylogenetic tree of the viral B1R kinase family. A BLAST search (http://blast.ncbi.nlm.nih.gov) for the 32 highest scored proteins that belong to the B1R kinase family is shown. The query protein used is protein kinase CMLV190 from Camelpox virus. All viruses that were identified belong to dsDNA Class I of different genera. The tree branches are color coded for viruses and mammals (including platypus). All the 21 viral sequences belong to dsDNA Class I from different genera. Representatives are of Orthopoxvirus (Variola, cowpox virus) Capripoxvirus (e.g., Lumpy skin disease virus), Leporipoxvirus (Rabbit fibroma virus) and Yatapoxvirus (e.g., Yaba monkey tumor virus) and more. (PPT) Figure S4 Linker lengths in Pfam families that contain viral and metazoan proteins. The cumulative fraction function for all analyzed Pfam families for TAIL and IDOL sequences. A zoomed section of this graph is shown in Figure 6 . Viral proteins are marked in red and metazoan proteins in blue. (PPTX)  Treatment of Neuroterrorism Bioterrorism is defined as the intentional use of biological, chemical, nuclear, or radiological agents to cause disease, death, or environmental damage. Early recognition of a bioterrorist attack is of utmost importance to minimize casualties and initiate appropriate therapy. The range of agents that could potentially be used as weapons is wide, however, only a few of these agents have all the characteristics making them ideal for that purpose. Many of the chemical and biological weapons can cause neurological symptoms and damage the nervous system in varying degrees. Therefore, preparedness among neurologists is important. The main challenge is to be cognizant of the clinical syndromes and to be able to differentiate diseases caused by bioterrorism from naturally occurring disorders. This review provides an overview of the biological and chemical warfare agents, with a focus on neurological manifestation and an approach to treatment from a perspective of neurological critical care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13311-011-0097-2) contains supplementary material, which is available to authorized users. Substances that can potentially be used as weapons of mass destruction or agents of terrorism may be chemical, biological, nuclear, and radiological [1] . Bioterrorism is defined as the intentional use of these substances to cause disease or death in humans and/or animals, and/or environmental damage [2] .
In case of an attack, a large number of victims could be affected in a very short period of time, putting an enormous strain on the healthcare system [3] . Personnel will be faced with enormous logistical problems, and medications and other resources are likely to be insufficient [4] . Therefore, the United States (U.S.) Centers for Disease Control (CDC) urges healthcare professionals to be familiar with warfare agents, and in conjunction with governmental organizations have an implemented "Bioterrorism Preparedness and Response Program" to quickly detect and appropriately respond to a potential bioterrorist attack [5] . Early recognition is key in minimizing casualties, initiating appropriate therapy, and preserving resources. However, symptoms and signs caused by those warfare agents are often nonspecific and can easily be mistaken for common diseases. An important concept in differentiating a naturally occurring epidemic from a terrorist attack consists of recognizing an epidemiologic pattern [6] . Clues that suggest an attack include unusual age distribution or clustering of an illness [7] , a rapidly increasing incidence of an illness [8] , as well as an increased occurrence of an unusual illness or death in animals.
The range of biological agents or chemical substances that potentially could be used as weapons of mass destruction is wide. The ideal agent can be produced and stored easily, in adequate amounts that are easy to disseminate, capable of producing a disease in great proportion to the exposed, and remains effective, despite environmental exposure and change of environmental conditions, and is challenging to detect [9] . Very few agents have all of these characteristics [10] .
The CDC classifies potential bioterrorism agents into 3 categories: 1) A, 2) B, and 3) C. These categories are based on the agents' potential as weapons, such as their ability to be disseminated, transmitted, and to cause disease; the mortality rate; the expected impact on public health; and the potential for panic and social disruption [11, 12] . Category A agents, judged to have the greatest risk, include anthrax, plague, tularemia, smallpox, the hemorrhagic fever viruses, and botulinum toxin [11] . Most experts in the field believe that anthrax and smallpox would be the agents most likely to be used by terrorists [13] . Although these are the most easily fatal, terrorists could also reach their goals by simply causing illness on a large scale [10] . Category B agents are ones that would cause moderate morbidity and low mortality. Category C agents are pathogens in the emerging phase, [10] ( Table 1 ). The relative toxicity of selected agents for comparison is shown in Table 2 .
Chemical and biological weapons can cause a wide range of nervous system damage and neurobehavioral effects. Therefore, preparedness among neurologists is as important as it is for emergency, infectious diseases, and critical care personnel [14] . The main challenge is to be cognizant of the clinical syndromes and to be able to distinguish diseases caused by bioterrorism from more commonly occurring natural disorders [15] .
Nervous system complications in victims of warfare include penetration injuries to the brain and spine, contusions and concussions of the nervous tissue, meningitis and encephalitis, seizures, myelopathies, radiculopathies, peripheral neuropathies, post-traumatic encephalopathy, hypoxic brain injury, and behavioral changes [16] . Often, psychological symptoms would need differentiation from early manifestation of organic disease. In addition, vaccines against some categorized agents have neurological side effects (e.g., encephalitis after smallpox vaccination) [17] . In general, neurological disease tends to manifest somewhat later on in case of a biological attack, as compared to chemical weapons [15] . Prompt death, however, might occur following exposure to botulinum toxin, tetrodotoxin, saxitoxin, and nerve agents.
Of the many agents that may be used, prominent neurological features occur with cyanide, cholinesterase inhibitors, botulinum toxin, anthrax [14] , and paralyzing toxins, as well as nerve agents (Table 3) .
Anthrax is caused by Bacillus anthracis, a large, nonmotile, spore-forming, gram-positive rod. B. anthracis is common among domestic animals. It can be passed to humans by direct skin contact or inhalation of anthrax spores. Although the vegetative form survives poorly outside of a host [18] , the spore form can survive for decades [19] . It has many characteristics of an ideal biological weapon, its production is simple and cheap, and it can be stored for long periods of time. It is highly effective, with a morbidity rate of 65 to 80% if treatment is not promptly initiated. Weaponized anthrax can be produced as insoluble, liquid slurry, or dry powder. Although the most likely method of deployment is aerosolization of dry spores [20] , contamination of food and water supplies is conceivable [21] . The most serious terrorist threat posed by anthrax is infection by inhalation. For humans, the dose sufficient to kill half of the exposed persons ranges from 2500 to 55,000 inhaled spores [18, 22] . According to a U.S. government estimate, the outdoor release of 100 kg of B. anthracis in Washington, D.C. could produce between 130,000 and 3 million deaths [23] . Anthrax has been weaponized at various times in the past, most recently in October and November of 2002 in the U.S., which led to 18 confirmed and 4 suspected cases of disease [24] . Infection is acquired by ingestion, inhalation, or absorption of the spores through breaks in the skin and mucous membranes. Depending on the route of exposure, cutaneous, gastrointestinal (GI), or inhalation anthrax ensues.
Most naturally occurring human infections are cutaneous from contact with infected animals or contaminated material. Naturally occurring inhalational anthrax is rare, particularly in the industrial world [25] ; therefore, the occurrence of anthrax should raise concerns of an intentional dissemination.
Cutaneous transmission of the hands, arms, and face are the most common routes of clinical infection in humans. A pruritic papule evolves into an ulcer, followed by the development of a large painless black eschar. The eschar dries and desquamates after 1 to 2 weeks. Painful lymphadenopathy and sepsis can arise. With treatment, local cutaneous anthrax has a mortality rate of less than 1%; if the disease becomes systemic, mortality may be as high as 20% [26] . GI anthrax, while not common, occurs naturally as a result of ingesting poorly cooked, contaminated meat. Ulcers in the mouth or esophagus, or lesions lower in the intestinal tract may develop, and presenting symptoms include nausea, vomiting, diarrhea, abdominal pain, or an acute abdomen progressing to a sepsis syndrome with high mortality.
Inhalational anthrax follows the deposition of sporebearing particles into alveolar spaces. From there, they are transported to the mediastinal lymph nodes. Subsequent germination within the lymph nodes leads to a massive release of bacteria and toxins into the bloodstream. The incubation period is usually less than 1 week, but it can be as much as 6 weeks. Initial symptoms of the clinically and fairly consistent 2-stage disease are nonspecific, with fever, chills, myalgia, cough, and sore throat [26] . Substernal chest pain, dyspnea, abdominal pain, nausea, and vomiting are common. With disease progression for 2 to 3 days, severe pneumonitis develops, and abruptly, sepsis, hypoxemia, cyanosis, and shock follow. Prominent shortness of breath reflects thoracic lymphadenitis and mediastinitis rather than bronchopneumonia. However, inhalation anthrax can sometimes present without the usual symptoms of chest pain and shortness of breath [27] .
Weaponized anthrax presents with these inhalational findings. However, the epidemiology of weaponized anthrax is similar to that of a single point toxin exposure, with those exposed starting to become ill in relatively large numbers during a short period of time. In the Sverdlosk (now known as Ekaterinaburg) accidental release of 1979, most of the 68 known victims became ill within 2 weeks of exposure [28] .
Death ensues approximately 24 to 36 h after the appearance of respiratory distress, but sometimes it occurs within hours [26] . Untreated, mortality reaches 95%. Among the confirmed inhalational cases from the attack in the fall of 2001, the case fatality rate was 45% [29] .
For diagnosis, the organism may be detected by cultures and gram stain of blood or aspiration of skin lesions. Sputum 
Although the primary clinical presentation is a systemic or pulmonary illness, which is unlikely to be solely or initially neurological [15] , all 3 forms of anthrax can be complicated by meningitis, mostly in the second stage of the disease [30] . The risk of hemorrhagic meningitis in cases of inhalational anthrax is estimated to be as high as 50% [18] . There was 20% of the known patients who developed meningitis after the mail-borne inhalational anthrax attack [29] . The most common neurological manifestations are headache and confusion [29] . In the closely studied cases in the U. S. in 2001, neurological abnormalities were noted in 80% [29] .
Meningitis presents with fever, headache, nausea, vomiting, and altered mental status. Clinical signs include meningeal signs, long-tract signs, hyperreflexia, seizures, myoclonus, fasciculations, rigidity, stupor, or coma. Untreated, mortality is high, but early diagnosis and prompt initiation of antibiotics can halt disease progression.
Cerebrospinal fluid (CSF) shows neutrophilic pleocytosis often greater than 500 ml, elevated erythrocyte count, and elevated protein [14] , which are findings similar to the profile expected in herpes simplex virus (HSV) encephalitis or subarachnoid hemorrhage [30] . Gram-stain shows copious largegram positive rods with or without endospores. Blood cultures are positive in most patients with meningoencephalitis.
Neuroimaging reveals diffuse cerebral edema, prominent leptomeningeal enhancement, focal intracerebral, subarachnoid, or intraventricular hemorrhage [31] . An electroencephalogram may show disorganized, low-amplitude slow waves of 1 to 7 Hz. At autopsy, the meninges show extensive fresh hemorrhage, sometimes described as a "cardinal's cap" [32] .
Unless engineered, B. anthracis is susceptible to penicillin, amoxicillin, chloramphenicol, doxycycline, erythromycin, streptomycin, ciprofloxacin, and other quinolones. It is resistant to ceftriaxone and other 3 rd generation cephalosporins. Treatment for anthrax consists of a multi-drug regimen of ciprofloxacin, and at least one other agent of vancomycin, chloramphenicol, or penicillin [14] . For inhalational anthrax, the recommended regimen is ciprofloxacin or doxycycline, plus clindamycin and rifampin. Doxycycline and clindamycin, however, exhibit poor cerebrospinal fluid penetration and should be avoided in cases of anthrax meningoencephalitis. The addition of rifampin serves for the prevention or treatment of neurological manifestations [14] in cases treated with doxycycline or clindamycin. Treatment duration is long; a 60-day course is not unusual, given that spores can remain dormant for a long time. Corticosteriods are recommended in all patients who have pulmonary edema, respiratory failure, and meningitis [12] . In anthrax meningitis, steroids have been reported to improve survival [26] ; however, their use is controversial in adults, but it has improved outcome in children.
Mortality rates of as much as 20% for the cutaneous form, 60 to 80% for the GI form, and 90 to 99% for the pulmonary form make prompt treatment essential [33] . With multi-drug antibiotic regimens and supportive care, survival rates have improved. If there is a delay in treatment initiation from 2 to 4.8 days, the mortality would be expected to double [34] .
Vaccination is available for military personnel and civilian workers at risk for exposure [22] . The 2 types of vaccines for humans are both directed against the protective antigen of B. anthracis, and should protect against cutaneous and inhalational anthrax. It is given in 6 does of 0.5 ml for 18 months, followed by yearly boosters. Although the incidence of adverse reactions is low [18] , neurological side effects, such as optic neuritis have been reported [35] .
If a bioterrorist attack is suspected, or after an exposure, prophylaxis with ciprofloxacin 500 mg twice a day or doxycyline 100 mg twice a day is recommended for the target population. Treatment duration should be 4 weeks, while the effects of simultaneous vaccination take effect [36] . Resistance to penicillin and tetracycline should be assumed until proved otherwise by susceptibility testing [20] .
The differential diagnosis for anthrax includes mycoplasma pneumonia, Legionnaire's disease, psittacosis, tularemia, Q fever, viral pneumonia, histoplasmosis (fibrosing mediastinitis), and coccidioidomycosis.
The spores can be inactivated in water of near boiling temperature (25 minutes at 95°C). Formaldehyde or 5 to 10% chlorine bleach can be used to destroy spores on contaminated surfaces [37] . Filtration removes spores if the pore size is less than 1micrometer μm.
Given that there is no person-to-person transmission, standard infection control precautions are sufficient. In case of contact with spores, vigorous washing with soap and water is recommended, and the affected clothing should be placed in a plastic bag.
The plague is caused by the gram-negative bacillus Yersinia pestis. It is a zoonotic infection of rodents that can be transmitted through flea bites, but also person-to-person. The plague is more difficult to use as a biological weapon than anthrax, because Y. pestis does not form spores, it is susceptible to drying, heat, and ultraviolet light, and does not survive well outside the host body. Therefore, so far there has not been an effective bioweapon using aerosolized bacteria [38] . Unlike anthrax, secondary cases may result from person-to-person transmission [10] , however this requires close contact with a patient during the final stage of the illness [39] . Experts believe that the danger of terrorists using this organism may be greatly exaggerated [40] .
The plague can manifest in 3 different major forms: 1) bubonic, 2) pneumonic, and 3) septicemic. The bubonic plague begins as painful adenopathy 2 to 10 days after the infecting flea bite [41] , usually in the groin, axilla, or cervical region. A bubo is a 1-to 10-cm large, acutely swollen, erythematous, extremely painful, lymph node with surrounding edema and warmth [42] . Fever, chills, headache, and weakness occur with acute onset, and can transition to the septicemic form of the plague [42] in a quarter of patients. There are 80% of patients with the bubonic plague, which are bacteremic. There are 5 to 15% of bubonic plague victims who develop pneumonic plague, and hence become contagious. The overall mortality is estimated to be 60%, but can be less than 5% with prompt initiation of treatment.
The pneumonic plague usually manifests after an incubation period of 2 to 3 days with fulminant pneumonia, malaise, high fever, cough, hemoptysis, and septicemia with ecchymoses, and extremity necrosis. The disease progresses rapidly, leading to dyspnea, stridor, cyanosis, and septic shock. Death is normally the result of respiratory failure and circulatory collapse [42] . The pneumonic plague is highly contagious via inhalational exposure or secondary hematogenous spread, and therefore the most likely form to be used in a bioterrorist attack. It is invariably fatal, unless treated within the first day of onset.
Early diagnosis is important in initiating treatment within 24 h of symptom onset, which is crucial for survival. Aspiration of a bubo or sputum and gram stain analysis can provide a rapid bedside diagnosis. Definite diagnosis is made by culture; the cultures are often negative for 24 h, but turn positive at 48 h. The anti-Y. pestis titer rises fourfold or greater. Blood count shows a leukocytosis with left shift, and bilirubin and aminotransferase levels are elevated. The central nervous system (CNS) manifestations with meningeal involvement can complicate any of the forms and occur in approximately 6 to 7% of plague cases. Cerebrospinal fluid analysis reveals a neutrophilic pleocytosis [43] .
The differential diagnosis for the pneumonic plague includes disease caused by other biowarfare agents, such as anthrax, tularemia, and melloidosis (glanders), and other pneumonias such as severe community-acquired pneumonia, hantavirus pulmonary syndrome, influenza, or leptospirosis. The septicemic plague has to be differentiated from meningococcemia, Rocky Mountain spotted fever, other gram-negative sepsis, and thrombotic thrombocytopenic purpura.
Symptomatic patients should be isolated with strict respiratory isolation until treatment for at least 3 days [10] . The treatment of choice is streptomycin, alternatively doxycycline, gentamicin, ceftriaxone, chloramphenicol, or fluoroquinolones can be used. Treatment duration is for 10 days at a minimum. If exposed to aerosolized plague or to a patient with suspected pneumonic plague, prophylaxis with ciprofloxacin, doxycycline, tetracycline, or chloramphenicol should be given [42] .
Francisella tularensis is a nonmotile, aerobic, gramnegative coccobacillus [44] . There are 4 subspecies. Usually it is associated with zoonoses in rural areas [8] . In North America, type A, which is believed to be the most virulent strain, is predominant [45] . F. tularensis is highly infectious: only 10 to 50 organisms are needed to cause human disease [8] if inhaled or injected. Oral ingestion requires approximately 108 organisms that lead to disease. Human-to-human transmission has not been reported. The most likely method of deployment, therefore, would be made via aerosol, although contamination of food and water sources seem possible [8] . In a World Health Organization report from 1969, it was reported that 50 kg of aerosolized F. tularensis in an area inhabited by 5 million people would result in 19,000 deaths and 250,000 persons with severe illness [46] . F. tularensis can survive for weeks in the environment and for years in temperatures of freezing and below [10] ; however, it is easily destroyed by heat (55°C for 10 minutes) or standard disinfectant solutions, such as 10% bleach [8] .
The clinical manifestations depend on the route of infection. It can be transmitted through a bite from an infected arthropod or handling of infected animal carcass, ingestion of contaminated food or water, or inhalation of droplets [44] , and respectively patients can present with ulceroglandular, glandular, oculoglandular, oropharyngeal, typhoidal, or pneumonic tularemia [45] . The incubation period usually comprises 3 to 6 days.
Ulceroglandular tularemia, which is the most common form and makes for 80% of patients, starts with the infected suppurative skin lesion, most commonly the hands, and localized lymphadenopathy. The original skin lesion erupts and ulcerates with raised edges. Glandular tularemia in confined to lymphadenopathy [47] . Oculoglandular tularemia ensues after inoculation of the organism through the conjunctiva, with painful conjunctivitis, and preauricular, submandibular, and cervical lymphadenopathy. Oropharyngeal tularemia occurs after consuming contaminated food, with painful exudative pharyngitis and tonsillitis [44, 45, 47] . Pneumonic tularemia is similar to an atypical pneumonia with abrupt onset of constitutional symptoms and a nonproductive cough [48] .
Typhoidal tularemia is the systemic form that occurs in 30% of cases after any form of acquisition, but most commonly after inhalation of infectious aerosols. From the regional lymph nodes, the organisms spread to various organs, such as the liver, spleen, lungs, kidneys, intestines, CNS [44, 45] . It would be the most likely form to be encountered after use of francisella tularensis as a bioweapon. It is characterized by high fevers, headache, myalgias, prostration, vomiting, diarrhea; renal failure, rhabdomyolysis, pericarditis, meningitis, and erythema nodosum [47] . Approximately 80% of patients have pneumonia.
Neurological manifestations with severe meningitis or encephalitis are rare and only occur with widespread dissemination and sepsis [48] .
Case fatality rates of untreated naturally acquired typhoidal cases is approximately 35% compared with 1 to 3% for appropriately treated cases [10] .
Diagnosis is usually made by serology. A high antibody titer can be detected by enzyme-linked immunosorbent assay (ELISA), but is not very sensitive in the first week [49] . Titers become positive during the second week of infection in 50 to 70% of cases, and reach their highest level after 4 to 8 weeks [50] . Definitive diagnosis can also be made by culture of oropharyngeal specimens or fasting gastric fluid; however, the organism rarely can be isolated from blood [48] . PCR from wound swabs is 78% sensitive and 96% specific [51] .
Treatment regimens according to the Working Group on Civilian Biodefense [48] are streptomycin (1 g intramuscularly twice a day×10 days) or gentamicin (5 mg/kg intravenously or intramuscularly every day×10 days) for isolated cases, and ciprofloxacin (500 mg by mouth twice a day×10 days), or doxycycline (100 mg by mouth twice a day×10-14 days) in the setting of a mass casualty. Postexposure prophylaxis is with ciprofloxacin or doxycycline for 2 weeks.
Given that person-to-person transmission is rare, standard precautions are sufficient.
Q fever is caused by the intracellular coccobacillus Coxiella burnetii [52] after exposure to infected sheep, cattle, goats, or other livestock [8] . The bacterium's spore-like form is resistant to heat and desiccation, and it can persist for months [8] . This form can be distributed easily by wind [8] . It is highly infective; only 1 to 100 organisms are necessary to produce disease [52] . It cannot be transmitted human-to-human, but tissue may pose a risk [10] . Exposed surfaces can be decontaminated with 5% hydrogen peroxide or 70% ethyl alcohol for 30 minutes [52] .
The incubation period lasts from days to several weeks. The presenting symptoms are nonspecific; most patients experience a febrile flu-like illness with or without cough, which resolves within 1 to 2 weeks [8] .
Neurological manifestations occur in as much as one fourth of patients, and include severe retrobulbar headache, meningitis, and encephalitis [53] .
Mortality is reported to be 2.4% [54] . Chronic morbidity is low as well [8] ; however, endocarditis, intravascular infection, hepatitis, or osteomyelitis may persist.
Diagnosis can be made by ELISA. Treatment options are tetracycline, doxycycline, or macrolides; fluoroquinolone are to be considered in meningitis [55] . Treatment should be continued until the fever has subsided for 1 week [52] . Postexposure prophylaxis with a 5-day course of tetracycline or doxycycline may be effective if initiated within 8 to 12 days of exposure [52] .
Brucellosis is caused by Brucella species, small, aerobic, slow-growing gram-negative coccobacilli. There are 4 of 6 species (B. abortus, B. melitensis, B. suis, and B. canis) that can cause human disease. Brucella species can survive for many weeks in water or soil. It could be spread as a dry aerosol or in bomblets [8] . Infection occurs most often after ingestion of unpasteurized dairy products or contact with infected meat or animals [56] .
Most infections remain asymptomatic. Depending on the organism, symptoms begin as early as 2 weeks after exposure, but can occur as late as months after exposure. The organism tends to seed tissues with large numbers of macrophages, such as lung, spleen, liver, CNS, bone marrow, and synovium. The disease most often starts with a nonspecific prodrome, which is, however, absent in infection with B. melitensis. This is followed by the bacteremic stage, with intermittent fever, lasting for several weeks before subsiding, and then recurring in addition to other symptoms. This pattern of periodic febrile waves and remission can last for months or even years. Common manifestations in naturally acquired disease include joint pain, which is often incapacitating, and most commonly affects the sacroiliac joint, but also ankles, knees, and hips. Low back pain is seen in 60% of infected people and can be associated with vertebral osteomyelitis, intervertebral disc, or sacroiliac infection, or paravertebral abscess. Although pneumonia is not a common complication of brucellosis, 20% of patients develop cough and pleuritic chest pain. GI symptoms develop in 70% of adult cases. Hepatomegaly or splenomegaly is the result of granuloma formation and occurs in 45 to 63% of cases [57] . Endocarditis occurs in fewer than 2% of cases.
Neurobrucellosis with direct invasion of the CNS complicates less than 5% of infected individuals [58] . It may manifest as meningitis or meningoencephalitis, demyelination, cranial neuropathies, myeloradiculitis, cerebral arteritis, or spinal peripheral entrapment neuropathy [59] . Diagnosis can be made by blood culture, bone marrow aspiration, or serology. In patients with neurological symptoms, CSF analysis reveals a lymphocytic pleocytosis and elevated protein. CSF cultures are positive in 13% of cases [60] .
Although most patients will recover without treatment, antibiosis reduces the severity and duration of the disease. The most commonly used regimen consists of doxycycline plus rifampin for 6 weeks, but up to 3 to 4 months. Gentamicin or streptomycin is sometimes added in more severe infections [10] . Steroids may be beneficial in patients with encephalitis or meningitis.
There is no human vaccine available for brucellosis. The mortality rate for untreated brucellosis is estimated to be 5%; death occurs in severe cases with meningitis or endocarditis.
Glanders is caused by the nonmotile gram-negative bacillus Burkholderia mallei. Due to its ability to result in serious infection and the possibility of it being spread through aerosol, B. mallei may have potential as a bioweapon [10] .
Infection from inoculation through skin break typically results in a tender nodule with local lymphangitis. If transmitted through mucosa of the eyes, nose, or oropharynx, mucopurulent discharge with ulcerating granulomas may occur. If inhaled and causing systemic invasion, septicemia develops after 1 to 2 weeks, and the disease commonly manifests as pneumonia [61] . The most common manifestations include fever, myalgias, headache, and pleuritic chest pain. Lymphadenopathy or splenomegaly can often be found. A generalized papular or pustular rash is frequent. The septicemic form frequently results in death within 7 to 10 days.
Distinct neurological manifestation is not expected, but nonspecific symptoms, such as headaches are encountered as part of the common manifestation.
The organism is difficult to identify. Cultures usually remain negative. Antibiotics used to treat human melioidosis include tetracyclines, trimethoprim, and sulfamethoxazole, amoxicillin clavulanate, and chloramphenicol. Strict isolation of infected patients is indicated due to the possibility of person-to-person transmission.
Smallpox is caused by a DNA virus of the orthopox family. It can be transmitted by aerosols, droplets, direct contact with infected skin lesions, or even contaminated clothing or linens, and spreads easily from person-to-person [62] . Humans are the only reservoir for the virus [8] . Smallpox was declared eradicated by the WHO in 1980 [62] , and routine vaccination was stopped soon afterward. The virus is officially stored at 2 laboratories of the WHO, in the U.S. and in Russia [8] , although it is possible that clandestine samples are held elsewhere. As aerosolized smallpox is extremely virulent with a low infectious dose and the easy transmission from person-to-person even in asymptomatic stages, smallpox is 1 of the most feared agents that could be used in a biological attack [62, 63] .
Smallpox infection occurs as major and minor form (variola maior, variola minor). The major form has 3 clinical phases: 1) the incubation period, 2) a prodromal illness, followed by 3) a fulminant infection [63] . The asymptomatic period lasts from 7 to 17 days (usually 12 to 14 days) after the initial exposure [62] . Asymptomatic viremia develops 3 to 4 days after infection. After multiplication of the virus in the spleen, bone marrow, and lymph nodes, a secondary viremia develops on approximately day 8 of infection [62] . During this prodromal phase, nonspecific symptoms, such as malaise, headache, backache, myalgias, fever, and vomiting develop. The overt smallpox syndrome occurs 2 to 3 days later, while the prodromal symptoms are subsiding. Infected leucocytes transport the virus to dermis and oropharyngeal mucosa, leading to the characteristic skin lesions [62] . Within 2 to 3 more days, a maculopapular rash appears; the greatest concentration of the lesions is in the face and distal extremities. The rash spreads from there in a centrifugal pattern [8] . Macules transform to papules to vesicles to pustules, each stage lasting 1 to 2 days. Vesicles and pustules are deep-seated, firm, round, well-circumscribed lesions; they are sharply raised and feel like small round objects embedded under the skin. Eventually, the lesions crust over and form scabs, leaving deep pitting scars that are unique to variola. Unlike varicella, all smallpox lesions are at the same stage of development.
A more fulminant form, hemorrhagic smallpox or blackpox, occurs in approximately 3 to 10% of cases. The incubation period is shorter, and the characteristic rash presents as a dark, dusky erythema followed by petechiae and frank hemorrhage into the skin and GI tract. This form is almost uniformly fatal [64] ; death occurs 5 to 6 days after the onset of the rash [62] . This illness could be confused with meningococcemia or acute leukemia.
During the phase of the rash, patients are most infectious as virus particles are released from the lesions or infected mucosa [62] . Patients stay contagious until all scabs separated [62] .
Infectivity is low during the incubation period and the first 2 days of fever and increases during the febrile period. Carriers can even be asymptomatic, shedding infectious virions without ever manifesting the disease [8] .
Complications of smallpox infections include panophthalmitis, keratitis, corneal ulcers, blindness, osteomyelitis, arthritis, orchitis, and encephalitis [65] . Delirium occurs in approximately 15% of patients [8] . Encephalitis is reported to occur in 1 of 500 cases of variola major, and 1 of 2000 of variola minor, usually developing during the stages of the rash. Psychosis and seizures may occur [66] .
Mortality is reported as approximately 30% for variola major among unvaccinated persons, but this reflects historical data. Mortality in the minor form is less than 1% [66] . Previously vaccinated patients experience a milder disease, a shorter course, and a lower mortality rate.
Diagnosis is usually clinical, but must be confirmed by laboratory testing. PCR, antibody detection, or virus isolation are possible. Specimens should be handled under biosafety level 4 conditions if smallpox is a consideration [10] .
The most important aspect, once the disease is suspected, is prevention of further disease spread by strict isolation of patients and quarantine with respiratory isolation for 17 days of people with direct contact to patients.
In patients with neurological complications, CSF usually shows a neutrophilic pleocytosis by day 2 to 4, which later turns into a lymphocytic pleocytosis.
Treatment is mostly supportive. Cidofovir has shown antiviral activity in vitro, but is not approved for use in humans with smallpox [67] .
Unlike many other vaccines, the smallpox vaccine can be effective in preventing or attenuating disease, even when administered within 4 days after exposure [62] . As vaccinia is a live virus, secondary transmission after vaccination is possible. The vaccine provides 90 to 97% protection for at least 3 years. Smallpox vaccination is not without risk. There may be cardiac adverse events, so the vaccine is not recommended for people with cardiac disease.
Workers in the former Soviet Union developed a weaponized form of smallpox in which the onset of the disease is shortened, decreasing the likelihood that postexposure vaccination would be effective [68] .
The most feared complications are CNS complications, such as encephalitis and encephalopathy [69] , which occur in 1 in 100,000 to 500,000 [67] . Postvaccinal encephalitis presents with headache, meningismus, fever, drowsiness, and vomiting; some cases are accompanied by spastic paralysis. A second form, postvaccinal encephalomyelitis, may present in 11 to 15 days after vaccination, similar to encephalitis with fever, mental status change, meningeal signs, seizures, and additional spinal cord dysfunction. Mortality of these complications is as high as 25% [62] , and 25% of survivors develop persistent deficits [67] .
Viruses that cause hemorrhagic fevers and are category A agents in the CDC classification are the Ebola, Marburg, Lassa, Junin, Machupo, Guanarito, and Sabia viruses [70] . They are widely distributed in nature. Humans are highly susceptible [71] . Many are spread by airborne transmission, and although humans are not natural hosts for any of the viral hemorrhagic fevers, infected humans can spread the disease from person-to-person [12] .
All of those cause fever, malaise, vomiting, and may evolve into diffuse hemorrhage and bleeding diathesis [10] , but they all have a unique set of clinical complications [70] .
The incubation period varies from 4 to 21 days until the nonspecific prodrome develops. Within hours or days after initial presentation, the clinical condition rapidly deteriorates, which results from the affinity for the vascular system of the virus. Increased vascular permeability leads to flushing, petechial hemorrhages, mucus membrane hemorrhage, and shock, often with neurological, pulmonary, or hepatic involvement [64] .
Signs of CNS involvement, such as delirium, seizures, or coma, usually indicate a poor prognosis. Patients who survive this disease may be left with hearing or vision loss, impaired motor coordination, transverse myelitis, uveitis, pericarditis, orchitis, parotitis, hepatitis, or pancreatitis.
Laboratory evaluation shows thrombocytopenia, disseminated intravascular coagulation (DIC), elevated liver enzymes, and elevated creatinine. A diagnosis can be made by ELISA in specialized laboratories. Treatment is mainly supportive.
Infection control includes contact precautions and careful handling of all bodily fluids. Ribavirin is effective against arenaviruses (Lassa and New World arenaviruses) and bunyaviruses (Rift Valley fever, Crimean-Congo hemorrhagic fever, and Hantavirus) [64] .
Alphaviruses are categorized as category B agents by the CDC, as they are stable during storage and can be fairly easily produced in large amounts [8] .
Diseases caused by alphaviruses are mainly neurological and include Venezuelan equine encephalomyelitis and Eastern and Western equine encephalomyelitis. This disease occurs naturally in North, Central, or South America, but human illness is rare, and most infections result in nonspecific symptoms of fever, headache, and myalgia. Less than 6% of infected adults or children will develop encephalitis, however the mortality rate of those can be as high as 50 to 75% for Eastern equine encephalitis [72] , which is the most severe of these infections, and survivors frequently have neurological sequelae [73] .
Diagnosis is made by serological testing of CSF or serum. Treatment is supportive. There is no person-to-person spread.
Venezuelan equine encephalitis virus is an alphavirus that is most commonly found in Central and South America. It is transmitted to humans by mosquitoes. In case of a bioterrorist attack, the distribution would be made through aerosols [17] . The virus usually leads to an initial severe febrile illness in nearly everyone exposed at 1 to 6 days after exposure.
Naturally, only few patients (4% in children and less than 1% in adults) develop a severe encephalitis in a second phase a few days later [74] , but in case of an attack, increased numbers of encephalitis cases would be expected.
Diagnosis is made by isolation of the virus in serum or throat culture. CSF shows a pleocytosis. Viremia is typically absent in patients with encephalitis. Preventative and postexposure treatments are limited. Vaccines that have been shown to have some protective efficacy [75] are available for laboratory personnel at high risk of exposure. Pegylated interferon-α (IFN-α) improves survival in mice [76] , but data for humans are not available.
The overall mortality rate in a natural epidemic is estimated to be less than 1%, however, this increases to 20% if encephalitis develops [77] .
Botulinum toxins are the most toxic substances known, and thus a potentially devastating weapon if efficiently dispersed [1] . The lethal dose of botulinum toxin for a 70 kg human is estimated to be 0.7 to 0.9 μg inhaled or 70 μg ingested [78] . Enough toxin is present in a single gram of crystallized botulinum toxin to kill more than 1 million people [14] . It is 15,000 times more lethal than the highly potent chemical agent VX and 100,000 times more lethal than sarin [8] .
Botulinum toxin is produced by the obligatory anaerobic, gram-positive spore-forming soil bacterium Clostridium botulinum, and some strains of C. baratii and C. butyricum. There are 7 types of botulinum toxin (A-G), all of which use the same mechanism of action and can cause botulism. The toxin subtype is A in 50%, the remainder is usually B or E [79] . Types A, B, E, and F cause human disease, primarily affecting the nervous system [80] , and thus are of importance to neurologists.
The toxin is readily absorbed by mucosal membranes, but it does not penetrate intact skin [14] . The bloodstream carries the toxin to the peripheral cholinergic synapses. It enters neurons by endocytosis at the nerve terminal and prevents synaptic vesicles from fusing with the nerve terminal, preventing their release of acetylcholine [81] . As few as 10 molecules of botulinum toxin can irreversibly stop acetylcholine release. The result is complete failure of neuromuscular junction transmission, followed by degeneration of the motor end plate and denervation of the muscle fiber.
Most cases of naturally occurring botulism result from the ingestion of improperly prepared or inadequately homecanned food [82] . Although rarely, the disease is also associated with infected wounds or abscesses related to injection drug use. In infants, the toxin can be produced during growth of C. botulinum in the bowel.
There is no natural inhalation botulism. The toxin is colorless and odorless, such that terrorists could contaminate food supplies [78] . Aerolization of preformed botulinum toxin is believed the most likely means of deployment of botulinum toxin in a warfare scenario [78] .
Despite its high toxicity, the toxin is easily destroyed by heat; a temperature of 80°C for 30 minutes or 85°C for 5 minutes effectively degrades and inactivates the toxin [78] . Decontamination of exposed objects can be accomplished by washing them in a 0.5% sodium hypochlorite solution [83] . As there is no person-to-person toxin transmission, standard precautions are sufficient when caring for exposed individuals.
Botulism has a characteristic presentation [10] . Unlike other threat toxins, botulinum toxin appears to cause the same disease independent from its route of exposure. The neurological syndrome is caused by presynaptic blockade of neuromuscular and autonomic cholinergic junctions [1] .
The time of onset of symptoms varies with route of intoxication, and it is also dose-dependent [8] . Incubation time following ingestion is 12 to 36 h, with a range from 2 h to 8 days [79] . Symptoms after inhalation usually start 18 to 72 h after exposure. The rapidity and severity of paralysis depends on the amount of toxin absorbed.
The clinical hallmark of botulism is an acute, afebrile, descending, symmetric, flaccid paralysis that always begins in the bulbar musculature [78] . Cranial nerve palsies invariably occur, making bulbar symptoms, such as ptosis, diplopia, dysphonia, and dysarthria, some of the earliest and most indicative symptoms of contamination [79] [14] . The earliest clinical signs are usually blurred vision from dilated pupils, ptosis, dry mouth, dysarthria, and dysphagia, as well as generalized weakness, fatigue, and dizziness. By the third day after exposure, patients will pool mucous in the throat, experience difficulty swallowing solid food, and have a sense of catching a cold, but without fever. Bilateral facial palsy is common.
The cranial nerve palsies are followed by a symmetric, descending paralysis of skeletal muscles, which can quickly lead to respiratory failure. Severe weakness tends to occur by day 4 after exposure. Pharyngeal and upper airway paralysis may result in obstruction, and diaphragmatic and accessory muscle paralysis may render ventilation inadequate [78] . Death is usually a consequence of respiratory muscle failure or upper airway obstruction.
Ascending weakness has not been reported. True sensory changes are not encountered, but hyperventilation may produce paraesthesias. Patients remain fully conscious, as the toxin does not penetrate the blood brain barrier; however, mental numbness may occur, and patients may appear lethargic because of diffuse muscle weakness and difficulty communicating due to bulbar weakness [78] . Urinary retention or GI ileus may occur with abdominal cramping. Postural hypotension may be present. Deep tendon reflexes are intact in the beginning, but decline during a period of days. There are no dermatologic abnormalities.
The classic triad of botulism, according to the Working Group on Civilian includes [1] symmetric, descending flaccid paralysis with prominent bulbar palsies in [2] an afebrile patient with [3] a clear sensorium [78] .
The diagnosis of botulism is primarily clinical. Descending paralysis with prominent cranial nerve involvement and autonomic dysfunction (especially the gastrointestinal (GI)) should raise suspicion [15] . CSF and routine blood studies are typically normal, as are imaging studies of the brain, and thus they have limited value in the acute setting [83] . Definitive diagnosis requires detection of botulinum toxin in serum or stool, gastric aspirate, and if possible the suspected source [78] . For serum confirmation, testing must be done on ≥30 ml of blood in adults before therapy with antitoxin. However, toxin in serum or stool is identified in less than half of clinically diagnosed cases [79] . A mouse bioassay is the standard laboratory diagnostic method, in which the toxin type is identified by protecting mice with specific antitoxins against individual strains. The test takes days to be arranged and performed; therapy and notification of public health authorities must be based on clinical suspicion [78] . An antibody response is not mounted in most patients, because the amount of toxin required to produce a clinical syndrome is not large enough to generate an immunological response [8] .
On electrophysiological testing, motor conduction velocities and sensory nerve conduction remain normal. Compound muscle action potentials from affected muscles are diminished [84] . High frequency repetitive nerve stimulation produces an incremental muscle response similar to the Eaton-Lambert syndrome [85] . Autonomic function studies show an absent sympathetic skin response and significantly decreased heart rate variation [86] . Differential Diagnosis. Botulism may be confused with Guillain-Barré syndrome (especially the Miller Fisher variant), myasthenia gravis, or a pontine stroke. Furthermore, the differential diagnosis includes drug intoxication, poliomyelitis, tick paralysis, diphtheria, and paralytic shellfish poisoning. Botulism and atropine poisoning can both cause dilated pupils, dry mouth, constipation, urine retention, and prompt vomiting after food ingestion.
The only specific treatment for botulism is passive immunization with an equine antitoxin. A trivalent antitoxin, which is active against the 3 most common types of food borne botulism (A, B, and E) is available from the CDC [8] . A pentavalent toxoid vaccine for types A, B, C, D, and E is only available to military personnel [8] . The U.S. Army possesses limited quantities of a heptavalent antitoxin, which might be available in a terrorist attack [87] .
Although the antitoxin does not reverse existing symptoms, the deficits may stabilize and stop progressing [14] . Retrospective studies showed that early administration (within 24 h of symptom onset) reduced mortality and duration of hospital stay [88] . Animal studies suggest, if administered before clinical effects appear, the antitoxin might prevent symptoms from occurring [8] . The antitoxin is not generally recommended if a patient's exposure is greater than 72 h before administration [78] .
The antitoxin is provided in a 10 cc vial that provides 5500 to 8500 international units of each type of specific antitoxin. It has to be diluted 1:10 in isotonic sodium chloride solution and must be slowly infused intravenously. Because it is of equine origin, hypersensitivity reactions are possible, and antitoxin administration should be preceded by a small challenge dose. Diphenhydramine and epinephrine should be available during administration of the antitoxin in case of a severe hypersensitivity reaction. Patients who respond to the test dose with a substantial wheal and flare can be desensitized for more than 3 to 4 h [78] .
Antibiotics are not useful in the setting of inhalation or toxin ingestion, as it is not the bacterium itself, but the preformed toxin that is causing the illness [83] . Antibiotics may be useful for wound botulism, and for GI colonization with C. botulinum.
The mainstay of therapy is supportive. Severe morbidity and death due to botulism is mostly attributable to aspiration or to respiratory failure. Close monitoring of cough and gag reflexes, assessment of oropharyngeal secretions, respiratory mechanics, and oxygenation is necessary. Mechanical ventilation should be strongly considered if the vital capacity falls below 15 ml/kg or negative inspiratory force measures less than 20 cm of water. Placement of a nasogastric tube to prevent aspiration and to permit nutrition in the setting of bulbar palsy often becomes necessary. When treating secondary infections, aminoglycosides and clindamycin should be avoided as they may exacerbate the existing neuromuscular blockade [78, 89] .
Prognosis. Damage to the synapse and thus the neuromuscular blockade is permanent. Recovery only occurs with the sprouting of a new axon, which reinnervates the paralyzed muscle fibers [15] . In adults, this process may require many weeks or months or as much as a year or longer [14] . After many months, the original neuromuscular junction may regain activity. If respiratory paralysis has resulted, the patient usually remains ventilator-dependent during the recovery period, usually for 2 to 8 weeks [82] . In the case of a bioterrorist attack, supplying large numbers of patients with intensive care and mechanical ventilation would present tremendous logistical problems [10] .
The fatality rate has been reported to be 25% for index patients and 4% for subsequently identified patients. Mainly, mortality is attributable to delayed recognition of the disease, or to the complications of prolonged intensive care [15] .
Given that patients with botulism are not infectious to others, standard universal procedures but no barrier nursing are required [15] .
Anatoxin A is a bicyclic amine produced by Anabaena flosaquae, a filamentous, freshwater bacterium found in pond scum worldwide [37] . A. flosaquae exhibits 2 mechanisms of action as an acetylcholine agonist by: 1) binding to postsynaptic acetylcholine receptors and 2) stimulating muscle contraction. As the binding to the receptor is permanent, continuous contraction of the affected muscle ensues [90] . Secondly, anatoxin A inhibits acetylcholinesterase, increasing the amount of acetylcholine in the synaptic cleft. Symptom onset occurs within a few minutes, and the combined effect of the 2 mechanisms of action of the toxins results in a flaccid paralysis [90] . Initially, symptoms may mimic organophaosphate poisoning, with miosis, excess oral and lacrimal secretions, and muscle fasciculations, [90] . Death results from respiratory arrest [37] . Supportive care is the mainstay of treatment. 2-pyridine aldoxime methyl (2-PAM) and physostigmine have shown some effect when used as pretreatment in animals [90] .
During a terrorist attack, the toxin could conceivably be distributed by contamination of water supplies.
Trichothecene mycotoxins are produced by the Alternaria, Fusarium, Aspergillus, Claviceps, Penicillium, and Stachybotrys species of fungi [37] . The best known toxin is T-2. Due to the simplicity obtaining the toxins, their resistance to autoclaving and ultraviolet light, and their rapid lethal effect, they have potential for use as biological weapons [91] .
Inhalation, ingestion, or absorption through skin and mucous membranes leads to infection [91] . The toxins act by inhibiting protein synthesis and disrupting mitochondrial electron transport [37] . The main symptoms depend on the route of infection, and are cutaneous with blistering and skin necrosis, or respiratory with cough, dyspnea, and epistaxis. Neurological symptoms can include lethargy and incoordination. No rapid test is available for diagnosis; however, antigens and toxin metabolites can be detected in blood and urine within 1 month after exposure [91] . Treatment includes careful decontamination by washing with soap water, and 1% sodium hypochlorite solution with sodium hydroxide [91] , and is otherwise supportive.
Ricin is a protein cytotoxin derived from the bean of the castor plant. Ricin acts by inhibition of DNA replication and protein synthesis, leading to cell death within 8 to 12 h [91] , and producing symptoms usually after 12 h [37] . Distribution of the toxin would most likely occur as an aerosol or droplet [91] .
Clinical symptoms of the toxin depend on the route of exposure. Nonspecific symptoms include fever, nausea, arthralgias, and profuse sweating. After inhalation, chest tightness, cough, and dyspnea are prominent, and necrosis of the respiratory epithelium leads to tracheitis, bronchitis, bronchiolitis, and interstitial pneumonia [1] . When ingested, ricin causes nausea, vomiting, and diarrhea. If exposed to a sublethal dose, symptoms improve within several hours. Lethal doses produce necrosis of the respiratory tract and alveolar filling, or GI hemorrhage and hepatic, splenic, and renal necrosis [92] . Death from ricin toxin is dose-dependent, occurring 36 to 72 h after inhalation [1] . Death can be a consequence of pulmonary edema, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation, microcirculatory failure, or GI hemorrhage [93] . Injection of the toxin produces the most severe symptoms, and the CNS is affected early with convulsions [37] .
Overall, the toxicity of ricin is much lower compared to botulinum toxin or Staphylococcus Enterotoxin B (SEB) [93] . The toxin can be inactivated by heat; 80°C for 10 minutes or 50°C for approximately 1 h is sufficient for neutralization of the toxin [37] . There is no specific treatment.
This toxin is produced by the ubiquitous anaerobic, grampositive, spore-forming bacillus Clostridium perfringens. It can be found in the stool of every vertebrate. After accidental exposure, epsilon toxin causes increased vascular permeability leading to edema in various organs, and can result in a rapidly fatal acute toxemia. Inhalation can result in high permeability pulmonary edema, followed by circulatory spread with resultant renal, cardiac, and CNS damage. After ingestion, GI symptoms, such as watery diarrhea, nausea, and abdominal cramps will develop. Fever is rare. Spontaneous resolution typically occurs within a day. Fatality is rare, however, if delivered in high doses, epsilon toxin theoretically could rapidly debilitate civilian or military populations in large numbers [1] .
There are at least 11 different enterotoxin serotypes, produced by various biotypes of Staphylococcus aureus. All subtypes are structurally similar and produce the same clinical syndrome [94] . Enterotoxin B is a potent T-cell activator, and the clinical symptoms are largely mediated by the immune system rather than direct toxic effects. The toxin is heat-stable and relatively stable in aerosols. It is the second most common cause of food poisoning, and when inhaled, even low doses can produce symptoms. Although the fatality rate is only approximately 5%, a high percentage of those exposed could become seriously ill within a few hours [95] . In naturally occurring disease, approximately 15% become ill enough to require hospitalization. Contamination of food or water supplies with enterotoxin could debilitate a population or army within hours [1] .
Symptom onset is usually within 1 to 4 h, but can occur up to 12 h after exposure. Ingestions leads to nausea, vomiting, abdominal cramping, and diarrhea [10] . Less commonly, high fever, headache, myalgia, prostration, and dry cough develop. Symptoms resolve after a day [95] , but patients may be incapacitated for as much as 2 weeks. In severe cases, pulmonary edema or respiratory distress syndrome may develop. Death also may occur from dehydration [10] .
Diagnosis can be made with a toxin assay. Treatment mainly consists of fluid and electrolyte replacement.
There are 2 naturally occurring seafood neurotoxins: 1) tetrodotoxin produced by puffer fish, and 2) saxitoxin produced by microalgae in bivalve shellfish [37] . The toxins bind to voltage-gated sodium channels, inhibiting membrane depolarization and the conduction of action potentials [96, 97] . Both cause a severe paralysis of rapid onset. Numbness and tingling are often prominent, starting periorally before spreading to the limbs; GI distress, anxiety, headache, and mild peripheral weakness may appear within minutes to a few hours after ingestion. Successively, an ascending paralysis develops. Bulbar symptoms, hypersalivation, and sweating are commonly encountered. Hypotension (tetrodotoxin [97] ) or hypertension (saxitoxin [96] ), convulsions, and cardiac arrhythmias can occur. Death ensues secondary to respiratory failure within 24 h [37] . The victims may remain fully conscious.
There is no specific treatment or antidote. Gastric lavage with activated charcoal and administration of anticholinergic agents has been suggested [98] . Intoxication can be survived with supportive treatment, as clearance of the toxin is fast. Recovery of survivors takes as much as 2 weeks [15] .
The toxins can be deployed by contaminated food or water. They are not affected by temperature extremes and survive boiling [37] . Inactivation can be accomplished by chlorine under acidic and alkalinic conditions [37] . The toxins are highly potent, a thousand times more toxic than the chemical warfare agent sarin [99] . Inhalation is believed to produce the most severe effects [37] .
Nerve agents are substances that cause their effects by inhibition of acetylcholinesterase and accumulation of acetylcholine. Medically used substances that cause these effects include carbamates (physostigmine, neostigmine, and pyridostigmine). In agriculture, insecticides (sevin) and organophosphates (malathion, diazinon) are used.
The militarized nerve agents were originally synthesized as insecticides, before being used in World War II, and subsequently by Iraq against Iranian troops and Kurdish civilians, and by terrorists in Japan in 1994 in Matsumoto, and 1995 in Tokyo. They are the most toxic of the known chemical warfare agents. They are named Tabun, or "German agent A" (GA), "Sarin" (GB), Soman (GD), Cyclosarin (GF), and "Venemous" (VX) [1] . Their toxicity increases from GA to VX. They cause morbidity and mortality at extremely low doses [100] , persist in the environment for long periods of time, and can be released from contaminated clothing, skin, and secretions.
The G type gases are clear colorless liquids, when fresh. VX is amber-colored and oily. Distribution occurs in gas form, with inhalation and absorption through the skin as the most common forms of intoxication [1] . They have no taste, and most are odorless; tabun has a slightly fruity odor, and soman's odor resembles camphor. The volatility is greatest for GB, followed by GD, GA, GF, and VX. Subsequent to binding to cholinesterase, sarin, soman, and cyclosarin lose fluorine; tabun, VX, and Russian VX lose cyanide and the thiol groups.
The principal effect of nerve agents is exerted by inhibition of the enzyme acetylcholinesterase (AChE), which results in cholinergic overt stimulation with both muscarinic and nicotinic effects [101] . Pathophysiologically, their effects are the opposite of botulinum toxin; nerve agents result in increased acetylcholine in the synaptic cleft, while botulinum toxin results in decreased acetylcholine. The clinical manifestations of nerve agent intoxication are those of cholinergic excess. Muscarinic effects mainly manifest with symptoms from affected smooth muscles (Table 4 ) of airways, GI tract and eyes, glands, and the heart. Nicotinic effects concern skeletal muscles and pre-ganglionic nerves [1] . The mnemonic Salivation, Lacrimation, Urination, Defecation, GI hypermotility, Emesis (SLUDGE) summarizes the commonly experienced early symptoms of salivation, lacrimation, urination, defecation, GI hypermotility, and emesis [14] .
The initial effects of nerve gas exposure depend on the dose and route of exposure. With exposure to vapor in small amounts, smooth muscles and glands of eyes, ear-nosethroat (ENT), and GI tracts and airways are mostly affected with miosis, rhinorrhea, salivation, and shortness of breath. The onset of those effects is within seconds to minutes. There is no worsening after the removal from the exposure, and no late-onset effects. After a large exposure to vapor, all symptoms of a small exposure are more prominent, and the CNS is affected. CNS symptoms range from irritability to convulsions and coma [102] . Nicotinic symptoms include weakness of skeletal muscles, fasciculations (localized in areas where droplets penetrated skin, generalized with respiratory or large transdermal exposures [102] ), and paralysis. Muscarinic symptoms include profuse exocrine secretions (tearing, rhinorrhea, salivation, bronchorrhea, and sweating), in addition to ophthalmic symptoms, such as miosis, dim vision, headache, and eye pain. Large doses may lead to seizures and coma.
Cardiovascular effects initially are due to nicotinic stimulation, leading to tachycardia and hypertension [103] , but hypotension and cardiac conduction abnormalities are seen as well.
Pulmonary symptoms include chest tightness, labored breathing, wheezes, and copious secretions. Acute respiratory failure is a combined effect from bronchoconstriction, marked increase in airway secretions, and respiratory muscle weakness.
With dermal exposure, there is a delay of symptom onset for as much as several hours, and symptoms may persist even after decontamination due to the rapid absorption. The most sensitive indicator is miosis. Miosis is almost always present after vapor exposure and after large liquid exposure, and possibly after exposure to medium amounts of liquid nerve agents.
High-dose exposure can produce rapidly (seconds to minutes) fatal systemic effects. If patients survive a large exposure because death from hypoxia is averted by atropine and 2-PAM, the CNS cholinergic effects become overt in form of convulsions.
Seizures may evolve into status epilepticus, which can be prevented by giving large quantities of atropine early on Activity of plasma butyrylcholinesterase is more sensitive for most insecticides. Neurophysiological studies may assist in the diagnostic process. In acute organophosphate poisoning, nerve conduction velocities and distal latencies are normal, even in severely paralyzed patients [104] . The earliest and most sensitive indicator of the AChE inhibition is a small amplitude of compound muscle action potential after single supramaximal stimulation with often repetitive activity [104, 105] . On repetitive nerve stimulation, there is usually no decrement when stimulating at 3 Hz, and only occasional decrement at 10 Hz. At 30 or 50 Hz, there may be a decrement-increment response [104] in less severe stages of poisoning [105] .
One of the most important principles in management of nerve agent exposure is self-protection with protective gear. Initial treatment of the victim consists of physical removal of clothing or other exposed objects, and decontamination and forceful wash with soap and water or 0.5% sodium hypochlorite [5] . Early skin decontamination, within 1 to 2 minutes, is best. There is little benefit after 30 minutes.
The principle of antidotes is to reverse the effects of excess acetylcholine by inhibiting cholinergic effects and by reactivating the enzyme. Atropine may help to reverse bronchial constriction, which is given a starting dose of 2 to 6 mg followed by 2 mg every 5 to 10 minutes until the secretions halt and ventilation is improved. High cumulative doses (10-20 mg) in the first hours are not uncommon. Monitoring for atropine toxicity (delirium, hyperthermia, increased fasciculations) is necessary. Atropine may also cause arrhythmias and may even result in ventricular fibrillation if given intravenously in the presence of hypoxia. Electrocardiographic changes (ST depression and T-wave flattening) and cardiac arrhythmias reflect atropine toxicity and may be treated with propranolol. The combination of atropine with benactyzine is believed to be more effective, presumably by increasing central anticholinergic activity [5] .
Oximes work by reactivating AChE. They bind to the organophosphate-inactivated AChE and displace and hydrolyze the organophosphate. They must be administered rapidly to be effective, due to a process called "aging," which refers to the organophosphoryl moiety and the amino acids of the active site becoming covalent and changing their structure. Once this has happened, the enzyme cannot be reactivated. The aging times depend on the nerve agent: GD has the fastest aging time, with a half-time of 2 minutes [106] . GB ages in 3 to 4 h, others take longer; VX ages very little. The oximes affect nicotinic sites; there is no clinical effect at muscarinic sites, and available oximes do not cross the blood brain barrier. Pralidoxime is the compound most frequently used in a dosing of 1 to 2 mg in 100 cc normal saline for 15 to 30 minutes, followed by a second dose after an hour if paralysis persists [102] . In critically ill patients, a pralidoxime infusion at 7.5 mg/kg/h is safe [107] . Very rapid administration of pralidoxime, on the other hand, can worsen motor weakness. Oximes are mostly given in conjunction with atropine and benzodiazepines.
The dosing for 2-PAM chloride is simplified by combipen, which contains 600 mg. Infusion of intravenous doses of 25 mg/kg for approximately 25 minutes produces marked hypertension, which is rapidly but transiently reversed by phentolamine (5 mg).
Apart from oximes, exogenous butyrylcholinesterase (Protexia TM) is available.
For seizures, which may evolve into status epilepticus after pyridostigmine treatment leads to survival of an exposure, high quantities of benzodiazepines (usually diazepam in the military setting) may be required. The "convulsive antidote nerve agent autoinjector" (CANA) contains 10 mg diazepam.
Pretreatment with physostigmine could help prevent the CNS consequences, but could also cause its own CNS toxicity. In animal studies of soman intoxication, ketamine in combination with atropine and benzodiazepines proved effective in stopping seizure, reducing brain damage, and increasing survival [108] The weakness usually resolves within 5 to 18 days. Ventilatory support in survivors is often required for several days or weeks. Table 4 provides an overview of the recommended therapy for casualties of nerve agents.
Apart from the acute presentation, neurological sequelae of organophosphate poisoning may arise. A relapse of the weakness can occur 1 to 4 days after a seemingly welltreated and resolved course. This so-called intermediate syndrome has an incidence of 8% and presents with respiratory paralysis, cranial motor nerve palsies, and proximal limb and neck flexor muscles weakness [109] . Therapy is supportive, but patients may require (re)-intubation. Recurrent weakness typically resolves within 5 to 18 days [109] .
Furthermore, an organophosphate-induced delayed polyneuropathy (OPIDP) may result from a distal dying back axonopathy [110] , believed to be caused by phosphorylation of the enzyme neuropathy target esterase (NTE) [111] . OPIDP appears 1 to 3 weeks after exposure with cramping pain in the legs, paresthesias, and motor weakness. This is rare after nerve agent exposure, but more common with insecticide overdose. Apart from neuropathy, pyramidal signs and symptoms can develop [112] . If exposure is low grade, but persistent, pervasive effects of nerve agent exposure on human emotion, learning, and memory may be ensue [113] .
Gases, vapors, and other particles with a diameter of less than 2 mm can injure the entire airway [1] . Agents with highly water solubility (e.g., ammonia, sulfur dioxide) affect the upper airways with immediate burning sensation [114] , while low solubility agents (e.g., phosgene and nitrogen oxides), produce less immediate injury to mucous membranes and upper airways, and thus provide fewer warning signs of the exposure [114] . Massive exposure may lead to death from acute respiratory failure by destroying the alveoli and adjacent capillary endothelial cells. Delayed onset (up to 24 h) of acute lung injury is more common [114] .
Vesicant agents are oily, clear to yellow-brown liquid alkylating agents. They lead to cell damage by alkylation of DNA [115] . Symptom onset ranges from 1 to 12 h after exposure in a dose-dependent fashion [116] , but it can be delayed.
There is 20% absorbed from the skin [115] , and symptoms range from erythema and edema to necrosis and vesicles [116] . Groin and axillla are vulnerable due to their moisture and warmth [116] . Apart from the skin, the eyes and respiratory tract are affected [117] . Additional clinical effects include GI upset. Bone marrow suppression after high-dose exposure can be seen [117] .
Long-term clinical consequences include blindness, chronic bronchitis, and cancers of the respiratory tract [118] . There is no known antidote. Fatality rates are low with 2 to 4% [116] . Sodium thiosulfate may prevent death by acting as a mustard scavenger if given within minutes of exposure.
Hydrogen cyanide and cyanogen chloride are widely available, colorless, and come in gas or liquid form with high volatility. Hydrogen cyanide has an odor of bitter almonds; however, many people are not able to detect this distinctive odor [119] . Cyanogen chloride has a pungent, biting odor. They are absorbed through skin and respiratory mucosa. Mechanism of action is by interruption of the citric acid cycle and halting oxidative phosphorylation, inhibiting aerobic energy production and leading to rapid cell death [1] .
Severity and types of symptoms depend on the level of exposure. Duration of exposure and ambient concentration of the substance influence whether a symptomatic threshold is reached. Therefore, the use of these agents in a terrorist attack is limited to a closed environment (e.g., an office space or a subway system) [14] . In confined spaces, these agents are highly lethal [1] .
Mild exposure will cause headache, dizziness, drowsiness, mucosal irritation, and GI upset. Progression to coma can occur for several hours. Severe exposure leads to impaired consciousness and coma, arrhythmias, hypotension, cardiovascular collapse, respiratory irritation, and death [120] . The death can occur within minutes of inhalation [1] . Because hydrogen cyanide is excreted by the lungs, a patient's breath may have the characteristic of a bitter almond odor. The pupillary light reflex may be delayed [120] . Focal neurological signs are usually not prominent.
Overall fatality rates are estimated at 11 to 34% [120] . If survived, sequelae are rare, but anoxic encephalopathy can occur [1] .
Diagnosis should be suspected in an acyanotic patient with severe hypoxia. As differential diagnosis, carbon monoxide poisoning, exposure to organic solvents, drug intoxication, hypoglycemia, electrolyte disturbances, and postictal state should be considered. Cyanogen chloride induces mucosal irritation and excessive respiratory secretions, which are reminiscent of organophosphate poisoning. Laboratory findings are lactic acidosis and a decrease in the arterial-venous difference in partial pressure of oxygen [120] . Plasma thiocyanate levels can be measured, but not acutely.
Treatment in mild cases can be limited to decontamination and observation and oxygen supplementation. In severe cases, treatment includes several antidotes, which are available in a prepackaged cyanide antidote kit [121] . Sodium thiosulfate promotes the formation of thiocyanate by the enzyme rhodanese and leads to excretion of thiocyanate in urine. Sodium nitrate and amyl nitrate lead to formation of methemoglobin, which has an affinity for cyanide, and thus helps to reduce its active presence. The desired methemoglobin level is between 20 and 30% of total hemoglobin [122] . Sodium nitrate is given intravenously, and amyl nitrate vapor can be administered by inhalation through saturated gauze or by emptying an ampule in a respirator reservoir.
Delayed toxicity may affect the basal ganglia and present with Parkinsonian features. Dysarthria, eye movement abnormalities, dystonia, and ataxia have also been described [123] .
Magnetic resonance imaging may show cavitation of the putamen and globus pallidus. Cortical, cerebellar, and diencephalic changes have also been reported.
Disclaimer This article, including its tables, is intended to serve as a review of possible agents or biochemical warfare from a perspective of neurocritical care. It is in no way complete, nor is it intended to be complete. The appropriate agencies need to be consulted in case of a suspected attack or casualty. New Cardiovascular and Pulmonary Therapeutic Strategies Based on the Angiotensin-Converting Enzyme 2/Angiotensin-(1–7)/Mas Receptor Axis Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT(1) receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis. The renin-angiotensin system (RAS) plays a key role in several target organs, such as heart, blood vessels, and lungs, exerting a powerful control in the maintenance of the homeostasis [1] [2] [3] [4] . This system is activated by the conversion of the angiotensinogen to the inactive peptide angiotensin (Ang) I through the renin action [5] . Subsequently, Ang I is cleaved by the angiotensin-converting enzyme (ACE) generating Ang-(1-7) by ACE2 is important to regulate the RAS activity since Ang-(1-7) induces opposite effects to those elicited by Ang II [16] [17] [18] [19] [20] [21] [22] [23] [24] . Additionally, ACE2 can form Ang-(1-7) less efficiently through hydrolysis of Ang I to Ang-(1-9) with subsequent Ang-(1-7) formation [24] .
The relevance of the RAS is highlighted by the success obtained in therapeutic strategies based on the pharmacological inhibition of this system in cardiovascular and respiratory diseases [27] [28] [29] [30] [31] [32] . Blockade of the RAS with ACE inhibitors (ACEi) or AT 1 receptor antagonists (ARBs) improves the outcomes of patients with hypertension, acute myocardial infarction, and chronic systolic heart failure [33] [34] [35] . Furthermore, based on the involvement of the ACE/Ang II/AT 1 axis in respiratory diseases and the crucial role of the lungs in the RAS metabolism, several studies have reported the contribution of the RAS in lung pathophysiology [28, 30, 31, [36] [37] [38] [39] [40] . Importantly, it has been shown that administration of ACEi and ARBs causes substantial increases in plasma Ang-(1-7) levels, leading to the assumption that part of their clinical effects might be mediated by this heptapeptide [41] [42] [43] . Indeed, some effects of ACEi and ARBs can be blocked or attenuated by A-779, a Mas antagonist, confirming the role of Ang- (1) (2) (3) (4) (5) (6) (7) in the actions of these compounds [44] . The beneficial effects of Ang-(1-7), as well as its likely participation in the effects of the ACEi and ARBs, represent evidences for the potential of the ACE2/Ang-(1-7)/Mas axis as a therapeutic target.
In this review, we will focus on the recent findings related to the pathophysiology actions of the ACE2/Ang-(1-7)/Mas axis in the cardiovascular and respiratory system. Also, we will discuss the promising initiatives to develop new therapeutic strategies based on this axis to treat pathological conditions.
The heart is one of the most important targets for the actions of the ACE2/Ang-(1-7)/Mas axis. In the heart, ACE2 International Journal of Hypertension 3 is expressed in the endothelium [45] , myofibroblasts [46] , cardiomyocytes, and fibroblasts [47, 48] . Classical pharmacotherapeutic agents used to treat heart failure, including ACEi, ARBs, and aldosterone receptor blockers, increase ACE2 activity and/or expression, indicating its importance in the cardiac diseases establishment and progression [49] [50] [51] .
Additionally, pharmacological and genetic (transgenic animals and gene transfer) approaches have evidenced the significance of ACE2 in cardiac pathologies. Despite some controversies concerning the consequences of the ACE2 deficiency, in general, evidences indicate a protective role of ACE2 in the heart [48, [52] [53] [54] [55] [56] [57] . Crackower and colleagues [52] were the first to demonstrate that genetic ablation of ACE2 results in severe blood-pressure-independent systolic impairment. Also, disruption of ACE2 was able to accelerate cardiac hypertrophy and shortened the transition period to heart failure in response to pressure overload by increasing local Ang II [54] . Recently, it has been demonstrated that loss of ACE2 enhances the susceptibility to myocardial infarction, with increased mortality, infarct expansion and adverse ventricular remodeling [56] . In keeping with these genetic findings, pharmacological inhibition of ACE2 exacerbated cardiac hypertrophy and fibrosis in Ren-2 hypertensive rats [58] . On the other hand, cardiac overexpression of ACE2 prevented hypertension-induced cardiac hypertrophy and fibrosis in spontaneously hypertensive rats (SHR) and in Ang-II-infused rats [59, 60] . Indeed, transfection of Lenti-ACE2 (lentivirus containing ACE2 cDNA) or Ad-ACE2 (recombinant adenovirus carrying the murine ACE2) into the surrounding area of the infarcted myocardium was protective against pathological remodeling and cardiac systolic dysfunction in a rat model of myocardial infarction [61, 62] . This effect was associated with decreased expression of ACE and Ang II and increased expression of Ang-(1-7) [62] . Collectively, these observations reveal that ACE2 effectively plays a protective role in the cardiac structure and function.
Since the discovery of Ang-(1-7) in the late 1980s [63, 64] , several studies have demonstrated important effects of this peptide in hearts. The presence of Ang-(1-7) and its receptor Mas in the heart [65, 66] and the ability of this organ to produce Ang-(1-7) [55, 67] are evidences of the role of this peptide in cardiac tissues. Functionally, Ang-(1-7) induces an antiarrhythmogenic effect against ischemia/reperfusion injuries in rats [17, 68] as well as prevents atrial tachycardia and fibrillation in rats and dogs [69, 70] . Treatment with Ang-(1-7) improved the coronary perfusion and cardiac function in rats after myocardial infarction [71] and after ischemia/reperfusion injury [72] . Increases in circulating Ang-(1-7) levels in transgenic rats reduced the cardiac hypertrophy [17] and fibrosis [20, 22] induced by isoproterenol administration. These effects are apparently independent of changes in blood pressure since Grobe and colleagues [18] have demonstrated that the antifibrotic and antihypertrophic actions of Ang-(1-7) are still observed in Ang-II-infused hypertensive rats. Local overexpression of Ang-(1-7) in hearts of mice and rats improved the myocardial contractility and prevented the isoproterenol-and hypertension-induced cardiac remodeling [19, 21] . Altogether, these findings support a direct effect of Ang-(1-7) in the heart.
Further evidence for the role of Ang-(1-7)/Mas in the pathophysiology of the heart came from experimental protocols utilizing mice with genetic deficiency of Mas. They revealed that the cardiac function is impaired in Mas knockout mice likely due to the increased extracellular matrix proteins deposition in the heart [66, 73] . This profibrotic phenotype may be related to changes in matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) levels and/or activities [74, 75] .
Although further elucidations regarding the signaling pathways involved in Mas activation are necessary, some mechanisms have been proposed. Overexpression of Ang-(1-7) in hearts of rats causes an improvement in the [Ca 2+ ] handling in cardiomyocytes and increases the expression of SERCA2a [21] . In keeping with these results, cardiomyocytes from Mas-deficient mice present slower [Ca 2+ ] i transients accompanied by a lower Ca 2+ ATPase expression in the sarcoplasmic reticulum [66, 76] . Although acute Ang-(1-7) treatment failed to alter Ca 2+ handling in ventricular myocytes of rats [76] , these findings suggest an important role of the Ang-(1-7)/Mas in the long-term maintenance of the Ca 2+ homeostasis in the heart.
One of the mechanisms by which Ang-(1-7) plays its effects in the heart is stimulating the nitric oxide (NO) production. Indeed, it has been demonstrated that Ang-(1-7) via Mas increases the synthesis of NO through a mechanism involving the activation of the endothelial NO synthase (eNOS). These effects were abolished by A-779 and are absent in cardiomyocytes from Mas-deficient mice [76] . Recently, Gomes et al. [77] found that the treatment of isolated cardiomyocytes of rats with Ang-(1-7) efficiently prevents the Ang-II-induced hypertrophy by modulating the calcineurin/NFAT signaling cascade. These effects were blocked by NO synthase inhibition and by guanylyl cyclase inhibitors, indicating that these effects are mediated by the NO/cGMP pathway.
Also, Ang-(1-7) inhibits serum-stimulated mitogen-activated protein kinase (MAPK) activation in cardiac myocytes [78] and prevents the Ang-II-mediated phosphorylation of ERK1/2 and Rho kinase in hearts in a dosedependent manner [79] . In line with these data, activation of endogenous ACE2 significantly reduced the phosphorylation of ERK1/2 in hearts of hypertensive rats (SHRs) [48] . However, Mercure et al. [19] reported that overexpression of Ang-(1-7) in hearts of rats decreases the Ang-II-induced phosphorylation of c-Src and p38 kinase, whereas the increase in ERK1/2 phosphorylation was unaffected by the expression of the transgene, thereby suggesting a selective effect of Ang-(1-7) on intracellular signaling pathways related to cardiac remodeling.
Overall, these data reveal a key role of the ACE2/Ang-(1-7)/Mas axis in the pathophysiology of the cardiac structure and function. Activation of this axis might be an important strategy to develop a new generation of cardiovascular therapeutic agents against cardiac dysfunction and pathological remodeling of the heart.
Early studies have reported the endothelium as the major site for generation [67] and metabolism [41] of Ang- (1) (2) (3) (4) (5) (6) (7) . In addition to Ang-(1-7), endothelial cells also express ACE2 and Mas [80, 81] . Thus, now it is recognized that the ACE2/Ang-(1-7)/Mas axis is present in vascular endothelial cells and modulates its function promoting vasorelaxation [82] , reduction of the oxidative stress [83, 84] , and antiproliferative effects [85, 86] .
The vasodilatory actions of Ang-(1-7) have been reported in many studies in several vascular beds and preparations, including mouse [16, 23] and rat [15] aortic rings, canine [87] and porcine [88] coronary arteries, canine middle cerebral artery [89] , porcine piglet pial arterioles [90] , feline mesenteric vascular bed [91] , rabbit renal afferent arterioles [92] , and mesenteric microvessels of normotensive [93] and hypertensive [94] rats. Vascular Ang-(1-7) actions are still controversial in human. For example, it has been shown that Ang-(1-7) causes vasodilation in forearm circulation of normotensive subjects and patients with essential hypertension [95] while other studies were unable to report any significant effect of Ang-(1-7) in the same vascular territory in ACEi-treated patients [43] .
The Mas receptor is critically involved in the vascular effects of Ang-(1-7). In fact, many of these actions are completely abolished by A-779 or partially blocked by this antagonist [3, 86, 96] . Importantly, the endothelium-dependent relaxation induced by Ang-(1-7) in mouse aortic rings is absent in vessels derived from Mas-knockout mice [16] . However, other studies have shown that Ang-(1-7) also interacts with ACE, AT 1 , and AT 2 -like receptors, suggesting the existence of additional sites of interaction for Ang-(1-7) [3, 97, 98] . Indeed, Silva et al. [99] reported evidence for the presence of a distinct subtype of Ang-(1-7) receptor sensible to D-pro 7 -Ang-(1-7), a second Mas antagonist, but not to A-779 in aortas of Sprague-Dawley rats.
The vascular effects of Ang-(1-7) are endothelium dependent and involve the production of vasodilator products, such as prostanoids, NO, and endothelium-derived hyperpolarizing factor (EDHF) [16, 81, 100] . Pinheiro and coworkers [101] found that Ang-(1-7) promotes an increase in NO release in Mas-transfected chinese hamster ovary (CHO) cells [101] . Furthermore, short-term infusion of Ang-(1-7) improved the endothelial function by a mechanism involving NO release in rats [102] . Mas deletion resulted in endothelial dysfunction associated with an unbalance between NO and oxidative stress [83] . Also, Mas activation by Ang-(1-7) in human endothelial cells stimulated eNOS phosphorylation/activation via the Aktdependent pathway [81] . Other mechanisms appear to be involved in the Ang-(1-7) vascular actions. Roks et al. [103] have shown that Ang-(1-7) inhibits the vasoconstriction induced by Ang II in human internal mammary arteries, thereby suggesting that Ang-(1-7) can regulate the Ang II effects [103] . In fact, Ang-(1-7) negatively modulates the Ang II type 1 receptor-mediated activation of c-Src, and its downstream targets ERK1/2 and NAD(P)H oxidase [104] . The counterregulatory action of Ang-(1-7) on Ang II signaling has been also observed in cardiomyocytes [77] , vascular smooth muscle cells [105] , and fibroblasts [106] . Additionally, an interaction between Mas and bradykinin (Bk) type 2 (B 2 ) receptors may modulate some of the Ang-(1-7) effects in blood vessels [107] . Indeed, it has been demonstrated that Ang-(1-7) potentiates the vasodilator and hypotensive effects of Bk in several vascular beds [93, [108] [109] [110] .
As the major enzyme involved in Ang-(1-7) formation, ACE2 has also a crucial role in vessels. Lovren et al. [111] have demonstrated that ACE2 ameliorates the endothelial homeostasis via a mechanism involving reduction of the reactive oxygen species production [111] . Of note, this effect was attenuated by A-779 [111] . Moreover, overexpression of ACE2 in vessels of hypertensive rats resulted in reduction in the arterial blood pressure and improvement of the endothelial function associated with increased circulating Ang-(1-7) levels [112] . Overall, these data indicate that the beneficial effects of ACE2 are, at least in part, mediated by Ang- (1-7) . Recently, we have demonstrated that activation of endogenous ACE2 causes a dose-dependent hypotensive effect in normotensive and hypertensive rats [113] . Also, the response to Bk administration was augmented in rats chronically treated with XNT, an ACE2 activator [113] . However, we were unable to demonstrate any significant effect of XNT on blood pressure in response to the administration of Ang II or Losartan in normotensive and hypertensive rats ( Figure 2 ).
In the past few years, the participation of the ACE2/Ang-(1-7)/Mas axis in the establishment and progression of pulmonary diseases has become evident. Indeed, the important role of the RAS in the lung pathophysiology and the side effects and pulmonary toxicity induced by the ACEi raised the interest to evaluate the activation of the ACE2/Ang-(1-7)/Mas axis as an alternative target to treat pulmonary pathologies. Thus, it has been reported beneficial outcomes induced by the activation of this axis in animal models of acute respiratory distress syndrome (ARDS), pulmonary hypertension (PH), fibrosis, and lung cancer [31, 37, [114] [115] [116] [117] . These studies pointed out that the imbalance between the ACE/Ang II/AT 1 and the ACE2/Ang-(1-7)/Mas axes of the RAS might be relevant in lung diseases. Taking into account that systemic hypotension is an important limitation to the use of ACEi and ARBs in pulmonary patients, therapies based on the ACE2/Ang-(1-7)/Mas axis emerge as a safe and efficient approach since studies using the ACE2 activator XNT or ACE2 gene transfer have shown that these strategies induce beneficial pulmonary outcome without changes in systemic blood pressure in rats and mice [39, 117, 118] .
Imai and colleagues [37] demonstrated the role of ACE2 in ARDS pathogenesis. They found that a more severe ARDS was reached in ACE2 knockout mice, and this phenotype was reversed by double genetic deletion of the ACE2 and ACE genes or by the treatment with recombinant human ACE2 (rhACE2). Furthermore, Ang II levels were related International Journal of Hypertension to the severity of the lung injury. Of note, ACE2 is widely expressed in the pulmonary endothelium, vasculature, and pneumocytes [119, 120] . Also, rhACE2 inhibited the increase of Ang II and TNF-α levels, attenuated the arterial hypoxemia and PH, and ameliorated the distribution of the pulmonary blood flow in lipopolysaccharide-induced lung injury in piglets [121] . Therefore, these studies suggest that ACE2 is a suitable target to arrest the development of ARDS in patients at risk.
The stimulation of the ACE2/Ang-(1-7)/Mas axis has been successful used to prevent and reverse PH and fibrosis in animals. ACE2 activation using the compound XNT or induction of ACE2 overexpression by gene transfer efficiently prevented and, more importantly, reversed the increase of the right systolic ventricular pressure (RSVP), pulmonary fibrosis, imbalance of the RAS, and inflammation in animals (rats and mice) with PH induced by monocrotaline (MCT) or in rats with pulmonary fibrosis caused by bleomycin treatment [39, 117, 118] . In keeping with these findings, Ang-(1-7) gene transfer into the lungs triggered similar protective actions in MCT-treated rats [39] . In addition, Ang-(1-7) via Mas prevented the apoptosis of alveolar epithelial cells and the Jun N-terminal kinase (JNK) activation induced by bleomycin [122] . The involvement of the Ang-(1-7)/Mas in PH was further evidenced by the observation that the XNT effects are blocked by A-779 [117] . Furthermore, in both lung specimens from patients with idiopathic pulmonary fibrosis and from animals with bleomycin-induced pulmonary fibrosis were reported a reduction in mRNA, protein, and activity of ACE2 with a reciprocal increase in Ang II level [116] .
A growing body of studies has focused on the relevance of the ACE2/Ang-(1-7)/Mas axis in the pulmonary cancer pathophysiology. The protein expression of ACE2 is reduced in non-small-cell lung carcinoma (NSCLC) along with an increase in Ang II levels. Moreover, overexpression of ACE2 in cultured A549 lung cancer cells and in human lung cancer xenografs inhibited the cell growth and the vascular endothelial growth factor-a (VEGFa) expression induced by Ang II [123, 124] . Gallagher and Tallant [125] evaluated the effects of several angiotensin peptides [Ang I, Ang II, Ang-(2-8), Ang- (3) (4) (5) (6) (7) (8) , and Ang-(3-7)] in SK-LU-1 cancer cells growth, and only Ang-(1-7) showed significant attenuation of the DNA synthesis and proliferation. The antiproliferative effect of Ang-(1-7) was mediated by its receptor Mas and inhibition of the ERK1/2 pathway. Neither the blockage of AT 1 nor AT 2 succeeded in inhibiting the action of Ang-(1-7). In keeping with these data, the antiproliferative effect of Ang-(1-7) was observed in human A549 lung tumor xenograft growth along with a marked decrease in the vessel density in mice through a mechanism involving cyclooxygenase-2 (COX-2) [126, 127] . Of note, in a nonrandomized phase I clinical trial conducted by Petty and colleagues [38] , subcutaneous injections of Ang-(1-7) were administered in 18 patients with advanced solid tumors refractory to standard therapy. Despite the mild adverse effects observed with the Ang-(1-7) treatment, generally it was well tolerated. There were no treatment-related deaths. Clinical benefits were observed in 27% of the patients. Altogether, these studies provide insights into the involvement of the ACE2/Ang-(1-7)/Mas axis in lung cancer.
Many advances have been achieved regarding the therapeutic regulation of the RAS. Current therapies based on the modulation of the RAS include the ACEi, ARBs, and renin inhibitors. In general, these drugs prevent or reverse endothelial dysfunction and atherosclerosis, reduce cardiovascular mortality and morbidity of patients with coronary artery disease, and hold antihypertensive effects [128] . Classically, the mechanisms of action of the ACEi and ARBs involve the blockade of the synthesis and actions of Ang II, respectively. However, the RAS is a complex hormonal system and, consequently, other mechanisms are likely implicated in the actions of these drugs [42, 86, 129] . They cause substantial increase in plasma levels of Ang-(1-7), leading to the assumption that their clinical effects might be partly mediated by this heptapeptide [42, 130] . Indeed, a variety of effects of the ACEi and ARBs can be abolished or attenuated by Mas antagonism, confirming the role of Ang-(1-7) in the actions of these compounds [129, 131] . The beneficial effects of Ang-(1-7) as well as its likely involvement in the effects of the ACEi and ARBs represent a strong evidence for the therapeutic potential of the activation of the ACE2/Ang-(1-7)/Mas axis (Figure 3 ). Ang-(1-7) Formulations. The beneficial effects of Ang-(1-7) are well known; however, the therapeutic utilization of this peptide is limited due to its unfavorable pharmacokinetic properties. Ang-(1-7) has a short half-life (approximately 10 seconds) since it is rapidly cleaved by peptidases [132] . Furthermore, Ang-(1-7) is degraded during its passage through the gastrointestinal tract when orally administrated. Thus, new strategies are crucial to make feasible the clinical application of Ang-(1-7) .
Recently, a formulation based on the Ang-(1-7) included into hydroxypropyl β-cyclodextrin [HPβCD/Ang-(1-7)] was developed by Lula and colleagues [133] . Cyclodextrins are pharmaceutical tools used for design and evaluation of drug formulations, and they enhance the drug stability and absorption across biological barriers and offer gastric protection [134] . The amphiphilic character of cyclodextrins allows the possibility of formation of supramolecular inclusion complexes stabilized by noncovalent interactions with a variety of guest molecules [133, 134] . In this regard, the formulation HPβCD/Ang-(1-7) allowed the oral administration of Ang-(1-7). Pharmacokinetic and functional studies showed that oral HPβCD/Ang-(1-7) administration significantly increases plasma Ang-(1-7) levels and promotes an antithrombotic effect that was blunted in Mas deficient mice [135] . Marques and colleagues [136] have found that chronic oral administration of HPβCD/Ang-(1-7) significantly attenuates the heart function impairment and cardiac remodeling induced by isoproterenol treatment and myocardial infarction in rats [136] .
In addition, liposomal delivery systems represent an alternative method to administer Ang-(1-7) [137] . Administration of liposomes containing Ang-(1-7) in rats led to prolonged hypotensive effect for several days in contrast to the response observed when the free peptide was used [137, 138] .
A strategy used to protect the Ang-(1-7) against proteolytic degradation was proposed by Kluskens and coworkers [139] . Using the ability of prokaryotes to cyclize peptides, they synthesized a cyclic Ang-(1-7) derivative [thioetherbridged Ang-(1-7)] which presented an increased stability in homogenates of different organs and plasma and enhanced the Ang-(1-7) bioavailability in rats [139] . Furthermore, cyclized Ang-(1-7) induced a relaxation in precontracted aorta rings of rats which was blocked by the Ang-(1-7) receptor antagonist D-Pro 7 -Ang-(1-7), providing evidence that cyclized Ang-(1-7) also interacts with Mas [139] .
Agonists. AVE 0991 was the first nonpeptide synthetic compound developed with the intention of stimulating the Mas receptor. This compound mimics the Ang-(1-7) effects in several organs such as vessels [140, 141] , kidney [101] , and heart [142, 143] . Similar to Ang-(1-7), AVE 0991 induced a vasodilation effect which was absent in aortic rings of Mas-deficient mice [140] . Moreover, its effects in aortic rings were blocked by the two Ang-(1-7) receptor antagonists, A-779 and D-Pro 7 -Ang-(1-7) [140] . AVE 0991 potentiated the acetylcholine-induced vasodilation in conscious normotensive rats, and this effect was abolished by A-779 and L-NAME [102] . Similarly, it was able to increase the hypotensive effect of Bk in normotensive rats, and A-779 also blocked this effect [107] . Ferreira et al. [142, 143] reported that AVE 0991 protects the heart against cardiac dysfunction and remodeling caused by isoproterenol treatment or by myocardial infarction in rats [142, 143] . In Mas-transfected cells, AVE 0991 induced NO release which was blunted by A-779 and not by AT 2 or AT 1 antagonists [101] . All these data support the concept that AVE 0991 is an Ang-(1-7) mimetic and that its actions are mediated by the interaction with Mas. Using a computational discovery platform for predicting novel naturally occurring peptides that may activate GPCR, two novel peptides, designated as CGEN-856 and CGEN-857, with amino acid sequence unrelated to angiotensin peptides, were found to display high specificity for Mas [23] . These peptides elicited Ca +2 influx in CHO cells overexpressing Mas without any activity in AT 1 or AT 2 receptors [144] . CGEN-856S, a derivative of the CGEN-856 peptide, induced beneficial cardiovascular effects similar to those caused by Ang-(1-7) [23] . This compound competes with Ang-(1-7) for the same bind site in Mas-transfected cells. Furthermore, similar to Ang-(1-7), CGEN-856S produced a vasodilation effect which was absence in Mas-deficient mice, indicating that this compound also acts via Mas [23] . This was confirmed by the inhibition of the CGEN-856S effects by the Mas antagonist A-779. Importantly, Savergnini et al. [23] showed that CGEN-856S promotes antiarrhythmogenic effects and produces a small dose-dependent decrease in arterial pressure of conscious SHR [23] .
A new approach addressing the therapeutic potential of the activation of the ACE2/Ang-(1-7)/Mas axis was proposed by Hernández Prada et al. [113] . Based on the crystal structure of ACE2 and using a virtual screening strategy, it was identified small molecules that may interact with this enzyme leading to changes in its conformation and, consequently, enhancing its activity [113] . Thus, the ACE2 activator, namely XNT, was identified and its administration in SHR decreased blood pressure, induced an improvement in cardiac function, and reversed the myocardial and perivascular fibrosis observed in these animals [48, 113] . The beneficial effects of XNT were also observed in rats with PH induced by MCT [117] . Furthermore, this compound attenuated the thrombus formation and reduced the platelet attachment to vessels in hypertensive rats [145] .
It appears that the pharmacological activation of ACE2 promotes its beneficial effects due to an increased Ang-(1-7) production with concomitant degradation of Ang II. In fact, coadministration of A-779 abolished the protective effects of XNT on PH [117] . In addition, the antifibrotic effect of XNT observed in hearts of SHR was associated with increases in cardiac Ang-(1-7) expression [48] . However, it is also pertinent to point out that off-target effects of XNT on these beneficial outcomes cannot be ruled out at the present time.
The complexity of the RAS is far beyond we could suspect few years ago. There is growing evidence that changes in the novel components of the RAS [Ang-(1-7), ACE2, and Mas] may take part of the establishment and progression of cardiovascular and respiratory diseases. Importantly, these new components of the RAS, due to their counter regulatory actions, are candidates to serve as a concept to develop new cardiovascular and respiratory drugs. Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields. Single-stranded RNAs (ssRNAs), typically located at the ends of RNA hairpins and consisting of more than three unpaired residues, serve diverse structural and functional roles. They can fold onto neighboring RNA hairpins to form pseudoknots, essential architectural RNA elements involved in ribosomal frameshifting (1, 2) , hepatitis C internal ribosomal entry site (IRES) recognition (3, 4) and telomerase activity (5) . Messenger RNA (mRNA) degradation is prevented or promoted by 3 0 addition of a polyadenylated tail, which recruits essential protein cofactors (6) . Cleavage of the 5 0 transfer RNA (tRNA) leader by RNase P is a key step in tRNA maturation (7) . In riboswitches, ssRNA links the ligand-binding aptamer domain to the expression platform, providing the basis for communication between the two (8) (9) (10) .
Much of our understanding of the conformational behavior of ssRNA comes from high-resolution NMR and X-ray structures of RNA, in which ssRNA directly engages in tertiary or RNA-protein interactions. However, the atomic-level structural and dynamic behavior of these elements in the absence of these interactions remains unclear, in large part due to their high degree of flexibility. Several studies suggest that ssRNA polynucleotides adopt stacked and partially helical conformations, particularly adenine-rich sequences; however, the biological relevance of these structures is unclear (11) (12) (13) (14) (15) (16) (17) . Atomic-resolution studies of ssRNA are scarce: at present only one isosequential ssRNA and ssDNA sequence has been characterized by homonuclear NMR methods and shown to possess properties reminiscent of A-form and B-form helices, respectively (18) . Few MD studies have been performed on ssRNA, the majority of which use the AMBER force field (19, 20) to explore the impact of chemical modifications such as peptide nucleic acids (PNA) and O2 0 -methylation (21) (22) (23) (24) .
The class I prequeuosine riboswitch (queC), typically found in firmicute bacterial species, is commonly located in the 5 0 -untranslated region (UTR) of the queCDEF operon, which expresses proteins directly involved in the queuosine biosynthetic pathway (25) . The aptamer binds preQ 1 , an intermediate in queuosine synthesis, with high affinity to attenuate protein expression at either the transcription or translation level (25) . This class has the smallest minimal aptamer domain (34 nucleotides, nt) discovered to date, consisting of a small hairpin followed by a 12 nt ssRNA tail ( Figure 1A ). Upon ligand recognition, the highly conserved adenine-rich tail condenses into a pseudoknot, forming a host of interactions to both the hairpin and ligand, including A-minor 'kissing' interactions between the ssRNA polyadenine tract and the minor groove (26) (27) (28) (29) (30) . The activity of transcriptionregulating riboswitches, such as the Bacillus subtilis queC riboswitch, has been shown to depend on the kinetics of ligand binding as well as the rate of transcription (8) . Notably, the very small size of the queC riboswitch leaves very little time, in comparison to other switches, for ligand binding to take place prior to formation of the anti-terminator helix which, when formed, prevents terminator helix formation, thereby allowing gene expression to continue. For example, the B. subtilis FMN riboswitch, which is highly dependent upon the rate of polymerase and contains sites that locally pause polymerase to lengthen the ligand-binding window, has $70 nt between the minimal aptamer sequence and complete formation of the anti-terminator helix (8) . In comparison, the ligand-binding window for the queC riboswitch is $20 nt (26, 27) . How efficient ligand binding is achieved is unclear given that the ssRNA tail is thought to be highly disordered, and therefore capable of sampling a wide range of competing conformations.
Here, we use NMR chemical shifts, spin relaxation, and residual dipolar couplings (RDCs) in conjunction with REMD simulations using the recently updated CHARMM27 nucleic acid force field (31, 32) to explore the conformational properties of the 12 nt ssRNA tail from the queC aptamer domain and the impact of a single A-to-C mutation targeting the polyadenine tract. Our study unmasks a previously unappreciated level of complexity in ssRNA and suggests that these structures can serve as excellent model systems for testing and developing computational force fields.
Uniformly 13 C/ 15 N-labeled queC36 and C14U/C17U constructs were prepared by in vitro transcription as described previously (33) . Unlabeled wild-type (WT, 5 0 -AUAAAAAACUAA-3 0 ) and A29C (5 0 -AUAACAAA CUAA-3 0 ) RNAs were purchased from Integrated DNA Technologies (IDT) and purified using a C18 column (Waters) followed by lyophilization and reconstitution in NMR buffer (15 mM sodium phosphate, pH 6.4; 25 mM sodium chloride, 0.1 mM EDTA) containing 10% D 2 O by volume. 100% D 2 O samples were prepared by repeatedly lyophilizing the sample and replacing with 99.99% pure D 2 O (Sigma) three times. RNA concentrations ranged from 1.5 to 2.8 mM. AMP, UMP and CMP (Sigma) were directly dissolved into NMR buffer with no additional purification to 5 mM. For RDC measurements, samples were dialyzed into Millipore-purified ddH 2 O using 1 kDa dialysis tubing (Spectrum Labs), lyophilized, and reconstituted into 52.4 mg/ml Pf1 phage solution (34) (35) (36) in NMR buffer with 100% D 2 O (Asla Biotech). RNA concentrations in Pf1 phage ranged from 1.5 to 2 mM.
UV/Vis melting RNA samples (0.25-0.5 mM) were prepared in NMR buffer and the melting profiles measured between 275 K and 368 K using a Varian Bio 300 UV/Vis instrument equipped with a Cary Temperature Controller. The absorbance at 260 nm was recorded every 0.5 with a ramp rate of 0.5 /min. The two-state helix to coil melting transition was analyzed using
where A is the absorbance value at a given temperature T, A H is the absorbance of the fully helical ssRNA, A C is the absorbance of the fully random coil ssRNA, ÁS and ÁH are the entropy and enthalpy of the melting transition respectively, and R is the gas constant (37, 38) . Absorbance values were fitted to the above equation using the non-linear least squares fitting function in Origin 7 to determine thermodynamic parameters. The melting temperature (T m ) was determined by dividing the enthalpy by the entropy.
All NMR experiments were performed on a Avance Bruker 600 MHz NMR spectrometer equipped with a triple-resonance 5-mm cryogenic probe. NOESY experiments were performed at 277 K and 298 K using a mixing time of 350 ms (39) . 13 C spin relaxation experiments were performed at natural abundance and 298 K (40) . Relative order parameters were calculated by normalizing (2R 2 -R 1 ) to either A31 (C8) or C33 (C6). Relaxation parameters were computed using HydroNMR (41, 42) , assuming an idealized A-form structure, to obtain diffusion tensor parameters (t m and D ratio ), and in-house written software was used to compute R 2 /R 1 values as previously reported (33, 40) . Motionally averaged bond lengths of 1.104 Å were used for both C8 and C6 moieties as previously described (40, 43) . The following experimentally derived CSAs (s xx , s yy , s zz ) were used in the analysis: (89, 15, À104); (80, 5, À85); and (98.4, 9.2, À107.5) for C2, C8 and C6 moeties (43, 44) . IP-COSY experiments were performed at 277 K and 298 K to observe relative 3 J H1 0 -H2 0 scalar coupling crosspeak intensities (45) . Base and sugar 1 H-13 C splittings were measured from the difference between the upfield and downfield components of the 1 H-13 C doublet along the 1 H component using the narrow transverse relaxation-optimized spectroscopy (TROSY) component in the 13 C dimension as implemented in 2D 1 H-13 C S 3 CT-heteronuclear single quantum correlation (HSQC) experiments (46) . 2 H splittings were 71 and 69 Hz for WT and A29C, respectively. Idealized A-form structures were constructed using Insight II (Molecular Simulations, Inc.) correcting the propeller twist angles from +15 to À15 using an in-house program, as previously described (47) . The complementary strand was removed and the resulting ssRNA used in NMR data analysis. B-form helices were constructed using W3DNA (48) .
Simulation. REMD simulations were performed with the CHARMM simulation package (49) using the recently updated CHARMM27 nucleic acid force field (31, 32) and the MMTSB (50) tool set. Each REMD simulation comprised 40 replicas exponentially distributed over a temperature range from 278 K to 330 K, resulting in an average exchange acceptance ratio of 30%. Each replica was first equilibrated for 0.5 ns, restraining nucleotide heavy atoms, and subsequently run without any restraints for 10 ns, with exchange moves attempted every 0.5 ps.
Both WT and A29C RNAs were initially built in an ideal A-form helical configuration and served as the starting conformation in every simulation of REMD. The RNA was solvated in an 80-Å cubic box of pre-equilibrated TIP3P water (approximately 50 000 atoms). Twelve pairs of sodium chloride with an additional 11 sodium ions were added to the box, corresponding to the experimental ionic concentration of 40 mM.
Analysis. We utilized the last 5 ns of the REMD trajectory at 298 K for the following analysis. Base stacking energies were defined as the electrostatic and van der Waals interaction energies between the adjacent bases. The molecular orientation was expressed by the order parameters S 2 of the C-H bond vectors employing the model-free approach of Lipari and Szabo (51) . After a translational and rotational fit of each RNA snapshot to the ideal A-form helical structure, the order parameters were taken from the plateau phase of the correlation function, given by
where P 2 is the second order Legendre polynomial and * is the unit vector along the C-H dipole. Additionally, from the atomic coordinates we constructed the RDC values by first orienting an idealized A-form ssRNA helix into the principal axis system determined from the order tensor analysis of the experimental RDCs. Each frame of the trajectory was superimposed with this ideal helix followed by calculating the average of , 3 cos 2 À1 D E , where is the angle between a given bond vector (e.g. C1 0 H1 0 ) and the z-axis. The RDC values were then scaled by À82/r 3 , in which r is the C-H bond length and a factor of À82 is applied to shift the computed RDCs to the same scale as the NMR values. The average structure of the ssRNA was calculated as the structure with the minimal root-mean-square deviations from all RNA conformations in the 5 ns REMD trajectory.
Previous studies have shown that in the absence of ligand, the queC aptamer domain folds into a non-native hairpin, in which the 5 0 -strand frame-shifts to allow the first two guanine residues to base pair, with the 12 nt ssRNA tail lacking any tertiary interactions (26) . The 2D C-H NMR spectra of the 36 nt queC minimal aptamer domain ( Figure 1A ), in the absence of ligand, show severe resonance overlap and large variations in resonance intensities indicating a highly disordered conformation ( Figure 1B ). Excess imino proton resonances as well as 1 H-15 N NOE data indicate that the unbound queC aptamer domain is in equilibrium between native and non-native hairpin conformations (data not shown), consistent with previous NMR studies (26) .
The NMR spectra suggest that the unbound 36 nt queC minimal aptamer domain is highly disordered and that the ssRNA tail is not involved significantly in any tertiary interactions. To test this hypothesis further, we compared NMR spectra of the isolated 12 nt ssRNA tail with the corresponding spectra of the unbound queC aptamer. Remarkably, NMR spectra of the isolated 12 nt ssRNA tail overlay almost perfectly with the queC aptamer domain and specifically onto the highly intense resonances corresponding to highly disordered residues ( Figure 1B ). The only significant deviations are observed for A25 and U26, which are located at the junction site between the hairpin and the tail ( Figure 1B ). This indicates that in the absence of ligand, the ssRNA tail is not involved in any significant tertiary interactions under the NMR conditions (1 mM RNA, 25 mM sodium chloride, 15 mM sodium phosphate, pH 6.4, 0.1 mM EDTA, 298 K).
Similarly to the Fusobacterium nucleatum queC riboswitch, the B. subtilis queC aptamer forms kissing dimers, as observed in non-denaturing polyacrylamide gels (Supplementary Figure S1 ) (52) . To ensure that the dimer does not obstruct hairpin-tail interactions, we compared a mutant C14U/C17U construct characterized previously by Kang and coworkers to generate a ligandbound solution NMR structure (26) to the WT aptamer. MFold predicts the C14U/C17U mutations will reduce the dimer stability from À6.1 kcal/mol to À0.9 kcal/mol (53). While we observe removal of the kissing dimer, chemical shifts overall overlay extremely well between the WT queC aptamer and the C14U/C17U mutant (Supplementary Figure S1 ). Specifically, tail chemical shifts correspond extremely well to the 12 nt ssRNA, further suggesting that the tail does not participate in tertiary interactions in the absence of ligand under our NMR conditions. Strikingly, the spectra of the 12 nt ssRNA are well resolved, indicating that it does not adopt a completely random conformation ( Figure 1B and Supplementary Figure S2 ). This stands in stark contrast to corresponding spectra of a 12 nt polyuridine (polyU) ssRNA, well established to have a random-coil conformation (16) , which exhibits severe spectral overlap indicative of a highly disordered conformation (Supplementary Figure S2) . This structural order is observed in the ssRNA despite the lack of any observable imino protons and therefore any base pairing or secondary structure (Supplementary Figure S3) .
The 2D 1 H-1 H NOESY spectrum of the ssRNA shows abundant nuclear Overhauser effect (NOE) connectivities expected for a helical conformation, allowing the near complete assignment of base and sugar (H1 0 ) protons at 298 K (Supplementary Figure S3) . Particularly noteworthy are inter-base NOEs observed between adenine H8 protons within the polyadenine tract and between C33-U34 H6 protons, indicating significant base stacking within the polyadenine core at 277 K and decreased at 298 K (Supplementary Figure S3) (54) . Sequential NOEs are only observed for A25, U26, A35, and A36 upon decreasing the temperature from 298 K to 277 K, indicating a higher level of disorder at the terminal ends ( Figure 1C and Supplementary Figure S3 ). Furthermore, homonuclear three bond scalar couplings ( 3 J H1 0 -H2 0 ) indicate that residues within the polyadenine core adopt a C3 0 -endo sugar pucker conformation, consistent with an A-form-like geometry, with the tendency to adopt alternative sugar pucker conformations increasing towards terminal residues (Supplementary Figure S2) .
NMR chemical shifts are extremely sensitive probes of the local electronic environment for a given bond vector and can provide useful structural information (55) (56) (57) (58) . Highly disordered residues are expected to have chemical shifts similar to nucleotide monophosphates (NMPs). While the chemical shifts of terminal residues are similar to their NMP analogs, increasing differences are observed when approaching the polyadenine core with the greatest differences observed for A30-32 (Supplementary Figure S2 ). The directionality of the chemical shifts is consistent with increased formation of stacking interactions towards the center of the tail (57) . This is further supported by chemical shift perturbations in a trajectory toward the NMPs with increasing temperature (data not shown). Alternatively, addition of magnesium up to 4 mM results in slight chemical shift perturbations farther from NMPs, consistent with previous studies suggesting that increases in ionic strength stabilize ssRNA stacking interactions (59) (data not shown). In contrast polyU has near-identical ( 0.1 ppm) chemical shifts to UMP (Supplementary Figure S2) . Thus, consistent with NOE data, the chemical shift data suggest a comparatively stacked core with a growing level of disorder towards the terminal ends. Normalized resonance intensities (33) further support these observations, which gradually increase towards the terminal ends, consistent with a higher level of pico-to nanosecond motions (Supplementary Figure S2) .
The abundance of NOEs indicates significant base stacking interactions, which likely contribute to ordering of the tail. To probe the thermodynamic stability of the tail, we performed UV/Vis melting experiments to determine the melting temperature of the helix to coil transition. Consistent with previous studies of single-stranded nucleic acids, the melting profile of the ssRNA is extremely broad, characteristic of a non-cooperative transition (Figure 2A ) (37) . Previous studies of a 7 nt polyadenine ssRNA in similar buffer conditions yield analogous melting temperatures to those observed ($35 C compared to 31.7 ± 1.90 C) (37) .
We then used our REMD simulations to explore the temperature dependence of base stacking compared to the described UV/Vis melting curves. Base stacking energies from the REMD simulation between temperatures 278-330 K show a similar gradual decrease with increasing temperature and a similar, although reduced, T m value (experimental 31.7 ± 1.90 C compared to computed 20-25 C, as estimating the T 50 value from melting curve, Figure 2A) . However, the calculated base stacking energy plateaus around 320 K while the experimental slope begins to plateau around 330 K, indicating that stacking energies may be under-estimated in the REMD simulation or that additional unaccounted-for factors contribute to the ssRNA stability. Nevertheless, our data suggest that base stacking is the guiding force behind ssRNA stability, consistent with previous studies.
To gain further insights into the dynamic properties of the ssRNA at pico-to nanosecond timescales, we measured longitudinal (R 1 ) and transverse (R 2 ) carbon relaxation data for the nucleobases (C2 C6 C8) using 2D 13 C relaxation R 1 and R 1r NMR experiments (40) , where R 1 and R 2 values are determined using in-house software (Supplementary Figure S4) . These measurements represent the first nucleobase 13 C relaxation measurements performed on a ssRNA. The measured R 1 and R 2 values were used to compute order parameters (51) using S 2 = (2R 2 -R 1 ) (60), and normalized to yield a relative order parameter (S 2 rel ) describing the relative degree of order within a molecule ranging from 0 to 1, where 0 and 1 represent minimum and maximum order, respectively. The S 2 rel values were normalized against central residues A31 (C8) and C33 (C6). Resonance overlap prevented the normalization of C2 spins. Again, we observe a gradual reduction in S 2 rel indicating higher levels of disorder moving from central polyadenine residues (A28-C33) towards the terminal ends ( Figure 2B ).
We also computed the S 2 rel values based on the REMD simulation described above. The REMD simulations reproduce the general trends observed in the experiments; however, the simulations show significantly increased dynamics at the terminal ends compared to experimental values, with S 2 rel values approaching the dynamic limit ( Figure 2B ). Additionally, while experimental values have similar relative order parameters from A28-C33, large variations are observed in the REMD simulation, with A29-A30 being more ordered and A32 less ordered than experimentally observed ( Figure 2B ). These differences may reflect shortcomings in the force field and/or mismatch in the experimental/computational timescales since the REMD simulations likely probe fluctuations that extend beyond the picosecond timescales sensed by spin relaxation data.
The high level of disorder and motional coupling in the ssRNA prevents quantitative analysis of relaxation data using the model-free formalism, which assumes that internal and overall motions are decoupled from one another (51) . This makes it difficult if not impossible to assess the absolute level of disorder in the ssRNA; one can only make qualitative assessments about the relative disorder across different residues. However, it is noteworthy that even the comparatively high R 2 /R 1 values measured in the rigid core ($2.9, Figure 2C ) remain significantly lower than values predicted for a perfectly rigid helical ssRNA ($6.4, Supplementary Figure S4 ) as estimated using the program HYDRONMR (41, 42) . If we assume an overall diffusion tensor predicted by HYDRONMR, we find that central polyadenine residues are highly flexible with an estimated average NMR spin relaxation order parameter S 2 of $0.45 ( Figure 2C and Supplementary Figure S4) . Interestingly, similar though slightly smaller absolute S 2 values are calculated from the REMD simulations (on average S 2 $ 0.36 for core residues, Figure 2D ). These data indicate that despite measurable stacking interactions and a helical-like average conformation, the polyadenine core is highly disordered with residues experiencing fluctuations on the order of a ±40 cone angle (61) at pico-to nanosecond timescales.
To further probe the conformation of the ssRNA and extend the NMR timescale sensitivity to milliseconds, we measured RDCs (62,63) using 52.4 mg/ml Pf1 phage as an ordering medium. While most RNAs align optimally in $25 mg/ml of phage, a much higher concentration of phage was used for the ssRNA to ensure optimal alignment. To our knowledge, these are the first RDC measurements reported on a single-stranded nucleic acid. The RDCs measured between two nuclei depend on 3 cos 2 À1 2 D E , where is the angle between the inter-nuclear vector and the magnetic field and the angular bracket denotes a timeaverage over all orientations sampled at sub-millisecond timescales (62, 63) . RDCs were measured for base C5H5, C6H6, C8H8, C2H2 and sugar C1 0 H1 0 moieties (47) .
In general, isotropic motions tend to reduce the observed RDC value, approaching zero at the limit of spatially unrestricted isotropic motions (61, 64, 65) . In the ssRNA, large base C-H RDCs are measured in the polyadenine tract residues that gradually decrease at the termini ( Figure 3A ). Although small RDC values can also arise from static placement of the bond vector near the magic angle relative to the principal direction of order, the overall trends observed are consistent with NMR chemical shift and S 2 rel data suggesting that the RDCs indicate increased dynamic averaging at the termini ( Figure 3A) . Interestingly, the near-zero RDCs measured at terminal residues ( Figure 3A and Supplementary Figure S5 ) agree more closely to the REMD simulations compared to the S 2 rel values, indicating that the discrepancy between the measured and computed S 2 rel values may be due to truncation of the S 2 sensitivity to motions faster than nanoseconds. These results add to a growing number of NMR studies on different types of RNA showing that RDC data are capable of probing motions that are incompletely sensed by spin relaxation due to truncation of the time-sensitivity by overall correlation time of the molecule (64, (66) (67) (68) . Unfortunately, severe spectral overlap, particularly pronounced in the Pf1 phage sample, prevented measurement of several C1 0 H1 0 RDCs for the polyadenine core.
We subjected the RDCs (excluding RDCs for the two flexible residues from the terminal ends) to an order tensor analysis (47, 69, 70) assuming different input structures including single strands derived from idealized A-form and B-form helices, the REMD-averaged structure, and available ligand-bound X-ray and NMR structures (26, 27) . Despite the relatively small number of RDCs used in this analysis, we clearly observe a better fit with an A-form geometry (Q-factor 4.77%) as compared to all other conformations (Q-factor ! 16%) ( Figure 3B ). This is consistent with independently observed 3 J H1 0 -H2 0 scalar coupling crosspeaks, which indicate a C3 0 -endo sugar conformation for core residues in the tail, suggesting an A-form (and not B-form) helical geometry. The RDCs are in strong disagreement with preQ 1 -bound X-ray and NMR structures (PDBID: 3FU2 and 2L1V) indicating that the tail must undergo a transition from an A-form helical geometry towards the distinct helical conformation observed in the X-ray and NMR structures in which the A-form geometry is perturbed at the hairpin-tail junction, likely due to torsional strain from the ssRNA folding back upon the hairpin. The REMD-averaged structure has a Q-factor of 30%, indicating a better fit than ligand-bound structures, but is still outside the range considered to represent a good fit. Together, these data suggest that, on average, the ssRNA tail adopts an A-form like conformation. The good RDC fit to the A-form structure also suggests that averaging of the RDCs due to internal motions is largely isotropic in nature, causing a semi-uniform attenuation of the RDCs relative to values expected for an A-form structure. The dynamics could involve exchange between a stacked ordered conformation and unstacked highly disordered conformation, or local isotropic motions about the average A-form conformation.
As a further check on the accuracy of the A-form structure, we compared the principal direction of alignment (S zz ) determined experimentally using RDCs assuming a ssRNA A-form structure with the orientation predicted by PALES (71) using a ssRNA A-form structure. Surprisingly, we find that the experimentally determined S zz deviates from the helix axis by $19.8 ( Figure 3C ). Interestingly, PALES predicts a principal direction of order that deviates from the helix axis by 14.4 ; the S zz orientation predicted using PALES is in good agreement from that measured experimentally (deviation $ 5 ). The deviation from the helix axis can be attributed to the absence of the complementary strand, resulting in an overall shape with a long axis that is not coincident with the helical axis, as reported previously for a quadruplex DNA topology (72) .
To further test the conformational distribution from the REMD simulations, we used a number of simplifying assumptions to compute RDCs from the REMD trajectory. Snapshots from the REMD simulations were superimposed onto an idealized A-form helix oriented in the principal axis system determined using the experimental RDCs and the order tensor fit. RDCs were then arbitrarily scaled by À82/r 3 , in which r is the C-H bond length and accounts for bond length variations during the dynamics. We find excellent agreement between experimental and computed nucleobase RDCs; however, computed C1 0 H1 0 RDCs fail to reproduce observed RDCs, particularly for A32: while the magnitude is similar (18 Hz compared to À30 Hz) the sign differs, suggesting the orientation of the C1 0 H1 0 bond vector differs between experiment and simulation (Supplementary Figure S5) . C1 0 H1 0 RDCs are generally opposite in sign to base RDCs in a double-stranded A-form helix. However, back-calculated C1 0 H1 0 RDCs from the order tensor analysis assuming a ssRNA A-form helix are positive in sign, suggesting the C1 0 H1 0 orientation in the REMD simulations deviates from an A-form structure (Supplementary Figure S5) .
Taken together, the data show that the polyadenine tract is relatively ordered at 298 K, with a gradual reduction in order approaching the termini and that the base stacking interactions are the guiding force behind this order. To determine whether disrupting the polyadenine tract will destabilize the global structure, we substituted A29 within the polyadenine tract with a cytosine residue (referred to as A29C). Other types of mutations involving placements of uridine were not explored as these were expected to yield partially base paired conformations. As with the WT construct, we observed no imino protons, indicating the absence of any detectable base pairing and secondary structure (Supplementary Figure S7) . The 2D C-H spectra for the A29C mutant remain highly disperse, and the chemical shift perturbations relative to WT are clustered around the site of mutation (A28 and A30) ( Figure 4A and Supplementary Figure S6) . However, small but significant chemical shift perturbations relative to WT are also observed at more distant residues, including A27, A31, C33 and U34. These perturbations diminish when moving away from the center of the ssRNA and are basically absent in the highly flexible terminal residues ( Figure 4A and Supplementary Figure S6 ). Such longer-range perturbations suggest that the mutation may have a long-range effect possibly by influencing the stacking interactions of several nucleobases. A perturbation to stacking interactions is also supported by distinct NOE connectivities in A29C, which show weakened cross peaks to C29, and new crosspeaks between A28 (H2) and A30 (H1 0 ) that indicate C29 partially loops out to allow A28 to stack onto A30 (Supplementary Figure S7) . The melting temperature of the mutant is reduced by $5 C, and the base stacking energies are computed to be $2 kcal/mol lower compared to WT, indicating that the mutation likely destabilizes the stacking interactions (Supplementary Figure S6) .
Interestingly, many of the residues that experience chemical shift perturbations following the A29 to C29 mutation also exhibit a greater degree of dynamics as assessed by normalized resonance peak intensities in 2D C-H HSQC spectra (Supplementary Figure S6 ) and carbon relaxation data (R 1 and R 2 ) ( Figure 4B and Supplementary Figure S8 ). In particular, severe line broadening consistent with a slow exchange process occurring at micro-to millisecond timescales manifesting as reduced resonance intensities in 2D spectra and higher R 2 values is observed for C29 in the A29C mutant ( Supplementary  Figures S6 and S8 ). This is not observed for A29 in WT. Smaller but significant line broadening is also observed for residues A31, A32 and U34 (Supplementary Figure S8 ). This line broadening across several residues may reflect exchange between stacked and unstacked conformations. Higher intensities as well as reduced S 2 rel values are observed for residues A27 and A28, indicating a greater degree of fast pico-to nanosecond dynamics ( Supplementary Figures S6 and S8) . Note that the high R 2 and weak signal intensity leads to a higher error in the R 2 /R 1 measurements, particularly for C29.
Although the A29C RDCs are generally in good agreement with the WT RDCs, variations are observed for a number of residues (U26, A27, A31) that indicate differences in conformation and/or dynamic behavior ( Figure 4C ). Though an order tensor analysis of 13 RDCs shows best agreement with an A-form structure, the quality of the fit is not as good as that observed for WT (Q-factor = 8.77%, Figure 4D ). The S zz direction measured for A29C when assuming an A-form structure deviates substantially from that predicted using PALES ($11 , Supplementary Figure S9 ). These data suggest that A29C deviates from an idealized A-form structure as compared to WT. These deviations may reflect static and/or dynamic bending about the C29 pivot point, possibly arising from looping out of this residue from the helical stack. Such a conformation is observed in the REMD simulations of A29C $1% but not in WT (data not shown).
In general, the REMD simulations predict the NMR data measured for A29C with reduced quality to that noted for WT. Interestingly, the computed absolute S 2 values indicate a global reduction in order for A29C, particularly for residues A27-C33 (Supplementary Figure  S8) , whereas NMR relaxation parameters between WT and A29C are more similar, suggesting comparable global order parameters. The REMD simulations reveal enhanced dynamics at C29 consistent with the NMR chemical exchange data. The REMD simulation also suggests increased dynamics at A32, which is not observed experimentally: although slightly reduced, the S 2 rel is within error of A29-A31 values (S 2 rel of 1) (Supplementary Figure S8) . Computed RDCs agree reasonably with measured RDCs, although the C1 0 H1 0 RDCs are opposite in sign as observed in the comparison between WT NMR and REMD-calculated RDCs. The Q-factor comparing the average REMD structure to measured RDCs is 70%; however, removal of A28 C8H8, A30 C2H2 and A30 C1 0 H1 0 RDCs improves the Q-factor significantly. This improvement is observed only for the REMD structure ( Figure 4D ), indicating that these residues, localized about the mutation site, adopt non-Aform conformations and likely experience perturbations from the increased dynamics at C29. The difference in timescales between the REMD simulations and NMR may be another factor leading to the observed discrepancies. Nevertheless, MD and NMR data both indicate significant dynamics at the mutation site with perturbations extending toward the 3 0 end of the ssRNA. ssRNA tail conformation and dynamics optimized for ligand docking in queC aptamer
One of the main questions we set out to explore during the course of our studies was how the queC aptamer manages to efficiently bind its cognate ligand despite the small commitment time available in the kinetic switch and the large conformational space that may be available to a highly disordered ssRNA, which would have to search many competing conformations before arriving at the ligand bound pseudoknot conformation. Our study reveals that the ssRNA is not entirely disordered, but rather, has the character of a stacked A-form-like helical conformation which may effectively reduce the conformational search of the ssRNA, promoting efficient docking onto the hairpin to form the pseudoknot. Moreover, our study uncovers a greater degree of flexibility towards the terminal ends, particularly the 5 0 -end which forms the pivot point for docking the ssRNA tail onto the hairpin.
The NMR data clearly show the absence of any pre-existing tertiary interactions involving the ssRNA tail in the unbound queC aptamer domain. This together with our findings regarding the conformational behavior of the unbound ssRNA tail suggests the following model for ligand binding ( Figure 5 ). In the absence of ligand, the ssRNA tail is disordered but on average forms an A-form helix-like conformation, which can efficiently explore conformational space about a highly flexible junction. The ligand may transiently form encounter complexes when the tail is close in space to the P1 hairpin, and possibly with the help of divalent ions such as calcium (27, 73) , triggering the necessary conformational changes required to form the pseudoknot and binding pocket. This finding is consistent with computational modeling of the ligand binding mechanism in which A-minor tertiary interactions form first, followed by pseudoknot formation (30) and may explain the fast ligand binding rate observed in the related F. nucleatum queC riboswitch (52). Our results, including the observation of greater dynamics in the mutant, provide a framework for more rigorous testing of this proposed model with future in vitro and in vivo studies.
Our study shows that ssRNA can exhibit complex conformational behavior, including variable levels of stacking and propensities to form an A-form helical conformation across the polynucleotide chain, and also, the ability to interrupt stacked residues by introducing sequence-specific kinks and/or distortions. While it has been known for some time that polyadenine stretches tend to stack and form helical conformations (13, 14, 16, 18, 37) , the details of this helical geometry were difficult to decipher based solely on NOE-based NMR data. Our RDC measurements on the ssRNA, together with scalar coupling constant measurements, strongly suggest that the polyadenine tract forms an A-form-like conformation in the WT ssRNA. Our results also unveil dynamic complexity in ssRNA, including a gradual increase in disorder occurring towards the terminal ends that is reminiscent of unfolded polypeptide chains (74) , and also, slower sequence-specific dynamics occurring at micro-to millisecond timescales that may involve transient stacking/unstacking motions that may result in kinking of the ssRNA. Altogether, our studies show that 'structured' ssRNA exhibits exquisite quality spectra and can be studied quantitatively using NMR-based structure and dynamics measurements.
The REMD simulations recapitulate many of the key features and trends observed based on the melting and NMR data, including the existence of stacking interactions that are weakened by the A29C mutation, the formation of helical geometry that may be kinked in A29C at the mutation site, and an increase in dynamic disorder towards the terminal ends and localized about C29 in the mutant. However, the REMD simulations showed weaker agreement with sugar RDCs or sugar conformation, particularly in the A29C mutant, and had increased dynamics compared to the NMR data. Prior studies on HIV-1 TAR RNA noted higher levels of dynamics in CHARMM simulations compared to NMR measurements (75) . Our studies indicate that suboptimal base stacking energies may be a source of these excess dynamics. However, a quantitative assessment of the simulations requires the application of domain-elongation methods to rigorously decouple internal and overall motions, and make it possible to quantitatively predict NMR measurements (33, (75) (76) (77) . In addition, MD simulations that retain aspects of time are required to compare the rates of dynamics observed by relaxation and exchange broadening type measurements. The simplicity of ssRNA offers a much needed model system for such studies directed at rigorously examining currently used nucleic acid force fields.
Finally, our results suggest that the conformational properties of the ssRNA tail are optimized to allow the queC riboswitch to efficiently bind ligands within the short commitment time available to this kinetic switch. In particular, the pre-stacked ssRNA tail can efficiently rotate about a flexible hinge against the hairpin loop, and explore conformational space efficiently for rapid ligand binding. This pre-stacking about dynamic hinges may be a general feature of many ssRNAs that can play different architectural roles in a variety of RNA contexts.
Supplementary Data are available at NAR Online: Supplementary Figures S1-S9.  Soluble RAGE as a severity marker in community acquired pneumonia associated sepsis BACKGROUND: Community-acquired pneumonia (CAP) is considered the most important cause of death from infectious disease in developed countries. Severity assessment scores partially address the difficulties in identifying high-risk patients. A lack of specific and valid pathophysiologic severity markers affect early and effective sepsis therapy. HMGB-1, sRAGE and RAGE have been involved in sepsis and their potential as severity markers has been proposed. The aim of this study was to evaluate HMGB-1, RAGE and sRAGE levels in patients with CAP-associated sepsis and determine their possible association with clinical outcome. METHOD: We evaluated 33 patients with CAP-associated sepsis admitted to the emergency room and followed in the medical wards. Severity assessment scores (CURB-65, PSI, APACHE II, SOFA) and serologic markers (HMGB-1, RAGE, sRAGE) were evaluated on admission. RESULTS: Thirty patients with a diagnosis of CAP-associated sepsis were enrolled in the study within 24 hours after admission. Fourteen (46.6%) had pandemic (H1N1) influenza A virus, 2 (6.6%) had seasonal influenza A and 14 other diagnoses. Of the patients in the study group, 16 (53.3%) had a fatal outcome. ARDS was observed in 17 (56.6%) and a total of 22 patients had severe sepsis on admission (73%). The SOFA score showed the greatest difference between surviving and non-surviving groups (P = .003) with similar results in ARDS patients (P = .005). sRAGE levels tended to be higher in non-surviving (P = .058) and ARDS patients (P = .058). Logistic regression modeling demonstrated that SOFA (P = .013) and sRAGE (P = .05) were the only variables that modified the probability of a fatal outcome. CONCLUSION: The association of elevated sRAGE with a fatal outcome suggests that it may have an independent causal effect in CAP. SOFA scores were the only clinical factor with the ability to identify surviving and ARDS patients. Community-acquired pneumonia (CAP) is considered the leading cause of death from infectious disease in developed countries [1] . In Mexico, the annual estimated incidence is100 to 230 cases per 100, 000 inhabitants, causing an alarming impact on public health since 25% of these cases require hospitalization [2] . Severity assessment scores help identify high-risk patients that need hospital therapy; however, the lack of specific and valid pathophysiologic severity markers affects early effective interventions. The recent H1N1 influenza pandemic (p2009A H1N1 or S-OIV) was associated with an increase in cases of CAP that required hospitalization and continues to be a national public health threat [3] [4] [5] [6] . Although the mortality rate was only 1.8%, 31% of patients with severe disease were admitted to an intensive care unit, and 14%-46% died [7] [8] [9] [10] . The first 18 cases, seen from March 24 to April 24, 2009 were reported at the National Institute of Respiratory Diseases in Mexico City. More than half of the patients were between 13 and 47 years of age. Twelve patients required mechanical ventilation and seven died (38%) [3] . Increased mortality was associated with systemic manifestations and complications of CAP with sepsis being the most common and challenging.
Physicians may underestimate the severity of CAP, which can lead to insufficiently aggressive interventions inpatients with a high risk of complications [11, 12] . Scoring systems have been used to calculate the probability of morbidity or mortality. The most studied scoring system, the Pneumonia Severity Index (PSI), is a 20-point score that classifies patients into five risk categories based on their percentage of risk of death within 30 days. This score was useful in patients with a low risk of death (0.1%-0.7%) and was recommended for outpatient therapy [13] . However, PSI is limited by its number of variables, making it complex for the emergency room setting [14] .
The British Thoracic Society subsequently designed a simpler prediction tool, the confusion, urea, respiration, and blood pressure (CURB) score, also based on the risk of 30 day mortality [12] . In 2003, Lim and colleagues added age ≥65 years as a risk factor to create CURB-65 [15] . CURB-65 is significantly easier to use than PSI since it has only five variables with a single point awarded for each. CURB-65 is recommended together with PSI.
Other severity assessments, such as the Acute Physiology and Chronic Health Evaluation II (APACHE II), are commonly used in intensive care units to determine a patient's outcome. The Sequential Organ Failure Assessment score (SOFA) on admission has also been used with results similar to APACHE II. The combination of these may improve sensitivity [16] .
Current severity assessment scores only partially overcome the difficulties in identifying patients with severe disease, providing objective classifications of patients into high-risk categories [17] . Thus, there is increasing interest in improving diagnostic accuracy by measuring inflammatory mediators that participate in sepsis. In 1999, Wang et al. reported that high-mobility group box 1 (HMGB1) was detectable in plasma of mice exposed to a lipopolysaccharide. Removal of circulating HMGB1 with a specific antibody improved survival. HMGB1 has delayed kinetics and remains in circulation longer than the initial studied immunologic mediators. HMGB-1 induces the release of proinflammatory and procoagulant factors and when injected into mice, leads to the development of clinical features of sepsis and multiorgan dysfunction [18] .
Angus and colleagues studied serum HMGB1 levels in a subgroup of 122 patients with CAP and observed elevated levels more than a week after presentation with high circulating levels associated with greater mortality [19] . These data differ from Sunden-Cullberg et al., who found lower HMGB1 serum levels in non-survivors of severe sepsis [20] . There have also been studies evaluating the role of HMGB-1's receptor, the receptor for advanced glycation end products (RAGE) [21] . Experimental studies demonstrate that RAGE-dependent activation of nuclear factor-kappa B (NF-B) plays a central role in modulating mortality after cecal ligation and puncture [22] . RAGE possesses a secretory isoform known as soluble RAGE (sRAGE), which maintains the extracellular ligand-binding domain but lacks the cytosolic and transmembrane domains. sRAGE has the same ligand binding specificity and competes with cell-bound RAGE, serving as a decoy that abolishes cell activation. In sepsis models, the administration of exogenous sRAGE slightly improved survival [22] . Evidence suggests that human endogenous sRAGE is generated by alternative splicing of RAGE mRNA, or alternatively, by proteolytic cleavage from membranous RAGE [23] . This former mechanism was considered to be a cell regulating mechanism, permitting restoration of homeostasis and survival.
Since there is very little knowledge of the role of HMGB-1/RAGE in the clinical setting of CAP-associated sepsis, we decided to perform a pilot study to investigate of HMGB-1, RAGE and sRAGE levels in septic patients with CAP and identify if there is a correlation with severity assessment scores.
This observational clinical study included patients evaluated at the UANL University Hospital in Monterrey, Mexico. The Bioethics Committee of the School of Medicine of the Universidad Autonoma de Nuevo Leon previously approved this project and written informed consent was obtained from the patient or a legal representative. Thirtythree consecutive patients, from July 2009 through August 2010, were enrolled in the study within the first 24 hours of their arrival to the emergency room with sepsis secondary to CAP. They were followed-up either in the general ward or in the intensive care unit. Patients were classified according to the Sepsis Consensus Conference of 1992 [24] and the Infectious Diseases Society of America. Clinical data, diagnosis, treatment modalities, and blood samples were collected.
The severity of CAP was estimated using the following scores: CURB-65, PSI, APACHE II, and SOFA. To be enrolled, subjects had to be ≥ 18 yrs of age and have both a clinical diagnosis of pneumonia and a new pulmonary infiltrate on chest X-ray. Patients with hospital-acquired pneumonia, an episode of pneumonia in the last 30 days, pulmonary tuberculosis, pregnancy, palliative care, cancer, human immunodeficiency virus infection, chronic steroid use, acute or chronic viral liver disease, and chronic renal disorders were excluded from the study.
At enrollment, blood samples were taken, and RAGE receptor was immediately detected by flow cytometry, determining its mean fluorescence intensity. Subsequently HMGB-1 and sRAGE antigen were determined in plasma by enzyme-linked immunosorbent assay (ELISA). At the same time, CURB-65, PSI, APACHE II score, and SOFA score were documented. During the patient's hospital stay we evaluated the presence of acute respiratory distress syndrome (ARDS). Also, a follow-up at 28 days was performed to distinguish between survivors and non-survivors. After enrollment of patients, data was blinded to avoid potential bias.
Blood samples were obtained from each patient and sera were recovered to test HMGB1 and soluble RAGE levels. The HMGB1 ELISA kit (IBL International, Germany) and the soluble RAGE ELISA kit (R&D system, Minneapolis, Mn) were used according to the manufacturer's recommendations.
A sample of whole blood, anticoagulated with EDTA, from each patient was used for flow cytometry analysis. One hundred microliters of whole blood was incubated with a rabbit anti-human RAGE antibody (Chemicon, Billerica, MA) for 15 min at room temperature. A Goat anti-Rabbit IgG FITC conjugate (Chemicon) was used for flow cytometry detection. Samples were incubated for 15 min at room temperature in darkness. Lysis solution was then added to eliminate erythrocytes and two washes with PBS (0.1 M, pH 7.2) were done centrifuging at 220-240 × g for10 min in each time. Leukocytes were recovered by centrifugation in the same condition and the samples were resuspended in 1 ml of FACS flow (BD Biosciences, Pharmingen) for cytofluorometric analysis (FACS SortCalibur, BD, San Jose, CA). Then 10, 000 cells, in which mean fluorescence intensity (MFI) was obtained and nonspecific fluorescence was deleted, were analyzed.
All statistical analyses were performed in SPSS (SPSS, version 13.0), assuming a statistical significance of P ≤ .05. The general descriptive characteristics are presented as means, standard deviations, medians, and percentages. We compared the severity assessment scores and HMGB-1, RAGE and sRAGE levels in surviving and non-surviving patients at 28 days, and between ARDS and non-ARDS patients using a statistical inferential analysis with the U Mann-Whitney nonparametric test. Using the normality tests Kolmogorov-Smirnov with the Lilliefors correction and Shapiro-Wilk, we determined if the obtained values came from a normally distributed population. We present data as plots of admission day medians.
We used multivariate logistic regression and Cox regression models with a backward technique to select variables that predicted a fatal outcome, including clinical severity and the inflammatory markers studied, such as age, gender, CURB-65, SOFA score, APACHE II, pneumonia severity index, HMGB-1, sRAGE and RAGE. Correlation between clinical severity scores and immunologic markers at admission, and between the markers, was detected using Spearman's correlation coefficient.
Acute organ dysfunction was defined as a new Sequential Organ Failure Assessment score [25] ≥3 in any of six organ systems, following the European Society of Intensive Care Medicine sepsis occurrence in the acutely ill patient study criteria [26] . We also added patients that met the following alternate definition to the analyses: an increase of 1 Sequential Organ Failure Assessment point in any two organ systems, 2 points in one system, or an absolute score of ≥3 in the respiratory system, similar to criteria used in several large trials of antisepsis agents [27] [28] [29] .
Thirty-three patients with confirmed CAP were included in the study; three were excluded (one was pregnant and two because of problems with their blood sample). Of the remaining 30 patients, 14 (46.6%) had pandemic (H1N1) 2009 influenza virus confirmed by PCR and 2 patients (6.9%) had seasonal influenza A. No etiologic agent was found in the other 14 patients. Twenty-two patients (73.3%) had severe sepsis or septic shock detected at admission; of these, 17 developed acute respiratory distress syndrome (ARDS). The mortality rate of the study group was a total of 16 patients (53.3%) at the end of the 28 days. There were eight who never developed severe sepsis and survived to hospital discharge, six who developed severe sepsis and survived to discharge, and 16 who developed severe sepsis and died in the hospital.
There were no significant differences between survival and non-survival patients with respect to age, gender, ethnicity, microbiological etiology, initial CURB-65, initial PSI class, initial APACHE II score, or emergency room length of stay (P value range, .07-.99) nor between ARDS and non-ARDS patients with respect to gender, ethnicity, microbiological etiology, initial CURB-65, initial PSI class, initial APACHE II score, or emergency room length of stay (P value range, .36-.77) ( Figure 1A) . Group characteristics are provided in Table 1 . Compared with those who did not survive, those who survived had lower SOFA scores (5.5, CI: 4.9-7.7 versus 3, CI: 2.3-4.2) ( Figure 1B) . Compared with patients that did not develop ARDS, those with ARDS had higher SOFA scores (3, CI: 2.1-4.9 versus 5, CI: 4.6-7.1) and were younger ( Figure 2B and Table 1 ).
There were no statistically different RAGE, sRAGE and HMGB-1 levels found during early CAP-associated sepsis in ARDS or non-surviving patients ( Figure 1C , Figure 1E , Figure 2A and Table 2 ). No difference was found between influenza A H1N1 infected patients and the rest of the study group (2767 ± 1655 vs 2174 ± 1344, P = .327). We did not find a correlation between immunological molecules and severity assessment scores using Spearman's correlation coefficient (P value range = .16-.99). Finally, none of the studied severity assessment scores correlated with each other (P value range = .18-.79).
Using a logistic regression model involving age, gender, APACHE II, SOFA, HMGB-1, sRAGE and RAGE, we found that the only variables that modified the probability of the patient having a fatal outcome were SOFA (P = .013) with a relative risk of surviving of .347 (CI: .151-.797); and sRAGE (P = .05) with a relative risk of surviving of .998 (CI: .998-1) ( Table 3 ).
According to multivariate Cox regression analysis we found that a high SOFA score was an independent predictor of non-survival (hazard ratio 1.53, CI: 1.2-1.97, P = .001) ( Table 4 ).
We found that SOFA scores and the measurement of sRAGE levels in patients with CAP-associated sepsis helped predict survival. To date, this is the first study that analyses the levels of both of these molecules (the "HMGB-1" ligand and the "RAGE" receptor) in the inflammatory cascade of patients with CAP-associated sepsis. In Mexico, as in developed countries, CAP continues to be an important cause of death from infectious disease [1] with an elevated cost to public health. This was particularly evident with the H1N1 (2009, S-OIV) influenza virus pandemic [8] . Overall mortality is about 50% in patients with CAP that develop septic shock [25] . Although there has been intense research on the pathophysiology of CAP and its severe forms, such as ARDS, only slight improvements in new and effective treatment strategies have occurred.
Despite the identification of several recent molecules in patients with infection, such as the receptor expressed on myeloid cells-1 (TREM-1), these lack specificity in sepsis pathophysiology [26] [27] [28] . Discovery of markers may add additional information, increasing the validity of clinical estimates and permitting early, aggressive, and effective sepsis therapy. This justifies every effort to further explore the paradigm of biomarkers in the area of pulmonary infections [29] . We still lack efficient tools to identify patients with CAP who are likely to develop severe complications. Current clinical severity scores partially limit these difficulties, but are far from perfect. In our study, CURB-65, APACHE II and PSI .001 demonstrated no difference between groups (fatal outcome and ARDS). Recently published studies have found that CURB-65 dose not reliably distinguish patients with pandemic influenza CAP who will have good or poor outcomes [30, 31] . In the case of PSI, this could represent its higher ability to detect mild cases; although, this could be explained by the small number of patients in our study. In contrast, we noticed that SOFA scores, although not specific for CAP, were significantly higher in non-surviving or ARDS patients. Thus, in spite of the wide variety of etiologies, this last organ dysfunction score seems to be useful in patients with CAP.
It is well known that the recognition receptor "RAGE" and HMGB-1 play a central role in the innate immune system with an impact on its perpetuation and amplification [22] . RAGE stimulation results in sustained NF-B activation, which may be a predictor of severity in sepsis [32] . Conditions that induce NF-B also increase RAGE expression, which in turn produces sustained inflammation; this is seen in CAP, where RAGE ligands are abundantly present. Angus et al. found that CAP patients had higher HMGB-1 concentrations, and this correlated with mortality [19] . Gaini et al. also found higher levels of HMGB-1 in CAP [33] . Studies of severe influenza CAP demonstrated an association between excessive release of cytokines and increased mortality [34, 35] . However, Alleva et al. found in a murine model of severe influenza that HMGB-1 concentrations were not increased in plasma at the time of peak mortality, and peak levels of HMGB1 did not occur until relatively late in infection [36] .
Recently, Bopp et al. demonstrated that sRAGE concentrations in sepsis patients were higher in non-survivors when compared with survivors. They concluded that larger clinical trials should study the potential role of sRAGE as a new sepsis marker [37] . However, sRAGE has been used in animal models to block HMGB-1's binding to the RAGE receptor, leading to increased survival. This data indicates that HMGB-1 and RAGE participate in sepsis, including sepsis patients with CAP.
After developing multivariate regression models using backward selection techniques, we found that sRAGE and SOFA predicted survival; although the statistical significance was greater for SOFA, a limitation of our study is the small number of patients. One explanation for the elevated sRAGE levels could be an increased gene expression of RAGE in patients with sepsis [22, 38] . Since we know that RAGE participates in tissue damage [39] , it could represent a marker for cellular damage in sepsis.
As mentioned previously, there were elevated concentrations of sRAGE on admission in those with a fatal outcome, but without statistical significance. The same was observed in those patients who developed ARDS. On the other hand, receptor RAGE and HMGB-1 demonstrated lower differences between groups. Larger studies will be necessary to investigate the role of these potential sepsis markers.
The elevated levels of sRAGE found in our study, as in others, might represent the septic status of the patients as splice-variants of RAGE or shed variants of cell surface RAGE. In contrast to animal studies where a protective effect of sRAGE was seen, we found that sRAGE levels were higher in patients with more inflammation and in non-survivors. This finding could be related to shed variants of cell surface RAGE but this aspect was not one of our objectives. The ELISA we used did not differentiate between splicing variants and the shed variants of RAGE.
To the best of our knowledge, this is the second study that finds higher sRAGE levels in plasma of sepsis nonsurvivors compared with survivors [37] . This has Variables included in equation: RAGE, sRAGE, HMGB-1, SOFA score, APACHE II score, gender, age discrepancies with mouse model studies of sepsis after CLP [22] . This could be in part explained by different kinds of sepsis, different etiologic agents, and what was difficult to determine in our study, the time of measurement after the immunologic process started. We do not know if sRAGE concentrations were enough to bind HMGB-1, after they had scavenged AGEs and other RAGE ligands. Moreover, the higher concentrations found in sicker patients could represent sRAGE modified structurally and functionally during sepsis, diminishing its binding and neutralizing capacity.
Plasma sRAGE levels are elevated in CAP patients. sRAGE performed as an independent factor affecting the probability of a fatal outcome. Interestingly, the SOFA score demonstrated greater accuracy with the ability to differentiate between surviving/non-surviving and ARDS/non-ARDS groups.
Abbreviations APACHE II score: Acute Physiology and Chronic Health Evaluation II; ARDS: Acute respiratory distress syndrome; CAP: Community-acquired pneumonia; CURB-65: Confusion, urea, respiration, blood pressure and age ≥65 years; EDTA: Ethylenediaminetetraacetic acid: ELISA: Enzyme-Linked ImmunoSorbent Assay; FACS flow: Fluorescence-Activated Cell Sorting, flow cytometry; FITC: Fluorescein isothiocyanate; HMGB-1: High-mobility group box 1; MFI: Mean fluorescence intensity; NF-κB: Nuclear factor-kappa B; PSI: Pneumonia Severity Index; RAGE: Receptor for advanced glycation end products; sRAGE: Soluble RAGE. Virus Identification in Unknown Tropical Febrile Illness Cases Using Deep Sequencing Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness. Viral infections pose a significant global health burden, especially in the developing world where most infectious disease deaths occur in children and are commonly due to preventable or treatable agents. Effective diagnostic and surveillance tools are crucial for reducing disability-adjusted-life-years (DALYs) due to infectious agents and for bolstering elimination and treatment programs [1] . Previously unrecognized and novel pathogens continually emerge due to globalization, climate change, and environmental encroachment, and pose important diagnostic challenges [2, 3] .
Dengue virus (DENV) infection is the most common arthropodborne viral disease of humans, with an estimated 50-100 million clinical infections occurring annually worldwide [4] . DENV infection manifests clinically as dengue fever or the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) [4] . The increased spread of dengue virus and its mosquito vectors in many subtropical regions over the past several decades, especially in Latin America and Asia [5] , highlights the need for additional methods of dengue virus surveillance. Diagnosing dengue relies on detecting viral nucleic acid or antigens in the blood or confirming the presence of anti-DENV IgM and IgG antibodies and therefore traditionally depends on RT-PCR, ELISA, and viral cell culture methods [5] [6] [7] . Dengue diagnostics are of crucial importance due to its broad spectrum of clinical presentations, global emergence and spread, unique disease epidemiology, and possible clinical relation to other as-yet unknown tropical febrile pathogens.
Traditional viral detection methods, such as serology, virus isolation, and PCR, are optimized for the detection of known agents [2] . However, novel and highly divergent viruses are not easily detected by approaches that rely on a priori sequence, antigen, or cell tropism knowledge. PCR-based assays that employ degenerate primers may successfully target conserved regions within related virus groups, but unlike bacteria, viruses lack universally conserved genetic regions, such as ribosomal RNA, that can be exploited to amplify all viruses [8] .
Metagenomic analysis enables more systemic detection of both known and novel viral pathogens [9] [10] [11] [12] and is approached through a variety of microarray and sequencing strategies [13, 14] . The Virochip is a pan-viral microarray platform that has been previously utilized in the detection and discovery of viruses from both human and animal samples [15] [16] [17] [18] [19] . Deep sequencing and shotgun sequencing of human clinical samples has been used for viral detection [20] [21] [22] [23] , novel virus discovery [24] [25] [26] [27] , and divergent virus genome recovery [28] . Viral metagenomic approaches have also been employed as a diagnostic supplement to pathogen detection as part of public health monitoring systems [22] , but have been limited to shotgun sequencing of viral-enriched libraries and have yet to utilize deep sequencing data. Currently available sequencing platforms can generate millions to billions of sequencing reads per run, far exceeding large-scale shotgun sequencing [13] . Deep sequencing of clinical samples, in which hundreds of thousands to millions of sequencing reads are generated per sample, can be incorporated into stepwise virus detection pipelines [29] . Database searches using Basic Local Alignment Search Tool (BLAST) and other alignment tools [30] can be used to identify sequences in samples that correspond to known and novel viruses, including those present at low concentrations or deriving from viruses that may be too divergent to be detected with PCR or microarray methods. Deep sequencing represents an unbiased, highly sensitive method for identifying viral nucleic acid in clinical samples.
This study describes the use of the Virochip microarray and deep sequencing for the direct viral diagnosis of serum from cases of acute pediatric febrile illness in a tropical urban setting. Patient clinical data and serum samples were collected between 2005 and 2009 as part of an ongoing pediatric dengue study in Managua, Nicaragua [31] . Virochip and deep sequencing were performed on positive control samples and on 123 dengue virus-negative serum samples. Using these methods, viruses were detected in 45 of 123 (37%) previously negative samples. Sequences derived from known and apparently divergent viruses. The viruses identified in some of the cases are known to induce symptoms consistent with those observed, though the definitive causative agent of these infections remains to be determined.
Acute serum samples were collected from suspected dengue cases at the Hospital Infantil Manuel de Jesús Rivera (HIMJR), the National Pediatric Reference Hospital in Managua, Nicaragua, after undergoing informed consent or the informed consent procedure. Patients were enrolled in the study if they presented with fever or history of fever less than 7 days and one or more of the following signs and symptoms: headache, arthralgia, myalgia, retro-orbital pain, positive tourniquet test, petechiae, or signs of bleeding. Patients with a defined diagnosis other than dengue, e.g. pneumonia, were excluded. Suspected dengue cases were tested for dengue virus (DENV) infection at the Centro Nacional de Diagnóstico y Referencia (CNDR) of the Nicaraguan Ministry of Health and were considered laboratory-confirmed if: 1) DENV was isolated, 2) DENV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), 3) seroconversion was observed by IgM capture enzyme-linked immunosorbent assay (ELISA) of paired acute and convalescent sera, or 4) a $4-fold increase in DENV-specific antibodies was demonstrated by inhibition ELISA in paired acute and convalescent sera [32] . All patients were aged 6 months to 14 years and presented between August 2005 and January 2009.
Approximately one half of the suspected dengue cases testing negative by all four dengue diagnostic assays were included in the metagenomics analysis described here. 34 cases (pools 1-4, see below) corresponded to the subset of patients who presented within 4 days of symptom onset and who reported both fever or history of fever and rash. 89 of the samples (pool 5) were selected randomly from among the remaining samples. As positive controls, seven samples (pool 5) that had been clinically diagnosed as virus positive were included. The study protocol was reviewed and approved by the Institutional Review Boards (IRB) of the University of California, Berkeley, and of the Nicaraguan Ministry of Health.
Total nucleic acid from 140 ml of serum was extracted using the QIAamp Viral RNA Isolation Kit (Qiagen), which co-purifies RNA and DNA. End-tagged dsDNA libraries were created essentially as previously described [28] . RNA was reverse transcribed in reactions containing 16 reaction buffer, 5 mM dithiothreitol, 1.25 mM dNTPs, 20 pmoles primer (59-CGC TCT TCC GAT CTN NNN NN-39), 100 U Superscript III (Invitrogen), and ,20 ng template. Following reverse transcription, Sequenase reaction buffer and 2 U of Sequenase DNA polymerase (Affymetrix) were added to samples for second strand synthesis. The Sequenase reactions were performed twice so that starting DNA templates would be converted into end-tagged library molecules. The resulting libraries were amplified by PCR using primer 59-CGC TCT TCC GAT CT-39. PCRs contained 16 reaction buffer, 2 mM primer, 0.25 mM dNTPs, 2 U Taq DNA polymerase, and 2 ml library template. Thermocycling conditions were 95uC for 2 min; 25 cycles of 95uC for 30 sec, 40uC for 30 sec, and 72uC for 1 minute, with a final extension of 5 minutes. These libraries were further processed for microarray hybridization and deep sequencing as described below.
For microarray hybridization, a fraction of each library was amplified by PCR as above but with a modified dNTP mixture including 5-(3-aminoallyl)-dUTP (Ambion) in lieu of 75% of the dTTP normally in the mixture. The resulting amino-allylcontaining DNA was purified using a DNA Clean and Concentrator-5 column (Zymo Research). The eluate was heat
Dengue virus infection is a global health concern, affecting as many as 100 million people annually worldwide. A critical first step to proper treatment and control of any virus infection is a correct diagnosis. Traditional diagnostic tests for viruses depend on amplification of conserved portions of the viral genome, detection of the binding of antibodies to viral proteins, or replication of the virus in cell cultures. These methods have a major shortcoming: they are unable to detect divergent or novel viruses for which a priori sequence, serological, or cellular tropism information is not known. In our study, we use two approaches, microarrays and deep sequencing, to virus identification that are less susceptible to such shortcomings. We used these unbiased tools to search for viruses in blood collected from Nicaraguan children with clinical symptoms indicating dengue virus infection, but for whom current dengue virus detection assays yielded negative results. We were able to identify both known and divergent viruses in about one third of previously negative samples, demonstrating the utility of these approaches to detect viruses in cases of unknown dengue-like illness. denatured at 95uC for 2 min, cooled briefly on ice, then fluorescently labeled in reactions containing 100 mM sodium bicarbonate pH 9, 10% DMSO, and 667 mM Cy3 mono NHS ester (GE Healthcare) for 1 hour at 25uC. Labeled DNA was purified using DNA-CC-5 columns and added to hybridization reactions containing 36SSC, 25 mM HEPES pH 7.4, and 0.25% SDS. Hybridization mixtures were heated at 95uC for 2 minutes, applied to microarrays, and hybridized overnight at 65uC. Following hybridization, arrays were washed twice in 0.576 SSC and 0.028% SDS and twice in 0.0576SSC, then scanned on an Axon GenePix 4000B microarray scanner. Three analysis tools were used to analyze Virochip data: E-predict [33] , Z-score analysis [34] , and cluster analysis [35] . An array was deemed positive for a particular virus if the virus was identified by at least two of these methods. Virochip results were deposited in the NCBI GEO database (GEO accession series: GSE28142).
For deep sequencing, the Illumina paired-end adapter sequences were appended to library molecules using PCR, essentially as previously described [28] . Library generation primers (Table S1) were modified from adapter A and adapter B sequences (Illumina). Samples were reverse transcribed and libraries were created and amplified as described above for the Virochip. Library molecules of approximately 300 bp were purified on a 4% native polyacrylamide gel, ethanol precipitated, and PCR amplified for 17 additional cycles using a 22-nt-long primer consisting of the 39-end of Illumina adapter A (primer 2) and the full-length 61-bp Illumina adapter B (primer 4) under the following conditions: 2 cycles of 94uC for 30 s, 40uC for 30 s, and 72uC for 1 min, followed by 15 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 1 min. Amplicons generated with the correct adapter topology (one end with adapter A and the other with adapter B) were approximately 355 bp and were separated by polyacrylamide gel electrophoresis from adapter A/A and adapter B/B amplicons, which migrate differently (approximately 40 bp smaller or larger than the expected size). An additional 10 cycles of PCR were then performed using the full-length adapter sequences as primers (primers 3 and 4). Libraries were validated by Sanger sequencing before high throughput sequencing. Following validation, samples were combined into five pools for sequencing. For pools one through four reverse transcription primers included a three or four-nucleotide barcode sequence at the 39-end. For pool five, barcodes were located internally in the adapter sequence. Each pool was sequenced on one lane of a flowcell on the Illumina Genome Analyzer II (pools 1-4) or HiSeq 2000 (pool 5). Pools' 1-4 molecules were sequenced as 67 nucleotide paired ends, and pool 5 molecules as 97 nucleotide paired ends. Paired-end sequencing was performed for several reasons: (1) to double the overall amount of data generated, (2) to double the amount of sequence information per molecule, and (3) to provide anchors from which additional sequence could be recovered by subsequent PCR.
In some cases, PCR and Sanger sequencing was used to confirm Virochip and deep sequencing calls and to recover additional sequence. Primer sequences are listed in Table S1 . PCR conditions were: 95uC for 2 minutes, 35 cycles of 95uC for 30 seconds, 50-60uC for 30 seconds (primer dependent), 72uC for 1 minute, and 72uC for 2 minutes. PCR products were sizeselected on an agarose gel, purified with the Purelink gel extraction kit (Invitrogen), cloned, and Sanger sequenced.
Full-length poliovirus genomic RNA was transcribed from MluI-linearized plasmid prib(+)XpA using T7 RNA polymerase as previously described [36] . Poliovirus RNA was mixed with HeLa total RNA in a dilution series ranging from 10 22 to 10 26 poliovirus gRNA per HeLa RNA. Randomly-primed dsDNA libraries were prepared, hybridized to the Virochip, and analyzed as described above.
Predicted circovirus-like replicase sequences were searched against the NCBI non-redundant protein database (BLASTx, E value 10 22 ). Aligning sequences were retrieved and consolidated using CD-HIT into a set of representative sequences [37] (CD-HIT version 4.5.4; parameters: -c 0.7). These sequences were aligned in Geneious [38] as a global alignment with free end gaps and trimmed to the 47 amino acid overlap shared by the two recovered sequences. A neighbor-joining tree was generated by Geneious Tree Builder [38] .
The initial FASTQ data from each pool's lane were binned by barcode. The barcode-split reads were trimmed of non-template deriving and potentially error-prone sequence: a randomly incorporated nucleotide (N), the barcode bases, and the sequence corresponding to the random hexamer, leaving 55 (pools 1, 2, and 4), 54 (pool 3), or 90 (pool 5) bases per read. The lowest complexity fraction was identified by sequences with LZW ratios (compressed size/uncompressed size) less than 0.45 [39] . Reads were aligned to the human genome (build hg18) first using BLAT [40] with the ''-fastMap'' flag, and after filtering, the remaining reads were aligned using BLAT without the flag. Paired reads for which at least one of the reads in the pair had at least 80% identity to the database were marked as human and removed from subsequent analyses. After removal of reads identified as human by BLAT, remaining reads were aligned and filtered by mapping to the human transcriptome using nucleotide BLAST (BLASTn version 2.2.21, word size 30, E value 10 23 ). Remaining reads were next aligned to the human genome using BLASTn (word size 30, E value 10 23 ), filtered, and again aligned to the human genome by BLASTn (word size 11, E value 10). After all human filtering, we reanalyzed the distribution of the complexity of reads and observed a relative enrichment of reads with LZW ratios lower than 0.54 (pools1-4) or 0.48 (pool5; different LZW ratio distributions are an inherent property of different read lengths), and those reads were removed from further analysis. To look for reads with viral homology, we searched the non-redundant nucleotide database (nt) using BLASTn (word size 20, E value 10 23 ). Reads that did not map to nt were aligned to the non-redundant protein database (nr) using translated BLAST (BLASTx, word size 4, E value 10).
In order to make specific virus-positive calls, we implemented a set of rules to minimize false positives while maintaining sensitivity. In order to reduce the number of false positive sequences that may share identity equally with both viral and non-viral genomes, we restricted our analysis to those queries whose best alignments were only to animal viral sequences. In a number of datasets, we detected human klassevirus 1, a virus identified and studied in our lab [26] , human poliovirus, used in our Virochip sensitivity experiments, sequences from mosquito densoviruses, also studied in the lab, as well as Moloney murine leukemia virus (MMLV), the polymerase of which was used in the sequence library preparation. We believe these reads represent lab contaminants, and others studies that prepared sequence libraries in the same location have reported similar findings [41] . To account for these contaminants, positive calls were only made on viruses for which there were more supporting reads than there were reads to any known contami-nant. Finally, in order to avoid making calls based on potentially spurious alignments, we considered only those viruses for which there were at least 10 reads supporting their presence.
We initially screened the serum samples with the Virochip pan viral detection microarray. This was done as a complement to the deep sequencing analysis and in order to compare the sensitivity of the two approaches. We included 7 blinded positive control samples that had been previously diagnosed in the clinic as being positive for DENV-2 (n = 4), DENV-1 (n = 1), or hepatitis A virus (HAV; n = 2). The Virochip successfully identified the correct virus in all of these positive controls, and in the case of the dengue virus positive samples, the correct serotype as well (Table 1) . We also identified ten samples positive for torque teno virus (TTV).
We applied in vitro transcribed poliovirus RNA diluted into HeLa cell total RNA to the Virochip as an additional positive control to quantify Virochip sensitivity. Using the E-predict analysis tool, the lowest detectable concentration of poliovirus was 1 viral RNA per 10 5 HeLa RNA molecules (approximately 10 polio gRNAs per cell equivalent of HeLa RNA; Figure S1 ). The raw reads were first separated by barcode and analyzed as individual data sets as described in the Methods. The bioinformatic filtering process consisted of removing low complexity and low quality sequences, then filtering sequences of human origin ( Figure 1 ). After the filtering steps, an average of 1.9% of the initial reads remained, with an absolute average of 60,000 reads remaining per sample ( Figure 1 and Table 1 ). A few of the barcode datasets appeared to have a larger non-human fraction. Upon further inspection, the non-human components were accounted for by known library preparation contaminants, such as E. coli and S. cerevisiae.
The reads remaining after filtering were then compared to sequences in the NCBI non-redundant nucleotide and protein databases using BLASTn and BLASTx respectively. Virus-derived sequences were detected in all 7 positive control samples and in 45/123 (37%) of previously negative serum samples (Table 1 ). In 78/123 (63%) samples, we were unable to identify virus sequence by our detection criteria (Methods).
We recovered virus sequences matching the expected viral genomes in all of the positive control samples. The fraction of viral sequences in the controls spanned 4 orders of magnitude, from 0.002% to 2.8% of total reads. The two HAV positive control samples (#401) were aliquots of the same serum sample and were processed and analyzed independently. The fraction of viral reads in the duplicates was within 4-fold (0.4% and 1.2%). This demonstrates that our library preparation, sequencing, and bioinformatics pipeline is capable of reproducibly detecting evidence of clinically relevant infections.
In addition to the controls, two non-control samples contained evidence of RNA virus sequence. Both samples had reads deriving from GB Virus C (GBV-C, also known as Hepatitis G Virus) and were essentially identical to GBV-C database sequences. We detected no sequences that best aligned to dsRNA viruses or to retroviruses (except for human endogenous retrovirus and contaminating MLV RT-derived sequences, see Methods).
Human Herpesvirus 6 (HHV-6) sequence was detected in 13/ 123 previously negative samples (10.6%). The HHV-6 positive samples had an average normalized read count of 145 HHV-6 reads per sample (range: 24-411), representing 0.002% to 0.02% of the datasets (Table 1) , and all of these reads possessed high sequence identity to the HHV-6B reference genome sequence (gi: 9633069). We generated alignments to the reference genome to investigate the depth and genomic position of the sequence coverage across the HHV-6 genome ( Figure 2) . Although the reads only constitute a relatively small fraction of each dataset, there is coverage across the entire genome and over many genes in most of the HHV-6 positive samples.
In addition to HHV-6, we detected Human Herpesvirus 4 (HHV-4, also known as Epstein Barr Virus) sequences in one sample. As with HHV-6, The HHV-4 sequences were virtually identical to previously reported sequences. One sample also contained reads similar to another dsDNA virus, African Swine Fever Virus (ASFV), which has been previously detected in human serum [42] . In this case, the reads best matched ASFV capsid sequences and were relatively divergent (47-51% amino acid identity; no similarity to non-ASFV sequences by BLASTx). Attempts to recover additional ASFV sequence by PCR were unsuccessful.
We also identified sequences derived from single-stranded DNA viruses in some samples. In one sample we detected Parvovirus B19-derived reads with high identity to database sequences. Sequences related to various members of the Anelloviridae virus family (TTVs) were detected in 21 (17%) samples. This frequency of detection is within the range reported previously for human serum [43, 44] . The TTV sequences ranged from 40-97% amino acid identity to their closest database matches. We did not pursue these sequences further, because TTVs are known to form a divergent family of viruses and are commonly detected in apparently healthy individuals.
Sequences similar to members of the Circoviridae family of ssDNA viruses were detected in 13/123 samples (10.6%). All of the sequences aligned to circovirus or circovirus-like replicase protein sequences. The alignments ranged from 36-84% amino acid identity, and appeared to derive from the replicase genes from multiple related species (Table 1) . Circovirus-like replicase sequences have been detected in human stool, animals, and environmental samples [45] [46] [47] [48] . We detected a range of 12 to 205 circovirus-like reads per positive sample ( Table 1 ). The low sequence coverage prohibited complete genome sequence assembly but informed sequence-specific primer design, from which we were often able to recover larger continuous regions of the replicase genes by PCR and Sanger sequencing (GenBank accessions JF781513, JN837698, and see Table S2 ).
We termed the extended replicase-like sequences Circovirus-like NI/2007 1-3 (Cvl-NI 1-3), and compared them to a representative set of other replicase sequences (Figure 3) . The Cvl-NI-1 sequence is most closely related to Circovirus-like virus RW-E (gi: 254688530), a circular single-stranded DNA virus previously found in reclaimed water samples in Florida [45] . The Cvl-NI-2 sequence is most closely related to a replicase sequence recovered [48] . The Cvl-NI-3 sequence did not overlap with the other sequences enough to be included in the phylogenetic analysis, but was most similar to Circovirus-like CB-A, a circovirus-like genome identified in a Chesapeake Bay environmental sample (gi: 229562105) [45] . A subset of the positive samples (Table 1) contained sequences from more than one virus, which may be evidence of co-infection. Almost all of the cases with multiple viruses involved TTV-derived sequences along with HHV-6, DENV-2, or circovirus-like sequences (samples 282, 235, 183, 270, 350, and 377). Two samples contained circovirus-like sequences with ASFV-like (sample 315) or GBV-C sequences (sample 387).
In this study, we examined the virus diversity in serum samples from Nicaraguan children with unknown acute febrile illness. We performed Virochip microarray and deep sequencing analyses on 7 positive control and 123 undiagnosed samples. Both of these methods succeeded in detecting the expected virus in the positive control samples. Virochip analysis produced putative viral hits in 10/123 (8%) of the previously negative samples, whereas deep sequencing revealed virus or virus-like sequences in 45/123 (37%). This study demonstrates the utility of these metagenomic strategies to detect virus sequence in multiple human serum samples and is the first to utilize second-generation sequencing to simultaneously investigate many cases of acute unknown tropical illness.
Monitoring the emergence and spread of novel human pathogens in tropical regions is a central public health concern. Metagenomic analysis enables more systemic viral detection of both known and novel viral pathogens [42] and can be employed as diagnostic supplements to pathogen detection as part of public health monitoring systems and epidemiologic surveys [9] [10] [11] [12] [15] [16] [17] 19, 21, 23] . Despite the headway, metagenomic virus detection studies will have to confront several remaining difficulties concerning diagnostic accuracy. Foremost concerns include enhancing the sensitivity and specificity of deep sequencing-based diagnostic methods and re-evaluating the evidence for disease causality in light of increasingly sensitive nucleic acid detection and pathogen discovery methods. The former will require improved strategies to biochemically enrich and computationally identify viral sequences while reducing host background sequences. The latter will require a cautious reconsideration of criteria used to establish causal links between microbes and disease, as well as extensive case-by-case follow-up studies employing classical laboratory methods, such as serological analysis and cell culture amplification. It is important to highlight that observing viral sequence in sequencing data is insufficient to establish the role of a virus in disease causality. Like other detection strategies, deep sequencing will serve to inform secondary tests, including seroconversion assays, further nucleic acid testing, cell culture amplification, and additional investigations into plausible disease mechanisms.
We detected virus sequence at concentrations as low as ,2 in 10 6 reads. Virus sequence detected in a clinical sample at vanishingly low copy numbers may reflect several possible hostmicrobe scenarios. The sequence detected may be that of a pathogenic virus capable of causing illness at low copy number or through indirect effects, a ubiquitous non-disease causing microbe, a virus outside of its primary replication site, low-level contamination, an artifact of sample collection timing/processing, or remains of incomplete immune clearance. Additional evidence must be considered in each case to define the host-microbe relationship.
In this study, we compared the performance of the Virochip and deep sequencing for detecting virus sequence in human serum. The limit of detection of the Virochip was approximately one part in 10 5 for the poliovirus controls, for which there are microarray probes with perfect sequence complementarity ( Figure S1 ). The sensitivity of deep sequencing is limited by the number of reads generated per sample, or read depth. In this study, we detected virus sequences down to two parts per million. Nearly every virus that was detected on the microarray was also detected by deep sequencing; additionally, in numerous samples (n = 44), sequencing revealed viruses not detected by the Virochip (Table 1) . There were two instances where Virochip analysis identified a virus (TTV) that was not detected by deep sequencing (Table 1) . Deep sequencing, therefore, is a superior method for novel virus discovery, because it is more sensitive and provides more conclusive genotypic information than the Virochip. The NCBI TaxID and name of the virus species with the highest number of hits among those viruses with BLAST hits is given.
These two samples were prepared from aliquots of the same serum sample.
(c)
In its deep sequencing dataset, Sample 168 had 9 reads matching TTV, just below our positive identification threshold. doi:10.1371/journal.pntd.0001485.t001 the Virochip is a relatively fast and inexpensive method that is best applied to samples with expected virus copy numbers present at levels greater than 1 in 10 5 host sequences.
We were unable to detect a virus in two thirds of the 123 dengue-like illness samples. These results could reflect true negative status, which would result from a non-viral infection, illness due to non-infectious agent, or complete immunologic clearance. Alternatively, the negative results could reflect failures in our diagnostic approaches due to imperfect sensitivity, unsatisfactory sample preparation, improper sample type, or failure to recognize highly divergent viral sequences. The presence of sequences that lack even remote similarities to known species also highlights the need for further development of de novo assembly methods for metagenomic data. Assembled data, increased depth, and enhanced sequenced comparison methods should enable more sensitive detection of divergent viruses in metagenomic samples.
Determining the etiology of human diseases with symptoms that overlap with dengue-like illness is important for understanding the full spectrum of emerging or previously uncharacterized pathogens in tropical populations. In this study, 10% of acute serum samples negative for dengue virus from cases of pediatric dengue-like illness were positive for HHV-6. Primary HHV-6 infection causes undifferentiated febrile illness and exanthem subitum (roseola infantum or sixth disease), an acute illness with high fever and rash that typically resolves in three to seven days [49] . Exanthem subitum is a common disease of infants worldwide, and HHV-6 infection most frequently occurs between 6 and 12 months of age [50] , with seropositivity estimates of .95% in adult populations in developed countries [51] . The HHV-6 positive patients in this study were between 7-12 months old, and presented with fever and rash (Table S3) . We detected multiple kilobases of HHV-6 sequence in each positive sample, with sequence deriving from multiple viral genomic regions (Figure 2 ).
After acute infection, HHV-6 can latently persist in the host quiescently, with no production of infectious virions or with low levels of viral replication. Latency is believed to endure in several cell types, including monocytes and bone marrow progenitor cells [52, 53] , and may undergo chromosomal integration that can be vertically transmitted [54] . The confounding effects of chromosomal integration make differentiating between active and latent HHV-6 infections difficult when detecting HHV-6 sequence in Sequencing Viruses in Tropical Dengue-like Illness www.plosntds.org serum DNA [55, 56] . A previous study detected integrated HHV-6 genomic sequence in ,1% of healthy blood samples [57] . Since detection of HHV-6 nucleic acid in serum alone does not prove active viral infection, we cannot definitively confirm that the HHV-6 sequences in these samples were not derived from the vertical transmission of chromosomally integrated virus. However, the clinical, epidemiological, and virus sequence data suggest HHV-6 may be the etiologic agent in these febrile illness cases.
Primary HHV-6 infection is a major cause (,20%) of infant hospitalizations in the United States [58] , a clinical burden likely shared throughout the tropical world given similar seroprevalence rates [59] . The results of this study illustrate the importance of administering HHV-6 diagnostic tests to cases of suspected dengue-like illness in infants from dengue-endemic regions to differentiate between cases of exanthem subitum, a ubiquitous selflimiting childhood illness, and dengue fever, which carries a greater risk of severe clinical complications and death.
Similarly, the one sample positive for Parvovirus B19 sequence may be a case of acute infection with a commonly acquired childhood virus. Parvovirus B19 can manifest as erythema infectiosum (fifth disease), a condition associated with characteristic ''slapped cheek'' rash [60] . Infection can also be subclinical or result in mild nonspecific symptoms. It is possible that Parvovirus B19 infection caused the symptoms in this case (Table S3) , though as with HHV-6, the identification of viral sequences does not definitively demonstrate causality. Histograms of HHV-6B genome coverage generated by aligning reads with minimum 90% identity over the total read length to the genome. The depth of sequence coverage was calculated as the total Kb of aligned sequence per 1 Kb bin over the HHV-6B reference genome. Genome track representation adapted from Dominguez et al [65] . The blue box represents conserved genes across the betaherpesvirus subfamily, the orange boxes represent core genes across the herpesvirus family, the green box represents the late structural genes (gp82-105), and the asterisk denotes the origin of lytic gene replication. Inset text for each histogram is the sample code. Coverage is shown for samples with greater than 80 HHV-6 reads. doi:10.1371/journal.pntd.0001485.g002
Sequencing Viruses in Tropical Dengue-like Illness www.plosntds.org Epstein Barr Virus (HHV-4) sequences were found in the serum of one patient who presented with relatively severe symptoms, and died during hospitalization (Table S3) . HHV-4 infection is a nearly universal occurrence in the first two decades of life [61, 62] . Primary infection in adolescents or adults can manifest as infectious mononucleosis, and chronic infection is associated with various malignancies later in life. Primary infection during childhood, however, is usually asymptomatic or produces only mild symptoms. It is not clear that HHV-4 infection or HHV-4 alone caused the illness in this case.
In addition to the viruses for which a plausible disease association exists, many samples contained sequences from viruses with no well-established link to human disease. These included the two samples positive for GBV-C and those containing ASFV-like, TTV-like, and circovirus-like sequences.
The Circoviridae family is an extraordinarily diverse group of small, single-stranded circular DNA viruses that includes cycloviruses (genus Cyclovirus) and circoviruses (genus Circovirus), which are commonly detected in human stool and blood, and also in environmental samples [43] [44] [45] [46] [47] [48] . Some circovirus species, such as beak and feather disease virus and porcine circovirus 2, have been associated with disease in bird and pig hosts, respectively, but the pathogenic potential of circoviruses in humans remains unconfirmed [63, 64] . The circovirus-like sequences reported here were detected in nucleic acid libraries prepared from acute human serum and were most closely related to circovirus-like viruses (Figure 3 ), which were first reported in environmental samples and in bats [45, 48] . We were unsuccessful in recovering a full genome sequence corresponding to any of the circovirus-like sequences, and it has not yet been possible to prove that these sequences were not an environmental artifact introduced during sample preparation. It is also possible that these sequences derive from other organisms, such as Giardia intestinalis or Entamoeba dispar, whose genomes encode proteins that share amino acid similarity with circovirus replicase proteins ( Figure 3) . Furthermore, it has yet to be established whether circoviruses are capable of replicating in humans. Pending additional screening and serologic studies, the detection of circovirus-like sequences from human serum should be interpreted with caution.
Metagenomic approaches provide an effective high-throughput method to detect uncharacterized virus diversity in a tropical setting from many samples simultaneously. The findings presented in this study further our knowledge of well-characterized and previously unknown viruses present in serum collected from pediatric dengue-like illness patients and advance our understanding of the application of metagenomic approaches to human pathogen detection. Deep sequencing analysis of clinical samples holds tremendous promise as a diagnostic tool by permitting the detection of many different viruses simultaneously, including those present at low-copy numbers and of divergent origin. Major remaining barriers to high-throughput sequencing strategies becoming standard diagnostic practice include prohibitive cost, lengthy sample preparation time, and computationally intensive data analysis requirements. These challenges are magnified in resource-limited settings, such as Nicaragua, but are gradually being addressed. Industry hardware and technical advancements have steadily decreased the per-base cost of deep sequencing, and the results presented here strengthen our expectations of multiplexed sample preparation and bioinformatic data filtering within the framework of current secondgeneration sequencing platforms. Long-term bi-directional partnerships with developing country collaborators facilitate easier access to techniques not currently available on-site, such as deep sequencing, and are also important in providing training opportunities for local scientists and developing relevant pathogen tests and diagnostic policies. This study expands our understanding of the virus diversity in pediatric dengue-like illness in Nicaragua and the application of genomic detection techniques in a tropical setting, findings that are particularly valuable given the pressing need for improved global emerging pathogen surveillance. Figure S1 Virochip sensitivity using poliovirus control RNA. The Virochip can detect one poliovirus gRNA in a background of 10 5 HeLa RNA molecules. Poliovirus RNA was mixed with HeLa total RNA and analyzed on the Virochip. Eighty enterovirus Virochip oligos were found to be responsive to the poliovirus RNA and the mean fold above background of the normalized intensity of these oligos is plotted. Background is defined as the normalized intensity for each oligo in the HeLa-only control sample. The top E-predict hit in the 10 25 to 10 22 samples was human enterovirus C. (PDF) Figure 3 . (PDF)  Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20 We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(−1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme. Glycyrrhizin (GL), the main constituent of licorice extract (Glycyrrhiza glabra), is a natural edulcorant as well as an important ingredient of traditional Chinese medicine [1, 2, 3] . By hydrolyzing one or two distal glucuronides, GL can be transformed into glycyrrhetinic acid monoglucuronide (GAMG) or glycyrrhetinic acid (GA) ( Figure 1 ). As an important derivative of GL, GAMG displayed stronger physiological functions than GL such as antiviral, anti-inflammatory, anti-tumor functions, and so on; and it is also 1000-fold sweeter than saccharose [4] . On the other hand, GA is the bioactive substance of GL well known for its pharmacological features [4, 5, 6] . The research on GL biotransformation catalyzed by b-glucuronidase (GUS, EC 3.2.1.31) was reported mainly in animal tissues such as duck [7] and human [8] , whereas studies on GL biotransformation in fungal species are few [9] . In our previous work, a fungal strain, Aspergillus terreus Li-20 was screened, which can use GL as a carbon source and produce GAMG and GA after catalysis by b-glucuronidase (AtGUS). The main disadvantages of AtGUS were low enzyme productivity, low catalytic efficiency, and pathogenicity, which rendered it unsafe for use in the food and medical industries.
To overcome disadvantages of a natural enzyme, many methods was applied to obtain artificial evolution enzymes, and this approach is not only faster than natural evolution but also provides a deeper understanding of enzyme evolution. Several methods of designing new enzymes are available, and gene sequence truncation is also investigated for its effects on enzymatic properties. The non-conservative N-terminal domain of the protein phosphatase1 (PP1), with 1-8 residues deleted, showed higher sensitivity to three substrates and influenced the structure and properties of PP1 [10] , whereas the truncation of the Cterminal region improved the thermal stability of endo-bglucanase from Bacillus subtilis JA18 [11] . However, the loss of the C-terminal regulatory domain resulted in a loss of the ability to catalyze the aldol reaction [12] .
With development of molecular biology and bioinformatics characterization, an increasing number of sequence data have been cloned and applied in the biotransformation industry. Bioinformatics characterization from the National Center for Biotechnology Information (NCBI) showed that most b-glucuronidases belong to the glycoside hydrolase family (GHF) 2, and all of them consist of sugar-binding, immunoglobulin-like b-sandwich, and TIM barrel domains (triosephosphate isomerase, TIM) [13, 14, 15] . The TIM barrel domain, which is one of the most common catalytic domains, is adopted by about 10% of the enzymes; thus, sequence modification inside or outside the domain to improve the enzymatic property and determine the catalytic mechanism was reported in many studies. The site-directed mutagenesis of seven amino acids (aa) in the TIM barrel domain was performed to investigate the importance of the residue in the catalysis of an exo-b-d-glucosaminidase from Trichodema reesei [16] . Heparanase is an endo-b-d-glucuronidase, and its C-terminal region, which is not an integral part of the TIM barrel domain, is essential for the enzymatic activity and secretion of heparanase [17] .
Although b-glucuronidases from many species have been registered in Genbank, only a few genes have been published for GL biotransformation [18] . Three fungal strains, namely, A. terreus Li-20, P. purpurogenum Li-3, and A. ustus Li-62, were screened in our previous studies and represented three modes of GL biotransformation: (1) GLRGA+GAMG; (2) GLRGAMG; and (3) GLRGA [9] . The three b-glucuronidase genes were cloned in our laboratory, and the aa sequence alignment showed that the bglucuronidase from A. terreus Li-20 (AtGUS) was quite different from the other two b-glucuronidases(PGUS and AuGUS) in Cterminal non-conservative sequence. AtGUS can hydrolyze GL into two products; thus, the different modes of GL biotransformation of the b-glucuronidases from the other two fungi may be related to the natural evolution in the sequence. In the present research, Atgus and the partial sequence [Atgus(-3t)] without Cterminal non-conservative sequence behind the TIM barrel domain were amplified in order to investigate effects of nonconservative sequence on enzymatic property.
No specific permits were required for the described field studies. No specific permissions were required for these locations/ activities. No location is privately-owned or protected in any way. The field studies did not involve endangered or protected species.
In our previous work, A. terreus Li-20 was isolated and screened from a G. glabra planting field in Shihizi, Xinjiang. It was incubated in 100 ml liquid Czapek's medium in a 500 ml Erlenmeyer flask at 30uC and placed in a shaker incubator at 170 rpm.
Escerichia coli DH5a and E. coli BL21 were used as hosts for plasmid amplification and expression, respectively. The plasmids pMD19-T (TaKaRa, Japan) and pET28a (+) (Invitrogen, U.S.) were used as vectors. The recombinant cells were inoculated in a lysogeny broth (LB) medium with kanamycin (50 mM) and operated at 37uC for 3 h. The recombinant protein was induced by adding 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG).
GL was purchased from Xinjiang Tianshan Pharmaceutical Co. (China). GA was purchased from Sigma Chemical Co. (U.S.), whereas GAMG was obtained from the Nanjing University of Technology, China. Methanol was of chromatographic grade. All other chemicals used were of analytical grade. The DL2000 marker and the protein low weight marker were purchased from TaKaRa, Japan.
An intron in an AtGUS genomic sequence was removed via three-step polymerase chain reaction (PCR) to express the gene in E. coli BL21 (Figure 2A ). According to the database of A. terreus NIH2624, a primer set containing P1(59-CCGTACgTAATGCT-GAAGCCCCGACAAACACCTT-39) and P2(59-CATGCGG-CCGCTTAAGCGCCAAATAGGAAGTATAGT-39) was designed to obtain the sequence with an intron from the A. terreus Li-20 genome under the following conditions: 94uC for 10 min, 30 cycles of 94uC for 1 min, 58uC for 1 min, 72uC for 2 min, and a final extension at 72uC for 10 min with Ex Tag (TaKaRa, Japan). After ligated into PMD19-T, a primer set containing P3(59-CACTCCACCGTGTTTTCAATGTATGAGCTGCAGC-39) and P4(59-CCGGCTTCGCAGCTATGTGTCTTGAGCATC-39) was used for the second PCR, and the reaction was performed by Pfu polymerase (Shenggong, China) under the following conditions: 94uC for 10 min, 30 cycles of 94uC for 1 min, 55uC for 1 min, and 72uC for 5 min. The fragment amplified in the second PCR was ligated by T4 DNA ligase after a terminal phosphation with T4 polynucleotide kinase (Takara, Japan), and the positive clones were screened in an LB plate with 100 mg/mL ampicillin. The primer set containing P1 and P5(CATGCG-GCCGCTTAACTCCACCGTGTTTTCAATGTATG-39) was used for AtGUS(-3t) under the same conditions as those of P3 and P4.
After IPTG introduction and the ultrasonication of the recombinant E. coli BL21 cells, supernatant was brought to 70% saturation with (NH4) 2 SO 4 and stored overnight at 4uC, and then again centrifuged. The enzymes expressed by pET28a(+) vector were fused to an N-terminal six-histidine tag and purified via nickel chelate affinity chromatography (GE, U.S.), which was eluted with 150 mM imidazol. The quality of the purified protein was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining.
HPLC for analysis of GL, GAMG, and GA GL, GAMG, and GA concentrations were measured via reverse-phase high performance liquid chromatography (HPLC) on a C18 column (4.6 mm6250 mm, 5 mm particle size, Kromasil) at 40uC. The sample (injection volume, 10 ml) was separated with a mobile phase consisting of 6% acetic acid/ methanol (19:81 v/v), and the elution was monitored via ultraviolet detection at 254 nm. The GL, GAMG, and GA amounts were calculated from the standard curve of the peak area and concentration. Determination of pH and temperature profiles
The activity of b-glucuronidase was determined using GL as the substrate. The reaction mixture consisted of the enzyme and substrate (2 g/L GL) at a 1:4 (v/v) ratio.
50 Mm Na 2 HPO 4 -citric acid buffer at pH 4.0-8.0 was used for determine the pH effects of the enzyme. The catalytic activity of the enzyme was examined at 30 to 70uC in 50 mM Na 2 HPO 4citric acid buffer (pH 7.0). The enzyme activity under the optimal temperature and pH was defined as 100%.
The temperature stability of the enzyme was determined by incubating the enzyme samples at different temperatures (45, 55, 65, and 75uC) for 15, 30, 45, 60, and 120 min at optimum pH without the substrate GL, and the residual activity was determined at the optimum temperature.
The effect of several metal ions on the activity of AtGUS(-3t)-E and AtGUS-E was investigated. The enzyme activity was determined in the reaction mixture consisting of K + (KCl), Na + (NaCl), Mg 2+ (MgCl 2 ), Mn 2+ (MnCl 2 ), Co 2+ (CoCl 2 ), Ca 2+ (-CaCl 2 ), Ni 2+ (NiSO 4 ), Cu 2+ (CuSO 4 ), and Al 3+ (AlCl 3 ) ions at final concentrations of 1 and 5 mM. The enzyme activity was subsequently determined at the optimum temperature after incubation for 30 min.
Different concentrations of the substrate GL, ranging from 0.375 to 4 mM, were prepared to determine the kinetic constants. The catalytic reactions were continuously monitored, and the initial velocities were fitted to the Michaelis-Menten equation using the Origin 7.5 software (OriginLab). The values of the Michaelis-Menten constant (Km), maximal velocity (Vmax), catalytic turnover rate (Kcat), and catalytic efficiency (Kcat/Km) were evaluated.
Far-UV Circular dichroism (CD) spectra were recorded at 25uC in the range from 190 to 260 nm with a spectral resolution of 0.2 nm using a Jasco J-715 spectropolarimeter. The scan speed was 100 nm/min and the response time was 0.125 s with a bandwidth of 1 nm. Quartz cells with an optical path of 0.1 cm were used. Typically, scans were accumulated and subsequently averaged. The spectra were corrected for the corresponding protein-free control.
The protein three-dimensional structural was modeled by modeler 9v7 to analyze three domains of the protein.
The 2,193 bp product was amplified and sequenced using a genomic template ( Figure 2B) , and its 219 bp intron was analyzed by NCBI. After a three-step PCR, the full encoding sequence was cloned. The results show that the open reading frame of this gene was 1,974 bp ( Figure 2B) , which encodes for 657 aa.
The conserved domain database (CDD) was performed to analyze domains of AtGUS, and there were sugar-binding domain, immunoglobulin-like beta-sandwich domain, and TIM barrel domains in it which all belonged to glycoside hydrolase family (GHF) 2. GHF 2 comprised b-galactosidase (EC 3.2.1.23), bmannosidase (EC 3.2.1.25), and b-glucuronidase (EC 3.2.1.31), so the phylogenetic tree was constructed according to it (Figure 3) . It showed that the gene cloned was a b-glucuronidase gene named AtGUS (Genbank accession No. JF894133), which was found very similar to PGUS(Genbank accession No. EU095019) from P. purpurogenum Li-3 and AuGUS (Genbank accession No. JN247805) from A. ustus Li-62, especially in the sugar-binding, immunoglobulin-like beta-sandwich, and TIM barrel domain ( Table 1 ). The obvious difference among them lied in the non-conservative sequence of the C-terminal behind the TIM barrel domain which may result in the difference enzymatic properties. Therefore, the 1-1,776 bp segment, named Atgus(-3t), was amplified in the 
Both pET28a(+)-AtGUS and pET28a(+)-AtGUS(-3t) were constructed and transformed into the E. coli BL21 strain, and the recombinant proteins AtGUS-E and AtGUS(-3t)-E were successfully expressed ( Figure 4) . The induction condition for the optimum production of the two recombinant proteins was 20uC with 0.4 mM IPTG. Both AtGUS-E and AtGUS(-3t)-E were purified through Ni-NTA sepharose (Figure 4) . The target protein was eluted with 150 mM imidazole. Furthermore, the concentrations of the soluble purified proteins of AtGUS-E and AtGUS(-3t)-E were determined as ,7 and ,12 mg/L, respectively. Both purified enzymes could hydrolyze GL into GAMG and GA.
We investigated the enzymatic properties to determine the effect of the non-conserved sequence on the enzyme. The optimal pH for the bioconversion reaction by AtGUS-E was 6.6, whereas that for AtGUS(-3t)-E was 7.0 ( Figure 5A) .The optimal temperatures for AtGUS-E and AtGUS(-3t)-E were both 55uC ( Figure 5B) .
Enzyme thermal stability experiments showed that the enzymes remained more than 80% residual activity at 45uC and 55uC for 120 min heat treatment, respectively ( Figure 5C and 5D) . At 65uC, the residual activity of AtGUS(-3t)-E remained almost 60% of enzymatic activity after 30 min heat treatment, which was comparatively higher than that of AtGUS-E with less than 5% residual activity after the same treatment. At a higher temperature (75uC), both enzyme residual activity rapidly vanished, and within 15 min heat treatment, almost all enzymatic activity was lost.
The effect of various metal ions with different concentration gradients (from 1 to 5 mM final concentration) on the activities of AtGUS(-3t)-E and AtGUS-E was evaluated, and the results are presented in Table 2 . The enzymatic activity assayed in the absence of metal ions was taken as 100%.
The effect of monovalent cations on the two enzymes was similar: the 1 and 5 mM K + and 1 mM Na + exhibited no obviously affecting effects on the activity of AtGUS(-3t)-E and AtGUS-E, while the 5 mM Na + inhibited the enzymatic activity. The divalent cations Mg 2+ and Mn 2+ discretely promoted the activities of AtGUS-E and AtGUS(-3t)-E. With increasing concentration of Co 2+ , AtGUS-E was firstly activated and then inhibited while AtGUS(-3t)-E showed an inverse effect. Ca 2+ and Ni 2+ also exhibited opposite effects on the two enzymes. Ca 2+ at 5 mM final concentration enhanced the activity of AtGUS-E by 107% but inhibited AtGUS(-3t)-E activity by 61%. In the presence of 5 mM Ni 2+ buffer, AtGUS(-3t)-E was increased by 78%, whereas AtGUS-E was decreased by 40%. Cu 2+ distinctively inhibited the enzyme activity, while Al 3+ showed activation at 1 mM concentration and inhabitation at 5 mM concentration to both enzymes. These results reveal that K + , Na + , Mg 2+ , Mn 2+ , Cu 2+ , and Al 3+ exhibited nearly similar effects on the activity of AtGUS-E and AtGUS(-3t)-E; however, Co 2+ , Ca 2+ , and Ni 2+ , showed opposite effects on both enzymes, respectively. The data reported here have been taken from three replicate samples from three independent experiments.
The reaction kinetics of AtGUS-E and AtGUS(-3t)-E were determined. The Vmax of the AtGUS-E and AtGUS(-3t)-E enzymes toward GL were calculated using Lineweaver-Burk plots ( Table 3 ) and were determined as 1.84 and 0.97 mmolmin 21 mg 21 , respectively. The Km of the recombinant AtGUS(-3t)-E was 1.95 mM, which was approximately one-seventh that of AtGUS-E (12.9 mM), indicating that a higher affinity of AtGUS(-3t)-E for GL than AtGUS-E. In addition, the catalytic efficiency (kcat/Km) of AtGUS(-3t)-E (7.16 mM s 21 ) was 3.2 folds higher than that of AtGUS-E (2.24 mM s 21 ). The enzymatic activities were determined at different concentrations of the substrate GL from three independent experiments.
To determine the impact of the sequence truncation on the structure of the protein, a circular dichroism (CD) spectra was amplified. The far-UV spectra for AtGUS-E and AtGUS(-3t)-E have been presented in Figure 6 . It illustrated that the curves exhibited significant difference between the two proteins. These results suggest that the secondary structure of AtGUS-E has changed after deletion of non-conservative sequence.
The b-glucuronidase (GUS) gene was first cloned in 1987 [19] , and in subsequent years, many GUS genes were cloned and registered in the GenBank. However, this gene has never been cloned for the hydrolysis research of GL into GAMG or/and GA, with more valuable merits. Based on CDD analysis, the three domains of the enzyme AtGUS were well investigated, and the main aim of the present study is to modify the non-conservative sequence of AtGUS and try to obtain an artificial evolution enzyme with better enzymatic properties.
The TIM barrel domain is a canonical (b/a) 8 -barrel composed of eight units, each of which consists of a b-strand and an a-helix [20] . There was a non-conservative segment behind the catalytic domain (TIM barrel domain) of AtGUS which showed low identity with PGUS and AuGUS after the primary sequence alignment. A model of the three-dimensional structure of AtGUS was presented in the current research ( Figure 7) . The deleted sequence exhibited no involvement in the TIM barrel domain, locating near the ''stability face'' rather than the ''catalytic face'' [21] .
AtGUS-E and AtGUS(-3t)-E were very similar with each other at some enzymatic properties, such as optimal pH and optimal temperature. It was reported in previous studies that many modified enzymes maintained some original enzymatic properties even though some sequence has been modulated [10] . Furthermore, we could also speculate that the non-conservative sequence lied outside of the catalytic face of the TIM barrel domain which may not affect the catalytically active residues and the GL biotransformation mode.
Interestingly, the stability of AtGUS(-3t)-E was slightly higher than that of AtGUS-E at 65uC. Similar results have been reported that the modification of the C-terminal region could improve the thermal stability of endo-b-glucanase from Bacillus subtilis JA18 [11] . In addition, previous study showed that ab-loops in ''stability face'' are important for the stability [22] . The truncation of the non-conservative sequence lies near ab-loops of the stability face, therefore, we can predicted that the deletion of the C-terminal region outside the TIM barrel domain has influence on thermal stability of AtGUS. The effect of nine metal ions with different concentration gradients on the activities of AtGUS(-3t)-E and AtGUS-E was evaluated, and Co 2+ , Ca 2+ , and Ni 2+ showed opposite effects on the two enzymes, respectively. In addition, AtGUS(-3t)-E showed higher affinity and catalytic efficiency than AtGUS(-3t)-E. Both of the result might suggest that the spatial structural rearrangement, and the speculation has been proved by CD spectra, which showed great difference between the secondary structure of the two enzymes. It has been reported that the loops above the catalytic face was very important for substrate hydrolysis [23] , so we can conclude that the truncation of the non-conservative domain firstly changed the secondary structure of the enzyme and then influenced the substrate affinity, catalytic efficiency and metal ions effects. Moreover, the crystal structure of human bglucuronidase was firstly reported in 1996 [24] , and the structure of bacterial b-glucuronidase has also been published recently [25] . Both b-glucuronidase structures were tetramers. The deleted region of the AtGUS non-conservative sequence lies outside the main three domains, so it was predicted that the non-conservative might change the combination pattern of each monomer.
Different methods have been applied in creating new enzyme such as error-prone PCR [26] , DNA shuffling [27] and staggered extension process (StEP) [28] . Some efforts have been made to modify the enzyme by directed screening but high ratio of negative mutated forms of enzyme in the initial screening is a big hurdle and requires further screening for positive mutated forms of enzyme. Every method has its own advantages and disadvantages that determines the feasibility of a particular method so as sequence truncation [10, 11, 12] . Based on the same hydrolyzing mode, relatively higher thermal stability, and especially the enhanced affinity and catalytic efficiency for GL, deletion of the non-conservative sequence behind the TIM barrel domain was a successful evolution of AtGUS. The truncation of non-conservative region based on sequence alignment could be an effective way of artificial enzyme evolution as it can alter and influence the stability and catalytic efficiency of enzyme, and could help in understanding the relationship between the structural modulation and enzymatic properties.  Protein sequence analysis based on hydropathy profile of amino acids Biology sequence comparison is a fundamental task in computational biology. According to the hydropathy profile of amino acids, a protein sequence is taken as a string with three letters. Three curves of the new protein sequence were defined to describe the protein sequence. A new method to analyze the similarity/dissimilarity of protein sequence was proposed based on the conditional probability of the protein sequence. Finally, the protein sequences of ND6 (NADH dehydrogenase subunit 6) protein of eight species were taken as an example to illustrate the new approach. The results demonstrated that the method is convenient and efficient. The comparative biological sequence is one of the issues in bioinformatics when analyzing similarities of function and properties of different sequences. Similarly, evolutionary homology is analyzed by comparing DNA and protein sequences. In general, there are two types of methodologies to conduct the comparison. One is an alignment-based method, and the other is an alignment-free method.
Sequence alignment is based on computeroriented and computer-intensive comparisons of sequences, and then a distance function or a score function is obtained. Using the distance function, one can compare biological sequences. However, multiple sequence alignment of several hundred sequences always produces a bottleneck, firstly due to long computational time, and secondly due to possible bias of multiple sequence alignments for multiple occurrences of highly similar sequences (Pham and Zuegg, 2004) . Therefore, the emergence of a study on alignment-free sequence analysis is obvious. Until now, alignment-free sequence analysis is still in its early development. For most alignment-free methods, a biological sequence should be transformed into an object for which a linear algebra and statistical theory already has useful analytical tools. Since 1983, DNA sequence has been represented in different dimension spaces (Hamori and Ruskin, 1983; Hamori, 1985; Nandy, 1994; 1996; Nandy and Basak, 2000; Randić et al., 2001; Randić, 2003; Randić and Balaban, 2003; Zhang et al., 2003; Liao and Wang, 2004; Liao et al., 2005; Nandy et al., 2006; Bai et al., 2007; Feng and Wang, 2008) . Each nucleotide of a given DNA sequence is a point in different dimension spaces, and these graphical representations can allow us to qualitatively analyze DNA sequences, and provide a way of viewing, sorting and comparing various genomic sequences. Based on the graphical representation, it is possible to numerically characterize DNA sequence and further quantitatively measure similarity of different DNA sequences. Although protein sequence and DNA sequence belong to symbolic sequences, compared with DNA sequence, there are fewer methods for the graphical representation of protein sequence. This is mainly because extension of DNA graphical representation to protein sequences would enormously increase the number of possible alternative assignments for the 20 amino acids. The amino acid sequence is the key to understanding protein structure and function in the cell, so analysis of amino acid sequence is an important part of post-genomic studies. Recently, several schemes have been proposed in protein graphical representation (Randić and Krilov, 1997; Vinga and Almeida, 2003; Bai and Wang, 2005; Li J. et al., 2006; Li C. et al., 2008; Munteanu et al., 2008; Yau et al., 2008; Yao et al., 2008; Wen and Zhang, 2009) . In order to plot amino acid sequence, 20 amino acids in protein sequences are divided into different types, including protein sequence regarded as a word with three, four, or five different letters. Since ordering amino acids based on their physicochemical properties may offer better insights into comparative study of protein than representation of protein based on the random ordering of amino acid, Randić (2007) and Yao et al. (2008; outlined different 2D graphical representations of protein sequence based on different physicochemical properties. The graphical representation of protein sequence cannot only describe amino acid sequence, but also measure similarity/ dissimilarity of different protein sequences. However, the methods only consider the string's information of protein, and do not consider adjacent string's information of amino acid sequence. Here, we choose conditional probability to measure adjacent string's information.
In this paper, we converted a protein sequence into three-letter sequence based on hydropathy profile of amino acid and defined the three curves to represent different hydropathy features. We then selected conditional probability as a new invariant for the protein sequences. To illustrate the proposed method, we made a comparison of the sequences belonging to eight ND6 (NADH dehydrogenase subunit 6) proteins from http://www.ncbi.nlm.nih.gov/: human
, rat (AP_004903), and mouse (NP_904339).
According to the hydropathy profile of amino acids, the amino acids can be classified into three groups (Nei and Kumar, 2002; Liu and Wang, 2006) : internal group (F, I, L, M, V), external group (D, E, H, K, N, Q, R), and ambivalent group (S, T, Y, C, W, G, P, A). The amino acid of internal group tends to occur in the inner side of the protein's spatial structure, while the amino acid of external group tends to appear at the surface. In order to characterize the hydropathicity of a protein primary structure, we defined a primary protein sequence as a symbolic sequence including three letters according to the following rule:
where S(i) is the letter in the ith position in the protein primary sequence, and F(S(i)) is the substitution for S(i). Since the hydropathy profile can detect more evolutionary relationships, in the next section, we analyzed the new protein sequence containing three letters through different mathematical methods.
Given a protein primary sequence with length N, we transformed it into a new sequence according to the above definition. For example, for the protein sequence, S=MMYALFLLSVGLVMGFVGFS, then F(S)=IIAAIIIIAIAIIIAIIAIA. To obtain more information, we defined three curves of the sequence. Firstly, we let IE EA IA 1 if ( ( )) I, 0 otherwise,
where i ranges from 1 to N. Then, let
Y n u and n are Y axis and X axis, respectively, and then we can draw three different curves, which are named as IE, IA, and EA curves of the protein sequence. The three different curves can give us some information about the protein sequence. According to the IE curve, we can compare the numbers of the amino acids belonging to the internal group and the external group at different positions. The IA curve can then be used to compare the numbers of the amino acids belonging to the internal group and the ambivalent group at different positions. Finally, the EA curve can compare the numbers of the amino acids of the external group and the ambivalent group at different positions. According to the above definitions of three different curves, we drew three curves of ND6 proteins for the eight species (Fig. 1 ). Fig. 1 shows that the amino acids of the internal group in ND6 protein sequences are more than the amino acids of the external group, and the amino acids of the ambivalent group are more than the amino acids of the external group. Furthermore, it is evident that G. seal and H. seal have similar curves, rat and mouse's curves are almost identical, and the three curves of human, gorilla, and chimpanzee are similar, but wallaroo's curve is different from curves of other species.
Protein sequence is composed of three parts, internal group, external group and ambivalent group, so we regard the random numerical sequence to be composed of three parts (+1, 0, −1). We calculated the conditional probability, which was invariant to quantity protein sequences.
For example, let X i IE represents the state of the ith (i=1, 2, ..., N) moment, state space S={+1, 0, −1}.
There are nine conditional probabilities as follows: ( 1 1 ),
According to the above definition, we can obtain these conditional probabilities of a given protein sequence. The conditional probability of each of ND6 proteins is listed in Table 1 . 
Given two protein sequences, we can obtain two nine-component vectors whose elements are conditional probabilities for each protein sequence. Based on the vectors, we can compare different protein sequences. In general, similarities of the two vectors can be obtained by calculating Euclidean distance. The smaller the Euclidean distance of two vectors is, the more similar are the protein sequences. The Euclidean distance of two vectors u and v is as follows:
where u i and v i denote the components of vectors u and v, respectively. k is the dimension of vectors u and v. Yao et al. (2009) proposed a new similarity measure of sequences, and coefficient of determination (r 2 ), which is defined as:
r 2 can vary from 0 to 1, and represents the percent of the data, which is the closest to the line of best fit. The larger the coefficient of determination of two vectors is, the more similar are the protein sequences. In Tables 2 and 3, we give the similarity/dissimilarity matrices for the eight ND6 sequences based on Euclidean distance and coefficient of determination amongst nine-component vectors. As shown in Tables 2 and 3, it is obvious that ND6 proteins of human, gorilla, and chimpanzee are more similar to each other. In addition, ND6 proteins are more similar for (G. seal, H. seal) and (mouse, rat). However, ND6 protein of wallaroo is very dissimilar to others amongst the eight species. The results are consistent with the known fact of evolution (Yao et al., 2009) .
Biology sequence analysis is a fundamental task in computational biology, whose aim is to detect similarity/dissimilarity relationships between molecular sequences. Some alignment-free methods to analyze similarities/dissimilarities of DNA sequences have been proposed. However, there are few alignmentfree methods to analyze protein sequences. The amino acid sequence of a protein is the key to understanding its structure and function in the cell, so we present a new method to analyze protein primary sequence in this paper. The method is based on the graphical representation and conditional probability taken as the numerical characterization of the protein sequence. The demonstrable significance of the new method is that it cannot only analyze similarity/dissimilarity of protein sequences, but also provide more biological information about the protein sequences. According to the IE curve, we can compare the numbers of amino acids of the internal and external groups at different positions. Also the IA curve can be used to compare the numbers of amino acids of the internal and ambivalent groups at different positions. The EA curve can be used to compare the numbers of amino acids in the external and ambivalent groups at different positions. Therefore the three curves show the distribution of the three types of amino acids. Furthermore, the conditional probability reflected the distribution of the two adjacent amino acids. The new approach was applied to ND6 protein sequences of several species and results have shown that the introduction of hydropathy profile of amino acids into protein sequence is effectual and feasible. Filovirus Tropism: Cellular Molecules for Viral Entry In human and non-human primates, filoviruses (Ebola and Marburg viruses) cause severe hemorrhagic fever. Recently, other animals such as pigs and some species of fruit bats have also been shown to be susceptible to these viruses. While having a preference for some cell types such as hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages, filoviruses are known to be pantropic in infection of primates. The envelope glycoprotein (GP) is responsible for both receptor binding and fusion of the virus envelope with the host cell membrane. It has been demonstrated that filovirus GP interacts with multiple molecules for entry into host cells, whereas none of the cellular molecules so far identified as a receptor/co-receptor fully explains filovirus tissue tropism and host range. Available data suggest that the mucin-like region (MLR) on GP plays an important role in attachment to the preferred target cells, whose infection is likely involved in filovirus pathogenesis, whereas the MLR is not essential for the fundamental function of the GP in viral entry into cells in vitro. Further studies elucidating the mechanisms of cellular entry of filoviruses may shed light on the development of strategies for prophylaxis and treatment of Ebola and Marburg hemorrhagic fevers. Ebola virus (EBOV) and Marburg virus (MARV), classified as biosafety level 4 agents, belong to the Family Filoviridae. Whereas MARV consists of a single species, Lake Victoria Marburgvirus, there are four distinct EBOV species, including Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Côte d'Ivoire ebolavirus (CIEBOV), Reston ebolavirus (REBOV), and the proposed new species Bundibugyo ebolavirus (BEBOV) Towner et al., 2008) (Figure 1 left). Among these, ZEBOV, first identified in 1976, seems to be the most virulent, killing approximately up to 90% of infected individuals, whereas REBOV, which was initially isolated from cynomolgus monkeys imported from the Philippines into the USA in 1989, is less pathogenic in experimentally infected non-human primates (Fisher-Hoch and McCormick, 1999) and has never caused lethal infection in humans .
Ebola virus and Marburg virus are filamentous, enveloped, non-segmented, single-stranded, negative-sense RNA viruses (Figure 2) . The viral genome encodes seven structural proteins, nucleoprotein (NP), polymerase cofactor (VP35), matrix protein (VP40), glycoprotein (GP), replication-transcription protein (VP30), minor matrix protein (VP24), and RNA-dependent RNA polymerase (L). EBOV also expresses at least one secreted nonstructural glycoprotein (sGP). Figure 3 summarizes filovirus replication in cells. At the first step of replication, viral attachment through interaction between GP and some cellular molecules is followed by endocytosis, including macropinocytosis (Nanbo et al., 2010; Saeed et al., 2010) . Subsequent fusion of the viral envelope with the host cell endosomal membrane releases the viral proteins (i.e., NP, VP35, VP30, and L) and RNA genome into the cytoplasm, the site of replication. Transcription of the negative-sense viral RNA by the viral polymerase complex (VP35 and L) yields mRNAs that are translated at cellular ribosomes. During replication, full-length positive-sense copies of the viral genome are synthesized. They subsequently serve as templates for replication of negative-sense viral RNA synthesis. At the plasma membrane, NP-encapsidated full-length viral RNAs and the other viral structural proteins are assembled with VP40 and GP and incorporated into enveloped virus particles that bud from the cellsurface (Noda et al., 2006; Bharat et al., 2011) . Though filoviruses show broad tissue tropism, hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages are thought to be their preferred target cells, and infection of these cells is important for hemorrhagic manifestation and immune disorders (Geisbert and Hensley, 2004) .
Filoviruses are known to cause severe hemorrhagic fever in human and non-human primates, but recent studies suggest that quadrupeds are also naturally susceptible to EBOV infection (Figure 1, right) . In 2008-2009, REBOV infection was confirmed for the first time in pigs in the Philippines (Barrette et al., 2009) . REBOV was occasionally isolated from the samples subjected to the diagnostic investigation of multiple outbreaks of a respiratory and abortion disease syndrome in swine, which were caused by porcine reproductive and respiratory syndrome virus, common in pigs in Asia. It is speculated that REBOV became detectable, most likely due to the coinfection with this porcine virus. Although pathogenicity of these swine REBOV strains to humans, non-human primates, or even pigs remains unclear, other EBOV species (i.e., ZEBOV) was shown to cause severe respiratory disease in experimentally infected pigs (Kobinger et al., 2011) . During the 2001-2003 ZEBOV outbreaks in Gabon and the Democratic Republic of the Congo (DRC), when large numbers of FIGURE 1 | Phylogenetic analysis of filovirus GP amino acid sequences. The phylogenetic tree was constructed using the neighbor-joining method. For construction of this tree, ten complete GP amino acid sequences were used. Infectious viruses were isolated or viral genome and/or specific antibodies were detected (in parentheses) from the animals shown on the right.
gorillas and chimpanzees were infected, the viral genome was also detected in duikers, medium-sized Bovid related to antelopes and gazelles (Leroy et al., 2004) . It was also reported that several dogs in the ZEBOV-epidemic area might have been highly exposed to the virus by eating infected dead animals, as suggested by high seroprevalence, but the putative infection seems to be asymptomatic (Allela et al., 2005) .
Infectious MARV was recently isolated from Egyptian fruit bats (Rousettus aegyptiacus) in Uganda, indicating that this species is susceptible to MARV infection and potentially acts as the natural reservoir of the virus . Phylogenetic analysis showed that viruses in the bats were closely related to those isolated from victims of the 2007 MARV outbreak in Uganda, providing the first evidence for an epidemiological link between viruses in bats and hemorrhagic fever outbreak in humans. On the other hand, EBOV has not been isolated from any bat species. During the 2001-2003 EBOV outbreaks in Gabon and DRC, however, fruit bats (Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata) captured in the outbreak area were found to have EBOV genomic RNA and virus-specific antibodies , suggesting they are potential natural reservoirs for EBOV. However, it is still unclear whether these bats continuously maintain EBOV and/or MARV and act as a potential source of filovirus transmission to humans.
It has been shown that laboratory animals, including mice and guinea pigs, are susceptible to filovirus infection. However, these animals infected with filoviruses obtained from patients normally develop a non-lethal illness, though the viruses have the ability to replicate in the animals. Guinea pigs have been used as an animal model for filovirus infection since serial passage of MARV and EBOV in the animals results in a substantial increase in lethality (Bowen et al., 1980; Hevey et al., 1997; Volchkov et al., 2000; Subbotina et al., 2010) . It was also demonstrated that passages of ZEBOV through young mice resulted in the selection of variants with pathogenicity associated with mutations in viral internal genes (e.g., NP and VP40) (Bray et al., 1998; Ebihara et al., 2006) . This mouse-adapted ZEBOV is highly lethal to mice. Similarly, a mouse model for MARV infection has been established (Warfield et al., 2009) . Interestingly, mutations found in the GP gene of these mouse-or guinea pig-adapted viruses were not the primary factor for efficient replication in mice and guinea pigs, suggesting the importance of some other mechanisms underlying in viral replication and/or immune evasion, as shown in the pathogenesis of influenza virus (Fukuyama and Kawaoka, 2011) .
The fourth gene from the 3 end of the filovirus genome encodes the viral envelope GP (Figure 2) , which is responsible for both receptor binding and fusion of the virus envelope with the host cell membrane (Takada et al., 1997; Wool-Lewis and Bates, 1998) (Figures 3 and 4) . GP is highly glycosylated with large amounts of N-and O-linked glycans, most of which are uniformly located in the middle one-third of the GP, designated the mucin-like region (MLR) Manicassamy et al., 2007) . The amino acid sequences of the MLR are highly variable among filovirus species (Sanchez et al., 1996 (Sanchez et al., , 1998 . GP undergoes proteolytic cleavage by host proteases such as furin (Volchkov et al., 1998) , which produces two subunits, GP1 and GP2, linked by a disulfide bond. The GP1 subunit mediates viral attachment, most likely through the MLR or the putative receptor binding region (RBR; Kuhn et al., 2006; Dube et al., 2009) . The GP2 subunit has the heptad repeat regions required for assembling GP as a trimer. The hydrophobic fusion loop on GP2 is thought to catalyze fusion of the viral envelope and host cell membrane (Weissenhorn et al., 1998; Ito et al., 1999) . Although the trigger to promote the conformational change leading to membrane fusion is not fully understood, it was recently suggested that endosomal proteolysis of EBOV and MARV GPs by cysteine proteases such as cathepsins B and L plays an important role in inducing membrane fusion (Chandran et al., 2005; Schornberg et al., 2006; Matsuno et al., 2010a) . Since GP is the only viral surface GP, it is believed to have an important role in controlling the tropism and pathogenesis of filovirus infection Hoenen et al., 2006; Sanchez et al., 2007) .
In the early years, studies of filoviruses were hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, pseudotype virus systems for functional analysis of filovirus GPs have been established (Takada et al., 1997; Wool-Lewis and Bates, 1998) . The systems rely on recombinant viruses (e.g., replication-competent or -incompetent vesicular stomatitis virus and retroviruses) that contain filovirus GP instead of their own GPs (Figure 5 ). Such pseudotype virus systems enable us to investigate cell tropism mediated by simple interaction between filovirus GP and its cellular ligands. Using such a system, it was shown that pseudotyped viruses infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, and that cell-surface GPs with N-linked oligosaccharide chains might contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry (Takada et al., 1997) . Furthermore, filovirus receptor-deficient cell lines that have been used in expression cloning strategies searching for filovirus entry mediators were discovered in an early study (Wool-Lewis and Bates, 1998) . Thus, Frontiers in Microbiology | Virology FIGURE 2 | Structure of Ebola virus particle and genome organization. Electron micrograph of Ebola virus particle (A), its diagram (B), and negative-sense genome organization (C) are shown. Viral protein names and functions are described in the text. Transcribing the glycoprotein (GP) gene produces a soluble GP (sGP). Transcriptional editing accompanied by frame shifting is required to produce full-length, membrane-anchored GP, which shares its first 295 amino acid residues with sGP. pseudotype virus systems are an essential tool for recent filovirus receptor research.
Although filoviruses can replicate in various tissues and cell types, the molecular mechanisms of their broad tropism remain poorly understood (Figure 6) . By using an expression cloning strategy that has been used to identify several virus receptors, human folate receptor-α was first identified as a ubiquitous cellular cofactor that mediates infection by both MARV and ZEBOV (Chan et al., 2001) . This molecule is a glycosyl-phosphatidylinositollinked protein expressed on the cell-surface. However, a human immunodeficiency virus pseudotyped with EBOV GP could not www.frontiersin.org 
pseudotyped with filovirus GP. The pseudotype virus relies on a recombinant virus that contains a reporter gene instead of the viral envelope protein gene responsible for receptor-binding and membrane fusion. Since filovirus glycoprotein is efficiently incorporated into VSV particles, a recombinant VSV that contains the green fluorescent protein (GFP) gene, instead of the G protein gene can be generated. This virus is not infectious unless the envelope protein responsible for receptor binding and membrane fusion is provided in trans.
infect T-cell lines stably expressing this protein, suggesting that folate receptor-α is not sufficient to mediate entry (i.e., some other molecules are required) (Simmons et al., 2003b; Sinn et al., 2003) . A similar approach identified members of the Tyro3 receptor tyrosine kinase family (Axl, Dtk, and Mer) as molecules involved in cell entry of filoviruses (Shimojima et al., 2006) . Expression of these family members in lymphoid cells, which are originally non-permissive to filoviruses, enhanced infection by pseudotype viruses bearing filovirus GPs on their envelopes. These molecules are widely distributed in many types of cells throughout the body, though not on lymphocytes and granulocytes (Linger et al., 2008) . A more recent study demonstrated that reduction of Axl expression by RNAi treatment resulted in decreased ZEBOV entry via. macropinocytosis but had no effect on the clathrin-dependent or caveola/lipid raft-mediated endocytic mechanisms, suggesting that Axl enhances macropinocytosis . However, direct interactions between these cellular molecules and the GP RBR remain to be demonstrated.
Recently, a bioinformatics approach, comparative genetics analysis, was used to screen the candidate genes involved in ZEBOV entry and T-cell immunoglobulin and mucin domain 1 (TIM-1) was identified as a candidate ZEBOV and MARV cellular receptor by correlation analysis between the gene expression profiles and permissiveness to viral infection (Kondratowicz et al., 2011) . TIM-1 was shown to bind to the RBR of ZEBOV GP, and ectopic TIM-1 expression in poorly permissive cells enhanced EBOV infection. In addition, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells, which are commonly used for filovirus propagation. However, the fact that not all cell types that are naturally permissive for filoviruses express the above-mentioned molecules implies that filoviruses may utilize multiple cellular proteins for infection of a wide variety of cells. More recent studies suggest that endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1) is essential for filovirus infection, providing a model of EBOV infection in which cleavage of the GP1 subunit by endosomal cathepsin removes heavily glycosylated regions to expose the putative RBR, which is a ligand for NPC1 and mediates membrane fusion by the GP2 subunit (Carette et al., 2011; Côté et al., 2011) . Available data indicate that the cellular tropism of filoviruses does not necessarily match the distribution of any cellular molecules so far identified. Importantly, it remains elusive whether these molecules act as functional receptors that mediate both viral attachment and membrane fusion or as so-called co-receptors whose interaction with viral GP is required only for membrane fusion.
Both MARV and EBOV GPs contain both N-and O-linked carbohydrate chains with different terminal sialylation patterns that seem to depend on the virus strains and cell lines used for their propagation. The MLR contains a number of potential N-and Olinked glycosylation sites as mentioned above. Though the MLR is found in all known filovirus GPs, its highly variable amino acid sequences and sugar chain structure suggest different GP properties among filovirus species. Interestingly, it is well documented that deletion of the MLR does not affect the fundamental function of GP in viral entry into cells in vitro, as indicated by the observation that pseudotyped viruses bearing GP lacking the MLR infect primate epithelial cells (e.g.,Vero E6 cells) similarly or rather more efficiently than viruses with wild-type GP (Simmons et al., 2002; Takada et al., 2004; Matsuno et al., 2010a) . According to the crystal structure of ZEBOV GP in its trimeric, prefusion conformation, the MLR may restrict access of the putative RBR to virus receptors (Lee et al., 2008) . Thus, pseudotyped viruses bearing MLR-deletion mutant GP have often been used for approaches to identify filovirus-specific receptors (Shimojima et al., 2006; Kondratowicz et al., 2011) . However, the MLR plays an important role in filovirus entry into preferred target cells such as endothelial cells, hepatocytes, and antigen-presenting cells, whose infection is likely involved in tropism and pathogenesis of filoviruses, as described below.
Virus particles attach to the cell-surface through the interaction between GP and some cellular molecules (e.g., putative ubiquitous receptors, C-type lectins). Following virus uptake and trafficking to late endosomes, GP is cleaved by cellular proteases such as cathepsins to remove heavily glycosylated regions including the MLR and expose the RBR of GP1. Binding of cleaved GP1 to a coreceptor (e.g., NPC1) might be necessary for the GP conformational change leading to membrane fusion.
C-type lectins are a family of Ca 2+ -dependent carbohydraterecognition proteins that play crucial roles in innate immunity. It has been demonstrated that membrane-anchored cellular C-type lectins facilitate filovirus infection in vitro by binding to glycans focused on the MLR (Figure 6) . The asialoglycoprotein receptor, a C-type lectin found exclusively in hepatocytes, initially proposed as a receptor for Marburg virus (Becker et al., 1995) , recognizes GPs displaying N-linked sugar chains with terminal galactose residues on the GP molecule and enhances filovirus infectivity. It was subsequently shown that carbohydrate chains on filovirus GP, especially on the MLR, are recognized by other cellular C-type lectins such as dendritic cell-and liver/lymph node-specific ICAM-3-grabbing non-integrin (DC/L-SIGN) (Alvarez et al., 2002; Lin et al., 2003; Simmons et al., 2003a; Marzi et al., 2004; Gramberg et al., 2008) , human macrophage galactose-type C-type lectin (hMGL) (Takada et al., 2004; Matsuno et al., 2010a) , and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin) (Gramberg et al., 2005; Dominguez-Soto et al., 2007; Powlesland et al., 2008) . Though these C-type lectins show different specificities, depending on the structures of target glycans, and thus MLR may not be the only binding site for the lectins, all have been reported to promote filovirus entry. It should be noted that C-type lectins enhance filovirus infectivity when expressed on the target cell-surface, but are unlikely to act as functional receptors mediating both attachment and membrane fusion (Simmons et al., 2003a; Marzi et al., 2007; Matsuno et al., 2010b) . The fact that interaction between the GP MLR and C-type lectins is not essential for viral entry into cells lacking C-type lectins (e.g., Vero E6 cells) may also suggest that C-type lectins facilitate viral attachment but not infectious entry.
Hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages, all of which express C-type lectins, are thought to be the preferred target cells of filoviruses Geisbert and Hensley, 2004; Hoenen et al., 2006) . Indeed, primary macrophage and dendritic cell cultures transduced for C-type lectin expression greatly increased their susceptibility to virus infection (Simmons et al., 2003a; Marzi et al., 2007) . While C-type lectins do not directly mediate filovirus entry, their pattern of expression in vivo and their ability to enhance infection indicate that C-type lectins can play an important role in filovirus transmission and tissue tropism. Thus, increased infection of these cells might be directly involved in the pathogenesis of filoviruses. Accordingly, it was shown that soluble mannose-binding C-type lectin played a role in protection from lethal Ebola virus infection in a mouse model (Michelow et al., 2011) . It should be noted that the ability to utilize the C-type lectins (i.e., DC-SIGN and hMGL) to promote cellular entry was correlated with the different pathogenicities among filoviruses (Takada et al., 2004; Marzi et al., 2006; Matsuno et al., 2010a) . Interestingly, the MLR amino acid sequence does not seem to be the primary factor contributing to the difference (Marzi et al., 2006; Matsuno et al., 2010a; Usami et al., 2011) . Although there might be some distinct mechanisms of entry between MARV and EBOV (Chan et al., 2000) , the similarity of tissue tropism and pathological features of infection between these viruses suggests that C-type lectins are one of www.frontiersin.org the important molecules, likely as attachment factors, for filovirus entry into cells, and that they are directly involved in filovirus tropism at the cellular level.
In addition to the common receptor/co-receptor-dependent mechanism of cellular attachment and membrane fusion, some viruses utilize antiviral antibodies for their efficient entry into target cells (Takada and Kawaoka, 2003) . This mechanism is known as antibody-dependent enhancement (ADE) of viral infection. Filoviruses utilize virus-specific antibodies for their entry into cells in vitro through interaction between anti-GP antibodies and the cellular Fc receptor (FcR) or complement component C1q and its ligand, which likely promotes viral attachment to cells (Takada et al., , 2003a (Takada et al., , 2007 Nakayama et al., 2011) (Figure 6) . FcR are expressed exclusively on the cells of the immune system such as monocytes/macrophages, neutrophils, B-cells, and granulocytes (Fanger and Guyre, 1992) , whereas C1q ligands have been identified in most mammalian cells (Eggleton et al., 1998; Nicholson-Weller and Klickstein, 1999) , suggesting a ubiquitous mechanism for ADE of filovirus infection.
By using GP-specific monoclonal antibodies, several epitopes recognized by ADE antibodies were identified and these epitopes were mostly located in the MLR of the GP1 subunit (Takada et al., 2007; Nakayama et al., 2011) . It should be noted that neutralizing antibodies appear to recognize different epitopes that are not located on the MLR (Takada et al., 2003b; Lee et al., 2008) . As reflected by the high variability of the MLR amino acid sequences and limited overall cross-reactivity of anti-sera among filovirus species (i.e., ZEBOV, SEBOV, CIEBOV, BEBOV, REBOV, and MARV), ADE activities of the anti-sera to GP are virus-species-specific (Takada et al., , 2007 Nakayama et al., 2010) . Interestingly, potential viral pathogenicity is correlated with the ability to induce ADE antibodies, suggesting the possible contribution of ADE to different pathogenicity between filoviruses Nakayama et al., 2011) . More importantly, the demonstration of ADE of filovirus infection raises fundamental questions about the development of GP-based vaccines and the use of anti-GP antibodies for passive immunization.
Recently, GP has been used for viral vector-based or DNA vaccines that were shown to protect animals effectively. Replicationincompetent adenovirus expressing GP, a replication-competent vesicular stomatitis virus expressing GP, and a recombinant paramyxovirus expressing GP have been shown to protect nonhuman primates from lethal infections of filoviruses (Sullivan et al., , 2003 Jones et al., 2005; Bukreyev et al., 2007; Feldmann et al., 2007) . It should be noted that these vaccines potentially induce cytotoxic cellular response (i.e., CD8+ T lymphocytes) as well as antibody production, suggesting that activating cytotoxic T-cells is a key protective mechanism (Olinger et al., 2005; Sullivan et al., 2006; Reed and Mohamadzadeh, 2007) . Since cytotoxic T-cell response cannot be fully induced by immunization with non-replicative protein antigens such as inactivated virus and subunit vaccines, viral vector-based, or DNA vaccines may be promising in preventing filovirus infection.
All enveloped viruses initiate infection by attaching to host cells followed by membrane fusion via interaction between viral surface proteins and receptor/co-receptor molecules on target cells, and this interaction is often a key determinant controlling viral tissue tropism and/or host range. As described above, it has been demonstrated that filoviruses utilize multiple molecules for their entry into cells. However, it remains elusive whether these molecules serve as functional receptors mediating both viral attachment and membrane fusion or play independent roles as either attachment receptors or fusion receptors. More importantly, none of the cellular molecules identified so far explains filovirus tissue tropism and host range reasonably. It might also be hypothesized that filoviruses do not use a single common receptor to infect a broad range of cells and, unlike many other viruses, may not need a "specific" receptor. Although the overall tropism and pathogenicity of filoviruses is controlled by multiple host cell factors (e.g., interactions with the host immune system), further studies aimed at identification of cellular molecules interacting with GP are needed to fully understand the mechanisms of cellular entry of filoviruses, and may shed light on the development of strategies for prophylaxis and treatment of Marburg and Ebola hemorrhagic fevers.
I thank Kim Barrymore for editing the manuscript. This work was supported by the Takeda Science Foundation, and done within the framework of the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) and the Global COE Program "Establishment of International Collaboration Centers for Zoonosis Control" of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. The work was further supported by a Grant-in-aid from the Ministry of Health, Labor, and Welfare, Japan. Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS) Viruses Expressing Indicator Proteins and Antiviral Cytokines Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection. More than 20 years after initial reports [1] [2] [3] , porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a global swine industry problem with losses in the U.S. approaching $6 billion over the last decade [4] . Belonging to the arteriviridae family in the order nidovirales, PRRSV is an enveloped RNA virus containing a single positive-strand RNA genome. The 15 kb viral RNA genome consists of seven open reading frames (ORF1- 7) . ORF1 comprises about 80% of the genome and encodes proteins with protease, replicase and regulatory functions. The smaller overlapping ORF2-7 encode five minor (GP2a, GP3, GP4, 5a and E proteins) and three major (GP5, M and N proteins) structural proteins [5] [6] [7] . Several studies have shown that PRRSV possesses the capacity to subvert early innate immune responses in pigs by suppressing the production of antiviral cytokines [8] [9] [10] [11] [12] [13] [14] [15] , which also contributes to ineffective B-and T-cell responses [16] [17] [18] [19] . Superimposed on this suppressive activity is a high viral mutation rate, which has made the development of vaccines challenging [20] . Modified live vaccines (MLV) used for control of PRRSV in the U.S. are based on only two virus isolates [20, 21] . Although MLV protect against some homologous field strains, their efficacy is not satisfactory due to failure to protect against infections of heterologous strains, as well as the potential risk for reversion to virulence [20, 21] . To develop successful vaccines against PRRSV infections, particularly those by heterologous strains, it is necessary to develop novel vector systems and to extensively categorize host factors critical in the virus-host interaction.
Several viral vectors, including those based on pseudorabies virus, poxvirus, adenovirus and transmissible gastroenteritis coronavirus (TGEV) have been used to express PRRSV structural proteins [22] or host immune factors [23] [24] [25] for developing anti-PRRSV immunity. For example, humoral immunity against PRRSV GP5 protein was detected in pigs immunized with fowlpoxviruscoexpressing PRRSV GP5/GP3 and porcine IL-18 [23] , and in mice immunized with adenovirusexpressing GP5/GP3 fused with swine granulocyte-macrophage colony stimulating factor (GM-CSF) [25] . In this context, we and others have proposed to use PRRSV infectious cDNA clones [26, 27] or virus replicons [28] as vectors for the expression of immune effectors that potentiate innate and adaptive immunity against a broad range of PRRSV isolates. Here we show that PRRSV infectious clones are effective vector systems to express exogenous antigens and host immune effectors. Specifically, we have constructed serial replication-competent viruses from a PRRSV infectious clone-based vector expressing indicator proteins and porcine type I interferons (IFNs). The indicator protein-expressing PRRS viruses efficiently produce progeny viruses and provide an efficient means for real-time monitoring of viral replication, thus allowing high-throughput elucidation of the role of host factors in PRRSV infection. In addition, the replication of some IFN-incorporated viruses is associated with the expression of active IFN peptides, which are capable of counteracting the subverted innate immune response and with potential to induce more effective adaptive immunity against PRRSV infection.
To investigate the potential of infectious PRRSV cDNA clones as a platform for gene manipulation [29, 30] , we first engineered an established infectious clone to express several indicator proteins including Renilla luciferase (Rluc), and green and red fluorescent proteins (GFP and DsRed, respectively). Coding regions of the indicator proteins were constructed in the junction regions surrounding ORF1b/ORF2a through introduced restrictive digestion sites as described [29, 30] ( Figure 1A ). The construct was designed to express the recombinant protein gene through the creation of an additional subgenomic mRNA. Plasmids of selected authentic clones were transfected into MARC-145 cells for the production of progeny viruses. As shown in Figure 1 , infectious clones efficiently produced progeny viruses with successful expression of indictor proteins ( Figure 1B ). Replication rates of these engineered viruses were similar to their parental virus as judged by monitoring ratios of virus-positive cells; and they produced comparable infectious virons shown by similar viral titers ( Figure 1C ). Further experiments with GFP-PRRSV have shown their similar infectivity as a parental strain in porcine alveolar macrophages (PAMs) as well as monocyte-derived dendritic cells (mDCs) ( Figure 1B and data not shown). The indicator-expressing viruses provide an efficient means for real-time monitoring of viral replication. Whereas GFP-and DsRed-PRRSV facilitated detection of fluorescent proteins after 16 h, the Rluc-PRRSV was useful for measuring Rluc activity from 5-20 h.
These viruses allowed us to efficiently elucidate the role of some host factors in PRRSV infection. Several infectious cDNA clones have been generated from field PRRSV strains [31] [32] [33] [34] [35] [36] [37] , which provide efficient means for molecular manipulation of PRRSV genome and evaluation of molecular evolution of this RNA virus with a high mutation rate. The vector cassette used in this study was generated from an infectious clone of North American PRRSV strain P129 with two unique restriction sites and a copy of the transcription regulatory sequence of ORF6 (TRS6) inserted between ORFs 1b and 2a [26, 27, 29, 30] . In addition to GFP, which was introduced in PRRSV infectious clones in several previous studies [26, 27, 29, 31] , two other indicator proteins of DsRed and Rluc were introduced in the PRRSV cDNA clone in this study. The construction of DsRed and particularly Rluc into the PRRSV cDNA clone was intended to produce laboratory viruses that mimic their parental PRRSV with similar replication kinetics but are easily detected and quantified during the early phase of virus infection/replication. As shown in both MARC-145 cells and PAMs, all GFP, DsRed and Rluc expressing progeny viruses had similar replication kinetics as their parental PRRSV. For detection, the red fluorescence of DsRed labeling not only provided a counterstaining choice but also was more sensitive for microscopic observation in real-time rather than traditional immunostaining procedures in fixed cells (Rural Technologies, Brookings, SD, USA). In contrast to GFP and DsRed viruses, which were generally detectable after 12 h in infected cells using microscopy, the Rluc PRRSV in conjunction with an EnduRen™ in vivo substrate (Promega, Madison, WI) facilitated real-time detection of the virus replication as early as 5 h post infection in cells. This Rluc expressing PRRSV thus provides an efficient means for genome-wide examination of host factors involved in the early stages of virus infection [38, 39] . However, our attempts to clone the firefly luciferase gene (~1.6 kb) into the same PRRSV cDNA vector were unsuccessful, suggesting that the PRRSV clone vector has a limited capacity for incorporation of exogenous genes at about 2 kb. 
TSG101 is a housekeeping protein and has been implicated in a number of cellular functions, including mitotic spindle formation, genome stability and endosomal sorting [40] . Essential in endosomal sorting, TSG101 interacts directly with ubiquitinated proteins and internalizes them into the multivesicular body pathway for degradation. During replication, viruses require a retrograde movement from the cell interior to the outer membrane [40] . A number of reports have shown that TSG101 is involved in the virus fusion/budding process from the cellular membrane. These viruses include human immunodeficiency virus (HIV) and hepatitis C virus (HCV) [40] [41] [42] . Recently, a tentative TSG101-targeting peptide, FGI-104, was shown to have a broad-spectrum ability to inhibit infections by several viruses including HIV, HCV and PRRSV [43] . However, there is no direct mechanistic evidence that has demonstrated the involvement of TSG101 in the control of PRRSV replication. To study this possibility, we produced MARC-145 cell lines with targeted suppression of endogenous TSG101 expression. MARC-145 cells were transfected with a pGFP-V-RS vector expressing a 29 nt shRNA (OriGene, Rockville, MD, USA) against a conserved region of TSG101. Two puromycin-resistant colonies showing significant suppression of TSG101 at both RNA and protein levels, were selected. As shown in Figure 2A , cells from colony 1 had less than 10% endogenous expression of tsg101 RNA, and those from colony 9 about 20% of endogenous expression of tsg101 RNA, as well as 70-80% reduction in TSG101 protein. We then compared PRRSV replication in these TSG101-suppressed cells with control MARC-145 cells or cells transfected with scrambled shRNA constructs. Using either GFP-or DsRed-expressing PRRSV, the retarded replication of viruses in TSG101-suppressed cells was demonstrated between 12 and 48 h after infection ( Figure 2B ,C). However, by 72 h, viral replication among TSG101-suppressed and control cells was not significantly different ( Figure 2D ). The return of PRRSV replication in TSG101-suppressed cells might result from virus-stimulated expression of TSG101 and/or incomplete suppression. The earlier and more quantitative comparison of PRRSV replication among TSG101-suppressed and control cells was conducted with the Rluc-expressing PRRSV. Significantly retarded viral replication was detected as early as 5 h post Rluc-PRRSV infection with the in vivo Rluc substrate. The difference in PRRSV replication was quantitatively correlated to the TSG101 levels in different group of cells (Figure 2A and 2E), and could be monitored until 20 h post infection when the substrate was limited. Notably, the replication retardation of indicator protein-expressing viruses in TSG101-silent cells was mostly due to TSG101 suppression because these bioengineered viruses have similar replication kinetics as their parental viruses in normal cells (Figure 1 ). Using the kinetics (i.e., 12-20 h post infection) defined by the indicator protein-expressing viruses, we reproducibly detected retarded replication rates of wildtype PRRS viruses in TSG101 suppressed cells (data no shown). To further test the role of TSG101 in PRRSV infection in porcine cells or pigs, we have characterized the porcine TSG101 cDNA sequence (GenBank TM accession numbers JN882576). The full-length porcine TSG101 cDNA is 1580 bp encoding a precursor protein of 391 residues. TSG101 genes are conserved with most mammalian homologs sharing >94% identity at both RNA and protein levels. The shRNA template sequence we used for loss-of-function studies in MARC-145 cells is identical among human, monkey and porcine TSG101 cDNA. Transient transformation of the shRNA into porcine mDCs similarly suppressed GFP, DsRed-and Rluc-expressing PRRSV until 48 h post infection (data not shown). 
To determine the potential of host factors as a means of counteracting viral immunomodulating activity and stimulating effective antiviral immunity [44] , we bioengineered the infectious clones to express a variety of type I IFNs, which were selected because of their well-documented role in PRRSV infections and activity in mediation of antiviral immunity [9, 15, [45] [46] [47] . In addition, the suppression of type I IFN expression by PRRSV infection has been well documented [15] . Shown in Figure 3 are data from the infectious clone expressing four type I IFNs (IFNα1, IFNβ, IFNδ3 and IFNω5). At 4 d post transfection with equivalent cDNA clones, virus-positive cells were detected in cells transfected with all four infectious clones but with different densities. Where IFNδ3-PRRSV propagated similarly as the control GFP-virus, the replication rates of IFN-β-, IFNα1-and IFNω5-viruses were attenuated by approximately 70% (β-type) or 90% (α1-& ω5-types) ( Figure 3A-H) . Attenuation of IFN-expressing viruses could be caused by viral genome alteration or more likely by the replication-associated expression of active IFN peptides, which was consistent with the anti-PRRSV activity of these IFN subtypes [15] ( Figure 3C ). In contrast, IFNδ3-and GFP-type viruses replicated well. To confirm these findings, we counterstained IFNα1-virus infected cells with antibodies against porcine IFNα (R&D, Minneapolis, MN, USA) and PRRSV nucleocapsid (N) protein ( Figure 4A-D) . Co-localization of IFNα-and PRRSV-labeling ( Figure 4C ) indicated that IFN polypeptides were expressed by the engineered viruses during infection. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected; and again, the intensity of the inhibition was consistent with the anti-PRRSV activity of these IFN subtypes, i.e., IFNω5IFNα1>IFN-β>IFNδ3 ( Figure 4E ). However, because the expression of IFN inhibits virus replication [22, 44] , engineered viruses with the most active IFN subtypes against PRRSV may not be good candidates for a modified live virus (MLV) vaccine. This limitation would prevent the preparation of a high titer virus as a vaccine. Several measures may be used to overcome these limitations, such as incorporating IFN subtypes that do not inhibit replication of a MLV strain but may up-regulate B-and T-cell responses [44] , or controlled expression/activation of the incorporated IFN peptides within certain temporal windows or cell types. Pigs have at least 35 functional type I IFN genes with diverse antiviral or immunoregulatory activity [47] , which provides several candidates for balancing antiviral and immunoregulatory activity to optimize the replication-competent recombinant viruses. Type I IFNs mediate antiviral responses through induction of IFN stimulated genes, such as MxA and RNase L. Direct incorporation of some ISGs (or their functional domains) into the PRRSV infectious clone provides an attractive alternative given that PRRSV-specific ISGs have been identified [15] . In addition, incorporating an exogenous gene tag (a compliance marker) at the vector backbone of a MLV will also allow differentiation of vaccinated from non-vaccinated animals [27] .
As for the stability of the bioengineered viruses, we passed viruses through MARC-145 cells and PAMs for 5-6 generations. Authentic progeny viruses were rescued after three generations, but viruses with a titer higher than 10 3 were only rescued with the indicator protein expressing group and the one expressing IFNδ3. DsRed-and IFNδ3-expressing viruses remained stable for 6 generations (Data not shown). 
Viruses and cells: All virus and animal procedures were approved by the Kansas State University Biosafety and Institutional Animal Care and Use committees. The wild type strains of PRRSV used here were two North American PRRSV strains: NVSL97-7895 and SDSU28983 [26, 27] . The expression cassette was created by insertion of two unique restriction sites and a copy of the transcription regulatory sequence of ORF6 (TRS6) between ORFs 1b and 2a in the infectious cDNA clone of pCMV-P129 ( Figure 1A ) [29, 30] (gift from Dr. Jay G. Calvert, Pfizer Animal Health, Kalamazoo, MI, USA). All bioengineered viruses originated from the backbone of pCMV-P129 with incorporation of an exogenous gene between the two cloning sites. All PRRSV strains were propagated in a simian kidney cell line (MARC-145) or porcine alveolar macrophages (PAMs). MARC-145 cells were maintained in minimum essential medium (MEM) with 8% fetal bovine serum (FBS) and 1× penicillin/streptomycin and fungizone (Invitrogen, Grand Island, NY, USA) PAMs were obtained from lungs of 4-to 6-week-old pigs by lung lavage with PBS and cryopreserved in liquid nitrogen until use. In use, PAMs were plated in RPMI medium with 10% FBS plus 1X penicillin/streptomycin and fungizone. After 2 d, cells were infected with the virus [47] . Monocyte-origin dendritic cells (mDCs) were induced from porcine peripheral blood mononuclear cells (PBMCs) and infected with PRRS viruses as described in Loving et al. [48] .
Production and titration of recombinant PRRS viruses with expression of exogenous genes: In brief, two restriction enzyme digestion sites (Afl II and Mlu I) were introduced into 5'-and 3'-ends of the coding regions, which were amplified from authentic cDNA clones using a high-fidelity PCR [15, 47] . The cloning primers used for this study are listed in the Supplemental Table 1 . The amplified coding regions were purified and cloned into the expression cassette. MARC-145 cells were transfected with authentic plasmids purified from E. coli clones for examination of the production of progeny viruses and expression of indicator proteins or IFNs. Transfection of MARC-145 in 24-well plates was performed with 2 μg of plasmid DNA using Lipofectamine™ 2000 (Invitrogen). Subsequent virus yields were measured by end-point titration of culture media on MARC-145 cells and PAMs. Serial 10-fold dilutions of virus were placed in 96-well tissue culture plates containing confluent MARC-145 cells or PAMs. Cells were fixed in 4% formaldehyde in PBS and the virus was detected by staining for the presence of nucleocapsid antigen using a mAb (SDOW-17, Rural Technologies, Brookings, SD, USA) labeled with TRITC-conjugated secondary antibodies. In addition, the recombinant viruses could be distinctly detected by the expression of fluorescent proteins. Results were reported as log TCID50/mL [15, 47] . The replication of Rluc-expressing PRRSV was monitored with the addition of an in vivo Renilla luciferase substrate (Promega, Madison, WI, USA) at 60 μM in cell culture medium and measured the luminescence after 2 h [49] .
Determination of growth kinetics and stability of the recombinant viruses in cells: Culture supernatants from cells transfected with infectious clones were harvested at 5 d post-transfection and designated 'passage (P) 1'. The P1 virus was used to inoculate fresh MARC-145 cells to collect P2 then P3 at an interval of 4-5 d between successive passages. Each passage virus was titrated, aliquoted and stored at −80 °C until use. Growth curves of the rescued viruses were evaluated by inoculating MARC-145 cells with P3 viruses at a MOI of 0.1. Aliquots of the supernatants of infected cells were collected at points with 10 h intervals until 100 h and the virus was titrated by determining log TCID50/mL to monitor growth kinetics [26] . To determine the stability of the recombinant virus, the rescued P1 virus was passaged on MARC-145 and PAM cells until P6. Expression of indicator proteins and IFN along with virus replication was detected by RT-PCR, western blotting and immunofluorescence as described above [15, 47] .
Producing MARC-145 cell lines with shRNA-mediated silencing of the Tsg101 gene: Three g of pGFP-V-RS vector expressing a scrambled shRNA or a 29 nt shRNA (OriGene, Rockville, MD, USA) against a conservative region between human and simian TSG101 cDNA, was used to transfect MARC-145 cells growing in 6-well culture plates. Transfected cells were selected with 0.8 µg/mL of puromycin (Invitrogen) to obtain individual colonies. Approximately 30 puromycin-resistant colonies were picked and two of them showing significant suppression of TSG101 at both RNA and protein levels were subcultured for loss-of-function studies of the role of TSG101 in PRRSV infection. Primers used for RT-PCR detection of tsg101 were generated against consensus sequence of monkey, human and porcine tsg101 cDNAs (GenBank TM accession numbers, NM_001195481, NM_006292 and JN882576, respectively), which allowed us to detect both monkey and porcine tsg101 with the same pair of primers. The primers were 5′-ATACCCTCCCAATCCCAGTGGTTA-3′ (sense) and 5′-ATCCATYTCCTCCTTCATCCGCCA-3′ (antisense, Y = C or T). Anti-TSG101 monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The involvement of TSG101 in PRRSV infection was evaluated by comparing the replication kinetics of PRRS viruses between cells with or without TSG101-suppression [47] .
Data analyses: Virus titrations were done with at least three repeats to report log TCID50/mL . Relative gene-expression data of real-time PCR was normalized against C t values of the housekeeping gene (GAPDH) and the relative expression index (2 −ΔΔCt ) was determined in comparison to the base levels of control samples. Growth curves were generated with Sigmaplot 11.0 (Systat, San Jose, CA, USA) and the densitometry analysis of images were done by using AlphaEase FC Software (Alpha Inotech, Santa Clara, CA, USA) as described [47, 50] .
The PRRSV cDNA infectious clone, pCMV-129, has vector capacity to express exogenous genes of less than 2 kb, which allows reconstruction of the virus for bioengineering manipulation [29, 30] .
Recombinant PRRS viruses expressing several indicator proteins have replication and infection dynamics similar to the parental strain in both MARC-145 and porcine cells. Therefore, they may be used to decipher the role of host factors in PRRSV infection [38, 39] . Using several indicator protein-expressing viruses, in particular the Rluc-expressing PRRSV, we showed that TSG101 was significantly involved in PRRSV infection at the early phase of the infection.
Recombinant PRRS viruses expressing antiviral cytokines produce active cytokines in the infected cells and alter the replication of co-infected PRRSV. These constructs may be candidates for modified live virus vaccines, which could ameliorate subverted innate immune responses and potentially enhance adaptive immunity against PRRSV infection. A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells. All eukaryotic plus-stranded (+)RNA viruses have similar replication cycles in infected cells. After translation of their mRNA-sense genomic RNA(s), the viral RNA and the viral replication proteins are recruited to the site of viral replication in membranous compartments. After the assembly of the membranebound viral replicase complexes (VRC), the viral replicase uses the viral RNA as a template to produce complementary (2) RNA. This is then followed by (+)-strand synthesis in an asymmetric manner, producing excess amounts of (+)-strand progeny, which is released from replication for other viral processes. For efficient replication, (+)RNA viruses recruit numerous host proteins [1] [2] [3] [4] [5] . Among the identified host proteins are RNA-binding proteins, such as translation factors, ribosomal proteins and RNAmodifying enzymes [1] . The co-opted host proteins likely affect several steps in viral RNA replication, including the assembly of the replicase complex and/or viral RNA synthesis. However, the functions of host factors in (+)RNA virus replication are known only for a small number of host factors [1] [2] [3] [4] [5] [6] [7] [8] [9] .
Tomato bushy stunt virus (TBSV) is a model plant RNA virus that is used intensively for identification and characterization of host factors [1, [10] [11] [12] . The single genomic RNA codes for two replication proteins, p33 and p92 pol , which are sufficient to support TBSV replicon (rep)RNA replication in yeast (Saccharomyces cerevisiae) model host [13, 14] . p33 and p92 pol are components of the membrane-bound VRC that also requires the tombusviral (+)repRNA as a platform during its assembly and activation [11, [15] [16] [17] . Recent genome-wide screens and global proteomics approaches with TBSV based on yeast revealed a large number of host factors interacting with viral components or affecting TBSV replication. A large fraction of the identified host proteins are RNA binding proteins, which might affect viral RNA synthesis [10, [18] [19] [20] .
The highly purified tombusvirus VRC is known to contain at least six permanent resident host proteins, including the heat shock protein 70 chaperones (Hsp70, Ssa1/2p in yeast) [21] [22] [23] [24] , glyceraldehyde-3-phosphate dehydrogenase (GAPDH, encoded by TDH2 and TDH3 in yeast) [25] , pyruvate decarboxylase (Pdc1p) [24] , Cdc34p E2 ubiquitin conjugating enzyme [24] [25] [26] , eukaryotic translation elongation factor 1A (eEF1A) [27, 28] , eukaryotic translation elongation factor 1Bgamma (eEF1Bc) [29] and two temporary resident proteins, Pex19p shuttle protein [30] and the Vps23p adaptor ESCRT protein [28, 31, 32] . Although the functions of several of these proteins have been studied in some details, additional host proteins might be also present in tombusvirus VRC [5, 10, [21] [22] [23] 25] . One of the intriguing questions is if tombusviruses use host-coded helicases for their replication. This is because, unlike larger RNA viruses, the tombusvirus genome does not code for a protein with helicase function. Thus, can tombusviruses and other small RNA viruses replicate without the use of a helicase or they subvert a cellular helicase(s) to assist their replication? DEAD-box proteins constitute the largest family of RNA helicases that are involved in all aspects of cellular metabolism and perform RNA duplex unwinding and remodeling of RNA-protein complexes in cells [33] [34] [35] . Ded1p, which is essential for yeast growth, is among the best-characterized DEAD-box helicases [36] . It is involved in initiation of translation of every yeast mRNA [37] [38] [39] and down-regulation of Ded1p level also reduced p33 and p92 levels in yeast [20] . Therefore, not surprisingly, TBSV replication also decreased significantly in yeast with reduced level of Ded1p. Due to its essential role in translation, it is critical to separate the possible role of Ded1p in viral replication from its effect on viral translation.
In this paper, a yeast-based cell-free TBSV replication assay and recombinant Ded1p was used to test the role of Ded1p in TBSV replication. We define that Ded1p plays a role in enhancing plusstrand (+)RNA synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus VRC and binds to the 39-end of the viral minus-stranded (2)RNA. The obtained data support the model that Ded1p unwinds the 39-end of the TBSV (2)RNA, rendering the RNA compatible for initiation of (+)strand synthesis. Interestingly, we find that Ded1p and GAPDH play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (2)RNA and the replication proteins.
Reduced level of TBSV RNA replication in yeast cell-free extract depleted for Ded1p DEAD-box RNA helicase Since Ded1p is an essential translation factor for all yeast mRNAs [38, 39] , it is difficult to separate its direct versus indirect effect on TBSV replication in yeast model host. To circumvent this problem, we used whole cell extracts (CFE) prepared from yeast containing reduced level of Ded1p ( Figure S1 ) to support cell-free TBSV replication. As we have shown previously, TBSV (+)RNA can perform one complete cycle of replication in the CFE-based replication assay when purified recombinant p33 and p92 pol replication proteins are provided [23, 40] . The CFE-based replication assay showed that both (+) and (2)RNA synthesis decreased by over 4-fold when Ded1p was down-regulated as compared with the control CFE prepared from yeast with high level of Ded1p expression ( Figure S1 , lanes 4-6 versus 1-3). Thus, these data confirm that Ded1p is important for TBSV replication. However, the observed decrease in the CFE-based TBSV replication could be due to either reduced replication when Ded1p is limiting (direct effect) or lesser amounts of other critical host factors needed for TBSV replication in the CFE with reduced level of Ded1p (indirect effect due to Ded1p's role in host translation). To address these points, we first studied if Ded1p is present within the tombusvirus replicase and then, what is the mechanistic role of Ded1p during TBSV RNA synthesis.
To examine if Ded1p is present within the tombusvirus replicase complex, we FLAG affinity-purified the tombusvirus replicase from yeast cells actively replicating TBSV repRNA [14, 28] . This yeast also expressed HA-tagged Ded1p from yeast chromosome based on the native promoter. We found that the tombusvirus replicase preparation, which is highly active on added templates in vitro (not shown), contained Ded1p ( Figure 1 , lane 4), while Ded1p was undetectable in the control yeast sample obtained using the same affinity purification ( Figure 1 , lane 3). The control yeast also expressed the tombusvirus replication proteins (including the 66His-tagged p33, but lacking FLAG-tag) and the HA-tagged Ded1p from yeast chromosome based on the native promoter and supported TBSV repRNA replication (not shown).
We also FLAG-affinity-purified 66His/FLAG-tagged Ded1p from the membrane-fraction of yeast co-expressing HA-p33 and HA-p92 and found that the purified Ded1p preparation contained HA-tagged p33 ( Figure S2A , lane 1). The affinity-purified Ded1p preparation also showed TBSV replicase activity in vitro on added TBSV template ( Figure S2B , lane 2), suggesting that the membrane-bound Ded1p is associated with the active tombusvirus replicase. Altogether, these data support the model that Ded1p is part of the tombusvirus replicase. Figure 1 . Co-purification of Ded1p with the p33 replication protein from yeast. The FLAG/66His-tagged p33HF or 66His-tagged p33H was purified from yeast extracts using a FLAG-affinity column (lanes 3-4). Top panel: Western blot analysis of Ded1p tagged with 66HA, expressed from the natural promoter in the chromosome, with anti-HA antibody in the purified p33 preparations. Bottom panel: Western blot analysis of HF-tagged p33 or 66His-tagged p33 with anti-His antibody. Note that ''total'' represents the total protein extract from yeast expressing the shown proteins. Each experiment was repeated three times. doi:10.1371/journal.ppat.1002537.g001
Subverted host factors play a role in plus-strand RNA virus replication. Small RNA viruses do not code for their own helicases and they might recruit host RNA helicases to aid their replication in infected cells. In this paper, the authors show that the Ded1p DEAD-box helicase, which is an essential translation factor in yeast, is recruited by Tomato bushy stunt virus (TBSV) into its replicase complex. They also show that Ded1p binds to the viral (2)RNA and promotes (+)-strand TBSV synthesis when added to a yeast-based cell-free extract depleted for Ded1p. An ATPase defective Ded1p mutant failed to promote TBSV replication in vitro, suggesting that the helicase activity of Ded1p is essential for its function during TBSV replication. In addition, the authors also show that another host protein, which also binds to the (2)RNA, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), further enhances TBSV (+)RNA when added together with Ded1p to yeast-based cell-free extract. In summary, the authors show that the major functions of Ded1p and GAPDH host proteins are to promote TBSV replication via selectively enhancing (+)-strand synthesis.
Recombinant Ded1p DEAD-box helicase enhances TBSV RNA replication in yeast cell-free extract To address if Ded1p could play a direct role in TBSV replication, we added purified recombinant Ded1p to yeast CFE with reduced level of Ded1p (Figure 2A ). We observed a ,2-fold increased level of TBSV RNA replication (based on total singlestranded repRNA level) in the presence of recombinant Ded1p when compared with the MBP control ( Figure 2B , lane 2 versus 4). Interestingly, the amount of double-stranded RNA, which correlates with (2)-strand levels [23] , did not increase in the presence of Ded1p in the CFE assay ( Figure 2B, lanes 1 versus 3) . This finding suggests that (2)-strand RNA did not contribute to the 2-fold increase in total TBSV RNA levels when recombinant Ded1p was added to the CFE-based replication assay. Therefore, we conclude that the added recombinant Ded1p selectively increased TBSV (+)-strand RNA synthesis in vitro.
To demonstrate if the ATPase/helicase activity of Ded1p is important for the enhancement of TBSV RNA replication in the CFE-based replication assay, we tested an ATPase defective mutant of Ded1p (D1 mutant) [41] . This mutant could not enhance TBSV replication in vitro ( Figure 2C , lanes 1-2 versus 3-4), suggesting that the ATPase activity of Ded1p is important for TBSV replication.
To further test what step(s) Ded1p promotes during TBSV replication, we performed a two-step replication assay based on yeast CFE. In this assay, the first step includes the assembly of the replicase complex on the endogenous membranes present in CFE in the presence of the viral (+)repRNA, the p33/p92 replication proteins and ATP/GTP [23] ( Figure 3A , step 1). Under these conditions, the viral replication proteins recruit the repRNA to the membrane and the replicase becomes partially RNase and protease insensitive, but it cannot initiate minus-strand synthesis yet, due to the absence of CTP/UTP [23] . Then, centrifugation and washing the membranes will remove all the proteins and molecules not bound to the membrane. This is followed by addition of ATP/CTP/GTP/UTP to initiate RNA synthesis during the second step.
Addition of purified Ded1p to the CFE during the first step did not increase TBSV replication ( Figure 3B , lane 4 versus 5). This suggests that Ded1p did not facilitate the assembly of the replicase complex, unlike other host factors, such as Hsp70 and eEF1A [23, 27] . Moreover, Ded1p was likely lost during the centrifugation/washing step in this assay due to its low association with the membrane. However, addition of Ded1p exclusively during the second step of the CFE assay resulted in ,2-fold increase in TBSV RNA replication ( Figure 3B , lane 14 versus 15), similar to the stimulatory effect of Ded1p during standard CFE replication assay ( Figure 2 ). Interestingly, a Ded1p mutant (D1) deficient in ATPase activity could not stimulate TBSV RNA synthesis in this assay ( Figure 3B , lane 13), while a mutant (D11, Figure S3C ) with increased ATPase activity [41] promoted TBSV RNA synthesis by ,2-fold ( Figure 3B , lane 11). Altogether, these findings indicate that Ded1p has a direct stimulatory function during TBSV RNA synthesis, while Ded1p is unlikely to affect viral RNA recruitment for replication or VRC assembly.
To further test the direct effect of Ded1p on RNA synthesis, we utilized detergent-solubilized and affinity-purified tombusvirus -stranded RNA products produced in the cell-free TBSV replication assay. The dsRNA products are also shown on the right subpanel with high contrast for better visualization. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (only ssRNA is present). The % of dsRNA and ssRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right replicase from Ded1p-depleted yeast ( Figure 4A ). This purified replicase can only synthesize complementary RNA products on added TBSV templates, but, unlike the above membrane-bound replicase in the CFE-based assay, it cannot perform a complete cycle of RNA synthesis [14, 15] .
We found that addition of purified recombinant Ded1p to the purified tombusvirus replicase programmed with the (2)repRNA stimulated (+)-strand synthesis by ,2.5-3-fold ( Figure 4B , lanes 3-4 versus 1-2; and Figure S3D , lane 3 versus 2). Interestingly, Ded1p stimulated the production of the full-length (+)-strand RNA product, while the amount of 39-terminal extension product (39TEX; due to self-priming by the 39 end of the template [42] [43] [44] , Figure S3B ) decreased in the presence of added recombinant Ded1p. Therefore, we suggest that Ded1p facilitates the de novo initiation on the (2)RNA template by the tombusvirus replicase.
The ATPase inactive mutant D1 ( Figure 4B , lanes 5-6 and S3D, lane 4), D5 and D10 ( Figure S3D , lanes 6 and 8) could not promote (+)-strand synthesis, suggesting that the ATPase activity of Ded1p is required for the above stimulatory effect on TBSV RNA synthesis. Also, two active ATPase mutants, D3 and D11 ( Figure S3C ), did facilitate (+)-strand synthesis ( Figure S3D , lanes 5 and 9), confirming that the ATPase activity of Ded1p is required during TBSV (+)-strand RNA synthesis. Interestingly, Ded1p mutant D11, albeit has increased ATPase activity, has wt-like strand displacement activity in vitro [41] . We found that D11 behaved similarly in TBSV replicase assay ( Figure S3D ) to wt Ded1p, suggesting that the unwinding activity of Ded1p is important during TBSV replication.
To test if Ded1p can also stimulate the activity of the tombusvirus replicase on (+)-stranded RNA templates, we used DI-72(+) RNA in the purified tombusvirus replicase assay ( Figure  S4A ). The TBSV (+)RNA is known to carry a replication silencer element (RSE) at the 39 end that inhibits (2)RNA synthesis in vitro [45] . Addition of Ded1p to the tombusvirus replicase assay did not enhance (2)RNA synthesis ( Figure S4B , lane 3 versus 1-2). Also, D1 mutant deficient in ATPase activity had not much effect on (2)RNA synthesis ( Figure S4B , lane 4 versus 1-2). Based on these data, we suggest that Ded1p does not affect (2)RNA synthesis by the tombusvirus replicase in vitro.
To identify the region(s) of the TBSV RNA bound by the recombinant Ded1p, we performed electrophoresis mobility shift assay (EMSA) with purified components. Comparison of 32 Plabeled TBSV (+) and (2)RNAs in binding to purified recombinant Ded1p revealed that (2)RNA bound more readily to Ded1p in vitro than (+)RNA ( Figure 5B , lanes 2-6 versus 8-12). Additional EMSA experiments using the four regions in DI-72 (2)repRNA ( Figure 5A ) as unlabeled competitors revealed that RI(2) was the most efficient in outcompeting the 32 P-labeled TBSV (2)repRNA in binding to Ded1p ( Figure 5C , lanes [8] [9] . This is important since RI(2) is the 39 end of (2)repRNA and contains important cisacting elements, such as the promoter and a short enhancer sequence for (+)RNA synthesis [46, 47] .
Further testing of Ded1p binding to (+)repRNA regions revealed that RIV(+), representing the 39 noncoding region in TBSV RNA, was bound more efficiently by Ded1p ( Figure S5 , Step-wise cell-free TBSV replication assay does not support a role for Ded1p helicase in the assembly of the TBSV VRC. (A) Scheme of the CFE-based TBSV replicase assembly and replication assays. Purified recombinant p33 and p92 pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the whole cell extract prepared from Ded1p-depleted yeast strain in step 1. The assay either contained or lacked the purified recombinant Ded1p (0.3 mg) or MBP during step 1. Note that the assay was performed in the presence of ATP/GTP to facilitate TBSV VRC assembly, but prevent RNA synthesis in step 1. After step 1, centrifugation was used to collect the membrane fraction of the CFE, and after washing the membranes, step 2 was performed in the presence of ATP/CTP/GTP and 32 P-UTP to allow TBSV RNA replication. In the samples presented in right panel, Ded1p or MBP were added at the beginning of step 2. (B) Denaturing PAGE analysis of the 32 P-labeled TBSV repRNA products obtained in the CFE assays in the presence of wt and various Ded1p mutants (0.3 mg each) ( Figure S3C ) or MBP. See further details in Figure 2 . Note that the various preps of D3 mutant of Ded1p showed high variation in in vitro activity (for reasons currently unknown)-see Figure 4B . Each experiment was repeated at least three times and the data were used to calculate standard deviation. To test if Ded1p can interact with the TBSV p33 replication protein, we used the membrane-based split-ubiquitin assay [31] . We found that Ded1p interacted with p33 protein in yeast ( Figure  S6A ). The pull-down experiments with MBP-p33 and MBP-p92 also showed that Ded1p bound to the tombusvirus replication proteins in vitro ( Figure S6B ). This was further supported by the reverse pull-down experiments with GST-tagged Ded1p, which resulted in the co-purification of p33 and p92 ( Figure S6C ).
To test if a DEAD-box helicase from plants might have similar stimulatory function on the activity of tombusvirus replicase, we have cloned and purified RH20 cytosolic DEAD-box helicase from Arabidosis thaliana, which shows high degree of similarity to yeast Ded1p ( Figure S7 ). Addition of the recombinant AtRH20 to the CFE prepared from Ded1p-depleted yeast strain increased TBSV repRNA replication by ,2-fold ( Figure 6A, lane 3) . Also, adding the recombinant RH20 to the replicase assay led to almost Yeast with depleted Ded1p co-expressing p33 and p92 pol replication proteins and DI-72 (+)repRNA were used to affinitypurify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (2)repRNA, and they also contained purified recombinant Ded1p, mutants or MBP in addition to ATP/CTP/GTP and 32 P-UTP. (B) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 0.5 mg or 1.0 mg of purified recombinant Ded1p or its mutants is shown. The level of complementary RNA synthesis producing ''repRNA'' (marked as ''FL'', the full-length product, made via de novo initiation from the 39-terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein (lane 1). Note that this replicase preparation also synthesizes de novo internal initiation products (''ii'') and 39-terminal extension products (''39TEX''). Each experiment was repeated three times. (C) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p or its mutants is shown. Note that the FHV replicase can use TBSV (2)repRNA as a template in vitro. Each experiment was repeated three times. (D) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p or its mutants is shown. Note that the FHV DI-634 (2)repRNA was used as a template in the FHV replicase assay. doi:10.1371/journal.ppat.1002537.g004
3-fold increase of the activity of the purified tombusvirus replicase on TBSV (2)RNA template ( Figure 6B , lanes 2-3 versus 1). To test if AtRH20 can interact with the TBSV p33 replication protein, we used the membrane-based split-ubiquitin assay [31] . We found that AtRH20, similar to Ded1p, interacted with p33 protein in yeast ( Figure S6A ). Altogether, these data strongly suggest that plants also have DEAD-box helicases that could play similar role to the yeast Ded1p DEAD-box helicase in tombusvirus (+)RNA synthesis.
To test if Flock house virus (FHV) replicase is affected by Ded1p, first we have developed an in vitro FHV replication assay based on affinity-purified FHV replicase preparation that can be programmed with exogenously added RNAs ( Figure 4C , lanes 1 versus 10). The in vitro FHV replicase assay revealed that the purified recombinant Ded1p increased (+)-strand RNA synthesis on the (2)-stranded FHV template by ,3-fold ( Figure 4D Figure  S4D , lane 2 versus 1). It is likely that the observed stimulation of FHV replicase activity by Ded1p is direct, since we found that Ded1p bound to the FHV repRNA ( Figure S8A ) and protein A RdRp protein in vitro ( Figure S8B ). Altogether, these data show that, similar to the tombusvirus replicase, the activity of FHV replicase is stimulated by Ded1p only on the (2)RNA templates, but not when using the (+)RNA templates.
Ded1p facilitates initiation by the tombusvirus replicase on RNA/DNA duplex To gain further insights into the mechanism of Ded1p-driven stimulation of TBSV and FHV RNA synthesis, we exploited template structures, such as an RNA/DNA duplex, that are known to hinder RdRp-driven RNA synthesis [48, 49] . Since Ded1p is an RNA helicase [39, 41] and the ATPase/helicase function of Ded1p is needed for stimulation of (+)RNA synthesis by the tombusvirus and FHV replicases, we wanted to examine if Ded1p might facilitate RNA synthesis on a partial DNA/RNA duplex. We chose partial duplex for this assay, since Ded1p and other DEAD-box helicases are not processive enzymes and can only unwind short duplexes [34] . Also, we have shown previously that short DNA oligos hybridized to the promoter region of (2)RNA can inhibit (+)RNA synthesis by the tombusvirus replicase in vitro [42, 47, 48] . Interestingly, addition of purified Ded1p to the tombusvirus replicase assay containing the short RNA/DNA duplex ( Figure 7A Non-overlapping functions of Ded1p and GAPDH in promoting initiation by the tombusvirus replicase GAPDH (Tdh2p in yeast) RNA binding protein is also a host factor stimulating (+)RNA synthesis by the tombusvirus replicase [25, 50] . To test if Ded1p and GAPDH could play a complementary role during (+)RNA synthesis, we added the purified recombinant Ded1p and Tdh2p to the in vitro tombusvirus replicase assay based on the purified preparation ( Figure 8A ). While Ded1p mostly stimulated de novo (+)RNA synthesis initiated from the 39 end of the (2)repRNA up to ,3-fold ( Figure 8B , lane 6), Tdh2p enhanced both de novo (+)RNA synthesis and 39TEX by ,2-and ,3-fold, respectively ( Figure 8B, lane 7) . Interestingly, the two host proteins together had the largest (4.5-fold) effect on (+)RNA synthesis, while their effect on 39TEX was only ,2-fold. Based on these data, we suggest that Ded1p and Tdh2p have a We also performed similar experiments with a short partial RNA/DNA duplex as a template. Both Ded1p and Tdh2p alone could enhance (+)RNA synthesis on the short RNA/DNA duplex ( Figure 8C , lanes 2-3 versus 1) but the largest stimulatory effect (i.e., close to 5-fold increase) was seen when both host factors were included in the assay ( Figure 8C, lane 4) . Altogether, these data further support that Ded1p and GAPDH play synergistic roles in (+)RNA synthesis by the tombusvirus replicase. [9] . Since the viral RNA plays multiple roles during infection, it is likely that remodeling of the viral RNAs and RNP complexes during the switch from one step to another requires RNA helicases or RNA chaperones. Accordingly, the larger RNA viruses all code for RNA helicases [51, 52] . However, RNA viruses with shorter than 6 kB genomes usually do not code for RNA helicases. They could still use RNA helicases during infections if they can subvert selected host helicases for viral purposes. Indeed, we show here that Ded1p helicase is recruited for TBSV replication to aid (+)strand RNA synthesis.
It is not yet known if Ded1p is the only host helicase needed for TBSV replication, since genome-wide screens and global proteomics approaches with TBSV have identified additional host helicases as well [9, 10, 28] . However, Ded1p helicase seems to be an ideal host factor to be recruited for viral replication because it is involved in mRNA and viral RNA translation, thus it is colocalized with the viral RNA prior to replication. Also, by recruiting Ded1p, viruses could affect RNA degradation (P-body formation; [53] ) and initiation of translation of new RNAs, likely affecting subsequent host translation (including virus-induced mRNAs coding for anti-viral proteins or required for anti-viral signaling).
The essential nature of Ded1p for host mRNA translation, however, makes characterization of Ded1p function as a host factor difficult. Therefore, the combined use of in vivo and in vitro approaches might be necessary to dissect the function of Ded1p in RNA virus replication as shown in this paper. By using a Ded1pdepleted yeast CFE in combination with recombinant Ded1p allowed us to define that the ATPase (helicase) activity of Ded1p is required for efficient TBSV (+)-strand synthesis (see below). Ded1p also increased the RdRp activity of the FHV replicase, suggesting that recruitment of DEAD-box helicases might also be useful for additional small RNA viruses (see below).
Co-purification experiments revealed that Ded1p is present in the tombusviral VRC (Figure 1 ). In addition, Ded1p affected (+)strand synthesis, but not the assembly of the VRC in vitro (Figures 2-4) , indicating that Ded1p is likely present in the VRC. Surprisingly, Ded1p, unlike other previously tested host factors Hsp70, GAPDH, or eEF1A [21] [22] [23] 27, 54] , was able to affect TBSV repRNA replication even after the replicase assembly step took place in vitro (Figure 3 ). This suggests that Ded1p can enter the membrane-bound VRC, possibly due to its interaction with p33 ( Figure S6 ) and the helicase activity of Ded1p might lead to some remodeling of VRC.
It is not yet known if additional members of the large helicase family could perform similar function to Ded1p during TBSV replication. Our in vitro data with AtRH20 plant helicase protein, which is very similar to Ded1p ( Figure S7 ), suggests that this protein can likely perform similar function in plant infections. For example, AtRH20 has been shown to boost TBSV (+)RNA synthesis in vitro ( Figure 6 ) and bind to p33 replication protein ( Figure S6A ), which can facilitate its recruitment into the VRC. Since more than 50 RNA helicases are present in plants, further experiments will be needed to define if additional host helicases might also be involved in TBSV replication.
The in vitro data, based on the CFE assay containing the membrane-bound VRC as well as the solubilized/purified tombusvirus replicase, showed the Ded1p can mainly stimulate TBSV (+)-strand synthesis, while its effect on (2)RNA synthesis is less pronounced. The ATPase activity of Ded1p is required for this stimulatory effect, suggesting that the helicase function of Ded1p is likely important for unwinding the secondary structure of (2)RNA template with the purified tombusvirus replicase or destabilizing the replication intermediate with the membrane-bound VRC, which might contain dsRNA structure. This function of Ded1p can explain why yeast with down-regulated Ded1p level produced small amount of (+)-stranded RNA progeny, albeit it has also shown that depleted Ded1p reduced the level of p33/p92 in yeast [20] . However, the decreased p33/p92 levels are expected to reduce both (+) and (2)RNA levels [55] ( Figure S1 ). Since the recombinant Ded1p enhanced (+)-strand synthesis by the purified recombinant tombusvirus replicase, we propose that Ded1p directly affect TBSV RNA synthesis via affecting the structure of the RNA templates. However, we cannot exclude that Ded1p could also affect the activity of the VRC due to its interaction with p33, albeit the assembly of the VRC was not affected by Ded1p in the CFE-based assay ( Figure 3) . Overall, the recruitment of a host DEAD-box helicase for replication of a small RNA virus is remarkable, since RNA viruses with less than 6 kB genomes are usually do not code for their own helicases [51] . These viruses are thought to replicate without needing a helicase by using RNA chaperones or possibly recruiting host helicases. The emerging picture with TBSV is that this virus utilizes both the viral-coded p33 RNA chaperone [48] and the host Ded1p helicase for replication by promoting (+)-strand synthesis. Both p33 and Ded1p have been shown to open up short DNA/RNA duplexes, although the activity of Ded1p was more robust than that of p33 in vitro [48] . Why would TBSV utilize both an RNA chaperone and an RNA helicase? It is possible that both proteins are needed for robust (+)RNA synthesis to make excess amount of progeny RNA. Also, Ded1p helicase could be involved in remodeling the viral RNA bound by the viral RdRp or host proteins prior or during RNA synthesis. Since Ded1p can work in both 59-to-39 and 39-to-59 directions [56] , it could be used for multiple purposes during RNA synthesis.
Based on the available data, it seems that TBSV (+)RNA synthesis is not only affected by the p33/p92 replication proteins, but by GAPDH and Ded1p host proteins as well. Since the viral replication proteins were shown to bind to TBSV (2)RNA nonspecifically [57] , we propose that the above host proteins, which bind strongly to the TBSV (2)repRNA, are involved in facilitating the proper and efficient recruitment of the p92 RdRp protein to the (2)RNA template (or alternatively to the dsRNA intermediate) within the VRC as shown in Figure 9 . For example, Ded1p might unwind either local secondary structure in (2)repRNA or dsRNA region and that, in turn, could favor binding of GAPDH or p92 to the (2)RNA template. This is followed by binding of GAPDH to an AU-rich internal site and proper positioning of the p92 RdRp (bound to GAPDH, [50] ) over the (+)-strand initiation promoter, leading to (+)RNA synthesis.
The synergistic effect of these host proteins could promote efficient recycling of the viral RdRp resulting in multiple rounds of (+)RNA synthesis ( Figure 9 ). Thus, the roles of these host proteins are to serve as ''matchmakers'' between the viral RNA template and the viral RdRp. It is intriguing that two host proteins, eEF1A and eEF1Bc, are proposed to serve somewhat similar functions during (2)RNA initiation for TBSV [27, 29] . Therefore, the emerging picture is that TBSV utilizes different host proteins for promoting (2) versus (+)RNA synthesis. This strategy could be beneficial for the virus by allowing asymmetric RNA synthesis, thus leading to excess amount of progeny (+)RNA.
Overall, host DEAD-box or related RNA helicases have been shown to affect many different aspects of virus infections, including translation of viral proteins [58] [59] [60] ; viral RNA replication [61] [62] [63] [64] ; reverse transcription [65] ; the activity of anti-viral proteins [66, 67] , and virus-mediated regulation of host gene transcription [68] . Interestingly, Ded1p is known to affect minus-strand synthesis during replication of the L-A dsRNA virus of yeast [69] , suggesting that the use of DEAD-box helicases is wide-spread among RNA viruses.
Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) and TET::DED1 yeast strain (yTHC library, MATa his3D1 leu2D0 met15D0 URA3::CMV-tTA) was obtained from Open Biosystems (Huntsville, AL, USA). The plasmid pESC-HIS-Gal-His33/Gal-DI-72 expressing Cucumber necrosis virus (CNV) 66His-tagged p33 and the TBSV DI-72 repRNA was described earlier [30] . The CNV p33 protein has very high sequence identity with the closely related TBSV p33, but the CNV p33 is expressed better and more active in yeast than TBSV p33. Recombinant yeast Tdh2p protein was produced in E. coli as GST fusion using plasmid pGEX-TDH2, described earlier [50] . Recombinant Ded1p helicase proteins were produced in E. coli as maltose binding protein (MBP) fusions [48] . The expression plasmid pMal-DED1 was prepared by PCR using primers #3956 (CCAGCTG-CAGTCACCACCAAGAAGAGTTG)/ #3957(CCAGGAATT-TGGCTGAACTGAGCGAACAAG) and the yeast genomic DNA as a template. The plasmids pMal-Dx, expressing different Ded1p mutants, were prepared by PCR using #3956/ #3957 primers and plasmids containing mutated DED1 sequences [41, 53] . The PCR products obtained from DED1 wt and mutant sequences were digested with EcoRI and PstI and inserted between EcoRI and PstI sites in pMalc-26 (New England Biolab).
To express recombinant FHV protein A in yeast, we generated plasmid pGAD/Cup/FHV/protein A/C-term/HA/FLAG. The following sequence was fused downstream to the full length FHV protein A coding region by repeated PCR and cloning steps:
The PCR product was amplified with oligos #3629 (CCGAT-CATGACTCTAAAAGTTATTCTTGGAG) and #3716 (GG-AGCTCGAGTTACTTATCGTCATCGTC) followed by digestion with PagI and XhoI. It was cloned into NcoI and XhoI digested pCupHis92 [70] .
Expression and purification of the recombinant MBP-tagged host proteins and the MBP-tagged TBSV p33 and p92 replication proteins and Ded1p helicase from E. coli were carried out as described earlier with modifications [71] . Briefly, the expression plasmids were transformed separately into E. coli strain BL21(DE3) CodonPlus. Protein expression was induced using isopropyl b-Dthiogalactopyranoside (IPTG) for 8 h at 23uC in the case of host proteins and at 16uC in the case of p33 and p92, then the cells were collected by centrifugation (5,000 rpm for 5 min). The cells were suspended and sonicated in MBP column buffer containing 30 mM HEPES-KOH pH 7.4, 25 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol. The extract was then centrifuged at 16,000 g for 10 min, followed by incubation with amylose resin (NEB) for 15 min at 4uC. After washing the resin 2 times with the column buffer, the proteins were eluted with column buffer containing 0.18% (V/W) maltose. Purification of GST-tagged TDH2 (pGEX-TDH2) [50] was carried out using glutathione resin and eluted with 10 mM glutathione, 10 mM ß-mercaptoethanol in the column buffer following the same protocol as MBPproteins. Eluted proteins were aliquoted for storage at 280uC. Protein fractions used for the replication assays were at least 95% pure, as determined by SDS-PAGE (not shown). We have previously shown that our Ded1p preparation has helicase activity on short RNA/DNA duplexes in vitro [48] .
The 32 P-labeled or unlabeled full-length DI-72 (+) and (2)RNAs and the four separate regions (RI-IV), were generated as described [57] . Transcripts for replicase or CFE replication assays were purified as described earlier [57] . The amounts of transcripts were quantified by UV spectrophotometer (Beckman). To obtain fulllength FHV-derived DI-634 RNA [72] , we used primers #3842 (GTAATACGACTCACTATAGTAAACAATTCCAAGTTCC-AAAATGG) and #3509 (ACCTTAGTCTGTTGACTTAAA-CTGG) or #3519 (GTAAACAATTCCAAGTTCC) and #3527 (GTAATACGACTCACTATAGGGAACCTTAGTCTGTTG-ACTTAAAC) for (+) or (2)DI-634 RNAs, respectively, and pDI634 [72] as a template in PCR reactions. The RNA transcripts were synthesized on the PCR templates using T7based transcription [47] .
In vitro TBSV replication assay in cell-free yeast extract CFEs from BY4741 or TET::DED1 strains capable of supporting TBSV replication in vitro were prepared as described earlier [23, 40] . Briefly, the in vitro TBSV replication assays were performed in 20-ml total volume containing 2 ml of CFE, 0. [23, 40] . Host proteins were added in different amounts as indicated in the Figure legends. The reaction was performed as described [23, 40] . The newly synthesized 32 P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.56 Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85uC for 5 min in the presence of 50% formamide [27] .
Fractionation of the whole cell extract was done according to [23, 40] . The total extract was centrifuged at 21,0006 g at 4uC for 10 min to separate the ''soluble'' (supernatant) and ''membrane'' (pellet) fraction. The pellet was re-suspended and washed with buffer A (30 mM HEPES-KOH pH 7.4, 150 mM potassium acetate, and 5 mM magnesium acetate) followed by centrifugation at 21,0006 g at 4uC for 10 min and re-suspension of the pellet in buffer A. In vitro TBSV replication in the fractions was performed as described [23, 40] .
Yeast strains (BY4741 and TET::DED1) were transformed with plasmids pGBK-HisFlagp33 (or pGBKHisp33 as a control), pGAD-HisFlagp92 (or pGAD-Hisp92 as a control) and pYC-DI72. The 66His-Flag double-tagged HF-p33 and p92 were expressed from the ADH1 promoter and DI-72 repRNA was under the Gal1 promoter. Transformed yeast were pre-grown on SC-ULH 2 media containing 2% glucose at 29uC. After centrifugation at 2,000 rpm for 3 min and washing pellet with selective media containing 2% galactose and 1 mg/ml Doxycycline, yeast were grown for 24 hours in SC-ULH 2 media containing 2% galactose at 23uC. The replicase purification was done according to a previously described procedure [24] with the following modification. Briefly, 200 mg of yeast cells were resuspended and homogenized in TG buffer [50 mM Tris-HCl [pH 7.5], 10% glycerol, 15 mM MgCl 2 , 10 mM KCl, 0.5 M NaCl, , and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] by glass beads using FastPrep Homogenizer (MP Biomedicals). The membrane fraction containing the viral replicase complex was solubilized with 1 ml TG buffer containing 1% Triton X-100, 1% [V/V] Ypic as described [14, 15] . After affinity purification of HF-p33 on anti-FLAG M2-agarose affinity resin (Sigma), the resinbound replicase complex was eluted in 100 ml elution buffer , 10% glycerol, 15 mM MgCl 2 , 10 mM KCl, 50 mM NaCl, 1% Triton X-100, and 0.15 mg/ml Flag peptide (sigma)].
In vitro RdRp activity assay was performed by using DI-72(2) or (+) RNA template transcribed in vitro by T7 transcription [14] . To measure the effect of host proteins in the replicase assay with DI72(2) RNA containing short double-stranded region at the 39end, a heat denatured RNA transcript (94uC for 2 min) was annealed with a 21-nt oligodeoxynucleotide (#20) (in 1:10 molar ratio) complementary to the 39 end of DI-72(2)RNA in STE buffer (10 mM TRIS, pH 8.0, 1 mM EDTA, and 100 mM NaCl) and then slowly (in 30 min) cooled them down to 25uC . RNase ONE digestion to remove single-stranded 32 P-labeled RNA was performed at 37uC for 30 min in a 16 RNase ONE buffer containing 0.1 ml of RNase ONE (Promega).
To obtain FHV replicase preparation, BY4741 yeast strain was transformed with plasmid pGAD/Cup/FHV/proteinA/C-term/ HA/FLAG and pESC-His-GAL1::FHVRNA1framshift. After selection of transformed yeast on SC-LH 2 plates, yeast were pre-grown overnight in selective media containing 2% glucose at 29uC. After centrifugation at 2,000 rpm for 3 min and washing pellet with selective media containing 2% galactose, yeast were grown 36 hours at 29uC in SC-LH 2 media containing 2% galactose and 50 mM CuSO 4 to induce FHV RNA replication. Affinity-purification was done similarly as for tombusvirus, except for using different buffers: homogenization buffer consisted of 
The split-ubiquitin assay was based on the Dualmembrane kit3 (Dualsystems). The bait construct, pGAD-BT2-N-His33, expressing the CNV p33 replication protein has been described earlier [26] . The prey constructs were made by PCR amplification of individual genes using gene specific primers: #3957 (CCAGCTG-CAGTCACCACCAAGAAGAGTTG) / #4602 (CCAGCCAT-GGCCACCAAGAAGAGTTG) followed by digestion with EcoR1 and Nco1 for DED1 and #4312(CCAGGGATCCATGAC-TTACGGTGGTAGAG) / #4603 (CCAGCCATGGATAGT-TTGAACGACCTC), #4318 (CCAGGGATCCATGAGTCGC-TACGATAGCCG) / #4604 (CCAGCCATGGGCTCCACCC-TCTTCTGCTC) followed by digestion with BamH1 and Nco1 in the case of RH20 gene, respectively. Digested PCR products were fused to NubG at either the 59-or 39-termini (NubG-x and x-NubG) by cloning into pPRN-N-RE or pPRN-C-RE vectors [26] , respectively, using the same enzymes. Yeast strain NMY51 was co-transformed with pGAD-BT2-N-His33 and pPR-N-RE or one of the prey constructs carrying the cDNA for a given helicase and plated onto Trp 2 /Leu 2 synthetic minimal medium plates. Transformed colonies were picked with a loop, re-suspended in water, and streaked onto TLHA 2 (Trp 2 /Leu 2 /His 2 /Ade 2 ) plates to test for p33-helicase protein interactions as described [26] .
Protein co-purification with the viral replicase S. cerevisiae strain DED1::66HA-hphNT1 was generated by homologous recombination using strain BY4741. PCR was performed using plasmid pYM-16 (EUROSCARF) [73] as template and primers #2493 (GCAGAAAACGAAGAATCCT-CACCCTAGTTTGTCTGAAATCAATCGATGAATTCGAG-CTCG) / #2494 (GGCTGGGGTAACAGCGGTGGTTCAAA-CAACTCTTCTTGGTGGCGTACGCTGCAGGTCGAC). The PCR products were transformed to BY4741 and recombinant yeast colonies were selected in YPD plates supplemented with hygromycin. Recombinant yeast strains were transformed with plasmids pGBK-HisFlagp33, pGAD-HisFlagp92 and pYC-DI72 [24] . 66His/Flag-tagged HF-p33 and HF-p92 were expressed from ADH1 promoter and DI-72 transcript was under GAL1 promoter. After selection of transformed yeast on SC-ULH 2 plates, yeast were pre-grown overnight in selective media containing 2% glucose at 29uC. After centrifugation at 2,000 rpm for 3 min and washing the pellet with selective media containing 2% galactose, yeast were grown for 36 hours in SC-ULH 2 media containing 2% galactose at 23uC. 200 ml of pelleted yeast were used to affinity-purify HF-p33 and HF-p92 with anti-FLAG M2 agarose as described previously (see also Text S1) [14, 15] . HF-p33 and HF-p92 were detected with anti-His 6 antibody (1/5,000 dilution) and AP-conjugated anti-mouse antibody (1/5,000). DED1-66HA protein was detected with anti-HA antibody from rabbit (Bethyl; 1/10,000 dilution) and AP-conjugated anti-rabbit (1/10,000) followed by NBT-BCIP detection. Figure S1 Reduced TBSV replication in CFE prepared from Ded1-depleted yeast. CFEs were prepared from TET::DED1 yeast strain cultured in the absence of doxycycline (i.e., Ded1p is expressed) or in its presence (10 mg/ml) (i.e., Ded1p is depleted). The CFEs were programmed with recombinant p33/p92 and DI-72(+) repRNA as described in REF 21. Note that the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. The CFEs contained comparable amounts of host proteins (not shown). Figure S3 Ded1p mutants with ATPase activity promote plusstrand synthesis by the affinity-purified tombusvirus replicase. (A) Scheme of the tombusvirus replicase assay. Yeast with depleted Ded1p co-expressing p33 and p92 pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (2)repRNA, and they also contained purified recombinant Ded1p. (B) Representative denaturing gel of 32 P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 mg of purified recombinant Ded1p. The level of complementary RNA synthesis producing ''repRNA'' (marked as ''FL'', the full-length product, made via initiation from the 39-terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein. Note that this replicase preparation also synthesizes internal initiation products (''ii'') and 39-terminal extension products (''39TEX''). RNAse One digestion was used to confirm the replicase products: the de novo products are insensitive, while the 39TEX product changes migration after RNase treatment  Transmission of Infectious Diseases En Route to Habitat Hotspots BACKGROUND: The spread of infectious diseases in wildlife populations is influenced by patterns of between-host contacts. Habitat “hotspots” - places attracting a large numbers of individuals or social groups - can significantly alter contact patterns and, hence, disease propagation. Research on the importance of habitat hotspots in wildlife epidemiology has primarily focused on how inter-individual contacts occurring at the hotspot itself increase disease transmission. However, in territorial animals, epidemiologically important contacts may primarily occur as animals cross through territories of conspecifics en route to habitat hotspots. So far, the phenomenon has received little attention. Here, we investigate the importance of these contacts in the case where infectious individuals keep visiting the hotspots and in the case where these individuals are not able to travel to the hotspot any more. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a simulation epidemiological model to investigate both cases in a scenario when transmission at the hotspot does not occur. We find that (i) hotspots still exacerbate epidemics, (ii) when infectious individuals do not travel to the hotspot, the most vulnerable individuals are those residing at intermediate distances from the hotspot rather than nearby, and (iii) the epidemiological vulnerability of a population is the highest when the number of hotspots is intermediate. CONCLUSIONS AND SIGNIFICANCE: By altering animal movements in their vicinity, habitat hotspots can thus strongly increase the spread of infectious diseases, even when disease transmission does not occur at the hotspot itself. Interestingly, when animals only visit the nearest hotspot, creating additional artificial hotspots, rather than reducing their number, may be an efficient disease control measure. The spread of infectious diseases strongly depends on how habitat characteristics shape patterns of between-host interactions [1, 2] . In particular, habitat heterogeneity influences patterns of between-individual contacts and hence, disease dynamics [1, 3] . For example, ''habitat hotspots'', sites that attract individuals or social groups over long distances, can be visited by a large subset of a population. Around hotspots, between-individual contact rates often increase in frequency, which amplifies disease transmission. In humans, schools and working places are typical examples of hotspots and have been shown to accelerate the spread of measles, influenza and SARS [4, 5, 6] . Thus, limiting transmission at hotspots has become a promising strategy for mitigating epidemics (e.g., influenza [7] ) although the efficiency of such strategies also depends on the role hotspots plays relative to other sources of local transmission (e.g., influenza [6, 7] )
In wild animal populations, high quality feeding spots (e.g., fruit trees), breeding sites, waterholes or sleeping sites can exacerbate direct physical contacts. Empirical and theoretical studies on the epidemiological importance of habitat hotspots have mainly focused on how the spatial aggregation of animals favors disease transmission at the hotspot itself [8, 9] . For example, the aggregation of wild boar at watering sites significantly increases the transmission of tuberculosis-like lesions [8] . However, interindividual contacts may not always significantly increase at the hotspot itself. This is for example the case of habitat hotspots that some animal species only visited occasionally, such as some mineral licks [10, 11] . Also, animals present at the same time at a particularly large hotspot may not be close enough to each other to transmit infectious diseases. This is the case of large forest clearings [12, 13] or large waterholes. Finally, species such as primates and ungulates might avoid defecating in hotspots of high food resources, limiting the transmission of fecal-oral parasites at hotspots [14, 15] .
When disease transmission does not occur at the hotspot, it can still occur at a certain distance from the hotspot. This phenomenon has received little attention so far. Specifically, infective contacts may be observed when infectious individuals travel to the hotspot and cross the territory of susceptible individuals and, reversely, when susceptible individuals cross the territory of infectious individuals. This second type of transmission may be prominent when the disease reduces the mobility of sick individuals (i.e., sickness behavior [16, 17, 18] ). For example, in humans, sick individuals often stay home, which alters disease dynamics [19, 20] . Sick wild animals also commonly reduce their rate of search for food or water [21] . Such transmission may particularly apply to parasites that can survive in the environment (e.g., gastrointestinal parasites) for which the spatial overlap of the home ranges of sympatric hosts favors transmission [22] .
To investigate these transmission mechanisms, we developed an agent-based model exploring patterns of disease spread in a large closed population composed of territorial social groups, in which one or more hotspots influence group movement patterns, but where direct disease transmission at the hotspot itself is negligible. Our hypothesis is that terrestrial animals necessarily cross conspecifics' home ranges on their way to a hotspot, which modifies the contact network of the population and may subsequently alter disease transmission. We assumed that between-group disease transmission can occur both between groups having neighbouring territories and between groups travelling to a hotspot and groups whose territories are crossed en route. We also assumed that only groups which territory lies within a certain distance from the hotspot (further referred as ''radius of attraction'') can visit it, and that their visitation rate decreases as this distance increases.
The relationship between the radius of attraction and the disease dynamics was then investigated under two scenarios: i) when groups including sick individuals do not travel to the hotspot, and ii) when these groups still travel to the hotspot. The first scenario corresponds to the case of virulent parasites that can strongly decrease the mobility of infected individuals, such as Ebola virus in western lowland gorillas [23] , whereas the second scenario applies to pathogens that do not strongly modify the behavior of their host, such as some gastro-intestinal macroparasites and bacteria [24] . Under both scenarios, we investigated the relationship between the disease attack rate and the hotspot radius of attraction, identified the groups in the population that have the highest risk of infection and explored the relationship between the number of hotspots and the magnitude of an epidemic.
The model has a 51651 lattice structure, where each cell of the lattice corresponds to a group's territory. We assumed disease transmission can occur between each group and its eight neighbours (Fig. 1) . We use N i to denote the list of indices of the eight neighbours of group i. Initially, a single habitat hotspot is placed at the center of the lattice. All groups are assumed to include ten individuals. At each daily time step, each group either visits the hotspot or stays in its territory. The probability P visit i ð Þ of a visit by group i is a decreasing function of the Euclidean distance, d i , between the group's territory and the hotspot. We assume that all groups gain the same benefit from visiting the hotspot and that the travel cost is proportional to d i , leading to:
where P max is the probability of a visit for the eight groups directly neighbouring the hotspot, and R is the hotspot radius of attraction. Groups occupying cells that are farther from the hotspot than R never visit it. When a group visits the hotspot, it follows a Biased Random Walk from its home cell to the hotspot (BRW [25] ) and returns to its home cell on the same day. The length of each step of the BRW (denoted S) is held constant and the direction of the step is consistently biased towards the hotspot during the approach to the hotspot and towards the group's home cell during the return from the hotspot. Each turning angle is randomly drawn from a normal distribution N (0, s 2 ), where s is a standard deviation parameter. The list of groups residing in cells encountered along each BRW to the hotspot is recorded. Groups travel to the hotspot and come back within a single time step.
At each time step, each group interacts with (i) groups occupying neighbouring cells (neighbour-neighbour contact), and (ii) if the group travels to the hotspot, all of the groups it encounters along the BRW (traveler-resident contact).
We model infectious disease dynamics using a simple stochastic susceptible-infectious-removed (SIR) epidemic model, with oneday time steps. Each individual moves independently through the three states: Susceptible (at risk of contracting the disease), Infectious (capable of transmitting the disease), or Removed (recovered or dead). Susceptible individuals can be infected by infected individuals from either its own group or other groups. The latter can occur during neighbour-neighbour contacts or travelerresident contacts.
The local transmission probability P i is defined as the per-timestep probability that a susceptible individual in group i is infected by an infectious individual from its own group or a neighbouring group. Let I i denote the number of infectious individuals in group i. The probability that the focal individual is infected by at least one of the infectious individuals in its own group is 1{ 1{P w ð Þ Ii , where P w is the within-group transmission probability from an infected individual to a susceptible individual of the same group. Likewise, the probability of a susceptible individual being infected by an infectious individual from one of the eight neighbouring groups is 1{ P
where P B is the between-group transmission probability from an infected individual to a susceptible individual of a neighbouring group. Combining these two sources of infection, the local transmission probability is given by
The per-time-step probability of becoming infected during transit to a hotspot P H1 depends on the number of infectious individuals I c in each group c encountered en route and the P T ''travelling'' probability of transmission during one of these transient contacts between a resident and traveling group. Specifically, during a one-day trip to a hotspot, the probability that a susceptible individual in the group is infected along the way is given by
where BRW i denotes the list of indices of the groups encountered by group i as it travels to and from the hotspot.
In a second version of the model, infected groups are assumed to travel to the hotspot. Transmission from infected travelers to susceptible residents encountered en route can then occur. In this case, the per day probability that a susceptible individual in group i is infected by a traveler depends on the numbers of infected individuals in each of the groups that travels through the territory of i en route to the hotspot, and is given by
where PT i denotes the set of groups passing through i's territory. At the end of each time step, each infected individual is removed with probability c. We assume that no transmission occurs between groups travelling to the hotspot simultaneously.
We explored two versions of the transmission model. In the ''Sick-stay'' model, groups that included at least one infected individual -infected groups -were assumed to stop travelling to the hotspot; in the ''Sick-travel'' model, infected groups continue to travel to the hotspot as if uninfected. In the Sick-stay model, disease transmission between travelers and residents can only occur from an infected resident to a susceptible traveler, while in the Sick-travel model, transmission can be bi-directional.
We also considered models with multiple hotspots. A specified number of hotspots are randomly placed on the lattice, and groups visit only their nearest hotspot (according to P visit , described above).
At the beginning of each simulation, all individuals were susceptible. Epidemics were started with a single infected individual. Unless stated otherwise, the first case was introduced into one of the eight groups adjacent to the hotspot. At each time step, the number of infected and removed individuals (and groups) was recorded until no individual in the population was infected, which indicated the end of the epidemic. For each parameter combination, we ran 1000 simulations.
For all simulations, the recovery rate c was set to 0.1, the maximum probability of a visit to the hotspot, P max , was set to 0.1, and the BRW step length S was set to 0.25 (i.e., 1/4 of the distance between the center of neigbouring group's territories). We also assumed that the within-group transmission probability, P w , was at least ten times higher than the between-group transmission probabilities (P B and P T ). The model was implemented in Delphi 7 (Borland Software Corporation, 2002). Table 1 summarizes parameter definition and values, and a sample run of the model is shown in Video S1.
The attack rate (proportion of groups becoming infected following a single disease introduction) generally increases with the hotspot radius of attraction R, and the traveler-resident transmission probability P T (Fig. 2 ). This occurs whether or not infected groups are assumed to travel during infection ( Fig. 2 and Fig. S1 in supplemental materials). As predicted by percolation theory [26] , the attack rate also increases with both the withingroup transmission parameter P W and the between-group transmission parameter P B . Interestingly, the highest impact of the hotspot radius of attraction on the attack rate was observed for intermediate values of P W and P B . For low values of P W and P B , inter-group disease transmission primarily occurred between travelling and resident groups and was often not sufficient to sustain an epidemic. For high values of P W and P B , the disease always percolated, even in the absence of the hotspot. For intermediate values of P W and P B , groups infected en route to the hotspot then stochastically triggered small outbreaks around their territories. The epidemiological impact of the hotspot was amplified by this interaction between traveler-resident transmission and neighbour-neighbour transmission.
In both models, the attack rate decreased as the distance between the hotspot and the point of disease introduction increased (Fig. 3) . The greater this distance, the lower the probability that a group visiting the hotspot encountered the group initially infected. The Sick-travel model yields higher attack rates than the Sick-stay model, particularly for groups ranging at intermediate distances between the location of the first disease case and the hotspot.
In the Sick-travel model, groups ranging closer to the hotspot exhibited higher probabilities of infection ( Fig. 4 and Fig. S2 in supplemental materials), since their territories are crossed by large numbers of infected groups travelling to the hotspot (Fig. S4a) . The relationship is more complex in the Sick-stay model: groups ranging at intermediate distances from the hotspot experience the highest risks of infection ( Fig. 4 and Fig. S3 in supplemental materials). In this case, hotspot-mediated infection occurs only from infected residents to susceptible travelers. Groups ranging close to the hotspot travelled more often, but encountered only a small number of potentially infected groups. Groups ranging far from the hotspot encountered larger numbers of groups when visiting the hotspot, but did so only rarely. Thus groups ranging at intermediate distances experienced the greatest number of potentially infective contacts with resident groups encountered en route to the hotspot (Fig. S4b) . When epidemics occur, the difference in spatial pattern of disease spread observed between the models is insensitive to the parameter values ( Fig. S2 and Fig.  S3 ).
The fact that groups ranging at an intermediate distance from the hotspot display a higher number of potentially infective contacts can easily be understood using a simple mathematical model. Indeed, the expected number of groups encountered by a group i visiting the hotspot, per time unit, can be assumed to be approximately proportional to P visit and to the territory-hotspot distance d i :
This approximation holds as long as d i is large or the turning angle is low. The derivative of this second-order polynomial has a maximum in R=2. Second, we assessed the epidemiological impact of the hotspot on groups that never visit it because they range at a distance larger than R from the hotspot. For both models, when P B is high enough to allow some between-neighbour transmission, the attack rate for these groups was found to be larger than expected under a model with no hotspot (R = 0) (Fig. 4) . Stochastic, local between-group transmission events allow the spread of the disease beyond the radius of attraction. Groups that never visit the hotspot are thus indirectly impacted by the hotspot.
Finally, outside of the radius of attraction, disease spreads as expected for a lattice model [27] whereas inside the radius of attraction, disease spread rapidly among the groups, with no apparent spatial structure.
For both models, the relationship between the number of hotspots and the attack rate is bell-shaped (Fig. 5) . For a low number of hotspots, adding new hotspots increases the fraction of the population ranging within the radius of attraction of these hotspots and, thereby, increases the overall attack rate. Beyond a certain number of hotspots, however, all groups are already attracted by at least one hotspot on the landscape. Under the assumption that these groups travel exclusively to the nearest hotspot, adding more hotspots decreases the distance travelled by the groups and the number of infective contacts they can have en route, and thereby lowers the attack rate.
Spatial features of the landscape such as habitat hotspots can profoundly influence the spread of infectious diseases [28, 29] . Our model extends previous studies focusing on transmission at the hotspot, and reveals that hotspots can also strongly alter disease transmission by generating infective contacts between animals travelling towards or from the hotspot and animals whose territories are traversed. Our results show that even when sick groups stay in their territory, hotspots may increase the size of an epidemic. When infected animals cease to visit the hotspot, groups ranging at intermediate distances to the hotspot are the most vulnerable. We also found that the epidemiological impact of hotspots extends far beyond the subset of the population that visits it; even groups having no contact with those visiting the hotspot display elevated risks of infection. Finally, our model predicts that when groups visit their nearest hotspots, the epidemiological impact of hotspots is most severe when the number of hotspots is intermediate. Hotspots impact disease transmission via a combination of both local between-neighbour and long-range traveler-resident transmissions, which is characteristic of a small-world network [30] . Disease dynamics in our model resemble those in a classic smallworld network in several aspects. First, the attack rate increases with long-distance interactions, determined by the hotspot radius of attraction (Fig. 2) . Second, new foci of infection established by long-distance traveler-resident contacts only spread when the local transmission rate, between neighbours, is sufficiently high. This phenomenon extends the influence of the hotspot beyond the radius of attraction (Fig. 4) . Finally, as in small-world networks [31] , all groups within the hotspot radius of attraction were infected almost at the same time. Thus, habitat hotspots potentially play a significant role in fuelling disease outbreaks, much like other natural mechanisms that generate small-world networks, such as the movement of vectors between plants [32, 33] .
We find that hotspots are expected to influence disease dynamics significantly, even when infected groups do not travel to the hotspot at all. However, in this case, the hotspot effect strongly decreases as the distance between disease introduction and the hotspot increases. The reduction of mobility in infected groups also generates an unexpected spatio-temporal pattern: groups ranging at intermediate distance from the hotspot have the highest risk of infection, even if the disease is introduced immediately next to the hotspot. This counterintuitive result highlights the importance of understanding the behavioral effects Figure 2 . Influence of multiple model parameters on attack rate, when infected groups do not travel (Sick-stay model). The fraction of groups infected increases with the hotspot radius of attraction, but varies with the traveler-resident transmission probability P T (four lines in each graph), within-group transmission probability P w (three different columns of graphs), and between-neighbour transmission probability P B (four different rows of graphs). Each value is based on 1000 simulations in which disease was introduced randomly in one of the eigth groups adjacent to the hotspot. doi:10.1371/journal.pone.0031290.g002 of disease in wild animal populations. For example, as in humans, predicting the impact of hotspots on disease dynamics will strongly depend on understanding whether infectious individuals still travel to hotspots because disease symptoms appear after an infectious state (e.g., influenza H1N1 [6, 19] ), or whether infectious individuals do not visit hotspots because disease symptoms appear before the infectious state (e.g., SARS [34, 35] ). Furthermore, our results suggest that when transmission does not directly occur at hotspots, disease control measures targeting groups residing around the hotspot might not necessarily be the most efficient ones. Further simulation work is needed to identify optimal disease control measures. The habitat of wild animal populations often includes more than one hotspot. For example, the habitat of terrestrial mammals can include a small number of high-value hotspots attracting dozens of groups (e.g., salt licks or forest clearings) and more numerous low-value hotspots attracting only a few groups (e.g., fruiting trees). Our model reveals that, when groups are assumed to travel to their nearest hotspot, the impact of disease outbreaks is a bell-shaped function of the number of hotspots (Fig. 5) . This result challenges the hypothesis that the number of hotspots and disease prevalence will correlate positively [8] , and could be used to optimize strategies for controlling disease in wild animal populations. Thus, wildlife managers may consider increasing, rather than decreasing [36] , the number of water holes in order to reduce the number of highly-connected individuals or social groups, and hence the impact of an outbreak. However, additional studies are needed to determine if our result still holds when each animal visits more than one hotspot.
The values of the parameters of our model can be estimated from empirical data. The relationship between the distance from a group's territory and the hotspot visitation rate can be estimated using capture-mark recapture and telemetric data, between-group contact rates can be estimated from direct observation or telemetric data, and plausible distributions of disease transmission rates can be found in the literature. The step length of the biased random walk is assumed to have a fixed value (here, 0.25 times the size of a territory). This parameter does not need to be estimated accurately since it is redundant with another parameter, the traveler-resident contact rate, which is allowed to vary. Thus, the model can be applied to a broad range of host-parasite systems, from primate groups travelling to waterholes on a daily basis [37, 38] to large mammals visiting every few weeks mineral-rich Figure 5 . Number of hotspots. Each line graphs the change in attack rate as a function of the number of hotspots, for a different value of P B (from 4e-04 to 16e-04). Results are presented for hotspots ranging from 1-100 (left) and 1-500 (right) in the Sick-stay model (top) and the Sick-travel model (bottom). Each value is averaged over 1000 stochastic simulations assuming R = 30, P w = 0.06, P T = 4e-04. Each hotspot was located randomly in the population, and disease was introduced into the group ranging in the middle of the habitat. doi:10.1371/journal.pone.0031290.g005 areas [12, 13, 39] . In our model, the impact of the hotspot is particularly sensitive to the ratio between the local and the traveler-resident between-group transmissions. When the local between-neighbour transmission is high compared to the travelerresident transmission, the impact of the hotspot is minimal.
We considered two discrete transmission scenarios, the Sicktravel and the Sick-stay scenarios. However, intermediate scenarios are also possible. For example, infected groups may fission such that only healthy individuals travel to the hotspot. In this case, we expect that although the overall disease transmission will increase compared to the pure Sick-stay scenario, the spatial pattern of the disease impact will be qualitatively similar to that observed for the Sick-stay model.
In this study, we have shown how transmission occurring around habitat hotspots influences disease transmission patterns, while previous studies have focused on disease transmission occurring at the hotspot itself. In some ecological systems, both transmission modes may coexist. For example, some fecal-orally transmitted parasites can infect both the soil and waterholes, and spore-forming bacteria such as Bacillus anthracis can persist for extended periods of time in animal carcasses, water and soil [40] . Additional works are needed to understand such epidemiological systems. Figure S1 Influence of multiple model parameters on attack rate, when infected groups travel (Sick-travel model). The fraction of groups infected increases with the hotspot radius of attraction, but varies with the traveler-resident transmission probability P T (four lines in each graph), withingroup transmission probability P w (three different columns of graphs), and between-neighbour transmission probability P B (four different rows of graphs). Each value is based on 1000 simulations in which disease was introduced randomly in one of the eigth groups adjacent to the hotspot. (TIF) Figure S2 Group's probability of infection in relation to the distance to the hotspot, predicted by the Sick-travel model. The relationship is presented for different values of the hotspot radius of attraction (R). Each graph represents a combination of the between-neighbour (P B ) and the travelerresident (P T ) transmission. The disease was introduced randomly in one of the eight groups adjacent to the hotspot. For all simulations, P w = 0.06. (TIF) Figure S3 Group's probability of infection in relation to the distance to the hotspot, predicted by the Sick-stay model. The relationship is presented for different values of the hotspot radius of attraction (R). Each graph represents a combination of the between-neighbour (P B ) and the travelerresident (P T ) transmission. The disease was introduced randomly in one of the eight groups adjacent to the hotspot. For all simulations, P w = 0.06. (TIF) Figure S4 Traveler-resident contact patterns. Each graph shows the relationship between the distance of a group from the hotspot and (a) the number of other groups that travel through its territory when travelling to and from the hotspot, (b) the number of resident groups it encounters when travelling to and from the hotspot. Values are based on encounters occurring during 100 time steps, in the absence of disease transmission. (TIF)
Video S1 Model dynamics. The model shows one simulation run corresponding to the Sick-travel model. White, red and black squares represent susceptible, infected and removed groups, respectively. Blue squares represent groups travelling to the hotspot at each time step. The hotspot, in green, is in the middle of the lattice. The disease is introduced at the periphery. (WMV) The Dispanins: A Novel Gene Family of Ancient Origin That Contains 14 Human Members The Interferon induced transmembrane proteins (IFITM) are a family of transmembrane proteins that is known to inhibit cell invasion of viruses such as HIV-1 and influenza. We show that the IFITM genes are a subfamily in a larger family of transmembrane (TM) proteins that we call Dispanins, which refers to a common 2TM structure. We mined the Dispanins in 36 eukaryotic species, covering all major eukaryotic groups, and investigated their evolutionary history using Bayesian and maximum likelihood approaches to infer a phylogenetic tree. We identified ten human genes that together with the known IFITM genes form the Dispanin family. We show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and metazoa, and that the family later expanded in vertebrates where it forms four subfamilies (A–D). Interestingly, we also find that the family is found in several different phyla of bacteria and propose that it was horizontally transferred to eukaryotes from bacteria in the common ancestor of choanoflagellates and metazoa. The bacterial and eukaryotic sequences have a considerably conserved protein structure. In conclusion, we introduce a novel family, the Dispanins, together with a nomenclature based on the evolutionary origin. Membrane proteins are essential for the ability of all cellular organisms to respond and interact with their environment. Therefore they have attained large research interest and are one of the major groups of drug targets [1] . We have previously estimated that 27% of the human genes codes for alpha-helical membrane proteins and provided a comprehensive classification based on their function and evolutionary origin [2] . However, the identification and annotation of many membrane bound protein families is still being revised. We have during recent years worked on the annotation of both G protein-coupled receptors [3, 4] and solute carriers [5] and most of the genes of these large superfamilies now have a clear identity and annotation. There is however still large work to be done to clarify the identity, annotation and the evolutionary history of several families of membrane bound proteins. Establishing a rigid nomenclature based on evolutionary information and structural features of the predicted proteins facilitates prediction of the functional role of these genes that often have only have been studied in large gene or transcription consortia.
In previous studies we have found that membrane proteins with few transmembrane (TM) helices are less studied than other. This is particularly true for 2TM proteins where more than 70% of the about 700 proteins remained unclassified. Interestingly, we found evidence for several uncharacterized homologues to a small group of genes known as the Interferon-induced transmembrane proteins (IFITM) family. The IFITMs constitute a group with four human members (IFITM1-3, 5) that are found in a consecutive order on chromosome 11, having two transmembrane (2TM) helices. The IFITM4 gene is not present in human, but is located in proximity to the other four genes in the mouse genome. The IFITM1-3 proteins were identified 25 years ago as being upregulated by interferons (IFN) [6] . Recently they received considerable attentions as IFITM1-3 were found to prevent infection of a growing list of viruses such as HIV-1, SARS influenza A H1N1, West Nile and Dengue fever viruses [7, 8, 9, 10] . Hence, proteins of the IFITM family mediate part of the antiviral response orchestrated by IFNs. However, the IFITM family is also involved in other processes such as oncogenesis, bone mineralization (IFITM5) and germ cell development (IFITM1 and 3) and IFITM5 has not been identified as interferon-inducible [11, 12, 13, 14] . Although the biological roles of the IFITM genes are emerging, no thorough evolutionary analysis has been performed on this group.
In this study, we sought to infer the evolutionary history of the human IFITM genes and identify potential homologues. We mined 36 eukaryotic species, covering all major eukaryotic groups, and found that the IFITMs form a subfamily in a larger novel family that has ten human members in addition to the four IFITM genes. We propose Dispanins as a novel name for this family, which refers to their common 2TM structure. Further, we find that the eukaryotic Dispanins first appeared before the radiation of metazoa and that they branch out into four subfamilies (A-D). More surprisingly, we also discover that the Dispanins are found in a large range of bacteria and in brown alga.
In total, we collected 87 eukaryotic IFITM homologues from H. Sapiens (14 genes) M. musculus (17 genes), G. gallus (6 genes), X. tropicalis (13 genes), D. rerio (7 genes), P. marinus (1 gene), C. intestinalis (1 gene), B.floridae (12 genes), S. manosoni (1 gene), S. purpuratus (9 genes), N. vectensis (4 genes) and M. brevicollis (1 gene). No IFITM genes could be detected in any of the remaining 24 analyzed proteomes, which covers all other major eukaryotic groups. The search of the nr database with HMMER did not get any hits outside metazoa except bacteria, choanoflagellates and the brown alga Ectocarpus siliculosus (2 genes). Nine genes were deemed as pseudogenes based on annotation and sequence analysis and removed from further analysis. In addition to the four previously identified human IFITM genes, ten novel human homologous genes were detected. These ten genes together with the four IFITM genes form a human gene family that we choose to call Dispanins based of their common 2TM structure. In UniProt we identified 65 annotated IFITM homologues from full bacteria proteome sets spread over seven different phyla (See table S1): Acidobacteria (2 genes), Actinobacteria (43 genes), Cyanobacteria (3 genes), TG1 (1 gene), Bacteroidetes (2 genes), Firmicutes (1 gene) and Proteobacteria (13 genes). Out of these, 46 bacterial sequences from 32 species were included for further analysis. No viral or Archaean genes were annotated as IFITM homologues in UniProt.
The phylogeny of the vertebrate Dispanins ( Figure 1 ) allows the division of the Dispanins into four subfamilies A-D that are supported by strong confidence with respect to posterior probabilities (pp) or bootstraps (pp.0.75 and bs.90% for all nodes). We propose a common nomenclature for the Dispanins that are based on their subclass and a number (DSPA1 etc). The proposed names together with previous gene symbols and accession number can be found in Table S1 .
The finding of two Dispanin homologs in the brown alga E. siliculosus, which is evolutionary distant to metazoa, and the single Dispanin in the close metazoan relative M.brevicollis are the two only non-metazoan eukaryotic Dispanins. A BLAST search gives that the E. siliculosus proteins have a higher similarity to metazoan family members (best hit E-value,10 210 ) than bacterial Dispanins and M. brevicollis. The M. brevicollis Dispanin share a conserved splice site with all metazoan family members, which suggest that the eukaryotic Dispanins first emerged in a common ancestor of M. brevicollis and the metazoan lineage. Within the metazoan lineage the Dispanins have been lost in at least two separate occasions, i.e. T. adhaerens and in the ecdysozoan lineage (D. melanogaster and C. elegans). The vertebrate Dispanins sort into subfamilies A-D ( Figure 1 ). The DSPA subfamily has six human genes (DSPA1, DSPA2a-d and DSPA3) of which the DSPA2a-c corresponds to IFITM1-3 and DSPA1 to IFITM5. DSPA2d (AC023157) and DSPA3 (AC068580) are two novel identified genes, closely related to the IFITM family. The phylogenetic tree indicates that the DSPA2 genes have undergone an independent duplication in H. Sapiens and M. musculus and these were given a species specific nomenclature, e.g. DSPA2a-d in human. The Dspa4, Dspa5, Dspa6 genes in the phylogenetic tree do not have any clear human orthologs. The DSPB subfamily is only found in tetrapoda and contains three human genes called DSPB1 (TUSC5), DSPB2 (TMEM233) and DSPB3 (PRRT2) whereas Dspb4 is only present in G. gallus. DSPC1 (TMEM90A), DSPC2 (TMEM90B) and DSPC3 (TMEM91) make up the human DSPC subfamily, which is represented in all investigated vertebrates. The DSPD1 (PPRT1) gene is found in all the vertebrate species except the basal organism P. marinus whereas DSPD2 (AL160276) is mammalian specific.
Five vertebrate genes and all invertebrate were excluded from the phylogenetic analysis and instead classified into subfamilies by using a BLAST approach (Table S1) . Some genes could not unambiguously be classified into the vertebrate subfamilies: C. intestinalis (1 gene), S. mansoni (1 gene), B. floridae (2 genes), S. purpuratus (3 genes), N. vectensis (1 gene) and M. brevicollis (1 gene). By combining the results of the phylogenetic analysis and BLAST classification, we created a schematic overview of the organisms' gene repertoire and a schematic picture of the Dispanin family's evolutionary history, which suggests that the invertebrate Dispanins share more similarity towards the DSPC and D subfamilies than DSPA and B ( Figure 2 ).
All the members of the human DSPA are located on chromosome 11 except for DSPA2d which resides on chromosome 12. The genes of the other subfamilies are not enriched on any chromosome. Several features are common to all the eukaryotic Dispanin proteins ( Figure 3 ). They comprise two transmembrane helices that are predicted between 20 and 30 amino acids in length with the second helices often being slightly longer. The Dispanins are rich in both glycosylation-and phosphorylation sites that predominantly are found on the Nterminus. The N-terminus is often long (.100 amino acids) compared to the C-terminal (,10 amino acids) and both are always oriented towards the outside of the cell.
The Dispanins contain several conserved motifs ( Figure 3 and 4), which are found both among eukaryotes and bacteria. The most conserved pattern is the G-D motif and the A-X(6)-A motif, both situated in the intracellular loop between the transmembrane helices that also is frequently rich in positive amino acids (K and R). The first helix is the most conserved with an alanine (A) residue and double cysteine C-C (C-F-C in the DSPB family) motif whereas a glycine (G) residue is the most conserved in the second helix. Another highly conserved motif, though only amongst the eukaryotes, is the single aspartic acid (D) on the N-terminus, flanking the first helix.
Analysis of the exon structure in the protein sequences was made for all eukaryotic Dispanins except E. siliculosus where no such information was found. The eukaryotic Dispanins have a conserved splice site in the intracellular loop that separates the two transmembrane helices into different exons ( Figure 4 ). This site is only missing in the S. mansoni Dispanin and the mouse Dspa2f genes. The vertebrate proteins of the DSPA and DSPB subfamilies only have these two exons whereas the whole DSPC subfamily and the DSPD1 proteins have an additional exon that codes for their N-terminus. The DSPD2 proteins that only are found in mammals seem to have lost their N-termini exon. All the classified B. floridae and S. purpuratus sequences has the corresponding splice site in the N-terminus, whereas the N. vectensis and M. brevicollis proteins has 3-6 and 7 exons respectively.
We provide evidence that the four IFITM genes together with ten additional human genes, known as TUSC5, TMEM233, PRRT2, TMEM90A, DSPC2, TMEM90B, TMEM91, AC023157, AL160276 and AC068580, form a novel gene family that we call the Dispanins, which refers to the 2TM membrane topology that is common to all identified members. This family is the second largest 2TM family in the human genome, superseded only by the Inwardly rectifying potassium channel family that has 15 members [2] . Except for the 2TM memebrane topology the Dispanins are not homologous or share domains with any other 2TM proteins in the human genome and constitute a distinct gene family. We have discovered that this family is found in metazoan, the choanoflagellate M. brevicollis and the brown alga E. siliculosus, but not in other eukaryotes. Surprisingly it is widely present in bacteria where it is found in several different phyla such as Actinobacteridae, Acidobacteria, Cyanobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The highest number of bacterial Dispanins is detected in Actinobacteria and Proteobacteria, which diverged around three billion years ago [15] . We find that Dispanins in eukaryotes and bacteria have high sequence similarities and share several conserved sequence motifs (Figure 4) , which is strong evidence for a common evolutionary origin and possibly a functional relationship. As the family is found in several bacterial phyla we suggest that it first emerged in bacteria to later be introduced in eukaryotes through a horizontal gene transfer event. However, we were not able to construct a stable phylogenetic tree including bacterial and eukaryotic Dispanins. As the eukaryotic Dispanins only is widespread in metazoa it was unexpected to find the family in the evolutionary distant brown alga E. siliculosus (Figure 2 ). Our sequence analysis supports that all metazoan Dispanins have their origin in the common ancestor of M. brevicollis and metazoa as the choanoflagellate share a conserved splice site in the intracellular loop with nearly all metazoan Dispanins (Figure 4) . However, the finding of the family in E. siliculosus suggests that the family has undergone two horizontal gene transfer events. As the E. siliculosus Dispanins are more similar to metazoan family members than M. brevicollis and bacteria we propose that the first horizontal gene transfer event was from bacteria to a common ancestor of choanoflagellates and metazoa followed by a second transfer between metazoa and brown alga.
During the course of metazoan evolution the Dispanins have expanded and diverged into four distinct subfamilies. However, it has also been lost in the basal metazoa T. adhaerens and the ecdysozoan lineage, which show that it is not essential for all metazoan life. Although we were unable to create a stable phylogenetic tree that include both vertebrate and invertebrate sequences BLAST searches suggest that the DSPC and D subfamilies are the oldest of the vertebrate subfamilies as the invertebrate sequences has higher resemblance to these two subfamilies ( Figure 2) . Moreover, the DSPC and D subfamilies forms a separate cluster from DSPA-B (Figure 1) . Hence, the phylogenetic analysis suggests that the DSPA and B subfamilies have their origin close to the radiation of teleost, although DSPB have been lost in D. rerio (Figure 1 and 4) . The DSPC family is found in two to three copies in all vertebrates and is the most widespread family as the BLAST classification suggest that it is present in two invertebrate species and E. siliculosus. In mouse, the family members are expressed predominantly in brain tissues (Dspc1/Tmem90a, Dspc2/Tmem90b) or ubiquitously (Dspc3/ Tmem91). DSPC1 (TMEM90A) has been proposed to have a role in striatial functioning and the pathophysiology of Huntington's disease and is localized to the Golgi apparatus [16] . The DSPD family has been lost at several occasions, both in vertebrates and invertebrates ( Figure 2 ). The mouse Dspd1 (Prrt1) gene is ubiquitously expressed with the highest expression in B-cells according to BioGPS [17] . However, no previous studies have been performed on the DSPD subfamily. The DSPB subfamily is found in tetrapoda and has three members in human and mouse. Mice expression profiles from BioGPS shows that Dspb3 (Prrt2) is exclusively expressed in brain tissues and that Dspb1 (Tusc5) is expressed in dorsal root ganglia and adipose tissues. In agreement with this expression data, Dspb1 (Tusc5) has been suggested to be involved in neural regulation of adipocyte differentiation and is regulated by PPARc [18, 19] . The DSPA/IFITM subfamily is the most numerous and the mouse and human genes are all clustered in a consecutive manner on chromosome six and eleven, respectively. These regions share a conserved synteny (http:// cinteny.cchmc.org/) and are flanked by the ATHL1 and B4GALNT4 genes on each side, which is strong evidence for the genes to have their origin in common evolutionary gene duplications. This is supported by the phylogenetic analysis ( Figure 1 ) for DSPA1 (IFITM5) and DSPA3 (AC068580), which have orthologs in all tetrapoda. However, for the DSPA2 group (DSPA2A-D/IFITM1-3 and AC068580) the phylogeny suggests that M. musculus and H. sapiens have undergone independent expansions of the group. Rather than being created by independent gene duplications in the two species, it is possible that these genes are subject to concerted evolution, where paralogous genes within a species are more conserved towards each other than towards orthologs in other species. This phenomenon is most common in tandemly repeated genes, such as the DSPA2 group, and is believed to primarily be the result of recombination mechanisms [20] . Interestingly, also the Dspa4a-f genes of X. tropicalis seem to have undergone and independent expansion. However, the phylogeny is not strong enough to prove that the Dspa4a-f genes are orthologous to the mammalian DSPA2 genes (Figure 1 ). The DSPA/IFITM subfamily is the most well studied and is a multifunctional family of which its antiviral properties are best understood [8] . The family is expressed in many mouse tissues with the highest expression in mast cells, macrophages and osteoblasts according to BioGPS. We add two novel human members to this subfamily: DSPA2D, which is closely related to DSPA2C (IFITM3) and DSPA3, which forms a distinct cluster (Figure 1 ). Both these genes are poorly characterized. Microarray data from Array Express (www.ebi.ac.uk/ arrayexpress/) shows that DSPA3 is upregulated by interferon after exposure of macrophages to interferon-gamma in a study (E-GEOD-5099) where DSPA2a-c (IFITM1-3) also were induced [21] . The mouse Dspa3 gene is like the other genes of this subfamily situated on chromosome seven, but is 1.5 Mbp away from the DSPA (IFITM) cluster where the other genes reside. Hence, Dspa3 could explain the mild phenotypes and be responsible for the suggested functional redundancy that was found when deleting the whole DSPA (IFITM) loci [22] .
The Dispanin family has several conserved motifs across subfamilies that are also detected in bacteria (Figure 4) . One of the most prominent is the double cysteine motif (C-C) in the first transmembrane helix. This motif has recently been shown to undergo post-translational modification through S-palmitoylation in DSPA2C (IFITM3), which increases hydrophobicity [23] . Further, Yount and colleagues shows that the antiviral activity of DSPA2C (IFITM3) is dependent on this modification, which induces clustering of the proteins. As this motif is highly conserved, it is likely that S-palmitoylation is an important regulatory mechanism also among the other subfamilies. Intriguingly, this motif is also found in the bacterial Dispanins even though bacterial proteins do not undergo S-palmitoylation. Hence, the cysteine motif of the may have other means of structural and functional importance apart of from the S-palmitoylation.
In this study, we introduce the Dispanin family, of which the IFITM genes constitute a subfamily. In addition to the IFITM genes we identify 10 novel human Dispanins and investigate the family's evolutionary history and suggest that the eukaryotic members are descending from bacteria through a horizontal gene transfer. Thus, the expansion and diversification of Dispanins in vertebrates may reflect the evolution of a larger functional repertoire, which is a supported by the distinct expression profiles Figure 3 . The protein features and topology of the Dispanin subfamilies. The picture shows the membrane topology and sequences features of a representative human member of each subfamily. Conserved motifs and residues are shown and those which have a sequence identity of more than 90% are framed in black and those with 80-90% sequence similarity are framed in blue. Predicted phosphorylation (Green) and glycosylation (orange) sites are shown. doi:10.1371/journal.pone.0031961.g003 of the subfamilies. By identifying homologs to the IFITM genes and establishing the Dispanins as a family together with a solid detailed and evolutionary based nomenclature for the vertebrate genes, we provide a fundament for future functional characterization these genes.
The whole proteome dataset for the following eukaryotic species was included in the analysis: Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Petromyzon marinus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerviciae, Schistosoma mansoni, Apis mellifera, Anopheles gambiae, Pediculus humanus, Ixodes scapularis, Daphnia pulex, Oryza sativa, Pristionchus pacificus, Acyrthosiphon pisum, Trypanosoma brucei, Leishmania braziliensis and Ciona instestinalis were downloaded from Ensembl; Strongylocentrous purpuratus was downloaded from Spbase (www.spabase.org); Branchiostoma floridae, Nematostella vectensis, Trichoplax adhaerens, Phytophtera soyae, Thalassiosira pseudonana, Naegleria gruberi and Monosiga brevicollis were downloaded from the Joint Genome Institute; Dictyostelium discoideum was downloaded from dictyBase (www.dictybase.org); Arabidopsis thaliana was downloaded from TAIR (http://www.arabidopsis.org/); Entamoeba histolytica was downloaded from amoebaDB (http://amoebadb.org); Paramecium tetraurelia was downloaded from NCBI; Tetrahymena thermophila was downloaded from UniProt; Trichomonas vaginalis was downloaded from TrichDB (http://trichdb.org); Giardia lamblia was downloaded fromGiardiaDB (http://giardiadb.org).
All proteomes were searched against a local installation of the Pfam database (v.23) [24] using HMMER3 [25] and the script pfam_scan.pl, which was obtained from the Pfam ftp-site (ftp://ftp. sanger.ac.uk/pub/databases/Pfam/Tools/), with Pfam's default settings. In Pfam, the IFITM family is represented by a specific hidden Markov model [Pfam: P04505]. All the proteins that were assigned to this model in the Pfam-search were considered to be homologous to the IFITM family and were therefore included for further analysis. The script pfam_scan.pl uses the homology criterion set by the Pfam database, which is based on a manually curated gathering threshold for each model. The gathering threshold for PF04505 is a score of 20.6. The sequence datasets were controlled for annotated pseudogenes and transcript variants from the same gene. In the case of multiple transcript variants, the longest sequence was kept. The resulting non-redundant datasets were used for the analysis. The bacterial sequences were obtained by querying Uniprot (www.uniprot.org) for the Pfam ID [Pfam: PF04505]. Thereafter, the sequence set was downloaded by browsing by taxonomy and restricting it to species with a full proteome set. To assure that no lineages were missing in the selection of proteomes the nr protein dataset from NCBI was downloaded. The nr datasets contained 15,322,545 (20-09-11) protein sequences from a wide range of organisms. The dataset was searched against the PF04505 Pfam model using HMMER3 with default settings and sequences with a score above the Pfam gathering threshold (20.6) were deemed as homologous to IFITM.
Mafft-einsi was used, with default settings, to create a multiple sequence alignment (MSA) for the vertebrate protein sequences [26] . The MSAs were thereafter examined and refined in Jalview 2.5.1 [27] , i.e. the sequences were trimmed and well conserved and aligned regions were kept, which included 82 aligned amino acid columns. Phylogenetic analysis was performed with a Bayesian approach implemented in MrBayes [28] . The following settings for the eukaryote proteins were adjusted: The analysis was run using a gamma shaped model for the variation of evolutionary rates across sites (rates = gamma) and the mixed option (aamodelpr = mixed) was used to estimate the best amino acid substitution model. We generated 5 000 000 trees and the Markov chain Monte Carlo analysis reached well below a standard deviation of split frequencies of 0.01. Each hundred tree was sampled from the mcmc run and the first 25% of the sampled trees were discarded (burnin = 0.25) to reassure a good sample from the posterior probability distribution. A consensus tree was built from the remaining 37 500 trees with the MrBayes sumt command using the 50% majority rule method. The sump command was used to assure that an adequate sample of the posterior probability distribution was reached during the mcmc procedure. To validate the phylogenetic inference with MrBayes a maximum likelihood method implmemented in RAxML was used [29] . The combined rapid bootstrapping and search for the bestscoring ML tree option (-f a) in RAxML was used to create 1000 bootstraps (-# 1000) using a gamma model of evolutionary rates and the JTT substitution model (-m PROTGAMMAJTT). The JTT substitution model was identified as the most suitable model in the Bayesian analysis and therefore selected for the maximum likelihood phylogeny. The consensus phylogenetic tree found with MrBayes was drawn in Dendroscope 3.0 and the bootstrap support values from RAxML were annotated on the corresponding nodes [30] . The phylogenetic tree was used to determine subfamilies by identifying clusters with a high posterior probability and bootstrap support. Invertebrate sequences were excluded from the phylogenetic analysis as they induced highly unstable topologies together with 1 M. musculus, 2 D. rerio and 3 X. tropicalis sequences. These excluded sequences were categorized into their respective subfamilies by using a BLAST search towards the categorized sequences. The top five hits were examined to classify the invertebrate and excluded sequences into subfamilies. A sequence was assigned to the subfamily if four out of the five top hits are from the same subfamily.
Several resources were used to identify the protein sequence features of the Dispanins. NetPhos 2.0 [31] identified potential phosphorylation sites. NetNGlyc 1.0 and NetOGlyc 3.1 [32] were used to find possible N-and O-glycosylation sites respectively. Transmembrane helix prediction was made using TMHMM 2.0 [33] . Motifs were found manually through Jalview 2.5.1 and the server MEME 4.4.0. Finally, EMBOSS:cons was used to create consensus sequences of the different Dispanin families that emerged from the phylogenetic analysis and the bacterial sequences. The consensus sequences were aligned together with the M. brevicollis and the invertebrate sequences. The resulting MSA was viewed and trimmed in Jalview and the conserved region around the TM helices was kept. Splice sites were detected in the eukaryotic Dispanins by studying their annotation in the respective databases and align them to their genome using BLAT at the UCSC Genome Browser website (http://genome.ucsc.edu).
Table S1 This is a record of all identified Dispanins together with their accession numbers, nomenclature and species belonging.
(XLS)  Health System Resource Gaps and Associated Mortality from Pandemic Influenza across Six Asian Territories BACKGROUND: Southeast Asia has been the focus of considerable investment in pandemic influenza preparedness. Given the wide variation in socio-economic conditions, health system capacity across the region is likely to impact to varying degrees on pandemic mitigation operations. We aimed to estimate and compare the resource gaps, and potential mortalities associated with those gaps, for responding to pandemic influenza within and between six territories in Asia. METHODS AND FINDINGS: We collected health system resource data from Cambodia, Indonesia (Jakarta and Bali), Lao PDR, Taiwan, Thailand and Vietnam. We applied a mathematical transmission model to simulate a “mild-to-moderate” pandemic influenza scenario to estimate resource needs, gaps, and attributable mortalities at province level within each territory. The results show that wide variations exist in resource capacities between and within the six territories, with substantial mortalities predicted as a result of resource gaps (referred to here as “avoidable” mortalities), particularly in poorer areas. Severe nationwide shortages of mechanical ventilators were estimated to be a major cause of avoidable mortalities in all territories except Taiwan. Other resources (oseltamivir, hospital beds and human resources) are inequitably distributed within countries. Estimates of resource gaps and avoidable mortalities were highly sensitive to model parameters defining the transmissibility and clinical severity of the pandemic scenario. However, geographic patterns observed within and across territories remained similar for the range of parameter values explored. CONCLUSIONS: The findings have important implications for where (both geographically and in terms of which resource types) investment is most needed, and the potential impact of resource mobilization for mitigating the disease burden of an influenza pandemic. Effective mobilization of resources across administrative boundaries could go some way towards minimizing avoidable deaths. Recent experience from the 2009-H1N1 pandemic highlights how health system capacities, even in developed countries, can be stretched by relatively mild pandemic scenarios [1] [2] [3] . Indeed, the vast majority of previous health system analyses in relation to pandemic influenza have focused on developed countries [1, [3] [4] [5] [6] [7] [8] [9] [10] [11] , while the capacity of health systems in low and middle income countries remains largely unstudied. Paradoxically, understanding outbreak response capacity in low and middle-income countries is arguably of greater importance than that in developed countries, not only because health systems are weaker [12] , but also because many of these countries are in regions where the risk of emerging infectious diseases is highest [13] . Moreover, these countries may suffer disproportionately because of associations between morbidity, pandemic influenza and poverty [14] .
Proposed strategies for pandemic preparedness in many countries frequently focus on development and acquisition of pandemic vaccines and stockpiling and distribution of antiviral drugs [15] [16] . In the Southeast Asia region, while surveillance and outbreak response capacities have been strengthened in hope of early detection and control of outbreaks, there has been much less investment into preparing health systems for pandemic mitigation [17] . Modeling studies have been used to inform optimum intervention strategies for responding to pandemic influenza, but often neglect to take into account feasibility of health systems to implement such a response and the potential impact of resource shortages on the pandemic burden. Investigation of health system capacity in East and Southeast Asia is of particular interest, not only given the fertile conditions for the emergence and spread of new diseases [13, 18] , but also the wide socio-economic inequalities within the region, and focus of investment by the international community into pandemic influenza preparedness [19] . Resource gaps for a pandemic response are likely to be wide and vary greatly between and within countries in Asia [20] . But exactly how wide are these gaps, what are the consequences of the gaps in terms of the pandemic disease burden, and to what extent could these consequences be mitigated by improving resource allocation and mobilization?
To address these questions, we conducted a health systems analysis across six Asian countries and territories with widely varying socioeconomic conditions: Cambodia, Indonesia, Lao PDR, Taiwan, Thailand and Vietnam. In this analysis, mathematical modeling and health system resource data collected across the six territories were used to estimate and compare, within and across countries, the resource gaps, and potential consequences of those gaps in terms of expected mortalities, for a hypothetical pandemic influenza scenario.
This study was conducted as part of the AsiaFluCap project (www.asiaflucap.org), the overall aim of which is to conduct health systems analyses to support capacity development for responding to pandemic influenza across six countries and territories in Asia, specifically: Cambodia, Indonesia, Lao PDR, Taiwan, Thailand and Vietnam.
For this comparative analysis we focus on four key health system resources: antiviral drugs (specifically oseltamivir), hospital beds, mechanical ventilators and healthcare workers (doctors and nurses), chosen due to their critical importance for responding to pandemic influenza. These resources, along with over 50 other resource items relating to health system infrastructure, equipment, materials, and human resources, were selected through a systematic literature review and a Delphi consensus process by a panel of 24 experts, as described in [21] . Quantities of these resource items were enumerated during March to September 2009 through questionnaires administered to hospitals and health offices in all districts of each of the six study countries (except Indonesia, where data were collected only from districts in Jakarta and Bali due to the vast geographic scale of the country). Additional questionnaires were sent to ministries of health to capture central stockpiles.
We received 100% response rates from hospitals and district health offices in Cambodia. Overall response rates from district health offices were more than 95% in Viet Nam and Indonesia (Jakarta and Bali), 86% in Lao PDR, 72% in Taiwan, and 59% in Thailand. The response rates for hospital questionnaires were slightly lower at 93% for Indonesia and Vietnam, 70% for Lao PDR, and approximately 46% for Thailand and Taiwan. Data from the questionnaires were double-entered into an Excel database. Missing values, due to non-responses or incomplete questionnaires, were extrapolated using linear prediction models, specific to each country and resource item, based on a number of district characteristics such as total number of hospital beds or public hospital beds, population size, and geographic location (region/province). For oseltamivir and ventilators, two-step models were used to first estimate the likelihood of having any oseltamivir or ventilators, and then to predict the number of these items. Extrapolation of missing data was carried out in STATA version 11.
In order to estimate health system resource needs and gaps for a pandemic influenza scenario, we used a mathematical model previously developed as part of the AsiaFluCap project to simulate the transmission dynamics of a pandemic influenza outbreak [22] . Full details and equations of the model can be found in [22] , and are summarized in Text S1. Briefly, the model is based on a deterministic SEIR (Susceptible-Exposed-Infectious-Recovered/removed) model described by differential equations tracking number of people in each compartment over time. Given that the primary aim of this analysis was to provide relative estimates of resource gaps within and across countries, rather than to accurately simulate the spread of pandemic influenza throughout each country, the model structure was kept relatively simple, with homogeneous mixing patterns and no age-structure assumed within the modeled population. However, novel complexity is incorporated through making parameters describing the clinical course of infected individuals conditional upon the availability of certain key health system resources (antivirals, beds, and ventilators). Thus we could obtain relative indications of the consequences of resource shortages on the pandemic disease burden, specifically in terms of ''avoidable deaths'', which we define as deaths that would not have occurred in the presence of sufficient resources.
The infectious compartment of the model was subdivided into three groups based on clinical severity: asymptomatic, mild and severe infections. All asymptomatically and mildly infected patients were assumed to recover, while severe cases were at risk of death and were assumed to need antiviral treatment and hospitalized care, although whether they received either of these depended upon the availability of oseltamivir and hospital beds, respectively. Treatment with antivirals and hospitalization were assumed to reduce the infectious period and the probability of death for severe cases. Furthermore, a proportion of severe cases are assumed to require mechanical ventilation, which they will receive as long as ventilators are available, otherwise these cases would die.
The model parameters describing transmissibility and clinical severity were chosen based on data from the 2009-H1N1 pandemic, with a basic reproduction number of 1.32 [23] [24] . Under the parameter values chosen (see Table S1 and [22] ), in a population with sufficient resources (i.e. when there are no shortages of oseltamivir, hospital beds, or ventilators), the scenario predicts an overall attack rate of 35.6%, a clinical attack rate of 24.9%, a peak prevalence (of symptomatic cases) of 0.94%, and a case fatality rate of 0.018%. In the absence of any resources, the case fatality rate is substantially higher at 0.029%, while attack rates and peak prevalence remain very similar.
Estimating resource needs, gaps, and associated mortality In our baseline scenario, resource gaps were estimated assuming that 12% of ''general'' hospital resources (beds, ventilators and human resources) are available for care of pandemic influenza cases, with the remaining 88% required for maintaining essential healthcare services, as in a previous pilot study for Thailand [20] , and based on previous reports [25] [26] . We also assumed that, in the event of a pandemic all available oseltamivir doses would be dedicated to severe influenza cases. These assumptions regarding resource spare capacity and oseltamivir usage were relaxed in a multivariate uncertainty analysis (described below). The resource data were aggregated at provincial level for all countries except Taiwan, where they were aggregated at county level, and Indonesia, where the data were kept at district level. (Counties in Taiwan and districts in Indonesia are of comparable population size to the provinces of the other four countries.) We then ran the model separately for each province (Lao PDR, Cambodia, Thailand, Vietnam), county (Taiwan) and district (Indonesia), with the appropriate resources for each of these administrative areas, assuming a closed population and that resources could not be shared between these areas in a timely manner. For the purposes of narrative flow, we henceforth use the term ''province'' for counties in Taiwan. All simulations started with one mild case entering a completely susceptible population.
In addition to running the model with the available resources, based on the survey data, we also ran the model with unlimited resources, in order to calculate resource needs, and thus also the resource gaps (or indeed surpluses) by comparing with the resource availability data. The needed number of hospital beds, ventilators and humans resources were estimated from the peak number of cases requiring hospitalization and ventilation, while the needed number of oseltamivir doses was calculated from total number of severe cases occurring over the duration of the outbreak (full details on assumptions of resource depletion rates are detailed in [22] , and summarized in Text S1). By comparing the number of deaths predicted by simulations with sufficient resources with those from simulations using actual resource data, we also estimated the number of deaths due to resource gaps, which we term as ''avoidable deaths''.
A multivariate uncertainty analysis was conducted to approximate uncertainty surrounding avoidable deaths in light of uncertainty in resource spare capacity and effectiveness, which may vary between settings. Specifically, the proportion of ''general'' healthcare resources within each province/district that would be available to care for pandemic influenza patients was allowed to vary between 5-20%, and parameters describing the effectiveness of each resource for reducing the risk of death in cases requiring those resources were allowed to vary independently between a wide range of 20-80%. We also explored the impact of relaxing the assumption that oseltamivir administration is restricted to severe influenza cases, by allowing between 0-5% of mild cases to be treated. One hundred combinations of values were chosen randomly from these ranges using Latin hypercube sampling, and simulations were run using each combination. The medians, interquartile ranges (IQR) and 95 th percentile ranges of model outcomes were then calculated across the simulations.
Since the aim of this study was to compare resource capacities across geographic areas and resource types, rather than to evaluate how transmission dynamics may vary across geographic areas, epidemiological parameters of the model were kept fixed in the multivariate uncertainty analysis to ensure comparability of resource capacity outcomes. However, due to the unpredictability of pandemic scenarios, we also explored model outcomes for a range of values for R 0 and for the severe clinical attack rate in a separate univariate sensitivity analysis.
The model was coded and run in R version 2.10.1, using the ''simecol'' package [27] with the Runge-Kutta 4 th order algorithm for numerical integration of the differential equations. ArcGIS version 10 was used to map the calculated resource gaps and avoidable mortalities at provincial level. Figure 1 presents the geographical distribution of estimated resource gaps across provinces (or districts in the case of Indonesia) in each study country for the modeled pandemic influenza scenario, under our baseline assumptions and point estimates for parameter values. The corresponding statistical distributions of resource capacities across areas within each country can be found in Figure S1 . A summary of overall resource gaps for each country is presented in Table 1 . There was substantial variation in resource gaps both between and within countries, and across resources types (Figures 1 and S1) . Overall, the biggest gaps were generally seen in Cambodia and Lao PDR, particularly when standardized by population size, with almost all provinces in these countries displaying gaps in all resources, with the exception of nurses which were estimated to be sufficient in approximately half of the provinces in these countries. In contrast, relatively few provinces in Taiwan were estimated to have gaps, at least in general health system resources (beds, ventilators, and human resources), with quantities of these resources often considerably above those predicted to be needed for this scenario. Nevertheless, almost half of provinces in Taiwan were predicted to have insufficient oseltamivir supplies to treat all severe cases (although it should be noted that the results in Figures 1 and S1 do not account for central stockpiles which might be mobilized in the event of a pandemic, as discussed later).
Thailand, Indonesia and Vietnam generally displayed a more mixed picture. Results were comparable between Vietnam and Indonesia, with relatively few provinces of Vietnam (7?9%) and only one district of Jakarta (and none in Bali), estimated to have insufficient oseltamivir to treat all severe cases, with supplies of this antiviral drug comparably high across most other areas in these countries. Healthcare workers were also predicted to be mostly sufficient in Vietnam, with only 3?1% and 1?6% of provinces predicted to have a shortage of doctors and nurses, respectively, for this scenario. However, gaps in hospital beds were observed in over half of provinces in Vietnam and districts of Jakarta and Bali, and all of these provinces displayed a shortage of mechanical ventilators. Indeed, of all the resources, the largest gaps were observed in ventilators across all countries except Taiwan (Table 1) .
Thailand had the second highest number of ventilators (absolute and per capita) after Taiwan, but a shortage of this resource was nevertheless predicted in over 80% of Thai provinces. A very heterogeneous pattern was observed in Thailand, with around 50% of provinces showing a shortage of hospital beds and oseltamivir, while many other provinces showed a clear ''surplus'' of the latter. Meanwhile, a shortage of medical doctors was predicted in over 80% of Thai provinces, with gaps in doctors comparable to those Lao PDR and Cambodia ( Table 1 ). The number of nurses in Thai provinces was estimated to be somewhat more sufficient, however. A fairly distinct geographical pattern of resource gaps was evident in Thailand, particularly for oseltamivir and ventilators, with north-eastern (and some southern) provinces showing shortages more comparable with those in neighboring Lao PDR and Cambodia than with other Thai provinces ( Figure 1 ).
It should be noted that estimates of gaps in beds, ventilators and human resources were highly dependent on the spare capacity of resources assumed to be available to care for pandemic influenza patients. For example, if spare capacity was assumed to be 5%, rather than 12%, then even in Taiwan most areas would suffer gaps in these resources for the modeled scenario. Furthermore, use of oseltamivir on even a fairly small proportion (5%) of mild cases, resulted in much faster depletion of this resource, such that all countries are predicted to experience a shortage of oseltamivir for treating all severe cases.
The geographic distribution of avoidable deaths, estimated by calculating the number of deaths that would be prevented by filling all resource gaps in each province, and standardized by population size, is presented in Figure 2 . (A corresponding map showing absolute numbers of estimated avoidable death is given in Figure S2 .) Figure 3 shows estimated avoidable death rates attributable to gaps in each resource type (antivirals, beds and ventilators) aggregated across all provinces in each country, and accounting for uncertainty in resource effectiveness and spare capacity. Avoidable deaths for a given resource gap were estimated by calculating the number of deaths that would be prevented by filling that resource gap only. Figures 2 and 3 highlight how resource gaps could have a substantial impact on mortality rates during an influenza pandemic. A combination of the large population size and shortage of ventilators results in the estimation that, out of the five countries for which nationwide data were collected, Vietnam would have the highest total number of avoidable deaths. However, the results for Jakarta and Bali suggest that Indonesia would have the highest avoidable death toll if the data is extrapolated across the entire population of this country. When standardized by population size, the highest rates of avoidable deaths were estimated in Cambodia and Lao PDR, accounting for over half of all pandemic-associated mortalities in these countries. The median avoidable death rates for these countries were over 15 times higher than that for Taiwan, where a relatively low proportion (median: 7.6%, IQR: 5.5-10.7%) of total deaths was estimated to be due to resource gaps. Almost all avoidable deaths in Taiwan were predicted to be due to local shortages of oseltamivir. In all other countries shortages of ventilators were estimated to be the biggest cause of avoidable deaths (Figure 3 ). In Indonesia, Lao PDR, and Vietnam, this result was largely robust to uncertainty surrounding resource effectiveness and spare capacity. For Cambodia and Thailand, however, the uncertainty analysis suggested that gaps in oseltamivir might also be a main cause of avoidable deaths (Figure 3 ). When adding an additional layer of uncertainty to the model assumptions, by allowing for up to 5% of mild cases to be treated with oseltamivir, the increased shortages of the latter further increased uncertainty surrounding the relative importance of gaps in oseltamivir ( Figure S3 ). Given such uncertainties and the sensitivity of results to model assumptions, the proportion of mortalities that can be attributed to gaps in specific resources should be interpreted with some caution.
A clear negative correlation was observed between estimated avoidable mortality rates and GDP per capita at country level ( Figure 4A ). Total funds per capita committed by donors towards avian and human influenza for each country, up to December 2009 [28] were positively correlated with avoidable mortality rates ( Figure 4B ).
Within many countries it was evident that, while at least some provinces displayed resources gaps, other provinces were estimated to have more than sufficient resources for responding to the modeled scenario, with an overall ''surplus'' of some resources in several countries (Table 1) . Furthermore, central stockpiles of oseltamivir were present in all countries from which data on this could be obtained. Thus we also investigated the proportion of avoidable deaths that might be averted in each country if the total available resources were equitably distributed across provinces according to provincial population size ( Figure 5 ). When accounting for central stockpiles of oseltamivir, the overall supply of this drug was estimated to be sufficient to treat all severe cases in all countries (Table 1) . Thus it was estimated that, in each country (except for Vietnam, and Jakarta and Bali, where provincial supplies of oseltamivir are already relatively high), effective mobilization of oseltamivir across administrative areas could potentially avert a significant proportion of the avoidable deaths estimated under current resource distributions (up to 100% of avoidable mortalities in Taiwan; Figure 5 ).
The (less feasible) scenario of redistributing available beds and ventilators according to provincial need within each country was generally estimated to have less of an impact on the number of avoidable deaths, compared to mobilization of oseltamivir ( Figure 5 ). In the case of ventilators, this highlights how the large numbers of deaths attributed to gaps in this resource (Figure 3 ) are mostly due to overall nationwide shortages of ventilators, rather than an inequitable distribution of ventilators within most countries. In Thailand, however, if all ventilators were distributed in proportion to provincial population sizes, the model predicts around 30% (IQR: 21-41%) fewer avoidable deaths than the number predicted under the observed ventilator distribution.
Estimates of resource gaps, and thus also avoidable mortalities, were very sensitive to the severity of the modeled pandemic scenario in relation to transmissibility and proportion of cases requiring hospitalization ( Figure S4) . A sensitivity analysis showed that under more severe (yet still plausible) pandemic scenarios, even Taiwan could experience substantial deaths due to shortages of hospital resources ( Figure S4A and S4C) . Furthermore, as the severity of the scenario increased, so too did the proportion of avoidable deaths that were attributable to gaps in hospital bed capacity (shown for Cambodia in Figure S4B and S4D) . It is important to note, however, that for the ranges of values explored for the basic reproduction number and the proportion of cases that become severely ill, consistent patterns were observed when comparing relative magnitudes of avoidable mortality rates across countries (and also across provinces within countries).
Our results indicate that health system resource gaps for responding to a mild to moderate pandemic influenza scenario are wide and vary greatly, both within and between countries in Southeast Asia, and that these gaps could have a profound impact on pandemic-associated mortalities. Our estimates of resource gaps and avoidable mortality rates at country level show a clear association with national GDP. This result is consistent with a previous analysis of data from the 1918 influenza pandemic, which found that per capita income explained a large proportion of the variation in mortality across countries during the pandemic period [14] . Moreover, extrapolation of these mortality rates to the 2004 world population suggested that around 96% of deaths from pandemic influenza would occur in developing countries [14] . Our results suggest that, due to inequitable distribution of resources, the variation in pandemic burden is likely to be profound within, as well as between, countries. Countries which have experienced the highest burden of Highly Pathogenic Avian Influenza (H5N1), namely Vietnam and Indonesia, appear to be most prepared in terms of the availability and geographical distribution of oseltamivir. In the other study countries, we estimated that central stockpiles of oseltamivir would be sufficient to cover any provincial gaps for treating all severe cases from the modeled scenario, and thus mobilization of this resource could potentially avert a large number of avoidable mortalities in these countries. Indeed, in all countries except Vietnam, we estimated that optimum mobilization of resources across administrative boundaries could save more than 10% of avoidable deaths. While timely mobilization of resources may be possible in Taiwan, with its small geographical size and relatively developed infrastructure, the feasibility of this scenario is questionable in the poorer, and larger, countries of the Mekong region, where it might be prudent to disburse central stockpiles of antiviral drugs to provincial and district health facilities prior to an outbreak.
Gaps in mechanical ventilators were predicted to be a major cause of avoidable deaths, with almost all provinces across all countries estimated to have severe shortages of this of this resource, with the exception of Taiwan and some Thai provinces. This pattern likely reflects the relatively high cost and human resource skills associated with acquisition and operation of ventilators, and highlights the importance of developing robust triage criteria as part of pandemic preparedness plans to ensure that this resource is allocated to the patients who are most likely benefit [29] [30] . A previous analysis similarly suggested that a dire shortage of mechanical ventilators would be a major limiting factor in responding to a pandemic influenza outbreak in the United States [31] . We found particularly wide variation in the availability of ventilators, and indeed other hospital resources, in Thailand, where our results suggest that inequitable distribution of health system resources [32] , rather than simply an overall nationwide shortage, could lead to a high number of avoidable deaths from pandemic influenza.
Of course, hospital equipment such as beds and ventilators are useless unless sufficient and qualified human resources are available to treat influenza patients, and our results suggest that gaps in healthcare workers would also be an important limiting factor for responding to pandemic influenza in many countries, particularly for Cambodia, Lao PDR and Thailand.
It is encouraging that total donor funds committed to avian and human influenza broadly correspond to avoidable mortality rates estimated at country level. A recent paper on Financial and Technical Assistance from the 2010 International Ministerial Conference on Animal and Pandemic Influenza (IMCAPI) reports that over 50% of total donor funding committed towards avian and human influenza worldwide between 2005 and 2009 was allocated towards ''human health and pandemic preparedness'' (with other funds committed towards sectors such as animal health; monitoring, information, and internal coordination; and information, education and communication) [28] . However, the extent to which these funds have been, or will be, allocated towards mitigating the resource gaps identified in our study is unknown to us and beyond the scope of this analysis.
This study is subject to several limitations, many of which relate to assumptions that were necessary for the modeled scenario. For example, in our baseline scenario we assumed that 12% of hospital capacity, across all provinces and countries, would be available to care for the surge of patients with influenza infections. In reality, surge capacity is likely to vary substantially between and within countries (and over time), but few data on this are available. Robust analytical frameworks are urgently needed to define and measure health system surge capacity in order to inform analyses of resource gaps for emergency response scenarios. The effectiveness of resources such as antiviral drugs, ventilators, and general hospital care for improving the survival rates among severe influenza cases is also surrounded by considerable uncertainty and may vary between settings. Our results show that, even if epidemiological parameters describing transmission and pathogenicity are kept constant, uncertainties in spare capacity and resource effectiveness lead to considerable uncertainties in estimates of avoidable mortalities rates. Nevertheless, the distributions of model outputs from our multivariate uncertainty analysis still showed some significant differences when compared across countries and across resource types (Figures 3 and 5) . Furthermore, it seems likely that spare capacity and resource effectiveness would be higher in more resource-rich settings, which would only strengthen the findings of this study in terms of the geographic distribution of resource gaps and avoidable mortalities.
Estimates of resource gaps and avoidable mortality rates were also very sensitive to parameters describing pandemic severity, although similar patterns were observed across geographic areas for the range of values explored. However, the same cannot be said for the relative importance of gaps in different resource types. Thus, although our results generally suggest that shortages of ventilators could be a major cause of avoidable deaths in low-and middle-income countries in Southeast Asia, investment in this resource should not necessarily be prioritized over other healthcare resources. A natural extension of this study would be to investigate the cost-effectiveness of investing different types of health systems resources for mitigating the burden of an influenza pandemic. However, more data on the effectiveness of different resources for managing severe influenza cases is needed before such assessments can be made.
Other limitations relate to the simplicity of the model structure. We assumed homogenous mixing and a constant basic reproduction number across all populations. In reality, heterogeneities in factors such as age-structure, geographic structure, population density, human behavior, and the underlying health of the population are all likely to play a role in transmission dynamics and burden of influenza. A previous modeling analysis, for example, has shown that higher levels of population heterogeneities, such as in age and spatial structuring of contacts, result in lower overall attack rates and peak prevalence for a given basic reproduction number [33] . However, there is a lack of data on such heterogeneities and how they might affect patterns of pandemic progression for our study region. Ongoing studies, such as contact pattern surveys in Asia similar to those undertaken in Europe [34] , are attempting to rectify this. Another limitation is that the resource data were collected between May and September 2009, which includes the first wave of the H1N1-2009 pandemic; thus some resource data (particularly for antiviral stockpiles) may be influenced by the time point within this period at which the data were recorded.
Given the above caveats, it is important to emphasize that we do not advocate these results to be accurate quantitative reflections of resources shortages or deaths that are likely to occur in any given pandemic scenario. Rather, they highlight the scale of health system inequalities within and across countries in Asia, and the considerable impact such inequalities could have on the pandemic disease burden. By indicating the relative disparities in resource availability within and across countries, and the potential consequences of resource shortages, these results could help guide investment decisions in scaling up resources to mitigate the burden Figure 5 . Estimated impact of resource mobilization/redistribution across provinces on avoidable mortality rates within each territory. Data were calculated by estimating the number of avoidable deaths if available resources (including central stockpiles for oseltamivir) within each territory were geographically distributed in proportion to provincial population size, and comparing with the total number of avoidable deaths predicted given actual resource distribution. Boxplots show medians, interquartile ranges, and 95 th percentile ranges derived from a multivariate uncertainty analysis. Data are aggregated across provinces for Cambodia, Lao PDR, Thailand, Vietnam, and across counties for Taiwan. Data for Indonesia are aggregated across districts of Jakarta and Bali only. doi:10.1371/journal.pone.0031800.g005 of future pandemics. As many of these resources have a generic healthcare function beyond pandemic influenza, they may also be useful to guide health system strengthening. Figure S1 Variation in estimated resource capacities across provinces within each territory for the modeled pandemic scenario. (DOCX) Figure S2 Geographical distribution of estimated avoidable deaths due to resource gaps for a modeled pandemic influenza scenario.  Epidemiology and viral etiologies of Severe Acute Respiratory Infections (SARI) in the Northern Vietnam   Impact of different influenza cultivation conditions on HA N-Glycosylation  Jana V Roedig 1 Background Influenza virus is a highly contagious human and animal pathogen causing infections of the respiratory track. Prevention such as high standard hygiene and vaccination still represent the best measures for protection. Beside the traditional egg-based influenza vaccine production, numerous cell culture-based processes are currently being established. Due to its ability to induce strong and protective immune responses, the highly abundant glycoprotein hemagglutinin (HA) represents the major component in influenza vaccines. Since variations in Nglycosylation of glycoproteins such as HA can alter quality characteristics of antigens, the impact of cell lines and process parameters for vaccine manufacturing needs to be addressed. This study investigates the impact of virus adaptation and different harvest time points on HA N-glycosylation. Therefore, the HA of influenza A virus Uruguay/716/2007 (H3N2, high growth reassortant), in the following referred to as IVA-Uruguay, was purified and N-glycans analyzed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF).
Cell culture and virus production IVA-Uruguay (H3N2, #07/360, NIBSC, South Mimms, UK) was produced in either adherently growing MDCK (No. 84121903) or Vero (No. 88020401) cells purchased from ECACC (Salisbury, UK) . For cell growth GMEM (Invitrogen, #22100-093, Darmstadt, Germany) was supplemented with 5.5 g/L glucose (Roth, #X997.3, Karlsruhe, Germany), 2 g/L peptone (IDG, #MC33, Lancashire, UK), 10 % FCS (Invitrogen, #10270-106) and 4 mg/mL NaHCO 3 (Roth, #6885.3). Infections were performed in the same medium without addition of FCS but supplemented with trypsin (Invitrogen, #27250-018) at a final concentration of 5 U/mL. Virus was quantified by a hemagglutination assay according to Kalbfuss et al. [1] and is expressed in HAU (log HA/100 µL).
Virus was harvested and processed for HA N-glycosylation pattern analysis according to Schwarzer et al. [2] applying an optimized work-flow [3] and data evaluation [4] . Finally, the samples were separated by CGE-LIF using an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems, Foster City, California, USA). For data processing and evaluation the x-axis of capillary electropherograms was normalized using an internal standard, resulting in N-glycosylation patterns, in which each peak corresponds to at least one distinct N-glycan structure. This allowed a direct qualitative comparison regarding N-glycan structure presence in different samples. For quantitative comparison, the relative peak height (RPH: the ratio of peak height to the total height of all peaks) was determined for each peak and sample. Low abundant peaks were defined with RPH < 5 %.
MDCK cell-derived virus seed, exhibiting 2.6 HAU at 24 hours post infection (hpi; data not shown), was used to infect five consecutive passages of Vero cells. Adaptation of the virus to Vero cells resulted in increased virus yields within shorter time frames in the new host system: in the first passage of Vero cells 2.1 HAU were obtained at 96 hpi, whereas in the fifth passage a titer of 2.7 HAU at 72 hpi was reached (data not shown). The HA N-glycosylation pattern of the MDCK cell-derived IVA-Uruguay seed exhibited 25 different characteristic peaks in the range of 160 bp to 400 bp. Of these a total ure 1A) . In comparison to MDCK cell-derived HA, the Vero cell-derived antigen showed a tendency towards smaller glycan structures. The relative abundance of each peak over all Vero passages only varied marginally with standard deviations (SD) ≤ 2.1 % (table 1). The increase in virus titers within shorter time frames suggests increased viral fitness, during adaptation from MDCK to Vero cells. An impact of HA N-glycosylation on properties of the virus, i.e. virus replication, has already been descried [8] [9] [10] [11] [12] . Interestingly, the glycan pattern stabilized soon after the first passage in Vero cells. This clearly indicates that further increase in HA titer did not depend on changes in the HA N-glycosylation pattern.
The impact of harvest time on the HA N-glycosylation pattern of MDCK cell-derived IVA-Uruguay is shown in figure 1B . Virus harvested at either 24 hpi or 72 hpi exhibited the 25 different MDCK cell-specific peaks between 160 bp and 400 bp. At 72 hpi one additional, but very low abundant peak was detected (numbered 23). Overall, differences in relative structure abundance were rather small with a maximal difference of 1.7 % (table 1) . This indicates that HA of virus particles released in the supernatant is rather stable over the time window relevant for influenza virus production [5, 6] ]. However, there are minor variations in RPH from passage to passage during adaptation and between harvesting time points. Possible explanations are varying ratios either of completely/incompletely processed or of intact/ degraded N-glycan structures or a combination of both. In 2009, Schwarzer et al. [7] characterized the MDCK cell-derived HA N-glycosylation pattern of a H3N2 influenza virus subtype as a mixture of complex N-glycan structures with terminal αand β-galactose and high mannose type structures. In contrast, the Vero cellderived HA was characterized by complex N-glycans with exclusively terminal β-galactose and structures of the high mannose type. For final evaluation of the results presented here, determination of the N-glycan structure of all peaks would be required.
In this study, the impact of adaptation and harvesting time point on HA N-glycosylation of IVA-Uruguay was investigated. So far, it is not clear whether differences in the HA N-glycosylation have an impact on immunogenicity or other properties of influenza vaccines. Other factors, e.g. differences in cell culture media, cell density, etc. may also contribute to variations in HA N-glycosylation. Nevertheless, monitoring N-glycosylation patterns during vaccine production processes allows not only to evaluate antigen quality and the impact of process modifications on lot-to-lot consistency but also to critically assess consequences of unwanted process variations or process failure.  Isolation of active peptides from plant hydrolysates that promote Vero cells growth in stirred cultures  Background Vero cells are adherent cell lines commonly used for the production of viral vaccines. We had developed an animal component free medium that allows an optimal growth of this cell line in stirred bioreactor [1, 2] . We had also showed that Vero cells grown in this medium (called IPT-AFM) sustained rabies virus replication, and resulted in an overall yield comparable to the level obtained in serum-supplemented medium.
IPT-AF medium contains plant hydrolysates, namely soy (Hypep 1510) and wheat gluten hydrolysates (Hypeps 4601 and 4605). These peptones were shown to promote cell attachment and growth. However, although these components are of non-animal origin, their use in vaccine production process has several drawbacks, mainly due variability between lots.
The aim of this work is to identify active peptides from these hydrolysates that show a positive effect on cell adhesion, attachment and growth. For this purpose, the hydrolysates were fractionated using chromatography and precipitation techniques. The effect of the isolated fractions on Vero cells growth were tested in 24 and 6-well cell culture plates using experimental design approach. Fractions that sustain cell growth, were further tested in stirred culture on Cytodex1 microcarriers, to confirm their positive effect on Vero cells growth.
Cell line: Vero cells adapted to IPT-AF medium as described in Rourou et al. [2] were used in this study.
Media: M199 medium was purchased from Invitrogen, IPT-AFM was prepared as detailed in Rourou et al. [2] . Hypeps were provided by Sheffield Bio-Science.
Static cultures: Cells were grown in 24-well plates (Falcon) and 6-well plate experiments « Nunclon Δ». The inoculation density was 2x10 5 cells/ml and the working volume was equal to 1 ml for the 24-well plate and 3 ml when the cells were grown in 6-well plate. Cells were grown at 37°C in 5% CO 2 incubator.
Stirred cultures: Cells were grown in 6-well low binding plates (Costar) and in spinner flasks; cultures were inoculated at a cell density of 2x10 5 cells/ml. The Working volume was equal to 3 ml for the 6-well plate and 200 ml when cultures were performed in spinner flask. Cells were grown on 2 g/l Cytodex 1 at 37°C and 30 rpm.
Fractionnation protocols: Hypeps 1510, 4601 and 4605 were fractionated by either chromatography methods or sequential precipitation with different ethanol concentrations as described in Shen et al. [3] .
Sephadex G-25 (GE Healthcare), BioGel P-2 fine (Biorad), Sephadex G-10 (GE Heathcare), HiTrap Q HP (GE Healthcare) and HiTrap SP HP (GE Healthcare) matrixes were tested for the fractionation of the different solutions of Hypeps. Fractionation was monitored by measuring the absorbance of the collected fractions at 214 nm. Collected fractions were desalted, then frozen at -70°C and lyophilized. After lyophilization the fractions were resuspended in M199 so they would be compatible with cell culture.
Analytical methods: Cells grown in static cultures were first detached with the TrypLe Select (Invitrogen) then counted according to the Trypan blue method.
Experimental design and statistical analysis: The software Modde 6.0 (Umetrics, Sweden) was used in this study for the design of the experiments and the statistical analysis of the data.
Sephadex G-10, Sephadex G-25 and Biogel fine-2 were used to fractionate Hypeps 4605 and 4601. However, none of these matrixes was efficient.
Anion and cation exchange chromatography were therefore used as an alternative method; two pH levels were tested: 5 and 8. Although these methods allowed the isolation of different fractions for each peptone, none of them had enhanced Vero cells when the cells were cultivated in 24-well culture plates. In addition, most of these fractions showed a toxic effect on cell growth.
Sequential precipitation with different ethanol concentration was also applied for the fractionation of the three peptones. The fractionation was conducted as described by Shen et al. [3] ; 5 fractions were obtained for Hypep 4605 whereas for Hypep 4601 and Hypep 1510, 4 fractions were obtained for each. The effects of the isolated fractions on Vero cells growth were investigated in 24-well plates using a full factorial experimental design; 83 combinations were assessed in duplicate. Two combinations of fractions that show a cell growth comparable to that obtained in IPT-AF medium (positive control), were selected (combinations 1 and 2). The identified combinations were also tested in 6-well plates on 2 g/l Cytodex 1 microcarriers. The highest cell density level reached under these conditions was similar to that achieved in IPT-AF medium.
These combinations were further tested in spinner flask on Cytodex1 microcarriers, to confirm their positive effect on Vero cells growth. Data shown in Figure 1 , indicate that cell density level reached 2x10 6 cells/ml after 5 days of culture when Vero cells were grown in combination 1. Such level was slightly lower than that obtained in IPT-AF medium (2x10 6 cells/ml versus 2.4x10 6 cells/ml). However, Vero cell growth in combination 2 was less efficient; the highest cell density Figure 1 Vero cells on 2 g/l Cytodex 1 in spinner flask, in different media obtained in this medium was equal to 1.7x10 6 cells/ml. Thus, combination 1 appears to be more suitable for Vero cells on Cytodex 1 microcarriers.
Sephadex G-25, Sephadex G-10, Biogel-fine and ion exchange chromatography matrixes were not efficient for Hypeps fractionation. Sequential precipitation with ethanol appears to be the best method to isolate various fractions that promote Vero cells growth. Two Combinations (1 & 2) were identified as the best in terms of cell density and cell attachment. Spinner cultures demonstrated that these combinations are suitable for Vero cells growth on Cytodex 1. Further fractionation and characterization of these compounds are ongoing to identify the active components.  First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination. The mammalian tick-borne flavivirus group (MTBFG) includes viruses associated with important human and animal diseases such as encephalitis (Tick-borne The corresponding polyprotein is proteolysed and processed into structural, Capsid (C), Pre-Membrane (PrM), Envelope (E) and nonstructural proteins NS1 (glycoprotein), NS2A, NS2B (protease component), NS3 (protease, helicase, RNA triphosphatase activity and NTPase activity), NS4A, NS4B and NS5 (methyltransferase, RNA-dependant RNA polymerase) [1] .
Several viruses bear close evolutionary relationships to TBEV [2] [3] [4] [5] [6] viz. LIV, Spanish sheep encephalomyelitis virus (SSEV), Turkish sheep encephalitis virus (TSEV) and Greek goat encephalitis virus (GGEV). These four lineages have recently been assigned to a single species dubbed Tick-borne encephalitis virus [4] , whose members are primarily associated with ixodic hard-tick vectors. Within this species, the TBEV lineage is further divided into three evolutionary distinct subtypes, the Western European-(W-), the Far Eastern-(FE-) and the Siberian-(S-) TBEV [1, 7] .
In this contribution, our attention is mainly focused on the evolutionary relationships between W-TBEV, SSEV and LIV. W-TBEV is widely distributed throughout continental Europe and Russia, SSEV is endemic to Spain [2] [3] [4] [5] [6] 8] , and LIV, initially considered to be restricted to the British Isles and Ireland [2] [3] [4] [5] [6] , has now been reported from Norway [2] and Denmark [9, 10] . The ecology and pathogenesis of both W-TBEV and LIV have been intensively investigated [11, 12] , whereas studies dedicated to SSEV are scarce.
In contrast to mosquito-borne flaviviruses where recombination events are frequent [13, 14] , evolution in the MTBFG was considered to be clonal. This perception changed recently with reports of several putative recombinations in Tick-borne encephalitis virus [15, 16] . We aim to investigate the strength of the recombination signals reported by Yun et al. [16] , since if proved valid their discovery would lead to a radical departure from the classical understanding of the evolutionary dynamic in MTBFG. Although, we could not confirm the previously described recombinations, we did identify a strong recombinant signal in the LIV lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods.
The second aspect of this study pertains to date the recombination event. We used the available full length coding genomes for dating, but this small sample may limit the power of the molecular-clock analysis. There are a large number of Esequences available from molecular epidemiological studies.
Unfortunately, E-sequence cannot be used directly to date recombination events, as we estimated that the substitution rates for the E-gene is significantly lower than for other viral genes. This means that dates obtained from E-sequences alone tend to be younger and do not represent accurately the temporal dynamic of this viral lineage. We suggest that a large dataset that includes sonly E-sequences could nevertheless be used to date additional divergence events by specifying informative priors on the ages of some important nodes. We describe an incremental analytical strategy that bases these priors on posterior distributions derived from the analysis of full-length coding sequences following removal of the E-sequences.
Alignments were generated from GenBank sequences retrieved in January 2011, aligned using Muscle [17] , rechecked and improved manually in the UTR regions. Sequences were numbered from the start of the ORFs using Neudoerfl (U27495) as reference. Details on the included sequences are provided in Table S1 . ALN1 contains 41 complete nucleotide sequences of Tick-borne encephalitis virus and three out-groups selected among LGTV and OHFV. This initial alignment was scanned for recombination events and then down sampled to an alignment (ALN2) of 28 complete sequences of known collection dates (from 1937 to 2008), with the deletion of out-groups and strains with unusual sampling locations. UTRs and gap columns were deleted. ALN2 was further partitioned by individual genes resulting in alignments ALN2_C, ALN2_PrM, ALN2_E, ALN2_NS1, ALN2_NS2A, ALN2_NS2B, ALN2_NS3, ALN2_NS4A, ALN2_NS4B and ALN2_NS5. Next, we produced ALN3 from ALN2 with the deletion of the E gene and the region of NS3 identified as a possible recombinant fragment. Finally, E_161 was compiled from the 161 longest Esequences available in Genbank (1033 to 1491 nt in length) endowed with sampling dates (from 1931 to 2008).
An analysis of the entire species (ALN1) was conducted with split networks using the neighbor-net method [18] . Evolutionary distances were estimated using maximum likelihood (ML) with a GTR+C 4 +I as the best-fit substitution model as determined by MODELTEST v.3.7 [19] , according to the Akaike Information Criterion.
Several methods were used to extract recombination signal from ALN1 with the RDP3beta36 package [20] , because inspection of the split network had established the possibility of recombination within the species (see results). All analyses were carried out with Bonferroni correction (P-value,0.05) and signals reported by more than one method were retained. RDP [21] , GENECONV [22] , BootScan [23] , MaxChi [24] , Chimaera [20] , and SiScan [25] were used for screenings the alignment. For this initial phase, the following settings were modified to balance sensitivity and statistical significance: RDP: window size 25, detect recombination between sequences sharing 90% to 100% identity; GENECONV: G-scale 5; BootScan: windows size 100, use NJ trees, 200 bootstrap replicates, cutoff percentage at 95% and Jin and Nei 1990 model; Chimaera: 40 variable sites per window; SisScan: window size 80, slow exhaustive scan. As all methods detected the presence of significant recombinant signals in the NS3 gene, the dataset was further evaluated for phylogenetic evidence of recombination based on an alignment of NS3-sequences derived from ALN1.
For the phylogenetic analysis, the NS3 partitions 59 and 39 of the putative recombinant fragment were concatenated. Trees were inferred separately for the recombinant region alone and for the concatenated region.
Maximum likelihood analyses were performed with RAxML VI-HPC v.2.2. [26] via the RAxML Web server [27] . The proportion of invariable sites and the number of bootstrap runs were automatically determined.
Bayesian phylogenetic trees were constructed with a GTR+I+G nucleotide substitution model for the concatenated alignment of NS3 and a GTR+G model for the recombinant partition. Model selection was based on the corrected Akaike information criterion in MrAic [28] . For each alignment, two separate analyses were run simultaneously with MrBayes v.3.2-cvs [29] (source code accessed with CVS 22 January 2009) for 5000000 generations using the default settings for priors and MCMC proposals. Trees were sampled every 1000 th generation, and standard deviation of split frequencies was below 0.01 at the end of each analysis. For all Bayesian analyses (i.e. MrBayes and BEAST), mixing of the MCMC chains and effective sample size (ESS) for each parameter estimate were investigated using Tracer v.1.5 [30] which showed convergence and larger than 200 ESS for each summary statistic. For both MrBayes analyses, the first 2500 trees where discarded as burn-in and the 7500 remaining trees were summarized in a majority-rule consensus tree.
For each of the two partitions, we tested alternative topological placement for the putative recombinant strain. Constraining the topology in ML analyses yielded likelihoods for alternative placements that were compared with the likelihood of the best ML tree using the approximately unbiased (AU) test [31] in CONSEL [32] . For this step, ML analyses were performed with PAUP* v.4.0b10 [33] and best trees were sought by heuristic searches (10 random addition replicates, TBR branch swapping, Multrees in effect).
Throughout the study, node support was estimated by nonparametric bootstrap (BS, bootstrap support) in ML and with multiple samples from the posterior distribution (PP, posterior probability) in BI.
Each separate gene alignment (ALN2_C, ALN2_PrM, ALN2_E, ALN2_NS1, ALN2_NS2A, ALN2_NS2B, ALN2_NS3, ALN2_NS4A, ALN2_NS4B and ALN2_NS5) was investigated for signs of positive selection. To that end, the dN/dS ratio for the whole gene, and for each codon in the alignment, was inferred using the M3 model [34] implemented in MrBayes, otherwise using default settings. Mixing of the MCMC chains, as well as the ESS of each estimated parameter was assessed by analyzing the resulting parameter files with Tracer. Each analysis was run until the ESS exceeded 200 for all parameters, after which the probability for the whole gene, or individual codons in the sequence, to have evolved under positive selection was analyzed with Tracer.
Substitution rates and dates of ancient divergence were estimated with Bayesian MCMC in BEAST version 1.5.3 [35] , with collection times in years used as calibration points in the clock model. The youngest strain was collected in 2008, which sets this year as the origin for past time estimates. Each dataset was evaluated individually for best fitting substitution model, which ranged from HKY+C 4 +I to GTR+C 4 +I. However, analyses performed under GTR family models neither converged nor mixed well, possibly due to an insufficiency of data to estimate these highly parametric substitution models. Hence, the simpler, less parameter rich, HKY+C 4 +I model was used throughout the BEAST investigation. We tested the impact of using a GTR model by running an analysis for 20610 6 generations. Estimates for the parameters of interest were largely concordant (data not shown), albeit the analyses returned very low ESS and much wider confidence intervals. Pairwise comparisons of Bayes factors calculated in Tracer selected the uncorrelated lognormally distributed relaxed-clock (UCLN) and the Bayesian Skyline coalescence model [36] as the best fitting clock and demographic models following the procedure in Hon et al. [37] . We defined two partitions that separated first and second positions from third codon positions. For each analysis, four independent MCMC chains were run for 20610 6 generations and their log output combined with 10% burn-in samples discarded. Tracer was used to determine the degree of mixing, shape of the probability density distribution, median and highest posterior density regions at 95% (HPD) for the relevant parameters. The modes and parameters of the posterior distributions were estimated using the distribution fitting software EasyFit 5.3 (MathWave Technology). For all analyzed parameters, we modeled the posterior distributions with gamma distributions. The analytical framework of the BEAST analyses is presented in Figure 1 and the details are explained below.
We compared the mean substitution rates derived from BEAST analyses for ten individual genes obtained from ALN2. Settings were as described above with an additional uniform prior distribution on the time interval [360-10000] fitted to the root height. This prior captures the background knowledge that crown radiation of flaviviruses occurred after the end of the last glaciations (placed 10,000 years ago) and that the Tick-borne encephalitis virus emerged before the divergence of two of its inclusive clades namely LIV and W-TBEV whose split was estimated to be earlier than 360 years ago [38] , placing the species divergence within this rather wide interval.
To estimate the time of the recombination event (tRE), as well as the time of the most recent common ancestor (tMRCA) for each parental strain, we studied separately the genomic partition spanning the recombinant element from nt 5787 to 5991 (ALN2-1) and the partition covering the rest of the ORF (ALN2-2) that is the 59 region (nt 1 to 5786) together with the 39 region (nt 5992 to 10245) flanking the recombinant portion. The same uniform prior was fitted on the root height as before. For individual genes in ALN2-2, prior distributions for the MeanRate parameter were derived from posteriors in BEAST inference 1 with substitution and clock models unlinked during the analysis.
This step was designed to provide posterior distributions for the BEAST inference 4. ALN3 (28 full length ORFs with both E-gene and the recombinant fragment omitted) was analyzed the same way as ALN2-2. The mode and parameters of posterior distributions for the root height, tMRCA(Neudoerfl-Hypr) and tMRCA(LIV & SSEV) were estimated in order to be incorporated as priors in the following step.
BEAST inference 4: refining estimates for tMRCA(Neudoerfl-Hypr) Due to its sampling, the E_161 alignment allows access to the antiquity of additional divergence events. Posteriors obtained from BEAST inference 3 were included as priors, with an additional uniform prior distribution over [7.28610 25 -6.29610 24 substitutions/site/year] set on the meanRate parameter. This value reflects previously observed substitution rates for the E gene in the Tick-borne encephalitis virus: the lower bound comes from the value of 7.28610 25 substitutions/site/year estimated for nonsynonymous substitutions [38] , while the upper bound comes from an estimation of 4.78610 24 substitutions/site/year with a standard error of 1.51610 24 [39] for synonymous substitutions. Because the analysis of selection pressure (see results) inferred that a strong purifying selection acts on the proteins, we expect to see higher rate of synonymous substitutions than of nonsynonymous substitutions. As the mean rate takes both types of substitutions into account, its estimate should be intermediate between their two values.
All alignments, xml-files for the BEAST analyses and all phylogenetic trees have been deposited at Dryad Repository: doi:10.5061/dryad.504636cd.
On the inferred network (Figure 2 ), the region of the split-graph separating the four main clusters exhibits a significant ''tree-like'' structure that rules out frequent recombination between the clusters. Nevertheless, a prominent split associated with SSEV (DQ235152) and LIV (Y07863) indicates a marked conflicting and/or ambiguous signal that could be associated with a recombination event. This hypothesis was first examined with RDP3, wherein all methods identified the LIV strain as displaying signs of homologous recombination between the SSEV strain as the major parent and a strain belonging to W-TBEV as the minor parent ( Figures S1 a-b) . All methods recognized with significance that an insert within the NS3 gene of LIV originated from a W-TBEV strain, but they were not consistent with respect to the precise location of the two recombination methods. When run simultaneously, all methods bar, Chimaera and MaxChi, identified Neudoerfl (U27495) as the minor parent and estimated the breaking points at nt 5787 and 5991. When the data were analyzed with Chimaera or MaxChi as single primary detection methods, they instead proposed, with significance (P-value ,3.10 22 ), slightly different breakpoints (Table 1) .
No significant evidence for recombination was found in the other strains or genes. We compared our result to the outcome of the screening performed by Yun et al. [16] that identified 11 recombinations within the 39UTR and the 39 end of the NS5 region, but did not include a LIV strain. Their observations could only be replicated when we used exactly the same settings, i.e. when detection was performed on ClustalW [40] aligned sequences, without Bonferroni correction for multiple comparisons. This suggests that the previously reported signal was not strongly supported and could have been caused by alignment problems, as UTRs are notoriously difficult to align due to spontaneous variations in length during laboratory passages [41, 42] .
To evaluate phylogenetic evidence of recombination, trees were constructed for the putative recombinant region and for the concatenated regions of NS3 from both sides of the crossover points. As shown in Figure 3 , ML and Bayesian reconstructions contrast the placement of LIV in the two partitions: In the nonrecombinant partition, LIV groups with SSEV with maximum support and falls outside the highly supported W-TBEV clade (BS 99%, PP 1.00). In contrast, LIV is well embedded within the W-TBEV clade and is placed together with Neudoerfl for the recombinant partition. Although the two most supported nodes that identify close evolutionary relationships between LIV and a strain from W-TBEV display moderate BS and PP (78% and 0.92 for the branching with Neudoerfl, 89% and 0.99 for the inclusion of LIV within W-TBEV), they are among the most significantly supported nodes in this tree. We tested the three putative recombinant fragments obtained by different methods in RDP3 and found that the shorter segment branched together with Neudoerfl with higher support values. Hence, we proceed with further characterization of this mosaic history under the assumption that crossovers occurred at nucleotides 5787 and 5991, which places the 204 nt long recombination in the highly conserved helicase domain of NS3 (subdomain 3). At the nucleotide level, the comparison of the daughter with its parental strains revealed 23 variable sites within the putative recombinant element, while the rest of the NS3 gene contained 274 variable sites. A comparison of genetic distances based on nucleotide sequence is reported in Table 2 .
Phylogenetic discrepancies were assessed statistically with the AU test. For the combined (non-recombined) NS3 partition, the topological constraints forced LIV and W-TBEV into a monophyletic group with SSEV as sister taxon. Conversely, for the recombinant partition we imposed the grouping of SSEV and LIV outside the W-TBEV clade. Both alternative placements were rejected by the AU test (see Table 3 ), confirming that the different placements of LIV between partitions expresses genuine phylogenetic information rather than mere stochastic effects.
Results of BEAST inference 1 are summarized in Figure 4a , showing that under the same set of priors, the posterior substitution rate (meanRate) varies up to five-fold between the different genes. The estimates distinguish the E-gene; it is both the most clearly separated and narrowly distributed, with a median of 3. 
In order to increase precision, dating the recombination was carried out using the recombinant region ALN2-1 and the recombination free ORF (ALN2-2). Overall, when compared to the outcome of ALN2-2, divergence times for ALN2-1 were younger and less precise, probably due to the low amount of informative data. For the sake of studying the recombination event, ideally three nodes should be scrutinized: Node ''r'' on Figure 3 estimates the actual recombination. It refers to the clustering of the LIV recombinant segment with the Neudoerfl strain which places tRE at a median of 76 (HPD: 45-160) years before origin. Accordingly, this time point is paramount and constitutes the lower bound of the estimate, but caution is advised when interpreting it as the definitive estimate. Indeed, it suffers from being inferred from a dataset comprising very short sequences. Moreover this analysis, carried out with a low level of prior enforcement, demonstrates a systematic bias towards younger antiquity. ''M'' and ''m'' are time points that refer respectively to the oldest estimate for the emergence of the major and minor parental lineages. Point ''M'' corresponds to the split of SSEV and LIV lineages, dated at a median of 1017 (HPD: 664 to 1510). Point ''m'' refers to the emergence of Neudoerfl, which corresponds to its split with the most closely related strain Hypr. However, few substitutions among the nine W-TBEV strains leads to poor phylogenetic resolution. Hence, a more conservative estimate for the onset of the minor parent would coincide with the divergence of the W-TBEV clade, placed at a median of 307 (HPD: 208 to 444) years. The next step aimed to retrieve divergence times for ''M'' and ''m'' with both increased accuracy (better locate the events in time) and increased precision (achieve narrower confidence intervals). We analyzed the largest available dataset for TBEV strains with collections dates (161 sequences); unfortunately, it only covers the Envelope glycoprotein (E) obtained from epidemiological studies. This brings on two problems: Firstly, this dataset is unable to target the actual recombination that occurred within the NS3 gene. Secondly, inference 1 has demonstrated that the E-gene presents the lowest rate of substitution among the viral genes, therefore it estimates older divergence dates than other portions of the genome. Although the former issue cannot be avoided, Esequences can nevertheless pinpoint which lineages would carry the recombinant element in a much larger tree. The latter issue can be tackled in a Bayesian framework by incorporating posterior information on divergence times derived from full-length coding sequences as prior distributions in an E-sequence analysis. The underlying rational is that by injecting information that pertains to all genes, bar E and the recombinant segment, we would be able to downplay the influence of the low substitution rate, while still combining all available evidence and avoiding circularity. We used BEAST inference 3 to calculate prior distributions for the root age and tMRCA(W-TBEV). The prior on the substitution rate was derived from the literature and not from BEAST inference 1 in order to avoid circularity in the use of data. Figure 5 depicts the outcome of BEAST inference 4, wherein the general tree summarizes the entire TBEV species evolutionary history and the enlarged chronogram gives median divergence dates within the W-TBEV, LIV, SSEV, GGEV, TSEV cluster.
Dates for the principal nodes are indicated in Table 4 . Within the cluster concerned with the recombination (Figure 5b tMRCAs estimated from ALN2-2 and E_161 are consistent, suggesting that the appropriate priors have successfully counterbalanced the influence of a lower substitution rate in the E-gene. Inference 2 placed tRE at 76 (HPD: 45-160) years before origin, which localizes the recombination within the Neudoerfl lineage and after the split with the Scharl lineage. The upper (older) bound for the tRE corresponds to the youngest of the parental divergence times in the tree Figure 3 . As the estimate for M is much older than time point m, the latter can be considered as the theoretical upper bound for the observed recombination event. The lower bound leaves open the possibility that recombination occurred after the LIV 369/T2 -LI/G divergence, whereas the upper bound sets it within a clade comprising LI/G, LIV 69/T2, LI/ 261, LI/K, LI/A, LI/NOR and LI/917. Based on previous phylogenetic dispersal reconstructions [39] , the first bound places the event in Scotland, whereas the second allows a wide range of locations within the UK, after the initial virus emergence in Ireland.
The possibility of recombination within tick-borne flaviviruses was raised by Twiddy et al. [14] , but given the low amount of genetic variation in this group, they pointed out that detection would prove difficult. A recent report [16] would indicate that tickborne flaviviruses have the potential to obtain and spread advantageous traits, and to remove deleterious genes [43] by homologous recombination. Alas, re-analysis of the published data did not recover that signal using a more accurate alignment method and more stringent detection conditions, but found evidences for a different event. Therefore our study shows for the first time that demonstrable recombination (that is, with sufficient statistical support and with strong phylogenetic evidences) has occurred in the mammalian tick-borne flavivirus group.
Substitution rates are compound products of at least four factors: generation time, effective population size, underlying mutation rate and mutation fitness [44] . The last factor can be assessed indirectly by studying the level of selection pressure on the variable sites. The low positive selection is a well documented aspect of the mode of evolution of vector-borne RNA viruses [45] [46] [47] , which demonstrate a lack of immune-driven positive selection [46] and a very effective purifying selection [48] . Our analyses did not identify any site under positive selection. Moreover, the substitution rate analysis yielded a median of 3.3610 25 (HPD: 1.5610 25 -6.1610 25 ) subs./site/year for E, significantly lower than the previously reported rates of 1.6610 24 , within S-TBEV [49] and FE-TBEV [50] and the 8.0610 24 found W-TBEV [21] . The main difference with the previous studies can be pinpointed to our use of a relaxed clock, which was chosen because Bayes factor comparisons indicated that the strict clock performed significantly worse than relaxed models. It is known that incorrect clock assumption may lead to spurious rate estimates [51] and dating analyses effectuated under a strict clock and the same set of priors as in inference 1, yielded a mean rate estimate twice as high as under a relaxed clock and, consequently underestimated all divergence times by about a factor two (data not shown).
Woelk et al. [47] suggested that the reduced positive selection in vector borne RNA viruses, results from three possible trade-offs associated with the life cycle carried in both mammalian and The third addresses the differences in immune response in the two host types: Mutations facilitating immune escape or tolerance in the first host might cause the opposite effect in the second. In the present analysis the Envelope gene displays the lowest substitution rate. As it encodes the protein responsible for the induction of protective antibody response in mammals [52] , the reported rate could be explained by the third trade-off mechanism. Accordingly, the other surface-exposed structural proteins do not interact with hosts environment as strongly as the E protein (the M protein is buried under a scaffold of E dimmers and the Capsid is covered by the Envelope) and could accumulate more mutations. We conjecture that the C-gene reaches the highest substitution rates because the Capsid is not directly involved in the replication or in the mounting of an anti-viral immune response. Rate differences for nonstructural proteins could in turn be explained by the first and second trade-offs. Finally, it has been proposed that rate of replication governs the long-term substitution rate; for instance in dsDNA viruses very high replication rates may inflate the observed substitution rate [53, 54] . Within tick-borne arboviruses, the tempo of replication is the compound of phases of high replication rates following mammalian infection and phases of low to very low rates in the arthropod environment, with the phase transition commanded by a putative termo sensitive ribo-switch [55] . It is unclear how this rate shift would impact our estimate of a global rate and, as opposed to dsDNA viruses, whether the long periods of latency could deflate the observed rates.
The core idea of Bayesian approaches consists in updating our degree of belief in the truth of a hypothesis in light of new pieces of evidence pertaining to it. It is a form of incremental induction wherein the belief at the end of an investigative step is injected as a prior belief for the next step. This new belief will in turn be modified by conditionalizing upon new evidence. In order to reach credibility interval for drawing conclusions about the temporal setting of the RE, we were compelled to apply several informative priors on our final BEAST analysis. In the first step it is beneficial to place a weakly informative prior on the root [56] . This prior obtained from the literature had the effect of concentrating the probability density around its mean so it could be captured by a narrow shaped gamma distribution. In the following steps, formal probability distributions were retrieved from posteriors in the previous step and used as prior assumptions about rates and node antiquity. Although, the overlap of datasets between iterations was kept minimal, our strategy imposes to maintain some sequences across datasets in order to identify the nodes to which the derived prior should be applied. The reduction of the credibility intervals for the date parameters indicates that our approach succeeded to improve the accuracy of the time estimates by combining different lines of information coming from informative data and from the literature.
Our use of relaxed-molecular clocks is the main cause for discrepancies between our estimates and previously published divergence dates. Using a strict clock Zanotto et al. found four to five times younger divergences than those presently reported (Table 5 ) [38] . Due to the rejection of the strict clock model, we argue that our approach provides a better estimate of divergence times given the data at hand, although some notes of caution should be raised. Our molecular dating could be hampered by sequencing errors, especially since sequence variation is low. In addition, the low substitution rates, could lead to inaccurate rate estimations [57] . Indeed, our estimates for individual genes approaches the limit of 1610 25 substitutions/site/year below which the temporal signal for heterochronous sampled virus begins to break down [58] and tend not to converge on the true rate when analyzed with BEAST. On that account, the least reliable time estimates are produced by the shortest alignment, which casts doubts on the tRE lower bound that was derived from 204 nt long sequences from the recombinant region. Therefore, although our dating estimates are more accurate than those relying on a poorly fitted molecular clock, more full-length genomes with a wide temporal sampling are required for a definitive assessment of divergence events in the Tick-borne encephalitis virus.
Our dating locates the tRE after LIV's colonization of the British Isles. Little is known about the modes of Tick-borne encephalitis virus dispersal over long distance. Birds on a longitudinal migrating route have been found to carry infected ticks through Scandinavia [59] . However, phylogenetic analyses have not shown any clear admixture of Northern and Southern strains that would point towards bird distribution. Therefore, livestock importation from central Europe to the UK seems a more likely explanation for the footprint of past W-TBEV presence observed in the LIV genome. It is not clear why W-TBEV strains did not form stable foci in the British Isles; possibly the number of continental strains was too small to find its way from infected sheep to the small rodents that are their natural vertebrate hosts.
The ecology of the tick vector, which feeds only occasionally and is relatively immobile, the rarity of infected ticks, implying that the probability of multiple strains co-infecting the same tick must be low, the short mammalian viraemia and high mortality rate, are all plausible factors that would explain that no recombination has hitherto been reported in TBEV [14] . For recombination to occur, Table 4 . Times of origin (in years before 2008) for selected clades in the phylogenetic tree of Tick-borne encephalitis virus, obtained from the BEAST inference 4 based on a large Esequences dataset. a vector can become infected with multiple strains during cofeeding in close proximity on the host skin with other ticks carrying different strains. Co-infection is then mediated via the tick saliva [60] . Alternatively, ticks can engage in multiple feeding on viraemic hosts that have been previously infected with different strains [14] . For both situations, sheep are an ideal milieu for recombination to occur when they are fed upon by several vectors carrying both W-TBEV and LIV strains. Indeed, unlike TBEV, LIV can induce a high-titer viraemia in sheep which enables tick re-infection during bloodsucking [8, 11] .
Given the high similarity between strains within a sub-type, recombinant sequences in Tick-borne encephalitis virus species can probably only be detected between sub-types. Dating recombination events is challenging, due to high sequence similarity, low substation rate and condensed temporal sampling. In order to refine this analysis, additional full-length genomes of LIV strains are necessary. Now that the recombining fragment has been identified, it can readily be researched in LIV genomes. Finally, although sequencing the E-gene in order identify strains is a standard practice, the low substitution rate observed in this gene does not supply enough information for robust phylogenetic/ phylogeographic studies. We would therefore recommend to sequence, together with E, a faster evolving marker such as the Capsid-gene.
Figures S1 a-b RDP3 analyses results. The x axis shows genome length in nucleotides, numbered form the start of ORFs after alignment with Neudoerfl (U27495) as reference. The y axis represents the metric used by each method for detecting recombination. Detected recombination signals appear as colored rectangles.
(TIF)  Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1 BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.
Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.
To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .
LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .
The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.
Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).
DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).
Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).
HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .
The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.
The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.
To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.
The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.
To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.
The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC.
Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. 
In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.
Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.
We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.
For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.
The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC. Design and management of an orthopaedic bone bank in the Netherlands The design and management of an orthopaedic bone bank is a complex process in which medical organisation and legislation intertwine. Neither in the Netherlands, nor in any other European country, there are official guidelines for the organisation and management of an orthopaedic bone bank. In the Netherlands, the recently modified ‘law of security and quality for using human materials’ (WVKL) dictates requirements for technical and organisational aspects for the use of human tissue and cells. The bone bank procedures include a thorough questionnaire for donor selection, extensive serological, bacteriological and histopathological examination, as well as standard procedures for registration, processing, preservation, storage and distribution of bone allografts. This article describes the organisation of an accredited bone bank and can be used as a proposition for an official guideline or can be useful as an example for other orthopaedic bone banks in Europe. For reconstruction of bone defects, donor bone from orthopaedic bone banks is often necessary. Bone grafts are used in bone defects that arise from trauma (Friedlaender 1987) , infection, resection of bone tumours or it is used in spinal fusion (Raizman et al. 2009 ) and as impaction grafting in revision of total joint arthroplasty (Slooff et al. 1996) .
Autologous bone is preferred because of its osteoconductive and osteoinducive activity, but it is often not sufficiently available and it repeatedly involves donor site morbidity (Summers and Eisenstein 1989) . Allogenic bone exclusively has osteoconductive activity; it serves as a frame against which newly formed bone gets deposited (Elves and Pratt 1975; Urist 1953) . Allogenic bone is provided by an orthopaedic bone bank.
In Leiden, the Netherlands, the Dutch Bone Bank Foundation (NBF) was founded in 1988 (Veen et al. 1990) . In this central bone bank, bone-and tendon transplant material of deceased donor patients is stored (Veen et al. 1991) . When needed, hospitals may order such material from the NBF.
A number of hospitals manage their own bone banks, such as the VU university medical center in Amsterdam, where an orthopaedic bone bank has been established. This VUmc orthopaedic bone bank contains only femoral heads of suitable patients who underwent total hip replacement surgery. The advantage of possessing a bone bank is that the hospital always has its own supply of donor bone material; this may also be a financially viable strategy for hospitals carrying out many procedures for which donor bone material is required.
Up to now, nationally recognized guidelines for maintenance and management of bone banks do not yet exist in the Netherlands. In this paper we describe the VUmc orthopaedic bone bank procedure, which recently gained official approval and recognition and could serve as a potential outline for other hospitals.
From October 2008, the Ministry of Health, Welfare and Sport (VWS) officially recognized the orthopaedic bone bank of the VUmc (Inspectie voor de Gezondheidszorg 2008). A biannual inspection is performed by the Health Care Inspectorate (IGZ) as a requirement to maintain this recognition. The bone bank procedure has to meet the requirements of the adjusted 'law of security and quality for using human materials' (Wet Veiligheid en Kwaliteit Lichaamsmateriaal 2003) . This law became effective from mid-2007 in the Netherlands as a result of European guidelines 2004/23/EC and 2006/86/EC. These guidelines state the technical requirements for coding, processing, preserving, storing, and distributing of human tissue and cells. Human tissue should be traceable, and serious side effects and incidents with human tissue and cells should be reported.
The procedure of our orthopaedic bone bank is based on guidelines of The American Association of Tissue Banks (AATB), and the criteria of the Council for Blood Transfusion of the Netherlands Red Cross (Richtlijn Bloedtransfusie 2004), together with the recently merged Netherlands Bone Bank Foundation (NBF) and Bio Implant Services (BIS) (NBF-BIS Foundation 2010).
Previously, we followed the guidelines of the European Association of Musculoskeletal Transplantation. As a result of diverging European legislations, this organization has been discontinued as an European umbrella organization; currently only national associations prevail. To date, the Netherlands has not possessed such an association; consequently there is no national guideline with regards to maintenance and management of an orthopaedic bone bank.
The bone bank procedure should be carefully described in an extensive protocol consisting of the following five components: organization, donor selection, documentation, storage and processing, and implementation. The HOD (Head of Department) of the Department of Orthopaedics and the bone bank administrator compose this protocol.
In an organization chart we describe the responsibilities of different stakeholders: the HOD of the Department of Orthopaedics, a bone bank administrator, a theatre nurse, a medical microbiologist, an anatomic pathologist, a clinical chemical analyst, a haematological laboratory technician, and a trainer. The HOD is the main responsible of the bone bank, whereas the bone bank administrator is responsible for the daily management. The knowledge and skills concerning surgical techniques and clinical hygiene are guaranteed by the orthopaedic surgeon and theatre nurse. The bone bank administrators' responsibilities include administration as well as storage and allocation of donor bone. Additionally, the administrator takes care of the maintenance and cleaning of the storage facilities (freezers, etc.), and verifies the registration forms of femoral heads meeting the requirements for storage in the bone bank. Both the bone bank administrator and the trainer are responsible for training of bone bank employees.
Apart from an orientation module for new employees, the training program consists of regular refresher courses for all members of the staff, in order to keep the knowledge of the procedures updated.
Preceding the hip replacement procedure, the attending orthopaedic surgeon requests the patient for his permission to store any removed tissue for donation. It concerns patients whose femoral head grafts will be retrieved in order to be replaced by a total hip prosthesis. Corticospongious bone tissue can not be sufficiently obtained in knee or shoulder arthroplasty; therefore patients undergoing such procedures cannot be taken into consideration for donor bone tissue donation.
The attending orthopaedic surgeon informs the patient both orally and in written. In case the patient grants permission he or she signs the consent forms, and fills out a standard survey (see Table 1 ). The orthopaedic surgeon now decides whether the patient is suitable for being a donor; he uses general and specific exclusion criteria (see Tables 2, 3 ). All criteria must be met; if not, exclusion necessarily follows. The orthopaedic surgeon examines the patient thoroughly: blood samples are collected to determine blood type, Rh-factor and erythrocyte sedimentation rate (ESR) ( Tables 4, 5) .
During surgery, bacterial culture swab samples from hip ligament are collected and a biopsy of corticospongious bone is sent off for histopathological analysis. Serological screening for infectious diseases is performed 6 months after surgery. Once all requirements are met (Tables 1, 2 In the past 3 months, did you have any vaccination or inoculation, or have you been injected with narcotic drugs?
In the past 6 months, did you have a malaria attack or did you use anti-malarial medication?
Have you ever been infected with a sexually transmitted disease?
Have you ever been diagnosed with jaundice or liver illness?
In the past 6 months, have you been in contact with patients diagnosed with jaundice/hepatitis? Are you a sexual partner of an individual for which any of the abovementioned questions can be answered with 'yes'?
Have you been actively involved in prostitution after 1977, or have you been a sexual partner of a person involved in prostitution in the past 6 months?
Have you ever been diagnosed with a haematological disease or any malignant disorder?
Have you ever been treated for diabetes mellitus?
Have you ever been treated for chronic brain-or neurological diseases?
Have you ever received radiation therapy?
Have you ever been diagnosed with rheumatoid arthritis?
Have you ever been diagnosed with tuberculosis? Have you ever been diagnosed with any disease, other than the abovementioned?
Have you ever received hormonal treatment?
Do you use any prescribed medication?
Have you ever used any narcotic drugs?
Have you recently been exposed to hazardous or toxic materials? If yes, please specify.
What is your alcohol consumption per week?
Have you recently been in surgery? If so, when? Did you receive blood from a blood transfusion?
In the past 14 days, have you been traveling through or staying in a region exposed to a SARS epidemic, or have you been in contact with patients infected with SARS?
In the past 6 months, have you tattooed yourself or did you get a piercing?
Cell Tissue Bank (2012) 13:63-69 Documentation Accurate documentation and coding are a necessity for a well functioning bone bank. A unique registration code is allocated to each femoral head. Only the bone bank administrator is able to trace the donor based on this code. Of every registered femoral head, a file, containing the consent forms and results of ESR, bacteriological and histopathological examination, is kept updated. Other relevant data, such as the size of the femoral head and the allocation date are also documented and stored in this file. When the file is completed (which takes at least 6 months due to the serological examination), and no abnormalities are recorded, both bone bank administrator and the responsible orthopaedic surgeon sign the forms. The femoral head is now available for transplantation. In case a file cannot be completed in full, or any Individuals who stayed in a SARS epidemic area or individuals who had face-to-face contact with a SARS patient abnormal values are recorded, the femoral head will be destroyed according to hospitals' protocol.
The femoral head is surgically removed under sterilized conditions. The ligament and synovial tissue are cultured on aerobic and anaerobic bacteria. In order to exclude malignancies, auto-immune processes, or infections, a biopsy of 1 cm 3 corticospongious bone and ligament is collected for histopathological examination. After determining its size, the femoral head is wrapped in a sterile plastic bag and in three layers parcelled in sterile packing material, labelled and stored in the freezer within 30 min. The freezer has a temperature of -80°C, and has a continuous temperature registration device installed. Should the temperature fall outside the acceptable range of -90 and -70°C, an alarm system gives off a warning signal to the Technical Service, guaranteeing a 24-h security against temperature-induced damage to the tissue. A nitrogen tank is fitted onto the freezer, as a backup cooling mechanism in case of mechanical breakdown of the freezer. In deep frozen condition, the allogenic bone tissue can be preserved for a maximum of 5 years. The temperature data is stored and managed by the bone bank administrator for a period of at least 5 years.
If during surgery a surgeon decides to use a femoral head as an allograft, a femoral head from the freezer together with its documents are handed over to the orthopaedic surgeon and surgery team. The orthopaedic surgeon and theatre nurse verify the file and expiration date of the femoral head. The femoral head is thawed in physiological saline; after being defrosted the theatre nurse takes a bacterial culture swab.
The hospital or care institution warrant fulfilment of the traceability requirements, which implies storing the file of the femoral head and records of the receiving patient for 30 years post implantation.
Macewen first describes the use of allogenic human bone tissue in 1881 (Macewen 1881). From that year onwards, the use of allogenic bone transplantation has been increasingly applied and is nowadays a standard orthopaedic procedure (Tomford et al. 1987) . However, much has changed in the past decade: donor selection, clinical hygiene, storage and processing, allocation, implantation, and documentation are bound to strict rules. Donor selection takes place by a thorough broad survey and supplementary physical examination. The survey should be regularly revised to comprise the latest knowledge and developments, and as a response to the spread of new infectious diseases. For instance, after the outbreak of the SARS epidemic in certain countries, a question was added to the survey; prospective donors were asked whether they had been in SARS-infected regions or whether they had been in contact with an infected person. It is not unthinkable that newly arising infectious diseases will be included in the survey and henceforth become exclusion criteria. Laboratory examination is performed before surgery, including ESR determination. Elevated values are often encountered, often without clinical implications. However, the exclusion criteria are strictly enforced. Preoperatively, blood type and Rh-factor are determined. The Rh-factor only becomes an issue if the receiving patient is a young woman. Additionally, the donor is serologically examined, to exclude possible transmission of infections, such as HIV and hepatitis (Strong et al. 1996; Shutkin 1954; Patel and Trampuz 2004; Karcher 1997) . The serological tests are carried out at least 6 months after surgery to avoid type II error (false negative): if the patient was infected at the time of surgery, the test will provide conclusive evidence of this. Normally, the donor will not be informed of the results, unless specifically requested by the donor.
In addition to existing national and international guidelines and procedures of the AATB, NBF-BIS, and former EAMST, histopathological examination is added to the current bone bank procedure. Previous studies have found pathological abnormalities in 8%, and B-cell lymphomas in 2,2% of donor femoral heads (Palmer et al. 1999; Zwitser et al. 2009; Sugihara et al. 1999) . Though transmission of malign cells following transplantation has never been shown, femoral heads with histopathological abnormalities are excluded from allogenic bone transplantation.
Apart from an orthopaedic bone bank, many hospitals often have more organ banks, such as a haematological bone marrow bank for stem cell transplantation or the IVF laboratory of the department of Gynaecology and Obstetrics. Therefore, in every hospital a central system for tissue vigilance should be established and all departments that require cells and/or tissue for transplantation purposes should participate. Thus, highest cell and tissue security levels within the hospital are warranted by controlling the complete transplantation chain from donation to transplantation, including appropriate reporting and follow-up of incidents and side effects.
The design and management of an orthopaedic bone bank as an organ bank is a very complex process in which aspects of medical organization and legislation intertwine. In this paper we describe our bone bank procedure, which is approved and recognized by the Ministry of Health, Welfare and Sport (VWS) of the Netherlands. This procedure could serve as a provisional Dutch protocol for the setup and maintenance of other orthopaedic bone banks. Dectin-1 and DC-SIGN Polymorphisms Associated with Invasive Pulmonary Aspergillosis Infection The recognition of pathogen-derived structures by C-type lectins and the chemotactic activity mediated by the CCL2/CCR2 axis are critical steps in determining the host immune response to fungi. The present study was designed to investigate whether the presence of single nucleotide polymorphisms (SNPs) within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes influence the risk of developing Invasive Pulmonary Aspergillosis (IPA). Twenty-seven SNPs were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable IPA according to the 2008 EORTC/MSG criteria. Association analysis revealed that carriers of the Dectin-1 (rs3901533 T/T) and Dectin-1 (rs7309123 G/G) genotypes and DC-SIGN (rs4804800 G), DC-SIGN (rs11465384 T), DC-SIGN (7248637 A) and DC-SIGN (7252229 C) alleles had a significantly increased risk of IPA infection (OR = 5.59 95%CI 1.37–22.77; OR = 4.91 95%CI 1.52–15.89; OR = 2.75 95%CI 1.27–5.95; OR = 2.70 95%CI 1.24–5.90; OR = 2.39 95%CI 1.09–5.22 and OR = 2.05 95%CI 1.00–4.22, respectively). There was also a significantly increased frequency of galactomannan positivity among patients carrying the Dectin-1 (rs3901533_T) allele and Dectin-1 (rs7309123_G/G) genotype. In addition, healthy individuals with this latter genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers, suggesting a role of the Dectin-1 (rs7309123) polymorphism in determining the levels of Dectin-1 and, consequently, the level of susceptibility to IPA infection. SNP-SNP interaction (epistasis) analysis revealed significant interactions models including SNPs in Dectin-1, Dectin-2, CCL2 and CCR2 genes, with synergistic genetic effects. Although these results need to be further validated in larger cohorts, they suggest that Dectin-1, DC-SIGN, Dectin-2, CCL2 and CCR2 genetic variants influence the risk of IPA infection and might be useful in developing a risk-adapted prophylaxis. Invasive Pulmonary Aspergillosis (IPA) is a life-threatening infection caused mainly by Aspergillus fumigatus, an opportunistic fungal pathogen that frequently colonizes respiratory tracts and rapidly spreads to blood vessels and tissues [1] . The incidence of IPA infection has increased in the last years due to the use of immunosuppressive and immunomodulatory drugs and is still causing significant morbidity and mortality worldwide, especially in immunosuppressed and hematopoietic stem cell transplantation (HSCT) patients [2] .
Early recognition of A. fumigatus by myeloid leukocytes is a crucial for down-stream immune response and conidia clearance and, therefore, in the development and progression of IPA infection [3] . Myeloid leukocytes are activated by patternrecognition receptors (PRRs), which directly recognize fungal cell wall structures and pathogen-associated molecular patterns (PAMPs) [4] . Among the PRRs expressed in neutrophils, pulmonary macrophages and dendritic cells, C-type lectin family members such as DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), Dectin-1 (Dendritic cell-associated C-type lectin-1) and Dectin-2 (Dendritic cellassociated C-type lectin-2) seem to be important in the effectiveness of innate immune response against A. fumigatus [5, 6] . Recent studies have reported that the C-type lectins mediate A. Fumigatus recognition, capture and internalization [7] and that their binding to fungal cell wall carbohydrates is highly specific and selective [8] . DC-SIGN and Dectin-1 recognize b-(1-3)-glucans and galactomannans (GM) in the cell wall of A. fumigatus [7, 9] while Dectin-2 interacts with a-mannans [10] . However, numerous studies have shown that C-type lectins are not only involved in the recognition of fungal pathogens but also in the induction of antifungal Th1 and Th17 immune responses [5, 11] .
Although the mechanisms underlying DC-SIGN-mediated immune response are still highly speculative, it has been suggested that DC-SIGN promotes dendritic cell (DC) migration and T-cell activation through the ICAM-3 binding [12, 13] and modulates TLR signalling by targeting Raf-1, which regulates NFkB p65 activation and, consequently the production of pro-inflammatory cytokines [14] . Likewise, Dectin-1 and Dectin-2 induce Sykdependent canonical and noncanonical NFkB pathways [15, 16] promoting the production of some pro-inflammatory cytokines (IL1a, IL1b, IL12 and IL8), chemokines (CCL2/MCP-1, CCL3/ MIP-1, CXCL2/MIP-2 and CXCL10) [17] and the IL17-mediated neutrophil recruitment [18] . It has also been described that Dectin-1 and Dectin-2 are able to work in collaboration with TLRs (mainly TLR2, TLR4 and TLR6) modulating immune responses through the production of IL6, IL12p70 and TNF [3, 16] . These findings support the hypothesis that C-type lectins, cytokines or chemokines are important mediators during infection with A. fumigatus.
Several polymorphisms in human PRRs [19] [20] [21] [22] [23] [24] as well as in some cytokines [25] [26] [27] , chemokines [28] and their receptors [29, 30] have hitherto been associated with an increased risk of invasive aspergillosis in susceptible hosts. Based on these observations, the objective of the present study was to investigate the role of tagging and potentially functional single-nucleotide polymorphisms (SNPs) located within the DC-SIGN, Dectin-1, Dectin-2, MCP-1/CCL2 and CCR2 genes on IPA susceptibility.
All participants enrolled were Caucasian and recruited in the University Hospital Virgen de las Nieves (Granada, Spain) or in the University Hospital of Salamanca (Salamanca, Spain). All determinations and genetic analyses in hematological patients were performed with fully informed written consent, and anonymity of the data was guaranteed. The study protocol was approved by the Ethical Review Committee of Virgen de las Nieves University Hospital, Granada, Spain. The population included 182 hematological patients recruited between January 2004 and January 2011. All hematological patients in this study received a prolonged chemotherapy treatment or underwent HSCT and were therefore considered susceptible to develop IPA infection. Demographic information and clinical data were obtained by detailed review of hospital records. Data were gathered on: site of infection; host factor criteria (severe neutropenia for .10 days, persistent fever for .96 h refractory to appropriate broad-spectrum antibacterial treatment, signs and symptoms indicating graft-versus-host disease [GVHD], corticoid therapy [.0.3 mg/kg per day], and invasive fungal infection during a previous episode of neutropenia), microbiological criteria (positive result for Aspergillus antigen in $2 consecutive blood samples when considering an index of 0.5 or in only 1 sample when the index was higher than 0.8), and clinical criteria of lower respiratory tract infection [major criteria: any of the following new infiltrates on computed tomography (CT) imaging: halo sign, aircrescent sign, or cavity within area of consolidation; minor criteria: cough, thoracic pain, hemoptysis, pathologic pulmonary sound, and radiological evidence suggestive of invasive infection]. Laboratory data were also recorded. Proven and probable IPA was diagnosed based on the updated criteria (2008) reported by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group (EORTC/ IFICG) [31] .
Serum GM detection has been shown to be a useful test for the early diagnosis and follow-up of IPA and is now included in IPA diagnosis criteria [31] . In the present study, serum GM antigen was determined twice weekly during the hospital stay and at each outpatient visit until the end of their immunosuppressant or chemotherapeutic treatment. Serum GM concentrations were determined by Platelia Aspergillus ELISA kit (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer's instructions. This commercial kit has proven to offer good sensitivity to detect GM [32] , and GM concentration was found to correlate with the fungal tissue burden [33, 34] . A test sample was classified as positive when the optical density ratio was $0.5 in two consecutive positive samples or .0.8 in a unique serum sample. A careful review of concomitant treatments (piperacillin-tazobactam or amoxicillin-clavulonic acid) in each patient was necessary to detect possible false-positive GM determinations. Likewise, tests were performed on the same day to avoid sample contamination and to ensure accuracy of results.
Twenty-seven tagging/functional SNPs within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 were selected to genotype the entire panel of individuals ( Table 1 ). The aim of the SNP tagging was to identify a set of SNPs that efficiently tags all the known SNPs while the functional approach was used to determine the net impact of potentially functional variants within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes on IPA risk. Tagging SNPs were selected using Haploview Tagger program (http://www.broad.mit.edu/ mpg/haploview/; http://www.broad.mit.edu/mpg/tagger/) and a pairwise tagging with a minimum r2 of 0.8. In this selection we forced the inclusion of the DC-SIGN rs4804803 , CCL2 rs1024610 and CCL2 rs1024611 polymorphisms as their functionality has been suggested [35] [36] [37] . Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) Qiagen Mini Kit (Qiagen, Valencia, CA, USA). Genotyping of DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 polymorphisms was carried out using KASPar assays (KBiosciences, Hoddesdon, Hertfordshire, UK) in a 384well plate format (Applied Biosystems, Foster City, CA, USA) where hematological patient samples were randomly distributed. KASPar reactions were performed using KASPar assay mix (containing probes) and KASPar kit containing 26 Reaction Mix and MgCl2 (50 mM). PCR conditions were: denaturation at 94uC for 15 min, 10 cycles of denaturation at 94uC for 10 sec, annealing at 57uC for 5 sec and elongation at 72uC for 10 sec and 20 cycles of denaturation at 94uC for 10 sec, annealing at 57uC for 20 sec and elongation at 72uC for 40 sec. Recycling conditions were 94uC for 10 sec, annealing and elongation at 60uC for 60 sec. PCR products were analyzed with the ABI Prism 7900HT detection system using the SDS 2.4 software (Applied Biosystems). For internal quality control, about 5% of samples were randomly selected and included as duplicate. Concordance between the original and the duplicate samples for the 27 SNPs analyzed was $99.5%. Call rates for all SNPs were $97.8% with the exception of the Dectin-1 rs11053599 SNP with a call rate of 94.5%.
The Hardy-Weinberg Equilibrium (HWE) tests were performed in the control group (non-IPA patients) by a standard observedexpected chi-square (x 2 ) test at each polymorphic site (http://ihg2. helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Unconditional logistic regression was used to assess the main effects of the genetic polymorphisms on IPA risk using co-dominant, dominant and recessive inheritance models. For each SNP, the more common allele in the control group was assigned as the reference category. All analyses were adjusted for age, gender, hematological malignancy and established risk factors for IPA infection (HSCT, neutropenia, GVHD and corticoid therapy use) and were conducted using the statistical software SSPS (version 14.0, SPSS Inc., Chicago, USA). All tests were considered to be statistically significant with a p value of ,0.05. Adjustment for multiple testing was carried out following a conservative threshold for statistical significance, based on a revised version of the Bonferroni method: we calculated for each gene the ''number of effective independent variables'' (M eff ) by use of the SNP Spectral Decomposition approach (http://gump.qimr. edu.au/general/daleN/SNPSpDsuperlite/) [38] . We obtained a study-wise M eff value by adding up the gene M eff 's. SNPtool (http://www.dkfz.de/de/molgen_epidemiology/tools/ SNPtool.html) [39] and the Haploview v4.2 software were used for LD blocks reconstruction and haplotype association statistics. Block structures were determined according to the method of Gabriel et al. [40] .
We used the Web-based tool FastSNP [41] available at http:// fastsnp.ibms.sinica.edu.tw for predicting the functional significance of the SNPs associated with IPA infection. FastSNP utilizes information from another online program PolyPhen (http://www. bork.embl-heidelberg.de/PolyPhen/) and from four different web resources (TFSearch, ESEfinder, Rescue-ESE and FAS-ESS) to determine whether SNPs are located at exonic splicing regulatory sites, cause a non-conservative amino acid change or whether they alter the transcription factor-binding site of a gene (for instance, acting as intronic enhancer). The score was given by this tool on the basis of levels of risk with a ranking of 0 (no effect), 1 (very low), 2 (low), 3 (medium), 4 (high), or 5 (very high).
Whole blood samples from 21 healthy donors were collected into PAXGENE RNA tubes and stored at 280uC until use. Total RNAs were extracted using a PAXGENE Blood RNA Isolation Kit (PreAnalytiX) and reverse transcribed to cDNA using QuantiTect Reverse Transcription Kit (catalog number: 205311; Qiagen). Real-time quantitative PCR was carried out using an ABI PRISMH 7500 HT Sequence Detection System (Applied Biosystems) according to manufacturer's instructions. Briefly, 2 ml of the cDNA were loaded in a PCR reaction containing 12.5 ml of 26 QuantiTect SYBR Green PCR Master Mix with an appropriate concentration of MgCl 2 , 2.5 ml of primers (Hs_CLE-C7A_1_SG QuantiTect Primer Assay, catalog number: QT00024059; Geneglobe, Qiagen) and 8 ml of RNase-free water. PCR cycling conditions were as follows: 95uC for 15 minutes, followed by 40 cycles of denaturation at 95uC for 15 seconds combined with annealing at 60uC for 30 seconds and extension at 72uC for 30 seconds. All samples were run in duplicate. Relative quantification of Dectin-1 mRNA expression was calculated with the 2 2DDCt method. We obtained the fold changes in gene expression normalized to an internal control gene (GAPDH, Hs_GAPDH_2_SG QuantiTect Primer Assay, Catalog number: QT01192646; Qiagen) and relative to one calibrator (First- 
We also analyzed high-order interactions between SNPs using the multifactor dimensionality reduction (MDR) constructive induction algorithm. A detailed explanation on the MDR method has been described elsewhere [42, 43] . SNPs in LD (cut-off of r 2 ,0.8) were excluded from the MDR analysis. Cross-validation and permutation testing were used to identify the best models. All possible two-, three-and four-way SNP interactions were tested using 100-fold cross-validation and the exhaustive search. The model with the highest testing balanced accuracy (TA) and cross validation consistency (CVC) was selected as ''best model''. Statistical significance was evaluated using a 1.000-fold permutation test to compare observed testing balanced accuracies with those expected under the null hypothesis of no association (using the MDR permutation testing module 0.4.9 alpha). MDR results were considered statistically significant at the 0.05 level. Finally, interactions were visualized by constructing an interaction dendrogram according to the method described by Moore et al [43] . MDR software and MDR permutation testing module are open-source and freely available from http://www.epistasis.org.
Population characteristics are described in Table 2 . Compared to non-IPA, IPA cases were more likely to have cough and pathologic pulmonary sound (p,0.001 and 0.011, respectively) and presented more often pathological chest radiographies and CT scans (p,0.001). Established risk factors for IPA infection such as HSCT, neutropenia, GVHD and corticoid therapy use were homogenously distributed between IPA and non-IPA patient groups.
Fifty-seven patients were diagnosed with proven or probable IPA infection according to the revised EORTC/MSG criteria (2008). The association of risk of IPA infection with the individual 27 SNPs in the C-type lectin and chemokine genes is shown in the Table S1 . All analyzed polymorphisms fulfilled Hardy-Weinberg expectations for the control group (non-IPA patients). Several polymorphisms were found to be associated with IPA infection ( Table 3) Table 3 and Table S1 ). After correction for multiple testing using SNPSpD (number of independent marker loci, 21; p = 0.05/21 = 0.002), none of the SNPs retained significance although Dectin-1 rs7309123 showed significance when the carrier status was analyzed (CC+CG vs. GG, P = 0.001; Table 3 ).
In order to assess the degree to which the selected SNPs and the positivity of GM correlated, we also performed lineal regression analysis. In the whole population, 3784 assays were performed in duplicate and 531 were considered as positive (14.03%). We found a significantly higher percentage of positive GM among patients carrying the Dectin-1 rs3901533_T allele and among those patients bearing the Dectin-1 rs7309123_G/G genotype suggesting a role of these polymorphisms in determining a defective recognition and clearance of Aspergillus conidia (Figure 1 ). No correlation was observed for the polymorphisms within DC-SIGN that were associated with the infection.
We subsequently assessed whether SNPs associated with IPA infection showed capacity to change putative transcription factor binding sites using FastSNP. The predictive functional analysis suggested an intronic enhancer function for the Dectin-1 rs7309123 SNP due to its location in transcription factor Cdxa (caudal type homeo-box transcription factor 1) binding site (GAAAGAC; score 1-2). These data suggest a central role of the rs7309123 in the susceptibility to IPA infection. None of the remaining SNPs associated with IPA infection was predicted to affect transcription factor binding sites or splicing or to introduce a damaging amino acid change.
To explore the potential consequences of Dectin-1 rs7309123 SNP, Dectin-1 mRNA expression was measured in 21 healthy donors and was correlated with genotypes. Our results showed that subjects harbouring GG genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers (p,0.001; Figure 2 ). These results further supported our hypothesis suggesting that Dectin-1 rs7309123_G allele may disrupt binding sites for potential transcription factors. The MDR analysis of the SNPs tested revealed statistically significant interactions. Two SNPs (rs1024611 and rs4264222) were excluded from the MDR analysis as they were in LD with other SNPs using a cut-off value of r 2 ,0.80 in our sample set. The best interaction models selected by the TuRF filter algorithm along with its testing accuracy and cross-validation consistency are shown in Table 4 . The overall best model with the highest crossvalidation consistency (CVC) consisted of a model that included the CCL2 rs4586 , Dectin-1 rs3901533 , CCR2 rs3918358 and Dectin-2 rs7134303 SNPs. This model had a significant testing accuracy of 0.7735 (permutation p,0.001) and a cross-validation consistency of 100/ 100. Of note is that two SNPs showing genetic interaction in this model were not significantly associated with an increased risk of IPA infection in the univariate analysis (CCR2 rs3918358 and Dectin-2 rs7134303 ). The best two-locus model consisted of the Dectin-1 rs3901533 and DC-SIGN rs4804800 SNPs, two variants that showed also association in the single-locus analysis. This model had a testing accuracy of 0.6409 and a cross-validation consistency of 76/100. This model was not any more significant after 1.000-fold permutation testing. However, the entropy based information gain calculated for this pair of SNPs indicated strong synergy, which may be interpreted as the two SNPs acting together to increase the risk of IPA infection. The best three-locus model included the CCL2 rs4586 , Dectin-1 rs3901533 and Dectin-2 rs7134303 SNPs. In this model, testing accuracy raised to 0.7085 (permutation p = 0.025) whereas the cross-validation consistency was of 68/100. Figure 3 illustrates an interaction dendogram that summarizes the estimates of interactions.
The marked differences in susceptibility to IPA infection among hematological patients (with or without allo-HSCT) suggest that the effective immune response against Aspergillus is determined by both environmental and host genetic factors [44, 45] . Studies on genetic polymorphisms in genes coding for components of the innate immunity have supported this hypothesis [19, [23] [24] [25] [26] [27] [28] [29] [30] 46] .
In this report, we studied the influence of the tagging and potentially functional polymorphisms of DC-SIGN, Dectin-1, Dectin-2, CCL2/MCP-1 and CCR2 in susceptibility to IPA infection in a Spanish population. Polymorphisms in these genes have been reported to influence a number of infectious diseases including HIV-1 [47] [48] [49] , HTLV-1 [50] , CMV [51] , Tuberculosis [52] [53] [54] [55] , HCV [56, 57] , HBV [58] , Dengue [35] and SARS [59] among others, revealing their potential role in host defense against pathogens.
We found that polymorphisms in Dectin-1 (rs3901533 and rs7309123) and DC-SIGN (rs4804800, rs11465384, rs7248637 and rs7252229) were associated with an increased risk to develop IPA infection, which points towards their critical involvement in the pathogenesis of this invasive fungal infection. The highest risk of IPA infection was found for carriers of the Dectin-1 rs3901533_T/T and Dectin-1 rs7309123_G/G genotypes and the DC-SIGN rs4804800_G allele. Patients carrying these genotypes/alleles had from 2 to 6 times increased risk of IPA infection. Additionally, patients carrying the DC-SIGN rs11465384_T , DC-SIGN 7248637_A and DC-SIGN rs7252229_C alleles showed a 2-fold increased risk in comparison with patients harboring the wild-type allele. Although it was not statistically significant, we also found that the DC-SIGN rs2287886 SNP may be associated with a reduced risk of IPA infection. Interestingly, this latter result was in agreement with our previously reported findings using the former EORTC/MSG classification criteria, 2005 [60] . The apparent effect of these SNPs on IPA susceptibility persisted even after adjustment for age, gender, hematological malignancy and several known risk factors (HSCT, neutropenia, GVHD and corticoid therapy use), indicating that Dectin-1 and DC-SIGN variants contribute independently to the risk of infection.
Another interesting finding of this study was the significantly greater positive GM percentage of patients carrying the Dectin-1 rs3901533_T allele than those with the wild-type allele. Additionally, patients harboring the G/G genotype for the Dectin-1 rs7903123 SNP showed an increased percentage of positive GM compared to those carrying the C allele (C/C+C/G). No differences were found when positive GM determinations were correlated with DC-SIGN polymorphisms. These data along with the remarkable degree of association of Dectin-1 and DC-SIGN variants with risk of IPA infection provides a compelling evidence for a critical role for these PRRs in immune response to IPA infection. In this regard, numerous studies have shown that Dectin-1 and DC-SIGN are not only involved in the recognition of fungal pathogens but also in the induction of anti-fungal Th1 and Th17 immune responses [5, 11] .
Mezger et al. also demonstrated that Dectin-1 is involved in the induction of several pro-inflamatory cytokines, chemokines and immune receptors [18] while Werner et al. showed that Dectin-1 is also regulating Th17-mediated immune response in the lungs [61] . Furthermore, Dennehy and Brown suggested a role of Dectin-1 mediating its own signaling, as well as synergizing with TLRs to trigger NFkB-mediated immune response against fungal pathogens [62] .
Although it is now well recognized that SNPs in genes modulating immune response are likely to be determinants of host susceptibility to fungal infections, so far, little is known regarding the biological significance of these variants. In order to shed light into the potential functionality of Dectin-1 (rs3901533 and rs7309123) and DC-SIGN (rs4804800, rs11465384, rs7248637, rs7252229 and rs2287886) variants, we investigated whether they were involved in disruption of a binding site for critical transcription factors that might influence transcription level of these genes. Our predictive analysis showed that the carriage of the C allele for the Dectin-1 rs7309123 SNP creates a putative binding site for Cdxa, a relatively unknown transcription factor, which might be involved in the control of Dectin-1 gene expression. To assess whether the Dectin-1 rs7309123 polymorphism might be associated with a decreased expression of Dectin-1, we correlated Dectin-1 mRNA expression levels with Dectin-1 rs7309123 genotypes. Interestingly, we observed that individuals harbouring the GG genotype showed a relatively lower expression than those carrying the C allele (CC+GC). These results further supported our hypothesis suggesting that Dectin-1 rs7309123 SNP may have an effect on the Dectin-1-mediated recognition of Aspergillus conidia and subsequent immune responses. However, this predicted change in transcription activity is only suggestive at this stage and will need further validation using in vitro functional assays.
Several lines of evidence point to the relevance of epistatic effects in the etiology of complex diseases but, up to now, no studies have been carried out to analyze the presence of SNP-SNP interactions in IPA infection. For this reason, we decide to assess interactions among genetic polymorphisms within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2, genes and the risk of IPA infection. The MDR approach used in this study was able to determine two multilocus combinations associated with high risk to develop IPA infection. Of the interactions identified, MDR indicated that the type of interaction in the two significant models was synergistic. These results support the hypothesis that multiple SNP-SNP interactions may play a role in determining the risk of IPA infection. This hypothesis is biologically plausible since the immune system would warrant prevention of fungal infection even when some genetic variants were present.
Recent population-based studies have even led to the identification of several SNPs involved in the early recognition of Aspergillus and associated them with an increased risk for invasive fungal infection. It has previously been suggested that SNPs within C-type lectin genes are associated with fungal infections. Platinga et al. (2009) and Cunha et al. (2010) suggested that patients carrying the Y238X (rs16910526) polymorphism in the Dectin-1 gene were more likely to be colonized with Aspergillus and Candida species [19, 63] , compared with those harboring the wild-type allele. However, these results were not replicated in a very recent study [24] . In our study, such findings were neither evidenced even when HSCT and non-HSCT patients were analyzed separately (data not shown). This puzzling finding suggests that, although intronic polymorphisms within Dectin-1 and DC-SIGN are indeed themselves a strong indication that these genes play an important role in the susceptibility to IPA infection, we cannot rule out the Figure 3 . Interaction dendrogram generated by the MDR software. The interaction dendrogram was used to confirm, visualize, and interpret the interaction model. The MDR analysis was performed by using the open-source MDR software package. The colors used depict the degree of synergy, ranging from red (highest information gain) to blue (highest information redundancy). Note that the interaction between Dectin-1 (rs3901533) and DC-SIGN (rs4804800) SNPs showed the highest degree of synergy (gain of information). doi:10.1371/journal.pone.0032273.g003 possibility that these SNPs are part of a bigger haplotype containing important other genetic variants in the neighboring genes. In any case, because all these population-based studies have been conducted using relatively small cohorts, additional studies in larger set of patients are needed to definitely establish the role of these variants in the susceptibility to invasive fungal infection.
In summary, this study provides evidence of association between Dectin-1 and DC-SIGN polymorphisms and the risk of IPA infection. By the inclusion of functional prediction analyses, the correlation of genotypes with positive GM determinations and Dectin-1 mRNA expression levels, the study strongly supported the role of Dectin-1 gene variants in determining susceptibility to IPA infection. Epistatic analyses also suggested the presence of a gene-gene interaction involving Dectin-1 with CCL2 and CCR2 variants to determine IPA infection. Despite all these evidences, additional studies using larger cohorts will be necessary to confirm the effect of these polymorphisms on the susceptibility to IPA infection.
Table S1 Associations of polymorphisms involved in the phagocyte-immune related response against Aspergil-lus. 1 Models adjusted for age, gender, hematological malignancy, HSCT, neutropenia (defined as absolute neutrophil count ,500 cells/mm3 for a period of more than 10 days), GVHD and corticoid therapy use (.0.3 mg/Kg/day). { Assuming a recessive model of inheritance. Abbreviations: OR, odds ratio; CI, confidence interval. Differences in samples numbers are due to failures in genotyping. (DOC)  Lethal Nipah Virus Infection Induces Rapid Overexpression of CXCL10 Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis. Nipah virus (NiV) is a recently emerged zoonotic pathogen of the family Paramyxoviridae that is distinguished by its ability to cause fatal disease in both animals and humans. NiV along with Hendra virus (HeV) comprise the new genus Henipavirus (reviewed in [1] ). NiV was first identified during an outbreak of severe encephalitis in Malaysia and Singapore in 1998-99, with pigs serving as the intermediate amplifying host [2] . Since 1998 NiV has caused regular outbreaks, primarily in Bangladesh and India, with the most recent occurrences at the beginning of 2011 [3] . In the majority of subsequent spillover events, the mortality rate among humans has been higher (,75%) along with evidence of multiple rounds of person-to-person transmission [4, 5, 6] .
The endothelial cells represent one of the major targets of NiV infection, which is characterized by a systemic vasculitis and discrete parenchymal necrosis and inflammation in most organs, particularly in the central nervous system (CNS). The high pathogenicity of NiV infection appears to be primarily due to endothelial damage, syncytia and vasculitis-induced thrombosis, ischemia and vascular microinfarction in the CNS, allowing the virus to overcome the blood-brain-barrier (BBB) and to subsequently infect neurons and glia cells in the brain parenchyma [2, 7] .
Pathogenesis of NiV infection is still poorly understood. As the endothelium forms the primary barrier of the circulatory system, endothelial dysfunction during infection could broadly affect immune cell function by regulating cytokines, chemokines and cell receptors and influencing vascular permeability. To gain new insights into virus-host interaction, we analyzed the transcriptome signature of NiV infection in primary human umbilical vein endothelial cells (HUVECs). Among the ten most strongly upregulated genes 8 h after NiV infection, we identified CXCL10 (interferon gamma-induced protein 10, IP-10), a chemokine that promotes leukocyte trafficking by acting on T lymphocytes, NK and dendritic cells via its receptor CXCR3 [8] . In addition to its protective role during the viral infection, CXCL10 may enhance the severity of virus infection and cause neuronal apoptosis and calcium dysregulation [9, 10] and enhance autoreactive lymphoproliferation and brain injury [11] . We have confirmed secretion of CXCL10 by NiV-infected HUVECs by ELISA and showed that increased production of CXCL10 follows NIV replication in experimentally infected hamsters. Finally, we demonstrated production of CXCL10 in brain endothelial cells of patients with fatal acute NiV encephalitis. Altogether, these results suggest that CXCL10 may be an important regulator of NiV-induced pathogenesis as well as a potential marker for NiV infection.
As endohelial cells are the first target of NiV infection, we have initially studied a permissivity of HUVEC cultures to NiV infection. We established the HUVEC cell culture and assessed their purity at passage 2, either by cell immunostaining or flow cytometry using two endothelial-specific markers, Von Willebrand Factor (VWF) stored in cytoplasmic Weibel-Palade bodies, and CD31 (PECAM-1) localized in homophilic intercellular contacts, which gave us reproducibly satisfactory results (Fig. S1 ). We then analyzed the expression of NiV receptors, production of viral specific RNAs, generation of the cytopatic effect and production of viral particles (Fig. 1) . The expression of mRNA specific for NiV entry receptors ephrinB2 and B3 [12, 13, 14] was readily detected in these cells, at levels close to those in the astroglioma cell line U373, which was also highly permissive to NiV infection [15] (Fig. 1C ). Production of NiV structural genes coding for nucleoprotein, matrix, fusion protein, glycoprotein and polymerase increased rapidly during the infection, following a gradient characteristic of paramyxoviruses, with highest expression of the first gene represented in the genome, N, and lowest for the gene coding for the polymerase (Fig. 1D ). Formation of large syncytia was rapidly detected in HUVECs (Fig. 1B ) and the highest level of the production of infectious particles was obtained 24H after infection, as most of the cells were lysed 48 h p.i. in this cell type (Fig. 1E) . Therefore, NiV infects rapidly HUVECs and induces a significant cytopathic effect.
To better understand virus-host interaction, we have analysed the effect of NiV infection at the level of gene expression in HUVECs, using Codelink microarray (see Materials and Methods). Cells were infected with either NiV or treated with mock preparation and RNA was taken 8 h p.i., to obtain the information about the early changes in NiV-induced gene expression in HUVEC in the conditions when cell viability was still preserved. Among 55.000 analyzed genes, pair-wise comparisons between infected and uninfected samples revealed 807 deregulated genes (fold change cut-off = 1.3). NiV infection down-regulated galectin 3 gene expression, a member of the lectin family, involved in cell adhesion, cell activation and chemoatraction [16] , but most of the other down-regulated genes were not found associated with any known function (table 1) . The 538 up-regulated genes were classified according to their Gene Ontology (GO) biological processes and molecular functions. This analysis revealed that NiV-infection up-regulated 34 genes implicated in the immune response, particularly those associated with the interferon pathway, including MxA, RIGI, MDA, 2959-OAS 1 and 2 ( Fig. 2) . Interestingly, among the top ten up-regulated genes was CXCL10 (interferon gamma-induced protein 10, IP10), an important chemokine secreted by endothelial cells (table 1). These results have been further confirmed by RT-qPCR at 8 and 24 h post-infection for several genes linked to interferon pathway, including MXA, OAS1, IFN beta and CXCL10 (Fig. 3A ). In addition to stimulation of CXCL10, which was observed at different MOI of infection, MOI of 1 as well as 3, the induction of the closely related chemokine CXCL11 (Interferon-inducible Tcell alpha chemoattractant, I-TAC or IP-9), being at 19 th position among up-regulated genes, was also confirmed by RT-qPCR (Fig. 3A) . However, the production of the third closely related chemokine CXCL9 (MIG), which shares the same receptor CXCR3 with CXCL10 and CXCL11, was not detected. Finally, 
As CXCL10 is known to have an important immunobiological function in the organism, which received a great deal of attention in recent years, we focused further studies on this chemokine. Thus, we analyzed whether NiV-infection induces production of CXCL10 in vivo, using the hamster animal model, which closely reproduces human infection and induces lethal outcome starting from day 5 p.i. [17] . Hamsters were infected by NiV and sacrificed on a daily basis for analysis of RNA in different organs (Fig. 4) . The baseline expression of CXCL10 was observed in all organs and increased during the course of infection, particularly in brain and kidney. In lung and spleen the level of CXCL10 initially increased, but decreased on the last day that preceded lethal outcome of the infection. The most rapid and highest induction of CXCL10 mRNA expression was observed in the spleen (16 times more than the baseline expression, already 24 h p.i.), which may correlate with the abundance of cells capable of secreting CXCL10 in the spleen (leukocytes, endothelial cells and splenic stromal cells [18] . The highest NiV replication was observed in lungs of infected hamsters. The level of CXCL10 expression significantly correlated to the level of NiV N expression in analysed organs (p,0.001), suggesting that production of this chemokine closely follows the NIV replication.
We have next performed immunohistochemical analysis of brain tissues from patients that succumbed to NiV-infection during the outbreak in Malaysia in 1999. Widespread vasculitis and perivascular infiltration were regularly detected after hematoxylin (Fig. 5A) . Altogether, these results indicated that NiV-infection activates cellular pathways leading to an increase of CXCL10 expression that may play a role in the pathogenesis of this highly lethal emergent infection.
The inflammatory cellular infiltrates found in the CNS of patients with acute NiV encephalitis include neutrophils, macrophages, lymphocytes and reactive microglia [7] , which may all play an important role in NiV pathogenesis. Recruitment of these cells indicates a possible action of chemokines, known as main regulators of leukocyte trafficking. Our analysis of the transcriptome profile of NiV-infected primary endothelial cells revealed the induction of several genes involved in the interferon type I pathway as well as the overexpression of CXCL10, an important chemoattractant chemokine with proinflammatory activity [19] .
Although expression of IFN alpha and beta was shown to be inhibited in human cell lines after NiV infection [20] , human primary endothelial cells seem to be resistant to this inhibition [21] . Our study demonstrated that a few other members of interferon type I pathway, including MxA, RIGI, 2959-OAS 1 and 2, are highly expressed early after NiV infection, suggesting a functional interferon pathway in NiV-infected human endothelial cells.
CXCL10 is a secreted polypeptide of 10 kDa that was first identified as an early response gene induced after interferon gamma treatment in a variety of cells, and was thus named interferon gamma-inducible peptide, IP-10 [22, 23] . Furthermore, it was shown that interferon alpha could also induce CXCL10 in primary cultured neurons [24] and seminiferous tubules [25] . In addition to interferons, HIV envelope glycoprotein gp120 was shown to induce expression of CXCL10 in brains of mice [26] . HIV Tat protein can also induce CXCL10 in astrocytes [27] . Furthermore, CXCL10 is expressed early in the CNS in response to a wide variety of viruses [11, 13, 28, 29, 30] . Our results demonstrate that NiV-infection induces rapidly production of CXCL10 in primary endothelial cells. As production of interferon gamma by these cells is contested, it is possible that the generation of CXCL10 is either a direct effect of NiV proteins or is induced via NiV-activation of interferon beta, following infection. Moreover, our results demonstrate induction of CXCL10 in vivo in different organs of NiV-infected hamsters, as well as in patients that succumbed to lethal NiV-infection, thus revealing an important association between NiV-infection and CXCL10 production. Although endothelial cells were clearly secreting this chemokine, the perivascular inflammatory cells and some neurons are most probably participating in the production of CXCL10 as well.
Individual chemokines, including CXCL10, play opposing roles in neuroinflammation in different experimental models of infectious disease, making it difficult to predict whether they have a protective role by contributing to immune eradication of the microbial attack or they cause inflammatory damage and disease [19] . Therefore, potential effect of CXCL10 in NiV infection could be either beneficial or harmful and the balance between these two effects may be critical for the final outcome of the disease. Infection with RNA viruses that may cause encephalitis in humans, such as HIV or lymphocytic choriomeningitis viruses, or in rodent models, such as mouse hepatitis virus and Theiler's virus, can directly induce the expression of chemokines by astrocytes and microglia and establish chemokine gradients that promote leukocyte trafficking within the CNS [11, 31, 32, 33] . In addition, neutralization of CXCL10 decreased leukocyte migration to areas of infection in measles virus-infected brain tissue [34] . Although several of these viruses directly infect neurons, these cells have not been observed to participate in the inflammatory response, suggesting the indirect induction of inflammatory response via cytokines. Nevertheless, in a transgenic mouse model of measles virus encephalitis, neuronal expression of CXCL10 was associated with T-cell recruitment, suggesting that neurons may play a role in the induction of immune responses to viral invasion [35] . In recent study Lo et al. showed that NiV infection could induce production of CXCL10 and several other inflammatory chemokines in microvascular, but not in macrovascular endothelial cells [21] . However, in contrast to our study, they cultured endothelial cells in the presence of hydrocortisone, known to have important antiinflammatory activity and to reduce the expression of proinflammatory cytokines [36] , which could account to the differences with our results. In accord to our results, NiV was shown to induce CXCL10 expression as well as several other inflammatory chemokines in brain and lungs of NiV infected hamsters [37] suggesting altogether that the other cytokines may contribute to the CXCL10 proinflammatory effect. CXCL10 has been detected in the cerebrospinal fluid of individuals with HIV-1 infection [38, 39] and in the brains of individuals with HIV-associated dementia [33] , but was absent in uninfected control individuals. These authors also reported that CXCL10 levels were closely associated with the progression of HIV-1-related CNS infection and neuropsychiatric impairment. Moreover, CXCL10 and its receptor CXCR3 were shown to be present in the brains of macaques with SIV/SHIV-E [40, 41] and to elicit apoptosis in fetal neurons [9, 42] . The mechanisms of neuronal injury mediated by the CXCL10 was suggested to be associated to calcium dysregulation during CXCL10 mediated apoptosis [10] , and we hypothesize that overexpression of CXCL10 in Nipah-infected patients may be directly involved in NiV-induced neuropathology. Lack of the appropriate regaents for hamster animal model prevented us from further in vivo analysis of the role of CXCL10 during Nipah infection.
CXCL10 is the substrate for serine protease CD26/dipeptidylpeptidase 4, which cleaves its aminoterminal part and alters its receptor binding and signaling, producing therefore an antagonistic protein with dominant negative function [43] . This antagonist form of CXCL10 was very recently shown to play an important role in patients chronically infected with hepatitis C virus [44] and to present an important negative prognostic marker for the response to therapy [45, 46] . Whether this cleaved antagonist form of CXCL10 is generated during NiV-infection remains to be elucidated. If produced, its action on inhibition of the physiological role of CXCL10, may play an important role in the pathogenesis of NiV-encephalitis.
These results suggest that NiV-infection in endothelial cells induces CXCL10 production both in vitro and in vivo and highlight the use of molecular profiling of virus-infected cells as a powerful tool to define novel mechanisms of virus-host cell interaction. As equilibrium in cytokine production is essential in the generation of the adequate immune response, CXCL10 overexpression may be crucial for the development of NiV-associated encephalitis and could be a target for therapeutic approaches.
Umbilical cords were obtained between 2006 and 2008 from healthy full-term newborns with written parental informed consent according to the guidelines of the medical and ethical committees of Hospices Civils de Lyon and of Agence de Biomedecine, Paris, France. Ethics Committee approval for this study was not required according to institutional guidelines and French law Nu2004-800, from 6 August 2004 -art 12 ORF 7.08.2008, Article L1245-2, allowing the utilization of the placenta and umbilical cords in scientific and therapeutic purpose when their donors do not express any opposition.
All animals were handled in strict accordance with good animal practice as defined by the French national charte on the ethics of animal experimentation. Animal work was approved by the Regional ethical committee (Comité Régional d'Ethique pour l'Experimentation Animal de la Région Rhone-Alpes, CREEA, protocol Nu 220) and experiments were performed in the INSERM Jean Mérieux BSL-4 laboratory in Lyon, France (French Animal regulation comittée Nu A69 387 05 02).
Autopsies of human brain tissue were performed after receiving written patient's relatives consent for autopsy studies at the Pathology Department of University of Malaya, Kuala Lumpur, Malaysia and their analysis was approved by review board of Faculty of Medicine of University.
Primary HUVECs were isolated from human umbilical cords of 22 donors by treating the umbilical vein with 0.1% collagenase for 30 min at 37uC as described previously [47] . Cell cultures were pooled by sets of three donors for experiments. Cells were cultured in flasks coated with 0.2% gelatin in complete medium containing M199 medium (Gibco), 20% of fetal calf serum (Gibco), 100 mg/ml bovine brain extract [48] , 14 mM Hepes (Gibco), 10 UI/ml heparin (Pfizer), and a cocktail of antibiotic/antimycotic (Gibco). At passage 4, cells were seeded at 30 000 cells/cm 2 for 8 h, then serum deprived for 16 h prior to NiV-infection in complete medium. Immunofluorescent staining and analysis of HUVEC culture was performed as described in Methods S1. U373 astroglioma and Vero cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 0.1 mg streptomycin, 10 mM HEPES and 2 mM L-Glutamine at 37uC in 5% CO 2 .
Nipah virus (isolate UMMC1, Genebank AY029767) [49] was prepared on Vero-E6 cells as described previously [50] . HUVECs were infected with a multiplicity of infection (MOI) of 1 and harvested for RNA isolation 8 and 24 hours post infection (p.i.). At indicated times p.i. 150 ml of cell culture supernatant were collected and frozen and viral titration was performed as detailed elsewhere [50] .
Eight-week-old golden hamsters (Mesocricetus auratus, Janvier, France) were anesthetized and infected intraperitoneally (i.p.) with 0.4 ml of wild-type NiV (10 000 PFU) in the BSL-4 laboratory in Lyon. Each day post infection (p.i.), one hamster was euthanised and organs were frozen at 280uC.
Total RNA was extracted from uninfected or NiV-infected HUVECs 8 h p.i., both prepared from two different pools, each containing 3 donors and cultured at same conditions, using RNeasy kit (Qiagen) according to the manufacturer's protocol. Quality of total RNA was checked on nanochips with the Agilent 2100 Bioanalyzer 2100 (Agilent Technologies,).
Amplified and biotin-labeled RNAs were obtained from 2 mg of total RNA, by a round of in vitro transcription (dIVT) using the Message Amp a RNA kit version II (Ambion), following the manufacturer's protocol. Different quantities of positive synthetic mRNA controls (spikes, corresponding to 6 bacterial RNAs, used to control sensitivity, quality of hybridization and data normalization) were added to all samples, during the first step of reverse transcription of total RNAs. Hybridization was performed on Codelink Uniset Human Whole Genome bioarrays (http://www. codelink bioarrays.com). 10 mg of biotin-labeled RNA were fragmented using 5 ml fragmentation buffer in a final volume of 20 ml, then mixed with 240 ml Amersham hybridization solution and injected onto Codelink Uniset Human Whole Genome bioarrays, containing approximately 55.000 30-mer probes based on the NCBI/Unigene RefSeq database that permit the expression analysis of 57,347 transcripts and ESTs (GE Healthcare Europe GmbH). Arrays were hybridized overnight at 37uC, then washed in stringent TNT buffer at 46uC for 1 h before performing a streptavidin-cy5 detection step. Each array was incubated for 30 min in 3,4 ml streptavidin-cy5 solution, washed four times in 240 ml TNT buffer, rinsed twice in 240 ml water containing 0.2 M Triton X-100, and then, dried by centrifugation at 650 g. Arrays were then scanned at 635 nm using an Axon Genepix 4000B Scanner (Axon).
Data extraction and raw data normalization were performed using the CodeLink Gene Expression Analysis v4.0 software. Normalization was performed by the global method. The threshold was calculated using the normalized signal intensity of the negative controls supplemented by 3 times the standard deviation. Spots with signal intensity below this threshold were referred to as ''absent''. Obtained datasets were deposited in GEO database in accord to MIAME guidelines (Accession number: GSE 32902: GSM813064, GSM813065, GSM813066 and GSM813067). Finally, data were converted to the excel format and data analysis was performed by using the Gene Spring v7.0 software from Agilent and pariwise comparisons were performed between infected and uninfected samples.
Total RNA was extracted from mock and NiV-infected HUVECs 8 and 24 hours post-infection using RNeasy Mini Kit according to the manufacturer's instructions including additional step with RNase-free DNase (Qiagen). RNA was isolated from hamster organs 10-30 mg using a tissue lyser (Qiagen) in RLT buffer containing 1% ß-mercaptoethanol, according to manufacturer recommendations.
Reverse transcriptions were performed on 0.5 mg of total RNA using the Transcriptor first strand cDNA synthesis kit (Roche) and run in BiometraH T-GRADIENT PCR devise. Obtained cDNAs were diluted 1:10. Quantitative PCR was performed using PlatinumH SYBRH Green qPCR SuperMix-UDG with ROX kit (Invitrogen TM ). qPCR was run on the ABI 7000 PCR system (Applied biosystems) using the following protocol: 95uC 59, and 40 cycles of 95uC 150, 60uC 19, followed by a melting curve up to 95uC at 0.8uC intervals. All samples were run in duplicate and results were analyzed using ABI Prism 7000 SDS software available in the genetic analysis platform (IFR128 BioSciences Lyon-Gerland). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene for mRNA quantification and normalization. GAPDH and standard references for the corresponding genes were included in each run to standardize results in respect to RNA integrity, loaded quantity and inter-PCR variations. Primers used were designed using Beacon 7.0 software, and validated for their efficacy close to 100% in control PCR amplification: EFN B2 for: TCGGGCTAGTTAAGGTGTGC, EFN B2 rev: ATGAGTGTTCCATGAGTGATGC, EFN B3 for: TCACCCTCTTGGCTTCTTATCC, EFN B3 rev: GGGGA-GTGGTTGGTATGAGAG, NiV N for: GGCAGGATTCT-TCGCAACCATC, NiV N rev: GGCTCTTGGGCCAATT-TCTCTG, NiV L for: ATGGTGCTGTGCTGTCTCAGG, NiV L rev: AGCCGACATTTCTTGACAACCC, IFNß for: CTCCTAGCCTGTGCCTCTGG, IFNß rev: TGCAGTACAT-TAGCCATCAGTCAC, MxA for: AGCCACTGGACTGAC-GACTTG, MxA rev: AAATCACCACGGCTAACGGATAAG, OAS1 for: AGAACTTACCTCTTGCCAAAGG, OAS1 rev: GGACAAGGGATGTGAAAATTCC, CXCL10 for: GGAAG-GTTAATGTTCATCATCCTAAGC, CXCL10 rev: TAGTAC-CCTTGGAAGATGGGAAAG, CXCL11 for: GGATGAAAG-GTGGGTGAAAGGAC, CXCL11 rev: AACGTGAAAGCAC-TTTGTAAACTCC, GAPDH for: CACCCACTCCTCCACC-TTTGAC, GAPDH rev: GTCCACCACCCTGTTGCTGTAG. Primers used for hamster study: hamster CXCL10 for: AGA-CAACAGTAACTCCAGTGACAAG, hamster CXCL10 rev: A-GTGTAGCACCTCAGCGTAGC, murine GAPDH for: GC-ATGGCCTTCCGTGTCC, murine GAPDH rev: TGTCAT-CATACTTGGCAGGTTTCT. The relative expression represents the ratio of the number of copy of mRNA of interest versus mRNA of GAPDH and expressed in relationship to the quantity of RNA analyzed All calculations were done using the 2 DDCT model of [51] and experiments were performed according to the MIQE guideline [52] .
Supernatants of mock and NiV-infected HUVECs were collected 8, 24, 48, 72 h p.i. and used to quantify the induction of CXCL10 using human IP-10 Immunoassay (Invitrogen TM ), according to manufacturer's instructions. All tests were performed on 2 sets of 3 donors, in duplicates.
Autopsy brain tissue was obtained from 3 fatal acute Nipah cases from the outbreak in Malaysia in 1999, fixed in 10% buffered formalin and paraffin-embedded and from two control brain tissues from patients that succumbed to non-neurological diseases. Sections (4 mm) were deparaffinized with xylene and rehydrated through graded alcohol and distilled water. Antigen retrieval was performed in 10 mM trisodium citrate buffer, pH = 6, using waterbath (99uC, 20 min). Slides were then washed 3 times in PBS 16. Endogenous peroxidase was blocked using H2O2, 3% in PBS, for 20 min at Room temperature (RT). Slides were then washed 2 times in PBS 16 and additional blocking was performed using goat serum (1:20 in PBS) for 20 min at RT. Primary rabbit anti-human CXCL10 (Peprotech), was applied diluted at 4 mg/ml, overnight at 4uC. After 3 washes in PBS, slides were incubated with secondary goat anti-rabbit conjugated with HRP (Promega) 1:500 for 1-2 hours at RT. Sections were then developed with AEC kit (VECTOR) for 25 min, washed in distilled water and counterstained with Harris hematoxylin solution (Sigma-Aldrich) 1:3 in PBS, 30 s. After one wash in water (3 min) sections were mounted with DakoCytomation faramount aqueous mounting medium and coverslipped. Slides were analysed using Axioscope microscope equipped with Zeiss Axiovision software (Zeiss Geramny).
Data were expressed as mean6standard deviation (SD). Statistic analyses were performed using Mann-Whitney U-test and Pearson correlation test. Figure S1 Expression of the endothelial-specific markers. Von Willebrand Factor (VWF) and CD31 (PECAM-1) were analyzed in 2 nd day HUVEC cultures by immunostaining with anti-CD31 (green) and anti-VWF (red). Nuclei were stained with DAPI (blue). Analysis was performed as described in Methods S1. (TIF)
Methods S1 Supplementary methods. (DOCX) Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes. Despite reports of conjunctivitis following infection with numerous respiratory pathogens (including influenza, adenovirus, respiratory syncytial virus, and others), research investigating the role of ocular infection in virus pathogenicity and transmissibility has been underrepresented [1] [2] [3] [4] . Influenza virus represents a highly transmissible respiratory pathogen, resulting in .200,000 hospitalizations in the United States annually [5] . While ocular disease is generally rare following influenza virus infection in humans, viruses within the H7 subtype have demonstrated an apparent ocular tropism, with the majority of human infections with H7 influenza viruses associated with conjunctivitis [6] . Moreover, ocular complications have been sporadically documented following seasonal, 2009 H1N1 pandemic, and avian H5N1 virus infections in humans [7] [8] [9] [10] [11] [12] [13] .
Numerous properties allow the eye to serve as both a potential site of influenza virus replication as well as a gateway for the establishment of a respiratory infection. Similar to epithelial cells within the human respiratory tract, human ocular tissue and secreted mucins express sialic acids, the cellular receptor for influenza viruses [14] [15] [16] . The anatomical proximity between the eye and nasal passages, notably the linkage of both systems via the nasolacrimal duct, facilitates aqueous exchange and provides shared lymphoid tissue between these sites [17, 18] . Influenza virus can rapidly spread between ocular and respiratory tissues, as was demonstrated in a recent study which detected by RT-PCR live attenuated influenza vaccine (LAIV) in nasal washes in humans within 30 minutes of experimental ocular exposure to LAIVcontaining aerosols [19] .
Well-characterized mammalian models to study extraocular spread following ocular inoculation with influenza virus have been limited to the mouse [20] . The ferret, widely used to study influenza pathogenesis and transmission following intranasal inoculation, has also been recognized as an appropriate experimental model for studies involving the visual system [21] [22] [23] . A previous study demonstrated H7N3 virus replication in nasal, ocular, and rectal tissue following ocular inoculation in ferrets, but did not comprehensively examine the ability of multiple subtypes to use the eye as a portal of entry or examine virus transmissibility following inoculation by this route [24] .
It is clear from epidemiological and laboratory data that ocular exposure to influenza virus can manifest in both ocular and respiratory disease. However, the properties that contribute towards the ocular tropism of select influenza virus subtypes, and the mechanisms of virus spread from ocular to respiratory tract tissue following ocular exposure to influenza virus, are not well understood. Here, we present a ferret model where influenza virus in a liquid suspension is placed on the surface of the eye and massaged across the surface of the eye within the conjunctival sac (ocular inoculation) to study the ability of human and avian influenza viruses to cause disease and transmit to naïve animals. We found that both human and avian influenza viruses can mount a productive virus infection following ocular inoculation, attributable to replication-independent drainage of virus inoculum from the site of inoculation to respiratory tract tissue. The viral infection following ocular inoculation was capable of causing severe and fatal disease (in the case of H5N1 virus), but was less transmissible by respiratory droplets (in the case of seasonal influenza viruses) compared with infection following inoculation by the traditional intranasal route.
Due to a high degree of similarity in lung physiology and distribution of sialic acids in respiratory tract tissues, the ferret model is frequently utilized to model the kinetics and severity of respiratory disease following administration of human and avian influenza viruses by the intranasal route [23, 25] . To determine if this homology extends to ocular tissue, we examined the composition of sialic acids on the ferret cornea, which represents a potential site of influenza virus replication following ocular inoculation. Staining of ferret corneal epithelial sheets with biotinylated lectins specific for a2-3 and a2-6 sialic acids revealed a predominance of a2-3-linked sialic acids with relatively weak expression of a2-6 sialic acids on the epithelial surface, an expression pattern similar to human corneal and conjunctival tissue (data not shown) [2, 20] .
To assess the pathogenicity of influenza viruses of multiple subtypes following ocular inoculation (i.o.) in ferrets, we administered 10 6 EID 50 of each indicated virus in a volume in 100 ml on the corneal epithelial surface of the right eye of an anesthetized ferret and massaged the inoculum across the surface of the eye with the eyelid (Table 1) . Ferrets were observed daily for two weeks for clinical signs of illness (including fever, weight loss, nasal or ocular discharge). Nasal wash (NW) and rectal swab (RS) samples were collected on alternate days post-inoculation (p.i.) and were titered for infectious virus, while conjunctival wash (CW) samples were collected alternate days p.i. to measure the incidence and kinetics of infectious virus replication and levels of viral RNA from inoculated eyes (Tables 1 and 2 , Figures 1 and 2 ).
All virus subtypes tested replicated in ferrets following ocular inoculation, as measured by detectable virus in NW samples as early as day 1 p.i. (Table 2 and Figure 1 ). The duration of virus shedding from NW samples and transient fever and weight loss generally mirrored that seen following intranasal (i.n.) inoculation for each virus [26] [27] [28] [29] . However, in comparison to i.n. inoculation, the incidence of nasal discharge was reduced following i.o. inoculation with all influenza viruses tested, and
Most infections with influenza virus result in respiratory disease. However, influenza viruses of the H7 subtype frequently cause ocular and not respiratory symptoms during human infection, demonstrating that the eye represents an alternate location for influenza viruses to infect humans. Using a ferret model, we studied the ability of influenza viruses to cause disease following ocular inoculation. We found that both human and avian influenza viruses could use the eye as a portal of entry to establish a respiratory infection in ferrets. Influenza viruses were also detected in ocular samples taken from ferrets during virus infection. We identified that influenza viruses spread to different tissues in ferrets when inoculated by ocular or respiratory routes, and that these differences affected the transmissibility of influenza viruses in this model. This study is the first to confirm that virus can spread from the eye to the respiratory tract in a replication-independent manner, and offers greater insight in understanding the ability of influenza viruses of all subtypes to cause human infection by the ocular route. Figure 3 ). Ocular inoculation with all H7 influenza viruses tested resulted in elevated peak mean viral titers in NW samples and a higher incidence of detection in CW samples compared with H5N1 viruses (45% positive among H7 virus samples compared with 25% of H5 virus samples) (Table 2, Figure 2 ). However, despite reduced viral replication in NW samples, H5N1 viruses were capable of causing .20% weight loss and lethal disease in ferrets following i.o. inoculation (Table 1) . Interestingly, all seasonal H1N1 and H3N2 viruses evaluated were detected at high titer in both NW and CW samples; i.o. inoculation with Mex/4108, Brisbane, and Panama viruses, along with the H7N7 NL/230 virus, resulted in the highest peak mean titers in CW samples (.10 3 EID 50 /ml) compared with all viruses examined (Table 2, Figure 2 ). All viruses with the exception of Thai/16 virus were also detected at low titer in RS samples, with peak titers 10 1.8 -10 2.7 EID 50 /ml observed days 3-7 p.i. (Table 2 ).
In summary, we found that both avian and human influenza viruses were capable of mounting a productive infection in ferrets following i.o. inoculation, with virus replication observed in samples collected from both ocular and respiratory tract locations regardless of virus subtype. H7 influenza viruses replicated to peak titers .2 logs higher compared with H5 influenza viruses in NW samples following i.o. inoculation, yet H5N1 influenza viruses were capable of maintaining a lethal phenotype following introduction by the ocular route. Seasonal and 2009 H1N1 pandemic influenza viruses efficiently used the eye as a portal of entry to replicate efficiently in the upper respiratory tract as well as ocular tissue.
To examine the capacity of influenza viruses to cause severe disease following ocular inoculation, and to better identify those features specific to ocular inoculation, we inoculated ferrets by either the traditional i.n. route (using a 1 mL inoculation volume) or the i.o. route (using a 100 ml inoculation volume) with 10 6 EID 50 of NL/219, NL/230, or Brisbane virus and collected systemic tissues on day 3 p.i. (Table 3 and 4). While i.n. inoculation with the H7N7 viruses tested in this study results in high virus titers throughout the respiratory tract of ferrets, H7N7 virus dissemination following i.o. inoculation was generally restricted to the upper respiratory tract, with a .3 log reduction in titers in nasal turbinates (p,0.05) and only sporadic virus isolation in trachea and lung samples compared with intranasal inoculation (p,0.005) ( Table 3) . A similar pattern of virus dissemination following H7N7 i.o. virus infection was observed when ferrets were inoculated by the i.n. route using a 100 ml and not 1 mL inoculation volume ( Table 3 ). The H1N1 virus Brisbane replicated with comparable efficiency in nasal turbinate samples regardless of the inoculation route or volume chosen, but similar to H7N7 virus ocular infections, did not consistently replicate to high titers in lower respiratory tract tissues. Unlike virus dissemination to the respiratory tract, virus spread to the intestinal tract was not contingent on the route or volume of inoculation.
Despite restriction of virus following i.o. inoculation to upper respiratory tract tissues compared with traditional 1 mL intranasal inoculation through day 3 p.i., virus introduced by the ocular route was still capable of causing lethal disease, as ferrets inoculated with the HPAI H5N1 virus Thai/16 by the ocular route required euthanasia days 7-8 p.i. due to development of neurological signs (Table 1) . Ferrets which succumbed to Thai/16 virus infection following ocular inoculation exhibited pronounced lymphopenia in peripheral blood and systemic spread of virus to all regions of the respiratory tract and brain comparable to 1 mL intranasal inoculation, albeit with reduced lethargy and a delayed time-to-death ( [32] and data not shown). These data suggest that ferrets inoculated by the ocular route succumb to a similar course of disease as intranasally inoculated ferrets, however following i.o. inoculation there is a delay in both the kinetics of virus dissemination and the development of neurological signs and severe disease, potentially owing to differences in virus inoculum reaching lower respiratory tract tissues at the time of ocular inoculation.
Ocular tissue is not routinely titrated following i.n. inoculation of influenza virus in ferrets, making it difficult to elucidate if viral titers in ocular tissue are a function of i.o. inoculation or are detected regardless of the inoculation route. Therefore, we collected both left and right whole ferret eyes and all surrounding conjunctiva/eyelid for virus titration from ferrets inoculated by the intranasal or ocular route with NL/219, NL/230, or Brisbane viruses (Table 4) . Surprisingly, sporadic viral titers from both left and right eye and conjunctival tissue were detected following HPAI H7N7 virus infection by both i.n. (using either a 1 mL or 100 ml inoculation volume) or i.o. inoculation routes (Table 4) . While the magnitude of viral titers and viral RNA was generally similar between intranasal and ocular routes of inoculation, realtime RT-PCR detected CW-positive samples with a greater sensitivity compared with viral culture. Isolation of virus from ocular tissue may be a reflection of the ability of these HPAI viruses to spread to extra-pulmonary tissues post-inoculation as previously described [26] . However, virus was also detected in left and right conjunctival tissue following i.n. or i.o. inoculation of the H1N1 virus Brisbane, a virus which lacks a high capacity for systemic spread [28] . Comparable levels of viral RNA were isolated from CW samples from ferrets inoculated with Brisbane virus by either intranasal or ocular routes, although infectious virus was only detected in CW samples collected from the eyes of ferrets inoculated by the ocular route. To confirm that virus detected in the eye and conjunctiva was associated with tissue-specific virus replication, immunohistochemistry (IHC) was performed to visualize the presence of influenza A nucleoprotein (NP) in ferret ocular tissues. As shown in Figure 3 , influenza virus antigen was detected in epithelial cells from both the lacrimal glands in the conjunctiva and the ciliary processes in the eye collected day 3 p.i. from ferrets inoculated by the ocular route. These results indicate that the route of virus inoculation in ferrets can affect the extent of virus dissemination in respiratory tract tissue, but extra-pulmonary spread, notably to ocular tissue, is present regardless of the point of entry once an infection is established.
The detection of high viral titers in NW samples as early as day 1 p.i. following i.o. inoculation suggests replication-independent Figure 1 . Comparison of mean titers of influenza viruses recovered from nasal wash following ocular inoculation of ferrets. Ferrets were inoculated by the ocular route with 10 6 EID 50 /ml of each virus shown. Viral titers were measured in nasal washes collected on indicated days following serial titration in eggs; endpoint titers are expressed as mean log 10 EID 50 /ml plus standard deviation. The limit of virus detection was 10 1.5 EID 50 /ml. {, ferrets did not survive to day 9 p.i. doi:10.1371/journal.ppat.1002569.g001 spread of virus from the eye to the respiratory tract ( Figure 1 ); this has been similarly hypothesized in previous studies, but has yet to be proven experimentally [20, 33, 34] . Reduced viral titers in the lungs of ferrets on day 3 p.i. following ocular compared with intranasal administration further indicates differential patterns of virus spread following inoculation (Table 3) . To visualize the deposition of virus immediately following different routes of inoculation, we labeled NL/219 virus with an AF680 fluorescent tag (NL/219-FL) and inoculated ferrets with equal quantities of NL/219-FL virus by the ocular (100 ml total volume) or intranasal (1 ml total volume diluted in PBS) route ( Figure 4 ). Ferrets were euthanized 15 minutes following virus inoculation for ex vivo imaging. In ferrets inoculated by the traditional intranasal route, the majority of virus was deposited in the nasal turbinates and lungs, consistent with a previous study demonstrating virus dissemination throughout upper and lower respiratory tract tissue following this route of inoculation [32] . In contrast, virus deposition in ferrets inoculated by the ocular route (right side only) was primarily localized in the nasal turbinates and right conjunctiva. Lower relative quantities of virus inoculum were present in the upper trachea and esophagus following either intranasal or ocular inoculation. These findings demonstrate that, following i.o. inoculation in ferrets, influenza virus rapidly spreads to the nasal turbinates and upper trachea in a replicationindependent manner, but in contrast to i.n. inoculation, does not immediately deposit in peripheral lung tissue. Furthermore, initial deposition of virus inoculum following ocular inoculation occurs not on the corneal surface of the eye but is rather concentrated in the surrounding conjunctival tissue.
To determine if ocular exposure to influenza virus results in a transmissible respiratory infection, we inoculated ferrets by the Figure 2 . Comparison of influenza virus recovery in conjunctival wash samples following ocular inoculation of ferrets. Ferrets were inoculated by the ocular route with 10 6 EID 50 /ml of each virus shown. Viral titers were measured in conjunctival washes (CW) collected on indicated days following serial titration in eggs; endpoint titers are expressed as mean log 10 EID 50 /ml plus standard deviation (left y-axis and bars). Relative viral RNA copy number in conjunctival washes was determined by real-time PCR using a universal M1 primer and extrapolated using a standard curve based on samples of known virus (right y-axis and lines). The limit of virus detection was 10 1.5 EID 50 /ml. {, no ferrets survived until day 9 p.i. R-squared values are shown for those viruses where a statistically significant (p,0.05) correlation between viral titer and viral RNA copy number exists. NS, not significant. doi:10.1371/journal.ppat.1002569.g002 PLoS Pathogens | www.plospathogens.org ocular route with selected influenza viruses known to transmit following traditional i.n. inoculation to naïve contacts in the presence of direct contact or by respiratory droplets ( Figure 5 ). Transmission was assessed by the detection of virus in NW samples and seroconversion of contact ferrets. To assess virus transmission in the presence of direct contact, ferrets were inoculated by the ocular route with the H7N2 virus NY/107 or the H7N7 virus NL/ 230, both viruses which transmit efficiently by this route following i.n. inoculation in ferrets [27] . Twenty-four hours later, a naïve ferret was placed in the same cage as each inoculated ferret to assess transmission. Both NY/107 and NL/230 viruses replicated efficiently in the inoculated ferrets following ocular inoculation as expected, and spread to 2/3 and 3/3 contact ferrets by day 7 postcontact (p.c.), respectively ( Figure 5A ). To assess virus transmissibility by respiratory droplets in the absence of direct contact, ferrets were inoculated by the ocular route with the H1N1 virus Brisbane or the H3N2 virus Panama, both viruses which transmit efficiently by this route following traditional i.n. inoculation [28, 30] . Twenty-four hours following i.o. inoculation, a naïve ferret was placed in an adjacent cage with modified side walls, so that air exchange was permitted between inoculated and contact ferrets in the absence of direct or indirect contact. Unlike the efficient transmission observed with these viruses following traditional i.n. inoculation, ferrets inoculated by the ocular route with either Brisbane or Panama only transmitted virus by respiratory droplets to 1/3 contact ferrets ( Figure 5B ). Virus was not detected in CW or RS samples from the infected Brisbane contact ferret; the infected Panama contact ferret had a peak CW titer of 10 2.75 EID 50 day 5 p.c. and peak RS titer of 10 2.5 EID 50 day 7 p.c. While RD contacts with detectable virus in NW samples seroconverted to homologous virus at the end of the experimental period, contact ferrets which did not have detectable virus in NW samples did not exhibit seroconversion (data not shown). Ferrets inoculated intranasally with 10 6 EID 50 of Brisbane virus in a reduced 100 ml volume and tested for their ability to transmit virus by respiratory droplets exhibited a similar pattern of virus transmissibility as ferrets inoculated by the ocular route, with virus shedding and seroconversion detected in only 1/3 contact ferrets (data not shown). These findings suggest that despite high titers of virus in NW samples, the respiratory infection resulting from inoculation of ferrets with a reduced volume, by either ocular or intranasal inoculation routes, is distinct from that following traditional i.n. inoculation, characterized by a diminished incidence of sneezing and nasal discharge and resulting in reduced transmission of virus by respiratory droplets.
While the ferret has proved essential for the study of influenza virus pathogenesis and transmission, the use of this species to examine alternate inoculation methods has been limited [32, [35] [36] [37] [38] . Characterizing the progression of disease following alternate routes of inoculation with influenza virus will assist in the better understanding of unique features and the relative severity and risk associated with different exposure routes. In this study, we established an in vivo model using the ferret to assess the ability of influenza viruses of multiple subtypes to use the eye as a gateway to establish a productive infection. Both human and avian influenza viruses were capable of mounting a respiratory virus infection in ferrets following i.o. inoculation. The detection of virus in ocular samples collected from ferrets inoculated by either ocular or intranasal routes demonstrates the importance of studying ocular involvement in respiratory virus infection. Divergent patterns of virus transmissibility by respiratory droplets following use of different inoculum volumes and routes of inoculation highlights the complexity of properties which govern virus transmission.
The high similarity of respiratory tract tissue between humans and ferrets makes the ferret model an attractive one for modeling human respiratory disease and investigating the role of receptor specificity in influenza virus pathogenesis, providing an advantage over murine models [25] . We found that the sialic acid composition of ferret corneal epithelial sheets more closely mimics that of humans compared with a mouse model, demonstrating another physiological parallel between ferrets and humans [20] . Bridging the a2-3 rich corneal and conjunctival epithelial surfaces with a2-6 rich upper respiratory tract tissue is the lacrimal duct, which expresses both a2-3 and a2-6 linked sialic acids [14, 34] . Characterization of the distribution of sialic acids in the ferret conjunctiva and lacrimal duct, in addition to the composition of ferret ocular mucins, will allow for a better understanding of virus attachment and replication in these locations. However, in a previous study, we demonstrated that the ability of influenza viruses to bind to or replicate in ocular tissue cannot be explained by sialic acid binding specificity alone [20] . Our detection in ferret ocular tissue of both human and avian influenza viruses with distinct binding specificities further underscores this point (Table 4 , Figure 3 ).
Macroscopic signs of ocular disease in ferrets were not observed during the course of infection with any virus tested, similar to prior observations in mouse and rabbit models following deposition of influenza virus on the corneal surface [20, 39] . A previous study in ferrets reported mild conjunctival inflammation following i.o. inoculation with an H7N3 virus, however this may be attributable to strain-specific differences or the use of younger (3-5 month old) ferrets [24] . Despite the absence of visible ocular complications, virus was consistently detected in CW samples from ferrets inoculated by the intranasal or ocular route (Table 2, Figure 2 ). Levels of viral M1 RNA generally correlated with the magnitude of virus isolation, and were a more sensitive detection method compared with virus isolation in CW samples, similar to that observed in human eye swabs (Table 4, Figure 2 ) [40] . The presence of virus in RS samples following both i.n. and i.o. inoculation with influenza virus has been previously reported and likely originates from virus swallowed during inoculation [24, 32, 41] , as indicated by deposition of virus in the esophagus following initial virus inoculation by both inoculation routes (Table 3, Figure 3 ).
Unlike in a murine model, the ferret model supported virus replication of both human and avian influenza viruses following i.o. inoculation [20] . In this ferret model, H7 viruses were detected at higher titer in NW samples and with a higher frequency in ocular CW samples compared with H5N1 viruses, suggesting a recapitulation of the tropism of the H7 virus subtype observed in humans (Table 2) . However, the permissiveness of multiple virus subtypes to cause a productive infection following i.o. inoculation in the ferret points to a greater capacity of influenza viruses to use the eye as a portal of entry in an experimental in vivo setting, just as previous in vitro studies have demonstrated that numerous human ocular cell types distributed throughout the ocular area support infection and replication with both avian and human influenza viruses [42] [43] [44] . Cumulatively, these previous in vitro studies suggest that the apparent ocular tropism associated with viruses of the H7 subtype is not due to a superior ability to replicate in ocular cells compared with other virus subtypes. Future studies evaluating potential fine receptor specificity differences on the ocular surface and the composition of ocular mucins which may restrict exposure to the ocular epithelial surface to selected virus subtypes may provide a greater understanding of this property.
Consistently high titers of human influenza viruses in ocular samples following both i.n. and i.o. inoculation indicates that these H1N1 and H3N2 viruses are not exhibiting a preferential tropism for ocular infection but more likely are a reflection of the high titers observed in the upper respiratory tract in these tissues independent of the initial inoculation route. Specifically, the nasolacrimal duct which links the ocular lacrimal sac to the nasal meatus could serve as a conduit for virus-containing fluid exchange between ocular and respiratory tract tissue [18] . Numerous reports have documented drainage of vaccine or immunizing agents to nasal tissue following topical ocular administration as well as the spread of intranasally administered solutions to the conjunctival mucosal surface [18, 45] . However, spread of virus from respiratory tract tissue to ocular tissue following i.n. inoculation with human or avian influenza viruses has not been observed previously in the ferret and only sporadically reported in the mouse, possibly due to relatively low titers of virus in nasal tissue or other anatomical differences [20, 24, 26, 46] . Detection of both human and avian influenza viruses in ferret eyes and conjunctival tissue following i.n. inoculation indicates that virus can circulate proximal to the nasal cavity and nasolacrimal canal more readily than previously considered and that more routine collection of ocular tissue during standard virus pathotyping in mammalian models is warranted to better understand the extent of viral ocular dissemination (Table 4 ). While it is unlikely that ferret grooming practices are solely responsible for virus spread between these locations, human studies with numerous respiratory viruses have demonstrated the ability to self-inoculate between ocular and respiratory sites, and it is possible that self-inoculation could be further contributing to virus spread in this model [47, 48] . Inoculation of ferrets by the ocular route with 10 6 EID 50 of selected human and avian influenza viruses in a 20 ml volume resulted in comparable results to those obtained employing a 100 ml inoculation volume, indicating that replication-independent drainage of virus to respiratory tract tissue and subsequent virus detection in NW and CW samples reported in this study was not contingent on the inoculum volume (data not shown).
Visualization of virus deposition using fluorescently-tagged virus has allowed for a new understanding of dissemination patterns following in vivo inoculation. Using this technique, we confirmed previously hypothesized reports of replication-independent drainage from ocular to respiratory tract tissue [20, 33] . Viral load measured in respiratory tract tissues day 3 p.i. following i.n. or i.o. inoculation largely mirrored initial virus deposition patterns, with tissues possessing the greatest quantity of virus reflecting those sites of greatest initial virus deposition during virus inoculation (Table 3 -4, Figure 4 ). Both i.o. and i.n. inoculation using a 100 ml volume resulted in detection of virus at high titers in upper and not lower respiratory tract tissues day 3 p.i., indicating that inoculation with a reduced volume leads to limited initial virus deposition in respiratory tract tissues regardless of inoculation route. The delay in high virus titer recovery from lower respiratory tract tissues during HPAI virus infection after i.o. inoculation and the delay in onset of severe disease and lethal outcome with Thai/ 16 virus is likely due to replication-dependent spread (and not deposition) of virus to the lower respiratory tract, suggesting that during inoculation, the majority of virus was retained in conjunctival tissues, drained to the nasal turbinates, or swallowed and diverted away from the lower respiratory tract; future study of ferret lacrimal tissue in this role is warranted. Comparable delays in onset of severe disease compared with traditional i.n. inoculation were observed in a murine model of i.o. inoculation and a ferret model of aerosol inoculation, despite ultimately similar lethal outcomes [20, 32] . The finding here of H5N1 subtype viruses using the eye as a portal of entry to initiate a lethal infection, shown previously in a mouse model, underscores the risk of ocular exposure to influenza viruses, even those subtypes not typically considered to have a tropism for this tissue [20] .
Despite efficient replication of seasonal H1N1 and H3N2 viruses in the upper respiratory tract of ferrets following i.o. inoculation, these viruses did not result in frequent detection of sneezing and nasal discharge, and did not transmit efficiently to naïve contacts by respiratory droplets (Table 1, Figure 5 ). Infrequent sneezing is commonly observed among influenza viruses which do not transmit efficiently by respiratory droplets and could be contributing to the reduced transmissibility seen here [28, 30, 31] . Further research is needed to better understand the virologic and immunologic properties which confer the incidence of sneezing and nasal discharge and the role of these properties in virus transmissibility [49] . Additionally, the efficiency of virus transmission by respiratory droplets following i.o. inoculation was likely influenced by the reduced initial virus deposition and subsequent limited replication in the ferret trachea, as reduced virus transmissibility by respiratory droplets was observed following i.n. inoculation when using a 100 ml but not 1 mL volume (Table 3 , Figure 4 , and data not shown). Despite similarly high virus titers and duration of virus shedding in NW samples, the presence of expelled virus particles originating from tracheal replication which was present during traditional i.n., but not i.o. or i.n. inoculation using a reduced volume, may have contributed to differing transmission efficiencies between inoculation routes and may represent a previously unrecognized role for virus replication in tracheal tissue in virus transmissibility by respiratory droplets. In contrast, virus transmission in the presence of direct contact did not differ between inoculation routes. The reduced transmissibility of virus following i.o. inoculation is in agreement with epidemiological studies which demonstrate that the majority of human cases of conjunctivitis following H7 influenza virus exposure are self-limiting [50] . Further study evaluating the shedding of virus into the environment among persons infected with influenza viruses which cause respiratory or ocular disease will shed light on potential differences in transmission dynamics independent of virus subtype.
The diversity of potential exposures to influenza virus underscores the importance of studying the development of respiratory disease resulting from alternate exposure routes. This knowledge is critical for both a greater understanding of the establishment of influenza virus respiratory disease as well as differences in virus transmission dynamics following differing exposure routes. The facile dissemination of virus inoculum from ocular to nasal tissue, and the detection of virus in both NW and CW samples throughout the acute phase of ferret infection, highlights the ability for concurrent ocular and respiratory disease following influenza virus infection; not surprisingly, reports of conjunctivitis and influenza-like illness in the same individual have been documented during H7 outbreaks resulting in human infection [40, 51] . While much regarding the properties which regulate the ocular tropism of influenza viruses remains to be determined, our results highlight the potential for a range of influenza A subtypes to initiate infection through the eye and support the use of eye protection during occupational exposure to aerosols containing influenza viruses [19, 52, 53] .
This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All ferret procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. Animal studies were performed in accordance with the IACUC guidelines under protocol #2195TUMFERC-A3: ''Studies on the Pathogenesis and Transmission of Recombinant Influenza Viruses in Ferrets''.
Influenza A viruses of the H7, H5, and H1 subtype used in this study are shown in Table 1 . Virus stocks were propagated in the allantoic fluid cavity of 10 day old embryonated hens' eggs as previously described [26] ; virus stocks were confirmed by sequencing to be free of mutations. The 50% egg infectious dose (EID 50 ) for each virus stock was calculated by the method of Reed and Muench [54] following serial titration in eggs. Fluorescenttagged virus (NL/219-FL) was generated using formalin-inactivated NL/219 virus and a SAIVI Antibody Alexa Fluor 680 Labeling kit (Invitrogen) per manufacturer's instructions as previously described [32] . All experiments with HPAI viruses were conducted under biosafety level 3 containment, including enhancements required by the U.S. Department of Agriculture and the Select Agent Program [55] .
Male Fitch ferrets (Triple F Farms), 7 to 10 months old and serologically negative by hemagglutination inhibition to currently circulating influenza viruses, were used in this study. Ferrets were housed in a Duo-Flow Bioclean mobile clean room (Lab Products) for the duration of each experiment. Intranasal (i.n.) inoculations were performed under anesthesia as previously described using 10 6 EID 50 of virus diluted in PBS in a 1 ml or 100 ml volume [29] . Ocular (i.o.) inoculations were performed under anesthesia using 10 6 EID 50 of virus diluted in PBS in a 100 ml or 20 ml volume. Virus inoculum was administered dropwise to the surface of the right eye of each ferret and massaged over the surface of the eye by the eyelid.
Ferrets were monitored daily post-inoculation for morbidity and clinical signs of infection as previously described [29] . Any ferret which lost .25% of its pre-inoculation body weight or exhibited neurological dysfunction was euthanized. Virus shedding was measured on alternate days post-inoculation (p.i.) in nasal washes (NW), conjunctival washes (CW), and rectal swabs (RS). NW and RS samples were collected as previously described [28, 29] . CW were obtained by bathing the inoculated (right) ferret eye three times with 500 ml wash solution (PBS containing Pen/Strep, Gentamycin, and BSA) and collecting the run-off in a small petri dish, then swabbing the surface and surrounding conjunctival tissue of the right eye with a pre-wettened cotton swab for 5 seconds, and placing the swab in a collection tube containing the run-off liquid. All samples were immediately frozen on dry ice and stored at 270uC until processed.
To assess virus dissemination following i.n. or i.o. inoculation, three ferrets per group were inoculated with indicated viruses and euthanized 3 days p.i. for postmortem examination and collection of tissues for virus titration as previously described [29] . Tissue specimens were collected for virus titration were immediately frozen on dry ice and stored at 270uC until processed. Blood samples collected during necropsy were subjected to complete blood counts (CBCs) and serum chemistry analyses performed per manufacturer's instructions as previously described [56] .
Virus transmissibility following i.o. inoculation was assessed by inoculating ferrets by the ocular route with indicated viruses and, 24 hrs p.i., placing a naïve ferret in the same cage as an inoculated ferret [to assess transmission in the presence of direct contact (DC)] or in an adjacent cage with modified side-walls to allow air exchange between inoculated and contact animals via perforations but inhibiting direct or indirect contact between animals [to assess transmission by respiratory droplets (RD)] as previously described [30] . For each i.o. transmission experiment, an aliquot of each virus stock used to characterize transmissibility in previous publications by the i.n. route was tested. NW, CW, and RS samples were collected on alternate days p.i./post-contact (p.c.) to assess kinetics of virus shedding. Serum was collected days 17-21 p.i./p.c. to measure seroconverison. Animal research was conducted under the guidance of the Centers for Disease Control and Prevention's Institutional Animal Care and Use Committee in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited animal facility.
Sample titration and processing NW, CW, and RS samples were serially titrated in eggs, starting at a 1:10 dilution (NW, RS; limit of detection, 10 1.5 EID 50 /ml) or 1:2 dilution (CW; limit of detection, 10 0.8 EID 50 /ml). Virus infectivity for all samples was calculated by the method of Reed and Muench [54] . At the time samples were thawed for virus titration, RNA was extracted from CW samples using a QIAamp Viral RNA kit (Qiagen). Real-time RT-PCR was performed with a QuantiTect SYBR Green RT-PCR kit (Qiagen) using an influenza A virus M1 gene primer set to determine viral load [32] . Viral RNA copy numbers were extrapolated using a standard curve based on samples of known virus as previously described [32, 57] . Baseline levels were determined by collecting CW samples from uninfected ferrets.
Tissue specimens were homogenized in 1 ml cold PBS using disposable sterile tissue grinders and clarified by centrifugation before serial titration in eggs, starting at a 1:10 dilution. Ferret eye and conjunctival tissues were rinsed with PBS prior to virus titration. Eye, conjunctival, and nasal turbinate tissues are expressed as EID 50 /ml, while all other tissues are expressed as EID 50 /g.
Uninfected ferret corneal epithelial sheets were dissociated from excised whole ferret eyes following incubation in tetrasodium EDTA for determination of expression of surface sialoligosaccharides as previously described [20, 58] . To assess virus dissemination, ferrets were inoculated with NL/219-FL virus either i.o. (100 ml) or i.n. (1 ml) as previously described [32] . Fifteen minutes p.i., ferrets were euthanized and respiratory and ocular tissues were excised for ex vivo imaging using a Spectrum in vivo imaging system and Living Image 4.0 Software (Caliper Life Sciences). All ex vivo imaging was performed in triplicate. To quantify the presence of NL/219-FL virus in excised tissues, regions of interest were drawn around each tissue using Living Image 4.0 Software to obtain maximum relative efficiency values for each tissue, expressed as photons/second/cm 2 /steradian, the mean of which was generated from three ferrets per tissue as expressed in Figure 3 .
Tissues for immunohistochemistry (IHC) were collected day 3 p.i with the viruses indicated. or from uninfected ferrets, fixed by submersion in 10% neutral buffered formalin for 3 days, routinely processed, and embedded in paraffin. Immunohistochemical detection of influenza A virus nucleoprotein was performed as described previously [59] .
The Pearson product-moment correlation coefficient was generated to measure the correlation between viral titer and viral RNA copy number in CW sample using GraphPad Prism 5.0 (GraphPad Software, Inc.). Statistical significance for all other experiments was determined using Student's t test. Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation. Human immunodeficiency virus type 1 (HIV-1) gag encodes a polypeptide Pr55 gag that can self-assemble into virus-like particles (VLPs) [1] . During or soon after virus release from cells, Pr55 gag is cleaved by viral protease (PR) into four major products: matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and p6 domains [1] . PR is encoded by pol, which is initially translated as a Pr160 gag-pol polyprotein by a ribosomal frameshift event that occurs at a frequency of 5%, resulting in the expression of Pr160 gag-pol to Pr55 gag at a ratio of approximately 1:20 [2] . Pr160 gag-pol is incorporated into virions via interactions with assembling Pr55 gag [3, 4, 5, 6, 7, 8] . Pr160 gag-pol cleavage by PR yields reverse transcriptase (RT) and integrase (IN) in addition to Gag products. The PRmediated proteolytic cleavage of Pr55 gag and Pr160 gag-pol , known as virus maturation, is essential for the acquisition of viral infectivity [9, 10, 11, 12, 13] .
How PR is activated to mediate virus maturation is not completely clear. One proposal is that interaction among Pr160 gagpol molecules triggers the activation of embedded PR, which in homodimeric form mediates Gag and Gag-Pol cleavage following PR autocleavage from Pr160 gag-pol . Maintenance of the Pr55 gag / Pr160 gag-pol expression ratio is critical to virus assembly; the artificial overexpression of Pr160 gag-pol or PR drastically reduces virion production as a result of enhanced Gag processing by overexpressed PR activity [14, 15, 16, 17, 18, 19, 20] . Equally important is the Pr160 gag-pol sequence and structure, since sequence mutations upstream or downstream of PR often result in defective virus maturation or Gag cleavage [4, 21, 22, 23, 24] . Impaired Gag cleavage is assumed as being due, at least in part, to impaired PR activation, which is likely secondary to inadequate PR dimer interaction. Since natural RT is heterodimeric [25, 26] , there is speculation that RT in the Gag-Pol context facilitates Pr160 gag-pol -Pr160 gag-pol interaction via RT-RT interaction, which in turn influences PR activation. Consistent with this scenario, RT deletion mutations can lead to severely impaired PR-mediated Gag processing [23] . In addition, efavirenz (EFV), a nonnucleoside reverse transcriptase inhibitor that enhances RT dimerization in vitro [27, 28] , reduces virus production as a result of greatly enhanced Gag and Gag-Pol cleavage [29, 30] . Furthermore, a single amino acid substitution in RT (W402A) leads to significantly reduced virus production due to markedly enhanced PR-mediated Gag cleavage [31] . Combined, these data suggest that the RT domain plays an important role in PR activation by influencing PR dimer interaction.
It is likely that altered conformation induced by the RT mutation significantly impacts PR dimer interaction, resulting in premature or impaired PR activation. Accordingly, structural conformations rather than specific sequences may be major determinants of the PR activation process. A protein sequence unrelated to HIV-1 but possessing dimerization capacity may therefore promote PR activation by facilitating PR dimer interaction when fused to the end of PR. To test this possibility, we removed the RT and IN sequences and placed a leucine zipper (LZ)-coding sequence at the C-terminus of PR. Results indicate that LZ placement significantly reduced virion release due to enhanced Gag cleavage, similar to observations for RT W402A mutations. These results support the hypothesis that the placement of heterologous protein dimerization sequences downstream of PR can significantly enhance Gag processing efficiency by promoting PR activation.
To determine whether forced PR dimer interactions affect virus assembly and processing, we fused a LZ protein dimerization domain either singly or in tandem repeat to the C-terminus of an HIV-1 Gag-Pol truncated construct (Gag/PR), which is virusassembly competent but processing-defective [23] . The resulting constructs were designated PRWz and PRWWz (Fig. 1) . We used PRKz and PRKKz constructs containing the dimerizationdefective LZ mutant version (Kz) as controls. Kz fusion to PRWz at the wt LZ C-and N-termini yielded constructs PRWKz and PRKWz, respectively. Each mutant was transiently expressed in 293T cells. Virus particle assembly and processing were analyzed by Western immunoblotting. The results shown in Figure 2A indicate that Gag/PR transfectants produced substantial quantities of VLPs at levels that were near wild-type. Unprocessed Gag (the Gag precursor Pr55) and incompletely processed Gag (the intermediate p41gag) represent two major Gag products compared to wt in our supernatant and cell samples ( Fig. 2A , lanes 1 vs. 2). This is consistent with a report stating that a deletion in a downstream pol sequence significantly impairs PR-mediated virus maturation [23] .
PRWz and PRWWz transfectants expressed readily detectable Gag, but produced barely detectable virus-associated Gag, suggesting a severe defect in virus assembly or release ( Fig. 2A , lanes 3 and 4). In contrast, cells transfected with PRKz or PRKKz released readily detectable (though incompletely processed) Gag (lanes 5 and 6), similar to the Gag/PR scenario. VLP levels produced by PRWKz and PRKWz (lanes 7 and 8) were inbetween those produced by PRWz and PRKz. Since enhanced or premature Gag cleavage by PR can lead to significantly reduced virus release, and since the wt LZ fusion-containing constructs exhibited higher ratios of cellular p24 gag to Pr55 gag compared to those found in Gag/PR cell lysates ( Fig. 2A , lanes 3-4 vs. lane 2), we suggest that the LZ-associated virus production defect was largely due to enhanced Pr55 gag cleavage efficiency.
Since cell samples were collected between 48 and 72 h posttransfection, it is possible that Gag processing reached a level of stability that prevented us from detecting any differences in efficiency between the wt form and mutants. To test this possibility, and to confirm the effect of LZ domain placement on Gag processing efficiency, we collected samples at 24 and 48 h following the transient expression of wt and mutants. We observed that both PRWz and PRWWz showed significantly higher cellular p24 gag /Pr55 gag ratios compared to those of wt or Gag/PR (Fig. 2B) .
Although PRWKz and PRKWz showed cellular Gag processing profiles similar to those of PRKz and PRKKz at 48 h, they displayed higher p24 gag /Pr55 gag ratios compared to Gag/PR at 24 h post-transfection ( Fig. 2B upper panel, lanes 15-16 vs. lane 10) . This suggests that LZ domain placement significantly enhanced Gag processing efficiency. The virus-associated Gag precursor that we detected may reflect, at least in part, the release of assembled Gag molecules that escaped PR-mediated cleavage (Fig. 2B, lanes 7-8) .
To determine whether LZ placement affected virion production by wt or assembly-competent mutants in trans, we coexpressed PRWz or PRWWz with the wt or the HIV-1 protease-defective mutant D25, and observed that virus-associated Gag was markedly reduced when D25 was cotransfected with either PRWz or PRWWz at a 1:1 ratio (Fig. 2C ). Similar results were observed when the PRWz or PRWWz was coexpressed with a wt HIV-1 expression vector (data not shown). Combined, these results suggest that (a) PRWz and PRWWz both provided functional PR, and (b) the LZ-triggered virion assembly defect was primarily due to a higher Gag cleavage efficiency. The PRWWz transfectant frequently expressed a lower Gag level compared to other constructs (Fig. 2B, lane 12) , likely the result of increased proteolytic degradation of Gag mediated by the PR.
To determine whether the PRWz or PRWWz assembly defect is directly associated with viral PR activity, we treated PRWz and PRWWz transfectants with Saquinavier, an HIV-1 PR inhibitor (designated as PI). As expected, virus-associated PRWWz Gag (Pr55 gag and p41 gag ) that was previously undetectable (or barely detectable) became readily detectable when PI concentrations were gradually increased (Fig. 3A) . We noticed that wt cellular p24 gag was still easily detected, even though PRWWz p24 gag was undetectable under the same treatment conditions (Fig. 3A , lanes 3 vs. 7). This suggests that PRWWz is more susceptible to a protease inhibitor than wt, despite having higher Gag cleavage efficiency.
Since we centrifuged the culture supernatant through 20% sucrose cushions, we assumed that the recovered Gag would be present in pelleted particles. To confirm that the recovered Gag was from VLPs, we observed supernatant samples ( Fig. 3B ) with a transmission electron microscope, and found spherical wt and mutant Gag particles with electron-dense cores in PI-treated transfectant samples (Figs. 3D and 3F). However, mature virions with cone-shaped cores were only detected in non-PI-treated wt transfectant samples (Fig. 3C ). Some vesicles lacking cores were noted, but virion-size particles containing electron-dense cores were not detected in mock-transfected samples, or barely detected in PRWWz transfectant supernatant that had not been treated with PI (Figs. 3E and 3G). These data support the hypothesis that the LZ-incurred assembly defect is PR activity-dependent.
We looked for correlations between enhanced PR-mediated Pr55 gag cleavage efficiency and increased Gag-PR-LZ multimerization capacity. Believing that the potent Gag assembly domain might determine chimera multimerization status, we predicted that the contribution of LZ to enhanced chimera multimerization would be barely (if at all) detectable. We therefore assessed chimera multimerization capacity in a Gag assemblydefective context. After constructing an assembly-defective mutant (designated MoGag) and confirming that the mutation significant-ly impaired Gag assembly (Fig. 4B ), we cloned PR-LZ chimeras into MoGag. To block the effect of PR activity on Gag-PR-LZ chimera assembly assays, all chimeras were introduced into a PRinactivated HIV-1 Pr160 gag-pol -expression plasmid GPfs [4] , with gag and pol in the same reading frame (Fig. 4A ). Results from repeated independent experiments indicate that chimeras with predicted molecular weights were detected in both supernatants and cell lysates following transient expression in 293T cells, suggesting that all chimeras were capable of assembly and release to some extent. However, we noted that MoGagfsWz transfectants produced more chimeric VLPs than MoGagfsD or MoGagfsKz (Figs. 4C and 4D). To confirm that MoGag was multimerizationdefective and that LZ did enhance assembly-defective Gag multimerization, we subjected wt Gag and each mutant to velocity sedimentation analyses. A non-myristylated (myr-) Gag mutant [32] known to be severely defective in both membrane binding and multimerization served as a negative control. Our data indicate that most of the wt Gag was recovered at fractions 3 to 5; in contrast, most myr-Gag and substantial amounts of MoGag were recovered at fractions 1 and 2. Portions of MoGagfsD and MoGagfsKz were detected at lower sucrose density fractions, whereas MoGagfsWz was almost completely recovered at higher sucrose density fractions (Fig. 4E ). Unlike MoGagfsWz, which mostly sedimented at fractions 4 and 5, considerable amounts of MoGagfsWKz and MoGagfsKWz were also recovered at fraction 3, and low but detectable amounts were observed in fraction 2. This sedimentation pattern was similar to that of MoGagfsKz (Fig. 4E , three bottom panels). Also similar to MoGagfsKz, both MoGagfsWKz and MoGagfsKWz were incapable of efficiently assembling into chimeric VLPs (data not shown). We observed this result in repeated independent experiments. This finding suggests that when fused to the PR C-terminus, a LZ tandem repeat containing a wt and mutant LZ (WKz or KWz) does not enhance Gag-PR multimerization as effectively as a wt LZ tandem repeat (Wz). This may partly explain why PRWKz and PRKWz showed relatively lower Gag cleavage efficiency compared to PRWWz (Fig. 2) .
Compared to MoGag, which had significant amounts of Gag detected at lower sucrose density fractions (1 and 2), MoGagfsD molecules were mostly sedimented at fractions 3 to 5, a difference that may be explained in part by p6 pol -PR contributing to MoGagfsD multimerization via PR dimer interaction. Although GagfsD presented an efficient multimerization profile, it produced VLPs at a relatively lower level compared to WtGag (Figs. 4C and 4D). This may have been due to its lack of p6 gag , which is required for efficient virus budding [33, 34] .
Our next question was whether LZ enhanced the cleavage efficiency of the assembly-defective mutant MoGag. MoGag with an LZ fusion was expressed in a Gag/PR context, thus expressing both Pr55 gag and PR containing Gag-Pol or Gag-PR(-LZ) fusions (Fig. 5A ). As expected, the insertion of LZ into MoGag/PR at the PR C-terminus resulted in significantly enhanced Gag cleavage efficiency (Fig. 5C, lanes 11 vs. 15 ), suggesting that the LZ enhancement of Gag-PR multimerization is correlated with increased PR-mediated Gag cleavage efficiency. Inefficient Gag cleavage, likely due to either impaired PR activation as a result of Pol truncation or to inhibited PR activity due to a protease inhibitor, resulted in improved MoGag VLP assembly (Fig. 5C , 12 and 15-18 ). This suggests that the MoGag assembly defect is PR activity-dependent, at least in part. The MoPRWWz had significantly higher cellular p24 gag /Pr55 gag ratios compared to MoGag/PR, but slightly lower compared to PRWWz (Figs. 5B and 5C), suggesting that the multimerization-defective Gag mutation reduced the LZ-mediated enhancement of Gag cleavage. It also supports the proposal that the Gag assembly domain plays a role in PR activation.
Although myristylation is not required for Gag-Pol viral incorporation [7, 35] , it is essential for Gag multimerization and virus assembly [11, 36] . To determine if a myristylation signal is necessary for the LZ enhancement effect on Gag cleavage, we introduced myr-into wt, Gag/PR, PRWWz, and PRKKz, and measured the Gag processing efficiency of each mutant. Our results indicate an efficient Gag processing profile for myr-PRWWz, similar to that of its myristylation-positive counterpart, PRWWz (Fig. 6 ). Since myristylation is essential to Gag membrane binding and virus assembly, we failed to detect virus-associated Gag products in any of the myr-supernatant samples (data not shown). This suggests that the LZ insertion made a significant contribution to enhanced Gag cleavage, regardless of the presence or absence of a myristylation signal.
Despite a lack of direct evidence, it is generally accepted that Gag-Pol molecule dimerization or multimerization triggers HIV-1 PR activation, which mediates Gag and Gag-Pol cleavage. Here we demonstrated that the insertion of LZ at the C-terminus of PR triggers markedly enhanced PR-mediated Gag cleavage efficiency, which is associated with increased Gag-PR multimerization capacity. This suggests that an HIV-1-unrelated protein sequence capable of self-association can enhance Gag cleavage efficiency when fused to the PR C-terminus. It also provides evidence in support of the assumption that Gag-Pol/Gag-Pol interaction triggers PR activation.
In an assembly-competent Gag/PR context, the wt LZ insertion resulted in markedly reduced, PR activity-dependent virus production (Fig. 2) . Cellular Pr55 gag was barely detected in PRWWz at 12 and 24 h post-transfection, while most Pr55 gag remained unprocessed in wt transfectants 12 h post-transfection (data not shown). Combined, these results suggest that LZenhanced Gag cleavage is associated with premature Gag processing due to premature PR activation. We suggest that when fused at the PR C-terminus, the LZ domain facilitates PR-PR interaction via enhanced interactions among MA-CA-NC-PR-LZ chimeras. Gag is subsequently cleaved by PR in trans, either by PR embedded in the chimera, or by a mature and fully processed PR dimer.
Several researchers have suggested that the PR-mediated initial cleavage occurs via an intramolecular mechanism [37, 38, 39, 40, 41] . Although we did not search for the presence of a PR dimer, PR was theoretically capable of being released from the chimera as a fully processed dimer, since the cleavage site at the PR/LZ junction remained intact. Compared to the wt LZ fusion, the placement of a mutant LZ (Kz) at either the N-or Cterminus of the wt LZ (Wz) resulted in reduced Gag cleavage efficiency (Fig. 2) . This may be attributable to the inability of WKz or KWz to promote Gag-PR multimerization (Fig. 4E) .
Although myristylation is required for Gag membrane binding (which may in turn promote efficient Gag multimerization [32] ), we found evidence that myr-Gag/Pol or MoGag/Pol were capable of mediating Pr55 gag processing (Figs. 5 and 6 ). This finding suggests that neither membrane association nor an assembly-competent Gag domain is essential for the activation of PR embedded in Gag-Pol. It is likely that myr-Gag-Pol can still undergo dimerization to a level that is sufficient to trigger PR activation. Previous studies have shown that myr-Gag-Pol can efficiently cleave Pr55 gag in trans and be packaged into Pr55 gag VLPs [4, 7, 35] . The multimerization defect as a result of membrane binding apparently does not significantly compromise the LZ enhancement of PR-mediated Gag cleavage.
The next question is why premature PR-mediated Gag and Gag-Pol cleavage do not occur during virus assembly, given that multiple assembly domains outside of protease promote Gag-Pol multimerization. One possibility is that the transframe peptide p6 pol may play a role in modulating PR dimer interface interaction, thus preventing premature PR activation. Due to a blocking mutation at the p6 pol /PR cleavage, p6 pol -retaining PR is defective in mediating virus maturation [42, 43, 44, 45] , suggesting that a fully functional PR requires the removal of p6 pol . However, to our knowledge there are no reports on the role of p6 pol in PR dimer interaction in a Gag-Pol context. We found that a Gag-Pol mutant with deleted p6 pol was incapable of efficiently processing coexpressed Pr55 gag , which argues against the possibility of p6 pol playing a role in suppressing PR activation [46] . According to one recent study, the insertion of a larger reporter sequence in the p6 pol region markedly impairs virus maturation, whereas partial substitution with a heterologous sequence does not [47] . This suggests that structural conformation, rather than a specific sequence upstream of PR, is important for determining PR activation.
Although the RT domain is essential for proper PR-mediated Gag cleavage, the RT homodimerization domain has no enhancement effect on Gag cleavage [48] unless EFV (an HIV-1 RT-dimerization enhancer) is added to culture medium [23, 49] . In contrast, we found that LZ is capable of triggering Gag cleavage enhancement when placed at the PR C-terminus. The RT domain apparently plays a role in preventing premature PR activation. Previous studies suggest that the downstream structural conformation of PR is important in terms of modulating PR activation. First, a single RT amino acid substitution (W402A) that is not known to have major impacts on in vitro RT dimerization [50] markedly enhances Gag processing [31] . An additional partial deletion at the C-terminus of p66RT not only reverses W402Atriggered Gag cleavage enhancement, but also markedly impairs virus maturation. In contrast, truncated Gag-Pol mutants that retain intact p66 or p51 RT domains still respond to the enhancement effect of W402A on Gag processing [31] . Second, substitution mutations in RT are capable of neutralizing the enhancement effect of EFV on Gag processing [49] . Combined, these data suggest that conformational changes in Gag-Pol may significantly affect PR dimer interface interactions, leading to either premature or insufficient PR activation. Our finding that the fusion of HIV-1-unrelated dimerization protein sequences at the C-terminus of PR sharply enhanced Gag cleavage strongly supports the hypothesis that structural conformation, rather than specific sequences, largely determines PR activation status. The RT structure domain in the Gag-Pol context may help prevent PR from premature activation via a conformation that prevents the Gag and Gag-LZ chimeras. HIV-1 Gag domains, pol-encoded PR, and the transframe peptide p6 pol are indicated; fs denotes a frameshift mutation that forces gag and pol into the same reading frame. The fused leucine zipper (LZ) domains wt (Wz) or mutant (Kz) at the PR C-terminus are indicated as described in the Figure 1 legend; x denotes substitution mutations in CA, NC (NC15A), and PR that blocked either Gag assembly or PR activity. (B-E) Assembly and multimerization of HIV-1 Gag mutants. 293T cells were transfected with designated constructs. At 48-72 h post-transfection, culture supernatants and cells were collected and subjected to Western immunoblotting (panels B and C). (D) Gag-associated proteins from medium or cell samples were quantified by scanning immunoblot band densities (C). Ratios of Gag in media to Gag in cells were determined for each construct, and compared with wt release level by dividing the release ratio for each chimera by the wt ratio. (E) Velocity sedimentation analysis of cytoplasmic Gag precursor and Gag-LZ chimeras. Homogenized and extracted cytoplasmic lysates were centrifuged through consecutive 25%, 35%, and 45% sucrose gradients at 130,0006g for 1 hour. Fractions were collected from the top of each gradient. Aliquots of each fraction were subjected to SDS-PAGE (10%) and probed with a monoclonal antibody directed against HIV-1 CA. doi:10.1371/journal.pone.0032845.g004 PR dimer interface from interacting efficiently. This conformation may change during Gag-Pol packaging, thus supporting more efficient PR dimer interaction.
Pettit et al. [41] have demonstrated that inactivated PR dimer interface mutations can be compensated for to some extent by extra PR sequences in the Gag-Pol context. Altered PR dimerization kinetics or activity has been identified in several studies of PR-containing C-or N-terminal extensions into Gag-Pol [21, 23, 24, 38, 40, 43, 51, 52] . In most cases, it is not feasible to assay the impacts of these constructs on virus assembly or PR-mediated virus maturation, due to overlapping gag and pol reading frames. Our approach provides a convenient system for analyzing the impacts of mutations on PR dimer interactions by assessing virus particle assembly and processing. Studies are underway to determine if LZ sufficiently compensates for the inactivation of PR dimer interface mutations.
To place a leucine zipper (LZ) in frame into the HIV-1 PR Cterminus, we engineered a pBRCla-Sal plasmid cassette containing an HIV-1 coding sequence (from ClaI-nt.831 to SalI-nt.5786) and a pcDNA3.1-myc/hisA polylinker inserted at the IN Cterminus. DNC(wtZip) and DNC(Kzip) [53] served as templates for amplifying the respective wt and mutant LZ domains of human CREB [54] using the forward primer 59-CGGGATCCTGGAG-GAGGACGAGAGTGTCGTAG-AAAGAAG-39 and reverse primer 59-CCAAGCGGCCGCGATTTGTGGCAG-TAT-39. Plasmids containing the wt and mutant LZ (provided by E. Barklis [55] ) were used to construct the DNC(wtZip) and DNC(Kzip). The human CREB LZ sequence is 284-RE-CRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYC-HKSD-327. The underlined amino acid residues E298, R300, E305, Q307, I312, E314, and L321 were mutated to Lys, and N308 was mutated to His, yielding the mutant LZ [54] . Amplified fragments were digested with BamHI and NotI and ligated into the pBRCla-Sal cassette, yielding constructs PRWz and PRKz, respectively. To add the LZ copy, PCR-amplified wt and mutant LZ fragments were purified, digested with a restriction enzyme, and ligated into the PRWz and PRKz, yielding the constructs PRWWz, PRWKz, PRKKz, and PRKWz.
The Gag assembly-defective mutant MoGag was constructed by recombining the CA mutant M39A/W184A/M185A with NC mutant NC15A, which was kindly provided by P. Spearman [56] . NC15A has 15 NC-basic residues replaced with alanine. M39A/ W184A/M185A was created by overlapping PCR with the following mutagenic primers: for M39A, 59-CTGATAGCGCT-GAAAATGCGGGTATCA-39, and for W184A/M185A, 59-CAACAACGTTTCTGTAGCCGCATTTTTTAC-39. The Mo-Gag mutation was cloned into the indicated PR-leucine zipper constructs. As described previously, Gag/PR has deleted RT and IN coding sequences [23] . In D25, Arg is substituted for the PR catalytic residue Asp [20] . The backbone of all expression constructs is the HIV-1 proviral plasmid HIVgpt [57] .
293T cells were maintained in DMEM supplemented with 10% fetal calf serum. Confluent 293T cells were trypsinized, split 1:10 and seeded onto 10-cm dish plates 24 hours before transfection. For each construct, 293T cells were transfected with 20 mg of plasmid DNA by the calcium phosphate precipitation method (18) , with the addition of 50 mM chloroquine to enhance transfection efficiency.
Culture media from transfected 293T cells were filtered through 0.45 mm-pore-size filters, followed by centrifugation through 2 ml of 20% sucrose in TSE (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA) plus 0.1 mM phenylmethylsulfonyl fluoride [PMSF]) at 4uC for 40 min at 274,0006g (SW41 rotor at 40,000 rpm). Viral pellets then were suspended in IPB (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.02% sodium azide) plus 0.1 mM PMSF. Cells were rinsed with ice-cold PBS (phosphate-buffered saline), scraped from the plates, collected in 1 ml of PBS and pelleted at 2,500 rpm for 5 min. The cell pellets were resuspended in 250 ml of IPB plus 0.1 mM PMSF, and then subjected to microcentrifugation at 4uC for 15 min at 13,7006g (14,000 r.p.m.) to remove cell debris. Either supernatant or cell sample was then mixed with equal volumes of 26 sample buffer (12.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue) and 5% b-mercaptoethanol and boiled for 5 min. Samples were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes. Membrane-bound Gag proteins were immunodetected using an anti-p24 gag (mouse hybridoma clone 183-H12-5C) monoclonal antibody at a 1:5,000 dilution from ascites. The secondary antibody was a rabbit anti-mouse (HRP)-conjugated antibody at 1:15,000 dilution as appropriate and the procedures used for HRP activity detection followed the manufacturer's protocol (Pierce). Immunodetected bands on film were quantified by using AlphaImager 2000 (Alpha Innotech Corp.) and Image J software.
Cells were rinsed twice with PBS, pelleted and resuspended in 1 ml TEN buffer containing Complete protease inhibitor cocktail followed by homogenization. The cell lysates then were centrifuged at 3,000 rpm for 20 min at 4uC. Five hundred ml of the postnuclear supernatants were mixed with an equal amount of TEN buffer, and were then applied to the top of a pre-made 25-45% discontinuous sucrose gradient. This gradient was prepared in TEN buffer containing 1 ml of each of 25%, 35%, and 45% sucrose. The gradient was then centrifuged at 130,0006g for 1 hour at 4uC. Five 0.8-ml fractions were collected from the top of the centrifuge tubes. The proteins present in aliquots of each fraction were precipitated with 10% TCA and subjected to western blot analysis as described in the membrane flotation assay.
Virus-containing supernatants were centrifuged through 20% sucrose cushion. Concentrated viral sample was placed for 2 min onto a carbon-coated, UV-treated 200 mesh copper grid as described [58] . Sample-containing grids were rinsed 15 s in water, drained off water with filter paper, and stained for 1 min in filtered 1.3% uranyl acetate. Staining solution was drained off by applying filter paper to the edge of the grid. Grids were left to dry before viewing in a JOEL JEM-2000 EXII transmission electron microscope. Images were collected at 20,0006 and 60,0006. Year in review in Intensive Care Medicine 2011: I. Nephrology, epidemiology, nutrition and therapeutics, neurology, ethical and legal issues, experimentals  Acute kidney injury continued to be hot topic in 2011. Early diagnosis remains an issue of increasing importance, with the aim being to install early preventive measures in critically ill patients. Several very promising biomarkers turned out to have serious limitations in specific disease states, e.g. NGAL in septic shock [1, 2] . Cystatin C is another biomarker that is presumed to allow the early detection of small declines in glomerular filtration rate (GFR), but may also reflect tubular damage if measured in the urine. Royakkers et al. [3] investigated both urine and serum cystatin c in a multicentre prospective observational trial including 151 patients, and could not detect any diagnostic value for urinary cystatin c, only a very moderate performance of serum cystatin c for the prediction of AKI (AUC 0.72), and a very poor one for predicting the requirement for renal replacement therapy RRT (renal replacement therapy) (AUC B0. 66) . Serum cystatin C may also be futile as a biomarker for AKI (acute kidney injury) in the setting of renal obstruction [4] .
Discrimination between transient and persistent AKI is of clinical relevance but currently often based on urinary indices which are not very reliable in critically ill patients. Darmon et al. [5] used a different approach by determining Doppler resistive indices in 51 patients. They found that a RI [0.795 predicted persistent AKI with an OR of 1.85 (95% CI 1.2-2.85) upon applying logistic regression analysis. Considering the large influence of interobserver variability on this method, larger multicentre trials are required to prove the utility of this method in daily clinical practice.
The question of the optimal mean arterial pressure to optimise renal perfusion and renal function is still unresolved. Redfors et al. [6] approached this question in an elegant crossover study including 12 postcardiac surgery patients with norepinephrine-dependent vasodilatory shock. Increasing the target mean arterial pressure (MAP) from 60 to 75 mmHg resulted in significantly improved GFR (27%) and urinary output associated with increased oxygen delivery (13%) and reduced renal oxygen consumption. A further increase of MAP to 90 mmHg was ineffective or even detrimental for these parameters. The results indicate severely disturbed autoregulation of the kidney in vasodilatory shock and possible benefits from targeting a higher MAP of around 75 mmHg.
Three studies investigated the epidemiology of AKI of specific aetiologies. Hoste et al. [7] examined the incidence of contrast-media induced AKI (CI-AKI) in 787 predominantly surgical (*75%) patients. As opposed to data obtained from non-critically ill patients, the authors found a near-threefold increased incidence, with rates of between 16 and 23% depending on the definition used. Contrast-induced AKI was associated with a significantly increased length of stay, higher requirements for renal replacement therapy and mortality (1-year mortality 55.5 vs. 24% in patients without CI-AKI). Decreased renal function at the time of intervention, lower mean arterial pressure, vasoactive therapy, and administration of diuretics were found to be major risk factors for CI-AKI. This study, currently the largest of its kind, underlines the importance of nephrotoxic medication in this specifically vulnerable group of patients, and sets the stage for future randomised trials investigating effective preventive measures against CI-AKI.
Pettila et al. [8] and Nin et al. [9] reported the epidemiology of AKI associated with severe H1N1 infections in different parts of the world. They found incidence rates of AKI of between 34 and 51%, demonstrating the high frequency of this complication in patients suffering from H1N1 infection. The occurrence of AKI was associated with a significantly increased risk of hospital mortality. Nin et al. specifically addressed the question of early AKI versus late AKI (after ICU day 2), and found a worse outcome for the latter, indicating that a large proportion of early AKI occurring in the context of H1N1 infection is presumably due to pre-renal factors like dehydration or hypotension, which can be easily corrected, resulting in rapid renal recovery. However, as demonstrated in accompanying correspondence by Nin et al. [10] , viral particles could also be found within renal tissue obtained from a few biopsies, which possibly contributed to AKI. These studies provide further insight into the pathophysiology of AKI in the setting of severe viral infections; it is most probably a mixture of pre-renal causes combined with endothelial damage and severe disturbance of the coagulation system caused by exaggerated inflammatory response and finally complicated by secondary bacterial infections. Direct renal effects of viral infiltration of the kidney, however, appear to be a very rare mechanism for AKI [11] .
Sepsis is considered a well-established risk factor for AKI, with sepsis-associated AKI showing a very modest prognosis, substantiated by mortality rates of up to 75% [12] . On the other hand, AKI itself is considered a systemic disease associated with impaired immune function. Mehta et al. [13] investigated the frequency and the clinical consequences when sepsis developed as a complication of AKI in 618 critically ill patients from the multicentre PICARD study. They found that about 40% of the patients developed sepsis at a median of 5 days after AKI, with similar consequences for outcome as seen in sepsis-associated AKI itself. This study demonstrates the importance of preventing AKI as well as taking measures against infections in patients already suffering from AKI to avoid consecutive development of sepsis.
Another condition in which alterations in patients' immune response may significantly influence outcome is liver cirrhosis. Berry et al. [14] showed that a reduction in HLA-DR expression by monocytes to less than 40% of admission values was highly correlated with increased 90-day mortality. The changes in HLA-DR expression were also associated with reduced Th1 response to antigenic stimulation and exaggerated secretion of the antiinflammatory cytokines IL-10 and IFN-c.
Three epidemiologic studies investigated the outcomes of postsurgical patients admitted to the ICU. Analysing the data for 88,504 patients collected from several Austrian ICUs over a period of 11 years, Rhodes et al. [15] found that pre-existing morbidities such as chronic renal disease, respiratory or cardiac failure, cirrhosis, alcoholism, acute kidney injury or nonmetastatic cancer significantly impaired outcome. Independent from those risks, reason for admission, urgency of operation, age and SAPS II score turned out to be major factors associated with hospital mortality. An interesting observation was the fact that survival had improved over the investigated period of 11 years.
Another important risk factor for increased morbidity and mortality appears to be postoperative red blood cell transfusion, as shown by Mohnle et al. [16] , who analysed a cohort of 945 patients with a low-to-moderate risk profile after coronary artery bypass grafting among 5,436 patients enrolled in an international multicentre trial. Patients receiving red blood cell transfusions had a higher likelihood of experiencing cardiac events or harvest site infections, and suffered from impaired composite morbidity outcome which included hospital mortality, renal failure, pneumonia and mediastinitis.
An increased body mass index may be protective in surgical intensive care unit patients. Hutagalung et al. [17, 18] analysed ICU and hospital mortality in 9,935 patients, and found that both overweight (BMI 25-29.9 kg/m 2 ) and obese (BMI 30-39.9 kg/m 2 ) patients showed a significantly lower 60-day hospital mortality than very obese and even normal weight patients. Interestingly, the improved survival prevailed despite a higher incidence of AKI in these specific weight groups, as already shown in another recent study [18] .
The association of older age and outcome with prolonged ICU stay was investigated in a cohort of 11,395 cardiac surgery patients by Ettema et al. [19] . The authors were able to demonstrate that all three major currently applied prediction models for outcome-the Parsonnet model, the Huijskes model and the EuroSCORE-showed poor calibration with age [70 and decreasing discrimination, which was worst for age [80 (AUCs were 0.68, 0.69 and 0.64, respectively). Obviously, outcome and recovery in older patients is rather more dependent on their general condition than on the conventional parameters used for scoring systems.
Simple, widely available lab tests could represent useful markers and predictors of outcome in ICU patients, especially when a strong rationale supports their potential prognostic value. For instance, eosinophil count is tightly and readily regulated by cytokines and is related to the stimulation of the adrenal axis. Likewise, the presence of eosinopenia has already been reported as a marker of infection. In a large prospective study, Abidi et al. [20] evaluated the prognostic value of serial eosinophil count measured daily from ICU admission until the seventh day of stay in 200 patients. The absolute eosinophil count was lower over the entire ICU stay in nonsurvivors. Eosinopenia (\40 cells/mm 3 ) was found to be an independent predictor of ICU mortality, with an area under the ROC curve of 0.82. These findings highlight the usefulness of the inexpensive and probably underused leukocyte formula in the ICU.
Similarly, the presence of hypoxic hepatitis-defined as the presence of a transient increase in serum aminotransferase activity of over 20 times the upper limit of the normal range in the absence of putative causes of liver necrosis (virus, drugs)-in patients with cardiocirculatory or respiratory failure was carefully scrutinised in a large cohort of 1,066 patients admitted to a medical ICU [21] . The mortality of the 118 patients who fulfilled the criteria of hypoxic hepatitis was indeed increased, especially in the subgroup of patients who required vasopressor therapy. After adjustment, the presence of hypoxic hepatitis was found to be a strong independent risk factor for mortality in patients receiving vasopressor therapy.
Another common finding is corticosteroid insufficiency related to critical illness, a condition associated with increased mortality. The diagnosis of this condition is challenging, as the usefulness of the ACTH test as well as the type of cortisol assay that should be used are still debated. Molenaar et al. [22] compared the values of total and free cortisol after an ACTH test. They found in a set of 112 patients (49 with sepsis) that the diagnosis of adrenal failure was not affected by the type of test used, as free and total cortisol measurements were closely related. These findings could have important implications for daily practice.
The search for biomarkers of sepsis outcome is an intense field of clinical research. Several new potential markers were assessed and validated by different teams who published their reports in the journal in 2011. An early rise in circulating endothelial protein C was found to be associated with a high mortality [23] . This finding confirms the activation of coagulation at the initial stage of sepsis, and suggests a pathogenetic role for endothelial protein C in the pathway.
In another study [24] , the concentration of cardiac troponin T detected by a high-sensitivity assay was found to be elevated in all patients with severe sepsis, and this level was correlated with other indices of poor outcome of sepsis, even though it was not found to be an independent predictor of survival. These findings further support the presence of myocardial injury in sepsis, in proportion to the severity of the sepsis, consistent with other data.
Likewise, the risk of acquisition of a second infection in patients with sepsis was found to be increased when the dendritic cell count was persistently low [25] . A selective depletion of circulating plasmacytoid and myeloid dendritic cells was demonstrated by these authors and by others early after the onset of sepsis. The study suggests that the persistence of low levels increases susceptibility to the development of a second infection, and opens up a large field for further research.
High-mobility group box 1 is a histone that plays a role in the coordination of inflammatory pathways. Barnay-Verdier et al. [26] demonstrated the presence of antibodies directed against high-mobility group box 1 in plasma samples from patients with sepsis. The presence of these antibodies could play a protective role, since the mortality was found to be lower in patients with the highest levels. Moreover, the emergence of these antibodies during the course of sepsis was detected more often in survivors than in nonsurvivors.
The pathogenesis of encephalopathy associated with sepsis could include mitochondrial dysfunction. Therefore, mutations and polymorphisms of mitochondrial DNA might affect the cerebral response to sepsis. In a welldefined cohort of Chinese patients with sepsis, Yang et al. [27] found that a haplogroup of mitochondrial DNA was a strong predictive factor for the development of septic encephalopathy. If confirmed in other populations, such findings pave the way for genomic research in the ICU.
In another exciting line of clinical research, the metabolic alterations of subcutaneous fat tissue were characterised by microdialysis [28] . These researchers found that tissue glycerol and glucose levels were higher during septic shock than during severe sepsis. In contrast, lactate, pyruvate and lactate-to-pyruvate ratios were similar in both conditions. These important findings are likely to change our understanding of the role of fat tissue in the metabolic response to sepsis.
In 2011, several important findings in the fields of metabolism and therapy were reported in the journal. These were new physiopathological insights based on commonly recorded data, or more sophisticated approaches using cutting-edge technologies. New therapeutic advances were presented as well.
Blood glucose concentration and glucose metabolism continuous to be a major topic of interest and is still controversial. During the last decade, neither the most cited study in Intensive Care Medicine [29] nor the largest interventional study published in Intensive Care Medicine [30] has been able to settle the issue of tight glucose control. During 2011, Mackenzie et al.
[31] contributed with a systematic analysis of a local database containing blood glucose concentrations and outcomes using a metric methodology. In principle, three different measures that influenced outcomes in terms of mortality were identified out of the blood glucose concentrations; hyperglycaemia, hypoglycaemia, and glucose variability. Needless to say, the study did not include the possibility of differentiating the relative predictive values of the three measures, but as pointed out by Krinsley [32] in an accompanying editorial, future studies of blood glucose control cannot avoid including all three measures.
Meyfroidt et al.
[33] from the working group who presented the initial report on tight glucose control, reported the advantage of an automatic predefined blood glucose threshold. The incidence of hypoglycaemia was reduced by 1/3, but the variability of blood glucose was not affected.
The difficulties involved in feeding critically ill patients by the enteral route are well recognised. Often it is gastric emptying that is the problem. Nguyen et al. [34] have investigated the absorption of fat and carbohydrates in the small intestine in relation to motility following surgical repair of abdominal aortic aneurysms. Motility was impaired as compared to controls. This resulted in reduced absorbtion of both fat and carbohydrate. Over time, it was demonstrated that the reduced fat absorbtion was more prolonged after the surgical procedure. This technique opens up interesting possibilities and seems suited for use in evaluations of other groups of critically ill patients, and perhaps to guide the composition of enteral formulations as suggested by the authors.
The optimal nutrition of critical ill patients is a really hot topic, in particular after the EPaNIC study favouring delayed parenteral nutrition [35] . The TICACOS study by Singer et al.
[36] is a pilot study in mechanically ventilated patients (n = 112) employing a bundle comprising supervised nutrition intervention including indirect calorimetry, as compared to a body weight guided protocol. The indirect calorimetry group received a higher intake of calories and protein, demonstrating the feasibility of indirect calorimetry. The fact that higher intake was also associated with a longer ICU stay is in total accord with the abovementioned EPaNIC study. As pointed out in an accompanying editorial, the use of indirect calorimetry should be recommended to individualise nutrition, extending follow-up beyond the doors of the hospital [37] .
Another important part of nutrition is selenium supplementation, as most countries in Europe have a low natural abundance of selenium, and inflammatory states lower the plasma concentration further. Recent studies have demonstrated a survival advantage of selenium supplementation [38] . Manzanares et al.
[39] report a study with high selenium supplementation: 2.0 mg as a bolus followed by 1.6 mg/24 h for 10 days. The high dose of selenium resulted in stimulation of glutathione peroxidase and a lower incidence of hospital-acquired pneumonias. The clinical advantage in terms of morbidity in this comparatively small study (n = 35) indicates that prompt restoration of selenium status early in the course of critical illness is important.
The muscle protein depletion seen during critical illness is present in all age groups. Tuvdendorj et al. [40] report quantitative measurements of skeletal muscle protein turnover, including separate measurements of the synthesis rate and the degradation rate in patients with extensive burn injuries. Muscle protein degradation was increased regardless of age, but the synthesis rate was higher in children, resulting in a significantly better protein net balance in children. This finding is in line with earlier clinical reports of separate studies in children and adults of protein turnover in critical illness, but this finding of Tuvdendorj et al. is original, as it has never before been demonstrated in a homogeneous patient population dichotomised for age. Still, the generalisability beyond burn injury remains to be demonstrated.
Modulating oxidative stress by increasing endogenous antioxidant defence mechanisms has already been intensively investigated. A new set of data was presented in the journal this year [41] . The administration of pharmacological doses of selenium for 14 days in 75 patients with systemic inflammation or sepsis allowed an increase in glutathione peroxidase activity as compared to 75 untreated patients. However, no change was found in mortality, even in subgroups of patients stratified by severity of disease or organ failures. The modalities and indications for antioxidant strategies should definitely be carefully scrutinised in the future. Paraquat intoxication is a prototypical example of oxidative stress associated with high mortality. In such cases, therapeutic options and antidotes are scarce. A new treatment algorithm (high-dose cyclophosphamide ? high-dose methylprednisolone) has been assessed and compared to the previous treatment (cyclophosphamide ? dexamethasone) in a very large cohort of 111 patients with paraquat poisoning [42] . Mortality was decreased from 92 to 66% in patients randomised to the new algorithm. These important results obviously bring a major contribution to the field of toxicology.
The occurrence of symptomatic cerebral vasospasm (SCV) after aneurysmal subarachnoid haemorrhage (aSAH) is a frequent clinical condition that is unfortunately associated with worsened morbidity and mortality [43, 44] . The ability to predict patients at high risk for vasospasm would help in targeting early therapeutical efforts. So far, this has remained an unsatisfied plea [45] . In the ICU, clinical assessment by bedside evaluation is of limited usefulness due to sedation or clinical severity. Transcranial Doppler is a useful screening tool for middle cerebral artery vasospasm, with less utility in evaluating other intracranial vessels, and computed tomographic perfusion may help predict vasospasm when used early during the clinical course [46] . Due to the difficulties involved in identifying SCV, several early markers that could be used to provide therapies focused on early brain damage signs have been search for, with the aims being to prevent but also reduce the intensity of later developing neurological complications [47] . Turck [48] used an awkward panel including four brain injury-related proteins, one cardiac marker and a clinical score, and demonstrated that this is a valuable albeit complex tool for identifying aSAH patients at risk of poor outcome.
Recently, pentraxin 3 (PTX3), a protein induced by proinflammatory signals produced by neutrophils, macrophages, myeloid dendritic and endothelial cells, has emerged as a key player in local inflammatory and ischaemic events, and it has been proposed as a valuable prognostic and diagnostic tool in many conditions, substantiating the evidence that PTX3 has useful features for a potential biomarker. In this context, Zanier [49] investigated the concentrations of PTX3 and its relation with SCV in the plasma and cerebrospinal fluid (CSF) of 38 aSAH patients. PTX3 was elevated in all SAH patients in both plasma and CSF. Early, acute peak CSF PTX3 was significantly higher in patients who later developed vasospasm [median 13.6 (range 2.3-51.9) ng/ml] compared to those who did not [3.2 (0.1-50.5) ng/ml, p = 0.03]. The temporal pattern of CSF PTX3 in patients with vasospasm was triphasic, with a peak during the first 48 h after SAH, a subsequent decrease in the following 48-96 h, and a secondary significant increase with the occurrence of vasospasm. The measurement of CSF PTX3 can improve early diagnosis of this complication, and will be evaluated as a potential early biomarker in further researches. Even if we are improving in the identification of potential biomarkers to use as an early warning for CVS, this search is not yet concluded, and in the future we will probably ameliorate our comprehension in this exciting field.
Barbiturate therapy for refractory ICP High intracranial pressure (HICP) frequently complicates severe traumatic brain injury, aSAH and other neurological disorders. Raised intracranial pressure has been consistently associated with poor neurological outcome. Treggiari [50], in a systematic review of trauma patients, evaluated the management of raised ICP and related neurological outcomes. ICP refractory to therapy and the response to treatment are very good predictors of neurological outcome compared to absolute ICP values. Relative to normal ICP, raised ICP was associated with a 3.5-to 6.9-fold increase in mortality. Raised but reducible ICP was associated with a 3-to 4-fold increase in the probability of death or poor neurological outcome. Refractory ICP pattern was associated with a dramatic increase in the relative risk of death (odds ratio [110) . The same applies to aSAH patients [51] .
Our efforts at controlling HICP encompass the use of first-line therapies (CSF withdrawal, sedation, osmotics, hyperventilation, CPP optimisation). If all of these fail and the patient is salvable, we are obliged to use ''second-tier'' therapies, including barbiturates, hypothermia and decompressive craniectomy. High doses of barbiturates are still recommended to control HICP refractory after the failure of maximal standard medical and surgical therapy [52] . Along with many other severe side effects, the most important being haemodynamic instability and pulmonary complications, dyskalaemia has been described as a severe adverse effect in case reports [53, 54] . Thiopentone inhibits voltage-dependent potassium currents and inhibits phosphofructokinase, causing intracellular sequestration of potassium during its administration and an extracellular release when it is stopped. For this reason, the discontinuation of the drug must not be abrupt; it has to be slowly tapered. However, the real incidence and characteristics of dyskalaemia during and after barbiturate coma have not been well described. Ng et al.
[55] performed a retrospective review of all 47 patients who received barbiturate therapy for refractory HICP during an 18-month period in a neurosurgical ICU. 89.4% of the barbiturate patients' cohort developed hypokalaemia after the induction of barbiturate therapy. The median time to onset of hypokalaemia was 11 h and the time to nadir of serum potassium levels was 25 h. More importantly, 34% patients developed hyperkalaemia on weaning off barbiturate therapy. All patients who developed hyperkalaemia had previously been hypokalaemic. The mean potassium replaced during hypokalaemia was higher in patients who developed hyperkalaemia compared to those who did not. Therefore, hypokalaemia and hyperkalaemia are frequently associated with induction and cessation of barbiturate coma. Serum potassium levels must be monitored vigilantly. Patients who develop hypokalaemia and receive large potassium replacement may be at greater risk of hyperkalaemia on cessation. Hyperkalaemia at cessation has been described as cause of cardiac arrest in this population.
Not all monitors give you the same numbers! The physiological idea behind cerebral oxygen monitoring is that the PbrO 2 value accurately represents the balance between oxygen delivery and oxygen consumption in brain cells, and that changes in PbrO 2 will therefore reflect pathophysiological alterations. These changes could then be used to guide treatments to prevent or mitigate hypoxic injury [56] . PbrO 2 has been frequently monitored using a Licox system (LX, Integra Neuroscience, Biot, France), the only available device until a couple of years ago. Recently, a different manufacturer (Raumedic, Münchberg, Germany) introduced a new probe, the Neurovent-PTO (NV). Dengler [57] compared the performance of the two systems in 11 comatose traumatic brain injury (TBI) or aSAH patients during dynamic changes in inspirational oxygen fraction and mean arterial pressure. PbrO 2 was recorded continuously in patients using the two probes which were placed side-by-side in the same cerebrovascular region. Once a steady baseline value was reached, FiO 2 was increased by 20% for 10 min. Once the baseline was again achieved, MAP was increased by 20 mmHg for 10 min. The PbrO 2 values of both probes differed significantly at all times. The LX probe reacted significantly faster to changes in FiO 2 and MAP. Limits of agreement ranged between -32.1 and 20.0 mmHg. Mean LX values were 6.1 mmHg lower than NV values. Even if the examined patient cohort was rather small, the data suggest that LX and NV probes measure different PbrO 2 values in routine monitoring as well as during phases of dynamic changes in FiO 2 and MAP. These data therefore do not support the view that both probes can be used interchangeably. The practical advice for the clinician is to be aware of the performance of the system they are using and to adapt treatment thresholds accordingly.
Lescot and co-workers [58] tested intracranial pressure monitors in a study on neurosurgical intensive care patients. They used the Pressio and the Codman devices and compared the results with direct intraventricular measurement. They found that both pressure monitors approximated intraventricular cerebrospinal fluid pressure with an accuracy of ±7 mmHg, a result that may be acceptable under many clinical conditions.
In another study, the brain temperature was measured using two different magnetic resonance techniques [59] . Healthy volunteers were exposed to intranasal cooling, and brain temperature changes were measured and mapped using MR spectroscopic imaging and phase-mapping techniques. The decrease in brain temperature assessed by MR was -1.7°C by spectroscopic imaging and -1.8°C by phase mapping at the end of 1 h of nasal cooling. The rectal temperature fell by 0.5°C. Thus, the simple nasal cooling technique had a substantial effect on brain temperature.
End-of-life care in specific countries During 2011 in Intensive Care Medicine, there were three articles that focused on end-of-life care in specific countries. First, Solarino and colleagues conducted a national survey of Italian physicians following the a high profile death of a 36-year-old woman who had been in a vegetative state for 17 years [60] . The Italian Supreme Court granted the woman's father his wish to discontinue nutrition and hydration. The authors of this paper emailed a questionnaire to 70,000 physicians working for the Italian Public Health System and University Medical Hospitals. 22,219 doctors responded, representing a response rate of 32%. Approximately three-quarters had experience in treating patients in a persistent vegetative state. Approximately 60% of them considered tube feeding to be a medical therapy, and 66% believed that withdrawing artificial nutrition and hydration could be appropriate if based on the patient's wishes. There was broad consensus among Italian physicians that a clear legislative position regarding the withdrawal of artificial nutrition and hydration is needed.
Second, Weng and colleagues [61] conducted an anonymous survey of the attitude of Chinese intensivists toward end-of-life care in the ICU. Although no list of Chinese intensivists exists, they used an innovative snowball method to identify 534 potential participants from 21 regions in China. The response rate was 59%. The authors found that approximately 60% of physicians reported admitting patients with very poor prognosis to the ICU, and only 19% reported giving complete information to the patients and their families. The use of donot-resuscitate orders or the limitation of life-sustaining therapy in terminally ill patients was uncommon. Interestingly, the authors compared their results to studies using the same survey in Hong Kong and in Europe. Intensivists in China were equally likely to admit patients with poor prognosis to the ICU as those in Hong Kong and Europe, but were less likely to report withholding or withdrawing life support.
Finally, Kubler and colleagues [62] reported the results of a survey of intensivists attending a national intensive care congress in Poland. Of the 400 questionnaires distributed, 54% were returned. Almost all respondents (93%) reported that they had withheld life support, and 75% of the respondents reported that they had withdrawn life support. Older physicians and those who reported no religious affiliation were more likely to report withholding life support. Respondents from large hospitals ([400 beds) were also more likely to report foregoing life support in ICU patients. These results were similar to a study conducted in Western Europe using the same questionnaire.
A qualitative study by Lind and colleagues [63] studied family members' experiences of end-of-life decisionmaking in Norwegian ICUs, in order to ascertain the degree to which the family felt informed and included in decision-making. The authors used a grounded theory method of qualitative analysis with interviews of 27 bereaved family members. This study found that family members wanted a more active role in decision-making, in order to be able to communicate the values and preferences of the patient. The authors also found that clinicians often used a ''wait and see'' strategy to address decision-making early in the ICU stay, which many family members experienced as unnecessarily delaying the family members' ability to come to terms with the severity of the patient's illness and to participate in decision-making. Family members also discussed the importance of communication with ICU nurses. The authors suggest that ICU clinicians need more training in the knowledge and skills of effective communication with families of critically ill patients.
Jensen and colleagues [64] conducted a survey of nurses and physicians in the ICU as well as physicians in primary care to examine communication and collaboration between ICU and primary care clinicians regarding decisions to forego life support in the ICU. The study was conducted in seven hospitals in Southern Denmark. Participants included 495 ICU nurses, 135 ICU physicians, and 146 primary physicians, with an overall response rate of 84%. Among primary care physicians, approximately 60% found their experience of collaboration to be satisfactory, compared to only 36% of ICU physicians and 27% of ICU nurses. ICU physicians and nurses were more likely than primary care physicians to perceive that decisions regarding withdrawal of life support were unnecessarily postponed due to communication between ICU and primary care clinicians. The authors suggest that multidisciplinary patient conferences, nurse involvement in the decision-making process, and guidelines for foregoing life support might improve communication and collaboration.
Cremer and colleagues [65] conducted a multicentre prospective observational study to assess the prevalence of questioning about the appropriateness of initiating or maintaining life support in 15 French-speaking paediatric ICUs, and to evaluate decision-making processes. Among the 5,602 children admitted, 410 died (7%) and 175 of those (43% of the deaths) died after foregoing life support. The utility of life support was questioned in 308 children (6%) with a prevalence of 13 per 100 patientdays. More than 30% of the children survived despite the appropriateness of life support being questioned, and 23% survived despite a decision to forego life support. The median clinician time spent on making and presenting decisions about the utility of life support was considerable at 11 h per child. On any given day in each paediatric ICU, there was more than one child for whom a decision to forego life support was being considered, representing a significant amount of paediatric ICU clinicians' time, and suggesting that this is an important part of paediatric intensive care.
Devictor and colleagues [66] conducted a multicenter prospective study, the Eurydice II study, in 45 paediatric ICUs in Europe. They identified decisions to forego life support in 166 children, representing 41% of the paediatric deaths. They found some regional variability, with more patients dying after CPR in Eastern and Central Europe than in Northern and Western Europe. The vast majority of decisions to forego life support were discussed by clinicians and families during a formal family meeting, after which the medical staff generally made the final decision. The decision was almost always documented in the medical record. The majority of parents were informed of the final decision and were at the bedside during their child's death. Decisions were made to forego life-sustaining treatment in 41% of children who died, which was higher than the 33% seen in Eurydice I conducted in 2002, 6-7 years earlier. Eurydice II found that a higher percentage of parents were now informed about the meeting and its conclusion as compared with the earlier Eurydice I. This study shows a trend towards the standardisation of paediatric end-of-life practices across European countries in the past decade, with increased communication with the parents of critically ill children.
Kohn and colleagues [67] sent questionnaires to a national sample of US ICU clinicians, soliciting their preferences for allocating their last bed to a gravely ill patient with little chance of surviving versus a deceased or dying patient for whom aggressive management could help others through organ donation. The authors received completed surveys from 684 of 2,206 physicians (31%) and 438 of 988 nurses (44%). They found that physicians were more likely than nurses to adhere to the ''rule of rescue'' by allocating the last bed to the gravely ill patient (46 vs. 33%). The magnitude of the social benefit to be obtained through organ donor management (5 or 30 lifeyears added for transplant recipients) had small and inconsistent effects on clinicians' willingness to prioritise the donor. The most common reason for allocating the last bed to an identifiable patient was that clinicians perceived strong obligations to identifiable living patients.
The authors argue that this allegiance to the rule of rescue will be a major challenge for efforts to develop ethical and standardised ICU triage practices.
In order to identify a potential organ donor as early as possible and maximise the donor conversion rate, it is important to study this conversion rate. However, the donor conversion rate is calculated with different assessment tools, making it difficult to compare across studies and centres. de Groot and colleagues [68] conducted a study to determine which assessment tool can be used for a realistic estimation of a potential organ donor pool, and how they compare to each other with regard to the donor conversion rate. They conducted a retrospective chart review of patients diagnosed with a subarachnoid haemorrhage, traumatic brain injury, or intracerebral haemorrhage, and applied three different assessment tools. In their cohort of patients, 179 out of 564 (32%) died. After applying three different assessment tools, the number of patients found to be brain dead, before exclusion due to medical reasons or age, ranged from 76 to 107 patients. The authors identified one tool that was the most practical for identifying patients with a realistic chance of becoming brain dead and therefore identifying the patients most likely to become a potential organ donor.
Most previous studies have focused on the propagation of epithelial and endothelial injury in ALI/ARDS; the ability of bacteria to colonise host defense mechanisms are not well studied in this pathophysiologic process. Piccin and colleagues [69] examined the hypothesis that mechanical ventilation directly affects respiratory defense mechanisms (the mucociliary system). The authors used different modes of mechanical ventilation in rabbits, and demonstrated that mechanical ventilation using high volume and high pressure led to detrimental changes in the large proximal airway mucociliary system compared with lower tidal volume ventilation. Although a significant decrease in tracheal mucus secretion was noted across all ventilated groups, only animals ventilated with high pressures showed a significant reduction in ciliary beating frequency. The authors speculated that the mucociliary alterations occurred as a result of tissue hypoperfusion caused by mechanical ventilation with high pressures and high airway flows. An editorial [70] pointed out that the relevance of the observation is yet to be investigated in patients under mechanical ventilation. It is nevertheless important to understand the effects of changes in mucociliary clearance in the context of ventilator-induced lung injury that may alter host susceptibility to infections or prolonged intubations due to secretion retention.
Mechanical ventilation with high tidal volume increased the generation of reactive oxygen stress (ROS), cytokine responses, and the activation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-jB associated with lung injury [71] . In an isolated, perfused rat model, the administration of apocynin, a strong oxidative inhibitor known to block nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils, macrophages, and endothelium [72] [73] [74] , depressed oxidative stress, attenuated inflammatory responses, and reduced ventilatorinduced lung injury (VILI) [71] .
Resolution of the alveolar epithelial/capillary membrane damage that occurs during acute lung injury (ALI) requires a coordinated and effective tissue reconstruction program to re-establish a functional barrier [75] . Regeneration of alveolar epithelial cells and matrix turnover are controlled by regulatory pathways, while dysregulation of these pathways may result in amplification of the initial injury and disorderly repair, with sustained inflammation and development of pulmonary fibrosis [76, 77] . Since WNT/b-catenin signaling has been reported to be involved in epithelial cell injury [78] , Villar et al. [79] demonstrated that this pathway is modulated early during ventilator-induced lung injury. This modulation may represent a promising therapeutic target for attenuating or preventing the pathological consequences of acute lung injury.
The mechanisms of ventilator-induced diaphragmatic damage (VIDD) are not fully elucidated [80] . Previous studies have shown that high tidal volume ventilation leads to the activation of serine/threonine kinase/protein kinase B (Akt) and c-Jun NH2-terminal kinase (JNKs) [81] . Activation of the phosphoinositide 3-OH kinase (PI3-K)/Akt pathway also results in the phosphorylation of the class O of forkhead box transcription factor (Foxo) proteins [82] . Li et al. demonstrated that a high tidal volume ventilation-induced diaphragmatic damage model was associated with the activation of JNK and Foxo4, and this process was dependent on the Akt and JNK pathways. These data help us to understand the effects of mechanical forces on the diaphragmatic injury, as well as suggest whether inhibiting the Akt and JNK pathways offers possible treatment options [83] .
Previous studies have shown that high-dose corticosteroids or neuromuscular blocking agents (NMBA) [84] may affect diaphragm function [85] . Maes et al. [86] hypothesised that the combination of rocuronium (NMBA) and corticosteroids would result in a further deterioration of diaphragm function in a model of ventilator-induced diaphragm dysfunction (VIDD). They observed that the negative effect of rocuronium infusion with controlled mechanical ventilation was absent with the administration of a high dose of corticosteroid due to inhibition of the calpain and caspase-3 system.
It has been suggested that the airway pressure used in the mechanical ventilation of patients with ALI/ARDS should be in-between the low and upper inflection points of the pressure-volume (P-V) curve. During constant inspiratory flow, analysis of the airway pressure-time profile (P-t) can replace the static P-V curve [87] . From the mathematical point of view, the airway pressure can be described as a function of inspiratory time by the following equation: airway pressure = at b ? c, where the coefficient b, called the stress index, describes the shape of the P-t curve during a tidal breath. If b \ 1, it means that the compliance increases, suggesting tidal recruitment; if b = 1, it suggests that the compliance does not change, while if b [ 1, it indicates compliance decreases, suggesting tidal overinflation [88] . Formenti et al. [89] tested the predictive ability of the stress index compared with quantitative lung CT scan in paralysed, mechanically ventilated pigs. The tests were conducted at PEEP values of 1 and 10 cm H 2 O in the absence and presence of pleural effusion by fluid instillation in pleural space. The investigators demonstrated a dissociation between the stress index, which suggested lung overinflation, and the CT scan, which showed lung recruitment. This observation suggests that the ability of the stress index to predict lung recruitment and overdistension was significantly reduced in the pathophysiological conditions. However, the authors did not measure the transpulmonary pressure in their study. An editorial [90] suggests that computation of the stress index as usually obtained from the airway pressure curve should be replaced by analysis of the transpulmonary pressure curve profile. In the heterogeneous airspace environment of ALI/ARDS, a reliable tool at the bedside to set desirable PEEP and VT is critical [91] . Traditional methods of monitoring lung mechanics using the pressure-volume curve, static compliance and the stress index have shown drawbacks, especially when the compliance of the chest wall is altered. Computed tomography (CT) scan is a powerful tool for mapping the distribution of the gas volume, but it is yet to be a part of bedside technology. The forced oscillation technique (FOT) may offer several potential advantages over traditional methods because it applies very small volume displacement, it minimises artifacts resulting from nonlinearity of the respiratory system, and it does not require deep sedation and muscle paralysis. Using FOT in a surfactant depletion model of ALI, Dellaca and colleagues [92] performed incremental and decremental PEEP trials, and measured both conventional and FOTderived respiratory system compliance at each step of PEEP changes. CT scan analysis was performed as a gold standard for monitoring lung compartments at end expiration. FOT was able to detect the minimal PEEP value needed to keep the lung open, and this was in good agreement with CT scan analysis with respect to sensitivity and specificity, suggesting that FOT may have potential value in defining adequate levels of PEEP to prevent/reduce the occurrence of de-recruitment. However, the validation of FOT to detect the extent of recruitment/de-recruitment and hyperinflation remains to be determined in more complicated clinical situations [93] . Variable ventilation is a new mode delivering volume-controlled ventilation with variation of the tidal volume around an average value. Pillow et al. [94] found that variable ventilation increased dynamic compliance but not static compliance or oxygenation in preterm lambs. They also found decreased PaCO 2 and concluded that variable ventilation was more efficient than conventional ventilation. The authors speculated that the lack of improvement in oxygenation between the groups was due to right to left shunt. However, one may argue that the pulmonary vascular resistance might have decreased with the observed improvement in compliance and reduced CO 2 levels, thus resulting in better pulmonary blood flow and oxygenation. Further investigations are needed to address the effects of variable ventilation in physiological and pathological conditions.
The gradient between end-tidal partial pressure of CO 2 (PETCO 2 ) and arterial partial pressure of CO 2 (PaCO 2 ) (PET-aCO 2 ) in mechanically ventilated patients presents a wide range of variation [95] . The reduction of the PET-aCO 2 gradient can be achieved in spontaneously breathing healthy humans using an end-inspiratory rebreathing technique, which equilibrates end-tidal, alveolar, arterial and venous PCO 2 . Based on the aforementioned, Fierstra et al. [96] investigated whether this method would reduce the PET-aCO 2 gradient in a ventilated animal model. This technique led to a reduction in the PET-aCO 2 gradient, while the precision of PETCO 2 as a surrogate for PaCO 2 was independent of the PaCO 2 , PaO 2 , and SaO 2 as well as the extent of lung disease. Therefore, the end-inspiratory rebreathing technique may allow precise, noninvasive monitoring of PaCO 2 in ventilated patients, but further studies are required to identify the limitations of the method.
Bohr's dead space (VD Bohr ) measurement is commonly calculated using end-tidal CO 2 instead of the true alveolar partial pressure of CO 2 (PACO 2 ). Tusman et al. [97] compared the measurement of VD Bohr using PACO 2 derived from either volumetric capnography or the standard alveolar air formula. The authors concluded that VD Bohr can be calculated with accuracy using volumetric capnography. In a correspondence letter, Graf [98] expressed concerns about using Bohr's formula for measuring physiological dead space. The measurement may refer to airway (VD aw ) but not alveolar (VD alv ) fraction. The letter also argued that using CO 2 as a tracer of overall ventilatory efficiency still requires the measurement of VCO 2 , expired minute ventilation and PaCO 2 . The value of PaCO 2 may be increased by shunt and/or V/Q inequalities, leading to physiological dead space overestimation. This is an interesting topic, and more studies and discussion are encouraged to clarify this issue. Despite all of the technical advances of recent years, auscultation provides both useful physiological information and close patient-physician interaction. Today, there are automated systems that are available to detect, analyse, and interpret lung sounds. Image-based techniques of lung sound analysis such as vibration response imaging have been introduced and tested clinically. However, studies are warranted to determine their potential role in clinical practice. Vena et al. [99] report an analysis of the spectral characteristics of lung sounds. They observed significant correlation between intratidal recruitment measured with dynamic CT and the degree of crackles in the sound analysis, suggesting that air passage through atelectasis as well as poorly aerated areas is crucial for generating lung crackle sounds. They concluded that the computer-based analysis of crackle sounds is more sensitive for differentiating between small differences in healthy and injured conditions, especially at higher positive end-expiratory pressure (PEEP) levels, compared to conventional clinical auscultation. This simple and noninvasive technique of computer-based lung sound analysis may help intensivists monitor mechanical ventilation and diagnostic or therapeutic procedures such as recruitment manoeuvres in real time at the bedside. Yet the validation of the technique, and the variation of frequent measurements and interpretation of complex respiratory signals to optimise ventilator settings or to detect alveolar recruitment, especially in injured lungs, remain to be further investigated at the bedside [100] . Endotracheal tubes at high volume and low-pressure cuffs may fail to protect the lower airway from leakage of potentially contaminated secretions below the longitudinal folds. Ouanes et al. [101] studied the effects of PEEP levels, inspiratory effort intensity, peak pressures, tracheal tube cuff materials and sizes on the leakage of fluids past the cuff in an in vitro model of tracheal intubation and mechanical ventilation. The authors observed that leakage occurred more frequently at lower levels of PEEP, higher inspiratory efforts were associated with higher leakage, and polyurethane cuffs performed better than polyvinylchloride cuffs. In theory, most aspiration during mechanical ventilation occurs when tracheal pressure falls below hydrostatic pressure. Thus, the most important dependent variables are PEEP, inspiratory efforts and duty cycles. More studies are required to examine the effects of mechanical ventilation on leakage across the cuff.
It is becoming increasingly apparent that lung and brain represent an integrated physiological ensemble such that insults involving one will compromise the other and vice versa [102] . Severe neurological injury is associated with concurrent lung injury in clinical settings. Recent studies have shown that acute lung injury may be responsible for brain injury and poor neurocognitive outcomes. However, the underlying biological mechanisms are yet to be elucidated. Heuer et al. [103] evaluated the independent and combined effects of sustained acute intracranial hypertension (AICH) and ARDS on lung injury and brain damage in a porcine model. They observed that markers of lung injury were augmented with AICH and rose further with concurrent AICH/ARDS. These observations shed valuable light on the complex process of signaling, involving neural, inflammatory, immunological, and neuroendocrine pathways. The fundamental physiological mechanisms still need to be addressed in future studies investigating whether the brain injury was due to increased capillary permeability, and whether the brain edema was vasogenic or cytotoxic. Given the significant epidemiologic and mechanistic overlap between sepsis and ALI/ARDS, it seems plausible that some of the known effects of sepsis on the brain may be relevant to brain dysfunction encountered in ALI/ARDS [102] .
The brain is one of the first organs affected during sepsis development; however, the mechanisms associated with septic encephalopathy are not well known [104] . Comim et al. hypothesised that during sepsis, brain cytokines and chemokines may lead to alterations in the blood-brain barrier (BBB) permeability, resulting in an increase in the flux of inflammatory cells and toxic mediators into the brain, thus contributing to injury. They demonstrated that the brain's production of cytokines and chemokines was an early event during sepsis, and seemed to participate in both central nervous system dysfunction and BBB permeability alterations [105] .
Studies are needed to demonstrate these links to enable the development of specific therapeutic strategies.
Since there is increasing interest in the use of both pulse pressure variation (PPV) and stroke volume variation (SVV) to predict preload responsiveness and drive resuscitation protocols, Mesguida et al. [106] evaluated the impact of increasing tidal volume, decreased chest wall compliance, and left ventricular contractility during intermittent positive-pressure ventilation (IPPV) on the relation between PPV and left ventricular stroke volume variation and intrathoracic blood volume changes in dogs. This study provides a systematic examination of the changes in the right and left heart pressure and stroke volumes during the ventilator cycle. The author's major conclusion is that there is tight coupling between pulse pressure and left ventricular SVV. The authors have also added some important new information that shows the effect of changes in chest wall compliance, tidal volume and left ventricular function on the measurements. It is noteworthy that a proper quantitative analysis would require integrating the area under the stroke volume curves of all the stroke volumes in inspiration and expiration. What the author is really showing is that there are transient differences between the right and left side peak stroke volumes that must be associated with gains or losses of pulmonary vascular volume to fulfil the conservation of mass.
Reliable biomarkers of sepsis are needed for early diagnosis and guidance of appropriate treatment.
Pentraxin 3 (PTX3) is expressed in a variety of cells, including inflammatory (e.g. macrophages, neutrophils, dendritic cells), endothelial, and epithelial cells. Additionally, the PTX3 level is well correlated with the severity of lung injury and multiple organ failure, and may serve as a biomarker for ALI/ARDS [107] . In order to analyse the role of PTX3 in an LPS-induced ALI model, PTX3 knock-out (PTX3-KO) mice were used, and more severe lung tissue injuries, neutrophil infiltration, cell death, activation of the coagulation cascade, and inflammatory responses were observed, indicating that PTX3 plays a protective role in the pathogenesis of ALI [108] .
Izquierdo-Garcia et al. [109] conducted a study in rats where lung tissue, lung lavage fluids and serum samples were profiled from caecal ligation and punctureinduced sepsis and control groups using NMR and highresolution magic angle spinning detection. Predictive PLS-DA models were constructed based on the NMR spectra of the three types of biological samples and employed to diagnose sepsis in other samples. The authors further reported that the predictive power obtained by combining the three types of samples was 100%. An advantage of employing metabolomics for potential diagnosis is the utilisation of biofluids and/or easily accessible tissues. Because lung tissue collection is invasive, while sepsis often appears in critically ill patients, the use of lung tissue for metabolomic analysis is not practical at the bedside. Moreover, bacteria play a key role in sepsis, particularly in conditions like peritonitis-associated sepsis. However, bacterium-related information was absent from their study. Therefore, the diagnostic value of the clinical treatment provided by this study is limited. Future studies may need to include urine samples for metabolomic analysis in order to understand bacterium-specific metabolites.
Several mechanisms have been proposed to explain vascular dysfunction induced by sepsis [110] . Experimental studies have shown that selective and nonselective inhibitors of vascular potassium (K ? ) channels increase arterial pressure or reverse shock-induced vascular hyporeactivity [111] . However, while the use of channel inhibitors remains an attractive option to counteract systemic vasodilation, it may also impair microcirculatory adaptation to shock [112] . Collin et al. [113] demonstrated that vascular K ? channels are activated and overexpressed, while their inhibition restores arterial pressure and vascular reactivity, and decreases lactate concentration, thus offering potential therapeutic perspectives for septic shock.
In the search for treatments for critical illnesses, levosimendan has been found to be interesting candidate, as it is a potent stimulator of vascular ATP-dependent potassium channels (K ? -ATP channels), inducing systemic vasodilation and reducing afterload. Schwarte et al. [114] examined the effect of levosimendan on cardiac output and tissue perfusion in the presence of hypoxia in a canine model. They found that when levosimendan was infused before hypoxia was induced, myocardial contractility, stroke volume and cardiac output were preserved in spite of the hypoxic insult. The novelty of this study is the examination of the antagonising effect of glibenclamide on the action of levosimendan. When given alone, glibenclamide caused a rise in systemic vascular resistance, suggesting that glibenclamide was acting to block K ? -ATP channels. When levosimendan was administered in the presence of glibenclamide, levosimendan caused a significant rise in cardiac output by improving myocardial contractility. This is indicative of levosimendan acting through a calcium-sensitising effect rather than on K ? -ATP channels. In a separate study, Revermann et al. [115] reported that the administration of levosimendan and nicorandil (a K ? -channel opener) attenuated the increased pulmonary vascular medial wall thickness in a model of pulmonary hypertension induced by monocrotaline challenge. Levosimendan significantly diminished the proliferation of pulmonary arterial smooth muscle cells, and this effect was attenuated by glibenclamide. In cell culture, levosimendan had a direct inhibitory effect on the platelet-derived growth factor induced proliferation of pulmonary arterial smooth muscle cells. These findings of levosimendan exerting inotropic, vasodilatory and anti-inflammatory properties under experimental conditions are exciting, but the real promise for levosimendan as a therapeutic candidate to support critical illness remains to be elucidated. Further in vitro and animal studies are required to understand the vasodilatory action of levosimendan, and the consequent effects-positive or negative-on end-organ perfusion [116] . Acute kidney injury is a common complication in patients with sepsis.
The primary resuscitation strategy for these patients is fluid resuscitation to improve organ perfusion and oxygenation and thereby prevent distal organ failure. Legrand et al. [117] examined the effects of fluid resuscitation on renal perfusion in relation to renal microcirculatory function in endotoxemic rats. The authors observed that infusion of endotoxin resulted in altered microvascular perfusion and oxygenation distributions. Early fluid resuscitation greatly improved renal perfusion compared to late resuscitation, but it did not influence oxygenation distribution. Serum cytokine levels decreased in the resuscitated groups, no matter whether early or late resuscitation was involved. Although the results are interesting, one has to keep in mind that the study was conducted at 300 min after endotoxin administration, and it is unknown whether this time frame is long enough to fully assess the therapeutic value of immediate versus delayed resuscitation in clinical situations. There are several issues that need to be addressed in future studies. For example, the volume and rate of fluid administration could simply have altered the disposition of endotoxin in the kidney. It is unclear how dramatic differences in aortic and renal artery flow and microvascular flow between endotoxin alone and endotoxin with fluid resuscitation had no impact on microvascular oxygen tension. Since the human erythropoietin fusion protein (EPO) gene was cloned over 25 years ago, several variants of the EPO molecule have been developed to improve its pharmacokinetic and pharmacodynamic characteristics and to separate its haemopoietic and neuroprotective properties [118] . For example, carbamylated erythropoietin fusion protein (cEPO-FC) contains two recombinant human EPO (rhEPO) molecules connected by the Fc region of human IgG 1 , and is thought to improve the cytoprotective effects while reducing side effects including hypertension and thrombosis. Simon et al. [119] report that pretreatment with a cEPO-FC is as effective as rhEPO in ameliorating spinal cord injury in a porcine model of acute spinal cord ischaemia and reperfusion. This study assessed short-term outcomes, including motor-evoked potentials and histological damage. Future studies will need to focus on the longer-term neurological outcomes and demonstrate an absence of systemic toxicity, including thrombosis. It is noteworthy that clinical studies raised the possibility that rhEPO could improve some clinical outcomes, including neuronal recovery, but rhEPO failed to show any clinical benefit with respect to the Barthel index, modified Rankin scale and mortality rate in patients with stroke [120, 121] . Thus, further experimental and clinical research is needed to determine if derivatives of rhEPO such as cEPO-FC can improve long-term neurological outcome without having potential adverse effects such as hypertension, thrombosis and mortality. Reactive oxygen species are produced by activated neutrophils during the inflammatory response to stimuli such as endotoxins, can directly or indirectly injure host cells, and have been implicated in the pathogenesis of ALI/ARDS. Hassett et al. [122] described the result of pulmonary overexpression of superoxide dismutase in the response to endotoxin administration in animals. Endotoxin produced a severe lung injury compared to a sham injury. Their results support the conclusion that superoxide dismutase plays an important role in lung injury in terms of neutrophil infiltration, cytokine responses, lung edema and histology. However, the delivery technique for the superoxide dismutase transgene-using adeno-associated virusremains controversial. The conclusion that gene therapy may be appropriate for ALI is a bit premature and need further studies.
Sepsis affects both oxygen delivery to tissues, through cardiac and macro-and microcirculatory alterations [123] , and oxygen consumption, through effects on mitochondrial respiration [124] . Dyson et al. reported that the early fall in tissue oxygenation was associated with both macro-and microcirculatory impairment, the latter persisting despite restoration of the macrocirculation. However, by 24 h, this impairment had largely recovered, even though the animal predicted to have a poor prognosis were then manifesting clinical and biochemical signs of organ dysfunction, suggesting that cellular abnormalities were enhanced at this later stage [125] . Therefore, the utility of tissue PO 2 monitoring to highlight the local oxygen supply-demand balance, and dynamic O 2 challenge testing to assess microcirculatory function, merits further investigation.
The benefits of stress-dose steroid therapy and recombinant activated protein C (APC) in septic shock remain controversial [126] . Bouazza et al. [127] hypothesised that the combination of APC and steroids would be beneficial compared with their individual use in resuscitated septic shock induced by caecal ligation and puncture. They observed that either APC or dexamethasone improved arterial contractility and endothelial dysfunction resulting from septic shock; however, their combination increased survival, thus recommending the re-evaluation of the combined use of APC and steroids. The conclusion of the PROWESS-SHOCK study (still unpublished) that led to the retraction of the APC from the market has eliminated this molecule from clinical use.
Sepsis is associated with massive discharges of catecholamine and consequent persistent stimulation of the badrenergic receptor [128] . Selective b1-adrenergic blockers might present a new therapeutic capability against sepsis [129] , although the exact mechanism remains to be elucidated. Mori et al. [130] observed that esmolol, a selective b1-blocker, improved outcome in a rat model of sepsis by preventing gut barrier dysfunction and thereby bacterial translocation.
Modulating the adrenergic system may be a new approach to the treatment of sepsis [131] . In rats with peritonitis, b1-blockade decreased proinflammatory cytokines and improved cardiac function and haemodynamics [129] . However, so far, no study has evaluated the haemodynamic tolerance of esmolol-a selective ultrashort-acting b1-blocker-in large animals with endotoxemic shock. Therefore, Aboab et al. [132] observed that selective b1-blockade was well tolerated in endotoxemic pigs treated with a continuous infusion of esmolol, and prevented sepsis-induced cardiac dysfunction, confirming findings reported in small animals.
Oxidative stress has an important role in the development of the systemic inflammatory response [133] . Honokiol, a low molecular weight natural product, is an effective antioxidant and also presents anti-inflammatory and antitumor properties [134] . However, the precise mechanism of action of honokiol on septic acute lung injury remains unclear. Weng et al. [135] observed that honokiol administered after the onset of sepsis reduced acute lung injury and prolonged survival via the amelioration of oxidative stress in endotoxemic mice.
Tumor necrosis factor (TNF)-a has been implicated in the pathogenesis of septic shock. TNF inhibitors resulted in increased survival in experimental sepsis [136] ; however, no anti-TNF agent modified survival in clinical sepsis trials [137] . Fluid therapy may itself have antiinflammatory effects [138] , but is rarely employed in preclinical sepsis models. Therefore, Qiu et al. [139] analysed whether the combination of TNFsr and fluids would be beneficial. They reported that the individual survival benefits of TNFsr and fluids were not additive in a rat sepsis model.
Multiple organ dysfunction syndrome (MODS) is defined as the progressive deterioration of function that occurs in several organs or systems in patients with septic shock. Zymosan-induced generalised inflammation reproduces the characteristics of human MODS, and has been used to evaluate new therapies [140] . Rinaldi et al. investigated the effects of hyperbaric oxygen (HBO) exposure on the expression of Toll-like receptors (TLR) 2 and 4, on their signal transduction, and on organ dysfunction during zymosan-induced MODS. They reported that the anti-inflammatory effect of HBO is associated with the inhibition of TLR signaling, and suggest that HBO therapy is effective at reducing systemic inflammation and associated organ dysfunction in this model [141] .
Reactive oxygen species (ROS) are believed to be involved in electrical shock (ES) related myocardial injury [142] , ischaemia/reperfusion injury, reperfusion arrhythmia, and cardiogenic shock. Ascorbic acid, a potent water-soluble antioxidant, has been found to attenuate oxidative damage, myocardial injury, and arrhythmia during reperfusion [143] . However, so far, no in vivo study has evaluated whether ascorbic acid administration benefits defibrillation and resuscitation. Therefore, Tsai et al. [144] , in a rat model of ventricular fibrillation and electrical shock, observed that the intravenous administration of ascorbic acid at the start of cardiopulmonary resuscitation reduced lipid peroxidation and myocardial necrosis, diminished mitochondrial damage, facilitated resuscitation, and improved outcome.  Using Support Vector Machine and Evolutionary Profiles to Predict Antifreeze Protein Sequences Antifreeze proteins (AFPs) are ice-binding proteins. Accurate identification of new AFPs is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. In this paper, an accurate method, called AFP_PSSM, has been developed for predicting antifreeze proteins using a support vector machine (SVM) and position specific scoring matrix (PSSM) profiles. This is the first study in which evolutionary information in the form of PSSM profiles has been successfully used for predicting antifreeze proteins. Tested by 10-fold cross validation and independent test, the accuracy of the proposed method reaches 82.67% for the training dataset and 93.01% for the testing dataset, respectively. These results indicate that our predictor is a useful tool for predicting antifreeze proteins. A web server (AFP_PSSM) that implements the proposed predictor is freely available. Antifreeze proteins (AFPs) are functional proteins in a cell. With special antifreeze activity, AFPs make the organisms less sensitive to cold temperatures. AFPs bind to small ice crystals to inhibit growth and recrystallization of ice that would otherwise be fatal [1] . By contributing to both freeze resistance and freeze tolerance, AFPs have helped to increase species diversity in some of the harshest and most inhospitable environments. Freeze resistance involves the inactivation or removal of ice-nucleating agents in freeze-avoiding species, whereas freeze tolerance involves the activation or synthesis of ice-nucleating agents in winter in freeze-tolerant species [2, 3] .
AFPs have been found in various insects, fish, bacteria, fungi, and overwintering plants such as gymnosperms, ferns, monocotyledonous, angiosperms, etc. [4] [5] [6] [7] [8] [9] [10] [11] [12] . Relational analyses show that there is low sequence or structure similarity for an ice-binding domain, and lack of common features among different AFPs [7] [8] [9] [10] . One reason for this phenomenon is that ice can present many different surfaces with different arrangements of oxygen atoms [8] . So it is difficult to establish powerful prediction methods to identify AFPs. However, AFPs play important roles in different fields, such as freeze-resistant transgenic plants and animals, food technology, preservation of cell lines, organs and cryosurgery [13, 14] . How to discriminate AFPs from other proteins is important in understanding protein-ice interactions and creating new ice-binding domains in other proteins.
Many lines of evidences have indicated that computational approaches can provide useful information for both drug discovery and basic research in a timely manner [15] , such as protein subcellular location prediction [16, 17] , structural bioinformatics [18] , identification of proteases and their types [19] , identification of membrane proteins and their types [20] , molecular docking [21] [22] [23] , identification of enzymes and their functional classes [24] , and signal peptide prediction [25, 26] . Up until now, there are few studies using computational approaches to discriminate AFPs and non-AFPs. Kandaswamy et al. [27] investigated this problem using the predictor of Random Forest. That is the first and the only method utilizing machine learning technique to deal with the prediction of AFPs. With the model AFP-Pred, they obtained 81.33% accuracy from training and 83.38% from testing. Although high accuracy has been achieved, the problem is worthy of further investigation because the performance of the aforementioned method is still not fully satisfactory and they do not provide an online web server for predicting antifreeze proteins.
In this study, we focus on developing a new antifreeze protein predictor by seeking a more informative encoding scheme. After a preliminary evaluation of different encoding schemes, we found that the evolutionary information in the form of PSSM profiles is suitable for representing the antifreeze protein sequence. Then a predictor called AFP_PSSM is established using the feature PSSM-400 as the input of support vector machine (SVM). AFP_PSSM yields 82.67% accuracy from training dataset and 93.01% accuracy from test dataset. This indicates that our predictor is very promising and may at least play an important complementary role to existing methods. The proposed predictor is freely available at the web server AFP_PSSM [28] . For a query protein sequence of 500 amino acids, it will take about 20 s for the web server to yield the predicted result; the longer the sequence is, the more time it needs.
According to a recent review [29] , to establish a really useful statistical predictor, the following four procedures need to be considered: (i) construct or select a valid benchmark dataset to train and test the predictor; (ii) formulate the protein samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the attribute to be predicted; (iii) introduce or develop a powerful algorithm to conduct the prediction; (iv) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor; (v) establish a user-friendly web server for the predictor that is accessible to public. Below, let us describe how to cope with these procedures one by one.
The datasets used in this paper is retrieved from Kandaswamy et al. [27] which consists of 481 antifreeze proteins and 9493 non-antifreeze proteins. To get rid of redundancy and homology bias, the sequences with ≥40% sequence similarity have been removed using program CD-HIT [30] . Then the training dataset contains 300 antifreeze proteins randomly selected from the 481 antifreeze proteins and 300 non-antifreeze proteins randomly selected from the 9493 non-antifreeze proteins. The test dataset contains the remaining 181 antifreeze proteins and 9193 non-antifreeze proteins. These datasets can be freely downloaded from [31] .
To develop a powerful predictor, one of the keys is to formulate the protein sequences with an effective mathematical expression that can truly reflect their intrinsic correlation with the attribute to be predicted [17] . To realize this, some popular sequence-based encoding schemes have been investigated to represent each protein sequence.
Evolutionary information, one of the most important types of information in assessing functionality in biological analysis, has been successfully used to encode protein in many applications, such as our previous work of lysine ubiquitylation site prediction [32] , transmembrane protein topology prediction [33] and malaria parasite mitochondrial protein prediction [34] . To extract the evolutionary information, the profile of each protein sequence is generated by running Position Specific Iterated BLAST (PSI-BLAST) program [35, 36] . Then this information can be represented as a two dimensional matrix which is known as the PSSM of the protein.
In this paper, the PSSM of each protein sequence in the constructed dataset is generated against the non-redundant Swiss-Prot database [37] (version 56, released on 22 July, 2008) using the PSI-BLAST program with three iterations (−j 3) and e-value threshold 0.0001 (−h 0.0001). This matrix is composed of L  20 elements, where L is the total number of residues in a peptide. The rows of the matrix represent the protein residues and the columns of the matrix represent the 20 naive amino acids. Each element represents the probability of the occurrence of each 20 amino acid when it's mutated to the others at one position during the evolution process.
In the view of the fact that SVM requires the fixed length feature vectors as their inputs for training, we generate a vector of dimension 400, called PSSM-400 from the PSSM. PSSM-400 is composition of occurrences of each type of amino acid corresponding to each type of amino acids in protein sequence [38] . Thus for each column we have a vector of dimension 20. Figure 1 shows the schematic representation of transformation each protein sequence into PSSM-400. 
The purpose of calculating composition of proteins is to transform the variable length of protein sequence into fixed length feature vectors [33] . This is a necessary step during classification of proteins using SVM. The transformation of each protein sequence into a vector of 20 dimensions using amino acid composition will encapsulate the information of protein. Besides amino acid composition, dipeptide composition is also utilized, which gives a fixed pattern length of 400. The advantage of dipeptide composition compared with amino acid composition is that it encapsulates both the fraction information of amino acids and the local order information of protein sequence. 
PROTEIN A R N ∷ V T -4 -1 0 ∷ 0 V -1 -3 -3 ∷ 4 V 0 -3 -3 ∷ 4 F -2 -3 -3 ∷ -1 C 0 -4 -3 ∷ -1 G 0 -3 0 ∷ -3 K -1 5 0 ∷ -3 L -2 -3 -4 ∷ -1 S 1 -1 1 ∷ -2 G -1 -3 0 ∷ -3 K -1 2 0 ∷ -3 P -1 -2 -2 ∷ -3 ∷ ∷ ∷ ∷ ∷ ∷ R -2 6 -1 ∷ -3
The Chou's pseudo amino acid composition (PseAAC) encoding scheme feature has been widely used to predict various properties of proteins [39] [40] [41] [42] [43] . It can be calculated as following: 20 
Where ω is a weighting factor (default ω = 0.1). 
Support vector machine (SVM) [45] belongs to the family of margin-based classifier and is assumed to be a very powerful method to deal with prediction, classification, and regression problems. SVM look for optimal hyperplane which maximizes the distance between the hyperplane and the nearest samples from each of the two classes. Formally, given a training vector x i R n and their class values y i {−1, 1}, i = 1, …, N, SVM solve the following optimization problems:
where w is a normal vector perpendicular to the hyperplane and ξ i are slake variables for allowing misclassifications. Here C (>0) is the penalty parameter which balances the trade-off between the margin and the training error. In this study, LIBSVM package [46, 47] with radial basis kernel function is used. Two parameters, the regularization parameter C and the kernel width parameter γ are optimized based on 5-fold cross-validation using a grid search strategy.
Ten-fold cross validation [48] is used in this work. The dataset is randomly divided into ten equal sets, out of which nine sets are used for training and the remaining one for testing. This procedure is repeated ten times and the final prediction result is the average accuracy of the ten testing sets. To reduce the computational time, we also adopt the independent testing dataset cross validation in this study as done by [49] to evaluate our model. Three parameters, sensitivity (S n ), specificity (S p ), and accuracy (Acc) are used to measure the performance of our model. They are defined by the following formulas:
where TP, TN, FP and FN stand for true positive, true negative, false positive and false negative, respectively. Moreover, we create ROC (receiver operating curve) for all of the models in order to evaluate the performance of models using different encoding schemes.
The detailed flowchart of our work is shown in Figure 2 . First, sequential evolution information in form of PSSM profiles for the input sequence is generated by PSI-BLAST. Second, the obtained PSSM is further transformed into PSSM-400 vector. Finally, the predictor AFP_PSSM is applied to output the test results. 
For the convenience of experimental scientists, we give a step-by-step guide on how to use it to get the desired results as follows: (i) Open the web server AFP_PSSM [28] and you can see the prediction page on your computer screen, as shown in Figure 3 . You must input your email address since the prediction process may take a long time; (ii) Input your query protein sequence to the text box in Figure 3 . Note that the input protein sequence should be in the FASTA format. The FASTA format sequence consists of a single initial line beginning with a greater-than symbol (">"), followed by lines of amino acid sequence. You can click on the "example and note" button to see the example protein sequence; (iii) Choose a threshold value in the drop-down list. For prediction with high confidence (less probability of false positive prediction), high threshold should be chosen; (iv) Click on the submit button to see the predicted result. For example, if you use the first sequence in the example page, the predicted result will be "0.847538, yes" as can be seen in Figure 4 , which means that the protein is an antifreeze protein with the probability of 0.847538. It takes about 15 s for a protein sequence of 300 amino acids before the predicted result appears. 
In this section, four SVM models based on amino acids composition, dipeptides composition, Chou's PseAAC and PSSM-400 are constructed respectively. The accuracies and receiver operating characteristic (ROC) curves for these four SVM models are shown in Table 1 and Figure 5 . One can see that PSSM-400 encoding scheme performs better than the others with accuracy of 82.67% and AUC (Area Under Curve) of 0.926. Thus we use it as our final encoding scheme to represent antifreeze protein sequences. In order to further examine the prediction of power of the current classifier, we compare our predictor AFP_PSSM with the recent work of Kandaswamy et al. [27] on the testing dataset. The number of antifreeze proteins and non-antifreeze proteins in the testing dataset are highly imbalanced, and this situation is close to reality. The compared results are shown in Table 2 . As can be seen from the table, the predictor proposed in this study obtains accuracy of 90.17%, higher than the accuracy of 83.38% gained by [27] . The better prediction performance may be credited to the appropriate protein sequence encoding scheme adopted in our prediction model. 
Accurate identification of new antifreeze proteins is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. Though some researchers have focused on this problem, the accuracy of prediction is still not satisfied, and there are few online web servers for predicting antifreeze protein sequences. In this paper, a highly accurate method is developed for predicting antifreeze proteins using support vector machine and evolutional profiles. This is the first paper in which evolutionary information in the form of PSSM profiles has been utilized to predict antifreeze proteins. The proposed predictor is freely available at the web serve AFP_PSSM [28] . Reliability and External Validity of AMSTAR in Assessing Quality of TCM Systematic Reviews Objective. The aim of this study is to measure the reliability and external validity of AMSTAR by applying it to a sample of TCM systematic reviews. Study Design and Methods. We tested the agreement, reliability, construct validity, and feasibility of AMSTAR through comparisons with OQAQ. Statistical analyses were performed by using SPSS 13.0. Results. A random of sample with 41 TCM systematic reviews was selected from a database. The interrater agreement of the individual items of AMSTAR was moderate with a mean kappa of 0.50 (95% CI: 0.26, 0.73). The ICC for AMSTAR against OQAQ (total score of 9 items, excluding item 10) was 0.87 (95% CI: 0.76, 0.93). Conclusions. Although there is room for improvement on few items, the new tool is reliable, valid, and easy to use for methodological quality assessment of systematic reviews on TCM. Traditional Chinese medicine (TCM) is one of the rarely existing traditional medicines that hold systematic theories as well as preventative and therapeutic methods for diseases in practice [1] . Since the 1950s, research methods in modern medicine (Western medicine) have been gradually introduced to TCM studies; the first TCM systematic review appeared in medical journals in the late 1990s, and now hundreds to thousands of reviews have been published on the area of TCM [2, 3] ; systematic reviews have become the standard approach in assessing and summarizing primary studies. However, even though systematic reviews proved to be useful, serious consideration must be given to how they were conducted; high methodological quality of systematic reviews is a prerequisite for recommendation of the use or avoidance of a TCM intervention. At present, either researches or methods for assessing quality of systematic reviews are still not fully developed in TCM and there is substantial room for improvement [2, 3] ; even in Western medicine current instruments for assessing methodological quality of systematic reviews are still suboptimal and need revision and updating [4, 5] . A new tool termed AMSTAR, an acronym for "a measurement tool to assess systematic reviews," was developed strictly upon the OQAQ (Overview Quality Assessment Questionnaire) [6] , the Sack checklist (quality assessment checklist) [7] , and three additional items. This tool is an 11-item questionnaire requiring assessors to answer "yes," "no," "cannot answer," or "not applicable"; and a recent study reported that AMSTAR has good agreement, reliability, construct validity, and feasibility to assess the quality of systematic reviews [8] . However, these psychometric properties within AMSTAR were tested by having it apply to only a limited set of systematic reviews from Western medicine; a further step is needed to assess its validity and reliability, by a broader range of assessors and more samples of reviews in diverse circumstances [8] . Will the results be reproducible when assessing methodological quality of systematic reviews on traditional chinese medicine (TCM)? The answer is not clear yet, further validation is necessary. The aim of this study is to validate the reliability and external validity of AMSTAR by applying it to a sample of TCM systematic reviews.
We have adopted the definitions used by the Cochrane 2 Evidence-Based Complementary and Alternative Medicine Collaboration: a systematic review is a review of a clearly formulated question, that which uses systematic and explicit methods to identify, select, and critically appraise relevant researches, and to collect and analyze data from the studies included in the review [9] . Then a search strategy for locating systematic reviews on TCM was formulated by using the definitions; search terms included "Chinese medicine", "Chinese herb", "plant preparations", "Chinese medical formula," "meta analysis," "meta-analysis," "meta-analyses," and "systematic review." We performed a comprehensive search on CNKI (China National Knowledge Infrastructure), CBM (Chinese Biomedical Database), VIP (Chongqing VIP periodicals Database), Medline and EMbase databases (January 1, 1999 to the end of 2008) in addition to hand search on Chinese Journal of Evidence-Based Medicine (incept to the end issue of 2008), Chinese Journal of Integrated Traditional and Western Medicine (from the first issue in 1999 to the end issue in 2008), and Journal of Chinese Integrative Medicine (from the first issue in 1999 to the end issue in 2008). Two reviewers screened the titles and abstracts of identified studies independently, disagreement was resolved by discussion. A total of 165 systematic reviews on TCM were included, full detail of evaluations has been reported separately [10] . We used a computer-generated random sample (approximately 25% of the 165 systematic reviews) as a test set for validation.
We tested the agreement, reliability, construct validity, and feasibility of AMSTAR through comparisons with OQAQ, the latter was a validated scale (overview quality assessment questionnaire, OQAQ) developed by Oxman and Guyatt in 1991 [6] . The OQAQ scale measures across a continuum using nine questions (items 1-9) designed to assess various aspects of the methodological quality of systematic reviews and the tenth item requires assessors to assign an overall quality score on a seven-point scale [6] .
In order to adhere more faithfully to the guidance provided by AMSTAR and OQAQ, two assessors (KDY, WY) performed a separate translation and conducted a pilot test independently. Each translator prepared a separate translation and the difficulty in obtaining conceptually equivalent expressions in Chinese was assessed too; subsequently, a sample of 2 systematic reviews on TCM was taken to perform pilot test by two authors independently; based on the results and consensus from reviewers, the final Chinese versions of AMSTAR and OQAQ were then developed for formally evaluating methodological quality of Chinese systematic reviews; all inconsistencies identified either in translation or in application were resolved by discussion. On the basis of this, we constructed a data extraction form in Chinese for this study, in which 11 items of AMSATR and 10 items of OQAQ were adopted directly. In addition, publication status and reporting characteristics of systematic reviews, such as publish language, number of pages, funding source, update or not, Cochrane systematic review or not, target disease, and institution of first author, were also incorporated; besides, the time required to complete an assessment while applying AMSTAR and OQAQ was recorded too.
Assessors were required to answer "yes" score or any other scores for each of items (AMSTAR and OQAQ). If an item was scored "yes," it would be given one point, otherwise, 0 point. We added up these to calculate a total score, the reliability of this total score was assessed through calculating intraclass correlation coefficients; the agreement for each item and the overall tool was explained by percentage of actual agreement as well as Kappa coefficient [5, 8] . Kappa coefficient is a popular measure for chance-corrected nominal scale agreement between two raters. We adopted the Kappa values of <0 rates as less than chance agreement, 0.01-0.20 as slight agreement, 0.21-0.40 as fair agreement, 0.41-0.60 as moderate agreement, 0.61-0.80 as substantial agreement, and 0.81-0.99 as almost perfect agreement [8] .
The OQAQ was selected as a criterion tool because it had been rigorously developed, its face validity was strong, and its validity had been thoroughly tested [6] . We assessed construct validity by comparing AMSTAR, OQAQ, and a self-developed global assessment scale. Construct validity was showed by intraclass correlation coefficient (ICC); for the purpose of calculating ICC, we adopted methods used by the AMSTAR group [8] , and converted the mean total scores (mean of two assessors) of per review to the percentage of maximum score (11 points in AMSTAR and 9 points in OQAQ accordingly); in addition, we developed a 100-point rating scale for overall quality assessment based on answers to the eleven questions in AMSTAR, in which two assessors indicated his or her judgments by checking tick-marks on a horizontal line (0 to 100 point), an SR without any flaws would be scored 100 points. Meanwhile, we also adopted the item 10 in OQAQ as a validated global assessment instrument. The overall mean scores (mean of two assessors), either using the self-developed 100point rating scale or using the item 10 from OQAQ, were also taken to verify the construct validity of AMSTAR. The feasibility of AMSTAR was assessed by recording the time it took to complete scoring, and paired t-test or nonparametric test was applied when comparing with OQAQ.
Database was established by using an electronic form on Microsoft Excel 2003 (Microsoft Corp., Redmond, WA); the data set extracted contained two quality ratings for each review, yielding a total of four ratings per review. Data analysis was performed by SPSS 13.0 (SPSS, Chicago, IL). P < 0.05 was considered significant.
A random sample with 41 TCM systematic reviews was selected from a database developed in a previous study [10] . Of which, only 9 reviews were written in English, and the majority (78%) was published in Chinese journals . The sample included 35 paper-based reviews and 6 Cochrane reviews; there was only one updated Cochrane review. According to the International Classification of Diseases 10 (ICD-10), the topics of the reviews ranged across 9 systems, and mainly focused on diseases of circulatory system Evidence-Based Complementary and Alternative Medicine 3 (15 SRs or 37% of the sample, such as stroke and other cardiovascular diseases), infectious and parasitic diseases (7 SRs, like HBV, SARS), genitourinary system (5 SRs, such as ectopic pregnancy), digestive system (4 SRs, like ulcerative colitis), nervous system (3 SRs, such as Parkinson's disease), and musculoskeletal system and connective tissue (3 SRs, osteoporosis). The number of pages of included TCM SRs ranged widely from 2 to 80 with a median of 6 pages, of which, Cochrane systematic reviews had more pages than non-Cochrane reviews (P < 0.001), with medians of 31 (range: 16-80) and of 5 (range: 2-11), respectively. Less than half of the reviews (41.5%) were presented by clinicians.
Total mean scores on AMSTAR ranged from 2 to 10 (out of a maximum score of 11) with a mean percentage score of 55.1%. The total mean quality scores on OQAQ ranged from 3 to 8 (out of a maximum score of 9) with a mean percentage score of 63.6%. The overall scores for the global assessment instrument (item 10 in OQAQ) ranged from 1 to 6 (out of a maximum score of seven) with a mean of 3.3 (95% CI: 2.9, 3.6), and overall scores using the self-developed 100-point rating scale ranged from 15 to 73 with a mean of 47.6 (95% CI: 43.4, 51.7).
3.1. Agreement and Reliability. Substantial agreements (>80% or nearly 80%) were observed in majority of the individual items (item 1, 2, 3, 4, 6, 7, 8, 10, 11) on AMSTAR, with a mean percentage of 84% (95% CI: 71.1%-91.9%); agreements on item 5 and item 9 were mild at a percentage of less than 60%. Kappa ranged widely from −0.03 to 1.00 with a mean of 0.50 (95% CI: 0.26, 0.73); five items (item 2, 3, 4, 10, 11) scored a kappa of >0.70 (0.70 to 1.00); the highly agreements in four items (item 1, item 6, item 7, item 8) ranged from 80% to 95% and inversely low kappa (−0.034, 0.40, 0.36, 0.36) may be explained by a skewed distribution of responses, that is, approximately 80% (in item 6) to 90% (in item 1, 7, 8) of reviews in which the assessors agreed on the score "yes." However, items 5 (list of included and excluded studies) and 9 (appropriate method to combine studies) scored relative low either in agreement percentage or in kappa parameter (Table 1) . Agreements on individual items of OQAQ were inferior to AMSTAR, with a mean percentage of 79% (95% CI: 64%, 89.5%), and the mean of kappa was relatively low too, with a mean of 0.35 (95% CI: 0.08, 0.62).
The interobserver ICC to the total score was excellent for AMSTAR at 0.84 (95% CI: 0.70, 0.91), and superior to OQAQ, 0.67 (95% CI: 0.38, 0.82). The interrater agreement (kappa) between two assessors for the global assessment (item 10 in OQAQ) was 0.72 (95% CI: 0.48 to 0.85), and better than the self-developed 100-point rating scale: 0.50 (95% CI: 0.07-0.74).
Total mean score was converted into the percentage of the maximum score for each of the instruments, the ICC for AMSTAR against OQAQ (total score of 9 items, excluding item 10) was 0.87 (95% CI: 0.76, 0.93), that is, the results of AMSTAR were highly convergence with the results of OQAQ. Besides, both overall scores were converted into the percentage of maximum score.
ICC obtained when comparing AMSTAR with the item 10 in OQAQ was 0.84 (95% CI: 0.69, 0.91), and when comparing with the 100-point rating scale, ICC was at 0.81 (95% CI: 0.65 to 0.90) respectively; thus AMSTAR showed well convergence with global assessment instruments too.
In addition, we compared the total scores obtained by applying AMSTAR on Cochrane reviews (n = 6, 8.42 ± 1.02) with that of non-Cochrane reviews (n = 35, 5.66 ± 1.31), and the former had higher quality score than the latter (mean difference = 2.76, P < 0.001, 95% CI: 1.62, 3.90).
The relationship between quality scores and publish year was explored too, reviews published after 2005 had similar AMSTAR scores comparing to earlier reviews (5.98±1.51 versus 6.18 ± 1.76, P = 0.70). As the methodological quality and the reporting quality were not mutually exclusive addressing in AMSTAR [8] , we explored whether the number of pages had a positive or negative effect on the AMSTAR score, the result showed there was a statistical association between AMSTAR score and the number of pages (Spearman's rho = 0.67, P < 0.001).
It took 13.2 (95% CI: 12.2, 14.2) minutes to complete use of AMSTAR for each review, while it took less time to complete scoring of OQAQ, averagely 9.3 (95% CI: 8.8, 9.9) minutes per review (paired difference = 3.9, P < 0.001). Besides, a linear regression analysis was performed (time = 6.35 + 2.94 × langrage + 1.75 × log(pages), P < 0.001), revealed the time needed to complete using AMSTAR had significant associations with logarithm of the number of pages (unstandardized coefficients = 1.75, 95% CI: 0.61 to 2.90) and langrage (unstandardized coefficients = 2.94, 95% CI: 0.73 to 5.15); that is, systematic reviews with more pages or written in English need more time to complete scoring. The two assessors found there's difficulty in approaching a final decision on item 9 "were the methods used to combine the findings of studies appropriate" and item 5 "was a list of studies (included and excluded) provided"; for the latter, more detailed guidance for scoring "yes" are required.
A considerable amount of systematic reviews on traditional Chinese medicine have been conducted since the first TCM systematic review was published in the late 1990s [2, 3] . We selected a random sample of TCM systematic reviews from a database developed in a previous study [10] , the sample covered a wide variety of health topics, and thus, we believe that we had a representative sample of TCM reviews of which the AMSTAR was ready to apply.
Our findings in this research revealed that the AMSTAR is a good choice for evaluating quality of TCM systematic reviews. The AMSTAR showed satisfactory interrater agreement, convergent validity, and feasibility in assessing methodological quality of TCM systematic reviews.
Interrater reliability was evaluated by assessing the degree to which different individuals agreed on the scientific quality of a set of reports [7, 8] , the performance of AMSTAR in terms of agreement and reliability was better than that of OQAQ; overall agreement and kappa of items in AMSTAR ranged from moderate to perfect, the reliability of its total score was excellent. However, fair agreement and relatively low kappa were observed in item 9 "were the methods used to combine the findings of studies appropriate" and item 5 "was a list of studies (included and excluded) provided," indicated that there is a room for improvement on AMSTAR when applying this new tool to assess methodological quality of systematic reviews on TCM.
In the absence of a gold standard, we assessed construct validity by comparing AMSTAR with OQAQ as well as two global assessments. Construct validity was shown by intraclass correlation coefficient (ICC); this statistic reflects the extent to which the results of AMSTAR converge with the results of other "criterion" instruments. The analysis revealed that the construct validity was excellent, that is, the AMSTAR is a reliable and valid tool. Given the extremely strict implementation of Cochrane systematic review, such reviews conducted with high methodological quality have been widely recognized [52] ; the AMSTAR revealed Cochrane systematic reviews have higher quality scores than non-Cochrane systematic reviews, that is, the AMSTAR has an ability to discriminate methodological quality, so it is sensitive when applying it to a sample of systematic reviews in diverse quality. The relationship between AMSTAR quality score and the number of pages can be explained by the fact that Cochrane reviews always have considerable amount of pages, these extreme outliers determined the direction and strength of the association.
Compared with the results reported by Shea et al. [8] , considerable differences exists either on ICC for AMSTAR contrast to the OQAQ, or on items with low kappa values. Shea et al. reported a lower ICC of 0.66 (95% CI: 0.28, 0.84), and different items (item 4 and item 7) with relatively low kappa parameter. Possible explanations for these differences include (i) the different samples of systematic reviews being evaluated, most SRs in our sample were short, published in Chinese, and more likely to be rated as low quality, such less conversely evaluations could produce a higher ICC value in our study; (ii) the extra procedure of translation and the conducts of evaluations by applying two tools simultaneously may act as a kind of consensus training to make the evaluations of AMSTAR more likely convergent with the results of OQAQ, that lead to a higher ICC value too in the present study; (iii) the background, skills, and expertise of the assessors were different from that of Shea et al. study.
Regarding the feasibility of AMSTAR, the time needed to complete scoring showed this new tool is feasible in assessing quality of TCM systematic reviews too; it took about 13 minutes on average to complete an assessment and showed well applicability. The statistical analysis revealed that the AMSTAR was slightly more time consuming in contrast to OQAQ; there may be several explanations for this. First, the AMSTAR has 11 items, longer than the 10 items in OQAQ; second, the AMSTAR was developed based on two instruments, including OQAQ itself, so items in the instruments may be overlapped, and it would take less time to complete scoring when filling replicated items; third, the sequence of applying tools may be another explanation, as we conducted the assessment in the order of first AMSTAR and then OQAQ, assessors need more time to look through a systematic review to facilitate it in the first round of assessment by using AMSTAR, while in the second round by applying OQAQ, such time would be saved.
The reliability analysis revealed that the Kappa was poor to fair in some items on AMSTAR. As kappa coefficient shows the proportion of agreement beyond that expected by chance alone, it is a popular measure for chance-corrected nominal scale agreement between two raters [5, 8] ; however, if distribution of item responses is skewed or over concentrated on either the "yes" or the "no" category, the kappa coefficients will become unstable and invalid, and no longer suitable for measuring agreement between two raters, so new methods for calculating valid agreement coefficients are needed to explore in future study. Such items shown in our study included item 1 "was an 'a priori' design provided," item 6 "were the characteristics of the included studies provided," item 7 "was the scientific quality of the included studies assessed and documented," and item 8 "was the scientific quality of the included studies used appropriately in formulating conclusions"; however, the relatively low kappa in item 5 and item 9 cannot be explained by the limitation of kappa statistic.
AMSTAR proved to be feasible to apply in quality assessment of TCM systematic reviews; the main problems emerged were the absence of guidance for certain item response, such as item 5 "was a list of studies (included and excluded) provided," to get "yes" score, four situations may be encountered: list of included studies provided, list of excluded studies provided, both lists of included and excluded studies provided in the same time, and the characteristics of the included studies presented; it is difficult to reach a final conclusion without more detailed directions regarding its use. Those items (4 and 7) with relatively low kappa values identified by Shea et al. [8] , on the contrast, presented more precisely guidance, were easy to apply and easily reached consensus among raters.
As the assessment was undertaken by two assessors, one assessor was with expertise in clinical epidemiology and clinical research methods, and the other was a novice user to these quality assessment instruments, thus it could possibly result in underestimation of the reliability of AMSTAR. Another limitation in this study is lack of backward translation for the adapted tools, the translation into Chinese may produce a different measurement instrument with different properties. The current Chinese version should be translated back into English by a third party, and the back translations would be compared with the original tools to ensure the conceptual equivalence. However, the absence of back translation may offset somewhat in present study by a check of accuracy with a previous Chinese version of two instruments tools [10] .
Although both instruments proved to be useful in this study, the performance of AMSTAR in terms of reliability and validity was better than OQAQ; the new tool is reliable, valid, and easy to use when applied to assess methodological quality of systematic reviews on TCM, although there is room for improvement on a few items. Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo. Alveolar macrophages (AM) reside at the air-tissue interface in the lung and are one of the first lines of defense that interact with inhaled microorganisms and particles [1] . They play a critical role in homeostasis, host defense, and tissue remodeling [2] , and they are readily infected by influenza [3] . AM express many pattern recognition receptors (PRRs) to help recognize the pathogenassociated molecular patterns (PAMPs) on the surface of microorganisms [4, 5] . They are important in initiating response to influenza, regulating the inflammatory response, and potentially limiting secondary bacterial infections [6] .
Influenza A virus causes seasonal and pandemic flu, both of which pose significant public health burdens. Influenza viral antigens have been detected in AM from humans and many animal species [7] [8] [9] [10] [11] [12] [13] [14] [15] , and AM are critical for controlling viral replication in vivo [11, 14] .
Recently, several groups have explored the responses of human monocyte-derived macrophages to avian and/or seasonal flu viral infection using a genome-wide approach [16] [17] [18] . Avian H5N1 and human H1N1 and H3N2 viruses induce increases in similar groups of genes despite the stronger response induced by pathogenic avian viruses compared to seasonal flu viruses in human monocyte-derived macrophages [16] [17] [18] [19] . However, the genome-wide response of resident human AM to influenza infection has not been reported. Our previous study showed that cultured primary human AM support a productive infection with H5N1 but not H1N1 and H3N2 influenza viruses though AM express both avian and human influenza receptors [3, 19] . However, human monocyte-derived macrophages support productive infection with both human and avian viruses [19] [20] [21] . These results suggest that the response of human AM to influenza might be different from the response of human macrophages derived from peripheral blood [22] .
The purpose of our study was to use a genome-wide approach to define the innate immune response of human AM to influenza. Using H1N1 influenza virus PR/8, we performed gene profiling of virus-infected human AM at 4 and 24 h post inoculation (hpi) and verified the alterations in IFN-related genes by real-time RT-PCR and cytokine response by ELISA. We investigated the kinetics of infection-induced cytokine response in human primary AM infected with both live and UV-inactivated PR/8 and the contemporary H3N2 virus A/New York/238/2005 (NY/238) [23] . We also determined if the cytokine response was amplified by paracrine proinflammatory cytokines, TNF-a and IL-1b. In addition, we explored whether influenza infection diminishes gene expression of macrophage scavenger receptors, which could contribute to the impaired ability of AM to clear other pathogens after influenza.
Viral infection resulted in significant alterations of mRNA levels in 1,347 transcripts at 4 hpi and 2,152 transcripts at 24 hpi; these transcripts mapped to 1,077 (4 hpi) and 1,493 (24 hpi) known genes. Tables 1 and 2 show the top 25 genes that were up-regulated or down-regulated by influenza virus. The complete list of altered genes is listed in Data S1. To identify the cellular functions and pathways affected by the infection, the array data were processed by Ingenuity Pathway Analysis (IPA) using IPA version 8.0 (IngenuityH Systems, Redwood City, CA), which associates differentially regulated genes with known specific biological pathways based on information from published literature (www.ingenuity.com). The results from IPA indicate some functional groups of genes were changed at both time points. These genes are involved in antimicrobial and inflammatory responses, cell death, cancer, infection mechanisms, cellular growth and proliferation, cell-mediated immune responses, and immune cell trafficking. Interferon regulatory factor (IRF) activation and PRR signaling were the most prominent pathways activated by viral infection at both time points. In addition, retinoic acid-induced gene-1 (RIG-I) and interferon (IFN) signaling were dominant at 4 hpi, whereas homeostasis-related pathways such as IL-10 and IL-6 were activated at 24 hpi.
As shown in Table 1 at 4 hpi, seven out of the top ten PR/8 upregulated genes were type I IFN family members, and another two up-regulated genes, IFN-stimulated gene (ISG) 20 and CXCL11, were IFN-stimulated genes [24] . At 24 hpi, IFN-stimulated genes CXCL9-11, and IFITM1 were among the top ten genes upregulated by PR/8 (Table 2) . Therefore, we verified the microarray data with a focus on IFN-associated genes by real-time RT-PCR ( Figure 1 ). As shown in Figure 1A , PR/8 infection induces an early response in type I IFN genes IFNA1 and IFNB as well as type III IFN genes IL-29 and IL-28A, although the degree of increase was slightly less than that of most type I IFN genes (Tables 1 and 2) . Along with the increased IFN gene expression, the infection also increased expression of well-known PRR genes associated with IFN production. mRNA levels of RIG-I and melanoma differentiation associated protein-5 (MDA-5) were mainly increased at 4 hpi, whereas TLR3 and 7 were mainly stimulated at 24 hpi ( Figure 1B ). The infection also significantly increased mRNA of IFN-stimulated anti-viral genes myxovirus (influenza virus) resistance 1 (MX1), 2959 oligoadenylate synthase (OAS), and IFN-stimulated gene 56 (ISG56) ( Figure 1C and Data S1), as compared to control cells.
In addition to IFN related genes, PR/8 significantly increased the expression of many cytokine genes and cytokine-regulated genes. These included the proinflammatory cytokines TNF-a (7.3-fold at 4 hpi and 25-fold at 24 hpi), IL-1a (6.9-fold at 24 hpi), and IL-1b (2.3-fold at 4 hpi and 16.3-fold at 24 hpi). We verified the alteration in IL-1a and IL-1b by real-time RT-PCR (data not shown). The infection also upregulated expression of TNF-a induced proteins 2, 3, 6, and 8, as well as TNF receptor family members 9 and 10. Expression of IL-1 family members interleukin 1 receptor 1 (IL-1R1) and the IL-1 receptor antagonist (IL-1Ra) was also increased (Data S1). In addition, PR/8 infection up-regulated mRNA expression of many chemokine genes including CC chemokines CCL2-5, and CCL20, as well as CXC chemokines CXCL9-11 (Data S1 and Tables 1 and 2). CXCL9-11 were markedly increased when compared to controls both in the microarray studies and in additional verification studies (Tables 1 and 2 and Figure 1D ). CCL5 was the most increased CC chemokine (232-fold at 4 hpi, 234-fold at 24 hpi) (Tables 1  and 2) .
Besides the increased mRNA expression of proinflammatory mediators, PR/8 also increased mRNA expression of the antiinflammatory cytokine IL-10 (1.9-fold at 4 hpi and 2.8-fold at 24 hpi), its receptor (8-fold at 4 hpi and 6.8-fold at 24 hpi), and suppressor of cytokine signaling (SOCS)1 (9.4-fold at 4 hpi and 12.1-fold at 24 hpi) and SOCS3 (5.4-fold at 4 hpi), which have been shown to be important in turning off inflammatory responses and dampening a robust innate immune response [25] . PR/8 also upregulated expression of IL-6 (36.9-fold at 4 hpi and 66.6-fold at 24 hpi), another important cytokine responsible for homeostasis, and several cytokines that activate and regulate adaptive immune response, especially IL-15 and its receptor, IL-23A, and IL-27 [26] [27] [28] (Data S1).
We verified the putative increases in secreted cytokines and chemokines at the protein level by ELISA in 8-14 additional donors. As shown in Figure 2 , PR/8 infection significantly increased secretion of cytokines of TNF-a, IL-6, IFN-a, and IL-29, and CXC chemokines CXCL8-11 as well as CC chemokines CCL2, 4 and 5. Consistent with the mRNA data, CXCL10, CXCL11 and CCL5 were the main chemokines induced by the virus, and AM secreted slightly more IFN-a than IL-29 ( Figure 2 ). Secretion of cytokines and chemokines occurs without the release of a significant amount of infectious viral particles From our previous studies we knew that AM do not release a significant amount of infectious virus particles after infection with human influenza viruses [3, 19] . To investigate whether the infected macrophages were synthesizing viral proteins, we performed a time-course infection experiment in AM from an additional 4 donors using both live ( Figure 3A -D) and UVinactivated PR/8 viruses ( Figure 3E ), examined the kinetics of viral antigen synthesis by staining hemagglutinin (HA) or nucleoprotein, and measured secretion of selected cytokines by ELISA. As shown in Figure 3F , there was a slight increase in viral production at 6 hpi, when about 20% of the cells expressed viral antigens and then no more net increase in viral release as up to 80% of the cells expressed viral proteins by 48 hpi. The viral antigen staining was due to viral replication, since there was no signal with UV-inactivated virus ( Figure 3E ). Despite the abortive release of infectious virus, PR/8 infection induced a time dependent cytokine and chemokine response in human AM ( Figure 3G -K). Viruses triggered an early and rapid secretion of IFN-a and CXCL10 at 6 hpi. Secretion of CCL5 and CXCL8 followed the pattern of the viral protein synthesis increasing with time. The virus-induced increase of TNF-a peaked at 24 hpi and then declined. UV-inactivation abolished the virus-stimulated TNF-a production, significantly decreased secretion of IFN-a, CXCL8, and CCL5. However, the inactive virus was able to stimulate a strong CXCL10 response, although the degree was slightly smaller than that from live PR/8 ( Figure 3I ). The different patterns of the induction suggest that the cytokine response may involve different regulatory mechanisms. In addition, we compared the alterations in mRNA levels of selected innate immune response genes at 3 and 24 hpi for both UV-inactivated PR/8 and live PR/8 infections. Consistent with the protein data, both live and UV-inactivated PR/8 stimulated a large increase in CXCL10 mRNA at both time points. UV-inactivated PR/8 stimulated an up to 4 fold increase of CCL5 and IFNA1. UV-inactivated virus did not alter mRNA levels of RIG-I, TLR7, or ISG56 at either time point (data not shown). These results indicate that viral replication is required for most selected innate immune responses but not required for the CXCL10 response.
To investigate whether the results observed with PR/8 can be extended to contemporary human influenza virus infection, we performed a time-course experiment using a H3N2 virus NY/238, a influenza virus rescued by reverse genetics technology based on a swab sample from a patient from New York during the winter of 2005 [23] . Consistent with the results from PR/8 infection, human AM do not support a productive NY/238 infection as verified by no increase in infectious viral particles released from infected culture as measured by plaque assay (data not shown). As shown in Figure 4A , NY/238 infection markedly stimulated CXCL10 mRNA, NY/238 virus also triggered an early increase in the expression of RIG-I and IFNA1 genes and increased mRNA levels of antiviral gene ISG56 and CCL chemokine CCL5. Inoculation with the same amount of UV-inactivated NY/238 virus was able to stimulate an IFNA1 and CXCL10 response. However, the response was smaller than that observed with live virus. Unlike PR/8, NY/238 virus did not induce a significant increase in TLR7 mRNA ( Figure 4A) . At the protein level, NY/238 virus induced a similar response as PR/8 virus in terms of cytokine and IFN production ( Figure 4B ). Consistent with the finding with PR/ 8, viral replication was required for most chemokine and cytokine response and but was not requisite for CXCL10 release.
Because PR/8 infection increased secretion of TNF-a and increased gene expression of IL-1 family members, well-known proinflammatory mediators that cause release of inflammatory chemokines, we were interested in the impact of these proinflammatory mediators on the overall chemokine response during the infection in human AM. Our hypothesis was that TNF and IL-1 signaling would augment chemokine secretion in a paracrine manner [29] . As shown in Figure 5 , neutralization of TNF pathway by its soluble receptor significantly decreased secretion of CXCL8 by 65% (P,0.001) and CCL5 by 53% (P,0.05), but did not alter secretion of IFNs, CXCL10, or TNF-a itself. Blockade of the IL-1 receptor by its naturally occurring receptor antagonist IL-1Ra [30] had a similar effect. When the activity of both cytokines was inhibited, there was no further reduction in chemokines greater than that of a single inhibitor, although there was a slight decrease in CXCL10 response in the presence of both inhibitors, the response was not statistically significant.
AM are important phagocytes and express many scavenger receptors. The microarray experiments indicated that PR/8 infection also significantly decreased mRNA levels of many macrophage receptors especially at 24 hpi (Table 2 and Data S1). We, therefore, investigated the impact of influenza infection on expression of scavenger receptors by real-time RT-PCR. Consistent with the results from microarray experiments, PR/8 infection significantly decreased the mRNA levels of CLEC7A (Dectin 1), macrophage scavenger receptor 1 (MSR1), CD36, and the mannose receptor C type 1 (MRC1) but did not change the expression of MRC2. However, we were not able to confirm the decrease of MARCO, due to the large variation in responses among different donors ( Figure 6A ). To further investigate if the decrease in macrophage receptor expression was associated with functional consequences, we evaluated the uptake of zymosan, which are yeast walls recognized by CLEC7A, and heat-killed S. aureus. As shown in Figure 6B , PR/8 infection reduced uptake of zymosan by AM at 24 hpi in a dose dependent manner. We did not observe a significant cell loss or cytopathic effect at 24 or 48 hpi, although most cells were infected as seen in Figure 3A -C. In addition, PR/8 infection did not affect uptake of heat-killed S. aureus until 72 hpi, when the infection induced a significant cytopathic effect (data not shown).
Alveolar macrophages produce a robust innate immune response to influenza. This includes a significant induction of cytokines and chemokines, pathogen recognition, and apoptotic responses, which are similar to the responses of human monocyte derived macrophages [16, 17] . Consistent with other studies of avian or human influenza infections in humans and animals [16, 17, [31] [32] [33] , PR/8 stimulated an early and prominent IFN response in human AM despite of the failure to release infectious viral particles. Human AM produce both type I and type III interferons (Figures 1 and 2) . In contrast, alveolar epithelial cells do not produce any type I interferon IFN-a in response to influenza [34] . These results indicate a cell-specific pattern in producing IFN in response to viral infection. It is well known that RIG-I like RNA helicases (RLHs) and TLRs are the two main PRRs responsible for IFN production against RNA viruses including influenza. RLHs (RIG-I and MDA-5) recognize cytoplasmic viral double-stranded RNA, whereas TLRs (TLR3 and TLR7) sense viral nucleic acid in the endosomal compartment [35, 36] . In the current study, PR/8 infection up-regulated mRNA levels of RIG-I and MDA-5 mainly at 4 hpi, but the mRNAs of TLR3 and 7 mainly at 24 hpi ( Figure 1B) , which suggests that RLHs might be the early sensors and TLRs might be the late sensors for PR/8 in human AM. These results correlate well with those reported by Takeuchi and Thompson that RLHs were responsible for local production of IFNs, whereas TLRs were mainly involved in the late stages of systemic infection [35, 36] . At early times PR/8 triggered mainly pro-inflammatory responses, whereas at later times PR/8 also activated pathways involved in the maintenance of homeostasis such as the activation of IL-10 and IL-6, as well as up-regulation of SOCS genes (Data S1). Therefore, therapeutic regulation of the inflammatory response in acute lung injury should consider both strategies to inhibit secreted cytokines but also strategies to dampen the innate immune response by stimulating IL-10 and SOCS genes. We were able to confirm the results found with PR/8 in contemporary influenza virus NY/238-infected human AM with the exception of an increase in TLR7 mRNA. This might be due to a lower MOI of virus used in the experiments because of the limitation of the viral titer, but it could also be due to differences in the natures of these two viruses or the difference in methods for propagating these two viruses.
CXCL9-11 were the most highly induced chemokines by influenza viruses as verified at both mRNA and protein levels (Figures 1 and 2) . These three chemokines bind to a common receptor CXCR3, and the importance of CXCR3 signaling has been shown in the pathogenesis of several viruses including influenza [32, [37] [38] [39] . CXCL10 is highly induced in avian flu (H5N1)-infected ferrets, non-human primates, and human cells including alveolar epithelial cells and monocyte-derived macrophages [16] [17] [18] 32, 33, 40] , and has been viewed as a prognostic marker for several viral infections [37, 39, 41, 42] . In mice, the peak level of CXCL11 mRNA coincides with the peak of the viremia [43] , and the CXCL11 protein has been reported to have antiviral activity [44] . In addition, all three CXCR3 ligands can induce epithelial cell chemotaxis and proliferation and perhaps accelerate epithelial wound repair during the resolution of viral infections [45, 46] . The robust induction of CXCL9, 10, and 11 in both AM (Figures 1 and 2) and human alveolar type II cells [34] as well as the distinct CXCL10 response induced by both live and UV-inactivated influenza virus PR/8 and contemporary virus NY/238 (Figure 3 and 4) suggest that this family of proteins likely plays an important role in the human lung alveolar defense against influenza infection, which will require further study.
The response of alveolar macrophages was different in a several ways from that reported for human monocyte derived macrophages. The major difference is that alveolar macrophages infected with human influenza viruses do not release much infectious virus, whereas human monocyte-derived macrophages do ( [19, 20] and Figure 3 ). The mechanism for the non-productive infection was not investigated in this study and is likely complicated. One of the possible mechanisms might be related to the lack of gene expression of transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT) by human AM (microarray data not shown). Both TMPRSS2 and HAT are type II transmembrane serine proteases [47] possessing trypsin-like activity and are known to be important for cleaving influenza HA required for productive infection [48] . In recent studies Bottcher et al suggest that TMPRSS2 is mainly responsible for cleavage of newly synthesized HA, whereas HAT cleaves both endocytosed and newly synthesized HA [49] . Therefore, lack of these two gene products in human AM may partially explain the lack of released infectious virus by these cells. In addition, both PR/8 and NY/238 viruses induced an early activation of type I IFN, especially IFN-a (Table 1 and Figures 1,  3, and 4) . The strong anti-viral property of type I IFN [50] may also contribute to the non-productive infection in these cells. Further studies will be required to understand the mechanism for the failure of release of infectious viral particles by human AM. In addition, inactivation of influenza by UV did not abolish the influenza viruses-stimulated CXCL10 secretion by AM (Figures 3  and 4) , which is different from studies with human monocytederived macrophages [21, 51] and with human alveolar type II epithelial cells isolated from the same donors ( [34] and data not shown). In those studies, release of CXCL10 is totally dependent on viral replication. The mechanism for the distinct CXCL10 response in human AM will require additional and carefully designed studies. The differences between human AM and monocyte-derived macrophages indicate the importance of investigating the response of AM to influenza infection during the initial phases of infection in the lung because AM are main targets for both human and avian influenza viruses [19] .
Chemokine and cytokine responses are required for protection of the host against viral infection. However, an exuberant response contributes to the influenza-induced morbidity and mortality, especially in severe pandemic and avian influenza infections [16, 52] . In the current study, PR/8 infection induced an increase in TNF-a and IL-1b, well-known paracrine proinflammatory factors. Therefore, we hypothesized that inhibiting these factors might reduce the influenza-induced-inflammatory response. Since the contemporary virus NY/238 induced a similar cytokine and chemokine response as PR/8, it would be reasonable to expect that the regulation of chemokine and cytokine in contemporary influenza infection might also be similar to PR/8 infection. As shown in Figure 5 , inhibiting TNF and/or IL-1 decreased more than 50% of the PR/8-induced secretion of inflammatory chemokines CXCL8 and CCL5 but did not truly affect type I interferon or CXCL10 response, although we observed a decrease of CXCL10 in the presence of both inhibitors ( Figure 5 ). TNF and IL-1 signaling are known to be regulated by NF-kB and there are several NF-kB binding sites in the promoter of CXCL10 [53] , despite of the fact that CXCL10 is an IFN-induced protein [24] . This may explain why inhibiting both pathways slightly decreased the amount of CXCL10 from infected AM. Our results suggest that short term targeting the critical paracrine factors might be beneficial for controlling the excessive infiltration of inflammatory cells and acute lung injury during pandemic or avian flu infection in vivo. Of course, this would require careful consideration of time and dose so as not to increase secondary bacterial infections. Influenza infection significantly decreased mRNA level of macrophage receptors CLEC7A, MSR1, CD36, and MRC1 ( Figure 6A and Table 2 ). CLEC7A belongs to the C-type lectin family and functions as a PRR that recognizes a variety of beta-1, 3-linked and beta-1, 6-linked glucans from fungi. A decrease of CLEC7A in infected AM suggests that these cells might not efficiently recognize and engulf fungi after influenza infection. As shown in Figure 6B , the uptake of zymosan, a yeast cell wall component containing beta-1-3-glycosolic linkeages, was decreased in a dose-dependent manner in PR/8-infected human AM. This effect was not associated with cell loss or cytopathic effect because we did not observe a significant cytopathic effect ( Figure 3B ) even at a MOI of 1 (data not shown). However, the explanation of the decreased uptake might be more complicated than simply the loss of this receptor. In addition, other macrophage receptors MSR1, MARCO, CD36, as well as mannose receptor MRC1 are important for bacterial and particle uptake [54] [55] [56] . Mice with deletions of MSR1 or CD36 have increased susceptibility to pneumococcal or staphylococcal pneumonia [57] [58] [59] . Although impairment of macrophage phagocytosis of bacteria after influenza in mice is well recognized [60, 61] and secondary bacterial infection after influenza is a common clinical problem, we were not able to detect a significant decrease in uptake of heat-inactivated S. aureus in human AM until 72 hpi, at which time the cytopathic effect was significant. We did not observe a consistent decrease of MSR1 protein by flow cytometry in PR/8-infected AM, which might explain why the infection did not impair the bacterial uptake (data not shown). We were also not able to verify the decrease of mRNA level of MARCO, another important macrophage scavenger receptor for influenza infections in mice and human cells [54, 56, 58, 62] . Nine of 11 donors showed a decrease in mRNA levels of MARCO after infection with PR/8 ( Figure 6A ). Two other donors had an increase in levels of MARCO mRNA. Therefore, changes of bacteria-related receptors in human AM after influenza require additional studies, and there may be variations in response among individuals.
In summary, we performed a global profiling of innate immune response and regulation with a focus on chemokine and cytokine response in influenza-infected human AM. Human AM are apparently different from human monocyte derived macrophages in their ability to release infectious virus and the CXCL10 response to UV inactivated virus. Future studies should compare these responses in peripheral and alveolar macrophages from the same donors. In addition, during acute lung injury, short term targeting of paracrine inflammatory factors such as TNF and IL-1 as well as targeting IL-10 and SOCS genes might decrease the acute injury and allow for better gas exchange.
Isolation and culture of human alveolar macrophages AM were isolated from deidentified human donor lungs, which were not suitable for transplantation and donated for medical research. We obtained the donor lungs through the International Institute for the Advancement of Medicine (Edison, NJ) and the National Disease Research Interchange (Philadelphia, PA) [3] . The Committee for the Protection of Human Subjects at National Jewish Health approved this research and has designated this research as non-human project. The isolated AM could be frozen and recovered in 90% FBS and 10% DMSO. There was no apparent difference in response with frozen or freshly isolated macrophages in terms of the level of infection and virus-induced TNF-a secretion (data not shown). AM were plated in DMEM/ 10% FBS with antibiotics, and cultured at 37uC in 10% CO 2 overnight. The cells were then washed and cultured for another day in DMEM and 1% charcoal stripped FBS with antibiotics before viral infection. Their purity was measured by staining for CD68 (Dakocytomation, Carpinteria, CA) [3] .
Influenza A virus A/PR/8/34 (PR/8) was grown in 10-day-old SPF Premium Eggs (Charles River SPAFAS. North Franklin, CT) and prepared as described previously [3] . Contemporary influenza H3N2 virus, A/New York/238/2005 (NY/238), was created by reverse genetics using plasmids that corresponded to the consensuses sequence obtained from a human swab specimen collected in New York State in the winter of 2005 [23] . NY/238 was passaged in Madin-Darby Canine Kidney (MDCK) cells and the viral titer was measured by plaque assay on MDCK cells as described previously [34] . Briefly, stocks of purified virus was serially diluted in DMEM with 1 mg/ml TPCK trypsin (Sigma-Aldrich, St. Louis, MO) and used to inoculate triplicate wells of near confluent MDCK cells. After a 1 h inoculation, the inoculum was removed and the cells were overlaid with MEM with 4% FBS and 0.5% SeaKem LE agarose (Cambrex, Rockland, ME). Plaques were stained and counted after 72 h incubation at 37uC, with the agarose overlay medium containing 10% neutral red (Sigma-Aldrich). For UV-inactivation of PR/8 or NY/238, 500 ml diluted virus was placed in a 35-mm 2 petri dish on ice and irradiated twice in a UV Stratalinker (Stratagene, La Jolla, CA) at a cumulative dose of 120 mJ/cm 2 . Viral inactivation was demonstrated by plaque assay on MDCK cells as described above.
On the day of infection, AM were inoculated with live PR/8 at a designated multiplicity of infection (MOI) or with the same amount of UV-inactivated PR/8 for 1 h. After inoculation, cells were washed and then cultured until harvest. Influenza infection was verified by immuno-fluorescent staining with goat antibody to the hemagglutinin of PR/8 (kindly provided by BEI Resources, Manassas, VA). For NY/238 infection, AM was inoculated with a MOI of 0.1 instead of 0.5 due to the limitation of viral titer and infection was confirmed by immuno-fluorescent staining with mouse antibody to influenza nucleoprotein (Millipore, Billerica, MA).
For the inhibition experiments, cells were treated with 10 mg/ml human IL-1 receptor antagonist (IL-1Ra) [63] and extracellular TNF neutralization was achieved by treating cells with 10 mg/ml recombinant human soluble TNF receptor (sTNFR) [64] . Both IL-1Ra and/or sTNFR were added to the cells for 45 min before virus inoculation. DMEM alone was used as vehicle control for both inhibitors. After inoculation, cells were washed and cultured with the inhibitors for an additional 24 h.
At 4 and 24 hpi, total RNA from virus-infected and noninfected AM from three donors was extracted and purified using RNeasy kit (QIAGEN, Valencia, CA). The samples were run on Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA) and processed as indicated by the manufacturer in the Microarray Core of the University of Colorado Denver. All data is MIAME compliant and the raw data had been deposited in a MIAME compliant database Gene Express Omnibus (GEO). The GEO accession numbers are GSM762686, GSM762687, GSM762688, GSM762689, GSM762694, GSM762695, GSM762696, GSM762697, GSM762702, GSM762703, GSM762704, GSM762705. Analyses of microarray data were performed using R statistical package from Bioconductor open source software for bioinformatics. Prior to statistical analyses, raw data from array scans were processed using the Robust Multi-chip Average (RMA) normalization method to subtract a background value [34] . After normalization, data were filtered to exclude all probe sets with an ''absent'' call in all samples and to remove transcripts that demonstrated little variation across all arrays by comparing the variances of the log-intensities for each gene with the median of all variances for the entire array. The filtered gene list was generated using the Student's T test to select statistically significant genes and corrected using the False Discovery Rate approach. Genes that had at least a 2-fold change in comparison to the uninfected controls for all three subjects were used for further analyses.
Real-time RT-PCR mRNA expression of selected genes that were significantly upregulated by PR/8 or NY/238 were validated by quantitative realtime RT-PCR [34] . These genes include IFNs, PRRs, chemokines, and SOCSs. Except for IFN-b and IL-29 genes whose probes were synthesized in house [34] , the specific probes for other genes were purchased from Applied Biosystems (Applied Biosystems Inc. Foster City, CA). The expression level of each specific gene was normalized to the level of a constitutive probe cyclophilin B [34] .
Supernatant from PR/8 OR ny/238-infected and non-infected cells were harvested at designated times after inoculation for the measurement of chemokine and cytokine secretion by ELISA. The ELISA kits for human CXCL9, CXCL10, CXCL11, CCL5, CXCL8, and IL-29 were purchased from ELISA Tech (ELISA Tech, Aurora, CO). The ELISA kit for IFN-a was purchased from Invitrogen (Invitrogen, Carlsbad, CA).
Human AM were cultured and infected with PR/8 at the designated MOI. Uptake of zymosan or heat-killed S. aureus were performed according to manufacturer's instructions. For uptake of zymosan, cells were incubated with fluorescent-labeled zymosan A Bioparticles (Invitrogen) at a ratio of 10 particles per cell for 2 h, then cells were washed and fixed with 4% paraformaldehyde for 10 min. The uptake was analyzed by fluorescent microscopy. For uptake of heat-killed S. aureus, cells were incubated with pHrodolabeled, heat-killed S. aureus (pHrodo-SA) (Invitrogen) at a ratio of 20 particles per cell for 2 h. The cells were then washed to remove non-internalized particles, collected, and fixed with 4% paraformaldehyde. Uptake of the pHrodo-SA was analyzed on the LSR II flow cytometer (BD Biosciences) in the National Jewish Health Flow Cytometry Core, and the data were analyzed using FlowJo software (TreeStar, Ashland, OR). In addition to the PR/8 infected cells, positive control uninfected cells and negative control paraformaldehyde fixed cells were also used.
Statistical analyses were conducted in GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). Pair-wise comparisons were tested for significance using Wilcoxon matched pairs test or Paired T test. Comparison among three or more groups was performed using one-way ANOVA with Tukey's post test analysis.
Data S1 Human AM from 3 non-smoking donors were isolated, cultured, and infected by PR/8 virus at MOI of 0.5. The gene profiling of infected and non-infected cells at 4 and 24 hpi from each donor was examined by microarray experiments using Affymetrix HG-U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA). The filtered gene list was generated as described in the Section of Methods and Materials. The data show probe ID, gene symbol, gene name, and fold change at 4 and 24 hpi. Red indicates similar results from multiple probes for the same gene, and the probe ID is the representative probe ID from several probes. (XLS) Successful treatment of Chlamydophila pneumoniae acute respiratory distress syndrome with extracorporeal membrane oxygenator: a case report and diagnostic review INTRODUCTION: Chlamydophila pneumoniae is a respiratory pathogen known to infect the upper and lower respiratory tracts. Infection severity can range from sub-clinical pulmonary infection to acute respiratory distress syndrome. CASE PRESENTATION: A previously healthy 62-year-old Caucasian man was admitted to our hospital for acute respiratory failure. Serum samples obtained every week starting from the day of admission showed clear-cut seroconversion for C. pneumoniae antibodies. All other cultures obtained during the first days of hospitalization were negative. Despite maximal ventilatory support (high positive end expiratory pressure, fraction of inspired oxygen of 1.0, nitric oxide inhalation, neuromuscular blocking agents and prone positioning), our patient remained severely hypoxemic, which led us to initiate an extracorporeal membrane oxygenation treatment. Extracorporeal membrane oxygenation and hemodiafiltration were withdrawn on day 12. Our patient was extubated on day 18 and discharged from our Intensive Care Unit on day 20. He went home a month later. CONCLUSION: We describe the first published case of acute respiratory distress syndrome due to C. pneumoniae infection successfully treated by extracorporeal membrane oxygenation, a very useful tool in this syndrome. A quick and specific method for the definite diagnosis of Chlamydophila infection should be developed. Chlamydophila pneumoniae is an obligate intracellular Gram-negative bacterium. The spectrum of disease, in addition to pneumonia and influenza-like illness, includes pharyngitis, sinusitis, bronchitis, exacerbation of chronic obstructive pulmonary diseases and reactive arthritis [1] [2] [3] [4] [5] . C. pneumoniae accounts for 6% to 20% of cases of community-acquired pneumonia (CAP) [1, 2] . Many of these cases have few symptoms and don't require hospitalization ('walking pneumonia'). However, more severe cases may occur, with up to 18% requiring hospitalization [6] and even mechanical ventilation, especially in elderly, immunocompromised hosts and patients with coexisting cardiopulmonary disease [7] , but also, rarely, in previously healthy adults [8] .
Old and new macrolides are effective against C. pneumoniae and have been recommended as first-line treatment. New fluoroquinolones are also effective in vitro against C. pneumoniae and can be used. Studies have shown that 35% to 47% of C. pneumoniae pneumonia is mixed with other pathogens, the most common being Streptococcus pneumoniae [9, 10] .
We describe the case of severe CAP due to C. pneumoniae infection in a previously healthy adult patient, with acute respiratory distress syndrome (ARDS) necessitating extracorporeal membrane oxygenation (ECMO).
A previously healthy 62-year-old Caucasian man was admitted to our hospital for acute respiratory failure.
Our patient developed a fever of up to 40°C seven days earlier and a non-productive cough three days later. He had not received any antimicrobial treatment prior to his hospitalization, the diagnosis of his primary care physician being influenza (A/H1N1v), given the ongoing outbreak.
His medical history was remarkable for possible viral pericarditis without any consequence in 2007 and a gastric ulcer 30 years earlier. He had no drinking habits. He did not smoke cigarettes. He had not travelled abroad recently. He did not have any bird or pet.
On hospital admission, our patient was in acute respiratory distress. His respiratory rate was 40 breaths/ minute, his temperature 38.3°C, his pulse 98 beats/minute and his blood pressure 114/60 mmHg. Auscultation revealed crackles over his whole left lung and over his right lower lung field. A computed tomography scan showed diffuse alveolar type infiltrates in his two lung fields with air bronchograms (Figure 1 ).
Arterial blood gas analysis (under 100% oxygen through a non-rebreathing mask) showed pH 7.54, a partial pressure of carbon dioxide (PaCO 2 ) 44 mmHg, partial pressure of oxygen (PaO 2 ) 38 mmHg and an arterial blood oxygen saturation of 84%. His white blood cell count was 5780 cells/μL (86% neutrophils) and the erythrocyte sedimentation rate was 92 mm/h. Laboratory values showed serum creatinine at 1.7 mg/ dL, potassium at 2.8 mEq/L, creatine phosphokinase at 644 IU/L, liver test alterations (alanine transaminase at 87 IU/L), lactate dehydrogenase elevation (1708 IU/L) and D-Dimers at 7420 ng/mL, activated partial thromboplastin time of 72 seconds, normal international normalized ratio and blood platelets at 166.000/μL. His urine output was 0.4 mL/kg/h over six hours. An electrocardiogram showed a sinus tachycardia with a complete right bundle branch block.
Serum samples obtained every week as from the day of admission showed a clear-cut seroconversion for C. pneumoniae antibodies (the course of the antibody titers shown in Table 1 ).
Paired serum samples and antigens against the most common microorganisms, including atypical bacteria and common viruses such as A/H1N1v, were negative. Blood, sputum and urine tests for bacterial cultures obtained during the first day of hospitalization were negative.
Our patient was treated by amoxycillin-clavulanic acid, moxifloxacin and oseltamivir. His respiratory status necessitated endotracheal intubation and mechanical ventilation. Severe arterial hypotension prompted norepinephrine infusion and the insertion of a pulmonary artery catheter. The initial hemodynamic pattern was typical for sepsis (hemodynamic values are shown in Table 2 ). Metabolic data showed a mixed venous saturation of 56%.
Despite maximal ventilatory support (high positive end expiratory pressure, an inspired oxygen fraction (FiO 2 ) of 1.0, nitric oxide inhalation of 20 ppm, neuromuscular blocking agents and prone positioning), our patient remained severely hypoxemic (PaO 2 /FiO 2 = 38) which led us to initiate ECMO treatment.
Venovenous ECMO (a Sorin Revolution centrifugal pump, a Sorin ECCO oxygenator and a Sorin Satcrit console from Sorin Group, Milano, Italy) was put in place on the fifth day of hospitalization, with a left femoral 22-Fr drainage cannula and a right femoral 23-Fr return cannula, inducing a drastic improvement of our patient's oxygenation parameters (PaO 2 = 120 mmHg). The mixed venous oxygen saturation (SVO 2 ) increased from 56% to 86%. Continuous veno-venous hemodiafiltration (CVVHDF) renal replacement therapy was also initiated on day three because of acute renal failure.
There were no severe complications of the ECMO treatment except for hemorrhagic suffusion of the two femoral catheter insertion points, requiring a blood transfusion.
ECMO and CVVHDF were withdrawn on day 12. Our patient was extubated on day 18 and discharged from our Intensive Care Unit on day 20. He went home a month later. He is now in good physical condition and has returned to work and to a normal social life.
This case illustrates the polymorphism in the presentation of C. pneumoniae infection, which can cause severe CAP complicated by ARDS, even in immunocompetent patients. Pneumonia and bronchitis are the most common clinical infections associated with C. pneumoniae. The classical pulmonary presentation is a single subsegmental infiltrate, even though lobar consolidation or bilateral infiltrates can also be seen.
Two major aspects are discussed below: the rationale for the use of ECMO in ARDS patients as well as its different techniques, and the difficulties of diagnosing C. pneumoniae pneumonia.
There are two main ECMO techniques according to the type of vascular access that is used: venovenous and venoarterial. Each one has a specific indication. Venoarterial ECMO gives cardiac and respiratory support. Indications for venoarterial ECMO include postcardiac surgery (heart surgery or heart transplantation), cardiogenic shock due to acute myocardial infarction or acute myocarditis and intoxication. For cardiac support, the goal is to optimize organ perfusion (by obtaining a SVO 2 greater than 70%, which usually needs an output index of about 3 L/ min/m 2 ). This is achieved by choosing the appropriate sizes of cannula according to the patient's body surface area.
In isolated respiratory failure, a venovenous access is preferred. The objective is CO 2 removal at least equal to the patient's metabolism (roughly 3 cm 3 /kg/min in adults). The purpose of venovenous ECMO use in ARDS patients is lung protection (reducing ventilatorinduced lung injury) through a decrease in alveolar distention permitted by the reduction in ventilator conditions.
Even though a lot of progress has been done in technical issues, the risk-benefit ratio must be taken into account when ECMO is proposed to a patient with severe respiratory failure. As in all extracorporeal devices, anticoagulation is mandatory. Many patients will experience bleeding, which can be very severe. Contraindication for anticoagulation remains the most important limitation of this technique. Previous severe disability or poor prognosis due to underlying disease constitutes the other main contraindication for ECMO initiation. The main complications are bleeding, thromboembolism, cannula-related complications, pulmonary embolism or infarction, aortic thrombosis and coronary or cerebral hypoxemia; the latter three being more frequent in a venoarterial montage type. Discussion still stands in the literature as to whether mechanical ventilation of more than seven days is or is not a relative contraindication for ECMO because of ventilation-induced lung injury [11] .
The first randomized clinical trials failed to demonstrate beneficial effect of ECMO for severe respiratory failure in the 1970s and 1980s. Since then, technical improvements for ECMO on one hand, and better treatment of the ARDS (protective ventilation and others) on the other hand, have renewed interest in ECMO [12] . The recent CESAR trial (Conventional Ventilation or ECMO for Severer Adult Respiratory Failure [13] ) demonstrated a possible beneficial effect of ECMO. Of those patients referred for ECMO, there was 63% survival rate at six months without disability, compared to 47% in those who were assigned to conventional management. This translates to one extra survivor without disability for every six patients treated.
A recent Italian experience, in patients with ARDS due to the influenza A/H1N1 virus, based on pre-emptive patient centralization showed a 77% survival rate if ECMO was started within seven days of initiation of mechanical ventilation [11] . Indication for ECMO in this study was refractory hypoxia or an oxygenation index below 30 despite a PaO 2 /FiO 2 ratio greater than 100 mmHg.
Treatment of critically ill patients affected by the 2009 Influenza A (H1N1v) outbreak in Australia and New Zealand [14] included ECMO, with a 71% survival rate at Intensive Care Unit discharge, an excellent result. These three studies emphasize the renewed place of ECMO in the treatment of severe ARDS with very good survival rates, considering the severity of the initial insult.
The limitation of plateau airway pressures and the low tidal volume used in ARDS patients have been at the cost of an increased PaCO 2 . There has therefore been an increase in interest for extracorporeal CO 2 removal techniques. Different techniques have been developed but most of them have not gone to relevant, sufficiently powered clinical trials due to technical problems or insufficient CO 2 removal or oxygenation [15] . The pumpless extracorporeal lung assist (PECLA), or Novalung ® [Novalung GmbH, Heilbronn, Germany], is a compact pumpless device driven by the pressure gradient between arterial and venous blood. A femorofemoral setting is most commonly used. The main advantage of a pumpless device is a reduction in mechanical blood trauma, bleeding and hemolysis. The Novalung ® has been widely studied in ARDS. Three studies have demonstrated the clinical efficacy of this PECLA device [16] [17] [18] . CO 2 removal is efficient but oxygenation may be insufficient in these critically ill patients.
Diagnosis of C. pneumoniae pneumonia is still debated in the current literature. According to Grayston et al. and Saikku et al. [19, 20] , who first described this Chlamydia species, almost everybody is infected and reinfected with C. pneumoniae throughout his or her life. Unspecific symptoms of C. pneumoniae infections make the diagnosis even more difficult, with a possible underestimation of its frequency. Undetected infections may lead to chronic disease with serious consequences, such as atherosclerotic cardiovascular disease [21] or asthma. Its incidence in CAP ranges from 3% to 22%, varying according to the diagnostic method used [22] . The most frequent routinely used diagnostic techniques are serological, including complement fixation test, immunofluorescence assay, microimmunofluorescence (MIF) and genus-and species-specific enzyme-linked immunosorbent assay (ELISA) systems. No currently available serologic tests of a single serum specimen will provide conclusive evidence of a current infection with C. pneumoniae.
Since the publication of the Centre for Disease Control (CDC) 2001 recommendations for Nucleic Acid Amplification Tests (NAATs) [23] , a multitude of inhouse polymerase chain reaction (PCR) tests have been described, though very few of them have been validated by the CDC. Results of NAATs may be unreliable because of cross-contamination, inappropriate treatment of the clinical samples (leading to the loss of the target nucleic acid) or the presence of inhibiting substances [24] . Additionally, validation of these tests is primarily analytical and not against clinically obtained specimens.
Bacterial culture has traditionally been considered the 'gold standard' for diagnosis; however, its sensitivity, even in excellent laboratories, seldom exceeds 90% and is typically between 75% and 85%. The culture is technically difficult to implement and is only available in a few laboratories worldwide [25] .
Even though MIF is still actually considered as the 'serological gold standard' technique for the detection of species-specific antibodies, there is a large discrepancy between MIF testing for C. pneumonia and detection of these organisms by culture or PCR. The MIF test is reliable for detection of a prior exposure to Chlamydiae by the presence of immunoglobulin G (IgG) antibodies and is relatively sensitive for the detection of IgM. IgM is an unreliable marker of acute infection in adolescents and adults since it is often not present, presumably because of previous infection by a chlamydial species [24] . Additionally, MIF is quite a laborious and subjective technique that requires much experience, is not standardized and has significant laboratory-to-laboratory variations.
In our institution, in order to get better sensitivity (for example, after reinfections), better reproducibility, greater objectivity and less cross-reactivity in the serology tests for Chlamydiaceae, two-level serological testing has been implemented for C. pneumoniae. The first level test is a search by ELISA-technique for anti-major outer membrane protein (MOMP) IgG and IgA antibodies (C. pneumoniae-IgG & IgA-ELISA plus, Medac, Hamburg, Germany) which are species-specific. This permits us to screen patients. The disadvantage of anti-MOMP antibodies is their late appearance in acute primary infection (three weeks for IgA, six to eight weeks for IgG) and their considerable persistence in human serum afterwards, independent of the bacterial eradication. In general, the prevalence of C. pneumoniae IgG antibody reaches 50% in adults above 20 years of age, and 80% in the elderly population (> 70 years old). The second level test is another ELISA assay, rELISA (Medac), for the detection of genus-specific anti-lipopolysaccharide(LPS) antibodies. Chlamydiae contain, as a common immunodominant antigen, the LPS to which the first immune reaction is directed. These anti-LPSs have the main advantage of rising quickly during acute infection (five to ten days for IgA), allowing early diagnosis, and returning to normal a few weeks after the infection and are thus associated to the eradication of the bacterium [26] . The use of paired sera enables the discrimination of current infections, reinfections and reactivations (defined by titer increase) from chronic persistent ones (constant LPS, IgG and IgA antibody titers, present in 5% to 10% of the adult population) for which no antibacterial treatment is needed. The latter is due to the chronic presence of bacteria in the organism in a latent phase (monocytes). Criteria for acute infections are a four-fold increase in IgA or IgG or a doubling of IgA and IgG at 10 to 15 days.
Moreover, the described consecutive sera of our patient underwent an additional retrospective examination by a MIF assay (Chlamydia MIF IgG & Chlamydia MIF IgM, Focus Diagnostics, Cypress, CA, USA). A straightforward IgG seroconversion could also be observed (Table 1 ). Thanks to the detection of IgA and of IgG antibodies to C. pneumoniae in various combinations, serology allows a classification of the state of infection. In our patient, reinfection was diagnosed because MIF were positive on admission; there was a four-fold increase in anti-MOMP IgG and, finally, an important rise and a rapid decline in anti-LPS IgA.
C. pneumoniae can induce very severe ARDS. We describe the first published case of ARDS due to C. pneumoniae infection successfully treated by ECMO. Definite Chlamydophila diagnosis remains a challenge but should be sought for in severe ARDS patients without evidence of other infectious cause. A quick and specific method for the definite diagnosis of Chlamydophila infections should be developed.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.  Environmental Sampling for Avian Influenza Virus A (H5N1) in Live-Bird Markets, Indonesia To identify environmental sites commonly contaminated by avian influenza virus A (H5N1) in live-bird markets in Indonesia, we investigated 83 markets in 3 provinces in Indonesia. At each market, samples were collected from up to 27 poultry-related sites to assess the extent of contamination. Samples were tested by using real-time reverse transcription–PCR and virus isolation. A questionnaire was used to ascertain types of birds in the market, general infrastructure, and work practices. Thirty-nine (47%) markets showed contamination with avian influenza virus in >1 of the sites sampled. Risk factors were slaughtering birds in the market and being located in West Java province. Protective factors included daily removal of waste and zoning that segregated poultry-related work flow areas. These results can aid in the design of evidence-based programs concerning environmental sanitation, food safety, and surveillance to reduce the risk for avian influenza virus A (H5N1) transmission in live-bird markets. F ood markets that offer both poultry meat and live birds either for sale or for slaughter are collectively referred to as live-bird markets (LBMs). LBMs are part of the supply chain and are essential for maintaining the health and nutritional status of rural and urban populations, especially in developing countries (1, 2) . However, LBMs provide op-timal conditions for the zoonotic transfer and evolution of infectious disease pathogens because they provide major contact points between humans and live animals (3, 4) .
Studies in Hong Kong Special Administrative Region, People's Republic of China; other areas of China; Indonesia; and the United States have shown that LBMs can harbor avian infl uenza viruses (AIVs), including highly pathogenic infl uenza virus A (H5N1), and have been associated with human infection (4) (5) (6) (7) (8) (9) . Continual movement of birds into, through, and out of markets provides opportunity for the introduction, entrenchment, and dissemination of AIVs. Most studies have focused on testing live birds rather than environmental sites in the LBMs (6, 7, 10) . However, a study in New York, NY, that tested environmental sites for AIV (H7N2) found that virus could be isolated from samples from fl oors, walls, and drains from the poultry areas of LBMs (8) . The study also found that despite the ongoing infl ux of infected birds into LBMs, the level of environmental contamination decreased with routine cleaning and disinfection. Another study in Hong Kong LBMs showed that AIV (H9N2) could be isolated at higher rates from poultry drinking water than from samples of bird fecal droppings (11) . Environmental aspects of LBMs are needed for an avian infl uenza control program for 2 reasons. First, a contaminated environment can provide a continuing source of virus transmission, in which healthy birds coming into the market may become infected and persons working in or visiting the market may also be exposed. Second, ongoing surveillance programs in LBMs based on environmental sampling are more likely than those based on invasive bird testing to be acceptable to traders and stall vendors. Environmental sampling is also safer for public health offi cers and veterinary health offi cers than handling and sampling live birds that may be infected with AIV.
Markets, Indonesia
In this study, we aimed to identify the environmental sites commonly contaminated by AIV (H5N1) in LBMs in Indonesia. Identifying these sites is the fi rst step in the design of evidence-based environmental sanitation, food safety, and surveillance programs to reduce the risk for virus transmission and to develop environmental surveillance programs to monitor LBM contamination status.
Three provinces in the western part of Java Island in Indonesia participated in the study: Jakarta, Banten, and West Java (Figure) . Eighteen districts in these provinces were selected on the basis of their proximity to the laboratory, high levels of avian infl uenza activity in farmed birds (Ministry of Agriculture, unpub. data), and high number of LBMs available for study (n = 300). The required sample size was 73 markets based on an estimated disease prevalence of 50% and a maximum error of 10% at 95% confi dence. We based our assumption that 50% of LBMs would be contaminated with AIV (H5N1) on results from a previous study in US LBMs in 2001 (12) . This study found that 60% of markets tested positive for AIV (H7N2) virus in areas in which the virus was endemic. To account for nonresponse, we increased the total sample size to 83 LBMs. We selected markets for inclusion in the study using systematic sampling. On the basis of a sampling frame of 300 markets, every fourth market (the sampling interval) was selected from a list of all the markets. A random numbers table was used to determine the starting point for selection of the 83 markets from the list. Diagnostic specimens and data were collected during October 2007-March 2008. These months have high rainfall and high AIV transmission according to data gathered during 2005-2007 about AIV (H5N1) outbreaks in farmed birds (Ministry of Agriculture, unpub. data).
A structured questionnaire containing 42 questions to assess risk factors for AIV (H5N1) contamination was developed. Responses to questions were obtained through visual inspection of each LBM and through an interview with the manager of the participating LBM. The questions sought information about volume of poultry in the LBM and the infrastructure in the delivery, holding, slaughter, sale, and waste-disposal zones of the market. These 5 zones refl ect general demarcation of work fl ow and activities relating to poultry in LBMs (13) . Questions about the sanitation and slaughtering practices were also included.
Questionnaire validation was conducted by members of a study advisory team. The team comprised 2 food safety/environmental health offi cers from the Ministry of Health, a communicable disease epidemiologist from the World Health Organization, a veterinary epidemiologist from the Food and Agriculture Organization, and 2 virologists from the Ministry of Agriculture in Indonesia. The questionnaire was tested in 3 LBMs in West Java province to ensure coherence, appropriate use of terminology, and high face validity. The same markets were also inspected to ensure that the questionnaire addressed all aspects of the poultry-related work fl ow in the 5 poultry zones and relevant infrastructure. Members of the study advisory team trained 3 study data collection teams in questionnaire administration and sample collection procedures.
To select the environmental sites to be sampled in each LBM, the study advisory team visually inspected 3 markets and reviewed the literature to identify LBM sites commonly contaminated with AIVs or similar pathogens. Sites sampled in previous studies for AIV included fl oors, drains, and water troughs (8, 11, 12) . In this study, 27 sites were selected for environmental sampling ( Table 1) . The sites represented different poultry-related work activities: 3 sites related to delivery of birds into LBMs, 7 in the birdholding zone, 9 in the slaughter zone, 6 in the sale zone, and 2 in the waste-disposal zone. Because of variation in LBM infrastructure and processes, each LBM did not necessarily have all 27 sites. Samples were collected from as many of the 27 sites as were available in each LBM.
For each of the 27 sites, 6 swab specimens were collected and pooled. Each pool (vial) consisted of a maximum of 3 swabs. The data collection teams were instructed to increase the representativeness of the samples by swabbing different locations for each environmental site. For example, if the market had 6 poultry stalls, each with its own scale for weighing poultry, then teams collected 1 swab from each scale and pooled them into 2 pools of 3 swabs each. Swab specimens were pooled in the market, and swabs remained inside the vials until testing. The data collection teams were instructed to focus on visibly dirty, moist, or diffi cult-to-clean surfaces in an effort to increase the sensitivity of the sampling.
Sample collection, pooling, transportation, and storage were based on techniques used in previous studies (10, 12) . Each data collection team comprised 3 persons, 2 of whom collected samples and 1 administered the questionnaire. To reduce the risk for cross-contamination during sample collection, teams changed disposable gloves and shoe covers between each of the 5 LBM poultry zones. Sterile cottontipped swabs were used to collect all samples, and samples were placed in viral transport media and transported immediately back to the laboratory on frozen gel packs. The viral transport media consisted of Dulbecco modifi ed Eagle medium (Sigma-Aldrich, St. Louis, MO, USA) with 1,000 IU penicillin and gentamicin, and 1% fetal buffer serum (14) . Samples were stored in the laboratory at -70°C until tested.
RNA extraction, cDNA synthesis, and real-time reverse transcription-PCR (RT-PCR) were used as described (15) . Virus isolation methods have also been described (16) but in general involved supernatants from a 1,000-μL sample homogenized by vortex and centrifuged at 2,500-3,000 rpm into 9-to 10-day-old specifi c pathogen-free eggs. Those positive in the hemagglutination assay were tested by hemagglutination-inhibition test with reference antiserum (A/chicken/West Java/Hamd/2006).
The degree of association between AIV (H5N1) positivity in the 5 LBM poultry zones was determined by using Spearman rank correlation. To assess risk factors for environmental virus (H5N1) contamination, we estimated odds ratios (ORs) using multivariable logistic regression analyses, where variables with p<0.1 from the univariate analyses were included in the initial model. A backward stepwise variable-selection strategy was used to construct a fi nal model with a signifi cance level of p<0.05. The Hosmer and Lemeshow test and the residual χ 2 goodness-of-fi t test were used to assess model stability. Microsoft Excel (Microsoft, Redmond, WA, USA), Epi Info (Centers for Disease Control and Prevention, Atlanta, GA, USA), and 
All 83 LBMs selected participated in the study; 62 (75%) were located in urban and 21 in rural areas. LBMs were from 16 districts in 3 provinces: 31 (38%) from Jakarta province, 11 (13%) from Banten province, and 41 (49%) from West Java province (Figure) . Most (49 [59%]) LBMs were retail markets, 10 (12%) were wholesale only, and 24 (29%) were a combination of retail and wholesale. Most Forty-eight (58%) LBMs reported monthly or more frequent visits from animal/human health personnel to inspect the poultry zones. Eight (10%) LBMs reported that live birds were tested periodically (less frequently than weekly) for AIV infection. For cleaning and sanitation, 80 (96%) LBMs reported washing poultry zones daily, and 55 (66%) applied detergent or disinfectant daily.
Thirty-nine (47%) LBMs had evidence of contamination. For 17 (44%) of these, <5 environmental sites were positive for AIV (H5N1) by real-time RT-PCR. For each of 22 (56%) LBMs, >6 environmental sites were positive.
The environmental sites most heavily contaminated were in the slaughter and sale zones (Table 1 ). In the slaughter zone, the most contaminated sites were the poultry-processing tables (21%), baskets holding poultry meat (20%), and chopping boards (20%). In the sale zone, the most contaminated sites were the tables for carcass display (24%) and scales (21%). Another commonly contaminated site was the waste-disposal bin in the waste-disposal zone (19%). In most cases, this bin is not an enclosed bin but rather was a dedicated uncovered fl oor space where remnants are dumped daily and collected weekly by the local government rubbish collection team.
Thirteen viruses were isolated from LBMs, most frequently from the slaughter zone (7 of 13 viruses isolated, Table 1 ). All isolated viruses came from 6 LBMs, from which 1-4 viruses were isolated per LBM.
From the zones contaminated in each LBM (Table 1) , we calculated correlations between different zones. Contamination in preceding LBM poultry zones correlated with contamination in the subsequent zones ( Table 2 ). Correlations were high between holding and slaughter zones, slaughter and sale zones, and sale and waste-disposal zones.
We assessed risk factors for AIV (H5N1) contamination in LBMs. We compared exposures in 39 LBMs with a minimum of 1 contaminated environmental site to 44 LBMs with no contamination. From the univariate analyses, several exposures predicted AIV (H5N1) contamination in LBMs (Table 3) . LBMs with wooden tables, Muscovy ducks, or >200 ducks other than Muscovy were at greater risk for AIV (H5N1) contamination, as were LBMs in West Java province.
Six other exposures approached signifi cance, either as protective factors or as risk factors. LBMs that disposed and removed solid waste daily (OR 0.41, 95% confi dence interval [CI] 0.16-1.09); had zoning that clearly segregated poultry delivery, holding, slaughter, sale, and wastedisposal areas (OR 0.28, 95% CI 0.07-1.11); or stacked poultry cages vertically rather than side by side (OR 0.38, 95% CI 0.13-1.10) had less risk for avian infl uenza virus (H5N1) contamination. LBMs with pigeons (OR 3.06, 95% CI 0.96-9.81), mixed bird species in the same cages (OR 2.92, 95% CI 0.98-8.70), or slaughtered birds in the market (OR 3.53, 95% CI 0.89-13.93) were more likely to be contaminated.
None of the 9 other variables considered in the study were associated with AIV (H5N1) contamination in LBMs (data not shown). These included the LBM trading category (wholesale, retail, or combination), days operational per week, chicken population in LBM, source of chickens (small-scale backyard farmers, commercial farms, or com- bination), inspection from authorities, use of detergent during cleaning, mixing poultry arriving on different days in the same cages, average length of poultry stay in LBM, and whether poultry were removed from stalls before cleaning. From the univariate analyses, 10 variables were signifi cant at p<0.1. However, the ducks other than Muscovy variable was removed from the multivariate analyses because of its collinearity with another variable (presence of Muscovy ducks, r>0.4). Nine variables were considered for the multivariate analyses. The fi nal multivariable logistic regression model had 4 variables, of which 2 were independent risk factors for subtype H5N1 contamination in LBMs (Table 3) . They were location in West Java province (adjusted OR [aOR] 6.83, 95% CI 2.01-23. 19 ) and bird slaughtering in the LBM (aOR 6.43, 95% CI 1.01-40.82). Two variables were independent protective factors: zoning of poultry activities in LBMs (aOR 0.16, 95% CI 0.03-0.86) and daily disposal of solid waste (aOR 0.2, CI 95% 0.06-0.69).
We have demonstrated extensive environmental contamination in LBMs with the AIV (H5N1) in Indonesia. Nearly 50% of LBMs in AIV (H5N1)-endemic districts were positive, with all 5 poultry zones affected. The study identifi ed environmental points of contamination and protective and risk factors for contamination. This study provides baseline information for 2 aspects that can aid in control of AIV (H5N1) in LBMs: 1) development of routine monitoring and surveillance programs and 2) structural interventions and work fl ow modifi cations to minimize risk for contamination.
Our fi ndings provide further evidence that environmental contamination with AIVs is not uncommon (8, 14) . Poultry water, drains, tabletops, cages, tablecloths, utensils, bins, and fl oors were all contaminated. Environmental sites most commonly contaminated were located in slaughter zones and zones where carcasses were taken after slaughtering, such as the sale and waste-disposal zones. This contamination can be expected because slaughtering generates droplets that may contain viral particles and exposes internal organs with potentially high viral loads. Even if slaughtering is conducted in a separate zone, contamination can spread to the sale and waste-disposal zone through the carcasses and through the process of evisceration usually conducted in both slaughter and sale stalls.
We found rates of contamination in water from poultry feeding bottles similar to those from the study in Hong Kong on AIV (H9N2) (11% and 7% markets with contamination respectively, p = 0.12) (11) . Even though AIVs were detected from poultry drinking water, our study suggests that other environmental sites are more effi cient for monitoring AIV (H5N1) in markets. Processing tables and baskets holding freshly cut poultry meat in the slaughter area, as well as display tables and scales in the sale area, were positive in 20 (24%) LBMs surveyed. The risk and protective factors we identifi ed complement fi ndings from previous studies. Daily disposal and removal of waste from the market is part of routine environmental cleaning and sanitation and eliminates AIV reservoirs (8) . Segregating poultry-related activities into zones limits virus spread (17) . Vertical stacking of cages can limit transmission because trays between layers of birds prevent the scatter of fecal matter. These results add evidence to the World Health Organization current recommendation that waste trays should be used to segregate stacked cages in markets to prevent cross-contamination (13) .
LBMs in West Java province had a higher risk for contamination than did other provinces. This risk probably is due to greater AIV (H5N1) disease activity in the province. Surveillance activities during 2006-2008 showed that West Java had a 4.7% outbreak detection rate compared with rates in Banten (4%) and Jakarta (0.2%) (18) . Furthermore, in West Java province chicken density is high: 14,000 birds/km 2 compared with densities in the neighboring provinces Banten and Jakarta (3,900 birds/km 2 and 400 birds/km 2 , respectively) (19) . Poultry density data are commonly used as a proxy for disease activity where areas of high poultry density have the highest risk for an outbreak (20, 21) .
Several issues need to be considered regarding our fi nding of low virus isolation rates compared with realtime RT-PCR-positive rates. Virus isolation detects viable virus, whereas real-time RT-PCR detects small stretches of nucleic acid, even if the larger genomic RNA is inactivated. This makes real-time RT-PCR a more sensitive detection tool but does not provide information about virus viability. Samples obtained from the environment may be less suitable than animal samples for virus isolation techniques. Organic matter, duration and temperature of exposure, and humidity can all affect virus survival outside the animal host (22) . Three studies conducted in LBMs tested environmental samples and bird samples by using virus isolation (8, 10, 23) . Only 1 of these studies stratifi ed the avian infl uenza detection rates by type of sample (bird vs. environment) (8) ; that study found that from 12 LBMs, 11 were positive for avian infl uenza in bird samples compared with only 5 positive in environmental samples. These results were based on a small sample of LBMs, and real-time RT-PCR was not conducted. Therefore, to determine the suitability of virus isolation for environmental samples, we recommend that future studies compare real-time RT-PCR-positive rates to virus isolation rates in both environmental swab and bird samples.
Risk and protective factors identifi ed in this study, together with fi ndings from other studies, can assist in developing environmental or behavioral interventions to reduce AIV transmission in LBMs. Previous studies have shown that regular cleaning with detergents, including free chlorine concentrations typically used in drinking water treatment, can rapidly decontaminate surfaces from AIVs (8, 24) . Previous studies also have shown that periodic market rest days coupled with thorough cleaning can minimize the reservoir of AIV in LBMs (4, 12, 25) . These messages have been disseminated to LBMs throughout Indonesia and formed the basis of the Ministry of Health Decree in 2008 on building healthy food markets (26) .
For a more systematic food safety monitoring system, this study will be used to develop a risk-based approach for AIV risk reduction in LBMs in Indonesia (27) . The contamination sites and risk factors will be used to determine critical control points and critical limits for intervention. LBM operators, stall vendors, and other stakeholders (e.g., sanitarians and public health offi cers) will need to be provided with simple monitoring plans to reduce the risk for contamination. Such monitoring plans are expected to have an impact not only on AIV (H5N1) but also on other viruses and bacteria commonly associated with food safety for poultry products.
In addition to tools for disease control, the study fi ndings can aid AIV (H5N1) surveillance activities in LBMs. Commonly contaminated environmental sites in LBMs can form the basis of an environmental sampling strategy for detection of AIV (H5N1) in LBMs. Environmental sampling is more benefi cial than live-bird sampling because it is less time and labor intensive and eliminates the need to handle and restrain live birds. Environmental sampling reduces the potential for virus aerosolization and the risk for infection for persons collecting the samples or standing nearby. Further work is needed to assess the adequacy of environmental sampling for surveillance in LBMs under different conditions, especially because detection sensitivity will vary by AIV (H5N1) prevalence in farms supplying the birds.
A limitation of this study is that the observation of environmental contamination was based on a cross-sectional survey in which LBMs were sampled only once. We recommend that future studies observe persistence of the virus over time in the various environmental sites. Reports from market managers and vendors about inspection and cleaning practices in the LBMs were not verifi ed during the course of the study. These activities may have been overreported because respondents may have wanted to report what they perceived interviewers wanted to hear. Be-cause of the high cost associated with the fi eld and laboratory work for such studies, studies should focus on a small number of markets and collect in-depth information about contamination trends and associated risk factors, as well as data on other indicator organisms, such as Escherichia coli or Enterobacteriaceae, that provide information about general market hygiene. Future work also should evaluate the effects of interventions in markets especially in lowresource settings because this would be of most benefi t to low-income and middle-income countries. Manifesting Ecologic and Microbial Connections  became an influential part of his childhood. The artists who created the dioramas, he later said, were guided by the same painters who inspired him, Thomas Cole, Frederic Church, Albert Bierstadt (3) .
His interest in zoology and botany was rivaled by other interests, among them film making and animation. He studied at the Rhode Island School of Design and graduated from New York's School of Visual Arts with a major in illustration. He worked as columnist and illustrator for Natural History magazine, while he gradually moved toward fine arts and started to show his work in solo and group exhibitions. A major influence was artist Ross Bleckner, whom he served as assistant for a time and who advised him to move toward modernism (4) .
A leader among contemporary artists returning to figurative content, Rockman wants to paint what he sees (5) . Taken with the natural world, he studies not only nature's creatures but also the puzzles surrounding them: their origins, survival, adaptability, evolution. Plants and animals are photographed and researched in libraries, their native habitats, or the Bronx Zoo. He delves into taxonomy and molecular biology and has enlisted the help and gained the following of paleontologists, biologists, ecologists, ichthyologists, and other scientists, who provide him clues to the accuracy of his exacting images. He has traveled to the rainforests of Brazil and Guyana in search of authentic specimens and to the South Pacific to sketch the extinct Tasmanian tiger in a local laboratory (6) . He counts Charles Darwin as a mentor. A combination of natural science and fantasy, his work explores the predatory relationship between nature and culture. Inspired equally by scientific curiosity and artistic compulsion, his startling images are at once literal, naturalistic, and entirely imaginary. Challenging the way we see and categorize the world, he questions human-animalnature interaction by creating "in your face" scenarios based on vital popular culture dilemmas, among them genetic engineering and global warming.
"He tweaks my cerebrum," late professor Stephen Jay Gould said of Rockman (7) . His snakes grow legs and chickens sport multiple sets of wings. Kangaroo-sized rats stroll across futuristic landscapes. A pig harbors human organs for harvest, and grossly oversized parasites, ticks, ants, and viruses populate his large surreal scenes. Botanical compositions, swarming with nature's less appreciated creatures and extinct or mutant forms feature aquatic or tree-sized dandelions. Humans are rarely present, though human handiwork always is. Riddles and humor are mixed in with actual soil, mud, sand, vegetation, and other collage materials, adding tactile interest to rich layers of color and varnish, which create a highly finished, luminous effect.
For nearly two decades, Rockman has worked from his studio in TriBeCa (Triangle Below Canal, between the Hudson River and Broadway), transforming historical culture into naturalistic images. Referring to himself as a "paleogeek," he favors large prehistoric landscapes reinterpreting the ecologic past and still lifes exploring the evolutionary future.
In Manifest Destiny, on this month's cover, Rockman imagines Brooklyn 3,000 years in the future. Fueled by exhaustive research, his artistic imagination produces a panoramic view of Brooklyn Bridge and environs. The polar ice caps have melted and the borough is under water. An eerie orange glow permeates layers of underwater ruins covered with slime and inhabited by weird creatures. In what the painter has referred to as "democratic space," prehistoric beasts paddle with newfangled mutants and everyday pests.
"I'm dying to see what scientists will think," Rockman said, while still working on the painting, transforming technical information from his research into visual language (3) . In this restructured environment, geology is turned on its ear, along with the food chain. Large fish with snake heads or oversized whiskers swim by a tirelike cell infected with giant HIV. Jellyfish tentacles stretch halfway across the seascape, past a two-tailed salmon. Flocks of wild birds hover above the waterline. Minute life forms, enlarged against the ruins, signal the survival of the unexpected. A galleon rests near the wreckage of a nuclear submarine. And the grand bridge lies broken, a fossil amidst decaying structures and vegetation.
Rockman's haunting vision of the future is rife with cultural and evolutionary undertones. The geologic, botanical, and zoologic clues to the future, rooted in the past and buried in the lurid reds of rust and pollution, are well understood by scientists. For ecologic disaster and disease emergence evolve along the same path, guided by the same factors: human demographics and behavior, technology and industry, economic development and land use, international travel and commerce, microbial adaptation and change, and the breakdown of public health measures (8) .
And while short-term risk for epidemics after geophysical disasters may be low (9), long-term effects of ecologic change on disease emergence, aptly shown in the exaggerated size of viruses (e.g., HIV), are huge. Rockman's meticulously drawn mutants, alluding to genetic engineering or environmental pollution, also articulate dilemmas inherent in disease control: because of microbes' evolutionary potential, our very drugs or pesticides, may contribute to selection of mutations, adaptations, and migrations that enable pathogens to proliferate and nonpathogens to become virulent. Manifest or not, the destiny of humans, animals, and the natural environment is inextricably interlinked. Influenza in Refugees on the Thailand–Myanmar Border, May–October 2009 We describe the epidemiology of influenza virus infections in refugees in a camp in rural Southeast Asia during May–October 2009, the first 6 months after identification of pandemic (H1N1) 2009 in Thailand. Influenza A viruses were detected in 20% of patients who had influenza-like illness and in 23% of those who had clinical pneumonia. Seasonal influenza A (H1N1) was the predominant virus circulating during weeks 26–33 (June 25–August 29) and was subsequently replaced by the pandemic strain. A review of passive surveillance for acute respiratory infection did not show an increase in acute respiratory tract infection incidence associated with the arrival of pandemic (H1N1) 2009 in the camp. We describe the epidemiology of infl uenza virus infections in refugees in a camp in rural Southeast Asia during May-October 2009, the fi rst 6 months after identifi cation of pandemic (H1N1) 2009 in Thailand. Infl uenza A viruses were detected in 20% of patients who had infl uenza-like illness and in 23% of those who had clinical pneumonia. Seasonal infl uenza A (H1N1) was the predominant virus circulating during weeks 26-33 (June 25-August 29) and was subsequently replaced by the pandemic strain. A review of passive surveillance for acute respiratory infection did not show an increase in acute respiratory tract infection incidence associated with the arrival of pandemic (H1N1) 2009 in the camp. P andemic (H1N1) 2009 emerged in April 2009 and subsequently spread around the globe. The World Health Organization issued a pandemic declaration on June 11, 2009 (1,2). By October 25, 2009 , >440,000 laboratory-confi rmed cases, including >5,700 deaths, had been reported to WHO (3) . The fi rst case of pandemic (H1N1) 2009 infection was diagnosed in Thailand on April 28, 2009 , and subsequently the virus was detected in all provinces. The Thailand Ministry of Public Health reported 27,639 confi rmed cases and 170 deaths as of October 10, 2009 (4) . Myanmar (Burma) reported its fi rst confi rmed case of pandemic (H1N1) 2009 infection during the week beginning July 5, 2009 , and by the end of October 2009 had reported <100 confi rmed cases with no deaths (5) . Although most infections caused by this new virus have been mild, severe disease has been reported, particularly in young adults (6) .
Data regarding the effect of infl uenza in rural areas of the developing world are scarce, as are etiologic data from refugee populations (7) (8) (9) . A recent review of published reports from Southeast Asia concluded that infl uenza infection may be identifi ed in up to 26% of outpatients with febrile illness and in 14% of hospitalized patients with pneumonia (10) . In Thailand, seasonal infl uenza virus activity peaks during the rainy season (June-September), with smaller peaks occurring during the cold months (January and February) (11) . Incidence of infl uenza infections in Thailand was 64-91 cases/100,000 persons per year during 1999-2002; the infl uenza-related hospitalization rate was 21/100,000 persons during 1999 (11) . Infl uenza infections in Myanmar are also seasonal; cases are documented predominantly in the rainy season (May-October) (12) (13) (14) . Incidence data for infl uenza virus infections in Myanmar are not readily available.
Of 15.2 million refugees worldwide, approximately one third live in camps (15). These refugees often live in crowded conditions and have contact with populations from the host country and the country of origin, where public health infrastructure and surveillance may be poor (16, 17) .
Approximately 150,000 refugees from Myanmar are housed in several camps on the Thailand-Myanmar border. Maela Temporary Shelter (Maela, Thailand) is the largest of these camps, with a population of >40,000, predominantly of the Karen ethnic group, housed in a 4-km 2 area (18) . This camp is located in the hills adjoining the Myanmar border, ≈500 km northwest of Bangkok, and has been in operation since 1984. Primary health and sanitation services are provided by nongovernmental or- 
From May 1 through October 31, 2009, trained local fi eld workers visited the hospital in Maela daily (Monday-Saturday). Patients whose illnesses met clinical case defi nitions for infl uenza-like illness (ILI) or pneumonia (Table  1) were identifi ed by clinic staff at the time of examination, and these patients were asked to complete an additional clinical interview. Inpatient and outpatient department cases were included in the surveillance. From July 27 through October 31, 2009, original clinical case defi nitions were modifi ed to capture each patient who had a history of fever during the current illness but who was not febrile at the clinic visit (either because of the intermittent nature of fever or self-administration of antipyretics).
A nasopharyngeal aspirate (NPA) was collected from each patient; a sterile 8-French infant feeding tube was inserted into the nasopharynx and then withdrawn while suction was applied with a 20-mL syringe attached to the feeding tube. The nasopharyngeal secretions and the tip of the feeding tube were transferred to a 1-mL tube of viral transport medium and stored in a cool box until transfer, within 24 h, to a -80°C freezer before analysis.
All NPA specimens were subjected to a panel of realtime reverse transcription-PCR (rRT-PCR) assays for the following viruses: infl uenza A (separate primer/probe sets for infl uenza A [universal], pandemic [H1N1] 2009, seasonal subtype H1N1, and seasonal subtype H3N1 detection) (20) ; infl uenza B (CDC in-house assay [details available on request]); respiratory syncytial virus (RSV; CDC in-house assay [details available on request]); and human metapneumovirus (HMPV) (21) . An internal control PCR specifi c for the human RNAseP gene was used to monitor sample adequacy and to detect the presence of PCR inhibitors (22) . Positive and negative controls were included in each PCR run. A Rotorgene 6000 real-time PCR thermocycler (Corbett Life Science, Mortlake, New South Wales, Australia) and SuperScript III One-Step RT-PCR Kits (Invitrogen, Carlsbad, CA, USA) were used throughout. All laboratory work was conducted at the Shoklo Malaria Research Unit microbiology laboratory in Mae Sot, Tak Province, Thailand.
To compare virologic results from 2009 with our surveillance data from 2008, we subsequently restricted the 2009 dataset to match data collected in 2008 (i.e., we included only patients whose illnesses met the strict case defi nitions and who were sampled on either Monday or Tuesday in the outpatient department). Clinical and laboratory data collected in 2008 were identical to data collected in 2009.
To estimate the incidence of infl uenza-associated illness, we reviewed passive disease surveillance data collected by the hospital in Maela and collated by the Com- mittee for Coordination of Services for Displaced Persons in Thailand. This surveillance system captured data only on patients visiting the hospital for treatment. The number and incidence rate (calculated by using monthly camp population census data) of clinically diagnosed upper respiratory tract infections (URTIs) and lower respiratory tract infections (LRTIs) were reported by month. No information was available to determine the number of ILI cases; therefore, we could not estimate the proportion of URTIs caused by infl uenza viruses in Maela. However, because most LRTIs reported are likely to be clinical pneumonia, we estimated the incidence of infl uenza-associated pneumonia as the incidence of LRTI multiplied by the percentage of pneumonia patients with specimens positive for infl uenza A. To determine the effect of pandemic (H1N1) 2009 on overall case numbers, we compared 2008 data with 2009 data.
The Human Studies Oversight and Review Team of CDC reviewed the surveillance project and declared it to be a nonresearch activity, as defi ned by US 45 CFR 46.102(d). Therefore our study was exempt from the need for full review by an institutional review board.
All statistical analyses were performed by using STATA version 10.1 software (StataCorp, College Station, TX, USA). Categorical variables were analyzed by using the Fisher exact test; continuous variables were analyzed by using the Wilcoxon rank-sum test (because none were normally distributed). Two-tailed p values <0.05 were considered signifi cant. Epidemiologic week numbers were calculated by using standard criteria (23) .
During May 1-October 31, 2009, a total of 324 patients were included in the surveillance. Of these, 19 were excluded from further analysis; 18 patients did not meet the clinical case defi nitions, and no NPA specimen was received for 1 patient (Figure 1) .
Pneumonia was diagnosed for 234 (77%) of the 305 eligible patients, and ILI was diagnosed for 71 (24%). For patients with pneumonia, median age was 2.0 years (range 0.1-68 years) and 55% were male; for those with ILI, median age was 1.4 years (range 0.2-10 years) and 54% were male.
Fifty seasonal infl uenza A infections and 17 pandemic (H1N1) 2009 infections were detected by rRT-PCR. Fortynine of the 50 seasonal infl uenza A infections were subtyped as H1N1; one was subtype H3N1 (Figure 2 ; with dual virus infection were not signifi cantly more ill than those with infl uenza A infection alone (3/7 vs. 26/60; p = 1.0). Illnesses for 205 (67%) patients met the strict case definition for ILI or pneumonia; 100 (33%) met only the expanded case defi nitions. Age distribution and proportion of infl uenza A viruses did not differ signifi cantly between the strict and expanded case defi nition groups. However, a signifi cantly higher proportion of patients with ILI (18/25 vs. 6/46; p<0.001) or pneumonia (99/180 vs. 5/54; p<0.001) whose illnesses met the strict case defi nition were hospitalized, which suggests that the expanded case defi nitions captured patients with milder illnesses.
Overall, at least 1 virus was detected in 175 (57%) patients (37/71 ILI, 138/234 pneumonia). HMPV and RSV accounted for 120/187 (54%) viruses detected. These viruses were detected in ILI cases (HMPV 23%,; RSV 17%) and pneumonia (HMPV, 21%; RSV 18%). RSV was detected signifi cantly more often in children <5 years of age (48/221 vs. 6/84; p = 0.003) and was more age restricted than all other viruses.
In Figure 4 ) (R. Sedhain, pers. comm).
Our study demonstrates that infl uenza virus infections are common etiologic agents of respiratory infection in a Southeast Asian refugee population living in crowded conditions. During the 6 months of surveillance in 2009, infl uenza A viruses were detected by rRT-PCR in 23% of clinical pneumonia and 20% of ILI cases sampled, representing a considerable impact that this vaccine-preventable disease has among patients with ARI.
Maela is an overcrowded and relatively closed refugee camp and therefore might be considered an ideal location for a novel infl uenza virus to cause an explosive outbreak. However, the number of confi rmed cases indicated that no major outbreak occurred in 2009. After the fi rst case of pandemic (H1N1) 2009 was identifi ed in August, these cases increased modestly in September, then substantially declined during October. Overall, only 25% of all infl uenza A viruses were determined to be the pandemic strain. However, supportive data show a change of the predominant infl uenza virus. In late August 2009, seasonal infl uenza A (H1N1) was the predominant circulating virus; during the subsequent 2 months, only cases of pandemic (H1N1)
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16 2009 were detected. During May-August, the incidence of LRTI and URTI in cases captured by the passive surveillance system was higher each month in 2009 than in 2008. The rates of URTI were similar in September and October of both years, whereas the LRTI rate was higher in October 2008 than in October 2009. Pandemic (H1N1) 2009 did not clearly increase in case-patients with ARI after its fi rst detection in the camp in August 2009. However, surveillance did not capture mild infections that did not result in visits to the outpatient department. The occurrence of most infl uenza A infections in patients who had pneumonia most likely refl ects a sampling bias, although infl uenza is a generally underrecognized cause of pneumonia in the tropics (24) . ILI is not a routinely used diagnosis for the clinic staff at Maela, so most of the ILI case-patients likely were not interviewed and sampled. However, when infl uenza A was identifi ed, pandemic (H1N1) 2009 case-patients were less likely than seasonal infl uenza case-patients to have been hospitalized. This information suggests that, in this population, illness caused by pandemic (H1N1) 2009 was no more severe than illness associated with seasonal infl uenza A. Several confounding factors, unrelated to the innate pathogenicity of the viruses, may account for this fi nding: 1) the timing of the modifi cation of case defi nitions in relation to the appearance of pandemic (H1N1) 2009; 2) differences in age distribution; and 3) presence of underlying illnesses in the patient groups. Data regarding underlying medical conditions were not collected as part of this surveillance so the effect of other conditions cannot be assessed. To prevent spread of infection, public health systems may request persons with ILI to self-quarantine, which might result in underestimation of the number of cases identifi ed in clinic-or hospital-based surveillance systems. During the 2009 infl uenza season, announcements regarding infl uenza and the need for good hygiene were made on the Maela public address system; healthcare workers reinforced these messages by home visits. Whether this intervention had any effect on healthseeking behavior remains unclear. An infl uenza triage system was in operation at the hospital, but our surveillance staff had access to patients seen and treated in this area.
Our study has several limitations. Most importantly, not every patient eligible for sampling was included, frequently because the patient refused or clinic staff failed to identify patients with illnesses that met the case criteria. These data were not recorded, so the effect of this bias cannot be estimated. As previously discussed, ILI is not a frequently used diagnosis outside this surveillance program, and most cases with this clinical syndrome were diagnosed as common cold. Many of the ILI cases documented were miscategorized in the clinic as pneumonia but were subsequently found not to meet the case defi nition, explaining the presence of persons hospitalized with ILI. Overall, these factors may bias toward sampling of case-patients who had more severe symptoms. Also, screening took place in only 1 of the 2 hospital outpatient clinics. However, because both are general clinics, the impact of this screening is likely to be refl ected in the absolute number of cases detected rather than in the proportion of ILI and pneumonia cases caused by infl uenza viruses. Regarding laboratory data, the likelihood of confi rmation of infl uenza infection is associated with the clinical case defi nitions in use: the strict ILI case defi nition used in our surveillance has a sensitivity of 98.4%-100% but a specifi city of only 7.1%-12.9% (25) . In another study, the probability of having a positive infl uenza virus PCR was directly related to magnitude of fever (26) . Therefore, given the bias toward severe cases, we may have considerably underestimated the impact of infl uenza in Maela.
As a result of the limitations noted above, we could (27) . Given the likely health inequalities between our refugee population and rural provinces in Thailand, direct comparison of these datasets is diffi cult. However, the incidence of infl uenza-associated pneumonia in Maela was ≈5× higher than in the Thai provinces (27) .
Population structure, such as the number of young children and elderly persons, may account for some of this difference, because the incidence of infl uenza infection is highest in these age groups. As with ILI, the case defi nitions used may have affected the data or the use of different laboratory confi rmation tests for infl uenza infection may have resulted in considerable variation in disease rates between studies; the study in Thailand used RT-PCR for laboratory confi rmation. Although the rates of infl uenzaassociated pneumonia were different in the refugee camp, the proportions of pneumonia cases associated with infl uenza were similar (23% vs. 18%).
Methods of preventing or mitigating infl uenza outbreaks in a community include vaccination; use of antiviral drugs; and basic infection control measures, particularly good respiratory etiquette, hand washing, and social distancing (28) . The World Health Organization has devised a specifi c infl uenza pandemic preparedness and mitigation plan for refugee and displaced populations, but implementation requires the coordinated efforts of healthcare providers (frequently nongovernmental organizations) and governments to ensure that control measures are available and used effectively (29) . Because resources are likely to be strained during an infl uenza pandemic, refugee and displaced populations might not be adequately represented in a country's pandemic preparedness plan. Availability of items required to control infl uenza transmission (personal protective equipment, vaccines, and antiviral medication) may be limited for this population without robust planning at the local and national levels. In addition to pandemic preparedness, camp administrators and donor agencies should consider routine vaccination for seasonal infl uenza in these populations.
Continuation and refi nement of this surveillance as the pandemic continues may provide further insight into the epidemiology of infl uenza in resource-poor rural Asian populations. Work such as this solidifi es the need of inclusion of refugee populations in infl uenza vaccine strategies and pandemic planning.  Early Clinical Features of Dengue Virus Infection in Nicaraguan Children: A Longitudinal Analysis BACKGROUND: Tens of millions of dengue cases and approximately 500,000 life-threatening complications occur annually. New tools are needed to distinguish dengue from other febrile illnesses. In addition, the natural history of pediatric dengue early in illness in a community-based setting has not been well-defined. METHODS: Data from the multi-year, ongoing Pediatric Dengue Cohort Study of approximately 3,800 children aged 2–14 years in Managua, Nicaragua, were used to examine the frequency of clinical signs and symptoms by day of illness and to generate models for the association of signs and symptoms during the early phase of illness and over the entire course of illness with testing dengue-positive. Odds ratios (ORs) and 95% confidence intervals were calculated using generalized estimating equations (GEE) for repeated measures, adjusting for age and gender. RESULTS: One-fourth of children who tested dengue-positive did not meet the WHO case definition for suspected dengue. The frequency of signs and symptoms varied by day of illness, dengue status, and disease severity. Multivariable GEE models showed increased odds of testing dengue-positive associated with fever, headache, retro-orbital pain, myalgia, arthralgia, rash, petechiae, positive tourniquet test, vomiting, leukopenia, platelets ≤150,000 cells/mL, poor capillary refill, cold extremities and hypotension. Estimated ORs tended to be higher for signs and symptoms over the course of illness compared to the early phase of illness. CONCLUSIONS: Day-by-day analysis of clinical signs and symptoms together with longitudinal statistical analysis showed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These findings stress the importance of considering day of illness when developing prediction algorithms for real-time clinical management. Dengue virus (DENV) causes the most prevalent mosquitoborne viral disease affecting humans, with 2.5-3 billion people at risk for infection and approximately 50 million cases of dengue each year [1, 2] . The four DENV serotypes are transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes, primarily in urban and peri-urban areas in tropical and subtropical countries worldwide. Most cases present as classic dengue fever (DF), a debilitating but self-limited illness that manifests with high fever, retro-orbital pain, severe myalgia/arthralgia, and rash. However, in some cases, mainly children, illness progresses to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), characterized by vascular leakage leading to hypovolemic shock and a case fatality rate up to 5% [1, 3, 4] . Currently, no licensed vaccine or antiviral therapy exists for dengue. Early identification of patients at risk of developing severe dengue is critical to provide timely supportive care, which can reduce the risk of mortality to ,1% [1, 2] . However, distinguishing dengue from other febrile illnesses (OFIs) early in illness is challenging, since symptoms are non-specific and common to other febrile illnesses such as malaria, leptospirosis, rickettsiosis, and typhoid fever [5] [6] [7] in dengueendemic countries. In addition, many distinguishing clinical features of DHF/DSS generally emerge only after 4-5 days, at defervescence, when the patient is already critically ill.
Although the World Health Organization (WHO) has recently established new clinical guidelines to classify dengue severity [1] , serological, virological, and molecular biological tests are required to definitively diagnose DENV infection. In many endemic countries, laboratory diagnosis of dengue is often problematic due to lack of reagents, expense, or delay in obtaining results. Patients with suspected dengue are often hospitalized for close monitoring to ensure proper treatment if they begin to develop severe dengue; however, up to 38-52% are later diagnosed with OFIs [8, 9] and thus were hospitalized unnecessarily at great financial cost to their family and society [10] . New tools are therefore needed to distinguish dengue from OFIs to prevent deaths from severe dengue and to mitigate the economic burden of excess hospitalization.
Recent approaches using multivariable logistic or linear regression models have shown that petechiae, thrombocytopenia (platelet count #100,000 cells/mm 3 ), positive tourniquet test, rash, and other signs and symptoms can distinguish dengue from OFIs [11] [12] [13] [14] [15] [16] [17] ; however, results were not consistent across studies. Only two studies considered clinical and laboratory features according to day of illness [18] [19] [20] , but as these were hospital-based studies, the results likely reflect patients with more severe symptoms and not the clinical spectrum of all symptomatic cases in dengue-endemic populations. Furthermore, none of these studies analyzed data using longitudinal statistical methods, which account for correlations between repeated measures on individuals over time. The use of longitudinal statistical methods to analyze cohort data is essential to utilize all of the data available for analysis and appropriately estimate the within-person and betweenperson variance in measures over time.
In this study, we used five years of data from an ongoing prospective cohort study of approximately 3,800 children aged 2-14 years in Managua, Nicaragua, to examine the frequency of clinical signs and symptoms by day of illness and to generate models for the association of signs and symptoms during the early phase of illness and over the entire course of illness with testing dengue-positive. In order to account for the longitudinal structure of the data, odds ratios (ORs) and 95% confidence intervals were calculated using generalized estimating equations (GEE), adjusting for age and gender.
In August and September 2004, a community-based pediatric cohort was established in District II of Managua, a low-to-middle income area with a population of approximately 62,500 [21] . Study activity was based in the Health Center Sócrates Flores Vivas (HCSFV), a public facility that is the primary source of health care for District II residents. Briefly, participants aged 2-9 years were recruited through house-to-house visits, and additional two year-olds were enrolled each year to maintain the age structure of the cohort [21] . Children were eligible to remain in the study until age 12 or until they moved from the study area. The parent/legal guardian of each participant signed an informed consent form, and children $6 years old provided verbal assent. In 2007, participants #11 years old were given the opportunity to continue for an additional 3 years, and a second informed consent was performed.
The study was approved by the Institutional Review Boards of the University of California, Berkeley, the Nicaraguan Ministry of Health, and the International Vaccine Institute in Seoul, Korea. Parents or legal guardians of all subjects in both studies provided written informed consent, and subjects 6 years of age and older provided assent.
Upon enrollment, parents/legal guardians of all participants were encouraged to bring their child(ren) to the HCSFV at first sign of illness or fever. Study physicians and nurses, trained in identification of possible dengue cases, provided medical care for study participants. Febrile illnesses that met the WHO criteria for suspected dengue (Table 1 ) and those without other apparent origin (undifferentiated febrile illnesses) were treated as possible dengue cases and followed daily while fever or symptoms persisted through visits with study medical personnel ( Figure 1 ). Complete blood counts (CBCs) were completed every 48 hours or more frequently as necessary, as indicated by the physician. Cases were monitored closely for severe manifestations and were transferred by study personnel to the Infectious Disease Ward of the Manuel de Jesús Rivera Children's Hospital, the national pediatric reference hospital, when they presented with any sign of alarm (Table 1 ). In addition, an annual healthy blood sample was collected to identify all DENV infections during the previous year and for baseline CBC values. Study physicians in both the hospital and HCSFV completed systematic data collection forms that contained approximately 80 variables (Table 1 ). In the hospital, additional clinical data, including fluid balance and treatment, were collected daily during hospitalization or through ambulatory follow-up visits by a team of study physicians and nurses. Data were also recorded on medical tests ordered and treatments prescribed.
A case was considered laboratory-confirmed dengue when acute DENV infection was demonstrated by: detection of DENV RNA by RT-PCR; isolation of DENV; seroconversion of DENVspecific IgM antibodies observed by MAC-ELISA in paired acuteand convalescent-phase samples; and/or a $4-fold increase in anti-DENV antibody titer measured using Inhibition ELISA [22] [23] [24] [25] in paired acute and convalescent samples. DENV serotypes were identified by RT-PCR and/or virus isolation.
Laboratory-confirmed dengue cases were further classified by severity. DHF and DSS were defined according to the traditional WHO criteria (Table 1 ) [26] . Additional categories of severity were included for those cases presenting with shock without thrombocytopenia and/or hemoconcentration (dengue with signs associated with shock (DSAS)) [23] or dengue fever with compensated shock (DFCS) [27] (Table 1) . Laboratory-confirmed
Dengue virus causes an estimated 50 million dengue cases and approximately 500,000 life-threatening complications annually. New tools are needed to distinguish dengue from other febrile illnesses. In addition, the natural history of pediatric dengue early in illness in a community-based setting has not been well-defined. Here, we describe the clinical spectrum of pediatric dengue over the course of illness in a community setting by using five years of data from an ongoing prospective cohort study of children in Managua, Nicaragua. Day-by-day analysis of clinical signs and symptoms together with longitudinal statistical analysis showed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These findings are important for clinical practice since outside of the hospital setting, clinicians may see dengue patients toward the beginning of their illness and utilize that information to decide whether their patient has dengue or another febrile illness. The results of these models should be extended for the development of prediction algorithms to aid clinicians in diagnosing suspected dengue.
Early Clinical Features of Pediatric Dengue www.plosntds.org cases were defined as primary DENV infections if acute-phase antibody titer, as measured by Inhibition ELISA, was ,1:10 or if convalescent phase antibody titer was ,1:2560, and as secondary infections if the acute titer was $1:10 or convalescent titer was $1:2560 [22] [23] [24] [25] .
Data from the first five years of the study (August 30, 2004-June 30, 2009) were used for analysis. The first three days after onset of fever were considered the early febrile phase of illness. Day of illness at presentation was determined by the date of fever onset, which was defined as the first day of illness as reported by the parent/guardian. Variable definitions are described in Table 1 . Positive tourniquet test was examined using cut-offs of $10 petechiae/in 2 and $20 petechiae/in 2 . Platelet count was dichotomized using a cut-off of #150,000 cells/mm 3 to enable comparisons during days 1-3. Only data from days 1-8 of illness were included for analysis.
The frequency of dengue testing results (laboratory-confirmed dengue-positive versus dengue-negative) and disease severity (DF versus severe dengue) was examined by year, demographics, serotype and immune response. The frequency of the WHO case definition for suspected dengue was examined by dengue testing results and age, and a chi-square test for trend was performed. The frequency of clinical signs and symptoms by day of illness and dengue severity was also examined using chisquare tests.
To examine the association between clinical signs and symptoms and the odds of testing dengue-positive versus dengue-negative, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using GEE models assuming an exchangeable correlation structure with robust standard errors to account for the correlations between repeated measures on the same patients over time. First, ORs were calculated using bivariable models that included only dengue testing results and each of the signs or symptoms. All signs and symptoms were then examined in multivariable models that adjusted for age and gender. Data from the first three days of illness and from all days of illness only were analyzed separately. Finally, for comparison, we used traditional logistic regression models to analyze the association between signs and symptoms and testing dengue-positive with data collapsed by illness episode to disregard repeated measures on the same 
From August 2004 to June 2009, 22,778 episodes of febrile illness were evaluated, of which 1,974 episodes were suspected dengue or undifferentiated fever (Figure 1 ). Of the 1,974 possible dengue cases, 1,793 (91%) tested negative and 181 (9%) were laboratory-confirmed as dengue-positive, of which 161 (89%) were classified as DF, 9 (5%) as DHF, 4 (2%) as DSS, 3 (2%) as DSAS and 4 (2%) as DFCS (Table 1) . Nearly all (95%) of the severe dengue cases but only 116 (72%) of the DF cases met the WHO case definition for dengue. The proportion of laboratoryconfirmed DENV infections that met the WHO case definition significantly increased by age (chi-square test for trend 5.977, p = 0.01), while younger children experienced significantly more undifferentiated febrile illness due to DENV infection ( Figure 2 ). The median age for cases meeting the dengue case definition was 8 years (range 2-13) and that of undifferentiated febrile illness due to DENV infection was 6 years (range 2-10).
The number of confirmed dengue-positive cases varied by year, as expected (Table 2 ) [28] . Both genders were equally represented, with a slightly higher percentage of females experiencing severe dengue, though this difference was not statistically significant. The majority of DF cases were DENV-2 (58%), followed by DENV-1 (21%) and DENV-3 (9%), while 60% of severe dengue cases were DENV-2, followed by DENV-3 (25%) and DENV-1 (10%). In addition, there were nearly equal proportions of primary and secondary immune responses among DF cases, whereas the majority (70%) of severe dengue cases were secondary DENV Figure 2 . Frequency of dengue-positive episodes that met the WHO classification criteria for suspected dengue by age (n = 181). Upon presentation to the health center or hospital, children with a febrile illness were classified according to whether or not they met the WHO classification criteria for suspected dengue. One patient had two dengue virus infections over the course of the study and is represented twice. n = 6 for age 2, n = 10 for age 3, n = 18 for age 4, n = 23 for age 5, n = 21 for age 6, n = 16 for age 7, n = 21 for age 8, n = 23 for age 9, n = 24 for age 10, n = 19 for age 11+. Chi-square test for trend 5.977, p = 0.01. WHO, World Health Organization. doi:10.1371/journal.pntd.0001562.g002 Table 2 . Characteristics of study participants by dengue testing results and disease severity (n = 1,974). Table 2 ). The median day of illness at presentation was day 2 for all patients, and almost all presented on days 1-3 of illness (90%). The total follow-up time of all children in the cohort was 17,931 person-years with a median follow-up of 3.9 years per child. As shown in Figure 3 , several signs and symptoms appeared to differentiate OFIs from DF cases, and DF cases from severe dengue cases, according to day of illness. In particular, higher proportions of DF and severe dengue cases experienced petechiae, platelets #150,000 cells/mm 3 , leukopenia, and positive tourniquet test compared to patients with OFIs. Higher proportions of severe cases experienced petechiae, platelets #150,000 cells/mm 3 , myalgia/arthralgia and abdominal pain compared to DF cases and patients with OFIs. Abdominal pain differentiated severe dengue cases from DF and OFI only beginning on day 3 of illness (for severe dengue compared to DF: chi-square 0.144, p = 0.70 for days 1-2 versus chi-square 16.910, p,0.0001 for day $3).
Bivariable and multivariable analyses were performed using GEE models to examine signs and symptoms early in illness and over the course of illness (Table 3) . On days 1-3 of illness, dengue-positive cases had up to 2-fold increased odds of fever, headache, retro-orbital pain, myalgia, arthralgia, and vomiting compared to patients with OFIs. They also had from 3-fold to 9fold increased odds of rash, petechiae, positive tourniquet test with cut-offs of $10 and $20 petechiae/in 2 , leukopenia, platelets #150,000 cells/mm 3 , poor capillary refill, cold extremities and hypotension compared to patients with OFIs. In contrast, they had decreased odds of abdominal pain, likely Figure 3 . Frequency of signs and symptoms by day in patients with OFI, DF and severe dengue. Over the course of an episode of febrile illness, signs and symptoms were observed by medical personnel or reported by children and/or their parent/guardian. Selected signs and symptoms are shown here. A, Petechiae; OFI versus DF: chi-square test for trend 21.313, p,0.0001; day 1, n = 606; day 2, n = 1,243; day 3, n = 1,066; day 4, n = 876; day 5, n = 675; day 6, n = 481; day 7, n = 291; day 8, n = 175; B, Platelet count #150,000 cells/mm 3 ; OFI versus DF: chi-square test for trend 14.928, p = 0.0001; day 1, n = 604; day 2, n = 970; day 3, n = 615; day 4, n = 568; day 5, n = 348; day 6, n = 234; day 7, n = 122; day 8, n = 65; C, Myalgia/ arthralgia; OFI versus DF: chi-square test for trend 4.569, p = 0.03; day 1, n = 612; day 2, n = 1,253; day 3, n = 1,075; day 4, n = 877; day 5, n = 671; day 6, n = 477; day 7, n = 289; day 8, n = 181; D, Leukopenia; OFI versus DF: chi-square test for trend 6.449, p = 0.01; day 1, n = 604; day 2, n = 971; day 3, n = 615; day 4, n = 568; day 5, n = 348; day 6, n = 234; day 7, n = 122; day 8, n = 65; E, Positive tourniquet test; OFI versus DF: chi-square test for trend 20.124, p,0.0001; day 1, n = 256; day 2, n = 496; day 3, n = 402; day 4, n = 308; day 5, n = 202; day 6, n = 156; day 7, n = 78; day 8, n = 38; F, Abdominal pain; OFI versus DF: chi-square test for trend 9.149, p = 0.002; DF versus severe dengue: chi-square test for trend 4.127, p = 0.04; day 1, n = 609; day 2, n = 1,245; day 3, n = 1,066; day 4, n = 877; day 5, n = 675; day 6, n = 482; day 7, n = 290; day 8, n = 174; All other chi-square tests for trend comparing DF to severe dengue were non-significant. OFI, other febrile illness; DF, dengue fever; Severe dengue = dengue hemorrhagic fever, dengue shock syndrome, dengue with signs associated with shock, or dengue fever with compensated shock. Leukopenia is defined as WBC #5000 cells/mm 3 because this feature appears later in the entire course of dengue illness. On all days of illness, dengue-positive cases had increased odds of the same signs and symptoms as on days 1-3 of illness; however, the magnitude of the point estimates tended to be higher. This difference was most pronounced for rash and platelets #150,000 cells/mm 3 , which had ORs approximately double in magnitude. In addition, denguepositive cases had increased odds of three additional signs and symptoms: poor appetite, absence of cough, and increased hematocrit. When GEE analyses on data with the longitudinal structure preserved were compared to traditional logistic regression analyses on data collapsed on febrile episode, the point estimates for the ORs were similar, although the 95% confidence intervals for the logistic regression models tended to be slightly narrower (data not shown).
In this study, we describe the clinical spectrum of pediatric dengue starting early in illness in a community setting. Longitudinal statistical analysis of day-by-day clinical signs and symptoms revealed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These results stress the importance of considering day of illness when developing prediction algorithms for real-time clinical management.
The early identification of dengue cases and particularly those at risk for severe dengue is critical for preventing severe illness and death. We found that 25% of laboratory-confirmed dengue cases did not meet the WHO case definition, suggesting that the WHO criteria are not sufficient to identify dengue at younger ages. Younger children may experience different signs and symptoms from adults or may be unable to communicate their symptoms to their parents, health care providers, or both. Previous studies demonstrated that children may experience significantly more cough, vomiting, abdominal pain, rash, epistaxis, oliguria, thrombocytopenia, hepatomegaly, and shock compared to adults, although the direction of these differences was not consistent across studies [13, 15, [29] [30] [31] [32] [33] [34] . A recent study of dengue in adults showed significant differences in clinical features and outcomes across ten-year age groups, indicating that signs and symptoms associated with DENV infection may continue to evolve past childhood [12] . If these differences are confirmed, the WHO case Table 3 . Signs and symptoms associated with testing DENV-positive among patients using generalized estimating equation models. Days Generalized estimating equation models assume an exchangeable correlation structure with robust standard errors. DENV, dengue virus; OR, odds ratio; CI, confidence interval; aOR, adjusted odds ratio. definition may need to be adjusted to be age-specific to function effectively for younger children and older age groups. Retro-orbital pain and low platelets were among the clinical features independently associated with DENV infection in this study. These results are supported by a study of dengue patients in Puerto Rico in which data were recorded at the time of initial consult rather than at hospitalization [15] , and by a study of Thai children [11] . Moreover, our results showing increased frequency of abdominal pain in patients beginning at day 3 of illness are consistent with a prospective study of adults admitted to an emergency department in Martinique [35] . A positive tourniquet test using cut-offs of $10 and $20 petechiae/in 2 was also independently associated with DENV infection. Both cut-offs were used because studies have indicated that a cut-off of $10 may improve discrimination of DENV infection [20, 36] ; however, the 1997 WHO classification scheme specified a cut-off of $20 [26] . Our results support using a cut-off of $10 petechiae/in 2 , and this cut-off has been specified in the 2011 WHO clinical guidelines [37] .
A major strength of this study is the use of statistical models designed for analysis of longitudinal data. Few other prospective community-based cohort studies have analyzed early clinical features in pediatric dengue compared to OFI [20, [38] [39] [40] , and none that we are aware of were analyzed using longitudinal statistical methods that account for correlations between repeated measures on patients. Here, we preserved the longitudinal structure of the dataset by using statistical models that support repeated measurements on subjects over time and account for correlations between signs and symptoms experienced within the same individual on different days of illness and in multiple episodes. Longitudinal data have long been collected in dengue research but have rarely been analyzed using appropriate statistical methods. This may introduce bias into findings, as studies may overestimate the magnitude of association or reduce the statistical power of the study as data are lost when they are collapsed for non-longitudinal analysis.
An additional strength of this study is that it is community-based [21] , enabling day-by-day capture of information on the early course of illness and on the full clinical spectrum of symptomatic dengue. In contrast, nearly all previous studies enrolled patients upon presentation to a hospital [18] , where patients present later; thus, these studies were unable to capture information on the early days of illness or on mild disease. By examining the clinical spectrum of dengue by day of illness, we were able to detect differences in the prevalence of signs and symptoms that could not be revealed by simply analyzing whether they ever occurred over the course of illness. In addition, through multivariable longitudinal models, we were able to identify distinguishing features of dengue during the early phase of illness compared to the entire course of illness. These findings are important for clinical practice since outside of the hospital setting, clinicians may see dengue patients toward the beginning of their illness and utilize that information to decide whether their patient has dengue or another febrile illness. The results of these models should be extended for the development of prediction algorithms to aid clinicians in diagnosing suspected dengue.
This study was not without its limitations. Some participants migrated out of the study area or withdrew from the study; however, our retention rate was approximately 95% per year [21] , suggesting that any bias from loss to follow-up would be minimal. It is also possible that we did not capture all symptomatic dengue cases. However, in yearly participant surveys, only an average of 2-3% of participants reported having attended a health-care provider outside of the study or having an illness and not attending any medical provider [21] , and approximately 20-fold more laboratory-confirmed dengue cases were captured in the cohort study than by the National Surveillance System [41] . Unfortunately, due to the low number of severe dengue cases, this study did not have sufficient statistical power to compare severe dengue cases to DF cases using GEE models, and these low numbers may have influenced the lack of significant association of signs of severe dengue with testing dengue-positive. For this study, we used the 1997 WHO classification scheme for disease severity. In 2009, the WHO updated its guidelines for classification of dengue disease severity [1, 37] ; it would be interesting to re-analyze the data in a future study using the new classification scheme. Studies of the usefulness and applicability of the revised guidelines have been recently performed [42, 43] .
In summary, this study is one of the few cohort studies to provide early data on the full clinical spectrum of pediatric dengue. Though we found significantly increased odds for association of several clinical signs and symptoms with testing dengue-positive, these increases were more modest for the early phase of illness compared to the course of illness, suggesting that caution should be taken when using the results from the entire course of illness to develop prediction algorithms. Non-parametric methods such as decision tree analysis overcome some of the limitations of traditional logistic regression models and have recently been applied to develop algorithms for prediction of dengue diagnosis and disease severity [9, 44, 45] . These and other data-adaptive approaches such as Super Learner [46] that are less subject to bias should be further explored to develop prediction algorithms for early identification of dengue cases and improved clinical management.
Checklist S1 STROBE checklist for cohort studies. (DOC) Influenza A H1N1 2009 (Swine Flu) and Pregnancy The Influenza A H1N1 pandemic (A H1N1) occurred between June 2009 and August 2010. Although the pandemic is now over, the virus has emerged as the predominant strain in the current seasonal influenza phase in the northern hemisphere. The A H1N1 influenza is a novel strain of the influenza A virus and is widely known as swine flu. The virus contains a mixture of genetic material from human, pig and bird flu virus. It is a new variety of flu which people have not had much immunity to. Much has been learnt from the Pandemic of 2009/2010 but the messages about vaccination and treatment seem to be taken slowly by the clinical profession. Most people affected by the virus, including pregnant women, suffer a mild viral illness, and make a full recovery. The median duration of illness is around seven days. This influenza typically affects the younger age group i.e. from the ages of 5–65 years. Current experience shows that the age group experiencing increased morbidity and mortality rates are in those under 65 years of age. Pregnant women, because of their altered immunity and physiological adaptations, are at higher risk of developing pulmonary complications, especially in the second and third trimesters. In the United Kingdom, twelve maternal deaths were reported to be associated with the H1N1 virus during the pandemic and clear avoidable factors were identified (Modder, Review of Maternal Deaths in the UK related to A H1N1 2009 influenza (CMACE). www.cmace.org.uk, 2010). The pregnancy outcomes were also poor for women who were affected by the virus with a fivefold increase in the perinatal mortality rate and threefold increase in the preterm delivery rate (Yates et al. Health Technol Assess 14(34):109–182, 2010). There continues to be a low uptake of the flu vaccine and commencement of antiviral treatment for pregnant women. Although the outbreak of influenza A H1N1 2009 appeared first in Mexico in April 2009, this was followed by a growing number of cases reported across the globe. The outbreak of the novel A H1N1 virus (swine flu) was declared a global pandemic by the World Health Organisation (WHO) from 11 June 2009 until 10 August 2010. These pandemics happen when a new influenza virus, to which the population has little or no immunity emerges and starts to spread. Unlike seasonal influenza, high rates of disease due to a pandemic virus may occur throughout the year. Swine flu is a novel strain of the influenza A virus affecting humans and contains segments of genes from pig, bird and human influenza viruses.
The WHO classifies the spread of infections such as influenza in terms of phases. Phase 6 or a global pandemic is declared when the infection is characterized by humanto-human spread of the virus with community level outbreaks in at least two WHO regions, meaning that there is widespread global transmission.
The influenza A virus has been responsible for three global pandemics in the last century: the Spanish Flu in 1918, Asian Flu in 1957 and the Hong Kong Flu in 1968. These pandemics were responsible for a large number of fatalities, the Spanish Flu being the most severe and caused severe pneumonias, particularly among pregnant women (Table 1) . A recently published epidemiological study about cohorts born in and around the pandemic estimated that the individuals born had a[20% excess cardiovascular disease at 60-82 years of age, relative to those born without exposure to the influenza epidemic, thus suggesting that prenatal exposure to even uncomplicated maternal influenza may have lasting consequences later in life [3] .
The 1918 pandemic was associated with a high maternal mortality rate of 27% and also associated with high rates of spontaneous miscarriage and preterm labour. In the 1957 pandemic, a 20% maternal mortality rate was reported and increased incidences of birth defects such as neural tube defects and cardiac abnormalities were reported.
Although we are now in the post pandemic phase, the A H1N1 virus has now emerged as the predominant strain of virus in the seasonal influenza season that is currently affecting the northern hemisphere. Current experience in the United Kingdom shows that the population below the age of 65 years are worst affected by the complications of the flu and that the deaths associated with the flu are predominantly associated with the H1N1 virus. From October 2010 to early January 2011, 50 deaths were reported by the Health Protection Agency. Forty-five of these people died with the H1N1 (2009) strain and 5 with Influenza B. The majority were under 65 years of age and five were under the age of five [4] .
Every year, we experience an outbreak of the seasonal flu during the winter months. It affects around 5-15% of the population and most of the complications and deaths tend to affect the elderly population. The swine flu virus, however, typically affects the younger population, i.e. from 5 to 65 years. Most will experience a mild illness and make a full recovery. Children and young adults appear to be most susceptible to clinical infection with the highest incidence in 5-9 and 10-14 year olds, with a smaller number of cases in adults older than 50 years of age (born before 1957). The low number of cases seen in those aged over 50 is thought to be due to previous exposure in this age group to similar strains of H1N1 that circulated between 1918 and 1957. The median duration of illness is estimated at 7 days. However, people who are in the at risk group such as those with underlying medical conditions e.g. chronic respiratory disease, asthma, diabetes, chronic heart disease, chronic renal disease, chronic liver disease, morbidly obese, those who are on medication to suppress their immune system and children under the age of 5 years. Pregnant women are all at higher risk of developing complications should they catch the infection. The illness may be complicated by bronchitis, or viral or secondary bacterial pneumonia.
This virus is an Orthomyxovirus. This is an enveloped virus with the characteristic spike-like viral glycoproteins called Haemagglutinin and Neuraminidase, hence their description of H1N1, H5N1 etc. depending on the type of H or N antigens.
Haemagglutinin is an antigenic glycoprotein which causes erythrocytes to clump together. It is responsible for The influenza virus is known for its ability to mutate. This new strain appears to be a result of reassortment of human influenza and swine influenza viruses, in all four different strains of subtype H1N1. Reassortment is the mixing of genetic material of a species into new combinations in different individuals. This process has been responsible for some of the major genetic shifts experienced in previous pandemics. This novel strain of the H1N1 Influenza A subtype is a result of a mixing of the virus from humans, birds and pigs.
The typical symptoms of swine flu are a sudden fever of at least 38°C and sudden cough with at least one other symptom of chills, lethargy, dehydration, headache, sorethroat, coryza, diarrhoea, vomiting, abdominal pain, myalgia or arthralgia. Gastro intestinal symptoms (vomiting and diarrhoea) have also been reported more often with H1N1 than with seasonal flu (Table 2A) .
The likelihood of infection with pandemic H1N1 2009 influenza is strongly influenced by age; the young are at most risk and those aged [60 years at least risk. Approximately 60% of patients with confirmed H1N1 2009 influenza virus infection are aged 16-64 years. However, case fatality increases with age especially those with associated co-morbidities, the young children \5 years of age [5] and pregnant where it is also high (Table 2B) .
In the UK, the first case of Influenza A H1N1 was confirmed on 27th April 2009. The first wave of the pandemic in UK started in July 2009 with the second wave from September 2009 and carrying on over the winter months. The vast majority of patients in the first wave in the UK experienced mild illness with 50% of patients recovering within 7 days of symptom onset and a further 25% within 10 days. The main symptoms reported were fever, fatigue, dry cough, sore throat and headache. However, severe gastrointestinal disease (nausea, vomiting, diarrhoea, abdominal pain) has been a feature of H1N1 infection in children and adults requiring admission [6, 7] .
Complications of swine flu in the general population appear similar to seasonal influenza. Myocarditis has been observed, usually associated with a marked tachycardia. The prognosis is unclear, though influenza-related myocarditis usually has a good prognosis for recovery.
As with seasonal influenza, neurologic complications such as seizures and encephalitis, with altered mental state, can occur. The prognosis for patients with isolated neurological symptoms which can be optimised without requirement for ICU admission appears good. The initial symptoms of rapidly-lethal infections such as meningitis, encephalitis and bacteraemia can resemble those of influenza [8] .
Although in the majority of cases of H1N1 2009 influenza affects the upper respiratory system, the most common complication is that of influenza-related pneumonia. This has occurred in 40% of hospitalised patients in the United States, usually with bilateral infiltrates apparent in the chest X-ray [9, 10] . Amongst patients with H1N1 influenza admitted to ICUs in New Zealand and Australia, 49% had viral pneumonitis or ARDS and 20% had secondary bacterial pneumonia. The most common bacterial pathogens reported included Streptococcus pneumonia, S. pyogenes, Staphylococcus aureus, S. mitis and Haemophilus influenzae [11] .
The virus is highly contagious and is contracted by aerosol transmission from inhaling the droplets from an infected person coughing and sneezing, and also touching the nose and mouth after being in contact with contaminated surfaces. The best way to reduce the risk of spreading the virus is to observe good respiratory hygiene by covering the mouth and nose with a piece of tissue paper when coughing and sneezing and disposing of it immediately. In addition, it is important to observe good hand hygiene by washing There is no evidence to show that the risk is reduced by avoiding crowded places as long as these good hygiene measures are observed. Naturally, if anyone is down with the flu, they should avoid going to crowded places themselves (Table 3) .
There is no evidence that face masks are effective in reducing the risk of catching or spreading the virus and are not recommended for use by the general population. They are only effective for use in a healthcare setting. In most clinical situations, a patient admitted to hospital with suspected swine flu or its complication, they will be managed in isolation and barrier nursed. Healthcare professionals should don a plastic apron and surgical face masks, observing strict hygiene measures. The respirator masks (FFP 3 masks) should only be used when potentially aerosol generating procedures are used e.g. in ventilated patients or those using nebulisers.
Patients suffering from the following symptoms are more likely to be admitted to the Intensive Care Unit These are:
• dyspnoea (strongly predictive of both death and ICU requirement), respiratory rate [30 per minutes • requirement for supplemental oxygen (strongly predictive of ICU care and death) • pneumonia on admission (strongly predictive of significant complications after admission-including ICU multi-organ support and death) • tachycardia (the higher the pulse the greater the chances of ICU care being required) • altered conscious level
It is commonly thought that a pregnant woman's immunity is suppressed. In pregnancy, there is modification of the immune system to prevent rejection of the fetus and placenta. It is thought that the cell mediated immunity is modified by an increased in the production of T-helper cells as opposed to T-killer cells to reduce the risk of rejection. There appears to be a tendency towards humoral immunity. As the virus replicates intracellularly, the ability to fight off an infection is reduced. It is known that infections such as chicken pox get worse in pregnancy, particularly in the later half of pregnancy, but this is due to the risk of pneumonitis from local spread to the respiratory system of the virus and also the mechanical effects of the enlarged uterus on the diaphragm and the respiratory system. There is no evidence that Human Immunodeficiency Virus (HIV) or tuberculosis get worse in pregnancy. Hence, there is no evidence to suggest that pregnant women are at increased susceptibility of catching the flu virus [13] .
However, due to the modification in their immune systems to accommodate their developing fetus and adaptations in their body as a result of the hormonal and physical changes, they are at greater risk of developing complications should they acquire the illness. The enlarging uterus presses on the diaphragm and together with changes in the lungs such as reduced tidal volume, congestion and localized oedema, make the woman more prone to complications such as pneumonia and Adult Respiratory Distress Syndrome (ARDS). Recent reports have shown that the percentage of hospitalised cases requiring transfer to ICU within each gestational band is similar (27, 25, 24%); and the death rate amongst hospitalised patients within each gestational age group is also remarkably uniform (10, 6, 9%). This may imply that outcome in severely ill pregnant women is independent of gestational age after severe illness is established [14] .
There is still much to learn about the virus. Studies from the USA showed that pregnant women with swine flu were four times more likely to be hospitalised for complications compared to the non-pregnant population. In separate studies pregnant women are over represented in the group of patients admitted to hospital requiring Level 2 (High Dependency Care) or Level 3 care (Critical/Intensive Care). Observations from the USA, Canada and Australasia showed that pregnant women formed between 7 and 9% of admissions to Intensive Care Units (ICU).
In the United Kingdom, the United Kingdom Obstetric Surveillance System (UKOSS), working with the Department of Health and Health Protection Agency, collected information on pregnancies reported to be affected by A H1N1 from 221 of the 223 (99%) obstetric units in the UK during the pandemic. UKOSS assessed the data on morbidity in pregnancy. The Centre for Maternal and Child Morbidity UKOSS was able to obtain complete data on 241 women admitted to hospital with confirmed A H1N1. During that period, there were 314,135 maternities, representing an incidence of 7.7 hospitalised cases per 10,000 maternities. Twenty percent (1.6 per 10,000 maternities) of these women were admitted to the Intensive Care Unit.
Only 2% of women admitted had received the A H1N1 vaccine but it has to be noted that the vaccination programme in the UK was rolled out after the peak of the hospital admissions in the series.
Sixty percent of the hospitalised women commenced antiviral treatment within 48 h of onset of their symptoms . (Table 4) Obesity and delay in treatment in antiviral treatment were significant factors associated with admission to ICUs. Treatment with antiviral agents within 2 days of onset of symptoms was associated with an 84% decrease in the odds of admission to an ICU.
As for pregnancy outcomes, a three fold increase in preterm delivery and a five fold increase in stillbirth rate was observed.
Between 1 April 2009 and 13 January 2010, 12 maternal deaths in the United Kingdom and 1 maternal death in the Republic of Ireland with confirmed A H1N1 were reported to the Centre for Maternal and Child Enquiries (CMACE). Eight of the maternal deaths were investigated in detail by CMACE through their confidential enquiry panel.
As in the observations noted by UKOSS, Black and ethnic Minority (BME) women were over-represented (4/8) . Clinical co-morbidities such as asthma, paraplegia, previous stroke and stroke were present. Obesity was found to be a risk factor for severe morbidity and mortality.
Smoking did not appear to be a risk factor although both studies noted them to be a factor in the younger women.
Clear avoidable factors were noted and they are:
• Delayed admission to hospital and/or consideration of the likelihood of A H1N1 infection in the differential diagnosis; • Delayed confirmation of A H1N1; wrong swabs used or inappropriate storage/carriage to laboratory; • Lack of clear clinical leadership in overall case management (1 case); • Delay in administering antiviral medication.
Pregnant women admitted with respiratory complications should be managed jointly between the obstetric and medical teams. An assessment needs to be made with respect to the best place to manage the woman. There should be early involvement of obstetric anaesthetists, respiratory physicians and haematologists and a clear management plan needs to be set out from the outset.
Women requiring more respiratory support may be best managed in the Respiratory Unit with close input by the obstetric and midwifery teams. If a woman is in labour, she would be best managed in the Delivery Unit with input from the Respiratory team and the Obstetric Anaesthetic team. Following delivery, she may need to be transferred to the clinical area that would be best to provide expert care for her.
It is important to bear in mind that pregnancy related pathologies such as chorioamnionitis, severe urinary tract infections, group A and B streptococcus infections and even malaria may present with similar symptoms. Careful clinical assessment is of vital importance in order not to miss an important infection. The patient needs to be carefully monitored with the modified early warning scores, taking into account the pregnancy. Complications of obstetric problems such as pre-eclampsia, venous thromboembolism and pulmonary embolism have also to be excluded.
The most common complication of A H1N1 influenza is pneumonia. The plan of management in such cases has been summarized in Table 5 . Complications must be recognized and treated appropriately. In order to treat pneumonia effectively, the antimicrobial therapy should be based on bacteriological sensitivities. However, pending bacteriological confirmation, co-amoxiclav (Augmentin) is the recommended empiric antibiotic. In penicillin sensitive patients, Clarithromycin is a suggested alternative. Co-amoxiclav is not contraindicated in pregnancy. In situations where there is preterm prelabour rupture of membranes concomitant with pneumonia, the use of co-amoxiclav is not recommended because of the increased risk of necrotizing enterocolitis observed in the ORACLE study. An alternative antibiotic such as piperacillin is recommended and the microbiologist should be involved in management discussions. In addition, antiviral treatment should be started on clinical grounds whilst awaiting test results. It is also important to treat maternal pyrexia with paracetamol and not Non Steroidal Anti Inflammatory Drugs (NSAID). Epidemiological studies have linked uncontrolled maternal pyrexia to miscarriage and fetal abnormalities such as neural tube and cardiac defects. Maternal pyrexia is also a recognised risk factor for preterm delivery. The importance of control of maternal pyrexia with regular and effective doses of paracetamol and hydration should be emphasised.
Current obstetric practice is to administer corticosteroids e.g. 2 doses of betamethasone or dexamethasone 12 or 24 h apart to promote fetal lung maturity in situations of threatened pre-term labour or where a decision is made to deliver the fetus prematurely for maternal or fetal reasons. The effects on the maternal immune system from a single course of corticosteroids are unclear but the evidence does not suggest that it results in sufficient immuno-suppression to cause maternal harm or exacerbation of infection.
The administration of corticosteroids is important for the promotion of fetal lung maturity, and the benefits outweigh the risks. Recent evidence suggests that repeated (rescue) doses of corticosteroids may be beneficial for fetal reasons, but studies have also shown that these may lead to maternal secondary adrenal insufficiency and fetal complications. The practice of repeated (rescue) doses of corticosteroids is not recommended.
The use of high dose steroids for the treatment of pulmonary complications is not recommended by ICU specialists for fear of immune suppression and prolonged viral shedding.
Most mothers with symptoms of influenza in labour will be able to tolerate labour with adequate pain relief and hydration. In most cases, the decision to deliver will be made for an obstetric indication. In the event of a critically ill woman close to term, it is not unusual to deliver her baby, usually by caesarean section, to help with mechanical ventilation of the lungs to improve her recovery. This should be done once her clinical condition is stabilised and other potential complications such as coagulopathy have been excluded or corrected.
As indicated above, most of the respiratory complications have been shown to occur in the second and third trimesters. There may be situations where a preterm baby needs to be delivered in order to improve the outcome for ventilation of a very ill mother. The decision is made in conjunction with the obstetric, critical care and neonatal teams. The pregnant woman should be informed but if unable to participate in clinical decision making, the partner or close relatives should usually be involved in discussions. In order to improve the outcome for the premature infant, corticosteroids (in accordance with the guidance above) should ideally be administered at least 24 h prior to delivery. It is unlikely that pregnancies in the 1st or early second trimesters will need to be terminated unless it is felt that continuation of the pregnancy will be detrimental to the woman's condition. There may be occasions where a woman who is booked for elective caesarean section becomes symptomatic at the time of the planned procedure. If possible, it would be advisable to commence her on antivirals and to delay the procedure by about 5 days, to allow her to recover, in order not to increase her risks of respiratory complications, and also to reduce the risk of spread to other patients and staff. In severe cases of respiratory complications where the woman has developed Adult Respiratory Distress Syndrome (ARDS) and where she is not responding to mechanical ventilation, she may have to be considered for Extra Corporeal Membrane Oxygenation (ECMO). This is highly Be prepared to deal with: DIC, post viremia encephalitis Anti microbial therapy should be based upon bacteriological sensitivities-be cautious with the use of Augmentin in women with ruptured membranes
The anti viral treatment should be started ASAP Maternal pyrexia should be treated with paracetamol Consider antenatal steroids for preterm labour specialized treatment and the decision is made by the Intensive Care specialists. As above, it may not always be necessary to terminate a pregnancy in the first or early second trimesters as there is no benefit in doing so for maternal ventilation. It is of vital importance to maintain the maternal physiology in the best possible state in order to allow the satisfactory progress of the pregnancy. There is now more capacity for ECMO treatment in the UK and more pregnant women are being referred to ECMO units for treatment. It is vitally important that the obstetric teams in the ECMO units are jointly involved in the mother's care and good communication is essential.
Women in the postnatal period are probably at lower risk of respiratory complications because the effects of the gravid uterus on the lungs have been removed. However, they may still experience similar complications if they are infected and there is a risk of transmission to the newborn infant. They should observe the same strict hygiene measures and be offered antiviral medication i.e. oseltamivir if clinically indicated. Mothers should be encouraged to breastfeed. Breast feeding is important and should be continued as long as possible.
The benefits of breastfeeding are significant and are twofold: (i) it gives babies the most appropriate nutrition for health and promotes attachment between mother and baby and (ii) colostrum is rich in antibodies which will help to protect the baby from many infections. Women who are breastfeeding and have symptoms of influenza should be treated with an antiviral medicine. The preferred medicine is oseltamivir, as for other adults. However, if a baby is born and breastfeeding is started while the woman is taking zanamivir, she should complete the course of zanamivir; it is not necessary to switch to oseltamivir.
If the woman is too tired to breastfeed, she should be encouraged to express her breast milk in order to feed her baby. She should get help with breastfeeding and her baby should be with her as much as possible. She should observe strict hygiene measures to avoid spread of the virus to her baby.
The two types of antiviral drugs known to be effective against the swine flu virus are oseltamivir (Tamiflu Ò ) and zanamir (Relenza Ò ). They are neuraminidase inhibitors and act by preventing the virus from budding and escaping from the host cells. Oseltamivir is given in the form of oral capsules and zanamivir is given as an inhaler (Diskhaler). The safety of these antiviral drugs in pregnancy has been looked at from experience with their usage in seasonal flu in some countries and from recent data from Canada. Although oseltamivir has been shown to cross the placenta and breast milk in small amounts, no adverse effects on the fetus or pregnancy have been recorded. As zanamivir acts in the respiratory tract with no absorption into the blood stream, it is recommended as first line treatment of swine flu in pregnancy in the United Kingdom. However, if the woman is unable to tolerate the inhaler e.g. if she has severe asthma or is admitted to hospital with complications such as pneumonia, oseltamivir is the recommended drug for treatment. The benefits of antiviral treatment are most noticeable if it is commenced within 48 h of symptom onset but recent experience with hospitalised patients reveals that antivirals given more than 48 h after symptom onset also confer benefit.
Treatment with antivirals should be started on clinical grounds whilst awaiting test results. Waiting for confirmatory tests will only lead to a delay in commencement of treatment. Clinical judgement should be exercised in the initiation of treatment. A negative test does not necessarily exclude H1N1 influenza as the sensitivity of rapid influenza diagnostic tests can range from 10 to 70% for 2009 H1N1 virus.
Pre or post exposure prophylaxis with antivirals has been shown to be effective in preventing the development of the influenza infection. Unlike in treatment after symptoms, prophylaxis may inhibit the development of immunity and prolonged or repeated prophylaxis may predispose to development of resistance. Prophylaxis is only recommended for very high-risk individuals for whom prompt treatment may not avoid the development of severe disease. Most pregnant women do not require prophylaxis.
In the United Kingdom, the vaccination programme was launched in October 2009. Healthcare workers were the first group to be offered the vaccine. Following this, at risk groups, pregnant women and children between 6 months and 5 years were invited to be vaccinated.
It is of no doubt that any programme to vaccinate pregnant women will raise concerns and controversy. New vaccines cannot be tested on pregnant women but the experience on its safety has been drawn from data from seasonal flu vaccines. The swine flu vaccine is similar to the seasonal flu vaccine in many respects. The vaccine contains the inactivated (killed) H1N1 virus which is developed from the bird flu H5N1 virus-a virus to which most people have no immunity. The inactivated virus will not cause any harm to the fetus or mother and active immunity will develop. The antibody response to vaccines in pregnant women has been shown to be as effective as those who are not pregnant.
The reluctance to be vaccinated is mainly due to worries about the side effects. Experience from the 2009/2010 pandemic confirmed the safety profile of the vaccine. Over 350 million doses of the vaccine were administered worldwide during the pandemic with no adverse long term effects noted in both the pregnant and non pregnant populations [15] .
In the UK, the seasonal influenza vaccines for 2010 have incorporated the A H1N1 strain as part of the trivalent vaccine. In spite of the proven safety profile, until the end of December 2010, the uptake of the vaccine in the younger population who are at risk, including pregnant women, remained low with less than 50% uptake [4] . Communication strategies have been stepped up to promote the value of the vaccine and to heighten awareness of the severity of complications affecting the younger at risk population.
Although we are now experiencing the post pandemic phase, the A H1N1 virus has emerged as the predominant virus in the current seasonal influenza season affecting Europe and the UK, with significant morbidity and mortality rates affecting the younger population and pregnant women. Most people who catch swine flu experience mild symptoms and make a full recovery. However, pregnant women are more likely to develop life-threatening complications should they catch the flu. It is important for pregnant women seek medical advice early and are started on antiviral treatment, ideally within 48 h, but they can also be effective up to 7 days. If, in spite of treatment, they do not get better, they must contact their doctor or midwife. It is vitally important to treat any fever in pregnancy with paracetamol and drinking plenty of fluids.
Strict hygiene measures are effective in preventing the spread of the infection. Vaccines are the most effective way to avoid catching the virus and pregnant women should consider taking up the offer of vaccination against swine flu. Use and Evaluation of Molecular Diagnostics for Pneumonia Etiology Studies Comprehensive microbiological testing will be a core function of the Pneumonia Etiology Research for Child Health (PERCH) project. The development stage of PERCH provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. These efforts allowed us to articulate the unique requirements of PERCH, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. This process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. Although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of PERCH, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies. Comprehensive microbiological testing will be a core function of the Pneumonia Etiology Research for Child Health (PERCH) project. The development stage of PERCH provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. These efforts allowed us to articulate the unique requirements of PERCH, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. This process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. Although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of PERCH, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies.
The development of a comprehensive microbiological testing strategy has been a core principle in the conception and design of the Pneumonia Etiology Research for Child Health (PERCH) project [1] . In formulating the most effective approach for respiratory diagnosis, we determined that a multiplex molecular diagnostic platform would be an essential component in our approach. Many of the technical and operational considerations encountered through this process proved relevant to the overall design of the project. We describe here the theoretical and practical challenges encountered in the evaluation and selection of a molecular platform for the diagnosis of pneumonia.
As described elsewhere in this issue [2, 3] , microbiological evidence of infection must be considered in the context of several fundamental difficulties found in respiratory diagnostics, including the frequent lack of access to the site of infection, the insensitivity of available tests, insufficient assay validation, and complexities in determining whether a detected pathogen has a causal role in the illness. The specific research-related demands of PERCH added to these constraints, requiring that our diagnostic strategy must exclude any prior assumptions regarding the likely importance of specific pathogens; must include a full range of respiratory tract specimens, including upper respiratory swab or aspirate, induced sputum, lung aspirate, bronchoalveolar lavage, and pleural fluid; must be comprehensive, yet realistic; must appropriately balance the demands of accuracy and efficiency; must account for both clinical and research ethical issues; and must be feasible for use and support at all participating field sites.
To begin the selection process, the PERCH investigators conducted an extensive review of the microbiologic diagnosis of respiratory infections. Using published and unpublished data, as well as user and developer experiences, our team prepared a strategic summary of the available technologies that could detect pathogens from respiratory tract specimens. We evaluated each major assay category, including traditional bacteriology and viral culture, direct antigen and immunofluorescent antibody detection, and nucleic detection acid tests. It was evident that molecular diagnostics should be among the mix of diagnostic tools required to meet the needs of PERCH.
Nucleic acid detection tests (NADTs) have a number of advantages over other diagnostic platforms for the evaluation of respiratory specimens [4] . They demonstrate superior sensitivity in detecting organisms that are fastidious, less viable, or present in only small amounts [5] . Molecular diagnostics can also be quickly adapted to detect evolving or emerging pathogens and are amenable to efficiencies of scale such as automation. They also allow the simultaneous detection of multiple targets (multiplexing), which in turn allows for testing by clinical syndrome and the detection of co-infections. NADT methods present less of a safety hazard for laboratory personnel compared with culture, typically require less time compared with bacterial culture, and require less technical capacity compared with viral culture. Given these advantages, NADTs have been extensively evaluated in the detection of several viruses and bacteria of the respiratory tract and have become the diagnostic tool of choice for many agents that are difficult to isolate [4] .
Molecular diagnostic platforms are not without their disadvantages. Cost and complexity remain significant barriers to adoption in many laboratories, and NADTs often risk problems of laboratory contamination with amplified products, particularly if the assay procedure requires opening of the reaction tube prior to the target detection step [6] . Measures to limit contamination often require additional laboratory space that may not be available in resource-constrained settings. Nevertheless, NADT methods represent one of the more productive areas of diagnostics research, promising future improvements in automation and speed, smaller devices, improved cost efficiencies, and better detection of emerging pathogens [7] .
The focus on molecular methods for respiratory pathogen detection yielded a large variety of potential technologies for consideration in PERCH (Table 1) . Polymerase chain reaction (PCR) technology is more common at research sites worldwide, can be adapted to various platforms, and easily allows for multiplex amplification. Multiplexing, in which several targets are assayed for simultaneously, is commonly employed in PCR-based assays and offers significant advantages over single-pathogen assays in terms of efficiency and pathogen coverage. Still, developers must overcome considerable complexities in harmonizing the reaction requirements of each individual target and limiting potential competition among the analytes. These factors may result in a measurable decrease in sensitivity compared with single-plex assays. Several techniques have been developed to address such factors, such as alterations in cycling protocols [8] , nested primer combinations [9, 10] , complex primer structures and concentrations [11, 12] , and the use of nontraditional nucleotides [13] [14] [15] . Other NADT technologies, such as nucleic acid sequence-based amplification and loop-mediated isothermal amplification, have been used for the detection of respiratory pathogens, but experience with multiplexing is limited [16] [17] [18] [19] .
Technologies for target detection take on an even larger variety of formats. Older methods include agarose gel electrophoresis, reverse-transcription PCR enzyme hybridization assay [20] [21] [22] and enzyme-linked oligonucleotide capture [18] . More recently, solid-and liquid-phase array platforms have become more useful for the detection of multiple targets. Solid-phase arrays use a variety of formats, typically embedding target-specific oligonucleotides onto a glass or silicon microchip [23] [24] [25] [26] [27] [28] [29] [30] [31] to detect anywhere between dozens to hundreds of thousands of amplified sequences. Several respiratory diagnostic systems have been based on a liquid-phase technology using polystyrene microbeads (Luminex) [9, 10, [13] [14] [15] 32] or mass spectroscopy [33, 34] for amplicon discrimination. Although these approaches have greatly expanded the versatility and sensitivity of multiplex PCR, their complexity, specialized equipment, and high start-up costs have limited their widespread adoption to date. Moreover, these platforms typically require separate steps for amplification and detection, increasing both the workload and the risk of operator error or amplicon contamination.
Real-time PCR assays address these issues by combining amplification and detection in one reaction tube, thus facilitating automation and reducing contamination. In addition, this technique allows for the quantification of pathogens and the assessment of replication efficiency. As with conventional PCR, multiplex real-time assays are subject to competition and inhibition among primers [5, 35] . Real-time assays are also restricted in the number of reaction products that can be detected in parallel [35] , although this problem can be partially circumvented using arrays of uniplex real-time reactions at very small volumes [36] . Successful in-house realtime assays directed against respiratory pathogens have been developed using uniplex [37] and multiplex [38, 39] approaches, but data on the performance of commercialized versions are not readily available.
Much of the effort in NADT development for respiratory diagnostics has focused on the detection of viruses, given the advantages of these techniques over conventional methods in terms of speed, sensitivity, and versatility for detecting this class of pathogens. Multiplex approaches for viral detection have become more common as technologies have improved (reviewed by [5, 7, 40, 41] ). In addition, NADTs have now become the gold standard for the detection of Mycoplasma pneumoniae [42] and Chlamydophila pneumoniae [43] and a useful addition to antigen testing for Legionella species [44] .
Multiplex assays for the detection of more traditional bacterial pathogens have not been studied as frequently in respiratory specimens, primarily because culture techniques are usually adequate for clinical practice. Moreover, molecular methods provide no additional advantage over culture in differentiating infection from colonization of the upper respiratory tract. Nevertheless, multiplex NADTs for bacteria such as S. pneumoniae, Haemophilus influenzae, and Streptococcus pyogenes have been evaluated in respiratory specimens [45] and will likely be incorporated into larger multiplexing assays.
Having conducted our survey of the field, we narrowed our list of candidate molecular diagnostic platforms even further on the basis of the unique needs of our research study. As with clinical laboratories, we closely examined factors such as cost, feasibility, quality assurance, capital investment, platform versatility, and future utility. In contrast with clinical laboratories, we considered issues such as the rapid return of results or regulatory approval for use in patient care to be less crucial to our objectives. Moreover, our approach emphasized comprehensive pathogen detection, rather than focusing primarily on pathogens relevant for clinical management or infection control. Finally, our selected platform would be deployed in low-resource settings, where requirements for a reliable or continuous power supply, adequate access to reagents, sensitivity to extreme environmental conditions, and access to technical support would be highly relevant.
As our appraisals progressed, we encountered many challenges in interpretation that are common to the field of diagnostics evaluation. Most basic among these was confusion regarding the usage of the terms ''sensitivity'' and ''specificity,'' and the evaluations needed to measure these parameters [46] . The distinction between ''analytic'' performance characteristics, as opposed to ''diagnostic'' or ''clinical'' performance characteristics, is essential for properly assessing the validation of any assay, but it is particularly true in the field of molecular diagnostics. For NADTs, analytic sensitivity refers to the lowest concentration of target that can be detected, whereas analytic specificity measures the ability of the test to exclude undesired targets despite similar genetic sequences. In contrast, diagnostic or clinical sensitivity of a nucleic acid detection test refers to the appropriate identification of all patients carrying the agent, and diagnostic or clinical specificity describes the assay's ability to exclude uninfected patients. Clinical performance characteristics are subject to a number of factors, including the patient's disease status, variations in the concentration of the target throughout the course of illness, inhibition by other substances present in the specimen, sample quality, sampling variability, and specimen degradation. Generally, assays should be tested against a reference or gold standard. For tests of microbial detection, the reference standard typically is culture, but molecular diagnostics are often much more sensitive in detecting nucleic acid than is culture for viable organisms, leading to difficulties in interpreting the clinical relevance of false-positive results. The challenges of assessing diagnostic tests have been increasingly recognized in recent years. For instance, the Standards for Reporting of Diagnostic Accuracy initiative [47, 48] offers guidelines on the reporting of diagnostic studies, whereas the Quality Assessment tool for Diagnostic Accuracy Studies provides corresponding guidance on their evaluation [49] .
Nevertheless, respiratory diagnostics are particularly limited by the inability to determine whether the detection of a particular pathogen in a symptomatic patient indicates that it is causative of the illness or results from contamination, colonization, or prolonged shedding from a prior unrelated infection, particularly when testing specimens from the upper respiratory tract. This issue is not typically addressed in diagnostic evaluation studies, but it has become more relevant as molecular diagnostics have expanded the lower limits of pathogen detection by several orders of magnitude. Attempts to answer this question have suggested an additional category of test performance, the ''epidemiological'' specificity of a test, to describe the ability of an assay to assign true etiologic status to a pathogen for a specific illness. Ultimately, determination of the epidemiologic specificity of a respiratory diagnostic would require the interpretation of microbiologic results in conjunction with all other clinical and laboratory data, perhaps in the form of a predictive model. Such analyses are uncommon but will be a main focus of the PERCH study.
Respiratory diagnostics are further complicated by the absence of a perfect gold standard. Culture is difficult or insensitive for some pathogens and unavailable for others (eg, human metapneumovirus, parainfluenzavirus type 4, rhinovirus group C, or Pneumocystis jiroveci). Serologic tests are often not available and usually require paired serum specimens for accurate results. Statistical methods to adjust for such alloyed gold standards, such as discrepant analysis, have been frequently employed, but they can be susceptible to significant bias [50] . Comparative evaluations of respiratory diagnostic assays must also take into account variations in which panel of pathogens is selected, which genetic sequences are targeted, what specimen sources are used [3, 51] , and even what methods are used for nucleic acid extraction [52] . The US Food and Drug Administration has recently published industry guidance that may encourage additional work in this area [53] .
As the PERCH evaluation progressed, the concepts derived from our deliberations were distilled into a list of desirable and essential attributes summarizing our strategy for evaluation ( Table 2 ). This list addressed issues such as assay performance (range of targets, acceptable specimen sources, sensitivity, and specificity), operational concerns (space requirements, assay throughput, quality assurance programs, maintenance requirements, and reagent availability), and strategic issues (capacity for automation, versatility and future utility, start-up and maintenance costs, and developer engagement). For additional input, we presented our summary to the Pneumonia Methods Working Group, an expert committee formed to advise PERCH. Ultimately, this outline of key qualities and data allowed us to articulate our thoughts and communicate our strategy more effectively to collaborators, advisors, and assay developers.
We applied our list of attributes to more than a dozen candidate diagnostic systems that met our initial criteria, and developed a short list of candidate platforms. We then tested these final assays in our PERCH-affiliated laboratories, using a standardized set of mock specimens. This process allowed us to engage with the assay manufacturers and their academic partners, directly compare the performance characteristics of Nucleic acid extraction procedure included in overall process (and automated)
Ability to process a variety of respiratory tract specimens Small specimen volume requirements Specimen collection requirements well-characterized and suitable for field studies Readily available reagents with long expiry dates and room-temperature storage requirements the platforms, and gain essential information that could only be acquired through hands-on experience, such as capabilities for technology transfer, ease of use, and workflow. Details of this evaluation will be the subject of a separate article.
By including a phase for protocol development, the PERCH investigators were able to perform an extensive literature review of respiratory diagnostics, clearly outline the major theoretical and practical concerns, and engage a group of experts for critical input. Through this process, we confirmed the suitability of molecular diagnostics for our needs and identified critical information gaps. Our evaluation highlighted numerous advantages of this technology, including excellent sensitivity and adaptability for a full range of respiratory pathogens and specimen sources, as well as clear capabilities for multiplexing and automation. We nevertheless realized that our conclusions represent but a snapshot in time, and the field of molecular diagnostics is rapidly evolving, with constant improvements in accuracy, speed, automation, and cost. Yet it can also be expected that methods for evaluating respiratory diagnostics will continue to evolve in parallel, providing new answers to the practical and conceptual challenges that shaped the development of a diagnostic testing strategy for PERCH. Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1 In the field of herpesvirus research, the exact molecular mechanism by which such viruses reactivate from latency remains elusive. Kaposi's sarcoma-associated herpesvirus (KSHV) primarily exists in a latent state, while only 1–3% of cells support lytic infection at any specific time. KSHV reactivation from latency is an exceedingly intricate process mediated by the integration of viral and cellular factors. Previously, our lab has described early growth response-1 (Egr-1) as an essential component for the KSHV reactivation process via its ability to mediate transcription of KSHV ORF50, the gene encoding for replication and transcription activator (RTA), a viral component known to control the switch from latent to lytic infection. In here, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments revealed that Egr-1 binds KSHV ORF50 promoter (ORF50P) in at least two different GC-rich binding domains. Expression profiles of cellular egr-1 and KSHV-encoded ORF50 follow a similar pattern during de novo KSHV infection. Over-expressing Egr-1, a signaling component downstream of Raf>MEK>ERK1/2, in KSHV-infected cells activates KSHV lytic replication. Through performing more physiologically relevant experiments, we analyzed the effect of a dietary supplement containing resveratrol on KSHV-infected cells. Our results, for the first time, demonstrate resveratrol to act in lowering ERK1/2 activity and expression of Egr-1 in KSHV-infected cells, resulting in the suppression of virus reactivation from latency. Taken together, these findings will undoubtedly contribute to future studies on not only combating KSHV related disease conditions, but also on other herpesviruses-induced pathogenesis. Significant strides have been made since the discovery of Kaposi's sarcoma-associated herpesvirus (KSHV) by Chang et al [1] nearly 20 years ago that have helped to increase our understanding of this infectious agent. KSHV is a c2-herpesvirus that has been directly linked to the development of Kaposi's sarcoma (KS), primary effusion (PEL), and multicentric Castleman disease (MCD). This virus is closely related to Epstein-Barr virus (EBV), murine gammaherpesvirus-68, and herpesvirus saimiri [2] . The prevalence of KSHV infection varies depending on the geographical location with highest levels observed in Africa, where it has been reported to be greater than 40% [3] . As KSHV displays several characteristics shared among other herpesviruses, its ability to switch between latent and lytic stages of infection is of particular concern. The virus remains predominantly in a latent state, while 1-3% of cells may support a lytic infection at any given time [4] . Regulation of the switch between the two stages of infection is mediated by viral and cellular factors. Specifically, the KSHV protein, replication and transcription activator (RTA), is known to be a crucial viral component controlling the transition from latency to a lytic infection [5] . Recently, cellular early growth response-1 (Egr-1) protein was also shown to be an important factor involved in KSHV reactivation through its ability to mediate transcription of KSHV ORF50, the gene encoding RTA [6] .
Egr-1 is a transcription factor that is also known as zif268, Krox-24, NGFI-A, and TIS8 [7] . It is induced by several external stimuli including growth factors, different forms of stress, and hormones. As a result of stimulation from various factors, egr-1 gene products advance to play a role in several cellular functions such as, but not limited to, growth, proliferation, and differentiation [8] . Egr-1 is part of a zinc-finger gene family that includes Egr-2, Egr-3, Egr-4, and the Wilms tumor suppressor (WT1) [9] . TPA is used to activate a lytic infection in KSHV-infected cells [10] . Egr-1 mediates the effect of TPA activation and is a downstream target of MAPK signaling [9, 11] . Furthermore, MAPK signaling is crucial for triggering KSHV reactivation from latency [12, 13] . However, despite the ability of Egr-1 and KSHV ORF50 to interact with each other, there is little information available describing this association.
In a recent study, the ability of Egr-1 to bind KSHV ORF50 promoter (ORF50P) was described [6] . In this report, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments were employed to determine the locations on ORF50P that have an affinity for Egr-1 binding. Our results demonstrated at least two targets that are likely crucial for mediating Egr-1 binding to ORF50P. These findings were confirmed through the use of mutation studies. In addition, we tested the ability of resveratrol, a naturally occurring product found in a variety of fruits and nuts [14] , in regulating MAPK signaling.Egr-1 expression.promoting virus latency. As such, the ramifications on the ability of Egr-1-induced transcription of ORF50 in viral pathogenesis are discussed.
Egr-1 binds at least two different sites within the ORF50P Egr-1 is said to bind a GC-rich DNA template (such as GCGC(G/T)GGGCG, GCGGGGGCG, and CGCCCATGC) on the promoter and initiate gene transcription [15, 16] . Eight possible GC-rich Egr-1 binding sequences have been identified by us in the promoter region of KSHV ORF50. In order to determine the sites where Egr-1 bound ORF50P, EMSA experiments were performed using 8 different DIG-labeled probes, referring to identified locations on ORF50P, (Table 1) and Egr-1 in vitro transcribed and translated (IVT) proteins. IVT of egr-1/ pcDNA3.1(+) construct yielded a protein of roughly 78 kda (data not shown) [6] . Of all the probes tested, IVT-synthesized Egr-1 proteins were able to bind and form separate protein:DNA complexes with the ORF50P3 and ORF50P8 probes, respectively; displaying distinct band shifts when compared to controls using the probes alone ( Fig. 1A ; lanes 6 and 16). It is important to note that the sequence for ORF50P3 is the same for the probe used in earlier study [6] . Band shifts were not observed when ORF50P probes were incubated with IVT-synthesized KSHV glycoprotein L (gL) (data not shown). Additionally, experiments using the ORF50PNP probe (does not contain the GC-rich binding domain) did not form a complex with Egr-1 protein, thus confirming the specificity of Egr-1 binding (Fig. 1A, lane 18) . Finally, competition experiments using unlabeled ORF50P probes reduced band shifts by preventing Egr-1 binding to DIG-labeled probes (data not shown).
A second set of EMSA studies were conducted using ORF50P probes carrying mutations (ORF50P3m and ORF50P8m; Table 1 ) in the putative Egr-1 binding region to further confirm the binding ability of Egr-1 proteins. Briefly, ORF50P3 and ORF50P8 were mutated to carry the 5 bp ATATA sequence in the GC-rich binding domain and then incubated in binding buffer alone or with IVT-synthesized Egr-1. Samples consisting of wildtype (wt) or mutated probes alone did not display a shift in the respective ORF50P3 and ORF50P8 probes ( Fig. 1B ; lanes 1, 3, 5. and 7). Following incubation of wt probes with Egr-1, complexes were formed producing separate band shifts (Fig. 1B , lane 2 and 6). Interestingly, Egr-1 did not bind ORF50P3m or ORF50P8m probes that displayed mutations in the Egr-1 binding domain, thus confirming the necessity for the consensus GC-rich binding domain mediating Egr-1/ORF50P interactions (Fig. 1B, lanes 4  and 8) .
Finally, gel shift assays were performed using the nuclear extract from KSHV-infected cells to verify the ability of Egr-1 to bind ORF50P3 and ORF50P8. As expected, there was no hindrance in the migration of ORF50P probes without the addition of cell lysate ( Fig. 1C; lanes 1 and 5) . However, the presence of the lysate in the samples resulted in the formation of protein:DNA complexes indicated by a band shift (Fig. 1C , lanes 2 and 6). Egr-1 binding to ORF50P was confirmed by incubating lysates with specific Abs and performing a supershift. Samples that were incubated with nonspecific IgG Abs displayed band shifts that were similar to samples containing only Egr-1 and the respective probes (Fig. 1C, lanes 3 and 7) . Alternatively, a supershift occurred exhibiting a discrete band when nuclear lysates were pre-treated with Egr-1 specific Abs (Fig. 1C, lanes 4 and 8). Taken together, these experiments provide support for the ability of Egr-1 to specifically bind to two separate locations on KSHV ORF50P. IVT-synthesized Egr-1 binds to ORF50P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIGlabeled ORF50P probes (see Table 1 ). (B) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50P3 and ORF50P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain (ORF50P3m and ORF50P8m). (C) Nuclear lysates from KSHV-infected cells formed a complex with ORF50P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols [28] , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50P3 and ORF50P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 (lanes 4 and 8) or nonspecific IgGs (lanes 3 and 7). The arrowhead indicates protein/ DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk. doi:10.1371/journal.pone.0033364.g001
A semi-quantitative chromatin immunoprecipitation (ChIP) assay was performed to analyze Egr-1 binding to ORF50P in a chromatin context (in vivo) using specific antibodies. TPA-induced KSHV-infected cells were used to assess the binding ability of Egr-1 to ORF50P via ChIP assays. The presence of specific ORF50P in the IP samples was analyzed by semiquantitative PCR using specific primers covering the regions of ORF50P3 or ORF50P8. As expected, when Egr-1 was expressed in BCBL-1 cells it was recruited to the promoter of KSHV ORF50 and specifically targeted both ORF50P3 and ORF50P8 ( Fig. 2A, cycle 30 ). Recruitment of Egr-1 to the nonspecific ORF50NP region was not detectable in our experiments (data not shown). For negative controls, samples were IP with nonspecific (NS) IgG Abs and recruitment of Egr-1 to ORF50P was not observed ( Fig. 2A , cycle 30 on control gels). However, positive controls using specific Abs to histone proteins recovered ORF50P targets ( Fig. 2A) . These results help us confirm that Egr-1 binds to two separate domains on ORF50P, in vivo.
To establish a critical role for these interactions between Egr-1 and ORF50P, luciferase reporter constructs were used to investigate the necessity of ORF50P3 and ORF50P8 during Egr-1-mediated activation of the ORF50P. Empty vector (pGL3) or vectors encoding a deletion series of ORF50P (Fig. 2B ) along with the downsteam luciferase gene were transiently transfected into target cells in conjunction with empty vector or egr-1/ pcDNA3.1(+). Cells transfected with pcDNA3.1(+) did not induce significant luciferase activity (Fig. 2C) . However, following incubation of cells transfected with egr-1/pcDNA3.1(+), we noticed the luciferase activity to be significantly greater in cells that were also transfected with constructs encoding the full length (FL) ORF50P compared to cells transfected with pGL3 ( Fig. 2C) , Furthermore, we observed a decrease in relative luciferase activity following deletion of the fragment containing the ORF50P3 domain (D-2922 to -2044; D-2922 to -1322; D-2922 to -894; and D-2922 to -169) when compared to the construct encoding full length ORF50P (Fig. 2C) . Although the absence of ORF50P3 contributed to a decrease in luciferase activity, this activity was never completely abolished ( Fig. 2C) suggesting the need for an intact ORF50P3 and ORF50P8 for an optimal Egr-1-induced transcription of ORF50. Taken together, these results suggest a role for Egr-1 to specifically bind and activate ORF50P to trigger a lytic infection in KSHV-infected cells.
Cellular Egr-1 and virus-encoded KSHV RTA follow a similar expression pattern during de novo KSHV infection Several different viruses are known to activate Egr-1 expression upon infection [17, 18, 19, 20] . Since BCBL-1 cells already carry KSHV DNA, KSHV-infected HEK293 cells were used to evaluate the expression pattern of Egr-1 and KSHV RTA during early stages of de novo infection. Expression of Egr-1 and RTA proteins were significantly elevated by 1 hour post infection (hPI) and continued to maintain increased expression until roughly 6-8 hPI (Fig. 3A, lanes 2-4) . In contrast, a considerable decrease in the expression of these proteins was observed from 12-48 hPI (Fig. 3A , lanes 5-7). A significant difference in expression of total bactin was not observed (Fig. 3A ) during the course of KSHV infection demonstrating the specificity of the results on Egr-1 and RTA expression.
To further support our findings, mRNA extracted from KSHVinfected HEK293 cells were subjected to quantitative real-time PCR (qRT-PCR) analysis in order to evaluate egr-1 and ORF50 transcriptional activity. Uninfected cells (0 hPI) did not show detectable ORF50 expression (Fig. 3B ). On the other hand, a low baseline level of egr-1 expression was observed in the uninfected samples ( Fig. 3B ). With the onset of a primary infection, expression levels of both egr-1 and ORF50 increased up to 6hPI (Fig. 3B ). These elevated levels of egr-1 and ORF50 decreased substantially by 12-24 hPI (Fig. 3B ). No significant changes in the expression of the internal control gene encoding M6PR was observed indicating specificity of the results (data not shown). Taken together, the expression profiles of Egr-1 and RTA seem to follow an identical pattern during primary infection of cells.
Elevated Egr-1 expression activates lytic genes in KSHVinfected cells BCBL-1 cells have turned out to be a blessing in disguise for this study as they harbor KSHV DNA in a predominantly latent state [21] . BCBL-1 cells were transiently transfected using egr-1/ pCDNA3.1(+) for 24, 48, and 72 h ( Next, qRT-PCR studies were performed to identify changes in egr-1 and virus-encoded ORF50 gene expression. We did not observe any noticeable alterations in egr-1 and ORF50 transcription in target cells that were untransfected (UT), mock transfected, or transfected with empty vectors (Fig. 4B ). As expected, levels of egr-1 were significantly increased in cells transfected with egr-1/pCDNA3.1(+) over controls (Fig. 4B) . Furthermore, elevated egr-1 expression coincided with an increase in KSHV ORF50 transcription (Fig. 4B ). Peak expression for both genes was observed by 48 h post transfection (Fig. 4B) . Incidentally, we also observed an elevated expression of ORF8 (Fig. 4B ), encoding the late structural virus protein termed as gB, in response to an enhanced egr-1 and ORF50 expression. All these changes implicate Egr-1 expression to significantly induce virus reactivation.
Immunofluorescence assay (IFA) was conducted to determine a possible role for elevated Egr-1 on the expression of KSHVencoded lytic proteins in the above transfected cells (Fig. 4A, B ). KSHV-encoded ORF59, a processivity factor for KSHV DNA polymerase, is expressed in the nucleus of infected cells during early stages of virus reactivation [22] . Target cells transfected with empty vectors showed low levels of ORF59 expression (Fig. 4C) . However, transfection cells with egr-1/pcDNA3.1(+) augmented the level of ORF59 protein expression in KSHV-infected cells (Fig. 4C ); clearly implicating a critical role for Egr-1 in inducing KSHV reactivation. Finally, we analyzed MAPK signaling in the above cells relative to Egr-1 expression levels. Our results suggest that cells transfected with egr-1/pCDNA3.1(+) displayed elevated levels of Egr-1 (Fig. 4D, lane 7) . Transfection of cells with pCDNA3.1(+) (Fig. 4D , lane 5) or mock transfection (Fig. 4D, lane 3) of cells did not significantly alter the expression levels of Egr-1 and phosphorylation state of ERK1/2 compared to untransfected cells (Fig. 4D, lane 1) . Interestingly, treatment of cells with a known inhibitor of MEK1/2 (10 mM of U0126) significantly lowered both the expression levels of Egr-1 and ERK1/2 activity (Fig. 4D, lane  8) . We observed U0126 to dose dependently inhibit ERK1/2 activity and Egr-1 levels in the above cells confirming the specificity of U0126 in targeting MAPK.Egr-1 signaling. The inhibition of ERK1/2 activity and Egr-1 levels was greatest following treatment of infected cells with 10 mM of U0126 (Fig. 4E,  lane 4) . These results suggest Egr-1 to be downstream of the MAPK signaling cascade. [28] and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 mg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50P3 or ORF50P8 (see Table 1 ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. (B) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. (C) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1/pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (D-2922 to -2044; D-2922 to -1322; D-2922 to -894; and D-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1/pcDNA3.1(+) was represented as 1-fold. Each point denotes the average 6 SD of three experiments. Columns with different alphabets are statistically significant (P,0.05) by least significant difference (LSD). doi:10.1371/journal.pone.0033364.g002
Resveratrol inhibits Egr-1 and ORF50 during early and late stages of infection Due to the vital role of MAPK signaling on Egr-1 expression and KSHV reactivation, the effect of resveratrol on KSHV replication was analyzed. We chose to use resveratrol because: (i) it is a naturally occurring product; and (ii) it is a known regulator of MAPK signaling and Egr-1 expression [14] . Furthermore, resveratrol inhibits ERK1/2 activity in virus-infected cells [23] . It is important to understand that even though KSHV-encoded ORF50 is a gene crucial for reactivation, it is also expressed during early stages of KSHV infection and may play a role in the establishment of virus latency [6, 24] . Therefore, it was necessary to determine the expression pattern of cellular egr-1 and virusencoded ORF50 during both early stages of infection as well as during virus reactivation (late stages). In this study, effect of resveratrol on early stages of infection was analyzed in HEK293 cells while its effect on late stages (reactivation using TPA) was analyzed predominantly in BCBl-1 cells, for convenience. In this study, resveratrol was able to inhibit egr-1 expression in a dose dependent manner during early stages of de novo KSHV infection of HEK293 cells (Fig. 5A) . The doses tested in this study were confirmed by trypan blue test to be non-toxic to cells (data not shown). The resveratrol doses used by us are also those that have been published previously [25, 26, 27] . Resveratrol (100 mM) was able to suppress the expression of phospho-ERK1/2 and Egr-1 proteins during de novo infection of cells (Fig. 5B, lanes 3, 6, and 9 ). Additionally, resveratrol was able to inhibit the expression Egr-1 and phophorylated ERK1/2 in mock-infected cells ( Figure S1,  lanes 2, 4, and 6 ). On the other hand, it was not able to significantly alter the expression of endogenous ERK1/2 and actin controls (Fig. 5B, lanes 3, 6, and 9 ; Figure S1, lanes 2, 4, and 6) . DMSO, the vehicle for resveratrol, did not significantly alter the phosphorylation of ERK1/2 and the expression of Egr-1 (data not shown).
In order to present more physiologically relevant studies, an over-the-counter resveratrol dietary supplement (RDS) was used to treat KSHV-infected BCBL-1 cells under TPA-induced conditions. RDS containing 100 mM resveratrol did not significantly induce cell death as monitored by the lactate dehydrogenase assay (data not shown). These results were confirmed by the conventional trypan blue test. More than 95% of the target cells were found to be viable when the target cells were treated with RDS (data not shown). As shown in earlier reports [12] , TPA treatment augments phospho-ERK1/2 expression (Fig. 6A, lane 2) . The effect of TPA also resulted in an increase in Egr-1 and KSHV RTA expression when compared to uninduced cells (Fig. 6A, lane  2) . Unfiltered RDS successfully inhibited phospho-ERK1/2, Egr-1, and RTA expression in TPA-induced KSHV-infected cells (Fig. 6A, lane 3) . A slightly lesser inhibitory effect was observed in cells that were treated with RDS that had particulates removed using a 0.2 mm filter (Fig. 6A, lane 4) . We did not discover either form of RDS to have noticeable effect on endogenous ERK1/2 and actin controls (Fig. 6A, lanes 3 and 4) . The data from Western blotting (Fig. 6A ) was further confirmed in HEK293 cells by IFA (Fig. 6B) . To authenticate the results from monitoring the effect of RDS on protein levels of Egr-1 and RTA, we analyzed the effect of RDS on (i) uninduced cells transfected with vectors encoding egr-1, and (ii) TPA-induced KSHV reactivation in BCBL-1 cells by performing qRT-PCR. Our results clearly demonstrate the ability of RDS to lower the expression of both egr-1 and ORF50 under both circumstances (Fig. 6C, D) . Finally, ChIP assay was performed to discern the specificity of RDS on virus reactivation using primers specific to ORF50P8 region. Under TPA-induced conditions, Egr-1 specifically targeted ORF50P8 (Fig. 6E . cycles 25 and 30). However, under RDS treated conditions the binding of Egr-1 to ORF50P8 was significantly decreased (Fig. 6E, cycles 25  and 30 ). For negative controls, samples were IP with (NS) IgGs and recruitment of Egr-1 to ORF50P was not observed (Fig. 6E) . However, positive controls using specific Abs to histone proteins recovered ORF50P targets (Fig. 6E) . These results suggest resveratrol in its chemical form and RDS may lower Egr-1 expression to inhibit KSHV reactivation from latency.
Egr-1 regulates expression of several viral genes and plays a crucial role in the replication of different viruses Our results from employing the EMSA and ChIP assay ( Fig. 2A) demonstrate that Egr-1 may bind ORF50P via at least two different GC-rich binding domains: at positions between 2 100 -2 76 bp (ORF50P8) and 2 2173 -2 2149 bp (ORF50P3). The results from employing the ChIP assay ( Fig. 2A) demonstrate that Egr-1 may bind ORF50P with a greater affinity at positions between 2 100 -2 76 bp (ORF50P8) compared to 2 2173 -2 2149 bp (ORF50P3). However, we did not observe any such differences in the binding affinity of Egr-1 to ORF50P3 and ORF50P8 by EMSA using IVT Egr-1 (Fig. 1B, C) . All the more, our data supports the need for Egr-1 to bind both ORF50P3 and ORF50P8 for an optimal transcriptional activity in luciferase reporter assays (Fig. 2C) . This difference observed in Egr-1 binding to both these domains could be due to one or both the reasons: (i) IVT synthesized Egr-1 was used in EMSA experiments; and (ii) the design of ChIP assay conducted in this study was not to decipher the relative binding affinity of Egr-1 to these domains; instead was performed to just confirm if Egr-1 bound these domains, in vivo.
Although we previously noticed that the egr-1 and KSHVencoded ORF50 followed a similar expression pattern, the experiments were conducted in TPA-induced cells to evaluate their expression during the reactivation process [6] . The present study discovered that the transcriptional activity of egr-1 and ORF50 and their subsequent translation is comparable and followed a similar pattern during de novo KSHV infection (Fig. 3) . However, enhanced cellular Egr-1 and viral RTA expression during early stages of primary infection (Fig. 3) is not sufficient to trigger a lytic infection [6] . These results suggest the following: (i) the role of Egr-1.RTA signaling in initiating a lytic cycle of infection during the course of initial infection of cells is limited; and (ii) there is a missing element in the Egr-1.RTA driven cellular events critical for inducing a productive replication.
In this study, transfection of cells with egr-1/pCDNA3.1(+) resulted in a significant increase in virus-encoded ORF50 transcription followed by the expression of early-lytic ORF59 protein and late-lytic gene (ORF8) encoding gB (Fig. 4B, C) ; all of which are indicators of an active lytic replication of KSHV [12, 28, 29] . MAPK signaling was observed to regulate Egr-1 expression in cells transfected with egr-1/pCDNA3.1(+) (Fig. 4D) . Interestingly, treatment of KSHV-infected cells with TPA induces a lytic replication via MAPK signaling [12, 13] . In addition, Egr-1 is a downstream target of Raf/MEK/ERK signaling (Fig. 4D ) [6, 30] . It is unclear at this time if the effect of egr-1/pcDNA3.1 over-expression resembles the milieu supporting a lytic infection in vivo. It is important to remember that KSHV reactivation can be regulated by other cellular factors including STAT6, NFkB, and XBP-1 [31, 32, 33] . Thus, activation of a lytic infection may require unique cellular factors under specific conditions or a combination of factors. Further investigation is required to unravel the environment(s) supporting virus reactivation under physiologically relevant conditions; especially in terms of the different transcription factors.
In order to support these findings, more physiological relevant studies were performed by analyzing the effect of resveratrol on KSHV-infected cells. Resveratrol, or trans-3,5,49-trihydroxystilbene, is a phytoalexin that is produced in various plants such as grapes, berries, and peanuts in response to attacks by pathogens [14] . Several reports provide evidence for resveratrol to exhibit antiviral effects [27, 34, 35] . On the other hand, resveratrol has also been shown to induce virus replication [36, 37] . We have demonstrated that resveratrol, in its chemical form, inhibits 1-3) were incubated at 37uC for 48 h while the cells transfected with egr-1/pcDNA3.1 were incubated for 24, 48, and 72 h, respectively. At the end of incubation, the cells were lysed and the lysates were used to perform Western blotting. (B) Effect of elevated Egr-1 on KSHV ORF50 and ORF8 expression. BCBL-1 cells were untransfected or transfected as described above. RNA was extracted, cDNA was synthesized, and the expression of cellular egr-1; and KSHV encoded ORF50 and ORF8 were analyzed by qRT-PCR. Baseline expression of genes was considered as 1-fold for comparisons. Each point denotes the average6S.D. (error bars) of three experiments. (C) Expression of lytic proteins in BCBL-1 cells transfected with Egr-1. KSHV-infected cells were untransfected, transfected with pcDNA3.1, or egr-1/pcDNA3.1 for 48 h. The stained cells examined using a confocal microscope (magnification 662). The average number of fluorescent cells were counted over five random fields and used for comparisons. (D) Enhanced egr-1 activates MAPK signaling in BCBL-1 cells. KSHV-infected cells were untransfected, mock-transfected, transfected with pcDNA3.1(+), or egr-1/pcDNA3.1(+) for 48 h. Each group of cells was left untreated or they were treated with 10 mM of U0126 1 h prior to transfection and remained throughout the incubation period. Cell lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. (E) U0126 inhibits phosho-ERK1/2 and Egr-1. Briefly, BCBL-1 cells were treated with different concentrations of U0126. Following 24 h incubation, the cells were lysed and the lysates were used to perform Western blotting as per earlier protocols using specific antibodies. doi:10.1371/journal.pone.0033364.g004 Egr-1 and phospho-ERK1/2 in KSHV-infected cells (Fig. 5B) . RDS significantly inhibited KSHV reactivation in uninduced and TPA-induced cells (Fig. 6A , B, C, D). While performing these experiments we noticed that unfiltered RDS was able to inhibit KSHV reactivation to a greater extent when compared to cells treated with RDS that was passed through a 0.2 mm filter. However, both treatments were able to significantly inhibit gene products associated with KSHV reactivation (Fig. 6A, D) . These differences are likely due to the presence of unknown factors in unfiltered RDS that may act in combination with resveratrol. Incidentally, the decrease in the expression of RTA by RDS coincided with a sharp decline in the ability of Egr-1 to bind ORF50P as shown by the semi-quantitative ChIP assay (Fig. 6E) . Taken together, this is the first report to describe the role of physiologically relevant RDS on KSHV infection.
Several cellular pathways are regulated by resveratrol including apoptotic, NFkB, and all forms of MAPK signaling [14, 38] . We found resveratrol to inhibit expression of Egr-1 and phosphorylation of ERK1/2 resulting in suppression of KSHV reactivation ( Fig. 5 and 6 ). Very recent studies have established the fact that resveratrol significantly lowers phosphorylation of ERK1/2 (directly upstream of Egr-1) in target cells, in vivo and in vitro [25, 26, 39, 40] . At this point in time, we are certain about the ability of RDS to block TPA-induced virus reactivation. However, further research is required to confirm if this ability of RDS to promote viral latency is by its direct inhibitory effect on the expression Egr-1 or the upstream MAPK signaling component(s), namely ERK1/2 activity.
KSHV reactivation from latency is a complicated process which is regulated by an intricate relationship between viral and cellular factors. The method in which resveratrol may regulate KSHV reactivation has yet to be fully understood. However, we propose that resveratrol may inhibit KSHV reactivation by altering the interactions between cellular Egr-1 and viral ORF50P in a Raf.MEK.ERK-dependent manner. All three MAPKs (ERK, p38, and JNK) have been shown to positively regulate Egr-1 expression [41] . However, the role for active p38 MAPK is not fully understood as it has been shown to reduce Egr-1 expression in B-lymphocytes unlike ERK and JNK [42] . Further studies are required to better understand the involvement of different MAPKs on Egr-1 expression during KSHV infection. The findings presented in this study open a Pandora's Box of questions pertaining to treating/managing a variety of viral infections using RDS. Future studies are aimed at appreciating the cellular milieu critical for the effectiveness of the MAPK associated signaling in inducing virus reactivation. These findings may provide for more useful applications to combat a variety of viral lytic infections.
Cells HEK293 cells and BCBL-1 cells were cultured in DMEM and RPMI (Invitrogen, Carlsbad, CA), respectively, as per earlier studies [6, 43] .
Rabbit antibodies to gB [44] , RTA [6] ; and mouse antibodies to ORF59 were used in this study. Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study. Mouse (S-25) and rabbit (15F7) antibodies to Egr-1 purchased from Santa Cruz biotechnologies, Inc. (Santa Cruz, CA) were used in Immunofluorescence assay (IFA) and Western blotting experiments, respectively. Additionally, rabbit polyclonal antibodies (588) to Egr-1 was used in gel shift and chromatin immunoprecipitation assays (Santa Cruz Biotechnology, Santa Cruz, CA).
We used egr-1/pCDNA3.1(+) and gL/pCDNA3.1(+) vectors in this study. Both these vectors have been described elsewhere [6] . 
IVT of egr-1/pCDNA3.1(+) and gL/pCDNA3.1(+) was conducted as per earlier studies [45] using the TNT-coupled rabbit reticulocyte lysate system (Promega).
HEK293 cells were infected as per earlier procedures (25) . Figure 6 . RDS reduces the Egr-1/ORF50 association in vivo. (A) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 mM of resveratrol. The cells were incubated at 37uC for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. (B) RDS reduces the number of KSHVinfected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 mM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification 6100). (C) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1/pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 mM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average6S.D. (error bars) of three experiments. (D) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 mM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average6S.D. (error bars) of three experiments. Columns with different alphabets are statistically significant (P,0.05) by LSD. (E) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo. BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 mM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 mg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively. doi:10.1371/journal.pone.0033364.g006
In this study, we synchronized BCBL-1 cells in S phase of cell cycle as per earlier protocols [46] .
Equal amounts (20 mg) of protein was used in Western blotting experiments as per earlier studies [47] .
The qRT-PCR was performed using the synthesized cDNA in a 25 ml reaction volume to analyze the expression of ORF50, egr-1, and M6PR as per earlier protocols [47] .
Target cells were transfected with pCDNA3.1(+), egr-1/ pCDNA3.1(+), gL/pCDNA3.1(+) using GeneJammer transfection reagent (Stratagene, La Jolla, CA) as per earlier studies [12] .
Target cells were fixed for 10 min in ice cold acetone and washed thrice in phosphate buffered saline (PBS). These cells were sequentially stained with mouse anti-ORF59 antibodies and goat anti-mouse FITC as per earlier studies [12] . The stained cells were further incubated for 20 min on ice with 5 mM SYTO Red (a nuclear stain; Invitrogen) before being analyzed with a laserscanning LSM 510 Carl Zeiss confocal microscope. In another set of experiments, acetone fixed cells were incubated with a combination of mouse anti-Egr-1 IgGs and rabbit anti-RTA for 45 min at room temperature (RT), and incubated with a combination of goat anti-mouse FITC and goat anti-rabbit TRITC) for 30 min at RT. Stained cells were washed in PBS, mounted by using an anti-fade reagent containing DAPI (4,6diamidino-2-phenylindole; Molecular Probes) and examined under a Nikon fluorescent microscope with appropriate filters.
IVT products of Egr-1 or KSHV gL were evaluated by EMSA for DNA binding using several 25 bp digoxygenin (DIG)-labeled probes containing sequences from the ORF50P (Table 1) as per earlier studies [6] . For a supershift, the cellular lysate was incubated with rabbit monoclonal antibodies to Egr-1 or nonspecific IgG at 37uC for 30 min prior to the addition of the DIG-labeled probe. All samples were run on a 4% nondenaturing gel for approximately 1.5 h and transferred to a PVDF membrane. The protein:DNA interaction was detected using the CSPD detection system (Roche Applied Science).
Target cells were transiently co-transfected using appropriate pGL3 and internal control pRL-TK contructs (Promega) and pcDNA3.1(+) vectors (Invitrogen). The total amount of DNA used per sample was approximately 2 mg. Following 48 h post-transfection, cells were harvested and Firefly and Renilla luciferase activities were analyzed using the dual luciferase system (Promega). Luciferase activity was monitored using a Turner Systems Luminometer (Sunnyvale, CA) as per earlier protocols [6] . The relative luciferase activity was calculated by normalizing ORF50P luciferase activity to control Renilla luciferase activity. The results were plotted as a percentage of the activity of the empty pGL3 vector.
BCBL-1 cells were treated with a final concentration of 1% formaldehyde and crosslinked for 10 min at RT. Crosslinking was stopped by addition of glycine at a final concentration of 0.125 M for 5 min. The cross-linked cells were washed in 16 PBS and counted so that approximately 10 7 cells were used in each immunoprecipitation (IP) reactions. Nuclei from the cells were purified and lysed to collect chromatin. Chromatin was sheared to approximately 500 bp using a Bioruptor sonicator (Diagenode, Sparta, NJ). Lysates containing the chromatin were pre-cleared using 35 ml of Protein A sepharose beads (Amersham Biosciences) in pre-IP dilution buffer for 30 min at 4uC. The samples were centrifuged to remove beads and the lysate was recovered. After setting aside input controls, primary antibodies were added to the samples and incubated overnight at 4uC. The DNA/protein complexes were IP using protein A beads for 4 h at 4uC and then washed using various ChIP wash buffers. Following elution, proteinase K was added to the complexes and incubated at 65uC overnight in order to reverse the crosslinks. The DNA samples were purified using phenol/chloroform extraction, resuspended in 16 TE buffer, and finally analyzed by polymerase chain reaction (PCR). PCR was performed using platinum pfx polymerase (Invitrogen, Carlsbad, CA) as per standard protocols. PCR amplification of the precipitated DNA was performed using primers that targeted ORF50P3, ORF50P8, and ORF50PNP regions ( Table 1) Figure S1 Resveratrol inhibits Egr-1 expression in the absence of KSHV infection. HEK293 cells were mockinfected by incubating with growth medium for 2 h at 37uC. These cells were washed and cultured in growth medium in the presence or absence of 100 mM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. (TIF) Common Variants in CDKN2B-AS1 Associated with Optic-Nerve Vulnerability of Glaucoma Identified by Genome-Wide Association Studies in Japanese BACKGROUND: To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. METHODS AND PRINCIPAL FINDINGS: We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10(−10)). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma. Glaucoma is a neurodegenerative ocular disease and one of the leading causes of irreversible blindness worldwide [1] . It is characterized by the progressive loss of retinal ganglion cells and optic nerve axons, resulting in visual field defects [2] . One of the well-known major risk factors for glaucoma is elevated intraocular pressure (IOP) [2] . Thus, the measurement of IOP is routinely involved in the diagnosis of glaucoma. In fact, the IOP level has been applied to subdivide the most common form of glaucoma, primary open-angle glaucoma (POAG), into two subtypes [3] : POAG with high ($22 mmHg) IOP (POAG/HPG, high pressure glaucoma; hereafter referred to as ''HPG'') and with normal (,22 mmHg) IOP (POAG/NPG, normal pressure glaucoma; hereafter referred to as ''NPG''). Interestingly, ,92% of the Japanese POAG patients are categorized into the NPG subtype [4] , whereas ,41% of Caucasian POAG patients are categorized as NPG [5] , thus showing a unique epidemiological distribution of Japanese patients compared with other ethnic groups.
Aside from IOP measurements, the diagnosis of glaucoma is commonly made by observing optic nerve degeneration and visual field defects by means of fundus examinations and visual field tests, respectively. In the case of HPG, early drug treatment to lower IOP immediately following the onset of visual field damage has been shown to be quite effective in slowing the irreversible progression toward blindness [6, 7] . In contrast, since NPG patients show normal IOP, they are often misdiagnosed. Therefore, both fundus examinations of the optic nerve and visual field tests are critical for the proper diagnosis of NPG patients. However, due to the restriction of the healthcare expenditure to include those examinations into a person's regular medical checkup, especially in the preclinical state of glaucoma, it would be of great benefit if the risk of developing glaucoma could be ascertained based on a simple blood test to assess the genetic markers for the disease.
Since glaucoma shows familial aggregation and its prevalence varies between individuals of different ethnicities, it has been theorized that genetic factors play a significant role in the pathogenesis of glaucoma [8, 9] . Therefore, several institutions are making profound efforts to discover single-nucleotide variants for glaucoma by conducting a genome-wide association study (GWAS) [10] [11] [12] [13] [14] , and there are a few published reports of the association of particular loci, such as the CAV1/CAV2 locus (7q31.1) [13] and the TMCO1 or CDKN2B-AS1 loci (1q24.1 or 9p21.3, respectively) [14] , with POAG using Caucasian subjects. However, it appears that a controversy still exists as to determining the authentic variants associating with POAG, even within the same ethnicity of European descent [14, 15] . We previously reported a GWAS and the subsequent follow-up study focused on the high-ranked variants identified in the initial population using in total of 1,575 Japanese subjects [10] . We identified six variants located in three loci, 1q43, 10p12.31, and 12q21.31, on the chromosome which were modestly associated with POAG, although the association results were not reproducible with a different population of different ethnicities [13, 16, 17] . In that previous study, we combined the patients from both HPG and NPG subtypes as a single case group in order to increase the statistical power [10] . Therefore, the genetic loci that were identified are most likely to be components of the molecular mechanism shared by both subtypes. Another Japanese group performed a GWAS [12] and discovered variants in SRBD1 on 2p21 and ELOVL5 on 6p12.1 that were associated with NPG (in that study, NPG was referred to as ''normal tension glaucoma'') [18] . However, since the findings in that study were derived from a single population, replication studies to support those findings still need to be conducted.
In this present study, we examined two independent GWAS data sets, and then performed a meta-analysis by combining the data sets in order to discover authentic genetic markers for POAG, HPG, or NPG. We succeeded in identifying some significant variants in the CDKN2B-AS1 locus using a Japanese population, as that locus has also been shown to be significantly associated with POAG in Caucasians [14] . Moreover, the variants that passed the Bonferroni correction seemed to be associated with POAG and POAG/NPG, thus suggesting that the locus potentially affects the NPG, rather than the HPG, subtype of POAG. Although we still need to confirm our findings by use of a larger HPG cohort in order to increase the statistical power, the variants identified in this study could be associated specifically with the vulnerability of the optic nerve to IOP, thus enabling us to investigate the etiology of glaucoma.
We first performed a GWAS in 833 POAG patients and 686 control subjects (hereafter referred to as ''Present GWAS''; Figure 1A ) who were selected and divided into case and control groups based on our strict diagnosis criteria. After genotyping, 653,519 SNPs passed the quality controls and were used for the association study ( Figure 1A) . In a quantile-quantile (Q-Q) plot ( Figure S1A ), the genomic inflation factor (l) showed 1.021, suggesting that the population substructure should not have any substantial effects on the association analysis. Under these conditions, we obtained 8 genome-wide significant SNPs (Table  S1 ) that passed the Bonferroni correction threshold (0.05/ 653,519 = 7.65610 28 ), of which 5 SNPs were located in CDKN2B-AS1 on chromosome 9p21.3 ( Figure 2 ). A few SNPs that showed modest to strong association were distributed across a single linkage disequilibrium (LD) block located in the locus ( Figure S2 ). The remaining genome-wide significant SNPs identified from different chromosomes turned out to be genotyping errors due to the poor 2D clusters ( Figure S3B-D) .
Next, we updated the case and control groups of the previous GWAS population [10] with the latest clinical information and ended up with 411 POAG patients and 289 controls (hereafter referred to as ''Previous GWAS''; Figure 1A ). One SNP (rs8181047) of 5 genome-wide significant SNPs in CDKN2B-AS1 identified from the Present GWAS was not able to be assessed in the Previous GWAS because a DNA probe of that particular SNP was not designed for the Affymetrix 500 K platform. Consequently, we combined the two sets of GWAS genotype data for the remaining 4 SNPs by use of the Mantel-Haenszel test [19] , and the level of significance of all 4 SNPs increased from P,5.2610 29 to P,5.8610 210 (Table 1 ). These 4 SNPs were highly correlated with one another (r 2 .0.98), and were considered to have a similar effect for POAG based on the conditional analysis. These results suggested that it was effective to combine the data since the population stratification between the two data sets seemed to be ignorable, which was also supported by the results of the heterogeneity test. Moreover, we assessed the confounding effects for these SNPs with respect to age and gender, and none were found to be statistically significant (Table S2) , thus suggesting that the obtained association results were specific to the case-control comparison. In addition, the two sets of GWAS data for 40 SNPs surrounding the significant 4 SNPs in the 9p21.3 locus were also combined and analyzed (Table S3, Table S4 ). Since these 44 SNPs, in total, were genotyped in both the Present and Previous GWAS, and all of the SNPs passed the genotyping quality controls, these SNPs should prove useful in providing a broader overview of the significance of the locus. The results showed that the significance of combined SNPs was generally high between 22.0-22.1 Mb ( Figure 3A ). Within the particular locus, there seemed to be two distinct LD blocks; one in the side including genome-wide significant SNPs (''LD Block 1'' in Figure 3D ) and one in the other side with modestly associated SNPs (''LD Block 2'' in Figure 3D ). For the subtype analyses ( Figure 1B) , the POAG patients from both the Present and Previous GWAS populations were divided into two subtypes based on the clinical record of IOP measurement: 1) POAG patients with HPG (IOP$22 mmHg) and 2) those with NPG (i.e., patients who consistently showed an IOP of less than 22 mmHg) [3] . The number of subjects in each subtype was found to be 330 HPG and 503 NPG patients and 215 HPG and 196 NPG patients in the Present and Previous GWAS, respectively ( Figure 1B) . When we performed the analysis separately using the Present GWAS data set, the Q-Q plots of HPG vs control and NPG vs control appeared to be quite different from one another ( Figure S1B , C). In particular, the HPG results indicated non-deviated Q-Q plots ( Figure S1B ). In fact, although none of the SNPs were genome-wide significant for HPG ( Figure  S4A ), we obtained 4 genome-wide significant SNPs that passed the Bonferroni correction threshold for NPG (Table S1, Figure S4B ). As for NPG, the significance level of all 4 SNPs also increased when the two data sets were combined ( Figure 3C , Table S3 , Table S4 ), however, the significance was still far from the Bonferroni threshold of significance in relation to HPG ( Figure 3B , Table S3, Table S4) . Although the significance level of SNPs residing in LD Block 2 showed slight differences among the different subtypes, the level in LD Block 1 seemed to be determining the difference of significance between the two subtypes, thus suggesting that the variants on LD Block 1 are closely associated with the susceptibility to glaucoma in the 9p21.3 locus ( Figure 3D) . Surprisingly, all of the significant SNPs from NPG overlapped with those obtained from POAG (Table S1) . However, when we performed the heterogeneity test between the HPG and NPG groups, the results were not significant (P = 0.21-0.25, Table S3 ), suggesting that the significant differences between the two subtypes could be due to the small sample size of the HPG group. Although we need to confirm the above results using a larger HPG cohort, these results suggested that the significant SNPs identified in CDKN2B-AS1 on the 9p21.3 locus probably serve as genetic markers of glaucoma, which could be useful for investigating the etiology of glaucoma with respect to the vulnerability of the optic nerve to IOP.
In this study, we successfully identified genetic markers in CDKN2B-AS1 strongly associated with POAG and POAG/NPG, but not with HPG, by analyzing two GWAS data sets using independent study populations totaling 2,219 Japanese subjects. For the case-control study of the Present GWAS, we excluded the samples without hesitation that didn't meet our strict quality control (see Supporting Information S1). Since we succeeded in excluding the samples possessing a fair amount of no-call or missed-call genotype data, our more stringent filters certainly improved the actual genotyping results for the association studies performed later. Thus, we were able to obtain an increased level of significance without any substructure effects of the two populations based on the definite diagnosis when combining the genotyping results of the Present and Previous GWAS data sets (Figure 3 ).
Using our polished data sets derived from distinct case and control subjects (see Methods and Supporting Information S1), we succeeded in identifying a cluster of genome-wide significant SNPs associated with POAG in CDKN2B-AS1, a non-coding gene with an unknown function, on chromosome 9p21.3 (Figure 2A, B) . Several variants in chromosome 9p21 have been found to be associated with a variety of common diseases, and 9p21 was initially identified as a locus for coronary artery disease (CAD) [20] [21] [22] [23] and type-2 diabetes [24] [25] [26] . However, little is known about the biological meanings underlying the locus, as 9p21 is a ''gene desert'' locus and most of the variants identified were noncoding. Recently, Harismendy et al. [27] reported that they have identified 33 enhancers in 9p21, some of which being within CDKN2B-AS1, and it turned out to be the second densest gene desert for predicted enhancers, thus suggesting the regulatory role of sequences residing within non-coding loci. They finally determined a few adjacent, as well as distant (.45 kb), target gene regions relevant to CAD biology physically interacting with the enhancer by 3D-DSL (also known as ''4C''), a chromatin conformation capture technology, in human vascular endothelial cells. Overall, their study has provided an excellent example of a solution to link the unknown meanings of statistical association to a biological function. Since the POAG variants were also identified in CDKN2B-AS1, the variants would probably affect the expression level of the downstream genes CDKN2A and CDKN2B. In fact, Burdon et al. reported up-regulated CDKN2A and CDKN2B expression in response to the elevated IOP [14] , suggesting the involvement of the locus in molecular pathways leading to glaucoma development. Moreover, the possibility that the variants would also affect the distant unidentified target genes in the context of the complex etiology of glaucoma, as well as the nature of the identified locus [27] , cannot be ruled out.
To date, several institutions have attempted to discover variants for glaucoma by conducting a GWAS, and a few published studies have reported the association of particular loci with POAG using Caucasian subjects (Table S5) . Thorleifsson et al. reported the association of common variants near CAV1/CAV2 on 7q31.1 using a population of European ancestry [13] . In contrast, Kuehn et al. reported that they failed to replicate their results in a United States cohort [15] . Moreover, meta-analysis of the association results derived from several institutions of Northern Europe, including the data from Thorleifsson et al. [13] , identified a few new loci associated with POAG, including the CDKN2B-AS1 locus on 9p21.3 [17] . Recently, the new loci at TMCO1 on 1q24.1 and CDKN2B-AS1 were reported to be associated with Australian populations [14] . Thus far, only the association of the CDKN2B-AS1 locus was replicated, even within the same ethnicity of European descent. Interestingly, Thorleifsson et al. also showed a modest association with the CAV1/CAV2 locus using Chinese subjects [13] , although the allele frequency of the particular variant in Asian subjects was quite low when compared with that in Caucasians, suggesting etiological differences due to the genetic background. In fact, according to the results of our Present GWAS for POAG, we were unable to replicate the association with the CAV1/CAV2 and TMCO1 loci ( Figure S5F , G, Table S5 ). Moreover, in the Present GWAS, we were unable to replicate the association results of the 6 SNPs identified in the previous study [10] (Table S5) , even though the populations were of the same ethnic background, thus suggesting that we still need to discover authentic variants, irrespective of the difference in ethnicities, to elucidate the complex etiology of glaucoma. Consequently, it should be noted that the variants identified in the CDKN2B-AS1 locus in this study using a Japanese population seemed to be shared with the Caucasian subjects (Table S5) , thus showing that we have successfully obtained one of the authentic variants for POAG that is not ethnicity related.
In this study, we also subdivided the POAG subjects into two subtypes based on the IOP level measurements in an attempt to discover subtype-specific variants, which would be useful for investigating the different mechanism of each pathogenesis. Since the clinical states of both subtypes overlap almost completely, they are usually categorized as a single disease. However, as a unique epidemiological distribution compared with other ethnic groups, ,92% of the Japanese POAG patients fall into the NPG category, as determined by the Tajimi study [4] , a robust epidemiology study. In fact, another Japanese group performed a GWAS focused on discovering NPG-specific variants [12] . They reported that the SNPs in SRBD1 on 7q31.1 and ELOVL5 on 6p12.1 were associated with NPG, although our group was unable to replicate those results ( Figure S5D, E) . On the contrary, we obtained an unexpected result that the variants associated with NPG were completely identical to those associated with POAG in CDKN2B-AS1 identified in this study (Table S1 ). In contrast, none of the variants were significant for HPG ( Figure 3B ). Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG cases according to the heterogeneity test (P = 0.21-0.25; Table S3 ), the results suggested that the genetic loci identified are most likely components of the molecular mechanism specific for NPG. It has been reported that there are differences in vulnerability of the optic nerve to IOP between HPG and NPG [18] . It has also been theorized that CDKN2B-AS1 affects the susceptibility of the optic nerve [14, 28] . Since the variants identified in this study seemed to be shared among different ethnicities in functional aspects as well, the other variants should be contributing to the unique epidemiology of NPG in Japanese. By continuing to build upon the detailed investigation, such as obtaining in-depth sequencing data of the non-coding 9p21 locus, it might be possible to reveal not only the molecular mechanism of glaucoma pathogenesis but also the genetic diversity that resides within the locus among different ethnic backgrounds.
This study was approved by the Institutional Review Board of Kyoto Prefectural University of Medicine and all procedures were conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent after an explanation of the nature and possible consequences of the study.
All participants were interviewed to determine their familial history of glaucoma and other ocular or general diseases. A total of 2,126 Japanese participants, including POAG patients, healthy volunteers, and patients with other ocular diseases, were recruited between March 2005 and December 2010 at the University Hospital of Kyoto Prefectural University of Medicine (Kyoto, Japan) to give peripheral blood samples for this study. A third person who was blind to both the blood sampling and genotyping assigned an anonymous code to each blood sample. Genomic DNA used for the genotyping experiments was isolated from the blood, and Epstein-Barr virus (EBV)-transformed lymphocytes were prepared from the remaining blood as previously described [29] to serve as the future resource of genomic DNA. POAG patients and controls suitable for this study were precisely selected based on the strict diagnosis as previously described [10] . In particular, subtype selection was performed by dividing POAG into two categories according to peak IOP without treatment; POAG/High Pressure Glaucoma (POAG/HPG, defined as $22 mmHg) and POAG/Normal Pressure Glaucoma (POAG/ NPG) [3] . All of the diagnostic procedures, including case-control selection and subtype classification, were performed by three ophthalmologists (YI, MU, and KM) from a single institution. The age and female/male ratio of all of the subjects used in the Present and Previous GWAS are shown in Table S6 . To examine the possible confounding effects of age and gender, we assessed the correlations between the values and the genotype data from the case and control samples by one-way ANOVA or chi-square test (Table S2) .
First, 906,600 SNPs were genotyped for 2,126 Japanese subjects by Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. As for the Present GWAS population, 839 POAG patients and 708 controls were selected after performing the quality control (QC) as described (Supporting Information S1). To exclude the potential inclusion of genetically related subjects into the population, identity-by-descent (IBD) estimates were performed for all possible combinations by PLINK v1.07 (http://pngu.mgh.harvard.edu/ ,purcell/plink/). In total, 6 POAG patients and 22 controls were assumed to be in first-degree relationships or in relationships with a few more-distant relatives, and were thus excluded from the Present GWAS population. Finally, the Present GWAS population ended up with 833 POAG patients, comprised of 330 HPG and 503 NPG patients, and 686 controls. Population stratification for the present GWAS population was examined by principal component analysis using EIGENSTRAT software v3.0 (http:// genepath.med.harvard.edu/,reich/Software.htm). As for the reference genomic population, the four HapMap populations (CEU, YRI, JPT, and CHB) were simultaneously applied to EIGENSTRAT. The generated cluster plots indicated that our POAG and control population was genetically clustered within the JPT population, and there was no outlier sample ( Figure S6 ). We performed SNP quality control for the population on the autosomal SNPs based on the following QC filters: (i) call rate per SNPs in case and control samples $95%, (ii) minor allele frequency (MAF) in case and control samples $1%, and (iii) Hardy-Weinberg equilibrium (HWE) in control samples P$0.001. Consequently, we analyzed the remaining 653,519 SNPs for the Present GWAS population.
Since 6 years had passed since the patients and healthy volunteers who participated in our Previous GWAS [10] were first recruited, we updated the case (n = 418) and control (n = 300) groups based on the latest clinical information. In total, 7 samples from the case group and 11 samples from the healthy control group were removed. The population for the reanalysis finally ended up with 411 POAG patients, comprised of 215 HPG and 196 NPG patients, and 289 controls. The Previous GWAS data was obtained by GeneChipH Human Mapping 500 K Array platform (Affymetrix) containing 500,568 SNPs. The quality control for the reanalysis was performed by using the same QC filters as used in the Present GWAS. Genotype data derived from the particular locus for combining with the Present GWAS data was extracted from these filtered SNPs.
To manage and analyze all of the genotype data, we used our in-house Genoika Server System (SASA Plus Co., Ltd., Fukuoka, Japan), which was well improved from the system used in the Previous GWAS [10, 30] by the same system engineers. In addition, to manage the Previous GWAS data effectively, the Labo Server System (World Fusion Co., Ltd.) was used simultaneously. The Genoika Server System comes with PLINK v1.07 (http://pngu.mgh.harvard.edu/,purcell/plink/), the R program v 2.9.2 and 2.10.1 (http://www.r-project.org/), EIGEN-STRAT software v3.0 (http://genepath.med.harvard.edu/ ,reich/Software.htm), and Haploview 4.2 (http://www.broad institute.org/scientific-community/science/programs/medical-andpopulation-genetics/haploview/haploview) built in, and all the analyses were performed by use of this system. In addition, Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA) was used for preparing the data sets and statistical analysis. The frequency of alleles in the case and control samples was compared by use of the basic allele test. The odds ratio (OR) and the upper and lower limit of the 95% confidence interval (CI) of each SNP were calculated for the allele possessing a higher frequency in the case samples than in the control samples. The HWE was evaluated by the chi-square test. Q-Q plots were generated by ranking the observed values from minimum to maximum and plotting them against their expected chisquare values using the ''snpMatrix'' package ver 1.14.6 in the R program (http://www.r-project.org/). We applied the Mantel-Haenszel test [19] to combine the data derived from the two data sets in order to reduce potential negative effects arising from the biases in age and female/male ratio among the populations (Table  S6 ). Figure S1 Q-Q plots for the Present GWAS. Quantilequantile (Q-Q) plots for the Present GWAS of POAG (A), HPG (B), and NPG (C) were generated by ranking the observed chisquare values from minimum to maximum and plotting them against their expected values. Genomic inflation factors (l) are also shown. These plots were created using the R-package snpMatrix. (PDF) Figure S2 LD plots in the 9p21 locus. LD plots were generated from the Present GWAS data. The SNPs applied to these plots and the span of the region are the same as shown in Figure 2B . Upper and lower plots indicate the value of pairwise D9 and r2, respectively. The positions of 5 SNPs, which passed the Bonferroni correction threshold in the Present GWAS, are drawn in vertical dashed lines. These LD plots were generated using Haploview v4. Figure S6 Population stratification analysis. Population stratification analysis by EIGENSTRAT in the Present GWAS data set. POAG samples separated with HPG and NPG groups were applied. The version of HapMap reference data is release22 (Build36). Since Control, NPG, HPG, and HapMap JPT samples were tightly overlapped, only the plots for NPG (yellow cross) are visible. (PDF)
Table S1 Genome-wide significant SNPs that passed the Bonferroni correction threshold in the Present GWAS. (PDF) Supporting Information S1 Quality control for genotyping. (PDF) Interleukin-18 expression and the response to treatment in patients with psoriasis INTRODUCTION: The aim of the study was to demonstrate Interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions. MATERIAL AND METHODS: This study included 16 patients of different clinical subtypes of psoriasis. IL-18 gene expression analysis was performed using real-time quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of two months' treatment course. The treatment was in the form of topical steroids or oral systemic methotrexate. RESULTS: Of all 16 studied patients significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (P = 0.001 and 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (P = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and P = 0.01) and clinical severity of psoriasis (r = 0.72 and P = 0.001). CONCLUSIONS: Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept which views psoriasis as a T-cell-mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients. Psoriasis is a chronic inflammatory condition which is the result of persistent stimulation of T cells by antigens of epidermal origin [1] . This persistent stimulation of T cells is the result of an interaction between T cells, antigen-presenting cells, i.e. Langerhans cells, and antigens, and comprises the following steps: primary signals (signal 1) including T-cell receptor stimulation by peptide antigen, co-stimulation (so-called accessory signals) (signal 2), and T helper type 1 (Th 1) differentiation and proliferation [2] .
In 1986, it was proposed that cytokines released by activated T-lymphocytes initiate and maintain the psoriatic process by stimulating keratinocyte proliferation [3] . Since that time it has become clear that, upon stimulation, keratinocytes are also able to secrete an array of different cytokines with a variety of functional effects on both themselves and other cell types, including T-lymphocytes [4] .
Interleukin-18 (IL-18) is related to the IL-1 family in terms of both structure and function. At the receptor level, the activity of IL-18 takes place through a signalling chain of a putative IL-18 receptor (IL-18R) complex [5] .
Interleukin-18 was first described as an interferon-γ (IFN-γ) inducing factor. In general, IL-18 induction of IFN-γ is similar to that of IL-12, when it is a sole cytokine. IL-18 induces low levels of IFNγ, but in the presence of co-stimulants, IFN-γ production is greatly enhanced [6] . However, because antibodies to IL-18 also reduced the hepatotoxicity of endotoxaemia, IL-18 was considered to possess other biological properties beyond that of inducing IFN-γ [7] . IL-18 activates T cells to synthesize IL-2, GM-CSF, and TNF-α. There are also reports that IL-18 suppresses the production of IL-10 and does not induce the production of IL-1Ra. In general, the ability of IL-18 to induce different cytokines depends on the cellular targets [8] . Flisiak et al. confirmed an association between plasma IL-18 concentration and psoriasis severity. Moreover, it was shown that combined measurement of IL-18 and TGF-beta1 in plasma can be considered as a possible biomarker of psoriasis activity [9] . Kato et al. indicated that a single nucleotide polymorphism (SNP) in the promoter of the interleukin-18 gene is associated with the presence of psoriasis, but not atopic dermatitis. This suggests that the SNP is associated with susceptibility to psoriasis vulgaris, presumably by affecting the production of IL-18 [10] .
A few studies have analysed IL-18 expression in psoriasis vulgaris [11] [12] [13] [14] . Much less information is available on the ability of different therapeutic interventions to modulate ongoing changes in expression in chronic disorders, especially in a human disease like psoriasis.
The aim of this study was to demonstrate expression of IL-18 in keratinocytes from psoriatic lesions in comparison to keratinocytes from nonlesional skin and to study the change of IL-18 expression after therapeutic interventions.
This study included 16 patients. The patients were selected from the outpatient clinic of Benha University Hospital, Al-Haud Al-Marsoud and The National Research Center, Cairo, Egypt. The patients included 4 (25%) females and 12 (75%) males, and their ages ranged from 21 to 62 years with a mean of 40.87 ±13.17 years. They had different clinical subtypes of psoriasis and exhibited various degrees of severity. History of present illness included the onset, course, duration, previous treatment and date of last topical application or systemic therapy. Inclusion criteria: those included in the study were adult psoriatic patients ≥ 18 years who should not have received a topical therapeutic modality apart from petrolatum for the last four weeks or systemic therapy for the last eight weeks.
Presence or absence of any coexisting noncutaneous conditions was established. Three patients were hypertensive, three had psoriatic arthropathy, one had hepatitis C virus (HCV) with anaemia of chronic disorder type and one had diabetes mellitus with hypertension. A positive family history was encountered only in one case. Two patients had upper respiratory tract infections; cold exposure worsened five patients' clinical presentation; sweating aggravated one patient's condition; three patients had psychogenic factors precipitating their cases. Only five of the studied patients were not affected by predisposing factors (31.3%).
The psoriasis area and severity index (PASI) score sheet was adopted according to Fredriksson and Pettersson and Marks et al. [15, 16] for all patients as an indication of degree of severity. In mild cases, the extent of the disease does not exceed 10, while in severe cases, the extent of the disease is more than 30 based on the PASI score [17] . Informed consent was obtained after complete description of the study from each participant, according to the guidelines of the local ethical committee of the National Research Center.
Two biopsies were taken from each patient in the first set. The first biopsy was taken from the lesional psoriatic skin and the second from nonlesional skin. All patients were subjected to continuous treatment (in the form of topical steroids and oral systemic methotrexate) and followed up for 2 months. Six patients with moderate to severe degrees of psoriasis and two with palmoplantar subtype of psoriasis (who did not respond to topical steroids) received oral systemic methotrexate. The dosage used was: 15 mg per week in three divided doses taken at 12-hour intervals during a 24-hour period with folate supplementation. Patients receiving systemic methotrexate were subjected to monitoring liver blood tests monthly. Eight patients with mild to moderate degrees of psoriasis were maintained on topical steroid creams and ointments in the form of betamethasone dipropionate, twice daily. At the end of the course of two months, a third psoriatic lesional skin biopsy was taken from all patients.
To prevent degradation by intracellular RNases, tissues were embedded soon after excision in RNA stabilizing reagent RNAlater provided from QIAGEN worldwide companies according to the manufacturer's instructions to be stored at -70°C before sample processing. Frozen tissues were not allowed to thaw during handling to be ready for Interleukin-18 expression and the response to treatment in patients with psoriasis quantitative PCR determination of IL-18. Each biopsy was weighed before processing. Biopsies weighed up to 20 mg. Efficient disruption and homogenization of the starting material was performed using a rotor-stator homogenizer.
Total RNA was extracted using QIA amp RNA extraction kit provided from QIAGEN worldwide companies. The concentration and purity of RNA were determined by measuring its absorbance at 260 nm (A 260 ) and 280 nm (A280) in a UV visible spectrophotometer. Total RNA (2 μg) was reversetranscribed to cDNA with High-Capacity cDNA Archive kit provided from Applied Biosystems. cDNAs were stored at -20°C until real-time quantitative PCR was performed.
Real-time quantitative PCR was performed using Applied Biosystems Perkin Elmer 7300 sequence detection system. Primers were IL-18 sense primer 5'-AGG AAT AAA GAT GGC TGC TGA AC-3' and antisense primer 5'-GCT CAC CAC AAC CTC TAC CTC C-3'. Each PCR reaction (in a 50 uL volume) contained 1 x of TaqMan universal PCR master mix, (2 x) 50 to 900 nM of forward primer, 50 to 900 nM of reverse primer, 250 nM of TaqMan probe labelled with 6-carboxyfluorescein (FAM) and 10 to 100 ng of cDNA. The amplification protocol was 2 min at 50°C, 10 min at 95°C, then 40 cycles for 15 sec at 95°C and 1 min at 60°C. Relative gene expression was determined for each sample. Amplification of the gene for glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a constitutively expressed housekeeping gene, was performed on all samples. All genes were subsequently normalized against GAPDH levels. In this study mRNA levels were expressed in picograms/microlitre (pg/μl).
All data were collected from the patient charts and entered into a computerized spreadsheet. The fit of the data to the normal distribution was tested with the Kolmogorov-Smirnov test. Since the distribution of the data was significantly different from normal, nonparametric statistics were further used. Comparisons were made using Mann-Whitney U, Kruskal Wallis and Wilcoxon signed ranks tests between variables adjusted for appropriate covariates, and Spearman rank correlation coefficients were calculated between the assessed variables. The null hypothesis was rejected with a two-sided P value of < 0.05. All analyses were performed with SPSS 11.0 for Macintosh (Statistical Package for the Social Sciences. SPSS Inc., Chicago, IL, U.S.A.).
Demographic and behavioural characteristics of included patients are presented in Table I . PASI score used to evaluate the severity of psoriasis varied from 1.4 to 39 with a mean of 14.96 ±11.82. According to Ramsay and Lawrence [17] , the PASI score grades in the different subtypes of psoriasis are demonstrated in Table II .
All patients had detectable levels of IL-18 mRNA in the uninvolved skin. Table 3 shows the range, mean ± SD and median levels of cytokine gene expression in each skin biopsy at the different times before and after receiving medication whether topical or systemic. Comparisons between median IL-18 expression levels with percentiles in all studied patients are presented in Figure 1 . Significantly lower IL-18 expression in uninvolved skin than lesional skin before treatment (P = 0.001) and lesional skin after treatment (P = 0.002) was observed in all studied patients as one group. The IL-18 expression in the psoriatic skin lesions after treatment was significantly lower than lesional skin before treatment (P = 0.023).
IL-18 mean expression levels in non-lesional and lesional skin (before and after treatment) in the different subtypes of psoriasis are shown in Figure 2 . The highest expression level was noted in plaque and nails clinical subtype of psoriasis.
Among patients receiving topical steroids significantly lower IL-18 expression was detected in non-lesional skin than lesional skin before treatment (P = 0.012) and after treatment (P = 0.012). No statistical significance was obtained when comparing the change of expression before and after topical treatment (P = 0.069).
Individual variations of the IL-18 expression levels in non-lesional and lesional skin before and significantly lower IL-18 expression was detected in non lesional skin than lesional skin before treatment (P = 0.018). The change of expression levels in lesional skin after treatment was not statistically significant when compared to non lesional skin (P = 0.069) and lesional skin before treatment (P = 0. 12) after receiving topical steroids are presented in Figure 3 .
In patients receiving methotrexate, significantly lower IL-18 expression was detected in non-lesional skin than lesional skin before treatment (P = 0.018). The change of expression levels in lesional skin after treatment was not statistically significant when compared to nonlesional skin (P = 0.069) and lesional skin before treatment (P = 0.12).
Individual variations of the IL-18 expression levels in non-lesional and lesional skin before and after receiving systemic methotrexate are shown in Figure 4 .
In the correlation between IL-18 expression and different demographic and behavioural characteristics of the studied patients as one group, a significant correlation was obtained between IL-18 expression levels in psoriatic lesions and the severity of psoriasis expressed by PASI score (r = 0.72 and P = 0.001) and disease duration (r = 0.40 and P = 0.01). No significant correlation was revealed between IL-18 expression levels in psoriatic lesions and patients' age, smoking status, predisposing factors or coexisting diseases.
In the present study our patients with psoriasis showed significantly increased IL-18 expression in keratinocytes from psoriatic lesions before and after receiving medication whether topical or systemic in comparison to keratinocytes from non-lesional skin. This supports the concept viewing psoriasis as a T-cell-mediated autoimmune disease. Krueger [18] reported, in his study on a large series of skin biopsies, increased protein expression in lesional as opposed to non-lesional skin samples. Other investigators stated that lesional skin might be the source of the elevated plasma levels of IL-18 [19] . In the present work quantitative PCR using mRNA taken from uninvolved skin revealed low but detectable levels of IL-18 mRNA expression. This finding is consistent with previous studies showing that human keratinocytes are capable of synthesizing low levels of IL-18 mRNA even under non-stimulated conditions [20] . Chang et al. [21] found that genes specifically modulated in uninvolved skin play a major role in triggering disease progression, and in being the first candidate for early disease diagnosis or development of new therapies. This finding could allow selection of the treatment regimen for individual patients.
In the present study the IL-18 expression in patients receiving topical steroids was significantly lower in non-lesional skin than lesional skin before and after treatment. After 2 months of treatment, IL-18 expression levels returned back to low values but not significantly lower than the lesional skin levels of expression before treatment. In patients receiving methotrexate non-lesional skin expression was significantly lower than lesional skin before treatment. Also the expression levels decreased after treatment but still not statistically significantly when compared to lesional skin before treatment.
Otkjaer et al. [22] determined IL-19 and IL-20 in nonlesional and lesional psoriatic skin after 28 days of treatment using topical calcipotriol ointment for mild cases and oral cyclosporine for moderate and severe cases. They found that in patients treated with calcipotriol, IL-19 and IL-20 mRNA expression levels in lesional psoriatic skin were reduced after 14 days, but the reduction became statistically significant only after 28 days. So the timing for mRNA expression analysis could affect the degree of significance. This is further supported by studies demonstrating a decrease of IL-18 levels after narrowband UVB therapy together with other parameters' characteristics for the T helper 1 (Th1) response [23] . Apart from stimulating the Th1 response, IL-18 can regulate the Th2 response depending on the local cytokine network. IL-12 enhances IFN-γ production induced by IL-18, whereas IL-18 alone induces IL-4 and IL-13 production [24] .
Classic systemic treatments for psoriasis have not fully met the needs of patients for permanent improvement. Antibody-based or fusion proteinbased selective targeting of key mediators of inflammation has been added to the treatment approaches for psoriasis. Kong Our study demonstrated that IL-18 expression in the psoriatic lesions before treatment was significantly correlated with the severity of psoriasis and disease duration. No correlation was noted between IL-18 expression and patients' age, smoking status, predisposing factors or coexisting diseases. Flisiak et al. [9] found a significant correlation between plasma IL-18 levels and PASI values. But they did not find any correlation between IL-18 concentration in scales and PASI score. Trinquet et al. [28] studied the influence of disease duration on gene expression profiles in psoriasis. They did not reveal any correlation. However, Craven et al. [29] studied IL-10 in early and late onset psoriasis and confirmed that IL-10 expression was correlated significantly with disease duration. Sampogna et al. [30] studied 380 patients with psoriasis and did not find any significant difference between older and younger patients regarding IL-18 levels. On the other hand, Chen et al. [31] found a statistically significant correlation between IL-18 expression levels and age, with a P value of 0.015.
In conclusion, based on the increased IL-18 expression levels in psoriatic skin lesions relative to uninvolved skin, this cytokine appears to be crucial in the development of the active psoriatic lesion itself, where it is produced locally by a step in the evolution of the psoriatic lesion. This could be supported by its correlation with clinical severity and disease duration, and reduction of its expression after treatment. The decreased expression after treatment was not to an extent indicating that the skin had returned to normal. The small size of the study population and the heterogeneous group of patients participating in the study preclude the drawing of firm conclusions. Future studies on genes involved and array-based technologies should allow confirmation of the results obtained. Also further studies are recommended to try other lines of treatment for psoriasis. One of these lines of therapy is to try r-h IL-18 BP to neutralize IL-18 activity on a wide scale that will ultimately lead to improved outcomes for patients. Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. Tick-borne encephalitis virus (TBEV) is a human pathogen that causes about 16 000 human cases of tick-borne encephalitis (TBE) across Europe and Asia annually (1) (2) (3) . Taxonomically, TBEV is a species within the mammalian tick-borne flaviviruses (mTBFV). Together with the seabird tick-borne flavivirus group (sTBFV), they comprise one ecological group of tick-borne flaviviruses (TBFV) within the genus Flavivirus, family Flaviviridae. Two other ecological groups within the genus Flavivirus are the mosquito-borne flaviviruses (MBFV) and flaviviruses with no-known vector (NKV) (4) . A fourth group including Kamiti River virus (KRV) (5) , cell fusion agent virus (CFAV) (6) and Culex flavivirus (CuFV) (7) have been isolated only from mosquitoes with no demonstrated capacity to replicate in mammals and are under consideration by the ICTV Committee for classification as 'probably arthropod-borne viruses' (PABV).
Flavivirus virions are $50-nm particles with a nucleocapsid composed of capsid (C) protein surrounding a positive-sense single-stranded RNA genome of $11 kb. The capsid is enclosed in a lipid membrane within which the viral membrane (M) and envelope (E) proteins are embedded. The genome encodes a single polyprotein of approximately 3400 amino acids from which the three structural (C, M and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins are processed by cellular and viral proteases (8) .
Flavivirus genome replication involves synthesis of a negative-sense template strand by the RNA-dependent RNA polymerase (RdRp; NS5 pol ) from which additional genome-sense strands are transcribed. This process is controlled by numerous RNA-RNA and RNA-protein interactions determined by virus RNA sequence motifs and secondary structures, called cis-acting replication *To whom correspondence should be addressed. Tel: +44 0118 378 6368; Fax +44 (0)118 378 6537; Email: t.s.gritsun@reading.ac.uk elements (CRE), mapped to the 5 0 -and 3 0 -untranslated regions (UTR) that flank the single open reading frame (ORF) of the genome (9) (10) (11) (12) (13) (14) (15) .
The concept of promoter and enhancer function during replication has been introduced recently in relation to the flavivirus CREs (16) . The promoter has been identified as a complex of highly conserved interacting RNA structures recruited from the 5 0 -and 3 0 -UTR to assemble viral and cellular proteins into a functional RdRp complex. In evolutionary terms, the 3 0 -UTR of the TBFV group is formed by four conserved long imperfectly repeated sequences (LSRs), genetic remnants of which are revealed in the MBFV, NKV and PABV groups (17) . It has been proposed that the 5 0 -UTR may have evolved from a trans-terminal duplication of the archival flavivirus 3 0 -UTR (16) .
An additional complexity in flavivirus replication is the presence of replication enhancer elements (REEs) in the 3 0 -UTR that, while not obligatory for replication of laboratory-maintained viruses, are likely essential for virus circulation and transmission in nature (16, 18) . Engineered deletions or modifications of the REEs enable the recovery of viable viruses that are attenuated as a result of reduced RNA synthesis (10, (19) (20) (21) (22) . The cumulative effect of several REEs enhances the assembly of the RdRp complex and is probably critical to the survival of flaviviruses in nature (23) . The REEs identified for MBFV have become an important target for the development of a live attenuated vaccine for dengue virus (24, 25) .
The relatively compact nature of the flavivirus genome, together with constraints imposed by the need to replicate in vertebrate and invertebrate hosts, means that additional CRE sequences may be present in parts of the genome other than the non-coding regions. Indeed, RNA secondary structures have been predicted within the coding region of several flaviviruses (26) (27) (28) . Here, using bioinformatic and reverse genetic analysis we demonstrate that the capsid-encoding region of TBEV contains an REE which we designate SL6 (26, 27) . Phylogenetic evidence suggests that the MBFV group also contains at least a partial SL6-like structure, though it is absent in the NKV or PABV groups. The significance of these findings in the context of flavivirus evolution and adaptation to transmission is discussed.
Genbank accession numbers for sequences from all four groups of flaviviruses (TBFV, MBFV, NKV and PABV) used for in silico analysis are listed in Supplementary  Table S1 . RNA nucleotide sequences were aligned using ClustalX (29) and then edited manually. Nucleotide and dinucleotide scans and analysis of suppression of synonymous site variability (SSSV) were determined by mean pair-wise distance comparison at each codon within the ORF using the Simmonics 1.6 package (http://www.picornavirus.org/), as previously described (30) . SSSV was calculated only at aligned codon positions in which over 40% of sequence comparisons were synonymous and averaged over a sliding window of 21 codons; consequently, data point are only produced from codon 11.
RNA secondary structures were predicted using the MFold 3.2 and DINAMELT packages (http://mfold .bioinfo.rpi.edu/) with default settings (31, 32) . Phylogenetically conserved RNA structures were predicted using STRUCTURE_DIST (http://www. picornavirus.org/) to analyze connect files generated using hybrid-ss-min from the UNAFold suite of programs (32) .
Porcine embryo kidney cells (PS) have been used in experiments with TBEV strain Vasilchenko (Vs) and its infectious clone (pGGVs) as described previously (33) (34) (35) .
The construction of the infectious clone pGGVs for Vs virus and methods of mutagenesis have been described (34, 36) . Briefly, the pGGVs was subcloned into two plasmids; one, pGGVs 660 contained the first 660 nt of the virus genome and the second pGGVs 660-10927 included the remainder. Site-directed mutagenesis was accomplished by PCR (details of primers are available on request). Mutated PCR products were cloned into the pGGVs 660 between MluI and EcoRI sites followed by sequencing.
The recovery of virus from the two plasmids representing the infectious clone has been described previously (34) (35) (36) . Briefly, plasmid pGGVs 660 (or mutated derivatives) was digested with PspAI, dephosphorylated with Shrimp Alkaline Phosphatase (SAP; USB) and, after heatinactivation of SAP, digested with AgeI. Similarly, pGGVs 660-10927 was digested with NotI, dephosphorylated and then digested with AgeI. The excised linker DNA fragments from pGGVs 660 and pGGVs 660-10927 were removed using MicroSpin TM S-400 columns (Pharmacia Biotech) and ligated at the AgeI site generating full-length cDNA which was linearized with SmaI and used as a template for SP6 transcription (34) . In vitro-synthesized RNA was inoculated intracerebrally into suckling mice to recover the mutant viruses which were not passaged further prior to phenotype evaluation (35) . Recovered viruses were amplified by RT-PCR between nucleotides 1-940 (5 0 -UTR-C-prM region of the TBEV genome) and 10206-10927 (3 0 -UTR), and sequenced to validate the presence of the introduced mutations and to exclude extraneous mutations at the 5 0 -UTR and 3 0 -UTR (36) .
For growth curves, monolayers of PS cells in 96-well plates were infected with viruses at a multiplicity of infection (moi) of 1 PFU/cell, in quadruplicates. The inoculum (30 ml) was removed after 1 h, the monolayer washed thoroughly and replaced with 200 ml of media containing 2% serum. Media (10 ml) was collected at different time-points (8, 12, 16 and 24 h post-infection) and stored frozen at À70 C, before virus quantification by plaque assay. For cytopathic effect (cpe), PS cell monolayers were infected in 96-well plates at an moi of 1 PFU/cell, in quadruplicates, and stained with naphthalene black after 72 h.
Statistical analysis was performed on the data obtained from the virus growth curve studies and the evaluation of cpe in PS cells. For growth curves, the data were plotted to include the standard error of the mean (SEM) for each data set. At any given time point divergence by at least 2 SD from the mean, between wild-type and mutant viruses, was taken as significant. Measurement of cpe was done visually by three independent evaluators in a 'blind' manner. The cpe of viruses were estimated on a scale of 1-4 corresponding to 20-40, 40-60, 60-80 and 80-100% of monolayer destruction following microscopic examination. The interevaluator consistency error was verified using F-test which revealed no one evaluation was significantly different from that of the others.
Previous in silico studies have predicted a stable RNA structure designated SL6, in the C protein-encoding region, for a limited number of viruses within the mTBFV subgroup (16, (26) (27) (28) . Structural RNA elements were also revealed in the C region of some MBFV (28) although their homology to SL6 of TBFV had not been established. Here, we utilized a variety of independent structure prediction methods and a much larger sample of viral sequences to analyze whether or not the SL6-like structure was conserved throughout the entire genus Flavivirus.
In silico analysis of SL6 in the TBFV subgroup It was found that the overall folding of the first 333 nt of TBFV was highly conserved among several members of the mTBFV subgroup (16, (26) (27) (28) , with six stable SLs (enumerated 1-6 in Figure 1A ). This analysis was extended to investigate the conservation of SL6 in the larger group of distantly related mTBFV, sTBFV and KADV (37) . A nucleotide alignment of the C region was generated and optimized by the introduction of numerous gaps (Supplementary Figure S1A) ; it shows that divergent RFV, GGYV and KSIV (distant virus species of the mTBFV) maintained homology in the SL6 region. However, some nucleotide perturbations in the SL6 region were observed between mTBFV, sTBFV (MEAV, TYUV and SREV) and KADV proving that the region between the initiation codon and SL6 had evolved with frame shifts as we previously demonstrated (16) . We conducted MFold analysis to investigate the presence of SL6-like structures in the distantly related mTBFV (RFV, GGYV and KSIV), sTBFV and KADV groups ( Figure 1B) .
Despite sequence divergence, all viruses in the mTBFV group formed similar SL6-like structures when the 333 nt or a longer nucleotide region (up to 1000 nt) was used for MFold analysis (data not shown). The SL6-like folds contained a remarkably high number of co-variant and semi-covariant substitutions which maintained the general conformation across divergent viruses ( Figure 1B ). The minimum free energy dG of folding for SL6 varied between À32.3 and À17.2 kcal/mol with RFV and LIV/GGYV as extremes in this range. Although KADV had a shorter SL6 compared with other TBFVs, the energy of folding was À17.32 kcal/mol, within the range found for mTBFV.
In comparison to SL6 of the mTBFV, the SL6 of sTBFV was shorter and less stable, with a dG in the range À12.5 to À10.6 kcal/mol ( Figure 1B) . However, the SL6-like structures of sTBFVs were observed as elements of longer and branched RNA conformations (data not shown).
A smaller terminal loop was revealed in the KFDV/ AHFV and KSIV sequences resulting in the formation of the tetraloop U(GCCA)A ( Figure 1B ). The presence of U:A as a loop-closing base pair has been shown to decrease tetraloop stability considerably; in combination with some intraloop sequences this results in intermolecular tensions that prevent the folded tetraloop from achieving a global thermodynamic minimum (38, 39) . Thus, despite the MFold-mediated predictions, a tetraloop may not form for KFDV/AHFV and KSIV or at least not be sufficiently stable for biologically significant (RNA-RNA or RNA-protein) interactions.
The conservation of a UGCCAA hexanucleotide motif in the terminal loop of SL6 in all the divergent TBFVs was striking. Both TYUV and KADV showed one substitution in the hexanucleotide UGCCUA; TYUV has also lost the first nucleotide (Supplementary Figure S1A ).
In the minus-sense orientation, the conservation of an SL6-like structure was not as robust as in the positivesense. Although most of the TBFVs formed a structure in the minus-sense RNA, the number of hydrogen bonds, the lengths of the stems and free minimal energy of folding varied significantly even between closely related viruses (data not shown). Consequently, the formation of SL6 is likely to be biologically significant only in the positive-sense RNA.
Structure predictions correlated with evidence for SSSV in TBFV genomes ( Figure 2) . A remarkable drop in SSSV was observed in the SL6 region between positions 209 and 254 of the Vs sequence. The most extreme drop in variability was observed in a window centred on position 221 within the apical stem of SL6. The levels of SSSV within the remainder of the structural protein-encoding region (positions 295-2435) were higher than the upstream portion. Similarly, high levels of SSSV were observed across the non-structural portion of the genome between positions 322 and 2425 (data not shown).
We excluded the possibility that SSSV in the C-coding region was due to codon bias by analyzing nucleotide composition at each position within the codons. Comparison of SL6 between TBFV species. Numeration in brackets corresponds to SL6 numbered from the start codon of each virus (abbreviated in Table S1 ). Free energy dG values of folding are shown in kcal/mol. Covariant and semi-covariant substitutions are underlined on Vs virus.
No unusual variation of G/C or purine/pyrimidine composition was observed at the third codon position or at positions one or two of the codon (not shown). Likewise, we analyzed the dinucleotide composition at all three possible positions. Although there was a general under-representation of CpG and UpA, and overrepresentation of CpA and UpG, there was no correlation between areas of SSSV and regions of unusual dinucleotide frequencies (data not shown). These results indicated that evolutionary constraints restrict nucleotide variation within the 5 0 -coding regions of flavivirus genomes.
The phylogenetic conservation of thermodynamically stable RNA structures across all TBFV group ORFs was further analyzed using the program STRUCTURE_DIST ( Figure 2 ) (40) . This method quantifies phylogenetically concordant structures predicted using the widely accepted MFold or UNAFold algorithms, which can then be aligned and overlaid with SSSV results (31, 32) . Analysis of the entire ORF showed the most striking evidence for conserved base-pairing between the initiation codon at position 133 and position 318, after which a large drop in the frequency of base-paired nucleotides was observed. Within this region SL6 was predicted to be the most significant structure, with conserved pairing between 209 and 254 centred on a region with a conserved lack of base pairing between positions 228 to 236, representing the unpaired apical loop of SL6. The base-paired stem of SL6 contained conserved short single-stranded regions between positions 218-220 and nucleotides 244 and 245 consistent with the unpaired bulge, either side of the paired stem. This corresponds exactly to the position and structure of SL6 predicted by MFold (Figure 1 and Supplementary Figure S1A ).
An annotated nucleotide alignment of the C-coding region between TBFV and three MBFV groups (JEV, DENV and YFV) was constructed based on a previously presented alignment (16) but modified to include newly sequenced distantly related mTBFV, sTBFV and KADV isolates (Supplementary Figure S1A ). The C protein TBFV/MBFV alignment (available on request) was used to anchor the divergent nucleotide sequences. The annotations include the 5 0 -CYCL of MBFV, an 8-nt long cyclisation domain highly conserved between all MBFVs (16) . The 5 0 -CYCL interacts with a complementary sequence 3 0 -CYCL in the 3 0 -UTR to form a dsRNA panhandle, a vital element of the replication promoter that initiates viral RNA synthesis (16) . For the TBFV the 21-nt long 5 0 -CYCL is located in the 5 0 UTR (i.e. outside the alignment in Supplementary Figure S1A ; highlighted in Figure 1A ). The 5 0 -CYCL for MBFV mapped to the capsid gene and, among the TBFV, aligns optimally with a region that is identified only in TUYV (Supplementary Figure S1A) .
Nucleotide sequence homology was observed between the TBFVs and MBFVs particularly in the SL6 region of some JEV group viruses. For example, WNV was observed to share both the stem and loop sequences of TBFV SL6 (Supplementary Figure S1A) . It is of note that the SL6-like region of MBFV maps directly downstream of the highly conserved 5 0 CYCL (Supplementary Figure S1A) .
MFold was used to test the ability of these regions to form SL6-like structures within each MBFV group and the stem and loop elements of these SL6-like structures were superimposed onto the TBFV/MBFV alignment (Supplementary Figure S1A) . This comparison revealed that structures predicted within each MBFV group show not only sequence but also structural homology with SL6 of the TBFV group. This alignment was further annotated with RNA structures predicted by the ALIDOT-based analysis of entire flavivirus genomes of 11 000 nt (28), i.e. JE2, JE3 and JE4 for JEV; DV2 and DV3 for DENV and YF4 for YFV (Supplementary Figure S1A) . For all MBFVs with the exception of YFV the MFold predictions were somewhat different from those made using ALIDOT, most likely due to the shorter length of the regions (60-80 nt) used for the MFold analysis. Additional statistical methods, SSSV and STRUCTURE_DIST were used to assess the conservation of the SL6 homologous structures for each of the major MBFV groups (Figure 2) .
For the JEV group the mean SSSV between positions 117-358 (start codon at position 97) was consistent with ALIDOT-predicted RNA structures JE2, JE3 and JE4 (Supplementary Figure S1A) (28) . However, the SL6-like structure for the JEV group was clearly predicted by STRUCTURE_DIST analysis (brown box in Supplementary Figure S1A ), in accordance with MFold and alignment analysis.
For the DENV subgroup, a marked region of SSSV was revealed in the C-coding region between positions 155-257 (start codon at position 95) when compared with the rest of the structural coding region (Figure 2) , consistent with RNA structure DV3 previously predicted between nucleotides 163-183 (28) (Supplementary Figure S1A) . Both ALIDOT-predicted DV2 and DV4 (28) fall immediately either side of the region of maximum SSSV suggesting that they are less conserved than DV3 (Supplementary Figure S1A) . STRUCTURE_DIST also predicts the formation of the DV2 and DV3 but not the SL6-like structure (Supplementary Figure S1A and Figure 2 ). However, a truncated SL6-like structure was predicted to form in all DENV serotypes, albeit at a suboptimal energy level, when the SL6-like region was folded independently from neighboring regions that form more stable overlapping structures (Supplementary Figure  S1A) . Taken together, these data indicate that the DENV SL6-like structure was the least stable conformation among the MBFVs, potentially preventing its prediction by statistical approaches used here and elsewhere (28) . Despite this, the short-stem region of putative DENV SL6-like structures is highly conserved within the DENV group (DENV serotypes [1] [2] [3] [4] and also between DENV and JEV (Supplementary Figure S1A) suggesting that a linear or conformational signal at this location might have some functionality.
A similar restriction in SSSV was observed in the YFV C-coding region, with maximum SSSV corresponding to the ALIDOT-predicted structure YF4 ( Figure 2 ) (28). Among the MBFVs, only the YFV SL6-like structure was predicted by both thermodynamic and phylogenetic methods.
In summary, a proximally truncated SL6-like structure was predicted in all MBFV groups, although it was less stable in the DENV group, particularly the DENV3 serotype.
In silico prediction of SL6 in the NKV and PABV groups
In contrast to TBFV and MBFV, the NKV and PABV groups are not arboviruses and their replication is limited to only one natural host, i.e. rodents/bats (NKV) or mosquitoes (PABV). The high nucleotide divergence (Supplementary Figure S1B and S1C) and limited number of complete published sequences for members of the NKV and PABV groups precluded the use of both phylogenetic and thermodynamic approaches to RNA structure prediction. When MFold analysis was performed with available sequences, no thermodynamically stable RNA structures were observed in the region corresponding to the TBFV SL6 region. However, an SL6-like structure, with a similar apical loop CCAA motif was observed in KRV (PABV), upstream of the analogous TBFV SL6.
Strategy of mutagenesis on stem-loop 6. Initial design of mutations focused on synonymous codon positions. However, in all but a few instances, this was limited due to the distinctive sequence organization of the apical loop and base paired stem. The first and third codons of the conserved MPN tripeptide (loop region) are limited in respect of variation; M could not be changed and N has two possible silent variations both of which are outside the apical loop ( Figure 3) . Consequently, when mutating the terminal loop sequence UGCCAAAU, silent substitutions could only be introduced into the P codon. Similar difficulties were encountered with mutagenesis of the stem, in which the vast majority of possible synonymous and non-synonymous mutations resulted in no significant conformational changes. The MFold-simulated folding of numerous SL6-mutants revealed a high level of evolutionary 'protection' of SL6 against spontaneous single mutations (not shown) and provides additional evidence for the maintenance of SL6 functionality.
In order to resolve the difficulties with design of mutations, three different approaches were adopted ( Figure 3) . First, we introduced all possible silent substitutions, to target the conserved hexanucleotide and the stem (mutants C12, C13, C14, C16 and C33). Second, we introduced mutations (C10, C15, C17, C19 and C34) that mimicked 'natural' amino acid substitutions observed in this region of other mTBFV spp. Third, as a control for mutations that changed amino acids we also introduced compensatory substitutions encoding the same mutated amino acids while restoring the SL6 structure. Accordingly, mutations R32, S31, N 28, V 39, V 39 and P 28 were designed as controls for non-synonymous mutants C22, C23, C27 and C34 (Figure 3) .
The predicted impact of each substitution (Figure 3 ) on the secondary structure of SL6 is shown in Figure 4 . The plaque characteristics, cpe and growth dynamics of each mutant compared with those of original pGGVs virus (Table 1 and Figure 5 ). Single-step growth curves revealed differences of $1 log 10 between the mutants early after infection (12-16 h p.i.) which were reproducible and statistically significant ( Figure 5) .
To exclude the effect of spontaneous mutations in the 5 0 -and 3 0 -UTRs which contain TBFV promoter and enhancer elements (16) that might compensate for the effect of the SL6-mutations, rescued virus was not passaged prior to phenotype evaluation and key regions of the genome (1-940 and 10206-10927) were sequenced following recovery of each SL6-mutated virus. Only the intended substitutions were present, with no reversions or other compensatory mutations were observed. The effect of each mutation (reduction from large wild-type plaques of the pGGVs virus to medium, small or pin pointed) was scored if the SL6-mutated strain contained >90% of plaques with the altered morphology. The presence of a minor plaque population (between 1 and 10%) was considered as the inevitable result of the variation inherent R  1  4  5  3  3  3  6  2  4  5  2  6  3  2  8  2  2  9  0  2  |  |  |  |  |  |  |  |  TBEV  L40361  ACG CGU CAA UCC AGA GUC CAA AUG CCA AAU GGA CUC GUG UUG AUG CGC  TRQSRVQMPNGLVLMR TBEV  C10 . in all RNA viruses, the consequence of a high error rate in the virus RdRp (41) .
Sequence changes in the apical loop of SL6. In mutants C12, C13, C14, C16 and C19 substitutions within the apical loop changed the nucleotide sequence without altering the overall conformation (Figures 3 and 4) . Four of these mutations C12, C13, C14 and C19 were introduced into the conserved hexanucleotide UGCCAA. Silent mutations C12, C13 and C14 changed plaque morphology; the C13 and C14 mutants that contain purine-to-pyrimidine substitutions also showed reduced growth characteristics and cpe (Table 1 and Figure 5 ). Silent substitution C16, located outside the conserved hexanucleotide in SL6, did not affect the virus phenotype. Two purines were changed for two pyrimidines in mutant C19, one in the conserved hexanucleotide. This mutant was highly attenuated in cell culture producing no cpe and a small turbid plaque phenotype (Figures 3-5 and Table 1 ). These two purine-pyrimidine substitutions resulted in the amino acid substitution M 33 !L that mimicked the corresponding natural amino acid in KFDV and AHFV (Figure 3) . Nevertheless, M 33 !L, mutant C15, produced by different nucleotide substitutions had only a moderate effect on virus replication (below). Therefore, the biological consequences of mutation C19 may be attributed, at least partially, to the nucleotide substitutions.
Conformational changes to the apical loop of SL6. Mutations C10, C11, C15, C21, C22 and C23 changed the shape of the loop and base-paired stem within SL6 (Figure 4) . Replication of mutant C10 (with an enlarged loop and shortened stem) and C17 (restored wild type conformation due to the second compensatory mutation) was delayed in the early stage of the infection cycle; C17 caused slightly reduced cpe but the plaque morphology of both was equivalent to that of the parental pGGVs virus ( Figure 5 and Table 1 ). The minor phenotypic changes resulting from these mutations could be explained by the accompanying amino acid substitutions N 35 !K and Q 32 !P imitating POWV ( Figure 3 ). However, a silent substitution A 234 !G that also enlarged the apical loop of mutant C11 (Figure 4 ) Table 1 . Affect of mutations within the SL6 on TBEV phenotype Plaque size for each mutant was defined as large (5-6 mm), medium (3-4 mm), small (1-2 mm) or pinpointed (>1 mm). Some plaques, in comparison with parent Vs virus, were described as turbid. The cpe produced by each mutant in comparison to the wild-type virus was evaluated on a scale of 0-4 where 0 indicates no cpe and 4 is maximum cpe (i.e. 80% cell lysis as observed for the control pGGVs virus) in five repeated experiments, each in quadruplicates. Nt/AA* -Nucleotide/amino acid substitutions. caused similar biological effects; it did not affect virus plaque size or level of cpe, but reduced virus replication rate early after infection ( Figure 5 and Table 1 ).
Mutation C15, which shortened the apical loop (Figure 4) , did not affect virus growth but changed the plaque morphology and delayed the development of cpe (Table 1 and Figure 5 ). The C15 mutation altered the amino acid M 33 !L, which imitates the KFDV/AHFV group (Figure 3) , potentially contributing to the observed biological effect. Mutation C21 that reduced the apical loop size (Figure 4 ) interfering with exposure of the hexanucleotide, also had a moderate affect on virus growth although the accompanying effect of amino acid substitution Q 32 !H (Figure 3 ) cannot be excluded (Table 1 and Figure 5 ).
Mutant C22 contained three substitutions that considerably increased the size of the apical loop thus shortening the base paired stem. Three nucleotide substitutions present in mutant C23 had the opposite affect in shrinking the apical loop (Figures 4) . Both C22 and C23 had altered growth dynamics, plaque morphology and cpe (Table 1 and Figure 5 ). The nucleotide substitutions of both led to amino acid substitutions Q 32 !R and V 31 !S, respectively. To exclude their influence on virus growth, counterpart 'control' mutants R 32 and S 31 were analyzed, with the same amino acid substitutions but without alteration of the SL6 conformation (Figures 3 and 4) . Both of these control mutants exhibited wild-type plaque morphology and cpe characteristics (Table 1) .
Substitutions in the stem of SL6. Three mutants were designed to investigate the influence of SL6 stem length. Most attempts to design synonymous substitutions had little effect on the stem folding conformation. Only silent mutant C33 (C 253 !A and C 255 !G) exhibited a significantly shortened duplex stem, with a corresponding elevated level of dG folding energy. These positions are highly conserved among the mTBFV (Figure 3 ) and, as expected, had a profound effect on virus replication; C33 displayed pinpoint plaques, reduced growth characteristics and almost no cpe (Table 1 and Figure 5 ).
Two other mutants C27 and C34 had shortened stems due to the formation of a large internal bulge (Figure 4) , and exhibited profoundly altered biological characteristics (Table 1 and Figure 5 ). However, C27 and C34 included amino acid substitutions Q 28 !N and Q 28 !P, respectively, the latter resembling POWV (Figure 3 ). To rule out the amino acid change as influential, two mutants were designed as a control for C27; double mutant N 28 V 39 and single mutant V 39 , neither of which affected SL6 conformation (Figure 4 ). Similarly mutant P 28 , a control for C34, contained the same amino acid substitution Q 28 !P but maintained SL6 conformation (Figures 3  and 4 ). All three control mutants, N 28 V 39 , V 39 and P 28, displayed wild-type large plaque phenotype and cpe (Table 1) .
In a previous study using MFold-simulated RNA structures for a limited number of mTBFV species, we predicted the existence of SL6 in the C-coding region of TBFV. A conserved hexanucleotide UGCCAA in the apical loop and compensatory mutations in the duplex stem of the SL6 implied the formation of the stable RNA structure in ORF of the TBFV genomes (26, 27) . However, contradicting these findings a deletion within the C-coding region, which included SL6, did not prevent recovery of viable, albeit attenuated, TBEV (42) . In this study, we employed a variety of complementary phylogenetic and thermodynamic methods to examine the evolutionary conservation of SL6 using a much larger sample of significantly divergent TBFVs, including new members of the mTBFV, sTBFV and Kadam subgroups (37) . The viruses in the other ecological groups, namely MBFV, NKV and PABV were also included in this analysis to trace the evolution of SL6 throughout the entire genus Flavivirus. In addition, we used a reverse genetic system (34, 36) to engineer TBEV strains with mutated SL6 to reveal the biological significance of this structure.
Thermodynamic and phylogenetic analysis of large sequence data sets indicated that all TBFVs including even the distantly related mTBFV, KADV and sTBFV form an SL6-like structure with an exposed conserved hexanucleotide although the molecular details of the predicted stem-loop varied among the mTBFV, sTBFV and KADV subgroups ( Figure 1B) . In similar manner, an SL6-like structure has been predicted for MBFV although with less stability in comparison to SL6 in TBFV. Two other flavivirus groups NKV and PABV demonstrated no significant sequence homology with the TBFV SL6 region although the genome of KRV (PABV group) formed a thermodynamically stable structure in close vicinity to the TBFV SL6 with a similar terminal loop motif CCAA (TBFV-UGCCAA) (Supplementary Figure S1) .
To test the biological significance of SL6 in the TBFV group we engineered 21 mutant viruses with point mutations that altered the linear sequence of the unpaired apical loop or destabilized the base-paired stem. Substitutions within the conserved hexanucleotide loop down-regulated virus growth kinetics whereas changes in the terminal loop outside the hexanucleotide sequence did not alter the observed phenotype. The most significant changes of virus phenotype resulted from substitutions that distorted the stem of SL6; mutations that influenced the length or stability of the stem resulted in the recovery of viruses that formed small and/or turbid plaques. Increasing or decreasing the size of the apical loop had a minor biological effect on virus replication although this could also be interpreted as an effect of the altered stem length. However, the changes in replication kinetics from all modifications of SL6 were moderate and manifested themselves predominantly during the early stage of the virus replication cycle (Table 1) .
Previous analysis of RNA secondary structure across the Flavivirus genus led to the concept of promoter and enhancer elements that initiate assembly of the virus polymerase complex (16) (17) (18) 23, 27, 43, 44) . Enhancers were identified as RNA structures that individually produce only small biological effects on virus replication. However, the significance of enhancers as targets for the attenuation of flaviviruses to engineer live vaccines is evident from the example of dengue virus (24, 25) . Moreover, sequence and structural conservation of flavivirus enhancers is consistent with a role as key players in virus survival in the natural environment. We previously proposed that the cumulative action of several enhancer elements could contribute significantly to the overall rate of assembly of polymerase complexes, thereby enhancing virus survival across a range of natural hosts (17, 18, 23, 27, 43, 44) . In this respect, the presented experimental data indicate that SL6 belongs to the category of REEs, i.e. RNA structures that accelerate the replication of viruses (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) . This eliminates the apparent contradictions between extremely high levels of SL6 conservation across divergent TBFV virus species and the redundancy of this element for the replication of laboratory-maintained TBEV strains (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) . However, the specific mechanism by which SL6 functions to enhance virus replication remains to be elucidated.
It has recently been demonstrated that a short but highly conserved RNA hairpin (sHP) localized in the 3 0 -UTR of DENV2 RNA regulates the transition from a circular (required for the initiation of RNA replication) to linear RNA form during the progress of viral RNA synthesis (57) . The SL6-like structure of MBFV is localized immediately downstream of the 5 0 -CYCL (i.e. within the capsid gene, Supplementary Figure S1A ) suggesting it could also contribute to genome circularization. It is possible that in accord with the 3 0 sHP (highly conserved throughout the genus Flavivirus), it contributes to the unpairing of the 5 0 -3 0 -CYCL panhandle, to promote RNA elongation on the linear template. In contrast, the 5 0 -CYCL of TBFV is mapped to the 5 0 -UTR (i.e. upstream of the capsid gene, Figure 1A ) and therefore other tentative functions of the TBFV SL6 are not excluded, such as enhancing virus translation, RNA replication or playing a role in regulation between these processes; the possibility of a kissing-loop enhancer of genome circularization was previously discussed (17, 18, 23, 27, 43, 44) .
The C protein of flaviviruses is highly basic at the N-terminus, specifically binding virus genomic RNA during encapsidation and plausibly acting as an RNA chaperone as shown for other viruses (58) . The sequence of SL6 within the C coding region localizes to the junction of the positively charged domain and a following hydrophobic domain that interacts with the virus envelope proteins during assembly (42) . It is possible that additional synonymous codon flexibility may be accommodated in this region due to the requirement to conserve the charge or hydrophobic characteristics of the domain, rather than any specific amino acid sequence.
Although our studies provide support for the REE role of SL6 in TBEV it is unclear if SL6-like structures of MBFV act similarly as functionally significant REE. However, the remarkable resemblance of the WNV SL6-like structure to TBFV SL6 suggests that it might serve a similar function, at least in one virus group. However, a final conclusion for the MBFV and also for the more distant NKV or PABV groups is not possible ahead of further functional studies.
Being arboviruses, MBFVs and TBFVs are adapted for transmission between distantly related vertebrate hosts and invertebrate vectors. The requirement to adapt to different molecular environments might result in the evolution of enhancer elements essential for virus replication in one host while being redundant in another. This could explain the contradiction between strict conservation of the different flavivirus enhancers and their apparent redundancy in laboratory systems, which are largely based on mammalian cells (17, 18, 23, 27, 43, 44) . Mutations in SL6 described here have demonstrated its enhancer properties in mammalian cells and it will be interesting to evaluate SL6 enhancer activity in ticks, the major host for maintenance of the TBFV group in the environment (59) (60) (61) .
In conclusion, bioinformatic analysis demonstrated the presence of a conserved RNA secondary structure in the C coding region of the divergent TBFV group. Disruption of this structure compromised virus replication implying an REE function for SL6. By homology with the TBFVs, SL6-like structures were observed in the genomes of some MBFVs and plausibly indicate a similar role as replication enhancers. Future studies using sub-genomic replicons will allow direct measurement of the influence of these sequences on RNA replication and on interaction with viral and host proteins. Both TLR2 and TRIF Contribute to Interferon-β Production during Listeria Infection Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression. Detection of microbial pathogens by pattern recognition receptors, such as Toll-like receptors (TLRs) triggers innate immune responses as a first line of defense against infections [1] [2] [3] . Pathogen-associated molecular patterns (PAMPs) such as bacterial cell walls and their structural components induce a vast variety of biological effects in host organisms. The innate response against infection with intracellular pathogens includes the synthesis of type I IFNs (IFN-I). Whereas this cytokine family generally protects against viruses, its impact on bacterial infections can be either detrimental or advantageous for the host organism [4] .
Listeria monocytogenes is a bacterial pathogen which replicates in the cytoplasm of infected cells. Cytosolic pattern recognition receptors (PRRs) respond to cytosolic bacterial products and contribute to the induction of the innate immune response [5, 6] . Previous studies in bone marrow-derived macrophages (BMM) and epithelial cells show that in these cell types the synthesis of IFN-I in response to infection with L. monocytogenes is independent of TLRs and their adapters, relying exclusively on signals originating from cytosolic sensors [5] [6] [7] [8] . DNA as well as cyclic dinucleotides released from lysed bacteria were suggested to function as the relevant L. monocytogenes PAMPs [9] [10] [11] . Several cytosolic proteins with the ability to sense pathogen-derived nucleic acids have recently been described [11] [12] [13] [14] [15] [16] [17] [18] [19] . Cytosolic recognition of L. monocytogenes causes the activation of the serine/threonine kinase TBK1 and the phosphorylation of its substrate transcription factors IRF3 and IRF7 [7, 8] . Both IRF3 and IRF7 participate in the formation of an enhanceosome at the IFN-b promoter [20] .
During uptake by host cells L. monocytogenes is exposed to plasma membrane and endosomal TLRs. Among these, TLR2 which recognizes lipotechoic acids and lipopeptides, contributes to the innate response against infection [21] [22] [23] . Reportedly, TLR2 signals through the interacting adapter proteins Mal/TIRAP and MyD88 and does not contribute to the synthesis of type I IFN in Listeria-infected BMM [7] [8] [9] . Signaling through TRIF, an adapter protein known to connect TLRs 3 and 4 with the IFN-I genes was similarly ruled out for Listeria-infected BMM [7] .
In order to establish a successful infection, pathogens must survive host defense systems or else mitigate the activities of PRRs. Consequently, they have evolved to modify the structural components which normally trigger PRR responses. Bacterial PGN is a hetero-polymer consisting of alternating residues of b-1,4-linked N-acetylglucosamine and N-acetylmuramic acid to which a peptide chain is attached [24] . Interestingly, L. monocytogenes modifies its PGN, with fifty per cent of the muropeptide composition being N-deacetylated [25] . We previously reported that a PGN N-deacetylase gene, pgdA, is responsible for this modification [25] . PGN deacetylation confers resistance to the action of lysozyme, one of the most important and widespread antimicrobial agents of the innate defense system, thus preventing degradation and release of immunostimulants. A strain of L. monocytogenes mutated in its ability to alter its PGN, DpgdA, is sensitive to lysozyme and induces an enhanced IFN-b response in macrophages compared to the isogenic parental strain [25] .
The aim of the present study was to decipher the signaling pathways involved in this response to DpgdA infection. We reveal that IFN-b production in peritoneal macrophages requires TLR2 signaling and the TRIF adapter protein.
Listeria DpgdA mutant in a TLR2-dependent manner A L. monocytogenes pgdA mutant induced a much higher IFN-b response than the parental strain [25] . To definitively establish a role for the peptidoglycan deacetylase PgdA in the downregulation of IFN-b production, we complemented our original pgdA mutant with the wild-type gene and we measured IFN-b secretion of peptone elicited peritoneal macrophages (PEM) infected with wild-type EGDe, DpgdA and a complemented DpgdA strain (Fig. 1) . Inactivation of pgdA led to a strong induction of IFNb secretion in wild-type macrophages. In contrast, the complemented strain did not induce any massive IFN-b secretion, similar to wild-type EGDe. Thus, PgdA directly contributes to downregulation of IFN-b production.
Consistent with our previous report measuring secretion of IFNb protein in PEM, IFN-b mRNA synthesis induced by L. monocytogenes infection of PEM required TLR2 ( Fig. 2A) , while TLR2-deficient BMM showed no impairment in their synthesis of IFN-b mRNA (Fig. 2B) . Moreover, IFN-b secretion was strongly reduced in tlr2 2/2 PEM infected with both the DpgdA mutant ( Fig. 2C ) and the complemented DpgdA strain (Fig. 2D) , definitively establishing the TLR2 dependence of IFN-b production.
We next analyzed the pathways by which Listeria induces IFN-b. Our previous study and the above results strongly suggested the critical involvement of TLR2 [25] . TLR2 signaling depends on Mal/TIRAP and MyD88 adaptor proteins. We had previously shown that MyD88 contributed to full IFN-b induction by Listeria [25] . We then compared IFN-b production by wild-type and mal/ tirap 2/2 macrophages infected with EGDe or DpgdA (Fig. 3A) . Surprisingly, production of IFN-b was not decreased in infected macrophages deficient in Mal/TIRAP, indicating that the normal TLR2 adaptor Mal/TIRAP was not required for Listeria-mediated induction of IFN-b.
The adapter TRIF is employed by TLRs 3 and 4 to signal through the TBK1-IRF3/7-IFN-b pathway. There is no previous evidence of an association or functional interaction between TRIF and TLR2. In spite of this, the link between TRIF and the IRF pathway on the one hand, and the unusual employment of TLR2 for signaling to the IFN-b gene in PEM on the other suggested the possibility of a role for TRIF. To test this hypothesis we compared induction of IFN-b expression in wild-type and trif 2/2 PEM or BMM infected with EGDe or DpgdA strains. IFN-b induction strongly decreased in TRIF-deficient macrophages infected with any of the two Listeria strains compared to wild-type PEM, showing the requirement for TRIF (Fig. 3B ). In contrast, BMM showed a TRIF-independent IFN-b production (Fig. S1 ).
The PEM used in our studies are recruited to the peritoneal cavity by injection of the sterile irritant proteose peptone. Hence they differ from BMM not only regarding their anatomical location, but also their partially inflammatory character. To distinguish which of these differences was responsible for the TLR2 and TRIF signaling pathways, we examined IFN-b production by resident PEM. Figure 3C demonstrates a requirement for TLR2 and TRIF by the resident macrophage population. Thus, location to the peritoneal cavity rather than inflammatory character determines the difference in signaling to the IFN-b gene between BMM and PEM.
To examine the role of TLR3, which uses TRIF to trigger IFNb synthesis, we compared induction of IFN-b in wild-type and tlr3 2/2 PEM infected with EGDe or DpgdA strains. IFN-b production was decreased in TLR3-deficient PEM infected with EGDe or DpgdA (Fig. 4A) . We also compared induction of IFN-b in wild-type and tlr4 2/2 PEM infected with EGDe or DpgdA strains, as TLR4 can mediate TRIF-dependent synthesis of IFN-b. In contrast to TLR3-deficient PEM, TLR4-deficient PEM did not show a decrease in IFN-b response to EGDe or DpgdA (Fig. 4B ). Thus, IFN-b induction in response to Listeria infection relies in part on TLR3 and does not require TLR4.
Induction of IFN-b via TLR2 is no longer an exception. It has recently been shown that vaccinia virus-induced IFN-b production was dependent on TLR2 signaling and it was reported that this was occuring from late endosomes [26] . To investigate if an intracellular localization was also required in the case of Listeria, we pretreated cells with cytochalasin D to prevent internalization and measured IFN-b secretion by macrophages infected with EGDe or the DpgdA mutant (Fig. 5A ). In both cases, IFN-b induction was strongly reduced. Thus, internalization is critical for Listeriamediated IFN-b production. We also used dynasore, a dynamin inhibitor and chloroquine, which inhibits endosome acidification, and measured IFN-b induction in macrophages infected with EGDe or the DpgdA mutant ( Fig. 5B -C). IFN-b synthesis was strongly diminished by both dynasore and chloroquine treatments. Together, these results suggest that the TLR2-dependent IFN-b induction is triggered intracellularly.
In BMM rapid synthesis of IFN-b is entirely dependent on IRF3, but not on IRF7, whereas in bone marrow-derived myeloid DC IFN-b synthesis requires both IRF3 and IRF7 [27] . We investigated the role of IRF3 and IRF7 in the production of IFN-b by PEM. To this end we infected irf3 2/2 and irf7 2/2 macrophages with EGDe or the DpgdA strains. Inactivation of IRF3 totally abrogated IFN-b mRNA induction in response to both strains (Fig. 6 ). IFN-b induction in IRF7-deficient macrophages was also strongly affected highlighting the important role of both transcription factors in response to Listeria infection (Fig. 6 ). PEM thus resemble bone marrow-derived myeloid DC, not BMM, in relation to their IRF requirement for Listeria-mediated IFN-b synthesis.
In addition to IRF3/7, NFkB contributes to the formation of the IFN-b enhanceosome [20, 28] . We therefore examined the involvement of the NFkB pathway by measuring induced synthesis of an NFkB-dependent mRNA. IkB is an NFkB-dependent gene and thus a read-out for NFkB activation in response to Listeria infection. We measured the induction of IkB expression in PEM infected with EGDe or the DpgdA mutant. Both strains induced IkB expression and this required internalization as treatment with dynasore reduced the level of IkB induction (Fig. 7A) . Degradation of the IkB protein was examined in PEM infected with EGDe by immunoblot using anti-IkB antibodies. IkB level was reduced rapidly after infection of wild-type PEM (Fig. S2A ). In contrast, IkB degradation was not observed in tlr2 2/2 PEM infected with Listeria (Fig. S2B ). Infection of wild-type, tlr2 2/2 and trif 2/2 macrophages with EGDe or DpgdA showed that both TLR2 and the adaptor were required for full induction of IkB mRNA in response to EGDe and DpgdA strains (Fig. 7B) . These results suggest that TLR2 and TRIF contribute to NFkB activation. The comparison between EGDe and DpgdA strains showed that both caused similar magnitudes of IkB mRNA synthesis. Thus, the activation of NFkB by Listeria is independent of PgdA, suggesting that the increased IFN-b production after infection with DpgdA relies on activation of other transcription factors such as IRFs.
Nucleic acids released intracellularly are critical for IFN-b induction TLR2 or TRIF deficiency strongly reduced, but did not completely shut off IFN-b synthesis. This suggested a potential contribution of intracellular, nucleic acid-dependent pathways to IFN-b synthesis, particularly after infection with DpgdA. We therefore examined whether these pathways are able to signal in PEM.
Since inactivation of PgdA increases Listeria sensitivity to peptidoglycan-targeting antimicrobials such as lysozyme, and thus induces bacterial degradation, we measured the DNA and RNA released by EGDe and DpgdA strains following lysozyme exposure. As expected, DpgdA released significantly higher amounts of DNA and RNA than wild-type and complemented DpgdA strains, raising the possibility that both DNA and RNA could be involved in IFNb production (Fig. 8A) . We thus measured IFN-b induction in THP1 macrophages transfected with Listeria DNA, either undigested or treated with DNase. Intact but not DNase-treated DNA significantly induced IFN-b (Fig. 8B ). Macrophages were then transfected with lysozyme-digested EGDe or DpgdA, either untreated or digested with DNase. Treatment with DNase significantly reduced IFN-b production (Fig. 8C) . Taken together, these results show that Listeria DNA can induce IFN-b, strongly indicating that destruction of DpgdA bacteria intracellularly activates DNA sensors.
We had recently reported that a PGN modification involving a N-deacetylase gene, pgdA, was playing a key role in L. monocytogenes virulence [25] . A DpgdA strain of L. monocytogenes which is unable to modify its PGN, was shown to be extremely sensitive to the bacteriolytic activity of lysozyme, normally found within macrophage vacuoles and its virulence was strongly attenuated [25] . Furthermore, this mutant induced a much higher TLR2dependent IFN-b response than the parental strain [25] . We hypothesised that this unconventional IFN-b response induced by the pgdA mutant was due to an enhanced accessibility of bacterial cell wall components to TLR2. Here we have shown that IFN-b production requires bacterial internalization and is triggered by Mal/TIRAP-independent pathways which involve TLR2, TRIF, IRF3 and IRF7.
It was surprising to see a role for TLR2, as, based on results in BMM and epithelial cells, type I IFNs production is usually not known to result from TLR2 signaling [5] [6] [7] [8] . Classical TLR2 signaling leads to NF-kB-dependent production of inflammatory cytokines [21] . However, in support of an unconventional role for TLR2, recent studies reported roles for TLR2-dependent induction of IFN-b in response to vaccinia virus or synthetic ligands [26, 29] . In the vaccinia virus study, a specific inflammatory monocyte population -Ly6C hi -was shown to be the source of IFNb [26] . In the present study we show that TLR2-dependent IFN-b synthesis is a property of both resident and recruited inflammatory PEM. Furthermore, the two previous studies documented that TLR2 activation of type I IFN responses to TLR ligands occurs within intracellular compartments, and that TLR2 signals from the phagosome in response to viral infection or synthetic TLR2 ligands [26, 29] . These results challenged the view that TLR2 signals solely from the plasma membrane. In our experiments, pretreatment of PEM with either cytochalasin D, an inhibitor of actin polymerization and thus internalization, dynasore, an inhibitor of the endocytic effector dynamin, or chloroquine, which inhibits endosome acidification [30, 31] , significantly impaired the induction of IFN-b following Listeria infection, strongly suggesting that phagocytosis of L. monocytogenes and intracellular location of TLR2 trigger this response. These observations also correlate with our early hypothesis that the inflammatory response induced by DpgdA is due to an enhanced release or accessibility of bacterial cell wall components to TLR2.
Induction of the IFN-b gene was independent of the TLR adapter Mal/TIRAP, but, unexpectedly required the TLR3/4 adapter TRIF. Francisella tularensis has recently been shown to signal through TLR2 from the phagosome in a Mal/TIRAP independent manner [32] , and it was shown that Mal/TIRAP is dispensable in TLR2 signaling at high concentrations of ligands [33] . Thus our study reinforces the view that TLR2 can act independently from Mal/TIRAP. In addition our report suggests a synergy between a TLR2 pathway and TRIF, an adapter previously known to trigger the synthesis of pro-inflammatory cytokines and type I IFNs upon engagement of TLR3 and TLR4. TLR3 is known to bind viral dsRNA to induce secretion of type I IFN and lead to control of viral infections [3, 34, 35] . To our knowledge Chlamydia muridarum is the only bacterium reported to induce a TLR3-dependent IFN-b response specifically in murine oviduct epithelial cells [36] . We tested whether the dual requirement for TLR2 and TRIF resulted from a functional or physical interaction between TLR2 and TLR3. In fact, IFN-b production was reduced in TLR3-deficient macrophages, but significantly less so than in trif2/2 PEM. Therefore, there is no evidence for a putative TLR2/TLR3 interaction. Another possibility to incorporate TRIF into the pathway stimulated in PEM by Listeria would be a cooperation of TLR2 and TLR4. This was ruled out by showing that Listeria-infected tlr4 2/2 PEM produced a similar amount of IFN-b as their wild-type counterparts. TRIF could possibly orchestrate an additional pathway. Along these lines, TRIF has recently been shown to be required for IFN-b synthesis by dendritic cells upon activation of the cytosolic receptor complex DDX1/DDX21/DDX36 by viral RNA [19] .
Engagement of TLRs by various microbe-associated molecular patterns induces activation and translocation to the nucleus of NF-kB, IRF3, IRF7 and/or activator protein-1 (AP-1), which collaborate to induce transcription of type I IFNs [37] . We addressed the role of these transcriptional activators in the IFN-b response to wild-type Listeria and DpgdA, and revealed that inactivation of IRF3 totally abrogated this response to both strains while IFN-b induction was significantly but not totally impaired in IRF7-deficient macrophages, indicating that both of these transcription factors are required for induction of IFN-b following infection with L. monocytogenes. We also assessed the involvement of NF-kB in this response using induction of the IkB gene as a readout. We observed an induction of IkB expression in macrophages which was similar after infection with EGDe or DpgdA. Thus, activation of NF-kB by Listeria is independent of PgdA, strongly suggesting that the elevated IFN-b production by the DpgdA mutant mostly relies on IRF3.
The increased IFN-b response to the DpgdA strain probably results from the fact that within the phagosome, its lysozymesensitive cell wall is degraded, releasing PAMPs able to interact with TLR2 and other PRRs, including cytoplasmic ones. As recent studies have highlighted novel DNA-sensing pathways in the induction of type I IFNs [9, [14] [15] [16] [17] 38] , we thus also investigated the involvement of bacterial nucleic acids in the IFN-b induction, Firstly, we showed that inactivation of PgdA, which confers a higher susceptibility to lysozyme, leads to increased release of DNA. We then showed that DNA from L. monocytogenes can induce IFN-b expression in PEM, suggesting that this macrophage population employs cytoplasmic nucleic acid sensing similar to macrophages or macrophage lines derived from different anatomical locations [9, 38] . Which -if any-of the recently described nucleic acid sensors are used by PEM for the recognition of Listeria DNA remains subject to future investigation. Nevertheless, other bacterial components could participate in IFN-b production upon infection with the DpgdA mutant. For example, the second messenger molecule cyclic diadenosine monophosphate (c-di-AMP), was shown to be secreted by Listeria multidrug efflux pumps triggering type I IFN response [10] and could be involved in the process.
In conclusion, this study describes a novel mechanism leading to induction of type I IFNs in which intracellular sensing plays an important role, ultimately showing how these different recognition pathways can synergise to induce innate immune responses which are required to control infection. In this regard cooperation between TLR2 and TRIF may reflect the need for convergence of the NF-kB and IRF pathways at the IFN-b promoter, with TLR2 being responsible mainly for NF-kB activation and TRIF being instrumental for activation of IRF3 and IRF7. By employing the strategy of PGN modification, L. monocytogenes can avoid immune detection by TLR and evade the innate immune response, thus enabling the infectious process to occur. It is important to recall that pgdA orthologs are found in other pathogenic bacteria, such as Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis and Helicobacter pylori, strongly suggesting that PGN N-deacetylation is a general mechanism evolved by microbes to escape from pattern recognition receptor-mediated immune recognition [39] [40] [41] [42] .
Bacterial strains and growth conditions L. monocytogenes EGDe (BUG1600, ATCC BAA-679), L. monocytogenes isogenic mutant DpgdA (BUG2288, [25] ) and L. monocytogenes DpgdA complemented strain (BUG2382) were grown in brain heart infusion (BHI, Oxoid), aerobically at 37uC and 200 rpm.
A DNA fragment containing the pgdA gene (lmo0415) and its promoter was generated by PCR using oligonucleotides lmo0415-1 (59-AAGGATCCCACAATATGTTAGTTTTCAGGGG-39) and lmo0415-2 (59-AAGGATCCTTATTTCACCATTCTT-GAATCTG-39). The fragment was integrated into pCR-Blunt-II-TOPO (Invitrogen) and the construct was verified by sequencing. After digestion of the construct by BamHI, the fragment was purified on agarose gel and cloned into the integrative vector pPL2 [43] , previously digested by BamHI, constructing pOD98. The pOD98 was electroporated into DpgdA at 2,500 V, 200 V and 25 mF. Transformants were selected at 37uC on BHI agar containing chloramphenicol (7 mg/mL). The presence of the pgdA gene in the complemented strain was confirmed by PCR using oligonucleotides lmo0415-1 and lmo0415-2.
Mice were used for obtaining peptone-elicited peritoneal macrophages, resident peritoneal macrophages and bone marrow-derived macrophages. Animal experiments were performed in accordance with protocols approved by the Animal Experimentation Ethics Committee of the Institut Pasteur (permit #03-49) and following Austrian law in accordance with protocols approved by the Ethics Committee of the University of Veterinary Medicine, Vienna (#GZ680 205/67-BrGt/2003).
Isolation and culture of murine peptone-elicited peritoneal macrophages (PEM) PEM were isolated from 7 to 10 week-old C57BL/6J and genetically-matched tlr2 2/2 , tlr3 2/2 , tlr4 2/2 , mal/tirap 2/2 , trif 2/2 , irf3 2/2 and irf7 2/2 mice as previously described [44] . The percentage of macrophages was determined by flow cytometry using CD11b (1:100, eBiosciences) and F4/80 (1:100, eBiosciences) antibodies. More than 90% of the cells were macrophages. PEM were seeded onto 6-well plates at a concentration of 2610 6 cells per well in DMEM (PAA) supplemented with 10% FCS, 10% L929 conditioned medium (LCM) and 1% penicillin-streptomycin or RPMI-1640 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin.
Resident macrophages were isolated from 6 to 8 week-old C57BL/6J and genetically-matched tlr2 2/2 , trif 2/2 mice by washing the peritoneum twice with 10 mL DMEM (PAA) supplemented with 10% FBS, 10% LCM and 1% penicillinstreptomycin. Harvested cells were centrifuged at 300 g for 5 minutes and resuspended in complete medium. The percentage of macrophages was determined by flow cytometry analysis as above. Cells were seeded onto 6-well plates (Nunc) at a concentration of 2610 6 cells per well.
Tibia and femur from 6 to 8 week-old C57BL/6J and genetically-matched tlr2 2/2 , trif 2/2 mice were collected in ice cold PBS. Bones were sterilized with 70% ethanol and flushed with a 25-G needle using cold DMEM supplemented with 10% FCS, 10% LCM and 1% penicillin-streptomycin. Cells were seeded onto 6-well plates (Nunc) at a concentration of 10 6 cells per well and incubated at 37uC with 5% CO 2 . After 4 days, complete medium was added and cells were split at a ratio of 1:2. After 8 days, macrophages were fully differentiated.
Human acute monocytic leukemia THP-1 cells (ATCC TIB202) were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded onto a 24-well plate at a concentration of 4610 5 cells per well in antibiotic-free media supplemented with 12.5 ng/mL phorbol myristate acetate and incubated for 24 h at 37uC with 5% CO 2 . Differentiation was determined to be successful upon formation of a confluent adherent monolayer. HEK-blue type I IFN cells (Invivogen) were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded at a concentration of 5.6610 4 cells per well onto a 96-well plate.
For cytokine analysis, macrophages were infected with Listeria strains at MOI 10:1, centrifuged at 300 g for 2 min and incubated at 37uC for 15 min. Following phagocytosis, monolayers were washed twice followed by incubation in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and gentamicin (20 mg/mL). Supernatants were collected at various time points, for detection of IFN-b by ELISA. For transcript analysis, macrophages were infected with Listeria strains at MOI 20:1 and incubated at 37uC for 1 h to allow phagocytosis. Monolayers were washed and incubated in DMEM supplemented with 10% FCS and gentamicin (5 mg/mL). After 2 h, medium was changed to DMEM supplemented with 10% FCS and gentamicin (1 mg/mL). Cells were lysed at various time points and RNA collected for qPCR analysis.
For inhibition of bacterial internalization, cell monolayers were pretreated either for 2 h with 100 mM cytochalasin-D (Sigma-Aldrich), or 30 min with 80 mM dynasore (Sigma-Aldrich) or 30 min with 100 mM chloroquine (Sigma-Aldrich) prior to infection assays.
Listeria were grown overnight in BHI at 37uC and cultures were centrifuged at 8000 g for 5 min. Bacterial pellets were resuspended in 75 mg/mL lysozyme and incubated at 37uC for 1 h. DNA was then extracted using the DNeasy blood and tissue kit (Qiagen) and quantified by spectrophotometry (Nanodrop). For transfection assays, THP-1 macrophages were transfected with 200 ng/mL DNA with 2% lipofectamine 2000 (Invitrogen) and incubated for 24 h. Following incubation, supernatants were collected for IFN-b analysis. For pretreatment of DNA with DNase, DNase was added at final concentration of 100 mg/mL for 45 min at 37uC.
Bacterial cultures were treated with 10 mg/mL lysozyme, a concentration leading to lysis of DpgdA but not EGDe, and incubated at 37uC and 200 rpm for 1 h. Following lysozyme treatment, lysed bacterial cultures were centrifuged at 5000 rpm during 10 min. Two types of experiments were performed on supernatants. First, nucleic acid release was quantified. DNA was purified using the Qiagen DNeasy blood and tissue kit omitting lysis steps and quantified by spectrophotometry (Nanodrop). RNA was purified using Qiagen RNeasy kit and quantified by spectrophotometry (Nanodrop). Data shown are representatives of at least three independent experiments. Second, 100 mL of each supernatants were treated by DNase during 30 min at 37uC. Enzymes were inactivated and treated-or untreated-supernatants were transfected in PEM. 8 h after transfection, supernatants of cells were recovered and the IFNb was quantified.
Detection of type I IFN by ELISA and HEK-blue type I IFN cell assay Murine IFN-b production was detected in macrophage supernatants by ELISA according to the manufacturer's procedure (PBL Biomedical Laboratories). For the HEK-blue type I IFN assay, supernatant from THP-1 macrophage assays was collected and 20 mL added onto HEK-blue type I IFN cells plated in 96-well plates, which were incubated at 37uC overnight. Supernatant from HEK-blue cells was collected and 40 mL added to 160 mL of Quanti-blue reagent (Invivogen) for 20 min at 37uC. The colorimetric reaction was measured at 625 nm on a plate reader. Data was normalised against absorbance for the untreated cells and plotted as relative fold increases. Data shown are representatives of at least three independent experiments.
PEM from WT or tlr2 2/2 C57BL/6J mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. IkB and tubulin were detected in lysates by immunoblotting using anti-IkB (Santa Cruz, 1:100) and anti-a-tubulin (Sigma, 1:5000) antibodies.
RNA preparation was performed using NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions. Quantitative real-time PCR was performed on a Mastercycler EP realplex S (Eppendorf). Primers for HPRT (housekeeping gene control), IFNb and IkBa mRNA expression were as follows: HPRT forward GTTGGATACAGGCCAGACTTTGTTG, HPRT reverse GAGGGTAGGCTGGCCTATTGGCT, IFNb forward 59-TCAGAATGAGTGGTGGTTGC-39, IFNb reverse 59-GACCTTTCAAATGCAGTAGATTCA-39; IkBa forward 59-GCAATTTCTGGCTGGTGGG-39, IkBa reverse 59GATCC-GCCAGGTGAAGGG-39. Data shown are representatives of at least three independent experiments.
Results are expressed as means of at least three values, with error bars representing standard deviations. Student's t tests were performed to determine statistical significance where * indicates P,0.05, ** indicates P,0.01 and *** indicates P,0.0001. Figure S1 TRIF is not required for IFN-b response to Listeria in bone marrow macrophages. BMM from C57BL/6J or trif 2/2 mice were infected with the parental EGDe strain (black bars) or the DpgdA mutant (grey bars). After 4 h of infection, IFN-b induction was measured by qRT-PCR. Data are mean 6 SD (NS, non significant, n = 3). (EPS) Figure S2 TLR2 is required for optimal activation of NF-kB. (A) PEM from WT C57BL/6J mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. Activation of NF-kB was measured by determination of IkB degradation relative to tubulin following immunodetection. (B) PEM from tlr2 2/2 mice were infected with EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. Activation of NF-kB was measured by determination of IkB degradation relative to tubulin following immunodetection. (EPS) Protein Reporter Bioassay Systems for the Phenotypic Screening of Candidate Drugs: A Mouse Platform for Anti-Aging Drug Screening Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery. have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery. Keywords: drug development; high throughput screening; reporter mice; age-related disorders
Reporter assays have been widely used in biological research [1] [2] [3] . Such assays are a powerful means of investigating the signaling pathways involved in various biological functions induced by the activation of specific transcription factors. The reporter construct usually consists of a promoter (enhancer) that drives the transcription of a gene that is easily detected by its activity or expression in the assay system. The promoter region binds transcription factors that are activated in response to the stimulation of an upstream, receptor-mediated or receptor-independent signaling cascade. Binding of the transcription factor to the promoter results in the induction of reporter gene (and subsequent reporter protein) expression in response to signaling pathway activation by external stimuli, including drug candidates. It is important that the exogenous protein encoded by the reporter gene has unique enzymatic activity or another property that distinguishes it from endogenous proteins [4] .
The activity of the reporter protein is typically assayed by photometry, colorimetry or fluorometry. Recently, detection systems based on bioluminescence, chemiluminescence and fluorescence have become widely used because of their signal detection sensitivity and ease of use. Improved detection equipment, such as charge-coupled device (CCD) cameras, has also enabled the development of convenient reporter assay systems for drug screening. There are two major types of reporters, i.e., intracellular and extracellular reporters. Intracellular reporter gene products are expressed and retained inside the cells. On the other hand, extracellular reporter genes encode proteins that are secreted into the medium of cultured cells or the blood stream in animals. Some intra-and extracellular reporter gene assays may detect the reporter protein's activity without killing the cells or animals in which the assay is performed. This allows time-course experimentation by sampling of the medium of cultured cells, or the blood plasma of research subjects. As such, report bioassays are potentially suitable for high throughput screening (HTS) of small molecule drug candidates. Bioassays are used to determine the concentration or biological activity of molecules such as hormones, growth factors, and enzymes. Moreover, they can be used for measuring the effects of candidate drugs on an organism, cultured cells, or recombinant receptors by comparison to gold standard molecules with known target activity. The present review provides an overview of the basic properties, efficacies and limitations of protein reporter assays, with an emphasis on their application as bioassays for drug screening.
Chloramphenicol O-acetyltransferase (CAT) has, until recently, been widely used as a reporter protein [5] . CAT is a bacterial enzyme that is not expressed in eukaryotes, and it therefore provides a highly specific detection system. CAT catalyzes the reaction between acetyl-CoA and chloramphenicol to form CoA. The CAT assay has been widely used but is difficult to adapt to HTS platforms because its activity is not easy to detect directly in cells.
β-Galactosidase (β-Gal) is derived from the bacterial lacZ gene and is widely used as an intracellular reporter gene. There are a number of detection options depending on which substrate is used. However, β-Gal is affected by endogenous enzymatic activity in most mammalian cells [4] . Therefore, it is important to distinguish between the endogenous and exogenous (reporter) enzymes in the assay system. This usually requires the pH of the assay mixture to be adjusted, although this may not completely eliminate endogenous enzyme activity. Therefore, the utility of β-Gal for HTS assays is limited.
Genes encoding luciferase enzymes have been cloned from several species, including firefly and sea pansy [1] . Firefly luciferase catalyzes the oxidation of firefly luciferin, which produces a short-lived flash of light that decays within a few seconds. However, the sensitivity of the luciferase assay is significantly higher than the CAT assay [6] . The high sensitivity of luciferase-based bioluminescence assays is better suited to HTS platforms than CAT or β-Gal assays. Moreover, various luciferase substrates are available that have a long half-life and provide linear results over eight orders of magnitude [4] . The enzyme has also been used in a dual-luciferase reporter assay system, which allows both firefly and sea pansy luciferase reactions to be monitored independently in the same cell [7] . This enables the activation of two different signaling pathways to be measured simultaneously in response to the same stimulus.
Rather than being a reporter enzyme, green fluorescent protein (GFP) (and its engineered derivatives) is particularly suited to bioimaging assays in living cells or live animals. GFP is produced by the jellyfish, Aequorea victoria. Upon excitation by UV light, GFP emits green light with an emission maximum at 509 nm [4] . The intensity of light emissions has been improved by protein engineering in the fluorophore region of the protein. GFP-based reporter assays provide a distinct advantage because they do not require cell permeabilization or the addition of exogenous substrates. Therefore, these proteins are suitable for drug screening in HTS assays. Recent improvements in GFP detection technologies allow changes in the localization of GFP-tagged proteins, in response to small molecules, to be detected at the subcellular level in living cells cultured in 384-well plates. Therefore, GFP is suitable as both a reporter of transcriptional activation, and of functional protein translocation in the cell, in response to various stimuli.
The secreted alkaline phosphatase (SEAP) reporter system has been used to investigate the activity of known or putative promoter/enhancer elements [8] . As a reporter, SEAP has several important advantages over other reporters, such as high sensitivity and specificity both in vitro and in vivo [9] [10] [11] [12] . SEAP is a C-terminal truncated mutant of the membrane-anchored, placental alkaline phosphatase [13] . Removal of the anchoring domain results in secretion of the enzyme into the culture medium or blood stream, thus enabling repeated sampling from living cells or animals. Expression of the gene can be detected using chemiluminometry [14] . The secreted form of alkaline phosphatase is extremely heat stable, and its activity can be detected following the inactivation of endogenous alkaline phosphatases by heating. Therefore, it is easy to distinguish between exogenous and endogenous SEAP activity. SEAP assays can also be coupled to a luciferase reaction by incubation with the substrate, firefly D-luciferin-O-phosphate. The product of the first reaction, luciferin, then becomes a substrate for luciferase. This assay protocol improves the sensitivity of the reporter assay system, thus enabling its broader application [15] .
A recently-identified secreted form of luciferase also serves as a useful reporter for HTS assays. Luciferase from the marine copepod, Metridia longa, has unique secretary features that make it suitable for extracellular reporter assays. Markova et al. reported the substrate specificity of Metridia luciferase (MetLuc) and demonstrated its suitability for use as a reporter for monitoring gene expression [16] . In common with SEAP, secreted MetLuc is well suited to monitoring gene expression in time-course reporter assays. Both SEAP and MetLuc are currently available in a commercial dual reporter system that has been successfully applied in drug screening research [17] [18] [19] [20] .
Reporter assay systems are suitable for the multi-parameter phenotype screening of drug candidates [21] . In contrast to the single molecular target approach, cell-based reporter assays reflect the complexity of the living organism. Therefore, they are becoming widely used to screen the effects of small molecules on signaling pathways [22] . However, drug discovery by HTS in cell-based assays presents its own difficulties. For instance, isolation of a hit compound often does not suggest a single, particular molecular target, preventing further optimization of hit compounds by medicinal chemistry [23] . This has led to cell-based screens being termed 'black-box' screens [21] . However, in most cases, target pathways may be predicted and investigated further. Nonetheless, drug companies prefer the well-characterized molecular target approach rather than phenotypic screening to accelerate the early phase of drug development. In fact, cell-based screens tend to identify more false positives than molecular target screens, presumably because of the existence of many potential targets in the biological pathway of interest [24] . Nonetheless, hit compounds from molecular target screens can fail to generate the expected effect on cells or have undesirable side effects that are potentially harmful to humans. In the context of the ongoing paradigm shift towards pathway-driven drug discovery, particularly in the academic field, it becomes increasingly important to accomplish promising candidate isolation earlier in the drug discovery pipeline and with higher throughput. Currently, many large pharmaceutical companies have established open innovation research programs through collaboration with non-profit research organizations to overcome these problems. Academic research teams typically utilize cell-based reporter assay systems for primary drug screening. Furthermore, research is also being conducted using living animals to monitor the more complex dynamics of the hit compounds identified by screening.
Animal-based reporter assays using reporter mice are a potentially powerful tool for the identification of drug candidates. To our knowledge, the ERE-Luc (estrogen responsive element-luciferase gene) mouse is the first example of a reporter animal to be used for drug development [25] , although several studies with simple reporter mice have been documented previously [26] [27] [28] . The ERE-Luc model is a unique reporter animal for drug discovery that was developed to identify ligands that bind estrogen receptors (ERs). Because ERs are ubiquitously expressed in mammals, this mouse model was required to be generally applicable to the activity of ERs [29] .
Validation of the animal model is important to establish whether the reporter activity correlates with receptor activation through the targeted pathways. Nonetheless, the ERE-Luc mouse demonstrated the existence of unliganded activation of ERs [30] . Thus, animal-based reporter assays may provide new insights into the physiology of target pathways. Indeed, the use of the reporter mice revealed differential mechanisms of ER activation in reproductive and non-reproductive tissues [31] . These results indicate that reporter mice are useful for biological research. Precise toxicology studies may also be performed by crossing another mouse model, such as the humanized liver metabolism model, with reporter mice [32] . Such studies might require that the high costs of compound provision are met, and that animal welfare conditions are appropriate. Table 1 summarizes the properties of reporter proteins. 
A A: suitable, B: applicable, C: not applicable. 1 Time-course assay is suitable for extra-cellular reporters compared to intra-cellular reporters. 2 An in vivo imaging system is needed to detect luciferase and GFP. 3 A tissue specific transgenic mouse is needed to detect specific tissue expression of extra-cellular reporters. 4 Chemiluminescence is more sensitive than fluorescence. CAT: Chloramphenicol acetyltransferase; β-Gal: β-Galactosidase; GFP: green fluorescent protein; SEAP: secreted alkaline phosphatase; MetLuc: Metridia longa luciferase; HTS: high throughput screening; CCD: charge-coupled device.
Calorie restriction (CR) extends the lifespan of many organisms [33] . The identification of CR mimetic compounds may therefore enable therapeutic retardation of the onset of various age-related disorders, including metabolic syndrome, cancers, neurodegenerative diseases, and other rare disorders [34] . The development of CR mimetics would therefore fulfill unmet medical needs. Although SIRT1-activating compounds (STACs) may offer a promising approach to the development of CR mimetics, a novel approach to finding potential CR mimetics is required [9] . For this purpose, we developed a cell-and mouse-based screening system using an extracellular reporter assay [10] .
Using microarray analysis, we have identified putative regulatory elements in pro-longevity genes (dwarfism and CR-response elements: DFCR-RE) [9, 35] . Using one of the DFCR-REs, we have developed a cell-and animal-based reporter screening system, named CRISP: the CR-Imitating anti-aging agent Screening Platform. Because the DFCR-REs were isolated from long-lived mice, the unidentified transcriptional regulators of this sequence may be beneficial for longevity and delay the onset of age-related diseases. We found that reporter construct transgenic mice (CRISP mice) showed increased reporter activity upon CR ( [9] and our unpublished data). Hence, compounds that activate this reporter may be CR mimetic pro-longevity drug candidates. As revealed in the ERE-Luc mice, CRISP mice may also reveal novel longevity signaling pathways and target molecules. The activity of candidate compounds can be easily studied on cells by their addition to culture medium, or in mice by their addition to food and water. In addition to isolating the activators of pro-longevity elements, our CRISP method may also identify inhibitors or negative regulators of these elements. In this respect, CRISP is advantageous compared to single molecular target screening. Therefore, a two-step screen in which a primary HTS in CRISP cells is used to identify hit compounds, followed by a secondary screen to verify the activity, metabolism and toxicology of the hits in CRISP mice, is a potentially powerful tool for identifying promising novel drug candidates.
Current reporter assay technologies enabled us to perform high throughput screening of chemical libraries. The isolation of candidate molecules is dependent on the choice of reporter protein and the construction of efficient cell-or animal-based assays. The identification of CR mimetics is attractive for the development of anti-aging therapeutics. While CR mimetics may not be a 'cure-all' for ageassociated disorders, they may suppress and/or delay the progression of various kinds of refractory diseases. Importantly, pharmaceutical companies do not have a high incentive to develop drugs for rare diseases. Therefore, academic laboratories are perfectly placed to develop drugs for rare disorders by using phenotypic cell-or animal-based reporter assay systems. Although candidate CR mimetics identified in such systems must still be validated to show that their beneficial effects are similar to those provided by CR, CR mimetics offer great potential to solve the unmet medical needs of rare diseases and common age-related disorders. We have focused on bioassay (protein reporter assay) screening systems in this review, but biosensor technologies (analytical devices consisting of biological components) are also important for drug discovery [36] . Therefore, the development of biosensors to screen CR mimetics will also be an important approach to future drug discovery efforts. Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC)) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B) adenosine receptor (A(2B)AR), largely abolished the adenosine-stimulated chloride transport, suggesting that A(2B)AR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2B)AR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections. Alcohol abuse is a risk factor for pulmonary infections. It is not fully understood how alcohol exposure compromises the lung host defense. Previous studies suggest that multiple pathophysiological mechanisms may be involved [1, 2, 3] . Airway mucosal immunity and mucociliary clearance are the two primary host defense mechanisms, which take place in a thin layer of liquid on the top of airway epithelia, known as airway surface liquid (ASL). ASL, composed of a gel-like mucus layer and a sol-like periciliary liquid layer [4, 5, 6] , is the ''battlefield'' for pulmonary infection and immunity. The viscous mucous blanket traps inhaled microorganisms and particles to restrict their spreading in the lung. In contrast, the watery periciliary liquid (PCL) underneath pools antimicrobial substances, antibodies, cytokines, chemokines and other immune modulators [7, 8, 9] . More importantly, PCL provides the milieu for innate and adaptive immune cells including neutrophils and macrophages to home and function. Moreover, PCL prevents cilia from being entrapped in viscous mucus and bathes them for mechanical movement for mucociliary clearance [5, 10] .
ASL composition and volume are collectively regulated by epithelial chloride secretion, sodium absorption and secondarily water secretion and absorption [6, 11] . Mounting evidence indicates that paracrine/autocrine purinergic signaling is critical to airway epithelial ion transport and ASL hydration [12] #. Adenosine has been shown to be a potent regulator in the process, which can be directly released by local epithelial cells and immune cells [13] or from extracellular metabolism of ATP [12] #. It is known that ATP is constitutively released by epithelia due to various stimuli including mechanical stretch and shear stress due to respiration [14] . The released ATP is then converted to adenosine by extracellular ectonucleotidases [15] . Thus, ASL has relatively high levels of adenosine. Further studies demonstrate that adenosine largely regulates epithelial CFTR channel function by acting on A 2B AR [16, 17, 18] . Thus, the adenosine-A 2B AR signaling pathway is a crucial element in lung host defense [19, 20] .
Previous alcohol studies have documented that ethanol exposure decreases cAMP signaling and protein kinase A (PKA) activation [3, 21] . Ethanol also up-regulates phosphodiesterase 4 (PDE4), which increases cAMP degradation [22] #. In spite of the clear link between alcohol exposure and alteration of adenosine signaling, no published data are currently available concerning alcohol effects on airway ion transport through this signaling pathway. The current report directly measured the adenosineinduced chloride secretion of airway epithelia under the exposure of physiologically relevant concentrations of alcohol and found that ethanol attenuates epithelial CFTR-mediated chloride transport by modulating cellular cAMP levels.
No human subjects or animals were used in this study.
Calu-3 cells, a human airway epithelial cell line (ATCC, Manassas, VA), were seeded on collagen-coated MillicellH-PCF membrane inserts (Millipore, Billerica, MA) at a density of 1610 6 cells per insert of 0.6 cm 2 surface area. Two days after the initial submerged culture, the apical media were aspirated off and the cells cultured at an air-liquid interface according to previously published protocol [23] . Regardless of submerged culture or air-liquid interface culture, the media used were the same, consisting of Advanced-MEM (Gibco, Carlsbad, CA) containing 10% fetal bovine serum, 1% L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B. After 2 weeks at 37uC in presence of 5% CO 2 , the epithelia established a dry apical surface and had a transepithelial electrical resistance greater than 1000 V/cm 2 . The cystic fibrosis (CF) airway epithelial cells CFBE41o- [24] were similarly cultured. The fully differentiated CF epithelia after 2 weeks exhibited a transepithelial resistance greater than 700 V/cm 2 .
Air-liquid interface cultures were basolaterally exposed to different concentrations of ethanol (200 Proof; AAPER Alcohol and Chemical Co., Shelbyville, KY), as indicated in individual experiments. All the cultures were kept at 37uC, 5% CO 2 in incubators that had been pre-saturated with specified concentrations of ethanol.
The TEER of the airway epithelial cell cultures was measured by using a ''chop stick'' epithelial ohmmeter (World Precision Instruments, Sarasota, FL), as described previously [25, 26] .
Calu-3 cells, cultured at the air-liquid interface for 2 weeks, were exposed to different concentrations of ethanol for 24 hours and mounted on an Ussing chamber apparatus (World Precision Instruments, Sarasota, FL). The cells were bathed with an apical low chloride buffer (135 mM sodium gluconate, 5.0 mM HEPES, 1.2 mM MgCl 2 , 0.6 mM KH 2 PO 4 , 4 mM CaCl 2 , 2.4 mM K 2 HPO 4 3H 2 O, and 10 mM Dextrose, pH 7.4) and with a basal high chloride buffer (135 mM NaCl, 5.0 mM HEPES, 1.2 mM MgCl 2 , 0.6 mM KH 2 PO 4 , 1.2 mM CaCl 2 , 2.4 mM, K 2 HPO 4 3H 2 O, and 10 mM Dextrose, pH 7.4). Both buffers were continuously stirred, gassed with 95% O 2 and 5% CO 2 and maintained at 37uC. These buffers were to maintain a chloride gradient across the epithelial monolayer. The TEER was measured with an open circuit by applying an electrical pulse across the epithelial monolayer. Short circuit currents (I SC ) were measured by applying an epithelial voltage clamp. For all the experiments, 100 mM of amiloride was added to the apical side to block sodium channels. Anion currents were induced by apical addition of 100 mM adenosine. The A 2B AR blockade was achieved by apical application of 50 mM alloxazine. To differentiate between chloride and bicarbonate currents, 100 mM bumetanide was used basally to block chloride transport. Acetazolamide (20 mM) or DNDS (4,49dinitro stilbene-2,29-disulfonate) (100 mM) was employed apically to block bicarbonate transport. Further, to inhibit epithelial phosphodiesterases, 100 mM of IBMX (3-isobutyl-1-methylxanthine) or 50 mM papaverine was added apically.
The air-liquid interface cultures of Calu-3 cells were exposed to either 0 mM or 100 mM of ethanol at the basolateral side for 24 hours. On the apical surface the cells were stimulated with 100 mM of adenosine. The cells were washed with PBS containing 100 mM IBMX to inhibit phosphodiesterases that might degrade cAMP. The cells were lysed and cAMP was measured by cAMP immunoassay (R&D Systems, Minneapolis, MN). IBMX (100 mM) was also included in the lysis buffer. The samples were read at 450 nm using a spectrophotometer (BioTEK, Winooski, VT). All the treatments were carried out in quadruplicates.
Data presented represent mean of multiple experiments and error bars indicate standard deviation from the mean. Where indicated, the data points were analyzed by Student's t-test or One-way ANOVA test. The P values smaller than 0.05 were considered statistically significant.
To explore if ethanol affects adenosine-activated ion transport function of airway epithelium, we employed air-liquid interface cultures of Calu-3 cells, a system widely used to investigate airway epithelial electrophysiological properties [27, 28] . The cultured epithelia were exposed basolaterally for 24 hours to different concentrations of ethanol (0, 25, 50 and 100 mM) and adenosineinduced transepithelial ion transport was assessed by measuring I SC with an Ussing chamber apparatus. Two buffers with asymmetric chloride were applied: apical low chloride (10.4 mM) and basolateral high chloride (139.8 mM). After voltage clamp, sodium channels were blocked by apical amiloride (100 mM) and I SC was stimulated by apical addition of adenosine (100 mM). As shown (Fig. 1) , the ethanol exposure decreased adenosine-activated I SC in a dose-dependent manner. Significant differences were detected by Figure 1 . Effect of ethanol pre-exposure on adenosine-induced epithelial ion transport. Calu-3 cells were cultured at an air-liquid interface on membrane filters and basolaterally exposed to 0, 25, 50 and 100 mM of ethanol for 24 hours. These Calu-3 epithelia were placed in an Ussing chamber with asymmetrical buffers of chloride (apical 10.4 mM and basolateral 139.8 mM). Following voltage clamp, stable short circuit baselines were attained in 15 to 20 min. I SC was measured after blocking Na + channels with 100 mM of apical amiloride and stimulated with 100 mM of apical adenosine. Alterations in ion transport are expressed as difference in I SC from their baseline. Ethanol preexposure decreased 100 mM adenosine mediated epithelial ion transport in a dose-dependent manner. Asterisks indicate significant differences between groups by One-way ANOVA test (p,0.05, n = 5 for each condition). doi:10.1371/journal.pone.0032112.g001
One-way ANOVA test between the control and the alcoholexposed (50 mM and 100 mM) groups (p,0.05, n = 5).
It was previously documented that adenosine-stimulated anion secretion in airway epithelia is largely mediated by chloride transport [17, 29] . To explore if the alcohol-suppressed I SC , as identified above, is actually a chloride current, we first confirmed chloride transport by Calu-3 epithelia in our experimental setting. To this end, the cultured Calu-3 epithelia without any alcohol exposure were subjected to I SC measurement in the presence of various inhibitors to block different ion channels. Bumetanide (100 mM), a specific inhibitor for the Na + -K + -2Cl 2 cotransporter, was applied to the basolateral side of the Calu-3 epithelia after adenosine stimulation. This drug decreased I SC by ,58%, while acetazolamide, a carbonic anhydrase inhibitor, and DNDS, an inhibitor for Na + /HCO 3 2 cotransporters and Cl 2 /HCO 3 2 exchangers, had no effect on I SC (Fig. 2A) . These data suggest that the adenosine-stimulated anion current that is suppressed by alcohol is the chloride channel conductance. The result was further validated by measuring the adenosine-stimulated I SC with identical apical and basal chloride buffers. Without chloride gradient (Fig. 2B) , adenosine-induced I SC was dropped significantly by Student's t-test (p,0.01, n = 5). The I SC was only ,8% of that measured with the asymmetric chloride buffers.
The adenosine-stimulated chloride transport is mediated through the CFTR channel CFTR has been found to be a major chloride channel in the airway epithelium responsible for adenosine-induced ion transport [30] #. To validate if CFTR is responsible for the observed adenosine-stimulated chloride transport in the Calu-3 epithelia, we applied CFTR channel inhibitor CFTR inh 172 to the apical side of Calu-3 epithelia for 30 minutes, followed by adenosine stimulation. The chloride conductance was decreased by ,76% when CFTR was inhibited (Fig. 3) , which is significantly lower than that of the no drug control (p,0.01, n = 5). To seek a second approach to confirm the result, CFBE41o cells, an airway epithelial cell line derived from a cystic fibrosis (CF) patient with homozygous DF508 mutations in CFTR, were used [24] . Strikingly, adenosine failed to elicit any chloride currents across the CF epithelia (p,0.01, N = 4). Thus, the currents under our experimental condition and drug profile are CFTR-mediated chloride conductance. These data altogether confirmed that in airway epithelial cells ethanolsuppression of chloride transport is mediated through CFTR, a cAMP-activated chloride channel.
The aforementioned data suggest that the negative modulation of epithelial chloride secretion by ethanol is likely through the adenosine-adenosine receptor signaling pathway. Up to date, four different adenosine receptors have been identified: A1, A 2A , A 2B and A3 receptors [31] . In airway epithelial cells, A 2B AR predominantly regulate CFTR function [18] . Here, we wanted to examine if the same signaling pathway is involved in the alcohol-induced inhibition of CFTR-mediated chloride transport. To this end, alloxazine (50 mM), a commonly used A 2B AR specific blocker, was applied to the apical buffer after adenosine stimulation. The data ( Figure 4) demonstrate that ,75% adenosine-stimulated epithelial I SC was reduced by alloxazine, indicating that A 2B AR was largely responsible for the adenosinemediated chloride secretion. Figure 2 . Adenosine-stimulated short circuit current is chloride conductance. A) I SC of Calu-3 epithelia was measured by using the asymmetric chloride buffers. After stimulated with 100 mM of apical adenosine, the cells gave rise to I SC of ,75 mA/cm 2 . The I SC was blocked by ,58% by basolateral addition of bumetanide (100 mM), but not by acetazolamide (20 mM) or DNDS (100 mM). B) I SC of Calu-3 epithelia was measured with either asymmetric chloride buffers or symmetric chloride buffers. The adenosine-stimulated I SC with no chloride-gradient buffers was ,92% lower than that with chloride-gradient buffers. Significance of the difference was determined by Student's t-test (p,0.01, n = 5). doi:10.1371/journal.pone.0032112.g002 Figure 3 . Adenosine-stimulated transepithelial I SC was mediated by CFTR channel. Calu-3 epithelia were stimulated with 100 mM of apical adenosine, resulting in a significant increase in I SC above baseline. Such an adenosine-induced I SC was mostly absent in CFBE41o cells in which CFTR channel was dysfunctional or was significantly inhibited with 25 mM of CFTR inhibitor CFTR inh 172. Student's t-test was performed to determine the statistic significance (p,0.05, n = 5). doi:10.1371/journal.pone.0032112.g003
Ethanol exposure affects CFTR-mediated chloride secretion through modulation of cellular cAMP level Upon adenosine binding, the A 2B AR signals through G s protein to activate adenylyl cyclase and raises intracellular cAMP [13, 32] . Because CFTR is a cAMP-activated chloride channel, we hypothesized that ethanol inhibits adenosine-stimulated cAMP production to cause a reduced CFTR channel activity. To test this hypothesis, air-liquid interface cultures of Calu-3 cells were exposed basolaterally to either 0 mM or 100 mM of ethanol for 24 hours. These cells were apically stimulated with 100 mM of adenosine as described above for 10 minutes, the time point at which the epithelial I SC was at its peak levels. Then, the cells were lysed and the levels of cellular cAMP assayed. The results demonstrate that ethanol pre-treatment significantly decreased adenosine-stimulated cAMP levels (p,0.05, n = 5) (Fig. 5A ). In addition, to test whether the decrease in cAMP was in fact causing the suppression of adenosine-stimulated chloride transport by ethanol, Sp-cAMPS, a cell permeable and phosphodiesteraseresistant cAMP analogue, was used to directly activate PKA which then activates the CFTR channel. As shown in Figure 5B , the alcohol-treated Calu-3 cells in the presence of 10 mM Sp-cAMPS obliterated the ethanol suppressive effect on adenosine-induced chloride secretion (p,0.05, n = 6). Thus, we conclude that ethanol inhibits CFTR-mediated chloride secretion by directly affecting the cellular cAMP level instead of the downstream PKA enzyme.
Based on the data that ethanol impairs the adenosine-activated chloride secretion by reducing the cellular cAMP level, we chose to block endogenous cAMP degradation pharmacologically to counteract the alcohol-suppressive effect on chloride secretion. Air-liquid interface cultures of Calu-3 cells were similarly exposed to ethanol for 24 hours and treated with 100 mM of non-specific phosphodiesterase inhibitor IBMX along with adenosine stimulation. The data in Figure 6 indicate that IBMX almost completely restored the ethanol suppression of Calu-3 chloride secretion (p,0.05, n = 4). Papaverine is a clinically used phosphodiesterase inhibitor. This drug also overcame the inhibitory effect of ethanol on adenosine-induced transepithelial chloride conductance (p,0.05, n = 4) (Fig. 6) . These results not only confirm that ethanol modulates adenosine-cAMP signaling but also suggest that phosphodiesterase inhibitors may be useful as the potential therapeutic agents for improving the airway epithelial ion transport and mucociliary clearance in alcoholic patients.
Airway epithelial cells not only constitute the physical barrier that separates airway lumen from interstitial compartments, but also actively participate in innate and adaptive immunity to protect the host from pulmonary infections [33] . Multiple defense systems have been evolved in airways. First, the polarized airway epithelia have a mechanical clearance mechanism. Goblet cells or submucosal glands secrete mucus that entraps airborne particles and inhaled infectious agents. Synchronized ciliary movement sweeps inhaled particulate matter toward the mouth to be expectorated or swallowed [34] #. Second, airway epithelia secrete antimicrobial factors, such as lysozyme and b-defensins [35] . Third, airways and alveoli are patrolled by phagocytic cells, most notably the alveolar macrophage, which can engulf microbes [36] . Fourth, airways can recruit neutrophils and monocytes to sites of inflammation [37] . Fifth, the acquired immune system, including antigen-stimulated T and B lymphocytes, provides cellular and humoral defenses for the airways [38, 39] . It is noteworthy that all the above-mentioned pulmonary defense mechanisms are executed in ASL, the thin film of liquid on top of the airway epithelia. Therefore, ASL homeostasis is pivotal to lung host defense. Here we provide the first evidence suggesting that ethanol exposure suppresses adenosine-stimulated chloride secretion that regulates ASL.
Our data demonstrate that ethanol affects adenosine-stimulated chloride secretion through CFTR. On epithelial apical surface, adenosine binds to A 2b AR, a predominant adenosine receptor present in airway epithelia [16] . This receptor is coupled to G s protein to activate adenylyl cyclase, which elevates intracellular cAMP and consequently activates PKA. Then, CFTR is phosphorylated by PKA and the channel opens to permeate chloride [40] #. More chloride in ASL will cause less Na + absorption and more water retention, thus increasing ASL height and volume [15, 20] . It is even speculated that adenosine is a sensor for ASL homeostasis. When ASL falls, adenosine levels increase in airways beyond their basal levels and activate A 2B AR and CFTR channels [16, 29] . Our data have linked ethanol-suppression of cAMP levels to attenuation of chloride secretion by airway epithelia. Previous studies have documented that ethanol decreases ciliary beating and epithelial cell migration during wound repair which are associated with compromised cAMP signaling and PKA activation [21, 41] . Moreover, alcohol is reported to upregulate the PDE4 enzyme expression as well as the enzymatic activity in epithelia [22] #, which results in accelerated cAMP degradation. Thus, our data are consistent with the data reported, indicating that alcohol modulates various cellular pathways by reducing cellular cAMP levels.
Pulmonary mucociliary clearance is largely affected by three factors: 1) mucus production, 2) ciliary sweeping and, 3) ASL state. Studies on human bronchial epithelial cells have revealed that a 24-hour exposure with 100 mM of alcohol caused an 8-fold increase in trachea-bronchial mucin gene expression [42] . Interestingly, experiments using bovine bronchial epithelial cells have established that alcohol exposure at 100 mM beyond 6 hours decreases ciliary beating frequency [22, 41] . A recent publication by Allen-Gipson and colleagues reports that purinergic stimulation of CBF requires A 2B AR activation [43] . Similarly, in rats chronic alcohol administration decreased ciliary beating frequency and enhanced lung colonization of nasally administered S. pneumoniae [44] #. Our current results demonstrate that 24-hour ethanol exposure reduces chloride secretion, which consequently alters ASL composition and volume. Thus, alcohol affects all three mucociliary clearance components.
Pharmacologically, blocking cAMP degradation has the potential of restoring ethanol-suppressed cellular cAMP levels and therefore epithelial functions. This finding is of clinical implications. Phosphodiesterase inhibitors such as papaverine may improve mucociliary clearance in alcoholics by enhancing airway epithelial ion secretion and ciliary beating. Moreover, enhancement of airway epithelial ion secretion alters the composition of ASL. Our laboratory has reported that extracellular chloride levels affect neutrophil microbial killing ability [45] . Thus, increasing ASL chloride level by phosphodiesterase inhibitors may also improve phagocytic innate immunity in airways of alcoholics.
In summary, ethanol exposure compromises chloride ion secretion which is pivotal to maintain ASL volume and composition. Such an effect may alter the properties of airway host defenses predisposing ethanol abusers to an increased risk of infection in the lung. Restoration of cellular cAMP level with phosphodiesterase inhibitors could potentially ameliorate mucociliary clearance by improving epithelial ion secretion and hence lung host defense. Figure 6 . Phosphodiesterase inhibitors restore the ethanol suppression of adenosine-induced transepithelial chloride conductance. The air-liquid interface cultures of Calu-3 cells were exposed to 0 and 100 mM of ethanol for 24 hours. Adenosinestimulated I SC was measured, showing that ethanol-exposed Calu-3 epithelia had significantly lower adenosine-stimulated I SC than the no ethanol control. However, phosphodiesterase inhibitors, IBMX (100 mM) or papaverine (50 mM), fully restored the ethanol-suppressed adenosine-stimulated I SC . Asterisks indicate statistically significant differences between groups (p,0.05, n = 4 for each group). doi:10.1371/journal.pone.0032112.g006 High Fidelity Processing and Activation of the Human α-Defensin HNP1 Precursor by Neutrophil Elastase and Proteinase 3 The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1–4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (“folded proHNP1”) and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro. Antimicrobial peptides (AMPs) produced by animal cells provide a first line of defense against potentially infectious microorganisms. In mammals, defensins and cathelicidins are the primary AMPs expressed in professional phagocytes such as neutrophils and macrophages [1] . Mammalian defensins comprise three structural subfamilies termed a-, band h-defensins. While all defensins are arginine rich, tridisulfide containing peptides, they are distinguished from each other by the presence of unique disulfide motifs [2] . In addition, h-defensins, which are approximately half the size of aand b-defensins, are macrocyclic molecules which have only been isolated from leukocytes of Old World monkeys (reviewed in [2, 3] ). Defensins have antimicrobial activities against diverse microbial targets that include bacteria [4] , fungi [5, 6] , viruses [7, 8, 9] , and protozoa [10] . Certain a-, b-, and h-defensins function as regulators of adaptive immune cells and thus function to modulate inflammation [11, 12, 13] and bridge innate and adaptive immunity [14] .
In humans, four a-defensins, human neutrophil peptides 1-4 (HNP [1] [2] [3] [4] , are expressed in neutrophils and monocytes of myeloid lineage, and a-defensins (HD5 and HD6) are produced primarily in Paneth cells of the small intestine. In neutrophils, HNP 1-4 are stored in the cytoplasmic azurophil granules which are mobilized during phagocytosis resulting in the exposure of ingested microorganisms to high concentrations of HNPs in the phagolysosomes [2] . In the setting of bacteremic sepsis, HNPs also accumulate in the extracellular compartment [15, 16] . Several studies have demonstrated an extracellular role for myeloid adefensins in regulating cellular activities including cell proliferation [17, 18, 19] , neovascularization [20, 21] , chemotaxis [22] , and regulation of cytokines [23, 24] and signal transduction pathways [17, 19, 24, 25] . To date, these activities have been attributed to the mature, folded a-defensin.
Defensins of all three subfamilies are expressed as preprodefensins containing an N-terminal signal peptide followed by the propeptide that contains the prosegment and the folded/oxidized mature peptide sequence [26] . The prosegment in aand hdefensin precursors is ,45 amino acids in length, but that of bdefensin precursors is typically very short. In a-defensins, studies suggest that the anionic prosegment interacts with residues present in the mature peptide to maintain it in an inactive state until it reaches the correct cellular location, possibly to protect cellular compartments from peptide-mediated cytotoxic effects [27, 28, 29] .
Processing of the human enteric a-defensin, HD5 occurs extracellularly and is mediated by trypsin [30] , whereas mouse Paneth cell a-defensins are processed intracellularly by matrix metalloprotease 7 (MMP7, matrilysin) [31] . Neutrophil elastase (NE), an azurophil granule protease, was shown to correctly process RMAD4, a rhesus macaque neutrophil a-defensin in vitro [32] . Bovine and human cathelicidins, a distinct class of antimicrobial peptides, are processed by the azurophil granule proteases, NE [33] and proteinase 3 (PR3) [34] respectively. Studies with HL-60 cells, a human acute promyelocytic leukemia cell line, show that HNPs are biosynthesized and processed to mature peptides in these cells [35] , but the proteases involved in maturation of human myeloid a-defensins have not been identified. Because HNPs co-localize to neutrophil granules containing the serine proteases NE, PR3 and cathepsin G (CG) [36] , we hypothesized that HNP maturation is mediated by one or more of these proteinases. In this study, recombinant proHNP1 was used as substrate to analyze the human a-defensin convertase activities of NE, PR3, and CG. The requirement for the native HNP1 fold was analyzed by assessing the processing of folded and misfolded preparations of proHNP1. In addition, the antimicrobial activity of the resulting HNP-1 peptides was analyzed utilizing Staphylococcus aureus as the test organism.
Recombinant hexa-His-tagged pro-HNP-1 ( Fig. 1 ) was expressed at approximately 2 mg/L in E. coli cells. The imidazole eluant from the nickel affinity column (Ni-eluant) contained a major band of ,12 kDa, consistent with the molecular weight of His 6 -proHNP1 (theory = 12647 A.M.U.; Fig. 2A ). C18 reversed phase high performance liquid chromatography (RP-HPLC) of the Ni-eluant produced two major peaks (A and B; Fig. 2B ). On reducing SDStricine PAGE, peaks A and B contained the ,12 kDa band (Fig. 2C ) and both were detected with anti-His 6 antibody confirming the presence of His 6 -proHNP1 (Fig. 2D) . On AU-PAGE, His 6 -proHNP1 present in peak A (proHNP1A) migrated more rapidly than that contained in peak B (proHNP1B; Fig. 2E ), suggesting that proHNP1A and proHNP1B are differently folded forms of the recombinant protein. This was confirmed by the finding that DTTreduced proHNP1A and proHNP1B co-eluted on RP-HPLC (data not shown) and by demonstrating that folding of proHNP1B in the presence of reduced/oxidized glutathione led to the formation of proHNP1A (Text S1 and Figure S1 ). Thus, proHNP1A and proHNP1B contain folded and misfolded His 6 -proHNP1 respectively. ProHNP1A and proHNP1B-containing fractions were pooled separately and purified to homogeneity by a second round of RP-HPLC (Fig. 2F ).
The presence of a Met residue at the junction of the prosegment and mature peptide in proHNP1 sequence (see Fig. 1 ) offered a non-enzymatic approach to generate HNP1 from proHNP1 utilizing cyanogen bromide (CNBr) cleavage [28] . Cleavage of proHNP1A with CNBr, followed by C18 RP-HPLC, gave rise to a major peptide product (Fig. 3A ) with a mass (Fig. 3B ) and HPLC retention time (Fig. 3C ) matching that of natural HNP1. However, CNBr digestion of proHNP1B generated multiple species (Fig. 3A ) that had masses consistent with oxidized HNP1 (Fig. 3B ), but which eluted later than native HNP1 in RP-HPLC (Fig. 3C) . On AU PAGE, HNP1 derived from proHNP1A comigrated with natural HNP1. However, HNP1 derived from proHNP1B migrated more rapidly than the natural standard (Fig. 3D) . These data demonstrate that proHNP1A contained folded HNP1 with native disulfide linkages but the mature peptide in proHNP1B, although oxidized, is misfolded and contains non-native disulfide linkages. In solution, HNP1 exists as a non-covalent dimer [37] and the dimeric form corresponds to the single band observed in AU PAGE (Selsted et al., unpublished data) . Therefore, it is likely that the higher mobility of proHNP1B-derived HNP1 (Fig. 3D ) is due to the monomeric state of this conformer.
To analyze the microbicidal properties of the HNP1 conformers produced by CNBr cleavage of proHNP1A and proHNP1B, we tested the respective peptides for killing of S. aureus in vitro. HNP1 derived from proHNP1A had microbicidal activity similar to that of native HNP1 (Fig. 4) . However, HNP1 derived from proHNP1B was devoid of staphylocidal activity (Fig. 4) . Thus, formation of native disulfide linkages in HNP1 is essential for the staphylocidal activity of the peptide, and may reflect a further requirement for dimerization.
Previous studies have demonstrated that NE, PR3, and CG colocalize with HNPs in the azurophil granules of neutrophils. Kamdar et al. demonstrated that NE digestion of proRMAD4, precursor of rhesus myeloid a-defensin-4 (RMAD4), converted proRMAD4 to mature RMAD4. However, digestion of pro-RMAD4 with PR3 and CG generated RMAD4 variants with Nterminal extensions (Fig. 5D ) [32] . To test for the role of these granule serine proteases in human HNP processing, proHNP1A was incubated in separate reactions with NE, PR3, or CG, and the RP-HPLC-purified cleavage products were analyzed by MALDI-TOF MS and analytical HPLC ( Fig. 5 and Table 1 ). Incubation of His 6 -proHNP1 with NE or PR3 generated peptide products that coeluted with native HNP1 in RP-HPLC (34. Table 1 ). The identities of HNP1 derivatives were also confirmed by MALDI-TOF MS of each peptide following reduction and S-carboxyamidomethylation. In each case reduction and alkylation produced a mass increase of 348 A.M.U., confirming the presence of 6 disulfide-linked cysteine residues ( Table 1 ). The NE-and PR3-derived peptides also comigrated with native HNP1 on AU-gels ( Fig. 5C ; lanes 1-3). Thus, NE and PR3 processing of His 6 -proHNP1 generated a peptide that was indistinguishable from natural HNP1.
Incubation of His 6 -proHNP1 with CG did not generate mature HNP1 but instead a digestion product that eluted from C18 HPLC later than HNP1 (Fig. 5A ) and had a mass of 3685.4 A.M.U. This mass corresponds to HNP1 with a dipeptidyl (Asn-Met-) aminoterminal extension, consistent with Lys62QAsn63 cleavage by CG (Fig. 5D ). The CG liberated HNP1 product eluted as a single peak (Fig. 5A) , which was resolved into two species by high resolution RP-HPLC (Alliance analytical HPLC system; Fig. 5B ). Peptide in the second peak corresponded to the mass of Asn-Met-HNP-1 (3685. 
Although the junctional sequences recognized by NE, PR3, and CG in His 6 -proHNP1 are identical to those in the natural precursor, we questioned whether the 41-residue extension of the proHNP-1 sequence resulting from the hexa-His and DDDDK tags and spacer sequences might alter protease interactions with the target. To test this, we produced proHNP1 without the Nterminal tag by incubating proHNP1A with enterokinase, thereby releasing the 41-residue tag sequence. This peptide was then treated with NE, PR3, or CG which liberated peptides with monoisotopic masses of 3439.7, 3439.7 and 3684.5 A.M.U., respectively, products equivalent to those generated from His 6 -proHNP1A. Thus the 41-residue N-terminal fusion tag did not alter the specificity of cleavage of proHNP1A by NE, PR3, or CG.
Protease processing of misfolded His 6 -proHNP1 NE, PR3, and CG degraded proHNP1B, the misfolded form of the HNP1 precursor. In contrast to the discrete products generated by digestion of proHNP1A by the serine proteases ( Fig. 5) , NE, PR3, or CG treatment of proHNP1B yielded no mature HNP1 or HNP1 derivatives in the digestion mixtures detectable by C18 RP-HPLC or MALDI-TOF MS (data not shown). These results indicate that the correct fold and disulfide linkages in proHNP1 are required for processing of the precursor to mature peptide and to confer resistance to these activating convertases.
Microbicidal activities of HNP1 products of proHNP1A processing by NE, PR3 and CG
The functional properties of HNP1 generated by serine protease processing of proHNP1A were tested in microbicidal assays against S. aureus. Natural HNP1 and NE, PR3 and CG processed preparations possessed nearly identical dose-dependent bacteri- cidal activities against this organism (Fig. 6) . These data demonstrate that HNP1 generated by NE and PR3 processing of proHNP1A is functionally equivalent to native HNP1. Of note, the Asn-Met-HNP1, produced by CG processing of proHNP1A, had staphylocidal activity equivalent to that of the fully processed HNP1 isoform.
To study the processing of human myeloid a-defensins, we established an in vitro assay utilizing recombinant proHNP1.
Approximately 60% of the recombinant His 6 -proHNP1 was folded with native disulfide linkages (Fig. 2) and misfolded propeptide was readily converted to the folded form in buffer containing urea and reduced/oxidized glutathione (Fig. S1 ). The expression of folded and misfolded forms of proHNP1 allowed for the analysis of the structural requirements for HNP1-mediated bactericidal activity in vitro. CNBr treatment of folded (proHNP1A) or misfolded (proHNP1B) precursor generated different HNP1 conformers (Fig. 3) . Despite the generation of oxidized peptides with masses matching that of natural HNP1, only the conformer generated by CNBr cleavage of proHNP1A had microbicidal activity against S. aureus. Processing of proHNP1A by NE or PR3 generated HNP1 peptide identical to natural HNP1, but proHNP1B was heterogeneous and was degraded by NE, PR3 and CG. These data demonstrate that the native HNP1 fold is necessary for proteolytic stability and accurate processing of the HNP1 propeptide by the three azurophil granule serine proteases investigated.
The requirement for the native HNP1 fold for microbicidal activity is consistent with a previous report demonstrating that an all Cys-to-Ala analog of HNP1 was nearly 10-fold less potent in bactericidal assays than the native peptide [38] . Similarly, disruption of disulfide linkages by mutagenesis of Cys residues to Ala or Ser in the human Paneth cell a-defensin HD5 reduced in vitro bactericidal activity demonstrating the requirement for the disulfides for in vitro bactericidal activity of this peptide [39] . In contrast, disruption of disulfide linkages in mouse enteric defensin, Crp4, by Cys-to-Ala mutagenesis did not eliminate microbicidal activities of the peptide [40] .
Results presented here support the hypothesis that NE and PR3, proteases that co-localize with myeloid a-defensins, participate in the intracellular maturation of HNP1. In previous studies with HL-60 and chronic myelogenous leukemia cells, Valore and Ganz demonstrated sequential proteolytic processing of proHNP with the formation of a 56-residue intermediate that is subsequently processed to mature HNP. While proHNPs were found exclusively in the cytoplasmic/microsomal fraction, the granule fraction contained only the intermediate and mature HNP [35] . Therefore it is likely that proHNP-processing by NE and PR3 occurs during the packaging of HNPs in azurophil granules. CG processing of proHNP1A produced a peptide that was extended at the amino terminus by two residues (Asn-Met) expressed at the carboxyl terminus of the HNP1 prosegment. Harwig et al. identified a minor form of HNP3 in fully differentiated neutrophils with the same Asn-Met dipeptide extension [41] reported here. Thus, CG processing of proHNP1 may also occur in vivo.
In contrast to the processing of folded proHNP1 by the azurophilic proteases, misfolded His 6 -proHNP1 was degraded by these enzymes. These results are consistent with studies demonstrating that Cys substitutions in mutants of RMAD4 [32] , Crp4 [40] and HD5 [39] are protease sensitive compared to the respective native peptides. However, in contrast to the examples cited above, misfolded His 6 -proHNP1B was disulfide-linked but still susceptible to proteolysis. These data demonstrate that the native disulfide motif is essential for proteolytic stability of the proHNP1 as well as the mature peptide.
Wilson et al. demonstrated that proHNP1 could be processed by matrix metalloproteinase 7 (MMP7; matrilysin) to generate a 59residue molecule with microbicidal activity, but no mature HNP1 (30 amino acids) was detected [42] . Since MMP7 is not present in neutrophils, it was proposed that MMP7 may be involved in the extracellular processing of proHNP1 released from the specific granules of neutrophils. In this regard, Borregaard and co-workers showed that proHNP1 is present in the specific granules of mature human neutrophils and that the propeptide is secreted by activated cells [43] . Since enzymatically active NE is present in inflammatory exudates [44] , we hypothesize that proHNP1, elaborated by activated PMNs, may be activated by NE (and possibly other enzymes such as PR3 and CG) in the extracellular milieu. Therefore this process may regulate the extracellular expression of mature a-defensin in the setting of inflammation.
The proHNP1 cDNA sequence encoding the natural 75 amino acid residue sequence extended by 5 amino acids (DDDDK; enterokinase recognition site) at the N-terminus was amplified using oligonucleotides (59-GCGAATTCGATGACGATGACAAGGAGCCACTCCAGG-CAAGAGCTG) and (59-GCGGATCCGGTACCTCGAGTCAG-CAGCAGAATGCCCAGAGTC) with plasmid PEC2081 (proHNP1 cloned in pCR2.1 Topo vector) as template. EcoRIand XhoI-digested DNA was ligated into pET28a+ vector to generate plasmid PEC2129 which encodes proHNP1 with a 41 amino acid residue N-terminal sequence containing the His 6 -tag (His 6 -proHNP1; Fig. 1 ). The plasmid was used to transform Escherichia coli BL21(DE3)(codon+), generating strain PEC2132-1 which was used for expression and purification of His 6 -proHNP1.
PEC2132-1 cells were cultured for 3 h at 37uC in 3 L of LB+Kanamycin (50 mg/ml) medium and expression of His 6 -proHNP1 was induced by addition of 0.5 mM isopropyl b-D-1thiogalactopyranoside (IPTG) for an additional 3 h. Cells were harvested by centrifugation and lysed by stirring in 60 ml guanidine lysis buffer (10 mM Tris HCl, 6 M guanidine, 100 mM NaH 2 PO 4 , pH 8.0) at room temperature for 2 h. The lysate was clarified by centrifugation and supernatant was mixed with 6 ml of Ni-resin (His-Bind resin; Novagen) overnight at 4uC. Resin was transferred to a chromatography column, washed with 10 volumes of wash buffer 2 (20 mM Tris HCl, 450 mM NaCl, 0.1% Tween, pH 8.0), and bound proteins were eluted with 3.5 column volumes of elution buffer (20 mM Tris-HCl, 450 mM NaCl, 0.1% Tween, 250 mM imidazole, pH 8.0). The eluate was dialyzed in Spectrapor 3 membrane against 2 L of dialysis buffer (4 M guanidine, 20 mM Tris, pH 8.0), overnight at 4uC.
The dialysate was acidified by addition of acetic acid to 10% (vol/vol) and fractionated on a 10 mm6250 mm Vydac C18 reversed phase high performance liquid chromatography (RP-HPLC) column equilibrated in aqueous 0.1% trifluoroacetic acid (TFA). Proteins were eluted using a water-acetonitrile (ACN) gradient containing 0.1% TFA. As discussed in Results, His 6 - 
Protein preparations, quantified using the BioRad protein assay (BSA standard) or by measuring absorbance at 280 nm, were analyzed by sodium dodecyl sulfate (SDS)-tricine and acid-urea (AU) gel polyacrylamide gel electrophoresis (PAGE) as described [45] . For western blotting, proteins resolved by SDS-tricine PAGE were transferred to nitrocellulose membranes using a GE Novablot/Pharmacia Biotech Multiphor II system. The mem-brane was blocked with 5% milk in TTBS (100 mM Tris HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) and incubated with 0.02 mg/ml anti-His 6 mouse IgG (Qiagen). The membrane was washed with TTBS and probed with goat anti-Fc mouse IgGhorse radish peroxidase conjugate (1:20,000 dilution; Sigma) for 1 h, washed with TTBS, and developed by incubation with Supersignal West Pico chemiluminescent substrate (Pierce) and the resulting chemiluminesence was visualized on x-ray film.
Matrix assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) was employed for analysis of HPLC-purified peptides. Typically, a 0.8 ml sample was mixed 1:1 with sample matrix (15% w/v a-cyano-4-hydroxycinnamic acid dissolved in 50% ACN+0.1% TFA), spotted and dried on the MALDI-TOF MS sample plate. In some cases, samples were first purified using C18 Zip Tips (Millipore) as recommended by the manufacturer. MALDI-TOF MS was performed on a Microflex LRF MALDI-TOF system (Bruker Daltonics) [46] .
Samples of lyophilized His 6 -proHNP1A (150 mg) or His 6 -proHNP1B (550 mg) were dissolved in 2 ml 80% formic acid containing 20 mg CNBr. The reaction mixtures were sparged with N 2 and incubated for 18 h in the dark at room temperature after which the solutions were diluted 10-fold with water and lyophilized. The reaction products were dissolved in 0.1% acetic acid and purified by RP-HPLC on an analytical C18 column and analyzed by MALDI-TOF MS. Fractions containing HNP1 were purified further by C18 RP-HPLC.
Human NE, PR3, and CG were purchased from Elastin Products Company. Reactions were performed in 50 ml volumes containing 15 mg His 6 -proHNP1 and 2 mg of protease. Buffer for NE and PR3 reactions was 50 mM Tris-HCl pH 7.5, 150 mM NaCl and CG reactions were performed in 50 mM Tris-HCl pH 8.3, 150 mM NaCl [32] . Incubation was performed at 37uC for 18 h after which 5 ml samples were acidified by addition of 10 ml 5% acetic acid, and desalted with C18 Zip Tips (see above) prior to MALDI-TOF MS analysis. The remainder of the reaction mixture was subjected to further purification by RP-HPLC on an analytical C18 column using a 0-60% water-ACN gradient containing 0.1% TFA. The identity of putative HNP-containing species was confirmed by MALDI-TOF MS analysis of reduced (DTT) and alkylated (iodoacetamide) samples which produced a new species with the mass of hexa-S-carboxyamidomethylated peptide derivative (+348 amu) [47] .
To generate enzymatically-processed HNP1 for functional studies, 150 mg of His 6 -proHNP1A was digested with 0.1 mg/ml PR3 or CG in 200 ml or with NE in 400 ml reactions for 22 h at 37uC and purified by C18 RP-HPLC as described above.
Analytical HPLC HNP1 preparations derived by enzymatic or CNBr treatment were analyzed on an Alliance 2690 Separations Module using a C18 column (3 mm particle, 180 Å pore, 2.0 mm6150 mm, Varian Polaris 5A) with a flow rate of 0.2 ml/min using a 0-to-60% ACN linear gradient in 0.1% aqueous TFA at 1% per minute. Eluting peaks were detected at 210 nm and 280 nm using a Waters 2487 Dual l absorbance detector using Clarity software. Natural (human neutrophil-derived) HNP1 was used as standard in these analyses.
The antibacterial activity of HNP1 preparations was evaluated using Staphylococcus aureus 502a as the test organism employing a microbicidal assay format described previously [46] . Briefly, log phase cultures of S. aureus (,1610 4 CFU/ml) were suspended in 100 ml buffer (20 mM Tris pH 8.0, 28 mM NaCl) plus 0.03% TSB with dilutions of HNP1 ranging from 0 to 8 mg/ml in 0.01% acetic acid for 2 h at 37uC. Aliquots were plated on TSB plates and colonies were counted after overnight incubation at 37uC. Native HNP1 from human leukocytes was used as control. The source of human leukocytes was discarded, de-identified buffy coat preparations from The University of California Irvine-Medical Center clinical labs. Figure S1 Folding of His 6 -proHNP1B. His 6 -proHNP1A and His 6 -proHNP1B were analyzed by C18 RP-HPLC and AU-PAGE. Shown are chromatograms and corresponding Coomassie stained gels of His 6 -proHNP1A and His 6 -proHNP1B analyzed A) without additional treatment, B) after 4 h incubation in TUN buffer alone, and C) after 4 h incubation in TUN buffer supplemented with reduced and oxidized glutathione. (TIF)
Text S1 Folding of His 6 -proHNP1B in redox buffer system. In a previous study, Wu et al. demonstrated efficient folding of proHNP1 in vitro using reduced/oxidized glutathione to mediate thiol-disulfide exchange [29] . Employing this approach, 10 mg of HPLC purified His 6 -proHNP1 was dissolved in 500 ml 50 mM Tris HCl pH 7.5, 0.8 M urea and 100 mM NaCl (TUN buffer) with or without 2 mM reduced and 0.4 mM oxidized glutathione and incubated at 37uC for 4 h at which point the reaction was stopped by addition of 50 ml acetic acid. Reaction products were purified by RP-HPLC on a Vydac analytical C18 column (4.6 mm6250 mm) using a 20-to-55% (1% per min; 1 ml/min flow rate) linear gradient of ACN in 0.1% TFA acetonitrile. Peak fractions were collected and analyzed by AU-PAGE. In the absence of glutathione, the retention time during RP-HPLC and electrophoretic pattern was unchanged compared to the respective starting materials (Fig. S1A and B ). After incubation with glutathione, the behavior of proHNP1A was unchanged on C18 RP-HPLC (Fig. S1C) and on AU PAGE (Fig.  S1C, lane 5) . However, treatment of proHNP1B with glutathione generated a new species that behaved like proHNP1A on both C18 RP-HPLC and AU-PAGE (Fig. S1C) . These data indicate that proHNP1A and proHNP1B are folded and misfolded forms of His 6 -proHNP1respectively and that proHNP1B can be folded in the presence of reduced/oxidized glutathione. (DOC) Foxp3(+) Regulatory T Cells Control Persistence of Viral CNS Infection We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4(+) CD25(+) Foxp3(+) Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8(+) T cells predominantly recognising the H-2D(b)-presented viral hemagglutinin epitope MV-H(22–30) (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8(+) effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS. The role of CD4 + CD25 + regulatory T cells (Tregs) in autoimmune and pathogen-induced immune responses has been studied intensively during recent years. An important tool was provided by the discovery of the transcription factor Foxp3 (forkhead box P3) as a marker for Tregs and their suppressive activity [1, 2, 3, 4] . During acute viral infections depletion of Tregs was found to prevent the development of exhausted T cells and to improve the immune response. In addition, transient depletion of Tregs in several persistent viral infections led to reactivation of virus-specific T cells and reduction of the virus load [5, 6, 7, 8, 9] . The important protective role of Tregs against an overshooting immune response in the CNS became obvious in animal models of stroke and experimental autoimmune encephalitis [10, 11] , and human immunodeficiency virus-1 (HIV-1)-associated neuro-degeneration, where they reduce astrogliosis and microglia-mediated inflammation [12] . Interestingly, some viruses even developed the strategy to support the expansion of Tregs in order to suppress anti-viral cytotoxic T cell (CTL) responses and to limit viral immunopathogenesis [13, 14, 15, 16, 17, 18] . Defects in regulation of numbers or the activity of Tregs are also involved in a number of human autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis [19] .
Viral infections of the brain mostly represent clinically important, often life-threatening complications of systemic viral infections. For example, after acute measles, CNS complications may occur early as acute post-infectious encephalitis, or after years of viral persistence as subacute sclerosing panencephalitis (SSPE). Epidemiological studies indicated that the primary MV infection of SSPE patients takes place predominantly below the age of two years, when the immune system of the host is still immature and residual maternal antibodies may be absent or not sufficient for complete virus neutralization [20, 21] . For intracerebral MV infection of mice, a transgenic human receptor for MV is not necessarily required, and various infection models exist depending on the age of the mice, the virus strain, and the infectious dose [22, 23, 24, 25, 26] . As found in genetically unmodified and MVreceptor transgenic mice, T cells and IFN-c have a critical role for protection and clearance of virus from the brain [27, 28, 29, 30] . Transient immunosuppression during MV-infection enhanced virus replication and facilitated persistence [31] . After intranasal infection of MV-receptor CD150-transgenic mice, a specific antiviral cellular immune response including an increased proportion of Foxp3 + Tregs in the spleen was observed [32] . Although the presence and activity of Tregs has been demonstrated, their actual role in viral immunosuppression or immunopathogenesis in the brain remains to be elucidated.
Here we investigated the role of Tregs for virus persistence in the CNS. According to our model, two week old C57BL/6 mice (an age in which these mice survive infection, while the immune system is still not fully matured) were intracerebrally infected and virus persists in a limited number of neurons in most animals for more than 10 weeks [33] . We expanded and depleted Tregs in the periphery during the persistent phase of the viral infection, and investigated whether this can be exploited to modulate the ''hidden'' CNS infection. Our data indicate that this is indeed the case and that manipulation of Tregs can be utilized to regulate virus-specific CD8 + effector T cells and virus persistence in the brain.
Regulatory T cells are present predominantly in spleen and lymph nodes, but at a low frequency also in the brain Two week old mice were intracerebrally (i.c.) infected with 10 3 PFU recombinant MV expressing the rodent brain-adapted haemagglutinin CAMH and eGFP, or, when indicated, the recombinant MV without eGFP. Both recombinant viruses (rMV Edtag EGFP-CAMH and rMV Edtag CAMH) have the same distinct tropism for mouse neurons, and infections cause similar acute and persistent CNS infections [24, 33, 34, 35] . These recombinant viruses were designated throughout the manuscript as rMV-green and rMV.
To determine the number of Tregs present in our infection model in secondary lymphoid organs and the brain, C57BL/6 mice were infected with rMV-green and analyzed 3, 7, 10, 14 and 28 days post infection. Lymphocytes were isolated from 6 draining cervical lymph nodes (LN), the spleen, and the brain of MVinfected and control (i.c. injected PBS) animals. The fraction of CD4 + CD25 + Foxp3 + T cells of all lymphocytes was determined to be 1.5-3% in the spleen and 4-6% in LN ( Fig. 1 A, B ) with no significant difference between infected and control animals. This corresponds to a total number of Tregs of approximately 1610 6 in the spleen and 2610 5 in the prepared LN at day 28 post infection. Only a small statistically not significant number of Foxp3 + T cells was detected in the brain, irrespective of the infection (data not shown). To obtain statistically significant results and to reduce standard errors caused by the staining procedure, we used DEREG mice, which express GFP in Foxp3 + cells. After infection of these mice with rMV (not expressing GFP), a small but significant number of GFP + cells, approximately 800 per brain, was detected in the brains, whereas almost no GFP + cells were detected in controls ( 
In order to find out whether Tregs have an influence on the persistent CNS infection we first expanded and functionally activated Tregs using the superagonistic anti-CD28 monoclonal antibody clone D665 (mAb D665) [36] . This mAb, while causing a transient expansion of the total lymph node and splenic T cell population, predominantly expands functional CD4 + Tregs without causing systemic cytokine release [37] . In order to test the efficiency of mAb D665 in young mice, we measured Tregs in a preliminary experiment in uninfected mice 3 days after a single i.p. injection of 100 mg D665 by flow cytometry. The proportion of CD4 + Foxp3 + Tregs increased in the spleen and lymph nodes approximately 2fold ( Fig. 2 A; n = 4; P,0.01). This result indicated that mAb D665 can be used for expansion of Tregs also in young mice.
For the following experiments, to investigate the effect of mAb D665 on the persistent viral infection, we applied 100 mg at day 14 and 21 post infection, and analyzed the brains at 28 dpi (Fig. 2 B ). Under these conditions the total number of lymphocytes increased significantly after D665 treatment in spleen and LN, but not in the brain (Fig. 2 C) . Furthermore, the proportion of Foxp3 + Tregs increased significantly (approximately 2-fold, P,0.01) in spleen in LN (Fig. 2 D) . Interestingly, histological analyzes revealed that the extent of the viral infection increased considerably after D665 treatment. Large clusters and groups of bright GFP-positive (directly reflecting virus replication) infected neurons emerged in the brains of these mice (Fig. 3 A) . This was in striking contrast to the brains of untreated mice at 28 dpi, which contain only a limited number of infected neurons (Fig. 3 B) . It is also strikingly different from brains at 14 dpi, when the treatment with D665 begins. At this time point areas with weak (vanishing) GFP expression are observed, that represent areas from which virus is being eliminated (Fig. 3 C) . The quantification demonstrated approximately 100-fold more infected cells per brain in D665treated animals in comparison to control animals at 28 dpi ( Fig. 3 D, compare lanes 3 and 5; differences were highly significant: P,0.0001). Two weeks later, at 42 dpi, the number of infected cells was reduced, but still higher than in control animals ( 
For the depletion of Tregs we used transgenic mice expressing the human diphtheria toxin (DT) receptor under the control of the Foxp3-promoter, which can be treated with DT to eliminate specifically Foxp3 + Tregs (DEREG-mice) [38] . To test the activity of the DT-batch used, adult DEREG mice were in a pilot experiment treated with DT under standard conditions (1 mg DT i.p. injected at 6 consecutive days) and analyzed the next day. Lymphocytes isolated from the spleen and lymph nodes were analyzed by flow cytometry to demonstrate the successful depletion of Foxp3 + GFP + Tregs by more than 90% (Fig. 4 A) . In order to assess the effect of Treg depletion on virus persistence in young mice, persistently infected mice were treated at 3 consecutive days (at day 17, 18, and 20 post infection) with 1 mg DT and analyzed at day 28 post infection ( Fig. 4 B) . In these mice, the viral infection was again quantified histologically in subsequent brain slices through the complete cerebrum as described earlier [33] . Treg depletion in DEREG 2/+ mice (compared to DT- treated DEREG 2/2 mice as control animals) led to a significant reduction of the number of infected neurons from brains of persistently infected animals (n = 6, P = 0.0098; Fig. 4 C) .
In order to investigate whether this increase in virus clearance correlates with the presence of higher numbers of virus-specific CD8 + T cells, we first identified T cell receptor recognized peptides presented by MHC class I of C57BL/6 mice (H-2 b ). Two CD8 + T cells were detected in the brain at 7, 10, 14, and 28 dpi (Fig. 5 A) . Interestingly, high percentages of D b MV-H 22-30specific CD8 + T cells are present in the brain during the persistent phase of the infection (at 28 dpi approximately 18% of all CD8 + T lymphocytes). Mock-treated animals (i.c. PBS injection; ctrl) did not contain virus-specific CD8 + T cells. After treatment of DEREG mice with DT, the number of Tregs decreased by approximately 95%, while the number of CD8 + T cells in brains slightly increased (Fig. 5 B) . Interestingly, after depletion of Tregs, the fraction and absolute number of D b MV-H 22-30 -specific CD8 + T cells increased from 2,000 to 8,000 cells per brain, or 5% to 23% of CD8 + T cells (n = 3, P = 0,05; Fig. 5 C) . Thus, depletion of Tregs during the persistent phase of infection led to an increase of virus-specific CD8 + T cells and a significant reduction of the persistent infection in the brain.
Our results indicate that the immune system keeps the ''hidden'' persistent viral CNS infection under permanent control. Manipulation of Tregs in the periphery had significant consequences for the fate of the viral infection in the brain, although only few infected neurons are present during the persistent phase of the infection. Expanding the number of Tregs by superagonistic CD28 antibody D665 led to an activation of virus replication and dramatic increase of the number of infected neurons, whereas transient depletion of Tregs by DT led to a significant reduction of the number of infected neurons. Interestingly, complete elimination of virus and clearance of the infection was not achieved by transient depletion of Tregs suggesting that additional means of an antiviral immune response are required for complete clearance.
Looking for Tregs in the brain, we did not detect a significant number of CD4 + Foxp3 + Tregs by FACS-analysis after staining with Foxp3-specific antibodies, and only a small number of GFP- Figure 3 . Expansion of T lymphocytes with the superagonistic CD28 antibody D665 induces virus replication and spread. Consecutive coronal brain sections (100 mm sections) were prepared from complete rMV-green-infected mouse cerebra and analyzed using the UV microscope. Overviews and details of a typical section of an infected brain of a mouse treated with mAb D665 and analyzed at 28 dpi (A), and sections of infected control animals in the absence of mAb D665 at 28 dpi (B), and 14 dpi (C) are shown. The numbers of infected eGFP + cells per brain (sections through the complete cerebrum of each animal were evaluated as described [33] ) were determined microscopically in infected control C57BL/6 mice at 7, 14, 28 and 42 dpi (D, lanes 1-4) and in D665-treated mice at 28 and 42 dpi (D, lanes 5 and 6). The difference between control and D665-treated mice at 28 dpi was highly significant (P,0,0001). doi:10.1371/journal.pone.0033989.g003 expressing Foxp3 + cells in infected DEREG mice. These findings suggest that only very few Tregs, if any at all, are required within the brain for the regulation of effector immune cells, and that this regulation predominantly takes place in secondary lymphoid organs. Sellin et al. observed an increase of Foxp3 + Tregs as a consequence of MV infection of CD150-transgenic mice in spleen and brain [32] , which suggests that also the MV-infection itself supports the expansion and migration of Tregs, and that these infection-induced Tregs may be part of the multifactorial MVinduced immunosuppression [3, 4] . Tregs can restrain effector T cell responses through the production of immunomodulatory cytokines, such as TGF-b, IL-10, and IL-35, expression of inhibitory ligands, such as CTLA-4 and LAG-3, cytokine consumption, and direct cytolysis. It remains to be elucidated which of these mechanisms are involved in the persistent brain infection with MV.
Several reports support the view that T cells play a decisive role in control of viral CNS infections. Using primary human lymphocytes, in vitro experiments demonstrated that CD8 + T lymphocytes control the dissemination of MV [39] . In resistant mouse strains the depletion of the CD4 + T cell subset by monoclonal antibodies led to a breakdown of resistance to the infection, whereas depletion of CD8 + T cells had no effect [23, 40] . In TAP-transporter deficient mice, which cannot present antigen on MHC class I molecules, MV was found to spread more transneuronally than in brains of normal mice [41] . These findings indicated that infected neurons are target cells of CD8 + lymphocytes, and that brain infections to some extent can be inhibited by CTL activity. Further investigations revealed that CD4 + T cells are able to protect either alone (in resistant mouse strains), or through cooperation with CD8 + T cells (in mice with intermediate susceptibility), and that CD8 + T cells are able to protect alone after immunization of the mice [42, 43] . Using CD46transgenic Rag-deficient mice, adoptive transfer of lymphocytes revealed that the combined activity of CD4 + T lymphocytes with CD8 + T cells or B cells is required to control the intracerebral infection [44] . Thus, most findings support the view that CD8 + T cells play an important role in the control of transneuronal virus spread, and our findings suggest that MV-specific CD8 + T cells are involved in maintaining the steady state and control of infection during the persistent phase of CNS infection.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of and control (DEREG 2/2 ) mice both infected i.c. with rMV-green and treated with DT. The reduction of mean values from 50 to 8 was significant, with P = 0,0098. The number of infected eGFP + cells per brain was determined by microscopic evaluation of 100 mm sections through the complete cerebrum as described [33] . doi:10.1371/journal.pone.0033989.g004
Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Würzburg (Permit Number: 55.2-2531.01-67/06). Specific pathogen free C57BL/6 mice were purchased from Harlan-Winkelmann, Germany. DEREG mice [38] were breeded in the animal facilities of the Institute for Virology and Immunobiology and the Centre for Experimental Medicine, Würzburg. Mice were anesthetized using isofluran and infected intracerebrally (i.c.) into the left hemisphere with 20 ml virus suspension containing 1610 3 PFU at an age of two weeks. To expand Tregs, mice were treated intraperitoneally (i.p.) with 100 mg superagonistic anti-CD28 monoclonal antibody (mAb) D665 [36] , and to deplete Tregs, DEREG mice were treated i.p. with 1 mg diphtheria toxin (DT; Merck) [38] .
Vero cells (African green monkey; ATCC CRL 6318) were cultured in Eagle's minimal essential medium (MEM) containing 5% fetal calf serum (FCS), 100 U/mL penicillin and 100 mg/mL streptomycin. Recombinant measles viruses expressing the rodent adapted haemagglutinin of the strain CAM/RB (CAMH) and/or not the enhanced green fluorescent protein (eGFP) rMV Edtag EGFP-CAMH (rMV-green) and rMV Edtag CAMH (rMV) [35, 45] were propagated using Vero cells.
For analyzes, animals were anesthetized with CO 2 and perfused with 4% (w/v) paraformaldehyde (PFA). Brains were fixed in 4% PFA at least 18 h, and free-floating sections (100 mm) were prepared using a vibratome (Technical Products International) as described [33, 35] . Slices were analyzed directly by UV microscopy. Photomicrographs were taken with a digital camera (Leica). Numbers of infected eGFP-positive neurons were counted and statistical analyzes done using the student's t test and the program Prism (GraphPad, Inc.).
Isolation of lymphocytes from lymph nodes, the spleen, and the brain Draining cervical lymph nodes and the spleen were pressed through a steel sieve in 4 ml HBSS and homogenized in a total volume of 13 ml HBSS. After a centrifugation step at 310 g for 10 min the cell pellets were resuspended in an adequate volume of HBSS (approximately 10 7 /cells/ml). Spleen cells were additionally treated with erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA) and washed with HBSS. Brains were pressed through a steel sieve in 5 ml HBSS 3% FCS and homogenized in a total volume of 20 ml HBSS 3% FCS. After a centrifugation step at 170 g for 10 min the cell pellet was resuspended in 1.4 ml dissociation buffer (23 mM CaCl 2 , 50 mM KCl, 42 mM MgCl 2 , 153 mM NaCl) containing 0.4 U collagenase (Serva) and 50 U Benzonase (Novagen) and incubated at 37uC for 1 h. Afterwards the cells were washed with HBSS and applied on a percoll density gradient to separate the lymphocytes from the rest like myelin debris or neuronal cells as described [46] . The lymphocytes were isolated and washed to remove the percoll for subsequent analyzes. mouse CD3 (clone 145-2C11)-, CD4 (clone RM4-5)-, CD8 (clone Ly-2)-, CD19 (clone 1D3)-and CD25 (clone 7D4)-antibodies were purchased from Becton Dickinson. PE-conjugated anti-mouse Foxp3 (clone FJK-16s)-antibody was purchased from NatuTec. Lymphocytes were stained in FACS buffer (PBS containing 0.4% BSA and 0.02% sodium azide) at 4uC for 20 min. Intracellular staining of Foxp3 was performed using the Foxp3 Staining Buffer Set (NatuTec) according to the manufacture's protocol. Briefly, cells were fixed and permeabilized in 500 ml fixation/permeabilization buffer (Concentrate/Diluent 1:4) at RT for 1 h and stained afterwards in permeabilization buffer at RT for 30 min. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson).
For identification of peptides presented by MHC class I that can be used in ELISPOT and pentamer staining experiments we used the software programs SYFPEITHI (University of Tübingen, Germany) and BIMAS (BioInformatics and Molecular Analysis Section, National Health Instituts, Bethesda, USA) to establish a ranking of potential peptides. From 12 potential H-2 K b and D bpresented peptides of MV-N and MV-H with the highest probability scores, we found that D b MV-H 22-30 and D b MV-H 446-454 (RIVINREHL and SNHNNVYWL, respectively) were most efficiently recognized. ELISPOT experiments were performed using the Mouse IFN-c ELISPOT set (BD Biosciences) according to the manufacture's protocol.
MHC class I (H-2D b ) pentamers presenting the selected peptide MV-H 22-30 (D b MV-H 22-30 -pentamers) were ordered from ProImmune Ltd (Oxford, UK). Cells were washed with FACS buffer and stained with 5 mL pentamer-solution diluted in 100 ml FACS buffer at 4uC for 30 min. After one washing step the cells were analyzed using the FACSCalibur. MV-specific cells were gated as CD8 + and CD19-negative lymphocytes to exclude pentamer + CD19 + cells. Superimposed high-frequency jet ventilation combined with continuous positive airway pressure/assisted spontaneous breathing improves oxygenation in patients with H1N1-associated ARDS BACKGROUND: Numerous cases of swine-origin 2009 H1N1 influenza A virus (H1N1)-associated acute respiratory distress syndrome (ARDS) bridged by extracorporeal membrane oxygenation (ECMO) therapy have been reported; however, complication rates are high. We present our experience with H1N1-associated ARDS and successful bridging of lung function using superimposed high-frequency jet ventilation (SHFJV) in combination with continuous positive airway pressure/assisted spontaneous breathing (CPAP/ASB). METHODS: We admitted five patients with H1N1 infection and ARDS to our intensive care unit. Although all patients required pure oxygen and controlled ventilation, oxygenation was insufficient. We applied SHFJV/CPAP/ASB to improve oxygenation. RESULTS: Initial PaO(2)/FiO(2 )ratio prior SHFJV was 58-79 mmHg. In all patients, successful oxygenation was achieved by SHFJV (PaO(2)/FiO(2 )ratio 105-306 mmHg within 24 h). Spontaneous breathing was set during first hours after admission. SHFJV could be stopped after 39, 40, 72, 100, or 240 h. Concomitant pulmonary herpes simplex virus (HSV) infection was observed in all patients. Two patients were successfully discharged. The other three patients relapsed and died within 7 weeks mainly due to combined HSV infection and in two cases reoccurring H1N1 infection. CONCLUSIONS: SHFJV represents an alternative to bridge lung function successfully and improve oxygenation in the critically ill. The swine-origin 2009 H1N1 influenza A virus has become the predominant influenza virus worldwide since its identification. H1N1 influenza might cause acute respiratory distress syndrome (ARDS) and potentially result in extracorporeal membrane oxygenation (ECMO) therapy [1] . Experience from Australia and New Zealand describes an incidence of mechanical ventilation in 64.6% of H1N1 patients and 11.6% ECMO (with a mortality of approximately 21%) treated within the intensive care unit (ICU) [2] .
Mortality rates of ARDS patients suffering from hypoxemia and/or hypercapnia remains high [3] . In this situation, ECMO therapy represents the standard of bridging lung function [2] . However, ECMO therapy is associated with unfavorable complications and high cost. In this report, we suggest an alternative strategy to bridge lung function: the use of superimposed high-frequency jet ventilation (SHFJV) in combination with continuous positive airway pressure/assisted spontaneous breathing (CPAP/ASB). This alternative ventilation strategy is based on jet ventilation to improve oxygenation at lower plateau pressure combined with assisted spontaneous breathing by CPAP/ASB to improve CO 2 removal.
In winter 2009-2010, five patients with ARDS (H1N1) were admitted to our ICU due to inadequate oxygenation by conventional mechanical ventilation from outside hospitals. ARDS was defined according to the definition of the American-European Consensus Conference on ARDS [4] .
Virological and microbiological tests were performed in bronchioalveolar lavage under sterile conditions. Influenza A H1N1 infection was determined by realtime reverse transcriptase-polymerase chain reaction (RT-PCR) assay. In addition, herpes simplex virus (HSV)-DNA, cytomegalovirus (CMV)-DNA, Epstein-Barr virus (EBV)-DNA, RSV, and adenovirus were tested by TAQMAN PCR. Atypical species, such as Mycoplasma, Pneumocystis jiroveci, or Legionella were screened through PCR. BAL, swabs, and blood cultures were taken routinely on admission and depending on clinical need.
Patients were ventilated with a Monsoon I or Monsoon III jet ventilator (Acutronic Medical Systems, Hirzel, Switzerland, distributed by IfM GmbH, Wettenberg, Germany) combined with a standard respirator, Evita XL (Draeger, Lübeck, Germany) or Hamilton G5 (Hamilton Medical, Bonaduz/Switzerland, distributed by Heinen & Löwenstein GmbH, Bad Ems, Germany).
• Spontaneous breathing within 12 h after admission • Positive end-expiratory pressure (PEEP) primary 12-15 mbar • Ppeak (peak pressure) < 30 mbar • Pmean < 20 mbar • FiO 2 , work pressure, and frequency of jet, ASB in adaption to blood gas analyses; goal: oxygen delivery (DO 2 ) normal, lactate normal, pH 7.3-7.45 • Supine position 135°up to 12-14 h as long as oxygenation benefits • ECMO (QUADROX PLS and ROTAFLOW RF 32, MAQUET GmbH &Co. KG, Rastatt, Germany) Interventional Lung Assist (ILA Membranventilator ® , Novalung GmbH, Talheim, Germany) when CO 2 > 50 mmHg, pH < 7.3 and no progress by conservative ventilation and sedation management
To achieve a RASS (Richmond agitation and sedation scale) of -3 to -4 propofol or midazolam, in two cases additionally with γ-hydroxybutanoic acid was used in combination with remifentanil or sufentanil and clonidine.
All patients received oseltamivir, two patients in combination with ribavirin and amantadine up to 9 days. HSV was treated as standard with acyclovir; two patients received foscarnet-sodium when CMV coinfection was suspected or proven.
Five patients with severe influenza A H1N1 infection and refractory hypoxemia were ventilated between 2 and 16 days before admission. The transfer of these patients to our ICU was due to failure to improve during conventional ventilation (PEEP 10-15 mbar, pressure-controlled ventilation with P peak up to 35 mbar, FiO 2 1.0).
On admission, patients presented with a paO 2 /FiO 2 ratio of 58 to 79 (Murray score > 3.0; Table 1 ). After institution of SHFJV, oxygenation improved in all patients within 24 h (paO 2 /FiO 2 ratio 105-306) with a PEEP of 8 to 15 mbar, P peak < 30 mbar, and P mean 15-18 mbar. Therefore, the institution of SHFJV allowed improved oxygenation with adequate DO 2 and a simultaneous reduction of lung injury, inducing high peak and mean pressures at the same time. Spontaneous breathing was set during 8 h after admission. Three patients required jet ventilation for up to 72 h. One patient required prolonged jet ventilation due to concomitant mycoplasma pneumonia, and the other due to persistent H1N1 pneumonia.
FiO 2 was reduced from initially 1.0 to 0.4-0.6 in four cases. However, one patient with a ventilation period of 14 days before admission due to H1N1 and concomitant Pneumocystis jiroveci infection needed a FiO 2 of 0.9 up to day 7.
Jet ventilation was performed in three cases with a work pressure of 1.2-1.7 bar, a frequency of 140-200/ min (Monsoon I Jet ventilator); in two patients a novel jet ventilator was used and the work pressure was set to 0.5-0.9 bar and the frequency adjusted to 500-600/min (Monsoon III Jet ventilator).
When only H1N1 infection was present, the oxygenation deficit was the leading clinical symptom. In cases with prolonged treatment (reinfection or concomitant infection with HSV, Mycoplasma) hypercapnia also was notable. Therefore, one patient received ECMO and one patient ILA therapy (Figure 1 ). The patient with ECMO therapy demonstrated improved oxygenation and normocapnia but deceased due to intracranial bleeding. This patient had histologic confirmed HSV pneumonia for 4 weeks without improvement after acyclovir/foscarnet treatment. A similar clinical pathway was observed in the two other patients who died.
Despite pulmonary failure, organ dysfunction was moderate. Some patients showed liver dysfunction; two of three nonsurvivors had acute renal failure. Vasoactive drugs were needed initially in all patients.
From anamnesis, four patients were obese; one patient had pre-existing medical complications (acute CMV colitis and acute Pneumocystis jiroveci pneumonia by ulcerative colitis). None of the patients had pre-existing immunodeficiency, such as HIV.
During ARDS, ECMO is seen as the standard therapy to improve oxygenation. The CESAR study demonstrated that in highly specialized centers, ECMO therapy may improve long-term outcome. However, this study had limitations within the study design, because the intervention and control group were treated at different centers [5] . In a review of 62 cases with ECMO therapy, the survival rate was 55% [6] ; 17.8% of patients died as a result of ECMO-related complications, translated into a mortality of 8% due to ECMO alone. A retrospective U. S. database analysis (1986-2006) demonstrated a mortality rate of 50% in patients treated with ECMO. Although technical improvement of ECMO equipment translated into fewer cases of circuit rupture, renal insufficiency, pulmonary hemorrhage, inotropic medications, hyperglycemia, extremes of pH, arrhythmias, or hypertension became more common [7] .
The primary goal for ARDS patients is to preserve an acceptable gas exchange without further injury to the lungs. High-frequency jet ventilation (HFJV) and highfrequency oscillatory ventilation (HFOV) are characterized by rapid delivery of small tidal volumes (V t ) (1-3 ml/kg predicted body weight) at high frequencies (2.5-5 Hz). As such, P mean during jet ventilation is comparable to the pressure level of PEEP in conventional mechanical ventilation due to its minimal V t . Yet, P peak and P mean are markedly reduced with a reduction of tidal hyperinflation and tidal decruitment [8] . Furthermore, oxygen toxicity may be reduced as lower FiO 2 often are required compared with mandatory ventilation [8] . This beneficial effect could be demonstrated in animal models of HFOV. In addition, pulmonary compliance improved, the infiltration of polymorph nuclear leukocytes was reduced, and tumor necrosis factor-α levels attenuated. As a result, injury to alveoli and membranous bronchioles was reduced [9] .
HFOV mainly differs from HFJV by active in-and expiration, whereas HFJV only uses active inspiration and passive expiration. Thus, HFJV allows patients spontaneous breathing, preserving muscles of diaphragm and intercostal musculature. This is a major advantage, because early spontaneous breathing significantly reduces deleterious organ-organ interactions, e.g., lungliver interactions, and improves liver perfusion [10] .
One major problem of jet ventilation is CO 2 clearance due to very low V t . There are two options in resolving this problem. The simplest approach is to establish spontaneous breathing. However, patients might be sedated and therefore need superimposed CPAP/ASB to improve tidal inflation until sedation is adapted. In the case of severe hypercapnia, the use of Interventional Lung Assist (ILA) can improve CO 2 clearance. We observed a high mortality rate, which can be -explained, in part, by patient selection. All of our patients presented in this report were admitted with fixed hypoxemia despite optimized conventional ventilation. Two patients presented a prolonged period of hypoxemia and mechanical ventilation before admission. One of these patients was already on mechanical ventilation 14 days due to septic CMV-colitis and Pneumocystis jiroveci pneumonia with poor prognosis. The other patient was admitted to our ICU after a prolonged period of severe hypoxemia and septic shock with hypoperfusion. All five patients had a HSV-PCR-positive BAL, confirmed via lung biopsy in one patient. Viral coinfection with HSV may cause and prolongs a status of persistent immunosuppression during severe H1N1 infection, which is associated with a high risk of death. This hypothesis is supported by findings of Monsalvo and colleagues, who found pathogenic immune complexes as previously unknown biological mechanism for the unusual age distribution of severe H1N1 infections [11] .
Although jet ventilation is actually not in the focus of ARDS treatment, we were able to demonstrate that SHFJV represents an alternative of lung-protective ventilation during ARDS. Compared with ECMO, it is easier to use, associated with fewer complications, cost-effective, and can be used in secondary and tertiary centers. Therefore, SHFJV is an alternative approach to improve lung function and oxygenation in patients suffering from ARDS. Nevertheless, this report is limited by the small study size, including a heterogeneous patient collective. Therefore, a controlled study with SHFJV to treat patients with ARDS is required for an evidencebased conclusion. Serious Invasive Saffold Virus Infections in Children, 2009 The first human virus in the genus Cardiovirus was described in 2007 and named Saffold virus (SAFV). Cardioviruses can cause severe infections of the myocardium and central nervous system in animals, but SAFV has not yet been convincingly associated with disease in humans. To study a possible association between SAFV and infections in the human central nervous system, we designed a real-time PCR for SAFV and tested cerebrospinal fluid (CSF) samples from children <4 years of age. SAFV was detected in 2 children: in the CSF and a fecal sample from 1 child with monosymptomatic ataxia caused by cerebellitis; and in the CSF, blood, and myocardium of another child who died suddenly with no history of illness. Virus from each child was sequenced and shown to be SAFV type 2. These findings demonstrate that SAFV can cause serious invasive infection in children. M olecular biology has revolutionized the diagnostics of infectious diseases through the introduction of more sensitive and specifi c diagnostic tests. Despite these advances, the etiologic agents of many apparent infections are still unidentifi ed. For example, the etiologic agent is unknown for many cases of apparent pneumonia (1); in a study conducted in California, USA, despite extensive testing and evaluation, an underlying cause of encephalitis was unidentifi ed for 207 (62%) of 334 patients (2) .
During the past few years, intensive searches for new viruses, using conventional virologic methods and metagenomics, have resulted in the discovery of several new viruses. During the past decade, the family Picornaviridae has grown as the number of recognized genera has increased from 6 to 12 (3, 4) ; the numbers of species, types, and subtypes have increased even more. However, only viruses from 3 genera (Enterovirus, Hepatovirus, and Parechovirus) have been fi rmly established as being capable of causing clinically signifi cant disease in humans. Viruses from other genera (Cardiovirus, Cosavirus, and Kobuvirus) have so far been detected only in noninvasively collected human sample material such as fecal and respiratory samples, and their clinical signifi cance remains to be fully elucidated. (Invasively collected sample material is that from tissues considered sterile, i.e., devoid of microorganisms.)
The phylogenetic relationships of human picornaviruses are shown in Figure 1 . Most picornaviruses that are pathogenic to humans are ubiquitous viruses capable of causing a variety of diseases, from monosymptomatic febrile infection to severe infection in the central nervous system and myocardium. However, most infections with these viruses are asymptomatic (5) .
Saffold virus (SAFV) was discovered by Jones et al. in 2007 by sequence-independent genomic amplifi cation of virus isolated from a fecal sample (6) . The sample had been obtained in 1981 from an 8-month-old child with fever of unknown origin. The genetic sequence of the virus indicated that the virus belonged to the species Theilovirus of the genus Cardiovirus, which contains 3 other members: Theiler's murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), and Thera virus. In mice, TMEV is capable of causing infection in the central nervous system, and some variants of this virus cause a persistent infection and even multiple sclerosis -like disease (7) . VHEV was isolated in the 1950s from cerebrospinal fl uid (CSF) from a patient with Vilyuisk encephalomyelitis, a progressive neurologic disorder that occurs in indigenous populations of an isolated part of eastern Siberia (8) . However, the correlation between VHEV and Vilyuisk encephalomyelitis is still uncertain because VHEV has been isolated by multiple passages in mice and thus may represent a highly divergent strain of TMEV. Thera virus (previously named Theilerlike rat virus) has been isolated from rats, but the clinical signifi cance of infections with this virus is unknown (9, 10) . The genus Cardiovirus also contains a second species called encephalomyocarditis virus. Only 1 serotype is known, and it is capable of causing encephalitis and myocarditis in various animals (11, 12) .
Since the discovery of SAFV, several articles have provided insight into its epidemiologic and, to a minor degree, clinical signifi cance. Saffold viruses are distributed worldwide (6, (13) (14) (15) (16) (17) (18) (19) , and 2 serologic studies have demonstrated that infection occurs early in life (14, 20) . However, fi nding an association with human disease has thus far been elusive. Most studies (13, 15, 17, (20) (21) (22) have tried to associate SAFV with gastroenteritis, but no convincing results have been produced. A few studies (16, 18, 21, 23) analyzed the clinical signifi cance of SAFV virus in the respiratory system, but no substantial association between the virus and respiratory symptoms or disease has been made. Only 1 study (21) reports having tested invasively collected sample material (CSF samples), but no fi ndings were positive.
To investigate the possible invasive potential of SAFV in humans, we developed a diagnostic PCR and tested CSF samples from a group of children. SAFV was detected in 2 of these children.
We tested previously submitted CSF samples for SAFV, reviewed the patients' medical records, and sequenced the viruses isolated. 
We selected fecal samples from 479 children <5 years of age with gastroenteritis that had been submitted for viral diagnostic testing from September 2009 through February 2010 and tested them for SAFV. Nucleic acid extracted from these samples was combined into 48 pools, with 9 or 10 samples per pool. Samples from pools with positive results were identifi ed, and new extractions from these pools were tested individually. However, enough sample material for new extractions was available for only about half of the samples.
Nucleic acids were extracted from 200 μL of CSF or blood (from SAFV-positive patients) by using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and semiautomatic extraction on the QIAcube instrument (QIAGEN). Nucleic acid was extracted from 200-μL fecal suspension (10% in phosphate-buffered saline) by using the Total Nucleic Acid Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) on the MagnaPure LC instrument (Roche Diagnostics GmbH).
Five microliters of extracted material was used per reverse transcription PCR (total volume 25 μL) by using the OneStep RT-PCR Kit (QIAGEN). The reaction mixtures contained 1 μmol/L of each primer and 0.2 μmol/L of probe. Design of the primers and probe was based on an alignment of all available SAFV sequences in GenBank (www.ncbi.nlm.nih.gov/genbank) in July 2010 by using ClustalW (www.clustal.org) and Primer3 (http:// frodo.wi.mit.edu/primer3) software. The primers and probe are selective for a highly conserved part of the 5′ untranslated region (Table) . The Strategene Mx3005P real-time thermocycler instrument (Agilent Technologies A/S, Horsholm, Denmark) was used for amplifi cation and detection with the following settings: 50°C for 20 min, 95°C for 15 min, followed by 45 cycles of 95°C for 15 s and 55°C for 1 min.
Genotyping was conducted by nested PCR and sequencing of parts of the viral protein (VP) 1 and VP2 regions of the capsid gene by using primers listed in the Table. The inner VP1 and VP2 primers amplifi ed DNA fragments of ≈599 and 577 bp, respectively. PCR products were purifi ed by using the High Pure PCR Purifi cation Kit (Roche Diagnostics GmbH) before sequencing, which was performed by using the inner PCR primers on an ABI automated sequencer and BigDye version 1.1 chemistry (both from Applied Biosystems, Darmstadt, Germany). Sequences were aligned, and phylogenetic analysis with known reference sequences was performed by using MEGA4 software (www.megasoftware.net). The sequences have been submitted to GenBank under accession nos. JF693612-23.
SAFV was detected in CSF from 2 of the 319 children. Additional sample material from these 2 children was subsequently obtained and tested. From child 1, blood and CSF collected at the same time and a fecal sample collected 2 weeks later were tested; only test results for the fecal sample were positive for SAFV. From child 2, a postmortem blood sample and a myocardial biopsy sample were tested; test results for each sample were positive for SAFV.
Child 1 was a 16-month-old, previously healthy boy who became ill in May 2009. The boy had a fever 6 days before hospital admission, followed 1 day later by sudden onset of monosymptomatic ataxia, with no fever. The ataxia fl uctuated from causing an insecure gait to walking into things and falling. The patient also had intermittent diffi culty controlling his arms when trying to eat. No history of recent travel was reported. The boy was in otherwise good health; he had no abnormal psychological symptoms and retained a normal degree of consciousness throughout the acute phase of the disease. Differential diagnoses at hospital admission were intracranial tumor or viral cerebellitis. The boy's 4-year-old sister remained healthy.
Laboratory test results are listed in the online Appendix Table  ( wwwnc.cdc.gov/EID/article/18/1/11-0725-TA1. htm). At hospital admission, CSF values (leukocyte count, protein level, and glucose level) were within reference ranges, and no microorganisms were detected. A magnetic resonance imaging scan of the brain showed a small venous anomaly in the left frontal lobe but no tumor, hemorrhage, or infl ammation. A fecal sample collected 2 weeks later yielded positive test results for parechovirus type 3 and negative results for enterovirus and adenovirus. Parechovirus was not found in the CSF or blood.
During the next 2 months, the ataxia remitted completely without sequelae. The diagnosis at this time was viral encephalitis, possibly caused by parechovirus type 3.
Child 2 was a 27-month-old, previously healthy girl who was found dead in her bed in August 2009; she had no Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 1, January 2012 9 known history of disease or symptoms. During necropsy, signs of cerebral herniation were detected. A small vascular malformation surrounded by edema was found in the brain. No signs of encephalitis or hemolytic uremic syndrome were found. The following sample materials were collected: CSF, blood, feces, myocardium, pericardial aspirate, lung tissue, and respiratory secretions (online Appendix  Table) . In the CSF, mononuclear pleocytosis was noted. Results of routine bacteriologic culture found coagulasenegative staphylococci in the CSF, a few nonhemolytic streptococci in the lung tissue, and a few nonhemolytic streptococci and a few Staphylococcus aureus organisms in the pericardial aspirate. Verotoxin-producing Escherichia coli was cultured from the fecal sample. The conclusion of the autopsy and laboratory fi ndings was that cerebral herniation was the immediate cause of death.
Virus from each of the 2 children was characterized by sequencing part of the VP1 region of the capsid gene, and the sequences were compared with those of other SAFVs detected in fecal samples from the patients with gastroenteritis ( Figure 2 ). Of the 48 fecal sample pools, 10 were positive for SAFV by PCR. From these pools, 6 individual samples were available for further testing; the VP2 capsid region was successfully sequenced for 4 of these 6 samples, and they were all SAFV type 2. Later, an SAFV type 2-specifi c VP1 PCR was designed, which provided VP1 sequences from all 6 fecal samples and samples from the 2 children reported here: the fecal sample from child 1 and the blood sample from child 2. The phylogenetic analysis ( Figure 2) showed that all viruses were SAFV type 2 and that the sequences arranged in 2 clusters with 8% nt differences and 5% aa differences between the clusters.
Several novel viruses have recently been discovered by using microarrays or mass-sequencing methods (6, 21) . Almost all of these viruses have been found in noninvasively collected patient materials, making the correlation to clinical disease diffi cult to establish. For the picornavirus group, this issue is further complicated by knowledge that this virus family can cause a wide variety of diseases with a high proportion of nonspecifi c symptoms or asymptomatic infections (5) .
We report 2 cases of invasive infection with SAFV type 2 in children. For each child, SAFV was detected in at least 1 compartment other than the central nervous system. This fi nding strengthens the evidence of an acute infection as the cause of clinical disease. In child 1, SAFV virus was found in the CSF and a fecal sample. In child 2, fi ndings were even more convincing because SAFV was found in 3 invasively collected samples: CSF, myocardium, and blood. No other credible cause of infection was found for either of the 2 children. In child 1, the only other positive fi nding was parechovirus type 3 in a fecal sample taken 2 weeks after onset of disease. Parechovirus was not found in the CSF and therefore seems unlikely as the cause of the acute symptoms. From child 2, several types of bacteria were identifi ed, but these seem unlikely to be the cause of death because culture of postmortem samples often grows commensal organisms. The bacteria from this child were not found consistently in the tissue samples, thus making systemic bacterial infection less likely. Verotoxinproducing E. coli also seems an unlikely cause of death because no diarrhea or signs of hemolytic uremic syndrome were present before the child's death or at autopsy.
At autopsy of child 2, a small vascular malformation surrounded by edema was found. This edema could be the cause of the herniation. However, the edema might also have been caused by cerebral infection or septicemia. This possibility is supported by the fi nding of mononuclear 10 Emerging cells in the spinal fl uid and the direct virus identifi cation in the CSF and blood. The exact cause of death and whether there is a connection between the infection and the changes surrounding the malformation are unclear. However, our investigations show that before her death, the child's blood contained SAFV. Child 1 had monosymptomatic ataxia preceded by 1 day of fever. The symptoms receded over the next few months, and the patient recovered fully. Child 2 died without any preceding symptoms or any known predisposing factors; this clinical picture is sometimes found for patients with enteroviral infections (24) .
The fi nding of SAFV in invasively collected samples from the 2 children described here fi ts well with the knowledge about the picornavirus group. SAFV probably behaves similarly to the viruses in the enterovirus group, producing mainly asymptomatic infections but also producing nonspecifi c symptoms in other patients and severe disease in a few patients. Enteroviruses are known to cause many more or less organ-specifi c diseases such central nervous system infection (meningitis, encephalitis, and myelitis), myocarditis, enanthema, exanthema, and septicemia. These 2 cases fi t well within the expected range of diseases attributed to picornaviruses, but further studies are needed to determine the exact correlation of SAFV to disease in humans.
In a previous study, Chiu et al. looked for SAFV in 400 CSF samples but found none (21) . Lack of detection could be explained by different assay sensitivities or by the selection of samples tested. In their study, patient selection was based on neurologic disease and patient ages were not reported.
Our study has also shown that Saffold virus type 2 circulated in Denmark in 2009, the same year that the children reported here became ill. Other studies have shown type 2 to be a common type of SAFV and to be circulating worldwide (13, 15, 16, 18, 20, 21, 23) . Because SAFVs are single-stranded RNA viruses, a nucleotide variation of 8% among the restricted number of samples in this study is expected.
In conclusion, we have established SAFV virus as a cause of invasive infection and a highly probable cause of severe disease in children. More studies are needed to further illuminate the role of SAFV as a human pathogen.  Pandemic (H1N1) 2009 among Quarantined Close Contacts, Beijing, People’s Republic of China We estimated the attack rate of pandemic (H1N1) 2009 and assessed risk factors for infection among close contacts quarantined in Beijing, People’s Republic of China. The first 613 confirmed cases detected between May 16 and September 15, 2009, were investigated; 7,099 close contacts were located and quarantined. The attack rate of confirmed infection in close contacts was 2.4% overall, ranging from 0.9% among aircraft passengers to >5% among household members. Risk factors for infection among close contacts were younger age, being a household member of an index case-patient, exposure during the index case-patient’s symptomatic phase, and longer exposure. Among close contacts with positive test results at the start of quarantine, 17.2% had subclinical infection. Having contact with a household member and younger age were the major risk factors for acquiring pandemic (H1N1) 2009 influenza virus infection. One person in 6 with confirmed pandemic (H1N1) 2009 was asymptomatic. I n early April 2009, human cases of infection with a novel infl uenza virus of swine origin, pandemic (H1N1) 2009 virus, were identifi ed in the United States and Mexico, and this virus spread rapidly across the world (1-3). On June 11, 2009 , the World Health Organization raised the pandemic level to 6, the highest level for pandemic alert (4) .
Estimating attack rates is a major task in characterizing pandemic (H1N1) 2009. Some studies have reported attack rates of pandemic (H1N1) 2009 among household members and aircraft passengers (5) (6) (7) . These studies suggested that the transmissibility of pandemic (H1N1) 2009 virus was low. These studies were conducted in outbreak settings, and attack rates were calculated on the basis of clinical diseases that included infl uenza-like illness (ILI) or acute respiratory illness (ARI) of close contacts rather than confi rmed infection with pandemic (H1N1) 2009 virus. In addition, in these studies only symptomatic index and secondary cases were included. Although most infections of pandemic (H1N1) 2009 infl uenza virus produce ILI or ARI symptoms (8) (9) (10) (11) (12) , subclinical infection can occur and can change the estimate of attack rate. In addition, the infectivity of asymptomatic case-patients has not been clearly defi ned (13) .
Because of the high rates of illness and death among the initial case-patients with pandemic (H1N1) 2009 (14) , the Chinese government decided to prevent and contain the rapid spread of disease through tracing and quarantine of persons who had close contact with persons with confi rmed cases of pandemic (H1N1) 2009. Beijing, the capital of the People's Republic of China, took strict containment and control measures through October 2009. The Beijing municipal government implemented border entry screening, ILI screening in hospitals, health follow-up of travelers from overseas, and quarantine and testing of close contacts to identify new introduction of cases and local transmission. Public health workers conducted epidemiologic investigation of all index case-patients (including those with subclinical infections) and traced and quarantined close contacts whose residence was within the jurisdiction of Beijing. We estimated the attack rate of pandemic (H1N1) 2009 virus infection and assessed risk factors or correlates for infection among different types of close contacts, including household members and aircraft passengers.
In 2009, under the guidance of the Beijing Center for Disease Prevention and Control (Beijing CDC), a network of 55 collaborating laboratories was established to perform reverse transcription PCR testing to confi rm cases of pandemic (H1N1) 2009 (15) . The confi rmed cases included symptomatic and asymptomatic cases, and these cases were detected mainly by border entry screening, ILI screening in hospitals, health follow-up of travelers from overseas, and quarantine and testing of close contacts. Once confi rmed, index case-patients were immediately quarantined in designated hospitals to receive treatment while in isolation. All the confi rmed cases were required by law to be reported to Beijing and local CDCs. From May through October 2009, a detailed epidemiologic investigation was conducted for each confi rmed case of pandemic (H1N1) 2009 (including symptomatic and asymptomatic cases) by Beijing and local CDCs within 6 hours after confi rmation of infection. Patients with confi rmed cases were interviewed about demographic characteristics, course of illness, travel and contact history, and information about close contacts. Patients with confi rmed cases were categorized as having imported cases (travelers) and locally acquired cases (no travel history) on the basis of where the infection was acquired.
Close contacts were defi ned as anyone who ever came within 2 meters of an index case-patient without the use of effective personal protective equipment (PPE) (including masks and gloves, with or without gowns or goggles) during the presumed infectious period. Trained staff from local CDCs made the determinations on the basis of fi eld investigation. The relationships of close contacts to index case-patients were categorized as 1) spouses, 2) other household members, 3) nonrelated roommates, 4) contacts at workplace or school, 5) nonhousehold relatives, 6) passengers on the same fl ight, 7) friends, and 8) service persons met at public places. A close contact on an aircraft was defi ned as a passenger sitting within 3 rows in front and 3 rows behind the index case-patient.
All close contacts were traced and quarantined for 7 days after the most recent exposure to the index casepatient. All index case-patients detected between May 16 (the fi rst case, the date of confi rmation) and September 15, 2009 (before widespread transmission in Beijing), and their close contacts were included in this study. We excluded cluster or outbreak cases for which close contacts could not be determined clearly by epidemiologic investigation (the transmission chain was obscure).
For each close contact, before quarantine, a pharyngeal swab specimen was collected for reverse transcription PCR testing, regardless of symptoms. A second pharyngeal swab specimen was collected for testing for pandemic (H1N1) 2009 virus if any of the following symptoms developed in a close contact during quarantine: axillary temperature >37.3°C, cough, sore throat, nasal congestion, or rhinorrhea.
Data were analyzed by using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Median and range values were calculated for continuous variables, and percentages were calculated for categorical variables. Differences in attack rates were compared between subgroups of close contacts by using the χ 2 test. For the signifi cant difference found in multiple subgroups, this test does not enable identifi cation of which multiple subgroups are signifi cantly different, only that across all the subgroups there are differences. The variables with p<0.10 in χ 2 test were included in multivariate analysis. Multivariate unconditional logistic regression analysis was conducted to determine risk factors associated with infection in close contacts. Backward logistic regression was conducted by removing variables with p>0.10. Odds ratios (ORs) and 95% confi dence intervals were calculated for potential risk factors of infection. The Hosmer-Lemeshow goodness-of-fi t test was used to assess the model fi t for logistic regression. All statistical tests were 2-sided, and signifi cance was defi ned as p<0.05.
A total of 613 eligible index case-patients, detected from May 16 through September 15, 2009, were included in this study. Through fi eld epidemiologic investigations, 7,099 close contacts were traced and quarantined in Beijing. The median number of close contacts per index case per day was 7.0 persons (range 2.0-95.0 persons); the median number for an imported index case was 7.0 persons (range 1.7-95.0 persons) and for a locally acquired index case was 5.3 persons (range 1.0-25.0 persons). For the 601 symptomatic index case-patients, the median interval between illness onset and sample collection was 1.0 days (range −1.9 to 7.0 days).
Among close contacts with symptomatic infection, the median interval between illness onset and sample collection was 0.5 days. More than 85% of close contacts were quarantined within 72 hours after interview of the index case-patients. The median interval between fi rst exposure and quarantine was 3.4 days for the close contacts, and it was shorter, on average, for fl ight passenger contacts than nonpassenger contacts (1.7 days vs. 3.8 days). For symptomatic close contacts infected with pandemic (H1N1) 2009, the median of generation time (i.e., the time from illness onset in an index case to illness onset in a secondary case) were 2.4 days; it was shorter for fl ight passenger contacts than nonpassenger contacts (1.6 days vs. 2.5 days) ( Table 1) .
Approximately 43% of the index case-patients were women; the median age was 20 years, and 38% likely contracted pandemic (H1N1) 2009 virus locally because they had not traveled recently. Among the index casepatientss, 2% had subclinical infection. Only 18% of index case-patients had close contacts with confi rmed pandemic (H1N1) 2009 (Table 2) , and the total number of close contacts who were infected by the virus from 110 index case-patients was 167.
Fifty percent (3,514 of 7,032) of close contacts were women, and the median age was 27 years. Approximately 12% of close contacts were household member of index case-patients (spouse or other household member), and aircraft passengers accounted for 44% of close contacts. Approximately 61% of close contacts were exposed to symptomatic index case-patients during their symptomatic phase. About 70% were quarantined in a quarantine station ( Table 2 ).
The overall attack rate for infection among close contacts (positive test result) was 2.4% (167 of 7,099), indicating that 1 index case-patient transmitted infection to 0.27 close contacts (167 of 613) on average (reproduction number = 0.27). Among those close contacts with a positive test result, 14.4% (24 of 167) had subclinical infection; among the close contacts with positive test results at the start of quarantine, 17.2% (20 of 116) had subclinical infection.
Attack rates did not differ by index case-patient's sex (p = 0.225). However, attack rates differed signifi cantly by index case-patient's age (p = 0.022), and the lower attack rate was found for older index case-patients. There was no signifi cant difference in attack rates between close contacts of patients with imported cases and those with locally acquired cases (p = 0.282). No infection was found in close contacts exposed to index case-patients with subclinical infection, and the attack rate observed in close contacts exposed to symptomatic index case-patients during their symptomatic phase was higher (p<0.001). Almost identical attack rates were found among male and female close contacts (p = 0.808). However, attack rates were signifi cantly different among different age groups of close contacts (p<0.001), and the lowest attack rate was found for those >50 years of age. The attack rates were signifi cantly different across 8 contact types (p<0.001). The attack rate was 5.3% among spouses and 6.6% among other family members in the household, and was lower among other types of close contacts ( Table 3 ).The attack rate among passengers on the same fl ight was low, 0.9% overall, and 1 index case-patient transmitted infection to 0.19 close contacts on a fl ight on average (28 of 147), and the attack rate was higher among the passengers with longer fl ight times (>12 hours, p = 0.001). The attack rate among close contacts of service persons at public places was 0.2%, and 1 index case-patient transmitted infection to 0.01 close contacts of service persons on average (1 of 113). Nonpassenger close contacts with longer exposure duration (>12 hours), compared with those with shorter duration (>12 hours), recorded the higher attack rate (p<0.001) ( Table 3) .
By multivariate analysis, age and type of contact were the major predictors of infection ( (OR 3.42; p = 0.002) and 0-19 years of age (OR 7.76; p< 0.001) were at higher risk for infection. Other signifi cant independent risk factors associated with infection included being a household member of a person with an index case (OR 3.83; p<0.001), being exposed to index case-patients during their symptomatic phase (OR 1.86; p = 0.003), and exposure duration >12 hours (OR 1.83; p = 0.002). Similar risk factors were observed among aircraft passengers.
We estimated that pandemic (H1N1) 2009 virus was transmitted by 18% of index case-patients to their close contacts and that 2.4% (167 of 7,099) of close contacts we traced were infected. Our data indicate that pandemic (H1N1) 2009 virus has low transmissibility in nonoutbreak settings.
We found that 1 index case-patient transmitted infection to 0.27 close contacts on average, i.e., reproduction number = 0.27. This fi nding suggests that among those quarantined index case-patients, the number of persons with secondary cases who could be traced through rigorous fi eld investigation was small and far less than the number needed for the sustainable transmission of infectious disease in the population (reproduction number >1). However, the fact that the pandemic eventually spread in Beijing indicates that contact and case tracing were far from complete, especially later in the summer and early fall of 2009. The strict control measures may have worked to some extent at the beginning but were outpaced by local transmission (16) ; the percentage of locally acquired infections ranged from <10% in June 2009 to >80% in September 2009 (data not shown).
In this study, the median number of close contacts per index case-patient per day was 7.0 persons. Although locating and quarantining these close contacts was done quickly, and stringent quarantine measures were used, which hindered implementation of control measures, the real number of close contacts was unknown and probably exceeded this number. Many close contacts were persons met in public places, including public transportation, theaters or cinemas, and shopping malls, and it is nearly impossible to trace all of the contacts. In addition, some persons who had worn PPE during contact with index casepatients were excluded from close contacts management (i.e., they were not quarantined), but because wearing PPE might not protect (or fully protect) against infection, some persons excluded might have become infected. In addition, many persons with mild and asymptomatic cases cannot be detected, but they may transmit the virus. Furthermore, the short generation time of pandemic (H1N1) 2009 shown in this study and in a previous study (13) could lead to the rapid accumulation of infection sources and close contacts. This rapid compounding could overwhelm response capacity and would have resulted in compromised effectiveness of containment measures. It should also be mentioned that we did not include persons with cluster or outbreak cases for whom close contacts could not be determined clearly by epidemiologic investigation to examine the basic feature of pandemic (H1N1) 2009 (e.g., generation time), and the reproduction number obtained from our data is an underestimate.
Attack rates of infection differed signifi cantly by contact type. Among household members of index casepatients, the attack rate was the highest, as shown in the multivariate analysis after controlling for age and other factors. The most likely reason for this fi nding is that household members are more likely to have come into closer contact with index case-patients for a longer period with shorter distance and longer duration. Another possible reason is that household members may have some certain linkage with index cases in genetic susceptibility or living habits that would cause higher predisposition in household members than in other close contacts. This fi nding is similar to fi ndings in other investigations of respiratory infectious disease (17) .
Close contacts on fl ights accounted for the highest proportion of all the close contacts, in part because of how the index cases were detected and the broad defi nition we used for close contacts. However, the attack rate was much lower than that for other close contacts; 1 index case infected only 0.19 close contacts on fl ights on average. This fi nding indicated that the possibility of transmission of pandemic (H1N1) 2009 virus on fl ights was low, and the yield of tracing and quarantining of close contacts on fl ights was limited. Tracing contacts of service persons at public places was more diffi cult than tracing other categories of contacts, and the lowest attack rate (0.2%) was recorded in this category. Despite extensive measures, on average, only 0.01 infected close contacts per index case-patient were identifi ed among service persons. Tracing the contacts of service persons at public places seems far less cost-effective. Criteria for close contacts on fl ights and those of service persons should be refi ned with respect to exposure duration and age of those exposed. Exposure to index case-patients for >12 hours was a signifi cant independent risk factor for infection in fl ight passenger contacts. This fi nding suggests that limiting the time of contact with persons with ILI on aircraft can reduce risk for transmission, and a long duration of exposure may be necessary for transmission to occur on aircraft.
Younger close contacts were at higher risk for infection than older ones. The possible reason was that younger persons had much closer contact with index case-patients than did older persons; another reason may be that younger persons were more susceptible to infection with pandemic (H1N1) 2009 virus (18) . This fi nding was consistent with fi ndings reported in other studies (5, 6) . No secondary cases were found among close contacts exposed to index case-patients with subclinical infection. The attack rate among close contacts who were exposed to symptomatic index case-patients during their symptomatic phase was much higher than that among those exposed to these case-patients before their illness onset. Exposure to index case-patients during the symptomatic phase was a signifi cant independent risk factor for infection among close contacts. These fi ndings indicate that the infectivity of pandemic (H1N1) 2009 virus was higher after illness onset, and that the infectivity of symptomatic pandemic (H1N1) 2009 case-patients before illness onset was higher than that of persons with subclinical cases, although persons in each group were asymptomatic when in contact with other persons.
In general, the earliest infectious time for pandemic (H1N1) 2009 was considered as 1 day before illness onset (19) . We found that index case-patients and infected close contacts shed pandemic (H1N1) 2009 virus <1 day before illness onset, which suggests that the infectious period of symptomatic persons with pandemic (H1N1) 2009 might be <1 day before illness onset.
Among close contacts with pandemic (H1N1) 2009, ≈14.4% were asymptomatic. It is noteworthy that specimens from some close contacts tested negative for pandemic (H1N1) 2009 virus before quarantine, but those persons could shed the virus during quarantine without symptoms. Such infection could not be detected, and the proportion of subclinical infection was underestimated. Therefore, we calculated the proportion of subclinical infection by cross-sectional analysis of the subclinical infection of close contacts before quarantine, and we found that ≈17% of case-patients with pandemic (H1N1) 2009 were asymptomatic.
This study has several limitations. We could not fi nd all close contacts of persons with pandemic (H1N1) 2009 and did not know their infection status, so the infection parameters of pandemic (H1N1) 2009 that we found in this study might not be precise, especially for reproduction number, which may be underestimated to some extent. Furthermore, we could not exclude the possibility that the infected close contacts had been infected from another unknown source before quarantine started, which might infl uence our conclusion to some extent.
In summary, the attack rate among close contacts was low, even among household contacts. Household member and younger age were the major risk factors for infection with pandemic (H1N1) 2009 virus among close contacts. Approximately 17% of cases of pandemic (H1N1) 2009 were asymptomatic. Table 2 were included in multivariate unconditional logistic regression analysis. Hosmer-Lemeshow goodness-of-fit test was used to assess the model fit for logistic regression. OR, odd ratio; CI, confidence interval; NA, not available, indicating not included in the final model. †One dependent variable (infection with pandemic [H1N1] 2009 virus) and 5 independent variables (age of index case-patient, type of exposure to index case-patients, age of close contacts, relationships to index case-patients, and exposure duration of close contacts) were included in multivariate analysis. One independent variable (age of index case-patient) was removed in the stepwise regression equation. The goodness-of-fit test suggested that the logistic regression model fitted well (p = 0.631).
‡One dependent variable (infection with pandemic [H1N1] 2009 virus) and 4 independent variables (age of index case-patient, type of exposure to index case-patient, age of close contacts, and exposure duration of close contacts) were included in multivariate analysis. Two independent variables (age of index case-patient and type of exposure to index case-patient) were removed in the stepwise regression equation. The goodness-of-fit test suggested that the logistic regression model fitted well (p = 0.982).
§One dependent variable (infection with pandemic [H1N1] 2009 virus) and 5 independent variables (age of index case-patient, type of exposure to index case-patient, age of close contacts, relationships to index case-patient, and exposure duration of close contacts) were included in multivariate analysis. Two independent variables (age of index case-patient and exposure duration of close contacts) were removed in the stepwise regression equation. The goodness-of-fit test suggested the logistic regression model fitted well (p = 0.751).
¶Exposed to symptomatic index case-patients before their illness onset or exposed to index case-patients who had subclinical infections. Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture. In the past 20 years, it has become increasingly evident that lipids are important bioactive molecules that mediate signalling cascades and regulatory events in the cell. The ability to synthesize lipids predisposes an organism to function as a host to parasites that have lost or lack this trait [1] . Viruses as obligate parasites rely exclusively on the host to fulfill their membrane and lipid requirements. This is especially true for enveloped viruses since they utilize host-derived lipid membranes to facilitate release from infected cells by budding as well as to enter cells through membrane fusion. Lipids also form an integral structural component of the virus particle.
For most viruses that replicate in the cytoplasm of infected cells, lipids are essential for the replication of viral genomes. Both enveloped and non-enveloped viruses induce extensive ultrastructural changes in infected cells. Host-derived membranes are rearranged to provide extensive platforms that help to assemble arrays of replication factories [2] [3] [4] [5] [6] . Some of these factories are housed in specialized membrane compartments that assist in evading host antiviral defense mechanisms [2] [3] [4] 7] . These compartments also function to increase the local concentration of molecules necessary for efficient viral RNA replication and particle assembly. Recent advances in electron tomography techniques have been instrumental in providing a three-dimensional perspective of these virus-induced membranes [2] [3] [4] 7] . However, the metabolic cost to the host or vector and the contribution of lipid biosynthesis and trafficking to the formation of these replication factories is yet in its early stages of investigation [8] [9] [10] [11] [12] .
In this study, we have focused on the importance of lipid biosynthesis on dengue virus (DENV) replication. DENV is one of the most aggressive re-emerging pathogens worldwide [13] . Over two and a half billion people in more than 100 endemic countries are at risk for contracting dengue fever. Currently 50-100 million cases of dengue fever are estimated annually [14] . Since DENV replicates within the mosquito vector as well as the human host, the spread of the virus can be greatly reduced by controlling the vector. Much effort has been placed in understanding the dynamics of virus transmission and replication in the mosquito vector, including identification of host proteins in the midgut and salivary glands that are regulated by DENV infection [15] [16] [17] . Less is known about the global impact of DENV on host metabolic pathways.
Previous electron microscopy studies on DENV infected mosquito cells have shown that virus-induced membrane structures similar to those observed in human cells are prevalent [18] [19] . This extensive requirement in both host and vector, for intracellular membranes that support viral RNA replication and assembly suggest that quantifiable changes may exist in the lipid repertoire of the infected cell to assist in the formation of these membranes. Identifying these lipid changes that occur during infection is a necessary first step to discovering how DENV and its constituent proteins modify the lipid metabolism of cells. The reason(s) for such modifications has yet to be described, but can be pursued with a knowledge of which lipid changes occur. Furthermore, novel therapeutics that modify or inhibit these lipid changes and lipid-protein interactions could conceivably result in inhibition of virus replication.
To investigate these possibilities, we used high-resolution mass spectrometry methods to profile the lipidome of DENV infected mosquito cells. We have identified several lipid classes that are regulated by DENV infection. Many of these lipids have characteristic roles in influencing membrane architecture as well as functioning in cellular signal transduction pathways. Specifically, we have identified differences induced in the lipid profile upon virus binding and entry alone compared to those induced by viral RNA replication, assembly and egress. We have also profiled the lipidome of cells treated with an inhibitor of de novo phospholipid biosynthesis. Through this we have identified a lipid environment that supports cell survival but is yet inhibitory to DENV replication.
We had previously shown that fatty acid biosynthesis was a key target of DENV in human cells and that the rate limiting enzyme, fatty acid synthase (FAS), was both required and re-localized to sites of viral RNA replication during DENV infection [20] . In this study, using an inhibitor of FAS, C75, we determined that this requirement for fatty acid biosynthesis was conserved between the host and its vector during DENV infection. In the presence of C75 DENV replication was significantly reduced in C6/36 mosquito cells indicating that fatty acid biosynthesis is important for virus viability ( Figure 1A ). Furthermore, a time course of addition of C75 ( Figure 1B ) indicated that while pre-treatment or treatment of cells with C75 during viral adsorption reduced virus replication by ,10-100 fold, the most significant effect occurred upon addition of the drug at 4 and 8 hr post-infection (,1000 fold). This suggested that a post-entry step was affected by the inhibition of FAS. A comparison of virus released into the supernatant to intracellular virus indicated that an accumulation of intracellular virus was not occurring in the C75 treated cells (data not shown). Thus, the block in replication was not at the level of virus assembly or release.
Based on the observation that DENV induces significant rearrangements in the membrane architecture of infected cells, together with its distinct susceptibility to inhibitors of FAS, it was our hypothesis that lipid biosynthesis was important to DENV replication. Therefore, to investigate whether the intracellular lipid composition was altered during DENV infection, we carried out LC-MS-based analyses of the global lipidome of mosquito cells infected with DENV. To differentiate between changes to the lipid profile that occurred upon exposure to infectious virus versus that induced by virus entry alone, we included UV-inactivated DENV (UV-DENV) in the studies. This inactivated virus is only capable of binding, entry and initial rounds of viral RNA translation but does not have the ability to replicate its genome. Figure 2 shows a principle component analysis (PCA) plot of the overall lipid abundance in C6/36 mosquito cells infected with either DENV or UV-DENV at 36 and 60 hr post-infection. The 36 hr time point was chosen to represent a peak in viral replication while the 60 hr time point represents late stages of replication as well as increased cellular stress. Our previous analysis of earlier time points (ie. 24 hr post-infection) indicated that concurrent with increasing RNA synthesis activity and virus release, there were substantial changes in the lipidome of infected cells. Some of these changes were greater (higher fold changes) than those observed at the 36 hr time point. However, the overall intensities of the expressed lipids was lower contributing to a low signal to noise ratio. Furthermore, the number of species expressed at significant levels (p,0.05) were limited. Therefore, we chose to pursue the later time points. Under conditions that ensured that all cells were infected (multiplicity of infection of 20), optimal viral RNA synthesis occurred between 24-36 hr post infection (data not shown). A total of 7217 features observed in the LC-MS analyses were included in the PCA analyses. The plot shows specific segregation of lipid profiles between DENV and UV-DENV exposed cells compared to uninfected cells (mock). A temporal regulation of the lipid profile was also observed. However, this is more discernible upon analysis of individual lipid species (Figure 3 ) rather than in the PCA analysis. Overall, in this whole cell analysis
Dengue virus is one of the most aggressive human pathogens worldwide. It causes 50-100 million infections per year but there is no vaccine or antiviral that is currently effective against the disease. The virus is spread by Aedes aegyptii and Aedes albopictus mosquitoes and viral replication within the mosquito vector is required for transmission to a new human host. During this replication cycle, the virus causes significant changes to the membrane organization of infected cells. These virus-induced membrane alterations help to assemble arrays of viral replication factories and aid the virus to evade host antiviral defense mechanisms. Previously, much effort has been placed in trying to identify viral and cellular protein effectors that aid virus replication. In this study we have explored the role of lipids in the formation of these extensive membrane platforms in mosquito cells. Using high-resolution mass spectrometry we have profiled the lipid composition of dengue virus infected mosquito cells and compared it to uninfected cells. Through this we have identified several lipid classes that are differentially regulated during dengue virus replication. Using inhibitors of lipid biosynthesis we have also identified a lipid repertoire that is inhibitory to viral replication. Knowledge of how dengue virus utilizes cellular lipids and downstream signaling pathways to facilitate its replication will provide novel targets that could be utilized for developing effective antivirals. This study is also a forerunner for future comparative analyses of the human host and vector membrane environments required for viral replication. 15% of the metabolites identified were significantly (Anova p,0.05) different between virus infected cells and the mock control.
Based on the total number of lipids that were significantly regulated between groups (uninfected and infected, Anova p,0.05) and subsequently structurally identified (Table S1 and Methods) the overall abundance of lipid classes is as follows: phosphotidylcholine (PC), 39.2%; phosphatidylethanolamine (PE), 31.2%; phosphatidylserine (PS), 0.8%; sphingomyelins (SM), 18.5%; ceramide (CER), 5.3%; lysophospholipids, 0.8% and ceramide phosphoethanolamine (CER-PE), 4%. This distribution is similar to the membrane lipid composition of eukaryotic cells where the most abundant phospholipid is PC [21] . It is also consistent with the membrane lipid composition of the Diptera species where PE is a predominant PL [22] . CER-PE, which is preferentially expressed in insect cells, is also observed here [23] [24] . Several of these lipids are differentially regulated upon exposure of cells to DENV or UV-DENV ( Figure 3 ). It was noticeable however, that several negatively charged lipid classes such as phosphatidylglycerol (PG), phosphatidylinositol (PI), and Cardiolipin, were absent from this list. These lipids have previously been reported to account for 6-13% of the lipidome of mosquito cells [25, 26] . However, in this whole cell analysis, these lipids were not significantly regulated during virus infection.
A comparative analysis of the overall abundance of lipid species (relative to the mock control) in DENV and UV-DENV exposed cells is shown in Figure 3A and B. In all of the lipid classes with significantly regulated lipids (Anova p,0.05), DENV-infected cells have a unique expression pattern (expressed lipid molecular species) compared to UV-DENV infected cells. For a complete list of the differentially expressed lipids see Table S1 .
Phospholipids (PL). In this whole cell analysis, the primary PLs that were significantly regulated (Anova p,0.05) (and subsequently structurally identified) were mostly neutral or zwitterionic. PS was the only acidic lipid significantly regulated. At 36 hr post-infection, there was an ,2 fold up-regulation of PC species in DENV-infected cells compared to UV-DENV and mock controls ( Figure 3A ). At the later time point (60 hr post-infection), the relative levels of PC remained elevated in DENV-infected cells, however the differential expression between DENV and UV-DENV-exposed cells was not as evident ( Figure 3B ). Selected PC species were up regulated only at the 36 hr time point while others were up regulated at both time points ( Figure 3C ). Interestingly, a majority (,80%) of the PC species that were up regulated had unsaturated fatty acyl chains.
Analysis of the overall fold change in PE at the 36 hr time point ( Figure 3A ) does not show a difference between the virus-exposed (DENV and UV-DENV) cells and the mock. This is due to an equal number of individual lipid molecular species being up regulated as were down regulated ( Figure 3D ). At the 60 hr time point the relative levels of PE in virus exposed cells were lower than mock levels ( Figure 3B and D). There was also limited overlap between the specific PE molecular species regulated between DENV-and UV-DENV-exposed cells (Table S1 ). Given that insect cells have a high abundance of PE in their membranes (40-50%), it is interesting that there is a selective requirement for PC (over PE) in DENV-infected mosquito cells [22] .
Another group of PLs that were preferentially expressed in DENV-infected cells were the lysophospholipids (LPLs) ( Figure 3A and B). These lipids result primarily from the hydrolysis of PC by phospholipase A2-type enzymes (PLA 2 ) and represent a PL that is missing an acyl chain [27] [28] [29] [30] . In DENV-infected cells, there was a preferential up regulation (,3 fold) of these lipids compared to both UV-DENV and mock controls at the 36 hr time point. This expression was slightly down regulated at the 60 hr time point to about ,1.5 fold above the mock. In UV-DENV exposed cells, LPLs were undetectable at the 36 hr time point, while at the later time point, they were similar or slightly above the mock levels. As shown in figure 3H , LPLs with C16 acyl chains were up regulated at both time points while there was a selective up regulation of LPLs containing C18 acyl chains only at the early time point ( Figure 3H ). The C16 LPLs have been implicated in pro-apoptotic signaling pathways. This observation of selected LPL expression presents an attractive hypothesis that chain length differences may dictate specific roles for LPLs during virus infection.
Since LPLs result from the activity of PLA 2 -type enzymes, we investigated whether DENV infected cells displayed a higher activity of PLA 2 compared to uninfected cells. Utilizing a fluorescent PC substrate (BODIPY-PC) we used mass spectrometry to monitor the hydrolysis of PC to LPC by intracellular PLA 2 . Essentially, we monitored the production of BODIPY-LPC (resulting from PLA 2 mediated metabolism of PC) during a time course of DENV infection ( Figure S1 ) (method from [31] ). The assay indicated that following 24 hr of infection and longer, PLA 2 activity was elevated in DENV-infected cells compared to the controls, with the highest activity occurring at 48 and 72 hr postinfection. Therefore, the elevated levels of PLA 2 could be the source of the LPL detected in DENV-infected cells.
Sphingolipids. Although originally known as vital components of barrier membranes, sphingolipids are also potent bioactive molecules that regulate cell death, growth, differentiation and intracellular trafficking [32] [33] . The primary sphingolipids regulated during DENV infection were sphingomyelin (SM) and ceramide (CER). In DENV infected cells, SM was up regulated by ,2-fold compared to the controls (UV-DENV and mock) and remained elevated at both 36 hr and 60 hr time points ( Figure 3A and B). Furthermore, this temporal expression varied depending on the specific molecular species being expressed ( Figure 3E and Table S1 ). UV-DENV exposed cells showed similar levels of SM compared to the mock at the 36 hr time point, but these levels decreased at the later time point. Interestingly, an analog of SM known as ceramide phosphoethanolamine (CER-PE), was preferentially expressed (up regulated by ,2 fold) in UV-DENV exposed cells at the 36 hr time point (compared to DENV and mock) ( Figure 3G ). However, this expression level dropped below the mock at the later time point. In comparison, DENV infected cells showed very low expression of this lipid at the early time point (,0.7 fold compared to mock) and its expression was undetectable at the later time point ( Figure 3A and B). This lipid is expressed more abundantly in insect cells rather than in mammalian cells [23] [24] .
Another bioactive sphingolipid, CER, results from either the degradation of SM by sphingomyelinases or de novo synthesis through the condensation of palmitate and serine [32, 34] . These lipids were preferentially up regulated in DENV-infected cells at both the 36 and 60 hr time points (,2-fold compared to the mock). Since SM was also up regulated similarly in DENV infected cells, the rise in CER is possibly the result of de novo synthesis rather than SM degradation. UV-DENV-exposed cells showed undetectable CER levels at 36 hr, while at 60 hr the expression was (,2 fold) above the mock control. Since there was a concurrent decrease in SM in UV-DENV exposed cells at 60 hr, the increased levels of CER in this case could be due to the degradation of SM resulting from increased cellular stress (resulting from incubation of the cells for 60 hr). Similar to other lipid species, CER also showed temporal regulation depending on the molecular species expressed ( Figure 3F ).
An overall comparison of the PLs regulated at the 36 and 60 hr time points indicated that in all lipid classes that were significantly regulated (Anova p,0.05) during DENV infection, selected molecular species were regulated only at the 36 hr time point while others were regulated at both time points. Representative examples for each lipid class are shown ( Figure 3C -H). These observations may represent lipidome differences in cells sustaining early but active DENV replication (36 hr) compared to those experiencing advanced cellular stress (60 hr). It is also interesting to note that any up regulation observed in UV-DENV exposed cells (at either time point) was only in the range of 1-1.5 fold compared to the mock control, while DENV infected cells showed much higher levels of expression.
In addition to evaluating the lipid composition of whole cells, we also explored the possibility of profiling the lipid repertoire of specific membranes induced by DENV. Given the extensive interconnectivity of the membranes induced in DENV infected cells, isolation of morphologically distinct membranes proved challenging. Therefore, we carried out subcellular fractionation of C6/36 cells and isolated post-nuclear supernatants that were further fractionated to provide a total membrane fraction (16K pellet) enriched in viral replication components and a remaining cytoplasmic extract (CE) [35] . To confirm that the 16K pellet was indeed the membrane fraction enriched in the replication complex components, we used quantitative RT-PCR to measure the ratio The fold changes represent DENV-infected cells or UV-DENV exposed cells compared to the mock control. A lack of cones indicates that the expression level of those specific lipids were not significant (p,0.05). Panels C-H are representative lipid molecular species from specific lipid classes significantly regulated at the two different time points. The data are plotted as the integrated LC-MS peak abundance, in log 2 scale with standard deviation. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; CER, ceramide; CER-PE, ceramide phosphoethanolamine; Lyso, lysophospholipids. See supplementary table S1 for a complete list of lipid features detected in this study. Four replicates were included in the lipidomic analyses. The error bars represent standard deviation of the mean. The blue dashed line separates species that remain elevated at both time points (36 and 60 hr) from species that are only elevated at the 36 hr time point. Infections were carried out using an MOI of 20 in C6/36 cells. Significantly expressed lipids species are shown denoted with an asterisk (*). doi:10.1371/journal.ppat.1002584.g003 of viral RNA to lipid in both 16K and CE fractions. Lipids were quantified by pulse-chase analysis using 14 C-acetate to label newly synthesized lipids and the results are shown in Figure 4 . In postnuclear supernatants, there was increase in the incorporation of 14 C-acetate into lipids in the DENV infected cells compared to the mock control ( Figure 4A ). This increase was observed at both 36 hr and 60 hr post-infection with the latter time point showing a greater difference between DENV and mock. A more detailed analysis of the lipid distribution in the subcellular fractions (16K and CE) is shown in Figure 4B . At the 36 hr time point, the CE fraction was enriched in newly synthesized lipids (compared to the 16K fraction) in both the DENV and mock samples, with DENV showing an almost 2-fold increase compared to mock. This distribution changed at the 60 hr time point where the 16K pellet was more enriched in newly synthesized lipids, however, the fold change (DENV compared to mock) was not as significant.
To identify the primary fraction containing viral induced membranes that included the viral replication complex, we carried out a comparison of the viral RNA genome copies to labeled lipid in the 16K and CE subcellular fractions ( Figure 4C ). The results indicated that the 16K membrane fraction was enriched (a ,3fold increase) in viral RNA (compared to CE) at the 36 hr time point while at the 60 hr time point the CE fraction had slightly more viral RNA per labeled lipid (compared to the 16K fraction).
Based on the 14 C-acetate labeling studies above, the CE fraction showed an increased amount of newly synthesized lipids (compared to the 16K fraction at the 36 hr time point). In lipid biosynthesis pathways, acetate is a precursor to both phospholipid as well as sterol biosynthesis. Therefore, the labeled lipids (in CE) represent not only phospholipids in membranes, but also cholesterol and triglycerides that form lipid droplets [36] . In this study, lipidomic analyses were carried out on both the 16K and CE subcellular fractions isolated from the 36 hr time point from DENV infected cells, UV-DENV exposed cells and the mock control. The primary lipids observed in the CE fraction were sphingolipids. Cholesterol and triglycerides were not successfully separated using our mass spectrometry analyses and very few phospholipids were observed to be statistically significant (p,0.05) in virus infected cells over the mock control (data not shown). Therefore, we focused our primary lipidomic analysis on lipids extracted from the 16K pellet at the 36 hr time point. Similar to the whole cell analysis, DENV infected cells displayed a unique expression profile compared to UV-DENV and mock infected cells ( Figure S2 ). Over 85% of the metabolites detected were significantly regulated in virus-infected cells compared to the controls. Analysis of the membrane fraction also highlighted several lipid species not detected in our whole cell analysis. An overall fold change (compared to mock) of the significantly (p,0.05) expressed lipid classes in the membrane fraction is shown in Figure 5 . For a complete list of differentially expressed lipids see Table S2 .
Phospholipids. There was an up regulation of phospholipids, sphingolipids and several bioactive intermediates in DENV infected cells compared to UV-DENV and mock controls ( Figure 5A ). Amongst the specific phospholipids expressed, PE was selectively up regulated in membrane fractions isolated from DENV infected cells ( Figure 5B ). However, the primary PE molecular species up regulated was a lysophospholipid (PE P-16:0e). Both DENV and UV-DENV exposed cells showed similar expression of fatty acids ( Figure 5A ). However, the individual molecular species expressed differed between the two viruses ( Figure 5C ). For instance, stearic acid was up regulated in DENV infected cells while oleic acid was up regulated in UV-DENV exposed cells. Palmitic acid had a similar level of expression in both DENV and UV-DENV exposed cells. Several bioactive intermediates such as monoacylglycerol (MG), diacylglycerol (DG) and phosphatidic acid (PA) were also prominently up regulated in DENV infected cells compared to the controls ( Figure 5D ).
Sphingolipids. Although the Blygh and Dyer method [37] utilized for lipidomic analyses of whole cells or membrane fractions detected the expression of sphingolipids, Merrill et al optimized a protocol dedicated to analyzing sphingolipids without the interference from co-purifying PLs [38] . Utilizing this protocol, we carried out mass spectrometry analysis of these bioactive lipids from mosquito cells fractionated to provide three subcellular fractions: nuclear (N), cytoplasmic (CE) and membrane (16K). Similar to the above analyses, UV-DENV and mock infected cells were included as controls. The specific sphingolipids monitored included CER, SM, monohexosylceramide (GlcCER) and dihexosylceramide (GalCER).
As shown in Figure 6A , CER levels in C6/36 cells showed clear elevation in DENV-infected cells compared to mock-infected or UV-DENV exposed cells. Comparison of the different subcellular fractions indicated that the elevated CER was primarily concentrated in the CE fraction with the exception of two species (d18:1/ 16:0 and d18:0/16:0) which were enriched in the 16K pellet. These two species consist of the most abundant fatty acids observed in cells. The analysis of SM levels in mosquito cells ( Figure 6B ) indicated that virus exposed cells have a higher level of SM compared to mock-infected cells. This is most evident in the 16K pellet with the exception of the most abundant species (d18:1/20:0) that showed the highest levels in the CE. Comparison of infectious DENV to UV-DENV exposed cells indicates that the latter which is incapable of replication maintained higher levels of SM overall.
GlcCER were primarily detected in the 16K pellet of both DENV infected and UV-DENV exposed cells ( Figure 6C ). However, the overall levels were down-regulated compared to the mock control with the exception of d18:1/16:0, which showed slight elevation in the UV-DENV exposed cells. Dihexosylceramides were not detected to significant levels in any of the samples.
The FAS inhibitor, C75 is a potent inhibitor of DENV replication in both mosquito ( Figure 1 ) and mammalian cells [20] . Cells treated with non-cytotoxic concentrations of C75 (,50 mM) induce an environment that has redistributed its lipid repertoire to support cell survival in the absence of FAS activity, and yet, this environment does not support viral replication. To determine the basis for this exclusion of virus replication, we profiled the lipidome of cells treated with 25 mM C75. Membrane fractions isolated from DENV infected and mock control cells in the presence or absence of C75 were profiled. A hierarchical clustering analysis of the lipidomic data ( Figure S3 ) indicated that the lipid environment of DENV infected cells treated with C75 clustered very closely to the mock C75 treated control and was furthest from DENV infected cells suggesting that the two represent significantly different environments. A comparative analysis of the lipid species expressed in the two environments indicated that a majority of the lipids that were up regulated in DENV infected cells were down regulated upon treatment of those cells with C75.
Amongst the phospholipids, several phosphatidylinositol species were up regulated 3-4 fold in the untreated DENV infected cells compared to the C75 treated cells ( Figure 7B ). However, none of these PI species were phosphorylated. A single species of PG was also up regulated by over 5 fold in the untreated cells. Since C75 disrupts the formation of lipids down stream of FAS, several fatty acids (stearic and palmitic acid) were down regulated in the drug treated cells. An enrichment of these fatty acids was observed in the untreated cells upon comparison with C75 treated cells ( Figure 7C ). In contrast, as observed in the previous comparison of DENV infected cells to uninfected cells ( Figure 5 ), oleic acid was down-regulated in untreated DENV infected cells and therefore, showed enrichment in C75 treated cells. Interestingly, C75 treated cells also showed a down regulation of metabolic intermediates such as mono-and diacylglycerol as well as phosphatidic acid. Enrichment of these lipids was observed in untreated DENV infected cells compared to C75 treated cells ( Figure 7D ). In addition to the phospholipids, de novo synthesis of sphingolipids was also disrupted by C75. Key intermediates in this pathway such as N-palmitoylesphingosine and N-stearoylesphingosine were down regulated upon treatment of cells with C75 and up regulated in untreated DENV infected cells ( Figure 7A and Table S3 ).
The architecture of biological membranes is defined by the composition and distribution of lipids and proteins in the bilayer.
Optimizing this composition and distribution to facilitate the formation of virus-induced intracellular membrane structures has to be a requirement for efficient DENV replication. Previously we showed that FAS, the rate-limiting enzyme in lipid biosynthesis was both recruited and activated during DENV replication in human cells [20] . In this study we demonstrated that this requirement for FAS activity was also conserved in the mosquito vector-derived cells. Furthermore, inhibition of FAS activity seemed to be most effective between 4-12 hr post-infection suggesting an early requirement for FAS activity during the replication cycle.
As a next step in investigating the role of lipids in DENV induced membrane expansion we used high-resolution mass spectrometry to profile the lipid composition of DENV infected mosquito cells. We observed a very distinct segregation in the lipid composition between DENV infected and uninfected cells. Specific lipid changes that occurred upon virus binding and entry alone were also identified. In DENV infected cells, there was a selective enrichment of lipids that have characteristic functions in influencing membrane structure as well as those that have potent signaling functions. Among these lipids were bioactive sphingolipids such as sphingomyelin and ceramide, lysophospholipids and several intermediates such as mono-and diacylglycerol and phosphatidic acid. The phospholipids that were enriched in DENV infected cells were primarily unsaturated. There was also evidence for the up regulation of de novo phospholipid and sphingolipid biosynthesis as well as triacylglyceride metabolism ( Figure 8 ). Furthermore, we identified a unique lipid environment that supported cell survival but did not support DENV replication. This environment was created by the addition of C75, an inhibitor of fatty acid synthase, the rate-limiting step in phospholipid biosynthesis.
Analysis of the whole cell lipidome indicated that unsaturated PC was the most up regulated phospholipid in DENV infected cells. Membranes that are similar or derived from the ER are enriched in unsaturated lipids, primarily PC [39] . Although DENV-induced membranes in mammalian systems are ER-derived, it is unknown if the same is true in mosquito cells. While PC is known to form planar membranes, unsaturated PC induces membrane curvature which may be important for maintaining the highly curved membranes observed in DENV-infected cells [40] . PC enriched membranes are also more fluidic compared to the rigid membranes enriched in SM and cholesterol, which may be a feature attractive to DENV infection [41] .
Bioactive sphingolipids such as SM and CER are also key lipids that were up regulated during DENV infection. In cells exposed to replication competent virus, CER was up regulated in both the whole cell lipidome as well as replication complex membranes. This is either a cellular response to virus infection, or a direct need for CER in the virus replication cycle. The latter hypothesis is particularly attractive considering the enrichment of specific molecular species of CER in the 16K pellet. CER is a cone-shaped Figure 6 . Bioactive sphingolipid species are differentially regulated in replication complex membranes isolated from DENVinfected mosquito cells. Multiple Reaction Monitoring (MRM) analysis of sphingolipids species differentially regulated in DENV-infected cells (MOI 20) or UV-DENV exposed cells compared to the mock control (see also supplementary table S3). The data represent fold changes observed in three subcellular fractions that were analyzed in this study; 16K, replication complex membranes; CE, cytoplasmic extracts following removal of replication complex membranes and nuclei; N, nuclear fraction. Panels A-C represent ceramide, sphingomyelin and monohexosylceramide species respectively. The dashed line highlights values equal to the mock. The data represent three independent experiments. The error bars represent standard deviation of the mean. doi:10.1371/journal.ppat.1002584.g006 molecule that promotes inward budding of membranes (negative curvature) [42] . Some DENV-induced membranes show a double membrane morphology indicating that negative curvature modifying lipids such as CER may be active in their formation.
As a second messenger, CER can also induce apoptosis and autophagy [34] . However in mosquito cells, DENV maintains a persistent infection and antagonizes apoptotic pathways [19, 43] .
Therefore, CER up regulation may not be due to the prevalence of an apoptotic response but may be utilized to up regulate autophagy during DENV infection. It has been shown in mammalian cells that autophagy is up regulated and necessary for DENV replication. The prevalence of several intermediates in CER biogenesis, including N-palmitoylsphingosine and diacylglycerol, suggest that the observed CER could result from either de [20] . 2. Inhibition of this process with C75 disrupts the cellular lipid repertoire in mosquito cells to be unfavorable for virus replication. 3. The lipidomic analyses reveal an up regulation of fatty acids such as palmitic (C16) and stearic (C18) acid. These fatty acids are intermediates in the biosynthesis of phospholipids, which is up regulated during DENV infection. Interestingly, in DENV infected cells the prevalent phospholipids primarily consist of C16 and C18 unsaturated acyl chains. Very long chain fatty acids are not significantly up regulated during infection. 4. FAS activity also stimulates de novo sphingolipid biosynthesis. In the lipidomic analyses, the up regulation of intermediates such as N-palmitoylesphingosine suggests sphingolipid biosynthesis is activated during DENV infection. Specifically, SM and CER are enriched in DENV infected cells. Alternately, the up regulation in CER (and DG) during infection could result from the degradation of SM through the activity of sphingomyelinases (Smase). The resulting CER and DG could be redirected into several signaling pathways or be utilized for de novo phospholipid biosynthesis. The glycopshingolipids, GlcCER and GalCER are down regulated during DENV infection, which suggest that they are catabolized to produce CER. 5. Lipidomic analyses also suggest the up regulation of triacylglycerol catabolism (Lipolysis) in DENV infected cells. This pathway results in the generation of MG, DG and palmitic acid. These intermediates are all up regulated in DENV infected cells and could be utilized for downstream signaling or de novo phospholipid biosynthesis. It has also been shown that TG catabolism is necessary for mitochondrial b-oxidation during DENV infection [50] . 6. Elevated levels of LPC in DENV infected cells also suggest activation of PC hydrolysis by PLA 2 . This enzyme is activated during DENV infection. The elevated levels of other phospholipids such as PA, PI, PE, PG as well as PC suggest that the CDG-DG pathway for phospholipid biosynthesis could also be activated. FAS, fatty acid synthase; DENV, dengue virus; C75, inhibitor of FAS; SM, sphingomyelin; CER, ceramide; MG, monoacylglycerol; DG, diacylglycerol; TG, triacylglycerol; LPC, lysophosphatidylcholine; PLA 2 , phospholipase A 2 ; PA, phosphatidic acid; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PC, phosphatidylcholine. doi:10.1371/journal.ppat.1002584.g008 novo synthesis by serine palmitoyltransferase (SPTLC) in the ER or catabolism of SM by sphingomyelinases (SMases) at the plasma membrane [44] . The lipidomic analyses also indicated a down regulation of GlcCER, which may suggest that a salvage pathway that metabolizes GlcCER to CER may also be active.
Inverted cone-shaped lipids such as LPLs were also up regulated during DENV infection. Primarily, LPC was up regulated in the whole cell analysis. It results from the hydrolysis of PC by PLA 2 [45] . We have shown that this enzyme is activated during DENV infection. Many other viruses have also shown a dependency for PLA 2 in their life cycle [31, 46] . Due to its inverted cone-shaped structure, asymmetric incorporation of LPC into membranes causes positive curvature and induces vesicle fission and budding [47] . Structural analyses of DENV-induced membrane structures in mosquito cells indicate the presence of highly curved membranes and smaller vesicles [18] [19] . Therefore, vesiculation through fission and budding as well as enrichment of molecules that increase membrane curvature may be an underlying mechanism for forming these membrane structures.
LPC also increases the permeability of membranes [48] . This is an attractive concept given that DENV replication complexes are encased within membrane barriers that need to facilitate the exchange of components to and from the cytosol for genome replication and virus assembly. Therefore, leaky membranes may be favored in DENV-infected cells through the selective incorporation of molecules such as LPC. Another observation that gives credence to this hypothesis is that the transmembrane permeabilizing effect of LPC is synergistically enhanced in the presence of palmitic acid, which is also up regulated in DENVinfected cells. As a signaling molecule, LPC is both a pro-survival as well as an inflammatory molecule [46] . Therefore, its expression in DENV infected cells could be a cellular response to viral infection.
It has been previously reported that de novo lipid biosynthesis was up regulated during DENV infection [20] . Consistent with these observations, several primary fatty acids (palmitic, stearic and oleic acid) involved in the biosynthesis of higher order PLs were detected in our lipidomic analyses. Palmitic acid is the first fatty acid of the lipogenesis pathway and stearic acid (C18:0), is immediately downstream of palmitic acid. They are both up regulated in DENV infected cells. The latter is the precursor to the biosynthesis of oleic acid (C18:1) or long chain fatty acids (with acyl chains of C20 or greater). Interestingly, since oleic acid is down regulated in DENV-infected cells stearic acid may be stimulating long chain FA biosynthesis. However, a majority of the PLs expressed in DENV infected cells have C16-or C18-acyl chains with varying degrees of unsaturation. Therefore, it seems that desaturation of palmitic and stearic acid rather than elongation may be the chosen pathway for lipogenesis during DENV infection.
Several intermediates in lipid catabolism were also detected in the replication complex membranes (palmitic acid, MG and DG). Analysis of these intermediates suggested the up regulation of pathways for PC hydrolysis and triacylglycerol (TG) metabolism. The latter pathway is responsible for regulating lipid homeostasis and energy production in the cell through the metabolism of lipid droplets [49] . Through the action of lipases the degradation of lipid droplets results in the release of palmitic acid, DG and MG [36] . These metabolic products are substrates for mitochondrial boxidation as well as intermediates (fatty acids) in the synthesis of new lipids. DENV infected cells show a high prevalence of these species compared to mock infected cells.
While several acidic phospholipid species were detected in the 16K membrane fraction very few were significantly regulated during infection. Previous studies in mammalian cells have indicated the importance of acidic lipids in DENV infection [46, 50] . These negatively charged lipids are also implicated in influencing membrane structure.
The most striking results were obtained by the comparison of DENV infected cells in the presence and absence of the FAS inhibitor, C75. It is well established that treatment of cells with this inhibitor disrupts de novo phospholipid biosynthesis [51] . To circumvent this inhibition, the cell redistributes its lipid repertoire to ensure cell survival. However, this redistribution does not support DENV replication. A comparison of the different lipid environments indicated that many of the lipids that were up regulated in DENV infected cells compared to cells treated with C75, were similar to those previously observed when infected cells were compared to mock (uninfected) controls. Therefore, comparison of DENV infected cells to two different controls (uninfected cells and C75 treated infected cells) highlighted the same lipids as being up regulated during an active DENV infection.
In these studies we utilized UV-DENV as a control to identify lipid metabolic changes that occurred upon virus binding and entry alone. This virus is incapable of replication but immunofluorescence microscopy with anti-NS3 antibodies have indicated that there is a very low level of translation that occurs within the first 24 hr (data not shown). Analysis of the lipidome of UV-DENV exposed cells showed a significant difference in the phospholipid and sphingolipid content in comparison to DENV infected cells and the mock control. It is possible that binding, membrane fusion and entry alone, or the expression of viral proteins from the incoming viral RNA triggers the activation of lipases or lipid transport mechanisms that result in the differences observed in the lipidome of the cells. This has been shown for other viruses. Jan et al. have shown that binding and entry alone of Sindbis virus triggered the activation of sphingomyelinases that degraded membrane bound SM to Cer [52] . It has also been shown that viruses induced apoptotic-signaling cascades upon binding and entry alone [53] [54] [55] . These cascades may either result from or induce lipolysis or lipid transfer between compartments in the cell.
The most pronounced changes are those observed in the 16K pellet analyses where a significant decrease in phospholipids, sphingolipids and lipid intermediates are observed (compared to mock cells, Figure 5A) . A comparison of SM and CER in the whole cell analysis at the 36 hr and 60 hr time points ( Figure 1A and 1B) indicated a reduction in SM with a corresponding increase in CER with time. This could be due to a similar scenario to SINV where a non-replicating UV-DENV is capable of inducing lipid conversion in membranes [52] . Therefore, SM and other sphingolipids may be converted to CER that is transported away from the 16K membrane fraction to other locations in the cell. This is also evident from the increased content of fatty acids compared to mock cells that may result from the degradation of lipids. UV-DENV exposed cells do not show significant regulation of MG or DG, but show an up regulation of palmitic acid (C16:0). Virus binding and entry alone could stimulate lipid droplet hydrolysis (catabolism of TG) resulting in the release of palmitic acid. These studies have shown that UV-DENV could be a versatile tool to pursue mechanistic studies on the metabolic pertabations that occur upon virus binding, membrane fusion, and entry.
In summary, using high-resolution mass spectrometry, we have determined that DENV drastically alters the lipid profile of infected cells. Specifically, DENV infection elevates the expression of lipids that have the capacity to change the physical properties of the bilayer such as bilayer curvature, permeability, and the recruitment and assembly of protein complexes in the membrane. Several of the identified molecules also function as bioactive messengers that control signaling and membrane trafficking pathways in the cells. They represent molecules that result from the activation of cellular stress pathways that respond to viral infections. Based on these findings, the next steps will be to investigate the mechanisms of how these lipid species play a role in DENV replication, as well as identify the control points in these pathways that may be influenced by viral gene products.
Cell culture and virus infections C6/36 (Aedes albopictus) cells (ATCC) were maintained in Minimal Essential Medium (MEM) supplemented with glutamine (2 mM), non-essential amino acids, 25 mM Hepes and 10% heat inactivated fetal calf serum. Dengue virus 2, strain 16681 (obtained from Richard Kinney, CDC. Ft. Collins) stocks were amplified in C6/36 cells in MEM (supplemented as above) and 2% heat inactivated fetal calf serum. UV-inactivated DENV was obtained by exposing the same virus stock (used for infection) to UV-light for 3 hr and then conformation of inactivation by two blind passages of the virus on cells for 60 hr per passage. Lack of infectivity was confirmed by plaque assay and immunofluorescence assay.
Sample preparation. For infection, ,5610 8 C6/36 cells were infected with DENV at a MOI of 20 at 30uC. Infection of all cells (required for lipidomic analyses) was confirmed by immunofluorescence analyses. Each experiment included three biological replicates. Following infection, cells were harvested at 36 and 60 hr post-infection, pelleted and resuspended in 100 ml of 100 mM ammonium bicarbonate. Lipids were extracted from an equal number of cells using a modified Bligh and Dyer protocol. Briefly, a mixture of 2:1 Chloroform:methanol, 0.1% acetic acid and 0.01% butylated hydroxy toluene (BHT) were added to the cell suspension in ammonium bicarbonate such that there was a 4:1 ratio of organic solvent to cells. Following lipid extraction, the organic phase was separated from the aqueous phase by centrifugation and dried down under a N 2 stream in low retention microfuge tubes (Fisher). The dried lipids were resuspended in 75 ml of methanol and vortexed for 10 s. The samples were then centrifuged at 13,4006 g for 5 min to remove any particulates.
chromatography/mass spectrometry. Lipid molecular species were profiled using a dual-column nanocapillary LC system equipped with 75 mm665 cm columns, each packed with 3 mm Jupiter particles (Phenomenex, Torrance, CA). The mobile phases were (A) 10 mM ammonium acetate in 50:50 water/methanol (v/v) and (B) 10 mM ammonium acetate in 50:50 methanol/acetonitrile (v/v). The LC system was equilibrated at 10,000 psi with mobile phase A prior to injecting 0.4 mL of sample. Exponential gradient elution was initiated 50 min after sample loading with an initial column flow of ,300 nL/min. After 90 min of gradient separation, the mobile phase mixer was purged with 3 mL of mobile phase B, followed by a 5 min column wash. Finally, the mobile phase mixer was purged with 10 mL of mobile phase A, which represented the end of one separation cycle. While gradient elution was performed on one column, the other column was equilibrated with mobile phase A. The LC system was coupled to a hybrid linear ion-trap-Orbitrap mass spectrometer (ThermoFisher, San Jose, CA), and the capillary temperature and electrospray voltage were 200uC and +2.2 kV, respectively. The Orbitrap was used as the mass analyzer during MS survey scans over the m/z range 200-2000 with a duty cycle of ,1.2 s. Data-dependant MS/MS was performed in the LTQ for the top 5 ions, with a normalized collision energy of ,35%. Dynamic exclusion in the LTQ during data-dependant MS/MS experiments was enabled as follows: repeat count of 2, repeat duration of 30 s, exclusion list size of 250, and exclusion duration of 60 s.
Data processing. LC-MS datasets, defined as the data obtained from a single LC-MS analysis, were processed using the PRISM Data Analysis system [56] , a series of software tools freely available at http://ncrr.pnl.gov/software/ and developed in-house. The first step involved deisotoping of the raw MS data to give the monoisotopic mass, charge state, and intensity of the major peaks in each mass spectrum using Decon2LS [16] . The data were next examined in a 2-D fashion using MultiAlign to identify groups of mass spectral peaks that were observed in sequential spectra using an algorithm [57] that computes a Euclidean distance in n-dimensional space for combinations of peaks. Each group, generally ascribed to one detected species and referred to as a ''feature'', has a median monoisotopic mass, central normalized elution time (NET), and abundance estimate computed by summing the intensities of the MS peaks that comprise the entire LC-MS feature. LC-MS features were then chromatographically aligned across all datasets using the LCMSWARP algorithm [58] in MultiAlign, and the lipid identities of detected features were initially determined by comparing their measured monoisotopic masses and NETs to calculated monoisotopic masses and observed NETs for lipids in an AMT tag database [59] within search tolerances of 63 ppm and 60.03 NET for monoisotopic mass and elution time, respectively. Subsequent identifications of lipid features that did not match to entries in the AMT tag database were made by searching their molecular weights against entries in the Lipid MAPS database followed by manual confirmation based on MS/ MS spectra.
Statistical analysis of processed data. Following chromatographic alignment and database matching, the abundances of all detected features (both AMT tag database matched and unmatched) were loaded into DAnTE [60] for statistical analysis. Feature abundances were transformed to log2 scale then subjected to central tendency normalization [61] . Comparative data analysis was then performed and statistically significant differences between the lipid profiles of the samples were determined using ANOVA. Partial least-squares (PLS) [62] analysis was also performed using the data matrices containing either AMT tag database matched features alone or all features (both database matched and unmatched). homogenized and centrifuged at 15006 g, at 4uC for 5 min to remove nuclei. Post-nuclear supernatants were centrifuged at 160006g for 30 min at 4uC to obtain a membrane pellet (referred to as the 16K pellet). The supernatant post nuclear and membrane fractionation is referred to as the cytoplasmic extract (CE). Total RNA was extracted from each fraction (16K and CE) using Trizol reagent (Sigma) according to the manufacturer's instructions. Following RNA extraction, total lipids were extracted using the Bligh and Dyer method mentioned above and counted using a Beckman LS 6000 scintillation counter. The viral RNA in each fraction was measured using the SuperScript III Platinum SYBR Green One-Step qPCR Kit with ROX and DENV specific primers; (forward) 59 ACAAGTCGAACAACCTGGTCCAT 39 and (reverse) 59 GCCGCACCATTGGTCTTCTC 39 on a Applied Biosystems 7300 Real Time PCR machine. The labeled lipid in subcellular fractions was standardized to the total RNA isolated from each fraction prior to determining the viral RNA genome copies per labeled lipid in subcellular fractions.
Sample preparation. For profiling the lipidome of the replication complex membranes, lipids were extracted from the 16K pellet using the same modified Blygh and Dyer protocol described above.
Liquid chromatography/mass spectrometry profiling analysis of lipid content. A LTQ Orbitrap XL instrument (Thermo Fisher Scientific San Jose, CA) was used for the analysis. It was coupled to an Agilent 1100 series LC (Agilent Technologies, Santa Clara, CA) equipped with a micro well plate auto sampler and binary pumping device. Reverse phase liquid chromatography was used to analyze the samples. An Agilent Eclipse XDB-C8 column with 2.16150 mm, 3.5 mm dimensions was used for the separation. Solvent A consisted of water +0.1% piperidine. Solvent B contained acetonitrile : methanol (50:50 v/v) +0.1% piperidine. The flow rate was 300 mL/minute. A volume of 10 mL was loaded onto the column. The gradient was as follows: time 0 minutes, 50% B; time 25 minutes, 95% B; time 45 minutes, 95% B; time 50 minutes, 50% B; time 60 minutes, 50% B. The MS analysis used negative polarity electrospray ionization. The source voltage was 4.0 kV, source current 100 mA, capillary voltage 230.0 V, tube lens voltage 2100.0 V. The capillary temperature was 200uC, sheath gas flow was 35, auxiliary and sweep gas were both set to 0. Data were acquired using data dependent scanning mode. FTMS resolution of 60,000 with a mass range of 70-1200 was used for full scan analysis and the ITMS was used for MS/MS data acquisition. The top three most intense ions were acquired with a minimum signal of 500, isolation width of 2, normalized collision energy of 35, default charge state of 1, activation Q of 0.250, and an activation time of 30.0. The samples were evaluated with Thermo XCalibur software (version 2.1.0) and downstream alignment done with an in-house data processing package called Omics Discovery Pipeline [63, 64] .
Liquid chromatography/mass spectrometry for targeted sphingolipid analysis. The method was slightly modified from [65] . Briefly, each sample was extracted according to published protocol then dried and reconstituted in 100 mL of methanol : water : formic acid (74:25:1) containing 5 mM ammonium formate. An Agilent 6400 QQQ (Agilent Technologies, Santa Clara, CA) was used for analysis coupled to an 1100 Series LC equipped with HPLC Chip interface (Agilent Technologies Santa Clara, CA). Solvent A consisted of methanol : water : formic acid [65] with the following source conditions: gas temperature 300uC, gas flow 4 L/minute, capillary voltage 1900 V. The fragmentor voltage was set to 180 V in all cases. Data were processed with Agilent Mass Hunter software version B03.01.
C6/36 cells were infected with DENV at an MOI of 3. Following adsorption, virus was removed and the cells were washed and overlayed with media containing varying concentrations of C75 [51] . The vehicle for C75 was ethanol. Virus supernatants were harvested at 24 hr post-infection and virus titer assayed by plaque assay. Cytotoxicity of C75 was simultaneously assayed using the Quick Cell Proliferation Kit (Abcam). TIME OF ADDITION OF C75: C6/36 cells were infected with DENV as described above. At the indicated time points, 6.3 mM C75 was added to the cells. Cells were harvested at 24 hr post-infection and the amount of released virus was determined by plaque assay. For the lipidomic studies, cells were infected with DENV (as described above) or left uninfected. Following adsorption of the virus at room temperature for 2 hr, C75 was added to the media in the overlay, and cells were incubated at 30uC for 36 hr. Sample preparation, lipid extraction and mass spectrometry analyses were carried out as described above for the fractionated (16K pellet) samples.
Phospholipase A2 activity was monitored using a Red/Green BODIPY PC-A2 substrate (Invitrogen) using a similar method as described in [31] . C6/36 cells were infected with DENV at an MOI = 3 in 6-well plates. Following virus adsorption, the cells were overlayed with MEM (supplemented as above) and 10% heat inactivated fetal calf serum (2 ml/well). At the indicated time points, the media were removed, and new media (1 ml/well) containing 6 mM of the fluorogenic phospholipase A substrate (BODIPY-PC) were added to the cells. The cells were incubated at 30uC for 30 min. Following incubation, media was removed and the cells were washed with 16PBS. The lipids were then extracted using butanol-1 (1 ml/well). The aqueous fraction was discarded and the lipids in the butanol phase were analyzed by mass spectrometry to monitor the conversion of BODIPY-PC to BODIPY LCP by PLA 2 .
Phospholipase A2 activity analysis. The lipid samples were reconstituted in 100 mL of methanol : water : formic acid (74:25:1) containing 5 mM ammonium formate and analyzed with an Agilent 6400 QQQ (Agilent Technologies, Santa Clara, CA) coupled to an 1100 Series LC equipped with HPLC Chip interface (Agilent Technologies Santa Clara, CA). Solvent A consisted of methanol : water : formic acid (74 : 25 : 1) containing 5 mM ammonium formate and solvent B methanol : formic acid (99 : 1) containing 5 mM ammonium formate. The gradient was as follows: time 0 minutes, 40% B; time 5 minute, 70% B; time 7 minutes, 100% B; time 9 minutes, 100% B; time 9 minutes, 40% B; time 13 minutes, 40% B. The flow rate was 0.4 mL/min. Data were acquired for parent ions corresponding to the BODIPY-PC (986.9 m/z) and BODIPY LCP (320.2 m/z) in single ion monitoring (SIM) mode with the following source conditions: gas temperature 300uC, gas flow 4 L/minute, capillary voltage 1900 V. The fragmentation voltage was set to 200 V in all cases. Data were processed with Agilent Mass Hunter software version B03.01.
Supplemental data include three figure and three tables. carried out on 100 lipids. For each condition and time point, the following information is provided: Treatment P; p-values from the Anova analysis on minimum observations data (p,0.1), Exact mass; mass of each lipid from http://www.lipidmaps.org, [M+H] + ; Protonated molecular ion, NET; normalized elution time, total carbon/double bond; corresponds to each lipid species detected, LM_ID; identification for each lipid as displayed in LIPID MAPS, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/Mock; fold change of average abundance from 4 replicates of each treatment. Identity abbreviations were made for phoshatidylcholine (PC; O-fatty acid chain number means that an alkyl acyl linkage to the glycerol chain is present for the respective PC), phosphatidylethnolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), ceramide (Cer), ceramide phosphoethanolamine (Cer-PE), lysophosphatidylcholine (LPC). The notation further indicates total number of carbons and double bonds however it does not discern redundancy associated with varying fatty acid composition for the same molecular weight. Accession numbers (LM_ID) were obtained from the Lipid Maps Gateway (lipidmaps.org). (PDF)
Table S2 Select list of lipid species from the analysis of the 16K membrane fraction (isolated from mosquito cells treated with conditions described below) regulated across conditions (p,0.05). Conditions: Mock (uninfected cells), DENV (infectious dengue virus type 2, strain 16681), UV-DENV (UV-inactivated dengue virus type 2, strain 16681). A total of 484 features were detected in the mass spectrometry analysis following normalization of Mock, DENV and UV-DENV treated samples and 415 of these features were significantly regulated (p,0.05). Identification was successful for 68 lipids (ppm error ,10). For each condition the following information is provided: pttest and pwilcox; p-values on minimum observation data (a measurement must be present in two of three replicates to be considered present), Exact mass; mass of each lipid from http:// www.lipidmaps.org, M/Z; mass to charge ratio, RT; retention time, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/Mock; Fold UV-DENV/Mock; fold change of average abundance from 3 replicates of each treatment. Identity abbreviations were made for phoshatidylcholine (PC), phosphatidylethnolamine (PE), phosphatidylglycerol (PG, O-fatty acid chain number means that an alkyl acyl linkage to the glycerol chain is present for the respective PG), sphingomyelin (SM), monoacylglycerol (MG), diacylglycerol (DG), phosphatidic acid (PA). Accession numbers were obtained from the Lipid Maps Gateway (lipidmaps.org) and the Human Metabolome Database (hmdb.ca). (PDF)
Table S3 Select list of lipid species from the analysis of the 16K membrane fraction (isolated from mosquito cells treated with conditions described below) regulated across conditions (p,0.05). Conditions: DENV (cells infected with DENV in the presence of vehicle only), DENV C75 (cells infected with DENV and treated with the FAS inhibitor C75). A total of 366 features were detected in the mass spectrometry analysis following normalization of DENV and DENV C75 treated samples and 314 of these features were significantly regulated (p,0.05). Identification was successful for 27 lipids (ppm error ,10). For each condition the following information is provided: pttest and pwilcox; p-values on minimum observation data (a measurement must be present in two of three replicates to be considered present), Exact mass; mass of each lipid from http://www.lipidmaps.org, M/Z; mass to charge ratio, RT; retention time, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/DENV C75; fold change of average abundance from 3 replicates of each treatment. Identity abbreviations were made for phoshatidylinositol (PI), phosphatidyllglycerol (PG), monoacylglycerol (MG), diacylglycerol (DG), phosphatidic acid (PA). Accession numbers were obtained from the Lipid Maps Gateway (lipidmaps.org) and the Human Metabolome Database (hmdb.ca). (PDF) Human Cardioviruses, Meningitis, and Sudden Infant Death Syndrome in Children Cardioviruses cause myocarditis and encephalomyelitis in rodents; human cardioviruses have not been ascribed to any disease. We screened 6,854 cerebrospinal fluid and 10 myocardium specimens from children and adults. A genotype 2 cardiovirus was detected from a child who died of sudden infant death syndrome, and 2 untypeable cardioviruses were detected from 2 children with meningitis. Cardioviruses cause myocarditis and encephalomyelitis in rodents; human cardioviruses have not been ascribed to any disease. We screened 6,854 cerebrospinal fl uid and 10 myocardium specimens from children and adults. A genotype 2 cardiovirus was detected from a child who died of sudden infant death syndrome, and 2 untypeable cardioviruses were detected from 2 children with meningitis.
Cardiovirus) are pathogens of rodents and include a murine encephalomyocarditis virus and Theiler's virus and related strains (species Theilovirus), the latter serving as laboratory models of the pathogenesis of multiple sclerosis in mice (1) . The existence of specifi c human cardioviruses was suspected in the 1960s in conjunction with a rare infectious neurodegenerative disease known as Vilyuisk encephalitis (2, 3) . Recently, human cardioviruses (hCVs) were identifi ed in archived diagnostic cell culture supernatants (4) and in clinical samples from children with diarrhea or respiratory infection (5, 6) . Up to 8 different putative hCV types have since been characterized in human feces (7) .
Despite the remarkable pathogenicity of rodent cardioviruses, specifi c disease associations of hCV could not be made. An initial clinical study yielded no evidence of hCV in cerebrospinal fl uid (CSF) of 400 patients with aseptic meningitis, encephalitis, or multiple sclerosis (8) .
To evaluate the pathogenetic potential of these emerging viruses, we investigated 6,854 CSF specimens from adults and children with neurologic disease and 10 myocardium specimens from infants who had died of sudden infant death syndrome (SIDS).
CSF specimens were collected from 3 cohorts. The fi rst cohort comprised 2,562 specimens sent during 1998-2008 to the Institute of Virology, University of Bonn Medical Center (UBMC), Bonn, Germany, for routine investigation of meningoencephalitis (333 from the Department of Pediatrics and 2,229 from other departments). The second cohort comprised 3,960 specimens collected during 1982-2008 at the UBMC children's hospital from children with cancer and neurologic complications during chemotherapy. The third cohort comprised 348 specimens from hospitalized children with clinical meningitis or encephalitis in which no etiologic agent had been found; the specimens were sent for virologic investigation to the Institute for Hygiene and the Environment in Hamburg, ≈400 km from UBMC, during 2006-2008. Myocardium specimens were collected during 2010 at the UBMC Institute for Forensic Medicine from 10 epidemiologically unlinked children who died of SIDS.
Viral RNA was purifi ed from clinical specimens by using the Viral RNA Mini and RNeasy Mini kits (QIAGEN, Hilden, Germany). Detection of hCV RNA was done in pools of 2-10 specimens by using quantitative realtime reverse transcription PCR (RT-PCR) and nested RT-PCR specifi c for the viral 5′ untranslated region (5′-UTR), as described (6) . Amplifi cation of further hCV genomic regions from individual positive specimens was conducted by using ≈20 sets of different nested RT-PCRs (primers available on request from C.D.).
In 2 of 681 CSF specimens (n = 333 and n = 348 from cohorts 1 and 3, respectively) from children with meningitis (online Appendix Table, wwwnc.cdc.gov/EID/ article/17/12/11-1037-TA1.htm), hCV RNA was detected at low concentrations (1.14 × 10 4 and 9.63 × 10 2 copies/ mL). In 1 of these patients, hCV was also detectable in feces (9.50 × 10 2 copies/g). In 1 of 10 myocardium specimens, hCV was detected by nested RT-PCR, and results of quantitative real-time RT-PCR were negative. Underquantifi cation because of nucleotide mismatches below oligonucleotide binding sites and contamination of nested RT-PCR was excluded by sequence comparison (up to 5% nt divergence from other hCV strains, including the positive control). Serum and liver specimens from the patient who died of SIDS were negative according to realtime RT-PCR. No histopathologic alterations could be observed in myocardial tissue from this same patient.
To evaluate whether detected hCV strains differed from previously described genotypes, amplifi cation and nucleotide sequencing of additional genomic regions was attempted. In a case of meningoencephalitis (specimen 07/03981), we sequenced a 1,297-nt fragment comprising the near complete 5′-UTR and the fi rst 489 nt of the structural protein gene (leader, viral protein [VP] 4 domain, and upstream VP2 domain, GenBank accession no. JN209931). Despite repeated trials, further sequence fragments could be amplifi ed neither from the specimen from this patient nor from that from the second patient with meningoencephalitis that showed very low virus concentrations (specimen VI1607). From the specimen from the SIDS patient (specimen 347/10), amplifi cation of the complete structural genome and partial nonstructural genome was successful (5,333 nt, GenBank accession no. JN209932). This virus belonged to hCV genotype 2 in the VP1 genomic region (i.e., the region used for the designation of genotypes) (Figure, panel A) . The CSF specimen 07/03981 was also phylogenetically related to genotype 2 viruses in the 5′-UTR and Leader-VP2 genomic regions ( Figure, panels B and C). On the basis of the 5′-UTR sequences, the closest known relative to both viruses was D/VI2229, obtained in Germany in 2004 (nucleotide percentage distance 4.7% for the SIDS specimen and 0.9% for the CSF specimen). In the structural protein gene fragment, the closest relative of both viruses was a strain obtained in the Netherlands in 2008 (Nijmegen2008, nucleotide distance 13.9% for the SIDS specimen and 3.5% for the CSF specimen). This suggested geographic rather than phylogenetic clustering of viruses detected within and beyond the respiratory and enteric tracts. However, formal and fi nal virus typing is pending because VP1 regions could not be sequenced from 2 viruses.
Absence of other detectable pathogens in 1 of the meningoencephalitis case-patients (07/03981) made causation by hCV plausible (online Appendix Table) . For the second case (VI1607), an enterovirus was co-detected by real-time RT-PCR in CSF and feces. Serotyping from feces classifi ed this virus as echovirus type 30, known to cause aseptic meningitis. For the specimen from the child who died of SIDS, a rhinovirus was co-detected at low concentrations (real-time RT-PCR threshold cycle value >40), most compatible with shedding after previous respiratory infection (9) .
The detection of hCVs in body compartments beyond the respiratory and enteric tracts is novel and suggests a role of these viruses in organ-related disease. A low detection rate in CSF does not contradict a general potential of these viruses to cause meningoencephalitis, as exemplifi ed by enteroviruses for which lack of detection in CSF despite clear association with disease is not uncommon (10) . Considering links between the related Theilovirus and demyelinating disease in laboratory models (1) outcomes of patients with hCV infection of the central nervous system should be followed up. Such longitudinal studies should include suffi cient numbers of patients because natural infections with Theilovirus in rodents are common and will less frequently result in multiple sclerosis-like disease than in laboratory models (1) . The rarity of hCV detection in our study suggests the assembly of such cohorts to be a diffi cult and lengthy task that could benefi t greatly from international coordination. Despite the absence of histopathologic alterations, the detection of hCV in a child who died of SIDS is remarkable because the related encephalomyocarditis virus constitutes a prototypic model for myocarditis in mammals (11) . Again, the high human seroprevalence against hCV (12) will complicate epidemiologic studies, yet investigations of links between hCV and SIDS are highly justifi ed because diarrhea is an acknowledged risk factor for SIDS (13) .
A limitation of our study is that the VP1 genomic region of the viruses detected in CSF could not be obtained. In analogy to enteroviruses and parechoviruses, certain genotypes may be associated with distinct disease profi les, like polioviruses with encephalitis or parechovirus 3 with meningitis (14) . Although we were able to classify the virus detected in the child who died of SIDS as a common genotype 2, the partial hCV sequence from a patient with meningitis did not permit typing because hCVs, as all picornaviruses, recombine frequently (15) . We thus cannot exclude that the viruses detected in the meningitis cases may have acquired distinct features in their capsid protein or elsewhere that might infl uence pathogenicity. Knowledge of Avian Influenza (H5N1) among Poultry Workers, Hong Kong, China In 2009, a cross-sectional survey of 360 poultry workers in Hong Kong, China, showed that workers had inadequate levels of avian influenza (H5N1) risk knowledge, preventive behavior, and outbreak preparedness. The main barriers to preventive practices were low perceived benefits and interference with work. Poultry workers require occupation-specific health promotion. In 2009, a cross-sectional survey of 360 poultry workers in Hong Kong, China, showed that workers had inadequate levels of avian infl uenza (H5N1) risk knowledge, preventive behavior, and outbreak preparedness. The main barriers to preventive practices were low perceived benefi ts and interference with work. Poultry workers require occupationspecifi c health promotion. I n 1997, a zoonosis in humans caused by a highly lethal strain of avian infl uenza virus (H5N1) was reported in Hong Kong. Live-poultry markets were the source of this outbreak (1) . As one of the world's most densely populated regions (16,000 persons/mile 2 [>6,300 persons/km 2 ]) (2), Hong Kong is a city at high risk for a large-scale outbreak of avian infl uenza caused by live poultry in large-volume wholesale markets and within neighborhood wet markets (open food stall markets).
Because members of the average household in Hong Kong shop in wet markets on a habitual basis, these markets are located in the most densely populated areas ( Figure) and are commonly multistory complexes or in basement levels of shopping centers. Because poultry workers are a potential bridge population (3, 4) , the government has instigated voluntary avian infl uenza training since 2001 that reviews regulations for workplace disinfection, waste disposal, poultry storage, and personal hygiene measures (5,6).
Despite occupational risk for exposure to avian infl uenza (7, 8) , there have been few studies of poultry workers (8) (9) (10) (11) (12) . Most studies were conducted in rural settings in developing countries (9) (10) (11) (12) , but fi ndings cannot be readily extrapolated to cities such as Hong Kong because of differences in food-handling practices and occupational settings. Knowledge, perceptions, and work practices of live-poultry workers in Hong Kong have not been examined. Therefore, a survey of these workers is timely and warranted, given confi rmed persistence of avian infl uenza in Asia. (13) The Study
An anonymous, cross-sectional survey was conducted during June-November 2009. Interviewers approached 132 licensed live-poultry retail businesses in wet markets and 23 wholesale establishments. The fi nal sample was 360 poultry workers (194 retailers and 166 wholesalers; response rate 68.1%).
Respondents were asked about their demographics, past month's work and preventive behavior, and avian infl uenza-related knowledge on the basis of a World Health Organization factsheet (14) . We asked perception questions based on the Health Belief Model and the likelihood of adopting certain behavior patterns in the event of a local bird-to-bird or bird-to-human outbreak of avian infl uenza.
Summative scores were computed for avian infl uenzarelated knowledge, current preventive behavior patterns, outbreak preparedness, and various perception domains. Higher scores refl ected more benefi cial levels of each domain. Unconditional multilevel regression indicated no evidence of clustering effect by poultry market. Standard multivariable linear regression was conducted by using SAS version 9.1.3 (SAS Institute, Cary, NC, USA) with knowledge, practice, and preparedness scores as outcomes and potential predictors showing p<0.25 in unadjusted analyses as input variables. Distribution of standardized residuals and their association with predicted values were examined to assess model assumptions.
Most (208, 60.1%) respondents were men 35-54 years of age, of whom 192 (55.3%) had worked a mean of 16.1 years in the poultry industry. Respondents showed low mean summative scores for knowledge of avian infl uenza (online Appendix incorrectly believed that a human vaccine for avian infl uenza was available. Most (208, 89.9%) respondents were familiar with infl uenza-like symptoms of avian infl uenza virus (H5N1) infection such as fever, but fewer workers were aware of respiratory and gastrointestinal symptoms of virus infection.
The Internet and other sources (e.g., health talks) of information about avian infl uenza were strong independent predictors of greater knowledge. However, training did not result in higher knowledge levels.
Poultry workers reported low-to-moderate levels of compliance with hand hygiene and other preventive measures (ranging from 7.3% [36] using eye protection to 65.2% [245] using handwashing with soap after slaughtering poultry). Working in the poultry industry ≥10 years, lower perceived barriers to preventive behavior, and retail poultry work were independent predictors of higher preventive behavior scores.
With regard to avian infl uenza-related perceptions, lack of training (277, 83.4%) and the view that compliance with all infection regulations is diffi cult during peak hours (218, 64.9%) were the most frequently cited barriers to adoption of preventive behavior. A total of 154 (46.4%) workers believed that face masks reduced business, and 153 (46.1%) believed that vaccination was expensive.
Low anxiety about illness was reported by 242 (76.6%) respondents. In the event of a local outbreak, workers expressed various levels of acceptance for precautionary actions, ranging from 15.8% (56) for reducing work hours to 82.4% (290) for seeking medical care for infl uenza-like symptoms. Ninety-six (27.4%) respondents anticipated taking oseltamivir. Greater perceived benefi t of preventive behavior was the strongest independent predictor of higher preparedness scores (online Appendix Table 2 , wwwnc.cdc. gov/EID/article/17/12/11-0321-TA2.htm).
Similar to other regions (8) (9) (10) (11) , poultry workers in Hong Kong showed low risk perceptions for avian infl uenza, inadequate knowledge, and a wide range of compliance with preventive measures. Because training (6) was not associated with overall preventive behavior or preparedness, there may be an unmet need for occupationspecifi c health information.
Higher levels of knowledge demonstrated by workers who accessed health information sources (e.g., Internet) that provide detailed information suggest that comprehensive, occupation-relevant information should be more widely accessible. However, occupational practices of animal workers might not be amenable to change solely on the basis of improvements in knowledge. Only 129 (42.1%) respondents reported that poultry workers could realistically adhere to all government guidelines (6) . Interference with work, high cost, and reduction of business were repeatedly cited as impediments to the adoption of preventive behavior. Even in the event of local outbreaks of avian infl uenza, most workers were not amenable to actions having adverse economic effects such as reducing work hours. Animal workers are thereby unlikely to widely adopt preventive behavior if these measures confl ict with their economic interests.
Despite the ongoing government regulations regarding avian infl uenza in Hong Kong (6), a complete ban on live poultry is unrealistic because of the culturally entrenched demand for fresh poultry. Increasing knowledge and risk perceptions while simultaneously reducing occupational barriers to preventive behavior thereby continues to be the cornerstone of effective zoonotic infection control among animal workers.
Implications of these fi ndings extend to other poultryborne pathogens, such as Campylobacter spp. and Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 Salmonella spp., which share common preventive measures. Close adherence to workplace measures will likely reduce outbreak risk for other poultry-borne diseases. Therefore, a framework for greater integration of risk management strategies and worker education of these poultry-borne infections tailored to the local context is worthwhile and cost-effective.
In the spirit of the One Health Commission, which calls for an integrated, interdisciplinary approach to human-veterinary-environmental health challenges (15), the fi ght against global pandemics, such as those of avian infl uenza virus (H5N1), necessitates greater dialogue and collaborative leadership between governments and livestock industries. Development of realistic occupational safety measures is an ongoing challenge for national governments. A Pilot Study of Host Genetic Variants Associated with Influenza-associated Deaths among Children and Young Adults We compared the prevalence of 8 polymorphisms in the tumor necrosis factor and mannose-binding lectin genes among 105 children and young adults with fatal influenza with US population estimates and determined in subanalyses whether these polymorphisms were associated with sudden death and bacterial co-infection among persons with fatal influenza. No differences were observed in genotype prevalence or minor allele frequencies between persons with fatal influenza and the reference sample. Fatal cases with low-producing MBL2 genotypes had a 7-fold increased risk for invasive methicillin-resistant Staphylococcus aureus (MRSA) co-infection compared with fatal cases with high- and intermediate-producing MBL2 genotypes (odds ratio 7.1, 95% confidence interval 1.6–32.1). Limited analysis of 2 genes important to the innate immune response found no association between genetic variants and fatal influenza infection. Among children and young adults who died of influenza, low-producing MBL2 genotypes may have increased risk for MRSA co-infection. I t is unknown why some apparently healthy persons become severely ill after infl uenza infection while others infected by the same strain remain asymptomatic or become only mildly ill. The presence of neutralizing antibody to a specifi c infl uenza strain is protective, and certain chronic medical conditions increase the risk for severe outcomes of infl uenza infections, but the risk factors for infl uenzaassociated deaths among previously healthy persons remain largely unknown (1) .
Infectious disease mortality risk has a heritable component; children of parents who died of an infectious disease are ≈6× more likely to die of an infectious cause compared with the general population (2) . A recent large family study that used genealogic databases found an elevated risk for infl uenza death among relatives of persons who died of infl uenza (3) . By comparing the infl uenza mortality rate for relatives of persons who died of infl uenza with the infl uenza mortality rate for relatives of spouses of persons who died, the authors showed that the increased risk was not explained by shared exposure to infl uenza virus and thus may have a genetic component. However, to our knowledge, no published studies have examined the association between specifi c host genetic variants and severe infl uenza disease outcomes.
To address the paucity of research on host genomics and infl uenza, the Centers for Disease Control and Prevention (CDC) convened a meeting of experts in 2007 to solicit opinions on how to explore the role of host genomics in public health activities for infl uenza conducted by the agency. A study of host genomic factors related to severe infl uenza outcomes in children was recommended as an activity that CDC was well positioned to pursue. This article reports the fi ndings of the study implemented in response to that recommendation.
We conducted a hypothesis-generating pilot study to examine if host genetic variants were associated with fatal infl uenza virus infection by comparing prevalence of selected host genetic variants among children and A Pilot Study of Host Genetic Variants Associated with Infl uenzaassociated Deaths among Children and Young Adults 1 young adults who died of infl uenza with population-based prevalence estimates. We focused on 8 single-nucleotide polymorphisms (SNPs) in 2 candidate genes important in the innate immune response to infl uenza infection and for which national prevalence estimates were available: the gene for tumor necrosis factor superfamily, member 2 (offi cial symbol TNF) and the mannose-binding lectin gene (offi cial symbol MBL2).
Because infl uenza-associated deaths in children, but not adults, are nationally reportable in the United States, most cases in this study were pediatric cases reported to CDC through the Infl uenza-associated Pediatric Mortality Surveillance system. This system requires state public health authorities to report to CDC any infl uenza-associated death among persons <18 years old that occurred within their jurisdiction. Information collected by this surveillance system constitutes the primary phenotypic information used in this study and includes underlying health status and chronic medical conditions, infl uenza vaccination status, clinical course and features, and results of microbiologic and virologic testing. Reporting to this surveillance system does not require submission of tissue samples; however, CDC routinely receives tissue samples for a subset of fatal pediatric infl uenza cases for diagnostic confi rmation. For some cases, medical records and autopsy reports provided additional information.
A total of 442 infl uenza-associated deaths among children (<18 years old) and young adults (18-40 years old) residing in the United States were reported to CDC for the 1998-99 through 2007-08 infl uenza seasons; of these, 105 cases with laboratory-confi rmed infl uenza infection had suffi cient tissue specimens available for DNA extraction and constitute the analytic dataset for this study. Fatal infl uenza cases were considered laboratory confi rmed if a positive test result for infl uenza by viral culture, immunohistochemical analysis, or reverse transcription PCR (RT-PCR) had been documented. These represented 1) fatal pediatric cases reported to CDC during the 2003-04 infl uenza season when CDC conducted surveillance for infl uenza-associated pediatric deaths as part of an emergency response effort; 2) fatal pediatric cases identifi ed through national surveillance since 2004 when pediatric infl uenza-associated death was made nationally notifi able in the United States; or 3) fatal cases of infl uenza among young adults at any point in time or among children before 2003 whose case reports and specimens were received by the CDC Infectious Diseases Pathology Branch on a case-by-case basis.
To obtain DNA for genotyping, a 10-μm section from blocks containing formalin-fi xed, paraffi n-embedded tissues was deparaffi nized with xylene and washed twice with absolute ethanol. After residual ethanol evaporated, tissues were digested overnight at 56°C in 200 μL Buffer PKD with 20 μL proteinase K (QIAGEN, Valencia, CA, USA). Extraction of the supernatant was completed with an EZ1 DNA Tissue Kit or a MagAttract DNA Mini M48 Kit (QIAGEN), with DNA eluted into a fi nal 100-μL volume. DNA quality was assessed with a human RNase P real-time PCR in 25-μL volumes by using Agilent Brilliant II QPCR Master Mix as described (4) . Validated TaqMan assays were used to genotype each SNP (protocols, primers, and probes available at http://snp500cancer.nci.nih.gov). Each 25-μL real-time PCR consisted of 12.5 μL of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 900 nmol of assay-specifi c primer, 200 nmol of assay-specifi c probe, and 5 μL of DNA. All controls (extraction blanks, no template controls, and positive controls for each genotype used at 5 ng per PCR; Coriell Institute for Medical Research, Camden, NJ, USA) and unknown samples were assayed in duplicate. Thermal cycling conditions consisted of 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 50 cycles of 92°C for 30 s and 60°C for 1 min. Data were collected during the annealing plateau.
For TNF, we examined 3 promoter SNPs: −308G>A (rs1800629), −238G>A (rs361525), and −555G>A (rs1800750) (5,6); we were unable to infer TNF haplotypes. For MBL2, we examined 5 SNPS, 3 in the coding region of exon 1 and 2 in the promoter region. The 3 structural SNPs in MBL2 that we examined encode variant alleles known as D (codon 52, rs5030737), B (codon 54, rs1800450), and C (codon 57, rs1800451); the wild-type is A (7, 8) . These variants are typically pooled and designated as the O allele. The MBL2 genotype A/A refers to wild-type homozygotes, A/O refers to heterozygotes, and O/O refers to homozygotes or compound heterozygotes. Promoter polymorphisms at positions −550 (H/L variant, rs11003125) and −221 (X/Y variant, rs7096206) encode variants that mediate MBL2 expression. Case-patients were classifi ed as low, intermediate, or high producers of MBL on the basis of their structural and promoter variants (referred to as a "truncated haplotype") (7). Case-patients homozygous or compound heterozygous for any of the 3 variant structural alleles and case-patients with a variant structural allele on 1 chromosome and the X variant on the other were categorized as low MBL producers. Case-patients homozygous for the wild-type structural allele were categorized as high MBL producers except for those also homozygous for the X variant, who were classifi ed as intermediate MBL producers on the basis of evidence that possession of the X/X promoter genotype signifi cantly down-regulates MBL production (9) . Case-patients with the YA/O genotype were classifi ed as intermediate MBL producers on the basis of analyses indicating that this genotype confers intermediate levels of functional MBL (9) . For some analyses, the intermediate and high producers were combined into 1 group and compared with MBL low-producers.
The prevalence of genetic variants among cases was compared with population-based prevalence estimates for the same genetic variants for the 12-19-year age group available from the National Health and Nutrition Examination Survey (NHANES) III CDC-National Cancer Institute Collaborative Genomics Project databank (10) . NHANES is a nationally representative survey of the US population conducted by the CDC National Center for Health Statistics. During the second phase of NHANES III (1991-1994), leukocytes from participants were used to create a DNA bank maintained by CDC's National Center for Environmental Health that contains specimens from >7,000 participants, including ≈1,200 children. To our knowledge, the NHANES DNA bank is the only currently available source of nationally representative prevalence estimates for genetic variants among US residents. The 12-19-year age group is the youngest age group available in the NHANES DNA bank.
Cases were stratifi ed by presence or absence of any chronic medical conditions in the patients known to increase the risk for infl uenza-associated complications (including moderate to severe developmental delay; hemoglobinopathy, immunosuppressive disorders, asthma or reactive airway disease, diabetes mellitus, history of febrile seizures, seizure disorder, cystic fi brosis, or cardiac, renal, chronic pulmonary, metabolic, or neuromuscular disorders) (11) . Case-patients without chronic medical conditions were classifi ed as "previously healthy." Casepatients who were admitted to an inpatient ward or intensive care unit were classifi ed as "hospitalized." Length of illness was defi ned as the duration of time between the reported date of illness onset and death. Case-patients with length of illness <3 days were classifi ed as having "sudden death." Bacterial co-infection was defi ned as at least 1 positive culture for a bacterial pathogen from a normally sterile site (e.g., blood, cerebrospinal fl uid).
Minor allele frequencies between groups were compared with a test of binomial proportions. The null hypothesis was that there was no difference in minor allele frequency between the cases and the reference sample. A priori groups examined in subgroup analyses included previously healthy case-patients, case-patients <5 years old, case-patients with invasive bacterial co-infection, and case-patients with sudden death. Differences in length of illness were evaluated with the Kaplan-Meier estimator with differences tested with the log-rank statistic. Tests of signifi cance were based on a 2-sided test with α = 0.05. Tests of departure from Hardy-Weinberg equilibrium for the reference sample have been published (10) . Analyses were conducted in SAS version 9.2 (SAS Institute, Cary, NC, USA).
This study was exempted from institutional review board review for approval of human subjects research. Data were obtained only from deceased case-patients, and reference sample data were used only in a de-identifi ed and aggregate manner.
Of 442 cases of fatal infl uenza in children and young adults reported to CDC during the 1998-99 through 2007-08 infl uenza seasons, 105 (24%) cases had available autopsy specimens with suffi cient DNA for genotyping. Case-patient characteristics are summarized in Table 1 . Genotyped casepatients had a median age of 6.0 years (range 1 month-40 years) and 52% were female. Sixty-one percent of casepatients were white, and 17% were black. Seventy-four percent of cases occurred during 3 infl uenza seasons: 2003-04 (31%), 2006-07 (21%), and 2007-08 (22%). Eighty-one (77%) of 105 case-patients were infected with infl uenza A and 24 (23%) with infl uenza B. There were no signifi cant differences in the distribution of infl uenza types by season between cases and the national pattern of types found in the US viral surveillance system (data not shown).
Compared with case-patients who were not genotyped, the 105 case-patients with DNA available for genotyping were slightly older (median age 6 years vs. 4 years; p<0.05), less likely to have had a preexisting medical condition (28% vs. 61%; p<0.001), and less likely to have been vaccinated for infl uenza during the season of death (7% vs. 16%; p<0.01). Case-patients genotyped were more likely to have experienced sudden death (31% vs. 22%; p<0.05) and to have died before reaching medical care (34% vs. 22%; p<0.001). It is not surprising that case-patients with sudden death were more likely to have undergone autopsy and, hence, to have had tissues available for DNA extraction. Genotyped case-patients were less likely to have had pneumonia evident on chest radiograph (22% vs. 46%; p<0.05) and about equally likely to have had invasive bacterial co-infection (21% vs. 23%; not signifi cant), but differences in these characteristics are diffi cult to interpret because genotyped case-patients were less likely to have received medical care for their illnesses (presumably because of a greater frequency of sudden death).
Genotype and minor allele frequencies among casepatients are summarized in Table 2 . Minor allele frequencies comparing case-patients to the NHANES reference sample are shown in Figure 1 .
No statistically signifi cant differences were observed in minor allele frequencies or genotype prevalence between the case-patients and the NHANES reference sample for the 3 TNF variants with all case-patients examined together or with black and white racial groups examined separately. 
No statistically signifi cant differences were observed in minor allele frequencies for the 5 MBL2 SNPs examined (Figure 1 ) or the prevalence of pooled MBL2 genotypes (Figure 2 ) between the case-patients and the NHANES reference sample with all case-patients examined together or with black and white racial groups examined separately. In a subgroup analysis, the minor allele frequency of rs5030737 was signifi cantly less common among case-patients <5 years old than in the reference sample (2% vs. 7.2%; p = 0.02). Among low producers of MBL, we observed an estimated odds ratio of 7. Table 3 ). Low-producing MBL2 genotypes were also associated with an approximate 3-fold increased risk for bacterial coinfection in general and with S. aureus infection overall, but these associations did not reach statistical signifi cance. Characteristics of case-patients with invasive MRSA coinfection are shown in Table 4 .
We found no signifi cant differences in allele frequencies or genotype prevalence for variants in the TNF and MBL2 genes between fatal infl uenza cases in patients <40 years old and a nationally representative reference sample. However, among the case-patients who died, most of whom died in childhood, variants of MBL2 responsible for low production of MBL were associated with MRSA co-infection. This observation should be viewed cautiously as a hypothesis for further exploration, given the small number of case-patients with MRSA in our study (n = 8). This fi nding is consistent with results from previous studies that found associations between MBL insuffi ciency (defi ned by genotype) and respiratory infection in children (12) (13) (14) , severe and fatal sepsis (9, (15) (16) (17) , and systemic infl ammatory response syndrome in children (18) .
TNF is a potent proinfl ammatory cytokine produced early in the innate immune response to infection that promotes a wide range of immunologic responses. Excessive systemic TNF is responsible for many symptoms of clinical infection and may lead to fatal complications. Studies have demonstrated a signifi cant genetic contribution to circulating TNF levels, with 50%-60% of variance in TNF levels genetically determined (19) (20) (21) . The most studied SNP is at position −308 (rs1800629), with the A allele associated with 20%-40% greater TNF production (22) (23) (24) and with susceptibility to and severity of numerous infectious diseases (20, 22, 25, 26) . Carriage of the A allele at the −238 position (rs361525) also has been associated with a variety of diseases (20, 22) . MBL, another key component of the innate immune system, is a soluble protein of the collectin family that binds to microbial surfaces and promotes phago-opsonization directly and indirectly by activating the lectin complement pathway. Low serum MBL levels are common and associated with an increased risk for a variety of infections and autoimmune diseases (15, (27) (28) (29) , including acute respiratory infection in young children (12) . MBL levels are strongly infl uenced by genetic factors, with >75% of variation in MBL levels explained by a small number of polymorphisms in the MBL2 gene (30) . Variant proteins are unstable and of lower oligomeric form, which decreases affi nity for microbial ligands and complement-activating ability. Each variant produces signifi cantly reduced serum MBL levels.
MBL has been shown to strongly bind S. aureus (31) and susceptibility to fatal S. aureus infection due to MBL defi ciency has been convincingly demonstrated in murine models (32) . Phase I clinical trials of MBL replacement therapy indicate that this therapy is well tolerated and effective at improving MBL defi ciency in healthy persons (33) . Reports of MBL replacement therapy administered to severely ill persons (34) (35) (36) or to patients with S. aureus sepsis (37) suggest that therapy can improve clinical conditions, although results of these studies were mixed, and in some cases, clinical improvements were temporary. The clinical implications of MBL replacement therapy for infl uenza treatment or prevention are unknown. Among persons with fatal cases, we observed an increased risk for sudden death in carriers of the variant allele of TNF rs1800750. We are unaware of previous literature reporting a similar association; there is no obvious biologic mechanism to explain the fi nding. The TNF rs1800750 variant is in linkage disequilibrium with other TNF variants (http://pga.gs.washington.edu), some of which (including TNF rs361525) have been associated with increased TNF serum levels. Therefore, it is possible that the observed association may be due to linkage disequilibrium with unmeasured polymorphisms that are the causal variants, and more exhaustive analysis of TNF variants is worthy of future study.
A strength of this study is its use of a cohort of case-patients particularly well-suited for investigation of potential host genetic risk factors-these case-patients died with active infl uenza infections, yet were predominantly children and young adults without severe preexisting medical conditions. In such a group, other factors associated with severe infl uenza are less likely to obscure possible genetic associations. An additional strength was access to postmortem lung tissue for immunohistochemistry and/or RT-PCR confi rmation of infl uenza infection.
We recognize that this study has several limitations. Although the study cohort is, to our knowledge, the largest sample of fatal infl uenza cases in children and young adults, the analysis has limited statistical power to detect associations because of small sample sizes, especially when examining subsamples. We had access to limited information about racial and ethnic background of casepatients. Clinical data were obtained primarily from a US surveillance system and were not validated with medical chart review. Although we were able to infer truncated haplotypes for MBL2, haplotype information for TNF was unavailable. Despite these shortcomings, the possibility that specifi c variants of the MBL2 gene known to infl uence serum MBL levels appear to be associated with severe bacterial co-infection is an intriguing fi nding deserving of additional study, especially given the prevalence of co-infection among case-patients who died of pandemic (H1N1) 2009 virus infection (38) and observations that children co-infected with infl uenza and S. aureus may have higher case-fatality rates (39) .
That we observed a stronger relationship between low-producing MBL genotypes and MRSA infection than between those genotypes and S. aureus infection in general is puzzling. We are unaware of an obvious physiologic explanation for why low MBL would predispose more strongly to infection with methicillin-resistant versus methicillin-sensitive S. aureus. One possibility is that MRSA is a marker for other strain characteristics. For example, such an association could arise if MRSA infections were predominantly the USA300 strain while other S. aureus infections were predominantly the USA100 strain. Unfortunately, we do not have data on S. aureus genetic strain types. We also found that of the 4 fatal infl uenza cases in which patients had both MRSA co-infection and low-producing MBL genotypes, 2 patients reportedly also had asthma. It is well-established that asthma increases the risk for serious complications of infl uenza, and although we know of no evidence suggesting that low-producing MBL genotypes are associated with increased risk for asthma (40) , this fi nding may be worth further exploration in future studies. Our fi ndings suggest several opportunities for additional infl uenza-related research. An obvious next step is examination of all functional variants of the MBL2 gene in conjunction with gene expression and functional assays in a larger group of severely ill infl uenza case-patients with suffi ciently detailed clinical data to defi ne important phenotypes (e.g., MRSA co-infection). Interest in association studies of rare variants, the availability of new sequencing technologies that dramatically decrease the cost of sequencing, and access to reference human sequence data suggest that investigating rare variants in candidate genes (including MBL2 and TNF) and their functional effects may be a promising avenue of research. Large-scale genotyping of a sample of case-patients to look for common variants by using methods such as genomewide association studies may be possible if a network of collaborators capable of pooling a suffi cient number of case-patients is developed. Recent initiatives such as the Genome-based Research and Population Health International Network (www.graphint. org/ver2) are aimed at encouraging such networks. Given the rapid acceleration in laboratory technologies, enhancement in bioinformatics methods and capacity, and trends toward collaborative research within large consortia, exploration of the role of host genomic factors in serious illness associated with infl uenza and other viral pathogens is increasingly feasible. We believe that host genomics is a promising area for future research regarding who is at risk for severe complications of acute infectious diseases, including infl uenza.  Clinical features and risk factors for severe and critical pregnant women with 2009 pandemic H1N1 influenza infection in China BACKGROUND: 2009 pandemic H1N1 (pH1N1) influenza posed an increased risk of severe illness among pregnant women. Data on risk factors associated with death of pregnant women and neonates with pH1N1 infections are limited outside of developed countries. METHODS: Retrospective observational study in 394 severe or critical pregnant women admitted to a hospital with pH1N1 influenza from Sep. 1, 2009 to Dec. 31, 2009. rRT-PCR testing was used to confirm infection. In-hospital mortality was the primary endpoint of this study. Univariable logistic analysis and multivariate logistic regression analysis were used to investigate the potential factors on admission that might be associated with the maternal and neonatal mortality. RESULTS: 394 pregnant women were included, 286 were infected with pH1N1 in the third trimester. 351 had pneumonia, and 77 died. A PaO(2)/FiO(2 )≤ 200 (odds ratio (OR), 27.16; 95% confidence interval (CI), 2.64-279.70) and higher BMI (i.e. ≥ 30) on admission (OR, 1.26; 95% CI, 1.09 to 1.47) were independent risk factors for maternal death. Of 211 deliveries, 146 neonates survived. Premature delivery (OR, 4.17; 95% CI, 1.19-14.56) was associated neonatal mortality. Among 186 patients who received mechanical ventilation, 83 patients were treated with non-invasive ventilation (NIV) and 38 were successful with NIV. The death rate was lower among patients who initially received NIV than those who were initially intubated (24/83, 28.9% vs 43/87, 49.4%; p = 0.006). Septic shock was an independent risk factor for failure of NIV. CONCLUSIONS: Severe hypoxemia and higher BMI on admission were associated with adverse outcomes for pregnant women. Preterm delivery was a risk factor for neonatal death among pregnant women with pH1N1 influenza infection. NIV may be useful in selected pregnant women without septic shock. Pregnant women are at an increased risk for contracting influenza and its complications associated with influenza [1] . Like previous epidemic and pandemic diseases, 2009 pandemic H1N1 (pH1N1) influenza posed an increased risk of severe illness among pregnant women [2] [3] [4] [5] [6] [7] [8] [9] . A report from the first month of the pH1N1 outbreak noted that the rate of hospitalization among pregnant women was approximately four times the rate in the general population in the USA [3] . As reported by the California Department of Public Health (CDPH), a total of 10% of the 1088 patients who were hospitalized or died from the 2009 pH1N1 influenza were pregnant [10] . According to the Ministry of Health (MOH) of the People's Republic of China, pregnant women accounted for 13.7% of deaths associated with 2009 pH1N1 influenza [11] . Pregnant women with influenza appear to have an increased risk of miscarriage, premature birth and stillbirth [2, 12, 13] . Reports from Victoria in Australia [14, 15] , New York [16] , and California [17] , demonstrate that 2009 pH1N1 infection was associated with substantial maternal and fetal morbidity and mortality. However, information is limited concerning the risk factors for maternal and neonatal death when pregnancy is complicated by severe or critical illness related to 2009 pH1N1 influenza.
In this report, we described the characteristics of pH1N1 influenza in pregnant women and the risk factors for maternal and neonatal death.
All patients who were admitted to hospitals with confirmed 2009 pH1N1 influenza from Sep. 1 to Dec. 31, 2009 from 27 Chinese provinces were screened if they fulfilled the diagnostic criteria for severe or critical cases. A confirmed case was a person whose pH1N1 virus infection was verified by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) with or without the presentation of other clinical symptoms. Patients were excluded if they had been treated as outpatients or in emergency rooms or duration of hospitalization < 24 h, or if they had incomplete records of clinical outcomes. Severe and critical cases were defined according to the H1N1 2009 Clinical guidelines (Third Edition, 2009) released by the MOH (Additional file 1: Table S1 ). Our research retrospectively collected the patient's clinical information and did not involve the patient's personal information and samples, so there was no informed consent.
The case report form included demographic information, underlying conditions, gestational age, vaccination status, treatment, intensive care unit (ICU) admission, complications, and maternal and neonatal outcomes. Body mass index (BMI) was calculated using height and weight recorded in the case report form, patients with BMI ≥ 30 were categorized as obesity. Indications for applying noninvasive ventilation (NIV): pregnant women who complained shortness of breath or blood gas analysis confirmed hypoxemia PaO 2 to FiO 2 < 300. One nonpulmonary major organ dysfunction or unconsciousness was contraindications for NIV. Indications to change from NIV to invasive ventilation: A cautious trial of NIV was attempted and response to NIV was monitored after the first hour or two. If there was a deterioration of oxygenation, invasive ventilation was considered. Definition of successful NIV: PaO 2 to FiO 2 improved and respiratory rate decreased during one or two hour NIV therapy. The patients successfully weaned off NIV and survived. Definition of failed NIV: During the one or two NIV trial, a deterioration of oxygenation was observed and invasive ventilation was needed. Data collection and analysis were coordinated by the MOH. A standard data collection form was used for each study site. Site investigators were primarily infectious disease physicians closely involved in taking care of such patients at their centers. The data were entered in duplicate into a computerized database. Patient confidentiality was maintained by recording only patient date of birth and gender on the data collection form. The research ethics board at Beijing Chao-Yang Hospital and The First Affiliated Hospital, School of Medicine, Zhejiang University approved the study.
We analyzed the reported demographic characteristics, underlying conditions, symptoms, treatments, complications, clinical course and maternal and neonatal outcomes. Means (standard deviations, SD) or medians (interquartiles, IQR) were calculated as summaries of continuous variables. For categorical variables, percentages of patients in each category were calculated. We compared clinical characteristics and clinical outcomes by using an ANOVA test, chi-square test, or Fisher's exact test or Wilcoxon rank-sum test as necessary. The primary outcome was in-hospital mortality. We performed univariable logistic analysis to investigate the potential factors on admission that might be associated with the maternal mortality. Factors with statistical significance (p < 0.05) in the univariate analyses were included in the multivariate logistic regression analysis. A p value of less than 0.05 was considered to indicate statistical significance. All analysis was carried out using SPSS for Windows (release 13.0).
Clinical description of cohort 3570 severe or critical cases were screened and 394 cases involved pregnant women ( Figure 1 ). Demographic characteristics, underlying conditions, symptoms, and lab findings of the 394 pregnant women are illustrated in diseases were rare in this analysis. None of the patients had been immunized against seasonal influenza or 2009 pH1N1. The median APACHE II score was 7.0 (IQR, [4] [5] [6] [7] [8] [9] [10] [11] . At the time of admission, 351 patients (90.0%) had pneumonia with an abnormal chest radiography or chest computed tomography. The most common symptoms were cough (372; 94.7%) and dyspnoea (199; 50.6%). The median PaO 2 /FiO 2 on admission was 154.7 (IQR, 89.5-320.5) ( Table 1 ). Of the 394 hospitalized patients, 246 (63.7%) were admitted to an ICU at a median of 8 days from onset of illness (IQR 5 to 14; Table 2 ).
Medication 378 (95.9%) patients received oseltamivir. The median time from onset of illness to oseltamivir therapy was 5 days (IQR 3 to 7), among them only 52 patients (14.0%) received oseltamivir within 48 h of onset of illness. 387 out of 394 patients received antibiotics. 244 received traditional Chinese medicine. Corticosteroid therapy was administered to 242 patients ( Table 2) .
The most commonly reported complication in this study was acute respiratory disease syndrome (ARDS) (151; 53.4%) ( Table 2) . 211 (59.4%) women delivered at a median of 6 days (IQR 3 to 12) after pH1N1 symptom onset. 122 out of 211 women delivered prematurely (Additional file 2: Table S2 ). The most common delivery method was cesarean delivery (172 patients, 82.7%) ( Table 2 ). Among 143 live-birth deliveries for which the gestational age was known, 68 were premature (Additional file 2: Table S2 ). Among the 394 pregnant women in the study, 77 died (Table 2) , 56 out of the 77 patients who died were in their third trimester. The main cause of death was refractory hypoxemia (66 patients, 85.7%). Of 5 patients with secondary infection, three patients had Acinetobacter baumannii, one patient had Aspergillus spp, and one patient had both Acinetobacter baumannii and Aspergillus spp.
62.4% of women included in the study required intensive care and 47.2% required mechanical ventilation. 83 (Table 5 ).
The first case of 2009 pH1N1 virus infection in China was documented on May 10, the virus has rapidly spread throughout the mainland. A total of 126,000 confirmed cases were reported by Mar 31, 2010, including 7414 patients severe and 800 patients died. Among all these severe cases, about 13.7% of patients were pregnant women [18] .
In this large study of pregnant women who were hospitalized with severe 2009 pH1N1 influenza, the clinical characteristics were similar to those reported by others [3, 4, 17, 19] . 95.6% of patients were infected in the second or third trimester. In our study, the most common comorbidities were cardiovascular diseases (3.3%), diabetes mellitus (1.0%), respiratory diseases (2.8%), and obesity (18.5%). In our study, the prevalence of underlying diseases was much lower than reports from the United States (49.3%) [19] , 56% in Australia [14] , 34% in California [17] , 22 .8% in Brazil [20] , and 62% in France [4] . In those studies, the main cause of underlying disease was asthma. A study compared asthma prevalence of Chinese adolescents living in Canada and in China. The authors found that for girls, the range of asthma was 4.3% in Guangzhou to 9.8% in Canadian-born Chinese adolescents. These results suggest that the lower prevalence of pre-existing asthma in our samples reflects prevalence of the disease in the Chinese population [21] . The mortality rate for severe or critically infected pregnant women in our study was 20%, similar to what was reported in Canada, Mexico, and New Zealand [22] [23] [24] [25] , but higher than in France (8% death in ICUhospitalized pregnancy women) [4] . Risk analysis showed that a PaO 2 /FiO 2 ≤ 200 and higher BMI (i.e. ≥ 30) on admission were risk factors for maternal death. Pregnancy and ARDS are associated with increased oxygen consumption, which can result in hypoxemia in the mothers and the neonates. We reported that a higher BMI was associated with maternal mortality after adjusting for baseline clinical factors. Observations of a high prevalence of obesity in severe and fatal cases of 2009 pH1N1 infection have been reported in Chile, Canada, the United Kingdom and Mexico [10, 26, 27] . As observed in Australia, 42% of patients had a BMI of more than 30 and 22% of patients more than 35, while the corresponding proportions in the general Australian pregnant population was 24% and 10% respectively [28] . However, our research retrospectively collected the patient's clinical information recorded in CRFs. Proportion of obesity has been overestimated based on BMI in the 3 rd trimester of pregnancy.
Data from previous pandemics and seasonal influenza epidemics suggested that the risk of complications associated with influenza might be higher in the second and third trimester of pregnancy than in the first trimester [2, 3, 17] . We also observed a higher proportion of maternal death occurring in the second and third trimester. During the 2009 H1N1 influenza pandemic, in the United States, the rate of premature birth (30.2%) was higher than the rate of premature births (13%) reported [29] , consistent with data demonstrating a higher rate of premature delivery during previous pandemics [2] . Among women in our study for whom data on pregnancy outcomes was available, the rate of premature birth was 57.8%. In a multivariable analysis, preterm delivery contributed to fetal mortality. Delivery in severe and critically infected women after 37 weeks' of gestation had improved neonatal outcomes compared to similar patients who delivered before 37 weeks of gestation.
Evidence on the useful role of NIV in pregnant patients with ARDS secondary H1N1 viral infection was lacking. Dr. Amit Banga [30] reported a 28-year-old pregnant female with ARDS (PaO 2 /FiO 2 155) due to community-acquired severe pneumonia who successfully treated with NIV. In 2009, Dr. Michel Djibre and collegues [31] reported a 38-year-old pregnant woman at 31 weeks' gestation with PaO 2 /FiO 2 98 who was successfully treated with NIV. In our study, the success rate among pregnant women with H1N1 infection for NIV was 45.8%. A recent prospective multicenter survey also found that when NIV was used as first-line therapy for selected ALI/ARDS patients (those with 2 organ failures, hemodynamic instability, or encephalopathy were excluded), 54% avoided intubation and had excellent outcomes [32] .
Apart from previous findings that major organ dysfunction and obtunded sensorium would obviously be unsuitable candidates for NIV, we found that pregnant women complicated by septic shock were less likely to be successfully treated by NIV. Our data also support that cautious selection of appropriate patients is important for successful application of NIV. Patients should be monitored closely for signs of NIV failure until stabilized. If there are signs of NIV failure, patients should be intubated promptly before a crisis develops.
Our investigation has several limitations. Firstly, we only evaluated pregnant women admitted to a hospital who fulfilled the diagnostic criteria of severe or critical cases. Secondly, it was an observational study, and could therefore only demonstrate associations and could not infer cause. Thirdly, we lacked follow up visits for maternal and neonatal outcomes. Lastly, despite the use of a standardized data-collection form, not all information was collected for all patients.
The clinical data reported herein is consistent with previous studies that demonstrate that pregnant women with influenza are at an increased risk of serious illness and death. Our novel findings included: 1) NIV was useful for some selected pregnant women with pH1N1 virus infection complicated by respiratory failure, but septic shock should be considered a contraindication; 2) a PaO 2 /FiO 2 ≤ 200 and higher BMI (i.e. ≥ 30) on admission were independent risk factors for maternal death; 3) Premature delivery was an independent risk factor for neonatal death.
Additional file 1: The diagnosis criteria for severe and critical cases.
Additional file 2: Maternal and neonatal outcomes by different delivery methods in different trimesters. Data are presented as no.
(%)/total no.(%), if otherwise stated. Percentages are based on patients with complete information in the respective categories. * Two patients missed the detailed information in maternal outcomes. Neonatal outcomes were unknown in four cases. ** One patient missed the detailed information in maternal outcomes. Neonatal outcomes were unknown in two cases.  A scientometric analysis of Indian research output in medicine during 1999–2008 OBJECTIVE: This study analyzes the research activities of India in medicine during 1999–2008, based on the total publication output, its growth rate, quality of papers published and rank of India in the global context. Patterns of international collaborative research output and the major partner countries of India are also discussed. This study also evaluates the research performance of different types of Indian medical colleges, hospitals, research institutes, universities and research foundations and the characteristics of published literature in Indian and foreign journals. It also analyzes the medical research output by disease and organs. MATERIALS AND METHODS: The publication data on medicine has been retrieved by using SCOPUS database. RESULTS: India holds 12th rank among the productive countries in medicine research consisting of 65,745 papers with a global publication share of 1.59% and registering a growth rate of 76.68% for the papers published during 1999–2003 to 2004–2008. CONCLUSION: High quality research in India is grossly inadequate and requires strategic planning, investment and resource support. There is also a need to improve the existing medical education system, which should foster research culture.  Membrane Fusion and Cell Entry of XMRV Are pH-Independent and Modulated by the Envelope Glycoprotein's Cytoplasmic Tail Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry. Enveloped viruses must fuse with host cell membranes in order to gain entry and initiate infection. For retroviruses, this process is mediated by the envelope glycoprotein (Env) acquired from the viral producer cells. The Env is initially synthesized as a precursor in the endoplasmic reticulum (ER) and subsequently cleaved by cellular proteases in the trans-Golgi complex into the surface (SU) and transmembrane (TM) subunits [1] . The SU subunit contains a receptor binding domain (RBD) that is responsible for interactions with specific cellular receptors or coreceptors, and the TM subunit possesses a fusion peptide, two heptad repeats (HRs), a membranespanning domain (MSD), and a cytoplasmic tail (CT), all of which have been shown to control or regulate membrane fusion [2] . Upon proper triggering, the TM subunit undergoes a large scale conformational rearrangement, leading to the formation of a stable helix bundle (6-HB) that drives fusion between the viral and cellular membranes [3] .
The retroviral Env-mediated fusion is controlled at multiple steps to prevent premature activation [2, 4] . First, the cleavage of retroviral Env precursor into SU and TM is a pre-requisite for fusion as it liberates the fusion peptide located at the amino terminus of TM so that it can insert into the target membrane upon triggering [3] . Second, post-translational modifications, such as glycosylation, are also critical for proper folding and receptor binding of Env thereby influencing membrane fusion and cell entry [5, 6, 7] . In addition, several retroviruses, such as murine leukemia virus (MLV), Mason-Pfizer monkey virus (M-PMV), equine infectious anemia virus (EIAV), etc, contain a ,16 aminoacid stretch in the CT of Env, known as R peptide, that intrinsically restricts membrane fusion [8, 9, 10] . In the latter case, the Env proteins containing the full length CT are not fusogenic in the virus-producer cells, but become fully fusogenic after viral protease cleavage of the R peptide upon budding from host cells [9, 11, 12] . The mechanism underlying the R peptide-mediated control of retroviral Env fusion is still not known. Whereas fusion of most retroviruses is triggered by receptor binding, increasing numbers of retroviruses have been shown to require a low pH, or receptor binding plus low pH, for membrane fusion [13, 14, 15, 16, 17, 18, 19, 20] . It is interesting that infection by ecotropic murine leukemia virus (E-MLV) has been shown to be blocked by inhibitors of cellular cathepsins [21] , suggesting host proteases are involved in the fusion activation of E-MLV and perhaps of other retroviruses. Similar mechanisms have been reported for other enveloped viruses [22, 23, 24, 25, 26] .
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome (CFS) [27, 28] . However, recent studies have shown that this virus is a recombinant mouse retrovirus that was likely generated during the passages of a human prostate tumor in nude mice [29, 30] . Moreover, numerous groups have failed to detect XMRV from human prostate cancer samples as well as CFS patients, making the claim of its association with these human diseases questionable [31, 32] . Regardless, it is still important to understand how the Env protein of XMRV mediates membrane fusion and cell entry from the virology perspective, especially in light of the emerging diverse mechanisms of retroviral Env-mediated fusion activation and cell entry [2] . The Env of XMRV shares significant sequence homology with that of other xenotropic and polytropic MLVs (X/P-MLV), especially in the SU subunit, and these viruses share the same xenotropic and polytropic retrovirus receptor 1 (XPR1) for entry [27, 33, 34, 35, 36] . XMRV has been shown to infect a wide range of cell lines derived from different species including humans, with the notable exception of hamster and mouse cells; overexpression of XPR1 in NIH 3T3 and CHO cells renders these cells susceptible to XMRV infection, indicating that XPR1 is the key cellular receptor for XMRV [37, 38, 39, 40, 41] . In this study, we aimed to understand the mechanisms of membrane fusion and cell entry mediated by the XMRV Env protein, particularly the possible role of its relatively long CT (compared to Mo-MLV) and of the viral receptor, XPR1, in modulating this process.
Retroviruses have been historically believed to fuse directly at the plasma membrane of target cells for entry and infection [42] . However, recent studies have shown that some retroviruses, including avian sarcoma leukosis virus (ASLV), mouse mammary tumor virus (MMTV), Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor virus (ENTV), foamy virus, EIAV, and ecotropic Moloney MLV (MoMLV) require a low pH or low pH-dependent proteases for cell entry [13, 14, 15, 16, 17, 18, 19, 20, 21] . Here, we produced MoMLV pseudotypes bearing XMRV Env, and investigated the cell entry of XMRV by using classical chemical inhibitors that block pH-dependent viral entry [4] . We first treated human HTX cells (a subclone of HT1080) with a lysosomotropic agent, NH4Cl, and observed that it did not inhibit but rather somewhat enhanced XMRV infection (p.0.05). As expected, the infection of pH-dependent vesicular stomatitis virus (VSV) pseudovirions was dramatically decreased (p,0.01, Fig. 1A ). We next treated cells with a proton-pump inhibitor, Bafilomycin A1 (BafA1), and found interestingly that XMRV infection was again increased (p.0.05), yet that VSV entry was almost completely blocked by BafA1 even at the 5 nM concentration (p,0.01, Fig. 1B ). We noted that entry of 10A1 MLV was also slightly enhanced by BafA1 (p.0.05), but the effect was not dosedependent (Fig. 1B) . Similar effects of NH4Cl and BafA1 on XMRV entry were also observed in 293 and a human prostate cancer cell line, DU145 (data not shown), together supporting the idea that XMRV entry does not require a low pH as do the typical pH-dependent viruses, such as VSV and influenza A [4] .
The modest but reproducible enhancement of XMRV infection in the presence of NH4Cl and BafA1 could be explained by a block of viral particle degradation in the endosomes or lysosomes. To investigate this possibility and explore if XMRV entry requires cellular proteases, we performed pseudoviral infection in the presence or absence of leupeptin or cathepsin III inhibitor, both of which are broad spectra, lysosomal protease inhibitors. XMRV infection was enhanced by both protease inhibitors, albeit the increase was not statistically significant (p.0.05); however, infection of Ebola pseudovirions was dramatically impaired (p,0.01, Fig. 1C and 1D ). We noted that VSV infection was also slightly enhanced by these two protease inhibitors but the effect was not dose-dependent. The effect of these protease inhibitors on Ebola infection was consistent with the notion that Ebola GP-mediated membrane fusion with endosome requires cellular cathepsin B and L [22, 26] . Taken together, these results show that XMRV entry does not require a low pH or low pHdependent cellular proteases, and that endocytosis XMRV may occur for XMRV but this would likely result in virions inactivation through pH-dependent host proteases.
In order to investigate the role of interactions between XMRV Env and its receptor XPR1 in modulating membrane fusion and cell entry of XMRV, we created a soluble form of XMRV SU fused to the human IgG Fc fragment ( Fig. 2A) . The fusion protein was produced by transient transfection of 293T cells and purified using protein A beads using a procedure we had previously described for the JSRV SU fusion protein [43] . As shown in Figure 2B , incubation of XMRV SU-human IgG fusion protein with the permissive HTX cells resulted in an apparent fluorescence shift relative to that of secondary antibody alone (which served as a negative control), and overexpression of XPR1 receptor in HTX cells substantially increased the XMRV SU binding to the cells, indicating that the binding was specific. Similar results were also obtained in the permissive human 293, DU145, A549, dog MDCK, and monkey Vero cells (data not shown). The specific binding of XMRV SU for XPR1 was further confirmed in CHO/XPR1 cells which were established by transduction using a retroviral vector expressing XPR1; but surprisingly, we reproducibly detected a fluorescent shift in the parental CHO cells (Fig. 2B) , which are known to be nonpermissive for XMRV infection [37, 41] (also see Table 1 below).
We next assessed the effects of purified XMRV SU fusion protein on pseudoviral infection in HTX cells. Cells were pre- incubated with different amounts of XMRV SU fusion proteins for 1 h at 4uC, followed by switching the temperature to 37uC to initiate infection in the constant presence of the fusion protein for 6 h. As shown in Figure 2C , the XMRV SU fusion proteins substantially blocked the XMRV pseudoviral infection (p,0.05) in a dose-dependent manner, with the JSRV SU having no apparent effect (p.0.05). As would be expected, the JSRV SU fusion protein specifically blocked the JSRV pseudoviral infection but not that of XMRV (p,0.05) ( Fig. 2C and 2D ). The concentration of soluble XMRV SU required to block 50% of XMRV infection was ,10 ug/ml, which was relatively higher compared that of JSRV SU (,5 mg/ml, which is necessary to block 50% of JSRV infection) ( Fig. 2C and 2D ). Together, these results demonstrate that the soluble XMRV SU fusion protein interacts with the XPR1 receptor on the cell surface and functionally blocks the XMRV pseudoviral infection.
Truncation of XMRV Env from the C-terminal cytoplasmic tail (CT) promotes SU shedding and syncytia-forming activity While identical in the N-terminal and central regions, including the conserved R peptide cleavage site between 624 and 625, the Cterminal CT of XMRV Env differs from that of MoMLV Env, with a relatively longer length (Fig. 3A ). Here we sought to determine the membrane fusion property of XMRV Env, particularly the effect of CT truncation on cell fusion. We first created a series of truncation mutants in the CT and examined the Env processing and expression by metabolic labeling. 293T cells were pulse-labeled with [ 35-S] Met-Cys for 1 h and chased for 4 h; the XMRV Env proteins in the cell lysates and their SU shed into the culture media were immunoprecipitated with anti-FLAG beads (FLAG is tagged at the N-terminus of SU). As shown in Figure 3B , all the Env constructs were properly processed and expressed in the transfected cells, except CT635 which consistently showed a decreased level of expression of the processed SU (,30% of wildtype) (note the SU subunits of CT624, CT613, CT609, CT608 and CT606 co-migrated with their full length precursors because of their reduced size of precursor, Fig. 3B , upper panel). Of note, CT624, CT613 and CT609 exhibited enhanced SU shedding into culture media as compared to that of wildtype and other mutants (Fig. 3B , lower panel). We also examined the SU surface expression of these Env constructs in 293T cells by flow cytometry using an anti-FLAG antibody, and observed that CT624 exhibited a wildtype level of expression whereas all the other truncation mutants had reduced SU on the cell surface (,50%) (Fig. 3C) . Altogether, these results demonstrate that truncation of the CT of XMRV Env affects SU shedding and surface expression.
We next performed syncytia-forming assay in 293 cells and assessed the membrane fusion properties of XMRV Env and mutants. 293 cells were chosen because they are highly transfectable and also permissive to XMRV infection [41] . The full length XMRV Env was unable to induce syncytia formation, presumably due to the presence of an R peptide in the CT (Fig. 4A ). CT624 and CT613, in which the CT of XMRV Env was truncated at the putative R peptide cleavage site and further towards the N-terminus (Fig. 3A) , respectively, elicited apparent syncytia (typically ,30 syncytia per mg DNA, with .6 nuclei per syncytium) (Fig. 4A) . Interestingly, CT609, which contains the first arginine residue of the CT only, showed a much reduced fusion activity as compared to CT624 and CT613 (,5-10 syncytia per mg DNA, with smaller size) (Fig. 4A ). These results were somewhat different from what had been reported for MoMLV, where an identical mutant largely retained the fusogenicity of R peptide-minus mutant [11, 44] .
Noticeably, the increased fusion activity of CT624 and CT613, and to lesser extent of CT609, correlated with the enhanced SU shedding of these mutants in culture media (Fig. 3B) . These results are similar to our previous findings made on JSRV Env, severe truncation of which led to pronounced SU shedding accompanied with greatly increased fusogenicity [19] . Interestingly, we observed that the tailless CT608 and CT606 mutants were virtually fusiondefective, possibly due to their truncation into the MSD and/or reduced surface expression ( Fig. 3A and 3C ). We further treated the individual Env-expressing cells with a low pH buffer (pH 5.0) for 1 min or 5 min (pictures not shown), but did not observe apparent effect on syncytia induction of any of these constructs, supporting the above conclusion that XMRV Env-mediated fusion and cellular entry is pH-independent.
The finding that syncytia induction can be observed in cells expressing XMRV Env truncation mutants prompted us to further quantitatively measure their membrane fusion activities using a flow cytometry-based cell-cell fusion assay adapted from our previous studies on JSRV [18, 19, 45] . In this assay, the effector 293T/GFP cells were transfected with Env-encoding plasmids, and the target 293 cells were labeled with a red-fluorescent dye, CMTMR. Consistent with the syncytia formation data, the XMRV Env wildtype and CT635 were unable to induce cellcell fusion as evidenced by no fluorescent dye transfer (,1.8%, similar to the No-Env background), whereas truncation at the putative R-peptide cleavage site or further upstream towards the MSD, i.e., CT624 or CT613, induced apparent cell-cell fusion (4,5%) (p,0.05) ( Fig. 4B and 4C ). Again, CT609 showed a relatively low cell-cell fusion activity (,3%), and the tailless CT608 and CT606 mutants were incapable of inducing fusion with background signals (Fig. 4B and 4C ). We also examined the fusion activities of all constructs in 293 cells overexpressing the XPR1 receptor (293/XPR1), but observed only modest increases for CT624, CT613 and CT609 (,10-20%). The titers of XMRV wildtype and mutants in 293/XPR1 cells were approximately 5fold higher than those in the parental 293 cells, and the overexpression of XPR1 in 293/XPR1 cells was confirmed by flow cytometry using the soluble XMRV SU (data not shown). The differential fusion activities of CT624, CT613 and CT609 could be due to their different levels of Env expression on the cell surface or/and intrinsic fusogenicity. To distinguish these possibilities, we transfected effector 293T cells with different amounts of plasmid DNA encoding individual truncated Envs, and determined their cell-cell fusion activities and SU surface expression in parallel. As shown in Figure 5A , the fusion profiles of CT624 and CT613 were almost identical, as evidenced by their similar slopes (,0.095 and ,0.010, respectively, R 2 = 0.97-0.99). In contrast, CT609 exhibited a slightly decreased slope (,0.068, R 2 = 0.93), implying that its reduced fusogenicity relative to CT624 and CT613 cannot be fully attributable to its low level of surface expression. We further performed cell-cell fusion using different co-culture periods, i.e., 0, 2, 4, and 8 h, and again observed faster fusion kinetics for CT624 and CT613 (Fig. 5B ) as compared to CT609, further confirming that additional truncation of XMRV Env beyond the R peptide cleavage site does not increase fusion activity as we had seen for JSRV Env and that CT609 has an intrinsically relatively low fusogenicity. The expression of XMRV Env on the 293T cell surface was measured using anti-FLAG and flow cytometry. Fluorescence geometric means were normalized to XMRV Env (100%). Shown are the averages of 3 independent experiments 6 S.D. XMRV Env: XMRV Env tagged with a FLAG sequence only at the N-terminus. All truncations were also tagged similarly with an N-terminal FLAG. F-XMRV-F: an XMRV Env construct that is tagged by FLAG sequences on both N-and C-termini. Mock: untransfected 293T cells. doi:10.1371/journal.pone.0033734.g003
To examine the possibility that the lack or reduced fusion for CT609, CT608 and CT606 might be due to a block at hemifusion, we treated co-cultured target and effector cells with chlorpromazine (CPZ, 0.2-0.5 mM), a membrane permeable reagent that promotes the transition from hemifusion to full fusion [46] , but observed no apparent increase in fusion for any of these Env constructs (data not shown). These results suggest that the fusion suppression in these XMRV Env constructs unlikely takes place at the hemifusion step. Overall we conclude that, distinct from JSRV Env, severe truncation of the CT of XMRV Env towards the MSD does not further enhance but rather impairs the Env fusogenicity. The reason for the decreased fusogenicity of CT609, CT608 and CT606 remains unclear, but is likely related to reduced surface expression or/and altered Env conformation (see Discussion).
We next determined the role of XPR1 in XMRV Env-induced membrane fusion by using nonpermissive CHO cells and CHO cells expressing human XPR1 (CHO/XPR1). The CHO/XPR1 cell line was established by transducing CHO cells with a LXSN retroviral vector encoding XPR1 [33] . Expression of XPR1 in the CHO/XPR1 cell line was demonstrated by the specific binding of soluble XMRV SU fusion protein to those cells as shown in Figure 2B , and was further confirmed by immunostaining using an anti-XPR1 antibody (Fig. 6A) . The titers of XMRV wildtype and CT truncation mutants in these two cell lines are shown in Table 1 . CHO cells were apparently not susceptible to XMRV Env pseudoviral infection (Table 1) , consistent with previous reports from other groups [38, 39, 40, 47] . Overexpression of XPR1 in CHO cells resulted in a titer of 10 4 IU/ml for the wildtype and somewhat reduced titers for the CT truncation mutants (Table 1) . Overall, these data support the notion that XPR1 is a critical cellular receptor for XMRV.
The cell-cell fusion activities of XMRV Env and mutants in CHO and CHO/XPR1-expressing cells were then examined. For this purpose, we labeled CHO or CHO/XPR1 cells with CMTMR, and co-cultured them with the effector 293T/GFP cells expressing XMRV Env or truncation mutants plus GFP. We observed that, surprisingly, CT624 and CT613 reproducibly induced a detectable level of cell-cell fusion activity in the nonpermissive CHO cells (p,0.05) (Fig. 6B) , despite the fact that this cell line is nonpermissive for XMRV infection (Table 1) . Interestingly, overexpression of human XPR1 in CHO cells only slightly increased the fusion activities of XMRV Env CT mutants, CT624 and CT613 (Fig. 6B) , despite their significantly increased pseudoviral titers (Table 1) . These results, along with the data using 293/XPR1 cells described above, imply that XPR1 may not be the sole trigger for XMRV Env-mediated membrane fusion and cell entry.
The CT of retrovirus Env plays various roles in the replication cycle, including entry and assembly; this has been mostly studied in HIV-1 [48] . Here, we wished to determine the ability of XMRV Env and CT truncation mutants to pseudotype the MoMLV retroviral and HIV-1 lentiviral vectors as well as its relationship to membrane fusion and cell entry. As shown in Table 2 , all the XMRV Env constructs (tagged with a FLAG sequence at the N- terminus) were able to pseudotype both types of vectors but with distinct efficiencies. The full length XMRV Env exhibited approximately 2610 4 infectious units per ml for both vectors ( Table 2 , and data not shown), similar to a recent report [49] . The titers of MoMLV retroviral pseudotypes harbouring CT635 or CT624 were slightly reduced as compared to that of the wildtype Env (,4-6 fold), whereas the other more severely truncated mutants exhibited a 2,3-log decrease in the infectious titer ( Table 2) . Similar patterns were also observed for the HIV-1 lentiviral pseudotypes (Table 2 ), but interestingly we found that CT635 consistently exhibited pronounced reductions in the lentiviral pseudoviral titers (,100 fold) as compared that of MoMLV retroviral pseudotypes (,6-fold). The generally reduced viral titers for the CT truncations cannot be fully explained by the enhanced SU shedding, at least for some of these mutants, but appeared to correlate with the differential levels of SU surface expression (Fig. 2C) . We also examined the incorporation efficiencies of these Envs into the MLV pseudovirions by Western blot using concentrated pseudoviral particles, and detected similar levels of SU for CT624, CT613, CT609 and the wildtype Env, as compared to CT635, CT608 and CT606 for which the SU incorporation efficiency was greatly reduced (Fig. 7) . We have attempted to detect the XMRV TM in viral producer cells and the viral particles using an antibody against the MoMLV TM but without success (a gift from Marc Johnson, data not shown). Nevertheless, the Env incorporation data based on the SU (Fig. 7 ) and the XMRV pseudotype titers shown in Table 2 correlated with the SU expression profiles shown in Figure 3C . We noticed that the titers of pseudoviral infection for CT624 and CT613 did not correlate with their enhanced Env fusogenicity based on the syncytia formation and cell-cell fusion assays, and this was particularly the case for CT613, which showed a strongly enhanced fusogenicity (Figs. 4 and 5) but a much reduced titer relative to the wildtype Env (Tables 1 and 2 ).
Retroviruses use distinct mechanisms for membrane fusion and cell entry, the mechanisms of which are still poorly defined. In this report, we provided evidence that XMRV entry does not require a low pH or pH-dependent host proteases, but uses a mechanism that is similar to that of typical pH-independent viruses. Interestingly, we find that XMRV entry is enhanced by NH4Cl and BafA1, the two most commonly used agents that neutralize acidic endosomal environments, as well as by leupeptin and cathepsin inhibitor III, which broadly inhibit the lysosomal protease activities. Together, these observations suggest that endocytosis may occur in non-productive entry of XMRV, leading to viral particle degradation. Consistent with this notion, we did not observe specific block of XMRV entry by Dynasore or a dominant negative mutant of Dynamin (K44A) in 293T cells (data not shown). Previous studies have shown that different endocytic pathways mediate entry of some pH-independent retroviruses, including amphotropic and ecotropic MLV as well as HIV-1 [21, 50, 51] , however the exact mechanisms and the underlying significance remain largely unknown. It should be added that, while we have not observed any inhibitory effects of leupeptin and cathepsin III inhibitor on XMRV infection in HTX and 293 cells, we cannot rule out the possibility that cellular proteases may be involved in the XMRV entry of other cell types. In this sense, it is interesting to note that endocytosis and cathepsins were recently shown to affect the entry of several gammaretroviruses, including XMRV, in human TE671 and rat XC cells [52] . Additional studies are warranted to clarify this issue and further characterize the entry pathway of XMRV, perhaps with assistance of the recently developed single molecule labeling and confocal imaging technique.
One important objective of this study was to understand the possible roles of the CT of XMRV Env in modulating membrane fusion and cell entry. We showed here that CT624 and CT613, which are truncated at or beyond the putative R peptide cleavage site of the XMRV Env (Fig. 3A) , induced apparent syncytia formation and cell-cell fusion in permissive 293 cells (Figs. 4 and 5), presumably due to the removal of the putative R peptide. Surprisingly, we observed apparent cell-cell fusion of CT624 and CT613 also in CHO cells (Fig. 6B) , which are known to be nonpermissive for XMRV infection, including these two truncation mutants (Table 1) . These results suggest two possibilities: first, CHO cells may express a low but functional level of XPR1 that permits cell-cell fusion of XMRV Env mutants and that the resistance of CHO cells to XMRV infection may be due to a block at the post-fusion steps. This possibility is supported in part by our observation that a soluble form of XMRV SU fusion protein reproducibly binds CHO cells relative to the negative control using secondary antibody alone (Fig. 2) . These binding results also argue against the possibility that potential N-linked glycosylation of XPR1 in CHO accounts for its resistance to XMRV infection, a situation that has been previously shown to be the case for several retroviruses [53, 54] . Second, the XPR1-mediated binding may The MLV titers were determined as described in Table 1 not be the sole trigger for XMRV Env-mediated membrane fusion. This scenario is in line with our finding that overexpression of XPR1 in 293 and CHO cells did not significantly increase the cell-cell fusion activities of XMRV Env truncation mutants despite their increased infection in these cells (Fig. 6B and Table 1 ; data not shown). We also considered the possibility that the XMRV Env truncation mutants may have acquired a receptor-independent, spontaneous cell-cell fusion or are pre-activated in 293T/ GFP cells due to their reduced kinetic barrier required for membrane fusion; however, the lack of infection in the CHO cells for the truncation mutants did not support this hypothesis ( Table 2) . Taken together, we favour the notion that, while XPR1 is a critical receptor for XMRV and is required for membrane fusion and cell entry, other cellular factors as yet to be identified are likely to be involved in cell entry and membrane fusion of XMRV. Consistent with this idea, it has been recently reported that XMRV does not infect BHK cells even when XPR1 is overexpressed in this cell line [47] , and that XMRV can infect A549 cells even though this cell line does not express a functional XPR1 receptor [55] . Hence, identification of additional cellular factors involved in XMRV entry would help to better understand the mechanisms of membrane fusion and cell entry mediated by XMRV Env.
Previous studies from HIV and other simple retroviruses have suggested that the enhanced fusion activities of some retroviral Env truncations in the CT may be due to increased steady-state levels of Env expression on the cell surface [56, 57, 58, 59] . However, here we have found little evidence that suggests that this might be the case for XMRV (Fig. 3C ) -despite the highly conserved endocytosis motif, YXXh (Y = tyrosine, X = any amino acid, h = residue with hydrophobic side chain) present in the CT of XMRV Env (Fig. 3A) . Another commonly assumed mechanism is that truncation of the retroviral Env CT can somehow alter the conformation of Env ectodomain, resulting in a reduced association between SU and TM thereby promoting membrane fusion [60] . Indeed, we observed that all three truncation mutants with enhanced fusogenicities, i.e., CT624, CT613 and CT609, exhibited increased levels of SU shedding, which was in sharp contrast to that of wildtype Env and other mutants (CT635, CT608 and CT606) having minimal cell-cell fusion activity (Fig. 3B, Figs. 4 and 5) . Future studies will focus on how the CT of XMRV Env structurally modulates the Env fusion activation.
Another surprising finding of this study is that CT609, which harbours the single arginine residue in the CT possesses a reduced fusogenicity relative to that of CT624 and CT613, which cannot be solely explained by its reduced surface expression (Figs. 4, 5, and 6). This observation is clearly different from what we had seen for JSRV Env [19] and is also somewhat different from some though not all of the previous studies on MoMLV [11, 44, 61] . Importantly, the tailless mutants, CT608 and CT606, are virtually fusion-defective (Fig. 4) , collectively leading us to propose that the N-terminal CT proximal to the MSD of XMRV Env is critical for Env-mediated membrane fusion. One possible mechanism is that residues in this region, including the highly conserved arginine present in many transmembrane proteins including the retroviral Envs, may interact with cell membrane and thus modulate lipid movement during the membrane fusion process [62, 63] .
Despite enhanced fusogenicity of CT624 and CT613, we found that the infection efficiency of their pseudovirions were rather low compared to those of wildtype Env (Tables 1 and 2 ). One plausible explanation is that the incorporations of these truncations into the MoMLV and HIV-1 vectors might be reduced as compared to those of wildtype; however, based on our immunoblot analysis using an anti-FLAG antibody to detect the XMRV SU, we found no evidence to support this scenario (Fig. 7) . Alternatively, the reduced pseudoviral titers for the truncation mutants might result from their altered ability to bind to the viral receptor, XPR1. While we do not have direct evidence in support of this possibility, the apparent SU shedding induced by the CT truncations in the Env-expressing cells (Fig. 3B ) strongly suggests that conformational changes likely occurs in the ectodomain of the truncated Env, including their SU subunits. Indeed, prior studies from HIV and other retroviruses have demonstrated that CT truncation of retroviral Env can alter the Env receptor binding capability thus affect viral infection [60, 64] . In this regard, it would be interesting to explore the role of the CT of XMRV Env in the infectious virus system.
The HTX (a subclone of HT1080), 293T, 293, 293T/GFP (293T cell line stably expressing GFP) and 293/GP-LAPSN (293 cells stably expressing MoMLV Gag-Pol and alkaline phosphatase or AP) cell lines have been previously described [19, 65] . The 293/ XPR1, HTX/XPR1 and CHO/XPR1 cell lines were generated by transducing the 293, HTX or CHO cells using a retroviral vector, LXSN, encoding the XPR1 receptor (LhXPR1SN, kind gift of Dusty Miller) [33] and bearing VSV-G. Infected cells were selected using G418 (Invitrogen, Carlsbad, CA) for ,10 days. All cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37uC at 10% CO 2 -air atmosphere at 100% relative humidity.
The anti-FLAG monoclonal antibody, the EZview Red anti-FLAG affinity gel, and anti-mouse immunoglobulin G (IgG) coupled to phycoerythrin (PE) were purchased from Sigma (St. 
XMRV Env was initially engineered to contain a FLAG tag at both N-and C-termini by using pcDNA3.1-VP62 (gift of Robert Silverman) [37] as a template for PCR, and cloned in a pCIneo expression vector (Promega, Madison, WI), the resulting construct was referred to as pCIneo-F-Xenv-F. To create the N-terminal FLAG-tagged XMRV Env wildtype and CT truncations, the pCIneo-F-Xenv-F construct was used as a template, with the following lower primers being used for PCR amplification (Not I sites are underlined): XMRV Env, 59-ATCGGCGGCCGCT-CATTCACGTGATTCCACTTC-39; CT635, 59-TTCTGCG-GCCGCTCATGATTTGAGTTGGTGATA-39; CT624, 59-C-TGTGCGGCCGCTCACAGGGCCTGCACTACCGA-39; CT-613, 59-AATTGCGGCCGCTCAAAACTGGACCAAGCGGT-TG-39; CT609, 59-TACAGCGGCCGCTCAGCGGTTGAGA-ATACAGGGTCCGA-39; CT608, 59-AAACGCGGCCGCT-CAGTTGAGAATACAGGGTCCGA-39; CT606, 59-GACC-GCGGCCGCTCAAATACAGGGTCCGAAGA-39. The pCI-neo-10A1, pMD.G and pCIneo-Ebola GP expression vectors have been previously described [18] .
The soluble XMRV SU construct was generated by overlapping PCR using pcDNA3.1-VP62 [37] and the previously described pCSI-JSU (for JSRV SU fusion protein) [43] as templates. The first fragment containing XMRV SU was amplified using the following primers: upstream primer (Not I underlined), 59-GCATGCGGCCGCATGGAAAGTCCAGCGTTCTC-39; downstream primer, 59-CCTAGGCCTGTCGACGCCTTTTCA-AACTGGCC-39. The second fragment containing human IgG Fc was generated using the following primers: upstream primer, 59-GGCCAGTTTGAAAAGCTGTCGACAGGCCTAGG -39; downstream primer, 59-TGTATCTTATCATGTCTGGATC-CCC-39. The XMRV SU fused to the human IgG Fc was generated using the two fragments as templates, and the upstream and downstream primer of the first and second fragment, respectively, and then the PCR product was cloned into the pCSI vector.
The MoMLV retroviral pseudotypes encoding the alkaline phosphatase (AP) were produced by transfection of 293/GP-LAPSN cells with plasmid DNA encoding individual XMRV Env, CT truncations, or JSRV Env. The MoMLV retroviral pseudotypes encoding the green fluorescent protein (GFP) were generated by co-transfection of 293T cells with pCMV-gag-pol-MLV, pCMV-GFP-MLV (both vectors are kind gifts of François-Loïc Cosset) and plasmids encoding XMRV Env, XMRV Env CT truncations, Ebola GP (pCIneo-Ebola GP) [18] , VSV-G (pMD.G), or MLV 10A1 Env (pCIneo-10A1) [18] . The HIV-1 lentiviral pseudotypes encoding AP were produced by co-transfecting 293T cells with pCMV-HIVD8.2, pHR'CMVAP [66] and plasmid DNA encoding individual Envs. All pseudotypes were harvested 48 and 72 h post-transfection and cell debris were removed by centrifugation at 2,5006 g. MLV pseudovirions were purified by ultracentrifugation on a 20% sucrose cushion for 2 h at 185,0006 g and 4uC, and Western blot was performed to examine SU incorporation using an anti-FLAG antibody. All viral infections were carried out in the presence of 5 mg/ml polybrene (Sigma) and viral titers were determined by AP staining or flow cytometry analysis to measure GFP + cells 48-72 h post-infection. For infection in the presence of drugs or soluble XMRV SU or JSRV SU, cells were first pre-treated with the indicated concentrations of drugs at 37uC or the soluble proteins at 4uC for 1 h, and then incubated with retroviral pseudotypes for 6 h in the presence of drugs or fusion proteins before inactivation using citrate buffer (40 mM sodium citrate, 10 mM KCl, 135 mM NaCl, pH 3.15).
The syncytium induction assay was performed as described previously with some modifications [6, 19] . 293 cells were cotransfected with plasmids encoding XMRV Env or CT truncation mutants plus a GFP-encoding plasmid in order to monitor the transfection efficiency and syncytia formation. Syncytia formation was typically observed and photographed 24 h post-transfection. Where applicable, cells were treated for 5 minutes at 37uC with pre-warmed pH 5.0 buffer (phosphate-buffered saline (PBS), 10 mM MES, 10 m M HEPES) or 0.2-0.5 mM CPZ for 1 min and incubated in normal growth media at 37uC for 1 h.
The cell-cell fusion assay was performed as described previously [19, 45] . Briefly, effector 293T/GFP cells were transfected with plasmid DNA encoding XMRV Env or CT truncation mutants using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, cells were washed with PBS and detached using PBS containing 5 mM EDTA. Target 293, 293/XPR1, CHO, or CHO/XPR1 cells were detached using PBS-5 mM EDTA and labeled with 3.5 mM CMTMR in serum-free media for 30 min at 37uC, washed, incubated for an additional 30 min at 37uC in fresh media and washed 3 times with media. Effector cells and target cells were cocultured on 24-well plates for the indicated time periods. Cell-cell fusion was measured by flow cytometry using FACSCalibur (BD Bioscience, Missauga, Canada). The surface expression of XMRV Env in the 293T/GFP cells was measured by flow cytometry using the anti-FLAG antibody and anti-mouse IgG coupled to PE.
Production of XMRV SU fusion protein and its binding to cells Soluble XMRV SU and JSRV SU fusion proteins were produced as described previously [43] . 293T cells were transfected using the calcium-phosphate method with plasmids encoding the different SU. Twelve hours post-transfection, media were replaced with DMEM supplemented with 2% ultra-low IgG FBS (Invitrogen). The proteins in the media were purified using protein A beads (GE Healthcare, Uppsala, Sweden) and analyzed by SDS-PAGE and Sypro Ruby staining (Bio-Rad, Hercules, CA).
XMRV SU binding assays were performed as described previously [6, 43] . Cells were incubated with 2-10 mg of soluble XMRV SU-IgG fusion protein for 3-4 h on ice, washed 3 times with PBS-2% FBS and incubated with anti-human IgG coupled to FITC for detection. Fluorescence was measured by flow cytometry using FACSCalibur (BD Bioscience, Missauga, Canada).
Immunostaining CHO or CHO/XPR1 cells were fixed using 4% paraformaldehyde in PBS, permeabilized using 0.5% Triton X-100 and stained using anti-XPR1 and anti-rabbit IgG coupled to FITC. Before mounting the slides, cells were counterstained with the nuclear stain DAPI. Pictures were taken using a fluorescence microscope (Carl Zeiss, Goettingen, Germany) and images were processed using the ImageJ software (U.S., National Institutes of Health).
Metabolic labeling was performed as previously described [19, 45] . Briefly, 293T cells were transfected using the calciumphosphate method with plasmid DNA encoding individual Env. Twenty-four hours later, cells were starved in cysteine and methionine-free DMEM for 30 minutes, pulse-labeled with 62.5 mCi 35 S-cysteine and -methionine for 1 h at 37uC, washed with fresh media and chased for 4 h at 37uC in complete growth medium. Media were then collected and cells washed and lysed (50 mM Tris pH 8.0, 150 mM NaCl, 0.4 mM EDTA, 1% Triton X-100, 0.1% NP-40, 10 mg/ml aprotinin (Sigma), 10 mg/ml leupeptin (Sigma) and 1 mM phenylmethylsulfonyl fluoride (Sigma). The XMRV Env proteins in media and in cell lysates were immunoprecipitated using anti-FLAG beads and resolved by SDS-PAGE. Dried gels were autoradiographed and band intensities of XMRV SU in the cultured media were quantified using the Quantity One software (Bio-Rad, Hercules, CA). The Impact of Weather on Influenza and Pneumonia Mortality in New York City, 1975–2002: A Retrospective Study The substantial winter influenza peak in temperate climates has lead to the hypothesis that cold and/or dry air is a causal factor in influenza variability. We examined the relationship between cold and/or dry air and daily influenza and pneumonia mortality in the cold season in the New York metropolitan area from 1975–2002. We conducted a retrospective study relating daily pneumonia and influenza mortality for New York City and surroundings from 1975–2002 to daily air temperature, dew point temperature (a measure of atmospheric humidity), and daily air mass type. We identified high mortality days and periods and employed temporal smoothers and lags to account for the latency period and the time between infection and death. Unpaired t-tests were used to compare high mortality events to non-events and nonparametric bootstrapped regression analysis was used to examine the characteristics of longer mortality episodes. We found a statistically significant (p = 0.003) association between periods of low dew point temperature and above normal pneumonia and influenza mortality 17 days later. The duration (r = −0.61) and severity (r = −0.56) of high mortality episodes was inversely correlated with morning dew point temperature prior to and during the episodes. Weeks in which moist polar air masses were common (air masses characterized by low dew point temperatures) were likewise followed by above normal mortality 17 days later (p = 0.019). This research supports the contention that cold, dry air may be related to influenza mortality and suggests that warning systems could provide enough lead time to be effective in mitigating the effects. It is well known that intra-annual mortality exhibits a pronounced winter peak in locations with seasonal climates [1] , [2] . In the United States, mortality arising from respiratory disease is 50% higher in winter than in summer [1] . One contributor to this excess winter mortality is influenza [3] , the timing and severity of which can vary significantly from year to year. The association between the annual influenza peak and winter weather in temperate locations has lead to the hypothesis that weather variability could influence influenza mortality variations [4] , [5] .
Recent research examining climatic influences on influenza transmission has galvanized interest in this topic. Airborne transmission of Influenza A/Panama virus between guinea pigs was more likely at low temperatures and relative humidities [4] . Because temperature is physically/mathematically linked to relative humidity and thus confounds interpretation, a mass-based humidity measure (such as specific humidity or vapor pressure) provides a stronger relationship to influenza transmission [5] . Given the cold-season decline in specific humidity in temperate climates, a retrospective study [6] showed an association between year-to-year humidity variations and the timing of the influenza seasonal onset in U.S. states. This differs from tropical locations which generally lack a seasonal influenza peak [7] . One theory is that tropical climates are dominated by direct contact transmission which, unlike airborne transmission, is not influenced by air temperature and humidity [7] .
These recent studies [4] , [5] , [6] , [7] have motivated several reviews on climate and influenza seasonality [8] . [9] , [10] . These reviews, which approach the issue from atmospheric sciences, virological, and epidemiological perspectives, do not reach firm conclusions on the causes of influenza seasonality but suggest that those causes are complex and multifactorial and that the solution will require interdisciplinary cooperation.
A variety of theories exist as to how weather and climate might exert some influence on influenza seasonality. Low temperatures enhance viral stability [4] , reduce mucosal blood flow [11] , and/or diminish mucociliary clearance [12] . Correlations exist between the number of upper respiratory infections and cold-air outbreaks [13] . It is commonly assumed that winter indoor crowding enhances influenza virus transmission, though direct evidence is lacking [8] . Low humidity conditions, which are often but not always accompanied by low temperatures, enhance survival times of viral aerosols [14] , [15] , [16] . If correct, these relationships suggest that indoor winter heating without humidification could enhance influenza transmission [8] , as indoor absolute humidity tends to be correlated with outdoor values and thus is typically lower in winter [6] .
From a micro-physical perspective, there is evidence that both droplet size and transmission mode depend on ambient environmental factors [17] , [18] . Small droplets can remain airborne longer whereas large drops tend to precipitate, suggesting that ambient conditions would influence whether airborne-or contactmode transmission predominates. The virulence of the influenza virus changes constantly as the virus undergoes antigenic shift and antigenic drift [8] . Although virulence depends upon a variety of factors, one possible atmospheric influence is that cold, dry air allows the lipid envelope encasing the influenza virus to remain intact longer, increasing the likelihood of infection [19] .
Theories proposing that factors other than weather/climate are responsible for influenza seasonality include cycles in viral interference [20] and intrinsic temporal viral dynamics that operate independent of external forcing factors [21] . Some research suggests that the seasonality is driven by the school calendar in which students reconvene after a summer break, but this timing is not consistent with the typical onset of influenza in early winter [22] .
We examine the hypothesis that cold and/or dry weather enhances human pneumonia and influenza (P&I) mortality through a retrospective study of daily mortality in New York City and environs from 1975-2002. We hypothesize that periods with colder and/or less humid conditions exhibit excess P&I mortality for a period of time following those climatic conditions. Our research differs from recent work on this topic that examined the timing of the influenza season onset over large geographic areas [6] -our focus is on the influence of daily weather on influenza characteristics for a single, large metropolitan area.
We selected New York City for this study for several reasons. Our study examines daily mortality, and statistical robustness is enhanced when the daily sample size is sufficiently large. Weather obviously has a high spatial variability, so it is important that the observed weather be representative of the environmental conditions likely experienced by the decedents. In addition, New York City's mid-latitude location provides a high degree of both interannual and intra-annual variability in weather and climate, so this variability provides a wider range of sample conditions. Thus, New York City has both a large enough population to provide a consistent daily mortality signal while the population density is high enough that the weather observed at a single station is sufficiently representative of conditions experienced throughout the metropolitan area.
We conducted a retrospective cohort study of pneumonia and influenza (P&I) mortality of residents of the New York City metropolitan area. Daily frequencies of P&I mortality were tallied from National Center for Health Statistics archives for the New York City Consolidated Metropolitan Statistical Area (which, as defined in the year 2000, includes 30 counties in New York, New Jersey, Connecticut, and Pennsylvania). This period of record spans three revisions of the International Classification of Diseases (ICD) codes (Table 1 ).
In the National Center for Health Statistics mortality files that we used for this research, all information that could allow an individual to be identified has been removed. This research utilized only mortality counts for a large metropolitan area. These de-identified counts are stored in governmental archives for the purposes of retrospective research; because all personal identifying information is redacted, consent is not required. Thus, this research is exempt from IRB review under the auspices of Title 45 Part 46 exemption category 4.
Pneumonia or influenza must be listed as the primary cause of death to be included in this analysis. These diseases are commonly combined as an endpoint because of specific challenges associated with influenza. First, the number of deaths attributable to influenza is difficult to estimate directly because of a lack of virologically-confirmed infections. Second, many influenza-associated deaths occur from secondary complications when influenza viruses are no longer detectable by laboratory means [6] , [23] , [24] , [25] . Third, the use of P&I mortality in the study of influenza also reflects the problems associated with other measures of prevalence of influenza. Reporting of cases of influenza through routine channels is unsatisfactory because mild influenza may be under-diagnosed and the use of laboratory confirmation is skewed by the impact of variable testing patterns based on prevalence of disease [26] . Finally, multiple studies have shown that there exists a relationship between influenza morbidity and P&I mortality that can be mathematically described and used in epidemiological studies [3] , [27] , [28] , [29] , [30] , [31] .
Daily deaths were aggregated into ten age groups (0-4, 5-14, 15-24, 25-34, 35-44, 45-54, 55-64, 65-74, 75-84 and .84) and standardized via direct standardization [32] based upon the age distribution of population of the United States in the year 2000. This procedure adjusts for temporal changes in age demographics over the period of record based on data for each county using U.S. Census archives, thereby allowing for consistent comparisons of mortality rates over time. After age standardization, the long-term mean for each day (within ICD period) was subtracted from that day's standardized P&I mortality to remove the inherent seasonal signal from the time series. Because our goal was to determine if cold and/or dry days or periods within a year were related to P&I mortality peaks, this level of granularity requires the examination of daily data with the seasonal signal removed.
Examination of the P&I mortality time series exhibits obvious temporal discontinuities that are exactly coincident with the dates of ICD revision code changes. We removed this artifact by converting each day's P&I mortality to a z-score by dividing the mean departure by the standard deviation separately for each of the three relevant ICD periods ( Table 1 ). The time series of agestandardized mortality prior to and after deseasoning and z-score adjustment is shown in Figure 1a and Figure 1b , respectively. Because of the general lack of influenza in the summer months, June, July, and August were removed from the analysis as they had the potential to distort any relationships during the primary influenza season.
Mortality data were smoothed using a 17-day leading moving average (e.g., mortality on January 1 is the mean from January 1-17). This smoother was selected after testing a variety of filter lengths and based upon prior research [6] . We examined both daily P&I mortality ''events'' and longer mortality ''episodes.'' ''Events'' are days with (smoothed) mortality at least one standard deviation above the long-term (smoothed) mean for that date. This z$1 criterion was chosen because the frequency distribution of smoothed mortality is positively skewed with the tail beginning at approximately one standard deviation. After smoothing, there is an obvious tendency for high mortality events to cluster into prolonged periods when the z$1 threshold is (Figure 2 ). We thus identified 12 P&I mortality episodes over the 28-year period of record (Table 2) .
To summarize the outcome data treatment, after age standardization the daily mortality data were deseasoned to remove the large influence of season on respiratory infection and converted to z-scores to adjust for discontinuities related to ICD coding. These data were then smoothed using a 17-day leading smoother to account for the inherent lag between infection and mortality. Days or periods with z-scores.1 were identified as mortality ''events'' or ''episodes,'' respectively ( Figure S1 ).
Hourly climate data from La Guardia Airport, New York, were retrieved from National Climatic Data Center archives. We utilize 1200 and 1900 Universal Time Coordinate (UTC) air temperature (T) and dew point temperature (T d ) to approximate the typical times of the warmest and coldest hours of the day (7 or 8 a.m. and 2 or 3 p.m. local time). The dew point temperature is the temperature at which water vapor begins to condense via cooling at constant pressure. We use dew point as a measure of the amount of moisture in the air because, unlike relative humidity, it is independent of air temperature [5] .
In addition to dew point temperature, we employ an air mass classification, which has the advantage of incorporating a variety of weather variables into a single nominal variable. Specifically, we utilize the Spatial Synoptic Climatology (SSC) [33] which classifies each day's weather into one of six air mass types [dry moderate (DM), dry polar (DP), dry tropical (DT), moist moderate (MM), moist polar (MP), moist tropical (MT)]. A seventh transition (TR) category identifies days characterized by a significant change in weather-typically a frontal passage [34] . The SSC uses air temperature, dew point temperature, wind, surface air pressure, and total cloud cover observations taken four times per day as input to the classification. Thus, the SSC is a multivariate nominal classification of daily weather conditions. The approach has been utilized in a variety of human health applications [35] , [36] , [37] , [38] , [39] , [40] , [41] .
The temperature and dew point time series were converted to zscores to remove seasonality and then smoothed using a centered 5-day moving average filter. This filter length was employed after examining various options because it represents a balance between Figure 1b smoothed with a 17-day centered moving average filter. A centered smoother is used here to more clearly present the peak times of the mortality episodes (see Table 2 ). doi:10.1371/journal.pone.0034091.g002 high frequency weather events and more long-term (monthly to seasonal) trends. The seasonality of the SSC air mass types was removed by comparing the presence (coded 1) or absence (coded 0) of each air mass type on each day of the year to the long-term average frequency. For example, if Moist Moderate air was present on average 30% of the time on January 1 over the period of record, then its occurrence on January 1, 2000 would result in a value of +0.7 for that day. These daily anomalies were then converted into continuous variables using a centered 7-day moving average filter for each SSC category.
In summary, raw dew point observations were first de-seasoned by conversion to z-scores and then smoothed using a 5-day filter. Daily air mass frequencies were converted from a nominal to a continuous variable by first adjusting for the long-term frequency on each day and then smoothing those frequencies using a 7-day moving average ( Figure S2 ).
Daily Analyses. A series of t-tests were employed to determine if temperature, dew point, and air mass frequency differed between high P&I mortality events (z$1) and non-events. To address the temporal autocorrelation in the weather variable time series and the resulting overestimate of the true degrees of freedom, the sample size was adjusted based upon the lag one temporal autocorrelation [42] (Wilks 2006) as follows:
where N = number of observations N9 = adjusted degrees of freedom P = lag one temporal autocorrelation. N9 was then adjusted again based on the length of the smoother employed to determine the final effective sample size. For these and all other tests, a Type I error rate of 0.05 was employed and Levene's test for equality of variances was used to determine if pooling of the samples was required.
The following tests were performed: 1) smoothed temperature, dew point, and air mass frequency, lagged 17-days, between mortality events (z$1) and nonevents (z,1) using an unpaired two-sample t-test ( Figure S3 ); 2) same as in 1 for unsmoothed temperature and dew point temperature (to determine if a strict 17-day lag exists); and 3) same as in 1 but using a one-sample t-test (to account for the possible influence of disparate sample sizes between groups).
Episodic Analyses. For each high P&I mortality episode, we calculated the duration (in days), the summed total mortality over the entire episode, and the average daily episode mortality ( Table 2 ). These quantities served as dependent variables in a linear regression analysis vs. the independent (weather) variables (1200 and 1900 UTC T and T d and the deseasoned SSC frequencies). We used a 17-day lag in which the mean for each variable was calculated across the days in the episode and the preceding 17 days. This lag was selected based upon prior research that examined absolute humidity [6] and the results of other studies [28] , [43] .
Given the relatively small number of episodes, we used bootstrapped regression analysis to generate a robust estimate of the regression coefficients. Based on the initial full sample, data sets of the same size were generated by randomly sampling variable pairs, with replacement, and estimating the regression parameters from that sample using ordinary least-squares. This procedure was repeated 10,000 times and the resulting suite of regression coefficients was examined to determine if the 2.5 percentile and 97.5 percentile observations were of the same sign. If so, the regression slope was deemed to be statistically significant [44] .
In the daily analysis, 1200 UTC dew point temperature was significantly lower for events than for non-events (p = 0.003), a result that is consistent with the hypothesis that drier conditions are related to enhanced P&I mortality. However, 1900 UTC dew point was higher during events (p = 0.036), a result that contradicts the underlying hypothesis. Table 3 . Results of t-tests comparing weather variables between mortality event days to non-event days. When this test was repeated without smoothing the weather variables, only 1200 UTC dew point was significant (p = 0.028; Table 3 ). The lack of a relationship for 1900 UTC dew point (p = 0.181) suggests that the finding of high afternoon dew points during mortality events using smoothed weather data was not robust.
Because the high number of non-event days vs. event days can bias the t-test, an additional test was performed comparing the 5day smoothed weather variables during events to the long-term mean. Here, 1200 UTC temperature and dew point were both significantly lower during high P&I mortality events (Table 3) .
For the daily SSC analysis, lower frequencies of moist moderate (MM) air (p = 0.019) and higher frequencies of moist polar (MP) air (p,0.001) occurred 15-19 days before high mortality events (Table 3) . No relationship was found for dry polar (DP) air (p = 0.277).
Temperature, dew point temperature, and air mass frequencies were examined 17 days prior to and throughout each of the 12 high P&I mortality episodes identified from 1975-2002. There is a statistically significant negative relationship between episode duration and mean 1200 UTC dew point (r = 20.61, p,0.05; Figure 3a ). The longest episode (57 days in 1981) was associated with a dry period during which the mean dew point was more than 0.8uC below normal for that time of year. Total episode mortality is likewise negatively correlated with 1200 UTC dew point (r = 20.56, p,0.05; Figure 3b ). Two of the three episodes with the lowest mortality also had dew points that were near normal, whereas the higher mortality episodes exhibited drier conditions. No significant relationships were found for afternoon variables, air temperature, or any of the SSC air mass types.
Dew point temperature is commonly used by atmospheric scientists to measure humidity because it is relatively invariant to pressure and temperature changes and thus is a conservative quantity. In New York City from 1975-2002, periods with high P&I mortality were preceded 2-3 weeks by periods with low morning dew points. Furthermore, for the 12 high mortality episodes identified in that period, morning dew point was negatively correlated with both episode duration (r = 20.61) and total episode mortality (r = 20.56). This finding of a linkage between dry air and influenza mortality is consistent with the results of recent research [6] showing that absolute humidity (a correlate of T d ) influences the timing of the onset of the influenza season in various U.S. states (including New York state). Our results provide some limited evidence supporting laboratory studies linking dry air to higher airborne infection rates among guinea pigs [4] .
The association between high frequencies of Moist Polar air masses prior to high mortality events is consistent with the dew point results. The average morning dew point in Moist Polar air in New York City is lower than for any air mass other than Dry Polar (Table 4 ). Although it may seem counter-intuitive that a ''moist'' air mass has a low dew point temperature, dew point must (physically) be less than or equal to air temperature, so cold air masses typically have low dew points, especially in winter. In general, Moist Polar air masses are relatively uncommon in the cold season in New York, occurring only 7.3% of the time (Table 4 ) and are associated with cold, overcast and often stormy conditions with moist air arriving from the Atlantic Ocean [33] . We also identified a significant relationship between low Moist Moderate air mass frequencies 3-4 weeks before mortality episodes. In an effort to understand this association, we calculated the correlation between Moist Moderate frequencies and the other air mass types during that period. Moist Moderate is negatively correlated with the two driest air masses-Dry Polar (r = 20.47) and Moist Polar (r = 20.31) (Table 4 ). Thus, the significant association between low Moist Moderate frequencies prior to P&I mortality events appears to be a proxy for the cool, low dew point conditions that are common when Moist Polar and Dry Polar air masses are present. On average, cold season dew points are 5.5uC higher in Moist Moderate air masses than in Moist Polar ( Table 4) .
The lack of a direct Dry Polar relationship is surprising, as Dry Polar air masses exhibit the most extreme combination of cold air and low humidity. Dry Polar is far more common than Moist Polar in New York winters, however, so its high daily frequency during the influenza season limits the likelihood of identifying an underlying relationship. It might be more fruitful to examine an extreme cold, dry subset of Dry Polar air masses to identify the coldest and driest days.
In New York City, high P&I mortality periods within a given year were preceded by multiple day periods with unusually low temperature and humidity. Over the 28-year period of this study, we identified 12 episodes of high P&I mortality and found that both the total mortality occurring during each episode and duration of each episode were inversely correlated with the average morning dew point temperature prior to and during the episodes. These results support the burgeoning hypothesis that unusually cold dry air enhances the airborne transmission of influenza virus.
The exploratory nature of this analysis was necessitated by the lack of an underlying theory of influenza seasonality, sociobehavioral factors, and inherent variability in disease transmission and virulence. The time between infection and a resulting mortality event (i.e. ''latency'') varies between individuals depending on age, overall health, co-morbid conditions, and other factors. Thus, lags must be estimated to best fit the overall data structure. Similarly, the high frequency variability in the variables requires some smoothing to elucidate relationships, and the selection of appropriate smoothers is somewhat subjective. Nevertheless, our findings are consistent with several others. For example, there is evidence supporting a two-week lag between rising influenza virus and pneumonia mortality [28] . Other research showed fairly convincingly the existence of a 2-4 week lag between laboratory-confirmed cases of influenza and increased incidence of invasive pneumococcal disease [43] . The weather variables used in this study do not directly account for the ambient conditions experienced by the influenza victims while indoors, but cold and/or stormy weather could result in the decedents spending more time in heated indoor environs with low humidity, thereby enhancing infection opportunities [8] .
For this study, P&I mortality was used to characterize the influenza time series in New York City. The limitation of this method is the potential for confounding as P&I mortality includes mortality from infections other than influenza. In addition, in nonpandemic years, P&I mortality is skewed by the extremes of age. This limitation is unlikely to be a major contributor in this study as 90% of influenza-related deaths involve persons over the age of 65 during seasonal epidemics [45] and our mortality data are ageadjusted to account for demographic changes in New York City over the period of the study.
We chose to focus on New York City because the large population (and thus large daily P&I mortality rate) enhances statistical robustness, and New York weather is highly variable owing to its midlatitude, coastal location. These results should be confirmed using a similar methodology in other cities worldwide to determine if the humidity-influenza linkage is pervasive. It would be particularly interesting to determine how these relationships evolve in subtropical or tropical climates where the P&I mortality seasonality is more muted or nonexistent.
It is likely that the underlying causes of influenza seasonality are multi-factorial, and we suspect that weather is but one of those factors. A predictive model for P&I mortality based on weather alone would likely be unsuccessful in accounting for most of the short-term influenza variability. Nevertheless, our results confirm recent emerging hypotheses of a relationship between cold, dry air and influenza transmission or virulence [6] , [8] . Identifying periods of low dew point temperatures a few days or even weeks in advance is well within the skill of existing weather forecast models. Given the lag between infection and mortality, it seems reasonable to propose that, during the prime influenza season, skillful forecasts of high P&I mortality periods could be made weeks to months in advance. Figure S1 Sample of mortality data from December 1, 1998 through March 10, 1999 . Daily mortality (z-scores) (red dashed lines) shows evidence of the beginning of a prolonged peak starting around day 43. When these data are smoothed using a 17day centered moving average filter (solid red line), the mortality peak becomes more evident. In our analysis, we instead employ a leading 17-day smoother (blue line), which effectively shifts the red line forward by 8 days. High mortality episodes are classified when the z-scores exceeds 1, so the 1999 episodes begins on day 39 and ends on day 77. (TIF) The decline in dew point on day 10 preceded the start of the mortality increase by approximately 17 days. During the subsequent period of declining dew points, mortality continued to rise. When dew point reached its minimum for this period on day 31, 17-day lagged mortality was one standard deviation above the mean. Although there is no consistent 1:1 lagged relationship between dew point temperature and mortality, this example illustrates the procedure and shows a general linkage between a low dew point period in mid-late December, 1998 and a subsequent high pneumonia and influenza mortality anomaly several weeks later. (TIF)
Conceived and designed the experiments: RED CER KBE. Performed the experiments: CER. Analyzed the data: CER RED KBE. Wrote the paper: RED CER KBE. Finding and removing highly connected individuals using suboptimal vaccines BACKGROUND: Social networks are often highly skewed, meaning that the vast majority of the population has only few contacts whereas a small minority has a large number of contacts. These highly connected individuals may play an important role in case of an infectious disease outbreak. METHODS: We propose a novel strategy of finding and immunizing highly connected individuals and evaluate this strategy by computer simulations, using a stochastic, individual-and network-based simulation approach. A small random sample of the population is asked to list their acquaintances, and those who are mentioned most frequently are offered vaccination. This intervention is combined with case isolation and contact tracing. RESULTS: Asking only 10% of the population for 10 acquaintances each and vaccinating the most frequently named people strongly diminishes the magnitude of an outbreak which would otherwise have exhausted the available isolation units and gone out of control. It is extremely important to immunize all identified highly connected individuals. Omitting a few of them because of unsuccessful vaccination jeopardizes the overall success, unless non-immunized individuals are taken under surveillance. CONCLUSIONS: The strategy proposed in this paper is particularly successful because it attacks the very point from which the transmission network draws its strength: the highly connected individuals. Current preparedness and containment plans for smallpox and other infectious diseases may benefit from such knowledge. Super-spreader events (cf. [1] ) crucially influence the course of infectious disease outbreaks, as has been shown for SARS, measles and smallpox. Targeting control efforts on individuals with highest potential to spread disease is more effective than mass control [1] . This is very important for diseases like smallpox for which herd immunity is decreasing and stockpiled vaccines are of low eligibility or uncertain immunogenicity [2] [3] [4] [5] [6] . Specific information on social networks and on their contact structures is still scarce, but common properties have been revealed for many networks [7] : it has been shown that the degree distribution of social networks are frequently highly skewed, i.e. they are bound together by just a few very highly connected individuals. The frequency of contacts in such networks is not Poisson distributed (as would be expected in networks which originate from a random mixing process), but follows a skewed and long-tailed distribution [8] : the vast majority of the population has rather few contacts whereas a small minority has a huge number of contacts. Highly skewed networks are ubiquitous in nature and it seems that this particular topology confers "dynamical robustness and reliability to perform a certain function in the presence of perturbations" [9] .
Indeed, a highly skewed frequency distribution of the number of contacts per person has some astonishing effects on the transmission of infection diseases and on the effect of interventions [1] . In contrast to the results with an assumption of a homogeneous mixing population, the disease transmission is only marginally reduced if a percentage of individuals is immunized at random and, thus, "removed" from the transmission network. In the presence of highly connected individuals, even the celebrated basic reproduction number R 0 , originally defined as "the average number of secondary cases caused by a single index case in a completely nonimmune homogenously mixing population where no interventions are taken", no longer predicts whether an outbreak can occur or not [8, [10] [11] [12] [13] .
These same highly-connected individuals which stabilize the transmission properties of a skewed contact network in the case of random "removal" of individuals, also make these networks vulnerable, if they can be identified and "removed". One approach to identify them, considering a theoretical infection process spreading on a skewed contact network, has been termed "acquaintance immunization" [14, 15] . Here, people are picked at random and asked to name one contact each who will then be vaccinated. People with many contacts are most likely to be mentioned by somebody and are very likely to be vaccinated. An even more efficient strategy has been found by assuming that individuals can guess information about their neighbors and their contacts [16] . Acquaintances have also been shown to be good social network sensors for early detection of outbreaks [17] .
Inspired by Cohen et al. [14, 15] , the aim of the present paper is to explore the effect of such targeted immunization strategies on the course of an epidemic considering a real disease -smallpox-using pessimistic assumptions and suboptimal vaccines. We use computer simulations based on a stochastic transmission model where individuals are connected with each other in two superimposed networks, which allows us to distinguish between close contacts (comprising family members and close friends which can easily be traced) and casual contacts who will be more worrisome in case of an outbreak, because they are more difficult to detect and to be placed under surveillance. For the latter ones, we use a highly skewed network. Our baseline intervention scenario considers case isolation (with a limited capacity) and tracing of close contacts which we combine with a pre-emptive vaccination of highly connected individuals. In our simulations, we identify highly connected individuals by first "asking" a small random fraction of the population to supply the names of their casual contacts. The most frequently mentioned contacts are then offered to be vaccinated. Depending on the simulation scenario, only a fraction of the contacts can be named, or alternatively, only a fraction of the most frequently mentioned contacts can be immunized (this may be caused by combination of a low vaccination eligibility and an imperfect vaccine efficacy). In some of the considered scenarios, highly connected individuals are additionally placed under surveillance for an indefinite duration, so that they can be prevented from spreading the infection.
We use a stochastic, individual-and network-based simulation approach. Individuals have discrete states which can be changed by events that are scheduled on a continuous time scale and executed using a discrete event simulation algorithm. Executed events can trigger future events which affect the same individual or -through the contact network-other individuals.
The population consists of 100,000 fully susceptible individuals which form the nodes of a network graph. The contact network is a combination of two networks, representing two types of links: close contacts are represented by a two-dimensional toroidal square lattice with eight nearest neighbours as contacts, a so-called Moore neighbourhood; casual contacts are represented by a highly skewed network created with the Barabási-Albert algorithm that starts with a fully interconnected network of 12 nodes and then uses preferential attachment to add nodes [7] . Each individual of the population is characterized by two internal states: the infection state and the surveillance state. All individuals are 100% susceptible at the beginning of the simulation.
The infection model which controls the natural history of the disease is an extended SEIR-model (see Table 1 and Additional file 1: Figure S1 ). The simulation starts with all individuals being susceptible except for 100 randomly selected individuals who are newly infected with the virus. As their infection progresses, the infected individuals develop prodromal fever and then proceed through an early rash, a middle rash and a late rash state. A fraction of individuals dies in the early rash state, all others acquire a lasting immunity after recovery.
A detailed description of the infection process is given in the Appendix.
The surveillance model (Additional file 2: Figure S2 ) controls individual states related to case detection, contact tracing, observation, and interventions like isolation or seclusion. At the start of each simulation, all individuals are unobserved. Two days after a yet unobserved new case develops the earliest signs of a rash, he or she is detected. Immediately after case detection, all close contacts and 10% of the casual contacts of the case are traced and put under observation which lasts for a maximum of 21 days (which is longer than the maximum incubation period). Observed individuals are detected immediately after developing prodromal fever. After detection, cases ought to be isolated immediately, but the number of isolation units is limited. To deal with this limited resource, all detected cases first enter a waiting queue; as soon as free isolation units become available, they are isolated (isolation is assumed to prevent all further contacts). While no free isolation units are available, detected cases are asked to seclude themselves which means that they will prevent all casual and 50% of their close contacts. Secluded cases are immediately isolated when free isolation units become available.
Vaccination is implemented by first asking a fraction of the population to supply the names of their casual contacts. The most frequently mentioned contacts are then pre-emptively vaccinated, before the outbreak occurs. Technically, we first define the index cases and then define the 'vaccinated' individuals, different from those who are index cases. We do this to ensure a standard number of index cases (100), all of them unvaccinated. Depending on the simulation scenario, only a fraction of the contacts can be named, or alternatively, only a fraction of the most frequently mentioned contacts can be immunized (this may be caused by combination of a low vaccination eligibility and an imperfect vaccine efficacy). In some of the considered scenarios, vaccinated individuals are additionally placed under surveillance for an indefinite duration, so that they can be prevented from spreading the infection.
In Figure 1 , we explore the theoretical limits of what can be achieved by "removing" highly connected individuals from the contact network: the vertical axis shows the maximum eigenvalue of the next generation matrix of the casual contact network which predicts the initial spread of the epidemic. The horizontal axis shows the fraction of the population which is removed from the transmission network (e.g. by immunization), whereby the individuals are sorted by their number of contacts, such that the most highly connected individuals will be removed first (red curve). Even if only a small fraction of the population is removed, Figure 1 shows a steep decline in the eigenvalue, which clearly indicates that removing the most highly connected individuals has a huge effect on the index cases' capacity to spread the infection, as has been shown in other publications (1). The blue curve in Figure 1 shows a much less dramatic effect which is gained if individuals are removed at random -irrespective of their number of contacts.
The relevance of this finding can be seen in Figure 2 , which shows how the targeted immunization influences the simulation results of an outbreak which starts with 100 index cases in a population of 100,000 individuals. We start with a highly over-simplified situation in which we assume that we can ask everybody in the population to supply the names of all casual contacts, and later progress towards more realistic scenarios. The contacts are first sorted by frequency and then scheduled for vaccination, starting with the most frequently named person. As described in detail in the Methods section, detected cases are isolated or are asked to stay at home or to seclude themselves from contacts with other individuals (if all 500 isolation units are occupied); their contacts are traced and taken under surveillance. If only case isolation and contact tracing are used to fight against the outbreak, the large initial attack overwhelms the public health resources and the smallpox outbreak afflicts more than 50% of the population. When additionally targeting the vaccination on the most frequently named contacts, an immunization coverage of only 4% yields a median outbreak of less than 1,600 cases; a vaccination coverage of 6% can further reduce the median to less than 1,300 cases with a worst case scenario of under 1,700 cases. Asking everybody in the population is clearly an unrealistic task for any public health system. We will next explore how the results change, if only a small random fraction of the population is asked to name all casual contacts. Figure 3 shows how the median outbreak size changes if only a small random sample is asked. The horizontal axis again shows what percentage of the population is vaccinated, whereby immunization begins with the most frequently named contact. If we are willing to vaccinate 10% of the population, it suffices to ask a random sample of 4% of the population for their contacts in order to obtain a median outbreak size of less than 2,000 cases. For the highly optimistic goal of having a median outbreak size of less than 1,000 cases, we have to ask 10% of the population to supply the names of their contacts.
In reality, hardly anybody may be able to recall all casual contacts. In the next refinement, we assume that people are asked to supply only a limited number of casual contacts. Figure 4 shows that an immunization of 10% of the population still yields very optimistic results, even if the 10% randomly sampled individuals only name 6 to 10 contacts each.
In the previous scenarios, we have assumed a vaccine with 100% efficacy and we have assumed that every contact which was scheduled for vaccination was also eligible, but these assumptions cannot be made for the existing smallpox vaccines [2] . Figure 5 explores the influence of an imperfect vaccine on the median of the outbreak size.
Successfully immunizing as much as 80% or 90% of the scheduled individuals may only be possible with one of the new generation vaccines [2] [3] [4] [5] [6] , and even this may already be regarded as over-optimistic by some, yet this assumption already increases the median outbreak size considerably ( Figure 5 ). The disproportionate effect of a few vaccination failures on the simulation result can be Figure 2 Influence of Perfect Vaccination of Highly Connected Individuals on a Smallpox Outbreak. Efficacy = 100%. Simulated outbreak sizes, caused by 100 index cases in a population of 100,000: the boxes show 25%, 50%, 75% quantiles, the whiskers show 10% and 90% quantiles and the triangles show minimum and maximum results (for 2% vaccination, the upper limit is not given, as one of the 100 simulations afflicted more than half of the population, as did all simulations for 0% and 1% vaccination). Targeted vaccination: (a) everybody is asked to name all casual contacts; (b) these contacts are sorted by frequency; (c) vaccination starts with the most frequently named person and progresses towards less frequently named ones. The horizontal axis shows what percentage of the population is vaccinated. explained by a closer look at the maximum eigenvalues depicted in Figure 1 : The removal of all highly connected individuals (red curve) is necessary to really incapacitate the transmission network; if only a random sample of 90% of the highly connected individuals are removed (yellow curve), it practically stays intact.
In our final scenario, we combine immunization targeted to highly connected individuals (using an imperfect vaccine) with the surveillance of the vaccinated individuals: each contact which is scheduled for vaccination, will be taken under surveillance (if the vaccination success can be determined, it is sufficient to observe contacts with failed vaccination and contacts who were excluded from vaccination). Figure 6 assumes that a random sample of 90% of the individuals who are scheduled for vaccination can really be immunized. The blue curve shows the median outcome with, the red curve without additional surveillance of selected individuals.
Our results show that a combination of a novel strategy of finding and immunizing highly connected individuals with case isolation and contact tracing can prevent a large smallpox outbreak, with the advantage of vaccinating only about 10% of the population (Figure 2 ). This low vaccination coverage considerably reduces the number of severe side effects and deaths due to vaccination [2, 3, 18] . In the case of smallpox vaccines this is of paramount importance, as reported by Casey et al. 2006 , "after the inoculation of 37,901 people in the United States, three deaths, two permanent disabilities, and ten life-threatening illnesses were attributed to vaccination during 2003" [6] .
Successful control strategies proposed for an intentional release of smallpox, vary from targeting high-risk individuals to broader random vaccination campaigns and no one control method can be identified a priori as best [19] [20] [21] [22] . Uncertainty with respect to transmission before the onset of symptoms, residual herd immunity, demographics and mobility exist, so their differing conclusions can largely be attributed to underlying differences in model structures and parameter assignments. Our study is the first that uses a highly skewed contact structure for a smallpox outbreak.
In comparison to previous modeling studies, and in order to show the strength of this strategy, we focus on a highly pessimistic scenario: (a) the outbreak starts with the simultaneous infection of 100 independent index cases on a fully susceptible population; (b) a high proportion of transmission takes place during the prodromal fever phase which does not yet reveal the nature of the infection and which does not trigger case detection except for suspects who are already under surveillance; (c) we consider a limited number of isolation units which are easily exhausted by a major outbreak; and (d) we consider suboptimal vaccines.
We have made a big effort to design a realistic model and to use plausible parameter values, yet we made a number of inevitable simplifying assumptions on aspects that we consider out of the scope of the present paper. Worth of mention is that we use a pre-assigned and bidirectional contact network, to which we superimpose a stochastic transmission process (see Appendix). The contact network constitutes 'potential contacts' and the actual transmissions occur according to the transmission process. In reality, contact structures change over time so it is uncertain whether all pre-emptively identified highly connected individuals would play a role in a future outbreak. Specially in acts of bioterrorism, superspreading events (cf. [1] ) might be difficult to foresee unless awareness is increased. We assume, however, that highly connected individuals would have a potential for disease spread and constitute a good first guess.
Our study is the first that addresses the problem of suboptimal vaccines in the case of smallpox. We show that a network-based vaccination strategy strongly depends on a very high vaccine efficacy and on a high eligibility and compliance of the people selected for vaccination. It can quickly lose its effect if some of the most highly connected individuals cannot be vaccinated or if vaccination fails protect them ( Figure 5 ). If only a few of them are left unprotected, they can fuel a super-spreading event (cf. [1] ) multiplying the infection in the population.
Adopting the described containment strategy may require to overcome political, social and ethical hurdles.
A vaccination campaign against smallpox will not be initiated anywhere before a strong suspicion of bioterror attack occurs or the appearance of smallpox cases inside or outside a country has been confirmed. As long as a smallpox bioterror attack is regarded to be highly unlikely, most people would not accept to receive a potentially harmful smallpox vaccination anyway. This perception may change drastically after smallpox have reappeared somewhere in the world. Individuals who have been identified to be under the highest risk of contracting the infection may gladly accept to be chosen to receive a protective vaccine. Yet, due to their health condition, not all of them may be eligible for vaccination. As it is very important to remove all potential highly infectious individuals (Figure 5) , highly connected individuals who cannot be vaccinated or whose immunization has failed must be taken under surveillance ( Figure 6 ).
Considering the described containment strategy in preparedness plans would shift their focus to identifying potential highly infectious individuals and to preparing for pre-emptive vaccination of a pre-selected fraction of the population. It would also put emphasis on the availability of isolation units and of people trained in contact tracing and surveillance. If the public health system is overtaxed by an outbreak despite this targeted vaccination, containment strategies involving ring vaccination or large-scale vaccination should be implemented. Public health emergency plans have to provide instructions on (1) the observation of the dynamics of an outbreak and (2) which observations trigger the transition from a targeted containment strategy to a population-based one.
Infections which share the same route of transmission may also have the same highly connected individuals. Knowing which individuals have many contacts (whereby a "contact" depends on the mode of transmission of an infectious agent) may help public health agencies to control whole groups of infectious diseases, including newly emerging ones, rather than individual diseases. In specific settings, highly connected individuals may be identified using other approaches: in hospitals and companies, an analysis of the team structure and its meeting schedules can hint at who has most contacts. This may also affect the focus of business continuity plans, written to prepare for pandemic influenza or similar events. Simulation studies show that highly connected individuals are reached rather early by newly introduced infections. Thus, the knowledge of such people can also be used to improve outbreak detection [17] .
Current preparedness and containment plans for smallpox and other directly transmitted diseases like measles and pandemic influenza can benefit from considering the described pre-emptive strategy which effectively targets the fraction of the population fuelling disease spread and minimizes vaccine related side effects.
We define eight expected numbers E s,n of secondary infections per index case which depend on the index case's infectious stage s {prodromal, earlyrash, middlerash, laterash} and on the network n {close, casual} in which the secondary cases are produced (Table S1 ). This distinction enables us to consider a more intense exposure of close contacts and to modify the cases' contagiousness in their different disease stages, as suggested by previous smallpox studies [19, 23] . We set the sum of these expected values to 5, the estimate at smallpox's eradication [24] . In a homogeneously mixing population, this would be called the basic reproduction number R 0 . We have decided to parameterize our model with these expected values rather than the maximum eigenvalue of the next generation matrix, not only because expected values are more intuitive, but also because estimates of R 0 are frequently based on secondary cases [14, 23] or on homogeneous mixing assumptions [25, 26] . As the maximum eigenvalue better predicts the initial spread of an epidemic, we use it in Figure 3 in the results. In the simulation model, we implement the following infection process: when an individual enters one of the four contagious states, it triggers future infection events for each of its contacts. This is done in the following way: (1) we chose the effective contact rate b s,n such that 
where D s is the duration of the specific contagious stage s and c n is the average number of contacts per person in network n (for close contacts, c n is 8, for casual ones, it is 24). Solving Equation 1 yields β s,n = − ln(1 − E s,n /c n ) D s (2) (2) we draw a random number r from an exponential distribution with mean 1/b s,n , (3) if r is smaller than the duration of the current contagious state D s , we schedule an infection event for the corresponding contact with delay r, otherwise the infection event is discarded. Infection events are stored in an event queue. At the scheduled time, it is checked whether the infection source is still contagious, the prospective victim is still susceptible and the contact between the two has not yet been impeded by interventions (e.g. isolation or seclusion) (see Table 1 ). The infection only takes place if all of these three conditions are fulfilled.
Additional file 1: Figure S1 . Infection model.
Additional file 2: Figure S2 . Surveillance model.  The impact of media coverage on the transmission dynamics of human influenza BACKGROUND: There is an urgent need to understand how the provision of information influences individual risk perception and how this in turn shapes the evolution of epidemics. Individuals are influenced by information in complex and unpredictable ways. Emerging infectious diseases, such as the recent swine flu epidemic, may be particular hotspots for a media-fueled rush to vaccination; conversely, seasonal diseases may receive little media attention, despite their high mortality rate, due to their perceived lack of newness. METHODS: We formulate a deterministic transmission and vaccination model to investigate the effects of media coverage on the transmission dynamics of influenza. The population is subdivided into different classes according to their disease status. The compartmental model includes the effect of media coverage on reporting the number of infections as well as the number of individuals successfully vaccinated. RESULTS: A threshold parameter (the basic reproductive ratio) is analytically derived and used to discuss the local stability of the disease-free steady state. The impact of costs that can be incurred, which include vaccination, education, implementation and campaigns on media coverage, are also investigated using optimal control theory. A simplified version of the model with pulse vaccination shows that the media can trigger a vaccinating panic if the vaccine is imperfect and simplified messages result in the vaccinated mixing with the infectives without regard to disease risk. CONCLUSIONS: The effects of media on an outbreak are complex. Simplified understandings of disease epidemiology, propogated through media soundbites, may make the disease significantly worse. Infectious diseases are responsible for a quarter of all deaths in the world annually, the vast majority occurring in low-and middle-income countries [1] . There are diseases such as SARS and flu that exhibit some distinct features such as rapid spatial spread and visible symptoms [2] . These features, associated with the increasing trend of globalization and the development of information technology, are expected to be shared by other emerging/ re-emerging infectious diseases. It is therefore important to refine classical mathematical models to reflect these features by adding the dimensions of massive news coverage that have great influence not only on the individual behaviours but also on the formation and implementation of public intervention and control policies [2] .
People's response to the threat of disease is dependent on their perception of risk, which is influenced by public and private information disseminated widely by the media. While government agencies for disease control and prevention may attempt to contain the disease [3] , the general information disseminated to the public is often restricted to simply reporting the number of infections and deaths. Mass media are widely acknowleged as key tools in risk communication [4, 5] , but have been criticised for making risk a spectacle to capitalise on audience anxiety [6, 7] .
The original interpretation of media effects in communication theory was a "hypodermic needle" or "magic bullet" theory of the mass media. Early communication theorists [8, 9] imagined that a particular media message would be directly injected into the minds of media spectators. This theory of media effects, in which the mass media has a direct and rapid influence on everyday understanding, has been substantially revised. Contemporary media studies analyses how media consumers might only partially accept a particular media message [10] , how the media is shaped by dominant cultural norms [11, 12] and how media consumers resist dominant media messages [13, 14] . It follows that media effects may sway people into panic (eg swine flu), especially with a disease where scientific evidence is thin or nonexistent. Conversely, media may have little effect on seasonal diseases (eg regular influenza).
Media reporting plays a key role in the perception, management and even creation of crisis [6] . Since media reports are retrievable and because the messages are widely distributed, they gain authority as an intersubjective anchorage for personal recollection [4] . At times of crisis, non-state-controlled media thrive, while statecontrolled media are usually rewarded for creating an illusion of normalcy [6] . Media exposure and attention partially mediate the effects of variables such as demographics and personal experience on risk judgments [5] . The role of media coverage on disease outbreaks is thus crucial and should be given prominence in the study of disease dynamics.
Klein et al., [15] noted that much more research is needed to understand how provision of information influences individual risk perception and how it shapes the evolution of epidemics; for example, individuals may overprotect, which can have additional consequences for the spread of disease. An example of such complex dynamics is the 1994 outbreak of plague in a state in India: after the announcement of the disease, many people fled the state of Surat in an effort to escape the disease, thus carrying the disease to other parts of the country [16] . Even though information on the number of cases and deaths can have an adverse effect, the number of those vaccinated has not been given prominence.
A handful of mathematical models have described the impact of media coverage on the transmission dynamics of infectious diseases. Liu et al. [2] examined the potential for multiple outbreaks and sustained oscillations of emerging infectious diseases due to the psychological impact from reported numbers of infectious and hospitalized individuals. Liu & Cui [3] analysed a compartment model that described the spread and control of an infectious disease under the influence of media coverage. Li & Cui [17] incorporated constant and pulse vaccination in SIS epidemic models with media coverage. Cui et al., [18] showed that when the media impact is sufficiently strong, their modelwith incidence rate being of the exponential form capturing the alertness to the disease of each susceptible individual in the populationexhibits multiple positive equilibria (also see [2] ) which poses a challenge to the prediction and control of the outbreaks of infectious diseases.
The aim of this study is to investigate the impact of media coverage on the spread and control of an influenza strain when a vaccine is available, and where the media reporting of both disease dynamics and vaccination is high. Vaccination is one of the most effective tools for reducing the burden of infectious diseases [19] . However, despite their public-health benefit, vaccination programs face obstacles. Individuals often refuse or avoid vaccinations they perceive to be risky. Recently, rumours that the polio vaccine could cause sterility and spread HIV have hampered polio eradication in Nigeria [20] , while misplaced fears of autism in the developed world have stoked vaccination fears [21] . Reporting the number of individuals who vaccinate may have a positive effect on the disease transmission by increasing the vaccination rate.
Conversely, behavioural interventions can also have an enormous effect on the course of a disease [22, 23] Our model considers the same contact rate after a media alert, as proposed by Liu & Cui [3] , but there are fundamental differences in both models. They consider the classical SIR type model, while vaccination is included in ours to reflect transmission dynamics of human influenza.
We divide the population (N) into four sub-populations, according to their disease status: susceptible (S), vaccinated (V ), infected (I), and recovered (R). Our model monitors the dynamics of influenza based on a single strain without effective cross-immunity against the strain. The susceptible population is increased by recruitment of individuals (either by birth or immigration), and by the loss of immunity, acquired through previous vaccination or natural infection. This population is reduced through vaccination (moving to class V ), infection (moving to class I) and by natural death or emigration. The population of vaccinated individuals is increased by vaccination of susceptible individuals. Since the vaccine does not confer immunity to all vaccine recipients, vaccinated individuals may become infected, but at a lower rate than unvaccinated. The vaccinated class is thus diminished by this infection (moving to class I) by waning of vaccine-based immunity (moving to class S) and by natural death. The population of infected individuals is increased by infection of susceptibles, including those who remain susceptible despite being vaccinated. It is diminished by natural death, death due to disease and by recovery from the disease (moving to class R). The recovered class is increased by individuals recovering from their infection and is decreased as individuals succumb to natural death. Media coverage is introduced into the model via a saturated incidence function.
A schematic model flowchart is depicted in Figure 1 .
The transmission model with media coverage is given by the following deterministic system of nonlinear ordinary differential equations: 
where Λ is the rate at which individuals are recruited into the population (recruitment of infectives is ignored for now); θ is the rate at which susceptible individuals receive the vaccine; µ is the the rate at which people leave the population, through natural death or emigration. We assume this rate to be the same for all sub-populations. b 1 is the rate at which susceptibles get infected; ω is the rate at which vaccine-based immunity wanes; g is the vaccine efficacy; a is the death rate due to the infection; and l is the recovery rate from infection. The terms b 2 I m I I + and b 3 I m I I + measure the effect of reduction of the contact rate when infectious and vaccinated individuals are reported in the media [2, 3, 18] . The half-saturation constant m I > 0 reflects the impact of media coverage on the contact transmission. The function g I I m I I ( ) = + is a continuous bounded function which takes into account disease saturation or psychological effects [24] . We note that recovered individuals cannot be vaccinated. Also, a vaccinated individual who gets infected and then recovers will return to the susceptible class with no vaccine protection. This is true even if ω is quite small but s and l are large. For example, if vaccination lasts three years, but recovery and loss of immunity takes 6 months, then we are assuming this person is subsequently unvaccinated.
In the Michaelis-Menten functional response, the rate at which information is spread by the media rises as infectives increase, but eventually levels off at a plateau (or asymptote) at which the information (rate) remains constant (i.e. it has reached a maximum number of individuals due to information saturation) regardless of the increase in infections. Such dynamics can easily be observed in the spread of rumours, gossip and jokes (also known as randomized broadcast) [25, 26] . This constant coverage is extended by examining more complex effects which involve more than just reducing contacts down the line. The news in particular is extremely fickle so that what is news one day may be forgotten about next week; including the media effects in some more sophisticated way such as by an impulsive pulsing is also investigated. The limited power of the infection due to contact is accounted for by the saturation incidence. The first available information is the reported number of infected individuals when the disease is emerging. We assume that media coverage can slow but not prevent disease spread, so b 1 ≥ b 2 and b 1 ≥ b 3 .
The above model is closely related to those in [27, 28] to analyze the transmission dynamics of human influenza, but there are some differences. In [27] , the authors consider the inflow of infective immigrants, while in [28] the model includes treatment. Neither of these are considered here. Our model is clearly a crude reflection of the complicated nonlinear phenomena of the transmission dynamics, and it does not incorporate the self-control property due to the change of avoidance patterns of individuals at different stages of the infectious process [2] . News coverage may have a significant impact on avoidance behaviours at both individual and society levels, which may reduce the effective contact between susceptible and infectious individuals; we include this via a saturation incidence functional response.
Since the model monitors human populations, all the variables and parameters of the model are nonnegative. Based on biological considerations the system of equations (1)-(4) will be studied in the following region,
which is positively invariant and attracting (thus, the model is mathematically and epidemiologically wellposed); it is therefore sufficient to consider solutions in Ω. Existence, uniqueness and continuation results for model system (1)-(4) hold in this region and all solutions of this system starting in Ω remain in Ω for all t ≥ 0.
The disease-free equilibrium of the system is given by
The endemic equilibrium of the system is given by 
Substituting the above into the second equation at equilibrium will yield the expression for Î after some rearrangement. 
The basic reproductive ratio, R v , is defined as the expected number of secondary infections caused by an infective individual upon entering a totally susceptible population [29] [30] [31] . This quantity is not only important in describing the infectious power of the disease, but can also can supply information for controlling the spread of the disease [32] . The linear stability of E v0 is governed by the basic reproductive ratio R v . Using the next-generation method [31] , we have
The basic reproductive ratio is the spectral radius r(FV -1 ) which is
Local stability of the disease-free equilibrium
Proof. The Jacobian of the system evaluated at E v0 is given by
For local stability of the disease-free equilibrium, we require that all the eigenvalues be negative. Three of the eigenvalues satisfy this condition while ς 2 < 0 implies that R v < 1 and, consequently, all the eigenvalues of the Jacobian matrix above have negative real part. Thus, the disease-free equilibrium is locally asymptotically stable.
We adopt the method of Castillo-Chavez et al, [33] and we rewrite the set of model equations in the form
with G(X G ,0) = 0. X G ℝ 3 denotes the number of uninfected classes and Z G ℝ denotes the number of infected classes. U X
For the set of equations in (1)-(4), we set X G = (S, V, R) and Z G = (I). The conditions (H1) and (H2) below must be met for global stability.
(H1) For
* 0 is an M-matrix (the off-diagonal elements of A are nonnegative) and Ω is the region where the model makes biological sense.
If the above two conditions are satisfied, then the following theorem holds.
Theorem 2 (Castillo-Chavez et al, [33] ): The fixed point U X
is a globally stable equilibrium of (2.28) provided that R v < 1 and that assumptions (H1) and (H2) are satisfied.
Therefore, E v0 is globally asymptotically stable (GAS) since Ĝ(X G ,Z G ) > 0. The GAS of E v0 excludes any possibility of the phenomenon of backward bifurcation. We note that the GAS of the DFE E v0 when s = 0 is straightforward.
Our objective in this section is to extend the initial model to include two intervention methods, called controls, represented as functions of time and assigned reasonable upper and lower bounds, each representing a possible method of influenza intervention. Using optimal control theory and numerical simulations, we determine the benefit of vaccination and media coverage when the latter has positive or negative effect on the former.
We will integrate the essential components into one SIVR-type model to accommodate the dynamics of an influenza outbreak determined by population-specific parameters such as the effect of contact reduction when infectious and vaccinated individuals are reported in the media.
Let u v and u m be the control variables for vaccination and media coverage respectively. Thus, model (1)-(4) now reads 
A balance of multiple intervention methods can differ between populations. A successful mitigation scheme is one which reduces influenza-related deaths with minimal cost. A control scheme is assumed to be optimal if it maximizes the objective functional
The first two terms represent the benefit of the susceptible and vaccinated populations. The parameters B 1 and B 2 represent the weight constraints for the infected population and the control, respectively. They can also represent balancing coefficients transforming the integral into dollars expended over a finite time period of T days [34] . The goal is to maximize the populations of susceptible and vaccinated individuals, minimize the population of infectives, maximize the benefits of media coverage and vaccination, while minimizing the systemic costs of both media coverage and vaccination. The value u v (t) = u m (t) = 1 represents the maximal control due to vaccination and media coverage, respectively. The terms B u t v 2 2 ( ) and B u t m 2 2 ( ) represent the maximal cost of education, implementation and campaigns on both vaccination and media coverage. S(t) and V(t) account for the fitness of the susceptible and the vaccinated groups as a result of a reduction in the rate at which the vaccine wanes, and vaccination and treatment efforts are implemented [35] . We thus seek optimal controls u t v * ( ) and u t m * ( ) such that
The basic framework of this problem is to characterize the optimal control.
The existence of an optimal control can be obtained by using a result by Joshi [36] and Fister et al. [37] .
Theorem 3Consider the control problem with the system of Equations (4.1)-(4.4). There exists an optimal control ( )
Proof. To prove this theorem on the existence of an optimal control, we use a result from Fleming and Rishel [38] (Theorem 4.1 pp. 68-69), where the following properties must be satisfied.
1. The set of controls and corresponding state variables is nonempty.
2. The control set U is closed and convex.
3. The right-hand side of the state system is bounded above by a linear function in the state and control.
4. The integrand of the functional is concave on U and is bounded above by
An existence result in Lukes [39] (Theorem 9.2.1) for the system of equations (6)-(9) for bounded coefficients is used to give the first condition. The control set is closed and convex by definition. The right-hand side of the state system (Equations (4.1)-(4.4)) satisfies Condition 3 since the state solutions are a priori bounded. The integrand in the objective functional,
, is concave on U. Furthermore, c 1 , c 2 > 0 and k > 1, so
Therefore, the optimal control exists, since the lefthand side of (11) is bounded; consequently, the states are bounded.
Since there exists an optimal control for maximizing the functional (10) subject to equations (6)-(9), we use Pontryagin's Maximum Principle to derive the necessary conditions for this optimal control. Pontryagin's Maximum Principle introduces adjoint functions that allow us to attach our state system (of differential equations), to our objective functional. After first showing existence of optimal controls, this principle can be used to obtain the differential equations for the adjoint variables, corresponding boundary conditions and the characterization of an optimal control u v * and u m * . This characterization gives a representation of an optimal control in terms of the state and adjoint functions. Also, this principle converts the problem of minimizing the objective functional subject to the state system into minimizing either the Lagrangian or the Hamiltonian with respect to the controls (bounded measurable functions) at each time t [40] . The Lagrangian is defined as where w 11 (t) ≥ 0, w 12 (t) ≥ 0 are penalty multipliers satisfying w 11 (t)(a 11u v (t)) + w 12 (t)(u v (t)b 11 ) at the optimal u v * , and w 21 (t) ≥ 0, w 22 (t) ≥ 0 are penalty multipliers satisfying w 21 (t)(a 22u m (t)) + w 22 (t)(u m (t)b 22 ) at the optimal u m * . Given optimal controls u v * and u m * , and solutions of the corresponding state system (6) with transversality conditions l i [t f ] = 0, for i = 1, 2, 3, 4. To determine the interior maximum of our Lagrangian, we take the partial derivatives of L with respect to u v and u m , respectively, and set it to zero. Thus, To determine an explicit expression for our controls u m * , u m * (without w 11 , w 12 , w 21 , w 22 ), a standard optimality technique is utilized. The following cases are considered to determine a specific characterization of the optimal control. 
The optimal system
The optimality system consists of the state system coupled with the adjoint system, with the initial conditions, the transversality conditions and the characterization of the optimal control: [36] . The uniqueness of the optimal control follows from the uniqueness of the optimality system.
The state system of differential equations and the adjoint system of differential equations together with the control characterization above form the optimality system solved numerically and depicted in Figures 2, 3 , 4, 5.
The general model with pulse vaccination is given as In this model, vaccination occurs at fixed times, not continuously. This is closer to reality, since vaccination centres are only open at certain times, when people may get vaccinated in waves. Similarly, media stories tend to clump together, so that a big news story occurs on one day, which may trigger a short period of intense vaccination. We shall use a simplified version of this model to illustrate the possibility that media may have an adverse effect.
Consider the following scenario. At the onset of the outbreak, the media -and hence the general population -is unaware of the disease and thus nobody gets the vaccine, allowing the disease to spread in its initial stages. At some point, there is a critical number of infected individuals, whereupon people are sufficiently aware of the infection to change their behaviour. We suppose that, initially, new infected people arrive at fixed times.
We further assume that vaccinated people mix more than susceptibles. In this case, people who are vaccinated feel confident enough to mix with the infected, even though they may still have the possibility to contract the virus. This might be the case for health-care workers, for instance, who get vaccinated and then have to tend to the sick.
Mathematically, we have a threshold for the critical number of infectives, I crit .
For I <I crit , this model would look like For I >I crit , the model becomes However, to illustrate the adverse affect, we shall simplify the model even further. For a short timescale, we can assume recovery is permanent, so s = 0. Thus, we can ignore the R equation.
For I <I crit , we assume that there is no mixing, but rather that new infectives arrive impulsively into the system at fixed times t k and in numbers I i , where I i ≪ I crit .
(If the new infectives arrive at irregular times, then the broad results will be unchanged.)
For I >I crit , fear of the disease keeps susceptibles from mixing with the infected, but the vaccinated will.
Thus b 4 = b 6 = 0. Since I i ≪ I crit , we can assume that, for I >I crit , the effects of new infectives are negligible.
The model then becomes for I <I crit and
for I >I crit . Thus, the effects of the media are to trigger a vaccinating panic whenever the number of infectives is large enough. We kept the model with impulse vaccination as simple as possible since even this simplified version shows that media reports could have an adverse effect.
Suppose new infectives appear regularly, so that t k+1t k = τ. (If not, the analysis generalizes quite easily.) For t k <t <t k+1 , we have It follows that the endemic equilibrium is stable if Î >I crit . Thus, even in an extremely simplified version of the model, the media may make things significantly worse than if no media effect were included. We kept this model deliberately simple, partly for mathematical tractability and partly to show that the media effects apply even in this idealised scenario.
Note that, in reality, the fluctuations would apply in the upper region as well, making the actual value even larger. In the lower region, we ignored interaction between susceptibles and infectives (ie we assume b 4 = b 6 = 0). The effect of including these terms would be to slow the exponential decay between impulses (or possibly cause it to increase). This would only increase the effect seen here.
In summary, a small series of outbreaks that would equilibrate at some maximum level m + >I crit will, as a result of the media, instead equilibrate at a much larger value I >m + >I crit . The driving factor here is if an imperfect vaccine causes overconfidence, so that people who have been vaccinated mix significantly more with infectives than susceptibles do. If this happens (as would be quite likely; most people who have been vaccinated feel invulnerable, even if the vaccine is not perfect, largely thanks to media oversimplifications), then the media effect is likely to be adverse. A simplified version of the model with pulse vaccination shows that the media can make things worse, if the vaccine is imperfect because the vaccinated mix over-confidently with the infectives.
We now return to model (6)- (9) and illustrate some of the properties discussed in the previous sections. The parameter values that we use for numerical simulations are in Table 1 [41] . We consider an imperfect flu vaccine for which the waning rate is about 0.15. The relationship between b 2 and b 3 is not very obvious; consequently, we can either assume equality or that the former is slightly greater than the latter. Transmission dynamics of infectious diseases with and without media coverage have already been carried out in previous studies, but these models do not account for the vaccination coverage. Therefore, we illustrate some numerical results for the model with optimal control when media coverage has (i) a beneficial effect (see Figures 2 and 3) (ii) and adverse effect on the vaccination rate (see Figures 4 and 5 ).
The optimality system is solved using an iterative method with a fourth order Runge-Kutta scheme. Starting with a guess for the adjoint variables, the state equations are solved forward in time. Then the state values obtained are used to solve the adjoint equations backward in time; the iterations continue until convergence. Simulations are carried out to determine how maximizing media coverage enhances vaccination. The effects of costs that can be incurred, which include education, implementation and campaigns on media coverage, are also studied to evaluate how these costs can affect the transmission of human influenza. We increase the value of B2 (the cost weight) in Figure 2 to assess how the populations of susceptibles, infectives, vaccinated and recovered individuals are altered. In Figure 3 , we investigate how increasing minimization of infectives through increasing the weight B1 affects the control of human influenza transmission. We do the same in Figures 4  and 5 , respectively, to see how, if media coverage has an adverse effect, the various populations behave. In Figure   4 , we vary the cost weight, while in Figure 5 , we vary the weight of minimizing infectives.
We note from Figure 2 that, during the initial days, there is a very sharp drop in the population of infected individuals, while other populations show increases. Increasing minimization of infectives, while keeping costs low, can lead to the disease being controllable. The slight rise and fall, after the initial 20 days, in the population of infectives could be attributed to complacency on the part of some individuals (or may be due to oscillations in the system independent of external factors). We find that people tend to relax after the initial shock of the disease threat. However, we note that this is not for long, and this could be attributed to the fact that vaccination levels continue to rise, so as people continue to receive vaccination, infection is controlled. Thus, if costs are kept minimal, and more people are able to access media and vaccination, then infection can be controlled. Both vaccination and media coverage continue at optimum levels as a result of the low costs and minimization of infectives.
From Figure 3 , as costs are increased, few people have access to media and vaccination; as a result, low numbers get vaccinated against the disease. In the long run, the infection levels rise. The degree of media coverage and vaccination also decrease as a result of the exorbitant costs. With the little available media coverage and the few vaccinated individuals, we find that, due to information filtration, there is a jump in the vaccination levels, though these only last briefly; as the degree of media coverage and vaccination decrease, so do the vaccination levels.
From Figure 4 , even though costs are kept at minimal levels, the negative reports concerning vaccination result in a drastic reduction in the vaccination levels. After some time, we note a slight increase in the vaccination levels; however, these numbers remain very low. This could be due to the fact that, as infection rises, a few will risk getting vaccinated in the hope of being cured. Thus, media coverage can have adverse effects if people's perception towards the vaccine is negatively influenced by the media.
In Figure 5 , both media coverage and vaccination are eventually withdrawn. Very low numbers get vaccinated. It is only when infection escalates that vaccination levels also increase as some might find it better to try to prevent the infection, despite the negativity towards vaccination in the media. Figure 6 illustrates other potential adverse effects that media may have, if the effect is to trigger a vaccinating panic where vaccinated individuals are not fully protected and mix with infected individuals but susceptible individuals do not. In this case, the number of infected individuals may increase sharply as a result of the media. Figure 7 illustrates the long-term 
Media simplifications can lead to overconfidence in the idea of a vaccine as a cure-all. The result is not just a vaccinating panic and a blow-out epidemic, but a net increase in the endemic equilibrium. Thus, media coverage of an emerging epidemic can fan the flames of fear and also implicitly reinforce an imperfect solution as the only answer.
We have formulated and investigated a simple deterministic vaccination model describing the effects of media coverage on the transmission dynamics of influenza. The media effect due to reporting the number of infections as well as the number of individuals successfully vaccinated is introduced into the compartmental model via a saturated incidence-type function. The With media effects Figure 6 The vaccination panic threshold The effect of the vaccination panic threshold using the simplified model (14)- (20) . Without media triggering a vaccinating panic, the number of infected individuals remains low (solid purple curve). However, if the media triggers a vaccinating panic, then the number of infected individuals rises sharply (dashed green curve). Inset: Comparison of the two outcomes around the vaccination threshold.
impact of costs that can be incurred, which include vaccination, education, implementation and campaigns on media coverage, are also investigated using optimal control theory applied via the Pontryagin's maximum principle. A simplified version of the model with pulse vaccination shows that the media can have an adverse effect if the vaccine is imperfect and the vaccinated mix over-confidently with the infectives. Numerical simulations are carried out to support the analytical results. We note, however, that our caricature model is not complete; a more comprehensive study will require interdisciplinary research across traditional boundaries of social, natural, medical sciences and mathematics [2] . Nevertheless, our work provides some insights into the effects of media reporting on the transmission dynamics of infectious diseases for which a vaccine exists. The present study is in no way exhaustive and can be extended in various ways: for example, to investigate the case in which there is media coverage but people ignore it (in which case the vaccination rate is unchanged despite the control). Thus, the effects of media on an outbreak of influenza with a partially effective vaccine may be complicated. While the media may encourage more people to get vaccinated, they may also trigger a vaccinating panic or promote overconfidence in the ability of a vaccine to fully protect against the disease. This may have potentially disastrous consequences in the face of a new pandemic.  Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method BACKGROUND: Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. METHODOLOGY: We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. PRINCIPAL FINDINGS: The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. CONCLUSION/SIGNIFICANCE: The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin disease endemic mostly in rural wetland of tropical countries of Africa, America, Asia and Australia. The disease also occurs in non-tropical areas of Australia, China and Japan. Globally, BU has been reported in over 30 countries [1] [2] [3] . The burden of BU is however most severe in sub Saharan Africa where the true incidence of the disease is difficult to determine as a result of poor surveillance measures and case confirmation [2] . Available data however reveals an increase in BU incidence over the last several years in the west African countries of Ivory Coast, Ghana and Benin. In these countries BU has replaced leprosy as the second most prevalent mycobacterial disease [1] , [3] [4] [5] .
BU begins as painless nodule, papule, plaque or edema that evolves into characteristic ulcers with undermined edges. If untreated, extensive ulceration (that can cover 15% of the body), scarring and contractures may cause serious functional disabilities in patients [5] [6] [7] .
Unfortunately most patients seek treatment late and present with large ulcers [8] [9] [10] . Previously treatment of such lesions involved surgical removal of all the affected tissue and part of the surrounding tissues, eventually followed by skin grafting [9] [10] [11] [12] .
In 2004 antimycobacterial treatment alone (if necessary in combination with surgery) was introduced and has since been considered as the treatment of choice for BU [6] , [13] [14] [15] [16] . Laboratory confirmation of clinically suspected BU cases has therefore become crucial for the clinical management of BU [17] .
Four laboratory tests are recommended for the diagnosis of BU. These include microscopic examination, culture, IS2404 PCR and histopathological analysis. Microscopic examination detects 29%-78% of clinically suspected BU cases and is currently the only rapid and affordable test available for BU diagnosis in many endemic areas. The detection rate of culture is between 34%-79% and takes an average of 9-12 weeks to yield positive results. Culture therefore cannot be used for rapid laboratory confirmation of BU. Histopathological analysis is reported to detect 30% additional cases than other confirmatory tests, however this technique is restricted to external reference laboratories and are unavailable in peripheral health centres or district or regional hospitals. IS2404 PCR has close to 96% sensitivity and is considered the method of choice for laboratory confirmation of BU [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] .
The WHO recommends that at least 50% of cases must be confirmed by IS2404 PCR before commencement of antibiotic therapy [22] [23] [24] [25] .
However technical difficulties (eg, cold chain requirement, stable power supply and qualified laboratory staff) limit the use of this diagnostic test in BU endemic areas.
A dry reagent PCR consisting of lyophilized PCR mix which is reconstituted with water for testing DNA was developed to simplify BU diagnosis by PCR [26] but this method also requires the use of a thermocycler, electrophoresis and gel imaging equipment and therefore similarly makes the use of this diagnostic test for BU diagnosis in endemic areas unlikely.
The Loop Mediated Isothermal Amplification (LAMP) is a novel nucleic acid amplification method for molecular detection and identification [27] . The principle of LAMP is autocycling strand displacement DNA synthesis in the presence of Bst DNA polymerase with high strand displacement activity under isothermal conditions between 60-65uC within 60 minutes [28] . The assay is highly specific due to the recognition of target DNA by 4 to 6 independent sequences and the amplification efficiency of LAMP is equivalent to that of PCR based methods ( [27] , [29] , [30] ).
The LAMP reaction enables easy identification of positive tests due to the accumulation of high amounts of amplification products in the reaction tubes. Further improvement in visual identification can be realized through the addition of intercalating dyes such as SYBR green or hydroxynapthtol blue to reaction tubes [31] . This therefore precludes the need for post amplification analysis and hence reduces cost and labour. LAMP has also been shown to be less affected by a number of inhibitors of conventional PCR [32] . Additionally the closed tube format of this assay reduces problem of carry over contamination which is likely in less controlled environments [33] . With all of these characteristics LAMP of DNA has emerged as a powerful tool to facilitate point of care diagnostic test [31] .
In order to develop a field applicable technique that offers high detection sensitivity and specificity for the diagnosis of BU, we explored the use of the pocket warmer LAMP (pwLAMP) technique, a DNA amplification method using isothermal conditions (60uC) provided by a disposable pocket warmer [34] .
Ethical approval for analysing patients' specimens was obtained from the ethical review board of the Noguchi Memorial Institute for Medical Research. Specimens used were anonymously taken from an already existing collection of patients' specimens processed for diagnosis of BU from Agogo Presbyterian Hospital in Ghana.
Thirty clinical specimens consisting of 20 swabs and 10 fine needle aspirates taken respectively from ulcers and pre-ulcerative lesions of suspected BU patients were used in this study. The fine needle aspirate specimens were kept in 1 ml phosphate buffered saline (PBS) and swabs were stored dry in sterile tubes. Each swab was transferred into a tube containing 2 ml milli-Q purified water (Millipore Corporation, Billerica, MA) and gently vortexed for 5 sec and then removed. Portions 250 ml of the sample suspensions were transferred to separate new sterile eppendorf tubes containing 250 ml of lysis buffer (1.6 M GuHCl, 60 mM Tris pH 7.4, 1% Triton X-100, 60 mM EDTA, Tween-20 10%), 50 ml proteinase-K and 250 ml glass beads.
The mixtures were incubated horizontally in a shaker (200 rpm) at 60uC overnight. To capture the DNA, 40 ml of diatomaceous earth solution (10 g diatomaceous earth obtained from Sigma Aldrich Chemi GmbH in 50 ml of H 2 O containing 500 ml of 37% (wt/vol) HCl) was added to the suspensions and incubated at 37uC with shaking (200 rpm) for 60 min. The mixtures were centrifuged at 14,000 rpm for 10 sec and the resulting pellets were washed twice with 900 ml of 70% ethanol (2-8uC) followed by 900 ml of acetone. The pellets were dried at 50uC for 20 min and resuspended in 100 ml milli Q purified water and centrifuged at 14,000 rpm for 10 sec. The purified DNA was used as templates for both IS2404 PCR and LAMP assays to detect M. ulcerans.
To investigate the performance of the LAMP assay on crude DNA preparations, we obtained 2 types of DNA extracts for each clinical specimen. One crude extract consisted of 250 ml suspensions of the specimen boiled for 10 min followed by centrifugation at 14,000 rpm for 5 min (boiled extract). The other crude extract used was a 250 ml suspension of the unboiled specimen.
Ten M. ulcerans strains grown on LJ slants were harvested and DNA was extracted as previously described [35] . Serial dilutions of purified M. ulcerans DNA containing 300,000, 30,000, 300, 30 and 3 copies of IS2404 element per 5 ml were prepared. The number of copies of the insertion sequence element was determined based on the genome size of 5,806 kb and presence of an average number of 207 copies of IS2404. This was used to determine the detection limit of the LAMP assays.
In order to develop a simple and rapid test that can be used to diagnose Buruli ulcer under field conditions, we modified the conventional LAMP assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified DNA as the diagnostic specimen. Thirty clinical specimens from suspected Buruli ulcer patients were investigated by the modified LAMP (or pocket warmer LAMP) and the conventional LAMP, as well as IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans. There was no significant difference in the detection rate (63-70%) in all of the methods when purified samples were used for the tests. On the other hand the use of crude specimen preparation resulted in a drop in detection rate (30-40%) . This study demonstrates that the LAMP test can be used for rapid detection of M. ulcerans when purified DNA preparations are used. With further improvements in the sample reaction, as well as in specimen purification, the pocket warmer LAMP may provide a simple and rapid diagnostic test for Buruli ulcer.
LAMP for Detection of Mycobacterium ulcerans www.plosntds.org PCR for IS2404 PCR targeting IS2404 was performed as described previously [21] . The first and second round PCRs used primers pGp1: 59-AGGGCAGCGCGGTGATACGG-39and pGp2: 59-CAGTG-GATTGGTGCCGATCGAG-39 and pGp3: 59-GGCGCAGAT-CAACTTCGCGGT-39 and pGp4: 59-CTGCGTGGTGCTTT-ACGCGC-39, respectively.
For the First round, the 30 ml reaction volume contained 3 ml DNA, 25 pmol/ml of each primer (pGp1 and pGp2), 3 ml of 106 PCR buffer (containing 1.5 mM magnesium chloride), 6.0 ml Qsolution, 0.2 mM deoxynucleotide triphosphates (dNTPs) and 1.0 U HotStar Taq polymerase (QIAGEN).
For the second run, 1 ml of the first run product was added to 24 ml reaction volume containing, 25 pmol/ml of each primer (pGp3 and pGp4), 2. Pocket warmer LAMP (pwLAMP) was performed using a loopamp DNA amplification kit (Eiken Chemical) described previously [32] . Each 25 ml reaction mixture contained 1.6 mM each of FIP and BIP, 0.2 mM each of F3 and B3, 0.8 mM each of LF and LB, 26 reaction mixture (12.5 ml), 1 ml of Bst DNA polymerase, 1 ml of fluorescence detection reagent (Eiken Chemical), 3.5 ml distilled water and 1 ml sample.
Reaction tubes were incubated at 60uC for 60 min in the heat block (GeneAmp 9700, Applied Biosystems, Foster City, CA) while with the pwLAMP, the tubes were sandwiched in a twofold pocket warmer (Hokaron Haru-type, Lotte Health Products, Tokyo, Japan) surrounded by a paper towel, and put in a Styrofoam box for 120 min (60 min reaction incubation). The reaction was terminated at 85uC for 5 min and the results were read by eye in ambient light and also using UV illumination. 
A chi-squared test was performed to reveal the statistical difference using SPSS (version 16.0; SPSS Inc., Chicago, IL) software.
LAMP reaction requires a constant temperature of about 60u-65uC for 60 min for amplification of DNA [27] [28] [29] [30] [31] [32] [33] [34] . In a previous study a pocket warmer reached 58uC in 30 min and stayed around 60uC for more than 60 min in a Styrofoam box [34] . The 3 pocket warmers (of a pack of 30 hand warmers) tested in this study achieved a temperature of 60uC after 60 min and maintained this temperature for about 90 min. The pocket warmer thus provided a suitable temperature (60uC) and time range (60 min) for amplification. Both pwLAMP and the conventional LAMP assays were able to detect to the limit of 300 copies of the target sequence after 60 min of amplification. This limit improved to 30 copies when the conventional LAMP was carried out at 65uC (the pocket warmer was not able to attain this temperature and was therefore not investigated). 
The sensitivity and specificity of the LAMP assays for the detection of M. ulcerans is shown in tables 1 and 2. Under ambient illumination, positive specimens in the LAMP assay produced greenish colouration (Figure 2 ). When purified DNA extracts were used, 21 (16 swabs, 5 fine needle aspirates) (70%) of 30 clinical specimens were positive by IS2404 PCR as well as by the conventional LAMP. None of the PCR positive specimens were negative by conventional LAMP. However 19 samples of purified DNA extracts were positive with the pwLAMP, but the 90.5% sensitivity (19/21) of the pwLAMP compared to the results by IS2404 PCR was not statistically significant (p = 0.58, Chi-square test).
All negative specimens in IS2404 PCR were negative in both LAMP assays, indicating specificities of both LAMP assays to the reference method were 100%. Twelve unboiled (9 swabs and 3 fine needle aspirates) and 9 boiled (6 swabs and 3 fine needle aspirates) extracts were positive by all 3 detection assays with sensitivities of 57.1% (unboiled, 12/21) and 42.9% (boiled, 9/21) compared to results using purified DNA extracts respectively for both LAMP and IS2404 PCR assays. The positivity of swabs was found to be in the range of 30% to 80% compared to 30% to 50% for fine needle aspirates.
When the positivities in crude DNA specimens were compared with those in purified DNA, the differences were statistically significant by chi-square test (unboiled vs purified DNA, p = 0.0195, and boiled vs purified DNA, p = 0.0019). None of the IS2404 PCR negatives was positive in the LAMP assays irrespective of the DNA extracts type used. These data suggest that sensitivity of LAMP and PCR assays for the detection of M. ulcerans in clinical specimens is enhanced when purified DNA extracts are used.
BU is a neglected tropical disease that mostly affects the poor in resource limited communities in sub-Saharan Africa [1] [2] [3] . IS2404PCR, the method of choice for confirmation of BU diagnosis cannot be operational in BU endemic areas [16] , [17] , [24] [25] . The development of rapid and reliable point of care diagnostic assays is of high priority to BU management and prevention.
This study explored the potential use of the LAMP method for field diagnosis of BU. Some important limitations to the use of this assay in the field include specimen purification and difficulty in maintaining isothermal condition for the reaction. A study suggested that omission of DNA extraction or the use of crude DNA extracts have no effect on the LAMP test [32] . Hatano et al used a disposable pocket warmer to provide isothermal condition for LAMP reaction [34] .
Based on this knowledge, we applied the pwLAMP to crude and purified DNA extracts in order to determine whether this method will be suitable for use as a point of care diagnostic test for BU.
Although the pocket warmers used in this study reached 60uC after 1 hr (instead of 30 min in previous studies [34] ), both devices achieved the requisite temperature and holding time for executing LAMP reaction, a major advantage in the use of amplification based assay for the detection of an infectious agent under field condition. The pwLAMP did not cross-react with other mycobacteria ( Figure 1) . Moreover, the experiment on clinical specimens demonstrated that the pwLAMP had a 100% specificity in clinical specimens of BU.
The pwLAMP was found to have comparable sensitivity as the conventional LAMP at 60uC as both assays were able to detect 300 copies of IS2404 element (equivalent of 1.5 genomes of M. ulcerans). The detection limit of the conventional LAMP at 65uC improved to 30 copies of IS2404 and this level of sensitivity may probably be achieved with a pocket warmer that can attain a temperature of 65uC and maintain a holding time of 60 min. When applied to purified DNA extracts of clinical specimens, the pwLAMP, conventional LAMP and IS2404 PCR yielded concordant results (Tables 1 and 2) .
However, 2 of the samples that were positive by IS2404 PCR/ conventional LAMP were negative by the pw LAMP. None of the IS2404 PCR negative samples were positive in both types of LAMP assays. On the other hand we observed a drop in detection rate from 63-70% to 30-40% when crude extracts of clinical specimens were used (Tables 1 and 2) .
This indicates that the use of crude DNA extracts as template may not be appropriate for the detection of M. ulcerans by the LAMP method. This observation contradicts a previous study that suggested omission of DNA extraction had no effect on sensitivity of the LAMP assay [32] .
For the crude preparations however, it is noteworthy that the detection rate of the LAMP assay was significantly higher for the unboiled extracts than for the boiled extracts. Explanations for these results were not explored. The observation that the LAMP assay was not inhibited especially for the unboiled specimens is quite consistent with previous work that have shown LAMP to be tolerant to culture medium and to certain biological substances including phosphate buffered saline, serum, plasma, urine and vitreous [32] .
In conclusion, the study demonstrates that the LAMP assay yields comparable results as IS2404 PCR when it is performed at 60u-65uC for 60 min on purified DNA extracts and further supports the use of the pocket warmer as a device for providing isothermal amplification condition for the LAMP assay. This therefore is a potential boost to the application of pwLAMP in resource poor settings.
Challenges of obtaining pure DNA extracts of clinical specimen as well as the use of a pocket warmer capable of maintaining 65uC for 1 hr, however needs to be addressed in order to improve the performance of the pwLAMP assay.
Further development and testing in larger numbers of specimens is therefore necessary to access the potential use of pwLAMP as a simple and rapid point of care diagnostic test for BU. A Human PrM Antibody That Recognizes a Novel Cryptic Epitope on Dengue E Glycoprotein Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins. Dengue virus (DENV) is a flavivirus with four related but antigenically distinct serotypes (DENV1-4). It infects approximately 50-100 million people each year, of which 500,000 people exhibit the life-threatening form of severe dengue -dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) [1] . The current lack of treatment or licensed vaccine means dengue poses a serious public health threat [2] . Infection by one serotype of DENV confers lifelong immunity against the homologous serotype, but only limited crossprotection to the remaining three serotypes [3, 4] . The presence of cross-reactive, non-neutralizing antibodies generated during a primary infection has been suggested to enhance the pathogenicity of subsequent infections via the process of antibody-dependent enhancement (ADE) [5] . A successful and safe vaccine candidate must therefore elicit a protective long-lasting immune response to all four serotypes [4, [6] [7] [8] .
Recent immunological studies have shown the human anti-DENV immune response to be dominated by prM-specific antibodies in both primary and secondary infections [9, 10] . These prM-specific antibodies are highly cross-reactive and non-neutralizing. When complexed with immature DENV (imDENV), it has the ability to render normally non-infectious imDENV highly infectious [11, 12] . This has caused concern over current vaccine candidates that contain native dengue prM [9] -which is a component of most current vaccine strategies whether naturally attenuated, recombinantly attenuated, yellow fever-dengue-virus chimeras, chemically inacti-vated virus, DNA vaccine or recombinant subunit protein vaccines [13, 14] . Vaccine candidates that do not contain prM proteins, such as soluble recombinant Envelope (E) protein or E domain subunit vaccines may thus become increasingly important.
To gain a deeper understanding of the early DENV-specific immune response in humans, the four serotypes of DENV were sequentially screened with a non-immunized human Fab phage display library. Broadly cross-reactive prM-specific antibodies dominated the screen and the Fab with highest affinity, D29 Fab-IgG, was converted into full-length human IgG1 format for thorough characterization. This antibody (D29 Fab-IgG) was found to have high affinity for a conformational epitope on prM and, like other prM antibodies [11, 12] , was capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FccR-bearing K562 cells. The antibody also cross-reacted with E protein -fine mapping and site-directed mutagenesis studies localized the epitope to the DI/ DII junction of E, which would be inaccessible in a native virus particle. This suggests the possibility that immunization strategies employing E protein alone or subunits of E proteins may not be able to completely avoid generation of anti-prM antibodies that enhance DENV infection.
Aedes albopictus C6/36 cells (ATCC) were maintained in Leibovitz L-15 media (GIBCO, Invitrogen) supplemented with 8% fetal bovine serum (FBS), at 28 o C, 5% CO 2 . BHK-21 cells (ATCC) and human erythroleukemic K562 cells (ATCC) were maintained in RPMI 1640 GlutaMAX medium (RPMI) (GIBCO, Invitrogen) containing 10% FBS, and incubated at 37 o C, 5% CO 2 . Vero cells (ATCC) were grown in 199 medium (M199) (GIBCO, Invitrogen) supplemented with 8% FBS, 1% sodium pyruvate and 1% NEAA. HEK293 T cells (ATCC) was cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Invitrogen) supplemented with 10% FBS at 37 o C in 5% CO 2 .
Mouse monoclonal antibodies 3H5 (m3H5), 4G2 (m4G2) and 2H2 (m2H2) are specific to EDIII of DENV2, EDII of flaviviruses and prM of DENV1-4 respectively. Chimeric humanized 3H5 (h3H5) and 4G2 (h4G2) were constructed by cloning the Fab portion (variable light and heavy chains) into an expression vector containing the human IgG1 framework for expression in HEK293 T cells [15] . Conjugation of HRP to antibodies-D29, -m3H5 and -m2H2, was performed using the Lightning-Link HRP Conjugation Kit (Innova Biosciences).
Dengue 1 (DENV1) strain Hawaii, Dengue 2 (DENV2) strains New Guinea C (NGC) and ST, Dengue 3 (DENV3) strain H87 and Dengue 4 (DENV4) strain H241 were passaged in Vero cells for all assays unless otherwise stated. Virus titers were determined by plaque assay on BHK cells. Immature DENV (imDENV) was produced as described previously [16] . Briefly, the media of infected Vero cells was replaced 2 dpi with M199 NH 4 Cl medium (M199 with 10% FBS, 1% sodium pyruvate, 1% non essential amino acids, 1% PS, 2.5 mM L-Glutathione and 20 mM NH 4 Cl). Culture supernatants were harvested after 5 days and imDENV was purified by PEG precipitation. Virus titer was determined as genome containing particles (GCP)/ml by quantifying real-time PCR (qPCR) described previously [17] .
To ascertain the binding specificity of D29 Fab-IgG, direct binding ELISA against dengue virus was performed using standard ELISA methods. Briefly, 2610 5 pfu/well purified DENV1-4 was coated on Maxisorb plate followed by blocking using 5% SM. Following incubation with 1 mg/ml dengue-specific antibodies, plates were probed with HRP-conjugated anti-human IgG-Fc secondary antibody or anti-mouse IgG-Fc secondary antibody (Pierce). Detection steps in subsequent ELISAs followed this method unless otherwise stated. To determine the binding affinity of D29 Fab-IgG direct binding ELISA was performed with serially diluted D29 Fab-IgG. Results were fitted to a one-site binding hyperbola using Prism 5.03 (GraphPad, San Diego, USA).
For Peptide phage ELISA, 2 mg of D29 and non-related control antibodies were coated on Maxisorb plate and incubated with phage, followed by detection with HRP-conjugated anti-M13 secondary antibody.
DENV-infected Vero cells or C6/36 cells were fixed and permeablilized with 80% acetone at 220 o C for 10 min. The presence of virus was detected by D29 Fab-IgG, h3H5 and h4G2 at 1 mg/ml, followed by staining with FITC-conjugated antihuman IgG (ZyMax, Invitrogen). Cells were mounted with MOWIOL (Sigma) and images were captured using a fluorescent microscope (Olympus).
DENV-infected Vero cells were harvested 5 days post-infection and lysed with cold lysis buffer (1.5% Triton X-100, 0.2 mM Phenylmethylsulfonyl Fluoride (PMSF), 1 mg/ml pepstatin A and 75 mM KCl in PBS) for 40 min on ice. An equal volume of 56 loading dye was added to the cell lysate before 12% SDS-PAGE and transferred onto nitrocellulose membranes. Blocked membranes were probed with dengue-specific antibodies at RT for 1 hr and detected with HRP-conjugated anti-human IgG-Fc or antimouse IgG-Fc secondary antibody and developed with chemiluminescence (Supersignal West Pico Chemiluminescent Substrate, Pierce).
To establish the binding target of D29 Fab-IgG, competition ELISA against epitope-characterized monoclonal antibodies was carried out. Plates were prepared as for direct binding ELISA. After blocking, serially diluted m3H5, m4G2 or m2H2 was added to the plates and incubated for 1 hr at RT, followed by addition of D29 Fab-IgG at 2.5 mg/ml and incubation for 1 hr at RT. Bound antigen-antibody complexes were detected by HRP-conjugated anti-human IgG-Fc secondary antibody (Pierce), and developed as described above.
For competition Western, Fab-IgG D29 was incubated with m3H5, m4G2 or m2H2 for 1 hr at RT before applying to blocked membranes for 30 min at RT. HRP-conjugated anti-Human IgG-Fc secondary antibody was applied to detect bound D29 for 45 min at RT after 4 washes with PBST.
For radioactive immunoprecipitation, DENV2-infected BHK-21 cells were harvested 48 hr later and prepared as described previously [18] . For immunoprecipitation with SDS to disrupt protein complexes, lysate from infected cells was incubated with 1.25% SDS for 30 min at 4 o C. The SDS concentration was diluted to 0.2% before immunoprecipitation and precipitated proteins were analysed by silver stained SDS-PAGE (Silver stain Plus Kit, Bio-Rad) and Western blot.
The phage-displayed-12 random dodecapeptide (Ph.D-12) library (New England Biolabs) was panned against D29-Fab-IgG to identify the antibody epitope as per instruction manual. The first round of panning was carried out with 100 mg/ml D29 Fab-IgG immobilized on Maxisorb Immunotube (Nunc). The amplified phage was enriched by three further rounds of panning in solution using Protein A or G sepharose in alternate rounds of panning. One round of negative selection with Protein A and G sepharose was carried out to minimize non-specific binders. The isolated peptide phage sequences were mapped against 3D protein structure using the Peptiope server with crystal structures of prM-E heterodimer at neutral pH (Protein Data Bank (PDB) accession code 3C6E). Graphic visualization was carried out using MSI WebLab ViewLite (Accelrys).
To test whether isolated peptide phage inhibits binding of D29 to its natural epitope, inhibition ELISA was carried out. 10-fold serially diluted peptide phage (starting at 10 12 pfu/ml) or 2610 5 pfu DENV2 was incubated with D29 Fab-IgG for 1 hr before applying to DENV2-coated plate for 5 min at RT. Bound antibody was detected with HRP-conjugated anti-human IgG-Fc secondary antibody for 1 hr at RT.
To test the inhibition capacity of peptide phage by Western blot, pre-incubation of 0.1 mg/ml of D29 Fab-IgG with 4610 11 pfu peptide phage; 8610 6 pfu DENV2 or 5%SM for 1 hr was carried out before applying to membrane transblotted with DENV2 lysate for 30 min. HRP-conjugated anti-Human IgG-Fc secondary antibody was applied to detect bound D29 Fab-IgG for 45 min at RT and the membrane was processed as described previously to visualize the reactive bands.
The gene portion containing prM-E, as described in Puttikhunt, et al [24] , was amplified from DENV2 (NGC) cDNA using the primers D2-FW (59-AATTAATACGACCGTCTCCCATGAA-TAGAAGACGCAGATCTGCAGGC-39) and D2-RV (59-CGC-CCGTTTGATCTCGAGCTACTAGGCCTGCACCATGACT-CCC-39) and cloned into pCMV-myc-ER vector via the restriction sites Nco I and Xho I to create plasmid construct pCMV-prM-E. Using this as template, selected amino acids residues of the predicted epitopes, P3 and P9, were mutated by site-directed mutagenesis using Quikchange Multi Site-directed Mutagenesis kit (Stratagene, Agilent Technologies). PCR reactions and subsequent cloning steps were carried out according to manufacturer's instructions. Substitution of amino acids in all mutant constructs was confirmed by sequencing. Mutants were expressed in HEK293-T cells and harvested 48 hr posttransfection for immunoblot analysis and immunoprecipitation experiments.
To determine the ability of D29 Fab-IgG to neutralize Dengue infection in vitro, PRNT was carried out on BHK cells as described previously [19] .
The ADE assay was performed as described previously [20] . Briefly, pre-formed immune complexes were prepared by incubating serially diluted antibody with DENV2 or imDENV2 at a multiplicity of 25 GCP per cell (MOG 25) in RPMI MM at 37 o C for 1 hr before applying to 2610 4 K562 cells in 96-well U-bottom plate (Nunc). Supernatant was harvested 48 hr postinfection and virus titer was quantified by plaque assay.
Following isolation from the human non-immune Fab-phage library by panning against the 4 serotypes of DENV sequentially (Fig. S1 ), D29 Fab-IgG was converted into full length human IgG1 (Materials and Methods S1). To ensure the antibody was specific for DENV viral proteins, ELISA and IFA were performed with all 4 serotypes of DENV grown in C6/36 and Vero cell lines. D29 Fab-IgG reacted with infected cells from DENV1-4 without crossreaction to the non-infected cell controls, indicating that the determinant for D29 Fab binding is indeed viral and not host (Fig. 1A , B, C).
Using direct ELISA, D29 Fab-IgG was found to have high affinity for all 4 serotypes of DENV ( Fig. 1D hypermutation. This suggests that antibodies like D29 can be generated in the early immune response against DENV.
To broadly characterize the epitope of D29 Fab-IgG on DENV, we used competition ELISA with well characterized DENV antibodies including mouse-derived 3H5 (m3H5) which is specific to DENV2 EDIII; m4G2, a cross-reactive antibody that recognizes EDII of all flaviviruses and m2H2, a cross-DENV reactive anti-prM antibody [21, 22] . Making use of humanized versus parental murine antibodies for 3H5 and 4G2, and HRPconjugated m2H2 versus non-conjugated for m2H2, each of the reference antibodies was shown to self-compete ( Fig. 2A) . No significant competition was observed between D29 Fab-IgG and m3H5, m4G2 or non-DENV specific control antibody. However, binding of D29 Fab-IgG to DENV2 decreased significantly in the presence of m2H2 in a dose-dependent manner, indicating that D29 Fab-IgG recognized an epitope on prM similar to that of m2H2 ( Fig. 2A) .
To confirm the target protein of D29 Fab-IgG, Western blot analysis was carried out with DENV-infected Vero cell lysates and the recombinant ectodomain of DENV2 E protein (rE) under reducing and non-reducing conditions. Under non-reducing conditions, the control antibodies recognized their respective target proteins from the cell lysates: h4G2 detected the ,55 kDa E protein of all 4 serotypes and the ,50 kDa rE protein, and m2H2 detected the ,20 kDa prM of all 4 serotypes (Fig. 2B) . Remarkably, D29 Fab-IgG detected both E and prM of all 4 serotypes; however, the binding was abolished in the presence of reducing agent, indicating that the D29 epitope is conformational. All control antibodies except h3H5 lost binding to their target proteins under reducing condition (Fig. 2C) , which is expected since h3H5 recognizes a linear epitope on EDIII [22, 23] , whereas both h4G2 and m2H2 are conformationally sensitive [10, 24] . In agreement with the competition ELISA data, h3H5 and h4G2 did not compete with D29 Fab-IgG for epitopes in the Western blot format, whereas incubation with m2H2 abolished D29 binding to prM but not to E protein (Fig. 2D) .
Given the ability of D29 Fab-IgG to bind both prM and E on western blots, immunoprecipitation was carried out with DENV2infected BHK cell lysate to elucidate if D29 Fab-IgG binds both proteins in solution. An initial radioactive immunoprecipitation showed D29 Fab-IgG binding to both E (,55 kDa) and prM (,20 kDa), as both proteins are precipitated whereas h4G2 only precipitated E protein (Fig. 3A) . However, as prM intrinsically interacts with E to form heterodimers, it is possible that antibody binding to one of the components in the heterodimer would pull down the other. To address this, dissociation of the heterodimer with 1.25% SDS was performed prior to the assay. Immunoprecipitated proteins were resolved by reducing SDS-PAGE and visualized by silver staining. Identity of the precipitated proteins was verified by non-reducing Western blot analysis with h3H5 and m2H2, respectively (Fig. 3B ). The control antibodies were able to precipitate their corresponding binding partners. Despite the ability to recognize both prM and E on western blots, D29 Fab-IgG only precipitated prM. Taken collectively, our data indicate that D29 binds both prM and E but the epitope on E is not accessible on the native protein.
As the DTT-sensitive m2H2 had previously been mapped to its epitope using linear peptides [21] , we attempted a similar approach to locate the binding epitope of D29 Fab-IgG. An inhibition ELISA was carried out using synthetic ,20 mer linear peptides covering DENV2 prM [25] as well as a library of short 15-mer overlapping linear peptides (Mimotope) corresponding to aa1-166 of prM and aa1-495 of E (Materials and Methods S1). However, none of the linear peptides were able to significantly inhibit the binding of D29 Fab-IgG to DENV2 (Fig. S3) , suggesting that the epitope of D29 Fab-IgG1 is discontinuous. Next, D29 Fab-IgG was screened using a random dodecapeptide (Ph.D-12)-phage library as this approach has previously been used to map antibody-binding motifs [26, 27] . Peptide-phage clones were randomly selected from the second, third and fourth round of panning, and their binding specificity was confirmed by direct binding ELISA against D29 Fab-IgG. Reactive clones were sequenced and checked against a list of common target-unrelated peptides (TUPs) [28] to eliminate peptides that react with constant regions of antibodies, protein A/G sepharose or plastic surfaces. No TUPs were detected and the resulting 20 unique clones fall into three main consensus groups: 26% contained the motif W(TL/SV)K(L/X)PXW; 14% contained the motif AKTMP and 33% contained the motif KXPXW ( Table 1) .
Mapping of peptide sequences using the Peptiope server against the 3D crystal structure of the prM-E heterodimer at neutral pH (PDB 3C6E) identified three main clusters of predicted epitope location. Since Pepitope assumes all input peptides mimic surface residues; all buried residues are eliminated from the search. The Figure 4 . Localization of D29 Fab-IgG predicted epitopes in 3D crystal structures of DENV2 prM-E heterodimer (PDB 3C6E) and the binding specificity of peptide-phages. (A) Clusters of conformational epitopes predicted by Pepitope server are displayed on the prM-E heterodimer crystal structure at neutral pH with their solvent-accessible surfaces highlighted. The prM is pink, EDI is red, EDII is yellow, EDIII is blue, FP is cyan. (B) The binding specificity of peptide-phages (P1-P9) to D29 Fab-IgG was tested in a direct ELISA format with 10 mg/ml of D29 Fab-IgG, h3H5, m2H2 and non-DENV specific human antibody (Hu) immobilized on a Maxisorb plate. Ability of peptide-phages to inhibit D29 Fab-IgG binding to DENV2 was investigated by (C) ELISA and (D) Western blot analysis. For ELISA, peptide-phages (10 12 pfu/ml) were incubated with D29 Fab-IgG for 1 hr at RT before application to immobilized DENV2 for 5 min at RT. Bound D29 Fab-IgG was detected with HRP-conjugated anti-Human IgG-Fc. The percentage of inhibition of D29 Fab-IgG binding by the peptide-phages shown is the average of three experiments. Error bars represent the standard errors of the mean (***p-value ,0.005). For Western blot analysis, 0.5 mg/ml of D29 Fab-IgG was incubated with 8610 6 pfu of purified DENV2, 5% SM or 4610 11 pfu of peptide-phage clones for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT. doi:10.1371/journal.pone.0033451.g004 peptides with the KXPXW motif could mostly be mapped to the highest scoring Cluster 1 located at the prM/EDII interface. A few exceptions mapped to Cluster 3, most probably due to poorer sequence match and a lower alignment score. The remaining two motifs were mapped to the second highest scoring Cluster 2 that spans EDI and EDII (Fig. 4A) .
To verify the specificity of the identified motifs, nine representative clones were selected (Table 1) and assessed for their ability to bind D29 Fab-IgG by direct ELISA. No crossreaction by these peptide-phage clones with control antibodies was observed, confirming that the binding to D29 Fab-IgG was specific (Fig. 4B) . The peptide-phage clones were then tested for their ability to inhibit D29 Fab-IgG's binding to immobilized DENV2. Although most of the selected peptide-phage clones only weakly inhibited D29 Fab-IgG's binding to DENV2, clone P3 (Cluster 1) and P9 (Cluster 2) significantly competed by 30% and 70%, respectively (Fig. 4C ). Significant inhibition of binding to both prM and E was also observed on Western blot for P9, however no noticeable inhibition was observed for P3 (Fig. 4D) . Alignment of P3 and P9 peptide sequences to their respective clusters revealed 9 and 11 aa match, respectively (Fig. 5 ). This may explain the difference in the ability of the two peptide phage clones to inhibit binding of D29 Fab-IgG.
To confirm the residue assignation of the predicted epitopes for D29 on prM and E, clusters of 2-3 residue mutants were made on a prM-E construct that Puttikhunt and co-workers had previously shown to fold correctly when expressed in HEK293 [24] For the P3 (prM-E) epitope, two mutants, M1 (P56L-A, P58Q-G, P59N-G) and M2 (E246K-A, E247K-A) were generated (Fig. 6A ). Their reactivity with D29 Fab-IgG was tested by Western blot analysis, using h4G2 and m2H2 as control antibodies. All antibodies reacted with their respective protein targets for the non-mutated prM-E protein; however, binding of m2H2 and D29 Fab-IgG to the prM protein was almost completely abolished by mutation at the prM residues in M1 (Fig. 6B) . Mutation at the E residues within the P3 sequence (M2) on the other hand, did not have significant impact on the reactivity with D29 Fab-IgG (Fig. 6B) . Detection of both M1 and M2 by h4G2 verified the expression of both mutants. To verify the P9 (E) epitope, eight mutants were generated:
E57R-G, E129V-A, E131Q-G) and M5/6 (E129V-A, E131Q-G, E133E-G, E134N-G) (Fig. 6A ). Both control antibodies were able detect their respective protein targets for all mutants, apart from Figure 5 . Localization of P3 and P9 peptide sequence on 3D crystal structure of prM-E heterodimer (PDB 3C6E). P3 (Purple) and P9 (green) peptide sequences were aligned with the predicted clusters (Cluster 1 -Navy blue; Cluster 2 -Black) on the prM-E crystal structure. The path of peptide phage sequences was highlighted with the participating residues from the cluster and the peptide phage labeled. Matched residues were purple (P3) or green (P9) and numbered accordingly; mis-matched residues were grey. The respective proteins and domains were highlighted as above. doi:10.1371/journal.pone.0033451.g005 M3/4 and M4/5 which appeared to be non-viable (Fig. 6B) . The binding of h4G2 to E of M4 also appeared to be affected by the mutation introduced (Fig. 6B) ; likely to be caused by a change of local conformation, rather than a drop of expression level since prM-binding by m2H2 was normal. Notably, the binding of D29 Fab-IgG to the E protein of the M3, M5 and M6 mutants were severely affected; in particular, D29 Fab-IgG failed to detect E of M4 and M5/6, whereas binding of prM for all these mutants was not affected at all (Fig. 6B ). Together these results confirm the epitopes identified through peptide-phage display.
Previous studies with anti-prM antibodies have shown that while the antibodies are generally non-neutralizing [9, 25] , they are capable of rendering non-infectious immature DENV (imDENV) particles infectious through an FccR-mediated process [9, 12] . Indeed, D29 Fab-IgG failed to neutralize any of the 4 serotypes of DENV at the highest concentration tested (400 mg/ml) in a PRNT assay (Table S1 ). Next, the ability of D29 Fab-IgG to enhance infection of DENV2 and imDENV2 was determined by carrying out ADE assays with FccR-bearing K562 cells. The specific infectivity of the viruses was established by qPCR and plaque assay to be 8.5610 5 :1 for imDENV2, which was significantly higher than that of the standard-grown DENV2 at 240:1 (Fig. 7A) .
Enhancement of DENV2 infection was observed for all antibodies, with the exception of the non-DENV specific antibody. All the DENV-specific antibodies caused an approximately 20-fold increase in infection compared with IgG control (Fig 7B) . As D29 Fab-IgG is non-neutralizing, it was capable of enhancing infection of prM-containing virus in the preparation for the entire range of concentrations tested.
Infection of virtually non-infectious imDENV was also significantly enhanced by D29 Fab-IgG to a level similar to m2H2, causing an 80-100 fold increase in infection relative to control IgG (Fig. 7C) . The enhancement of imDENV infection by h3H5 was similar to that observed for DENV2, since its epitope was not affected by the maturity state hence it enhanced both equally well; h4G2 failed to enhance imDENV2 infection in K562 cells possibly be due to the occlusion of its epitope by the pr peptide [29, 30] , and indicated by the immunoprecipitation protein profile (Fig. 3B ).
To gain a deeper understanding of the early immune response against DENV, we have isolated and characterised a highly crossreactive antibody fragment -D29, from a non-immune human Fab-phage library that shows near germline sequence. Upon conversion into full-length IgG, D29 showed cross-reactivity against all four serotypes of DENV and could be competed with the prM-specific mouse antibody 2H2. Intriguingly, D29 Fab-IgG was found to recognize both E and prM during Western blot analysis in a conformational dependant manner. Cross-reactivity between E and prM has been observed in antibodies isolated from mice as well as human patients [10, 11, 21] . However, the binding epitopes of these antibodies remain unknown and the observation is usually explained by general cross-reactivity of anti-prM antibodies.
Initial immunoprecipitation experiments with D29 Fab-IgG precipitated both prM and E proteins. However, upon dissociation of the prM interaction with E by the addition of SDS, only prM was precipitated by D29 Fab-IgG, pinpointing prM as the native binding target. Similarly, h3H5 only precipitated E upon addition of SDS, whereas the protein profile precipitated by h4G2 was not affected possibly due to its epitope at the fusion loop of E being occluded by the presence of prM [29, 30] , and only 'free' E proteins were pulled down in both the presence and absence of SDS.
The epitope of D29 was mapped using a random-peptide phage display library, an approach which has been widely used for the identification of both linear and conformational epitopes [26, [31] [32] [33] [34] . Two peptide phage clones, P3 (Cluster 1) and P9 (Cluster 2), displayed significant inhibition of D29 Fab-IgG binding to immobilized DENV2 by ELISA; P9 was also able to significantly block the binding of D29 Fab-IgG in a nonreducing Western blot analysis. This may be due to a better sequence match of P9 to its predicted cluster compared with P3 (Fig. 5) , hence, a higher affinity of the peptide to D29 Fab-IgG. To ascertain the authenticity of the predicted epitopes, the predicted residues were confirmed by site-directed mutagenesis of the P3 and P9 peptide sequence. The residues P56L, P58Q and P59N within the P3 epitope were found to be the principle residues involved in the interaction between prM and D29 Fab-IgG; they were also critical for the binding of m2H2, corroborating with results of competition assays. The binding for E protein was not really affected by mutation of aa residues within the P3 epitope; contrasting to the significant impact caused by mutation of 1-2 residues within the P9 epitope. The combined mutation of E129V, E131Q, E133E, and E134N completely abolished binding of D29 Fab-IgG to E protein, establishing the critical residues within the P9 epiotpe. Taken together, these results suggest that D29 Fab-IgG recognises two epitopes on DENV -a solvent-accessible epitope on prM as the principle binding epitope, and a cryptic epitope on E which mimics the prM epitope but is not available on a native, functionally-folded E protein. The P3 and P9 epitopes are highly conserved across the four DENV serotypes, as well as two other flaviviruses, Japanese encephalitis virus and West nile virus; but with increasing divergence in Yellow fever virus and Tick-borne encephalitis virus (Fig. S4) .
Most current dengue vaccines consist of both prM and E proteins [12, 13, 35] ; studies performed with tick-borne encephalitis virus indicate that proper folding of E requires the chaperone function of prM [36] . However, as demonstrated in this study and recent reports, prM-specific antibodies are able to restore and enhance the infectivity of imDENV and partially mature DENV [9, 11, 12] ; it may be worthwhile to consider alternative vaccine approaches that minimize anti-prM responses during vaccine design [9] . The findings from our study also suggest that any partial denaturation or unfolding of an E protein vaccine preparation may result in the exposure of a cryptic P9-like epitope, which has the potential to induce D29-like antibodies with threatening ADE capability.
A recent study investigating the acute and early convalescent B cell response in dengue patients has found dengue virus to have a significant B cell activation capacity, causing a transient but high appearance of plasmablast and plasma cell, coinciding with that of dengue-specific IgG antibodies at day 4-7 after onset of fever [37] . The similarity of D29 Fab-IgG to germline sequence suggests that such antibodies are present in the immune repertoire of naïve individuals and given the inherent high affinity of D29, such antibodies may be preferentially selected during clonal expansion and maintained as memory. Indeed, antibodies that cross-react between prM and E has been isolated from memory B cells in primary and secondary dengue patients [9, 11] . The original immunogens for these cross-reactive antibodies are difficult to determine since they were generated in the course of natural infections, but it would be interesting to identify the epitope and germline of these antibodies and compare the sequences with D29 Fab-IgG. and prM, 10 mg/ml of synthetic peptides corresponding to prM (A) and E (B) was incubated with D29 Fab-IgG. Peptides corresponding to E were tested individually but presented as groups of 5. For all experiments, 2610 6 pfu/ml of DENV2 or imDENV2 was included as control antigen. M1-32: 15 mer peptides corresponding to prM. Pr1-5: .20 mer custom-made peptides covering parts of prM. E1-73: 15 mer peptides corresponding to E. (**p-value,0.005) (TIF)  Lack of Association between CLEC5A Gene Single-Nucleotide Polymorphisms and Kawasaki Disease in Taiwanese Children Background. Kawasaki disease is characterized by systemic vasculitis of unknown etiology. Previous genetic studies have identified certain candidate genes associated with susceptibility to KD and coronary artery lesions. Host innate immune response factors are involved in modulating the disease outcome. The aim of this study was to investigate CLEC5A (C-type lectin domain family 5) genetic polymorphisms with regards to the susceptibility and outcome of KD. Methods. A total of 1045 subjects (381 KD patients and 664 controls) were enrolled to identify 4 tagging single-nucleotide polymorphisms (tSNPs) of CLEC5A (rs1285968, rs11770855, rs1285935, rs1285933) by using the TaqMan Allelic Discrimination Assay. The Hardy-Weinberg equilibrium was assessed in cases and controls, and genetic effects were evaluated by the chi-square test. Results. No significant associations were noted between the genotypes and allele frequency of the 4 CLEC5A tSNPs between controls and patients. In the patients, polymorphisms of CLEC5A showed no significant association with coronary artery lesion formation and intravenous immunoglobulin treatment response. Conclusions. This study showed for the first time that polymorphisms of CLEC5A are not associated with susceptibility to KD, coronary artery lesion formation, and intravenous immunoglobulin treatment response in a Taiwanese population. Kawasaki disease (KD) is characterized by acute, febrile, and systemic vasculitis and was first described by Kawasaki et al. in 1974 [1] . In developed countries, KD is the leading cause of acquired heart diseases in children [2, 3] . KD occurs worldwide and particularly in Japan, Korea, and Taiwan and mainly affects children less than 5 years of age [4] [5] [6] . The most serious complication of KD is the occurrence of coronary artery lesions (CALs) [7, 8] . The prevalence of KD in children younger than 5 years is the highest in Japan, followed by Korea and Taiwan, and lowest in Europe. Previous studies have either failed to identify causative pathogens for KD or reported discrepant results [9] [10] [11] . Therefore, it is possible that a genetic background plays an important role in the pathogenesis of KD.
CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) Journal of Biomedicine and Biotechnology contains a C-type lectin-like fold similar to the naturalkiller T-cell C-type lectin domains and is associated with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells [12] [13] [14] . Signaling via this complex constitutes a significant activation pathway in myeloid cells and plays an important role in immune defense. Recently, it has been demonstrated that CLEC5A acts as a signaling receptor for proinflammatory cytokine release, and that blockade of CLEC5A-mediated signaling attenuates the production of proinflammatory cytokines by macrophages infected with dengue virus [14] . In contrast, it has been demonstrated that MDL-1 stimulation induces a significant amount of RANTES and macrophage-derived chemokine (MDC) production in cooperation with signaling through TLR in mouse myeloid cells [15] . Furthermore, there is ample evidence that activation of peripheral blood monocytes/macrophages [16] [17] [18] , proinflammatory cytokines [16] , and the RANTES gene play a central role during acute KD [19, 20] . A persistent or increased expression of chemokine genes in the convalescent phase in patients is associated with coronary artery lesions [17, 19] . In addition, infiltration by the cells is notable in affected tissues in autopsy cases and in skin biopsy specimens from KD patients [21] .
However, no CLEC5A genetic association with KD has previously been reported. To gain further understanding of the genetic role of CLEC5A in the pathogenesis of KD, the aim of our study was to determine if any CLEC5A SNPs are associated with susceptibility to KD, CAL formation, or IVIG treatment response in Taiwanese children.
All study cases were children enrolled from Chang Gung Memorial Hospital, Kaohsiung Medical Center, between 2001 and 2009, who fulfilled the diagnostic criteria for KD. All patients were treated with IVIG (2 g/kg) and aspirin as per our previous studies [7, 8, 18] . This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Blood samples were collected after informed consent was obtained from parents or guardians. CAL formation was defined as the internal diameter of the coronary artery measuring at least 3 mm (4 mm if the subject was over the age of 5 years) or the internal diameter of a segment at least 1.5 times that of an adjacent segment, as observed in echocardiography [8, 22] . IVIG responsiveness was defined as defervescence within 48 h after the completion of IVIG treatment and no recurrence of fever (temperature > 38 • C) for at least 7 days after IVIG with marked improvement or normalization of inflammatory signs [7, 8] .
Blood cells were subjected to DNA extraction by treating them first with 0.5% SDS lysis buffer and then protease K (1 mg/mL) for digestion of nuclear protein for 4 h at 60 • C. Total DNA was harvested by using a Gentra extraction kit followed by 70% alcohol precipitation as described in our previous report [23] . 
Susceptibility of KD. A total of 381 KD patients and 664 controls were included in this study ( Table 1 ). The distribution of CLEC5A genotypes was in accordance with the Hardy-Weinberg equilibrium for both cases and controls (Table 2) . However, none of the tSNPs was significantly associated with the genotype or allele frequency of the controls or KD patients under 3 genetic models (dominant, recessive, or allelic models) ( Table 2) . 
In this study, 37 patients (9.9%) had CAL formation and 49 patients (13.1%) had resistance to the initial IVIG treatment (Table 1) . However, no tSNPs were significantly associated with genotype or allele frequency in the KD patients with or without CAL formation (Table 3) .
Additionally, the CLEC5A polymorphisms tested in this study failed to show any significant associations with genotype or allele frequency in the KD patients who showed a response to IVIG treatment (Table 4 ). Table 1 ), CAL formation (Supplemental Table 2 ) and IVIG treatment response (Supplemental Table 3 ) in the KD patients. However, none was significantly associated with the phenotype.
The C-type lectin-like super domain (CTLD) family has diverse functions, and in particular, is important in innate immunity including nature killer (NK) function or pathogen recognition [25] . CLEC5A belongs to the Group V "NK cell receptors" family, and MDL-1 expression is upregulated in activated myeloid cells [26] and acts as a signaling receptor for proinflammatory cytokine and chemokine release [14] . Even though a number of reports have demonstrated that KD involves activation of a wide array of immunological elements such as T cells and macrophages [16] [17] [18] 27] , with the subsequent release of several cytokines [28] , only a few reports have addressed the role of lectin in the pathogenesis of KD. Several genetic associations with susceptibility to KD and CAL formation have been reported, but the results are inconsistent [29] [30] [31] [32] . Previous genetic association studies have indicated that the intronic SNP (rs28493229) of ITPKC, 1,4,5-trisphosphate 3-kinase C, reduces gene expression by altering splicing efficiency, and the C allele contributes to immune hyperreactivity in KD patients [29] . Recently, it has been demonstrated that rs28493229 is associated with susceptibility to KD and CAL formation [24, 29] . ITPKC is able to regulate the immune system via calcium-dependent NFAT pathways [29] . Similarly, previous studies have indicated that C-type lectin receptors (CLRs) are critical in the activation of the Syk-mediated NFAT signaling pathway [33] . In addition, CLEC5A has been shown to play a key role in host defense and to be involved in dengue virusmediated disease [14] . This finding suggests CLEC5A may be a potential target protein that involves calcium-dependent immune regulation and contributes to the development of coronary artery lesions. However, we did not find evidence to support a genetic role of CLEC5A in the pathogenesis of KD. Since we picked tagging SNPs from the HapMap database, only the tagging SNPs with a minor allele frequency of more than 10% were selected. Although our tSNP could capture majority of the underlying genetic variances with MAF > 10% across the CLEC5A gene, the rare causal genetic polymorphisms in CLEC5A may not have been detected in this study. Therefore, we cannot rule out or exclude rare causal genetic polymorphisms in CLEC5A. In addition, there are, at least, seventeen groups of CLRs in vertebrates. Indeed, it has been reported that mannose-binding lectin gene polymorphisms are associated with susceptibility to KD [34] and CAL formation [35] . Thus, large-scale DNA sequencing to CLR family is needed to better understand KD.
In conclusion, this study showed for the first time that tSNPs of CLEC5A are not associated with susceptibility to KD, CAL formation, and IVIG treatment response in a Taiwanese population. A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections. Seasonal influenza causes up to 500,000 deaths worldwide each year [1] . Infants, immunocompromised individuals and the elderly are particularly susceptible, with 90% of deaths occurring in the latter group [2] . Influenza viruses can also cause pandemics that, although rare, are recurrent events historically associated with high levels of morbidity and mortality [3, 4, 5, 6] . Preventive vaccination has historically been the most efficient measure of influenza control, but this approach presents important limitations due to the accumulation of antigenic mutations in the virus, known as antigenic drift. Vaccines typically elicit a potent neutralizing antibody response limited to the specific viral strains included in the preparation and to closely related viruses [2] . For this reason, seasonal vaccines need to be annually reformulated based upon the forecasting of viral strains that will circulate in the coming influenza season. Furthermore, influenza vaccines have suboptimal immunogenicity and efficacy in the groups at highest risk of severe disease [7] . Moreover in the case of a pandemic, the use of vaccine is limited by the time required for its development and deployment [8] .
The current therapeutic regimen for influenza A viruses is limited to two classes of drugs: the adamantanes (amantadine and rimantadine) and the neuraminidase inhibitors (oseltamivir and zanamivir). However, the natural and/or acquired resistance to these drugs has been reported [9, 10] . Resistance to adamantanes is prevalent among seasonal and avian influenza A viruses significantly reducing their usefulness [11, 12] . The sudden and widespread emergence of resistance to oseltamivir among prepandemic H1N1 viruses has raised further concerns over the current therapeutic options [13, 14] . Oseltamivir resistance was reported in patients infected with the pandemic H1N1 viruses and highly virulent H5N1 viruses [15, 16] . The resistance to zanamivir is rare [17] , but its use is limited to patients who can actively inhale it, which often excludes young children, impaired older adults or patients with underlying airway disease [14] , that is the groups of patients most vulnerable to serious influenza infection complications.
Alternative strategies are needed to combat the constant threats posed by influenza. One of such strategies may come from passive immunoprophylaxis with monoclonal antibodies (mAbs) recognizing broadly conserved influenza epitopes and endowed with broad-range neutralizing activity [18] .
The most important protective antigen on the surface of influenza virus is HA, whose structure can be divided in two distinct regions: the globular head, responsible for the binding to the sialic acid, and the stem region that contains the fusion peptide and the membrane anchor domain. On the globular head, constituted by the HA1 subunit, lie several epitopes targeted by neutralizing antibodies [18] . However, mAbs recognizing this region are of restricted application due to the antigenic drift that this region encounters [18] . In contrast, the stem region of HA, formed mostly by the HA2 subunit, is relatively conserved among different influenza A subtypes [19] and indeed could represent an universal target for the development of cross-neutralizing monoclonal antibodies.
Several human heterosubtypic neutralizing mAbs, directed against HA stem region and with protective features in animal models, have been recently described [20, 21, 22, 23, 24, 25] . All these mAbs recognize epitopes located in the most conserved region of influenza viruses HA and neutralize influenza viruses by blocking fusion of the viral and the host endosomal membranes. Many of these anti-influenza heterosubtypic neutralizing mAbs utilize the VH1-69 germline gene and bind to a hydrophobic region on the HA stem using their complementary determining region 2 (CDR2).
In this study, we describe a human monoclonal antibody named PN-SIA49 recognizing a highly conserved epitope on the stem region of HA and featuring one of the strongest in-vitro neutralizing activity described so far against a broad spectrum of viruses belonging to different influenza subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenges with H1N1 and H5N1 viruses suggesting its potential as a broad-spectrum monoclonal antibody for treatment of influenza virus infection.
It was previously described that the Fab fragment of PN-SIA49 binds to the stem region of HA and neutralizes all tested H1N1 isolates [26, 27] . The heavy chain variable region of PN-SIA49 uses the VH3-23 gene and is paired with a VL1-39 light chain. The nucleotide sequence homology of PN-SIA49 with the germline sequence is 93.11% for the VH gene and 92.30% for the VL gene, demonstrating its origin from a somatic mutation process.
In this study, in order to better characterize its neutralizing activity, PN-SIA49 was produced as whole IgG1 molecule using the BD BaculoGold System. The whole IgG molecule was tested in fluorescence inhibition assay, infectious foci formation reduction assay and plaque reduction assay against human, swine and avian influenza A viruses belonging to phylogenetic group 1 (H1N1, H2N2, H5N1 and H9N2) and group 2 (H3N2 and H7N2). The results obtained showed that IgG PN-SIA49 has a stronger neutralizing activity compared to Fab PN-SIA49 (Table 1) [26, 27] . Indeed, IgG PN-SIA49 neutralized all tested viruses belonging to group 1, except the virus strain belonging to H9N2 subtype, with a half maximal inhibitory concentration (IC50) ranging between 0.1-1.9 mg/ml (Table 1 and Figure S1 ). On the contrary, PN-SIA49 showed no neutralizing activity against the viruses belonging to group 2 ( Figure S2 ). These data suggest that the epitope recognized by PN-SIA49 is conserved among group 1 viruses.
Therapeutic efficacy of PN-SIA49 against H1N1 and H5N1 challenge in a mouse model
To determine whether the in-vitro neutralizing activity displayed by PN-SIA49 would be predictive of its protective efficacy in-vivo, BALB/c mice were inoculated intranasally with 3 fifty percent lethal dose (LD50) of A/Wilson Smith/33(H1N1) (WS33) or A/Vietnam/1203/2004 (H5N1) (VN04) virus, and were treated 24 hours later with 10, 1 or 0.1 mg/kg of PN-SIA49. An anti-HCV/E2 mAb (e137) [28] was used as control at 10 mg/ kg. PN-SIA49 protected mice from lethal challenge with WS33 in a dose-dependent manner providing 100% protection (6/6) against death in animals that received 10 mg/kg of the antibody and 83.3% protection (5/6) in animals that received 1 mg/kg ( Figure 1C ). Consistent with the in-vitro neutralizing activity, PN-SIA49 at 10 mg/kg afforded 66.6% protection against lethal H5N1 virus challenge ( Figure 1D ) whereas control mice rapidly succumbed to infection by day 8 post-challenge (p.c.). Surviving mice remained healthy and showed minimal body weight loss (maximum weight loss: 7.2% in WS33 group, 14.4% in VN04 group) over the 2-week observation period. At the conclusion of the experiment, The mean body weight loss was 2.2% in the treated mice challenged with VN04 virus ( Figure 1B ) while mice infected with WS33 regained their full body weight ( Figure 1A ).
To gain further insights into the kinetics of viral neutralization in vivo, 4 mice from each group were euthanized on day 4 p.c. and viral titer was determined in whole lung tissues. PN-SIA49 significantly reduced virus titer in the lungs of mice infected with WS33 by approximately 2,000 fold at 10 mg/kg and about 700 fold at 1 mg/kg ( Figure 1E ). A 10-fold reduction in pulmonary virus titer was noted in mice challenged with VN04 virus that received PN-SIA49 at 10 mg/kg ( Figure 1F ).
Taken together, these results indicate that the survival was associated with an important reduction of the virus burden in the lungs of mice treated with PN-SIA49 and that the reduction is concordant with its in-vivo activity.
In order to better define the HA region recognized by PN-SIA49, several approaches were used.
Firstly, the hemagglutination inhibition (HI) activity for PN-SIA49 was evaluated and the resulting HI titre was 2.5 mg/ml and 0.039 mg/ml for VN04 and WS33 virus, respectively.
Secondly, to evaluate if PN-SIA49 recognizes an epitope on the HA stem region, a competition assay on A/Puerto Rico/8/1934-HA (A/PR/8/34-HA) human epithelial kidney HEK293T transfected cells was performed between PN-SIA49 and the commercially available mouse monoclonal antibody C179 (Takara Bio inc., Otsu, Shiga, Japan), which binds to an epitope on the HA stem region [29] . The results obtained showed that PN-SIA49 completely inhibited C179 binding to the A/PR/8/34-HA ( Figure  S3 ).
Further evidence that PN-SIA49 binds to an epitope on the HA stem region is given by lack of protease susceptibility of the HA at low pH in the presence of PN-SIA49. Exposure to low pH followed by trypsin digestion results in degradation of HA. In contrast, when the HA is pre-treated with PN-SIA49, most HA is retained in a protease resistant, pre-fusion form ( Figure 2 ).
Taken together, these preliminary data suggest that the epitope recognized by PN-SIA49 is localized in the HA stem region, but in close proximity of the HA globular head.
Based on the PN-SIA49-C179 competition assay results, a large panel of HA mutants carrying an alanine substitution were generated [29, 30, 31, 32] . The binding of PN-SIA49 to these mutants was then evaluated by FACS analysis. Data obtained revealed that the binding of PN-SIA49 was decreased by His25Ala, Asn336Ala, Pro338Ala mutants on HA1 and Me-t360Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala, Va-l395Ala, Asn396Ala, Glu400Ala mutants located on HA2 (sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709). Importantly, these residues are extremely conserved among viruses belonging to H1N1 subtype spanning from 1918 and 2009 and also highly conserved in H2N2 and H5N1 subtypes ( Figure 3 ). All the other mutations did not have any effect on the antibody PN-SIA49 binding to the HA ( Figure 3 ). To exclude the possiblity that the reduced PN-SIA49 binding to HA was due to reduced expression of HA on the cell surface, we performed a FACS analysis in which wild-type HA and mutants HA were stained with a mouse anti-influenza A HA (H1 subtype) monoclonal antibody. This antibody was directed against a linear epitope in order to evaluate the expression level for each HA on cell surface. As shown in figure S4 , the HA mutants are expressed on the cell surface at a similar levels to that of wild type HA.
Consistent with the great phylogenetic distance, the amino acidic difference in positions 25, 360 and 362 between the tested viral strains belonging to H1N1, H2N2, H5N1 subtypes and the viral strains belonging to H9N2 subtype may at least partially explain the lack of neutralizing activity against the H9N2 strain.
Based on the results obtained from the alanine scanning study, an in-silico analysis on the HA crystal structure of A/PR/8/34 (PDB ID code 1RU7) was carried out. The analysis confirmed that the residues identified lie on the stem region of HA, that they belong to the HA1 and HA2 subunits and that they are exposed on the surface of the HA molecule ( Figure 4 ).
In this study, we characterize a human mAb, IgG PN-SIA49, directed against influenza viruses and previously described as Fab fragment [26, 27] . IgG PN-SIA49 neutralizes a broad spectrum of viruses belonging to different influenza subtypes (H1, H2 and H5) and is characterized by the lowest mean IC50 values described to date for a heterosubtypic mAb [20, 21, 22, 23, 24, 25] against the influenza subtypes tested in this study (Table S1 ). Furthermore, IgG PN-SIA49 is endowed with a stronger neutralizing activity compared to its monovalent molecule (Table 1) . Indeed, it is well documented in the literature that bivalency of IgG molecule may be an essential feature for the biological activity of a mAb, mostly due to an increase in antibody avidity [33, 34, 35, 36] . The increased avidity may also play an important role in the prophylactic and therapeutic application of an antibody, allowing the administration of smaller amount compared to the Fab fragment. Additionally, the whole IgG molecule has a longer half-life than its fragments, with a consequent prolonged effect of the molecule. For all these reasons, we used IgG PN-SIA49 to treat mice infected with H1N1 and H5N1 viruses and data obtained show that PN-SIA49 protects mice from H1N1 and H5N1 virus lethal challenge suggesting its potential application in treatment of influenza virus infections.
Most of the anti-influenza heterosubtypic neutralizing mAbs described thus far, derived from the VH1-69 germline, bind to a conserved epitope in the HA stem region present only on group 1 influenza A viruses. The binding is mainly through highly hydrophobic amino acidic residues in the heavy chain CDR2 [20, 21, 22, 23, 24, 25] . Instead, the heavy chain variable region of PN-SIA49 results from VH3-23 gene rearrangement with the D3-3 and JH6 gene segments demonstrating that, despite the preferential usage of VH1-69 in the heterosubtypic response to influenza HA [20] , an in-vivo heterosubtypic protection may be conferred also by non VH1-69 derived Abs.
PN-SIA49 recognizes a novel broadly neutralizing epitope on HA stem region, which is highly conserved among group 1 subtypes that have been confirmed in humans. In particular, this region is broadly shared among H1N1 isolates spanning from 1918 to 2009, H2N2 subtype responsible for the 1957 pandemic, and highly pathogenic and potentially pandemic H5N1 influenza virus. Finally, PN-SIA49 is protective in mice when given after lethal challenge with either H1N1 or H5N1 virus. This suggests its great potential as broad-spectrum monoclonal antibody for in vivo treatment of influenza virus infections.
Taken together, these data underline the importance of PN-SIA49 for the development of anti-influenza strategies based on passive immunization. Further studies will be necessary to define the most effective prophylactic and therapeutic administration protocols. Finally, these data and a more detailed definition of the epitope recognized by PN-SIA49 could also be useful to develop new vaccination strategies able to elicit a humoral immune response directed against key regions of the influenza HA protein.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of . Amino acidic sequence conservation in hemagglutinin groups and subtypes at the region bound by PN-SIA49. Circles below residues indicate PN-SIA49 percentage binding to each HA alanine mutant compared to binding to the wild-type HA: red 25% binding; yellow 50-75% binding, blue 100% binding. Sequence numbering is based on H1N1 A/Puerto Rico/8/1934 coding region (GenBank accession number ABO21709). Subtypes that can be neutralized by PN-SIA49 are indicated with a green '+' on the left, while the ones that can not be neutralized are indicate with a red '2'. a Recombinant HA from H1N1 A/South Carolina/1/1918 pandemic strain was previously shown to be bound by PN-SIA49 [26, 27] . b H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA49 as Fab fragment [26, 27] . doi:10.1371/journal.pone.0034415.g003
We previously described the molecular cloning of a human monoclonal antibody Fab fragment named PN-SIA49 [26, 37] . An anti-influenza A antibody directed against H1N1 subtype HA, named RB62, and an anti-HCV E2 glycoprotein antibody, named e137 [28] , produced and purified with an identical procedure were used as controls in all experiments.
The following human H1N1 and H3N2 reference strains were acquired from the American Type and Culture Collection , 100 mg/ml of streptomycin (Gibco Invitrogen, Carlsbad, CA, USA) and 2 mg/ml TPCK-trypsin (Roche Applied Science). The A/swine/Parma/1/97 isolate was analogously grown on Newborn Swine Kidney (NSK) cells, kindly provided by the Zooprophylactic Institute of Brescia, Italy. At 80% confluence, cells in MEM supplemented with 2 mg/ml serum-free TPCK-trypsin, were infected with each strain at a MOI of 0.001. After 1 hour of infection, cells were washed with phosphate buffered saline (PBS); MEM supplemented with 2 mg/ml TPCK-trypsin was then added and cells were incubated at 37uC in 5% CO2 atmosphere. Cells were observed daily to monitor the cytopathic effect and, usually after 72-96 hours, the supernatant was collected, centrifuged at 1000 rcf for 10 minutes to eliminate cells debris and filtered with 0.22 mm filters (Millipore, Billerica, MA, USA). The supernatant was then aliquoted and stored at 280uC as cell-free virus. well). Serial dilutions, 10 mg/ml-0.03 mg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37uC with 100 median tissue culture infective doses (TCID50) of virus. Following incubation, 100 ml of the mix antibody-virus were added to the cells and incubated for another hour at 37uC in 5% CO2. At the end of this incubation, cells were washed with PBS and 100 ml of MEM TPCK-Trypsin (2 mg/ml) were added in each well. Cells were incubated for 7 hours at 37uC in 5% CO2 and then washed with PBS, fixed and permeabilized with ice-cold ethanol. Cells were incubated with anti-influenza A mouse antibody (Argene, Shirley, NY, USA) for 30 minutes at 37uC in a humid chamber. The cells were then washed with PBS and incubated for 30 minutes at 37uC in a dark humid chamber with a FITC-conjugated secondary antibody (Argene, Shirley, NY, USA). Nuclei staining was obtained with Hoechst 33342 (Sigma Aldrich). An infection control without antibody was included, as well as a negative control with the anti-HCV/E2 antibody e137. Each neutralization assay was performed in triplicate and repeated in two different sessions.
The neutralization activity for each antibody concentration was expressed as the percentage reduction of fluorescent nuclei compared with the nuclei count in the infection control. Nuclei counting was performed by using the GE Healthcare's IN Cell Analyzer 1000, an automated epifluorescence based microscope system. The neutralization curves were then fit by non-linear regression with the GraphPad Prism software, allowing IC50 calculation.
The A/PR/8/34 (H1N1) and A/Milan/UHSR1/2009 (H1N1) viruses were also tested in plaque reduction assay as previously described [26] . Briefly, neutralizing assays were carried out in 6 wells plates using MDCK cells (56105 cells/well). Two dilutions, 1-0.1 mg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37uC with 100 TCID50 of virus. Following this incubation, 1 ml of each virus-antibody mix was added on MDCK monolayer and the plate was incubated 1 hour at 37uC in 5% CO2. Then, the medium was removed and the monolayer washed twice with PBS. Two ml of MEM-agarose 0.8% supplemented with penicillin (50 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), streptomycin (100 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM) (Gibco Invitrogen, Carlsbad, CA, USA) and trypsin (2 mg/ml) (Roche Applied Sciences) were added to each well and the plates were incubated 48 hours at 37uC in 5% CO2. After this incubation, the agarose medium was removed from each well and 1 ml of 70% methanol-crystal violet 1% (w/v) was added to each well at room temperature. Finally, the wells were washed with tap water and dried. An infection control without antibody was added as well as a negative control with anti-HCV/E2 e137 mAb. The neutralization was determined counting the PFU reduction in presence of antibodies compared to the infection control.
Infectious foci formation reduction assay. The following H1N1, H2N2, H3N2, H5N1, H7N2 and H9N2 viruses were . Each viral isolate was titrated to establish working dilution that produces 15-30 foci forming units per well in 96 tissue culture plates. Neutralizing assays were carried out in 96 wells plate using MDCK/SIAT-1 cells. Serial dilutions, 30 mg/ml-0.37 mg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37uC with the subset of viruses. Following this incubation, 100 ml of the antibody-virus mix was added to the cells and incubated for another hour at 37uC in 5% CO2. At the end of this incubation, the cells were washed twice in PBS and 100 ml of virus growth media containing 2 mg/ml of TPCK treated trypsin was added. Cells were incubated for 12-16 hours at 37uC in 5% CO2 and then washed with PBS, fixed and permeabilized with ice cold methanol/acetic acid (95:5) for 30 min at 220uC. Cells were incubated with anti-NP antibodies (Millipore, Billerica, MA, USA) for 30 minutes at 37u. The cells were then washed and incubated for 30 minutes at 37uC with a mouse HRP-conjugated secondary antibody. True Blue chromogenic substrate (KPL) was used to count the number of foci.
Female BALB/c mice were purchased at 6 to 8 weeks of age from Charles River Co. (Wilmington, MA). All mice were maintained in specific pathogen-free barrier facilities. All animal experiments and procedures conformed to protocols approved by the Centers for Diseases Control and Prevention (CDC), Atlanta, GA, USA.
For each virus, four groups of 10 mice were inoculated intranasally with 3 LD50 of A/Wilson Smith/33 or A/ Vietnam/1203/2004 virus in a 50 ml volume. At 24 h after inoculation, graded doses (10, 1, 0.1 mg/Kg) of PN-SIA49 or the control antibody (e137, 10 mg/Kg) were administrated to mice by intraperitoneal injection in a final volume of 0.2 ml. A subset of six mice in each group were weighed on the day of virus challenge and then observed and weighed every 2 days for 2 weeks after inoculation. Mice that lost more than 25% of their initial body weight were euthanized.
A subset of four animals treated with mAbs were euthanized on day 4 after inoculation, and whole lungs were homogenized in 1 ml of sterile PBS. Virus titers in lung tissue homogenates were determined by plaque titration in MDCK cell monolayer cultures.
Hemagglutination (HI) assay. HI tests using mAb PN-SIA49 or e137 control antibody against live WS33 and VN04 viruses were performed according to standard protocols [38] . Briefly, serial dilutions of purified mAbs in PBS were performed from initial concentration of 5 mg/mL. Positive and negative control ferret sera were diluted initially 1:10 in receptor-destroying enzyme from Vibrio cholerae (Denka Seiken, Tokyo). Serial dilutions of control sera or mAbs were pre-incubated with 4 HA units of virus per well. For WS33 virus, turkey red blood cells (RBCs) were added to a final concentration of 0.5%, whereas horse RBCs were used at a 1% suspension for VN04 virus. Normal ferret serum gave a value of less than 10. Specific HI activity of mAbs was calculated as the lowest concentration of mAb that displayed HI activity.
Protease susceptibility assay. Each reaction contained 3 ml of the anti-influenza vaccine season 2011-2012 (InflexalV-Crucell), which contains 30 ng of A/California/7/09 (H1N1) HA or 3 ml of the anti-influenza vaccine season 2011-2012 combined with 2 fold molar excess of PN-SIA49. Titron X-100 was added to prevent aggregation of the post-fusion HA. The pH was lowered in all samples except controls using citric acid 0.1 M pH 3. Reactions were mixed, briefly centrifuged and incubated at 37uC for one hour. After incubation, reactions were equilibrated to room temperature and the pH was neutralized by addition of 1 M Tris, pH 9. The actual pH reached was determined in parallel using larger buffer volumes without protein. Trypsin was added to all samples except controls at a final ratio of 1:25 by mass and samples were digested overnight at 37uC. Non-reducing SDS buffer was added to each reaction. Samples were boiled for ,2 minutes and loaded on a nonreducing 4-15% polyacrilamide pre-casted gel (Biorad, Italy). After running, samples were transferred on a PVDF membrane (PerkinElmer, Belgium) for 2 hours at 350 mA. The membrane was then blocked with 5% not fat milk in PBS-Tween20 0,1% (PBST) for 1 h at room temperature and then washed three times with PBST. PN-SIA28, a human anti-HA monoclonal antibody recognizing HA0 [39] , was used as primary antibody at 1 mg/ml in 5% not fat milk-PBST. The membrane was incubated for 1 hour at room temperature and then was washed three times with PBST. Secondary anti-human antibody was added and incubated for 1 h at room temperature. After incubation the membrane was washed, the substrate solution (SuperSignalH West Pico Chemiluminescent Substrate, PIERCE) was added and incubated for 2 min.
To determine the pH required to convert the HA to the postfusion form, pH titrations using the assay describing above was performed. Samples were exposed to a range of pH conditions (pH 4.5, 4.9, 5.3, 5.7, 6.1, 6.5, 7 and 8), neutralized and processed as described above.
A/Puerto Rico/8/1934 (H1N1) hemagglutinin (A/PR/8/34-HA) was amplified as previously described [26, 37] using the following PCR primers:
APR834_fw:CACCATGAAGGCAAACC-TACTGGTCCTGTTATGTG.
APR834_rev:TCAGATGCATATTCTGCACTGCAAA-GATCCATTAGA.
The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, HA alanine mutants were generated using Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 A/PR/8/34-HA mutants were generated (His25Ala, His45Ala, Thr315Ala, Asn336Ala, Ile337Ala, Pro338Ala on the HA1 subunit and Trp357, Thr358, Gly359, Met360, Ile361, Asp362, Gly363, Trp364, Thr384, Ile388, Thr392, Val395, Asn396, Glu400 on the HA2 subunit. Sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709).
The binding activity of PN-SIA49 was assayed using full-length wild type and mutants HAs. Human epithelial kidney HEK293T cells (ATCC CRL-1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) (16106 cells/well) with 4 mg of pcDNA 3.1D/V5-His-TOPO vector containing the HA nucleotide sequences described above. After centrifugation and fixation with 4% paraformaldehyde for 15 minutes at RT, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA49 or conformational controls for H1N1 (RB62) at 10 mg/ml. Additionally, the isotype control, e137 (10 mg/ml) was introduced as well as untransfected cells and a mouse anti-influenza A HA (H1 subtype) monoclonal antibody (GeneTex Inc., Irvine, CA, USA) directed against a linear epitope to evaluate the transfection efficiency and the expression level for each HA. The cells were then washed with PBS and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) antibody. Afterwards, the cells were washed with PBS and analyzed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA49 to the different HA-mutants was then expressed as a binding percentage compared to wild-type. The data showing the PN-SIA 49 binding decrease between H1N1 wild type HA and H1N1 HA mutants, were obtained normalizing each PN-SIA 49 binding value to corresponding anti-H1 expression control values.
For the competition assay, serial dilutions of PN-SIA49 were used in combination with a fixed concentration (1 mg/ml) of mouse monoclonal antibody C179 (Takara Bio inc., Otsu, Shiga, Japan) which binds to an epitope on the HA stem region [29] .
For sequences analysis the following software packages were used: SeqScape (Applied Biosystems), ClustalX (Toby Gibson), Bio Edit (Tom Hall, Ibis Therapeutics) and Treeview (GubuSoft). For molecular visualization and rendering UCSF Chimera package from the Resource for Biocomputing Visualization and Informatics at University of California, RasMol (Roger Sayle), Jmol (Jmol: an open-source Java viewer for chemical structures in 3D. http:// www.jmol.org/), Cn3D (United States National Library of Medicine, NLM) were used. Finally for data analysis and graphical editing GraphPad Prism was used.
Figure S1 Neutralization assays against group 1 influenza viruses. Dose-response curve fit nonlinear regression is reported for IgG PN-SIA49 against neutralized group 1 influenza viruses. (A) Results from fluorescence inhibition assays, (B) plaque reduction assays and (C) infectious foci formation reduction assays. Data from at least two different experiments for each virus are reported. Each point was performed in triplicate. (PDF) Figure S2 Influenza hemagglutinin unrooted phylogenetic tree of all the viral strains tested in neutralization assays with PN-SIA49. Viral isolates belonging to group 1 and group 2 are divided into two different boxes. Subtypes that can be neutralized by PN-SIA49 are indicated with a green '+', while the ones that cannot be neutralized are indicate with a red '2'. As reported in the text, PN-SIA49 is able to neutralize all of the group 1 viruses tested in this study except for the H9N2 strain. No neutralizing activity was detected against the H3N2 viruses tested. * The recombinant HA from A/South Carolina/1/1918 (H1N1) pandemic strain was previously shown to be bound by PN-SIA49 [26, 27] . # H1N1 A/New Caledonia/20/1999 was previously shown to be neutralized by PN-SIA28 as Fab fragment [26, 27] . (PDF) Figure S3 C179/PN-SIA49 competition assay. Graphic representation of cell staining and flow cytometric analysis of HEK293T cells transfected with the pcDNA 3.1D/V5-His-TOPO vector containing the HA-A/PR/8/34 were performed. Serial dilutions of PN-SIA49 were used in combination with a fixed concentration (1 mg/ml) of C179 (blue line). A monoclonal antibody directed against the HA globular head was used as competition negative control (pink line). (PDF) Figure S4 HA mutants that determine a decrease of PN-SIA49 binding to HA are expressed at the same level of wild type HA on cell surface. FACS curves showing the binding of anti-H1N1 HA antibody (directed against a linear epitope) to untransfected cells, HA wild-type and HA-mutants. White and red curves represent, for each graph, respectively the binding of anti-HA expression control to untransfected cells and wild type H1N1-HA. The different colour curves represent the different mutants. (PDF)  Web-based Investigation of Multistate Salmonellosis Outbreak We investigated a large outbreak of Salmonella enterica serotype Javiana among attendees of the 2002 U.S. Transplant Games, including 1,500 organ transplant recipients. Web-based survey methods identified pre-diced tomatoes as the source of this outbreak, which highlights the utility of such investigative tools to cope with the changing epidemiology of foodborne diseases. T he epidemiology of foodborne illnesses is influenced by a variety of factors, some of which have changed dramatically in recent years. The increased availability of preprocessed foods and the improved survival of persons with immune defects have affected the sources and nature of foodborne illness (1) (2) (3) (4) . Increased mobility of Americans through interstate travel has complicated the identification and investigation of outbreaks. New technologies for outbreak investigation have the potential to greatly assist public health officials in successfully managing these changing factors. We describe an outbreak of Salmonella enterica serotype Javiana infections affecting a large group of geographically dispersed organ transplant recipients. The prompt and successful investigation of this outbreak was facilitated by the use of Web-based surveys.
On To identify additional cases, state health departments were asked to report any S. Javiana isolates with a PFGE pattern indistinguishable from the outbreak strain. To develop hypotheses about potential sources of infection, we conducted in-depth telephone interviews with several persons who were identified with culture-confirmed illness.
On the basis of interview results, we conducted a Webbased cohort study among Transplant Games attendees to identify risk factors for infection by using eQuest, a software package developed by the Centers for Disease Control and Prevention (CDC) that allows rapid development of Web-based surveys (5) . Using email addresses provided to us by the Transplant Games organizers, we electronically distributed a message on July 20 to attendees (including athletes and spectators), requesting that they complete an outbreak survey. We included information about salmonellosis and its treatment and provided a link in the email to the secure Web site containing the outbreak survey. In each survey respondent's household, we collected information for a single person visiting Orlando, regardless of whether he or she had been ill. A case was defined as fever or diarrhea with onset between June 25 and July 7 in a person who visited Orlando. Submitted answers were automatically stored in a secure electronic database and linked only to a random survey number.
To identify the specific food item responsible for illness, we performed a Web-based case-control study. On July 31, we distributed a survey containing detailed questions about specific food items available in theme park A to persons who had responded to the first survey. Casepatients were questioned about food items eaten in the 3 days before illness onset. Controls were defined as well survey respondents, and all were questioned about the middle 3 days of the Transplant Games (June 26-28).
Plant X, the processing plant that supplied tomatoes to theme park A, was inspected on August 13. Molecular subtyping of confirmed S. Javiana isolates was performed at state public health laboratories (6) . Diced Roma tomatoes from unopened boxes that had been stored frozen at theme park A were cultured at the Florida State Public Health Laboratory.
Statistical analyses were conducted by using SAS software version 8.2 (SAS Institute Inc., Cary, NC, USA) to calculate odds ratios (OR) and 95% confidence intervals (CI). Multivariable logistic regression analyses were conducted for variables that were significantly associated with illness.
Through laboratory surveillance, 21 additional S. Javiana infections with indistinguishable PFGE patterns were identified in 10 states, for a total of 23 identified culture-confirmed cases. Dates of illness onset were from June 24 to July 8. Of 22 patients for whom travel information was collected, 19 reported visiting theme park A in the last week of June; 16 visited theme park A but did not report any contact with the Transplant Games, which suggests a true outbreak of S. Javiana infections among visitors to the theme park.
An electronic link to the Web-based cohort study survey was distributed on July 20, 2002, to 1,100 Transplant Games attendees. Among these 1,100, we received survey responses from 369 persons (34%) in 42 states; 80% responded within 48 hours. Of the 369, a total of 82 (22%) reported illness and 41 (53%) were female. The median age of ill respondents was 47 years (range 4-71 years); 48 (59%) were transplant recipients. Dates of symptom onset were June 26-July 7 (Figure) . Predominant symptoms included diarrhea (93%), abdominal pain (79%), and fever (51%). Three respondents (4%) had been hospitalized. No deaths were reported to the organizers of the Transplant Games or CDC. Among ill respondents, 75 (91%) reported eating food items at specific food courts in theme park A.
The Web-based case-control study was distributed on July 31 to the 369 persons who responded to the first survey. By August 2, a total of 222 persons (60%) responded. Of 217 valid responses, 41(19%) were ill persons who met the case definition; the remaining 176 were healthy controls. Ill persons were significantly more likely to report eating dishes containing diced Roma tomatoes than were well persons (44% of ill vs. 15% of well, OR = 4.3, 95% CI 2.1-9.1). Other food items that were significantly associated with illness on univariate analysis were dishes containing shredded iceberg lettuce (OR = 3.7, 95% CI 1. 8-7.4) , pre-shredded cheddar cheese (OR = 2.9, 95% CI 1.5-5.9), fresh ground beef (OR = 3.0, 95% CI 1.4-6.4), and pre-sliced beefsteak tomatoes (OR = 4.6, 95% CI 1.1-19.4) ( Table) . In multivariable logistic regression modeling, only diced Roma tomatoes remained independently associated with illness at the 0.05 significance level.
Diced Roma tomatoes were supplied to theme park A from plant X, where whole Roma tomatoes were mechanically diced and washed in a manually chlorinated recycled water tank. Levels of chlorine in the tank were variable (≈1.5-3.5 ppm free chlorine), providing potential opportunity for the amplification of any existent microbial contamination. Review of invoices showed that diced Roma tomatoes used in food courts patronized by persons with outbreak-related illness were processed at plant X from June 20 through July 3. No diced tomatoes from the implicated lots were available for testing. Microbiologic evaluation of an unopened box of plant X diced Roma tomatoes processed on July 12 indicated the presence of fecal coliforms (150-1,000 CFU/g).
The nature of this outbreak highlights several changing features of foodborne disease epidemiology, including the enhanced mobility of persons through air travel, an increasing reliance on pre-processed foods, and an expanding immunocompromised population at risk. Through a Web-based investigation, we were able to rapidly identify the source of this outbreak and inform an immunocompromised population of its potential risk for illness. Our approach allowed us to contact and question several hundred geographically dispersed persons in a matter of days. Survey respondents' answers were automatically stored in a secure electronic database, eliminating the need for data entry. With the development of questionnaire templates, a public health official could select sets of questions and precoded answers from pull-down menus and modify them to design an outbreak-specific, Web-based questionnaire that is automatically linked to an electronic database (5) . These methods make it increasingly possible to design and post a Web-based investigative tool for wide distribution within hours.
Our investigation has several limitations. Use of a Web-based investigation tool limited responses to only those Transplant Games attendees with known email addresses and Internet access. The initial response rate to our survey was only 34%; households with ill persons may have been more likely to respond to our Web-based survey. However, most (>75%) respondents to both surveys were from households in which no one had experienced illness, which provided us with a sufficient number of responses from both well and ill persons to identify the source of the outbreak. Hospitalized and severely ill persons may have been too sick to respond to the survey or may have been unable to access the Internet, which limited our ability to calculate accurate hospitalization or attack rates among persons attending the Transplant Games. Although isolation of S. Javiana from plant X diced tomatoes would have strengthened our findings, the tomatoes tested were processed well after the outbreak period, when levels of contamination may have differed considerably.
As use of the Internet becomes more widespread for participation in regional, national, and international conferences, groups, and listservs, electronic mail cohorts are becoming more commonplace. The development of Webbased public health investigative tools can facilitate future investigations of outbreaks affecting geographically dispersed persons who may be part of an electronic mail cohort. A Web-based approach to data collection can also play a critical role in rapidly sharing data in outbreaks involving multiple jurisdictions (7) . The growing use of Web-based technologies in public health investigations will have to be balanced with the need to protect the privacy of personal information in the online environment (8, 9) . The careful application of emerging technologies and conventional epidemiologic techniques can help public health officials effectively cope with the multitude of changing factors that shape public health in the United States. Dengue Fever, Hawaii, 2001–2002 Autochthonous dengue infections were last reported in Hawaii in 1944. In September 2001, the Hawaii Department of Health was notified of an unusual febrile illness in a resident with no travel history; dengue fever was confirmed. During the investigation, 1,644 persons with locally acquired denguelike illness were evaluated, and 122 (7%) laboratory-positive dengue infections were identified; dengue virus serotype 1 was isolated from 15 patients. No cases of dengue hemorrhagic fever or shock syndrome were reported. In 3 instances autochthonous infections were linked to a person who reported denguelike illness after travel to French Polynesia. Phylogenetic analyses showed the Hawaiian isolates were closely associated with contemporaneous isolates from Tahiti. Aedes albopictus was present in all communities surveyed on Oahu, Maui, Molokai, and Kauai; no Ae. aegypti were found. This outbreak underscores the importance of maintaining surveillance and control of potential disease vectors even in the absence of an imminent disease threat. D engue viruses cause a wide range of illness, including dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Four dengue serotypes, known as DENV-1, -2, -3, and -4, can cause severe and fatal disease. Dengue typically occurs in tropical and subtropical areas in the world and is transmitted by Aedes mosquitoes; Aedes aegypti is the principal vector worldwide (1) . DF and DHF are the most important arboviral diseases of humans; ≈50-100 million dengue infections and several hundred thousand cases of DHF occur annually (2) .
The first large-scale dengue fever epidemic in Hawaii occurred in the late 1840s; a second outbreak occurred at the turn of the century, with an estimated 30,000 cases (1, 3) . During those periods Ae. aegypti was widespread in Hawaii (4) . Epidemic dengue occurred again on Oahu in 1943 to 1944, when 1,498 infections were reported, mostly in urban areas of Honolulu (5) . Ae. albopictus had been introduced into Hawaii at the beginning of the century, and by 1940 it was the dominant day-biting Stegomyia mosquito species in the islands (4, 5) .
After the Second World War, no confirmed autochthonous dengue infections were reported in Hawaii. Nevertheless, dengue illnesses were occasionally identified among travelers to Hawaii who had been infected overseas. The annual number of imported cases was low, with 20 infections recorded during the 10-year period from 1991 through 2000 (P. Effler, unpub. data).
On September 12, 2001 , the Hawaii State Department of Health (HDOH) received a call from a physician in Hana, Maui, who had seen a patient with febrile illness and rash 1 week earlier. The physician indicated that several of the patient's family members had become symptomatic; none had a history of recent foreign travel. On investigation by HDOH staff, dengue fever was suspected, and clinical specimens were collected and forwarded to the Centers for Disease Control and Prevention (CDC) for diagnosis. On September 21, CDC confirmed recent dengue infection in the index patient. We report the results of an investigation into the first outbreak of dengue fever in Hawaii in 56 years.
From September 23 to 28, 2001 , HDOH contacted all licensed physicians in the state by email or facsimile to request that any patient with a denguelike illness (DLI) be tested for dengue, regardless of travel history. DLI was defined as fever or chills plus 2 or more of the following symptoms (6): myalgia, headache, arthralgia, eye or retroorbital pain, rash, or hemorrhagic manifestation (e.g., petechiae, hematuria, hematemesis, menorrhagia, melena).
On September 24, 2001 , active surveillance was established at 51 clinical settings across the state. All acute-care hospitals and major clinics were contacted daily to determine the number of clinically compatible illnesses seen in the previous 24 hours and to arrange for laboratory evaluation of suspected cases. Although HDOH recommended dengue testing only for patients meeting DLI criteria, it was performed whenever requested by a physician.
HDOH staff interviewed persons with suspected dengue infection to obtain symptom and travel histories. Visits to residences and work sites were conducted. Patients' household contacts or co-workers with a history of illness were urged to be tested for dengue.
All clinical laboratories in Hawaii were asked to report any requests for dengue diagnostic testing and to forward aliquots of serum samples obtained for dengue testing to the HDOH State Laboratories Division. Laboratory analyses to detect anti-dengue immunoglobulin (Ig) M and IgG and to isolate and identify the virus were performed by methods previously described (7) (8) (9) (10) (11) (12) .
RNA was extracted by using QIAmp Viral RNA Mini kits (Qiagen GmbH, Hilden, Germany). Sequencing was performed by using the Taq DyeDeoxy Terminator Cycle Sequencing kits (Applied Biosystems, Foster City, CA, USA). Sequencing products were cleaned by using agarose gel electrophoresis and silica gel adsorption (Qiagen PCR purification columns) and analyzed on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Sequences were assembled and aligned with Lasergene software (DNAStar, Madison, WI, USA), and phylogenetic trees were generated with PHYLIP v. 3.5c (University of Washington, Seattle, WA, USA).
Laboratory-positive recent dengue infection was defined as a person who had 1) dengue virus isolated from serum, 2) a positive dengue IgM antibody test result, or 3) a positive IgG antibody test result in a person initially tested for dengue >60 days after onset of DLI and who was epidemiologically linked to another person with recent dengue infection identified by virus isolation or positive IgM serologic test result.
Persons were classified as negative for dengue infection if they had at least 1 specimen collected 6-60 days after illness onset that was IgM negative or a first specimen collected >60 days after illness onset that was IgG negative. Persons were classified as indeterminate for dengue infection if all specimens were collected <6 days after illness onset and were negative for virus isolation and for anti-dengue IgM. Imported dengue was defined as illness in a person with laboratory evidence of recent dengue infection and a history of international travel within 14 days of illness onset.
During the outbreak investigation, a CDC entomology team conducted spot checks of potential breeding sites in 29 communities (at least 20 sites per community) on all islands except Hawaii and Lanai. From March to May 2002, HDOH vector-control staff placed ovitraps at 295 sites throughout the state; local vector-control staff relied on prior experience to select sites with known populations of day-biting mosquitoes. In both surveys, larvae were collected from breeding sites and identified to species. In the second survey, eggs were reared to the fourth larvae or adult stage before speciation. Adult mosquitoes attracted to humans were also captured and identified at many of these sites; in the outbreak areas; landing counts were obtained by recording the number of mosquitoes landing on a stationary person during a 5-minute period.
Univariate analyses were conducted by using EpiInfo Version 6.4c (CDC, Atlanta, GA, USA). A difference in proportions was considered significant if the chi-square p value was <0.05.
From September 12, 2001 , to April 30, 2002, a total of 1,644 persons in Hawaii without a history of recent foreign travel were tested for possible dengue infection. Of these, 122 (7%) had laboratory evidence of a recent dengue infection: 15 (12%) were positive by virus isolation; 99 (81%) had anti-dengue IgM; and 8 (7%) had a history of DLI, anti-dengue IgG, and an epidemiologic link to a patient with recent infection (Table 1) . Testing was indeterminate for 422 (26%) persons, and the remaining 1,100 (67%) did not have dengue infection. The median age was 41 years (range 1-77), 35 years (range 0-89), and 29 years (range 0-81) for persons who were laboratory positive, negative, and indeterminate for dengue infection, respectively.
Autochthonous dengue infections were identified on 3 of 6 islands (Table 1) . Exposures on Maui, Oahu, and Kauai accounted for 76%, 21%, and 3% of all recent dengue infections, respectively. Eighty (66%) of the laboratory-positive infections were from persons who stayed in the Hana area of Maui, an area with <2% of the island resident population (Figure 1 ). On Oahu, 20 (77%) of the infections occurred among residents of 2 nearly adjacent communities on the windward side with a combined population of 25,709 (<3% of the island's total). The heavily affected areas of Maui and Oahu both have thick vegetation and heavy precipitation (average annual rainfall >177 cm/year, 4 times the annual rainfall in Honolulu).
The outbreak spanned >8 months, with a peak incidence in late September 2001. (Figure 2 ) The first suspected dengue illness was reported with an onset date September 5, 2001; subsequent investigations identified an additional 31 laboratory-positive patients with illness onset before that date, and the earliest was May 27, 2001 .
Of laboratory-positive cases, 89% met the clinical criteria for DLI (Table 2) . Patients with recent dengue infection reported a greater number of symptoms than those who did not have dengue. One or more hemorrhagic manifestations were reported in 42 (34%) persons with dengue infection. Myalgia, chills, arthralgia, and rash were significantly more common among patients with laboratory-positive dengue infection than in persons with negative or indeterminate results.
No cases of DHF or DSS, as defined by the World Health Organization, were reported, and no deaths occurred (14) . Three patients with laboratory-positive dengue infection were hospitalized for their illness.
Eighty-one (66%) of the recent infections were initially reported by physicians treating acutely ill patients, while the remaining 41 (34%) were identified through HDOH field investigations. Thirteen household clusters accounted for 53 (43%) of the 122 patients.
One-hundred and fifteen (95%) of the 122 persons with laboratory-positive infection were residents of the state of Hawaii. All 7 visitors with dengue stayed at rental properties in the Hana area of Maui. Another 70 nonresidents with possible dengue infections who visited Hawaii during the outbreak were reported to HDOH; 30 of these nonresidents were serologically tested, and results for all were negative.
From January 1, 2001, to April 30, 2002, a total of 43 cases of imported dengue infection were reported to HDOH ( Figure 3) . Oahu had the greatest number of imported infections (31 infections), followed by Maui (6 infections), Hawaii (4 infections), and Kauai (2 infections). Eighteen (42%) of the imported dengue infections were from the Society Islands, 13 (30%) were from American or Western Samoa, 7 (16%) were from the Philippines, and 1 each was from Cambodia, Easter Island, Indonesia, Thailand, and Vietnam. Imported dengue peaked in July and August 2001; exposures in the Society Islands accounted for the largest proportion of cases during this time (n = 9, 47%).
All 15 dengue virus isolates obtained from patients with exposure in Hawaii were DENV-1. Phylogenetic analysis of envelope glycoprotein sequences showed that the Hawaiian isolates belonged to a group composed primarily of Pacific Island isolates from recent years (Figure 4 ). High bootstrap values showed the Hawaiian isolates were associated more closely with contemporaneous Tahiti and subsequent Easter Island isolates than with a 2001 isolate from American Samoa.
In entomologic surveys conducted during the outbreak, Ae. albopictus was present in all 29 communities surveyed on Oahu, Maui, Molokai, and Kauai, but no Ae. aegypti were found at any site. In drier areas, on the leeward sides of the islands, container indices were high (>50%), but landing rates were generally low. However, in Nahiku, a small community in densely vegetated woodland near Hana, Maui, that was heavily affected during the outbreak, adult Ae. albopictus populations were high, with landing rates of 70 to 90 mosquitoes per person in 5 minutes. In the surveys conducted at 300 sites in 2002, Ae. albopictus larvae were ubiquitous on all islands, including Lanai and Hawaii, but Ae. aegypti was only found in 3 communities in the southern part of the island of Hawaii.
This report describes the first outbreak of dengue fever in Hawaii since the mid-1940s. Understanding the factors that contributed to the reemergence of dengue after such a prolonged absence and to the cessation of transmission will help public health authorities develop future prevention and control strategies. At the time of the 2001 Hawaii outbreak, a large DENV-1 epidemic was occurring in the Society Islands, 4,400 km south of Hawaii. More than 33,000 dengue illnesses were recorded in the Society Islands from February to November 2001, and of the 1,400 persons hospitalized, DHF was diagnosed in 45%, and 20% had symptoms of DHF or DSS. Ae. aegypti was identified as the vector (15, 16) .
Virologic and epidemiologic data strongly suggest that the Hawaii dengue outbreak was directly linked to the one in French Polynesia. Travelers are a potential source for dengue outbreaks; many epidemic introductions are thought to result from the arrival of a single viremic person into an Ae. aegypti-or Ae. albopictus-infested area (17) . DENV may have been introduced to Maui when a group of >30 persons from Hana visited Tahiti during April-May 2001. One of the travelers (patient A) became ill shortly after returning to Hana and later tested positive for anti-DENV IgM and IgG. Patient A was a close associate of the first known autochthonous case-patient in the Hawaii outbreak, whose illness onset occurred ≈2-3 weeks later.
Although patient A may have been the source for the Hana outbreak on Maui, available information suggests that additional separate virus introductions led to independent foci of autochthonous cases on the other 2 affected islands. In Kauai, only 1 of 4 dengue case-patients had any known exposure to persons from Maui. Moreover, the first identified case-patient in Kauai shared accommodations with a person in whom a febrile illness developed shortly after the patient returned from Tahiti. On Oahu, none of the 26 confirmed infections could be epidemiologically linked to exposures on Kauai or Maui. Furthermore, during an investigation of an autochthonous cluster on Oahu, the likely index patient was as an IgM-positive family member who had a DLI 4 days after returning from a trip to Tahiti.
Ae. albopictus was the vector responsible for the 2001 Hawaii outbreak. Both entomologic surveys support that Ae. albopictus is ubiquitous, often common on all the islands, whereas Ae. aegypti is restricted to a few small foci on the relatively sparsely inhabited island of Hawaii.
Several factors may explain why the outbreak in Hawaii followed a much different course than the concurrent epidemic caused by an apparently similar DENV-1 strain elsewhere in the Pacific. First, differences in mosquito species, behavior, and ecology are critical to understanding why the Hawaii outbreak was less severe than that described in the Society Islands, where Ae. aegypti was the principal mosquito vector. Ae. aegypti females are highly anthropophilic and often feed on several persons before obtaining enough blood to complete a gonotrophic cycle. This tendency towards multiple feeding may contribute to the explosive nature of dengue outbreaks in areas where Ae. aegypti is present. Compared with Ae aegypti, Ae. albopictus is considered to be an inefficient epidemic dengue vector because it is less anthropophilic and not as well adapted to urban domestic environments (18) . Ae. albopictus will readily feed on humans, but usually only on a single person, and it also feeds on other animals, which decreases the probability of human contact (19, 20) . Lifestyle factors may also help explain why Hawaii's dengue outbreak was limited (21) . Residences in many affected areas often had dense, uncultivated vegetation near housing and, not infrequently, an abundance of items that could serve as suitable Aedes breeding sites: tires, buckets, and discarded vehicles. Furthermore, dwellings in these areas often lacked window screens and doors. The combination of ample mosquito breeding sites and relatively unrestricted access to residents in some sections of windward Oahu and Hana, Maui, probably enhanced opportunities for mosquito-human contact beyond levels that existed in Hawaii's major population centers.
Public health measures may also have helped mitigate the spread of Hawaii's outbreak. This response consisted of 4 simultaneous, integrated initiatives: 1) enhanced surveillance to detect new foci of transmission; 2) rapid education of healthcare providers to improve the diagnosis and treatment of dengue; 3) health promotion activities directed toward the general public, including visitors; and 4) vector-control efforts, which included a combination of source reduction activities, limited use of larvicides, and area spraying (Appendix).
Worth noting is that most of the illnesses in the Hawaii outbreak were mild, given that an apparently similar DENV-1 strain caused a major epidemic of DHF and DSS in French Polynesia. One possible explanation for the difference in illness severity observed between these locations is that the number of cases in Hawaii was too small to manifest the extremes of the clinical spectrum. A second explanation is that a history of dengue infection, i.e., antibody-dependent enhancement, may have been important in French Polynesia (22) . A third explanation is that the Hawaiian virus had changed genetically and became less virulent or lost its epidemic potential. This loss of epidemic potential occurred in the 1970s when both DENV-1 and DENV-2 were reintroduced into the Pacific after an absence of 25 years (23) . Despite close similarities in the envelope protein sequences of the 2001 Tahiti and Hawaii viruses, important differences may exist in other areas of the genome that could influence these properties. Recent studies in Sri Lanka and Puerto Rico suggest that the genetic changes associated with epidemic potential occur in the nonstructural virus genes and not the envelope gene commonly usually used to show genetic relatedness between dengue viruses (24, 25) . Full-length genomic sequencing of DENV-1 viruses is pending.
The Hawaii experience demonstrates the potential of Ae. albopictus, under suitable conditions, to transmit small outbreaks of dengue within the United States. During the last 15-20 years, this mosquito has expanded its geographic range within the United States and now is found in at least 24 states on the mainland (26, 27) . From 1986 to 2000, a total 516 laboratory-confirmed and 2,128 suspected dengue infections were imported into the United States (28) (29) (30) (31) (32) (33) . The true incidence of imported dengue infection is probably higher, since dengue may often go undiagnosed in areas where the virus is not endemic (4, 20, 23, 34, 35) . Given the high volume of travel between the US mainland and dengue-endemic areas of the world (an estimated 14 million passengers to and from the Caribbean, Central and South America, and Oceania in 2001), we recommend that health officials keep local clinicians informed of dengue activity in these regions and that clinicians consider the possibility of autochthonous transmission when evaluating febrile rash illnesses, particularly when local vector surveillance indicates high populations of Ae. aegypti or Ae. albopictus mosquitoes (36,37). This investigation has several limitations. First, despite extraordinary efforts to obtain specimens, ≈25% of all persons initially evaluated for dengue did not submit a convalescent-phase specimen (>5 days after illness onset) required for definitive case classification. During followup attempts to obtain convalescent-phase sera, we often learned that patients or their physicians had decided that dengue was unlikely and no further testing was necessary; however, some dengue infections may have been missed. Secondly, because persons acquire dengue from mosquitoes that feed during the daytime, infection might have occurred at a location other than where the patient lived. We mapped the distribution of residences, however, because this information is not subject to recall bias. Thirdly, when investigating newly reported cases, we did not routinely elicit the number of household members and obtain serum samples from them in a standardized manner. Therefore, we cannot calculate the proportion of close contacts who were infected.
The Hawaii dengue experience is another example of how readily pathogens can cross great expanses of ocean to cause outbreaks in new territory (1, (38) (39) (40) . Important lessons learned from this episode include the need to closely monitor and respond to disease developments in the global community and the need to maintain surveillance and control of potential disease vectors even in the absence of an imminent disease threat. samples, 4) creating a patient-tracking system, and 5) notifying all state epidemiologists though Epi-X to identify any possible dengue cases exported from Hawaii.
Provider education included 1) issuing medical alerts to physicians, 2) conducting grand rounds and other lectures on dengue at local medical centers, and 3) distributing CDC video tapes on dengue diagnosis and treatment to physicians.
Health promotion efforts included 1) issuing frequent press releases, including daily case counts and messages about eliminating mosquito breeding sites around the home; 2) giving multiple news interviews by HDOH staff with radio, television, and print media; producing public service announcements by HDOH for radio and television; 3) conducting joint town meetings by HDOH and Department of Education health educators; 4) distributing >600,000 dengue brochures through high-volume stores and other venues; 5) developing a dengue education Web site, which provided the public and officials with information on the latest developments; 6) distributing educational brochures to Maui rental car agencies and hotels; and 7) establishing checkpoints along the Hana Highway staffed by public health nurses and others who distributed educational materials and mosquito repellent.
Vector control efforts included 1) inspecting private and public properties for mosquitoes, larvae, and potential breeding sites; 2) conducting door-to-door source reduction campaigns by HDOH staff and community volunteers in Hana and windward Oahu; and 3) treating >2,500 residences statewide with insecticides or larvicides. Estimating SARS Incubation Period   Syndromic Surveillance  a large number of cases within 24 hours. A fully functional hospital syndromic surveillance system that uses automated analysis (such as the daily emergency department-based surveillance with SaTScan in New York City) should identify a substantial increase in a relevant syndrome within 12 to 24 hours after data submission (2) . A continued daily rise in any disease category would most certainly set off alarms in a syndromic surveillance network. If active statewide laboratory surveillance is included in syndromic surveillance, such as the gram-positive rod surveillance conducted in Connecticut (3), this surveillance should rapidly detect even single cases of anthrax concurrent with the presumptive diagnosis within the hospital.
The authors also state that syndromic surveillance would not detect outbreaks too small to trigger statistical alarms. The combination of active and passive surveillance in the hospital admissions-based syndromic surveillance in Connecticut allows a number of syndromes to be tracked immediately upon notification; these syndromes include pneumonia and acute respiratory disease in healthcare workers admitted to a hospital, all disease clusters, and fever with rash illness. This system is very flexible, and active surveillance of other syndromes can be quickly instituted as required. This active surveillance component has been proven useful. The first 2 of Connecticut's 17 confirmed human cases of West Nile virus during 2002 were discovered in August when a health director, who regularly monitored the syndromic admissions data for the hospital in his municipality, requested immediate West Nile virus testing from the hospital's infection-control department when he received two late summer reports of neurologic illness.
Buehler et al. state that specificity for distinguishing bioterrorism-related epidemics from more ordinary illness may be low because the early symptoms of bioterrorism-related illness overlap with those of many common infections. Illness specificity can be modulated within a syndromic surveillance system by making changes in the definition of the information requested, the method of analysis used, or by incorporating varying amounts of active surveillance into a passive reporting system. In Connecticut, annual rates of hospital admissions for pneumonia and respiratory illness have significantly increased (>3 standard deviations) during winter months. These increases have corresponded temporally with peaks in laboratory-confirmed influenza reports and in our state-based and the national sentinel physician influenzalike illness reports. Similarly, in the militarybased syndromic surveillance system, respiratory outbreaks are detected by monitoring routine outpatient visits and pharmacy prescriptions. Absolute numbers of visits, as well as percentage of visits, to primary care clinics for influenzalike illness provide upto-date information on respiratory disease conditions at military installations in both active-duty personnel and family members.
Connecticut has added additional active surveillance categories to its syndromic surveillance for potential SARS cases by gathering extensive data on all healthcare providers hospitalized with respiratory illness. In the absence of an identified pathogen, the entire United States was conducting syndromic surveillance for SARS during the spring of 2003.
What are existing alternatives to rapid, patient-based reporting through syndromic surveillance for bioterrorism and emerging illness? Will individual physicians (i.e., the "astute clinicians") truly recognize an increase of nonspecific symptoms among their patients in time to warn public health authorities of an impending bioterrorism event? During the past 4 years in the U.S. military population, unless disease was extremely severe with high rates of hospitalization, virtually no outbreaks of infectious diseases detected by syndromic surveillance were reported to public health officials, even when effective preventive measures existed. Our experience leads us to encourage states and municipalities to develop functional, patient-based syndromic surveillance systems and discover both their limitations and their possibilities.
In reply: The thoughtful letter of Drs. Dembek, Cochrane, and Pavlin draws attention to several key themes emerging in the ongoing dialogue about the utility and role of syndromic surveillance. First, as illustrated by their work, the growing body of experience in conducting syndromic surveillance should advance this dialogue beyond the hypothetical framework described in our manuscript to a more evidence-based assessment of epidemic detection. Second, in the absence of a bioterrorism-related illness since 2001, the utility of syndromic surveillance for detecting naturally occurring events is coming into greater focus, particularly for detecting the onset of anticipated seasonal upswings in infectious diseases, including West Nile virus disease, gastrointestinal illness, and influenza.
Syndromic surveillance coupled with follow-up investigations can assist clinicians by alerting them to communitywide problems likely to be manifest among their patients. This recognition may occur at the hospital level, as reported by Dembek et al. in the initial recognition of West Nile virus disease in Connecticut in 2002, or at the community level, as illustrated by public health alerts in New York City to notify clinicians about viral gastrointestinal illness (1). Multiple studies have documented that newer syndromic surveillance systems can recognize the onset of the annual influenza season (2), but it is not clear what these systems add to existing syndrome-based systems that track "influenzalike illness" as part of a larger array of influenza-specific surveillance methods. While Dembek et al. note that syndromic surveillance has detected multiple outbreaks that would have been otherwise unrecognized, Sichel et al. observed that syndromic surveillance did not detect outbreaks recognized through more traditional means (1). This discrepancy emphasizes the need to further assess the characteristics of epidemics and surveillance systems that favor detection by using syndromic methods.
We recommend distinguishing between the increasing practice, prompted by concerns about bioterrorism, of syndromic surveillance for epidemic detection and the longstanding and common practice of using syndrome-based case definitions in public health surveillance. Such case definitions have been used in situations in which a wide net is cast to identify potential cases of a particular disease (e.g., acute flaccid paralysis as part of global efforts to eradicate poliomyelitis [3] , liver disease associated with a new therapy for latent tuberculosis infection [4] , and inhalational anthrax in New Jersey in 2001, after bioterrorism-related cases were clinically detected [5] ), when resource and infrastructure constraints do not allow routine use of laboratory-based definitions (e.g., surveillance for sexually transmitted diseases in infrastructure-weak countries [6] ), and when surveillance is initiated for a new disease of unknown origin (e.g., toxic shock syndrome [7] , AIDS [8] , and severe acute respiratory syndrome [SARS] [9]). Although SARS surveillance did not represent syndromic surveillance according to this distinction, relationships between health departments and hospitals, fostered in establishing syndromic surveillance, likely facilitated SARS surveillance.
New guidelines offer an approach for evaluating syndromic surveillance systems, including what is learned from follow-up of statistical alarms and whether syndromic surveillance or other methods lead to the earliest detection of outbreaks (10) . These guidelines also provide a framework for modeling exercises to test syndromic surveillance under various bioterrorism scenarios, supplementing experience gained from real-life, but typically less severe, seasonal illness or community epidemics. Eventually, this information should be useful in developing guidance for health departments seeking to determine whether and how to implement syndromic surveillance.
To the Editor: Polymicrobial invasive infections are infrequent, representing <10% of the invasive infections of known etiology (1). They are often correlated with a predisposing factor: immunodeficiency (e.g., diabetes mellitus, malignancies, extremes of age) or use of a central catheter. Escherichia hermanii is an extremely rare etiologic agent for invasive infections; only four cases were published from 1980 to 2002. We report the first case of double invasive infection by E. hermanii and Staphylococcus aureus and emphasize the importance of screening of all the septic foci for demonstrating a polymicrobial invasive infection.
In August 2000, a 54-year-old comatose man was admitted to our infectious diseases department with a 10-day history of fever. He had a medical history of vertebral arthrosis (lumbar laminectomy in 1989) and insulin-dependent diabetes mellitus. Six weeks before, he had received for 3 days gluteal injections with kebusone (an intramuscular nonsteriodal antiinflammatory drug [NSAID]) for acute lower back pain. Twenty-eight days after the first injection, a gluteal abscess developed, which was surgically drained, without perioperative antimicrobial therapy. Three days later, he became febrile, and pyrexia persisted despite local wound management and treatment with oxacillin, 4 g/day for 3 days; cefuroxime, 3 g/day, and gentamicin, 160 mg/day for another 7 days.
The patient became comatose and was transferred to our department. At that time, the physical examination showed fever (40.2°C), neck stiffness, Brudzinski sign, thoracic dullness, and bilateral crackling rales. The level of C-reactive protein was 123 mg/L. Renal failure was noted with a creatinine blood level of 312 mmol/L and uncontrolled diabetes with fasting glucose of 24.75 mmol/L. Computed tomographic (CT) scan of the brain did not show brain abscesses or tumors. Examination of the cerebrospinal fluid (CSF) indicated a protein level of 2.67 g/L, decreased glucose concentration of 0.55 mmol/L, and a leukocyte count of 2.3 x 10 9 /L with 96% neutrophils; no microbial pathogens were demonstrable under direct examination of CSF. Chest xray identified bronchopneumonia and bilateral pleural effusion. The pleural fluid analysis revealed a purulent exudate-protein, 4.5 g/L-containing 55% neutrophils. A urine specimen and three blood samples were obtained for cultures over the first 4 hours after admission. A bacterial invasive infection was considered and the antibiotic therapy was started with ceftriaxone, 2 g/day, and rifampin, 1,200 mg/day. Concomitantly, the patient received colloids to reestablish blood volume, intravenous dexamethasone, 6 mg four times daily, to diminish the cerebral edema; and fast- Human Metapneumovirus and Severity of Respiratory Syncytial Virus Disease  I n the United States, 100,000 infants and young children are hospitalized each year with respiratory syncytial virus (RSV) bronchiolitis (1) . Although the risk factors for severe RSV disease, such as prematurity and bronchopulmonary dysplasia, are well defined, severe RSV disease may develop in otherwise healthy children. The pathogenesis of severe RSV disease is poorly defined.
In 2001, van den Hoogen et al. reported the isolation of a novel paramyxovirus, human metapneumovirus (HMPV) from children with respiratory tract disease (2) . HMPV has been identified worldwide (3) (4) (5) (6) (7) and appears to have a seasonal distribution (winter and spring) (8) . Since the circulation of HMPV may overlap with that of RSV, simultaneous infection with both RSV and HMPV may contribute to severe disease. Greensill et al. (9) reported that 70% of RSV-infected children who required admission to the Pediatric Intensive Care Unit (PICU) in Liverpool, U.K. were co-infected with HMPV.
We sought to determine whether infection with HMPV was associated with the severity of RSV disease. We determined the frequency of HMPV infection in children with either mild or severe RSV disease. Disease severity was assessed by both the disposition (PICU vs. non-PICU) and by a clinical severity score.
As part of an ongoing epidemiologic study of viral respiratory infections in children, we collected all the RSV direct fluorescent antibody (DFA)-positive respiratory specimens from the Clinical Virology Laboratory at Yale-New Haven Hospital from November 1, 2001, to October 31, 2002. All respiratory specimens were also screened by DFA for parainfluenza viruses 1-3, influenza A and B viruses, and adenoviruses (10) . Any RSV-positive patient who also tested positive for one of the viruses listed above was excluded from the study. All RSV-positive children admitted to PICU during this yearlong period were identified. Because the peak time of infection with RSV and with HMPV may differ, each RSV-positive child from PICU was matched by date of diagnosis with a child with mild RSV disease. All RSV-positive children who were not admitted to PICU and diagnosed within 2 days of the diagnosis of the PICU-admitted child were identified. Of these, the child whose age most closely matched the age of the PICU-admitted child was selected. If no child diagnosed with RSV was identified within 2 days of the PICU-admitted child, the child with the closest date of diagnosis was identified and matched to the PICU-admitted patient.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) were performed as previously described (4) . Primers used for the amplification of the RSV L gene were as follows: forward primer 5′-GGTA-GAATCTACATATCCTTACCTAAGTG-3′ (genome location 14881-14909, [reference strain RSV A Melbourne, Australia/2/'61] GenBank accession no. M74568), reverse primer 5′-CGAGATATTAGTTTTTGACAC-3′ (genome location 15190-15210, GenBank accession no. M74568). The HMPV forward primer, 5′-GCGCGTTCTGAG-GACAGGTTGG-3′ (HMPV genome location 3163-3180, GenBank accession no. AF371367, G/C clamps are underlined) and reverse primer, 5′-GCGCTCAAGCCGGATG-GTTTTGG-3′ (3425-3444, GenBank accession no. AF371367, G/C clamps are underlined) used for the amplification of the HMPV F gene were based on regions of the F gene conserved in New Haven, Netherlands, and Australian strains (4). PCR reactions were performed by using HotStar (Qiagen, Inc., Valencia, CA) and amplification cycles were as follows: 95°C for 15 min followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and a final 10-min cycle at 72°C. Each set of reactions contained appropriate negative (water) and positive control samples for the RT (HMPV-positive nasal wash) and the PCR (HMPV cDNA) steps.
A clinical severity score (CSS) was adapted from the severity score described by Martinello et al. (11) . Two points were assigned if the patient required positive-pressure ventilatory support during the illness, and one point was assigned for each of the following: hospital admission, hospitalization for >5 days, oxygen saturation <87% (at least one measurement), and any use of supplemental oxygen. Therefore, CSS ranged from 0 to 6. Medical records from all patients were abstracted and scored by a reviewer who did not know the patient's HMPV status.
On the basis of the published literature (9), we expected the frequency of RSV/HMPV co-infection in the PICU patients to be approximately 70%, and in the non-PICU patients to be closer to the DFA-negative-HMPV-positive infection rate of 6.4% found in our population (DFA-negative refers to samples that tested negative for RSV, parainfluenza viruses, influenza viruses, and adenovirus) (4). Power calculations were performed by using PASS 2002 (J. Hintze, NCSS and PASS, Number Cruncher Statistical Systems, Kaysville, Utah). By using a power of 90% and α of 0.05, a sample of 12 patients in each group would be sufficient to support these findings (70% vs. 6.4%). By using the same power calculations, 23 patients in each group would provide adequate power to show as little as a 45% difference in proportions with co-infection between the two groups. Comparisons were made by using the chisquare test and Wilcoxon rank sum tests, as appropriate (Table) . Exact 95% confidence intervals were calculated in SAS V8.2 (SAS Institute, Cary, NC).
Twenty-three RSV DFA-positive patients were admitted to PICU during the study period, and 23 matched patients were identified. Demographic and clinical information were obtained for each patient from the medical record. Of the children admitted to PICU, 7 (30.4%) of 23 had a known predisposing risk factor for severe RSV disease. All the PICU-admitted children had CSS >3 and most (16 [70.0%] of 23) had a CSS >5. Eighteen (78.2%) of 23 PICU patients were hospitalized for >5 days. None of the RSV-positive patients admitted to the PICU tested positive for HMPV by RT-PCR.
Children with mild RSV disease were initially seen in the emergency department, and according to their severity of illness, either were discharged or were admitted to the pediatric ward. Eight patients (34.8%) of the 23 mild RSV disease group were admitted to the hospital, although only 4 (17.4%) of these children were hospitalized for >5 days. The CSS range for this group was 0-4. Most patients (14 [61.0%] of 23) had a CSS of <3. None of these patients were positive for HMPV by RT-PCR. Statistically significant differences between the PICU group and the mild disease group were observed in admission age (median age 7 weeks vs. 54 weeks, p = 0.025), hospital admission rate (23/23 vs. 8/23, p = 0.004) and CSS (median CSS 5 vs. 1, p = 8 x 10 -12 ) (Table) . Positive pressure ventilation was required by 17 (73.9%) of 23 PICU patients and 10 of these 17 patients needed it for >5 days. None of the patients screened in either group of RSV-DFA positive patients had evidence of HMPV infection (p = 1.0). To ensure that the methods for RT-PCR were adequate, we performed RT-PCR for RSV for each patient's respiratory specimen. Overall, 44 (95.7%) of 46 of children had a positive RT-PCR test for RSV.
The possibility that HMPV plays a role in the pathogenesis of infections with other respiratory viruses is not known. The importance of identifying HMPV in persons with SARS remains to be explained (5, 12) . Greensill et al. observed a 70% co-infection rate with HMPV and RSV and a 90% co-infection rate among intubated infants with HMPV and RSV admitted to their PICU (9) . Although Greensill et al. did not include an appropriate control group in their study, these findings suggest that co-infection with both HMPV and RSV is common and that together the two viruses may contribute to increase the severity of disease.
We did not observe HMPV infection in children with either mild or severe RSV disease. Our findings cannot be explained by the absence of HMPV in Connecticut. From (13) . Furthermore, we matched children with mild RSV disease to children with severe RSV disease by date of diagnosis to eliminate the possibility that the temporal distribution of the viruses might influence our results.
The potential difference in the sensitivity of the screening tests used by Greensill et al. and our group likely does not account for the differences in the observed rates of coinfection. We used a similar RT-PCR-based approach. We are confident that our methods to detect HMPV are both sensitive and specific (4, 13) . The observed rate of HMPV in respiratory specimens in our previous study (8.1%) (13) is comparable to rates observed elsewhere (6, 14) . Furthermore, in our previous studies, we have used sequence analysis of RT-PCR amplicons to confirm the identification of HMPV (4, 13) . An increased prevalence of HMPV in Liverpool, UK, may account for the high rate of RSV/HMPV co-infection observed by Greensill et al., although no data at this point support this hypothesis.
Our relatively small sample size limited the power of our analysis. However, on the basis of the sample size calculations with 90% power, our patient numbers were sufficient to detect a difference of >45% above the rate of HMPV infection in the non-PICU group (6.4%). Nonetheless, our results demonstrate that the rate of coinfection is low (0% of 23 patients, 95% confidence interval 0%-14.8%). Other studies also support our findings that the frequency of co-infection with HMPV and RSV is rare (7, 14) The basis of the pathogenesis of severe RSV disease is multifactorial. Since severe RSV disease may develop in apparently healthy children, known host risk factors cannot completely account for instances of severe illness. Preexisting or maternally acquired immunity, innate immunity, viral factors and genotypes and environment all likely contribute to disease pathogenesis. Although we did not detect co-infection, HMPV may worsen RSV disease in a small percentage of infants. Nonetheless, HMPV most likely does not play an important role in the severity of RSV disease in the population. Community responses to communication campaigns for influenza A (H1N1): a focus group study BACKGROUND: This research was a part of a contestable rapid response initiative launched by the Health Research Council of New Zealand and the Ministry of Health in response to the 2009 influenza A pandemic. The aim was to provide health authorities in New Zealand with evidence-based practical information to guide the development and delivery of effective health messages for H1N1 and other health campaigns. This study contributed to the initiative by providing qualitative data about community responses to key health messages in the 2009 and 2010 H1N1 campaigns, the impact of messages on behavioural change and the differential impact on vulnerable groups in New Zealand. METHODS: Qualitative data were collected on community responses to key health messages in the 2009 and 2010 Ministry of Health H1N1 campaigns, the impact of messages on behaviour and the differential impact on vulnerable groups. Eight focus groups were held in the winter of 2010 with 80 participants from groups identified by the Ministry of Health as vulnerable to the H1N1 virus, such as people with chronic health conditions, pregnant women, children, Pacific Peoples and Māori. Because this study was part of a rapid response initiative, focus groups were selected as the most efficient means of data collection in the time available. For Māori, focus group discussion (hui) is a culturally appropriate methodology. RESULTS: Thematic analysis of data identified four major themes: personal and community risk, building community strategies, responsibility and information sources. People wanted messages about specific actions that they could take to protect themselves and their families and to mitigate any consequences. They wanted transparent and factual communication where both good and bad news is conveyed by people who they could trust. CONCLUSIONS: The responses from all groups endorsed the need for community based risk management including information dissemination. Engaging with communities will be essential to facilitate preparedness and build community resilience to future pandemic events. This research provides an illustration of the complexities of how people understand and respond to health messages related to the H1N1 pandemic. The importance of the differences identified in the analysis is not the differences per se but highlight problems with a "one size fits all" pandemic warning strategy. During an influenza pandemic the lack of experience of dealing with such hazards increases public reliance on information from government, public health agencies, employers, the community and the media. A challenge for government and health agencies is sustaining public awareness and alertness over a protracted period [1, 2] . Responding to the challenges posed by managing the risk from relatively unknown health hazards requires recognition of the fact that promoting sustained action involves not only getting the information right but also ensuring that it is communicated in ways that accommodate diversity in community characteristics, needs and expectations. The lack of experience also calls for research to provide the evidence base health authorities need to respond effectively in circumstances (e.g. rapid onset of disease outbreak) that precludes learning in situ.
There is evidence that greater levels of perceived susceptibility to and perceived severity of the disease and greater belief in the effectiveness of recommended preventative and avoidant measures are important predictors of behavior [3] [4] [5] . It is important to accommodate the fact that disbelief in the effectiveness of measures can result in people failing to act and developing distrust of sources of information [6] . Previous New Zealand research identified a general belief that local agencies will manage a pandemic well and that New Zealand is a relatively safe place to be in the event of a pandemic. However, there was also a lack of trust in information providers as well doubt that the health system would cope [4] . Trust is a crucial component of effective risk communication when people face uncertainty and levels of trust are correlated with people's perceptions of the integrity of information. Lack of trust can be exacerbated by scepticism about the veracity of health risk warnings and the view that media are sensationalist and untrustworthy [7, 8] . Being uncertain about an outbreak, and thinking it, and its consequences had been exaggerated have been associated with a lower likelihood of behavioural change [5] . Increasing trust is a function of the degree to which agencies engage with and empower communities [6] .
Lack of trust in authorities may also affect how people process and interpret health messages and advice, increase concerns and interfere with the way that the risk messages are interpreted and acted on [9] . Transparency and honest communication where both good and bad news is conveyed can empower the public to make their own decisions [1] . The public are more likely to take appropriate action and accept the recommendations if they have been involved in the decision-making process, and the quality of the relationship between authorities and the community has a direct effect on the uptake of risk messages, and trust in the message providers [2] .
The primary objective of this study was to provide health authorities with evidence-based practical information to guide the development and delivery of key health messages for H1N1 and other health campaigns. The study focused on community responses to key health messages in the 2009 and 2010 H1N1 campaigns.
The study was part of a rapid response initiative; therefore focus groups were selected as the most efficient means of data collection in the time available. Eight semi-structured focus groups were recruited between May and July 2010 (the New Zealand winter season) comprising 7 to 13 participants each and lasting approximately 1 hour. Separate focus groups were conducted for each of the target groups with a total of 80 participants representative of five target populations groups identified in consultation with Ministry of Health staff: Māori, Pacific Peoples, children (or parents of children), general population, and vulnerable people with chronic conditions (defined as those who are eligible for subsidised vaccinations, such as pregnant women, those with diabetes, using asthma inhalers, with heart disease or kidney problems. Participant characteristics are summarised in Table 1 .
Purposive sampling methods were used to ensure the sample met the criteria specified by the Ministry of Health. The period of data collection was selected to coincide with the period in which people are most aware of flu and the need for preventive and protective actions. Participants were recruited using posters in a community library and through contacts within key agencies (e.g. university research centres, Pacific health services, district health boards, and the Ministry of the Social Development). Staff of the Research Centre for Māori Health and Development, Massey University were responsible for the Māori components of the research to ensure a culturally appropriate methodology given the time restraints of the study. In contrast to New Zealanders of European descent, Māori possess different cultural characteristics. For example, based on cultural dimensions [10] Māori score higher on dimensions such as individualism-collectivism, power distance and uncertainty avoidance compared with their counterparts. Because these differences have significant implications for social influences on behaviour and for the nature of the relationships that exist between community members and health authorities, it was essential to ensure that data were collected in culturally sensitive and competent ways. Hence Māori were treated differently from the perspective of the data collection approach adopted. This is consistent with effective cross cultural research methods [11] .
All focus group sessions (except Māori groups) were recorded and independently, professionally transcribed. Transcriptions were mainly verbatim, with verbal padding and hesitations omitted. Apart from the facilitators, specific individuals were not identified in the transcripts or any subsequent reports. The extracts used to illustrate the content of each theme are identified by codes which correspond to the focus group transcripts from which they were taken ( Table 1) .
The analysis of the focus group data (excluding Māori groups) was undertaken by a single researcher who was neither present at the focus groups nor had read any preliminary findings. This work was verified by the focus group facilitators to ensure that any "contextual richness" had not been missed in the data. Thematic analysis was used to identify themes and concepts across the entire data set (6 transcripts) to "identify repeated patterns of meaning" [12] . The process involved working through the six phases of thematic analyses as identified by Braun and Clarke [12] .
This study was approved by the Massey University Human Ethics Committee: Southern A (ref 10/32, 21 May 2010) . Written informed consent was obtained from all participants.
The final coding scheme consisted of 17 themes which were grouped into four main categories: risk, building community understanding, responsiveness and information preferences. Transcript extracts were selected on the basis of their relevance to the theme under discussion and all identifying information has been removed.
People's perception of risk is a product of the perceived likelihood of a pandemic and its perceived consequences. The analysis identified that people made judgements about both likelihood and consequences. Understanding these inputs helps tailor risk communication strategies. Previous research identified a general belief that New Zealand, as a result of factors such as geographic isolation, reduce the likelihood of a pandemic and thus increase a belief that New Zealand is a relatively safe place to be in the event of a pandemic [4] . Perceived risk was also attenuated by a belief that border control agencies will prevent pandemic flu entering New Zealand, with this possibly resulting in people transferring responsibility for preparing from themselves to border control agencies [6] .
"It's pretty much a one-border control place. That's why that's better. We're lucky, because we've got only that one contact point coming in." (G3)
Other participants were aware that our perceived geographical isolation does not in fact translate to a decrease in risk. In fact H1N1 arrived in New Zealand early in the global pandemic of 2009 with students returning from a school trip to Mexico and the USA.
"Well we think that we're different because we're far away. But actually, if you think of how people travelled here, it's the biggest factor for it always, because everyone who comes here comes in an aeroplane, pretty much. And they come from everywhere." (G3)
Discussion reflected variable levels of awareness of and reaction to pandemics. Many regarded the 2009 H1N1 pandemic as an overreaction that was not taken very seriously by many people. The following extract illustrates the concept of "normalisation bias" in which people extrapolate from the current experiences (H1N1) to define what a future pandemic would look like and therefore underestimate their future risk which makes them less respective to current health risk messages [6] .
"I thought it was scaremongering, personally. I thought the way it was handled was quite interesting, and I think it also made a lot of people very paranoid about things that sometimes people don't need to be paranoid about." (G3) For some participants it was the use of term "pandemic" that gave rise to the sense of overreaction by suggesting something more serious than what actually occurred.
"It became reasonably clear reasonably quickly last time that hundreds and thousands and millions weren't dying. Even when they kept on sort of saying things were happening, and then you saw the numbers, it just didn't add up." (G1) There is evidence that higher levels of general anxiety and perceptions of high risk severity are related to a greater likelihood of carrying out preventive and avoidant protective behaviours [3, 5] . However, it is important to note that this relationship is only present when people with high risk perceptions also know what to do to manage their risk. If people do not know what to do, or question the veracity of the advice offered, they are more likely to respond by denying the risk or transferring it to others [13] . Whilst most of the participants in the general population groups regarded swine flu as "bit of a joke, really" others reported feelings of anxiety, fear or panic. In particular, reports of flu-related deaths gave rise to more serious concern.
For those who felt that geographical remoteness provided some form of protection, it was not until cases were reported within New Zealand that the threat became more real. This shift in thinking is important as the perceived relevancy and immediacy of risk affects the decision to act or not to act on information [4, 8] .
"Because you kind of go, well, we're just little old New Zealand, where nothing happens. You know, we'll be right. And it wasn't until you actually heard that people in New Zealand had brought it back from overseas. And that's when you really does go "Ooh! Alright." (G3) Concern about news reports from overseas also related to the reliability of the information in terms of both its trustworthiness and relevance to "us".
'The other thing, people are a bit cynical about the whole thing. Because there are these people dying in Mexico, you think, well, there are people sick in hospital. You know, I've got no idea what the health system's like there." (G2) Such comments reflect a belief "it won't happen to me because -it happens to others". Through the process of "othering", individuals focus on differences in others, effectively creating a separation between "us" and "them" [14] . By projecting the risk of infection and death onto "them" the sense of powerlessness and vulnerability is reduced for "us" [15] .
Cynicism about the veracity of media reports was not limited to news from overseas. Indeed there was a strong feeling that the New Zealand media had a central role in the "overhyping" of the 2009 and 2010 pandemic risk. Media are credited with amplifying the risk perceptions in such a way that risk communicated may not be an accurate reflection of the true risk [7] .
"I think the news media have a lot to blame, because they want to make news. They sensationalise stuff. That's not helpful." (G2)
Public distrust in journalists and the sensationalising of health related stories can also be a hindrance to taking the risk seriously and of undertaking precautionary measures [5] . A belief that risk has been exaggerated is associated with an increased sense of helplessness and frustration and a reduction in the likelihood that people will prepare in the short-term [5, 7, 16] .
In an attempt to understand potential risks people situate risk-knowledge in historical and local contexts [17] . This can be seen in the focus group discussions in which participants were more concerned about other risks, including illnesses such as heart disease, cancer, meningitis, and respiratory disease.
Much of the discussion in the general population groups focused on their reassuring themselves that the pandemic was indeed an overreaction and a "media beat-up" as they believe that this had happened before with other events. In this condition, people preferentially seek information that reinforces this belief and this may reduce the likelihood of their attending to public information. Maintaining a sceptical and cynical perspective is a means of "distancing" themselves from the threat.
"Like the bird flu that was... that, I think, killed some... a few people in Canada. And people said it was going to be the next big flu, and it wasn't." (G2)
There is evidence that individual perceptions of risk are important determinants in undertaking preventive, and/ or protective behaviours for events such as pandemics as long as people know how to manage their risk [3, 5, 12, 21] . Some participants appeared to have a sense of "personal invulnerability" based on their history of rarely having experienced flu's or colds. The comments were also indicative of the phenomenon of unrealistic optimism. This means that while people accept the existence of a risk to the community in general, they see themselves as less vulnerable or more capable than others. This bias results in their transferring risk from themselves to others and seeing risk communications as applying to others rather than to themselves [6] . This bias represents a significant constraint on the effectiveness of risk communication. However, encouraging active discussion of pandemic issues in community groups can reduce its influence.
"I've been fortunate enough to... I don't know that I've even actually ever had the flu, even ordinary flu, in my life... So I seem to have a bit of a natural immunity to it, luckily enough, so... yeah." (G2)
In contrast there were others who expressed a heightened sense of risk due to their personal circumstances and health history. Assessing their personal risk in this way supports the view that people's understanding of risk is developed not only through cultural and sub-cultural membership, but also through personal experience [17] . This highlights the importance of risk communication encouraging people to personalize information. While all groups were aware of the concept of "high risk" groups the relationship between knowledge of "high risk factors" and individual perceptions of risk was less clear. Although some participants identified themselves as "high risk" they appeared to be uncertain about what this meant for them. Others used "othering" to minimise their own perception of risk by differentiating themselves from those who they identified as "high risk". This social construction of boundaries of "self" and "other" and their relationship to boundaries of "safety" and "danger" are particularly relevant to understanding notions of health and disease [18] .
However, the self/other divide can be used to facilitate preparing [13] . By considering whether something can be done to assist those more vulnerable, people are more likely to also consider what they can do for themselves.
Recall of key health messages was varied, however most participants were aware of hygiene self-efficacy measures such as hand washing, sanitiser use, covering of coughs and sneezes, and staying at home. This was particularly strong in one of the Pacific Peoples groups and for many appeared to be translated into action.
"Mainly they tell you to wash your hands....Cover your mouth when you cough.......And don't share hankies, they say, yeah." (P2)
There was some recall of the 2009 posters and the messages which they contained. Although some who recalled the posters expressed concern that they were inaccurate and overused. There was generally less recall of the 2009 television advertisements, and even less recall of the messages they were conveying. Overall, participants did not feel that they were better prepared or had changed their behaviour as a result of the information which they recalled from 2009. Where lessons had been learnt from the previous year, these were mostly related to improved personal hygiene measures.
Awareness of the 2010 campaign was scant and what was recalled tended to be from commercial advertising, for example, from private commercial companies promoting flu vaccinations and household hygiene products.
Participants reported receiving pandemic information from a variety of media sources including newspapers, TV, radio and the internet. However the primary source of information for participants was their workplace and/ or community. This differs from previous research in which Google was listed as a primary source of information [8] and television was the preferred means of receiving information during a pandemic [19] .
The general population groups tended to report workplace as a key source of information, including workplace intranets. In contrast there was general agreement in the Pacific Peoples groups that their primary sources of information were the community. This included social networks and family, regular forums and meetings, church groups and health centres. This supports the view that when faced with uncertainty, people turn to others to reduce their uncertainty and guide their preparation; often these are family and friends, but also health agencies with whom they have a direct relationship [2] . This highlights the importance of both recognising the existence of diverse communities that facilitate developing understanding and engaging with these communities to ensure that information can be tailored to meet the needs, goals and expectations of each group.
"And even in the church. Because one thing in the church is, our people fear God, and they always go church no matter what. And the pastor is one of the key people that talks into the community." (P2)
The 2010 campaign had largely gone unnoticed by Māori tribal elders (kaumātua). They did not believe that information was readily accessible, no one had seen articles in the local press regarding H1N1 and pamphlets and posters "were not freely available". This view was mirrored in the young Māori mothers (Tamariki Ora) focus group.
The kaumātua group felt that information is best disseminated to places of work, school and family and to Māori health providers to ensure coverage of the Māori population. It appeared that the messages in the media did not make an impact with Tamariki Ora mothers.
Previous research has shown that community participation and trust in emergency management agencies played significant roles in increasing community preparedness, willingness to take responsibility for own safety, risk acceptance and satisfaction with communication [2] . Public are more likely to take appropriate action and accept the recommended actions if they have been engaged in all aspects of the risk management and decision-making processes through mechanisms such as focus groups or forums in ways that empower people to take action [6, 20] .
Kaumātua were of the view that the 2007/08 campaign had been successful because the District Health Board had come out into the community and involved them in planning or providing information, but "it had not been effectively followed up on". A number of other comments also highlighted the importance of engaging with communities and disseminating information through community mechanisms.
"Civil Defence needs to be proactive, and actually make sure that they've got the right community people, and the right community organisations on the board." (G3)
With respect to workplace pandemic or disaster response plans, most participants had little or no knowledge of these. Some were aware of general plans or the existence of emergency supplies at work. There was a general belief in one group that emergency preparedness was seen as an "individual responsibility" by their employers. The apparent lack of clearly-articulated workplace response strategies is consistent with previous research [20] .
As with previous research [2, 20, 21] few participants had stocked up on emergency supplies or prepared for the pandemic any other way. Some individuals had stocked up on food and essential supplies and/or had a family disaster plan.
"I've certainly thought about how would we get food, or how much food did we have in the house, if we were to get quarantined." (G1)
While some participants reported that they had already prepared emergency boxes, others were yet to act on their intentions to do so; it was on the list of things to do. Participants were aware of advice to stockup on pharmaceutical products but a number found the array of over the counter products available very confusing.
Perceived or actual economic impact influences psychological and behavioural responses [22] . Many participants expressed concern that the cost of emergency kits could be a barrier for low income families and that some sort of financial assistance should be available, particularly as they may believe that the costs of acting will only incur a benefit in the event of a pandemic and this may not occur until some future date.
"And the thing is that it would affect... I mean, imagine someone on a really base-level income... isn't going to have a preparedness kit. So they're the ones that are going to suffer, through in some ways, no fault of their own." (G2)
Attitudes towards having the flu vaccination varied greatly as did uptake. Discussion concerned H1N1, H5N1, pandemic flu and seasonal influenza. There were those who routinely have them and those who "don't do flu shots". Uptake rates ranged from none in the Tamariki Ora mothers group to over 90% of those present in the kaumātua group.
For some participants the decision whether or not to have the vaccination was related to their perception of risk.
"Yes. I normally do have it. But I insisted on having it early, even though the GP was only giving it to people who were in vulnerable groups, because of the fact that I was travelling ..." (G1) There were others who, having identified themselves as being in an "at risk" group, still chose not to be vaccinated. For these and others it appeared to be a balancing act between their perceived risk of influenza against the perceived risks associated with the vaccine itself. The "costs", psychological and health "benefits", and expected outcome are all important issues that influence people when making a decision about whether to act on advice about a pandemic [2] .
"I'm kind of stuck on that one. I've certainly thought more about it this year, as to whether I should just take the risk; but I'm not going to. I'm still not having one." (G1) Several of the Tamariki Ora mothers were apprehensive and confused about the flu injection and wanted more information about "immunisation for their children".
"Will the current flu injection help us to stay immune for several years?" (M2) Whilst some participants elected not to have the vaccination even if it was free, for others cost was a significant issue. There was also a degree of cynicism about the vaccination and flu treatment amongst those that reported not having had one. In particular cynicism was expressed with respect to Tamiflu ® .
"The whole Tamiflu... again, it makes me cynical, you know. Somebody was making a heck of a lot of money out of that, you know?" (G2)
The reluctance to be vaccinated and the cynicism illustrated by these extracts is consistent with research showing that decisions to engage in preventive and avoidant behaviours is influenced by attitudes towards public health interventions [9] including having confidence in the efficacy of the behaviour [3] . It is worth noting that the latter beliefs influence the level of trust in health agencies and that specifically advising people (about all preparedness measures and not just antivirals) about why specific preparations are required increases the likelihood of adoption and helps maintain trust in health agency sources of information [12] .
Participants had heard the social isolation/distancing message.
"If you're sick, go home. And if you're sick and you're at home, stay there....That's right. I'm quite vocal about that anyway. I hate seeing people sick around me." (G1 with agreement from several other participants)
Although participants recognised that isolation is an important response strategy, the economic pressures to go to work instead of staying at home was a major concern.
"That's a big push, yeah, that's a big reason why people still go out, even though they know they have a cough. ...Yeah, that pushes me (into employment). ......You send your kids to school sick. .........Yeah, because you haven't the time, yeah." (P2) This is consistent with previous research showing that perceived or actual economic impact is a major factor in decisions around avoiding the flu [9, 19, 21, 22] . These issues can be compounded by not preparing, being unaware of workplace policies and plans (e.g., policies about wage payments if people are advised to stay home).
Kaumātua believed should a person develop flu-like symptoms they should isolate themselves from other members of the community, however they were concerned that when individuals are isolated they may not be contacted by members of the community or whānau. Concerns were also expressed by this group about observing specific cultural practices and greeting protocols, reflecting the role that cultural and sub-cultural membership has in people's understanding of risk [17] .
Participants wanted guidelines about who should stay home and when, and they wanted backing from their employers with respect to this issue. Participants felt that they had been given contradictory information about when to stay home and when to go to work or school which left them feeling uncertain about what to do. Consistency of advice is a significant important factor in communications from key agencies [3] .
Information preferences "Knowing the difference" between swine flu and other flu emerged as a significant issue. This reflects previous reports of a strong desire from the public for symptom details about influenza [19] and the finding that public information about signs and symptoms are beneficial to public understanding of a pandemic [8] . Participants across all groups wanted to know about the specific swine flu symptoms which they could use to identify it and protect themselves from possible infection. There is a view that surveillance combined with good scientific information and operational research is crucial in limiting the spread of H1N1 [23] . A number of participants were concerned about testing and monitoring. They felt that the Ministry should not have "cut off monitoring quite so early" and that the failure to test for the H1N1 after health services became inundated with patients undermined the advice that the Ministry of Health was giving.
In addition to the desire for symptom details many participants wanted concrete facts, such as how many people were diagnosed with or dying from swine flu. Some participants wanted to know percentages; others preferred the information to be given as numbers because they found percentages confusing. For many participants it was not necessarily numbers they wanted but information that helped them judge the "seriousness" of the pandemic and their level of personal risk.
There was general agreement in a Pacific Peoples group that simplicity in the framing of messages was important. This aligns with the contention people can absorb only a small amount of information at a time and have difficulty understanding some kinds of information. Risk communication should take this into account and identify the most critical facts [24] .
It is apparent from previous research that trust in authorities and satisfaction with communications received are associated with compliance of preventive, avoidant, and management behaviours [2, 3, 22] . People want the truth, even if it is worrisome, so honesty is crucial [24] even if that meant being told "we don't know, at this stage".
"Give it to us the way it is. Because I'm sure adults are capable of dealing with that information, and then, you know, making their own choices later of how they deal with the information, but to actually be given that information, without any drama, and yet not being, you know, pushed under the rug somewhere." (P3) While some participants expressed confidence in the organisations providing information, others felt that they were not being given all the facts and that this affected their ability to make informed decisions. Trust in the information given is important because it affects the perceived credibility of risk assessments from authorities which in turn can influence response behaviours [3] . It is important to note that trust can be easily lost if people believe that agencies are not acting in their interests or do not provide information that meets their needs and once lost, trust is difficult to regain [6] .
"Well, I'm always dubious about the... particularly the death rates that... that was not real... from what I understand, a lot of people that died had pre-existing conditions." (G2) Transparency and honest communication where both good and bad news is conveyed empowers the public to make their own decision [1] and that openness of government communication and acknowledging uncertainty is important for fostering trust [3] .
Consistency of advice also appears to be an important factor in communications from key agencies [3] . Participants provided a number of examples where conflicting information or advice led to a feeling of confusion and frustration and loss of trust in key sources.
"...at the beginning of the swine flu, the communication breakdown between the hospital and the local GP services. Because people were coming to the GPs, and they were referring them to the hospital, and then they were... (saying "No, we only take emergencies"), there's a lot of confusion." (P1) Dissatisfaction with health providers was not limited to poor inter-agency communication. Just getting an appointment with a GP was difficult for some and others had concerns about how they were treated and the advice which they were given. The issue of financial impact is also relevant to seeking medical assistance with a number of participants reporting that the high cost of a doctor's appointment was definitely a deterrent to seeking treatment. The issue of trust is illustrated by this extract from a woman who believes she was misdiagnosed in the emergency department putting vulnerable family members at risk.
"I don't think she knew what I had. I just think she wanted to go tick, "Goodbye, here's the antibiotic." ... and I believed her. And that's how naive I was. I should have gone again, but I thought, "Oh no, she's got the ticket. She's got the certificate that says 'doctor'." I trusted her. ... when I came here further, I had the swine flu, I just wanted to get out and go back up there and find that little lady, and taking her and her certificate, and bang her up the side of the head and say, "You could have taken out my granddaughter, my daughter, and my father, because you were in too much of a hurry to go tick, tick, tick." (P2)
Focus group participants expressed a desire for practical information to guide their responses to the influenza threat. Such views are consistent with previous reports of research participants preferring risk messages that empower with information about actions that could reduce risk and/or mitigate consequences [4, 5, 12] . People want to know how to protect themselves and their families during an influenza pandemic and the ability to act provides a sense of relief that "they could do something" [8] . It has been reported that participants who received little or no information about protective actions they could take, expressed helplessness and frustration [8] ,
Language preference has been shown to be an important factor in satisfaction with risk communications [4] . Participants in the Pacific Peoples groups stated strongly that messages should be communicated in an appropriate language.
"Sometimes our older folk, they don't understand English. They have to be in original languages. The diversity of the Pacific -I mean, for some of our older people, because it's their cultural background, and it's so hard for them to understand." (P2)
The acceptance of public health messages can be affected by factors such as socio-cultural behaviours, gender roles, generational differences, religious beliefs and language preferences [9] . The following extract also supports the argument that emotions can also cloud people's decision making, so communicators must treat audiences respectfully [24] . In order to accommodate these cultural and demographic factors it is important to work through communities.
"Sometimes I don't think it's just not only their language....it's the way you use your language. You speak too fast, your English words are beyond me. And they're not dumb, the people. .... Sometimes it's just the way you... your tone. If you talk to them like they're dumb, well they'll just...They'll shut up." (P2)
The following comment from a kaumātua also supports the argument that key health information and advice must take into account factors such as socio-cultural behaviours, spiritual beliefs and language preferences. In the event of an outbreak or pandemic, it would be difficult for kaumātua not to observe the protocols that are very much part of their traditional practices.
If there was a outbreak we would have been ok, we were concerned at the lack of knowledge of tikanga Māori and it is not usual to have to stay away from marae or to stay at home, the thought of mass burials in a pandemic was culturally irresponsible." (M1)
Discussions around preferences for the timing and frequency of key health messages were contradictory. On one hand participants, especially those in one Pacific Peoples group expressed a desire to be given early information through frequent messages. Other participants however, felt that they were being "bombarded" with too much information too soon.
Participants wanted to be warned about potential risk well in advance to allow time for them to prepare. This supports the view that occasional media reports are insufficient to adequately inform individuals about pandemic preparedness, and interventions are needed before a pandemic occurs to improve public awareness, promote effective coping responses and help in the successful implementation of plans [25] .
In contrast others thought it would be better if information was given much closer to the actual event. This is consistent with previous research that found participants preferred "just in time" delivery of information to avoid having to think about pandemic influenza unless they had to or unless a pandemic was imminent [8] . "Just-in-time" messaging that included technical terms, risks, health benefits and protective actions has been shown to help align public perception with realistic assessments of pandemic threat [24] . Both of these perspectives reflect problems with people's risk beliefs. Those who desire advance warnings may be unaware that a pandemic could be in New Zealand very quickly and possibly before its existence is formally identified. They may not have the time they expect to prepare. Those who adopt a "just in time" approach may overestimate their capacity to prepare in a short time frame (e. g., food supplies in supermarkets being rapidly depleted and not restocked).
"I don't know we should say anything unless there's a clear danger, because otherwise you just get used to it. I mean you ignore it." (G1)
The extract above illustrates the serious problem that arises from warning fatigue. Too many early warnings can result in cynicism, disengagement and a decline in trust [26] . Warning fatigue is more likely to result from potential threats with a 'long-lead-time'like pandemics, where there are many warnings in the absence of the actual threat. People "get sick of hearing it" and are likely to "switch off" and ignore future warnings.
"But I think part of it is, that we've had so many health scares in the past decade. Like SARS, and different types of flu, and it's all this big media hype thing....and then it just kind of disappears." (G3)
Focus group discussion about other information campaigns highlighted differences between what participants considered to be effective communications and from who they wanted to hear important messages. Some advertisements were seen as very effective because they were "straight-up, to the point" and included people to whom the participants could relate.
"Because they're using the Pacific People, and the different languages. Like after the Cook Island one, they have a Cook Island lady saying, ("Don't have hesitation")." (P2)
The perceived success of other campaigns was associated with the person fronting that campaign as much as the quality of the presentation.
"Going back to those first John Kirwan (sporting celebrity) ads, they were just really nicely shot, with someone that... most New Zealand men would respect John Kirwan. He was the ideal person." (G1) Such comments confirm previous research that shows that people are more likely to act when information comes from within their own community; community leaders are highly credible sources of information and people would prefer them to be trained in issues of pandemic risk management [27] .
The front person was an important factor in presenting important messages. There was some interesting debate about who could "be trusted" and who was "believable". Some participants felt that important messages should come from medical professionals or official agencies such as the Ministry of Health, District Health Board or WHO. This view highlights the importance of credibility which is regarded in the literature as a critical element in effective risk management and communication [28, 29] . Communicators who are also scientists and perceived as being impartial and knowledgeable are more likely to be regarded as credible [28] .
"Well, the Ministry of Health, ideally, should front it, because you know, they're the national organisation." (G3)
Others argued that the front person should be a role model, or someone recognisable to the public at large. "...I think it might stick in your mind a lot more if you'd got someone that looks familiar to you telling you the information. And I think that we do actually trust someone that maybe is a household name, more than someone that they have no idea who they are". (P1)
As some participants pointed out, it may be that a variety of communicators may be best. Indeed there is support in the literature for employing a multidisciplinary approach to risk communication with input from a range of experts [1, 24] .
Any conclusions drawn from this study should be considered tentative as the findings cannot be generalised to the population at large. It is not known whether the individuals who chose to participate differed from those who were eligible but chose not to participate. Whilst this study intentionally involved participants with diverse cultural and ethnic backgrounds, and included individuals from vulnerable groups, the sample does not permit conclusions regarding the effect of socio-demographic factors such as age or gender. Further research is needed to explore the complexities involved in the way in which the framing of risk messages impacts on people's perception of risk and subsequent preparedness and response behaviours.
The results of this study highlight the problem with a "one size fits all" pandemic warning strategy that risks antagonising and distancing communities and thereby reducing trust in agencies and the likelihood that advice will be followed. Agencies must acknowledge that the public are diverse and need to be involved in the development and management of pandemic response initiatives appropriate for different communities and sensitive to existing cultural and/or spiritual practices.
Pacific Peoples and Māori focus groups identified community institutions with established mechanisms which could provide useful vehicles for the dissemination of information and engaging with the community. With a community engagement perspective the role of the health agencies would be that of consultant to the community or a change agent rather than trying to disseminate directly to the public in a top-down approach. For all segments of the population, the effectiveness of risk communication can be increased by using community engagement and empowerment principles that help tailor information to the needs and expectations of diverse groups.
Pandemic management strategies need to be seen in relation to the wider context of overall societal emergency preparedness. Preparedness for specific events, such as pandemics, might be better situated within more general risk campaigns rather than as stand alone approaches. Such a collaborative approach would also help reduce the sense that people have of being bombarded with information from multiple sources with frequently conflicting messages.
Information alone is insufficient to motivate people to prepare. The way in which information is presented or conveyed is an important factor in determining an individual's response. People wanted messages about specific actions that they could take to protect themselves and their families and to mitigate any consequences. They wanted transparent and honest communication where both good and bad news is conveyed. There was a desire across all groups for clear and specific information, such as infection and/or death rates and defining symptoms. This reflects a failure to distinguish between the pandemic and its consequences and highlights the importance of doing so for risk communication. Quantifying Type-Specific Reproduction Numbers for Nosocomial Pathogens: Evidence for Heightened Transmission of an Asian Sequence Type 239 MRSA Clone An important determinant of a pathogen's success is the rate at which it is transmitted from infected to susceptible hosts. Although there are anecdotal reports that methicillin-resistant Staphylococcus aureus (MRSA) clones vary in their transmissibility in hospital settings, attempts to quantify such variation are lacking for common subtypes, as are methods for addressing this question using routinely-collected MRSA screening data in endemic settings. Here we present a method to quantify the time-varying transmissibility of different subtypes of common bacterial nosocomial pathogens using routine surveillance data. The method adapts approaches for estimating reproduction numbers based on the probabilistic reconstruction of epidemic trees, but uses relative hazards rather than serial intervals to assign probabilities to different sources for observed transmission events. The method is applied to data collected as part of a retrospective observational study of a concurrent MRSA outbreak in the United Kingdom with dominant endemic MRSA clones (ST22 and ST36) and an Asian ST239 MRSA strain (ST239-TW) in two linked adult intensive care units, and compared with an approach based on a fully parametric transmission model. The results provide support for the hypothesis that the clones responded differently to an infection control measure based on the use of topical antiseptics, which was more effective at reducing transmission of endemic clones. They also suggest that in one of the two ICUs patients colonized or infected with the ST239-TW MRSA clone had consistently higher risks of transmitting MRSA to patients free of MRSA. These findings represent some of the first quantitative evidence of enhanced transmissibility of a pandemic MRSA lineage, and highlight the potential value of tailoring hospital infection control measures to specific pathogen subtypes. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a high burden of morbidity and mortality worldwide [1] [2] [3] [4] . While community-associated MRSA is becoming increasingly important globally [5, 6] , in many countries, including the United Kingdom, MRSA remains predominantly a nosocomial pathogen [7, 8] . The dominant sequence type (ST) in Asia is ST239, and recent analysis of whole-genome sequence data has shown that this ST has distinct lineages in Asia, Europe and South America which probably share a European ancestor [9] [10] [11] . Little is known about what has enabled this ST to be so successful, or whether its propensity to transmit between hosts differs from other MRSA types in certain settings. A recent concurrent outbreak due to an ST239 MRSA strain (ST239-TW, subsequently referred to as TW) and the two dominant endemic UK MRSA types (ST22 and ST36, which we refer to as non-TW) in two linked adult intensive care units in a London teaching hospital provided a rare opportunity to compare the transmissibility of different MRSA types in the same clinical setting [12] .
The transmissibility of a potentially emerging pathogen (the rate at which it spreads from an infected host to exposed susceptible hosts) is an important factor in determining its success and, in the case of an established pathogen, for estimating how effective interventions must be to bring an epidemic under control [13, 14] . Quantifying the degree to which strains of a nosocomial pathogen differ in their transmissibility in a particular setting could lead to a better understanding of why major clonal replacements occur. Measuring how such transmissibility changes in response to interventions would allow us to quantify the value of specific control measures, which may vary according to the strain [15] . This could lead to better resource use by allowing us to choose control measures appropriate for the specific strain. Such an analysis has greatest relevance for predominantly clonal organisms, such as S. aureus, where distinct lineages cocirculate over extended periods of time [10] .
A fundamental measure of the overall transmission potential of a pathogen in a given setting is the basic reproduction number, R 0 . This is defined as the mean number of secondary cases generated by a typical case in a fully susceptible population [16, 17] . If the transmissibility of each infected host remains constant throughout its infectious period, and if each infected host has an equal chance of infecting each susceptible host, then R 0 is simply the product of the mean rate at which an infected host generates secondary infections and the mean infectious period (provided the two are not correlated).
The self-sustaining chain reaction that constitutes a major epidemic is possible only if R 0 is greater than one. If it is less than one, although there may be some self-limiting chains of secondary transmission following the introduction of an index case (and quite large clusters become possible as R 0 approaches one), this will not lead to a sustained increase in cases and, in a large population, only a small proportion of susceptible hosts will be infected [18] . An important related number is the net (or effective) reproduction number, R t . This is defined as the average number of secondary cases generated by a case infected at time t, accounting for incomplete host susceptibility to infection and control measures in place. If R t is greater than one at time t, the epidemic will (on average) be growing. If R t is less than one, it will be declining [16, 19] .
These reproduction numbers are central to a mechanistic understanding of infectious disease epidemiology, and a number of methods for estimating them from different types of surveillance data have been devised [16, [19] [20] [21] [22] [23] . However, epidemics that predominantly affect hospitalized patients require some special considerations. First, unlike the community setting, the population of those exposed to infection changes rapidly over time as patients are admitted and discharged. Second, most common nosocomial pathogens are bacteria which can be carried asymptomatically over long periods, during which time colonized hosts may have several hospital admissions. This can give rise to distinctive dynamics: in addition to the usual explosive outbreaks, we also see epidemic patterns characterized by a sequence of self-limiting clusters of transmission which, over time, become more frequent and eventually coalesce into an exponentially growing epidemic [24, 25] .
The concept of the single admission reproduction number, R a , can help in the understanding of these features of hospital epidemics [25, 26] . R a is defined as the mean number of secondary cases caused by a typical infectious patient during a single admission to a particular hospital or ward otherwise free of the pathogen. Necessarily, R a is less than or equal to R 0 . However, if R a v1 and R 0 w1 then every outbreak will be locally controlled in the short term, but, with repeated challenges to the hospital, longterm control failure will be inevitable. This results from the persistence of carriage following discharge which, over time, leads to a gradual increase in numbers colonized on admission. To account for changing numbers of susceptibles, we can also define a net single admission reproduction number, R a,t . This is analogous to R t and represents the average number of secondary cases generated during a single hospital/ward admission where not everyone is necessarily susceptible.
Direct ascertainment of R a and R a,t would be possible if we could reliably assess who infected whom during a hospital outbreak. In practice, even with detailed surveillance and molecular typing data, there is almost always considerable uncertainty about the true transmission tree. Instead, computationally-intensive approaches based on fitting mechanistic mathematical models to data which account for uncertainty in transmission routes and screening data represent the state-of-the art for analysing nosocomial transmission dynamics [27] [28] [29] [30] [31] [32] . However, such approaches require detailed data on both susceptible and colonized or infected patients, and an assumption that temporal changes in the transmissibility can be described parametrically by some standard functional form (most commonly, piecewise constant). As currently implemented they do not allow direct estimates of the number of transmission events associated with each patient.
The aims of this paper are twofold: to describe a new approach (method 1) for estimating R a,t using hospital surveillance data; and to use it to analyse MRSA data from concurrent outbreaks with different MRSA types (TW and non-TW) in two linked adult intensive care units (ICUs). The method is simple to use and enables us to track how R a,t changes over time without the assumption that changes in transmissibility follow a fixed functional form, and without requiring data on susceptible patients. The method extends techniques for the probabilistic reconstruction of epidemic trees developed for analyzing foot and mouth disease and SARS data [21, [33] [34] [35] . We contrast results using this approach with that from a fully parametric mechanistic model (method 2), which represents an adaptation of previously described parametric models for nosocomial infection to a multistrain system [31, 32] . This second approach allows R a to be estimated. It requires more detailed data and stronger assumptions, but allows us to explicitly test hypotheses about how transmissibility is affected by interventions, and how it varies between different wards and subtypes of MRSA. While betweenclone and between-ward differences in single admission effective reproduction numbers, R a,t , calculated using method 1 may be caused by differences in transmissibility, number of susceptibles, and lengths of stays, with method 2 we assume all MRSA positive patients have the same length of stay distribution and explicitly adjust for different numbers of susceptible patients when calculating R a .
Different strains of hospital pathogens may differ in their ability to spread between patients and respond differently to control measures. Attempts to quantify such betweenstrain variation are lacking in high prevalence settings. We analysed data from concurrent outbreaks with different MRSA strains in two adult intensive care units. MRSA is usually carried by patients asymptomatically, and most of our data came from routine screening swabs used to detect such carriage. We divided strains into two groups: common United Kingdom strains and strains from a type often found in Southeast Asia. We developed a new method to estimate how transmission changes over time and compared results with those from an adaptation of a previously described approach. An advantage of the new method is that it makes weaker assumptions about the process generating the data. The methods gave broadly similar results: the introduction of daily antiseptic bodywashes for all patients was the only intervention associated with a substantial fall in transmission, but this intervention was less effective for the Asian strain. This work should be useful for assessing the between-strain variation in the transmission of other hospital pathogens, and for assessing the impact of interventions on patientto-patient transmission.
Under baseline assumptions, on ICU1 there were 282 MRSA importation events (episodes where patients were assumed to be MRSA positive when admitted to the ICU) and 132 acquisition events. These comprised of 12 importations and 23 acquisitions with TW MRSA and 270 importations and 109 acquisitions with non-TW MRSA. On ICU2 there were 285 importations (25 with TW) and 166 acquisitions (43 with TW) (figure 1). Importations with non-TW to the respective ICUs decreased from 0.20 and 0.19 per day in phase 1 to 0.11 and 0.13 per day in phase 4. In contrast, importations with TW MRSA peaked in phase 2 in both ICUs (at 0.03 and 0.12 per day) and were at or below 0.01 per day in phases 1 and 4. Amongst patients who were MRSA positive on admission the median length of stay was 12 days (inter quartile range [IQR]4, 18) for TW-positive patients and 6 days (IQR 3, 13) for non-TW patients (p~0:01, Wilcoxon rank sum test with continuity correction). For patients who acquired MRSA the corresponding numbers were 26.5 (13.25, 42.5) for TW patients and 19 (12, 29.25) for non-TW patients (p~0:02). There was no evidence that length of stay differed by ward (p~0:79), or by study phase (p~0:44, Kruskal-Wallis rank sum test).
Over the study period (January 2002 to April 2006) there were three interventions (referred to as A, B and C) and these define four study phases. Estimated net single admission case reproduction numbers (expected number of secondary cases per case during a single ward admission) associated with each MRSA-positive patient episode are shown in figure 2 (bottom panel) together with histograms of case reproduction numbers for each ward and study phase (top panel). These highlight wide between-patient variability which decreases in the second half of phase 4 when transmission is reduced and the TW clone is eliminated. While most patients have a very low expected number of secondary cases, 22 out of 103 patients (21%) with TW MRSA are expected to transmit to at least one other patient. Corresponding numbers for non-TW MRSA are 40 out of 762 (4%). This proportion was consistently higher for TW MRSA in all four study phases: 25, 11, 18 and 31% versus 7, 0, 9 and 2% for non-TW MRSA.
Aggregating these reproduction numbers into four-week intervals highlights the temporal trends, differences between wards and impact of interventions (figure 3). In ICU1 these suggest similar patterns of transmission for the different MRSA types for the period prior to intervention C (a surface antiseptic protocol). In contrast, there were marked differences between MRSA types in ICU2 and throughout the study period the four-week averaged reproduction numbers for the TW clone usually exceeded those for non-TW clones when both types were present. These differences are also seen when reproduction numbers are averaged over study phases (table 1); the TW clone had a higher reproduction number than the non-TW MRSA in each phase in ICU2 but not in ICU1. Reproduction numbers for TW MRSA were also more volatile than those for non-TW MRSA in ICU2. There was evidence from both units to suggest differences between the MRSA types in their response to infection control interventions: while the net reproduction number of non-TW MRSA fell to a low level following intervention C in both ICUs, this was not the case for the TW clone which continued to transmit for several months at pre-intervention levels. Eventually, the TW outbreak came to an end after all patients with TW MRSA were treated empirically with systemic antibiotics (linezolid) from 1st September 2004 [12, 15] . After this intervention, although patients with TW MRSA continued to be imported into the ICUs, only three isolated apparent transmission events occurred (figure 1). When reproduction numbers for the two ICUs combined were estimated (allowing for cross transmission between ICUs) the results suggested the reproduction number of the TW clone was consistently higher than that for the non-TW MRSA and varied little throughout the study period (table 1) . Reproduction numbers for the non-TW clones, in contrast, fell in phase 2 and 4. These results were not 
Results from method 2 showed broad agreement with these findings, but in contrast to method 1 made a priori assumptions about the timing of the changes in transmissibility (tables 2-3, figure S1 ). On both wards, averaging over all phases, there was about a 1 in 400 chance of a given susceptible patient acquiring MRSA from a particular MRSA-positive patient on a particular day (table 2) . When estimates of daily transmission probabilities from a single MRSA positive patient were constrained to take the same values for the TW and non-TW clones but were allowed to vary by ward and study phase, we found clinically significant variation between the four study phases in both ICUs (table 2). In particular, while estimates were similar in phases 1 to 3, there was a marked reduction in phase 4. There was no strong evidence that these joint estimates (for all MRSA clones) varied by ICU in any of the study phases (table 2) . These findings were robust to the assumptions made about acquisition events and times (supplementary table S3).
Extending this analysis to allow transmission probabilities to vary with the MRSA type enabled differences between MRSA clones and wards to be quantified (table 3) and allowed hypothesis tests about whether the daily transmission probabilities differed between strains (thus allowing us to test whether the observed differences in transmissibility found using method 1 could be entirely explained by the longer length of stay of the TW patients). In ICU1 no consistent differences were seen. In ICU2 the TW clone had a higher daily transmission probability to susceptible patients in each of the four study phases under the baseline assumption of complete bacterial interference, though confidence intervals were wide and showed considerable overlap. Differences between TW and non-TW MRSA transmission probabilities reached statistical significance at the 5% level for both wards combined and for ICU1, but not for ICU2 alone, and in only one of the four phases (phase 4) using combined data from both ICUs. This phase corresponded to the introduction of the surface antiseptic bodywash protocol, which was associated with a more than halving of the transmission probability from a patient with non-TW MRSA compared to earlier phases. The fall in the transmission probability for TW MRSA in phase 4 was smaller, and likely to be confounded by the use of linezolid for TW carriers in this phase. Large differences in transmission probabilities for the two MRSA types were also seen in phase 2 (corresponding to the introduction of hand hygiene promotion), but in this case confidence intervals were wider reflecting the short duration of this phase. In other phases differences between TW and non-TW estimates were much smaller. The magnitude of the differences depended on which patients were assumed to be susceptible. Under the baseline assumption that patients colonized with one strain were not susceptible to acquiring another (complete bacterial interference) the differences were larger than in the sensitivity analysis where no bacterial interference was assumed (table 3) . This can be explained by the higher prevalence of non-TW MRSA clones; under the assumption of no bacterial interference all non-TW MRSA positive patients would be considered susceptible to infection or colonisation with TW MRSA and vice versa. Changing from complete interference to no interference therefore results in a greater increase in the number of susceptibles available for the TW clones to infect than it does for the non-TW clones. To accommodate these changes, a larger reduction in the daily transmission probability for TW clones is required. Overall, combining data from both wards, the TW clone was estimated to have a daily transmission probability that was between 63 and 100% higher than the non-TW clones in phase 4, and between 53 and 94% higher in phase 2 (the lower numbers corresponding to the no bacterial interference assumption) though the differences only reached significance at the 5% level in phase 4 and only under baseline interference assumptions. Transmission probabilities were broadly similar in the two other study phases. Estimates of the single-admission reproduction number (R a ) from the model without background transmission (and assuming TW and non-TW patients have the same length of stay distribution) are reported in the supplementary material (figure S1). Results were robust to the assumptions made about the number and timing of MRSA acquisition events (supplementary table S4), but fitting a more complex model allowing patient-to-patient transmission and transmission from background sources suggested that the relative importance of patient-to-patient and background transmission could not be reliably identified in such hyperendemic settings without additional data (supplementary table S5 ).
Common bacterial nosocomial pathogens have distinct dynamics from typical community pathogens and call for different analytical approaches. Important features of hospital epidemics with such organisms include: i) a host population that changes rapidly over time in comparison with the timescale of epidemic dynamics; ii) a high proportion of infected (or colonized) hosts who are already infected when they enter the population (the hospital or ward); iii) a dominant role for asymptomatic infection so infected hosts can usually only be identified using screening swabs, leading to large uncertainty in the timing of transmission events; iv) a lack of a well-defined serial interval or generation time (since asymptomatic carriage can persist for months or years, but transmission is only intermittently observed during hospital admissions). The probabilistic tree reconstruction approach described above (method 1) overcame these limitations by using a hazards-based approach applied to patient screening data to assign probabilities to potential source patients for observed Phase-specific estimates for the daily probability of a susceptible patient acquiring MRSA from an MRSA positive patient in the same ward, for ICU 1 and ICU 2 (without distinguishing between TW and non-TW strains). In the Combined row, the estimates are constrained to be the same in both wards, and the All phases column constrains the estimates to be the same in the four phases. acquisition events. Using hazards in this way to reconstruct epidemic trees and estimate reproduction numbers appears to have first been suggested by Kenah et al [36] . Results using this method were supplemented with a maximum likelihood approach (method 2) where the timing of cross-infection events was assumed to be known but which allowed estimation of the daily transmission probability, enabling us to study effects related to study phase and MRSA type while controlling for differences in length of stay. These methods were applied to data from two adjacent general ICUs in which admission and weekly MRSA screens and culture results from clinical samples identified patients admitted with and acquiring MRSA over a four year period. During that time there was sustained transmission with endemic MRSA and a newly introduced TW variant.
Both analytical methods supported the hypothesis that intervention C (the surface antiseptic protocol) was associated with a sustained reduction in MRSA transmission, and both indicated a reduced effect for the TW clone. Both methods gave point estimates that indicated elevated transmission of TW MRSA compared with endemic strains in all four study phases in ICU2 but not ICU1. There were, however, some differences: the wardlevel reproduction numbers (method 1) tended to indicate greater increased transmission for the TW compared to non-TW MRSA than was seen using method 2. This reflects the fact that the two methods are quantifying different things: method 1 estimates secondary cases per case, which depends both on transmissibility and the length of ICU stay while carrying MRSA; method 2, in contrast, estimates only the daily transmission probability from one MRSA carrier to one susceptible patient. This will not be affected by length of stay. Indeed, there was some evidence that patients colonised with TW MRSA (particularly those colonised on ICU admission), had a longer length of stay than those colonised with non-TW MRSA. This may reflect the link between MRSA infection and excess length of stay in this cohort [37] , and the increased virulence of the TW strain which was over four times more likely to cause blood stream infection in colonised patients compared to non-TW MRSA strains in the same ICUs [12] . Even in the absence of an increased rate of transmission to other patients, increased length of stay would lead to a higher singleadmission reproduction number. It is possible that such differences in length of stay reflect underlying differences in the characteristics of patients most vulnerable to acquiring the different MRSA types. For example, because the TW outbreak was centred on the two ICUs, patients carrying TW on ICU admission might be more likely than patients carrying non-TW MRSA to have had recent ICU admissions. The TW clones showed a far broader range of antibiotic-resistance than endemic MRSA clones and have previously been shown to preferentially colonise vascular catheters but not carriage sites compared with endemic strains [12] . Taken together, these observations suggest that the TW MRSA could represent a phenotype particularly adapted to transmission in Table 3 . Estimates of the daily transmission probability (q) from one exposed to one susceptible patient. Estimates of the daily transmission probability (q) from one exposed to one susceptible patient. settings, such as ICUs, with high levels of antibiotic usage and patient catheterisation, perhaps at the expense of persistence outside these areas. There is some evidence that such adaptation results from both increased persistence in the ICU (perhaps by targeting long-stay patients, and causing infections that increase length of stay) and from an increased daily transmission probability (particularly in the presence of widespread antiseptic use). Caveats, of course, apply: differences in lengths of stays between TW and non-TW colonized/infected patients could be confounded by exposure history (the recent arrival of the TW clone rather than its biological properties may account for the different patient characteristics). Differences in daily transmission probabilities could also be subject to such confounding and could also have arisen by chance (in all phases -even phase 4, where the effect size was largest -confidence intervals were wide). The mechanisms underlying variations in transmissibility of different MRSA (and S. aureus) strains are poorly understood. Reasons for the differences in the two ICUs are also unclear. Chance variation cannot be ruled out, as the formal investigation of transmission potential of different MRSA types was, in part, motivated by perceived differences in transmissibility (using the same data), and the usual limitations of post hoc analyses therefore apply. Also, although the analyses accounts for demographic stochasticity, there may also be important sources of environmental stochasticity which are not accounted for. It seems unlikely that the difference in TW transmission in the two ICUs can be explained by colonized staff: a universal staff screening programme failed to detect the TW clone during the outbreak [12] . Differences in infection control practice also seem unlikely but cannot be ruled out: the two wards share the same infection control policies and staff pool, with medical and nursing staff rotating between units at 3-6 monthly intervals, though only physiotherapy, radiology and pharmacy staff worked across both units at the same time. It is possible that the built environment influences MRSA transmission. ICU2 was last refurbished in 1969, retaining a mixture of original materials including wood, and has much less open space, only eight sinks, and one side room, whereas ICU1 was refurbished in 1999 to an open plan configuration with better space utilization, 19 sinks and three side rooms. The reduced availability of sinks, side rooms and space to circulate may have adversely affected the ability to carry out infection control practice or cleaning, although it is unclear why this should only affect TW MRSA, which was not detected on environmental screening during the outbreak [12] .
Despite anecdotal reports that some lineages of S. aureus strains have an enhanced epidemic potential in hospital settings [38] , objective assessments of between-strain variation in transmissibility are largely lacking. Such variation is nonetheless to be expected given the large degree of phenotypic variation in different S. aureus and MRSA clones, and the dominance of a small number of MRSA lineages [39] . One of the few instances where the nosocomial transmission potential of different subtypes of the same nosocomial pathogen have been quantified comes from a comparison of the onward transmission from patients admitted to hospitals in the Netherlands carrying MRSA [40] . In this case, because MRSA introductions were infrequent (as MRSA prevalence in hospitals in the Netherlands is below 1%) and contact tracing extensive, the secondary cases could be assigned to distinct clusters of transmission following identified introductions. This allowed the authors to use methods based on a branching process model to estimate the single admission reproduction number, R A [41] . It was found that newly admitted ST398 MRSA strains (which are commonly associated with livestock production) had a greatly reduced propensity to spread compared with other MRSA sequence types, with an R A value (95% CI) of only 0.16 (0.04-0.40), about one sixth of the corresponding value for non-ST398 MRSA. The authors concluded that less stringent control measures were likely to be sufficient to control ST398 MRSA clones than those needed for non-ST398 MRSA types.
Such methods would not have been applicable for our data, and the first method used here to quantify the transmissibility of different strains (method 1) instead built on recent approaches to estimate reproduction numbers by probabilistically reconstructing epidemic trees. Such tree-reconstructions have used simple rulebased methods, for example assigning sources from a candidate list based on proximity data [33] , more formal semi-parametric methods using partial likelihoods and assuming a known serial interval distribution [21, 34] , and, most recently, semi-parametric hazard-based approaches [36] . Hazard-based approaches have some advantages over the first two methods: they avoid some of the arbitrary assumptions of the rule-based approaches, do not require knowledge of the serial interval distribution, and can avoid biases that arise from the fact that the serial interval distribution changes over the course of an epidemic. Advantages over approaches based on fitting a full transmission model include fewer assumptions, in particular with regard to the functional form of changes in the transmission potential over time. In this respect, tree reconstruction approaches have some similarities with other semi-parametric approaches that make use of survival analytical methods, such as the approach adopted by Wolkewitz et al., who derived non-parametric estimates of a time-varying transmission rate changed over time using a Martingale-based method [42] . An important difference in the current approach is that we are specifically interested in estimating how the distribution of the number of secondary cases resulting from each case changes over time. The method 1 approach described here also makes relatively low demands for data (with no information required for patients who do not become colonized or infected), has a low computational burden, and can be easily adapted to cope with cocirculating subtypes as in the application here. This approach is appropriate when the daily probability of a patient acquiring MRSA is small, as in this case reconstructed epidemic trees will be approximately independent of this probability. This approximation is likely to be reasonable for all but the most explosive outbreaks. For example, using the exact formula we found that changing this probability from a baseline of 0.005 to 0.001 and 0.025 changed the estimated mean reproduction numbers for each phase and MRSA type by less than 3%.
Two assumptions underlying the analytical approaches used here are i) that new MRSA acquisitions can be explained by patient-to-patient spread within the units (which is likely to be mediated by contacts with transiently colonized healthcare workers) and ii) that risk of transmission increases in line with colonization pressure (the number of patients with MRSA on the ward). While these assumptions are supported by observational and quasi experimental studies [43, 44] , it would be desirable to more rigorously challenge them. Unfortunately, unpublished simulation studies and analysis here with a more complex model allowing different transmission routes (table S5) both suggest that the ability to identify the relative importance of background and patient-to-patient transmission may be limited in hyper-endemic settings in the absence of more discriminatory typing data. The inability of our typing methods to reliably distinguish between non-TW MRSA types, or to identify genetic variants of the TW clone therefore represent important limitations of this work. High resolution genotyping data would enable more definitive assessments of who infects whom, and therefore allow us to quantify the risks of transmission of different MRSA subtypes in different wards at different times with greater certainty. Figure 1 confirms that not all acquisition events can be explained by transmission from a known MRSA positive patient from the same ward. The combined-ICU analysis, allowing for between-ward transmission, is able to account for some MRSA acquisitions where no known source was present on the same ward, and this explains why combined ICU estimates of the reproduction number are sometimes outside the range of individual ICU estimates (table 1), but unknown MRSA sources are also likely to be present in the patient population [32] . A full model-based analysis using data augmentation (which estimates model parameters and latent parameters that represent ''unobserved'' -or augmented -data, typically using Markov chain Monte Carlo methods for fitting) could account for such unknown sources. Such an approach retains some important advantages for analysing typical surveillance data. These include the ability to account for imperfect swab sensitivity and for uncertainty in the number and timing of acquisition events, circumventing the need to make arbitrary assumptions about which patients were colonized on admission to a ward. In the present context such an analysis would allow us to explicitly account for the change in the screening protocol in November 2004. Since this involved screening more body sites, it is likely to have increased screening sensitivity and led to increased detection of MRSA, potentially biasing the estimated effect of intervention C. To date, however, no published work has adapted such approaches to cope with multiple co-circulating subtypes. The method 2 used here can be thought of as a simplified version of such an approach (in that it is based on a fully-specified mechanistic transmission model) but it avoids the complexities of data augmentation by assuming the epidemic process is perfectly observed. An important area for future work will be to extend data augmentation methods to cope with carriage of multiple types. Such approaches have been developed for the sequential carriage of community pathogen subtypes [45] . Addressing issues of co-colonisation with different subtypes may be particularly important for some nosocomial pathogens, and neglecting such effects is a potential source of bias. In our analysis here we considered two possibilities -complete bacterial interference (where one strain completely inhibits the acquisition of another), and no bacterial interference. The reality may lie somewhere between these two extremes. Such an analysis will be complicated by the fact that routinely-used laboratory methods are not well-suited to detecting the simultaneous carriage of multiple types [46] , and sensitivity for detecting a second type will not, in general, be the same as sensitivity for detecting a single type.
Ethical approval for this research was granted by the NHS National Research Ethics Service, South East Research Ethics Committee. All data were analyzed anonymously.
Anonymised data from two 15-bed adult general intensive care units (ICU) within a 1050-bed teaching hospital in London, United Kingdom, were collected between 1st January 2002 and 30th April 2006 as described elsewhere [12, 15] . Dates of admission and discharge and MRSA culture results from screen and clinical samples were analysed for all 4,570 consecutive patient admissions to both ICUs. Infection control policies were in place including specifying hand hygiene between patient contacts and use of contact precautions for known MRSA colonized patients throughout. On this background three main new MRSA control interventions were introduced: intervention A (introduced on 15th July 2003) was an education campaign to promote hand hygiene and barrier nursing; intervention B (introduced on 15th October 2003) was isolation of known MRSA colonized patients in side rooms or in patient and nursing cohort pairs; intervention C (introduced on 26th April 2004) was a surface antiseptic protocol which included daily chlorhexidine bodywashes for known MRSA positive patients, and daily triclosan bodywashes for other patients. The three interventions defined four study phases for analysis: phase 1 from 1st January 2002 to 14th July 2003; phase 2 from 15th July to 14th October 2003; phase 3 from 15th October 2003 to 25th April 2004; and phase 4 from 26th April 2004 to 30th April 2006. Patients were swabbed for MRSA carriage on admission and every Monday morning. Swabs were taken from nose, axillae and perineum until 1st November 2004, when additional rectal and throat samples were included (a change associated with an approximate 30% increase in the proportion of patients identified as carriers on admission to ICU) [47] . Clinical samples were collected when infection was suspected. S. aureus colonies were identified using a combination of catalase positivity, Staphaurex (Remel Europe Ltd., Dartford, England) and/or salt mannite positivity with confirmation by a tube coagulase test. Methicillin resistance was determined by disc testing. Screen samples were identified using a selective mannitol broth technique [47] .
TW MRSA was defined initially by its distinctive and extensive antimicrobial resistance pattern, sequence typing and microarray analysis [12] . More extensive typing of available admission and acquisition isolates has shown all antimicrobial resistance patterns defined TW isolates to belong to CC8/239 and non-TW isolates to be w90% ST22 and ST36 [15] . When TW and non-TW MRSA isolates were recovered from the same patient, only the first type recovered was considered. This was, however, rare: two patients had both types recovered from pooled screening sites; nine had both types from sputum; and seven had both types from wounds. Thirteen patients had both types recovered from different sites. Further details of patient characteristics, interventions, swabbing sites and microbiological procedures have been described elsewhere [12, 15] .
We analyse the data using two separate approaches which we refer to as method 1 and method 2. In both analyses we define a new MRSA acquisition to have occurred if a patient has a negative admission screening swab, a subsequent MRSA positive screen or clinical sample while in the ICU and more than 48 hours after being admitted to the ward, and no prior MRSA positive isolate in the 90 days preceding ICU admission. Patients with any MRSA positive samples taken within 48 hours of admission are assumed to be positive on admission (MRSA importations). A patient who is believed to be neither colonized nor infected on a given day is assumed to be susceptible to becoming colonized or infected by either MRSA type (see supplementary material for further details). In the first approach (method 1), which probabilistically reconstructs the epidemic tree, we assume that the acquisition occurred one or more days before the first positive screening swab. In the second approach (method 2) we assume a new acquisition to have occurred on day t{1 if a patient has his or her first MRSA positive swab on day t, following a negative MRSA admission screening swab during the same ward admission.
We also assume i) that once MRSA positive, a patient remains so until ward discharge (hence no information from swab results after the first positive is used), and ii) MRSA-positive patients only become potential sources for transmission to other patients after their first positive swab, unless they are assumed to be positive on admission, in which case they are potential sources from their date of admission. For patients readmitted to one of the wards following ward discharge, we apply the same criteria that we use for first time admission to determine admission colonisation status. We use a time unit of one day, and take dates of admission and discharge to represent the first and last whole days of a patient admission.
Notation. We introduce the following notation: let q ijt represent the daily probability of a single susceptible patient in ward i acquiring MRSA of type j from a single patient on the same ward at day t who is colonized or infected with MRSA type j. We also define the daily avoidance probability of acquiring MRSA, q' ijt~1 {q ijt .
Denote by S wjt , C wjt , and A wjt the number of patients on ward w on day t who are, respectively, susceptible to MRSA type j, known to be colonized or infected with type j, and found to be colonized or infected with type j for the first time on day t (having had a prior negative admission screening swab). Here we take C wjt to be the number of patients on ward w on day t who have had at least one previous positive swab with MRSA type j on or before day t. We take A wjt to be the number of patients on ward w who have their first positive swab with MRSA of type j on day t. In our default method 2 analysis we take S wjt as the number of remaining patients (i.e. those with no prior MRSA positive swabs during the current episode) excluding those patients who are discharged on day t since we assume that acquisitions on the day of discharge would be not be detected. The tree reconstruction approach (method 1), in contrast, does not require knowledge of S wjt to estimate reproduction numbers.
In the application we consider here there are two wards and we define two subtypes (TW and non-TW), so both w and j can take values 1 or 2. We also define N w to be the total number of patient episodes on ward w over the study period for which there was at least one MRSA positive swab and M w to be the total number of new MRSA acquisitions on ward w over the study period, i.e. M w~P t P j A wjt . Method 1: Reconstruction of the epidemic tree. The treereconstruction approach calculates the probability that each observed new MRSA acquisition was acquired from each of the other MRSA positive patients in one of the two ICUs. In this approach we condition on the probabilities q ijt and assume that all new acquisitions were acquired from a known patient source. A scaling factor, s, specifies the reduction in the daily risk of transmission from an MRSA positive patient in one ward to an MRSA negative patient in a different ward. We explore the sensitivity of the results to both the q ijt and the s values.
Let p kl represent the conditional probability that patient k acquired MRSA from patient l given that patient k acquired MRSA from one of the other P w N w {1 MRSA positive patients. We calculate the elements p kl of the P w M w | P w N w matrix P kl as follows. Define u ijt to be the probability that a susceptible patient on ward i at time t escapes cross-infection from one of the P w C wjt MRSA type j positive patients in the ICUs on that day. Therefore u ijt~Pw 1{s (1{ fwg (i)) q ijt À Á Cwjt where fwg (i) is an indicator function that equals one when w~i and zero otherwise. Now consider a single patient k on ward i who is free of MRSA on admission and whose first and last days on the ward are t k f and t k l . The probability that this patient is free of MRSA type j at the end of day s (sƒt k l ) is where the h wijt terms represent the hazards of transmission of MRSA type j from patients in ward w at time t to a patient in ward i and lijs is an indicator function that takes the value 1 if patient l is present on ward i and MRSA type j positive on day s and 0 otherwise. These hazards can be expressed in terms of the probabilities q ijt as h wijt~{ C wjt ln(1{q ijt s (1{ fwg (i))), which is approximated by C wjt q ijt s (1{ fwg (i)) when q ijt is small, as will usually be the case. The conditional probabilities, p kl , which represent the probability that patient k was infected by patient l given that patient k was infected by one other patient, are then given by the following expression, which is approximately independent of q ijt when q ijt is small:
Here m indexes all the patients who could potentially have infected patient l. The net single admission reproduction number for patient episode l (i.e. the expected number of secondary cases resulting from this episode) is then given by R l~X m p ml and corresponding reproduction numbers for a given time period are obtained by averaging over these patient reproduction numbers for all patient episodes starting in the given period. Associated confidence intervals are derived by simulation, by repeatedly drawing the source of infection for each of the P w M w new infections from a multinomial distribution with probability vectors given by the rows of P kl . All confidence intervals reported for reproduction numbers are based on the quantiles of 1000 such simulations.
By default, when analysing both wards together, we assume minimal cross-infection between the wards (s~0:0001), though we also consider the opposite extreme (s~1), representing complete ward mixing. We report results here where q ijt is fixed at 0.005, though also describe results of sensitivity analyses with values of 0.001 and 0.025.
Method 2: A likelihood-based approach. The second approach estimates the probabilities q ijt using maximum likelihood estimation (MLE). With two MRSA types, we assume that patients susceptible to type 1 are also susceptible to type 2, so S i1t~Si2t~Sit . This implicitly assumes complete bacterial interference (i.e. that colonisation with one type of MRSA prevents acquisition of another type one or more days after the first acquisition [48] ). As a sensitivity analysis we also considered the other extreme: complete lack of bacterial interference, so that a patient colonized with one MRSA type had the same daily risk of acquiring a different subtype as an uncolonized patient. With the additional assumption that new MRSA acquisitions occur the day before they are detected, the log likelihood of the new MRSA acquisition data in ward i on day t is given by:
where c is a constant and
Here P i1t (P i2t ) is given by the product of the probability of not acquiring MRSA type 2(1) on day t{1 and the probability of acquiring type 1 (2) . P 0 it is the product of the probabilities of not acquiring either MRSA type. For completeness we could add a term to represent acquisition of both types on the same day. In practice we had no such observations.
The overall log-likelihood is then given by the sum of LL it terms over i (the two wards) and is maximized using unconstrained optimization with the Nelder-Mead algorithm [49] as implemented in the function optim in R version 2.11.1 (www.project-r.org). Approximate 95% confidence intervals (CIs) were derived by inverting the square matrix of second-order partial derivatives of the loglikelihood function, i.e. the Hessian.
In practice, with method 2 rather than estimating separate q ijt terms for every day t (which would over-parameterize the model), we apply constraints. We consider constraints where i) q ijt terms from the same ward and same study phase are required to take the same value; ii) q ijt are the same across all time periods within each ward; iii) q ijt terms from the same study phase are constrained to take the same value and do not vary across study wards; and iv) q ijt terms are the same for different MRSA types, q i1t~qi2t . These constraints imply a series of nested models, and we apply likelihood ratio tests to determine whether there is evidence to reject the hypotheses that these equality constraints represent.
To obtain estimates of R a requires consideration of the length of stay distribution. To simplify matters we ignore any potential additional length of ICU stay caused by infection, but account for the facts that the longer a susceptible patient stays the greater the risk of acquiring MRSA, and the longer an MRSA-positive patient stays the greater the expected number of secondary transmission events they will cause. In simple models it is often assumed that there is constant hazard of hospital discharge and the length of stay distribution is exponential. In such cases the risk of a patient acquiring MRSA is unrelated to the subsequent length of stay and R a is trivially calculated as the product of mean length of stay, the mean number of exposed patients on the ward, and the daily probability of transmission from a single source to a single exposed patient. In practice, the hazard of ICU discharge is likely to be dependent on the day of stay, and typically decreases with increasing day of stay. In this case, the patients at greatest risk of acquiring MRSA (i.e. those who have stayed longest on the ward) will also have longer expected future stays and will therefore tend to cause more secondary infections. To account for this we partition patients into groups defined by the number of ICU days a randomly-selected patient on the ward would stay after becoming MRSA positive (assuming no additional stay due to MRSA). To do this we use the empirical length of stay distribution and calculate the probability, p i , that a randomly selected patient on the ICU is a member of group i. This is given by p i~P ? k~1 l kzi{1 = P ? k~1 kl k , where l k is the probability that a newly admitted patient stays for k days. Assuming an average of n exposed patients per day on the ward, and that each patient has a daily probability, q, of acquiring MRSA from a single MRSA positive patient on the ward, an MRSA patient in group j will, on average, transmit MRSA to k ij~pi |q|j|n patients in group i (ignoring saturation effects, which will be negligible for sufficiently small q). These k ij values are the elements of the next generation matrix. The dominant eigenvalue of this matrix gives the reproduction number, R a [20] .
In contrast to method 1, which excludes susceptible patients from the analysis and enables estimates only of the net (or effective) reproduction number, this method accounts for susceptibles in the model and therefore allows us to estimate the single-admission reproduction number (the transmission potential of an MRSA positive patient in an otherwise fully susceptibility ward). This will be greater than or equal to the net single-admission reproduction number, and determines the threshold epidemic behaviour [25] . A further difference is that this method allows the transmission potential of an MRSA positive patient to change over time, according to the current study phase. In contrast, method 1 makes no explicit assumptions about the timing of changes in the transmission potential, and net reproduction numbers for a particular time period relate to the transmission potential of patients admitted to the ward during that time period even though the actual transmission events may occur at a later time.
Both methods 1 and 2 assume that MRSA acquisition events occur as a result of patient-to-patient transmission from known carriers and exclude observations where there are no potential source patients. An additional sensitivity analysis therefore extended method 2 by allowing for both patient-to-patient transmission and background transmission (for example, from colonized staff or persistent environmental contamination). If the daily probability of a susceptible patient on ward i at time t acquiring strain j from such background sources is q' ijt , then the P i1t term above is replaced by (1{q' i2t )(1{q i2t ) C i2(t{1) 1{(1{q' i1t )(1{q i1t ) C i1(t{1) À Á in the new model, with similar changes for other terms. When applying this model, q' i1t was assumed to vary by ward, MRSA type and study phase, but to remain constant within in a phase. Figure S1 Single admission reproduction numbers (R a ) estimated using method 2. Estimates (95% CIs) of the wardlevel reproduction number, R a , according to study phase, MRSA type and ward obtained using method 2 and assuming complete bacterial interference and no interaction between ICU 1 and ICU 2. (PDF) Protocol S1 Protocol for defining MRSA importation and acquisition events.
Table S1 TW and non-TW MRSA importation and acquisition events under different assumptions. The baseline assumption classifies all episodes where MRSA was recovered from an isolate taken within 48 hours of admission as importations. The SA1 assumption uses a 24 hour cutoff instead. See protocol S1 in supporting material for full details of baseline and SA1 assumptions. (PDF) Table S2 Estimated ward-level reproduction numbers (s.e.) for TW and non-TW MRSA clones under alternative assumptions. Phase-specific estimates of ward-level reproduction numbers for TW MRSA and Non-TW MRSA derived using Method 1 under baseline assumptions with perfect ward coupling (applies to combined ICU estimates only) and under SA1 assumptions (see protocol S1 in supporting material for details of baseline and SA1 assumptions). (PDF) Table S3 q estimates for TW and non-TW combined under SA1 and SA2 assumptions. Sensitivity analysis for phase-specific estimates for q, the daily probability of a susceptible patient acquiring MRSA from an MRSA positive patient in the same ward, for ICU 1 and ICU 2 (without distinguishing between TW and non-TW strains). In the Combined row, the estimates are constrained to be the same in both wards, and the All phases column constrains the estimates to be the same in the four phases. See protocol S1 in supporting material for details of the SA1 and SA2 assumptions used in the sensitivity analyses. 1 P-values test the null hypothesis that transmission does not vary between study phase (likelihood ratio test, df = 3). 2 P-values test the null hypothesis that transmission in the current phase does not differ between wards (likelihood ratio test, df = 1). (PDF) Table S4 Estimates of the daily transmission probability (q) from one exposed to one susceptible patient under SA1 and SA2 assumptions. Estimates of the daily transmission probability (q) from one exposed to one susceptible patient under assumptions SA1 and SA2. See protocol S1 in supporting material for details of the SA1 and SA2 assumptions used in thesse sensitivity analyses. 1. P-values test the null hypothesis that transmission varies between study phases but not MRSA types against the alternative that it varies between study phases and MRSA types (likelihood ratio test, df = 4). 2 P-values test the null hypothesis that transmission in the study phase does not differ between TW and Non-TW MRSA using combined data from both wards (likelihood ratio test, df = 1). (PDF) Table S5 Estimates of the daily transmission probability (q) from one exposed to one susceptible patient and from background transmission sources. 'Patient to patient' estimates corresponds to the daily transmission probability (q) from one exposed to one susceptible patient. Background estimates corresponds to the daily probability of acquisition from background sources (such as environmental contamination). This probability is assumed to remain constant within each phase for each of the two MRSA types. In some cases confidence intervals could not be estimated for numerical reasons, while in others the very wide confidence intervals indicate that parameters are weakly identifiable. For both ICUs we compared the model with background and patient-to-patient transmission (with both phase and MRSA-type specific parameters) with nested models with only background transmission (but still with both phase and MRSAtype specific parameters) using a likelihood ratio test based on the chi-squared distribution with eight degrees of freedom. The results gave strong evidence to prefer the more complex model in the case of ICU2 (p = 0.008), but no evidence to prefer it in the case of ICU 1 (p = 0.70). (PDF) Discovery and Genomic Characterization of a Novel Bat Sapovirus with Unusual Genomic Features and Phylogenetic Position Sapovirus is a genus of caliciviruses that are known to cause enteric disease in humans and animals. There is considerable genetic diversity among the sapoviruses, which are classified into different genogroups based on phylogenetic analysis of the full-length capsid protein sequence. While several mammalian species, including humans, pigs, minks, and dogs, have been identified as animal hosts for sapoviruses, there were no reports of sapoviruses in bats in spite of their biological diversity. In this report, we present the results of a targeted surveillance study in different bat species in Hong Kong. Five of the 321 specimens from the bat species, Hipposideros pomona, were found to be positive for sapoviruses by RT-PCR. Complete or nearly full-length genome sequences of approximately 7.7 kb in length were obtained for three strains, which showed similar organization of the genome compared to other sapoviruses. Interestingly, they possess many genomic features atypical of most sapoviruses, like high G+C content and minimal CpG suppression. Phylogenetic analysis of the viral proteins suggested that the bat sapovirus descended from an ancestral sapovirus lineage and is most closely related to the porcine sapoviruses. Codon usage analysis showed that the bat sapovirus genome has greater codon usage bias relative to other sapovirus genomes. In summary, we report the discovery and genomic characterization of the first bat calicivirus, which appears to have evolved under different conditions after early divergence from other sapovirus lineages. The caliciviruses are a family of small non-enveloped viruses, and can be classified into five genera: Vesivirus, Lagovirus, Norovirus, Sapovirus and Nebovirus. They possess a non-segmented, polyadenylated, positive-sense ssRNA genome of about 7.5 to 8.5 kb in length, enclosed in an icosahedral capsid of 27 to 40 nm in diameter. Among them, noroviruses and sapoviruses (SaVs) are well known to cause enteric disease in a range of mammals, including humans, while vesiviruses and lagoviruses cause systemic diseases in specific animal hosts. Nebovirus is the most recently established genus in the family Caliciviridae [1] , and its members are associated with enteric diseases in cattle [2, 3] . A putative sixth genus, Recovirus, has been proposed for a novel calicivirus detected in stool specimens from rhesus monkeys [4, 5] . Another new genus, Valovirus, has been proposed for a novel group of swine caliciviruses known as the St-Valérien-like viruses [6] . In addition, there exist other unclassified caliciviruses, such as the recently described chicken calicivirus [7] .
The genus Sapovirus currently contains only one recognized species, the Sapporo virus, which was discovered in 1977 in Sapporo, Japan [8] . The SaV genome is approximately 7.1 to 7.5 kb in length, and may have two or three ORFs. ORF1 encodes a polyprotein that undergoes proteolytic cleavage to form the non-structural proteins and the major capsid protein VP1. ORF2 encodes the minor structural protein VP2. ORF3 encodes a small basic protein of unknown function [9, 10] . Interestingly, it is located in an overlapping reading frame within ORF1, and is present only in SaVs from selected genogroups. At present, SaVs are classified formally into 5 genogroups based on phylogenetic analysis of the full-length VP1 sequence, though additional genogroups have been proposed to accommodate some novel SaVs discovered in recent years. Further classification of SaVs into genotypes has also been undertaken, though taxonomic assignment at the genotype level appears to be less well-defined than at the genogroup level [11] .
As mentioned above, both noroviruses and SaVs generally cause mild to asymptomatic enteric infections in human and animal hosts [12] . Human SaV infections are reported to be similar to or milder than human norovirus infections, but SaV infections have a shorter duration of viral shedding and are less associated with projectile vomiting [13] [14] [15] [16] . Incidence of SaV-associated gastroenteritis infections remains less than norovirusassociated infections for both sporadic and outbreak settings, though various studies have reported increasing rates of SaV infections around the world [17] [18] [19] [20] [21] . The genetic diversity of SaVs is comparable to that of noroviruses, and the diversity of reported animal hosts is also similar. Noroviruses have been discovered in specimens from humans, pigs, cattle, dogs, sea lions, African lion, and mice [22] [23] [24] [25] . In comparison, SaVs have been found in specimens from humans, pigs, dogs, minks and California sea lions [23, [25] [26] [27] .
Bats (order Chiroptera of class Mammalia) constitute a significant portion of biological diversity in many ecosystems and have a wide geographical distribution [28] . We have previously discovered novel viruses in several local bat species [29] [30] [31] [32] [33] [34] [35] , and there were many similar discoveries of novel bat viruses by researchers in other parts of the world. In particular, important human viral pathogens like the SARS virus, Nipah virus and Ebola virus were found to have originated from bats and contributed to substantial human morbidity and mortality in recent outbreaks. Taken together, these discoveries hint that these small mammals are important reservoirs of diverse and undiscovered animal viruses, with significant risk of zoonotic transmission to humans [36] .
In the present study, we investigated the presence of unknown calicivirus diversity in bats by targeted RT-PCR screening. Novel SaV sequences were amplified from several faecal samples of the bat species Hipposideros pomona, and genome sequences were obtained for three strains of the bat SaV. Sequence analysis indicated that the novel virus possesses several genomic features atypical of SaVs, and phylogenetic analysis revealed that it descended from a lineage that had diverged early from other SaV.
A total of 728 anal swabs from different bat species in Hong Kong were obtained. No obvious signs of enteric disease, like anorexia and diarrhoea, were observed in the bats during the brief period of captivity needed for sampling.
RT-PCR using broadly reactive degenerate primers for a 185 nt fragment in the 3D-like RNA-dependent RNA polymerase (RdRp) region of the calicivirus ORF1 gene was positive in two specimens. Repeated screening using more sensitive specific primers revealed three additional positive specimens. Further information on the species and RT-PCR screening results are presented in Table 1, Table S1 and Figure S1 . Sequence similarity search using BLASTN against the NCBI nonredundant nucleotide database did not reveal significant similarity to known SaV sequences. Another search using BLASTX against the NCBI non-redundant protein database produced hits to SaV sequences, with the most closely related sequence being the RdRp sequence of porcine SaV (GenBank accession number ACT98315) at 43% aa identity. A phylogenetic tree was constructed from the nucleotide alignment based on the length of the partial RdRp sequence obtained from bat SaV/TLC72 (GenBank accession number JQ267527) ( Figure S2 ).
Complete or nearly full-length genome sequences (with incomplete 59 ends) were obtained for three positive samples using the sequencing strategy as described in the Methods section. For two of the samples that were positive only for RT-PCR screening with specific primers, only sequences for short segments of the viral genome were obtained. Additional viral genome sequencing on these samples was unsuccessful due to limited clinical materials available and possibly low viral titres. The complete genome of bat SaV strain TLC58 (Genbank accession number JN899075) is 7696 nt in length and has a genomic G+C content of 60.2 mol%. Both the length and G+C content of the bat SaV genome are significantly higher than that of other known SaVs (Table 2) . Each genome is predicted to contain 3 overlapping ORFs, comparable to the genome organization of SaVs in GI, GIV and GV ( Figure 1 ). The 59-UTR and the 39-UTR are 9 nt and 225 nt in length, respectively. The length of the 39-UTR is considerably longer than other SaVs ( Table 2 ). The two other nearly full-length bat SaV genomes were found to be highly similar to that of the complete bat SaV/TLC58 genome in nucleotide sequence and genome organization, and were not analysed separately.
The complete ORF1 is 6855 nt long, and encodes a large precursor polyprotein with an estimated molecular mass of 246.8 kDa. The polyprotein contains characteristic amino acid motifs conserved in caliciviruses: 2C-like NTPase at residue 482 (GPPGIGKT), VPg at residues 958 (KGKTK) and 972 (SEYEE), 3C-like protease at residue 1183 (GDCG), 3D-like RNAdependent RNA polymerase at residues 1520 (GLPSG) and 1568 (YGDD), and VP1 at residue 1867 (PPG). It undergoes proteolytic processing to produce the nonstructural viral proteins and the major capsid protein VP1. Based on comparison with the ORF1 cleavage map of SaV/Mc10 [37, 38] , a human SaV GII strain, we predicted the cleavage site that generates the major capsid protein to be located between residues 1740 (E) and 1741 (G). An in-frame AUG start codon is located in a favourable context for translation initiation (GUGUUUGUGAUGGA) just upstream to the cleavage site, which has also been reported in other caliciviruses [6, 39] . This sequence is noted to be similar to the 59 UTR of the genome, and it was postulated that the site might permit internal translation initiation from subgenomic RNA [39] . The sequence identities of the bat SaV/TLC58 with other SaVs in the complete ORF1 protein sequence vary between 36.0% and 37.4% (Table 3) . While comparison with caliciviruses of other genera, the ORF1 sequence identities are overall lower (15.6%-22.8%) than those between different SaVs (Table S2) . For individual alignment of protease -polymerase, sequence identities with other SaVs (45.3%-48.4%) are overall higher than those with other genera (22.7%-32.1%) ( Table 3 and Table S2 ). The VP1 is predicted to be 546 aa long, and has a molecular mass of 56.6 kDa. It shares 36.1% to 39.2% amino acid identities with VP1 of other SaVs (Table 3) . Likewise analysis with caliciviruses of other genera reveals lower similarities with 14.9% to 23.4% sequence identities (Table S2 ). The complete ORF2 is 615 nt long, with an overlapping region of 8 nt with the 39 terminus of ORF1. Its reading frame is +1 relative to that of ORF1, unlike most other SaVs (Table 2) . ORF2 encodes the minor structural protein VP2, which has an estimated molecular mass of 21 kDa. The mechanism of translation initiation in ORF2 of SaVs has not been fully elucidated. In the present case, a translational upstream ribosome binding site (TURBS) motif (CAUGGGACC; underline indicates region complementary to 18 S ribosomal rRNA sequence) could be identified at 24 nt upstream of the ORF2 start codon. Sequence identities for VP2 with other SaVs vary from 15.5% to 19.9% (Table 3) . By comparison with caliciviruses of other genera, sequence identities are generally lower than those between SaVs. (4.8%-12.3%) (Table S2 ).
Phylogenetic trees were constructed using the predicted amino acid sequences of the ORF1 precursor polyprotein (Figure 2 ), VP1 and VP2 ( Figure 3 ). The LG+G+F model was found to be the bestfit substitution model in all cases. Phylogenetic analysis was not performed for the putative ORF3 product as no homologous sequences were available. Sequence analysis with the Recombination Analysis Tool did not reveal any potential recombination breakpoints in the bat SaV sequences.
There are subtle but important differences in the phylogenetic position of the bat SaV in the three phylogenetic trees. In the tree based on the full-length amino acid sequences of the ORF1 polyprotein, the bat SaVs are clustered tightly with the porcine SaVs in a monophyletic clade constituting the SaVs. However, in the VP1 tree, the bat SaVs are positioned just outside the clade of other SaVs. In the VP2 tree, the bat SaVs are located approximately equidistant from the GII noroviruses and porcine SaVs. The phylogenetic positions of the bat SaVs are supported by high Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-like aLRT) branch support values as calculated by PhyML.
Although the phylogenetic positions of the novel bat virus are slightly divergent in the three trees, they generally show the bat SaV as being most closely related to the SaVs. In our opinion, there is insufficient ground for proposing a new genus for the novel virus under the current framework of taxonomic classification. The ORF1 polyprotein and VP1 capsid protein sequences of the novel bat virus showed obvious phylogenetic clustering with other SaV sequences. It should also be noted that the VP2 protein sequences are shorter and more divergent, and therefore are considered to be less useful in the phylogenetic classification of caliciviruses [4] . Lastly, the genome organization of the bat SaV is highly similar to that of the SaVs as shown above. Hence, together with relatively high sequence identities with other SaVs rather than with calicivirues in other genera (Table S2) , we propose that the novel bat virus be classified as a new member of the genus Sapovirus in the family Caliciviridae. Codon usage and compositional bias analysis
As genomic nucleotide composition is strongly associated with codon usage bias in viruses, we examined the codon usage in the genomes of the novel bat SaV and other SaVs given their different nucleotide composition. The bat SaV genome was found to have significantly greater codon usage bias than the other SaV genomes, as measured by their effective number of codons (N c ) ( Figure 4 ). Adjusting N c for background nucleotide composition (N c 9) did not significantly affect the observed difference in codon usage bias.
Next, we examined CpG dinucleotide bias in the SaV genomes, as studies on other animal RNA viruses suggest that CpG suppression is a major factor in their genome evolution [40, 41] . Odds ratio of CpG and GpC dinucleotides (r CG and r GC ) and the CpG/GpC ratio were calculated to assess the degree of CpG suppression. Results confirm the presence of significant CpG suppression (r CG #0.78) in examined SaV genomes, with the only exception being the bat SaV genome (Table 4 ). r GC values are similar across examined SaV genomes, suggesting that the difference in CpG suppression is specific. All SaV genomes are found to have a slightly negative GC skew, and there is no major difference between the degree of GC skew in bat SaV and the other SaVs (Table 4 ). This suggests that the degree of cytosine deamination is not a major factor in the altered GC composition and CpG suppression in the bat SaV genome.
Although the taxonomic classification of caliciviruses has improved with the availability of full-length gene sequences and robust phylogenetic methods [42] , the increase in genetic diversity introduced by novel caliciviruses would necessitate further taxonomic revisions within the family. The International Committee on Taxonomy of Viruses has adopted a systematic polythetic approach towards virus taxonomy, but classification at or below the genus level may be complicated by the specific biology of diverse viruses. As a case in point, the proposed assignment of the novel bat virus to the genus Sapovirus might be opposed on the basis of an increased genomic G+C content, the different reading frame of ORF2, and the increased length of the 39 UTR. On the other hand, the polythetic criteria for inclusion in the genus are not fully clear, and phylogenetic distances between viral gene sequences have assumed overriding importance in previous and current classifications. It should be noted that even phylogenetic analysis may be confounded by other factors such as the cleavage pattern of ORF1 polyprotein, which has not been determined experimentally for many caliciviruses.
Among the various notable genomic features and properties in the novel bat SaV, we were most intrigued by its remarkably high G+C genomic content. Most caliciviruses have a genomic G+C content of 44.2-57.4 mol%. Among them, the genomic G+C content of the SaVs lie within the relatively narrow range of 49.0-53.6 mol% in spite of their genetic diversity. Hence, the presently observed G+C genomic content of 60.2% is significantly higher than that for other SaVs or caliciviruses, and indeed would rank amongst mammalian RNA viruses with the highest G+C genomic content [43] . Relatively little is known about the evolution of genome composition in caliciviruses. A number of factors have been postulated to exert selectional pressure on the G+C content of viral genomes, including host body temperature, immune pressure, codon and nucleotide usage patterns [44] [45] [46] [47] . Our results suggested that the increased G+C content is associated with a decrease in CpG suppression, but does not have a direct correlation with codon usage bias. We are unaware of any Table 3 . Comparison of genome identities and amino acid identities between the predicted polyproteins of bat SaV and the selected SaV. previous findings indicating that genomes of bat viruses are under less CpG suppression, thus the observed reduction in CpG suppresion is unlikely to result from host-related factors. The greater codon usage bias in the bat SaV genome is another interesting genome feature, which could be associated with altered dinucleotide frequencies. The association could be tested by Markov modelling of the dinucleotide and codon frequencies in the SaV genomes, although the small genome sizes and the presently small number of complete genomes would limit the usefulness of this approach [48] . The novel SaV described presently is the first known member of the Caliciviridae in bats. The approach to its discovery is based on the established strategy of targeted genetic screening informed by conserved sequences of related viruses. Although this ''homologybased'' strategy has been successful in the discovery of numerous viruses, the advent of affordable high-density microarrays and high-thoughput sequencing has given rise to virus discovery through metagenomics. Indeed, the first canine SaVs were discovered recently by metagenome sequencing of canine diarrhoea samples on a high-throughput pyrosequencer [23] . Important advantages of the new method include detection of novel viruses not closely related to known viruses, and the capacity to detect multiple divergent viruses in cases of co-infection. However, metagenomics sequencing can suffer from possible bias during sample preparation [49] , and it is unlikely to detect very low titres of viruses in a specimen, such as the three bat faecal samples that were positive upon repeat screening with specific PCR primers in the present study. While we anticipate the increasing utilization of the metagenomics approach, existing methods such as viral culture, electron microscopy and targeted nucleic acid amplification would continue to serve important roles in virus discovery.
As Hong Kong is a highly urbanized city, the local roosting sites of bats are mainly man-made structures, such as water tunnels and abandoned mines. Hipposideros pomona is very common and widespread throughout Hong Kong countryside areas. It is a small-sized leaf-nosed bat with body weight ranged from 6-8 g. It possesses a small nose leaf which is simple, small, and lacking of lateral leaflets (Figures S3 and S4 ). This species may aggregate in small chambers or enclosures where the air flow is relatively limited. The 5 SaV-infected specimens were all captured in a place called Tai Lam -Shek Kong located next to a major country park of Hong Kong, and this roosting site shares similar ecological characteristic with other sampled roosting sites. Due to the extremely high human population density in Hong Kong, direct contact between humans and bats is relatively frequent. Fortunately, no local case of bat zoonosis has ever been reported [36] . The relatively large genetic distance between the present bat SaV and other mammalian SaVs suggests that the zoonotic risk posed by this virus is likely to be low, though this should be confirmed with further in vitro and in vivo studies.
There are two main limitations in the current study. First and foremost, clinical information on the sampled bats is limited to the brief period of captivity needed for sample collection, which is unlikely to reflect the disease association of the virus accurately. In other words, the scope of the study is limited to surveillance of viral diversity and possible discovery of new viruses. Secondly, the number of samples for the novel virus is quite small, despite the use of specific PCR primers for screening and the relatively large number of samples collected. Thus, we were unable to draw conclusions on the seasonality of its detection or its host specificity. To address these limitations, long-term follow-up studies would be required to identify sufficient positive samples with associated clinical data. Increasing the scale of surveillance would also help, though there are practical geographical and logistic constraints in our locality.
In conclusion, we identified a novel bat SaV with several genomic features and properties that set it apart from other members of the genus Sapovirus. Phylogenetic analysis suggests that its ancestral lineage had diverged early from the other SaVs and evolved under different conditions. Further discovery and characterization of additional strains would enhance our understanding of the evolutionary history of the SaVs and other caliciviruses.
The study was approved by the Department of Agriculture, Fisheries and Conservation, HKSAR; and Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong. Bats from 14 different locations in rural areas of Hong Kong, including water tunnels, closed mines, sea caves and forested areas, were captured over a 36-month period. Anal swabs were collected by an experienced veterinary surgeon, and kept in viral transport medium at 4uC before processing.
Viral RNA was extracted from the anal swabs using a QIAamp Viral RNA mini kit (Qiagen). The RNA was eluted into 50 ml RNase-free water and was used as the template for RT-PCR.
Screening was performed by amplifying a 185 nt fragment in the RdRp region of the ORF1 gene of caliciviruses. Conserved degenerate primers (59-GAYTAYTCNMRRTGGGAYTC-39 and 59-GGCATNCCNGAKGGNAYNCC -39) were designed from the multiple sequence alignment of the available calicivirus gene sequences in NCBI GenBank. First-strand cDNA synthesis was performed using SuperScript III kit (Invitrogen) according to manufacturer's instructions. The PCR mixture (25 ml) contained cDNA, PCR buffer (10 mM Tris/HCl pH 8.3, 50 mM KCl, 2 mM MgCl 2 and 0.01% gelatin), 200 mM of each dNTP and 1.0 U AmpliTaq Gold polymerase (Applied Biosystems). PCR cycling conditions were as follows: hot start at 94uC for 7 min, followed by 50 cycles of 94uC for 1 min, 50uC for 1 min and 72uC for 1 min with a final extension at 72uC for 10 min in an automated thermal cycler (Applied Biosystems). Standard precau-tions were taken to avoid PCR contamination and no false-positive signal was observed in the negative controls. The PCR products were gel-purified using a QIAquick gel extraction kit (Qiagen). Both strands of the PCR products were sequenced twice with an ABI Prism 3730xl DNA Analyser (Applied Biosystems), using the two PCR primers.
Additional RT-PCR screening was performed on the same samples using specific primers designed from the RdRp nucleotide sequences of bat SaVs obtained from previous rounds of RT-PCR and sequencing, as RT-PCR screening with specific primers usually offers higher sensitivity than a comparable screening with consensus degenerate primers. Sequences of the specific primers are as follows: forward primer 59-CACAATGCAGCCAGCCA-39 and reverse primer 59-GGTGCGCGTGGTGAACAC-39. PCR cycling conditions were as follows: hot start at 94uC for 7 min, followed by 50 cycles of 94uC for 1 min, 52uC for 1 min and 72uC for 1 min with a final extension at 72uC for 10 min in an automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid PCR contamination and no falsepositive signal was observed in the negative controls. PCR product purification and sequencing were performed as above.
Purified PCR products were cloned into a pCR2.1-TOPO vector (Invitrogen) according to manufacturer's instructions. The vector was then used to transform the competent Escherichia coli strain DH5a by electroporation. Positive transformants were identified by blue-white screening, and eight colonies were selected for DNA sequencing of the construct using the M13 forward and reverse primers according to the manufacturer's instructions. Sequencing reactions were performed as described above.
Viral genome sequences were obtained using strategies we had previously used for other RNA viruses [50] [51] [52] . RNA extraction and cDNA generation were performed as described above. PCR primers were designed by targeting conserved regions, which were identified from the multiple alignment of genomes of related SaVs, as primer-binding sites. Additional primers for subsequent rounds of PCR were designed based on the results of earlier rounds of genome sequencing. The complete set of primer sequences is available from the authors upon request. The 59 and 39 ends of the viral genomes were sequenced following amplification of the segments by rapid amplification of cDNA ends, which was performed using the SMARTer RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions.
ORFs were located using the ORF Finder tool at NCBI (http:// www.ncbi.nlm.nih.gov/projects/gorf/). Annotation of the predicted proteins was performed by BLAST sequence similarity search against annotations in the NCBI RefSeq database. Multiple sequence alignments were constructed using MUSCLE version 3.8.31 [53] , and phylogenetic informative regions were extracted using BMGE [54] . Maximum-likelihood phylogenetic trees were constructed using PhyML version 3 [55] , under the best-fit protein evolution model as selected by ProtTest 3 [56] . Branch support values were estimated by calculation of SH-like aLRT values [57] . Recombination detection was performed by analysing the translated sequences of ORF1 and ORF2 separately using the Recombination Analysis Tool [58] .
The full-length ORF1 and ORF2 coding sequences were extracted from selected SaV genomes and concatenated for codon usage analysis (see Table 4 for the list of included genome sequences). Codon usage and summary statistic of codon usage bias (N c and N c 9) were calculated using the INCA package version 2.1 [59] , where N c is the effective number of codons in the coding regions of the genome [60] , and N c 9 is the effective number of codons adjusted for background nucleotide composition [61] . For CpG dinucleotide bias analysis, odds ratio of CpG and GpC dinucleotides and the CpG/GpC ratio were calculated as described in previous studies [40, 41] . Odds ratio of #0.78 indicates significant suppression of the dinucleotide, same as the interpretation criteria of previous studies. Symmetrized nucleotide frequencies and dinucleotide odds ratio were not considered in the present study, as SaV genomes consist of positive-sense ssRNA only. To investigate the possible effects of cytosine deamination, genomic GC skew, which is the ratio (G-C)/(G+C), was calculated for the SaV genomes. The strength of the GC skew had been suggested to correlate with the degree of cytosine deamination [41, 44, 62] .  Angiotensin Converting Enzyme (ACE) and ACE2 Bind Integrins and ACE2 Regulates Integrin Signalling The angiotensin converting enzymes (ACEs) are the key catalytic components of the renin-angiotensin system, mediating precise regulation of blood pressure by counterbalancing the effects of each other. Inhibition of ACE has been shown to improve pathology in cardiovascular disease, whilst ACE2 is cardioprotective in the failing heart. However, the mechanisms by which ACE2 mediates its cardioprotective functions have yet to be fully elucidated. Here we demonstrate that both ACE and ACE2 bind integrin subunits, in an RGD-independent manner, and that they can act as cell adhesion substrates. We show that cellular expression of ACE2 enhanced cell adhesion. Furthermore, we present evidence that soluble ACE2 (sACE2) is capable of suppressing integrin signalling mediated by FAK. In addition, sACE2 increases the expression of Akt, thereby lowering the proportion of the signalling molecule phosphorylated Akt. These results suggest that ACE2 plays a role in cell-cell interactions, possibly acting to fine-tune integrin signalling. Hence the expression and cleavage of ACE2 at the plasma membrane may influence cell-extracellular matrix interactions and the signalling that mediates cell survival and proliferation. As such, ectodomain shedding of ACE2 may play a role in the process of pathological cardiac remodelling. Heart failure is characterised as a decline in cardiac contractility, which is associated with structural changes, collectively termed cardiac remodelling. Cardiac myofibroblasts are key mediators of cardiac remodelling via their proliferation, invasion and secretion of extracellular matrix proteins. Angiotensin II (Ang II) stimulates cardiac myofibroblast transdifferentiation leading to fibrosis. Ang II also stimulates proliferation [1] , NADPH oxidase activation [2] (and thereby reactive oxygen species production), the production of proinflammatory cytokines [3] and the activation of matrix metalloproteinases (MMPs) [4] . As a result, Ang II is a major contributor to the pathology of cardiovascular diseases. Ang II is generated from the biologically inert peptide, Ang I, by the catalytic action of angiotensin converting enzyme (ACE), a key proteolytic step in the renin angiotensin system (RAS). Aberrant functioning of the RAS is a feature of a variety of cardiovascular, renal and other pathologies and ACE inhibitors and Ang II receptor 1 (AT1R) antagonists are widely used in the clinic. Accordingly, ACE inhibition has been shown to prevent cardiac remodelling after myocardial infarction (MI) and preserves cardiac function [5, 6] . A combination of ACE inhibitors and AT1R blockers has been shown to be more effective than either alone [7] . A decade ago a new member of this system was identified, termed angiotensin converting enzyme 2 (ACE2) [8, 9] . ACE2 acts to hydrolyse Ang II into the vasodilator Ang-(1-7), thereby contributing to reductions in blood pressure. Current models of the RAS are based on the concept that the two enzymes counterbalance each other.
The balance between the two angiotensin converting enzymes has been highlighted by ACE2 deletion murine models, which have a significantly higher mortality rate post-MI than wild-type mice. Mortality was associated with enhanced adverse ventricular remodelling following MI [10] , a state which was reversed by the use of an AT1R blocker and as such the pathology of ACE2 deletion was attributed to the increased levels of Ang II [10] . A mounting body of evidence is forming in support of a cardioprotective role for ACE2, through the metabolism of Ang II [10, 11] , but also through the direct action of Ang-(1-7) via its own receptor, Mas [12] . Like Ang II the actions of Ang-(1-7) extend beyond vasopressor control, and for the most part appear to counteract the effects of Ang II and therefore mediate cardioprotection [13] . Ang-(1-7) reduces interstitial fibrosis [14] , myocyte hypertrophy [15] and inhibits myocyte cell growth [16] . The reduction in myocyte hypertrophy resulting from expression of Ang-(1-7) was associated with a decrease in pro-inflammatory cytokines (TNF-a and IL-6) and also a reduction in exogenous ACE transcript [17] .
Both ACE and ACE2 are increased in the failing heart [18] [19] [20] . Over-expression of ACE2 and inhibition of ACE exert a protective influence on the heart post-myocardial infarction (MI) and prevent the pathological remodelling [21] . These data together suggest that the regulated activity of angiotensin converting enzymes may play a role in cardiac homeostasis.
In addition to its catalytic actions, ACE2 is the cellular receptor for the SARS virus; more recently, other regulatory actions of ACE2 through protein-protein interactions have been identified [22] . ACE2 acts as a chaperone protein for the neutral amino acid transporter, B 0 AT, [23] and binding of calmodulin to the cytoplasmic tail of ACE2 regulates its retention on the cell surface [24] . The reported observation that ACE2 binds integrin b1 (ITGB1) in the failing human heart [25] adds another dimension to the role of the ACE family in cardiac homeostasis.
Integrins are a family of ab heterodimeric cell surface receptors, which link extracellular matrix proteins with the intracellular cytoskeleton. Integrins have an important role in the regulation of gene expression, cell proliferation, differentiation, migration and apoptosis. Activated myofibroblasts develop specialised focal adhesions, containing high levels of a5, b1 and b3 integrins [26] . ITGB1 serves as a mechanotransducer and expression of this integrin increases in the heart after MI [27] . ITGB1 is highly implicated in left ventricular remodelling and MI models have shown it is essential for the adaptive remodelling response [28] leading to the suggestion that the functional activity of ACE2 in cardiac remodelling may, at least in part, be mediated through ITGB1.
Both the angiotensin converting enzymes and ITGB1 traverse the plasma membrane; the angiotensin converting enzymes also exist in soluble forms in the plasma when shed from the cell membrane. Recent studies of heart failure patients have linked elevated levels of soluble (shed) ACE2 (sACE2) to increased myocardial dysfunction and thus have indirectly identified a protective role for the cell surface-associated form [29] . Hence, the ectodomain of ACE2 may have a role in cardiac remodelling independent of its catalytic activity. Here we show that both angiotensin converting enzymes bind ITGB1 and integrin a5 (ITGA5) in vitro. The data presented provide evidence that the enzymes provide a substrate for cellular adhesion and that the interaction between ACE2 and an integrin increases cellular adhesion when both are present on the cell membrane. We demonstrate that the ectodomain of ACE2 regulates integrin induced cell signalling via modulation of the phosphorylation of focal adhesion kinase (FAK) and Akt expression levels.
A typical integrin binding motif is the tripeptide sequence RGD. Bioinformatic analysis was used to compare the protein sequences of ACE and ACE2; a highly conserved integrin binding domain was identified in the ectodomain of ACE2 but not ACE ( Figure 1A ). There are two isoforms of ACE: somatic ACE, that contains two homologous catalytic ectodomains (the N and Cdomains) and the testicular ACE (tACE), composed solely of the C-domain. The RGD sequence in ACE2 is replaced by the sequence RSW in the ACE N-domain and RSM in the C-domain.
To assess the ability of ACE2 to bind an integrin, immunoprecipitation was performed on HEK cells over-expressing ACE2 (HEK-ACE2 cells). Anti-ITGB1 antibody was used to pull down any interacting proteins and western blotting performed using an anti-ACE2 antibody ( Figure 1B ). An interaction was found to occur in the HEK-ACE2 cells, represented by a single band at 120 kDa corresponding to the fully glycosylated ACE2 protein ( Figure 1B ). This interaction was specific, since ACE2 was not detected when immunoprecipitation was performed with isotype control IgG antibody.
Immunoprecipitation was repeated in Huh7 cells, which endogenously express ACE2. Crosslinking was performed in order to fix any interaction of less than 9 Å in length. An interaction between ACE2 and ITGB1 was readily detected in these cells ( Figure 1C ). In addition an interaction between ACE2 and the common binding partner of ITGB1 in cardiac tissue, ITGA5, was probed by subjecting cell lysates to immunoprecipitation with antiintegrin a5 (ITGA5) antibody. ITGA5 was also found to bind to ACE2 in Huh7 cells ( Figure 1C ). The subcellular location of both ACE2 and ITGB1 was visualised in HEK-ACE2 cells by immunofluorescence microscopy ( Figure 1D ). Antibodies to ACE2 (green) and ITGB1 (red) located both proteins to the plasma membrane. Co-location of the two proteins was observed in some areas (yellow), highlighted by arrows.
Both isoforms of ACE lack the RGD motif present in the extracellular domain of ACE2 and we therefore hypothesised that ACE would not bind integrins. Cells over-expressing tACE were used to examine any potential interaction between ACE and ITGB1. Immunoprecipitation revealed that ACE does bind ITGB1 ( Figure 2A ) and that ACE also binds ITGA5 similarly ( Figure 2B ). In order to clarify any role of the RGD motif in the binding of ACE2 to integrins, cross-linked immunoprecipitation was repeated in Huh7 cells, in the presence and absence of an RGD peptide. The interaction between ACE2 and ITGB1 was not blocked by the presence of RGD peptide ( Figure 2C ). Hence, the ability of ACE2 to bind the RGD-independent integrin subunit ITGA2 was additionally investigated. ACE2 bound ITGA2 at a comparable level to ITGA5 and ITGB1 ( Figure 2D ).
We utilised molecular modelling to ascertain the location of the RGD motif in the structure of ACE2 to investigate the apparent functional redundancy of this motif in integrin binding. Examination of the ACE2 structure in silico [30] revealed that the motif was on the protein surface ( Figure 3A ). However, the space filling model revealed that the aspartate residue of the RGD motif faces into the active site cleft ( Figure 3B and 3C) and is, therefore, inaccessible for protein-protein interactions. The RSM sequence in tACE superimposes in exactly the same position as the RGD sequence in ACE2 and is therefore also inaccessible for proteinprotein interactions ( Figure 3D ). The similarity between the two proteins is illustrated by the close proximity of the outline trace and the degree of overlap. Variations in amino acid sequences are illustrated by the slight offset in the a-helical loops.
To examine the functional significance of an interaction between the angiotensin converting enzymes and integrins, adhesion assays were used to explore the possibility that they may act as a cell adhesion substrate. We designed an in vitro technique representative of an in vivo cellular environment to study the effect of ACE2 expression on cellular attachment.
A cell to cell adhesion assay was developed in order to examine the ability of membrane bound ACE or ACE2 to act as a ligand for cell adhesion. Cells over-expressing ACE or ACE2 or their mock transfected controls were used as cell adhesion substrates and Huh7 cells were labelled with BCECF fluorescent dye and allowed to adhere. Huh7 cell adhesion to the substrate cells was confirmed by immunofluorescence microscopy ( Figure 4A ). Calibrations confirmed that the relative fluorescence measured was proportional to the number of cells seeded ( Figure 4B ). A significant difference was seen between the adhesion of Huh7 cells to ACE2-expressing cells compared with non ACE2expressing cells ( Figure 4C ); the expression of ACE2 on the cell surface increased cell adhesion by approximately 25%. To further examine any role of RGD-mediated cell adhesion, cells were incubated with an RGD peptide prior to plating. Pre-incubation with RGD peptide significantly reduced the adhesion of Huh7 to HEK control cells ( Figure 4C ); cellular expression of ACE2 abolished the decrease mediated by pre-incubation with an RGD peptide ( Figure 4C ). Conversely, cellular expression of ACE conferred no enhanced adhesion properties compared to control cells ( Figure 4D ). In this model the presence of an RGD peptide reduced cellular adhesion by approximately 35% independent of tACE expression ( Figure 4D ).
To examine the physiological importance of the adhesion properties of ACE2 and ACE, adhesion assays were performed using primary human cardiac myofibroblast (CF) cells, important mediators of cardiac remodelling. All cells adhered strongly to fibronectin (data not shown), an important component of the extracellular matrix, adhesion to which is integrin-mediated [31] . A significant difference in cell binding was seen in the presence and absence of ACE2 ( Figure 5A ) in patient samples. The average fold increase in cell adhesion in the presence of ACE2 was 3.9 fold and p = 0.0035. Differences in adhesion to both fibronectin and ACE2 were seen in all patient cells ( Figure 5A ); this is likely due to an inherent variation in integrin and/or ACE2 expression levels since these were primary cells [32] . As previous experiments had shown ACE2 and ACE both bind integrins, investigations were performed to determine if ACE could also act as a cell substrate. Cells adhered comparably to both ACE and ACE2 ( Figure 5B ) and the presence of an RGD peptide had no effect on the adhesion to ACE or ACE2 (data not shown).
In light of the association of ACE2 with integrins, experiments were performed to determine if ACE2 could elicit integrin signalling. Focal adhesion kinase (FAK) is stimulated early in any integrin signalling cascade. Given that the extracellular domain of ACE2 binds integrin, cells were stimulated with the ectodomain of ACE2. The levels of phosphorylated FAK (pFAK) in the presence and absence of sACE2 were quantified by ELISA. At 0.1 mg/ml sACE2 significantly reduced levels of pFAK in Huh7 cells and in primary myofibroblasts ( Figure 6A and B). No further decrease was observed when cells were incubated with 1 mg/ml ACE2. Downstream translation of this signal to Akt was investigated by quantifying the levels of phosphorylated Akt. Western blot analysis revealed that levels of phosphorylated Akt in Huh7 cells increased in response to stimulation with sACE2 and Ang II for 30 min (data not shown). However, this increase was accounted for by an up-regulation in Akt protein expression ( Figure 6 ). The catalytic product of ACE2, Ang-(1-7), alone did not elicit the same effects as ACE2 ( Figure 6 ).
Further analysis revealed that this signal was not transmitted to the downstream effector of Akt signalling, NF-kB. Cells were transfected with NF-kB reporter vector. Treatment of cells with Figure 1 . ACE2 binds an integrin. A) Evolutionary alignment of a part of the ACE2 amino acid sequence. ACE2 protein sequence conservation surrounding and including the proposed integrin binding site. Amino acids are shown as their one letter codes. The predicted integrin binding site is highlighted in red, whilst the homologous region in ACE is coloured blue. This alignment of the ACE2 protein sequence was taken from the Uniprot database. B) Immunoprecipitation of ITGB1 with ACE2 in HEK-ACE2. HEK cell lysates incubated with ITGB1 antibody and eluted using protein G. Immunoblotting for ACE2 was performed with anti-ACE2 antibody. C) Immunoprecipitation of ITGB1 and ITGA5 with ACE2 in Huh7 cell monolayers. Cell monolayers were cross-linked using DTBP before lysis and immunoprecipitation as before. D) Immunocytochemical detection of ACE2 and ITGB1 location in HEK-ACE2 cells. Cell imaging shows ACE2 (green) and ITGB1 (red) are located together (yellow) on the cell membrane of HEK-ACE2 cells. doi:10.1371/journal.pone.0034747.g001 sACE2 or ACE for between 2 and 24 h resulted in no significant change in luminescence compared to control (Figure 7 ). Furthermore, neither Ang II nor Ang-(1-7) had any significant effect (data not shown).
The present study reveals that both ACE and ACE2 bind integrins in an RGD-independent manner. We have shown that ACE2, in particular, increases cellular adhesion and, moreover, affects integrin signalling. Shedding of the ACE2 protein may relieve repression of integrin signalling, exerted by the presence of ACE2 on the cell membrane. Using primary cardiac myofibroblasts we have demonstrated that the actions of ACE and ACE2 exerted on cell models are physiologically relevant to the diseased human heart.
Both ACE and ACE2 are increased in the failing heart [18] [19] [20] . ACE2 expression has consistently been seen to be up-regulated in the peri-infarct area after MI [10, 20, 33] and in end stage heart failure [34] indicative of a role in injury. Knockout of the ace2 gene increases MMP2 and MMP9 levels in the peri-infarct region of mice, resulting in disruption of the extracellular matrix and enhanced adverse remodelling [10] . ACE2 over-expression has been shown to inhibit collagen production in response to hypoxic injury [35] . Moreover, activation of remodelling pathways has been demonstrated in ace2 knockout animals in the absence of an increase in Ang II [36] . Cardiac remodelling is a key pathological process in the development of heart failure. Integrins play a key role in this process by mediating cell-ECM interactions and cellular signalling. ITGB1 has also been implicated in myocardial dysfunction [28] . An association between ITGB1 and ACE2 has previously been reported in the failing heart and attributed to the presence of an RGD motif in ACE2 [25] . We have established that both ACE and ACE2 binds ITGB1 and also its common cardiac binding partner, ITGA5 [37] , as well as the RGD-independent and liver rich ITGA2 [38] . However, in contrast to the study of Lin et al. [25] our data clearly suggest that these interactions occur independently of an RGD motif. 3-dimensional modelling has demonstrated that the RGD motif present in the ectodomain of ACE2 is inaccessible, which explains its redundancy in integrin binding. The aspartate residue is positioned facing the active site cleft of the protein and as such is not available to bind into the integrin binding pocket [39] . Given the structural homology between the two proteins, it is not surprising that ACE is also capable of binding integrins as the interaction appears to be independent of the RGD sequence, which is lacking in ACE.
Prothrombin, like ACE2, contains a partially buried RGD motif, however in prothrombin this sequence is exposed upon activation. The ability of prothrombin to bind integrin avb3 is key to its biological activity in the fibrotic cascade [40] . As in ACE2, the carboxylate group of the Asp of the RGD motif is directed towards the specificity pocket of the enzyme [41] . In its native state prothrombin does not exert strong adhesive properties. However, proteolytic maturation exposes the RGD motif and thereby enhances the adhesive properties of thrombin [42] . This mechanism is unlikely to occur in ACE2 which, when present on the cell membrane, is in its mature form (unlike thrombin it does not occur in an inactive proenzyme form during biosynthesis). What is more, any rearrangement of the active site of ACE2 is likely to inhibit its catalytic activity; sACE2 is catalytically active.
Although RGD motifs are the most common mechanism of integrin binding, there are other cell surface proteins which bind integrins despite lacking an RGD motif. ADAM 9, for example, binds through a hypervariable loop stabilised by disulphide bridges, which protrudes from the surface of the protein structure [43, 44] . The cellular adhesion molecule ICAM-1 is hypothesised to bind via immunoglobulin type domains [45] , whereas other proteins have been predicted to bind through a (D/E)ECD motif [46] . Neither ACE nor ACE2 contain an ECD motif.
We have shown that the integrin binding to ACE2, but not ACE, is essential for the role of ACE2 as a cellular anchor when expressed on the cell surface. What is more, the presence of ACE2 on the cell surface partially removed the requirement of RGDinteractions for cellular adhesion. Fibroblast motility is a key process in the development of scar tissue and thus cellular adhesion is an important homeostatic mechanism. In order to study the role of ACE and ACE2 in the remodelling response, primary cardiac myofibroblasts were used as a disease model. These cells play a key role in the maintenance of cardiac architecture under conditions of injury, by forming scar tissue through their ability to proliferate and adhere [47] . We demonstrate that both ACE and ACE2 exert comparable effects over myofibroblast adhesion, but that again this effect is not mediated through an RGD motif. Recent clinical investigations have highlighted that elevated levels of sACE2 in patient plasma correlated with increased myocardial dysfunction [29] and vascular compliactions in type 1 diabetes [48] . We have shown that both ACE and ACE2 interact with the cell surface in adhesion assays and, furthermore, ACE2, in particular, enhances cell adhesion and may modulate integrin signalling. Cellular retention of ACE2 is therefore required for its role as a cellular anchor and, as such, the cleavage of ACE2 by ADAM17 [49] may be a pathological step in the development of heart failure. The Figure 2 . Both angiotensin converting enzymes bind integrins independent of an RGD motif. A) Immunoprecipitation of ITGB1 with tACE in HEK-tACE cells. HEK cell lysates were incubated with ITGB1 antibody and eluted using protein G. Immunoblotting for ACE was performed with anti-ACE mC5 antibody. This interaction was similar to that between ACE2 and ITGB1 in ACE2 over-expressing cells. B) Immunoprecipitation of ITGB1 and ITGA5 with tACE in SHSY5Y cells over-expressing the testicular form of ACE. Immunoprecipitation was performed as before. C) Immunoprecipitation of ACE2 with ITGB1 in the presence of a RGD peptide. Huh7 cells were pre-incubated and crosslinked using DTBP, in the presence or absence of RGD peptide before lysis and immunoprecipitation as before. D) Immunoprecipitation of ITGA2, ITGA5, ITGB1 with ACE2 in Huh7 cell monolayers. Cell monolayers were cross-linked using DTBP before lysis and immunoprecipitation as before. doi:10.1371/journal.pone.0034747.g002 retention of ACE2 on the cell membrane is known to be regulated by cell signalling [24] and viral infection [50] . Shed ACE2 could activate integrins by binding to them and transducing activating signals or, additionally, by interacting with non-integrin sites given the multiple protein-protein interactions with which the ACE2 protein is involved.
FAK is a critical signalling component associated with areas of substratum adhesion; signalling via FAK is mediated through autophosphorylation of Tyr 397 [51] . We demonstrate that sACE2, at levels comparable to those reported in human plasma, significantly reduces FAK phosphorylation levels. Furthermore, we show that treatment with sACE2 increases the levels of Akt expression, a pro-survival, pro-proliferative protein. Changes in the level of phosphorylated Akt were also seen in response to sACE2; however, these were accounted for by the increase in the amount of total Akt. As such, signalling by Akt was not transmitted to its downstream effector NF-kB. sACE2 has no known function. We do not dismiss the possibility that this circulating ACE2 may bind integrins, when released from the plasma membrane, and elicit autocrine or paracrine signalling. In fact, one of the shed forms of the amyloid precursor protein, sAPPa, binds to ITGB1 and signals to enhance axon outgrowth [52] . What is more, cellular expression of full length APP inhibits axonal outgrowth and an excess of the shed form overcomes this inhibition [52] . We therefore propose some of the anti-proliferative actions of ACE2 may in part be mediated through a non-catalytic interaction with integrins, rather than by metabolism of Ang II per se. These data suggest that ACE2, through its integrin binding abilities, may have regulatory roles in cellular attachment and support a novel mechanism of integrin activation upon ACE2 shedding.
All routinely used reagents were purchased from Sigma unless otherwise stated. Cell culture reagents were purchased from Lonza (Slough, UK). Cell Extraction Buffer and Elisa (pFAK) kit were purchased from Invitrogen (Paisley, UK) along with lipofectamine and BCECF reagent (2979-bis-(2-carboxyethyl)-5-(and6)-carboxyfluorescein). Roche (Welwyn, UK) supplied protease inhibitor tablets. Fluorescent ACE2 substrate Mca-APK-Dpn was supplied by Enzo (Exeter, UK) and MTS reagent by Promega (Southampton, UK). Secondary antibodies, the chemiluminescence system used and Protein G Sepharose 4 fast flow were supplied by GE healthcare (Chalfont St. Giles, UK). DTBP (Dimethyl 3,39dithiobispropionimidate) was purchased from Pierce (Cramlington, UK). The ACE2 inhibitor 416F2 [53] was a generous gift from Prof V. Dive (CEA, Gif sur Yvette, France). Polyclonal ACE2 antibody raised in goat was purchased from R&D systems (Abingdon, UK). Integrin antibodies raised in mice against ITGB1 and ITGA5 antibodies were bought from Santa Cruz Biotech (Heidelberg, Germany). Polyclonal ADAM17 antibody raised in rabbit was purchased from Calbiochem (Nottingham, UK), while Akt antibody raised in rabbit was purchased from Cell Signaling (Hertfordshire, UK). Purified ACE enzyme was a kind gift from Prof N. Hooper (The University of Leeds, UK) and Prof S. Danilov (University of Illinois at Chicago, USA) generously provided the ACE monoclonal antibody [54, 55] .
HEK (human embryonic kidney) and Huh7 (hepatocellular carcinoma-derived) [24] cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% (v/ v) foetal bovine serum, 2 mM essential amino acids, 1% (v/v) nonessential amino acids. HEK cells stably transfected with full length ACE2, designated HEK-ACE2 [49] , and those overexpressing the testicular form of ACE (tACE), HEK-tACE [56] , were cultured in the same conditions with the addition of G418 (0.5 mg/ml) to the medium. SHSY5Y cells were cultured in DMEM-F12 media [57] , and SHSY5Y cells overexpressing tACE, SHSY5Y-tACE cells, were a kind gift from Dr. C. Rushworth, and were cultured in DMEM-F12 supplemented with G418 (0.5 mg/ml).
Cardiac myofibroblasts were obtained by enzymatic digestion of biopsies of human right atrial appendage. Patients were undergoing elective coronary artery bypass surgery and had normal ventricular function (ejection fraction normal ($50% by cardiac ultrasound and/or LV Angiography). Local (Leeds West) Research Ethical Committee (LREC) approval is in place for this study; written consent was given, reference number 01/040. Informed, written patient consent is obtained. The investigation conformed to the principles outlined in the Declaration of Helsinki, 1997. Primary cultures of cardiac fibroblasts were harvested, characterized as myofibroblasts co-expression of smooth muscle a-actin and vimentin and cultured as described previously [58] . Experiments were performed on cells from different patients at passages 2-5 [59] . Cell images were taken on a Nikon Eclipse TS100 microscope using a Nikon COOLPIX 4500 4.0 megapixel camera. Recombinant ACE2 purification HEK cells stably expressing a FLAG-tagged ACE2 ectodomain (sACE2) were created. HEK cells were transfected with plasmid DNA (pCl-neo containing nucleotides 104-2323 of ACE2 cDNA with the FLAG peptide conjugated to the C-terminus). Successfully transfected cells were selected by passage in media containing G418 (1 mg/ml). sACE2 was collected from the conditioned media of these cells and purified by affinity chromatography using an anti-FLAG M2-agarose column and eluted into tubes containing 25 ml 1M Tris pH 8.0 by addition of 0.1 M glycine, pH 3.5. Eluted fractions were analysed for ACE2 activity by fluorometric assay [24] and purity was checked by silver stain, using SilverXpress (Invitrogen) as per manufacturer's instructions, and then immunoblotted for ACE2.
Cells were treated at 80% confluency and all pharmacological reagents were diluted in OptiMEM. For pFAK quantifications and phospho-Akt quantifications, incubations with sACE2 were carried out with 100 ng/ml or 1 mg/ml sACE2 for the time indicated (pFAK, 20 min). After treatment, cells were placed on ice and lysed in either ice cold RIPA buffer containing protease inhibitor and phosphoSTOP (phosphor-Akt), or Cell Extraction Buffer (pFAK). Lysates were then analysed by pFAK ELISA as per manufacturer's instructions, or by western blot for Akt levels.
Huh7 cells were transfected with pGL4.32 [luc2P/NF-kB-RE/ Hygro] (NF-kB reporter) vector (500 ng) using Lipofectamine 2000 in OptiMEM. Renilla CMV (1 ng) was co-transfected. Cell medium was changed 4 h post-transfection and stimulated after 24 h with either sACE2/ACE, 100 ng/ml, IL-1b, 100 ng/ml, diluted in DMEM containing 1% FCS. After treatment, luciferase levels were analysed using Dual-Luciferase Reporter Assay System following manufacturer's instructions.
Lysates were routinely prepared by solubilisation of cells in RIPA buffer (0.1 M Tris-HCl, pH 7.4, 0.15 M NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) Nonidet P-40). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay [60] . Bovine serum albumin was used as a standard with a 50:1 ratio of 4% (w/v) CuSO 4 .5H 2 O.
All cells were washed twice and scraped into ice cold PBS, where crosslinking were preformed Huh7 cells were cross-linked with dimethyl 3,39 dithiopropionimidate (DTBP, 5 mM) for 30 min on ice prior to scraping. Cells were pelleted before resuspending in ice-cold RIPA lysis buffer (0.4% (v/v) with proteinase inhibitor cocktail). Lysates were passed through a 22G needle 5 times and re-cleared by centrifugation at 116006g for 2 min.
For immunoprecipitation, protein-G-Sepharose was pre-cleared by rotation in 5% (w/v) BSA in TBS for 1 h at 4uC, prior to washing 3 times with protein binding buffer (50 mM Tris-HCl pH 7, 50 mM NaCl, 1 mM ZnSO 4 ). Samples were incubated with monoclonal anti-b1 integrin antibody (4uC, overnight), pre-cleared protein-G-Sepharose was then added and samples rotated (2 h, 4uC). Bound samples were eluted by heating to 85uC with 1xSDS-PAGE sample buffer. Sepharose beads were pelleted and the eluted supernatant heated to 95uC with b-mercaptoethanol. Where cells had been crosslinked, crosslinking was denatured by heating with DTT before elution (30 min, 37uC).
Proteins were separated by SDS-PAGE and then transferred onto PVDF membranes using 5% (v/v) transfer buffer, 20% (v/v) Figure 5 . The angiotensin converting enzymes are cell adhesion substrates. Microtitre plates were coated with or without sACE2 and blocked with BSA (1%). Wells were washed; CmF cells plated and allowed to adhere (2.5 h at 37uC). After incubation unadhered cells were washed off and cell adhesion was quantified using MTS reagent and reference to a calibration curve. A) Tabulated cell adhesion results for different patients. B) Graphical representation of (A). C) Cell adhesion is comparable between ACE and ACE2. Plates were coated with sACE2, ACE or PBS and adhesion assays performed as before. doi:10.1371/journal.pone.0034747.g005 methanol. The membrane was saturated with blocking solution (TBS 0.1% (v/v) Tween 20, 2% (w/v) BSA, 5% (w/v) dried milk) (1 h, room temp). Membranes were incubated with primary antibody (4uC, overnight). After washing with TBST four times at 10 min intervals the membranes were incubated with secondary antibody for 1 h at room temperature and then washed as before. Bound antibody was detected using the enhanced chemiluminescence system following the manufacturer's instructions. Densitometric analysis was performed using AIDA software.
Immunostaining HEK-ACE2 cells were plated onto coverslips and fixed with 4% (w/v) paraformaldehyde (10 min), washed twice with PBS and incubated with blocking buffer (5% BSA in PBS) for 30 min at room temp. Blocking buffer was removed and cells were placed in primary antibodies (ACE2 and ITGB1) for 2 h. Antibody binding was visualised using anti-goat Alexa Fluor 488 and anti-mouse Alexa Fluor 594 (Molecular Probes) for 2 h. Coverslips were mounted using Vectashield (Vector Laboratories Ltd.). Cells were imaged using a Delta Vision microscope and SoftWoRx software.
Adhesion assays were carried out in 96 well plates. Wells were coated overnight (4uC) with protein (10 mg/ml) or PBS and washed in PBS. Wells were then blocked with 1% BSA in serum free media (1 h, 37uC.) Blocking solution was removed and wells washed twice with PBS. Cells were plated at a density of 10,000 or 20,000 cells per well in serum-free medium and allowed to attach by incubation at 37uC for 2.5 h. Non-adherent cells were removed by rinsing wells twice with PBS. Adhered cells were quantified using MTS reagent and measuring absorbance at 492 nm. Figure 6 . sACE2 inhibits integrin signalling. A &B) Treatment with sACE2 decreases the level of pFAK. Huh7 cells (A) or CF cells (B) were starved for 12 hours, then treated in the presence or absence of sACE2 at concentration indicated for 20 min. Significance determined by one-way ANOVA (Huh7) or Students' t-test (CF) where *p#0.05. Following treatment with sACE the level of pFAK within the cells was assayed using an ELISA kit as per manufacturer's instructions. C, D&E) Treatment with sACE2 increases cellular Akt expression. Cells were treated as above with either: sACE2, ACE, or Ang-(1-7), at the concentration and for the length of time indicated. Levels of Akt (C) and b-actin (B) were visualised by immunoblot and quantified by densitometric analysis (E). doi:10.1371/journal.pone.0034747.g006
Cell to cell adhesion assay HEK, HEK-ACE2 or HEK-tACE cells were seeded into a microtitre plate and starved in serum free medium (16 h). Huh7 cells were labelled with BCECF in OptiMEM (37uC, 30 min); labelled cells were washed three times with PBS. Huh7 cells were resuspended in serum free medium and plated onto the HEK, HEK-ACE2 or HEK-tACE cells for 2 h, non-adherent cells were removed by washing in PBS, PBS was added to each well and the fluorescence was read, (excitation 440 nm/emission 535 nm). Cell adhesion was examined using an inverted microscope (TE-2000E, Nikon) illuminated with a halogen lamp filtered through a GFP bandpass filter (450-480 nm excitation wavelength).
Protein structures were taken from the PDB.org, file 1r42 and manipulated with Discovery Studio 2.0 (DS2.0, Accelrys Inc.). Turquoise, ACE2 extracellular domain, residues 1-615; red, tACE; yellow, RGD motif; Mln-4760, green; zinc, lilac; active site residues, pink; all other colours in the ribbon structure are sections of the C-terminal domain disordered in solution. In the spacing filling model: Overall surface, light pink; hydrogen bond acceptors, red; hydrogen bond donors, blue.
Results are expressed as mean +/2 standard error of the mean (SEM). Significance was assessed by Student's t test or one-way ANOVA and p#0.05 was considered significant.  Altered Thymic Function during Interferon Therapy in HCV-Infected Patients Interferon alpha (IFNα) therapy, despite good efficacy in curing HCV infection, leads to major side effects, in particular inducement of a strong peripheral T-cell lymphocytopenia. We here analyze the early consequences of IFNα therapy on both thymic function and peripheral T-cell homeostasis in patients in the acute or chronic phase of HCV-infection as well as in HIV/HCV co-infected patients. The evolution of T-cell subsets and T-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of T-cell receptor excision circles (TREC) and estimation of intrathymic precursor T-cell proliferation during the first four months following the initiation of IFNα therapy. Beginning with the first month of therapy, a profound lymphocytopenia was observed for all T-cell subsets, including naïve T-cells and recent thymic emigrants (RTE), associated with inhibition of intrathymic precursor T-cell proliferation. Interleukin (IL)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. This was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble CD127. Decrease in IL-7 plasma concentration under IFNα therapy correlated with the decline in HCV viral load, thymic activity and RTE concentration in blood. These data demonstrate that IFNα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs T-cell homeostasis. Such a side effect might be detrimental for the continuation of IFNα therapy and may lead to an increased level of infectious risk, in particular in HIV/HCV co-infected patients. Altogether, this study suggests the therapeutic potential of IL-7 in the maintenance of peripheral T-cell homeostasis in IFNα-treated patients. The hepatitis C virus (HCV) causes persistent infection in approximately two thirds of cases leading to chronic liver disease, liver failure, and, eventually, hepatocellular carcinoma in a substantial proportion of infected individuals. The most common therapy for chronic hepatitis C consists of pegylated interferon-a (IFNa) and ribavirin administration which results in viral clearance in 43-46% (genotype 1) to 80%, (genotype 3) of treated patients [1] . Interferon will continue to be a major component of new direct acting antivirals for treatment of HCV [2] .
IFNa is produced in large amounts during the acute phase of many viral infections [3, 4, 5, 6] . Direct activation of interferonstimulated genes enhances naïve T-cell survival through increased Bcl-2 and reduced Bax activation [7] and contributes to clonal expansion of antigen-specific T-cells [8] . Recent data suggest that early therapeutic intervention with pegylated IFNa rescues polyfunctional memory T-cells expressing high levels of the IL-7 receptor alpha chain (CD127) and Bcl-2, allowing a higher rate of sustained viral response [9] . However, despite good efficacy, IFNa-based therapies lead to sustained anemia, thrombocytopenia, neutropenia and lymphocytopenia [10, 11, 12, 13, 14] . Moreover, pegylated IFNa therapy enhances the risk of infection in older HCV-infected patients and HIV-infected individuals, independently from neutropenia [15, 16, 17] .
The mechanisms of action of IFNa include inhibition of different hematopoietic growth factors [18, 19] , possibly affecting lymphoid differentiation at an early stage [20] , and modifications in cell homing [12, 21, 22] . The mechanisms involved in IFNa therapy-associated leukocyte depletion remain poorly understood.
Others and we have documented a strong reduction in the ability of HIV-infected patients to sustain thymic production as a direct consequence of a drop in intrathymic precursor T-cell proliferation [23, 24, 25] . Similar thymic impact was also seen during early SIV-infection in the rhesus macaque model [26] . The capacity of the thymus to produce recent thymic emigrants (RTEs) is, in large part, dependent on thymocyte proliferation [27] . Indeed, extensive thymocyte proliferation occurs between T-cell receptor beta (TCRB) and alpha (TCRA) chain rearrangements. The extent of this proliferation directly correlates with thymic export [28] . The extent of cell proliferation in the thymus can be measured in patients through estimating, in peripheral blood cells, the ratio (sj/bTREC ratio) between the frequency of signal joint T-cell receptor excision circles (sjTREC), produced during the excision of the TCRd locus prior to TCRa chain rearrangement, and that of DbJbTREC T-cell receptor excision circles (TRECs) produced during TCRBD to TCRBJ rearrangement [29] . These by-products of TCR rearrangement processes are generated by the circularization of the chromosomal DNA excised during TCR rearrangements respectively occurring at the DN3 (DbJbTREC) and DP (sjTREC) stages of differentiation. Due to the fact that TRECs do not replicate during mitosis, increased proliferation between DN3 and DP leads to the reduction of DbJbTREC frequency in RTEs as compared to sjTREC frequency and consequently to an increase of the sj/bTREC ratio [23] . The correlation between initial plasma IFNa levels and the speed of thymic dysfunction observed during HIV primary infection suggested that IFNa, produced as part of the innate immune response to infection, participates in the impairment of thymopoiesis. However, no direct evidence of the relationship between IFNa production and thymic dysfunction was provided by these studies. In contrast, Arizcorreta and colleagues showed that IFNa and ribavirin therapy induces a substantial reduction of circulating sjTRECs, in HIV/HCV co-infected patients, accompanied by sustained naïve CD4 + T-cell defect, suggesting thymic dysfunction [10] . Similarly, in the SIV-infected rhesus macaque model, we showed that IFNa therapy induced a strong decrease of circulating RTE numbers as defined either by sjTREC frequency and numbers or by CD31 hi expression on naïve T-cells [30] . Interestingly, in these animals, recombinant interleukin (IL)-7 therapy more than abrogated the deleterious effects of IFNa therapy [30] . IL-7 is a key cytokine implicated at various levels of thymocytes differentiation. It allows cell survival during the rearrangement processes, and is implicated in the extensive thymocyte proliferation, in particular in intermediate single positive (ISP) and early DP cells [31, 32, 33, 34] . This proliferation participates in the development of naïve T-cell diversity [35] . While up regulated by IFNa [36, 37] , the cyclin-dependent kinase inhibitor P27/Kip1 is negatively regulated by IL-7 [38] , allowing ISP and early DP thymocytes to proliferate. Moreover, IFNa also inhibit IL-7 dependent proliferation through down modulation of the common c chain, that participates, together with CD127 to the IL-7 receptor [39] . We here investigated the early impact of IFNa therapy on thymic function and naïve T-cell homeostasis in both HCV-infected and HIV/HCV co-infected patients who started IFNa therapy.
We first evaluated the evolution of naïve T-cell subsets in three groups of HCV infected individuals: 1) Acute HCV infection (n = 8), defined as ,6 months post estimated date of infection; 2) chronic HCV infection (n = 8), defined as .6 months post estimated date of infection; and 3) HIV/HCV co-infected individuals (n = 10). In all groups, patients were enrolled at the beginning of IFNa therapy and were followed for a total of 4 months. While, for any group of patient's, naïve CD4+ and CD8+ T-cell counts were not significantly different from healthy individuals (figure 1A), as early as one month following treatment initiation, naïve CD4+ T-cell counts were significantly reduced in chronically HCV-infected patients (39%, 58%, 46% and 35% decrease at M1, M2, M3 and M4 respectively; p#0.025; Figure 1B , top central panel). A similar trend was also observed in the CD8 compartment (40%, 39%, 33% and 33% decrease; Figure 1B , bottom central panel). A comparable effect was also observed in most co-infected patients (mean cell count declines were 19%, 32%, 52% and 43% at M1, M2, M3 and M4 in the CD4+ T-cell compartment and 9%, 21%, 41% and 42% in CD8+ T-cell subset; p#0.05 by M2-M3; Figure 1B , right panels). In contrast, naïve T-cell counts were only barely affected in acutely-HCV infected patients under IFNa therapy ( Figure 1B , left panels). Similarly, central memory CD4+ T-cells (CD45 RA-CCR7+; TCM) demonstrated 38% and 28% decrease in HCV and HIV/HCV patients respectively (59% and 60% in CD8+ TCM) while effector memory (CD45RA2 CCR7-; TEM) CD4+ T-cell counts declined by 45% and 10% in the same groups (61% and 65% in CD8+ TEM) ( Figure S1 ).
Within CD4+ naïve T-cells, RTEs can be identified by their higher expression of the platelet endothelial cell adhesion molecule-1 (PCAM-1 or CD31) [40] . While the number of RTEs was similar in HCV-infected patients at study entry and healthy individuals ( These data demonstrate that, as early as one month following treatment initiation, IFNa induces stronger alterations of naïve Tcell subsets, and more specifically in the RTE compartment than in any other T-cell subset, suggesting a specific effect on thymopoiesis. We thus analyzed the evolution of intrathymic precursor T-cell proliferation, peripheral T-cell cycling, IL-7 plasma concentration and IL-7 receptor alpha chain (CD127) expression, different factors affecting naive T-cell homeostasis.
Despite differences between the 3 groups at study entry, RTE cycling rate, as estimated through measurement of Ki-67 expression, did not change significantly during the follow-up period ( Figure 3A ). These data demonstrate that the observed changes in sjTREC frequencies were not a consequence of variations of RTE proliferation during IFNa therapy but more probably due to reduced thymic production.
We thus estimated thymic output through quantification of the sj/bTREC ratio in all groups of patients ( Figure 3B ). The sj/ bTREC ratio estimates the extent of thymocyte proliferation between TCRB rearrangement and the excision of the T-cell receptor delta (TCRD) locus [23] . This parameter directly reflects the extent of thymic production and, contrarily to sjTREC values, is independent from peripheral RTE proliferation or survival capacity [28] . The sj/bTREC ratio was already low in HIVinfected patients (p,0.005 as compared to healthy control donors; Figure 3B bottom left panel) and did not evolve further under IFNa therapy in co-infected patients ( Figure 3B , bottom right panel). In contrast, acutely HCV-infected patients demonstrated higher than normal sj/bTREC ratio at baseline (p,0.05 as compared to aged matched healthy controls), showed a significant reduction in sj/bTREC ratio at M1 (p = 0.014) and M2 (p = 0.001; Figure 3B , top panel). Finally, a similar decline in the sj/bTREC ratio was observed during IFNa therapy in chronically HCV-infected patients (p,0.02 at M1, M2 and M3; Figure 3B , central panel).
Precursor T-cell proliferation in the thymus is, at least in part, dependent upon IL-7. We thus quantified plasma IL-7 concentration in all groups of patients. At study entry, HCV-and HIV/ HCV-infected patients presented with elevated plasma IL-7 (median = 10.3 pg/mL, range (6.7-12.9) in acutely HCV-infected patients; 8.3 pg/mL (6.3-10.5) in chronic HCV-infected patients and 7.15 pg/mL (4.3-13.5) in co-infected subjects), as compared to that observed in healthy control individuals (p,0.001 for any patients' group; Figure 4A) . Surprisingly, while lymphocytopenia established, IL-7 plasma concentrations significantly decreased in both groups of HCV-infected patients (30, 54, 18 and 29% decrease at M1 to M4 in acute infection, p,0.05; 25, 46, 26 and 16% decrease at M1 to M4 in chronic infection, p,0.05; Figure 4B left and central panels). In contrast, IL-7 plasma levels did not significantly evolve in co-infected individuals during the first month of IFNa therapy ( Figure 4B right panel) . Only patients with the highest IL-7 plasma levels showed a reduction in the concentration of this cytokine.
Decreased plasma IL-7 concentrations could be a consequence of reduced IL-7 production, increased consumption by T-cells or sequestration by soluble IL-7 receptor (sCD127). In both HCVinfected and HIV/HCV co-infected patients, neither sCD127 
Considering the variations in all the parameters we used to evaluate thymic function, we then sought to evaluate the impact of changes in IL-7 plasma levels on de novo production from the thymus and on the number of both sjTREC and circulating CD4+ RTEs.
In a majority of patients, IL-7 plasma level, sj/bTREC ratio, sjTREC/ml and blood RTE concentration fluctuated in parallel ( Figure S2 ). Variation of IL-7 plasma concentration (DIL-7) during the first month of therapy correlated with variations in naïve T-cell counts (CD4+ + CD8+; DNaïve T-cell counts) and RTE CD4+ T-cell counts (DRTE T-cell counts) in both HCV (r = 0.521, p = 0.039 and r = 0.595, p = 0.025; Figure 5A and 5B, left panels) and, to a lesser extent, HIV/HCV co-infected patients (r = 0.636, p = 0.048 and r = 0.539, p = 0.108; Figure 5A and 5B, right panels). Moreover, in HCV-infected patients, DIL-7 also correlated with variations in intrathymic precursor T-cell proliferation (Dsj/bTREC ratio; r = 0.601, p = 0.020; Figure 5C ).
Variations in plasma IL-7 levels also correlated with changes in the proportions (D%Ki-67+ in CD4+RTEs; r = 0.806, p = 0.0002; Figure 5D , left panel) and numbers (DKi-67+RTEs; r = 0.706, p = 0.002; Figure 5E , left panel) of cycling RTEs in acute and chronic HCV infected patients and with D%Ki-67+RTE counts in co-infected patients (r = 0.709, p = 0.022; Figure 5E , right panel). Overall, IL-7 concentration was associated with reduced thymopoiesis and RTE proliferation, lower consequently leading to limited circulating RTE and naïve T-cell counts. These data strongly suggest that changes in IL-7 plasma levels during IFNa therapy directly impact the homeostasis of RTEs.
We herein demonstrated that IFNa-based therapy leads to major lymphocytopenia in naïve T-cell compartments, in particular in the RTE subset. Several mechanisms could be implicated in the establishment of such a lymphocytopenia [41] . Among these, enhanced apoptosis [42, 43] , cell sequestration in lymphoid or non-lymphoid organs [12, 21, 22] and regulation of peripheral T-cell homeostasis [20] . In our study, no major change in cell survival (Bcl-2 expression) or T-cell activation (CD25 and CD69 expression) was observed during the follow-up period (data not shown). Moreover, we did not observe any significant modification in Ki-67 expression in any T-cell subset during the first month of therapy (data not shown and Figure 3 ). Finally, IFNa-induced T-cell homing, although rapid and massive, is only a transient process [22] suggesting that this mechanism marginally contributes to the observed long lasting lymphocytopenia.
Interestingly, both sjTREC quantification (sjTREC/mL) and intrathymic precursor T-cell proliferation (sj/bTREC ratio) were affected very early on after initiation of therapy ( Figures 2B and  3B ). While sjTREC frequency and concentration in peripheral blood can be affected by modifications of parameters that impact on peripheral T-cell homeostasis (cycling, survival/apoptosis, homing), the sj/bTREC ratio is a marker of the intrathymic proliferation history of RTEs. Indeed, this parameter is generated by cell proliferation that occurs between TCRb chain rearrangement and the excision of TCRd locus. Further cell cycling after TCRa chain rearrangement does not modify the sj/bTREC ratio as both type of TRECs are similarly diluted upon cell proliferation. Accordingly, while exported to the periphery, the sj/bTREC ratio of mature T-cells cannot be modified. Therefore, while the observed decrease in sjTREC concentration (figure 2) can be a consequence of modifications of circulating T-cell homeostasis, the decline of the sj/bTREC ratio observed during the first months of IFNa therapy (figure 3) defines changes in thymocyte proliferation, thus in thymic output [28] . Acutely infected patients demonstrated a higher sj/bTREC ratio at baseline than patients in the chronic phase. However, this group was younger (Median = 31.5 (26-47)) versus Median = 53.5 (37-61)) than the chronic group (p,0.01; data not shown) and demonstrated normal sj/bTREC ratio for their age. Similar evolution of thymic function and circulating T-cell subsets were observed in both groups of HCV-infected patients, irrespective of the development stage of HCV pathology. The lack of effect of IFNa therapy in HIV/HCV co-infected patients might be due to the fact that, as expected for chronically HIV-infected individuals, these patients already had a low thymic function at study entry. The impairment of thymopoiesis in HCV-infected patients under IFNa therapy is reminiscent of that observed during the acute phase of HIV-1 infection [23] which suggested that long term production of IFNa, as part of the anti-HIV innate immune response, may play a role in the observed thymic defect. The correlation between decline in IL-7 plasma levels under IFNa therapy and both thymic dysfunction and reduced T-cell counts, in particular in the naïve and RTE compartments ( Figures 5A and  5B) , confirms this hypothesis. Finally, in a recent study, we showed that IFNa treatment leads to decreased sjTREC frequency as well as reduced naïve T-cell and RTE counts in SIV-infected rhesus macaques [30] . Such an effect was accompanied by a 30-40% decrease in IL-7 plasma levels in these animals and could be counteracted by injection of recombinant simian IL-7 [30] . One could expect that such an effect of type I IFNs is not restricted to HIV-infection as many viral infections induce IFNa responses and cause transient lymphocytopenia in the infected hosts [3, 4, 5, 6] .
Moreover, the IFNa-induced reduction of thymic function and its probable consequences on naïve T-cell diversity may contribute to the higher infectious risk associated with IFNa therapy, in particular observed in older patients [15, 16, 44] . There are multiple sources for circulating IL-7 during viral infections including lymphoid organs, epithelial cells and recently the liver was identified as a major source of IL-7. Moreover, increased plasma IL-7 levels can also be observed during viral infection in non-lymphopenic individuals ( [33] and unpublished data), suggesting a role in the development of immune responses. Indeed, this cytokine participates to T-cell homing in various lymphoid and non-lymphoid tissues through stimulation of local chemokine productions [45] . Increased IL-7 plasma levels in lymphopenic individuals is likely due to reduced consumption [46] yet augmented production to counteract lymphopenia cannot be excluded [33] . The recent identification of the liver as an IL-7 producing tissue upon TLR stimulation [47] makes it tempting to speculate that HCV-infection can also, through TLR activation, stimulate IL-7 production by the liver. Indeed, non-lymphopenic HCV-infected patients demonstrate similar IL-7 plasma levels than lymphopenic HIV-infected individuals [33, 48] suggesting that most of the IL-7 production in untreated HCV-infected patients was not linked to circulating T-cell counts. The reduction of IL-7 plasma levels while lymphocytopenia establishes under IFNa therapy, the absence of a correlation between IL-7 plasma levels and CD127 expression and the concomitance of decreases in IL-7 plasma levels and HCV viral load under therapy suggest that viremia might be driving IL-7 production before initiation of therapy. Our data suggest that, before initiation of IFNa therapy, actively replicating HCV leads to the overproduction of IL-7. Subsequent reduction of IL-7 production upon initiation of therapy probably reflects the elimination of IL-7 producing HCV-infected hepatocytes. This sudden reduction of IL-7 plasma levels may lead to diminished thymopoiesis. The fact that IL-7 plasma levels did not reach normal levels when HCV became undetectable may suggest that, after the initial decline that follows the drop in viremia, IL-7 plasma levels were regulated, as in HIVinfected patients [33] and in IFNa-treated SIV-infected rhesus macaques [30] , as a consequence of lymphocytopenia through either reduced consumption or increased production in lymphoid organs [49] . Future studies with a longer follow-up period, in particular after the end of IFNa therapy and recovery from lymphocytopenia are required to further elucidate this point.
We herein demonstrated that a substantial reduction in thymic export was observed in HCV-infected patients, during the first months of IFNa therapy. This effect directly paralleled IFNainduced lymphocytopenia and decreased IL-7 plasma levels, initially high in HCV-infected patients. These data suggest that IL-7 production by the liver, a consequence of active HCV replication, was reduced while patients controlled HCV viremia. Restricted IL-7 plasma levels might, in association with the antiproliferative effect of IFNa, limit T-cell production in the thymus. Our study highlights the therapeutic potential of IL-7 as a complement to the standard IFNa based treatment to help HCVinfected patients to sustain normal circulating T-cell counts, and restore the diversity of the peripheral T-cell repertoire through its central thymopoietic effect. Restoring the breadth and intensity of T-cell control over the HCV virus might be immediately beneficial for the HIV/HCV co-infected population and offer new promising avenues for chronic HCV in the context of massive drop of HCV viral load after short term treatment with new antiviral compounds that will continue to be administered in combination with IFNa [50] .
Sixteen HCV-infected patients (C-1 to C-16) and ten HIV/ HCV co-infected patients (I-1 to I-10) naïve to IFNa therapy were enrolled in this study. A summary of the virological and immunological status of patients at baseline is shown in table 1. All the HIV/HCV co-infected patients but one were under HAART with undetectable viremia (,40 HIV copies/mL). Chronically infected patients (C-9 to C-16 and I-1 to I-10) initiated pegylated IFNa/ribavirin treatment (IFNa-2a: Pegasys, 180 mg weekly, Ribavirin: Copegus, 800 mg to 1000 mg daily) and were followed over a 4 months period. Patients included in the acute phase of HCV infection (C-1 to C-8) were treated with pegylated IFNa (IFNa-2a: Pegasys, Roche, 180 mg weekly) [51, 52] . Blood samples were taken monthly on EDTA. Two milliliters of total blood were 2-fold diluted in FCS/20%DMSO frozen at 280uC and conserved in liquid nitrogen. These total blood samples were subsequently used for flow cytometry analyses. Plasma was separated from the remaining eight milliliters and mononuclear cells were purified on Ficoll Hypaque (Eurobio, Courtaboeuf, France) and frozen for further analyses. Patients from the HCV mono-infection group were followed at the Centre de Recherche du CHUM, Hôpital Saint Luc, Montreal, QC, Canada and its collaborators as previously described [9, 53] . Patients from the HIV-HCV groups were followed at the Hôpital Henri Mondor, Creteil, France. Clinical protocols conformed to Figure 5 . Variations in IL-7 plasma levels correlate with evolution of RTE production. Correlations. between variations in IL-7 plasma levels (DIL-7) and either variations in (A) total (CD4 + + CD8 + ) naïve T-cell counts (Dnaïve T-cell counts), (B) RTE defined as CD31 hi naïve CD4 + T-cells (DRTE CD4 counts), (C) the sj/bTREC ratio (Dsj/bTREC ratio), (D) the frequency of Ki-67 + cells in the RTE CD4 + T-cell subset (D%Ki-67 + in CD4 + RTEs) or (E) the number of circulating Ki-67 + CD4 + RTEs (DKi-67 + RTE counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) HCV-infected patients (left panels) and HIV/HCV co-infected patients (right panels). Correlation coefficients (Spearman's r) and the associated probabilities (p) are shown. doi:10.1371/journal.pone.0034326.g005 ethical guidelines of the authors' institutions and the US Department of Health and Human Services' human experimentation guidelines. This study was approved by both the Ethical committee of Centre Hospitalier de l'Université de Montreal (CHUM) and the ethical committee of Hôpital Henri Mondor, Créteil, France. Samples were obtained with the written subjects' informed consent.
Immunophenotyping and flow cytometry analysis FACS analyses were performed on cryopreserved samples. After thawing blood cells were incubated for 15 minutes at 4uC with conjugated monoclonal antibodies (mAbs). For intracellular labeling, cells were permeabilized with the Cytofix/Cytoperm Kit (Becton Dickinson) before incubation with specific mAbs according to the manufacturer's instructions. Samples were then washed, fixed in 2% paraformaldehyde phosphate-buffered saline (PBS/PFA 2%) and acquired using a Cyan cytofluorometer (Dako) and analyzed with FlowJo 8.7 software.
The monoclonal antibodies used in this study were: CD3-pacific blue (PB) (clone UCHT-1; Dako, Trappes, France), CD4-peridin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5) (clone L200; BD, Le-Pont-de-Claix, France), CD45RA-phycoerythrin (PE) (clone HI100; BD), CCR7-allophycocyanin (APC) (clone 150503; R&D Systems Europe, Lille, France); CD8-phycoerythrin-cyanine 7 (PE-Cy7) (RPA-T8; BD), CD31-biotin (clone WM59; AbDSerotec, Düsseldorf, Germany); Ki-67-fluorescein isothiocyanate (FITC) (clone MIB-1; Dako), Bcl-2-FITC (clone 124; Dako) and strepatavidin-PE-Texas-RED (BD).
IL-7 was quantified in the plasma using the IL-7 Quantikine HS kit according to the manufacturer's instructions (R&D Systems Europe). Plasma soluble-CD127 quantification Soluble plasma IL-7 receptor (sCD127) quantification was performed as previously described [54] .
Parallel quantification of the sjTREC and the 13 DJbTRECs, together with CD3c gene (used as a housekeeping gene) was performed for each sample using LightCyclerTM technology (Roche Diagnostics) with a technique adapted from [29] . Intrathymic precursor T-cell proliferation was evaluated through calculation of the sj/bTREC ratio as described [23] .
HCV RNA quantification was performed using an in-house quantitative real-time reverse transcription-PCR assay as previously described [9] , COBAS Amplicor HCV Monitor test TM , Version 2.0 (sensitivity 600 IU/ml)), qualitative COBAS Ampli-Prep/COBAS Amplicor HCV test TM , version 2.0 (sensitivity 50 IU/ml) or Abbott RealTime HCV assay TM (sensitivity 12 IU/ ml).
Statistical analyses (Spearmans rank correlations and Wilcoxon matched -paired signed-rank tests) were performed using the Stata/IC 10.0 (Stata corporation, College Station, Tx U.S.A.). Due to the exploratory nature of the study there was no correction for multiple comparisons, and calculated p values are reported herein.  Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile. METHODS: We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G. RESULTS: Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection. CONCLUSION: Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen. Respiratory syncytial virus (RSV) is the most important viral pathogen of causing serious bronchiolitis and pneumonia in infants and young children worldwide. RSV is also receiving increasing recognition as an important cause of lower respiratory tract illness in immunocompromised patients, the young children, and the elderly (1) (2) (3) . Despite the importance of RSV as a respiratory pathogen, there is no licensed vaccine currently available against RSV infection.
In the 1960s, the immunization of children with formalin inactivated-RSV (FI-RSV) vaccine not only failed to protect, but also yielded enhanced pulmonary disease in vaccinated infants following RSV infection (4, 5) . Studies with BALB/c mice have become a useful model for RSV pathogenesis, since FI-RSV-enhanced disease is also observed in vaccinated BALB/c mice. It is likely that the augmented lung disease and the development of pulmonary eosinophilia are attributed to an excessive Th2 type immune response (6) .
The RSV G protein is one of the major protective antigens and good inducer of strong serum and mucosal neutralizing antibody responses. It also has single immunodominant I-E d epitope spanning RSV G amino acid 183 to 198 and largely induces a specific subset of CD4 T cells (7, 8) . Previously, we have reported that RSV G protein fragment (spanning amino acid residues 131-230) delivered by recombinant adenoviral vector successfully elicited long-term protective immunity against RSV infection in mice (9) .
Baculoviruses are enveloped viruses possessing a rod-shaped nucleocapsids in which double stranded circular DNA genome of 88-135Kbp is packaged (10) . Baculoviruses are generally utilized as a vector for the high level production of recombinant proteins but they could be also employed in gene transfer to mammalian cells (11) (12) (13) . Several research groups have demonstrated that vaccination with recombinant baculovirus can induce high-level humoral and cell-mediated immunity against various antigens, suggesting that baculovirus could be used as a vaccine carrier (14, 15) . In addition, it has been reported that immunization with a recombinant baculovirus expressing the hemagglutinin gene of influenza virus elicited a strong innate immune response and protected mice against influenza virus challenge (16) . Moreover, it is known that there is no baculovirus-specific immunity in mammals that might interfere the action of baculovirus-based vaccine (17) (18) (19) .
In the present study, a recombinant baculovirus displaying the RSV G protein (Bac-RSV/G) was constructed. Our results clearly show that strong humoral and cellular immune responses were induced by intranasal administration of Bac-RSV/G in a mouse model. Importantly, complete protection against RSV challenge without vaccine-induced illness was observed in Bac-RSV/G-immune mice, suggesting that vaccination of Bac-RSV/G could be utilized as a prophylactic vaccination regimen against RSV infection.
Baculoviruses were propagated in Spodoptera frugiperda 9 (Sf9) insect cells using SF-900 serum-free medium (Invitrogen) at 27 o C. RSV A2 strain was propagated in HEp-2 cells (ATCC, Manassas, VA) in Dulbecco's modified Eagle's medium (Life Technologies, Gaithersburg, MD) supplemented with 3% heat-inactivated fetal calf serum, 2 mM glutamine, 20 mM HEPES, nonessential amino acid, penicillin, and streptomycin and titrated for infectivity by plaque assay as described elsewhere (20) .
The coding sequence of RSV G protein from RSV A2 strain was amplified from cDNA by PCR and cloned into the EcoR I and Xho I sites of pFastBac-1 vector (Fig. 1A) . The recombi-nant baculovirus was subsequently generated by using the Bac-to-Bac Ⓡ system (Invitrogen) following the manufacturer's instructions. The recombinant baculoviruses were purified from supernatants of infected Sf9 insect cells with 25% (w/v) sucrose in 5 mM NaCl, 10 mM EDTA in a SW28 rotor (Beckman, USA) at 24,000 rpm for 75 min at 4 o C. The supernatant was decanted, and the pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged for 4 h at 24,000 rpm, 4 o C. The viral pellet was resuspended in PBS and titrated by plaque assays on Sf9 cells.
Female BALB/c mice were purchased from Charles River Laboratories Inc. (Yokohama, Japan). Mice kept under specific-pathogen-free conditions. For immunization, 6-to 8-weekold mice were inoculated with baculoviruses via the intranasal (i.n.) route. For i.n. immunizations, mice were lightly anesthetized by ether/chloroform inhalation, and 2×10 8 PFU of Bac-RSV/G or Bac-control in a volume of 70μl was applied to the left nostril. Three to four weeks after second immunization, the mice were challenged i.n. with 1×10 6 PFU of live RSV A2 strain. All animal studies were performed according to the guidelines of our Institutional Animal Care and Use Committee (Approval No. 2010-9-4).
Blood was obtained from the retro-orbital plexus with a heparinized capillary tube, collected in an Eppendorf tube, and centrifuged, and serum was stored at −20 o C. RSV G protein-specific antibody titers in immunized mice were measured by a direct enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated overnight with 100μl/well of 0.5μg/ml of purified G protein fragment diluted in PBS and then blocked with PBS containing 1% skim milk and 0.05% Tween-20 for 2 h. Sera were then added in serial dilutions and incubated for 2 h. The plates were washed five times with PBS containing 0.05% Tween 20 and incubated for 30 min with various dilutions of horseradish peroxidase-conjugated affinity-purified rabbit anti-mouse total IgG, secondary antibody (Zymed Laboratories, San Francisco, CA). The plates were washed five times and developed with 3,3',5,5'-tetramethylbenzidine, and the reaction was stopped with 1 M H 3 PO 4 and analyzed at 450 nm with a Thermo ELISA plate reader. The wells receiving no serum were used to calculate cut-off values.
The lungs were perfused with 5 ml of PBS containing 10 U/ml heparin (Sigma-Aldrich, St. Louis, MO) through the right ventricle using a syringe fitted with 25-gauge needle. The lungs were then removed and placed in RPMI medium supplemented with glutamine, gentamicin, penicillin G, and 10% FBS (HyClone, Logan, UT). The tissue was then processed through a steel screen to obtain a single-cell suspension, and particulate matter was removed by passage through a 70-μm Falcon cell strainer (BD Labware, Franklin Lakes, NJ). Freshly explanted BAL fluid or lung cells were purified by density gradient centrifugation and stained in PBS-3% FBS-0.09% NaN 3 using fluorochrome-conjugated antibodies. The antibodies used were anti-CD4 (clone RM4-5), anti-CD44 (clone IM7) or anti-CD43 (clone 1B11). Both antibodies were purchased from BD PharMingen (San Diego, CA). After staining, cells were fixed in PBS-2% (wt/vol) paraformaldehyde, and events were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). To enumerate the number of cytokine-producing cells, intracellular cytokine staining was performed as described elsewhere (21) . In brief, 2×10 6 freshly explanted lung lymphocytes were cultured in a culture tube. Cells were left untreated or stimulated with 10μM G (183-195) peptide (WAICKRIPNKKPG) and then incubated for 5 h at 37 o C in 5% CO2. Brefeldin A (5μg/ml) (Sigma-Aldrich) was added for the duration of the culture period to facilitate intracellular cytokine accumulation. Cells were then stained for surface markers, washed, fixed, permeabilized with fluorescence-activated cell sorter buffer containing 0.5% saponin (Sigma-Aldrich, Seoul, Korea), and stained for cytokines. The antibodies used were anti-IFN-γ (clone XMG1.2) or anti-IL-17A (TC11-18H10.1). Dead cells were excluded on the basis of forward and side light scatter patterns. Data were collected using CELLQuest software (BD Biosciences) and analyzed with CELLQuest and WinMDI version 2.9 software (Scripps Research Institute, La Jolla, CA). Lung supernatants were also collected for analysis with the FlowCytomix (eBioscience), according to the protocol. Kits containing antibody beads (IL-4, IL-5, IL-6, IL-10, IL-13) were used to measure cytokine levels in each of the samples. 
Four or five days after RSV challenge, subsets of mice were euthanized and the lungs were removed into Eagle's modified essential medium. The tissues were then processed through a steel screen to obtain a single-cell suspension, and particulate matter was removed by passage through a 70-μm cell strainer (BD Labware, Franklin Lakes, NJ). The supernatants were collected, and RSV titers in the supernatants were measured by standard plaque assay on subconfluent HEp-2 monolayers. The data are expressed as the PFU per gram of lung tissue.
Comparison of differences was conducted by using an un-IMMUNE NETWORK http://www.ksimm.or.kr Volume 12 Number 1 February 2012 paired, two-tailed Student t test. The difference was considered statistically significant when the p value was ≤0.05.
Generation of recombinant baculovirus displaying RSV G The recombinant baculovirus expressing RSV G coding sequences under the control of the polyhedrin promoter, Bac-RSV/G, was generated using Bac-to-Bac system with a pFastBac-1 plasmid as shown in Fig. 1A . To examine whether Bac-RSV/G express and display RSV G in the virus particle, western blotting analysis was performed with sucrose gradient-purified baculoviruses. Using the G-specific monoclonal antibody, specific bands of approximately 70 kDa to 90 kDa were detected in the purified Bac-RSV/G particles, but not in Bac-control virus particles (Fig. 1B) . As a positive control, purified RSV particles produced in HEp-2 cells were used and slightly higher molecular weight bands were detected (Fig.  1B) . The size difference might be due to different patterns of glycosylation between insect cells and human cells.
To investigate the immunogenicity of Bac-RSV/G, the groups of mice were immunized i.n. with Bac-RSV/G, Bac-control, or PBS. RSV G-specific antibody levels in sera from the immune mice at 2 weeks after priming and 2 weeks after boost-ing were determined by ELISA. The specific antibody responses were barely detectable in all groups of mice at 14 days after priming (data not shown). Following booster immunization, however, the mean titers of serum antibodies increased significantly only in the group of mice immunized with Bac-RSV/G ( Fig. 2A) . Secretory IgA has been shown to directly mediate local immunity against aerial pathogens, implying an important role for antibody in protection in the upper respiratory tract (22) . Thus, for an effective RSV vaccine development, the induction of secretory IgA on respiratory mucosal surface is critical. To examine whether Bac-RSV/G vaccination elicits IgA response in the respiratory tract, bronchoalveolar lavage (BAL) was performed at day 5 after RSV challenge and levels of IgA were determined by RSV-specific ELISA. Each group of mice immunized with Bac-control or PBS did not exhibit any specific IgA response, whereas Bac-RSV/G-immune mice showed significantly enhanced level of specific IgA response (Fig. 2B) . These results suggest that intranasal immunization of Bac-RSV/G effectively induce both RSV G-specific serum IgG and respiratory IgA.
Since the RSV G contains I-E d -restricted CD4 T-cell epitope, we examined whether Bac-RSV/G immunization induces specific CD4 T cells or not. To this end, Bac-RSV/G, Bac-control, or PBS immune mice were challenged with RSV. CD4 T cell responses were evaluated at 5 days after challenge by measuring the production of IFN-γ in lung lymphocytes that had been stimulated with I-E d -restricted G (183-195) epitope peptide ex vivo. As shown in Fig. 3 , G-specific CD4＋IFN-γ＋ cells were detected in the lungs of Bac-RSV/G-immunized group (∼2.5% of gated CD4 T cells on average), while few specific cells (＜0.2% of CD4 T cells) were observed in the lungs of Bac-control or PBS-immunized group. We next investigated whether immunization of Bac-RSV/G induces other CD4＋ T cell subset such as Th2 and Th17. Bac-RSV/G-vaccinated mice showed increased frequency of G-specific IL-17-producing CD4 T cells in the lungs after RSV challenge (∼3.7% of gated CD4 T cells on average; Fig. 4 ). However, the levels of Th2-type cytokines such as IL-4, IL-5, IL-10, and IL-13 measured by multiplex antibody-based bead assays were not significantly different in the lungs from all groups of mice (Fig. 5) . Together, these results indicated that mixed G-specific Th1/Th17-cell responses were induced by Bac-RSV/G vaccination.
To determine whether Bac-RSV/G has protective efficacy against RSV infection, mice were challenged with live RSV A2 virus at four weeks after booster immunization. While there was active RSV replication in the lungs of the Bac-control or PBS immune mice, immunization of Bac-RSV/G prevented any detectable RSV replication in the lungs during the peak of infection (Fig. 6) . Interestingly, immunization of Bac-control resulted in partial decrease of viral replication at the peak. This might be due to nonspecific innate immunity induced by baculovirus inoculation. It was previously reported that intranasal administration of wild-type baculovirus induces strong innate immune responses and provides protection against lethal challenge of influenza virus (16) . Fig. 3 . BAL was performed five days after RSV challenge, and BAL cells were stained with antibodies to CD45, Siglec-F, and CD11c, and eosinophils were quantitated among CD45 
It was reported that immunization with vaccinia virus expressing the entire RSV G glycoprotein results in pulmonary eosinophilia following challenge with live RSV (6, 23, 24) . These studies indicated that RSV G expressed vaccines have a possibility for the development of pulmonary eosinophilia. To determine whether the intranasal immunization of Bac-RSV/G potentiates eosinophilia, the levels of eosinophils in the BAL fluids of the immune mice were examined by flow cytometry 5 days after RSV challenge using antibodies to Siglec-F, CD45, and CD11c as described previously (25) . Bac-RSV/G-immunized mice have a higher frequency of CD11c-SiglecF＋ in the BAL as compared with Bac-control or PBS group (Fig.  7A , p＜0.05), but the level of eosinophil influx in Bac-RSV/G immune mice was relatively weak (∼2% of the total CD45＋ BAL cells). These results suggest that intranasal Bac-RSV/G immunization barely increase the risk of vaccine-induced eosinophilia.
To evaluate other RSV-induced pathology by Bac-RSV/G immunization, we monitored weight loss in all immune-mice after RSV challenge. Following infection with live RSV, there was no significant weight loss in Bac-RSV/G-immune mice (Fig. 7B ) and disease score (data not shown). Taken together, these results suggest that intranasal Bac-RSV/G vaccination give rise to protective immunity in the absence of subsequent vaccine-enhanced disease.
To develop a safe and effective RSV vaccine, many strategies and platforms have been applied in pre-clinical and clinical phases (26) . Many RSV vaccine candidates specifying target antigens employed two envelop proteins, G attachment protein and F fusion protein, because these antigens are known to induce protective immunity against live RSV infection. However, in the BALB/c mouse model, immunization of G protein expressed from recombinant vaccinia virus elicited Th2-biased responses, which was responsible for the vaccine-enhanced diseases (27) . Thus, G protein has been falsely regarded as a bad target antigen for a long time, although it could induce strong neutralizing antibody responses upon immunization. Recently, it has been suggested that G protein itself is not the cause of vaccine-enhanced diseases and the proper balance between RSV-specific Th1 and Th2 responses is rather an important factor controlling both safety and efficacy of G-targeted RSV vaccine. Using appropriate platforms and/or adjuvants, this balance could be achieved with G-targeted vaccines. For example, we have recently demonstrated that mucosal immunization of recombinant adenovirus vac-cine expressing the core domain of G successfully induced protective immunity without vaccine-induced diseases (9) .
Baculovirus has recently been emerged as a new tool for vaccine vector development since it has many advantages as a vaccine platform (28) (29) (30) . First, baculovirus, which has been used to overexpress recombinant proteins in insect cells, is neither replication-competent nor pathogenic in mammalian cells, making it safer vaccine vector than other mammalian viral vectors. In addition, baculovirus-based vaccines produced in insect cells are not contaminated by LPS. Second, baculovirus-based vaccine possesses several advantages upon production, such as easy manipulation, relatively simple scale-up, and high titers (11, 31) . Thirdly, there is no pre-existing vector immunity against baculovirus in mammals, which enables baculovirus to escape vector neutralization by pre-existing immunity during in vivo delivery (19) . Lastly, baculovirus has the ability to stimulate strong innate immunity, exhibiting strong adjuvanticity itself. It has been previously shown that inoculation of wild-type baculovirus alone can stimulate the secretion of inflammatory cytokines from innate immune cells and confer protection from lethal virus infection in mice (16) . Consistent with this report, intranasal inoculation of control baculovirus also provided partial protection against live RSV challenge in our study (Fig. 6) . The baculovirus-mediated stimulation of innate immunity might be associated with the induction of type I interferons, mediated by both TLR9-dependent (32) and TLR9-independent pathways (33) . Thus, it becomes evident that baculovirus can act as a natural adjuvant by stimulating innate immunity and be used as a safe and effective vaccine carrier.
Our data demonstrate that intranasal immunization of Bac-RSV/G vaccine induces complete protection from RSV infection, which is associated with enhanced Th1 and Th17 responses in the absence of significant Th2 responses. Although the role of Th1 or CTL responses to RSV clearance is well delineated (34) , the role of Th17-mediated responses have not been fully elucidated in host defense against RSV. Th17 cells, which are characterized by massive production of IL-17, are thought to contribute to autoimmune diseases but also play a crucial role in host defense against extracellular bacteria (35, 36) . In addition, Th17-associated effector molecules seem to be necessary and sufficient for protection against several viral pathogens. For example, recent studies have reported that Tc17 cells are potently cytolytic against vaccinia virus-infected cells (37, 38) . In these studies, both IL-17-producing CD4＋ and CD8＋ T cells are necessary to confer pro-tection against viral infections, exhibiting cytotoxic activity for clearance of vaccinia virus-infected cells. Mice immunized with a recombinant vaccinia virus expressing the G protein of RSV (vvG) exhibit pulmonary eosinophilia induced by Th2 type cytokines after challenge RSV infection (6) . Th2 type cytokines are thought to promote eosinophil infiltration to the lungs of RSV-infected mice and may contribute to immunopathology associated with vaccine-enhanced diseases. Interestingly, we showed that the levels of Th2-type cytokines, such as IL-4, IL-5, IL-10, or IL-13, were very low in the lungs of Bac-RSV/G-immune mice following RSV challenge, not significantly different to those in the PBS control group. Several evidences suggest that IL-17 negatively regulates Th2 cytokine production (39, 40) . Thus, the induction of Th1/Th17 responses by Bac-RSV/G vaccination may have attenuated the development of Th2 responses. Furthermore, IL-17 may play certain roles in immune defense in the lungs, including augmentation of mucosal immunity by delivery of IgA and IgM into the airway lumen (41) . Though the exact mechanism by which the Th17 responses are induced by Bac-RSV/G vaccination is not clear, the G-specific Th17 response may be associated with clearance of RSV from the lungs. Further studies will be needed to elucidate the exact role of Th17 immunity in RSV infection.
In conclusion, our present study demonstrates that baculovirus displaying RSV G protein, Bac-RSV/G, induces antigen-specific humoral and cellular immunity, and provides protection against RSV challenge without vaccine-induced immunopathology. Thus, our study provides strong evidence that Bac-RSV/G vaccine could be further developed as a mucosal RSV vaccine. Divergent lineage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Côte d'Ivoire Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses. Discovery of phylogenetically divergent hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] raises the possibility that rodents (order Rodentia, family Muridae and Cricetidae) may not be the principal or primordial reservoirs. Moreover, newfound hantaviruses harbored by soricomorphs of multiple species, distributed in widely separated geographic regions across four continents, suggest that their host diversity may be far more expansive than previously assumed. Specifically, other mammals having shared ancestry or ecosystems with soricomorphs may serve as reservoirs and may be important in the evolutionary history and diversification of hantaviruses. In particular, bats (order Chiroptera) may be potential reservoirs by virtue of their rich diversity and vast geographical range, as well as their demonstrated ability to host myriad medically important, disease-causing viruses [14] [15] [16] [17] [18] . Surprisingly little attention, however, has been paid to this possibility.
As in our previous investigations on the spatial and temporal distribution of hantaviruses in soricomorphs [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] , we relied on the availability of archival tissues. Using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), total RNA was extracted from 168 frozen and 45 ethanol-fixed liver and other visceral tissues of 213 insectivorous bats (representing 13 genera), collected during May 1981 to June 2011 in Asia, Africa and the Americas (Table 1) . cDNA was then prepared with the SuperScript III First-Strand Synthesis System (Invitrogen) using random hexamers, and PCR was performed as described previously, using an extensive panel of oligonucleotide primers, designed on conserved genomic sequences of rodentand soricomorph-borne hantaviruses [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] 19, 20] . Each reaction mixture contained 250 μ dNTP, 2 mM MgCl 2 , 1 U AmpliTaq polymerase (Roche, Basel, Switzerland) and 0.25 μ oligonucleotide primers. Initial denaturation at 94°C for 5 min was followed by two cycles each of denaturation at 94°C for 40 s, two-degree step-down annealing from 48°C to 38°C for 40 s, and elongation at 72°C for 1 min or 1 min 20 s, then 32 cycles of denaturation at 94°C for 40 s, annealing at 42°C for 40 s, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA). Amplicons were purified using the QIAQuick Gel Extraction Kit (Qiagen, Hilden, Germany), and DNA sequencing was performed using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA).
After innumerable failed attempts, hantavirus RNA was detected by RT-PCR in ethanol-fixed liver tissues from two of 12 banana pipistrelles (Neoromicia nanus Peters 1852), captured during June 2011 near Mouyassué village (5°22'07"N, 3°05'37"W) in Aboisso District, 130 km from Abidjan, in the extreme southeastern region of Côte d'Ivoire in West Africa ( Figure 1 ). The taxonomic identity of the hantavirus-infected vesper bats was confirmed by phylogenetic analysis of the cytochrome b gene of mtDNA (GenBank JQ287717), amplified by PCR as previously described [8, 9] . Despite similarly exhaustive efforts, hantavirus RNA was not detected in any of the other bat species tested (Table 1) , including frozen liver tissue of six tiny pipistrelles (Pipistrellus nanulus), collected in Parc National du Mont Péko, 700 km northwest of Mouyassué, in February 1992, and ethanol-fixed liver tissue of three tiny pipistrelles, collected in December 2009 in Azagny, where a hantavirus was previously found in the West African pygmy shrew (Crocidura obscurior) [8] .
A 423-nucleotide region of the RNA-dependent RNA polymerase-encoding L segment, amplified using a hemi-nested primer set (outer: 5'-GAAAGGG-CATTNMGATGGGCNTCA GG-3', 5'-AACCADT-CWGTYCCRTCATC-3'; inner: 5'-GNAAAYTNATGT-ATGTNAGT GC-3', 5'-AACCADTCWGTYCCRT-CATC-3'), was aligned and compared with hantavirus sequences available in GenBank, using ClustalW (DNASTAR, Inc., Madison, WI) [21] and transAlign [22] . The newfound hantavirus, designated Mouyassué virus (MOUV), exhibited low nucleotide and amino acid sequence similarity of less than 69% to all representative soricomorph-and rodent-associated hantaviruses, except for the 76.3% sequence similarity with Nova virus (NVAV), previously reported in the European common mole (Talpa europaea) [12] . Interestingly, MOUV sequences were identical in the two banana pipistrelles (KB576 and KB577), a male-female pair captured simultaneously and presumed to be a mating couple, suggesting horizontal virus transmission or common-source infection.
MOUV formed a uniquely divergent lineage, distant from all other hantaviruses identified to date, except for NVAV (Figure 2 ), in phylogenetic trees based on L-segment sequences, generated by the maximum-likelihood and Bayesian methods, implemented in PAUP* (Phylogenetic Analysis Using Parsimony, 4.0b10) [23] , RAxML Blackbox webserver [24] and MrBayes 3.1 [25] , under the best-fit GTR+I+Γ model of evolution established using jModeltest 0.1.1 [26] . Topologies were well supported by bootstrap analysis of 100 iterations, and posterior node probabilities based on two runs each of 2 million generations sampled every 100 generations with burn-in of 25%.
Despite the overall success of our brute-force RT-PCR approach at identifying previously unrecognized hantaviruses in frozen tissues [2, 3, [5] [6] [7] [10] [11] [12] [13] and tissues preserved in RNAlater ® RNA Stabilization Reagent [4, 8] , designing universal primers for the amplification of soricomorph-borne hantaviruses has presented continuing challenges. Thus, while it is likely that many more hantaviruses await discovery, overcoming technical barriers is essential to facilitating their detection. Viewed in this context, the failure to detect hantavirus RNA in all but one bat species was not altogether unexpected and may be attributed simply to suboptimal primer design and imperfect cycling conditions. Also, low RNA yields and poor RNA preservation in tissues fixed in ethanol under field conditions may have thwarted our efforts at obtaining more of the MOUV genome. That said, the successful amplification of hantavirus RNA from ethanol-fixed tissues is highly instructive and augments the pool of archival tissues for future exploratory studies of hantaviruses in bats, and possibly other insectivorous small mammals that share ancestral lineages with soricomorphs, such as hedgehogs (order Erinaceomorpha, family Erinaceidae).
Dating to the seminal discovery of Hantaan virus in lung tissue of the striped field mouse (Apodemus agrarius) [27] , lung has been the preferred tissue in studies aimed at finding new hantaviruses [28] [29] [30] . However, lung is not the only tissue in which hantaviruses can be detected [27, 31] . In our search of genetically distinct hantaviruses in long-stored archival tissues from shrews and moles, lung tissue was frequently unavailable. Instead, liver tissue was more often accessible and proved to be quite suitable [4, 5, 12, 13] . Similarly, liver tissues were more often available in the present study. As in reservoir rodents and soricomorphs, hantavirus RNA is likely to be present in many tissues of persistently infected bats. Real-time quantitative RT-PCR analysis of lung, liver and other viscera will clarify the tissue distribution of MOUV in newly captured banana pipistrelles from Mouyassué.
Having their fossil origins in the Eocene epoch, approximately 50 million years before present, bats occur on every continent except Antarctica and are among the most speciose orders of mammals, with more than 1,100 extant species [32] . The banana pipistrelle, which is distributed widely in forests and savannas across sub-Saharan Africa ( Figure 1C, inset) , is one of 13 species in the genus Neoromicia of the family Vespertilionidae and subfamily Vespertilioninae. Like other vesper bats, the banana pipistrelle is insectivorous. Unlike large fruit bats, such as the straw-colored fruit bat (Eidolon helvum) and hammer-headed bat (Hypsignathus monstrosus), which are sold as bush meat, the banana pipistrelle, weighing approximately 3 g, is not consumed as food. However, because banana pipistrelles occasionally roost within houses or reside near human habitation, rare human encounters raise the possibility of hantavirus exposure.
Previously, serological evidence of hantavirus infection was reported in the common serotine (Eptesicus serotinus) and greater horseshoe bat (Rhinolophus ferrumequinum) captured in Korea [33] , but genetic analysis of hantaviral isolates from these insectivorous bat species proved to be indistinguishable from prototype Hantaan virus [34] , suggesting laboratory contamination. In the present study, the strikingly divergent lineage of MOUV precluded any possibility of contamination and lends support to our earlier conjecture that the ancient origins of hantaviruses may have involved insect-borne viruses [7, 10] , with subsequent adaptation to and host switching between early soricomorph and chiropteran ancestral hosts in the mammalian superorder Laurasiatheria. However, since the biological and evolutionary implications of bats as reservoirs of hantaviruses are considerable, studies are underway to establish that the banana pipistrelle is the natural host of MOUV. Moreover, high-throughput sequencing technology is being applied to obtain the full genome of MOUV and to ascertain the geographic range and genetic diversity of hantaviruses harbored by bats. Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus In this study we propose a novel bacterial vaccine strategy where non-pathogenic bacteria are complemented with traits desirable for the induction of protective immunity. To illustrate the proof of principle of this novel vaccination strategy, we use the model organism of intracellular immunity Listeria. We introduced a, low copy number BAC-plasmid harbouring the virulence gene cluster (vgc) of L. monocytogenes (Lm) into the non-pathogenic L. innocua (L.inn) strain and examined for its ability to induce protective cellular immunity. The resulting strain (L.inn::vgc) was attenuated for virulence in vivo and showed a strongly reduced host detrimental inflammatory response compared to Lm. Like Lm, L.inn::vgc induced the production of Type I Interferon's and protection was mediated by Listeria-specific CD8(+) T cells. Rational vaccine design whereby avirulent strains are equipped with the capabilities to induce protection but lack detrimental inflammatory effects offer great promise towards future studies using non-pathogenic bacteria as vectors for vaccination. Current state of the art vaccine technology focuses on three distinct strategies 1) the creation of live attenuated pathogens based on the deletion of virulence factors [1] 2) the use of subunit vaccines [2] which contain one or more semi-pure antigens that are critical in inducing an immune response and 3) the use of metabolically active but non-viable bacteria [3] .
For the first strategy, it must be considered that in today's medicine vaccines will often be administered to immunocompromised individuals and that the use of live vaccines in such subpopulations poses serious difficulties [4, 5] . The greatest disadvantage of subunit vaccines is their general requirement for strong adjuvants, as these adjuvants often induce detrimental tissue reactions. Lastly, the concept of so-called killed but metabolically active (KBMA) bacteria involves bacteria which are unable to form colonies on growth media but still have an intact protein synthesis and secretion machinery. Such mutants are reportedly capable of inducing CD4 + and CD8 + T cell responses and protection [3] . However this requires multiple injections.
Lm is a facultative intracellular microorganism and many of the bacterial determinants necessary for pathogenesis, including intracellular growth and spread of Lm, have been identified and are clustered on a 10-kb region of the chromosome termed the virulence gene cluster (vgc) which encodes the prfA, plcA, hly, mpl, actA and plcB genes organized in three transcriptional units [6] .
Being a facultative intracellular bacterium makes Lm particularly attractive as a potential live vaccine vector for the induction of cell-mediated immunity to foreign antigens [7, 8] . However, despite its capability to induce effective CD8 + T-cell responses the safety of recombinant Lm remains an important issue, as infections with Lm can cause severe and life-threatening infections [9] . Moreover, infection with Lm is mainly accompanied by undesired CD4 + T-cell mediated delayed type hypersensitivity (DTH) responses and granulomatous inflammation [10, 11] . Therefore the use of Lm in a clinical setting is associated with major risks limiting its potential as an effective vaccine vector.
An alternative strategy would entail the transfer of a core set of virulence genes from pathogenic Lm to create a strain that is attenuated for virulence but is capable of inducing an effective immune response. To explore this approach we have transferred the vgc locus of Lm into a non-pathogenic species of Listeria such as L. innocua (L.inn) as a carrier strain. Here we show that a single immunization with this recombinant strain (L.inn::vgc) fulfills the desired requirements for a successful bacterial vaccine vector. These include low virulence in association with induction of protective antigen-specific CD8 + T-cell responses and reduction of CD4 + T cell-mediated inflammation.
In vivo survival of the recombinant L.inn::vgc strain
The ability of Listeria to survive in vivo at the early stage of infection is crucial for the induction of cell-mediated immunity [12, 13] . We examined the ability of L.inn::vgc to survive in the spleen and liver in infected mice and compared it to that of the wild type Lm. BALB/c mice were infected intravenously (i.v.) with sub-lethal doses of wild type Lm EGD-e (10 3 ), L.inn::vgc (10 7 ), or the wild type L.inn strain (10 7 ). Time points correlating with the critical phases of host immune response to listerial infection were selected and used to compare bacterial growth and induction of immune effectors in wild-type Lm, L.inn and L.inn::vgc strains. Day 3 of a Listeria infection refers to the end of the pre-immune phase before the expansion of specific T cells in the mouse model of listeriosis [14] . The presence of viable bacteria on this day has been shown to be critical for the successful induction of T cell-mediated immunity [15] . Therefore on day 3, bacterial load as well as spleen morphology was analyzed. Day 9 corresponds to the primary immune effector phase. At this time point, DTH to soluble antigen was measured in vivo as an indicator of DTH reaction and CD4+ T cell activity. Moreover, the numbers of antigen specific IFN-c producing CD8+ cytotoxic T cells were analyzed. Day 60 postinfection as well as day 5 post-challenge were chosen to analyze the memory immune effector phase [16] . To this end the number of memory effector T-cells was determined quantitatively.
In vivo survival and growth kinetics of bacteria were followed by determining the number of bacteria in spleens and livers of infected mice. As expected, regardless of the dose of infection, the wild type L.inn strain was progressively cleared from both organs ( Fig. 1A) whereas the L.inn::vgc strain successfully survived in both spleen and liver during the first two days after infection as indicated by the bacterial numbers that increased in both spleen and liver till day 2 and gradually decreased over days 3 and 4 postinfection. On the other hand, the bacterial numbers of the wild type Lm, increased from day 1 till day 4 post-infection in both spleen and liver.
Stimulation of Type I interferon's by the L.inn::vgc strain A striking phenomenon for cytosolic resident microbes is the ability to induce expression of Type I interferons. In contrast to the wild type Lm, its isogenic mutant lacking listeriolysin remains trapped in vacuoles and does not induce Type I interferon's [17] . We have recently documented that the L.inn::vgc can successfully survive inside phagocytic cells, thereby egressing from the phagolysosome [18] . In order to confirm if cytosolic persistence of the recombinant L.inn::vgc strain is efficient enough to stimulate production of such cytokines, we examined the transcriptional responses of IFN-a2 and IFN-b1 in bone marrow-derived macrophages following infection with Lm, L.inn as well as the recombinant L.inn::vgc strain. L.inn::vgc and the wild type Lm showed significantly higher transcriptional induction of both IFN-a2 and IFN-b1 than wild type L.inn at 2 hours post-infection (Fig. 1B) . This effect was more pronounced at a later time point (8 hours) post-infection reflecting the efficient intracellular survival pattern of the L.inn::vgc strain.
The recombinant L.inn::vgc strain exhibits a lowered inflammatory response At the early stages of infection, wild type Lm is engulfed by professional phagocytes like macrophages, dendritic cells, or neutrophils. These cells produce a variety of proinflammatory cytokines which recruite or activate other inflammatory immune cells. The levels of IL-1ß, IL-6, IL-12, and TNF-alpha in mice sera were measured over the first 4 days after infection with Lm (10 3 ), L.inn (10 7 ), and the L.inn::vgc strain (10 7 ). Like L. inn, , L.inn::vgc was not able to produce significant amounts of these cytokines over the first 4 days post-infection in spite of high infection doses (10 7 ) while primary infection with Lm led to high proinflammatory cytokine production (Fig. 2) .
Both granuloma formation and delayed-type-hypersensitivity footpad responses have previously been shown to be CD4 + T cell dependent inflammatory responses following infection with Lm. Morphological changes were examined in the spleens on day 3 after i.v. infection. Although the numbers of bacteria in spleens at day 3 post-infection for both Lm and L.inn::vgc were approximately the same (Fig. 1A) , distinct differences in the morphological appearance between spleens isolated from mice infected with Lm and those isolated from mice infected with L.inn::vgc were observed (Fig. 3A ). Splenomegaly associated with extensive granuloma formation was observed in spleens of Lm infected mice, as a result of intensive leukocyte infiltration which was visualized in stained spleen sections (Fig. 3B) , whereas splenomegaly in the absence of granuloma formation was observed in spleens of L.inn::vgc infected mice. Infection with the wild type L.inn did not result in any morphological changes in spleens.
These observations were confirmed by antigen-elicited skin responses showing corresponding results (Fig. 3C ). Mice were injected into the left hind footpads with 50 ml of somatic soluble Lm EGD-e antigen (60 ng/ml) at day 9 post-infection. Twentyfour hours later, thickness of the left and right footpads of individual mice were measured. Footpads of mice pre-immunized with L.inn::vgc showed reduced thickness than those of mice preimmunized with the wild type Lm. The wild type L.inn strain did not induce a DTH response in the footpads of these mice. Moreover, antigen-induced CD4+ T cell-derived IFN-gamma production of spleen cells was measured as an indication for a proinflammatory T cell response. Spleen cells were isolated at day 9 post-infection and stimulated in vitro with the released soluble antigen of L. monocytogenes EGD-e (100 ng). Spleen cells from mice immunized with L.inn::vgc produced significantly lower levels of IFN-gamma when compared to spleen cells from mice immunized with wild type Lm. The wild type L.inn strain failed to prime T cells for the production of IFN-gamma (Fig. S1 , supplementary information).
Induction of T cell-mediated immunity by the recombinant L.inn::vgc strain A number of cell types are involved in host defense against Listeria. Antigen-specific T lymphocytes mediate recovery from primary listerial infections and protective immunity to subsequent infections [13, 19] . Both CD4 + (helper, MHC class II restricted) and CD8 + (cytotoxic, MHC class I restricted) T cell subpopulations have been implicated [20] . Experimental evidence indicates, however, that CD8 + T cells play the predominant role in mediating protective immunity [21] [22] [23] [24] . The ability of the recombinant L.inn::vgc to induce T-cell mediated immunity as a prerequisite for protective immunity was analyzed. Groups of BALB/c mice were infected with Lm (10 3 ), L.inn (10 7 ), or L.inn::vgc (10 7 ). Two months later, all mice were challenged with a lethal i.v. dose (10 5 ), corresponding to 206LD50, of the wild type Lm, and survival was monitored. As controls, a group of untreated BALB/c mice that received a similar lethal dose of the wild type Lm were included. A single pre-immunization with the L.inn::vgc strain led to a significant protection against subsequent lethal infection with Lm. As expected, all mice that were pre-immunized with sub-lethal doses of Lm were also protected against a lethal listerial infection and survived whereas all non-immunized mice as well as those preimmunized with L.inn died within 4 days after challenge (Fig. 4A) .
Entry of Listeria into the cytosol is a critical event for CD8 + T cell recognition and induction of immunity [22] . In order to establish the correlation between the protection of mice preinfected with Lm or the L.inn::vgc strain upon lethal challenge and the induction of CD8 + T cells in response to infection, the generation of antigen-specific MHC class I restricted CD8 + T cells were quantitatively examined. The numbers of antigen-specific MHC class I restricted effector CD8 + T cells induced in mice spleens 9 days after primary infection and 5 days after challenge with the wild type Lm (2610 3 ) was determined through evaluation of the number of IFN-c producing CD8+ T cells induced showing reactivity against the dominant H-2K d restricted LLO 91-99 epitope [25] in an in vitro ELISPOT assay. As shown in Fig. 4B , infection with wild type Lm as well as the L.inn::vgc strain induced significant numbers of LLO 91-99 specific CD8 + T-cells. After recall infection the numbers of LLO 91-99 specific CD8 + T-cells showed a significant increase. On the other hand, infection with L.inn failed to induce a significant number of CD8 + T-cells either after primary infection or after challenge.
To address the contribution of effector memory CD8+ T cells in mediating long-lasting immunity after re-infection with the wild type Lm, the expression level of the cell surface adhesion molecule CD62L was quantified. Expression of CD62L is down regulated on the surface of cytotoxic CD8 + T-cells when developed to protective memory T cells [21] . Two months after the primary infection, the number of CD8 + CD62L lo lymphocytes was approximately identical in all groups of primarily infected mice. This number increased dramatically upon re-infection with the wild type Lm (2610 3 ) in mice pre-immunized with Lm as well as with L.inn::vgc while pre-immunization with L.inn was not able to induce CD62L down-regulation seen in the other groups (Fig. 5 ). In addition we have monitored the expression of CD44 on CD8 + T-cells. CD44 is expressed at high levels on memory but not in naïve T-cells [26] . In mice that were primarily infected with Lm and L.inn::vgc, the expression of CD44 was upregulated on CD8 + T-cells 5 days post-challenge infection with the wild type Lm (2610 3 ) while primary infection with L.inn did not lead to a significant change in CD44 expression pattern (Fig. S3 ).
In this study, we define a unique vaccine strategy which is based on a rationally designed pathogen by complementation of a non- pathogenic strain with selected genes necessary to induce a vigorous immune response. To illustrate the proof of principle of this strategy we used the Listeria model by taking a non-pathogenic L.inn strain and complementing it with genes from pathogenic Lm which were previously shown to be a sine qua non requirement for intracellular growth and survival [18] . This novel vaccine strategy resulted in generation of a recombinant strain (L.inn::vgc) that possesses properties needed to induce the marked protective immunogenic properties of the wild type Lm but is attenuated in virulence as well as in its capacity to induce host detrimental cellmediated inflammation. The recombinant L.inn::vgc strain showed a significant in vivo survival rate in the first 3 days post-infection (Fig. 1A) . This observation is in accordance with our recent finding that the L.inn::vgc strain is able to survive in phagocytic host cells [18] . Moreover, it has the capability to induce identical Type I interferon at levels similar to wild type Lm. As previously shown by McAffrey et al., 2003, induction of type 1 IFN is a surrogate marker indicating access of Listeria into the cytosol of antigen presenting cells [17] . We have shown that the L.inn::vgc strain could use the complemented virulence factors to escape into the cytosol and subsequently be presented to CD8 T lymphocytes. In this context Zwaferink et al. 2008 have shown a role for IFNb in macrophage cell death. Treatment of macrophages with this cytokine could enhance host-cell membrane permeabilization by listeriolysin consequently leading to cell apoptosis [27] .
Especially encouraging was the observation that, although L.inn::vgc strain was injected at a high dose of 10 7 cfu, mice could still efficiently control the infection and showed very low blood levels of pro-inflammatory cytokines thus reducing the detrimental inflammatory responses caused by the wild type Lm. Morphological and histological analysis of spleen after infection with Lm have shown the induction of splenomegaly and granuloma as a result of monocytic infiltrations of the white pulp which were most pronounced on day 3 post-infection while infection with L.inn::vgc only resulted in a splenomegaly without any significant morphological changes detectable (Fig. 3A,3B) . The intensity of the morphological and histological alterations in spleens paralleled the level of Listeria-induced DTH responses, as the in vivo induction of DTH after L.inn::vgc infection was also significantly lower than DTH induction following Lm infection (Fig. 3 and Fig. S1 ). We therefore show that the recombinant L.inn::vgc strain shows a significantly reduced proinflammatory and CD4 + mediated inflammatory response compared to Lm, thereby addressing one major concern regarding the use of live Lm as a vaccine.
However the crucial question remained as to whether the L.inn::vgc strain elicits significant adaptive immune responses resembling those of the wild-type strain. Since Lm is located in both phagosomes and cytosol of professional antigen-presenting cells during infection, epitopes derived from Listeria proteins are presented by the MHC pathway thereby priming both effector CD4 + and CD8 + T cells [28, 29] resulting in full elimination of Listeria from the host. Previous experimental studies [30, 31] have revealed that persistence and number of viable microorganisms are important parameters for establishing efficient T cell-mediated immunity. Moreover, it has been shown that the presence of live bacteria in mice organs over the first 48 hours after immunization is critical for the induction of effector CD8 + T cell mechanisms [32] . Indeed we were able to show that all animals immunized with Lm or L.inn::vgc were protected against 206LD 50 of virulent Listeria (Fig. 4A) . Although the L.inn::vgc strain elicits lowered CD4 + mediated inflammatory responses as compared to infection with Lm, it is capable of mounting a successful anti-listerial protective response, indicating that the observed in vivo survival pattern of the L.inn::vgc strain was sufficient to induce protection.
The entry of effector T cells into a memory stage, however, is accompanied by the ability to rapidly expand their population during recall responses and to down regulate expression of cell surface markers such as CD62L and CCR7 [33] . It was previously reported that primary infection with the wild type Lm induces down regulation of CD62L on the surface of effector CD8 + T cells which reaches its lowest levels at day 8 post-infection [34] . However, over the following weeks, expression of CD62L is up regulated. During recall infection, CD62L is then rapidly down regulated on the surface of memory CD8 + T cells [32, 35] . In order to correlate protection against challenge with Listeria with antigen specific CD8 + T cells, we examined the induction of LLO 91-99 specific CD8 + T cells in response to primary infection with the different Listeria strains. Infection with L.inn::vgc induced a significant population of cytotoxic CD8 + T lymphocytes (Fig. 4B) which, upon challenge with the wild type Lm, showed a CD62L expression pattern similar to that presented in mice primarily infected with the wild type Lm (Fig. 5) . The identity of the memory T-cells induced in response to L.inn::vgc infection was confirmed by testing the CD44 expression on the CD8 + T-cells following recall infection with Lm where high expression of CD44 was observed on CD8 + T-cells derived from mice primarily infected with the recombinant L.inn::vgc strain but not with the wild type L.inn (Fig.  S3) . The inability of the Lm strain lacking listeriolysin O (LLO) [36] as well as the L.inn strain expressing only LLO [37] to induce a protective T cell response reflects the requirement of the entire virulence gene cluster in conferring a long lasting immunity. We therefore show that a non-pathogenic L.inn strain complemented with the entire vgc is capable of inducing a vigorous anti-listerial response.
Even though we have demonstrated a vigorous immune response following i.v. infection the immune response to Listeria can vary considerably depending on the route of administration. Using the intraperitoneal route of infection, we obtained a similar result i.e. protection following pre-infection with the Lm and L. inn::vgc strains but not with mice pre-immunized with the L. inn strain (Fig. S2) . Thus despite a different route of infection Lm::vgc is able to induce protection in-vivo. The mouse is not a suitable and reproducible model for evaluating oral immunization protocols because of the specificity of the listerial InlA molecule [38] . Therefore experiments examining mucosal immunity will have to be carried out in the guinea pig model of listerial infection.
Recently, highly attenuated mutants of Lm have been developed as candidates for vaccine vectors [3, 39] , however, a single immunization with these strains was not sufficient for the induction of protective cellular immunity.
Here a transcomplemented strain of a non-pathogenic L. inn strain expressing genes of the vgc cluster provides robust protection with a single dose of 10 7 cfu bacteria without causing any signs of overt illness. The LD50 of the wild type Lm is around 5000 cfu. As shown in figure 1A , the L.inn::vgc strain does not grow in-vivo beyond day 3 post-infection and is subsequently eliminated. These properties, imparting protective responses and rapid elimination from the host are considered to be desirable properties for successful vaccine vectors. Our results, namely, the in vivo survival pattern, the induction of interferon's and antigen specific CD8 + T cells, the lack of overt detrimental inflammatory reactions and most importantly the induction of protection against challenge with Listeria, allow the conclusion that the L.inn::vgc strain is potentially capable of inducing protection and that further development of this strain as a suitable live bacterial vaccine vector in clinical settings are warranted.
Mice experiments were done according to the requirements of Justus-Liebig University Giessen Animal Ethics Committees with ethics approval number: 63/2007. Animals were sacrificed using CO2 asphyxiation and the appropriate organs aseptically harvested.
Six to eight week-old female BALB/c mice, purchased from Harlan Winkelmann (Borchen, Germany), were kept at our breeding facilities in specific-pathogen-free conditions and used in all experiments.
Bacterial strains used in this study are wild type Listeria monocytogenes EGDe serotype 1/2a (Lm) [40] , wild type L. innocua strain (serotype 6a NCTC 11288) [41] transformed with either the recently characterized gram+ve/gram-ve shuttle pUvBBAC+vgc1 vector and referred to as (L.inn::vgc strain) or the pUvBBAC vector without the inserted vgc and referred to as L.inn [18] Bacteria were grown in brain-heart infusion (BHI) (Difco, Augsburg; Germany) broth in presence or absence of 5 mg/ml erythromycin. For each experiment, erythromycin was used as a selective antibiotic for growth of L.inn::vgc and the wild type L.inn harbouring the pUvBBAC vector. Wild type L. monocytogenes was grown in absence of erythromycin. In all experiments, fresh cultures of bacteria, prepared from an overnight culture, were used. Briefly, bacteria were grown in Brain Heart Infusion (BHI) at 37uC, harvested in the exponential growth phase and washed twice with PBS. The pellet was resuspended in PBS and the bacterial concentration was calibrated by optical absorption. Further dilutions were prepared in PBS to obtain required numbers of bacteria for infection.
The protocols for animal handling were previously approved by our institutional Animal Ethics Committee (protocol number 63/ 2007). Bone marrow-derived macrophages were isolated from 4 to 6 week old C57Bl/6 female mice and grown and differentiated for 7 days in L929 conditioned medium to an approximate concentration of 2,5610 5 cells/well in 6-well plates. On the day of infection the medium was exchanged against MDEM medium with 1% FCS and the cells were infected with 5610 6 cfu per well with the wild type Lm and L.inn strains as well as the recombinant L.inn::vgc strain for 2 h and 8 h. The cells were lysed and their total RNA was isolated.
For every bacterial strain and negative control the cells of at least two wells of a six well tissue culture plaque were lysed and total RNA was isolated. Prior to lysis culture medium was aspirated and cells were lysed using RLT lysis buffer (Qiagen, Germany). Total RNA was isolated using the RNeasy Mini Kit and the RNase free DNase I set (Qiagen) following the manufacturers protocol. The RNA was recovered in RNase free water, heat denatured for 10 min. at 65uC; quantified with the NanoDropH ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, USA) and a quality profile with the Agilent 2100 bioanalyzer (Agilent Technologies, Germany) was made.
First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the Quanti-Tect SYBR Green PCR Kit (Qiagen). Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler. The relative expression of the targets IFNa2 (Interferon alpha 2) and IFNb1 (Interferon beta) were normalized to that of two reference genes: SDHA (Succinate dehydrogenase alpha subunit) and PPIA (peptidylprolyl isomerase A). Finally a mean of the fold change of the target versus each of the reference genes was taken as the final value.
Somatic soluble antigen was prepared by culturing Lm in tryptic soy broth for 18 h, washing it in PBS, and subsequently subjecting it to ultrasonication.1 g (wet weight) of bacterial cells were suspended in 10 ml of PBS and sonicated five times for 1 min (87.5%, output, degree 7 on a sonifier model S-125; Branson Sonic Power, USA) on ice. The sonicated suspension was centrifuged at 39 000 U for 50 min, and the supernatant was filter sterilized (pore size,0.45 mm) and stored at 220uC at a dilution of 1:100 in PBS [42] .
Primary in vivo infection with Lm (10 3 ), the wild type L.inn (10 7 ), or the L.inn::vgc (10 7 ) strain was performed by an intravenous injection of viable bacteria in a volume of 0.2 ml PBS. Bacterial growth in spleens and livers was determined by plating 10-fold serial dilutions of the organ homogenates on BHI agar plates. The detection limit of this procedure was 10 2 colony forming units (CFU) per organ. Colonies were counted after 24 h of incubation at 37uC.
Cytokine production was assayed from the collected sera of infected mice using a multiplex cytokine assay kit and Luminex technology (Bio-Rad). Balb/C mice were infected with Lm (10 3 ), the wild type L.inn (10 7 ), or the L.inn::vgc (10 7 ) strain. Sera were aseptically isolated on days 1, 2, 3, and 4 post-infection. Four cytokines were tested: TNFa, IL-1b, IL-6, and IL-12(p70) and cytokine levels were presented as absolute concentrations in pg/ ml.
Spleens were aseptically isolated from mice previously infected with the different Listeria strains as mentioned above and examined for morphological alterations. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and 5 mm sections were stained with hematoxylin and eosin (HE). Pathoplogical foci in spleen sections were then microscopically examined (Keyence).
Spleens were aseptically removed from mice at day 9 postinfection with the wild type Lm, the wild type L.inn, or the L.inn::vgc strain. Spleen cells were isolated and antigen (LLO 91-99 ) specific IFN-c producing CD8+T cells were determined in the spleens of mice after i.v. infection with the same bacterial strains mentioned above by using an ELISPOT system as previously described [23, 35] .
Quantification of IFN-gamma in cell culture supernatants IFN-gamma was measured in the supernatants of splenocytes by using a mouse IFN-gamma ELISA kit, BD optEIA TM (BD Biosciences Pharmigen) according to the manufacturer instructions. The assay was performed in duplicates, and data represent means 6 standard error.
For flow cytometry analysis, approximately 1610 6 splenocytes, isolated from infected mice (Lm, L.inn, and L.inn::vgc strains) were stained with FITC labelled anti-CD8 and biotinylated anti-CD62L or anti-CD44 (pharMingen, Becton Dickinson). PEconjugated streptavidin was used to detect the binding of anti-CD62L or anti-CD44 on the cell surface. Flow cytometry was performed using a FACS Calibur flow cytometer and further analyzed with CELL Quest software (Becton Dickinson, CA).
All mice, pre-immunized with wild type Lm, the wild type L.inn strain and the L.inn::vgc strain were challenged 2 months later with a 206LD 50 (10 5 ) lethal dose of wild type Lm. A group of non preimmunized Balb/c mice were included as controls. Survival of mice was monitored for several days and expressed as percentage of animals surviving challenge with Lm.
Data are representative of at least three independent experiments. Significance of the represented data was calculated using ANOVA (analysis of variance). Data are expressed as mean 6 standard errors (S.E.). Figure S1 Listeria-induced IFN-gamma production by spleen cells 9 days after infection (i.v.). Mice were infected with 10 3 CFU of Lm, 10 7 CFU wild type L.inn, or with 10 7 CFU of L.inn::vgc strain. On day 9 after infection, mice were killed and spleens removed. Single cell suspensions were stimulated in vitro with secreted soluble Listeria antigen to produce IFN-gamma. After 48 hours, culture supernatants were tested for presence of IFNgamma by ELISA. *P,0.05 (EGD-e vs L.inn::vgc). (TIF) Figure S2 Intraperitoneal infection with the L.inn::vgc strain induces protective immunity. Mice were infected intraperitoneally with Lm, L.inn and the L.inn::vgc strain as described in figure 4A . After 2 months all mice were challenged i.v. with a lethal dose (206LD 50 ) of the wild type Lm. As a control, a group of uninfected normal mice was included. Survival was monitored up to 8 days after challenge. (TIF) Figure S3 Quantification of CD44 expression on CD8 + splenocytes following primary and recall infection with Lm, L.inn and the L.inn::vgc strain. Flow cytometry was performed on spleen cells, isolated from mice on day 60 after the primary infection or day 5 after the challenge. Cells were stained with FITC-labelled anti-Lyt-2 and PE-labelled anti-CD44. Numbers shown are gated CD8 + CD44 hi T cells and analyzed with CELLQuest software. (TIF) Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing BACKGROUND: The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. CONCLUSIONS: This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with important commercial value in Europe and Asia, and its culture has expanded in recent years. Nevertheless, diseases produced by a wide range of microorganisms, from viruses to metazoan parasites, can result in large economical losses. Among clam diseases, the majority of pathologies are associated with the Vibrio and Perkinsus genera [1] [2] [3] . Although molluscs lack a specific immune system, the innate response involving circulating hemocytes and a large variety of molecular effectors seems to be an efficient defense method to respond to external aggressions by detecting the molecular signatures of infection [4] [5] [6] [7] [8] ; however, not many immune pathways have been identified in these animals.
Although knowledge of bivalve immune-related genes has increased in the last few years, the available information is still scarce and fragmentary. Most of the data concern mussels and Eastern and Pacific oysters [9] [10] [11] [12] [13] [14] , and very limited information is available on the expressed immune genes of R. philippinarum. Recently, the expression of 13 immune-related genes of Ruditapes philippinarum and Ruditapes decussatus were characterized in response to a Vibrio alginolyticus challenge [15] . Also, a recent 454 pyrosequencing study was carried out by Milan et al. [16] , who sequenced two normalized cDNA libraries representing a mixture of adult tissues and larvae from R. philippinarum. Even more recently Ghiselli et al. [17] , have de novo assembled the R. philippinarum gonad transcriptome with the Illumina technology. Moreover, a few transcripts encoded by genes putatively involved in the clam immune response against Perkinsus olseni have been reported by cDNA library sequencing [18] . Currently (19/12/ 2011) , there are 5,662 ESTs belonging to R. philippinarum in the GenBank database.
The European Marine Genomics Network has increased the number of ESTs for marine mollusc species particularly for ecologically and commercially important groups that are less studied, such as mussels and clams [19] . Unfortunately, most of the available resources are not annotated or well described, limiting the identification of important genes and genetic markers for future aquaculture applications. The use of 454-pyrosequencing is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms [20] . This relatively low-cost technology facilitates the rapid production of a large volume of data, which is its main advantage over conventional sequencing methods [21] .
In the present work, we undertook an important effort to significantly increase the number of R. philippinarum ESTs in the public databases. Specially, the aim of this work was to discover new immune-related genes using pyrosequencing on the 454 GS FLX (Roche-454 Life Sciences) platform with the Titanium reagents. To achieve this goal, we sequenced the transcriptome of R. philippinarum hemocytes previously stimulated with different pathogen-associated molecular patterns (PAMPs) to obtain the greatest number of immune-related transcripts as possible. The raw data are accessible in the NCBI Short Read Archive (Accession number: SRA046855.1).
The R. philippinarum normalized cDNA library was sequenced with 454 GS FLX technology as shown in Figure 1 . Sequencing and assembly statistics are summarized in Table 1 . Briefly, a total of 975,190 raw nucleotide reads averaging 284.1 bp in length were obtained. Of these, 974,976 exceeded our minimum quality standards and were used in the MIRA assembly. A total of 842,917 quality reads were assembled into 51,265 contigs, corresponding to 29.9 megabases (Mb). The length of the contigs varied from 40 to 5565 bp, with an average length of 582.4 bp and an average coverage of 5.7 reads. Singletons were discarded, resulting in 37,093 contigs formed by at least 2 ESTs, and 26,675 of these contigs were longer than 500 bp. Clustering the contigs resulted in 1,689 clusters with more than one contig. The distribution of contig length and the number of ESTs per contig, as well as the contig distribution by cluster are all shown in Figure 2 .
Even though the knowledge of expressed genes in bivalves has increased in the last few years, it is still limited. Indeed, only 41,598 nucleotide sequences, 362,149 ESTs, 24,139 proteins and 704 genes from the class Bivalvia have been deposited in the GenBank public database (19/12/11) , and the top entries are for the Mytilus and Crassostrea genera. For Ruditapes philippinarum, these numbers are reduced to 5,662 ESTs, 612 proteins and 12 genes. This evidences the lack of information which prompted the recent efforts to increase the number of annotated sequences of bivalves in the databases. For non-model species, functional and comparative genomics is possible after obtaining good EST databases. These studies seem to be the best resource for deciphering the putative function of novel genes, which would otherwise remain ''unknown''.
NCBI Swissprot, NCBI Metazoan Refseq, the NCBI nonredundant and the UniprotKB/Trembl protein databases were chosen to annotate the contigs that were at least 100 bp long (49, 847) . The percentage of contigs annotated with a cut off evalue of 10e-3 was 44.7%. Contig sequences and annotations are included in Table S1 . Of these contigs, 3.26% matched sequences from bivalve species and the remaining matched to non-Bivalvia mollusc classes (4.13%), other animals (81.38%), plants (2.58%), fungi (1.78%), protozoa (1.50%), bacteria (4.95%), archaea (0.20%), viruses (0.21%) and undefined sequences (0.01%). As shown in Figure 3A , the species with the most sequence matches was Homo sapiens with 3,106 occurrences. The first mollusc in the top 35 list was Lymnaea stagnalis at position 11. The first bivalve, Meretrix lusoria, appeared at position 17. R. philippinarum was at position 25 with 124 occurrences. Notably, a high percentage of the sequences had homology with chordates, arthropods and gastropods ( Figure 3B and C), and only 343 contigs matched with sequences from the Veneroida order ( Figure 3D ). These values can be explained by the higher representation of those groups in the databases as compared to bivalves and the quality of the annotation in the databases, which has been reported in another bivalve transcriptomic study [22] . The data shown highlight, once again, the necessity of enriching the databases with bivalve sequences.
A detailed classification of predicted protein function is shown for the top 35 BLASTx hits ( Figure 4A ). The list is headed by actin with 903 occurrences, followed by ferritin, an angiopoietin-like protein and lysozyme. An abundance of proteins directly involved in the immune response was predicted for this 454 run; ferritin, lysozyme, C1q domain containing protein, galectin-3 and hemagglutinin/amebocyte aggregation factor precursor are immune-related proteins present on the top 35 list.
Ferritin has an important role in the immune response. It captures circulating iron to overcome an infection and also functions as a proinflammatory cytokine via the iron-independent nuclear factor kappa B (NF-kB) pathway [23] . Lysozyme is a key protein in the innate immune responses of invertebrates against Gram-negative bacterial infections and could also have antifungal properties. In addition, it provides nutrition through its digestive properties as it is a hydrolytic protein that can break the glycosidic union of the peptidoglycans of the bacteria cell wall [24] . The C1q domain containing proteins are a family of proteins that form part of the complement system. The C1q superfamily members have been found to be involved in pathogen recognition, inflammation, apoptosis, autoimmunity and cell differentiation. In fact, C1q can be produced in response to infection and it can promote cell survival through the NF-kB pathway [25] . Galectin-3 is a central regulator of acute and chronic inflammatory responses through its effects on cell activation, cell migration, and the regulation of apoptosis in immune cells [26] . The hemagglutinin/amebocyte aggregation factor is a single chain polypeptide involved in blood coagulation and adhesion processes such as self-nonself recognition, agglutination and aggregation processes. The hemagglutinin/ amebocyte aggregation factor and lectins play important roles in defense, specifically in the recognition and destruction of invading microorganisms [27] .
Other proteins that are not specifically related to the immune response but could play a role in defense mechanisms include the following: angiopoietin-like proteins, apolipoprotein D and the integral membrane protein 2B. In other animals, angiopoietin-like proteins (ANGPTL) potently regulate angiogenesis, but a subset also function in energy metabolism. Specifically, ANGPTL2, the most represented ANGPTL, promotes vascular inflammation rather than angiogenesis in skin and adipose tissues. Inflammation occurs via the a5b1 integrin/Rac1/NF-kB pathway, which is evidenced by an increase in leukocyte infiltration, blood vessel permeability and the expression of inflammatory cytokines (tumor necrosis factor-a, interleukin-6 and interleukin-1b) [28] . Apolipoprotein D (apoD) has been associated with inflammation. Pathological and stressful situations involving inflammation or growth arrest have the capacity to increase its expression. This effect seems to be triggered by LPS, interleukin-1, interleukin-6 and glucocorticoids and is likely mediated by the NF-kB pathway, as there are several conserved NF-kB binding sites in the apoD promoter (APRE-3 and AP-1 binding sites are also present). The highest affinity ligand for apoD is arachidonic acid, which apoD traps when it is released from the cellular membrane after inflammatory stimuli and, thus, prevents its subsequent conversion in pro-inflammatory eicosanoids. Within the cell, apoD could modulate signal transduction pathways and nuclear processes such as transcription activation, cell cycling and apoptosis. In summary, apoD induction is specific to ongoing cellular stress and could be part of the protective components of mild inflammation [29] [30] [31] . Finally, the short form of the integral membrane protein 2B (ITM2Bs) can induce apoptosis via a caspase-dependent mitochondrial pathway [32] .
To avoid redundancy, the longest contig of each cluster was used for Gene Ontology terms assignment. A total of 23.05% of the representative clusters matched with at least one GO term. Concerning cellular components ( Figure 4B ), the highest percentage of GO terms were in the groups of cell and cell part with 25.9% in each; organelle and organelle part represented 19.67% and 11.38%, respectively. Within the molecular function classification ( Figure 4C ), the most represented group was binding with 49.25% of the terms, which was followed by catalytic activity (29.12%) and structural molecular activity (4.60%). With regard to biological process ( Figure 4D ), cellular and metabolic processes were the highest represented groups with 16.78% and 12.43% of the terms, respectively, which was followed by biological regulation (10.18%).
Similarities between the R. philippinarum transcriptome and another four bivalve species sequences were analyzed by comparative genomics (Crassostrea gigas of the family Ostreidae, Bathymodiolus azoricus and Mytilus galloprovincialis of the family Mytilidae and Laternula elliptica of the family Laternulidae). This analysis could identify specific transcripts that are conserved in these five species. A Venn diagram was constructed using unique sequences from these databases according to the gene identifier (gi id number) of each sequence in its respective database: 207,764 from C. gigas, 76,055 from B. azoricus, 121,318 from M. galloprovincialis and 1,034,379 from L. elliptica. C. gigas was chosen because is the most represented bivalve species in the public databases. The other three species are bivalves that have been studied in transcriptomic assays. Figure 5 shows that of the total 29,679 clusters, 72% were found exclusively in the R. philippinarum group, while only 7.59% shared significant similarity with all five species. The number of coincidences among other groups was very low (4.14% to 0.31% of sequences), suggesting that 21,454 new sequences were discovered within the bivalve group. The percentage of new sequences is very high compared to previous transcriptomic studies [33] [34] , in which the fraction of new transcripts was approximately 45%. One possible explanation for this discrepancy is the low number of nucleotide and EST sequences currently available in public databases for R. philippinarum, but these transcripts could also be regions in which homology is not reached, such as 59 and 39 untranslated regions or genes with a high mutation rate. On the other hand, a comparison between our 454 results and the Milan et al. [16] transcriptome using a BLASTn approach is summarized in Table 2 Immune-related sequences R. philippinarum hemocytes were subjected to immune stimulation using several different PAMPs to enrich the EST collection with immune-related sequences. The objective was to obtain a more complete view of clam responses to pathogens. A keyword list and GO immune-related terms were used to find proteins putatively involved in the immune system. After this selection step, we found that more than 10% of the proteins predicted from the contig sequences had a possible immune function. Some sequences were found to be clustered in common, well-recognized immune pathways, such as the complement, apoptosis and toll-like receptors pathways, indicating conserved ancient mechanisms in bivalves ( Figures 6, 7, 8 ).
The complement system is composed of over 30 plasma proteins that collaborate to distinguish and eliminate pathogens. C3 is the central component in this system. In vertebrates, it is proteolytically activated by a C3 convertase through both the classic, lectininduced and alternative routes [35] . Although the complement pathway has not been extensively described in bivalves, there is evidence that supports the presence of this defense mechanism. ESTs with homology to the C1q domain have been detected in the American oyster, C. virginica [36] , the tropical clam Codakia orbicularis [37] , the Zhikong scallop Chlamys farreri [38] and the mussel M. galloprovincialis [39] [40] . More recently, a novel C1q adiponectin-like, a C3 and a factor B-like proteins have been identified in the carpet shell clam R. decussatus [41] [42] . These data support the putative presence of the complement system in bivalves.
Our pyrosequencing results, using the BLASTx similarity approach, showed that the complement pathway in R. philippinarum was almost complete as compared to the KEGG reference pathway ( Figure 6 ). Only the complement components C1r, C1s, C6, C7 and C8 were not detected.
i. Lectins. Lectins are a family of carbohydrate-recognition proteins that play crucial self-and non-self-recognition roles in innate immunity and can be found in soluble or membraneassociated forms. They may initiate effector mechanisms against pathogens, such as agglutination, immobilization and complement -mediated opsonization and lysis [43] .
Several types of lectins have been cloned or purified from the Manila clam, R. philippinarum [44] [45] [46] , and their function and expression were also studied [18, 47] . Also, a Manila clam tandemrepeat galectin, which is induced upon infection with Perkinsus olseni, has been characterized [46] .
Lectin sequences have been found in the stimulated hemocytes studied in our work: 23 of the contigs are homologous to C-type lectins (calcium-dependent carbohydrate-binding lectins that have characteristic carbohydrate-recognition domains), 115 are homologous to galectins (characterized by a conserved sequence motif in their carbohydrate recognition domain and a specific affinity for bgalactosides), 4 contigs have homology with ficolin A and B (a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains) and 34 contigs have homology with other groups of lectins such as lactose-, mannoseor sialic acid-binding lectins.
ii. b-glucan recognition proteins. b-glucan recognition proteins are involved in the recognition of invading fungal organisms. They bind specifically to b-1,3-glucan stimulating short-term immune responses. Although these receptors have been partially sequenced in several bivalves, there is only one complete description of them in the scallop Chlamys farreri [48] .
Two contigs with homology to the beta-1,3-glucan-binding protein were found in our study.
iii. Peptidoglycan recognition proteins. Peptidoglycan recognition proteins (PGRPs) specifically bind peptidoglycans, which is a major component of the bacterial cell wall. This family of proteins influences host-pathogen interactions through their pro-and anti-inflammatory properties that are independent of their hydrolytic and antibacterial activities. In bivalves, they were first identified in the scallops C. farreri and A. irradians [49, 50] and the Pacific oyster C. gigas, and from the latter four different types of PGRPs were identified [51] .
Peptidoglycan-recognition proteins and a peptidoglycan-binding domain containing protein have been found for the first time in R. philippinarum in our results and were present 4 and 1 times, respectively.
iv. Toll-like receptors. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting non-self substances and activating the immune system. The unique bivalve TLR was identified and characterized in the Zhikong Scallop, C. farreri [52] . TLR 2, 6 and 13 were present among the pyrosequencing results. TLR2 and TLR6 form a heterodimer, which senses and recognizes various components from bacteria, mycoplasma, fungi and viruses [53] . TLR13 is a novel and poorly characterized member of the Toll-like receptor family. Although the exact role of TLR13 is currently unknown, phylogenic analysis indicates that TLR13 is a member of the TLR11 subfamily [54] suggesting that it could recognize urinary pathogenic E. coli [55] . It has been demonstrated that TLR13 colocalizes and interacts with UNC93B1, a molecule located in the endoplasmic reticulum, which strongly suggests that TLR13 might be found inside cells and might play a role in recognizing viral infections [56] . Figure 7 summarizes the TLR signaling pathway with the corresponding molecules found in the R. philippinarum transcriptome. 
Pathogen proteases are important virulence factors that facilitate infection, diminish the activity of lysozymes and quench the agglutination capacity of hemocytes. Because protease inhibitors play important roles in invertebrate immunity by protecting hosts through the direct inactivation of pathogen proteases, many bivalves have developed protease inhibitors to regulate the activities of pathogen proteases [1] . Some genes encoding protease inhibitors were identified in C. gigas [57] , A. irradians [58] , C. farreri [59] and C. virginica; in the latter a novel family of serine protease inhibitors was also characterized [60] [61] [62] .
A total of 23 contigs with homology to Serine, Cystein, Kunitzand Kazal-type protease inhibitors and metalloprotease inhibitors were found among our results.
Lysozyme was one of the most represented groups of immune genes in this transcriptome study with 208 contigs present. It is an antibacterial molecule present in numerous animals including bivalves. Although lysozyme activity was first reported in molluscs over 30 years ago, complete sequences were published only recently including those of R. philippinarum [24] .
Antimicrobial peptides (AMPs) are small, gene-encoded, cationic peptides that constitute important innate immune effectors from organisms spanning most of the phylogenetic spectrum. AMPs alter the permeability of the pathogen membrane and cause cellular lysis [63] . In bivalves, they were first purified from mussel hemocyte granules [64, 65] . In mussels, the AMP myticin C was found to have a high polymorphic variability as well as chemotactic and immunoregulatory roles [66, 67] . In clams, two AMPs with similarity to mussel myticin and mytilin [68] and a big defensin [69] are known.
We were able to detect 36 contigs with homology to different defensins: defensin-1 (American oyster defensin), defensin MGD-1 (Mediterranean mussel defensin) and the big defensin previously mentioned. Four contigs were similar to an unpublished defensin sequence from Venerupis ( = Ruditapes) philippinarum.
The primary role of heat shock proteins (HSPs) is to function as molecular chaperones. Their up-regulation also represents an important mechanism in the stress response [70] , and their activity is closely linked to the innate immune system. HSPs mediate the mitochondrial apoptosis pathway and affect the regulation of NF-kB [71] . HSPs are well studied in bivalves. For R. philippinarum, several assays have been developed to better understand the HSPs profile in response to heavy metals and pathogen stresses [72] [73] [74] . The most important and well-studied groups of HSPs were present in our R. philippinarum transcriptome (HSP27, HSP40/ DnaJ, HSP70 and HSP90), but other, less common HSPs were also represented (HSP10, HSP22, HSP83 and some members from the HSP90 family).
Recently, several genes related to the inflammatory response against LPS stimulation have been detected in bivalves. Such is the case of the LPS-induced TNF-a factor (LITAF), which is a novel transcription factor that critically regulates the expression of TNFa and various inflammatory cytokines in response to LPS stimulation. It has been described in three bivalve species: Pinctada fucata [75] , C. gigas [76] and C. farreri [77] .
Other TNF-related genes have been identified in the Zhikong scallop, such as a TNFR homologue [78] and a tumor necrosis factor receptor-associated factor 6 (TRAF6), which is a key signaling adaptor molecule common to the TNFR superfamily and to the IL-1R/TLR family [79] . Figure 7 shows that several components of the TLR signaling pathway that are present in our transcriptomic sequences (MyD88, IRAK4, TRAF-3 and -6, TRAM, BTK, RAC-1, PI3K, AKT, BTK and TANK).
A total of 1,918 contigs, 8.43% of those annotated, had homology with the main groups of putatively pathogenic organisms such as viruses (47 hits), bacteria (1,126 hits), protozoa (341 hits) and fungi (404 hits). Figure 9 displays the taxonomic classification of these sequences and Table 3 summarizes a list of the known bivalve pathogens found in our results.
Bacteria constitute the main group found among the sequences not belonging to the clam. As filter-feeding animals, bivalves can concentrate a large amount of bacteria and it could be one of their sources of food [24] . Because Vibrio spp. are ubiquitous in aquatic ecosystems, it was expected that the Vibrionales order, with 141 hits, would be the most predominant. Several species of the Vibrio genus are among the main causes of disease in bivalves specifically causing bacillary necrosis in larval stages [80] . Is noticeable that sequences belonging to the causative agent of Brown Ring Disease in adults of Manila Clam, Vibrio tapetis, have not been found.
Perkinsus marinus, with 2 matches, is the only bivalve pathogen found within the protozoa (Alveolata) group. Perkinsosis is produced by species from the genus Perkinsus. Both P. marinus and P. olseni have been associated with mortalities in populations of various groups of molluscs around the world and are catalogued as notifiable pathogens by the OIE.
Viruses were the least represented among pathogens. The Baculoviridae family was the most predominant with 21 matches, but the corresponding sequences were inhibitors of apoptosis (IAPs) [81] that could also be part of the clam's transcriptome. Five viral families were found in our transcriptome study: Iridoviridae, Herpesviridae, Malacoherpesviridae, Picornaviridae and Retroviridae. A well-known bivalve pathogen was also identified, the ostreid herpesvirus 1, which has been previously been found to infect clams [82] .
Fungi had 404 matches in our results. It is known that bivalves are sensitive to fungal diseases, which can degrade the shell or affect the larval bivalve stages [83, 84] .
This study represents the first R. philippinarum transcriptome analysis focused on its immune system using a 454-pyrosequencing approach and complements the recent pyrosequencing assay carried out by Milan et al. [16] . The discovery of new immune sequences was effective, resulting in an enormous variety of contigs corresponding to molecules that could play a role in the defense mechanisms. More than 10% of our results had relationship with immunity. This new resource is now gathered in the NCBI Short Read Archive with the accession number: SRA046855.1.
Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and gene expression studies as well as for the identification of genetic markers for various applications including the selection of families in the aquaculture sector. We have found sequences from molecules never described in bivalves before like C2, C4, C5, C9, AIF, Bax, AKT, TLR6 and TLR13, among others. As a part of this work, three immune pathways in R.
philippinarum have been characterized, the apoptosis, the toll like signaling pathway and the complement cascade, which could help us to better understand the resistance mechanisms of this economically important aquaculture clam species.
Animal sampling and in vitro stimulation of hemocytes R. philippinarum clams were obtained from a commercial shellfish farm (Vigo, Galicia, Spain). Clams were maintained in open circuit filtered sea water tanks at 15uC with aeration and were fed A total of 100 clams were notched in the shell in the area adjacent to the anterior adductor muscle. A sample of 500 ul of hemolymph was withdrawn from the adductor muscle of each clam with an insulin syringe, pooled and then distributed in 6-well plates, 7 ml per well, in a total of 7 wells, one for each treatment. Hemocytes were allowed to settle to the base of the wells for 30 min at 15uC in the darkness. Then, the hemocytes were stimulated with 50 mg/ml of Polyinosinic:polycytidylic acid (Poly I:C), Peptidoglycans, ß-Glucan, Vibrio anguillarum DNA (CpG), Lipopolysaccharide (LPS), Lipoteichoic acid (LTA) or 1610 6 UFC/ml of heat-inactivated Vibrio anguillarum (one stimulus per well) for 3 h at 15uC. All stimuli were purchased from SIGMA.
Pyrosequencing. After stimulation, hemolymph was centrifuged at 1700 g at 4uC for 5 minutes, the pellet was resuspended in 1 ml of Trizol (Invitrogen) and RNA was extracted following the manufacturer's protocol. After RNA extraction, samples were treated with Turbo DNase free (Ambion) to eliminate DNA. Next, the concentration and purity of the RNA samples were measured using a NanoDrop ND1000 spectrophotometer. The RNA quality was assessed in a Bioanalyzer 2010 (Agilent Technologies). From each sample, 1 mg of RNA was pooled and used for the production of normalized cDNA for 454 sequencing in the Unitat de Genòmica (SCT-UB, Barcelona, Spain).
Full-length-enriched double stranded cDNA was synthesized from 1,5 mg of pooled total RNA using MINT cDNA synthesis kit (Evrogen, Moscow, Russia) according to manufacturer's protocol, and was subsequently purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA). The amplified cDNA was normalized using Trimmer kit (Evrogen, Moscow, Russia) to minimize differences in representation of transcripts. The method involves denaturation-reassociation of cDNA, followed by a digestion with a Duplex-Specific Nuclease (DSN) enzyme [85, 86] . The enzymatic degradation occurs primarily on the highly abundant cDNA fraction. The single-stranded cDNA fraction was then amplified twice by sequential PCR reactions according to the manufacturer's protocol. Normalized cDNA was purified using the QIAquick PCR Purification Kit (Qiagen USA, Valencia, CA).
To generate the 454 library, 500 ng of normalized cDNA were used. cDNA was fractionated into small, 300-to 800-basepair fragments and the specific A and B adaptors were ligated to both the 39 and 59 ends of the fragments. The A and B adaptors were domain; PKC: Protein kinase C; PTEN: Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN; RAIDD: Caspase and RIP adapter with death domain; TNF R1: Tumor necrosis factor receptor 1; TNF-a: Tumor necrosis factor alpha; TRADD: TNF receptor type 1-associated DEATH domain protein; TRAF2: TNF receptor-associated factor 2; TRAIL: TNF-related apoptosis-inducing ligand; TRAIL decoy: Decoy TRAIL receptor without death domain; TRAIL-R: TRAIL receptor. doi:10.1371/journal.pone.0035009.g008 used for purification, amplification, and sequencing steps. One sequencing run was performed on the GS-FLX using Titanium chemistry. 454 Sequencing is based on sequencing-by-synthesis, addition of one nucleotide, or more, complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera within the instrument. The signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow. All reagents and protocols used were from Roche 454 Life Sciences, USA.
Pyrosequencing raw data, comprised of 975,190 reads, were processed with the Roche quality control pipeline using the default settings. Seqclean (http://compbio.dfci.harvard.edu/tgi/software/) software was used to screen for and remove normalization adaptor sequences, homopolymers and reads shorter than 40 bp prior to assembly. A total of 974,973 quality reads were subjected to MIRA, version 3.2.0 [87] , to assemble the transcriptome. By default, MIRA takes into account only contigs with at least 2 reads. The other reads go into debris, which might include singletons, repeats, low complexity sequences and sequences shorter than 40 bp. NCBI Blastclust was used to group similar contigs into clusters (groups of transcripts from the same gene). Two sequences were grouped if at least 60% of the positions had at least 95% identity. The 51,265 contigs were grouped into a total of 29,679 clusters.
An iterative blast workflow was used to annotate the R. philippinarum contigs with at least 100 bp (49,847 contigs out of 51,265). Then, BLASTx [88] with a cut off value of 10e-3, was used to compare the R. philippinarum contigs with the NCBI Swissprot, the NCBI Metazoan Refseq, the NCBI nr and the UniprotKB/Trembl protein databases.
After annotation, Blast2GO software [89] was used to assign Gene Ontology terms [90] to the largest contig of a representative cluster (minimum of 100 bp). This strategy was used to avoid redundant results. Default values in Blast2GO were used to perform the analysis and ontology level 2 was selected to construct the level pie charts.
To make a comparison between R. philippinarum and other bivalve species, the nucleotide sequences and ESTs from C. gigas, M. galloprovincialis, L. elliptica and B. azoricus were obtained from GenBank and from dedicated databases, when available. [93] . Unique sequences from these databases (based on gi number) were used from each of the databases. These sequences were compared by BLASTn against the longest contig from each of 29,679 R. philippinarum clusters with a cut off e-value of 10e-05. Hits to R. philippinarum sequences were represented in a Venn diagram.
The comparison between our 454 results, the longest contig from each of 29,679 clusters, and the Milan et al. [16] transcriptome, contigs downloaded from RuphiBase (http:// compgen.bio.unipd.it/ruphibase/query/), was made by BLASTn with a cut off e-value of 10e-05. Another analysis was carried out to compare just the longest contig from each of 2,005 clusters identified as immune-related and the Milan et al. contigs as well. The results were summarized in a table ( Table 2 ). The percentage of coverage is the average % of query coverage by the best blast hit and the percentage of hits is the % of query with at least one hit in database, in parenthesis were added the total number of hits. Identification of immune-related genes All the contig annotations were revised based on an immunity and inflammation-related keyword list (i.e. apoptosis, bactericidal, C3, lectin, SOCS…) developed in our laboratory to select the candidate sequences putatively involved in immune response. The presence or absence of these words in the BLASTx hit descriptions was checked to identify putative immune-related contigs. The remaining non-selected contigs were revised using the GO terms at level 2, 3 and 4 assigned to each sequence after the annotation step that had a direct relationship with immunity. Selected contigs were checked again to eliminate non-immune ones and distributed into functional categories.
Immune-related genes were grouped in three reference immune pathways (complement cascade, TLR signaling pathway and apoptosis) to describe each route indicated by our pyrosequencing results.
To identify and classify the groups of organisms that had high similarity with our clam sequences, the Uniprot Taxonomy [94] was used except for the protozoa group. Because protozoa are a highly complex group, a specific taxonomy [95] was followed. Briefly, after the BLASTx annotation step all the hit descriptions included the species name (i.e. Homo sapiens) or a code (i.e. HUMAN) meaning that protein has been previously identified as belonging to that species. With such information sequences were classified in taxonomical groups and represented in pie charts.
Table S1 List of contigs (e-value,10-3) of Ruditapes philippinarum including sequence, length, description (Hit description), accession number of description (Hit ACC), e-value obtained and database used for annotation (Blast).  Distribution and Risk Factors of 2009 Pandemic Influenza A (H1N1) in Mainland China Data from all reported cases of 2009 pandemic influenza A (H1N1) were obtained from the China Information System for Disease Control and Prevention. The spatiotemporal distribution patterns of cases were characterized through spatial analysis. The impact of travel-related risk factors on invasion of the disease was analyzed using survival analysis, and climatic factors related to local transmission were identified using multilevel Poisson regression, both at the county level. The results showed that the epidemic spanned a large geographic area, with the most affected areas being in western China. Significant differences in incidence were found among age groups, with incidences peaking in school-age children. Overall, the epidemic spread from southeast to northwest. Proximity to airports and being intersected by national highways or freeways but not railways were variables associated with the presence of the disease in a county. Lower temperature and lower relative humidity were the climatic factors facilitating local transmission after correction for the effects of school summer vacation and public holidays, as well as population density and the density of medical facilities. These findings indicate that interventions focused on domestic travel, population density, and climatic factors could play a role in mitigating the public health impact of future influenza pandemics. In early April 2009, human cases of infection with 2009 pandemic influenza A (H1N1) virus were first identified in the United States and Mexico (1) . The virus then spread rapidly to other regions of the world. As of January 24, 2010, laboratory-confirmed cases of pandemic influenza (H1N1-2009) were being reported in more than 209 countries or regions worldwide, with 14,711 deaths among confirmed cases (2) . Pandemic influenza was introduced to mainland China on May 9, 2009 (3, 4) , and then spread across the whole country. By the end of 2009, more than 120,000 confirmed cases were reported to the Chinese Center for Disease Control and Prevention (CCDC), including 648 deaths (5) . Information on reported cases was released daily by the Ministry of Health of the People's Republic of China in the early stages of the pandemic and then twice weekly later on.
Analyzing the information gathered and unearthing underlying risk factors provide an opportunity to identify epidemic characteristics and transmission patterns of the pandemic in China, thereby producing useful information for prevention and control measures during future epidemics. In this study, we aimed to characterize the temporal and spatial distribution of pandemic influenza in mainland China, to understand the diffusion pattern of the disease within the country, and to identify risk factors for invasion and local transmission of this disease.
We used a database that included all cases of pandemic influenza (H1N1-2009) reported to the China Information System for Disease Control and Prevention (CISDCP) from May 9, 2009 , when the first confirmed case in China was reported, to December 31, 2009 (6) . The CISDCP covers all provincial, prefectural, and county centers for disease control and prevention, 95.3% of the provincial, prefectural, and county hospitals (9,084 in total), and 84.0% of township clinics (38,175 in total) across mainland China. After the World Health Organization issued an alert about the novel influenza virus (H1N1-2009), pandemic influenza was classified as a class B notifiable infectious disease on April 30, 2009 , by the Ministry of Health but was managed according to the criteria for class A notifiable infectious diseases. According to the Law for Prevention and Control of Infectious Diseases in China, information regarding each patient, once identified, must be reported to the CCDC within 2 hours through the Web-based CISDCP system. A suspected case was defined as a person with influenza-like symptoms who had had close contact with a confirmed case within the past 7 days, had a history of travel to affected areas within the past 7 days, or tested positive for influenza A virus, excluding other known subtypes of influenza A. A laboratory-confirmed case was defined according to World Health Organization criteria-that is, a person with influenza-like symptoms and laboratory-confirmed pandemic influenza A virus infection by one or more of the following tests: reverse-transcriptase polymerase chain reaction, real-time reverse-transcriptase polymerase chain reaction, viral culture, or a 4-fold rise in specific antibodies to pandemic influenza A virus (7) . Influenzalike symptoms were defined according to World Health Organization criteria: sudden onset of fever greater than 38°C, cough or sore throat, and absence of other diagnoses (8) . All laboratory-confirmed cases from 2009 were included in our database, including information about age, sex, occupation, residence address, work address, onset date and location, hospital admission date and address, and clinical outcome. Furthermore, census information was obtained from the National Bureau of Statistics of China (9) .
Epidemic curves were created by plotting the daily number of newly confirmed cases and the number of deaths. Mainland China is divided into 2,925 counties, which are political subdivisions of provinces, usually containing several townships. The incidences for different sex and age groups were calculated using 2009 census data. As was done previously (10, 11) , each case was linked to a digital map of China (1:100,000) according to its onset location. The incidence for each county was calculated and standardized using direct standardization for age and sex according to the overall composition of the 2009 Chinese population, using 5-year age-group categories. To explore the spatial and temporal diffusion trend of pandemic influenza in mainland China, a map of pandemic influenza spread was developed using trend surface analysis, which is a spatial smoothing method that uses polynomials with geographic coordinates, as defined by the central point of each county (12) (13) (14) . The time delay of the first confirmed case for each county was defined as the duration of time (in days) since May 9, 2009, the date of the first confirmed case in mainland China. A trend surface on these durations was created in ArcGIS 9.2 (ESRI Inc., Redlands, California) using a second-order trend surface model with a local polynomial method to explore the diffusion patterns of pandemic influenza over time. In addition, the epidemic curves were also plotted for 5 selected cities/provinces in different regions of mainland China: Guangdong (southern China), Shanghai (eastern China), Shaanxi (central China), Tibet (western China), and Beijing (northern China).
To assess the association between the invasion of pandemic influenza and different means of domestic travel, we performed a survival analysis (Cox analysis) of the time delay of the first confirmed case for each affected county, considering unaffected counties as right-censored. The time delay of the first confirmed case was defined as the duration (in days) since May 9, 2009, the date of the first confirmed case in mainland China. Domestic travel was expressed using 4 variables: distance (from the midpoint of each county) to the nearest civil airport and whether or not a county was intersected by national highways, freeways, or railways. This information was obtained from the National Bureau of Statistics of China (9, 15) . Spatial analyses were used to extract data on these variables in ArcGIS 9.2 (ESRI Inc.). Since population density is also linked to human activities and may facilitate influenza transmission (16, 17) and since density of medical facilities could be linked to patient reporting, we adjusted for the effects of these variables. In this study, the population density for each county was obtained from the National Bureau of Statistics of China (9, 15) , and the densities of medical facilities were based on the CISDCP, including all reporting sectors for notifiable infectious diseases, comprising provincial, prefectural, and county centers for disease control and prevention and provincial, prefectural, and county hospitals and township clinics. Hazard ratios and their 95% confidence intervals and P values were estimated using maximum likelihood methods. Hazard ratios for the continuous variables were calculated for the following units: distance to the nearest airport (in 50-km increments), population density (in 1,000 persons per km 2 ), and density of medical facilities (in number of reporting sectors for pandemic influenza per 10,000 persons).
To explore the effect of climatic factors on local transmission within counties, we performed multilevel Poisson regression. Climatic data (temperature, relative humidity, and precipitation) during May-December 2009 were obtained from the National Meteorological Bureau of China (18) . Owing to probable time lags, the climatic variables were processed by calculating the average value for the current day and a lag of 1-3 days, which is the observed incubation period of pandemic influenza (19) . Poisson regression deals with the daily number of laboratoryconfirmed cases per county. The inclusion of the population size for each county as an offset makes it an analysis of incidence. To account for possible confounding, we included school summer vacation and public holidays, the proportion of the school-age population (ages 6-19 years), population density, and the density of medical facilities as correction factors in the analysis. The percentage change in incidence in response to the change of the variable by a given amount (10°C for temperature, 10% for relative humidity, 1 mm for precipitation, 10% for school-age population, 1,000 persons per km 2 for population density, and number of facilities per 10,000 persons) was used to reflect the impact of each variable. The 95% confidence intervals and corresponding P values were estimated after correcting for overdispersion because of the nature of infectious diseases with spatial clustering patterns (20, 21) . For temperature, we also included a quadratic term in the analysis.
For all analyses, univariate analysis was performed first to examine the effect of each variable separately. Multivariate analysis was then performed by including all variables with P values less than 0.20 in univariate analysis and exclusion of variables with P values greater than 0.10, using a standard backward likelihood ratio method. For all continuous variables, we also presented categorical results in 3-5 categories to allow inspection of the data and determine whether or not the assumption regarding continuous variables (quadratic for temperature) was justified. Statistical analyses were performed using the Stata package (StataCorp LP, College Station, Texas) (20) . Readers interested in further research can contact the corresponding author to obtain the full data set used in this study.
A total of 121,805 cases of pandemic influenza (H1N1-2009), distributed in all 31 provinces in mainland China, were reported from May 9, 2009, to December 31, 2009. There was much variation in the numbers of confirmed cases in different provinces, ranging from 881 to 12,748, with a median of 2,958 cases, and in the incidence of confirmed cases in different provinces, ranging from 3.94 per 100,000 population to 71.72 per 100,000, with a median of 8.41 per 100,000. From the time profile, we found that the number of confirmed cases increased rapidly beginning at the end of August, when a new term began for school students, and peaked by the end of November. The first death caused by pandemic influenza was reported on October 4, 2009. The number of deaths eventually rose to 648 by the end of the year, and peaked in early December (Figure 1 ). The age-and sex-standardized incidence map shows that the epidemic spanned a large geographic area, and the most affected areas were in western China (see Web Figure 1 , which appears on the Journal's website (http://aje.oxfordjournals.org/)). Significant differences in incidence were found among age groups, with incidences peaking in school-age groups (Web Figure 1 ). Boys showed a higher incidence than girls (ages <20 years).
Web Figure 2 shows the trend of the spatial spread of pandemic influenza over time in mainland China and indicates that the epidemic areas during the first 120 days after May 9, 2009, were limited to the circumferences of cities with international airports, such as Beijing, Shanghai, Guangzhou, Shenzhen, Chengdu, and Changchun. Thereafter, it spread to the rest of mainland China, roughly from southeast to northwest. The largest-scale spread took place 150-180 days after the first case (Web Figure 2) . Figure 2 shows the large variation in the temporal patterns of pandemic influenza for the 5 selected cities/provinces, but there was a marked drop in incidence during the first week of October for all locations.
Survival analysis of the duration of time to the first confirmed case in each county indicated that all 4 factors related to domestic travel or human mobility were significantly associated with the invasion of pandemic influenza in the Cox univariate analysis (Table 1) . Population density and the density of medical facilities also showed a significant association. The significant effect of being intersected by railways disappeared, and the density of medical facilities showed borderline significance after correction for other factors in multivariate analysis, whereas being intersected by national highways and freeways and proximity to airports and higher population den-sity remained as significant factors, all showing a positive association (Table 1) . Table 2 shows that all climatic factors (except precipitation), school summer vacation and public holidays, proportion of the school-age population, population density, and the density of medical facilities were significantly associated with the extent of local transmission in univariate multilevel Poisson regression. School summer vacation and public holidays showed a significant negative association with the incidence of pandemic influenza. The significant effect of the proportion of schoolage children disappeared after correction for other factors; thus, temperature, relative humidity, school summer vacation and public holidays, population density, and the density of medical facilities remained as significant factors in multivariate analysis. Temperature showed a peak pattern, with the highest incidences for the range from 0°C to 10°C, which was also reflected in the statistically significant quadratic term.
Our study provides a complete overview of the spatial and temporal characteristics of the pandemic influenza (H1N1-2009) epidemic in mainland China in 2009. The epidemic spanned a large geographic area and presented spatial and temporal heterogeneity in different regions of mainland China. Our analyses of the invasion of pandemic influenza indicated that domestic travel by air and by national highways and freeways and population density contributed to the spread of the epidemic. Lower temperatures and lower relative humidity were climatic factors that facilitated local transmission after correction for the effects of school summer vacation and public holidays, as well as population density and the density of medical facilities. The density of medical facilities could have influenced pandemic influenza patient reporting, and this effect seemed more important for the reporting of local transmission (a highly significant positive association) than for reporting of the invasion (borderline significance). This indicates that the CISDCP can be further improved.
In the initial phase of the epidemic, the Chinese government took measures to prevent and control the spread of the novel influenza virus, declaring it a notifiable infectious disease in order to strengthen national surveillance and find newly confirmed cases quickly. In addition, quarantine measures were implemented at the international airports (e.g., Beijing, Shanghai, Guangzhou, and Fuzhou) to identify and isolate prob-able cases and close contacts in order to decrease the risk of local transmission caused by imported cases. It is possible that these measures were effective in achieving a slower pace of spread in the early stages of the epidemic, but the current evidence is inconclusive (22) . However, following the development of the global pandemic and the beginning of the new school term, a rapid spread of the epidemic occurred in mainland China, and local outbreaks increased at the end of August 2009. A reduction in incidence was observed during the first week of October, when there was an 8-day public holiday during the National Days from October 1 to October 8. This drop in incidence was largely due to a lower tendency for patients to visit medical facilities at that time, together with the fact that many hospitals had reduced the open hours of their outpatient clinics during the holidays. This is clearly visible in Figure 1 , where we see the drop beginning on September 28, 3 days (i.e., the average duration between onset and seeing a physician) before the start of the holiday period. Thus, the holiday period led to a reduction in the number of people being diagnosed with pandemic influenza. Apparently, most undiagnosed patients recovered in the following days, because there was no marked compensation visible in the days after the holiday period. Additionally, there Abbreviations: CI, confidence interval; NS, not significant. a Results were adjusted for school summer vacation and public holidays, population density, and the density of medical facilities. b For all continuous variables, categorical results are also reported to allow inspection of the data and assessment of whether or not the assumption regarding continuous variables was justified. may have been some reduced transmission because of school closure, as was observed in Japan, where transmission was substantially reduced during school closure (23) . In addition, the temporal death curve could reflect the massive rise in confirmed cases with approximately 1 week's delay following the peak of confirmed cases at the end of November.
The direction of the spread of pandemic influenza was from the southeast to the northwest, indicating how the virus benefited from entering the country through international airports in the coastal areas and spreading further along routes of long-distance domestic travel.
With the fast-growing public transportation infrastructure and increasing socioeconomic activities, travel has become an important issue in the prevention of emerging airborne infectious diseases such as influenza, especially during the introduction period. Obviously, the presence of airports and high densities of transportation routes coincide with more developed areas (i.e., those with a higher population density and more medical facilities); however, after we corrected for these factors, proximity to airports and the presence of national highways or freeways remained significantly associated with the spread of the infection.
Geoinformatics plays an important role in the study and control of infectious disease outbreaks, and it includes techniques such as geographic mapping and location-based alert services (16, (24) (25) (26) . As was recognized previously, the international spread of pandemic influenza and severe acute respiratory syndrome was largely related to air travel (27) (28) (29) . Our study confirms that air travel and transportation routes accelerated the spread of pandemic influenza between counties in mainland China. Air travel and travel by national highways and freeways especially appeared to play a role, whereas railways were less important. In mainland China, trains are mainly used for occasional long-distance travel, whereas highways are more often used for daily or weekly commuting, especially because bus schedules are more flexible. Our previous study on the geographic spread of the severe acute respiratory syndrome epidemic in China also demonstrated that domestic travel along national highways played a more important role than travel by railway (30) . Data from the National Bureau of Statistics of China show that passenger traffic by highways in 2009 was 18.2 times that of railway travel (9) . Transportation by highway remains an important mode of travel between Chinese cities and provinces and therefore is a potential target for controlling any future emerging airborne infections.
We also showed that lower temperature and lower relative humidity create a higher risk of local transmission of pandemic influenza. However, much lower temperature (e.g., <0°C) did not facilitate local transmission, as indicated by the daily incidence over categorical temperature in Table 2 , and required inclusion of a quadratic variable in the model. The observation of an influence of temperature and relative humidity on pandemic influenza is in accordance with animal experiments on seasonal influenza virus (31) . In addition, recent studies have suggested that absolute humidity could also play an important role in the transmission of pandemic influenza and seasonal variations in influenza epidemics in temperate regions (32) (33) (34) . As expected, population density further facilitated both invasion and local transmission, whereas holiday periods reduced spread (16, 17, 35, 36) . We also used population size as a correction factor instead of population density, which led to similar results (not shown).
In conclusion, this is the first complete documentation of pandemic influenza in mainland China, to our knowledge. The findings indicate that interventions focused on domestic travel, population density, and climatic factors could play a major role in mitigating the public health impact of future influenza pandemics. Replication, Neurotropism, and Pathogenicity of Avian Paramyxovirus Serotypes 1–9 in Chickens and Ducks Avian paramyxovirus (APMV) serotypes 1–9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2–9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2–9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2–9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2–9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1–9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes. The family Paramyxoviridae consists of enveloped viruses with a nonsegmented, single-stranded, negative-sense RNA genome [1] . These viruses have been isolated from a great variety of mammalian and avian species around the world. Many members of the family cause important human and animal diseases, while the disease potential of many other members is not known. The family is divided into two subfamilies: Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae comprises five genera: Rubulavirus, Respirovirus, Morbillivirus, Henipavirus, and Avulavirus. Subfamily Pneumovirinae is divided into two genera: Pneumovirus and Metapneumovirus. All paramyxoviruses isolated from avian species are classified into the genus Avulavirus, except avian metapneumoviruses, which are classified in the genus Metapneumovirus. Avian paramyxoviruses (APMVs) have been divided into nine different serotypes (APMV 1 to 9) based on Hemagglutination Inhibition (HI) and Neuraminidase Inhibition (NI) assays [2] . APMV-1 comprises all strains of Newcastle disease virus (NDV) and has been well characterized because of its economic importance in poultry industry. As an initial step towards characterizing other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 have been determined [3] [4] [5] [6] [7] [8] [9] [10] .
The genomes of all paramyxoviruses range between 15 and 19 kb in length and contain 6-10 genes that encode up to 12 different proteins [11] . For members of subfamily Paramyxovirinae, efficient RNA replication requires that the nucleotide (nt) length of the genome is an even multiple of six, known as 'rule of six', reflecting the precise packaging of the polynucleotide in the nucleocapsid [12] . The genomes of APMVs are very similar in organization. All the members contain six genes in the order of 39-N-P-M-F-HN-L-59 with the exception of APMV-6, which contains an additional small hydrophobic protein (SH) gene in its genome. APMVs encode a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutininneuraminidase protein (HN), and a large polymerase protein (L). Two additional proteins called, V and W, are produced by RNA editing of the P gene. The F and HN proteins form spike-like projections on the outer surface of the viral envelope and are the neutralizing and protective antigens of NDV. Significant sequence divergence in these two proteins exists among APMV serotypes [6] . The HN protein possesses receptor-binding and neuraminidase activity; whereas, the F protein is directly involved in membrane fusion which is necessary for the entry of the virus. Homotypic interactions between the HN and F proteins are hypothesized to control initiation of the fusion process for most paramyxoviruses [13, 14] . The M protein forms the inner layer of the envelope and plays a key role in assembly by interacting with the HN and F proteins as well as ribonucleocapsid [15, 16] .
APMVs have been isolated from many different avian hosts [17] . APMV-1 is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. NDV isolates vary greatly in their pathogenicity for chickens, ranging from no apparent disease to severe respiratory and neurological disease causing 100% mortality [18] . NDV strains are categorized into three main pathotypes: lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (virulent), based on their pathogenicity in chickens [19] . In contrast, the disease potential of APMV-2 to -9 is not well known because many of these viruses were isolated from birds dying in quarantine, hunter killed, trapped wild birds, apparently healthy poultry or exotic birds [20] . APMV-2 and -3 have been reported to cause significant disease in poultry, whereas the pathogenic potential of APMV-4 to -9 is generally unknown. In general, APMV-2 strains have been isolated from chickens, turkeys and wild birds across the globe and have been found to cause mild respiratory disease, decreases in egg production, and infertility [21] [22] [23] . APMV-3 strains have been isolated from wild and domestic birds and their infections have been associated with encephalitis and high mortality in caged birds [24] . APMV-5 strains have only been isolated from budgerigars (Melopsittacus undulatus) and cause depression, dyspnoea, diarrhea, torticollis, and acute fatal enteritis in immature budgerigars, leading to very high mortality [25] . Infections from APMV-4, -8, and -9 appear to be restricted to ducks and geese. APMV-6 and -7 infections in turkeys cause drops in egg production and induce respiratory disease. There are no reports of isolation of APMV-5, -8 and -9 from poultry [2] . But recent serosurveillance of commercial poultry farms in the U.S. indicated the possible prevalence of all APMV serotypes excluding APMV-5 in chickens [26] . The pathogenicity of APMV-2 and APMV-3 has been studied in experimentally infected chickens [27, 28] . However, replication, pathogenicity, and neurovirulence of APMV serotypes 2 through 9 have not been comprehensively studied. Therefore, we characterized in vitro replication of APMVs (growth kinetics and cytopatic effect in chicken fibroblast cells) and their in vivo replication and tropisms by infecting prototype strains of each serotype in two different ages of chickens (1-day-old and 2-week-old chickens) and 3-week-old ducks. Specifically, neurotropism of APMVs was evaluated in primary chicken neuronal cells and brain tissue of 1-day-old chicks.
The chicken embryo fibroblast cell line (DF1, ATCC, Manassas, VA, USA) was grown in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. The African green monkey kidney Vero cell line (ATCC, Manassas, VA, USA) was grown in Eagle's minimum essential medium (EMEM) containing 10% FBS and maintained in EMEM with 5% FBS. Primary chicken neuronal cells were grown in Neurobasal medium with B-27 supplement (Invitrogen).
The viruses used in this study were nine prototype strains of APMV serotypes 1 to 9: APMV-1 (NDV), lentogenic strain LaSota/46 and mesogenic strain Beaudette C (BC); APMV-2, APMV-2/Chicken/California/Yucaipa/56; APMV-3, APMV-3/ PKT/Netherland/449/75; APMV-4, APMV-4/duck/Hon-gKong/D3/75; APMV-5, APMV-5/budgerigar/Kunitachi/74; APMV-6, APMV-6/duck/HongKong/18/199/77; APMV-7, APMV-7/dove/Tennessee/4/75; APMV-8, APMV-8/goose/ Delaware/1053/76; and APMV-9, APMV-9/duck/New York/ 22/1978. All of the viruses were grown in 9-day-old embryonated specific-pathogen-free (SPF) chicken eggs inoculated by the allantoic route, except for APMV-5, which was grown in Vero cells. The ability of viruses to produce plaques was tested on Vero and DF1 cells under 0.8% methylcellulose overlay. Exogenous protease was supplemented into the cells for replication of APMV-1 LaSota, APMV-3 and -9 (10% allantoic fluid) and APMV-8 (1 mg/ml of acetyl trypsin) (4, 9, 10) . The monolayers were fixed with methanol and plaques were visualized by immunoperoxidase staining using virus specific antiserum raised against N protein.
Virus titers for in vitro and in vivo replication were quantified by immunoperoxidase staining with N-specific antibodies on DF1 cells (APMV-1, -2, -3, -4, -6, and -9) or Vero cells (APMV-5, -7, and -8) [3-10].
The multicycle growth kinetics of the viruses was evaluated in DF1 cells. Duplicate wells of six-well plates were infected with each APMV at an MOI of 0.01 PFU/cell. After 1 h of adsorption, the cells were washed and then covered with DMEM containing 2% FBS at 37uC in 5% CO 2 . APMV-1 LaSota, APMV-3, -8, and -9 were supplemented with protease, as described above. Supernatants were collected and replaced with an equal volume of fresh medium at 12-h intervals until 72 h post-infection (hpi). Virus titers in the collected supernatants were quantified in DF1 cells or Vero cells by limiting dilution and immunostaining with N-specific antibodies [29] [30] . Virus titers were expressed as 50% tissue culture infectious dose (TCID 50 /ml) by the end-point method of Reed and Muench [31] .
The pathogenicity of APMVs was determined by the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs and by the intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks [19] . Briefly, for the MDT, a series of 10fold dilutions of infected allantoic fluid (0.1 ml) was inoculated into the allantoic cavities of five 9-day-old eggs per dilution and incubated at 37uC. The eggs were examined once every 8 h for 7 days, and the time of embryo death was recorded. The MDT was determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos. The criteria for classifying the virulence of NDV isolates are: ,60 h, virulent strains; 60 to 90 h, intermediate virulent strains; and .90 h, avirulent strains. For the ICPI test, 0.05 ml of a 1:10 dilution of fresh infective allantoic fluid for each virus was inoculated into group of 10 1-day-old SPF chicks via the intracerebral route. The birds were observed for clinical symptoms and mortality once every 8 h for a period of 10 days. At each observation, the birds were scored as follows: 0 if normal; 1 if sick; and 2 if dead. The ICPI is the mean of the score per bird per observation over the 8-day period. Highly virulent velogenic viruses give values approaching 2, and avirulent or lentogenic strains give values at or close to 0. All experiments involving experimental animals were approved by the committee of IACUC, University of Maryland (protocol number R-09-81) and conducted following the guidelines.
To evaluate neurotropism of APMVs in vitro, primary chicken neuronal cells were prepared from 9-day-old chicken embryos for virus infection. Briefly, the section of hippocampus was dissected from embryos, digested with trypsin, passed through a cell strainer (40 mm nylon), seeded onto poly-L-lysine-coated plates, and then infected with each virus [32] . Spread of viruses in neuronal cells was determined by confocal microscopy analysis. Briefly, at 48 hpi, the cells were fixed, permeabilized, stained with polyclonal antibodies against the respective N protein and a neuronal marker (anti-neuron specific beta III tubulin antibody, abcamH, Cambridge, MA) followed by anti-Alexa Fluor 488 and 594 antibodies, and then analyzed by confocal microscopy. In addition, virus replication in neuronal cells was determined by collecting supernatants at 12-h intervals until 72 hpi. Virus titers in the collected supernatants were quantified in DF1 or Vero cells by limiting dilution and immunostaining with N-specific antibodies as described above.
To evaluate the ability of APMVs to replicate in chicken brains, ten 1-day-old SPF chicks were inoculated with 0.05 ml of a 1:10 dilution of fresh infective allantoic fluid for each virus via the intracerebral route. Two birds were sacrificed daily until 5 days post-infection (dpi). Brain tissue samples were collected from the sacrificed birds and processed for immunohistochemistry as described later. Brain tissue samples also were homogenized and virus titers were determined by limiting dilution and immunoperoxidase assay in DF1 cells or Vero cells using polyclonal antibodies against the respective N protein.
2.5. Replication of APMVs in 1-day-old Chicks, 2-week-old Chickens, and 3-week-old Ducks Following Intranasal Inoculation Three 1-day-old chicks per group were inoculated with 100 ml of each virus (256 HA units/bird) via the intranasal route. On day 3 post-infection, tissue samples (lung, trachea, spleen, and brain) were collected for virus titration by limiting dilution and immunoperoxidase assay as described above. To evaluate the tropism of APMVs in older birds, groups of ten 2week-old SPF chickens were inoculated with 200 ml of each virus (256 HA units/bird) by the intranasal route. Three birds from each group were sacrificed at 4 dpi and tissues samples (lung, trachea, spleen, and brain) were collected and processed for immunohistochemistry as described later. Tissue samples also were homogenized for virus titration. Virus titers in DF1 or Vero cells were determined by limiting dilution as described above. To confirm the replication of viruses in infected birds, the tissue samples were also inoculated into 9-day-old embryonated chicken eggs. On 3 dpi, virus growth was determined by HA assay. The remaining birds were observed daily for 10 days for any clinical signs and then sacrificed for virus titration of various tissues as described above. To evaluate virus replication in different hosts, we further determined replication of APMVs in 3-week-old mallard ducks. Six birds each were infected with 500 ml of individual APMVs (256 HA units/bird) via the combined intranasal and intratracheal routes and sacrificed at 4 dpi for collection of tissue samples (lung, trachea, spleen, and brain). Homogenates were prepared for virus titration in cell cultures and replication in eggs as described above. The remaining birds were observed daily for 10 days for any clinical signs.
From the experiments described above, brain tissue harvested 3 dpi from 1-day-old chicks infected by intracerebral route and various tissue samples harvested 4 dpi from 2-week-old chickens infected by the intranasal route were fixed in phosphate-buffered formalin (10%). Fixed tissues were embedded in paraffin and sectioned (Histoserv, Inc., Germantown, MD). Sections from mock-infected birds were used as controls. The tissues were deparaffinized, rehydrated, and subsequently, immunostained to detect viral N protein using the following protocol. Briefly, the sections were blocked with 1% BSA in PBS for 1 h at room temperature, incubated with a polyclonal antibody (1:200 dilution) against the respective N protein (29, 30) followed by horseradish peroxidase-conjugated goat anti-rabbit antibodies for 30 min, and then stained with AEC (3-amino-9-ethylcarbazole) substratechromogen.
Syncytium formation is a hallmark of the cytopathic effect (CPE) caused by many paramyxoviruses, including APMV-1, in cell culture [1] . To investigate syncytium formation by APMV serotypes 2-9 and to compare their CPE, Vero cells were infected with mesogenic APMV-1 strain BC or with representatives of the other APMV serotypes at a multiplicity of infection (MOI) of 0.1 PFU/cell, incubated for 48 h, and visualized directly by photomicroscopy (data not shown) and following immunostaining with rabbit antiserum to the respective N protein (Fig. 1 ). Exogenous protease was supplemented into the culture medium for replication of APMV-3, 8, and 9, based on our previous studies [4, 9, 10] . APMV-1 strain BC was known to be independent of protease supplementation. APMV-3 and -5 produced distinctive CPE with syncytium formation similar to those of APMV-1. In contrast, the rest of APMVs produced single cell infections leading to cell rounding and detachment of infected cells but a lack of evident syncytia. Similar results were observed in chicken embryo fibroblast DF1 cells (data not shown).
We further evaluated the multicycle replication of the APMVs in DF1 cells (Fig. 2 ). There was a great difference in the kinetics and magnitude of replication among different APMVs. APMV-1 strain BC and APMV-1 lentogenic strain LaSota grew better than any other APMVs and reached the highest titers (10 8 TCID 50 /ml) at 32 and 60 hpi, respectively. APMV-3 replicated relatively well, reaching 10 7 TCID 50 /ml at 72 hpi. The highest titers of APMV-2 and -5 also were detected at 72 hpi, but their titers were 4.0 log 10 lower than those of APMV-1. In general, replication of other APMV viruses was limited in DF1 cells (,10 2 TCID 50 /ml). Inefficient virus replication in vitro (i.e., APMV-4, 6, 7, 8, and -9) was correlated with the pattern of a single-cell infection as observed with CPE in infected cells (Fig. 1 ).
The pathogenicity of APMVs was evaluated with the standard pathogenicity tests used for APMV-1, namely MDT in embryonated chicken eggs and ICPI in 1-day-old chicks ( Table 1 ). The pathogenicity of the two strains of APMV-1 has been well characterized: strain BC is neurovirulent (MDT 58 h and ICPI 1.55), whereas strain LaSota is avirulent (MDT 112 h and ICPI 0.00) and is used as a vaccine strain. For the most part, APMV 2-9 were even more avirulent based on these assays than strain LaSota, regardless of their isolation sources. The one exception was APMV-3, which had a MDT value (117 h) that is similar to that of the LaSota strain, but had an ICPI value (0.53) that was higher than that of the LaSota strain (0.00), but nonetheless remained in the avirulent range. The MDT and ICPI values of other APMVs were .144 h and 0.00, respectively, consistent with being avirulent. Chicks infected with APMV serotypes 2-9 had no apparent clinical signs during the 8-day period of the ICPI test.
To evaluate neurotropism of APMVs, virus replication was evaluated in primary chicken neuronal cells in vitro. We first evaluated infection (MOI of 0.1 PFU) in neuronal cells by confocal microscopy analysis of cells that had been immunostained at 72 hpi with N protein against each respective APMV (Fig. 3A) . Expression of the N protein of APMV-1 strain BC was clearly detected in dendrites and axons. In contrast, APMV-1 strain LaSota failed to replicate in neuronal cells even after supplementation with allantoic fluid as a source of protease. Among serotypes 2-9, APMV-3 was able to replicate well without the addition of allantoic fluid, whereas the presence of APMV-7 and -8 antigens in infected cells was sporadically detected. Replication of the other APMVs was not detected up to 3 dpi. We also examined replication in the neuronal cell cultures by collecting supernatants from the cultures at 12 h intervals and assaying for infectious virus by titration. This confirmed the ability of APMV-3 to replicate in neuronal cells, although its titer was lower than that of neurovirulent APMV-1 strain BC (Fig. 3B) . Titers of BC and APMV-3 in neuronal cells increased gradually and reached 5.5 and 3 log TCID 50 /ml, respectively, at 72 hpi. In contrast, other APMVs, including APMV-1 strain LaSota, were not detected in the supernatants of neuronal cells, indicating their inability to replicate in these cultures of primary chicken neuronal cells.
We further determined the ability of the APMV serotypes to replicate in chicken brains by inoculating 1-day-old chicks via the intracerebral route. The infected chickens were sacrificed daily and brain tissue was collected for virus titration (Fig. 4) . In chicks infected with neurovirulent APMV-1 strain BC, the virus reached the highest titer (.6.0 log 10 TCID 50 /g) in the brain at 2 dpi, resulting in the death of all of the infected chicks on 3 dpi. The non-neurovirulent LaSota strain was detected at low titer on day 1, but was not detected on subsequent days and did not cause disease or death during the 5 days of observation. APMV-3 strain reached a titer of 5 log 10 TCID 50 /g on 3 dpi, but did not cause disease or death of any of the infected chicks during the 5 days of observation. We detected a low level of replication of APMV-8 (,2 log 10 TCID 50 /g), with no observed disease or death. Replication of the other APMVs was not detectable on any day, and there was no detectable disease or death. Thus, APMV-3 was identified as the only neurotropic virus among APMV serotypes 2-9. However, despite the ability of APMV-3 to replicate to moderate titer in the brain, it did not cause discernable disease or death and thus was non-neurovirulent.
The neurotropism of the APMVs in 1-day old chicks also was evaluated by immunohistochemistry analysis of brain tissue harvested on day 3 (Fig. 5) . Viral antigens were detected in the brain tissues that had been infected with APMV-1 strain BC and APMV-3, confirming the replication of the two viruses in chicken brain. Our result showed a more extensive distribution of viral antigens in brain tissue infected with BC compared to APMV-3. In contrast, presence of viral antigens was not detected in the brain tissues that had been infected with the other APMVs (Fig. 5) .
We next determined the replication and tissue tropism of APMVs in 1-day-old and 2-week-old chickens following intranasal inoculation. Chickens were sacrificed on 3 dpi for 1-day-old chicks and on 4 dpi for 2-week-old chickens, and the following tissues were harvested for quantitative virology: trachea, lungs, spleen, and brain (Fig. 6) . In 1-day-old chicks, APMV-1 strain BC replicated to high titers (.5.0 log 10 TCID 50 /g) in each of the sampled tissues in each of the birds, whereas LaSota replicated in trachea, lung and spleen, but not the brain, and its titers were less than those of BC (Fig. 6A) . APMV-3 replicated to moderate titers (.4.0 log 10 TCID 50 /g) in all of the collected samples, including the brain. However, replication of other APMVs was restricted to the trachea, and their titers were low, ranging from 1.5 to 3 log 10 TCID 50 /g (data for APMV-2 are shown in Fig. 6A ; the other serotypes are not shown). Chicks infected with APMV-1 strain BC began to show clinical signs on 1 dpi and distinctive neurological signs on 2 dpi, whereas chicks infected with other APMVs had no apparent clinical signs during the 3-day post-infection. In 2-week-old chickens, virus replication was more restricted and viral titers were lower compared to 1-day-old chickens (Fig. 6B) . APMV-1 strain BC was able to replicate in all of the collected tissues, but the virus titers in the brains of older birds (2.6 log 10 TCID 50 /g) were much lower than that of 1-day-old chickens (6.5 log 10 TCID 50 /g). APMV-1 strain LaSota replicated in the trachea, lungs, and spleen, but not the brain, as was observed in the 1-day-old chickens. Interestingly, APMV-3 replication was detected only in the trachea and the brain, indicating that it is neutrotropic in these older chickens despite this restricted replication. APMV-2 was also able to replicate in trachea of 2week-old chickens. However, replication of the other APMVs was not detected in any of the harvested tissue samples. Since APMV 2-9 can replicate much better in embryonated chicken eggs than in cell culture [20] , the collected tissue homogenates from this same experiment also were inoculated into 9-day-old embryonated chicken eggs to confirm the replication of viruses in various tissues ( Table 2 ; APMV-5 was not assayed because it does not replicate in the allantoic cavity of chicken eggs). This approach with increased sensitivity enabled us to detect replication of all of the tested APMVs in the trachea of most of the birds. In addition, APMV-2, 3, 6, 7, and 9 were detected in the lungs of at least one of the three chickens in each group, and APMV-3 was detected in the spleen of two chickens. Among the tested APMVs, APMV-4 showed the least replication in chickens both in vitro and in vivo ( Fig. 2 and Table 2 ), suggesting that it has a strong host range restriction in chickens. On 10 dpi, none of the APMVs were detected in any of the sampled tissues from any of the birds (not shown). Clinical signs of illnesses in any of the infected groups were not found up to 10 dpi.
Histopathological examinations of tissue samples collected on 4 dpi revealed similar microscopic findings in all APMVs. Specifically, the trachea showed mild lymphocytic tracheitis, mild to moderate multifocal mucosal attenuation, and loss of tracheal alveolar mucous glands (Fig. 7A) . Lung sections exhibit moderate, multifocal, lymphocytic to lymphohistiocytic bronchitis with mild to moderate perivascular and peribronchial interstitial inflammation and focal perivascular cuffing with varying severity (Fig. 7B) . Minimal lymphoid depletion was detected in the spleen, and microscopic lesions were not found in any of the brain tissues (data not shown). The presence of virus antigens in various tissues of infected chickens was further evaluated by immunohistochemistry analysis. Deparaffinized sections of the virus-infected and uninfected control tissue were immunostained using polyclonal antibodies against the N protein of the respective APMV. The presence of antigens for most APMVs was detected in the epithelial lining of trachea (Fig. 7C) . However, no antigen was detected in any tissues harvested from chickens infected with APMV-5. In the lungs, the viral antigens were mostly localized in the epithelium surrounding the medium and small bronchi (Fig. 7D) . Lung tissues showed extensive presence of antigens for APMV-1 strain BC followed by APMV-1 strain LaSota, APMV-3, and APMV-2. However, presence of antigens for other serotypes was not detected in lung tissues. Our results suggested that APMV 2-9 were avirulent, and their replication was mostly restricted to trachea and lungs of infected chickens.
Since several of the specific APMV strains under evaluation (i.e., APMV-4, -6, and -9) were isolated from other avian species including the mallard duck (see Table 1 ), their replication and pathogenicity were further evaluated in groups of 3-week-old ducks following intranasal inoculation. No clinical signs of illnesses in any of the infected groups were found up to 10 dpi. Three birds from each group were sacrificed on 4 dpi and the tissues were harvested for virus titration. None of the APMVs were detected in any of the tested tissues from any of the ducks by virus titration in DF1 cells (data not shown). However, APMVs were detected in some of the samples of trachea, lung, and spleens by inoculation into 9-day-old embryonated chicken eggs, although none of the APMVs (including APMV-1) were detected in any of the brain samples (Table 3 ; APMV-5 was not evaluated). APMV-1 strains BC and LaSota were the only APMVs that were detected in all three ducks in their respective groups, although replication was mostly restricted to the trachea. APMV-2 was found only in the trachea of a single duck, in contrast to its more efficient replication in chickens. APMV-3 was not detected in any samples from any duck, in contrast to its efficient replication and neurotropism in chickens. APMV-8 and -9 also were not detected in any duck. The other serotypes (APMV-4, -6, and -7) were each detected in the trachea of two ducks from its group, and in the lungs of one or two ducks from its group. APMV-4 replicated slightly better in ducks than in chickens. APMV-7 was the only serotype detected in the spleen (of a single duck), whereas it had not been detected in the spleen of infected chickens. Thus, the pattern of infection in chickens and ducks was substantially different and could not be predicted by the isolation history.
APMVs are frequently isolated from a wide variety of avian species and are grouped into nine serotypes based on antigenic analysis [33] , with a likely tenth serotype recently described in penguins [34] that was not evaluated in the present study. APMV-1 (NDV) is the most extensively characterized member of the APMV serotypes. APMV-2 to -9 have been isolated from both wild and domestic birds, but their disease potential in wild or domestic birds was largely unknown. APMV-1 also has been shown to infect a number of non-avian species [35] and presently is being evaluated as a potential human vaccine vector for human pathogens [36] . There is a possibility that APMV-2 to -9 could also be used as human vaccine vectors for human pathogens, providing multiple vectors with minimal cross-restriction from vector-specific immunity. Our previous studies demonstrated that APMV-2 to -9 can replicate in mammalian hosts, specifically in the hamster and mouse models [29, 30] . However, their replication and pathogenicity in avian host has not been comprehensively Chicks were inoculated with each virus by the intracerebral route, and brain tissue was harvested for immunohistopathology on 3 dpi. The tissues were fixed in phosphate-buffered formalin, and sections were prepared and stained using an antibody against the respective N protein (stained red). doi:10.1371/journal.pone.0034927.g005 evaluated and compared. Therefore, in this study, we evaluated in vitro and in vivo replication and pathogenicity of APMVs in chickens and their in vivo replication and pathogenicity in ducks and characterized differences in their tropisms, including neurotropism in chickens.
Virulent APMV-1 strains contain a multibasic cleavage site (with the general consensus sequence of RRQKRQF) with a polybasic furin motif (RX[R/K]RQ) that is readily cleaved in cell culture by intracellular furin or furin-like protease. Avirulent APMV-1 strains lack this polybasic site and depend on extracellular protease for cleavage, which in vitro can be supplied by exogenous protease and in vivo is supplied by secretory trypsinlike protease found in the lumen of the respiratory and enteric tracts [20] . Our study showed that APMV-1 strain BC readily forms syncytia in cell culture and replicates more efficiently in vitro than any of the other APMVs, followed by the APMV-1 LaSota strain. Characterization of the CPE of APMV types 2-9 demonstrated that only APMV-3 and -5 induced syncytium formation in infected cells. The cleavage site of APMV-3 (RPRGRQL) lacks the furin motif, and efficient growth and syncytium formation depended on supplementation with exogenous protease, resembling an avirulent APMV-1 strain. The cleavage site of APMV-5 (KRKKRQF) is the only example from APMV2-9 to have a furin motif [6] , and syncytium formation and growth were independent of added protease, resembling a virulent APMV-1 strain. Production of syncytia by APMV-3 and APMV-5 correlated with efficient multicycle replication in cell culture in the case of APMV-3 and a moderate level of replication in cell culture in the case of APMV-5. Although APMV-5 contains the greatest number of basic residues for any of the APMV except for some strains of APMV-1, its replication in DF1 cells was much less efficient compared to APMV-1 and -3. Interestingly, the level of replication of APMV-5 in cell culture was very similar to that of APMV-2, which lacks a furin motif (KPASRQF), does not form Figure 6 . Replication of APMVs in 1-day-and 2-week-old chickens. Groups of (A) 1-day-or (B) 2-week-old chickens were inoculated with each virus (256 HA units) by the intranasal route. Three birds from each group were sacrificed on 3 dpi (1-day-old chicks) or 4 dpi (2-week-old chickens), and virus titers in the collected tissues samples (brain, trachea, lung, and spleen) were determined by limiting dilution in DF1 cells and immunostaining with polyclonal antibodies raised against the respective N protein. doi:10.1371/journal.pone.0034927.g006 syncytia, and whose replication and lack of ability to form syncytia are independent of exogenous protease. Thus, for those two viruses (APMV-2 and -5), the growth phenotype in cell culture did not correlate well with the presence or absence of a furin motif in the cleavage sequence. The other APMVs (types 4, 6, 7, 8, and 9) have one or two basic amino acids in their cleavage site sequences and exhibited single cell infection and inefficient replication in vitro. However, only in the case of serotypes 8 and 9 did the addition of exogenous protease increase the efficiency of replication, and the overall level of replication remained very low and syncytia were not observed. Thus, APMV types 2, 4, 6, 7, 8, and 9 have cleavage sites that seemed similar to those of avirulent APMV-1 strains, but the inclusion of protease had little effect on growth in cell culture.
The pathogenicity of APMVs was first characterized by standard pathogenicity tests, namely the ICPI and MDT assays. We included two well-characterized strains of APMV-1, mesogenic BC and lentogenic LaSota, and compared their pathogenicity with that of APMV serotypes 2-9. This showed that APMV serotypes 2-9 were avirulent by both assays. Of types 2-9, only APMV-3 was associated with embryo death in the MTD assay and disease in the ICPI assay, although these effects were modest and APMV-3 was categorized as avirulent by the standards of these assays. By the MDT assay, the other types (types 2, 4, 5, 6, 7, 8, and 9) lacked detectable virulence and thus were less virulent than the well-known avirulent vaccine strain APMV-1 LaSota, and by the ICPI assay these APMVs were equivalent to LaSota in lacking detectable virulence. Thus, possession of a furin cleavage site and the ability to form syncytia by APMV-5 did not correlate with virulence in vivo, nor did the ability of APMV-2 to replicate to moderate titers in cell culture without the addition of trypsin.
We investigated the neurotropism of APMVs in chicken neuronal cells in vitro and in the brains of 1-day-old chicks infected via the intracerebral route. The neurovirulent APMV-1 strain BC was observed to replicate in chicken neuronal cells both on the basis of antigen expression and the production of infectious virus. In contrast, the non-neurovirulent LaSota strain did not detectably express viral antigens in these cells, and infectious virus was detected in the culture only at a low level on a single day early in infection. Among serotypes 2-9, expression of viral antigen in the neuronal cells was detected only with APMV-3, -7, and -8, and production of infectious virus was detected only with APMV-3. Thus, only APMV-1 strain BC and APMV-3 appeared to replicate productively in these neuronal cell cultures.
Following intracerebral inoculation of 1-day-old chicks, only APMV-1 strain BC, APMV-3, and (to a much lesser extent) APMV-8 could be detected in brain tissue samples. With APMV-1, virus neurotropism depends on the presence of a furin cleavage site, since secretory proteases are unavailable in the brain [37] . The lack of neurotropism of the APMV-1 strain LaSota is consistent with this idea. However, APMV-3 appears to be an exception, since it lacks a furin site and is dependent on added protease in cell culture and thus presumably dependent on secreted protease in vivo, yet it replicated efficiently in the chick brains and neutronal cells in vitro. Interestingly, however, while APMV-3 was clearly neurotropic, it caused no disease or death and thus was not neurovirulent. These findings indicate that the simple paradigm that neurotropism depends on the presence of a furin cleavage site does not hold for APMV-3. Furthermore, it shows that neurotropism by an APMV does not necessarily confer neurovirulence. Our previous study with replacement of the APMV-2 F protein with that of APMV-1 BC suggested an important role for the BC F protein in virus neurotropism, neuroinvasiveness, and neurovirulence in 1-day-old chicks [38] . Based on the generally held model for APMV-1 pathogenesis, it would have been reasonable to suggest that the presence of the furin site in the BC F protein was the major determinant of the observed neurotropism, neuroinvasiveness, and neurovirulence. However, the present study challenges the predominant role for the cleavage site, and suggests that other features of the F protein may be involved in the observed neurotropism, neuroinvasiveness, and neurovirulence. Evaluating the difference in the roles of the F proteins in virus neurotropism versus neurovirulence may provide information on these activities and may help design safer vaccines for neurotropic pathogens.
We also evaluated replication, tropism, and pathogenesis following intranasal infection in 1-day-old chicks and 2-week-old chickens. In 1-day-old chickens, only the APMV-1 strain BC and APMV-3 were able to spread to the brain and replicate, demonstrating neuroinvasiveness and neurotropism. These two viruses, plus APMV-1 strain LaSota, also were detected in the spleen of 1-day-old chicks. Conversely, replication of the other APMVs was mostly detected the in trachea of chicks, and the virus titers were moderate.
In 2-week-old birds, at 4 dpi, infection of APMV-1 strain BC was found in all of the tested tissues, including the brain, and LaSota was detected in all of the tested tissues except the brain. APMV-3 also was detected in all of the tested tissues including the brain. Replication of the other APMVs was restricted to the trachea and lungs. In general, virus titers in the 2-week-old chickens were lower than in the 1-day-old chicks. By 10 dpi, no virus could be detected in any of the tissues in any of the infected chickens for any serotype by virus isolation, suggesting that the virus was cleared from all tissues and disease was resolved, indicating the self-limited nature of the infections.
Histopathology analysis of APMV-infected 2-week-old chickens showed that infection by each of the APMVs, including APMV-5, produced tracheitis and mild pathology that was mainly restricted to the respiratory tract. Previous study with serologic assays in chickens infected with APMVs demonstrated a good humoral response on 14 dpi [39] . Thus, the birds indeed appeared to be infected even thought the recovery of infectious virus was often sporadic and low. A single APMV type, namely APMV-5, was not detected by virus isolation or by immunohistochemistry in any of the chickens in this study. However, the serologic assay of chickens that had been inoculated with APMV-5 also showed the development of virus specific antibodies detected by virus plaque reduction neutralization assay [39] , suggesting that infection had occurred. This suggests a low level of virus replication in chickens. Thus, although APMV-5 bears a furin cleavage site and can cause 100% mortality in budgerigars [25] , it was completely avirulent and replicated inefficiently in chickens. This is suggestive of a host range difference that was not greatly ameliorated by the presence of a multi-basic cleavage site.
Experimental infection of mallard ducks with APMVs indicated that all APMV serotypes, including duck isolates, are avirulent in ducks. Differences were observed in the replication of various APMVs between chickens and ducks. For example, replication of APMV-1 strain BC was low in ducks compared to its efficient replication chickens, although APMV-1 has shown to infect waterfowls, such as geese and Muscovy and Pekin ducks [40] [41] [42] . Several APMV serotypes (APMV-3, -8, and -9) did not replicate in ducks, even though APMV-3 replicated well in chickens and the APMV-9 strain had been isolated from ducks. In chickens, APMV-4 poorly replicated in both 1-day-old and 2-week-old chickens in our study. In addition, the virus has been shown to induce low HI titers in chickens compared to other APMVs, which was taken as evidence of a low level of replication of this virus in chickens [26, 39] . However, we detected replication of APMV-4 in trachea and lungs of infected ducks, indicating its preference to ducks from which this strain had been isolated. moderate multifocal mucosal attenuation, and loss of tracheal alveolar mucous glands. Lung sections (B) exhibit moderate, multifocal, lymphocytic to lymphohistiocytic bronchitis with mild to moderate perivascular and peribronchial interstitial inflammation and focal perivascular cuffing with varying severity. The presence of antigens for most APMVs was detected in the epithelial lining of trachea (C) and in the epithelium surrounding the medium and small bronchi of the lungs (D). doi:10.1371/journal.pone.0034927.g007 Table 3 . Replication of APMVs in 3-week-old ducks. Three birds from each group were sacrificed on day 4, and tissues samples (brain, trachea, lung, and spleen) were collected and homogenized. To confirm the virus replication, each sample (100 ml) in the collected samples was inoculated into three eggs, and allantoic fluids were collected on 3 dpi. Virus replication was determined by hemagglutination assay. Note that APMV-5 was not analyzed because it does not replicate in the allantoic cavity of chicken eggs. doi:10.1371/journal.pone.0034927.t003
In summary, our findings indicate that APMV serotypes 2-9 were avirulent in both chickens and ducks as well as in standard international assays. Each of the APMV serotypes replicated to low-to-moderate titers in the trachea of chickens, with APMV-3 having the highest titers. Among APMV types 2-9, only APMV3 replicated systemically and was neuroinvasive and neurotropic, although not neurovirulent. The sequence of the F protein cleavage site was not a reliable predictor of pathogenicity of APMVs in chickens, indicating incongruity with the well-known APMV-1 paradigm. For example, the systemic replication and neurotropism of APMV-3 is inconsistent with its lack of a furin cleavage site and dependence on exogenous trypsin for replication in cell culture. As another example, several of the APMV serotypes, including types 2, 4, 6, and 7, lacked a furin site, but replication of these viruses in cell culture was not improved by the addition of exogenous trypsin. As yet another example, APMV-5 was the only one of serotypes 2-9 to have a furin site, and yet it replicated to only moderate levels in cell culture, and replication in chickens could be detected only by seroconversion. Thus, the paradigm from studies with APMV-1 that virulence and systemic spread correlates with the presence of a furin site generally was not observed with APMV serotypes 2-9. Reverse genetics for different pathotypes of APMV-1 and for prototype strains of APMV-2, 3, and -7 have been developed and used for studying determinants of virus pathogenesis [38, [43] [44] [45] . Further characterization of virus replication and pathogenicity using reverse genetics will enhance our understanding of overall APMV pathogenesis. Patterns and influencing factor of synonymous codon usage in porcine circovirus BACKGROUND: Analysis of codon usage can reveal much about the molecular evolution of the viruses. Nevertheless, little information about synonymous codon usage pattern of porcine circovirus (PCV) genome in the process of its evolution is available. In this study, to give a new understanding on the evolutionary characteristics of PCV and the effects of natural selection from its host on the codon usage pattern of the virus, Patterns and the key determinants of codon usage in PCV were examined. METHODS: We carried out comprehensive analysis on codon usage pattern in the PCV genome, by calculating relative synonymous codon usage (RSCU), effective number of codons (ENC), dinucleotides and nucleic acid content of the PCV genome. RESULTS: PCV genomes have relatively much lower content of GC and codon preference, this result shows that nucleotide constraints have a major impact on its synonymous codon usage. The results of the correspondence analysis indicate codon usage patterns of PCV of various genotypes, various subgenotypes changed greatly, and significant differences in codon usage patterns of Each virus of Circoviridae.There is much comparability between PCV and its host in their synonymous codon usage, suggesting that the natural selection pressure from the host factor also affect the codon usage patterns of PCV. In particular, PCV genotype II is in synonymous codon usage more similar to pig than to PCV genotype I, which may be one of the most important molecular mechanisms of PCV genotype II to cause disease. The calculations results of the relative abundance of dinucleotides indicate that the composition of dinucleotides also plays a key role in the variation found in synonymous codon usage in PCV. Furthermore, geographic factors, the general average hydrophobicity and the aromaticity may be related to the formation of codon usage patterns of PCV. CONCLUSION: The results of these studies suggest that synonymous codon usage pattern of PCV genome are the result of interaction between mutation pressure and natural selection from its host. The information from this study may not only have theoretical value in understanding the characteristics of synonymous codon usage in PCV genomes, but also have significant value for the molecular evolution of PCV. Genetic information is transmitted from mRNA to protein in a mode of triplet codon. Each amino acid matches with at least one codon, at most six codons. The codons encoding the same amino acid is called synonymous codon. During biosynthesis of protein, usage probability of those synonymous codons is different. Some species or some genes are usually prone to use one or several particular synonymous codons. These codons are called preferable codons, which is called as codon bias. Usage bias of codons from various species has been studied, and it is found that during protein biosynthesis synonymous codons encoding amino acid is not used randomly [1] [2] [3] . Many studies have indicated that obvious bias exists between different genes from different species or the same species [4] [5] [6] . Usage bias of codons is influenced mainly by mutation bias, translation selection, secondary protein structure, replication and selective transcription, hydrophobia and hydrophilia of protein, and external environment [7] [8] [9] [10] [11] [12] [13] .
PCV belongs to genus of porcine circovirus, family of porcine circovirus. It has two genotype, PCV genotype I and PCV genotype II, and it is the smallest virus which has been discovered so far [14] . Among the different genotype, PCV genotype II infection and its related diseases have become one big problem across the globe for pig feeding, which threatens greatly to normal development of the industry of pig feeding. The PCV genome is a single-stranded negative circular DNA, and very small; full length of the PCV genotype I is only 1,759 bp, and PCV genotype II, 1,767 bp or 1,768 bp. The genome contains 11 open reading frames (ORF), among which, ORF1 encodes replication-associated proteins (Rep and Rep'); ORF2, structural proteins (viral capsid proteins, Cap); ORF3, toxicity-associated proteins, which can cause apoptosis [15, 16] . By analyzing the whole sequence of PCV genome, it is found that ORF2 has smaller selective pressure than ORF1, and more mutation. Nucleotide sequences among various strains in the same genotype are very conservative, their homology is over 90%, while similarity between nucleotide sequences from various strains respectively from the two genotypes is less than 80% [17, 18] . However, so far, studies have not related to usage of PCV codons. Explanation of codon usage pattern of PCV has significance on PCV evolution, gene prediction, gene classification, design of high expressed genes and viral vectors, and understanding of interaction between PCV and its host cells. Therefore, in this study, we first performed comprehensive analysis on codon usage pattern of PCV genome and the related factors affecting on codon usage. This study will play a major role in explanation of evolution process of PCV genome and further studies.
The characteristics of synonymous codon usage in PCV In order to investigate usage pattern of the PCV codons, we calculated various RSCU values of various codons from 28 different strains from different genotype. It can be seen from the three-dimension mesh plot of the analysis results of correspondence between 59 synonymous codons in the PCV genome (See Figure 1) , range of Z axis (f'1) is between 15 to 2.5, which indicates that synonymous codons usage in the PCV genome is not balanced, that is to say, among all the 59 synonymous codons, a part of codon is rarely used, while others have higher usage frequency. Additionally, it can be seen from Table 1 that among 18 preferable codons, 12 ones have the end base of G or U, only 6 have the end base of A or C, and so those codons with the end base of G or U are prone to use in PCV genome. Nevertheless, compared with other vertebrate DNA viruses, PCV genome has lower GC%, from 48.35% to 49.12%, with an average content of 48.61% and SD value of 0.19 (Table   2 ). And hence, the phenomenon, that in PCV genome GC content is lower while the condons with the end base of G is used in a way of bias, suggests that content of G or C as the end base of codons has effect on usage pattern of synonymous codons. Apart from this, we can also see from Table 2 , ENC values between PCV genomes has less fluctuation, with a range from 55.32 to 58.67, and an average value of 56.80 and SD value of 0.85, which indicates that codon bias of the PCV genome is stable.
Natural selection and mutation pressure has been considered to be two key factors which have effect on codon usage patterns of organisms [19] . In order to explore whether determinative factors for codon usage mutation in PCV is mutation pressure or natural selection, we compared correlation between A 3 %, U 3 %, G 3 %, C 3 %, GC 3 % and U 3 %, G 3 %, C 3 %, GC 3 % with correlation analysis ( Table 3 ). The analysis results show, except GC %, GC3% has marked correlation with A%, U%, G% and C%. This indicates that GC 3 % can reflect interaction between natural selection and mutation pressure to some extent.
In addition, the correlation between f'1,f'2 value and A %, U%, G%,C%, GC%, A 3 %, U 3 %, G 3 %, C 3 %, GC 3 % was analyzed ( Table 4 ). The results showed that significant correlations exist between synonymous codon usage pattern and nucleotide composition in PCV. This result further verifies the conclusion that during the shaping of synonymous codon usage pattern of PCV, Composition constraints play and very important role. 
In order to compare synonymous codon usage patterns between different PCV genomes from different genotype, we carried out analysis on codon usage of different PCV genotype with correspondence analysis (CA). In correspondence analysis, the first dimension variable f'1 and the second dimension variable f'2 can reflect 39.87 and 23.83 percent of total mutation respectively. We can see from Figure 2 , except for two strains of PCV genotype I in deviation from the cluster, other all the strains of PCV genotype II lie in the same cluster and overlap partly with each other. Obviously, PCV genotype I and PCV genotype II lay in two independent areas, which demonstrate that codon usage between the different PCV genotype is of great significance. Meanwhile, PCV genotype II-A and PCV genotype II-B almost lie in the same area, but they have tiny difference (Figure 2 ), which suggests that different sub-genotype from the same PCV genotype have difference in the aspect of codon usage.
From ENC values and corresponding relation distribution diagram of GC 3 % ( Figure 3 ) we can see, most points are near or under the theoretical curve, which suggests that apart from mutation pressure which influences on codon usage pattern in PCV, the usage pattern is also influenced by other factors. As parasitic organisms, virus's codon usage pattern would be subject to its host to some extent [6] . In this study, patterns of codon usage are compared in PCV and its natural host, and then found that there are high similarities between them. In detail, the high frequently used codons in the swine were also the non-preferred codons of PCV, such as CUG, GUG, CAG, AAU, GAC, GAA, UGU, AGA and UUU. Further more, all preferentially used codons of the genome of PCV and swine were all G-ended or U-ended codons (Table 1 ). These results suggest that the selection pressure from the host affects codon usage pattern of PCV. It is worthwhile to note that PCV genotype II have high similarities with swine than PCV genotype I ( Figure 4 ). In details, the values of RSCU in PCV genotype II and swine codon such as, AGA for Arg, GUG for Val, AGC for Ser, ACC for Thr were clearly different from that of PCV genotype I. It may be important one of Molecular mechanisms of infection and pathogenesis of PCV genotype II.
Researches in recent years indicated that dinucleotide biases can affect codon bias [20] . To study the possible effect of dinucleotide composition on codon usage of the PCV genome, the relative abundances of the 16 dinucleotides in genome of the 28 PCV strains were calculated ( Table 5 ). The result show that the occurrences of dinucleotides are not randomly distributed and no dinucleotides were present at the expected frequencies.
The relative abundance of CpG showed a strong deviation from the "normal range" (mean ± SD = 0.622 ± 0.029) and was markedly under represented. Correspondingly, the synonymous codon containing the CpG were inhibited because of CpG was present at the expected frequencies. In detail, low RSCU values were present in 7 of all 8 codon containing CpG and they are all not preferentially used codons (such as CCG, UCG, ACG, CGC, CGG, CGA, CGU) ( Table 1) . These observations indicate that the composition of dinucleotides also plays a key role in the variation found in synonymous codon usage among PCV. 
The correspondence analysis has been performed in order to compare the synonymous codon usage pattern between the viruses of Circoviridae. From which we could detect one major trend in the f'1 which accounted for 20.27% of the total variation, and another major trend in the f'1 for 15.46%of the total variation. A plot of the f'1 and the f'1 of each virus of Circoviridae was shown in Figure 5 . We can see from the plot that there were considerable differences for codon usage patterns among PCV, DCV, GCV, CoCV, CAV and BFDV. Concretely, PCV belong to the different genotype tends to come together. Moreover, DCV, GCV, CoCV and BFDV tend to come together and CAV was alone in separate area. It's clear that although different virus tends to come together, differences of synonymous codon usage pattern still exist between each virus. Therefore, the synonymous codon usage pattern of each virus of Circoviridae varies by the species of virus.
To investigate whether there is a correlation between the codon usage of PCV and geographic factor, 28 virus genes of PCV were divided into eleven groups according to obtained area, and correspondence analysis was also used. As can be seen from the plot, coordinate of virus isolates from different country is separated, and these relatively isolated spots tend to cluster into several groups according to the genotype ( Figure 6 ). All above 
During protein biosynthesis synonymous codon encoding amino acids are not used randomly, and some species or some gene always prefers to use of one or several particular synonymous codons, which is called as codon usage bias. Precious studies reveal that different genes from different species or the same one have obvious codon usage bias [21, 22] . Codon usage bias is influenced mainly by mutation bias [23, 24] , translation selection [25, 26] , secondary protein structure [20, 27] , replication and transcription selection [28] , secondary mRNA structure [29] , gene length [30] , tRNA abundance [31] , gene function and external environment [32] . However, most of these studies focus on some higher organism and many microorganisms with large genome and more genes, and there are few studies on virus with small genome and few genes or comparison between virus and host. Relatively, there are more reports on codon usage in genomes from viruses with great harm to mankind, such as SARS, human immunodeficiency virus, influenza virus A and hepatitis virus. PCV is a primary pathogen of postweaning multisystemic wasting syndrome (PMWS), which has threatened the development of pig feeding industry seriously because in recent year's occurrence of this disease has increased so as to bring about great economic loss in the world industry of pig feeding. Further studies on codon usage pattern in PCV have great significance on mutation pattern and molecular evolution of PCV. However, reports on codon usage pattern in PCV are rare, and this study is the first report. By comparison with reported DNA viruses such as Duck plague virus, Duck enteritis virus, Iridovirus, Herpesvirus [33] [34] [35] [36] , synonymous codon usage bias in the Table 4 The correlation analysis between the first two axes in CA and the nucleotide contents of PCV a b NS in superscript represent non-significant ***P-value < 0.001. **P-value < 0.01. *0.01 < P-value < 0.05 Figure 2 Correspondence analysis plot of relative synonymous codon usage in PCV. The first dimension variable f' 1 and the second variable f' 2 can reflect 39.87% and 23.83% of total mutation respectively. ). This suggests that low codon bias may result from increase in itself replication efficiency in PCV in order to adapt to replication system of its hosts. In this study, relation between main indices (f'1 and f'2)for the correspondence analysis on PCV usage cofon usage and its nucleotide composition (See Table 2 ) indicates, mutation pressure has a significant role in PCV codon usage. Other factors which can influence on PCV codon usage are also analysed and the initial results show that mutation pressure is the main factor to influence on PCV codon usage variation.
There were reports that natural selection can influence on synonymous codon usage pattern in viruses and the same conclusions are also obtained from this study. Three evidences support this conclusion. The first evidence is that PCV genome is GC3% -poor (average value = 47.08, SD = 2.88), but most of preferentially used codons are G/ T-ended codons. Meanwhile, average of A 3 % is higher than that of T 3 %, but among the codons which PCV prefers to using, there are only three preferable codons with the end base of A 3 % while six those with the end base of T 3 %. The second evidence is that the high similarities exist between PCV and its natural host. The third evidence is that CpG and the synonymous codon including it were inhibited. The three above evidences both state that natural selection is involved in formation of PCV synonymous codon usage pattern.
At present, according to pathogenicity, antigenicity and nucleotide sequence difference, PCV is divided into two genotypes, PCV genotype I and PCV genotype II, of which PCV genotype II includes various subtypes. From significance of PCV codon usage between different genotypes in Figure 1 , we can see that PCV codon bias may have association with genotypes. In addition to this, the results in this study also reveal that geological factor may almost have relation with codon usage in PCV. In some reports, gene length has certain correlation with codon usage [30] . Similarly, in some viruses, gene length has no effect on codon usage [22] . With correlation analysis we surveyed codon usage bias and gene length in PCV, and it is found that in these viral genes, codon usage bias has no notable correlation with gene length (Spearman, r = 0.075, p > 0.1). The results indicate that PCV gene length has no effect on synonymous codon usage. Other factors, including GRAVY and aromaticity may also significantly influence codon usage of PCV
Taken together, the codon usage patterns of PCV possibly result from interactions between natural selection and mutation pressure. These results not only provide an insight into the variation of codon usage pattern among the genomes of PCV, but also may help in understanding the processes governing the evolution of PCV.
The information of 28 PCV genomes, including the genotype, length value, the isolated area and GenBank accession numbers of these strains was listed in the Table 6 . In order to compare the differences between PCV and its host, twenty swine gene were gained and detailed information of these genes is listed in Table 7 . In addition, to compare the codon usage patterns among different viruses, twenty-five viral genomes of Circoviridae were taken into account ( Table 8 ). All of the sequences were downloaded from NCBI (http:// www.ncbi.nlm.nih.gov/Genbank/). Each general nucleotide composition (T%,A%,C% and G%) and each nucleotide composition in the third site of codon (T 3 %,A 3 %, C 3 % and G 3 %) in PCV coding sequence were calculated by biosoftware DNAStar7.0 for windows.
In order to eliminate the influence of amino acid composition on codon usage and directly reflect the usage Figure 4 Compare the codon the codon usage pattern among PCV genotype I, PCV genotype II and swine. characteristics of codon, the study evaluates synonymous codon usage bias through statistical estimation on relative synonymous codon usage frequency (RSCU) [37] . RSCU value refers to the ratio between the usage frequency of one codon in gene sample and expected frequency in the synonymous codon family. If the synonymous codon usage of one amino acid has no preferences, that is, codon usage frequency is close to expected frequency, the RSCU values of codons are equal to 1; if a codon RSCU value is greater than 1, the codon use frequency is higher than expected frequency, whereas it is less than expected value. The definition on a single gene codon bias is mainly based on effective number of codons (ENC) [38] . ENC values can reflect the preference degree of synonymous codon non-equilibrium use in codon family. The range of ENC values is from 20 (each amino acid only uses one codon) to 61 (all synonymous codons are equivalently used). ENC value is closer to 20, the degree of being used non-randomly is higher, and the bias is stronger. It is generally believed that the genes are provided with significant codon bias when ENC ≤ 35. The values of RSCU and ENC were obtained by codonW program.
A comparison of actual and expected dinucleotide frequencies of the 16 dinucleotides in coding region of PCV genomes was also undertaken using SPSS 17.0.
Correspondence analysis is mainly used for detecting the changes of codon RSCU values in genes [39] . It is an effective multivariate statistical method of studying the internal relation between the variables and samples, and it is successfully applied to the study of codon. In correspondence analysis, all genes in samples are distributed in a 59-dimensional (59 justice codons, in addition to the stop codon, Met, and Trp) vector space, each gene is described with 59 (f' 1 , f' 2, ..., f' 59 ) variables, the results can be applied for finding out the major factors affecting codon usage bias in genes [40, 41] . This was done using the CodonW program.
Correlation analysis of PCV was used to identify the relationship between nucleotide composition and synonymous codon usage pattern [42] . All statistical processes were carried out by with statistical software SPSS17.0 for windows. Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2 BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans. Dengue is the most important arthropod-borne viral disease in humans and an increasing public health concern in tropical and subtropical regions of the world. Approximately 50-100 million cases of dengue fever (DF) and 500,000 cases of dengue hemorrhagic fever (DHF) occur every year, and 2.5 billion people are at risk of dengue infection globally [1, 2] . Dengue infection may lead to fever, headache and joint pain in milder cases but may also lead to the more severe life-threatening DHF/dengue shock syndrome (DSS) has plasma leakage, thrombocytopenia, and hemorrhagic manifestations, possibly leading to shock [3, 4] .
Dengue virus (DENV) is positive-sense single-stranded RNA virus of approximately 11 kb genome of the genus Flavivirus, a family Flaviviridae. It has four genetically and antigenically related viral serotypes: DENV-1, -2, -3 and -4. Flaviviruses encode a single polyprotein processed by host and viral protease to produce three structural proteins, including capsid (C) protein, precursor membrane/membrane (prM/M) and envelope (E) protein, and seven nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 [5] . The E protein, a 53 kDa glycoprotein important for attachment, entry, and viral envelope fusion, can bind to cellular receptors and induce neutralizing antibodies [6, 7] .
The DENV consists of an icosahedral ectodomain, containing 180 copies of the E protein [8] . E protein monomer contains three structural and functional domains [9, 10] . E protein domain I (E-DI) is a central b-barrel structure. E protein domain II (E-DII) is organized into two long finger-like structures and contains the flaviviruses conserved fusion loop. E protein domain III (E-DIII) has an immunoglobulin-like fold and may mediate interactions between the virus and the receptors on the host cell [11] . Studies of the biological characteristics and epitope specificities of mouse monoclonal antibodies (mAbs) have elucidated the antigenic structure of flavivirus E proteins [12] [13] [14] [15] . Serotype-specific mAbs with neutralizing activity against DENV-2 have been found to be located on the lateral ridge of E-DIII and the subcomplex-specific mAbs recognized A-strand of E-DIII [14, 16, 17] . Antibody-mediated neutralization has been found to alter the arrangement of viral surface glycoproteins that prevent cells from viral attachment [16] . Binding of an antibody to the viral surface can interfere with virus internalization or membrane fusion [6] .
Primary DENV infection is believed to provide lifelong immunity against re-infection with the same serotype [18, 19] . However, humoral immune responses to DENV infection are complex [20] [21] [22] , and may exacerbate the disease during heterologous virus infection [18, 19] . Antibody-dependent enhancement (ADE) in dengue pathogenesis results from the increase in the efficiency of virus infection in the presence of nonneutralizing or sub-neutralizing concentrations of anti-E or anti-prM immunoglobulins [21, 23] . The attachment of antibody-virus complex to such Fcc receptor-bearing cells as monocytes and macrophages can lead to an increased virus replication [18, 24, 25] .
A better understanding of the neutralizing epitopes may facilitate the generation of new antibody-based therapeutics against DENV infection. In this study, we generated several mAbs against DENV-2. We found that serotype-specific anti-E-DIII mAbs played an important role in the neutralization of virus infectivity. Studies of the neutralizing epitopes found the strongest mAbs to be DB32-6 and DB25-2, both DENV-2 serotype-specific antibodies. These two mAbs recognized the A-strand of E-DIII at residues K310 and E311, respectively. Humanized DB32-6 mAb efficiently neutralized DENV-2 infection in a therapeutic mouse model and its variant version prevented enhancing activity.
BHK-21 cells were grown at 37uC with 5% CO 2 in Minimal Essential Medium (MEM, Gibco) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco) and 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B (Antibiotic-Antimycotic, Gibco). Aedes albopictus C6/36 cells were grown at 28uC in 1:1 Mitsuhashi and Maramorosch (MM) insect medium (Sigma-Aldrich)/Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% FBS and 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B (Antibiotic-Antimycotic, Gibco). The four DENVs (DENV-1 Hawaii, DENV-2 16681, DENV-3 H87 and DENV-4 H241) were provided by Dr. Duane J. Gubler from the Centers for Disease Control and Prevention, Fort Collins, U.S.A. The various DENV-2 strains including New Guinea-C (NGC), NGC-N (mouse-adapted neurovirulent), PL046 and Malaysia 07587 were used in this study [26, 27] . These viruses were passaged in C6/36 cells.
Anti-DENV-2 mAbs were generated according to previously described procedures [28, 29] . Female 4-to 6-week-old BALB/c mice were immunized with 10 7 plaque-forming units (pfu) of DENV-2 (16681). The DENV-2 was purified from viral culture supernatant using 4G2 (an anti-E protein mAb)-coupled protein G-Sepharose 4 Fast Flow gel. After four inoculations with the same concentration of antigens, the splenocytes from the immunized mouse spleen were harvested and then fused with mouse myeloma NS-1 cells. Fused cells were cultured in DMEM supplemented with 15% FBS, HAT medium and hybridoma cloning factor (Roche) in 96-well tissue culture plates. Two weeks after fusion, culture supernatants were screened by ELISA. Selected clones were subcloned by limiting dilutions. Hybridoma clones were isotyped using a commercially isotyping kit (Southern Biotech) by ELISA. Ascites fluids were produced in pristine-primed BALB/c mice. mAbs were affinity-purified by standard protein G-Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences) according to manufacturer's directions.
Screening of mAbs against DENV1-4 by ELISA C6/36 cells at 80% confluency in 96-well plates were infected with DENV-1 to -4 to produce viral antigens. These cells were then harvested 5-7 days after infection. One mg/ml mAbs was added to the plates and incubated at room temperature (RT) for 1 h. After washing with PBS, horseradish peroxidase (HRP)-conjugated antimouse IgG (Jackson ImmunoResearch Laboratories) was incubated at RT for 1 h. Finally, plates were incubated with peroxidase substrate o-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich). Reaction was stopped with 3N HCl and optical density was measured using a microplate reader set at 490 nm.
C6/36 cells were harvested after viral infection. Lysates or expression proteins were collected. Cell extracts were mixed with sample buffer (Bio-Rad Laboratories). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Hybond-C Super). Nonspecific antibody-binding sites were blocked with 5% skimmed milk in PBS, and membranes were incubated with primary antibody. Blot was then treated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories) and then developed with enhanced chemiluminescence reagents (ECL, Thermo Fisher Scientific).
Immunofluorescence assay (IFA) BHK-21 cells at 80% confluency were infected at a multiplicity of infection (MOI) of 0.5 with DENV-2 (16681). After 2 days infection, the cells were fixed with 1:1 methanol/acetone for 10 min at 220uC. Cells were blocked using PBS supplemented with 1% BSA for 1 h at RT. Primary anti-DENV antibodies or control antibodies (normal mouse IgG, Jackson ImmunoResearch Laboratories) were diluted (1:250) in block solution for 1 h at RT. Secondary antibody, FITC-conjugated goat anti-mouse IgG
Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients.
Humanized mAb against Dengue Virus www.plosntds.org (Jackson ImmunoResearch Laboratories) was diluted to 1:250 and supplemented with DAPI (Invitrogen) diluted 1:2,000 for 1 h at RT. The binding activity of antibodies to the DENV-2-infected or transfected cells were observed and photographed through a fluorescence microscope.
The expression constructs of E-DI-II and E-DIII were cloned into the pET21a vector (Merck). The E-DI-II, comprising amino acids 1-295 of the E protein, was tagged to flag and hexahistidine at the C terminus for affinity purification. The E-DIII, comprising amino acids 295-400 of the E protein, was tagged to flag and hexahistidine, too. The plasmids were expressed in Escherichia coli strain BL21 (DE3). The recombinant proteins E-DI-II and E-DIII were analyzed using 12% SDS-PAGE by Western blot analysis. The DNA fragments corresponding to E-DI-II and E-DIII were also cloned into a mammalian expression vector, pcDNA3.1 (Invitrogen). The expression constructs of DENV-2 C, prM, prM-E, E, NS1, NS2A, NS2B, NS2B-3, NS3, NS4A, NS4B and NS5 were obtained from Dr. Y.-L. Lin [30] . Transient expression of DENV-2 proteins in BHK-21 cells was transfected by PolyJet (SignaGen Laboratories) according to manufacturer's recommendations and then to test specificity of mAbs.
In vitro neutralization assay (i) For the plaque reduction neutralization test (PRNT), eight 3fold serial dilutions of mAbs (from 200 mg/ml to 0.1 mg/ml) were mixed with an equal volume of 200 pfu of DENV-2 (16681) and incubated at 4uC for 1 h. The final concentration of mAbs at the PRNT ranged from 100 to 0.05 mg/ml. Antibody-virus mixtures (100 ml) were added to BHK-21 cells at 80%-90% confluency in 12-well plates. After absorption of virus for 2 h, BHK-21 cells were washed and 2 ml of 1% (w/v) carboxyl methyl cellulose (Sigma-Aldrich) in MEM plus 2% (v/v) FBS was layered onto the infected cells. After incubation at 37uC for 5 to 7 days, the viral plaque that had formed on the cell monolayer was fixed by 1 ml 3.7% formaldehyde (Sigma-Aldrich) at RT for 1 h. The cells were then stained with 1% crystal violet. Percentage of plaque reduction was calculated as: %Inhibition = 1002[(plaque number incubated with mAb/plaque number without mAb)6100]. (ii) For flow cytometry, serial dilutions of DB32-6 mAb were incubated with DENV-2 (16681, NGC, PL046 and Malaysia 07587) at MOI of 0.5 at 4uC 1 h before adding BHK-21 cells. After 2 h absorption, the monolayers were washed and incubated with MEM (Gibco) plus 2% (v/v) FBS at 37uC for 2 days. The cells infected with DENV-2 were washed and fixed with 3.7% formaldehyde at 4uC for 10 min. They were then permeabilized in PBS supplemented with 1% FBS, 0.1% saponin (Sigma) at 4uC for 10 min. For staining, cells were incubated with 4G2 at a concentration of 1 mg/ml at 4uC for 30 min. After two washes, R-Phycoerythrin (PE)conjugated AffiniPure F(ab9) 2 fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratorie) diluted 1:250 was then added at 4uC for 30 min followed by two washes and analyzed by flow cytometry. % Infection = (the intensity of cells incubated with mAb/without mAb)6100.
This study was carried out following strict guidelines from the care and use manual of National Laboratory Animal Center. The protocol was approved by the Committee on the Ethics of Animal Experiments of Academia Sinica. (Permit Number: MMi-ZOOWH2009102). The mice were killed with 50% CO 2 containing 50% O 2 . All efforts were made to minimize suffering. (i) Breeder mice of the ICR strain were purchased from the Laboratory Animal Center National Taiwan University College of Medicine. Purified mAbs at doses of 1, 10 and 100 mg/ml were incubated with 1610 4 pfu (25-fold LD 50 ) of DENV-2 (16681) at 4uC for 30 mins. Two-day-old suckling mouse brain was inoculated with 20 ml of the reaction mixture by intracranial (i.c.) injection. Survival rate and signs of illness, including paralysis, were observed daily for 21 days following challenge. In post-exposure therapeutic experiments, mice were passively injected with 5 mg of mAb via i.c. route after 1 day of infection. (ii) Stat1-deficient mice (Stat1 2/2 ) [31] were bred in the specific-pathogen-free animal facility at the Institute of Biomedical Sciences, Academia Sinica. Mice were challenged intraperitoneally with 1610 5 pfu (300-fold LD 50 ) of DENV-2 (NGC-N) in 300 ml of PBS and simultaneously injected intracranially (i.c.) with 30 ml of PBS. In prophylaxis experiments, antibodies (100 mg per mouse, intraperitoneally) were administered 1 day before infection and administered on day 0, 1, 3, 5 and 7 after infection. In postexposure therapeutic experiments, antibodies (100 mg per mouse, intraperitoneally) were administered on day 1, 3, 5 and 7 after infection.
The phage display biopanning procedures were performed according to previous reports [28, 32] . Briefly, an ELISA plate was coated with mAbs at 100 mg/ml. Samples of 100 ml diluted mAb were then added to wells and incubated at 4uC for 6 h. After washing and blocking, the phage-displayed peptide library (New England BioLabs, Inc.) was diluted to 4610 10 pfu of phage and incubated for 50 mins at RT. After washing, bound phage was eluted with 100 ml 0.2 M glycine/HCl (pH 2.2) and neutralized with 15 ml 1 M Tris/HCl (pH 9.1). The eluted phage was amplified in ER2738 for subsequent rounds of selection. The phage was titrated onto LB medium plates containing IPTG and X-Gal. The biopanning protocol for the second and third rounds was identical to the first round except for the addition of 2610 11 pfu of amplified phage for biopanning.
An ELISA plate was coated with 50 ml mAbs 50 mg/ml. After washing and blocking, amplified phage diluted 5-fold was added to coated plate and incubated at RT for 1 h. After washing, 1:5000 diluted HRP-conjugated anti-M13 antibody (GE Healthcare) was added at RT for 1 h. OPD developed and was terminated with HCl. Optical density was measured at 490 nm.
We used the recombinant expression plasmid pCBD2-2J-2-9-1 [33] to generate VLP mutants. Various VLP mutants were generated by site-directed mutagenesis derived from pCBD2-2J-2-9-1 as a template. PCR was performed using pfu ultra DNA polymerase (MERCK) and all mutant constructs were confirmed by sequencing. BHK-21 cells at 80%-90% confluency in 48-well plates were transfected with plasmids of various VLPs. After two days transfection, the cells were washed with PBS supplemented with 1% FBS, fixed with 3.7% formaldehyde at 4uC for 10 min, and then permeabilized in PBS supplemented with 1% FBS, 0.1% saponin (Sigma-Aldrich) at 4uC for 10 min. For staining, cells were incubated with mAbs at 4uC for 30 min, DB32-6, DB25-2, 3H5 and mix mAbs (4G2, DB2-3, DB13-19, DB21-6 and DB42-3) at a concentration of 0.1, 1, 1 and 1 mg/ml, respectively. After being washed twice, R-Phycoerythrin (PE)-conjugated AffiniPure F(ab9) 2 fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories) diluted to 1:250 was then added at 4uC for 30 min and analyzed by flow cytometry. Relative recognition was performed according to previously described procedures and calculated as [intensity of mutant VLP/intensity of WT VLP] (recognized by a mAb)6[intensity of WT VLP/intensity of mutant VLP] (recognized by mixed mAbs) [34] .
Total RNA was extracted from hybridoma cells using the TRIzol reagent (Invitrogen) and mRNA was isolated with the NucleoTrap mRNA Mini Kit (Macherey-Nagel GmbH & Co. KG.). Purified mRNA was reverse transcribed using oligo (dT) as a primer in a ThermoScript RT-PCR system (Invitrogen). The variable heavy-and light-chain domains (V H and V L ) were amplified from the cDNA product by PCR with a variety of primer sets [35, 36] . The PCR products were cloned using the TA kit (Promega) and the V H and V L sequences were determined by DNA sequencing. Software Vector NTI was used for sequence analysis. From these sequences, the framework regions (FRs) and complementarity-determining regions (CDRs) were analyzed by comparing them with those found in the Kabat database and the ImMunoGeneTics database [37] .
Two human genes, GenBank accession DI084180 and DI075739, were 94.7% and 92.2% identical to DB32-6 V H and V L , respectively. Humanized DB32-6 V H consisted of the modified FR1 to FR4 from the accession DI084180 gene, and the CDR1 to CDR3 of the DB32-6 V H , respectively, while humanized DB32-6 V L consisted of the modified FRs from the accession DI075739 gene and the CDRs of the DB32-6 V L . Both were synthesized (GENEART) and amplified by PCR using pfu Turbo DNA polymerase (EMD Bioscience). The resulting V H was cloned into modified expression vector pcDNA3.1 (Invitrogen) with a signal peptide and human IgG1 constant region, while the V L was cloned into modified expression vector pSecTag (Invitrogen). We generated a variant of humanized DB32-6 (hDB32-6 variant) in which leucine residues at positions 1.2 and 1.3 of C H 2 domain were substituted with alanine residues [38] . The V H and V L plasmids were cotransfected into CHO-K1 cells and selected by G 418 and puromycin for 2-3 weeks. Transformed cells were limit diluted in 96-well plates. After two weeks, stable clones produced humanized antibodies in the McCoy's 5A medium (Sigma-Aldrich), as identified by ELISA. Humanized antibodies were produced by CELLine AD 1000 (INTEGRA Biosciences) according to manufacturer's directions.
Murine and humanized DB32-6 mAbs affinity analysis for E-DIII of DENV-2 was performed by surface plasmon resonance (BIAcore X, Biacore, Inc). Purified E-DIII (50 mg/ml) was immobilized on a CM5 sensor chip (Biacore, Inc) and injected at a flow rate of 10 ml/min. The mAbs were diluted to 4, 2, 1, 0.5, 0.25 and 0 nM in HBS-EP buffer (Biacore, Inc). mAbs were injected at a flow rate of 30 ml/min for 3 min and then allowed to dissociate over 1.5 min. Regeneration of the surface was achieved with an injection of 10 mM glycine HCl/0.2 M NaCl (pH 3.0) before each mAb injection. The data were analyzed by the BIAevaluation software with a global fit 1:1 binding model.
Serial dilutions of mAbs were mixed with DENV-2 (16681) at MOI of 1 at 4uC for 1 h. The 100 ml mixture were incubated with 5610 4 K562 cells [39] in 96-well plates at 37uC for 2 h. After infection, the cells were washed and incubated with RPMI (Gibco) plus 2% (v/v) FBS at 37uC for 2 days. The cells were washed with PBS supplemented with 1% FBS, fixed with 3.7% formaldehyde, and permeabilized in PBS supplemented with 1% FBS, 0.1% saponin (Sigma) at 4uC for 10 min. For staining, cells were incubated with DB42-3 at a concentration of 3 mg/ml at 4uC for 30 min. After two times washes, R-Phycoerythrin (PE)-conjugated AffiniPure F(ab9) 2 fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:250 was then added at 4uC for 30 min follow by two times wash steps and analyzed by flow cytometry.
Survival rate was expressed using Kaplan-Meier survival curve, and log rank test was used to determine the significant differences. For body weight change experiments, paired t-test was used to determine the significant differences, * P,0.05, ** P,0.01.
Seventeen mAbs with high reactivity against E protein of DENV-2 were generated after immunization of mice with DENV-2 strain 16681. We identified 17 mAbs belonging to the IgG isotype that reacted with DENV-2-infected cells but not with mock-infected cells using immunofluorescence assay (IFA) ( Figure  S1 ) and ELISA ( Figure 1A ). 4G2 was a pan-flavivirus mAb that could recognize the fusion loop of E-DI-II, and 3H5 (ATCC HB46) was a DENV-2 serotype-specific mAb that could recognize the lateral ridge of E-DIII [12, 17, 40] . Both 4G2 and 3H5 were used as positive controls (Figure 1 ). The specificities of the mAbs recognized as the four DENVs were further confirmed by ELISA and Western blotting (Figures 1A-1B and Table 1 ). Based on our Western blot analysis using a nonreducing condition, 14 of the mAbs recognized E protein (53 kDa) ( Figure 1B ). Three mAbs could not be identified by Western blotting. In order to identify the target proteins of these mAbs, we prepared BHK-21 cells transfected with plasmids expressing DENV-2 C, prM, prM-E, E, NS1, NS2A, NS2B, NS2B-3, NS3, NS4A, NS4B and NS5 ( Figure S2 ). Results indicated that three mAbs (DB21-6, DB22-4 and DB36-2) recognized E protein ( Figure 1C ). The identification and characterization of the 17 mAbs are summarized in Table 1 .
To characterize the antigenic structure of the DENV E protein and to study the relationship between epitopes and their neutralizing potency, we constructed and expressed the recombinant E-DI-II and E-DIII from DENV-2 in E. coli and mammalian expression systems. Western blot analysis and IFA showed that, of the 17 mAbs recognizing E protein, 10 mAbs (DB2-3, DB9-1, DB13-19, DB21-6, DB22-4, DB23-3, DB27-3, DB33-3, DB39-2 and DB42-3) targeted to E-DI-II and 2 mAbs (DB25-2 and DB32-6) recognized E-DIII ( Figures 1D-1E and Table 1 ). However, 5 mAbs could not be identified by these two assays.
We evaluated the ability of mAbs to inhibit DENV-2 infection in BHK-21 cells using a plaque reduction neutralization test (PRNT). Ten mAbs had neutralizing activity with 50% PRNT (PRNT 50 ) concentrations ranging from 0.14 mg/ml to 33 mg/ml (Table 1) . DB32-6 was found to be a DENV-2 serotype-specific mAb against E-DIII ( Figures 1A, 1B and 1E) and was the most efficient at neutralizing DENV-2 infection at a PRNT 50 concentration of 0.14 mg/ml (Figure 2A ). In addition, it could completely inhibit the infection at a lower concentration of 1.2 mg/ml (Figure 2A) . The mAb DB25-2 was found to be a DENV-2 serotype-specific mAb against E-DIII ( Figures 1A, 1B Humanized mAb against Dengue Virus www.plosntds.org and 1E) and to neutralize DENV-2 at a PRNT 50 titer of 1.2 mg/ ml (Figure 2A) . These findings indicate that serotype-specific mAb DB32-6 against E-DIII was the most potent in neutralizing DENV infection. Some serotype-specific mAbs, such as DB2-3 and DB23-3 against E-DI-II and DB25-2 against E-DIII showed strong neutralizing activity. Many complex reactive mAbs showed moderate-to-poor neutralizing activity (Table 1) . mAbs prevent DENV-2-induced lethality in suckling mice and Stat1 2/2 mice Two different mouse models were used to assess whether DB32-6 could efficiently protect mice against DENV-2 challenge.
Protection assay of neutralizing mAbs was performed with ICR strain 2-day-old suckling mice [41] . Mice were inoculated intracerebrally with 20 ml of DENV-2-mAb mixture containing 1610 4 pfu (25-fold LD 50 ) of DENV-2 with neutralizing mAbs at concentrations of 1, 10 or 100 mg/ml. Generally, the nonneutralizing antibody normal mouse IgG (NMIgG) treated group showed paralysis, ruffling, and slowing of activity around 6 to 9 days. This was followed by severe sickness leading to anorexia, asthenia and death within 9 to 17 days ( Figures 2B and 2C) . In contrast, mAbs DB32-6 at a concentration of 10 mg/ml protected 93% of the mice from the lethal challenge of DENV-2 ( Figure 2B ). mAbs 3H5, DB23-3, DB2-3 and DB25-2 had survival rates of Humanized mAb against Dengue Virus www.plosntds.org 75%, 76%, 72% and 71%, respectively. DB42-3 and DB13-19 had survival rates of 46% and 28%, respectively ( Figure 2B ). The neutralizing mAbs showed a significant delay of the onset of paralysis and death relative to the NMIgG. To evaluate the therapeutic potential of the highly protective mAb DB32-6, we administered 100 mg/ml or 1 mg/ml to infected suckling mice. The survival rates for DB32-6 at 100 mg/ml or 1 mg/ml were 100% and 89%, respectively ( Figure 2C ). In comparison, 3H5 showed 82% and 40% survival rates at 100 mg/ml or 1 mg/ml, respectively. Stat1 2/2 mice, which lack a transcription factor involved in interferons (IFNs) signaling were sensitive to lethality induced by DENV-2 infection [27, 31] . To test the potential therapeutic effects of the strongest neutralizing mAb DB32-6, we challenged Stat1 2/2 mice at a strict condition with 1610 5 pfu (300-fold LD 50 ) of DENV-2 (NGC-N). After 21 days observation, mice showed ruffled fur, mild paralysis and lost approximate 20% of their initial body weight at day 7 after infection (P,0.01), and then they all died within 7-18 days of infection ( Figures 2D and 2E ). In the prophylaxis experiments, antibodies (100 mg per mouse, intraperitoneally) were administered 1 day before infection and at day 0, 1, 3, 5 and 7 after infection. The DB32-6 prophylactically treated group showed 100% protection ( Figure 2D left) . Even in the postexposure therapeutic experiments, the DB32-6 treated mice had a survival rate of 50% (Figure 2E left) . The mAb DB32-6 had excellent neutralizing activity against different DENV-2 strains (16681 and NGC-N) in two mouse models.
To further evaluate whether the strongest mAb DB32-6 could broadly neutralize the diverse DENV-2 strains, we infected BHK-21 cells with four different DENV-2 Southeast Asian genotype strains, 16681, NGC, PL046 and Malaysia 07587. Remarkably, mAb DB32-6 exhibited effective neutralization against various DENV-2 strains ( Figure S3 ).
Epitopes recognized by neutralizing antibodies have been identified in all three domains of the E protein [42] [43] [44] . To find out more about the epitopes of these neutralizing antibodies, we used phage display [29, 45] to identify the neutralizing epitopes. After three rounds of phage display biopanning, the phage titers were increased to 85-fold (DB32-6) and 331-fold (DB25-2) compared to the phage display biopanning results from the first round ( Figure 3A ). Individual phage clones from the third round of biopanning were randomly selected. ELISA was performed to determine whether the mAbs could specifically recognize selected phage clones. Of 20 selected phage clones, 17 and 18 clones had significant enhancement of binding activity to DB32-6 and DB25-2, respectively ( Figure 3B ). The selected phage clones PC32-6 and PC25-14 were specific and dose dependently bound to DB32-6 and DB25-2, respectively. They did not react with control NMIgG ( Figure 3C) .
The 17 immunopositive phage clones that were highly reactive with DB32-6 were amplified and phage DNA was isolated for DNA sequencing. All of the phage clones displayed 12 amino acid (aa) residues ( Figure 3D left) . Phage-displayed peptide sequences selected by DB32-6 had the consensus motifs of histidine (H)-lysine (K)-glutamic acid (E)-tryptophan (W)/tyrosine (Y)-histidine (H) (Figure 3D left) . Similarly, 17 immunopositive phage clones selected by DB32-6 using phage library displayed 7 amino acid residues, which contained the consensus motif H-K-E-W/Y-H ( Figure 3D left) . Interestingly, all phage-displayed peptides selected by DB32-6 and DB25-2 contained lysine (K) and glutamic acid (E), respectively ( Figure 3D ). To further confirm the neutralizing epitopes, we developed various E protein epitope-specific variants VLPs and screened lossof-binding VLP mutants for identification of critical recognition residues. Using this strategy, we found that DB32-6 lost its VLP binding activity when the residue K310 in the A-strand of E-DIII was changed to alanine (K310A) or glutamine (K310Q) (Figure 4A  left) . Similarly, DB25-2 lost its VLP binding activity when E311 was changed to arginine (E311R) in the A-strand of E-DIII ( Figure 4A right). Both the critical recognition residues K310 and E311 were located in the A-strand of E-DIII (Figures 4B and 4C) . We found that mAb 3H5 recognized residues K305, E383 and P384 ( Figure  S4 ), as previously reported [17, 20] . Notably, even the adjacent residues (K310 and E311) induced antibodies with different levels of neutralizing activity. By comparing the amino acid sequences of E proteins from representing genotypes of DENV-2 (Table S1), we found residues K310 and E311 in E-DIII of the different genotypes (Southeast Asian, West African and American) ( Figure S5 ). Our data further showed epitopes in the A-strand of E-DIII were important for inducing neutralizing antibodies.
Murine mAbs have been shown to have limited clinical use because of their short serum half-life, inability to trigger human effector functions and the production of human anti-murine antibodies (HAMA) response [46] . mAbs have been humanized by grafting their CDRs onto the V H and V L FRs of human Ig molecules [47] . DB32-6 was the most potent mAb against DENV-2 and showed potential as a therapeutic antibody. To develop humanized mAbs, we sequenced V H and V L segment of the neutralizing mAbs from hybridoma cell lines. The CDRs of DB32-6 were grafted onto human IgG1 backbone to create humanized DB32-6 (hDB32-6) ( Figure 5A ). The hDB32-6 was expressed in CHO-K1 cells and purified from culture supernatants. Both hDB32-6 and mDB32-6 were able to against DENV-2 ( Figure 5B ). The hDB32-6 maintained the specificity of murine DB32-6 (mDB32-6). Furthermore, we established stable clones of hDB32-6. After selection, mAbs hDB32-6-30, hDB32-6-48 and hDB32-6-51 were found to have highly binding activity ( Figure 5C ). Comparing to these mAbs, we found hDB32-6-48 to have the highest production rate in cells. mAb hDB32-6-48 was dose-dependent against DENV-2 and E-DIII ( Figure 5D ). The affinity was analyzed by surface plasmon resonance. The mDB32-6 and hDB32-6-48 bound to E-DIII of DENV-2 with a similar affinity (0.12 nM and 0.18 nM, respectively) ( Figure 5E ). The results revealed that hDB32-6 maintained the same binding affinity to the E protein as mDB32-6. mAb hDB32-6 protected mice from DENV-2-induced mortality
We established a suckling mice model to determine the protective activity of mDB32-6 and hDB32-6. To evaluate Humanized mAb against Dengue Virus www.plosntds.org therapeutic effect of mAbs, we administered 5 mg of mAb at day one after 1610 4 pfu (25-fold LD 50 ) of DENV-2 (16681) infection. Through 21 days of observation, groups treated with mDB32-6, hDB32-6-48 and 3H5 mAbs were found to have survival rates of 96%, 94% and 56%, respectively ( Figure 6 ). However, none of the mice in control antibody normal human IgG (NHIgG)-treated group survived (Figures 6) . These results demonstrate that both mDB32-6 and hDB32-6 have excellent neutralizing activity against DENV-2. mAb hDB32-6 variant eliminate ADE phenomenon When developing the antibody-based therapy, ADE phenomenon is a major cause for concern in dengue pathogenesis because it might enhance DENV infection. Modification of Fc structure in an antibody can prevent Fcc receptors binding and lead to eliminate ADE [38, 39, 48] . We generated a variant of humanized DB32-6 (hDB32-6 variant) to prevent Fcc receptors binding while maintaining DENV neutralizing capability without enhancing infection (Figure 7) . The hDB32-6 variant retained the same neutralizing activity as unmodified mAb mDB32-6 at high concentrations (100 mg/ml and 10 mg/ml) but was completely devoid of enhancing activity at low concentrations (1 mg/ml and 0.1 mg/ml) (Figure 7) . The hDB32-6 variant eliminated the ADE phenomenon and holds great potential for being developed into therapeutic antibodies for the prevention and treatment of DENV-2 infection.
mAbs of DENV have served as powerful research tools for antiviral development and pathological investigations. Here, we newly generated and characterized 17 mAbs with high reactivity against E protein of DENV-2. Several mAbs had potent neutralizing activity. The neutralizing epitopes were identified using a combination of strategies, including phage display, computational structure analysis [49] , and high-throughput epitope mapping of VLPs. From these results, the A-strand of E-DIII was found to be important in neutralizing DENV-2 than the Humanized mAb against Dengue Virus www.plosntds.org lateral ridge of E-DIII. mAb DB32-6 which had the strongest neutralizing activity against various strains of DENV-2 was humanized and modified to abrogate the ADE phenomenon. The mAb DB32-6 was demonstrated to increase the survival rate in two mouse models even after DENV-2 infection.
Based on previous epitope mapping results, several epitopes have been shown to elicit strong neutralizing antibodies against individual flaviviruses that situated in E-DIII [14, 50] . Investigation of neutralizing epitopes on the E proteins may provide the framework for a detailed understanding of both specific mechanisms of the viral infection as well as the identification of the specific DENV domain that attaches to a cellular receptor. Phage display is useful in the identification of B-cell epitopes, including linear [32, 51] and conformational epitopes [29, 45] . However, these epitopes need to further elucidation using other methods. Combining different strategies provided a fast and reliable evidence for identifying epitopes (Figures 3 and 4) . To date, few mAbs possess better neutralizing activity than 3H5, which has been shown to bind to residues K305, E383 and P384 at the lateral ridge of E-DIII [17, 20] . DB32-6 had higher neutralizing activity than 3H5. Neutralizing epitope of DB32-6 was mapped on K310 residue in A-strand of E-DIII (Figure 4 ). Neutralizing epitope of another mAb DB25-2 was mapped on E311 residue in A-strand of E-DIII, too ( Figure 4) . These serotype-specific neutralizing epitopes located in the A-strand of E-DIII induced stronger neutralizing activity than those located on the lateral ridge of E-DIII. We aligned different DENV-2 genotypes and found that the K310 and E311 were frequently observed in DENV-2 ( Figure S5 ). The K310 may be important to DENV-2. Thus by binding DB32-6 to K310, it lead to dramatic neutralized DENV-2. To determine whether DB32-6 can neutralize diverse genotypes of DENV-2 is a critical step in evaluating the potential of therapeutic development in the future.
Previous studies have shown that the strongly neutralizing mAb, subcomplex-specific 1A1D-2 and cross-reactive 9F12 recognized residues at K305, K307 and K310 in A-strand [15, 17] . Our mAb DB32-6 is a serotype-specific neutralizing mAb that recognized residue K310 but not residues K305 or K307. Although K310 is considered as a subcomplex-specific epitope, DB32-6 is a serotypespecific mAb. There may be other regions that affect the binding of DB32-6 to DENV-2. We found that by mutating residue I312, DB32-6's binding activity was reduced by 50% (data not shown). Residue I312 may be a minor epitope of DB32-6. Moreover, 1A1D-2 is a temperature dependent mAb due to its needs for dynamic motion on the virion surface to neutralize virus [16] . Different from 1A1D-2, DB32-6 is temperature independent. When DB32-6 was incubated with DENV at 4uC, it still exhibited significant neutralizing activity (Figures 2 and 6 ). As expected, when incubating the DENV and DB32-6 at 37uC, DB32-6 showed better efficacy than it did at 4uC (data not shown). The residue K310 on the surface of DENV-2 may be accessible to DB32-6 binding. Additionally, DB32-6 had high binding affinity (0.12-0.18 nM) to DENV-2. Based on the above finding, the residue K310 induce serotype-specific mAbs and is crucial in the neutralization of virus infectivity.
Antibodies to E-DI-II tend to be more cross-reactive and less potent in neutralization of dengue infection [39] . However, there are fewer antibody concentrations capable of recognizing E-DIII than there are that recognize E-DI-II in dengue patients [20, 39] . Wahala et al. studied the human immune sera of DENV infection and found the E-DIII binding antibodies to play a minor role in DENV neutralization, similar to West Nile virus-infected human [52, 53] . The mAbs that bind to E-DIII expresses potent neutralizing activity, but only a few of them exist in serum of the patients infected with DENV or WNV. Combining the information from both mice and human mAbs studies of DENV infection is critical to understanding the complex mechanism behind the humoral immunity following natural DENV infection. According to one previous study, the immunoglobulin populations recognizing residues K310, E311 and P364 in dengue fever patients were much larger in IgM than in IgG [20] . The strong neutralizing IgG made up a small proportion of the antibody in dengue patients. de Alwis et al. has conducted an in-depth analysis of the human mAbs derived from memory B-cells of patients infected with primary DENV infections [54] . After the epitope mapping of anti-DENV-2 human mAbs, the strong neutralizing mAb 10.16 was mapped to K305, K310 and E311 in the A-strand. Together, the finding above suggest that the highly protective epitopes K310 and E311 in mouse play a role in humans as well.
We also identified several E-DI-II specific mAbs with high to no neutralizing activity. Serotype-specific mAbs (DB2-3 and DB23-3) with potent neutralizing activity were found to recognize E-DI-II of DENV-2 ( Figure 2 and Table 1 ). Some studies have identified highly neutralizing and protective antibodies against JEV and Figure 5 . Construction and characterization of humanized DB32-6 mAb. (A) Amino acid sequences of humanized DB32-6 (hDB32-6). FR, framework region; CDR, complementarity determining region. Red residues represent the different amino acids from murine DB32-6 (mDB32-6). (B) mDB32-6 and hDB32-6 mAbs recognized DENV-2-infected BHK-21 cells by IFA. Cells were counterstained with DAPI (blue) and observed at 4006 magnification. (C) Binding activity of hDB32-6 mAbs. Three stable clones of hDB32-6 (hDB32-6-30, hDB32-6-48 and hDB32-6-51) recognized DENV-2infected C6/36 cells and recombinant E-DIII of DENV-2 by ELISA. (D) Various concentrations of mDB32-6 and hDB32-6-48 mAbs were reactive to DENV-2 and recombinant E-DIII of DENV-2 but not to mock control. NMIgG and NHIgG were used as negative controls. (E) Binding affinities of mDB32-6 and hDB32-6-48 to E-DIII of DENV-2. mAbs affinity analysis was performed by surface plasmon resonance (SPR). Binding affinity was tested at the mAb concentrations ranging 0 to 4 nM. Binding curves and kinetic parameters are shown. doi:10.1371/journal.pntd.0001636.g005
Humanized mAb against Dengue Virus www.plosntds.org DENV located in E-DI [55, 56] Currently, we are in the process of identifying the neutralizing epitopes of DB2-3 and DB23-3. mAbs that broadly cross-react with other flaviviruses are in E-DII near the fusion loop, which is immunodominant antigenic [20, 34, 42] . Binding an antibody to DENV can change the rearrangement of the E protein, which may neutralize or enhance viral infection [16, 57] . The high or no neutralizing activity of our mAbs can be used help identify neutralizing or immunopathogenic epitopes in the E protein. Studies that explore the mAbs mediated neutralization mechanism and mAbs dependent enhancement are currently underway.
The mouse models for dengue infection developed to date do not represented the entirety of the pathogenesis of human dengue infection [58] . Developing of mouse models to studying its pathogenesis is important but challenging. We used two models, suckling mice protection assay and Stat1-deficient (Stat1 2/2 ) mouse model with different DENV-2 strains through intracerebral or intraperitoneal inoculation to evaluate the neutralizing activity of DB32-6 mAb (Figures 2 and 6 ). Our findings suggested that mAb DB32-6 might effectively block virus entry. However, disease manifestation of suckling mice is not relevant to dengue disease in humans since DENV infections in humans rarely involve the nervous system [58] . The Stat1-deficient mice are genetically mutated and not immunocompetent, hence they are not representative of the wild types' immune response to DENV. However, their survival rates might reflect the therapeutic potential of these mAbs. The results from these mouse models showed that the therapeutic potential of this newly generated mAb DB32-6 is worth further investigation.
In the absence of an effective dengue vaccine, neutralizing antibodies can be used as a passive immunotherapeutic strategy for treating dengue. Previous studies of humanized antibodies against DENV were derived from two chimpanzee Fab fragments: humanized IgG1 1A5 cross-neutralizing DENV-1 and DENV2 and humanized IgG1 5H2 specific against DENV-4 [42, 48, 56] . Our newly generated hDB32-6 was derived from murine mAb. However, when developing antibody-based therapy, ADE phenomenon is a major concern. Modification of Fc structure in an antibody can prevent Fcc receptors binding and inhibit ADE (Figure 7 ) [39, 48] .
Our studies show that the serotype-specific mAbs targeting the A-strand of E-DIII could serve as a dramatic neutralization determinant. Through testing in different mouse models, we have successfully generated a mAb hDB32-6 variant with high therapeutic potential against diverse DENV-2 strains without inducing ADE. Such an antibody-based therapy may help control severe dengue in the future. Figure S4 Identification of mAb 3H5 neutralizing epitopes by VLP mutants. BHK-21 cells expressed various DENV-2 VLP mutants. After fixation and permeabilization, mAbs were incubated with the cells. Binding activity was assessed Figure 6 . mAbs, mDB32-6 and hDB32-6-48, protected against DENV-2-induced mice mortality. Two-day-old suckling mice (ICR strain) were injected intracranially (i.c.) with 1610 4 pfu of DENV-2 (16681). After 1 day of infection, 5 mg of mAb were passively injected into mice through an i.c. route. Log rank test was used to determine significant differences in survival rate, and the mAbs with neutralizing activity were compared to the control group treated with NHIgG, P,0.001. doi:10.1371/journal.pntd.0001636.g006 Figure 7 . Antibody-mediated enhancement of DENV-2 infection by mAbs. Serial dilutions of NMIgG, 4G2, mDB32-6 and hDB32-6 variant were incubated with DENV-2 (16681) at MOI of 1 at 4uC for 1 h before they were added to K562 cells. After 2 days infection, cells were fixed, permeabilized, and stained with mAb DB42-3, and the percentage of cells infected with DENV-2 was detected by flow cytometry. doi:10.1371/journal.pntd.0001636.g007
Humanized mAb against Dengue Virus www.plosntds.org by flow cytometry. The fluorescence intensities were quantified to determine the relative recognition, calculated as [intensity of mutant VLP/intensity of WT VLP] (recognized by a mAb)6[intensity of WT VLP/intensity of mutant VLP] (recognized by mixed mAbs). Data shown are one representative experiment out of three independent experiments. (DOC) Figure S5 Sequence alignment of different DENV-2 genotypes and highlights of the neutralizing epitopes in E-DIII. The sequence of E-DIII from DENV-2 (strain 16681, Southeast Asian genotype) is aligned with other DENV-2 genotypes including NGC (Southeast Asian), PL046 (Southeast Asian), PM33974 (West African) and IQT2913 (American). Black blocks show residues of genotypic variation. The serotype-specific neutralizing epitopes located in E-DIII are K310 (green) and E311 (purple) which are recognized by DB32-6 and DB25-2, respectively. (DOC)  CD8(+) T Cells in Leishmania Infections: Friends or Foes? Host protection against several intracellular pathogens requires the induction of CD8(+) T cell responses. CD8(+) T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8(+) T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8(+) T cells during various types of Leishmania infections, following vaccination, and as potential immunotherapeutic targets. CD8 + T cells play a major role in protective immunity to a wide variety of pathogens, including viruses, bacteria, and protozoan parasites. However, the protective role of CD8 + T cells during Leishmania infections has been controversial, mainly because of the discrepancy among infections with different Leishmania species. Different Leishmania species have different tropisms and their diversity is reflected in the various clinical manifestations they induce. Hence, it is not surprising that the contribution of CD8 + T cells to the immune response against the parasite depends on the clinical form and the species that is causing it. Here, we discuss the literature on the contribution of CD8 + T cells to the immune response against Leishmania, taking into account the various clinical forms and experimental models.
CD8 + T cells recognize peptides that are presented in the context of major histocompatibility complex (MHC) class I molecules via the T cell receptor (TCR). Although peptides presented via MHCI mainly derive from endogenous antigens, various exogenous cellassociated antigens have also been shown to be uploaded onto the MHCI pathway, by a process referred to as cross-presentation. Leishmania antigens were also shown to be cross-presented (Bertholet et al., 2006) . During in vivo infections, cross-presentation of Leishmania antigens may result from several internalization pathways, such as direct infection, receptor-mediated uptake (Woelbing et al., 2006) , or internalization of apoptotic vesicles (Winau et al., 2006) . Thus far, two different processing pathways have been proposed. An early work demonstrated that a surface antigen of L. amazonensis was processed in a proteasome-dependent manner within the cytosol (Kima et al., 1997) . In contrast, a more recent study showed that cross-presentation of secreted leishmanial antigens is confined to an intraphagosomal processing pathway that is TAP-and proteasome-independent (Bertholet et al., 2006) .
After activation, antigen-specific CD8 + T cells differentiate into effector cells and acquire the capacity to kill target cells, and produce several cytokines and chemokines (Kaech et al., 2002; Harty and Badovinac, 2008) . Among the various CD8 + T cell subsets, Tc1 were shown to play a major role in the fight against several protozoan parasites (Jordan and Hunter, 2010) . The hallmark of this subset is the production of IFN-γ and TNF, and cytotoxic capacity (Woodland and Dutton, 2003) . The precise mechanism underlying cytotoxic T lymphocyte (CTL) killing of microbes is still poorly understood. CTLs can exert cytotoxicity through various mechanisms: via exocytosis of lytic granula containing perforin, granzyme A/B, and/or granulysin; through the interaction between FasL and Fas expressed on targets cells; via TNF; or via TRAIL (Trapani and Smyth, 2002) . A study has also shown that reactivated memory CD8 + T cells efficiently killed Listeria monocytogenes via a mechanism mediated by CCL3 and involving the induction of radical oxygen intermediates (Narni-Mancinelli et al., 2007) . Direct killing of extracellular pathogen by CTLs has also been described. For example, CTLs can mediate killing of Mycobacterium tuberculosis through the release of anti-bacterial products (Stenger et al., 1998; Canaday et al., 2001) . However, direct killing of Schistosoma mansoni (Ellner et al., 1982) and Entamoeba histolytica (Salata et al., 1987) is thought to be contact-dependent. CTLs have also been reported to directly kill extracellular Toxoplasma gondii (Khan et al., 1990) . Interestingly, killing in this case appeared to be antigen-specific. To date there is no evidence that CD8 + T cells can mediate protection against Leishmania parasites through their cytotoxic activity. However, since CTLs have been www.frontiersin.org observed in various mouse models and also in human patients, a possible protective role for Leishmania-specific cytotoxic T cells should not be excluded.
In addition to killing and releasing cytokines and chemokines, recent studies have ascribed a novel regulatory role for CD8 + T cells (Sun et al., 2009; Palmer et al., 2010; Trandem et al., 2011) . Regulatory CD8 + T cells represent a transient state of effector CD8 + T cells (Trandem et al., 2011) , which is possibly induced by potent TCR stimulation, which promotes the production of the immunosuppressive cytokine IL-10 (Zhang and Bevan, 2011) . Not only do these cells produce IL-10, but they are also excellent killers and produce normal to higher amounts of IFN-γ and TNF (Sun et al., 2009; Palmer et al., 2010; Trandem et al., 2011) . The main function of these cells is thought to lie in the prevention of immunopathology during infection without affecting the kinetics of pathogen clearance. IL-10-producing CD8 + T cells have also been observed in human patients infected with L. guyanensis (Bourreau et al., 2007) and in patients suffering from post-kalaazar dermal leishmaniasis (PKDL; Ganguly et al., 2008) . The role of regulatory CD8 + T cells in the immune response against parasitic infections is still unknown.
The role of CD8 + T cells in the immunity to L. major has always been controversial. Early studies in BALB/c mice reported that CD8 + T cells were the main mediators of protection following CD4 + T cell depletion in mice infected with L. major (Titus et al., 1987; Hill et al., 1989; Muller et al., 1991) . Interestingly, depletion of CD4 + T cells was rendering susceptible BALB/c mice resistant to L. major infection. The results obtained using the CD4 + T cell depletion model suggested that CD8 + T cells could potentially control L. major infection in mice. A few years later, experiments in β2-microgobulin-deficient mice contradicted these findings and revealed that CD8 + T cells were not essential in mediating protection in L. major-infected BALB/c mice (Wang et al., 1993) . Moreover, a study in CD8 + T cell-deficient mice demonstrated that Cd8 −/− mice were able to control L. major infection for at least 1 year, suggesting that CD8 + T cells were not required for long-lasting immunity (Huber et al., 1998) . The contribution of CD8 + T cells in the control of primary L. major infection became less important also because of the strong evidence that Th1 cells were the primary cells involved in mediating protection against cutaneous leishmaniasis (Reiner and Locksley, 1995; Louis et al., 1998; Sacks and Noben-Trauth, 2002) . Several studies have demonstrated that Th1 cells producing IFN-γ were essential in controlling L. major infection, and that failure to develop a Th1 response resulted in susceptibility to the diseases. Hence, a consensus was reached in that if a mouse generates Th2 responses, this will lead to susceptibility; in contrast, Th1 responses were successfully controlling infection without the help of CD8 + T cells.
This paradigm was later challenged when new findings arose from a more natural model of infection, where 100 metacyclic promastigotes were inoculated intradermally in the ears of C57BL/6 mice. In this model, Cd8 −/− and CD8 + T cell-depleted mice fail to control L. major infection, and CD8 + T cells were thought to be necessary for supporting Th1 responses (Belkaid et al., 2002) .
The discrepancy between the findings in the low-and the highparasite dose model was clarified by another work that compared the requirements of CD8 + T cells in both systems (Uzonna et al., 2004) . Interestingly, in the low infection model CD8 + T cells producing IFN-γ were essential for modulating CD4 + T cell responses toward a Th1 response. In contrast, C57BL/6 mice inoculated with a high L. major dose did not require CD8 + T cell help to generate protective Th1 responses. The CD8 + T cell requirement for optimal IFN-γ production by Th1 cells was also proposed in a high-dose L. major infection model in BALB/c mice (Herath et al., 2003) . Moreover, CD8 + T cell-derived IFN-γ was reported to contribute to the induction of nitric oxide production in macrophages during experimental cutaneous leishmaniasis (Stefani et al., 1994) .
Although the role of CD8 + T cells-derived IFN-γ has been clarified, little is known about the involvement of cytotoxic CD8 + T cells in cutaneous leishmaniasis. In a low-dose model of L. major infection, CD8 + T cell responses were shown not only to be protective, but also to mediate pathology (Belkaid et al., 2002) . Hence, it is possible that CTLs may be involved in the ulceration of skin lesions through tissue disruption. This suggests that perhaps two types of CD8 + T cell effectors are generated during L. major infection: antigen-specific CD8 + T cells that produce IFN-γ but lack cytotoxic activity; and CTLs that are potent killers but produce little to no IFN-γ and promote pathology.
Although the role of CD8 + T cells during primary immune responses is controversial, these cells appear to play a prominent role in protecting mice from a secondary challenge (Muller et al., 1993 . Indeed, antigen-specific CD8 + T cells were expanding up to 50-fold in the spleen and lymph nodes of reinfected BALB/c mice . This expansion correlated with a substantial production of IFN-γ, which is thought to be essential for controlling Leishmania infections. These observations have major implications for vaccine design.
In summary, during experimental cutaneous leishmaniasis, CD8 + T cells are necessary to support protective Th1 responses through IFN-γ production, but they are also involved in the development of immunopathology. Further investigations are needed to better identify various subtypes of CD8 + T cells that arise during cutaneous leishmaniasis.
In contrast to the cutaneous models, CD8 + T cells have always been thought to play a major role in experimental visceral leishmaniasis (VL). Over 20 years ago, Stern et al. (1988) demonstrated for the first time that CD8 + T cells significantly contribute to the formation of granulomas in the liver of L. donovani-infected mice. Indeed, CD8 + T cell depletion resulted in impaired granuloma formation and exacerbation of liver disease. In agreement with these results, Kaye et al. (1992) also reported a delayed onset and a decrease of the liver granulomatous response in non-obese, diabetic mice expressing transgenic I-E molecules, suggesting that antigen-specific CD8 + T cells are required for proper granuloma formation. CD8 + T cells appear to participate in controlling parasite growth in the spleen as well, since CD8 + T cell depletion during chronic VL significantly increased splenic parasite burden (Stäger, unpublished) . This observation was underscored by the fact that adoptive transfer of antigen-specific CD8 + T cells during Frontiers in Immunology | Microbial Immunology chronic L. donovani infection resulted in 90% reduction in the splenic parasite burden (Polley et al., 2006) . Moreover, therapeutic vaccination aimed at reactivating CD8 + T cells during chronic VL ensued in the control of parasite growth in the spleen (Joshi et al., 2009) .
Interestingly, CD8 + T cells do not only participate in primary responses to L. donovani, but are also the major mediators of resistance upon reinfection (Stern et al., 1988) . Indeed, protection was abrogated following CD8 + T cell but not CD4 + T cell depletion.
A prominent function for CD8 + T cells was also described in L. infantum-infected mice. Using an intradermal infection model, Ahmed et al. (2003) demonstrated that CD8 + T cells contribute to parasite clearance in the skin of L. infantum-infected mice.
Another study also showed that CD8 + T cells purified from L. infantum-infected mice expressed IFN-γ and TNF, and displayed considerable cytotoxic activity against cells expressing Leishmania antigens (Tsagozis et al., 2003) . Interestingly, killing of infected target cells was mediated by both the perforin and Fas/FasL pathways (Tsagozis et al., 2003) . The Fas/FasL pathway has also been implicated in the defense against L. donovani (Alexander et al., 2001) . Indeed, gld and lpr mice, which lack a functional Fas/FasL pathway, were shown to be more susceptible to L. donovani.
Additionally to the classical cytotoxic pathways, a novel counterregulatory function for a subset of cytotoxic CD8 + T cells has recently been proposed in the L. donovani infection model (Martin et al., 2010) . In this study, CD3 + CD8 + CD40 + T cells are shown to suppress regulatory T cells via CD40/CD40L interaction during the early stages of infection in BALB/c mice. CD40 signals through Ras, PI3K, and protein kinase C, leading to the induction of granzyme and perforin, and ultimately to the killing of Tregs.
CD8 + T cells may not be merely participating in the primary immune response by secreting IFN-γ and possibly killing infected target cells and/or Tregs, but they could also be involved in the recruitment of inflammatory cells and in the maintenance of granulomas. Indeed, a study using L. infantum demonstrated that CD8 + T cells expressed RANTES and MIP-1α (Tsagozis et al., 2003) , two chemokines that are involved in the recruitment of T cells at the inflammatory site and in the formation and maintenance of granulomas (Mackay, 2001) . The authors proposed that CD8 + T cells may thus be involved in granuloma formation. This hypothesis is in agreement with the depletion data (Stern et al., 1988) , showing that depletion of CD8 + T cells results in impaired granuloma formation and ultimately in disease exacerbation.
Despite the documented evidence that CD8 + T cells strongly participate in the immune response to L. donovani and L. infantum, our recent findings suggest that L. donovani induces defective antigen-specific CD8 + T cell responses (Joshi et al., 2009 ). Interestingly, mice infected with L. donovani generate CD8 + T cell responses with limited clonal expansion. The extension of the clonal expansion is thought to be correlated with the effectiveness in eliminating pathogens. It was calculated that a naïve CD8 + T cell may go through 19 cell divisions in the first week after pathogen inoculation (Badovinac et al., 2007) . Massive clonal expansions have not only been observed during viral infections, but also following the injection of irradiated Plasmodium berghei sporozoites (Sano et al., 2001) . During L. donovani infection, CD8 + T cells underwent at least 8-9 rounds of division, but failed to accumulate in the spleen (Joshi et al., 2009) . Moreover, only 10% of CD8 + T cells during clonal expansion expressed markers typically associated with end-differentiated effector cells, such as KLRG1, unpublished) . The cause of this limited expansion is yet unknown and may depend on several factors. One of the possible explanations is limited antigen availability that may result from poor antigen-processing and presentation. Processing of Leishmania antigens is thought to be confined to a TAP-independent, intraphagosomal pathway that is less efficient and requires higher amounts of antigen than the endoplasmic reticulum-based, TAP-dependent cross-presentation pathway (Bertholet et al., 2006) . Furthermore, the major surface protease of Leishmania, gp63, was shown to cleave epitopes within the parasitophorous vacuole, further reducing antigen availability (Garcia et al., 1997) . Hence, Leishmania antigens may be poorly presented and this poor presentation may not be enough to induce and sustain a massive clonal expansion of antigen-specific CD8 + T cells.
Nonetheless, antigen may be suddenly available in large amounts later on during L. donovani infection, since CD8 + T cells undergo a second round of activation, become dysfunctional, and ultimately die by "exhaustion" (Joshi et al., 2009) ; high antigen levels have been described as a cause of CD8 + T cell "exhaustion" during chronic viral infections (Mueller and Ahmed, 2009 ). Further research is needed to clarify the mechanisms involved in CD8 + T cell exhaustion during chronic VL.
In conclusion, CD8 + T cells are required to control parasite growth during experimental VL and reactivation of these responses results in a dramatic reduction in parasite burden. Therefore, immune interventions that target CD8 + T cell responses may have great therapeutic potential against VL.
The role of CD8 + T cells in human leishmaniasis patients is still unclear and seems to depend on the various species of parasites and the disease they cause.
Few studies have been conducted with human VL patients. However, most of the studies ascribe a protective role for CD8 + T cells, in agreement with results obtained from experimental models. Indeed, the control of L. infantum infection was shown not only to be associated with IFN-γ-producing CD4 + T cells, but also with CD8 + T cells (Mary et al., 1999) . Interestingly, during active VL, CD8 + T cells are less responsive to stimulation and a greater percentage stains positive for Annexin V compared to healthy controls (Clarencio et al., 2009 ). These observations correlate very well with what we observed in mice experimentally infected with L. donovani, where CD8 + T cells became increasingly dysfunctional during chronic infection and died by exhaustion (Joshi et al., 2009) . Whether human CD8 + T cells also display signs of exhaustion during active VL still remains to be tested.
Another study investigating CD8 + T cell responses in patients infected with L. chagasi has revealed that the frequency of CD18 + CD45RO + CD8 + T cells is significantly decreased in the spleen of patients with active VL (Clarencio et al., 2009 ). In contrast, CD18 + CD8 + T cells seem to be retained in the bone marrow of VL patients. CD18, or integrin β-2, is the β subunit of LFA-1 (CD11a), CD11b, CD11c, and CD11d. In humans, lack of CD18 www.frontiersin.org causes leukocyte adhesion deficiency, a disorder characterized by lack of leukocyte extravasation from blood into the tissue (Bunting et al., 2002) . With exception of the fact that CD18 + cells appear in the granulomas of dogs with asymptomatic VL (Sanchez et al., 2004) , very little is known about the function of CD18 + CD8 + T cells during VL and whether cells lacking CD18 expression have similar migratory capacity and effector functions to their CD18 + counterparts.
Not only is the frequency of CD18 + CD8 + T cells reduced in L. chagasi patients, but also, the level of circulating memory T cells is significantly decreased during active VL (Hailu et al., 2005; Clarencio et al., 2009 ). This observation is in agreement with our findings in the experimental model of VL, where the majority of the CD8 + T cells displayed an effector phenotype during chronic infection (Joshi et al., 2009) .
Although CD8 + T cells positively correlate with cure of VL patients, one report suggested that these cells may contribute to the immunopathogenesis of PKDL (Ganguly et al., 2008) . Indeed patients suffering from PKDL showed a significant increase in the percentage of CD8 + T cells producing IL-10, which disappeared after cure (Ganguly et al., 2008) . IL-10-secreting CD8 + T cells are thought to play a regulatory role in different viral infection models. This CD8 + T cell subset was shown to display great cytotoxicity and produce granzyme B, IFN-γ, and TNF (Sun et al., 2009; Palmer et al., 2010; Trandem et al., 2011) . IL-10 + CD8 T cells seem to represent a transient and reversible state of CD8 + effector T cell differentiation. Its primary function is to balance pathogen clearance with bystander tissue damage (Zhang and Bevan, 2011) . Interestingly, in viral model, this subset disappears after the infection is cleared. Hence, it is possible that the IL-10-producing CD8 + T cells in PKDL patients are actually killing parasites and protecting patients from tissue damage, rather than suppressing protective responses. Further studies are needed in order to define the nature of these cells.
CD8 + T cells also actively participate in the immune response to cutaneous infections in human. As observed in the low-dose model in mice (Belkaid et al., 2002; Uzonna et al., 2004) , L. major also induces Th1 and CD8 + T cells in human patients and both responses are associated with disease resolution (Nateghi Rostami et al., 2010) . CD8 + T cells were not only observed in large numbers in the lesions of L. major patients during the acute phase, but also during the healing process (Da-Cruz et al., 1994 Gaafar et al., 1999) . The exact role of CD8 + T cells in L. major infections in humans is not yet known. A major correlate of protection appears to be the high amounts of IFN-γ produced by CD8 + T cells after restimulation (Nateghi Rostami et al., 2010) . In vitro studies have also demonstrated that Leishmania-specific CTLs are generated upon co-culturing human naïve T cells with antigens from L. amazonensis promastigotes and IL-12 (Russo et al., 1999) , or with L. major parasites (Da . Moreover, increased granzyme B activity was also found in patients with an active infection and was associated with a good prognosis (Bousoffara et al., 2004) . In this study, in vitro cytotoxicity by peripheral blood lymphocytes on L. major-infected macrophages appeared to be mediated by granzyme B, suggesting that CTL activity may be involved in controlling parasite growth. It is possible, though, that the cytotoxic activity not only contributes to disease clearance, but also to the development of skin ulceration, as observed in L. major-infected mice (Belkaid et al., 2002) .
A strong CD8 + T cell expansion has also been observed in L. mexicana patients during the healing process (Salaiza-Suazo et al., 1999) . Interestingly, lesions of patients with localized cutaneous leishmaniasis (LCL) harbor a higher number of CD8 + T cells compared to patients with diffuse cutaneous leishmaniasis (DCL; Hernandez-Ruiz et al., 2010) . As already observed in VL patients, CD8 + T cells in DCL patients, unlike LCL patients, show a reduced capacity to respond to antigen-specific stimulation during active infection. In fact, these cells displayed low cytotoxicity and only produced little IFN-γ upon stimulation, therefore showing typical signs of functional exhaustion (Hernandez-Ruiz et al., 2010) . Strikingly, effector functions could be restored in vitro after stimulation with TLR2 agonists, highlighting the potential therapeutic benefit of the revival of CD8 + T cell functions in DCL patients.
In contrast to the cutaneous and visceral forms of leishmaniasis -where CD8 + T cells seem to correlate with cure and contribute to the immune response -in mucocutaneous infections (ML) CD8 + T cells seem to be implicated in the pathogenesis of the disease. Indeed, high numbers of cytotoxic CD8 + T cells were observed in ML patients (Barral-Netto et al., 1995; Brodskyn et al., 1997) . Moreover, the recruitment of granzyme A + CD8 + T cells is associated with lesion progression (Faria et al., 2009) , suggesting that CTLs may contribute to immunopathology. The development of ML is not only associated with the presence of CTL, but also with a high frequency of activated CD4 + T cells, an extreme IFNγ and TNF production, and a reduced control of inflammation due to low expression of the IL-10 receptor (Gaze et al., 2006; Faria et al., 2009) . Furthermore, IL-17-secreting CD4 + and CD8 + T cells were also found in ML patients (Boaventura et al., 2010) . Consequently, neutrophils, which are typically recruited during a TH17-mediated inflammatory response, were also detected in necrotic and perinecrotic areas (Boaventura et al., 2010) . This suggests that neutrophils, together with CTLs, may be involved in tissue injury and in the development of immunopathology.
Taken together, the literature shows that CD8 + T cells actively participate in the fight against most Leishmania infections in humans and their presence correlates with cure. In contrast, CD8 + T cells in ML patients contribute to disease exacerbation.
Vaccination of humans with heat-killed Leishmania or recombinant parasite proteins has so far failed to induce long-term immunity and only recovery from natural or experimental infection has provided proper protection. Several trials have analyzed the protective effect of autoclaved L. major plus Bacillus-Calmette-Guérin (BCG) versus BCG alone assessing the cumulative incidence of cutaneous leishmaniasis caused by L. tropica (Sharifi et al., 1998) or L. major (Momeni et al., 1999) , or of VL (Khalil et al., 2000) caused by L. donovani. Although no trial showed a significant effect on disease incidence, the vaccination induced skin test conversion and provided limited protection. Additionally, a study showed that immunization of Colombian soldiers with three doses of L. amazonensis alone was non-protective (Velez et al., 2005) .
In the human disease, there is evidence that mixed T helper cytokine profiles are present, while healing and protection against reinfection are associated with dominant Th1 and CD8 + T cells. These findings suggest that it is the cytokine balance that activates or suppresses activation of macrophages harboring Leishmania parasites. This, in turn, determines the outcome of the infection. Thus treatments or antigen/adjuvant formulations that can alter the type of T helper response may change the course of disease (Da-Cruz et al., 2002; Rogers and Titus, 2004; Mohajery, 2007) . For this purpose, different vaccination strategies have been examined in animal models including leishmanization (Modabber, 1990) , killed parasite (Grimaldi, 1995) , live attenuated parasite (Titus et al., 1995) , and subsequently, subunit vaccines composed of recombinant or native proteins from different stages of the parasite's life cycle, and DNA vaccines (Skeiky et al., 1998; Webb et al., 1998; Stager et al., 2000; Bottrel et al., 2001; Campos-Neto et al., 2001; Rafati et al., 2001; Coler et al., 2002) . The latter two strategies encompass candidates such as gp63, gp46, LACK, CPB, CPA, Kmp11, LmsTI1, TSA, LeIF, HASPB1, and LPG, and have shown promising results in murine models. Nonetheless, only Leish111f (a recombinant fusion protein of LmsTI1, TSA, and LeIF) progressed through phase I and II clinical trials (Llanos-Cuentas et al., 2010; Chakravarty et al., 2011) .
Nowadays, it is clear that CD8 + T cells play an important role in the mechanisms for cure of and resistance to Leishmania infection, either by production of IFN-γ and activation of macrophages, or by direct killing of parasitized macrophages, or a combination of both effects. CD8 + T cells have been associated with protection against Leishmania reinfection in murine models; however, the induction of these T cell subsets in humans seems to be also related to the healing process. Today, there are several reports about different leishmanial antigens eliciting CTL responses such as P8, gp46 , HASPB1 (Stager et al., 2000) , Kmp11 (Basu et al., 2007) , CPB (Rafati et al., 2002) , nucleosomal histones (Iborra et al., 2004) , LmaCIN (Farajnia et al., 2005) , LmsTI1, and TSA (Coler et al., 2002) .
The essential point to be considered in vaccine design for a heterogeneous population, such as that of humans, is the HLA polymorphism. Effective vaccination against a complex parasitic infection such as Leishmania would require a multivalent vaccine composed of several antigens to enhance the possibility of covering a good number of MHC types. This is possible either through recombinant fusion proteins encompassing the whole antigen or through vaccines composed of peptides from different antigens (Campos-Neto et al., 2001; Rafati et al., 2001; Mendez et al., 2002) . The latter strategy, called polytope vaccine, is finding its way in vaccinology because of its extraordinary properties, especially the ability to direct the immune response toward the induction of CTLs (Sbai et al., 2001; Schirmbeck et al., 2003; Robinson and Amara, 2005) .
As CTL responses play a pivotal role in defense against viruses and tumor cells, polytope vaccines have found their way in these fields but there is still no report on leishmaniasis even it has been shown that CTLs could be very important in protection and long-lasting resistance to infection. Recently, we took advantage of the potential of immunoinformatics tools to screen for L. major epitopes that could be presented in HLA A2, which is the most prevalent HLA supertype in the Iranian population. In vitro stimulation to recall memory CD8 + T cells from Leishmania-infected individuals and intracellular cytokine assays for IFN-γ-producing cells confirmed that HLA A2 positive individuals that recovered from an L. major infection successfully generated CD8 + T cell responses against peptides derived from LmsTI1 and LPG-3 (Seyed et al., 2011) . Furthermore, Walden and co-workers have mapped the T cell epitopes from kinetoplastid membrane protein of L. major (Kmp11) via classical mapping for different human HLA class I alleles (Basu et al., 2007) . Gazzinelli and co-workers have studied CD8 + T cell responses against the Leishmania A2 antigen and mapped the CD8 + T cell epitopes in BALB/c mice (Resende et al., 2008) . Laouini and co-workers (Guerfali et al., 2009 ) and Dumonteil and co-workers (Dumonteil, 2009 ) started genomewide screenings for novel epitopes. Using a combination of T cell epitope prediction tools, they successfully validated such epitopes in both BALB/c and C57BL/6 mice.
Although understudied, CD8 + T cells appear to play an important role in the immune response to most Leishmania infections. Pilot studies in the murine model of VL have also demonstrated that adoptive transfer of antigen-specific CD8 + T cells (Polley et al., 2006) or reactivation of CD8 + T cell responses through a therapeutic vaccine (Joshi et al., 2009) results in the control of parasite growth. A better understanding of the mode of activation, the specificity, and effector functions of the various CD8 + T cell subsets generated during Leishmania infections could ameliorate the design of vaccines and of novel therapeutic interventions.
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Barral-Netto, M., Barral, A., Brodskyn, C., Carvalho, E. M., and Reed, S. G. (1995) . Cytotoxicity in human mucosal and cutaneous leishmaniasis. Parasite Immunol. 17, 21-28. Basu, R., Roy, S., and Walden, P.
(2007). HLA class I-restricted T cell epitopes of the kinetoplastid membrane protein-11 presented by Leishmania donovani-infected human macrophages. J. Infect. Dis. 195, 1373 -1380 S., Lira, R., Caler, E., Bertholet, S., Udey, M. C., and . CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J. Immunol. 168, 3992-4000.
www.frontiersin.org Macroevolutionary Immunology: A Role for Immunity in the Diversification of Animal life An emerging picture of the nature of immune systems across animal phyla reveals both conservatism of some features and the appearance among and within phyla of novel, lineage-specific defense solutions. The latter collectively represent a major and underappreciated form of animal diversity. Factors influencing this macroevolutionary (above the species level) pattern of novelty are considered and include adoption of different life styles, life histories, and body plans; a general advantage of being distinctive with respect to immune defenses; and the responses required to cope with parasites, many of which afflict hosts in a lineage-specific manner. This large-scale pattern of novelty implies that immunological phenomena can affect microevolutionary processes (at the population level within species) that can eventually lead to macroevolutionary events such as speciation, radiations, or extinctions. Immunologically based phenomena play a role in favoring intraspecific diversification, specialization and host specificity of parasites, and mechanisms are discussed whereby this could lead to parasite speciation. Host switching – the acquisition of new host species by parasites – is a major mechanism that drives parasite diversity and is frequently involved in disease emergence. It is also one that can be favored by reductions in immune competence of new hosts. Mechanisms involving immune phenomena favoring intraspecific diversification and speciation of host species are also discussed. A macroevolutionary perspective on immunology is invaluable in today’s world, including the need to study a broader range of species with distinctive immune systems. Many of these species are faced with extinction, another macroevolutionary process influenced by immune phenomena. Recent years have witnessed a dramatic increase in our understanding of the diversity of immune systems across animal phyla (Flajnik and Kasahara, 2010; Messier-Solek et al., 2010; Rast and Litman, 2010; this volume) . Availability of genome sequences from a broad variety of animals coupled with an increased appreciation for the diversity of their defenses has given the study of immunity a much stronger evolutionary foundation, one that has been further enriched by studies of plant immunity and responses of bacteria and archaea to threats to their genomes (Horvath and Barrangou, 2010) . The increasing depth and breadth of immunological studies is also bringing to light a greater awareness of the impact that immunity has had on all forms of life, especially parasites. Here "parasite" is used inclusively, referring to infectious agents ranging from viruses to bacteria to protists to multicellular helminths. The features uniting parasites are that they infect hosts, provoke some degree of fitness-diminishing harm, prompt the deployment of immune responses, and undertake immune evasive actions. "Immune systems" are referred to as those molecules, cells, tissues, and organs that protect hosts from parasites (see caveats below). This discussion excludes a broad range of behavioral defenses like preening (Bush and Clayton, 2006) or avoidance (e.g., Mooring et al., 2003; Garnick et al., 2010) .
Here I attempt to draw together ideas that begin to put immunological phenomena into a broader macroevolutionary context. Macroevolution is the study of patterns, and the evolutionary processes that have generated them, at or above the species level (Stanley, 1998; Levinton, 2001) . It is the study of how and why life has diversified, and attempts to document how and why lineages of organisms have come into being and either given rise to additional lineages or gone extinct. The process of speciation is germane to macroevolutionary studies because it is the process responsible for increasing the diversity of life forms. Extinction and its causes are also an essential part of such studies.
The attributes of immune systems across the spectrum of animal diversity provide a new way to view and reinterpret the diversity of animals. Immune systems exhibit unforeseen novelty and thus offer new insights into major selective forces influencing animal life. Also, phenomena that are fundamentally immunological provide fertile ground for investigating the impact of immunity as a driver of biodiversity. The role of immune systems in macroevolutionary processes is one that deserves recognition and more study.
In considering what is to follow, several caveats should be borne in mind:
(1) We are just beginning to view molecular components of immune systems from a broad sampling of animal phyla. Detailed analyses are still few for how these presumptive immune components actually function in defense, and how critical their roles might be in protecting the organisms in question.
(2) Also poorly known are the specific parasite threats faced by the more obscure groups of animals serving as hosts.
(3) Many of the examples of immunological novelty presented below emphasize differences at the phylum level. Some phyla such as the Arthropoda, Mollusca, and Chordata are immense in species numbers and undoubtedly collectively employ as yet many undiscovered immune capabilities. Also, some of the smaller animal phyla are essentially unexplored with respect to their immune systems. Once understood, these additional examples will only add to the overall diversity of immune responses. (4) It is not always easy to circumscribe "the immune system" or an "immune response." This is particularly so in cases where potent defenses for parasites result from selection for variant alleles for genes like hemoglobin B or apolipoprotein L-1 that otherwise might not be considered a core part of the immune system (Anstee, 2010; Barreiro and Quintana-Murci, 2010; Genovese et al., 2010; Wheeler, 2010) .
Discoveries relating to the innate immune systems of plants, flies, and mammals have tended to accentuate the similarities among them, implying a grand conservatism even across kingdoms with respect to basic immune system design and function. Indeed, there are intriguing similarities between the membraneassociated and intracytoplasmic receptors of plants and animals suggestive that some basic solutions to recognition and response to parasites have been conserved since at least the time animal and plant lineages diverged. However, particularly given that some of these similarities are a likely result of convergent evolution rather than indicative of a common origin (Ausubel, 2005) , conserved immune features are not the emphasis here. Rather, this overview accentuates the emergence of immunological novelty among and within animal phyla (Figure 1 ; Table 1 ).
The most basal animal group is the phylum Porifera, the sponges (Srivastava et al., 2010) . Sponges lack the complex tissue and organ structure found in other animal phyla, and lack cells specialized for protection from parasites. Although sponge immunobiology is in its infancy, one of the best-known sponges, Suberites domuncula, possesses membrane-spanning molecules that contain an intracellular Toll-interleukin 1 receptor (TIR) domain, though it lacks an external leucine-rich repeat pattern recognition receptor more typical of TLRs. On the basis of having a MyD88 homolog, S. domuncula has at least the rudiments of an NF-κB signaling pathway. Sponges also have molecules for attacking bacterial membranes, presumptive antiviral responses (Schroder et al., 2008) , and diversified scavenger receptor cysteine-rich molecules of unknown function (Wiens et al., 2007) .
Among basal animals, it is members of the phylum Cnidaria (jellyfish, Hydra, anemones, and corals) that have proven most surprising with respect to the large size and content of their genomes, including their immune systems. Cnidarians have distinct tissues but lack organs and are generally considered to be diploblastic, meaning they have recognizable ecto-and endoderm, but lack well-developed mesoderm tissue. Like sponges, they lack recognizable specialized immune cells. However, the starlet sea anemone Nematostella vectensis has at least one TLR, an NF-κB signaling pathway, a homolog of a complement 3-like molecule, the likely presence of functioning intracellular NOD-like receptors (NLRs), perforin-like molecules, diverse C-type lectins (Wood-Charlson and Weis, 2009) , and even a recognizable homolog of the recombination activating gene, RAG1 Augustin et al., 2010) . Cnidarians often live in colonies and have to contend with encroaching competitors, including conspecifics. For this they have well-developed mechanisms to recognize self and non-self. One of the responsible molecules has been identified, and is surface expressed, polymorphic and possesses three external immunoglobulin superfamily (IgSF) domains (Nicotra et al., 2009) .
The remaining animals, the Bilateria, are bilaterally symmetrical and triploblastic, with well-developed tissues and organs. They often have specialized immune cells. Most fall into two major lineages, the protostomes and deuterostomes. Among the protostomes, representatives of the molting clade (Ecdysozoa) have been most extensively studied in an immune context, as this clade includes nematodes and arthropods, both containing well-studied model organisms. Caenorhabditis elegans and other nematodes have reduced genomes and from an immunological perspective are surprising for what they do not have. Although C. elegans has one bonafide TLR that plays a role in defense against some bacteria (Tenor and Aballay, 2008) , it lacks Myd88, NF-κB and several other components of the canonical Toll pathway. NLRs are also lacking. Nonetheless, C. elegans can mount inducible, parasite specific responses. It has several novel signaling pathways for defense (Irazoqui et al., 2010) and produces many distinctive antimicrobial peptides (AMPs) for protection from bacteria (Roeder et al., 2010) . C-type lectins may serve as recognition molecules in C. elegans. The preoccupation with production of AMPs by gut cells reflects their diet of bacteria, which could include potential parasites (Roeder et al., 2010) .
Another prominent model of ecdysozoan immunity is Drosophila (Lemaitre and Hoffmann, 2007) , but increasingly other insect are studied as well, such as mosquitoes because of their role in transmitting human parasites (Bartholomay et al., 2010) . Insects have dedicated immune cells such as plasmatocytes and lamellocytes that circulate through their open circulatory system and phagocytose or encapsulate foreign objects. The Drosophila immune system also shows evidence of gene loss: it lacks the C3-like complement component and NLR homologs found in cnidarians. Their TLRs are different from those of vertebrates in that they do not engage microbial ligands directly. Of their nine TLR genes, only one or two function in immunity, activating NF-κB signaling pathways in the fat body to produce AMPs (Lemaitre FIGURE 1 | An overview of some of the novel features associated with immune responses of representatives of major animal lineages (see text for details). TLR, Toll-like receptor; AMP, antimicrobial peptide; Dscam, Down syndrome cell adhesion protein; VCBPs, variable region-containing chitin-binding proteins; NLRs, intracellular NOD-like receptors; LRR, leucine-rich repeat; IgSF, immunoglobulin superfamily; Ab, antibodies; TCR, T cell receptor; MHC, major histocompatibility complex.
and Hoffmann, 2007). Insects are by no means immunologically bereft though. They have a number of other effective defense components not seen in many other organisms. They have elaborate cascades of CLIP-serine proteases that mediate and coordinate phagocytosis, nodule formation, encapsulation, and AMP formation, and they can deposit layers of melanin around foreign objects (Kanost et al., 2004) . They engage multimeric fibrinogen-related proteins (FREPs) in parasite recognition (Dong and Dimopoulos, 2009 ) and employ Down syndrome cell adhesion molecule (Dscam), a member of the IgSF, in antigen recognition. Tens of thousands of Dscam isoforms can potentially be generated by alternative splicing (Schmucker and Chen, 2009 ) and parasite challenge-specific Dscam splice form repertoires can be produced (Dong et al., 2006) .
Insect studies provide additional examples of immunological novelty, at the ordinal, family, or even genus level. One example is provided by Drosophila and Anopheles, both in the same order (Diptera), but representing very different life styles and having been separate lineages for 250 million years. Gene families involved in immunity have evolved rapidly and divergently in the two dipterans. For example, with respect to thioester containing proteins (TEPs), Anopheles has 10 genes and Drosophila only four, with only one orthologous pair between the two. Anopheles has 58 fibrinogen-like immunogenes whereas Drosophila has only 14, with only two shared orthologous pairs (Dong and Dimopoulos, 2009) .
At the family level, a comparison of three different mosquito genera (Aedes, Anopheles, and Culex, all in the Culicidae) has revealed prominent genus specific expansion of some immune gene families (Bartholomay et al., 2010) . Comparative studies of Drosophila species are particularly revealing, showing that novel immune genes and immune gene families have originated relatively recently, suggestive of a role of parasites in driving adaptive evolution in flies (Sackton et al., 2007) . Furthermore, for particular immune proteins, the amino acids under positive selection vary between Drosophila species groups, suggesting different fly species experience different parasite pressures (Morales-Hojas et al., 2009) . Insects with very different life styles, such as the social honey bees (Evans et al., 2006) and ants (Smith et al., 2011) , and symbiont-dependent aphids (Pennisi, 2009 ) likewise have immune systems that are surprisingly divergent from the Drosophila immune system.
The other major lineage of protostomes, the Lophotrochozoa, includes prominent groups such as the flatworms, annelids, and mollusks. In the polychete annelid Capitella capitata, TLRs have undergone an expansion to over 100 genes, most of which are similar, suggestive of recent duplication. Another annelid, the leech Helobdella robusta, has only 16 TLR homologs which are not only highly divergent from one another but also are not orthologous to any of the polychete sequences (Davidson et al., 2008) . In the freshwater snail Biomphalaria glabrata, FREPs are encoded by an expanded gene family, and are implicated in defense against gastropod parasites such as digenetic trematodes (Hanington et al., 2010a,b) . In B. glabrata, FREPs are particularly noteworthy for being comprised of juxtaposed IgSF and fibrinogen domains, and for the fact they are somatically diversified during the production of hemocytes by the snails (Zhang et al., 2004) . Expanded families of C-type lectins are present in other mollusks and the bivalve www.frontiersin.org Mytilus edulis is capable of generating diversified forms of the AMP myticin C both within and among individuals (Costa et al., 2009 ). Among invertebrate deuterostomes, the sea urchin has proven surprising in featuring dramatic echinoderm-specific expansion of several recognition molecules (Hibino et al., 2006) . Sea urchins possess >220 TLR genes (vertebrates usually have 21-25), >200 NLRs (mammals have 20-35), >200 SRCR genes (humans have 16; Messier-Solek et al., 2010) , and a novel Sp 185/333 gene family. The latter gene family produces a repertoire of defense proteins more diverse than the sequence diversity encoded in the genes, indicative of the presence of another mechanism to generate diversity (Ghosh et al., 2010) . Sea urchins also possess an NF-κB pathway, lectin and alternative complement pathways and homologs of RAG1 and RAG2, but do not produce immunoglobulins (Ig), T cell receptors (TCRs), or have a major histocompatibility complex (MHC; Hibino et al., 2006) .
With respect to our own phylum, the Chordata, the cephalochordate Branchiostoma (better known as Amphioxus or the lancelet), is novel in having expanded families of TLRs, NLRs, and SRCRs (Huang et al., 2008) , over 1,200 C-type lectins, and an extraordinary diversity in adaptors/facilitators and signaling/effector domains functioning downstream from their NLRs (Huang et al., 2008; Messier-Solek et al., 2010) . Amphioxus also possesses distinctive variable region-containing chitin-binding proteins (VCBPs; Dishaw et al., 2008; Cannon and Litman, 2009) which are further distinguished by high levels of polymorphism, resulting in yet another distinct "hyper-diversified," multigene immune receptors family (Dishaw et al., 2010) . Cephalochordates have a functioning complement system operating via the alternative and lectin pathways, including with a distinctive expanded number of C1q-like genes (Huang et al., 2008; Messier-Solek et al., 2010) . A RAG1 gene is present, and possibly a RAG2 gene as well (Dong et al., 2005) .
The urochordates, or tunicates, the sister group to the vertebrates, in the same immunological vein as nematodes and flies, are surprising for what they do not have. None of the genes playing a pivotal role in adaptive immunity in the jawed vertebrates are present. MHC, TCRs, Ig, RAG, and activation-induced cytidine deaminase (AID) genes are all lacking. V-like and C1-like domains are present and VCBPs have been identified (Cannon et al., 2004) , and they do have complement components, three TLRs, an expanded family of C-type lectins and FREPs. However, urochordates lack obvious expansions of any gene family highly relevant to vertebrate immunity (Azumi et al., 2003) . Based on what we know thus far, genome reduction is the hallmark of urochordate immunobiology.
Even closer to home are the agnathans or jawless vertebrates, lampreys and hagfish, the sister group to the jawed vertebrates or gnathostomes. We now know they lack RAG1 and RAG2 and do not produce TCRs or Ig, however, they have a remarkable ability to make highly diverse variable lymphocyte receptors (VLRs) that consist of somatically re-arranged modules containing leucine-rich repeats (Pancer et al., 2004) . It is striking that agnathans and gnathostomes have adopted divergent solutions to the same problem of generation of recognition capability, both of which involve rearrangements of germ-line encoded genes, but in entirely different ways with different starting sets of molecules.
The basic gnathostome immune system, the one most familiar to immunologists, features a close collaboration between innate and adaptive arms. As noted above, relative to some of the invertebrate deuterostomes such as echinoderms or cephalochordates, gnathostome innate immune components are modest in numbers, typically possessing 10-25 TLRs and 20-35 NLRs (Messier-Solek et al., 2010) . The gnathostome adaptive immune system features somatic diversification of both TCRs and Ig, requiring for this process RAG1 and RAG2, the former likely derived and modified from a transib-like transposon (Fugmann, 2010) . The gnathostome immune system works in conjunction with a unique antigen processing and presentation system, the MHC, to limit self-reactivity. It is notable for its specificity, its emphasis on expansion of relevant clones of lymphocytes, and for its memory and capacity to produce heightened secondary responses long after primary stimulation . The ongoing discovery of new types of immune cells (Neill et al., 2010; Saenz et al., 2010) and novel receptors (Parra et al., 2007) strongly suggests there are more fundamental insights to come with respect to gnathostome immunology. Furthermore, and a point relevant for the general discussion here, there is considerable variability among gnathostomes in how their immune systems function (Flajnik and Kasahara, 2010) .
To conclude this overview, it is indeed remarkable that organisms as diverse as cnidarians and humans have some immune architecture such as TLRs (and associated pathways) and NLRs in common. However, it is argued from the examples provided above that at least as compelling are the differences among and within phyla, even among species in a genus. Surprises abound, such as in the unexpectedly complete set of immune genes found in basal cnidarians and the immune genome reductions exhibited by nematodes, arthropods, and urochordates. Even the more familiar examples of conservatism such as TLRs and NLRs in arthropods and vertebrates may have been derived independently (Hughes, 1998; Hughes and Piontkivska, 2008; Zhang et al., 2010) . Large lineage-specific gene expansions such as noted for echinoderms, and domain reshuffling such as for invertebrate NLRs (Zhang et al., 2010) have occurred, creating remarkable heterogeneities among and within phyla. Layered on top of this are other forms of innovation such as elaboration of novel signaling pathways and production of associated AMPs in nematodes, distinct antigen recognition and melanin-deposition systems in arthropods, and the emergence of several distinct mechanisms for generating diverse antigen receptors in mollusks, arthropods, echinoderms, cephalochordates, agnathans, and gnathostomes. From this it is concluded that immune systems across and within phyla have a remarkable propensity to generate novelty and distinctiveness. As we learn about the immune systems of more animals, this diversity is bound to increase.
It is hardly surprising that immune systems are so variable. Animals have been extant and diversifying for up to 800 million years (Erwin et al., 2011) . They have adopted a diversity of life styles: sessile, colonial predators; inhabitants of extreme environments dependent on chemosynthetic symbionts; animals that are at times net producers of energy due to their photosynthetic symbionts; pelagic species that live in enormous schools; inhabitants of arid terrestrial environments; social species living in large colonies; and endoparasites that are so modified morphologically as to belie their origins, to name just a few. These different life styles will impose very different exposures to potential parasites.
Similarly, life histories vary radically from wind-dispersed organisms like tardigrades or rotifers that live in ephemeral habitats or that have life spans measured in hours, to sessile filter feeders like marine bivalves that routinely live for over 100 years in the same location. The role of life history traits such as survival rates and reproductive output are predicted to strongly influence the extent and kinds of particular immune responses that might be expected both among and within particular host species (Lee, 2006) .
Another factor likely to have influenced immune capability is the nature and extent of commitment to mutualistic symbionts. Some animals have established mutualistic associations with what are essentially monocultures of specialized bacteria (Nyholm and Nishiguchi, 2008; Pais et al., 2008) . Others, like ourselves, are dependent on a diversity of both archaeal and bacterial mutualists to which our immune system has made extensive accommodation. Third party symbionts have long played a role in educating, augmenting, and modulating animal immune systems (Turnbaugh et al., 2007) . The outcome of host-parasite interactions is often influenced by third party symbionts which probably play a far great role in host defense in many animal groups than customarily realized (Loker, 1994; Welchman et al., 2009; Gross et al., 2009; Eberl, 2010) .
The adoption of body plans differing in complexity and mass has also influenced immune system structure and function. It has been argued that evolution of the vertebrate jaw and an accompanying predatory life style introduced parasites into the gut and required a more elaborate adaptive immune system that now typifies gnathostomes (Matsunaga and Rahman, 1998) . It has also been argued that the complexities of adaptive immunity could not have evolved in animals with limited numbers of cells or with small size or simplified body architecture (Hauton and Smith, 2007) . With respect to body mass, for vertebrates, it has been suggested that the number of B and likely T cells in a clone scale with body mass as does the B cell repertoire (Wiegel and Perelson, 2004) . The general point is that the adoption of different habitats, life histories, symbionts, and bodies of differing body mass and complexity are all factors that will influence immune system design and mode of action.
In addition to the above considerations, all organisms have to contend with another category of symbionts, namely parasites. Because viruses, bacteria, and protists were present before animals arose, all animals from their inception would have had to contend with these parasites. Several modes of transmission of such parasites among early animals of disparate lineages were available, including: intimate proximity of many different kinds of animals (such as on a coral reef), predation, presence of vectors imbibing blood or plant juices containing parasites, and even one parasite serving as a vector for another, as for example a trematode vectoring a bacterium into new animal hosts.
In such a situation, where frequent transfer of parasites was possible among hosts from even disparate phyla, if all emerging animal lineages had the same defenses, it would be possible for an effective parasite that had overcome the defenses of one host group to simply spread into another host phylum. Consequently, having an immune system with distinctive means of antigen recognition and/or novel effector mechanisms would have been a distinct advantage when inevitably confronted with parasites that had evolved in other host groups (Figure 2) . Being immunologically different increases the odds that parasites from other inhabitants of the same coral reef are not as easily acquired. The notion that a parasite can track and exploit a common host genotype creating an advantage of rareness has been predicted and observed in specific host-parasite systems (Trachtenberg et al., 2003; Wolinska and Spaak, 2009 ), further suggestive of a similar dynamic favoring distinctiveness or "avoidance of commonness" among members of different host lineages.
Once animals began to diversify, a major trend was for some animals to parasitize others. Some animal parasites became wholly or largely committed to particular lineages of animal hosts, in which they subsequently diversified. Such lineage-specific parasites (some examples in Table 2 ) are another general factor expected to drive immunological novelty. These parasites often establish prolonged, intimate, and extensive infections in their chosen hosts that have profound fitness consequences such as castration or death (Lafferty and Kuris, 2009a) . Furthermore, given the phylogenetic diversity represented among these parasites, it is not surprising they would evolve novel methods of infectivity. For example, ichneumonid and braconid hymenopteran parasitoids have acquired mutualistic polydnaviruses that function to suppress the immune responsiveness of their hosts and facilitate parasitoid infection (Webb et al., 2009) . In contrast, without the aid of viral symbionts, larvae of digenetic trematodes secrete both anti-oxidants and immunosuppressive factors that down-regulate snail host immune components for a period sufficient to enable them to complete their lengthy period of larval development (Hanington et al., 2010a) . Therefore, the immune response devised by a particular host lineage afflicted with its own phylogenetically distinct, host specific, and harmful parasites would likely be divergent from the responses mounted by a different host group experiencing its own lineage-specific parasites (Figure 2) . This is not to imply that only animal parasites have developed lineage-specific associations with hosts, but merely serves to show that specialized parasites can help us understand the origins of immunological novelties.
The macroevolutionary patterns noted above with respect to novelty in defense strategies among animal lineages could not occur if there were not microevolutionary processes ultimately involved in www.frontiersin.org FIGURE 2 | One scenario for early in the divergence of animals is that different lineages (A-D) have fundamentally similar immune systems such that they all are colonized by the same parasites. In the case shown at the top, an immunological innovation occurred in lineage A, that allowed it to resist these parasites. This may have permitted a subsequent radiation in "parasite-free space" in this host lineage. At the bottom, lineage D has acquired a lineage-specific parasite different from those previously experienced. This requires an immunological accommodation that causes the immune system of lineage D1 to diverge. Both the host lineage and the lineage-specific parasites along with them may subsequently diverge. generating them. These microevolutionary processes occur below the species level, within and among populations of either host or parasite species, and might culminate in speciation of either participant. Speciation may be accompanied by colonization of new habitats, and further divergence to create major new lineages. Starting with the process of parasite diversification, the sections below discuss how the involvement of immunology in microevolutionary processes could lead to events that can help explain the macroevolutionary patterns discussed above.
The following quotes outline some of the key ideas for how immunity can play a role in generation of parasite diversity.
"For a pathogen, the selective pressures arising from the host immune system are a major influence on the evolution of mechanisms of infectivity and of immune-recognition avoidance" (Acevedo-Whitehouse and Cunningham, 2006) .
"In parasitism an essential factor in survival is immune escape, which allows a parasite to resist host attack. Immune escape is a mechanism for reducing gene flow at the level of the compatibility filter because its result is assortative survival (Combes and Théron, 2000) as opposed to assortative mating." (Combes, 2001, p. 154) .
"The stepwise coevolutionary process results in extreme specialization and complex defense mechanisms. . .specialization is likely to increase the rate of speciation that may occur in both host and parasite" (Price, 1980) "Host specificity thus is an ideal prerequisite for rapid speciation" (Mayr, 1963) .
Parasites are often cited as examples of specialists because they have limited ranges of host species, often with restricted ranges of habitats within their chosen hosts. For example, the lineage-specific parasites mentioned above, often show considerable specificity to particular species or genera within their adopted host lineages. Most animal parasites are host specific (Poulin and Keeney, 2008; Agosta et al., 2010) , but this by no means is to suggest that generalists do not occur: parasites like Schistosoma japonicum, Toxoplasma gondii, Borrelia burgdorferi, or the rabies virus routinely infect a remarkably broad range of host species. Using molecular techniques to identify parasites, species formerly considered to be generalists have in some cases been shown to be complexes of cryptic, host specific species (Poulin and Keeney, 2008) .
As eloquently documented by Combes (2001) , both encounter and compatibility filters operate to restrict the spectrum of host usage. Encounter filters pertain in situations where host and parasite live in different geographic localities, have different ecological circumstances, or where host or parasite behavioral tendencies preclude contact. The compatibility filter refers to barriers imposed by the host that prevent infection once contact has occurred. The compatibility filter includes both physiological and biochemical suitability of the host to support the parasite, and the active defense provided by the immune system.
Encounter filters are undeniably important in restricting parasite host range. Many examples of emerging infections (Goss et al., 2009; Gray et al., 2009; Pfeffer and Dobler, 2010) owe their emergence to a change in the encounter filter such that a new combination of parasite (often including its vector) and host are juxtaposed (Daszak et al., 2000; Parrish et al., 2008; Weissenbock et al., 2010) . Emerging diseases are an indication that parasite infectivity is not always dependent on a long accommodation to a particular host species: lack of contact may have prevented prior infections. Similarly, experimental infections of new hosts with parasites essentially bypass the encounter filter, and are sometimes successful (Poulin and Keeney, 2008) , affirming the reality and importance of the encounter filter.
With respect to the compatibility filter, a role for unsuitability should not be discounted and could be manifested in several ways, such as a lack of receptors needed for efficient viral entry into cells (Parrish et al., 2008) , lack of appropriate structures for parasite attachment (Tompkins and Clayton, 1999) , or by a general failure to provide the biochemical environment needed for the parasite to survive (Sullivan and Richards, 1981) .
Although unsuitability is probably underappreciated with respect to preventing infections, relying on the possibility of being unsuitable is not a cogent defense strategy. The importance of active immunity to the compatibility filter is illustrated by several lines of evidence.
(1) As illustrated by HIV, when the immune system is compromised, the door is opened to opportunists that themselves can become life-threatening (Holmes et al., 2003) .
(2) Genetic defects in the immune system, such as with TLRs, are associated with increased susceptibility to several different pathogens (Qureshi and Medzhitov, 2003) . (3) Experimental exposures of hosts to parasites they have not previously encountered often fail (Bowen, 1976; Bozeman et al., 1981; Vidal-Martinez et al., 1994; Philips and Clarkson, 1998; Sapp and Loker, 2000; Duke, 2004; de Vienne et al., 2009; Giraud et al., 2010) or the parasite replicates poorly or is inefficiently transmitted in a new host (Komar et al., 2003; Parrish et al., 2008) . Table 3 provides examples of parasites in novel hosts that are engaged and killed by immune responses. (4) Host defense genes are under strong selection and are conspicuous for evolving quickly (Sackton et al., 2007; Viljakainen et al., 2009; Barreiro and Quintana-Murci, 2010; Schulte et al., 2010) . (5) The extraordinary diversity of strategies undertaken by parasites to evade, manipulate, or suppress the immune system is testament to the impact of immunity on their success (Schmid-Hempel, 2008) . These evasive strategies have been shown in some cases to be specific with respect to the particular hosts involved (Table 4) , providing a mechanistic basis for the connection between immunity and parasite specialization.
Given that the consequences to a parasite for engaging the compatibility filter of an atypical host could be disastrous and result in its death, strong selection to avoid colonization of such hosts would be expected in some cases. Similar considerations may also apply to the host as well, as mounting an immune response can be costly and detrimental (Graham et al., 2011) . This means that some avoidance behaviors attributed to the encounter filter may actually be a consequence of the operation of a strong host immune response (Kuris et al., 2007; Keesing et al., 2009) .
To conclude this section, specialization and attendant host specificity is a central, emergent property of parasitism and has multiple underlying determinants, involving both encounter and compatibility filters. The ubiquity of host defenses and the evidence that they often eliminate novel parasites argue that host immune systems play a critical role in limiting parasite host www.frontiersin.org Table 3 | Examples of colonizing parasites, or parasites placed in novel hosts, that are killed or limited by immune responses.
Infection of the crab Pachygrapsus marmoratus with the rhizocephalan barnacle Sacculina carcini results in melanization of larvae in thoracic ganglia (Kuris et al., 2007) Antibody/factor that activates complement in serum of the non-host Raja radiata kills the tapeworm Acanthobothrium quadripartitum whereas larvae survive in serum of the normal host, Raja naevus (McVicar and Fletcher, 1970) Destruction of cercariae of avian schistosomes in the skin of mammals associated with a mixed Th1/Th2 lymphocyte cytokine response followed by more polarized Th2 response upon repeated exposures (Horak and Kolarova, 2005) Encapsulation of hymenopteran parasitoids by hemocytes of non-permissive insect hosts (Schmidt et al., 2001) Lysis of the trypanosome Trypanosoma brucei brucei by apolipoprotein L-1 in serum of humans who are refractory to this subspecies (Wheeler, 2010) .
Disruption of the Erk-STAT1 signaling pathway allows cross species transmission of the normally rabbit-specific myxoma virus to mice (Wang et al., 2004) Animal handlers who were exposed to a new coronavirus developed antibodies to the new virus and did not develop clinical infections (Guan et al., 2003) Species specific forms of APOBEC3G and other innate, intracellular defense components, can prevent cross species transfer of lentiviruses (Mangeat et al., 2004; VandeWoude et al., 2010) 
A staphylococcal complement inhibitor that specifically blocks human C4b2a and C3bBb, interfering with additional C3b deposition through classical, lectin or alternative pathways (Rooijakkers et al., 2005) . Sung et al. (2008) found several genes conserved in all Staphylococcus aureus isolates from humans were variable or missing in one or more animal isolates, including fnbA, fnbB, and coa. Human and murine chlamydial infections depend on different virulence factor genes that coevolved to counter host species specific IFN-γ-mediated effector responses mounted by the particular host species (Nelson et al., 2005) .
Orf virus is evolutionarily adapted to sheep as its primary host (Seet et al., 2003) . Different strains of influenza A virus likely have NS1 genes adapted to antagonize the IFNα/β antiviral system of their specific host species (Garcia-Sastre,
In a review of the interactions between monogenean parasites and their fish hosts, Buchmann and Lindenstrøm (2002) concluded that "immune evasion mechanisms are probably a main factor in host specificity." Rosengard et al. (2002) noted that the smallpox inhibitor of complement enzymes (SPICE) is nearly 100-fold more potent than the vaccinia homolog in inactivating human C3b and sixfold more potent at inactivating C4b, providing evidence for how variola proteins are particularly adept at overcoming human immunity relative to vaccinia.
The host specificity of three species of Bacillus (B. cereus, B. thuringiensis, and B. anthracis) is determined by the presence of virulence plasmids that determine the type of particular virulence factors produced (Gohar et al., 2005). ranges and thereby at least in part dictate the specialization so characteristic of parasitic organisms. Also in support of this claim is that some patterns of parasite host specificity can be attributed to the operation of specific immune evasion strategies, and that such strategies are pervasive among parasites.
As exemplified by the parasites indicated in Table 2 , in addition to being specialized to exploit particular host groups, they are remarkably diverse in species, as are the lineages of many parasites. One of the potential consequences of specialization, including in an immunological context, is diversification in species, of both parasite and host lineages. The mechanisms involved in promoting speciation remain a matter of active investigation and for the discussion below, the purpose is to indicate that immunological phenomena may play a role in this process that deserves further attention. One prominent mechanism of parasite speciation is switching to a new host species, and the role of accommodation to the immune system of new hosts to permit such switches is discussed in a separate section below.
A second mechanism is co-speciation. For some parasite groups closely tied to their hosts and with limited options for colonization of new hosts, such as sucking lice on burrowing mammals (Light and Hafner, 2008) , speciation may occur if the hosts upon which they are found themselves speciate, often following a physical separation of populations of the host species. In such cases, persistence of new daughter parasite species should be favored by the fact that the parental species had already achieved successful accommodation to the parental host species. Although the actual role of specific immune phenomena in influencing the persistence of incipient parasite species in co-speciating systems is not known, an important underlying role for a preexisting immunological accommodation between parental host and parasite species that also favors persistence of the new parasite species should not be discounted.
Another important way in which specialization dictated by immunological phenomena can increase the probability of formation of new parasite species is by promoting intraspecific diversification. The interactions between a particular host and parasite species can be expected to be variable across space (Wood et al., 2007) . Parasite abundance will vary across local scales, possibly because of the variable presence of other hosts needed to complete its life cycle (Byers et al., 2008) . Other parasite species impacting the same host may be present or absent, such that the host experiences different overall parasite pressure in different locations within its range. Furthermore, the host itself will be variable across its range owing to its responses to other local circumstances. All of these factors conspire to create heterogeneities with respect to how the host potentially mounts immune responses to the parasite (Kraaijeveld and Godfray, 1999; Thomas et al., 2000; Lindstrom et al., 2004; Kalbe and Kurtz, 2006; Blais et al., 2007; Bryan-Walker et al., 2007; Scharsack et al., 2007; Matthews et al., 2010) . Variability within parasite species with respect to infectivity to their hosts is a pervasive phenomenon (Carius et al., 2001; Schulenburg and Ewbank, 2004; Seppala et al., 2007; Vorburger et al., 2009) and this is likely driven in part by variations in immune evasive measures taken by parasites (Hammerschmidt and Kurtz, 2005; Cornet et al., 2009; Vorburger et al., 2009 ). These dynamics are compatible with general theoretical predictions that parasite variation is driven by immunity, and hosts themselves are variable with respect to immunity due to pressure posed by parasitism (Frank, 2002) . Immune responses are drivers for parasite diversification (Summers et al., 2003; Lazzaro and Little, 2009; McKeever, 2009 ). An overall increase in intraspecific genetic variability, with that variation partitioned into regionally differentiated parasite populations accommodated to local host populations provide rich opportunities for further divergence.
One possibility for further divergence is that local adaptation to host immunity could potentially lead to "assortative survival" (Combes and Théron, 2000; Combes, 2001, p. 154) , meaning that the only options for mating (parasites frequently seek mates and undergo sexual reproduction within their hosts) occur between individuals able to survive in hosts with similar immune capability that are vulnerable to the same parasite immune evasive capacity (Giraud et al., 2010) . This would further accentuate local differentiation of parasites, potentially leading to ecological speciation of the parasite, particularly if subsequent gene flow is prevented by failure of immune adapted parasites to thrive in hosts (from other localities) with different immune capacities.
It must be noted that fluctuations in local patterns of abundance of hosts and parasites may diminish the strength of local adaptation and promote gene flow such that speciation is precluded (Lazzaro and Little, 2009) , and that in general, evidence that parasite speciation is effected by underlying immune mechanisms is sparse. However, given the need for parasites to accommodate to a host's internal environment and that a host species is likely to be confronted with varying parasite pressure, it seems host immune responses will favor diversification in parasite lineages. To add an additional dimension to the concept that spatially variable relationships favor parasite diversification, it has recently been argued a general underlying mechanism favoring biological diversification is the existence of localized parasite-coevolutionary races that select hosts to prefer immunologically similar conspecifics and to avoid out-group individuals, thereby minimizing the risk of exotic disease acquisition (Fincher and Thornhill, 2008) . By promoting strong intraspecific diversification within host species based on avoidance of contagion, this mechanism has also been predicted to lead to parasite diversification (Fincher and Thornhill, 2008) .
To conclude this section, all of these observations fit into the more generalized geographic mosaic theory of coevolved relationships (Thompson, 2005) : in this particular case, local adaptations based on immunological accommodation of host and parasite can lead to diversification of parasites and potentially speciation.
An important way diversity in parasite lineages is generated, one that has increasingly come to light from molecular phylogenetic studies and the study of emerging diseases, is via switching to new hosts ( Table 5) . Although successful host switching cannot be a ubiquitous process, otherwise we might expect to find only a few species of generalist parasites instead of a predominance of host specific parasites, clearly it has been an important factor historically and examples continue to be regularly documented. A priori, it seems logical that most successful switches would be to hosts not phylogenetically distant from the original host species. Such close range switches are likely favored by a degree of phenotypic plasticity and preadaptation (exaptation) of the parasite and its use of phylogenetically conserved resources in the new host species such that new attributes are not needed to overcome a new host's immune system (de Vienne et al., 2009; Agosta et al., 2010) . For example, in a study of host switches in bats involving the fast-evolving RNA virus causing rabies, the success of cross species transfers diminished as the phylogenetic distances among the hosts involved increased (Streicker et al., 2010) .
It is also possible for parasite switches to occur when the original and new host species are not closely related (Brant and Loker, 2005) . This has been observed for emerging human parasites for which ungulates and carnivores were more likely originating host species than primates, and it was concluded that an already broad host range as opposed to the phylogenetic relatedness of the new and old host species was the more important factor dictating success in interspecific parasite jumps (Woolhouse et al., 2005) .
In any case, a host switch can lead to a speciation event if the parasite in the new host becomes isolated from the founding stock, or can have even more profound effects if the switch is into a new host lineage and leads to the founding of a diverse new parasite lineage (Agosta, 2006; Janz et al., 2006; Hoberg and Brooks, 2008; Martinsen et al., 2008; Refrégier et al., 2008; Winkler et al., 2009; Giraud et al., 2010; Nyman, 2010) . The isolation of the switching parasite from the founding stock is reinforced because even a single individual may be able to establish a new population and because differing ecological circumstances of the new host may preclude mixing of parasite progeny with the source population: the new parasites may never get back into the original host and thus mate only with other parasites in the new host. Assortative survival and mating would again be factors favoring isolation of the founding parasites. Host switching is also relevant to the idea stated above that if a host lineage acquires a new parasite, it may then have its immune system substantially altered. Particularly if the parasite is successful and radiates, then the immune system of the new host lineage may be forced to diverge to adjust to the new challenge.
www.frontiersin.org Table 5 | Examples of parasite groups exhibiting hosts switches likely to have played a major role in diversification of that group.
With respect to Plasmodium and related genera of blood parasites, major clades are associated with shifts into different families of dipteran vectors, and the Plasmodium species of birds and squamate reptiles show evidence of repeated switching back and forth (Martinsen et al., 2008) . Major lineages within the blood fluke genus Schistosoma are defined by acquisition of different genera of even families of snail intermediate hosts, by host switching (Morgan et al., 2003) . Long-range host shifts involving acquisition of both new snail and vertebrate hosts appear to have occurred during the history of schistosomes (Brant and Loker, 2005) . Zietara and Lumme (2002) note that as many as 20,000 species of the monogenean genus Gyrodactylus may exist, and note that in a study of one subgenus (Limnonephrotus) that several host switch events were statistically confirmed, including into new host families, supporting the idea that host switching is a means to drive innovation and adaptive radiation in these ectoparasites.
It appears that host switching has been common in trypanorhynch tapeworms, one of the most diverse and abundant groups of metazoan parasites of elasmobranchs (Olson et al., 2010) Coronaviruses have likely undergone several host switches, between mouse and rat, chicken and turkey, birds and mammals, and between humans and other mammals (Rest and Mindell, 2003) .
Braconid wasps of the subfamily Euphorinae have undergone extensive host switching among phylogenetically distantly related insect host groups, often followed by adaptive radiations of the parasitoids within a particular host lineage (Shaw, 1988) .
"Infection of a novel host is the most frequent cause of fungal emerging disease" (Stukenbrock and McDonald, 2008; Giraud et al., 2010) Several examples from the literature of emerging infectious disease indicate that switches are often favored by changes in the encounter and not the compatibility filter (Woolhouse et al., 2005) . Ecological circumstances have exposed humans to a parasite they previously did not encounter. Such examples of host switches, particularly if the new host is distantly related to the original host, would seem to argue against the points made in the preceding sections regarding the importance of parasite accommodation to the idiosyncrasies of their host's immune system. If immunity is important in restricting parasite host ranges, how can such switches occur?
First, these conspicuous successes need to be weighed against all the encounters between novel parasite and host combinations that fail and therefore go unnoticed, which is likely a far more frequent outcome (de Vienne et al., 2009; Tunaz and Stanley, 2009; Giraud et al., 2010 ; see also Table 3 ). In cross species transfers of rabies into bats, the vast majority are dead ends: they did not establish sustained infections (Streicker et al., 2010) . Although some of the failures could be explicable because of less frequent contact among more distantly related bats (the encounter filter), increasingly divergent defense systems leading to higher levels of innate resistance were also invoked as an explanation (Daszak, 2010; Streicker et al., 2010) . The role of immune systems in preventing such infections would be easy to underestimate because the result is a failed experiment that in all probability we never even knew had happened. In a similar vein, a survey of fieldtrapped insects in turkey revealed that 98% exhibited some kind of melanotic hemocyte nodule (Tunaz and Stanley, 2009 ). Such host reactions provide a convenient historical record of previous parasite encounters (Kuris et al., 2007) . It was concluded that insects are regularly challenged by infections from which they recover. The action of innate immunity in routinely preventing acquisition of new parasites is probably considerable and easy to underestimate.
Secondly, host switches would be favored if the new parasite, as exemplified by HIV, directly attacks the host's immune system and compromises it, or if the new host is immunocompromised by some other means. Diminished levels of immune competence can occur for several reasons, including ones likely to have been in operation throughout animal evolution. One possible means is that the host's indigenous parasites might use immunosuppression to favor initiation and persistence of their own long-term infections ( Table 6 ) and thereby facilitate colonization of that host by other parasites (Krasnov et al., 2005) . An intriguing possibility is that the successful colonization of a host species by one or more immunosuppressive parasites might then favor colonization by opportunist parasites, resulting in an unusually diverse parasite fauna supported by that host. The large number of species of larval digenetic trematodes known to be supported by some snail species (Loker et al., 1981; Lafferty and Kuris, 2009b) might exemplify this possibility.
High host density, stressful thermal (Bruno et al., 2007) or oxygen regimes (Aeby and Santavy, 2006) , and even mating (Rolff and Siva-Jothy, 2002) are some natural situations that can also lower immune competence. To this list can be added a number of human-imposed immune stressors including crowded aquaculture conditions (Flemming et al., 2007) , use of harmful chemicals on fields or roads (Rohr et al., 2008; Karraker and Ruthig, 2009) , altered diets (Sahu et al., 2008) , elevated or altered environmental nutrient conditions (McKenzie and Townsend, 2007; Wedekind et al., 2010) , and deliberate implementation of immunosuppressive therapies. Not only can these situations lower host immune competence, they may also increase parasite virulence and thereby alter probabilities of successful colonization in a new host species (Wedekind et al., 2010) .
A switch into an immunocompromised individual of a new host species is likely to be temporary and not lead to speciation unless the new parasite can better adapt to its new host, at the same time with minimal gene flow occurring with conspecifics from its ancestral host. Availability of populations of similarly immunocompromised new hosts that allow continued transmission and adaptation of the new parasite host could favor divergence from the founding parasite and speciation. Varroa mites (Varroa destructor ) in honey bees (Apis mellifera) suppress the activity of several immune-related genes (encoding both antimicrobial peptides and enzymes) and favor higher infection titers with the deformed wing virus (Yang and Cox-Foster, 2005) . Drosophila simulans infected with Wolbachia have reduced ability to encapsulate eggs of the parasitoid Leptopilina heterotoma (Fytrou et al., 2006) .
The malaria parasite Plasmodium gallinaceum suppresses the encapsulation response of the mosquito Aedes aegypti (Boëte et al., 2004) .
Two acanthocephalan parasites Pomphorhynchus laevis and Polymorphus minutus both have the effect of decreasing the standing level of immune defense (as measured by reduced phenoloxidase enzyme activity) in their local gammarid hosts, Gammarus pulex, but not in their more recently introduced host Gammarus roeseli (Rigaud and Moret, 2003) .
Hymenopteran parasitoids induce immunosuppression in their host insects in part by the injection of polydnaviruses which target and inhibit both cellular and humoral components of the host response (e.g., Labropoulou et al., 2008) and the parasitized hosts become increasingly susceptible to opportunistic infections by viruses (Rivkin et al., 2006) , bacteria (Shelby et al., 1998) , and other parasitoids (Guzo and Stoltz, 1985) .
As noted by Lie (1982) , interference by trematode larvae with gastropod defense responses appears to be a common consequence of infection (Hanington et al., 2010a) , and the presence of one trematode infection can facilitate the colonization of an infected snail by trematodes that would not ordinarily be able to infect that species of host (Walker, 1979; Southgate et al., 1989) .
HIV in people was associated with parasites that rarely if ever had been implicated in causing human disease including microsporidia, cryptosporidia, JC virus, and Mycobacterium avium (Kovacs and Masur, 2008) .
Studies of parasite communities suggest that taxonomic distinctiveness of ectoparasites and endoparasite richness are positively correlated across species of rodent hosts, indicative of immune responses to some parasites depleting energy reserves and facilitating colonization by others (Krasnov et al., 2005) .
How might immunological phenomena influence the degree of diversity shown among the hosts contending with parasites? As highlighted below, parasite pressure clearly favors immunological diversification at microevolutionary scales. Whether this diversification contributes in a meaningful way to host speciation remains controversial, but has attracted considerable attention and is gaining some support, as discussed below.
As early as Haldane (1949) , and as more recently underscored (Frank, 2002; Lazzaro and Little, 2009) , it has been recognized that parasites drive polymorphisms in host immune competence, particularly in variable environments. This can occur by balancing (Wegner, 2008) or disruptive selection (Duffy et al., 2008; Matthews et al., 2010) . High levels of polymorphism are found in several genes of both the innate and adaptive immune systems (Hill, 2001; Trowsdale and Parham, 2004; Acevedo-Whitehouse and Cunningham, 2006) . MHC genes show a predominance of non-synonymous over synonymous mutations in their peptidebinding regions, and have extensive allelic diversity, indicative of strong role of selection, generally considered to be mediated by parasites. For example, parasite pressure is believed to have promoted maintenance of high MHC diversity in sticklebacks (Wegner et al., 2003) and Atlantic salmon (Dionne et al., 2007) . In humans, regional differences in HLA class I diversity has been associated with intracellular parasite richness (Prugnolle et al., 2005) . Across species comparisons of rodents have associated helminth species richness with increased MHC class II polymorphisms (de Bellocq et al., 2008) . MHC genes are also known for their role in mediating mate choice through olfactory systems in humans (McClintock et al., 2002) , rodents (Sommer, 2005) , and fishes (Landry et al., 2001; Milinski et al., 2005) .
The evidence supporting the idea that variability in immune response driven by parasites can be a factor favoring speciation of host lineages is mostly correlational, but is supported by a growing body of literature. One general factor favoring this is the development of strong local immunological accommodations of particular host populations to the distinctive parasites they encounter, such that across a broader host range different host populations differ significantly with respect to the nature of their responses (Wheatley, 1980; Blais et al., 2007; Scharsack et al., 2007; Matthews et al., 2010) . For example, malaria might be encountered in some but not all parts of the host range where appropriate mosquitoes were present, or different suites of parasites might be encountered in different foraging habitats such as rivers or lakes (Eizaguirre et al., 2011) . Differences among populations of the same host species with respect to their immune defenses have been noted in Drosophila against parasitoids (Kraaijeveld and Godfray, 1999) , Darwin's finches (Lindstrom et al., 2004) , sticklebacks coping with eye flukes (Kalbe and Kurtz, 2006) , and marine amphipods infected with trematodes (Thomas et al., 2000; Bryan-Walker et al., 2007) . Differentiation resulting from spatially variable antagonistic interactions with parasites would in this case provide the substrate for further diversification of host lineages (Thompson, 2005 (Thompson, , 2009 .
The impact of this local adaptation could be augmented by assortative mating mediated by sexual selection to favor further divergence (van Doorn et al., 2009 ). According to this line of thought, those locally adapted hosts that best resist parasites are able to elaborate ornaments that signal superior resistance to parasites, such that local mates preferentially breed with them. If these hosts were transplanted to other host populations with their own distinct parasite challenges, they would not be as resistant, their sexual ornamentation would suffer, and they would not be selected for mating. Thus a combination of natural and sexual selection could favor divergence of new host species.
Parasites can exert strong selection on traits known to affect mate choice (Hamilton and Zuk, 1982; Poulin and Thomas, 1999; MacColl, 2009) and in some cases the genes involved also have immune functions, such as genes of the MHC (Milinski et al., 2005; Milinski, 2006; Eizaguirre et al., 2009 Eizaguirre et al., , 2011 . MHC genes www.frontiersin.org have been considered as possible "magic traits" in sticklebacks, influencing both defense and mate choice (Matthews et al., 2010) . MHC divergence in a closely related and sympatric pair of cichlid species from Lake Malawi has been proposed to be a result of local host-parasite-coevolutionary associations, and to have influenced odor-mediated mate choice, and ultimately to have favored speciation (Blais et al., 2007) .
Diverging cichlids of the genus Pundamilia in Lake Victoria provide another example of how local adaptation and assortative mating (both potentially influenced by immune traits) may work together to promote host speciation. In this system, females have a preference for conspicuously colored males. These bright colors seem to be reliable indicators of male fitness, including resistance to parasites. Conditions of light penetration favor blue males in shallow depths and red males in deeper waters. The parasites encountered at different depths also vary in density and composition such that habitat-specific defenses could occur. Females from the depths prefer brighter red males whereas those from shallow water prefer brighter blue males, potentially leading to divergence, with visual cues playing a key role. This example points out the immunology may work in concert with a number of other forces such as predator avoidance or dietary preferences which all conspire to reinforce divergence of the two incipient species by visual means (Maan and Seehausen, 2010) . Mating among hosts between different locations would break down these differences, but might be disfavored if the progeny had reduced resistance to any local set of parasites.
"Infectious speciation" as exemplified by interactions between Drosophila species and inherited, endosymbiotic Wolbachia bacteria provides another possible mechanism for the involvement of immune processes in host speciation. For a group of six related species in the D. paulistorum complex, each species has its own distinct host specific obligatory and mutualistic Wolbachia with which it has achieved accommodation. This accommodation likely involves suppression of immune pathways involving apoptosis of infected cells. In hybrids, the Wolbachia over-replicate and cause embryonic inviability and male sterility, suggesting the unique host accommodation has been lost. In addition to such postmating isolation, it has also been shown that females can detect and will reject males harboring the wrong symbiont, thus further reinforcing isolation .
Among animal hosts, hybrids are often more susceptible to parasites than parental species (see overviews provided by Fritz, 1999; Wolinska et al., 2008) , potentially favoring isolation of the parental host species, but other outcomes have also been noted and the responsiveness of hybrids to infection recorded 1 year might differ from those reported the next. This implies that the interactions between hybrids and the parasites they experience exhibit complex temporal dynamics and that the parasites themselves have undergone complicated changes as a result of their hosts' hybridization history that have not been sufficiently investigated (Detwiler and Criscione, 2010) . Certainly some studies suggest that isolation of incipient parental species might be reinforced by a breakdown of co-adapted immune gene complexes among their hybrids. Also, in some cases, the act of hybridization contributes to the formation of new species by allopolyploidy, as has been postulated for anurans of the genus Xenopus. Hybrids in this case often have increased resistance for parasites, potentially providing a selective advantage to favor the persistence of new species of recent hybrid origin (Jackson and Tinsley, 2003) .
Another hypothesized mechanism favoring diversification of host lineages is that localized interactions with particular parasites allows immunological accommodation to them, resulting in strong preference for interactions with individuals with similar immune accommodation and philopatry (limited host dispersal). This is coupled with avoidance of out-group individuals that might lead to introduction of exotic parasites (Fincher and Thornhill, 2008) . Diverse parasite populations are thus hypothesized to drive diverse host populations, and ultimately speciation, leading to a general correlation between host and parasite biodiversity (Fincher and Thornhill, 2008) .
Extinction too is a macroevolutionary process, including the major pulse of extinction events currently underway. Habitat destruction, human overpopulation, industrialization, threats from introduced species, emerging diseases, and global climate change, have lead to predictions that up to 50% of all species will be lost in the next 50 years (Pimm and Raven, 2000; Koh et al., 2004; Thomas et al., 2004) . The study of immunology is relevant to extinction in at least three broad contexts outlined below.
First, as noted above, the interactions between parasite and host often lead the parasite to specialization and host specificity which may in part be dictated by interactions with the host immune system. It has long been argued that specialization leads to a greater probability of extinction because if the host on which the specialized parasite is dependent undergoes severe population fluctuations or itself goes extinct, the parasite will soon follow: a co-extinction event has occurred. Co-extinctions involving pairs of mutualists or host-parasite units may be the most common forms of loss of biodiversity (Koh et al., 2004; Dunn et al., 2009) . By comparison, a generalist parasite able to exploit alternative hosts would have a greater likelihood of surviving under similar circumstances. The evolutionary trajectories taken by parasites have been much debated, and specialized parasites have been shown to give rise to large lineages of specialized, or even to more generalized descendents (Johnson et al., 2009) . However, host specificity remains a salient feature affecting the odds of extinction (Koh et al., 2004; Poulin and Keeney, 2008) .
Second, an inescapable feature of the modern world is the frequency with which invasions of exotic species occur (Torchin et al., 2002) . Invasive species might be either hosts or parasites (possibly including their vectors), and all can present dire and often unpredictable consequences, including extinction, for indigenous organisms (Wyatt et al., 2008) . A role for immunology in influencing invasions can occur in at least three ways:
(a) Introduced host species often leave their native parasites behind and although they are likely to be colonized by parasites in their new environment, in some cases this colonization is slow to occur, such that they experience relatively low parasite burdens for a long time. Insofar as immune responses are costly to mount and the harmful effects of parasites are avoided, the invading species may enjoy a distinct advantage over its competitors in its relative freedom from parasites, particularly if they can adopt relatively low cost defense measures against their maladapted parasites ( Lee and Klasing, 2004) . The relative inability of parasites to colonize the new intruder is a testament to the specialization often required to achieve infectivity, as noted above. (b) If an introduced host is accompanied by some of its indigenous parasite fauna, then related hosts in the newly colonized area may then have to contend with a parasite to which they are not accustomed, a process that may take decades to achieve Taraschewski, 2006) . This is particularly likely to cause problems if the colonized area is an isolated habitat like an island where hosts have simply not encountered comparable parasites previously. The devastating impact of the introduction of mosquitoes followed by avian malaria and avian pox on the endemic honeycreepers of the Hawaiian islands, likely causing both extinctions and slowing recovery of still extant species, is an iconic example (Atkinson and Samuel, 2010) . Islands often favor speciation yet the subsequent colonization of mainland habitats by island species is likely to be limited and unsuccessful due to active transmission of parasites there that a migrant is unable to handle immunologically. (c) In some cases, invasion of a parasite can occur even without the benefit of a simultaneous introduction of its indigenous host. An example is the eel swimbladder nematode Anguillicola crassus which has been introduced from the Orient into Europe where it provokes intense tissue reactions from European eel species (Taraschewski, 2006) . By contrast, Oriental eels mount immune responses that prevent a high and robust population of worms from building up.
All of these examples centered on introductions have macroevolutionary implications as they might lead to expansion and radiation of hosts and their parasites into new habitats, or may directly cause extirpations of indigenous hosts (and possibly their co-adapted parasites as well). Immunological phenomena provide proximal causal explanations. Once again, as with the discussion above regarding host switching, there may be significance to what we do not see as well: many parasite introductions fail because the colonist is unable to breach indigenous host defenses and host introductions fail because they are ill-prepared for indigenous parasites. For example, the introduction of the American rainbow trout Oncorhynchus mykiss into Europe failed because they encountered the parasite Myxobolus cerebralis which is normally transmitted by the indigenous brown trout, Salmo trutta: rainbows succumbed to whirling disease in Europe . When this parasite was introduced into North America, brown trout (themselves also introduced) were already well-adapted to it but indigenous rainbows and other salmonids were not and have suffered outbreaks of whirling disease as a consequence. A third broad context in which immunology becomes relevant to extinction, and one that is much in evidence today, is the role played by diminution of diversity in key immune loci such as the MHC. This can result in vulnerability of endangered host species to general parasite attack and thus extinction (Radwan et al., 2010) . Loci other than the MHC may also play major roles in dictating susceptibility to particular groups of parasites (Behnke et al., 2003) , and polymorphism in non-MHC genes are relevant in resistance to both tuberculosis (Ottenhoff et al., 2005) and malaria (Hill, 2001) . MHC genes are estimated to account for only about half of the genetic variability for resistance traits (Acevedo-Whitehouse and Cunningham, 2006) .
Regardless of the immune locus involved, the role of random drift in diminishing allelic diversity in bottlenecked or fragmented host populations would seem to increase the risk of successful parasite attack and greatly increase extinction probabilities. There is a need to determine if other factors like genome-wide inbreeding depression are more important in causing extinction, to understand why some species depauperate in MHC diversity seemingly continue to thrive (Hedrick, 2003; Radwan et al., 2010) , for increased transfer of information from the study of the main immunological models to endangered species, and to study other polymorphic genes involved in effective defense (Acevedo-Whitehouse and Cunningham, 2006) .
A picture of the structure and function of immune systems across animal phyla is slowly emerging, but so many organisms remain unstudied we lack perspective on how representative our current picture is. Do all tunicates, for example, reveal evidence of pronounced genome reduction with respect to immune function, or is this a characteristic of just the few species studied to any extent? What kind of pressure from parasites or otherwise has driven the expansion of all the innate immunity genes evident in organisms like sea urchins, and how do these animals regulate and orchestrate effective responses given the multiplicity of defense molecules they possess? In their 100+ years life span, how frequently must marine bivalves mount immune responses and how do they avoid the problem of fast-evolving parasites from "locking on" and overrunning them? In the meantime, it is clear that the approaches taken to achieve immune defense are diverse, frequently innovative and often capable of generating diverse repertoires of defense molecules that blur the distinctions commonly made to distinguish adaptive from innate immunity. The diversity in immune systems among and within phyla is in and of itself a major macroevolutionary pattern that should become a more central part of how we characterize animal diversity, including in textbooks. We need to know what the full pattern actually is, and the pattern begs an explanation for the processes involved in generating it.
Study of model parasite-host systems shows that the strength of selection imposed on particular immune genes is strong, and can result in some of the fastest evolutionary rates known for metazoans. In other words, parasite-host immune interactions strongly influence anagenesis, the evolutionary changes occurring within parasite or host lineages. If we adopt a broader perspective with a longer time frame, it seems likely such intense interactions will be seen to have an impact on cladogenesis, the origin of lineages, as well. It seems the overall impact of infection and immunity should attract as much attention as predation, competition, or other biotic interactions in shaping the overall diversity of animal life. Needed are more empirical studies over longer time www.frontiersin.org frames to provide more specific mechanistic insights as to how host immune responses drive diversification of parasites and how this can lead to speciation events, potentially in both parasite and host lineages. Further revelation of specific genes, often not considered part of the immune system per se, and how they facilitate defense against particular parasites and might favor evolutionary divergence among parasites are needed. Finally, placing immunology in a macroevolutionary perspective can hopefully provide insights for understanding today's world in which a host of rapid changes greatly increase the odds for extinction of many animals.
This work was supported by NIH grants AI024340, and by 1P20RR18754, the latter from the Institute Development Award (IDeA) Program of the National Center for Research Resources, NIH.
immunodeficiency virus type 1related opportunistic infections in sub-Saharan Africa. Clin. Infect. Dis. 36, 652-662. Horak, P., and Kolarova, L. (2005) . Molluscan and vertebrate immune responses to bird schistosomes. Parasite Immunol. 27, Recent Advances in the Diagnosis and Treatment of Influenza Pneumonia A potentially fatal complication of influenza infection is the development of pneumonia, caused either directly by the influenza virus, or by secondary bacterial infection. Pneumonia related to the 2009 influenza A pandemic was found to be underestimated by commonly used pneumonia severity scores in many cases, and to be rapidly progressive, leading to respiratory failure. Confirmation of etiology by laboratory testing is warranted in such cases. Rapid antigen and immunofluorescence testing are useful screening tests, but have limited sensitivity. Confirmation of pandemic H1N1 influenza A infection can only be made by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) or viral culture. The most effective preventive measure is annual influenza vaccination in selected individuals. Decisions to administer antiviral medications for influenza treatment or chemoprophylaxis should be based upon clinical and epidemiological factors, and should not be delayed by confirmatory laboratory testing results. Neuraminidase inhibitors (NI) are the agents of choice. Influenza is acute respiratory illness caused by influenza A or B viruses with seasonal circulation during the winter months. However, outbreaks of novel recombinant strains that take place in animals have produced, along the years, many worldwide outbreaks with serious public health issues. Although normally a self-limited process in the general population, high risk groups for complications and death have been identified (Table 1) . During an outbreak, in otherwise healthy subjects the diagnosis of influenza infection can be made confidently based on clinical manifestations alone. However, in certain situations, such as sporadic cases, in patients at an increased risk for complications, or in hospitalized patients with severe pulmonary compromise, confirmation of etiology by laboratory testing is required to guide treatment and for surveillance purposes [1••, 2••]. A potentially fatal complication of influenza infection is the involvement of the lower respiratory tract caused directly by the influenza virus, and the development of secondary bacterial pneumonia. Novel recombinant influenza A strains carry the risk for more severe disease and have the potential to cause widespread illness and a large number of deaths, regardless of age or previous health status. Examples of this are the H5N1 "avian" influenza A outbreak since 2004, and the more recent H1N1 "swine" influenza A pandemic in 2009, both of which have prompted the development of quick and reliable laboratory test in an effort to optimize their management and reduce morbidity and mortality. In this article, we review the latest available data for the diagnosis of influenza lower respiratory tract infection, as well as for treatment and prevention strategies.
Signs and symptoms of upper and/or lower respiratory tract infection, along with systemic involvement in the form of fever, myalgia, and headache, are usually the main presenting features of the disease. In the context of an outbreak, otherwise healthy subjects presenting with a self-limited acute febrile respiratory illness usually require no further diagnostic procedures. In two retrospective studies that examined which clinical signs and symptoms are most predictive of influenza infection in patients with influenza-like illness, cough and fever were the only symptoms significantly associated with a positive PCR test for influenza [3, 4] . In another study, no isolated symptom or sign was able to accurately predict influenza infection, though the absence of fever, cough and nasal congestion significantly decreased its likelihood [5] . In general, patients diagnosed with pandemic H1N1 influenza A virus had similar signs and symptoms compared to those with seasonal influenza. However, these patients had gastrointestinal manifestations more frequently [6, 7] , were more likely to have pneumonia [8] , and also had higher rates of extrapulmonary complications, intensive care unit admission, and death [9] .
Pneumonia is the most frequent and severe complication of influenza, most commonly presenting in high risk patients (Table 1) . Primary influenza pneumonia represents direct lung involvement by influenza virus, and should be suspected in non-resolving influenza infections. Typically, primary influenza pneumonia presents in chest x-rays with bilateral reticular or reticulonodular opacities. Less frequently, focal areas of consolidation can be seen, particularly in the lower lobes. High-resolution computed tomography may show ground glass opacities with or without multifocal peribronchovascular and subpleural consolidation [10] .
The cytopathic effect of the influenza virus on the tracheobronchial epithelium may predispose to secondary bacterial pneumonia [11, 12] . Secondary bacterial pneumonia must be suspected whenever there is an exacerbation of fever and respiratory symptoms after initial improvement in a patient diagnosed with acute influenza. Leukocytosis, instead of a normal or low white blood cell count, and lobar consolidation on chest imaging, instead of the diffuse pattern that is typical of viral pneumonia, are also suggestive [13] .
In an observational study of 543 hospitalized patients with H1N1 influenza A infection in Spain, 43 % of the 243 patients in which chest radiographs were performed had pneumonia, 83 % of the 210 patients who had microbiologic confirmation had primary influenza pneumonia, and the remaining 17 % had concomitant secondary bacterial pneumonia. Bilateral pneumonia occurred in 48.3 % of patients; Streptococcus pneumoniae being the most frequent pathogen [14] . Several reports have identified methicillinresistant Staphylococcus aureus (MRSA) as the etiologic agent for severe community acquired pneumonia (CAP) in otherwise healthy young patients with influenza [15] [16] [17] . In another study that investigated the incidence of communityacquired MRSA pneumonia in H1N1 influenza patients, 50 patients of 4491 (1 %) laboratory-confirmed pandemic influenza A (H1N1) cases had a bacterial respiratory tract pathogen. The most commonly cultured organisms were S. pneumoniae (16 patients), S. aureus (13 patients) and Haemophilus influenzae (9 patients); MRSA was detected in only 2 patients [18] . In contrast, among 838 children and adolescents admitted to 35 intensive care units in the U.S. with confirmed or probable severe H1N1 influenza A infection, 48 % of the 71 patients with suspected diagnosis of early S. aureus coinfection had MRSA [19] .
Non-seasonal influenza infections have specific clinical manifestations. Pneumonia related to the 2009 H1N1 influenza A pandemic was also found in many cases to be rapidly progressive, leading to respiratory failure and ARDS [20•, 21•] . Additionally, the risk for complications and death due to that (2) Including disorders of the brain, spinal cord, peripheral nerve, and muscle such as cerebral palsy, seizure disorders, stroke, intellectual disability, moderate to severe developmental delay, muscular dystrophy, or spinal cord injury Adapted from the recommendations of the Advisory Committee on Immunization Practices (ACIP) [40••] pandemic influenza was found to be underestimated by commonly used pneumonia severity scores [22•, 23] . Avian influenza (H5N1) frequently presents as severe primary pneumonia that often progresses rapidly to the acute respiratory distress syndrome (ARDS), having caused high rates of death, especially among infants and young children in Southeast Asian countries [24] .
In certain situations, confirmation of etiology by laboratory testing is required in order to guide the initiation and duration of antiviral therapy, and for the implementation of infection control measures and surveillance. Other benefits of influenza virus detection are the reduction of inappropriate antibiotic use, decreased length of stay in emergency departments, and fewer additional laboratory studies, all leading to a reduction in health care costs [1••] . The Centers for Disease Control and Prevention (CDC) and the Infectious Diseases Society of America (IDSA) have published guidelines to better define patients who should undergo influenza testing [1••, 2••]. The available methods include immunological techniques (i.e. rapid antigen-based tests, immunofluorescence assays, serologic testing), molecular techniques (i.e. reverse-transcriptase polymerase chain reaction [RT-PCR]), and microbiological techniques (i.e. viral cultures). While RT-PCR has the highest sensitivity and specificity, rapid antigen and immunofluorescence testing, though very useful as initial screening tests, are considerably less sensitive. Besides, the sensitivity and specificity of any of these techniques will vary depending on the laboratory equipment, personnel expertise, the timing of recollection, and the appropriate handling of the samples [1••, 2••]. Respiratory specimens can be obtained by many different methods including throat swabs, nasal aspirates, and nasopharyngeal swabs, aspirates and washing [2••] . In mechanically ventilated subjects, more invasive maneuvers such as endotracheal aspirates and bronchoscopic or non-bronchoscopic bronchoalveolar lavage (BAL) may be required in order to obtain adequate lower respiratory tract samples. While rapid antigen tests, immunofluorescence and RT-PCR all can yield results quick enough to guide point-of-care clinical decisionmaking, serologic tests and viral cultures provide retrospective diagnosis, availability only in reference laboratories, and usefulness when confirming screening test. For this reason, they are normally reserved for epidemiological and research purposes (Table 2) .
Rapid influenza diagnostic test (RIDT) are designed to detect influenza A and B nucleoproteic antigens in respiratory specimens, with results expressed qualitatively as positive or negative in no more than 15 min. Many different FDAapproved tests are available; while some assays are capable of distinguishing between influenza A or B viruses, none can distinguish between pandemic and seasonal strains of influenza A [1••, 2••]. Compared to reference methods (i.e. RT-PCR and viral culture), they have low sensibility (10 % to 80 %) and high specificity (95 % to 100 %), and there is also great variability between the different commercially available kits [25] [26] [27] . Sensitivity appears to be somewhat lower for influenza B than that seen for influenza A [26] . Also, the reported sensitivity for rapid tests is higher for nasopharyngeal samples than for throat swabs [27] . There are two main factors that influence RIDT's negative predictive value. First, negative results obtained during periods of high viral activity in the community are more likely to be false negatives; alternatively, false positives, though much less frequent, are more likely during periods of less viral circulation. Second, respiratory samples collected within the first 48 to 72 h of symptom onset positively influence RIDT's sensitivity. For these reasons, when rapid tests are negative, confirmation by means of RT-PCR or viral culture should be considered [1••, 2••]. Compared to RT-PCR, the sensitivity of RIDTs for detecting novel influenza A (H1N1) was equal to or lower than the sensitivity to detect seasonal influenza viruses [29] [30] [31] [32] , so RIDT results need to be interpreted with caution when evaluating patients suspected of having pandemic H1N1 influenza A.
Immunofluorescence Direct (DFA) or indirect (IFA) immunofluorescence antibody staining techniques are capable of detecting influenza A and B viruses, and distinguish the viruses from each other as well as from other respiratory viruses [1••, 2••]. They have levels of sensitivity and specificity that come close to those of RT-PCR [28, 33] , and results are often available in a few hours [1••, 2••]. Although these tests have improved sensitivity over RIDT, they are more technically complex and require expertise in obtaining quality respiratory samples ( Table 2) .
This is the reference influenza detection method and has the highest sensitivity and specificity [34, 35] . Several modalities of RT-PCR have been designed: conventional gel-based PCR (cRT-PCR), multiplex PCR (mRT-PCR), and real-time RT-PCR (rRT-PCR). They can differentiate between influenza types (A or B) and subtypes (including pandemic H1N1 influenza and avian H5N1 influenza), and results are available in 2-6 h (although due to transportation of batched specimens to reference centers for processing, it may take longer for results to be available). During the 2009 H1N1 influenza A pandemic, it became clear that rapid case identification was essential for timely management of patients and for adequate public health actions to be taken. To answer to this threat, the CDC optimized the previously developed rRT-PCR procedures for detection of the A/H1N1 2009 pandemic influenza virus [36• ]. Yang et al. compared the performance of 12 rRT-PCR primer-probe sets designed for detecting the hemagglutinin (HA) or the neuraminidase (NA) gene of the pandemic influenza A/H1N1 2009 virus, using the primer-probe set developed at the CDC as reference. They found that although all primer-probe sets had specificity levels as high as 98.4 % to 100 %, some of the primer-probe sets had better specificity, sensitivity, and amplification efficiency than others, and that a combination of primer-probe sets targeted to the HA and NA genes had higher detection sensitivity than those targeting HA or NA individually [37] . In another study, Lam et al. showed that although rRT-PCR assays can be 10-fold more sensitive than cRT-PCR, newly developed cRT-PCR assays targeting the HA gene are a reliable alternative for laboratories where a rRT-PCR machine is not available [38] . Sensitivity and specificity of these assays also depend on the type of respiratory sample employed. For example, in a Spanish study that assessed the utility of rRT-PCR for the diagnosis of the novel influenza A/H1N1 virus, the authors reported that the diagnostic yield of combined nose and throat swabs was higher than that of nasopharyngeal aspirates [39] .
Even the fastest viral cultures techniques can take days to demonstrate influenza cytopathic effects. Since they are not suitable for initial clinical management, their utility during an influenza outbreak is to confirm some negative test results from RIDT and immunofluorescence. Viral cultures also provide information about circulating influenza strains and its subtypes; this information is required for next season vaccine production, for surveillance of the emergence of new influenza A strains, and for the detection of antiviral resistance. Similarly, serologic tests (i.e., hemagglutinin inhibition, ELISA, complement-fixation) that demonstrate a four-fold increase in serum antibody titers between acute and convalescence phases of the disease, are only useful for retrospective diagnosis or research purposes ( Table 2) .
Annual immunization is the most important preventive measure [40••] . However, two classes of antiviral drugs are available and play an important role in the treatment and prevention of influenza [41••] : the neuraminidase inhibitors (NI), oseltamivir and zanamivir, which are active against both influenza A and B viruses; and the M2 inhibitors, amantadine and rimantadine, which are active against all influenza A strains, but have no activity against influenza B viruses. In general, the duration for therapy with an NI is 5 days, and with the M2 inhibitors is three to 5 days.
NI are sialic acid analogs that competitively inhibit neuraminidase on the surface of both influenza A and B, thus interfering with the release of virus from infected cells. Oseltamivir phosphate is an orally bioavailable prodrug that is rapidly absorbed from the gastrointestinal tract and is converted by hepatic esterases to the active metabolite, oseltamivir carboxylate. It has an elimination half-life of approximately 8 h, primarily . These side effects are usually mild and limited to the first days of treatment, although more serious side effects have been described. Oseltamivir has been linked to selfinjury and delirium in pediatric populations, although no causal association could be demonstrated [42, 43] . The use of inhaled zanamivir has been associated with bronchospasm, sometimes severe or fatal, particularly in patients with underlying airways disease such as chronic obstructive pulmonary disease (COPD) or asthma [44] . Amantadine and rimantadine target the M2 protein of influenza A, which forms a proton channel in the viral membrane that is essential for viral replication. Amantadine is primarily excreted unchanged in the urine. In patients older than 65 years and in those with an estimated creatinine clearance of less than 50 mL/min, the daily dose should be reduced. Rimantadine is extensively metabolized in the liver, and dose reduction is recommended for patients with severe hepatic dysfunction, renal failure (creatinine clearance <10 mL/min), and the elderly. Central nervous system (CNS) side effects are well described with amantadine, particularly in elderly patients. In comparison, rimantadine is associated with a considerably reduced rate of CNS side effects [45] .
In mild to moderate uncomplicated disease, the reported benefits of early treatment (< 48 h of symptom onset) with NI have been a shorter duration and severity of flu-like symptoms, and a reduced duration of viral shedding [46] [47] [48] [49] . More importantly, several trials and a few systematic reviews have shown that treatment with NI may reduce illness severity and the rate of lower respiratory tract complications [47, [50] [51] [52] . In a meta-analysis of 10 randomizedcontrolled trials, Kaiser et al. found that therapy with oseltamivir was effective in reducing the incidence of influenzarelated lower respiratory tract complications that required antibiotic use (4.6 % for oseltamivir vs. 10.3 % for placebo, P<0.001), independent of the presence of risk factors for complications [51] . More recently, Hernán et al. conducted another meta-analysis of 11 controlled trials, most of which were included in the previous meta-analysis by Kaiser et al. They found that treatment with oseltamivir significantly reduced influenza-related lower respiratory tract complications by 37 % (CI 95 % ) [52] . NI have also been reported to reduce the duration of hospitalization in severely-ill cases [53] , and also to reduce influenza-related mortality [54] . During the 2009 H1N1 Influenza A pandemic, several observational studies of hospitalized and critically-ill patients reported that treatment with oseltamivir reduced disease severity, complications and mortality [55] [56] [57] . In a Chinese study of 1291 patients with confirmed H1N1 influenza A infection, oseltamivir reduced the risk of developing pneumonia, even when administered after the first 48 h of symptom onset (OR 0.12, 95 % CI [0.08-0.18]) [58] . In another retrospective study of 304 hematopoietic stem cell transplant recipients with influenza infection, 161 patients had H1N1 influenza A confirmed infection, and both early and delayed administration of antiviral therapy was shown to be beneficial in terms of decreased risk of lower respiratory tract compromise (OR00.04, 95 % CI [0-0.2] vs. OR00.14, 95 % CI [0-0.7]) [59] . An intravenous form of zanamivir is under development, and was made available during the 2009 H1N1 influenza A pandemic for severely ill patients with highly suspected or confirmed oseltamivir-resistant infection that could not tolerate inhaled zanamivir. Several studies reported favorable outcomes with the use of IV zanamivir [60] [61] [62] [63] .
During the 2009 swine influenza pandemic, the FDA briefly authorized the emergency use of peramivir, an investigational NI that is administered intravenously [64] . In one study, a single dose of 300 mg or 600 mg of IV peramivir significantly reduced the time to alleviation of symptoms compared with placebo (HR 0.68 and 0.67 for the 300 mg and 600 mg doses respectively) [65] . Laninamivir octanoate, another long acting NI in development, was non-inferior to a five-day course of oral oseltamivir in adults with seasonal influenza in one study [66] . Experience with avian influenza H5N1, which has caused sporadic cases since 2004 with an elevated mortality rate, is much more limited and recommendations for its treatment are in most cases extrapolated from trials of seasonal influenza. There are, however, reports that suggest reduced severity and mortality with the administration of oseltamivir in patients with avian influenza [67] [68] [69] . In patients with pneumonia or with clinical failure to standard regime with oseltamivir, a higher dose of 150 mg bid for 10 days should be considered [24, 67] . In regions with adamantine-susceptible strains, combination therapy with a NI and an adamantine may also be considered with pneumonia or clinical worsening [24] . Therapy with oseltamivir should be administered even in the late course of influenza A (H5N1) infection since viral replication is more prolonged than with seasonal influenza [24] .
The United States Advisory Committee on Immunization Practices (ACIP) and the CDC have recently published updated guidelines for the treatment of patients with confirmed or suspected influenza virus infections caused by either pandemic H1N1 or seasonal strains [41••, 70••] . According to these recommendations, those individuals with severe disease (requiring hospitalization or with evidence of lower respiratory tract infection) or those at high risk for complications (Table 1) should receive antiviral therapy even after 48 h of symptom onset. Adults with mild illness without high risk conditions who are younger than 65 years of age do not require treatment. If such individuals present within the first 48 h of illness, antiviral treatment can be considered in order to reduce the duration of illness. In all cases, decisions to administer antiviral medications for influenza treatment should be based on clinical and epidemiologic grounds, and never be delayed because of confirmatory laboratory tests [41••] . The usual dosing of oseltamivir for the treatment of influenza is 75 mg orally twice daily and of zanamivir is 10 mg (2 inhalations) twice daily. The recommended duration for antiviral treatment is 5 days, although longer treatment courses for patients who fail to improve after 5 days of treatment can be considered [70••] . Also, doubling the dose of oseltamivir to 150 mg orally twice daily has been suggested to be beneficial for some severely ill patients with H5N1 avian influenza [24, 67] . The FDA licensed oseltamivir for use in children 1 year of age and older in a liquid formulation at a dosage of 2 mg/kg/dose twice daily for 5 days. Zanamivir is not approved for use in children under 5. Oseltamivir and zanamivir are Pregnancy Category C drugs, but since influenza causes more severe disease and an increased rate of mortality among pregnant women, they should receive antiviral therapy with oseltamivir when indicated, since the potential benefit outweighs the theoretical risk to the fetus (Table 1) .
Although oseltamivir-resistant seasonal H1N1 influenza A viruses have been identified since 2007 (the H274Y mutation) [71] , in 2009 the CDC reported that most circulating strains of the novel H1N1 influenza A virus were sensitive to the NI, oseltamivir and zanamivir, but that nearly all strains were resistant to the amantadines [41••, 72] . Consequently, the ACIP has advised against the use of M2 inhibitors for treatment of influenza, except in selected circumstances [41••] .
It is recommended that patients with pandemic H1N1 influenza A who develop pneumonia be treated empirically for CAP according to published evidence-based guidelines, given the risk of secondary bacterial pneumonia [73] . In the presence of profound hypoxemia that has been refractory to routine mechanical ventilation, salvage therapies include neuromuscular blockade, inhaled nitric oxide, high-frequency oscillatory ventilation, extracorporeal membrane oxygenation (ECMO), and prone positioning ventilation [74•, 75] . Corticosteroids should not be used routinely, but may be considered for septic shock with suspected adrenal insufficiency requiring vasopressors [76] . Therapy for influenza-associated ARDS should be based upon published evidence-based guidelines for sepsis-associated ARDS, specifically including lung protective mechanical ventilation strategies [76] .
The CDC recommends routine annual influenza vaccination for all persons 6 months of age and older. When vaccine supply is limited, vaccination efforts should focus on those groups with health conditions associated with increased risk of influenza complications [40••] . Antiviral drugs should not be used as a substitute for influenza vaccination. Their adjunctive use is appropriate in certain targeted populations at high risk for complications of influenza who are close contacts with suspected or confirmed cases [ Table 1 ]. Postexposure prophylaxis should only be used when antivirals can be started within 48 h of the most recent exposure. Recommended duration is 7 days after exposure, and the CDC recommends a minimum of 2 weeks for control of influenza outbreaks in long-term care facilities (i.e., nursing homes with elderly) and hospitals [70••] . The choice to offer post-exposure prophylaxis to otherwise healthy unvaccinated adults should be weighed against the risk of promoting antiviral drug resistance [41••, 1••].
A potentially fatal complication of influenza infection is the involvement of the lower respiratory tract caused directly by the influenza virus, and the development of secondary bacterial pneumonia. In these cases, and in patients who are at an increased risk for influenza infection complications, confirmation of etiology by laboratory testing is required in order to guide the initiation and duration of antiviral treatment, and for the implementation of infection control measures and surveillance. Recently, the emergence of novel influenza A strains that carry the risk for more severe disease regardless of age or previous health status, has prompted the development of quick and reliable laboratory tests in an effort to optimize their management and reduce morbidity and mortality. Pneumonia related to the 2009 influenza A pandemic was found in many cases to be rapidly progressive, leading to respiratory failure which in many cases was underestimated by commonly used pneumonia severity scores. Given the limited sensitivity of RIDT and immunofluorescence assays, confirmation of pandemic H1N1 influenza A infection can only be made by rRT-PCR or viral culture. Although annual immunization is the most important preventive measure, NI are the agents of choice for chemoprophylaxis in selected high risk patients, and for treatment. Treatment with NI beyond 48 h of symptoms should be considered only for patients with severe disease.
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• Of importance, •• Of major importance 1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-one In the title pyrazolone derivative, C(15)H(12)N(2)O, the five-membered ring is approximately planar (r.m.s. deviation = 0.018 Å), and the N- and C-bound benzene rings are inclined to this plane [dihedral angles = 21.45 (10) and 6.96 (10)°, respectively] and form a dihedral angle of 20.42 (10)° with each other. Supra­molecular layers are formed in the crystal structure via C—H⋯O and C—H⋯N inter­actions, and these are assembled into double layers by C—H⋯π and π–π inter­actions between the pyrazole and C-bound benzene rings [ring centroid–centroid distance = 3.6476 (12) Å]. The double layers stack along the a axis being connected by π–π inter­actions between the N- and C-bound benzene rings [ring centroid–centroid distance = 3.7718 (12) Å]. In the title pyrazolone derivative, C 15 H 12 N 2 O, the fivemembered ring is approximately planar (r.m.s. deviation = 0.018 Å ), and the N-and C-bound benzene rings are inclined to this plane [dihedral angles = 21.45 (10) and 6.96 (10) , respectively] and form a dihedral angle of 20.42 (10) with each other. Supramolecular layers are formed in the crystal structure via C-HÁ Á ÁO and C-HÁ Á ÁN interactions, and these are assembled into double layers by C-HÁ Á Á andinteractions between the pyrazole and C-bound benzene rings [ring centroid-centroid distance = 3.6476 (12) Å ]. The double layers stack along the a axis being connected byinteractions between the N-and C-bound benzene rings [ring centroid-centroid distance = 3.7718 (12) Å ].
For the therapeutic importance of pyrazoles, see: Sil et al. (2005) ; Haddad et al. (2004) . For their diverse pharmacological activities, see: Bekhit et al. (2012) ; Castagnolo et al. (2008) ; Ramajayam et al. (2010) . For background to the synthesis, see: Nef (1891) ; Katritzky et al. (1997) ; Wardell et al. (2007) ; de Lima et al. (2010) . For evaluation of tautomeric forms using NMR MO calculations and crystallography, see: Feeney et al. (1970) ; Hawkes et al. (1977); Freyer et al. (1983) ; Dardonville et al. (1998) ; Kleinpeter & Koch (2001) ; Bechtel et al. (1973a,b) ; Chmutova et al. (2001); Wardell et al. (2007) ; Gallardo et al. (2009); Ding & Zhao (2010) . For a previous synthesis, see : Kimata et al. (2007) . For a recently reported structure, see: Wardell et al. (2012) . Table 1 Hydrogen-bond geometry (Å , ).
Cg1 is the centroid of the C10-C15 ring. Symmetry codes: (i) x; Ày þ 1 2 ; z þ 1 2 ; (ii) x; Ày þ 3 2 ; z þ 1 2 ; (iii) Àx þ 1; Ày þ 1; Àz þ 2.
Data collection: COLLECT (Hooft, 1998) ; cell refinement: DENZO (Otwinowski & Minor, 1997) and COLLECT; data reduction: DENZO and COLLECT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008) ; program(s) used to refine structure: SHELXL97 (Sheldrick, 2008) ; molecular graphics: ORTEP-3 (Farrugia, 1997) and DIAMOND (Brandenburg, 2006) ; software used to prepare material for publication: publCIF (Westrip, 2010) . 
Pyrazoles are key structures in numerous compounds of therapeutic importance (Sil et al., 2005 , Haddad et al., 2004 .
Compounds containing this ring system are known to display diverse pharmacological activities, for example as antimalarial agents (Bekhit et al., 2012) , anti-tuberculosis agents (Castagnolo et al., 2008) , and as SARS-coronavirus protease inhibitors (Ramajayam et al., 2010) .
A general route to pyrazole derivatives involves reaction of an arylhydrazine, ArNHNH 2 , with a β-dicarbonyl compound, R/COCH 2 COX.. This reaction provides initially a hydrazone derivative, RNHN=CR/CH 2 COX, I ( Fig. 1 ), which can be isolated but which readily undergoes cyclization to a pyrazone derivative, II ( Fig.1 ), (Nef, 1891; Katritzky et al., 1997; Wardell et al., 2007; de Lima et al., 2010) . Equilibrium involving tautomers of II in solution have been variously studied using NMR and IR spectroscopy and ab initio calculations. (Feeney et al., 1970; Hawkes et al., 1977; Freyer et al., 1983; Dardonville et al., 1998; Kleinpeter & Koch, 2001) . Crystal structures of various pyrazone compounds of forms IIa, IIb and IIc have been reported (see for example, Bechtel et al., 1973a; Bechtel et al., 1973b; Chmutova et al., 2001; Wardell et al., 2007; Gallardo et al., 2009; Ding & Zhao, 2010) . In continuation of recent studies (Wardell et al., 2012) , herein, the isolation the title compound [i.e. form IIc ( Fig. 1) ] from the reaction between PhNHNH 2 and PhCOCH 2 CO 2 Et in EtOH is described as is its crystal structure. The same tautomer was also isolated in the reaction between PhNHNH 2 and PhCOCH 2 CONHPh in EtOH.
In the title compound, Fig. 2 , crystallography proves the IIc tautomer in the solid-state. The pyrazole ring is planar with a r.m.s. deviation for the fitted atoms of 0.018 Å; the maximum deviations from this plane are 0.015 (1) Å (for the N1 atom) and -0.015 (1) Å (C8). The N-and C-bound benzene rings are inclined to this plane forming dihedral angles of 21.45 (10) and 6.96 (10)°, respectively; the dihedral angle between the benzene rings is 20.42 (10)° consistent with a nonplanar molecule.
In the crystal structure, supramolecular layers are formed in the bc plane through C-H···O and C-H···N interactions, Fig. 3 and Table 1 . Large 21-membered {···NC 4 H···N 2 CO···HC 2 O···HC 5 H} synthons are formed through these interactions. Layers are connected into double layers by C-H···π, Table 1 , and π-π interactions formed between the pyrazole and C-bound benzene rings [ring centroid···centroid distance = 3.6476 (12) Å, angle of inclination of 6.96 (10)° for symmetry operation 1 -x, 1 -y, 2 -z]. The double layers stack along the a axis being connected by π-π interactions between the N-and C-bound benzene rings [ring centroid···centroid distance = 3.7718 (12) Å, angle of inclination of 21.45 (10)° for symmetry operation -x, 1 -y, 1 -z].
A solution of PhNHNH 2 (1 mmol) and PhCOCH 2 CO 2 Et (1 mmol) in EtOH (15 ml) was refluxed for 1 h. The reaction mixture was maintained at room temperature and crystals of the titled compound were collected after 2 days, M.pt: 408-410 K: lit. value 409-411 K (Kimata et al., 2007) . IR and NMR spectra are in agreement with published data (Castagnolo 
The C-bound H atoms were geometrically placed (C-H = 0.95-0.99 Å) and refined as riding with U iso (H) = 1.2U eq (C).
Owing to poor agreement one reflection, i.e. (3 1 1) , was removed from the final cycles of refinement. The maximum and minimum residual electron density peaks of 0.74 and 0.20 e Å -3 , respectively, were located 1.02 Å and 0.58 Å from the H8a and O1 atoms, respectively.
Data collection: COLLECT (Hooft, 1998) ; cell refinement: DENZO (Otwinowski & Minor, 1997) and COLLECT (Hooft, 1998) ; data reduction: DENZO (Otwinowski & Minor, 1997) and COLLECT (Hooft, 1998) ; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008) ; program(s) used to refine structure: SHELXL97 (Sheldrick, 2008) ; molecular graphics: ORTEP-3 (Farrugia, 1997) and DIAMOND (Brandenburg, 2006) ; software used to prepare material for publication: publCIF (Westrip, 2010) . The molecular structure of (I) showing the atom-labelling scheme and displacement ellipsoids at the 50% probability level. A view in projection down the c axis of the crystal packing in (I). The C-H···O, C-H···N, C-H···π and π-π interactions shown as orange, blue, brown and purple dashed lines, respectively. where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.74 e Å −3 Δρ min = −0.20 e Å −3 Special details Geometry. All s.u.'s (except the s.u. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell s.u.'s are taken into account individually in the estimation of s.u.'s in distances, angles and torsion angles; correlations between s.u.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell s.u.'s is used for estimating s.u.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.
x y z U iso */U eq O1 0.22694 ( (9) 0.0201 (9) −0.0037 (6) 0.0061 (7) 0.0006 (7) C2 0.0271 (9) 0.0236 (9) 0.0244 (9) −0.0011 (7) 0.0113 (7) 0.0003 (7) (7) C5 0.0235 (9) 0.0233 (9) 0.0294 (10) −0.0014 (7) 0.0053 (7) 0.0042 (7) C6 0.0216 (9) 0.0230 (9) 0.0243 (9) −0.0013 (7) 0.0086 (7) −0.0014 (7) C7 0.0217 (9) 0.0170 (8) 0.0221 (9) −0.0005 (7) 0.0099 (7) −0.0007 (7) C8 0.0231 (9) 0.0174 (9) 0.0207 (8) 0.0006 (7) 0.0094 (7) 0.0010 (7) C9 0.0170 (8) 0.0201 (9) 0.0195 (8) 0.0000 (6) 0.0079 (7) 0.0015 (7) C10 0.0191 (8) 0.0230 (9) 0.0214 (9) −0.0013 (7) 0.0106 (7) 0.0000 (7) C11 0.0214 (9) 0.0241 (9) 0.0233 (9) 0.0026 (7) 0.0086 (7) 0.0018 (7) C12 0.0272 (9) 0.0235 (9) 0.0294 (10) 0.0012 (7) 0.0140 (8) −0.0040 (7) C13 0.0286 (10) 0.0316 (11) 0.0217 (9) −0.0024 (8) 0.0108 (8) −0.0062 (7) C14 0.0273 (10) 0.0305 (10) 0.0199 (9) 0.0018 (8) 0.0093 (7) 0.0035 (7) C15 0.0257 (9) 0.0205 (9) 0.0232 (9) 0.0012 (7) 0.0120 (7) 0.0016 (7) Geometric parameters (Å, º) Symmetry codes: (i) x, −y+1/2, z+1/2; (ii) x, −y+3/2, z+1/2; (iii) −x+1, −y+1, −z+2. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F(326), T(328), N(333), V(388), G(389), P(390), E(392), I(408), and N(410). Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. Carcinoembryonic antigen (CEA; CEACAM5; CD66e) is a glycosylated human oncofetal antigen that belongs to the CEArelated cell adhesion molecule (CEACAM) family of the immunoglobulin (Ig) gene superfamily [1, 2] . CEA is closely related to CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8. Carcinoembryonic antigen has been suggested to mediate cell-cell adhesion, facilitate bacterial colonization of the intestine, and protect the colon from microbial infection by binding and trapping infectious microorganisms [3] .
CEA is expressed at low levels in normal tissues of epithelial origin in a polarized manner; found only at the luminal portion of the cell but not at the basolateral surface [3] . In contrast, expression of CEA is frequently high in carcinomas, including colon, pancreatic, gastric, esophageal, lung, breast, uterine, ovarian, and endometrial cancers [3] . Cancer cells not only lose polarized (luminal) expression of CEA, but actively cleave CEA from their surface by phospholipases, an action that results in serum concentrations of CEA that can approach 5 mg/mL [3, 4, 5] . Serum CEA levels may be monitored to detect a response to anticancer therapy, or disease recurrence in colorectal cancer [6] , and serves as a prognostic indicator in patients with gastrointestinal cancers, where elevated levels indicate a poor prognosis and correlate with reduced overall survival [7, 8, 9] .
Cell-bound CEA has served as a target for tumor imaging and anti-cancer therapies. Clinical studies have demonstrated that radiolabeled anti-CEA antibodies and antibody fragments, such as the Food and Drug Administration-approved, Tc-99m-labeled, anti-CEA Fab arcitumomab (CEA-ScanH), can be successfully used as imaging reagents to specifically localize CEA-expressing solid cancers [10, 11, 12] . Anti-CEA radio-immunoconjugate anti-bodies have also been shown to be potentially efficacious for the treatment of patients with metastatic colorectal cancer [13] . In addition, CEA-specific antibody-directed enzyme prodrug therapy and CEA-based vaccine approaches have been developed to treat cancers [14, 15] .
As a novel CEA-directed therapy, we have developed a CEAtargeting bispecific single-chain antibody of the bispecific T-cell engager (BiTE) class termed MEDI-565 (also known as MT111) [16, 17, 18] . BiTE antibodies are a unique subclass of bispecific antibodies that contain one single chain variable fragment (scFv) with specificity for a tumor associated antigen molecularly fused to another scFv with specificity for CD3 on T cells [19] . Highly potent and specific tumor cell lysis is triggered only when BiTE antibodies bind both epitopes simultaneously, resulting in directing T cells to the tumor cells and activating the T cell through the CD3/T cell receptor (TCR) complex [20] . Notably, activation of T cells is independent of TCR specificity, peptide antigen presentation, and co-stimulatory signals [21] . As a result of T cell activation, granzymes and perforin are mobilized to the tumor cell-T cell interface and mediate an apoptotic killing of target cells; FAS ligand expression may also contribute to the induction of apoptosis through engagement of FAS on tumor cells [22, 23, 24] . BiTE antibodies activate both CD4+ and CD8+ T cell subsets [23, 25, 26, 27, 28] ; both subsets of T cells contribute to tumor cell killing at relatively low effector T cell:target tumor cell (E:T) ratios [22, 29] . Cytokine secretion and the upregulation of cell surface IL-2 receptor by T cells (measured experimentally using antibodies recognizing the IL-2Ra chain/CD25) occur simultaneously and accompany tumor cell killing [18, 22, 25] . Both potentiate further T cell activation and proliferation, facilitating the subsequent engagement and killing of additional tumor cells bound by a BiTE antibody. BiTE antibodies not only induce the potent lysis of human cancer cell lines in vitro, but have also been shown to mediate the killing of primary human tumor cells by cancer patient T cells ex vivo [17, 25, 30] . Furthermore, BiTE antibodies promote in vivo tumor regression in numerous pre-clinical animal models [16, 31, 32, 33, 34, 35, 36] and have shown signs of clinical benefit in patients with cancer [37, 38, 39] .
MEDI-565 itself is composed of a humanized single-chain antibody recognizing human CEA connected by a short flexible linker to a de-immunized single-chain antibody specific for CD3, and shares the characteristics of the BiTE antibody class as described above [16, 17, 18] . It specifically binds to CEA and not to other CEACAM family members [16] . Upon concurrent binding to CEA on cancer cells and CD3 on T cells, MEDI-565 mediates T cell activation and the subsequent killing of CEA-expressing target cells in a perforin-and granzyme-dependent manner that is insensitive to concentrations up to 5 mg/mL of soluble CEA [16, 17, 18] . Furthermore, intravenous and subcutaneous administration of MEDI-565 had anticancer activity in severe combined immunodeficient mouse xenograft models of various human cancers [18] ; growth inhibition of the cancers was contingent on both the presence of un-stimulated human T cells and the expression of CEA on the cancer cells [18] . MEDI-565 is currently in phase I clinical trials (clinicaltrials.gov identifier: NCT01284231) for the treatment of gastrointestinal adenocarcinomas.
To understand the molecular basis of human CEA recognition by MEDI-565 and the impact of amino acid polymorphisms and splice variants of CEA on the activity of MEDI-565, we sought to identify the CEA domain and the critical residues involved in MEDI-565 binding. The CEA-specific arm of MEDI-565 is a humanized version of the murine antibody A5B7 [40, 41] the binding epitope of which is unknown [40, 42] . We mapped its epitope using mutagenesis and computational modeling approaches. We further investigated what impact a CEA splice variant which lacks a large portion of the identified epitope could have on the binding and activity of MEDI-565.
The plasmid containing the full-length CEA sequence was purchased from Open Biosystems (Thermo Fisher Scientific, Huntsville, AL). The CEA complementary DNA (cDNA) was subcloned using sequence specific primers into a modified version of the lentiviral vector pCDH1-CMV-MCS-EF1-Puro (System Biosciences, Mountain View, CA) in which the puromycin-resistance casette was replaced with a blasticidin-resistance cassette (pCDH1-CMV-MCS-EF1-Blast) and the CEA sequence was verified by DNA sequencing. The plasmid containing the sequences of the CEA splice variant (cDNA clone DKFZp781M2392) was purchased from ImaGenes GmbH (Source BioScience UK Limited, Nottingham, UK) in association with B Bridge International (Cupertino, CA). The CEA splice variant was cloned into the puromycin resistance lentiviral vector pCDH1-HCS1-EF1-Puro (System Biosciences) using sequence-specific primers and the resulting clones were sequence-verified.
FreeStyle human embryonic kidney (HEK293) F cells (American Type Culture Collection, Manassas, VA) were cultured in FreeStyle 293 expression medium (Life Technologies/Invitrogen, Carlsbad, CA). Dihydrofolate reductase deficient (DHFR-) Chinese hamster ovary (CHO) cells or CHO DHFR-cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 media (Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen) in a humidified cell culture incubator at 37uC and 5% CO 2 . CHO cells expressing the CEA splice variant (CHO SV CEA) were created by infection of CHO DHFR-cells with the pCDH1-HCS1-EF1-Puro-CEA splice variant lentiviral vector, and selected by culturing in medium containing 5 mg/mL puromycin (EMD Chemicals Group, Merck KGaA, Darmstadt, Germany) for 72 hours. CHO cells expressing the full-length CEA sequence (CHO FL CEA) were created by infecting CHO DHFRcells with the lentiviral expression vector pCDH1-CMV-MCS-EF1-Blast-CEA, and selected by culturing in medium containing 10 mg/mL blasticidin (Invitrogen) for 10 days. Likewise, cells expressing both the CEA splice variant and full-length CEA (CHO FL+SV CEA) were created by infecting CEA splice variantexpressing cells with the pCDH1-CMV-MCS-EF1-Blast-CEA lentiviral vector followed by blasticidin selection. The anti-CEACAM5 specific monoclonal antibody (mAb) clone 26/3/13 was purchased from Genovac GmbH (Aldevron, Freiburg, Germany). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO). Construction and production of MEDI-565 and the Control BiTE (MEC14 BiTE) has been described [17] .
Twenty pancreatic primary tumors were purchased from Asterand, Inc (Detroit, MI). The panel included 13 adenocarcinomas (stage I-IV), 5 pancreatic endocrine tumors and 2 benign pancreatic adenomas. Four of the 20 pancreatic tumor tissue samples had matched normal adjacent pancreatic tissue samples. Patient age ranged from 23 to 77 years. All samples were freshly frozen and collected before the initiation of any cancer treatment. Tumor samples were macrodissected to remove normal tissue, and tumor purity in all samples was greater than 85%. Normal samples were macrodissected to remove non-glandular tissue.
Cancer tissue cDNA arrays: HCRT101 (Colon Cancer), HGRT101 (Gastroesophageal Cancer), PNRT101 (Pancreatic Cancer), HLRT101 (lung cancer), and BCRT101 (breast cancer) were purchased from OriGene Technologies (Rockville, MD). Each array contains cDNAs from 5 to 8 normal tissues and 19 to 42 cancer tissues. The tumor stage ranged from stage I to IV and the tumor tissues were comprised of 50-90% tumor.
Cells were lysed in RIPA lysis buffer (Boston Bioproducts, Ashland, MA) and the resulting protein was quantified using the BCA protein assay kit (Thermo Fisher Scientific, Huntsville, AL). Cell lysates were mixed with NuPAGEH LDS 4X LDS sample buffer (Invitrogen) to 1X concentration; ten micrograms of total protein from each lysate was analyzed on a pre-cast NuPAGEH NovexH 10% polyacrylamide Bis-Tris gel (Invitrogen) together with the NovexH Sharp Pre-stained Protein Standard (Invitrogen) using NuPAGEH Tris-acetate SDS running buffer (Invitrogen). Protein from each gel was transferred to a polyvinylidene fluoride membrane using an iBlotH Dry Blotting System (Invitrogen) and 2X NuPAGEH transfer buffer (Invitrogen) containing 20% methanol and blocked with 3% bovine serum albumin (Sigma) in PBS pH 7.4 (Invitrogen). To detect CEA, each blot was probed overnight with 1 mg/mL of the CEACAM5-specific mAb clone 26/3/13 (Genovac), washed with PBS pH 7.4 containing 0.1% Tween-20, and subsequently probed for 1 hour with a horseradish peroxidase-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). After 5 washes with PBS pH 7.4 containing 0.1% Tween-20, each blot was exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and exposed to BioMax MS Kodak film (Sigma) for detection of chemiluminescence resulting from bound anti-CEACAM5 antibody. Equal amounts of protein loaded into each lane of the gel were controlled by detecting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein using a rabbit anti-human GAPDH polyclonal antibody (Trevigen, Gaithersburg, MD) followed by a horseradish peroxidaseconjugated donkey anti-rabbit IgG (Jackson ImmunoResearch).
The cDNA encoding the CEA deletion variants DelA1-A2, Del A1, DelB1, DelA2, and DelB2-A3 and the swap variants KO_A, KO_B, KO_C, KI_A, KI_B, and KI_C (see Figures 1, 2, 3, and 4 for domain definition and variant nomenclature) were assembled and amplified by overlapping extension polymerase chain reaction (PCR) using an in-house full-length CEA cDNA clone, pNEO human CEA-GPI, as a template. The CEA variants and the fulllength CEA cDNA were cloned into a mammalian expression vector pcDNA3.1 (Invitrogen).
Expression vectors encoding the CEA deletion and swap mutants were transiently transfected for expression of glycophosphatidyl inositol (GPI)-anchored proteins using FreeStyle HEK293 F cells (Invitrogen). One day prior to transfection, HEK293 F cells were seeded at a density of 0.7610 6 cells/mL. Three and a half micrograms of each expression vector were transfected into 5 mL of a suspension of HEK293 F cells using 5 mL of 293fectin transfection reagent (Invitrogen). Transfected cells were incubated with 5 mg/mL of MEDI-565 for 1 hour. For the detection of bound MEDI-565, which possesses a six-histidine tag at its carboxy-terminus, the cells were washed three times with PBS and incubated with 1 mg/mL of an Alexa FluorH 488conjugated anti-penta-His antibody (Qiagen, Valencia, CA) for 30 minutes, and then analyzed for binding using the LSRII flow cytometer (BD Biosciences, San Jose, CA). Expression of CEA deletion mutants was monitored with a goat anti-CEA polyclonal antibody (R&D Systems, Minneapolis, MN) that was detected by an R-phycoerythrin-conjugated anti-goat IgG (Invitrogen).
Site-directed mutagenesis and computational homology modeling were implemented to identify amino acids within CEA that are critical for MEDI-565 binding. The following amino acids in the epitope-containing segments A and C of the A2 domain (see Results and Discussion section) that differ from those in the A3 domain were mutated in clusters or individually to encode the corresponding amino acids of the [44] . This model was used as a template to engineer two additional mutants. These two mutants 1) substituted clusters V 388 G 389 P 390 E 392 and I 408 N 410 of the A2 domain with the corresponding clusters of the A3 domain (KO_VGPE+IN), and 2) grafted clusters F 326 T 328 N 333 , V 388 G 389 P 390 E 392 , and I 408 N 410 of the A2 domain into the A3 domain (KI_FTN+VGPE+IN), respectively. All mutants were assembled by overlapping extension PCR using the truncated mutants A2 or A3 as a template, which encodes for the N-terminal domain, the A2 or A3 domain, and the GPI region. They were cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). Expression of these mutants and binding analyses were conducted as listed above for the deletion and swap mutants.
Full-length mRNA transcript sequences for CEA (NM_004363.2) and the CEA splice variant (DKFZp781M2392) were retrieved from the NCBI Reference Sequences database. For the full-length CEA assay we targeted the splice junctions of exons 3 and 4 to design the gene specific probe. For the CEA splice variant assay we targeted the splice junctions of exons 2 and 5 exon to design the gene specific probe. All primer/probes were imported into the Primer Express (Applied Biosystems, Foster City, CA) software tool to ensure the optimal design for utilization in the TaqMan Gene Expression assay procedure. All probes were designed to incorporate a minor groove binding moiety (MGB), and were labeled with a fluorescent dye (FAM) for detection and as a non-fluorescent quencher. Primers and probes were custom ordered from Applied Biosystems. Sequences for all primer/probe combinations are as follows:
CEA full-length: The sequence of the probe is 59-CAGGCG-CAGTGATTCA-39.
The sequence of the forward primer 59-GAAACCCAGAACC-CAGTGAGT-39.
The sequence of the reverse primer is 59 GCCATAGAGGA-CATTCAGGATGAC-39.
CEA splicing variant: The sequence of the probe is; CEA 59-ATGCATCCCTGCTGATCC-39.
The sequence of the forward primer is 59-CGCATA-CAGTGGTCGAGAGATAATA-39.
The sequence of the reverse primer is 59-CGCTGTGGTCAA-CACTTAATTTGT-39.
Real-time qPCR was conducted with the BioMark TM Dynamic Array Microfluidics System (Fluidigm Corporation, South San Francisco, CA) using RNA isolated from frozen pancreatic tissues. First, total RNA was extracted from frozen slide sections of pancreatic tissue classified as normal (NAT, normal adjacent to tumor), adenocarcinoma, benign adenoma, or endocrine tumor using the ZR RNA MicroPrep kit (Zymo Research, Orange, CA). RNA purity and concentration were determined spectrophotometrically. RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChipH (Agilent Technologies, Santa Clara, CA). Single stranded cDNA was generated from total pancreas RNA using the SuperScriptH III First-Strand Synthesis SuperMix (Invitrogen). cDNA samples were pre-amplified using TaqMan Pre-Amp Master Mix (Invitrogen), according to the manufacturer's instructions. Reactions were cycled with the recommended program for 14 cycles and then diluted 1:5 with TE buffer. Preamplified cDNA was either utilized immediately or stored at 220uC until processed. Samples and TaqMan Gene Expression assays (Applied Biosystems, Carlsbad, CA) were load onto a 96.96 dynamic array (Fluidigm) according to the manufacturer's instructions. The prepared array was loaded on the BioMark TM Real-Time PCR System (Fluidigm) for thermal cycling (10 min at 95uC followed by 40 cycles of 95uC for 15 sec and 1 min at 60uC). Upon completion of the qPCR, Fluidigm Real-Time PCR Analysis software was used to generate threshold cycle (Ct) values. A Ct cutoff value of 24, below which the samples were considered to be positive and above which the samples were considered to be negative for transcript expression, was empirically selected.
TissueScan TM Disease Tissue quantitative polymerase chain reaction (qPCR) arrays (Origene Technologies, Rockville, MD) were employed to determine the expression of full-length and CEA splice variant (SV) transcripts in normal and cancerous tissues of various stages and grades. For each tissue cDNA array, lyophilized cDNA was resuspended in 2.5 mL of ribonuclease-free water. Plates were sealed, vortexed, and centrifuged (1200 g for 1 min) to ensure resuspension of the full cDNA sample. A 0.2X mixture of the TaqMan assays (Applied Biosystems) was then created according to protocols supplied by the manufacturer for pre-amplification (one cycle of 95uC, 10 min followed by 14 cycles of 95uC 15 sec/60uC 4 min). Subsequent amplification and transcript detection (one cycle of 95uC, 20 sec followed by 40 cycles of 95uC, 1 sec/60uC, 20 sec) of the specific target transcripts was carried out using an ABI 7900HT Fast Real Time PCR Instrument (Applied Biosystems). Upon completion of the qPCR, SDS 2.2 Software (Ericsson, Stockholm, Sweden) was used to generate threshold cycle (Ct) values. A Ct cutoff value of 30, below which the samples were considered to be positive and above which the samples were considered to be negative for transcript expression, was empirically selected. As positive controls, cDNAs from CHO cells expressing either full-length CEA (CHO FL CEA) or the CEA splice variant (CHO SV CEA) were used. Complementary DNA isolated from DHFR-deficient CHO cells lacking CEA expression was used as a negative control for qPCR. To measure the killing of human tumor cell lines by MEDI-565 or the control BiTE, cell lines were co-cultured with enriched CD3+ T cells and BiTE antibody as described above, and cell lysis was measured by the release of caspase-3 into the tissue culture media via a caspase-3 specific electrochemiluminescence assay (Meso Scale Discovery, Gaithersburg, MD).
The statistical significance of differences in affinity (apparent K D ) and cytotoxicity (EC 50 ) values was calculated using two-tailed, parametric t tests calculated in GraphPad Prism software version 5.01 for Windows (GraphPad Software).
MEDI-565 recognizes full length CEA; however, the corresponding epitope is unknown. CEA deletion mutants were generated to identify the domain of CEA to which MEDI-565 binds. The full-length CEA transcript (NCBI accession number NM_002483) contains 10 exons and 9 introns encoding a 702 amino acid (aa) protein composed of a 34 aa processed leader sequence, one IgV-like N-terminal domain, six immunoglobulin constant (IgC)-like domains denoted A1, B1, A2, B2, A3 and B3, and a C-terminal 17 aa peptide which is removed during GPI linkage [1, 2, 45, 46] (Figure 1, 2) . Five deletion mutants were constructed by removing the following IgC-like domains: A1, B1 and A2 domains (DelA1-A2), A1 domain (DelA1), B1 domain (DelB1), A2 domain (DelA2), and B2 and A3 domains (DelB2-A3) as shown in Figure 3A . The mutants were transiently expressed as GPI-anchored proteins on HEK293 F cells. The binding of MEDI-565 or the anti-CEA polyclonal control antibody to each of the deletion mutants was analyzed using flow cytometry. MEDI-565 did not recognize any of the deletion mutants which lack the A2 domain (DelA1-A2 and DelA2, Figure 3B ), but bound well to all mutants that contained it (DelA1, DelB1 and DelB2-A3, Figure 3B ). Additionally, when all but the A2 IgC-like domains were removed (A2; Figure 4B ), MEDI-565 still recognized the corresponding truncated CEA protein at a level similar to that measured for full-length CEA protein ( Figure 4C ). These results suggested that the epitope of MEDI-565 is localized in the A2 domain of CEA.
The A3 domain is not involved in MEDI-565 binding despite being highly homologous with the epitope-containing A2 domain; therefore, swap-mutants were constructed by exchanging short segments of the A2 domain with the corresponding portions of the A3 domain to further refine the MEDI-565 binding epitope. Two truncated CEA mutants (A2 and A3) were engineered and used as templates for the construction of swap mutants. The A2 or A3 mutant is comprised of the N-domain, the A2 or A3 domain, and the GPI region. An alignment of the amino acid sequences of the A2 and A3 domains identified differences in their aa composition. These regions of sequence diversity were divided into three shorter segments labeled as A (aa 326 to 349), B (aa 360 to 382) and C (aa 388 to 410) ( Figure 4A ). Six swap mutants were generated by swapping segments A, B or C of the A3 domain into the A2 domain (KO mutants), or by swapping segments of the A2 domain into the A3 domain (KI mutants) as shown in Figure 4B . MEDI-565 did not bind to any of the KO or KI mutants lacking either the A or C segment of the A2 domain (KO_A, KO_C, KI_A, KI_B and KI_C, Figure 4B , C), but bound well to the swap variant which encoded both the A and C segments of the A2 domain (KO_B, Figure 4B , C). Some residual binding of MEDI-565 to the KO_C mutant could be detected. Together with . Swap-mutants of human CEA. A, amino acid sequence alignment of the A2 and A3 domains of CEA. Sequence homology analysis revealed 21 amino acids that differed between these two domains (amino acids boxed). Three segments, A, B, and C, were defined in the A2 and A3 domains to generate swap-mutants. B, a schematic display of swap mutants that were constructed by exchanging segments A, B, or C between the A2 (open boxes) and A3 (grey boxes) domains using the truncated mutant A2 or A3 as a template which encodes the N-domain, the A2 or A3 domain, and the GPI region. C, flow cytometry analysis of binding of MEDI-565 to deletion mutants expressed on the surface of HEK293 F cells. All mutants were expressed well as monitored by anti-CEA polyclonal antibody. MEDI-565 did not bind well to any of the knock-out (KO) or knock-in (KI) mutants which lack either the A or C segment of the A2 domain (KO_A, KO_C, KI_A, KI_B and KI_C), but bound well to the one variant which encoded both the A and C segments of the A2 domain (KO_B). doi:10.1371/journal.pone.0036412.g004 MEDI-565 lack of binding to KO_A mutant, this data indicated that segment C significantly contributed to MEDI-565 binding, but to a lesser degree than segment A. Due to the high degree of identity between segment B in the A2 and the A3 domains, direct involvement of this segment in MEDI-565 binding could not be entirely ruled out using a swap mutant-based approach. It is however unlikely for the following reasons 1) MEDI-565 did not bind to the mutants encoding the B segment of A2 domain in conjunction with either A or C segment of the A2 domain (KO_A or KO_C, Figure 4 ); 2) The binding level of MEDI-565 to the mutant encoding both the A and the C segments of A2 domain (KO_B) was comparable to the signal of both the full-length CEA and the A2 deletion mutant (Figure 4) ; and 3) The modeled structure of the A2 domain using murine CEACAM1 as a template revealed that segment B was spatially distal from critical residue N 333 (see following section). Therefore, the swap-mutants revealed that MEDI-565 bound to a nonlinear epitope in the A2 domain of CEA, which is comprised of two segments, namely 326-349 (segment A) and 388-410 (segment C).
Site-directed mutagenesis and computational homology modeling were employed to identify critical residues within segments A and C that are essential for the binding of MEDI-565. The amino acids of segments A and C of the A2 domain that differed from the A3 domains were replaced with the corresponding A3 residues encoding several substitutions at a time: Figure 4A ). The binding of MEDI-565 was substantially decreased to the variants in which residue N 333 was mutated (KO_FTN and KO_NE), but bound well to the other mutants ( Figure 5A ). Furthermore, replacing only residue N 333 with either its counterpart residue Lys in the A3 domain (KO_N) or with Ala (N 333 to A) abolished MEDI-565 binding ( Figure 5C ). Mutating the residue F 326 or T 328 to Ala substantially decreased the binding of MEDI-565 (F 326 to A, T 328 to A), suggesting that they were also involved in the interaction with MEDI-565 but to a lesser extent than residue N 333 ( Figure 5C ). Taken together, our data demonstrated the importance of F 326 , T 328 , and N 333 . Finally, since E 338 is encoded in the KO_ELI variant which binds well to MEDI-565, we concluded that this residue is not energetically involved in MEDI-565 epitope. We had previously noted that grafting only segment A that includes the F 326 , T 328 , and N 333 residues of the A2 domain into the A3 domain (KI_A) did not result in MEDI-565 binding. This suggested that MEDI-565 binding epitope comprises additional critical residues and pointed towards it being nonlinear and conformational.
The modeled structure of the A2 domain also revealed two clusters of amino acids V 388 G 389 P 390 E 392 and I 408 N 410 in segment C that were spatially close to the already identified critical residue N 333 in segment A ( Figure 5B ). This observation suggested that these amino acids could contribute to the binding of MEDI-565 in concert with N 333 . Knocking-out amino acids V 388 G 389 P 390 E 392 (KO_VGPE) or I 408 N 410 (KO_IN) separately ( Figure 5A ), or knocking-in the entire segment C which includes both clusters of V 388 G 389 P 390 E 392 and I 408 N 410 (KI_C) ( Figure 4C ) had no effect in abolishing or restoring MEDI-565 binding, respectively. However, knocking-out these amino acids together reduced (but did not completely abolish) MEDI-565 binding (KO_VGPE+IN, Figure 5C ). Thus, amino acids V 388 , G 389 , P 390 , E 392 , I 408 , and N 410 in segment C of CEA could also be involved in MEDI-565 binding.
It is possible that some knock-out CEA variants exhibited an incorrect fold in or near the MEDI-565 epitope region, thereby losing their binding capacity. Therefore, we further confirmed the potential epitope regions identified with knock-out variants by using gain of function (knock-in) mutants. Indeed, ''knocking in'' segments A (326-349) and C (388-410) of the A2 domain into the A3 domain (KO_B/KI_A+C, KO_B encodes the same amino acids as KI_A+C) resulted in MEDI-565 binding comparable to full length CEA ( Figure 4C, 5C) . Furthermore, only grafting the three amino acids F 326 T 328 N 333 of segment A with the six amino acids V 388 G 389 P 390 E 392 and I 408 N 410 of segment C into the A3 domain (KI_FTN+VGPE+IN) also resulted in MEDI-565 binding comparable to full length CEA ( Figure 5C ). In summary, these results demonstrate that the epitope of CEA bound by MEDI-565 is a nonlinear, conformational epitope located in the A2 domain of CEA; it is comprised of two segments of amino acids 326 to 349 and 388 to 410 with critical amino acids F 326 , T 328 , N 333 , V 388 , G 389 , P 390 , E 392 , I 408 , and N 410 . The residue N 333 may contribute more to the binding of MEDI-565, since mutating it alone completely disrupted the interaction ( Figure 5C ).
MEDI-565 binds to mature full-length CEA. However, polymorphisms and/or isoforms of CEA may alter the binding epitope and negatively affect the ability of MEDI-565 to bind. Once the epitope of CEA bound by MEDI-565 had been identified, the ability of MEDI-565 to recognize cancerous cells expressing polymorphisms and isoforms of CEA could be evaluated.
Polymorphisms of CEA were surveyed using the NCBI singlenucleotide polymorphism (SNP) database (http://www.ncbi.nlm. nih.gov/projects/SNP). Two non-synonymous coding SNPs of CEA (rs10407503, rs7249230) were identified in the binding epitope of MEDI-565 (shown in Figure 2 ). The single-nucleotide C to A change in the SNP rs10407503 resulted in the amino acid change of Ala to Asp at the aa position 340. The single-nucleotide A to G change in the SNP rs7249230 encoded an aa change of Glu to Lys at the aa position 398. According to the SNP database, the minor allele frequencies in the population for rs10407503 are 0.014,0.267 and minor allele frequencies for rs7249230 are 0.03,0.3 in the population, respectively. However, the minor allele homozygosity rate for rs10407503 is close to 0 in both European and Asian populations and is 0.083 in Sub-Saharan African populations. The minor allele homozygosity rate for rs7249230 was from 0 to 0.068 in different populations. Since the homozygosity rates of both SNPs are very low, we anticipated the identified CEA SNPs to have little or no impact on MEDI-565 binding to CEA for these populations.
In addition, the National Center for Biotechnology Information (NCBI) GenBankH database (http://www.ncbi.nlm.nih.gov/ genbank) was searched for splice variants of CEA. A single splice variant (NCBI accession number CR749337) from colon cancer tissue was identified. This transcript uses an alternative splice donor site in exon 2 and skips exons 3 and 4; thus, the translation of the transcript results in a 420 aa protein with an in-frame truncation from amino acids 116 to 396 of the full-length CEA. This truncation deletes a small portion of the N-terminal domain, the entire A1 and B1 domains, and a large portion of the A2 domain (Figure 1, 2) . The splice variant sequence codes for the same 34-aa processed leader sequence and C-terminal 17-aa peptide which is expected to be removed during GPI linkage in a similar manner as full-length CEA, and, after undergoing similar post-translational modifications as full-length CEA, is predicted to be a mature, GPI-anchored membrane protein of 369 amino acids.
Another putative CEA splice variant involving novel splicing of exons 9, 10 and the intervening intron sequence has been detected in the peripheral blood of colon cancer patients by reverse transcription PCR [47] . However, sequences within the middle part of a 152 bp PCR product (base pairs 26-99) of the putative CEA isoform isolated from white blood cells did not match any part of the genomic sequence of CEA. Therefore, this PCR product more likely represents a PCR artifact and not a real CEA splice form. Consequently, we did not investigate this putative CEA splice variant further.
The biological function and distribution of the CEA splice variant in normal and cancerous tissues is unknown. To determine the expression frequency of full-length and CEA splice variant transcripts in normal and cancerous human tissues, real-time qPCR was performed using both RNA isolated from frozen primary human pancreatic tissues and cDNA generated from frozen colorectal, lung, breast and pancreatic tissues purchased in the form of multi-tissue cDNA arrays. Each analysis used sequence-specific primers that specifically amplified either fulllength CEA or CEA splice variant sequences.
Results (Table 1 ) of qPCR analysis using frozen primary pancreatic tissue specimens demonstrated that full-length CEA transcript was detected in normal human pancreatic tissues (3 of 4). Among diseased tissue, the full length transcript was found frequently in pancreatic adenocarcinoma (12 of 13) and less frequently in benign adenomas (1 of 2) and in pancreatic endocrine tumors (3 of 5), although the total number of specimens representing the latter two categories were small. Transcripts of the CEA splice variant were not detected in normal pancreatic tissues (0 of 4), benign adenomas (0 of 2) nor in endocrine tumors (0 of 5), and were rarely detected in pancreatic adenocarcinomas (1 of 13). Expression of the CEA splice variant transcript in the single positive adenocarcinoma specimen was concordant with full-length CEA transcript expression (Table S1) .
Results from qPCR analysis using human tissue cDNA arrays ( Table 2) (Table  S1 ). Thus, expression of the CEA splice variant transcripts varied in different cancers; however, it was always coexpressed with fulllength CEA transcripts. 
Although the CEA splice variant was infrequently found in pancreatic tumors, it was found at a high frequency in colorectal (98%) and gastroesophageal (50%) cancers, and to a lesser degree in lung (30%) and breast (12%) tumors. Due to its concordant expression with full length CEA in human tumors, we sought to understand the binding of MEDI-565 to the CEA splice variant and the role that this CEA isoform might play in targeting CEApositive tumors. The amino acids in full-length CEA important for MEDI-565 binding were found to be largely absent in the CEA splice variant, except amino acids I 408 and N 410 in segment C ( Figure 2 ). This observation suggested that binding of MEDI-565 to the CEA splice variant was unlikely to occur. To test this hypothesis, CHO cells were infected with lentivirus constructs directing the expression of full-length CEA (CHO FL CEA) or the CEA splice variant (CHO SV CEA), or both concurrently after sequential infection of cells with the CEA splice variant then the full-length CEA (CHO FL+SV CEA); full-length CEA and CEA splice variant protein expression were verified by western blotting ( Figure S1 ). As anticipated, MEDI-565 bound to cells expressing full-length CEA but not to cells expressing the CEA splice variant ( Figure 6A ). We note that a higher level of MEDI-565 binding was observed for cells expressing both the CEA splice variant and full length CEA together relative to cells expressing only full length CEA. This is likely due to different levels of expression efficiency in each independently infected cell line, although effects of the CEA splice variant protein on full length CEA protein levels or epitope accessibility in cells cannot be ruled out.
Zhou et al [48] showed that the both the N-terminal domain and A3 mediated the interaction between CEA molecues on apposing cell surfaces; both domains are present within the CEA splice variant. Thus, we tested the hypothesis that co-expression of the CEA splice variant and full-length CEA proteins may, through heterophilic interactions, result in the MEDI-565 binding epitope being masked in the full-length form and subsequently prevent or reduce MEDI-565 binding. Co-expression of the CEA splice variant with full-length CEA on the same cells did not significantly affect the apparent binding affinity of MEDI-565 to full-length CEA (CHO FL CEA, apparent K D = 5.060.15 nM; CHO FL+SV CEA, apparent K D = 5.663.0 nM; p = 0.86). These results were expected since homophilic interactions between fulllength CEA proteins also do not appear to prevent the binding of MEDI-565 [17, 18] or other CEA-specific BiTE antibodies [16] .
Consistent with the MEDI-565 binding data, CHO SV CEA did not trigger the activation of T cells from healthy donors in the presence of MEDI-565, as measured by the up-regulation of the IL-2Ra chain/CD25 protein on either CD8 or CD4 T cells ( Figure 6C and D) . This T cell activation marker has been shown previously to correlate temporally with the release of cytokines from T cells activated by MEDI-565 [18] and other BiTE antibodies [23, 25] . Likewise MEDI-565 also did not mediate the lysis of target cells expressing the splice variant ( Figure 6B ). In contrast, MEDI-565 activated T cells and induced killing of CHO FL CEA or CHO FL+SV CEA cells with similar levels of potency (EC 50 values: CHO FL CEA, EC 50 = 75635 ng/mL; CHO FL+SV CEA, EC 50 = 59643 ng/mL; p = 0.79). Somewhat higher maximal T cell activation and maximum achievable cytotoxicity levels were observed with CHO FL+SV CEA cells relative to CHO FL CEA cells, and may be explained by the higher full length CEA expression in the former, as described above.
Studies with full length CEA and the splice variant were carried out in CHO cells due to their ease of infection with lentiviral constructs and their subsequent selection of CEA-expressing cells using flow cytometry. To demonstrate the killing specificity of MEDI-565 for CEA positive human tumor cells, we examined numerous human cancer cell lines for CEA expression and for their ability to be killed by MEDI-565 activated T cells. Consistent with the specificity observed for CHO cells, MEDI-565 mediated the killing of human tumor cells that expressed CEA, but not those that did not express cell surface CEA ( Figure S2 ). Additionally, a control BiTE did not induce T cell lysis of CEA positive or negative cell lines.
To provide insights for CEA-targeted diagnostics and therapy and understand the specificity of anti-CEA mAbs, many efforts have been made to characterize the binding epitopes of these antibodies using various approaches [49, 50, 51, 52] . The most extensive investigations involve the classification of 52 anti-CEA mAbs into five distinct epitopes, Gold epitopes 1-5, by competitive binding analysis [50] . Further studies have correlated the Gold epitopes to the domains of CEA using different CEA fragment constructs [51, 53, 54] , however there is no precise localization of the epitopes to the amino acid sequence level. An attempt to identify the amino acid sequences corresponding to Gold epitopes using synthetic overlapping fifteen-mer peptides has failed [55] , suggesting that the Gold epitopes are conformational. In addition, immunohistochemistry and immuno flow cytometry were used to demonstrate a good correlation between the Gold epitope groups and their binding specificity: 1) mAbs that bind to Gold epitopes 2 and 3 were generally specific to CEA, reacting only with colon carcinoma, normal colon mucosa and normal gastric foveola; 2) mAbs in epitopes 4 and 5 were highly cross-reactive with different normal tissues possibly due to binding to CEA-related antigens; and 3) both specific and cross-reactive mAbs were found in epitope 1 [56] . We have mapped the epitope of the CEA-specific arm of MEDI-565 to the A2 domain comprised of two stretches of amino acids, 326-349 and 388-410. Interestingly, the MEDI-565 epitope on CEA belongs to the Gold epitope 2, which has been localized in the A2-B2 domains [53] and identified as a CEA-specific epitope group [56] .
CEA comprises highly repetitive immunoglobulin domains of A1-B1, A2-B2, and A3-B3. Some anti-CEA mAbs bind repetitive epitopes of CEA [51] and some are highly cross-reactive, lacking specificity to CEA [56] . The anti-CEA arm of MEDI-565 is a humanized version of the murine antibody A5B7, which has been shown to specifically bind to CEA [40, 56] . Characterizing the epitope of MEDI-565 has led us to propose that the critical residues F 326 , T 328 , and N 333 mediate the fine specificity to CEA of MEDI-565 due to their uniqueness in the A2 domain of CEA when compared with all other immunoglobulin domains of CEA and other related molecules of the CEACAM family, including CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8.
After identifying the binding epitope of MEDI-565, we evaluated the impact of polymorphisms and isoforms in the A2 domain of CEA on the activity of MEDI-565. Two nonsynonymous SNPs were identified in the binding epitope of MEDI-565 but occurred at a very low frequency in the general population and were considered to have little or no impact on MEDI-565 binding to CEA. In addition, a single splice variant was identified lacking a portion of the A2 domain critical for MEDI-565 binding. Efforts to understand the association of splice variant expression to disease found that its expression occurred in a substantial percentage of primary human colorectal and gastro-esophageal tumors, and to a lesser extent in lung and breast tumors in a pattern that was concordant with full length CEA expression. However, since MEDI-565 did not bind the CEA splice variant due to loss of the antibody-binding epitope, it was clear that the splice variant itself is not a target for MEDI-565 in primary human tumors that also express full length CEA. Because CEA could participate in homotypic interactions on adjacent cells, it remained possible that expression of the CEA splice variant may interfere with the binding of MEDI-565 and the subsequent tumor cell lysis through its interaction with full length CEA. By coexpressing the CEA splice variant with full length CEA, we formally demonstrated that this was not the case, as MEDI-565 binding to full length CEA and potency of CEA-directed lysis were not significantly affected by simultaneous expression of the splice variant and full length CEA. Therefore, the expression of the CEA splice variant by primary human tumor cells is not anticipated to interfere with MEDI-565 binding to full-length CEA, nor should it inhibit MEDI-565-mediated T-cell killing of tumor cells expressing full-length CEA. However, discrimination of full-length CEA from the CEA splice variant may be important while monitoring the status of CEA positive tumors via changes in serum CEA levels in clinical study patients that receive MEDI-565. Figure S1 Western blot of CEA protein expressed by CHO cell lines. The full-length CEA protein is indicated by a filled arrowhead, and the CEA splice variant by an open arrowhead; both proteins were detected using a CEACAM5-specific mAb. Molecular weights (kilodaltons; KDa) of the protein standard are indicated to the left of the image. Lanes of CHO FL and CHO FL+SV cell lysates contain a band at ,100 kDa that is presumed to be a non-glycosylated form of CEA. Equal amounts of protein loaded into each lane of the gel were controlled by detecting  High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit. There has been significant progress in the development of vaccines and therapeutics against viruses, but there are still major gaps in medical therapy for some of the most common types of viral infections. For these types of infections, vaccines can still be ineffective due to new and emergent strains and can exhibit significant off-target effects [1, 2] . Similarly, the efficacy of antiviral therapeutics can often be limited by pathogen resistance as another sign of the difficulty in keeping up with rapidly evolving viral genomes [3] [4] [5] [6] [7] [8] [9] . An alternative to agents that specifically and directly target the virus itself is the possibility of improving natural host defense against a broad range of viruses. Although antiviral defense exhibits significant complexity and redundancy, one system that stands out as a useful target for improvement is the one based on the action of interferons (IFNs). And within this IFN system, which is similarly complex, the STAT1 transcription factor is remarkable as a central component that is critical for the functional activity of each type of IFN ( Figure 1 ). Consequently, genetic loss of STAT1 function causes a marked susceptibility to viral infection in mice and humans [10] [11] [12] . Moreover, modification of STAT1 to a form) that improves the efficiency of IFN signal transduction can result in improved control of viral infection [13] . These observations indicate that the IFN-signaling pathway is subject to a so-called ''rheo-STAT'' adjustment wherein downregulation causes increased susceptibility to viral infection whereas up-regulation might lead to increased efficiencies for IFNstimulated gene (ISG) expression and control of infection [14] .
In the present study, we aimed to mimic the beneficial actions of STAT1 modification with a small molecule that also enhances the activity of the IFN signaling pathway. We describe here the development of a high-throughput screening (HTS) system for novel small molecular weight compounds (so-called ''small molecules'') that might increase ISG expression and antiviral activity. To develop this screening system, we generated cell lines that stably express the human interferon-stimulated response element (ISRE) driving a luciferase reporter gene. The ISRE gene promoter element is responsible for type I IFN signaling that mediates host defense against a wide range of viruses [15, 16] . After establishing that the ISRE-reporter cell line responded linearly to IFN-b concentration and treatment time, we converted the assay to an automated format for a screen against already approved or approvable drugs. We also screened a library of phosphatase inhibitors that might mediate increased STAT1 phosphorylationactivation. Our analysis identified a series of diverse compounds capable of significantly increasing ISRE activity. One compound in particular, the anthracycline antibiotic idarubicin hydrochloride, was used to explore mechanism of action and to validate the proposal that small molecules can enhance ISRE activity to drive higher levels of ISG expression and improved control of viral level. The findings provide for the concept that current antiviral therapeutics act directly and specifically on viral proteins whereas next-generation antivirals might act to enhance host immunity against a broad range of viruses. Either alone or together, these approaches might better address the current need for more effective treatment against common as well as new and emergent viral infections.
Based on the observation that STAT1-CC-expressing cells show increased activity of the endogenous ISRE promoter element [13] , we established cell lines that stably expressed an ISRE-containing gene promoter driving a click beetle luciferase reporter gene CBG99luc (Figure 2A ). We used 2fTGH cells (the parental line for STAT1-deficient U3A cells) as well as HEK293T cells that both express endogenous STAT1. Clonal lines showing IFN-b-inducible ISRE-promoter driven luciferase activity were designated 2fTGH-or HEK293T-ISRE-CBG99 cells. For initial assay development, we used the 2fTGH-and HEK293T-ISRE-CBG99 cells to establish an optimal luciferase light reaction time for both cell-lines. Based on luminescence signal stability over the time course of the reaction, an optimal readout window of 40-70 min after the start of the reaction was chosen for subsequent experiments ( Figure 2B ). After optimization of cell growth time, response to various IFN-b treatment times and concentrations were tested in 2fTGH and HEK293T cell lines. Each cell line exhibited a distinct IFN-b treatment time for maximal signal: 7-12 h for 2fTGH-ISRE-CBG99 cells and 14-24 h for HEK293T-ISRE-CBG99 cells. Although a lower signal magnitude was obtained with 2fTGH-ISRE-CBG99 cells compared to HEK293T-ISRE-CBG99 cells, the 2fTGH-ISRE-CBG99 cells show more specificity for IFN-b (compared to IFN-c) treatment at all IFN-b treatment time periods tested ( Figure 2E ) and over a range of IFN-b (and IFN-c) concentrations ( Figure 2F , G). Thus, the 2fTGH-ISRE-CBG99 cells were chosen for further assay development.
To achieve assay automation and miniaturization, the ISRE activity assay was first automated in 96-well plates and then reformatted for 384-well plates. In the 384-well format, the assay exhibited a near-maximal signal at 8000 cells per well and consistent well-to-well and plate-to-plate reproducibility (Figure S1). In 96-and 384-well formats, signal to background (S/B) ratios, coefficients of variance, and Z9-factors achieved excellent performance in comparison to published standards [17, 18] . Representative results for 2fTGH-ISRE-CBG99 cells treated with IFN-b (1000 U/ml) for 7 h compared to 1% DMSO vehicle alone are provided in Table 1 . The results indicate the development of a quantitative and specific cell-based HTS assay of IFN-responsive gene promoter activity.
We used the automated ISRE-activity assay to perform a screen of a 2240 chemical compound library. This library consisted of 2160 compounds from the Johns Hopkins Clinical Compound Library (JHCCL) of FDA approved or approvable drugs [19, 20] . In addition, we included 33 compounds from the Screen-Well Phosphatase Inhibitor Library based on the observation that the improvement in IFN signaling in STAT1-CC-expressing cells correlated with prolonged phosphorylation of STAT1 and STAT2 [13] . Each compound was tested at 4 different concentrations (0.24, 1.2, 6 and 30 mM) and simultaneous treatment with IFN-b at 5 U/ml, the concentration at the initial inflexion of the IFN concentration-response curve (as shown in Figure 2F ). These treatment conditions were duplicated on a second plate. Each assay plate also contained control wells containing a range of IFN- Figure 1 . Scheme for IFN signal transduction. Type I IFN signaling starts by activation of the IFN-a/b receptor (IFNAR) and subsequent activation of the IFNAR1-associated TYK2 and IFNAR2-associated JAK1, with consequent recruitment of STAT2. Phosphorylation of STAT2 enables reruitment of STAT1 and release of the phosphorylated STAT1-STAT2 heterodimer bound to IRF-9. This complex binds to the IFN stimulated response element (ISRE) and in concert with recruited transcriptional co-activators such as p300/CBP then drives IFN-stimulated gene (ISG) transcription. doi:10.1371/journal.pone.0036594.g001 b concentrations (0-200 U/ml) in quadruplicate ( Figure 3A ). This arrangement achieved excellent signal reproducibility between duplicate compound plates as well as signal consistency through the full screening run of 56 assay plates (28 duplicate pairs) ( Figure 3B , C).
After raw data were normalized, scaled to z-scores, and summarized, we found that 321 data points (out of a total of 8960 data points representing the 2240 compounds tested at 4 concentrations) had an ISRE activity z-score $2 ( Figure 4A ). This data set represented 285 individual compounds, as some compounds had an ISRE activity z-score $2 at more than one dose. Of these 285 compounds, 64 hit compounds (2.9% of the total compound library) were selected for validation based on a combination of dose-response characteristics and inter-replicate reproducibility. This approach captured all 20 of the compounds with the highest z-scores. In support of re-purposing as a drug discovery strategy, the 64 screening hits were found in a broad range of drug classes ( Figure 4B ).
Each of the 64 primary hits was subjected to primary validation for ISRE activity over a broader range of concentrations of drug and IFN-b (0-15 U/ml). Among the primary and confirmed screening hits, idarubicin hydrochloride ranked highest in potency for enhancing ISRE activity (i.e., idarubicin exhibited a significant effect at a lower concentration than other compounds). During the ISRE validation, we found that idarubicin caused a concentrationdependent increase in ISRE activity over a range of IFN-b treatment concentrations, with highly significant effects as low as 25 nM idarubicin in combination with 15 U/ml IFN-b ( Figure 5A) .
The structure for idarubicin shows characteristic features of an anthracycline antibiotic unrelated to any other antiviral compound in clinical use. To further validate the effect of idarubicin on ISRE activity, we tested three other anthracyclines (daunorubicin, doxorubicin, epirubicin) with very similar chemical structures to idarubicin. Each of these compounds also showed a capacity to significantly increase ISRE activity ( Figure 5B ). In addition, we found that the immune activators DMXAA (Vadimezan) and Imiquimod did not cause any increase in ISRE activity in the same concentration range ( Figure 5C ). These compounds appear to directly activate immune cells (including increased IFN production) [21, 22] . However, for the present work, we specifically studied non-hematopoietic cells since that population appears critical for STAT1-mediated defense against at least some types of viruses [11] . We also found a cytotoxic effect of idarubicin that is consistent with previous observations [23, 24] . Under the present conditions, the major effect of idarubicin on cell viability was detected at drug concentrations .3 mM, so that the ISRE-activating effect of idarubicin occurred at concentrations below those that cause a major effect on cell viability ( Figure 5D ). Nonetheless, idarubicin is best known as an anti-neoplastic agent that acts via DNA intercalation and topoisomerase II inhibition [25] . To determine whether the effect of idarubicin on ISRE activity is related to topoisomerase II inhibition, we tested three other potent topoisomerase inhibitors Etoposide, Hu-0331, and ICRF-193 up to concentrations known to cause topoisomerase II inhibition [26] [27] [28] . In contrast to idarubicin, these other compounds caused no significant increase in ISRE activity ( Figure 5E ). In fact, two of the topoisomerase inhibitors (Etoposide and Hu-0331) caused a decrease in ISRE activity in concert with cytotoxic effects at higher concentrations. These findings indicate that the capacity of idarubicin to activate the ISRE component of the IFN signaling pathway occurs independently of topoisomerase inhibition. Together, the findings provide evidence of idarubicin capacity to increase ISRE activity independent of the anti-neoplastic properties of the drug. We also observed that the effect of idarubin and the other anthracyclines on ISRE activity occurred at a lower concentration of drug when IFN-b was co-administered, particularly at the highest concentration of IFN-b (15 U/ml) ( Figure 5A ). For example, the EC 50 for idarubicin decreased as the concentration of IFN-b increased ( Table 2 ). These findings suggested that idarubicin might somehow interact with the IFN signaling pathway. In that regard, we also found that the effect of idarubicin on ISRE activity was observed under baseline conditions when there was no detectable production of endogenous IFN-b and no administration of exogenous IFN-b (data not shown and Figure 5A ). These results suggested that the effect of idarubicin on ISRE activity is independent of IFN production or action. Indeed, we also found that the effect of idarubicin on ISRE activity persisted without change during effective IFN-a/b receptor 2 (IFNAR2) blockade ( Figure 6 ). Together, the findings indicate that idarubicin causes an increase in ISRE activity independent of IFN production or IFN-IFN-receptor interaction and instead acts downstream of ligandreceptor binding in the IFN signaling pathway.
We subjected idarubicin to further validation as an ISRE activator in assays of ISG expression and antiviral activity. For ISG expression, U3A (STAT1-null) and U3A-STAT1 cells were treated with a range of concentrations of idarubicin and IFN-b and then harvested for gene expression using quantitative realtime PCR assay. We found that idarubicin increased the expression of the antiviral gene 29,59-oligoadenylate synthetase 1 (OAS1), particularly with IFN-b treatment ( Figure 7 ). There was no effect of drug on ISG expression in STAT1-null U3A cells, indicating that the drug is specific for STAT1-dependent gene expression. We found similar results for the antiviral ISG guanylate-binding protein 1 (GBP1) and three other ISGs (MX1, PARP9, and IRF1) ( Figure 6 and data not shown).
For antiviral activity, 2fTGH cells were treated with idarubicin along with or without IFN-b and then assessed for control of encephalomyocarditis virus (EMCV) levels and virus-induced cytopathic effect. We selected EMCV since it was previously found to be sensitive to STAT1-CC-dependent improvement in IFN signaling [13] . In the present experiments, we found that idarubicin treatment (at a relatively low concentration of 25 nM) caused a significant decrease in EMCV titer at baseline and with IFN-b treatment (at a relatively low concentration of 5 U/ml) ( Figure 8A ). In addition, we observed that idarubicin-dependent improvement in viral control translates into a significant decrease in viral cytopathic effect under those same treatment conditions ( Figure 8B ). Higher concentrations of idarubicin in combination with IFN-b treatment caused a significant cytotoxic effect (data not shown), consistent with the antineoplastic properties of the drug. Nonetheless, the results with a relatively low concentration of idarubicin provide proof-of-concept that a small molecule activator of the ISRE component of the IFN signaling pathway will allow for increased ISG expression and improved control of viral level.
The present study was undertaken to discover antiviral therapeutics that broadly increase host defense. We focused on the IFN system that is central to the antiviral response, although we recognized that other labs have pursued this target with limited success in the past. Some of these previous investigators have used administration of IFN itself to increase the antiviral response, but for this therapeutic goal and others, the side effects of IFN administration have proven to be rate-limiting [29] . Similarly, other investigators have attempted to boost IFN production, e.g., through administration Toll-like receptor (TLR) agonists CpG or Imiquimod, but these agents have also caused similar side effects [30] [31] [32] [33] . The small molecule DMXAA activates multiple immune pathways (NF-kB, TBK1/IRF3, NOD, and MAP kinase) but was ineffective as an antiviral unless it was administered before infection [34] [35] [36] [37] . To circumvent at least some of these issues with IFN production, toxicity, and specificity, we therefore pursued the goal of antiviral drug discovery with a novel screening approach for identifying small molecule enhancers that might selectively boost the activity or efficiency of the IFN signaling pathway.
Our specific approach was based on previous success with the use of a modified STAT1 signaling pathway. In this work, we demonstrated that a designer form of STAT1 (designated STAT1-CC based on double-cysteine substitutions) was able to enhance IFN signaling and better control viral replication [13] . Although STAT1-CC gene expression would be challenging to translate to practical application, the mechanism of action served as a guide to design a screening strategy to identify small molecules that could mimic the antiviral benefit. In that regard, we also recognized that phenotypic screening approaches have proven to be more effective than target-based approaches for the discovery of first-in-class small molecule drugs [38] . Thus, target-based approaches (defined as direct drug action on a particular target) allow for analysis and refinement of structure and function but can also waste resources when the molecular hypotheses used to design screening assays may not be relevant to the disease. Meanwhile, phenotypic screening can take longer in terms of hit-to-lead development but provide more proteins in the pathway to be targeted and do not require prior knowledge of molecular mechanisms of action. Most importantly, the activity found in phenotypic-based approaches is often more effectively translated into therapeutic impact in disease models. For the present work, we take advantage of both of these strategies to some extent and devise a screen that incorporates molecular mechanism (i.e., enhancing a specific type of IFN signaling pathway) and the need to achieve phenotype (i.e., identifying any compound that could increase this type of signaling pathway regardless of specific mechanism).
Considering these factors and observations in the STAT1-CC model system, we designed a cell-based luciferase reporter assay for measuring type I-dependent ISRE activity. This assay proved to yield excellent signal-to-background and Z factors, specificity for IFN-b treatment over IFN-c treatment, and suitability for automation and screening. Furthermore, because the construct design uses the Click Beetle Green luciferase, the assay can be paired with other luciferase reporter genes to develop dual color assays to report activity of other signaling pathways, including the type II IFN-c activated sequence (GAS) promoter activity that mediates defense against intracellular bacteria. A related approach was used to screen for small molecules that increase GAS activity for anti-proliferative and pro-apoptotic effects in cancer cells [39] . Others screened for compounds that inhibit Type I IFN production and signaling [40] . An ISRE-RFP reporter system has also been used to screen for assessing the effects of immunostimulatory RNA [41] . Others have used a less directed approach to screen for compounds that might use any mechanism to decrease viral levels [40, [42] [43] [44] [45] . However, to our knowledge, the present study conducts the first semi-quantitative screen measuring ISRE activity to discover small molecule enhancers of the type I IFN signaling pathway as broad-spectrum antiviral therapeutics.
Our primary screen identified idarubicin on the basis of its capacity to significantly increase ISRE activity. Subsequent validation assays demonstrated that idarubicin facilitates STAT1-dependent ISG expression and STAT1-directed control of viral replication and cytopathic effect. While others previously reported the antiviral properties of anthracyclines some time ago, no mechanism of their antiviral action was elucidated [46] [47] [48] . In the present study, we observed drug-induced cytotoxicity in a dose-range similar to those reported previously [23, 24] , however, we establish that the effect of idarubicin on the antiviral IFN pathway is independent of cytotoxicity and topoisomerase inhibition. Because idarubicin enhances the IFN signaling pathway output, we questioned whether the drug might also cause IFN-driven cell death. However, we found no increase in cytotoxicity in cells treated with idarubicin and IFN together compared to cells treated with idarubicin alone. We also found that the idarubicin concentrations for activating the ISRE component of the IFN signaling pathway were significantly less than those required for major cytotoxicity. Thus, we conclude that idarubicin effect on IFN signaling is distinct from the effect on DNA-based cytotoxicity. The dose dependency of these effects underlines the need to conduct screening at multiple concentrations of test compounds, particularly lower concentrations that prevent false negative hits due to cytotoxic effects.
In a further analysis of drug mechanism, our study demonstrates that the antiviral activity of idarubicin and other closely related anthracyclines is derived from enhancing the activity of the type I IFN signaling pathway. Our data further show that the enhancing effect of idarubicin is based on ISRE activation and ISG expression independent of IFN production or IFN-IFN-receptor interaction, since the effect of idarubicin is unchanged by IFNreceptor blockade. These findings suggest that idarubicin activation of the ISRE is due to an action in the IFN signaling pathway distal to ligand-receptor binding, e.g., at the level of receptorassociated JAK kinases or further downstream at the formation, transport, binding, and/or assembly of the ISRE transcriptional complex. In that regard, anthracyclines are known to inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand to prevent replication, but whether this mechanism can affect ISRE or other gene promoter elements still needs to be defined. The present screening approach overcomes the uncertainty in molecular mechanism by using a phenotypic (rather than a target-based) screening approach and thereby captures compounds that increase the activity of the IFN signaling pathway by either established or undefined mechanisms.
In sum, we describe and validate a phenotypic screening strategy to identify small molecules that enhance the activity of the type I IFN signaling pathway and consequently improve antiviral host defense. This approach is designed to lead to discovery of drugs with activity against a broad range of viruses for clinical application as well as experimental tool compounds to further understand IFN-dependent immune mechanisms. Current approaches to defining the basis for IFN signal transduction, particularly in vivo, often rely on complex transgenic and gene targeting approaches. Thus, the use of small molecule enhancers (SMEs) of the IFN signaling pathway may provide much greater flexibility and ease of application to achieve transient adjustment of IFN-related actions and consequent scientific and clinical benefit. Our approach should thereby prove useful to discover drugs with activity against a broad range of viruses as well as effectiveness in other conditions (e.g., multiple sclerosis and melanoma) where the efficacy of IFN treatment might benefit from enhancing the IFN signaling pathway.
Stimulating agents and chemical compounds IFN-b and IFN-c were obtained from PBL Interferon source (Piscataway, NJ), diluted and aliquoted according of manufacturer's recommendation, and stored at 280uC. The Johns Hopkins Clinical Compound Library (JHCCL) was obtained from Dr. David Sullivan at the Johns Hopkins University [19, 20] . The Screen-Well Phosphatase Inhibitor Library was obtained from Enzo Life Sciences (Farmingdale, NY). All other chemical compounds were obtained from Sigma Aldrich (St. Louis, MO).
To construct the pISRE-CBG99 vector, we first generated a 5xrepeat of the ISRE sequence and a TATAA box (5xISRE-TATAA) in the pUCMinusMCS vector from Blue Heron Biotechnology (Bothell, WA) and then cloned this sequence into the Chroma-Luc pCBG99-Basic reporter vector from Promega (Madison, WI) and ligated into Xma1 and Nco1 sites using T4 DNA ligase from Life Technologies (Carlsbad, CA). The DNA sequence of the resultant pISRE-CBG99 vector was confirmed by carrying out BigDye Terminator v3.1 sequencing reactions (Life Technologies) on an ABI capillary sequencer. This vector and the pPUR selection vector from Clontech (Mountain View, CA) were co-transfected at a 9:1 ratio into 2fTGH or HEK293T cells to increase the likelihood that cells tolerating puromycin selection (0.5 mg/ml) contained one or more copies of pISRE-CBG99 in addition to pPUR. The 2fTGH cells [49] were obtained from G. Stark (Cleveland Clinic), and HEK293T cells [50, 51] were obtained from T. Brett (Washington University). Transfection was performed using Fugene6 transfection reagent from Roche Applied Science (Indianapolis, IN) . Limiting dilution was used to obtain individual cell clones that were then screened for luciferasemediated luminescence after treatment with IFN-b (1000 U/ml) on a BioTek Synergy 4 multimode plate reader (BioTek, Winooski, VT). Clonal cells exhibiting stable expression were then used for further assay development.
To optimize the luciferase light reaction, we tested a series of flash and glow luminescent substrate systems in both lysed and live 2fTGH-ISRE-CBG99 and HEK293T-ISRE-CBG99 cells, including D-luciferin (Fisher Scientific, Pittsburgh, PA), the Chroma-Glo Luciferase assay system from Promega and the steadylite plus reporter gene assay system from Perkin Elmer (Waltham, MA) under a range of incubation conditions. The steadylite plus system was selected based on kinetic profile. Effects of IFN-b concentra-tion and treatment time were assessed with all other variables constant.
The assay was automated in a 96-well format with a customized and fully integrated robotic system. The system equipment included: a Caliper Sciclone ALH 3000 workstation (Perkin Elmer) and a EL406 washer (BioTek) for liquid handling, an automated Liconic incubator (Thermo Scientific) for cold storage of plates, an automated Cytomat incubator (Thermo Scientific) for cell culture environment, a separate hotel for storage of plates at room temperature, a Synergy 4 plate reader, a Flexiseal plate heat sealer (K Biosciences, Beverly, MA), a Caliper Twister II, and a Beckman Sagian Orca robotic arm on a linear rail (Beckman Coulter, Fullerton, CA). Construction allowed for transfer of plates, reagents, and plasticware between all instruments, so that there was no need for any manual interference during screening assays. This entire system was enclosed in a custom-made laminar flow hood to allow for HTS screening capability under BSL2 sterile conditions. After the system demonstrated satisfactory performance in a 96-well format, the assay was miniaturized to a 384-well format and re-tested for reproducibility and stability under IFN-b and vehicle (1% DMSO) treatment conditions.
To achieve simultaneous treatment of cells with IFN-b and various compound concentrations and to avoid reagent degradation over time, the screen was run in a modular manner with a precise timeline ( Figure S2 ). The first step included production of plates with appropriate concentrations of compound and IFN-b and then storage at 4uC. A separate plate was made for each of the four compound concentrations (0.24, 1.2, 6 and 30 mM). The Twister II, Sciclone, Orca, and Liconic cold storage incubator handled this step. For the second step, cells were plated at 8000 cells per well in 384-well assay plates (n = 56). This step was accomplished in seven batches (8 assay plates per batch) using the Sciclone. A uniform suspension of cells was maintained by intermittent mixing on the Sciclone deck between cell plating. Simultaneously, the compound stock plates were sealed using the Flexiseal and stacked back into a Twister II rack for storage. For the third step, cells were allowed to grow for 11 h and then were treated with compound and IFN solutions. This step required that a plate containing cells be brought from the Cytomat incubator to the Sciclone deck in concert with a set of compound/IFN dilutions plates from the cold storage incubator. Cell treatments were timed so that each assay plate would be incubated for 10.3 h before the final step of performing the luciferase assay. For this last step, robotics were programmed so that each assay plate developed the luciferase light reaction for 40 min at 25uC in the plate hotel and then was delivered to the Synergy 4 plate reader for determination of luminescence. For this assay, the BioTek EL406 washer was used for aspiration of media and dispersion of substrate. In entirety, the screen took 41.6 h to complete.
The raw data from the HTS assay were subjected to statistical analysis using cellHTS2 [52, 54] , a software package designed for the analysis of HTS data as part of the Bioconductor project for statistical computing [55] . Raw data were normalized using the plate median method [52, 56] . Next, a z-score transformation was applied to center and scale the data across the experiment. Replicates for a given compound at a given dose (N = 2 for each dose/compound combination) were then mean summarized. A zscore threshold of $2 was chosen to identify potential hits.
Thereafter, to reach a smaller and tractable set of hits to validate experimentally, we took advantage of testing each compound at four concentrations. Specifically, we used self-organizing maps analysis to cluster hit compounds by shape of the dose-response curve. The significance of change from dose to dose (0.24 to 1.2, 1.2 to 6, and 6 to 30) was also analyzed using linear models and moderated F-statistics as implemented in the limma package [57] in Bioconductor [55] . The concentration-response curves for each compound were then visually inspected, using scatter plots generated in TIBCO Spotfire DecisionSite (TIBCO, Palo Alto, CA), with respect to the shape of the curve and reproducibility between replicates. Compounds showing an erratic concentrationresponse (e.g. increase, then decrease, and increase again in ISRE activity with increasing concentration) were rejected. Compounds with a consistent increase or decrease in response with increasing drug concentration or good efficacy at any concentration were included for further validation. This approach led to selection of 64 compounds for further validation, including compounds with the 20 highest z-scores.
Hits from the primary screen were validated using the ISRE activity-luciferase reporter assay over a broad range of compound concentrations (0.01-25 mM) in the absence or presence of IFN-b (1, 5, and 15 U/ml). To determine drug potency, as defined by halfmaximal effective concentration (EC 50 ), this data was fit to a fourparameter concentration-response curve as described previously [58] using the log agonist concentration versus response, variable slope algorithm in GraphPad Prism 5 software (La Jolla, CA) where Y = Bottom + (Top-Bottom)/(1+10((LogEC50-X)*HillSlope)). To determine whether compound effect depended on IFN production, the ISRE activity-luciferase reporter assay was also performed in the presence of mouse anti-human IFN-a/b receptor chain 2 (IFNAR2) blocking mAb (clone MMHAE-2; Millipore, Billerica, MA) at a concentration of 4 mg/ml.
A resazurin (Alamar Blue) metabolism assay was used to assess cell toxicity during compound treatment [59] . For these experiments, cells were treated with compound or an equivalent concentration of vehicle (DMSO) for 12 h, and the medium was replaced with fresh medium containing resazurin (80% dilution of Tox-8 kit, Sigma-Aldrich, St. Louis, MO). After 1.5 h at 37uC under standard culture conditions, the fluorescence of the resultant product resorufin was measured using the Synergy 4 plate reader. Wells with cells containing no compound (DMSO alone) and wells containing no cells were used as 100% and 0% viability controls. Data were normalized to calculate percentage viability.
Expression of ISG's was assessed with real-time quantitative PCR assay for the corresponding mRNA level. For these experiments, U3A and U3A-STAT1 cells were first treated with the programmed combination of compound and IFN-b for 12 h and then were washed twice with cold Dulbecco's PBS followed by lysis with Cells-to-cDNA II lysis buffer (Life Technologies) and treatment with DNase according to the manufacturer's instructions. The U3A cells were obtained from G. Stark (Cleveland Clinic) and complemented with STAT1 to generate U3A-STAT1 cells as described previously [13] . A 25-ml aliquot of the cell lysate was used to generate cDNA using the High Capacity Reverse Transcription Kit (Life Technologies). The resulting cDNA was quantified using the Quant-iT OliGreen ssDNA kit (Life Technologies). Average cDNA concentration was 71625 ng/ml. Subsequent PCR assays were performed in adherence with MIQE guidelines [60, 61] , including the design of assays for ISGs (OAS1 and GBP1) and normalizer gene ornithine decarboxylase antizyme (OAZ1). The normalizer gene OAZ1 was selected and validated for cell samples treated with and without IFN-b. In brief, candidate normalizer genes were selected from a combination of invariant genes selected from previous microarray data [11, 62, 63] and prior large-scale analyses of publicly available microarray data [64, 65] . These candidates were then tested using real-time quantitative PCR assays. Comparison of candidate normalizer gene expression between various IFN treatment and infection conditions using multiple software packages [66 68] led to the selection of the OAZ1 as the normalizer gene. Primers and probes for real-time quantitative PCR assays were designed using the ProbeFinder design algorithm (Roche Applied Science). For OAS1, 59ggtggagttcgatgtgctg-39 and 59-aggtttatagccgccagtca-39 were used as forward and reverse primers, along with UPL probe #37 (Roche Applied Science). A plasmid containing OAS1 transcript variant 2 cDNA (Ref ID NM_002534, Origene, Rockville, MD) was used as a standard for absolute quantitation of OAS1 copy number. For GBP1, 59-ttccaaaactaaaactctttcagga-39 and 59tgctgatggcattgacgtag-39 were used as forward and reverse primers, along with UPL probe #85. A plasmid containing the GBP1 cDNA (Clone ID: 3606865, Thermo Open Biosystems, Huntsville, AL) was used as a standard. The IDT PrimeTime pre-designed assay Hs.PT.42.328511.g (Integrated DNA Technologies, Coralville, IA) was used for OAZ1. A cDNA vector was used for OAZ1 as well (Clone ID: LIFESEQ913650, Thermo Open Biosystems). Data were collected on a LightCycler 480 instrument (Roche Applied Science). Quantification cycle values were calculated using a second derivative maxima algorithm as implemented in the Lightcycler 480 software.
Cells were cultured overnight and then treated with compound and IFN-b for 6 h. Thereafter, cells were washed and then were inoculated with EMCV (strain VR-129B, ATCC, Manassas, VA) for 1 h at MOI 1 as described previously [13] . Cells were then washed twice and cultured in medium containing 2% fetal bovine serum for 28 h. At that time, cell supernatants were used to determine viral titer based on real-time quantitative PCR assay for EMCV RNA with 59-ctgccttcggtgtcgc-39 (forward primer), 59tgggtcgaatcaaagttggag-39 (reverse primer), and 59caaggttttgagcgtgtctacgatgtgg-39 (probe). A TA plasmid containing the EMCV 3D protein was used as a standard for absolute quantitation of viral copy number. In addition, cell viability was determined using the Cellomics Arrayscan VTi high content imager (Thermo Scientific). For this assay, 15 images per well were obtained with a 10x objective. After background subtraction, cells were identified by nuclei stained by cell permeable dye Hoechst 33342. Propidium iodide fluorescence was quantified by defining a boundary of 2 pixels around the nuclei and then gating on a cell population that showed higher staining. For each sample replicate, cytotoxicity was calculated as the percentage of cells that showed increased propidium iodide staining based on samples of at least 5000 cells per well.  Feline Immunodeficiency Virus in South America The rapid emergence of AIDS in humans during the period between 1980 and 2000 has led to extensive efforts to understand more fully similar etiologic agents of chronic and progressive acquired immunodeficiency disease in several mammalian species. Lentiviruses that have gene sequence homology with human immunodeficiency virus (HIV) have been found in different species (including sheep, goats, horses, cattle, cats, and several Old World monkey species). Lentiviruses, comprising a genus of the Retroviridae family, cause persistent infection that can lead to varying degrees of morbidity and mortality depending on the virus and the host species involved. Feline immunodeficiency virus (FIV) causes an immune system disease in domestic cats (Felis catus) involving depletion of the CD4+ population of T lymphocytes, increased susceptibility to opportunistic infections, and sometimes death. Viruses related to domestic cat FIV occur also in a variety of nondomestic felids. This is a brief overview of the current state of knowledge of this large and ancient group of viruses (FIVs) in South America. Feline immunodeficiency virus (FIV) is a Lentivirus, closely related to HIV and SIV, which infects members of Felidae family. FIV is an important viral pathogen worldwide in the domestic cat (Felis catus), causes a slow progressive degeneration of immune functions that eventually leads to a disease. FIV is unique among the nonprimate lentiviruses because in its natural host species it induces a disease similar to AIDS in humans infected with human immunodeficiency virus type 1 (HIV-1), characterized by a progressive depletion of CD4 + T lymphocytes [5, 28, 35, 48, 77] . Species-specific strains, related to domestic cat FIV, have been isolated from a variety of nondomestic Felidae [11, 43] . Like HIV, FIV can be transmitted via mucosal exposure, blood transfer, and vertically either prenatally or postnatally [26] . For these reasons, FIV has been studied widely as both an important veterinary pathogen and an animal model for HIV/AIDS. Although FIV was first recognized in 1993 in Brazil [23] and in 1994 in Argentina [65] , there are few data describing the prevalence, ecology, clinical aspects, or genetic analyses of FIV in South America ( Figure 1 ). The prevalence of FIV within the continent is summarized in Table 1 . A better characterization of FIV strains circulating within South America will be required to augment our understanding of the importance of this lentivirus in felids. This paper provides an overview of the current state of knowledge of this large and ancient group of viruses (FIVs) in South America, grouped according to domestic and nondomestic felids. The data obtained allow a better understanding on FIV epidemiology and distribution. Efforts were made to gather and review all of the available information for each country. 
FIV infection, in domestic cats, causes a variable immunodeficiency syndrome characterized by recurrent gingivitis-stomatitis, cachexia, wasting, neurology, and an increased incidence of tumor development [1, 4, 48, 76] . In contrast, the ungulate lentiviruses cause diseases reminiscent of chronic inflammatory conditions while infection with the bovine lentivirus seems to be inapparent [71] . The rate of progression of the disease can depend on the genotype of the infecting FIV and is also likely influenced by undefined genetic determinants of the particular host [16] . FIV infection in domestic cats is associated with early robust humoral and cellular anti-viral immune responses, followed by a progressive immune suppression that results eventually in AIDS. The outcome of infection depends on the balance between the viral destruction of the immune system and the ability of the remaining immune system to eliminate the virus. Although the decrease in numbers of CD4+ cells is the hallmark of FIV infection, the virus has been shown to infect a variety of cell types in their respective hosts including CD4 + and CD8 + lymphocytes, B lymphocytes, cells of neuronal lineage and monocyte/macrophage lineage [15, 17, 29] . Joshi et al. (2005) have characterized feline CD4 + CD25 + T regulatory cells that support FIV replication. Recently, Reggeti, Ackerley and Bienzle (2008) have shown that feline dendritic cells express specific viral receptors and are infected productively by FIV [53] . FIV shares a similar pattern of receptor usage to HIV-1; however, CD 134 rather than CD4 is the primary binding partner, and subsequent interaction with the secondary receptor CXCR4 permits cells entry [58, 72, 73] . Differences in pathogenicity have been demonstrated among genetically distinct subtypes of FIV that circulate in domestic cats [14, 16, 49, 63, 68, 73] . On the basis of the analysis of envelope glycoprotein (Env), focusing on the third to fifth variable regions (V3-V5), FIV has been classified into five subtypes [30, 46, 60] a number that should be expected to increase as further studies reveal additional diversity. Recent studies identified distinct groups of FIV isolates from the United States and New Zealand [24, 69] (Figure 2 ).
Data regarding FIV infection in domestic felids in South America are sparse and have not been well evaluated. Expanded surveys of South American isolates will be required to determine the FIV isolates in the continent since only few studies have been published. Although there are no doubts about the presence of FIV in South America, prevalence data obtained using different techniques cannot be compared amongst countries or studies (Table 1) . Knowing the prevalence and variability of FIV is important for designing and testing vaccines under field conditions [27, 77] . Also, identification of circulating subtypes is essential to develop strategies for molecular diagnosis, since the genetic diversity of this virus is high [44, 54] which may lead to false negative diagnoses if inappropriate primers are used. In South America, only subtype B and E viruses have been found. It is important to remember that subtype B viruses are distributed worldwide and that the subtype E viruses have been more consistently identified only in Argentina ( Figure 1 ).
Preliminary studies suggested that FIV infection is widespread in the domestic cat population of Brazil [9, 12, 40, 52, 61, 64 ] . A published review indicated that subtype E was the only prevalent in Brazil [77] . Nevertheless, all studies indentified B as the only subtype circulating in FIV positive animals in Brazil, [12, 32, 40, 62] . Here an analysis was conducted with 473 bp of sequence encoding 157 amino acids comprising the V3-V4 region of the envelope glycoprotein from different subtypes, including those reported previously from South America (Figure 2 ). In this study we used this region of env in order to permit us to include more samples from South America. For this phylogenetic tree, the GenBank accession numbers, names, country and subtype for the FIV env sequences included were: M25381. It is important to state that all phylogenetic studies carried out in Brazil were performed in the same area, namely the south-east, and that Brazil is a huge country (Figure 1 ). More widespread surveys of Brazilian isolates are required to determine whether a single subtype of FIV predominates in Brazil. In Brazilian domestic cats, FIV infected cats have been observed over a prolonged period. During this time, few clinical signs were observed, although the virus was replicating and inducing changes in the immune system, leading to a progressive decline in immune function and the development later of overt clinical signs [51, 78, Hagiwara and Teixeira, unpublished data]. Previously, Brazilian studies established relationships between FIV infection and Toxoplasma gondii and Mycoplasma haemofelis [37, 39] . Otherwise, no association with disease has been recorded in cases of Brazilian FIV infection. It has been suggested that clade B viruses may be more ancient and relatively host adapted and thus may be less virulent [2, 50, 63] .
Preliminary seroepidemiological studies carried out on clinical cases suggested that FIV infection is widespread in the domestic cat population of Argentina [65] . The genetic diversity of FIV isolates from Argentine domestic cats has been well characterized [47, 75] . FIV isolates were isolated from peripheral blood mononuclear cells of four domestic cats. Phylogenetic analysis revealed that one isolate clustered with subtype B and the others formed subtype E [47] , prototype sequence for this group (Figure 2) .
In the north of the continent a single study was performed in 52 domestic cats on Isabela Island, Galapagos, Ecuador's coast. It was demonstrated using serological methods that none of the tested animals was infected with FIV [36] .
Viruses related to domestic cat FIV occur also in nondomestic felids, indeed FIV strains have been present in the nondomestic cat population for longer than domestic cats [45] . Carpenter et al. (1996) comment that members of at least eighteen of the 37 species in the family Felidae carry an FIV-related virus, as has been shown by the presence in their sera of antibodies which react with FIV antigens. A further twelve species were reported in another study that employed a three-antigen Western blot screening (cat, puma and lion FIV antigens) and a multigene PCR amplification of FIV genes [66] . In South America, 12 [74] . Lentiviruses in eight of these species have been detected in South America [6, 10, 19, 20, 33, 55, 66] .
Data regarding FIV infections in South American wild felids are sparse and studies have concentrated primarily on Brazil. The presence of antibodies against FIV in puma, detected by Western blotting, was found in Argentina (5 in 22, 23%), Bolivia (5 in 5, 100%), Brazil (2 in 13, 15%), Peru (1 in 5, 20%) and Venezuela (4 in 8, 50%) [10] . Further studies have reported antibodies recognizing FIV and the puma lentivirus (PLV in Brazilian free-ranging puma) [6, 19] . Troyer et al. (2005) concluded that most of the South American felids maintain a low level of FIV infection throughout their population. Within wild populations, the seroprevalence in South American felids varies from 5 to 28%. Unfortunately, the authors did not describe the regions of the continent where the samples originated. FIV pol genes from a Peruvian and a Brazilian zoo puma have been sequenced, the former being classified as subtype B and the latter as a distinct group, neither A nor B [10] . Additionally, FIV provirus has been reported in Brazilian jaguars (Panthera onca), pumas, jaguarondis (Puma yagouaroundi), oncelots (Leopardus pardalis), margays (Leopardus wiedii), pampas cat (Leopardus colocolo), geoffroy's cat (Leopardus geoffroyi) and little spotted cats (Leopardus tigrinus) [20, 33, 55] . The finding of these FIV infected species highlights the need for additional monitoring. Although the implications of these infections for wild felid conservation are difficult to assess, it is generally accepted that monitoring these infections is an important component for the management of endangered populations [13] .
It is important to emphasize that FIV strains infecting 9 species of the Felidae have been at least partially sequenced and molecularly characterized [3, 10, 11, 25, 34, 38, 42, 43, 66] . Genetic analysis indicates that different felid species are infected by different strains of FIV [8, 11] . Analysis of pol gene sequence of FIV from lions (Panthera leo), pumas (Puma concolor) and domestic cats indicated that each species has a specific strain of FIV and that the strains are related but distinct [7, 43] . Also, strains from African lions (subtype B and E) differ in their abilities to replicate in feline cell lines [59] , their sensitivity to receptor antagonists [71] , and their requirement for ectopic expression of CD134, the primary cellular receptor, for productive infection [41] .
It remains to be demonstrated that FIV-related viruses cause severe disease in species other than the domestic cat [6, 38] . The apparent absence of clinical signs in pumas and lions may reflect a longer period of coevolution between virus and host in these species, whereas in the domestic cat, the virus and host have not yet had time to reach a similar state of nonpathogenic coexistence [6, 7, 57] . However, it is by no means certain that FIV does not cause disease in non-domestic cats. Not long ago, reports have shown immune depletion associated with FIV infection in lions and pumas [56, 57] and another recent study reported evidence of immune suppression in the Pallas' cat (Otocolobus manul), including histopatological changes [8] . In addition, interspecies transmission (although is rare) may occur [22, 67] . For example, a leopard cat (Felis bengalensis) was found to be infected with a domestic cat virus [42] and FIV infecting one puma was more characteristic of domestic cat FIV rather than puma FIV [10] .
The prevalence of FIV infection is South America has not been well evaluated and regional variations remain largely unexplored in domestic and wild cats. Considering that FIV has been detected in domestic cats in South America and that wild and domestic cats have overlapping territories in the communities and buffer zone, there is the potential for domestic felids to transmit this virus to naive wild felids in zoologic as well as free-range settings. The isolation and molecular characterization of these pathogens, both in domestic and a variety of wild felines, would be helpful and may provide important baseline data to develop effective programs aimed at infectious disease prevention. We believe that the feline population should be continually monitored for FIV infection and that clinical correlates to FIV infection should be further investigated. As recently proposed [70] , researchers could consider early surveillance programs across defined populations and detailed, cohort studies of naturally infected animals to provide further insights. Such studies would provide an opportunity to track retrospectively the pattern and consequences of an ongoing epizootic. There are technical reasons that hinder such studies, there is an urgent need for increased capacity in South American laboratories in order to conduct FIV screening and the apparent absence of FIV infection in some countries of the continent may merely reflect an absence of investigations. In addition, it is not easy to study FIV in wild cats as it is difficult to obtain samples from wild populations and only when these difficulties are overcome will it be possible to analyze and characterize FIV strains from the continent. Chinese Medicine Shenfu Injection for Heart Failure: A Systematic Review and Meta-Analysis Objective. Heart failure (HF) is a global public health problem. Early literature studies manifested that Shenfu injection (SFI) is one of the most commonly used traditional Chinese patent medicine for HF in China. This article intended to systematically evaluate the efficacy and safety of SFI for HF. Methods. An extensive search was performed within 6 English and Chinese electronic database up to November 2011. Ninety-nine randomized controlled trails (RCTs) were collected, irrespective of languages. Two authors extracted data and assessed the trial quality independently. RevMan 5.0.2 was used for data analysis. Results. Compared with routine treatment and/or device support, SFI combined with routine treatment and/or device support showed better effect on clinical effect rate, mortality, heart rate, NT-proBNP and 6-minute walk distance. Results in ultrasonic cardiography also showed that SFI combined with routine treatment improved heart function of HF patients. There were no significant difference in blood pressure between SFI and routine treatment groups. Adverse events were reported in thirteen trails with thirteen specific symptoms, while no serious adverse effect was reported. Conclusion. SFI appear to be effective for treating HF. However, further rigorously designed RCTs are warranted because of insufficient methodological rigor in the majority of included trials. Heart failure (HF) is a leading cause of death, hospitalization, and rehospitalization worldwide. Despite advances in the treatment of HF, including use of drugs, devices, and heart transplantation, the condition remains associated with substantial morbidity and mortality [1] .
International cooperation research program on cardiovascular disease in Asia showed that, on a total of 15,518 Chinese adults (35-74 years old) survey, the prevalence of HF was 0.9%, 0.7% for the males, and 1.0% for the females [2] . In the United States, HF incidence approaches 10 per 1,000 of the population over 65 years of age [3] . A report from the European Society of Cardiology (ESC) indicated at least 10 million patients with HF in these representing countries with a population of over 900 million. Half of the HF patients will die within 4 years, and more than half of those with severe HF will die within 1 year [4] .
At present, the conventional therapeutic approaches in HF management include angiotensin-converting enzyme (ACE) inhibitors, β-blockers, and diuretics. Although several of them have led to an important effectiveness, HF remains the leading cardiovascular disease with an increasing hospitalization burden and an ongoing drain on health care expenditure [5] . Therefore, it remains necessary to search alternative and complementary treatment, in which Traditional Chinese Medicine takes a good proportion [6] .
In TCM theory, pathogenesis of HF is related to deficiency of heart yang and heart qi and stasis of blood and excessive water (fluid), as well as interaction within these pathological factors. Under physiological conditions, yang can promote water metabolism, while qi can accelerate blood circulation, so yang and qi are the vital elements for human body to maintain life activity. TCM theory holds that patients suffered from HF are in deficiency of heart yang and qi for a long course, which directly leads to excessive fluid retention and blood stasis (Figure 1 ).
Two Chinese herbal medicines, namely, Radix Ginseng (ginseng) and Radix Aconiti Lateralis Preparata (prepared aconite root), are used in treating HF over 2000 years. Ginseng invigorates qi, while prepared aconite root can warm and strengthen yang and lead to diuresis. Long-term clinical practice has proved that compatibility of ginseng and prepared aconite root can effectively ameliorate patients' symptom of HF and improve quality of life ( Figure 1 ). Shenfu injection (SFI) has been used in treating cardiac diseases for a long time in China [7] . The main active components of SFI are extraction of traditional Chinese herbs, namely, ginsenosides and higenamine. Modern pharmacological research shows that ginsenosides can improve ischemic myocardium metabolism, scavenge free radicals, protect myocardial ultrastructure, and reduce Ca 2+ overload, and higenamine can enhance heart contractility, improve coronary circulation, and decrease the effect of acute myocardial ischemia [8] .
Currently, SFI used alone or integrated with routine treatments has been widely accepted as an effective method for the treatment of HF in China. Many clinical studies reported the effectiveness ranging from case reports and case series to controlled observational studies and randomized clinical trials, but the evidence for its effect is not clear. This paper aims to evaluate the beneficial and harmful effects of SFI for treatment of HF in randomized controlled trials. (Shenfu injection or Shenfu or Shen-fu) AND (heart failure or cardiac dysfunction or cardiac inadequacy or cardiac failure or congestive heart failure). All of those searches ended before November 2011. And the bibliographies of included trials were searched for thorough references, irrespective of languages.
All the randomized controlled trails (RCTs) of SFI compared with routine or conventional treatment (control group) in adult patients with HF were included. RCTs combined SFI with conventional treatment and/or invasive respiratory support (SFI group) compared with conventional treatments and/or invasive respiratory support (control group) were included. Both acute heart failure and chronic heart failure were included. Outcome measures include clinical effect rate, death and adverse events, ultrasonic cardiography, heart rate and blood pressure, and quality of life.
Wen-Ting and C. Fa-Feng) extracted the data from the included trials independently, based on the inclusion criteria outlined above. Nonrandomized evaluations, pharmacokinetic studies, animal/laboratory studies, and general reviews were excluded, and duplicated publications reporting the same groups of patients were also excluded ( Figure 2 ).
Extracted data was entered into an electronic database by two authors, S. Wen-Ting and C. Fa-Feng independently. The methodological quality of RCTs was assessed by using criteria from the Cochrane Handbook for Systematic Reviews of Interventions, Version 5.0.1. The quality of trials was categorized into low risk of bias, unclear risk of bias, or high risk of bias according to the risk for each important outcome within included trials, including adequacy of generation of the allocation sequence, allocation concealment, blinding, whether there were incomplete outcome data or selective outcome, or other sources of bias. 
The statistical package (RevMan 5.0.2), which is provided by The Cochrane Collaboration, was used to analyze collected data. Dichotomous data was presented as risk ratio (RR), with 95% confidence intervals (CIs). Continuous outcomes were presented as mean difference (MD), with 95% CI. Analyses were performed by intention-to-treat where possible. Heterogeneity between trials results was tested, and heterogeneity was presented as significant when I 2 is over 50% or P < 0.1. Random effect model was used for the meta-analysis if there was significant heterogeneity, and fixed effect model was used when the heterogeneity was not significant [21] . Publication bias was explored via a funnelplot analysis.
According to the search strategy, we screened out 903 potentially relevant studies for further identification ( Figure 2 ). By reading titles and abstracts, we excluded 701 studies that were obviously ineligible, including review articles, case reports, animal/experimental studies, and nonrandomized trials. 202 studies with full text papers were retrieved. After the full text reading, 6 studies were excluded because of duplicated publication. 84 studies were excluded due to lack of clinical effect rate which is the primary outcome evaluated in present study. 4 studies were excluded because the reported groups of participants were same as previous trials. In 108 RCTs, 11 studies were excluded due to other herbal intervention which was combined with SFI as treatment arm. Thus, 97 RCTs [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] were included for systematic review.
Ninety seven RCTs involved a total of 8,202 patients with HF, including 92 trails (7854 patients) of chronic HF and 5 trials (348 patients) of acute HF. The sample size varied from 24 to 248 participants, with an average of 42 patients per group. Since RCTs of HF on children were excluded, patients are adults (ranged from 28 to 89 years old). More males were included than females (52% males and 48% females). Disease duration was reported in 31 trials, ranging from 3 months to 26 years. 49 trials were observed in inpatients, 5 outpatients [22] [23] [24] [25] [26] , 5 both inpatients and outpatients [27] [28] [29] [30] [31] , and 39 unclear. All studies were published in Chinese.
Mortality was reported in eleven studies, while the rest of the eighty eight trials did not mention death. Effect rate was assessed in all the trials, based on the improvement of heart function. Ninety one trials used New York Heart Association (NYHA) Classification of Clinical Status, and six trials used Killip's Rating Standards [22, 25, 26, [33] [34] [35] for diagnosing HF and rating the patients. Patients in fifty one trails ranged from II to IV, seven trials II to III, twenty one trials III to IV, and five trials IV according to NYHA Classification; patients in five trials ranged from II to IV and one trial IV according to Killip's Standard.
Results of ultrasonic cardiography were reported in 61 trails (5135 patients) with left ventricular ejection fraction (LVEF) as main parameter. Other parameters such as left ventricular diastolic diameter (LVDd), cardiac output (CO), cardiac index (CI), stroke volume (SV), and A peak E-wave velocity ratio (E/A) were reported in 16, 17, 20, 18 , and 11 trials, respectively. N-terminal pro-B-type nature tripeptide (NT-proBNP) level in blood was reported in 12 studies of 887 patients, and 6-minute walk distance (6-MWD) was reported 4 Evidence-Based Complementary and Alternative Medicine in 8 trials of 630 patients. Heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were reported in 27, 15, and 13 trials, respectively (Table 1) .
According to our predefined quality assessment criteria, all of 97 included trials were evaluated as having unclear risk of bias (Table 2, Figure 3 ). None of the 97 trials reported sample size calculation. Eleven trials described randomization procedures, nine trials [9-11, 20, 30, 38-41] used a random number table, one drew lots [19] , and one trial separated patients by odd and even number of patient ID as a quasirandomization [42] . Only one trial [43] blinded both patients and outcome assessors, and three trials [44] [45] [46] blinded patients. None of the trials reported adequate allocation concealment. Five out of ninety seven trials mentioned that followup ranged from 3 months to 12 months after treatment. One trial [47] followed all the patients for 12 months, one trail [38] for 6 month, and the rest [9, 11, 12] for 3 months. However, neither of them used intention to treat method.
The primary outcomes were effect rate and mortality. Secondary outcome measures included LVEF, LVDd, SV, CO, CI, HR, systolic blood pressure (SBP), diastolic blood pressure (DBP), NT-proBNP, and 6-MWD.
Effect Rate. All the trials reported clinical effect rate to evaluate the outcome, which was based on NYHA Classification of Clinical Status and Killip's Rating Standards. Killip's Rating Standards were used by six trials with patients of myocardial infarction-induced HF, while other trials used NYHA Classification. Most of trails used three categories to evaluate treatment effect including markedly effective (an improvement of two classes on the classification), effective (an improvement of one class), and ineffective (no improvement, deterioration or death), and others only reported total effect. Total effect rate is the combination of markedly effect rate and effect rate. Trials of myocardial infarction-induced HF and nonmyocardial infarction-induced HF were separated into two subgroups. The meta-analysis showed a total significant difference between SFI and control groups on total effect rate (RR: (Figure 4) .
Death. Eleven studies reported mortality data, and total death number was 142 out of 978. Two trials [12, 38] assessed the mortality with 3-and 6-month followup, respectively, and other trials reported death at the end of treatment course. Trials were also separated into two subgroups depending on whether HF was induced by myocardial infarction. The result of meta-analysis indicated that SFI can significantly reduce mortality of patients of myocardial Figure 5 ).
NT-proBNP. NT-proBNP level is used for screening and diagnosis of acute HF and may be useful to establish prognosis in HF, as it is typically higher in patients with worse outcome [109] . It was reported in 12 studies [20, 22, 38, 45, 49, 52, [54] [55] [56] [57] [58] [59] Heart Rate and Blood Pressure. Heart rate and blood pressure were reported in 27 and 15 trials, respectively. Metaanalysis showed that there was statistical significance between SFI group and control group (WMD: 6.31; 95% CI [5.18, 7.44] , P < 0.01) (see Supplementary Figure 1 Results of Ultrasonic Cardiography. LVEF is the ratio of the stroke volume and the left ventricular end-diastolic volume [107] . It is usually used for the assessment of HF and drug efficacy. Sixty-one studies reported the outcomes for LVEF. Meta-analysis showed that SFI group was better than control group in increasing LVEF (WMD: 6.31; 95% CI [5.18, 7.44] , Figure 4) .
SV is the volume per stroke by left ventricle, and CO is the volume of blood being pumped by the heart in the time interval of one minute [107] . CI is a vasodynamic parameter that is relating CO to body surface area [107] . All the three parameters indicate left ventricular systolic function, as LVEF does. This paper made meta-analysis of these outcomes, respectively; results showed that SFI group was better than control group in these three parameters: SV (WMD: 7.25; 95% CI [4.60, 9. E/A ratio is widely accepted as a clinical marker of diastolic HF, and E/A ratio is reduced in diastolic dysfunction [108] . The result of meta-analysis of E/A ratio was WMD: 0.15; 95% CI [0.08, 0.22], P < 0.01, which indicated that SFI better improved diastolic function of heart on HF patients Evidence-Based Complementary and Alternative Medicine 5 than conventional medicine treatment did (Supplementary Figure 8) .
LVDd is the end-diastolic dimension of the left ventricle. There was no statistical significance between SFI combined with conventional medicine treatment and conventional medicine treatment groups (WMD: −1.59; 95% CI [−5.29, 2.12], P = 0.40) (Supplementary Figure 9) .
None of the trials reported quality of life.
Funnel plots based on the data of effect rate were elaborated in Figure 8 . The figure was asymmetrical, which indicated that potential publication bias might influence the results of this paper. Although we conducted comprehensive searches and tried to avoid bias, since all trials were published in Chinese, we could not exclude potential publication bias.
3.6. Adverse Effect. Thirty seven out of ninety seven trials mentioned the adverse effect except in sixty-two trials which was unclear. Thirteen trials [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 60] reported the following thirteen specific symptoms of side effects including dry mouth, dryness heat, fullness of the head, insomnia, dysphoria, skin itching, tachycardia, feverish dysphoria, flushing of face, tidal fever, dizziness due to low blood pressure, gastrointestinal discomfort, and palpitation. Among these side effects, dry mouth and fullness of the head were reported in 4 trails with 14 and 10 cases, respectively. These symptoms were regarded to be mild and recovered spontaneously after SFI withdrawal. Twenty four trials reported that no side effects were observed in the SFI group (Table 3) .
The above side effects might be related to higenamine, which is the active ingredient of prepared aconite root. In TCM books and papers, prepared aconite root is frequently mentioned with adverse effects as dry mouth, dryness heat, fullness of the head, and dysphoria due to its strong effect of strengthening yang.
In many years, western medicine has made tremendous progress and has become the dominating medical treatment worldwide. However, it has been increasingly recognized that Events   27  26  31  37  32  28  79  49  122  37  58  36  25  30  51  26  33  71  46  33  55  17  42  36  36  40  57  58  68  28  47  56  33  54  32  46  63  23  24  68  45  36  36  40  31  44  29  40  54  47  28  77  23  56  33  39  18  36  34  13  52  49  45  18  26  22  54  37  34  41  28  28  37  27  50  37  37  41  36  54  29  11  45  28  70  53  17  52  28  55  18   3678   22  24  21  22  40  24   153   3831   Total   30  30  35  40  35  30  90  56  127  41  62  40  30  40  55  31  36  76  48  35  60  18  48  38  38  46  62  64  80  30  50  60  42  58  35  48  66  25  28  74  50  42  40  43  32  50  31  42  60  50  32  85  24  60  34  45  22  38  37  16  62  58  50  20  31  24  58  40  40  44  33  30  40  30  56  39  40  46  40  60  30  12  50  30  78  56  19  56  30  55  20  4047   35  36  23  28  54  36  212   4259   Events   20  22  22  32  26  22  61  45  97  14  48  30  16  12  15  23  19  42  25  22  44  15  26  28  27  28  56  47  40  23  33  54  21  41  24  41  52  17  14  63  39  33  33  32  29  38  21  24  40  46  19  67  21  50  28  33  4  22  29  10  31  25  22  15  24  20  43  30  24  27  18  22  32  21  21  27  28  28  30  38  22  8  34  22  59  40  13  46  22  40  13   2770   17  16  14  12  29  16   104   2874   Total   39  28  33  40  35  27  90  56  121  22  62  40  30  20  30  28  26  76  30  30  60  18  39  38  38  40  62  64  60  30  40  60  41  54  32  48  64  25  20  78  50  40  40  42  32  50  30  42  52  50  30  85  24  60  34  42  10  30  36  16  61  38  26  20  31  24  58  40  34  44  29  30  40  30  30  37  40  32  40  50  26  12  50  30  80  56  18  56  30  50  20  3731   35  38  22  23 western medicine may sometimes fail to treat an illness, whereas such illness is reportedly improved by the so-called complementary medicine based on a different theory [110, 111] . Although conventional therapeutic approaches were used in HF, it remained a cardiovascular disease with an increasing hospitalization burden and an ongoing drain on health care expenditures [2] . TCM plays an important role in treating HF in China. SFI was a traditional Chinese Patent Medicine based on TCM theory, which was approved by the Chinese State Food and Drug Administration. In recent 10 years, it has been widely used for HF in many hospitals and clinics. However, few RCTs of SFI were reported in English journals, and it was difficult for western doctors to accept SFI as an alternative medicine. Although there were two systematic reviews about SFI for HR published in Chinese journal [112, 113] , only 16 and 8 trials were included in their study. Therefore, the present study aimed to systematically assess the efficacy and safety of SFI for HR. Data from the 97 RCTs demonstrated that SFI combined with conventional medication may be more effective on HF than conventional medication only. With improvement of cardiofunction of patients, based on NYHA Classification of Total (95% CI) Test for overall effect: Z = 7.14 (P < 0.00001) Mean   443  250  497  329  218  216  445  330   SD   66  112  74  64  17  18  65  18   Total   40  35  36  38  50  42  40  46   327   Mean   395  200  413  280  211  203  395  316   SD   64  90  67  52  15  16  63  17   Total   40  35  26  38  50  42  40 Clinical Status and Killip's Rating Standards, the effect rate of SFI group was, on average, 17 percent more than control group (RR, 1.19; 95% CI, 1.17 to 1.21). Mortality data was another primary outcome. In eleven trials in which death was recorded, meta-analysis showed that mortality was significantly lower in SFI group than control group. This result was mainly contributed by subgroup of HF induced by myocardial infarction, for patients in this subgroup were more vulnerable.
Ultrasonic cardiography is widely used in inspection for HF patients. From results of ultrasonic cardiography, the systolic and diastolic functions of heart can be interpreted. LVEF, CO, CI, SV, LVDd, and E/A were reviewed by us, respectively. There was significant difference between SFI group and control group in all of the outcomes except LVDd. Since SV, CO, CI, and LVEF indicate heart systolic function, and E/A indicate heart diastolic function, conclusion can be drawn that SFI benefits both systolic and diastolic functions of heart. But it did not have significant effect on expansion of heart. NT-proBNP level in serum of SFI group was significantly lower than the control group, which is inconsistent with effect rate. 6-MWD results of patients of SFI group also are better than thos of control group. It indicates that SFI had a tendency to improve life status. Furthermore, heart rate was obviously reduced in SFI group, which could be related to alleviation of HF.
Meta-analysis on LVEF, CO, CI, SV, LVDd, E/A, heart rate, and NT-proBNP all showed significant heterogeneity. Several possible explanations can be given, for example, different complications, different instruments employed for test, and difference in methodological rigor.
However, we should consider the following limitations before accepting the findings of this paper.
Firstly, the methodological quality of the included studies is generally poor. Although all trials claimed to perform randomization, only eleven trials reported the procedure to generate the sequence, while the rest of trials did not give any details of the randomization method. Thus, whether randomization was effectively conducted in these trials was doubtful. Blinding was mentioned in four trials, with one trial blinded patients and outcome assessors [43] and three blinded patients only [44] [45] [46] . Neither of them described the methods of allocation concealment. Dropouts account and intention to treat analysis were not mentioned in all the trails. Due to inadequate reporting of methodological design, it was possible that there was performance bias and detection bias due to patients and researchers being aware of the therapeutic interventions for the subjective outcome measures. Therefore, we cannot draw a confident conclusion that there were significant beneficial effects of SFI combined with conventional medicine treatment compared with conventional medicine treatment.
Secondly, limited outcomes were reported, especially death and adverse events. Since HF is a disease with high mortality, death is the most important primary outcome. However, only eleven studies out of ninety seven trials reported death, and most of the eleven trials assessed mortality at the end of treatment, without followup. Another outcome was adverse events, to which more attention should be attached. Only 37.4% of the trials described the occurrence of adverse events, indicating an incomplete evaluation of the safety profile of SFI, as well as poor quality of reporting. In most trials, the duration of therapy and followup was 22 Evidence-Based Complementary and Alternative Medicine too short to achieve conclusive results, except that only one trial had a treatment of 10 months [47] . Only 6 included trials had a followup period (ranged from 3 to 12 months), while in rest of studies, the outcomes were evaluated at the end of the treatment (mostly range from 14 to 21 days). In order to evaluate drug efficacy for chronic HF, long-term improvement (at least 6 months) of chronic HF-specific clinical symptoms is needed [114] , because some drugs have shown to increase mortality in the long-term application despite a short-term improvement in clinical symptoms [115] . In addition, long-term toxicity assessment was also important for drug safety evaluation.
Next, although irrespective of languages, all the trials included in this paper were published in Chinese journals, Zhang et al. and Liu et al. [115, 116] found that some Asian countries including China unusually publish high proportions of positive results. Wu et al. [117] and Jin et al. [118] accounted that RCTs in Chinese journals often had problems of low methodological quality and selective publication of positive results. Considering that all of the ninety seven trials were published in Chinese, the publication bias possibly existed.
Additionally, none of the ninety seven trials reported sample size calculation, and in most trials, the sample size was limited. Further high-quality studies with larger sample size are needed to confirm the effectiveness of SFI in treating HF. Quality of life was not reported in all the including trials. Although 6-MWD showed a tendency of SFI to improve life status for HF patients, we advise future RCTs to select outcomes of life quality according to international practice.
Considering that there was no sufficient amount of highquality trials on SFI treating patients with HF, the effectiveness and safety of SFI need further rigorous trials to prove, which should be consistent with the CONSORT statement on the reporting of the results of randomized trials (http:// www.consort-statement.org/).
The preliminary conclusion of the current study suggests that SFI might be beneficial to patients with HF. More rigorously designed trails with high methodological quality are necessary for further proof. Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential. Influenza A viruses belong to the Orthomyxoviridae family, and have a genome composed of eight segments of single-stranded, negative-sense RNA. Their surfaces are composed by a lipid envelope, originated from the plasmatic membrane of infected epithelial cells, and two antigenic proteins: Hemagglutinin (HA) and Neuraminidase (NA); these two antigens exhibit higher variability compared with their remaining proteins [1] . Depending on the extent of variability of two surface proteins, until now are known 16 HA (H1-H16), and 9 NA genotypes (N1-N9), respectively, which can be combined in different combinations [1, 2] .
In early April 2009, authorities from the Mexican public health observed a high number of influenza-like illnesses in their territory, and informed about this outbreak to the regional office of the World Health Organization (WHO). In mid April, the Centers for Disease Control from USA identified the new virus in two cases from California. The new virus spread rapidly throughout the world, and as a consequence the WHO authorities declared the "Pandemic (H1N1) 2009" on June 11, 2009 [3] . It is thought that the new 2009 H1N1 pandemic virus (from here, 2009 H1N1pdm) has emerged through at least four reassortment and transmission events among swine, avian and human H1N1 lineages, probably in Asia and North America [4] . Particularly, the HA segment of 2009 H1N1pdm was originated from American swine lineage, whereas the NA segment derived from the European swine lineage [5, 6] . It is believed that the ancestors of this pandemic strain remained undetected for approximately one decade due to lack of a *Address correspondence to this author at the Rio de la Plata y Lagerenza, CP 1120 Asunción, Paraguay; Tel: +595 21 424 520; Fax: +595 21 480 185; E-mail: emilioespinola@hotmail.com surveillance system in pigs, the historical "mixing vessel" for new influenza viruses. Furthermore, the closest ancestors of the new pandemic strains emerged probably in January 2009 [4] .
The objective of this study was to analyze a dataset of complete nucleotide (nt) sequences of HA and NA genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs.
Complete CoDing Sequences (CDS) of HA (1701 nt) and NA (1410 nt) genes corresponding to 2009 H1N1pdm, isolated from humans, were downloaded from the Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU /SwineFlu.html) from the National Center for Biotechnology Information, by the year of sequence repository. The first dataset consisted of 3765 HA and 2996 NA sequences, respectively, which were reported in the period 2009-2010. After discarding exact duplicates in sequence using a Perl script, we obtained 2038 HA and 1273 NA sequences, respectively; these sequences were different in at least one nucleotide among all representatives. Reassortant strains were discarded, as well as incomplete CDS sequences.
Nucleotide sequences were manually edited in FASTA format, using BioEdit v7.0.5 [7] , and aligned with CLUSTAL W [8] . Sequence information (GenBank accession number, strain, and year of isolation) for each sample used in this study are available for HA (Table S1) and NA genes (Table S2) , respectively. Pairwise distances were calculated with MEGA v5 [9] . The percentages of identities were calculated by applying the formula 100 -(pairwise distance value x 100). A graph was constructed by plotting the percentage identities in the abscissa (x axis) vs the frequency of each of the calculated pairwise identities in the ordinate (y axis). The graphs were prepared in the R environment, using ggplot2 package (www.r-project.org).
The models of nucleotide substitution that best fitted each dataset were determined with MEGA v5 [9] , and were: GTR+I model for HA genes, and T92+G model for NA genes, respectively. Phylogenetic relationships were reconstructed by the Neighbor-Joining method [10] , with the appropriate models of nucleotide substitution for each dataset (as described above) and bootstrap analysis of 1000 replicates, as incorporated in MEGA v5 [9] . Outgroup sequences for HA and NA genes corresponded to strain A/Puerto Rico/8/1934.
Mutations in each CDS were analyzed by the method of Nei and Gojobori [11] . Codon aligned sequences for each dataset were analyzed using the Perl-based SNAP program (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.htm l) [12] in order to calculate the variability of each CDS. The selective pressure was measured by comparing the rate of non-synonymous nucleotide substitutions per nonsynonymous site (d N ) against that of synonymous substitutions per synonymous site (d S ). The ratio d N /d S was used as an index to assess positive selection. A ratio d N /d S >1 means positive (diversifying) selection, =1 means neutral selection, and <1 means negative (purifying) selection.
The analysis of pairwise identity frequencies showed high percentage of similarities among circulating 2009-2010 pandemic influenza strains (Fig. 1) . The average percentage of identity was 99.7% for both HA and NA genes. Thus, in this period of pandemic circulation, both genes did not segregate into different clusters, but on the contrary showed a constant and stable evolution.
The high percentages of nucleotide identity were in accordance with the single clustering of all 2009-2010 strains in the phylogenetic tree of HA and NA genes (Fig. 2) , without temporal or geographical distribution. It is interesting to note that the overall genetic diversity among 2009 H1N1pdm was less than typically observed among seasonal influenza. This is in accordance with its short period of time of circulation in humans [13] . The single clustering of 2009 H1N1pdm observed in this report, however, is in contrast with other studies [14] , in which the authors observe differences by using small datasets of sequences. The single clustering of 2009 H1N1pdm, furthermore, agrees with serological data in which it was observed that antigenically, the new pandemic viruses were all similar [6] , and thus not requiring a new update of the vaccine (strain A/California/07/2009) until now.
Given that 2009 H1N1pdm constituted a homogeneous phylogenetic group, it was hypothesized that the diversity in nucleotide sequences localized (in average) in the 0.3% of differences within each analyzed gene. Taking into account the complete CDS for HA and NA genes, this percentage of differences constitutes approximately four to five nucleotide random variations among circulating strains.
Calculation of average d N /d S rates of evolution showed that both HA and NA genes evolved through negative (purifying) selection (Table 1) , with d N /d S values of 0.2762 and 0.1939, respectively. Even though in general, both genes underwent negative selection, some positions can evolve through positive selection. For example, an early study showed that two sites involved in receptor binding specificity of HA (220 and 278) were under positive selection, and these sites were not found in swine or seasonal H1N1 viruses [15] . Thus, changes in receptor binding sites could lead to alterations in receptor binding specificities.
In other viruses such as SARS-CoV, it was observed that they can develop through positive selection through the Fig. (1) . Pairwise identity frequencies for (a) Hemagglutinin and (b) Neuraminidase genes, respectively. cross-species transmission in early epidemics, and negative selection during late epidemics [16] . It is possible that the same mechanism was the driven force of evolution of 2009
H1N1pdm, with positive selection at least during crossspecies transmission. A number of different amino acid mutations that could confer new functionalities to the new virus were reported worldwide, including those related to increased pathogenicity or antiviral resistance ( Table 2) . Polymorphism at position 239 in HA has been associated with severe clinical outcomes, especially in immunocompromised patients; in particular, substitution 239G was found to correlate with fatal outcomes in different countries [17, 18] . Furthermore, this mutation can arise de novo from wildtype (D239) virus in the same patient throughout the disease course [19] . Mutation at position 239 can induce alterations in the receptor binding site, and 239G mutants bind a broader range of 2-3-linked sialyl receptors sequences expressed on cells from the lower respiratory tract, which suggested that its presence could be responsible for the exacerbation of disease [20] . Mutants 239E target mainly non-ciliated cells. We found no significant difference between sequences bearing mutations 239G (2.6%) and 239E (5.5%). The low percentage of global circulation of mutants 239G found in this study is in accordance with its lower potential to transmit to other individuals [21] .
Positions 239 and 220 are localized within the HA antigenic site called Ca. The amino acid S220, though not exposed to the surface, is localized in the receptor binding domain (RBD), and its change could affect the transmissibility and infectivity of H1N1 in humans. The fixed mutation, S220T, has been found at high percentage (76.7%) in this study. To test whether change 220T could contribute to antigenic drift, it would be interesting to compare its antigenic profile against a wildtype isolate (S220). This mutation, probably, has become fixed in all pandemic strains through optimization of viral fitness, rather than immune selection or adaptation to the host. Substitution S101N has been proposed previously as a reversion to the seasonal H1N1 residue 101N and thus possibly an adaptation to the human host, being found in some studies at high frequencies. Its global impact, however, is controvertible because it was found in only 0.2% of our sequences. Substitution E391K, found at 15.6% in our study, has been identified as part of a highly conserved epitope in the 1918 H1N1 virus with a possible role in membrane fusion [22] . Another proximal substitution found in other studies, N387H, was found in only 1.7% of our sequences.
In the NA gene, it was showed that mutations V106I and N248D were present in samples at increasing numbers through early pandemic month (April to December 2009) [23] . We found both mutations at high percentages, 85.1% and 85.9% respectively, in our dataset. Change 106I was present in the 20th century cases of H1N1 (in 1918 [pandemic] , and 1977), as well as 248D (in 1977). Since residue at position 248 is located at the drug target domain (DTD) region, as residue 275, it could potentially affect the sensitivity to NA inhibitors. Another substitution of possible interest in NA sequences is D199N, which was previously associated with an increase in oseltamivir resistance in both seasonal and H5N1 virus strains [24] . We found, however, only 4 out of 1273 NA sequences (0.3%) containing this change. The rare substitution I223R, which was reported in association with resistance to oseltamivir, zanamivir, and peramivir [25] , was also found in only 2 out of 1273 NA sequences (0.2%). Substitution H275Y has been related to oseltamivir resistance, especially in immunocompromised or severely ill persons [26] . It was found, however, in sporadic cases in most of the countries at low frequencies (~1%) [27] . In our study, we found 2% of sequences containing this change.
In conclusion, the stable evolution of 2009 H1N1pdm offers an opportunity to control its spread and prevent infections. Reports about new mutations, however, will still be important if those changes can confer an enhanced transmissibility or resistance to drugs. Furthermore, a continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.
This work (project code: INV11) was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) Programa de Apoyo al Desarrollo de la Ciencia, Tecnología e Innovación en Paraguay (BID 1698/OC-PR).
Declared none.
Supplementary material is available on the publisher's web site along with the published article. Severe imported malaria in an intensive care unit: a review of 59 cases BACKGROUND: In view of the close relationship of Portugal with African countries, particularly former Portuguese colonies, the diagnosis of malaria is not a rare thing. When a traveller returns ill from endemic areas, malaria should be the number one suspect. World Health Organization treatment guidelines recommend that adults with severe malaria should be admitted to an intensive care unit (ICU). METHODS: Severe cases of malaria in patients admitted to an ICU were reviewed retrospectively (1990-2011) and identification of variables associated with in-ICU mortality performed. Malaria prediction score (MPS), malaria score for adults (MSA), simplified acute physiology score (SAPSII) and a score based on WHO's malaria severe criteria were applied. Statistical analysis was performed using StataV12. RESULTS: Fifty nine patients were included in the study, all but three were adults; 47 (79,6%) were male; parasitaemia on admission, quantified in 48/59 (81.3%) patients, was equal or greater than 2% in 47 of them (97.9%); the most common complications were thrombocytopaenia in 54 (91.5%) patients, associated with disseminated intravascular coagulation (DIC) in seven (11.8%), renal failure in 31 (52.5%) patients, 18 of which (30.5%) oliguric, shock in 29 (49.1%) patients, liver dysfunction in 27 (45.7%) patients, acidaemia in 23 (38.9%) patients, cerebral dysfunction in 22 (37.2%) patients, 11 of whom with unrousable coma, pulmonary oedema/ARDS in 22 (37.2%) patients, hypoglycaemia in 18 (30.5%) patients; 29 (49.1%) patients presented five or more dysfunctions. The case fatality rate was 15.2%. Comparing the four scores, the SAPS II and the WHO score were the most sensitive to death prediction. In the univariate analysis, death was associated with the SAPS II score, cerebral malaria, acute renal and respiratory failure, DIC, spontaneous bleeding, acidosis and hypoglycaemia. Age, partial immunity to malaria, delay in malaria diagnosis and the level of parasitaemia were not associated with death in this cohort. CONCLUSION: Severe malaria cases should be continued monitored in the ICUs. SAPS II and the WHO score are good predictors of mortality in malaria patients, but other specific scores deserve to be studied prospectively. The number of malarial infections acquired by international travellers, thought to be nearly 25,000 cases annually, remains very small compared with the annual global incidence of nearly 250 million malaria cases and about 700,000 related deaths in endemic areas, mainly among young children in Africa. Malaria in non-endemic countries is a challenge since, due to the fact that it is infrequent, experience in diagnosis and treatment is scarce and delays in diagnosis and adequate treatment may be fatal to the patient [1 -3] .
The diagnosis of malaria requires a high index of suspicion, as symptoms can mimic many other diseases. Portugal maintains a close relationship with African countries for several reasons and so malaria must be suspected in travellers that return from those countries.
The present study includes expatriates (emigrants), people that were born and live in Africa travelling to Portugal to visit friends and relatives, immigrants (people that were born in an endemic area and that live in Portugal) and those travelling for business or tourism.
Malaria is a notifiable disease in Portugal; about 50 cases are reported annually to the Public Health System, but cases are under-reported [4] . The notification implies filling-in a form that needs to be posted, which leads to forgetfulness. A person from a non-endemic country (emigrant or expatriate) who stays in malariaaffected areas for less than two years is considered nonimmune, and also at greater risk of more severe forms of the disease [5] . Non-immune people can develop symptoms of falciparum malaria within a month after leaving the endemic area (average 10 days) but, sometimes, after longer periods as can be observed in pregnant women, immigrants, people that were prescribed mefloquine and HIV-infected people [6] [7] [8] .
Severe forms of malaria are almost always caused by Plasmodium falciparum; though rare, vivax malaria can also cause severe disease. The object of this study is falciparum malaria solely. Severe forms of malaria should be regarded as a medical emergency and managed in intensive care units (ICU) [9] [10] [11] . Clinical deterioration usually develops three to seven days after fever onset, but it can sometimes develop in the first 24 hours. A high standard of care and continued monitoring in the acute stage is very important to reduce mortality [12, 13] .
In 1990, the World Health Organization (WHO) established the criteria for severe malaria which were revised in 2010 (Table 1) , with the purpose of identifying individuals at risk of dying and specifying the risk factors of severe forms of the disease. The guidelines for the treatment of malaria published in 2006 were also revised in 2010 [14] [15] [16] .
In general, there is a correlation between the parasite density in the peripheral blood and the severity of the disease and its complications, especially among nonimmune people. In such cases, the patients' clinical condition can deteriorate even after initial appropriate treatment due to exacerbation of systemic inflammatory response, leading to organ dysfunction [17] [18] [19] .
The aim of this retrospective study is to describe the clinical spectrum of severe malaria cases admitted to an ICU and identify factors associated with in-ICU mortality. Parasitological diagnosis was done by thin smear and light microscopic observation; parasitaemia quantification was done whenever possible. Daily smears were done until the Plasmodium smear became negative. Since 2000, a immunochromatographic assay for the qualitative detection of Plasmodium antigens were also used at the emergency department (Malaria Now-Binax ® ). Clinical and laboratory parameters were analysed in order to determine which of them were associated with ICU mortality. To evaluate severity at the ICU admission, the Simplified Acute Physiology Score II (SAPS II) was applied.
The degree of immunity to malaria was estimated as follows: a) people who had been living in an endemic area for at least two years (emigrant) at the time of the diagnosis were presumed semi-immune, as well as adult migrant Africans that come to Portugal to visit friends or relatives; b) Europeans who travelled occasionally to endemic areas were considered non-immune. WHO's definition for severe malaria was applied and major and minor indicators considered according to the definitions presented in Table 1 . The number of severity criteria was quantified and analysed.
Two prognostic scores of malaria were applied: (1) Malaria Prediction Score (MPS) determined by: 2.13 + 0.02 × (age) + 0.25 × (creatinine) -0.24 × (haemoglobin) + 3.05 (malaria cerebral criteria) + 0.8 (presence of pregnancy) + 0.8 (ventilated) (where age = age in years; creatinine is in mg/dl, haemoglobin in g/dl; presence of pregnancy, cerebral malaria or ventilatory support, when present = 1, when absent = 0) [20] .; (2) Malaria Score for Adults (MSA) was applied to all but three children and the score was determined by: 1 (severe anaemia) + 2 (acute renal failure) + 3 (respiratory distress) + 4 (cerebral malaria). The MSA ranges from 0 to 10 [20] .
Variables' definitions were: severe anaemia (Hgb < 5 g/dL), thrombocytopaenia (platelets < than 100 x10 5 / uL); acute respiratory distress syndrome (ARDS) (acute respiratory failure defined by an acute hypoxaemia with the ratio of the partial pressure of oxygen in the patients' arterial blood (PaO 2 ) to the fraction of oxygen in the inspired air (FIO 2 ) (PaO 2 /FIO 2 ratio), less than 200 after exclusion of cardiogenic pulmonary oedema by clinical criteria or by a pulmonary capillary wedge pressure (PCWP) < 18 mm Hg in patients with a pulmonary artery (Swan-Ganz catheter); cerebral malaria (unrousable coma), acute renal failure (ARF) (creatinine > 3 mg/ dl or urine output < 400 mL/day in adults), liver dysfunction (ALT at least two times the normal value IU/ mL), hyperbilirubinaemia (bilirubin value > 2.5 mg/dL), hyponatraemia (Na < 125 mEq/mL), acidaemia (pH < 7.25), and hypoglycaemia (blood glucose < 40 mg/dL).
Community-acquired co-infection was defined as any infection diagnosed within the first two days of hospitalization. Infections occurring later than this were considered nosocomial. ICU admission criteria other than high parasitaemia were any cerebral dysfunction (GCS < 12), haemodynamic instability (SBP < 80 mmHg), respiratory distress (respiratory rate > 20/min or pCO2 < 32 mmHg), jaundice (bilirubine > 2.5 mg/dl) or renal dysfunction (oliguria or creatinine > 3 mg/dl).
Before 1992, the only available treatment for malaria was oral triple therapy (quinine sulphate plus pyrimethamine plus sulphadiazine), whereas after 1992, intravenous quinine dihydrochloride plus clindamycin or doxycycline has been chosen for the treatment of severe cases of malaria. Artemisinin derivatives are not yet available in Portugal. Treatment support includes: blood products to treat severe anaemia and coagulation disorders, intravenous glucose for hypoglycaemia, acetaminophen for hyperpyrexia, if moderate hypoxaemia oxygen by facial mask, antibiotics for bacterial co-infection; haemodynamic and life support according to SSC guidelines since 2005 [21] .
Endotracheal intubation and ventilatory support would be done if the patient had respiratory failure or neurologic dysfunction, needing airway protection; if acute oliguric renal failure or severe metabolic acidosis and/or severe electrolytic imbalance were present, renal support, usually a continuous technique, would be started.
Statistical analysis was performed with Stata V12. Descriptive statistics included frequency analysis (percentages) for categorical variables and median and interquartile ranges (IQR) for continuous variables. The differences in characteristics according to the outcome were tested using Fisher exact tests or chi-squared test for categorical variables and Wilcoxon tests for continuous variables.
From January 1990 to May 2011, a total of 284 patients suffering from malaria were admitted. Fifty nine (20.7%) patients were within the criteria of severe malaria and were admitted in the ICU-ID, a level III medical-surgical unit. Three patients were children aged one, nine and 11. Adults' ages varied from 20 to 71, with a median value (IQR) of 42 (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) , and six were older than 60; 47 (79.6%) were male.
Forty one (69%) were Portuguese emigrants working in Africa (for a period of two or more years in 20 patients and varying from two to 34 years; for a period of up to two years in14 patients and for a period that has not been defined in 7); seven patients were tourists and other seven lived in Africa and came to Portugal (five were Africans that came to visit friends and relatives and two were immigrants), and four worked in boats that had made several stops in seaports along the way. All but three had returned from sub-Saharan Africa: 48 (81.3%) from former Portuguese colonies in Africa -Angola (31), Mozambique (9), Guinea-Bissau (7) and Sao Tome and Principe (1); the others had returned from Senegal (2) and Gabon, Malawi, Burkina Faso, Central African Republic, Nigeria, and Côte d'Ivoire, one patient from each country; two other patients had returned from Thailand and one from the Philippines. Fifty one patients (86.4%) were white, six black and two Asian. Malaria immune status was evaluated in 54 (91.5%) patients: 25 (46.3%) were nonimmune and 29 (53.7%) semi-immune, according to the defined criteria. Chemoprophylaxis was prescribed to 14 (23.7%) patients, but discontinued or not taken properly, except from one patient with cerebral malaria that was on mefloquine chemoprophylaxis.
Co-morbidities were present in 15 (25.4%) patients: alcoholic hepatic disease, diabetes mellitus and ischemic cardiac disease in five patients; sickle cell anaemia, nephrolithiasis, Still disease and hepatitis C were observed in one patient each. One had been bone marrow transplanted seven years before and was asymptomatic, with no immunomodulatory therapy. An HIV primary infection was diagnosed simultaneously with malaria in one patient, being all other patients HIV negative. Some patients presented more than one comorbidity.
The diagnosis was made between the day of return from the endemic area up to the 47th day in 54 patients; in one patient, diagnosis was done on the 120th day after leaving Angola, where he had been for 16 consecutive months; in other four patients, the gap was impossible to determine.
Symptoms, namely fever before admission, varied from one day to three weeks, the median (IQR) duration being more than seven days in 44% of the patients. SAPS II was registered in 56 patients and the median value (IQR) was 33 . Malaria diagnosis was established by positive smears for asexual P. falciparum forms. In 48 (81.3%) patients, parasitaemia quantification was ≥ 2% and varied from 2-90%, with a median (IQR) of 15 . Parasitaemia was considered high but was not quantified in 13 (22%) patients. The immunochromatographic assay in whole blood for the qualitative detection of Plasmodium antigens, used at emergency department, was positive in all the samples tested.
The most common analytic changes observed in these patients were: thrombocytopaenia in 54 (91.5%) patients associated with DIC in seven (11.8%) and bleeding dis- Considering the major WHO criteria of severe malaria, 29 (49.1%) patients presented five or more dysfunctions.
Forty one (69.5%) patients were treated with quinine dihydrochloride, with a 20 mg/Kg loading dose followed by 10 mg/Kg tid, in a four-hour infusion associated with endovenous clindamycine. The other 18 patients (30.5%) were treated with oral quinine sulphate associated with doxycycline or pyrimethamine plus sulphadiazine in the first period of the study. Blood smears became negative for Plasmodium from the second to eighth day of treatment, except in a patient whose parasitaemia was 90% and who died with positive smears in the second day after admission.
All patients admitted to the ICU were close monitored and had treatment support according to the dysfunctions presented, namely transfusions of packed red cells whenever haemoglobin was lower than 7.5 g/ dL, associated with platelets and fresh frozen plasma in patients with bleeding disorders; blood products were also given before an invasive technique whenever platelets were lower than 50 × 10 5 /uL or if coagulopathy was present. Fluids were administered to correct hypovolaemia monitored by central venous pressure, mean arterial pressure and urinary output. Even so, 29 (49.1%) patients needed vasopressor support, with noradrenaline or dopamine or both. Ventilatory support was needed in 22 (37.3%) patients, for a period varying from two to 74 days (median 10 days). Two patients with severe ARDS with refractory hypoxaemia had been put on venovenous Extracorporeal Membrane Oxygenation (ECMO). In one of the patients, ECMO was completely successful and the patient is now doing well, without significant respiratory sequels; the other patient died on ICU in refractory shock. Eight (13.6%) patients required renal replacement therapy. Six patients had upper gastrointestinal bleeding and did an endoscopy for further study. All patients began enteric nutrition in the first 48 hours, if not contraindicated. Glycaemia control was monitored, being hypoglycaemia registered and treated in 18 patients (30.5%).
Other simultaneous infections occurred in some patients. Thirteen (22.0%) patients had pneumonia: in two of them, pneumonia was considered communityacquired; Pneumococcus and Klebsiella spp. were isolated from the tracheal aspirate, in each patient. In the remaining 11 cases, pneumonia was nosocomial and, apart from one isolate (an MDR Pseudomonas aeruginosa), no agent was identified; six of these patients were ventilator associated pneumonia. All patients but one recovered.
Another seven (11.9%) patients developed acute acalcoulous cholecystitis, which was managed with medical treatment in five and, in the other two, a percutaneous drainage was done, with a favourable evolution in all of them. Two (3.3%) patients had nosocomial central venous catheter-associated sepsis, having Acinetobacter baumanni been identified (from central catheter and peripheral venipuncture) in one patient and Pseudomonas aeruginosa MDR in the other; both were treated with antibiotics and had a favourable outcome.
The UCI stay ranged from one to 81 days, being the average (IQR) four days (two to twelve) in survivors and eight days (three to twenty seven) in the patients that died (p = 0.185). All patients who came out alive from the ICU were discharged from the hospital.
In two of them, an acute disseminated encephalomyelitis (ADEM) was diagnosed based on neurologic dysfunction and cerebral resonance magnetic image; both had a favourable outcome. All patients were also evaluated after hospital discharge for follow-up of residual dysfunctions and no other sequelae were detected; in those whose rapid malaria test had been consistent with coinfection with P vivax, primaquine was prescribed after excluding Glucose-6-Phosphate Dehydrogenase deficiency.
The global case fatality rate of the infectious diseases department was 3.1%, but the ICU fatality rate was 15.2% (nine patients). Eight were workers: seven returned from Angola, one from Thailand and the 9th was a tourist who returned from Mozambique. Death occurred between the second and 28th day. In all but three patients death occurred in the acute phase of the illness. One of them was a male nurse who, despite compliance to mefloquine chemoprophylaxis, had malaria with 30% of parasitaemia; he was conscious when he was admitted but had a rapid neurologic deterioration in some hours with severe intracranial hypertension and an irreversible brain swelling on cerebral computerized tomography image, with brain death in the first 24 hours. In the other three patients, who died later on, death was related to ARDS complications.
The variables associated with death ( ; p = 0.08) and hyperbilirubinaemia (77.8% vs 40.4%; p = 0.066), although more frequent in patients who died, did not reach statistical significance. The duration of the symptoms before admission has no statistical significance when considering the risk of death (p = 0.82), but the same was not true when we analysed the time since the patients' arrival from the endemic area to diagnosis, which was statistically associated with a worse prognosis (p = 0.02).
The number of organ dysfunctions as described in septic shock was correlated with prognosis and this is well documented by the presence of more than four WHO major criteria of severe falciparum malaria and the risk of death (p < 0.001). Renal replacement (88.9% vs 46.0%; p = 0.027) and mechanical ventilation (88.8% vs 28.0%; p < 0.001) were also significantly more frequent in those patients who died. The median (IQR) duration of the ICU stay increased, but without reaching statistical significance in the nine patients who died vs 4 (2-13); p = 0.575).
The MPS ranged from 1.78-5.20 (median, IQR: 1.8; 0.31-3.0) and the MSA from 3-9 (median; IQR: 2; 0-5). Comparing the median value of the two prognostic scores, both were good predictors of death in this cohort (Table 2) . When the four scores were compared, MPS (AUC 0.77; IC95% 0.64-0.90), MSA (0.84; IC95% 0.70-0.98), SAPS II (0.90; IC95% 0.81-0.99) and WHO score (0.91; IC95%0.82-1.0), based on the area under the ROC curve, the best death predictors were SAPS II and the WHO scores ( Figure 3 ).
In this retrospective study 59 consecutive patients with severe malaria admitted in a Portuguese infectious disease UCI over a 22 year period were considered. Obviously, the longer the period, the greater the impact of lost data, but the sample did not change over time and it includes mostly adult white males, middle-aged, travelling for work reasons. Malaria is a complex disease and it is well known that the higher the parasitaemia the greater the systemic inflammatory response and the production of cytokines and so, a major risk of organ dysfunctions, but some aspects concerning severe forms need to be clarified [21] [22] [23] . The WHO has redefined the criteria of severe malaria and pointed out a list of complications, but prognostic importance of each complication has not been well established. So, parameters usually associated with poor prognosis should not be forgotten and the patients' monitoring according to their presence or possibility, including the admission to an ICU [22] .
In this paper, the ICU admission was based on the presence of at least a life-threatening organ dysfunction, haemodynamic instability, as well as high parasitaemia, which often precedes clinical deterioration. Delay in diagnosis of malaria has been associated with a worse prognosis [24, 25] , but this was not found in this review, where 49% of the patients had the symptoms for more than a week before diagnosis, without any correlation with a worse prognosis. It is difficult to explain the reasons why. The validity of the variable (duration of symptoms) may be questioned because the time since arrival to diagnosis was statistically significant for the risk of death. A delay in diagnosis can be caused by patient ignorance about the potential severity of malaria, auto medication for fever with antipyretics before seeking medical attention or the doctor's delay in considering the diagnosis, only overcome with proper training and experience. In this review, the case fatality rate was 15.2% (nine patients) and factors statistically associated with death were time since arrival to diagnosis (p = 0.027), SAPS II score (p < 0.001), cerebral malaria (p = 0.019), acute renal failure (p < 0.001), ARDS (p < 0.001), hypoglycaemia (p = 0.02), DIC or spontaneous bleeding (p = 0.007) and acidaemia (p < 0.001) ( Table 2) ; the need for renal replacement (p = 0.027) and invasive ventilation (p = 0.027) was significantly more frequent in those who died. These data are in agreement with literature review, where the fatality rate is higher in cerebral malaria and acute respiratory distress syndrome (ARDS). Two of the patients were put on ECMO, used as a rescue therapy [26] [27] [28] . They had been working in Angola and Mozambique for some years, had parasitaemia 2% and 3% respectively and severe ARDS and multiorganic dysfunction. One of them survived and recovered without respiratory sequelae, the other died in refractory shock. These cases show the importance of a clinical surveillance of all patients, because the evolution is sometimes unfavourable in cases that initially seemed benign. The patient on mefloquine chemoprophylaxis that died had a parasitaemia of 30% and a fulminant evolution. Unfortunately he was not autopsied and it was impossible to investigate if it was a problem of resistance to mefloquine.
The number of organ dysfunctions may be directly correlated with mortality as was observed in this cohort, where the presence of more than four WHO major criteria for severe falciparum malaria significantly increased the risk of death (100% vs 40%; p < 0.001) [11, 29, 30] .
Unexpectedly, age, duration of symptoms, immune status, parasitaemia level, bilirubin value and the presence of shock were not statistically associated with mortality, even though hyperbilirubinaemia and shock were more frequent in patients who died, but diverse results are found in different studies [31] . These results should be interpreted with some caution because the study covers a small number of patients over a long period of time, although this does not necessarily explain the discrepancies between this and other studies. Previous papers reporting malaria parameters associated with mortality, mainly retrospective, have different aims and different degrees of severity and only a few of them take ICU cohorts into consideration [12, 13, 32, 33] . This is the case of a study reported in 2003 from a French group that evaluated 188 ICU patients with imported falciparum malaria over a 12-year period [10] with an overall mortality rate of 5.3% but, in most severe patients, the mortality rate was 11%. Factors associated with death were SAPS II, shock, acidosis, coma, pulmonary oedema and coagulation disorders.
No predictive malaria score system had been used as routine. Two malaria prognostic scores (MSA and MPS) used in this cohort were good death predictors [20] . When the four scores applied, SAPS II, MPS, MSA and WHO score were compared, SAPS II and the WHO score were the most sensitive to predict death ( Figure  1) .
Patients with severe forms of malaria are highly susceptible to bacterial infections and WHO recommends concomitant antibiotic therapy. In this review, some infections were documented: 13 patients had pneumonia, two of them community-acquired and 11 nosocomial, seven had acute acalculous cholecystitis and, in another two patients, central venous catheter-associated sepsis occurred. Although malaria acute acalculous cholecystitis is rarely reported, seven cases (11.9%) were found in this cohort [34, 35] .
The HIV primary infection was considered as the patient has been tested six months before, when he was treated for a cellulitis, having on that date a negative test for the HIV virus. The patient had a favourable evolution without concomitant antiretroviral therapy. There is no consensus about severity or complicated malaria in HIV co-infected adults but the fact that fever is a common symptom to both diseases can delay both diagnoses [7] .
Post-malaria neurological syndrome, another rare complication, was reported in two patients who recovered completely. This neurological syndrome has been defined as the acute neuropsychiatric manifestation in patients recently recovered from malaria [36] .
The prevention of malaria is essential with easy interventions such as different barrier methods and chemoprophylaxis whenever justified. Pre-travel advice and general practitians should stress prevention measures. Although the study does not point out that a delay in diagnosis is directly connected with a worsening of malaria, people at risk should be informed about the malaria symptoms, allowing an early diagnosis and treatment.
Although anti-malarial drug medication is the only intervention of proven efficacy to treat severe malaria, it is very important to monitor and treat severe forms with organic dysfunctions in ICU [37] . Use of controlled low dose gamma irradiation to sterilize allograft tendons for ACL reconstruction: biomechanical and clinical perspective As reviewed here, numerous biomechanical and clinical studies support the use of controlled, low temperature irradiation of allograft tendons, to provide both excellent clinical results and medical-device grade sterile allografts with minimal risk of disease transmission. The preferred method of terminal sterilization for tendon allografts, gamma irradiation, remains a concern to some surgeons. While some older studies have shown that higher, uncontrolled doses of gamma irradiation ([3 Mrad) can have detrimental effects on the strength of allograft tissue, numerous studies suggest that the currently used practices of low dose, controlled, low temperature gamma irradiation are effective to achieve terminal sterilization without detrimental impact on allograft tissue strength. In this review, irradiation methods are presented as well as biomechanical, clinical, and safety assessments of irradiated tendons used for ACL reconstructive procedures.
Prior to assessing studies regarding irradiated tendons, it is important to understand how irradiation methods are reported. Key variables of irradiation include:
• Target dose • Dose range • Temperature of irradiation • Tissue treatment prior to irradiation
The targeted dose is that (e.g., 22 kGy) which the tissue is intended to be exposed. However, the manner in which tissue is irradiated in its container or chamber does not allow all tissues to receive an exact similar dose. If only a single targeted dose is reported for tissue treatment, then that is likely the minimal exposure dose. For example, an exposure reported as 25 kGy (2.5 Mrad) likely indicates that all materials received at least 25 kGy exposure and that some may have received a much higher dosage (e.g., the outer grafts in a container). Thus, when only a single dose is reported it is fair to consider that to represent the minimal exposure and that some or most tissues will receive a higher dose.
The most accurate way to report an irradiation dose is as a dose range, e.g. 15-18 kGy (1.5-1.8 Mrad), which should indicate both the minimum and maximum dose exposure throughout the irradiated container. In order to minimize any negative material impact, it is important that both low doses and tightly controlled dose ranges be employed. It is very difficult to interpret any study that does not include a dose range. Again, if only a single dose is given, it should be considered the minimum exposure.
The third variable, temperature has been shown to be important as low (dry ice) temperatures serve to minimize any free radical generation and subsequent tissue damage (Anderson et al. 1992; Hamer et al. 1999) . Prior to knowledge of the importance of a controlled irradiation dose range at low temperatures, it is difficult to rely on the findings of studies where the details are not reported. Ideally a low average dose, at a narrow dose range, and at low temperatures are all factors in minimizing any irradiation-mediated alteration of material properties.
In addition, the treatment of any tendon prior to irradiation could play a role in how the tissue is potentially impacted. There are certain cleaning and disinfection methods that involve harsh chemicals or physical forces on the tissue. These may damage grafts independent of irradiation or pre-dispose grafts to alteration by irradiation.
To best interpret study results involving irradiated tissue, it is important to know how a tissue was treated prior to irradiation and exactly how it was irradiated.
In testing biomechanical properties of irradiated tendons, Balsly et al. (2008) reported no change in graft strength or elastic modulus for bone-patellar tendon-bone (BPTB) grafts, anterior tibialis tendons, semitendinosus tendons, or fascia lata soft tissue grafts when treated with sterilizing, low dose (18.3-21.8 kGy) gamma irradiation at dry ice temperatures. Likewise, Greaves et al. (2008) investigated the biomechanical properties of low dose (14.6-18.0 kGy) gamma irradiation on tibialis tendon allografts. In this matched pair study, 63 tibialis tendons were irradiated on dry ice while the contralateral tendons from the same respective donors were not irradiated. The study found that low dose irradiation did not significantly affect the failure load of either single stranded or double stranded tibialis tendon grafts (Table 1 ).
In a similar study, Roche et al. (2005) investigated the ultimate tensile strength of low dose gamma irradiation (15.4-15.5 kGy) on patellar ligaments and fascia lata allografts. Each irradiated allograft was matched with a control non-irradiated graft from the same donor to limit biomechanical variability resulting from different donors. The study did not find a statistical difference in the tensile strength between the matched low dose irradiated and non-irradiated allografts. In addition, Gibbons et al. (1991) showed in a biomechanical study that maximum stress, maximum strain, and strain energy density to maximum stress was not significantly reduced in goat BPTB grafts irradiated with 20 kGy of gamma irradiation. Further, Goertzen et al. (1995) found no significant difference in strength between canine BPTB grafts irradiated with 25 kGy and non-irradiated BTPB grafts after being implanted in an ACL reconstruction for (Haut and Powlison 1990; Mae et al. 2003; Maeda et al. 1993 Maeda et al. , 1998 Smith et al. 1996) of irradiated tendons indicate that treatment below 20-25 kGy have minimal impact on biomechanical properties.
There is also clinical evidence supporting the utility of low-dose irradiated tendons, which also have the advantage of minimizing any risk of disease transmission. Fanelli et al. (1996) compared irradiated BPTB and Achilles tendon allografts versus BPTB autografts for ACL reconstruction in patients who had combined ACL/PCL instability. No irradiation levels were given, so the presumption is of at least a 15-25 kGy dose. Although the sample size was low (20 patients), the prospective study was the largest study to date (1996) that evaluated ACL/PCL instability. This clinical study found equivalent results between irradiated allografts and autograft tendons. In a technique article, Harner and Elkousy, along with lead author Sekiya et al. (2002) , noted they only use patellar tendon allografts that have been irradiated for ACL reconstruction. In further support, Rihn et al. (2006) investigated the irradiation variable for ACL reconstruction in a clinical study involving 102 patients with an average follow-up of 4.2 years. The study found that 2.5 Mrad of gamma irradiation on BPTB allografts, which is effective in eliminating bacteria, does not compromise the clinical effectiveness of the grafts ( Table 2 ). The authors concluded ''[t]hese data suggest that irradiation can be used to sterilize BPTB allograft without adversely affecting clinical outcome.'' Taken together, the above articles suggest that a sterilizing dose of irradiation may not be of clinically significant concern when using allografts for ACL replacement. While three reports in particular have presented higher failure rates for irradiated allografts in ACL reconstructions (Rappe et al. 2007; Sun et al. 2009; Prodromos et al. 2007) , there are significant questions regarding these studies or data analysis.
Those study limitations are detailed here. The most significant issues with the first study (Rappe et al. 2007) include questionable follow-up methodology and a lack of information regarding the irradiation process used. The study followed up with *73% of irradiated graft patients compared with *93% of non-irradiated graft patients. It is unclear why a substantially fewer number of patients in the irradiated graft group returned for follow up care especially since they reportedly had significant graft failure (33%). Also, the method used to irradiate the grafts is unknown, e.g., the temperature of irradiation. The irradiation dose is given as 20-25 kGy and it is unknown if this is a true dose range and what the dose range of irradiation exposure actually was. In addition, other processing methods are unknown, including whether these grafts were also exposed to harsh solvents or cyclic pressures as performed by some banks, such that they may have been prone to damage from the higher irradiation dose used. Also, one surgeon reported twice the failure rate as the other in the study and the recipient age in relation to failures in both groups was not reported, making interpretation difficult. Finally, many tissue providers will routinely treat 'non-irradiated' or 'non-sterilized' grafts with 10-15 kGy doses of pre-processing irradiation for safety reasons so it remains unknown whether the control grafts were truly non-irradiated and also whether the irradiated grafts were double-dosed. There are also numerous concerns with the Sun et al. study (2009) . Only the target dosage is given (25 kGy) with unknown dose range. Also, these grafts were irradiated at room temperature and the grafts were not disinfected prior to irradiation (author, private communication). In addition, grafts were soaked in iodine prior to use. These graft treatments compromise any meaningful interpretation of the results. As significantly, the study has inconsistent results that possibly indicate an issue in either the surgery or measuring techniques. Unfortunately, the allograft patients exhibited a significant increase in duration of post-operative fever over autograft patients. The average duration of fever for an irradiated allograft patient was over 1 week (8.8 days) versus 4.7 days for the autograft patients. In the discussion, the authors state that this high fever rate ''was associated with…[different possibilities, or]…the real ability of tissue banks in our country [China] to process allografts''. The study also concludes that irradiated bone patellar-tendon bone (BPTB) allografts are clinically inferior to both nonirradiated BPTB allografts and BPTB autografts because of the laxity measurements with a KT-2000 arthrometer. The study reports only 31.3% of irradiated BPTB allografts had less than 3 mm of laxity while 85.3% of the non-irradiated and 87.8% of the autograft group had less than 3 mm of laxity. This is a surprising difference made even larger by the report of 34.4% of the irradiated group exhibiting more than 5 mm of laxity (defined as graft failure by the authors). These extreme percentages should indicate noteworthy irradiated graft patient dissatisfaction as well as significantly different results in other subjective and objective tests. In contrast, however, there were no significant differences in the overall IKDC scores between any of the three groups. Irrgang et al. (1998) noted that the IKDC was an especially rigorous evaluation tool because the lowest score received in any given area becomes the overall score instead of combining the averages like other evaluation systems. This makes the validity of the Sun et al. study even more uncertain since laxity measurement is a component of the overall IKDC score. It is unclear how there was no significant difference in the overall IKDC score among the 3 groups when the irradiated graft group had such extreme laxity measurements. Furthermore, the objective range of motion (ROM), vertical jump, and one-leg hop tests found no significant difference in any of the groups. There were also no significant differences among the 3 groups for mean Lysholm, Tegner, or Cincinatti knee scores. Moreover, there was not a significant difference in patients' satisfaction with their ability to participate in sports in any of the groups. One would expect significant differences in all or most of these tests and responses if 68.7% of irradiated allograft patients had greater than 3 mm laxity. The IKDC system is one of the best evaluation tools to measure ACL reconstruction results (Foster et al. 2010 ) and the results of this test and all the others should have been balanced against the arthrometer measurements. This balance is particularly important because there may be no correlation between laxity measurements and functional outcome (Mirzayan 2005) . Pollet et al. (2005) investigated this correlation in a prospective, clinical study of 29 ACL deficient patients with an average 33 month followup. After comparing anterior knee laxity, questionnaire based on IKDC score, sports activity rating scale (SARS), activities of daily living (ADL), and other tests, the study found ''no correlation between the joint laxity and the functional outcome score.'' This lack of correlation is actually supported by the Sun et al. (2009) study in which, according to the authors, almost 4 times as many irradiated allograft patients had graft failure based on laxity measurements but there was no statistical difference in patients' satisfaction in their postoperative sports activity or overall IKDC score. At the very least, the inconsistent results and non-standard tissue treatment methods should have given the authors pause before making the recommendation to completely discontinue use of irradiation to sterilize allografts. Prodromos et al. (2007) performed a meta-analysis on stability of autografts and allografts for ACL reconstruction. This included a sub-analysis of nonirradiated vs. irradiated grafts. They came to the following conclusion: ''The direct deleterious effects of graft radiation are an additional area of concern. The stability rate in the radiation-sterilized grafts in this study was strikingly low.'' However, the data used to draw this conclusion is heavily skewed by one particular study. In detail, to examine irradiated tendons, the authors included three studies, here called Noyes, Gorschewsky, and Rihn. They based conclusions on normal stability rate (which was 43% for irradiated vs. 63% for non-irradiated allograft) and abnormal stability rates (which was 31% for irradiated and 12% for non-irradiated allografts). This certainly appears negative for irradiated allografts. However, the irradiated group included in the Gorschewsky study included more grafts, and thus was more heavily weighted, than the other two studies combined and, most importantly, included a process method with steps containing acetone, sodium hydroxide, and hydrogen peroxide. These harsh chemicals can be quite damaging to soft tissues and no conclusion can be drawn from the fact that these grafts were also irradiated unless the proper controls were included (treatment with these chemicals without irradiation). If this single study is removed from the meta-analysis, then the comparisons become: normal stability rates of 62% for irradiated vs. 63% for non-irradiated allografts and abnormal stability rates of 15% for irradiated and 12% for non-irradiated allografts, respectively. Thus, the exclusion of the harsh chemically treated graft data set, yields results suggesting equivalent performance for irradiated vs. non-irradiated grafts. Further, note that this study that was, in fact positive regarding irradiation reported on tissue irradiated with 2.5 Mrad, which is even above commonly used levels of 13-18 kGy, further suggesting the utility of terminal sterilization via gamma irradiation.
It appears that there still exists confusion as to the definitions of sterility and processing methods. Sterile or aseptic tissue recovery by itself is mistakenly considered as a method that will result in the supply of sterile grafts hence making terminal sterilization unnecessary (Marrale et al. 2007) . Recovery under aseptic conditions seeks to ensure that no further bioburden is introduced from the environment but does not remove existing bioburden in the tissue (Vangsness 2004; Vangsness et al. 2003) . Tissue banks must use disinfection steps and/or terminal sterilization to accomplish sterilization of existing bioburden. Furthermore, aseptic recovery occurring in a surgical operating room can only result in, at best, a sterility assurance level (SAL) of 10 -3 , and then only if properly validated, versus a terminal sterilization SAL of 10 -6 . Sterility assurance level gives the probability of there being viable microorganisms on a particular graft unit, instrument, etc. A SAL of 10 -6 indicates there is only at most a 1 out of 1,000,000 chance that a viable organism exists with any single graft compared with an SAL of 10 -3 which indicates a 1 out of 1,000 chance (Vangsness et al. 2003) . Some tissue banks choose to use terminal sterilization to increase the likelihood of the safety of their tissue. If the allograft can be guaranteed to a SAL of 10 -6 then it may possibly possess an even lower degree of infection risk than an autograft procedure (Bryans et al. 2010; Katz et al. 2008) .
While the potential risk of viral transmission is extremely serious, it should be kept in perspective that this risk is virtually non-existent if the allograft is procured from a bank using intensive donor screening, tissue disinfection procedures, and terminal sterilization methods that inactivate viruses. While some studies reported that at least 30 kGy of gamma irradiation is needed to inactivate HIV, these studies have assumed HIV is present in high density levels (Fideler et al. 1994; Hernigou et al. 2000) . At least 30 kGy may be necessary to inactivate high density amounts of HIV but is excessive for lower density levels of the virus. If in the extremely unlikely event that HIV is present at all, the donor screening and tissue disinfection procedures help ensure that the virus will only be present in extremely low density amounts. The low 10-20 kGy dosage used for terminal sterilization is able to deactivate 99.9% of any remaining low-density HIV in allograft tissue (Moore 2010 ).
The preferred method of terminal sterilization for allografts, gamma irradiation, remains a concern to some surgeons. However, while some studies have shown high dose gamma irradiation ([3 Mrad) can have detrimental effects on the strength of allograft tissue, numerous studies have shown that the currently used controlled and low doses of gamma irradiation are effective in terminal sterilization and have no detrimental effect on allograft tissue strength. Rihn et al. (2006) determined that not only is using 25 kGy of gamma irradiation on BPTB allografts effective in preventing bacterial infection but it does not compromise the clinical effectiveness of the graft. The results are also comparable for soft tissue allografts. Balsly Cell Tissue Bank (2012 ) 13:217-223 221 et al. (2008 reported no change in graft strength for patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata soft tissue grafts when low dose gamma irradiation was used to terminally sterilize at low temperatures. Greaves et al. (2008) found low dose gamma irradiation did not affect the strength or stiffness of soft tissue tibialis tendon allografts. The terminal gamma irradiation is necessary in order to provide allograft tissue with a SAL of 10 -6 which is equivalent with implantable medical devices. Low dose gamma irradiation (10-15 kGy) in combination with donor screening and tissue processing procedures allows for thorough bactericidal treatment while maintaining intrinsic biomechanical properties and ensuring successful clinical performance (Block 2006) .
As reviewed here, numerous biomechanical and clinical studies support the use of controlled, low temperature irradiation of allograft tendons, to provide both excellent clinical results and medical-device grade sterile allografts with minimal risk of disease transmission.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Distinct immunoregulatory cytokine pattern in Egyptian patients with occult Hepatitis C infection and unexplained persistently elevated liver transaminases BACKGROUND/AIM: The immunopathogenesis of occult Hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro- and anti-inflammatory activity. This work aimed at studying the serum Th1 and Th2 cytokine production in patients with occult HCV infection. MATERIALS AND METHODS: Serum levels of cytokines of Th1 (interleukin [IL]-2, INF-γ) and Th2 (IL-4) were measured in 27 patients with occult HCV infection and 28 patients with chronic hepatitis C infection. RESULTS: The levels of IL-2 and interferon-γ were highly significantly increased in patients with chronic HCV infection (P<0.001). IL-4 was highly significantly increased in occult HCV infection (P<0.001). Significant increases were noted in chronic HCV infection regarding bilirubin (P<0.001), ALT (P = 0.009), AST (P = 0.013), AFP (P<0.001), while serum albumin was significantly higher in occult HCV infection (P<0.001). Necroinflammation (P<0.001), fibrosis (P<0.001), and cirrhosis (P = 0.03) were significantly increased in chronic HCV infection. CONCLUSION: Our data revealed a high prevalence of occult HCV infection (25%) in patients with unexplained persistently abnormal liver function test results. Those patients exhibited a distinct immunoregulatory cytokine pattern, favoring viral persistence and explaining the less aggressive course of this disease entity than chronic HCV infection.  DC-SIGN (CD209) Promoter −336 A/G (rs4804803) Polymorphism Associated with Susceptibility of Kawasaki Disease Kawasaki disease (KD) is characterized by systemic vasculitis of unknown etiology. High-dose intravenous immunoglobulin (IVIG) is the most effective therapy for KD to reduce the prevalence of coronary artery lesion (CAL) formation. Recently, the α2, 6 sialylated IgG was reported to interact with a lectin receptor, specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) in mice and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) in human, and to trigger an anti-inflammatory cascade. This study was conducted to investigate whether the polymorphism of DC-SIGN (CD209) promoter −336 A/G (rs4804803) is responsible for susceptibility and CAL formation in KD patients using Custom TaqMan SNP Genotyping Assays. A total of 521 subjects (278 KD patients and 243 controls) were investigated to identify an SNP of rs4804803, and they were studied and showed a significant association between the genotypes and allele frequency of rs4804803 in control subjects and KD patients (P = 0.004 under the dominant model). However, the promoter variant of DC-SIGN gene was not associated with the occurrence of IVIG resistance, CAL formation in KD. The G allele of DC-SIGN promoter −336 (rs4804803) is a risk allele in the development of KD. Kawasaki disease (KD), mucocutaneous lymphnode syndrome, is a systemic vasculitis that predominantly affects children under the age of five years. Although the cause is still unknown, KD is the most common cause of acquired heart disease during childhood in the developed countries at this time. Coronary artery lesions (CAL) are the major complications of KD. There is a 15-25% chance of CAL developing in KD patients without early treatment [1] . Although the exact therapeutic mechanisms have not been fully established, high-dose intravenous immunoglobulin (IVIG) is the most effective therapy for KD to reduce the prevalence of CAL [2] .
Many potential mechanisms of action for IVIG have been suggested [3] . Of them, at least three main mechanisms are suggested to explain the anti-inflammatory function of high dose IVIG: first, high-dose IgG saturates the neonatal FcRs (FcRn) and leads to the increased catabolism of autoantibodies; second, high-dose IgG saturates the activating Fcγ receptors (FcγRs) and prevents autoantibody-mediated activation of leukocytes; third, high-dose IgG increases the cell surface expression of inhibitory Fcγ receptors [4] . A single, N-linked glycosylation site exists at the amino acid 297 in the heavy chain of all IgG subclasses with approximately 10% terminating in sialic acid [5] . Recently, the α2, 6 sialylated IgG was reported to interact with a lectin receptor, specific 2
The Scientific World Journal intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) in mice and dendritic cellspecific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) in humans, and to trigger an anti-inflammatory cascade that promotes the upregulation of inhibitory FcγRs on inflammatory macrophages [6] .
There has been some evidence demonstrating the role of DC-SIGN promoter variants in the susceptibility to or the protection against various infectious diseases, such as dengue fever, tuberculosis, and AIDS [7] [8] [9] . However, whether DC-SIGN promoter variants have effects on susceptibility to KD is still unknown. Since DC-SIGN, also known as CD209, is so important for the anti-inflammatory functions of IVIG, it is reasonable to hypothesize that a functional single nucleotide polymorphism (SNP) in the CD209 molecule will be involved in the pathogenesis of KD or response to IVIG treatment. We hypothesized that the SNP rs4804803 of DC-SIGN promoter may be involved in the susceptibility to KD, CAL formation, coronary artery fistula formation, and IVIG treatment response in KD patients. To test this hypothesis, we conducted a case-control study involving 278 patients with KD and 243 controls.
All patients studied were children who fulfilled the diagnostic criteria for KD and were admitted for IVIG treatment at Chang Gung Memorial Hospital-Kaohsiung Medical Center, from 2002 and 2009. All patients were treated with a single high-dose IVIG (2 g/kg) over a 12hour period [10] [11] [12] . This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital with written consent statement. We excluded patients who did not fit the diagnostic criteria of KD. CAL was defined by the internal diameter of the coronary artery being greater than 3 mm (4 mm, if the subject was over the age of 5 years) or the internal diameter of a segment being at least 1.5 times that of an adjacent segment, as observed in the echocardiogram [13, 14] . KD patients with coronary artery ectasia or dilatation which was disappearing within the initial 6-8 weeks after the onset of illness was defined as transient CAL [3] . The diagnosis of coronary artery fistula (CAF) was made mainly by pulsed Doppler and color flow imaging [15] . IVIG treatment responsiveness was defined as defevrscence 48 hrs after the completion of IVIG treatment and no fever (temperature, >38 • C) recurrence for at least 7 days after the initial IVIG treatment with marked improvement of inflammatory signs [16] . Patients with IVIG resistance received another dose of IVIG (1-2 g/kg) or other anti-inflammatory regiments. Children who were admitted for upper and/or lower respiratory tract infections (including acute bronchiolitis, acute pharyngitis, acute bronchitis, croup, and acute tonsillitis) were also collected as control subjects for comparison during the same study period, as we have previously described [12] .
Genotyping of CD209 rs4804803 SNP. Genomic DNA was isolated from heparin-anticoagulated blood samples using a standard phenol-chloroform extraction followed by 70% alcohol precipitation. Genotyping for the CD209 variant (−336 A/G; rs4804803) was carried out using Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA). The primer sequences were 5 -GGACAGTGCTTCCAGGAACT-3 (forward) and 5 -TGTGTTACACCCCCTCCACTAG-3 (reverse). The Taq-Man minor groove binder probe sequences were 5 -TACCTGCCTACCCTTG-3 and 5 -CTGCCCACCCTTG-3 . The probes were labeled with the TaqMan fluorescent dyes VIC and FAM, respectively. The PCR was conducted in total volume of 15 μL using the following amplification protocol: denaturation at 95 • C for 10 min, followed by 40 cycles of denaturation at 94 • C for 20 s, followed by annealing and extension at 60 • C for one minute. After the PCR, the genotype of each sample was determined by measuring the allele-specific fluorescence in the ABI Prism 7500 Sequence Detection System, using SDS 1.1 software for allele discrimination (both Applied Biosystems). To validate the genotyping by real-time PCR analysis, 100 PCR products were subject to restriction fragment length polymorphism (RFLP) analysis with MscI restriction enzyme (New England Biolabs, Beverly, MA, USA) and showed a 100% identical result between these two genotyping systems as noted in our previous report [17] .
The Hardy-Weinberg equilibrium was first checked. The statistical differences between case and control in genotype and allele frequency were assessed by chi-square test. The statistical differences in the genotype and allele frequency of KD patients with and those without CAL formation, aneurysm formation, patients responding to IVIG, and those showing resistance were assessed using chisquare test. SAS 9.1 for Windows was used for data analysis.
Associated with the Susceptibility of Kawasaki Disease. In this study, a total of 278 KD patients were included, of which 35 patients (12.6%) were resistant to initial IVIG treatment, 42 patients (15.1%) had CAL formation and 13 patients (4.7%) developed coronary artery fistula. In this study, as shown in Table 1 , the difference of rs4804803 genotype between KD patients and controls was statistically significant (P = 0.004, dominant model, Table 1 ). Minor G allele of rs4804803 was over represented in the KD patients as compared with the controls (8.1 versus 3.5%).
We further evaluate the relationship between rs4804803 and the risk of IVIG resistance or CAL formation. As shown in the Tables 2 and 3, the frequency of AA genotype was higher in the patients with IVIG responsiveness (86.0 versus 77.1%) and without CAL formation (85.2 versus 83.3%). The genotype or allele frequency of rs4804803, however, was not statistically associated with IVIG resistance ( Table 2 ) or CAL formation (Table 3) . 
To further identify the role of rs4804803 of CD209 in the pathogenesis of coronary artery fistula in KD patients, we performed a subset analysis in cases that were reported as having fistula formation (13/278, 4.7%). Subset analysis between cases with coronary artery fistula and rs4804803 did not yield any significant results (Table 4 ).
DC-SIGN is a transmembrane lectin receptor on dendritic cells with multiple immune modulation function [18] DC-SIGN can recognize many pathogens, such as viruses (HIV-1, dengue, and measles virus) [19] [20] [21] , bacteria (Helicobacter pylori, Mycobacterium tuberculosis) [22] , and fungi (Candida albicans and Aspergillus fumigatus) [23] contributing to generation of pathogen-tailored immune responses and immunosuppressive response by the MAPK pathway in DCs [24] . The real cause of KD remains unknown. It is generally accepted that KD results from an undefined infectious process trigger in a genetically predisposed individual [25] .
A genetic predisposition is suggested based on clinical and epidemiologic features [1, 26] . In this study, we investigated whether the polymorphism of DC-SIGN (CD209) promoter −336 A/G (rs4804803) was associated with susceptibility and CAL formation in KD. Our study showed that the allele −336G was associated with susceptibility to KD. To the best of our knowledge, this is the first study to explore the association between DC-SIGN polymorphisms and susceptibility to KD. Immunoglobulin is well known for its defensive role in pyogenic infection. In addition to its protective role, immunoglobulin G (IgG) was also noted to have anti-inflammatory effects at high doses. Recently, the α2, 6 sialylated IgG was reported to interact with a lectin receptor, SIGN-R1 in mice and DC-SIGN in humans, and to trigger an antiinflammatory cascade. Thus it is reasonable to hypothesize that a functional SNP in the DC-SIGN molecule will be involved in the response to IVIG treatment. However, in our study, we found the variant and haplotype of −336A/G in the DC-SIGN gene did not associate with the occurrence of IVIG resistance or CAL formation in KD. Because of its highly polymorphic nature and numerous SNPs of DC-SIGN gene 4
The Scientific World Journal [27] [28] [29] , further investigation into other candidate SNPs contributing to KD morbidity is needed. Besides dendritic cells, IVIG was observed to affect many other cells, including endothelial cells, monocytes, neutrophils, and T and B cells [30] [31] [32] . The numerous effects of IVIG therapy also partly explain there not being an association of −336A/G SNP of the DC-SIGN gene with IVIG resistance in KD.
There were some limitations with regards to this study. First, the relatively small sample size of this study might prevent some of the detected associations from being statistically significant. Second, our study results need to be validated across different populations. Since the incidence of KD in Asian populations is much greater than among Caucasians [1] , the host's genetic background must be considered in the study of KD. In our control group, the frequency of the −336G DC-SIGN gene allele was 3.5%. This result agreed with previous reports showing very low −336G DC-SIGN gene allelic frequency in Asians [33, 34] . The highest −336G allelic frequency was found in African populations (35-48%), next in Caucasian populations (20%), and the lowest was observed in Asians [8, 33] .
In conclusion, from our study, we found the G allele of DC-SIGN promoter −336 (rs4804803) to be a risk allele in the development of KD in a Chinese population. Further studies to explore the effects of other SNPs of DC-SIGN or a combination of genes are needed. Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 (−/−)) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 (−/−) mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2(−/−) mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 (−/−) mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 (−/−) mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 (−/−) mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2(−/−) mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 (−/−) mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon. Virus infection of mammals induces the synthesis of type I interferons (IFN), which, in turn, inhibit virus replication. The high susceptibility of type I IFN receptor knockout (IFNAR 2/2 ) mice to infection by a variety of viruses [1] [2] [3] provides strong evidence for the major role of the IFN system in protecting from viral pathogenesis. In these mice, although IFN is induced by virus infection, it cannot act on target cells. Similarly, in genetically altered mice that are defective in IFN production due to the absence of specific pathogen-associated pattern recognition receptors, signaling proteins or specific transcription factors, viral pathogenesis is enhanced [4] [5] [6] . Although the critical importance of the IFN system in regulating viral pathogenesis is now well established, in many cases it is still unclear how IFN inhibits the replication and spread of a specific virus in vivo. In this context, activation of different components of the immune system plays a major role in controlling viral diseases that are relatively slow to develop [7] [8] [9] . In contrast, in acute infection by viruses that cause severe pathogenesis and death within a few days after infection, protection is primarily provided by the intrinsic antiviral actions of IFN-induced proteins encoded by the hundreds of IFN-stimulated genes (ISGs) [10] [11] [12] , several of which often contribute to the overall effect of IFN against a given virus. Our knowledge of the antiviral and the biochemical properties of individual ISG products is mostly limited to a few intensively studied examples such as PKR, OAS/RNase L or Mx [13] . However, recent systematic investigation of the antiviral functions of the entire family of ISGs has started producing exciting new information [14] .
In the above context, we have been investigating the biochemical and biological functions of the members of the Ifit family of ISGs, which are very strongly induced by IFN. There are three members of this family of genes in mice: Ifit1/ISG56, Ifit2/ ISG54 and Ifit3/ISG49; all of the encoded proteins contain multiple tetratricopeptide repeats (TPR), which mediate proteinprotein and protein-RNA interactions [15] . In vitro, P56 and P54, the products of Ifit1 and Ifit2, respectively, bind to the translation initiation factor eIF3 and inhibit protein synthesis [16] . The third member, P49, the product of Ifit3, does not share this property [17] . Recently, it has been reported that Ifit proteins form a multiprotein complex that can bind to the triphosphorylated 59 end of RNAs, an RNA-species produced during the replication of some, but not all, viruses [18] . In vivo, these genes are strongly induced in brains of mice infected with West Nile virus (WNV) or Lymphocytic choriomeningitis virus (LCMV); surprisingly, different Ifit genes are differentially induced in different regions of the brain, suggesting non-redundant functions [19] . To further explore the antiviral properties of the Ifit proteins, we generated Ifit1 knockout (Ifit1 2/2 ) mice and challenged them with different viruses. We observed that Ifit1 2/2 mice were particularly susceptible to a WNV mutant that is defective in its mRNA cap 29-O methylation; the mutant virus killed Ifit1 2/2 mice but not the wild-type (wt) mice [20] .
Here, we report on the antiviral properties of the newly generated Ifit2 2/2 mice; these mice, but not Ifit1 2/2 mice, were highly susceptible to neuropathogenesis after intranasal infection with vesicular stomatitis virus (VSV), a negative sense, singlestranded RNA rhabdovirus. VSV replication is highly sensitive to the inhibitory action of IFN and is routinely used to assay the antiviral activity of IFN in vitro [21] . As expected, IFNAR 2/2 mice are highly susceptible to VSV pathogenesis and the same is true for mice that specifically lack expression of IFNAR on the cells of their central nervous system (CNS) [1] . In spite of these observations, little is known about how IFN inhibits VSV replication in vivo. Our new results indicate that in the brain, but not in other organs, Ifit2 is a major mediator of IFN's protective effect against VSV. In contrast, Ifit2 could not protect mice from neuropathogenesis caused by encephalomyocarditis virus (EMCV), a picornavirus. Thus, we have uncovered a virusspecific, tissue-specific and ISG-specific antiviral effect of the IFN system.
Generation of Ifit2/ISG54 and Ifit1/ISG56 knockout mice Ifit2 gene knockout (Ifit2 2/2 ) mice were generated by deleting the entire protein-encoding region of the gene, which was achieved by flanking exons 2 and 3 with frt recombinase sites in C57BL/6 embryonic stem cells and excising the flanked region with Flp recombinase ( Figure 1A ). Ifit2 2/2 mice were bred to homozygosity ( Figure 1B) , and deficiency for induced expression of Ifit2 protein was confirmed in lysates of IFN-b-treated primary murine embryonic fibroblasts (MEF) ( Figure 1C ). Mice deficient for Ifit1 (Ifit1 2/2 ) were derived from C57BL/6 embryonic stem cells lacking the entire Ifit1 coding region ( Figure 1A ). Genotypic homozygosity of the Ifit1 2/2 mice and deficiency for Ifit1 protein induction were confirmed ( Figure 1B and 1C ). Both knockout mouse lines were healthy and fertile. Moreover, deletion of one gene within the Ifit locus did not alter the pattern of induction of other adjacent gene family members, as compared to wild-type (wt) mice ( Figure 1C ).
To determine the impact of Ifit2 on the outcome of viral infections in vivo, we compared susceptibilities of Ifit2 2/2 and wt mice to VSV infection, using IFNAR 2/2 mice as positive controls of enhanced susceptibility. Virus was administered at a low dose [4610 2 plaque forming units (pfu)], intranasally, reflecting a natural route of infection for VSV [22] . As seen previously, 100% of IFNAR 2/2 mice rapidly succumbed to VSV infection within 2 days ( Figure 2A , and [1] ), after suffering symptoms of lethargy. On the other hand, 79% of wt mice survived, the remaining 21% succumbed to VSV, and this occurred later, at 7-10 days post infection (d.p.i.). In contrast, 100% of Ifit2 2/2 mice died by 7 d.p.i. (Figure 2A ), with most succumbing by 6 d.p.i.; thus, we observed uniform and more rapidly occurring death of Ifit2 2/2 compared to wt mice after VSV infection. Within 24 h before death, both wt and Ifit2 2/2 mice developed neurological signs including ataxia, hind limb paralysis, and hyper-excitability. Ifit2 +/2 mice displayed an intermediate survival curve, demonstrating a gene dosage effect ( Figure 2B ). Next, the role of a related gene, Ifit1, in VSV pathogenesis was evaluated by infecting Ifit1 2/2 mice. Unlike the results observed with Ifit2 2/2 mice, no statistically significant increase in mortality was observed in Ifit1 2/2 mice ( Figure 2B , 21% death for wt versus 36% for Ifit1 2/2 , respectively; p.0.25). Consistent with this, survival kinetics of Ifit1 2/2 and wt mice were similar. Increasing the virus dose by 10,000-fold (to 4610 6 pfu) did not appreciably change the survival curves of wt, Ifit1 2/2 , or Ifit2 2/2 mice ( Figure 2C ). These results demonstrate functional differences between the two closely related proteins encoded by Ifit1 and Ifit2. The virus-specificity of the antiviral action of Ifit2 was evaluated by infecting Ifit2 2/2 mice with EMCV, an unrelated neurovirulent positive-strand RNA virus of the picornavirus family ( Figure 2D ). IFNAR 2/2 mice were highly susceptible to EMCV infection with all mice succumbing within 2 d.p.i.; in contrast, wt mice died with a slower kinetics and at a rate of only 80%. Notably, Ifit2 2/2 mice behaved similarly to the wt mice, without enhanced or accelerated mortality ( Figure 2D ). The same conclusion was true for a lower dose of EMCV ( Figure S1 ). The survival pattern of EMCV-infected Ifit1 2/2 mice also was similar to that of the wt mice ( Figure 2D ). Mice of all genotypes either succumbed after developing neurological symptoms, mainly hind limb paralysis, or survived without symptoms. These results demonstrate that the antiviral action of Ifit2 is both virus-and Ifitspecific.
The uniform penetrance of neuropathogenesis and lethality of VSV-infected Ifit2 2/2 mice, even at a low virus dose, prompted us to examine viral spread along its route from the nasal cavity into the CNS ( Figure 3A ). After intranasal administration, VSV infects
In mammals, the first line of defense against virus infection is the interferon system. Viruses induce synthesis of interferon in the infected cells and its secretion to circulation. Interferon acts upon the as yet uninfected cells and protects them from oncoming infection by inducing the synthesis of hundreds of new proteins, many of which interfere with virus replication. Vesicular stomatitis virus (VSV), a virus similar to rabies virus, is very sensitive to interferon but it is not known which interferon-induced protein inhibits its replication. Here, we have identified a single interferon-induced protein as the protector of mice from death by VSV infection. Knocking out the gene encoding this protein, Ifit2, made mice very vulnerable to neuropathogenesis caused by VSV infection; a related protein, Ifit1, did not share this property. Moreover, Ifit2 failed to protect mice from another neurotropic virus, encephalomyocarditis virus, nor was it necessary for protecting organs other than brain from infection by VSV. Our observation that a single IFN-induced protein protects a specific organ from infection by a specific virus revealed an unexpected degree of specificity of the antiviral action of IFN. shared wt mice (n = number of animals used). C, survival of Ifit2 2/2 , Ifit1 2/2 and wt mice after intranasal infection with a higher dose of VSV (4610 6 pfu). D, survival of Ifit2 2/2 , Ifit1 2/2 , IFNAR 2/2 and wt mice after infection with 5610 2 pfu of EMCV. In A-D, data are cumulative from at least two independent experiments (exceptions: Figure 2B , Ifit2 +/2 mice and Figure 2D , Ifit1 2/2 mice infected in a single experiment). Statistical significance of survival differences relative to wt mice is indicated by p-values; n.s., not significant; i.n., intranasal. doi:10.1371/journal.ppat.1002712.g002 the nasal epithelia including olfactory sensor neurons, which project to the outer layer of the olfactory bulbs (OB) [23] . This represents the entry step into the CNS, which we examined by immunostaining of OB sections. In wt mice, VSV P protein was detected exclusively within the glomeruli of the OB at 2 d.p.i. ( Figure 3B , upper right panel and [1] ), whereas in IFNAR 2/2 mice, VSV antigen had spread into deeper layers of the OB ( Figure 3B , lower left panel). In Ifit2 2/2 mice OB, viral antigen was restricted to the glomeruli, as seen in wt mice ( Figure 3B , lower right panel). This similar pattern of viral antigen expression between wt and Ifit2 2/2 mice was reflected in the equivalent levels of viral RNA in OB at 2 d.p.i. ( Figure 3C ). In contrast, ,10 times more VSV RNA was present in OB of IFNAR 2/2 mice ( Figure 3C , right panel, p,0.05). A comparison of the infectious viral burden between wt and Ifit2 2/2 mice in the OB confirmed these findings: at 2 d.p.i., ,10 6 pfu/g of VSV was present in both wt and Ifit2 2/2 mice ( Figure 3D , p = 1.0).
However, later in the course of infection, by day 6, viral OB titers in Ifit2 2/2 mice were not significantly changed, whereas in wt mice average titers of infectious VSV as well as viral RNA levels had decreased by ,10-fold ( Figure 3C and D, both p,0.05). These results suggest that VSV initially enters and replicates with similar efficiency in both wt and Ifit2 2/2 OB before spreading into the rest of the brain.
The efficiency of VSV replication in the brain, excluding the OB, was examined by quantifying infectious VSV as well as viral RNA. Early after infection, at 2 d.p.i., virus titers in brains were low (,10 4 to 10 5 pfu/g) and roughly equivalent in wt and Ifit2 2/2 mice ( Figure 4A , p.0.25). Similarly, viral RNA levels at the same Figure 3 . Ifit2 does not inhibit VSV entry and replication in olfactory bulbs. A, schematic entry route of VSV into the central nervous system of wt mice after intranasal infection, and VSV spread within brain, as reported in the literature. OB, olfactory bulbs; CX, cortex; MB, midbrain; CB, cerebellum; BS, brain stem; SC, spinal cord. B, VSV P protein in OB of VSV-infected wt, Ifit2 2/2 and IFNAR 2/2 mice at 2 d.p.i., detected by immunohistofluorescence. C, VSV RNA levels in OB of uninfected or VSV-infected wt, Ifit2 2/2 and IFNAR 2/2 mice at 1, 2 or 6 d.p.i., plotted as mean+SD on log scale; ND, none detected. D, infectious VSV titers in wt and Ifit2 2/2 OB at 2 and 6 d.p.i.; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. In C and D, n = 4-8 mice per infected group accumulated from three independent experiments; in B, n = 2 mice from two independent experiments. All infections were 4610 2 pfu of VSV administered intranasally. Asterisks indicate statistical significance: ** p = 0.006, * p,0.05; n.s.: not significant. doi:10.1371/journal.ppat.1002712.g003 time were low and comparable between wt and Ifit2 2/2 ( Figure 4B , p.0.25). However, at the same time, levels of VSV RNA (380-fold, p,0.05) were much higher in the brains of IFNAR 2/2 mice ( Figure 4B , right panel). Later in the course of infection (6 d.p.i.), brains of wt mice accumulated only ,5-fold more infectious VSV, with occasional clearance of the virus. In contrast, we detected markedly higher VSV titers in the brains of Ifit2 2/2 mice (,350-fold higher compared to wt mice, p = 0.0009), reaching ,10 8 pfu/g ( Figure 4A ); the high virus load likely caused the pronounced lethality. Differences in viral RNA levels in brains of wt and Ifit2 2/2 mice at 6 d.p.i. correlated well with levels of infectious VSV ( Figure 4B ). To determine whether Ifit2 selectively restricts replication of VSV in particular regions of the brain, we measured viral RNA levels in cortex, midbrain, cerebellum and brain stem at 6 d.p.i. In wt mice, VSV RNA was present prominently in the cortex, midbrain and brainstem, but not in the cerebellum ( Figure 4C ), which is consistent with published results [24] . However, in Ifit2 2/2 mice, viral RNA was 200-fold or more (p,0.05) abundant in all regions of the brain examined, including the cerebellum. The increase of VSV replication in Ifit2 2/2 brains was not due to a broadened cell tropism of the virus; immunostaining for viral P protein showed exclusive localization to neurons and not other cell types, such as astrocytes ( Figure 4D ). From the above observations, we conclude that after intranasal infection by VSV, Ifit2 protects mice from neuropathogenesis by suppressing replication or spread of the virus in brain neurons.
Ifit2 and Ifit1 are induced in VSV-infected regions of OB and brain
The protective effect of type I IFN signaling and in particular, Ifit2, against VSV neuropathogenesis prompted us to confirm its expression in OB and brain of wt mice, and whether it was induced in a type I IFN-dependent manner. In wt OB, Ifit2, Ifit1, and IFN-b mRNA was induced strongly by 2 d.p.i., and Ifit2 and Ifit1 RNA remained abundant until day 6 d.p.i. (Figure 5A ). The induction of these genes was dependent on type I IFN receptor in OB as well as in brain ( Figure 5B and 5E, and data not shown). Ifit2 suppresses VSV replication in the brain after intranasal infection. A, infectious VSV titers in wt and Ifit2 2/2 brains at 2 and 6 days after intranasal infection, plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. B, VSV RNA levels in brains of uninfected or VSV-infected wt, Ifit2 2/2 and IFNAR 2/2 mice at 2 or 6 d.p.i., plotted as mean+SD on log scale. C, VSV RNA levels in different regions of the brains of uninfected or VSV-infected wt and Ifit2 2/2 mice at 6 d.p.i., plotted as mean+SD on log scale. D, VSV P protein in midbrain neurons of Ifit2 2/2 mice at 6 d.p.i.; detection by immunohistofluorescence-labeling of VSV-P (red) and neuron (NeuN) or astrocyte (GFAP) markers (green); in A and B: n = 4-8 mice per infected group accumulated from three independent experiments; in C: n = 4 mice per infected group; in D: n = 2 mice per infected group; all infections in A-D were intranasal with 4610 2 pfu of VSV. ND, none detected. Brains in A and B were separated from OBs assayed in Figure 3D and 3C, respectively. Asterisks indicate statistical significance: *** p#0.0009; n.s.: not significant. doi:10.1371/journal.ppat.1002712.g004
Furthermore, expression of Ifit2 mRNA in wt OB coincided with the presence of detectable levels of the encoded Ifit2 protein ( = P54) at 2 d.p.i. and 6 d.p.i., as seen by immunohistochemistry ( Figure 5C , and data not shown). Ifit2 protein staining was observed in VSV-infected cells within OB glomeruli as well as in surrounding and distant viral antigen-free cells, consistent with a remote IFN-dependent induction of Ifit2 expression ( Figure 5C , arrowheads in magnified images of right panel). Ifit1 and IFN-b mRNAs were induced as strongly in OB of Ifit22/2 as in wt mice, which correlated well with similar abundance of VSV RNA in wt and Ifit2 2/2 OB (Figure 5A compared to Figure 3C ). In brains, at 6 d.p.i., in contrast to OB, induction of Ifit1 and IFN-b mRNAs was considerably stronger in Ifit2 2/2 mice compared to wt mice ( Figure 5D , 5-fold and 27-fold, respectively, both p,0.005). The enhanced gene induction in VSV-infected Ifit2 2/2 mice was not restricted to specific regions of the brain ( Figure S2 ). Enhanced cellular gene expression also was observed for several virusinduced cytokine and chemokine genes, as measured by quantitative RT-PCR ( Figure S3A ). Gene expression profiling of brain tissue at day 6 d.p.i., using microarray analysis, revealed that many other genes, including ISGs, were also more strongly induced (Table S1 ). These results demonstrated that enhanced virus replication in the brains of Ifit2 2/2 mice led to enhanced type I IFN, other cytokines and ISG induction, which nevertheless failed to restrict VSV replication in the absence of Ifit2.
Wt mice are as susceptible as Ifit2 2/2 mice to intracranial VSV infection
Our results from intranasal VSV infection indicated that Ifit2 induction in the brain was mediated by type I IFN that was, in all likelihood, produced by infected cells in the OB ( Figure 5A ). Virus replication and resultant IFN induction at 2 d.p.i. were similar in the OBs of wt and Ifit2 2/2 mice (Figs. 3C, 3D and 5A); presumably, the newly produced IFN diffused into the rest of the brain and induced local Ifit2 expression in the wt mouse brains, prior to the arrival of the infectious virus. If this were the case, one would anticipate that direct infection of the brain, without prior action of IFN produced in infected OB, would minimize the difference between the phenotypes of wt and Ifit2 2/2 mice. To test this idea, we injected a very low dose (10 pfu) of VSV intracranially. As hypothesized, wt and Ifit2 2/2 mice were now equally susceptible; almost all mice died by 3 d.p.i. even at this low dose ( Figure 6A ) and there were equally high virus titers and viral RNA levels in the brains of mice of both genotypes ( Figure 6B and 6C). Concomitant with virus replication, there was similar induction of Ifit1 and IFN-b ( Figure 6C ) and other cytokines and chemokines ( Figure S3B ). These results indicate that in the absence of prior induction of Ifit2 by IFN, brain neurons are highly susceptible to VSV infection.
Unlike the brain, other organs of Ifit2 2/2 mice are not more susceptible to intranasal VSV infection IFNAR 2/2 mice succumbed within two days after VSV infection without accumulating very high VSV RNA levels in the brain ( Figure 4B ). These mice did not develop CNS-related signs of disease, but showed severe lethargy before death, suggesting that death was due to efficient replication of the virus in peripheral organs, due to the absence of an otherwise effective type I IFN-mediated antiviral protection of the same organs in wt mice. To test this, we assessed the kinetics of VSV accumulation in brains, livers and lungs of wt, IFNAR 2/2 and Ifit2 2/2 mice (Figure 7) . At 2 d.p.i., VSV titers were very high in the liver of IFNAR 2/2 mice, reaching 10 9 pfu/g ( Figure 7A ). In contrast, no or little infectious virus was detected in the liver of wt mice at 2 or 6 d.p.i., indicating efficient IFN-dependent suppression of VSV replication; intriguingly, this was also observed in Ifit2 2/2 mice, demonstrating that Ifit2 did not mediate the anti-VSV effects of type I IFN in the liver. In lungs, which directly received a part of the virus inoculum from intranasal inhalation of VSV, the virus also replicated efficiently in IFNAR 2/2 mice, reaching 10 8 pfu/g before death ( Figure 7B ). In comparison, lungs of wt and Ifit2 2/2 mice exhibited much lower levels of VSV at 2 and 4 d.p.i. (3,000 to 10,000-fold lower for wt and Ifit2 2/2 compared to IFNAR 2/2 mice, all p,0.05). By days 5 and 6 d.p.i., the virus was cleared from the lungs of a subset of wt and Ifit2 2/2 mice. In contrast, in brains from the same animals, 10 to 100-fold higher average titers (p,0.05) of VSV accumulated in Ifit2 2/2 compared to wt mice at all time points between 2 and 6 d.p.i. ( Figure 7C ). As expected, in wt mice, both Ifit1 and Ifit2 were induced not only in brains ( Figure 5D ), but also in livers ( Figure 7D ) and lungs ( Figure 7E ); IFN-b was also induced in lungs, but not livers. Ifit1, Ifit2 and IFN-b mRNAs were also induced in the brains of EMCV-infected wt mice ( Figure S3C ). These findings demonstrate an unexpected brain-restricted and virus-restricted function of Ifit2 in the context of the type I IFN-mediated antiviral response to VSV infection. They also indicate that in Ifit2 2/2 mice, other ISGs, which presumably protect the peripheral organs of VSV-infected wt mice, are either not induced in neurons or insufficient to protect them.
IFNs are defined by their antiviral activities. They inhibit the replication of many, if not all, viruses mostly by direct inhibition of replication in the infected cells but also by promoting the ability of immune cells to recognize and eliminate the virus-infected cells [25] . The direct effects are mediated by ISGs, which number in the hundreds, and different ISGs are thought to have more potent antiviral activities toward different families of viruses [13] . However, in most cases, it is not known which ISG inhibits the replication of a given virus; the rare exception is the Mx-mediated inhibition of influenza viruses, the underlying effect which allowed for the discovery of IFNs [26] . The task of connecting a specific IFN-induced protein to a specific antiviral action is compounded by the fact that often several IFN-induced proteins act in concert to inhibit the same virus at different stages of its life cycle. Moreover, a specific IFN-induced protein may be more relevant for inhibiting a virus in one specific cell-type than another. Recent systematic investigation of the specific antiviral effects of different ISGs has started providing significant insight into this problem [14] . Such findings are complemented by the analyses of the spectra of the antiviral effects of a specific ISG or a family of ISGs [27] . We have undertaken an investigation of the Ifit family of mouse ISGs. The corresponding human proteins are known to have antiviral activities against human papillomavirus (HPV) and hepatitis C virus (HCV), neither of which replicate in mouse cells. The anti-HPV activity of human IFIT1 ( = P56) has been attributed to its ability to bind HPV E1 protein and to inhibit its helicase activity, which is essential for HPV DNA replication [28, 29] . The antiviral effect on HCV, on the other hand, is manifested at the level of inhibiting viral protein synthesis as a consequence of the ability of IFIT1 to bind the translation initiation factor eIF3 and inhibit its various actions in translation initiation [30] . It has been reported recently that the IFIT1 protein can form a complex and bind to RNAs with triphosphorylated 59 ends, presumably providing another means to inhibit specific viruses that produce such RNAs [18] .
The Ifit genes are clustered at a single locus in both human and mouse. In the latter species, two alleles of Ifit3 genes are flanked on two sides by one allele of Ifit2 and one allele of Ifit1 [15] . To identify their physiological functions, we have separately deleted the entire coding regions of Ifit1 or Ifit2 genes. The Ifit1 2/2 mice exhibited an interesting phenotype in allowing the replication of and resultant pathogenesis by a WNV mutant, which failed to replicate in wt mice [20] . Because this mutant is defective in 29-O methylation of the cap structure of viral mRNAs, its rescue in the Ifit1 2/2 mouse indicates that this antiviral protein recognizes the 59 ends of mRNAs, a conclusion that is consistent with the observation that, in vitro, it can bind to RNAs having specific structures at the 59 ends [18] . It remains to be seen whether the proposed property of Ifit proteins to recognize 59 ends of RNA is connected in any way to their ability to inhibit the functions of eIF3 [16] , which participates in several steps of translation initiation taking place at or near the 59 ends of mRNAs.
Replication of VSV is highly sensitive to the antiviral activity of IFNs, and VSV is widely used to determine the specific activities of IFN preparations quantitatively [21] . In spite of this strong connection, it is unclear how IFN inhibits VSV replication. An early report indicated that viral primary transcription is inhibited by IFN, but it is not known which IFN-induced protein mediates this inhibition [31] . The observed sensitivity of VSV replication in vitro is reflected in vivo. IFNAR 2/2 mice are extremely susceptible to VSV infection; they rapidly die within 2 days after infection and the virus replicates to very high titers in many organs of the infected mice. The extreme sensitivity of IFNAR 2/2 mice to VSV infection suggests that type I IFN provides the majority, if not all, of the protective innate immune defense. Eventually, protection may be facilitated by immune cell-mediated antiviral actions, but this is a slow process that does not appear to function before 6-10 days post-infection [32, 33] . Thus, it is likely that one or more ISGs directly inhibit VSV replication in vivo. In this context, it has been reported that mice lacking PKR, a well-studied ISG, display higher susceptibility to VSV pathogenesis [34] . However, detailed investigation of the underlying mechanism revealed that PKR did not execute IFN's antiviral action; rather, it was required for efficient induction of IFN-a/b in the infected mice [35] . In vivo VSV-infection induces IFN synthesis in many cell types, using either the cytoplasmic RIG-I pathway or the endosomal TLR7 pathway [4, 36] ; however, it is unclear how PKR aids this process.
Our results show that Ifit2 2/2 mice are highly susceptible to intranasal VSV infection and the effect is gene dosage-dependent: Ifit2 +/2 mice had an intermediate susceptibility phenotype. Infected Ifit2 2/2 mice displayed symptoms of severe neuropathogenesis late after VSV infection accompanied by efficient replication of the virus in many regions of the brain. However, virus replication was restricted to neurons and did not spread to other types of cells in the brain, such as astrocytes. Our results are consistent with the hypothesis that prior, IFN-induced, Ifit2 expression in the brain restricts VSV replication. Supporting genetic evidence for the requirement of IFN action is provided by the high susceptibility of the IFNAR 2/2 mice, which possess the functional Ifit2 gene but Ifit2 is not induced by VSV infection because these mice cannot respond to type I IFN. Additional evidence comes from a previous study using brain-specific IFNAR 2/2 mice, which displayed a pattern of susceptibility to intranasal VSV infection similar to that of our Ifit2 2/2 mice [1] . In our experimental system, the source of the IFN production was Figure 5C ). Ifit2 was also induced at this time in the rest of the brain, without any induction of IFN mRNA ( Figure 5D ) suggesting that the source of IFN was the OB. In accord with the well-established concept of IFN action, pre-induction of Ifit2 in neurons, before the onset of infection, was essential for the antiviral effect. In comparison, induction of IFN and Ifit2 that was concomitant with VSV infection failed to have an appreciable antiviral effect, as manifested by robust virus replication at directly infected sites, such as the OBs of wt mice infected intranasally ( Figure 3D ) or the brain of wt mice infected intracranially ( Figure 6B ). High mortality of the infected mice correlated with high virus titers in the brains of intranasally infected Ifit2 2/2 mice or intracranially infected wt and Ifit2 2/2 mice. In the intranasally infected Ifit2 2/2 mice, death was not preceded by widespread apoptosis in the brain ( Figure S4 ). However, as expected with high viral loads, IFN and other cytokines and chemokines were strongly induced ( Figures 5D, S2 and S3A) ; consequently, many ISGs, except Ifit2, were also induced (Table S1) .
Pre-induced Ifit2 prevents efficient VSV replication in the brain, most probably by blocking one or more essential step of the viral life cycle including viral entry, uncoating, primary transcription, viral protein synthesis, RNA replication, virion assembly or egress. It also might block trans-synaptic spread of the virus, although unlike another rhabdovirus, rabies virus, VSV is not known to depend on transit from neuron to neuron. In this context, it is important to note the observations made by Iannacone et al. [37] using a footpad VSV infection model. They concluded that type I IFN, produced by infected macrophages and plasmacytoid dendritic cells in infected mice, blocked infection of peripheral neurons resulting in lowered infection of the CNS and prevention of neuropathogenesis. It is worth noting that in our studies, the absence of Ifit2 did not affect IFN induction by VSV (Figures 5A  and 6C ). Further investigation of the biochemical mechanism behind the observed in vivo effect of Ifit2 2/2 is hampered by the absence of a suitable cell culture model of the phenomenon. For example, Ifit2 was not required for mediating the anti-VSV effect of IFN in mouse embryonic fibroblasts ( Figure S5 ), in primary fetal neurons or in Ifit2-ablated neuroblastoma cells (data not shown), results that are not surprising given the strong tissuespecificity of Ifit2 action observed in vivo (Figure 7) . Specific RNAbinding properties of Ifit proteins have been recently reported [18] . Following this lead, we examined the RNA-binding properties of recombinant murine Ifit1 and Ifit2 using VSV leader RNA as the probe in an electrophoretic mobility shift assay: Ifit1 bound RNA with a 59-ppp end but not with a 59-OH end; however, Ifit2 bound neither ( Figure S6 ). To obtain meaningful leads, future investigation of this kind may require using brain extracts from infected mice to detect protein-viral RNA complexes that may contain Ifit2 along with adult neuronspecific proteins.
Our results revealed several layers of specificity of IFN action, some of which were not anticipated. First, compared to Ifit2 2/2 mice, Ifit1 2/2 mice were much less susceptible to intranasal VSV infection; this was true for both low and high doses of virus. This finding was surprising in view of a recent report on VSV susceptibility of Ifit1 2/2 mice [18] and the observation that Ifit1, but not Ifit2, could bind VSV leader RNA in vitro ( Figure S6) . The above results demonstrate that different Ifit proteins have nonredundant functions in vivo. The second layer of specificity was directed toward the nature of the infecting virus. Although both VSV and EMCV caused neuroinvasive disease, induced IFN-b, Ifit1 and Ifit2 in the brain and type I IFN action was required for protection against both viruses, Ifit2 was critical only for protection against VSV; the absence of either Ifit1 or Ifit2 did not exacerbate susceptibility to EMCV. The third layer of specificity was revealed by the organ-specific action of Ifit2. In the complete absence of type I IFN action in the IFNAR 2/2 mice, intranasally infected VSV replicated vigorously not only in brains, but also in livers and lungs ( Figure 7A-C) . In contrast, in Ifit2 2/2 mice, efficient VSV replication was restricted to the brain suggesting that Ifit2 does not act as an anti-VSV ISG in the liver or the lung because its absence did not impact virus titers, even though Ifit2 was induced in these organs of infected wt mice ( Figure 7D and 7E) . The efficient VSV replication in livers and lungs of IFNAR 2/2 mice, but not wt and Ifit2 2/2 mice, indicates that other ISGs must have anti-VSV effects in those organs. Further investigation is needed to determine the basis of neuronal specificity of Ifit2 action and the identities of other ISGs that inhibit VSV replication in other organs.
All animal experiments were performed in strict accordance with all provisions of the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the PHS Policy on Humane Care and Use of Laboratory Animals. The protocol was approved by the Cleveland Clinic Institutional Animal Care and Use Committee (IACUC), PHS Assurance number A3047-01. All experimental manipulations or intranasal instillations of mice were performed under anesthesia induced by pentobarbital sodium or isofluorane, respectively, and all efforts were made to minimize suffering.
All mice used were of C57BL/6 background and of both sexes; Ifit2 2/2 mice were custom-generated by Taconic Farms, Inc. by flanking exons 2 and 3 of Ifit2, encompassing the complete protein-encoding region, with frt sites in C57BL/6 embryonic stem (ES) cells, and deleting the flanked region by transfection of Flp recombinase. ES cell clones were injected into BL/6 blastocysts, and heterozygous offspring mice were crossed to homozygosity. Ifit1 2/2 mice were generated from C57BL/6 ES cells lacking the whole coding region of Ifit1 (20); ES cells were obtained from the NIH Knockout mouse project (KOMP, allele Ifit1 tm1(KOMP)Vlcg ). The same ES cell line was independently used to generate mice in another study [18] . IFNAR 2/2 mice (lacking Ifnar1) were a gift of Murali-Krishna Kaja (Emory University, Atlanta, GA). Congenic wild-type mice were obtained from Taconic Farms.
Vesicular stomatitis virus (VSV) Indiana was a gift from Amiya K. Banerjee, Lerner Research Institute, Cleveland, Ohio. For intranasal infections, between 4610 2 and 4610 6 pfu of VSV in 35 ml of endotoxin-free PBS were inhaled by isofluorane-anesthetized 8-12 week-old mice, with PBS-only as control. For intracranial infections, 10 pfu of VSV in 30 ml of endotoxin-free PBS were injected into the brains of 6-7 week-old mice, with PBSonly as control. Thereafter, mice were monitored daily (twice daily after i.c. injection) for weight loss and symptoms of disease. Encephalomyocarditis virus (EMCV) K strain was a gift from Robert H. Silverman, Lerner Research Institute, Cleveland, Ohio. For intraperitoneal infections, between 25 and 5610 2 pfu of EMCV in 500 ml of PBS were injected into the peritoneal cavity of mice. Mice were monitored daily for weight loss and symptoms of disease.
Mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs by cardiac perfusion with 10 ml of PBS, followed by perfusion with 10 ml of 4% paraformaldehyde/PBS for fixation. Brains were placed in 4% paraformaldehyde overnight for complete fixation, submerged in 30% sucrose/ PBS overnight for cryoprotection, and frozen in O.C.T. compound (Sakura Finetek USA, Torrance, CA, USA). 10 mm sagittal sections were cut at 220uC in a Leica CM1900 cryostat, mounted on coated slides (Superfrost Plus, Fisherbrand, Fisher Scientific); membranes were permeabilized by 0.2% Triton X-100/PBS treatment for 15 min. For immunohistochemistry, the Envision+ DAB kit (Dako, Carpinteria, CA) was used with antimouse Ifit2/P54 [38] or anti-VSV-P protein (a gift from Amiya K. Banerjee, Lerner Research Institute, Cleveland, Ohio) as primary antibodies. For immunohistofluorescence, anti-VSV-P or anti-NeuN (Chemicon Intl./Millipore, Billerica, MA) or anti-GFAP (Sigma-Aldrich, St. Louis, MO) were used; labeled brain sections were stained with AlexaFluor-594 secondary antibody (Invitrogen/Molecular Probes, Carlsbad, CA). For detection of apoptotic cells in brain sections, the DeadEnd fluorometric TUNEL system (Promega) was used according to manufacturer's instructions. All objects were then mounted with VectaShield (with DAPI, Vector Labs, Burlingame, CA), and examined with a Leica DRM fluorescence microscope.
Mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs after cardiac perfusion with 10 ml of PBS. Brains were separated into olfactory bulbs and the remainder of the brain, snap-frozen in liquid nitrogen (as well as livers and lungs) and RNA was extracted using TRIzol reagent (Invitrogen). DNase I treatment (DNAfree, Applied Biosystems/ Ambion) and reverse transcription with random hexamers (ImProm-II, Promega) were performed according to manufacturer's instructions. 0.5 ng of RNA was used in 384 well-format realtime PCRs in a Roche LightCycler 480 II using Applied Biosystem's SYBR Green PCR core reagents. PCR primers for murine ISG49/Ifit3, ISG54/Ifit2, ISG56/Ifit1 and 18S rRNA have been published previously [17] ; primers targeting murine Ifnb1 [59-CTTCTCCGTCATCTCCATAGGG-39 [39] , with the alternative reverse primer: 59-CACAGCCCTCTCCATCAACT-39], VSV N RNA [40] or EMCV 3D polymerase genomic region [41] were described previously. Primers for Ccl2, Il1b, Il6, Tnf, Il12b and Nos2 have been described previously [42, 43] . Average expression levels, relative to 18S rRNA and normalized by use of calibrator samples, were graphed with Prism 5.02 software. For analysis of different regions of the brain, brains without OB of perfused mice were separated into cortex, cerebellum, brain stem and remaining ''midbrain'', and tissue was submerged into RNAlater stabilizing reagent (Qiagen) overnight and frozen. RNA was then extracted via TRIzol and further processed and assayed by realtime RT-PCR as described above. For microarray analysis, TRIzol-extracted and DNase I-treated RNA was additionally purified using spin columns (RNeasy Mini kit, Qiagen) before subjection to mRNA expression microarray analysis via Illumina Mouse Ref-8 V2 beadchip and GenomeStudio software V2010.2 (Illumina, Inc.); RNA hybridization to chips was performed by the Lerner Research Institute Genomics Core at the Cleveland Clinic. Microarray raw data were deposited in the NCBI Gene Expression Omnibus (GEO), accession number GSE33678.
For quantification of infectious VSV in organs, mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs by cardiac perfusion with 10 ml of PBS. Organs were snap-frozen in liquid nitrogen, weighed, pestle/tubehomogenized (Kimble/Kontes) in 1 ml of PBS per brain or peripheral organ or 0.1 ml per pair of olfactory bulbs, and virus was titered in 10-fold serial dilutions on Vero cells by plaque assay. Results are expressed as plaque-forming units (pfu) per gram of tissue. For quantification of infectious VSV yields in MEF, cells (2/+IFN-b pretreatment as indicated) were infected with VSV inoculum for 1 h, and after another 12 h, cells were freeze/ thawed, and cleared supernatants of lysates were assayed for VSV by plaque assay on Vero cells.
Primary murine embryonic fibroblasts (MEFs) were stimulated with 1000 U/ml murine IFN-b (PBL, Inc., Piscataway, NJ) for 16 h and lysed in lysis buffer [50 mM Tris pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM bglycerophosphate and 16 complete EDTA-free protease inhibitor (Roche, Indianapolis, IN)]. 10 mg of whole cell extract were separated via 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% dry milk in Tris-buffered saline/0.05% Tween-20 overnight and labeled with anti-Ifit3/P49, anti-Ifit2/P54 or anti-Ifit1/P56 polyclonal rabbit sera [17, 38] .
Single-stranded VSV leader RNA (nucleotides 1-18) was T7 polymerase-transcribed in presence of [a-32 P]-CTP, yielding radiolabeled 59-triphosphorylated (ppp-) RNA, followed by alkaline phosphatase treatment for generation of 59-hydroxyl (HO-) RNA. ppp-RNA or HO-RNA were added to bacterially expressed and purified 6xHis-tagged Ifit1 or Ifit2 protein in reaction buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.05% Triton X-100, 10% glycerol) and incubated for 30 min on ice. Reaction products were separated by 6% native polyacrylamide gel electrophoresis followed by exposure to film.
Statistical significance of mouse survival differences was calculated by Mantel-Cox log rank test. To assess significance of differences in gene expressions or virus titers, the two-tailed Mann-Whitney test was used. All calculations were performed using GraphPad Prism 5.02 software.
Previously published transcript sequences in the NCBI Entrez Nucleotide database: Ifit2, NM_008332; Ifit1, NM_008331; Ifit3, NM_010501; Ifnb1, NM_010510; Ifnar1, NM_010508. Figure S1 Survival of wt and Ifit2 2/2 mice after infection with low EMCV dose (25 pfu). Statistical significance of survival differences is indicated by p-value; n.s., not significant. (PDF) Figure S2 Enhanced ISG and IFN-b induction in intranasally VSV-infected Ifit2 2/2 brain regions. IFN-b-, and Ifit3/2/1 mRNA levels in different regions of brains of uninfected or VSV-infected wt and Ifit2 2/2 mice at 6 d.p.i., plotted as mean+SD. n = 4 mice per infected group; ND, not done. Infections were intranasal with 4610 2 pfu of VSV. (PDF) Figure S3 Gene induction in brains after VSV or EMCV infections. A, mRNA levels of select genes in brains (without OBs) of uninfected or intranasally VSV-infected wt and Ifit2 2/2 mice at 6 d.p.i., plotted as mean+SD; n = 3 mice per infected group; infection was intranasal with 4610 2 pfu of VSV. B, mRNA levels of select genes in brains (without OBs) of uninfected or intracranially VSV-infected wt and Ifit2 2/2 mice at 24 h post injection, plotted as mean+SD; n = 4 mice per infected group; infection was intracranial injection with 10 pfu of VSV. C, Ifit2, Ifit1, IFN-b and EMCV RNA levels in brains 4 days after EMCV infection (5610 2 pfu, n = 3 mice per infected group). (PDF) Figure S4 Region-selective induction of apoptosis in brains of intranasally VSV-infected Ifit2 2/2 mice. Ifit2 2/2 mice were i.n. infected with 4610 2 pfu of VSV; at 6 d.p.i., adjacent sections of fixed brains were labeled to detect apoptotic cells (TUNEL) or VSV P protein (immunohistofluorescence), n = 2 mice; only few regions such as striatum show positive TUNEL; infected wt brains and uninfected control brains of either genotype did not show appreciable signals, hence data not shown). (PDF) Figure S5 VSV yields from infected wt and Ifit2 2/2 MEF. Immortalized MEF were treated for 16 h with 10 U/ml IFN-b and infected with VSV at moi 10. 12 hours after infection, virus yields were determined by plaque assay. Results are plotted as mean+SD on log scale, representing one of two independent experiments. (PDF) Figure S6 Murine Ifit2 protein does not bind ppp-RNA. Single-stranded radiolabeled VSV leader RNAs (nt 1-18) with either 59-triphosphorylated or free 59-hydroxyl-ends (ppp-RNA or HO-RNA) were in vitro incubated with purified murine Ifit1 ( = P56) or Ifit2 ( = P54) proteins; formation of protein/RNA complex was detected by electrophoretic mobility shift assay. (PDF)
Table S1 Enhanced gene expression in brains incl. OBs of intranasally VSV-infected Ifit2 2/2 versus wt mice at 6 d.p.i. Wt or Ifit2 2/2 mice were intranasally VSV-infected with 4610 2 pfu, and at 2 or 6 d.p.i., brain (incl. OB) RNA expression profiles were obtained by microarray. Genes are ranked by their ''fold expression level in Ifit2 2/2 over wt at 6 d.p.i.''. Only genes with at least 3-fold higher expression level in Ifit2 2/2 are included. Note: The Ifit1/ISG56 probe of the Illumina mouse Ref-8 chip is defective and therefore the gene is not included in this list. (PDF) Immunogenetic Mechanisms Driving Norovirus GII.4 Antigenic Variation Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987–1997), contemporary (2004–2009), and broad (1987–2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294–298 and 368–372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393–395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution. Noroviruses (NoVs) are the leading cause of severe viral gastroenteritis and are responsible for 50% of all acute gastroenteritis outbreaks in the United States and Europe [1] . Although the severity of disease is usually moderate, lasting 1-3 days, infection can be especially virulent in young children, the elderly, and the immunocompromised, with the latter group experiencing chronic diarrhea and virus shedding for over a year [2] [3] [4] [5] [6] [7] [8] . Importantly, it is estimated that 200,000 people die each year from norovirus infections, primarily children in the developing world [9] . An effective vaccine would be particularly advantageous for the very young and aged populations, military personnel, children and healthcare providers, food handlers, cruise ship passengers, and populations of the developing world [10] . Immunotherapeutics are especially needed for treating immunosuppressed populations experiencing long-term infections with chronic diarrhea. The lack of understanding of the extensive antigenic relationships among the large number of norovirus strains and the complex relationship between host protective immunity and virus antigenic heterogeneity are the primary obstacles to norovirus vaccine development.
Noroviruses are ,38 nm icosahedral viruses with a ,7.5 kb single-stranded, positive-sense RNA genome that contains three large open reading frames (ORFs). ORF1 encodes the non-structural proteins, while ORFs 2 and 3 encode the major and minor capsid proteins respectively. Expression of the major capsid protein (ORF2) in Venezuelan equine encephalitis (VEE) virus or baculovirus results in the formation of virus-like particles (VLPs) composed of 90 copies of the major capsid protein dimer [11] . Noroviruses are grouped by the amino acid sequence of the major capsid protein: viruses with less than 14.3% difference are classified as the same strain, 14.3-43.8% difference as the same genotype, and 45-61.4% difference as the same genogroup [12] . Currently, noroviruses are grouped into five genogroups (GI-GV). Genogroups GI and GII are responsible for most human infections and are further subdivided into 8 and 21 different genotypes, respectively [1, 12] .
Structurally, the capsid monomer is divided into three domains. The shell domain (S) forms the core of the particle and the protruding domain (P) extends away from the core. The P domain is further subdivided into the P1 subdomain (residues 226-278 and 406-520) and the P2 subdomain (residues 279-405) [11] . The P2 subdomain is the most exposed region of the viral particle and is well positioned to interact with potential neutralizing antibodies and histoblood group antigen (HBGA) ligands [13] [14] [15] [16] [17] . Previous studies have shown that the P2 subdomain of the major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered carbohydrate ligand binding properties and antigenicity [13, [18] [19] [20] [21] [22] [23] .
For the past two decades, the majority of norovirus outbreaks have been caused by strains within the genogroup II, genotype 4 (GII.4 strains) subcluster. Between 1995 and 2006, four major norovirus pandemics associated with GII.4 strains were characterized using molecular epidemiologic methods. During the mid-1990's [24] strain US95/96 was responsible for ,55% of the norovirus outbreaks in the USA and 85% of the outbreaks in the Netherlands [25] . In 2002, the US95/96 strain was replaced by the Farmington Hills strain [26] , which was associated with ,80% of norovirus outbreaks [27] in the USA. In 2004, the Hunter GII.4 variant was detected in Australia, Europe, and Asia [28] [29] [30] . Hunter strains were largely replaced in 2006 by two new cocirculating GII.4 variants in the USA and Europe, Laurens (2006a) and Minerva (2006b) [5, 29, 31] . Although similar to Minerva, Apeldoorn317 (GII.4.2008, GenBank accession no. AB445395) represents a new evolutionary cluster in the phylogeny of the GII.4 viruses. Most recently, a new GII. 4.2006 4. variant, GII.4.2009 New Orleans, has been the predominate outbreak strain, although GII.4.2006 Minerva continues to circulate at low levels [1, 32] .
A variety of studies using time ordered human outbreak sera and mouse monoclonal antibodies support the hypothesis that the GII.4 noroviruses are undergoing antigenic variation and that this variation contributes to the emergence of new outbreak strains over time [13, 22, 33, 34] . However, the lack of a cell culture or small animal model for human norovirus cultivation restricts study of neutralization antibodies and epitopes. To circumvent this problem, highly informative in vitro assays have been developed that measure the ability of an antibody to ''block'' binding of a VLP to a carbohydrate ligand [13, 35, 36] . This assay is highly sensitive, as it differentiates between norovirus strains too similar to be distinguished by enzyme immunoassay (EIA). The clinical relevance of the blockade assay, as a surrogate neutralization assay, has been confirmed in both infected chimpanzees [37] and Norwalk virus-infected humans [10, 38] . For NoV strains that hemagglutinate RBCs, high blockade antibody titers that prevent hemagglutination also correlate with protection from disease in humans [39] . Using human norovirus outbreak sera, VLPimmunized mouse sera, and mouse mAbs [13, 33, 34] , the early GII. 4 strains (1987 and 1997) were antigenically indistinguishable from each other by EIA and surrogate neutralization assays. VLPs of strains circulating post 2002 had significantly less reactivity with sera directed against earlier strains and no reactivity to mouse mAbs directed to GII.4.1987 . Conversely, select mouse mAbs generated against GII.4.2006 reacted with VLPs that circulated only from 2002 or later. No blockade epitopes were found to be in common between GII. 4.1987 and GII.4.2006. Prior to this work, we and others had predicted GII.4 antibody epitopes using bioinformatic techniques. As expected, discreet amino acids are repeatedly identified as potential evolving epitopes. In particular, residues 296-298 and 393-395 are consistently identified as putative epitopes that change between epidemic GII.4 strains. Additional surface residues at 333, 340, 356, 368, 372, 407 and 412-413 were also predicted as potential antibody epitopes [13, 18, 19, 22, 28, [40] [41] [42] . These amino acids tend to cluster on loops and ridges of the P2 subdomain where antibody interaction would be most accessible. Beyond bioinformatics predictions, only a few studies have shown empirical evidence mapping GII.4 antigenic change. Allen et al. [42] compared the reactivity of five monoclonal antibodies against a pre and post-2002 epidemic GII.4 strain, and identified conformational epitopes composed of residues 294-296 and 393-395. The carbohydrate blockade potential of these antibodies was not reported, so the role of these sites in escape from herd immunity was unclear. However, the finding that residues 393-395 were antigenically important supported our previously published work identifying amino acid 395 as an antigenic determinant in the GII.4.2002 Farmington Hills strain [13] . Using mouse mAbs and molecular biology approaches to exchange predicted epitopes between GII.4 strain backbones, we have clearly identified amino acids 294, 296-298, 368 and 372 to comprise an evolving blockade epitope, as exchange of these amino acids from GII.4.1987 into GII.4.2006 conferred binding of mAbs that recognize GII.4.1987 but not GII.2006 [43] . Extending this approach, we have also confirmed amino acids 407, 412 and 413 to constitute a GII.4.2002 Farmington Hills-specific blockade epitope [17] . These empirical studies support the validity of using computational analysis to guide norovirus epitope studies.
Comparing reactivity of polyclonal sera collected from immunized mice and infected humans suggested antigenic variation within the GII.4 noroviruses [13, 33] . The development of mouse mAbs to different time-ordered GII.4 VLPs has greatly facilitated progress towards understanding the complex antigenic relations among these strains by clearly demonstrating antigenic variation over time and epidemic strain [34, 42, 43] . However, to maximally define the mechanistic relationships that exist between antigenic variation, immunity, and HBGA binding patterns noted in the GII.4 noroviruses in the context of natural infection history, the cross reactivity patterns, blockade responses, and epitope targets of human anti-GII.4 monoclonal antibodies are needed. Robust approaches exist for the isolation of human monoclonal antibodies that are elicited following virus infection. Using human PBMCs as
Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic sites. To define these sites we prepared the first human monoclonal antibodies (Hu mAbs) against GII.4 noroviruses by immortalizing memory B cells and characterizing antibody reactivity and carbohydrate blockade responses across a ,20 year panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the patient, human anti-GII.4 mAbs grouped into three VLP reactivity patterns: broad (1987-2009), contemporary (2004) (2005) (2006) (2007) (2008) (2009) , and ancestral (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) . We also identified the location of several defined epitopes which evolve over time and drive antigenic change. Our data indicate that antibodies targeting these sites block carbohydrate binding and likely select for the emergence of new strains that escape herd immunity and recognize unique carbohydrates for entry, resulting in new outbreaks of disease in vulnerable human populations. Importantly, these studies critically inform the rational design of broadly active vaccines and immunotherapeutics for the treatment of norovirus disease.
a source of memory B cells, we created a panel of human mAbs directed against GII.4 strains and compared the reactivity of these mAbs to a panel of time-ordered GII.4 VLPs using EIAs and surrogate neutralization assays. We identified one novel, broadly cross reactive antibody that differentially blocks GII.4.1987 through 2009 VLP interactions with carbohydrate ligands, a potential immunotherapeutic for the treatment of acute or chronic GII.4 disease. We also defined unique antibody interactions with two different surface exposed epitopes that evolve over time. Importantly, antigenic variation in one of these epitopes correlated with changing carbohydrate ligand binding patterns over time, supporting the proposed relationship between epitope escape from human herd immunity and changing HBGA usage for virus docking [13] . In addition to defining the first human monoclonal antibodies with therapeutic potential for treating acute and chronic NoV GII.4 infections, these data support the hypothesis that GII.4 norovirus evolution results in antigenic drift of neutralizing epitopes and consequently, antibody-driven HBGA receptor switching; thus, protective herd immunity is a driving force in norovirus evolution.
The development of mouse mAbs against different time-ordered GII.4 VLPs has greatly facilitated understanding of the complex antigenic relations between these strains by clearly demonstrating antigenic variation over time and by epidemic strain [34] . However, understanding of the human anti-GII.4 norovirus antibody response is essential not only for understanding the complex relationships between host immunity and virus antigenic change, but also for rational vaccine design based on defined neutralizing epitopes. Therefore, we developed a panel of human anti-GII.4 norovirus monoclonal antibodies to begin to characterize GII.4 antibody reactivity in the native virus host under natural infection conditions, noting that the norovirus preexposure histories in human volunteers are unknown and can only be inferred by human sera cross-reactive antibody binding and blockade patterns using time-ordered VLPs representing different outbreak and pandemic strains (Table 1) [13, 33] . Plasma and PBMC samples from 63 healthy individuals were collected in early 2009 and plasma binding titers (ED50) were measured by EIA against a panel of 6 different norovirus VLPs representing GI and GII strains ( Figure 1 ). The majority of tested samples reacted by EIA with variable ED50 titers to the panel of VLPs tested ( Figure S1 ). One sample (Donor NVB) was shown to react strongly with GII VLPs and was therefore chosen for further character- Figure 3 , and Figure S2 ). Significantly more antibody was needed to block GII.4.1997-PGM binding (EC50 0.4152 mg/ml) than GII.4.1987-PGM binding (EC50 0.3414 mg/ml) ( Figure 3B ) (p,0.05), supporting the hypothesis that subtle antigenic differences exist between these strains. 
The difference in blockade sensitivity of GII. 4.2006 and GII.4.2009 to NVB 97 provides the first evidence of subtle antigenic divergence between two Minerva variants, each of which caused widespread outbreaks globally [1] . This observation is further supported by Human mAbs NVB 111 and NVB 43.9 reactivity profiles. By single-dilution EIA, NVB 111 specifically reacted to 2006 but minimally with the 2009 variant of Minerva and other tested VLPs (Table 2 and Figure S2 ). Accordingly, NVB 111 required 13-fold more antibody to block GII.4.2009-PGM interaction (EC50 9.953 mg/ml) than it required to block GII.4.2006-PGM interaction (EC50 0.7376 mg/ml) ( Figure 5A and B) (p,0.05). In comparison, NVB 43.9 specifically recognized both the 2006 and 2009 Minerva variants by EIA (Table 2 and Figure S2 ). The interaction of both variants with PGM ligand was efficiently blocked by NVB 43.9 ( Figure 5C (Table 2 and Figure S2 ). Despite broad EIA reactivity, NVB 37.10 Figure 6D ). These three human mAbs indicate the existence of conserved GII.4 epitopes over the past twenty-five years and across three pandemic strains that could serve as targets for broad-based vaccine design. Importantly, NVB 71.4 represents the first potential, broad spectrum immune-therapeutic for any NoV.
In addition to VLP-PGM interaction blockade assay, human mAbs were also tested for blockade of VLP-synthetic biotinylated HBGA (Bi-HBGA) interaction and ability to block VLP hemagglutination of O+ RBCs. Regardless of substrate (PGM or Bi-HBGA), the dose-response profiles for all blockade antibodies and VLPs were similar (compare Figure 7E and F), although EC50 titers were relatively high (EC50 0.9753 and 1.581 mg/ml for NVB 37.10 and 61.3, respectively), compared to the amount of antibody needed to block GII.4.2009 by the strain-specific mAbs (EC50 0.1140 and 0.1732 mg/ml for NVB 43.9 and 97). Repeated testing with PGM as substrate did not replicate the findings with synthetic carbohydrate substrates ( Figure 6A and B ).
An additional measurement of antibody blockade ability uses RBCs as the VLP binding substrate. Previous work has demonstrated that Norwalk virus VLPs hemagglutinate (HA) O+ RBCs, that this interaction can be disrupted by antibodies found in polyclonal serum (hemagglutination inhibition; HAI), and that the HAI titer of serum correlates with antibody blockade of VLP-Bi-HBGA interaction [38, 39, 45] . To determine if these findings could be extended to study GII. 4 
Our previous work with mouse-derived anti-norovirus mAbs suggested that blockade epitopes are conformation dependent [17, 34] . To test the effect of protein conformation of human mAb binding, we used both Western blot and EIA analysis to compare antibody binding to GII.4.2006 VLPs and P proteins. P proteins of GII.4.2006 are composed of the C-terminal portion of the major capsid protein (amino acids 221-531) [21] . Expression of the P protein in E. coli results in small particle formation estimated to consist of 12 P dimers that reportedly maintains VLP characteristics in carbohydrate and antibody binding studies [46, 47] . None of the human anti-GII.4 mAbs recognized either the denatured VLP or P protein by Western blot analysis, suggesting that the epitopes for these antibodies are conformation dependent (data not shown). Surprisingly, only half of the mAbs that recognized GII.4.2006 VLP ( Figure 8A ) by EIA also recognized GII.4.2006 P protein by EIA ( Figure 8B ). NVB 71.4 and 61.3 extended their broad reactivity to P proteins, whereas NVB 37.10 did not, indicating that a minimum of three GII.4 cross-reactive epitopes must exist. NVB 97 also detected P protein by EIA. Neither of the Minerva variant mAbs recognized P protein even at protein concentrations 8-fold above standard EIA conditions (1 mg/ml coating protein). Further, all seven mAbs detected increasing concentrations of VLP in a linear dose response with signals saturating at 4 mg/ml of VLP when the mAb concentration was held at 1 mg/ml, which is our standard EIA antibody titer ( Figure 8A ). Antibody reactivity to the P protein saturated at a lower protein concentration than VLP and at optical densities below the linear range of the assay (compare Figure 8A and 8B), suggesting that even among the mAbs that bind to P proteins conformation-based epitopes may be limited in a way not observed with VLPs. These data suggest two important points. First, some of the mAb epitopes are highly sensitive to conformation, and secondly, that the principle P protein conformation is not identical to VLPs at least for some critical blockade epitopes.
The evolution of the GII.4 noroviruses was assessed over a 36year period of time by comparing strains from 1974 to 2010. In comparing these sequences, sites of variation in the P2 subdomain were noted, and these sites were mapped onto the crystal structure of the P-domain dimer for the 1997 strain VA387. Surfaceexposed sites of variation were then examined to determine which residues may be close enough to constitute a single epitope, and five epitopes were predicted based upon this variation ( Figure 9A , and [40] ).
Epitope A encodes significant amino acid changes over time and has also been demonstrated to be an evolving GII.4 blockade epitope using mouse mAbs ( Figure 9A, 9B and [43] ). Epitope A is conformational and is located on the top of the capsid proximal to the HBGA binding pocket. Six variable sites were close to each other in the region of this putative epitope, suggesting that these residues may work in concert to change the local structure of Epitope A. The variable, surface-exposed residues include positions 294, 296-298, 368 and 372. Of note, Epitope A is continuing to evolve in extant strains, whereby the amino acid at position 294 seems to vary extensively in strains from 2008-2010 (amino acid replacements P294A, P294S and P294T have been observed at this position). Epitope B was predicted based upon two variable residues at positions 333 and 382. While these residues are buried in the dimer interface between two chains, the patterns of variation at these sites suggest that they play an important role in the evolution of novel strains, perhaps by evolving replacements that allow the more surface exposed residues in other surface exposed epitopes to dramatically change the physiochemical properties of the amino acid replacements. Residues 340 and 376 make up the variable residues of putative Epitope C. This putative conformation dependent epitope is on the surface and lateral edge of the capsid and is directly proximal to the HBGA binding pocket, suggesting that this epitope may play a role in receptor switching along with Epitope D. Epitope D is comprised Table S2 . doi:10.1371/journal.ppat.1002705.g007 of three variable residues from positions 393-395. In the first reported crystal structure for the GII.4 noroviruses, this region was reported to be a secondary HBGA binding site [16] . However, the location of this epitope on the surface of the capsid, directly proximal to the HBGA binding site, suggests that it likely plays a role in both receptor switching and in escape from herd immunity and perhaps both, simultaneously [13, 21, 40, 43] . Epitope D is close enough to the HBGA binding pocket to contribute to or inhibit carbohydrate binding, and yet variable enough to suggest that it is targeted by the immune response. Putative Epitope E is comprised of variable residues 407, 412 and 413, which are surface exposed regions lateral to the HBGA binding pockets and the other epitopes. These residues vary with every major epidemic strain after 2002, suggesting that it is a hot spot for the emergence of immunologically novel GII.4 strains. Epitope E is a GII.4.2002 blockade antibody epitope [17] . This putative epitope is lateral to the HBGA binding pockets indicating that antibodies are targeting interior regions below the capsid surface, which suggests that other epitopes may be present in the P1 subdomain. A few variable residues do not necessarily identify the boundaries of a putative epitope. Moreover, it is nearly impossible to predict the surface area of a putative epitope by sequence analysis alone. Therefore, we expanded the putative epitopes to include residues within 8 Å of the variable sites from which the epitopes were predicted ( Figure 9B ).
The described mAbs indicate at least five unique or overlapping GII.4 blockade epitopes with different specificities: 1) early GII.4 strain specific, 2) contemporary GII.4 strain specific, 3) Minervavariant strain specific, 4) genogroup II strain specific, and 5) GII.4 strain specific. Using capsid sequences as a guide, mutant VLPs were designed to contain chimeric combinations of the predicted evolving GII.4 epitopes ( Figure 10) All epitope exchange VLPs were morphologically intact by electron microscopy visualization and retained the ability to bind PGM ( Figure 10A and B, [43] ), confirming chimeric VLP structural integrity.
Epitope mutant VLPs were compared to wild type strain VLPs for reactivity to the donor plasma sample. Consistent with high EIA reactivity to GII.4.1987 and 2006 VLPs (Table 2 and Figure  S2 ), donor plasma reacted across the panel of epitope-exchange mutant VLPs ( Figure 10B) . Donor plasma was able to block each epitope-exchanged VLP binding to PGM ( Figure 11A and C) . Exchange of all of the epitopes, except Epitope D into either backbone and GII.4.1987 C into GII.4.2006 resulted in significantly different EC50 values compared to the parental strains ( Figure 11B , 11D, and Table S4 ) (p,0.05). Only the exchange of Epitope A between the backbones resulted in an exchanged blockade phenotype, as observed with epitope-specific mAbs [43] . Exchange of Epitope A between the two parental backbones resulted in a chimeric VLP (GII.4.1987/2006A) that was blocked with significantly less plasma than the parental GII.4.1987 (EC50 0.0167 mg/ml compared to 0.0673 mg/ml) (p,0.05) and a chimeric VLP (GII.4.2006/1987A) that was blocked with signif- Figure S2 and Table 2 ). Bars are SEM. doi:10.1371/journal.ppat.1002705.g008 Table S4 ) (p,0.05). In this individual, these data suggest that Epitope A may be an important evolving GII.4 neutralization epitope as the blockade response is significant enough to be detected in the polyclonal antibody response.
Agreeing with the assumption that epitope-exchange mutants are unlikely to identify epitopes of cross-reactive mAbs, NVB 37.10, 61.3 and 71.4 reacted with the entire panel of chimeric VLPs by EIA ( Figure 10B ). In contrast, each of the strain-specific mAbs displayed differential EIA reactivity to exchanged epitopes Table S4 ). Exchange of the other GII.4.1987 epitopes did not eliminate NVB 114 blockade potential ( Figure 12A and B) Figure 13A and B) . Binding was not restored to wild type levels as the EC50 of GII.4.1987/2006D was 0.6349 mg/ml, significantly higher than the blockade EC50 for GII.4.2006 -PGM (0.1195 mg/ml) ( Figure 13B and Table S4 ) (p,0.05). Both EIA and blockade data clearly indicate that Epitope D is critical for the binding of NVB 97 and suggest that amino acids 393-395 are important components of a GII.4 evolving blockade epitope in addition to modulating VLP-carbohydrate ligand binding [13, 21] . Interestingly, Epitope D has a single amino acid change in GII.4.2004, explaining the highly conserved binding and blockade responses noted across GII.4.2005 to 2009 VLPs, while ancestral strains display significant antigenic variation across these residues. Together, these data map the GII.4 evolving blockade epitopes recognized by each of the four strain-exclusive human mAbs described in this study ( Figure 14 ).
Noroviruses are recognized as a leading cause of viral foodborne gastroenteritis. With the successful vaccination program being developed against rotavirus, focus is shifting to norovirus as the primary causative agent of severe childhood diarrhea resulting in a yearly estimate of 1.1 million episodes of pediatric gastroenteritis in developed nations and 218,000 deaths in developing nations [9] . The elderly and immunocompromised also suffer sometimes life-threatening or chronic long-term norovirus diarrheal disease characterized by malnutrition and dehydration [48] . In some HIV infected patients, chronic norovirus diarrheal disease is associated with persistent norovirus infection [49] . The economic disease burden of a norovirus outbreak within a care facility has been estimated at over $657,000 for a single event [50] . These statistics emphasize the critical need for a norovirus vaccine. Although recent reports strongly support the development and use of an efficacious norovirus vaccine in humans [38, 51] , a primary obstacle to a successful vaccine is the lack of a definitive correlate to protection coupled with the extreme antigenic variation across the many norovirus strains. In fact, the existence of long-term protective immunity to norovirus infection remains controversial within the field [52] . Human challenge studies conducted before molecular diagnostics of infection and refined immune response assays had indicated that some volunteers could be reinfected with the same norovirus strain, suggesting that norovirus infection did not induce long-term protection [53] . However, recent reports identifying immune responses in norovirus-challenged but uninfected volunteers [44, 54] have necessitated qualification of these early observations to acknowledge that the findings may be compromised by assay limitations and/or the overwhelming challenge dose in comparison to the very low norovirus infectious dose [44, 54] . Clearly defining the relationships between pre-exposure history, blockade antibody responses, T cell immunity, virus evolution, and the components of protective immunity represent key challenges for future vaccine and therapeutic design.
In addition to the clinical applications of therapy and diagnosis, monoclonal antibodies have also proven to be superior tools for studying viral antigenicity, evolution, and for the treatment of acute viral disease in humans [13, 34, 55, 56] . Characterization of neutralizing mAb escape-mutants has been fundamental to identifying epitopes associated with virus receptor usage, pathogenesis, and fitness [57] [58] [59] . In this manuscript, we isolated and characterized the first human mAbs against noroviruses, derived from a healthy donor whose pre-exposure history was unknown. The number of unique GII.4 human monoclonal antibodies in this patient accurately reflects the high prevalence of GII.4 norovirus infection seen in human populations over the past 25 years. Moreover, distinct antibody cross reactivity patterns support the hypothesis that the GII.4 genotype is undergoing antigenic variation which not only correlates with loss of antibody blockade activity and emergence of new epidemic norovirus strains, but also changing carbohydrate ligand binding patterns over time.
Importantly, antibody-mediated antigenic drift of GII.4 strains coupled with both mucosal IgA and T cell responses in challenged but uninfected volunteers strongly support the existence of longterm protective immunity against norovirus strains. In fact, the unique antibody reactivity patterns characterized herein are most likely explained as an archeological immune record of successive waves of contemporary GII.4 infections in this individual over time, implying that successful vaccine design is possible, as previously proposed by our group and others [37, 44, 54] .
Previously, we have identified two immunological responses associated with protection from infection in norovirus-challenged volunteers. An early (day 1-3) post-exposure salivary IgA response in genetically susceptible volunteers correlated with protection from Norwalk virus infection [54] and an early T h 1 response correlated with protection from Snow Mountain virus infection [44] . Historically, the role of IgG in norovirus protection has been unclear. By adulthood, .90% of the population [48] is positive for anti-norovirus IgG, and norovirus strains within a genogroup share a high degree of antigenic cross-reactivity as measured by EIA [44, 60] . These facts likely skew functional interpretations of the role of serum IgG titers on susceptibility to infection and/or infection outcomes. Although carbohydrate ligand blockade antibody responses have been suggested as correlates of protective immunity [13, 35, 36] , it wasn't until recently that these blockade responses were correlated definitively with protection from clinical disease and infection [10, 38] . Importantly, our surrogate neutralization assay is specific enough to differentiate GII.4 norovirus strains too similar to be distinguished by EIA but different at key antigenic sites. While human re-challenge studies using the same viral inoculum are necessary to confirm an association between a blockade IgG response and protection from repeat norovirus infection, our findings support the clinical relevance of the antibody blockade assay as a correlate of protective immunity [38] .
Monoclonal antibodies coupled with the blockade assay are powerful tools for elucidating the antigenic relationship between GII.4 strains. While mouse mAbs provide insight into GII.4 antigenic structure, data in this manuscript argues that human mAbs offer considerable advantages, including: a) immunologic record of B-cell immunity following repeat GII.4 infection, b) relationships between antibody blockade responses and antigenic variation, c) relationships between immune selection and carbohydrate ligand reactivity, d) relationships between early infection and downstream immunity, and e) epitope mapping. We focused on anti-GII.4 mAbs because of the clinical relevance of the GII.4 strains. One key finding is a direct relationship between anti-GII. (Figure 3) . Given the lack of cross reactivity with later strains, the most likely explanation is that NVB 114 was derived from a long-term memory plasma cell that had been elicited some 12-22 years earlier. In support of this idea, examination of human monoclonal antibodies against 1918 H1N1 influenza also identified antibody variants that recognized ancestral or contemporary isolates [61] . The exclusive blockade reactivity of NVB 114 with GII.4.1987 and 1997 supports potential long-term protective immunity. Further, NVB 114 is the first antibody identified to clearly demonstrate antigenic difference between GII. 4.1987 and GII.4.1997 , suggesting that antigenic variation may have subtly contributed to the emergence of the GII.4 US 95/96 pandemic strain from ancestral strains.
Human monoclonal antibodies clearly identified two evolving epitopes on the surface of the GII.4 VLP. Epitopes A and D were both confirmed as evolving GII.4 antibody blockade epitopes using chimeric VLPs containing a mixture of epitopes derived from early or contemporary strains. We recognize that Epitopes A and D have not been structurally defined as epitopes, supporting the need for structural studies to define the exact mAb binding site on the VLPs. Moreover, we focused on discrete regions of varying residues and all of the residues within 8 Å (representing approximately 201 Å 2 ) to the primary sites that may be influenced directly by an amino acid replacement ( Figure 9B ). However, Ab binding sites have been reported to be much larger (700-800 Å 2 ), suggesting that the epitopes that we have predicted may actually work in concert to form larger Ab recognition sites. By expanding the putative epitopes, we identified other residues that were less variable; however, the exact role that these varying residues play in evolution is less clear. In some cases variation may be required to encode changes that are necessary for the replacement at a primary site. In addition, all of our structural analyses have been conducted using models of the P dimer, representing about 1/90 th of the VLP structural surface. Interactions between the epitopes in the context of the superstructure have not been determined. Therefore, these observations indicate that the complex nature of the NoV Ab epitope requires further research to define the specific boundaries and residues that regulate Ab binding.
The prediction of five putative epitopes allowed us to gain several important insights into GII.4 norovirus evolution: 1) Discrete sites of variation occur on the GII.4 norovirus capsid, either directly on the surface or lateral to the HBGA binding sites; 2) Secondary variable sites are within 8 Å of the primary variable sites, and these secondary sites could also contribute to epitope remodeling; 3) Many of the putative expanded epitopes overlap, suggesting that two or more highly variable epitopes may work in concert to escape from an antibody response; 4) Putative epitopes that are buried may exert an effect on the structure by altering the interior fold space, allowing unconventional replacements to be tolerated; 5) An underlying amino acid network likely preserves the functional core of the capsid proteins by regulating the variable residues above them; and 6) Escape and HBGA binding may be intimately linked via the underlying regulatory network of amino acids that preserve the functional integrity of the capsid core.
Epitope A, which likely includes varying amino acid residues 294, 296-298 and 368 and 372 and potentially other undefined nearby residues, has been mapped as a blockade epitope in both GII Figure 9A ). At this time it is unclear how many and which amino acids in Epitope A are needed to mediate an escape mutant phenotype that is completely resistant to GII.4.2006 antibody blockade. However, it is clear that Epitope A varies, and that the site is conserved as a major target for blockade antibody response between 1987 and 2009. Although correlative, comparisons of Epitope A variation along with residues that are proximal to those that appear to be evolving over time suggest that changes at positions 292, 293, 294, 295, 296, 297, 298, 300, 365, 367, 368 and 372 might contribute to an escape phenotype, with residues 294, 296, 297, 298, 300, 368 and 372 playing direct roles in this variation ( Figure 9 ). However, the minor replacements at other positions are likely essential for remodeling the local structural neighborhood such that more profound changes can be tolerated. Supporting the sensitivity of epitopes to the local environment, mAbs that recognized Epitope A differentiated between GII.4.2006 VLPs and GII.4.2006 P dimers. P protein (P dimer) is a dimeric, truncated form of the major capsid protein composed of residues 214-539 [62] . P-dimers have been widely used to determine the crystallographic structure of NoV-HBGA interactions and are considered accurate reflections of the VLP surface topology [16, 21, 63] . P -particles can assemble as higher ordered structures composed of varying copies of the P dimer [46, 64] . These subviral particles are reported to have similar characteristics to VLPs [62, [65] [66] [67] and have been proposed as a candidate vaccine platforms [68] . To our knowledge, this is the first immunologic characterization of P-dimers vs. VLPs using monoclonal antibodies derived from human infections. Here, P-dimers derived from GII.4.2006 lost binding of mAbs NVB 111 and 43.9, the mAbs that recognize Epitope A in GII. 4.2006 . P dimer binding to the Epitope D binding mAb NVB 97 and the broadly cross-blockade mAb NVB 71.4 were retained. Recently, P-particle vaccines were shown to be less robust at inducing strong blockade responses, as compared with intact VLP [69] , perhaps because of the loss of blockade Epitope A in this higher ordered structure. While speculative, it is recognized that virions ''breathe'' suggesting that the possibility that P-dimers and P-particles may become ''locked'' in a slightly less immunologically reactive state that affects some but not all blockade epitopes on the virion surface [70, 71] . These data absolutely underscore the critical importance of determining the structures of several of these human mAbs with their appropriate GII.4 VLP epitopes by either cryoEM or crystallography, for informing targeted mutagenesis to identify the role of key residues in regulating antigenicity and antibody escape.
Epitope D is a conformational epitope comprised of varying amino acids 393-395 and likely other nearby residues that are less clearly defined; however, additional mapping and crystallographic studies will be needed to clarify this epitope structure. With the emergence of the pandemic GII. 4 (Figure 4) . Previously, these residues have been implicated in regulating norovirus VLPcarbohydrate ligand binding interactions [13, 21, 43] . The identi-fication of Epitope D as a human antibody blockade epitope that changes over time provides direct support for our previous hypothesis that escape from protective herd immunity may drive changes in carbohydrate ligand binding affinities over time and potential retargeting of virus infection in different human populations [13, 40] .
These data indicate that like influenza, a successful norovirus vaccine regimen will require periodic population sampling to identify future strains for inclusion into the next year's vaccine formulation not unlike the strategy employed by the Influenza Virus Global Surveillance Program. Norovirus population sampling has already begun as monitoring systems for detection of NoV infections have been established in the United States, Europe, and Japan. Identification of GII.4 evolving antigenic epitopes furthers our understanding of norovirus pathogenesis and provides target epitopes that may be useful for surveillance and prediction of new strain emergence. Identification of GII.4 conserved epitopes also informs diagnostic and potential therapeutic reagent development and design. Monoclonal antibodies NVB 37.10, 61.3 and 71.4 all recognize epitopes conserved among the GII.4 strains from 1987 through 2009. NVB 37.10 and 61.3 have enhanced GII VLP recognition, binding not only GII.4 VLPs but also other GII VLPs, but are unable to block VLP-PGM interaction. The high conservation of the NVB 37.10 and 61.3 epitopes suggest that these epitopes are highly resistant to antigenic variation within the GII strains, making these mAbs potentially valuable diagnostic reagents as GII strains cause up to 95% of norovirus outbreaks [27, 72] . The unidentified epitope for NVB 71.4 is clearly different from the epitopes recognized by NVB 37.10 and 61.3 and is conserved throughout the GII.4 strains. NVB 71.4 did not recognize any non-GII.4 VLPs but, importantly, it exhibited blockade activity for the entire panel of timeordered GII.4 VLPs with PGM and Bi-HBGAs. Emphasizing the difference between the two quantitative blockade assays and the qualitative HAI assay, GII.4.2002 HA was not inhibited by NVB 71.4. Noting that blockade assays are not true measurements of neutralization, NVB 71.4 has potential as a therapeutic reagent based on its broad GII.4 blockade potential and the fact that it is by nature a human antibody. Clearly, the effectiveness of NVB 71.4 at preventing or treating illness can only be determined empirically. Although one of the blockade-epitope specific mAbs with lower EC50 values/steeper Hill constants may be more effective at select strain neutralization, the breadth of strains neutralized is likely to be limited for these mAbs. There are a number of viral diseases currently being treated with mAbs including RSV, CMV and enterovirus; however, only the anti-RSV humanized mAb palivizumab has FDA approval for prophylactic use in humans [73, 74] . In outbreak settings or in chronically infected patients, an anti-NoV mAb that could be delivered before symptoms begin and protect from illness could be very useful in care facilities, the military and the cruise industry. Given the acute clinical disease window, it is less likely that therapeutic antibodies will provide relief in those individuals experiencing acute infections, however, therapeutic antibodies may offer opportunities for ameliorating symptomatic disease in chronic infections. The discovery of broadly cross reactive and cross blockade human GII.4 mAbs dictates the need for a new approach to map epitopes. Our current experimental approach was designed to identify GII.4 epitopes that change over time and provide insight into broadly conserved epitope locations. Because the identification of the epitopes recognized by NVB 37.10, 61.3, and 71.4 has important implications for successful vaccine design, new panels of mutated VLPs and other approaches will be needed to characterize these epitopes in the future.
GII.4 NoVs are significant human pathogens that cause considerable morbidity and mortality, worldwide. The development of mouse mAbs to different time-ordered GII.4 VLPs has greatly facilitated progress towards understanding the complex antigenic relationships between these strains by clearly demonstrating antigenic variation over time and epidemic strain [34] . Here we have expanded these observations using human anti-GII.4 mAbs isolated from a healthy adult donor, who has likely experienced multiple norovirus infections throughout his/her lifetime. The identification of highly significant, varying antigenic epitopes that influence VLP-carbohydrate ligand interaction provides important new insights into vaccine design and the development of therapeutics that target norovirus virions. For example, these antibodies represent the first anti-norovirus human mAbs to be characterized, and they confirm findings from studies using mouse mAbs supporting antigenic drift and its linkage with varying carbohydrate ligand binding profiles within the GII.4 noroviruses. Further, we have demonstrated that the GII.4 NoV varying epitopes can be exchanged between time-ordered VLPs, providing a robust platform for expanding the antigenic and blockade cross reactivity of future vaccine candidates. Using this approach, we have identified two surface-exposed antibody blockade epitopes that vary over time and were differentially recognized by four of the seven human mAbs. We also identified three antibodies which recognize either overlapping or three unique highly conserved epitopes within the GII.4 VLP. These data continue to support the hypothesis that norovirus long-term protective immune responses are elicited following acute infection, a concept essential for effective vaccine design. We anticipate that a full understanding of the varying antigenic and blockade epitopes of GII.4 NoVs may not only help to predict the emergence of new epidemic strains but simultaneously identify key reformulations in vaccine design that will protect public health against contemporary and emerging epidemic strains in the future.
A diverse panel of VLPs representing G1 and GII norovirus strains and epitope mutants was assembled as previously reported [13, 17, 75] . To design epitope exchange chimeric VLPs, we first identified surface exposed residue clusters that varied over time. Then, we synthesized a series of chimeric GII.4 ORF2 genes that exchanged ''putative'' epitopes between GII. 4.1987 and GII.4.2006 VLPs [43] . For all constructs except GII.4.2009 ORF2 [17] , the synthetically derived constructs were inserted directly into the VEE replicon vector for the production of virus replicon particles (VRPs) as previously described by our group. VLPs were expressed in VRPinfected BHK cells and purified by velocity sedimentation in sucrose and stored at 280uC. The GII.4.2009 (New Orleans [17] ) VLPs were expressed in the baculovirus system and purified by cesium chloride gradient centrifugation and were the kind gift of Dr. Jan Vinje, Centers for Disease Control and Prevention, Atlanta, GA. VLP protein concentrations were determined by the BCA Protein Assay (Pierce, Rockford, IL). VLP preparation purity averaged .80% by SDS-Page analysis.
In early 2009, following written consent, blood samples from 63 donors were collected from adult healthy donors at the Lugano and Basel Blood banks (Switzerland). Peripheral blood mononuclear cells (PBMCs) and plasma were isolated and cryopreserved. On the day of use, PBMCs from Donor 302898 (Figure 1 ), an individual born in 1948, were thawed and IgG + memory B cells were isolated using CD22 microbeads (Miltenyi) followed by cell sorting, as described [76] . Cells were immortalized at 5 cells/well in multiple cultures using EBV in the presence of CpG oligodeoxynucleotide 2006 (Microsynth) and irradiated allogeneic PBMC. After 2 weeks, culture supernatants were screened for the presence of norovirus-specific mAbs by EIA against VLPs and positive cultures were cloned by limiting dilution. Antibodies were recovered from supernatants and purified using protein A affinity chromatography and finally desalted against PBS using a HiTrap FastDesalting column.
Human mAb reactivity was determined by EIA, as reported [34] . Briefly, plates were coated at 1 mg/ml VLP in PBS before the addition 1 mg/ml purified IgG or donor plasma (0.2%). Primary antibody incubation was followed by anti-human IgG-alkaline phosphatase and color development with pNPP substrate solution (Sigma Chemicals, St. Louis, MO). Each step was followed by washing with PBS-0.05% Tween-20 and all antibodies were diluted in 5% dry milk in PBS-0.05% Tween-20. Data shown represent the average of at least three replicates and are representative of similar data from at least two independent trials. Establishment of EIAs using new mAbs included PBS-coated wells as negative controls and polyclonal anti-norovirus human sera as positive controls. Antibodies were considered positive for reactivity if the mean optical density after background subtraction for VLPcoated wells was greater than three times the mean optical density for PBS-coated wells [34] . For screening donor plasma samples, the binding titers of plasma to respective coated VLPs were determined by EIA as described above by measuring the plasma dilution required to achieve 50% maximal binding (ED50). EIA reactivity to GII.4.2006 P protein (amino acids 221-531 [21] ) was measured similarly to reactivity to VLP. GII.4.2006 P protein was the kind gift of B.V. Prasad, Baylor College of Medicine, Houston, TX).
Pig Gastric Mucin Type III (PGM) (Sigma Chemicals) has been validated as a substrate for NoV VLP antibody-blockade assays [17] . PGM contains relatively high levels of H and A antigen and more moderate levels of Lewis Y antigen [17] . All of the GII.4 VLPs used in the blockade assays in this study bind to both PGM and synthetic Bi-HBGA, and binding to PGM is consistent with synthetic Bi-HBGA binding profiles for a-1,2-fucose (H antigen) and a-1,4-fucose (Lewis antigen) containing molecules [13, 17, 77] . For blockade assays, PGM was solvated in PBS at 5 mg/ml and coated onto EIA plates at 10 mg/ml in PBS for 4 hours and blocked over night at 4uC in 5% dry milk in PBS-0.05% Tween-20. VLPs (0.5 mg/ml) were pretreated with decreasing concentrations of test mAb or donor plasma for 1 hour at room temperature before being added to the carbohydrate ligand-coated plates for 1 hour. Bound VLP was detected by a rabbit anti-GII norovirus polyclonal sera made from hyperimmunization with either GII.4.2009 or a cocktail of GII. 4.1997 , GII.3.1999 , GII.1.1976 , and GII.2.1976 VLPs, followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). The percent control binding was defined as the binding level in the presence of antibody pretreatment compared to the binding level in the absence of antibody pretreatment multiplied by 100. All incubations were done at room temperature. Each step was followed by washing with PBS-0.05% Tween 20 and all reagents were diluted in 5% dry milk in PBS-0.05% Tween-20. All antibodies were tested for blockade potential against the panel of GII.4 VLPs at two-fold serial dilutions ranging from 0.08 to 2 mg/ ml. Additional concentrations of blockade antibodies were tested if needed to complete the sigmoid dose-response curve. Blockade of synthetic Bi-HBGAs (Glycotech, Gaithersburg, MD) assays were done as described for PGM with the following exception. Bi-HBGAs were bound to Neutri-avidin coated plates (Pierce) at 10 mg/ml for one hour prior to the addition of 1 mg/ml VLP for 1.5 h. Reported mean % control binding reflects the results of at least two independent experiments with each dilution tested at least in duplicate. An antibody was designated as a ''blockade'' antibody for a VLP if at least 50% of control binding was inhibited by 2 mg/ ml antibody. Blockade data were fitted and EC50 values calculated using Sigmoidal dose response analysis of non-linear data in GraphPad Prism 5 (www.graphpad.com). EC50 values between VLPs were compared using the One-way ANOVA with Dunnett post test, when at least three values were compared or the unpaired t-test when two values were compared. A difference was considered significant if the P value was ,0.05. To test for antibody binding that prevents detector antibody from binding to the VLP instead of the VLP binding to the PGM, select blockade assays are performed without using a detector antibody and instead developed directly with an anti-human IgG-HRP. Antibodies tested this way give two responses; 1) a bell-shaped response curve for antibodies that are blockade and 2) a sigmoidal shaped curve for antibodies that are not blockade. These data indicate that it is the amount of human mAb that is directly blocking the VLP from binding to PGM. Of note, VLP concentrations in blockade assays are in the low nanomolar range and therefore cannot discriminate between antibodies with sub-nanomolar affinities.
HAI assays were performed as reported [38, 39, 45] . VLPs at 50 ng/reaction were pretreated with antibody as described above for the blockade assays before addition to O+ RBCs at 4uC, pH 5.5. An HAI titer was determined as the lowest antibody concentration that completely prevented NoV VLP-induced HA by visualization.
The amino acid sequences of GII. 4.1987 , GII.4.2002 , and GII.4.2006 capsids were individually aligned to the VA387 P domain sequence using Clustalx1.86 [78] , and the GII.4.2002 domain dimer X-ray crystal structure (PDB accession: 2OBT) [16] was used as a template for generating homology models. Homology models were generated using the program Modeller available via the Max Planck Institute Bioinformatics Toolkit (http://toolkit.tuebingen.mpg.de/). The structural models were analyzed and compared, and figures were generated using Mac Pymol (Delano Scientific).  Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection. During their replication in host cells, RNA and DNA viruses generate RNA intermediates, which elicit antiviral responses mostly through type-I interferon (IFN) production [1, 2] . Several families of proteins are known to sense double-stranded RNA (dsRNA), including endocytic Toll-like receptor 3 (TLR3) [3] , the dsRNA-dependent protein kinase (PKR) [4] and the interferoninducible 29-59-oligoadenylates and endoribonuclease L system (OAS/2-5A/RNase L) [5] . Viral dsRNA and the synthetic dsRNA analog polyriboinosinic:polyribocytidylic acid (poly I:C) are also detected by different cytosolic DExD/H box RNA helicases such as the melanoma differentiation-associated gene 5 (MDA5), DDX1, DDX21, and DHX36, which, once activated, trigger indirectly the phosphorylation and the nuclear translocation of transcription factors such as IRF-3 and IRF-7, resulting predominantly in abundant type-I IFN and pro-inflammatory cytokines production by the infected cells [1, 6, 7] .
Alphaviruses such as Chikungunya virus (CHIKV) are small enveloped viruses with a message-sense RNA genome, which are known to be strong inducers of type-I IFN in vivo [8, 9] , a key response for the host to control the infection [10, 11, 12] . In vitro, however, response to RNA viruses is heterogeneous, since Sindbis virus (SINV), do not elicit detectable IFN-a/b production in infected of murine embryonic fibroblasts (MEFs) [13] . The specific points of blockage of type-I IFN production during infection are still not well delineated, but SINV and other alphaviruses could antagonize IFN production by shut-off of host macromolecular synthesis in infected cells [14, 15, 16] . Recently, human fibroblasts infection by CHIKV was shown to trigger abundant IFN-a/b mRNA transcription, while preventing mRNA translation and secretion of these antiviral cytokines [13, 15] . Contrasting with these reports, other groups using different CHIKV strains have observed abundant type-I IFNs release in the culture supernatants of CHIKV-infected human monocytes [17] , human lung cells , human foreskin fibroblasts and MEFs [10] . Type-I IFN stimulation of non-hematopoietic cells has also been shown to be essential to clear infection upon CHIKV inoculation in mouse, but CHIKV was found to be a poor inducer of IFN secretion by human plasmacytoïd dendritic cells [10] . Thus, great disparities regarding alphavirus-triggered IFN responses exist between viral strains and the nature of host cells or animal models.
Once bound to their receptor on the cell surface (IFNAR), type-I IFNs activate the Janus tyrosine kinase pathway, which induces the expression of a wide spectrum of cellular genes including Pkr [18] . These different genes participate in the cellular defense against the viral infection. PKR is a serine-threonine kinase that binds dsRNA in its N-terminal regulatory region and induces phosphorylation of translation initiation factor 2-alpha (eIF2a) on serine 51 [19, 20] , leading to protein synthesis shut-off and apoptosis. PKR has been also been shown to participate in several important signaling cascades, including the p38 and JNK pathways [21] , as well as type-I IFN production [22, 23] . Inhibition of translation, IFN responses and triggering of apoptosis combine to make PKR a powerful antiviral molecule, and many viruses have evolved strategies to antagonize it [24, 25] . Interestingly, several positive RNA-strand viruses (eg. Togaviridae or Picornaviridae) have been shown to activate PKR, resulting in phosphorylation of eIF2a and host translation arrest [26] , while viral mRNA could initiate translation in an eIF2-independent manner by means of a dedicated RNA structure, that stalls the scanning 40S ribosome on the initiation codon [25] .
Despite the existence of these viral PKR-evading strategies, the importance of PKR for type-I IFN production has been strongly debated over the years and even considered dispensable since the discovery of the innate immunity function of the DExD/H box RNA helicases [27, 28] . However, several PKR-deficient cell types have reduced type-I IFN production in response to poly I:C [23, 29, 30] , while PKR was demonstrated to be required for IFNa/b production in response to a subset of RNA viruses including Theiler's murine encephalomyelitis, West Nile (WNV) and Semliki Forest virus (SFV), but not influenza, Newcastle disease, nor Sendai virus [31, 32, 33, 34] . These studies raise therefore the possibility that some but not all viruses induce IFN-a/b in a PKRdependent and cell specific manner. Infection of PKR or RNAse L deficient mice demonstrated that these enzymes were not absolutely necessary for type I IFN-mediated protection from alphaviruses such as SFV or WNV, but still contributed to levels of serum IFN and clearance of infectious virus from the central nervous system [25, 35] . Similarly, deficient mice for both PKR and RNAse L showed no increase in morbidity following SINV infection, although, like during WNV infection, increased viral loads in draining lymph nodes were observed [35, 36] . These observations support a non-redundant and cell specific role for these enzymes in the amplification of type-I IFN responses to viral infection more than in their initiation [31, 32, 35] . Nevertheless, the exacerbated phenotypes observed upon alphavirus infection of mice deficient for type-I IFN receptor (IFNAR), underlines the limits of the individual contributions of PKR and RNAse L to the anti-viral resistance of adult animals [10, 35, 36] .
In addition to dsRNA detection, different stress signals trigger eIF2a phosphorylation, thus attenuating mRNA translation and activating gene expression programs known globally as the integrated stress response (ISR) [37] . To date, four kinases have been identified to mediate eIF2a phosphorylation: PKR, PERK (protein kinase RNA (PKR)-like ER kinase) [38] , GCN2 (general control non-derepressible-2) [39, 40] and HRI (heme-regulated inhibitor) [41, 42] . ER stress-mediated eIF2a phosphorylation is carried out by PERK, which is activated by an excess of unfolded proteins accumulating in the ER lumen [38] . Activated PERK phosphorylates eIF2a, attenuating protein synthesis and triggering the translation of specific molecules such as the transcription factor ATF4, which is necessary to mount part of a particular ISR, known as the unfolded protein response (UPR) [43, 44] . Interestingly DNA viruses, such as HSV, that use the ER as a part of its replication cycle, have been reported to interfere with the ER stress response through different mechanisms, such as the dephosphorylation of eIF2a by the viral phosphatase 1 activator, ICP34.5 [45, 46] .
We show here, using SUnSET, a non-radioactive method to monitor protein synthesis [47] , that independently of any active viral replication, cytosolic poly I:C detection in mouse embryonic fibroblasts (MEFs) promotes a PKR-dependent mRNA translation arrest and an ISR-like response. During the course of this response, ATF4 and its downstream target, the phosphatase-1 (PP1) cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34, also known as MyD116 and Ppp1r15a) [48] , are strongly up-regulated. Importantly, although the translation of most mRNAs is strongly inhibited by poly I:C, that of IFN-ß and Interleukin-6 (IL-6) is considerably increased under these conditions. We further demonstrate that PKR-dependent expression of GADD34 is critically required for the normal translation of IFN-ß and IL-6 mRNAs. We prove the relevance of these observations for antiviral responses using CHIKV as a model: we show that GADD34-deficient MEFs are unable to produce IFN-ß during infection and become permissive to CHIKV. We further show that CHIKV induces 100% lethality in 12-day-old GADD34-deficient mice, whereas WT controls do not succumb to infection. Our observations demonstrate that induction of GADD34 is part of the anti-viral response and imply the existence of distinct and segregated groups of mRNA, which require GADD34 for their efficient translation upon dsRNA-induced eIF2a phosphorylation.
We monitored protein synthesis in MEFs and NIH-3T3 cells after poly I:C stimulation, using puromycin labeling followed by immunodetection with the anti-puromycin mAb 12D10 [47] . Poly I:C delivery to MEFs and NIH-3T3, rapidly and durably inhibited protein synthesis, concomitant with increased eIF2a phosphorylation (P-eIF2a) ( Fig. 1A and Fig. S1A ). In MEFs, a strong eIF2a phosphorylation was observed after 4 h of poly I:C treatment, followed by a steady dephosphorylation at later times (Fig. 1A) . Protein synthesis arrest was confirmed in individual cells by concomitant imaging of poly I:C delivery, mRNA translation and P-eIF2a ( Fig. 1B and Fig. S1B ), and with a wide range of dsRNA concentrations (Fig. S1C ). Poly I:C-induced eIF2a phosphorylation and subsequent translation arrest were not observed in PKRdeficient MEFs ( Fig. 1C and 1D ), while eIF2a phosphorylation induced by the UPR-inducing drug thapsigargin (th) (an inhibitor of SERCA ATPases) or arsenite (as) was unchanged in PKR2/2 cells (Fig. 1C) . PKR is therefore necessary to induce protein synthesis inhibition in response to cytosolic poly I:C.
Nucleic acids detection by multiple molecular sensors results in type-I interferon production, which protects cells and tissues from viral infections. At the intracellular level, the detection of double-stranded RNA by one of these sensors, the dsRNA-dependent protein kinase also leads to the profound inhibition of protein synthesis. We describe here that the inducible phosphatase 1 co-factor Ppp1r15a/ GADD34, a well known player in the endoplasmic reticulum unfolded protein response (UPR), is activated during double-stranded RNA detection and is absolutely necessary to allow cytokine production in cells exposed to poly I:C or Chikungunya virus. Our data shows that the cellular response to nucleic acids can reveal unanticipated connections between innate immunity and fundamental stress pathways, such as the ATF4 branch of the UPR. When levels of IFN-ß were quantified in culture supernatants and compared to total protein synthesis intensity, we found that most of the cytokine production occurred after 4 to 8 h of pIC delivery (Fig. 1E , WT, and S1D), a time at which mRNA translation was already considerably decreased ( Fig. 1A and S1E). We measured the amount of cytokine produced in NIH-3T3 cells at a time (7 h) at which translation was already strongly inhibited ( Fig. 1G and 1F ). To prove that IFN-b production truly occurred during this poly I:C-induced translation arrest, cells exposed for 7 h to poly I:C were washed and old culture supernatants replaced with fresh media for 1 h (with or without CHX), prior translation monitoring (Fig. 1F , right) and IFN-ß dosage (Fig. 1G , right). We observed that close to 30% of the total IFN-ß produced over 8 h of poly I:C stimulation is achieved during this 1 h period, despite a close to undetectable protein synthesis in the dsRNA-treated cells (Fig. 1F ). The neo synthetic nature of this IFN was further demonstrated by the absence of the cytokine in CHX-treated cell supernatants. IFN-b production in response to poly I:C is therefore likely to be specifically regulated and occurs to a large extent independently of the globally repressed translational context. As previously observed in MEFs, IFN-ß production in response to poly I:C was independent of PKR ( Fig. 1E ) [31] . This suggests that although its production occurs during cap-mediated translation inhibition, it does not directly depend on a specialized open reading frame organization, as described for the translation of the mRNAs coding for the UPR transcription factor ATF4 or the SV 26S mRNA upon eIF2a phosphorylation [26, 49] . This hypothesis is also supported by the ability of MEFs expressing the non-phosphorylatable eIF2a Ser 51 to Ala mutant (eIF2a A/A), to produce normal levels of IFN-ß in response to poly I:C (Fig. 1E) , while global translation was not inhibited by poly I:C in these cells (Fig. S2 ).
We went on to investigate the molecular mechanisms promoting this paradoxical IFN-ß synthesis in an otherwise translationally repressed environment. Induction of eIF2a phosphorylation by PERK during ER stress promotes rapid ATF4 synthesis and nuclear translocation, followed by the transcription of many downstream target genes important for the UPR [50] . Similarly, in presence of PKR, nuclear ATF4 levels were found to be upregulated in MEFs responding to cytosolic poly I:C, albeit less importantly than upon a bona fide UPR induced by thapsigargin ( Fig. 2A) .
One of the key molecules involved in the control of eIF2a phosphorylation is the protein phosphatase 1 co-factor GADD34, which relieves translation repression during ER stress by promoting eIF2a dephosphorylation [50, 51] , [52] . GADD34 is a direct downstream transcription target of ATF4 [53] . Expression of GADD34 was quantified by qPCR and immunoblot in WT and PKR 2/2 MEFs (Fig. 2B) . In WT cells GADD34 mRNA expression was clearly up-regulated (20 fold) in response to poly I:C, while GADD34 protein induction was equivalent in poly I:Cand thapsigargin-treated cells. GADD34 mRNA transcription and translation were not observed in PKR 2/2 cells responding to poly I:C, but occurred normally upon thapsigargin treatment, paragoning eIF2a phosphorylation (Fig. 2B, right) .
We next investigated the importance of ATF4 for GADD34 transcription by monitoring the levels of GADD34 mRNA in ATF4-deficient cells. ATF4 2/2 MEFs displayed higher basal levels of GADD34 mRNA than WT cells. However, in absence of ATF4, MEFs were unable to efficiently induce GADD34 mRNA transcription in response to any of the stimuli tested (Fig. S3 ). GADD34 mRNA expression was induced only 2 fold in ATF4 2/2 MEFs exposed to poly I:C, suggesting that its transcription is mostly dependent on ATF4 in this context. We further investigated P-eIF2a requirement for GADD34 expression and found that eIF2a A/A expressing MEFs were incapable of up-regulating GADD34 in response to poly I:C (Fig. 2C ). Phosphorylation of eIF2a by PKR in response to cytosolic poly I:C induces therefore a specific integrated stress response (ISR), that allows ATF4 translation, its nuclear translocation and subsequent GADD34 mRNA transcription.
GADD34 expression is required for global translation recovery in response to thapsigargin but not to poly I:C We next evaluated the relevance of GADD34 induction, by treating WT and GADD34 DC/DC fibroblasts with poly I:C or with drugs known to induce ER stress, such as thapsigargin and the Nglycosylation inhibitor tunicamycin [52] . As expected, in WT cells eIF2a phosphorylation was rapidly increased in response to all ISR-inducing stimuli and decreased concomitantly with the expression of GADD34 over time ( Fig. 3A and S4 ) [52] . Consequently eIF2a phosphorylation was greatly increased in GADD34 DC/DC MEFs in all the conditions tested ( Fig. 3A and  S4A ).
In thapsigargin-treated cells, protein synthesis was reduced in the first hour of treatment and rapidly recovered (Fig. 3B ) [54] . Poly I:C, however, nearly completely inhibited translation despite active eIF2a dephosphorylation. This was particularly obvious when poly I:C was co-administrated together with thapsigargin. Indeed, poly I:C dominated the response by preventing the translation recovery normally observed after few hours of drug treatment (Fig. 3B) . Surprisingly, in absence of functional GADD34, although eIF2a phosphorylation induction by poly I:C was augmented dramatically, no further decrease in protein synthesis was observed upon treatment of GADD34 DC/DC cells with the dsRNA mimic ( Fig. 3A and 3C ). The functionality of GADD34 in translation restoration was, however, fully demonstrated, when the same cells were treated with thapsigargin, and protein synthesis was completely inhibited by this treatment [52] control. Quantification of puromycin signal was quantified with ImageJ software and is represented above the immunoblot. Phosphorylation of eIF2a (P-eIF2a) was assessed in the same MEFs extracts. B) Immunofluorescence staining for puromycin, P-eIF2a and dsRNA of MEFs treated with poly I:C for 4 h and labeled with puromycin for 1 h. Scale bar, 10 mm. C) WT and PKR 2/2 MEFs were stimulated for 8 h with poly I:C (pI:C), thapsigargin (th) or arsenite (as). PKR and P-eIF2a were detected by immunoblot. D) WT and PKR 2/2 MEFs were stimulated for 8 h with poly I:C and protein synthesis was monitored like in (A). b-actin immunoblot is shown for equal loading control. E) IFN-b levels were measured, by ELISA, in cell culture supernatants of WT, PKR 2/2 , eIF2aA/A and control eIF2aS/S MEFs after 4 and 8 h of poly I:C stimulation. Data are mean 6 standard deviation of 3 independent experiments. F) Protein synthesis was measured in NIH3T3 cells by puromycin incorporation after 7 h of poly I:C treatment. Where indicated, a chase of 1 h with fresh media was performed prior to puromycin labeling and immunoblotting. Samples with cycloheximide (chx) and arsenite (as) added respectively 5 min and 30 min before the puromycin pulse are shown as controls. G) IFN-b was quantified by ELISA in culture supernatants in the conditions described above after 7 h of poly I:C stimulation or 7 h of poly I:C stimulation followed by 1 h with fresh media (chase). Data are mean 6 standard deviation of 4 independent experiments. doi:10.1371/journal.ppat.1002708.g001 (Fig. 3C) . Thus, cytosolic dsRNA delivery induces a type of protein synthesis inhibition, which requires eIF2a phosphorylation for its initiation, but conversely cannot be reverted by GADD34 induction and subsequent GADD34-dependent eIF2a dephosphorylation.
The potential contribution of the OAS/2-5A/RNAse L system to this P-eIF2a-independent inhibitory process was evaluated by investigating RNA integrity in MEFs exposed to poly I:C. We used capillary electrophoresis to establish precise RNA integrity numbers (RIN) computed from different electrophoretic traces (pre-, 5S-, fast-, inter-, precursor-, post-region, 18S, 28S, marker) and quantify the degradation level of mRNA and rRNA potentially resulting from the activation of this well characterized anti-viral pathway. No major RNA degradation could be observed upon poly I:C delivery (Fig. S5 ), suggesting that global RNA degradation does not contribute extensively to the long term translation inhibition observed upon poly I:C delivery in our experimental system.
We have observed that GADD34 expression counterbalances PKR activation by promoting eIF2a dephosphorylation, however it has little impact on reversing the global translation inhibition initiated by poly I:C. We next monitored the production of specific proteins and cytokines in WT and GADD34 DC/DC MEFs (Fig. 4) . . PKR is required for ATF4 and GADD34 expression in response to poly I:C. A) WT and PKR 2/2 MEFs were stimulated for 8 h with poly I:C (pI:C), or the UPR-inducing drug, thapsigargin (th) for 6 h. ATF4 protein expression was detected by immunoblot on nuclear extracts. Nuclear HDAC1 immunoblot is shown for equal loading control. * indicates unspecific band. B) GADD34 mRNA levels were quantified by qPCR after 6 h of poly I:C (pI:C) treatment in WT and PKR 2/2 MEFs. For the same cell extracts, immunoblots of GADD34 (middle panel), PKR and P-eIF2a (right panel) were performed. C) The same analysis was performed in eIF2a A/A and control eIF2a S/S MEFs. Treatment with thapsigargin (th), for 6 h was used as control to induce GADD34 and P-eIF2a. eIF2a and b-actin immunoblots are shown for equal loading control. Quantitative PCR data are the mean 6 standard deviation of 3 independent experiments. doi:10.1371/journal.ppat.1002708.g002
Cystatin C, a cysteine protease inhibitor was chosen as a model protein, since its secretion ensures a relative short intracellular residency time so that its intracellular levels directly reflect its synthesis rate [55] . This is confirmed by the N-glycosylated-and total Cystatin C accumulation in cells treated with brefeldin A (Fig. 4A , left panel). Cystatin C levels were found to follow a similar trend to that observed with total translation, being strongly reduced upon poly I:C exposure and not profoundly influenced by GADD34 inactivation (Fig. 4A, right panel) . Thapsigargin treatment induced a brief drop in cystatin C levels, prior to some levels of GADD34-dependent recovery. 6 hours of tunicamycin treatment affected more cystatin C accumulation than anticipated Figure 3 . GADD34 mediates eIF2a dephosphorylation but not global translation recovery in response to poly I:C. A) After treatment with poly I:C, protein extracts of WT and GADD34 DC/DC MEFs were immunobloted for GADD34 and P-eIF2a. B) Protein synthesis was analyzed in WT cells treated for 1 to 6 hours with poly I:C (pI:C) alone or together with thapsigargin (th). Controls are cells not treated with puromycin (co) and cells treated with cycloheximide (chx) 5 min before puromycin incorporation. C) Protein synthesis was analyzed in GADD34 DC/DC cells treated for 1 to 6 hours with poly I:C (pI:C) alone or together with thapsigargin (th). Tubulin or b-actin immunoblot are shown for equal loading control. In GADD34 DC/DC cells translation is strongly impacted by thapsigargin, but not poly I:C. doi:10.1371/journal.ppat.1002708.g003 Fig. 4A, right panel) , probably due to interference with the Nglycosylation and associated folding of this di-sulfide bridge containing protein [55] , thereby promoting its degradation by endoplasmic reticulum-associated protein degradation (ERAD) [56] .
We next turned towards PKR, which displayed a pattern of expression completely different from cystatin C (Fig. 4B ). As expected from its IFN-inducible transcription, levels of PKR were increased in poly I:C-treated MEFs (Fig. 4B) , despite the strong global translation inhibition observed in these cells (Fig. 3) . GADD34 inactivation appeared to influence the accumulation of PKR, since the cytoplasmic dsRNA sensor levels were not upregulated and even decreased in poly I:C-treated GADD34 DC/DC MEFs (Fig. 4B ). Control treatment with tunicamycin and thapsigargin did not alter significantly PKR levels (Fig. 4B) , suggesting that ER stress did not influence the kinase expression. The absence of PKR up-regulation in the poly I:C-treated GADD34 DC/DC MEFs led us to investigate the capacity of these cells to produce anti-viral and inflammatory cytokines, which normally drive PKR expression through an autocrine loop. We ruled out any interference from the UPR in triggering IFN-ß production in our experimental system, since, as anticipated from PKR expression, tunicamycin and thapsigargin treatments were not sufficient to promote cytokine production in MEFs (Fig. S6 ) [43, 44] .
We therefore investigated IFN-ß and IL-6 production in response to dsRNA in WT, GADD34 DC/DC and CReP 2/2 MEFs. CReP 2/2 MEFs were used as a control, since CReP (Ppp1r15b) is a non-inducible co-factor of PP1 and displays some functional redundancy with GADD34 [57] . Although basal levels of eIF2a phosphorylation were higher in CReP 2/2 , PKR expression and translation inhibition upon poly I:C delivery were equivalent in WT and CReP 2/2 MEFs ( Fig. S7A and S7B ). Quantification of IFN-ß and IL-6 levels in culture supernatants indicated that, although abundant and comparable amounts of these cytokines were secreted by WT and CReP 2/2 cells, they were both absent in poly I:C-treated GADD34 DC/DC MEFS ( Fig. 4C and S7C) .
Quantitative PCR analysis revealed that, IFN-ß, IL-6 and PKR transcripts were potently induced in poly I:C treated GADD34 DC/ DC MEFs (Fig. 4D ), thus excluding any major transcriptional alterations in these cells, as confirmed by the normal levels of cystatin C mRNA, which remained constant in all conditions studied. Moreover, using confocal immunofluorescence microscopy, we could not detect intracellular IFN-b in poly I:C-stimulated GADD34 DC/DC MEFs, in contrast to WT cells, which abundantly expressed the cytokine, despite the global translation arrest (Fig.  S8) . Thus, we could attribute the deficit in cytokine secretion of the GADD34 DC/DC MEFs to a profound inability of these cells to synthesize cytokines, rather than to a defect in transcription or general protein secretion.
GADD34 induction by poly I:C is therefore absolutely necessary to maintain the synthesis of specific cytokines and probably several other proteins in an otherwise translationally repressed context. Importantly, GADD34 exerts its rescuing activity only on a selected group of mRNAs including those coding for IFN-ß and IL-6, but not on all ER-translocated proteins, since cystatin C synthesis was strongly inhibited by poly I:C in all conditions tested.
Interestingly, in GADD34 DC/DC MEFs, PKR mRNA strongly accumulated in response to poly I:C (Fig. 4D) , despite the absence of detectable IFN-ß production and PKR protein increase (Fig. 4B) . This continuous accumulation of PKR mRNA in response to poly I:C suggests the existence of alternative molecular mechanisms, capable of promoting PKR mRNA transcription and stabilization independently of autocrine IFN-b detection. Nevertheless in these conditions PKR expression, like IFN-b, was found to be dependent on the presence of GADD34 for its synthesis (Fig. 4B) .
Recent results indicate that PKR participates to the production of IFN-a/ß proteins in response to a subset of RNA viruses including encephalomyocarditis, Theiler's murine encephalomyelitis, and Semliki Forest virus [31] . Even though IFN-a/ß mRNA induction is normal in PKR-deficient cells, a high proportion of mRNA transcripts lack their poly(A) tail [31] . As GADD34 induction by poly I:C was completely PKR-dependent, we wondered whether the phenotypes observed in PKR 2/2 cells and GADD34 DC/DC MEFs could be related. Oligo-dT purified mRNA extracted from cells exposed to poly I:C were therefore analyzed by qPCR. PolyA+ mRNAs coding for IFN-ß and IL-6 were equivalently purified and amplified from WT and GADD34 DC/DC MEFs (Fig. S9 ). This confirms that albeit the phenotypes of PKR 2/2 and GADD34 DC/DC cells might be linked, mRNA instability is not the primary cause of the cytokine production defect observed in GADD34 DC/DC . Taken together these observations suggest the existence of a specific mRNAs pool, encompassing cardinal immune effectors such as IFN-ß, IL-6, and PKR, which are specifically translated in response to dsRNA sensing and increased levels of P-eIF2a. This mRNAs pool requires GADD34 for their translation during the global protein synthesis shut-down triggered by dsRNA detection.
We verified that GADD34 inactivation, and no other deficiency, was truly responsible for the loss of cytokine production by complementing GADD34 DC/DC MEFs with GADD34 cDNA prior poly I:C delivery. IFN-ß secretion was partially restored in transfected GADD34 DC/DC cells while eIF2a was efficiently dephosphorylated in both WT and GADD34 DC/DC transfected MEFs (Fig. 4E) . To further demonstrate that the phosphatase activity of GADD34 controls cytokine production upon dsRNA detection, we treated WT MEFs with guanabenz, a small molecule, which selectively impairs GADD34-dependent eIF2a dephosphorylation [58] . Upon treatment with this compound, a dose dependent inhibition of IFN-ß secretion was observed in poly I:C-treated MEFs, confirming the importance of GADD34 in this process (Fig. S10) .
as controls to induce GADD34. Immunoblot of tubulin is shown as equal loading control. C) Amount of IFN-b (left panel) and IL-6 (right panel) in cell culture supernatants of WT and GADD34 DC/DC MEFs after 6 h of poly I:C stimulation. Mock are samples treated with lipofectamine alone. Data are mean 6 standard deviation of five (IFN-b) and three (IL-6) independent experiments. D) Transcription of IFN-b, IL-6, PKR and Cystatin C was analyzed by qPCR in samples of WT and GADD34 DC/DC MEFs treated with poly I:C (pI:C). Mock represent samples treated with lipofectamine alone. E) WT and GADD34 DC/DC MEFs were transfected overnight with an expression plasmid carrying the murine GADD34 (G34) cDNA and then treated with poly I:C for 6 h. IFN-b production was quantified by ELISA, left panel, in cell culture supernatants and plotted as a ratio of IFN-b to total cell proteins to compensate for different cell mortality levels induced by the transfection. In the right panel immunoblots for GADD34 and P-eIF2a in the same experimental conditions are shown. One representative analysis of 3 independent experiments is shown. doi:10.1371/journal.ppat.1002708.g004 GADD34 is necessary for IFN production and to control Chikungunya virus infection Fibroblasts of both human and mouse origin constitute a major target cell of Chikungunya virus (CHIKV) during the acute phase of infection [59] . In adult mice with a totally abrogated type-I IFN signaling, CHIKV-associated disease is particularly severe and correlates with higher viral loads. Importantly, mice with one copy of the IFN-a/ß receptor (IFNAR) gene develop a mild disease, strengthening the implication of type-I IFN signaling in the control of CHIKV replication [59] . Recently, human fibroblasts infection by CHIKV was shown to induce IFN-a/ß mRNA transcription, while preventing mRNA translation and secretion of these antiviral cytokines. CHIKV was found to trigger eIF2a phosphorylation through PKR activation, however this response is not required for the block of host protein synthesis [15] .
We tested the importance of PKR during CHIKV infection by infecting WT and PKR 2/2 MEFs with CHIKV-GFP, at a multiplicity of infection (MOI) of 10 and 50. Productive infection was estimated by GFP expression (Fig. 5A, left panel) , while culture supernatants were monitored for the presence of IFN-b (5A, right panel). PKR was found to be necessary to control CHIKV infection in vitro, since at least 60% of PKR-inactivated cells were infected after 24 of viral exposure, compared to only 15% in the control fibroblasts population. WT MEFs produced efficiently IFN-b, while the hypersensitivity to infection of the PKR 2/2 MEFs was correlated to a reduced type-I IFN production capacity after infection. Thus, during CHIKV infection, PKR is required for normal IFN production by MEFs. We also monitored protein synthesis in infected WT and PKR 2/2 fibroblasts using puromycin labeling followed by immunofluorescence confocal microscopy (Fig. 5B) . CHIKV-GFP positive PKR 2/2 MEFs were found to incorporate efficiently puromycin, while in their infected WT counterpart protein synthesis was efficiently inhibited. Thus CHIKV, in this experimental model, induces a PKR-dependent protein synthesis inhibition and is therefore particularly relevant to further confirm our observations on the role of GADD34 in controlling type-I IFN production during response to viral RNAs.
GADD34 DC/DC MEFs were exposed to CHIKV-GFP (MOI of 10 or 50) for 24 and 48 h. Productive infection was estimated by GFP expression and virus titration (Fig. 6A) , and culture supernatants monitored for the presence of type-I IFN (Fig. 6B , left). Only minimal CHIKV infection (15%) could be observed at maximum MOI in WT MEFs (Fig. 6A, left) , while robust IFN-b amounts were already produced at the lowest MOI (Fig. 6B) . Contrasting with WT cells and regardless of the MOI used, a higher level of viral replication was observed in GADD34 DC/DC MEFs (Fig. 6A) . The GADD34-inactivated cells were clearly more sensitive to CHIKV, displaying a 50% infection rate after 24 h of infection (MOI 50) and a log more of virus titer in culture supernatants (Fig. 6A, right) . Correlated with their susceptibility to CHIKV infection, IFN-b production was nearly undetectable in GADD34 DC/DC MEFs (Fig. 6B) . Such observation confirms the incapacity of GADD34-deficient cells to produce cytokines in response to cytosolic dsRNA, a deficiency likely to facilitate viral replication. This interpretation is further supported by the abrogation of viral replication in both WT and GADD34 DC/DC MEFs briefly treated with IFN-b (Fig. 6C) . Thus, GADD34 inactivation does not favor viral replication per se, but is critical for type-I IFN production. Interestingly infection levels were found to be higher in PKR2/2 than in GADD34 DC/DC MEFs, although this difference could be attributed to clonal MEFs variation, it more likely suggests that PKR-dependent translation arrest could be key in preventing early viral replication in this system. In addition, the relatively lower permissivity of GADD34 DC/DC MEFs to infection at high MOI could indicate the existence of GADD34-dependent defense mechanisms, which could be independent from IFN production and eIF2-a dephosphorylation. To strengthen and generalize these observations, we treated a different strain of WT MEFs with guanabenz and examined the consequences for CHIKV infection. Biochemically, GADD34 expression was induced upon CHIKV infection, and guanabenz treatment resulted in a clear increase in eIF2a phosphorylation, demonstrating the importance of GADD34 in limiting this process during infection (Fig. 6D, right) . As observed with GADD34 DC/DC cells, pharmacological and RNAi inhibition of GADD34 was found to increase significantly the sensitivity of MEFs to infection, while reducing their IFN-b production ( Fig. 6D and S10) . Thus, induction of GADD34 and its phosphatase activity during CHIKV infection, in vitro, participates to normal type-I IFN production and control of viral dissemination.
Several components of the innate immune response have been shown to impact on the resistance of adult mice and to restrict efficiently CHIKV infection and its consequences in vivo [10] . We decided to investigate the importance of GADD34 upon intradermal injections of CHIKV to WT (FVB) and GADD34 DC/DC mice. Neither strain of adult mice was affected by intradermal injections of CHIKV, with little statistically significant differences in the virus titers found in the different organs. Thus, GADD34 deficiency does not annihilate all the sources of type-I IFN in the infected adult animals, a situation exemplified by the capacity of GADD34 DC/DC bone-marrow derived dendritic cells to produce reduced, but measurable IFN-b in response to poly I:C [60] . This also infers that the light impact of GADD34 inactivation on mouse development [61] does not render these animals more sensitive to CHIKV infection.
As in Humans, CHIKV pathogenicity is strongly agedependent in mice, and in less than 12 day-old mouse neonates, CHIKV induces a severe disease accompanied with a high mortality rate [59] . GADD34 function was therefore evaluated in this more sensitive context by injecting intradermally CHIKV to FVB (WT) and GADD34 DC/DC neonatal mice. As previously observed for C57/BL6 mice [59] , when CHIKV was inoculated to FVB neonates, a rate of 50% of mortality was observed 3 days after the infection of 9-day-old mice, while 12-day-old pups were found essentially resistant to the virus lethal effect (Fig. 7A ). Strongly contrasting with these results, all CHIKV infected GADD34 DC/DC neonates died within 3-5 days post inoculation whatever their age (Fig. 7A ). When infection was monitored 5 days post-inoculation of 12-day-old mice at, GADD34 DC/DC pups displayed considerably more elevated CHIKV titers (10-100 folds) in most organs tested, including liver, muscle, spleen and joints, the later being primarily targeted by the virus (Fig. 7B, left) . As expected, and in full agreement with the in vitro data, infected GADD34 DC/DC tissues showed a considerably reduced IFN-ß production (40-50%) compared to control tissues ( Figure 7B, right) , while serum levels were reduced by 20% (not shown). Although Infectious virus was poorly detected in the heart of WT animals, elevated titers of virus were observed in the heart of GADD34-deficient pups, matching the limited production of IFN in this organ. We further investigated the possible pathological consequences of cardiac tissue infection by carrying-out comparative histopathology. Hearts of infected GADD34-deficient animals displayed severe cardiomyocytes necrosis with inflammatory infiltrates by monocytes/ macrophages and very important calcium deposition (Fig. 8) , all being indicative signs of grave necrotic myocarditis. As a consequence, the left ventricles were strongly dilated, being probably the cause of acute cardiac failures and of the important death rate observed in GADD34 DC/DC infected pups. Histology of infected FVB mice hearts was, however, normal with only few inflammatory cells (mainly lymphocytes) observed in the close vicinity of capillaries.
GADD34 expression is therefore necessary to allow normal type-I interferon production during viral infection and to promote the survival of young infected animals. We could circumvent the age-related acquisition of viral resistance in GADD34 DC/DC mice to 17 days, since mice inoculated at that age survived CHIKV inoculation. In these animals, 3 days post-infection, enhanced viral replication was observed in the spleen and muscles, matching the relatively low level of type-IFN production in these tissues ( Figure 7C ). Functional GADD34 is therefore required to mount a normal innate response against the virus, but in older mice type-I IFN production by non-infected innate cells is probably capable to gradually overcome GADD34-deficiency and limit viral proliferation in vital organs, such as the heart.
Translation inhibition occurs in response to stress, when other cellular activities have to be reassigned or suspended momentarily. We demonstrate here that the activation of PKR by cytosolic dsRNA results in a stress response, leading to ATF4 and GADD34 induction. GADD34 expression has been observed during the infection of cells by different types of viruses [62] or intracellular bacteria such as Listeria monocytogenes [63] . Our observations demonstrate that GADD34 expression is a direct consequence of PKR activation and dsRNA sensing. Interestingly, although GADD34 induction by poly I:C promotes eIF2a dephosphorylation, this is not sufficient to prevent global protein synthesis arrest. The uncoupling of efficient eIF2a dephosphorylation from global translation recovery in response to cytosolic poly I:C implies therefore the existence of additional mechanisms inhibiting global translation. The 2-5A/RNAse L pathway does not seem to be sufficiently active in our experimental setting to explain this prolonged protein synthesis inhibition. The cleavage or the inactivation of other translation factors could work in concert with eIF2a to block or affect the efficiency of other individual steps of mRNA translation [64] . For instance, the phosphorylation of translation elongation factor 2 (eEF-2) is also controlled by eIF2a phosphorylation. Thus, Thr56 phosphorylation of eEF-2, which is known to inhibit its translational function by reducing its affinity for ribosomes, could contribute directly to the protein synthesis inhibition induced by PKR activation [65] . Independently of general protein synthesis inhibition, eIF2a dephosphorylation is necessary for the production of specific proteins upon dsRNA-induced translation inhibition. As demonstrated for ATF4, translation of a given mRNA during stress could rely on the structure and organization of its coding sequence, as well as the presence of multiple alternative initiation codons [49] . Surprisingly, functional GADD34 expression was found necessary for the translation of IL-6, IFN-b, and PKR. This observation points to the existence of a distinct group of mRNAs efficiently translated upon dsRNA detection and dependent on GADD34 activity. GADD34 is extremely short lived and has been shown to accumulate on the ER, when over-expressed [51] . GADD34 could mediate its activity at the ER level and influence differently eIF2a sub-cellular distribution according to the type, localization, and level of activity displayed by the different eIF2a kinases. The strong eIF2a phosphorylation mediated by PKR in response to poly I:C or viral infection and leading to the initiation of translation inhibition, could be circumvented through GADD34 activity solely at the ER level, thereby allowing local cytokine production in absence of other functional protein synthesis. This selectivity for translation of several specific mRNAs among other ER-secreted molecules suggests further that GADD34 dependent mRNAs might display specific features allowing their efficient identification by GADD34 and associated molecules, as well as allowing their translation in presence of minimal levels of active guanine nucleotide exchange factor eIF2B.
GADD34 and PKR are necessary to produce anti-viral cytokines during CHIKV infection, and probably other types of infection. PKR, ATF4 and GADD34 should therefore be considered as an essential module of the innate anti-viral response machinery. The importance of PKR in anti-viral type-I IFN responses has been the object of contradictory reports [30, 31, 66, 67] . Our observations, however, suggest that PKR function should be re-evaluated by integrating the impact of viral detection on cellular translation. In eIF2A/A and PKR 2/2 cells, cytokine transcription is induced normally following poly I:C detection by DExD/H box RNA helicases, while as expected in these cells, no eIF2a phosphorylation and subsequent host translation inhibition are observed. This lack of translation arrest in the absence of potent eIF2a phosphorylation allows for normal cytokine production during dsRNA detection, with no requirement for an operational GADD34 feedback loop. The importance of PKR and GADD34 for IFN-b and other cytokines production could therefore be directly linked to the efficiency of the cellular translation inhibition induced by RNA viruses, as exemplified here with CHIKV, which in MEFs strongly activates PKR and subsequent protein synthesis inhibition.
GADD34 DC/DC neonates are extremely sensitive to CHIKV infection and display signs of acute myocarditis and ventricles dilatation probably causing recurrent cardiac failures. CHIKV cardiac tropism is not normally observed in WT mouse and inability of heart tissues to produce sufficient type-I IFN in GADD34 DC/DC could allow abnormally high viral replication, myocarditis and dilated cardiomyopathy. Interestingly many cases of myopericarditis induced by CHIKV and leading to dilated cardiomyopathies in infected patients have been reported since the Figure 7 . CHIKV infection in mouse neonates. A) Kaplan-Meier plots representing the survival of FVB (WT) and GADD34 DC/DC mouse neonates 9-day-old (n = 11 per group) (upper panel) or 12-day-old (n = 14 per group) (lower panel) after intradermal inoculation with 10 6 PFU of CHIKV and observed for 21 days. B) Left panel, viral titers in different tissues and serum of 12-day-old mice inoculated with 10 6 PFU of CHIKV via the intradermal route. Mice were sacrificed 5 days after infection and the amount of infectious virus in serum and tissues quantified by TCID50 (see methods) (n = 5). In addition of considerably increased levels of viral replication in CHIKV target tissues, GADD34 DC/DC neonates also display signs of heart infection. Right panel, Quantification of IFN-b for the same different tissues, CHIKV-infected target tissues of GADD34 DC/DC mice produced less IFN-b than WT. C) 17-day-old mice were infected with 10 6 PFU of CHIKV via the intradermal route, and sacrificed 72 h later. Quantification of viral titers and IFN-b/ viral titers ratio is presented for different tissues. A broken line indicates the detection threshold. In B and C represented data are arithmetic mean 6 standard deviation, n = 5. In B and C p values were calculated using a Student's t test, *p#0.1, **p#0.05. doi:10.1371/journal.ppat.1002708.g007 Figure 8 . CHIKV infection causes severe myocarditis in mouse neonates. Histological appearance of horizontal sections of the heart through left and right ventricles of 12-day FVB (A, C and E) and GADD34 DC/DC mice at D5 pi (B, D and F). Normal appearance of heart of FVB infected mice, at low magnification (A, 610) with normal cardiomyocytes (C, 6100) and exceptional small foci of lymphocytes (E, 6400). Numerous foci of necrosis in the heart of GADD34 DC/DC infected mice, at low magnification (B, 610) and extensive through the ventricular wall (D, 6100). Higher magnification shows few residual cardiomyocytes (arrow head) and inflammation mainly composed of monocytes as well as extensive deposition of calcium (F, 6400). The mice were inoculated with 10 6 PFU of CHIKV via the intradermal route. doi:10.1371/journal.ppat.1002708.g008
1970s after the different western Indian Ocean islands and Indian subcontinent disease outbreaks [68, 69] . These particular symptoms and complications might therefore be the consequences of great variation in the tissue-specific type-I IFN levels induced in CHIKV-infected patients, who might display particular polymorphisms in their innate viral sensing pathways increasing their peculiar susceptibility to viral dissemination in the heart. Importantly, our data reveal a link between pathogen-associated molecular patterns (PAMPs) and the UPR through the activation of the eIF2-a/ATF4 branch [70] . Similarly, several laboratories have reported that TLR stimulation activates the XBP-1 branch of the UPR and that XBP-1 production was needed to promote a sustained production of inflammatory mediators, including IL-6 [71, 72] . Here, we identify GADD34 as a novel functional link between ISR and PAMPs detection in MEFs, required for the production of cytokines including type-I IFN. It will now be important to explore the therapeutic potential of targeting GADD34 to reduce cytokines overproduction during inflammatory conditions. 
Puromycin labelling for measuring the intensity of translation was performed as previously described [47] . For immunoblots, 10 mg/ml puromycin (Sigma, min 98% TLC, cell culture tested, P8833, diluted in PBS) was added in the culture medium and the cells were incubated for 10 min at 37uC and 5% CO 2 . Where indicated, 25 mM cycloheximide (Sigma) was added 5 min before puromycin. Cells were then harvested, centrifuged at 4uC and washed with cold PBS prior to cell lysis and immunoblotting with the 12D10 antibody.
Cells were lysed in 1% Triton X-100, 50 mM Hepes, 10 mM NaCl, 2.5 mM MgCl 2 , 2 mM EDTA, 10% glycerol, supplemented with Complete Mini Protease Inhibitor Cocktail Tablets (Roche). Protein quantification was performed using the BCA Protein Assay (Pierce). 25-50 mg of Triton X-100-soluble material was loaded on 2%-12% gradient or 8% SDS-PAGE before immunoblotting and chemiluminescence detection (SuperSignal West Pico Chemiluminescent Substrate, Pierce). Nuclear extraction was performed using the Nuclear Complex Co-IP kit (Active Motif). Rabbit polyclonal antibodies recognizing ATF4 (CREB-2, C-20), GADD34 (C-19), Lamin A (H-102) and eIF2-a (FL-315) were from Santa Cruz Biotechnology, as well as mouse monoclonal anti-PKR (B-10). GADD34/PPP1R15A (Catalog No. 10449-1-AP) rabbit polyclonal antibody was purchased from PROTEINTECH.
Rabbit polyclonal anti-eIF2a[pS 52 ] and Cystatin C were from Invitrogen and Upstate Biotechnology, respectively. Mouse monoclonal antibodies for b-actin and HDAC1 (10E2) were purchased from Sigma and Cell Signaling Technologies. Secondary antibodies were from Jackson ImmunoResearch Laboratories.
MEFs and NIH3T3 were grown on coverslips overnight and stimulated for the indicated time with poly I:C complexed with Lipofectamine 2000. Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0,5% saponin in 5% FCS PBS with 100 mM glycine, for 15 min at room temperature and stained for 1 h with indicated primary antibodies. Anti-P-eIF2a was from BioSource; anti-dsRNA (clone K1) from English & Scientific Consulting Bt.; anti-IFN-b-FITCconjugated from PBL Interferon Source; anti-puromycin (clone 2G11, mouse IgG1) has been previously described [47] . Alexaconjugated secondary antibodies (30 min staining) were from Molecular Probes (Invitrogen). Coverslips were mounted on a slide and images taken with a laser-scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging) using a 636 objective and accompanying imaging software. When PKR WT and PKR 2/2 were infected with CHIKV, protocol was performed as follows: cells were fixed with 4% paraformaldehyde in PBS for 20 min, then permeabilized for 30 min in 0.1% Triton 100X (Sigma) and blocked in 10% of normal goat serum (Vector Laboratories). Cells were stained with a mouse monoclonal antibody directed against CHIKV capsid coupled to Alexa-488 and a mouse antibody against puromycin coupled to Alexa-555 and a rabbit antibody anti-eIF2a[pS 52 ] (Invitrogen) and a Cyanin-3 secondary antibody, and finally counterstained with Hoechst (Vector Lab). Cells were observed with an AxioObserver microscope (Zeiss). Pictures and Z-stacks were obtained using the AxioVision 4.5 software.
ELISA IFN-b and IL-6 quantification in culture supernatant was performed using the Mouse Interferon Beta ELISA kit (PBL InterferonSource) and Mouse Interleukin-6 ELISA kit (eBioscience) respectively, according to manufacturer instructions.
Total RNA was isolated from cells using the RNeasy miniprep kit (QIAGEN) combined with a DNA digestion step (RNase-free DNase set, QIAGEN). cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen) and random hexamer primers. Quantitative PCR amplification was carried out using complete SYBR Green PCR master mix (Applied Biosystems) and 200 nM of each specific primer. 5 ml of cDNA template was added to 20 ml of PCR mix, and the amplification was tracked via SYBR Green incorporation by an Applied Biosystems thermal cycler. cDNA concentration in each sample were normalized by using HPRT. A nontemplate control was also routinely performed. The primers used for gene amplification (designed with Primer3 software) were the following: mRNA isolation from total RNA was performed with oligodT columns (Genelute mRNA miniprep kit (Sigma). Data were analyzed using the 7500 Fast System Appled Biosystems software.
RNA integrity upon poly I:C stimulation was measured by capillary electrophoresis using the the Agilent RNA 6000 Pico Chip kit (Agilent Technologies) in an Agilent 2100 Bioanalyser, according to manufacturer instructions.
GADD34 DC/DC and the corresponding WT control MEFs were infected at a multiplicity of infection (MOI) of 10 or 50 with CHIKV-GFP generated using a full-length infectious cDNA clone provided by S. Higgs [71] . By 24 h and 48 h post infection, 30 000 cells were analyzed in triplicate by FACS for expression of GFP. At the same time-points, culture supernatants were collected and IFN-b protein assessed by ELISA. In experiments with exogenous IFN-b, cells were treated with mouse IFN-b (PBL InterferonSource) for 3 h before infection with CHIKV-GFP. When guanabenz was used to specifically inhibit GADD34, MEFs cells were treated for 2 h with 10 mM of Guanabenz or DMSO and then infected in the same medium. Three hours post infection the inoculum was removed and fresh medium with Guanabenz or DMSO was added and maintained all along the experiment. RNAi for GADD34 was performed as described in [60] .
FVB WT mice were obtained from Charles River Laboratories (France). GADD34 DC/DC FVB mice were obtained from L. Wrabetz (Milan). Mice were anesthetized and inoculated via the intradermal route with 10 6 PFU of CHIKV-21 isolate [72] . Viral titers in tissues and serum were determined as described before [59] , and expressed as tissue cytopathic infectious dose 50 (TCID50)/g or TCID50/ml, respectively. Organs including heart, liver, skeletal muscles and spleen were collected for histopathological procedures. organs were then fixed in 4% paraformalde-hyde solution, paraffin-embedded, sectioned coronally in 5-10 mm thickness and stained with hematoxylin-eosin. Figure S1 Poly I:C stimulation induces protein translation inhibition and IFN-b production in NIH3T3 cells. A) Protein synthesis was quantified in poly I:C-stimulated NIH3T3 using puromycin labeling followed by immunoblot with antipuromycin mAb 12D10. Protein synthesis was strongly reduced upon poly I:C stimulation. Immunoblot for phosphorylated (P-eIF2a) and total eIF2a were performed on the same NIH3T3 extract. Cycloheximide (chx) was added 5 min before puromycin incorporation. b-actin immunoblot is shown for equal loading control. B) Puromycin integration was analysed by immunofluorescence in NIH3T3 cells treated for 4 h with poly I:C and labeled with puromycin in the last 10 min. Figure S2 Protein translation in cells with non-phosphorylatable eIF2a. Protein synthesis was quantified in MEFs with non-phosphorylatable eIF2a, eIF2aA/A and the corresponding control cells, eIF2aS/S. After poly I:C or thapsigargin treatment, puromycin labeling followed by immunoblot, was performed. Puromycin labeling was quantified with ImageJ software and protein translation was depicted as percentage of steady state. Cycloheximide (chx) was added 5 min before puromycin incorporation. Tubulin immunoblot is shown for equal loading control. One of two independent experiments with similar results is shown. (TIF) Figure S3 GADD34 mRNA induction in ATF4-deficient MEFs stimulated with cytosolic poly I:C. The levels of GADD34 transcript were determined by qPCR in WT and ATF4 2/2 cells after 8 h of poly I:C stimulation. Treatment with tunicamycin and thapsigargin were used as positive controls for GADD34 induction. Results are displayed according to both WT internal reference (left) and ATF4 2/2 internal reference (right). (TIF) Figure S4 GADD34 mediates eIF2a dephosphorylation in MEFs stimulated with poly I:C. A) Wild-type and GADD34 DC/DC MEFs were treated for the indicated times with poly I:C (pI:C), tunicamycin (tun) or thapsigargin (th) and eIF2a phosphorylation was monitored by immunoblot. B) GADD34 expression was analyzed by immunoblot in samples treated for 1 or 6 hours with poly I:C alone or together with tunicamycin (tun) or thapsigargin (th). Data shown in (A) and (B) are representative of three independent experiments with similar results. (TIF) Figure S5 RNA integrity upon poly I:C exposure. WT MEFs were treated with poly I:C for the indicated times and RNA integrity evaluated by capillary electrophoresis (Agilent RNA 6000). RNA Integrity Numbers (RIN) between 8.2 and 9.2 were obtained, indicating a high level of RNA integrity. Data shown are representative of three independent experiments with similar results. (TIF) Figure S6 UPR-inducing drugs do not elicit IFN-b production. Cell culture supernatants of murine embryonic fibroblasts were tested for the presence of IFN-b, after treatment with poly I:C (8 h), tunicamycin and thapsigargin (6 h). The results shown are representative of 4 experiments. (TIF) Figure S7 Deletion of the constitutively-expressed PP1 co-factor, CReP, does not impact protein translation and IFN-b production in MEFs. A) WT and CReP 2/2 MEFs were treated with poly I:C (pI:C) for the indicated times and the levels of P-eIF2a and PKR were analyzed by immunoblot. Although basal levels of P-eIF2a were higher in CReP 2/2 MEFs, increase of phosphorylation upon poly I:C exposure was similar to the WT. PKR expression upon poly I:C treatment was equivalent in CReP 2/2 and WT MEFs. B) Protein synthesis was quantified using puromycin labeling followed by immunoblot with the antipuromycin mAb 12D10. Where indicated, cells were treated with cycloheximide (chx) 5 min before puromycin incorporation. No major differences were found between WT and CReP 2/2 cells at the level of translation inhibition following poly I:C exposure.  Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. The trichothecenes, a group of sesquiterpenoid mycotoxins produced by Fusarium that contaminate wheat, barley, and corn globally, are problematic because of their resistance to degradation during processing and their potential to adversely affect human and animal health (Pestka, 2010) . Among the over 200 trichothecenes discovered to date, deoxynivalenol (DON) is most frequently encountered in food, and human exposure to this toxin throughout the world has been well documented in a recent series of elegant biomarker studies (Hepworth et al., 2012; Turner et al., 2010a Turner et al., ,b, 2011 . A potential target for adverse health effects for DON is the innate immune system, with low doses of the toxin having immunostimulatory effects and high doses causing immunosuppression (Pestka, 2010) .
At the mechanistic level, DON has been shown in vitro and in vivo to activate mitogen-activated protein kinases (MAPKs), including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in macrophages and monocytes (Islam et al. 2006; Shifrin and Anderson, 1999; Zhou et al., 2003a) . These MAPKs mediate upregulation of proinflammatory cytokine and chemokine expression as well as apoptosis (Chung et al., 2003; Islam et al., 2006; Moon and Pestka, 2002) . Notably, DON concentration dependently induces competing survival (ERK/AKT/p90Rsk/Bad) and apoptotic (p38/p53/Bax/ mitochondria/caspase-3) pathways in the macrophage (Zhou et al., 2005a) . Accordingly, MAPK activation is critical to both stimulation and suppression of the innate immune system.
Two signal transducers that have been identified to be upstream of DON-induced MAPK activation are doublestranded RNA-(dsRNA) activated protein kinase (PKR) (Zhou et al., 2003b) and hematopoietic cell kinase (Hck) (Zhou et al., 2005b) . PKR is a widely distributed constitutively expressed serine/threonine protein kinase that can be activated by dsRNA, interferon, proinflammatory stimuli, cytokines, and oxidative stress (Garcia et al., 2006; Williams, 2001) . PKR also activates p53, p38, JNK, Nuclear Factor-kappaB, signal transducer and activator of transcription, and interferon regulatory factor-1 (Williams, 1999) . Hck, a member of Src kinase family, is expressed specifically in myelomonocytic cell lineages and transduces extracellular signals that regulate proliferation, differentiation, and migration (Ernst et al., 2002; Tsygankov, 2003) . Upon DON exposure, both PKR and Hck are activated prior to the MAPKs and their respective inhibitors suppress downstream MAPK activation (Zhou et al., 2005b) . Although crosstalk between PKR and Hck is not clearly understood, PKR appears to be required for Hck interaction with the ribosome in the human monocyte U937 cell line (Bae et al., 2010) .
A prominent consequence of DON exposure in macrophages is ribosomal RNA (rRNA) cleavage, which could negatively impact innate immune function (Li and Pestka, 2008) . Two fundamental questions arising from this work relate to the specificity and mechanisms for this cleavage. Using oligonucleotide extension, we identified one site of DON-induced rRNA cleavage is within the peptidyl transferase center of 28S rRNA; however, further identification of additional sites was precluded by the relatively poor resolution of conventional gel electrophoresis and limitations on this techniques imposed by RNA hairpin structures (Li and Pestka, 2008) . Regarding mechanisms, it is known that ricin and other ribosomeinactivating proteins (RIPs) cleave rRNA via a highly specific mechanism that involves N-glycosidase-mediated adenine depurination at highly conserved sarin/ricin (S/R) loop (Endo and Tsurugi, 1986; Hartley and Lord, 2004) . However, DON and other trichothecenes are low molecular weight chemicals and are devoid of inherent enzyme activities (Li and Pestka, 2008) . Induction of rRNA cleavage by chemicals or viruses has previously been suggested to be linked to apoptosis (Banerjee et al., 2000; Naito et al., 2009) raising the possibility that DONinduced rRNA cleavage might be similarly linked to this mechanism of cell death.
The purpose of this study was to characterize DON-induced rRNA fragmentation with respect to (1) specific sites of 18S and 28S rRNA cleavage and (2) identity of upstream signaling events that mediate this process. The results demonstrate that DON promotes cleavage of 18S rRNA and 28S rRNA into a minimum of five and six fragments, respectively. Furthermore, DON-induced rRNA cleavage occurred concurrently with apoptosis was mediated by sequential activation of PKR, Hck, p38, p53, and several caspases. Three other translational inhibitors, satratoxin G (SG), anisomycin, and ricin, evoked comparable selective rRNA cleavage to DON, suggesting that a common mechanism might exist for other ribotoxins.
Chemicals. DON, anisomycin, PKR inhibitor C-16, and Hck inhibitor PP1 were purchased from Sigma-Aldrich (St Louis, MO). Ricin was obtained from Vector Labs Inc. (Burlingame, CA). SG was purified as described previously (Islam et al., 2009) . The p38 inhibitor SB 203580, JNK inhibitor SP600125, ERK inhibitor PD 98059, RNase L Activator, p53 inhibitor pifithrin-a, and pifithrin-l were purchased from EMD Chemicals Inc. (Gibbstown, NJ). Pan caspase inhibitor Z-VAD-FMK was supplied by BD Biosciences (San Diego, CA). [c-32 P]ATP was purchased from PerkinElmer. All other chemicals and media components were obtained from Sigma-Aldrich, except where noted.
Cell culture. RAW 264.7 cells (ATCC, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) heatinactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), streptomycin (100 lg/ml), and penicillin (100 U/ml) at 37°C in a humidified incubator with 5% CO 2 . Cell number and viability were assessed by trypan blue dye exclusion using a hematocytometer. Prior to exposure of toxins (DON, SG, anisomycin, and ricin) or inhibitors, cells (2.5 3 10 6 ) were seeded and cultured in 100-mm tissue culture plates for 24 h to achieve approximately 80% confluency.
RNA purification and fragmentation analysis. RNAs were extracted by TRIZOL (Invitrogen, Carlsbad, CA) following the manufacturer's protocol and their concentrations were measured using a NanoDrop reader (ThermoFisher, Wilmington, DE). Fragmentation of RNA was assessed by denaturing agarose gel electrophoresis (Li and Pestka, 2008) or capillary electrophoresis using an Agilent 2100 Bioanalyzer with a NanoChip (Agilent, Santa Clara, CA) according to manufacturer's instructions.
Northern blot analysis. Northern blot analysis was performed by a modification of a previously described procedure (Li and Pestka, 2008) . Briefly, probes (Table 1) were labeled with c-32 P by DNA 5# end-labeling system (Promega, Madison, WI) and purified by Micro-Bio-Spin6 chromatography column (Bio-Rad, Hercules, CA). Label incorporation was measured using a TopCount NXT (PerkinElmer, Shelton, CT). Total RNA (10 lg per lane) was separated on a 1.2% (wt/vol) formaldehyde denaturing agarose gel and transferred to a Biodyne membrane (Pall Gelman Laboratory, Ann Arbor, MI). After ultraviolet (UV) crosslinking (Stratagene, Cedar Creek, TX), membranes containing immobilized RNA were prehybridized for 1 h at 68°C and then incubated with [c-32 P]-labeled probes (1 3 10 6 cpm/ml) in Quickhyb solution (Stratagene) containing 200 lg/ml of herring sperm DNA at 68°C for 2 h. Blots were washed twice with 23 Side Scatter (SSC) containing 0.1% (wt/ vol) SDS at room temperature and once for 15 min with 0.13 SSC containing 0.1% (wt/vol) SDS at 50°C. The membranes were assembled with the Hyblot autoradiography film (Denville, Metuchen, NJ) into an X-ray exposure cassette and the film was developed after 24 h.
Immunoblotting. Western blot analyses were conducted using primary antibodies specific for murine forms of total/cleaved caspase 9, cleaved caspase 3 (Asp 175), total caspase 8, and cleaved caspase 8 (Asp387) (Cell Signaling, Beverly, MA). Mouse b-actin antibody (Sigma) was also used to verify equal loading. Cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed in boiling lysis buffer (1% [wt/vol] SDS, 1 mM sodium orthovanadate, and 10 mM Tris, pH 7.4), boiled for 5 min, and sonicated briefly, the resultant lysate centrifuged at 12,000 3 g for 10 min at 4°C and protein concentration measured with a BCA Protein Assay Kit (ThermoFisher, Pittsburgh, PA). Total cellular proteins (40 lg) were separated on Bio-Rad precast 4-20% polyacrylamide gels (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL). After incubating with blocking buffer (Li-Cor, Lincoln, NE) for 1 h at 25°C, membranes were incubated with murine and/or rabbit primary antibodies (1:1000 dilution in Li-Cor blocking buffer) to immobilized proteins of interest overnight at 4°C. Blots were washed three times of 10 min with Tris-buffered saline and Tween 20 (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) and then incubated with secondary IRDye 680 goat anti-rabbit and/or IRDye 800CW goat anti-mouse IgG antibodies (Li-Cor) (1:2000 dilution in Li-Cor blocking buffer) for 1 h at 25°C. After washing three times, infrared fluorescence from these two antibody conjugates were simultaneously measured using a Li-Cor Odyssey Infrared Imaging System.
Apoptosis measurement by acridine orange/ethidium bromide staining. Acridine orange/ethidium bromide (AO/EB) staining was carried out using an adaptation of a previously described procedure (Muppidi et al., 2004) . Briefly, slides were cleaned and sterilized by UV light, placed into 100-mm tissue culture plates, and cultured with RAW 264.7 cells (2.5 3 10 6 ) for 24 h to achieve approximately 80% confluency. DON-exposed RAW 264.7 cells were stained for 2 min with 100 lg/ml AO and 100 lg/ml EB in PBS. The slides were washed twice with cold PBS and then covered with coverslip and examined at 3400 under Nikon fluorescence microscope equipped with a wide-band fluorescein isothiocyanate filter. Cells ( 200) were classified based on their nuclear morphology (bright chromatin, highly condensed, or fragmented nuclei) in to four categories: viable normal (VN), DON-INDUCED RRNA CLEAVAGE viable apoptotic (VA), nonviable apoptotic (NVA), and nonviable necrotic (NVN). The apoptotic index was calculated as follows:
Apoptosis measurement by flow cytometry. RAW 264.7 cells were treated with vehicle PBS or DON (1000 ng/ml) for 6 h. Total DON-treated cells and adherent DON-treated cells were collected separately. After washing twice with cold PBS, annexin staining was performed according to the manufacturer's protocol (BD Biosciences). Briefly, cells were resuspended to 13 binding buffer at a concentration of 2.5 3 10 6 cells/ml, and 100 ll of the cell suspension was transferred to fluorescence activated cell sorter tubes. Cells were incubated with 5 ll of annexin V and propidium iodide (PI) for 15 min at room temperature in dark. Finally, samples were resuspended to 400 ll volume with binding buffer, acquired on the Accuri C6 Flow Cytometer (Ann Arbor, MI), and analyzed using FlowJo software (Tree Star, Ashland, OR). Live Raw 264.7 cells were gated using Forward Scatter versus SSC.
Statistics. Data were analyzed by Student's t-test using Sigma Stat 3.11 (Jandel Scientific, San Rafael, CA). Data sets were considered significantly different when p < 0.05.
The capacity of DON to induce rRNA cleavage in RAW 264.7 macrophages was assessed by denaturing gel electrophoresis (Fig. 1A) and capillary electrophoresis (Figs. 1B and 1C). Distinct 18S and 28S rRNA bands were evident in both control and DON-treated RAW 264.7 cells, whereas over five additional rRNA fragment bands were detected in DON-treated cells. Based on its high resolution and reproducibility as compared with conventional electrophoresis, capillary electrophoresis was employed to monitor cleavage in subsequent experiments. When the kinetics of the response were measured, DON at 1000 ng/ml was found to cause rRNA cleavage as early as 2 h that was very robust by 6 h (Figure 2A ). Cleavage at 6 h was concentration dependent with 200 ng/ml of DON evoking modest rRNA cleavage as compared with more marked rRNA cleavage induced by 1000 ng/ml ( Figure 2B ). Based on these findings, incubation with DON at 1000 ng/ml for 6 h was uniformly employed for subsequent mechanistic studies.
Northern blot analyses using 32 P-labeled oligonucleotide probes (Table 1) for 18S and 28S rRNAs were performed to identify and map sites of rRNA cleavage. Incubation with five probes complementary to 28S rRNA revealed six fragments (a-f) with sizes approximating 4000, 3200, 2800, 1. Detection of DON-induced rRNA cleavage in RAW 264.7 by agarose gel and capillary electrophoresis. Cells were treated with or without 1000 ng/ml DON for 6 h. RNAs were purified and analyzed either (A) on 1.2% formaldehyde denaturing agarose gel (10 lg) or (B and C) by capillary electrophoresis (300 ng). The x-axis indicates the size of the fragments in nucleotide (nts) and the y-axis indicates the relative peak intensity in fluorescence units (FU). The two major peaks represent 18S rRNA (~2000 nts) and 28S rRNA (~4000 nts). Arrows designate three significant cleavage peaks between 28S and 18S rRNA and two peaks below 18S rRNA. Rectangle in C indicates chart region to be shown in subsequent figures. 384 HE, ZHOU, AND PESTKA 1500, 1000, and 500 nts (Fig. 3A) . Based on rRNA fragment sizes, probe position and Northern blotting, putative cleavage sites of 28S rRNA were deduced ( Figure 3B ). DON appeared to cleave 28S rRNA into one of two pairs of fragments: a þ e (approximately 5000 nts) and b þ d (approximately 4700 nts). Because no complementary fragment (approximately 2000 nts) to fragment c was detected, it was likely to be a product of the subsequent degradation of a or b.
Autoradiography following hybridization with three probes complementary to 18S rRNA indicated the presence of five fragments (A-E) with approximate sizes of 1000, 800, 600, 500, and 300 nts (Fig. 4 A) . The putative 18S rRNA cleavage pattern suggested that fragments A (approximately 1000 nts) and B (approximately 800 nts) are derived from intact 18S rRNA, whereas the remaining fragments (C, D, and E, approximately 600, 500, and 300 nts, respectively) resulted from further secondary cleavage of the two primary fragments (C and E from A and D from B) (Fig. 4B) .
AO/EB staining of adherent RAW 264.7 cells revealed that DON exposure (1000 ng/ml, 6 h) induced significant apoptosis concurrent with rRNA degradation (Fig. 5A) . Because RAW 264.7 cells attach to the bottom of the cell culture plate under normal physiological conditions but detach during apoptosis, flow cytometry was additionally employed to measure apoptosis in adherent and suspended cells following DON treatment (Fig. 5B) . Both adherent and total populations contained markedly higher annexin V-positive cells (early apoptotic) (60 and 78%, respectively) than the control total cells (31%). Similarly, the percentage of annexin V/PI-double positive cells (late apoptotic) were much higher in adherent (4.8%) and total (10.8%) populations than that of the control (1.2%).
Because DON-induced apoptosis involves activation of PKR, Hck, and MAPKs (Zhou et al., 2003b; Zhou et al., 2005b) , the role of these kinases in rRNA cleavage was determined using selective inhibitors. Both the PKR inhibitor C-16 (0.1 and 0.3 lM) (Fig. 6A ) and the Hck inhibitor PP1 (5 and 25 lM) (Fig. 6B) were found to concentration dependently suppress DON-induced rRNA cleavage. Although the p38 inhibitor (SB 203580; 1 and 5 lM) also inhibited DON-induced rRNA cleavage concentration dependently (Fig. 6C) , inhibitors of JNK (SP600125; 0.2, 1, and 5 lM) and ERK (PO 98059; 20 and 100 lM) did not (data not shown) have suppressive effects. Thus, as has been observed for DONinduced apoptosis, PKR, Hck, and p38 appeared to be key upstream elements for DON-induced rRNA cleavage. 
It was previously shown that p38 mediates the sequential activation of p53 and caspase 3 to induce apoptosis in RAW 264.7 cells (Zhou et al., 2005a) . Suppression of DON-induced rRNA cleavage was observed here both for pifithrin-a (80 and 100 lM) (Fig. 6D) , which can reversibly inhibit p53-dependent transactivation of p53-responsive genes and apoptosis and for pifithrin-l (10 and 25 lM) (Fig. 6E) , which blocks p53 interaction with Bcl-2 family proteins and selectively inhibits p53 translocation to mitochondria. The caspase inhibitor Z-VAD-FMK also caused concentration-dependent inhibition of DON-induced rRNA cleavage (Fig. 6F) . Accordingly, both p53 and caspase activation are additional upstream elements in the signaling pathway leading to DON-induced rRNA cleavage.
Extrinsic and intrinsic apoptotic pathways activate caspase 3 through caspases 8 and 9, respectively. To discern possible contributions of these two pathways, RAW 264.7 cells were treated with DON for 3 and 6 h and the presence of cleaved caspases 8, 9, and 3 was determined by Western blot analysis. Cleavage of caspases 3, 8, and 9 was observed (Figs. 7A and 7B), suggesting that extrinsic and intrinsic pathways could potentially be involved in DON-induced rRNA fragmentation (Fig. 8) .
Evoke rRNA Cleavages Lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria that can activate macrophages via the Toll-like receptor 4 receptor, did not affect rRNA integrity indicating that the macrophage activation per se was insufficient to induce RNA cleavage. Because DON is a translational inhibitor and causes rRNA cleavage indirectly, we questioned whether this might be a common effect for other ribotoxins. Cells were incubated with SG and anisomycin, which can freely diffuse through cell membrane and ricin, a RIP, which can enter the cells by endocytosis and retrograde translocation to ER and cytosol. Unlike SG and anisomycin that directly bind to ribosome to inhibit translation, ricin possesses inherent RNA N-glycosidase activity to depurinate RNA. Although these toxins have apparently different mechanisms for inhibiting translation, SG, anisomycin, and ricin induced a similar apoptosis-mediated cleavage profile to that of DON (Fig. 8) , suggesting that ribotoxins might share conserved pathway for mediating rRNA cleavage. 
Understanding how DON induces rRNA degradation will provide insight into the mechanisms by which trichothecenes and other ribotoxins incapacitate or kill immunocompetent cells. The results presented here establish for the first time that (1) DON-induced cleavage at a limited number of sites in both 18S rRNA and 28S rRNA, (2) these cleavages result from PKR-driven p38 activation, (3) these events closely parallel induction of apoptosis by this mycotoxin, and (4) DON's effects are shared with other ribotoxic agents.
In higher eukaryotes, 28S rRNA contains 12 conserved but variable divergent domains (D1-D12), originating from evolutionary large-scale length and diversity expansions (Michot et al., 1984) . Although the functions of D domains are not clearly understood, D2 and D8 have higher divergency rates than other domains (Houge et al., 1995) . Mouse 28S rRNA cleavage sites have been mapped to be within D2 (approximately 400-1140 nts) and/or D8 domains (approximately 2700-3280 nts) (Houge et al., 1993; Houge et al., 1995; Houge and Doskeland, 1996; Naito et al., 2009) . Apoptosisassociated cleavage pathways were previously reported to target D2 and D8 followed by secondary cleavage in other domains. As demonstrated here, DON-induced rRNA cleavage fragments (b-f) were from the D8 region, whereas two other cleavage sites (a and e) were within D10 (approximately 3785-3822 nts) (Michot et al., 1984) rather than canonical D2. It might be speculated that DON treatment rendered D8 and D10 accessible to constitutive and/or inducible RNases, whereas D2 did not.
We have previously reported that upregulated expression of several RNases in DON-treated RAW 264.7 cells occurs prior to or simultaneously with rRNA degradation (Li and Pestka, 2008) . Particularly notable was RNase L, an enzyme that can mediate inhibition of protein synthesis, apoptosis induction, and antiviral activity (Stark et al., 1998) . Although RNase L is activated by a number of stressors, its only known endogenous direct activator is 2-5A, a product of oligoadenylate synthetase (Pandey et al., 2004; Liang et al., 2006) . Upon binding to 2-5A, RNase L is activated by dimerization and possibly inhibits protein synthesis via the degradation of both messenger RNA and 28S rRNA (Clemens and Vaquero, 1978; Wreschner et al., 1981) . We found that under cell-free conditions in the presence of 2-5A, RNase L readily cleaved both FRET probe and purified rRNA; however, it did not degrade rRNA in The observation that p38 but not JNK mediated DONinduced rRNA cleavage is consistent with previous observations that p38 is a central regulator of cell fate in DON-exposed macrophages (Zhou et al., 2005a) . Our findings further demonstrate that PKR and Hck also mediate DON-induced rRNA cleavage. Although crosstalk between PKR and Hck is still not completely understood, these kinases are wellestablished upstream mediators of DON-induced p38 activation (Zhou et al., 2003b (Zhou et al., , 2005b . These data are consistent with our previous observations that 40S ribosome subunit-associated PKR, Hck, and p38 are activated upon DON exposure (Bae et al., 2010) .
We have previously hypothesized that rRNA cleavage might yield a double-stranded (ds) hairpin fragments capable of activating ribosome-associated PKR through its dsRNAbinding sites (Li and Pestka, 2008) . The data presented here do not support this hypothesis. First, PKR and Hck are activated by DON within minutes (Zhou et al., 2003b (Zhou et al., , 2005b , whereas DON-induced rRNA cleavage was detectable only after 2 h and required relatively higher DON concentrations. Second, suppression of DON-induced rRNA cleavage by PKR, Hck, and p38 inhibitors suggests that their activation is an upstream rather than downstream event. We therefore propose an alternative hypothesis in which entry of DON into the cell rapidly disrupts the conformation of rRNA yielding accessible intact hairpin loops at the surface of ribosome that would be capable of activating PKR or other double-stranded RNAbinding proteins. These events would initiate a stress response that would, especially at high toxin concentrations, ultimately drive rRNA cleavage in concert with apoptosis. This hypothesis is highly consistent with the growing recognition that damage-associated molecular patterns (DAMPs) initiate noninfectious innate immune and inflammatory responses (Newton and Dixit, 2012) . Bulavin et al. (1999) have previously shown that p38 mediates p53 phosphorylation. It was thus notable that rRNA cleavage was found here to be inhibited by pifithrin-a, a compound that reversibly inhibits both p53-dependent transactivation of p53-responsive genes and apoptosis, as well as by pifithrin-l, which blocks p53 interaction with Bcl-2 family proteins and selectively inhibits p53 translocation to mitochondria. Relatedly, we have previously demonstrated that DON induces translocation of BAX (a member of Bcl-2 family) to mitochondria and release of cytochrome c leading to apoptosis (Zhou et al., 2005a) . Finally, DON-induced rRNA cleavage was completely suppressed by the pan caspase inhibitor Z-VAD-FMK, indicating that activation of caspases was a prerequisite of rRNA cleavage.
Caspases, proteolytic enzymes that play important roles in inflammation and cell death, are independently activated via either an intrinsic pathway involving caspase 9 or an extrinsic pathway through caspase 8 (Fuentes-Prior and Salvesen, 2004) . It is thus important to note that DON activated caspases 8 and 9, both of which can activate caspase 3, suggesting the involvement of both apoptotic pathways, respectively, in rRNA cleavage. Caspases mediate degradation of some ribosomalassociated proteins, including eukaryotic translation initiation factor (eIF)2a, eIF3/p35, eIF4B, and eIF4G family (Clemens et al., 2000) . This proteolytic action might expose ribosome- FIG. 7 . DON induces cleavage of caspases 3, 8, and 9 in RAW 264.7. Cells were treated with DON (1000 ng/ml) for 3 and 6 h. Western blotting was used to detect (A) caspase 9, cleaved caspase 9, and cleaved caspase 3 and (B) caspase 8 and cleaved caspase 8. b-Actin was used as loading control. Data are representative of three separate experiments.
FIG. 8. SG, anisomycin, and ricin but not lipopolysaccharide induce rRNA cleavage patterns identical to DON in RAW 264.7. Cells were treated with DON (1000 ng/ml), SG (10 ng/ml), anisomycin (25 ng/ml), ricin (300 ng/ml), and lipopolysaccharide (10 lg/ml) for 6 h. RNAs were purified and analyzed by capillary electrophoresis. Results are representative of three separate experiments. 388 HE, ZHOU, AND PESTKA embedded rRNA to constitutive and inducible RNases, resulting in the highly selective cleavage observed here.
The DON concentrations used in this study (200-1000 ng/ ml) are consistent with those predicted to be in plasma and tissues (e.g., spleen, liver, and small intestine) of mice treated with immunosuppressive doses ( 2 mg/kg bw) of this toxin (Azcona-Olivera et al., 1995; Li et al., 2005 Li et al., , 2007 . Turner et al. (2010) estimated daily intake of DON in male Normandy farmers to range between 27 and 1088 ng/kg/day. Thus, the concentrations employed in the present study might only likely be encountered in humans that have consumed a large bolus dose of DON in a heavily contaminated cereal.
The results presented here and previously demonstrate that DON-induced rRNA cleavage requires prior sequential activation of PKR/Hck, p38, p53, and caspases (Fig. 9) . It was remarkable that ricin, which possesses N-glycosidase enzymatic activity for 28S rRNA depurination, induced the identical rRNA cleavage profile to that of DON and two other low molecular weight translational inhibitors, SG and anisomycin. Possibly, in all four cases, toxin-mediated ribosome damage activated a canonical apoptosis-associated rRNA cleavage pathway. Future work should focus on understanding how DON mediates PKR activation, identifying critical executing caspases and RNases, ascertaining whether induction of rRNA cleavage by other ribotoxins or apoptosis-inducing agents is mediated by the same intracellular signaling pathways as well as validating these findings in animal models.
Supplementary data are available online at http://toxsci. oxfordjournals.org/. FUNDING National Institutes of Health (PHS grant ES003358); United States Department of Agriculture (grant 59-0206-9-058). Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology. This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. The World Health Organization (WHO) Global Influenza Surveillance and Response System provides essential information on the types and variants of influenza viruses circulating worldwide. Direct detection and identification of influenza strains from clinical samples are mostly performed by using specific real-time PCR assays and, less frequently, by culture-based methods [1, 2] . Genotyping or phenotyping characterization of positive cases is performed by sequencing classical PCR amplicons (requiring multiple primer pairs), followed by phylogenetic analysis, or hemagglutinin inhibition assay, respectively.
The influenza A(H1N1)pdm pandemic in 2009 resulted in the development of a number of assays, such as single/multiplex real-time RT-PCR [3] [4] [5] [6] or microarray systems [7, 8] , allowing the rapid detection and typing of influenza virus in human specimens. Sensitive and rapid diagnostic or typing assays are essential for appropriate patient management, particularly high risk patients, and the use of appropriate antiviral therapy.
RT-PCR/electrospray ionization mass spectrometry (ESI-MS) assays were developed recently to enable the potential detection and typing of microbial agents, including influenza [9] [10] [11] [12] [13] [14] , during the same flow procedure [15] [16] [17] . This technology relies on the analysis of nucleotide base *Address correspondence to this author at the Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland; Tel: ++41 22 3724079; Fax: ++41 22 3724097; E-mail: samuel.cordey@hcuge.ch composition signatures of highly variable selected regions based on the measurement of the molecular weight of PCR amplicons [12, 18] .
A first version of the influenza assay (Abbott Molecular, Des Plaines, IL, USA) performed on the T5000 instrument (Ibis/Abbott, Carlsbad, CA, USA) was validated for influenza A (including A(H3N2) and H5N1) and B isolates collected between 1999 and 2006 [13] . Sensitivity and specificity were estimated to reach 97% and 98%, respectively. ESI-MS analysis was also capable of identifying viral reassortments or co-infections (i.e., a mixed population). More recently, this assay reported a sensitivity and specificity of 94.1% and 97%, respectively, for A(H1N1)pdm detection [11] . Since then, the assay has been redesigned and updated. This "PLEX-ID/Flu assay" includes one pan-influenza primer set targeting the PB1 segment, five pan-influenza-A primer pairs targeting individually NP, M1, PA, PB2 and NS1 genes, one pan-influenza B primer pair targeting the PB2 segment, and two additional primer pairs targeting two surface antigens HA (H1) and NA (N1) genes [12] .
The PLEX-ID platform was used to perform a pilot evaluation for the detection and subtyping or lineage characterization of human influenza virus types A and B, respectively. For this, nasopharyngeal swab specimens (NPS) collected from a network of more than 80 practitioners participating actively to the clinical surveillance of influenza cases in Switzerland and screened for influenza during the 2010-2011 season were used as follows. After viral genome extraction using the NucliSENS easyMAG (bioMérieux, Geneva, Switzerland), the following four onestep real-time RT-PCR assays were applied for the routine screening: the CDC pan-influenza A specific real-time RT-PCR assay [19] considered as a reference assay for influenza type A surveillance by the WHO, and three in-house developed assays specific for A(H1N1)pdm (Swine H1 GE), influenza A(H3N2) (A/H3), and influenza B (InfB MP) detection (supplementary Table 1 ) all validated on WHO quality controls. Each week, a batch of 22 or 46 influenzapositive specimens (according to the number of available specimens) were tested with the PLEX-ID/Flu assay in parallel to the usual real-time RT-PCR assays performed by the Swiss National Reference Centre for Influenza. NPS were selected blind of the real-time RT-PCR threshold cycle (C T ) values and the type of influenza (influenza A or B). For each assay, specific positive and negative internal controls were included systematically in each run to rule out any potential PCR inhibitors or contaminations, respectively. and Wisconsin/01/10 strains, respectively). Of note, the PLEX-ID/Flu assay's subtyping or lineage characterization for A(H3N2) and type B specimens, respectively, is less accurate than for A(H1N1)pdm specimens since HA and NA genes are not included in the analysis. Taken together, the PLEX-ID/Flu assay detected positively and gave a typing/lineage characterization result for 93% of all NPS detected positively by real-time RT-PCR. Positive results obtained by both methods were always in agreement. All non-typeable influenza A and B specimens by the PLEX-ID/Flu assay showed relatively low viral loads with C T values 33 obtained with the screening real-time RT-PCR assays.
The typing performance of the PLEX-ID/Flu assay versus the Sanger-based sequencing method was then compared for all influenza specimens with low viral loads (C T values 30; 19 influenza A and 18 influenza B-positive NPS; Table 2 ). Briefly, for the Sanger-based sequencing method, reverse transcription was performed in the presence of Uni12w or BUni11w primers updated from [20] and [21] , respectively, provided by Prof. Rod Daniels (MRC, London). Part of the influenza HA-1 gene was then amplified as follows: for influenza A viruses, a first PCR reaction was performed with cswHAF1 and cswHAR1264 primers, followed by two nested PCR reactions with cswHAF31/cswHAR873 and cswHAF451/cswHAR1264 primer pairs (supplementary Table 1 ). For influenza B viruses, BHA1F1 and BHA1R1 primers were used for the first PCR reaction, followed by a nested PCR using the BHA25/BHAF primer pair. Sequencing was performed with ABI Prism 3130XL DNA Sequencer (Applied Biosystems, Rotkreuz, Switzerland) and analyzed with the Geneious program (Biomatters, Auckland, New Zealand). The same 29 negative controls are used in both comparisons (marked *). ND: not done.
Among these 37 specimens, the PLEX-ID/Flu assay was able to subtype 9/19 influenza A (47.5%) and characterize the lineage for 14/18 influenza B (77.8%). The Sanger-based sequencing method was able to subtype 13/19 influenza A (68.4%) and identify the lineage for 9/18 influenza B (50%). Therefore, for these selected specimens, the PLEX-ID/Flu assay demonstrates a sensitivity for influenza B virus lineage characterization that is at least similar to the Sanger-based method, which is directly dependent on conditions used for viral genome amplification by classical PCR. However, the latter appears more sensitive for influenza A subtyping under these conditions. This observation can be explained by the PLEX-ID/Flu assay algorithm that requires the analysis of nucleotide base composition signatures of a minimum of three positive PCRs out of eight independent primer pairs present in the assay, with the mandatory coupling of detection and subtyping processes, for influenza A specimen analysis. Therefore, the typing constraint of the PLEX-ID/Flu assay explains partially the higher influenza A subtyping performance of the Sanger-based method for low viral load specimens. Indeed, 9/10 influenza A NPS detected positive by real-time RT-PCR could not be subtyped with the PLEX-ID/Flu assay, although one or two positive PCR signals were observed. Although the minimal requirement of three independent PCR signals for subtyping was not obtained, specific influenza A PCR amplifications were detected, suggesting a sensitivity threshold close to the realtime RT-PCR.
Based on a selection of positive NPS collected in Switzerland during the 2010-2011 influenza season, this study suggests that the PLEX-ID/Flu assay is a convenient platform for the detection, typing, and subtyping (lineage characterization for influenza B) of circulating influenza viruses. The detection rate observed with the PLEX-ID platform, as well as the subtyping or lineage characterization of human influenza virus types A and B tends to suggest that it might play a role in the near future in most routine laboratories, but this needs to be confirmed in prospective and large comparative studies. In addition, the sensitivity of the PLEX-ID/Flu observed in this study could be increased if any positive PCR results were considered and if the typing result is not a requirement. However, this pilot study has intrinsic limitations mainly related to the fact that only realtime RT-PCR-positive specimens were selected initially. This precludes any appropriate evaluation of the respective sensitivity or specificity of the method. Compared to sequence analysis, the PLEX-ID/Flu assay has also limitations in terms of identification of specific mutations potentially involved in antiviral resistance, as well as drifted strains. Finally, the lack of accurate typing concerning the HA and NA genes is a major drawback and possibly limits the use of this assay as a first line diagnostic tests.  Evaluation of atherosclerotic lesions using dextran- and mannan–dextran-coated USPIO: MRI analysis and pathological findings Magnetic resonance imaging (MRI) can detect atherosclerotic lesions containing accumulations of ultrasmall superparamagnetic iron oxides (USPIO). Positing that improved USPIO with a higher affinity for atherosclerotic plaques would yield better plaque images, we performed MRI and histologic studies to compare the uptake of dextran- and mannan–dextran-coated USPIO (D-USPIO and DM-USPIO, respectively) by the atherosclerotic walls of rabbits. We intravenously injected atherosclerotic rabbits with DM-USPIO (n = 5) or D-USPIO (n = 5). Two rabbits were the controls. The doses delivered were 0.08 (dose 1) (n = 1), 0.4 (dose 2) (n = 1), or 0.8 (dose 3) (n = 3) mmol iron/Kg. The dose 3 rabbits underwent in vivo contrast-enhanced magnetic resonance angiography (MRA) before and 5 days after USPIO administration. Afterwards, all animals were euthanized, the aortae were removed and subjected to in vitro MRI study. The signal-to-noise ratio (SNR) of the aortic wall in the same region of interest (ROI) was calculated in both in vivo and in vitro studies. Histological assessment through measurement of iron-positive regions in Prussian blue-stained specimens showed that iron-positive regions were significantly larger in rabbits injected with DM- rather than D-USPIO (P < 0.05) for all doses. In vivo MRA showed that the SNR-reducing effect of DM- was greater than that of D-USPIO (P < 0.05). With in vitro MRI scans, SNR was significantly lower in rabbits treated with dose 2 of DM-USPIO compared with D-USPIO treatment (P < 0.05), and it tended to be lower at dose 3 (P < 0.1). In conclusion, we suggest that DM-USPIO is superior to D-USPIO for the study of atherosclerotic lesions in rabbits. Atherosclerosis is a chronic inflammatory response to vessel wall injury, leading potentially, to acute coronary syndrome and cerebral vascular disorders, induced by plaque rupture. The noninvasive imaging of atherosclerotic plaque progression and of therapeutic response is very important. In atherosclerosis, macrophage accumulation initiates lesion development and triggers clinical events by producing several molecules that promote inflammation, plaque disruption, and subsequent thrombus formation. [1] [2] [3] [4] However, conventional anatomic measurements of advanced lesions (eg, plaque size or luminal narrowing) do not necessarily correlate with macrophage content. It is desirable that imaging of macrophages permits the identification of highly activated plaques, to help in the prediction of acute thrombotic events, and to facilitate assessment of the therapeutic effects of antiatherosclerotic drugs, such as statins. In hyperlipidemic rabbits and humans, atherosclerotic plaques have been investigated using magnetic resonance imaging (MRI), using ultrasmall superparamagnetic iron oxides (USPIO): iron oxide nanoparticles stabilized with low-molecular-weight dextran, having a mean diameter of 30 nm. [5] [6] [7] [8] As these relatively small particles are not immediately recognized by the hepatic and splenic mononuclear phagocytic systems (MPS), 9, 10 the prolongation of their intravascular half-life permits their uptake, via macrophages, by the whole body, including the lymph nodes, lungs, and the walls of atherosclerotic vessels. [5] [6] [7] [8] 11, 12 With respect to the excretion pathway, USPIO particles are internalized by receptor-mediated endocytosis, and metabolized via the lysosomal pathway. 13 On internalization by macrophages, the dextran coating is progressively degraded; 89% is eliminated in urine, and the rest is excreted in feces. The iron contained in USPIO is incorporated into the body's iron store and used in hemoglobin manufacture. Like endogenous iron, it is eliminated very slowly, predominantly via the feces. 14 Based on USPIO-associated T2-and T2*shortening effects, atherosclerotic lesions with accumulated USPIO can be detected on MRI scans. Higher doses tended to be administered in earlier animal studies. We posited that improved USPIO with higher affinity for atherosclerotic plaques would make it possible to obtain better images of plaques at lower doses.
The effectiveness of iron oxide nanoparticles, which conjugate with various biomolecules, has been studied previously in investigations of novel therapeutic actions and drug delivery, for example, in blood purification therapy and drug treatments for cancer and hyperthermia. [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] According to Bouhlel et al, 26 the mannose receptors of M2 macrophages are more abundantly expressed in pathological tissues of atherosclerotic lesions than in the adjacent zone. Therefore, we focused on mannan-dextran-coated USPIO (DM-USPIO), prepared in-house by adding alkalitreated mannan to dextran-coated USPIO (D-USPIO). Our working hypothesis posited that the addition of mannan to D-USPIO would facilitate its uptake by macrophage-rich atherosclerotic plaques. Here, we performed MRI and histopathological studies to compare the uptake of DM-USPIO with that of D-USPIO in atherosclerotic plaques of rabbits.
All experimental protocols were approved by our Animal Experimentation Committee. All experiments were conducted in accordance with the Animal Care Guidelines of Shiga University of Medical Science.
We studied two kinds of iron oxide: DM-USPIO and D-USPIO (supplied by Meito Sangyo, Kiyosu, Aichi, Japan). Superparamagnetic iron oxide (SPIO) is a widely-used, safe, liver-specific contrast medium (Resovist ® ; Bayer Health Care Japan, Osaka, Japan); 27 carboxydextran covers the iron core of the iron oxide particles. The particle size of the D-USPIO used in this study is different from that of SPIO but its composition is similar. We prepared DM-USPIO by adding alkali-treated mannan to D-USPIO ( Figure 1) were the same for D-and DM-USPIO (28 nm, 15 mg/mL, 0.028 erg ⋅ gauss -2 ⋅ g -1 , respectively).
To obtain D-USPIO, 84 g of carboxydextran (molecular weight: 2500, approximately) was dissolved in 150 mL of water. To this was added an aqueous solution obtained by dissolving 17 g of ferrous chloride tetrahydrate in 130 mL of a 1 M aqueous ferric chloride solution under a nitrogen gas stream. Then, 220 mL of a 3 N aqueous solution of sodium hydroxide was added, with heating and stirring. The mixture was adjusted to pH 7.0 by adding 6 N hydrochloric acid, and then refluxed for 1.5 hours. The reaction mixture was subjected to centrifugation at 2500 × g for 1 hour, and the supernatant liquid then subjected to ultrafiltration (Centramate™; Pall Corporation, Long Island, NY) (molecular weight cut-off [MWCO]: 100,000) to obtain 1 L of D-USPIO.
To create DM-USPIO we added 500 mL of D-USPIO solution (iron concentration: 30 mg/mL) to 15 g of mannan prepared by extraction from beer yeast and refluxed for 5 hours. The reaction mixture was then subjected to ultrafiltration (MWCO: 100,000) to obtain 500 mL of DM-USPIO.
We used six Watanabe heritable hyperlipidemic (WHHL) rabbits obtained from the Institute for Experimental Animals at Kobe University, Hyogo, Japan. Each was 9-12 months old and weighed approximately 3 kg. At this age, WHHL rabbits harbor active plaque formations in the aortic wall. 28 They were divided into two equal groups and injected intravenously with 0.8 mmol iron (Fe)/kg (dose 3) of D-or DM-USPIO. Gadolinium-enhanced magnetic resonance angiography (MRA) scans were obtained before and 5 days after the injection. For each MRI session, the rabbits were fully anesthetized with ketamine (Ketalar; Daiichi Sankyo, Tokyo, Japan), at 25 mg/kg body weight, and medetomidine (Domitor; Nippon Zenyaku Kogyo, Koriyama, Fukushima, Japan), at 0.1 mg/kg body weight, then injected intravenously with 2 mL gadopentetate dimeglumine (Magnevist ® ; Bayer Health Care Japan), diluted in 10 mL saline.
All scans were obtained using a 1.5T MRI system, using a transmit-receive coil (Magnetom Sonata; Siemens Medical Solutions, Erlangen, Germany) (maximum amplitude: 40 mT m -1 ; slew rate: 200 mT m -1 msec -1 ). For imaging, we used a fast low-angle shot (FLASH) protocol. The parameters used were: TR, 4.3 ms; TE, 1.7 ms; flip angle, 25°; field of view (FOV), 320 × 200 mm; matrix size, 512 × 320; and slice thickness, 1 mm. The source images were made available for analysis on a workstation that allowed interactive multiplanar reformatting of the data sets.
For quantitative analysis, we calculated the signal-to-noise ratio (SNR) in the same three regions of interest (ROI) (3 × 20 pixels) in the aortic wall of the lower intrathoracic aorta in three different coronal reformatted images. We compared the SNR yielded by both types of USPIO.
Of the twelve WHHL rabbits, ten were divided into two equal groups, and injected with D-or DM-USPIO at three different doses: 0.08 (dose 1) (n = 1), 0.4 (dose 2) (n = 1), or 0.8 (dose 3) (n = 3) mmol Fe/kg. Two were untreated and served as controls. The animals were euthanized 5 days post-injection, and MRI scans of resected aortic specimens were obtained.
The specimens were placed in centrifuge tubes filled with gadopentetate dimeglumine (1:50 dilution) and 10% gelatin by weight. MRI was performed using a 1.5T MRI scanner, using a 1-channel loop coil (Signa HDxt; GE Healthcare, Little Chalfont, UK). Imaging was achieved using the three-dimensional (3D) first-spoiled GRASS protocol. The parameters used were: TR, 11.8 ms; TE, 4.1 ms; flip angle, 30°; FOV, 5 × 5 cm; matrix size, 512 × 512; slice thickness, 1 mm. Source images were made available for analysis on a workstation that enabled interactive multiplanar reformatting of the data sets.
For quantitative analysis we calculated the SNR at three ROIs (3 × 20 pixels) in the wall of the lower intrathoracic aorta in three different coronal reformatted images. We compared the results obtained after three different doses of D-and DM-USPIO.
Aortic specimens were fixed in 10% paraformaldehyde. From five different lesions on the lower thoracic aorta, we cut paraffin-embedded 4 µm thick sections on the axial plane. These were stained with Prussian blue to identify the accumulation of iron oxide. We also stained macrophages immunohistochemically with RAM11, and compared Prussian blue-and RAM11-stained areas. For quantitative assessment, we measured the Prussian blue-stained area in one FOV (magnification, 200×) in three different lesions for each histological section of the lower intrathoracic aorta (using Image-Pro Plus; Media Cybernetics, Silver Spring, MD), and calculated the average values. In addition, the amount of iron per unit weight (300 µg) of the lower submit your manuscript | www.dovepress.com Dovepress Dovepress intrathoracic aortic specimens of dose 3 rabbits was measured by nuclear magnetic resonance (Minispec MQ20; Bruker Optics, Billerica, MA) (20 MHz, 0.47 T).
To acquire additional data on the biodistribution of Dand DM-USPIO, we examined tissues from the liver, spleen, lung, kidney, and heart of rabbits which had undergone in vivo MRI study and had received dose 3 of either D-or DM-USPIO (n = 3, respectively); two rabbits treated with neither D-nor DM-USPIO were the controls. These specimens were fixed in 10% paraformaldehyde. Paraffin-embedded 4-µm-thick sections were cut in the axial plane and stained with Prussian blue. We assessed one section for each organ. Prussian blue-stained areas were measured and evaluated as described above.
To compare the phagocytosis of D-and DM-USPIO, we used the murine macrophage cell line, J774.1 (RIKEN Cell Bank; Wako, Saitama, Japan), which is widely used in research on macrophages. [29] [30] [31] Cells (8 × 10 5 ) were seeded in 12 multiwell cluster plates (Corning Inc., Corning, NY) and grown at 37°C for 24 hours in 1 mL growth medium (RPMI; Nacalai Tesque, Kyoto, Japan), supplemented with 10% bovine fetal calf serum by volume (Invitrogen, Carlsbad, CA) and 1% by weight mixed penicillin and streptomycin solution (Nacalai Tesque). The medium was then replaced with fresh medium. D-USPIO or DM-USPIO (10 µg iron oxide and 100 µL, respectively) were added, and the plates incubated for 1 hour for cell labeling. The medium was again replaced and the wells incubated for 24 hours. To identify intracellular iron oxide accumulations, the cells were stained with Prussian blue. The amount of iron contained in cell lysates was measured by atomic absorption photometry (AA-6800; Shimadzu, Kyoto, Japan). Cell labeling was performed as described in a previous study. 32
We used SPSS for Windows software (SPSS Japan, Tokyo, Japan) for statistical analysis. Differences in the uptake of D-and DM-USPIO in the arterial wall, in the uptake of iron by cultured macrophages, and in the SNR values from MRA scans obtained in vivo and in vitro were determined using the one-tailed Student's t-test. A P-value of P , 0.05 was considered statistically significant.
In rabbits treated with dose 3 of D-or DM-USPIO (n = 3, respectively), MRA images obtained in vivo showed irregularities, seen as spotty signal voids in the aortic wall. These were interpreted as indicating iron deposits. Before treatment with either agent, the aortic wall appeared smooth, without any evidence of atherosclerotic plaque formation. The SNR of the aortic wall was significantly lower after treatment with D-and DM-USPIO than before treatment. The difference between the SNR obtained before and after the injection of nanoparticles was significantly greater in rabbits treated with DM-rather than D-USPIO (P , 0.05) (Figure 2 ).
The aortic walls of the control animals were smooth, and the signal emitted was relatively high. In contrast, signal strength decreased in a dose-dependent fashion in rabbits injected with D-or DM-USPIO. At dose 3, the SNR tended to be lower in rabbits injected with DM-than with D-USPIO (P , 0.1). At dose 2 DM-USPIO, the SNRs obtained from three ROIs on three different aortic images were significantly lower than those of the rabbit treated with an equivalent dose of D-USPIO (P , 0.05). At dose 1, there was no statistically significant difference (P . 0.1) (Figure 3) . These findings indicate that the uptake of either type of USPIO produced a decrease in SNR.
Prussian blue-stained histopathological sections showed marked iron uptake by macrophages embedded in atherosclerotic plaques in the aortic walls of rabbits injected with either type of USPIO. This was not the case in the control animals. Iron uptake was dose-dependent ( Figure 4 ). Immunohistochemical staining (RAM11) showed that the localization of iron deposits and of macrophages coincided ( Figure 5 ). Quantitative analysis of iron-positive areas in the aortic wall revealed that they were significantly larger in rabbits treated with dose 3 DM-than in those treated with dose 3 D-USPIO (P , 0.05). In addition, in different aortic sections, these areas were significantly larger in the rabbit treated with dose 1 or dose 2 DM-USPIO than in that treated with an equivalent dose of D-USPIO (P , 0.05) (Figure 6 ). At dose 3 of D-and DM-USPIO, the amount of iron accumulated per unit weight of aortic specimen was not different (Figure 7) .
There was no statistically significant difference in the biodistribution of D-and DM-USPIO in tissues from the spleen and kidney. The values determined by Prussian blue staining in tissues of the spleen were 40,088 ± 10,480 vs 43,607 ± 9456 for D-and DM-USPIO, respectively, and submit your manuscript | www.dovepress.com Dovepress Dovepress were 1711 ± 1508 vs 758 ± 583, respectively, in kidney tissues. In the liver and lung, these values tended to be higher in DM-than D-USPIO-treated rabbits (18,118 ± 5027 vs 14,421 ± 5920 in the liver, and 1261 ± 1363 vs 169 ± 245 in the lung). There was no significant iron accumulation in cardiac tissues. In the controls, only the spleen (the ironstoring organ) showed significant iron deposits. In both D-and DM-USPIO-treated rabbits these levels were increased.
Microscopically, almost all cultured (J774.1) cells phagocytosed both D-and DM-USPIO. However, the amount of intracellular iron measured by atomic absorption photometry was significantly higher in cells treated with DM-rather than D-USPIO ( Figure 8 ) (P < 0.05).
Our experimental study showed that, in WHHL rabbits, both D-and DM-USPIO particles were phagocytosed by macrophages embedded in atherosclerotic plaques. This leads to a susceptibility-induced signal reduction in the atherosclerotic vessel wall on T1-weighted 3D gradient echo (GRE) images. In the T1-weighted fast 3D GRE sequence, T1 shortening is a characteristic of lower USPIO concentration. This renders the signal bright, and produces a predominant T2/T2* shortening at higher concentrations, resulting in a completely dark signal. 33 Consequently, at the initial stage of USPIO administration, the signal of the aortic lumen is completely dark due to a high blood concentration of USPIO. According to Ruehm et al, 5 the uptake by the MPS of USPIO particles over a 4-to 5-day period creates an ideal situation for imaging the vascular wall. The iron concentration in inflammatory cells in plaques is sufficiently high for the production of dominant T2 and T2* effects, while its concentration in the blood pool is at levels at which T1 shortening effects predominate. In that study, iron particles were observed electron microscopically in actively phagocytosing cells, but not in inactive foam cells filled with fat vacuoles. These, and other observations, suggest that both D-and DM-USPIO are potential contrast agents for the diagnosis of atherosclerotic plaques -which exhibit high activity before the onset of luminal narrowing -and are potentially useful for the assessment of therapeutic effects of anti-atherosclerotic drugs, such as statins.
Macrophages and dendritic cells can be targeted by mannosylated nanoparticles because immune cells, including alveolar and peritoneal macrophages, monocyte-derived dendritic cells, and Kupffer cells, constitutively express high levels of the mannose receptor (MR). Hashida and colleagues took the lead in the development of mannosylated liposomes to target macrophages and dendritic cells for delivery (in animal models) of the anti-inflammatory agent dexamethasone palmitate and the CpG DNA complex. 34, 35 The study showed that intratracheally administered mannosylated liposomes, with various ratios of mannosylated cholesterol derivatives, were preferentially taken up by alveolar macrophages. Inhibition studies showed that mediation occurred via mannose receptor endocytosis. Mannosylation significantly improved liposome internalization by macrophages. 36 Our histological analyses showed that the uptake of DM-was higher than that of D-USPIO in the atherosclerotic wall of rabbits, resulting in a greater reduction of signal from the aortic wall in MRI scans. As the amount of intracellular iron was higher in macrophage cells treated with DM-rather than D-USPIO, we posit that macrophages contained in atherosclerotic plaques are more sensitive to DM-than to D-USPIO, making it possible to acquire plaque images at lower doses of contrast agent. We also found that the distribution of DM-USPIO in the liver and lung tended to be higher than that of D-USPIO, suggesting that receptor-mediated endocytosis had a strong role in the internalization of DM-USPIO by macrophages. As mannose receptors are expressed in the liver and lung, [34] [35] [36] it is not surprising that the distribution of DM-USPIO was higher than that of D-USPIO in these organs. We cannot rule out the possibility that increased distribution in these organs results in a reduction in the amount of circulating USPIO particles, and a consequent reduction in their presence in the aortic wall. However, based on our current findings, we suggest that the affinity of DM-USPIO particles for the aortic wall affects their uptake positively, and that the effect of reduced circulation of DM-USPIO due to its distribution in the liver and lungs is not strong. This is because the circulation lifetime of USPIOs is sufficiently prolonged, in accordance with particle size. On the other hand, the amount of iron per unit weight was not different irrespective of the type of USPIO used. We attributed this to technical difficulties with our preliminary arrangements for making NMR measurements. For our NMR measurements, the uniformity of the samples was the most important issue because we used minute quantities of sample for measurement. Nevertheless, it was very difficult to obtain vessel homogenates because the vessels contained abundant fibers. Consequently, insufficient preparation of uniform homogenate samples may have impacted our NMR measurements and caused inconsistency between NMR measurements and other examinations, such as imaging and histological analysis.
Our study has some limitations. First, the number of rabbits was too small to confirm the reproducibility of the observed aortic wall changes, and assessment of inter-individual differences was not possible. Second, the USPIO doses we used were too high for clinical applications; additional studies are necessary to identify appropriate human dose levels. Also, the imaging sequence was not optimized. (The use of a more T2*-weighted GRE sequence with longer echo times may enhance the sensitivity for iron-induced susceptibility effects. With such a sequence, even smaller iron accumulations can be detected. This may facilitate a further reduction in the contrast dose). Third, after the injection of D-and DM-USPIO particles, the signal from the aortic wall was very low and not uniform among the sites examined. ROI placement affects the results of imaging analysis, and ROI analysis of the aortic wall is subject to greater dispersions. In efforts to avoid this potential bias, we used the average result from three different ROIs in our SNR measurements. Fourth, the imaging effects we observed may be specific for WHHL rabbits.
The similarity between atherosclerotic plaque formation in rabbits and humans has been documented elsewhere. [38] [39] [40] [41] We suggest that the observed differences in the uptake of DM-and D-USPIO by rabbit atherosclerotic lesions are attributable to the binding of mannan to USPIO. Further study is required to investigate the stability of bound mannan and to determine optimum dosage amounts.
The trapping of DM-and D-USPIO particles in the atherosclerotic wall of rabbits was indicated by a remarkable decrease in SNR. Our histological and imaging analyses showed that DM-USPIO was taken up to a greater degree than D-USPIO. Based on our observations, we suggest that DM-USPIO is superior to D-USPIO for the study of atherosclerotic lesions. IL-15 Participates in the Respiratory Innate Immune Response to Influenza Virus Infection Following influenza infection, natural killer (NK) cells function as interim effectors by suppressing viral replication until CD8 T cells are activated, proliferate, and are mobilized within the respiratory tract. Thus, NK cells are an important first line of defense against influenza virus. Here, in a murine model of influenza, we show that virally-induced IL-15 facilitates the trafficking of NK cells into the lung airways. Blocking IL-15 delays NK cell entry to the site of infection and results in a disregulated control of early viral replication. By the same principle, viral control by NK cells can be therapeutically enhanced via intranasal administration of exogenous IL-15 in the early days post influenza infection. In addition to controlling early viral replication, this IL-15-induced mobilization of NK cells to the lung airways has important downstream consequences on adaptive responses. Primarily, depletion of responding NK1.1+ NK cells is associated with reduced immigration of influenza-specific CD8 T cells to the site of infection. Together this work suggests that local deposits of IL-15 in the lung airways regulate the coordinated innate and adaptive immune responses to influenza infection and may represent an important point of immune intervention. Influenza virus is a major human pathogen that causes substantial morbidity and mortality-approximately 36,000 deaths annually in the United States alone [1] . Combined with the severe economic burden imposed from seasonal influenza outbreaks and growing concerns over potential imminent influenza pandemics, there is considerable need for a firm understanding of the disease pathology, prevention strategies, and mechanisms of host defense against the virus [2] .
Influenza virus is primarily transmitted via inhaled aerosols and results in an infection localized to the upper respiratory tract, with viral replication largely limited to epithelial cells [3] . Mechanisms by which the immune system eliminates influenza have been well studied and are known to involve the coordinated actions of the innate and adaptive immune systems. Namely, the cytolytic action of influenza-specific CD8 T cells has been shown to be the primary mediator of complete viral clearance, but important roles have also been described for CD4 T cells [4, 5, 6] . In addition to T cells, a crucial role has also been established for innate immune effectors including natural killer (NK) cells, which provide short-term control of viral replication prior to T cell activation [7] . NK cells become activated following the loss of inhibitory signals coupled with positive activating signals resulting in direct (via release of cytotoxic granules and interferon c) or indirect (via activation of macrophages and dendritic cells) target cell lysis [8] . NK cells are vital in limiting influenza viral replication as depletion of NK cells dramatically increases morbidity and mortality in hamsters and mice [9] , and in humans severe infections with the 2009 pandemic H1N1 virus positively correlated with reduced numbers of NK cells in the lungs [10] . Studies have indicated that the natural cytotoxicity receptors NKp44 and NKp46, which recognize hemagglutinin proteins of several different influenza strains [11, 12] is one mechanism used by NK cells to protect against lethal viral challenge [13] . Secondarily, NK cells also aid in viral clearance indirectly through the production and secretion of cytokines which both amplifies local inflammation and recruits antigen-specific CD8 T cells to sites of inflammation [14] . Implicit in both of these functions is the ability of NK cells to accumulate within the respiratory tract to contact infected cells and provide a source of chemotactic signals to recruit recently activated CD8 T cells.
Type I IFNs expressed within hours after viral infection have been documented to induce expression of the chemokines CXCL9 and 10 which function to recruit CXCR3 expressing NK cells to sites of infection [15] . However, Type I IFNs also modulate the expression of the common gamma chain cytokine interleukin 15 [16, 17, 18] , which we recently reported to be temporally and locally increased following influenza infection [19] . This expression of IL-15 in the respiratory tract facilitates the recruitment of antigen-specific CD8 T cells to the respiratory tract. However, it is unclear whether the chemotactic properties of IL-15 uniquely affect migratory CD8 T cells or could be extended to other IL-15-sensitive immune cells. NK and NKT cells are nearly absent in IL-15 2/2 animals [20] , highlighting the important role of IL-15 on NK cell development and homeostasis in the steady-state. Following viral infections, de novo production of IL-15 by dendritic cells results in the activation and proliferation of NK cells [21, 22] , and transient systemic stimulation of NK cells with soluble IL-15/IL-15Ra complexes also results in an accumulation of phenotypically and functionally mature NK cells [23, 24] . In addition to these roles, IL-15 also can stimulate the migration of NK cells in vitro and enhances their adhesion to cultured endothelial cells [25] . We therefore hypothesized that virally induced IL-15 functionally assists in the migration of NK cells into the lung airways. We show here that an IL-15 deficiency results in a site-specific reduction in NK cells from the lung airway and an exacerbation of viral load at early time points post influenza infection. Additionally, exogenous IL-15 induces the specific migration of NK cells in vitro and in vivo. This IL-15dependent enhanced mobilization of NK cells to the lung airways correlates with decreased viral loads. Importantly, in the absence of NK cells, antigen-specific CD8 T cells fail to accumulate at the site of infection, providing a possible link between IL-15-mediated migratory effects of both the innate and adaptive immune responses to influenza infection and suggest therapeutic possibilities regarding the use of IL-15 to simultaneously regulate both arms of the immune system for improved responses to viral infection.
All animals were handled in strict accordance with good animal practice as defined by the American Association for Accreditation of Laboratory Animal Care as well as federal and state agencies. All animal work presented here was approved by Institutional Animal Care and Use Committee of University of Georgia (AUP No. A2009-6-114).
Mice, Viruses, IL-15 Blocking, and NK Cell Depletion C57BL/6 mice were purchased from Charles River (Wilmington, MA) through the NCI program. Influenza A/HK-x31 (x31, H3N2) was generously donated by Dr. S. Mark Tompkins (University of Georgia, Athens, GA). Animals were infected intranasally (i.n.) with 10 3 PFU HKx31 diluted in 50 mL sterile PBS. IL-15 was blocked using 25 mg anti-IL-15 mAb (clone AIO3) (eBioscience, San Diego, CA) administered daily via intraperitoneal injection (i.p.) in 200 mL sterile PBS. IL-15 depletion was confirmed by reductions in frequencies of both NK and CD44 hi CD8 + T cell in antibody-treated animals compared to untreated animals 7 days after the initiation of mAb treatment. NK1.1+ cells were depleted via intravenous (i.v.) injections of 200 uL PBS containing 200 mg anti-NK1.1/mouse (clone PK136) every other day (UCSF monoclonal antibody core facility, San Francisco, CA), and depletion of NK cells was verified using an anti-NKp46 mAb (Clone 21A9.4, eBioscience) throughout the experiment and 2 days after the last injection of the NK1.1 depleting mAb.
Lung airway-resident cells were harvested by bronchioalveolar lavage (BAL) with 3 consecutive washes of 1 mL PBS. To isolate cells from the lung parenchyma, lungs were perfused with ,25 mL PBS/heparin sodium solution, harvested, minced and incubated at 37uC for 30 minutes in 1.25 mM EDTA. The tissue was subsequently incubated in collagenase diluted in RPMI (6 mg/mL) at 37uC for 1 hour and passed through a 5 mm cell strainer. Isolated cells were subjected to separation via density gradient centrifugation by resuspending cells in 47% Percoll underlain with 67% Percoll. The gradients were then centrifuged at 2800 rpm for 20 minutes, and lymphocytes at the interface were collected. Spleen and lymph nodes were harvested from animals, homogenized, and then passed through a cell strainer. Spleen homogenate was depleted of erythrocytes by incubation in tris-buffered ammonium chloride.
Single cell suspensions were stained with combinations of cocktails containing anti-CD3, NK1.1, NKp46, CD11c, CD11b, CD122, and CD132 (eBioscience, San Diego, CA) as indicated for 20 minutes at 4uC. Where indicated, cells were concurrently stained with or without biotinylated anti-IL-15Ra (R&D Systems, Minneapolis, MN) followed by 20 minute incubation with APCconjugated Streptavidin at 4uC. Influenza nuclear protein (NP) MHC class I tetramer [H2-D b /ASNENMETM] were generated by the National Institute of Allergy and Infectious Diseases Tetramer Facility (Emory University, Atlanta, GA). Tetramer staining was conducted at room temperature for 1 hour concurrently with anti-CD3, NK1.1, NKp46, CD8, and CD44 (eBioscience, San Diego, CA). Stained cells were analyzed using a BD LSRII digital flow cytometer (BD Biosciences, San Jose, CA) and either BD FACSDiva or FlowJo software (Tree Star, Inc., Ashland, OR).
IL-15 complexes (IL-15c) were generated on the day of use by incubating 1.5 mg recombinant mIL-15 with 7 mg IL-15Ra Fcchimera (R&D Systems, Minneapolis, MN) at 37uC for 20 minutes followed by 4uC for at least 10 minutes. For Ra only controls, 7 mg IL-15Ra Fc-chimera (R&D Systems, Minneapolis, MN) was incubated similarly to complexes without addition of the cytokine. Complexes or Ra alone were administered via passive inhalation into both nostrils using a micropipette delivering 36.25 mL (for daily treatments) or 72.5 mL (for one time treatments) of complexes in sterile PBS. For assessment of cell proliferation, animals received 2 mg of BrdU (Sigma-Aldrich, St. Louis, MO) administered i.p. in a 200 mL volume of PBS. Cells were isolated from these animals 12 hours after treatment and stained with 20 mL aminoactinomycin D (7-AAD; BD Pharmingen, San Jose, CA) for 20 minutes at 4uC. Cells were surface stained as previously described and stained intracellularly with FITC-labeled anti-Ki-67 and APC-labeled anti-BrdU monoclonal antibodies (BD Pharmingen, San Jose, CA) according to manufacturer's instructions.
In vitro migration assays were performed by placing bulk populations of lymphocytes containing predetermined numbers of NK cells (verified by FACs analysis) from the indicated tissues on the top insert of a 5 mm chemotaxis transwell (Fisher Scientific, Waltham, MA) in which the bottom well contained warm media alone or supplemented with 100 ng/mL IL-15c. IL-15c was generated by incubating 100 ng of IL-15 with 500 ng IL-15Ra Fcchimeric protein at 37u for 20 minutes and 4u for 10 minutes. Plates containing transwells were then incubated at 37uC with CO 2 exchange, and 90 minutes after plating, cells were harvested from bottom chambers and the percent migration of NK cells was calculated as the ratio of the number of NK cells in the bottom chamber compared to the number of NK cells determined in the input sample.
Plaque assays were preformed as previously described [26] . Briefly, lungs from HK-x31 infected animals were collected and homogenized using a tissue lyser (Qiagen, Hilden, Germany). Monolayers of Madin-Darby kidney cells were incubated with 10fold serial dilutions of 10% homogenate in dilution media (16MEM, 1 mg/mL TPCK-treated trypsin) for 1 hour at 37uC. Cells were washed with 16 sterile PBS and overlaid with MEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPoly-mer, Philadelphia, PA), 0.04 M HEPES, 0.02 mM L-glutamine, 0.15% NaHCO 3 (w/v), and 1 mg/mL TPCK-treated trypsin. After 72 hours, the overlay was removed, and the cells were washed with 16 sterile PBS, fixed by incubation with cold methanol/acteone (60:40%), and stained with crystal violet.
Statistical significance was determined by Student's T test using Prism 5 software (GraphPad Software). Significance was determined to be any p-value where p,0.05.
We and others have shown that following influenza infection, IL-15 message and protein is increased in the lung airways [19, 27] . Because this IL-15 expression was rapidly induced by influenza infection and reached significant levels as early as day 3 post infection (p.i.) [19] , we hypothesized that influenza-induced IL-15 expression may be an important mediator of NK cell responses to influenza infection. We therefore first sought to determine whether NK cells responding to influenza infection were capable of receiving signals from this locally produced IL-15. To this end, lymphocytes were isolated from the lung airways of influenzainfected animals via BAL, and CD3 2 , NK1.1 + NK cells were analyzed for the expression of IL-15 receptor components by flow cytometry. The IL-15 receptor is a heterotrimer, consisting of the common gamma chain (CD132), the shared IL-2/IL-15Rb chain (CD122), and the specific IL-15Ra chain [28] . To date the majority of biological effects of IL-15 on NK cells, however, are mediated through the paired co-expression of CD122 and CD132 [29, 30, 31] , while IL-15Ra is only required by accessory cells which present IL-15 to respondent cells, a mechanism referred to as trans-presentation [32] . However, some groups have suggested that IL-15Ra expression alone contains some signaling moieties which may participate in distinct biological functions [27] . Therefore it is important to establish the kinetics of IL-15 receptor component expression and IL-15 signaling potential on NK cells in our model. NK cells are known to respond rapidly to influenza infection and continue accumulating in the lung airways through day 5 p.i. ( [13] and data not shown). Since we wished to specifically evaluate recent NK cell immigrants responding to the airway inflammation resulting from influenza infection, we restricted our analyses of NK cell kinetics to before and up to day 4 p.i.. NK cells were first detected in the BAL at day 2 post influenza infection (albeit at a low frequency, ,0.1-0.6% of lymphocytes, Figure 1A and Figure 2A ) but were completely absent in control mock-infected animals (data not shown). Despite the low frequency of NK cells in the lung airway at this early time point, one fifth consistently expressed IL-15Ra. Additionally, 30-40% of them expressed CD122 and CD132 ( Figure 1B ). By day 3 p.i., when NK cells represented a much more discernible population (,2-3% of lymphocytes, Figure 1A & 2A), greater than 90% of these gated cells expressed CD122 and CD132, and expression levels of these receptors on a per-cell basis increased over time as indicated by a higher median fluorescence intensity by day 4 p.i. ( Figure 1B ) and consistent with evidence that expression of IL-15R components is induced by activating stimuli [33] . Expression of IL-15Ra however, was variable but consistently much lower that the expression levels of CD122 and CD132. This biased expression of CD122/132 receptor chains over IL-15Ra and enhanced IL-15 levels [19] following influenza infection, indicate that NK cells accumulating at the site of influenza infection are capable of responding to locally produced IL-15 via the trans-presentation pathway.
Since co-expression of CD122 and CD132 render NK cells responsive to IL-15 signals, we next wished to determine whether virally induced IL-15 and subsequent signaling through these receptors affected the accumulation of these cells in the respiratory tract. Because IL-15 2/2 mice exhibit a severe developmental defect in both NK and NKT cell lineages and are nearly devoid of these cell populations [20] , we chose to use an anti-IL-15 blocking antibody to selectively deplete IL-15 concurrent with infection. Therefore, we monitored the influx of NK cells in animals receiving either PBS or anti-IL-15 mAb administered i.p. daily from day 0 through day 4 post influenza infection. In order to more accurately define bona fide NK cells, particularly in the lung airways harboring few NK cells at very early time points post influenza infection, we included NKp46 reactivity in our staining protocol and henceforth define NK cells as CD3 2 lymphocytes positive for both NK1.1 and NKp46. While NK cells accumulated in the lung airways of untreated mice as expected, by day 2 p.i. the overall frequency of NK cells in IL-15 blocked animals was reduced by half and remained this low through day 4 p.i. (Figure 2A) . Concordantly, total numbers of NK cells in IL-15blocked mice were partially reduced at day 2 p.i., and substantially (Figure 2A ). In fact, whereas the numbers of NK cells continued to accumulate in the lung airways of control-treated animals through day 4 p.i., numbers of NK cells in the lung airways of treated animals plateaued by day 3 p.i. (Figure 2A ). In the lung parenchyma of control-treated animals, NK cells also accumulated over time post infection, similar to those in the lung airways, but the frequencies of NK cells in this site remained unchanged in IL-15-blocked mice. Numbers of NK cells in the lung parenchyma of anti-IL-15 treated animals also remained similar to control animals with only a slight reduction at day 2 p.i. ( Figure 2B ). Importantly, while the numbers of NK cells were significantly reduced at the site of infection as a result of an IL-15 deficiency, anti-IL-15 treatment had little effect on the frequency or number of NK cells found in anatomical sites distal to the site of infection such as the spleen ( Figure 2C ). In order to determine whether an absence of IL-15 in the lung airways resulted in the reduced frequencies and numbers of NK cells specifically, numbers of other populations of innate cells in the airways at these early time points post infection were analyzed. CD11c 2 CD11b + cells (granulocytes) or CD11c + CD11b 2 (dendritic cells) in the airways following infection were mostly unaffected by the IL-15 deficiency, with only a small reduction observed at day 4 p.i. (Figure 2D ). In contrast, NK1.1 + CD3 + (NKT cells) were markedly reduced in the lung airways of IL-15blocked mice ( Figure 2D ), but overall, these cells represented a low proportion of the innate cells responding to influenza infection at these early time points following infection ( Figure 1A ). Together, these data indicated that short term blockade of IL-15 did not result in global defects in NK cell homeostasis or survival in peripheral tissues and the effects of IL-15 were largely specific to NK cells as blocking IL-15 selectively resulted in a significant loss of NK cells recovered from the site of infection.
To test whether this local reduction in NK cells impairs the control of viral replication, we performed plaque assays on control-and anti-IL-15 treated mice to quantify viral load in the lungs of animals with intact or diminished IL-15 and NK cell responses. Viral load was quantified on days 1-5 and 7-8 p.i. to specifically look at control during the time frame of NK cell entry and accumulation in the lung airways and the kinetics of subsequent viral clearance. In IL-15 blocked animals, differences in viral load were apparent as early as d2 p.i. where viral titers were about 36 higher through day 3 p.i. ( Figure 2E) ; however, these animals seemed to regain control of viral replication by day 4 p.i., which perhaps corresponds with the early entry of cells of the adaptive immune response as anti-influenza specific CD8 T cells are first detectable in the lung airways by d6 post infection by flow cytometry ( [34] and data not shown). Thus, while viral elimination is not ultimately dependent on IL-15, early control of the virus is impaired in the absence of IL-15, which correlates with the arrival of a significant number of NK cells in the lung airways. We thus hypothesized that IL-15 was important for the migration of NK cells in the lung airways following influenza infection.
We observed significant reductions in the numbers of NK cells in the lung airways of influenza-infected animals in which IL-15 was blocked at time points associated with their arrival at the site of infection, and failure of these cell populations to accumulate had implications in early viral control (Figure 2) . In order to determine whether IL-15 might be an important signal for NK cells in the migration to and/or the proliferation within the site of infection, we chose to provide exogenous IL-15 in an attempt to enhance any IL-15-dependent NK cell migration and/or in situ proliferation within the lung airways of influenza-infected animals. To this end, either PBS or recombinant IL-15/IL-15Ra fusion protein complexes (IL-15c) were administered intranasally to mice three days following influenza infection. To ensure that any biological effects of the IL-15c could be attributed to activity of the cytokine (which is merely stabilized by complexing to IL-15Ra), a control group of mice received the IL-15Ra only. Concurrent with treatment, mice received an i.p. pulse of the thymidine analog BrdU to identify proliferating cells. Twelve hours post treatment, the frequency and total number of NK cells in the BAL was quantified as well as the percentage of these cells incorporating BrdU. Isolated cells were simultaneously stained with 7-AAD as an indicator of cell viability, as only cells with disrupted membranes stain positive for this fluorescent dye. Importantly, neither the IL-15Ra alone nor the IL-15c affected cell viability, as cells isolated from animals receiving these treatments had similar percentages of 7-AAD + NK cells as those from PBS-treated mice (data not shown).
Upon introduction of IL-15c to the lung airways, the overall frequency of NK cells isolated from the BAL was significantly increased ( Figure 3A and B) , and the total number of NK cells isolated from this site was nearly three times that of PBS-treated control animals ( Figure 3B ). Interestingly, the percentage of NK cells expressing CD122, the IL-2/15 Rb chain, was reduced in IL-15c-treated animals ( Figure 3B ), perhaps indicative of increased signaling through and subsequent internalization of this receptor complex by IL-15 responsive cells. Unlike T cells, which require large clonal bursts of proliferation to achieve effector status, the effector function of NK cells is more related to activation and mobilization to the site of inflammation [35] . Nevertheless, we wished to test whether the large increases in NK cell number in the lung airways following IL-15c administration could be attributed to IL-15-induced proliferation of NK cells at the site. To assess the potential role of proliferation in this observed increase in NK cell frequency and number in the BAL of treated mice, these NK cells were analyzed for BrdU incorporation. Concomitantly, cells isolated from the BAL were assessed for expression of the cellcycle-specific protein Ki-67. Since BrdU is incorporated into the DNA of only cells in S phase whereas Ki-67 is expressed by cells in any stage of the cell cycle, it was unsurprising that BrdU + cells were only a fraction of Ki-67 + cells ( Figure 3A) . Therefore, we considered only cells positive for both markers as cells undergoing proliferation at the time of treatment. Although the percentage of BrdU-incorporating cells was modestly increased in IL-15c-treated animals, the overall frequency of proliferating cells was low in untreated (,10%) and treated (,12%) animals ( Figure 3B ). These data suggest that IL-15c may trigger the proliferation of NK cells, but proliferation of this cell population in the lung airways is, in general, low and not likely to be solely responsible for the total number of cells extracted from the lung airways. IL-15Ra alone had no significant impact on the accumulation or proliferation of this cell population ( Figure 3A and B) . Finally, differences in cell of CD11c 2 CD11b + cells, CD11c + CD11b 2 cells, and CD3 + NK1.1 + cells from BAL are shown 6 SEM in PBS control (solid lines) or aIL-15-treated (dashed lines) mice (n = 3 mice/group). (E) At the indicated times p.i. with 10 3 pfu HKx31, lung viral titers from PBS and aIL-15-treated mice were determined by plaque assay. Mean viral titer is plotted 6 SEM (n = 2 mice/group on days 1 and 5 p.i. or 3 mice/group for remaining time points; day 2 p = 0.0503). Data are representative of two independent experiments. doi:10.1371/journal.pone.0037539.g002 frequencies, numbers, CD122 expression, and BrdU incorporation were specific to the lung airways (the sight of treatment), as NK cells isolated from spleens were similar in control and IL-15ctreated animals ( Figure 3C and data not shown). Together, these data demonstrate that exogenous IL-15 results in increased numbers of NK cells in the lung airways. Since this increase appeared to be independent of IL-15-mediated effects on cell survival, and proliferation was low, we hypothesized that IL-15 may be responsible for a substantial amount of migration of NK cells into the lung airways following influenza infection similar to its effects on CD8 T cells [19] .
Intranasal administration of IL-15c resulted in increased numbers of NK cells in the lung airways that appeared to be due to increased migration into that site. Previous studies have indicated that IL-15 is indeed chemotactic for NK cells. In vitro checkerboard assays revealed that freshly isolated NK cells migrated to IL-15 gradients, and IL-15 stimulation increased LFA-1-dependent binding of NK cells to cultured endothelial cells [25] . IL-15 has also been shown to play a central role in the recruitment of CD16 2 human NK cells into the endometrium following ovulation [36] . To test the direct chemotactic potential of IL-15c for NK cells in our own system, we employed an in vitro chemotaxis transwell assay. Bulk lymphocytes (or purified splenic NK cells, data not shown) from the BAL, lung, and spleen of mice collected three days after infection with influenza were placed in the top chamber of a transwell filter support with either media alone or media supplemented with IL-15c in the bottom chamber. After 90 minutes of incubation, IL-15c significantly enriched NK cells isolated from the lung and spleen ( Figure 4A ). Consistent with our findings with CD8 T cells [19] , NK cells from the lung airways did not migrate to IL-15c, perhaps because this site represents the terminal destination for these cell populations. Unlike CD8 T cells, which lose expression of CD122 upon residence in the lung airways following influenza infection [19] [37] , nearly 100% of the NK cells residing in the BAL of influenza-infected mice express CD122 ( Figure 1A and B) . Nonetheless, these data indicate that NK cells in the lung parenchyma or the general circulation (as represented by the spleen) migrate to IL-15 in vitro. Intranasal administration of IL-15c during the innate phase enhances early viral control A temporal cessation in IL-15 bioavailability reduced the numbers of NK cells (Figure 2A ) in the respiratory tract resulting in increased viral titers ( Figure 2E) , presumably due to impaired NK cell responses. Conversely, exogenous IL-15c promoted the migration of NK cells ( Figure 4 ) and resulted in increased numbers of NK cells in the lung airways ( Figure 3B ). We therefore hypothesized that intranasal administration of IL-15c early after infection could be used to enhance the early innate immune response to influenza and augment viral control. To this end, influenza-infected animals received either PBS or IL-15c intranasally on days 1-4 p.i., a time frame corresponding to the migration of NK cells into the lung airways and limiting any confounding effects IL-15c might have on adaptive immune cells entering the lung airways at later time points. Every other day, from day 2-8 p.i., whole lungs were collected and viral titers were quantified via plaque assay ( Figure 5A ). Early (d2) p.i., we observed no difference in viral load between PBS and IL-15c-treated mice, but as viral replication reached more significant levels at day 4 p.i., animals receiving the IL-15c had 2.46 less viral load than animals receiving only PBS ( Figure 5B and C) . Although viral titers dropped 10 fold in both groups of animals by day 6 p.i., as expected, surprisingly, there remained a greater than 2 fold significant difference in viral load ( Figure 5B and D) . Although both groups of animals cleared the influenza virus completely by day 8 p.i. (Figure 5B ), our data suggest administration of IL-15c on days 1-4 p.i. can enhance early control of influenza virus by cells of the innate immune system.
Because we have previously reported that IL-15 is important for the migration of influenza-specific CD8 T cells into the lung airways [19] , it was possible that those observed effects were secondary to the recruitment of NK cells to the lung. To test whether the accumulation of NK cells in the lung was required for the subsequent immigration of influenza-specific CD8 T cells to the respiratory tract, influenza-infected animals were assayed for the accumulation of anti-influenza specific CD8 T cells in NK deficient animals, generated by administration of the aNK1.1 depleting mAb PK136 every other day ( Figure 6A ). Flow cytometric analyses of NKp46 expression of day 4 p.i. lymphocytes isolated from the lung, BAL, spleen, and mediastinal lymph nodes (MdLN) of aNK1.1-treated animals revealed robust depletion of NK cells as the number of CD3 2 NKp46 + cells were reduced to less than one third of those observed in PBS-treated control animals ( Figure 6B and data not shown). On days 6 and 8 p.i., the numbers of influenza-specific CD8 T cells in these same tissues were quantified through the identification of cells staining positive for a tetrameric reagent loaded with the immunodominant peptide derived from the influenza nucleoprotein (NP). Tetramer positive CD8 T cells could first be detected in the lung airways at day 6 p.i., and although numbers were low, they were somewhat reduced in the BAL of PK136-treated mice ( Figure 6C ). No reduction in influenza-specific CD8 T cell numbers could be observed in any other tissue. In fact, CD8 T cells seemed to accumulate in other tissues of animals depleted of NK1.1expressing cells. By day 8 p.i., the frequency of influenza-specific CD8 T cells in the BAL of PK136-injected animals was less than half than that of control animals, and the total numbers were reduced nearly threefold ( Figure 6D ). Again, this effect was specific to the lung airways, since frequencies and numbers of NPtetramer + CD8 T cells were unchanged in other tissues examined ( Figure 6D ). Therefore, the largely tissue-specific nature of the dependence of influenza-specific CD8 T cells on the presence of NK1.1-expressing cells in the lung airways partially indicates that lung-resident NK and possibly NKT cells are requisite for the subsequent migration of influenza-specific CD8 T cells into this site. These data suggest that IL-15-mediated migration of CD8 T cells to the lung airways may be, at least partially, an indirect effect of NK1.1 + cells moving into the lung airway space in response to IL-15 and that subsequent tissue remodeling or production of an intermediate factor is responsible for the subsequent recruitment of the CD8 T cells. To our knowledge, these data for the first time describe a role for IL-15 in linking the innate and adaptive responses to influenza infection necessitating the further inquiry of IL-15 as a potential vaccine adjuvant.
Here, we have demonstrated that NK cells, which accumulate in the lung airways early after influenza infection, are dependent on IL-15 for this accumulation and subsequent ability to control viral load. NK cells in the lung airways express high levels of the common gamma chain (CD132) and CD122, receptors responsible for imparting IL-15 responsiveness. Since IL-15 is known to be produced in the lung airways following influenza infection, we investigated the role of IL-15 in NK cell responses to influenza. In the absence of IL-15, NK cell frequencies and numbers were significantly reduced in the BAL, resulting in impaired early control of influenza virus. Although numbers of CD3 + NK1.1 + cells were also substantially reduced following anti IL-15 treatment, it is unclear whether this cell population plays a relevant role in controlling primary infections with influenza. While NK cells are thought to be very important participants in the control of influenza replication, evidence for NKT cells playing a similar role is more controversial [38] . Models of NKT cell activation using aGal-Cer revealed enhanced innate responses to influenza and improved disease outcome [39] , but challenge of CD1d 2/2 mice with influenza led to increased survival, implying an immuno-regulatory role for NKT cells in this model [40] . Whether or not these cells are critically involved in viral clearance, in our model, they represent a very small percentage (,1%) of the lymphocytes in the lung airways in the first five days following infection. Therefore, we believe that an abrogation of virallyinduced IL-15 in the lung airways most dramatically affects CD3 2 , NK1.1, NKp46 double positive NK cells responding rapidly to infection. By the same principle, exogenous IL-15 could be used therapeutically to increase NK cell populations in the BAL-primarily as a result of IL-15-induced migration-to enhance viral control. Therefore, these combined data indicate that influenza-induced IL-15 is an important signal for the migration of NK cells to the lung airways where they help limit viral replication.
IL-15 is produced by a variety of cell types within and outside of the immune system, including dendritic cells, monocytes and macrophages, stromal cells, endothelial cells, and epithelial cells [41] , and pathogenic stimuli are known to induce this expression above constitutive levels [17, 42, 43, 44] . Although dendritic cells are known to be an important source of IL-15 at day 6 post influenza infection [27] , it is unclear whether DCs or other cell types produce IL-15 to facilitate specific immune responses to influenza. For example, lung epithelial cells constitutively express IL-15 and IL-15Ra [45] , and neutrophils and macrophages can be a major source of IL-15 that is produced during a variety of lung inflammatory diseases including sarcoidosis, tuberculosis, bronchitis, and asthma [46] . Following influenza infection, the IL-15producing cell type(s) is/are still unknown, but influenza-induced expression of IL-15 is clearly an important regulator of the NK and CD8 T cell responses to this virus [19] .
Not only might there be different cellular sources of IL-15 at different times after infection, but IL-15 signaling in NK cells could also be regulated by the context in which IL-15 is presented. At least one isoform of IL-15 is known to enter the cell secretory pathway and could therefore be released as a soluble molecule [47, 48] . These data lend the idea that soluble IL-15 could bind heterotrimeric receptors on NK cells. However, IL-15 is also known to be transpresented to NK cells, and indeed, this mode of signaling appears to be most important for IL-15-mediated NK cell survival [49] . While IL-15Ra is dispensable for NK cell survival, IL-15Ra is thought to be an important component in signaling the migration of CD16 2 human NK cells into the endometrium, since this particular NK cell population expresses higher levels of this receptor component than CD16 + NK cells that do not migrate to IL-15 [36] . In our studies, IL-15Ra was only expressed on a relatively low proportion of NK cells in the lung airways of influenza-infected animals ( Figure 1 ). In contrast, CD122 signaling appears to be important for NK cell migration to IL-15 as this receptor chain is down regulated on NK cells exposed to IL-15c in vivo ( Figure 3 ). Thus, we consider it likely that signaling through IL-15Ra is not essential to IL-15-mediated of migration of NK cells to the lung airways. Therefore, future work is needed to identify the IL-15-producing cell population(s) and the mechanism of migration to this cytokine in order to target this response for eventual applications in influenza vaccines and treatments.
A significant finding of our work is that NK cells immigrating into the lung airways are partially required for substantial trafficking of influenza-specific effector CD8 T cells to this location. We found that NK cells are necessary for the optimal accumulation of antigen-specific CD8 T cells, important effectors of eventual viral clearance [4, 5] , at the site of infection, since depletion of NK1.1-expressing cells resulted in a significant decrease in the number of influenza-specific CD8 T cells in the BAL. A subpopulation of CD8 T cells has been shown to express NK1.1 upon activation [50] , and while these cells were detected in the lung parenchyma of influenza-infected C57Bl/6 mice, they comprised less than 1% of the NP-tetramer+ CD8 T cells at this site alone and were never detected in the lung airways. This and the fact that the reduction of activated, influenza-specific CD8 T cells was observed only in the lung airways, and only at later time points post infection lead us to believe that direct depletion of NK1.1-expressing CD8 T cells cannot account for this reduction in treated mice. These data strongly suggest that NK1.1 + cells in the lung airways are requisite for the subsequent accumulation of influenza-specific CD8 T cells in the BAL and implicate IL-15mediated NK cell migration into the lung airways as a link between the innate and adaptive responses to influenza infection. We currently favor a model in which NK cells migrate directly to influenza-induced IL-15 in the lung airways, and, CD8 T cell trafficking, in turn, occurs to both IL-15 directly and to chemotactic factors produced by NK cells already present at the site. A positive feedback loop in which NK cells responding to IL-15 induce further expression of IL-15 by dendritic cells for the stimulation of CD8 T cells is an additional possibility [51] . Moreover, IL-15 has also been shown to modulate chemokine and chemokine receptor expression by NK cells and T cells [52, 53, 54] . As IL-15 deficiencies also cause reductions in CD8 T cell accumulation in the BAL [19] , chemotactic potential of IL-15 for NK cells presented here provides a possible link between IL-15-mediated effects of both the innate and adaptive immune responses to influenza infection. Future work will attempt to tease out the roles of direct and indirect migration of both NK cells and CD8 T cells to IL-15 following influenza infection.
Regardless of mechanism, IL-15-induced trafficking of lymphocytes is an important part of our overall understanding of influenza immunobiology. The studies presented here emphasize the importance of IL-15 in mediating the innate response to influenza via the trafficking of NK cells, as viral titers were not as efficiently controlled early after infection in IL-15-blocked animals. These and previous studies also suggest that exogenous IL-15c could be used to modulate the immune response at both the innate and adaptive phases. Overall, we believe that IL-15 may be in important point of eventual immune intervention for treatment of primary infection and for adjuvanting vaccines. Enterovirus 104 Infection in Adult, Japan, 2011   Screening for Influenza A(H1N1)pdm09, Auckland International Airport, New Zealand Entry screening for influenza A(H1N1)pdm09 at Auckland International Airport, New Zealand, detected 4 cases, which were later confirmed, among 456,518 passengers arriving April 27–June 22, 2009. On the basis of national influenza surveillance data, which suggest that ≈69 infected travelers passed through the airport, sensitivity for screening was only 5.8%. T he virus that caused the 2009 infl uenza pandemic, hereafter referred to as infl uenza A(H1N1)pdm09, is mainly spread internationally by air travel (1) . To prevent or delay such spread, during the pandemic many countries initiated screening of air travelers arriving at airports, even though these measures have not been recommended by the World Health Organization (2) . On April 25, 2009, New Zealand was one of the fi rst countries outside the Americas to confi rm infl uenza A(H1N1)pdm09 in arriving airline passengers (3) . During April 27-June 22, 2009, at the direction of the Ministry of Health, the Auckland Regional Public Health Service began a screening program at Auckland International Airport.
Protocols for border screening were updated throughout the pandemic and evolved as new information became available. Screening was initially applied to all passengers arriving or transferring on fl ights from countries where community transmission of infl uenza A(H1N1)pdm09 virus was occurring. The screening program included the following ( Figure) :
• All fl ights notifi ed New Zealand of the health of passengers and crew on board before landing; if indicated, the aircraft was met by public health offi cials who triaged these travelers.
• Cabin crew announced a scripted health message requesting passengers to identify themselves if symptomatic; after disembarkation, all passengers passed through a public health checkpoint where signage encouraged ill travelers to seek screening.
• Information about infl uenza A(H1N1)pdm09 was available, those with symptoms could self-declare, and public health offi cials visually inspected arriving passengers and approached those with apparent symptoms.
• Neither thermal scanning nor active screening of every arriving passenger was used.
Each unwell passenger and crew member was screened for infl uenza-like illness by a nurse and assessed by a medical offi cer if illness met the defi nition of a suspected case. Those whose illness met the case defi nition had nasopharyngeal swabs taken, were offered oseltamivir, and were sent home or to a facility for isolation. Reverse transcription PCR (RT-PCR) was used to confi rm infection. Screening was escalated on April 29 to include all passengers arriving from other countries and stopped on June 22.
We quantifi ed the results of entry screening for infl uenza A(H1N1)pdm09 at Auckland International Airport. Using the information generated during screening, we retrospectively estimated the number of infected travelers who actually passed through the airport. To estimate the sensitivity of screening, we then compared screening fi ndings with the expected number of infected travelers who passed through the airport. Ethical approval was received from the Northern X Regional Ethics Committee of the New Zealand Ministry of Health.
The number of screened passengers was obtained from airport records. The numbers of crew members on inbound international aircraft were estimated by using averages for fl ights into Auckland. The number of travelers detected at each step and referred to the next step of the screening process was obtained from Auckland Regional Public Health Service records. Virologic test results were extracted from laboratory information systems. A confi rmed case was one that met the current case defi nition (as published on the Ministry of Health website, www.health.govt.nz) and one for which RT-PCR result was positive.
We estimated the number of infected travelers screened as the total number of confi rmed cases in New Zealand during this period, multiplied by the proportion of overseas-acquired cases, and the proportion of international travelers arriving at the airport. On April 30, 2009, nonseasonal infl uenza A (H1N1) was made notifi able, (4). During the screening period, 456,518 international travelers were screened; 406 (0.09%) of these were referred for medical assessment. Of those, 109 (27%) met the case defi nition and received virologic testing. RT-PCR results were located for 89 (82%), among which 4 were positive. A total of 312 cases were confi rmed. The proportion of travelers who acquired the infection overseas was 32%. The proportion who passed through the airport was 69%. The expected number of infected travelers estimated to have passed through the border during the screening program was therefore 69, giving an estimated sensitivity of 5.8% (95% CI 2.3%-14.0%).
The infl uenza A(H1N1)pdm09 screening program at Auckland International Airport had low sensitivity. This form of border screening is therefore unlikely to have substantially delayed spread of the pandemic into New Zealand in 2009.
Limitations of infl uenza screening include the high proportion of asymptomatic infected travelers (5), incubation of infections acquired before or during a fl ight (3), reliance on self-identifi cation, limitations of case defi nitions, and limitations of thermal scanning (6) . Modeling data have shown that the ability of border screening to delay global pandemic infl uenza is closely linked to the effectiveness of the screening process or travel restriction used. To delay infl uenza spread by 1.5 weeks, border restrictions need to reduce imported infections by 90% (7) . The entry screening program we describe does not meet these standards.
The potential effectiveness of screening arriving travelers to prevent or delay infl uenza epidemics has been debated. Mathematical models and literature reviews have argued for (7, 8) and against (9-11) this approach. Some authors have found that entry screening for respiratory conditions or infl uenza A(H1N1)pdm09 is insensitive and not cost-effective (12) . Border screening did not substantially delay local transmission of infl uenza A(H1N1) pdm09 (13) .
This study has several limitations, particularly with regard to estimating the number of infected travelers who would have passed through the airport during the screening period. Most cases of illness acquired overseas would probably not have been notifi ed, particularly those in patients with mild illness who did not see a doctor or who saw a doctor but did not receive a diagnosis. The estimated proportion of overseas-acquired cases was based on data from the fi rst 100 cases and would have decreased as the pandemic progressed. The net effect of these factors is unknown, but they would probably have increased the estimated number of undetected infected travelers passing through screening, thereby further reducing the estimated sensitivity of screening.
Border screening might be conducted for reasons other than preventing or delaying an epidemic. It might provide public assurance and confi dence that something is being done (14) . The communication of health information and advice on how to seek treatment is consistently recommended as a pandemic prevention strategy (12, 15) and is usually delivered as part of border screening programs. These benefi ts need to be balanced against the considerable resources used, opportunity cost (resources used for this activity and thereby unavailable for other activities), uncertain effectiveness, and inconvenience of border screening.
To delay or prevent infl uenza entry at borders, infl uenza screening needs to be considerably more effective than the mostly passive program described here. We hope that during this interepidemic period, a major international review of the role of international air travel in the dissemination of emerging infectious diseases will be conducted to identify effective interventions. Such a review should consider systemwide approaches, including exit screening, standardized health declarations, active screening of individual passengers (including use of rapid laboratory tests and thermal scanning), passenger tracking, policies and practices that support sick travelers wishing to defer travel, and circumstances where airline travel should be suspended entirely. Immunologic Changes during Pandemic (H1N1) 2009, China We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-γ, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later. mm 3 in 9 (32.1%) of 28 patients and represented <55% of total lymphocyte counts in 4 (14.3%) patients (Table). CD4 T-lymphocyte counts were <400 cells/mm 3 in 13 (46.4%) patients and represented <31% of total lymphocyte counts in 8 (28.6%) patients. CD8 T-lymphocyte counts were <190 cells/mm 3 in 3 (10.7%) patients. B-cell counts were <90 cells/mm 3 in 1 (3.6%) patient. Natural killer cells represented >27% of lymphocyte counts in 4 (14.3%) patients. Fourteen (50.0%) patients had a CD4:CD8 ratio less than the standard reference ratio of 1.4.
Flow cytometric results showing development of peripheral blood lymphocyte subsets during the disease course were divided into 3 groups on the basis of time of illness onset until date of blood sample collection: 1-3, 4-6, or 7-10 days. These groups were used because the longest time from onset of illness to hospitalization was 3 days for all patients, and the peak temperature for patients was observed 3 days after illness onset.
Mean counts and percentages of all T-and B-lymphocyte subsets increased after 3 days of illness compared with results obtained during the fi rst 3 days of illness ( Figure 1 ). However, the increase in CD8 and B cells was not signifi cant. Another study showed a decrease in CD4, CD8, and B cells ≤2 days of symptom onset in patients with pandemic (H1N1) 2009 than in healthy persons (4). Our results show impaired adaptive immune responses and a gradual increase during recovery in mildly affected patients. We measured serum cytokine concentrations and hemagglutination inhibition (HAI) antibody titers in patients during hospitalization and the follow-up period ( Figure  2 ). We observed an increase in interleulin-6 (IL-6) levels 1 day after illness onset, which were 6.0-fold higher than the baseline level, and a 2.3-fold increase in interferon-γ (IFN-γ) levels. These levels decreased to baseline levels 5 days after illness onset, although the IL-6 level 5 days after illness onset was higher than levels 15 and 37 days after illness onset. The maximum IL-10 level 1 day after symptom onset was 3.2-fold higher than the baseline level. This level decreased to a value lower than the baseline level within 4 days, and then gradually increased to the baseline level 37 days after illness onset. Serum IL-6, IFN-γ, and IL-10 levels were not related to patient temperature 1 day after symptom onset, peak temperature during the disease, or period of fever. These levels showed minor differences that were not related to cough or sore throat in patients.
Only 1 patient had an HAI antibody titer ≥10 (titer 20) 1 day after illness onset. The HAI geometric mean titer increased 5 days after symptom onset compared with that 1 day after symptom onset and continued to increase until it reached a peak level of 137.9 at 37 days after symptom onset (25.5-fold increase). Peak HAI antibody titers ≥40 and ≥4-fold increases were observed in 27 (96.4%) patients.
Bermejo-Martin et al. reported increased serum levels of IL-6, IFN-γ, and tumor necrosis factor-α in patients with pandemic (H1N1) 2009 during the fi rst 5 days after symptom onset; no difference in levels of these 3 cytokines was observed in patients with mild disease and controls (5) . However, similar to another report (4), we detected increases of IL-6 and IFN-γ levels in patients with mild disease during the fi rst 3 days after symptom onset. These different patterns may be caused by different intervals from time of symptom onset to date of sample collection (5 days vs. 3 days) because IL-6 and IFN-γ levels in our study quickly decreased to baseline levels ≤7 days after symptom onset. These results suggest that serum IL-6 and IFN-γ , and IL-6 are medians (pg/mL). Serum HAI antibody titers were transformed by using the natural logarithm and are shown as means. Baseline cytokine concentrations on the y-axis are values for healthy persons. *p<0.05 when IL-6 or HAI antibody levels were compared with those at day 5; †p<0.05 when IL-10 level was compared with those at baseline; ‡p<0.05 when IL-10 level was compared with those at days 5 or 15; §p<0.05 when value was compared with that at any other time point; ¶p<0.05 when value was compared with those at days 5, 15, or 49. levels may be increased in patients with pandemic (H1N1) 2009 within the fi rst 3 days after symptom onset, followed by a decrease to baseline levels ≤5 days after symptom onset in patients with mild disease or a continuous increase in severely affected patients. IL-6 and IFN-γ are associated with antiviral immune responses during infl uenza infection (6) (7) (8) . However, continuous, excessive release of IL-6 three days after illness onset likely contributed to serious pulmonary infl ammation and tissue injury, as has been documented for severe acute respiratory syndrome and 1918 pandemic infl uenza, but this release could be tempered by production of IL-10 (6, 7, (9) (10) (11) .
The proportion of persons 18-60 years of age with a ≥4-fold increase in HAI titer who received 1 dose (15 μg) of monovalent pandemic (H1N1) 2009 nonadjuvant vaccine was 96.2%, and the proportion with an increased HAI titer ≥40 was 97.1%, results similar to those of a recent study (12) . However, the geometric mean titer in healthy vaccinated persons was 237.8, a 34.5-fold increase over the prevaccination titer, which was greater than that for patients naturally infected with pandemic (H1N1) 2009 virus (12) . This fi nding may have resulted from impaired adaptive immune responses against pandemic (H1N1) 2009 virus in the initial phase, which included decreased numbers of CD4 and B lymphocytes and an increase in T regulatory cells (4) .
In conclusion, our data indicated changes in cellular profi les during pandemic (H1N1) 2009 virus infection; showed that transient production of IL-6, IFN-γ, and IL-10 are main effectors of the early innate immune response against pandemic (H1N1) 2009 virus; and indicated that adaptive immune responses are impaired in the initial phase after infection. These factors may help clarify the pathogenesis of pandemic (H1N1) 2009 virus and provide new approaches in overcoming severe infections. Pandemic (H1N1) 2009 Risk for Frontline Health Care Workers To determine whether frontline health care workers (HCWs) are at greater risk for contracting pandemic (H1N1) 2009 than nonclinical staff, we conducted a study of 231 HCWs and 215 controls. Overall, 79 (17.7%) of 446 had a positive antibody titer by hemagglutination inhibition, with 46 (19.9%) of 231 HCWs and 33 (15.3%) of 215 controls positive (OR 1.37, 95% confidence interval 0.84–2.22). Of 87 participants who provided a second serum sample, 1 showed a 4-fold rise in antibody titer; of 45 patients who had a nose swab sample taken during a respiratory illness, 7 had positive results. Higher numbers of children in a participant’s family and working in an intensive care unit were risk factors for infection; increasing age, working at hospital 2, and wearing gloves were protective factors. This highly exposed group of frontline HCWs was no more likely to contract pandemic (H1N1) 2009 influenza infection than nonclinical staff, which suggests that personal protective measures were adequate in preventing transmission. A ustralia was affected early in the (H1N1) 2009 infl uenza pandemic with 37,636 cases and 191 deaths reported. The state of Victoria was the fi rst to observe a substantial peak in the number of persons infected (1) . The pandemic was managed within the framework of the Australian Health Management Plan for Pandemic Infl uenza (2) . Guidelines for use of personal protective equipment (PPE) were established in the Victorian Health Management Plan for Pandemic Infl uenza (3) . Recommendations included use of N95 masks, gloves, protective eyewear, and longsleeved gowns.
Infl uenza in health care workers (HCWs) is common, and acquisition in the workplace is well documented. An uncontrolled study found that after an infl uenza epidemic in Glasgow, Scotland, 120 (23.2%) of 518 HCWs seroconverted (4) . Early in 2009, twelve HCWs with probable or possible work place acquisition of pandemic infl uenza were reported in the United States. None had worn full PPE (5) .
That HCWs may be concerned about attending work during a potentially serious infl uenza pandemic is not surprising. During the severe acute respiratory syndrome outbreak of 2003, some HCWs reportedly stayed at home for fear of becoming infected and transmitting infection to family members. A number of surveys have found that 16%-33% of HCWs may not report to work in the event of an infl uenza pandemic (6) (7) (8) (9) .
HCWs need to know the transmission risks to make rational decisions about working during an infl uenza pandemic. Because HCWs are exposed in the community as well as the workplace, they should know about the additional risks for contracting infl uenza at work. This information is also imperative for pandemic workforce planning.
We sought to determine whether frontline HCWs were at greater risk for contracting pandemic (H1N1) 2009 infl uenza than the control group. Additionally, we sought information on factors that may have increased or decreased the risk for infection.
We conducted a cohort study, comparing frontline HCWs with intensive patient contact (clinical) and staff with no patient contact (nonclinical). Frontline HCWs were defi ned as those who worked >1 shift per week and had likely exposure to patients with pandemic infl uenza infection. These workers included doctors, nurses, and physiotherapists, as well as others in the emergency department, intensive care unit, infectious diseases units, and respiratory and other wards where patients with suspected pandemic infl uenza were housed. Staff members who had no clinical contact were chosen as a convenient surrogate for a community control group. These workers included university and hospital staff in nonpatient contact areas such as the library, information technology, and administration. This study was approved by the Human Research Ethics Committees at each of the hospitals and all participants gave written informed consent. The study was conducted from August 24, 2009, through December 16, 2009. Four tertiary referral hospitals in metropolitan Melbourne were involved: Royal Melbourne, St Vincent's, Austin, and Alfred Hospitals. At all sites, patients with suspected or confi rmed pandemic infl uenza infection were cared for in negative pressure isolation rooms when they were available, and in private rooms when they were not. Institutional infection control policies directed that gloves, gowns, goggles, and masks be used when caring for these patients. Use of N95 masks was initially recommended in all hospitals, although hospital 1 changed to surgical masks after June 16, 2009 . Hand hygiene with an alcohol-based product and respiratory etiquette were promoted at all hospitals.
The progression of the pandemic in Victoria is shown in Figure 1 . The original research plan was to obtain 2 serum samples, 3 months apart, from all participants to test for seroconversion and also to obtain weekly nose swabs for pandemic infl uenza detection by using real-time PCR. By the time the study commenced, the pandemic was waning, infl uenza cases were decreasing in Victoria, and following the original study plan was not considered feasible.
The plan was thus modifi ed. An initial serum sample was obtained from all participants to measure for pandemic infl uenza antibodies. At study entry, participants completed a Web-or paper-based questionnaire that requested information on demographic characteristics, known infl uenza exposures outside the workplace, and any history of fever or respiratory symptoms occurring during the pandemic but before the study. In addition, the clinical group was asked about work exposure to patients with suspected pandemic infl uenza and their usual use of PPE when caring for these patients. Participants were also asked about use of neuraminidase inhibitors (NIs) and specifi cally whether they received prophylaxis after exposure to a patient with confi rmed infl uenza.
Participants were instructed to provide nose swab specimens for viral testing if they experienced signs and symptoms, including cough, sore throat, rhinorrhea, laryngitis, fever, myalgias, or headache. All were asked to complete a weekly questionnaire regarding symptoms, infl uenza exposure, and use of NIs. If a participant reported respiratory illness, a second serum sample was requested for antibody testing to document possible seroconversion.
Serum was tested for antibodies to pandemic (H1N1) 2009 infl uenza virus by using the hemagglutination inhibition assay with A/California/7/2009 virus and turkey red blood cells (10) . A titer of <40 was defi ned as negative and >40 as positive. Nucleic acid detection was performed on nasal swabs by using reverse transcription PCR (RT-PCR) for infl uenza-specifi c and pandemic (H1N1) 2009 virus-specifi c sequences on swabs; kits were provided by the Centers for Disease Control and Prevention (Atlanta, GA, USA) (11) and an ABI-7500FAST instrument at the World Health Organization (WHO) Collaborating Centre for Reference and Research on Infl uenza in Melbourne.
On the basis of early estimates of antibody positivity to pandemic infl uenza virus in the community, we assumed 20% infection rates in clinical staff and 10% rates in nonclinical staff. We calculated that 438 participants were required to achieve 80% power to detect this difference using a 0.05 two-tailed signifi cance level. The primary outcome was the presence of a positive antibody titer in the fi rst serum sample, indicating likely pandemic infl uenza infection.
We performed 2 separate univariate and multivariate analyses to delineate putative risk and protective factors (1 included all participants and the other included clinical participants only) to investigate any association between health care-specifi c risk factors and pandemic infl uenza. Multivariate analysis was performed by using forward and backward stepwise logistic regression, including all variables in the model initially and a p value for removal 
The study took place from August 24, 2009, through December 16, 2009, largely before release of the pandemic infl uenza vaccine, and no participant was vaccinated during the study. Table 1 shows the number of patients who had confi rmed pandemic infl uenza infection (by PCR) and were treated in each of the hospitals. Characteristics of study participants are shown in Table 2 . The median participant age was 38 years (range 18-74 years); 27% were <30 years of age, 20% were 30-39 years of age, 25% were 40-49 years of age, and 20% were >50 years of age. Figure 2 shows the reverse cumulative distribution of fi rst serum antibody titers, according to age. We found no statistically signifi cant difference between the curves (p = 0.11 by ordinal logistic regression).
On multivariate logistic regression, the only factor associated with a higher risk for pandemic infl uenza among all participants was younger age (OR 0.96, 95% CI 0.94-0.99) after adjustment for participant status (clinical vs. nonclinical), age, gender, hospital, seasonal infl uenza vaccination, confi rmed pandemic infl uenza, reported respiratory illness, community contact with infl uenza, oseltamivir prophylaxis, number of children in the household <18 years of age, and hours worked per week. On univariate analysis, the only factors that were signifi cantly associated with protection against infection in the clinical group were use of any mask (OR 0.16, 95% CI 0.03-0.97) and use of gloves (OR 0.09, 95% CI 0.02-0.5) for patients in droplet precautions. Adjusted odds ratios are shown in Table 3 .
Of the 395 participants, 140 (35%) reported a respiratory illness and 46 had nose swabs taken. Seven were positive for pandemic (H1N1) 2009 virus by PCR, 1 for subtype H3N2 infl uenza, and 38 were negative. One of the 46 had 2 swabs taken during different illnesses; the fi rst was positive and the second was negative for pandemic (H1N1) 2009 virus. PCR cycle threshold values for swab specimens were from 30 to 40, indicating low viral loads. This fi nding may indicate that poor swabbing techniques were used, that the sample had been taken as infection was waning, or that level of infection was low (data not shown).
For 87 participants, a second serum sample was taken because of a reported respiratory illness. The average number of days between the fi rst and second sample was 60 days (range 28 to 122 days, median 54) days. Thirtysix participants who had nose swabs performed also had a second serum sample taken. Seroconversion occurred in only 1/87 workers, with an initial titer of <10 and a subsequent titer of 40 (76 days later). This participant had a nose swab taken during a respiratory infection, which was negative for infl uenza virus. Seroconversion did not occur in any of the participants with a positive nose swab specimen. The mean number of days from obtaining a One participant with a positive nose swab sample did not have a second serum sample taken. None of the participants with a positive nose swab or seroconversion reported taking NIs in their weekly survey. Four of the 7 participants with a positive PCR result and the 1 in whom seroconversion occurred were in the clinical group (3 doctors, 1 pharmacist, 1 nurse, 1 physiotherapist). The participant who showed seroconversion was 29 years of age; participants with a positive PCR result ranged from 24-63 years of age. Two of the participants with a positive PCR result worked on the infectious disease ward, 2 in the emergency department, and 1 in the intensive care unit; seroconversion occurred in the participant who worked in a medical ward. Five of the participants with positive PCR results and the participant in whom seroconversion occurred had received the 2009 and previous seasonal infl uenza vaccines. None of the participants with confi rmed infl uenza reported taking oseltamivir for either prophylaxis or treatment.
In total, 395 participants completed 1-13 weekly questionnaires each. Eighty-nine clinical and 51 nonclinical participants reported 139 and 91 respiratory illnesses, respectively. No participant reported having laboratoryconfi rmed pandemic (H1N1) 2009 infl uenza. Six reported community contact with someone who had laboratoryconfi rmed infection. One reported taking oseltamivir after contact with an infected person in the workplace. This person had 2 serum samples taken 88 days apart; both had an antibody titer of <10.
In this study, we evaluated the risk for pandemic (H1N1) 2009 in HCWs compared with the risk for such infection in a control group, as well as the factors associated with infection. HCWs had slightly higher rates of seropositivity than nonclinical staff; however, this difference was not statistically signifi cant. Our data are supported by results of another recent study, which found that being a HCW was not a risk factor for serologically confi rmed seasonal infl uenza virus infection and that the risk of HCWs acquiring infl uenza was more strongly associated with household than workplace exposure (12) . That study found a seroconversion rate of 11.2% in HCWs and 10.3% in non-HCWs. However, it examined only doctors and nurses, whereas our study included other types of frontline HCWs. Another study reported a seroprevalence for pandemic (H1N1) 2009 of 26.7% in HCWs, which was not signifi cantly different from the seroprevalence of the general population (13) . Neither of these studies examined use of PPE.
Overall, we found that 17.7% of participants had serologic evidence of pandemic (H1N1) 2009 virus infection after the peak of the outbreak. This proportion refl ects the observed 16% seroprevalence in adults in Melbourne (14) . These rates are lower, however, than the 31.7% antibody positivity found in South Australia during a prelicensure study of pandemic infl uenza vaccine in July 2009, which excluded subjects with confi rmed or suspected pandemic (H1N1) 2009 infl uenza (15) . This difference in titers may have refl ected geographic differences in infection rates or differences between the populations sampled.
In the analysis of all participants, we found that older age was associated with lower rates of pandemic (H1N1) 2009 infl uenza infection. We did not observe higher levels of preexisting antibodies against pandemic (H1N1) 2009 infl uenza with increasing age, which has previously been reported. However, results of other studies examining the relationship between seroprevalence and increasing age are confl icting (15) (16) (17) (18) . Immune mechanisms other than typespecifi c antibodies may be providing protection for older participants. Other possibilities are that older persons have older children who may be less likely to acquire or transmit infl uenza or that older participants were more conscientious with respiratory etiquette and hand hygiene; attempts to measure these factors were not included in this study.
Among the HCWs we studied, working at hospital 2 conferred protection against pandemic (H1N1) 2009 virus infection. This hospital was in a geographic area with fewer cases than the others, but if this were the explanation, then a similar fi nding might have been expected in the nonclinical group, which was not demonstrated. Furthermore, at least as many cases of confi rmed pandemic (H1N1) 2009 infl uenza were seen at hospital 2 as were seen at the other hospitals (Table 1 ). Factors such as reported compliance with PPE, were adjusted for in the multivariable analysis to reduce the effect of hospital type on infl uenza risk. The reason for the lower risk associated with hospital 2 has not been identifi ed but may relate to other unmeasured factors, such as compliance with hand hygiene procedures.
Wearing gloves while caring for patients as part of droplet precautions was strongly associated with a lower risk of having had pandemic (H1N1) 2009 virus infection. Use of gloves was highly correlated with use of gowns, masks, and eye protection on logistic regression (results not shown). This fi nding confi rms the great importance of PPE in preventing transmission of respiratory viruses in the health care setting and may explain why HCWs with defi nite exposure to infl uenza in the workplace, in addition to probable exposure in the community, do not have higher rates of infection than those with only community exposure.
The risk for pandemic (H1N1) 2009 virus infection increased with the number of children <18 years of age living in the participant's household, which has previously been reported as a risk factor (12) . In Victoria, the median age of persons with reported pandemic (H1N1) 2009 virus infection was 15 years, with 67% of all notifi ed casepatients being 5-17 years of age (1). Miller et al. also found that children were predominantly infected (17) . This fi nding, coupled with the diffi culties of maintaining good respiratory etiquette in young children, is a plausible explanation for the effect of child number on infection risk.
Working in the ICU was also identifi ed as a risk factor for pandemic infl uenza; patients in ICU may be severely ill, with high viral loads, and staff may be heavily exposed during multiple aerosol-generating procedures. In addition, use of PPE and hand hygiene compliance may have been lower than in other wards or patients with pandemic infl uenza may have been unrecognized and therefore appropriate PPE not used.
Exposure of HCWs to suspected or proven pandemic infl uenza in the community was protective against having a positive antibody test result. This fi nding is counterintuitive and diffi cult to explain. One hypothesis is that HCWs who knew that they had had community exposure may have been more attentive to hand hygiene and other infection control precautions while at work or were more likely to enact social distancing.
We found only 1 instance of seroconversion among the 87 participants (including the 6 with PCR-confi rmed infection), each of whom had 2 serum samples taken for antibody measurement. Miller et al. reported that 89.1% of participants with pandemic (H1N1) 2009 had an antibody titer of >32 three weeks after infection, although a baseline serum sample was not taken; therefore, seroconversion could not be demonstrated (17) . None of the participants with positive PCR results reported taking NIs, and all had serum samples taken >2 weeks after the positive nose swab specimen, allowing suffi cient time for seroconversion. Our results are likely to be true positives, as all swabs were only taken when patients were symptomatic. Previously, virus isolation has been the gold standard for infl uenza detection but RT-PCR is now considered to be more sensitive and specifi c. A previous study by some of the current authors has shown that seroconversion occurs in 80%-90% of serum samples if they are tested a suffi cient time after infection (confi rmed by RT-PCR) (19) . Nasal swabs are a relatively peripheral type of sample (20) . If viral load is low in the nose, the sample may be insuffi cient as an antigenic stimulus to induce a detectable level of seroconversion in the serum. This may be the explanation for the lack of seroconversion seen in some PCR-positive cases in this study. Because the number of pandemic (H1N1) 2009 cases in Victoria was low by the time this study commenced, we used a single antibody measurement for diagnosis in most patients. This is not ideal, because some participants may have had preexisting cross-reactive antibodies, as reported by others (15, 16) . However, this cross-reactivity has been most commonly found in older persons >65 years of age, a population which was underrepresented in our study. The explanation given for presence of cross-reactive antibodies in older persons has been past exposure to other antigenically similar viruses or a lifetime exposure to infl uenza A virus (17) . Because this exposure could not have occurred in our younger study participants (median age 38 years) and serum samples were collected toward the end of the pandemic wave when many would have already been exposed, reactivity likely was specifi c to pandemic (H1N1) 2009. These factors support the use of a single antibody measurement for diagnosis.
This study relied on self-reported symptoms and risk factors, including use of PPE, making it subject to recall bias. This is a particular problem potentially for recall of exposures (e.g., to others with infl uenza or for use of PPE). However, many of the predictor variables were not subject to recall bias (e.g., clinical or nonclinical status, work place, age, gender, occupation, and number of children in the household). In addition, in order to infl uence the results, the 2 exposure groups would have had to exhibit differential recall. Although it could be postulated that HCWs may have perceived that they were at greater risk for exposure and may have therefore been more conscientious when fi lling out questionnaires, we believe that because of the large amount of public awareness of pandemic (H1N1) 2009 at that time, it is unlikely that this group would have been more conscientious than the nonclinical group.
In conclusion, we found that HCWs did not have a substantially increased risk of contracting pandemic (H1N1) 2009 in a health care setting with high availability of PPE. We conclude that use of PPE was highly protective against acquiring pandemic (H1N1) 2009 virus infection, and we therefore encourage its use, along with scrupulous hand hygiene and respiratory etiquette. Coronavirus HKU1 in Children, Brazil, 1995  with a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) by using random primers according to the manufacturer's instructions. The cDNA obtained was screened with primers able to amplify a 220-bp product in the conserved polymerase region of all known HCoVs (5) and other coronaviruses (e.g., bovine coronavirus). We used cDNA obtained from cultured human rectal tumor cell line HRT-18G cells inoculated with bovine coronavirus strain Kakegawa as positive controls for PCR.
In an attempt to improve the sensitivity of HKU1 detection, we analyzed samples with negative results by PCR with a nested PCR specifi c for coronavirus HKU1. This nested assay was designed on an alignment of our HKU1-positive sample sequence (BRA169) and different HKU1 genotype sequences deposited in GenBank. Primers Fn-HKU1 (forward 5′-CGTGCYA TGCCAAATATTTTGCG-3′, HKU1-NC_006577, nt 15433-15454) and Rn-HKU1 (reverse 5′-TAGCAACC GCCACACATAAC-3′, HKU1-NC_ 006577, nt 15562-15581) produced an amplicon of 149 bp. The nested PCR was run in a 50-μL reaction comprising 10 μL of PCR product, 1.5 units of DNA Polymerase (Biotools, Madrid, Spain), 1 μmol/L of each primer, 200 μmol/L of each dNTP (Applied Biosystems), 2 mmol/L MgCl 2 , and 1× buffer. PCR mixtures were heated to 95°C for 5 min, followed by 35 cycles of 1 min at 95°C, 30 s at 62°C, and 40 s at 72°C, followed by a fi nal 10 min at 72°C. Clinical samples positive for HKU1 were used as positive controls in the HKU1 nested PCR. All fragments obtained from PCR and nested PCR were analyzed in a 2% (wt/ vol) agarose gel by electrophoresis, stained with 0.5 μg/mL of ethidium bromide, and subsequently sequenced to confi rm the type of coronavirus. Nucleotide sequencing reactions were performed on both amplicon strands by using an ABI PRISM Big Dye Cycle Sequencing Kit with the ABI PRISM 3100 automatic sequencer (Applied Biosystems).
Six (3.6%) samples tested positive for HCoV-HKU1: 2 samples by PCR and 4 by nested PCR. HCoV types 229E, OC43, and NL63 were not detected in any sample by PCR. Samples positive for HCoV were associated with pertussis, pneumonia, bronchiolitis, and diarrhea (Table) .
In a recent review, an analysis of 18 studies indicated that the median (range) incidence of HCoV-HKU1 was 0.9% (0%-4.4%) (2), which is similar to the detection rate in our study. To our knowledge, the only study that has screened for HKU1 in Brazil found that 0.48% of children were positive for HKU1 (3), which is lower than our results.
Although we did not detect other HCoV types, all HCoV types were detected previously in Brazil in samples collected during 2006-2008 (3,6) . The absence of detection of 229E, OC43, and NL63 HCoV might have resulted from the seasonality and natural viral year cycle or from the characteristics of the children studied This may be the oldest collection of human samples in which HKU1 has been detected. To our knowledge, the oldest previous sample positive for HCoV-HKU1 was detected in children in Finland during 1996-1998, without an exact date specifi ed (7) . Retrospective studies also have been conducted in the United States and Greece that showed the HKU1 virus in different countries in Europe and North America before its discovery (8, 9) . We have confi rmed the circulation of HKU1 coronaviruses in children in Brazil in 1995.
Resistanceassociated 23S rRNA Mutation in Mycoplasma genitalium, Japan To the Editor: Mycoplasma genitalium is now recognized as a serious pathogen in sexually transmitted infections (1, 2) . Azithromycin regimens have been commonly used for treatment of M. genitalium infections (3). However, failure of azithromycin treatment has been reported in cases of M. genitalium-positive nongonococcal urethritis (NGU) (4, 5) , and macrolideresistant strains of M. genitalium have been isolated from case-patients in Australia, Sweden, and Norway for whom azithromycin treatment has failed (4, 5) . In these strains, mutations in the 23S rRNA gene were associated with macrolide resistance, and mutations in ribosomal protein genes L4 and L22 were also found (5) . Surveillance for antimicrobial resistance of M. genitalium is essential to identify antimicrobial resistant strains and to then determine appropriate treatment. Coculture of patient specimens with Vero cells has improved the primary isolation rate of M. genitalium from clinical specimens and offered some current clinical strains for antimicrobial drug susceptibility testing (6) . To determine their antimicrobial susceptibilities, a molecular real-time PCR method has been developed (7, 8) . However, isolating M. genitalium from clinical specimens and antimicrobial drug susceptibility testing of clinical isolates remain labor-intensive, time-consuming tasks. In addition, no methods are available to directly determine antimicrobial drug susceptibilities of M. genitalium in clinical specimens. To monitor macrolide susceptibilities in clinical Increased Urinary Angiotensin-Converting Enzyme 2 in Renal Transplant Patients with Diabetes Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients. Angiotensin-converting enzyme 2 (ACE2) is a recently identified member of the renin-angiotensin system (RAS) that degrades angiotensin (Ang) II to the seven amino acid peptide fragment Ang-(1-7) [1, 2] . ACE2 is a homologue of angiotensin-converting enzyme (ACE), but is not blocked by ACE inhibitors. Although ACE2 is found in many tissues, expression is especially high in the kidney, particularly within cells of the proximal tubule [3] [4] [5] . In mice deletion of the ACE2 gene is associated with development of late-stage glomerulosclerosis, and acceleration of diabetic nephropathy, in the absence of hypertension [6, 7] . In spontaneously hypertensive rats, administration of human recombinant ACE2 reduces blood pressure [8] , and in diabetic mice, exogenous human ACE2 diminishes blood pressure and glomerular injury [9] . Thus, ACE2 may be an endogenous protector against the progression of chronic kidney disease (CKD).
In kidney tubular epithelial cells, ACE2 is localized to the apical membrane and also appears in the cytoplasm [3, 10] . ACE2 is shed at its carboxy-terminus from the plasma membrane in cultured human embryonic kidney cells and airway epithelial cells, a process catalyzed by the enzyme ''a disintegrin and metalloproteinase-17'' (ADAM-17) [11] [12] [13] . Whether this process occurs in the proximal tubule is unclear, although soluble ACE2 has been detected in human urine [10] . In a recent study, urinary levels of ACE2 protein were significantly increased in humans with CKD (the majority with chronic glomerulonephritis), compared to healthy controls, as determined by enzyme-linked immunosorbent assay (ELISA) [14] . Urinary ACE2 was also higher in diabetics with CKD [14] . These results suggest that ACE2 may be shed into the urine, and could be a biomarker in CKD patients. However, the presence of urinary ACE2 has not been studied in renal transplant recipients, and the factors associated with elevated urinary ACE2 remain unclear. Accordingly, the principle objective of the present study was to determine if urinary ACE2 activity, protein, and mRNA can be detected in renal transplant patients, and to identify factors associated with the presence of ACE2. In addition, we examined factors associated with urinary ACE activity, protein and mRNA in these patients. Our data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells.
This study involved recruitment of human subjects as described below, with written informed consent, and the study was conducted according to the principles expressed in the Declaration of Helsinki. The study protocols were approved by the Research 
The subjects in this study were 50 healthy controls (age .18 yrs), recruited from the hospital or research centre staff, with no history of kidney disease, hypertension, or diabetes, and 100 renal transplant recipients from The Ottawa Hospital Renal Transplant Program, age .18 yrs, and .3 months posttransplant. At the time of enrollment, half of the transplant subjects (n = 50) were also enrolled in an ongoing randomized controlled trial to determine the effect of the ACE inhibitor ramipril on transplant outcomes (ACE inhibition for the preservation of renal function and survival in kidney transplantation; International Standard Randomized Controlled Trial Number Registry ISRCTN-78129473), but had not yet received either placebo or ramipril. These subjects had documented significant proteinuria (.200 mg urinary protein/day) at baseline. The 50 other transplant recipients were patients without significant proteinuria (spot urine ACR in the normal range, or ,200 mg proteinuria/day). Subjects were excluded if they were taking ACE inhibitors, angiotensin receptor antagonists, or renin inhibitors, or if they were pregnant or currently had a urinary tract infection.
After obtaining informed consent, a spot urine sample was collected from each subject, and a blood sample was drawn for measurement of serum creatinine (Cr) and determination of estimated glomerular filtration rate (eGFR), using the Modification of Diet in Renal Disease (MDRD) calculation [15] . Demographic information was obtained via interview with the subject and/or from the hospital chart. For transplant recipients, a diagnosis of diabetes mellitus and the primary renal diagnoses were obtained directly from survey of the hospital chart.
Urine samples were placed on ice, aliquoted and then centrifuged at 12000 g for 5 min at 4uC. Measurements of urinary albumin were performed on supernatant fractions, using an ELISA kit (Exocell Inc., Philadelphia, PA, USA). Results were corrected for urinary Cr concentration, using a kit specific for human Cr (Creatinine Companion, Exocell Inc.).
The enzyme activities of urinary ACE2 and ACE were measured using synthetic substrates, essentially as we previously reported [16] . All results were corrected for the Cr concentration in the urine samples. Details on the assays can be found in Text S1.
The amount of ACE2 present in urine specimens was quantified using a commercial ELISA kit (Cat. No. AG-45A-0022EK-KI01, AdipoGen, Seoul, Korea) according to the protocol provided by the supplier (http://www.adipogen.com/ ag-45a-0022/ace2-human-elisa-kit.html). A standard curve was generated by performing 1:2 serial dilutions of human recombinant ACE2 (50 ng/ml), provided with the kit, with the limit of detection ranging from 0.391 to 25 ng/mL. In preliminary experiments, the average intra-assay coefficient of variation (CV) for the assay was 2.9%, and the average interassay CV value was 8.7% (n = 10). The amount of ACE2 obtained by ELISA was normalized to the subject's urine Cr concentration, and is reported as ng/mg Cr.
Urine aliquots (supernatant fraction, 15 mL) were subjected to immunoblot analysis for ACE2 and ACE, using commercially available antibodies. To control for variations in urine concentration, the values obtained by densitometry were divided by the corresponding Cr concentration for that urine sample. Details on the assays can be found in Text S1.
Peptide N-Glycosidase F (PNGase F) Treatment Urine aliquots (supernatant fraction, 20 mL) were subject to deglycosylation reaction using PNGase F (Cat No. P0704S, New England Biolabs, Ipswich, MA, USA). Deglycosylated urinary ACE2 protein fragments were detected by western analysis. Details of the assays can be found in Text S1.
Urine samples (40 mL) were centrifuged at 1000 g for 20 min at 4uC. Total RNA was isolated from pellet fractions and then subjected to real-time RT-PCR for quantitation of ACE2 and ACE (see Text S1 for assay details).
Urinary levels of Ang II were measured using a commercial peptide radioimmunoassay (RIA) kit that contains an Ang IIselective polyclonal antibody and 125 I-Ang II (Peninsula Laboratories, San Carlos, CA, USA), essentially as described [16, 17] . Ang-(1-7) levels were measured using a commercial peptide enzyme immunoassay (EIA) kit that contains an Ang-(1-7)-selective polyclonal antibody (Peninsula Laboratories) [16] . For assay details, see Text S1.
Data are presented visually as box plots. Continuous variables are reported as the median value, with the interquartile range in parentheses. Statistical analysis was performed by the nonparametric Mann-Whitney U test for unpaired samples to compare data in healthy controls and transplant recipients, and diabetic vs non-diabetic transplant recipients. A linear regression model was constructed to identify potential explanatory variables for urinary ACE2 and ACE levels in the 100 transplant subjects. The dependent variable was the measure under study (e.g. ACE2 activity) while the following explanatory variables were entered into the model: age, gender, diabetes, eGFR, albuminuria, hypertension, and use of calcineurin inhibitors. Variables were forced in simultaneously and removed from the model if not statistically significant. Standardized regression coefficients (b) are presented for each dependent variable in the model. Data were analyzed using SigmaStat (version 3.01 A; SYSTAT) and JMP (version 8.0.1, SAS Inc.). For all data, a p value ,0.05 was considered significant.
The characteristics of the 50 healthy controls and 100 transplant subjects are depicted in Table 1 . The median age for control subjects was 45 [interquartile range (IQR), 36-50] yrs, and 26% were male. The median age for transplant subjects was 54 (IQR, 42-62) yrs, 65% were male, 38% had diabetes, and 88% had hypertension. Urinary ACE2 Activity, Protein, and mRNA As shown in Figure 1A , urinary ACE2 activity was significantly increased in transplant patients, compared to control subjects [control; median: 1.90 (IQR, 1.04-2.70) ng-eq/mg Cr610 2 vs transplants; median: 3.65 (IQR, 1.13-9.35) ng-eq/mg Cr610 2 ; p = 0.003]. Similarly urinary ACE2 protein by ELISA was significantly increased in transplant patients, compared to controls ( Figure 1B ) [control; median: 1.41 (IQR, 0.00-4.49) ng/mg Cr vs transplants; median: 8.04 (IQR, 0.00-21.11) ng/mg Cr; p,0.001]. In 96% of unconcentrated urine samples from control and 94% from transplant subjects, ACE2 was identified by western analysis as a protein doublet of 120 kDa and 90 kDa. As with urinary ACE2 activity and ELISA measurements, western analyses revealed a significant increase in urinary ACE2 protein in transplant patients compared to controls ( Figure 1C : p = 0.001).
Within the group of 100 transplant patients, multiple linear regression using primary renal diseases as explanatory variables revealed a significant association between the diagnosis of diabetic nephropathy (n = 21) and urinary ACE2 protein by ELISA or western analyses (p,0.001 for both). In contrast, there was no significant association between urinary ACE2 levels and other primary causes of renal disease in these patients. Further studies were performed on the association between diabetes mellitus and urinary ACE2 in the transplant patients. Of the 100 transplant patients, 38 had a diagnosis of diabetes mellitus listed on their hospital chart. The majority of these patients were taking insulin (31 out of 38, 81.6%), 6 (15.8%) were taking oral hypoglycemic agents, and only 1 patient (2.6%) was on dietary therapy alone.
Transplant patients with diabetes (n = 38) had significantly higher levels of urinary ACE2 activity, compared to non-diabetic (n = 62) subjects [ Figure 2A : diabetics; 8.75 (IQR, 3.09-15.54) vs non-diabetics; 2.32 (IQR, 0.63-5.37) ng-eq/mg Cr610 2 ; p,0.001]. Similarly, ACE2 protein levels by ELISA and western analysis were significantly increased in diabetic subjects, compared to non-diabetics ( Figure 2B , C; p,0.001 vs non-diabetics for both). In transplant patients with diabetes, both the 120 kDa and the 90 kDa ACE2 protein bands were significantly increased on westerns, compared to non-diabetics (p = 0.002 vs non-diabetics for the 120 kDa band, and p,0.001 vs non-diabetics for the 90 kDa band). Moreover, urinary ACE2 protein levels by ELISA were significantly increased in subjects with diabetes pre-transplant (n = 21), compared to subjects who developed diabetes after transplant (n = 17; p = 0.024).
The state of glycosylation of the urinary ACE2 proteins at 120 kDa and 90 kDa was studied by treating urine samples with the deglycosylase enzyme PNGase F. Urinary ACE2 fragment sizes were reduced to ,85 kDa and ,65 kDa by PNGase F treatment, indicating that both fragments were originally glycosylated ( Figure 2D ).
RNA was extracted from urinary pellets and subjected to realtime PCR for ACE2. ACE2 mRNA was detected in 45% of urine samples of transplant patients, with no significant difference between diabetic [0.669 (IQR, 0.00-6.15) pg mRNA/mg Cr610 25 ] and non-diabetic subjects [0.00 (IQR, 0.00-2.06) pg mRNA/mg Cr610 25 ; p = 0.091].
After adjusting for potential confounding factors [age, gender, diabetes, eGFR, albuminuria, hypertension, and use of calcineurin inhibitors] only diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p,0.001, Table 2 ) in transplant patients. An increase in urinary ACR was associated with urinary ACE2 protein by ELISA (p = 0.026), but not with ACE2 activity or ACE2 protein by western. Female gender was associated with higher urinary ACE2 mRNA levels in transplant patients (p = 0.004, Table 2 ).
Since the numbers of patients taking calcineurin inhibitors (95/ 100 patients) were highly skewed (Table 1) , multivariate analysis was also performed with exclusion of this variable. This did not alter the significance levels for any parameters reported in Table 2 .
Urinary ACE Activity, Protein, and mRNA Urinary ACE activity was detected in 65% of transplant patients [median for all 100 patients: 1.17 (IQR, 0.00-3.43) ng-eq/mg Cr610 2 ]. By western analysis, ACE was detected in 94% of urine samples of transplant patients as a protein band of approximately 190 kDa, consistent with previous studies [18] (Figure 3 ). Urinary ACE activity and protein levels were significantly increased in diabetic transplant subjects, compared to non-diabetic patients ( Figure 3A After adjusting for potential confounding variables, increased urinary albumin/Cr was associated with urinary ACE protein by western analysis in transplant patients (p,0.001, Table 2 ). However, diabetes did not associate with increased urinary ACE activity or ACE protein. Female gender and urinary albumin/Cr were significantly associated with increased urinary ACE mRNA levels in transplant patients ( Table 2 ).
Transplant patient with diabetes had significantly higher levels of urinary Ang II, compared to non-diabetics ( Figure 4A : p = 0.027). Urinary Ang-(1-7) levels did not differ between the two groups ( Figure 4B : p = 0.13). However, a significant positive linear correlation was found between urinary ACE2 activity and urinary Ang-(1-7) (r = 0.260, p = 0.009). In the multivariate analysis, diabetes remained significantly associated with urinary Ang II levels (p = 0.042, Table 2 ). Neither diabetes, nor any other clinical factor (eGFR, urinary albumin/Cr, etc.) was associated with urinary Ang-(1-7) levels in the multiple linear regression model.
The major finding of this study is that urinary ACE2 activity and ACE2 protein are increased in kidney transplant recipients, compared to healthy control subjects, and the presence of diabetes strongly associates with urinary ACE2 levels in the patient population, by multivariate analysis. Female gender associates significantly with urinary ACE2 mRNA and ACE mRNA levels, which are detected in 45% and 65% of transplant recipients, respectively.
ACE2 is expressed at relatively high levels in the kidney, particularly in the proximal tubule, and is thought to be renoprotective via its ability to reduce Ang II levels. ACE2 also increases production of Ang-(1-7), which can oppose the delete- rious non-hemodynamic actions of Ang II in tubular cells [19] . Kidney expression of ACE2 is reduced in experimental models, including the remnant kidney model of CKD [16, 20, 21] . In humans with diabetic nephropathy, expression of ACE2 is decreased in glomeruli and proximal tubules [22] , suggesting a predisposition to Ang II-mediated injury.
Our results support the hypothesis that transplant patients with diabetes exhibit increased urinary excretion of ACE2 protein. This was independent of eGFR as determined by multivariate analysis. Both soluble ACE2 and ACE have been detected in human urine [10] , and in humans with CKD, Mizuiri et al. reported increased urinary levels of ACE2 protein by ELISA, compared to healthy subjects [14] . Patients with diabetic nephropathy had higher urinary levels of ACE2 than those without diabetic nephropathy, and use of ACE inhibitor and/or angiotensin receptor blockers did not affect urinary ACE2 levels [14] . In our study, no subjects were taking inhibitors of the RAS. Using three independent assays for urinary ACE2 (enzyme activity, ELISA, and immunoblot), multiple linear regression revealed that diabetes was the only variable consistently predictive of higher urinary ACE2 levels. Age and gender had no influence on urinary ACE2 activity or protein levels. Similarly, in patients with CKD Mizuiri et al. found no difference in urinary ACE2 levels between males and females [14] .
Although gender did not influence urinary ACE2 activity or protein, in the present studies females had significantly higher urinary ACE2 mRNA levels. The gene for ACE2 is located on the X chromosome [2] , and in the rat renal wrap model of hypertension, intrarenal ACE2 activity and expression are upregulated by estrogens [23] , raising the possibility that female transplant recipients may express ACE2 mRNA at higher levels in the kidney compared to males. Female gender was also associated with higher urinary ACE mRNA levels, as was albuminuria, the latter being in agreement with studies in patients with diabetic nephropathy [24] . The cellular origin of urinary mRNA for ACE2 or ACE is unknown, although it has been speculated that proximal tubular cells may be the source, since they express all components of the RAS, and are found in the urine sediment [24] . However, the physiological significance of urinary ACE2 or ACE mRNA levels is unclear. In this regard, diabetes, albuminuria and eGFR did not influence urinary ACE2 mRNA levels in our transplant recipients, a result that differs from diabetic nephropathy patients, where proteinuria was found to correlate positively with urinary ACE2 mRNA, and eGFR to correlate negatively with ACE2 mRNA [24] . Factors related to transplant could therefore have an independent influence on urinary ACE2 mRNA. As one possibility, the use of immunosuppressive drugs in transplant subjects might regulate shedding of cells expressing ACE2 mRNA into the urine.
Urinary ACE2 protein could originate at least partly from plasma (via filtration at the glomerulus), or it could be derived via excretion from renal cells. Although Mizuiri et al. found higher urinary ACE2 levels in CKD patients, there was no difference in plasma ACE2 protein between CKD patients and healthy subjects [14] . However, the CKD patients in their study had significant albuminuria, suggesting that ACE2 may have leaked into the urine across the glomerular barrier. In the present study, we found no association between albuminuria and either urinary ACE2 activity or protein levels by western analysis. The data suggest that urinary ACE2 protein likely derives via shedding from cells along the nephron, and not from filtration from plasma. In support of this hypothesis, soluble ACE2 is shed from the plasma membrane in cell culture systems, via cleavage at its ectodomain by the protease enzyme ADAM-17 [11] [12] [13] . Interestingly, renal ADAM-17 is upregulated in Ang II-induced kidney injury [25] , and de novo expression occurs in human kidney disease, in proximal tubule, podocytes, and mesangial cells [26] . Whether ADAM-17 is activated in the transplant kidney in the presence of diabetes, and mediates shedding of soluble ACE2 into the urine requires further study. Two bands for ACE2 were detected by immunoblot of urine samples in the current study. One band was found at 120 kDA, consistent with the full-length protein, and the other at 90 kDa, which is likely a cleaved ACE2 fragment. Thus, the 90 kDa fragment does not simply represent a deglycosylated form of fulllength ACE2, since incubation with PNGase F resulted in a further reduction in its size, as shown in Figure 2D . To determine if this 90 kDa fragment is generated by ADAM-17-mediated cleavage of ACE2, further characterization is required, including use of antibodies directed against the cytoplasmic carboxyterminus of ACE2 (which may be lacking in this fragment), and/or direct sequence analysis of the polypeptide. Furthermore, although shedding of ACE2 fragments may occur via ADAM-17 in transplant subjects, proteomic analysis has identified ACE2 as one of the 1132 proteins present in urinary exosomes isolated from human urine [27] , suggesting that membrane-bound full-length ACE2 may also be a source of urinary ACE2.
By multivariate analysis, diabetes was significantly associated with urinary Ang II levels in transplant subjects, consistent with intrarenal RAS activation in diabetes [28] . In this regard, although diabetic patients had significantly higher urinary ACE activity and ACE protein levels by immunoblot, this association was not confirmed in the multivariate analysis. Finally, although diabetics had higher urinary ACE2 activity and protein levels, there was no significant association between diabetes and urinary levels of Ang-(1-7) by multivariate analysis, suggesting that urinary Ang-(1-7) may be influenced by other factors. A potential limitation of the urinary angiotensin peptide measurements is that urine specimens were not treated with protease inhibitors, which could have affected the sensitivity for detecting differences. Interestingly, however, we did observe a significant correlation between urinary ACE2 activity and Ang-(1-7) levels.
Our study has a number of strengths, including the reliability and reproducibility of the assays, the inclusion of data on mRNA, protein, and ACE2/ACE products, and the use of multiple linear regression to adjust for confounding variables. Limitations include the relatively small number of subjects, the absence of plasma ACE2 or ACE measurements, and the single time point for urinary ACE2, ACE and peptide assays. Furthermore, it is possible that diabetes alone may contribute to increased urinary ACE2 levels, independent of transplant status. Studies in patients with diabetes and normal kidney function are required to answer this question. Larger studies are also needed to determine if urinary ACE2 or ACE are biomarkers of transplant function or if they may predict responsiveness to blockade of the RAS.
In summary, in renal transplant recipients diabetes is a strong independent predictor of increased urinary levels of ACE2 activity and protein. Our data further suggest that ACE2 may be shed into the urine in transplant recipients, and could represent a marker to assess the role of the kidney RAS in these patients.
Text S1 Detailed methods for enzyme activity assays, immunoblots, real-time PCR assays, and measurements of Ang II and Ang-(1-7). (DOC) PepMapper: A Collaborative Web Tool for Mapping Epitopes from Affinity-Selected Peptides Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/ Epitope mapping from affinity-selected peptides has been proven to be a useful approach in identifying native epitopes for immunological applications in recent years [1, 2, 3, 4, 5, 6, 7, 8] . Affinity-selected peptides which are derived from phage-display experiments, also known as mimotopes, are assumed to have similar components with the native epitope [9] . Various ways have been proposed to map the mimotopes back to the genuine epitope. These methods were reviewed and compared in some recent literature [10, 11] . In general, they can be categorized as sequence based [12] , motif based [7, 13] , physicochemical properties based [14] , and graph search based [4, 15, 16] methods. Graph search methods are among the most efficient ways in epitope mapping demonstrated in many recent publications [4, 15, 16] because they take advantages of more information provided by using both the 3D structure of a protein than using the traditional amino acid sequence and the information from mimotope set.
The essential idea of graph search methods is to find a group of simple paths on a graph generated from the residues on the surface of a protein and find out some paths from the graph best matched to the query peptides derived from in vitro screening against a target antibody [6] . Searching in Pep-3D-Search [4] is achieved through an algorithm based on ant colony optimization (ACO) whereas PepSurf [6] realizes the mapping using a dynamic programming based stochastic color-coding algorithm. However, finding a simple path on a graph is computationally intractable for any large scale of searching problem. For example, PepSurf takes a few hours to get the result for a peptide of 14 or 15 amino acids.
MimoPro [16] has brought improvement on processing speed and sensitivity over both PepSurf and Pep-3D-Search. It uses an adaptable distance threshold (ADT) regulated by an appropriate compactness factor to define a graph from a small patch on the surface of a protein. Such a regulated graph contains a certain number of edges, which can guarantee that searching through the graph is more efficient. On average, MimoPro achieved the best performance among the three, but individual cases produced mixed outcomes. This indicates that no one dominates over others in all circumstances but each has its advantage in dealing with particular cases.
Perhaps the best strategy is to combine two or more methods together to deal with various cases of epitope-peptide mapping in practice. Pepitope [17] combined both PepSurf and Mapitope [1] together as a Web tool for epitope-peptide mapping so as to complement with each other. The algorithm used in PepSurf maps the affinity-selected peptides directly back to the protein surface. The most significant alignments are then clustered into a patch, from which the epitope location is inferred. In Mapitope, each peptide is first deconvoluted to amino acid pairs, and those pairs of residues that are significantly overrepresented in the panel of peptides are then identified. Epitopic regions are finally predicted through searching for a cluster of those enriched pairs on the 3D structure of the antigen.
Although significant progress has been made in epitope prediction through mimotope mapping, we must acknowledge that the performance of any algorithm devised and any tool developed so far was evaluated based on the outcomes of very limited test cases in which the epitopic region must be known and both the structure of the antigen and the peptide set derived from high-throughput screening must be available. If a single method is applied to a case in which the epitopic region is unknown, the mapping simply returns a candidate epitope (or none) with aligned paths formed by the antigen surface residues (or none). Such candidate epitope will become the focus of further investigation through other means.
If no any single experimentally derived peptide is related to any region on the antigen, it only indicates that this method is not applicable for the case through the mapping. However, it does not mean that no interacting epitope exists on the antigen, which may be detected by other methods. In this regard, the likelihood of finding a genuine epitopic region on an antigen should be higher if more associated peptides can be detected through the mapping. Furthermore, if more mapping methods can be combined together for exploring as many associated peptides as possible through the mapping, the likelihood of finding a genuine epitopic region on the antigen should be enhanced. On the other hand, users of a mapping tool would prefer to know some sort of certainty about the candidate epitope determined by the associated peptides through the mapping in relation to the likelihood of being a genuine epitope. In other words, some kind of verification on the candidate epitope, if not a confirmation, will be much helpful for the users to make an initial assessment on the quality of the candidate. A single method cannot achieve this goal by self verification, but a combined approach of two or more independent methods would be able to provide mutual verification on the candidate of the same case. Web tools realizing such collaborative concept have not been tried so far.
In this paper, we report our effort on combining both MimoPro and a modified version of Pep-3D-Search together to realize such a collaborative Web tool for supporting users in peptide-epitope mapping. In addition to the process of either MimoPro or Pep-3D-Search alone, the combined operation of Union captures the concept of exploring as many associated peptides as possible from both methods. The concept of mutual verification is realized by the combined operation of Intersection from both methods.
In the next section, we introduce the processes of MimoPro, Pep-3D-Search, and the combined approach of PepMapper. Their online implementations are then briefly outlined. Construction of test cases and assessment of mapping are incorporated with discussions of the experimental results. Conclusions are finally drawn.
The process of Pep-3D-Search [4] is illustrated in Figure 1a . Given a 3D structure of an antigen, Pep-3D-Search identifies all the surface residues and creates a surface graph using those residues. An ACO algorithm is then used to search the matched paths on the antigen surface with respect to the query peptides or motif. Each matched path is then rated by its P-value score [4] . A set of highly rated paths are selected to create a weighted graph of resultant paths. The Depth-First Search (DFS) algorithm is finally used to screen and cluster this weighted graph to define the candidate epitopes.
The process of Pep-3D-Search has two unique features. Firstly, Pep-3D-Search is able to deal with both mimotope searching and motif mapping on the residue surface graph. Secondly, the adoption of ACO algorithm allows longer mimotopes or motifs to be processed with reasonable efficiency. The performance of Pep-3D-Search assessed by a few comparative studies [16] seems to be above the average level.
The process of MimoPro [16] is illustrated in Figure 1b . Initially, the surface of a protein is divided into some overlapping patches and each patch is centered at atom C b of a surface residue with a radius of 15 Å . This radius allows most epitopes to be encompassed in such a patch [16] . Secondly, each surface patch is further transformed to a graph bounded by neighbor amino acids that are determined using a parameter called adaptive distance threshold (ADT). Afterwards a patch-based complete graph search algorithm is utilized to find the best alignment for each mimotope sequence in each graph. During this iteration, similarity between a path and the corresponding mimotope is rated. Finally the patch with the highest score is selected as a potential candidate for the native epitope.
This approach has some new features different from other similar methods. Firstly, the ADT that is changeable in different regions of a protein is introduced in generating a graph from a surface patch. Such a distance threshold is adjustable so that a longer distance is used in loose regions of an antigen to include more useful connections whereas a shorter distance is adopted in dense regions to preclude some insignificant connections. Secondly, a compactness factor is introduced to make sure that all resultant graphs share a uniform compactness so that searching over any regulated graph is simpler and faster compared with previous methods. Thirdly, the adopted algorithm not only employs dynamic programming (DP) to reduce repeating searches and prune some insignificant paths encountered in the traditional search algorithm, but also introduces the branch and bound method to optimize the candidate set of rated paths during the DP process. The performance of MimoPro assessed by a few comparative studies [16, 18, 19] shows that MimoPro seems to be the most sensitive tool on average among the compared tools.
PepMapper provides users with a united platform to conduct peptide-epitope mapping through either MimoPro or Pep-3D-Search or both for different purposes. The processes of PepMapper are illustrated in Figure 2 .
If a user selects either MimoPro or Pep-3D-Search, PepMapper works almost exactly as either does individually, except some possible minor variations in results of this modified version of Pep-3D-Search from its original version [4] . If a user selects the Both option, PepMapper executes both MimoPro and Pep-3D-Search concurrently without mutual interference. The user will get a complete report of processed results through the emailed link. The user can access the normal result of either MimoPro or Pep-3D-Search as each works alone. To view the results of the Both option, the user has to press Compare on the left side of the result Webpage, which will produce a new Webpage showing the text results of both Intersection and Union of the two methods ( Figure 3 ). By clicking Jmol button on this Webpage, the 3D image of the result from either Intersection (by default) or Union can be displayed ( Figure 4 ).
The combined operations of Union and Intersection are defined as
where A and B are two sets of epitopic amino acids predicted by MimoPro and Pep-3D-Search respectively. Union captures the concept of exploring as many associated peptides as possible from both methods by constructing a new set that consists of not only the common epitopic amino acids in both A and B, but also all the distinctive epitopic amino acids in either A or B. Therefore, Union should be more sensitive than either method in epitope detection.
Intersection realizes the concept of mutual verification from both methods by creating a new set that consists of only the common epitopic amino acids in both A and B. Hence, Intersection should be more reliable than either method in epitope detection if its outcome is positive. Without confirmation from real experiment results, mutual verification from artificial prediction methods can only provide an indication of where the true epitopic region is likely located on the antigen surface. Ideally if both methods produce exactly the same epitopic amino acids, both Intersection and Union should return the same set of epitopic amino acids. Hence MimoPro and Pep-3D-Search share a consistency of 100% to each other on the case However, this failure in mutual verification only means that the two methods cannot support each other on the case under study, but it does not mean that the predicted epitopic regions by either method are not related to the genuine epitope. Other approaches It can be clearly seen on these images that Intersection provides more confined prediction as most of the residues lie on the interface whereas Union outlines a larger area that may cover (part of) the potential epitopic region. doi:10.1371/journal.pone.0037869.g004 are needed to verify the epitopic regions predicted by either method.
Commonly, consistency of epitope prediction from the two methods falls between 0 and 1. To present this indication numerically, we define the Consistency of the two methods in epitope prediction as
The higher the Consistency, the larger the overlapped area of predicted epitopic regions by both methods; hence it is an indirect indication that a genuine epitope is more likely to be found around the overlapped area on the antigen surface under study.
PepMapper has been implemented using C++ as a Web-based tool located at http://informatics.nenu.edu.cn/PepMapper. It is currently deployed on Linux using tomcat server 6.0 and has been tested using many popular Web browsers, such as IE7-9, Firefox, and Opera. Three options, MimoPro, Pep-3D-Search, and their combination, are available for the users to choose for the purpose of their applications. Note that the original Web tool of Pep-3D-Search was implemented using VB.NET. Pep-3D-Search in PepMapper is re-implemented using C++ and a modified ACO algorithm is adopted for more efficient searching (See Table S1 for details).
If a user has multiple requests and needs the results to be returned fairly quickly, it is suggested to choose MimoPro for meeting such purpose because MimoPro is arguably the fastest in processing [20] . If the user wants to verify the results, it is suggested to choose the combination mode.
When accessing PepMapper online, Mapping is the default interface displayed. The input to PepMapper is the structure of a chosen antigen and the peptide library screened from the corresponding antibody. The user needs to specify both the identifier of an antigen in the PDB database through its PDB_ID and the identifier of the interacting chain through Chain No. The user then needs to specify at least one peptide in the box labeled as Mimotopes. The peptides should be grouped in the FASTA format or just in separated lines of sequences. At last, the user needs to provide a valid email address in the text box. By clicking Query, PepMapper begins processing and the results will be sent to the user through the email provided.
The result from PepMapper is a candidate epitope along with the alignment for each peptide sequence. Users can see the result in three ways: text/table, 3D graphics, and Rasmol scripts.
In text/table format, texts are used to list all potential amino acids. The resultant alignments for individual peptide sequences are tabulated with corresponding P-values. In 3D graphics through Jmol, the candidate epitope is shown in filled balls and the other amino acids are shown as backbones by default (Figure 4) . Results can also be presented in Rasmol script that can be downloaded by clicking the link provided. This is useful when the network connection is poor.
A new function Compare is also provided to make mutual verification easier between the results of the two methods. By clicking Compare on the left in the result Webpage, the peptides constituting the candidate epitope are displayed in two boxes corresponding to both methods. Clicking Compare under the left box will return a new Webpage that shows the results of both Intersection and Union from both methods (Figure 3) , which can also be viewed as 3D images by clicking Jmol button on this Webpage (Figure 4 ).
The task of epitope prediction based on the peptide set is to map it back to the epitopic region on an antigen that interacts with the target molecule during in vitro screening. Although there may be other epitopes on the antigen surface, we only consider the active epitope in the designated context and regard the rest part of the antigen as nonepitope.
For the test cases that correspond to the same epitope and same reference antigen structure in PDB database [18, 21] but different mimotope sets, we retain only one representative to avoid the possible bias caused by the duplication. Those cases with antigen smaller than 80 amino acids are excluded because they are too small to reflect the performance. Based on these rules, the final dataset was constructed by 27 test cases (Table 1 ).
In order to analyze the performances of Pep-3D-Search, MimoPro and PepMapper, the outcome is assessed by a number of measurements, including sensitivity (Se), specificity (Sp), and precision (Pr) defined as follows:
Pr~T P TPzFP
:
In these expressions, TP is the number of predicted epitopic amino acids proven to be the true epitopic amino acids. FP is the number of predicted epitopic amino acids proven not to be the true epitopic amino acids. TN is the predicted non-epitopic amino acids proven not to be the true epitopic amino acids. FN is the number of predicted non-epitopic amino acids proven to be the true epitopic amino acids. We use PE to denote the number of all predicted epitopic amino acids (the sum of TP and FP).
To demonstrate the improved performance of PepMapper over either MimoPro or Pep-3D-Search alone, we first present the results from MimoPro and Pep-3D-Search run separately and then the results from the combined operations. Table 2 presents the evaluation results from the two methods run separately. The performances of prediction from MimoPro and Pep-3D-Search varied in different test cases. MimoPro provided some good results on 2ADF_A, 3EZE_B, 1JRH_I, 1BJ1_H, 1N8Z_C and 1ZTX_E with sensitivity exceeding 0.8 and specificity higher than 0.6; meanwhile the worst results were observed in 1YY9_A, 2NY7_G, 2GRX_A, 1D4V_B, 3BT1_A and 1HX1_A with sensitivity approaching to 0. For the rest cases, the sensitivity of prediction was between 0.25 and 0.6 and the specificity is consistently higher than 0.8. Comparatively, Pep-3D-Search gave better results in 1HX1_A and 1D4V_B, in which MimoPro failed to predict any epitopic amino acids. However, Pep-3D-Search failed in 2ADF_A, 1EER_A and 1MQ8_B whereas MimoPro produced useful results. On average, MimoPro gives better results in terms of sensitivity (0.446) and precision (0.267), but slightly worse than Pep-3D-Search in specificity.
Both MimoPro and Pep-3D-Search failed in 1YY9_A, 2GRX_A, 1EER_A, and 3EZE_B. Consequently, PepMapper failed as well. We think that this failure could be attributed to a number of factors, including the quality of the experimental data and the complexity of the predicting tasks. For instance, we found that the mimotope set used for predicting the epitopic region of 2GRX_A is screened against the whole complementary protein rather than the restricted region of the two interacting proteins. Therefore it is reasonable to suppose that there may be multiple regions on the surface of the target antigen to which the mimotope can bind. As a result, the mimotopes may bind to the regions that are different from the preferable region.
Additionally, the limited number of mimotopes (1D4V_B, 1EER_A) and surface amino acids may also complicate the matter, since the small number of mimotopes contains little information for locating the epitopic region, especially where too many surface amino acids exist. Furthermore, even though the dataset for our experiments has been the largest ever reported publicly, a few bad results can still greatly influence the statistical results.
The Intersection operation of PepMapper captures the idea of mutual verification of epitope prediction. Intuitively, the more the commonly shared peptides in the same area are, the more likely the area to be a part of an epitope is. On average, this operation has the highest specificity of 0.930 and a high precision of 0.256 compared to that of the Union, MimoPro, and Pep-3D-Search (Tables 2 & 3) . However, its sensitivity is the lowest because some epitopic amino acids predicted by either method but not in common are left out in the calculation. This also reveals the weakness of the Intersection operation of PepMapper, i.e., in case of no overlapping between the two methods, it does not mean that no epitopic sites may be predicted by either MimoPro or Pep-3D-Search alone. 1MQ8 is such a case without common peptides, but MimoPro still predicts some positive epitopic sites.
Fortunately, the union operation of PepMapper complements the weakness of Intersection operation by joining the results from the two methods together to increase the size of potential epitopic sites. The Union operation produced the best performance in sensitivity but the worst in precision and specificity compared to that of the Intersection, MimoPro, and Pep-3D-Search (Tables 2  & 3) . This is because the increased size of potential epitopic sites brought in by the Union operation also contains more false positives in the candidates. Using Consistency defined in Equation (3) as an indirect sufficient condition to judge the likelihood of successful prediction of epitope by combining both Intersection and Union, our tests tend to support its usefulness in indicating the likelihood of successful prediction (Table 3) . Although the number of tests is still insufficient for us to draw any exclusive conclusion on its implication on epitope prediction, our initial analysis leads to the following indications: 
PepMapper, a combination of both MimoPro and a modified version of Pep-3D-Search together, sets a collaborative Web platform, on which users can conveniently conduct peptideepitope mappings. In addition to the normal process of either MimoPro or Pep-3D-Search alone, the combined operation of Union captures the concept of exploring as many associated peptides as possible from both methods and thus increases sensitivity in finding potential epitopic regions on a given antigen surface. The Intersection operation of PepMapper realizes largely the concept of mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen with respect to the interacting peptides. The Consistency between Intersection and Union can be used as an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping.
In the future, we will consider to ensemble more methods in more rationalized ways to minimize the occurrence of nil Consistency, which should enhance the effectiveness of PepMapper in peptide-epitope mapping. Effort should also be made on refining the indication of Consistency in epitope prediction by conducting more tests for various conditions. We will try to improve the efficiency of the server through utilizing distributed and/or cloud computing as well.
Availability. We introduced a new server, PepMapper, to incorporate both MimoPro and Pep-3D-Search which is implemented in C++ and deployed at http://informatics.nenu.edu.cn/ PepMapper. It is free for the science community and academic research. However, for commercial purposes, permission must be granted by the owner of the Web tool.
Table S1 Adaption from former Pep-3D-Search. To improve the time efficiency of Pep-3D-Search, we made few adaptations from the former one. These includes a quicker approach in the generating a random background distribution for scoring the best aligned paths from graph search as well as the adjustment of the key parameters. As is shown in the Table S1 , the performance improved on 3IU3_I, 1D4V_B in the adapted Pep-3D-Search on which the former Pep-3D-Search failed to predict any epitopic amino acids. On average, the new Pep-3D-Search has similar sensitivity and specificity, but higher precision. (DOC)  Autophagy: More Than a Nonselective Pathway Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs). For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy. Three different pathways can deliver cytoplasmic components into the lumen of the lysosome for degradation. They are commonly referred to as autophagy (cell "self-eating") and include chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy. CMA involves the direct translocation of specific proteins containing the KFERQ pentapeptide sequence across the lysosome membrane [1, 2] . Microautophagy, on the other hand, entails the invagination and pinching off of the lysosomal limiting membrane, which allows the sequestration and elimination of cytoplasmic components. The molecular mechanism underlying this pathway remains largely unknown. The only cellular function that so far has been indisputably assigned to microautophagy is the turnover of peroxisomes under specific conditions in fungi [3] . Recently, it has been reported the existence of a microautophagy-like process at the late endosomes, where proteins are selectively incorporated into the vesicles that bud inward at the limiting membrane of these organelles during the multivesicular bodies biogenesis [4] . In contrast to CMA and microautophagy, macroautophagy (hereafter referred to as autophagy) entails the formation of a new organelle, the autophagosome, which allows the delivery of a large number of different cargo molecules into the lysosome.
Autophagy is a primordial and highly conserved intracellular process that occurs in most eukaryotic cells and participates in stress management. This pathway involves the de novo formation of vesicles called autophagosomes, which can engulf entire regions of the cytoplasm, individual organelles, protein aggregates, and invading pathogens ( Figure 1 ). The autophagosomes fuse with endosomal compartments to form amphisomes prior to fusion with the lysosome, where their contents are degraded and the resulting metabolites are recycled back to the cytoplasm (Figure 1 ). Unique features of the pathway include the double-membrane structure of the autophagosomes, which were originally characterized over 50 years ago from detailed electron microscopy studies [5] . Starting in the 1990s yeast mutational studies began the genetic and molecular characterization of the key components required to initiate and build an autophagosome In response to inactivation of mTORC1 (but also other cellular and environmental cues), the ULK1 complex is activated and translocates in proximity of the endoplasmic reticulum (ER). Thereafter, the ULK1 complex regulates the class III PI3K complex. Atg9L, a multimembrane spanning protein, is also involved in an early stage of autophagosome formation by probably supplying part of the membranes necessary for the formation and/or expansion. Local formation of PI3P at sites called omegasomes promotes the formation of the phagophore, from which autophagosomes appear to be generated. The PI3P-binding WIPI proteins (yeast Atg18 homolog), as well as the Atg12-Atg5-Atg16L1 complex and the LC3-phosphatidylethanolamine (PE) conjugate play important roles in the elongation and closure of the isolation membrane. Finally, the complete autophagosome fuses with endosomes or endosome-derived vesicles forming the amphisome, which subsequently fuses with lysosomes to form autolysosomes. In the lysosomes, the cytoplasmic materials engulfed by the autophagosomes are degraded by resident hydrolases. The resulting amino acids and other basic cellular constituents are reused by the cell; when in high levels they also reactivate mTORC1 and then suppress autophagy. [6] . Subsequently, genetic and transgenic studies in plants, worms, fruit flies, mice, and humans have underscored the pathway's conservation and have begun to unveil the intricate vital role that autophagy plays in the physiology of cells and multicellular organisms.
For a long time, autophagy was considered a nonselective pathway induced as a survival mechanism in response to cellular stresses. Over the past several years, however, it has become increasingly evident that autophagy also is a highly selective process involved in clearance of excess or dysfunctional organelles, protein aggregates and intracellular pathogens. In this introductory piece, we will briefly discuss the molecular mechanisms of selective types of autophagy and their emerging importance as a quality control to maintain cellular and organismal health, aspects that will be presented in deep in the reviews of this special issue of the International Journal of Cell Biology and highlighted by the research papers.
2.1. The Function of the Atg Proteins. Autophagosomes are formed by expansion and sealing of a small cistern known as the phagophore or isolation membrane ( Figure 1 ). Once complete, they deliver their cargo into the hydrolytic lumen of lysosomes for degradation. A diverse set of components are involved in the biogenesis of autophagosomes, which primarily includes the proteins encoded by the autophagyrelated genes (ATG). Most ATG genes have initially been identified and characterized in yeast. Subsequent studies in higher eukaryotes have revealed that these key factors are highly conserved. To date, 36 Atg proteins have been identified and 16 are part of the core Atg machinery essential for all autophagy-related pathways [7] . Upon autophagy induction, these proteins associate following a hierarchical order [8, 9] to first mediate the formation of the phagophore and then to expand it into an autophagosome [10, 11] . While their molecular functions and their precise contribution during the biogenesis of double-membrane vesicles remain largely unknown, they have been classified in 4 functional groups of genes: (1) the Atg1/ULK complex, (2) the phosphatidylinositol 3-kinase (PI3K) complex, (3) the Atg9 trafficking system, and (4) the two parallel ubiquitinlike conjugation systems ( Figure 1 ).
The Atg1/ULK complex consists of Atg1, Atg13, and Atg17 in yeast, and ULK1/2, Atg13, FIP200 and Atg101 in mammals [12] [13] [14] [15] . This complex is central in mediating the induction of autophagosome biogenesis and as a result it is the terminal target of various signaling cascades regulating autophagy, such as the TOR, insulin, PKA, and AMPK pathways [16] (Figure 1 ). Increased activity of the Atg1/ULK kinase is the primary event that determines the acute induction and upregulation of autophagy. It is important to note that ULK1 is part of a protein family and two other members, ULK2 and ULK3, have been shown play a role in autophagy induction as well [14, 17] . The expansion of this gene family may reflect the complex regulation and requirements of the pathway in multicellular long-lived organisms. Stimulation of the ULK kinases is achieved through an intricate network of phosphorylation and dephosphorylation modifications of the various subunits of the Atg1/ULK complex. For example, Atg13 is directly phosphorylated by TOR and the phosphorylation state of Atg13 modulates its binding to Atg1 and Atg17. Inactivation of TOR leads to a rapid dephosphorylation of Atg13, which increases Atg1-Atg13-Atg17 complex formation, stimulates the Atg1 kinase activity and induces autophagy [18, 19] . The mAtg13 is also essential for autophagy, but seems to directly interact with ULK1, ULK2 and FIP200 independently of its phosphorylation state [13, 14] . In addition, there are several phosphorylation events within this complex as well, including phosphorylation of mAtg13 by ULK1, ULK2, and TOR; phosphorylation of FIP200 by ULK1 and ULK2; phosphorylation of ULK1 and ULK2 by TOR [13, 14] . Additional studies are required to fully characterize the functional significance of these posttranslational modifications.
Autophagy is also regulated by the activity of PI3K complexes. Yeast contains a single PI3K, Vps34, which is present in two different tetrameric complexes that share 3 common subunits, Vps34, Vps15, and Atg6 [20] . Complex I is required for the induction of autophagy and through its fourth component, Atg14, associates to the autophagosomal membranes where the lipid kinase activity of Vps34 is essential for generating the phosphatidylinositol-3-phosphate (PI3P) that permits the recruitment of other Atg proteins [9, 21] (Figure 1 ). Complex II contains Vps38 as the fourth subunit and it is involved in endosomal trafficking and vacuole biogenesis [20] . There are three types of PI3K in mammals: class I, II, and III. The functions of class II PI3K remains largely unknown, but both classes I and III PI3Ks are involved in autophagy. While class I PI3K is principally implicated in the modulation of signalling cascades, class III PI3K complexes regulate organelle biogenesis and, like yeast, contain three common components: hVps34, p150 (Vps15 ortholog), and Beclin 1 (Atg6 ortholog). The counterparts of Atg14 and Vps38 are called Atg14L/Barkor and UVRAG, respectively [22] [23] [24] . The Atg14L-containing complex plays a central role in autophagy and functions very similarly as the yeast complex I by directing the class III PI3K complex I to the phagophore to produce PI3P and initiate the recruitment of the Atg machinery ( Figure 1 ). Atg14L is thought to be present on the ER irrespective of autophagy induction [25] . Upon starvation, Atg14L localizes to autophagosomal membranes [8] . Importantly, depletion of Atg14L reduces PI3P production, impairs the formation of autophagosomal precursor structures, and inhibits autophagy [8, 24, 26, 27] . The UVRAG-containing class III PI3K complex also regulates autophagy but it appears to act at the intersection between autophagy and the endosomal transport pathways. UVRAG initially associates with the BAR-domain protein Bif-1, which may regulate mAtg9 trafficking from the trans-Golgi network (TGN) [28, 29] . UVRAG then interacts with the class C Vps/HOPS protein complex, promoting the fusion of autophagosomes with late endosomes and/or lysosomes [30] . Finally, the UVRAG-containing class III protein complex binds to Rubicon, a late endosomal and lysosomal protein that suppresses autophagosome maturation by reducing hVps34 activity [26, 31] . Importantly, both the Atg14L-and UVRAG-containing complexes interact through Beclin 1 with Ambra1, which in turn tethers these protein complexes to the cytoskeleton via an interaction with dynein [32, 33] . Following the induction of autophagy, ULK1 phosphorylates Ambra1 thus releasing the class III PI3K complexes from dynein and their subsequent relocalization triggers autophagosome formation. Therefore, Ambra1 constitutes a direct regulatory link between the Atg1/ULK1 and the PI3K complexes [32] .
Together with the Atg1/ULK and the PI3K complexes, Atg9 is one of the first factors localizing to the preautophagosomal structure or phagophore assembly site (PAS), the structure believed to be the precursor of the phagophore [9, 34] (Figure 1 ). Atg9 is the only conserved transmembrane protein that is essential for autophagy. It is distributed to the PAS and multiple additional cytoplasmic tubulovesicular compartments derived from the Golgi [35] [36] [37] . Atg9 cycles between these two locations and consequently it is thought to serve as a membrane carrier providing the lipid building blocks for the expanding phagophore [37] . One of the established functions of Atg9 is that it leads to the formation of the yeast PAS when at least one of the cytoplasmic tubulovesicular compartments translocates near the vacuole [34] . Atg9 is also essential to recruit the PI3K Complex I to the PAS [9] . Retrieval transport of yeast Atg9 from the PAS and/or complete autophagosome is mediated by the 4 International Journal of Cell Biology
Atg2-Atg18 complex [38] and appears to be regulated by the Atg1/ULK and PI3K complexes [37] . Mammalian Atg9 (mAtg9) has similar characteristics to its yeast counterpart. mAtg9 localizes to the TGN and late endosomes and redistributes to autophagosomal structures upon the induction of autophagy (Figure 1 ) [39] , further promoting pathway activity [29, [40] [41] [42] . As in yeast, cycling of mAtg9 between locations also requires the Atg1/ULK complex and kinase activity hVps34 [39, 43] .
The core Atg machinery also entails two ubiquitin-like proteins, Atg12 and Atg8/microtubule-associated protein 1 (MAP1)-light chain 3 (LC3), and their respective, partially overlapping, conjugation systems [44] [45] [46] (Figure 1 ). Atg12 is conjugated to Atg5 through the activity of the Atg7 (E1like) and the Atg10 (E2-like) enzymes. The Atg12-Atg5 conjugate then interacts with Atg16, which oligomerizes to form a large multimeric complex. Atg8/LC3 is cleaved at its C terminus by the Atg4 protease to generate the cytosolic LC3-I with a C-terminal glycine residue, which is then conjugated to phosphatidylethanolamine (PE) in a reaction that requires Atg7 and the E2-like enzyme Atg3. This lipidated form of LC3 (LC3-II) is attached to both faces of the phagophore membrane. Once the autophagosome is completed, Atg4 removes LC3-II from the outer autophagosome surface. These two ubiquitination-like systems appear to be closely interconnected. On one hand, the multimeric Atg12-Atg5-Atg16 complex localizes to the phagophore and acts as an E3-like enzyme, determining the site of Atg8/LC3 lipidation [47, 48] . On the other hand, the Atg8/LC3 conjugation machinery seems to be essential for the optimal functioning of the Atg12 conjugation system. In Atg3-deficient mice, Atg12-Atg5 conjugation is markedly reduced, and normal dissociation of the Atg12-Atg5-Atg16 complex from the phagophore is delayed [49] . Some evidences suggest that these two conjugation systems also function together during the expansion and closure of the phagophore. For example, overexpression of an inactive mutant of Atg4 inhibits the lipidation of LC3 and leads to the accumulation of a number of nearly complete autophagosomes [47] . While controversial [50] , it has been postulated that Atg8/LC3 also possesses fusogenic properties, thus mediating the assembly of the autophagic membrane [51, 52] .
It has to be noted that mammals possess at least 7 genes coding for LC3/Atg8 proteins that can be grouped into three subfamilies: (1) the LC3 subfamily containing LC3A, LC3B, LC3B2 and LC3C; (2) the gammaaminobutyrate receptor-associated protein (GABARAP) subfamily comprising GABARAP and GABARAPL1 (also called GEC-1); (3) the Golgi-associated ATPase enhancer of 16 kDa (GATE-16) protein (also called GABARAP-L2/GEF2) [53] . Although in vivo studies show that they are all conjugated to PE, they appear to have evolved complex nonredundant functions [54] .
Membranes. The origin of the membranes composing autophagosomes is a long-standing mystery in the field of autophagy. A major difficulty in addressing this question has been that phagophores as well as autophagosomes do not contain marker proteins of other subcellular compartments [55, 56] . A series of new studies has implicated several cellular organelles as the possible source for the autophagosomal lipid bilayers. The plasma membrane and elements of the trafficking machinery to the cell surface have been linked to the formation of an early autophagosomal intermediate, perhaps the phagophore [57] [58] [59] [60] [61] . It is possible that early endosomal-and/or Golgiderived membranes are also key factors in the initial steps of autophagy [34, 36, 39] . The Golgi, moreover, appears also important for autophagy by supplying at least in part the extra lipids required for the phagophore expansion [29, [62] [63] [64] [65] . The endoplasmic reticulum (ER) is also central in this latter event. While the relevance of the ER in autophagosome biogenesis was already pointed out a long time ago [5, 55, 66, 67] , recently two electron tomography studies have demonstrated the existence of a physical connection between the ER and the forming autophagosomes [68, 69] . These analyses have revealed that the ER is connected to the outer as well as the inner membrane of the phagophore through points of contact, supporting the notion that lipids could be supplied via direct transfer at the sites of membrane contact. In line with this view, it has been found that Atg14L is associated to the ER and PI3P is generated on specific subdomains of this organelle from where autophagosomes emerge under autophagy-inducing conditions [25, 70] (Figure 1 ). It has also been proposed that the outer membrane of the mitochondria is the main source of the autophagosomal lipid bilayers, but while the experimental evidences appear to show that mitochondria are essential for the phagophore expansion, it remains unclear whether these organelles play a key role in the phagophore biogenesis [71] . The discrepancy between the conclusions of the various studies has not allowed yet drawing a model about the membrane dynamics during autophagosome biogenesis. The different results could be due to the different experimental conditions and model systems used by the various laboratories. Alternatively, the lipids forming the autophagosomes could have different sources depending on the cell and the conditions inducing autophagy [72, 73] . A third possibility is that the source of phagophore membrane could depend on the nature of the double-membrane vesicle cargo. Additional investigations are required to shed light on these issues.
Despite the potential of curing, quite a substantial range of specific pathological conditions by inducting autophagy, there are currently no small molecules that allow to exclusively stimulate this pathway [74] . Nevertheless, there is a variety of chemicals that by acting on signaling cascades that also regulate autophagy permit to trigger this degradative process. These agents fall into two distinct categories based on the mechanism of action; whether they work through an mTORdependent (Rapamycin or Torin) or mTOR-independent pathway (e.g., lithium or resveratrol) [74] . In addition to these compounds, there are biological molecules such as interferon γ (IFNγ) and vitamin D that can be used to stimulate autophagy especially in experimental setups [75, 76] .
International Journal of Cell Biology 5 Inhibition of autophagy can also be beneficial in specific diseases but as for the inducers there are no compounds that exclusively block this pathway without affecting other cellular processes. The small molecules inhibiting autophagy include wortmannin and 3-methyladenine, which hamper the activity of the PI3K; Bafilomycin A and chloroquine, which impair the degradative activity of lysosomes [77] . They are currently solely used in the basic research on autophagy.
It is becoming increasingly evident that autophagy is a highly selective quality control mechanism whose basal levels are important to maintain cellular homeostasis (see below). A number of organelles have been found to be selectively turned over by autophagy and cargo-specific names have been given to distinguish the various selective pathways, including the ER (reticulophagy or ERphagy), peroxisomes (pexophagy), mitochondria (mitophagy), lipid droplets (lipophagy), secretory granules (zymophagy), and even parts of the nucleus (nucleophagy). Moreover, pathogens (xenophagy), ribosomes (ribophagy), and aggregate-prone proteins (aggrephagy) are specifically targeted for degradation by autophagy [78] .
Selective types of autophagy perform a cellular quality control function and therefore they must be able to distinguish their substrates, such as protein aggregates or dysfunctional mitochondria, from their functional counterparts. The molecular mechanisms underlying cargo selection and regulation of selective types of autophagy are still largely unknown. This has been an area of intense research during the last years and our understanding of the various selective types of autophagy is starting to unravel. A recent genomewide small interfering RNA screen aimed at identifying mammalian genes required for selective autophagy found 141 candidate genes to be required for viral autophagy and 96 of those genes were also required for Parkin-mediated mitophagy [79] .
In general, these pathways appear to rely upon specific cargo-recognizing autophagy receptors, which connect the cargo to the autophagic membranes. The autophagy receptors might also interact with specificity adaptors, which function as scaffolding proteins that bring the cargo-receptor complex in contact with the core Atg machinery to allow the specific sequestration of the substrate. The selective types of autophagy appear to rely on the same molecular core machinery as non-selective (starvation-induced) bulk autophagy. In contrast, the autophagy receptors and specificity adapters do not seem to be required for nonselective autophagy.
Autophagy receptors are defined as proteins being able to interact directly with both the autophagosome cargo and the Atg8/LC3 family members through a specific (WxxL) sequence [80] , commonly referred to as the LC3-interacting region (LIR) motif [81] or the LC3 recognition sequences (LRS) [82] . Based on comparison of LIR domains from more than 20 autophagy receptors it was found that the LIR consensus motif is an eight amino acids long sequence that can be written D/E-D/E-D/E-W/F/Y-X-X-L/I/V. Although not an absolute requirement, usually there is at least one acidic residue upstream of the W-site. The terminal L-site is occupied by a hydrophobic residue, either L, I, or V [83] . The LIR motifs of several autophagy receptors have been found to interact both with LC3 and GABARAP family members in vitro, but whether this reflects a physiological interaction remains to be clarified in most cases. It should be pointed out that not all LIR-containing proteins are autophagy cargo receptors. Some LIR-containing proteins, like Atg3 and Atg4B, are recruited to autophagic membranes to perform their function in autophagosome formation [84, 85] , whereas others like FYVE and coiled-coil domain-containing protein 1 (FYCO1) interact with LC3 to facilitate autophagosome transport and maturation [86] . Others might use an LIR motif to become degraded, like Dishevelled, an adaptor protein in the Wnt signalling pathway [87] . The adaptor proteins are less well-described, but seem to interact with autophagy receptors and work as scaffold proteins recruiting and assembling the Atg machinery required to generate autophagosomes around the cargo targeted to degradation. Examples of autophagy adaptors are Atg11 and ALFY [88, 89] .
The list of specific autophagy receptors is rapidly growing and the role of several of them in different types of selective autophagy will be described in detail in the reviews of this special issue. Here we will briefly discuss the best studied form of selective autophagy, the yeast cytosol to vacuole targeting (Cvt) pathway, as well as the best studied mammalian autophagy receptor, p62/sequestosome 1 (SQSTM1) (Figure 2 ).
The Cvt pathway is a biosynthetic process mediating the transport of the three vacuolar hydrolases, aminopeptidase 1 (Ape1), aminopeptidase 4 (Ape4) and α-mannosidase (Ams1), and the Ty1 transposome into the vacuole [90, 91] . Ape1 is synthesized as a cytosolic precursor (prApe1), which multimerizes into the higher order Ape1 oligomer, to which Ape4, Ams1, and Ty1 associate to form the socalled Cvt complex, prior to being sequestered into a small autophagosome-like Cvt vesicle. Sequestration of the Cvt complex into Cvt vesicles is a multistep process, which requires the autophagy receptor Atg19, which facilitates binding to Atg8 at the PAS, as well as the adaptor protein Atg11 (Figure 2(a) ) [92] . Atg11 acts as a scaffold protein by directing the Cvt complex and Atg9 reservoirs translocation to the PAS in an actin-dependent way and then recruiting the Atg1/ULK complex [40, 93] . The PI3P-binding proteins Atg20, Atg21, and Atg24 are also required for the Cvt pathway [94, 95] , but their precise function remains to be elucidated. Interestingly, Atg11 overexpression was found to recruit more Atg8 and Atg9 to the PAS resulting in more Cvt vesicles. This observation indicates that Atg11 levels could regulate the rate of selective autophagy, and maybe also the size of the cargo-containing autophagosomes in yeast [90, 96] . Indeed, a series of studies has revealed that Atg11 is also involved in other types of selective autophagy such as mitophagy and pexophagy. However, the autophagy receptors involved in the different Atg11-dependent types Figure 2 : Representative selective autophagy. (a) The cytoplasm-to-vacuole targeting (Cvt) pathway. Ape1 is synthesized as a cytoplasmic precursor protein with a propeptide and rapidly oligomerizes into dodecamers that subsequently associate with each other to form a higher order complex. The autophagy receptor Atg19 directly binds to the complex and mediates the recruitment of another Cvt pathway cargo, Ams1, leading to the formation of the so-called Cvt complex. Atg19 also interacts with the autophagy adaptor Atg11 and this protein allows the transport of the Cvt complex to the site where the double-membrane vesicle will be generated. At this location, Atg11 tethers the Atg proteins essential for the Cvt vesicle formation and the direct binding of Atg19 to Atg8 permits the exclusive sequestration of the Cvt complex into the vesicle. (b) A model for p62 and NBR1 as autophagy receptors for ubiquitinated cargos. p62 and NBR1 bind with ubiquitinated cargos via their ubiquitin-associated (UBA) domain and this interaction triggers the aggregate formation through the oligomerization of p62 via its Phox and Bem1p (PB1) domain. Furthermore, p62 interacts with both autophagy-linked FYVE protein (ALFY), which serves to recruit Atg5 and to bind PI3P, and directly with LC3. This latter event appears to organize and activate the Atg machinery in close proximity of the ubiquitinated cargos, which allows to selectively sequester them in the autophagosomes in analogous to the Cvt pathway.
of selective autophagy are different as Atg32 is required for mitophagy [97, 98] , whereas Atg30 is essential for pexophagy [99] . Like Atg19, these two proteins have an Atg8-binding LIR motif and directly interact with Atg11. Mammalian cells appear to not possess an Atg11 homologue, and further studies are necessary to delineate the molecular machinery involved in sequestration and targeting of different cargoes for degradation by autophagy in higher eukaryotes. The mechanism of the Cvt pathway is reminiscent of the selective form of mammalian autophagy called aggrephagy, which involves degradation of misfolded and unwanted proteins by packing them into ubiquitinated aggregates. In both cases aggregation of the substrate (prApe1 or misfolded proteins) is required prior to sequestration into Cvt vesicles or autophagosomes, respectively [100] [101] [102] . Similar to Cvt vesicles, aggregate-containing autophagosomes appear to be largely devoid of cytosolic components suggesting that the vesicle membrane expands tightly around its cargo [88] . Aggrephagy also depends on proteins with exclusive functions in substrate selection and targeting [81, 88, 100, 103] .
The autophagy receptors p62 and neighbour of BRCA1 gene (NBR1) bind both ubiquitinated protein aggregates through an ubiquitin-associated (UBA) domain and to LC3 via their LIR motifs and, thereby, promote the specific autophagic degradation of ubiquitinated proteins (Figure 2(b) ) [81, 82, 100, 103, 104] . NBR1 and p62 also contain an Nterminal Phox and Bem1p (PB1) domain through which they can oligomerize, or interact with other PB1-containing binding partners [83] . In addition to being a cargo receptor for protein aggregates, p62 has been implicated in autophagic degradation of other ubiquitinated substrates such as intracellular bacteria [105] , viral capsid proteins [106] , the midbody remnant formed after cytokinesis [107] , peroxisomes [108, 109] , damaged mitochondria [110, 111] , and bacteriocidal precursor proteins [112] . The PB1 domain was recently found to be required for p62 to localize to the autophagosome formation site adjacent to the ER [113] , suggesting that it could target ubiquitinated cargo to the site of autophagosome formation or alternatively promote the assembly of the Atg machinery at this location.
International Journal of Cell Biology 7
The large scaffolding protein autophagy-linked FYVE (ALFY) appears to have a similar function as the specificity adaptor Atg11. ALFY is recruited to aggregate-prone proteins through its interaction with p62 [101] and through a direct interaction with Atg5 and PI3P it serves to recruit the core Atg machinery and allow formation of autophagic membranes around the protein aggregate [88] (Figure 2(b) ). Interestingly, ALFY is recruited from the nucleus to cytoplasmic ubiquitin-positive structures upon cell stress suggesting that it might regulate the level of aggrephagy [114] . In line with this, it was found that overexpression of ALFY in mouse and fly models of Huntington's disease reduced the number of protein inclusions [88] . It will be interesting to determine whether ALFY, as p62, is involved in other selective types of autophagy such as the one eliminating midbody ring structures or mitochondria.
It is well known that posttranslational modifications like phosphorylation and ubiquitination are involved in the regulation of the activity of proteins involved in autophagy and degradation of autophagic cargo proteins, respectively. However, little is known about how these modifications may regulate selective autophagy. The fact that the core Atg machinery is required for both nonselective and selective types of autophagy gives raise to the question of whether these two types of autophagy may compete for the same molecular machinery. Such a competition could be detrimental for the cells undergoing starvation and to avoid this, there might be a tight regulation of the expression level and/or activity of the proteins specifically involved the selective autophagy. It has recently been proposed that phosphorylation of autophagy receptors might be a general mechanism for the regulation of selective autophagy. Dikic and coworkers noted that several autophagy receptors contain conserved serine residues adjacent to their LIR motifs and indeed, the TANK binding kinase 1 (TBK1) was found to phosphorylate a serine residue close to the LIR motif of the autophagy receptor optineurin. This modification enhances the LC3 binding affinity of optineurin and promotes selective autophagy of ubiquitinated cytosolic Salmonella enterica [115] . In yeast, phosphorylation of Atg32, the autophagy receptor for mitophagy, by mitogen-activated protein kinases was found to be required for mitophagy [116, 117] .
The Atg8/LC3 proteins themselves have also been found to become phosphorylated and recent works have identified specific phosphorylation sites for protein kinase A (PKA) [118] and protein kinase C (PKC) [119] in the Nterminal region of LC3. Interestingly, the N-terminal of LC3 is involved in the binding of LC3 to LIR-containing proteins [120] . It is therefore tempting to speculate that phosphorylation of the PKA and PKC sites might facilitate or prevent the interaction of LC3 with LIR-containing proteins such as p62. It has been found that phosphorylation of the PKA site, which is conserved in all mammalian LC3 isoforms, but not in GABARAP, inhibits recruitment of LC3 into autophagosomes [118] .
The role of ubiquitin in autophagy has so far been ascribed as a signal for cargo degradation. Ubiquitination of aggregate prone proteins, as well as bacteria and mitochondria, has been found to serve as a signal for recognition by autophagy receptors like p62 and NBR1, which are themselves also degraded together with the cargo that they associate with [83] . The in vivo specificity of p62 and NBR1 toward ubiquitin signals remains to be established under the different physiological conditions. Interestingly, it was recently found that casein kinase 2-(CK2-) mediated phosphorylation of the p62 UBA domain increases the binding affinity of this motif for polyubiquitin chains leading to more efficient targeting of polyubiquitinated proteins to autophagy [121] . CK2 overexpression or phosphatase inhibition reduced the formation of aggregates containing the polyglutamine-expanded huntingtin exon1 fragments in a p62-dependent manner. The E3 ligases involved in ubiquitination of different autophagic cargo largely remains to be identified. However, it is known that the E3 ligases Parkin and RNF185 both regulate mitophagy [122, 123] . SMURF1 (SMAD-specific E3 ubiquitin protein ligase 1) was recently also implicated in mitophagy, as well as in autophagic targeting of viral particles [79] . Interestingly, the role of SMURF1 in selective autophagy seems to be independent of its E3 ligase activity, but it rather depends on its membrane-targeting C2 domain, although the exact mechanism involved remains to be elucidated. It is also not clear whether ubiquitination could serve as a signal to regulate the activity or binding selectivity of proteins directly involved in autophagy, and whether this in some way could regulate selective autophagy. The role of ubiquitinlike proteins as SUMO and Nedd in autophagy is also unexplored.
Acetylation is another posttranslational modification that only recently has been implicated in selective autophagy. The histone de-acetylase 6 (HDAC6), initially found to mediate transport of misfolded proteins to the aggresome [124] , was lately implicated in maturation of ubiquitinpositive autophagosomes [125] . The fact that HDAC6 overproduction in fly eyes expressing expanded polyQ proteins is neuroprotective further indicates that HDAC6 activity stimulates aggrephagy [126] . Furthermore, the acetylation of an aggrephagy cargo protein, muntant huntingtin, the protein causing Huntington's disease, is important for its degradation by autophagy [127] . HDAC6 has been also implicated in Parkin-mediated clearance of damaged mitochondria [128] . The acetyl transferase(s) involved in these forms of selective autophagy is currently unknown, but understanding the role of acetylation in relation to various aspects of autophagy is an emerging field and it will very likely provide more mechanistic insights into these pathways.
Basal autophagy acts as the quality control pathway for cytoplasmic components and it is crucial to maintain the homeostasis of various postmitotic cells [129] . While this quality control could be partially achieved by nonselective autophagy, growing lines of evidence have demonstrated 8
International Journal of Cell Biology that specific proteins, organelles, and invading bacteria are specifically degraded by autophagy (Figure 3 ).
Mice deficient in autophagy die either in utero (e.g., Beclin 1 and Fip200 knockout mice) [130] [131] [132] or within 24 hours after birth due, at least in part, to a deficiency in the mobilization of amino acids from various tissues (e.g., Atg3, Atg5, Atg7, Atg9, and Atg16L knockout mice) [49, [133] [134] [135] [136] . As a result, to investigate the physiological roles of autophagy, conditional knockout mice for Atg5, Atg7, or FIP200 and various tissue-specific Atg knockout mice have been established and analyzed [133, 137, 138] . For example, the liver-specific Atg7-deficient mouse displayed severe hepatomegaly accompanied by hepatocyte hypertrophy, resulting in severe liver injuries [133] . Mice lacking Atg5, Atg7, or FIP200 in the central nervous system exhibited behavioral deficits, such as abnormal limb-clasping reflexes and reduction of coordinated movement as well as massive neuronal loss in the cerebral and cerebellar cortices [137] [138] [139] . Loss of Atg5 in cardiac muscle caused cardiac hypertrophy, left ventricular dilatation, and systolic dysfunction [140] . Skeletal muscle-specific Atg5 or Atg7 knockout mice showed age-dependent muscle atrophy [141, 142] . Pancreatic β cell-specific Atg7 knockout animals exhibited degeneration of islets and impaired glucose tolerance with reduced insulin secretion [143, 144] . Podocytespecific deletion of Atg5 caused glomerulosclerosis in aging mice and these animals displayed increased susceptibility to proteinuric diseases caused by puromycin aminonucleoside and adriamycin [145] . Proximal tubule-specific Atg5 knockout mice were susceptible to ischemia-reperfusion injury [146] . Finally, deletion of Atg7 in bronchial epithelial cells resulted in hyperresponsiveness to cholinergic stimuli [147] . All together, these results undoubtedly indicate that basal autophagy prevents numerous life-threatening diseases.
How does impairment of autophagy lead to diseases? Ultrastructural analyses of the mutant mice revealed a marked accumulation of swollen and deformed mitochondria in the mutant hepatocytes [133] , pancreatic β cells [143, 144] , cardiac and skeletal myocytes [140, 141] and neurons [138] , but also the appearance of concentric membranous structures consisting of ER or sarcoplasmic reticulum in hepatocytes [133] , neuronal axons [137, 139] and skeletal myocytes [141] , as well as an increased number of peroxisomes and lipid droplets in hepatocytes [133, 148] . In addition to the accumulation of aberrant organelles, histological analyses of tissues with defective autophagy showed the amassment of polyubiquitylated proteins in almost all tissues (although the level varied from one region to another) forming inclusion bodies whose size and number increased with aging [149] . Consequently, basal autophagy also acts as the quality control machinery for cytoplasmic organelles (Figure 3(a) ). Although this could be partially achieved by bulk autophagy, these observations point to the existence of selective types of autophagy, a notion that is now supported by experimental data.
p62/SQSTM1 is the best-characterized disease-related autophagy receptor and a ubiquitously expressed cellular protein conserved among metazoan but not in plants and fungi [83] . Besides a role of p62 as the receptor, this protein itself is specific substrate for autophagy. Suppression of autophagy is usually accompanied by an accumulation of p62 mostly in large aggregates also positive for ubiquitin (Figure 3(a) ) [104, 150] . Ubiquitin and p62-positive inclusion bodies have been detected in numerous neurodegenerative diseases (i.e., Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis), liver disorders (i.e., alcoholic hepatitis and steatohepatitis), and cancers (i.e., malignant glioma and hepatocellular carcinoma) [151] . Very interestingly, the p62positive aggregates observed in hepatocytes and neurons of liver-and brain-specific Atg7 deficient mice, respectively, as well as in human hepatocellular carcinoma cells, are completely dispersed by the additional loss of p62 strongly implicating involvement of p62 in the formation of diseaserelated inclusion bodies [104, 152] .
Through its self-oligomerization, p62 is involved in several signal transduction pathways. For example, this protein functions as a signaling hub that may determine whether cells survive by activating the TRAF6-NF-κB pathway, or die by facilitating the aggregation of caspase 8 and the downstream effector caspases [153, 154] . On the other hand, p62 interacts with the Nrf2-binding site on Keap1, a component of the Cullin 3-type ubiquitin ligase for Nrf2, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes including a battery of antioxidant proteins [155] [156] [157] [158] [159] . It is thus plausible that excess accumulation or mutation of p62 leads to hyperactivation of these signaling pathways, resulting in a disease onset (Figure 3(b) ).
Paget's disease of bone is a chronic and metabolic bone disorder that is characterized by an increased bone turnover within discrete lesions throughout the skeleton. Mutations in the p62 gene, in particular in its UBA domain, can cause this illness [160] . A proposed model explaining how p62 mutations lead to the Paget's disease of bone is the following: mutations of the UBA domain cause an impairment in the interaction between p62 and ubiquitinated TRAF6 and/or CYLD, an enzyme deubiquitinating TRAF6, which in turn enhances the activation of the NF-κB signaling pathway and the resulting increased osteoclastogenesis (Figure 3(b) ) [160] . If proven, this molecular scenario could open the possibility of using autophagy enhancers as a therapy to cure Paget's disease of bone.
It is established that autophagy has a tumor-suppressor role and several autophagy gene products including Beclin1 and UVRAG are known to function as tumor suppressor proteins [161] . The tumor-suppressor role of autophagy appears to be important particularly in the liver. Spontaneous tumorigenesis is observed in the livers of mice with either a systemic mosaic deletion of Atg5 or a hepatocytespecific Atg7 deletion [152, 162] . Importantly, no tumors are formed in other organs in Atg5 mosaically deleted mice. Enlarged mitochondria, whose functions are at least partially impaired, accumulate in Atg5-or Atg7-deficient hepatocytes [152, 162] . This observation is in line with the previous data obtained in iBMK cell lines showing that both the oxidative stress and genomic damage responses are activated by loss of autophagy [163, 164] . Again, it is clear that accumulation of p62, at least partially, contributes to tumor growth because the size of the Atg7 −/− liver tumors is reduced by the additional deletion of p62 [162] , which may cause a dysregulation of NF-κB signaling [165] and/or a persistent activation of Nrf2 [166] .
Almost all tissues with defective autophagy are usually displaying an accumulation of polyubiquitinated proteins [149] . Loss of autophagy is considered to lead to a delay in the global turnover of cytoplasmic components [137] and/or to an impaired degradation of substrates destined for the proteasome [167] . Both observations could partially explain the accumulation of misfolded and/or unfolded proteins that is followed by the formation of inclusion bodies. As discussed above, p62 and NBR1 act as autophagy receptors for ubiquitinated cargos such as protein aggregates, mitochondria, midbody rings, bacteria, ribosomal proteins and virus capsids [83, 168] (Figure 3 ). Although these studies suggest the role of p62 as an ubiquitin receptor, it remains to be established whether soluble ubiquitinated proteins are also degraded one-by-one by p62 and possibly NBR1. A mass spectrometric analysis has clearly demonstrated the accumulation of all detectable topologies of ubiquitin chain in Atg deficient livers and brains, indicating that specific polyubiquitin chain linkage is not the decisive signal for autophagic degradation [169] . Because the increase in ubiquitin conjugates in the Atg7 deficient liver and brain is completely suppressed by additional knockout of either p62 or Nrf2 [169] , accumulation of ubiquitinated proteins in tissues defective in autophagy might be attributed to p62mediated activation of Nrf2, resulting in global transcriptional changes to ubiquitin-associated genes. Further studies are needed to precisely elucidate the degradation mechanism of soluble ubiquitinated proteins by autophagy.
Concomitant with the energy production through oxidative phosphorylation, mitochondria also generate reactive oxygen species (ROS), which cause damage through the oxidation of proteins, lipids and DNA often inducing cell death. Therefore, the quality control of mitochondria is essential to maintain cellular homeostasis and this process appears to be achieved via autophagy.
It has been postulated that mitophagy contributes to differentiation and development by participating to the intracellular remodelling that occurs for example during haematopoiesis and adipogenesis. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Nix/Bnip3L, an autophagy receptor whose structure resembles that of Atg32, is also an outer mitochondrial membrane protein that interacts with GABARAP [170, 171] and plays an important role in mitophagy during erythroid differentiation [172, 173] (Figure 3(c) ). Although autophagosome formation probably still occurs in Nix/Bnip3L deficient reticulocytes, mitochondrial elimination is severely impaired. Consequently, mutant reticulocytes are exposed to increased levels of ROS and die, and Nix/Bnip3L knockout mice suffer severe anemia. Depolarization of the mitochondrial membrane potential of mutant reticulocytes by treatment with an uncoupling agent results in restoration of mitophagy [172] , emphasizing the importance of Nix/Binp3L for the mitochondrial depolarization and implying that mitophagy targets uncoupled mitochondria. Haematopoietic-specific Atg7 knockout mice also exhibited severe anaemia as well as lymphopenia, and the mutant erythrocytes markedly accumulated degenerated mitochondria but not other organelles [174] . The mitochondrial content is regulated during the development of the T cells as well; that is, the high mitochondrial content in thymocytes is shifted to a low mitochondrial content in mature T cells. Atg5 or Atg7 deleted T cells fail to reduce their mitochondrial content resulting in increased ROS production as well as an imbalance in pro-and antiapoptotic protein expression [175] [176] [177] . All together, these evidences demonstrate the essential role of mitophagy in haematopoiesis.
Recent studies have described the molecular mechanism by which damaged mitochondria are selectively targeted for autophagy, and have suggested that the defect is implicated in the familial Parkinson's disease (PD) [178] (Figure 3(c) ). PINK1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, have been genetically linked to both PD and a pathway that prevents progressive mitochondrial damage and dysfunction. When mitochondria are damaged and depolarized, PINK1 becomes stabilized and recruits Parkin to the damaged mitochondria [122, [179] [180] [181] . Various mitochondrial outer membrane proteins are ubiquitinated by Parkin and mitophagy is then induced. Of note, PD-related mutations in PINK1 and Parkin impair mitophagy [122, [179] [180] [181] , suggesting that there is a link between defective mitophagy and PD. How these ubiquitinated mitochondria are recognized by the autophagosome remains unknown. Although p62 has been implicated in the recognition of ubiquitinated mitochondria, elimination of the mitochondria occurs normally in p62-deficient cells [182, 183] .
When specific bacteria invade host cells through endocytosis/phagocytosis, a selective type of autophagy termed xenophagy, engulfs them to restrict their growth [184] (Figure 3(d) ). Although neither the target proteins nor the E3 ligases have yet been identified, invading bacteria such as Salmonella enterica, Listeria monocytogenes, or Shigella flexneri become positive for ubiquitin when they access the cytosol by rupturing the endosome/phagosome limiting membrane [185, 186] . These findings raise the possibility that ubiquitin also serves as a tag during xenophagy. In fact, to date, three proteins, p62 [105, 185, 187] , NDP52 [188] , and optineurin [115] have been proposed to be autophagy receptors linking ubiquitinated bacteria and LC3. An ubiquitin-independent mechanism has recently been revealed; recognition of a Shigella mutant that lacks the icsB gene requires the tectonin domaincontaining protein 1 (Tecpr1), which appears to be a new type of autophagy adaptor targeting Shigella to Atg5-and WIPI-2-positive membranes [189] . Interestingly, the Shigella icsB normally prevents autophagic sequestration of this bacterium by inhibiting the interaction of Shigella VirG with Atg5 indicating that some bacteria have developed mechanism to inhibit or subvert autophagy to their advantage [190] . This latter category of pathogens also includes viruses such as Herpes simplex virus-1 (HSV-1), which express an inhibitor (ICP34.5) of Atg6/Beclin1 [106] . However, it was recently shown that a mutant HSV-1 strain lacking ICP34.5 becomes degraded by selective autophagy in a SMURF1dependent manner [79] , suggesting that selective autophagy plays an important role in our immune system.
Recently, a different antimicrobial function has been assigned to autophagy and this function appears to be selective. During infection, ribosomal protein precursors are transported by autophagy in a p62-dependent manner into lysosomes [112] . These ribosomal protein precursors are subsequently processed by lysosomal protease into small antimicrobial peptides. Importantly, it has been shown that induction of autophagy during a Mycobacterium tuberculosis infection leads to the fusion between phagosomes containing this bacterium and autophagosomes, and the production of the antimicrobial peptides in this compartment kills M. tuberculosis [112] .
While the molecular mechanism is largely unknown, autophagy contributes at least partially to the supply of free fatty acids in response to fasting (Figure 3(e) ). Fasting provokes the increase of the levels of free fatty acids circulating in the blood, which are mobilized from adipose tissues. These free fatty acids are rapidly captured by various organs including hepatocytes and then transformed into triglycerides by esterification within lipid droplets. These lipid droplets appear to be turned over by a selective type of autophagy that has been named lipophagy in order to provide endogenous free fatty acids for energy production through β-oxidation [148] . Indeed, liver-specific Atg7 deficient mice display massive accumulation of triglycerides and cholesterol in the form of lipid droplets [191] . Agoutirelated peptide-(AgRP-) expressing neurons also respond to increased circulating levels of free fatty acids after fasting and then induce autophagy to degrade the lipid droplets [192] . Similar to the case in hepatocytes, autophagy in the neurons supplies endogenous free fatty acids for energy production and seems to be necessary for gene expression of AgPR, which is a neuropeptide that increases appetite and decreases metabolism and energy expenditure [192] .
Originally, it was assumed that autophagy was exclusively a bulk process. Recent experimental evidences have demonstrated that through the use of autophagy receptors and adaptors, this pathway can be selective by exclusively degrading specific cellular constituents. The list of physiological and pathological situations where autophagy is selective is constantly growing and this fact challenges the earliest concept whether autophagy can be nonselective. It is believe that under starvation, cytoplasmic structures are randomly engulfed by autophagosomes and delivered into the lysosome to be degraded and thus generate an internal pool of nutrients. In yeast Saccharomyces cerevisiae, however, the degradation of ribosomes, for example, ribophagy, as well as mitophagy and pexophagy, and the transport of the prApe1 oligomer into the vacuole under the same conditions requires the presence of autophagy receptors [97, [193] [194] [195] . As a result, these observations suggest that autophagy could potentially always operate selectively. This is a conceivable hypothesis because this process allows the cell to survive stress conditions and the casual elimination of cytoplasmic structure in the same scenario could lead to the lethal depletion of an organelle crucial for cell survival. Future studies will certainly provide more molecular insights into the regulation and mechanism of the selective types of autophagy, and this information will also be important to determine if indeed bulk autophagy exists.
AgRP:
Agouti-related peptide AMPK:
AMP-activated protein kinase ALFY:
Autophagy-linked FYVE protein Ams1:
α-mannosidase 1 Ape1: Aminopeptidase 1 Ape4: Aminopeptidase 4 Atg:
Autophagy-related gene Bnip3L:
B-cell leukemia/lymphoma 2 (BCL-2)/adenovirus E1B interacting protein 3 CK2:
Casein kinase 2 CMA:
Chaperone-mediated autophagy Cvt:
Cytoplasm to vacuole targeting ER:
Endoplasmic reticulum FIP200:
Focal adhesion kinase family interacting protein of 200 kD FYCO1:
FYVE and coiled-coil domain-containing protein 1 GABARAP: Gamma-aminobutyrate receptor-associated protein GATE-16: Golgi-associated ATPase enhancer of 16 kDa HDAC6:
Histone de-acetylase 6 HOPS:
Homotypic fusion and protein sorting HSV-1:
Herpes simplex virus-1 Keap1:
Kelch-like ECH-associated protein 1 LC3:
Microtubule-associated protein 1 (MAP1)-light chain 3 LIR:
LC3-interacting region LRS:
LC3 recognition sequences NBR1:
Neighbour of BRCA1 gene NDP52:
Nuclear dot protein (NDP) 52 NF-κB:
Nuclear factor κB NIX:
Nip-like protein X Nrf2:
NF-E2 related factor 2 PAS:
Phagophore assembly site PB1:
Phox and Bem1p
International Journal of Cell Biology PE: Phosphatidylethanolamine PD:
Parkinson's disease PI3K:
Phosphatidylinositol 3-kinase PI3P:
Phosphatidylinositol 3-phosphate PKA:
Protein kinase A PKC:
Protein kinase C ROS:
Reactive oxygen species Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein SMURF1: SMAD-specific E3 ubiquitin protein ligase 1 SUMO: Small ubiquitin-like modifier SQSTM1: p62/sequestosome 1 TBK1:
TANK binding kinase 1 Tecpr1: Tectonin domain-containing protein 1 TRAF6: Tumour necrosis factor receptor-associated factor 6 TOR:
Target of Rapamycin TGN:
Trans-Golgi network UBA:
Ubiquitin associated ULK1:
Unc-51-like kinase 1 UVRAG: UV-resistance associated gen Vps:
Vacuolar protein sorting. The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica BACKGROUND: Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. CONCLUSIONS/SIGNIFICANCE: We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity. The liver flukes, Fasciola hepatica and Fasciola gigantica are the causative agents of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide [1] . Liver fluke causes economic losses of over US$ 3 billion worldwide per annum to livestock via mortality, reduction in host fecundity, susceptibility to other infections, decrease in meat, milk and wool production and condemnation of livers [1] . The disease is increasing in livestock worldwide with contributing factors such as climate change (warmer winters and wetter summers supporting larger intermediate mud snail host populations); fragmented disease management (only treating sheep not cattle and limiting veterinary interaction); encouragement of wet-lands; livestock movement; and/or failure/ resistance of chemical control treatments in the absence of commercial vaccines [1, 2] . Fasciolosis is also a re-emerging human disease with estimates of between 2.4 and 17 million people infected worldwide [3] . In response, the World Health Organisation have added fasciolosis to the preventative chemotherapy concept [4] .
There are currently no commercial vaccines and triclabendazole (TCBZ) is the most important fasciolicide, as the only drug with significant efficacy against adult worms and juveniles [5] . Evidence from developed countries where TCBZ has been used widely exposes the reliance on this drug as an Achilles heel of liver fluke chemotherapeutic control, with well-established evidence of drug-resistance [5] . Therefore, TCBZ does not offer a long-term sustainable option for livestock farmers worldwide. The need for a liver fluke vaccine is further underscored by the fact that the costs associated with anthelmintic intervention for fluke control make short-lived chemotherapy an unsustainable option in developing countries. Protein superfamily studies in liver fluke have provided a number of leading vaccine candidates. High quality one-gene based vaccine discovery research has identified several vaccine candidates from protein superfamilies that provide significant, but often variable protection rates in challenge animal trials against liver fluke.
For example, Mu class Glutathione transferase (GSTs) have been widely investigated as vaccine candidates for fasciolosis [6] [7] [8] [9] . The Mu class GSTs have established roles in general Phase II detoxification of xenobiotic and endogenously derived toxins in F. hepatica within the host bile environment [10] . The general detoxification role is supported by GSTs contributing to 4% of the total soluble protein in F. hepatica, with a widespread tissue distribution. Proteomics and EST sequencing approaches have now delineated what members of the GST family are expressed in F. hepatica and two new classes of GST, Sigma and Omega, have been uncovered [11] . In the related trematode, Schistosoma mansoni, the Sigma class GST (Sm28) has generally shown more robust protection in vaccine trials against schistosome infection [12] , than the F. hepatica Mu GSTs against F. hepatica infection.
Sigma class GSTs, unlike Mu Class GSTs, have been characterized as GSH-dependent hematopoietic prostaglandin synthases responsible for the production of prostaglandins in both mammals and parasitic worms [13] [14] [15] [16] [17] [18] . Prostaglandins have been extensively studied in mammals and are shown to be involved in a range of physiological and pathological responses [19] [20] [21] [22] [23] . Parasite-produced prostaglandins may be involved in parasite development and reproduction as well as the modulation of host immunity, allergy and inflammation during establishment and maintenance of a host infection [16, [24] [25] [26] [27] [28] ]. The host protection success of Sigma GST based vaccinations in schistosomiasis may therefore be related to neutralising specific functions in host-parasite interplay, such as prostaglandin synthase activity.
In this manuscript we follow four work pathways to functionally characterise the newly identified Sigma GST from F. hepatica. 1) We confirm its designation as a Sigma class GST using substrate profiling, 2) we assess prostaglandin synthase activity and its effect on host immune cells, 3) we localise the Sigma GST within adult fluke and between ontogenic stages and 4) assess its potential as a vaccine candidate.
GST proteins representative of recognised GST superfamily classes were obtained from European Bioinformatics Institute Interpro database (http://www.ebi.ac.uk/ interpro/), and from non-redundant databases at NCBI (http://www.ncbi.nlm.nih. gov/). A mammalian and a helminth or invertebrate GST sequence were selected for each GST class where available. Sequences were aligned via ClustalW program [29] in BioEdit Sequence Alignment Editor Version 7.0.5.2. [30] and sequence identity matrices produced from multiple alignments. Phylogenetic bootstrap neighbour-joining trees were produced as PHYLIP output files in ClustalX Version 1.83 [31] according to the neighbour-joining method of Saitou and Nei [32] . ClustalX default settings for alignments were accepted using the GONNET protein weight matrices with PHYLIP tree format files viewed within TREEVIEW [33] .
Recombinant Fasciola hepatica glutathione transferase Sigma class (rFhGST-S1) production Full-length cDNA for FhGST-S1 was available in the form of an expressed sequence tag (EST) clone Fhep24h03, details of which can be obtained from the previously published Sigma class GST [11] and is identical to the submitted GenBank accession No. DQ974116.1 (NCBI http://www.ncbi.nlm.nih.gov/).
FhGST-S1 was amplified via PCR using the following primer pair: rFhGST-S1 forward primer, 59 GGAATTCCATATGGA-CAAACAGCATTTCAAGTT 39;rFhGST-S1 reverse primer, 59 ATAAGAATGCGGCCGCCTAGAATGGAGTTTTTGCAC-GTTTTTT 39. Restriction enzyme sites (in bold type and underlined) for NdeI (forward primer) and NotI (reverse primer) were included so that the entire ORF could be directionally cloned into the pET23a (Novagen) vector. Recombinant protein was produced in Escherichia coli BL21(DE3) cells (Novagen).
Protein purification of rFhGST-S1 and native F. hepatica GSTs rFhGST-S1 protein was purified according to the glutathione affinity chromatography method of Simons and Vander Jagt [34] from transformed E. coli cytosol following protein expression. Native GSTs were purified from F. hepatica soluble cytosolic supernatants as previously described [11] . Purity of rFhGST-S1 was assessed by electrospray ionisation (ESI) mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2DE according to LaCourse et al. [35] .
A range of model and natural substrates (see Table 1 for details) were used to profile the Sigma GST. A number of ligands were also assessed for their ability to inhibit GST activity with 1-chloro-2, 4-dinitrobenzene (CDNB) as the second substrate [36] . Values were reported as the concentration of inhibitor required to bring GST specific activity to 50% of its original activity (IC50). At least six different inhibitor concentrations were used in each IC50 determination in triplicate. Inhibitors were pre-incubated for 5 minutes prior to starting reactions. IC50 values were estimated graphically [37] .
Prostaglandin synthase activity was assessed via an adapted method based upon those of Sommer et al. [26] and Meyer et al. [16, 38] , with extraction modifications based upon Schmidt et al. [39] . In brief, reactions were performed in glass vials in 2 mM sodium phosphate buffer, pH 7.4, containing 10 mM glutathione,
Combating neglected parasitic diseases is of paramount importance to improve the health of human populations and/or their domestic animals. Uncovering key roles in host-parasite interactions may support the vaccine potential portfolio of a parasite protein. Fasciola hepatica causes global disease in humans and their livestock but no commercial vaccines are available. Members of the Sigma class glutathione transferase (GST) family have long been highlighted as vaccine candidates towards parasitic flatworms. To this end, a Sigma class GST is currently undergoing phase II clinical trials to protect against infection from the schistosomes. In this study we characterise the protein from F. hepatica following four work pathways that 1) confirm its designation as a Sigma class GST using substrate profiling, 2) assess prostaglandin synthase activity and its effect on host immune cells, 3) localise the Sigma GST within adult fluke and between ontogenic stages and 4) measure its potential as a vaccine candidate. The work presented here shows F. hepatica Sigma class GST to have key host-parasite roles and we suggest, warrants further investigation for inclusion into vaccine formulations. Recombinant FhGST-S1 shows activity towards a broad range of model and natural GST substrates with a similar enzymatic profile to the Schistosomiasis vaccine trialist (Sm28GST -P09792). rFhGST-S1 also displays high glutathione-dependent lipid peroxidase activity compared to both Sm28GST and Sj26GST (Q26513) [47] . Reasonably high GSH-dependent lipid peroxidase activity has also been seen in a 'weak affinity' fraction following chromatofocusing of GSH transferase activity that failed to bind GSH-sepharose [10] . ND -Not determined.
**Data taken from Walker et al. [47] . doi:10.1371/journal.pntd.0001666.t001
Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org 50 mM NaCl, 0.5 mM tryptophan, 1 mM hematin, 1 U COX-1 enzyme, 100 mM arachidonic acid (All Sigma, UK. COX-1 [C0733]) and rFhGST-S1 at final concentration ranges of 0.1-100.0 mg/ml. Negative control reactions lacking either GST or COX-1 were also prepared. Reactions were incubated for 5-10 min in a water bath at 37uC. This was followed by 4 minutes incubation at 25uC in a shaking water bath. Prostaglandins were extracted by adding 860 mL of ice-cold ethyl acetate. Reactions were vortexed for 30 s then centrifuged briefly at 10,0006g at 4uC for 2 min. The upper ethyl acetate layer was retained and solvent was evaporated under a nitrogen stream at 45uC. The remaining residue was reconstituted in 50 ml of methanol/water/fomic acid (25:75:0,1) mix at pH 2.8 and stored at 280uC until ready for mass spectrometry analysis. Standards of prostaglandins D2, E2 and F2a (Cayman, Ltd) were also prepared in methanol/water/ formic acid mix for analysis.
The nano LC-MS analyses were performed using a Waters Q-Tof micro mass spectrometer (Waters) coupled to a LC-Packings Ultimate nano LC system (Dionex). The pre-column used was a LC Packings C18 PepMap 100 and the nano LC column used was a LC Packings 15 cm PepMap 100 C18 (both Dionex). Samples were loaded on the pre-column with mobile phase A (25% methanol with 0.1% formic acid added). Loading flow rate was 0.03 ml/min for 6 min. The samples were eluted on to the nano LC column using mobile phases B (60% acetonitrile) and C (100% methanol). A typical gradient profile was 100% B to 100% C in 10 min (flow rate of 0.2 ml/min) with the column held at 100% C for 1 hour. The mass spectrometer was operated in the negative ion nano electrospray mode with a source temperature of 80uC and capillary voltage 2.8 kV. The scan range was 40 to 400 Da for 1.5 s.
Liver fluke extract and excretory/secretory (ES) product preparation F. hepatica adults were collected, cultured in vitro for 4 h and the ES products collected and prepared as previously described [40] . Newly excysted juveniles (NEJ) were excysted from metacercariea in vitro and cultured in Fasciola saline for 4 h post excystment as previously described [41] . F. hepatica (adult and NEJ) soluble fractions were obtained by homogenisation of frozen fluke at 4uC in a glass grinder in lysis buffer (20 mM KHPO 4 , pH 7.0, 0.1% Triton-X100 and a cocktail of protease inhibitors [Roche, Complete-Mini, EDTA-free]). Homogenates were centrifuged at 100,0006 g for 1 h at 4uC. Supernatants were considered as the soluble cytosolic fraction. Cytosolic protein extracts were treated and resolved by 2DE as described previously [11] . F. hepatica eggs were isolated, cultured and protein extracted as previously described [42] .
Recombinant F. hepatica Sigma GST (rFhGST-S1), and native F. hepatica S-hexylGSH-affinity purified GST samples (and human/rat recombinant PGD-synthase) were subjected to standard SDS-PAGE and 2DE, electro-transferred to membranes [43, 44] and western blotted with a polyclonal antibody (1:20,000 dilution) raised in rabbits to the recombinant F. hepatica Sigma GST by Lampire Biological Laboratories, USA. Membranes were also probed with Mu class GST antibody (represented by the anti-Schistosoma japonicum GST26 Mu class antibody [1:1,000 dilution] and an anti-rat PGD-synthase antibody [1:1,000 dilution], Pharmacia-Biotech 27-4577). F. hepatica eggs, NEJs (somatic and ES preparations) and adults (somatic and ES preparations) were subjected to SDS-PAGE and also electro-transferred as described above and probed with the polyclonal antibody raised in rabbits to the recombinant F. hepatica Sigma GST. All western blots were developed as described previously [11] .
Immunolocalisation studies F. hepatica Sigma class GST (FhGST-S1) was detected by immunohistology in tissue sections of whole adult F. hepatica extracted from bile ducts of sheep liver and also in situ from sections of liver.
Staining for FhGST-S1 was performed on formalin-fixed and paraffin-embedded tissue sections according to the method described previously [45] . Sections were washed in Tris-buffered saline (TBS; 0.1 M Tris-HCl with 0.9% NaCl [pH 7.2]), treated with 0.05% (w/v) protease (type XXIV, bacterial: Sigma) in TBS for 5 min at 37uC for antigen retrieval, before three further 5 min washes in ice-cold TBS. Following TBS washes, sections were incubated for 10 min in 50% (v/v) swine serum in TBS followed by incubation for 15-18 h at 4uC in rFhGST-S1 polyclonal antibody (diluted at 1:500 in 20% swine serum in TBS). Sections were again washed in TBS before further incubation at ambient temperature (approximately 20uC+/23uC) with anti-rabbit peroxidise anti-peroxidase (PAP; diluted at 1:100 in 20% swine serum in TBS). Following washes with TBS, sections were incubated, with stirring, for 10 min, with 3,3-diaminobenzidine tetrahydrochloride (DAB; Fluka, Buchs, Switzerland) with 0.01% v/v hydrogen peroxide in 0.1 M imidazole buffer pH 7.1, before counterstaining with Papanicolaou's hematoxylin for 30 s. Sections were then rinsed, dehydrated in alcohol, cleared in xylene, and mounted. Consecutive sections from each tissue were used as negative controls in which the rFhGST-S1 polyclonal antibody was replaced by TBS.
Animals. C57BL/6 mice were purchased from Harlan Ltd (UK) and TLR4KO (on a C57BL/6 background) bone marrow cells were a gift from Professor Padraic Fallon (Trinity College Dublin, Ireland). All mice were maintained according to the Irish Department of Children and Health.
Cell culturing and cytokine analysis. Bone marrowderived immature dendritic cells (DCs) were prepared by culturing bone marrow cells isolated from the femurs and tibia of C57BL/6j and TLR4 2/2 mice in complete RPMI 1640 (cRPMI; 5% [v/v] heat inactivated Fetal Calf Serum [FCS] [30 mins at 60uC], 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 50 mM 2-mercaptoethanol) with recombinant mouse GM-CSF (20 ng/ml; R&D Systems), at 37uC. On days 3 and 6 of culture, fresh medium with GM-CSF (20 ng/ml) was added to the cells. On day 8, cells were harvested, counted and stained with CD11c (Caltag Laboratories) for analysis by flow cytometry to determine purity (.90%). J774 cells and RAW264.7, murine macrophage cell lines were cultured in cRPMI 1640 medium containing 10% (v/v) FCS. All cells were used to conduct experiments when they reached ,90% confluence.
For all experiments, cells were seeded into 24-well plates (Nunc) at 10 6 /ml in complete RPMI 1640 except for DCs where GM-CSF (5 ng/ml) was also added. Cells were treated with medium only, rFhGST-S1 (10 mg) or LPS (Alexa; 100 ng/ml) for 18 h. Levels of total prostaglandin (PG), prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) were measured using the Cayman competitive EIA. The values were calculated using free data analysis software available at www.caymanchem.com/analysis/ eia. Data are presented as the mean 6 SEM following subtraction Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org of medium controls and are representative of two separate experiments. Prior to experimentation, rFhGST-S1 was assessed for endotoxin (i.e. LPS) contamination using the Pyrogene endotoxin detection system (Cambrex).
Experimental design. Nineteen 5-month old, Malagueña breed goats were used for a vaccine trial. The animals were free of parasitic and infectious diseases as indicated by fecal analysis and absence of clinical signs. Group 1 (n = 10) were immunized with two subcutaneous injections of 100 mg of rFhGST-S1 in 1 ml of Quil A in 1 ml of PBS each injection separated by 4 weeks. Group 2 (n = 9) served as an infected control and was immunized at the same time with 1 ml of Quil A in 1 ml of PBS. Twelve weeks after the first immunization, animals were orally infected with 100 F. hepatica metacercariae of bovine origin. Three animals from each group were killed at 7, 8 and 9 days post-infection to study hepatic changes and host response during the early stages post-infection; the remaining animals (7 and 6 goats per group) were killed 15 weeks after infection to study fluke burdens, fecal egg counts and hepatic lesions. All goats were sacrificed by intravenous injection of thiobarbital. The experiment was approved by the Bioethical Committee of the University of Cordoba (N. 7119) and it was carried out according to European (86/609/CEE) and Spanish (RD 223/1988) directives for animal experimentation.
Fluke burdens and morphometrics. At necropsy, gallbladders and bile ducts were opened and flukes recovered. Llivers were cut (,1 cm pieces) and washed in hot water to collect the remaining flukes. Flukes were counted, measured and weighed.
Fecal egg counts. Sedimentation techniques at 12 and 13 weeks after infection using four grams of feces were conducted to give eggs per gram (EPG).
Pathological assessment. At necropsy livers were photographed by the visceral and diaphragmatic aspects for gross pathology evaluation as described previously [46] . Gross hepatic lesions were scored as: absent [2] ; mild [+] (less than 10% of hepatic surface affected); moderate [++] (10-25% of hepatic surface affected); severe [+++] (25-50% of hepatic surface affected) and very severe [++++] (more than 50% of hepatic surface affected). Tissue samples were collected from the left (6 samples) and right (2 samples) hepatic lobes, fixed in 10% buffered formalin and embedded in paraffin wax. Tissue sections (4 mm) were stained with haematoxylin and eosin (HE) for the histopathological study.
Specific IgG response. Specific IgG anti-rFhGST-S1were measured using the ELISA method as described previously [46] . A total of 10 mg/ml of rFhGST-S1 was used to coat microtitre plates, 100 ml/well of goat sera diluted in blocking buffer and rabbit antigoat IgG peroxidase conjugated (whole molecule -Sigma) diluted in blocking buffer at 1:10000. Serum pools were from ten experimentally infected goats and ten uninfected goats as positive and negative controls, respectively. All samples were analysed in duplicate. Results were expressed as antibody titre (Log10).
Aligning Sigma class GSTs of trematodes shows the extent of identity and similarity across this class of GSTs ( Figure S1 ). An amino acid sequence comparison of FhGST-S1 with other trematode GSTs places FhGST-S1 into the Sigma class of GSTs, with identities averaging approximately 45%. Comparison with the most closely matching mammalian GSTs shows sequence identities averaging only approximately 28% (Table S1 ). Despite phylogenetic neighbour-joining trees place mammalian and trematode GSTs within the same broad Sigma class ( Figure S1 ) there remains a distinct separation of the trematode and mammalian clusters.
Full sequence length recombinant F. hepatica Sigma Class GST (rFhGST-S1) was shown to be purified to a high level from transformed E. coli cytosol following expression yielding 57.3 mg of rFhGST-S1 from a 1 litre culture of BL21 (DE3) cells. Purity was judged by the presence of a single band upon SDS-PAGE at the estimated size and a dominating single peak via ESI MS at the precise calculated theoretical mass for the complete protein sequence (Figure 1 ). Analysing this fraction by 2D SDS-PAGE revealed a single protein resolving into 3 protein spots. Western blotting of the 2DE profile with anti-rFhGST-S1 antibody confirmed all 3 resolved protein spots as rFhGST-S1 (2DE and western blot data not shown). No recognition was seen probing the 3 spots with an anti-Mu class antibody.
rFhGST-S1 was produced as an active protein, displaying significant enzymic activity towards the model GST substrate 1chloro-2,4-dinitrobenzene (CDNB) and a range of substrates commonly used to characterise GSTs (Table 1) . F. hepatica GST is very similar in terms of its enzymatic profile to the GST of S. Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org japonicum currently undergoing clinical vaccine trials. FhGST-S1 also displays higher glutathione-dependent lipid peroxidase activity compared to both Sm28GST and Sj26GST [47] . Interestingly, ligand inhibition studies on rFhGST-S1 showed the enzymic activity of rFhGST-S1 with CDNB was inhibited by the major pro-active form of the main liver fluke drug Triclabendazole. The sulphoxide derivative (TCBZ SO) gave an IC50 (50% enzyme inhibition) of 5765 mM (5 replicates). Bile acids, potentially natural ligands for liver fluke tegumental associated proteins in the host bile environment, were also assessed for activity inhibition. The rFhGST-S1 interacted with all three bile acids tested using five replicate assays: Cholic acid (IC50 302673 mM); Deoxycholic acid (IC50 223621 mM) and Chenodeoxycholic acid (IC50 6469 mM) .
Previous studies on the Sigma class GSTs from both mammals and helminth parasites have revealed a capacity to synthesise Prostaglandin D2 (PGD2) and PGE2. Since prostaglandin synthase activity may be a conserved role of Sigma class GSTs, we also tested the ability of rFhGST-S1 to synthesise prostaglandin eicosanoids using a coupled assay with COX-1. COX-1 catalyses the conversion of arachidonic acid to the H2 form before the prostaglandin isomer is converted to either the D or E form. Nano-LC/MS analysis enabled us to detect the presence of both PGD2 and PGE2 in the assay mixture with the PGD2 form being the more abundant of the two prostanoids ( Figure 2 ). While some PGE2 in the mixture could have arisen from rapid degradation of the unstable PGH2, nano-LC-MS was unable to detect either PGD2 or PGE2 in negative control reactions lacking either COX-1 or GST. The rFhGST-S1 catalyses PGD2 formation in a concentration-dependent manner as previously described for rOvGST-1 [26] . PGD2 was also detected in coupled assays with rFhGST-S1 and COX-1 using an Enzyme Immno Assay (EIA) detection kit (Cayman) and showed similar results (results not shown).
FhGST-S1 was first identified in adult liver fluke in S-hexyl-GSH affinity isolated fractions of cytosol [11] . Western blots confirmed the presence of FhGST-S1 in NEJs and adult flukes and further enabled us to identify the Sigma GST in relative abundance in egg extracts, suggesting that it may play a metabolic role in embryogenesis/reproduction (Figure 3) . Western blot analyses demonstrate that FhGST-S1 is consistently expressed during the course of in vitro parasite embryonation (days 1-9, only data for days 2, 7 and 9 shown in Figure 3 ). In contrast, immunoblot analysis of freshly voided (day 0) eggs reveals that expression of the Sigma class GST is greatly reduced at the time of voiding from the host (Figure 3) . However, immunolocalisation studies of adult parasites revealed an abundance of FhGST-S1 in the vitelline cells and eggs, emphasising the likely importance of this enzyme in egg formation and development. Some staining was also found in the parasite parenchyma and tegument, also suggesting a role at the hostparasite interface ( Figure 4) . Indeed, FhGST-S1 was detected in ES products of adult fluke cultured in vitro ( Figure 3 ) suggesting that the protein could, in principle, come into contact with the host immune system as it is released from the tegument during tegumental turnover and sloughing of the fluke body surface. Figure 2 . Detection of prostaglandin synthase activity of rFhGST-S1 via a mass spectrometry approach. A coupled assay with rFhGST-S1 and COX-1 catalyses the conversion of arachidonic acid to the H2 form before the prostaglandin isomer is converted to either the D or E form. Nano-LC/MS analysis allowed detection of both PGD2 (A) and PGE2 (B) in the assay mixture with the PGD2 form being the more abundant of the two prostanoids (C). Boxed figures above peaks show the fragmentation ions specific to detection of PGD2 (a) and PGE2 (b) according to the method of Schmidt et al. [39] . doi:10.1371/journal.pntd.0001666.g002
Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org Influence of rFhGST-S1 on prostaglandin synthesis in host immune cells rFhGST-S1 exhibited prostaglandin synthase activity producing PGE2 and PGD2. In addition, it has been shown previously that rFhGST-S1 activates DCs in vitro [48] . Therefore, an attempt to determine if rFhGST-S1 could induce the secretion of total prostaglandin, PGE2 and PGD2 from DCs was performed. Prior to experimentation, endotoxin levels in rFhGST-S1 were assessed and were similar to that of the media alone. Both of which were below the lower limit of detection (,0.01 EU/ml). When examining prostaglandin induction DCs stimulated with rFhGST-S1 secreted total prostaglandin and PGE2 (DC (WT); Figure 5 ) but not PGD2 (data not shown). Since it has been previously determined that the activation of DCs by rFhGST-S1 was dependent upon TLR4 [48] we repeated the experiment in DCs from TLR4KO mice and in keeping with previous findings demonstrated that the secretion of total prostaglandin and PGE2 by rFhGST-S1 was significantly reduced in the absence of the TLR4 receptor (DC (TLR4KO); Figure 5 ). rFhGST-S1 was then further assessed for its potential to induce prostaglandin secretion from macrophages by exposing two macrophage cell lines with rFhGST-S1. After 18 hours the levels of total prostaglandin, PGE2 and PGD2 were measured. In this assay, both macrophage cells lines stimulated with rFhGST-S1 secreted total prostaglandin, PGE2 and PGD2 ( Figure 6 ). However, the levels secreted by J744 cell line were higher when compared to the amount secreted by RAW264.7 cell line. In these experiments we included medium only as a negative control and LPS as a positive control. In all experiments the levels of prostaglandin in response to rFhGST-S1 was comparable to the levels secreted in responses to LPS.
Assessment of goat vaccinations with rFhGST-S1 challenged with F. hepatica Following the completion of the vaccine trial, liver fluke were recovered and the livers scored. The resulting data is summarised in Table 2 . When assessing fluke burdens, length, weight and fecal egg counts, no significant differences between rFhGST-S1 immunised and Quil A immunised groups were observed. Despite this lack of significance, at 7-9 days post-infection (dpi) the number of gross hepatic lesions appeared reduced in rFhGST-S1 immunised groups compared to the Quil A control group. At 15 weeks post-infection (wpi), a similar outcome is observed. Liver hepatic lesion scoring appeared to show reductions in the severity of damage occurred in the rFhGST-S1 immunised group compared to the Quil A only group, despite no significant differences in the aforementioned morphometric data.
Microscopically, at 7-9 dpi animals from the Quil A group showed tortuous necrotic tracts surrounded by a scarce inflammatory infiltration with occasional eosinophils ( Figure 7A ). Older necrotic areas were surrounded by macrophages, epithelioid cells and multinucleate giant cells and lymphocytes. Some migrating larvae were found in the liver parenchyma without inflammatory infiltrate associated to them. In goats immunised with rFhGST-S1 smaller necrotic areas associated to a heavy infiltration of eosinophils ( Figure 7B ) were seen. Unlike the Quil A immunised group, all migrating larvae found were surrounded by a heavy infiltration of eosinophils.
A significant increase of IgG anti-rFhGST-S1 was observed two weeks after vaccination with a strong increase after the second injection at week 4 in immunised animals ( Figure 8 ). The Quil A control group did not show any specific IgG response until 2 weeks after infection. Specific IgG titres increased during infection in both groups, but they were consistently higher in the immunised group throughout the duration of the the experiment.
Previous studies have highlighted the importance of parasite GSTs, including Sigma class GSTs, in host-parasite interactions and as potential vaccination candidates. With this in mind, we have studied the relatively newly identified Sigma class GST from F. hepatica to both enhance our understanding of this important enzyme in Fasciola and the Sigma class of GSTs as a whole.
Alignments and phylogenetics classified FhGST-S1 alongside trematode and mammalian Sigma class GSTs, yet there remains a Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org distinct divide between the parasites and their hosts, a phenomenon also observed for the recently reclassified 'Nu' class of GSTs from nematodes [49] . Therefore, it may be that trematode GSTs are sufficiently distinct to support a sub-classification within the broad Sigma class. The distinction of FhGST-S1 from fasciolosis host Sigma class GSTs enhances its potential as a therapeutic target.
Substrate activity profiling of rFhGST-S1 using model substrates showed the enzyme to have comparable activity to other trematode Sigma class GSTs such as Sm28GST [47] . However, rFhGST-S1 exhibits relatively high GSH-conjugating activity towards the potentially natural reactive aldehyde, 4-hydroxy-nonenal (4-HNE) toxin and high GSH-dependent peroxidase activity towards the tested lipid peroxides which includes the endogenous substrate linoleic acid hydroperoxide. 4-HNE is the major aldehydic end-product of lipid peroxidation that is involved in signalling of host immune cells leading to apoptosis of T-and Bcells [50] .
Assessing the inhibition of rFhGST-S1 activity with CDNB revealed that both bile acids and the flukicide TCBZ appear to bind to the enzyme. In particular, the interaction of the bile acid cholate with rFhGST-S1 is approximately ten fold higher than GSTs from the sheep intestinal cestode Moniezia expansa [51] . Host bile acids are known as triggers of physiological processes in trematodes including Fasciola sp. [52, 53] . Therefore, molecular interaction of bile acids with FhGST-S1 warrants further investigation especially, given that FhGST-S1 is localised to near the body surface of the fluke, where it could potentially bind cholate and other free bile acids found in abundance in host bile (cholate is found at approximately 100 mM in sheep bile) [54] . The hydroxy-TCBZ SO levels in the bile have been shown to be in excess of 100 mM [55] thus, the IC50 of 5765 mM for TCBZ SO suggests the abundant FhGST-S1 could be involved in TCBZ response in phase III sequestration based detoxification. This finding warrants further investigation to understand the role of FhGST-S1 in TCBZ action or detoxification.
Sigma class GSTs from both parasites and mammals have been known to exhibit prostaglandin synthase activity. To this end, the Sigma GST from F. hepatica shares a high sequence identity with recognised Sigma class GSTs with prostaglandin synthase activity, including rOvGST-1 from the filarial parasite, Onchocerca volvulus. Using a coupled assay with COX-1 we have shown that rFhGST-S1 is capable of synthesizing both PGD 2 and PGE 2 , with PGD 2 being the predominant prostanoid. Parasite-derived eicosanoids, including prostaglandins, are known to be important in the establishment of parasitic infection and the survival and proliferation within the host. Therefore, eicosanoids produced by parasitic helminths may play a role in pathophysiological changes during helminth infections. For example, chronic fasciolosis is associated with fever and changes in liver biochemistry, both of which could be associated with parasite-derived eicosanoids thromboxane B2 (TXB2), PGI 2 , PGE 2 and leukotriene B4 (LTB 4 ), detected in the ES products and homogenates of adult F. hepatica worms [56] . In addition, the migration of host epidermal Langerhans cells, which play a key part in immune defence mechanisms, has been shown to be inhibited by parasite-derived PGD 2 in the Schistosoma mansonimouse model of human infection, thus allowing schistosomes to manipulate the host immune system [57] . Earlier studies have revealed the presence of eicosanoids produced by S. mansoni cercariae which could also play a role in establishment of infections through loss of the cercarial tail following penetration of the skin [58] . It therefore seems likely that prostaglandins synthesised via FhGST-S1 will have a role in establishing the infection within the host.
In general, prostaglandins and eicosanoids have potent biological activities in reproduction. For example in the zebrafish egg, high levels of PGE 2 were seen post fertilisation coupled with high PGD 2 synthase transcript levels during the early stages of egg Figure 4 . Images of FhGST-S1 localisation within F. hepatica tissue. A) Anti-F. hepatica FhGST-S1 immunohistochemical stain of a fluke in cross section within the host sheep liver bile duct. Heavily stained eggs (E) are shown released from the fluke into the bile duct in the top left-hand corner. Brown stained areas show the presence of FhGST-S1 proteins. The lack of staining in the host liver (L) highlights the specificity of the antibody. Composite picture. B) Enlarged region of A showing the intense anti-F. hepatica FhGST-S1staining in the voided eggs (E). The spines (S) present in the tegument (T) can be clearly distinguished by their lack of FhGST-S1 presence. C-E) Cross sections of a F. hepatica adult highlighting staining of FhGST-S1 in the parenchyma (P), musculature (M),the tegument (T), basal membrane (Bm) and most intensely in the vitelline cells (V) and developing eggs (DE) . No staining can be seen in the tegumental spines (S), testes (T) or the intestinal caecum (IC). doi:10.1371/journal.pntd.0001666.g004 Figure 5 . rFhGST-SI stimulates the production total prostaglandin and PGE2 from dendritic cells (DCs) in a TLR4 dependent manner. DCs derived from the bone marrow from C57BL/ 6j mice were cultured in vitro with medium, rFhGST-S1 (10 mg/ml) or LPS (100 ng/ml) for 18 hours, and the production of total prostaglandin, PGE2 and PGD2 (data for PGD2 not shown) released into supernatants determined by competitive EIA. Data are presented as the mean 6 SEM following subtraction of medium controls and are representative of two experiments. WT -wild type; TLR4KO -Toll like receptor 4 knock out. doi:10.1371/journal.pntd.0001666.g005
Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org development concomitant with an exponential decrease of PGD 2 levels over the next 120 h post fertilisation [59] . However, in F. hepatica, eggs in gravid adults are released in an immature state in the bile duct, where they pass to the external environment via the host's excretory system and complete embryogenesis ex-host. Therefore, FhGST-S1 may have a secondary, or indeed primary, function in egg development and embyrogenesis. A role in egg development is further supported by proteomic studies of F. hepatica ontogenic stages which reveal the presence of FhGST-S1 in eggs ( [42] and the current study).
FhGST-S1 appears to be highly abundant in eggs with western blotting showing FhGST-S1 to be constitutively expressed, despite its association with a large spot consisting of multiple co-migrating proteins unresolved via 2DE (for association see [42] ). Immunolocalisation studies revealed that FhGST-S1 is closely associated with vitelline cells of mature adult worms. Given the importance of PGs in reproduction, we hypothesize that PG synthase activity exhibited by rFhGST-S1 contributes to developmental cues during egg formation. Interestingly, no FhGST-S1 was seen in day 0, unembryonated, eggs by western blotting yet in situ immunlocalisation showed freshly voided eggs, equivalent to day 0 eggs, to contain copious amounts of FhGST-S1. While it is most likely that FhGST-S1 is present in day 0 eggs, albeit at a reduced expression, the discrepancy seen between the two techniques is probably related to the antibody dilutions used for each method; in total a 40-fold difference in favour of immunolocalisation.
FhGST-S1 was also identified in both NEJs and adult worms using western blotting. This finding emphasises the multifunctionality of FhGST-S1, where in NEJs egg productions is not yet in process, suggesting its main function is in PG synthesis for host modulation or as a detoxification enzyme. In the adult worm, FhGST-S1 could also be localised, to a smaller extent, in the parenchyma and tegument. Given the high activity of FhGST-S1 towards the toxic 4-HNE and to lipid hydroperoxides this suggests a detoxification role at the host-parasite interface.
With near surface expression of FhGST-S1, in the parenchyma and tegument, there is the potential for this enzyme to be readily released into the host environment. Indeed, we have identified FhGST-S1 in the ES products of adult worms. With this in mind, previous studies have highlighted the importance of parasite Sigma class GSTs in immunomodulation of the host immune response. This includes our recent study implicating rFhGST-S1 in chronic inflammation through the activation of dendritic cells (DCs) [48] . While active rFhGST-S1 was able to induce levels of IL-12p40 and IL-6 cytokines in DCs in a dose-dependent manner, the previously described F. hepatica Mu-class GSTs failed to induce any cytokine secretion. Since denatured rFhGST-S1 also failed to induce any cytokines in DCs, activation of DCs is likely related to the structure and activity of the enzyme. However, inhibition of nitric oxide production, involved in driving a Th2 immune response, may also be a contributing factor in skewing the host response to fasciolosis [60] .
F. hepatica infections are associated with a T-helper-cell type 2 (Th2) immune response dominating during the chronic phases of infection [61] , but pro-inflammatory responses are suppressed [62] . Suppression of allergic responses during chronic parasitic worm infections has a mutually beneficial effect on the parasites' proliferation and the hosts' survival. Prostanoids, including PGD 2 , are important in mediating these allergic inflammatory responses. While generally regarded as pro-inflammatory molecules, these important lipid molecules are also involved in mediating antiinflammatory responses [63] . Helminth-derived molecules are thought to be involved in driving the Th2 response stereotypical of parasitic worm infections. DC and macrophage cell cultures Figure 6 . rFhGST-SI stimulates the production PGE2 and PGD2 from the macrophage cell lines J774 and RAW264.7. J744 and RAW264.7 macrophage cell lines were cultured in vitro with medium, rFhGST-S1 (10 mg/ml) or LPS (100 ng/ml) for 18 hours, and the production of total prostaglandin, PGE2 and PGD2 released into supernatants determined by competitive EIA. Data are presented as the mean 6 SEM following subtraction of medium controls and are representative of two experiments. doi:10.1371/journal.pntd.0001666.g006
Sigma Class Glutathione Transferase of F. hepatica www.plosntds.org exposed to rFhGST-S1 showed elevated levels of Th2 cytokines after 24 h [48] . In this study, the effects of rFhGST-S1 exposure onprostanoid synthesis in host immune cells was investigated. The results of which show the stimulation of PGD 2 and PGE 2 in both DCs and macrophage cell lines suggesting FhGST-S1 is one such helminth derived molecule capable of driving the Th2 response.
As we have shown FhGST-S1 to have key roles in F. hepatica, both in NEJs and adult worms, coupled with the near surface expression and release of the enzyme via the ES products, we assessed the potential of FhGST-S1 to be used as a vaccine candidate. This was especially poignant given that the S. mansoni Sigma GST homologue (Sm28) is in phase II clinical trials [12] . Unfortunately, the current goat based vaccine trial did not show any significant differences in fluke burdens between the rFhGST-S1 immunised and Quil A control group. However, a high individual variability was recorded, particularly in the vaccinated group also reported in previous trials using goats vaccinated with alternative candidates such as cathepsin L1 [64] and Sm14 [65] . The vaccine trial shown here using a target species with an acceptable adjuvant may have been adversely affected by the strain of F. hepatica used to challenge goats. Here we have shown an unusually high infectivity rate with the strain of F. hepatica used; which we have reported in a previous trial using goats [64] . Using an alternative strain of F. hepatica for experimental infections in this species has given normal infectivity rates ranging from 14% to 26.5% [65] .
In the present trial it appeared that goats immunised with rFhGST-S1, despite no variations in fluke burdens or morphometrics, showed reduced gross hepatic lesions during early infection, up to day 9 post infection, which continued to week 15 post infection where liver scores for hepatic lesions appeared reduced for rFhGST-S1 immunised animals. These results suggest that animals from the immunised group produced an early response to migrating larvae that has induced some partial protection from liver damage. The early and consistent specific IgG response found in the present work also agrees with the results obtained in a previous trial using naïve FhGST [46] . However, in both studies high levels of specific IgG did not induced a protective response reducing worm burdens.
A promising aspect of producing anti-helminth vaccines is developing multivalent vaccines. In many cases the greatest protection from challenge is by vaccinating with a combination of Fasciola antigens [66, 67] . Therefore, based on the immunisation with FhGST-S1 showing an early response reducing hepatica damage, could be considered for inclusion into a multivalent vaccine against Fasciolosis. In addition, in light of our findings showing FhGST-S1 to be highly prominent in egg production and the egg itself, as with previous vaccination trials [67] , it will be important to investigate the ability of eggs voided from vaccinated animals to embryonate. The potential to reduce pasture contamination by inhibiting egg embryonation, combined with the www.plosntds.org demonstrated reduction in liver damage, warrants further exploration using rFhGST-S1 as a vaccine candidate.
In summary, we have further promoted the concept that FhGST-S1 clearly demonstrates key host-parasite roles in synthesising PGs and stimulating PG release from host innate immune cells. In addition we have shown FhGST-S1 to be a key protein for detoxification, which may well be involved in TCBZ response. In line with current vaccine development theory we have shown FhGST-S1 to have multi-functional roles in the liver fluke physiology. Furthermore, we have shown FhGST-S1 to be expressed across ontogenic stages, localised to the fluke surface, and to the egg, both characteristics vital for vaccine development and success. Whilst no protection from fluke burden was seen in trials, the inclusion of rFhGST-S1 as a multivalent vaccine component should be investigated. However, it is important to fully characterise the host immune response during the early stages post-infection to better understand the mechanism mediating an effective host response. This will be essential to improve any future vaccine formulation. [36, 51, [68] [69] [70] [71] Table 2 Refs. Figure S1 Multiple sequence alignment and neighbourjoining phylogenetic tree across seven species-independent classes of GSTs. A) Alignment of the sigma class GSTs of trematodes shows the extent of identity and similarity across this class of GSTs. Boxed residues indicate complete identity between all sequences. Residues shaded in grey indicate conserved residues. B) Neighbour-joining tree placing mammalian and trematode GSTs within the same broad Sigma class. A distinct separation of clusters within this Sigma class is observed as with the recently reclassified 'Nu' class of GSTs from nematodes [49] . Sequences were aligned via the ClustalW program [29] in BioEdit Sequence Alignment Editor version 7.0.5.2. [30] . Phylogenetic neighbourjoining bootstrap trees were produced and viewed within TREE-VIEW [33] . Key to sequences in 1a and 1b. Author Contributions Discriminating Active from Latent Tuberculosis in Patients Presenting to Community Clinics BACKGROUND: Because of the high global prevalence of latent TB infection (LTBI), a key challenge in endemic settings is distinguishing patients with active TB from patients with overlapping clinical symptoms without active TB but with co-existing LTBI. Current methods are insufficiently accurate. Plasma proteomic fingerprinting can resolve this difficulty by providing a molecular snapshot defining disease state that can be used to develop point-of-care diagnostics. METHODS: Plasma and clinical data were obtained prospectively from patients attending community TB clinics in Peru and from household contacts. Plasma was subjected to high-throughput proteomic profiling by mass spectrometry. Statistical pattern recognition methods were used to define mass spectral patterns that distinguished patients with active TB from symptomatic controls with or without LTBI. RESULTS: 156 patients with active TB and 110 symptomatic controls (patients with respiratory symptoms without active TB) were investigated. Active TB patients were distinguishable from undifferentiated symptomatic controls with accuracy of 87% (sensitivity 84%, specificity 90%), from symptomatic controls with LTBI (accuracy of 87%, sensitivity 89%, specificity 82%) and from symptomatic controls without LTBI (accuracy 90%, sensitivity 90%, specificity 92%). CONCLUSIONS: We show that active TB can be distinguished accurately from LTBI in symptomatic clinic attenders using a plasma proteomic fingerprint. Translation of biomarkers derived from this study into a robust and affordable point-of-care format will have significant implications for recognition and control of active TB in high prevalence settings. Tuberculosis is the leading bacterial cause of death worldwide, with an estimated 8.8 million new cases of active disease and 1.6 million deaths per year [1] . Much of the burden of disease lies in the developing world, where annual incidence can reach 700 per 100,000 in certain regions [1] . New and unrecognised cases drive the epidemic, with transmission usually occurring before the index case is diagnosed. Multi-drug resistant cases and HIV co-infection further complicate control efforts [2] . Pulmonary TB is the most frequent clinical and transmissible manifestation of active disease. Rapid diagnosis and treatment are critical in the prevention of transmission.
The global burden of active TB occurs on a background of quiescent or latent TB infection (LTBI), affecting one third of the world's population and a higher proportion of the population of TB-endemic areas [3] . Respiratory and constitutional symptoms overlapping with those of pulmonary TB are very common in communities where TB is endemic [4] . In this scenario the challenge is to distinguish symptomatic patients with active TB from those with latent disease but whose presenting symptomatology is attributable to some other infectious or inflammatory process. In terms of rapid diagnosis, sputum microscopy will only identify approximately 50% of patients with active pulmonary TB. Conversely, while the interferon gamma release assays (IGRAs) represent a major advance in the detection of latent TB, they cannot distinguish active TB from symptomatic patients with latent infection in this context [5, 6] . This overlap between LTBI, active TB and non-specific clinical manifestations presents a formidable obstacle to the rapid recognition of active TB and the timely and appropriate targeting of anti-TB chemotherapy or chemoprophylaxis. In practice this difficulty may give rise to 2 types of therapeutic error. In the first instance, erroneous diagnosis of active TB in a symptomatic patient with LTBI may result in inappropriate administration of full course TB treatment. Conversely, offering chemoprophylaxis to a patient with supposed LTBI in whom active TB has not been recognized, will drive emergence of drug resistance.
Pulmonary TB is characterised by granuloma formation, caseation and ultimately cavitation, reflecting a complex interplay between distinctive components of the innate and acquired immune response and the pathogen [5] . Traditional serological analysis of single circulating proteins is notoriously unreliable for TB diagnosis [7] . In contrast, patterns of circulating proteins could provide an accessible readout of pathophysiological status. Discovery of such discriminatory biomarkers could open the way for the development of new point-of-care tests based on a lateral flow format such as dipsticks.
Proteomic analysis using Surface Enhanced Laser Desorption Ionisation Time of Flight (SELDI-ToF) mass spectrometry is a high throughput profiling methodology, which enables rapid comparison of protein patterns from large numbers of patients. The conceptual approach employed in the present study is termed proteomic fingerprinting. It is based on the principle that distinctive combinations of circulating proteins characterize different disease states. This strategy has been applied to the discovery of discriminatory proteomic patterns for a range of diseases including cancer [8] , vascular disease [9] [10] [11] and infectious diseases [12] [13] [14] [15] . Previously, we have demonstrated that proteomic patterns based on such profiles can distinguish active TB from healthy and symptomatic controls [12] .
In the present study we hypothesized that plasma proteomic differences would also distinguish patients with active TB from those without active TB but with overlapping clinical symptoms, irrespective of the co-existence of LTBI. Here we show that using this approach, we can indeed discriminate accurately between such patient groups.
All participants gave written informed consent and the research was approved by internationally accredited ethics committees including Universidad Peruana Cayetano Heredia (Lima, Peru) and Imperial College London (London, United Kingdom). The study involved adults from 15 years of age. Informed consent was obtained from the next of kin, carers or guardians on the behalf of the young adults involved in the study.
Participants were recruited over a period of two years from adults over the age of 15 years attending 16 community TB clinics serving a population of ,400,000 in the shantytown of Ventanilla on the outskirts of Lima, Peru ( Figure 1 ). All patients underwent the local standardized clinical workup for TB. This included up to 4 consecutive sputum samples for microscopy and culture. Participation in the study did not change patients' routine clinical management. The local incidence of TB in this population is ,130 per 100,000/year [16] and 95-97% of TB cases are HIV negative [17] . We recruited patients with active TB and individuals, termed symptomatic controls, presenting with respiratory symptoms suspicious of TB in whom TB was subsequently excluded.
Active TB cases were recruited on the basis of positive sputum microscopy with subsequent confirmation by culture. Mycobacterial culture was by automated liquid culture (BACTEC MGIT 960 TM , BD) as well as the Microscopic Observation Drug Susceptibility (MODS) assay which we have previously established as a standard local laboratory protocol [18] and which has since been adopted as the standard operating procedure by the national TB programme in Peru. Symptomatic controls, those patients with respiratory symptoms without active TB, were recruited if they had a persistent cough and one or more of the following clinical features: fever, weight loss, decreased appetite or haemoptysis. Symptomatic controls had 1-4 sputum smears and cultures to exclude active TB and were followed for 6 months to confirm that cultures had not become positive or were re-classified accordingly. Additional TB cases and symptomatic controls were identified through tracing household contacts, from whom sputum smears and cultures were obtained if symptomatic.
An IFN-c Release Assay (IGRA) (QuantiFERON-TB Gold In-TubeH) was performed on all participants. Latent TB was defined as a positive QuantiFERONH assay in the absence of clinical or microbiological evidence for active TB.
The Tuberculin Skin Test (TST) has limited value in the diagnosis of active TB and it was not carried out in our active TB patient group. We carried TST in the symptomatic controls group.
A 4 ml blood sample was obtained from each participant in an EDTA blood collection tube for subsequent plasma separation. Three additional aliquots were obtained at the same time for the QuantiFERONH-TB Gold in tube assay. Plasma was obtained before initiating TB treatment; otherwise plasma was taken within 1-2 days of treatment. Blood samples were transferred to the central laboratory on ice. Plasma was separated (3500 rpm, 10 minutes), aliquoted and frozen at 270uC at 6 hours following collection.
QuantiFERONH -TB Gold in Tube Assay This was performed according the manufacturer's instructions (Cellestis Plc, Sydney, Australia).
Plasma was profiled using Surface Enhanced Laser Desorption/ Ionisation-Time Of Flight (SELDI-TOF) mass spectrometry. All samples underwent a single freeze-thaw cycle prior to analysis. Samples were coded, blinded and randomised before application onto weak cation exchange (CM10) ProteinChipH arrays (Bio-Rad) in duplicate, as previously described [12] . Each ProteinCh-ipH included 1 quality control standard derived from a single healthy individual, placed at random. Liquid handling steps were automated using a Biomek 3000 Laboratory Automation Workstation (Beckman Coulter) and a 96 well BioprocessorH (Bio-Rad).
Mass spectra were generated on an automated System 4000 Bio-Rad ProteinChipH reader. Mass spectra data were collected and analysed using the ProteinChipH Data Manager Client 3.5 software (BioRad Inc.). Spectra were generated at both high (3,000 nJ) and low (1,600 nJ) laser energies with mass focus set to 40,000 Da and 6,000 Da respectively. Spectra were normalised by total ion current starting with a minimum mass/charge (m/z) of 2,500. Spectra with normalisation factor outside mean 62 standard deviations were removed. The remaining spectra were re-normalised by total ion current. Spectral peaks corresponding to mass/charge (m/z) clusters were detected and clustered using the ProteinChipH Data Manager Client 3.5 software (BioRad Inc.) by auto-detecting peaks to clusters in two steps. For the first step a signal to noise ratio of 5 and valley depth of 3 were used, with a minimum peak threshold of 20% of all spectra. For the second step a signal to noise ratio of 3 and valley depth of 1 were chosen. The cluster window was set at 1.0 peak width and expression difference mapping performed over m/z range of 2,500 to 200,000. Instrument calibration was performed using All-in-1 Peptide and Protein calibrants (Bio-Rad). Reproducibility was determined by measuring the inter-ProteinChipH coefficient of variation (CV) for the quality control spectra, based on all peaks in the spectrum with intensity .1 mA. Overall interchip CV for the quality control sample was 20%, consistent with similar studies.
Because highly abundant proteins/peptides suppress signal from lower abundance analytes in complex mixtures such as crude plasma, SELDI-ToF spectra were generated from both crude and pre-fractionated plasma to determine whether accessing the 'deeper' proteome yielded additional diagnostic information. Anion-ex- 
To visualize the covariance within the mass spectral profiles we used Principal Component Analysis (PCA). PCA encapsulates the covariance within a set of variables by extracting a ranked set of independent factors or principal components. The first 3 components encompass a high proportion (,95%) of the informational content of a multivariate dataset. We plotted each patient with respect to the first 3 components, in 3-dimensional space, color-coding according to patient group.
Although PCA is useful for visualizing data it cannot provide a classification rule for discriminating between patient categories. To find such discriminatory proteomic patterns, we adopted a supervised learning approach in which patient categories are used to train an algorithm to derive a classification rule. We used a Support Vector Machine (SVM) method [19] . Briefly, we used 10-fold cross validation to select parameters for the SVM. For the final model parameters, we selected those that gave the overall highest accuracy across the whole 10 fold cross validation. We next selected a subset of the most relevant mass clusters using the Recursive Feature Elimination (RFE) algorithm [20] which ranks variables based on their contribution to the classifier. To obtain accuracy estimates for the classifier, we took 1000 random resamplings of the original data, using 90% for training and 10% for testing. We selected as a final classifier the one that produced the highest accuracy while requiring the smallest number of m/z clusters. Results were expressed as sensitivity, specificity and accuracy (proportion of correct classifications) and as Receiver Operator Characteristic (ROC) curves. We assessed the different performances of classifiers derived from crude and pre-fractionated plasma by comparing mean values for sensitivity, specificity and accuracy using unpaired 2-tailed t tests. Comparisons of categorical data were by Fisher's exact test.
151 patients with active TB and 110 symptomatic controls were recruited (Figure 1 ). Of patients with active TB, 139 were both smear and culture positive, with the remainder either smear or culture positive. 48% of symptomatic controls had LTBI on the basis of a positive QuantiferonGold assay. Symptomatic controls had clinical features overlapping those of active TB patients, including cough, haemoptysis, fever, night sweats and weight loss, although symptom duration was generally longer among TB patients. Similar proportions of TB patients and symptomatic controls reported a previous history of TB (22% vs. 18%). The proportion reporting a history of TB was higher among controls with LTBI than among those without but did not reach statistical significance. Patients with active TB had lower BMIs at the time of recruitment compared with symptomatic controls (21.6 vs. 24.1 p,0.001). As expected, a higher proportion of patients with LTBI based on a positive IGRA had positive TSTs (.10 mm) compared with those without LTBI (62% vs. 30%, p,0.001). There was a higher proportion of female patients among the symptomatic controls than among the TB group. The effects of this potential bias are discussed below. Other key clinical features of the participant groups are given in Table 1 .
We plotted crude plasma global protein expression profiles in a heat map (Figure 2 ) that shows spectra patterns from active TB patients and unhealthy controls. The most striking area of upregulation in TB patients is seen in the 11 kDa region where a series of protein peaks are seen in red amongst TB patients ( Figure 2) . A parallel area of up-regulation is seen at 5 kDa and a third smaller area seen at the 21 kDa region (Figure 2 ). Inspecting in more detail the spectra in the 5.8 and 11.5 kDa regions ( Figure 3 ) reveals a complex of peaks at both these regions, which is more abundant in patients with active TB. We assessed overall separability of patient groups by PCA of mass spectra from crude and pre-fractionated plasma (Figure 4 ab) . In figure 4 , each patient sample is plotted in a 3-dimensional space defined by the first 3 principal components. The spectra from patients with active TB (purple spheres) cluster relatively tightly together and are well separated from symptomatic control patients (blue and green spheres) regardless of LTBI. This analysis, however, does not clearly separate symptomatic controls with or without LTBI (blue and green spheres, respectively).
The SVM classifiers distinguished active TB from both classes of symptomatic controls. The ROC curves in Figure 5 (a-f) summarize the performance of the classifiers, in terms of the tradeoff between sensitivity and specificity, for each of the different comparisons. In each case, the area under the curve (AUC) exceeded 0.9, irrespective of whether crude or pre-fractionated plasma was analyzed, indicating a high level of discrimination. Tables 2 and 3 and Tables S1 and S2 summarize the performance of the classifiers in discriminating active from latent tuberculosis in symptomatic patients using the number of selected relevant m/z clusters ( Table 3 in brackets). It was possible to distinguish patients with active TB from undifferentiated symptomatic controls with partially overlapping respiratory and constitutional symptoms with an overall accuracy of 85% using crude spectra with 98 relevant m/z clusters (Table 2, Table 3 , Figure 5a) . A higher specificity for active TB (90% vs. 84%, p,0.001) was achieved using prefractionated plasma with a total of 54 relevant m/z clusters ( Table 2, Table 3 , Figure 5b) . Notably, these levels of discrimination were achieved despite nearly half of the symptomatic controls having LTBI (Table 1) .
To further investigate the influence of background LTBI on classifier performance, separate comparisons were made between active TB and symptomatic controls either with or without LTBI. In both comparisons, active TB could be distinguished from symptomatic controls with overall classifier accuracies of at least 87% (Table 2, Table 3 , Figure 5 c-f, Tables S1 and 2). Active TB was readily distinguishable from symptomatic controls without LTBI using both crude and fractionated plasma, with overall accuracies, sensitivities and specificities of at least 90% (Table 2,  Table 3 , Figure 5 e,f and Table S1 and S2). The main influence of LTBI among the symptomatic controls was to reduce classifier specificity, reflected in a higher proportion of false positives. Strikingly, plasma pre-fractionation improved specificity from 75% to 82% only using four m/z clusters (Table 2, Table 3 , Figure 5 c,d, p,0.001) .
To address the issue of the gender bias in cases and controls we reanalysed the data to determine whether a classifier based on the proteomic profile could reliably discriminate males from females. This was found not to be the case, suggesting that gender is not a major confounder in our analysis. As a further test, a new classifier was trained on male patients alone, to discriminate active TB from symptomatic controls. When we applied the trained classifier to the female subjects, this classifier was nevertheless still capable of classifying TB to an accuracy of approximately 80%.
We also confirmed the presence of differential expression of the Serum Amyloid A (SAA, 11.5-11.8 kDa) and transthyretin (13.7-13.8 kDa ) peak complexes which emerged in our previous study [12] as important informative markers for active TB. SAA was identified by specific immunodepletion (data not shown).
In this study we have shown that a distinctive pattern of plasma proteins distinguishes patients with active TB from non-TB patients with overlapping clinical features, even in the presence of LTBI. This both reinforces and substantially extends our previous findings where we first showed that proteomic patterns could be used as a diagnostic approach for active TB [12] . We have now shown that the proteomic pattern does not merely reflect the presence of TB infection per se. Rather, it can be used to identify active TB even in a highly TB-endemic setting with high prevalence of both respiratory symptoms and background LTBI. (a,b) active TB vs. all symptomatic controls using crude or pre-fractionated plasma respectively; (c,d) active TB vs. symptomatic controls with latent TB using crude or pre-fractionated plasma respectively; (e,f) active TB vs. symptomatic controls without latent TB using crude or pre-fractionated plasma respectively. The ROCs are derived from 1000 random train/test resamplings of the data. Error bars show standard deviations. The Area Under the Curve (AUC) is shown in the centre of each plot. doi:10.1371/journal.pone.0038080.g005 Table 2 . Discrimination of active from latent tuberculosis in symptomatic patients. The ability to discriminate rapidly in a symptomatic patient between active TB and non-tuberculous disease has profound implications for both individual clinical management and TB control programs [21] . For example, current diagnostic limitations frequently result in many patients in resource-poor settings being treated empirically for community acquired pneumonia before eventual diagnosis of active TB. This may lead to on-going transmission during the interval preceding diagnosis as well as greater individual morbidity. The alternative strategy of empirical anti-TB chemotherapy is sometimes employed, but cost, toxicity and logistics often preclude this. Adjuncts to conventional microbiology for diagnosis of active TB in widespread use include the TST and IGRAs. The use of TSTs in the diagnosis of active TB in high prevalence settings is greatly limited by its poor specificity for active TB as reactivity is also seen in LTBI, previous BCG vaccination and exposure to environmental mycobacteria. Nor has the recent introduction of IGRAs into clinical practice resolved this key diagnostic issue. This is because of their inability to distinguish active TB from LTBI [6] and frequent false negative results in acute active TB [22] , limitations which are especially problematic in high prevalence settings [23] . Thus a diagnostic that overcomes these limitations is urgently required and would be a major advance in the management of the global TB pandemic. Recently it has been reported that a TNF-alpha + TB-specific CD4+response can be used to differentiate latent infection from active TB but the sensitivity was just 67% [24] . Moreover, that study relied on polychromatic flow cytometry limiting the feasibility of being translated in high prevalence settings. In contrast, our approach provides improved accuracy, 87%, by detecting relevant protein biomarkers in plasma. Despite the discovery-phase of our approach using sophisticated proteomic methodologies, the identification of relevant plasma proteins leads to a clear translational path for antibody-based point-of-care devices that can be used to measure these plasma proteins in the future.
There is increasing interest in the identification of novel biomarkers for TB -in the contexts of diagnosis, treatment response monitoring, prediction of relapse or re-activation and as surrogates for vaccine protection. Most studies have focused on individual markers such as secreted M. tuberculosis antigens, serological responses, microbiological indices and host inflammatory markers, with mixed results [7, 25] . There is growing recognition of the advantages of using combinatorial biomarker panels or 'omics'-based methods to achieve sufficient levels of accuracy [25] . However, relatively few studies have utilized such strategies.
Proteomic fingerprinting for biomarker discovery has been applied in the past decade to a variety of disease states, particularly in the sphere of cancer diagnostics [26, 27] . The power of this approach is reflected by the recent granting of FDA approval of a novel blood test derived from a SELDI-based fingerprinting method, for distinguishing malignant from benign ovarian tumours [27, 28] . In many infectious diseases, there are clinically important distinctions to be made between different manifestations associated with the same underlying pathogen. For example, distinguishing colonization or latent disease from active infection has obvious clinical and therapeutic implications. TB is a clear case in point. Proteomic fingerprinting has enormous potential for defining and distinguishing these disease states but has only recently received attention in this area [12] [13] [14] [29] [30] [31] . Because the circulation samples deep tissues throughout the body, local proteomic changes in organs such as the lungs can be reflected in the plasma proteome. Moreover, host modulation by the pathogen is likely to generate changing patterns of protein expression associated with different clinical manifestations. Thus the plasma proteomic response is a plausible index of disease state. Proteomic patterns are highly dynamic and it may be possible to define those that reflect stages in progression from latency to active disease. However, the complexity of the plasma proteome with its enormous dynamic range of solute concentrations means that detection of informative lower abundance proteins is particularly challenging. It is possible that differences between active TB and LTBI in symptomatic patients are reflected better by such lower abundance proteins not easily detectable in crude plasma. This may explain the higher specificity for active TB obtained from prefractionated as compared to the crude plasma spectra.
The gold standards used for defining patient groups in this study are notoriously imperfect. For example, while active TB was defined by positive microbiology, it is possible that some patients designated symptomatic controls may actually have had smear and culture negative TB. This might have resulted in an underestimate of the specificity of our diagnostic pattern for active TB, although our 6 months follow-up and appropriate re-labelling should have identified most of these. The lack of an adequate gold standard for defining LTBI must also be considered. While IGRAs show greater specificity than TSTs, sensitivity may be compromised especially in early active TB [22] . Thus some patients with unrecognized smear and culture negative TB may have been mislabeled as symptomatic controls without LTBI.
We did not perform routine HIV testing in our patient cohort and it is possible that over-representation of HIV seropositivity in our active TB group may have had a confounding effect. We believe this is unlikely in view of the low prevalence of HIV coinfection among TB patients in Peru (,5%) found in previous studies [17] . Important areas of future study will be to establish the applicability of this approach in the contexts of TB-HIV coinfection and smear-negative TB.
Our present findings confirm the utility of defining the host proteomic response in distinguishing clinically overlapping patient (5) Total number of mass/charge (m/z) clusters obtained from SELDI-ToF profiling of crude and pre-fractionated plasma. In brackets number of relevant discriminatory m/z clusters selected by the RFE algorithm. F1 = fraction 1 at pH 9; F2 = fraction 2 at pH 7; F3 = fraction 3 at pH 5; F4 = fraction 4 at pH 4; F5 = fraction 5 at pH 3; F6 = fraction 6 organic phase. doi:10.1371/journal.pone.0038080.t003 groups in a TB clinic setting. Moreover, this study shows that active TB can be identified by a blood test in a population of community TB clinic attenders, on a background of non-TB attributable symptoms, despite the coexistence of LTBI. Ultimately, a significant impact on control of TB in high prevalence settings will depend on the ability to translate these findings into a robust, affordable point-of-care format. Incorporation of a panel of biomarkers derived from this study into a lateral flow device or similar platform is the logical next step. Finally, the utility of defining proteomic patterns in TB may extend beyond diagnostics to provide new methods for monitoring treatment response and disease stage.
Table S1 Selected relevant m/z clusters from crude plasma.
(XLS)  Novel Evidence of HBV Recombination in Family Cluster Infections in Western China Two hepatitis B virus (HBV) C/D recombinants were isolated from western China. No direct evidence indicates that these new viruses arose as a result of recombination between genotype C and D or a result of convergence. In this study, we search for evidence of intra-individual recombination in the family cluster cases with co-circulation of genotype C, D and C/D recombinants. We studied 68 individuals from 15 families with HBV infections in 2006, identified individuals with mixed HBV genotype co-infections by restriction fragment length polymorphism and proceeded with cloning and DNA sequencing. Recombination signals were detected by RDP3 software and confirmed by split phylogenetic trees. Families with mixed HBV genotype co-infections were resampled in 2007. Three of 15 families had individuals with different HBV genotype co-infections in 2006. One individual (Y2) had a triple infection of HBV genotype C, D and C/D recombinant in 2006, but only genotype D in 2007. Further clonal analysis of this patient indicated that the C/D recombinant was not identical to previously isolated CD1 or CD2, but many novel recombinants with C2, D1 and CD1 were simultaneously found. All parental strains could recombine with each other to form new recombinant in this patient. This indicates that the detectable mixed infection and recombination have a limited time window. Also, as the recombinant nature of HBV precludes the possibility of a simple phylogenetic taxonomy, a new standard may be required for classifying HBV sequences. Not all viruses are equally prone to recombination. Recombination has not been detected in several viruses despite repeated searches [1] . Whether recombination does or does not exist is important for understanding the evolution and replication mechanism of a specific kind of virus. Hepatitis B virus (HBV), a major human pathogen, has been classified into 10 genotypes and several sub-genotypes [2, 3] . Many sub-genotypes were identified by polygenetic analysis as recombinants. But there is no direct evidence to indicate that these subgenotypes arose as a result of recombination or perhaps a result of convergence.
Coinfection with different HBV genotype strains is a prerequisite for recombination. As more than one genotype is predominant in most of the geographic regions, coinfection between the predominating HBV genotypes is not a rare finding, especially for B and C, or A and D. The prevalence of mixed HBV genotype infections has been reported using varied genotyping methods [4, 5, 6] .
Our previous study found two kinds of HBV C/D recombinants in northwest China [7] . In a further study of ethnic groups of five provinces, we confirmed the geographic and ethnic distribution of the HBV C/D recombinant in northwest China [8] , and found that family-cluster HBV infections were common in these endemic areas. We hypothesize that infected members of HBV family clusters would gain exposure to various genotypes through marriage, while at the same time; competent strains would be selected through vertical transmission. It would be useful to observe the mixed infection in family-cluster cases, especially in patients infected with C/D recombinants.
The aim of this study was to evaluate the possibility of recombination between two HBV genotypes within an individual by finding cluster-infected families in which individual members were infected with different HBV genotypes. We would then look for individuals within these families with multiple-genotypes that were likely to have been obtained from other family members as a result of vertical or horizontal transmission. Novel viral genomes within an individual with a multiple genotype infection that were mosaics of the known viral genotypes in the family, but not present in any of the other family members, would be consistent with the hypothesis that they arose within the individual with multiple genotype infections.
We enrolled 68 patients with a chronic HBV infection from 15 families. All the families were from a district located at the boundary of Gansu and Qinghai provinces, where the prevalence of genotype C2, D1 and C/D recombinant HBV were known to be high [8] . The families were initially identified with cluster HBV infection in an epidemiological survey in 2002. Sixty-eight individuals were sampled in June 2006 and December 2007 for the purpose of assigning HBV genotypes to chronically infected individuals and finding individuals with multiple HBV genotype co-infections. None of the patients received anti-viral therapy or immunosuppressant drugs. A written, informed consent was obtained from each family, and the study protocol was approved by the Southern Medical University Ethics Committee.
HBV DNA was extracted from 400 mL of serum by QIAamp UltraSens Virus Kit (Qiagen GmbH, Germany), then resuspended in 50 mL water and stored at 220uC until analysis. HBV genotypes, including C/D recombinant, were initially assigned using the PCR based restriction fragment length polymorphism (RFLP) methods described previously [9] , [8] .
For samples with mixed genotype infections, PCR cover HBV S gene (nt136-1110) was performed using the primers and thermocycling conditions descirbed by Sugauchi et al [10] . For samples needing further recombination analysis, PCR was performed using the primers and thermocycling conditions described by Günther to obtain full-length HBV genome [11] . Alternatively, a nested PCR was used to produce two overlapping fragments in subjects with low HBV DNA levels as described by Sugauchi et al [12] . The spanning of fragment A cover nucleotides 2813 to 1824, and fragment B included nucleotides 1821 to 237. LA-Taq (TAKARA, Japan) and high-fidelity polymerase COD-FX (TOYOBO, Japan) were used to produce amplimers for cloning and direct sequencing respectively. Finally, Fragment C (HBV nt56-nt1824) was obtained from a PCR amplification of Y2 HBV-DNA to which an aliquot of genotype B HBV-DNA had been added. The purpose of this experiment with in-tube control of genotype B was to determine if the recombinant clones were being generated during the PCR amplification. PCR products were gel-purified and cloned into the PMD19-T vector (TAKARA, Japan) according to the manufacturer's instructions, and used to transform JM109 competent cells (TAKARA, Japan). A minimum of 15 clones were sequenced from subjects with a mixed-strain infection and three clones were sequenced from family members with a single-strain infection. All sequencing of clones and PCR products was performed by Invitrogen Ltd. (Shanghai, China).
Genotypes of clones were determined by phylogenetic tree analysis and recombination analysis. The sequences were assembled using SeqMan II software (DNAStar Inc.). Sequence alignments were performed using ClustalW and confirmed by visual inspection. Phylogenetic trees were constructed by the neighbour-joining (NJ) method (Saitou & Nei, 1987) . To confirm the reliability of the phylogenetic tree analysis, bootstrap resampling and reconstruction were carried out 1000 times. A phylogenetic tree analysis of HBV strains isolated from the mixed infection family was compared with reference strains from GenBank. Accession numbers are indicated on the tree. Bootstrap values are shown along each main branch. The lengths of the horizontal bars indicate the number of nucleotide substitutions per site. The regions included in the analysis were the same with fragment A, B and C or a little shorter. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 (Tamura, Peterson, Stecher, Nei, and Kumar 2011).
Recombination signals were initially detected by RDP3.b.4 software [13, 14] . Bootscan, Geneconv and Siscan were used. The highest acceptable P-value was 0.05. Bootscan and Siscan window sizes were 300 bp, step size was 30, replicates for 100 times. A genotype F sequence (GenBank accession numbers is X75658 and X75663) was used as external reference. The precise map of recombination was determined by split phylogenetic tree and alignment. Split phylogenetic trees were constructed by the method same as above. In alignment, each clone was compared to reference C2, D1 and CD1 consensus sequences. We then inspected the alignments to determine the identical crossover sequences around the breakpoint within which the recombination occurred.
GenBank accession number of reference sequences of HBV genotype C2, D1, CD1 and CD2 are indicated in phylogenetic tree. Accession Numbers of Y2 clones are JX036326-JX036359.
Different HBV genotypes were found in three families among 15 families. The flow of participants in the study and family trees of families with mixed genotypes/subgenotypes of HBV infection are shown (Figure 1 ).
Family V had infected members across two generations and two genotypes: In 2006, the mother (V1W) and daughter (V2F) were infected with subgenotype D1 while the son (V2M) had a CD1 recombinant. In 2007, the daughter (V2F) had subgenotype D1 while other family members had HBV DNA levels below the detection limit of the nested PCR assay.
Family Q had infected members across three generations and two genotypes/subgenotypes. In 2006, the grandmother (Q1W) and grandson (Q3M) were infected with CD1 recombinant while father (Q2) and granddaughter (Q3F) had mixed infections of genotype C2 and CD1 recombinants. In 2007, the same genotypes were detected in all family members except that the granddaughter (Q3F) had an HBV DNA level below the detection limit of the nested PCR assay.
Family Y had affected members across three generations and three genotypes/subgenotypes. In 2006, the grandfather of family Y (Y1) was infected with genotype C2 while grandmother (Y1W) had mixed infections of CD1 and C2. Mother (Y2W) and granddaughter (Y3F) were infected with the CD1 recombinant. Father (Y2) had triplicate infections of genotype C2, D1 and CD recombinant. Grandson's (Y3M) serum was unavailable. In 2007, the grandfather (Y1) and mother (Y2W) had HBV DNA levels below the detection limit while the grandmother (Y1W) and granddaughter (Y3F) had genotype CD1. Father (Y2) and grandson (Y3M) had genotype D1.
A phylogenetic tree constructed from HBV nt 36-1110 from the clones of family Y is given (Figure 2A ). The clones (dotted) of family Y exhibits three clusters on genotype C2, D1 and CD1.
The phylogenetic tree construct from HBV nt136-1110 from the clones of families Q and V is given ( Figure 2B ). The clones of family Q (indicated by black dots) exhibit two clusters of subgenotypes C2, and CD1. The clones (indicated by black triangles) from family V exhibit two clusters of subgenotypes D1 and CD1.
A phylogenetic tree constructed from HBV nt 36-1110 of novel recombinants clones of Y2 is given in Figure 2C . The dotted clones are from Y2. The topology of phylogenetic tree with recombinants is totally different from typical trees. Recombinant sequences blurred the typical branch,in other words, blurred the typical genotype.
Results of recombination analysis of Y2 clones are as bellow: Three kinds of analytical methods certificated the same recombination map. The initial pictures of the three methods were all provided as supplemental figures. Recombination events detected by RDP software are shown in Figure S1 , S2, and S3. Split phylogenetic trees constructed by MEGA software are shown in Figure S4 , S5, and S6, (clone number and fragment used to construct tree are indicated beside each tree). Sequence alignments are shown in Figure S7 , S8, and S9.
The region where recombination breakpoints had the highest probabilities was recognized as crossover region, which is a region that one parental genotype switches to another. Upstream sequence of crossover region will have specific mutation of one genotype but with no specific mutation of another, downstream just opposite. At the same time, these two genotypes should share same sequence at crossover region. We indicated the crossover region in direct alignment by black bars in Figure S3 initially and marked it in recombination map by colorful bars in Figure 3A and black bars in Figure 3B . The clonal sequences of 2006 showed 17 unique crossover regions in fragments A, B and C. We could not identify any common motif within these sequences that might suggest a common mechanism for crossovers in the HBV. The size of switch region share the same sequence are different in different strains, from 6-174 bp (6 bp for Y2M-2 clone in Figure S7 and 174 bp for Y2M-29 clone in Figure S8 ).
To illustrate the recombination map in a simple way. An abbreviated alignment of fragment A, B and C are shown in Figure 3B . Green and pink bars indicated the genotype C2 and D1 respectively. Black bars showed the crossover region. The aligned sequences provide a snapshot of the recombinant HBV strains. Genotype C2, D1 and CD1 recombinant clones of Y2 were all used as parental sequences to recombine with each other to form new recombinants. A series of novel recombinants were found in three fragments.
In 15 clones of fragment A, there were five genotype C (Y2-6,9,13,14,15,); two genotype D (Y2-11,12); one CD1 (Y2-10) and seven novel different C/D recombination (Y2- 1, 2, 4, 7, 8, 3, 5) .
In Of the 56 clones of fragment C(in which genotype B HBVDNA were added as an in-tube control to exclude the recombination by PCR procedure), there were 32 pure genotype B clones; nine genotype C clones(Y2-B10,B5,B8,B9,B13,B16.B17.B18,B24); five genotype D clones(Y2-B22,B3,B4,B21,B23), two CD1 clones (Y2-B1,B11) and eight novel C/D recombinants (Y2-B6,B7,B14,B15,B19,B2,B12,B20). No recombinants of genotype B were found.
Recombination is one of the major mechanisms contributing to the evolution of retroviruses [15] . Since the HBV has a reverse transcription step in its life cycle, it is conceivable that recombination also contributes to diversity in HBV genomes. Although just four cases were observed with mixed genotype infections, we obtained a snapshot of naturally occurring HBV recombinants generated in the absence of selection and after selection. Our result showed direct evidence of HBV recombination, with new information of recombining crossovers compared with similar studies [16, 17, 18, 19] .
The recombination analysis of Y2 quasi-species showed variable types of recombinant between genotype C2, D1 and CD1 in 2006. Some studies show that hotspots of recombination most on the boundary of ORFs [12, 20] . Our results showed that two or more strains of HBV can recombine with each other at any region along the genome. Crossover regions can be hundreds or just several base pairs, The length of crossover region is depends on the location of it on HBV genome. If it is located in a conserved HBV region, for another word, where many different genotypes share the same sequence, the length of crossover region may be long. If it is located in a non-conserved region, it may be very short. At the same time, we found that the crossover region distributed totally at random on HBV genome. Consistent with our results, in vitro evidence showed the initial recombination events in a laboratory system of MHV were almost entirely randomly distributed along the sequence [21] . It was only after passage through cell culture, with the opportunity for selection to remove less fit variants, that crossover sites became ''localized'' to just a small area of the region examined. Crucially, they also suggested initial products of recombination may go undetected because of the action of strong purifying selection which will remove new, deleterious combinations of mutations. The conclusion is therefore an interpretation for the genotype change of Y2. The Y2 presented multiple strain infections of C2/D1/CD1 and many new recombinants with no obvious dominant genotype strain in 2006. After 18 months, however, all the type C2 and CD recombinant strains disappeared while the D strain became dominant. A similar case of mixed HBV genotype infection in which one genotype was lost and another prevailed was previously described in patients with HBeAg seroconversion [4, 22] .
Epidemiologically, HBV genotype CD1 and C2 are the most common strains in ethnic minorities of northwest China with CD2 and D1 as minor strains. Precise mapping of recombination suggests C2 and D1 are parental sequences of CD1 and CD2 recombinants. Virological differences among HBV genotypes were demonstrated in vitroand in CHiM mice, with genotype C having a higher replication capacity than D [23] . Why does the replication-deficient genotype D virus predominate over replication-competent genotype C? As mixed HBV infections together with recombination are rare, we have little knowledge about i this situation. On the one hand, we know little about host impact on different genotypes and recombinants. On the other hand, we know little about interference and competition in the quasispecies of mixed infection. In vitro results showed the replication capacity of individual clone, exclude the influence of host and other strains of quasi-species. An example from a ChiM mice study showed that monoinfection of HBV/G in ChiM mice display a very slow replication while coinfection with HBV/A remarkably enhanced the replication of HBV/G. The replication of HBV/G is heavily dependent on coinfection with other genotypes. When HBV/G superinfected on other genotypes, a rapidly takes over of HBV/G from original genotype were observed, though they are indispensable [24] . This study confirms that in a mixed infection system of different genotypes, the replication capacity of a genotype may be different from that of monoinfection. At the same time, replication capacity is not the only factor to influence which strain will become dominant. Variable recombinants found in our study may be mechanistically capable of genetic exchange, but strong selection guaranteed the elimination of hybrid genomes. The mechanism of selection in mixed infection also needs more investigation.
We found mixed HBV genotypes infection with many novel recombinants at one point in time, but just one genotype was found 18 months later. This may indicate that the detectable mixed infection and recombination has a limited time window due to the sensitivity of detection or strong selection power of the host. That's why in most studies, we can identify a major genotype in one patient. Even so, evolutionarily visible and invisible recombination of HBV could occur and play an important role by generating genetic variation or reducing mutational load. However, this study had limitation, because recombination signals were detected by RDP3 software and confirmed by split phylogenetic tree and alignment, indicating the recombinant or recombinantlike form should depend on the software. If we use another software, the results might be different.
Studies of HBV in endemic areas throughout the world have resulted in large numbers of full genome sequences available for phylogenetic analysis enabling the identification of novel, mosaic HBV genomes that appear to be the result of recombination between previously known sequences [7, 25, 26] . One of the most comprehensive analyses of putative HBV inter-genotype recombinants showed the existence of 24 phylogenetically independent HBV genomes involving all known human genotypes [27] . Some of these recombinants are unique to individual subjects, but some undergo expansion in specific populations and become recognized as new genotypes or subgenotypes [12, 28, 29, 30] . Four stages in the process of generating popular HBV recombinant genomes should be recognized. The first stage is the co-circulation of different HBV strains or genotypes in the same geographic area. The second is the existence of individuals who have been infected with more than one strain of HBV. The third is the generation of a novel recombinant strain(s) within an individual. The fourth is the selection of a recombined strain with the ability to replicate and be transmitted. Our data show the natural process of the formation and selection of recombination though the recombinant strains of Y2 that appeared in 2006 that were all removed from samples in 2007.
By using phylogenetic trees and homology calculations, HBV variants infecting humans are currently classified into ten genotypes that differ from each other in nucleotide sequence by 7.5 to 13% [2, 3] . There are some characteristic length differences between the genotypes that facilitates their detection and discrimination. However, as shown in Figure 2 , existence of a recombinant makes the topology of the phylogenetic tree totally different from one with no recombinant. Recombinant strains obscured the definition of genotypes. Based on the algorithm creating a phylogenetic tree, sequences with high homologues cluster together. With the same logic, recombinants always clustered with the backbone parental sequence, in other words, with which they have high similarity with the larger proportion of the recombination region. Therefore, recombinants always seem to be a subgenotype of their backbone parental sequence. Similar to Y2-8 clone in Figure 2C , for recombinants with similar proportion of both parental genotypes, the sequence shows a divergent trend different from both parental genotypes.
Based on phylogenetic topology changes of different regions of HBV, it was hypothesized that some of the genotypes that are conventionally regarded as ''pure,'' actually were recombinant. Genotype E strains show evidence of recombination with genotype D at 1950-2500. new reported genotype ''I'' actually belongs to genotype C. Furthermore, Subgenotype Ba possesses the recombination with genotype C at 1740 to 2485 [31, 32, 33] . Recombinants comprising regions with different histories have important implications for the way we think about HBV evolution. It means that there is no single phylogenetic tree that can describe the evolutionary relationships between genotypes.
In conclusion, mixed HBV genotypes infection with many novel recombinants at one point in time ended up with just one genotype 18 months later in this study. This may indicate that the detectable mixed infection and recombination have a limited time window due to the sensitivity of detection or strong selection power of the host. Also, as the recombinant or recombinant-like nature of HBV precludes the possibility of a ''true'' phylogenetic taxonomy, a new standard may be required for classifying HBV sequences.  Identifying Live Bird Markets with the Potential to Act as Reservoirs of Avian Influenza A (H5N1) Virus: A Survey in Northern Viet Nam and Cambodia Wet markets are common in many parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonoses. A survey was conducted in order to assess the potential of Vietnamese and Cambodian live bird markets (LBMs) to sustain circulation of highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). Thirty Vietnamese and 8 Cambodian LBMs were visited, and structured interviews were conducted with the market managers and 561 Vietnamese and 84 Cambodian traders. Multivariate and cluster analysis were used to construct a typology of traders based on their poultry management practices. As a result of those practices and large poultry surplus (unsold poultry reoffered for sale the following day), some poultry traders were shown to promote conditions favorable for perpetuating HPAIV H5N1 in LBMs. More than 80% of these traders operated in LBMs located in the most densely populated areas, Ha Noi and Phnom Penh. The profiles of sellers operating at a given LBM could be reliably predicted using basic information about the location and type of market. Consequently, LBMs with the largest combination of risk factors for becoming virus reservoirs could be easily identified, potentially allowing control strategies to be appropriately targeted. These findings are of particular relevance to resource-scarce settings with extensively developed LBM systems, commonly found in South-East Asia. First detected in 1996 [1] , highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1) has spread across 3 continents and is now considered to be endemic in several South-East Asian countries and Egypt. Due to its potential to recombine with human influenza strains to produce highly virulent reassortants [2] , ongoing circulation of HPAIV H5N1 continues to be a major public health concern.
Live bird markets (LBMs), in which the virus has been frequently detected in both disease-endemic and epidemic regions [3, 4, 5] , are suspected to play a major role in the epidemiology of HPAIV H5N1 [6, 7] . The LBM system provides consumers with freshly slaughtered birds. It is a dead-end for poultry, but not necessarily for viruses. LBMs have been shown to contribute to the spread of, and the possible maintenance of, HPAIV H5N1 within the poultry sector [8] . Zoonotic transfer to humans has also been documented in LBMs [9, 10] .
Birds are introduced into LBMs daily and stocked at a high density. If these birds remain in the market for a sufficient time to become infected and transmit virus to susceptible birds, LBMs would offer optimal conditions for amplifying and sustaining virus circulation and could, thus, become viral reservoirs themselves [11] .
Given the range of species present in typical LBMs and the variety of detected influenza viruses [12, 13, 14, 15, 16, 17, 18, 19] , LBMs could potentially act as drivers of viral evolution, promoting the emergence of new variants. Identifying those LBMs that could act as viral reservoirs is, therefore, crucial for improving surveillance and control.
Although the potential for an LBM to become a viral reservoir is determined by the management practices of poultry traders [11] , an understanding of these practices in high risk areas is lacking. In response to this need, a cross-sectional survey was conducted in northern Viet Nam and Cambodia to assess whether traders of live poultry engaged in practices that could sustain virus circulation in LBMs. Viet Nam is one of the most severely affected countries in the current HPAI H5N1 pandemic [20] , with the disease considered to be endemic in both northern and southern Viet Nam. Only sporadic outbreaks have been reported in Cambodia [20] . However, the reporting of isolated human cases without prior notification of poultry outbreaks [21] suggests that widespread, undetected virus circulation and persistence of HPAIV H5N1 in the Cambodian poultry population cannot be ruled out. In both countries, LBMs are common and potentially involved in virus spread.
An LBM for this study was defined as an open space with 2 or more traders selling live poultry at least once per week and with official government authorization to do so. ''Live poultry'' referred to finished birds, meaning birds intended to be slaughtered and eaten by the end-user.
Although LBMs are numerous in Viet Nam and Cambodia, the live poultry trade is only a small, irregular activity in most markets, necessitating a purposive sampling strategy. In the selected areas, only the largest LBMs in terms of the number of poultry sold were eligible. The selection of Cambodian LBMs was based on a previous cross-sectional survey on commercial poultry movements conducted in 2006-2007 [22] . Eight Cambodian LBMs with the highest volumes of poultry sales were recruited.
Data on the frequency and volume of live poultry sales in northern Vietnamese LBMs were not available. Study provinces were selected based on demographic features rather than outbreak reports, given that most disease events were presumed to be undetected [23, 24, 25] : Ha Noi, the most densely populated province in northern Viet Nam [26] , and Bac Giang, a rural province with a large poultry population [27] . A snow-ball sampling approach was adopted.
A first set of major LBMs were identified in each study area through meetings with trade and veterinary service officers. In these LBMs, traders were asked to identify the other LBMs of which they were aware and to rank them according to the number of live poultry sellers. These identified LBMs were then integrated into the survey and their traders were in turn asked to name the LBMs that they considered to be the biggest.
As there are 4 times as many people in Ha Noi as in Bac Giang [26] , the number of LBMs was also expected to be much higher, with traders likely to be aware only of markets located in the immediate vicinity of those from which they trade. For this reason, the snowball approach described above was applied only at the province level in Bac Giang, but also at the lower administrative level, the district level, in Ha Noi.
The present day province of Ha Noi is the result of the recent merging of an urban centre (former Ha Noi province) and a rural area (former Ha Tay province and a district from Vinh Phuc province). After exclusion of districts where live poultry marketing was prohibited, 4 of 5 districts in the urban centre and 2 of 13 districts in the rural area were randomly selected. Meetings held with Ha Noi veterinary services also identified the 4 main LBMs supplying the province, which were then integrated into the survey. In total, 30 markets were recruited in northern Viet Nam. Assuming that each commune (administrative division of district) had 1 or 2 LBMs, the LBM sampling rate per district ranged between 3% and 25%. Study areas are shown in Fig. 1 .
To participate in the survey, traders in the markets had to sell or purchase birds in an LBM at least 1 day per month. Live poultry traders consisted of sellers and middlemen. A seller was defined as selling live poultry mostly to the end-user (e.g. consumers, restaurants) or to another trader who would then sell the poultry at another location. A middleman was defined as selling live poultry to mostly sellers, or purchasing live poultry from sellers and then re-selling them at another location (e.g. market, restaurants). All traders that were present during the visit of the selected LBMs were interviewed, except in the largest LBM in Viet Nam where half were interviewed. Of note, in all other LBMs, the numbers of interviewed sellers corresponded well to numbers indicated by market managers.
The study periods, April-May 2009 in Viet Nam and June-July 2009 in Cambodia, did not include any seasonal festivals which could potentially influence live poultry sale patterns.
Three standardized questionnaires were designed for interviewing market managers, sellers, and middlemen. The questionnaires were translated into Vietnamese and Khmer and administered by trained interviewers. All questionnaires were piloted in Vietnamese LBMs that were not included in the survey. Informed oral consent was sought prior to interviewing. At the conclusion of each interview, the completed questionnaire was reviewed by the author for missing, unclear, or inconsistent answers. Questions for which the accuracy of the answer was doubtful were posed a second time. Market observations, such as counting the number of birds offered for sale and the number of birds left unsold at the end of the market day, were made during the visits. The interviewees' answers were consistent with these observations.
LBMs were described based on demographic features provided by market managers: days of the month and time of the day during which LBMs were open, average number of sellers, and seasonal variations. Sellers were classified as retailers or wholesalers if they reported selling most birds to consumers or traders, respectively. Markets were classified as either retail or wholesale markets if more than two thirds of sellers were retailers or wholesalers. Markets that did not fall into one of these categories were classified as mixed.
Traders' practices likely to influence the sustainability of virus circulation in LBMs were recorded; essentially, those that could impact the length of time during which birds remained in the market chain and the contact rate between birds. These factors included the number of days during which traders were active, and the length of time they spent at market in a day. The number of poultry sold within a day, and the type of poultry were also considered. Indeed, the susceptibility of chickens and ducks (muscovy or mallard duck derived breeds) to infection is known to differ [28] . Moreover, the supply management, and frequency and quantity of the surplus (unsold poultry reoffered for sale the following day) could impact on the length of time that poultry spent at market. The surplus frequency referred to the proportion of days traders reported having had surplus and the surplus volume to the proportion of birds left unsold when having a surplus. The surplus frequency was captured in 2 ways: the usual surplus frequency, recorded as a categorical variable (categories: never, sometimes, half of the time, often, always), and the surplus frequency in the last week, defined as the proportion of days with a surplus out of the number of trading days during the week preceding the interview. Since both variables were highly correlated (correlation ratio = 0.84), only the surplus frequency in the past week was kept in the analysis. The management of the supply described the frequency at which poultry were purchased, and whether poultry were purchased the day before being offered for sale and kept overnight at traders' homes. Changes in trade practices during festivals were also described.
Information regarding the origin of poultry and the number and type of visited farms and LBMs was also collected, as these contacts could influence the likelihood of spreading infection into and out of LBMs.
Questionnaire data were entered in Microsoft Access 2007H (Microsoft Corp., Redmond, WA, USA) database. The accuracy of the data entry was verified by cross-checking each questionnaire with the recorded entry.
Numerical variables were summarized as medians with interquartile ranges (IQR); binary and categorical variables as frequencies and percentages.
Multivariate analysis was performed to describe trader profiles, which were based on poultry management practices that could increase the risk of sustained virus circulation in LBMs ( Table 1) . As variables were both numerical and categorical, factor analysis for mixed data (FAMD) [29, 30] was used. This method allows a reduction in the dimensions of multivariate data, creating a smaller number of synthetic (and uncorrelated) factors accounting for most data variability. Further details are provided in Text S1.
Hierarchical cluster analysis (HCA) [31] was then used to group traders into clusters according to their level of similarity in the factors created by the FAMD. The Manhattan distance was used to assess the level of dissimilarity between 2 traders. The algorithm was agglomerative, and the Ward's criteria for linkage was adopted. Finally, a consolidation using the k-means algorithm was performed. FAMD and HCA were implemented in R 2.12.0 [32] , using the package FactoMineR 1.15 [33] .
There were 340 sellers and 221 middlemen interviewed in 30 Vietnamese LBMs, and 54 sellers and 30 middlemen in 8 Cambodian LBMs. The refusal rate was 8% among Vietnamese traders, with the principal reason being that they were too busy to participate. In Cambodia, only 2 (2%) people declined interviews.
LBM features are described in Table 2 . LBMs in Bac Giang province in Viet Nam were either open every day (n = 4) or periodically (n = 5). Periodic markets were open 6 or 12 days per month. LBMs in Ha Noi province were grouped as either retail and mixed markets (n = 17), hosting from 2 to 13 sellers, or wholesale markets (n = 4), which were the largest markets, hosting from 30 to 80 sellers. Cambodian LBMs were grouped as either urban markets (n = 4), located in Phnom Penh or its close proximity, or peri-urban markets (n = 4), in other provinces.
Urban markets were open throughout the day for 12-15 hours, whereas peri-urban markets were only open in the morning for 6 hours or less.
Most Vietnamese (80%, n = 340) and Cambodian (70%, n = 54) sellers reported having a surplus, at least occasionally. In contrast, most middlemen reported never having a surplus (Viet Nam: 76%, n = 221; Cambodia: 97%, n = 30). As they generally spent less than 1 hour in a LBM, middlemen kept their poultry in LBMs for only a very short time and were therefore excluded from the multivariate analysis. Following the FAMD and the HCA, Vietnamese sellers were divided into 4 clusters and Cambodian sellers into 2 clusters. Tables 3 and 4 present the distribution of poultry management and contact features for each seller profile in Viet Nam and Cambodia, respectively. A description of the factors is provided in Text S1.
Vietnamese sellers in Cluster V.1 were farmers or occasional sellers. Most farmers' flocks consisted of 50-500 birds and were located in the market vicinity. They were characterized by an infrequent and short presence at market and a very low number of sales. Encompassing 48% (n = 340) of Vietnamese sellers, Cluster V.2 was composed of sellers trading larger volumes and more frequently than Cluster V.1 sellers. However, they spent little time in LBMs, with 60% (n = 162) only trading 4 hours or less per day. Their surpluses were also low, with the median frequency of having a surplus being 14% and the median proportion of unsold chickens and ducks being 10% and 6%, respectively. Cluster V.3 was also composed of regular sellers. Although their number of sales was slightly lower than Cluster V.2 sellers, they reported higher surpluses than traders in other clusters. The proportion of traders reporting a surplus every day was 51% (n = 71), and the median proportion of unsold chickens and ducks was 32% and 17%, respectively. Moreover, 45% (n = 71) of Cluster V.3 sellers were not supplied every day and 66% (n = 71) purchased birds the day before offering them for sale, keeping them overnight at home. These proportions were higher than in other clusters. For traders who were not supplied every day when operating at a market, part of the newly purchased birds were stored at home for 1 to several days before bringing them to market. Cluster V.4 included sellers spending more time at market, with 64% (n = 64) trading at least 10 hours per day, and selling substantially more poultry than other clusters. However, the median surplus frequency of 29% and the median proportion of unsold birds, less than 10% for chickens and ducks, were much lower than those reported by Cluster V.3 sellers. Except for Cluster V.1, most Vietnamese sellers were supplied by farms, the majority of which were small commercial farms (50-500 birds). Cluster V.2 and V.3 sellers were also supplied by backyard farms (,50 birds), whilst Cluster V.4 sellers were also supplied by large farms (.500 birds). The number of sellers visiting several markets to purchase or sell poultry was higher in Cluster V.2 (48%, n = 162) than in Clusters V.3 (38%, n = 71) and V.4 (3%, n = 64).
Whilst the proportion of Cluster V.2 sellers was high in all market groups, 74% (n = 43) Cluster V.1 sellers were found in Bac Giang markets, 80% (n = 71) Cluster V.3 sellers were in Ha Noi retail and mixed markets and 95% (n = 64) Cluster V.4 sellers were in Ha Noi wholesale markets. All Bac Giang markets were strictly or predominantly populated by Cluster V.1 or V.2 sellers, or both (Fig. 2) . Cluster V.3 was the only, or the predominant, seller profile in 13 of the 17 Ha Noi retail and mixed markets. This seller profile was, however, absent in 2 markets located in peri-urban areas, far from the main urban centres. Contrary to other market groups, Ha Noi wholesale markets were highly heterogeneous in terms of seller composition. However, when considering the proportion of poultry traded by each seller profile in each market (Fig. S1 ), largescale sellers (Cluster V.4) were predominant in all Ha Noi wholesale markets but 1. The market location was, therefore, a good predictor of the seller composition. Cambodian sellers were classified into 2 clusters. Cluster C.1 sellers spent little time in LBMs and either rarely, or never, had a surplus. In contrast, most Cluster C.2 sellers spent all day in LBMs and reported high surpluses. The median length of time spent in LBMs was 4 hours for Cluster C.1, and 11 hours for Cluster C.2 sellers. Most Cluster C.1 sellers (58%, n = 26) never had any unsold poultry at the end of the market day, whereas all Cluster C.2 sellers but 1 reported surpluses. Whilst most Cluster C.1 sellers were supplied by farmers, most Cluster C.2 sellers were supplied by traders. Contrary to Viet Nam, all supplying farms were backyard farms (,50 birds), and none of the Cambodian sellers visited other markets to buy or sell poultry.
Cambodian seller profiles were also associated with market groups, with 85% (n = 26) Cluster C.1 sellers operating in periurban markets and 86% (n = 28) Cluster C.2 sellers operating in urban markets. Three of 4 peri-urban markets were exclusively populated by Cluster C.1 sellers, and Cluster C.2 predominated in 3 of 4 urban markets.
Chicken sales peaked in Viet Nam and Cambodia during the Tet and the Chinese New Year (late January or early February), respectively, with 72% (n = 340) and 96% (n = 54) sellers reporting an increase in chicken sales by 100% on average. Likewise, the number of sellers operating at markets increased. Sellers did not report any other changes in their practices during these periods.
Despite considerable variation in poultry management practices between sellers, some patterns were evident such that seller profiles of epidemiological importance could be identified. The high surplus frequency and volume reported by Clusters V.3 sellers (medium-scale sellers with high surplus) increased the time spent by birds in the LBM system. Moreover, their low supply frequency and the practice of purchasing birds the day before offering them for sale extended the time spent by birds in the sellers' flocks. This created opportunities for newly purchased birds to mix with unsold birds brought back from LBMs. These practices would make infection of susceptible birds more likely. This seller profile would thus be at high risk for contributing to the maintenance of HPAIV H5N1 in LBMs.
In contrast, surplus and supply features exhibited by Cluster V.2 (medium-scale sellers with low surplus) and V.4 sellers (large-scale sellers) meant that their birds spent little time in LBMs, limiting their potential to sustain virus circulation. However, these seller profiles may still play an active role in virus spread. A substantial proportion of Cluster V.2 sellers were mobile, visiting several markets to sell and/or purchase poultry. This could enable them to spread infection between LBMs. If infectious birds are Although these newly infected birds are unlikely to remain in LBMs for a sufficient period of time to infect others, their sale to traders operating in other LBMs could lead to virus spread between LBMs. Indeed, most large-scale sellers (V.4) operated in Ha Noi wholesale markets, from which a substantial part of the poultry population was then sold to retail market sellers (data not shown). In contrast, Cluster V.1 sellers (farmers and irregular sellers) were unlikely to play a major role in spatial virus spread as they only traded in markets located in their vicinity.
The distribution of seller profiles was associated with market groups. Therefore, knowing the type and location of a given LBM provides a good indicator of its seller profile composition, the proportion of poultry sold by each seller profile and, thus, the market's risk of sustaining HPAIV H5N1 circulation. LBMs located in the rural province of Bac Giang probably play a limited role in virus perpetuation. By contrast, most Ha Noi retail and mixed markets were dominated by Cluster V.3 sellers, which could permit virus maintenance. However, the low number of sellers and the low volume of sales might increase the risk of stochastic extinction of viruses, even with frequent surpluses. Two LBMs of this group were predominantly or exclusively populated by Cluster V.2 sellers and were located in districts which shared key features with Bac Giang province: rural areas with a large number of poultry farms. In contrast, other Ha Noi retail and mixed markets were located in, or close to, urban centres. This urban tropism of sellers at higher risk of maintaining virus circulation was also observed in Cambodia. The markets located in Phnom Penh or its outskirts were almost exclusively populated by sellers with high surpluses (C.2), similar to Vietnamese Cluster V.3 sellers, whilst Cambodian Cluster C.1 sellers, unlikely to allow virus maintenance, operated in provinces other than Phnom Penh.
Basic information on market type and location could be easily collected from each LBM to aid the identification of markets at high risk of virus maintenance, where risk mitigation strategies should be implemented. This information could be directly collected from market managers and would not require a laborintensive survey. The implementation of strategies aiming at breaking virus amplification cycles in all markets is unnecessary, and also impractical given that LBMs are ubiquitous. Targeting control measures to a few selected markets would not only reduce the overall cost, but would also allow closer monitoring and proper implementation. Simple hygiene measures and culling of unsold birds may be very effective in breaking the virus amplification cycle [11, 34, 35] . Successfully implemented in Hong Kong, such measures would, however, need to be adapted to each local setting in order to minimize their negative impact on trade.
HPAIV H5N1 has been isolated in northern Vietnamese LBMs [36, 37] , and poultry trade is suspected to spread the infection in Cambodia [38] . HPAIV H5N1 is likely to circulate in the study population. However, only the potential of LBMs to become virus reservoirs has been assessed in this survey. Complementing the findings with virological sampling of markets would be necessary to conclude that these high risk LBMs are truly virus reservoirs. Moreover, since the sampling frame is not representative of the population as a whole but of the largest markets in specific regions, inferences about the study population are necessarily limited. When asked to name the most populated markets, traders were more likely to name markets where they regularly operated, or of which they had personal knowledge, for example by being in their vicinity. However, as traders interviewed in Bac Giang province often named the same markets, regardless of the district where the interview took place, it is likely that most of the largest markets were identified. Three markets located in Bac Giang city were among the most commonly named markets, but authorization to visit was not possible as poultry sales at these sites had been officially prohibited. In Ha Noi province, some large retail and mixed markets may have been missed, as only a few districts were visited. However, the retail and mixed markets identified in Ha Noi were likely to be similar to those not identified, given similar population densities and housing.
In conclusion, this study was able to identify specific profiles of live bird sellers in Viet Nam and Cambodia which could play a key role in virus perpetuation. Moreover, the type and the location of an LBM could be a good predictor of its seller profile composition and, thus, of its potential for sustaining virus circulation. Therefore, results suggest that control strategies aiming at preventing HPAIV H5N1 maintenance in LBMs could potentially be targeted towards specific high risk LBM groups. This is of particular importance in resource-scarce countries with extensively developed LBM systems. Figure S1 Distribution of the number of poultry traded by seller clusters across markets and market groups. For each market group, a barplot (on the left) shows the proportion of the poultry flow (number of poultry sold) traded by each seller cluster in the market group, and a plot (on the right) shows the distribution of its markets according to the proportion of the poultry flow traded by each seller cluster in each market. Where the number of markets in a group is greater than 5, box plots are shown; otherwise each market (circle) and the median (line) are presented.
Text S1 Supplementary information includes further details on the approach used to construct a typology of traders, and on the results of the multivariate analysis and hierarchical cluster analysis. (DOC) Porcine Noroviruses Related to Human Noroviruses Detection of genogroup II (GII) norovirus (NoV) RNA from adult pigs in Japan and Europe and GII NoV antibodies in US swine raises public health concerns about zoonotic transmission of porcine NoVs to humans, although no NoVs have been detected in US swine. To detect porcine NoVs and to investigate their genetic diversity and relatedness to human NoVs, 275 fecal samples from normal US adult swine were screened by reverse transcription–polymerase chain reaction with calicivirus universal primers. Six samples were positive for NoV. Based on sequence analysis of 3 kb on the 3´ end of 5 porcine NoVs, 3 genotypes in GII and a potential recombinant were identified. One genotype of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. These results raise concerns of whether subclinically infected adult swine may be reservoirs of new human NoVs or if porcine/human GII recombinants could emerge. Detection of genogroup II (GII) norovirus (NoV) RNA from adult pigs in Japan and Europe and GII NoV antibodies in US swine raises public health concerns about zoonotic transmission of porcine NoVs to humans, although no NoVs have been detected in US swine. To detect porcine NoVs and to investigate their genetic diversity and relatedness to human NoVs, 275 fecal samples from normal US adult swine were screened by reverse transcription-polymerase chain reaction with calicivirus universal primers. Six samples were positive for NoV. Based on sequence analysis of 3 kb on the 3′ end of 5 porcine NoVs, 3 genotypes in GII and a potential recombinant were identified. One genotype of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. These results raise concerns of whether subclinically infected adult swine may be reservoirs of new human NoVs or if porcine/human GII recombinants could emerge.
Norovirus) cause diarrhea in humans and animals (1) (2) (3) . The NoV genome is 7.3-7.7 kb long with 3 open reading frames (ORFs) encoding a polyprotein that undergoes protease processing to produce several nonstructural proteins, including an RNA-dependent RNA polymerase (RdRp), a major capsid protein (VP1, capsid), and a minor capsid protein (VP2) (1, 4, 5) . The capsid protein contains a conserved shell (S) and hypervariable protruding (P) domains (6) . Noroviruses are genetically diverse and make up 27 genotypes within 5 genogroups, GI/1-8, GII/1-17, GIII/1-2, GIV, and GV, based on the capsid genes of 164 strains (7) . Human NoVs cause an estimated 23 million cases of illness annually in the United States (8) and >90% of nonbacterial epidemic gastroenteritis worldwide (1) . The low infectious dose, environmental resistance, strain diversity, shedding from asymptomatic persons, and varied transmission vehicles render human NoVs highly contagious.
Norovirus RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 4 of 1,017 normal slaughtered pigs in Japan (9) and in 2 of 100 pooled pig fecal samples in the Netherlands (10) . These porcine NoVs (Sw43/97/JP, Sw918/97/JP, and 34/98/NET) are genetically similar and are classified into GII (9,10), like most epidemic human NoVs (11) (12) (13) . Also, the viruslike particles (VLPs) of Sw918 strain cross-react with antibodies against human GII but not GI NoVs (14) . The close genetic and antigenic relationships between human and porcine NoVs raise public health concerns regarding their potential for zoonotic transmission and as reservoirs for emergence of new epidemic human strains.
Farkas et al. (14) reported that US swine sera react with Po/NoV/GII/Sw918 strain, but no direct detection of NoV from US swine has been reported. To detect porcine NoVs and assess their genetic diversity and relatedness to human NoVs, we screened 275 pig fecal samples from US swine by RT-PCR with a calicivirus universal primer pair p290/110 targeting the RdRp region (15, 16) , followed by sequencing the 3 kb on the 3′ end of the genome for 5 NoV strains. Gnotobiotic pigs were inoculated with porcine NoVs to examine their infectivity and to produce convalescent-phase antiserum for antigenic analysis.
Fecal RT-PCR was performed separately by using primer pair p290 (5′-GATTACTCCAAGTGGGACTCCAC-3′) (15) and p110 (5′-ACDATYTCATCATCACCATA-3′) (16) as previously described (15) but at 48°C for annealing (317 bp for NoV or 329 bp for sapovirus). To amplify the 3-kb 3′ end fragment, cDNA was synthesized by SuperScript III First-Strand cDNA synthesis kit (Invitrogen) with primer VN 3 T 20 (5′-GAGTGACCGCGGCCGCT 20 -3′). PCR was then performed with TaKaRa Ex Taq polymerase (TaKaRa Mirus Bio, Madison, WI, USA) with primers p290 and VN 3 T 20 . Quantitative (endpoint titration) RT-PCR (17) was performed with primer pair PNV7 (5′-AGGTGGTGGCC-GAGGAYCTCCT-3′) and PNV8 (5′-TCACCATAGAAG-GARAAGCA-3′) targeting the RdRp (211 bp) of QW101 strain.
RT-PCR products were purified with the QIAquick Gel Extraction kit (Qiagen) before cloning into pCR2.1-TOPO (T/A) or PCR XL cloning kit (Invitrogen). Five clones of each sample were sequenced. DNA sequencing was performed with BigDye Terminator Cycle and 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequence editing was performed by Lasergene software package (v5, DNASTAR Inc., Madison, WI, USA). The Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST) was used to find homologous hits. Multiple sequence alignment was performed with ClustalW (v1.83) at DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp). Phylogenetic and bootstrap (1,000 replicates) analyses were conducted by using MEGA (v2.1) (18) . Identification of recombinants was performed by using the Recombinant Identification Program (RIP, http://hivweb.lanl.gov/RIP/RIPsubmit.html) (19) . The classification and GenBank accession numbers of NoVs are listed in Table 1 .
Four gnotobiotic pigs were maintained and euthanized as previously described (25, 26) . The inoculate was a 20% fecal filtrate (0.2 µm) in EMEM of the QW126 or QW144 (QW101-like, GII-18) strains or EMEM only (2 negative control pigs). One pig was inoculated with QW126 orally and intranasally at 9 days of age, and convalescent-phase antiserum LL616 was collected at postinoculation day (PID) 26. A second pig was inoculated with QW144 orally at 35 days of age and euthanized at PID 5.
Immune electron microscopy (IEM) was performed as described previously (27) . For enzyme-linked immunosorbent assay (ELISA), the recombinant baculovirusexpressed human NoV VLPs and rotavirus VP2 and VP6 (2/6)-VLPs (negative control) (28) were CsCl-gradients purified. We coated 96-well microplates with VLPs (200 ng/well) in carbonate buffer (pH 9.6) and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS)-Tween 20 (0.05%). Serially diluted serum samples that included positive and negative controls were added to duplicate positive-and negative-coated wells, and the plates were incubated. After washing, horseradish peroxidase (HRP)labeled goat anti-pig immunoglobulin G (IgG) (H + L) for pig sera or goat anti-human IgG + IgA + IgM (H + L) (KPL, Gaithersburg, MD, USA) for human serum was added. After incubation and washing, the substrate 3,3′,5,5′-tetramethylbenzidine was added. The cutoff value was the mean absorbance of the negative coatings multiplied by 2.
Western blot was performed as described previously (29) . Nitrocellulose membranes were incubated with pig convalescent-phase antiserum LL616 against porcine GII-18 NoV or negative control serum in PBS containing 4% nonfat dry milk followed by goat anti-pig IgG (H + L)-HRP conjugate.
Porcine NoVs were classified into 3 genotypes within GII based on the complete capsid sequences: 1 genotype with prototype Japanese strains Sw43 and Sw918 and 2 new genotypes. A total of 19 of 275 samples showed a potential positive band after agarose gel electrophoresis of the RT-PCR products of primer pair p290/110. Fourteen samples representative of each potentially positive farm or the slaughterhouse were sequenced. After performing BLAST search, we identified 6 NoVs (QW48, Michigan farm A; QW101, QW125, and QW126, Ohio farm B; and QW170 and QW218, Ohio slaughterhouse), 3 sapoviruses, and 5 sequences that had no significant hit in the database. Because the QW126 shared 99% nucleotide (nt) identity with the QW101 and QW125 strains in the 274-nt RdRp region, it was not sequenced further.
We sequenced the 3-kb 3′ end of the genome containing the partial RdRp, VP1 and VP2 genes, and the 3′ untranslated region of the 5 strains. The porcine NoVs represented 3 distinct clusters: 1) Sw43, Sw918, and QW48; 2) QW101 and QW125; and 3) QW170 and QW218, on the basis of the size of each gene and the ORF1-ORF2 overlap region (Table 2 ). Across the 3 kb, the QW101 and QW125 strains and the QW170 and QW218 strains shared 99% nt identity.
The amino acid identity of the predicted complete and S and P domains of the capsid protein of the 5 porcine NoVs, the previously reported porcine NoVs (Sw43 and Sw918), and representative human, bovine, and murine NoV strains is summarized in Table 3 . In the complete capsid, the QW48 strain was most closely related to the porcine NoV prototype Sw43 strain (98% amino acid identity); the QW170 and QW218 strains shared the highest amino acid identities (81%) to porcine Sw43 and Sw918 strains; the QW101 and QW125 strains showed the highest amino acid identity to human GII-3/Mexico (71.4%), then to human GII-6/Baltimore (71.0%), porcine QW218 (71.0%), and porcine Sw43 (70.6%) strains. The S and P domains of these NoVs showed similar relationships. A neighbor-joining phylogenetic tree based on the amino acid sequences of the complete capsids ( Figure 1) showed that QW48 grouped with Sw43 and Sw918 strains into GII-11 and that QW170 and QW218 formed a new genotype (GII-19), which was closer to porcine than to human strains. However, QW101 and 125 formed a new genotype (GII-18) between human and porcine GII NoVs.
Further analysis of the predicted C-terminal ≈260 amino acids of the RdRp region ( Figure 2 ) showed similar grouping results for QW48, QW101, and QW125 strains but different for QW170 and QW218 strains, which were in the same cluster (GII-11) as Sw43, Sw918, and QW48 in the RdRp region. This finding suggested that a recombination event occurred between QW170/218-like and Sw43-like NoVs. The complete VP2 sequences of representative strains were also analyzed (data not shown). Results were similar to those of the capsid sequence classification.
A potential recombination event occurred between QW170/218-like and Sw43-like strains. To examine where the recombination occurred, we performed RIP analysis by placing the 3′-end RdRp and the capsid sequence of QW170 or QW218 as a query sequence and the corresponding sequences of Sw43 and QW101 as background sequences. The resulting diagram ( Figure 3A) showed that QW170 had high similarity to Sw43 in the RdRp but not in the capsid region. This abrupt change happened in the RdRp-capsid junction region. Therefore, we performed sequence alignments of the RdRp-capsid junction of NoVs, including the calicivirus genomic-subgenomic conserved 18-nt motif (20) (Figure 3B ). Between Sw43, QW170, and QW218, all 18 nt were identical, but identities decreased downstream of this motif. QW170 and QW218 grouped with Sw43 with a high bootstrap value of 95 in the RdRp tree (Figure 2 ), whereas they segregated from Sw43 with the highest bootstrap value of 100 in the capsid tree (Figure 1 ). We could not clarify which was the parent or progeny strain.
The porcine NoVs replicated in gnotobiotic pigs. Two pigs were inoculated with QW101-like GII-18 porcine NoVs (QW126 and QW144 strains) to verify their replication in pigs as confirmed by quantitative RT-PCR and IEM and to produce convalescent-phase serum to examine antigenic reactivity with human NoVs. These 2 strains were confirmed as QW101-like porcine NoVs in both the RdRp (169-nt) and the capsid S domain (363-nt) regions by sequence analysis of the RT-PCR products (Q.H. Wang and L.J. Saif, unpub. data). They shared 99% and 100% amino acid identities to the QW101 strain in the 2 regions, respectively. Porcine NoV shedding, assessed by quantitative RT-PCR with primer pair PNV7/8, was detected at PID 3-5 (euthanized) after QW144 exposure, coincident with mild diarrhea. The RT-PCR-detectable units of the rectal swab RNA increased from negative at PID <2, 10 3 at PID 3-4, and 10 4 at PID 5 (large intestinal contents). Norovirus shedding was detected only at PID 5 without diarrhea after QW126 exposure. Examination of the intestinal contents of the pig inoculated with QW144 by IEM with pig convalescent-phase antiserum LL616 showed clumps of ≈32-nm NoV particles (Figure 4) . The 2 control pigs had no virus shedding or diarrhea. Detailed studies of the pathogenesis of porcine NoVs in gnotobiotic pigs are in progress (S. Cheetham and L.J. Saif, unpub. data).
Antisera to QW101-like (QW126) porcine NoVs crossreacted with VLPs of human GII NoVs in ELISA and Western blot. In ELISA (Table 4) , the pig convalescentphase antiserum (LL616) to QW101-like porcine NoV QW126 strain showed higher titers (1:400-1:800) to GII-3/Toronto, GII-4/MD145, GII-4/HS66, and GII-6/Florida strains; a lower titer (1:100) to GII-1/Hawaii strain; and lowest titer (1:10) to GI-3/Desert Shield strain. In Western blot ( Figure 5 ), the capsid proteins (59-60 kDa) of Toronto, MD145, HS66, and Florida strains, but not the Hawaii and Desert Shield strains, were detected by pig antiserum LL616 but not the negative control serum (data not shown). Thus, 1-way antigenic cross-reactivity exists between human NoV antigens and porcine NoV (GII-18) antiserum, with moderate cross-reactivity to human NoVs GII-3, 4, and 6; low cross-reactivity to GII-1; and very low cross-reactivity to GI-3.
All porcine NoVs were detected from pigs without clinical signs (9, 10) . Subclinically infected pigs may be natural reservoirs for NoVs, and because porcine GII NoVs are genetically and antigenically related to human NoVs, concerns exist about their zoonotic potential. Whether human NoV strains similar to the QW101-like porcine NoVs circulate among people with occupational exposure to pigs is unknown, but such studies could provide information on the zoonotic potential of these porcine NoVs. The RdRp-capsid junction region of NoVs contains a highly conserved 18-nt motif in genomic and subgenomic RNA that is believed to be a transcription start signal (1, 20) . All 18 nt were identical within each genogroup except for the Hu/GII/J23, Po/GII/QW101, and Po/GII/QW125 strains ( Figure 3B , sequence alignments on other GI and GIII strains are not shown). This finding suggests that homologous recombination may occur within this motif between NoVs of different genotypes within the same genogroup. Recombinant human GII NoVs have been reported previously (20) (21) (22) (23) (24) . To our knowledge, this study is the first identification of a potential recombinant between pig NoVs. At present, NoV recombinants have been detected exclusively between viruses within the same genogroup and within the same host species, but few animal NoVs have been sequenced (RdRp and capsid) for comparative analysis, especially those from animals in developing countries, where humans and animals may be in close contact.
The QW101-like porcine NoVs replicated in gnotobiotic pigs with fecal shedding, documented by quantitative RT-PCR and IEM. No cell culture system or animal disease models are available for human NoVs, which impedes the study of their pathogenesis, replication strategies, host immune responses, and preventive approaches. The infection of pigs with porcine NoVs may provide a new infection or disease model to study NoV infections. In this study, 1-way antigenic cross-reactivity occurred between antiserum to QW101-like porcine NoVs and the capsid proteins of human NoVs, with highest cross-reactivity to GII-3, 4, and 6 NoVs. This finding coincides with the finding that the QW101 strain shares high amino acid identity with GII-3 (71%), GII-6 (71%), and GII-4 (63%) NoVs.
In summary, 3 genotypes of porcine NoVs were detected in US swine. One genotype (QW101-like, GII-18) was genetically and antigenically most closely related to human GII NoVs. Potential recombinant porcine NoV strains were identified. The QW101-like NoVs infected gnotobiotic pigs, and NoV particles were evident in intestinal contents. These results raise questions of whether pigs may be reservoirs for emergence of new human NoVs or if porcine/human GII recombinants could emerge. Figure 5 . Antigenic cross-reactivity between human genogroup (G) II norovirus (NoV) capsid proteins and a pig convalescent-phase antiserum (LL616) against porcine QW101-like (GII-18) NoV was determined by Western blot. The CsCl-gradient purified viruslike particles (1,250 ng) were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and tested with LL616. The sucrose-cushion (40%, wt/vol) purified Sf9 insect cell proteins acted as a negative control (lane 8). Lane 1, molecular weight marker (kDa); lanes 2-7, Hu/GI-3/Desert Shield, Hu/GII-1/Hawaii, Hu/GII-3/Toronto, Hu/GII-4/MD145, Hu/GII-4/HS66, and Hu/GII-6/Florida, respectively. Clinical Relevance and Discriminatory Value of Elevated Liver Aminotransferase Levels for Dengue Severity BACKGROUND: Elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is prominent in acute dengue illness. The World Health Organization (WHO) 2009 dengue guidelines defined AST or ALT≥1000 units/liter (U/L) as a criterion for severe dengue. We aimed to assess the clinical relevance and discriminatory value of AST or ALT for dengue hemorrhagic fever (DHF) and severe dengue. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at Tan Tock Seng Hospital, Singapore according to WHO 1997 and 2009 criteria for dengue severity. Of 690 dengue patients, 31% had DHF and 24% severe dengue. Elevated AST and ALT occurred in 86% and 46%, respectively. Seven had AST or ALT≥1000 U/L. None had acute liver failure but one patient died. Median AST and ALT values were significantly higher with increasing dengue severity by both WHO 1997 and 2009 criteria. However, they were poorly discriminatory between non-severe and severe dengue (e.g., AST area under the receiver operating characteristic [ROC] curve = 0.62; 95% confidence interval [CI]: 0.57–0.67) and between dengue fever (DF) and DHF (AST area under the ROC curve = 0.56; 95% CI: 0.52–0.61). There was significant overlap in AST and ALT values among patients with dengue with or without warning signs and severe dengue, and between those with DF and DHF. CONCLUSIONS: Although aminotransferase levels increased in conjunction with dengue severity, AST or ALT values did not discriminate between DF and DHF or non-severe and severe dengue. Dengue is a mosquito-borne arboviral infection endemic to most tropical and subtropical countries [1] . Elevation of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is common in acute dengue illness, occurring in 65-97% [2, 3, 4, 5] of dengue patients, peaking during the convalescent period of illness (days 7-10) [2, 4, 6] . In dengue-endemic countries, dengue is an important cause of acute viral hepatitis [7] .
Elevated AST and ALT levels have been associated with bleeding [2, 4, 6] and dengue hemorrhagic fever (DHF) [3, 8] . Liver failure has been recognized as a complication and unusual manifestation of dengue [9, 10] but occurred infrequently in 3 of 270 patients in Taiwan [6] and 5 of 644 patients in Vietnam [4] . In Malaysia, 8 of 20 pediatric DHF patients developed liver failure, 1 died, and the rest recovered completely [11] . In Singapore, AST or ALT levels were not independent predictors of DHF in 1973 adult dengue patients [12] .
In 2009, the World Health Organization (WHO) revised its dengue guidelines and proposed severe organ impairment as one category of severe dengue in addition to severe plasma leakage and severe bleeding [1] . Severe liver involvement was defined as AST or ALT$1000 units/liter (U/L). In Taiwan, AST.10 times the upper limit of normal (ULN) occurred in 11% of dengue patients [6] , while in Brazil this occurred in 4% of their cohort [3] . In this study, we aimed to evaluate the clinical relevance of elevated AST and ALT levels and correlate liver aminotransferase levels with dengue severity according to WHO 1997 and 2009 classifications.
All laboratory-confirmed dengue patients identified from our hospital microbiology database and treated using a standardized dengue clinical care path at the Department of Infectious Diseases, Tan Tock Seng Hospital (TTSH), Singapore from 2006 to 2008 were retrospectively reviewed for demographic, serial clinical and laboratory, radiological, treatment, and outcome data. These cases were positive by real-time polymerase chain reaction (PCR) [13] . We included patients with only positive dengue serology in only subgroup analyses, as we did not have paired sera, and other etiologies for elevated AST and ALT could not be excluded without more extensive evaluation.
Cases were categorized using serial clinical and laboratory data from the entire clinical course as dengue fever (DF), DHF, or dengue shock syndrome (DSS) using WHO 1997 classifications [9] . Dengue fever classification requires fever and at least two of the following: headache, eye pain, myalgia, arthralgia, rash, bleeding, and leukopenia. Dengue hemorrhagic fever requires all of the following: fever, platelet count #100610 9 /liter, bleeding, and plasma leakage [9] . Dengue shock syndrome is a case of DHF with either tachycardia and pulse pressure ,20 mmHg or systolic blood pressure ,90 mmHg [9] .
Cases were also categorized as dengue without warning signs (WS), dengue with WS, or severe dengue using WHO 2009 classifications [1] . Dengue (WHO 2009) requires fever and two of the following: nausea, vomiting, rash, aches and pains, leukopenia, or any warning sign [1] . Warning signs include abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, hepatomegaly, or hematocrit rise ($20%) with rapid drop in platelet count (,50,000/liter) [1, 14] . We modified the WHO 2009 warning sign of rise in hematocrit concurrent with rapid drop in platelet count by quantifying it as hematocrit $20% concurrent with platelet count ,50,000/liter, as this was shown to correlate significantly with dengue death in our adult dengue death study [14] . Severe dengue includes severe plasma leakage, severe bleeding, and severe organ impairment [1] .
We performed a subgroup analysis for median maximum AST and ALT values stratified by febrile (days 1-3 of illness), critical (days 4-6), and convalescent (days 7-10) phases as defined by WHO 2009 [1] and compared across dengue severity classification according to WHO 1997 [9] and 2009 [1] .
We excluded severe dengue due to isolated elevation of AST or ALT$1000 U/L from our definition of severe dengue outcome, as this would be a confounder in assessing the relevance of AST or ALT levels in defining dengue severity. Patients had AST/ALT taken at presentation and then throughout hospitalization at the physician's discretion. Maximum AST and ALT values recorded at a median of 4 days of illness (interquartile range [IQR]: 3-5 days) were used in this analysis. Those with pre-existing liver diseases were excluded. At TTSH, the ULN for AST is 41 U/L; for ALT, it is 63 U/L for males and 54 U/L for females.
We assessed the clinical relevance of elevated AST or ALT levels using four liver failure criteria-two for acute liver failure, and two that determine prognosis from chronic liver disease. The American Association for the Study of Liver Diseases (AASLD) recommends defining acute liver failure in a patient as: international normalized ratio (INR)$1.5, any degree of altered mental status, and illness ,26 weeks in duration without preexisting cirrhosis [15] . The King's College criteria assess prognoses in those with acute liver failure; the criteria are: prothrombin time .100 seconds or 3 of the following: age .40 years, prothrombin time .50 seconds, serum bilirubin .18 mg/dL, time from jaundice to encephalopathy .7 days [16] . The model for endstage liver disease (MELD) determines three-month mortality based on the following formula: 3.86(log serum bilirubin [mg/ dL])+11.26(log INR)+9.66(log serum creatinine [mg/dL])+6.4 [17] . The Child-Pugh criteria include assessment of degree of ascites, serum bilirubin and albumin, prothrombin time, and encephalopathy to determine one-and two-year survival [18] .
The Mann-Whitney U and Kruskal-Wallis tests were used to determine statistical significance for continuous variables, and chisquare or Fisher's exact test for categorical variables. Statistical tests were conducted at the 5% level of significance. Receiver operating characteristic (ROC) curves showing the area under the curve (AUC) were generated to determine the discriminatory performance of aminotransferase values. All statistical analyses were performed using Stata 10 (Stata Corp., College Station, TX).
This was a retrospective study involving data collection from medical records. All patient data were anonymized during analysis. This study was approved by the Institutional Review Board, National Healthcare Group, Singapore [DSRB E/08/ 567].
From 2006 to 2008, 690 dengue PCR positive cases were reviewed. Males comprised 493 (71%) of the cases, and the median age of the cohort was 35 years (IQR: 27-43 years). A Charlson comorbidity index $3, which predicts increased one-year mortality [19] , was noted in 5 (0.7%) patients. With WHO 1997 classification, 62% had DF, 31% DHF, and 7% DSS. With WHO 2009 classification, 14% had dengue, 62% had dengue with warning signs, and 24% had severe dengue. Hence, by WHO 1997 classification, 38% of patients with DHF/DSS needed close monitoring, while by WHO 2009 classification, 86% of patients with warning signs or severe dengue required close monitoring.
Median length of illness from onset to hospital presentation was 4 days (IQR: 3-5 days), while median length of hospital stay was 5 days (IQR: 4-6 days). Intravenous fluid was administered to 641 (93%) and platelet transfusion to 86 (12%). Intensive care unit (ICU) admission was required in 3 patients, and death occurred in 1 patient due to pneumonia.
(1) Elevation of AST/ALT and risk of liver failure Overall, 595 (86%) had AST above the ULN, and 316 (46%) had ALT above the ULN. Seven patients (1.0%) had severe dengue according to WHO 2009 criteria concurrent with AST or ALT$1000 U/L while three additional patients had severe dengue due to AST or ALT$1000 U/L only. Of the former seven patients, 86% had severe plasma leakage, 29% had severe bleeding, and none had severe organ impairment other than isolated AST or ALT$1000 U/L. Among the 3 patients admitted to the ICU, AST or ALT values were above the ULN but below 1000 U/L.
No patients in our cohort developed acute liver failure under AASLD or King's College criteria. With Child-Pugh scoring, 2 (0.3%) belonged to Child-Pugh class C. With MELD scoring, predicted three-month mortality of 6% were identified in 68 (10%)
Dengue is a global public health problem, as the incidence of the disease has reached hyperendemic proportions in recent decades. Infection with dengue can cause acute, febrile illness or severe disease, which can lead to plasma leakage, bleeding, and organ impairment. One of the most prominent clinical characteristics of dengue patients is increased aspartate and alanine aminotransferase liver enzyme levels. The significance of this is uncertain, as it is transient in the majority of cases, and most patients recover uneventfully without liver damage. In this study, we characterized this phenomenon in the context of dengue severity and found that, although liver enzyme levels increased concurrently with dengue severity, they could not sufficiently discriminate between dengue fever and dengue hemorrhagic fever or between non-severe and severe dengue. Therefore clinicians may need to use other parameters to distinguish dengue severity in patients during early illness.
Association between Transaminase Levels and Dengue www.plosntds.org patients in our cohort and 19.6% in 2 (0.3%) patients. The same two patients who were Child-Pugh class C also had a predicted 19.6% three-month mortality using MELD scoring; they both had DSS and severe dengue.
(2) Dengue severity and AST/ALT values Median AST and ALT values for dengue without warning signs, dengue with warning signs, and severe dengue (Table 1) In other hemorrhagic fevers, higher AST:ALT ratios correlated with disease fatality [20] . In our PCR-positive cohort, median AST:ALT ratios for DF, DHF, and DSS were 1.68, 1.68, and 1.88 (p = 0.29) and for dengue without WS, dengue with WS, and severe dengue, they were 1.60, 1.68, and 1.78 (p = 0.10), respectively.
The majority of our patients' maximum AST and ALT values were recorded during febrile (n = 258) and critical (n = 377) phases of acute dengue illness. By WHO 2009 dengue severity classification, the median AST and ALT values were significantly higher for severe dengue compared to dengue with and without warning signs during both the febrile and critical phases but not the convalescent phase (Table 3) . By WHO 1997 classification, the median AST and ALT values were significantly higher for DHF versus DF and DSS in the febrile phase only but not critical and convalescent phases (Table 4 ).
(4) Does a threshold AST or ALT value defining severe dengue exist?
In order to determine the reliability of AST and ALT values in defining dengue severity, ROC curves for AST and ALT against severe dengue excluding isolated transaminitis were determined (Figure 1 ). The AUC for AST was 0.62 (95% confidence interval [CI]: 0.57-0.67) and for ALT, 0.60 (95% CI: 0.54-0.64). This demonstrates that AST or ALT levels are insufficient to differentiate among the WHO 2009 dengue classifications. They were also poorly discriminatory between DF and DHF, as the areas under the curve (AUC) for AST and ALT were 0.56 (95% CI: 0.52-0.61) and 0.55 (95% CI: 0.51-0.59), respectively ( Figure 2 ). In our serology-positive cohort, the AUC values for AST and ALT were 0.56 and 0.54 for differentiating between DF and DHF. The AUC values for severe and non-severe dengue were 0.64 and 0.60 for AST and ALT, respectively.
The box plots in Figure 3 for the distributions of AST values show considerable overlap among the liver enzyme values for those with dengue with and without warning signs, and severe dengue. Because there were extreme outliers in our cohort, only those with AST below 1000 U/L were included in these plots. Figure 4 shows overlapping AST values among those with DF and DHF. Similarly, considerable overlap was observed in ALT values for patients with dengue with and without warning signs, and severe dengue, as well as for DF versus DHF (data not shown). 
Our analysis showed that liver aminotransferase levels were associated with but did not adequately differentiate between dengue severity. Although median AST and ALT values were significantly higher in those with DHF/DSS versus DF, and severe dengue versus non-severe dengue, very few (1.0%) had AST or ALT$1000 U/L. Notably, none developed liver failure, and death occurred in only 1 patient (0.1%). The majority of patients recovered uneventfully.
The lack of acute liver failure in our study was not unusual, as the incidence of acute liver failure in dengue patients was 1.1% in studies by Trung and Kuo [4, 6] . The largest study to date reported no acute fulminant hepatitis [3] . In contrast to these adult studies, it is noteworthy that in dengue-endemic countries, dengue may be an important cause of acute liver failure in children [21, 22] .
While some studies have shown that AST and ALT values differ between DF and DHF [3, 4, 8] , few studies support AST or ALT as an independent predictor of DHF [23] . Two studies in Singapore found liver aminotransferase levels to be significantly elevated among DF and DHF patients [12] and survivors and nonsurvivors of dengue [24] on univariate analysis, but this association was lost after adjusting for confounders on multivariate analysis.
Trung et al. showed significant differences comparing other febrile illness, dengue without plasma leakage, and dengue with plasma leakage with and without shock during critical and convalescent phases for AST but during critical phase for ALT only [4] . We made the novel finding that liver aminotransferase Association between Transaminase Levels and Dengue www.plosntds.org levels may significantly vary according to dengue severity during the febrile phase. For DHF by WHO 1997 classification, both AST and ALT were significantly higher during the febrile phase compared to DF or DSS, and for severe dengue by WHO 2009, AST and ALT were significantly higher during the febrile and critical phases.
The impact of co-infection with hepatitis viruses or concomitant hepatotoxic drugs was not assessed in our retrospective study, although we did exclude those with known liver comorbidities. Kuo et al. found that hepatitis B or C did not increase the extent of liver aminotransferase elevation in a retrospective adult dengue study in Taiwan [6] . In contrast, Trung et al. found that hepatitis B co-infection modestly increased ALT levels without significant clinical impact in a prospective adult dengue study in Vietnam [4] . Tang et al. showed that dengue and hepatitis B co-infected patients showed an aberrant cytokine secretion profile compared with those with dengue alone. [25] . In Singapore, seroprevalence for hepatitis B was 2.8% [26] and hepatitis C 0.37% [27] .
The etiology of elevated aminotransferase levels during acute dengue illness is unclear since AST is expressed in the heart, skeletal muscle, red blood cells, kidneys, brain, and liver, while ALT is secreted primarily by the liver [28, 29] . Because dengue infection can cause acute damage to these non-hepatic tissue types that express AST, raised aminotransferase levels may not be entirely due to severe liver involvement. It is therefore possible that the patients with high AST levels were also more likely to be classified as severe dengue under the 2009 criteria due to the common pathways to non-hepatic tissue damage, even though there is no association with poorer outcome. Our retrospective study has some limitations. Aspartate and alanine aminotransferase values were tracked according to clinical judgment rather than at regular intervals during illness. We did not have dengue serotype data for each patient, but in 2006, DENV-1 was predominant in Singapore with a switch to DENV-2 in 2007-2008 [30] . Serology-positive cases were not included in primary analyses because our clinical laboratory used a rapid diagnostic test with potential for false positive results [31] , we did not have paired sera to confirm dengue diagnosis [9] , and not every patient with elevated AST or ALT was comprehensively evaluated for other etiologies of viral and non-viral hepatitis. Although serology-positive cases presented later during illness, we saw no difference in outcome. Five serology-positive patients (0.34%) required ICU admission versus 0.43% of PCR-positive cases, while four patients (0.27%) died in the serology-positive cohort, versus 1 patient (0.14%) among PCR-positive cases. However, relative data accuracy in our retrospective study was made possible by using a standardized dengue clinical care path. Another limitation of this study is the relatively few cases with substantially elevated liver aminotransferase levels. At the same time, since our cohort comprised primarily adults, additional studies in pediatric populations will be useful to confirm our findings.
In patients with DHF/DSS or severe dengue, early diagnosis and proper management may improve outcome in most patients without comorbidities. However, in resource-limited countries, patients with severe disease may present late to the hospital with shock, with or without organ impairment at the time of admission. Our study highlights that early diagnosis and proper management of dengue patients may lead to excellent prognosis without organ injury.
In conclusion, elevated aminotransferase levels were associated with DHF/DSS and severe dengue in our cohort of adult patients with confirmed dengue. However, no threshold values discriminated between DF and DHF or between severe dengue and nonsevere dengue. Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies. Mesenchymal stem cells (MSCs) show promise for therapeutic recovery of function of damaged myocardium [1] [2] [3] [4] [5] . MSCs home to injured tissues [6, 7] and contribute to the structure or functional recovery of the myocardium via (1) secretion of paracrine factors that can inhibit immune responses [8] and/or facilitate angiogenesis [7, 9, 10] , (2) transdifferentiation/metaplasia [11, 12] , and (3) nuclear reprogramming through fusion with resident cardiomyocytes (CMs) [13] . The latter has been largely dismissed since the frequency at which fusion is detected is low relative to the number of transplanted MSCs. However, recent studies by us [14] and others [15] [16] [17] suggest that despite the low frequency cell fusion still may exert a dramatic impact on stem cell programming or reprogramming in the heart.
Cell fate determination was once thought to be unidirectional [18] , that is, as progenitor cells differentiate there is a progressive and permanent inactivation of specific genes that allow for their potency. However, technological advances suggest this is not strictly the case. Pioneering experiments of nuclear reprogramming utilized cell fusion to demonstrate that cytoplasmic elements of one fusion partner can impact nuclear transcription factors of the other fusion partner, inducing programming or reprogramming [19] [20] [21] . Later studies pinpointed specific transcription factors that, when activated exogenously, can fully reprogram somatic cells to an embryonic-like state [22] [23] [24] [25] [26] . Though successful reprogramming has been realized with this tailored in vitro approach, programming may require greater temporal control. Spontaneous physiologic cell-cell fusion is a temporally and spatially regulated process essential for programming or 2 Stem Cells International differentiation of certain cell types [27, 28] . Thus cell fusion may also confer a regulated transfer of transcriptional control necessary to drive stem cell or progenitor cell differentiation for repair of tissues in mature animals.
Cell-cell fusion occurs when the plasma membranes of neighboring cells fuse to form a multinucleated cell. To fuse, lipid bilayers of cell membranes must come into very close contact, in the range of several angstroms. To achieve this degree of close proximity, the two surfaces must become at least partially dehydrated as water bound to the membrane enhances polar repulsion of membranes. Next, one or both bilayers must be destabilized in some way, inducing a localized rearrangement of the bilayers. If both bilayers are destabilized, an aqueous bridge is formed and the cytoplasmic contents of both cells mix.
Destabilization of membranes can occur as the result of physical stress (e.g., electrofusion) or chemical interference (e.g., polyethylene glycol). Electrofusion utilizes short pulses of electricity to mechanically disrupt the lipid bilayer of a cell to form pores and if two disrupted membranes come into contact, cell fusion may occur [29] . Unfortunately, this process is toxic and the cells must be in contact with one another at the time the electric field is administered. Laser trapping prior to electrofusion has been used to more effectively position fusion partners, however the process is low throughput and cytotoxic [30, 31] . A less toxic, but also less effective and less reproducible approach uses polyethylene glycol (PEG) [32, 33] . The exact mechanism of PEG-induced fusion is unknown but is theorized to be due to either local dehydration leading to unfavorable molecular packing of the bilayer or to dehydration of the "water shell" near the lipid bilayer, causing the water molecules between cells to be displaced, thereby forcing the two membranes together and subsequently fusing the cells [34] . This technique has proven useful, but fusion only occurs during the time of administration of PEG, thus cell delivery with PEG would induce fusion immediately and nonselectively. A mechanism that would better regulate fusion either to specific cells or specific regions within tissues is necessary to study fusion in vivo.
In nature, destabilization of cell membranes and subsequent membrane fusion utilizes the activation of specific integral membrane proteins, termed fusogens. The primary source of information about fusogen architecture, receptor binding, and activation are from viruses. The most extensively characterized fusogens are influenza hemagglutinin (HA) and human immunodeficiency virus type 1 envelope protein (HIV-1 Env). Both fusion peptides are hydrophobic and require proteolytic cleavage, but HA is activated under acidic pH during endocytosis, while HIV-1 Env fuses at neutral pH (reviewed in [35] [36] [37] ). Less well-studied are the fusogens required for eukaryotic cell fusion such as the fusion of osteoclasts, myoblasts, and trophoblasts. The greatest challenge has been establishing which proteins are true fusogens and which proteins facilitate fusion by placing cells in close proximity. Many putative fusogens have been shown to be supporting proteins (i.e., essential for adhesion or migration). The identification of true fusogens is so difficult that groups have proposed ranking schemes to clarify the nature and function of these proteins [28] . Because putative fusogens for spontaneous stem cell fusion have not been identified, developing alternative strategies to direct stem cell fusion could augment our understanding of the biological impact of such fusion.
Here we utilize viral machinery from vesicular stomatitis virus (the glycoprotein, VSV-G), of the Rhabdoviridae family, to induce heterotypic fusion between human MSCs and mouse CMs in vitro and in an in vivo mouse model of myocardial infarction. Following MSC-CM fusion, we tracked the phenotype and morphology of fusion products for one week in vitro and 3 weeks in vivo. VSV-G was selected because it does not require proteolytic cleavage, is the sole mediator of receptor binding and fusion, and is pH dependent [38, 39] . In particular, VSV-G does not require facilitating proteins to either dock to the host membrane prior to fusion, or enzymes to prompt the activation of the fusogen. Furthermore, the pH dependence of VSV-G is advantageous as the local heart pH after acute ischemic injury [40] [41] [42] is within the acidic range needed to initiate a conformational change in VSV-G [38, 39] . In this way, selective activation of VSV-G on transfected MSCs (vMSCs) at the site of myocardial injury should induce local fusion in situ, thereby increasing donor cell engraftment and integration within the tissue and potentially facilitate cardiac differentiation.
MSCs derived from human embryonic stem cells (MSCs from WA-09, a gift of Dr. Peiman Hematti) and HL-1 cardiomyocytes (a gift of Dr. William Claycomb) were expanded and cultured as previously described [43, 44] . Briefly, MSCs were cultured on a 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA) pretreated flask containing α-minimum essential medium-(MEM-) complete. Alpha-MEM-complete consisted of α-MEM (Invitrogen, Carlsbad CA, USA), 10% fetal bovine serum (Hyclone, Logan UT), 0.1 mM nonessential amino acids (Invitrogen), and 2 mM Lglutamine (Invitrogen). MSC cultures were allowed to grow to 60-70% confluency and were replated at a concentration of 1,500 cells/cm 2 . CMs were cultured on fibronectin/gelatin (1.25 mg fibronectin/100 mL 0.02% gelatin) (Sigma-Aldrich) pretreated flasks containing Claycomb-complete. Claycombcomplete medium was comprised of Claycomb medium (SAFC Biosciences, St. Louis, MO, USA), 10% fetal bovine serum qualified for CMs (SAFC Biosciences), 100 U/mL: 100 μg/mL penicillin-streptomycin (Lonza, Walkersville, MD, USA), 0.1 mM norepinephrine (Sigma-Aldrich), and 2 mM L-glutamine (Invitrogen). CMs were passaged at 100% confluence and split 1 : 2. Experiments were performed using passages 7-10 and 60-110 for MSCs and CMs, respectively. All cultures were maintained at 37 • C in 5% CO 2 .
MSCs were transfected with a pCVSV-G-1 plasmid [45] that encodes VSV-G under a CAG promoter using the Neon Transfection System (Invitrogen), according to manufacturer's protocol. Briefly, 5 × 10 5 cells were transfected with 2 μg of plasmid with one 1,300 V pulse for 20 msec and plated in 6-well plates. To determine transfection efficiency, electroporated cells were cultured for 24 h and immunocytochemistry (ICC) was performed to detect VSV-G protein expression. Briefly, cells were washed with two rinses and two incubations of 1X PBS. Cell fixation was performed with 4% PFA, followed by another set of washes, and probed with the 1 : 50 dilution of FITCconjugated anti-VSV-G antibody (GeneTex, San Antonio TX, USA) in 3% BSA for 60 min. Cultures were washed a final time and mounted in DABCO/DAPI mounting medium (2. The Quantum MESF kit consists of 5 populations of microspheres with increasing surface-labeled fluorochrome, which have been standardized to specific concentrations of pure fluorophore per microsphere. Each population was analyzed via flow cytometry and a standard curve was generated by plotting population (i.e., concentration of fluorophore per microsphere) versus intensity. QuickCal software was used to verify the linearity of the standard curve. Next, vMSCs and corresponding control populations were labeled with an anti-VSV-G-FITC antibody and analyzed via flow cytometry. Using the standard curve and the measured intensity value for vMSC populations and corresponding controls, the number of fluorophores per cell was determined. This value was divided by the average number of fluorophores (4.2) that bind to a single antigen to determine the number of proteins expressed per cell. Ten thousand cells and three replicates were analyzed per population. Populations included vMSCs with anti-VSV-G antibody, MSCs with anti-VSV-G antibody and vMSCs without antibody.
To determine if vMSCs fuse more readily with cardiomyocytes than untreated MSCs, vMSCs and MSC controls were cocultured with CMs and analyzed for incidence of fusion. To distinguish cell types in cocultures, MSCs and CMs were stained with 1 μm Cell-Tracker Green CMFDA and 20 μm Red CMTPX (Molecular Probes Eugene, OR, USA), respectively, according to the manufacturer's protocol. Following labeling, 5 × 10 5 CMs were plated and cultured for 4 h followed by the addition of 1.5 × 10 5 MSCs or vMSCs per well in 6-well plates (BD Biosciences). After 14 h of coculture, suspensions were washed with 1X PBS and then bathed for 2 min in fusion media [46] of varying pH (i.e., pH 5.5, 6.5, or 7.5 that correspond to active and inactive forms of the VSV-G fusion protein) adjusted with HCl. For long-term characterization of fusion products, medium was changed 1 day and 4 days after coculture.
Cocultures of CMs with MSCs or vMSCs were maintained in culture medium for 4 h after incubation with fusion medium, followed by imaging and flow cytometry. Images were acquired with a 20X UPlanFluor objective (NA = 0.5), FITC and Texas Red filters, on an IX71 inverted deconvolution fluorescence microscope (Olympus Center Valley, PA, USA) and analyzed with Slidebook software (Intelligent Imaging Innovations Denver, CO, USA) and ImageJ (Fiji; open source software, http://pacific.mpi-cbg.de/wiki/index.php/Fiji). Images were normalized using unstained controls. Cells were analyzed at the UWCCC Flow Cytometry Facility on a FACSCalibur flow cytometer (BD Biosciences). Events were live/dead gated with forward scatter and side scatter plots. Fusion products were quantified by gating the region positive for FL1 and FL2 channels, corresponding to CellTracker Green CMFDA and Red CMTPX, respectively.
MSCs or CMs in monolayer were stained for proteins characteristic of MSCs (CD73, CD90, and CD105), as well as proteins characteristic of CMs (sarcomeric myosin (MF20)). Cell cultures were fixed with 4% paraformaldehyde for 10 min, followed by two washes with phosphate buffered saline (Fisher Scientific). Cells were probed with goat anti-CD73 (V-20, Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-CD90 (RB3970, Abgent, San Diego, CA, USA), goat anti-CD105 (GKY02, R&D Systems, Minneapolis, MN, USA), and mouse anti-MF20 (IgG2b, Developmental Studies Hybridoma Bank, Iowa City, IA) diluted 1 : 25, 1 : 50, 1 : 50, and no dilution, respectively, in diluting buffer (5% BSA (Fisher Scientific), 0.02% NaN3-(Acros Organics) in phosphate buffered saline (Fisher Scientific)) and incubated for 30 min at room temperature or overnight at 4 • C, followed by incubation with fluorescent secondary antibodies: donkey anti-goat Alexa Fluor (AF488, Invitrogen), goat anti-rabbit Alexa Fluor (AF647, Invitrogen), and donkey anti-mouse (AF546, Invitrogen) at a 1 : 200 dilution in preadsorption solution (90% diluting buffer, 5% human serum (Pelfreez, Brown Deer, WI, USA), and 5% mouse serum (Equitech-Bio, Inc, Kerrville, TX, USA)) for 45 min at room temperature. Samples were counterstained with DABCO/DAPI mounting solution. Fluorescence emission was detected on an IX71 inverted deconvolution fluorescence microscope (Olympus). Images were acquired with a 20X UPlanFluor objective (NA = 0.5), and analyzed using MSC-CM or vMSC-CM cocultures were probed with antibodies against CM marker (MF20) and MSC marker (CD105) to evaluate the morphology of fusion products and the phenotype of cells within coculture. Positive events for fusion were calculated as the percentage of CD105 and MF20 positive cells containing a nucleus divided by total number of nuclei obtained from analysis of at least eight optical fields per sample. Fields (3-10 fields) were selected based on cell number (minimum of 3 cells) and position within the wells (center of wells) n = 2.
Myocardial infarction was induced in C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) by left coronary artery ligation as previously described [47, 48] and as is routinely performed in the University of Wisconsin Cardiovascular Physiology Core Facility. All animal procedures were performed in accordance with the guidelines of the American Association for Laboratory Animal Science and the University of Wisconsin-Madison Animal Care and Use Committee.
to the Murine Myocardium. TissueMend (TEI Biosciences) was prepared and cells were seeded as previously described [48] . Briefly, TissueMend matrices (2 mm × 2 mm × 0.8 mm) were placed in wells of 24-well plates containing α-MEM-complete culture medium. Following electroporation, vMSCs were seeded on the TissueMend sections at a concentration 3 times greater than MSCs (cell control due to 30% cell viability after electroporation, yielding 1 × 10 6 cells/mL. Medium was changed at 24 and 48 h, at which point the TissueMend matrix containing ∼2.3 × 10 4 MSCs, vMSCs, or unseeded (matrix control) was tacked to the myocardium at each corner of the matrix. The matrix was placed such that it was in contact with both the infarct and the peri-infarct regions of the myocardium [48] .
Murine hearts were harvested three weeks after matrix implantation to assess the occurrence and, if detected, the frequency of in vivo fusion. Hearts were bisected longitudinally through the matrix. The tissues were immediately placed into 10% buffered formalin (pH = 7.2; Fisher Scientific) for 24 h followed by 24 h of fresh 10% buffered formalin, and a final 24 h of 70% ethanol. Samples were further processed for paraffin embedding and sectioning as previously described [49] . Fluorescent in situ hybridization (FISH) tissue digestion kit with all human centromere probe (red) and all mouse centromere probe (green) (Kreatech, Amsterdam, the Netherlands) was performed on sections to detect fusion events. Samples were processed by the Cytogenetics Laboratory (WiCell Research Institute, Madison, WI, USA) according to manufacturer's protocol. Briefly, slides with paraffin embedded sections were baked for 4 h at 56 • C. Specimens were incubated with pepsin for 70 min for tissue digestion prior to sequential hybridization of the human probe followed by mouse probe. Images were acquired with a 60X UPlanSApo (NA = 1.35 Oil), DAPI, Green, and Orange filters, on an Olympus BX41 Upright Fluorescence Microscope (Olympus Valley, PA, USA), and analyzed with FISHView Version 5.5 software (Applied Spectral Imaging, Vista CA). Fusion events were defined as nuclei with positive staining for both human centromeres (red) and mouse centromeres (green). The frequency of fusion events was reported as the number of fusion events per total nuclei for a given region of the heart tissue: myocardium (myo), myocardial infarct (MI), border region (border), within Tissuemend patch (TM), and in the healthy myocardium (healthy). Five to twelve fields were selected for each location and the number of hearts analyzed were, n = 1 for TM only and n = 3 for TM + vMSC.
For comparison of VSV-G expression, fusion frequency, and fusion product morphology versus controls, a normal distribution was assumed and oneway analyses of variance and Student's t-test were used. Data were analyzed with Microsoft Excel (Microsoft, Redmond, WA, USA).
MSCs were induced to express VSV-G via transfection by electroporation. Low transfection efficiency would limit the ability of VSV-G to promote fusion and so VSV-G expression on MSCs was determined following electroporation. Twenty-four hours after transfection control MSCs and MSCs transfected with VSV-G (vMSCs) were probed with an anti-VSV-G antibody conjugated to fluorescein isothiocyanate (FITC) and visualized with fluorescence microscopy to determine the percentage of cells expressing VSV-G. The average transfection efficiency was 32% ± 5% (n = at least 6 optical fields per sample per trial for 3 trials, Figure 1(a) ). Since vMSCs will be harvested for in vivo studies, we also assessed VSV-G expression via flow cytometry after removal from culture plates with trypsin. We found expression of VSV-G plummeted to 5% ± 2% (n = 1 replicate per sample per trial for 3 trials, Figure 1(b) ). This is perhaps not surprising as others have reported decreased stability of VSV-G with trypsin treatment [50, 51] . Trypsin is a serine protease that cleaves carboxyl groups on the cell surface to remove cells from a culture surface. VSV-G is a cell surface protein that would be exposed to the dissociation reagent [52] . The disruption to VSV-G by trypsin was corroborated by evaluating the number of VSV-G proteins per cell. The administration of trypsin significantly reduced the number of VSV-G proteins on the cell surface of vMSCs (Figure 1(d) ). Thus we replaced trypsin with Accutase, a mixture of proteases and collagenases that has been shown to improve cell viability compared to trypsin [53] . With Accutase treatment, the average number of cells expressing VSV-G after cell harvest was 21% ± 7%, a significant improvement over treatment with trypsin and at a level high enough to discern whether expression of VSV-G can impact MSC-CM fusion (n = 1 replicate per sample per trial for 3 trials). Fusogens such as VSV-G are most potent on the cell surface when there are adequate amounts of protein to facilitate fusion, but a low enough amount to avoid immune responses. Thus VSV-G protein expression per cell was determined using a Quantum MESF kit (Bangs Laboratories, Inc.), on the FACSCalibur. Using this method, the average number of VSV-G proteins per cell was 8 × 10 4 ± 2 × 10 4 with trypsin and 1 × 10 6 ± 8 × 10 3 with Accutase (n = 1 replicate per sample per trial for 3 trials, Figure 1(c) ). Thus, all further experiments were performed using Accutase as the dissociation reagent to prevent VSV-G cleavage.
To determine whether MSCs expressing VSV-G are better equipped to fuse with CMs than unmanipulated counterparts, vMSCs or MSCs were cocultured with CMs. To distinguish cell types in cocultures, MSCs and vMSCs were stained with Red Cell-Tracker, while CMs were stained with Green CellTracker fluorescent probes prior to being combined. Since VSV-G undergoes a conformational change from its inactive form to its active form at pH < 6.2 [54, 55] , cocultures were briefly incubated (2 min) with fusion medium of pH = 5.5. Image analysis of vMSC-CM cocultures treated with fusion medium of pH = 5.5 revealed cells with colocalization of green and red fluorescence, indicating fusion events, while MSC-CM cocultures under the same pH condition exhibited limited colocalization (Figure 2(a) ). To accurately assess the amount of cell fusion, cocultures were harvested 24 hours after seeding and analyzed via flow cytometry (double positive cells correspond to fusion events). vMSCs treated with acidic medium (pH 5.5) had significantly higher rates of fusion with CMs (4.7% ± 1.1%) than MSCs treated in the same way (i.e., spontaneous fusion, 1.4% ± 0.2%) (P < 0.05) (Figure 2(b) ). Further, the percentage of fusion products identified in vMSC-CM cocultures exposed to fusion medium of pH = 6.5 or 7.5 (maintaining VSV-G in the inactive form) did not differ from MSC-CM cultures (n = 3 replicates per sample per trial for 3 trials, Figure 2 (c)).
Many studies have demonstrated that stem cell programming is influenced by the microenvironment [56] [57] [58] . To determine whether the phenotypic fate of vMSC-CM fusion products could be regulated by the microenvironment, following treatment with fusion media, we cultured vMSC-CM fusion products under either MSCspecific or CM-specific culture conditions and examined the incidence of fusion and morphology of MSC-CM fusion products. At days 5 and 7 following the induction of cell fusion, cocultures were probed with CM and MSC specific antibodies (anti-MF20 and anti-CD105, respectively, n = 1 replicate per sample per trial for 3 trials). At day 5, vMSC-CM cocultures contained a relatively high number of cells that expressed both MF20 and CD105 and the percentage of MF20 + /CD105 + cells relative to the total cell number was significantly greater than that of MSC-CM cocultures for both culture conditions (P < 0.005) (Figure 3(a) ). Of note, the percentage of MF20 + /CD105 + cells was much higher than the percentage of double positive cells detected using CellTracker dyes and flow cytometry (Figure 2(b) ). This could reflect the loss of VSV-G sustained by cell harvest, the different analytical approach (i.e., flow cytometry versus image analysis) and/or the behavior of fusion products between day 1 and day 5 (i.e., proliferation). By day 7, the percentage of MF20 + /CD105 + cells decreased to levels not statistically different from controls for both culture conditions. At the same time, the number of cells expressing MF20 alone increased substantially for both culture conditions. The change in percentage of MF20 + /CD105 + cells from day 5 to day 7 could reflect death of fusion products, or programming of the MSC fusion partner to a cardiomyocyte phenotype or both. If death of fusion products occurred, one would expect unfused CMs and MSCs to proliferate to fill the voids of the culture space. Interestingly, only the CM population increased from day 5 to day 7 and at rates significantly higher than that of control cultures, suggesting at least a portion of fusion products were maintained, and ultimately adopted a cardiomyocyte-like phenotype. This result was observed independent of the culture conditions. Of note, this experimental approach does not exclude the possibility that metaplasia rather than fusion occurred, that is, MSCs differentiate into CMs as a consequence of soluble factors in the coculture medium and maintain (at least transiently) expression of each cell type. However, MF20 + /CD105 + cells were rare in MSC-CM cocultures, suggesting metaplasia alone cannot account for coexpression of MF20 + /CD105 + or subsequent loss of MF20 + /CD105 + cells. In addition, MF20 + /CD105 + cells exhibited two distinct morphologies; some were long and spread, displaying MSC-like morphology (MSC medium = 16.59% ± 6.32%; CM medium = 14.03% ± 1.59%) while the majority (P < 0.05) were round and cobblestone-like, indicative of CM-like morphology (MSC medium = 80.49%±10.45%; CM medium = 85.97%± 1.60%) (Figures 3(b) and 3(c), Supplementary Figure 1B) . These results further support the possibility that CM nuclear material and cytoplasmic elements direct programming of MSC-CM fusion products independent of culture conditions.
To determine whether MSCs expressing VSV-G could fuse with cardiac cell types in vivo, vMSCs were delivered to the damaged myocardium via a TissueMend patch. We have previously demonstrated that MSCs delivered in this way are maintained in the patch and in the tissue between the patch and the myocardium up to 3 weeks after delivery at higher percentages than with conventional delivery modalities [48] . Furthermore, Laflamme et al. have found one of the major factors for cell loss during transplantation is anoikis [59] , and thus providing anchorage support to transplanted cells increases viability and retention. In this study, we sought to determine whether VSV-G expressing MSCs (donor) would be able to migrate to the damaged myocardium and fuse with recipient cardiac cell types. Thus, one day following induction of infarction via ligation of the left anterior descending artery, a patch containing vMSCs was applied to the heart in contact with healthy and damaged tissue. Three weeks after cell transplantation, heart excision, and histology were performed on left ventricular tissue as previously reported [48] . Histological sections were probed using FISH for human-specific and mouse-specific centromeres and all nuclei containing both probes were considered fusion products. Human cells were found in the TissueMend patch and in the "border region" (the area between the patch and the myocardium). Donor-host cell fusion was evident in the TissueMend patch, the border zone, and in the infarcted myocardium of hearts receiving TissueMend with vMSCs. No human cells or fusion products were found in the healthy cardiac tissue of hearts receiving TissueMend with vMSCs. In addition, no human cells or fusion products were found in the TissueMend patch, border zone or infarcted myocardium of hearts receiving TissueMend only. In regions of hearts receiving TissueMend with vMSCs and selected for high density of fusion events, the frequency of cell fusion relative to the total number of nuclei in a given region was 22% ± 16%, TissueMend patch (n = 3 hearts, 12 fields); 14%±10%, border zone (n = 3 hearts, 5 fields); 19% ± 10%, infarcted myocardium (n = 3 hearts, 8 fields). Though these levels represent the maximum amount of fusion per region, they are substantially higher than those previously reported for
Merge CMs 
Fusion of transplanted stem cells with recipient cardiomyocytes has been observed in murine [13, 60] and porcine model systems [49] . But since these first observations, few have sought to unravel the mechanisms that govern stem cell fusion or to study the implications of cell fusion for stem cell programming. Lack of study reflects the overwhelming opinion that cell fusion occurs too infrequently to be of relevance for stem cell programming and, by corollary, for tissue repair. However, this opinion fails to appreciate the possibility that (1) detection methodologies may be insufficient to accurately gauge the contribution of cell fusion following stem cell transplantation and/or (2) that we might be able to control or increase the frequency of cell fusion to more effectively induce programming of stem cells following transplantation. We have begun to explore this second possibility by co-opting the well-described fusion machinery of viruses. We find that mesenchymal stem cells modified to express viral fusogen VSV-G are more apt to fuse with cardiomyocytes in a pH-dependent manner. vMSC-CM fusion products formed in this way are prone to adopt cardiomyocyte phenotype and morphology. In addition, vMSCs delivered to the myocardium of mice following infarction can fuse with resident cardiac cell types at rates much higher than previous reports of spontaneous fusion [13, 61] and are more apt to fuse at the site of infarction than in the healthy myocardium.
Increasing the frequency of MSC-CM cell fusion will aid in the study of cell fusion in vitro and may improve the therapeutic benefit of MSCs in vivo. One way that the therapeutic benefit may be improved is via induction of programming of MSCs to a cardiomyocyte fate. Differentiation of MSCs into CMs can be initiated in vitro via soluble factors including 5-azacytidine [62] [63] [64] or with exposure to insoluble factors including laminin [65] . However, functional differentiation of MSCs to cardiomyocytes has only been accomplished to date via cell fusion with mature cardiomyocytes. This result has been demonstrated in vitro [66] and in vivo wherein MSC-CM fusion products take on a cardiomyocyte morphology, express cardiomyocyte markers, and couple to adjacent cardiomyocytes [60] . Here we find that when MSC-CM fusion is induced with viral fusogens, the CM fusion partner is dominant in that the majority of fusion products (regardless of medium type) adopt a CM-like morphology and maintain expression of MF20 and lose CD105. These data further support the exciting possibility that induction of fusion with viral fusogens could enhance MSC programming to a CM fate in vivo. Of note, the CMs utilized here are HL-1 CMs. This cell line was used to enable largescale and long-term studies. However, the heterogeneity and immortal nature of these cells may account for the seeming dominance of the CM phenotype and future studies will utilize primary fetal cardiomyocytes or induced pluripotent stem cell-derived cardiomyocytes.
Our results suggest that the differentiation of MSCs to a CM fate can be promoted by cell-cell fusion. However, in certain circumstances in vitro, MSC-CM fusion products can reenter the cell cycle and proliferate suggesting cell-cell fusion can also promote reprogramming of the CM [67] [68] [69] . Proliferation of fusion products may be as advantageous for cardiac tissue repair as differentiation of functional cell types since more cells could be produced to replace lost cells. In addition, recent evidence has demonstrated that MSC-CM fusion includes mitochondrial exchange, which is essential for somatic reprogramming [69] . Understanding cell-cell fusion in conjunction with mitochondrial preservation may provide alternate, simple, and direct mechanisms to rescue cells following ischemia-induced damage. There is evidence indicating that the fusion product's proliferative capacity is regulated by the stem cell while the developmental direction is dictated by the somatic cell [70] [71] [72] , and the combination of both outcomes presented herein are means to repopulate the myocardium for functional improvement.
While we have utilized vMSCs to both understand and exploit the physiological role of MSC-CM fusion, induction of fusion of another stem cell, progenitor, or even mature cell types may augment our ability to repopulate and repair the damaged myocardium [59, [73] [74] [75] [76] [77] [78] [79] . In the case of mature or progenitor cell transplantation, the induction of fusion may be less beneficial from a differentiation standpoint and more beneficial from an engraftment or retention standpoint. One of the primary challenges for stem cell delivery is the ∼90% cell loss after transplantation [80] [81] [82] that has prompted the development of new methods to deliver and maintain cells in the heart [48, 83, 84] . This is particularly problematic for cardiac therapy as the heart is mechanically active, rapidly flushing cells from the intended target region. If stem cells transiently express a viral fusogen, they might rapidly adhere and so be maintained long term in the heart. The added advantage of pH sensitive fusogens, such as VSV-G, is the ability to control activity such that cells only fuse at pH lower than 6.5. This has major implications for inducing temporally (the window during ischemia) and spatially (the ischemic region) regulated fusion in vivo. In fact, vMSCs delivered to the heart were found in the patch and in damaged myocardium fused with mouse cells. The ability for VSV-G to induce fusion in the patch may be due to close proximity to the ischemic region, causing the environment to be more acidic or by the remodeling of the collagen patch [48] . Collagen remodeling has been shown to occur via MSC secretion of matrix metalloproteinases (matrixins), serine proteases, and cysteine proteases [85] . While matrixins are active at neutral pH, serine and cysteine proteases are active at acidic pH, indirectly demonstrating cells are able to make the microenvironment acidic [86] . Taken together, the induction of cell fusion in the heart could exert functional benefit via multiple mechanisms.
A primary limitation of this approach is introducing viral machinery to an already damaged recipient. The entire virion, VSV, is known to be immunogenic and, at high enough concentrations, is lethal to mice [87] . Purified VSV-G or VSV-G reconstituted in lipid bilayers administered to in vitro cell culture is mitogenic (>0.8 μg/mL) [88] . Interestingly, if the lipid concentrations were increased, while VSV-G concentration was held constant, the mitogenicity decreased, suggesting that the spacing of VSV-G in the membrane plays a role. Confirming the importance of VSV-G arrangement, Ochsenbein et al. demonstrated that 1,000 times more antibody is produced by C57BL/6 mice against highly organized VSV-G on the nucleocapsid of intact VSV versus poorly organized VSV-G in micelles [89] . The amount of viral proteins we delivered (based on the mass of the protein [39] , the proteins expressed per cell combined with the number of cells delivered) is 7 orders of magnitude below the reported amount to elicit an immune response [88] and we express only the fusogen and not the entire virion. Even if methods were developed to increase expression levels per cell and/or in combination with high numbers of cells, spacing could be evaluated to avoid immune responses. However, based on the reported concentration required to elicit a response, delivery of vMSCs as prepared in this study would not trigger a response.
While vMSCs may not be immunogenic, transfection itself may cause adverse genetic effects. For instance, stable transfection with most viral systems causes integration of the gene at a random site in the genome [90] [91] [92] . When mutagenesis occurs, integration may occur at a site that interferes with cells ability to regulate itself, resulting in deregulation of proliferation and tumorigenesis [93, 94] . In addition to experimental evidence of malignancy, this has been seen clinically ( [95, 96] , reviewed in [97] ). Here, transfection is largely transient and only rarely integrates into the genome. Clinical use would require further safeguards, perhaps including liposomal delivery of the protein.
The data presented support the utility of VSV-G-mediated fusion to study the effects of stem cell fusion on cell reprogramming and functional improvement of tissues including the heart. Future studies may also employ VSV-G to rescue damaged cells of other ischemic tissues in the body, or even selectively target cells for destruction. For example, the microenvironment of tumors and the overactive osteoclasts in Paget's disease are below the pH threshold necessary to activate the conformational change in VSV-G. Local administration of VSV-G in liposomes containing toxic factors or highly acidic pH to this microenvironment may fuse with these poorly regulated cells and dampen their detrimental effect.
This paper was supported by funding from the National Institutes of Health (HL089679). Design Novel Dual Agonists for Treating Type-2 Diabetes by Targeting Peroxisome Proliferator-Activated Receptors with Core Hopping Approach Owing to their unique functions in regulating glucose, lipid and cholesterol metabolism, PPARs (peroxisome proliferator-activated receptors) have drawn special attention for developing drugs to treat type-2 diabetes. By combining the lipid benefit of PPAR-alpha agonists (such as fibrates) with the glycemic advantages of the PPAR-gamma agonists (such as thiazolidinediones), the dual PPAR agonists approach can both improve the metabolic effects and minimize the side effects caused by either agent alone, and hence has become a promising strategy for designing effective drugs against type-2 diabetes. In this study, by means of the powerful “core hopping” and “glide docking” techniques, a novel class of PPAR dual agonists was discovered based on the compound GW409544, a well-known dual agonist for both PPAR-alpha and PPAR-gamma modified from the farglitazar structure. It was observed by molecular dynamics simulations that these novel agonists not only possessed the same function as GW409544 did in activating PPAR-alpha and PPAR-gamma, but also had more favorable conformation for binding to the two receptors. It was further validated by the outcomes of their ADME (absorption, distribution, metabolism, and excretion) predictions that the new agonists hold high potential to become drug candidates. Or at the very least, the findings reported here may stimulate new strategy or provide useful insights for discovering more effective dual agonists for treating type-2 diabetes. Since the “core hopping” technique allows for rapidly screening novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties, it has not escaped our notice that the current strategy along with the corresponding computational procedures can also be utilized to find novel and more effective drugs for treating other illnesses. Diabetes mellitus is a group of metabolic diseases that has been classified as a disease of glucose overproduction by tissues without enough insulin production, or a disease resulting from cells not responding to the insulin in human body [1] . Type-2 diabetes is the most common type among all the diabetes mellitus forms. The risk of developing type-2 diabetes (T2D) increases with age, obesity, cardiovascular disease (hypertension, dyslipidaemia), lack of physical activity, and family history of diabetes. Increasing dramatically in the US and worldwide, type-2 diabetes has reached epidemic scale. There are nearly 50 million individuals (US) and 314 million individuals (worldwide) with the metabolic syndrome [2] . People suffering from overweight or obesity are of huge risk for developing T2D.
Peroxisome Proliferator-Activated Receptor (PPAR) has drawn increased attention as a drug discovery target by regulating glucose and lipid metabolism [3] . PPAR, and its subtypes PPARa and PPARc, belong to the superfamily of nuclear receptors that function as transcription factors activated by several ligands. PPARs played a vitally important role in treating obesity, atherogenic dyslipidemia, hypertension, and insulin resistance as main therapeutic targets [4] . The primary function of PPARa is to act as regulator responding to transport and degradation of free fatty acids as well as reverse cholesterol transport by peroxisomal and beta-oxidation pathways [5] . A class of lipid-lowering drugs, such as fenofibrate and gemfibrozil, specially activate PPARa [6, 7, 8] . PPARc played a significant role in transcriptionally regulating lots of physiological pathways, including adipocyte differentiation and glucose homeostasis [9] . Thiazolidinediones (TZDs) are a class of the antidiabetic drugs, which act by activating the special PPARc [10] . If used alone, although each of the antidiabetic drugs could enhance the insulin sensitivity and hence lower glucose or fatty acid levels in type-2 diabetic patients [9] , some side effects would be caused, such as weight gain, fluid accumulation, and pulmonary edema [11] .
Recently, new dual agonists have received considerable attention for developing powerful drugs against diabetes. The strategy of dual agonists was aimed to treat both insulin resistance and dyslipidemia [12] . A critical challenge for developing dual agonists is how to identify the receptor subtype selectivity ratio [13] .
Many studies have indicated that computational approaches, such as structural bioinformatics [14, 15] , molecular docking [16, 17] , pharmacophore modelling [18] , QSAR techniques [19, 20, 21, 22, 23, 24] , as well as a series of user-friendly web-server predictors developed recently, such as GPCR-2L [25] for identifying G protein-coupled receptors and their types, En-zClassPred [26] for predicting enzyme class, iLoc-Euk [27] and iLoc-Hum [28] for predicting subcellular localization of eukaryotic and human proteins, NR-2L [29] and iNR-PhysChem [30] for identifying nuclear receptors and their subfamilies, and HIVcleave [31] for predicting HIV protease cleavage sites in proteins [32, 33] , can timely provide very useful information and insights for drug development. The software of ''Core Hopping'' [34] is another very powerful and cutting-edge computational technique that is particularly useful for de novel drug design [35] .
Encouraged by the aforementioned researches in successfully utilizing various computational approaches for drug development, the present study was initiated in an attempt to screen the fragment database for finding new PPAR dual agonists for treating type-2 diabetes. To realize this, the techniques of ''core hopping'' with glide docking [34, 36] as well as molecular dynamic simulation were utilized to analyze the binding interactions between the agonist and PPARs in hoping that the findings thus obtained may provide useful insights for developing new and powerful agonists against diabetes mellitus.
The L-tyrosine analogue GW409544 was obtained by modifying the structure of farglitazar, a dual agonist for both PPARa and PPARc [37] . The main difference between GW409544 and farglitazar is that the former contains a vinylogous amide as the Ltyrosine N-substituent [37] . That is why we chose GW409544 as a starting template structure for designing the new PPAR dual agonists.
The representative complex crystal structures of PPARa (PDB ID 1k7l) and PPARc (PDB ID 1k74) with the same ligand GW409544 [37] were download from the PDB Bank [38] , and were to be used for the molecular modelling studies.
All the calculations were carried out on Dell Precision TM T5500 computer with Schrodinger software package [34, 36] and Desmond 2.4 [39] .
The proteins with PDB codes 1k71 and 1k74 were chosen for modeling. In addition to the available knowledge of their 3D (dimensional) structures, the reasons of selecting the two proteins as receptors are as follows. (1) The two proteins contain the same ligand GW409544 as PPARa and PPARc do, and their binding affinities with the ligand are also quite similar; however, the former selectivity is about 10-fold weaker than the latter [37] . (2) The source organism of both PPARa and PPARc was from human.
In the process of preparing receptors for modelling, the protein preparation facility [40] was used that included the operations of assigning bond orders, adding hydrogen, treating metals, treating disulfides, deleting waters and alleviating potential steric clashes, adjusting bond order, building missing heavy atom and formal charges, as well as minimizing energy with the OPLS2005 force field [41] and refining the protein by imposing the 0.3 Å RMSD limit as the constraint.
The protein binding-site was identified by the SiteMap tool embedded in Schrodinger Suite 2009 (www.schrodinger.com) as described in [42, 43, 44] . The binding-site encompassed the ligand GW409544, which was observed in the crystal structures of both PPARa (1k71) and PPARc (1k74) as a ligand.
The information of the binding pocket of a receptor for its ligand is very important for drug design, particularly for conducting mutagenesis studies [14] . In the literatures, the binding pocket of a protein receptor to a ligand is usually defined by those residues that have at least one heavy atom (i.e., an atom other than hydrogen) with a distance #5 Å from a heavy atom of the ligand. Such a criterion was originally used to define the binding pocket of ATP in the Cdk5-Nck5a* complex [45] that has later proved quite useful in identifying functional domains and stimulating the relevant truncation experiments [46] . The similar approach has also been used to define the binding pockets of many other receptor-ligand interactions important for drug design [15, 17, 47, 48] . In this study, we also used the same criterion [45] to define the binding pockets of proteins 1k7l and 1k74 for the ligand GW409544. A close-up view for the protein-ligand interaction at the binding pocket thus defined is shown in Fig. 1 , where panel A is for the interaction between PPARa (1k71) and GW409544, while panel B for the interaction between PPARc (1k74) and GW409544.
Because the natural ligands of PPARs are fatty acids, the binding site of PPARs is almost hydrophobic. Several hydrophobic interactions with three arms of the Y-shaped ligand binding to the site are taken as the key point for designing the new PPARs agonist [49] . The PPAR binding site is composed of three arms, namely Arm I, Arm II, and Arm III, as explicitly marked in Fig. 1 . The first arm has mainly polar character including the AF2 (transcriptional activation function 2) helix indicated by red ribbon. The hydrophilic head group of the PPAR ligands forms a network of hydrogen bonds with AF2 of Arm-I; while the hydrophobic tail of PPAR agonist is either interacts with Arm II or Arm III. The network hydrogen bonds forms an important conformation for AF2-helix to generate a charge clamp, thus reducing the mobility of AF2 via binding a ligand and hence make it able to regulate the gene expression [4] . The drug-like database and the fragment database derived from ZINC [50] were used for virtual screening and core hopping searching, respectively.
Many useful insights for drug design could be gained via molecular docking studies (see, e.g., [14, 17, 51, 52] ). To acquire even more useful information for drug design, a new docking algorithm called ''Core Hopping'' [36] was adopted in this study that is featured by having the functions to perform both the fragment-based replacing and molecular docking.
Core Hopping [36] is a very powerful and cutting-edge technique for de novel drug design because it can significantly improve the binding affinity of the receptor with its ligands, e.g., GW409544 (Fig. 2) in the current study. The binding conformation thus obtained will be taken as an initial structure for further optimization by searching the fragment database to find the optimal cores that are attached to other parts of the template.
During the process of core hopping, the 1st step is to define the possible points at which the cores are attached. It is performed in the ''Define Combinations'' step from the Combinatorial Screening panel in Schrodinger2009 (www.schrodinger.com). The 2nd step is to define the ''receptor grid file'', which was done in the ''Receptor Preparation'' panel. The 3rd step is the cores preparation that was operated with the ''Protocore Preparation'' module to find the cores attaching to the scaffold using the fragment database derived from ZINC [50] . The 4th step is to align and dock the entire molecular structure built up by the core and scaffold. The cores are sorted and filtered by goodness of alignment and then redocked into the receptor after attaching the scaffold, followed by using the docking scores to sort the final molecules.
As the products of the core hopping operation, a total of 500 chemical compounds were prepared with the LigPre module [53] , which consists of the procedures of generating possible states by ionization at target pH 7.062.0, desalting, retaining chiralities from 3D structure and geometry minimization with the OPLS2005 force field [41] . When the above steps were accomplished, all investigated compounds were docked into the receptor pocket through the rigid protein docking model with the Stand-Precision (SP) scoring function [54, 55] to estimate the binding affinities.
Many marvelous biological functions in proteins and DNA as well as their profound dynamic mechanisms, such as switch between active and inactive states [56, 57] , cooperative effects [58] , allosteric transition [59, 60] , intercalation of drugs into DNA [61] , and assembly of microtubules [62] , can be revealed by studying their internal motions [63] . Likewise, to really understand the interaction mechanism of a receptor with its ligand, investigations should be aimed not only at their static structures but also at the dynamic process obtained by simulating their internal motions.
Here, the ''Desmond 2.4 Package'' [39] was adopted to study the internal motions of the receptor-ligand system. According to the software, the OPLS 2005 force fields [64, 65] was used to build aqueous biological systems, and the TIP3P model [66] was used to simulate water molecules. The orthorhombic periodic boundary conditions were set up to specify the shape and size of the repeating unit. In order to get an electrically neutral system, the minimum number of sodium and chloride ions needed to balance the system charge was placed randomly in the solvated system, and 0.15 mol/L sodium and chloride were then added to mimic the osmotic effect of water. Molecular dynamics simulations were carried out with the periodic boundary conditions in the NPT ensemble. The temperature and pressure were kept at 300 K and 1 atmospheric pressure using Nose-Hoover temperature coupling and isotropic scaling [67] . After all restrains were removed via the 3ns (nanosecond or 10 29 of a second) system minimization and relaxation, the operation was followed by running the 10 ns NPT production simulation and saving the configurations thus obtained in 2ps (picosecond or 10 212 of a second) intervals. All the molecular dynamics simulations were performed with a time step of 2fs (femtosecond or 10 215 of a second).
The QikProp [68, 69] is a program for predicting the ADME (absorption, distribution, metabolism, and excretion) properties of the compounds. With the QikProp software, a total of 44 properties of compounds can be predicted, including the principal descriptors and physiochemical properties.
All the compounds investigated need not the treatment for neutralization before using QikProp because it will be automatically done in QikProp. The normal mode was applied in the program. The property analyses for the partition coefficient (QP logP o/w), van der Waals surface area of polar nitrogen and oxygen atoms (PSA), predicted aqueous solubility (QP logS b ), and apparent MDCK permeability (QPP MDCK c ) [70] , were considered in the QikPro to evaluate the acceptability of the compounds.
The process of core hopping and the final agonists' structures thus selected are illustrated in Fig. 2 , from which we can see the following. The structure of GW409544, which is conceived as an agonist model to develop novel therapeutic agents for treating metabolic disorder, may be divided into three parts, Core A, Core B, and Core C, as marked by dash lines. Considering the great importance of the acidic head in Core A for activating PPARs receptors, let us retain the Core A part during the core hopping calculation as described below.
The 1st core hopping operation was aimed at the Core C part (see red part of Fig. 2) , generating five cores, Core C1, Core C2, Core C3, Core C4, and Core C5, respectively, to replace Core C. The 2nd core hopping operation was aimed at the Core B part (see (1k74) . The binding pocket is defined by those residues that have at least one heavy atom with a distance of 5 Å from the ligand [45] . The ligand GW409544 (in grey color) was extracted from the crystal structure while the ligand Comp#1 (rendered by three colors: grey for Core A; red for Core B; and blue for Core C) was generated by the ''core-hopping'' method. The hydrophobic surface of the receptor is colored in green. blue part of Fig. 2) , also respectively generating five cores, Core B1, Core B2, Core B3, Core B4, and Core B5 to replace Core B.
Consequently, we have a total of 1|5|5~25 different combinations for the GW409544 derivatives thus generated.
Subsequently, each of the 25 derivative compounds was docked into the two receptors PPARa (1k71) and PPARc (1k74), respectively. Listed in Table 1 are the 25 derivative compounds ranked roughly according to their docking scores to the receptors PPARa and PPARc, respectively. The top ten compounds highlighted with boldface type in Table 1 are those derivatives that are stronger than the original GW409544 in binding affinity with the two receptors. Of the top ten derivatives, the Comp#1, i.e., ''Core A-Core B1-Core C1'', has the strongest binding affinity with both PPARa (1k71) and PPARc (1k74), and hence it was singled out for further investigation.
Shown in Fig. 1 is the docked conformation of Comp#1 when aligned with GW409544 extracted from (A) the crystal complex in PPARa (1k7l) and (B) the crystal complex in PPARc (1k74), respectively.
As described in [37, 71] , the conversed H-bonding network formed by the polar acidic head of Core A in both GW409544 and Comp#1 to the four key residues of PPARa (or PPARc), such as Ser280 (or Ser289), Tyr314 (or His323), Tyr464 (or Tyr473) and His440 (or His449), were observed in our docking study. This Hbonding network played the role in stabilizing the conformation of the AF2-helix in arm I (red helix in Fig. 1) , which is vitally important for receptor-binding and activation [37, 72, 73, 74, 75, 76, 77] .
The hydrophobic tail of both Core A and Core C of agonists are buried well in the hydrophobic arm I and arm II that are formed by hydrophobic residues as shown by the green surface in Fig. 1 . Compared with GW409544 (shown with grey color in Fig. 1) , the compound of Comp#1 (purple color in Fig. 1 ) has more bulky molecular volume owing to the large hydrophobic Core C1, which is more fitted to the hydrophobic arm II, resulting in the much better binding affinity than GW409544 (cf. Table 1 ).
Molecular dynamics can provide useful information for characterizing the internal motions of biomacromolecules with time. For a comparison study, the 10 ns molecular dynamics simulations were performed, respectively, for the crystal structures of PPARa (1k7l), PPARc (1k74), as well as their complexes with GW409544 and Comp#1, i.e., PPARa-GW409544, PPARc-GW409544, PPARa-Comp#1, and PPARc-Comp#1. As we can see from Fig. 3 , all the characters concerned reached the simulation equilibrium within the 5ns (see panels A and C). Meanwhile, the corresponding root mean square deviation (RMSD) value curves of the protein backbone for PPARa, PPARc, PPARa-GW409544, PPARc-GW409544, PPARa-Comp#1, and PPARc-Comp#1 were also computed, respective-ly. It is interesting to see that the RMSD curves for PPARa-Comp#1 and PPARc-Comp#1 are remarkably more stable than those of PPARa-GW409544 and PPARc-GW409544, particularly for the case of PPARa (1k7l) system, where only a fluctuation of around 0.3 nm was observed when the complex system reached the plateau.
The detailed fluctuations of the aforementioned six different structures, as well as the root mean square fluctuations (RMSF) of their side-chain atoms, were also computed within 10 ns molecular dynamics simulations (see panels B and D of Fig. 3) .
It is instructive to point out that the RMSF curve of PPARa-Comp#1 or PPARc-Comp#1 is highly similar to that of PPARa-GW409544 or PPARc-GW409544, respectively. This is especially remarkable in the binding site of AF2 helix region with the residues 459-465 for PPARa-Comp#1 and residues 469-477 for PPARc-Comp#1 (see the grey frames in Fig. 3B and D) , indicating that the new designed compound, Comp#1, is very likely to have the same function for activating the AF2 helix as done by GW409544.
As a negative control, the similar molecular dynamics simulation was also performed for Comp#8 (A-B2-C3), which is ranked number 25 according to the strength of binding affinity with PPARa and PPARc (cf. Table 1 ). The corresponding simulation results thus obtained are shown in the Fig. 4 , from which we can see that the fluctuating magnitudes of molecular dynamics for PPARa-Comp#8 and PPARc-Comp#8, including the RMSD and RMSF, are much larger than those of PPARa-Comp#1 and PPARc-Comp#1, especially for the binding site of AF2 helix region (see the gray frames in Fig. 4B and D) . These phenomena indicate that Comp#8 is not as good as Comp#1 in stably binding to PPARa and PPARc, and hence Comp#8 might not have the same function for activating the AF2 helix as GW409544 had.
Some pharmaceutically relevant properties of the new designed agonist derivatives as well as the original GW409544 compound, such as the ''partition coefficient'' (logPo/w), ''van der Waals surface area of polar nitrogen and oxygen atoms'' (PSA), ''aqueous solubility'' (logS), and ''apparent MDCK permeability'' (PMDCK), were predicted by means of the QP program embedded in the ''Schrodinger2009 Software Package''. The results thus obtained are also listed in the Table 1 , respectively. Since PPARa and PPARc have a more spacious pocket (,1400 Å 3 ) than any other nuclear hormone receptors [37, 72] , it is quite natural that the agonist derivatives designed based on the two receptors by combining their three cores would have relatively large molecular weight (MW.500) and bulky volume, a trend quite similar to case in designing the inhibitors against the protein tyrosine phosphates (PTPase) [78] .
As shown in Table 1 , the values calculated by the QP program, such as PAS, logPo/w, logS, and PMDCK for the newly designed agonists are all within the reasonable ranges. Although the higher logPo/w value of a compound, the stronger its affinity to PPARs is, it is not a good idea to excessively enhance logPo/w because this would induce bad distribution of the compound on fat and body fluid [70] . It should be pointed out that, rather than the experiential values within the range between 26.5 and 0.50, most of the log S values for the new agonists are quite close to that of GW409544. Such a phenomenon might result from the core A part which was kept unchanged during the process of designing the newly compounds as mentioned above. If the core A part was modified as well, the log S value would be further improved accordingly. Also, as mentioned above, the values for the four ADME properties listed in Table 1 are all within the acceptable range for human beings, indicating that most of the 25 compounds, particularly the top 10 derivatives found in this study as highlighted in Table 1 , can be utilized as candidates for the purpose of developing new drugs.
The goal of this study was to find new and more powerful dual agonists for PPARa and PPARc. The new technique of ''core hopping'' adopted in this study allows for the rapid screening of novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties. A set of 10 novel compounds were found in this regard. Compared with the existing dual agonist, the new agonists not only had the similar function in activating PPARa and PPARc, but also assumed the conformation more favorable in binding to PPARa and PPARc. It is anticipated that the new agonists may become potential drug candidates. Or at the very least, they may stimulate new strategy for developing novel dual agonists against type-2 diabetes. Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors BACKGROUND: The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. METHODS: A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. RESULTS: The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). CONCLUSIONS: The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. Influenza A pandemic H1N1 2009 virus infections began to spread in Spain during spring 2009. Reports suggested high mortality in children and adults associated with the new virus in Mexico 1,2 and Argentina, 3 as well as in previously healthy young people. Analysis of cases hospitalized in the USA showed a mortality rate of 7%, with 25% of patients being admitted to the intensive care unit (ICU). 4 A study of 32 patients infected with the pandemic virus strain admitted to Spanish ICUs found a mortality rate of 25%, somewhat lower than in Latin American countries. 5 These findings suggest that the H1N1 virus is more virulent than previous strains.
As there was no specific targeted vaccine giving protection against the H1N1 influenza virus available at the beginning of the outbreak, health authorities began to recommend administration of neuraminidase inhibitors to reduce transmission and/or complications. Various studies have suggested that these drugs are also effective in reducing the severity of the infection. 4, 6, 7 We reviewed nationwide Spanish data on hospitalized patients with 2009 H1N1 influenza A in order to: (i) evaluate the frequency of adverse outcomes during hospitalization; and (ii) identify the factors influencing poor/good outcome, including the use of neuraminidase inhibitors shortly after the onset of symptoms.
We carried out a multicentre study in 36 hospitals from seven Spanish regions (Andalusia, Catalonia, Castile and Leon, Madrid, Navarre, the Basque Country and Valencia). Between July 2009 and February 2010 we selected hospitalized patients with influenza syndrome, acute respiratory infection, septic shock or multiple organ failure in whom influenza virus A (H1N1) 2009 infection was confirmed by real-time reversetranscription PCR (RT-PCR) from nasopharyngeal swabs; haemagglutinin (HA) sequencing was performed. We excluded patients who had nosocomial infection, defined as pandemic virus infection in a patient that appears ≥48 h after admission for another cause. All information collected was treated as confidential, in strict observance of legislation for observational studies. The study was approved by the Ethics Committees of the hospitals involved, following the Declaration of Helsinki principles. Written informed consent was obtained from all patients included in the study.
During the pandemic flu all patients suspected of having the disease, either in outpatient clinics or hospitals, were diagnosed by RT-PCR of samples from nasopharyngeal swabs. Within the next 48 h, hospitalized patients were interviewed at the centre. Of these, 23 rejected participation and 12 were excluded because flu had been acquired after hospital admission.
The following demographic variables and pre-existing medical conditions were recorded for all study participants: age, sex, ethnicity, educational level, smoking, alcoholism, pregnancy in women aged 15 -49 years, history of pneumonia in the previous two years, chronic obstructive pulmonary disease (COPD), asthma, cardiovascular disease, renal failure, diabetes, HIV infection, disabling neurological disease, cancer, transplantation, morbid obesity (body mass index ≥40), use of neuraminidase inhibitors before hospital admission (and their timing relative to the onset of symptoms, verified after contacting the prescribing general practitioner), use of other medications in the 90 days before hospital admission (corticosteroids, antibiotics etc.) and treatment received during hospitalization (medications, catheters and mechanical ventilation). For each vaccine, a case was considered vaccinated if the vaccine had been received ≥14 days before the onset of symptoms. Data were collected during hospital admission and the clinical chart was also reviewed after discharge.
The outcome variables were admission to an ICU, in-hospital death and length of hospital stay (in days). Given that the number of deaths was very low, a combined endpoint was classified as 'poor outcome': ICU admission and/or in-hospital death.
Bivariate comparisons were made using Pearson's x 2 test for categorical variables and Student's t-test for continuous variables. As a measure of association, the relative risk (OR) and 95% CI were calculated. Logistic regression was applied in the multivariate analysis for dichotomous adverse outcomes. To determine the variables to be included in the multivariate analysis, the procedure described by Sun et al. 8 was followed. Intermediate variables were discarded. We ran two stepwise models, one backward and another forward, including variables with P, 0.2. 9, 10 We constructed a list of predictors of mortality identified in other studies. Using information from stepwise models and the list of predictors, a saturated model was built, and by using a heuristic approach, variables that did not change the coefficient of the bundles by more than 10% were discarded, in order to construct a parsimonious model retaining all important confounders.
To analyse the impact of different variables on the length of hospital stay, patients who died were excluded from these analyses. Given that hospital stay did not follow the normal curve, natural logarithms were used. Firstly, to select potential variables related to length of stay, we used Cox regression in the same fashion as described above for the logistic regression analysis. The variables selected by this model were tested by including other potential candidates according to the logistic regression analyses. Secondly, an analysis of covariance was applied to estimate the adjusted means of hospital length of stay. All analyses were made using the Stata 10/SE package (College Station, TX, USA).
There were a total of 813 patients [410 (50.4%) were female, of which 51 (12%) were pregnant]. The mean age was 38.5 years (SD 22.8) and 24% were aged ,18 years. The use of neuraminidase inhibitors was reported by 495 patients (60.9%), with oseltamivir being administered in all cases but two (zanamivir). During hospitalization, 79 patients (9.7%) were admitted to the ICU and 10 died (1.2%), of whom 9 were not receiving intensive care. No death occurred in pregnant women, of whom only one was admitted to the ICU. The timings of the use of neuraminidase inhibitors before hospital admission were: 332 patients in the first 24 h after the onset of symptoms, 97 between 24 -48 h and 66 after 48 h ( Table 1) .
The relationship between study variables and ICU admission/ in-hospital death is shown in Table 2 . In the univariate analyses, age, most comorbidities (COPD, diabetes, liver failure and cardiovascular disease), ex-smoking, corticosteroid therapy and histamine-2 receptor antagonists were associated with an adverse outcome during hospitalization. In the multivariate models, the variables significantly associated with a poor outcome were diabetes (OR¼ 2.21, 95% CI¼ 1.21 -4.02), corticosteroid therapy (OR¼ 3.37, 95% CI ¼ 1.39-8.20) and use of histamine-2 receptor antagonists (OR ¼ 2.68, 95% CI ¼ 1.14 -6.36). Use of neuraminidase inhibitors was protective (OR ¼0.57, 95% CI ¼ 0.34 -0.94). Pneumonia at admission, COPD, ex-smoking and liver failure showed a trend to association.
Delgado-Rodríguez et al.
The trend analysis for age in the multivariable analysis yielded a P value of 0.11, with advanced age associated with a higher risk of adverse outcome. When the timing of treatment with neuraminidase inhibitors after the onset of influenza was analysed, the benefit was confined to administration within the first 48 h after the onset of symptoms. Table 3 shows the variables associated with length of hospital stay. The use of neuraminidase inhibitors within the first 2 days after the onset of influenza reduced hospital stay by a mean of 1.9 days (from 6.6 to 4.7, P,0.001), whereas delayed administration was associated with an increase in hospital stay. Pneumonia diagnosed at admission was clearly associated with longer hospital stay, as were comorbidities (COPD, neurological impairment and cardiovascular disease) and some therapies (proton pump inhibitors).
We found that traditional risk factors associated with hospitalization in patients with influenza (COPD and corticosteroid therapy before admission) were also found in our patients. Likewise, the use of neuraminidase inhibitors reduced the probability of adverse outcomes during hospital stay and significantly shortened the length of stay.
This study is observational and can be affected by several limitations. Kumar 11 has recently highlighted the drawbacks of observational studies in estimating the benefits of early viral treatment in the prognosis of flu. We agree that selection bias is difficult to avoid. Immortal time bias or survival-durationrelated selection bias imply that the late use of antivirals may be related to a better prognosis, whereas in fact our results suggest the opposite.
Our results show no benefit of late neuraminidase treatment. In Israel, a retrospective cohort study documented a higher rate of complications after admission. 12 Severe complications (excluding hypoxia and uncomplicated pneumonia) occurred more frequently with late oseltamivir. In the same way, a Spanish study of ICU patients showed that ICU length of stay, days of mechanical ventilation and mortality were reduced in patients receiving early treatment versus late treatment with oseltamivir. 13 These reports do not give comparisons with flu patients without antiviral treatment.
The mortality rate in our study (1.2%) was low in comparison with other studies. This may be due to the fact that our patients were not all admitted to the ICU, 5,14 and did not all have pneumonia at hospital admission. 15 Even so, the mortality rate was clearly lower than that found in the USA at the beginning of the pandemic (7%) 4 or the 4.9% reported in Canada. 16 Likewise, the rate of ICU admission (9.7%) was lower than that found in the USA (25%) 4 and Canada (16%), 16 although it was similar to the 8% reported in New Zealand Maoris. 17 Some form of selection bias cannot be completely ruled out as our study patients had to be interviewed to collect data on the use of medications before admission and other risk factors related to disease severity. In a study carried out in Catalonia (north-east Spain), of 773 cases hospitalized, 37.9% were admitted to the ICU. 18 In contrast, in Andalusia (southern Spain), 28 out of 311 hospitalized cases (9%) were admitted to an ICU. 19 In another Spanish study of patients admitted to the ICU, the mortality rate was 22%. 20 Taken together, these data suggest that patients who died shortly after admission were not picked up by our study.
The predisposing factors for a higher probability of adverse outcome during hospitalization were broadly similar to those found in other studies. 16, 21 In one international series of patients with community-acquired pneumonia, male sex and obesity were predictors of mortality, although we did not find similar results. 15 We found a significant association between reductions in ICU admission/death and the administration of neuraminidase inhibitors within the first 48 h after the onset of symptoms, similarly to the findings of Jain et al. 4 and other studies. 6, 7 In these reports none of the pregnant women who died had taken neuraminidase inhibitors within the first two days after the onset of illness.
Early use of neuraminidase inhibitors was associated with shorter hospital stay. Other reports have found no relationship between antiviral treatment and hospital stay. 22 In summary, we found that early treatment with neuraminidase inhibitors had a beneficial effect on outcomes during  Veillonella montpellierensis Endocarditis Veillonella spp. rarely cause infections in humans. We report a case of Veillonella endocarditis documented by isolating a slow-growing, gram-negative microbe in blood cultures. This microbe was identified as the newly recognized species Veillonella montpellierensis (100% homology) by 16S RNA gene sequence analysis. V eillonella are anaerobic, gram-negative cocci, part of the normal flora of the mouth, gastrointestinal tract, and vaginal tract. Veillonella dispar, V. atypica, and V. parvula have been cultured from human specimens. They are infrequently isolated in human infections. Rarely, Veillonella species have been the only etiologic agents identified in serious infections such as meningitis, osteomyelitis, prosthetic joint infection, pleuropulmonary infection, endocarditis, and bacteremia. A new species, V. montpellierensis, has recently been isolated from the gastric fluid of a newborn and from the amniotic fluid of 2 women (1). Its pathogenic role is still debated.
A 75-year-old woman was admitted to the intensive care unit with septic shock. She had a history of diabetes mellitus. A cardiac murmur had been noted 8 years earlier but was not investigated further. On physical examination, the patient had aortic and mitral murmur. Reagent strip for urinalysis detected leukocytes and nitrites. After 3 blood cultures and urinalysis, the patient was treated for septic shock secondary to upper urinary tract infection with ceftriaxone, 2 g/day intravenously. The patient's condition rapidly improved with antimicrobial drugs and dopamine. Three days after admission, she was afebrile and hemodynamically stable; she was transferred to the urology department for acute pyelonephritis, which had not been confirmed by computed tomographic (CT) scan. Urine culture yielded Gardnerella vaginalis. Chest radiograph showed a patchy density of the right inferior pulmonary lobe confirmed by chest CT scan that suggested either pneumonia or neoplasia. On day 6, a transesophageal echocardiograph, performed because of the cardiac murmur, showed oscillating intracardiac masses on the aortic and mitral valves. Because the blood cultures were still negative, we determined that the patient had culture-negative endocarditis and replaced ceftriaxone with amoxicillin, 12 g/day for 6 weeks, in addition to gentamicin, 3 mg/kg/day for 3 weeks. On day 26, another transesophageal echocardiograph was performed and showed that the vegetation on the aortic valve had disappeared and the mitral vegetation was greatly reduced. The patient was discharged after 42 days of antimicrobial drug treatment, and follow-up was not possible.
On day 14 after sampling, 2 of 3 anaerobic blood cultures (automated blood culture BACTEC 9240 system (Becton Dickinson, Le Pont de Claix, France) yielded a slow-growing, gram-negative microbe. Blood was subcultured onto Columbia agar with 5% sheep blood (Mérieux, Marcy l'Etoile, France) under 5% CO 2 and anaerobic atmosphere and resulted in small colonies. This slowgrowing microbe was lost after 2 subcultures, and no isolate is available for further description. The isolate retrieved in the blood culture was identified by 16S rRNA gene sequence analysis. The template DNA was prepared from a few colonies that were isolated on the blood agar incubated anaerobically. DNA was extracted by using Fastprep DNA extraction kit (Ozyme, St Quentin en Yvelines, France) according to the manufacturer's recommendations and was subjected to polymerase chain reaction (PCR) targeting the 16S rRNA gene as previously described (2) . Sequencing the PCR product (2) showed a 1,531-nucleotide sequence. This sequence shared 100% homology with that of V. montpellierensis (GenBank accession no. AY244769) and was already reported (GenBank accession no. AY244769) in a previous article (3) . In this article, the isolated Veillonella strain (that was isolated from our patient) was first identified as "candidatus V. atypica" since the sequencing of the amplicon disclosed 94% sequence similarity with that of V. atypica (3). V. montpellierensis had not yet been described. PCR contamination was unlikely since this organism had never been amplified in our laboratory and negative controls remained negative.
According to the modified Duke criteria (4), our patient had definite endocarditis. Anaerobic microbes do not commonly cause endocarditis (5) . Most cases of anaerobic endocarditis are caused by anaerobic cocci, Propionibacterium acnes, and Bacteroides fragilis group (5) . We describe the seventh reported case of well-documented infectious endocarditis in which a Veillonella species was the sole pathogen and the first due to V. montpellierensis. Characteristics of the 7 Veillonella endocarditis patients are summarized in the Table. Five of them fulfilled the Duke modified criteria for definite endocardi-tis; the 2 others were possible endocarditis. All previously reported cases of Veillonella endocarditis were due to either V. dispar (9,10), V. parvula (11) , or V. alcalescens (6) (7) (8) , currently considered V. parvula (12) . One patient had no history of fever (7), and 1 patient had no preexisting valvular disease (8) . Five patients had an infected mitral valve; 4 of the 5 had prosthetic valves. Our patient had mitral and aortic endocarditis. All patients had positive blood culture except 2, for whom the diagnosis was made by culturing the valve (6, 11) . Veillonella spp. had also been isolated from intravenous drug users with polymicrobial endocarditis (13); V. parvula was isolated from a lung abscess in a patient with echocardiographic vegetations, but blood cultures were negative (14) . We could not test the susceptibility of the organism because the bacterium was lost on subculture. In treating infections with Veillonella species, penicillin has been the antimicrobial agent of choice (10) . However, recent studies found a notably high resistance to penicillin G (MIC >2µg/mL) (15) . These penicillin G-resistant isolates showed generally reduced susceptibility to ampicillin or amoxicillin but remained susceptible to amoxicillin and clavulanate (15) . We treated our patient for culture-negative endocarditis with amoxicillin. As the clinical state of our patient improved, we did not change antimicrobial agents.
Our isolate has recently been compared with 3 other isolates and classified as a new Veillonella species named V. montpellierensis by Jumas-Bilak et al. (1) . We demonstrate here that V. montpellierensis is pathogenic for humans and may be included as a new agent of endocarditis caused by fastidious pathogens.
We report here the seventh case of endocarditis due to Veillonella spp. identified by PCR amplification and sequencing of 16S rDNA gene and the first case of endocarditis due to V. montpellierensis. This case reemphasizes the usefulness of molecular methods in identifying fastidious microorganisms and in describing new clinical entities (3) . Primate-to-Human Retroviral Transmission in Asia We describe the first reported transmission to a human of simian foamy virus (SFV) from a free-ranging population of nonhuman primates in Asia. The transmission of an exogenous retrovirus, SFV, from macaques (Macaca fascicularis) to a human at a monkey temple in Bali, Indonesia, was investigated with molecular and serologic techniques. Antibodies to SFV were detected by Western blotting of serum from 1 of 82 humans tested. SFV DNA was detected by nested polymerase chain reaction (PCR) from the blood of the same person. Cloning and sequencing of PCR products confirmed the virus's close phylogenetic relationship to SFV isolated from macaques at the same temple. This study raises concerns that persons who work at or live around monkey temples are at risk for infection with SFV. R ecent epidemics such as HIV and severe acute respiratory syndrome (SARS) have changed the way we view emerging infectious diseases; these epidemics show that animal reservoirs are important sources of new infectious threats to humans. Contact between humans and animals is a crucial rate-limiting step in this process, although data describing the variables that influence animal-tohuman transmission are relatively scarce. Nonhuman primates, by virtue of their genetic, physiologic, and sometimes social similarities to humans, are particularly likely sources of infectious agents that pose a threat to humans (1, 2) . Data on simian immunodeficiency virus (SIV)/HIV dramatize this point; scientists now theorize that SIVs were transmitted from primates to humans on several occasions (3, 4) . As a result, concern is increasing that other infectious agents enzootic in primate populations may endanger humans (5) .
The family of SIV is 1 of 4 primateborne retroviruses known to infect humans (6) . Simian T-cell lymphotropic viruses, enzootic in both Asian and African Old World monkeys and apes, may have repeatedly crossed the species barrier (7, 8) . The resulting human form of the virus, HTLV, is the etiologic agent of 2 human diseases, adult T-cell leukemia and tropical spastic paresis (9) . Serologic studies have demonstrated evidence of primateto-human transmission of simian retrovirus (SRV), a retrovirus enzootic among Old World monkeys, in laboratory workers exposed to captive primates (10) . To date, no disease has been linked to human infection with SRV. Finally, in the past decade, evidence of infection with simian foamy virus (SFV) has been identified in 1% to 4% of persons who come into frequent contact with primates in zoos and primate laboratories and among 1% of bushmeat hunters in Central Africa (11) (12) (13) (14) (15) .
SFVs are exogenous retroviruses enzootic in both New and Old World primates (16) (17) (18) . Phylogenetic analyses of SFVs indicate a species-specific distribution of virus strains not unlike that of SIV among some African primate species (19) (20) (21) (22) (23) . Among captive primate populations, seroprevalence of antibodies to SFV may reach 100% in adults, with many animals seroconverting before the onset of sexual maturity (19, 24 ; J. Allan, unpub. data). Fewer data are available on the seroprevalence of antibodies to SFV among free-ranging populations of primates. SFV is present in highest concentrations in the saliva of infected laboratory macaques (Macaca mulatta and M. fascicularis) and African green monkeys (Cercopithecus aethiops), which suggests that bites, scratches, and mucosal splashes with saliva from primates are likely mechanisms of transmission (25) (26) (27) .
Because SFV is not known to occur naturally in humans, detecting serologic or molecular evidence, or both, of infection in a human, along with a history of close contact with primates, constitutes strong evidence for primate-to-human transmission, i.e., a marker for crossspecies transmission (28) . Existing serologic and molecular techniques can sensitively and specifically detect human SFV infections (29) .
Primates and humans come into contact in a variety of contexts in Asia, including owning primate pets, observing performance monkeys, participating in ecotourism activities, hunting primates for bushmeat, and visiting monkey temples (30, 31) . Monkey temples are religious sites that have, over time, become associated with populations of free-ranging primates. Monkey temples are common throughout southern and Southeast Asia, where primates play an important role in culture. Asia's monkey temples annually bring millions of people, including hundreds of thousands of tourists, from around the world into close proximity with free-ranging primates (32) (33) (34) . Worldwide, monkey temples may account for more human-primate contact than any other context.
In Bali, ≈45 temple sites contain substantial populations of free-ranging macaques (33) . Those who spend the most time in or around monkey temples include workers who maintain the temples; nuns, monks, and others who live on or around temple grounds; merchants who sell a variety of goods to tourists; and farmers whose fields are raided by macaques. Worshippers and tourists may also come into contact with temple macaques. These temples are thus an important context in which to investigate crossspecies transmission of infectious agents.
Serologic data on free-ranging Southeast Asian macaques, though incomplete, suggest that SFV is enzootic among these macaque populations and that corresponding rates of SFV infection are high (80%-100%) (L. Jones-Engel, unpub. data). We hypothesized that humans who come into contact with these macaques might similarly show evidence of infection with SFV and investigated this proposition among a group of persons who worked at or around the Sangeh monkey temple in Bali, Indonesia.
The Sangeh monkey temple is in central Bali, Indonesia, ≈20 km north of Denpasar, Bali's most populous city. The 17th-century Hindu temple complex at Sangeh serves the surrounding community and is a popular domestic and international tourist destination. Approximately 200 free-ranging M. fascicularis roam throughout the 6-hectare temple complex and into the surrounding rice fields and farms. Most of the macaques' caloric intake is from daily provisions provided by temple workers and food given to them by visitors.
In July 2000, as part of a larger study on the epidemiology of exposure to primateborne viruses at the Sangeh monkey temple, 82 workers from Sangeh agreed to participate in the present study (32) . After informed consent was obtained, a questionnaire designed to elicit demographic data as well as data describing the frequency and type of exposure to Sangeh's macaques was administered in Bahasa Indonesia, the national language of Indonesia. Subsequently, 10 mL of blood was withdrawn from each participants antecubital vein, 6 mL was centrifuged to extract serum, and the remainder was mixed with EDTA. Serum specimens and whole blood were then stored at -20°C.
In July 2000, 38 macaques within the Sangeh monkey temple area and surrounding forest were darted opportunistically and sedated with 3 mg/kg of Telazol (tiletamine HCl/zolazepam HCl). Following universal precautions, researchers withdrew 10 mL of blood from each macaque's femoral vein. The macaques were closely monitored during anesthesia and recovery. Six milliliters of blood was placed in a serum separator tube and centrifuged in the field to extract the serum. The remaining blood was placed in a tube containing EDTA. Sera and whole blood were frozen and stored at -20°C.
Western blot immunoassays were performed with a few modifications (21) . Briefly, human foreskin fibroblast cells were infected with SFVbab1 (an isolate from a baboon) and maintained until notable cytopathologic changes were observed (19) . Culture supernatant fluid containing virus was harvested, and SFV was purified through a 20% sucrose cushion, separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and the antigens were blotted onto nitrocellulose sheets. The nitrocellulose paper was blocked with 3% bovine serum albumin and subsequently incubated with serum at a dilution of 1:40. Viral proteins were detected with the streptavidin-biotin system (Amersham Inc., Arlington Heights, IL, USA) by using diaminobenzidine as the substrate for color development. The criterion used for a positive sample was antibody reactivity to both p70 and p74 gag-related proteins (19) .
DNA was purified from whole blood by using the QIAamp DNA Blood Mini Kit (Qiagen, Inc., Valencia, CA, USA). Briefly, 20 µL protease and 200 µL buffer AL were combined with 200 µL whole blood and incubated at 56°C for 10 min. After incubation, 200 µL ethanol was added, and the entire mixture was applied to a QIAamp spin column. The purified DNA was eluted from the column with 70 µL nuclease-free water, and concentration was determined spectrophotometrically at optical density 260 nm.
The presence of SFV DNA was determined by using nested PCR. Five hundred ng purified DNA was combined with a PCR reaction mixture with a final concentration of 10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.1% Triton X-100, 2 mmol/L MgCl 2 , 200 µmol/L each dNTP, 0.15 mg/mL BSA, 1 µm Taq polymerase, and 400 nmol/L of each primer in a total volume of 50 µL. The following primer pairs were used: first round, forward 5′ CAG TGA ATT CCA GAA TCT CTT C 3′, reverse 5′ CAC TTA TCC CAC TAG ATG GTT C 3′; and second round, forward 5′ CCA GAA TCT CTT CAT ACT AAC TA 3′, reverse 5′ GAT GGT TCC CTA AGC AAG GC 3′ (29) . "Touchdown PCR" was used for both rounds with reaction conditions of initial denaturation at 94°C for 2 min, followed by 7 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a 2°C decrease in annealing temperature per cycle to 48°C, followed by 33 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 45 seconds with a final extension at 72°C for 2 min. Second-round conditions were the same, except 19 cycles were used instead of 33.
Twenty µL of the PCR reaction underwent electrophoresis on a 1% low melting point agarose gel. DNA bands (365-bp product) were excised from the gel and purified by using Wizard PCR Preps DNA Purification System (Promega Corp., Madison, WI, USA). The PCR product was ligated into the pCR 2.1-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, CA, USA.). An SFV DNA plasmid (pSFV-1Lgp), representing long terminal repeat, gag, and pol of SFV-1, was included as a positive PCR control and for determining sensitivity of detection by serial dilution (provided by A. Mergia). TOP10 cells were transformed with the ligation reaction, plated onto Luria broth agar plates containing 50 µg/mL kanamycin, and incubated overnight at 37°C. Miniscreen DNA was purified by using Wizard Plus Minipreps DNA Purification System (Promega). Samples were sequenced with the ABI 373 automated fluorescent sequencer using BigDye Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA, USA).
Five hundred ng purified DNA from whole blood was combined in a PCR reaction mixture with a final concentration of 10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.1% Triton X-100, 2.5 mmol/L MgCl 2 , 200 µmol/L each dNTP, 0.15 mg/mL BSA, 1 U Taq polymerase, and 400 nmol/L of each primer in a total volume of 50 µL. The following primers were used: forward, 12SA, 5´ CTG GGA TTA GAT ACC CAC TAT 3´, and reverse, 12SO, 5´ GTC GAT TAT AGG ACA GGT TCC TCT A 3´ (35) . Cycling conditions were the following: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min, with a final extension at 72°C for 5 min. The 101-bp product underwent electrophoresis and was processed for DNA sequencing essentially as described for SFV.
The alignments were made in Bioedit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html) and ClustalX 1.81 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). Columns in the alignment in which gaps had been inserted in regions with insertions, and deletions were stripped before the analyses. DNA trees were created with the neighbor-joining method by using the Phylip program (DNAdist; Neighbor), and the output was generated with Treeview (http://taxonomy. zoology.gla.ac.uk/rod/treeview.html). The GenBank accession numbers for the SFV and mitochondrial DNA sequences reported here are AY628152-69 and AY633510-39, respectively.
The seroprevalence of SFV among the Sangeh macaques is presented in Table 1 . Thirty-eight macaques (29 males and 9 females; 4 juveniles, 6 subadults, 28 adults) were sampled. Thirty-four (89.5%) of the 38 macaques were seropositive for SFV by Western blot; 2 (50%) of 4 juveniles, all 6 (100%) subadults, and 26 (93%) of the adults were antibody positive. All 9 females were SFV seropositive. SFV seroprevalence in this free-ranging macaque population was consistent with seroprevalence studies done in captive (25) and other free-ranging macaque populations (L. Jones-Engel, unpub. data).
Nearly two thirds of the study sample was male (62.2%), and the mean age of the study participants was 35 years (Table 2) . Of participants who reported being exposed, 23 (28.0%) of 82 persons reported having been bitten by the temple's macaques ( Table 3) . Five of the 23 reported being bitten more than once. Thirty-one (37.8%) of 82 reported having been scratched, including 9 persons who had been scratched more than once. In total, 37 (45.1%) of 82 reported having been either bitten or scratched.
Of the 82 persons whose serum was tested for antibodies to SFV, 81 had negative results by Western blot. Serum from a 47-year-old farmer (BH66) was reactive to SFV gag proteins, p70/p74 (Figure 1 ). This man reported that he visited the monkey temple every day and had previously been bitten once and scratched on more than one occasion by macaques there. He reported that the bite and scratches, which occurred on his hands and toes, had bled and that he had washed the wounds and applied traditional medicines. He denied owning or coming into contact with a pet primate. He also denied hunting primates or consuming primate meat.
To determine whether SFV was present in humans and macaques, we performed nested PCR amplification of SFV by using conserved primers designed to detect macaque SFV (29) . SFV was detected in the macaques tested, as well as in the 1 human participant (BH66), whose serum contained antibodies to SFV. Blood samples from all 81 human participants that were seronegative for antibodies to SFV were also negative for SFV by PCR. The limits of sensitivity for nested PCR were 1-10 SFV DNA copies per 500 ng cellular DNA, as determined by dilution of a positive control plasmid.
Quantitative PCR products of BH66, 5 of the Sangeh macaques (M. fascicularis), and 13 pet macaques (M. tonkeana, M. maura, and M. fascicularis) from Sulawesi, Indonesia, were cloned at least twice, sequenced, and compared with published sequences from a rhesus macaque (M. mulatta) (SFV-1mac, M55279), an African green monkey (Cercopithecus aethiops) (SFV-3agm, M74895), and a chimpanzee (Pan troglodytes) (SFVcpz, U04327). As shown in Figure 2 , SFV from BH66 was most closely related to an SFV sequence amplified from 1 of the macaques at the Sangeh Monkey Temple (BP6). To verify that the BH66 sample was of human origin and not a mislabeled sample from an SFV-infected monkey, we amplified a small fragment from the 12S ribosomal mitochondrial DNA from 2 healthy unexposed humans, BH66, several macaque species, and African green monkeys; cloned the products; and derived DNA sequences. Phylogenetic analysis (Figure 3) showed that the human DNA sample grouped with mitochondrial DNA sequences from humans, confirming that the BH66 sample was from a human. Lymphocytes from BH66 were not available for isolation of SFV directly.
This report documents the first case of SFV infection in a person with known exposure to free-ranging Asian primates. Antibodies to SFV were detected in serum, and SFV genomic segments were amplified from blood of the infected person. Because PCR is prone to contamination, which leads to false-positive results, PCR products were sequenced and compared to SFV from other macaque species. Phylogenetic analysis showed that SFV amplified from the infected human was most similar to SFV from M. fascicularis at the Sangeh monkey temple. Although ascertaining exactly how or when the person acquired his infection was not possible, he did report having been bitten once and scratched on a number of occasions by macaques at Sangeh. He denied other past contacts with primates.
Because only 1 infected person was detected and our sample size was limited (82 persons), we cannot estimate the prevalence of SFV infection in this human sample. Previous research on naturally acquired SFV infection among bushmeat hunters in Central Africa found 10 who seroconverted in a population of 1,099 (1%), of which 3 also had positive results by PCR. Surveillance of larger numbers of persons exposed to primates at monkey temples is necessary to estimate the risk of SFV infection in this context.
Human and macaque sympatry in Southeast Asia dates back as far as 25,000 years, as evidenced by remains at Niah Cave in Borneo (37) . Hindu and Buddhist temples are a relatively recent addition to the landscape, first appearing 1,000 to 4,000 years ago. Contemporary human-macaque commensalism at each monkey temple is shaped by the behavioral characteristics of the particular monkey population as well as the community's unique geographic, social, religious, and economic factors. Human-macaque contact differs at the various monkey temples. At Sangeh, where tourism has become an important economic resource, macaques depend on visitor feeding for most of their nutrition and have learned to climb on visitors' heads and shoulders to obtain food. Local photographers, who make a living photographing visitors with monkeys, encourage this behavior. Macaques, sometimes provoked by visitors, can become aggressive and will bite or scratch people.
The Sangeh Temple Committee, which manages the Sangeh Monkey Forest, estimates ≈250 persons work in and around the monkey forest. Of the 82 persons who participated in the present study, 28% had been bitten by a macaque, and 6% had been bitten more than once. In comparison, 700,000 tourists visit the 4 main monkey temples on the island of Bali (Padangtegal/Ubud, Sangeh, Alas Kedaton, and Uluwatu) annually and as many as 5% of these visitors are bitten by macaques (A. Fuentes, unpub. data). These data suggest that visitors receive the preponderance of macaque bites.
The literature describes ≈40 cases of human infection with SFV. Long-term follow-up data are unavailable for many of these cases. However, no disease has been linked to SFV among infected humans. Furthermore, no cases of human-to-human transmission have been described, even among those receiving transfusions of blood products from a worker at a primate center who was later shown to be infected with SFV (38) .
Notwithstanding, more data are needed before SFV can be proclaimed a "virus without a disease." First, SFV infection has not been extensively studied in immunocompromised persons, so whether SFV has a more aggressive course in an immunologically "permissive" environment is unknown. Two persons died shortly after receiving baboon Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 7, July 2005 ) , and SFV-3agm is a published sequence from an African green monkey (Cercopithecus aethiops). SFVcpz is a published sequence from a chimpanzee (Pan troglodytes) and was used an outgroup for this tree. The SFV human strain (BH66) clustered with an SFV sequence amplified from BP6 one of the macaques at the Sangeh monkey temple. The SFV DNA tree was created with the neighbor-joining method by using the PHYLIP program (DNAdist; Neighbor). Bootstrap replicates were 1,000. Bootstrap values were calculated by using Seqboot, DNAdist, Neighbor, and Consense (PHYLIP programs). Bootstrap values 60% are shown. The SFV tree was plotted in Treeview. liver transplants in 1994, and SFV was detected in the blood and tissues of both at autopsy (39) . More research in this area is needed. Moreover, little is known about the epizootiology of SFV among wild primates. Although SFV infection in humans has not culminated in any observable symptoms, SFV strains may differ in their capacity to infect or cause disease in humans. Finally, whether SFV can adapt in humans after transmission and potentially lead to disease needs to be examined.
The recent SARS epidemic vividly demonstrates how the economic infrastructure and dense population of Asia facilitate the rapid international spread of disease. The combination of large primate reservoirs, prevalent humanprimate contact, a growing immunocompromised population, and advanced infrastructure in Asia increases the likelihood of a primateborne zoonosis emerging on this continent.
The demonstration of SFV transmission in the context of a monkey temple in Bali points to a broad public health concern: other enzootic primate infectious agents may cross the species barrier and cause significant morbidity and mortality in human populations. Given our lack of knowledge of the effects of SFV, as well as the poorly defined risk of other primateborne zoonoses, steps should be taken to decrease the risks of cross-species transmission among the many persons who visit these sites. Previous data on both worker and visitor exposure to macaques at monkey temples suggest that human behaviors, especially the practice of feeding macaques, is a risk factor for being bitten or scratched (33, 34) . We have recommended that monkeys be fed only by specially trained personnel who minimize physical contact with monkeys. Such restrictions have been successfully employed at other monkey forests in Asia. For example, Singapore's Bukit Timah Nature Reserve has nearly 200 free-ranging macaques (M. fascicularis), and in 2002, an estimated 380,000 visitors made use of the reserve's trail system, yet contact between monkeys and visitors at Bukit Timah is rare. This finding may be ascribed to the park's policy, enforced by stiff fines, of prohibiting visitors from feeding macaques.
This study reports the first case of human SFV infection in Asia and also, for the first time, links natural transmission of SFV to a person to a specific population of primates. Our findings suggest that workers in and around monkey temples can become infected with SFV. By implication, visitors to monkey temples, especially those who are bitten by a macaque, may also be at risk for SFV infection. Because many visitors to monkey temples are international travelers, these findings have ramifications for the potential global spread of primateborne infectious agents. Demonstrating natural cross-species transmission in a context that does not involve hunting for bushmeat implies that other contexts of primate-human contact may also facilitate the transmission of simian pathogens. These data point to the need for further research into SFV transmission in other contexts, including pet ownership and performance animals, as well as in the diverse geographic areas where humans and primates come into contact. Such research will help describe the overall picture of the Bootstrap replicates were 1,000. Bootstrap values were calculated by using Seqboot, DNAdist, Neighbor, and Consense (PHYLIP programs). Bootstrap values >60% are shown. The mtDNA tree was plotted in Treeview. This analysis suggests that BH66 was of human origin. Although the phylogenetic tree constructed with mtDNA from a variety of monkey samples can be used to distinguish human from monkey mtDNA, a large number of nuclear mtDNA sequences, have evolved as pseudogenes (36) . These sequences can be highly divergent from mtDNA and resulted in some ambiguity as mtDNA amplified from several monkeys did not group with other members of the same species. Because of the nature and variability of these sequences, definitive conclusions about mtDNA phylogenies could not be determined; however, mtDNA trees were still useful for determining the origin of mtDNA material.
emergence of primateborne pathogens such as HIV/SIV and will form a scientific basis for guiding policies and programs to deter the spread of emerging zoonotic pathogens. Application of Consensus Scoring and Principal Component Analysis for Virtual Screening against β-Secretase (BACE-1) BACKGROUND: In order to identify novel chemical classes of β-secretase (BACE-1) inhibitors, an alternative scoring protocol, Principal Component Analysis (PCA), was proposed to summarize most of the information from the original scoring functions and re-rank the results from the virtual screening against BACE-1. METHOD: Given a training set (50 BACE-1 inhibitors and 9950 inactive diverse compounds), three rank-based virtual screening methods, individual scoring, conventional consensus scoring and PCA, were judged by the hit number in the top 1% of the ranked list. The docking poses were generated by Surflex, five scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, and PMF_Score) were used for pose extraction. For each pose group, twelve scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, PMF_Score, LigScore1, LigScore2, PLP1, PLP2, jain, Ludi_1, and Ludi_2) were used for the pose rank. For a test set, 113,228 chemical compounds (Sigma-Aldrich® corporate chemical directory) were docked by Surflex, then ranked by the same three ranking methods motioned above to select the potential active compounds for experimental test. RESULTS: For the training set, the PCA approach yielded consistently superior rankings compared to conventional consensus scoring and single scoring. For the test set, the top 20 compounds according to conventional consensus scoring were experimentally tested, no inhibitor was found. Then, we relied on PCA scoring protocol to test another different top 20 compounds and two low micromolar inhibitors (S450588 and 276065) were emerged through the BACE-1 fluorescence resonance energy transfer (FRET) assay. CONCLUSION: The PCA method extends the conventional consensus scoring in a quantitative statistical manner and would appear to have considerable potential for chemical screening applications. Molecular docking-based virtual screening is widely used to discover novel ligands in the early stages of drug development [1, 2, 3, 4] . Various docking programs, such as DOCK [5] , AutoDock [6] , Surflex [7] , FlexX [8] , GOLD [9] , and Glide [10, 11] , have been developed. As an essential component of these programs, the scoring function is able to evaluate the fitness between the ligand and receptor guiding the conformational and orientational search of ligand-binding poses. Since the 1990s, several dozens of scoring functions have been reported in the literature [12, 13] . Current scoring functions can be roughly classified as force-field-based methods [5, 14, 15] , empirical scoring functions [16, 17] , and knowledge-based statistical potentials [18] . The existing limitations in current docking and scoring include a lack of protein flexibility, inadequate treatment of solvation, and the simplistic nature of the energy function employed [19, 20, 21, 22] . In particular, the major weakness of docking programs lies in the scoring functions [12, 13] . Considering the computational cost and time required for virtual screening, all of the current scoring functions use various approximations resulting in inaccuracy in the score and rank of the ligand-binding poses [19] as well as in false positives mixed in with the top scorers in the ranking list when virtual screening was performed with only a single scoring function. Some studies focus on calculating proteinligand free binding energy, free energy perturbation (FEP), thermodynamic integration (TI) [23, 24, 25] , MM-PB/SA, MM-GB/SA [26, 27, 28] and linear interaction energy (LIE) [29, 30, 31] , which were used to perform post-docking processing. Although these methods are reported to be significantly more robust and more accurate than scoring functions, the accuracy is less than that usually required in typical lead optimization applications to differentiate highly similar compounds.
Attempts have been made to reduce the weakness of a single scoring function. In 1999, Charifson et al. introduced a consensus scoring method [20] . Many studies have suggested that employing consensus-scoring approaches can improve the performance by compensating for the deficiencies of the scoring functions with each other [19, 20, 21, 22] . Although the rationale for consensus scoring is still a subject of study, it has become a popular practice. Compared with the calculation of free binding energy mentioned above, the combination of three or four individual functions to perform consensus scoring is a relatively cheap computational method. Wang et al. carried out an idealized computer experiment with three different ranking strategies (''rank-by-number'', ''rankby-rank'', and ''rank-by-vote'') to explore why the consensus scoring method performs better than the single scoring function [32] . However, the application of consensus scoring approaches is not always practical under ideal conditions because many obstacles prevent us from obtaining satisfied enrichment rates. These obstacles are as follows: (1) the binding scores calculated by the different scoring functions are typically given in different units and signs; (2) the scoring functions employed in consensus scoring often come from different categories; and (3) the linear relationship between many scoring functions (i.e., one scoring function can be expressed linearly by one or some other scoring functions).
In addition to the three ranking strategies introduced by Wang et al., several groups employed another consensus scoring method involving the linear combination of several scoring functions. In the study by Guo et al., five commercially available scoring function were weighted and summed to build a consensus score [33] by training with a 53-molecule set. Verdonk et al. also employed a linear combination of three scoring functions to rerank the compounds [34] . Although an improvement was found for this consensus scoring method, the correlation between the scoring function and the experimental binding affinity is relatively poor. For a quantitative linear combination of the original scoring functions, the method for determining the appropriate weighting factors (correlation coefficients) for each scoring function is a complex problem.
In this study, we present an alternative method, principal component analysis (PCA) [35, 36, 37] , for performing a linear combination of multiple scoring functions, formulating a modified ranking score and PCscore, and re-scoring and re-ranking the compounds after virtual screening. PCA is a powerful tool for pattern recognition, classification, modeling, and other aspects of data evaluation [36] . In addition, PCA is a linear transformation technique used to simplify a data set by reducing the dimensionality of multivariate data while preserving as much of the relevant information as possible. The principal components (PCs) are linear combinations of the original variables. The linear coefficients of the inverse relationships of linear combinations are called the component loadings. It represents the correlation coefficients between the original variables and the PC. In the present study, the first principal component (PC1) accounts for the maximum variance (eigenvalue) in the original dataset. The second principal component (PC2) is orthogonal (uncorrelated) to the first one, and it accounts for most of the remaining variance. This procedure is continued until the total variance is accounted for. The method of PCA makes use of intercorrelations that originate from the covariance matrix of the variables.
This work was performed as part of a project aimed at identifying strong, selective inhibitors of b-secretase (BACE-1) to overcome the shortcomings of the existing drugs to treat Alzheimer's disease (AD) [38, 39, 40] . It is generally accepted that Alzheimer's disease is caused by extracellular senile plaque deposition and that the intracellular formation of neurofibrillary tangles in the brain. b-amyloid peptides, which form the senile plaques, are formed by the action of the b-secretase and csecretase enzyme on the amyloid precursor protein (APP) [41, 42, 43] . The design of a lead compound that can inhibit APP binding to the active site of BACE-1 will prevent the cleavage of APP from the b-amyloid peptide and thus eventually prevent senile plaque formation [44, 45] . In the present study, the training set is composed of 50 confirmed BACE-1 inhibitors and 9950 inactive compounds [46, 47, 48] . Three rank-based virtual screening methods, individual scoring, conventional consensus scoring and PCA scoring were examined to identify BACE-1 inhibitors. To validate the efficacy of PCA ranking method, after virtual screening of 113,228 compounds (Sigma-AldrichH corporate chemical directory) [49] and the BACE-1 fluorescence resonance energy transfer (FRET) assay, we found two drug-like and lowmicromolar inhibitors.
For the training set, in order to reduce artificial enrichment [34] , a subset of WDI (World Drug Index) was specifically designated as inactive molecules. Firstly, WDI was filtered to eliminate compounds whose molecular weight was either less than 200 or greater than 800. In addition, the compounds whose log P is larger than 7 and the number of rotatable bonds is more than 15 should be abandoned. Secondly, the remaining 37,843 WDI compounds were subjected to diverse selection based on 2D UNITY fingerprints. The dissimilarity selection was performed by the Selector module in SYBYL, which resulted in 9950 compounds with a maximum Tanimoto index of 0.69. The active set was compiled from a diverse selection of 50 BACE-1 inhibitors from the total compounds available in the Prous Integrity Drugs & Biologics database [50] . This library of 10,000 compounds as a training set has an active content of 0.5%, which mimics real-life screening situations.
In order to extend the application of the present study, a total of 113,228 compounds (Sigma-AldrichH corporate chemical directory, Z272000, 1997) [49] were used as the test set. Both the training set and test set compounds were stored as a SYBYL SLN list and converted to SYBYL mol2 format using Concord [51].
The ligand-bound (1W51) structure of BACE-1 was used [52] . The procedure used to prepare the structure was as follows: hydrogen was added, the protonation states were assigned, and a highly limited optimization was performed to reduce bad contacts and the overall strain energy in the protein structure. The aspartate located on the active site was adjusted to an ideal protonation state, the Asp32 was protonated, the Asp228 was ionized [46] .
Virtual screening experiments were performed using the Surflex docking program [7, 53, 54] with an empirical scoring function (based on the Hammerhead docking system). The empirical scoring function has been updated and re-parameterized with additional negative training data along with a search engine that relies on a surface-based molecular similarity method. Standard parameters were used as implemented in the SYBYL software (version 8.1) [51]. The search strategy of Surflex employs an idealized ligand (called protomol), which utilizes various molecular fragments. Molecular fragments were tessellated in the active site and optimized based on the scoring function. The search algorithm utilized the morphological similarity function, which is evaluated between the protomol and the putative ligands. For the docking algorithms, a post-dock minimization procedure was applied using the BFGS quasi-Newton method and an internal Dreiding force field. For each compound, the top 30 ranked poses were saved.
Five scoring functions in SYBYL, including D_Score [55] , G_Score [9] , ChemScore [56] , Surflex_Score [7, 53] , and PMF_Score [57, 58] , were applied to extract the stored poses. Next, five pose groups were produced, with each pose group containing 10,000 compounds.
For pose ranking, we use 12 scoring functions including the five scoring functions (D_Score, G_Score, ChemScore, Surflex_Score, and PMF_Score) from the SYBYL software and the seven scoring functions (LigScore1 [17] , LigScore2 [17] , PLP1 [59] , PLP2 [59] , jain [60] , Ludi_1, and Ludi_2 [61, 62] ) from the Discovery Studio software (version 2.1) [63] . Although the five pose groups generated by Surflex have been post-minimized using the internal Dreiding force field, these five pose groups were further minimized in the protein environment using the CFF force field [64] when the seven scoring functions were used for scoring by the Discovery Studio software.
All high-throughput docking calculations were performed on a Linux cluster using the CentOS 5.4 operating system.
In this study, we adopted the ''rank-by-number'' strategy in the consensus scoring to combine the results of multiple scoring functions. The ''rank-by-number'' strategy was previously found to outperform the ''rank-by-rank'' and ''rank-by-vote'' strategy because it can summarize most of the information [32] . For the ''rank-by-number'' strategy, the consensus score of each binding pose is an average of the values determined by each of the individual scoring functions in a given consensus scoring scheme. With this strategy, a moderate number of scoring functions (i.e., three or four) have been proposed to be sufficient for significantly improving the results. Therefore, we chose 4 of the 12 scoring functions (D_score, jain, and Ludi_1, Surflex_Score) to perform consensus scoring in the present study.
Because the binding scores calculated by the different scoring functions are typically given in different units, it is almost impossible to compute consensus scores simply by summing up the binding scores determined by each of the individual scoring functions. Therefore, we scaled the binding scores of each scoring function to unit variance and centered (i.e., the mean value is zero, the standard deviation is one). The Z-scaled scoring function values (ZScore) are computed by
where f i is the scoring value of a certain scoring function, m is the mean value and s is the standard deviation of this scoring function observed for the entire test set. The consensus score in a certain pose group is the average of ZScore by 4 individual scoring functions mentioned above in the given consensus-scoring scheme.
We describe PCA mathematically as described below. Consider p random variables X 1 , X 2 , …, X p , the original system can be rotated to form a new coordinate. Let S be the covariance matrix associated with the random vector X9 = [X 1 , X 2 , …, X p ]. The corresponding eigenvalue-eigenvector pairs are (l 1 , e 1 ), (l 2 , e 2 ), …, (l p , e p ), and the ith principal component is given by: PC i~e 0 i X~e i1 X 1 ze i2 X 2 z:::ze ip X p , i~1,2,:::,p ð1Þ
Then Var(PC i )~e 0 i Se i~li i~1,2,:::,p ð2Þ
Thus, the principal components are uncorrelated, and their variances are equal to the eigenvalues of S.
Another property of the principal components is:
Var(X 1 )z:::zVar(X p )~l 1 zl 2 z:::zl p~V ar(PC 1 )
Then the proportion of the total population variance due to the kth PC is:~l k =(l 1 zl 2 z:::zl p ) k~1,2,:::,p:
Consequently, if most of the total population variance for large p can be attributed to the first two or three components, then these first two or three components could serve as a substitute for the original variables with a minimal loss of information. Moreover, if the weight of the last PCs occupied a highly trivial part of the total population variance, then the last PCs can be neglected (i.e., set to zero).
In the present study, there were five extracted pose groups, and we used only eight scoring functions, which included LigScore1, PLP1, jain, Ludi_1, D_Score, G_Score, ChemScore, and Sur-flex_Score, to perform the PCA for each group (The details are mentioned in the results section). Thus, for the training set, the eight scoring functions were used as the variables (i.e., columns of the matrix) and 10000 compounds were arranged in the rows of the matrix. Then the 1000068 correlation matrix was established. Because the scoring functions in our test produce binding scores with different units and signs, the signs of the binding scores produced by LigScore1, PLP1, jain, Ludi_1, D_Score, G_Score, and ChemScore were reversed to ensure that positive binding scores always indicated higher binding affinities. All of the binding scores were scaled to unit variance and centered. Thus, each column of data had an average of zero and a standard deviation of one.
For each of the five scoring function extracted poses, we have calculated the eigenvalues and cumulative contribution rate. The first three principal components were extracted. Each principal component is a linear combination of eight Z-scaled scoring functions, which formulate a modified ranking score function, PCscore. PCscore is set to re-score and re-rank the extracted poses from each of the five scoring functions. PCscore can be written as follows:
WipÃZScore p i~1,2,:::,p
Where the n terms, ZScore p , are the Z-scaled scoring function, and the coefficients, w ip , are the loading values (i.e., the elements of p principal component eigenvector e p ). For example, for PC1score
The linear coefficient values (loading values) for w 11 , w 12 … w 18 were the elements of the first principal component eigenvector, e 1 .
In the present study, an SPSS version 16.0 statistical analysis package (SPSS Inc.) was used to normalize and calculate the principal components for all of the scoring data.
After virtual screening of the test set, 40 chemical compounds (Sigma-Aldrich Co. LLC) were purchased for experimental test through fluorescence resonance energy transfer (FRET) assay based on the results of conventional consensus scoring and PCA scoring. According to the previously described method [65] , during the assay, compounds were diluted to 8 different concentrations and incubated 60 minutes at room temperature. Additional measurements were performed in the presence of detergent or with an incubation time of only 3 min to check for nonspecific effects (e.g., compound aggregation [66, 67] ). Briefly, fluorescence progress curves of 30 mL reaction volumes were measured on a Gen5 TM ELISA reader (BioTekH Instruments, Inc.) upon excitation at 545 nm and emission at 580 nm in 384well microtiter plates (Corning, 3654). Linear regression analysis was calculated with the SPSS 16.0 software.
Upon docking 10,000 compounds of the training set with Surflex, every compound yields 30 poses in the active pocket of the target (1W51), and no solution was found on the outside of the active pocket. After docking, we used five scoring functions to extract the pose and twelve scoring functions to rank the extracted poses resulting in 60 different scoring combinations. The top 1% of the ranked database was set as the threshold value, that is, the evaluation of the effectiveness of the scoring protocols involved numbering the actives for the top 100 candidates. The enrichment rates of the scoring protocols are presented in Table 1 .
The best-scored pose was always used to represent the plausible binding mode of a particular compound. In many cases, the pose varied from one to another as dictated by the separate scoring functions. Inspection of each scoring function in Table 1 indicated that the quality of the extracted poses is similar. No single scoring function outperforms the others with respect to the extraction. The Surflex_Score provided reliable poses that were ranked best by D_Score and jain. The D_Score and ChemScore provided reliable poses that were ranked best by Ludi_1.
For the pose ranking, it appears that the ranking by Ludi_1 retrieved more actives than the other scoring functions. Ludi_1 retrieved 20 inhibitors with D_Score and ChemScore pose extraction and 18 inhibitors with Surflex_Score and G_Score pose extraction. Ludi_1 was derived by empirically fitting a set of protein-ligand complexes with experimentally measured binding affinities. It is a sum of the five contributions including hydrogen bonds, perturbed ionic interactions, lipophilic interactions, the freezing of internal degrees of freedom of the ligand, and the loss of translational and rotational entropy of the ligand.
At the same time, D_Score also performs well for ranking the docking poses. It retrieved 20 inhibitors with Surflex_Score and 19 inhibitors with PMF_Score pose extraction. This good performance can be attributed to D_Score providing the most accurate approximation of the binding energy where both the electrostatic and hydrophobic contributions to the binding energy are counted. In addition, a distance-dependent dielectric attenuates the chargecharge, and other polar interactions were considered.
PMF_Score also provided reliable poses that were ranked best by D_Score. It yields 19 actives in the top 1% of the ranked list. However, it failed to rank any sensible docking poses regardless of what poses were extracted by itself or by the other scoring functions. Thus, for the BACE-1 target, PMF_Score appears to be more capable of accurately docking and correctly identifying the true binding mode, but the disadvantage of PMF_Score is the enrichment of active compounds.
Inspection of Table 1 demonstrates that two paired scoring with LigScore1 & LigScore2 and PLP1 & PLP2 retrieved an equal number of active compounds. It is not surprising that both Ligscore1 & Ligscore and PLP1 & PLP2 use the same scoring functions with only slightly different algorithms and parameters sets [68] .
There are three different versions of Ludi (i.e., Ludi_1, Ludi_2, Ludi_3) [61, 62, 69] . According to the Discovery Studio user manual, only the weight factors employed by Ludi_2 for each term are derived by fitting to experimentally determined binding affinities. In fact, all three versions were tested for enrichment rates of virtual screening against BACE-1 in our study, and we found that Ludi_1 outperforms the other two versions.
Prior to re-ranking the results from the virtual screening using consensus scoring and PCA, the intercorrelations between the scoring functions mentioned above were investigated. The original data of each scoring function were scaled to unit variance and centered. The correlations between the binding scores computed by the 12 scoring functions are summarized in Table 2 .
For the four scoring functions (i.e., Ligscore1, Ligscore2, PLP1 and PLP2), Table 2 exhibited a high correlation between any two of them. The correlation was higher for LigScore and PLP (R = 0.97,0.98) because they belong to the empirical scoring function category and the sum of the pairwise linear potentials between the ligand and the protein heavy atoms with parameters is dependent on the interaction type.
In addition, Ludi_1 and Ludi_2 also exhibited a very high correlation (R = 0.911). However, the correlation coefficients between Ludi and the other functions, such as PLP and LigScore, were smaller because the master equations that describe the binding free energy used in Ludi are different from those used in the PLP and LigScore functions. In addition, the algorithms vary for the same term in the master equation, such as hydrogen bonding and hydrophobic effect.
Furthermore, there was a higher correlation between G_Score and D_Score (R = 0.771). We were not surprised by this result because both functions are in the same category, both of their algorithms adopted force-field-based methods that estimate the enthalpic contribution upon binding, and both of them use a very similar treatment of the energy terms.
As depicted in Table 2 , moderate correlation was exhibited by Surflex_Score and either the D_Score or the G_Score function; between D_Score and the ChemScore, LigScore1, LigScore2, PLP1, or PLP2 function; and between Jain and the LigScore1, LigScore2, or PLP2 function. This is consistent with virtually all of the scoring functions being designed to reflect the basic features in protein-ligand interactions including hydrogen bonds and hydrophobic contacts. Moreover, the binding scores computed by these scoring functions are all correlated, to some extent, to the known binding constants. Therefore, some intercorrelation between them is natural.
PMF shared the least with all of the other scoring functions. Its unique knowledge-based algorithm parameterized using crystal complexes is different from the rest of the scoring functions being considered [57, 58] .
The hit-rates observed among the top 1% of the screening set using the ''rank-by-number'' strategy are shown in Table 3 . By comparing the performance of all of the consensus ranking schemes tested, it appears that the consensus ranking does statistically outperform the best of the individual scoring function. The Surflex_Score pose extracted group produced 24 hits in the top 1% of the screening set when the quadruple-scoring scheme was applied. The improvements are not trivial. The best individual scoring function, jainScore, produces only 20 hits in the top 1% of the screening set.
Our results are in agreement with the previous study, which suggested that, in theory, combining multiple scoring functions should always provide improved performance over individual scoring functions in simulated virtual screening experiments [32] . According to the present results, we cannot definitively conclude that more scoring functions result in a better performance. For example, application of double-scoring schemes (e.g., Surflex_S-core&D_Score) could also obtain 24 hits in the top 1% of the screening set, which is the same result obtained using the quadruple-scoring scheme. However, it is important to note that double-scoring schemes do not outperform the best individual scoring function in all cases. For example, Surflex_Score&jain could obtain only 19 hits, which is slightly less than the 20 hits obtained from the single scoring function, jain. Therefore, it is largely unpredictable which combinations of scoring functions would produce the optimal results. In practice, it is better to test all possible combinations of scoring functions on the appropriate samples.
Some studies have shown that consensus ranking does not outperform the best individual scoring function [70, 71] . They argued that if one knew in advance which scoring functions worked best for a given target, the better performance could be achieved using this scoring function alone and by concentrating on only the highest ranking compounds. Given the contradiction between their arguments and our results, we explained as followed:
Firstly, the three scoring functions (D_Score, jain, Ludi_1) that we chose performed the best in single scoring. It is important to consider which scoring functions should be chosen to perform consensus scoring. We used an additional four scoring functions to perform consensus scoring, but the performance was not as good as the four functions that we chose. Due to the variation in the performance of the different scoring functions, blindly choosing scoring functions to perform consensus scoring will decrease the enrichment rates. Secondly, the four scoring functions that we chose were independent of each other. It is reasonable to expect that an effective consensus scoring scheme would combine complementary scoring functions rather than highly correlated ones. As indicated in Table 2 , if the consensus scoring schemes contained Ligscore1 and Ligscore2 as well as PLP1 and PLP2, it would perform poorly compared to the other schemes. Thirdly, Verdonk et al. performed a computational experiment on the simulated effect of consensus ranking with an increasing number of scoring functions using the rank-by-number protocol [34] . They noted that if the first scoring function performs well (standard deviation = 1.0), then adding additional scoring functions (standard deviation = 3.0) to perform consensus ranking can reduce the enrichment rates compared to the most accurate single scoring function. The main reason for this phenomenon was that noise was added to the protocol. However, if all of the scoring functions have a standard deviation of 2.0, then adding extra scoring functions to the consensus ranking protocol always improves the enrichment rates. In our study, all of the binding scores were scaled to unit variance and centered, which was consistent with the results reported by Verdonk et al [34] .
In summary, our present results suggest that application of triple-scoring and quadruple-scoring schemes are more robust and accurate than any single scoring procedure.
Principal component analysis (PCA) can extract information from large-scale scoring data and decompose multiple scoring functions into one or two scoring functions, which can be used to re-score and re-rank the compound binding poses. As mentioned above, the PLP1&PLP2, ligscore1&ligscore2 and Ludi_1&Ludi_2 have high relative between each other. In addition, PMF_Score failed to rank any sensible docking poses. Thus, we do not use these four scoring functions in the following study. We constructed five 1000068 matrices using the remaining eight scoring functions as the matrix column and the 10000 compounds as the raws. Then the matrix was transformed such that each column of data had an average of zero and a standard deviation of one. As observed from Table 4 , we can derive eight uncorrelated descriptors (the principal components) from each scoring matrix. The weight of each principal component was determined based on their contribution rate to the variance (eigenvalues, l). We found that the first three principal components (PC1, PC2, PC3) account for .80% of the total variance for each pose group. The PC4, PC5, PC6, PC7, and PC8 could be omitted in further studies due to their trivial contribution to the total variance. This result is in agreement with the aim of introducing PCA to significantly minimize the number of variables and to omit the principle components with low variance that will not affect the total variance.
These principal components may lack physical meaning by themselves because they may act as statistical descriptors. Nevertheless, we could still assess the physical meaning of each PC according to the energy terms of each scoring function. To the best of our knowledge, the physical meaning of PC1 could be attributed to van der Waals interactions, the physical meaning of PC2 could be attributed to electrostatic interactions, and the physical meaning of PC3 could be attributed to the hydrophobic interactions between the protein and ligands.
The loadings express how well the new abstract principal components correlate with the old variables. Loading values (i.e., correlation coefficients) .0.7 are marked in boldface type in Table 5 . PC3 accounts for approximately 11% of the total variance. It is interesting to note that PC3 negatively correlates with Surflex_-Score, D_Score, G_Score, and ChemScore (SYBYL software) but positively correlates with PLP1, LigScore1, jain, and Ludi_1 (Discovery Studio software).
The first two PC loadings against each other are shown in Figure 1 . Because the PCA is invariant to the mirroring through the origin, the data shown here indicate that there is a significant correlation between LigScore1 and PLP1. Likewise, the correlation between G_Score and D_Score in relation to the data is unambiguous and significant. There is no need to measure and evaluate all of the variables to achieve the same characterization in further studies. It is sufficient to measure one variable per group.
The present results show that Jain contains nearly the same information as D_Score and has low loading on PC2. Because PC2 could be attributed to electrostatic interactions between the protein and the ligands, Jain has no significant influence on the electrostatic interactions between the protein and the ligands. Among the eight loads in PC2, Ludi_1 exhibits the maximum Table 2 . The Correlation Matrix of each scoring function (poses extracted by Surflex_Score). value (Figure 1 ), which means Ludi_1 plays a significant role in the description of PC2. According to Eq 5, the PC is a linear combination of multiple original variables. Therefore, we can formulate the first three PCscore scoring functions for each pose group from the data in Table 5 . For example, for the poses extracted by Surflex_Score, the first PC scoring function was: 
Next, the docked compounds are re-scored and re-ranked using the PCscore scoring functions as mentioned above. The enrichment rates are also determined by noting the numbers of active compounds retrieved in the top 1% of the ranked database (Table 5) . When comparing the enrichment rates from PC1score to the results obtained from a single scoring function or conventional consensus scoring functions, PC1score exhibited better performance for the enrichment rates regardless of the scoring function employed to extract the compound pose. For example, PC1score yields 26 active compounds in the Surflex_Score and PMF_Score pose group, which outperforms both the single scoring function with 20 or less active compounds and the consensus scoring method with 24 active compounds. As indicated in Table 5 , application of PC1score results in more active compounds than the application of PC2score and PC3score for each of the five pose group due to the descriptiveness of the first principal component, which shares the maximum amount of the whole variance followed by the decreasing descriptiveness of the other PCs. We did not obtain any active compounds in the top 1% of the ranked database using PC3score because the values of the eigenvalue of PC3 were ,1.
The PCA can illustrate the relationship between the different compounds and the different scoring functions. The compounds can be plotted in the space defined by two PCs (score plot, Figure 2 ), which identifies active compounds as a function of inactive compounds. The values of the scores can be understood as the values of the compound in the new variable space, i.e., the principal component space. In Figure 2 , active compounds are depicted as red circles, and inactive compounds are depicted as black squares. The results showed that most of the data were scattered along the PC1 axis. The scattering variation along the PC1 axis is larger than that along the PC2 axis, which corresponds to the values of eigenvalue and reflects the descriptive power of first two PCs scores.
Because the new variable space is normalized with zero mean, the most active compounds, which are farther from the origin, have values significantly different from the mean and can be considered outliers. Moreover, we found that the scattering positions of the true BACE-1 inhibitors are located on the right side of the PC1 axis indicating that PC1 plays a significant discriminating role among active and inactive compounds. As for the PC2 axis, all of the data were scattered in a narrow area from -3 to 3, and the discriminating power among active and inactive compounds was weaker. 
To further investigate the validity of docking based virtual screening, after virtual screening of 113,228 compounds against BACE-1 by Surflex, we employed conventional consensus scoring and PCA scoring protocol to select compounds for experiment test against BACE-1. Standing the view of economic point, the number of compounds to be tested in computational docking studies should be restricted in a smaller and reasonable range, therefore, we used several filters to the select the final compounds in Surflex_score extracted pose for experiment.
In an initial attempt, we employed conventional consensus scoring protocol to select the potential inhibitors. Firstly, we selected the top 300 compounds according to the ranking results of the conventional consensus scoring protocol, e.g., quadruplescoring scheme (Surflex_Score&D_Score&jain&Ludi_1); Secondly, visual inspection has been given to all individual complexes for the top 300 compounds, Surflex provides interaction information between the protein and ligand for each docking experiment, only those compounds with interactions to the catalytic residues (Asp32 and Asp228) and other relevant residues are extracted; Thirdly, to remove unsuitable compounds that would not reach and pass the clinical trials due to undesired and toxic properties, the so-called Lipinski ''Rule-of-five'' [72] , a very popular method was used to evaluate the drug likeness of a candidate structure. Finally, 20 drug-like compounds were selected to purchase from Sigma-Aldrich Co. LLC. By the BACE-1 fluorescence resonance energy transfer (FRET) assay, disappointingly, no inhibitor was found among the compounds selected by the conventional consensus scoring protocol.
Based on the theory that PCA can summarize most of the information from the original scoring functions, we employed PC1score to re-rank the 113,228 Surflex_score extracted poses, as mentioned above, PC1score is a linear combination of eight scoring functions (Surflex_Score, G_Score, D_Score, ChemScore, LigScore1, PLP1, Jain and Ludi_1). By the same filter protocol as the conventional consensus scoring, another 20 drug-like compounds were select for purchase among the top 300 compounds. Excitingly, this time two compounds (S450588 and 276065), with a remarkable 10% hit rate, emerged as the BACE-1 inhibitors in the low-micromolar range, showing IC 50 values of 51.6 and 85.3 mM, respectively ( Table 6 ). The chemical structures of these two compounds were showed in Table S1 .
As depicted in Figure 3A , after compound 1 docking into 1W51 structure, the protonated 2-NH 3 group of the lysine moiety form hydrogen bond with Asp228, Gly230 and Thr231, respectively, the 6-NH group form hydrogen bond with Tyr198. The benzyl ring (P1) fills the S1 pocket shaped by the Tyr71, Phe108, and Trp115 residues, while carbobenzyloxy moiety (P29) fills the S29 pocket shaped by the Tyr71, R128, and Y198 residues, so as to allow the carbonyl group to form hydrogen bond with the Thr72 residue, the benzyl group to establish a cation-p interaction with the guanidine group of Arg128.
As depicted in Figure 3B , in the catalytic site, for the small size and symmetric overall shape of compound 2, one of the hydroxyl group of the tartaric diamide core are involved in hydrogen bonds with the side chain of the catalytic Asp32 and Asp228, respectively, whereas the other hydroxyl group form hydrogen bond with Thr72, and one of the amide group form hydrogen bond with Gly230. Both sides of compound 2 are benzyl groups, one of the benzyl group occupy the S2 pocket shaped by the Asn233, Arg235 and Ser325 residues, the other benzyl group occupy the S29 pocket, establish a cation-p interaction with the guanidine group of Arg128.
BACE-1 is one of the major Alzheimer's disease target [38, 39, 40] . To find novel BACE-1 inhibitors, a lot of academic research centres and pharmaceutical industries are quite active in this field. Merck research group performed in vitro highthroughput screening (HTS) and found a single molecule (a 1,3,5 trisubstituted benzene) as a hit from a multi-million compound library [73] . Johnson and Johnson also reported a novel cyclic guanidine screening lead, the initial screening lead had an IC 50 value of 900 nM [45] . Astex Therapeutics has taken a fragment-based lead generation approach [74] . After the virtual screening of a fragment library, a small number of potential structures were soaked with BACE-1 crystals in anticipation of obtaining a co-crystal with the enzyme. Huang et al. performed in silico Screening of 180,000 small chemicals, they found 10 diacylurea inhibitors showed an IC 50 value lower than 100 mM in a enzymatic assay and four of them were cell penetrant (EC 50 ,20 mM) [75] .
Despite the availability of many reliable in silico approaches and robust in vitro commercially available assays, discovering BACE-1 inhibitors still remains a challenging task. In the present study, based on the virtual screening of 10,000 compounds of training set, the PCA approach yielded consistently superior rankings compared to conventional consensus scoring and single scoring. By virtual screening of 113,228 compounds, and application of PCA approach to re-rank the score list, two drug like BACE-1 inhibitors were emerged as an effective low-micromolar inhibitors. It suggested that the application of PCA provides a more robust strategy for ranking compounds. The advantages of PCA are as follows.
First, PCA is efficient. For each five pose group, the application of PCA can result in superior enrichment of known inhibitors compared to either the conventional consensus scoring or the best individual scoring. In addition, the application of PCA for postprocessing of the scoring data from virtual screening was not timeconsuming. Second, PCA is reliable. PCA is mainly useful when there is limited knowledge about the target and its inhibitors. If we have no idea which scoring function would return the best enrichment rates (i.e., several known active compounds are required to determine the best scoring functions), then adopting PCA to formulate a new scoring function can provide better performance than blindly using a scoring function or some combination scoring functions when performing virtual screening 50 , and training with a data set were not necessary. When a training set was available, there are several other groups that perform a different type of post-docking processing using statistical methods and data mining. Wilton et al. discussed the use of several rank-based virtual screening methods, such as binary kernel discrimination, similarity searching, sub-structural analysis, support vector machine (SVM), and trend vector analysis, for prioritizing compounds in lead-discovery programs [76, 77] . Jacobsson et al. employed three different multivariate statistical methods including PLS discriminant analysis, rule-based methods, and Bayesian classification to analyze multidimensional scoring data from four different target proteins (i.e., the estrogen receptor R (ERR), matrix metalloprotease 3 (MMP3), factor Xa (fXa), and acetylcholine esterase (AChE)). The classifiers that they built showed that the precision is approximately 90% for three of the targets and approximately 25% for acetylcholine esterase for correctly predicting an active compound [78] . The difference between our work and their's is that we do not need a training set because PCA is a form of unsupervised learning and relies entirely on the input data itself. In addition, PCA is simpler than the methods mentioned above (SVM, trend vector analysis, PLS discriminant analysis, rule-based methods, and Bayesian classification).
With eight different scoring functions, Terp et al. docked a set of known inhibitors to three different matrix metalloproteases. They obtained scores analyzed using PCA and partial least-squares methods (PLS) [79] . The regression model they built has a good q 2 for predicting the activity of active compounds. The major difference between the present work and the work performed by Terp et al. is that we performed structure-based virtual screening on both active and inactive compounds. Terp et al. included only known inhibitors to quantitatively predict the binding affinity.
They did not discuss whether the docking scores have been calculated from a docking mode of an inactive compound that does not actually bind. In the virtual screening process, more attention is focused on how to identify promiscuous active compounds in a database of mainly inactive compounds rather than on how to rank a set of known binders (i.e., predicting the binding affinity of the different active compounds).
It should be emphasized the necessity of experimental validation for potential researchers, because no ranking method may help if not associated with verification in the experiment. In an initial attempt, we applied the conventional consensus scoring method to re-rank the score list and experimental test through BACE-1 FRET assay, no inhibitor was found. However, when we applied the PCA scoring method and experimental test through BACE-1 FRET assay, a remarkable 10% hit rate was achieved. On this basis, we summed up some experience as followed: when virtual screening of a new chemical database, the potential researchers usually do not know which kind of individual scoring function work best for the target protein, furthermore, for consensus scoring protocol, they are uncertain which kind of scoring functions should be used to combine for getting the best enrichment rates. Once trapped in this dilemma, the researchers could use PCA scoring protocol to re-rank the results from the virtual screening, a prominent advantage of application of PCA scoring protocol can summarize most of the information from the original scoring functions and improve the enrichment rate, which has been proved to be robust and reliable in the present study.
In conclusion, although the PCA approach is not intended to improve all aspects of virtual screening, such as generating more accurate binding poses, it extends conventional consensus scoring in a quantitative statistical manner, therefore, it has great potential for use in the virtual screening process. Future experiments are needed to further analyze the performance of PCA for other receptor binding sites. We believe that the two low-micromolar inhibitors described here may represent a starting point for finding potent and selective molecules capable of preventing BACE-1 activity for the treatment of Alzheimer's disease.  2,500-year Evolution of the Term Epidemic The term epidemic (from the Greek epi [on] plus demos [people]), first used by Homer, took its medical meaning when Hippocrates used it as the title of one of his famous treatises. At that time, epidemic was the name given to a collection of clinical syndromes, such as coughs or diarrheas, occurring and propagating in a given period at a given location. Over centuries, the form and meaning of the term have changed. Successive epidemics of plague in the Middle Ages contributed to the definition of an epidemic as the propagation of a single, well-defined disease. The meaning of the term continued to evolve in the 19th-century era of microbiology. Its most recent semantic evolution dates from the last quarter of the 20th century, and this evolution is likely to continue in the future. A t the start of the 21st century, epidemics of infectious diseases continue to be a threat to humanity. Severe acute respiratory syndrome, avian influenza, and HIV/AIDS have, in recent years, supported the reality of this threat. Civil wars and natural catastrophes are sometimes followed by epidemics. Climate change, tourism, the concentration of populations in refugee camps, the emergence of new human pathogens, and ecologic changes, which often accompany economic development, contribute to the emergence of infectious diseases and epidemics (1) . Epidemics, however, have occurred throughout human history and have influenced that history. The term epidemic is ≈2,500 years old, but where does it come from?
When works that put forward new ideas are translated, determining the original terminology (in Ancient Greek in this case) is not easy. In 430 BC, when Hippocrates was collecting the clinical observations he would publish in Epidemics, his treatise that forms the foundation of modern medicine, at least 3 terms were used in Ancient Greece to describe situations that resembled those described by Hippocrates: nosos, phtoros, and loimos (2) .
Nosos, meaning disease, was used by Plato in the 4th century BC and clearly had the same meaning 2 centuries earlier in the works of Homer and Aeschylus. Nosos encompasses disease of the mind, body, and soul: physical, including epilepsy, and moral (i.e., psychological and psychiatric). Phtoros or phthoros means ruin, destruction, deterioration, damage, unhappiness, and loss, after war for example. The word was frequently used by Aeschylus and Aristophanes, was known in the 8th century BC, and was later used by Plato and Thucydides. Its meaning has remained general. Bailly translates loimos as plague or contagious scourge. Used by Esiodus in the 7th century BC and later by Sophocles and Herodotus, this term is ancient. Its translation as plague should be interpreted in the sense of a scourge rather than as the disease plague. In the Septuagint, a translation of the Old Testament into Greek by 70 Greek Jews from Alexandria, this word is used in the book of Kings to describe the 10 plagues of Egypt.
But the term epidemic already existed in 430 BC. The Greek word epidemios is constructed by combining the preposition epi (on) with the noun demos (people), but demos originally meant "the country" (inhabited by its people) before taking the connotation "the people" in classical Greek. Indeed, the word epidemios was used by Homer, 2 centuries before Hippocrates, in the Odyssey (canto I, verses 194 and 230), where it was used to mean "who is back home" and "who is in his country" in contrast to a voyager who is not:
, "because someone said that your father was back (home)" (canto I, verse 194). In this context, epidemios means indigenous or endemic. In the Iliad, Homer confirmed this meaning (canto XXIV, verse 262), by using 2,500-year Evolution of the Term Epidemic also polemos epidemios to mean civil war: , "this one who liked passionately the frightening civil war" (canto IX, verse 64). Later, Plato and Xenophon (400 BC) used the word to describe a stay in a country or the arrival of a person:
, "a Parian who, I learned, was in town" (Plato, Apology, chapter I, paragraph 38). The verb epidemeo was used by Thucydides (460 BC-395 BC) to mean "to stay in one's own country," in contrast to apodemeo, "to be absent from one's country, to travel." For Plato, epidemeo meant "to return home after a voyage, to be in town." Later, the orators Demosthenes (384 BC-322 BC) and Eschines (390 BC-314 BC) used this word to refer to a stranger who came to a town with the intention of living there, and the verb epidemeo was used to mean "to reside." Typical of Greek semantics, epidemeo takes its meaning from the result of the action, rather than from the action itself. It relates to something that has already happened, with the implication that it had previously happened elsewhere. Authors before Hippocrates used epidemios for almost everything (persons, rain, rumors, war), except diseases. Hippocrates was the first to adapt this word as a medical term.
Written in the 5th century BC, Hippocrates' Corpus Hippocraticum contains 7 books, titled Epidemics (3). Hippocrates used the adjective epidemios (on the people) to mean "which circulates or propagates in a country" (4). This adjective gave rise to the noun in Greek, epidemia.
We do not know why Hippocrates chose epidemios to title his books instead of nosos, a well-established term meaning disease. Examining the meaning of the term before, during, and after his time may help us understand his choice. Schematically speaking, epidemios (or epidemeo) was used successively to mean "being at homeland" (Homer), "arriving in a country" or "going back to homeland" (Plato), and later "stranger coming in a city" (Demosthenes). Sophocles (495 BC-406 BC) used the adjective in Oedipus Tyrannos to refer to something (a rumor, noise, fame, or reputation) spreading in a country: ???' ???????? ??? ?'?? ???????? ?'??? , "I shall go (to make war) to Oedipus, against his fame which spread (in the country)" (verse 494). Oedipus Tyrannos was written at approximately the same time as Corpus Hippocraticum; consequently, we can infer that during Hippocrates' time, epidemios acquired a dynamic meaning, probably more adapted to describing a group of physical syndromes that circulate and propagate seasonally in a human population (i.e., on the people) than nosos, a term used to describe diseases at the individual level.
How epidemios, meaning "on the people," became adapted to mean "that which circulates or propagates in a country" is a crucial question. This evolution occurred during the second half of the 5th century (450 BC-400 BC), a period of intense activity in Greek literature, particularly with the prolificacy of Sophocles. But while nosos or loimos were frequently used, epidemios was not. In the Perseus Digital Library (www.perseus.tufts.edu), a database that does not yet include Hippocrates' works, the adjective epidemios was used only 9 times, including 4 times in Homer, in the 489 major referenced Greek texts (≈4.8 millions words, 0.02 occurrences per 10,000 words). Its Doric variants epidamos and epidemos were used 3 times. In comparison, nosos (disease) was used 712 times in the Perseus database (1.47 occurrences per 10,000 words). The verb epidemeo was used 144 times, primarily during the 4th century and always meaning "to live in or return to one's own country." This lack of material makes accurately exploring the reasons for the semantic evolution across centuries difficult. In Oedipus Tyrannos, Sophocles qualified the sense of epidemios as it referred to reputation or fame; fame naturally spreads in a country. , "It is a fact that the disease was propagating in the country" (Epidemics, book I, chapter 3). Although Sophocles used epidemios once in that new sense, Hippocrates established a medical meaning for the term.
In Epidemics, books I and III constitute lists of diseases describing clinical cases. Hippocrates compared these cases and grouped them to generate series of similar cases. He adopted a classification approach, initially seeking clinical similarities between cases, thereby discovering, in addition to the notion of epidemic, the more fundamental concepts of symptom and syndrome. However, Hippocrates believed that prognosis was a major aspect of medicine. This belief led him to consider disease a dynamic process with its own progression, a temporal dimension, that represents a first nosologic evolution: syndromic groupings become diseases. Another of the books written by the physician from Kos-Airs, Waters, and Placesdeals with the relationships between diseases and the environment, focusing particularly on the habitat of the patients and the season in which disease occurs. Hippocrates tried to determine the effect of environmental factors on what could be described as the distribution of diseases. He was, thus, more concerned about grouping together winter diseases or autumn diseases or diseases that occurred in a particular place or in persons whose way of life had changed than in identifying a large number of cases of the same disease in winter or autumn, at a particular place, or in association with a particular way of life. For Hippocrates, whose nosologic approach already contained a major element of preoccupation with the environment, the first meaning of epidemic was groups of cases 2,500-year Evolution of the Term Epidemic resembling each other clinically and the second meaning was groups of different diseases occurring at the same place or in the same season and sometimes spreading "on the people." Thus, Hippocrates applied the word epidemios to groupings of syndromes or diseases, with reference to atmospheric characteristics, seasons or geography, and sometimes propagation of a given syndrome in the human population.
Semantic confusion caused the great Emile Littré, who translated Hippocrates' works into French in the first half of the 19th century, to make a nosologic error. Hippocrates described what is known today, since the work of Littré, as the Cough of Perinthus. This account can be found in Epidemics book VI. Hippocrates described coughs that started toward the winter solstice and were accompanied by many symptoms: sore throat, leg paralysis, peripneumonia, problems with night vision, voice problems, difficulty swallowing, difficulty breathing, and aches. When Littré published his translation and commentaries on Epidemics in 1846, he mistakenly considered the Cough of Perinthus to be a single disease (5) . This error made retrospectively diagnosing the diseases of Perinthus difficult, if not impossible. Moreover, as Littré saw this collection of illnesses as a single disease, he essentially turned it into an epidemic, probably because he had the modern sense of the term in mind and thought that Hippocrates had observed and described an epidemic illness unknown to modern medicine (5).
According to Grmek, "Littré took chapter VI, 7.1 as a general description of an epidemic in the sense of this word in the medical language of the 19th century rather than in the sense intrinsic to the works of Hippocrates. In the Corpus Hippocraticum, the noun 'epidemic' designates a collection of diseases observed at a given place, during a given period. A disease described as epidemic, such as epidemic cough, is a condition occurring from time to time in a given place, the appearance of which is closely linked to changes in season and climatic variations from year to year" (5).
Historians of medicine and philologists have over the years attributed the Cough of Perinthus to diphtheria, influenza, epidemic encephalitis, dengue fever, acute poliomyelitis, and many other diseases. However, a French physician named Chamseru, who practiced in the 18th century, almost a century before Littré, finally got to the bottom of what may be meant by the Cough of Perinthus, probably because the term epidemic had not yet taken on the meaning it had in Littré's time. According to Chamseru, the Cough of Perinthus could have encompassed several diseases, among them diphtheria, influenza, and whooping cough (5) .
Thucydides (460 BC-395 BC) interrupted his account of the Peloponnesian War to describe the famous Plague of Athens, which occurred at the start of the summer in 430 BC. This description was long considered among the first descriptions of an epidemic. Indeed, whereas Thucydides used nosos, the term plague, which is used by all the translators of his work, is used in the sense of the Latin term pestis, a term with no clear etymology (4), meaning contagious disease, epidemic, or scourge. The description of the Plague of Athens, like that of the Cough of Perinthus by Hippocrates, is an essential text in the philologic and semantic study of epidemics (5) . We must therefore consider, as for Littré's translation, the meaning that translators have assigned to the original description by Thucydides. Thucydides never used the term epidemic that Hippocrates was in the process of establishing. Under the term nosos, Thucydides described a series of clinical signs, which originated in the south of Ethiopia and propagated throughout Egypt, Libya, and then Greece. Thucydides used the words nosos, kakos (evil), ponos (pain), phtoros (ruin, destruction), and loimos (scourge) to describe what his translators call plagues. In her translation of Thucydides' works (6) , published in 1991, in the chapter titled Second Invasion of Attica: the Plague of Athens (the original Greek work had no title), Jacqueline de Romilly translated nosos as disease or epidemic. Similarly, she translated loimos, kakos, and phtoros as disease or epidemic and the list of clinical signs (the original Greek meant "following these things") as symptoms. de Romilly rendered the text more elegant and accessible to 20th-century readers by this translation but gave the words used by Thucydides their 20th-century meaning rather than the meanings they had in the 5th century BC. Herein lies the principal problem of translation.
But was the Plague of Athens a true epidemic, in the modern sense? The death rate for the disease was extremely high, reaching up to 25% in 1 group of soldiers, and Pericles died of it. Historians have tried to understand the origin of this plague, and various diseases have been suggested, e.g., typhus, measles, smallpox, bubonic plague, ergotism, or an unknown disease. Thucydides wrote that all preexisting diseases were transformed into a plague and that persons in good health were affected in the absence of a predisposing cause (7) . The large number of symptoms and of possible and probable causes rules out the possibility of an epidemic in the modern sense of the term. Instead, the Plague of Athens seems to have been the appearance of a large number of diseases that affected the population at the same time. Plague therefore has the same meaning here as epidemic in the works of Hippocrates. These 2 terms have been used in association or confused throughout history. However, epidemic existed at this time, even if the notion of epidemic as we mean it in modern times had not yet emerged.
After the nonmedical use of the term epidemic by Homer, Sophocles, Plato, and Xenophon, Hippocrates gave it its medical meaning. However, the term has since undergone a long evolution. The adjective epidemios gave rise to the Greek noun epidemia. The Greek term epidemia in turn gave rise to the Latin term epidimia or epidemia. The term ypidime in Medieval French has its origins in these Latin words and went on to become épydime in the 14th century, epidimie in the 17th century, and then epidémie in the 18th century. Not until 22 centuries after Hippocrates, in the second half of the 19th century, were the terms épidémiologie (1855), épidémiologique (1878), and épidémiologiste (1896) coined in French and notions attached to them developed. Of course, at approximately the same time, corresponding terms appeared in the English language. The term epidemic and the terms linked to it therefore required an extremely long time to be constructed. This evolution is representative of the evolution of science and medicine over the centuries and reflects the semantic evolution of the term.
In parallel with the evolution of the term epidemic itself, its meaning also changed over time. If we limit ourselves to the meaning that epidemic has acquired with respect to infectious diseases, we can identify 4 major steps in its semantic evolution in the medical sense. For Hippocrates, an epidemic meant a collection of syndromes occurring at a given place over a given period, e.g., winter coughs on the island of Kos or summer diarrheas on other islands. Much later, in the Middle Ages, the long and dra-matic succession of waves of The Plague enabled physicians of the time to identify this disease with increasing precision and certainty; they began to recognize epidemics of the same, well-characterized disease. Then, with the historic contributions of Louis Pasteur and Robert Koch, epidemics of a characteristic disease could be attributed to the same microbe, which belonged to a given genus and species. The last stage in the semantic evolution of the term epidemic was the progressive acquisition of the notion that most epidemics were due to the expansion of a clone or clonal complex of bacteria or viruses known as the epidemic strain (8) . More recently, microevolution of a clone of a bacterium (the epidemic strain) was shown to occur during an epidemic with person-to-person transmission (9) . The Table summarizes these 4 major stages in the semantic evolution of the term epidemic.
In the second half of the 20th century, epidemic was also applied to noninfectious diseases, as in cancer epidemic or epidemic of obesity. The extension of the meaning to noninfectious causes refers to a disease that affects a large number of people, with a recent and substantial increase in the number of cases. This semantic extension of epidemic also concerns nonmedical events; the term is used by journalists to qualify anything that adversely affects a large number of persons or objects and propagates like a disease, such as crack cocaine or computer viruses.
What can we gain from investigating the origin and meaning of the word epidemic or from studying its semantic evolution? Beyond simply satisfying our curiosity, the slow evolution of the form and meaning of the term suggests that we still have much to learn about the concept of epidemic.
Dr Martin is a bacteriologist and chef de laboratoire at the Pasteur Institute and director of the Pasteur Institute of New Caledonia. His research interests focus on epidemics.
Ms Martin-Granel is a professeur certifiée de lettres classiques and teaches French literature, Latin, and Ancient Greek in secondary school in the south of France. She has recently translated a text by Petrarch into French. Human Bocavirus in Children  To the Editor: Respiratory tract infection is a major cause of illness in children. Despite the availability of sensitive diagnostic methods, detecting infectious agents is difficult in a substantial proportion of respiratory samples from children with respiratory tract disease (1) . This fact suggests the existence of currently unknown respiratory pathogens.
A new virus has been recently identified in respiratory samples from children with lower respiratory tract disease in Sweden (2) . Analysis of the full-length genome sequence showed that this virus is closely related to bovine parvovirus and canine minute virus and is a member of the genus Bocavirus, subfamily Parvovirinae, family Parvoviridae. This virus has been provisionally named human Bocavirus (HBoV) (2) . HBoV in respiratory samples from Australian children was also recently reported (3). Involvement of this new virus in respiratory tract diseases merits further investigation. We have therefore retrospectively tested for HBoV nasopharyngeal samples collected from children <5 years of age hospitalized with respiratory tract disease.
Samples were collected from 262 children from November 1, 2003, to January 31, 2004. The samples were tested for respiratory viruses by using direct immunofluorescence assays with monoclonal antibodies to respiratory syncytial virus; influenza virus types A and B; parainfluenza virus types 1, 2, and 3; and adenovirus. Samples were also placed on MRC5 cell monolayers for virus isolation and tested for human metapneumovirus by reverse transcriptionpolymerase chain reaction (RT-PCR). Nucleic acids were extracted from samples, stored at -80°C, and tested for HBoV DNA by PCR with primers specific for the predicted NP1 gene as previously described (2) . The expect-ed product size was 354 bp. In each experiment, a negative control was included, and positive samples were confirmed by analyzing a second sample. Amplification specificity was verified by sequencing.
Nine (3.4%) samples were positive. Comparison of PCR product sequences of these 9 isolates (GenBank accession nos. AM109958-AM109966) showed minor differences that occurred at 1 to 4 nucleotide positions, and a high level of sequence identity (99%-100%) was observed with the NP1 sequences of the previously identified ST1 and ST2 isolates (2) . This finding indicates that HBoV is a highly conserved virus.
HBoV was the only virus identified in 6 children and was associated with respiratory syncytial virus in 3 other children. An infection with other respiratory viruses was detected among 153 (60.5%) of the 253 HBoV-negative children. The viruses identified were respiratory syncytial virus in 114 (43.5%) samples, human metapneumovirus in 27 (10.3%) samples, influenza A virus in 14 (5.4%) samples, rhinovirus in 4 (1.5%) samples, adenovirus in 2 (0.8%) samples, and parainfluenza virus type 3 in 1 (0.4%) sample. Respiratory syncytial virus was associated with human metapneumovirus in 9 (3.4%) samples.
Clinical characteristics of the HBoV-infected children are shown in the Table. Children infected with only HBoV had mild-to-moderate fevers. Leukocyte counts and C-reactive protein levels were normal or moderately elevated. Chest radiographs obtained for 7 children showed abnormalities such as hyperinflation and interstitial infiltrates. Bronchiolitis was the major diagnosis. Dyspnea, respiratory distress, and cough were the most common respiratory symptoms observed. Four (44%) HBoV-infected children were born preterm, which suggests that these children have an increased susceptibility to HBoVassociated diseases. All children recovered and were discharged within 1 to 6 days.
The 3.4% incidence of HBoV observed in our study is similar to that (3.1%) reported by Allander et al. (2) . HBoV was the only infectious agent identified in 6 children, which suggests that it was the causative agent of the disease. However, more studies conducted in children with and without respiratory disease as well as in adults and elderly persons are needed to better assess the pathogenic role of HBoV. Extended-spectrum β-Lactamaseproducing Enterobacteriaceae,
To the Editor: Since the early 1980s, extended-spectrum β-lactamases (ESBLs) have been the largest source of resistance to broad-spectrum oxyimino-cephalosporins among Enterobacteriaceae (1). Molecular analysis techniques suggest that many ESBLs are derived from mutations in TEM-1, TEM-2, and SHV-1 β-lactamases and that these ESBLs can hydrolyze the extended-spectrum cephalosporins (particularly ceftazidime) and aztreonam (1). Members of a new group of ESBLs have been recently identified (1). Among them, CTX-M-type ESBLs are rapidly expanding and are derived from chromosomal class A β-lactamases of Kluyvera spp. (1, 2) . The CTX-M enzymes are not related to TEM or SHV enzymes, as they share only 40% identity with these ESBLs (2) .These ESBLS are usually characterized by a higher level of resistance to cefotaxime than ceftazidime, except for CTX-M-19 (2) . Most organisms that harbor ESBLs are also resistant to other classes of antimicrobial drugs, such as aminoglycosides, fluoroquinolones, chloramphenicol, and tetracyclines (1, 2) .
Reports concerning the existence of ESBL-producing Enterobacteriaceae in sub-Saharan Africa are scarce. We therefore conducted a study in the Central African Republic to determine the frequency of ESBLs in Enterobacteriaceae isolated at the Institut Pasteur de Bangui and to characterize their bla TEM , bla SHV , and bla CTX-M genes.
From January 2003 to March 2005, all Enterobacteriaceae isolated from human specimens at the Institut Pasteur de Bangui were screened for ESBLs. Antimicrobial drug susceptibility was determined by using the disk diffusion method (Bio-Rad, Marnes la Coquette, France) on Mueller-Hinton agar (MHA) and interpreted according to the recommendations of the Comité de l'Antibiogramme de la Société The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin Cytokine expression patterns of T cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. We showed previously that the epidermal growth factor family member Amphiregulin is expressed by T cell receptor-activated mouse CD4 T cells, particularly Th2 cells, and helps eliminate helminth infection. Here we report a detailed analysis of the regulation of Amphiregulin expression by human T cell subsets. Signaling through the T cell receptor induced Amphiregulin expression by most or all T cell subsets in human peripheral blood, including naive and memory CD4 and CD8 T cells, Th1 and Th2 in vitro T cell lines, and subsets of memory CD4 T cells expressing several different chemokine receptors and cytokines. In these different T cell types, Amphiregulin synthesis was inhibited by an antagonist of protein kinase A, a downstream component of the cAMP signaling pathway, and enhanced by ligands that increased cAMP or directly activated protein kinase A. Prostaglandin E2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced Amphiregulin synthesis while reducing synthesis of most other cytokines. Thus, in contrast to mouse T cells, Amphiregulin synthesis by human T cells is regulated more by acute signals than pre-commitment of T cells to a particular cytokine pattern. This may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses. Different functional subsets of CD4 T cells are crucially involved in immune defense against diverse pathogens. At least four effector subsets are derived by differentiation from naïve CD4 T cells, and each expresses a characteristic combination of transcription factors, soluble mediators and surface molecules [1, 2] . Th1 cells predominantly produce interferon-c (IFNc) and protect against intracellular pathogens; Th2 cells produce interleukin (IL)-4, IL-5 and IL-13 and help to eliminate extracellular parasites; Th17 cells produce IL-17a and IL-17f and are crucial in fighting against extracellular bacteria and fungi; whereas induced T regulatory cells (iTreg) produce IL-10 and Transforming Growth Factor b (TGFb), and suppress T and B cell effector responses.
Although the initial Th1 and Th2 subsets are relatively stable, recent studies have demonstrated some flexibility and plasticity, particularly in other CD4 T cell subsets. Cytokines and other soluble mediators in the lymph node and inflamed tissue can further affect the cytokine expression profile of effector CD4 T cells [3] , in some cases by changing the differentiation status of the effector CD4 T cells. Primed precursor CD4 T (Thpp) cells, that produce mainly IL-2 and chemokines when stimulated, remain uncommitted with respect to their effector cytokine pattern and can later differentiate into either Th1 or Th2 cells [4] [5] [6] . Suppressive Treg cells, expressing the forkhead transcription factor Foxp3, can lose the expression of Foxp3 and acquire the ability to produce pro-inflammatory cytokines during autoimmunity [7] . Th17 cells can acquire the ability to produce IFNc in Th1 polarizing conditions [8, 9] . Adoptively transferred IL-4-producing Th2 effector cells can produce IFNc during viral challenge infections [10] . Th9 cells develop from Th2 populations in the presence of TGFb [11, 12] and T follicular helper (Tfh) cells may represent a further differentiation step from several of the other subsets [13] .
Acute modifications of cytokine patterns can also occur. IL-12+ IL-18 enhance the secretion of IFNc by Th1 cells [14, 15] , and IL-2 enhances cytokine production [16, 17] . In contrast, IL-10, TGFb, prostaglandin E2 (PGE2) and adenosine inhibit inflammatory cytokine production [18] [19] [20] .
Mouse Th2 cells, but not naive or Th1 cells, express Amphiregulin (AR), a member of Epidermal Growth Factor (EGF) family. Like other EGF members, AR is expressed as a transmembrane precursor protein and released by proteolytic cleavage [21, 22] . Soluble AR binds to EGF receptors and promotes proliferation and differentiation of epithelial cells, fibroblasts and keratinocytes [23] [24] [25] . AR-deficient mice [26] showed slower kinetics of clearance [27] of the helminth parasite, Trichuris muris, that is cleared most effectively by Th2-biased responses. AR production is also induced in human mast cells by IgE cross-linking [28, 29] , in human eosinophils by IL-5 [30] , and in human basophils by IL-3 [31] . Thus AR is induced by activation of at least four cell types contributing to Type 2 inflammation, suggesting a role for AR during an allergic immune response. In addition, production of AR by immune cells is potentially important for tissue remodeling and repair [21, 26] during and after damaging immune responses.
Very little is known about the regulation of AR gene expression in human T cells. We examined the regulation of AR synthesis by human T cells, and found that in contrast to mice, many subsets of human T cells, including CD4 and CD8, naive and memory, Th1 and Th2, all express AR in response to TCR stimulation. Factors that elevate cAMP levels synergized with TCR stimulation to enhance AR expression, while inhibiting expression of most inflammatory cytokines. Thus in human T cells, AR production is regulated strongly by the environmental context during stimulation, but not restricted to particular precommitted effector subsets of T cells.
Biotinylated goat anti-human AR and biotinylated goat normal IgG (isotype control), and APC conjugated anti-human CCR4 (205410) were obtained from R&D Systems (Minneapolis, MN). LEAF TM purified anti-human CD3e (OKT3), APC-Cy7 conjugated anti-human CD4 (RPA-T4), Pacific Blue or APC-Cy7 conjugated anti-human CD69 (FN50), PE-Cy5 conjugated antihuman CD154 (24-31), PerCP-Cy5.5 conjugated anti-human CD27 (O323), Alexa Fluor 700 conjugated anti-human CD62L (DREG-56), Pacific Blue conjugated anti-human CXCR3 (TG1/ CXCR3), PE-Cy7 or PE-Cy5 conjugated anti-human CD123 (6H6), Alexa Fluor 700 conjugated anti-human IL-2 (MQ1-17H12), FITC conjugated anti-human IL-4 (MP4-25D2), and PerCP-Cy5.5 conjugated anti-human IL-17A (BL168) were purchased from BioLegend (San Diego, CA). Functional grade purified anti-human CD28 (CD28.2), PE conjugated anti-human CD45RA (HI100), FITC conjugated anti-human CD45RO (UCHL1), PE-Cy5 conjugated anti-human CD19 (HIB19), PE-Cy7 conjugated anti-human IFNc (4S.B3), and APC-conjugated streptavidin were obtained from eBioscience (San Diego, CA). Alexa Fluor 488 conjugated anti-human CXCR5 (RF8B2) and PE-Cy7 conjugated anti-human CCR7 (3D12) were purchased from BD Bioscience (San Jose, CA). QdotH 605 conjugated antihuman CD3 (UCHT1), PE-Texas Red and QdotH 705 conjugated anti-human CD8a (3B5), PE-Texas Red conjugated anti-human CD4 (S3.5), TRI-COLOR and QdotH 800 conjugated antihuman CD14 (TüK4), QdotH 655 conjugated anti-human CD45RA (MEM-56), Pacific Blue conjugated anti-human TNFa (MP9-20A4), and LIVE?DEAD Fixable Yellow Dead Cell Stain Kit were obtained from Invitrogen (Carlsbad, CA).
7-Aminoactinomycin D (7-AAD) and TAPI-1 was obtained from Calbiochem (Gibbstown, NJ). cAMP agonist (8-CPT-cAMP) and cAMP antagonist (Rp-8-Br-cAMP) were purchased from BioLog (Bremen, Germany). Phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, PGE2, forskolin and 3-Isobutyl-1methylxanthine (IBMX), adenosine were obtained from Sigma (St.Louis, MO).
Heparinized blood was obtained from healthy donors under a protocol approved by the University of Rochester Medical Center Research Subjects Review Board. Written, informed consent was obtained from all subjects. PBMC were isolated by Ficoll-Hypaque (Cellgro, Herndon, VA) density gradient centrifugation. Cells were suspended in complete RPMI-8 (RPMI-1640 medium containing 100U penicillin/streptomycin (Invitrogen) supplemented with 8% heat-inactivated fetal calf serum (FCS, HyClone, Logan, UT)). In the experiment treating cells with adenosine, serum-free medium X-VIVO TM 20 (Lonza, Walkersville, MD) was used.
To purify human naïve and memory CD4 T cells from PBMC, fresh PBMC were stained with antibodies specific for cell surface markers and CD4+CD8-CD14-CD123-CD45RA+CD45RO-(naïve CD4 T cells) and CD4+CD8-CD14-CD123-CD45RA-CD45RO+ (memory CD4 T cells) were sorted on a FACSAria (BD Bioscience, San Jose, CA).
Purified human naïve CD4 T cells were stimulated with irradiated (100Gy) allogeneic Epstein-Barr virus (EBV) -transformed B cells (1:1 ratio) in complete RPMI-8 medium at 10 5 cells/mL in round-bottom 96-well plate. Th1-biased cultures contained recombinant human IL-2 (5 ng/mL, PeproTech), recombinant human IL-12 (20 ng/mL, PeproTech) and anti-IL-4 (5 mg/ml, R&D Systems). Th2-biased cultures contained recombinant human IL-2 (5 ng/mL), recombinant human IL-4 (20 ng/mL, R&D Systems), anti-IL-12 (5 mg/ml, ebioscience) and anti-IFNc (5 mg/ml, R&D Systems). Fresh medium containing 5 ng/mL IL-2 was added if necessary to cultures showing strong proliferation. The cultures were restimulated and expanded every seven days.
To enrich for cells with the Th1 or Th2 phenotypes, after 14 days priming, Th1 and Th2 cells were stimulated with platebound anti-CD3+ anti-CD28 for 8 hours. IFNc+ Th1 cells and IL-5+ Th2 cells were stained and sorted by the MACS cytokine secretion assay (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The enriched IFNc+ Th1 cells and IL-5+ Th2 cells were expanded as previously for 14 days.
For intracellular staining (ICS) of AR, PBMC (10 6 per well) were stimulated with medium alone, anti-CD3 (5 mg/ml) + anti-CD28 (1 mg/ml), Staphylococcal enterotoxin B (SEB, 1 mg/ml), PMA (10 ng/mL) + ionomycin (500 ng/mL), influenza H1N1 peptides (H1N1 [New Caledonia/New York], 20 ng/mL/peptide), Fel d1 (50 mg/mL, INDOOR, Charlottesville, VA), Der p1 (50 mg/mL, INDOOR) or Tetanus peptides (3 mg/mL/peptide) in round-bottom 96-well plate (Costar, Corning Inc., Corning, NY). Th1 or Th2 cultures were treated with medium alone or PMA + ionomycin. After 10 hours stimulation (with 2 mM monensin present for the last 8 hours), the cells were first stained with LIVE?DEAD Fixable Yellow Dead Cell Stain Kit, and then stained for cell surface markers CD4, CD8, CD14, CD123, and CD45RA. After cells were fixed and permeabilized using Fix-Perm (BD Bioscience), the cells were stained with anti-AR, anti-IFNc, anti-IL-2, anti-IL-4, anti-IL-17A, anti-TNFa, anti-CD3 and anti-CD69 (or anti-CD154) intracellularly.
For cell surface staining of AR, PBMC were stimulated with medium alone or 1 mg/ml SEB in the presence or absence of 50 mM TAPI-1, an ADAM17 protease inhibitor [32] , for 6, 12, and 24 hours. After stimulation, the cells were stained with antibodies against AR, CD3, CD4, CD8, CD14, CD123, CD69 and 7-AAD. Data were acquired using an LSR II flow cytometer (BD Bioscience), and analyzed with FlowJo software (Tree Star Inc., Ashland, OR).
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was prepared by reverse transcription from total RNA using MultiScribe TM Reverse Transcriptase (Applied Biosystems, Foster City, CA) with random hexamer primers (Applied Biosystems). Quantitative real-time PCR (RT-PCR) was performed using the Applied Biosystems 7900HT Sequence Detection System. Primers and probes specific for AR, Heparin-binding EGF-like Growth Factor (HB-EGF), IL-2, IFNc, IL-3, IL-4, IL-5, IL-10, IL-13, CD3d, EGF, Neuregulin (NRG) 1-4, epiregulin (EREG), betacellulin (BTC) and TGFa were all obtained from TaqMan Gene Expression Assays (Applied Biosystems). CD3d gene expression was used as an endogenous control for normalizing mRNA amounts. All samples were run in duplicate and data were analyzed using SDS software (Applied Biosystems).
Purified CD4 T cells were treated with medium alone or CD3/ CD28 beads (cells:beads 2:1) in the presence or absence of 50 mM TAPI-1. After 24 hours, the supernatants were collected and AR was measured using the human Amphiregulin DuoSet ELISA Development kit (R&D Systems). The detection limit of the assay was 7.8 pg/mL. Because the antiserum for the ELISA was produced by immunization with bacterial recombinant human AR, and the standard is also non-glycosylated AR, this ELISA probably underestimates the concentration of normal human glycosylated AR.
Mouse Th2 cells produce AR in response to TCR-mediated activation [27] , and the expression of AR by hemopoietic cells contributes to the clearance of a helminth parasite. However, our recent studies showed that basophils were the major human PBMC type that produced AR in response to anti-CD3/CD28 stimulation [31] , whereas production of AR by T cells was much lower. Therefore we examined human T cells in more detail, to determine whether human T cells could produce AR, and if so, whether this was produced preferentially by human Th2 cells.
Human PBMCs were stimulated with soluble anti-CD3+ anti-CD28, SEB, or PMA + ionomycin for 10 hours (protein secretion inhibitors were added during the last 8 hours). CD69 staining increased on almost all anti-CD3/CD28-and P+I-stimulated cells, and a subset of SEB-stimulated cells ( Figure 1A ). AR staining was increased, only in the CD69+ population, and this increase was most obvious in the P+I-stimulated cells. For all three stimulation conditions, the staining intensity for AR increased for the whole CD69+ population, i.e. separate positive and negative populations were not resolved, and so the percentage of cells in the AR+ gate may be an underestimate of the total number of cells expressing AR. The specificity of AR staining was demonstrated by using a control goat antiserum (right column). Similar results were obtained with CD8 T cells ( Figure 1A) .
To independently confirm AR expression by human T cells, and to test whether T cells produced AR as a direct result of TCR stimulation, human CD4 and CD8 T cells were purified by sorting, and stimulated with beads coated with anti-CD3+ anti-CD28 antibodies. At different times, RNA was extracted from the cells, and levels of AR and IL-2 mRNA measured by RT-PCR. AR mRNA levels increased rapidly after stimulation, and returned to low levels after ten hours, whereas IL-2 showed slower kinetics ( Figure 1B) . The kinetics of AR production were similar in CD4 and CD8 T cells. Thus human T cells directly express AR in response to polyclonal TCR stimulation.
As demonstrated by other studies [33] , HB-EGF mRNA was also upregulated in activated human CD4 T cells (Figure 2A) , although the levels were lower than AR and peaked at a later time ( Figure 2B ). TGFa and EREG mRNA were also detected in resting CD4 T cells, but not increased during TCR activation. Other EGF members were undetectable. In our previous mouse experiments [27] , AR and HB-EGF were also the only EGF family members induced by TCR stimulation (data not shown). Expression of HB-EGF protein was confirmed by cell surface and intracellular staining (data not shown).
EGF family members (including AR) are initially expressed as transmembrane proteins and released into the extracellular region after cleavage by metalloproteases, particularly ADAM17 [22] . To determine whether T cells also initially expressed surface AR and then released the soluble cleavage product, surface AR was stained during TCR activation in the presence or absence of the ADAM17/TACE inhibitor TAPI-1 [32] . TAPI-1 increased AR expression on the surface of both CD4 and CD8 T cells measured by frequency ( Figure 3A ) or fluorescence intensity (data not shown). Conversely, TAPI-1 decreased soluble AR in the supernatant ( Figure 3B ). In the absence of TAPI-1, AR expression on T cells gradually decreased and was barely detectable after 24 hours. As ADAM17 mRNA was detected by RT-PCR in resting human T cells and upregulated on activation (data not shown), these results suggested that AR was first synthesized as a membrane protein on human T cells and then released by ADAM17 cleavage, as in other cell types [34, 35] .
In mice, AR was expressed selectively in TCR-activated Th2 cells [27] but not Th1 ( Figure S1 ) or naive CD4 T cells. This was a pre-committed, intrinsic property of the Th2 cells, as Th2 but not Th1 cells expressed AR even when in vitro-derived mouse Th1 and Th2 cell lines were activated together in the same culture (data not shown). However, most human CD4 (and CD8) T cells expressed AR in response to PMA plus ionomycin stimulation ( Figure 1A ). We therefore examined in more detail which human T cell subsets were responsible for AR production.
Naive and memory CD4 and CD8 T cells produce AR. To examine the ability of naive and memory T cell subpopulations to express AR, we stimulated PBMC with an allogeneic EBV-transformed B cell line, which would be expected to activate a small fraction of both memory and naive CD4 and CD8 T cells. Alloantigens stimulated a fraction of both CD4 and CD8 T cells to produce AR ( Figure 4A ), relative to the unstimulated control. The specificity of staining was confirmed by isotype control antibodies. The cells producing AR (and other cytokines) were included in the CD69+ population.
AR was induced by allogeneic stimulation in both CD45RAand CD45RA+ subsets of CD4 and CD8 T cells at frequencies ranging from 0.028% to 0.35%. These levels were comparable to the frequencies of CD4+ CD45RA+ or CD45RA-T cells producing IL-2, or CD4+ CD45RA-memory T cells producing IFNc. As expected, IL-2 was produced by both memory (CD45RA-) and naive (CD45RA+) CD4 T cells, whereas IFNc (and IL-4 at low levels) were produced mainly by memory cells ( Figure 4B ).
The expression of AR by both CD45RA-and CD45RA+ subsets of CD4 T cells was tested at the mRNA level in SEBstimulated cells sorted according to AR and CD45RA expression ( Figure 4C ). Confirming the specificity of the anti-AR antibody AR is produced by memory CD4 T cell subsets expressing different cytokine phenotypes. Although naive CD4 T cells are relatively homogeneous, the memory population includes a wide range of differentiated effector subsets. As AR is expressed selectively by mouse Th2 cells, we examined whether AR production by human CD4 memory T cells was preferentially associated with expression of a particular cytokine or surface marker pattern. Th1-and Th2-biased human CD4 T cell populations were induced by stimulation of sorted naive human CD4 T cells with an allogeneic B cell line in Th1-or Th2-biasing cytokine conditions. The populations were further enriched by using the Cytokine Secretion Assay to sort IFNc-or IL-5producing cells, respectively. The resulting populations were strongly polarized, but unlike mouse T cells, both Th1 and Th2 human cell lines expressed AR ( Figure 5A ).
These results were confirmed using ex vivo human CD4 T cell populations. Human PBMC were stimulated with SEB, and AR and other cytokines measured by intracellular staining. Naive cells (CD45RA+) expressed high levels of IL-2 and AR, but very low levels of either IFNc or IL-4 (data not shown). Memory cells produced all cytokines tested, at varying frequencies. To determine whether AR expression was associated positively or negatively with subset-specific cytokines, the frequencies of cells expressing AR plus each of the other cytokines were measured from the ICS results. These values were then compared with the double-producing frequencies predicted for random association of each cytokine pair, by multiplying the individual frequencies for each cytokine. Figure 5B shows that AR was expressed in association with TNFa, IL-2, IFNc, IL-4 and IL-17 at slightly higher frequencies than predicted by random association. Similarly, TNFa and IL-2 showed positive associations with all other cytokines. In contrast, the subset-specific cytokines IFNc, IL-4 and IL-17 showed mostly negative associations between each other, as expected. These results were confirmed at the RNA level by sorting SEB-stimulated human PBMC according to surface AR expression. Both AR+ and AR-memory CD4 T cell populations expressed similar levels of IL-4 and IFNc as measured by RT-PCR ( Figure 5C ). IL-2 mRNA levels were higher in AR+ T cells, in both CD45RA-and CD45RA+ cells.
AR is produced in response to antigen stimulation. We next tested whether human CD4 T cells expressed AR during antigen/APC stimulation in response to influenza peptides, allergens or tetanus antigens to stimulate Type 1, Type 2 and Thpp-biased recall responses, respectively [6] . PBMCs were stimulated with antigens for 10 hours, and AR and other cytokines measured by ICS.
Although these three antigens induced in vivo recall responses with characteristically different levels of IL-2, IFNc and IL-4, all three antigens induced substantial production of AR in the activated (CD154+) cells ( Figure 5D ). Similar results were obtained with cells from multiple subjects, although the magnitudes of the antigen responses were variable for all cytokines. Thus AR can be expressed by all the conventional defined subsets of T cells that we have tested, including CD4 and CD8, naïve and memory, Thpp, Th1 and Th2.
To confirm the protein results, influenza-specific CD69+ IFNc+ cells were sorted from two subjects (results from one subject are shown in Figure 5E) , and RT-PCR demonstrated that AR mRNA levels were strongly elevated in the IFNc+ influenza-specific cells AR is produced by T cell subsets expressing different chemokine receptors and surface markers. Chemokine receptors expressed selectively by T cell subsets lead to different homing and chemotactic properties. Expression patterns of chemokine receptors are partly but not entirely related to cytokine commitment patterns [36] [37] [38] [39] . Additional surface markers, including CD27 and the homing receptor CD62L are also expressed heterogeneously on human CD4 T cells. We therefore examined AR expression within subsets of memory CD4 T cells defined by the expression of these proteins. AR was produced at approximately similar frequencies by CD4 T cells positive or negative for the chemokine receptors CCR4, CCR7, CXCR3 and CXCR5, as well as CD62L and CD27 ( Figure 6 ). However, expression of the activation-induced protein CD69 was strongly correlated with AR expression, as seen in previous figures. Taken together with the data described above, AR expression appears to be a general ability of most or all subtypes of human T cells after TCR activation.
As our results had demonstrated that AR production was not limited to a pre-committed subset of T cells, we then tested whether AR production was regulated by acute signals in the immediate milieu during TCR stimulation. In many cell types AR is strongly regulated by the cAMP-PKA-CREB signaling pathway. AR expression was significantly up-regulated by cAMP-elevating agents in both resting and anti-CD3 stimulated human PBMC populations enriched for human T cells [40] . However, in that study the negatively-selected T cell population would also have contained basophils, and we have shown that basophils express AR rapidly in response to IL-3 and cAMP agonist ( [31] and unpublished data). Thus anti-CD3 stimulation of the CD4 T cell + basophil population could have induced IL-3 production by T cells, indirectly resulting in AR production by basophils. We have now re-examined the effect of cAMP elevation on the expression of AR by different T cell subsets.
TCR and cAMP signals synergize to induce AR expression. TCR activation alone (which transiently elevates cAMP [41] ) induced transient AR mRNA expression (Figure 7A) , and a strong cAMP agonist (PKA activator 8-CPT-cAMP) also induced low levels of AR in the absence of other signals. However, TCR and PKA signaling synergized to induce higher and more sustained levels of AR and HB-EGF mRNA ( Figure 7A ), as well as high levels of AR protein (supernatant plus cell-associated, Figure 7B ). This strong synergy contrasts with a previous study [40] , possibly due to the presence of basophils in the responding population in that study.
As both naive and memory CD4 T cells produce AR (Figure 4 ), we tested whether PKA activation would enhance AR expression in both populations. Figure 7C shows the response of purified Figure 5 . Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNc, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of doubleproducing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNc and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNc+ and CD69+IFNc-CD4 T cells were sorted from influenza peptidetreated PBMC using the cytokine secretion assay. The mRNA levels of IFNc and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments. doi:10.1371/journal.pone.0039072.g005 AR expression is modified by natural and synthetic modulators of cAMP signaling. In the experiments described above, cAMP signaling was altered by an agonist (8-CPT-cAMP) that directly targeted PKA to mimic the increase of intracellular cAMP levels. To further confirm that the cAMP-PKA-CREB signaling pathway regulates AR expression, we tested natural and pharmacological agents that increase the intracellular levels of cAMP by acting at two additional steps: PGE2 and adenosine are natural ligands for G-protein coupled receptors that activate adenylyl cyclase [20, [42] [43] [44] ; forskolin activates adenylyl cyclase directly [20] ; and IBMX is a broad inhibitor of cAMP-degrading phosphodiesterases [45] .
Consistently, all four cAMP elevating agents upregulated AR mRNA and protein expression in anti-CD3-stimulated T cells. In each case, the elevated signal was blocked by the cAMP antagonist ( Figure 7D ). The enhancement of AR by the PDE inhibitor suggested that PDE reduced the moderate levels of cAMP induced by TCR activation in CD4 T cells [41] .
AR and other cytokines are regulated reciprocally by cAMP signals. In contrast to the enhancement of AR expression, the cAMP agonist inhibited expression of many other cytokines ( Figure 7E ), and all four PKA-activating agents described in Figure 7 inhibited expression of IL-2 and IFNc (data not shown). These results are consistent with previous studies with cAMP agonists and natural cAMP elevating agents, such as PGE2 and adenosine [19, 20] . Thus AR expression in T cells is enhanced under conditions that suppress the production of many other cytokines.
In contrast to the preferential expression of AR by mouse Th2 cells, we have now shown that synthesis of human AR is not restricted to a particular human T cell subset. AR can be produced by activated naive and memory CD4 and CD8 T cells, including Th1 and Th2 phenotypes. Our results suggest that AR is not a specific product of certain pre-committed effector subsets of human CD4 T cells, but instead is regulated mainly by additional signals present during T cell activation, particularly signals influencing the cAMP signaling pathway. The lack of precommitment suggests that, in contrast to the memory of effector functions carried by T cells committed to Th1, Th2, Th17 etc phenotypes, the amount of AR produced in a particular immune response is regulated by the local environment during that response, but is less influenced by previous immune priming. The discrepancy we have identified between mouse and human T cell regulation highlights the importance of performing cross-species comparisons of effector T cell phenotypes.
AR production was not restricted to a defined T cell effector subset, but AR and IL-2 levels were moderately correlated in both naïve and memory CD4 T cells. Although this could indicate the existence of a previously-unrecognized subset, it is possible that the correlation could be the result of shared transcriptional or mRNA stability regulatory factors, or to similar activation thresholds for IL-2 and AR. Expression of AR also showed moderate correlation with the expression of TNFa.
High levels of AR mRNA and protein were induced by synergy between TCR signals and signals that elevated cAMP or activated PKA. This contrasts with a previous report suggesting that both resting and anti-CD3 stimulated T cells significantly up-regulated AR in response to a cAMP agonist [40] . However, the enriched T cell population used in that study was purified by negative selection and very likely included basophils, which we have shown are potent producers of AR in response to IL-3 [31] . AR expression is also strongly enhanced by cAMP agonists in basophils (Y. Qi and T.R. Mosmann, unpublished data) and so it is possible that basophils may have produced the AR in response to the cAMP agonist without TCR stimulation.
In contrast to the induction of AR by cAMP elevating agents, these mediators suppress inflammatory responses by inhibiting cytokine expression and T cell proliferation. Synthesis of several pro-inflammatory or Type 1 cytokines is inhibited by cAMP ( Figure 7E and [19, 20, 46, 47] , whereas cAMP can either inhibit or enhance production of Type 2 cytokines such as IL-4, IL-5 and IL-13 ( Figure 7E and [47] ) depending on the stimulation conditions [48, 49] .
Natural mediators that elevate the cAMP pathway and lead to PKA activation include PGE2 (mainly via the G protein-coupled receptors E2 and E4 on T cells) and adenosine (mainly via the A 2A receptor on T cells). Both mediators are produced at sites of immune inflammation, adenosine by degradation of ATP from dying cells, and PGE2 by activated macrophages. PKA activation signals also synergized with TCR signals to induce HB-EGF mRNA and protein expression in human CD4 T cells (data not shown). Thus during the progression of an inflammatory response, there may be a switch from pro-inflammatory cytokine production to AR (and HB-EGF) production.
Our findings allow us to construct a model of the role of T cell derived AR in adaptive immunity. During an immune response, initial immune attack mechanisms that destroy the pathogen are superseded at later times by suppression that reduces immunopathology, and tissue repair that restores normal structure and function. T lymphocytes are major cellular contributors to all three phases, and are thought to play a role in repair by producing HB-EGF and bFGF [33] . AR and HB-EGF, as members of the EGF family, promote the proliferation of fibroblasts, epithelial cells, and smooth muscle cells, which are major cell types repaired or remodeled at local tissue sites during an inflammatory response. Tissues with chronic inflammation show extensive cell proliferation, tissue thickening and reduced elasticity. Regulation of the balance between attack and repair cytokines produced by T cells is thus crucial to the successful outcome of the response. . TCR and cAMP synergize to induce AR production in human CD4 T cells. Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO-(naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 , 1000 mM) . The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments. doi:10.1371/journal.pone.0039072.g007
In this model, AR derived from human T cells would be expressed mainly in response to tissue injury, consistent with the importance of the local environmental signals for AR regulation. This contrasts with the requirement for specific effector mechanisms to combat different pathogens, in which pre-commitment to cytokine effector phenotypes (thus linking antigen and effector specificities) may be more effective for regulating clearance functions. Collectively, the coordinate inhibition of pro-inflammatory cytokines and induction of tissue-remodeling cytokines of the EGF family may represent a switch from pathogen clearance to tissue repair mechanisms by effector human T cells. Figure S1 Mouse Th2 but not Th1 cells express AR in response to TCR activation. In vitro induced allogeneic Th1 and Th2 cell lines [50] from B6PL or AR 2/2 mice were stimulated with plate-coated anti-CD3 (2 mg/mL) + anti-CD28 (1 mg/mL) antibodies for 6 hours. Expression of AR, IFNc and IL-4 in CD4 T cells was analyzed by ICS. Biotinylated goat antimouse AR antibodies were obtained from R&D Systems. LEAF TM purified anti-mouse CD3e (145-2C11) and LEAF TM purified antimouse CD28 (37.51) were purchased from BioLegend. APC-Cy7 conjugated anti-mouse CD3 (17A2), Alexa Fluor 700 conjugated anti-mouse CD4 (GK1.5), Pacific Blue conjugated anti-mouse CD44 (IM7), PerCP-Cy5.5 conjugated anti-mouse CD69 (H1.2F3), APC conjugated anti-mouse IL-2 (JES6-5H4), PE-Cy7 conjugated anti-mouse IL-4 (BVD6-24G2), PE conjugated antimouse IL-5 (TRFK5), and FITC-conjugated streptavidin were obtained from eBioscience. PE-Alexa Fluor 610 conjugated antimouse IFNc (XMG1.2) was obtained from Invitrogen. Similar results were obtained in at least three experiments. (TIF) Allo-SCT for multiple myeloma: a review of outcomes at a single transplant center Allogeneic stem cell transplant for multiple myeloma (MM) is one treatment associated with long-term disease-free survival. The high incidence of treatment-related mortality and relapses, however, are important reasons for controversy about the role of allografting in the management of MM. We reviewed our results of allografting for MM spanning a period of 34 years in order to better define long-term outcomes and identify areas of progress as well as areas requiring improvement. A total of 278 patients received allogeneic marrow or PBSCs after high-dose myeloablative (N=144) or reduced intensity, non-myeloablative (N=134) regimens. In multivariable analysis, adjusting for differences in patient groups, reduced intensity/non-myeloablative transplants were associated with significantly less acute GVHD, lower transplant mortality, better PFS and overall survival. There were no significant differences in relapse, progression or chronic GVHD, when adjusted. In multivariable analysis of patients receiving only non-myeloablative transplants, decreased overall survival and PFS were associated with relapse after a prior autograft and a β2 microglobulin >4.0. Transplant mortality was reduced and only influenced by a prior tandem autograft. The survival of patients with multiple myeloma (MM) has improved over the last decade as a result of melphalan-based high-dose therapy followed by auto-SCT, the introduction of novel anti-myeloma agents with increased efficacy in relapsed and refractory MM, and improvements in supportive care. 1 --6 Registry data indicate an improvement in median survival from 3 to 5 years, primarily among younger patients, as a result of these treatment innovations. 7 Despite these new developments, MM remains an incurable disease for the large majority, as all but a few patients will relapse. Allogeneic hematopoietic cell transplantation is currently one treatment with a potential for long-term disease control although its curative potential is debated. This is in part due to the graft-vs-myeloma effect, mediated by immune competent donor lymphocytes, best illustrated by the induction of sustained (molecular) remissions following donor lymphocyte infusions, 8 but could also be due in part to absence of contaminating myeloma cells in the donor graft and documented lower levels of residual disease. 9, 10 The role of allo-SCT in MM, however, is controversial due to the high mortality and morbidity associated with conventional myeloablative regimens and because convincing evidence for a survival benefit is lacking. 11 --13 In the last decade, non-myeloablative allo-SCT has gained in popularity due to significantly reduced TRM. 14, 15 Among four reports comparing auto-SCT with allo-SCT, two have shown survival advantages for the nonmyeloablative approach when compared with tandem autologous transplantation. 16 --19 A recently reported US clinical trial prospectively comparing tandem autologous transplant to autologous followed by non-myeloablative allo-SCTs found no differences in PFS or OS at 3 years. 20 In contrast, a European multicenter trial found than tandem autologous, non-myeloablative allo-SCT resulted in superior OS compared with single or tandem auto-SCT. 19 Furthermore, at least one registry report comparing conventional ablative with non-myeloabalative/reduced intensity allo-SCTs have shown similar survival outcomes with lower TRM for patients receiving non-ablative transplants yet higher rates of relapse and PFS inferior to ablative allo-SCT. 21 We reviewed our results of allo-SCT for patients with MM beginning in 1975 with the aim of identifying factors associated with improvements in disease-free survival and OS as preparative regimens have changed from ablative to non-myeloablative.
Beginning in 1975, patients with MM were referred to the University of Washington, Fred Hutchinson Cancer Research Center or the Seattle Veterans Hospital for consideration of allo-SCT. Patients were evaluated for suitability for transplant based on treatment protocols in effect at the time. Patient records, laboratory, X-rays and marrow aspirates were reviewed to confirm the diagnosis of MM. To be considered for marrow transplantation, patients had to meet the established criteria for active, symptomatic MM according to Durie and Salmon 22 and had to have received at least one cycle of conventional dose chemotherapy. Patients with a Karnofsky score of o50, a pulmonary diffusion capacity of o50% of predicted and symptomatic heart failure were excluded. Non-ablative transplant candidates were allowed to enroll with a diffusion capacity as low as 30%. Standard hematologic and chemistry studies were used to evaluate organ function. A suitable marrow donor was required, which included HLA identical relatives, HLA haplo-identical relatives or an unrelated donor who was phenotypically HLA identical, or single allele or Ag HLA-mismatched at class I with the patient. Transplants occurred between January 1975 and September 2008. The date of last follow-up was August 2011.
Initially, patients who had achieved a complete response (CR) to firstline therapy and were without any evidence of disease were excluded from transplantation. This policy changed, however, as non-myeloablative allo-SCT regimens were adopted. Ablative allo-SCT were utilized as standalone therapy. In contrast, non-abaltive allo-SCT were performed in the majority of patients, 2 --4 months following recovery from a standard auto-SCT utilizing high-dose melphalan. The auto-SCT was utilized to provide cyto-reduction before the non-ablative Allo-SCT, yet allow the patient time to recover from the effects of high-dose therapy used for auto-SCT. Maintenance therapies were not used following allo-SCT.
For purposes of this analysis, patients with at least a 50% reduction in monoclonal proteins in the blood or a 75% reduction in 24 h quantitative Bence Jones protein, to their most recent chemotherapy before allo-SCT or auto-SCT, in the case of tandem transplants, were categorized as having sensitive disease, whereas all other patients were judged to have chemotherapy-resistant disease.
Responses were categorized according to the IMWG criteria. 23 If certain data were missing that were required for response categorization, for example immunofixation for CR, the patient was classified as responding in the next lower category. An analysis of OS, PFS, TRM, relapse or progression, acute and chronic GVHD was undertaken. The initial analysis compared outcomes using non-myeloablative conditioning for the allogeneic transplant vs those with myeloablative conditioning. In the analysis of relapse or progression, time-dependent competing risks of treatment failure such as death from TRM were included. Cox regression models for these outcomes were adjusted for patient age (continuous), donor sex, chemotherapy responsive vs resistant disease, related vs unrelated donor, time from diagnosis to transplant (o2.5 years vs 42.5 years), prior radiation, prior number of chemotherapy regimens (continuous), b-2 microglobulin 44.0 either at diagnosis or transplant, and abnormal cytogenetics or FISH either at diagnosis or transplant. Abnormal cytogenetics included multiple abnormalities or any abnormality by conventional cytogenetics other than hyperdiploidy. Abnormal FISH included deletion 13, deletion 17, translocation 4;14, 14;16, or 14;20. Because data were missing for some patients, data available for abnormalities were compared with patients who had no abnormalities and patients with missing data. Subsequent multivariate analyses of risk factors for the same outcomes among patients receiving non-myeloablative conditioning included the factors noted above, plus single allo-SCT vs tandem autologous-allo SCT, and progression after a prior autologous SCT used as stand-alone treatment.
Patient characteristics are shown in Table 1 . Patients receiving non-myeloablative allo-SCTs were older by a median of 8 years. There were no important differences in the percentages of patients with advanced Durie Salmon staging, IgG or IgA subtypes, number of prior regimens, or total cycles of chemotherapy. Availability of data on beta-2 microglobulin levels, albumin and cytogenetic data were limited. A higher percentage of patients receiving ablative regimens had been given local radiation therapy, 50% compared with patients receiving non-ablative regimens, 33%. One third of the patients receiving non-ablative conditioning had progressed after an autologous transplant, while only four patients receiving ablative conditioning had progressed after an autologous transplant. A higher percentage of patients receiving ablative regimens were judged to have refractory disease, 77%, (based on less than a partial response to their last salvage chemotherapy), compared with 52% of patients who received non-ablative regimens. Relatively few patients were in remission before allografting; two patients undergoing myeloa- blative allografts were in 2 nd CR, whereas among the nonmyeloablative group, three were in first CR and four in 2 nd CR. The regimens used for transplant differed significantly by the time periods during which patients were transplanted with almost all ablative allo-SCTs occurring between 1975 and 2000, whereas the non-ablative approach was utilized from 1998 to 2008. (Table 2 ) The conditioning regimens given to ablative allo-SCT recipients consisted mostly of fractionated TBI 9 --12 Gy, plus CY, and/or BU. BU and CY without TBI were utilized for 69 patients. The non-ablative regimens were primarily TBI 2 Gy with or without fludarabine, whereas 14 patients received additional melphalan 100 mg/m 2 . Most donors were HLA-matched siblings (n ¼ 198) for both ablative and non-ablative transplants, however, a greater percentage of non-ablative transplants were performed from unrelated donors. Marrow was the primary stem cell source for most of the patients receiving ablative conditioning whereas PBSCs were used almost exclusively for non-ablative recipients.
The majority of regimens for GVHD prophylaxis in ablative recipients consisted of a calcineurin inhibitor with MTX or steroids. Almost all recipients of non-ablative regimens received a calcineurin inhibitor and mycophenolic acid for GVHD prophylaxis.
Response to transplant Among the 144 ablative transplant recipients, 33 (23%) achieved CRs, 33 (23%) a partial response, 12 (8%) did not respond and 67 (46%) were not evaluable owing to early death. Of 134 patients receiving non-ablative transplants, 51 (38%) achieved a CR, 48 (36%) a partial response, 31 (23%) did not respond, whereas 4 (3%) were not evaluable owing to early death. Patients who achieved a CR (n ¼ 84) had 5 and 10 year survivals of 62 and 53% compared with patients who did not achieve a CR (n ¼ 132) and excluding patients who were not evaluable owing to early death, who had 5 and 10 year survivals of 28% and 17%, respectively.
Among patients who received ablative conditioning, 104 developed acute GVHD; 7 grade 1, 44 grade 2, 34 grade 3 and 19 grade 4. Of patients who received non-ablative conditioning acute GVHD occurred in 90; grade 1 in 6, grade 2 in 72, grade 3 in 8 and grade 4 in 4. The cumulative incidences of chronic extensive GVHD were 27% and 66% for patients receiving ablative and non-ablative conditioning regimens, respectively.
Causes of death varied significantly between patients receiving ablative and non-ablative transplants. (Table 3 ) Among patients who died after receiving ablative conditioning, major causes included fungal infections (n ¼ 20), respiratory failure from diffuse alveolar damage or acute respiratory distress syndrome (n ¼ 8), acute GVHD (n ¼ 18), multi-organ failure (n ¼ 16), viral infections (n ¼ 13) and progressive disease (n ¼ 39). In contrast, only three patients receiving non-ablative transplants died of any of these causes. The major causes of death among recipients of nonablative transplants were mostly chronic GVHD (n ¼ 12) and progressive disease (n ¼ 50). At the time of last follow-up, August 2011, among 144 patients receiving ablative conditioning, 14 were alive a median of 15.1 years (3.6 --23.5) post transplant, of whom 6 had relapsed. Among 134 patients receiving non-ablative conditioning, 56 were alive a median of 7.1 years (2.9 --12.9) post transplant, of whom 25 had relapsed. At 2 years, the probabilities of non-relapse mortality were 18% and 55% for non-ablative and ablative regimens, respectively. At 6 years, the probabilities of relapse or disease progression were 55% and 34% for non-ablative and ablative regimens, respectively. For patients undergoing ablative allo-SCTs, the probabilities of OS and PFS are 11 and 8% at 15 years. For patients undergoing non-ablative transplants, the probabilities of OS and PFS are 39 and 16% at 10 years. (Figure 1 ) The best outcomes were found among 88 patients who received an autologous transplant, followed by a non-myeloablative allograft within 4 months of the autologous transplant and who had not progressed after a prior autologous transplant. (Figure 2 ) Their 10-year OS was 49% and PFS was 27%.
Cox regression analysis of overall mortality, PFS, TRM, relapse or progression and acute or chronic GVHD between non-myeloablative and ablative conditioning regimens are shown in Table 4 . When adjusted for patient and donor factors, non-myeloablative conditioning resulted in significantly lower overall mortality HR 0.40 (0.3 --0.6), improved PFS HR 0.55 (0.4 --0.8) and much lower TRM HR 0.22 (0.1 --0.4). The risks of acute GVHD grades 2 --4 were also significantly lower with non-myeloablative regimens HR 0.41 (0.3 --0.6). The risks of relapse or progression and chronic GVHD when adjusted for competing risks of death and patient and donor factors, were not significantly different between ablative and non-ablative conditioning, despite the almost exclusive use of PBSC for the non ablative recipients.
In a separate multivariable analysis, outcomes of only patients undergoing non-ablative allogeneic transplants were considered. (Table 5 ) The most important predictors of survival, PFS and In order to discern any association between chronic GVHD and disease progression, we examined this association and its effects on PFS, in a time-dependent fashion among recipients of nonablative transplants. We found only a weak association between patients with clinical extensive chronic GVHD and reduced rates of progression or relapse HR ¼ 0.74 (0.4 --1.3), P ¼ 0.32. This resulted in no net benefit on PFS HR ¼ 0.89 (0.5 --1.5), P ¼ 0.65.
In this retrospective review of allo-SCT for MM going back 34 years, significant improvements were observed in the TRM associated with the introduction of non-myeloablative conditioning. Mortality censored for relapse was 55% among the 144 patients receiving ablative transplants compared with only 18% in the non-myeloablative group. As a result, the survival at 10 years from transplant was significantly superior for non-ablative transplants, 35% compared with 15%. As these two groups were not prospectively studied and were not treated contemporaneously, it is likely that other factors including better antiinfectious prophylaxis and treatment, and the use of PBSCs may Bold numerals refer to number of patients for each heading.
Allo-SCT for multiple myeloma W Bensinger et al have contributed in part to these improvements. Indeed, there were almost no deaths due to viral or fungal pathogens among non-myeloablative recipients; a major cause of mortality among ablative transplant recipients. In addition, there were major differences between the groups in patient age, relapse after prior autologous transplant, and proportion of patients resistant to their last chemotherapy regimen just before transplant. Although the perception is that patients with MM tolerate allografting more poorly than patients with other hematologic malignancies, a recent analysis from the European Group for Blood and Marrow Transplantation suggested that when adjusted for risk factors including age, disease stage, interval from diagnosis to transplant and donor factors, outcomes for patients with MM were similar. 24 Additionally, there are now newer drugs available to treat relapse that were not available previously which would certainly affect survival after relapsed disease.
In univariate analysis, non-myeloablative transplants were associated with an apparent greater risk of disease progression or relapse, 55% at 6 years for non-myeloablative compared with 34% for ablative conditioning. When adjusted for competing risks of death due to higher TRM associated with ablative transplants, however, these differences were not statistically significant. Although this result does not appear to agree with the analysis of others such as the EBMT registry data, the study was only a univariate analysis and did not account for competing causes of death, as ours did. 25 Nevertheless, the amount of residual disease present at transplant, provides a greater challenge for clearance by the allogeneic donor graft when a non-myeloablative regimen is utilized and is still the primary cause of treatment failure. When comparing the incidences of chronic GVHD, 27% of the ablative recipients developed CGVHD compared with 66% for non-ablative recipients. As the risk of CGVHD is time-dependent, and more nonmyeloablative patients survived the early phases of transplant, this did not prove to be significantly higher when adjusted for competing causes of death.
In an attempt to overcome this limitation, many groups have employed a tandem autologous, non-myeloablative allogeneic transplant with the aim of providing major cytoreduction, but an opportunity for the patient to recover from high-dose chemotherapy before the Allo-SCT. 14, 16, 26 In multivariable analysis, patients receiving a tandem autologous, non-myeloablative allogeneic transplant had reduced non-relapse mortality, but did not independently affect other outcomes. This analysis also indicated that relapse after a prior autologous transplant is associated with inferior survival as well as other outcome measures. As seen in prior studies, a b-2 microglobulin 44 was also independently associated with increased risk of progression or relapse as well as inferior survival. Female donors were associated with a significantly reduced risk of relapse or progression, consistent with other analyses that have shown more of a graft-vs disease effect from female to male transplants.
These analyses agree with other studies showing prior autograft failure to be one of the major risk factors for disease progression after non-myeloablative allo-SCT. 27 The observation that prior autograft failures do poorly with an allo-SCT argues against the recommendation some have made to delay an allo-SCT until disease progression after initial treatment or autologous transplant. 7 In some retrospective analyses, a non-myeloablative allograft was able to overcome certain high-risk FISH characteristics such as the 4;14 translocation. 28 Our patient population contained too few patients with 4;14 to analyze this separately, however, in the multivariable analysis only high B2 microglobulin and not adverse cytogenetics were associated with inferior outcomes. This does not mean that cytogenetics are not important but merely reflect a limited number of observations in our database to directly address that question.
It is clear that reduced intensity allo-SCT regimens can result in reliable donor engraftment with a relatively low mortality compared with high-dose regimens. The immunologic effect of the allograft is, however, relatively modest requiring a prior autologous transplant for cytoreduction. Even with the tandem auto-non-myeloablative allo-SCT approach, relapses beyond 3--5 year continue to occur, making disease recurrence the primary cause of treatment failure after tandem auto, non-myeloablative allo-SCT.
Future studies of allo-SCT in MM should focus on regimens that are less toxic but able to preserve anti-tumor effects such as radioisotopes linked to antibodies that target myeloma cells or other marrow-based cells. It should be relatively easy to combine targeted radiotherapy with a non-myeloablative regimen to create a more tolerable cytoreductive protocol. It is also worth reconsidering more myeloablative regimens, as supportive care has improved greatly in the past 20 years. As previously noted, when younger patients are transplanted earlier from initial diagnosis, TRM is reduced.
Another strategy to make non-myeloablative regimens more effective would be to combine the donor graft with infusions of allogeneic donor lymphocytes or subsets of lymphocytes in the form of 'engineered grafts', for example CD4 lymphocytes, which may have a graft vs myeloma effect without increasing GVHD. 29 It may also be possible to exploit killer-Ig-like mismatching between donor and recipient, which has been shown to result in improved PFS due to a reduced rate of relapse. 30, 31 Maintenance strategies, which have been shown to delay disease progression after auto-SCT may also be effective after allo-SCT. 32, 33 Finally, it may be worthwhile to exploit monoclonal antibodies targeting myeloma cells such as the CD40 Ag or CS-1 Ag, in order to increase the ability of donor allogeneic cells to eliminate residual host disease. 34 In any case, due to the substantial morbidity and mortality associated with allografting as well as the uncertain benefits, future approaches to allografting for myeloma should only be performed within well-designed clinical trials.
The authors declare no conflict of interest.  Sequence Analysis of Feline Coronaviruses and the Circulating Virulent/Avirulent Theory  segregation. Hence, our data do not confirm the diagnostic potential of the M protein sequence nor do they support the suggested role of the membrane protein in FIP pathogenesis (9) .
Informative as it may be, comparative sequence analysis will eventually not be sufficient to answer the FECV/FIPV question. What will be needed is a reverse genetics system to generate and manipulate the FCoV genome as well as a cell culture system to propagate the viruses, both of which have thus far not been achieved.  UNC93B1 Mediates Innate Inflammation and Antiviral Defense in the Liver during Acute Murine Cytomegalovirus Infection Antiviral defense in the liver during acute infection with the hepatotropic virus murine cytomegalovirus (MCMV) involves complex cytokine and cellular interactions. However, the mechanism of viral sensing in the liver that promotes these cytokine and cellular responses has remained unclear. Studies here were undertaken to investigate the role of nucleic acid-sensing Toll-like receptors (TLRs) in initiating antiviral immunity in the liver during infection with MCMV. We examined the host response of UNC93B1 mutant mice, which do not signal properly through TLR3, TLR7 and TLR9, to acute MCMV infection to determine whether liver antiviral defense depends on signaling through these molecules. Infection of UNC93B1 mutant mice revealed reduced production of systemic and liver proinflammatory cytokines including IFN-α, IFN-γ, IL-12 and TNF-α when compared to wild-type. UNC93B1 deficiency also contributed to a transient hepatitis later in acute infection, evidenced by augmented liver pathology and elevated systemic alanine aminotransferase levels. Moreover, viral clearance was impaired in UNC93B1 mutant mice, despite intact virus-specific CD8+ T cell responses in the liver. Altogether, these results suggest a combined role for nucleic acid-sensing TLRs in promoting early liver antiviral defense during MCMV infection. Initiation of inflammation following infection requires recognition of the invading microbe by innate immune pattern recognition receptors (PRRs) that signal in response to pathogen-associated molecular patterns (PAMPs). PRRs recognize selfand microbe-associated molecules [1] [2] [3] [4] . Members of the Toll-like receptor (TLR) family of PRRs are transmembrane receptors that are expressed either on the cell surface or within the endosomal compartment and respond to a variety of PAMPs [1] . Murine TLR3, TLR7 and TLR9 are expressed in the endolysosome and are implicated in recognition of viral dsRNA, ssRNA and dsDNA, respectively [1, [5] [6] [7] [8] [9] [10] [11] . Ligation of the nucleic acid-sensing TLRs results in transcription of antiviral genes including type I IFNs (IFN-a/b) and proinflammatory cytokines [1] . TLR3 responses require signaling through the adaptor molecule Toll/IL-1R domain-containing adapter-inducing interferon-b (TRIF), while TLR7 and TLR9 are dependent on the adaptor molecule myeloid differentiation primary response gene 88 (MyD88) to activate transcription factors and induce gene transcription [1, [12] [13] [14] .
Murine cytomegalovirus (MCMV) is a betaherpesvirus that can establish acute infection in multiple organs including the liver. Acute MCMV infection induces an early systemic proinflammatory cytokine response including high levels of type I IFNs, IFN-c, IL-12 and TNF-a [15] [16] [17] [18] [19] . Infection in the liver induces early production of IFN-a, predominantly by plasmacytoid dendritic cells (pDCs), by 40 h post-infection [20, 21] . Type I IFN production mediates downstream responses including chemokine and cytokine production as well as monocyte/macrophage, natural killer (NK) cell and T cell recruitment [20] [21] [22] [23] . Early type I IFN signaling is necessary for NK cell recruitment to the liver, where they deliver the antiviral cytokine IFN-c within the first 48 h post-MCMV infection [23] . The NK cell IFN-c response is an important early step in the control of liver infection [24, 25] . This response induces IFN-c-dependent chemokines, which contribute to the recruitment of CD8+ T cells to the liver [26] . Liver CD8+ T cell responses occur by days 5 and 7 post-MCMV infection and are an important source of cytokines late in acute infection that contribute to resistance against MCMV [26] [27] [28] [29] [30] .
While early responses to MCMV infection in the liver are well understood, it remains unclear how the virus is sensed in this compartment. This is in contrast with other sites, namely the spleen, in which studies by our group and others have definitively shown a role for TLR9 and MyD88 signaling in IFN-a, proinflammatory cytokine and cellular responses in addition to restriction of virus replication [6, 20, 31, 32] . Although TLR7 alone does not appear to have a strong role in MCMV recognition, TLR7 and TLR9 combined deficiency was shown to severely impair pDC responses against MCMV in the spleen [33] . A significant but minor role for TLR3 signaling in the spleen has also been suggested in response to MCMV infection [6] . In the liver, however, studies by our group have demonstrated that early innate responses are TLR9-independent but MyD88-dependent [20] . Liver pDCs from mice genetically deficient in TLR9 produce wild-type (WT) levels of IFN-a at 40 h post-MCMV infection, with intact downstream cellular and proinflammatory cytokine responses. Further, TLR9-deficient mice do not exhibit elevated liver viral titers. Conversely, MyD88-deficient mice have severely impaired liver cytokine and cellular responses, and are unable to control virus replication in this compartment [20, 32] . MyD88 is a common adaptor molecule for TLR9 and TLR7 signaling; however, evaluation of TLR7-deficient mice also demonstrated that TLR7 signals alone were not required to initiate liver antiviral defense [20] .
These TLR-independent but MyD88-dependent antiviral responses suggested possible redundancies among TLR signals in the liver compartment in response to MCMV infection [20, 32] . To investigate this possibility, we utilized mice containing an H412R missense mutation in the endoplasmic reticulum protein UNC93B1 to address the combined function of nucleic acidsensing TLRs in the liver during acute MCMV infection. The UNC93B1 mutation (known as 'triple d' or '3d') impairs signaling through TLR3, TLR7 and TLR9 due to improper trafficking of these receptors to the endosomal compartment, and has been shown to affect exogenous antigen presentation [34, 35] . Our studies show that proinflammatory cytokine production after early infection with MCMV is dependent on UNC93B1. Further, UNC93B1 deficiency exacerbates liver disease and increases viral burden, although MCMV-specific CD8+ T cell responses are not impaired. Collectively, these results suggest a level of redundancy within the liver to promote viral recognition by demonstrating that a combination of nucleic acid-sensing TLRs contributes to innate inflammatory responses during MCMV infection.
Considering the potential of endosomal TLR signals to induce proinflammatory cytokine expression, UNC93B1 deficient 3d mice were first assessed for systemic IFN-a, IFN-c, IL-12 and TNF-a production during early infection with a moderate (5610 4 PFU) dose of MCMV. C57BL/6 (WT) and 3d mice were uninfected or MCMV-infected for 40 h or 48 h. Serum was collected at indicated time points and IFN-a, IFN-c, IL-12p70 and TNF-a were measured by enzyme-linked immunosorbent assay (ELISA). In WT mice, maximal production of IFN-a, IFN-c and IL-12p70 was detected at 40 h post-MCMV infection before declining by 48 h post-infection (Fig. 1 , A-C). In contrast, 3d mice exhibited lower serum levels of these cytokines in response to MCMV infection. Specifically, while serum IFN-a reached 13006420 pg/mL at 40 h post-MCMV infection in WT mice, IFN-a production was reduced by three-fold at this infection time point in 3d mice (4506300 pg/mL), with comparable levels maintained at 48 h post-infection (Fig. 1A) . Likewise, while average IFN-c concentrations in WT mice reached maximal levels of 5306245 pg/mL at 40 h post-MCMV infection, 3d mice failed to induce detectable levels of this cytokine (Fig. 1B) . IL-12p70 production similarly peaked at 40 h post-MCMV infection in WT mice, with levels reaching 10006600 pg/mL. 3d mice, however, produced 12-fold less IL-12p70 at this infection time point (Fig. 1C) . Serum TNF-a levels were elevated in response to MCMV infection at both 40 h and 48 h post-infection in WT mice (69610 and 63617 pg/mL, respectively, Fig. 1D ). In 3d mice, average concentrations of TNF-a were 6-fold lower than WT at 40 h and 2.5-fold lower than WT at 48 h after infection. These results indicate a requirement for endosomal TLR signals for early systemic proinflammatory cytokine production, and concur with previous reports [35] .
Having observed a reduction in the level of proinflammatory cytokines in the serum of 3d mice, cytokine responses in liver cells from 3d mice infected with MCMV were evaluated to address the impact of endosomal TLR signaling in a localized tissue site of infection. The best characterized liver cytokine responses are IFNa and IFN-c [20, 21, 36] . Therefore, to determine whether combined endosomal TLR signaling contributes to the production of these cytokines during MCMV infection, IFN-a and IFN-c in liver homogenates and in individual cell populations were measured in WT and 3d mice uninfected or infected with MCMV for 40 h and 48 h. As shown in Fig. 2A , WT mice displayed fourfold higher levels of IFN-a than 3d mice at 40 h post-infection, with increased levels still evident at 48 h following infection. Since pDCs expressing the marker PDCA-1 have been shown to produce the majority of liver IFN-a at 40 h post-MCMV infection [20] , this cell type was examined in WT and 3d livers during early infection. There was evidence of pDC accumulation in the livers of both WT and 3d mice (Fig. 2B ). However, liver pDCs from 3d mice were impaired in their ability to express IFN-a. There were 4-fold fewer PDCA-1+ pDCs expressing intracellular IFN-a at 40 h, and 3-fold fewer at 48 h post-infection, in 3d mice as compared to WT (Fig. 2C ). This trend was also reflected in the proportion of PDCA-1+ IFN-a+ pDCs at 40 h and 48 h after infection in 3d mice (0.8%60.4% and 2%61%) compared with WT (2%61% and 6%61%).
3d mice similarly demonstrated a defect in liver IFN-c production in response to MCMV infection. In WT mice, IFN-c reached maximal levels of 350061200 pg/g liver at 40 h before contracting by approximately half at 48 h post-infection. In contrast, at 40 h post-infection, 3d mice induced 5-fold less IFN-c than WT (Fig. 2D ). NK cells are an important early source of IFNc in the liver during MCMV infection [24, 25] , and accumulated at this site in both WT and 3d mice (Fig. 2E ). Using intracellular cytokine staining, results shown in Fig. 2F demonstrate a 7-fold reduction in the absolute numbers of NK1.1+ TCRb-liver cells expressing IFN-c in 3d mice at 40 h post-infection as compared to WT. There were also fewer IFN-c-expressing liver NK cells in 3d mice by proportion (0.8%60.3% and 1%60.4%) when compared to WT (6%62% and 4%60.4%) at 40 h and 48 h, respectively. Together, these results demonstrate that, in addition to an effect on systemic cytokine production, combined endosomal TLR signals can affect the expression of critical proinflammatory cytokines in the liver during MCMV infection.
The innate immune response is important both in establishing early control of virus replication and in coordinating downstream adaptive responses. Following MCMV infection, virus-specific CD8+ T cells are recruited to the liver within 5 days and control viral replication at this site through release of cytotoxic molecules and production of cytokines such as IFN-c and TNF-a [26, 27] . Given the abated liver cytokine responses observed in 3d mice, the effect of endosomal TLR signaling on liver CD8+ T cell responses was examined at late time points during acute MCMV infection. The results shown in Fig. 3A demonstrate comparable absolute numbers of CD8+ T cells in WT and 3d mice at days 5 and 7 post-MCMV infection, a trend that was also reflected in proportion (data not shown). To determine whether CD8+ T cells in 3d mice were properly activated against MCMV infection, intracellular expression of IFN-c and TNF-a in CD8+ T cells was examined following ex vivo restimulation with H-2D b M45 viral peptide, an immunodominant epitope of MCMV [29] . CD8+ T cells from 3d mice expressed these two cytokines at day 5 and day 7 post-MCMV infection by proportion and absolute numbers at levels that were comparable or slightly increased over WT (Fig. 3 , B-E).
As an indication of degranulation, surface expression of CD107a was also examined on liver CD8+ T cells from mice infected with MCMV; however, no differences in CD107a expression were detected between 3d and WT mice (data not shown). These results suggest that MCMV-specific CD8+ T cell responses in the liver are not compromised in the absence of endosomal TLR signals.
Previous studies have demonstrated resolution of virus-induced liver disease after 5 days of MCMV infection in WT mice [22, 24, 27, 37] . Therefore, given the impaired inflammatory responses observed in the absence of endosomal TLR signaling, liver sections prepared from 3d and WT mice that were uninfected or infected for 3, 5, or 7 days were hematoxylin and eosin (H&E) stained to evaluate pathology. The histological appearance of liver sections from uninfected 3d and WT mice appeared comparable (Fig. 4, A and B ). By day 3 post-MCMV infection, clusters of infiltrating cells or inflammatory foci, which have been shown to coincide with sites of MCMV antigen expression [23, 37] , were present in WT mice and persisted through day 5 before inflammation was resolved by day 7 post-infection (Table 1 , Fig. 4 , C, E and G). While the inflammatory foci per area of liver were equally apparent in liver sections from 3d mice infected for 3 days (Table 1) , there was an increased presence of cytomegalic inclusion bodies characteristic of MCMV-infected cells that were not readily apparent in WT mice (Fig. 4D) . By day 5 postinfection, livers from 3d mice were characterized by widespread areas of inflammation compared to the more punctate foci in WT livers (Fig. 4, E and F) . Moreover, the inflammatory foci per area of liver in 3d mice at day 5 post-infection were significantly more numerous and contained a greater number of nucleated cells compared to WT (Table 1) . However, by day 7 post-infection, inflammation in 3d mice showed signs of resolution that were similar to WT (Table 1, Fig. 4 , G and H).
To further evaluate the effects of endosomal TLR responses on overall liver function, the liver enzyme alanine aminotransferase (ALT) was measured in serum samples from WT and 3d mice that were uninfected or infected with MCMV for 3, 5 or 7 days. Uninfected mice had comparable baseline levels of systemic ALT (Fig. 5 ). By day 3 of infection, similar elevations in ALT levels were detected in both groups. In contrast, by day 5 post-infection, the levels of systemic ALT in 3d mice reached values of 9006400 U/ L and were three-fold higher than the ALT levels measured in WT mice (2706100 U/L; Fig. 5 ), indicating augmented liver disease. In contrast, by day 7 post-infection, 3d mice demonstrated a sharp decline in systemic ALT levels to values of 117663 U/L, which were comparable to ALT levels exhibited in WT mice (108685 U/L). Thus, at this time point of acute infection, both groups of mice exhibited ALT levels near baseline levels, consistent with the resolution of virus-induced liver pathology observed in 3d and WT mice (Fig. 4, G and H; Fig. 5 ). Taken together, these results suggest that in the absence of endosomal TLR signals, there is pronounced liver inflammation accompanied by a transient increase in liver damage during acute MCMV infection.
Given the diminished early cytokine responses and enhanced liver disease observed in 3d mice, the contribution of endosomal TLR responses to control of virus replication in the liver was assessed in WT and 3d mice infected with MCMV. Compared with those in WT mice, viral titers were elevated by 1 log on day 3 of infection in 3d mice, and remained significantly higher at day 5 post-infection when compared to WT (Fig. 6) . Ultimately, while WT livers showed evidence of viral clearance at days 7 and 9 postinfection, 3d liver virus titers remained approximately 2 logs higher at these times of infection as compared to WT mice. Thus, endosomal TLR signaling contributes to the control of MCMV replication in the liver during acute infection.
The aim of these studies was to identify the TLR signaling pathways required for the innate recognition of virus infection in the liver, a common target organ of many viruses that significantly contributes to innate immune defenses [38] [39] [40] . Moreover, the liver contains various innate immune cells that express TLRs [41, 42] ; however, the role of TLRs in host defense against infection at this site remains largely unclear. Because responses in the liver do not appear dependent on individual TLRs [6, 20, 32] , we utilized 3d mice, which lack endosomal TLR3, TLR7 and TLR9 signaling due to a mutation in the endoplasmic reticulumresident protein UNC93B1 [34, 35] , to address the contribution of endosomal TLRs in liver antiviral defenses against acute MCMV infection. The results demonstrated impaired production of proinflammatory cytokines by NK cells and pDCs in livers from MCMV-infected 3d mice. Additionally, 3d mice had elevated viral titers in the liver that coincided with transient but exacerbated liver disease, although virus-specific CD8+ T cell responses were not affected. Interestingly, a previous study demonstrated that TLR3 was not required for the generation of adaptive antiviral responses to MCMV [43] , although there is evidence that TLR3 signaling contributes in part to the early control of MCMV infection by the systemic induction of type I IFN [6] . Other studies have implicated a synergistic role for TLR7 and TLR9 in promoting MCMV recognition and immune defense in the spleen [33] . The impaired liver responses observed in 3d mice that were not apparent in TLR9 or TLR7-deficient mice [20] suggests that a level of redundancy unique to innate immunity is in place within infected tissue sites to rapidly respond to viral infection. Overall, these studies advance our understanding of the process of viral recognition in the complex liver environment and suggest that UNC93B1 is a critical intermediate factor in innate virus sensing activated by MCMV.
Previous studies have demonstrated impaired serum cytokine production and increased susceptibility to infection in 3d mice infected with a high lethal dose of MCMV [35] ; however, our study is the first report to document the contribution of the 3d mutation to impaired MCMV defense in the liver. In agreement with the study by Tabeta et al. [35] , our results, using a moderate dose of MCMV, demonstrated a diminished serum cytokine response (Fig. 1) , reduced splenic IFN-a production and elevated viral titers in the spleen (data not shown). These results were not unexpected given the known role for the nucleic acid-sensing TLRs in MCMV recognition and the subsequent production of proinflammatory cytokines and type I IFN by splenic pDCs [19, 31, 33, 44] . Production of type I IFN is a critical early step in antiviral defense, and we further demonstrated diminished levels of liver IFN-a in MCMV-infected 3d mice. This defect was in part due to an impaired ability of liver pDCs to express this cytokine and is consistent with previous reports identifying pDCs as an important early source of IFN-a in response to TLR7 and TLR9 ligands [10, 20, 31, 32, 33, [45] [46] [47] . Altogether, these results concur with previous studies indicating that pDCs are the predominant leukocyte producer of type I IFN in the liver during early MCMV infection [20] , and demonstrate that production of these cytokines by pDCs is influenced by the 3d mutation.
Despite impairments in liver IFN-a production in 3d mice, it is notable that this response was not totally abrogated. This suggests the presence of alternative pathways to type I IFN production in the liver, and may be the reason that IFNa/bR-deficient mice die by day 5 in response to infection with a moderate dose of MCMV, as reported previously [36] , while 3d mice do not. Studies have also demonstrated production of type I interferon from cells other than pDCs at 44-48 hours following MCMV infection [32, 48, 49] . In addition, the liver contains a variety of parenchymal and nonparenchymal cells that express TLRs and are capable of type I IFN production [38, [40] [41] [42] . There may also be TLR-independent pathways leading to the production of these cytokines in response to MCMV, including cytoplasmic RNA-and DNA-sensing receptors, as have been demonstrated with other virus models but have yet to be examined in the context of MCMV infection [1] [2] [3] [4] [50] [51] [52] [53] [54] [55] .
It has been established that NK cell inflammatory responses and production of IFN-c are essential to defense against MCMV in the liver [23] [24] [25] . In 3d mice, NK cell production of IFN-c in the liver was severely impaired during early MCMV infection and likely contributed to increased viral burden and liver pathology. The reduced levels of IFN-c in NK cells may reflect the deficiency of serum IL-12 seen in 3d mice. These results concur with previous reports that type I IFN regulates IL-12 production by conventional DCs and consequently the production of IFN-c by NK cells [19, 48, 56] . The defect in liver cytokine production in 3d mice is reminiscent of that reported for MyD88-deficient mice. Notably, however, MyD88-deficient mice exhibited more severe liver pathology and greater elevations in viral titers when compared to WT than we observed in 3d mice [20] . Interestingly, mice deficient in TLR7 and TLR9 exhibited decreased levels of systemic IFN-a/b and increased susceptibility to MCMV infection Data presented represent the mean 6 SD of two liver sections unless otherwise indicated. b Number of foci per 8650 mm 2 areas in day 5 infected 3d livers was significantly higher than WT at the same infection time point, p#0.04. C Nucleated cells from large areas of inflammation contained .60 nucleated cells in less defined foci, and do not include a calculation of SD. doi:10.1371/journal.pone.0039161.t001 Figure 5 . Assessment of virus-induced liver disease in 3d mice. Serum samples were collected from C57BL/6 (WT) or 3d mice that were either uninfected or infected for 3, 5, or 7 days with MCMV. Circulating levels of ALT were measured as described in Methods. Data are the combined results from three independent experiments and show the mean 6 SD (n = 6-8 mice per group). Asterisks denote p values#0.003. doi:10.1371/journal.pone.0039161.g005 [33] . Taken together, these observations further support the notion that the liver possesses compensatory mechanisms to combat viral infection in the combined absence of endosomal TLR signals. Accordingly, despite the early effects of endosomal TLR deficiency, 3d mice were able to mount robust CD8+ T cell cytokine responses. It should be noted that the 3d defect has previously been shown to impair exogenous antigen presentation, including cross presentation, which has a reported role in priming CD8+ T cell responses during MCMV infection [35] . However, we detected no overt defect in CD8+ T cell responses within the first seven days of MCMV infection in the liver. Further, examination of activation markers suggested that a similar proportion of CD8+ T cells from 3d mice were more highly activated when compared to WT (data not shown). Several studies have demonstrated the contribution of activated virus-specific CD8+ T cells to effective hepatic immunity against MCMV infection [26] [27] [28] . In addition, the normal development of adaptive responses despite impaired innate responses is well documented during MCMV infection. Studies have shown that reduced levels of type I IFN do not affect the accumulation or activation of antigenspecific CD8+ T cells in response to low or moderate MCMV inoculums [48] . Likewise, while IL-12 is critical in inducing NK cell IFN-c expression, T cell responses can occur in an IL-12independent manner [17, 18, [57] [58] [59] . NK cells have the potential to negatively regulate CD8+ T cell responses during MCMV infection [60] [61] [62] ; thus, it is probable that impaired NK cell function in the absence of endosomal TLR signals contributes to inflated T cell responses. The presence of increased virus in the liver may also contribute to the robust T cell recruitment and cytokine production observed at late infection time points in 3d mice. Interestingly, although the severity of viral liver pathology was more pronounced in 3d mice, inflammation and liver injury subsided late in infection. These observations suggest that CD8+ T cells in the liver can respond to limited amounts of type I interferon for activation in the presence of compromised innate responses, emphasizing the prevalence of compensatory mechanisms in place within the liver to deal with infection and promote adaptive immunity. In contrast, IFN-a/bR deficiency negatively impacts innate inflammatory responses and resistance to MCMV infection in the liver [21, 24, 36] .
In conclusion, this study indicates that UNC93B1, which is essential for combined endosomal TLR signaling, contributes to development of effective innate immune responses to an acute Figure 6 . Effect of the 3d mutation on viral clearance. Livers were harvested from C57BL/6 (WT) or 3d mice that were either uninfected or infected for 3, 5, 7 or 9 days with MCMV (5610 4 PFU). Viral titers were determined using a standard plaque assay. The level of detection of the plaque assay is 2 log PFU/g liver (dashed line). Each data point represents an individual WT (filled diamonds) or 3d mouse (open circles). Data from days 0, 3, 5, and 7 are the combined results of three independent experiments (n = 4-7 mice per group). In data from day 9, n = 5 mice per group. Asterisks denote a significant difference between WT and 3d mean PFU/g liver (p values#0.04). doi:10.1371/journal.pone.0039161.g006 virus infection in the liver. Our results show that this contribution involves modulation of early innate proinflammatory cytokine production from liver pDCs and NK cells and subsequent control of MCMV replication and pathology before activation of an adaptive immune response. Altogether, these results highlight a process of virus recognition with multiple pathways in place to promote host resistance to infection in the liver microenvironment.
Pathogen-free C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/6 Unc93b1 3d/3d mice were a kind gift from Dr. Bruce Beutler (The Scripps Research Institute, La Jolla, CA) and were generated as described [35] . C57BL/6J mice were housed and UNC93b1 3d/3d mice were bred in pathogenfree mouse facilities at Brown University. Male and female mice aged 8-10 weeks were used in experiments. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal work was approved by the Brown University Institutional Animal Care and Use Committee (Protocol Number: 0909082 and 0903035).
MCMV Smith strain was used in all experiments. This strain was prepared as a salivary gland-passaged stock from CD1 mice. Moderate dose infection (5610 4 PFU per mouse) was initiated on day 0 by intraperitoneal injection. In vivo responses were measured at indicated time points. For infectious viral titer quantification, organs were weighed, homogenized in cold supplemented DMEM (Invitrogen Life Technologies) and supernatants were collected following centrifugation. Serially diluted samples were used to inoculate confluent monolayers of bone marrow stromal cells (ATCC M2-10B4) in 24-well tissue culture plates and incubated for one hour at 37uC, 5% CO 2 . Following incubation, inoculums were removed and monolayers were overlaid with a 16DMEM/0.5% low-melt agarose solution. Cells were incubated for 7 days at 37uC, 5% CO 2 , then fixed in 10% buffered formalin and stained with crystal violet. Plaques were counted to determine viral titer as previously described [22, 24, 26] .
Liver leukocytes were prepared as previously described [23] [24] [25] . Briefly, following mechanical dissociation of tissue, red blood cells were removed by lysis with ammonium chloride and leukocytes separated by Percoll density gradient. To generate homogenates for cytokine analysis, the liver caudate lobe was homogenized in RPMI 1640 (Invitrogen Life Technologies) and supernatants collected following centrifugation. Serum was isolated from whole blood by centrifugation in the presence of heparin and stored at 280uC until further use in cytokine analyses or ALT assays.
Liver homogenates and serum were tested for cytokines by standard sandwich ELISA. IFN-c, IL-12p70 and TNF-a were assayed by DuoSet (R&D Systems). IFN-a was measured by VeriKine mouse ELISA kit (R&D Systems). Limits of detection were 15 pg/mL for DuoSets and 12.5 pg/mL for VeriKine ELISA kits.
The following fluorochrome-conjugated mAbs were used in flow cytometric analyses: NK1.1-PE and TCRb-APC to distinguish NK cells; CD8a-PECy7 and TCRb-FITC to distinguish CD8+ T cells; and PDCA-1-APC (Miltenyi Biotec) to identify pDCs. Prior to surface staining, cells were incubated with anti-CD16/CD32 mAb to block nonspecific binding of Abs to Fcc III/ II receptors (clone 2.4G2). Unless otherwise noted, antibodies were obtained from BD Biosciences or eBioscience. For intracellular staining of cytokines, cells were treated with Brefeldin A (eBioscience) for 4 hours at 37uC, 5% CO 2 and permeabilized prior to labeling with IFN-a-FITC (R&D Systems), IFN-c-PE, or TNF-a-APC (BD Biosciences). When indicated, leukocytes were stimulated for 5 hours with 100 ng/mL MCMV M45 peptide in addition to Brefeldin A treatment. Samples were acquired using a FACSCalibur and analyzed with BD Cell Quest software. For analysis, viable cells were gated by FSC and SSC. Isotype controls were used to set positive analysis gates.
Portions of the median liver lobes were isolated, fixed in 10% neutral buffered formalin, and paraffin embedded. Tissue sections (5 mm) were stained with H&E and analyzed microscopically. Images shown were photographed at the indicated magnification with a DP70 digital camera and software (Optical Analysis Corporation). Inflammatory foci, defined as discrete clusters containing between 6-60 nucleated cells, were quantitated as described previously [23] . In brief, inflammatory foci were identified, at a magnification of 200, as clusters of cells in totals of 8650 mm 2 areas per representative tissue. In some cases, liver sections had larger areas of inflammation with .60 cells with less defined foci, and are indicated as such. Numbers of nucleated cells per inflammatory foci were counted in 20 individual foci per representative tissue at a magnification of 400. Liver alanine aminotransferase (ALT) was measured in serum samples by Marshfield Labs (Marshfield, WI).
Student's t test was used to determine statistical significance of experimental results when indicated (p#0.05). Bioinformatics analysis of rabbit haemorrhagic disease virus genome BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Synonymous codons are not used randomly [1] . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors [2, 3] . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response [4] . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition [5] . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus [6] . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets [4] . Analysis of the bovine papillomavirus type 1 (BPV1) late genes has revealed a relationship between codon usage and tRNA availability [7] . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression [8] . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus [9] . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses [10, 11] . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression.
Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus (RHDV), which is the pathogen causing Rabbit haemorrhagic disease (RHD), also known as rabbit calicivirus disease (RCD) or viral haemorrhagic disease (VHD), a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames (ORFs) separately encoding a predicted polyprotein and a minor structural protein named VP10 [12] . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 [13, 14] . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 [15] . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: (i) the extent and causes of codon bias in RHDV; (ii) A possible genotyping of RHDV; (iii) Codon usage bias as a factor reducing the expression of VP10 and (iiii) the evolution of the ORFs.
The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number (SN), collection dates, isolated areas and GenBank accession numbers are listed in Table 1 .
To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports (2 Sharp, Tuohy et al. 1986 ) as the followed formula:
Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly.
The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons.
The ENC, as the best estimator of absolute synonymous codon usage bias [16] , was calculated for the quantification of the codon usage bias of each ORF [17] . The predicted values of ENC were calculated as ENC = 2 + s + 29
where s represents the given (G+C) 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program [18] . 
Analyses were conducted with the Nei-Gojobori model [19] , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω (dn/ds) were calculated in MEGA4.0 [20] .
Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon (excluding Met, Trp, and the termination codons) [21] .
Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern [22] . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows.
The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, (C+G)% content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and (C+G)% content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region (length > 250 bps) in the ORF1 of RHDV (30 isolates) genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω(dN/dS) values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints.
COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis (f' 1 ) which accounted for 42.967% of the total variation, and another major trend in the second axis (f' 2 ) which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is 
To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and (C+G)% were compared with A 3 %, U 3 %, C 3 %, G 3 % and (C 3 +G 3 )%, respectively (Table 5 ). There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes (f' 1 and f' 2 ) of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis (f' 1 ) accounts for 42.967% of the total variation, and the second axis (f' 2 ) accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples 
There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV (Table 2) , we obtained several conclusions and conjectures as followed.
4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values [18] . ENC values of RHDV genomes were plotted against its corresponding (C 3 +G 3 ) %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias.
As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 [5] . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10.
Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve.
value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome [23] [24] [25] [26] [27] [28] [29] , although the details remain elusive.
As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 (Table 7) , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different.
Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group (marked as Group 1) is positive value and the other one (marked as Group 2) is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes.
In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10
(encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference.  Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies BACKGROUND: Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). METHODOLOGY/PRINCIPAL FINDINGS: We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. CONCLUSIONS/SIGNIFICANCE: The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. Onchocerciasis, or river blindness has historically represented one of the most important neglected tropical diseases in the developing world as measured as a cause of socio-economic disruption [1] . It is also considered a candidate for elimination by the international community [2, 3] . As a result of these factors, the international community is currently supporting several programs whose goals are either to eliminate the disease as a public health problem, or to locally eliminate the causative agent of the disease, Onchocerca volvulus. These include the Onchocerciasis Elimination of the Americas (OEPA), the African Programme for Onchocerciasis Control (APOC), and the Ugandan Onchocerciasis Elimination Program (UOEP).
Entomological criteria play an important role in the elimination criteria recommended by the World Health Organization (WHO) [4] and those currently utilized by OEPA [5] . Entomological data play an especially important role in the certification of elimination following the cessation of treatment, as the prevalence of infective stages of the parasite in the fly population is the timeliest measure of transmission in a given area. However, demonstrating that transmission is interrupted requires that large numbers of flies be tested. For example, current OEPA guidelines require that the prevalence of flies carrying infective larvae (L3) be less than 1/2000 in every sentinel community for transmission to be interrupted [5] . In order to be able to state with a 95% confidence that the prevalence of infective flies is less that 1/2000 requires examining approximately 6000 flies from each sentinel community. Examining such large numbers of insects using conventional methods (dissection) is impractical. For this reason, the current guidelines recommend the use of pool screening PCR based methods to conduct the entomological studies necessary to document transmission interruption [4] .
Currently, the accepted method for pool screening vector black flies to detect O. volvulus relies upon screening DNA prepared from fly pools with a PCR assay targeting a repeated sequence family (the O-150 repeat [6] ) specific for parasites of the genus Onchocerca. Algorithms have been developed that permit one to derive a point estimate of the prevalence of infection in the fly population (and an associated confidence interval) from the number of PCR positive pools and the number of flies contained in each pool [7] . Furthermore, because the infective stage of the parasite is the only form found in the black fly head capsule, separated pools of heads and bodies may be screened to obtain estimates of the prevalence of infective flies (flies with infective stages in their head) and the prevalence of infected flies (flies with immature larval stages in their bodies). This approach has been used to monitor transmission of O. volvulus in many foci of Latin America and Africa [8] [9] [10] [11] , as well as to certify the interruption of transmission in foci on both continents [5, [12] [13] [14] .
Previous modeling studies have shown that increasing pool sizes has relatively little effect on the accuracy of the estimate of prevalence of infection obtained, so long as the proportion of positive pools remains less than the majority of pools screened [15] . Thus, in situations where pool screening is used to certify transmission interruption (where infective flies are extremely rare or non-existent) the pool size is only limited by the biochemical constraints of the assay. The current method of DNA extraction for the O-150 PCR assay is based upon adsorption to a silica matrix [16] . This preparation results in DNA samples that still contain inhibitors of the PCR, limiting the number of flies that may be included in each pool. Currently, pool sizes are limited to 50 individual heads or bodies (in the case of flies from Latin America) [9] or 100 individual heads or bodies (in the case of flies from Africa) [7] . Developing alternative methods to prepare DNA that would permit an increase in the maximum number of heads or bodies in each pool would decrease the cost and effort necessary to screen the requisite large numbers of flies necessary to certify transmission interruption.
Magnetic bead based purification protocols have been developed for many different pathogens. Most of these involve direct capture of the pathogen using beads coated with pathogen-specific antibodies. This method, known as immunomagnetic separation (IMS), has been successfully used to purify and concentrate viruses [17] , bacteria [18, 19] and fungal [20] pathogens. Similarly, methods have been developed which use oligonucleotides to magnetically purify pathogen genomic DNA [21] .
Here, we describe a magnetic bead capture method to isolate O. volvulus DNA from homogenates prepared from pools of Latin American and African Simulium vector black flies. This method is shown to be an improvement upon the current DNA purification method utilizing organic extraction and silica adsorption.
Simulium ochraceum s.l. females were collected in public areas of the community of José María Morelos y Pavón, Chiapas, México between the hours of 0700 and 1000. Previous studies have demonstrated that the majority of flies captured during this period were nulliparous, and the risk of infection was therefore minimal.
Simulium damnosum s.l. were obtained from breeding sites on public land located near the communities of Bodajugu and Sakora. These communities are located in the Region des Cascades in Southwestern Burkina Faso. This region is located within the area of the former Onchocerciasis Control Programme in West Africa, where onchocerciasis has been eliminated as a public health problem.
O. volvulus L3 were obtained from experimentally infected Simulium damnosum s.l. flies 7 days after infection with skin microfilariae. The flies were kept at 25uC and 80% relative humidity to allow the microfilariae to develop into L3. Larvae were isolated from the flies by dissection into dissecting medium (IMDM+10% FCS+2x Penicillin-Streptomycin-Fungizone) using a dissecting microscope. The cleaned larvae were frozen in 9% DMSO, 4 mM PVP, 10% FCS in Grace medium using Bio-Coll (freezing to 240uC at 1uC/minute followed by 30 minutes at 240uC) and then transferred to liquid nitrogen for long-term storage. The parasite material was prepared in the Tropical Medicine Research Station, Kumba, Cameroon, and is being stored at the New York Blood Center.
Pools containing varying numbers of black flies were prepared and the heads and bodies separated by freezing and agitation, as previously described [7] . A single O. volvulus L3 was added to each pool. Head and body pools were placed in a 1.5 ml microcentrifuge tube and purified using magnetic silica coated beads (Machery-Nagel GmbH & Co, Bethlehem, PA, USA) following the instructions provided by the manufacturer. In brief, the pools were homogenized in 200 ml of T1 buffer, 25 ml of proteinase K solution provided in the kit was added and the homogenates were incubated at 56uC for 30 minutes. The homogenates were subjected to centrifugation at 13,4006g for 5 minutes at room temperature. A total of 225 ml of the supernatant was transferred to a fresh tube containing 24 ml B-beads (Machery-Nagel) and 360 ml MB2 buffer (Machery-Nagel), and the tube shaken for 5 minutes at room temperature. The magnetic beads were isolated by placing the tubes in a six tube magnetic separator (Dynal MPC-S; Invitrogen). The supernatants were removed and discarded, and 600 ml of MB3 wash buffer was added to each sample. The bead/ DNA complexes were washed by shaking for 5 min at room temperature. The beads were collected in the magnetic separator
The absence of infective larvae of Onchocerca volvulus in the black fly vector of this parasite is a major criterion used to certify that transmission has been eliminated in a focus. This process requires screening large numbers of flies. Currently, this is accomplished by screening pools of flies using a PCR-based assay. The number of flies that may be included in each pool is currently limited by the DNA purification process to 50 flies for Latin American vectors and 100 flies for African vectors. Here, we describe a new method for DNA purification that relies upon a specific oligonucleotide to capture and immobilize the parasite DNA on a magnetic bead. This method permits the reliable detection of a single infective larva of O. volvulus in pools containing up to 200 individual flies. The method described here will dramatically improve the efficiency of pool screening of vector black flies, making the process of elimination certification easier and less expensive to implement.
as before, and the washing procedure repeated with successive washes with 600 ml of MB4 and MB5 wash buffers (Machery-Nagel). Following the wash in the MP5 buffer, the beads were exposed to air for one minute to permit the traces of ethanol to evaporate. DNA was eluted from the beads by the addition of 100 ml of elution buffer. The beads were shaken for 5 minutes at room temperature to elute the DNA, and the beads removed by placing the tubes in the magnetic separator. The supernatant containing the purified DNA was then transferred to a fresh tube.
Oligonucleotide capture of O. volvulus DNA Pools of spiked heads and bodies were prepared as described above. The head and body pools were homogenized in 500 ml of 10 mM Tris-HCl (pH 8.0) 1 mM EDTA, and proteinase K added to a final concentration of 2 mg/ml. The homogenates were incubated at 56uC for 2 hours, and dithiothreitol added to a final concentration of 20 mM. The samples were heated to 100uC for 30 minutes and subjected to three freeze-thaw cycles. The homogenates were subjected to centrifugation at 13,4006g for 5 minutes and the supernatant placed into a new tube. The solutions were brought to a final concentration of 100 mM Tris-HCl (pH 7.5) 100 mM NaCl. A total of 5 ml of a 0.5 mM solution of OVS2-biotin primer (59B-AATCTCAAAAAACGGGTA-CATA-39, where B = biotin) was added to each sample. The samples were then heated to 95uC for three minutes and allowed to cool slowly to room temperature.
While the probe was annealing to the DNA in the solution, 10 ml of Dynal M-280 strepavidin coated beads (Invitrogen) were placed in a single well of a 96 well tissue culture plate. The plate was placed on a magnetic capture unit (Dynal MPC-96, Invitrogen) and the beads collected for 2 minutes. The beads were then washed five times with 200 ml binding buffer (100 mM Tris-HCl (pH 7.5) 100 mM NaCl) per wash, resuspended in 10 ul and added to the sample. The samples were incubated at 4uC overnight on a roller to permit the oligonucleotide-DNA hybrids to bind to the beads. The samples were placed in the magnetic separator for two minutes to capture the beads and the supernatant discarded. The beads were resuspended in 150 ml of binding buffer by pipetting, and the beads captured by placing the tubes in the magnetic separator for two minutes. The wash step was repeated five times. The beads were then resuspended in 20 ml of sterile water, heated to 80uC for 2 minutes and cooled rapidly on ice for two minutes. The beads were removed by placing the tubes in the magnetic capture apparatus, and the supernatant containing the purified DNA transferred to a new tube.
A total of 2.5 ml of the purified genomic DNA was used as a template for the PCR amplifications carried out in a total volume of 50 mL containing 0.5 mmol/L of O-150 primer (59-GAT-TYTTCCGRCGAANARCGC-39) and 0.5 mmol/L of biotinylated O-150 primer (59-B-GCNRTRTAAATNTGNAAATTC-39, where B = biotin; N = A, G, C, or T; Y = C or T; and R = A or G). Reaction mixtures also contained 60 mM Tris-HCl, (pH 9.0), 15 mM (NH4) 2 SO4, 2 mM MgCl 2 , 0.2 mM each of dATP, dCTP, dGTP and dTTP, and 2.5 units of Taq polymerase (Invitrogen). Cycling conditions consisted of five cycles of one minute at 94uC, two minutes at 37uC, and 30 seconds at 72uC, followed by 35 cycles of 30 seconds each at 94uC , 37uC , and 72uC. The reaction was completed by incubating at 72uC for six minutes.
Amplification products were detected by PCR enzyme-linked immunosorbent assay (ELISA), essentially as previously described [10] . Briefly, 5 ml of each PCR reaction was bound to a streptavidin-coated ELISA plate, and the DNA strands denatured by treatment with alkali. The bound PCR fragments were then hybridized to a fluorescein-labeled O. volvulus-specific oligonucleotide probe (OVS2: 59-AATCTCAAAAAACGGGTACATA-FL-39), and the bound probe detected with an alkaline phosphataselabeled anti-fluorescein antibody (fragment FA; Roche Diagnostics). Bound antibody was detected using the ELISA amplification reagent (BluePhos) kit from KPL (Gaithersburg, USA) following the manufacturer's instructions. Color development was stopped by the addition of 100 ml AP stop solution, and the plates read in an ELISA plate reader set at 630 nm. Samples were scored positive if their optical density exceeded either the mean plus three standard deviations of ten negative control wells run in parallel or 0.1, whichever was greater.
An initial series of experiments were carried out with pools of heads and bodies of S. ochraceum s.l. (a major Latin American vector of onchocerciasis). Pools containing varying numbers of heads of bodies were spiked with a single O. volvulus L3, and DNA prepared from the pools using either the conventional method of organic extractions followed by adsorption to a silica matrix, or by oligonucleotide capture of O. volvulus genomic DNA followed by magnetic purification of the captured oligonucleotide-DNA complexes. The conventional method consistently produced a positive signal in pools containing up to 50 heads (Table 1) . Pools containing greater than 50 heads were not positive in the assay. All (Table 1 ). In contrast, positive signals were obtained in all pools containing up to 200 heads or bodies in the assays performed on the oligonucleotide capture purified DNA samples ( Table 1) .
The preliminary experiments suggested that the oligonucleotide capture method was capable of detecting one L3 in pools of up to 200 heads or bodies. To further explore the sensitivity of the assay, the experiment was repeated using 10 separate pools containing 200 heads or bodies spiked with a single L3. All pools were found to be positive, suggesting that the oligonucleotide capture assay was capable of consistently detecting a single L3 in pools of up to 200 heads or bodies of S. ochraceum s.l. (Table 2) .
Previous studies had demonstrated that the conventional silica adsorption method was capable of detecting a single infected S. damnosum s.l. (the major African vector of onchocerciasis) fly in a pool containing up to 99 uninfected flies [7] . To determine if the performance of the oligonucleotide capture assay was similar when applied to S. damnosum s.l., the spiking experiments were repeated employing pools containing 200 S. damnosum s.l. heads or bodies. All spiked pools were found to be positive ( Table 2 ), suggesting that the capture assay preformed equally well on both African and Latin American vectors of onchocerciasis.
For the capture assay to be cost effective, it should be competitive with the cost of the conventional silica adsorption assay. The two assays require roughly the same amount of technical time, so labor costs may be assumed to be equivalent per sample for the two assays. However, because it will be possible to increase the size of the pools 2-4 fold when using the capture assay, a reduction in labor costs of between 50% and 75% would be realized when costs are considered on a per-fly-tested basis. Similarly, the per-sample cost of carrying out the conventional assay is roughly $2.22 per pool, while the cost of the magnetic bead assay is $3.81 per pool. However, because the magnetic bead permits more flies to be tested per pool, cost savings in reagents are realized when the costs are amortized on a per fly basis (Table 3) .
Previous studies have demonstrated that the algorithms used to predict the prevalence of infection in a population from data derived from screening pools of samples are relatively insensitive to the size of the pool, so long as the proportion of positive pools does not represent a substantial majority of the samples screened [15] . Thus, the size of pools used in a pool screening protocol is more likely to be limited by the biochemistry of the detection assay than by the underlying statistical uncertainties associated with screening pools of samples. This is particularly true when positive samples are extremely rare, as in the case when monitoring for transmission interruption. The data presented above suggest that the oligonucleotide capture method of purifying O. volvulus DNA is superior to the conventional silica adsorption method. Mixing experiments have demonstrated that PCR inhibitors carried through the silica adsorption process limits the size of the pools that may be screened to 50 individual flies for S. ochraceum and 100 for S. damnosum s.l (data not shown). The oligonucleotide capture method appears to result in DNA preparations that are freer of PCR inhibitors than are those prepared using silica adsorption. The practical result of this improvement is that it permits a 2 to 4fold increase in the number of black fly heads or bodies that can be included in a single pool. This increase in pool size results in a dramatic cost savings in the per-unit cost of the O-150 pool screen assay. Using the oligonucleotide capture assay, labor costs are reduced by 50-75%, while the overall cost of reagents needed is $3.81 per pool of 200 flies or less than $0.02 per individual fly. The decrease in cost and corresponding increase in the efficiency of the assay will make it more practical to screen the large numbers of flies necessary to demonstrate transmission interruption and to certify elimination of onchocerciasis.
Because the oligonucleotide capture assay is a modification of the conventional silica adsorption assay, the equipment required to carry out both assays is quite similar. The only additional equipment necessary when replacing the conventional assay with the oligonucleotide assay is the magnetic capture apparatus. This unit is relatively inexpensive, costing less than $579 (Invitrogen's list price). This would be recovered in reagent costs alone after screening just 114 pools of flies.
The O-150 PCR is based upon the amplification of a genus specific tandemly repeated DNA sequence present in the genome of Onchocerca parasites [22] . Thus, the standard O-150 PCR will amplify sequences present in all Onchocerca species, including Onchocerca ochengi, a cattle parasite that is sympatric with O. volvulus in sub-Saharan Africa. Currently, the O-150 PCR is made species or strain specific by modifying the amplification conditions to limit amplification to species specific members of the O-150 repeat family [23] or by the use of species or strain specific probes to detect the resulting amplicons [8] . However the oligonucleotide used in the capture assay (OVS2) has previously Table 3 . Cost analysis of silica adsorption and oligonucleotide capture assays.
Step  Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89–97) and NP(198–206), respectively. The results indicate that peptides NP(89–97) (PKKTGGPIY) and NP(198–206) (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken. The introduction into the human population of animal-derived influenza A viruses with a novel haemagglutinin (HA), or a novel HA and neuraminidase (NA), and their subsequent spread could result in global influenza pandemics [1] . Since 2003, the highly pathogenic H5N1 avian influenza virus (AIV) has caused numerous cases of severe disease and death in humans [2] . An influenza pandemic could ensue if this virus developed the capacity to spread easily among humans [3] [4] [5] . Migratory birds constitute the natural reservoir for AIVs, but chickens may play a key role in the transmission to humans [6] . Epitopes can be used for accurately monitoring immune responses to AIV and for the rational design of protective vaccines. However, only two epitopes from the H5N1 avian influenza A/Vietnam/1194/2004 virus are included in the Immune Epitope Database and Analysis Resources (IEDB). The majority of T cell and B cell epitopes have been identified in mouse, human, or rabbit hosts. Few epitopes have been described in chicken [7] .
The structural basis of peptide binding to mammalian major histocompatibility complex (MHC) class I molecules is well understood [8] . The peptide-binding groove is formed by the a1 and a2 domains. Each domain contributes four strands to an eight -stranded anti-parallel b-sheet. Two long interrupted helices, one from each domain, pack against the side of this sheet in an orientation directed away from the cell membrane. There is a series of pockets (A-F) along the peptide-binding groove where highly polymorphic amino acids mediate recognition via haplotype-specific associations with antigens and T cell receptors. However, highly conserved residues are found at both ends of the peptide-binding groove that form a network of hydrogen bonds, which directly interact with hydrogen bonds at the peptide's Nterminus and C-terminus [9] [10] [11] . Peptides that bind to MHC class I molecules are usually octamers or nonamers, where only one or a few residues can interact with polymorphic residues in the groove. These residues are known as anchor residues when they are found in an anchoring position [12] [13] [14] [15] . Several public databases and prediction services are available for MHC molecular ligands and peptide motifs, including SYFPEITHI and RANKPEP [16, 17] .
Unlike mammalian studies, the majority of investigations of MHC class I molecules in chicken remain limited to primary sequences and the structure of MHC class I molecule from the B21 haplotype was solved only recently [18, 19] . The chicken MHC B system is located on chicken Chromosome 16 (Chr16) and is composed of tightly linked polymorphic regions: BF (class I) and BL (class IIb) and a large family of polymorphic Ig-superfamily (IgSF) genes called BG-the latter sharing sequence similarities with mammalian MHC butyrophilin, myelin oligodendrocyte glycoprotein, and TRIM genes [20, 21] . The MHC B-F molecules are structurally and functionally similar to mammalian MHC class I molecules. The two class I genes are known as BF1 and BF2, although the BF2 gene is mainly expressed. The MHC class I (B-F) molecules present antigen peptides to the CD8 + T lymphocytes, which have a central role in the immune system. The MHC class I molecules of different haplotypes have specific peptide-binding tropisms, and B-F-associated peptide-binding motifs have been determined for several haplotypes, including B4, B12, B15, and B19 [22] . Thirteen peptides derived from the v-src gene of the Rous sarcoma virus (RSV) Prague strain were predicted to fit the peptide-binding motif of the MHC class I molecules of B12 haplotype and all 13 synthetic peptides actually bound to the BF12 class I molecule in subsequent binding tests [23] . This demonstrates that peptide-binding motifs can be used to predict the antigen peptides presented by chicken MHC class I molecules.
However, no T cell epitope of H5N1 AIV NP has yet been identified in the chicken [7] , while no information is available on the structure of chicken MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes. For the first time, the current study reports the homology modelling structures of the peptidebinding domains of chicken MHC class I molecules for the B4, B12, B15, and B19 haplotypes. Potential T cell epitopes were predicted by molecular docking of 25 peptides in the H5N1 AIV NP in chicken. NP 89-97 and NP 198-206 were shown to be T cell epitopes of H5 AIV NP by analysing the CD8 + T cell proliferation and the interferon (IFN-c) expression. To our knowledge, this is the first report to describe the structure of chicken MHC class I molecules from the B4, B12, B15 and B19 haplotypes and the T cell epitopes of H5N1 AIV NP in chicken.
All computations were conducted using the Discovery Studio 2.5 (DS2.5) program developed by Accelrys Software Inc and the SYBYL 8.1 program developed by Tripos Inc on an SGI Fuel workstation running Red Hat Enterprise 5.3 and a Dell server running the Red Hat Enterprise 5.2 Linux operating system.
SPF chickens were housed in HEPA-filtered isolators.
Chicken lymphocytes collected from immunized chickens, which were approved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences and performed in accordance with animal ethics guidelines and approved protocols. The Animal Ethics Committee approval number is Heilongjiang-SYXK-2006-032.
DNA vaccine pCAGGS-NP was constructed and assessed according to Ma and Jiang [24, 25] . The synthetic gene for NP of the isolate A/Goose/Gongdong/1/96 (H5N1) with codons optimized for chicken usage was synthesized by PCR assembly of long single-strand DNA templates (100 bases in length) (the oligonucleotide sequences are available upon request). The synthetic optiNP was cloned into the plasmid vector pCAGGS under the control of the chicken b-actin promoter. The plasmid was named pCAGG-NP. The expression of the NP protein from the plasmid was confirmed by indirect immunofluorescence assay and Western blotting of plasmid transfected CEF cells.
Nine of the predicted peptides (Table 1) were selected to test their T cell responses in vitro. They were synthesized to .90% purity by the GenScript Corporation (Nanjing, China): NP [7] [8] [9] [10] [11] [12] [13] [14] (KRSYEQME), 
Amino sequence of H5N1 AIV(GD/1/96) NP was parsed into octamers or nonamers according to the peptide-binding motifs of MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes. The 3D structures of those peptides and the MHC class I molecules were predicted using homology modelling method and then the binding affinity between each peptide and MHC class I molecule was analysed using molecular docking. Those peptides, which have correct binding conformation and high binding affinity, were predicted to be potential T cell epitopes in chicken. Predicted peptides 9 of 25 were synthesized and used to stimulate splenic lymphocytes collected from NP DNA vaccineimmunized SPF chickens. MHC class I molecule-restricted potential T-cell epitopes were confirmed by detecting of CD8 + T cell proliferation and interferon (IFN-c) expression.
The amino acid sequences of chicken MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes were obtained from GenBank (B4, GenBankID: CAK54654.1; B12, GenBankID: BAG69386.1; B15, GenBankID: BAG69413.1; and B19, Gen-BankID: BAG69441.1). BLASTP was performed to search for homologous proteins in the Protein Data Bank (PDB) using the BlOSUM62 Scoring Matrix optimized with a gap penalty of 11 and a gap extension penalty of 1. The MODELER [27] program was then executed in DS2.5 to construct the 3D structures of chicken MHC class I molecules for the four haplotypes. The quality of the 3D models was evaluated with a Ramachandran Plot using the PROCHECK [28] program and the Verify Protein (Profiles-3D) program [29] in DS2.5.
To improve the quality of the models, unsatisfied loop regions in each model were refined using the loop-refinement protocol based on the MODELER energy in DS 2.5. All structures were then minimized using the CHARMm force field [30] and an explicit solvent model (TIP3P water) with a steepest descent method for 8000 steps and a conjugated gradient minimization for a further 8000 steps.
The cavity depth, lipophilic potential (LP), flexibility (FX), and electrostatic potential (EP) of the four structures was analysed using the MOLCAD program in SYBYL8.1. During this process, the Gasteiger-Hückel charges were assigned to all atoms, while surface maps were generated and visualized using SYBYL8.1.
The NP protein sequence of H5N1 isolate A/Goose/Gongdong/1/96 (H5N1) was downloaded from the UniProt database (UniProtID: NCAP_I96A0) and automatically parsed as octapep-tides or nonapeptides using a computer program, which was developed in our laboratory, based on the peptide-binding motifs of chicken MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes [22] . The motifs were as follows:
The structures of the octapeptides and nonapeptides were modelled using DS2.5. In total, seventy-five peptide structures were prepared for docking analysis.
Surflex-Dock [31] is used to dock ligands into a protein-binding site and it is particularly successful at eliminating false positive results [32] while offering unparalleled enrichment in virtual highthroughput screening combined with state-of-the-art speed, accuracy, and usability [33, 34] . To test the feasibility of using Surflex-Dock for docking MHC molecules and peptides, 50 peptide-MHC complexes retrieved from the PDB database were separated and re-docked using the Surflex-Dock program. The RMSD was calculated for each modelled pair and crystal structure pair to evaluate the docking program.
Surflex-Dock was used to dock the octapeptides and nonapeptides to their corresponding MHC class I molecule receptors, where the parameters of the threshold and bloat values of the program were optimized to 0.51 and 1. The interaction modes and binding energies of the docked complexes were analysed using DS2.5. Peptides with a higher docking score and rational conformation were predicted as candidate T cell epitopes for each haplotype.
For the DNA vaccine immunizations, 13 three-week-old SPF chickens were separated into two groups, eight were immunized twice with 100 mg of pCAGGS-NP in their leg muscle at threeweek intervals, while five chickens were injected with the same volume of PBS as controls. Sera were collected weekly for detecting of NP antibody.
Serum antibodies to H5N1 AIV NP were detected using an indirect ELISA method as described by [24] , which used prokaryotically-expressed NP as the antigen. The testing steps as follows, after washing of the plates, 50 ml of the test serum mixed in the test wells with 50 ml of antigen diluted 1:10 in ELISA buffer. After incubation at 37uC for 1 h, 100 ml horseradish peroxidase conjugate, diluted 1:1000 in ELISA buffer, was added to each well and plates were further incubated at 37uC for 1 h. After two washing steps, 100 ml of TMB substrate was added and incubated at room temperature for 10 min. The reaction was stopped by adding 100 ml H 2 SO 4 2 M. The extinctions were measured at 490 nm with a micro ELISA reader (Bio-Rad). 
To prepare splenic lymphocytes, all eight pCAGGS-NP immunized chickens were killed by cardiac puncture blood collection. Sterile spleens were collected and meshed through a sieve screen using a syringe plunger to obtain a single-cell suspension in tissue culture medium (RPMI 1640, Gibco BRL NY, USA). Cell suspensions were overlaid onto Histopaque 1077 density gradient medium and centrifuged at 1800 rpm for 20 min at 18uC. Lymphocytes were collected from the interface and washed three times in RPMI, before cells were counted using a trypan blue dye exclusion assay.
Splenic lymphocytes collected from the 8 immunized chickens were stained with 5 mM CFSE (Invitrogen) in pre-warmed PBS for 10 min, washed three times, and suspended in RPMI 1640 containing 2 mM L-glutamine, 100 IU mL -1 penicillin, 100 IU mL -1 streptomycin, and 10% foetal bovine serum (R10 medium). Cells were plated at 10 6 well -1 in 24-well plates with RPMI 1640 medium, before stimulation for 5d with 100 mg mL -1 of the nine peptides generated from NP and the unrelated peptide N 71-78 . Cells were then washed with PBS and stained using anti-CD8-PE before flow cytometric analysis, which was performed on Cytomics FC500 MCL(Beckman) and analysed with its embedded software CXP. Two repeats were performed simultaneously for each peptide during the flow cytometric analysis.
Splenic lymphocytes collected form the 8 vaccinated chickens were plated at 10 6 well -1 in 24-well plates with RPMI 1640 medium, before stimulation for 48 h using 100 mg mL -1 of the nine peptides generated from NP and the unrelated peptide N 71-78 . Cells were then collected and centrifuged at 1000 rpm for 5 min. The cell culture supernatants were then analysed to determine the chicken IFN-c using chicken IFN-c CytoSets TM (Invitrogen), according to the manufacturer's instructions [35] . The extinctions were measured at 450 nm with a micro ELISA reader (Bio-Rad). For each peptide, two copies of splenic lymphocytes from one chicken were treated and analysed simultaneously.
Results were expressed as mean 6 S.E.M. for two replicates. Statistical analyses were performed using SPSS 16.0 for Windows. Significant differences (P,0.05) between means were tested by one-way ANOVA, followed by Tukey's Honestly Significant Difference test.
The four haplotypes selected in this study belonged to 11 commonly encountered unique haplotypes (B2, B4, B5, B6, B7, B12, B13, B14, B15, B19, and B21) and the motifs of their binding peptides were previously reported [22, 23] . The peptide-binding groove of the chicken MHC class I molecule contains a1 and a2 domains [18] , so only those two domains were used for modelling and analysis in this study. BLASTP search results showed that the chicken MHC class I molecule 3BEV (PDB accession number) belonging to the B21 haplotype had the highest sequences identity and similarity with the four target sequences. The sequence identity and similarity between 3BEV and B4, B12, B15, and B19 were 84.3%, 84.3%, 87.1%, and 89.9%, and 89.3%, 89.9%, 91%, and 93.3%, respectively. The sequence alignments of the four target sequences and 3BEV are shown in Fig. 1. 3BEV has been crystallized to a high resolution of 2.1 Å , so it was selected as the template for modelling the four protein structures.
The initial crude homology models were built using MOD-ELLER and refined using modules for loop refinement and CHARMM minimization in DS2.5. Thus, the final structures were of high quality. The Profiles-3D scores of all amino acid residues in the four structures were greater than zero (Fig. 2) , while an evaluation of the stereochemical quality of the models using PROCHECK showed that no residues were in the disallowed regions of the Ramachandran plots (Fig. 3) . This indicated that the backbone dihedral angles, phi and psi, of the four structure models were reasonably accurate.
The four models are similar to the previously reported B21 haplotype. The peptide-binding domain is formed by two helices at the top and an eight-stranded sheet at the bottom (Fig. 4A) . The pairwise root mean-square differences (RMSD) between the Ca positions of 3BEV and the structures of the B4, B12, B15, and B19 haplotypes are very small (0.23, 0.20, 0.24, and 0.30 respectively). However, there are some variations around the peptide-binding grooves and these differences may determine differences in their peptide-binding properties. Specific residues in the binding groove have an essential role in peptide binding. Figure 4B shows the key residues comprising the binding site, when all the models and the template were superimposed.
Peptide binding to a given class I MHC molecule requires the presence of anchor residues that complement the physicochemical characteristics of the specificity pockets [36] . Anchor residues are usually found at peptide position 2 (P2), where they interact with pocket B, but sometimes at position 5 or 6 (P5 or P6), where they interact with pocket C or E. A C-terminal residue (PC) also typically binds in pocket F [8] . The anchor residues Glu or Asp (P2) in B4 have an electrostatically favourable interaction with the side-chain of the pocket B residue Aspa9, which points up from the b-sheet. Similarly, anchor residues P2 Arg in B15 and B19 provide an electrostatically favourable interaction with the negatively-charged residues Aspa34 and Glua62, the side-chains of which point towards the binding groove from the a-helix. The presence of Glu8 or Asp8 as major residues in the C-terminal position of the B4 motif is unprecedented, and there are no previous mammalian examples of negatively-charged residues in the C-terminal anchor position [16] . It is possible that the positively-charged Arga80 residue of the F pocket is the key to the binding of anchor residues P8Glu or P8Asp. There is an anchor residue in the central cavity region corresponding to pocket C in B12 and B4. This is very similar to the mammalian MHC class I molecules H-2 Kb and H-2 Db, and it may be related to the Glya68(69) residue without a side-chain located in the a-helix (the residue number in parentheses is derived from mammals, whereas that without parentheses is from the chicken). The volumes of the binding grooves in B4 and B12 are 662.45 Å 3 and 637.677 Å 3 , respectively, which are greater than those of B15 (582.595 Å 3 ) and B19 (581.676 Å 3 ) (Fig. 5) . This may explain why the former have anchor residues in the middle region of their binding peptides. In general, anchor residues point downwards into the binding groove, which allows them to fill the pocket and maintain the stability of the peptide-MHC complex.
The electrostatic potential of the peptide-binding groove of the B4 haplotype is highly positively-charged, whereas those of the B15 and B19 haplotypes are negatively-charged and that of B12 is neutral (Fig. 6) . The peptide-binding grooves are highly lipophilic in all models (Fig. 7) . The B pockets of the peptide-binding grooves . Verification score plots of the models (plots generated using DS2.5). The verification scores of all residues in B15 were greater than zero, whereas one residue in B12 and B19 and three residues in B4 were less than zero. doi:10.1371/journal.pone.0039344.g002 are more flexible than the F pockets in all structures (Fig. 8) , which suggests that the variation of the anchor residues in the B pocket is greater than in the F pocket.
As described in the Materials and Methods, 50 peptide-MHC complexes were selected from the PDB database to validate the docking process. All the peptides were extracted from the complexes, before they were re-docked to the corresponding MHC molecules using Surflex-Dock. When compared with the corresponding initial complexes, the average RMSD was 1.8 and the docking scores were all greater than 8.0 (Table 2) . Docking was also successfully performed with v-src C-tail peptide 517-524 (LPACVLEV) and an identified T cell epitope [23] to the modelled B12 MHC class I molecule, where the docking score was 10.91 and the docking complex conformation was rational. Thus, it was appropriate to use Surflex-Dock for screening T cell epitopes based on their homology-modelled structures.
Based on the assessment results, the criteria for T cell epitope prediction using Surflex-Dock were defined as follows: a) the peptide made close contact with the groove and it docked in the correct direction; b) the anchor residues bound to the anchor site in a rational conformation; and c) the docking score of the complex was greater than 8.0. A total of 25 potential T cell epitope peptides were predicted (eight for B4, six for B12, two for B15, and seven for B19). Nine peptides, which marked in bold in table 1, were selected to test their T cell responses in vitro. The binding energies of those complexes were also calculated. The results are shown in Table 1 . 
Peptide stimulation experiments were conducted using splenic lymphocytes derived from NP DNA vaccine-immunized chickens to verify some of the predicted potential T cell epitopes. To assess the immune effects of the vaccine, serum NP antibody was detected using the ELISA method. Compared to the control group, a significant increase (P,0.01) in blood NP antibody was observed in the immunized group two weeks after the first vaccination. After the boost, the blood NP antibody level of the immunized group increased further and it remained at a high level throughout the duration of the experiment (Fig. 9 ). This indicated that the immune systems of the chickens were activated by the vaccine immunization and that the splenic lymphocytes of chickens were sensitized.
Activation of Lymphocytes Using Peptides NP To verify the predicted T cell epitopes in NP, ten synthetic peptides (Top nine of the 25 predicted peptides from NP and one unrelated peptide) were incubated with sensitized splenic lymphocytes for 5 d. Flow cytometry analysis showed that the proliferation of CD8 + T lymphocytes increased by 13.7% and 11.9% in cells stimulated with the peptides NP 89-97 and NP 198-206, respectively (Fig. 10) . Chicken IFN-c concentration in cells stimulated using peptides NP 89-97 and NP 198-206 were significantly higher than the control and unrelated peptide-stimulated cells (Fig. 11) . These results demonstrate that the peptides NP 89-97 and NP 198-206 are NP T cell epitopes in chickens of certain haplotypes.
The objective of this study was to predict and verify T cell epitopes in the H5N1 AIV NP in chicken. Using a motif combined with a structure-based method, 25 potential T cell epitope peptides were predicted in the H5N1 AIV NP in chickens of B4, B12, B15, and B19 haplotypes. NP 89-97 and NP 198-206 were found to induce a significant proliferation of CD8 + T lymphocytes and they increased the secretion of chicken IFN-c in sensitized splenic lymphocytes. These data suggest that peptides NP 89-97 and NP 198-206 are NP T cell epitopes in chickens of certain haplotypes. This study is important for the following two reasons. First, this is the first study to determine the structural characteristics of the peptide-binding domains of chicken MHC class I molecules belonging to the B4, B12, B15, and B19 haplotypes using a combined motif-structure method to predict T cell epitopes in chickens. Second, NP 89-97 and NP 198-206 are the first two T cell epitopes to be identified in AIV NP in chickens of certain haplotypes.
Homology modelling is widely used in many areas of structure-based analysis and study [37, 38] . However, there are few studies of chicken MHC class I molecules compared with those of human or mouse. The only chicken MHC class I molecule structure that has been solved is the B21 haplotype [18] . Thus, there is little information available on the structure and function of chicken MHC class I molecules. Therefore, the current study used homology modelling to investigate the peptide-binding domains of chicken MHC class I molecules belonging to the B4, B12, B15 and B19 haplotypes. To the best of our knowledge, this is the first attempt to understand the peptide-binding properties of these molecules based on their structures. The assessment indicated that the models were of high quality in terms of their folding and they were suitable for structure-based T cell epitope prediction. The structural characteristics of the peptide-binding properties of these MHC class I molecules were described in the results section. Only four haplotypes with known motifs were selected in this study. However, it is possible to use this solution to predict T cell epitopes of MHC class I molecules belonging to different haplotypes for other antigens in chickens, because the only difference would be the increased number of peptides required for molecular docking.
This study found that 25 out of 75 peptides were potential T cell epitopes in the H5 AIV NP in chickens of the four haplotypes. An analysis of previous results showed that some of those peptides have been identified as T cell epitopes in humans. There is evidence that NP 91-99 (KTGGPIYKR), NP 361-375 (RGVQIAS-NENMETME), and NP 174-184 (RRSGAAGAAVK), which are derived from the H3N2, H3N2, and H1N1, respectively, are T cell epitopes, with MHC restriction alleles of HLA-A68, H-2Db, and HLA-B27, respectively [39] . Of these peptides, NP 89-97 (PKKTGGPIY) and NP 362-369 (GVQIASNE) are completely conserved in all influenza virus strains, while NP 179-186 (AGAAVKGV) is relatively conserved. Some potential epitopes were also shared by several haplotypes. Chickens used for meat and egg production are always heterozygotes, so the identification of these shared T cell epitopes will facilitate the development of broad-spectrum protective vaccines for chickens. Furthermore, epitopes shared by birds and humans are of great importance in the design of rational vaccines for protecting humans and birds from AIV infection.
This study used a DNA vaccine plasmid expressing the NP from A/Goose/Gong Dong/1/96 (H5N1). A DNA vaccine expressing HA from A/Goose/Gong Dong/1/96 (H5N1) has been found to elicit antibody responses and protect chickens against challenge with HPAI virus [25] . Methods used for producing the HA-expression DNA vaccine were adopted when preparing the DNA expression plasmid pCAGG-NP. NP antibody responses were detected in immunized chickens, suggesting that this vaccine elicits successful immune responses to the NP antigen in chickens. The T cell responses were not detected directly in this study, but it was inferred that a successful antibody response would have been accompanied by a successful T cell response based on our knowledge of the DNA vaccine. Nine of the peptides were synthesized and used to stimulate sensitized splenic lymphocytes to verify that they were epitopes. Increases in the proliferation of CD8 + T lymphocytes and the secretion of chicken IFN-c demonstrated the antigenicity of these peptides. NP 89-97 (PKKTGGPIY) and NP 198-206 (KRGINDRNF) induced significant T cell responses in splenic lymphocytes. An analysis of the prediction results showed that NP 89-97 was predicted to be a T cell epitope for both B15 and B19, while NP 198-206 also belonged to the B19 haplotype. Thus, it is suggested that the major haplotype of the experimental SPF chickens might be B19. NP 89-97 also overlapped with a previously identified HLA-A68 restriction T cell epitope NP 91-99 (KTGGPIYKR) of the H3N2 AIV [7] . This further verifies the significance of this epitope, which could be used in human and chicken vaccines to provide protection against different influenza virus subtypes.
Using in silico and in vitro approaches, this study identified two novel T cell epitopes NP 89-97 (PKKTGGPIY) and NP [198] [199] [200] [201] [202] [203] [204] [205] [206] (KRGINDRNF) in the H5N1 AIV NP in chickens of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chickens.  Novel Paramyxoviruses in Free-Ranging European Bats The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus. Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus–host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered. Members of the virus family Paramyxoviridae are divided into two subfamilies, Paramyxovirinae and Pneumovirinae, comprising a vast variety of animal-and human-pathogenic viruses [1] . Within the subfamily Paramyxovirinae, five genera have been classified, Respiro-, Morbilli-, Rubula-, Avula-, and Henipavirus, as well as a fastgrowing group of unclassified viruses. The increased molecular characterization of recently isolated paramyxoviruses indicates a much greater genetic diversity within the subfamily Paramyxovirinae than previously assumed. Furthermore, the detection of highly human-pathogenic paramyxoviruses has also influenced the attention drawn to paramyxovirus research and to the isolation of further novel paramyxoviruses from hosts that are suggested as likely species to transmit newly emerging viruses. Bats are among this highly suspected group of animals [2] . They belong to the most successful and diverse mammals on earth and comprise approximately 1,200 chiropteran species distributed worldwide. In the last two decades important zoonotic viruses including Ebola, Marburg, and SARS virus, but also paramyxoviruses such as Hendra and Nipah virus have been identified in various Pteropus spp. (flying foxes) fruit bats [3] [4] [5] [6] [7] [8] [9] [10] [11] . For Menangle virus, another paramyxovirus isolated from fruit bats, less pathogenic courses of disease in humans have been described [12] . For other bat paramyxoviruses isolated, infections in humans have yet to be associated, e.g. Tioman virus from flying fox [13] , bat parainfluenza virus from flying fox [14] , Tuhoko virus from flying fox [6] , Mapuera virus from non-pteropid fruit bat [15] , and Henipalike viruses also from non-pteropid fruit bat [4] . All viruses of the family Paramyxoviridae so far detected in bat species have been identified in fruit bats across Africa, Australia, South America, Asia, and Madagascar [3] [4] [5] [6] 10] . Only a few studies attempting the isolation of paramyxoviruses in bats concerned insectivorous bat species, and all of them turned out with negative results [16, 17] . The only indication of paramyxoviruses in this group of bat species was the detection of Nipah virus antibodies in lesser Asiatic yellow bats (Scotophilus kuhlii) [16] .
The present study aimed to detect and isolate novel paramyxoviruses in free-ranging European insectivorous bats and to estimate a possible impact of paramyxovirus infection on infected individual animals by comparing histo-pathological findings and virological results.
As part of a study to investigate diseases in free-ranging bats in Germany [18] , 120 deceased bats from 2009 of 15 different European vespertilionid species (Eptesicus nilssoni, E. serotinus, Myotis bechsteini, M. daubentonii, M. mystacinus, M. nattereri, Nyctalus leisleri, N. noctula, Pipistrellus kuhli, P. nathusii, P. pipistrellus, P. pygmaeus, Plecotus auritas, P. austriacus, Vespertilio murinus) were examined. The bat carcasses originated from 4 different geographic regions in Germany, i.e. Berlin greater metropolitan area (n = 83), Bavaria (n = 30), Brandenburg (n = 5), and Baden-Wuerttemberg (n = 2). Bat carcasses were stored at -20uC for transportation before performing a full necropsy. For histo-pathological examination, a small piece of tissue from all organs was fixed in buffered 4% formalin, processed routinely and embedded in liquid paraffin. Paraffin blocks were cut at 2-5 mm thickness and stained with hematoxylin-eosin [19] . Immuno-histochemistry was performed on all organs of PCR positive bats using rabbit immune sera against Beilong virus, J-virus, Menangle virus, Tioman virus and Nipah virus as described previously [20] . Samples of lung, liver, heart, and kidney, and conspicuous tissues (e.g. enlarged spleen) from each bat were homogenized in buffer and transferred to RNAlater (1:1).
Pooled organ tissue from each bat was used for RNA/DNA extraction (PureLink TM Viral RNA/DNA Mini Kit, Invitrogen, Germany) and further cDNA synthesis according to the manufacturer's instructions (TaqManH Reverse Transcription Reagents, Applied Biosystems, Germany). Broadly reactive paramyxovirusspecific RT-PCR assays were applied [21] , yielding amplicons of 538 base pairs (PAR primers) and 486 base pairs (RES-MOR-HEN primers) located across domains I and II of the RNA polymerase (L)-coding sequence, a region of the genome suitable for phylogenetic analyses [22] . Since cDNA was readily available, PCR conditions were modified using the optimization method by Taguchi [23] . For this, based on the use of orthogonal arrays representing individual reactions with components at different concentration levels, a minimal number of experiments is allowed. To increase PCR sensitivity, the product yield for each reaction is used to calculate the optimal concentration of each reaction component. By using this method, a novel PCR reaction mixture was determined and henceforth used. For first-round PCR in the seminested assay, PCR mixtures contained 3 pmol each of forward and reverse primers, 16PlatinumH Taq buffer (Invitrogen), 250 nmol MgCl 2 (Invitrogen), 2.5 pmol desoxynucleoside triphosphates (Invitrogen), 2 ml of cDNA, and 1.25 U of PlatinumH Taq polymerase (Invitrogen). Water was then added to a final volume of 25 ml. The PCR mixture was sequentially incubated at 94uC for 2 min for denaturation, and then 40 cycles at 94uC for 15 s, 50uC for 30 s, 72uC for 30 s, and a final extension at 72uC for 7 min. For the second amplification in the seminested PCR assay, 16PlatinumH Taq buffer, 25 nmol MgCl 2 , 2.5 pmol desoxynucleoside triphosphates, 3 pmol each of forward and reverse primers, 1.25 U PlatinumH Taq, 1 ml PCR product from the first reaction, adding water to a final volume of 25 ml. The cycling conditions were identical to the ones of the first round.
PCR products were run on an 1.5% agarose gel containing ethidium bromide. Images were captured on E.A.S.Y. RH-3 gel documentation system (Herolab, Germany). Amplicons from the PCR reaction were purified using the MSBH Spin PCRapace kit (Invitek, Germany). Both strands of the amplicons were sequenced with a BigDye Terminator v 3.1 Cycle Sequencing kit on an ABI Genetic Analyzer 3500 6l D6 automated sequencer (Applied Biosystems, Germany) using the corresponding PCR primers. Remaining reaction conditions were performed in accordance to the manufacturer's protocol.
On the basis of newly acquired sequence information, specific qPCR assays were designed (Table 1) to screen pooled organ tissues of all 120 bats. Cycler conditions for all qPCR assays were as follows: predenaturation (95uC for 10 min), 45 amplification cycles (95uC for 30 s, 60uC for 30 s, 72uC for 30 s), and final extension (72uC for 10 min).
Additional primers were designed using conserved regions between Jeilongviruses and Henipaviruses to extend the sequence obtained by PAR primers (primers and protocol are available on request).
Bayesian reconstruction of phylogenetic trees was performed in concordance with the current proposals of Paramyxoviridae taxonomy using MrBayes, version 3.1.2 [24, 25] . The underlying alignment by ClustalW was based on a 529 base pair fragment (PAR Primer) and 1,593 base pairs amplicon (long fragment) from PCR reactions. The evolutionary history was inferred using the bayesian MCMC method. First, a model selection for these calculations was performed with jModelTest [26] and model GTR+I+G (invariable sites, gamma distribution) was selected for the PAR and the RES-MOR-HEN fragment, and GTR+G to study the long fragment alignment. The calculation parameters were as follows: number of runs: four, number of generations: 1,000,000, sample frequency: 100, burn in: 25%. The results were finally visualized by the FigTree v1.2.1 program, a graphical viewer of phylogenetic trees. Based on the GTR substitution model the estimated transversion ratio, proportion of invariable sites and gamma distribution parameters were estimated automatically.
For confirmation of virus isolation and determination of the infected organs, RNA/DNA extraction and PCR analysis including sequencing was performed on all individual organs from infected bats. For two isolates, a second RT-PCR with primers RES-MOR-HEN [21] and the same PCR conditions as described above was conducted to acquire fragments comparable to previously isolated novel Henipa-like viruses from African fruit bats [4] . Bat species confirmation was achieved by sequencing and analyzing the mitochondrial DNA as described [27] .
The modified PCR protocol (PAR primers) resulted in a 10fold increase of sensitivity compared to the published protocol which was applied as a two-step PCR (Figure 1 ). With this optimization, three out of 120 pooled samples were PCR positive for paramyxoviruses using PAR primers ( Table 2 ). The identified viruses were termed after the infected bat species: BatPV/ Myo.mys/E20/09 (Accession number JN086950), BatPV/Pip.pip/E95/09 (Accession number JN086951), and BatPV/Nyc.noc/E155/09 (Accession number JN086952). Fragments of 529 bp length were aligned with homologous fragments of the partial polymerase gene of other members of the family Paramyxoviridae from GenBank ( Figure 2 ). Phylogenetic analysis confirmed three distinct isolates within the subfamily Paramyxovirinae. BatPV/Nyc.noc/E155/09 was in basal association to other members of the genus Rubulavirus. For both BatPV/ Myo.mys/E20/09 and BatPV/Pip.pip/E95/09, the closest association was observed to J-virus and Beilong virus (unclassified viruses) [28, 29] . A longer sequence of 1,593 bp was generated for BatPV/Pip.pip/E95/09 and used for an extended phylogenetic analysis ( Figure 3 ). The highest similarity was revealed for BatPV/Myo.mys/E20/09 to J-Virus with 66.4%, for BatPV/ Pip.pip/E95/09 also to J-Virus with 64.1%, and for BatPV/ Nyc.noc/E155/09 to Rubulavirus with 62.1% (Table 3) . Within the subfamily Paramyxovirinae the extent of minimal nucleotide homology for the partial polymerase gene between different viruses in the same genus ranges from 64.1% (Rubulavirus) to 76.8% (Henipavirus), whereas the extent of nucleotide similarity between viruses from different genera is between 40.3% (Morbillivirus) and 58.1% (Henipavirus). Analysing the nucleotide homology of the partial polymerase gene of the new insectivorous bat paramyxoviruses, no definite correlation to one of the other paramyxovirus genera could be obtained. The comparison of sequences of BatPV/Myo.mys/E20/09 (Accession number JN086953) and BatPV/Pip.pip/E95/09 (Accession number JN086954), obtained from the PCR assay with RES-MOR-HEN primers (Table 2) , confirmed the results of the above-mentioned phylogenetic analysis (data not shown).
The first paramyxovirus termed BatPV/Myo.mys/E20/09 was detected in pooled organs and was subsequently confirmed in the kidney only of one adult male whiskered bat (Myotis mystacinus) found in Bavaria. Histological examination of the internal organs revealed multifocal mild interstitial nephritis with lymphoplasmacytic infiltrates and occasional neutrophiles. Lungs had mild nonsuppurative interstitial pneumonia and marked leucocytostasis in most blood vessels. Additionally, there was distinct activation of the lymphoreticular tissue of the spleen with moderate follicular hyperplasia and sparse irregularly distributed small foci of lymphocytes and plasma cell aggregations within the liver.
The second virus (BatPV/Pip.pip/E95/09) was detected in the pooled organs of an adult female common pipistrelle bat (Pipistrellus pipistrellus) also found in Bavaria. No specific infected organ could subsequently be determined due to sample size limitations. Histologically the animal had multifocal moderate interstitial nephritis with segmental infiltrates of lymphocytes, plasma cells, and occasional single neutrophiles (Figure 4 ). There was mild generalized interstitial pneumonia and moderate follicular hyperplasia of the spleen. There were mild intrasinusoidal infiltrates of neutrophiles, lymphocytes, and plasma cells within the liver.
The third virus (BatPV/Nyc.noc/E155/09) was detected in pooled organs and was subsequently confirmed in the lung of only one adult female noctule bat (Nyctalus noctula) found in Berlin. Histologically the bat revealed marked follicular hyperplasia of the spleen without further inflammatory organ lesions. The lung was severely congested, and oedematous fluid was present in the lung parenchyma.
Using immune sera against Beilong virus, J-virus, Menangle virus, Tioman virus and Nipah virus in immuno-histochemistry, no stained antigens were visualized in any of the paramyxoviruspositive bats although all immune sera worked well against their homologous virus in corresponding positive controls.
After screening all 120 bats of 15 species with three virusspecific qPCR assays, an identical paramyxovirus to BatPV/ Myo.mys/E20/09 was detected in the spleen of one additional Myotis mystacinus (E120/09).
During the past decade, bats have increasingly been recognized as members of the animal group with the highest relative risk to harbour novel emerging zoonotic pathogens [2] . The emergence of Hendra and Nipah virus provided the first evidence of a zoonotic paramyxovirus originating from bats with a broad host range including humans. Interestingly, despite the enormously diverse chiropteran animal order, so far, with the exception of rabies, only fruit bats have been implicated as a reservoir of a number of new and emerging zoonotic viruses [3] [4] [5] [6] [7] [8] [9] [10] .
With this study, we were able to describe the detection and characterization of the first three paramyxoviruses in insectivorous bats. The genetic distance between these three novel paramyxoviruses and the closest related member known is higher than that of members within other paramyxovirus genera, suggesting that all three viruses might be considered as unassigned paramyxoviruses. Thus the two viruses BatPV/Myo.mys/E20/09 and BatPV/ Figure 1 . Improved detection sensitivity after Taguchi optimization of the Paramyxovirinae subfamily-specific PCR [21] . Gel electrophoresis of amplification products of the second round seminested PCR using 10-fold serial dilutions (10 0 to 10 24 ) of a cDNAsample (kidney of sample E20/09). (A) PCR protocol adopted for twostep PCR as previously published [21] using the pan-PAR-F1/PAR-R primer pair (1st run) and the pan-PAR-F2/PAR-R primer pair (2nd run). (B) Optimized protocol using the pan-PAR-F1/PAR-R primer pair (1st run) and the pan-PAR-F2/PAR-R primer pair (2nd run). doi:10.1371/journal.pone.0038688.g001 Figure 2 . Phylogenetic analysis of the partial L-gene sequence obtained from PCR fragments after Pan-Paramyxovirinae-PCR with PAR primers (529 bp) [21] . The revealed gap-free alignment was used to generate a phylogenetic tree of the novel bat paramyxoviruses (red) concordant with representatives from all known genera of paramyxoviruses with MrBayes. Posterior probability rates are given next to the tree nodes.
PLoS ONE | www.plosone.org Pip.pip/E95/09 might even be considered as members of a new putative genus, as they contain an amino acid identity of 79.5% of the partial L-gene, the highest conserved region of the paramyxovirus genome. Further precise genetic analyses will have to prove whether they ought to be integrated into the proposed new genus Jeilongvirus [29] , comprising J-virus and Beilong virus, with amino acid identities between 69.9% and 74%, respectively, although their viral antigens in PCR positive organs are not immunologically cross-reactive. The third novel paramyxovirus, BatPV/Nyc.noc/E155/09, has a basal association with the genus Rubulavirus and could therefore become a member of this genus, although nucleic acid identity was slightly lower than that between already classified members within this genus and no crossreactivity of viral antigens with immune sera of closely related rubulaviruses was obtained.
Besides virus detection, our study allowed a direct correlation of virology and histo-pathology results. In previous studies in which bats were examined for paramyxovirus infections, no overt clinical disease was noted [5, 30] , despite occasional high prevalences of antibodies against various paramyxoviruses (e.g. Hendra and Nipah virus) and the detection of paramyxovirus RNA in different bat organs [5, [30] [31] [32] . For Hendra virus infections of pteropid bats, the subclinical course has been confirmed by experimental infection. Kidneys are the only site of pathological lesions after Hendra virus infection of pteropid bats, with mild interstitial perivascular infiltrates by mononuclear inflammatory cells, while virus nucleic acids were also detected in lung, spleen, gastrointestinal tract, and urine [33] . In contrast, Nipah virus was only detected in kidneys (male) and uteri (female) of pteropid bats after experimental infection [34] . In all Hendra virus and Nipah virus experimental infection studies the amount of virus recovered reached the limit of detection level, a similar situation encountered in our study. In infected insectivorous bats, low band intensities of PCR products of organs tested positive for paramyxovirus infection indicated a low virus load. Likewise, the kidneys were the only organ infected in the male whiskered bat (BatPV/ Myo.mys/E20/09) and presumably in the female common pipistrelle. Interestingly, both animals had mononuclear inflammatory interstitial infiltrates similar to the reported experimental Hendra virus infections. Unfortunately, attempts to prove the evidence by specific immunohistochemistry with antibodies directed against Beilong virus, J-virus, Menangle virus, Tioman virus and Nipah virus were not successful. A number of reasons could account for this result. Either the noted inflammatory changes GenBank Accession numbers of novel paramyxoviruses PAR fragment: JN086950 (BatPV/Myo.mys/E20/09), JN086951 (BatPV/Pip.pip/E95/09), JN086952 (BatPV/Nyc.noc/E155/09). RSV = respiratory syncytial virus. doi:10.1371/journal.pone.0038688.g002 were indeed unrelated to paramyxovirus infection or the immunogenic epitopes of paramyxoviruses in European insectivorous bats differ significantly from their australo-asian relatives, hence prohibiting bonding between the reagents or, taking into account that molecular investigations indicated a low virus load, the number of infectious particles was too low to be picked up by immunohistochemistry. Although unequivocal evidence of the causative association between paramyxovirus infection and renal inflammation remains open it is important to note, that nephritis is a rare finding in insectivorous bats. Out of 500 examined deceased bats only 3% had inflammatory changes within their kidneys, while 20% of these respective cases were clearly associated to bacterial disease [19] . Considering this background information together with findings from experimental paramyovirus infections in fruit bats a possible link between the renal mononuclear infiltrates and the detected novel paramyxovirus nucleic acids seems feasible. With this, a possible transmission route via urine like in Hendra virus infection could also be assumed for these viruses. In contrast, the detection of BatPV/Nyc.noc/E155/09 limited to the lungs of the female noctule bat is suggestive of an oronasal and/or salivary route of transmission. Our findings confirm a promising approach for the ultra-sensitive detection of paramyxoviruses by applying a modified PCR protocol as a powerful tool, including non-invasive sampling (oral swabs, urine, faeces). However, it should be emphasized that substantial insights regarding the estimation of possible spill-over events can only be achived by a combination of virology, histo-pathology, and bat ecology investigations.
Emerging paramyxoviruses from fruit bats in spill-over hosts have regularly been associated with ecosystem and land-use changes resulting in an increased overlap of bats, domestic animals, and human ecologies and thereby increased opportunities for bat-borne zoonotic diseases to emerge [35] . As demonstrated in this study, paramyxoviruses basally related to Henipaviruses also exist in geographic areas distant to the distribution range of fruit bats, the suspected natural hosts for Henipaviruses, indicating a possible virus-host co-evolution beyond this animal group. Since infected bats were found in close proximity to heavily populated human habitations as well as intensive agricultural use, a potential risk for the emergence of zoonotic paramyxoviruses in Europe should be further elucidated. Although the three novel paramyxoviruses detected in three distinct European bat species cannot be readily assigned to any previously described paramyxovirus genus, they are associated to two very distinct genera (Rubulavirus and Jeilongvirus) [22] , indicating a similarly broad genetic diversity among paramyxoviruses in insectivorous bats compared to fruit bats. Given the much larger diversity amongst insectivorous bats with over 1,000 bat species in contrast to 186 fruit bat species [36] , we predict a far higher diversity of paramyxoviruses in insectivorous bats. Extensive phylogenetic studies of insectivorous bat-born paramyxoviruses will provide further insight into the suggested co-evolution of paramyxoviruses and bats [35] . In addition to Africa, Australia, South America, and Asia, the detection of novel paramyxoviruses in European bats extends the possible geographic overlap with other susceptible spill-over hosts.
Is there a possibility for paramyxoviruses of insectivorous bats to emerge as zoonotic pathogens? Before any answer to this question can be attempted, further research on paramyxovirus diversity and distribution combined with the understanding of dynamics of pathogen cycles within bat populations will be needed as well as investigations into pathogenicity factors of these viruses, like receptors for host invasion. Particularly as so far transmission of bat related paramyxoviruses did not occur directly between bats and humans but depended on a secondary host species like horses or pigs. Lipid Rafts and Alzheimer’s Disease: Protein-Lipid Interactions and Perturbation of Signaling Lipid rafts are membrane domains, more ordered than the bulk membrane and enriched in cholesterol and sphingolipids. They represent a platform for protein-lipid and protein–protein interactions and for cellular signaling events. In addition to their normal functions, including membrane trafficking, ligand binding (including viruses), axonal development and maintenance of synaptic integrity, rafts have also been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD). Lipid rafts promote interaction of the amyloid precursor protein (APP) with the secretase (BACE-1) responsible for generation of the amyloid β peptide, Aβ. Rafts also regulate cholinergic signaling as well as acetylcholinesterase and Aβ interaction. In addition, such major lipid raft components as cholesterol and GM1 ganglioside have been directly implicated in pathogenesis of the disease. Perturbation of lipid raft integrity can also affect various signaling pathways leading to cellular death and AD. In this review, we discuss modulation of APP cleavage by lipid rafts and their components, while also looking at more recent findings on the role of lipid rafts in signaling events. It has traditionally been difficult to reach any kind of consensus concerning the definition of lipid rafts. However, in 2006 at the Keystone Symposium on Lipid Rafts and Cell Function in Colorado it was agreed that "membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol, and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through proteinprotein and protein-lipid interactions" (Pike, 2006) . The long, saturated acyl chains of sphingolipids allow tight packing hence their juxtaposition with the kinked, unsaturated acyl chains of bulk membrane phospholipids leads to phase separation (Brown and London, 2000) . The cholesterol molecules can act as "spacers," filling any gaps in sphingolipid packing (Simons and Ikonen, 1997) .
The notion of lipid rafts, while not new, has never been far from controversy (Pike, 2009) , their existence frequently questioned (Munro, 2003) . It is almost 30 years since Karnovsky et al. (1982) suggested that protein diffusion in membranes is not free, but somehow constrained. They went on to detail the now familiar concept of the organization of lipids in domains, which may have functional significance. The differential detergent (Triton X-100) solubility of glycosylphosphatidylinositol (GPI)-anchored proteins and transmembrane proteins was first shown by Hooper and Turner (1988) . Later work showed the importance of lipid rafts in protein sorting and segregation, with GPI anchored proteins being preferentially localized in lipid rafts (Brown and Rose, 1992; Brown and London, 2000; Pike, 2009 ). Other lipid modifications of proteins have also been described, such as palmitoylation and myristoylation which may influence raft localization (Brown and London, 2000; Smotrys and Linder, 2004; Pike, 2009 ). In describing membrane lipid clusters as moving platforms, or rafts, which are enriched in both sphingolipids and cholesterol, perhaps the most important finding was that proteins could be segregated being selectively included or excluded from the rafts (Simons and Ikonen, 1997) . In this way, raft localization can serve to facilitate or obstruct protein interactions (Brown and London, 2000; Lingwood and Simons, 2009) or act as a protein scaffold while allowing diffusion (Maxfield and Tabas, 2005) . Despite significant efforts in developing methodology and techniques for lipid raft research, there are still some shortcomings in precise determination of their size, structure, and composition which were recently critically discussed by Simons et al. (2010) . The most important issue which still induces debate is related to application of detergents for isolation of lipid rafts and methylβ-cyclodextrin for extraction of cholesterol from cell membranes which could lead to formation of artificial complexes not existing in the natural environment. It has now been established that detergent-resistant membranes (DRMs) are not identical to lipid rafts and the data on proteins detected within DRMs from various types of cells should be treated with caution and not always considered to be functionally raft localized (Lichtenberg et al., 2005; Chen et al., 2009a) . Development of a new technique that purifies nano-meso scale DRMs at 37˚C in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids could be a significant step toward purifying individual physiologically relevant rafts (Morris et al., 2011) . Although Triton X-100 is still the standard detergent in preparation of lipid rafts some groups claim that use of Brij 96 produces more reliable results although comparing properties of both detergents in the same set of experiments www.frontiersin.org have not produced significantly different results. Moreover it has been suggested that preparation of the rafts in a buffer mimicking the cytoplasmic environment will preserve the structure of the rafts under physiological conditions by retaining the proteins associated with the intracellular part of the membrane (Chen et al., 2009b) .
Another source of controversy in lipid raft research which still has to be resolved is determined by the lack of suitable detection techniques in living cells. This resulted in estimation of the putative size of lipid rafts in the range of 5-700 nm. In the last decade a number of new techniques assisting in estimation of raft size and helping in their visualization has been developed and applied (for review see Simons et al., 2010) . They include single molecule spectroscopy and super-resolution microscopy such as fluorescence recovery after photobleaching (FRAP), stimulated emission depletion (STED), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and fluorescence correlation spectroscopy (FCS) techniques. However, even using these techniques the subwavelength lipid domains have never been directly visualized but their size was predicted to be <20 nm (Eggeling et al., 2009) . Despite the limitations these new techniques confirmed the existence of nanoscale cholesterol-based assemblies of lipids and proteins in the membranes of living cells and allowed to characterize further their dynamics and properties.
When comparing the data on the size and precise protein and lipid composition of lipid rafts it is important to bear in mind that they are determined not only by the type of detergent used, the conditions of the experiments and resolution of techniques applied, but also by the type of tissue and cells used for their isolation and visualization. This has to be taken into account when comparing the data and making conclusions about the physiological relevance of proteins detected in lipid rafts. From this point of view a systematic meta-analysis of existing data considering various experimental conditions, tissue specificity, and resolution of the techniques applied might provide a useful tool for understanding raft heterogeneity and for optimizing further research into this intriguing subject.
The best-characterized raft proteins include lipid-modified proteins containing saturated acyl chains, such as GPI-anchored proteins, and doubly acylated proteins, such as Src family kinases and the α subunits of heterotrimeric G proteins (Hooper, 1999; Liang et al., 2001; Oh et al., 2001) . Many other physiologically important proteins have been investigated for their possible raft localizations, including key proteins involved in Alzheimer's disease (AD), such as amyloid precursor protein (APP; Parkin et al., 1999) , β-site APP cleaving enzyme (BACE-1; Ehehalt et al., 2003; Kalvodova et al., 2005) , the γ-secretase complex (Hur et al., 2008) , a disentegrin and metalloprotease ADAM10 (Harris et al., 2009), acetylcholinesterase (AChE; Xie et al., 2010b) , angiotensinconverting enzyme (ACE; Parkin et al., 2003) , a ligand for the Notch receptor (Jagged1; Parr-Sturgess et al., 2010) , and most recently an amyloid β (Aβ)-degrading enzyme, neprilysin (NEP; Sato et al., 2012) . However the full list of raft-associated proteins is still far from completion.
The progress in understanding structural and functional diversity of lipid rafts led to the concept of caveolae as a sub-type of lipid rafts, appearing as 50-80 nm pits in the plasma membrane.
Although caveolae are sometimes considered to be synonymous with lipid rafts, it is now clear that they represent only a subset of rafts whose properties are defined by their major protein components belonging to the caveolin family and denoted Cav-1, Cav-2, and Cav-3 (Parton and Simons, 1995, 2007) . The characterization of PTRF (polymerase I and transcript release factor, originally identified as an RNA Pol I transcription factor, also called Cav-P60 and now termed cavin-1) and subsequently of other cavin family members as important constituents in caveolae formation has revealed new levels of complexity in the biogenesis of these plasma membrane invaginations (Briand et al., 2011) . Like the rafts themselves, the caveolae are enriched in cholesterol, glycosphingolipids, and SM. They are the site of several important protein-protein interactions, for example, the neurotrophin receptors, TrkA and p75(NTR), whose respective interactions with caveolin regulates neurotrophin signaling in the brain (Bilderback et al., 1999) . Caveolins also regulate G-proteins, MAPK, PI3K, and Src tyrosine kinases (Marin, 2011) .
Another group of proteins which was suggested as markers of caveolae are flotillins (also known as reggies) which were discovered as proteins involved in nerve regeneration (Schulte et al., 1997) . The flotillins are palmitoylated and myristoylated proteins, anchored to the plasma membrane and are considered to be exclusively raft localized (Schneider et al., 2008) . Flotillins belong to a larger class of integral membrane proteins that have an evolutionarily conserved domain called the prohibitin homology domain (PHB) which determines the affinity of proteins carrying it to the lipid rafts (Morrow and Parton, 2005) .
In the context of this review it is important to mention another cholesterol and ganglioside enriched membrane domain, which share some similarity but are physically and functionally distinct from the lipid rafts, the tetraspanin-enriched microdomains (TEMs). Tetraspanins are a large family of small membranespanning proteins (Yanez-Mo et al., 2011) , numbering at least 32 family members in mammals. They are involved in a whole range of cellular processes, from cell morphology and motility to signaling pathways (Hemler, 2005) .
In general, lipid rafts can be considered as signaling platforms that bring together various ingredients of the biological membranes determining specificity of the cells and their functioning. They include receptors, channels, recognition molecules, coupling factors and enzymes, facilitating their interaction and supporting signaling. Lipid rafts have been implicated in a plethora of both physiological and pathological processes. Among beneficial processes are axonal growth and branching (Kamiguchi, 2006; Grider et al., 2009; Munderloh et al., 2009 ) and hence raft disruption impedes axonogenesis (Petro and Schengrund, 2009) . Rafts are also involved in the stabilization of synapses (Willmann et al., 2006) . Raft components are also involved in cholera toxin entry via GM1 ganglioside (Holmgren et al., 1973) , HIV-1 entry (gp120), and conversion of prion protein (PrP c ) to its infectious form (PrP Sc ; Fantini et al., 2002; Vieira et al., 2010) . Lipid rafts play a critical role in entry, replication, assembly, and budding of various types of viruses (Suzuki and Suzuki, 2006) . In a more general sense, lipid rafts have been suggested to be involved in cardiovascular disease, carcinogenesis, and immune system diseases (Michel and Bakovic, 2007) . However, this review will focus on the role of lipid rafts in the pathogenesis of AD, a condition with which lipid rafts have been demonstrated to have multifarious links (Cordy et al., 2006; Cheng et al., 2007; Vetrivel and Thinakaran, 2010) .
In the last two decades the amyloid cascade hypothesis of AD (Hardy and Higgins, 1992) has prevailed leading to accumulation of a significant amount of data on the molecular basis of the disease which have recently been reviewed by those who originally proposed the concept (Selkoe, 2001; Hardy, 2009) . From the clinical point of view, the disease is characterized by global cognitive decline, associated with brain pathology involving accumulation of extracellular amyloid aggregates (also known as senile plaques) of amyloid β peptide (Aβ) and intracellular neurofibrillary tangles of hyper-phosphorylated tau protein (Bothwell and Giniger, 2000) . According to the amyloid cascade hypothesis, it is the Aβ which is principally responsible for many of the pathological features of the disease (Hardy and Higgins, 1992; Sakono and Zako, 2010) with Aβ oligomers representing the most toxic species (Haass and Selkoe, 2007; Walsh and Selkoe, 2007; Sakono and Zako, 2010) . Accumulation of amyloid plaques is accompanied by astrogliosis and microgliosis (Grilli et al., 2003) and the most affected brain areas are the neocortex and hippocampus (Sisodia and Gallagher, 1998) . Although there are strong genetic links, including APP and presenilin mutations (Bothwell and Giniger, 2000) , as well as the apolipoprotein ε4 allele (Saunders et al., 1993; Deane et al., 2008; Huang, 2010) , sporadic AD is the dominant form. From this point of view predominance of AD research based on the mechanisms of early onset disease versus the broader spectrum of the factors leading to the sporadic form might be one of the reasons for the failure of the majority of therapeutic trials and lack of any preventive measures 20 years since the amyloid hypothesis has been proposed.
APP is a type I integral membrane protein. It exists in three isoforms (APP 695 , APP 751 , and APP 770 ), generated from differential splicing of exons 7 and 8 (Sandbrink et al., 1996) . Exon 7 is homologous to protease inhibitors of the Kunitz type (KPI domain), while exon 8 is related to the MRC OX-2 antigen in thymocytes (Kitaguchi et al., 1988; Sandbrink et al., 1996) . APP 695 lacks both KPI and OX-2 domains, while APP 751 only lacks the OX-2 domain (Henriques et al., 2007) . In terms of distribution, APP mRNA is expressed in almost every tissue, where only the isoform ratio differs (Araki et al., 1991) . It is APP 695 that predominates in neurons (Gralle and Ferreira, 2007) . There are two proteolytic pathways of APP processing (Figure 1) . Amyloidogenic processing involves sequential cleavage of APP by βand γ-secretases (for review see Zhang et al., 2012) . This process ultimately releases Aβ peptide, responsible in large part for the pathogenesis of AD and a soluble ectodomain, sAPPβ. The second, non-amyloidogenic, pathway involves α-secretase cleavage of APP. This cleavage occurs between Lys16 and Leu17, within the Aβ region (Allinson et al., 2003) and precludes formation of Aβ. There is also the release of the large, soluble ectodomain, the neuroprotective sAPPα, which is shed from the cell surface (Allinson et al., 2004) and has been found in the CSF (Palmert et al., 1989) .
At the time when the terms were introduced, α-, β-, and γsecretases were activities known to cleave APP proteolytically, but not defined as specific enzymes performing the cleavage. It was later found that BACE-1 was responsible for β-secretase activity (Vassar et al., 1999) and that a complex of presenilin, nicastrin, Aph-1, and Pen-2 had γ-secretase activity Hooper, 2005) . The α-secretase cleavage was found to be mediated by zinc metalloproteases of the disintegrin and metalloprotease (ADAM) family, specifically ADAM10 and ADAM17 (also known as TNFα converting enzyme, TACE). ADAM10 was found to be the dominant enzyme of APP processing in SH-SY5Y cells, given a lesser effect of ADAM17 knockdown (Allinson et al., 2004) . Recent investigations have suggested that ADAM10 is the principal APP secretase in primary neurons, with ADAM17 playing more of an auxiliary role (Kuhn et al., 2010; Lichtenthaler, 2010) . Allinson et al. (2004) suggested a model whereby the group of metalloproteases would each contribute to greater or lesser extents to APP cleavage in different cells and under different conditions. The ADAMs have a wide number of substrates, including angiotensinconverting enzyme (ACE; Allinson et al., 2004) , ACE2 (Lambert et al., 2005) , the prion protein (Vincent et al., 2001) as well as each other ). The ADAMs have been reviewed in greater detail elsewhere (Allinson et al., 2003; Edwards et al., 2008; van Goor et al., 2009) .
The idea that lipid rafts may somehow modulate APP cleavage and hence affect the progression of AD has been around for over 10 years and previously reviewed (Cordy et al., 2006; Cheng et al., 2007; Vetrivel and Thinakaran, 2010) . APP itself is not generally a raft protein, although a small proportion of APP is localized in lipid rafts (Parkin et al., 1999) . Regulation of APP raft localization has been suggested to involve an interaction between the C-terminus of APP and flotillin-1 (Chen et al., 2006) . A more recent study suggested that flotillin-2 may also act as a scaffolding protein, clustering APP in lipid rafts. Schneider et al. (2008) postulated that a transient interaction between APP and flotillin-2 may regulate endocytosis of APP, which is important for its processing. It has also been suggested that another raft component, cholesterol, has a role in binding APP promoting its raft localization (Beel et al., 2010) although it was earlier proposed that one of the physiological functions of APP and Aβ is to control cholesterol transport (Yao and Papadopoulos, 2002) . There is also evidence that the adaptor protein Disabled1 (Dab1) interacts with APP regulating its processing and that the glycoprotein reelin promotes interaction of APP and Dab1 and their localization to lipid rafts involving phosphorylation by Fyn kinase (Hoe et al., 2009; Minami et al., 2011) . APP trafficking to the lipid rafts was shown to be dependent on the low-density lipoprotein receptor-related protein (LRP) which also promotes BACE1-APP interaction (Yoon et al., 2007) . On the other hand it was also demonstrated that ApoER2, a member of the low density lipoprotein receptor (LDL-R), negatively affects APP internalization and its expression stimulates Aβ production by shifting the proportion of APP from the non-raft region to the raft membrane domains (Fuentealba et al., 2007) . www.frontiersin.org FIGURE 1 | Schematic representation of APP processing and role of its products in AD pathology. The proteolytic processing of the large, transmembrane, amyloid precursor protein (APP) occurs in two distinct amyloidogenic and non-amyloidogenic pathways. The amyloidogenic pathway involves the sequential cleavage of APP by an aspartic proteinase, β-secretase, which releases a soluble ectodomain (sAPPβ) and the C-terminal fragment CTF99. This, in turn, is cleaved by another aspartic proteinase, γ-secretase, generating the transcriptional regulator APP intracellular domain (AICD), and releasing the 39-42 amino acid amyloid-β peptide (Aβ). Due to its very high ability to aggregation, Aβ forms dimers, trimers, and higher level oligomers which are toxic to cells and cause neuronal death. Formation of amyloid plaques from Aβ aggregates in complex with other proteins is a hallmark of AD but is considered as a scavenging process. In the non-amyloidogenic pathway APP molecules are cleaved at the α-secretase site within the Aβ-domain releasing a soluble ectodomain sAPPα and the C-terminal fragment CTF83. Proteolytic cleavage of CTF83 by γ-secretase releases AICD and p3 fragment whose functions are still unknown. The AICD fragment produced in the amyloidogenic pathway binds to a stabilizing factor Fe65 and in a complex with other factors (the histone acetyl transferase, Tip60, and a Mediator complex subunit Med12) can act as transcription factor regulating expression of a variety of genes, including an Aβ-degrading enzyme neprilysin. This process was found to be specific to the neuronal APP 695 isoform. AICD produced in the non-amyloidogenic pathway and from other APP isoforms (APP 751 and APP 770 ) is most likely to be degraded (e.g., by some intracellular proteases, e.g., insulin-degrading enzyme). Soluble APP ectodomains, sAPPα, and sAPPβ, have been shown to have neuroprotective properties.
In search of an explanation as to how APP could be cleaved in two distinct (amyloidogenic and non-amyloidogenic) pathways, it was suggested that APP was present in two cellular pools (Ehehalt et al., 2003) as schematically presented in Figure 2 . Amyloidogenic processing was suggested to be linked to lipid rafts as their integrity was critical for Aβ formation (Simons et al., 1998; Cordy et al., 2003; Ehehalt et al., 2003; Rushworth and Hooper, 2011) and Aβ production was also indicated to be raft-localized (Lee et al., 1998) . Furthermore, the precise protein/lipid composition of the rafts was shown to influence Aβ release (Lemkul and Bevan, 2011) and aggregation (Ikeda et al., 2011) . Tetraspanins were also shown to regulate APP processing (Yanez-Mo et al., 2011) and perturbation of tetraspanin enriched domains (TEMs) can affect cell signaling pathways (Hemler, 2005) . For example, the α-secretase ADAM10 is regulated by tetraspanins and in this way, tetraspanins can affect the non-amyloidogenic processing of APP (Arduise et al., 2008) . In addition to this, tetraspanins also regulate the amyloidogenic pathway of APP processing since γ-secretase was shown to be associated with TEMs (Wakabayashi et al., 2009 ).
The lipid raft is shown as part of the plasma membrane. The phospholipid domain (light blue) is separate from the lipid raft. The latter is enriched in glycosphingolipids and sphingomyelin (red) on the exofacial leaflet and glycerolipids (e.g., phosphatidylserine and phosphatidylethanolamine; green) on the cytofacial leaflet. Cholesterol (black) is enriched in both leaflets. The acyl chains in lipid rafts are more able to pack together. APP (Aβ region in maroon) is localized in raft and non-raft fractions, but predominates outside rafts. The α-secretase is not raft-associated, while the βand γ-secretases predominate in rafts. The cell surface is shown for clarity, although β-cleavage predominantly occurs in endosomes.
BACE-1 was found to be palmitoylated at three residues, which indicated possible raft localization and interaction with raftresident lipids (Kalvodova et al., 2005; Hattori et al., 2006) . When BACE-1 was targeted to lipid rafts via GPI-anchoring, production of Aβ was increased, indicating upregulation of amyloidogenic APP processing (Cordy et al., 2003) . A more recent study suggests that GPI-anchorage increases preferential cleavage at the β-site of APP, producing the full length Aβ, whereas wtBACE-1 cleaves APP at two sites (β and β' sites) producing full length and N-terminally truncated Aβ (Vetrivel et al., 2011) . Another study has reported the effects of GPI-anchorage of ADAM10, the principal α-secretase, and showed that no wtADAM10 was raft localized while all GPI-ADAM10 was in lipid rafts. It was associated with a reduction in Aβ as GPI-ADAM10 competed with BACE-1 for the APP substrate .
In addition to BACE1, it was shown that the subunits of the γ-secretase complex are also enriched in the lipid rafts (Lee et al., 1998; Hur et al., 2008) and that S-palmitoylation plays a role in localization and stability of nicastrin and Aph-1 within the rafts although not affecting the γ-secretase processing of APP (Cheng et al., 2009) . While caveolin-1 was shown to be an important regulator of γ-secretase spatial distribution and activity (Kapoor et al., 2010) , the proteins of the γ-secretase complex, e.g., PS1, in turn, can induce lipid raft formation and decrease the membrane fluidity . A subset of proteins, in particular voltage-dependent anion channel 1 and contact in associated protein 1, are also associated with γ-secretase in lipid rafts and affect APP processing (Hur et al., 2012) .
Another important lipid-raft associated protein which was shown to play an important role in APP processing is the GPIanchored prion protein (PrP; Naslavsky et al., 1997) . It was demonstrated that PrP c regulates APP processing by inhibiting BACE1 activity and that the effect of PrP c on the β-secretase cleavage of APP requires the localization of PrP c to cholesterol-rich lipid rafts and is mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans (Parkin et al., 2007) . This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves wild type APP but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP Swe ). Although deletion of PrP c in transgenic mice expressing human mutated APP Swe , Ind had no effect on APP processing and Aβ levels, deletion of PrP c in HEK293 cells expression wild type reduced APP cleavage by BACE1. Because there was no effect of PrP c deletion on processing of APP Swe in HEK cells the authors suggested that PrP c may be a key protective player against sporadic Alzheimer disease versus its less common familiar form (Griffiths et al., 2011) . Because there is no detected decrease of PrP c content in the AD brain (Saijo et al., 2011) it is possible to suggest that age-and disease-dependent disruption of lipid rafts might be a cause of decreased ability of PrP c to control BACE1 activity resulting in accumulation of Aβ peptide in the case of sporadic AD.
The amyloid-degrading enzyme, NEP, has been shown to be partially associated with lipid rafts in human synoviocytes in "ectopeptidase-rich membrane microdomains" (Riemann et al., 2001) , and also in pre-B and B cell lines (Nalm-6 and Raji; Angelisová et al., 1999) . By targeting NEP to different intracellular www.frontiersin.org compartments of neurons, including the lipid rafts, Hama and colleagues demonstrated that the endogenous targeting signal in wild-type NEP is well optimized for the overall neuronal clearance of Aβ (Hama et al., 2004) . Only the mature, fully glycosylated form of NEP, preferentially in its dimerized form, can be found in lipid rafts in direct association with phosphatidylserine (Hama et al., 2004) . These authors also suggested that the localization of NEP in different intracellular compartments may be involved in the metabolism of distinct pools of Aβ and that the endogenous targeting signal in wild-type NEP is well optimized for the overall neuronal clearance of Aβ. The partitioning of NEP into lipid rafts has also recently been confirmed by Sato and colleagues . Although another Aβ-degrading enzyme, insulysin (insulin-degrading enzyme, IDE), is primarily a cytosolic protein it is also, in part, associated with lipid rafts where it may facilitate Aβ clearance (Bulloj et al., 2008) .
Apart from compartmentalization within the lipid rafts, the amyloidogenic processing pathway was shown to be dependent on endocytosis (Ehehalt et al., 2003) . In addition to flotillin, caveolin-1 is responsible for partitioning of γ-secretase between the plasma membrane and endosomes and cells depleted of caveolin-1 had more γ-secretase localized within the clathrin-coated noncaveolar endocytic vesicles, although different caveolins have been shown to have different effects on APP processing (Nishiyama et al., 1999; Kapoor et al., 2010) .
Lipid rafts affect APP processing not only through favoring interactions between APP and BACE-1 but also by promoting endocytosis of APP. This process was shown to be APP isoform-dependent with the neuronal APP 695 isoform to be mostly processed via the β-secretase pathway whereas APP 751 and APP 770 mainly undergo α-secretase cleavage (Belyaev et al., 2010) . Some of the earliest work about subcellular localization of APP processing indicated that sAPPβ could be detected in neuronal NT2N cell lysates with the absence of sAPPα or p3 fragments. This suggested an intracellular β-secretase pathway (Chyung et al., 1997; Hartmann et al., 1997) . It is worth noting that the β-secretase pathway operates differently with wild-type APP (wtAPP) and APP carrying the Swedish mutation (APP Sw ; Haass et al., 1995; Griffiths et al., 2011 ). An alternative trafficking pathway has been recently reported, whereby APP can bypass endosomes and be trafficked directly to the lysosomes. This, though, does not occur with either APP Sw or APP London variants suggesting that these mutations affect APP transit (Lorenzen et al., 2010) .
Although there is quite a substantial amount of data on the physiological role of N-terminal soluble ectodomains of APP -sAPPα and sAPPβ, for review see Chasseigneaux and Allinquant (2012) , the C-terminal products of APP proteolytic cleavage have only recently started to attract special attention. It is now rather well documented (although not without some controversy) that the C-terminal fragment of APP, AICD, can act as a transcription factor (Cao and Sudhof, 2001; Leissring et al., 2002; Schettini et al., 2010) . The controversy of the AICD research has been underpinned by utilization of different cell types expressing different APP isoforms and by the difficulties in detecting AICD due to its very short half life (Cupers et al., 2001) . However, it was repeatedly demonstrated that functional AICD, which translocates to the cell nucleus and up-regulates expression of a reporter NEP gene, is produced only via the amyloidogenic pathway and only from the APP 695 isoform (Goodger et al., 2009; Belyaev et al., 2010) . This process was also shown to be neuronal cell specific and lipid raft dependent (Belyaev et al., 2010) . In an earlier work by Cao and Sudhof (2001) , a yeast 2-hybrid (Y2H) screen was used to identify binding partners of the C-terminal domain of APP which revealed the role of Fe65 and the histone acetyltransferase (HAT) Tip60 in formation of functionally active AICD.
AICD regulates the transcription of several target genes, some better characterized than others (Beckett et al., 2012; Pardossi-Piquard and Checler, 2012) . The most well documented gene upregulated by AICD is of the amyloid-degrading enzyme neprilysin (Pardossi-Piquard et al., 2005; Belyaev et al., 2009) . However, there is also evidence that APP itself (von Rotz et al., 2004) , BACE1 (von Rotz et al., 2004) , GSK-3β (Kim et al., 2003) and aquaporin-1 (Huysseune et al., 2009) can be regulated by AICD. In addition to APP, regulation of the GSK-3β can be considered as a link between AICD and AD pathology especially taking into account the data on elevated levels of AICD in the brain of AD patients (Ghosal et al., 2009) . Moreover, the ability of AICD to regulate expression of APP and BACE1 suggests a feedback mechanism of its own regulation by proteolytic processing of its precursor (Grimm et al., 2012a) .
AICD also has a direct link to lipid metabolism as it has been found to suppress the expression of the major lipoprotein receptor LRP1 and as such affect apoE/cholesterol metabolism (Liu et al., 2007) . On the other hand AICD controls expression of the alkyldihydroxyacetonephosphate-synthase which regulates plasmalogen synthesis in the cells (Grimm et al., 2011b) and reduced levels of these brain-specific lipids are characteristic of the AD brain (Han et al., 2001; Rothhaar et al., 2012) . Reduced plasmalogen levels in the AD brain might have direct effect on production of Aβ since they were shown to inhibit activity of γ-secretase (Rothhaar et al., 2012) . There is also evidence that AICD regulates sphingolipid synthesis via serine-palmitoyl transferase (Grimm et al., 2011a) , and as such may control composition of lipid rafts and APP processing. The wide range of putative AICD target genes highlights the role of APP signaling in normal brain functioning and in AD pathology. SPHINGOMYELIN The major component of lipid rafts, sphingomyelin (SM), is characteristic only for eukaryotic cells where it comprises about 10-15% of total phospholipids and even more in the brain and peripheral nervous tissue. SM and its metabolites play an important role as second messengers in signal transduction events during development, differentiation and immune response of the organisms (Nalivaeva et al., 2000; Hannun et al., 2001) . SM is essential for the activity of some types of receptors, including the α7 nicotinic receptor (Colón-Sáez and Yakel, 2011), NMDA receptors (Wheeler et al., 2009) , neurotrophic tyrosine kinase receptor type 2 (Trovò et al., 2011) , serotonin 1A receptor (Jafurulla et al., 2008) and the urokinase receptor (uPAR; Sahores et al., 2008) . It was also found Frontiers in Physiology | Membrane Physiology and Biophysics that some disease-related membrane proteins (APP, gp120, and PrP) have a common SM-recognition site which underscores the role of lipid rafts in AD, HIV, and prion diseases .
Investigation of lipid raft biology was enhanced by the discovery of SM-specific probes, e.g., lysenin, which serve as powerful tools to study the organization and biological function of this lipid in biological membranes (Hullin-Matsuda and Kobayashi, 2007; Shogomori and Kobayashi, 2008) . These studies have demonstrated functional and structural diversity of lipid rafts and characterized in the plasma membrane of Jurkat T cells the SM-rich domains which had spatial and functional specificity compared to the GM1-rich domains (Kiyokawa et al., 2005) . Formation of SM clusters in the membranes of neuronal cells was shown to depend on localization of SM synthase (SMS) isoforms in various cell compartments and that the activity of SMS is the rate-limiting step in SM cluster formation. In accordance with this it was also demonstrated that SM clusters were formed only in the vicinity of SM synthase proteins. In particular, it was also found that the SMS2 isoform is specific for the dendrites of hippocampal neurons (Kidani et al., 2012) . The pattern of individual species of sphingomeylin was also found to be different in various types of cells and even in cell compartments (e.g., in lipid rafts versus detergent-soluble fractions) which testifies to the role of these lipids in determining cell-specific membrane properties (Valsecchi et al., 2007) .
In the aging brain and especially in AD pathology, lipid metabolism, and in particular that of SM, undergoes significant changes. By using a shotgun lipidomics approach Han and colleagues have compared levels of over 800 lipid species in control and AD brains and demonstrated a significant decrease in SM and increases in ceramide levels in the affected brain. They suggested a model in which an AD-related increase in SMase activity results in faster SM hydrolysis (and increased ceramide production) which would lead to alterations in lipid raft formation (Han et al., 2011) . These authors also hypothesized that this would impair functions of GLUT4, which was previously shown to be functionally linked to lipid rafts (Michel and Bakovic, 2007) and lead to the dysfunctions in energy homeostasis characteristic of the disease. Moreover it was also shown that accumulation of the amyloid peptide in neuronal cells leads to SMase activation and SM depletion which, in turn, affects cellular trafficking and abnormal APP processing (Soreghan et al., 2003) . Further work demonstrated that the most toxic form of amyloid peptide Aβ 42 can also activate SMase (Grimm et al., 2005; Grosgen et al., 2010) . Since excessive SMasemediated cleavage of SM occurs early in AD it is likely to disrupt a range of protein-lipid interactions and hence downstream signaling pathways (Haughey et al., 2010) . In addition to AD-linked SMase over-activity, there have been reports of deficiencies of the enzymes responsible for sphingolipid synthesis (Piccinini et al., 2010) .
Gangliosides are glycosphingolipids with one or more sialic acid residues. They are essential components of all animal cell membranes where they are anchored in the external leaflet by the hydrophobic ceramide part of their molecule while the oligosaccharide chain protrudes into the extracellular medium. Gangliosides are particularly abundant in the plasma membranes of neuronal cells have been implicated in various cellular functions since the high heterogeneity of their oligosaccharide structures allows specific interactions with various proteins and oligosaccharide chains of other molecules at the cell membrane surface. Gangliosides to a great extent determine the fine structure of membranes, e.g., lateral diffusion of its components, as well as the organization of lipid rafts (Cantù et al., 2011) . Disruption of ganglioside metabolism leads to various neurological diseases (for review see Walkley, 2003) . Studies of ganglioside-knockout mice have demonstrated that depletion of various classes of gangliosides results in development of neurodegeneration and pathology similar to Alzheimer's or Parkinson's diseases Wu et al., 2011) . In the aging brain, and especially in AD patients, ganglioside content significantly decreases in various brain structures, especially in the areas of the brain related to the pathogenesis of the disease (Kracun et al., 1991) .
The link of gangliosides with AD pathology has been mostly related to their localization in lipid rafts which have been suggested to act as a platform for GM1-induced aggregation of Aβ peptide (Parton, 1994; Zha et al., 2004; Ariga et al., 2008) . Labeled Aβ shows high affinity for GM1 containing membranes, suggesting that GM1 acts as an Aβ binding molecule (Kakio et al., 2004) . It has been suggested that the N-terminal region of Aβ interacts with GM1 clusters through hydrogen bonding and electrostatic interactions (Lemkul and Bevan, 2011) . It was also suggested that cholesterol may facilitate GM1 clustering (Kakio et al., 2001) . Extraction of cortical lipid rafts from human AD brains showed an increase in both GM1 and GM2 (Molander-Melin et al., 2005) despite an overall reduction of gangliosides (Ariga et al., 2008) . There are data indicating that gangliosides accumulate in senile plaques and that they may be involved in the conversion of Aβ to a neurotoxic oligomeric form (Molander-Melin et al., 2005; Okada et al., 2008) .
Various synaptosomal and liposomal studies have suggested that GM1 accelerates formation of amyloid fibrils (Yamamoto et al., 2004; Zha et al., 2004) . Further analysis of the role of lipid rafts in Aβ aggregation have confirmed that raft proteins do not play a significant role in this process as proteinase K or SDS treatment did not affect the aggregation-promoting capacity of the rafts. Moreover, the cholesterol-depleting agent methyl-βcyclodextrin (MβCD) also did not have an effect on Aβ oligomerization (Kim et al., 2006) . Further cell studies reinforce the role of gangliosides in Aβ aggregation. Rafts taken from ganglioside rich cells (C 2 C 12 ) were able to induce Aβ aggregation more potently than ganglioside-poor cells (HeLa and SK-N-MC). Similarly, rafts isolated from brain tissue rich in gangliosides were able to increase Aβ aggregation compared to rafts isolated from the liver with lower ganglioside content. Also, CHO-K1 cells genetically deficient in the synthesis of complex gangliosides had lower levels of Aβ aggregation than the wild type cells (Kim et al., 2006) .
An examination of the effects of GM1 on APP processing in SH-SY5Y and COS7 cells showed an inhibition of α-cleavage (Zha et al., 2004) . On the other hand, Aβ 1-40 oligomers were found to stimulate the amyloidogenic processing of APP by reducing www.frontiersin.org membrane fluidity and complexing with GM1 ganglioside (Peters et al., 2009 ). Although precise mechanisms of GM1 changes with age are still unknown it was demonstrated that GM1 content in neuronal membranes, particularly in DRM microdomains, increases with age and this increase was more pronounced in the brain of apoE4 knock-in mice compared to apoE3 knock-in animals (Yamamoto et al., 2004) .
Recently two novel mechanisms linking gangliosides with AD have been described by Grimm et al. (2012b) . They discovered that Aβ binds to GD3-synthase (GD3S, the key-enzyme in converting a-series gangliosides to major brain specific b-series) and inhibits its activity. On the other hand, the APP intracellular domain AICD, together with Fe65, was found to down-regulate expression of the GD3S gene. This provides an explanation for age-dependent and AD-related changes in brain ganglioside patterns and supports an essential role of APP in ganglioside homeostasis. Another link between gangliosides and AD lies in their ability to alter APP processing. As observed by the authors, GM3 was able to decrease production of Aβ while GD3 was shown to increase its levels in COS7 cells. This might be related to the changes in the properties of lipid rafts containing different amounts of GM3 or GD3 ganglioside species and their ability to regulate activity of βand γ-secretases.
Taking into account the important role of gangliosides in normal brain function, their involvement in the pathogenesis of AD should be treated with caution since different species of gangliosides will have a different impact on the integrity and properties of neuronal membranes. Recently, by using G3 synthase KO mice (lacking only b-series gangliosides) and GM2/GD2 synthase KO mice (which lack almost all gangliosides except GM3 and GD3) it was found that these ganglioside species are important for neuroprotection and anti-inflammatory response via maintenance of lipid rafts . Intraventricular treatment of AD patients with GM1 was shown to stop the progression of cognitive deterioration and improve motor performance and neuropsychological assessments (Svennerholm et al., 2002) . Peripheral utilization of GM1 for prevention of Aβ aggregation in the brain via establishing peripheral/brain dynamics of Aβ was suggested as a possible therapeutic approach for AD since in the PS/APP mice this was shown to reduce Aβ accumulation in the brain (Matsuoka et al., 2003) . However, due to the antigenic properties of gangliosides this might provoke some side-effects (López-Requena et al., 2007) .
Another important component of cellular membranes and of lipid rafts, cholesterol, has been recently extensively reviewed in relation to its role in AD (Marzolo and Bu, 2009; Burns and Rebeck, 2010; Mathew et al., 2011) . The picture is by no means a simple one with cholesterol being ascribed both positive and negative roles. Indeed, other diseases are also linked to cholesterol, while it remains unclear which are specifically raft-associated. Such pathologies include Smith-Lemli-Opitz syndrome, Huntington's disease and Niemann-Pick Type C disease (Korade and Kenworthy, 2008) . In terms of normal aging, the data seem to show that the changes in cholesterol are highly dependent on the brain region and cell types used in the studies .
Cholesterol levels have been shown to be elevated in AD patients as well as the levels of cholesterol precursors in the mevalonate pathway, farnesylpyrophosphate, and geranylgeranylpyrophosphate (Hooff et al., 2010; Kolsch et al., 2010) . However, this view is by no means unanimous and other groups have found lower levels of cholesterol in AD brains (Kolsch et al., 2010; Leduc et al., 2010) , in addition to its precursors lanosterol and lathosterol (Kolsch et al., 2010) , with lower levels of its synthesizing enzyme, HMG CoA reductase (Leduc et al., 2010) . However, quantification of global cholesterol levels are not necessarily reflective of the number or distribution of lipid rafts (Leduc et al., 2010) .
As cholesterol is integral to ordered lipid rafts, the consequences of cholesterol depletion are widely regarded as effects of raft disruption (Hao et al., 2001; Mondal et al., 2009) . In this context the cholesterol content in the membranes has an inversely proportional relationship with the membrane-perturbing effects of Aβ oligomers (Cecchi et al., 2009 ). As such, if cholesterol increases do elevate lipid raft abundance, then it would increase Aβ formation (Simons et al., 1998; Ehehalt et al., 2003) and, on the contrary, low cholesterol levels will lead to up-regulation of the activity of the α-secretase, ADAM10 (Kojro et al., 2001) . Cholesterol depletion in cells by MβCD was shown to affect APP-processing and formation of functionally active AICD resulting in reduced levels of expression of the amyloid-degrading enzyme, neprilysin (Belyaev et al., 2010) . On the other hand, cholesterol has been shown to bind C99, which promotes amyloidogenic processing (Beel et al., 2010) and the increase of Aβ levels, in turn, can cause changes in cholesterol homeostasis in the Golgi and plasma membrane ). Formation of the Aβ "seed" and initiation of Aβ aggregation was also shown to be cholesterol dependent (Mizuno et al., 1999; Kakio et al., 2001; Simons et al., 2001) . In a more biophysical sense, raised cholesterol has been implicated in facilitating the insertion of Aβ into the plasma membrane. In so doing, Aβ then destroys the cells' membrane integrity (Ji et al., 2002) .
Just as cholesterol and lipid rafts have been shown to affect APP processing, APP-derived species have been shown to impact on cholesterol and lipid homeostasis. Aβ peptides modulate the metabolism of cholesterol, in particular its esterification rate, and of phospholipids in hepatocytes, neuronal cells, and in the entire brain Koudinova et al., 1996 Koudinova et al., , 2000 . It was also found that Aβ peptides alter vesicle trafficking and cholesterol homeostasis (Liu et al., 1998) . On the other hand, it was shown that cholesterol binds to APP at the α-secretase cleavage sites and Aβ itself can bind cholesterol and prevent its interaction with low-density lipoprotein (Yao and Papadopoulos, 2002) . This confirmed that Aβ might act as a component of lipoprotein complexes and affect reverse cholesterol transport from neuronal tissue to the periphery in addition to its role in cholesterol synthesis and intracellular dynamics (Koudinov et al., 2001; Michikawa et al., 2001) . In a later study Aβ 40 has been shown to inhibit a key enzyme in the biosynthesis of cholesterol, HMG CoA reductase (Grimm et al., 2005) . Further to this, APP intracellular domain AICD was found to regulate cholesterol levels via LRP1 (Grosgen et al., 2010) . However, there also are data reporting that cholesterol in physiological concentrations can protect neuronal cells Frontiers in Physiology | Membrane Physiology and Biophysics against Aβ-induced toxicity and slow down the process of formation of toxic aggregates of Aβ with metal ions, in particular with aluminum (Granzotto et al., 2011) . This correlates with the data suggesting that cholesterol may have a protective effect against membrane disruption by amyloid species (in this case, Aβ-derived diffusible ligands: ADDLs). Cholesterol supplemented SH-SY5Y cells were shown to display reduced binding of ADDLs to the plasma membrane, while oligomers increased in membrane presence after treatment with the cholesterol-depleting agent MβCD (Cecchi et al., 2009) .
Statins are a class of drugs which inhibit HMG CoA reductase, a key enzyme in the biosynthesis of cholesterol. Given the links between cholesterol and AD, it is not surprising that there are multiple investigations on the effects of statins in AD pathology. The current state of the field and problems have been recently discussed and the role of statins has been reviewed Wood et al., 2010) . The authors point out that the effects of statins are not only related to APP processing nor specific for the AD pathology. Furthermore, the physiological effects of statins are not solely due to inhibition of cholesterol biosynthesis but include perturbation of other mevalonate-dependent pathways such as protein prenylation.
A number of studies have shown positive effects of statins in AD (Jick et al., 2000; Buxbaum et al., 2001 Buxbaum et al., , 2002 . This is in agreement with the previous studies demonstrating that depletion of cholesterol reduces Aβ in cultured neurons (Simons et al., 1998) . One recent study has suggested that fluvastatin is able to modify the trafficking of APP. Use of this drug was stated to increase lysosomal degradation of APP C-terminal fragments (CTFs) and hence facilitate Aβ clearance (Shinohara et al., 2010) . However, the reduction in Aβ levels is not necessarily linked with the cognitive benefits. A recent randomized, double-blinded, placebo-controlled trial of simvastatin showed reduced Aβ in treated patients, but no corresponding improvement in cognitive performance (ADAS-Cog score; Sano et al., 2011) . The biochemical studies are fairly uniform in that cholesterol depletion results in reductions in key ADrelated markers. However, the results of epidemiological research are unequivocal. The Cochrane Dementia and Cognitive Improvement Group study reported that statin treatment had no effect on the prevention or treatment of dementia (McGuinness et al., 2009a,b) . However, taking into account the highly variable relationship between the initiation of statin therapy and the time and severity of the AD, it is very difficult to get a conclusive assessment of the accumulated data on the beneficial effect of statins and any such study should use a defined set of criteria during epidemiological meta-analysis (Shepardson et al., 2011a,b) .
Lipid rafts have been implicated as the sites for a great number of signaling pathways (Allen et al., 2007) . Perturbation of, or changes, in lipid rafts could therefore affect neuronal signaling, including cholinergic transmission. As cholinergic hypofunction is key to the pathogenesis of AD (Schliebs, 2005; Schliebs and Arendt, 2006) , there are strong links between lipid rafts, neuronal signaling pathways, and AD.
Various lipids, such as eicosanoids, docosanoids, and cannabinoids, can act as signaling mediators and their abnormal metabolism has implications in AD (Farooqui, 2011) . These lipid mediators can modulate the metabolism of sphingolipids through activation of SMases. Furthermore, sphingolipid-derived molecules such as ceramide and ceramide 1-phosphate can act as lipid mediators and accumulate in the AD brain. This accumulation of sphingolipid derivatives can activate cytosolic phospholipase A 2 (cPLA 2 ), which leads to changes in membrane fluidity and permeability with concomitant alterations in ion homeostasis. Furthermore, the degradation products of cPLA 2 metabolism are often pro-inflammatory (Frisardi et al., 2011) . This work shows how the lipid raft constituents, in this case sphingolipids, can affect AD processes in an APP-independent manner. Here, accumulation and action of these lipid mediators promotes inflammation, a process characteristic of AD (Akiyama et al., 2000) .
Numerous neurotransmitter signaling systems, especially receptor function, are influenced by lipid rafts. The range of receptors involved is all-encompassing, including ionotropic receptors such as α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA receptors), γ-aminobutyric acid (GABA) receptors, Nmethyl-d-aspartate (NMDA), and nicotinic acetylcholine (nACh) receptors. The influence of lipid rafts also extends to metabotropic G-protein coupled receptors, such as the muscarinic acetylcholine receptor (mAChR; Allen et al., 2007; Rushworth and Hooper, 2011) . Although all these signaling systems can be linked to AD, it is the cholinergic system which is of central importance, as its hypofunction is considered one of the hallmarks of the disease (Coyle et al., 1983; Auld et al., 2002; Schliebs and Arendt, 2006) and its involvement is therefore principally considered here.
Lipid rafts have been implicated in regulating the clustering of nAChRs via the myristoylated peripheral membrane protein, rapsyn, which is constitutively lipid raft localized. Perturbation of the rafts impedes the interaction between rapsyn and nAChRs, which reduces clustering of the receptors and hence their function (Zhu et al., 2006) . Protein reconstitution studies using lipid vesicles imply that the partitioning of the nAChR into raft domains is not solely due to the intrinsic biophysical properties of the receptor but requires a signaling event to translocate the protein into specific membrane domains (Bermúdez et al., 2010) . Furthermore, cholesterol affects the properties, both structural and functional, of some acetylcholine receptors . Agrin signaling represents one pathway which can enhance the translocation of the nAChR into lipid rafts (Campagna and Fallon, 2006; Zhu et al., 2006) .
In particular, one of the most prominent examples of a lipid raft-linked signaling protein is the α7 nicotinic acetylcholine receptor (nAChR), a Ca 2+ channel (Albuquerque et al., 2009 ), which has a close involvement with Aβ and cognitive decline (Albuquerque et al., 2009; Jurgensen and Ferreira, 2009; Hernandez et al., 2010) . On balance, the α7 nAChR seems to have a www.frontiersin.org neuroprotective role in model systems. Significant amounts of the α7 nAChR are associated with lipid rafts and these rafts are essential for maintenance of function (Bruses et al., 2001) . Lipid rafts appear to assist the interaction between soluble Aβ and the receptor (Khan et al., 2010) . Although α7 KO mice show no apparent cognitive defects, Aβ 42 is enriched and Aβ oligomers are enhanced in hippocampi from these animals (Hernandez et al., 2010) .
The α7 nAChR regulates, in part, the pleiotropic intracellular signaling effects of Ca 2+ . In addition to this, the receptor is closely linked to the signaling of cAMP (via adenylyl cyclase) as well as the kinases Fyn and PI3K (Oshikawa et al., 2003) . Disruption of lipid rafts with MβCD or SMase has significant effects on receptor desensitization kinetics (Colón-Sáez and Yakel, 2011) and hence the many important downstream signaling events. Additional interactions of the α7 nAChR have been assessed using a proteomic screen, which identified several proteins involved in neurite outgrowth and maintenance, namely α-catenin 2, BASP1/NAP-22, gelsolin, homer 1, and neuromodulin (Paulo et al., 2009 ). Further to this, the AD therapeutic donepezil protects against glutamate toxicity in part through stimulation of the α7 nAChR (Shen et al., 2010) . Given the central role of the α7 nAChR, it has been suggested promoting its activation via novel agonists may represent a new therapeutic approach in AD (Wang, 2010) .
Lipid raft localization has recently been linked to another protein of the cholinergic system, namely acetylcholinesterase (AChE), although the functional implications of this are as yet unclear (Xie et al., 2010b; Hicks et al., 2011) . AChE inhibition by compounds such as rivastigmine or galantamine represents the major therapeutic option for treating the cognitive impairment seen in the early stages of AD yet the relationship between AChE and AD represents something of a paradox. AChE exists in a number of different molecular forms (G 1 , G 2 , G 4 ) of which the tetrameric G 4 form is predominant in brain. In AD, brain G 4 AChE levels are seen to fall as the disease progresses, while G 1 and G 2 levels rise somewhat, as compared to normal brains (Atack et al., 1983; Garcia-Ayllon et al., 2010) . AChE is found associated with amyloid plaques, leading to the suggestion that AChE may promote Aβ aggregation (Moran et al., 1993; Inestrosa et al., 1996) . In some brain regions with AD pathology, virtually all of the AChE is localized in these complexes (Mesulam et al., 1987) . A direct interaction between Aβ and AChE has been proposed, with binding occurring at the peripheral anionic site (PAS) of the enzyme. Those AChE inhibitors which occupy the PAS (e.g., propidium) show the most significant reductions in fibril formation (Bartolini et al., 2003) since the catalytic site is not required for interaction with Aβ (Inestrosa et al., 1996) . Furthermore, monoclonal antibodies directed against the PAS inhibit fibril formation (Reyes et al., 1997) , which has led to the development of PAS blockers, such as the DUO compounds, that also occupy the active site. They show inhibitory activity on AChE as well as inhibition of Aβ 40 fibril formation (Alptuzun et al., 2010) and have been suggested as potential novel AD therapeutics targeting two facets of the disease. The related enzyme butyrylcholinesterase (BuChE), as well as a synthetic peptide derived from the BuChE C terminus (BSP41), have also been shown to reduce amyloid fibril formation (Diamant et al., 2006) . The corresponding AChE synthetic peptide, however, did not significantly affect Aβ fibril formation.
AChE is not a transmembrane protein, rather it is anchored to the plasma membrane by the proline rich membrane anchor (PRiMA) which is a 20-kDa type I transmembrane protein which can be acylated (Henderson et al., 2010; Xie et al., 2010b) . PRiMA contains a CRAC (cholesterol recognition amino acid consensus) motif which sequesters PRiMA into lipid rafts and hence AChE is also partly associated with rafts although the functional significance of this interaction is unclear (Xie et al., 2010a) . It has been suggested that the lipid raft localization and shedding of AChE may have a certain role in the pathology of AD (Hicks et al., 2011) .
PrP is another component of lipid rafts which was shown to act as a cellular receptor and change Ca 2+ signaling upon activation by some ligands, e.g., by antibody cross-linking in T-lymphocytes (Stuermer et al., 2005) . PrP c was also shown to be a receptor for Aβ oligomers at nanomolar concentrations and binding of Aβ oligomers to PrP c results in the blockage of hippocampal LTP. Incubation of hippocampal slices with PrP antibody abolishes the effect of Aβ oligomers and rescues synaptic plasticity (Lauren et al., 2009) . Most recently it was demonstrated PrP c also interacts with the NMDA receptor complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor and that Aβ(1-42), copper chelators or PrP c inactivation all enhance the activity of glycine at the receptor resulting in steady-state NMDAR currents and neurotoxicity. It was suggested that the physiological role of PrP c might be related to limitation of excessive NMDAR activity which might cause neuronal damage (You et al., 2012) . Together with the data on BACE1 regulation by PrP c this provides a unifying molecular mechanism explaining the interplay between toxic Aβ species, NMDA receptor-mediated toxicity and copper homeostasis in pathogenesis of AD (Rushworth and Hooper, 2011) .
It is possible for lipid rafts to modulate signaling in a general way by affecting the activities of signaling molecules involved in multiple pathways. These include, for example, the pleiotropic src kinases (Arcaro et al., 2007) with effects on the PI3K-Akt signaling pathway. Other raft-localized signaling proteins include the epidermal growth factor receptor (EGFR), which associates with caveolins (Couet et al., 1997) and is involved in diverse processes including cell cycle regulation, endocytosis, and the MAPK cascade (Oda et al., 2005) . This has led to the concept of the signalosome, containing interacting components of signaling pathways (e.g., EGFR) within lipid rafts along with other scaffolding proteins, such as caveolins, and sequestering the complex from other interacting proteins that may disrupt the signaling process. A prime example is the CD40 signalosome associated with cell growth in B cell lymphomas (Pham et al., 2002) .
Similar signaling platforms operate in neuronal systems, such as that involving estrogen receptor (ER) interactions (reviewed in Marin, 2011) . This signaling mechanism is linked to neurogenesis, neuronal differentiation, synaptic plasticity, and neuroprotection (including against Aβ). Recent research indicates that lipid rafts are the site of formation of a complex between the ER, insulin growth factor 1 receptor (IGF-1R), Cav-1, and a voltage gated anion channel, VDAC. The formation of this signaling complex is neuroprotective but also lipid raft dependent (Marin, 2011) . The "signalosome" paradigm was developed further by Chadwick et al. (2011) who isolated lipid rafts in control and 3xTg AD mice and compared their respective proteomes by mass spectrometry. Proteins so identified were then clustered into specific signaling pathways, which allowed an appraisal of which lipid raft signaling pathways may be altered in AD, rather than changes in individual proteins. This systems biology approach indicated that, in lipid rafts, wild-type mice had higher activation of prosurvival pathways such as PTEN and Wnt/β-catenin, whereas 3xTg mice showed activation of p53 and JNK signaling pathways. In addition, 3xTg mice had a deficit in growth factor signaling, neurodevelopmental signaling and signaling through the sonic hedgehog pathway (Chadwick et al., 2011) . Another proteomic analysis extracted post-synaptic lipid rafts and used LC-MS/MS to analyze their protein content. They found an enrichment of cell adhesion molecules, channels/transporters and G-protein related species. Their data linked lipid rafts to cell adhesion with cell-cell contact regions and cell adhesion points being enriched in rafts. Further to this, H + -ATPase and Na + -K + ATPase are enriched in lipid rafts, being responsible for maintaining ionic gradients and modulating neuronal excitability. This study also found the postsynaptic density to be associated with lipid rafts (Suzuki et al., 2011) . Among channels localized in lipid rafts and involved in maintenance of neuronal cell homeostasis, in particular of astrocytes, are K + -buffering inwardly rectifying Kir4.1 channels and the water channel AQP4 (Hibino and Kurachi, 2007) . Residence in the lipid rafts was also shown to be important for the activity of a member of the chloride channel family, ClC-2, which is widely expressed in the brain and other organs (Cornejo et al., 2009) .
Ever since the initial descriptions and characterizations of lipid rafts, the field has been beset by controversy (Pike, 2009 ) from their very existence down to the very specific aspects of how to isolate these structures in the laboratory. However, current consensus is that lipid rafts do represent dynamic structural components of cellular membranes integrating signaling events and regulating cell functioning and that their dysregulation can lead to disease. This review has largely concentrated on the links between lipid rafts and AD pathology since processing of AD-related APP and production of Aβ peptide are clearly affected by lipid rafts. Also Aβ signaling involves interactions of proteins resident to lipid rafts. The mechanism underlying these effects has been examined in some detail although numerous gaps in knowledge still remain. Although the attempts to modulate lipid rafts and hence amyloidogenic processing have failed to translate into successful drugs, some epidemiological studies still indicate that inhibition of cholesterol synthesis through statin treatment might be beneficial if applied early (Solomon and Kivipelto, 2009; Shepardson et al., 2011a,b) . The complexity of AD pathology and etiology dictates to consider involvement of lipid rafts in its pathogenesis in a more generic way, not only as a simplistic link between cholesterol levels, amyloid burden and cognition. More important for normal brain functioning is to maintain lipid metabolism in the aging brain at its normal rate and integrity.
In terms of future progress, lipid raft research might open new avenues in regulation of the proteolytic and signaling processes involved in AD pathology. The most recent discovery of the role of lipid raft disruption in decreased production of the functionally active transcriptional regulator AICD which might lead to an aberrant expression of its target genes including amyloid-degrading enzymes neprilysin (Howell et al., 1995; Carson and Turner, 2002; Belyaev et al., 2009; Liu et al., 2011) , suggests that any therapeutics aimed at manipulation of lipid raft composition should be treated with caution. The role of the lipid components in cell membrane functioning and their structural variability and adaptive potential is extremely important for normal functioning of cells and organisms and much of the recent work in this area is both novel and revealing. However, it is important to note that these discoveries should be recognized as important advances in cell science and not seen as stepping stones to a therapeutic panacea.
of (benzylidene-hydrazono)-1,4dihydropyridines with β-amyloid, acetylcholine, and butyrylcholine esterases. Bioorg. Med. Chem. 18, 2049 -2059 . Angelisová, P., Drbal, K., Horejsí, V., and Cerný, J. (1999 . Association of CD10/neutral endopeptidase 24.11 with membrane microdomains rich in glycosylphosphatidylinositolanchored proteins and Lyn kinase. Blood 93, 1437-1439. Araki, W., Kitaguchi, N., Tokushima, Y., Ishii, K., Aratake, H., Shimohama, S., Nakamura, S., and Kimura, J. (1991) . Trophic effect of β-amyloid www.frontiersin.org West Nile Virus Infection in Killer Whale, Texas, USA, 2007 In 2007, nonsuppurative encephalitis was identified in a killer whale at a Texas, USA, marine park. Panviral DNA microarray of brain tissue suggested West Nile virus (WNV); WNV was confirmed by reverse transcription PCR and sequencing. Immunohistochemistry demonstrated WNV antigen within neurons. WNV should be considered in cases of encephalitis in cetaceans. virus of the genus Flavivirus that is transmitted by mosquitoes. In humans and animals, WNV has been associated with a spectrum of clinical conditions from asymptomatic infections to sudden death. These have been identifi ed in a variety of animal species. Among marine mammals, WNV infection has been reported in a harbor seal (Phoca vitulina) (1) . We describe WNV infection in a killer whale (Orcinus orca) and seroprevalence in conspecifi c cohort and noncohort groups.
In 2007, a 14-year-old male killer whale at a marine park in San Antonio, Texas, USA, died suddenly without notable premonitory signs. On gross examination, mild multifocal meningeal hyperemia and petechial parenchymal hemorrhage were noted in the right cerebrum and cerebellum. The left hemisphere of the brain appeared normal. Focally extensive tan discoloration and fi brosis were present in the right accessory lung lobe with associated hemorrhage and congestion. Both lung lobes were mildly and diffusely heavy and wet. All thoracic and abdominal lymph nodes were moderately enlarged and edematous. The second gastric chamber displayed numerous chronic and active ulcerations of 1.5-2 cm. Fresh and buffered 10% formalin-fi xed specimens were collected. Fresh tissues were stored at -80°C. Tissues fi xed in 10% buffered formalin were processed routinely and stained with hematoxylin and eosin for histologic examination.
Histologic review demonstrated moderate multifocal subacute vasculitis and nonsuppurative encephalitis. Infl ammatory lesions of the central nervous system were focused in gray matter of the medulla oblongata, pons, mesencephalan, and cerebellum. Lesions were bilateral but more severe on the right side. Meninges demonstrated moderate focally extensive and multifocal areas of acute meningeal congestion and hemorrhage. Mild multifocal lymphocytic infi ltrates expanded the leptomeninges. Blood vessels demonstrated mild to moderate acute necrosis and lymphocytic and contained plasmacytic and neutrophilic infi ltrates within vascular walls. Encephalitis was characterized by perivascular lymphocytes and fewer plasma cells expanding the Virchow-Robbins spaces. Small, scattered, perivascular ring hemorrhages were noted. A few multifocal loosely arranged glial nodules were within cerebral white matter.
Predominant lesions in the lungs were areas of chronic and active abscessation amid a focally extensive area of mixed infl ammation and fi brosis. There was moderate diffuse acute pulmonary edema and congestion. Gastric ulcerations were present in the fi rst gastric chamber and were chronic and active. They were characterized by central ulcerations with necrosis and a mixed infl ammatory infi ltrate surrounded by variable fi brosis and a rim of epithelial hyperplasia. Changes in spleen, lymph node, and kidney included acute edema, congestion, and vascular dilation.
Conventional diagnostic assays were performed for aerobic, anaerobic, and fungal microbes in liver, lung, kidney, cerebrospinal fl uid, and brain. All yielded minimal growth of Escherichia coli.
The fi nal diagnosis was fulminant peracute bacteremia and septicemia secondary to a primary viral infection associated with nonsuppurative encephalitis. Published etiologic considerations for cetacean nonsuppurative encephalitis include morbillivirus and protozoal infections (2) . A DNA microarray with highly conserved sequences from >1,000 viruses was selected to screen for known and novel viruses (3). Total RNA was extracted from brain tissue and hybridized to a microarray as described (4) . Analysis of the resulting hybridization pattern demonstrated a strong hybridization signal to many oligonucleotide probes on the microarray from the family Flaviviridae, in particular to WNV. Consensus reverse transcription PCR primers (5) targeting WNV were used to confi rm the microarray results. Sequencing of the 261-bp amplicon (GenBank accession no. HQ610502) yielded a sequence with 99% nt identity and 100% aa acid identity to WNV strain OK03 (GenBank accession no. EU155484.1), a strain originally identifi ed in Oklahoma, USA.
To further support a WNV diagnosis, we performed immunohistochemical staining on brain tissue. The immunoperoxidase stain used was a commercial rabbit polyclonal antibody (BioRelience Corp., Rockville, MD, USA) with peroxidase-tagged goat antirabbit immunoglobulin G (DakoCytomation, Carpinteria, CA, USA) bridge and 3-amino-9-ethylcarbazole (DakoCytomation) as the chromogen. This staining demonstrated abundant WNV antigen within the cytoplasm of a small number of neurons and glial cells and in fewer macrophages in the brain tissue (Figure) .
We evaluated WNV exposure within the same cohort, as well as a geographically distant cohort of whales by using serologic testing. All testing was performed at the same laboratory by using a standard plaque-reduction neutralization test. In this assay, a 90% neutralization cutoff was used (6) . A 90% plaque-reduction titer >10 was considered positive. Serum from the affected whale and 5 cohort killer whales from the same marine park in San Antonio as well as 5 whales housed at another facility in Orlando, Florida, USA, were evaluated. In each facility, the animals have regular contact with each other. The facilities are geographically separated so the animals do not have exposure to those in the other park. All 6 animals from Texas had 90% plaque-reduction titers >10, ranging from 40 to 80. The 5 whales housed together in Orlando had no measurable titer.
We demonstrate that WNV can infect and cause disease in killer whales. These fi ndings broaden the known host tropism of WNV to include cetaceans in addition to previously known pinnipeds. Although we cannot defi nitively attribute the cause of death of this whale to WNV, the observed lesions are consistent with those caused by WNV in other animals. The serologic results demonstrate that subclinical infections can occur and that exposure can be variable. We did not determine specifi c dates of exposure for these populations. Both Bexar County, Texas, and Orange County, Florida, have had WNV in wildlife since 2002. We continue annual serology on previously negative animals to document seroconversion. Mosquito management practices are similar in both facilities and have been expanded since this diagnosis. Differences in WNV prevalence or mosquito numbers may have played a role in the different serologic results.
Health evaluations of free-ranging and captive cetaceans should include WNV serology to assess exposure rates. This report focuses on killer whales, but the "loafi ng" behavior (stationary positioning at the water's surface) is commonly seen in many coastal dolphins, thereby increasing the likelihood of mosquito bites and exposure to WNV. Serologic screening of bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon demonstrated WNV titers (7) . WNV-associated disease in these animals has not been reported. Active screening for WNV may enhance diagnostic investigations.
As with many species of birds and mammals, WNV infection carries a risk for zoonotic transmission. Until the implications of this infection in marine mammals are better understood, biologists and veterinarians working with cetaceans should consider this possibility. Potential viral shedding can occur through the oropharygeal cavity and feces as well as through blood and organs during necropsies.
Finally, our study demonstrates the broad applicability of using panviral microarray-based diagnostics. Even though PCR diagnostics are well developed for WNV, the agent was not initially considered as a potential pathogen in this species. Panviral microarray can be used not only to identify novel viruses but also to detect unsuspected agents. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 8, August 2011  Outbreak of Porcine Epidemic Diarrhea in Suckling Piglets, China  the genera Eptesicus and Rhinolophus in South Korea. However, nucleotide sequencing showed the presence of prototypical Hantaan virus indicating a spillover infection or laboratory contamination (7) .
Further screening is necessary to confi rm N. hispida as a natural reservoir host of the virus. Although the presented bat-associated sequence is obviously distinct from other hantaviruses, which suggests association with a novel natural host, a spillover infection from another, yet unrecognized host cannot be ruled out. However, detection of the virus exclusively in 1 organ (lung but not in liver, kidney, and spleen; data not shown) suggests a persistent infection that is typically observed in natural hosts of hantaviruses (8) .
To date, only a few reports exist on cases of hemorrhagic fever with renal syndrome in Africa (9,10). However, underreporting must be assumed because the symptoms resemble those of many other febrile infections. Moreover, in cases of infections by non-rodent-associated hantaviruses, cross-reactivity with routinely used rodent-borne virus antigens should be limited and may hamper human serodiagnostics (1). The results suggest that bats, which are hosts of many emerging pathogens (5), may act as natural reservoirs for hantavirus. The effect of this virus on public health remains to be determined.
To the Editor: Beginning in October 2010, porcine epidemic diarrhea (PED), caused by a coronaviral infection affecting pigs, emerged in China in an outbreak characterized by high mortality rates among suckling piglets. The outbreak overwhelmed >10 provinces in southern China, and >1,000,000 piglets died. This outbreak was distinguished by ≈100% illness among piglets after birth (predominantly within 7 days and sometimes within only a few hours) and death rates of 80%-100% (online Technical Appendix Table 1 , wwwnc. cdc.gov/EID/pdfs/11-1259-Techapp. pdf). Few sows or boars showed any clinical signs during the outbreak, which is not consistent with a recent report from Thailand (1). In that outbreak during late 2007, pigs of all ages were affected, exhibiting different degrees of diarrhea and no appetite. We characterized the genetic variation of the PED virus (PEDV) that caused a large-scale outbreak in China during 2010-2011 and compared it with viruses in other outbreaks. We also report a possible novel transmission pathway for PEDV.
A total of 177 samples (intestine, stool, and maternal milk) were collected from pigs from different farms who had diarrhea; 100% of farms had >1 porcine sample positive for PEDV. A total of 125/177 porcine samples were confi rmed as positive for PEDV by reverse transcription PCR using primers as described (2) . PEDV was detected in 105 (82.0%) of 128 fecal samples and 20 (40.8%) of 49 sow milk samples. Piglets infected with PEDV showed mild hemorrhage, undigested curdled milk in the stomach, and thin-walled intestines with severe mucosal atrophy and foamy fl uid (data not shown).
The spike (S) gene of the family Coronaviridae has a high degree of variation and can induce neutralizing antibody (3). Reverse transcription PCR products of the 651-bp partial S gene of PEDV and the deduced amino acid sequences were aligned by using ClustalW (www.genome. jp/tools/clustalw), and a neighborjoining tree with 1,000 bootstraps was constructed. Sequences of the S genes from this outbreak were 99.1%-100.0% homologous and had 88.7%-98.9% nt identity with all reference strains (online Technical Appendix Table 2 ), 98.5%-98.9% with Thailand strains, and 94.5%-95.1% with vaccine strain CV777. The partial S gene deduced amino acid sequences were compared and also showed a high degree of homology (98.0%-100.0%); they had 85.3%-98.7% identity with all reference strains listed in online Technical Appendix Table 2 , 98.0%-98.7% with Thailand strains, and 93.3%-94.7% with vaccine strain CV777 (data not shown).
Phylogenetic analysis indicated that the PEDV in the China outbreak was different from foreign and other domestic strains on the basis of the reported partial S gene sequences. All new strains were clustered in the same branch, close to the cluster of Thailand strains, and far from the cluster of vaccine strain CV777 (Figure) .
In the China outbreak, PEDV caused severe diarrheal disease in piglets; heavy economic losses in many provinces resulted, despite use of commercial vaccines (inactivated Phylogenic analysis showed that strain CV777 did not cluster with current common strains and showed 4) . To our knowledge, fecal-oral transmission is probably the main or only route of PEDV transmission (5) (6) (7) . In our study, if a fecal sample from a sick piglet was found to be positive for PEDV, we also collected and studied milk from its mother. These results showed that PEDV was present in sow milk (online Technical Appendix Table 3 ), but the detection rate was lower for these samples (40.8%) than for the fecal samples (82.0%).
On the basis of these results, we hypothesize that sow milk could represent a possible (and potentially major) route for the vertical transmission of PEDV from sow to suckling piglet. This hypothesis could be indirectly verifi ed by our fi eld observation that piglet death rates decreased as a result of fostering (data not shown). Our fi ndings show that PEDV was identifi ed not only in fecal samples from sick piglets, as expected, but also in the milk of the sow, which suggests vertical transmission of the virus. Differential Seroprevalence of Human Bocavirus Species 1-4 in Beijing, China BACKGROUND: Four species of human bocaviruses (HBoV1-4) have been identified based on phylogenetic analysis since its first report in 2005. HBoV1 has been associated with respiratory disease, whereas HBoV2-4 are mainly detected in enteric infections. Although the prevalence of HBoVs in humans has been studied in some regions, it has not been well addressed globally. METHODOLOGY/PRINCIPAL FINDINGS: Cross-reactivity of anti-VP2 antibodies was detected between HBoV1, 2, 3, and 4 in mouse and human serum. The prevalence of specific anti-VP2 IgG antibodies against HBoV1-4 was determined in different age groups of healthy individuals aged 0-70 years old in Beijing, China, using a competition ELISA assay based on virus-like particles of HBoV1-4. The seroprevalence of HBoV1-4 was 50%, 36.9%, 28.7%, and 0.8%, respectively, in children aged 0-14 years (n = 244); whereas the seroprevalence of HBoV1-4 was 66.9%, 49.3%, 38.7% and 1.4%, respectively, in healthy adults (≥15 years old; n = 142). The seropositive rate of HBoV1 was higher than that of HBoV2, HBoV3, and HBoV4 in individuals older than 0.5 years. Furthermore, IgG seroconversion of HBoV1 (10/31, 32.3%), HBoV2 (8/31, 25.8%), and HBoV3 (2/31, 6.5%) was found in paired sera collected from children with respiratory tract infections who were positive for HBoV1 according to PCR analysis. CONCLUSIONS/SIGNIFICANCE: Our data indicate that HBoV1 is more prevalent than HBoV2, HBoV3, and HBoV4 in the population we sampled in Beijing, China, suggesting that HBoV species may play differential roles in disease. Human bocavirus (HBoV), a member of the Parvoviridae family, is a potential etiologic agent of respiratory disease and of acute gastroenteritis [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . Based on phylogenetic analysis of viral genomes, four species of HBoVs (HBoV1-4) have been identified [1, [7] [8] [9] . HBoV1 is associated with respiratory tract diseases [1, 2, [12] [13] [14] . HBoV2 and 3 have been detected in the respiratory tract, but are associated mainly with stool samples [8] [9] [10] [11] 15, 16] . HBoV4 has been detected in enteric infections [9] . However, as HBoVs are frequently co-detected with other viral infections in patients with respiratory or enteric infections, the exact roles of HBoVs in pathogenicity are unclear.
HBoVs are small, non-enveloped viruses with a linear singlestranded DNA genome of approximately 5 kb in length. The genome consists of four open reading frames (ORFs), encoding two nonstructural proteins (NS1 and NP1) and two overlapping capsid proteins (VP1 and VP2) [1] . The lack of a well-established cell culture system or animal model to propagate HBoVs has hampered understanding of the infection and pathogenicity of HBoVs. Studies have shown that the VP2 protein harbors the major antigen of HBoV and can form the empty virus-like particles (VLPs) which mimic HBoV virions morphologically and antigenically. The VP2 VLPs have been successfully used as antigens for detecting antibodies against HBoVs [17] [18] [19] [20] [21] [22] .
Currently, detection of HBoVs nucleic acid is primarily used to estimate the prevalence of HBoV species in clinical samples. The prevalence of HBoV1, which is mainly detectable in children under two years old [23] , is 2-19% in patients suffering from acute respiratory tract infections (ARTIs) worldwide as detected by PCR analysis [1, 2, 10, 12, 13, 23, 24] . The detection rate of HBoV2, HBoV3, and HBoV4 DNA in stool samples have been reported as 1-26%, 0.4-5%, and 0-2%, respectively [8, 9, 23, 25] . The prevalence of HBoV2-4 is higher in children than in adults according to some, but not all studies [8, 9, 11, 16, 25] . However, the data collected from patients may not represent HBoV infection in the general population as subclinical infections can occur and HBoV persists in the nasopharynx [26] [27] [28] [29] .
Seroepidemilogical investigations of healthy populations may be more useful than patient studies in assessing the prevalence, spread, and exposure distribution of HBoVs in the population. The seroprevalence data also allow a comparison between the frequency of natural infection and the frequency of this virus in individuals with infections [30] . However, previous seroepidemiological studies of HBoVs have mainly focused on HBoV1. HBoV1 specific IgG antibodies were frequently detected in children, with a seropositive rate ranging from 40.7%-60% for children , four years old and up to .85% for those $ four years old [19] . The seropositive rate of HBoV1 VP2-specific IgG antibodies is about 94% in healthy adults [18] . However, Kantola et al. recently showed cross-reactivity between the VP2 VLPs of HBoV1-4, which can largely affect the seropositive data of HBoV species. They showed that after depletion of cross-reactive antibodies, the approximate seroprevalences of HBoV1-4 in adults were 59%, 34%, 15%, and 2%, respectively [22] . However, the samples used in that study were only collected from healthy young people with a narrow range of age, including 115 subjects aged 21-32 years from Finland and 80 subjects aged 18-20 years from Pakistan. Given that the prevalence of a virus infection may vary by age as well as geographically, there remains to be a need to estimate the seroprevalence of HBoVs based on the detection of antibodies against HBoV1-4 from data of a more complete age range and from other global, geographic regions.
In the present study, we used a competition ELISA (cELISA) assay to estimate the seroprevalence of HBoV1-4 in healthy Chinese individuals ranging in age from 0 to 70 years in Beijing, China. We also compared the seroconversion of anti-HBoV IgG antibodies in 31 paired serum samples from ARTI children who were positive for HBoV1 by PCR. Our findings provide informative data for evaluating the prevalence and pathologic roles of HBoVs.
To produce antigens that can be used to evaluate the seroprevalence of HBoVs, the VP2 genes of HBoV1-4 were expressed in baculovirus to generate VLPs. The VP2 genes of HBoV1, HBoV2, and HBoV3 were amplified from stool samples by PCR. The sequences of these genes were verified by phylogenetic analysis ( Figure 1A ). The VP2 genes from respiratory and stool specimens were very similar in sequences. The HBoV1 strain 111-BJ07 used in this study clustered with the reference HBoV1 strain ST2 [1] , with homologies of 99.8%. The HBoV2 strain 211-BJ07 has 97.9% identity with the reference HBoV2 strain PK-2255 [7] . The HBoV3 strain 46-BJ07 has 99.6% identity with reference HBoV3 strain W471 [8] . As we did not find any HBoV4 positive samples, the VP2 gene of HBoV4 was synthesized according to the sequence of HBoV4 strain NI-385 [9] .
The VP2 proteins of HBoV1-4 were expressed in insect cells using a baculovirus expression system. The generation of HBoV VLPs was verified by ultracentrifuge-purification and electron microscopy (EM). Typical parvovirus-like particles of 22-24 nm in size, similar to the particles of infectious parvovirus virions, were visualized under EM (data not shown). The purified VLPs of HBoV1, 2, 3 and 4 were also reactive to murine antisera specific for the VP2 protein of the respective HBoV species in Western blot assays ( Figure 1B ).
Sequence alignment showed a high amino acid identity of VP2 between HBoV species, with 77.5% identity between HBoV1 and HBoV2, 77.7% identity between HBoV1 and HBoV3, 77.5% identity between HBoV1 and HBoV4, 89.4% identity between HBoV2 and HBoV3, 88.5% identity between HBoV2 and HBoV4, and 90.9% identity between HBoV3 and HBoV4 (data not shown), indicating possible cross-reactivity between HBoV species. To evaluate the potential cross-reactivities, we examined the reactivity between the HBoV1-4 VLPs and purified mouse antisera against HBoV1, 2, 3, and 4 VP2 using Western blot and ELISA assays.
Western blot analysis showed that the antisera against HBoV1, 2, 3, and 4 reacted with 400 ng of VLPs of each HBoV species, as indicated by detection of specific bands of 60 kD in size ( Figure 2A) . Similar cross-reactivities were also detected by ELISA assays ( Figure 2B ). Mouse sera against HBoV1, 2, 3, and 4 reacted strongly with the homologous VLPs. Moreover, all four antisera reacted with the heterologous HBoV VLPs when the concentration of the mice antiserum was high (.0.25 mg/mL). No significant reactivity was observed with the human parvovirus B19 VP2 or influenza virus H5 hemaglutinin (H5) antiserum ( Figure 2B ). These results are consistent with those reported by Kantola et al [22] .
To develop ELISA assays that can detect antibodies against HBoV species, we first sought to identify HBoV-positive andnegative sera samples using Western blot analysis. To obtain negative sera samples, we tested the reactivity of sera samples collected from infants aged 0-6 months who visited Beijing Children's Hospital for regular health check-ups against HBoV VLPs. Only the samples that were negative for HBoV1, 2, 3, and 4 VLPs simultaneously were selected as the negative samples. The negativity of those samples was confirmed using a competition ELISA (cELISA) assay, in which the absorbance value at 450 nm did not change significantly with or without competition assays ( Figure 3 ). Serum samples identified by cELISA as positive for individual HBoV species were obtained from 3 children and 4 adults and used as positive controls. However, we did not obtain a serum sample that was positive only for HBoV4 based on the assay; we used a sample from an adult that was IgG positive for HBoV1, HBoV2 and HBoV4. To determine the concentration of VLPs required for exhaustive antibody competition of HBoV1-4, the serum samples were monocompeted with concentrations of HBoV1, 2, 3, or 4 VLPs ranging from 0-32 mg/mL ( Figure 3 ). We found that HBoV VLPs concentration of 16 mg/mL was the effective concentration to perform the VLP-based competition assays.
To determine the seroprevalence of HBoV1, 2, 3, and 4 in humans, we developed an ELISA protocol for detecting IgG antibodies against HBoV1-4 using VLPs as coating antigens. The concentration of the coated VLPs (0.125 mg/mL) for this ELISA assay was optimized using chessboard titration tests. The positive sera and negative sera showed different results at 1:200 serum dilutions. As the results obtained using lower serum dilutions were similar to those obtained at 1:200 (data not shown), the 1:200 dilution was used in subsequent ELISA analysis of serum samples.
To determine the cut-off values for the ELISA, we determined the mean values and standard deviation of negative sera for HBoV1, 2, 3, and 4 using HBoV VLP ELISA at dilutions of 1:200. We used the mean absorbance at 450 nm of the negative sera plus threefolds the standard deviation as the cut-off values, as previously described [19, 31] . For HBoV1, 2, 3 and 4, the cut-off values were 0.344, 0.304, 0.321, and 0.31, respectively. A sample was considered positive for HBoV1, 2, 3, or 4 if its absorbance at 450 nm was above the cut-off value of the respective species in ELISA.
As there are cross-reactivities between HBoV species in ELISA assay, we developed a cELISA assay to evaluate the seroprevalence of each HBoV species. We evaluated the specificity of this protocol using heterologous competition assays with parvovirus B19 and human parvovirus 4 (PARV4) VP2. Neither human parvovirus B19 nor PARV4 VP2 inhibited the reactivity of IgG against HBoV1, 2, 3, or 4 in the serum from adults or children ( Figure 3 ). These results suggest that there is no antigenic crossreactivity between HBoVs VP2 and human parvovirus B19 VP2 or between HBoVs VP2 and PARV4 VP2. To confirm the specificity of this ELISA protocol, we tested sera from 15 adults that were positive for human parvovirus B19. In the HBoV VLP ELISA, we did not detect false-positive signals for HBoVs (data not shown).
To determine the seroprevalence of HBoV1-4 in adults, we used the HBoV VLPs ELISA method to detect IgG antibodies against HBoV1-4 in 142 serum samples collected from healthy individuals 15-70 years old. Without competition, more than 90% samples were positive for HBoV1, HBoV2, and HBoV3; 73 (51.4%) samples were positive for HBoV4 (Table 1) . However, these IgG seroprevalences decreased with competition by VLPs of heterologous HBoV species (Figure 4 ). The cELISA resulted in seroprevalences of 66.9%, 49.3%, 38.7%, and 1.4%, for HBoV1, 2, 3, and 4 IgG, respectively. The seropositive rate of HBoV1 was higher than that of HBoV2, HBoV3, and HBoV4 (x 2 = 23,P,0.01) (Table 1 and Figure 5 ).
Twenty-five of the 142 (17.6%) adult samples that were positive for IgG antibody against HBoV1, 2, and 3, and 17 (12%) that were positive for HBoV4 in the ELISA were negative for IgG against all 4 HBoVs in the cELISA. Among positive serum samples against single HBoV species, there were 26 for HBoV1, eight for HBoV2, and two for HBoV3 based on the cELISA results.
The seropositive rates of HBoV1, 2, and 3 in children increased with age according to results from the ELISA without competition. The rates for HBoV1, 2, and 3 reached to 77.9%, 61.1%, and 58.9%, respectively, in the age group 5-14 years. The seroprevalence for HBoV4 among children aged 0-0.5 year and 5-14 years increased from 10.3% to 31.6% (Table 1) . However, according to results of the cELISA, the seroprevalences of HBoV1, 2, 3, and 4 IgG decreased about 10-27% (Table 1, Figure 5 ). The seroprevalence among children aged 0-0.5 years and children aged 5-14 years increased from 6.9% to 60%, 6.9% to 49.5%, and 6.9% to 38.9% for HBoV1, HBoV2, and HBoV3, respectively, in the cELISA.
Of note, among 29 infants aged 0-6 months, two (6.9%) were positive for HBoV1, 2, and 3 IgG, and one (3.4%) was positive for HBoV4. The detection of HBoVs IgG antibodies among children aged 0-6 months may be due to the presence of maternal antibodies. However, the seropositive rate of HBoV1 was higher than that of HBoV2, 3, or 4 in the age groups of 0.5-2 years, 2-5 years, and 5-14 years (x 2 = 10.1,P,0.01; x 2 = 9.3, P,0.01; x 2 = 8.42,P,0.05, respectively). Based on the results of the cELISA, there were 38 (15.6%) individuals positive for HBoV1, nine (3.7%) for HBoV2, and two (0.8%) for HBoV3 among positive serum samples against single HBoV species. Notably, the two samples positive for HBoV4 IgG were also positive for HBoV1, 2, and 3 IgG.
To characterize the antibody response after HBoV infection further, we measured the IgG antibody in 31 pairs of sera samples that were collected from children with ARTIs. All these children were positive for HBoV1 according to PCR analysis. Based on cELISA, the seroprevalences of HBoV1, 2, 3, and 4 in acute-phase sera were 29%, 25.8%, 16.1%, and 0%, respectively. Of the 31 samples, ten (32.3%) showed an IgG seroconversion for HBoV1, eight (25.8%) for HBoV2, two (6.5%) for HBoV3, and none for HBoV4 (Table2). Two pairs of sera showed concurrent seroconversions to HBoV1 and HBoV2, and one pair showed concurrent seroconversions to HBoV1, HBoV2, and HBoV3. In addition, absorbance at 450 nm increasing in the convalescent-phase serum was observed in seven children (22.6%) who were positive for HBoV1 IgG and eight (25.8%) children who were positive for HBoV2 IgG positive at the acute-phase.
In this study, we evaluated the cross-reactivity of mouse antisera against HBoV1, 2, 3, and 4 VP2 with VP2 VLPs for four species of human bocaviruses. Considerable cross-reactivity was found among the four HBoV species. However, human IgG antibodies against HBoV did not cross react with human parvovirus B19 and PARV4. These findings are consistent with those of a previous report [22] , suggesting that to obtain accurate data of the seroprevalences of the different HBoV species, it is necessary to correct for cross-reactivity between the four HBoVs. Comparison of the VP2 protein sequences of HBoV1-4 revealed a high degree of similarity. These similarities may account for the cross-reactivity of these viruses, and should be confirmed through epitope analysis. Human parvovirus B19 and PARV4 have no antigen crossreactivity with HBoVs, suggesting that human parvovirus B19 and PARV4 antibodies do not interfere with the HBoV seroprevalence results. The specificity of human bocavirus VLP-based ELISA with human sera has also been demonstrated with human parvovirus B19 VP2 and PARV 4 VP2 by Kantola et al [22] .
We used a VLP-based ELISA to assess the seroprevalence of HBoV1-4 in healthy individuals in Beijing, China, ranging from 0 to 70 years old. Our results show that the seroprevalences of HBoV1, 2, and 3 range from 40.3-67.8% in children 0.5-2 years old and are up to 100% in adults. To eliminate the interference of antibody cross-reactivity, the results were corrected with a cE-LISA. The results of our cELISA suggest that the seroprevalences of HBoVs are significantly lower than those obtained without competition, especially in adults. These findings further indicate a high degree of antigenic cross-reactivity between HBoV1-4. Our findings are consistent with those of a recent study of Finish and Pakistani individuals, in which the seroprevalences of HBoVs decreased after depletion of heterologous HBoV reactive antibodies [22] .
Our results from the cELISA are lower than those previously reported for HBoV1 in adults and children [18, 19, 32] , where HBoV1 antibodies against HBoV1 VP2 were detected by ELISA. Hence, the seroprevalence of HBoVs may be overestimated due to the serological cross-reactivity among the four HBoV species in previous studies. However, according to our cELISA results, the seroprevalence of HBoV1-3 in Beijing adults is somewhat higher than that reported in Finnish and Pakistani adults [22] . This disparity may be attributed to the difference of geographical location [22] .
Our age stratification data indicates that HBoVs circulate widely in the human population, and the primary infection with HBoV1-3 occurs in children aged six months and older after the maternal antibodies have waned. The seropositive rates of IgG antibodies against HBoV2, 3 and 4 were lower than that of HBoV1 in individuals of $0.5 years old. These data indicate that HBoV1 is the predominant circulating species of HBoV in Beijing, whereas HBoV2-4 do not seem to have a major impact on HBoV infections. This finding agrees with reports on HBoVs prevalence obtained from most parts of the world using DNA analysis [10, 14, 16] . Of note, our finding that the seroprevalence of HBoV4 is much lower than that of HBoV1-3 is consistent with the rare detection of HBoV4 DNA in clinical samples [9] . Overall, the seroprevalence of HBoVs indicated by results of the cELISA decreased less in children than in adults in comparison to the results without competition, indicating that HBoV infections are more specific in children, especially for HBoV1 [22] . These results suggest that HBoV species may play differential roles in disease.
Our results show that 17.6% of the adults who were positive for IgG against HBoV1, 2, 3, or 4 based on the ELISA results were negative for IgG against all four HBoVs based on the cELISA results. This finding may be due to antibody waning in the individuals with low IgG levels [22] or to low-affinity antibodies [20, 33] .
To assess the antibody response of HBoV1-4 IgG in children with acute respiratory tract infections, we used cELISA to determine the seroconversion rate of 31 paired sera from children who were positive for HBoV1 according to PCR analysis. We found that the HBoV1-specific seroprevalence was higher in convalescent sera than in acute sera. However, heterotypic seroconversion against HBoV2 and HBoV3 was also observed. Moreover, for some patients who were positive for HBoV1 and HBoV2 IgG at the acute phase, the absorbance value was higher in convalescent sera. These data may suggest the concurrent production of antibodies against HBoV2 and 3 during the infection of HBoV1, as it has been shown that B-cell memory can be boosted either by the homologous virus or by heterologous, yet immunological related virus [20, 34, 35] .
Overall, our findings suggest a differential prevalence of HBoV species in healthy individuals aged 0-70 years old. HBoV1 appears to be the dominant species responsible for HBoV infections among HBoV1, 2, 3, and 4 in Beijing, China. The seroprevalence of HBoV1-3 increased with age in children. Our study provides a basis for future evaluation of the epidemiology, genotype distribution, and pathogenesis of HBoVs worldwide.
Serum specimens were collected from 386 healthy individuals aged 0 to 70 years in 2008; 244 specimens were from infants and children who visited Beijing Children's Hospital for regular health check-ups. The 142 specimens from adults were provided by the Beijing Blood Center. Exclusion criteria for all subjects included pregnancy, any abnormalities in renal and liver function tests, HIV/AIDS, sexual transmitted diseases, tumor, recurrent or acute infection, and medication. None of the subjects had any respiratory infection for at least three months prior to the blood samples being taken. In addition, paired acute-phase (at the time of admission) and convalescent -phase (2 weeks after disease onset) serum samples were collected from 31 children (median age 17 months; range of 1 month to 9 years) with acute lower respiratory tract infections (ALRTIs) when they were hospitalized at the Beijing Children's Hospital. The DNA of HBoV1, but not HBoV2-4, was detected in the nasopharyngeal aspirates of these 31 patients at the time of admission by nested PCR and sequence analysis using primers targeting the viral proteins (VP) 1/2 region [9] . All serum samples were stored at 280uC prior to use.
Written informed consent was obtained from all participants or guardians on behalf of children. This study was approved by the ethical review committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences.
The full-length VP2 genes of HBoV1-4 were used for viruslike particle (VLP) production in this study. HBoV1-3 VP2 genes were amplified from HBoV-positive stool specimens (111-BJ07, 211-BJ07, and 46-BJ07; GenBank accession numbers: JQ240469, JQ240470 and HM132056) [15] . HBoV1 is 1,629 bp (nt 3,373-5,001 according to HBoV1 strain ST1, GenBank accession number DQ000495), HBoV2 is 1,617 bp (nt 3,306-4,922 according to HBoV2 strain PK2255, GenBank accession number FJ170279), and HBoV3 is 1,620 bp (nt 3,410-5,029 according to HBoV3 strain W471, GenBank accession number EU918736) in length. The VP2 genes of HBoV4 (1626 bp in length, nt3331-4956 based on HBoV4 strain NI-385, GenBank accession number NC012729) were synthesized by Sangon Biotech (Shanghai, China). These genes were verified by phylogenetic analysis using the Clustal W and MegAlign programs in the MEGA 4.0 software package [36] . The phylogenetic tree with 1,000 bootstrap replicates was generated based on the complete sequences of the VP2 genes used in this study and reference sequences from GenBank. HBoV1 strains ST1, ST2, and TW2888_06; HBoV2 strains W153, PK-2255, LZ55602, and 277-BJ07; HBoV3 strains W471 and W855; and HBoV4 strains NI-385 were used as reference sequences (GenBank accession numbers DQ000495, DQ000496, EU984237, EU082213, FJ170279, GU301645, JQ240471, EU918736, FJ948861, NC012729, respectively). 
The full-length VP2 genes of HBoV1, 2, 3, and 4 were cloned into the baculovirus expression vector pFastbac1 and expressed using Bac-to-BacH Baculovirus Expression System (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. VLPs were obtained as previously described [17, 37] . High Five cells (Invitrogen) were infected with recombinant baculoviruses at a multiplicity of infection (MOI) of five and harvested after three days. Cells were suspended in 25 mM NaHCO 3 solution at 2610 7 cells/mL and kept on ice for 30 min. After centrifugation at 18,000 rpm for 10 min at 4uC, (NH4) 2 SO 4 was added to the supernatants at a final concentration of 20% (w/v). Precipitants were harvested by centrifugation and dissolved in CsCl solutions with densities of 1.4 g/mL in Tris-EDTA buffer (10 mmol/L Tris, pH 8.7, 1 mmol/L EDTA, and 0.5% Triton X-100). After centrifugation at 35,000 rpm and 18uC for 39 h in a SW41 rotor centrifuge (Beckman Coulter, Fullerton, CA), the fractions were analyzed by SDS-PAGE and Western blot using mouse sera against HBoV VP2 proteins. A Tecnai12 transmission electron microscope (FEI, Hillsboro, OR) at 80 kV was used to verify the morphology of the VLPs. SDS-PAGE and Western blot analysis were used to identify the VLPs [18] .
Recombinant VP2 proteins of HBoV1, 2, 3 and 4 were also expressed in E. coli Rosetta (DE3) (Novagen, Madison,WI,) cells and purified as previously described [37] . The genes used in prokaryotic cloning were the same as those used in a baculovirus expression system. The recombinant antigens were used to immunize mice to produce antibodies (see below).
As controls, human parvovirus B19 VP2 was expressed, as described previously [38] , using the construct pFastBac1B19VP2 provided by Dr. Xiaohui Zou at the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease control and Prevention. Additionally, the PARV4 VP2 gene (1,659 bp in length, nt 3,464-5,122 based on the NC_007018 reference sequence) was synthesized by Sangon Biotech (Shanghai, China) and cloned into baculovirus vector pFBGP67-His [39] . The recombinant baculoviruses were generated in Sf9 cells using the Bac-to-BacH Baculovirus Expression System (Invitrogen) protocol provided by the manufacturer. High Five cells were infected with recombinant baculovirus expressing PARV4 VP2 gene at a MOI of five. The infected cells were collected three days post-infection and purified using a HisTrap HP 1 ml column (GE Healthcare, Waukesha, WI). The concentrations of all purified protein were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) and stored at 280uC prior to use.
ELISA was used to determine the anti-HBoV antibodies, as described elsewhere [40] . The purified HBoV VLPs were used as coating antigen (0.125 mg/mL). The absorbance of each serum sample was read at 450 nm and the mean values of the duplicate samples were calculated. Sera pooled from ten samples showing HBoV-specific IgG responses were used as the internal references in all experiments.
To minimize false positive results of the ELISA assay due to impurities in immunizing and coating antigens, the coating antigen and the protein used to prepare mouse antibodies were derived from a baculovirus expression system and a prokaryotic expression system. BALB/c mice were injected subcutaneously with the purified proteins obtained from E. coli. This study was carried out in accordance with the animal experiment regulations of the Chinese government. All animal experiments were performed in the facilities of the Institute of Laboratory Animal Sciences (ILAS), Chinese Academy of Medical Sciences (CAMS). All experimental procedures were approved (license number SCXKJ2009-0017) and supervised by the Animal Protection and Usage Committee of ILAS, CAMS. Sera collected from the treated mice were purified using protein-G sepharose columns (GE Healthcare, Waukesha, WI) and purified IgG antibody were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). The purified mouse IgG antibodies were serially diluted from 8 mg/mL to 0.008 mg/mL. Sera from pre-immune mice served as the negative control. Rabbit antiserum against human parvovirus B19 (a gift from Dr. Xia Xiao of National Institute for Viral Disease Control and Prevention, Chinese Center for Disease control and Prevention) and mice antiserum against influenza virus H5 hemaglutinin (HA) [39] were used as unrelated controls. The cross-reactivity between HBoV1, 2, 3, and 4 was tested using Western blot analysis and ELISA.
To measure antibodies specific to VP2 antigen of an individual HBoV species (HBoV1, 2, 3, or 4), antibodies in serum samples were absorbed with heterologous VLPs of the other three HBoVs, as previously described [22] . Briefly, HBoV VLPs were serially diluted from 32 mg/mL to 0.5 mg/mL to determine the concentration needed for effective competition between cross-reactive antibodies. For detection of specific HBoV antibodies, three heterologous HBoV VLPs of 16 mg/mL were added to a 1:200 dilution of plasma [17] and incubated for 2 hr at 4uC prior to performing the ELISA assay. In parallel with the heterologous competition, human sera samples were used to compete with VLP that was homologous to the immobilized antigen. The absorbance at 450 nm was residual absorbance value. Net absorbance values were calculated by subtracting the residual absorbance value from the raw absorbance value read at 450 nm [22] .
Seropositive rates were evaluated using x 2 tests. A P value #0.05 was considered significant. Clarithromycin Suppresses Human Respiratory Syncytial Virus Infection-Induced Streptococcus pneumoniae Adhesion and Cytokine Production in a Pulmonary Epithelial Cell Line Human respiratory syncytial virus (RSV) sometimes causes acute and severe lower respiratory tract illness in infants and young children. RSV strongly upregulates proinflammatory cytokines and the platelet-activating factor (PAF) receptor, which is a receptor for Streptococcus pneumoniae, in the pulmonary epithelial cell line A549. Clarithromycin (CAM), which is an antimicrobial agent and is also known as an immunomodulator, significantly suppressed RSV-induced production of interleukin-6, interleukin-8, and regulated on activation, normal T-cell expressed and secreted (RANTES). CAM also suppressed RSV-induced PAF receptor expression and adhesion of fluorescein-labeled S. pneumoniae cells to A549 cells. The RSV-induced S. pneumoniae adhesion was thought to be mediated by the host cell's PAF receptor. CAM, which exhibits antimicrobial and immunomodulatory activities, was found in this study to suppress the RSV-induced adhesion of respiratory disease-causing bacteria, S. pneumoniae, to host cells. Thus, CAM might suppress immunological disorders and prevent secondary bacterial infections during RSV infection. Human respiratory syncytial virus (RSV) is one of the most important infectious agents causing acute lower respiratory tract illness, such as bronchiolitis and pneumonia, in infants and young children [1, 2] . Viral RNA generated during RSV replication is recognized by host pattern recognition molecules, such as Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I), and it induces type I and type III interferon [3, 4] . Transcriptional induction of proinflammatory cytokines, chemokines, and interferons is mediated by NF-κB and interferon regulatory factors (IRFs) [5, 6] . These mediators are believed to contribute to the pathophysiology of RSV infection, such as mucous hypersecretion, swelling of submucous, and infiltration of lymphocytes, neutrophils, eosinophils, and macrophages [7] .
Frequently, there are coinfections with respiratory viruses, including RSV, and bacteria that cause communityacquired respiratory diseases, such as Streptococcus pneumoniae and Haemophilus influenzae. There is evidence for a positive correlation between infections with S. pneumoniae and RSV in the pathogenesis of otitis media, pneumonia, and meningitis [8] [9] [10] [11] . S. pneumoniae and H. influenzae colonize to the host respiratory epithelium via host cell surface receptors, such as the platelet-activating factor (PAF) receptor [12] [13] [14] . These bacteria interact with the PAF receptor via phosphorylcholine, which is a component of the bacterial cell surface. Both live and heat-killed S. pneumoniae cells show an increased adhesion to human epithelial cells infected with RSV [15] . The upregulation of PAF receptor expression that is induced by respiratory virus infections, including those caused by RSV, results in the enhanced adhesion of S. pneumoniae and H. influenzae to respiratory epithelial cells [15] [16] [17] . PAF receptor expression and S. pneumoniae cell adhesion are also upregulated by exposure to acid, which causes tissue injury and an inflammatory response [18] .
Clarithromycin (CAM) is 14-membered ring macrolide antibiotic that also acts as a biological reaction modifier with anti-inflammatory properties. In Japan, CAM is applied to diffuse panbronchiolitis, chronic bronchiolitis, otitis media, and chronic sinusitis as an immunomodulator [19] [20] [21] . The anti-inflammatory mechanism of CAM has not yet been completely clarified, but one of the important mechanisms for its anti-inflammatory action is considered to be the suppression of NF-κB [22] [23] [24] .
Recently, we reported that fosfomycin, which is an antibiotic, suppressed RSV-induced interleukin (IL)-8, regulated on activation, normal T-cell expressed and secreted (RANTES), and the PAF receptor by suppressing NF-κB activity [25, 26] . On the other hand, Wang et al. report that CAM suppressed rhinovirus-induced Staphylococcus aureus and H. influenzae adhesions to nasal epithelial cells [27] . So we anticipate that CAM suppresses RSV-induced bacterial adhesion to epithelial cells, because expression of PAF receptor is controlled by NF-κB [28, 29] In the present study, we examined the effect of CAM on cytokine production, PAF receptor expression, and RSV infection-induced S. pneumoniae adhesion to respiratory epithelial cells.
Lines, Bacteria, and Reagents. RSV strain Long, human type II pulmonary epithelial cell line A549 and S. pneumoniae strain R6 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). RSV was grown in HEp-2 cells. The virus titer of RSV was determined using a plaque-forming assay with HEp-2 cells as the indicator cells [25] . RSV infection to A549 cells was performed at multiplicity of infection (MOI) of 1. CAM was donated by Abbott Japan (Tokyo, Japan). A PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N,trimethyl)-hexanolamine, was purchased from Calbiochem-Merck (Darmstadt, Germany). An NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was purchased from Sigma-Aldrich (St. Louis, MO).
Production. A549 cells were infected with RSV at MOI of 1. After 24-hour infection, culture supernatants of RSV-infected and -uninfected cells were collected. The amounts of IL-6, IL-8, and RANTES in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) (DuoSet ELISA development kit, R&D systems, Minneapolis, MN).
(RT-PCR). Semiquantitative RT-PCR was carried out as described previously [4, 30] .
The cell surface expression of the PAF receptor was examined by flow cytometry as previously described [26] . The cells were harvested from culture flasks using a cell scraper and then incubated with 2.5 μg/mL of mouse anti-PAF receptor monoclonal antibody (11A4 (clone 21); Cayman Chemical, Ann Arbor, MI) or mouse IgG2a, κ isotype control antibody (eBioscience, San Diego, CA). After incubation at 4 • C for 30 min, cells were collected by centrifugation and washed once with Dulbecco's phosphate-buffered saline (PBS (−)). Cell suspensions were incubated with a phycoerythrin-conjugated goat anti-mouse IgG F(ab) 2 fragment antibody (1 : 100 dilution) (Abcam, Cambridge, UK) at 4 • C for 30 min, and the stained cells were assessed with FACSCalibur (BD Bioscience, San Jose, CA).
Assay. S. pneumoniae adhesion was assayed using fluorescein-isothiocyanate-(FITC-) labeled S. pneumoniae as previously described [26] . Briefly, a bacterial suspension in 0.1 M NaCl-50 mM sodium carbonate buffer (pH9.5) at 1 × 10 8 CFU/mL was prepared. FITC isomer-I (Dojindo Laboratories, Kumamoto, Japan) was added at a concentration of 1 mg/mL, and the mixture was incubated at 4 • C for 1 h. The cells were washed three times with PBS (−).
CAM was added to monolayers of A549 cells 1 h prior to RSV infection. The A549 cells infected with RSV at an MOI of 1 for 24 h and uninfected A549 cells were incubated with FITC-labeled S. pneumoniae cells at MOI of 10 for 30 min at 37 • C. For the control experiments, either 20 μg/mL of the PAF receptor antagonist or 10 μg/mL of the mouse anti-PAF receptor monoclonal antibody (11A4(clone 21)) was added to the A549 cells 1 h prior to the addition of the FITC-labeled bacteria. The cell monolayer was gently washed three times with PBS (−) and observed by fluorescence microscopy. Alternatively, the cells were harvested with cell scraper and then assessed by flow cytometry as previously described [26] .
First, we examined the effect of CAM on RSV replication in A549 cells. RSV infection to A549 cells was performed at MOI of 1. After 24 and 36 h of infection, significant alterations of the RSV titers or expression levels of G mRNA were not observed by the addition of CAM even at a concentration of 100 μg/mL (Figure 1 ).
When A549 cells were infected with RSV at MOI of 1, RANTES, IL-8, and IL-6 were markedly induced. These cytokine inductions were significantly suppressed in the presence of CAM in a dose-dependent manner ( Figure 2 ). The degree of suppression by CAM was less than that by an NF-κB inhibitor, PDTC.
PAF receptor expression on the cell surface is upregulated during RSV infection in A549 cells [26] . The RSV-induced upregulation of the PAF receptor was significantly suppressed by CAM and PDTC in a dose-dependent manner ( Figure 3 ). The degree of suppression by CAM was slightly less than that by PDTC. Suppression of the PAF receptor expression was also observed when A549 cells were posttreated with CAM (4 or 12 h after RSV infection) (data not shown). We examined the adhesion of FITC-labeled S. pneumoniae cells to A549 cells by fluorescence microscopy (Figure 4 ) and flow cytometry ( Figure 5 ). RSV infection significantly enhanced the adhesion of S. pneumoniae to A549 cells, and this enhancement was suppressed by adding a PAF receptor antagonist (Figures 4 and 5) or anti-PAF receptor monoclonal antibody (data not shown). This result indicated that the RSV-induced S. pneumoniae adhesion occurs via the PAF receptor on A549 cells. The bacterial adhesion was significantly suppressed by CAM, as well as PDTC.
These lines of evidence confirmed that the expression of the PAF receptor was induced by RSV infection and indicated that this induction, and subsequent RSV-induced S. pneumoniae adhesion, can be suppressed by CAM treatment.
Macrolides, with the exception of the 16-membered ring type, have both anti-inflammatory and antibacterial functions [20, 21] . One of the important mechanisms of antiinflammatory action is the suppression of NF-κB activation [22] [23] [24] . Our recent studies show that RSV upregulates proinflammatory cytokines, such as IL-6, and chemokines, such as IL-8 and RANTES, in the respiratory epithelial cell line A549. Furthermore, the induction of chemokines by RSV is significantly suppressed by an antibiotic, fosfomycin, via suppression of NF-κB activation [25] . In the present study, CAM was shown to suppress IL-6, IL-8, and RANTES, which are induced by RSV infection, at concentrations of 10 and 100 μg/mL. Patel et al. reported that the concentration of CAM in fluid of the bronchopulmonary epithelial lining was 34.2±5.16 μg/mL at 4 h, 23.01±11.9 μg/mL at 12 h in healthy adults orally administered CAM 500 mg [31] . We observed that CAM did not affect RSV replication even at a concentration of 100 μg/mL. However, it is reported that respiratory virus, such as RSV [32] , rhinovirus [33, 34] , and influenza virus [35] , replication is suppressed by 14-membered ring macrolides, including CAM. The reasons of contradictory results between the report of Asada et al. [32] and our present study have been unclear. These two studies used different types of epithelial cells and different experimental conditions of RSV infection. Asada et al. used primary human tracheal epithelial cells, and in contrast we used A549 cell line. Asada et al. carry out infection at a lower titer of RSV (10 −3 TCID 50 /cell) and measuring virus titer at a longer period (3-5 days) after infection. Our results indicated that suppression of the RSV-induced cytokines by CAM was not caused by the amount of replicated RSV. In other words, CAM was suggested to have suppressive activity of cytokine production independent of viral replication. Both IL-8 and RANTES, which are strongly upregulated during RSV infection, play important roles in pathogenesis [36, 37] . IL-8 primarily activates neutrophils and promotes their migration. RANTES is secreted from respiratory epithelial cells and promotes migration of eosinophils, basophils, monocytes, and neutrophils. In particular, RANTES is an efficient eosinophil chemoattractant involved in the pathogenesis of asthma [38] . CAM has been suggested to suppress the inflammatory disorders induced by RSV.
In the present study, we also observed that CAM suppressed enhanced S. pneumoniae adhesion by RSV infection in A549 cells. The RSV-induced S. pneumoniae adhesion was mainly mediated by host PAF receptor, as indicated by that suppressed by the PAF receptor antagonist and anti-PAF receptor monoclonal antibody. The PAF receptor acts as a receptor for S. pneumoniae and H. influenzae [12] [13] [14] . Transcription of the PAF receptor gene is controlled by NF-κB [28, 29] . We confirmed it by that the RSV-induced PAF receptor expression and S. pneumoniae adhesion were suppressed by an NF-κB inhibitor, PDTC. We revealed that CAM also suppressed PAF receptor expression induced by RSV infection and S. pneumoniae adhesion to RSV-infected A549 cells. It should be caused by the suppression of by inhibiting PAF receptor expression. CAM showed more potent suppression of RSV-induced S. pneumoniae adhesion and production of proinflammatory cytokines and chemokines than fosfomycin, as we reported previously [25, 26] . Notably, CAM significantly suppressed RSV-induced IL-6 production, whereas fosfomycin did not significantly [25] . This finding may be caused by that CAM is more potent than fosfomycin; however, the actual reason for this disparity is not clear. The upregulation of PAF receptor expression and the enhanced adhesion of pathogenic bacteria, such as S. pneumoniae, to respiratory epithelial cells are considered to be a major risk factor for secondary bacterial infections after primary respiratory viral infections. CAM may suppress both secondary bacterial infections and immunological disorders induced by RSV, without suppressing viral replication. Infection with other respiratory viruses, such as human parainfluenza virus 3 [16] and rhinovirus [17] , also upregulates known receptors for the pathogenic bacteria, including PAF receptor and S. pneumoniae adhesion. On the other hand, influenza virus does not upregulate the known receptors for bacteria, whereas bacterial adhesion is increased by the infection [16] . McCullers [39] reported that influenza-induced bacterial adhesion to A549 cells was not inhibited by PAF receptor antagonist, and the PAF receptor knock-out mice did not show lower susceptibility to experimental secondary pneumonia caused by S. pneunimoae following influenza infection compared to the parent mice. Lines of evidence suggest that adherent inducing mechanisms of S. pneumoniae to host respiratory epithelial cells are varied among viruses. So CAM may not always suppress virus-induced pathogenic bacteria adhesion.
We proposed that clarithromycin efficiently suppressed PAF receptor-mediated Streptococcus pneumoniae adhesion to respiratory epithelial cells as well as RSV-induced proinflammatory cytokine and chemokine production. Clarithromycin may suppress secondary bacterial infections and immunological disorders during RSV infection. Post-Transcriptional Control of Type I Interferon Induction by Porcine Reproductive and Respiratory Syndrome Virus in Its Natural Host Cells Porcine reproductive and respiratory syndrome virus (PRRSV) is not only a poor inducer of type I interferon but also inhibits the efficient induction of type I interferon by porcine transmissible gastroenteritis virus (TGEV) and synthetic dsRNA molecules, Poly I:C. However, the mechanistic basis by which PRRSV interferes with the induction of type I interferon in its natural host cells remains less well defined. The purposes of this review are to summarize the key findings in supporting the post-transcriptional control of type I interferon in its natural host cells and to propose the possible role of translational control in the regulation of type I interferon induction by PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded, positive-sense RNA virus with a genome size of approximately 15 kb. PRRSV belongs to the Arteriviridae in the order of Nidovirale. PRRSV causes acute respiratory disease in neonatal and young piglets and reproductive failure in pregnant sows. PRRSV primarily infects and destroys alveolar macrophages during acute infection of swine. In addition to macrophages, PRRSV has also been identified by immunohistochemistry in dendritic-like cells in tonsils and lymph nodes [1] , suggesting that dendritic cells may either be susceptible to PRRSV or capture PRRSV by taking up apoptotic infected cells.
tumor necrosis factor- (TNF-α), are absolutely dependent on activation of PI3K [9] . Although the nuclear factor kappa B (NF-B) pathway has been shown to contribute to the transcriptional activation of type I interferon by PRRSV [15] , one study shows that NF-B is more likely related to induction of inflammatory cytokines such as TNF- and IL-6, rather than type I interferon, after influenza A virus infection and CpG ODN stimulation [9] . This discrepancy may be due to the cell types and viruses used in different studies since different cell types and different viruses exhibit distinct features in the induction of type I interferon pathway. Virus replication and viral infectivity are usually not essential to the induction of type I interferon since both UV-inactivated and heat-inactivated influenza A viruses induce more abundant interferon- than their live virus counterparts [16] .
In 1998, Albina et al. first reported the failure of PRRSV in inducing the production of interferon- protein in the lung secretions of infected pigs and in the supernatants of PRRSV-infected alveolar macrophages and peripheral blood mononuclear cells [17] . Furthermore, they observed that PRRSV was also capable of blocking the production of interferon- protein in macrophages by a well-characterized and potent interferon- inducer, swine transmissible gastroenteritis virus (TGEV), a member of the coronaviridae family. Virus replication and infectivity is essential to the inhibition of interferon- production since UV-inactivated PRRSV fails to block the induction of interferon- by TGEV. Interestingly, a recent study reported that PRRSV infectivity is not essential to the inhibition of type I interferon in plasmacytoid dendritic cells, which are resistant to PRRSV infection [4] . The observation that PRRSV is a poor inducer of interferon- is further confirmed by Buddaert et al. [18] . In 2004, Chung et al. reported that PRRSV activated the transcription of interferon- and Mx1 in lung tissues at day 1 and peaked at day 7 after infection in acutely infected animals, suggesting that PRRSV does activate the transcription of interferon- and interferon induced genes such as Mx1 [19] . Others have further reported that different PRRSV isolates exhibit different capacities in inducing the production of interferon- in alveolar macrophages [20] . The activation of interferon- transcription in PRRSV-infected monocyte-derived dendritic cells, peripheral blood mononuclear cells, alveolar macrophages and in porcine alveolar macrophages of PRRSV-infected animals has also been reported [3, 21, 22, 23] . Taken together, the existing evidence clearly indicates that PRRSV activates the transcription of type I interferon in its natural susceptible cells. In MARC-145 cells, however, Miller reported that PRRSV not only failed to activate the transcription of interferon- and , but also suppressed the transcription of interferon- activated by Poly I:C [24] . This suggests a cell type dependent difference in activating the transcription of type I interferon by PRRSV. Marked differences in type I interferon induced antiviral activity against PRRSV between porcine alveolar macrophages and MARC-145 cells have also been reported recently [25] . Since MARC-145 cells are not the natural susceptible cells for PRRSV, the implication of the results in understanding the pathogenesis of PRRSV is debatable. Overall, the existing evidence clearly suggests that PRRSV may have intrinsic properties to inhibit or reduce the induction of interferon- and .
To further elucidate the molecular mechanisms by which PRRSV inhibits the induction of interferon- transcription in MARC-145 cells, Luo et al. examined the role of several key molecules in mediating the transcriptional activation of interferon- [26] . They reported that PRRSV interferes with the nuclear translocalization of IRF-3 by inactivating IPS-1, a downstream molecule of the RIG-I pathway [26] . Studies by Beura et al., suggested that the nonstructural proteins of PRRSV, including Nsp1, Nsp2, Nsp11 and Nsp4, when over-expressed in cell culture alone have the ability to antagonize the nuclear translocation of IRF-3 and the promoter activity of interferon- activated by Poly I:C and Sendai virus [27] . Similar studies have to some degree confirmed that over-expressed Nsp1 and Nsp2 of PRRSV in cell culture antagonize the promoter activity of interferon- as determined by using the luciferase reporter gene expression system [15, 28, 29, 30] . Sun et al. provided a detailed review on the role of PRRSV nonstructural and structural proteins in modulating the transcriptional activation of type I interferon in MARC-145 cells and human cell culture system including HeLa cells and 293T cells [12] . Despite the ease and convenience of using the luciferase reporter system to dissect the role of over-expressed viral proteins in regulating the transcription of interferon-, such results are often contradictory to the authentic virus infection of natural susceptible cells in vitro and in vivo [10, 16, 31] . Therefore, the implication of such results in natural virus infection is uncertain.
In 2004, Lee et al. first described the discrepancy between interferon- mRNA level and interferon- protein production in PRRSV-infected porcine alveolar macrophages [20] . They suggested that a post-transcriptional regulatory mechanism contributed to the inhibition of interferon- production. We have observed the same phenomenon in PRRSV-infected porcine monocyte-derived dendritic cells [32] . Despite the transient and abundant interferon- and  mRNA molecules, very little or no interferon- protein was detected in either cell lysates or supernatants of PRRSV-infected cells at different time points after virus infection, indicating a post-transcriptional control of type I interferon induction. Currently, very little is known about the translational control of type I interferon production by PRRSV. PRRSV inhibits the PI3K-dependent Akt (PI3K/Akt) pathway during late infection [33] , which makes the translation repressor, 4E-BP1, hyperactive and reduces global protein synthesis. Interestingly, a recent study has demonstrated that PI3K/Akt inhibition can also lead to the phosphorylation of eIF-2α to inhibit cellular translation [34] . We have indeed observed increased phosphorylation of eIF-2α in PRRSV-infected porcine Mo-DC during late infection (unpublished observation). Thus, it is possible that the translation of IFN-α is reduced partly by PI3K/Akt inhibition. However, it is more likely that PRRSV has employed multiple strategies to inhibit cellular protein synthesis as a simple way to evade the host defenses, which may also contribute to the inhibited type I interferon induction. In response to some virus infections, host dsRNA-activated protein kinase (PKR) phosphorylates eIF-2α to inhibit both cellular and viral translation [35, 36, 37] . PRRSV replication is known to trigger the stress-activated proteins kinases to modulate cytokine production in porcine alveolar macrophages [38] . It has also been demonstrated that cleavage of the eukaryotic translation initiation factor 4G (eIF4G) by either viral proteases or cellular proteases such as caspase 3 activated during apoptosis can result in rapid block of cellular protein synthesis [39, 40, 41, 42] . Furthermore, poly(A) tail elongation mediated by viruses may repress the efficient translation of type I interferon [43] .
Both UV-inactivated and heat-inactivated influenza A viruses are more potent than wild-type viruses in inducing the production of type I interferon [16] . Cytopathogenecity has been associated with translational shut-down of host genes including interferon [44] . PRRSV is highly pathogenic in alveolar macrophages and monocyte-derived dendritic cells and rapidly destroys these target cells by apoptosis and necrosis [2, 45, 46] . Studies have clearly shown that interferon mRNA has a short life span after induction [43, 47] . We speculate that the combination of high cytopathogenicity of virus and short half-life of interferon mRNAs may at least partially contribute to the low interferon proteins detected in virus-infected cells. It is also possible that the variability of different PRRSV isolates in inducing type I interferon is related to their varied cytopathogenicity [20, 23, 48] . For influenza A virus, different isolates can vary in their ability to induce interferon by up to 100-fold [16] . Finally, it remains to be determined whether PRRSV induces the shutoff of host protein synthesis to favor its own protein synthesis. More research efforts should be directed to the understanding of the translational control of type I interferon by PRRSV in its natural host cells.
PRRSV activates the transcription of type I interferon in porcine alveolar macrophages, peripheral blood mononuclear cells, and monocyte-derived dendritic cells. However, PRRSV interferes with the translation of type I interferon in these cells partly through cytopathogenicity since UV and heat-inactivated viruses lose their ability to interfere with the induction of type I interferon by porcine transmissible gastroenteritis virus or Poly I:C. Further studies are needed to delineate the exact mechanisms by which PRRSV interferes with the translation of type I interferon in its natural host cells. Clinical review: Special populations - critical illness and pregnancy Critical illness is an uncommon but potentially devastating complication of pregnancy. The majority of pregnancy-related critical care admissions occur postpartum. Antenatally, the pregnant patient is more likely to be admitted with diseases non-specific to pregnancy, such as pneumonia. Pregnancy-specific diseases resulting in ICU admission include obstetric hemorrhage, pre-eclampsia/eclampsia, HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, amniotic fluid embolus syndrome, acute fatty liver of pregnancy, and peripartum cardiomyopathy. Alternatively, critical illness may result from pregnancy-induced worsening of pre-existing diseases (for example, valvular heart disease, myasthenia gravis, and kidney disease). Pregnancy can also predispose women to diseases seen in the non-pregnant population, such as acute respiratory distress syndrome (for example, pneumonia and aspiration), sepsis (for example, chorioamnionitis and pyelonephritis) or pulmonary embolism. The pregnant patient may also develop conditions co-incidental to pregnancy such as trauma or appendicitis. Hemorrhage, particularly postpartum, and hypertensive disorders of pregnancy remain the most frequent indications for ICU admission. This review focuses on pregnancy-specific causes of critical illness. Management of the critically ill mother poses special challenges. The physiologic changes in pregnancy and the presence of a second, dependent, patient may necessitate adjustments to therapeutic and supportive strategies. The fetus is generally robust despite maternal illness, and therapeutically what is good for the mother is generally good for the fetus. For pregnancy-induced critical illnesses, delivery of the fetus helps resolve the disease process. Prognosis following pregnancy-related critical illness is generally better than for age-matched non-pregnant critically ill patients. an abnormally adherent placenta that implants in the uterine wall, usually in scar tissue following previous CS. With increasing severity there is placenta incretainvasion of the myometrium and placenta percreta -in which the placenta invades the extra-uterine pelvic tissues. Uterine rupture during labor is another potential complication and is infrequently associated with previous CS.
Postpartum hemorrhage (PPH) involves blood loss of greater than 500 mL within 24 hours regardless of the mode of birth. However, there is no universally accepted defi nition, and consideration should be given to physiologic response in addition to absolute blood loss. PPH is the most frequent indication for ICU admission. In 60% to 70% of cases, the cause of PPH is failure of uterine contraction following delivery. Th is uterine atony results in continuous bleeding that is often painless. Placental retention is the second most common cause of PPH (20% to 30% of cases). Genital trauma results in approximately 10% of cases of PPH and usually is associated with laceration of the vagina/cervix following instrumental delivery. Coagulation disorders may also result in PPH.
Th ese may be congenital -hemophilia or von Willebrand disease -or acquired: sepsis, amniotic fl uid embolus (AFE) syndrome, acute fatty liver of pregnancy, preeclampsia, or HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome.
An aggressive coordinated multidisciplinary approach between obstetricians, midwives, anesthesiologists, the labora tory, and blood bank is required (Figure 1 ). Initial management depends on the cause of the hemorrhage and whether delivery of the fetus has occurred. Where the hemorrhage occurs postpartum, uterine atony with or without retained products should be suspected. Oxytocin should be administered, the bladder should be emptied, and the uterus massaged. Intravenous access is obtained, blood is sent for crossmatching, and if crossmatched blood is unavailable, O negative blood is obtained. Th e obstetrician should examine the genital tract for evidence of trauma. If bleeding persists, prostaglandin therapy -either intravenous prostaglandin E 2 or 15-methyl prostaglandin F 2α -is administered. Consideration should be given to uterine tamponade, by packing or balloon compression. Continued bleeding should result in surgical intervention: arterial ligation, B-Lynch suture, Cesarean hyste rectomy, or uterine artery embolization [4] . It is important that these interventions occur without delay; with each advancing minute, continued blood loss leads to loss of clotting factors, coagulopathy, and the potential for exsanguination. It is imperative that the patient be kept warm and that blood products and fl uids be warmed through a rapid transfusion device. An arterial line should be placed to measure blood pressure and pulse pressure variability and to take blood samples to track hematocrit and acid-base status. Ideally, quali tative tests of clot quality (for example, thrombo elastography) would be combined with coagulation studies to guide blood component resuscitation. In the event of uncontrolled non-surgical bleeding, consideration should be given to the administration of recombinant factor VIIa (90 μg/kg, repeated at 20 minutes if there is no response) [5] [6] [7] .
Pre-eclampsia toxemia (PET) is a multisystem disease characterized by impaired organ perfusion resulting from vasospasm and activation of the coagulation system. PET is defi ned as hypertension and proteinuria appearing after the 20th week of gestation and resolving within 6 to 12 weeks of delivery. It occurs in 2% to 3% of all pregnancies and is more common in primigravida or the fi rst pregnancy with a particular partner. Other risk factors include a positive family history, pre-existing hyper tension, diabetes mellitus, multiple pregnancy, increasing maternal age, and obesity. Th e pathogenesis of PET appears to result from abnormal placenta formation (termed 'placentation'). Failure of the second phase of trophoblast invasion results in the lack of destruction of the muscularis layer of the spiral arterioles, impairing vasodilation in response to increases in blood fl ow, resulting in placental ischemia. Th e ischemic placenta releases vasoactive substances, leading to systemic endothelial injury and systemic organ dysfunction. As pregnancy progresses, placenta ischemia worsens, and the mother becomes hypovolemic and hypertensive and may develop renal dysfunction. Th ere is disordered prostaglandin metabolism, with an imbalance between vasoconstrictive thromboxane and vasodilatory prostacyclin, resulting in platelet dysfunction, endothelial damage, and further vasoconstriction.
PET is classifi ed as mild, moderate, or severe. Severe pre-eclampsia is defi ned as one of the following: (a) severe hypertension (systolic blood pressure of greater than 160 mm Hg or diastolic blood pressure of greater than 110 mm Hg), (b) proteinuria of greater than 5 g per 24 hours, (c) oliguria of less than 400 mL per 24 hours, (d) cerebral irritability, (e) epigastric or right upper quadrant pain (liver capsule distension), or (f ) pulmonary edema. PET is associated with signifi cant morbidity and mortality, for the mother and fetus. PET usually resolves following delivery of the fetus but may manifest postpartum. A variety of antihypertensive agents, including hydralazine, labetalol, sodium nitroprusside, alpha blockers, calcium channel blockers, and methyl dopa, have been advocated in PET. Hydralazine and labetalol are the most widely used of these in the critical care setting. Hydralazine is administered by slow intravenous injection: fi rst with 5 mg intravenously and repeated at 20-minute intervals (5 to 10 mg depending on the response). Once blood pressure control is achieved, hydrala zine can be repeated as needed (usually about every 3 hours). Labetolol is started at 20 mg intravenously as a bolus, and the dose is doubled every 10 minutes until blood pressure is under control. Th e maximum dose is 220 mg. Magnesium is usually co-administered to provide vaso dila tation and prevent seizures. Care should be taken with fl uid resuscitation because of the risk of pulmonary edema. Eclampsia is an extreme complication of PET and is defi ned by the occurrence of seizures in the absence of other neurologic disorders. Up to 40% of seizures occur following delivery. Convulsions are believed to result from severe intracranial vasospasm, local ischemia, intracranial hypertension, and endothelial dysfunction associated with vasogenic and cytotoxic edema. Seizures tend to be self-limiting, and status epilepticus is unusual. Magnesium sulphate (MgSO 4 ) is superior to phenytoin and benzodiazepines in the prevention of recurrent eclamptic seizures [8] . Magnesium toxicity is rare in the absence of renal failure. Respiratory arrest caused by magnesium toxicity can be reversed with calcium. While traditionally the mortality from eclampsia has been high, death is now uncommon, and much of the mortality is attributable to hepatic complications, including hepatic failure, hemorrhage, or infarction [9] .
HELLP syndrome is a constellation of fi ndings that include hemolysis with a microangiopathic hemolytic anemia (hemolysis), elevated liver function tests, and thrombo cytopenia (low platelets). HELLP syndrome complicates up to 3 per 1,000 pregnancies, may present as a severe manifestation of PET, and occurs in up to 20% of patients. Th e majority of patients are diagnosed before 37 weeks of gestation. Th ere is a clear overlap between PET and HELLP syndrome, and it is unclear whether the latter is a primary or secondary disease process. Th e syndrome is believed to be due to generalized endothelial and microvascular injury from activation of the complement and coagulation cascades, increased vascular tone, and platelet aggregation. Th is results in areas of hemorrhage and necrosis within the liver and may evolve to large hematomas, capsular tears, and intraperitoneal bleeding. Laboratory criteria for this syndrome include microangiopathic hemolysis with schistocytes present on the peripheral blood smear, platelet count of fewer than 50,000/mm 3 , serum total bilirubin of more than 20 μmol/L (1.2 mg/100 mL), serum lactate dehydrogenase of more than 600 U/L, and serum aspartate transaminase of more than 70 U/L.
Th e diff erential diagnosis includes thrombotic thrombo cytopenic purpura and hemolytic uremic syndrome, cold agglutinins, and acute fatty liver of pregnancy. In regard to distinguishing these conditions, it is likely that the patient with HELLP syndrome will have a more severe liver dysfunction, potentially complicated by hepatic infarction or subcapsular hematoma. HELLP syndrome is a medical emergency and the mother's blood pressure and coagulation status must be stabilized rapidly. Corticosteroids should be administered to the mother to advance the lung maturity of the fetus if the gestation age is below 34 weeks. In the majority of cases, liver, renal, and hematopoietic function normalizes after 5 days. In about 30% of cases, HELLP syndrome will develop in the postpartum period. Dexamethasone does not benefi t the mother with HELLP syndrome [10] . Delivery of the fetus can signifi cantly abrogate HELLP syndrome. Th e timing of delivery depends on fetal maturity and the severity of illness of the mother.
Th e major life-threatening complications of HELLP syndrome are hepatic hemorrhage, subcapsular hematoma, liver rupture, and multi-organ failure. Liver hemorrhage is managed conservatively where possible with aggressive blood product resuscitation to reverse the coagulopathy and ensure adequate oxygen-carrying capacity. Th e development of a subcapsular hematoma may lead to hepatic rupture, which is potentially life-threatening for the mother and fetus, and 50% maternal and up to 60% fetal mortality rates have been reported. Manage ment of threatened or actual hepatic rupture involves drainage of the hematoma, packing, oversewing of lacera tions, or partial hepatectomy. Consideration should be given to hepatic arterial embolization, either in the high-risk patient or in the period following operative stabilization.
Acute fatty liver disease of pregnancy (AFLP) occurs in about 1 per 10,000 pregnancies characterized by hepatic microvesicular steatosis and manifests in the third trimester. Without early diagnosis and treatment (fetal delivery), the patient may develop acute liver failure and hepatic encephalopathy. It is more common in primiparas, in twin pregnancies, and in patients who have preeclampsia. AFLP is a mitochondrial disorder [11] related to inherited mutations that cause a defi ciency of the long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD), a fatty acid beta-oxidation enzyme. When a heterozygous mother has a fetus that is homozygous for these mutations, the fetus is unable to metabolize longchain fatty acids; these acids accumulate in the fetus and spill over into the materna l circulation [12] . Th is mutant gene and defi cient coenzyme product leads to accumulation of long-chain fatty acid metabolites that are hepatotoxic [13] .
Th e patient usually presents with vague symptoms, vomiting, or abdominal pain and may develop preeclampsia. Often there are no specifi c clinical signs, except tenderness in the right upper quadrant. Serum aminotransferase and bilirubin levels are signifi cantly elevated, and in later stages there is a coagulopathy evidenced by low fi brinogen and a prolonged prothrombin time. Compared with HELLP, thrombocytopenia and hypertension are unusual. Urate levels may be extremely high and there may be signifi cant hypoglycemia. Conclusive diagnosis requires liver biopsy, although owing to coagulopathy thi s is rarely possible or practical. Th e diff erential diagnosis includes HELLP syndrome, pre-eclampsia, and acute hepatitis due to alcohol or a virus [14] . Th e treatment of choice is urgent delivery of the fetus. Th is stops the overload of the mother's fatty acid oxidation system from fetal production and leakage into the maternal circulation [13] .
AFE syndrome is a devastating complication that usually occurs within 24 hours of delivery. It manifests with acute severe hypoxic respiratory failure, associated with shock, disseminated intravascular coagulopathy (DIC), confusion, and seizures (Table 3 and Figure 2) . Th e incidence of AFE syndrome is unclear, and reports in the literature vary between 1:8,000 and 1:80,000 deliveries [15] . Th e disease is likely under-reported because of the absence of clear diagnostic criteria. Similarly, the mortality rate with AFE syndrome has been reported to be as high as 85%, and the majority of survivors suff er chronic neurologic defi cit [16] .
Th e pathophysiology of AFE syndrome is unclear. Previously, this syndrome was believed to result from embolization of amniotic fl uid into the pulmonary circulation. However, an anaphylactoid or hyper sensitivity reaction to the contents of this fl uid is more likely [17] . Th e presence of amniotic fl uid in the pulmonary circulation is neither sensitive nor specifi c, as this has been identifi ed in mothers who do not develop AFE syndrome. Patients may present with seizure-type activity or acute respiratory distress. Acute lung injury results in profound hypoxemia, intense hypoxic pulmonary vasoconstriction, and acute right heart failure (Table 4) , result ing in hemodynamic collapse. Bowing of the right ventricle into the left results in acute diastolic and then acute systolic failure of the left ventricle. Th ere is simultaneous DIC that may manifest with bleeding from the placental bed. Nausea, vomiting, headache, confusion, and seizures commonly follow. Death from AFE syndrome results from multi-organ failure, exsanguinations, or cardiac arrest. Neurologic injury is common in survivors.
Th ere is no diagnostic test for AFE syndrome. In a high-risk peripartum patient (Table 4 ) or one who has recently undergone termination of pregnancy (with hypertonic saline), the combination of coagulopathy, ARDS, and shock should be considered AFE syndrome until otherwise proven. Th e diff erential diagnosis includes sepsis, particularly due to chorioamniitis, thromboembolic pulmonary embolism, and aspiration pneumonitis. Th e fetus should be delivered emergently to avoid fetal demise. CS may be complicated by excess bleeding, requiring ligation of the uterine arteries and perhaps hysterectomy. Th ere is no specifi c treatment, although both aprotinin and activated protein C, compounds that modulate infl ammation and coagulation, may have some utility [18] . Critical care management should be directed at maintaining oxygen delivery and supporting the heart and circulation with inotropes and vasopressors. Early echocardiography is extremely useful to determine the nature of the cardiac injury (right versus left ventricular failure). Right ventricular failure can be worsened by high levels of positive end-expiratory pressure and vaso pressors and can be managed with milrinone, enoxamone, dobutamine, inhaled nitric oxide, and nebulized prostacyclin. Very large quantities of blood products may be required to control the coagulopathy, and this can result in signifi cant fl uid overload. Early consideration should be given to continuous renal replacement therapy. Although extracorporeal membrane oxygenation may appear to be an ideal approach to cardiopulmonary failure, excess bleeding may limit its application [19] .
Peripart um cardiomyopathy is defi ned as a dilated cardiomyopathy of unknown cause associated with pregnancy. It occurs in the last gestational month or in fi rst 5 months postpartum and is associated with no other cardiac disease [20] . Although this condition is relatively rare, its exact incidence is unclear (the reported incidence varies between 1 in 1,500 and 1 in 15,000 pregnancies), and the condition is more common among Africans and Haitians. It is associated with older maternal age, obesity, multiparity, multiple pregnancies, and pregnancy-induced hypertension. Th e patient typically presents with symptoms of congestive heart failure: dyspnea, orthopnea, shortness of breath on exertion, upper abdominal pain, and so on. Echocardiography demonstrates systolic dysfunction. Proposed pathogenic hypotheses for peripartum cardiomyopathy include viral myocarditis, auto-immunemediated injury, and prolonged tocolysis. Current evidence strongly suggests that the disease is triggered by a by-product of prolactin metabolism, resulting in unbalanced peri-/postpartum oxidative stress [21] . Th e hormone is proteolytically cleaved, and an antiangio-genic, proapoptotic, and proinfl ammatory 16-kDa byproduct appears to attack the myocardium [22] .
Medical therapy is eff ective in the majority of patients; treatment is commenced with a loop diuretic, and if the patient is postpartum, an angiotensin-converting enzyme inhibitor or antiotensin receptor blocker is added. In severe cases, placement of an intra-aortic balloon counter pulsation device or extracorporeal membrane oxygenation [23] may be necessary. Th ere is a very high incidence of thromboembolic disease associated with peripartum cardiomyopathy, and anticoagulation is essential. Early experience with prolactin inhibitors, such as bromocriptine, appears to be positive, and this may become the mainstay of treatment in the future [24] .
Mortality appears to be high; in the US, mortality occurs in 25% to 50% of cases, usually within 3 months of diagnosis [25] . Availability of quality health care for the vulnerable population may be a component of this. Approximately 50% of women recover their ventricular function within 6 months of delivery. In some cases, cardiac trans plantation is necessary. Despite recovery, cardiomyopathy may recur in subsequent pregnancies.
Pregnancy predisposes women to four specifi c infectious complications: pyelonephritis, chorio amnionitis (including septic abortion), endometritis (often following Cesarean delivery), and pneumonia. Pyelonephritis results from colonization of the kidney with Gram-negative bacteria secondary to loss of ureteral sphincter tone associated with progesterone. Pneumonia results, at least in part, from aspiration of gastric contents as a consequence of loss of lower esophageal sphincter tone and diaphragmatic elevation. Patients are also at elevated risk for viral and fungal pneumonia due to pregnancy-induced immunosup pres sion. Chorioamnionitis results from altera tions in the pH and increased glycogen content of the vagina, resulting in loss of the barrier for bacterial entry. It may complicate chorionic villus sampling, amnio centesis, or attempted instrumental (septic) abortion.
Bacteremia in pregnancy is relatively common (reportedly occurring in 8% to 9% of pregnancies), whereas progression to severe sepsis and septic shock is relatively rare [26] [27] [28] [29] ; the rate of sepsis ranges from 1 in 7,654 to 1 in 8,338 deliveries reported [30] . Kankuri and colleagues [31] reported that only 1 of 43,483 mothers developed septic shock during the peripartum period. Despite these low progression rates, the mortality from sepsis in pregnancy remains signifi cant. Infections may be Gramnegative, Gram-positive, or rarely anerobic in etiology. Th e most commonly isolated organisms are Escherichia coli, enterococci, and beta hemolytic streptococci.
Th e majority of infections occur postpartum; 'puerpural sepsis' or 'puerperal fever' is an umbrella term for a variety of infections that occur in the puerperium. Th e leading risk factor for puerpural sepsis is Cesarean delivery. Other signifi cant risk factors include retained products of conception, episiotomy, and prolonged rupture of the amniotic membranes. Infection may involve endometritis, parametritis (spread through the uterine wall), peritonitis, or thrombophlebitis of the pelvic veins. Endometritis is most commonly associated with group A streptococcal (GAS) infection, although Staphylococus, coliform, and anerobes may also be present. Hand washing and disinfectants dramatically decreased the incidence of puerperal fever. Pre-emptive antibiotics are administered if prolonged rupture of the membranes has occurred, to treat amnionitis, or if the woman has a fever and a foul-smelling vaginal discharge.
Th e most recent maternal mortality report from the UK revealed that sepsis was the leading cause of maternal death between 2006 and 2008 [32] . Sepsis resulted in 29 maternal deaths. Fifty percent of deaths occurred following Cesarean delivery, whereas 7 occurred after vaginal delivery. One third of these deaths occurred before 24 weeks of gestation. Th ere was a marked seasonal pattern; most deaths occurred between December and April. Associated factors included minority ethnic origin and the presence of sickle cell disease or trait. Interestingly, obesity was not a risk factor. Infant mortality in aff ected pregnancies was 45%. GAS was the principal pathogen, causing nearly 50% of deaths. All mothers who died from GAS either worked with or had children, and most of the mothers had a direct or family history of sore throat or respiratory infection. In the developing world, the mortality rate from puerperal sepsis remains extremely high (greater than 70%) and tetanus is a common cause of infection.
Th e clinical manifestations of sepsis include the hallmarks of systemic infl ammation that may be followed by coagulopathy, vasoplegia, and evolving multiple organ failure. Th e patient typically has a temperature of 38°C during the period from the end of the fi rst to the end of the 10th day after childbirth or abortion. Purpura fulminans may be associated with GAS infection. Physicians must have a high index of suspicion for sepsis in any peripartum patient who presents with fever and evidence of organ dysfunction: confusion, oliguria, tachycardia, and so on. Treatment includes fl uid resuscitation, empiric antibiotic therapy, and source control. Early aggressive volume resuscitation is essential and is followed with vasopressor therapy if necessary. Th ere is no evidence to suggest that norepinephrine has an adverse eff ect on fetal well-being. Th ere are no data on the use of vasopressin during pregnancy. Considering current data and the specifi c exclusion of pregnant (but not postpartum) patients from the PROWESS (Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis) trial and other trials, we cannot recommend the use of activated protein C during pregnancy. However, this agent may be of value in the postpartum period and clinicians should weigh up its value as a potent anti-infl ammatory/anticoagulant agent versus the risk of bleeding complications.
Goal-directed resuscitation of the pregnant patient is recommended. One must be aware of physiologic changes and adjust the goals of the resuscitation accordingly. Although central venous pressure remains essentially unchanged during pregnancy, venous oxygen saturation (SvO 2 ) progressively decreases in the later stages. Hence, achieving goals of 70% to 75% may not be possible -or necessary -in this setting. Antimicrobials are administered on the basis of the 'best guess' source of infection. In general, tetracycline and quinolone antibiotics should be avoided in early pregnancy. Penicillins, macrolides, and cephalosporins appear relatively safe within normal dosage range.
When cardiac arrest occurs in late pregnancy, cardiopulmonary resuscitation (CPR) is diffi cult [33] . Th e airway should be secured without delay, and the patient should be positioned to ensure left lateral displacement of the uterus. Unfortunately, this results in less eff ective CPR [33] . Drugs and defi brillation regimens should follow standard Advanced Cardiovascular Life Support guidelines. A decision regarding Cesarean delivery should be made immediately. Current data suggest that this should be performed within 5 minutes, to ensure viability of the mother [34] . Extraction of the fetus results in an increase in maternal blood volume and release of aortocaval compression [35] . Th is should be undertaken from the second trimester onwards. If the decision to perform Cesarean delivery is made, there should be no delay and CPR should be continued throughout surgery. Maternal brain death, due, for example, to an intracranial bleed, raises diffi cult ethical and legal issues [36, 37] . Depending on the gestational age, it may be possible, if the fetus is near viability, to maintain maternal organ support to allow the fetus to reach viability [38] . Although it may appear expensive to sustain the vegetative state of the brain-dead mother, this cost may be signifi cantly less than the long-term cost of care of a severely premature neonate, which would require neonatal ICU and a variety of health-care interventions.
Critical illness is an uncommon but potentially devastating complication of pregnancy. Th e fi rst priority for the intensivist is to stabilize the mother, with the understanding that what is good for the mother is good for the fetus. Th e intensivist must be aware of the physiologic changes associated with pregnancy and their timing. Critical care interventions are similar to those for the non-pregnant patient; however, adjustment of physiologic targets for metabolic, pulmonary, and hemodynamic control may be necessary. Th e majority of acquired pregnancy-related diseases, such as PET, AFLP, and cardiomyopathy, are abrogated by delivery. However, timing and the state of fetal maturation are crucial to decision making. PaCO(2 )and alveolar dead space are more relevant than PaO(2)/FiO(2 )ratio in monitoring the respiratory response to prone position in ARDS patients: a physiological study INTRODUCTION: Our aims in this study were to report changes in the ratio of alveolar dead space to tidal volume (VD(alv)/V(T)) in the prone position (PP) and to test whether changes in partial pressure of arterial CO(2 )(PaCO(2)) may be more relevant than changes in the ratio of partial pressure of arterial O(2 )to fraction of inspired O(2 )(PaO(2)/FiO(2)) in defining the respiratory response to PP. We also aimed to validate a recently proposed method of estimation of the physiological dead space (VD(physiol)/V(T)) without measurement of expired CO(2). METHODS: Thirteen patients with a PaO(2)/FiO(2 )ratio < 100 mmHg were included in the study. Plateau pressure (Pplat), positive end-expiratory pressure (PEEP), blood gas analysis and expiratory CO(2 )were recorded with patients in the supine position and after 3, 6, 9, 12 and 15 hours in the PP. Responders to PP were defined after 15 hours of PP either by an increase in PaO(2)/FiO(2 )ratio > 20 mmHg or by a decrease in PaCO(2 )> 2 mmHg. Estimated and measured VD(physiol)/V(T )ratios were compared. RESULTS: PP induced a decrease in Pplat, PaCO(2 )and VD(alv)/V(T )ratio and increases in PaO(2)/FiO(2 )ratios and compliance of the respiratory system (Crs). Maximal changes were observed after six to nine hours. Changes in VD(alv)/V(T )were correlated with changes in Crs, but not with changes in PaO(2)/FiO(2 )ratios. When the response was defined by PaO(2)/FiO(2 )ratio, no significant differences in Pplat, PaCO(2 )or VD(alv)/V(T )alterations between responders (n = 7) and nonresponders (n = 6) were observed. When the response was defined by PaCO(2), four patients were differently classified, and responders (n = 7) had a greater decrease in VD(alv)/V(T )ratio and in Pplat and a greater increase in PaO(2)/FiO(2 )ratio and in Crs than nonresponders (n = 6). Estimated VD(physiol)/V(T )ratios significantly underestimated measured VD(physiol)/V(T )ratios (concordance correlation coefficient 0.19 (interquartile ranges 0.091 to 0.28)), whereas changes during PP were more reliable (concordance correlation coefficient 0.51 (0.32 to 0.66)). CONCLUSIONS: PP induced a decrease in VD(alv)/V(T )ratio and an improvement in respiratory mechanics. The respiratory response to PP appeared more relevant when PaCO(2 )rather than the PaO(2)/FiO(2 )ratio was used. Estimated VD(physiol)/V(T )ratios systematically underestimated measured VD(physiol)/V(T )ratios. Since its first description in 1967 [1] , it has been accepted that acute respiratory distress syndrome (ARDS) includes a number of lung injuries of various origins whose consequences are decreased lung capacity available for ventilation, leading to the concept of "baby lung" [2] . Considerable progress has been made over the past decade in the ventilatory management of patients with ARDS. In particular, a strict limitation of tidal volume (V T ) and plateau pressure (Pplat) below 30 cmH 2 O reduces mortality [3] . The application of positive end-expiratory pressure (PEEP) is recognized to recruit the lung and to restore functional residual capacity [4] , but its optimum level is still widely debated [5] .
The prone position (PP) may also be part of the ventilatory strategy. This method was proposed more than 30 years ago, initially in pathophysiological studies [6, 7] . Recently, Sud et al. [8] suggested, on the basis of pooled data from randomized, controlled trials, that PP may improve survival in the subgroup of patients with the most severe ARDS, that is, those with a ratio of partial pressure of arterial O 2 to fraction of inspired O 2 (PaO 2 / FiO 2 ) < 100 mmHg. Many questions remain unresolved. In particular, response to PP is usually defined according to changes in PaO 2 , with responders being those in whom the PaO 2 /FiO 2 ratio increases > 20 mmHg after one to six hours in the PP [9] [10] [11] . However, we have previously reported that PP allows recruitment of a slow compartment previously excluded from ventilation [12] . This was associated with a decrease in partial pressure of arterial CO 2 (PaCO 2 ), an indirect reflection of the reduction of the alveolar dead space (VD alv ) [12] . Gattinoni et al. [10] also reported that the prognosis is improved in patients in whom PaCO 2 declines after an initial PP session. Finally, VD alv appears to be an independent risk factor for mortality in patients with ARDS [13] . In a recent study, Siddiki et al. [14] proposed evaluating the physiological dead space fraction (VD physiol / V T ) by using a rearranged alveolar gas equation for PaCO 2 without any expired CO 2 measurement.
In this context, we conducted a prospective physiological study to evaluate the impact of PP on ventilatory mechanics, gas exchange and VD alv . Our main objective was to validate our hypothesis that changes in PaCO 2 and VD alv might be more relevant than changes in PaO 2 in defining the respiratory response to PP. Our second objective was to validate the method of evaluation of the VD physiol /V T proposed by Siddiki et al. [14] .
In our unit, patients with a PaO 2 /FiO 2 ratio < 100 mmHg after 24 to 48 hours of mechanical ventilation are systematically turned to PP when hemodynamically stable [15] . Our study was approved by the Ethics Committee of the "Société de Réanimation de Langue Française" (SRLF-CE 07-213). After obtaining informed consent from the patients' relatives, 15 patients were included in the study between January 2008 and March 2010. Inclusion criteria were (1) the presence of ARDS according to the definition of the Acute Respiratory Distress Syndrome Network [3] ; (2) persistence of severe hypoxemia after 48 hours of mechanical ventilation, defined as a PaO 2 /FiO 2 ratio < 100 mmHg; and (3) hemodynamic stability, defined as systolic blood pressure > 90 mmHg with norepinephrine infusion at a rate < 0.5 μg/kg/minute. Patients with chronic obstructive pulmonary disease were excluded.
All patients were ventilated in volume-controlled mode (Servo-i; Maquet SA, Ardon, France), sedated and paralyzed by infusion of atracurium. The heat and moisture exchanger was routinely removed and replaced by a heated humidifier to reduce instrumental dead space as previously reported [16] . The ventilator settings included a "moderately restricted" V T of 6 to 8 mL/kg measured body weight, a respiratory rate allowing us to limit hypercapnia without generating intrinsic PEEP and an inspiration/expiration ratio of 1:2 with an end inspiratory pause of 0.5 seconds. Pplat was strictly limited < 30 cmH 2 O, and the PEEP selected was that which corrected the intrinsic PEEP, if any [17] . Ventilator settings were kept constant throughout the study. A recruitment maneuver was never used, and suction was not systematically performed. All patients were continuously monitored in terms of blood pressure with an arterial catheter, heart rate and O 2 saturation by pulse oximetry.
The study was conducted during the first session of PP. Our sessions routinely last 15 to 18 hours per day. Blood gas analysis, Pplat, total PEEP, end-tidal CO 2 (P etCO2 ) and mixed expired CO 2 (P ECO2 ) were recorded with the patient in the supine position, just before turning the patient to the PP, and every 3 hours in the PP until 15 hours had elapsed. Expired CO 2 was measured by a sensor positioned between the proximal end of the endotracheal tube and the Y piece of the ventilator circuit (COSMO; Novametrix, Wallingford, CT, USA). The ratio of VD/V T was calculated using the simplified Bohr equation [18] as follows: (1) VD alv /V T = 1 -P etCO2 / PaCO 2 and (2) VD physiol /V T = 1 -P ECO2 /PaCO 2 .
The estimated VD physiol /V T ratio was calculated as 1 -[(0.86 × VCO 2est )/(VE × PaCO 2 )], where VCO 2est is the estimated CO 2 production calculated using the Harris-Benedict equation [19] and VE is the expired minute ventilation.
Intrinsic PEEP was measured during a four-second end-expiratory occlusion period. Pplat was measured during a 0.5-second end-inspiratory pause. Respiratory system compliance (Crs) was calculated as Crs = V T / (Pplat -PEEP total ). Responders to PP were defined in two different ways: (1) an increase in PaO 2 /FiO 2 ratio > 20 mmHg after 15 hours of PP or (2) a decrease in PaCO 2 > 2 mmHg after 15 hours of PP.
Statistical analysis was performed using StatView 5 software (SAS Institute Inc., Cary, NC, USA). The continuous variables were expressed as medians (1st to 3rd interquartile range). Analysis of variance for repeated measurements was used for each parameter, and P < 0.05 was considered statistically significant. Measured VD physiol /V T and estimated VD physiol /V T were compared according to Bland-Altman analysis, together with the concordance correlation coefficient in 78 paired data. The same method was used to compare variations of measured and estimated VD physiol /V T every three hours while the patient was in PP.
Two patients were excluded from the study because of a history of severe chronic obstructive pulmonary disease, which left a study population of 13 patients. The patients' median age was 53 years (1st to 3rd interquartile range, 48 to 59 years), their median Simplified Acute Physiology Score II score was 62 (1st to 3rd interquartile range, 35 to 71) and their median Sequential Organ Failure Assessment score was 11 (1st to 3rd interquartile range, [8] [9] [10] [11] [12] [13] . All patients except one had ARDS of pulmonary origin. Eight patients had pneumonia, with six cases related to streptococcus pneumonia and two due to influenza (H1N1 virus). Two patients had aspiration, one had toxic shock syndrome and two had ARDS due to miscellaneous causes. No patient had abdominal hypertension or traumatic lung injury. Eleven patients required norepinephrine infusion. Respiratory parameters and blood gas analysis at the time of inclusion are reported in Table 1 .
A significant increase in PaO 2 /FiO 2 ratio occurred after 15 hours of PP, from 70 mmHg (51 to 77) in the supine position to 99 mmHg in the prone (83 to 139) (P < 0.0001) ( Table 2) . A significant decrease in PaCO 2 was also observed, from 58 mmHg (52 to 60) to 52 mmHg (47 to 56) (P = 0.04) ( Table 2) , with the lowest value occurring after nine hours of PP. As noted in Table 2 , Pplat was significantly reduced (P = 0.0004) and Crs improved (from 16 mL/cmH 2 O (13 to 30) to 18 mL/ cmH 2 O (15 to 30); P = 0.02). Finally, the VD alv /V T ratio was significantly reduced from 0.42 (0.35 to 0.47) to 0.40 (0.26 to 0.45), with the lowest value occurring after three hours in PP (hour 3) (0.31) ( Table 2) .
Seven patients were classified as "PaO 2 responders" and six were classified as "PaO 2 nonresponders" according to PaO 2 /FiO 2 ratio changes. No differences in VD alv /V T ratios or PaCO 2 or Pplat alterations during PP were observed between groups (Table 3 and Figure 1 ), whereas Crs increased more in the responders (Table 3) . Seven patients were also classified as "PaCO 2 responders" and six as "PaCO 2 nonresponders" according to the PaCO 2 changes. However, when compared with the PaO 2 /FiO 2 classification, four patients were classified differently. As shown in Table 4 and Figure 2 , VD alv /V T , PaO 2 /FiO 2 , PaCO 2 , Pplat and Crs were significantly more altered in responders than in nonresponders. As shown in Figure 3 , we found no correlation between changes in VD alv /V T and changes in PaO 2 /FiO 2 (P = 0.95), whereas we found a negative correlation between changes in VD alv /V T and changes in Crs (r = 0.29, P = 0.03).
As shown in Figure 4 , estimated VD physiol /V T systematically underestimated measured VD physiol /V T , with a poor concordance correlation coefficient of 0.19 (95% confidence interval (95% CI) 0.091 to 0.28), a bias of 0.16 and an agreement between -0.05 and 0.37. Concerning changes in VD physiol /V T during PP, estimated VD physiol /V T had a concordance correlation coefficient of 0.51 (95% CI 0.32 to 0.66) (Figure 4 ).
One of the objectives of our study was to describe alterations in VD alv induced by PP. ARDS is characterized by a heterogeneous lung with the existence of a slow compartment [18, 20] , defined as areas available for, but partially or totally excluded from, ventilation due in part to a bronchiolar collapse [12, 21] . In a previous study, we reported that PP may induce recruitment of this slow compartment, as suggested by its ability to counteract intrinsic PEEP and to decrease the expiratory time constant [12] . In the same study, we also reported that PP leads to a decrease in PaCO 2 , suggesting diminution of VD alv (alveolar dead space) [12] . Our present study demonstrates that PP may induce a decrease in VD alv . It occurred from the third hour and was maintained throughout the PP session. VD alv may be the consequence of nonperfused or poorly perfused lung areas in ventilated anterior areas, but also of a slow compartment partially excluded from ventilation. Our results suggest that PP induces functional lung recruitment, especially since decreases in VD alv related to PP were associated with a decrease in Pplat and strongly correlated with improvement in compliance. did not report a decrease in Pplat in PP, as we found, but after returning patients to the supine position [22] . This could be explained by the fact that they used roll under the upper part of the chest wall, leading to a significant impairment in chest wall compliance [22] , whereas we did not. The most beneficial reported effect of PP is oxygenation improvement [24, 25] . However, this better oxygenation can be due to (1) lung recruitment related to restoration of functional residual capacity [7] and improvement of the diaphragmatic movement in the posterior part [26] [27] [28] or (2) simply to an improvement in the ventilation/perfusion ratio due to a decreased hydrostatic gradient between the anterior and posterior parts of the lung [26, 29] . Whereas the first mechanism is crucial, one can say that the second mechanism is less important. This is why the second objective of our study was to test whether the response to PP in terms of PaCO 2 was physiologically more relevant than in terms of PaO 2 /FiO 2 ratio. Gattinoni et al. [10] reported that an increase in PaO 2 /FiO 2 ratio > 20 mmHg after six hours of PP is not predictive of the patient's prognosis, whereas a decline in PaCO 2 ≥1 mmHg is. In our present study, 7 of 13 patients were PaO 2 responders (increased PaO 2 /FiO 2 ratio > 20 mmHg after 15 hours of PP). However, changes in Pplat, PaCO 2 and VD alv did not differ between PaO 2 responders and PaO 2 nonresponders. On the other hand, 7 of 13 patients were PaCO 2 responders (decreased PaCO 2 > 2 mmHg after 15 hours of PP). PaCO 2 responders had a significant decrease in Pplat and VD alv , as well as a significant increase in oxygenation and compliance, compared with nonresponders. Our results are in accordance with a recent study of 32 ARDS patients [23] , in which the investigators reported that PaCO 2 variation induced by PP, and not PaO 2 /FiO 2 variation, is associated with lung recruitability. Interestingly, in our study, changes in VD alv were not correlated with changes in oxygenation but were strongly correlated with changes in compliance of the respiratory system.
An unexpected result of our work concerns the change over time of respiratory mechanics, blood gas analysis and VD alv . For many years, our PP protocol has been to turn patients to PP for up to 15 to 18 hours per day for 3 days [15] . In the study by Mancebo et al. [30] , which concluded that PP may reduce mortality in patients with severe ARDS, PP sessions lasted 20 hours/ day. In a recent study, we demonstrated that PP sessions that lasted 18 hours/day were independently associated with survival [31] . In the present study, the maximum effect of PP for VD alv , PaCO 2 and Pplat occurred six to nine hours after turning patients to PP. Later the effect seemed to be a decline. How this affects the effect of PP on patient prognosis remains to be elucidated.
The second objective of our study was to validate a recently proposed method to evaluate the VD physiol /V T ratio [14] . The method is based on CO 2 production calculated from the Harris-Benedict equation [19] and on the expired minute ventilation. Siddiki et al. [14] reported that it was associated with mortality in acute lung injury patients in a dose-response manner and proposed its routine use to estimate VD physiol /V T . However, they did not report any comparison with measured VD physiol /V T . In the present study, we have demonstrated that this method significantly underestimates VD physiol /V T , rendering it not accurate enough to assess the degree of lung injury. Interestingly, changes in estimated VD physiol /V T during PP appeared better correlated with changes in measured VD physiol /V T and could be proposed in the future in this field. Siddiki et al. [14] proposed the method in the context of a much larger series than ours and in patients with less severe ARDS, rendering it difficult to draw any definitive conclusions. Our work is limited by the small number of patients included. This is a consequence of our routine protocol, which strictly restricts PP to patients with the most severe ARDS, that is, those with a PaO 2 /FiO 2 ratio < 100 mmHg after 48 hours of ventilation. This also explains why it is not possible to link our results to outcomes. However, despite this limitation, we consider our results relevant from a physiological point of view.
In conclusion, our study demonstrates that PP induces a decrease in PaCO 2 and VD alv . This is related to an improvement in respiratory mechanics, with a decrease in Pplat and an increase in compliance. Testing the response to PP appeared to be physiologically more relevant using PaCO 2 changes than PaO 2 /FiO 2 changes. How this may affect management at the bedside remains to be studied. Estimated VD physiol /V T ratios systematically underestimated measured VD physiol /V T ratios.
• PP induced a decrease in VD alv /V T , which was correlated with an improvement in respiratory mechanics.
• Defining the respiratory response to PP appeared more relevant when using PaCO 2 changes rather than PaO 2 /FiO 2 changes. • Estimated VD physiol /V T using the Harris-Benedict equation systematically underestimated measured VD physiol /V T .
Abbreviations ARDS: acute respiratory distress syndrome; P ECO2 : mixed expired PCO 2 ; PEEP: positive end-expiratory pressure; P etCO2 : end-tidal PCO 2 ; PP: prone position; Pplat: plateau pressure; VD alv : alveolar dead space; VD physiol : physiological dead space. Lung Function and Organ Dysfunctions in 178 Patients Requiring Mechanical Ventilation During The 2009 Influenza A (H1N1) Pandemic INTRODUCTION: Most cases of the 2009 influenza A (H1N1) infection are self-limited, but occasionally the disease evolves to a severe condition needing hospitalization. Here we describe the evolution of the respiratory compromise, ventilatory management and laboratory variables of patients with diffuse viral pneumonitis caused by pandemic 2009 influenza A (H1N1) admitted to the ICU. METHOD: This was a multicenter, prospective inception cohort study including adult patients with acute respiratory failure requiring mechanical ventilation (MV) admitted to 20 ICUs in Argentina between June and September of 2009 during the influenza A (H1N1) pandemic. In a standard case-report form, we collected epidemiological characteristics, results of real-time reverse-transcriptase--polymerase-chain-reaction viral diagnostic tests, oxygenation variables, acid-base status, respiratory mechanics, ventilation management and laboratory tests. Variables were recorded on ICU admission and at days 3, 7 and 10. RESULTS: During the study period 178 patients with diffuse viral pneumonitis requiring MV were admitted. They were 44 ± 15 years of age, with Acute Physiology And Chronic Health Evaluation II (APACHE II) scores of 18 ± 7, and most frequent comorbidities were obesity (26%), previous respiratory disease (24%) and immunosuppression (16%). Non-invasive ventilation (NIV) was applied in 49 (28%) patients on admission, but 94% were later intubated. Acute respiratory distress syndrome (ARDS) was present throughout the entire ICU stay in the whole group (mean PaO(2)/FIO(2 )170 ± 25). Tidal-volumes used were 7.8 to 8.1 ml/kg (ideal body weight), plateau pressures always remained < 30 cmH(2)O, without differences between survivors and non-survivors; and mean positive end-expiratory pressure (PEEP) levels used were between 8 to 12 cm H(2)O. Rescue therapies, like recruitment maneuvers (8 to 35%), prone positioning (12 to 24%) and tracheal gas insufflation (3%) were frequently applied. At all time points, pH, platelet count, lactate dehydrogenase assay (LDH) and Sequential Organ Failure Assessment (SOFA) differed significantly between survivors and non-survivors. Lack of recovery of platelet count and persistence of leukocytosis were characteristic of non-survivors. Mortality was high (46%); and length of MV was 10 (6 to 17) days. CONCLUSIONS: These patients had severe, hypoxemic respiratory failure compatible with ARDS that persisted over time, frequently requiring rescue therapies to support oxygenation. NIV use is not warranted, given its high failure rate. Death and evolution to prolonged mechanical ventilation were common outcomes. Persistence of thrombocytopenia, acidosis and leukocytosis, and high LDH levels found in non-survivors during the course of the disease might be novel prognostic findings. On April 2009, a novel influenza A (H1N1) virus emerged in Mexico and spread rapidly across the world [1, 2] . As of 17 June 2010, more than 214 countries had reported confirmed cases of infection with pandemic 2009 influenza A (H1N1) virus, including at least 18,156 deaths [3] . Unlike seasonal influenza, in which hospitalizations occur among patients younger than 2 and older than 65 years, or in those with underlying diseases [4] , this novel virus affected otherwise healthy young and middle-aged adults and obese individuals [2, 5] . Patients with previous respiratory disease, immunocompromised hosts and pregnant women were affected as frequently as with seasonal influenza [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Although a mild form of the disease was prevalent, it soon became evident that the 2009 influenza A (H1N1) virus could also provoke severe, acute respiratory failure requiring admission to the intensive care unit (ICU) for mechanical ventilation [16] , which was reflected in the severe pathological injury found at autopsy [17] .
The Argentinian population was greatly affected during the pandemic, with a total of 1,390,566 cases of influenza-like illness requiring 14,034 hospitalizations. Of the 11,746 confirmed cases of patients infected with the new strain, 617 died [18] . This represents a death rate per infection of 4.3% in hospitalized cases; an intermediate figure compared to 3.6% in Brazil, 1.2% in Chile, and approximately 6% in Uruguay, Colombia and Venezuela [19] . It should be noted that these numbers reflect great uncertainty, particularly with regard to case diagnosis. Lack of testing of mild disease and difficulties due to laboratory overload have also been well described [15, 20] . These general problems have been acknowledged by experts [21] .
The severity of disease was rapidly perceived by health authorities and scientific societies. Hence, a committee of experts of the Argentinian Society of Intensive Care Medicine decided to focus on the most acutely ill patients: those presenting with diffuse viral pneumonitis requiring mechanical ventilation. They designed an epidemiological study, recently-published, to determine risk factors and outcomes [15] ; this is one of many series up to the present that have described epidemiological and clinical aspects of the 2009 influenza A (H1N1) pandemic [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .
There remains, however, a paucity of data published on physiological evolution during ICU stay [22] . This present study, concurrently planned with the first by the same committee of experts, thus aims to provide such information. Our objectives were: first, to characterize alterations of oxygenation, respiratory mechanics and the use of mechanical ventilation; second, to explore compliance with protective lung ventilation; and, finally, to assess the evolution of laboratory findings and organ dysfunctions throughout the course of the disease.
This was a multicenter, inception cohort study that included patients aged > 15 years admitted to the ICU with a previous history of influenza-like illness, evolving to acute respiratory failure that required mechanical ventilation during the 2009 winter in the Southern Hemisphere. These patients had confirmed or probable disease caused by the 2009 influenza A (H1N1) virus and were included in the Registry of Cases of the Argentinian Society of Intensive Care Medicine (SATI), created to characterize local aspects of the pandemic. On 27 June 2009, a form to collect online epidemiological data was posted on the official SATI website. A detailed description and analysis of this information was recently published [14] .
There was also an optional, more comprehensive casereport form to complete, developed by experts of the SATI's Respiratory Committee for recording certain prespecified variables throughout ICU stay, which included mechanical ventilation (MV), respiratory mechanics, oxygenation, blood chemistry and organ failure variables. This information was collected over 10 days and is analyzed in the present study.
Patients were characterized as confirmed, probable or possible cases of 2009 influenza A (H1N1) [20] according to the findings in the respiratory samples collected on admission. Some specimens, however, were not analyzed because laboratories soon became overloaded, especially at the beginning of the pandemic. As of 25 September 2009, the weekly update of the Ministry of Health reported that in patients ≥5 years with influenzalike illness, the 2009 influenza A (H1N1) virus had displaced other respiratory viruses in 93.4% of the samples processed [23, 24] . As a result of this, probable and suspected cases were considered as caused by the novel virus and were so included in the study.
We collected dates of hospital and ICU admission, and of MV onset; demographics; risk factors for influenza A; actual weight; height; severity of illness (Acute Physiology And Chronic Health Evaluation II, APACHE II), organ failures (Sequential Organ Failure Assessment, SOFA); type of MV used, as noninvasive (NIV) and invasive; and date of intubation. Ideal body weight (IBW, ml/kg) and body mass index (BMI) were calculated; obesity was defined as a BMI > 30.
At MV onset (Day 0) and on Days 3, 7 and 10, until death or discharge, whichever occurred first, we recorded: (1) MV-related variables. (2) MV modes: volume-controlled ventilation (VCV); pressure-controlled ventilation (PCV); bilevel mode; pressure support ventilation (PSV); other. (3) Tidal volume (Vt, in ml/kg of IBW) (4) Pressures: peak, plateau pressures, total positive end-expiratory pressure (PEEP) and driving pressure (plateau pressure -PEEP), in cmH2O. The main outcome measure was hospital mortality; secondary outcomes were length of MV, of ICU (LOSICU) and of hospital (LOSHOSP) stays.
In case of missing observations, local study coordinators were contacted to provide the corresponding values. Proportions were calculated as percentages of existing data.
No assumptions for missing data were made.
Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed for the entire population; for the subgroups of survivors vs. non-survivors; and for patients receiving NIV on admission vs. those who did not. Descriptive statistics used were: mean ± standard deviations (SD) and median and 25-75% interquartile ranges (IQR) for continuous data of normal and non-normal distribution, respectively; and percentages for categorical data. Differences between subgroups were analyzed with unpaired t test, Mann-Whitney U test, and Chi-square tests, as appropriate. A P-value of <.05 was considered statistically significant. A Kaplan-Meier curve was constructed to evaluate survival over the follow-up period. Over time, normally distributed data were analyzed with two-way repeated measures of ANOVA. At the pre-specified time points, differences within the entire group and subgroups, and between subgroups, were tested using paired and unpaired t tests, respectively.
In non-normally distributed data, differences over time within the entire group and the subgroups were analyzed with Friedman's and Wilcoxon tests. Comparisons between subgroups at the pre-specified time points were tested with Mann-Whitney U test. The Bonferroni correction was used to adjustments for multiple comparisons.
The local Institutional Review Boards waived the need for informed consent, given the general lack of knowledge on the clinical and outcome characteristics of the ongoing pandemic and to the non-interventional study design.
General characteristics (Table 1) Between 6 June and 28 August 2009, the SATI's online Registry included 337 patients admitted to 35 ICUs with confirmed/probable/possible diffuse viral pneumonitis caused by influenza A (H1N1), with acute respiratory failure requiring MV (14) . Of these, 178 consecutive patients admitted to 20 ICUs were followed over time, and are presented in this study. To address any potential concern that unconfirmed cases could belong to a different population of patients, we performed a sensitivity analysis of clinical and outcome characteristics data after exclusion of these patients. The results of this analysis did not differ from those of the primary assessments, so the 178 patients are considered for evaluation.
Briefly, patients were middle-aged, with no gender preponderance; they had a history of symptoms of nearly one-week duration and were ventilated at 1 [0 to -2] day after hospital admission. Pre-existent respiratory diseases, obesity, and diseases causing immunosuppression were the most frequent comorbid conditions; and prevalence of pregnancy was higher than in the general population, as expected [25] . Non-survivors were sicker on admission; duration of previous symptoms was longer; and organ failures were more severe. Obesity and immunosuppression were significantly more frequent as predisposing conditions. Ninety-three patients survived (52%) (See Figure 1 ). (Table 2) During the study period, the entire group had Vt values between 7.8 to 8.1 ml/kg of IBW, with plateau pressures remaining always < 30 cmH 2 O. Non-survivors displayed a trend towards lower Vt and higher plateau pressures, which differed significantly from survivors only at Day 7. Intermediate PEEP levels were used, and decreased in survivors from Day 3 onwards. Driving pressures were similar over time in all patients; only at admission did non-survivors exhibit higher values. PaO 2 /FIO 2 increased significantly over time in all patients and in survivors. It remained, however, < 200 in the whole group throughout the entire ICU stay due to non-survivor values. Non-survivors displayed significantly lower PaO 2 /FIO 2 at all time points.
Lung infiltrates (in quadrants) peaked at day 3 (3.1 ± 1.0 vs. 2.9 ± 1 at Day 0, P < 0.01) and then decreased during the study in the entire group, especially at Day 10 (2.8 ± 1.1, P < 0.83 vs. Day 0), which reflected the improvement in survivors (3.1 ± 1.0 at Day 3 vs. 2.9 ± 1.0 at Day 10, P < 0.01).
In Figure 2 , the utilization of ventilation modes and rescue therapies in the entire group are shown. Briefly, PCV use equaled VCV at Day 10, preceded by deterioration in oxygenation and respiratory mechanics: PaO 2 / FIO 2 78 ± 24 vs. 128 ± 33, (P = 0.03); PaCO 2 44 ± 4 vs. 35 ± 3 mmHg (P = 0.04); pH 7.29 ± 0.03 vs. 7.39 ± 0.05 (P = 0.05), and plateau pressures of 30 ± 2 vs. 25 ± 3 cmH 2 O (P = 0.03). Recruitment maneuvers became significantly more common in non-survivors at Day 3 (46%, vs. 29% in survivors; P = 0.03), as did prone positioning (24%, vs. 14%; P = 0.001). After that, only prone positioning remained significantly more used in nonsurvivors (at Day 7: 38%; vs. 14%, P = 0.004; and at Day 10: 25%; vs. 5%, P = 0.02). Six patients received tracheal gas insufflation; only one survived. Neuromuscular blockers were prescribed in 18% of patients on admission; and their use was subsequently more frequent in non-survivors (Day 3: 14% vs. 8%, P = 0.02; and Day 7: 14% vs. 8%, P = 0.04).
The main causes of death were refractory hypoxemia (64%); followed by multiorgan dysfunction syndrome (15%) and shock (10%). Prolonged mechanical ventilation and long ICU and hospital stays were frequent (Table 1) . Tracheostomy was performed in 29 patients (16%) at Day 14 [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . Acid-base variables and fluid balance (Table 3) Arterial pH increased over time in the whole cohort and in both subgroups, perhaps secondary to general resuscitation measures. Despite this, non-survivors displayed significantly lower pH at all time points, owing to changes in base excess on Days 0 and 3, and to pCO 2 elevations thereafter. Respiratory rates remained unchanged, only increasing at Day 10 in non-survivors; nevertheless, this corresponded to the highest pCO 2 values, indicating the more severe respiratory compromise. Bicarbonate paralleled pH behavior. Changes in fluid balance did not show clear trends: only at Day 10 they decreased significantly, expressing survivors' behavior.
Forty-nine patients (28%) underwent a trial of NIV on admission; they were significantly less ill and had a lower incidence of immunosuppression. Oxygenation and outcome variables were similar to those of patients not receiving NIV.
Sixty-one percent of patients (n = 30) receiving NIV survived; duration of NIV was of 8 (2 to 18) hours. There were no differences between survivors and nonsurvivors in the duration of the procedure, or in the type of interface or respirator used. Of note, most patients on NIV (46 out of 49; 94%) had to be intubated and ventilated invasively for hypoxemic failure. Characteristics associated to NIV success/failure are shown in Table 5 .
NIV was also used for treating post-extubation respiratory failure in 12 of 178 patients (7%), with success (reintubation not needed) in 8 cases (66%).
The most consistent changes over time were found in platelet count, which increased significantly in the whole cohort (P < 0.000 for Days 3, 7 and 10 vs. Day 0), secondary to elevations in survivors. At all time points, platelets differed between survivors and non-survivors. Conversely, white blood cell count showed a progressive Creatine-kinase and markers of liver injury (alanine/ aspartate aminotransferases, serum bilirubin; not shown) were mildly elevated and displayed no substantial changes. On the contrary, lactate-dehydrogenase levels were significantly higher in non-survivors throughout the study. Creatinine levels were stable over the period, but were significantly higher in non-survivors on Days 0 and 3. Finally, SOFA score diminished over time in all patients (P < 0.000 for Days 7 and 10 vs. Day 0), as a result of the decrease in survivors. SOFA was significantly lower in survivors throughout the study.
In Figure 3 , the differences between survivors and non-survivors are displayed.
We report on a large, prospective cohort of 2009 influenza A (H1N1) patients that were mechanically ventilated for acute respiratory failure due to diffuse pneumonitis during the pandemic in Argentina. Though most were middle-aged, previously healthy adults, patients with preexistent lung disease, immunosuppression, obesity and pregnancy were also affected. Mortality was high and evolution to chronic critical illness was common, as shown by prolonged mechanical ventilation, high needs of tracheostomy, and lengthened ICU and hospital stays.
Patients had characteristically a history of protracted symptoms and displayed severe compromise of oxygenation compatible with ARDS throughout the study period, which only improved in survivors. At all time points, PaO 2 /FIO 2 differed significantly between survivors and non-survivors, requiring higher FIO 2 and PEEP in this last subgroup. Yet the levels of applied PEEP were only in the intermediate range, similar to mean values of 8.7 cmH 2 O of PEEP in an international study on mechanical ventilation [26] , which may explain the relatively high FIO 2 used in our study. Driving pressures were similar in both subgroups most of the time, suggesting an intention to limit alveolar excursion as part of a protective strategy.
It is striking that, as has been described in similar studies on mechanical ventilation performed during the 2009 influenza A (H1N1) pandemic [6, 7] , tidal volumes used were between 7.5 and 8.3 ml/kg IBW, certainly higher than the 6 ml/kg demonstrated as being lungprotective [27] . Indeed, barriers to implementing lowtidal volume have been identified and might explain physician behavior [28] . Despite this, plateau pressures did remain below 30 cmH 2 O [29] , indicating that lung compliance might have been preserved. Perhaps clinicians focused on plateau pressures rather than on tidal volumes [30] since it still remains unclear which should be limited to avoid ventilator-induced lung injury [31] . We, like others [6, 7, 32, 33] , could not find differences in utilized tidal volumes between survivors and non-survivors. Even so, non-survivors tended to display lower values, probably reflecting physician efforts to intensify protective ventilation strategies in the most severely compromised. Some researchers [34, 35] have suggested that allowing higher tidal volumes in a population of young and previously healthy patients with strong ventilatory drive might reveal an attempt to restrain heavy sedation and neuromuscular blocker use. Notwithstanding this, we believe that these findings may also represent clinicians' inadequate prescription, as described in other scenarios [36] .
Not unexpectedly, VCV was the most common ventilator mode used. PCV use increased throughout the study period, peaking at Day 10. This is in contrast with the recently identified trend towards decreased PCV utilization. Transition to PCV mode was associated with preceding physiological worsening, so clinicians might have perceived PCV utilization as part of a global lungprotective strategy [37] .
Refractory hypoxemia was the main cause of death. As in other studies [6, 7, 11] , rescue therapies were frequently applied, with utilization highest 72 hours after admission. Recruitment maneuvers and prone positioning were the primary adjuvants utilized; ECMO and HFOV are currently not available in Argentina. A Table 3 Oxygenation and acid-base variables, and fluid balance in all patients, and in survivors and non-survivors. prolonged mechanical ventilation course was frequent as reported elsewhere [6] . NIV was the first ventilation approach in 28% of cases, with 94% later requiring invasive ventilation, as has been documented in other studies [6, 7, 11] . These common experiences should caution against delaying proper ventilatory support in this group, given that rapid deterioration is common. A recent meta-analysis suggests that NIV does not decrease the need for intubation, so evidence to support its use in severe ARDS is questionable [38] . In our study, improved outcomes with NIV could be due to milder disease, evidenced by APACHE II. The small number of patients that were not intubated precludes a statistical analysis; however, they were younger, with less severe disease and better oxygenation.
Significant changes in fluid balance were late and reflected changes in survivors. Negative fluid balances could never be obtained, perhaps suggesting a continuing need for hemodynamic support: 72% of patients presented with shock [14] . On the whole, fluid balances remained between those achieved by "liberal" and "conservative" strategies of the fluids and catheters treatment trial, depending on the day evaluated [39] . Thus far, it is not clear whether the negative fluid balance has a causal role in improving outcome in ALI/ARDS, or if it simply expresses the global recovery of patients.
Another important finding was that arterial pH consistently and significantly differed between survivors and non-survivors, as described elsewhere [40, 41] . During the first 72 hours acidosis had a major metabolic component, likely as a sign of hemodynamic impairment. After the first week, respiratory acidosis ensued, indicating either the effects of protective ventilation, or merely deterioration due to progressive shunt, profound ventilation/perfusion mismatch and increased deadspace.
With respect to blood chemistry, the usual findings of thrombocytopenia, leukocytosis and mildly elevated creatine-kinase blood levels were present [21, 42] . Regrettably, the lymphocyte count was not recorded. In viral infections, thrombocytopenia occurred frequently. Although the mechanisms by which the 2009 influenza A (H1N1) virus causes thrombocytopenia are unknown, its lack of resolution is a marker of poor prognosis. Both leukocytosis and leucopenia have been found in hospitalized patients with 2009 influenza A (H1N1) [2, 43] ; in our study, persistent leukocytosis was associated with increased mortality. LDH elevations have been previously described in fatal cases [2] , which corresponded to our finding of higher LDH levels in non-survivors at all time points. Such elevations have also been reported in seasonal influenza [44] . In experimental studies, increased LDH is a marker of human fetal membrane cell apoptosis induced by influenza virus [45] . Finally, multiorgan failure was frequent, and predictably more severe in non-survivors.
This study has several strengths: first, the clinical characteristics and time course of pandemic 2009 influenza A (H1N1) are thoroughly described and analyzed. Second, data were collected prospectively in consecutive patients and with a standardized casereporting form, representing a large, nationwide cohort. Third, temporal patterns of mechanical ventilation use, acid-base and blood chemistry variables, as well as fluid balance and organ failures, are carefully analyzed. Prognostic implications are highlighted. Finally, we present the largest experience with NIV use during the pandemic.
Study limitations include the focus on mechanically ventilated patients, excluding less severe cases also admitted to the ICU. Many cases could not be confirmed because laboratories were overwhelmed with clinical samples, which is also described elsewhere [7, 14] . Data about transmission to healthcare workers were not recorded, especially regarding NIV. Currently, most information about its use during an epidemic relies upon expert opinion [46] .
In 178 patients with diffuse viral pneumonitis caused by the 2009 influenza A (H1N1) virus admitted to the ICU and followed over time, ARDS was the rule, requiring high ventilation support and frequent use of rescue therapies. Death, organ failures, and evolution to prolonged mechanical ventilation were common. In most cases, noninvasive ventilation failed to prevent endotracheal intubation. Finally, elevated LDH levels, lack of recovery of platelet count and persistent acidosis and leukocytosis in non-survivors behaved as prognostic findings. 
• In 2009 influenza A (H1N1) patients, hospital admission with prompt indication of mechanical ventilation -a marker of severe disease -was associated with a history of symptoms of nearly one-week duration.
• An initial NIV trial was not effective to avoid intubation in most patients; thus, this ventilation approach should likely be discarded in this setting.
• Mortality and morbidity were frequent: death was common and was mainly caused by persistent, refractory hypoxemia. Prolonged mechanical ventilation and ICU and hospital stays were typical.
• pH, platelet count, LDH and SOFA differed significantly between survivors and non-survivors over time. Lack of recovery of platelet count and persistence of leukocytosis might be markers of poor prognosis.
• Every effort should be done to increase adherence to protective ventilation in the real world.
Abbreviations ALI: acute lung injury; ARDS: acute respiratory distress syndrome; BMI: body mass index; CXR: plain chest X-ray film; IBW: ideal body weight; ICU: Intensive Care Unit; LDH: lactate dehydrogenase assay; LOS: length of stay; MV: mechanical ventilation; NIV: non-invasive ventilation; PaO2/FIO2: relation between patient arterial pO 2 and inspired oxygen fraction used; PCV: pressure-controlled ventilation; PEEP: positive end-expiratory pressure; PSV: pressure support ventilation; RR: respiratory rate; RT-PCR: real-time reversetranscriptase-polymerase-chain-reaction; SATI: Argentinian Society of Intensive Care; SOFA: Sequential Organ Failure Assessment; VCV: volumecontrolled ventilation; Vt: tidal volume. Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis BACKGROUND: The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. RESULTS: Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. CONCLUSIONS: Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org. Tuberculosis is still a major killer worldwide, causing an estimated 2-3 million deaths per year [1] . The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for a decade [2, 3] , but the biology of the pathogen remains poorly understood. Available genome sequences from Mtb strains and other closely related Mycobacteria present an opportunity to bring the power of comparative genomics to the study of Mtb.
We report here the results of a comparative analysis of 31 publicly available genomes (http://www.tbdb.org, Figure 1 , Table 1 ). These include eight closely related members of the Mtb complex that can cause tuberculosis disease, (two M. bovis strains and six Mtb strains). To gain further insight into the Mycobacterium cluster, we also included a related Rhodococcus (also involved in bioremediation), a pathogenic Nocardia, four Corynebacteria (two pathogens and two that are commercially useful in amino acid production), two Streptomyces (antibiotic-producing soil bacteria), Acidothermus cellulolyticus (a thermophilic actinobacteria from the hot springs of Yellowstone), Propionibacterium acnes (causative agent of common acne), and Bifidobacterium longum (a digestive track commensal often found Mycobacterium tuberculosis F11 (ExPEC) Y Y Causes TB; isolated from TB patient in S. Africa [5] Mycobacterium bovis BCG str. Pasteur 1173P2 Y Y Causes bovine TB; attenuated vaccine strain [6] Mycobacterium bovis AF2122/97 Y Y Causes bovine TB [7] Mycobacterium tuberculosis Haarlem Y Y Causes TB; MDR strain [5] Mycobacterium tuberculosis C Y Y Causes TB; isolated in NY City [5] Mycobacterium tuberculosis CDC1551 Y Y Causes TB; highly contagious & virulent strain [8] Mycobacterium ulcerans AGY99 Y Causes Buruli ulcer [9] Mycobacterium marinum Y From fish; Skin lesions in human [10] Mycobacterium leprae TN Y Y Causes leprosy [11] Mycobacterium avium 104 Y Opportunistic pathogen; can causeTB-type pulmonary infection [12] Mycobacterium avium subsp. Paratuberculosis K-10 Y Y Causes paratuberculosis; obligate pathogen of cattle [13] Mycobacterium sp. MCS Soil bacteria; degrades PAH [14] Mycobacterium sp. KMS Soil bacteria; degrades PAH [14] Mycobacterium smegmatis MC2155 Y Widely used model for Mtb isolated from human smegma; causes soft tissue lesions [12] Mycobacterium vanbaalenii PYR-1 Soil bacteria; degrades PAH [14] Mycobacterium gilvum Soil bacteria; Degrades PAH + wide variety of organic compounds [14] Mycobacterium abscessus Y Skin & soft tissue infections [15] Rhodococcus jostii RHA1 Soil bacteria important for biofuels research and bioremediation; degrades PCB + wide variety of organic compounds [16] Nocardia farcinica IFM 10152 Y Causes nocardiosis [17] Corynebacterium glutamicum ATCC 13032
Produces amino acids (Glu) [18] Corynebacterium efficiens YS-314 Produces amino acids (Glu) [19] Corynebacterium diphtheriae NCTC13129 Y Causes diphtheria [20] Corynebacterium jeikeium K411 Y Causes nocosomial infections [21] Streptomyces avermitilis MA-4680 Soil bacteria;antibiotic-producing [22] Streptomyces coelicolor A3(2) Soil bacteria;antibiotic producing [23] Acidothermus cellulolyticus 11B Hot springs of Yellowstone [24] Rhodobacter sphaeroides Gram -, motile; photosyn.; fixes N 2 [14] in yogurt). We extend this comparative analysis to other more distantly related Actinobacteria to yield additional insight into evolutionary trends. We examined protein evolution across these 31 organisms, both at the nucleotide level and at the level of protein families, including studying gene families associated with the transition from nonpathogenic soil-dwelling bacteria to obligate pathogens. Our results highlight the importance of lipid metabolism and its regulation, and reveal differences in the evolutionary profiles for genes implicated in saturated and unsaturated fatty acid metabolism. Our analysis also suggests that DNA repair and molybdopterin cofactors are expanded in pathogenic Mycobacteria and Mtb.
We also identified highly conserved elements within noncoding regions using whole-genome multiple alignments and gene expression data. These conserved elements include 37 predicted conserved promoter regulatory motifs, of which 14 correspond to previously reported motifs. They also include approximately 50 predicted novel noncoding RNAs. Guided by our computational analysis, we tested and experimentally confirmed the expression of 4 novel small RNAs in Mtb.
The results of our analyses are available on our website, and provide a foundation for understanding the genome and biology of Mtb in a comparative context.
We used SYNERGY [27, 28] to reconstruct the phylogeny of proteins across all 31 organisms, define sets of orthologs ("orthogroups"), and construct a phylogenetic tree of the genomes (Figure 1 ). An orthogroup is defined as the set of genes descended from a single common ancestral gene in the last common ancestor of the species under consideration [28] , containing both orthologs and possibly paralogs (Methods). At each node in the phylogenetic tree, we tabulated orthogroup appearances, duplications, and losses ( Figure 2 ). Figure 2 gives an overview of the evolution of gene families within these species. Full listings of the events tabulated in Figure 2 , as well as additional information about each orthogroup, can be found on the Supplementary Information website:
To examine the evolution of entire pathways and gene families, we categorized orthogroups according to GO (Gene Ontology) and GO Slim terms [29] , PFAM domains [30] , metabolic pathways, predicted regulons (sets of genes predicted to be regulated by a common regulatory protein), and groups of genes upregulated under certain lipids (Methods). We also looked for orthogroups undergoing positive selection by calculating the ratio of nonsynonymous to synonymous mutations (the d N /d S ratio). Figure 3 shows several examples of pathway or gene family profiles and the predicted evolutionary events associated with the gene family. The sort of graphic presented in Figure 3 is browsable for every pathway, PFAM, and GO term in our Supplementary Information website. Tables 2 and 3 show the PFAM and GO categories most expanded (with the most orthogroup members) in the Mtb clade relative to the non-pathogenic Mycobacteria, and Tables 4 and 5 show those most expanded in the Mycobacteria relative to the non-Mycobacteria.
Despite the smaller genome sizes present in the pathogenic Mycobacteria, and the resulting background of orthogroup loss in the evolution towards pathogens, we observe significant expansions in certain gene families in the pathogenic Mycobacteria and the Mtb complex relative to non-pathogenic relatives. We also observe evidence for selection in certain families on branches leading to the pathogenic Mycobacteria, the Mtb complex, and the soildwelling Mycobacteria.
As expected, many genes known to be related to pathogenicity or antigenic variability are among the groups most expanded in the Mtb clade relative to soil dwelling Mycobacteria as well as being among the categories with the most variability in copy number in their category-level profiles overall, including toxin-antitoxin genes, genes containing PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, MCE (Mammalian Cell Entry) genes, genes involved in the synthesis of the mycolic acid coat, Esx genes, and gene involved in antibiotic resistance. Complete results for all groupings are available on our Supplementary Information website. Below we focus on specific additional families showing noteworthy expansions and trends.
The single most significant trend in our analysis of protein family evolution is that genes related to lipid metabolism are greatly expanded across all Mycobacteria and related organisms, consistent with previous observations [2, 31] (Table 5 ). Our analysis extends these previous observations by identifying the emergence of this expansion in lipid metabolism genes as occurring at the root node of the Mycobacteria and Rhodococcus (Figure 3 ).
Genes predicted to be involved in the metabolism of saturated fatty acids are more expanded than those involved in the metabolism of unsaturated fatty acids. Using a compendium of microarray expression experiments (Methods), we compiled a list of genes upregulated in the presence of different fatty acid sources. We found that genes upregulated under unsaturated conditions have more uniform phylogenetic profiles, while those upregulated under saturated conditions, cholesterol or ceramide have expanded through duplications in pathogenic Mycobacteria ( Figure 4 ). Saturated fatty acids and cholesterol are more prevalent in an animal host than in the soil, which contains mostly unsaturated fatty acid from plant inputs. Since it is believed that Mtb uses cholesterol as a carbon source within the host [32] , this could reflect an Figure 2 Summary of SYNERGY results: Number of gains, losses, and duplication at each node. For each node, the node number is marked in black; the total number of genes present at each node is indicated in red, and the numbers of gains, losses, and duplications are indicated in parenthesis in blue http://www.broadinstitute.org/ftp/pub/seq/msc/pub/SYNERGY/index.html.
adaptation to the host environment. Consistent with our observations in host-adapted Mycobacteria, Desulfovibrio desulfuricans intestinal strains contain a higher ratio of saturated to unsaturated fatty acids than soil strains of Desulfovibrio desulfuricans [33] .
Our analysis also reveals differences in evolutionary profiles between genes predicted to be involved in catabolism and anabolism of lipids. Both sets of genes are expanded in soil-dwelling and pathogenic Mycobacteria, but lipid synthesis genes are additionally expanded in pathogens relative to soil dwellers. General lipid synthesis genes are expanded across the Mycobacteria, but certain groups of lipid synthesis genes, including those related to cell wall synthesis, are further expanded in the Mtb complex (see Supplementary Information) . In pathogenic Mycobacteria, the waxy mycolic acid coat helps evade the host immune system [34] . Consistent with this, we see categories related to mycolic acid synthesis showing up among the most non-uniform categories, highly expanded in the Mtb complex (see Supplementary Information) .
In contrast, some lipid degradation gene families are more expanded in the soil-dwelling Mycobacteria than in the pathogens (Supplementary Data). The soil-dwellers have the unusual ability to degrade a vast array of compounds, including diverse lipids.
In addition to gene family expansions, we observe evidence for selection on the coding sequence of lipid metabolism genes. In our d N /d S calculations, we observe enrichment for positive selection in lipid degradation genes on the branch leading to the pathogenic Mycobacteria (Additional file 1: Table S2 ). For example, Rv2524c, the multifunctional FAS-I polypeptide utilized during de novo fatty synthesis [35] , has the second highest d N /d S value on this branch. Additional lipid metabolism genes with elevated d N /d S values include 15 genes predicted to be involved in the β-oxidation pathway of fatty acid degradation: seven fadE (acyl coA dehydrogenase) genes, three fadD (fatty acid CoA ligase) genes, two fadB (NADPH quinone oxidoreductase/3-hydroxybutyryl-CoA dehydrogenase) genes, one fadA (acetyl-CoA acyltransferase) gene, and two echA (enoyl-CoA hydratase) genes. Hence, we observe expansions of lipid biosynthesis genes, as well as observing evidence for positive selection acting on genes within the β-oxidation pathway. Both the lipid biosynthesis and lipid degradation pathways are specialized within the pathogenic Mycobacteria. This expansion could possibly benefit the pathogen in a manner to accommodate production and modification of cell wall lipids involved in manipulation of host immune response. The lipid degradation is particularly beneficial for the long term survival of the pathogen metabolizing host lipids encountered during infection.
KstR is a transcription factor known to be involved in lipid and cholesterol degradation [36, 37] . It has been recently shown that Mtb uses cholesterol as a carbon source within the host [32] . Strikingly, KstR exhibits an evolutionary history that parallels the expansion of lipid metabolism genes in the Mycobacteria, and displays a singular conservation in its regulatory binding sites. KstR appears to have evolved at the last common ancestor of the Mycobacteria and Rhodococcus. In all Figure S1 ). Remarkably, these paralogs of KstR are all absent in the pathogenic Mycobacteria. Thus, coincident with the expansion in lipid metabolism genes described above, the KstR gene appears to have emerged through gene duplication within the existing gene family of tetR-like transcriptional regulators at the last common ancestor of Mycobacteria and Rhodococcus.
All other members of this gene family were subsequently lost in the Mtb complex, while the KstR protein was maintained and underwent limited sequence divergence.
There is another homolog to KstR found in Mtb H37Rv (Rv3557c) that has previously been reported to also be involved in cholesterol metabolism, named KstR2 [38] . However, KstR is much more similar to the other members of the Mycobacterial tetR family discussed above than it is to KstR2. KstR2 is categorized into a separate orthogroup (orthogroup 32655) and is more distantly related to KstR.
The high sequence conservation of the KstR transcription factor is mirrored in the conservation of KstR binding sites across numerous promoters. KstR binding sites are known to be highly conserved across the Mycobacteria, out to Rhodococcus and Nocardia [36] . These sites are conserved in both sequence and position within their respective promoters. In our analysis, both in searches using known transcription factor binding motifs, as well as in our de novo motif searches, a subset of KstR binding sites are the most conserved transcription factor motifs observed. They are also among the most conserved of any noncoding sequence we identified. The conservation of the KstR gene and binding sites, the emergence of KstR at the ancestor of Rhodococcus and the Mycobacteria, and the loss of KstR paralogs within the pathogenic Mycobacteria, suggests that this transcription factor and its evolving regulon have played an important role in the expansion of lipid metabolism and its adaptation to pathogenicity in Mtb.
Mtb, as well as non-tuberculous Mycobacteria, differ from other bacteria in several key respects of DNA repair [ [39] [40] [41] [42] . Within the host, Mtb must combat damage to its DNA from macrophage-generated reactive oxygen and nitrogen intermediates. The mechanisms by which this is accomplished are not fully understood [43, 44] . Although genes implicated in DNA repair have not expanded in the Mtb lineage, we note that the set of genes showing positive selection on the Mtb lineage in our d N /d S analysis is enriched for genes involved in the COG category for DNA replication, recombination, and repair (Additional file 1: Table S2 ). Several of the genes in this set with highest d N /d S values are known DNA repair genes (including recA, recB, and dnaE2), and several additional genes are helicases (dnaB, helZ, and gyrB).
Interestingly, we observe that recA has the highest d N / d S score of all the genes in Mtb on the branch leading to the Mtb complex, and recB also has a very high score. Mycobacteria lack a mutSL-based mismatch repair (MMR) system [42] , and it is believed that recA may be involved in compensating pathways. dnaE2 (DNA polymerase III) also has one of the highest d N /d S values on the branch leading to Mtb, and both dnaE1 (DNA polymerase III) and dnaE2 show evidence of selection on the branch leading to the pathogenic Mycobacteria. In Mtb, damage-induced base-substitution mutagenesis is dependent on dnaE2. Loss of dnaE2 activity renders Mtb hypersensitive to DNA damage, eliminates induced mutagenesis, attenuates virulence, and reduces the frequency of drug resistance in vivo [39, 45] . dnaE1 provides essential, high-fidelity replicative polymerase function [39] , and is expressed in response to DNA damage, along with dnaE2 and recA [39, 45] .
We also observe positive selection for dinX (DNA polymerase IV) on the branch leading to the pathogenic Mycobacteria (branch-site model) in our d N /d S analysis (see Supplementary Information website). Most organisms use specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across sites of damage, including the dinB group of Y family polymerases. There are two dinB-family polymerases in Mtb (dinX and dinP). Unlike in other bacteria, dinX and dinP expression are not dependent on recA, the SOS response, or the presence of DNA damage, and could therefore serve a novel yet uncharacterized role in Mtb [46] [47] [48] [49] .
Genes involved in the first steps of pterin cofactor (a component of the molybdenum cofactor) biosynthesis are known to be expanded in the Mtb complex [50] . Molybdenum cofactor-requiring enzymes (such as xanthine oxidase and aldehyde oxidase) could have physiological functions in the metabolism of reactive oxygen species during stress response [51] . Molybdenum cofactor is an efficient catalyst in oxygen-transfer reactions, can be used in anaerobic respiration, and can catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. Recently, genes related to molybdenum cofactor protein synthesis have been shown to be upregulated under conditions of stress in Mtb [52] . Molybdenum cofactor biosynthesis has been previously linked to pathogenesis. The regulator of the moa1 locus, MoaR1, was identified as having a SNP in M. bovis BCG, but not in virulent M. bovis or Mtb [53] . In addition, moa3 is present with varying frequency in the RD1 region, which is absent in M. bovis BCG, of pathogenic strains [54] .
In agreement with previous observations of expansions of molybdopterin biosynthesis genes, we observe five protein domains related to pterin cofactor biosynthesis among the top protein domains expanded in the Mtb complex compared to the non-pathogenic Mycobacteria ( Table 2 , -"d"). Among the top GO terms expanded in the Mtb clade relative to the soil dwellers (Table 3) , there are also several groups involved in pterin and molybdopterin biosynthesis. Some of these gene copies (the moa1 locus) are believed to have been acquired by lateral gene transfer on the branch leading to the Mtb complex [10, 50] .
We also observe evidence for selection on molybdenumrelated genes in our d N /d S data. On the branch leading to the pathogenic Mycobacteria, several orthogroups with high log likelihood scores when testing for selection are related to molybdenum (see Supplementary Information website). The orthogroup containing BisC (biotin sulfoxide reductase, a molybdoenzyme), as well as the orthogroup containing ModA (an ABC-family molybdate transporter), are among those with the highest d N /d S values on the branch leading to the pathogens. MoaB2 is one of the highest-scoring genes on all three branches tested.
There are also many categories of unknown function that are greatly expanded in the Mtb clade relative to the nonpathogenic Mycobacteria (Tables 2 and 3 , red). For example, Rv0918 (in the Pfam group of unknown function PF08681) was found in a genetic screen that facilitates isolation of mutants defective in arresting the maturation of phagosomes [55] , helping Mtb to survive within host cells. PF07161 contains four lipoproteins (LprF, LprG, LprA, LppX). LprG and LppX were found to be in vivo essential genes by TraSH analysis [56] .
Sequence conservation -or phylogenetic footprintingprovides a powerful approach for identifying potential functional noncoding sequences, and has been used in a variety of eukaryotic and prokaryotic organisms to identify protein coding genes, noncoding RNAs, and regulatory elements [57, 58] . For optimal power, the organisms being analyzed must be sufficiently distant such that non-functional elements have diverged, but not so distant such that functional elements have evolved or re-arranged. Organisms within the Mtb complex are all highly similar at the sequence level, and thus by themselves do not allow for effective phylogenetic footprinting. By leveraging the evolutionary similarity of the most distantly related Mycobacteria and Actinomycetes, we gained additional power to allow us to detect functional sequences under purifying selection, albeit only those shared by at least a majority of Mycobacteria. We used this approach to predict two classes of conserved noncoding sequences: small noncoding RNAs and transcription factor binding motifs.
Small noncoding RNAs (sRNAs) have been shown to play a role in regulating gene expression in numerous bacterial species [59] , including Streptococcus [60, 61] . Yet only recently were sRNAs reported in Mycobacteria [60, 62] . Using a combination of direct isolation of small RNAs, and validation by Northern blotting and 5' and 3' RACE transcript mapping, Arnvig and Young [62] first described nine sRNAs in Mtb. Subsequently, DiChiara et al. [63] describe 34 small RNAs in M. bovis BCG, of which many were conserved in both Mtb and M. smegmatis.
To build on these results, we used a combination of comparative genomics, RNA-seq, and experimental validation by Northern blotting to identify additional sRNAs conserved among the Mycobacteria (Methods). Our computational results provide evidence for 50 conserved small RNAs in Mtb that have not been previously reported. It is likely that additional conserved regions are expressed under other diverse conditions. Figure 5a shows the expression and conservation map for one of our predicted RNAs in the GenomeView Browser [64] . Table 6 shows a listing of the top 12 candidate RNAs. To verify a subset of these candidate small RNAs, we used Northern blot analysis on four of the top predicted regions (Methods). The results (Figure 5b) show signals corresponding to small RNAs from each of four candidates (Table 6 , labeled 1, 2, 3, and 9). All transcripts were near the expected size, or slightly larger. Full-length gels are provided in Additional file 3: Figure S2 . Consistent with previous work, the majority of small RNAs were seen as more than one size transcript [62] . This suggests that small RNAs might be generated by processing of larger transcripts. In the RNAseq data, there are longer "tails" extending outside of the main peak that corresponds to the RNA prediction-different length RNAs could be responsible for the additional bands of higher mass.
Few transcription factor binding motifs have been identified in Mtb. Transcription factors for which binding motifs have been identified include KstR [36] , DosR [67] , IdeR [68] , ZurB [69] , Crp [70] , CsoR [71] , FurA [72] , MprAB [73] , and Acr [74] . Because of the limited knowledge of transcriptional regulation in Mtb, we searched for additional motifs computationally. We combined comparative sequence analysis with microarray data to identify a large number of motifs conserved in Mycobacteria.
We clustered microarray data contained in the TB database [75] and searched for upstream regulatory motifs shared in the upstream regions of the resulting clusters using AlignACE (Methods). Because of significant noise in the results, we used a set of stringent filters, including a requirement that candidate motifs be highly conserved. 37 motif instances passed our stringent filters ( Table 7 , Methods). 14 of the top 37 (38%) motif instances correspond to cases of known Mtb motifs (several known Mtb motifs were found more than once, in different clusters, or in clusters with different size parameters). In contrast, none of the top motifs showed similarity only to known E. coli or Corynebacteria motifs. Within these top motifs, we were able to identify four of the nine known Mtb motifs (DosR, IdeR, KstR, and ZurB).
As described above, the KstR motif shows a much stronger signal, in terms of both conservation and information content, than any of the other motifs (top of the ranked conservation list, Table 7 ). Based on the distribution of highly conserved predicted motif instances for KstR across the genome, we predict a more general role for KstR in lipid metabolism. We see KstR motif instances near many other lipid genes not related to cholesterol degradation, in support of the view that KstR is a more general lipid regulator controlling a large regulon [36] .
One of the most interesting new motif candidates that shows up in our analysis is a conserved palindromic motif, consisting of a highly conserved TAC... GTA separated by 6 bp of less well conserved sequence (marked with an X in Table 7 ) that is found in clusters of 2-3 closely spaced sites upstream of several genes related to fatty acid metabolism ( Figure 6 ). There is a cluster of 3 evenly spaced sites upstream of Rv3229c (linoeyl-coA desaturase), a cluster of 2 sites upstream of the adjacent Rv3230c (oxidoreductase), and a cluster of 3 sites upstream of Rv2524c (fatty acid synthase). This is the second highest-scoring new motif identified (Table  7) . This motif shows up as one of the top motifs associated with the clusters of genes upregulated under saturated fatty acid conditions (specifically palmitate).
To better understand Mtb, we performed a comparative analysis of 31 organisms from the Tuberculosis Database. We studied the evolution of protein families and metabolic pathways, looked for proteins with evidence Figure 5 New predicted RNAs. a) An example of a new predicted RNA. This is the RNA2 in Table 6 . This figure shows a screenshot from the GenomeView Browser [64] . The light blue bars show the coding regions (Rv1230c and Rv1231); the tan bar shows the conserved region predicted by Gumby [65] ; and the green bar shows the region predicted to fold by Evofold [66] . The yellow and green plots in the center show the RNA-seq data. Green signifies reads from the negative strand, and yellow shows the total reads (positive and negative strands). The multiple alignment is shown on the bottom (darker grey signifies a higher degree of conservation; red signifies no alignment at that position). You can see that this predicted RNA region is conserved through M. avium. The rulers at the top show the gene structure. Small red squares show where stop codons are present all six reading frames, indicating that this intergenic region is unlikely to be a protein-coding region missed in the annotation. b) Northern blots validating four of the new, predicted small RNAs (RNA1, RNA2, RNA3, and RNA9 in Table 6 ). 1 Conserved intergenic regions determined by Gumby. 2 Indicates whether this region is predicted to fold by Evofold. 3 Region in M. smegmatis that aligns with the conserved region in Mtb, and its corresponding RPKM value. 4 Tested experimentally 5 Orientation relative to neighboring genes. The first and last characters give the strands of the flanking genes; the middle character gives the strand for the predicted RNA. of selection, and searched for new noncoding RNAs and transcription factor binding site motifs. The most striking features of our analysis are related to lipid metabolism and its regulation. In addition to observing a general expansion of lipid metabolism genes in the Mycobacteria and Rhodococcus, we observe increased expansions of genes related to saturated fatty acid metabolism in the pathogenic Mycobacteria compared to the soil-dwelling Mycobacteria. We also note differences in evolutionary profiles for catabolic and anabolic lipid metabolism genes, and evidence for positive selection in lipid metabolism genes. The cis-regulatory elements bound by the KstR protein, a known regulator of lipid/cholesterol metabolism, are among the strongest, most highly conserved noncoding signals across the Mycobacteria. Both KstR and its binding sites are highly conserved, appearing at the last common ancestor between Rhodococcus and the Mycobacteria.
Within our set of organisms, we examine the evolution of pathogenicity, moving from the soil-dwelling Mycobacteria up to the intracellular parasites of the Mtb complex. We see expansions of many known gene families related to pathogenicity (PE/PPE genes, antibiotic resistance genes, genes involved in the synthesis of the mycolic acid coat, MCE genes, and Esx genes). By similarity of phylogenetic profiles, we can predict likely candidates for novel gene families related to pathogenicity. For example, we see similar expansions in gene families related to biosynthesis of molybdopterin. We further observe evidence of positive selection on molybdenum-related genes, providing further support for the importance of molybdenum in these pathogens. On the branch leading to the pathogenic Mycobacteria, we also observe evidence for positive selection in genes related to replication, recombination, and repair. It is possible that these DNA repair-related processes give the pathogenic Mycobacteria an advantage when dealing with the assault on its DNA by macrophage-generated reactive oxygen and nitrogen intermediates.
Our whole-genome alignments, coupled with RNA-seq and microarray data, allowed us to predict novel noncoding features, including small RNAs (four of which we have validated experimentally), and potential transcription factor binding sites.
The main forces driving genome evolution in prokaryotes include gene genesis, lateral gene transfer, and gene loss. Our analysis of protein evolution using SYNERGY does not examine whether orthogroups appearing have arisen by lateral gene transfer or by gene genesis involving duplication and divergence from other orthogroups. A detailed comparison to categorize these orthogroup appearances according to lateral or vertical gene transfer is beyond the scope of this study, but other studies indicate that lateral gene transfer has played a significant role in Mycobacterial evolution and the evolution of pathogenesis [79] [80] [81] [82] [83] .
A recent paper suggests that the Mycobacterial genome has been shaped by a biphasic process involving gene acquisition (including lateral gene transfer) and duplications followed by gene loss [79] . Other studies report numerous genes, including a large number involved in lipid metabolism, that have been acquired by horizontal gene transfer at different phylogenetic strata and have led to the emergence of pathogenesis in Mtb [80, 81] . Previous studies indicate a possible more ancient lateral gene transfer of fatty acid biosynthesis genes from α-proteobacteria to actinobacteria [84] . However, genetic studies show that the Mtb complex and pathogenic Mycobacteria do not exchange genetic material frequently [85, 86] , so there is limited lateral gene transfer within the Mtb complex.
We are currently performing high-throughput Chromatin Immunoprecipitation (ChIP)-Seq experiments in several different Mycobacteria, including Mtb, M. smegmatis, and M. vanbaalenii [87] . We plan to integrate the information obtained from our comparative analysis with data coming from these high-throughput experiments, as well as other 'omic datasets, using a systems biology approach. This will enable construction of gene regulatory networks for Mtb, and examination of their evolution across species.
The 31 organisms used in our analysis are described in Table 1 . These genome sequences are all contained in Table 7 Motifs passing our set of stringent filters, ranked by their degree of conservation (Continued) 50 k indicates the value of k in the k-means clustering process (50, 100, 200, or 250) b MAP score indicates the AlignACE MAP score [76] c Specificity score [77] d CompareACE score ≥ 0.7 to the alignment for this known motif e CompareACE score to its reverse complement f number of ScanACE hits in the genome that are conserved in ≥ 8 genomes g sequence logo [78] the TB database (TBDB) [75] . The three unpublished sequences generated at the Broad Institute (M. tuberculosis F11, M. tuberculosis Haarlem, and M. tuberculosis C) are high-quality genome sequences. M. tuberculosis F11 and M. tuberculosis Haarlem are finished, and M. tuberculosis C has 6.7× coverage and 4 scaffolds. The Broad Institute sequencing read pipeline interacts with the sample management system to ensure the read is associated with the correct sample. Vector identification, length checks and quality clipping were performed on all reads. Contamination checks and organism checks were also performed using a kmer-based algorithm that can compare sequence to a profile from any organism.
The SYNERGY algorithm [27, 28] was applied to the 31 genomes in Table 1 . SYNERGY organizes groups of genes across organisms into orthogroups, or groups of orthologs and paralogs, which consist of all the genes descended from a single ancestral gene in the species' last common ancestor. SYNERGY also associates orthogroups with a gene tree, from which we can derive an "extended phylogenetic profile", showing the gene copy number in each extant organism and at each ancestral node. Importantly, by reconciling an organism tree with each gene tree, SYNERGY provides an evolutionary scenario for each gene tree predicting where all losses, gains, and duplications occurred in its evolution. These lists of losses, gains, and duplications contain actual evolutionary events, as well as artifacts caused by genes that could not be properly categorized by SYNERGY. However, we observe that SYNERGY is effective at properly categorizing genes into orthogroups, and the SYNERGY orthogroups were very useful in our analysis. Analysis of the 31 genomes resulted in a total of 32,505 orthogroups, including those containing single genes from only a single genome (below). There were 177 "uniform" (1:1:1:1...) orthogroups representative of some of the most conserved and indispensible housekeeping genes. Additional file 4: Figure S3 summarizes the SYNERGY orthogroups.
We started running SYNERGY using an initial phylogenetic tree generated using orthologs based on bidirectional best BLAST hits. The list of uniform orthogroups from the first SYNERGY run was used to construct a refined phylogenetic tree. SYNERGY was then re-run using the refined phylogenetic trees. To generate our final phylogenetic tree, the final set of 177 31-way orthologs (31-way uniform orthogroups from the SYNERGY analysis) were aligned according to their nucleotide sequences with CLUSTALW [88] and concatenated, distances were computed with Phylip's DNADIST algorithm [89] , and Phylip's FITCH algorithm was used to create the tree.
Because of the similarity of the genomes within the Mtb complex, we were not able to resolve the phylogeny using only these 177 proteins that are uniform across all 31 organisms. In order to better resolve the tree within the Mtb cluster, we computed a separate tree using 1747 orthogroups that are uniform across the Mtb cluster and M. ulcerans, which we used as an outgroup. Using this expanded gene set, we were able to resolve the tree for the Mtb cluster.
Bootstrap analysis was performed to validate tree topologies. Phylip's SEQBOOT was used to create 1000 bootstrap input replicates for each tree. Phylip's CON-SENSE was used to obtain a bootstrap tree (Additional file 5: Figure S4 )
Metabolic pathways and functional groups EFICAZ [90] was used to assign EC numbers for proteins in all 31 organisms. Metabolic pathways were constructed in Biocyc [91, 92] . An orthogroup was considered to be part of a metabolic pathway if any of its component genes had been identified as part of that pathway using this pipeline.
We obtained the Gene Ontology (GO) [29] and GO Slim terms for each of the 31 organisms using BLAS-T2GO [93] . PFAM assignments [30] were taken from http://www.tbdb.org [75] . An orthogroup was associated with a GO, GO Slim, or PFAM descriptor if greater than half of its protein members were associated with that descriptor.
For each node in the phylogenetic tree, we tabulated orthogroups lost, gained, or duplicated. Using GO terms, GO Slim terms, and PFAM domain groupings with less than 500 members, we calculated over-representations within losses, gains, and duplications each of these groupings at each node using the hypergeometric test. A complete summary of gains, losses, and duplications for all nodes in the phylogenetic tree is available on our supplementary information website.
Extended phylogenetic profiles for each category (metabolic pathways, GO terms, GO Slim terms and PFAM categories) were obtained from SYNERGY output by summing the phylogenetic profiles from their component orthogroups. We define a category-level phylogenetic profile as the sum of its component orthogrouplevel phylogenetic profiles. The evolution of each of these categories can be quickly visualized on our website. Since genes with the same phylogenetic profile can be linked functionally [94] , the webpage for each category contains a link to other categories with similar phylogenetic profiles (Methods). Categories with the most similar profiles were obtained by calculating Euclidean distances to all other profiles.
Instances of expanded or missing pathways across the 31 organisms will have non-uniform pathway-level phylogenetic profiles. Thus we tabulated the number of genes in each genome for each category, and automatically searched for gene categories whose copy number (normalized for genome size) had the most non-uniform distribution across the 31 organisms in order to identify the most significant examples of expansions or losses. To identify categories with bimodal properties (such as a categories with a loss or a large expansion on only certain branches of the phylogenetic tree), we clustered each profile into two groups and looked for the pathways with the greatest separation between the two clusters. We used k-means (k = 2) to cluster the profile vectors, and compared the intra-and inter-cluster point-to-centroid distances to find the clusters with the greatest separation. We ranked categories by this separation to find bimodal categories. We further select those that have at least five organisms in the smallest of the two clusters, and an average of at least five genes per genome. P-values are calculated from a T-test between the values for the two groups, with Bonferroni correction applied. In our Supplementary Information website we list those categories with p < 0.05, ranked by the difference between their inter-to intra-centroid distances. When we select the metabolic pathways, PFAM domains, and GO terms with the most non-uniform category-level phylogenetic profiles overall, we find that many of the top categories are lipid metabolism-related categories expanded in the Mycobacteria.
We also measured the similarity between evolutionary profiles to find the PFAM categories and GO terms with the biggest difference between pre-defined sets of organisms. For example, we compared both the Mtb complex and a group consisting of other pathogenic Mycobacteria to the set of soil-dwelling Mycobacteria in order to examine the evolution of soil-dwelling, free-living Mycobacteria into more pathogenic Mycobacteria that require a host to survive. We used the following categories:
1. All Mycobacteria (excluding M. leprae because of its massive gene loss).
2. All non-Mycobacteria in our set (excluding Nocardia and Rhodococcus because of their similarity to Mycobacteria 6. R. jostii RHA1 and N. farcinia We calculated differences between two sets of organisms exactly as we calculated distances between clusters (above). However, rather than using different clusters of organisms determined by k-means clustering, we used these pre-defined clusters of organisms. We looked at distances between the following sets of organisms: 1-2, 3-4, 3-5, 3-6, 4-5, 4-6, 5-6. For each PFAM domain or GO term represented in at least two organisms in these pairings, we calculated p-values for the differences between the profile values by T-test (Bonferroni-corrected by the number of PFAM domains represented in that set of organisms) and computed inter-and intra-centroid distances (as described in the above paragraph). We compiled lists of those that are most expanded and a list of those most contracted across these pairings. On our website we have included complete lists of PFAM categories, including those that do not make the strict Bonferroni-corrected p-value cutoff. Many potentially interesting expansions do not make the overly conservative Bonferroni-corrected p-value cutoff [95, 96] .
Using a compendium of 946 microarray experiments from the TB database [75] , we used several different clustering methods to generate predicted regulons. We searched the upstream regions of these regulons for shared transcriptional regulatory motifs. We clustered microarray data by hierarchical and k-means clustering. Because real regulons can be of varying sizes, we performed k-means with k = 50, 100, 200, and 250, then used all the resulting clusters for further analysis. We found that the clusters obtained from hierarchical clustering were not very useful because their size distribution did not approximate that of real regulons as well as those from k-means; therefore we did not analyze clusters from hierarchical clustering further.
We used AlignACE [97] to search the upstream regions of the genes in these clusters for motifs. We used the methods for operon prediction, selecting upstream regions, and applying AlignACE to prokaryotic genomes as described in McGuire et al. [77] . Briefly, because of the presence of operons in prokaryotes, we must choose the upstream region of the operon head rather than the region immediately upstream of the gene of interest. Since it is more important to include the correct region than to erroneously include extra incorrect regions, we use a loose operon definition and include sequences for several different possibilities if there is any ambiguity. We look upstream of our gene of interest and select all intergenic sequences until we encounter either a divergent intergenic region or an intergenic region longer than 300 bp.
Motifs of interest were selected by applying a set of filters: specificity score [77] , quality of alignment (AlignACE MAP score) [97] , palindromicity [77] , and conservation. To determine the degree of conservation, a search matrix was constructed for each motif. Each of the other genomes was searched with this search matrix using CompareACE, and N-way conserved sites were identified. N-way conserved hits are hits identified upstream of orthologous genes in N genomes, where orthology is defined by membership in the same SYNERGY orthogroup. To select interesting motifs we required specificity score < 1e-10, palindromicity > 0.7, MAP score > 5, and at least 8 sites conserved in 8 genomes.
Motifs were compared to a library of search matrices for 9 known Mtb motifs (Acr, Crp, CsoR, DosR, FurA, IdeR, KstR, MprAB, and ZurB), as well as a library of 55 E. coli motifs [98] and 22 Corynebacterial motifs [99] . Comparison of motifs was done using CompareACE [76] . Within each experiment, we extracted a list of genes upregulated 1.5 and 2 standard deviations above the mean. For each category, we considered a gene to be upregulated if it was upregulated in more than 50% of the experiments making up that category. We then searched for genes that were only upregulated under certain conditions or sets of conditions. We looked at the evolution of these sets of Mtb H37Rv genes by taking the other members of their orthogroups across all 31 other organisms. Evolution of these groups can be visualized in our supplementary information http://www.broadinstitute.org/ftp/pub/seq/ msc/pub/SYNERGY/index.html.
We used PAML to calculate d N /d S values according to several different evolutionary models [100, 101] . Since orthogroups contain paralogs as well as orthologs, we used the gene trees output from SYNERGY when running PAML. Some orthogroups may contain single-copy orthologs in only two closely related organisms, whereas others could contain paralogs in all 31 organisms.
For the basic model, we used the following parameters: model = 0 and getSE = 1 (to calculate standard errors). This simple evolutionary model gives one value of d N /d S for each orthogroup, averaged over all lineages as well as all positions in the gene [102] . While this model does not reflect the evolutionary history that has taken place, it is nevertheless a very blunt yet efficient tool for observing selection.
To gain insight into the evolution of the three major clades of the phylogenetic tree we also used a "branch model" where a different d N /d S value is allowed on a "foreground" branch (but d N /d S is averaged along positions in the protein) [103, 104] . This was done in PAML by using "Model = 2". We compared this model to the basic model using a log-likelihood χ 2 test with d.o.f. = 1. For each of the three foreground branches, we used a Bonferroni correction equal to the number of orthogroups present at the branch. We ran this separately for three different "foreground" branches on the phylogenetic tree (labeled in Figure 1 ): A) The branch leading to the Mtb complex; B) The branch leading to pathogenic Mycobacteria; and C) The branch leading to soil-dwelling, non-pathogenic Mycobacteria. The loglikelihood model that we use here compares this branch model to the simple model with a single value of d N /d S described above, and tests whether the model allowing d N /d S to differ on the foreground branch fits the data better than the basic model.
Branch-site models allow d N /d S to vary across branches of the tree and among sites in the protein. We also used the branch-site model of Zhang and Nielsen [100] using Model = 2, NSsites = 2, and fix_blength = 2. We used the model = 0 calculations to determine branch lengths for the branch-site model calculations to save computational time. We compared the results for a subset of the orthogroups with and without fixed tree lengths and determined there was little difference in the results). We chose the same three sets of branches (A-C) that we used for the branch model described above. We compared this model to the corresponding null model using a log-likelihood χ 2 test with d.o.f. = 1 [100] . For each of the three foreground branches, we used a Bonferroni correction equal to the number of orthogroups present at the branch. The branch-site model was the most informative.
We calculated the functional group over-representations separately for each functional group dataset. These datasets included 21 COG categories, 168 KEGG categories, 749 metabolic pathways, and 7 additional Mycobacteria-specific groupings (PE genes, PPE genes, toxinantitoxin genes, DosR regulon, esx genes, Rv0474 regulon, and the KstR regulon). We multiplied the hypergeometric p-values by a Bonferroni correction equal to the number of categories or tests performed.
We constructed whole-genome alignments of all 31 organisms, as well as subsets including only Mycobacterial organisms, and organisms within the Mtb complex. These alignments can be downloaded from our website. Our whole genome multiple alignments reveal unannotated stretches of conservation in noncoding regions including transcription factor binding sites in promoter regions, noncoding RNAs, and mis-annotated proteins.
To generate whole-genome multiple alignments, we first aligned the reference genome to each target genome in a pairwise manner. The process of pairwise whole-genome alignment consists of using Pattern-Hunter [105] to identify anchors of local alignment, grouping collinear anchors separated by a limited distance into chains, filtering out smaller chains that shadow larger ones, and finally using LAGAN [106] to globally align the entire chain. Once all genomes have been aligned to the reference, we then identified intervals of the reference that map tightly to a single interval of some or all of the target genomes, and we consider these the endpoints of blocks of multiple alignment. These blocks are generally smaller than any precursor pairwise alignment, because a rearrangement or loss of detectable similarity in any genome will truncate the block for all member genomes. We then ran the multiple aligner MLAGAN on each block. Finally, to facilitate searches for constrained regions of the reference, we projected the blocks onto the reference genome, effectively unwinding all genome rearrangements in the target genomes relative to the reference. We visualized the alignments in the Geno-meView browser [64] .
We used Gumby [65] to select conserved regions in our multiple alignments using a value of p < 0.5. In a multiple alignment of all 19 Mycobacterial genomes, we identified 4697 regions of conservation overlapping coding genes in the reference annotation, and 394 regions in intergenic regions. We also used the method of Ruzzo and Tompa [107] to identify conserved regions. Scores were normalized to the background inferred from the 3 rd-base frequencies.
For all H37Rv coding sequences, all bases in the third position were extracted from the 31-way multiple alignment. These were concatenated in a new multiple alignment only containing third bases. From this new multiple alignment we calculated the baseline conservation which is used to normalize the conservation scores for the regular alignment. Both sets of highly conserved regions can be viewed as alignment tracks for the Geno-meView browser [64] , downloadable on our website.
We predicted regions likely to form RNAs within the conserved intergenic regions of our multiple alignment of 19 Mycobacteria, using Evofold [66] . We divided the intergenic region into 240-bp segments, tiled by 80 bp, to run Evofold. Looking within intergenic regions, we identified 536 regions with Evofold (regions greater than 5 bp in length with length-normalized folding potential score > 0.2).
We examined these 536 regions, as well as the 394 conserved intergenic regions found by Gumby, to see if any of these showed significant expression in our logphase Mtb RNA-seq data. We calculated RPKM [108] values for each of these regions. We examined the regions with RPKM value ≥200 and a number of RNAseq reads ≥ 20. We eliminated an additional 35 regions which corresponded to known RNAs from the Mtb annotation, or RNAs similar to those found in M. bovis and Streptococcus [60] [61] [62] [63] , including 26 tRNAs, 2 riboswitches, and 3 found in other organisms.
To select intergenic regions with high levels of expression that do not correspond to UTRs, we also calculated RPKM values for the 100 bp regions of the flanking genes closest to the intergenic regions. We selected those intergenic regions with the highest ratio of the RPKM value of the region of interest (within the intergenic region) to the RPKM of the start/stop of the flanking genes. We also looked for regions with a gap in expression between the gene and the region of interest. This will eliminate many regions that merely correspond to UTRs, and select for regions that are disproportionately expressed within the intergenic region only. We found this method to be most useful for selecting regions of interest, and successfully enriched our top hits for previously known small RNAs. The top 50 predicted RNAs can be viewed as a track in the GenomeView browser (see Supplementary  Information) .
We further examined log-phase RNA-seq data from M. smegmatis to confirm that many of the orthologous regions also show expression in M. smegmatis.
Mycobacterium tuberculosis H37Rv and M. smegmatis were grown at 37°c in 7H9 media supplemented with 10% ADC (Becton Dickinson), 0.2% glycerol and 0.05% Tween 80. For log phase, cells were grown to OD 540 0.2. Roller bottles were used for culturing M. tuberculosis, and shaker flasks for M. smegmatis.
Bacterial pellet from log-phase cultures of M. tuberculosis and M. smegmatis were resuspended in TRIzol reagent (Invitrogen) and immediately transferred to 2 ml screw-cap tubes containing 0.1 mm zirconia/silica beads (BioSpec Products). M. tuberculosis cells were lysed using a FastPrep-24 bead-beater (MP Biomedicals) 3 times for 30 seconds each at speed 6. M. smegmatis cells were lysed using MagNalyser (Roche). Samples were kept on ice for 1 min between pulses. The TRIzol extracted RNA was treated twice with DNAse and further purified using RNAeasy kit (Qiagen).
We generated mRNA-seq libraries for sequencing on Illumina's GA Sequencer (San Diego, CA). 2 μg purified RNA was depleted of ribosomal RNA using Ambion's MICROBExpress Kit (Austin, TX) as per manufacture's recommended protocol. The enriched mRNA was used to prepare libraries using Illumina's Directional mRNAseq Library Prep v1.0 protocol. Briefly, 100 ng mRNA was fragmented with cations and heat, end-repaired, adapted by sequential ligation of unique 5-prime and 3prime adapters, reverse transcribed, PCR amplified, and purified using Agencourt's AMPure Beads (Beverly, MA). The libraries were visualized on an Agilent 2100 Bioanalyzer (Santa Clara, CA) and found to have the expected average fragment length of~250 bp.
Total RNA was isolated from Mtb as described previously [109] with minor modifications. Briefly, logphase cells were pelleted, resuspended in TRIzol (Invitrogen), and transferred to Lysing Matrix B tube (QBiogene). The cells were lysed using MagNalyser (Roche), and RNA extracted with Trizol reagent as instructed by the manufacturer. RNA was treated with Turbo DNase (Ambion) for 30 minutes at 37°C twice and purified further using TRIzol solution and 100% Ethanol.
Total RNA was separated on 10% TBE-Urea acrylamide gels (Bio-Rad) and electroblotted onto Hybond N + membranes (GE Healthcare). After UV cross-linking the membranes were pre-hybridized and hybridized with labeled probes at 48°C as per the DIG manual (Roche). Probe sequences are CGATGGTCGAAAAGGAACTCGA-TACGGCTATGCGGTTCT (RNA1), AGTTCACGA Proteasome-Dependent Disruption of the E3 Ubiquitin Ligase Anaphase-Promoting Complex by HCMV Protein pUL21a The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity. Regulation of protein degradation plays a key role in many cellular processes ranging from cell cycle progression, innate immunity, and antigen presentation to the turnover of misfolded or oxidized proteins. Most degradation is carried out by the ubiquitin-proteasome system (UPS). Ubiquitin is added to proteins by a cascade of ubiquitin conjugating enzymes, resulting in a polyubiquitinated protein which is subsequently degraded by the 26S proteasome. As a means to regulate protein function, it is no surprise that many viruses have co-opted the UPS for their own benefit. Viruses can promote proteasome degradation of antiviral host proteins either by encoding their own E3 ubiquitin ligase, targeting proteins to a cellular E3 ligase, or even inducing ubiquitin-independent degradation of targets. Examples of viral E3 ligases include the herpes simplex virus-1 protein ICP0 [1] and Kaposi's sarcoma-associated herpesvirus proteins K3 and K5 (for a review, see [2] ). Viral proteins that can hijack a cellular E3 ligase include human immunodeficiency virus-1 vpr and vif (for a review, see [3] ), paramyxovirus V [4] , and human papillomavirus E6 and E7 (for a review, see [5] ). Finally, the human cytomegalovirus (HCMV) protein pp71 uses a ubiquitin-independent mechanism to target the Rb and hDaxx proteins [6, 7] . In fact, pharmacological inhibition of the proteasome blocks multiple stages of the viral life cycle, suggesting that viruses rely on activities of the UPS for their replication [8] [9] [10] [11] [12] . On the other hand, viruses must also modulate cellular E3 ligase activity in order to replicate because ubiquitination regulates many important cellular processes central to virus infection. The SV40 large T antigen inhibits the SCF fbw7 ubiquitin ligase to increase cyclin E levels [13] , and influenza virus NS1 inhibits TRIM 25-mediated ubiquitination of RIG-I, thereby attenuating interferon production [14] .
The anaphase-promoting complex (APC) or cyclosome is a macromolecular complex that contains cullin-ring E3 ubiquitin ligase activity and is conserved across all eukaryotes (for a review, see [15] ). It has at least eleven subunits and two co-activator proteins (CDC20 (cell-division cycle protein 20) and Cdh1 (CDC20 homologue 1)), which are separated into three subcomplexes. These include the cullin-ring ligase domain (composed of APC2, 10, and 11), the specificity arm (composed of APC3, 6, 7, and 8), and the bridge (composed of APC1, 4, and 5). Cdh1 and CDC20 activate APC activity to prevent premature entry into S phase and to promote progression through mitosis, respectively. The APC complex ubiquitinates more than 40 proteins, including A-and B-type cyclins, to regulate their stability. It also regulates degradation of its own coactivator proteins, Cdh1 and CDC20, as a form of feedback regulation. Due to its central role in cell cycle progression, the APC is also a promising target for anti-cancer therapeutics [16] .
Many viruses modulate the host cell cycle to establish optimal conditions for their replication. Several viral proteins have been reported to target the APC, possibly to force the cell into an S phaselike biochemical environment to promote efficient viral replication. Proteins from adenovirus, chicken anemia virus, human papillomavirus, human T-lymphotropic virus, hepatitis B virus, parapoxvirus, and HCMV have been reported to regulate the function of the APC [17] [18] [19] [20] [21] [22] [23] [24] . However, the mechanisms used by these viruses during infection to subvert the APC are largely unknown.
HCMV is a globally important opportunistic pathogen that causes severe diseases in immunocompromised individuals and is the leading viral cause of congenital diseases. This virus stimulates cell cycle progression of quiescent cells into an S phase-like environment but concurrently blocks host DNA synthesis [25] . HCMV promotes cell cycle progression likely in part by inactivating Rb [6, 26] and regulating the APC [24, 27, 28] . It has been reported that HCMV has two means to regulate the APC. The multifunctional viral kinase pUL97 phosphorylates the APC coactivator Cdh1, thus likely inhibiting its activity [24] . Nonetheless, abrogation of UL97 alone only results in a modest increase in APC activity during infection [24] . Independent of UL97mediated Cdh1 regulation, HCMV also induces degradation of two APC subunits, APC4 and APC5, leading to the dissociation of the complex during infection [24] . The viral factor and associated mechanism responsible for regulating degradation of the APC subunits have not been identified.
In this study, we demonstrate that the HCMV protein pUL21a interacts with the APC, resulting in proteasome-dependent degradation of APC4 and APC5. Expression of pUL21a dissociates the APC cullin-ring ligase subcomplex from its specificity arm. This regulation alters APC activity and increases levels of a subset of APC-regulated cell cycle proteins. We have identified residues proline-arginine (PR109-110) in pUL21a to be critical for its ability to bind and regulate the APC. A mutant virus in which the viral ability to regulate the APC is abrogated by both alanine substitution of proline-arginine residues in pUL21a and UL97 deletion is markedly more defective compared to the pUL97 deletion virus alone. This suggests that HCMV has evolved an invasive strategy of using both viral factors to regulate the APC to facilitate its infection. Our study has identified the HCMV protein pUL21a as a novel APC regulator and elucidated a unique mechanism to subvert APC activity.
pUL21a Interacts with the Anaphase-Promoting Complex (APC) HCMV pUL21a is a 15 kDa, highly unstable protein that is expressed with early kinetics [29] . One identified function of this protein is to facilitate efficient viral DNA synthesis [30] . However, this protein shares no significant homology with any known protein. To provide mechanistic insight into its activity, we used a proteomics approach to identify interacting partners of pUL21a during infection. We created a recombinant virus (ADgfpUL21a) in which the UL21a coding sequence was tagged with the green fluorescent protein (GFP) coding sequence. This virus grew with wild-type kinetics, and the tagged protein was fortuitously much more stable than native pUL21a [29] . A GFP tag can stabilize certain fusion proteins [31] , and made it possible to detect interacting proteins in our study. We infected fibroblasts with either ADgfpUL21a or control HCMV (ADgfp) that expressed free GFP only. At 48 hours post infection (hpi), we isolated the protein complexes from infected cells by a rapid one-step immunoaffinity purification on magnetic beads coated with GFP antibody-coupled protein A. Electrophoresis analysis revealed multiple protein bands that were specific to the pUL21a-containing sample ( Figure 1A ). We analyzed pUL21a-specific protein bands by mass spectrometry and identified the proteins depicted with arrows as APC specificity arm subunits, APC3, APC7, and APC8 (Table S1) .
We validated these interactions in HCMV infected cells by coimmunoprecipitation followed by immunoblot analysis. Here we used APC3 and APC8 as the marker for the APC complex. Pulldown of pGFP-UL21a, but not the GFP control, isolated both APC3 and APC8 ( Figure 1B ). The lower band detected by APC8 antibody was nonspecific as it neither co-immunoprecipitated with APC3 antibody ( Figure 1B ) nor was affected by shRNA knockdown of APC8 ( Figure S1 ). In the reciprocal experiment, APC3 antibody co-immunoprecipitated APC8 and pGFP-UL21a but not GFP. Neither GFP nor APC3 antibody co-immunoprecipitated cellular PCNA ( Figure 1B) , and an antibody against HA did not co-immunoprecipitate any of the proteins detected here (data not shown), thus providing additional evidence for the specificity of these interactions. As pGFP-UL21a is co-immunoprecipitated with multiple APC subunits, we interpret the result to suggest that pUL21a binds to the APC complex, even though the precise subunit where pUL21a directly interacts with remains unknown.
To determine if this interaction also occurred with native pUL21a, we performed co-immunoprecipitation assays on lysates from cells infected with wild-type virus (ADgfp), and we also included lysate from cells infected with UL21a deletion virus (ADsubUL21a) as a negative control ( Figure 1C ). Infected cells were treated with proteasome inhibitor, MG132, as pUL21a was highly unstable and otherwise could not accumulate to levels allowing reproducible detection of this interaction [29] . In the
In this study, we report an intriguing mechanism used by human cytomegalovirus (HCMV) to regulate a cellular E3 ubiquitin ligase, the anaphase promoting complex (APC). The ability to hijack the ubiquitin-proteasome system for regulating protein degradation and to manipulate the cell cycle for viral genome synthesis is critical in many viral infections. The APC is a master cell cycle modulator that targets a number of regulatory proteins for proteasomal degradation. It can prevent cells from entry into S-phase, thus creating a hindrance for viruses needing to coerce cells into a cellular environment favorable for viral DNA synthesis. We have identified an HCMV protein, pUL21a, which uses a seemingly counterintuitive mechanism to regulate the APC. It interacts with the APC to target the subunits of this ubiquitin ligase for proteasomal degradation. This causes disruption of the complex and reduces its activity. Furthermore, a virus lacking pUL21a and pUL97, which is another HCMV-encoded APC regulator, was highly attenuated when compared to loss of UL97 alone, suggesting that HCMV uses two proteins to fully disarm the APC. This study identifies a herpesviral protein that uses a unique, proteasome-dependent mechanism to regulate the activity of this prominent cellular E3 ubiquitin ligase. presence of MG132, the level of native pUL21a was markedly increased and could be co-immunoprecipitated with APC3 antibody. This interaction was specific as the antibody did not co-immunoprecipitate PCNA or the viral DNA polymerase accessory factor UL44.
To test if pUL21a was able to bind to the APC in the absence of other HCMV proteins, we performed co-immunoprecipitation assay on lysates from 293T cells transfected with constructs expressing the GFP-amino terminal tagged UL21a (gfpUL21a wt ) or UL21a carrying two stop codons at its amino terminus to abrogate pUL21a expression (gfpUL21a stop ) ( Figure 1D ). Both gfpUL21a wt and gfpUL21a stop were expressed at equal levels but only gfpUL21a wt associated with APC3 or APC8. Additionally, APC3 antibody co-immunoprecipitated gfpUL21a wt but not gfpUL21a stop . We conclude that pUL21a interacts with the APC and does not require other HCMV proteins for this interaction to occur. at an MOI of 5, collected at 48 hpi, and were immunoprecipitated with GFP antibody. Eluted proteins were run on an SDS-containing polyacrylamide gel and silver stained. The bands indicated with an arrow were identified by mass spectrometry as APC3 (100 kDa), APC7, and APC8 (both at 65 kDa). (B) GFP-tagged pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected as described in panel A, and lysates were subjected to coimmunoprecipitation with GFP or APC3 mouse monoclonal antibodies. Cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. GFP blots were cropped to save space but were from the same lane and exposed film. Non-specific cross-reacting bands are indicated by asterisk (see text). Partial proteolysis was often seen with the GFP-tagged UL21a protein, particularly in cell lysate samples. (C) Native pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected with ADgfp or ADsubUL21a (as described in Materials and Methods) in the presence (+) or absence (2) of 10 mM MG132. Cell lysates were prepared at 24 hpi and immunoprecipitated with APC3 antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. (D) Interaction of pUL21a with the APC does not require other viral proteins. 293T cells were transfected with plasmids expressing gfpUL21a wt or gfpUL21a stop . Cells were collected 72 hours later and cell lysates were immunoprecipitated as in panel B. Cell lysates and eluted proteins were analyzed by immunoblotting. PCNA and pUL44 were used as cellular and viral negative controls, respectively. doi:10.1371/journal.ppat.1002789.g001
The Carboxyl-Terminus of pUL21a Contains the APC Binding Site
To begin understanding the nature of this interaction, we identified the APC-binding domain of pUL21a. Sequence alignment of pUL21a with its homologues in chimpanzee CMV (CCMV) and Rhesus CMV (RhCMV) revealed a highly conserved N-terminus (residues 1-47), divergent middle region (residues 48-83), and C-terminus that contained several conserved residues (residues 84-123), including a proline-arginine (PR) pair at residues 109-110 ( Figure 2A ). We created a series of truncation mutations targeting each region in the GFP-tagged pUL21a, and tested the ability of mutant UL21a proteins to interact with the APC in 293T cells ( Figure 2B ). All mutants were expressed at similar levels and were efficiently immunoprecipitated by the GFP antibody ( Figure 2C , and data not shown). As expected, full-length gfpUL21a wt co-immunoprecipitated both APC3 and APC8 while the gfpUL21a stop mutant did not. Importantly, while the carboxylterminal fragment of pUL21a consistently co-immunoprecipitated APC3 and APC8, the amino-terminal and middle fragments were unable to do so. Thus the carboxyl-terminus of pUL21a contains the APC binding domain.
To define the precise sequence of the APC binding site, we made gfpUL21a mutants in which each of five conserved residue clusters within its carboxyl terminus were individually substituted with alanine residues (Figure 2A ). As a control, we also made alanine substitutions for the non-conserved proline-histidine pair at residues 111-112 (PH111-112AA) ( Figure 2A ). All mutants were stable and were efficiently pulled down by the GFP antibody ( Figure 2D , and data not shown). Among them, only the PR109-110AA mutant lost the ability to bind to the APC. Substitutions of the adjoining non-conserved residues (PH111-112AA) had no effect on APC binding. To validate the result in the context of infection, we constructed recombinant HCMV viruses expressing GFP-tagged or native forms of PR109-110AA or PH111-112AA pUL21a variants (ADgfpUL21a PR-AA , ADgfpUL21a PH-AA , AD-pmUL21a PR-AA , and ADpmUL21a PH-AA ). During infection, a reciprocal interaction between gfpUL21a PH-AA and APC3 could be detected while gfpUL21a PR-AA and APC3 did not interact ( Figure 2E ). Furthermore, untagged pUL21a PH-AA , but not pUL21a PR-AA , was co-immunoprecipitated with APC3 when stabilized by MG132 ( Figure S2 ). Together, these results indicate that the carboxyl terminus of pUL21a contains the APC binding domain and the residues PR 109-110 are critical for this binding.
It has recently been reported that the APC bridge subunits APC4 and APC5 are degraded during HCMV infection and the complex dissociates [24] . To test if pUL21a was required for these events, we first examined APC subunit accumulation during infection with or without pUL21a. Levels of APC4 and APC5 proteins were markedly reduced during wild-type infection relative to mock-infected cells at 24 hpi ( Figure 3A ). However, no reduction was observed in APC4 and APC5 levels during infection with the UL21a-deletion virus. The pUL21a-deficient virus fails to express late viral genes due to a defect in viral DNA synthesis [30] . To rule out any role of late genes in APC4 and APC5 degradation, we treated infected cells with phosphonoacetic acid (PAA) to block viral DNA synthesis and late gene expression. APC4 and APC5 levels were reduced during infection with wild-type virus but remained elevated during infection with the UL21a-deletion virus, even following PAA treatment. Furthermore, there was no appreciable difference in APC4 and APC5 transcript levels between wild-type and deletion virus infections ( Figure 3B ). These data suggest that the changes in APC4 and APC5 protein levels occur at the level of protein stability. Consistent with this hypothesis, MG132 enhanced APC4 and APC5 protein levels during infection with wild-type but not deletion virus ( Figure 3C ). Thus, pUL21a-mediated loss of APC4 and APC5 was due to proteasomal degradation. Moreover, the APC binding mutant virus ADpmUL21a PR-AA was unable to degrade APC4 and APC5 while the ADpmUL21a PH-AA virus was as efficient as the wild-type control virus. These data support the conclusion that pUL21a binding to the APC promotes proteasomal degradation of APC4 and APC5.
We next tested if the APC binding ability of pUL21a was also required for APC dissociation during infection. In this experiment, we used APC3 and APC10 as the marker for the specificity arm and cullin-ring ligase subcomplex of the APC, respectively. These two subcomplexes sit on opposite sides of the APC. APC10 has been proposed to bind APC substrates along with coactivator proteins, including Cdh1 [32] . APC10 associates with APC2 and APC11 of the ligase subcomplex, but its location in the inner cavity of the APC allows for contact with APC3 and APC6 of the specificity arm. In cells infected with ADpmUL21a PR-AA , total levels of APC3 and APC10 were similar to those in cells infected with ADpmUL21a PH-AA , allowing for a direct analysis of the efficiency of their association with the complex ( Figure 3D ). APC3 could not co-immunoprecipitate APC10 in ADpmUL21a PH-AAinfected cells, consistent with dissociation of the complex in the presence of functional pUL21a. In cells infected with ADpmU-L21a PR-AA , APC3 was able to pull down APC10 efficiently, indicating that the two subcomplexes remained associated. Finally, the integrity of the APC during ADpmUL21a PH-AA infection was largely restored upon addition of MG132, even though total protein levels were reduced likely due to MG132-induced cell death ( Figure 3D , and data not shown). These data were recapitulated during infection of wild-type and UL21a deletion viruses ( Figure S3 ). Our data provides strong evidence supporting the model that binding of pUL21a to the APC induces degradation of the APC bridge arm resulting in complex dissociation.
As APC8 was co-immunoprecipitated with pUL21a in our original screen, it raised the possibility that pUL21a might require APC8 to target APC4 and APC5. For instance, pUL21a might bind to APC8 to disrupt the structure of the APC leading to APC4 and APC5 degradation, or it might use APC8 as a docking site for recruiting protein degradation enzymes to target APC4 and APC5. To test this, we depleted APC8 in these cells by shRNA knockdown ( Figure S4 ). Following shRNA depletion of APC8, the APC4 and APC5 levels remained reduced in cells infected with wild-type virus compared to those with UL21a-deletion virus, even though APC knockdown did seem to affect the overall stability of APC4 and APC5 in pUL21a-independent manner ( Figure S4 ). This suggests that pUL21a-mediated degradation of APC4 and APC5 is independent of APC8.
To determine the functional consequence of pUL21a-dependent APC dissociation, we first analyzed the accumulation of APC substrates during wild-type or UL21a-deletion virus infection. The protein levels of APC substrates Cdh1 (that is also an APC coactivator) and geminin were markedly increased in wild-type virus infection as previously reported [27, 33] ( Figure 4A ). However, their levels were reduced during infection with the UL21a-deletion virus, suggesting increased APC activity. The geminin transcript accumulated to wild-type levels even without pUL21a, providing evidence that the difference in protein accumulation was not due to transcriptional regulation ( Figure 4B ). PAA treatment had no effect on substrate accumulation, ruling out pUL21a-mediated late gene expression as the source of the observed phenotype ( Figure  S5A ). MG132 largely restored substrate levels during UL21a deletion viral infection, indicating that the difference is likely due to increased proteasome degradation ( Figure 4C ). These results were also recapitulated during infection of APC binding mutant virus ADpmUL21a PR-AA and its control virus ADpmUL21a PH-AA ( Figure 4C ).
To confirm that decreased APC substrate accumulation during mutant virus infection was due to APC activity, we used shRNAs to knock down APC8 or the coactivator Cdh1 to deplete APC activity. Both APC8 and Cdh1 shRNAs efficiently reduced expression of their respective targets ( Figures 4D and S5B) . Importantly, APC8 knockdown restored geminin and Cdh1 levels in cells infected with ADpmUL21a PR-AA or ADsubUL21a virus to those with ADpmUL21a PH-AA or ADgfp virus. Likewise, Cdh1 knockdown restored geminin levels in cells infected with the pUL21a-deficient viruses. Thus, our results indicate that pUL21a association with the APC allows it to target APC4 and APC5 subunits for degradation to alter APC activity during infection.
It is noteworthy that not all APC substrates were subjected to pUL21a-mediated regulation. We did not observe significant difference in Cdc6 or a drastic reduction in thymidine kinase protein levels in the UL21a mutant relative to wild-type viral infection (data not shown). It is possible that these APC substrates are regulated by multiple mechanisms, including APC-independent viral regulation, pUL21a-mediated alteration in APC substrate specificity, and pUL97-mediated phosphorylation of the APC coactivator Cdh1. In fact, Cdh1 from both wild type and UL21a mutant virus infected cells migrated slower in an SDS-PAGE gel compared to that from mock cells, which was previously shown to be due to phosphorylation ( Figure 4E ) [28] . Therefore, virus-induced, Cdh1 phosphorylation-mediated APC regulation appears intact even without pUL21a during HCMV infection.
As the APC prevents the premature entry of the cell cycle into S phase, we predicted that increased APC activity in the absence of pUL21a would not compromise the ability of HCMV to arrest infected cells at G1/S phase boundary. Consistent with this hypothesis, cells infected with wild type, ADpmUL21a PH-AA , or ADpmUL21a PR-AA virus showed indistinguishable cell cycle profiles throughout infection, with the majority of cells phenotypically arrested in G1 phase ( Figure S6 ). To test if pUL21a was sufficient to alter APC activity, we first analyzed 293T cells that over-expressed pUL21a by transient transfection. Expression of pUL21a alone was sufficient to markedly reduce the levels of APC4 and APC5 ( Figure S7A) , and as expected, geminin and Cdh1 levels were elevated in these cells. These pUL21a-expressing cells were largely arrested in G2/ M phase ( Figure S7B ), failed to multiply, and ultimately died ( Figure S7C ). The biological characteristics of pUL21a-expressing cells are therefore consistent with reduced APC activity.
To more precisely test if pUL21a was able to regulate the APC in the absence of other HCMV proteins, we developed an inducible pUL21a expression system. We constructed a HeLa cell line stably expressing a GFP-tagged TetR (tetracycline repressor) gene. We then transduced this cell line with lentiviruses expressing pUL21a stop , pUL21a PH-AA , or pUL21a PR-AA under a CMV-TetO (tetracycline operator) promoter. pUL21a protein accumulation was only detected in the presence of tetracycline, suggesting tight regulation of pUL21a expression ( Figure 5A ), although its levels were significantly lower than those expressed in transiently transfected cells ( Figure S7A) . Importantly, the addition of tetracycline significantly reduced APC4 and APC5 protein levels in cells expressing pUL21a PH-AA , but not pUL21a stop or pUL21a PR-AA . To assess the consequence of pUL21a on APC activity, we synchronized cells expressing pUL21a PH-AA (i.e. wild-type pUL21a) in mitosis with nocodazole and then assayed their ability to progress out of mitosis after release from nocodazole treatment. In the absence of tetracycline and pUL21a, cells readily progressed through the mitotic phase following release. In the experiment shown in Figure 5B , 26% and 48% of cells entered the next G1 phase by 2 and 4 hours, respectively. In the presence of tetracycline where pUL21a was expressed, progression through the mitotic phase was clearly delayed. As the result, only 5% and 24% of cells reached G1 by 2 and 4 hours, even though by 8 hours most of pUL21a-expressing cells were able to enter G1, likely due to low expression of pUL21a in these cells as compared to those in transiently transfected cells. Additionally, following nocodazole withdrawal, APC substrates geminin and cyclin B1 remained elevated in the presence of tetracycline while their levels were reduced in its absence ( Figure 5C ). Our results provide strong evidence that pUL21a expression alone is sufficient to regulate APC activity.
In the final experiments, we tested the consequence of pUL21amediated APC regulation on HCMV replication in fibroblasts. We first tested if the ability of pUL21a to regulate the APC would be responsible for its previously reported role in promoting viral DNA replication [30] . We compared the growth of ADpmUL21a PR-AA mutant virus (i.e. pUL21a APC-binding deficient) to that of wildtype, ADpmUL21a PH-AA (i.e. pUL21a APC-binding competent), or UL21a deletion viruses in multi-step growth curve analysis. We found that ADpmUL21a PR-AA grew indistinguishably from wildtype and ADpmUL21a PH-AA viruses in both cycling and G0synchronized fibroblasts, whereas the UL21a deletion virus had a 100-fold defect ( Figure 6A ) [29] . Furthermore, knockdown of Cdh1 and APC8 was unable to enhance UL21a-deletion virus replication (data not shown). This suggests that pUL21a has at least two independent activities. One is to facilitate viral DNA replication via an unknown mechanism and is responsible for the growth defect of UL21a deletion virus. The second activity is to regulate the APC, whose impact on virus replication is not apparent under the aforementioned experimental conditions.
As two HCMV proteins, pUL97 and pUL21a, are capable of regulating the APC, we hypothesized that one of these two proteins acted to compensate for the loss of the other during infection. Consistent with this hypothesis, HCMV appeared to retain the ability, at least to some extent, to regulate the APC even when pUL21a or pUL97 is absent ( Figure 4E , and data not shown) [24] . To test this hypothesis more directly, we created recombinant HCMV viruses ADpmUL21a PH-AA /subUL97 and ADpmU-L21a PR-AA /subUL97. The two viruses were derived from AD-pmUL21a PH-AA and ADpmUL21a PR-AA , respectively, and both contained an additional deletion in UL97. Both recombinant viruses grew slower than wild-type virus due to lack of the multifunctional pUL97 protein ( Figure 6C ). However, reconstitution of ADpmUL21a PR-AA /subUL97 that lacked pUL21a APCbinding activity following BAC transfection was markedly slower than that of ADpmUL21a PH-AA /subUL97 ( Figure 6B ). At day 25 post transfection, while cells transfected with the BAC clone of ADpmUL21a PH-AA /subUL97 showed nearly 100% of CPE indicated by virus-driven GFP expression, GFP-positive foci in cells transfected with the BAC clone of ADpmUL21a PR-AA /subUL97 were distinctly smaller. Furthermore, multi-step growth curve analysis showed that titers of ADpmUL21a PR-AA /subUL97 were 13-and 14-fold lower than that of ADpmUL21a PH-AA /subUL97 at 14 and 21 days post infection (dpi), respectively ( Figure 6C ). As a control to show that this phenotype was not due to general viral attenuation resulting from the UL97 deletion, we also constructed double mutant viruses ADpmUL21a PH-AA /subUL117 and AD-pmUL21a PR-AA /subUL117. These two viruses were derived similarly from ADpmUL21a PH-AA and ADpmUL21a PR-AA , but also contained a deletion in viral gene UL117. We chose UL117 as the control because its mutation attenuated virus growth but not viral early or early-late gene expression so UL97 expression was unlikely affected [34] . BAC transfection reconstituted both mutant viruses at similar efficiency and produced viruses with similar titers (data not shown). Multi-step growth analysis demonstrated that ADpmUL21a PH-AA /subUL117 and ADpmUL21a PR-AA /subUL117 Figure 6 . Abrogation of both pUL21a APC regulatory activity and pUL97 results in a more severe attenuation in HCMV growth than pUL97 deletion alone. (A) Abrogation of pUL21a-APC binding alone is not sufficient to alter HCMV replication. MRC-5 cells in serum-containing (cycling condition) or serum-free (G0 condition) media were infected with ADgfp, ADsubUL21a, ADpmUL21a PH-AA , or ADpmUL21a PR-AA at an MOI of 0.01. Production of cell-free virus at indicated times was determined by plaque assay. (B) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced the efficiency of HCMV reconstitution as compared to abrogation of UL97 alone. To reconstitute ADpmUL21a PR-AA /subUL97 and pADpmUL21a PH-AA /subUL97 viruses, MRC-5 fibroblasts were transfected with their corresponding BAC clones. For each recombinant virus, three independent clones were tested. Shown are representative images of virus spread indicated by virus-driven GFP expression at indicated days post transfection of two of the three clones. Images were taken under a Leica fluorescent microscope. (C) Abrogation of both UL97 and the pUL21a-APC binding site markedly reduced HCMV replication as compared to abrogation of UL97 alone. MRC-5 cells were infected with indicated recombinant viruses at an input genome number equivalent to that of 0.03 infectious units of wild type virus/cell. Production of cell-free virion DNA at indicated times was determined by qPCR analysis and normalized to input levels of ADpmUL21a PH-AA , which was set to 1. (D) Multi-step growth analysis of double mutant viruses that carried the UL117 deletion and point mutation in the UL21a-APC binding site. Cells were infected with indicated recombinant viruses and analyzed as described in panel C. The input value of ADgfp was set to 1. doi:10.1371/journal.ppat.1002789.g006 replicated at similar kinetics ( Figure 6D ). At 14 dpi, the titer of ADpmUL21a PR-AA /subUL117 was slightly lower (e.g. 1.5-fold) than that of ADpmUL21a PH-AA /subUL117. However, growth of mutant virus carrying only the UL117 deletion tracked with ADpmUL21a PR-AA /subUL117, suggesting that the difference between the PH and PR mutants at 14 dpi, if any, is minimal. Together, our data provide evidence that disruptions of both pUL97 and the APC regulatory activity of pUL21a are synthetically lethal to HCMV replication. This is consistent with a working model that these two functions enable HCMV to cope with APC activity to promote virus replication (Figure 7) .
In sum, we have shown that the HCMV protein pUL21a antagonizes the APC by promoting proteasome-mediated disruption of this prominent cellular E3 ubiquitin ligase.
HCMV has been shown to have two different means to regulate the anaphase-promoting complex (APC) during infection [24, 27, 28] . It can induce phosphorylation of APC co-activator Cdh1, and it induces dissociation of the complex by promoting proteasomal degradation of two components of the bridge subcomplex, APC4 and APC5. The viral protein pUL97 appears to be responsible for Cdh1 phosphorylation [24] . However, pUL97 is an HCMV-encoded kinase that has many reported roles [26, 35] . How this particular pUL97 activity impacts HCMV infection remains elusive. Importantly, the viral factor or precise molecular mechanism mediating APC4 and APC5 degradation has not been identified, and how APC disruption contributes to HCMV replication is not known. Here, we have identified the HCMV protein pUL21a as the viral factor that mediates APC disruption. It does so by interacting with the APC and inducing proteasome-dependent degradation of APC4 and APC5, which results in complex dissociation. This is the first identified viral protein that modulates the APC in this manner. We also show, for the first time, the impact of viral modulation of the APC, particularly by pUL21a, on HCMV replication. Loss of pUL21amediated APC regulation has minimal impact on virus replication but the combined loss of both pUL97-and pUL21a-mediated regulation markedly attenuates growth of the virus relative to single loss of pUL21a-or pUL97-mediated regulation. Our studies support a working model in which HCMV uses pUL97mediated Cdh1 phosphorylation and pUL21a-mediated complex disruption to control APC activity for efficient virus infection (Figure 7 ). Why has HCMV developed these two distinct mechanisms that seemingly lead to a similar biological consequence? It is possible that these two mechanisms have differential roles in HCMV infection under different conditions or in particular cell types, even though either one seems sufficient and can compensate for loss of the other in fibroblasts. Alternatively, it is possible that these two mechanisms serve as the fallback for one another or act synergistically to maximize the ability of the virus to acquire a complete control of the APC during infection. In any event, the fact that HCMV uses multiple means to subvert the APC underlines its critical role in HCMV infection. This is particularly true for large DNA viruses such as HCMV, which often encode multiple viral factors to regulate the same or related cellular targets central to their infection [36] . However, it is often challenging to dissect these intertwined viral mechanisms during infection because of the presence of other factors targeting the same process. The regulation of the APC represents one such critical but complex viral regulatory strategy, and our studies shed light into its role and mechanism during HCMV infection.
Several viral factors from different viral families have been reported to use diverse mechanisms to regulate the APC. For instance, the human papillomavirus E2 protein binds to and inhibits the Cdh1 activator protein [20] , while the parapoxvirus virus protein PACR (poxviral APC regulator) functions as an enzymatically inactive APC11 mimic [23, 37] . The chicken anemia virus (CAV) protein apoptin can bind to the APC at the bridge and cause its dissociation using an unknown mechanism [19] . The fact that proteins from both HCMV and CAV target the APC bridge subcomplex suggests that viruses have evolved regulatory strategies converging on this sub-complex as an efficient means to disable APC activity. It is intriguing to speculate that modulating the APC complex by dissolving the bridge may allow viruses to alter substrate specificity of the APC instead of completely abolishing its activity, as the enzymatic portion of APC is known to have activity in vitro [23, 38] . HCMV does not appear to directly destroy the enzymatic subcomplex of APC, so it is of interest to determine if the APC retains some activity or is directed to target different substrates during virus infection.
Several viral proteins have now been reported to regulate the APC in overexpression, and evidence correlating the role of these factors and viral replication is emerging. Deletion of the parapoxvirus PACR or CAV protein apoptin markedly attenuated virus growth in tissue culture even though their ability and role in inhibiting the APC during infection has not been clarified [23, 39] . Recently, the UL97 kinase of HCMV has been shown to phosphorylate Cdh1 and partially inhibit the APC during infection but with unknown consequences for viral replication [24] . Our study elucidates the mechanism by which pUL21a regulates APC in the context of virus infection and indicates a role of this pUL21a activity in viral replication. Mutation abolishing the APC binding activity of pUL21a had no impact on viral growth in tissue culture, but the loss of both pUL21a-APC binding and pUL97 markedly attenuated viral replication relative to the loss of pUL97 alone. Our data suggest that HCMV has evolved a sophisticated strategy by encoding both pUL97 and pUL21a to overcome APC activity. However, further experiments are needed to unequivocally demonstrate the vital role of APC regulation in HCMV replication and provide mechanistic insight into how this regulation impacts its biology.
How does pUL21a target APC4 and APC5 for proteasome degradation? pUL21a does not contain a sequence domain that would suggest it as an E3 ligase, thus likely ruling out this possibility. Currently, we also do not know which subunit of the APC complex that pUL21a directly binds to so the precise mechanism that it uses to degrade APC4 and APC5 remains elusive. It is certainly possible that pUL21a may bind to a subunit neighboring to APC4 and APC5 so it can disrupt the APC structure leading to APC4 and APC5 degradation, or recruit a protein degradation enzyme (e.g. E3 ubiquitin ligase) to destabilize the subunits. However, knockdown of APC8 does not abrogate the ability of pUL21a to degrade APC4 and APC5, suggesting that APC8 is not involved and the presence of the entire complex is not required. Intriguingly, pUL21a itself is a highly unstable protein and likely degraded in a ubiquitin-independent manner [29, 40] . It is tempting to speculate that pUL21a may directly bind APC4 and APC5 and target them for degradation in a ubiquitin-independent manner. One focus of future work is to identify the APC component that pUL21a directly binds to and elucidate the mechanism of how pUL21a targets APC4 and APC5 to the proteasome.
What would be the benefit for the virus to alter APC activity? The APC may restrict HCMV replication via several mechanisms. The APC not only promotes cell cycle progression through M phase, it also prevents cells from prematurely entering S phase. Thus virus-mediated APC regulation may help HCMV maintain an S phase-like cellular environment for viral replication. The APC targets more than 40 proteins for degradation, so it may deplete host factors critical to viral replication. Consequently, viruses may need to alter the substrate specificity of the APC or allow accumulation of APC substrates critical for viral replication. Interestingly, the only viruses within the poxvirus and herpesvirus families that are known to modulate the APC (e.g. parapoxviruses and HCMV) are those that do not encode viral thymidine kinase (TK) and ribonucleotide reductase subunit M2 (RRM2). Both enzymes are APC substrates and critical for the production of deoxyribonucleotides. It is tempting to speculate that this viral regulation of the APC may provide viruses a means to produce sufficient nucleotides to replicate their genome [23, 27] . Nonetheless, the APC also targets proteins involved in cellular DNA synthesis, glycolysis and glutaminolysis, and cell cycle progression, all of which could impact viral replication [41] . Moreover, the APC may also promote ubiquitination and degradation of viral proteins to restrict infection [42] . Several HCMV proteins contain a putative destruction Box (D-box) motif, an APC recognition signal commonly found in its substrates [24] . Future work is needed to differentiate these possibilities and unravel the APC substrates that may be critical for viral replication.
Insight into the mechanism of pUL21a-mediated APC regulation may also have broad impact on cancer and neuronal disease. Due to its essential role in cell cycle progression, the APC is a promising target for novel anti-cancer therapeutics [16, 43] . In fact, we found in this study that overexpression of pUL21a essentially prevented the proliferation of a transformed cell line ( Figure S7 ), suggesting that pUL21a regulation of the APC could inhibit cancer cell growth. Furthermore, several recent studies have also highlighted a vital role of the APC in neuronal development (for a review, see [44] ). HCMV infects neuronal cells and congenital HCMV infection leads to neuronal disease and severe complications such as blindness, hearing loss, and mental retardation. It is reasonable to speculate that inhibition of the APC by pUL21a may play a role in promoting neuronal disease in congenitally infected infants. Therefore, an understanding of pUL21a-APC interaction may reveal novel mechanisms of APC assembly and regulation, give further impetus to target the APC for anti-cancer therapies, and uncover new insights into the molecular basis of HCMV pathogenesis.
Primary embryonic lung fibroblasts (MRC-5), human newborn foreskin fibroblasts (HFFs), 293T, and Hela cells were propagated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, non-essential amino acids, and penicillin-streptomycin. Transient transfection of expression constructs were carried out using lipofectamine according to the manufacturers' instructions.
pYD-C235 is a pLPCX-derived retroviral vector (Clontech) that expresses a DsRed gene driven by an internal ribosome entry site 2 (IRES2) [45] . pYD-C474 was created by PCR amplifying the coding sequence of the pGFP-UL21a fusion protein from pADgfpUL21a (see below) and ligating it into the multiple cloning site of pYD-C235. pYD-C580 was created by replacing the coding sequence of wild-type UL21a in pYD-C474 with that of mutant UL21a carrying two stop-codon mutations at the N-terminus (i.e. UL21a stop ) [30] . Vectors expressing pGFP-UL21a truncation mutants were derived from pYD-C235 while vectors expressing point mutants were derived from pYD-C474. Truncation mutants were made by PCR amplifying the targeted UL21a coding sequences and point mutants were created using a QuickChange XL kit (Stratagene). Primers used to create these mutants are listed in Table S2 . pYD-C160, pYD-C175, and pYD-C682 are pRetro-EBNA derived retroviral expression vectors that expressed GFP, UL21a, and UL21a stop , respectively. pYD-C648 and pYD-C649 are pLKO-based lentiviral vectors expressing GFP-TetR and carrying the CMV-TetO 2 promoter, respectively (generous gifts from Roger Everett, University of Glasgow Centre for Viral Research) [46] . YD-C665, YD-C667, and YD-C669 are lentiviral expression vectors created by cloning the UL21a stop , UL21a PH-AA , and UL21a PR-AA sequences into the multiple cloning site of YD-C649. To produce pLKO-based lentiviruses, 293T cells were transfected with corresponding pLKO vectors along with packaging plasmids. Lentivirus was collected at 48 and 72 hours and used to transduce MRC-5 cells. To create GFP-TetR expressing stable cells, Hela cells were transduced with pYD-C648 derived lentivirus and sorted for GFP expression 48 hours later. GFP-positive cells were collected, grown in the presence of G418 (500 mg/ml), and frozen as cells stably expressing GFP-TetR. These stable cells were then transduced with lentivirus derived from YD-C665, YD-C667, and YD-C669, selected with puromycin (2 mg/ml), and tested for tetracycline (1 mg/ml)-regulated expression of targeted genes.
For shRNA knockdown, MRC-5 cells were transduced with pLKO-based lentivirus expressing shRNA against the targeted gene for 48 hours. The shRNA sequence for Cdh1 knockdown was 59CCAGTCAGAACCGGAAAGCCA39 and the shRNA sequence for APC8 knockdown was 59GCAGGAGGTAA-TATGCTATAA39. All pLKO-based shRNA lentiviral vectors were purchased from the Washington University Children's Discovery Institute/Genome Center.
The primary antibodies used in this study included anti-b actin (AC-15, Abcam); anti-HA (HA.11, Covance); anti-GFP (3E6 and A6455, Invitrogen); anti-APC3 (AF3.1, Santa Cruz and 610454, BD); anti-APC8 (6114, Biolegend); anti-APC4 (A301-176A, Bethyl laboratories); anti-APC5 (A301-026A, Bethyl laboratories); antigeminin (sc-13015, Santa Cruz); anti-Cdh1 (DH01, Calbiochem); anti-cyclin B1 (ms868 P1, Thermo-Scientific); anti-UL21a [29] ; anti-IE2 (mAB8140, Chemicon); and anti-IE1 and anti-pp28 (generous gifts from Thomas Shenk, Princeton University) [45] . Phosphonoacetic acid (PAA), MG132, tetracycline, gancyclovir (GCV), and propidium iodide (PI) were purchased from Sigma-Aldrich. Lipofectamine 2000 and Protein A-conjugated Dynabeads were purchased from Invitrogen.
Recombinant HCMV AD169 viruses were reconstituted from transfection of corresponding BAC-HCMV clones as previously described [34] . Viral stocks were prepared by ultra-centrifugation of infected culture supernatant through 20% D-sorbitol cushion and re-suspending pelleted virus in serum-free medium. The following BAC-HCMV clones were used in the present study, and were constructed using PCR-based linear recombination as previously reported [29] , unless indicated otherwise. pAD-GFP, which carried the GFP-tagged genome of the HCMV AD169 strain, was used to produce wild-type virus ADgfp [45] . pADgfpUL21a, which carried an N-terminally GFP-tagged version of pUL21a, was used to produce ADgfpUL21a virus [29] . pADsubUL21a, which carried a GalK/kanamycin dual mutagenic cassette in place of the UL21a coding sequence, was used to produce UL21a-deletion virus ADsubUL21a [29] . pADgfpU-L21a PR-AA , pADgfpUL21a PH-AA , pADpmUL21a PR-AA , or pADp-mUL21a PH-AA carried point mutation PR109-110AA or PH111-112AA in the GFP tagged or native UL21a gene, respectively. These recombinant BAC clones were used to produce corresponding point mutant viruses. pADpmUL21a PH-AA /subUL97 and pADpmUL21a PR-AA /subUL97 carried the GalK/kanamycin mutagenic cassette in place of UL97 on the background of pADpmUL21a PR-AA and pADpmUL21a PH-AA BAC clones. Similarly, pADpmUL21a PH-AA /subUL117, pADpmUL21a PR-AA / subUL117, and pADsubUL117 carried the GalK/kanamycin mutagenic cassette in place of UL117 on the background of pADpmUL21a PR-AA , pADpmUL21a PH-AA , and pAD-GFP BAC clones, respectively. All BACs were confirmed by restriction digestion, PCR, and sequencing. HCMV virus titers were determined in duplicate in HFFs by tissue culture infectious dose 50 (TCID 50 ) assay or plaque assay. Relative viral genome numbers were determined by real-time quantitative PCR (qPCR) as described previously [29] .
For most infections, subconfluent MRC-5 cells in serumcontaining medium were inoculated with recombinant HCMV virus at an input genome number equivalent to that of 3-5 infectious units of wild type virus/cell for 1 hour, unless otherwise indicated. Inoculum was removed and fresh medium was replenished. For infection of G0-synchronized cells, MRC-5 cells were incubated in serum-free medium for 72 hours, infected as described above, and maintained in serum-free media throughout the infection. For shRNA knockdown experiments, subconfluent MRC-5 cells were transduced with lentivirus for 24 hours, incubated in fresh medium for additional 48 hours, and infected as described above. When necessary, PAA (100 mg/ml) was added immediately following infection, and MG132 (10 mM) was added 12-14 hours prior to harvest. For viral growth analysis, virus production in the media of infected cultures was determined by TCID 50 , plaque assay, or qPCR. For qPCR analysis, virion DNA was prepared as previously described [29] . Briefly, cell-free supernatants were treated with DNase I to remove contaminating DNA, and virions were lysed with proteinase K and SDS. DNA was extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The DNA was subjected to qPCR using primers and a taqman probe specific for UL54.
For immunoprecipitation, frozen cell pellets were lysed in lysis buffer (0.5% NP-40, 50 mM Tris-Cl pH 8.0, 125 mM NaCl, supplemented with protease and phosphatase inhibitors) using an end-over-end rotator at 4uC for 30 minutes. Cell extracts were cleared by centrifugation at 16,0006 g for 15 minutes. Supernatants were incubated with protein A-coated Dynabeads that were coupled to 1 mg anti-HA (HA.11, Covance), 1 mg anti-GFP (3E6, Invitrogen) or 2 mg anti-APC3 (AF3.1, Santa Cruz) mouse monoclonal antibodies at 4uC for 1-2 hours. Beads were washed with PBS and immunoprecipitated protein complexes were eluted by boiling beads in reducing sample buffer for 5 minutes. Cell extracts (pre-IP) were also collected and boiled in reducing sample buffer. For mass spectrometry analysis, protein complexes were resolved by SDS-polyacrylamide gel electrophoresis (Invitrogen) followed by staining with a silver stain kit (Sigma-Aldrich). Protein bands specific to immunoprecipitated pUL21a complex were excised for identification by MS/MS mass spectrometry [47] .
For immunoblotting, total cell or pre-IP extracts were lysed in sample buffer containing SDS and protease and phosphatase inhibitors. Proteins were resolved on a SDS polyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF) membrane, hybridized with a primary antibody, reacted with the horseradish peroxidase-conjugated secondary antibody, and visualized using chemiluminescent substrate (Thermo Scientific).
Total RNA was extracted with TRIzol (Invitrogen) and treated with Turbo DNA-free reagent (Ambion) to remove genomic DNA contaminants. cDNA was reverse transcribed from total RNA with random hexamer primers using the High Capacity cDNA reverse transcription kit (Applied Biosystems). cDNA was quantified using SYBR Advantage qPCR Premix (Clontech) and primers for the cellular genes geminin, APC4, APC5, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control (see below). cDNA from infected cells was used to generate a standard curve for each gene examined. The standard curve was then used to calculate the relative amount of specific RNA present in a sample.
Primers used for RT-qPCR are as follows: geminin, forward 59GCCTTCTGCATCTGGATCTCTT39 and reverse 59CGAT GTTTCCTTTTGGACAAGC39 [24] ; APC4, forward 59ATT CTCGTCCTTGGAGGAAGCTCT39 and reverse 59TTCTG GCCATCCGAGTTACTTCAG39 [24] ; APC5, forward 59GTG CCATGTTCTTAGTGGCCAAGT39 and reverse 59GATGCG CTCTTTGCAGTCAACCTT-39 [24] ; GAPDH, forward 59CT GTTGCTGTAGCCAAATTCGT39 and reverse 59ACCCACT CCTCCACCTTTGAC39 [30] .
To determine cellular DNA content, cells were trypsinized, collected by low-speed centrifugation, fixed, and permeabilized in ice-cold 70% ethanol overnight. Cells were stained with propidium iodide only, or double-stained with propidium iodide and anti-pUL44 antibody to identify HCMV-infected cells. Total or pUL44positive cells were determined for their DNA content by cell-cycle analysis with flow-cytometry. Percentages of cells in each cell cycle compartment were calculated using CellQuest or FlowJo software. Figure S1 APC8 knockdown by shRNA. MRC-5 cells were transduced with lentivirus expressing shRNA targeting either Luc (negative control) or APC8. Forty-eight hours post transduction, cells were infected with mock, wild-type (ADgfp), or UL21adeletion virus (ADsubUL21a). Cell lysates were collected at 72 hpi and analyzed by immunoblotting. Note that the asterisk-marked bottom band that reacted with the APC8 antibody was nonspecific as it was not affected by the APC8-targting shRNA. (TIF) Figure S2 Amino acid residues PR 109-110 of pUL21a are required for its APC binding during HCMV infection. Cells were infected with indicated virus and MG132 was added to the final concentration of 10 mM at 6 hpi. Cells were collected at 20 hpi and lysates were immunoprecipitated with APC3 antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. (TIF) Figure S3 pUL21a dissociates the APC by promoting degradation of the bridge subcomplex. MRC-5 cells were infected with ADgfp or ADsubUL21a, and MG132 was added to the final concentration of 10 mM at 6 hpi. Cells were collected at 20 hpi and lysates were immunoprecipitated with APC3 antibody. Both cell lysates and eluted proteins were analyzed by immunoblotting. (TIF) Figure S4 APC8 is not required for pUL21a-mediated degradation of APC4 and APC5. Knockdown and subsequent immunoblot were performed as described in the legend to Figure  S1 .  Deciphering Novel Host–Herpesvirus Interactions by Virion Proteomics Over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. However, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. One particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. Though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. The present mini review focuses on cellular proteins detected in herpesviruses. It highlights the common cellular functions of these proteins, their potential implications for host–pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues. Over the last decades, many host-pathogen interactions have been characterized using genetics, biochemical, and microscopy approaches. These discoveries relied on mutants, pharmacological reagents, immunoprecipitations, immunofluorescence, electron microscopy, cell fractionation, and Western blotting to name a few of the methods employed. These approaches provided much precious information but, given the typical focus of these approaches on individual molecules, likely only revealed a small portion of the proteins involved. Other methods such as high throughput two-hybrid and genetic screens, nucleic acid arrays, RNA interference, and proteomics are now proving essential tools to tackle the complexity of these interactions. The main advantages of mass spectrometry, for instance, are that it is a fast, sensitive and potentially a quantitative approach to identify putative novel players, particularly when coupled to efficient purification schemes. Already, proteomics revealed how viruses modulate the expression of host proteins (Rassmann et al., 2006; Sun et al., 2008; Tong et al., 2008; Antrobus et al., 2009; Pastorino et al., 2009; Thanthrige-Don et al., 2009; Zandi et al., 2009; Zhang et al., 2009 Zhang et al., , 2010 Coombs et al., 2010; Emmott et al., 2010; Lu et al., 2010 Lu et al., , 2012 Munday et al., 2010; Bartel et al., 2011; Lietzen et al., 2011; Ramirez-Boo et al., 2011; Chou et al., 2012) . A relatively new and interesting field is the characterization of host-pathogen interactions within mature purified virions. As reviewed on several occasions, several studies reported the presence of individual cellular proteins in viral particles (Bernhard et al., 2005; Maxwell and Frappier, 2007; Viswanathan and Fruh, 2007; Friedel and Haas, 2011; Zheng et al., 2011) . This includes vaccinia virus (Krauss et al., 2002) , influenza virus (Shaw et al., 2008) , HIV (Gurer et al., 2002; Cantin et al., 2005; Ott, 2008) , vesicular stomatitis virus (Moerdyk-Schauwecker et al., 2009) , and several herpesviruses (see below). Though these cellular components have often been considered purification contaminants, the presence of similar proteins in both related and unrelated viruses suggests that some of them may be biologically relevant. The identification of virion-associated host proteins could thus lead to the discovery of novel therapeutic tools against viruses. The present review focuses on their identification and putative roles with respect to the proteomics of herpesviruses.
Thus far, the protein composition of eight different herpesvirions has been studied by mass spectrometry. These studies include the alphaherpesvirinae herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV; Loret et al., 2008; Kramer et al., 2011) , the betaherpesvirinae human and murine cytomegaloviruses (HCMV and MCMV, respectively; Kattenhorn et al., 2004; Varnum et al., 2004) and the gammaherpesvirinae Kaposi sarcoma herpesvirus (KSHV), gamma herpesvirus 68 (γHV68), Epstein-Barr virus (EBV), and Alcelaphine (Bortz et al., 2003; Johannsen et al., 2004; Bechtel et al., 2005; Zhu et al., 2005; Dry et al., 2008) . Interestingly, host proteins were detected in all herpesvirions analyzed so far, as summarized in Table 1 . For instance, our laboratory previously reported the protein composition of mature extracellular HSV-1 viral particles and identified as many as 49 cellular proteins (Loret et al., 2008) . Similarly, studies focusing on PRV and EBV reported up to 48 and 43 cellular proteins, respectively (Johannsen et al., 2004; Kramer et al., 2011) . Meanwhile, Varnum et al. (2004) found as many as 70 different host proteins in extracellular HCMV virions. While fewer cellular proteins were reported for other viral particles, it is clear that herpesviruses can potentially incorporate many proteins from its host. Moreover, of the 173 different proteins detected in herpesvirions, nine protein groups are present in at least four distinct herpesvirions. This includes 14-3-3, actin, annexins, cofilin, translation factors, GAPDH, heat shock proteins, pyruvate kinase M2, and various Rab GTPases. These results indicate that, first of all, it is common for herpesviruses to incorporate cellular proteins into their viral particles and, secondly, that www.frontiersin.org ? different viruses share similar host proteins. Most excitingly, it also suggests that these host proteins may play common roles throughout the herpesviral family. This defines an interesting and novel set of host-pathogen interactions taking place within the virus itself, rather than the cell. It is tempting to speculate that some viruses might have a higher capacity to steal cellular proteins because of their size and symmetry. Herpesviruses are indeed large viruses containing a layer called the tegument between their capsids and envelopes that could accommodate non-viral proteins. Though some host proteins may randomly be incorporated into virions, others may rather be selected to insure the optimal replication of the viruses that carry them.
Bioinformatics databases such as the KEGG, Gene Ontology, or DAVID are useful tools to get an overview of the functional interplay of proteins (Ashburner et al., 2000; Huang Da et al., 2009; Kanehisa et al., 2010) . As pointed out by Friedel and Haas (2011) , complex statistical tools are available to quantitatively evaluate the implication of proteins in various processes but these are beyond the scope of the present review. Here an analysis of the proteins identified in herpesvirions was instead performed with the Ingenuity Pathways Analysis database (Ingenuity ® Systems), which contains all the known physical and functional links among cellular proteins and defines their most significant functions. That analysis indicates that many of the cellular proteins found in herpesvirions normally modulate trafficking, cell proliferation, cell death, cell migration, cell metabolism, or the cytoskeleton (Figure 1 , upper pie chart). Though subtle differences between family members are noticeable when looking at individual viruses, similar functions are found (Figure 1, other charts) . Immune-related molecules are also important constituents for several viruses, including HSV-1, KSHV, γHV68, Alcelaphine, and MCMV. Altogether, this provides an overall picture whereby herpesviruses, not surprisingly, modulate all of the important aspects of the cell but where each virus might deploy its energies slightly differently. The main surprise is that so many cellular proteins are detected within assembled viral particles, which raises an important question as to their biological significance and mode of action.
The overall picture that several important cellular functions might be modulated by the host proteins incorporated into viral particles is intriguing. This clever strategy is consistent with the parasitic nature of all viruses, including herpesviruses, which would presumably gain some replication advantage from stealing cellular modulators rather than coding for them in their own genomes. The most critical question is the benefit for the viruses to incorporate these cellular proteins in their assembled particles, particularly since these proteins also exist in the cells. While this is open to discussion, one possibility is that some of the incorporated cellular proteins may be remnants of the final capsid envelopment process. Alternatively, this may allow the prompt action of some of these proteins immediately upon viral entry. This could jumpstart the expression and/or duplication of the viral genome, as it is the case for the herpesviral VHS, VP16, ICP0, and ICP4 proteins that are present in virions (Lam et al., 1996; Everett, 2000; Halford and Schaffer, 2001; Ellison et al., 2005; Hancock et al., 2006; Loret et al., 2008; Sarma et al., 2008; Loret and Lippe, 2012) . Other early potential sites of action are the process of viral entry itself, intracellular capsid transport, import of the viral genome through the nuclear pore or immune modulation, all common steps among herpesviruses. Whatever the case might be, the question remains as to why the cellular pool of these proteins would not suffice. Several options may be considered. First, it may be that the virions incorporate specific isoforms, splice variants or post-translationally modified proteins that could have properties or functions distinct than their cellular counterparts. Second, the incorporation of a host protein from one cell type might permit the infection of a different cell type that does not express such protein. For example, alpha herpesviruses initially infect mucosal cells and could acquire host proteins that are beneficial to infect dormant neuronal cells. Finally, the host proteins might be in complex with viral proteins and it is those complexes that are active to promote the infection. These possibilities are of course speculative at this point and need to be explored.
One aspect where the incorporation of host proteins in mature virions might be beneficial is molecules involved in intracellular trafficking. Work by numerous laboratories demonstrated that the transport machinery used to move cellular proteins is also employed by viruses (Simons and Warren, 1984; Lodish et al., 2000; Sollner, 2004; Greber and Way, 2006; Mercer et al., 2010) . This is essential for their proteins and particles to reach their final destination, for example, the site of viral replication, assembly, and/or envelopment. Along with SNARES proteins, Rab and Arf GTPases are master regulators of molecular trafficking throughout the cell (Sollner and Rothman, 1996; Zerial and McBride, 2001; Mizuno-Yamasaki et al., 2012) . So far,VAMP3, a SNARE, was identified in PRV virions (Kramer et al., 2011) but it may only be a matter of time until other SNARES are discovered in other members of the herpes family. This is relevant as another SNARE was reported to facilitate the envelopment of MCMV capsids (Cepeda and Fraile-Ramos, 2011) . In contrast, a great number of Rab proteins have been identified in herpesvirions, particularly HSV-1 and PRV (Table 1) . One stimulating option is that these proteins regulate the displacement of viral capsids in the cell, which could justify their incorporation in the viral particles. As Rab and Arf proteins collectively modulate several intracellular transport steps within the cell, it is anticipated they may be involved in various stages of the infection. For instance, Rab1, which is present in HSV-1 extracellular virions (Loret et al., 2008) , and Rab43 were recently demonstrated to modulate the final envelopment of the virus (Zenner et al., 2011) . Similarly, Rab6, found in HSV-1 and PRV (Loret et al., 2008; Kramer et al., 2011) , is also necessary for the efficient assembly of the related HCMV (Indran and Britt, 2011) . It will now be of interest to determine if the virion-associated pool of these GTPases actively participates in the viral life cycle. Interestingly, several Rab proteins have been implicated in autophagosome formation and maturation (Chua et al., 2011) . While it is difficult to consider how virion-incorporated Rab proteins play a role at that stage, they might rather be incorporated into the virions as MCMV FIGURE 1 | The proteins from Table 1 were analyzed with the Ingenuity database to define their putative functions in the context of an infection.
To this end, the protein accession numbers (or GI numbers) were queried from the Ingenuity database. For the purpose of this figure, all known functions associated with these proteins were exported to Microsoft Excel and regrouped. In the top pie chart, the cellular proteins found in all the herpesvirions were analyzed collectively, while the other pie charts depict the host proteins incorporated into each virus. Since each protein can be associated with multiples functions in the database, the results of those analyses are expressed as relative values instead of raw numbers, which consequently exceeds the original number of proteins analyzed. The percentages therefore represent the number of proteins falling into a given category with the total of each pie chart being 100%. A graphical legend of the categories is provided at the bottom right corner of the figure.
a consequence of their involvement in autophagosome formation and concomitant viral envelopment. Given the vast impact of Rab proteins on the cell, it will be a major challenge to decipher all their roles in the life cycle of herpesviruses, particularly for the pool present in mature virions. Molecular trafficking is not only dependent on SNARES, Rab, and Arf proteins, it is also intimately linked to the cytoskeleton. It is thus not surprising that herpesviruses devote some of their resources toward regulating this central cellular machinery. For instance, herpesviruses significantly reorganize both cellular and nuclear actin as well as microtubules (Norrild et al., 1986; Avitabile et al., 1995; Sharma-Walia et al., 2004; Simpson-Holley et al., 2005; De Regge et al., 2006; Saksena et al., 2006) . They also travel along microtubules during both entry and egress and interact with several cellular molecular motors (Sodeik et al., 1997; Smith et al., 2001; Dohner et al., 2002; Marozin et al., 2004; Lee et al., 2006; Wolfstein et al., 2006; Radtke et al., 2010) as well as cortical and nuclear actin filaments (Forest et al., 2005; Feierbach et al., 2006; Roberts and Baines, 2011) . Furthermore, some members incorporate in their viral particles tubulin or actin-related components ( Table 1 ; Wong and Chen, 1998; Grunewald et al., 2003) . Actin has been reported to compensate the loss of various viral tegument proteins in PRV (del Rio et al., 2005; Michael et al., 2006) and may thus act as an abundant filling agent, so its significance in herpesviral particles remains enigmatic. Similarly, the relevance of intermediate filament components vimentin and keratins in some herpes virions ( Table 1) is difficult to assess given these filaments are not as well characterized as other cytoskeletal elements. It may nevertheless be important for herpesviruses, particularly since they are not all associated with the common skin or hair contaminants often detected in mass spectrometry (Hertel, 2011) .
Viruses tend to monopolize for their own purpose their host expression apparatus, including protein translation (Bushell and Sarnow, 2002) . For example, the prototypic HSV-1 ICP27 viral protein regulates all aspect of mRNAs including transcription, splicing, nuclear export, and translation for the benefit of the virus (Rice and Knipe, 1988; Sekulovich et al., 1988; Sandri-Goldin and Mendoza, 1992; Smith et al., 1992; Hardwicke and Sandri-Goldin, 1994; Hardy and Sandri-Goldin, 1994; Brown et al., 1995; Soliman et al., 1997; Chen et al., 2002; Lindberg and Kreivi, 2002; Ellison et al., 2005; Larralde et al., 2006; Fontaine-Rodriguez and Knipe, 2008) . As these cellular functions are highly regulated, the inclusion of DDX3X, a multifunctional RNA helicase that also regulates transcription, nuclear export, and translation that is used by several viruses (Schroder, 2010 (Schroder, , 2011 ) may be relevant. Its incorporation into mature virions could thus accelerate viral gene expression in the early stages of the infection. Similarly, the presence of translation initiation or elongation factors in virions (Table 1 ) may also jumpstart gene expression in favor of the viruses.
Interestingly, HSV-1 does not require cells to be in the S-phase and even arrests the cell cycle at the G1/S transition step (Shadan et al., 1994; Song et al., 2000) , which partly explains why it can grow in non-dividing neurons. While the precise mechanism of this arrest is unclear, it is known that the viral ICP0 protein and the VP16 cellular partner HCF modulate the cell cycle (Hobbs and DeLuca, 1999; Lomonte and Everett, 1999; Piluso et al., 2002) .
Moreover, ICP0 interacts with the host cyclin D3 (Kawaguchi et al., 1997) . However, it was recently reported that stress, rather than the cell cycle per se, may be a critical feature (Bringhurst and Schaffer, 2006) . Clearly, the interaction of herpesviruses with the cell proliferation apparatus is complex and likely involves several host and viral proteins. Identifying novel players that might be incorporated into mature virions may thus be very useful to clarify this process.
An interesting scenario is the possible regulation of apoptosis by host proteins loaded onto viral particles. Apoptosis is regulated both negatively and positively by several viruses (Teodoro and Branton, 1997; Goodkin et al., 2004) , presumably to insure their survival at the early stages of the infection but their efficient release later on. Conceptually, the presence of anti-apoptotic proteins in herpes particles might thus provide a mean to quickly evade death upon entry, while the presence of pro-apoptotic proteins on newly assembled/enveloped viral particles may trigger or stimulate their extracellular release. Only further work will resolve this open question.
Several factors generally contribute to variation among proteomic studies. Hence, the preparation of the samples (e.g., in-gel trypsin digestion versus liquid digestion and chromatography) may lead to the detection of different populations of tryptic peptides. Moreover, the sensitivity of the mass spectrometers and the abundance of the proteins in the samples also impact peptide detection. The relative abundance of a peptide is itself influenced by the complexity of the samples, where some proteins may evade identification. Finally, each protein differs in its properties (ionization, resolution in SDS-PAGE gels), which will be reflected in their detection. This includes SNARES, which are transmembrane proteins resistant to SDS extraction (Yang et al., 1999; Kubista et al., 2004) . It is thus likely that some of the proteins in Table 1 are present in more viral particles than reported and that additional proteins are indeed incorporated in herpesvirions. More specific aspects regarding herpesviruses includes the purification schemes employed to enrich the viral particles, which will directly influence the purity of the samples and hence the potential detection of contaminants. One important caveat is that some host proteins may simply stick to the large viral particles. Another one is common contaminants such as some hair/skin associated keratins or as mentioned above actin, which may simply fill the virions. However, even potential contaminants cannot simply be discarded since actin and even some keratins may indeed participate in viral life cycles. Moreover, the relative abundance of all the cellular proteins within the cell is unknown, so it is not possible to rule out potential contaminants on the sole basis of abundance. It is thus critical to orthogonally validate all proteomics hits.
Various tools are available to define the biological relevance of host proteins identified in viral particles, including Western blotting, immuno-electron microscopy or functional screens. One powerful method is RNA interference. However, given the dual presence of the host proteins within the viral particles and the cell itself, this becomes a challenging task. RNA interference also has its own caveats (false positives and negatives). Another common step is the expression of dominant positive or negative mutants.
In all cases, one major difficulty is that the host proteins may be essential for the cells and their depletion may lead to cytotoxicity, thus proper controls are needed. In addition, the host proteins might be essential for the virus within the cells but only accessory within the virions. Consequently, depletion of a protein may have limited impact on the virus since complemented by the other pool of that protein in the virus or the cell. Small reduction or stimulation in viral yields may thus result. It such cases, it may be necessary to produce the virus on cells that lack these proteins to see if this makes a difference. One should also consider animal models since tissue culture based screens may miss important players, for instance modulators of the immune system or virulence factors. Clearly, multiple experimental strategies are needed to ultimately insure the biological significance of the host proteins found in viral particles.
The identification and functions of host proteins in viral particles is an important step toward the elucidation of novel host-pathogen interactions. In the case of herpesvirions, this is well under way with eight different family members analyzed so far. One main aspect is to sort biologically relevant cellular proteins from sticky contaminants. The orthogonal validation of the host proteins found in herpesvirions using biologically relevant assays is thus critical. As pointed out above, it will necessary to analyze all these proteins in the background of two pools, one cellular and one virion-associated, which are likely to complement one another. An interesting possibility is that some isoforms or specific posttranslationally modified host proteins may be loaded into the capsids. Thus a detailed analysis of the host proteins present in viral particles will be important and a potential way to distinguish them from their cell-associated counterparts. Another issue is the expected variation among cell types. In that respect, it would be most interesting to examine the cellular protein content of HSV-1 produced in neurons in opposition to the virions produced on other cell types. Finally, the mechanisms by which all these host proteins are recruited to the viral particles will also need to be explored. Thus the proteomics of viral particles is only the beginning of the adventure, which should prove most exciting yet challenging.
I am indebted to the Canadian Institutes of Health Research (grant # MOP82921) for funding our proteomics research. I also wish to thank Kerstin Radtke for excellent suggestions and Daniel Henaff for critical reading of the manuscript. Naturally-Occurring Genetic Variants in Human DC-SIGN Increase HIV-1 Capture, Cell-Transfer and Risk of Mother-To-Child Transmission BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Dendritic cell–specific ICAM-3 grabbing-nonintegrin (DC-SIGN, also known as CD209) is an HIV-1 receptor that enhances its transmission to T cells and is expressed on placental macrophages. METHODS AND FINDINGS: We have investigated the association between DC-SIGN genetic variants and risk of MTCT of HIV-1 among Zimbabwean infants and characterized the impact of the associated mutations on DC-SIGN expression and interaction with HIV-1. DC-SIGN promoter (p-336C and p-201A) and exon 4 (198Q and 242V) variants were all significantly associated with increased risk of intrauterine (IU) HIV-1 infection. Promoter variants decreased DC-SIGN expression both in vitro and in placental CD163(+) macrophages (Hofbauer cells) of HIV-1 unexposed infants but not of HIV-1 exposed infants. The exon 4 protein-modifying mutations increased HIV-1 capture and transmission to T cells in vitro. CONCLUSION: This study provides compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection. In 2010, UNAIDS estimates that 390,000 children acquired HIV-1-infection worldwide mostly through mother-to-child transmission (MTCT) [1] . Overall transmission rates in the absence of any intervention vary from 12 to 42%. Although antiretroviral therapy (ART) can reduce MTCT to as low as 2% [2] , limited access to timely diagnostics and drugs in resource-poor settings blunts the potential impact of this strategy. A better understanding of the mechanisms acting in MTCT of HIV-1 is crucial for the design of interventions other than ART for transmission prevention.
MTCT of HIV-1 can occur during pregnancy (in utero, IU), at delivery (intrapartum, IP) and via breastfeeding (postpartum, PP). HIV-1 can cross the placental barrier in utero either by microtransfusion or by transcytosis across the trophoblast cell layer [2] . IP transmission may occur through direct contact between infant mucosa and HIV-1 infected maternal blood and/ or cervico-vaginal secretions [2] . Finally, HIV-1 in breast milk may result in PP infection of the newborn through mucosal exposure [2] . High maternal viral loads in serum and breast milk and low CD4 cell count as well as obstetric factors such as preterm delivery, vaginal delivery, and prolonged membrane rupture have been correlated with increased risk of MTCT of HIV-1 [2, 3] .
Genetic variations in HIV-1 co-receptors and determinants of immunity have been shown to influence the outcome of MTCT of HIV-1 [2, 4] . Variants that result in either increased CCR5 expression or a non-functional receptor (32 base-pair deletion variant) influenced risk of vertical transmission [5, 6] . The CCR5 32 base-pair deletion is absent in African populations [7] . Genetic polymorphism of innate immunity determinants such as toll-like receptor 9 and mannose-binding protein also increased the risk of MTCT [8] [9] [10] . Discordance at the human leucocyte antigen (HLA) class I loci between mother and child or specific HLA alleles also protect against MTCT [11, 12] .
Dendritic cell-specific ICAM-3 grabbing-nonintegrin (DC-SIGN, encoded by CD209) is a C-type lectin that binds to many pathogens including HIV-1 [13] . This interaction with HIV-1 leads to viral capture and subsequent transmission to adjacent T cells [14, 15] . DC-SIGN is expressed on the cell surface of myeloid dendritic cells and some macrophage subsets including Hofbauer cells present in the placenta [13, 16] . In the context of HIV-1, DC-SIGN may not only promote trans-infection of T cells but signalling initiated by HIV-1 binding may also influence immune responses and enhance productive infection of the dendritic cells themselves [17] [18] [19] .
Given the presence of DC-SIGN in the placenta and its known interaction with HIV-1, we hypothesized that polymorphism affecting its expression or function might influence the risk of MTCT of HIV-1. Here, we report significant associations between DC-SIGN genetic variants that modulate DC-SIGN expression in placental macrophages, promote HIV-1 capture and transmission to T cells and increase risk of MTCT among Zimbabwean infants.
We studied a subgroup of 197 infants born to ART-naive HIV-1-infected mothers recruited in the ZVITAMBO study, which enrolled 14,000 mother-baby pairs between November 1997 and January 2000 in Harare, Zimbabwe [20] . ART prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1positive mother/HIV-1-positive child pairs and 100 HIV-1positive mother/HIV-1-negative child pairs. Modes of infant HIV-1 transmission were determined using definitions adapted from Bryson and colleagues [21] and were described elsewhere [22] . Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere [20] . MTCT of HIV in the whole ZVITAMBO cohort occurred during the IU (22,9%), IP (48%) and PP periods (29.1%) [20] .
Haplotype reconstruction was performed as previously described [23] . Haplotype-tagged single nucleotide polymorphisms (htSNPs) were determined using the HaploBlockFinder software with a minor allele frequency over 5% [24] and numbers were redefined compared to our previous publication [23] for their frequency in the present study population. Ten htSNPs were selected corresponding to the 10 major haplotypes from the 20 SNPs (rs number in Table S1 ) found in the Zimbabwean population as we previously described [23] . These htSNPs along with the 3 others exon 4 mutations were genotyped in the 197 infants by direct PCR sequencing analysis as previously described [23] . Putative transcription factors binding sites in promoter region were analysed with TESS interface (http//:www.cbil. upenn.edu/tess) using the TRANSFAC database.
Genomic DNA from homozygous patients with or without mutation was amplified in the promoter region from nucleotide 2507 to 21 and cloned between the Bgl II and Hind III multiple cloning sites in the pGL2-Basic vectors (Invitrogen, Canada inc, Burlington, Canada). All recombinants clones were verified by DNA sequencing. Luciferase assay was performed as previously described [22, 25] . Firefly luciferase reporter vector was cotransfected with constitutive expressor of Renilla luciferase, phRL-CMV (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.
Site-directed mutagenesis was carried out using pcDNA3-DC-SIGN vectors obtained from Drs. S. Pöhlmann, F. Baribaud, F. Kirchhoff and R.W. Doms [26] through the AIDS Research and Reference Reagent Program, NIAID, NIH: pcDNA3-DC-SIGN exon 4 was amplified from genomic DNA of variants carriers and replaced between PspI and EspI (Fermantas, Burlington, Canada) restriction sites. All recombinants clones were verified by DNA sequencing for the presence of mutations of interest and conservation of the coding frame. Stably transfected cell lines were generated from Raji cells (ATCC, Manassas, USA) by nucleofection (Cell line Nucleofector Kit V, Amaxa, Walkersville, USA) and maintained in RPMI 1640 10% FBS containing 1 mg/ ml of G418 (Invitrogen). DC-SIGN-expressing cells were sorted (sorter BD ARIA, BD Biosciences, Mississauga, Canada) and limiting dilutions were performed. Cell lines were grown from a single clone. 3610 5 cells/well of each Raji transfectants were plated in 96-well plates and pre-incubated with mannan (200 mg/ ml, Sigma-Aldrich, St-Louis, USA), DC-SIGN blocking antibody (clone AZN-D1, R&D systems, Minneapolis, USA) (20 mg/ml), matching isotype control or medium for 30 minutes at 4uC before adding 50 ng of p24 equivalent of virus (HIV-1 HxBru-ADA or HIV-1 JRCSF ). Cells were incubated 2 h at 37uC and washed with PBS (Invitrogen). Cells were lysed and assayed for p24 Ag using ELISA. In co-culture experiment, Raji transfectants were pulsed as above and 3610 5 phytohemagglutinin-L activated human primary CD4 + T-lymphocytes (ratio 1:1) isolated from peripheral blood mononuclear cells were added. Cells were cultivated in RPMI 10% FBS 100 U/ml rIL-2 for 5 days. Supernatants were collected and p24 was measured using ELISA. CD4 + T-lymphocytes were isolated from healthy donors and activated as previously described [27] . HIV-1 stocks were generated by transient transfection of HEK293T cells with HxBRU ADA encoding the R5-tropic HIV-1ADA Env [28] or JRCSF proviral construct using the standard calcium-phosphate method. Viral stock was titrated using ELISA p24 Ag (BioChain, Hayward, USA).
DC-SIGN expression was monitored by flow cytometry analysis using FITC-labelled anti-DC-SIGN antibodies clones DCN46 (BD Biosciences, Missisaugua, Canada) and 5D7 (Santa Cruz Biotechnology, Santa Cruz, USA). The cells were also incubated with isotype-matched control antibodies. Flow cytometry was performed using a BD FACS-Scan (BD Biosciences). Full-term placentas were obtained following non-complicated pregnancies and deliveries at Hôpital St-Luc and Hôpital Bethesda in Cotonou, Benin. All infants were delivered vaginally except for two who were delivered by caesarean section and none of the mothers presented with signs of sexually transmitted infections or placental malaria infection. Placentas with signs of inflammation (chorioamnionitis) were excluded. These HIV-1-infected mothers received a combination of three antiviral drugs (full regimen) during pregnancy and delivery. Infants received a full regimen or a single dose nevirapine at delivery and none of them were HIVinfected. A small piece of each placenta was collected and processed within 3 hours following the delivery and washed extensively with PBS to remove blood and maternal cells. Mononuclear cells were mechanically isolated from placental tissue using a Medimachine (BD Biosciences) and purified on Histopaque gradient (Sigma-Aldrich, Oakville, Canada). Placental mononuclear cells were cryopreserved until flow cytometry analysis. Cells from 4 wild-type (WT) p-336T/p-201C and 11 homozygote or heterozygote p-336C/p-201A infants for the promoter variants born to HIV-1-negative mothers were analysed. Placental cells from 3 WT and 6 homozygote or heterozygote infants born to HIV-1-positive mothers were also analysed. Placental cells were analysed by flow cytometry using CD3-, CD19-(eBioscineces, San Diego, USA), and CD56-PerCPCy5.5 (BD Biosciences), CD14-Alexa700 (BioLegend, San Diego, USA), CD163-APC (R&D System, Minneapolis, USA), DC-SIGN-FITC, CD68-PE and HLA-DR-PECy7 (BD Biosciences) antibodies and isotype-matched controls. Dead cells stained with Live/ Death Aqua (Invitrogen) and lineage cells stained with CD3-, CD19-and CD56-PerCPCy5.5 antibodies were excluded. Placental macrophages were initially gated for CD14 expression and high granularity. Geometric mean fluorescence intensity (MFI) was calculated in DC-SIGN + CD163 + and DC-SIGN + CD163 2 subsets to assess the level of DC-SIGN expression and their level of maturation. Change geometric MFI represents the difference between specific marker expression and its FMO (fluorescence minus one). Flow cytometry was performed using a BD LSR-Fortessa (BD Biosciences).
Statistical analysis was performed using GraphPad PRISM 5.0 for Windows (GraphPad Software inc. San Diego, CA). In order to assess the association between each of the DC-SIGN hapotype ( Table 1) or htSNP (Table 2 ) alleles with MTCT of HIV-1, those subjects who were heterozygous and homozygous for the haplotype or htSNP alleles were compared separately with subjects who tested negatively for that allele (reference category). The association between each of the putative haplotype or htSNP alleles and risks of MTCT of HIV-1 was investigated using crude and adjusted multivariate logistic regression to derive odds ratio (OR) and 95% confidence interval (CI) as estimates of relative risks. Specifically, the models were adjusted for the maternal viral load. The analyses were restricted to those haplotypes found at a frequency above 5% in the study population (Table 1 ) or to those SNPs found only in H4 or H6 (Table 2) . Differences in frequencies of haplotypes and htSNPs were compared between groups using Fisher's exact test. All SNPs were in Hardy-Weinberg Equilibrium [23] . For luciferase DC-SIGN/HLA-DR/CD68 expression, capture and transmission assays comparisons between WT and variants were assessed with the unpaired two-tailed Student's t test.
Written informed consent was obtained from all mothers who participated in the study. 
We carried out an association study of DC-SIGN polymorphism in 197 infants born to ART-naive HIV-1-infected mothers recruited in Harare, Zimbabwe [20] . Among them, 97 were HIV-1-infected and 100 were uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 IP, and 17 PP. Timing of infection could not be determined for 12 HIV-1-infected infants as specimens were not available at some time points. Baseline characteristics of mothers and infants were reported previously [22] . Briefly, maternal age and CD4 + T cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. Maternal viral load .29 000 copies/ml was associated with increased risk of both IU and PP HIV-1 transmission, OR: 3.64, 95% CI: 1.82-7.31, P = 0.0002 and OR: 4.45, 95% CI: 1.50-13.2, P = 0.0045, respectively. Ten htSNPs from the 20 SNPs ( Figure 1A ) corresponding to the 10 major DC-SIGN haplotypes ( Figure 1B) previously described among Zimbabweans [23] , were genotyped in the study samples. Haplotypes with frequencies above 5% in the study population were analysed for their potential association with MTCT of HIV-1. Infants carrying H4 or H6 haplotypes had increased risk of IU HIV-1-infection, whereas H2 haplotype carriers were less likely to be infected during pregnancy compared to infant noncarriers ( Table 1) . None of the haplotypes were significantly associated with altered risks of IP or PP infections. The H4 and H6 hapotypes remained significantly associated with IU HIV-1 infection (OR: 4.98, 95% CI: 1.32-18.8, P = 0.0168 and OR: 2.93, 95% CI: 1.27-6.76, P = 0.0113, respectively) after adjustment was made for maternal viral load. H2 haplotype remained significantly associated with protection against IU infection after adjustment for maternal viral load (OR: 0.23, 95% CI: 0.10-0.51, P = 0.0003). To identify the causal SNPs associated with increased IU transmission of HIV-1, we determined the association between IU HIV-1 infection and each of H4 and H6 signature SNPs. Promoter p-201A (rs11465366) and exon 4 198Q (rs41374747) variants are found exclusively in H6 while exon 4 242V (rs11465380) variant tag H4 ( Figure 1B) . Both H4 and H6 haplotypes harbour promoter variant p-336C (rs4804803) that is known to influence DC-SIGN promoter activity [25] and increased risk of HIV-1 parenteral infection [29] . These variants were all associated with increased risk of IU HIV-1 infection after adjustment for maternal viral load (Table 2 ). In a step-wise logistic regression analysis including all DC-SIGN associated SNPs and maternal viral load, DC-SIGN 242V variant (OR: 4.87, 95% CI: 1.19-19.9, P = 0.0261) and maternal viral load (OR: 3.30, 95% CI: 1.48-7.37, P = 0.0033) remained independent predictors of HIV-1 IU acquisition. Maternal DC-SIGN haplotypes were not associated with MTCT of HIV-1 (Table S2) .
We have previously investigated the association between DC-SIGN-related (DC-SIGNR, encoded by CD209L) genetic variants and MTCT of HIV-1 in the same subset of infants [22] . DC-SIGNR is a DC-SIGN homologue expressed at the cell-surface of endothelial cells of placental capillaries [30] . DC-SIGNR promoter p-198A and intron 2 180A variants were significantly associated with increased risk of MTCT. When adjustment was made for all the significant DC-SIGN and DC-SIGNR associations in logistic regression analysis, DC-SIGN exon 4 242V (OR: 5.03, 95% CI: 1.18-21.4, P = 0.0275) and DC-SIGNR intron 2 180A (OR: 6.93, 1.51-31.7, P = 0.0119) variants remained associated with increased risk of IU transmission, suggesting that DC-SIGN and DC-SIGNR are independent predictors of IU of HIV-1 among Zimbabweans. 
We next investigated the impact of the HIV-1 associated promoter variants on both DC-SIGN transcriptional activity in vitro and expression in fetal macrophages (Hofbauer cells). Variant p-336C decreased the transcriptional activity of Sp1 binding site [25, 31] . Transcription factor binding site analysis predicted that variant p-201A would create a c-myc binding site. To test the effect of these promoter variants on transcription, we transiently transfected HeLa cells with a luciferase reporter gene under the control of DC-SIGN promoter region -507 to -1 containing AP-1, Sp1, Ets-1 and NF-KB transcription factors that are essential for promoter activity [32] and harbouring promoter WT p-336T/p-201C or variant p-336C/p-201A sequences ( Figure 2A ). As previously reported [25, 31] , the luciferase activity of the p-336C/p-201C variant construct was lower than that of WT p-336T/p-201C ( Figure 2B ) but the decreased did not reach significance in our assay. The p-201A variant either alone or in combination with p-336C significantly reduced DC-SIGN transcriptional activity in vitro (Ratio p-336T/p-201C/p-336C/p-201A = 3.13, P = 0.0039). Uninfected infants harboured more frequently H1 and H2 haplotypes reaching significance for H2 (Table 1) . H2 carries two promoter variants, p-939T and p-139C, that differ from WT H1 haplotype ( Figure 1B) . The promoter variants p-939T and p-139C did not show any influence on DC-SIGN transcriptional activity in vitro ( Figure S1 ).
However, HeLa cells derived from cervical carcinoma might not represent the best model to study the impact of promoter variants on DC-SIGN expression in macrophages. To address this issue, we further determine the net impact of susceptibilityassociated promoter mutations on DC-SIGN expression by measuring total DC-SIGN protein expression in Hofbauer cells. These cells are found within the chorionic villi beneath the syncytiotrophoblast layer at the maternal-fetal interface [33] . Term placentas contain a distinct population of Hofbauer cells that co-express DC-SIGN, CD163, CD14, CD68 and HLA-DR, a phenotype similar to alternatively activated macrophages (M2) known for their immunosuppressive properties [16, 33, 34] . Hofbauer cells were analysed by flow cytometry after isolation of mononuclear cells from term placentas of promoter WT p-336T/ p-201C and variants p-336C/p-201A carriers. CD14 + cells of high granularity that were negative for T, B and NK cell markers (CD3, CD19 and CD56) were identified as Hofbauer cells (CD14 + population) and two subsets of DC-SIGN + cells were observed (CD163 + and CD163 2 , Figure 2C ). CD163 + cells expressed significantly higher levels of DC-SIGN, HLA-DR and CD68 compared to CD163 2 cells ( Figure 2C ). We then compared levels of DC-SIGN expression in CD163 + and CD163 2 cells between infants carrying or not carrying promoter variants and born from HIV-1-negative or HIV-1-positive mothers ( Figure 2D ). In infants born to HIV-1-negative mothers, levels of DC-SIGN expression were reduced 1.9-fold (P = 0.0091) in CD163 + cells and 1.8-fold (P = 0.0305) in CD163 2 cells from infants carrying the promoter variants compared to infants harbouring the WT promoter sequence. Interestingly, DC-SIGN expression varied according to the mothers' HIV-1 status. In infants harbouring the WT sequence, levels of DC-SIGN expression were reduced 3.2-fold (P = 0.0402) by CD163 + cells and 2.2-fold (P = 0.0378) by CD163 2 cells in infants born from HIV-1-positive mothers compared to infants born from HIV-1-negative mothers. However, it remained unchanged in infants carrying the promoter variants. Hence, p-336C and p-201A altered DC-SIGN expression in placental Hofbauer cells and but their impact vary according maternal HIV-1 status.
Protein-modifying Variants Increase Viral Capture and Transfer to T cells DC-SIGN molecules on the cell surface enhance HIV-1 infection by capturing virions and transmitting them to CD4 + T-lymphocytes [14, 15] . The neck region, encoded by exon 4, is important for efficient binding to HIV-1 [35] . We hypothesized that the exon 4 protein-modifying variants associated with IU HIV-1 infection could affect the interaction between DC-SIGN and HIV-1. To assess viral capture, exon 4 from the DC-SIGN expression vector was replaced by exon 4 from infants carrying WT, 242V (designated as L242V) or 198Q, 214D and 221Q (designated as R198Q) variants ( Figure 3A ). Raji cells do not express endogenous DC-SIGN and allowed us to investigate the net impact of exon 4 mutations on DC-SIGN HIV-1 affinity. Raji cells ( Figure 3B) were stably transfected and cell lines grown from a single clone. Since viral capture is influenced by cell-surface expression of DC-SIGN [35] , we selected cell lines with similar baseline DC-SIGN surface expression ( Figure 3B ). The stable Raji transfectants were pulsed with equal amount of R5 tropic HIV-1 HXBRU-ADA or HIV-1 JRCSF strains extensively washed to remove the unbound virus, and then lysed. The parental Raji cells were used as controls. The number of virions used was not saturating since capture increased in a dosedependent manner ( Figure S2A ). Interestingly, DC-SIGN L242V and R198Q variants were more efficient at capturing viral particles than WT ( Figure 3C ). HIV-1 capture on the Raji transfectants was stable over time ( Figure S2B ) and dependent on DC-SIGN interaction since the capture was reduced to background levels following incubation with DC-SIGN antibody (AZN-D1) or mannan ( Figure 3C ). Similar results were obtained when cells were pulsed with HIV-1 JRCSF strain ( Figure S2C ). To investigate whether DC-SIGN exon 4 mutations could also enhance cell transmission of HIV-1, we co-cultivated activated primary human CD4 + T lymphocytes with HIV-1 pulsed Raji transfectants. Transmission was quantified by measuring HIV-1 p24 in the supernatants after 5 days. The DC-SIGN variants significantly increased viral transmission to CD4 + T-lymphocytes ( Figure 3D ). Transmission was dependent on DC-SIGN expression since Raji cells or transfectants pre-incubated with DC-SIGN antibody failed to transmit HIV-1 to CD4 + T lymphocytes. Moreover, cell infection was not due to viral particles shed into the supernatant since virus was undetectable in the absence of CD4 + T lymphocytes ( Figure 3D) . Thus, the DC-SIGN neck region variants associated with IU HIV-1 infection enhanced both the capture of HIV-1 by DC-SIGN and its subsequent transmission to the CD4 + T lymphocytes.
In vitro studies have shown that the interaction between DC-SIGN and HIV-1 can enhance short-term viral transfer to other susceptible cell types such as T lymphocytes [14, 15, 36] . Based on these findings, a Trojan horse model has been proposed whereby was compared in CD163+ and CD1632 subsets from infants bearing or not promoter variants and born from HIV-1-negative mothers (HIV-1 Unexposed; p-336T/p-201C group n = 4; p-336C or p-336C/p-201A group n = 11) or from HIV-1-positive mothers (HIV-1 Exposed; p-336T/p-201C group n = 3; p-336C or p-336C/p-201A group n = 6). Results in C and D are mean 6 S.E.M. values of MFI and difference between subsets or variants was calculated with Student's t test. doi:10.1371/journal.pone.0040706.g002 HIV-1 may subvert DC-SIGN-expressing submucosal dendritic cells to promote dissemination from the periphery to the lymphoid tissues [13] . To date, relatively few studies have assessed the potential impact of DC-SIGN polymorphism in adult HIV-1 infection and the findings have not been consistent. While some [29, 37, 38] have found a significant association, others have not [39] [40] [41] . Little is currently known about the mechanisms underlying HIV-1 passage across the placenta. We and others [16, 33, 34] have shown that DC-SIGN is expressed by placental Hofbauer cells. Moreover, the identification of natural and functional DC-SIGN genetic variants associated with an increased risk of IU HIV-1 infection further support the implication of DC-SIGN in HIV-1 dissemination across the placenta. DC-SIGN polymorphism was not associated with IP and PP HIV-1 infections. However, the relatively small number of subjects analysed in the IP (n = 11) and PP (n = 17) groups may have limited the power of the present study to detect any association and therefore we cannot rule out the possibility that DC-SIGN could also contribute to IP and PP transmission of HIV-1.
DC-SIGN promoter p-336C and exon 4 242V variants are observed on the H4 haplotype while p-336C, p-201A and 198Q variants are found on the H6 haplotype. Thus exon 4 variants are always transmitted with one or both promoter variants. These variants were all associated with IU HIV-1 infection and yet the promoter variants reduced DC-SIGN expression in Hofbauer cells whereas the exon 4 mutations enhanced capture and transmission of HIV-1 to CD4 + T lymphocytes. Compensatory mutations frequently evolve to dampen the effect of other mutations. Since the DC-SIGN gene has been under strong evolutionary pressure to conserve its function [42] it is not surprising that mutations increasing the affinity of DC-SIGN for pathogens have appeared that can compensate for mutations that reduce its expression. Interestingly, HIV-1 itself can also affect DC-SIGN expression. Indeed, HIV-or antibody-stimulated DC-SIGN signalling in monocytes-derived dendritic cells (MDDC) reduced DC-SIGN expression and prevented cell maturation [19, 43] . In infants harbouring the WT sequence, levels of DC-SIGN expression were significantly lower in infants born from HIV-1-positive mothers than those born from HIV-1-negative mothers ( Figure 2D ). However, the impact of HIV-1 was negligible or not noticeable in infants carrying the promoter variants since baseline DC-SIGN expression levels were already low in these subjects. Although we cannot exclude that ART may also modulate DC-SIGN expression, it is reasonable to believe that HIV affects DC-SIGN expression in the tissue since in vitro experiments support this hypothesis [19, 43] . In the present study, Zimbabwean infants were born from ART-naive HIV-1-positive mothers. Given the fact that they were all exposed to HIV during their intra-uterine life, they may have harboured similar levels of DC-SIGN expression ( Figure 2D) . Hence, the positive association observed between IU HIV-1 infection and DC-SIGN H4 and H6 haplotypes may thus result from exon 4 protein-modifying mutations found within these haplotypes that enhanced capture of HIV-1 by Hofbauer cells within the chorionic villi in close proximity to maternal infected cells and facilitate short-term transfer of the virus to the infant's T lymphocytes [16] . On the other hand, we cannot rule out the possibility that DC-SIGN variants might promote HIV-1 infection of Hofbauer cells and subsequently IU transmission of HIV-1. Hofbauer cells express both the HIV-1 CD4 receptor and the CCR5 co-receptor [16, 44] and HIV-1 genomic materials have been detected in placental macrophages [45] . Kumar et al observed compartmentalized HIV-1 replication within the placenta during IU transmission [46] and proposed that viral selection during IU transmission could be the manifestation of HIV-1 placental adaptation to the unique repertoire of cellular targets and increased adherence to C-type lectins which further support the implication of DC-SIGN in IU transmission of HIV-1. However, the net impact of this phenomenon on MTCT of HIV-1 remains to be determined since it has also been shown that placental macrophages can restrict HIV-1 replication [47] .
In addition to the enhancement of HIV-1 capture and transmission to target cells, DC-SIGN genetic variants may also contribute to a local immunological environment that promotes viral replication and dissemination of HIV-1 across the placenta [17, 19] . HIV-1 or HIV-1-derived products activated fetal macrophages and T lymphocytes and promoted the establishment of a productive infection within the placenta [46, [48] [49] [50] . In response to dengue infection, MDCCs from DC-SIGN p-336CT heterozygous individuals produced higher levels of pro-inflammatory factors such as TNF-alpha, IL-12 and IP-10 than those from WT p-336TT homozygotes [51] . Moreover, TNF-alpha enhanced HIV-1 replication and transcytosis within the placenta and TNFalpha level correlated with the amount of HIV-1 transcripts [52] [53] [54] . It is tempting to speculate that infants harbouring the p-336TT genotype, such as H1 and H2 carriers, may produce less TNF-alpha to reduce or thwart HIV-1 replication in the placenta.
In this study, we demonstrate for the first time, the impact of DC-SIGN natural polymorphisms on its expression in placental cells and interaction with HIV-1 and provide compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection. These findings raise the possibility that similar mechanisms may operate with other human pathogens known to interact with DC-SIGN and warrant further investigation. p24 Ag was measured by ELISA. Where indicated, cells were pre-incubated with anti-DC-SIGN (AZN-D1) or with mannan to inhibit DC-SIGN interaction with HIV-1. HIV-1 capture is shown relative to WT (WT = 100%). (D) HIV-1 transfer to T lymphocytes by DC-SIGN variants. Raji-transfectants were pulsed as in (C) and subsequently co-cultivated with activated human primary CD4 + T lymphocytes from two donors for 5 days. Virus release into the supernatant was measured by ELISA p24. Where indicated, cells were pre-incubated with AZN-D1. HIV-1 transmission is shown relative to WT (WT = 100%). Results are mean 6 SD of duplicates for each donor (D) or three independent experiments (C). Student's t test was used to calculate differences in % capture and transmission among Raji DC-SIGN transfectants L242V, R198Q and WT. doi:10.1371/journal.pone.0040706.g003 pGL2-Basic, the parental vector without a promoter. Expression of the DC-SIGN promoter constructs was calculated relative to the value of pGL2-Basic, which was arbitrarily set as 1. Data are mean 6 SD values of 3 independent experiments performed in triplicates and there were no significant differences in the relative expression between variants and wild-type (WT) as determined with Student's t test. (DOCX) Figure S2 HIV-1 capture by Raji transfectants (a) Dosedependent HIV-1 capture by Raji transfectants. Raji and Raji-H1 transfectants were incubated with 25, 50 and 100 ng of p24equivalent of HIV-1 HXBru-ADA for 2 h at 37uC, washed with cold PBS 1X and lysed in 0,5% Triton X-100. Cell-associated p24 contents were measured by ELISA. (b) Residual cell-associated HIV-1 over time. 3610 5 Raji-transfectants were exposed to 150 ng of p24-equivalent of HIV-1 HXBru-ADA for 2 h at 37uC, washed and incubated in fresh medium at 37uC for different time points (0, 1 h, 2 h, 4 h and 18 h). Cell-associated p24-contents were measured by ELISA after lysis in 0,5% Triton X-100. (c) Capture assay with HIV-1 JR-CSF . 3610 5 cells were incubated with 50 ng of p24-equivalent of HIV-1 JR-CSF for 2 h at 37uC, washed extensively with cold PBS 1X and lysed in 0,5% Triton X-100. Cell-associated p24 contents were measured by ELISA. Where indicated, cells were pre-incubated 30 min at 4uC with 20 mg/ml of anti-DC-SIGN (AZND1) or with mannan (200 mg/ml) to inhibit DC-SIGN interaction with HIV-1 before pulsing with HIV-1 JR-CSF . HIV-1 capture is shown relative to wild-type (WT = 100%). Data are mean 6 SD of 2 independent experiments performed in duplicates. Student's t test was used to calculate differences in % capture between the Raji DC-SIGN transfectants L242V, R198Q and WT. (DOCX) A New Model for Hendra Virus Encephalitis in the Mouse Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients. Hendra virus (HeV) causes serious systemic infection with pneumonia and encephalitis in humans, horses and various laboratory animals [1, 2, 3, 4] . It is a single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae and is classified within the genus Henipavirus which it shares with one other virus, Nipah virus (NiV). HeV first emerged in the Brisbane suburb of Hendra in 1994, where it caused the deaths of one human and fourteen horses [5] . Since then a further thirty four HeV outbreaks have been identified along the mid to north-eastern coast of Australia with infection of five more humans (of whom three died) and numerous horses [6, 7, 8, 9] . Pteropid bats have been identified as the reservoir host [10] , however epidemiological evidence does not support direct bat to human transmission. Horses have been an intermediate host in the transmission of disease to humans in all cases. There are as yet no readily available effective therapies or prophylaxis for HeV infection, either for use in humans or other susceptible animals.
Of necessity, HeV pathogenesis studies and evaluation of vaccine and therapeutic candidates must be carried out in animal infection models under Biosafety Level 4 (BSL4 conditions). Several species have been used for this purpose including: ferrets, hamsters, guinea pigs, pigs, cats, horses, and African green monkeys [3, 11, 12, 13, 14, 15, 16] . With bats, the list comprises species from six orders including; Rodentia, Primates, Chiroptera, Cetartiodactyla, Perrisodactyla and Carnivora. The broad species susceptibility is unusual for a member of the family Paramyxoviridae and is attributed largely to the highly conserved nature [17] of the host receptors for the virus, Ephrin B2 and B3 [18, 19] . Despite the possession of relevant receptors [19] , the laboratory mouse, a most useful host on account of their small size, ease of handling, and vast library of available reagents, is reported to be resistant to HeV infection and disease [20] .
Westbury et al in 1995 reported resistance of mice to HeV infection in a study that was designed to identify a suitable laboratory animal model of HeV disease. Juvenile BALB/c mice were inoculated with 5000 median tissue culture infective doses (TCID 50 ) of virus by a parenteral route and observed for clinical signs of infection. Mice remained clinically well throughout the 21 day study period and, after euthanasia, there was no evidence of infection by gross or histological examination, virus isolation or serology. Similar results were reported by Wong et al in 2003, who investigated the susceptibility of mice to the closely related Nipah virus [21] by inoculating juvenile Swiss brown mice by either parenteral or intranasal routes.
An understanding of the mechanisms of resistance of mice to HeV may provide novel targets for therapeutic and preventative intervention of human infections. Furthermore, circumvention of such mechanisms may induce a useful mouse model of HeV disease. Therefore, in view of the limited previous work, we decided to re-evaluate the apparent resistance of mice to HeV infection by investigating the outcome of HeV exposure by various routes to inbred mice of different ages and strains. Additionally, quantitative real-time polymerase chain reaction (qPCR), a technique not available at the time of the initial studies, would be used for detecting evidence of viral replication.
We found that mice are susceptible to HeV infection when exposed via the intranasal route, but resist infection when challenged by a parenteral route. Infection manifested as acute, transient, and asymptomatic virus replication in the upper and lower respiratory tracts, together with clinically significant encephalitis that has a longer incubation period than is reported for other models of fulminating HeV disease. The pattern of central nervous system involvement (CNS) supports neuroinvasion by the anterograde route (spread from the neuron cell body toward the axon terminus) and, importantly, transneuronal spread within the CNS. Over all, the study demonstrated that mice are susceptible to HeV infection and has provided a new and important model for HeV induced encephalitis.
Ten juvenile (8 weeks) and ten aged (12 months) C57BL/6 mice were each divided into two groups and challenged with 50,000 TCID 50 HeV using either an intranasal or subcutaneous route of exposure and monitored daily for 21 days post infection (DPI).
Weight loss and temperature changes were not observed for any animals beyond expected minor daily fluctuations. Aged and juvenile mice exposed by the subcutaneous route remained clinically well during the period of observation and at euthanasia there was no evidence of HeV infection by histology, immunohistochemistry or qPCR (Table 1) . Specific binding antibody to the soluble form of the HeV G glycoprotein (HeV sG) was detected in sera of two juvenile animals only, using a Luminex microsphere assay [22] . Low levels of serum neutralizing antibody was also detected in one of these animals by serum neutralisation test (SNT) ( Table 2) .
By contrast, aged animals exposed intranasally were either unexpectedly found dead (mice 14 and 15, 12 and 11 DPI) or developed peracute neurological disease during the study period (mice 11-13) necessitating euthanasia on 16, 20 and 21 DPI. Affected animals showed ataxia, muscle tremors and hypersensitivity. One juvenile mouse (mouse 5) with intranasal exposure also developed a neurological illness requiring euthanasia. Mouse 5 displayed a slower disease onset compared to the aged mice, with a three day waxing and waning depressive illness culminating in severe depression, lack of response to stimuli and hypothermia. In contrast to the systemic vasculitis and multi-organ involvement that are features of HeV infection in other animal species, inflammatory lesions and HeV antigen were only identified in the brain tissue of clinically affected mice and not in heart, lungs, spleen, liver, kidney, ovary, uterus, thymus, pharynx, or mesenteric lymph nodes of any mouse.
Encephalitis was confirmed in all five clinically affected mice (Table 1) , with meningitis in four of these (mice 5 and 11-13); vasculitis was not identified. Encephalitic lesions were characterised by neuronal degeneration, microglial activation, glial reaction, perivascular cuffing and, where present, non-suppurative meningitis ( Figures 1A and 1B) . Viral antigen and lesions were detected in the olfactory tract, cortex by the olfactory tract and piriform lobe in each case (Table 3 ). In mouse 12 lesions were more extensive and included the olfactory bulb, amygdala, thalamus, hippocampus and pons. Except for the pons, all neuroanatomical sites involved are associated with the afferent pathways of olfaction in the brain.
In addition, we analysed tissues and whole blood from each animal for the presence of viral genome using qPCR. All mice that developed clinical disease (excluding mouse 15 for which samples were not available) had high levels of viral genome present in brain tissue ranging from 10 6 . 3 to 10 11 . 8 HeV copies/10 12 18S copies ( Figure 2 ). All other mice were negative for viral genome in brain tissue. All tissues examined by qPCR including; heart, lung, thymus, pharynx, spleen, kidney, ovary, uterus and mesenteric lymph nodes, were negative for viral genome except in two cases; heart tissue of mouse 5 and lung tissue of mouse 14. In both cases C T values were high at 38.8 and 38.3 respectively, indicating low levels of genome and nearing the cut off for a positive sample set at a C T of 39.6. It is of interest to note that mouse 14 died comparatively early in the course of the study (day 12) and the viral RNA detected in the lungs was consistent with the results of mice that were euthanased in the earlier phases of the subsequent time course trial (described below). Low levels of viral genome (C T 39.2) were also detected in blood samples from one juvenile intranasally exposed mouse, mouse 3. Whether viral genome detected in the above three cases reflected replication at the time of euthanasia or residual genome from earlier transient infection could not be determined.
Virus reisolation was attempted on all tissues positive for viral RNA by qPCR with a C T value less than 39.6. Virus isolation was negative for all tissues assayed including brain tissue where high levels of viral genome, antigen, and lesions were detected with and without neutralizing antibody.
All animals that developed clinical disease were positive for a binding antibody response to HeV sG by Luminex assay. Neutralising antibody was also detected in two of these animals by SNT, however titres were evidently insufficient to control intracranial infection. In general, neutralising antibody titers were considerably lower than those reported in convalescent horses following field HeV infection [23] .
We wished to ascertain whether the aforementioned outcomes of exposure were restricted to the C57BL/6 mouse strain. Accordingly, the intranasal component of the above study was repeated in the widely used BALB/c strain. Five juvenile and five adult BALB/c mice were challenged with 50,000 TCID 50 HeV via the intranasal route and monitored daily for 21 days.
Clinical outcomes observed for BALB/c strain mice exposed to Hendra were generally similar to those observed for C57BL/6 mice. As with C57BL/6 mice, weight loss and temperature changes were not observed. All five juvenile mice remained healthy throughout the trial period, whereas three of the five aged mice (mouse 26, 27 and 29) developed clinical neurological disease necessitating euthanasia on 11, 17 and 18 DPI ( Table 1 ). Examination of tissues by histology and immuno-histochemistry revealed similarities and also differences to findings for C57BL/6 mice. In contrast to C57BL/6 mice, lesions and viral antigen were detected in nervous tissues of both symptomatic and asymptomatic animals ( Table 1 ). Two of the three animals that developed clinical disease had encephalitis and viral antigen in the brain: in the third animal (mouse 26) the olfactory bulb was not sampled, and only half the brain was available for histological assessment (the rest was used for qPCR and virus isolation). Four asymptomatic mice (one aged and three juvenile) had encephalitis and viral antigen in brain, with a further two asymptomatic, mice having encephalitis only. Encephalitic lesions and viral antigen deposition in BALB/c mice were also largely confined to neuro-anatomical sites associated with afferent pathways of olfaction ( Table 3 ). The lesions were generally more extensive in aged animals and, while this likely accounts for the differences in clinical signs observed here between old and young mice, it raises the possibility that following longer periods of observation illness may develop in young mice.
Viral genome was detected in brain tissue of all young and aged BALB/c mice, apart from one juvenile mouse in which neither antigen nor lesions were identified ( Figure 2 ). Viral genome loads in brain ranged from 10 5.1 -10 13.1 copies HeV/ 10 12 18S; viral genome was also present in lung tissue of four of the five aged animals and one of the five juvenile animals (Mouse 23, Figure 2 ), albeit at lower levels than seen in brain tissue.
Genome was also detected in pharyngeal tissue of one aged mouse (#29) at very low levels (C T 38.9), without lesions or HeV antigen by histology and immunohistochemistry. As with the C57BL/6 mice, virus could not be isolated from any samples including brain where high levels of viral genome, antigen, and lesions were detected and often in the absence of neutralizing antibody.
Specific antibodies to HeV sG were detected in all mice by Luminex assay and neutralising antibody was detected in five of the ten animals (see Table 2 ). Again, SNT titres were low l (1:5 to 1:20) and did not correlate with clinical outcome.
The above results suggested that aged mice were more susceptible to clinical disease following intranasal HeV exposure than juvenile animals. Using logistic regression analysis to determine if this trend was significant we found that 80% of aged animals developed clinical disease (s.e. = 12.7%) and only 10% of juvenile animals (s.e. = 9.5%) suggesting a real difference (70%, s.e. = 15.8%) in susceptibility to the development of disease between the aged and juvenile animals (p,0.01).
The study investigating the susceptibility of BALB/c strain mice to HeV infection described above revealed that four out of five aged mice had viral RNA in lung tissue as late as 21 DPI in the absence of clinically apparent respiratory disease or detectable pulmonary pathology at euthanasia. In light of these findings, a time-course study was performed to evaluate whether infection of the upper and lower respiratory tracts developed in aged mice following HeV exposure. Aged BALB/c mice exposed to HeV by the intranasal route were euthanased at each time point of the following schedule; two mice every 48 hours from 1-14 DPI, two mice every 72 hours from 15 to 23 DPI, and three mice at 28 days PI, or on development of clinical disease according to predetermined humane endpoints defined from the previous study. Whole brain, lung, nasal turbinates and other tissue samples were examined by histology and immunohistochemistry. Lung tissue was further analysed for presence of viral genome and live infectious virus by qPCR and virus isolation, respectively (Table 4) . Mice sampled between days 6 and 14 pi were positive for HeV viral antigen in bronchoalveolar tissue, but antigen deposits were both dense and focal and it was not possible to distinguish on morphologic grounds whether alveolar lining cells, alveolar interstitium, or endothelial cells were involved. Live virus was reisolated from the lungs of mice sampled between 4 and 10 DPI (Table 4 ) consistent with transient infection of the lower respiratory tract after intranasal exposure to HeV. The viral titres detected were comparatively low with a maximum titre (10 2.3 TCID 50) recovered from a mouse on day 6. Interestingly, HeV antigen deposition in lung ( Figure 1D ) was not associated with detectable bronchoalveolitis and pulmonary vasculitis was not identified. In the upper respiratory tract, immunopositive cellssometimes accompanied by small focal necrotising inflammatory lesions (Figure1E and 1F), were detected in olfactory mucosa of mice between 6 to 17 DPI. It was not determined whether cells involved were supporting cells, basal cells or olfactory sensory neurons.
Vasculitis and subsequent multi-organ infection is a feature of all reported animal models of HeV. In the initial observational study above there was little evidence of HeV virus spread to tissues other than brain apart from low levels of genome recovered from lung in some animals. To assess the extent of systemic involvement outside the respiratory tract, all other tissues for each animal collected in the time-course study were analysed for lesions and viral antigen/genome.
All tissues examined apart from the aforementioned nasal mucosa, lung, and brain were negative for both lesions and antigen. In brain, immunopositive cells were first detected 6 DPI (Table 4) , and from 8 DPI lesions characterised by neuronal degeneration, microglial activation, glial reaction, perivascular cuffing and non-suppurative meningitis were also identified. Vasculitis was not detected in any animal.
All other tissues excluding brain, which was fixed for histology and immuno-histochemistry in its entirety, were assessed for viral genome by qPCR and virus isolation was attempted from any positive samples. Some tissues, notably thymus that likely incorporated cranial mediastinal lymph node, were positive for viral genome (Table 5) , although negative for viral antigen and lesions. On occasion the relative genetic load was comparable to that seen in lung tissues although virus was not reisolated from any PCR positive tissues other than lung.
For assessment of transient viremia, blood samples were also collected at euthanasia from each animal in the time-course study and RNA extracted using a Ribopure blood extraction kit. Previous work showed this extraction method to be the most sensitive for qPCR detection of HeV RNA in experimentally infected horses (A. Foord, personal communication). In mice, a low level (C T of 39.2) of viral RNA was detected in blood from only one animal (#111); this level of genome was very close to our cut off of C T of 39.6 (equivalent to 1 copy RNA from the standard curve).
HeV Viral Antigen and Lesions in the Brain were Largely Confined to Neuroanatomical Sites Associated with the Afferent Olfactory Pathway As viremia and systemic spread was not an important feature of HeV in mice, neuroinvasion was unlikely to have been mediated via the haematogenous route. To better characterise the route of neuroinvasion, the distribution of viral antigen and lesions within the brain were examined in more detail. Brains collected at each point of the time-course study were transversely sectioned at 2 mm intervals; paraffin embedded sections were stained with haematoxylin and eosin for histology and with polyclonal anti-HeV N antibody for detection of HeV antigen (Table 6) .
HeV antigen was first detected in clinically healthy mice euthanased on day 6 and was located in the olfactory bulb involving periglomerular cells, mitral cells, granule cells and associated cell processes ( Figure 1C ). By day 8 there was histological evidence of an inflammatory response closely associated with immunopositive cells. On 9 and 10 DPI three mice showing clinical signs of disease were electively euthanased. In addition to antigen in the olfactory bulb of the brain, two of these mice had antigen detectable in the piriform lobe, olfactory tubercle and amygdala which are all components of the primary olfactory cortex and are connected with each other via synapses. Clinically healthy mice euthanased between days 12 and 20 post infection were uniformly positive for antigen and also inflammatory lesions in the olfactory bulb and piriform lobes. Occasionally, lesions and antigen were seen in the amygdala and more caudally in the hippocampus. The latter feature was especially seen in those mice euthanased in the latter part of the time-course study (day 17 onwards); both of these anatomic structures are associated with the afferent pathways of olfaction in the brain.
Three mice developed clinical disease between days 12 and 20 pi, one each on days 16, 17 and 18. As seen in Table 6 , lesions and antigen detected in the brains of these mice were more extensive than for the animals that did not develop clinical disease. In diseased mice, lesions and antigen were not only detected in the common structures described above, but also in higher processing structures of the cortex such as the thalamus and hypothalamus. Interestingly, two of these mice, mouse 116 and 120, were positive for antigen and lesions in the medulla and pons, and in mouse 116 the lesions and antigen were found in the vestibular nuclei proximal to the vestibulocochlear nerve.
Mice 122 and 123, euthanased 23 DPI and 28 DPI respectively, were clinically healthy. In these mice, lesions and antigen were confined to the olfactory bulb. Table 3 . Distribution of histologic lesions and viral antigen in the brain of intranasally HeV challenged mice. Viral Antigen is Largely Restricted to Neuronal Cells within the Brain As encephalitis was a major feature of Hendra infection in mice, co-localisation studies were performed using confocal imaging to identify affected cell types within the brain. Perfused brains were collected from three aged BALB/c mice, on DPI 9, 10, and 11 and stained for HeV antigen. Antigen was seen in brain sections from all three mice and with a similar distribution to that observed by immunohistochemistry. Antigen appeared to be present in cells with neuronal morphology only ( Figure 3A) . In order to confirm this observation, sections were labeled with antibodies recognizing neurons (NeuN and NFP1), astrocytes (GFAP) microglia (IBA1) and oligodendrocytes (MBP). Dual labeling of sections of infected mice with these markers and with HeV antibodies indicated that there was no co-localisation of Hendra antigen with GFAP or MBP ( Figure 3B and 3C) . On rare occasions there was evidence of low intensity staining for Hendra viral antigen and microglial markers in single cells ( Figure 3D) . The Hendra protein labelling appeared to be discrete and circumscribed which would be consistent with its presence within a cellular compartment such as lysosomes. Compared with uninfected control tissue, there was reduced labeling of neurons with neuronal markers (NeuN and NFP1) in infected tissue. Therefore, we could not use colocalisation studies with these antibodies to confirm HeV antigen in neurons. In order to explore whether viral proteins could be identified within endothelial cells of capillaries or larger vessels, differential interference contrast (DIC) images were taken of areas of Hendra replication. The capillary lumen and endothelial cell cytoplasm were clearly identified and in no case was Hendra antigen detected in the cytoplasm of endothelial cells ( Figure 3E ).
Taken together, this data suggest that HeV replication is restricted to neurons during infection of the brain.
This paper reports the successful infection of mice following intranasal exposure to HeV, with reliable induction of viral encephalitis in aged mice of two strains. A previous investigation by Westbury et al [20] reported that juvenile immunocompetent BALB/c mice exposed to HeV by a parenteral route did not develop clinical or pathological signs of infection although intracranial inoculation of suckling mice had been uniformly lethal (G. Crameri, personal communication). Infection studies were not pursued further and up to now a mouse model of HeV infection has not been available.
We have now confirmed progressive involvement of the mouse CNS by HeV which is most likely established via a nonhaematogenous route of neuroinvasion, as has been established or proposed for other paramyxoviruses including Sendai virus in mice [24] , Canine distemper virus in ferrets [25] , and the closely related Nipah virus in pigs [26] .
Our data are consistent with anterograde HeV entry into the brain, possibly via olfactory sensory neurons (OSN). Viral antigen was detected in both the olfactory mucosa and the olfactory bulbs of intranasally challenged mice as early as day 6 post infection and these two structures are connected by olfactory sensory neurons (OSN). OSNs are located within the olfactory mucosa of the nasal cavity and project cilia out into the lumen of the nasal cavity to detect odorants [27] . Their axons project to the olfactory bulb of the brain [28] and thereby provide pathogens with a potential route of direct transmission from the nasal cavity to the brain. Within the olfactory bulbs OSNs synapse with mitral, tufted (projection neurons) and peri/juxtaglomerular cells (interneurons) in the glomeruli [29] , and secondary dendrites of the mitral and tufted cells synapse with dendrites of granule cells, which reside largely in the granule cell layer [30] . The trigeminal nerve may provide another possible route of neuroinvasion from the nasal cavity to the glomeruli of the olfactory bulbs. Peripheral peptidergic fibres of the trigeminal nerve innervate the olfactory epithelium and at least some of these fibres will send collaterals to the olfactory bulb (fibres terminate in the glomeruli) [31] en route to the trigeminal ganglion and the contribution of these fibres to viral invasion of the olfactory bulbs requires closer investigation. Viral antigen and lesions were also identified in the pons and medulla in four mice. We suggest that in these cases brain infection occurred via another peripheral neural pathway that enters the CNS at the brainstem such as the trigeminal [27] or the vestibulocochlear nerve [32] and it is of interest that in one animal antigen was localised to the vestibular nuclei of the medulla, which would be consistent with neuroinvasion of the vestibulocochlear nerve [32] after infection arising in the inner ear.
The confinement of HeV antigen to neurons of mice, and the pattern and time course of viral antigen and lesions detected in their olfactory glomeruli, mitral and granule cell layers of the olfactory bulbs, as well as deeper structures in the olfactory pathway (such as elements of the olfactory cortex including the olfactory tubercles, piriform lobe and amygdala [33] , and then onto the hippocampus, thalamus and hypothalamus [27] ) suggests that HeV uses a transneuronal mode of spread within the murine brain that is mediated via synaptic connections. Transneuronal spread via synapses has been described for both H5 influenza virus [34] and Herpes simplex virus type 1 [35] .
Transneuronal spread via synapses using cell contact dependent processes without budding of infectious virions may explain how HeV could not be reisolated from infected mouse brains, despite the presence of encephalitis, viral antigen and recovery of substantial viral genome. Transneuronal virus movement within the brain using cell contact dependent processes at the synapse without budding of infectious virions or syncytial cell formation has been proposed for another member of the family Paramyxoviridae, measles virus [36] .
In humans, encephalitis represents a significant complication of HeV infection. Of the seven persons known to have contracted Hendra infection three have died from encephalitis and a fourth developed encephalitis and recovered [1, 4, 37] . Of the three patients that succumbed to encephalitis one case was a recrudescent infection, occurring 13 months after the acute disease [38] . Meningoencephalitis is also an important feature of the natural infection in horses, where it may persist weeks into convalescence, and is also a feature of other experimental infection models [2, 3, 39] . The pathogenesis of HeV infection of the central nervous system (CNS), including the means of spread throughout the brain and the mechanism of recrudescence, is poorly understood.
The mouse model of HeV encephalitis lends itself well to pathogenesis studies of this type as the animals do not succumb to fulminating systemic infection prior to the establishment of significant neuropathology, the CNS lesions develop over a sufficiently long period of time that their progression can be monitored in considerable detail, and high levels of genome persist in brain in spite of virus neutralizing antibody, albeit that it occurs at low level. A particular advantage is that mice are small and comparatively easy to handle at BSL4 and there is an established library of available reagents for neuropathogenetic studies. HeV infection has also been established in two strains of mice, BALB/c and C57BL/6, providing investigators with the opportunity to employ transgenic mouse systems on two genetic backgrounds to examine disease processes, particularly with regard to the immune response in CNS infection.
Although at present there is insufficient data available from human HeV cases to conclude whether neuroinvasion by the olfactory sensory neuron pathway is of primary pathogenetic importance in this species, we do note that the most likely route of human infection is contact droplet infection of the nasopharynx, and so exposure by this route is plausible. More importantly, the pattern of spread of HeV through the brain of mice is best explained by an ability of the virus to employ direct trans-neuronal transmission without concomitant generation of an infectious virion: these observations are of key pathogenetic significance to the human disease. Not only do they provide a possible explanation for failure to reisolate HeV from both sub-acute (I. Smith, unpublished data) and recrudescent [38] human cases of HeV using conventional techniques and in whom there was ample evidence of its ongoing replication, they raise important challenges to optimizing therapeutic interventions. A significant difference in response to infection is seen between aged and juvenile mice where aged mice appear to have a greater propensity to develop clinical disease compared with juvenile animals. A similar trend has been noted for other viral infections of mice, most notably SARS infection. In this case aged mice were susceptible to clinical disease development after challenge with SARS while their juvenile counterparts were not [40] . This observed difference in response to HeV infection between juvenile and aged mice can be used to model and investigate the mechanisms whereby age may affect clinical outcome.
This model may be of particular value in studying aspects of recrudescence. In most other animal models of HeV infection, exposure rapidly leads to acute and fatal disease. In juvenile mice, we have observed that clinical disease may not develop (up to at least day 21 post exposure) despite strong evidence of viral replication in brain that is also continuing in the face of circulating antibody to G protein. Furthermore evidence of encephalitis in these animals was observed in the absence of vasculitis. Some of these characteristics are a feature of both recrudescent human HeV [1] and also NiV [41, 42] infection; the mouse HeV model could be employed to explore the mechanisms by which the potential for virus replication may be sustained in convalescent individuals.
The HeV mouse infection model also provides an opportunity to study mechanisms of viral suppression. Our studies show that although intranasally exposed mice develop transient respiratory infection, the low viral titres detected suggest that infection is controlled in its early phase and cleared. Furthermore, this respiratory infection does not progress to a significant systemic disease as occurs in other animal species [2, 3, 13, 15] . Suppression of infection in the absence of evidence for a robust neutralising adaptive immune response suggests an important role for the mouse innate immune system in this process. HeV has been shown to employ various strategies to evade clearance and control by the innate immune system [43] and we suggest that in the mouse such strategies are rendered ineffective. It will be important in future research to elucidate the mechanism by which mice eliminate respiratory infection and resist systemic disease in order to guide the development of therapeutics that might mimic the process.
In summary, this paper reports three novel observations. Firstly, mice are susceptible to HeV infection after intranasal exposure with aged animals reliably developing encephalitic disease. Their small size and ease of handling, as well as the vast range of biological reagents available for use in the species, render them highly valuable infection models for HeV encephalitis. Secondly, our data strongly support a role for not only anterograde neuroinvasion along sensory neurones but also transneuronal viral spread of HeV within the brain. This finding is of particular importance as infection may be established within the nervous system before current systemic treatments are able to be delivered across the blood-brain barrier. Lastly, mice are resistant to systemic vasculitis and fulminating HeV disease, the mechanisms of which can be explored with therapeutic potential. These findings provide a significant contribution to the body of knowledge in the field and have opened up new areas of investigation, from which significant understanding and further research can arise of benefit to humans as well as animals.
For all studies reported in this paper, the animal husbandry and experimental design were approved by the CSIRO Australian Animal Health Laboratory's Animal Ethics Committee. All animal experimentation was conducted following the Australian National Health and Medical Research Council's Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Ten aged (12-13 months) and ten juvenile (7-8 weeks) C57BL/6 mice and thirty-one aged (10-13 months) and five juvenile (7-8 weeks) BALB/c mice were used for challenge experiments. Mice were housed in groups of four to five according to age, in cages in a room at BSL4. Animals were fed once daily with complete mouse chow and provided with water ad libitum. Animals were allowed 7 days to acclimatise before challenge and were implanted with a subcutaneous temperature chip 5 days prior to challenge. For this procedure and all others requiring restraint for manipulations, an intraperitoneal injection of ketamine (75mg/ kg; Ketamil; Ilium, Smithfield, Australia) and medetomidine (10mg/kg; Domitor; Novartis, Pendle Hill, Australia) was used to induce anaesthesia. Anaesthesia was reversed by intraperitoneal administration of atipemazole (1mg/kg; Antisedan; Novartis). Animals were monitored daily at which time temperature, weight and clinical data were collected and recorded. Staff wore fully encapsulating suits with an external air supply and all work with live virus was carried out at BSL4.
Mice were anaesthetised and exposed to a low passage clinical isolate of HeV (Hendra virus/Australia/Horse/2008/Redlands) [44] by either the intranasal or subcutaneous route. For the intranasal route, five aged (12 months) and five juvenile (8 weeks) Table 6 . Distribution of histologic lesions and viral antigen in the brain of intranasally HeV challenged mice. mice were anaesthetised, placed in dorsal recumbency and exposed to 50,000 TCID 50 in 50 ml saline by slow drop application to the nares. For the subcutaneous route, five aged and five juvenile mice were anaesthetised and exposed to 50,000 TCID 50 virus in 200 ml saline by subcutaneous injection. Animals were monitored daily thereafter and euthanased when reaching a previously determined end-point or at 21 days post infection (pi). The humane end-point was defined as a constant weight loss recorded over 3 days or reaching a 20% loss of pre-infection weight, and/or clinical signs consistent with neurological involvement including ataxia, tremors, depression and behavioural changes. As clinical signs of Hendra infection have not been observed in mice previously, the pre-determined endpoint was modelled on infection outcomes recorded for guinea pigs.
The intranasal component of the above study was repeated in five aged (12 months) and five juvenile (8 weeks) BALB/c strain mice.
Aged (10-13 months) BALB/c strain mice were challenged with 50,000 TCID 50 of HeV via the intranasal route. Mice were euthanased and samples collected every second day from days 2-14 pi, every 3 days from days 17-23 pi and, lastly, on day 28 pi; at least one replicate was used for each time point in case mice were lost from the study for reasons not associated with HeV, with three mice being sampled on day 28. If mice reached a pre-determined humane endpoint prior to the scheduled sampling day they were euthanased and samples collected and processed as for animals euthanased according to the schedule.
Three aged (12-13 months) BALB/c strain mice were exposed to 50,000 TCID 50 of HeV using the intranasal route described above. Deeply anesthetised mice were perfused with paraformaldehyde on 9, 10, and 11 DPI for confocal analysis of the brain.
At the end of the study, animals were anaesthetised and blood was collected via cardiac puncture, placed into EDTA and serum separator tubes. Following terminal exsanguination brain, heart, lungs, liver, kidney, pharynx, mesenteric lymph nodes, ovaries, uterus, spleen and thymus tissues were collected. For virus isolation and molecular studies samples were placed into 750 ml viral transport media [PBS with 1% bovine serum albumin and 1x antibiotics (Anti-anti, Invitrogen)] with 250 ml aluminium silicate beads (Biospec Products Inc., Bartlesville, OK, USA). The remaining tissues were fixed in 10% neutral buffered formalin for 48 hours prior to routine processing for histology and immunohistochemistry, the latter using a rabbit polyclonal antibody raised against the NiV N protein [45] . Nasal turbinate and oral swab samples were also collected at euthanasia, swabs were placed into 750 ml viral transport media. Tissue samples were homogenised and aliquots removed for RNA extraction and the remainder was stored at 280uC for subsequent processing.
For the time course study, the brains of all animals were fixed in their entirety and not sampled for virus isolation and molecular studies. Blood was collected via cardiac puncture and 500 ml whole blood placed into 1.3 ml RNAlater for Ribopure RNA extraction. Otherwise sampling was as described above.
For HeV antigen localisation in brain cell studies, animals were deeply anesthetised and euthanased by perfusion with paraformaldehyde to achieve optimal fixation of tissues for confocal microscopic studies. The thoracic cavity was opened and the right auricle of the heart removed. Three ml saline was injected into the left ventricle of the heart to achieve exsanguination. Following saline injection, 11 ml of 4% paraformaldehyde (volume/volume) was slowly injected into the left ventricle of the heart, perfusing the entire animal. Following perfusion, mice were placed under 4% paraformaldehyde soaked tissues and allowed to fix for 1 hour. After initial fixation, the brain was removed and immersion fixed in 4% paraformaldehyde (volume/volume) for a further 24 hours after which it was placed into PBSA for processing.
For RNA extraction, homogenised tissue samples or swab samples from studies 1-3 were centrifuged at 16 000 g for 2 minutes to pellet debris and 100 ml supernatant was mixed with 265 ml MagMAX Lysis/Binding solution (Ambion, Victoria, Australia) and removed from BSL4. RNA was extracted using the MagMax-96 viral RNA isolation kit (Ambion). For the susceptibility studies, 100 ml of EDTA blood was mixed with 265 ml MagMAX Lysis/Binding solution (Ambion, Victoria, Australia) and RNA extracted as described above. For the time course study, RNA was extracted from whole blood mixed in RNAlater using the Mouse RiboPure-Blood RNA Isolation Kit (Ambion). TaqMan qPCR was performed using the AgPath-ID one-step reverse transcription-PCR kit (Applied Biosystems, Victoria, Australia), targeting the N gene of HeV as previously described [46] . Positive results were defined by a cycle threshold (C T ) value of ,39.6 based on a standard curve where this C T represented one copy of target RNA. All samples were normalised against the housekeeping gene 18S and expressed as copy numbers HeV/10 12 copy numbers of 18S.
Virus isolation was performed on all HeV RNA positive samples detected in the HeV Taqman assay using the standard Vero cell line maintained in our institute and originally obtained from the American Tissue Culture Collection (ATCC) [10, 39] . Supernatants from the homogenised samples were inoculated onto Vero cell monolayers and scored positive if syncytia were present after 4 and 5 days.
Sera collected both prior to challenge and at euthanasia were analysed for binding to HeV sG protein using a Luminex microsphere assay. Assays were performed on a Bio-Plex Protein Array System (Bio-RadLaboratories, Inc., CA, USA) as previously described [22] . Bio-Plex Manager Software (v 4.1) (Bio-RadLaboratories, Inc., CA, USA) was used for data acquisition and analysis. All samples were assayed simultaneously for each mouse strain. A strong positive control was assayed alongside all samples as was a negative control. A positive result was defined as samples occasionally seen in microglia, identified with labelling for Iba1 (green) in olfactory bulb at day 9 PI. HeV antigen labeling (arrow) was discrete and circumscribed, consistent with its presence within a cellular compartment such as a lysosome. (E) Section of olfactory bulb showing a capillary amongst HeV infected cells at day 10 PI. HeV antigen (red) and GFAP (green) are not colocalised. The endothelial cell cytoplasm (arrow) is negative for HeV antigen. Scale bars = A) 50 mm, B) 10 mm, C) 15 mm, D) 10 mm and E) 10 mm. doi:10.1371/journal.pone.0040308.g003 with a median fluorescence intensity (MFI) greater than the mean of all pre-challenge samples plus 3 x the standard deviation, MFI .406.
Sera collected at euthanasia were gamma-irradiated to inactivate virus. Sera were serially-doubly diluted in a 96 well plate (final volume 50ul/well) to which 200 TCID 50 HeV was added and incubated for 1 hr at 37uC. Following incubation, 2x10 4 Vero cells/well were added and the assay read after 3 days incubation at 37uC with 5% CO 2 .
Whole brains, collected and fixed in 4% paraformaldehyde were immunolabelled as previously described [47] . In brief, they were sectioned at 50 mm thickness with a Leica Vibrating Microtome and stored in PBS at 4uC. Sections were treated with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 1 hour and blocked with 0.5% Bovine serum albumin (Sigma-Aldrich) in PBS (PBS/BSA) overnight at 4uC. Primary antibodies were diluted in 0.5% PBS/BSA and incubated on sections for 2 hours at 37uC. Antibodies and dilutions were: rabbit anti-IBA1-microglia marker PBS. Mouse Ig Blocking Reagent (Vector Laboratories) was used with mouse primary antibodies: anti-Human Neurofilament protein (DakoCytomation) 1:50, anti-NeuN, (Millipore) 1:50, and a mouse monoclonal antibody raised against whole HeV (AAHL). Bound primary antibody was detected with species-specific secondary antibodies conjugated to Alexa 488 or 568 (Invitrogen) diluted 1:200 in PBS/BSA for 2 hours at 37uC. Sections were washed 3 times for 5 minutes with PBS and nuclei labelled with DAPI diluted 1:1000 in dH 2 O for 30 minutes. Sections were rinsed twice with dH 2 O, mounted with Vectashield Mounting Medium (Vector Laboratories) and coverslips sealed with nail varnish. Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (K(A)) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. Plant defense is a complicated mechanism in response to mechanical wounding, herbivore and microorganism attack. Many proteins, namely wound-inducible proteins, are expressed to prevent pathogen infection, inhibit digestion by insects, and repair injured tissues [1, 2] . One group of wound-inducible proteins is lectin, the carbohydrate binding protein [3, 4] . Plant lectins are involved in the plant defense mechanism because of carbohydrate binding properties [5] [6] [7] [8] [9] . The toxicity of lectins was also confirmed in animal experiments [10, 11] . Plant lectins show resistance to digestive enzymes and can bind selectively to the carbohydrate moieties of gut epithelial cells to interfere in nutrient digestion and absorption [12] , so they could be a natural insecticide. In addition, plant lectins have been used for blood typing and immunological assay. The lectin concanavalin A is commercially used in affinity chromatography for purifying glycoproteins. Plant lectins have long been reported as potential inhibitors of viruses [13] [14] [15] [16] [17] .
Most plant lectins were originally isolated from seeds and vegetative storage tissues. Accumulating data have revealed that plants ubiquitously synthesize lectins in response to abiotic and biotic stresses. These inducible lectins are synthesized and then exported to vacuoles by signal peptides or reside in the cytoplasm [18, 19] . The physiological function of plant lectins for subcellular localization remains obscure. However, the major assumption is that lectins are involved in defense and may also have a role in signal transduction for response to stress [20] . Structure analysis of plant lectins demonstrated a diverse group of proteins that can be classified into 6 different groups (http:// www.cermav.cnrs.fr/lectines/): monocot lectin, hevein domain lectins, b-prism lectins, b-trefoil lectins, cyanovirin-N homologs, and legume lectin.
Jacalin-related lectins (JRL) have a b-prism fold. In 1996, the structure of Jacalin from seed of jackfruit (Artocarpus integrifolia) was first reported to have a tetrameric association for binding to galactose [21] . Later, Maclura pomifera seed agglutinin was reported to have the same tetrameric structure as Jacalin [22] . The other lectin, Artocarpin, from seed of jackfruit (Artocarpus heterophyllus) shares the same tetrameric association for binding to mannose [23] . Moringa M from black mulberry (Morus nigra) forms a tetrameric association like that of Jacalin [24] . JRLs were once thought to be confined to the Moraceae. However, increasing structural evidence reveals that the lectins with a bprism fold exist universally in plants and animals [25] but with different quaternary association. Heltuba is a plant tuber lectin from Helianthus tuberosus (Jerusalem artichoke) that has a donut shape with an octahedral assembly by the b-prism building block [26] . Caselpa is a rhizome lectin from Calystegia sepium (Hedge bindweed) that has a dimeric form [27] . PPL is a plant seed lectin from Parkia platycephala that contains 3 repetitive bprism domains and forms a dimeric form with hexahedral assembly [28] .
Ipomoelin (IPO), expressed in the leaves of sweet potato (Ipomoea batatas cv. Tainung 57), was found easily inducible by wounding and methyl jasmonate [29, 30] . Previous study showed that IPO can agglutinate human blood and bind to different carbohydrates, such as methyl a-D-mannopyranoside (Me-Man), methyl a-Dglucopyranoside (Me-Glc), mannose, glucose and galactose [10] . In this study, we resolved the crystal structures of IPO in the apo form and in complex with Me-Man, Me-Glc and methyl a-Dgalactopyranoside (Me-Gal) to reveal the different quaternary associations of IPO and its binding pocket for carbohydrates. A The packing diagram of apo IPO with 5 molecules in green. Four of 5 molecules form a tetramer and the 5th molecule can form another tetramer by the red one, the yellow one and the blue one in the center. The molecules in red, yellow and blue are generated by symmetric operations (-X, Y, -Z), (X, -Y, -Z), and (-X-1, -Y, Z). (B) The resolved IPO-Me-Man complex in green is 2 molecules in an asymmetric unit. However, the other 2 molecules in red are generated by the symmetric operation (X, -Y, -Z) to form a tetramer in the center. (C) The resolved IPO-Me-Glc complex is a tetrameric form, and (D) the IPO-Me-Gal complex is also a tetramer. All packing diagrams reveal its tetrameric nature. doi:10.1371/journal.pone.0040618.g001 The carbohydrate binding pockets are indicated by green mesh. Tetrameric IPO is represented by monomer A in blue, monomer B in purple, monomer C in light blue, and monomer D in pink. The symmetric axis is represented by a black ellipse in the center of tetramer. (C) Structure-based multiple sequence alignment of Jacalin family. Five homologs were selected for sequence comparison from resolved protein structures: Ipomoelin-tetramer from Ipomoea batatas (PDB: 3R52); Calsepa-dimer from Calystegia sepium (PDB: 1OUW); Banlec-dimer from Musa acuminate (PDB: 2BMZ); Jacalin-tetramer from Artocarpus hirsutus (PDB: 1TOQ); Parkia-hexamer from Parkia platycephala (PDB: 1ZGS); and Heltuba-octomer from Helianthus tuberosus (PDB: 1C3K). Positions of identical conserved residues are shown in white on dark grey background, and regions of similarly conserved residues in light grey are boxed. Representation of secondary structure elements and numbering above the alignment is based on the IPO structure. The secondary structure elements below the alignment are based on the Heltuba truncated IPO (DN10IPO) was prepared to reveal its role in tetrameric association in solution by gel filtration chromatography. In addition, the carbohydrate binding constants of IPO and DN10IPO were determined by isothermal titration calorimetry (ITC). DN10IPO showed a recovered mannose/glucose-specific lectin. Structural and functional analysis identified IPO as a member of the JRL family but with a different tetrameric association. The N-terminus of IPO plays a critical role in regulating broad carbohydrate binding.
The apo IPO showed an orthorhombic space group of I222. A reasonable volume of the unit cell (Vm) for the Matthew coefficient was estimated at 2.19 Å 3 /Da and 44% solvent content by 8 IPO molecules. However, only 5 IPO molecules in an asymmetric unit could be built after molecular replacement. We structure. The carbohydrates Me-Man, Me-Glc, and Me-Gal share 9 hydrogen-bonding interactions with Gly21, Tyr97, Gly141, Trp142, Tyr143 and Asp145 of IPO (blue triangle). The residues of IPO located at the interface are boxed in red. The two short b strands at the N terminus are also involved in the interface. The underlined Jacalin-tetramer representing the sequence is extracted from the C terminus of Jacalin (chain B). doi:10.1371/journal.pone.0040618.g002 obtained a higher Matthew coefficient with 3.51 Å 3 /Da and 65% solvent content. In the packing diagram for apo IPO, we observed a tetrameric association with an additional monomer in an asymmetric unit ( Figure 1A ). The additional monomer could form a tetrameric association with the other 3 neighboring molecules, which were generated by symmetric operations (-X, Y, -Z), (X, -Y, -Z), and (-X-1, -Y, Z). So 4 IPO molecules could form a tetramer.
To determine the carbohydrate binding pocket of IPO, carbohydrates such as Me-Man, Me-Glc and Me-Gal were used to co-crystallize with the IPO protein. The crystals of IPOcarbohydrate complexes were determined in different space groups. IPO-Me-Man belongs to an orthorhombic space group C222 1 . The Matthew coefficient and solvent content for IPO-Me-Man had a reasonable value of 2.21 Å 3 /Da and 44.4% for 2 molecules in an asymmetric unit. Although only 2 IPO molecules were built in the IPO-Me-Man complex, the other 2 IPO molecules could be generated by symmetric operation (X, -Y, -Z) and resulted in a tetrameric association ( Figure 1B ). The crystal of IPO-Me-Glc was determined to be a monoclinic space group P2 1 . The Matthews coefficient and solvent content was 2.26 Å 3 /Da and 45.5% for 4 molecules. The packing results for IPO-Me-Man and IPO-Me-Glc indicated that the carbohydrates binding to IPO might result in a compact packing as compared with that of apo IPO. In addition, the resolved structure of IPO-Me-Glc formed a tetrameric association ( Figure 1C ).
IPO-Me-Gal belongs to an orthorhombic space group P2 1 2 1 2 1 . The Matthews coefficient and solvent content were 2.25 Å 3 /Da and 45.2%, respectively, for 4 molecules in an asymmetric unit. The 4 IPO-Me-Gal molecules shown in Figure 1D form the same tetrameric association as that of IPO-Me-Glc. On the basis of crystal packings of apo IPO and IPO-carbohydrate complexes, IPO would form a tetrameric association.
The monomeric IPO from residues 1 to 154 shows a typical bprism fold found in the JRL family, with 12 b-sheets (b3-b14) and 2 additional short, extended, N-terminal b-strands (b1-b2) (Figure 2A and 2C) . Each b-prism fold comprises 3 Greek-key motifs forming 3 planes by 3 four-stranded b-sheets: plane 1 by b3 to b4 and b13 to b14; plane 2 by b5 to b8; plane 3 by b9 to b12.
Furthermore, the structure of these b-sheets comprises b1 from residues Gln4 to Leu5, b2 from residues His8 to Ser9, b3 from residues Ala11 to Gly17, b4 from residues Gln22 to Arg27, b5 from residues Lys34 to Gly41, b6 from residues Leu47 to Ser55, b7 from residues Ile61 to Gly65, b8 from residues Tyr74 to Asn79, b9 from residues Ile84 to Tyr94, b10 from residues Tyr97 to Thr107, b11 from residues Glu111 to Gly116, b12 from residues Thr121 to Lys126, b13 from residues Asn131 to Ser140, and b14 from residues Val144 to Ala153 (Figure 2A and 2C).
Four IPO protomers form a compact tetrameric association by swapping their extended N termini from residues 1 to 10. We analyzed the tetrameric association of IPO-Me-Glc. As shown in Figure 2B , the 2 extended N termini from monomer A in blue and monomer B in purple swap with each other. The interacting interface between the four IPO protomers is formed by the extended N termini. Consequently, a larger buried interface between monomers A and B is 1,522 Å 2 . The residues located at the interface are 2-10, 12, 15-30, 59-67, 91-92, 98, 121, 134, 137, 139-140, 146, 150, and 152 in monomer A (as shown in red box in Figure 2C ). In total, 13 hydrogen bonds are formed by the residues Leu5, His8, Asn19, Gln22, Ser25, Arg27, Asp60, Ile61, Thr63, Thr121, Asn139 and Tyr150 in the interface between monomers A and B. The buried interface between monomer C and monomer D is 1,554 Å 2 . Furthermore, the buried interface between monomers A and C is 755 Å 2 , which is mainly contributed by the interacting residues of N-terminal residues 4 to 17 and C-terminal residues 91, 121-126, 128, and 151. In addition, the interface between monomers D and B is 731 Å 2 .
The carbohydrate binding pocket of IPO was confirmed at loops b13 and b14 by the structures of IPO-Me-Glc, IPO-Me-Man and IPO-Me-Gal (as shown in Figure 2B with green mesh). In the chain A of IPO-Me-Glc, 9 hydrogen bonds are formed by the residues Gly21, Tyr97, Gly141, Trp142, Tyr143 and Asp145 of IPO and the atoms O1, O3, O4, O5, and O6 of Me-Glc ( Figure 3A and Table 1 ). The atom C7 of Me-Glc is involved in the methyl carbon (Me)…p interaction with Trp142 of IPO. The hydrogen bonds are slightly different between chain A and chains B to D. The hydrogen bonds of chains B to D are formed between the same residues of chain A and Me-Man, except for Table 1 ). The differences might result from the binding of cadmium ion (Cd 2+ ). In chains B to D, the Cd 2+ atom forms 5 coordinates by the O atom of the carbonyl group of Asn19, OG atom of Ser18, and 3 water molecules. One of the 3 water molecules forms a hydrogen bond with Asp145 ( Figure 3B ).
In the structure of IPO-Me-Man, two IPO protomers were built, and only one Me-Man molecule could be observed in chain A. The temperature factor of Me-Man in the structure of IPO-Me-Man is 66.5 Å 2 , which is higher than that of Me-Glc, with 34.5 Å 2 (Table 1) . This phenomenon might indicate that only a few Me-Man molecules bound to IPO proteins in IPO-Me-Man, which resulted in a higher temperature factor. Nine hydrogen bonds are formed by the residues Gly21, Tyr97, Gly141, Trp142, Tyr143, and Asp145 of IPO and the atoms O1, O3, O4, O5 and O6 of Me-Man ( Figure 3C and Table 1 ). The atom C7 of Me-Man is also involved in the Me…p interaction with Trp142 of IPO. The binding orientation of Me-Man is similar to that of Me-Glc. In the structure IPO-Me-Gal, 10 hydrogen bonds are formed by the same residues Gly21, Tyr97, Gly141, Trp142, Tyr143, and Asp145 of IPO ( Figure 3D and Table 1 ). The atom C7 of Me-Gal is shown in the Me…p interaction with Trp142 of IPO. This revealed the importance of the methyl group of carbohydrates for binding to IPO.
To validate that the quaternary association of IPO is also a tetrameric form in solution, purified IPO was used in gel filtration experiments. The molecular mass of IPO could be calculated according to the linear regression equation of the standard protein markers purchased from BioRad ( Figure 4C ). In the preliminary study, IPO protein was dissolved in running buffer (27 mM Tris-HCl pH 7.0, 2 M NaCl) without additional carbohydrates. We obtained a retarded result, with corresponding molecular mass 4.0 kDa (Peak 3 in Figure 4A ). Thus, IPO has the binding ability of dextran in the matrix of the Superdex 200 column. To eliminate the binding effect of IPO to dextran, running buffer was prepared with an additional 0.2 M Me-Glc, and a shift of the IPO peak could be observed, with corresponding molecular mass of 53.3 kDa (Peak 2 in Figure 4A ). Consequently, running buffer with an additional 1 M glucose was prepared to totally eliminate the binding effect of IPO. The corresponding molecular mass of IPO in solution was 64.7 kDa (Peak 1 in Figure 4A ). The molecular mass of recombinant IPO with a His tag was 17.3 kDa for a monomer and 69.2 kDa for a tetramer. The results from gel filtration experiments demonstrated that IPO shows a tetrameric association in solution.
To further identify the role of the N terminus in the tetramerization of IPO, we prepared a truncated IPO (DN10IPO) by removing residues 1 to 10 to monitor the change in quaternary association. The native IPO protein or the truncated IPO protein was dissolved in the running buffer with 1 M glucose. Peak 1 in Figure 4B represents the native IPO, with molecular mass 63.2 kDa, which is a tetrameric size. Peak 2 in Figure 4B represents the DN10IPO, with molecular mass 21.9 kDa, which is near the truncated monomer size (16.3 kDa). The results further confirmed that IPO has a tetrameric association and its N terminus plays an important role in forming a tetramer.
To determine the binding constants of IPO to Me-Man, Me-Glc and Me-Gal, 1 mM IPO solution was titrated with 25 mM carbohydrate solution. The interaction of IPO and carbohydrate was an exothermal reaction. The optimal curves and thermodynamics parameters could be fitting and calculated by Microcal Origin 7.0. The K A of IPO to Me-Man was the highest, 7.04610 3 M 21 . The K A values for Me-Gal and Me-Glc were 4.09610 3 M 21 and 2.01610 3 M 21 , respectively (Table 2 and Figure 5 ).
Subsequently, carbohydrates without the methyl group were used to determine the binding affinity of IPO. From preliminary study, 1 mM IPO titrated with 25 mM Man, Glc, and Gal revealed no obvious exothermal reaction. After increasing the concentration with 3 mM IPO titrated with 75 mM Man, Glc, and Gal, the exothermal curves could be observed and calculated. The K A values for IPO binding to Man, Gal and Glc were 1.05610 2 M 21 , 0.57610 2 M 21 , and 0.32610 2 M 21 (Table 2 and Figure 5 ). Thus, the interactions between IPO and carbohydrates were stronger with than without the methyl group. Figure 6 ). Thus, the N-terminus of IPO is involved in tetramerization in regulating the binding affinity to carbohydrates.
We submitted the coordinates of a monomer of apo IPO (e.g., chain A; Figure S1C ) to the web service Matras for 3-D protein structure comparison [31] . We found the highest Z-score, 124.5, for the template structure, a dimeric form of Calsepa from Calydyrgia sepium (PDB: 1OUW; Figure S1D ) [27] , in our molecular replacement procedure. The following structures were PPL from Parkia platycephala with a hexahedral ring (PDB: 1ZGR; Figure  S1E ) [28] , Heltuba from Helianthus tuberosus with an octahedral ring Figure S1F ) [26] , Banlec from banana with an another kind of dimeric form (PDB: 2BMZ; Figure S1B ) [32] , and Jacalin from jackfruit seeds with a tetrameric form (PDB:1UGW; Figure S1A ) [33] . These data indicate the various quaternary structures in the JRL family, despite the same b-prism fold of protomer.
The various quaternary associations in the JRL family exhibited different contacts between protomers. A previous report indicated that the buried interface of the Calsepa dimer is 1,327 Å 2 by a probe with 1.6 Å radius [27] . Here, we analyzed the buried interface of the selected structures from the above comparison by using the PDBe PISA service with 1.4 Å radius [34] . The buried interface area from tetrameric IPO encompasses 1,539 Å 2 , which is larger than that of Calsepa (1,202 Å 2 ), PPL (1,294 Å 2 ), Banlec (750 Å 2 ), Heltuba (736 Å 2 ), and Jacalin (1023 Å 2 ). The N terminus of the protomer in the JRL family has an important role in the quaternary association by swapping in the interface and then forming a dimer, tetramer, hexamer, and octomer. To compare the difference between the tetrameric Jacalin ( Figure  S1A ) and the tetrameric IPO ( Figure S1C ), the tetramer of Jacalin showed a looser interface than that of IPO. Therefore, IPO formed a different compact tetramer.
In this study, we resolved the crystal structures of IPO-Me-Man, IPO-Me-Glc and IPO-Me-Gal complexes. These monosaccharides showed similar orientation to bind to IPO. The binding pocket of IPO contains 6 residues such as Gly21, Tyr97, Gly141, Trp142, Tyr143 and Asp145, to form hydrogen bonds with different monosaccharides (Figure 3 Table 2 ). The carbohydrate binding manner of IPO is not confined as is the mannose-glucose-specific binding lectin.
In addition to determining monosaccharides with the methyl group, we used monosaccharides without a methyl group, such as mannose (Man), glucose (Glc), and galactose (Gal), to determine their binding constant to IPO. Since the lower binding affinity of IPO titrated with Man, Glc or Gal couldn't get the best fitting for the titration curves, the n value was consequently fixed at 1.0 for fitting the curves ( [36] . The results show no differences with or without the methyl group of monosaccharides for binding properties in Artocarpin and Banlec possibly because of no aromatic side chain of residues in Artocarpin and Banlec like the residue Trp142 in IPO ( Figure 7A and 7B) . Interestingly, IPO shared similar binding properties to Jacalin for its Tyr122, which [37] . To examine the binding mode of Me-Man for Artocarpin, Banlec, Jacalin, and IPO, the binding position of Me-Man with IPO showed a distant binding site as compared with that for Artocarpin, Banlec, and Jacalin ( Figure 7D ). DN10IPO could be recovered as the mannose/glucose specific lectin if DN10IPO represented the monomeric IPO and wild-type IPO represented the tetrameric IPO. The monomeric IPO showed 5 times and 6 times binding affinity to Me-Man and Me-Glc, respectively, as compared with those of tetrameric IPO. Therefore, the N terminus of IPO is involved in the carbohydrate recognition, which results in the carbohydrate binding polyspecificity of tetrameric IPO. From the tetrameric IPO structure, the residue Leu5 and His8 in the N terminus of monomer B (chain B) forms 3 hydrogen bonds with the residue Asn19 in the loop between b3 and b4 of monomer A (chain A) (Figure 8 ). The hydrogen bonds might pull out the loop of b3-b4 and form a larger binding cavity for different carbohydrates in monomer A. However, in DN10IPO, the hydrogen bonds would disappear and might relocate the b3-b4 loop to cause a smaller binding cavity. The axial O4 of Me-Gal would not easily enter into the smaller binding cavity. The results might be confirmed by the crystal structure of DN10IPO-Me-Man in further study.
In conclusion, we resolved the structures of apo IPO and IPO in complex with Me-Man, Me-Glc and Me-Gal. IPO is proposed to have a tetrameric association by 4 protomers of the b-prism with an additional N terminus, which shows a compact tetrameric association in the JRL family. From gel filtration experiments, we confirmed the tetrameric association of IPO in solution. The N terminus of IPO plays an important role in forming a tetramer. In addition, the binding pocket of IPO was identified and found to bind to Me-Glc, Me-Man, and Me-Gal with similar hydrogen bond networks. Furthermore, the binding constants of IPO were determined by ITC. The IPO structures further extend the diverse quaternary structures of the JRL family of plants and show versatile carbohydrate binding properties regulated by the N terminus. Thus, the wound-inducible protein IPO from sweet potato has versatile carbohydrate binding properties and might play a role in plant defense.
The expression vector pTZ18UH containing the IPO gene [GenBank: D89823.1] (pTZ18UH-IPO) of sweet potato (I. batatas cv. Tainung 57) was constructed previously [10] . A truncated form of IPO by removing 10 residues of N terminus (pTZ18UH- 
The pTZ18UH-IPO and pTZ18UH-DN10IPO vectors were transformed into Escherichia coli BL21 (DE3) cells (Novagen). A single colony was cultured in 5 ml LB medium containing 100 mg/ ml ampicillin (LB/Amp) at 37uC overnight. The medium was further transferred into 600 ml LB/Amp to an A 600 of about 0.5 to 0.7 and then induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 25uC for 6 hr. Cells harvested by centrifugation were resuspended in a loading buffer (20 mM sodium phosphate, pH 7.4, 0.5 M sodium chloride, 20 mM imidazole). After breaking cells by use of an ultrasonicator (Sonicator 3000, Misonix), the supernatant of the crude cell lysate was loaded onto a Histrap FF column (GE Healthcare) with use of an Ä kta Prime fast protein liquid chromatography (FPLC) system (GE Healthcare). After washing the Histrap FF column with 3x column volume of loading buffer (1x phosphate buffered saline, 5 mM adenosine triphosphate, 10 mM MgSO 4 ), the IPO protein was eluted by use of elution buffer (50 mM sodium phosphate, pH 7.4, Table 3 . Crystallography statistics for apo ipomoelin (IPO) and IPO in complex with carbohydrates methyl a-D-mannopyranoside (Me-Man), methyl a-D-glucopyranoside (Me-Glc) and methyl a-D-galactopyranoside (Me-Gal). sodium formate, 40% w/v polyethylene glycol 3,350. The crystals of IPO-Me-Gal appeared in 7 days. A mixture of the reservoir solution with 100% glycerol in a 4:1 volume ratio was used as cryo-protectant for data collection. The diffraction data were collected at 100K and detected by a Quantum 315 or Quantum 210 CCD detector at the BL13B1 or BL13C1 beamlines of NSRRC (Hsinchu, Taiwan). All diffraction data were processed and scaled with use of the HKL2000 program [38] . The diffraction statistics are in Table 3 .
We used a blastp search for the amino acid sequence of IPO [GenBank: BAA14024.1] against the algorithm of the National Center for Biotechnology Information (NCBI) protein databank database for searching structural templates. The amino acid sequence of Calystegia sepium agglutinin (Calsepa), a JRL (PDB: 1OUW), showed 53% sequence identity to that of IPO. The monomeric structure of Calsepa was further used in a search to determine the structure of apo IPO by molecular replacement with use of the program CNS [39] . After cross-rotation and translation of molecular replacement, 4 values were obtained. Initial rigid body refinement for the 4 monomeric structures gave a 48.8% R-factor. Clear continuous electron density could be observed after calculation of Fourier maps, and the 5 th molecule of apo IPO was further built accordingly. Because of different space groups for the structures of the IPO-Me-Man, IPO-Me-Glc and IPO-Me-Gal complexes, the resolved monomeric apo IPO was used as a search template in the following molecular replacement method. The solutions of cross-rotation and translation could be obtained with 2 molecules for the IPO-Me-Man complex, 4 molecules for IPO-Me-Glc and 4 molecules for IPO-Me-Gal. Those solutions were further applied to initial rigid body refinement, and reasonable values were obtained (e.g., 36 .7% Rfactor for IPO-Me-Man, 35.4% for IPO-Me-Glc, and 32.9% for IPO-Me-Gal).
Manual model rebuilding involved use of Coot [40] , alternating refinement by the CNS program, with 5% or 10% of the observed reflections randomly selected and set aside for calculation of the R free value. The final refined statistics are in Table 3 . For the protein interface of the tetrameric form, IPO-Me-Glc was used as a representative for analysis by the web service PDBe PISA [34] . All molecular representations were prepared with use of Deep-View [41] and PyMOL [42] . The coordinates of monomers of apo IPO (e.g., chain A) were subjected to the web service Matras for structure comparison [31] . 
The quantification of protein solution for binding assay was determined by the UV absorption method. The purified IPO protein was dialyzed against 20 mM Tris-HCl, 150 mM NaCl (pH 7.0) at 4uC overnight. The concentration of IPO and DN10IPO was determined by UV absorption spectroscopy at 280 nm with the specific extinction coefficient e of 22,920 M 21 cm 21 , which was determined from the prediction of IPO primary sequence. From Beer-Lambert law, A = e6b6C where A is the absorbance of the sample at 280 nm, b is the pathlength in 1 cm, and C is the protein concentration (M).
The protein concentration C could be calculated from the equation. Carbohydrates were prepared by weighting the amount on a microbalance before dissolving in dialysis buffer (20 mM Tris-HCl, 150 mM NaCl pH 7.0).
ITC measurements involved use of a MicroCal iTC200 microcalorimeter (GE Healthcare) at 25uC. In individual titration, 1-2 ml carbohydrate solution was added at 180-s intervals by use of a computer-controlled 40 ml syringe to a cell containing 280 ml IPO protein solution under constant stirring at 1,000 rpm. The concentration of IPO protein was 1-3 mM and that of Me-Man, Me-Glc, Me-Gal, Man, Glc and Gal 25-75 mM. The titration of carbohydrate solution in this range of concentration to the dialysis buffer was used as a control. Measurements of the heat change determined from the binding constant (K A ), reaction stoichiometry (n), and enthalpy (DH). The 18 experimental data were fitted for a 1:1 binding model (one-site of fitting) with Microcal Origin 7.0 software. Free energy (DG) and binding entropy (DS) were calculated by the equations DG = -RTlnK A and DG = DH -TDS. R is the gas constant and T the absolute temperature. The optimal c-value in ITC calculation varied between 1 and 10. However, for titrations with Man, Glc and Gal, the c-values were ,1.
The atomic coordinates and structure factors of apo IPO and IPO-carbohydrate structures have been deposited in the RCSB Protein Data bank, with 3R50 for apo IPO, 3R51 for IPO-Me-Man complex, 3R52 for IPO-Me-Glc complex and 4DDN for IPO-Me-Gal complex.  Retroviral Env Glycoprotein Trafficking and Incorporation into Virions Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins. All replication-competent retroviruses encode genes for three major proteins: Gag, Pol, and Env. Complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1), encode additional regulatory and accessory proteins required for efficient replication in host cell or the infected host organism. Gag, an essential retroviral protein, is necessary and sufficient for the assembly, budding, and release of virus-like particles (VLPs) in all types of retroviruses except the spumaviruses. Gag is synthesized on cytosolic ribosomes and is assembled as a polyprotein precursor. During and/or shortly after budding and release, the polyprotein is cleaved into several domains by the viral protease ( Figure 1 ) as reviewed in [1] [2] [3] . The major domains of the precursor Gag are the matrix (MA), capsid (CA), and nucleocapsid (NC). The primary role of the N-terminal MA domain is targeting of the Gag precursor protein to the site of assembly, typically the plasma membrane (PM). In general, electrostatic interactions between basic amino acid residues in MA and the acidic inner leaflet of the PM are important for Gag-membrane targeting [4, 5] . In the case of HIV-1, the N-terminal myristate group and a cluster of basic residues in the MA domain of the HIV-1 Gag that interacts with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) together target the Gag precursor Pr55 Gag to the PM [6, 7] . Although the Gag-membrane targeting of both murine leukemia virus (MLV) and Mason-Pfizer monkey virus (MPMV) is also affected by PI(4,5)P 2 modulation [8, 9] , it has been reported that the membrane targeting of Rous sarcoma virus (RSV) and human T-lymphotropic virus type 1 (HTLV-1) is largely independent of PI(4,5)P 2 [10, 11] . The MA domain also plays a role in the incorporation of the Env glycoprotein into virions. The CA domain is important for Gag-Gag interactions during virus assembly and constitutes the outer part of the viral core after Gag processing by the viral protease [12] [13] [14] . NC is the primary nucleic acid binding domain of Gag. This small, basic domain is responsible for the binding and incorporation of the viral RNA genome into virions, which is mediated by Gag interactions with genomic RNA.
Gag proteins are synthesized and transported to the PM. Many studies demonstrate that the major site of HIV-1 assembly is the PM [15] [16] [17] [18] , although late endosomes could be a platform for virus assembly under specific conditions [19] . In primary macrophages, HIV-1 has been shown to assemble in endosomal vesicles. However, studies have recently suggested that the above vesicles are not late endosomes but rather membrane invaginations connected to the PM [20] [21] [22] . In addition to Gag, the other major structural retroviral protein is the Env glycoprotein. Env proteins are required for virus entry into target cells and are thus essential for forming infectious retroviral particles. In this paper, we discuss current knowledge about the biosynthesis, intracellular trafficking, and virion incorporation of retroviral Env proteins, as well as the membrane microdomains involved in virus assembly and/or transfer. Most of this information was obtained from studies on HIV-1.
Retroviral Env glycoproteins are synthesized from a spliced form of the viral genomic RNA as reviewed in [23] [24] [25] (Figure 1 ). Translation of the Env protein occurs on ribosomes bound to the endoplasmic reticulum (ER) and starts with the leader sequence, which contains a small, N-terminal hydrophobic signal peptide. The Env protein is cotranslationally inserted into the lumen of the rough ER. In the ER, the leader sequence is removed by cellular signal peptidases. In addition, Env polypeptides are N-and O-glycosylated and subsequently trimmed [26, 27] . The number and location of glycosylated residues varies broadly among retroviruses. The hydrophobic transmembrane (TM) domain prevents Env proteins from being fully released into the lumen of the ER [28, 29]. The amino acid sequence following the TM is referred to as the cytoplasmic tail (CT), which varies from 30 to around 150 residues, depending on the virus. Env proteins are folded and assembled into oligomers in the RER. Retroviral Env proteins form trimers [30] [31] [32] [33] . The HIV-1 accessory protein Vpu binds to the CD4 receptor through its cytoplasmic domain and downregulates the receptor by transporting it to the proteasome for degradation, thereby preventing premature interactions between Env and its receptor [34] [35] [36] .
In the Golgi, cleavage of the retroviral Env precursor occurs at a polybasic (e.g., K/R-X-K/R-R) motif by cellular proteases such as furin or closely related enzymes probably within or near the trans-Golgi network (TGN) [37] [38] [39] [40] [41] [42] [43] . For HIV-1, the surface glycoprotein gp120 and the TM glycoprotein gp41, which bind together noncovalently, are both formed from the same precursor protein, gp160. Gp160 processing is essential for the activation of Env fusogenicity and virus infectivity [38, 42, [44] [45] [46] . Similarly, cleavage of Env is also essential for membrane fusion and virus infectivity in MLV [39, [47] [48] [49] [50] , in RSV [51, 52] , and in mouse mammary tumor virus (MMTV) [53] . A recent report showed that cleavage of MLV Env by furin also plays an important role in Env intracellular trafficking and incorporation [54] . Although most retroviral Env proteins including that of HIV-1 are associated with intracellular membranes [55] [56] [57] , at least part of the gp120/gp41 trimer complex traffics through the secretory pathway to the PM. It has been suggested that AP-1, one of adaptor proteins for clathrin-coated vesicle formation, is involved in the correct sorting of HIV-1 Env from the TGN to the PM, [58, 59] . It has been reported that intracellular CTLA-4-containing secretory granules are involved in the trafficking of HIV-1 Env to the PM although the subsequent trafficking of Env after the Golgi is not well understood [60] .
After reaching the PM, like those of other lentiviruses, HIV-1 Env undergoes rapid endocytosis, which is mediated by the interaction between the μ2 subunit of the clathrin adaptor AP-2 and a membrane-proximal, Tyr-based motif (YxxL) in the gp41 CT [58, 61, 62]. Although some of the endocytosed Env is recycled back to the PM, most retroviral Env is associated with intracellular membranes [63, 64] . The level of gp120-gp41 oligomers on HIV-1 virions is relatively low [33] . Maintaining low levels of Env at the cell surface allows the infected cells to evade the host immune response and to avoid induction of Env-mediated apoptosis. Gammaretroviruses such as MLV and MPMV also have dileucine-and Tyr-based motifs in their Env CT. These motifs are important to regulate intracellular trafficking of Env of both retroviruses via interactions with clathrin adaptors [65, 66].
As for pseudotyping of gammaretroviruses, it has been reported that the feline endogenous retrovirus RD114 Env does not allow pseudotyping with viral cores from lentiviruses such as SIV, whereas the RD114 Env is incorporated into MLV virions [67-69]. Intracellular trafficking of Gag and Env was examined using a set of chimeric viruses between MLV and RD114 [57]. Interestingly, it was found that the RD114 Env was mainly localized along the secretory pathway, whereas the MLV Env was mostly localized in endosomes, and that intracellular localization was dependent on specific motifs in the Env CT [57]. In addition, subsequent work revealed that an acidic cluster in the RD114 Env CT regulates assembly of not only the RD114 Env but also the MLV Env through the interaction with a host factor, phosphofurin acidic-cluster-sorting protein 1 [66].
Several models have been proposed for the incorporation of retroviral Env glycoprotein into virions as reviewed in [23, 70] (Figure 2 ).
Passive incorporation is the simplest model for the incorporation of Env proteins into virus particles (Figure 2(a) ). There are several lines of evidence supporting this model.
First, viral pseudotyping with a foreign glycoprotein can occur easily in many cases although there are some exceptions, one of which is the exclusion of HIV-1 or SIV Env with the long CT from most retrovirus cores [70] . With respect to HIV-1, the virus can be pseudotyped with Env glycoproteins not only from several other retroviruses but also with those from other virus families such as ortho (para) myxoviruses and flaviviruses [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] .
Second, retroviruses allow passive incorporation of host membrane proteins into virus particles [85] [86] [87] . Most cellular proteins are incorporated into the retrovirus envelope without significant sorting [88, 89] .
Finally, in the case of HIV-1, several studies have demonstrated that the gp41 CT can be removed without affecting incorporation of the Env into virions, although this has been shown to occur only for some laboratory cell lines such as HeLa or 293T [90] [91] [92] [93] [94] .
Although several lines of evidence support the passive incorporation model for retroviral Env, there is much evidence indicating that Env incorporation into virions is regulated by direct interactions between Gag and Env proteins (Figure 2(b) ). Although removal of the gp41 CT sequence of HIV-1 has little effect on Env incorporation in some cell types, as described above, smaller deletions in CT regions cause severe defects in Env incorporation [95] [96] [97] [98] [99] [100] . The MA domain of Gag has been shown to be important for Env incorporation into virions [91, 92, 101, 102] . The defect in Env incorporation caused by deletion of the gp41 CT is reversed by several MA mutations, indicating that an interaction between Env and the MA domain of Gag is required for incorporation of full-length Env into virions, at least in the case of HIV-1 [93, 98] .
More evidence for direct Gag-Env interaction comes from the finding that HIV-1 Env directs Gag budding to the basolateral surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells through the CT of HIV-1 Env, whereas Gag alone buds in a nonpolarized fashion [103] [104] [105] [106] . The Tyr-based motif in the gp41 CT is also utilized in polarized budding of HIV-1 in lymphocytes [107] . Surprisingly, the polarized budding of HIV-1 in MDCK cells could also be promoted by MLV and HTLV-1 Env through their CT [108] . It also has been reported that coexpression of Pr55 Gag inhibits endocytosis of HIV-1 Env through its interaction with the gp41 CT [63]. Another example of the specific Gag-Env interactions was demonstrated using Gag and Env proteins of MLV and HIV-1 in rat neurons [109] . Similarly, MLV Env is preferentially recruited onto MLV Gag through its CT domain in the presence of both MLV and HIV-1 cores although the authors also show an alternative mechanism by which the recruitment to HIV-1 budding sites is independent of the CT domain of MLV Env [110] . Furthermore, RSV Env is exclusively recruited to RSV budding sites through its CT, suggesting that the interaction between Env and Gag is direct in the case of this avian retrovirus [111] .
In addition to the circumstantial evidence discussed above, some biochemical data suggest a direct interaction between Gag and Env. In vitro binding between MA and a gp41 CT-GST fusion protein has been reported for both HIV-1 and SIV [112, 113] . Peptides corresponding to a large central domain of gp41 CT inhibited the capture of membrane-free Pr55 Gag with an anti-p24 antibody [114] . In addition, a stable, detergent-resistant gp41-Pr55 Gag interaction was detected in immature HIV-1 virions. The retention of gp41 in detergent-treated virions is dependent on the CT region, suggesting a direct or indirect interaction between Pr55 Gag and gp41 [115, 116] .
In the third model, it is assumed that host cellular factors (mostly proteins) play a role in bridging Gag and Env in virus-infected cells (Figure 2(c) ). Several host factors have been reported to bind to Gag and/or Env of HIV-1 or SIV however, only a couple of host factors were shown to be required for Env incorporation and/or viral replication.
The 47-kDa tail-interacting protein (TIP47) has been reported to bridge Gag and Env, allowing efficient Env incorporation in HIV-1 [117, 118] . The same group also showed that both the WE motif near the N-terminus of the MA domain and the YW motif in the gp41 CT domain are important for interactions between Gag or Env and TIP47 [118] . In a subsequent paper, the same group showed that mutations in either the WE motif of MA or the YW motif in the gp41 CT caused defects in virus replication in primary monocyte-derived macrophages [119] . Although this finding of an important role for TIP47 in Env incorporation in HIV-1 has received much attention from retrovirologists, no confirmatory data have been published by other researchers in this field.
Human discs large protein (hDlg1) has been reported to interact with the CT of HTLV-1 Env and to colocalize with both Env and Gag in virus-infected cells [120] . Subsequent work demonstrated that Dlg1 also binds HIV-1 Gag and that the expression level of Dlg1 is inversely correlated with HIV-1 Env expression and incorporation levels of the Env proteins, although the mechanism behind this phenomena needs to be investigated [121] .
Prenylated Rab acceptor 1 (PRA1), which was identified as a Rab regulatory protein, was reported to be a binding partner for the SIV gp41 CT in a mammalian yeast twohybrid (Y2H) assay [122] . Although colocalization of PRA1 and SIV Env was observed, changes in the endogenous levels of PRA1 did not affect virus production, Env incorporation, or infectivity of SIV or HIV-1 [123] .
A Prohibitin 1/Prohibitin 2 (Phb1/Phb2) heterodimer was identified as a binding partner of the gp41 CT of HIV-1 using human T-cell lines and tandem affinity chromatography [124] . Phb1 and Phb2 are members of the prohibitin superfamily of proteins, which are localized to several cellular compartments such as the mitochondria, nucleus, and the PM [125, 126] . Gp41 CT mutants, in which binding to Phb1/Phb2 is disrupted, could replicate well in permissive cell types such as MT-4, but could not replicate efficiently in nonpermissive H9 cells [124] . Further analysis is necessary to elucidate the mechanism by which these proteins regulate virus replication through interactions with Env.
Luman, a transcription factor that is mainly localized to the ER, was found to interact with the gp41 CT of HIV-1 in a Y2H screen using a cDNA library from human peripheral blood lymphocytes (PBL) [127] . Overexpression of a constitutively active form of this protein reduced the intracellular levels of Gag and Env, leading to a decrease in virus release. The mechanism for this negative effect on virus assembly involves Luman binding to Tat, which decreases Tat-medicated transcription [127] .
By using a Y2H screen with human cDNA libraries, p115-RhoGEF, an activator of Rho GTPase, was found to interact with the gp41 CT through its C-terminal regulatory domain [128] . The gp41 mutants that lost the ability to bind p115 showed impaired replication kinetics in T-cell lines such as SupT1, H9, and Jurkat, suggesting that the gp41 CT could modulate the activity of p115-RhoGEF to support virus replication [128] .
In addition to the host factors described above, calmodulin [129] [130] [131] [132] and α-catenin [133] [134] [135] have been reported to interact with HIV-1 and/or SIV. However, their roles in virus replication, especially with respect to the Env functions of both proteins, have not been clearly elucidated.
Regardless of whether direct or indirect interactions between retroviral Gag and Env proteins are required for Env incorporation into virions, a great deal of experimental evidence suggests that retroviruses assemble and bud from "membrane microdomains." The most well-known microdomains are "lipid raft(s)," which are enriched in cholesterol and sphingolipids [136, 137] . Lipid rafts are widely thought to function as a platform for the assembly of protein complexes and to allow various biological processes such as cellular transport and signal transduction to proceed efficiently [138, 139] . Lipid rafts are reportedly used as assembly platforms or entry scaffolds in the replication of enveloped viruses such as retroviruses [140] [141] [142] [143] [144] [145] [146] . The association of Gag/Env with lipid rafts is important for the regulation of Env incorporation and pseudotyping [143, 144, 147, 148] . Evidence that both the HIV-1 Pr55 Gag and Env proteins are preferentially localized to lipid rafts comes from biochemical studies as well as direct observations by microscopy [142, 149, 150] .
Another membrane microdomain for retrovirus assembly is the "tetraspanin-enriched microdomain (TEM)" [151] [152] [153] [154] . Tetraspanins are a superfamily of cell surface proteins that are ubiquitously expressed in mammalian cells. TEMs also act as platforms for signal transduction and immune responses. TEMs have been reported to be involved in the assembly and release of not only HIV-1, but also HTLV-1 and HCV [155] . When both HIV-1 and influenza virus were produced in the same cell, only HIV-1 colocalized with the TEM marker, and its release was inhibited by an anti-CD9 Ab, which led to extensive aggregation of tetraspanins [156] . Analysis of dynamics of both lipid rafts and TEMs by quantitative microscopy has revealed that components of both lipid rafts and TEMs are recruited during viral assembly to create a new microdomain that is different from preexisting membrane microdomains [153, 157] .
There have been three recent reports in which both pseudotyping and microdomain issues were discussed. In the first paper, the authors examined HIV-1 assembly under conditions where the Env proteins of HIV-1 and Ebola virus were coexpressed with HIV-1 Gag in the same cell [158] . They found that infectious HIV-1 virions were released with both types of Env proteins. Interestingly, however, the virions contained either HIV-1 Env or Ebola virus glycoprotein (GP), but not both Env proteins within a single virion. These results suggest that HIV-1 Env and Ebola virus GP localized to distinct microdomains on the surface of the same cell [158] . In the second paper, the subcellular localization of Gag and Env proteins was investigated using a combination of three different retroviral Env proteins (RSV Env, MLV Env, or vesicular stomatitis virus (VSV) G) and two different Gag proteins (RSV or HIV-1) [111] . Both VSV-G and MLV Env were redistributed to the virus budding sites when coexpressed with HIV-1 or RSV Gag. In contrast, RSV Env was mostly transported to RSV budding sites. A subsequent paper from the same group showed that the CT of MLV is not required for recruitment of MLV Env to HIV-1 budding sites, suggesting that there are no specific interactions between MLV Env and HIV-1 Gag [110] . Collectively, these results also suggest that retroviral Env glycoproteins are not recruited to preexisting membrane platforms but rather that they are actively recruited to newly formed microdomains on the cell surface [111] .
Human retroviruses such as HIV-1 and HTLV-1 spread more efficiently between target T cells by cell-cell infection than by cell-free infection [159, 160] . Sattentau et al. proposed, in analogy to the "immunological synapse", the "virological synapse (VS)" as a point of contact between virusinfected cells and uninfected cells [161, 162] . The molecular mechanisms of retroviral VS formation are as follows. (1) With respect to HIV-1 T-cell VS, initial contact between virus-infected cells and uninfected cells occurs through gp120-CD4 binding. Subsequent interactions between integrins and ICAMs enforce and maintain the stability of these junctions. (2) The gp120-CD4 interaction recruits CD4, coreceptors such as CXCR or CCR5, adhesion molecules, and filamentous actin into the synaptic area. (3) The cellular secretory machinery and microtubule organizing centers (MTOC) are polarized towards the HIV-1 assembly sites at the PM to form the VS. It has been reported that a socalled microsynapse formed by nanotubes between virusinfected cells and uninfected cells is also involved in cell-cell infection of HIV-1 [84, 163] . In cell-cell transfer of HTLV-1-infected cells, an extracellular matrix structure referred to as the "viral biofilm" was proposed as an alternative to the VS [164] . In addition to HIV-1 and HTLV-1, the spread of MLV between fibroblasts also occurs via the VS [165, 166] . It is noteworthy that assembly of MLV is directed towards cellcell contact sites through the interaction of the CT of MLV Env with Gag [167, 168] . Although the concept of cell-cell infection through the VS is now well appreciated, the detailed molecular mechanism of VS assembly and its relevance to viral spread in vivo will require further elucidation through the use of more advanced techniques.
Incorporation of Env glycoproteins into virions is crucial for producing infectious retroviral particles. Although this paper has introduced several experimental models for retroviral Env trafficking and/or incorporation, the correct mechanism for this process is still unclear. The following questions must be clearly addressed to not only gain a better understanding of this complex biological process, but also to develop new antiretroviral compounds that target Env incorporation.
(1) What are the structures of the CTs of retroviral Env proteins? The answers for this question will give useful information on elucidating a role of the Env CTs in the Env trafficking and/or incorporation in virus-infected cells. (2) What host factor(s) are necessary for the retroviral Env trafficking and/or incorporation into virions? (3) Where and how Env and Gag proteins of retroviruses are recruited to the assembly sites in order to form infectious virus particles?
[  Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins BACKGROUND: Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model. METHODOLOGY/PRINCIPAL FINDINGS: In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers. CONCLUSIONS/SIGNIFICANCE: Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii. Toxoplasma gondii is a well-adapted parasite of warm-blooded hosts, including human and production animals. The parasite has distinct developmental stages, such as the fast-replicating tachyzoites that are present in the acute phase of infection, while the slow-replicating bradyzoites form tissue cysts in muscular and nervous tissues during the chronic phase of infection [1] [2] . Infection usually takes place after accidental ingestion of raw or undercooked meat containing tissue cysts, oocyst-contaminated food or water, and by transplacental passage of tachyzoites in active infection during the gestational period [3] [4] . Congenital infection may lead to neurological disorders, ocular disease and/or fetal death [5] . T. gondii is also a major cause of infectious abortion and induces clinical disease in production animals, especially goat, sheep and swine, elevating production costs and resulting in considerable impact on the livestock industry as well as the public health [6] .
Immunized hens transfer circulating antibodies from blood to egg yolk by specific receptors on the surface of the ovarian follicle [7] . Immunized hens may continue to produce antibody-rich eggs for up to two years -also known as IgY, which shares great similarity to mammalian IgG, and is a reasonably stable bivalent protein with estimated molecular weight around 180 kDa [7] [8] . Egg yolk antibodies have been shown to be effective in immunohistochemical assays and for recognition of specific epitopes [9] . Polyclonal IgY have been previously described for the purification of polyepitope vaccine candidates against Plasmodium falciparum [10] , passive immunization against Eimeria acervulina [11] , immunoprophylactic protocols against influenza virus [12] , and analysis of tumoral biomarkers [13] , among others.
Specific polyclonal antibodies generated in mammals against T. gondii have been extremely useful in the last decades to unravel several aspects related to the parasite biology as host-parasite interactions, parasite invasion and replication mechanisms, candidate epitopes for diagnosis, characterization of T. gondii virulence factors and candidate protein for immunoprophylactic assays [14] [15] [16] [17] .
Here, we hypothesized whether polyclonal IgY could be a useful tool in the T. gondii model. In that sense, we produced, characterized and standardized distinct protocols for IgY application, using polyclonal IgY antibodies raised against T. gondii soluble tachyzoite antigen (STAg).
The first step consisted of the purification of IgY antibodies from egg yolks in order to obtain the highest yield and purity. Antibodies were separated from other constituents of the egg yolk by the addition of acid water and later precipitated by salting-out (19% sodium sulfate) (Fig. 1A) . Although enriched in IgY antibodies, the water soluble fraction (S1) still presented undesirable low molecular weight proteins after acid water precipitation of pooled egg yolk (Fig. 1B , lane S1) that were eliminated after salting-out step (Fig. 1B, lane P2) , presenting an average protein concentration of 4 mg/mL. We recovered 115 eggs from four STAg-immunized chickens during a 49-day interval. Each egg presented an average of 12 mL of yolk and the mean yield was 48 mg of polyclonal antibody per egg. Precipitated antibodies contained in P2 fraction were further submitted to purification by exclusion-size chromatography (Fig. 1C ), resulting in a protein peak between the 13th and 19th fractions, although elevated purity was detected mainly in the 14th fraction (F14), as it may be observed in its SDS-PAGE profile (Fig. 1D) . Thus, the used protocol was able to retrieve a mean of 4 mg of total IgY from 1 ml of crude egg yolk. Slot-blot assays confirmed IgY in fractions from salting-out and gel filtration purified fractions until the concentration of 110 ng of protein (Fig. 1E ).
After purification of an enriched IgY fraction, kinetics and avidity of the purified antibodies were evaluated by indirect ELISA. We verified that immunized hens presented detectable levels of specific IgY starting on day 21 after the first immunization (p.i.). The production of IgY anti-STAg increased significantly along the next weeks, and developed a progressive and sustained response, reaching its peak on day 42 p.i. ( Fig. 2A) . High avidity of IgY anti-STAg antibodies was already observed in the first reactive samples, at 21 days p.i., as demonstrated by the lack of significant differences in EI values between 6 M urea-treated and untreated samples (Fig. 2B) . The antigen recognition repertoire and avidity maturation of IgY anti-STAg were also verified by immunoblot. Similar to ELISA, reactivity was first observed on day 21 after immunization, with strong staining of a 30 kDa protein (Fig. 2C) . From the 35 th day onwards, IgY anti-STAg antibodies recognized a broad range of T. gondii antigens. The 6 M urea treatment presented a reduction in staining intensity of the described antigens between the 21 st and 35 th days p.i., with normal reactivity being observed after that period (Fig. 2C) .
Mice immunized by the same route present a similar antibody seroconversion kinetics, with reactivity to STAg being detected on 14 th day p.i., preceded by initial p30 recognition on 7 th day p.i. in serum samples (Fig. S1 ).
Once we were able to obtain pure, high avidity IgY anti-STAg antibodies, we aimed to verify potential applications of those antibodies in T. gondii model. The standardized immunohistochemical assay using IgY anti-STAg antibodies was able to detect antigenic distinct structures in brain tissue sections, as follows: (i) parasitophorous vacuoles, demonstrated as rounded and compartmentalized structures (Figs. 3A and 3B); (ii) tissue cysts, represented by a wall bright fluorescence (Figs. 3C and 3D); and (iii) free tachyzoites (Fig. 3C) .
In cell culture experiments, the antibodies were used in immunocytochemical assays, where monolayers of HeLa cells infected with T. gondii were incubated with IgY anti-STAg. In these experiments, we observed isolated or grouped intracellular tachyzoites closely to cell nucleus, detected by an intense fluorescence on the whole tachyzoite surface (Fig. 4) . Overlay of fluorescence emitted by the stained parasites and cell nucleus with phase contrast images of the fibroblast monolayer provided a valuable measure of the parasitism of the infected monolayers.
We next observed the repertoire of the polyclonal antibodies raised in avian and mammalian models. Although p30 was evenly recognized by antibodies from the distinct species by onedimensional immunoblotts (WB1D), differential protein recognition pattern was noted (Fig. 5A ). Only chicken-derived antibodies recognized proteins with approximate molecular weights of 40 kDa, while IgG anti-STAg from mice reacted strongly to ,50 kDa antigen. These differences were maintained independently of the route used to immunize the mice (i.m. or s.c., Fig. 5A ). In order to further observe those differences in recognition, we performed 2D-immunoblots (WB2D) using the polyclonal antibodies obtained from immunized hens and mice. The differences in recognition were clearly noted in WB2D assay, with IgY antibodies recognizing a sequence of five antigenic spots at ,40 kDa, however with distinct isoelectric points -from neutral to basic pH (Fig. 5B ). On the other hand, mouse IgG reacted strongly to two acidic antigens at ,50 kDa ( Fig. 5C ), while IgY reacted faintly to the same spots and a basic protein with similar molecular weight (Fig. 5B ). Such differences were also noted in the distinct recognition patterns in antigens with 60-80 kDa, as well as the absence of immunodominant p22 antigen by chicken IgY. Additionally, STAg immobilized in nitrocellulose strips were probed to anti-Neospora caninum e anti-Eimeria spp. IgY in order to check the specificity of the antibodies. The assay revealed that IgY antibodies against the closely related protozoa did not cross-react with Toxoplasma antigens ( Figure 5D ).
The use of specific antibodies produced in mammals against antigenic targets and cell markers are essential tools for the study of host-parasite relationships. Unfortunately, these antibodies may be painstaking to obtain, and depend on animal bleeding in order to retrieve the desired immunoglobulins [18] . Chickens present antibody production kinetics and avidity maturation similar to mammals, and IgY antibodies appear in serum approximately 4 to 7 days after inoculation of antigen [9] .
In the present study, we produced and purified polyclonal anti-T. gondii antibodies from STAg-immunized hens. These anti-STAg antibodies were obtained from the egg yolk, in a protocol which animal bleeding turned out to be unnecessary [19] . Generally, yolk IgY arise around 7 th to 10 th day after the sera antibodies, in a sera concentration-dependent manner [20] . In addition, the large amounts of polyclonal antibodies recovered from the egg yolk in this process, the high avidity of the produced IgY antibodies, and their potential applications are itself the great features of the protocol herein described. Egg yolks contain large amounts of IgY passively transferred by hens, with mass ranging between 2-8 mg per mL after purification, and it has been estimated that 2-10% of total egg yolk antibodies are antigen-specific [21, 9] . To obtain STAg-specific IgY we used simple protocols, which are easily reproduced without the need of refined equipment or reagents. Additionally, installations required for IgY production are smaller and easier to handle than those designed for small ruminants, commonly used for large scale antisera production [22] . The production of specific IgY has been previously described for the recognition of a broad range of targets, including Plasmodium falciparum [23] , hepatitis A virus [24] and tumor cells [25] [26] .
Avidity maturation of an antibody represents an increasing affinity interaction between antibodies and antigens. However, this maturation is slowly induced in mammals [27] . We demonstrated that STAg-immunized hens produced large amounts of high avidity polyclonal IgY anti-STAg antibodies after the first booster, with early recognition of p30 proteins. It is known that SAG1 protein (p30) triggers the human IgG maturation in latter stages of T. gondii infection [28] . Additionally, IgY antibodies recognized a broad range of proteins, with distinct isoelectric points determined by 2D-immunoblot. Previous studies have shown that serum samples from naturally infected humans react to a broad spectrum of T. gondii proteins [29] and experimentally infected mice produce specific antibodies against high and low molecular weight antigens of tachyzoites [30] . Consistently, our results demonstrated that IgY anti-STAg presented an antigen recognition profile similar to mouse serum, although some antigens were more immunogenic for hens than mice, probably due to the phylogenetic distance among these animals. In addition, IgY antibodies raised against closely related parasites did not recognize STAg proteins, which denotes an interesting feature of the IgY system, since crossreactivity is a normal feature of mammalian IgG.
Hassl and colleagues (1987) [31] , in a preliminary study, demonstrated some of characteristics of anti-T. gondii IgY and also compared protocols for antibody production and purification, however it was not demonstrated the possible applications of these antibodies to study the parasite. We herein demonstrated for the first time distinct applications for specific IgY antibodies in the T. gondii model. As previously stated, this antibody producing system offers high yield of specific antibodies [32] . We believe that immunization of hens with selected T. gondii targets could improve the range of applications for IgY antibodies. For the P. falciparum model, it has been demonstrated that monospecific and polyclonal IgY antibodies were advantageous [10] . In that sense, immunizations using single antigens of T. gondii could be employed to better understand the role of the selected molecules, as tools to improve the diagnosis of acute toxoplasmosis [33] or the knowledge of hostparasite interaction mechanisms [34] . Additionally, the identification of conserved surface antigens among apicomplexan organisms is usually performed by mammal specific IgG antibodies and contributes to our understanding of parasite evolution [35] [36] [37] .
As chickens are phylogenetically distant from mammals and have been shown to be refractory to T. gondii infection [38] , and differential recognition profile by antibodies from both classes of animals has already been described [39] . The comparative analysis of antigen recognition may be a useful tool to determine different virulence factors of the parasite, thorough proteomic studies for vaccine or diagnostic development. Moreover, the role of T. gondii antigens involved in parasite virulence has been largely addressed over the last decade [40] [41] . The most common protocols used for that purpose are the incubation of the parasite with specific antisera or the generation of strains with targeted depletions, preventing molecular interactions between host and parasite proteins [16] .
As shown in this study, IgY antibodies may also be used in this context and further studies are necessary to determine whether these specific antibodies could be employed in therapeutical protocols during acute toxoplasmosis, especially in immunizations performed with single antigens. In conclusion, our results demonstrated that polyclonal IgY anti-STAg antibodies are a promising complementary tool for studies of the T. gondii infection model.
Laying hens of the Isa Brown lineage, 25 weeks of age, were used for immunization protocols. Hens were kept in individual cages and received commercial rations and water ad libitum. C57BL/6 mice, 6-8 weeks old, were infected with T. gondii ME-49 strain to obtain parasite positive brain tissue sections [42] and immunized with STAg to obtain polyclonal IgG antibodies anti-T. gondii [43] . All animal procedures were approved by the institutional ethics committee in animal experimentation (Comissão de É tica no Uso de Animais da Universidade Federal de Uberlândia -Protocol No. 107/11), and were performed based on the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and instructions disposed in the 2000 Report of the AVMA Panel on Euthanasia [44] .
Soluble tachyzoite antigen (STAg) was obtained using a previously described protocol [45] . Briefly, tachyzoites of T. gondii (RH strain) were maintained by serial passage in BALB/c mice and obtained from peritoneal exudates from mice infected 48 h earlier. Only exudates containing 100% of free-ranging tachyzoites were used for antigen preparation. Parasite suspension was washed in phosphate buffered saline (PBS, pH 7.2), added to a protease inhibitor cocktail (Complete Ultra tablets, Roche, USA), processed through lysis by repeated freezing and thawing cycles, sonicated, and centrifuged at 14,0006g for 30 min at 4uC. After supernatant recovery, total protein was estimated by the Bicinchoninic acid kit (BCA, Sigma, St. Louis, MO, USA) and aliquots were stored at 280uC until use. The same protocol was performed with cells extracted from the peritoneal cavity of mice, used as experimental controls.
Hens (n = 4) were immunized by muscular route with emulsion composed of STAg and Freund's adjuvant, according to previously described protocol [46] . Primary immunization was performed with 100 mg of STAg in 250 mL of PBS and equal volume of Freund's complete adjuvant (Sigma). Two boosters were performed at 15 day intervals, with 100 mg of STAg plus Freund's incomplete adjuvant. In parallel, Neospora caninum (n = 4) and PBS (n = 2) -immunized hens were maintained for parasite-specific and Figure 1 . Purification of egg yolk antibodies. (A) Separation of the water soluble fraction (S1) from a lipid-rich precipitate (P1), after the incubation of crude egg yolk with acid water (pH 5.0-5.2); S1 precipitation by salting-out (19% Na 2 SO 4 ) produced an enriched IgY pellet (P2) and a supernatant with contaminants (S2). (B): Purity degree of IgY samples determined by SDS-PAGE (8%), demonstrating that salting-out protocol produced high purity IgY antibodies. (C) Size-exclusion chromatography of the P2 fraction, with peak of IgY between 13th and 19th fractions, where (D) the 14th fraction presented the highest degree of purity. (E): IgY enrichment conferred by Slot-blot assay, IgY was detected until the concentration of 0.01 mg of protein (box). Bovine sera albumin (BSA) was used as negative control. doi:10.1371/journal.pone.0040391.g001 irrelevant IgY purification, respectively. Hens were monitored daily for adverse effects and individual laid eggs were daily collected and stored at 4uC until further processing.
In addition, C57BL/6 mice were immunized by intramuscular and subcutaneous routes with STAg, for comparative purposes. Primary immunization was performed with 25 mg of STAg in 50 mL of PBS and equal volume of Freund's complete adjuvant (Sigma). Two boosters were performed at 15 day intervals, with 25 mg of STAg with Freund's incomplete adjuvant. Serum samples were collected weekly and stored at 220uC until use.
IgY was purified by the water-dilution method as previously described [19] . In order to obtain a representative sampling of antibody production along the weeks after immunization, eggs laid weekly from each hen were pooled prior to IgY extraction. Then, egg white was removed and egg yolk was diluted 10-fold in deionized ultrapure water adjusted to pH 5.0-5.2 with 1N HCl and homogenized thoroughly. After centrifugation at 10.0006g for 25 min at 4uC, the supernatant was collected, consisting of lipid-free fraction (S1). S1 was then precipitated with the addition of 19% sodium sulphate (w/v). After centrifugation (10.0006g, 25 min, 4uC), the pellet was retrieved and represented the IgYenriched fraction (P2). P2 samples were resuspended and dialyzed against PBS to eliminate residual salt. Additionally, P2 samples were submitted to exclusion-size gel chromatography using Sephacryl S-300 column (GE Healthcare, Uppsala, Sweden), at a flow rate of 3 mL/min, and the IgY-enriched protein fraction was determined by 280 nm readings. Actual protein concentration was measured by BCA kit (Sigma) and samples were stored at 220uC until use. This protocol was also applied to extract IgY antibodies from the eggs of hens immunized in house with N. caninum soluble antigen, as well as from commercially available egg yolk powder containing anti-Eimeria spp.(E. acervulia, E. maxima e E. tenella) IgY antibodies (Supracox, Investigación Aplicada, Sociedad Anónima de Capital Variable, Puebla, Mexico). Possible cross- Figure 3 . Immunohistochemistry for T. gondii detection using polyclonal IgY anti-STAg antibodies. Paraffin-embedded brain sections of mice chronically infected with ME-49 strain were incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). (A and B) , segmented parasitophorus vacuoles were detected into host cell cytoplasm around the DAPI-stained nucleus (blue). (C and D), IgY anti-STAg antibodies strongly recognized antigens on outer walls of tissue cysts and free tachyzoites (C, arrow). doi:10.1371/journal.pone.0040391.g003 reactivity of the purified IgY antibodies to mouse proteins were ruled out by immunoblots (Fig. S2) .
The quality of the purification protocols was analyzed in polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE) at 8% in non-reducing conditions. IgY enrichment was assessed by slot dot assays, as follows: proteins samples obtained during IgY extraction were sequentially diluted 10-fold (10 mg to 10 24 mg) and transferred to nitrocellulose membrane by a vacuum apparatus (Bio-Dot SF; Bio-Rad, EUA). The presence of detectable chicken antibodies was verified by an rabbit anti-IgY antibody conjugated to peroxidase (Sigma) as secondary antibody, revealed by H 2 O 2 and DAB (Sigma).
The kinetics of chicken IgY anti-STAg production and avidity maturation were evaluated by an indirect ELISA. The optimal conditions for ELISA were obtained through block titration of the reagents. Briefly, high affinity microtiter plates (Costar Corning Incorporated, USA) were coated with STAg (10 mg/mL) in 0.06 M carbonate buffer (pH 9.6) and incubated overnight at 4uC. Plates were washed 3 times with PBS-Tween 0.05% (PBS-T) and blocked with PBS-T plus 1% bovine serum albumin (PBS-T-BSA) for 1 h at room temperature. P2 samples were adjusted to 2 mg/well in PBS-T-BSA, added to the wells in duplicate and incubated for 1 h at 37uC. After washing, plates were incubated with rabbit anti-IgY antibody labeled with peroxidase, diluted 1:30.000 in PBS-T, for 1 h at 37uC. The reaction was revealed by adding 0.01 M 2,29-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS, Sigma) and 0.03% H 2 O 2 , and optical density (OD) was determined at 405 nm. Two positive quality-controls and six negative controls (irrelevant IgY) were included in each plate in order to calculate the cut off, which was established as the mean OD values for irrelevant IgY plus three standard deviations. The same protocol was applied to mouse IgG anti-STAg obtained after immunization, using a goat IgG anti-mouse IgG conjugated to peroxidase (Sigma), at 1:2000 dilution. Results were expressed as ELISA index (EI) as previously proposed [47] as follows: EI = OD sample /OD cut off , where values of EI .1.2 were considered positive.
To measure avidity maturation, plates were coated and blocked as described above. After the addition of P2 samples in quadruplicates, two of the wells were rinsed with 6 M urea in PBS-T, whereas the other duplicate wells were rinsed with PBS-T only for 10 min at room temperature. Next, all wells were washed 3 times in PBS-T and subsequent steps were performed as described for the regular indirect ELISA.
To investigate the antigen recognition repertoire of IgY anti-STAg antibodies, immunoblot assays were carried out along with avidity maturation analysis against STAg. In 1D-immunoblot, STAg was separated on 12% SDS-PAGE under non-reducing conditions, and electrotransferred to nitrocellulose membranes. Non-specific interactions were blocked by 5% skim milk-PBS-T incubation, for 2 h at room temperature. Nitrocellulose strips were then incubated with P2 samples from weekly egg yolk pools adjusted to 2 mg of IgY. In parallel, avidity maturation was measured by strip treatment with 6 M urea for 10 min at room temperature. IgY was detected by incubating the secondary antibody, rabbit anti-IgY labelled with peroxidase, diluted at 1:20.000, for 2 h at room temperature. Reaction was revealed by adding 10 mg of 3,39-diaminobenzidine tetrahydrochloride (DAB, Sigma) in 15 mL of Tris buffered saline (TBS) and 12 mL of 30% hydrogen peroxide (Sigma). Reaction was stopped with distilled water. Experiments with similar conditions were performed for IgY anti-N. caninum and anti-Eimeria spp., as well as for the establishment of recognition kinetics of mouse IgG obtained from mice immunized by i.m. and s.c. routes (1:100 sera dilution; 1:2000 anti-mouse IgG conjugated to peroxidase -Sigma).
A 2D-immunoblot assay was also carried out to evaluate the antigen recognition profile of immunized hens to STAg. Briefly, 60 mg of STAg dialysed in ultrapure water, was separated by isoelectric focusing (IEF) on 7-cm immobilized pH gradient strips (ReadyStrip TM IPG Strip pH 3-10) overnight at room temperature, following the manufacturer instructions for equipment and reagents (GE, Healthcare, Uppsala, Sweden). After IEF, strips were equilibrated and loaded onto precast 12% polyacrylamide gels. Electrophoresis was performed and 2D-gels were stained with Coomassie brilliant blue G-250H (Sigma) or electrotransferred to nitrocellulose membranes. 2D-immunoblot was performed as described above for 1D-immunoblot.
In parallel, to compare the STAg recognition profile between mammalian and avian hosts, immobilized STAg membranes were probed with sera of experimentally T. gondii immunized mice, diluted 1:100 in 1% skim milk in PBS-T. Primary antibodies were detected by goat anti-mouse IgG antibody labeled with peroxidase (1:1000, Sigma) for 2 h at room temperature. Reaction was revealed with DAB and stopped with distilled water.
In order to detect T. gondii forms on paraffin-embedded brain tissue sections of mice chronically infected with T. gondii ME-49 strain [42] , we carried out an immunohistochemical assay by using IgY anti-STAg as primary antibodies. Brain sections were deparaffinized and incubated with specific IgY (30 mg/mL) diluted in PBS for 30 min at 37uC. After washing, slides were incubated with rabbit anti-IgY labeled with fluorescein isothiocyanate (FITC, Sigma) diluted 1:300 in addition to DAPI for 60 min at 37uC. Slides were mounted with carbonate-buffered glycerin (pH 9.0) and read at an inverted fluorescence microscopy system (FSX-100, Olympus, Tokyo, Japan).
In order to detect intracellular T. gondii replication, we standardized a cell culture based immunocytochemical assay using IgY anti-STAg as primary antibodies. Briefly, HeLa cells (ATCC No. CCL-2) cultured in 24-well plates were incubated with tachyzoites of T. gondii RH strain in a multiplicity of infection (MOI) equal to 1, in RPMI-1640 medium supplemented with 10% fetal calf serum for 24 h at 37uC and 5% CO 2 atmosphere. After incubation, cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton-X 100, followed by the incubation with the specific IgY antibody (30 mg/mL) for 2 h at 37uC. The next step consisted of the incubation with rabbit anti-IgY antibody labelled with FITC (Sigma), diluted 1:600 in PBS plus 49,6diamidino-2-phenylindole (DAPI, Sigma) for 1 h at 37uC. The reaction was read in an inverted fluorescence microscope system (EVOS, AMG Microscopy Group, USA).
Statistical analysis was performed using the GraphPad Prism version 4.0 software (GraphPad Software Inc., La Jolla, CA, USA). The Student t test was used to investigate significant differences between groups. Values of P,0.05 were considered statistically significant.  Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol BACKGROUND: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa). METHODOLOGY/PRINCIPAL FINDINGS: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts. CONCLUSION/SIGNIFICANCE: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body. Momordica charantia L. (MC), a Momordica Linn. genus of the family Cucurbitaceae and commonly known as bitter melon, is a traditional medicine plant indigenous to China. 1 The fruit and seed extracts from MC have been used in China for centuries for antivirus, antitumor, and immunopotentiating agent purposes. 2 In recent years, several ribosome-inactivating proteins (RIPs), including momordica anti-HIV protein (MAP30) and α-momorcharin (MMC), β-MMC, and γ-MMC, a group of which belong to the family of single-chain RIPs, were found to have the ability to inhibit protein biosynthesis in tumor cells by catalytic inactivation of the 60S ribosomal subunit. 3, 4 These proteins were also found to be able to inhibit the multiplication of herpes simplex virus-1 (HSV-1), 4, 5 poliovirus I in Hep2 cells, 6 and acquired human immunodeficiency virus type-l (HIV-l). 7 However, the strong immunogenicity, allergic reaction, and short half-life of these proteins have been considered the major barriers for their application as therapeutic agents in vivo. 8, 9 In recent years, researchers have shifted their focus to other technologies. An established technology, polyethylene glycol (PEG) conjugation (PEGylation), can bestow on proteins several benefits, such as increasing plasma half-life, decreasing toxicity, and reducing immunogenicity and antigenicity. 9, 10 The Food and Drug Administration has approved the PEGylated forms of the therapeutic proteins such as uricase, erythropoietin, granulocyte-colony stimulating factor, interferon, adenosine deaminase, asparaginase, and a growth hormone antagonist. Another technology is nanotechnology, which uses nanomaterials for packing potential therapeutic proteins to extend the half-life period or to make them targeting drugs.
α-MMC and MAP30 as potential therapeutic proteins possess biological activities such as inhibiting protein biosynthesis (ribosome inactivation), 11 antitumor, antivirus, and, especially, anti-HIV replication. 7, 12, 13 However, as foreign proteins are like other potential therapeutic proteins, poor biocompatibility limits their further development and application. To overcome these problems, in this study we first purified the two main proteins from bitter melon seeds and carried out their PEGylation using a branched 20 kDa (mPEG) 2 -Lys-NHS directed specifically to lysil ε-amino groups. Homogeneous one-mer, two-mer, and three-mer PEG-RIPs were then identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Not only was their immunogenicity in vivo remarkably decreased but also, importantly, their antitumor and antivirus activities in vitro were moderately influenced when compared with the un-PEGylated counterparts. This work is just the beginning step towards them being used clinically. The application of PEGylation and nanotechnolgy may indicate that the potential application of both α-MMC and MAP30 can be developed for antitumor and antivirus agents in the future.
Bitter melon seeds were obtained from the Institute of Agricultural Science and Technique of Sichuan Province, China. Matrices for electrophoresis were products of Sigma-Aldrich (St Louis, MO) and Bio-Rad Laboratories (Hercules, CA). SP-Sepharose FF, Sephacryl S-100, Macro-Cap-SP, and ampholyte were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). (mPEG) 2 -Lys-NHS (20 kDa) was obtained from Shearwater Polymers (Huntsville, AL). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum used in cell culture were from Gibco BRL (Grand Island, NE). pUC18 DNA was purchased from TAKARA (Dalian, China). Nitrocellulose (NC) membrane was obtained from Bio-Rad Laboratories. Sheep-antimouse Ab-linked to alkaline phosphatase was purchased from Sigma-Aldrich. JAR choriocarcinoma cells were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China).
All steps tried either alone or in combination within the process of purification were carried out at 4-6°C unless specifically stated. Firstly, the powder from fresh bitter melon seeds was extracted in 0.15 M NaCl solution and then the pH of the solution was adjusted to 4.0. After simple centrifugation, the supernatant was neutralized and fractionated by 30%-65% ammonium sulfate. The precipitate was dialyzed against the pH 6.3, 0.05 M phosphate buffer. Secondly, the sample was applied onto a SP-Sepharose FF column and eluted with pH 6.3, 0.05 M phosphate buffer containing 0.15 M NaCl. The elution peak containing 30 kDa protein was collected. Thirdly, the portion was loaded onto a Sephacryl S-100 column and the elution peak with 30 kDa protein was pooled. Finally, the sample was applied onto a Macro-Cap-SP column. A linear gradient of 0-0.15 M NaCl in pH 7.0, 20 mM sodium phosphate buffer eluted the column and two peaks with 30 kDa proteins were respectively collected. 
Briefly, α-MMC was separated on SDS-PAGE and then transferred to an NC membrane. Nonspecific binding was blocked and washed by placing the membrane in a solution of BSA. NC membrane was incubated with a mouse anti-α-MMC McAb and was exposed to a sheep-antimouse Ab linked to alkaline phosphatase. After adding BCIP (5-bromo-4-chloro-3-indolyl phosphate), the colored bands were visualized and photographed. 16 In the analysis of MAP30, NC membrane was incubated with a diluted solution of rat anti-MAP30 PcAb under gentle agitation. Other conditions were the same with α-MMC.
In general, PEGylated α-MMC and PEGylated MAP30 can be obtained in the following optimal conditions: 10 mg/mL of α-MMC or MAP30 reacted with (mPEG) 2 -Lys-NHS (mass ratio of PEG:RIP was 2:1) in pH 8.5,100 mM borate buffer at room temperature for 30 minutes. The reaction mixture was applied with Sephacryl S-100 column. The PEGylated conjugates can be collected and assessed by gradient SDS-PAGE and MALDI-TOF-MS.
Topological inactivation activity was according to a previously described method. 17 Antitumor activity in vitro JAR choriocarcinoma cells were maintained in an incubator supplied with a humidified atmosphere of 5% CO 2 at 37°C. The culture medium was DMEM containing 20 mM HEPES and 10% fetal bovine serum. The cell viability and proliferation were determined by quantitative 3-(4,5dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A total of 3 × 10 4 cells/mL were applied into 96-well microtiter plates at 100 µL per well and exposed to PEGylated α-MMC, PEGylated MAP30, and un-PEGylated counterparts of 0.012, 0.06, 0.3, and 1.5 mg/mL at 24, 48, and 72 hours. Cells without drugs were used as controls. Each concentration was tested in quadruplicate. The determination procedure was carried out according to the previous description. 18 The optical density (OD) at 570 nm was measured using an ELISA plate reader (EL × 800, BIO-TEK, Atlanta, GA). Cell viability and proliferation were observed together with controls.
The inhibitory effect of PEgylated rIPs on herpes simplex virus-1 VERO cells were cultured to logarithmic growth phase in 10 mL DMEM containing 10% (v/v) fetal bovine serum, 100 µg/mL penicillin, and 100 U/mL streptomycin. After removing medium, cells were substituted to 5 mL of the above medium and infected with 100 µL of herpes simplex virus-1 (HSV-1) for 1.5 hours. Then, 5 mL maintenance medium containing 2% (v/v) fetal bovine serum, 100 µg/mL penicillin, and 100 U/mL streptomycin was added and cells were continually cultured for about 1-2 days when over 80% cells appeared with cytopathic effect (CPE). The infected supernatant was collected for testing of virus titers.
Determination of virus titer VERO cells of 1 × 10 5 cells/mL were applied 100 µL per well to 96-well microtiter plates and cultured for 24 hours. After discarding medium, 100 µL of infected supernatant, which was 10 −1 to 10 −10 diluted with medium, was added to each well (quadruplicate) until CPE did not increase. TCID 50 (50 percent tissue culture infective dose) was calculated according to Reed and Muench. 19, 20 Measurement of cytotoxicity The cytotoxicity of PEGylated α-MMC and PEGylated MAP30 was evaluated by the quantitative MTT test. 21 A total of 1 × 10 5 VERO cells/mL was applied to 96-well microtiter plates at 100 µL per well and was exposed to un-PEGylated proteins and PEGylated-proteins at 3.3 µmol ⋅ L −1 ∼3.3 nmol ⋅ L −1 (100 mg ⋅ L −1 ∼0.1 mg ⋅ L −1 ) and acyclovir (ACV) at 10 µmol ⋅ L −1 ∼0.01 µmol ⋅ L −1 for 48 hours as well as cells without drugs as control. Each concentration was tested in quadruplicate. The determination procedure was carried out according to the previous description. 18 The OD at 490 nm was measured using ELISA:
Inhibition of proteins on HSV-1 antigen secretion A total of 1 × 10 5 VERO cells/mL were seeded into 24-well plates at 1 mL/well and then incubated with 5% CO 2 at 37°C for 24 hours. After abandoning the supernatant, 300 µL HSV-1 stock solution with 100 TCID 50 /mL was added to each well and incubated for 1.5 hours. Then, 100 µL of culture medium containing proteins (concentration was mentioned previously in the cytotoxity method) was added to each well and negative and positive cell control established. After incubation for 48 hours, HSV-1 antigen in culture supernatant was tested by human HSV-1 antigen 1 (HSV-1 AG1) enzymelinked immunosorbent assay kit with indirect ELISA:
The analysis of virus inactivation VERO cells infected with HSV-1 were added with 20 µL (5 mg/mL) of MTT to each well and incubated at 37°C for 4 hours. Then, 150 µL of DMSO was added to each well. The OD 490 was read by a microplate spectrophotometer. The percentage of inhibition was calculated by the formula described under the heading "Measurement of cytotoxicity."
Fifty Sprague Dawley rats were randomly classified into control, α-MMC, PEGylated α-MMC, MAP30, and PEGylated MAP30 groups of ten each. The rats in the control group were subcutaneously injected with saline solution, and the RIP and PEGylated RIP groups were emulsified in Freund's complete adjuvant at a dose of 1.42 mg/kg at the infection frequency of 3 days for 17 days. Blood samples were collected from the capillary vessel in rats' eyes and separated sera were stored at -20°C. Antigen-specific serum IgG levels were measured by ELISA. Briefly, 96-well plates were coated with 100 µL of 30 µg/mL purified antigen (RIPs and PEG-RIPs) in 0.05 M carbonate-coating buffer, 
SPSS statistical software (SPSS, Inc, Chicago, IL) was used for analysis. P , 0.05 was considered statistically significant.
Purification and identification of α-MMC and MAP30
Purification of α-MMC and MAP30 was performed by applying 30%-65% ammonium sulfate precipitation, acidification, SP-Sepharose FF, Sephacryl S-100, and Macro-Cap-SP chromatography. In conclusion, 200 mg of α-MMC and 120 mg of MAP30 with the recoveries of 1.7% and 1.0%, respectively, was obtained from 200 g starting material seeds. The procedure of purification is summarized in Table 1 
Through optimized PEGylation reaction, a high total PEGylation ratio can be obtained (about 60%-70%). The gradient SDS-PAGE monitoring (Figure 4) showed that the un-PEGylated counterparts from the reaction mixture were successfully removed by Sephacryl S-100 chromatography or Superdex 75 chromatography. In the MALDI-TOF-MS analysis ( Figure 5 ), one-mer, two-mer, and three-mer PEGylated isomers with 49899.4 Da, 70430.9 Da, and 91057.6 Da (the molar ratios of 1:1, 2:1, and 3:1 of (mPEG) 2 -Lys-NHS:α-MMC) were detected from α-MMC-PEG conjugates.
Meanwhile, one-mer and two-mer PEGylated isomers with 50335.6 Da and 70614.2 Da (the molar ratios of 1:1 and 2:1 of (mPEG) 2 -Lys-NHS: MAP30) were found from MAP30-PEG conjugates.
To demonstrate their topological inactivation activity, supercoiled DNA (pUC18) was incubated with PEGylated RIPs and un-PEGylated counterparts. In suitable enzymatic digestion conditions, all of the experiment samples cleaved the supercoiled double-stranded DNA to produce nicked circular or linear DNA. As shown in Figure 6 , all of them exhibited DNase-like activity.
To investigate the effect of PEGylated α-MMC and PEGylated MAP30 on cell viability and proliferation, JAR cells were seeded on 96-well plates and were treated with increasing concentrations of PEGylated RIPs and un-PEGylated counterparts for 72 hours ( Figure 7A ) and for different times at 1.5 mg/mL ( Figure 7B ). Statistical analysis revealed that the concentrations of 1.5, 0.3, and 0.06 mg/mL started to significantly reduce the proliferation of cells after 48 and 72 hours of incubation. The analyses with the result of Figure 7A not prominent after 24 hours of treatment, but continued incubation for 48 or 72 hours with the proteins enhanced the cytotoxicity on cells.
α-MMC and PEGylated α-MMC induced alterations in the morphology of JAR cells (Figure 8 ). After processing for 72 hours, untreated JAR cells extended and flattened ( Figure 8A ), while the treated groups showed fewer cells and abnormal shapes such as shrinkage, blebbing, and loss of membrane asymmetry, indicating the cytotoxic effect of α-MMC and its modifier on JAR cells. In particular, α-MMC-treated groups showed extremely obvious morphological changes ( Figure 8C and D). MAP30 and PEGylated MAP30 also showed a similar cytotoxic effect to JAR cells.
The inhibitory effect of PEgylated α-MMC and PEgylated MAP30 on HSV-1 TCID 50 was calculated using the method of Reed and Muench, 19 and the emergence of CPE was used as a positive sign. The CPE of each dilution of HSV-1 cytopathic was tabulated as in Table 2 . Measurement of cytotoxicity was designed to establish appropriate dose range for the research of inhibitory effect of proteins on HSV-1 to VERO cells. The result (Figure 9 ) reflected the effects of different concentrations of test proteins on cell viability of VERO cells and showed that α-MMC/MAP30, PEG-α-MMC/MAP30, and ACV had no significant inhibition on VERO cells in the tested concentration range. The cell viability maintained above 97% from 0.003-0.03 µmol ⋅ L −1 and 85% from 0.3-3.3 µmol ⋅ L −1 . Therefore, the dose range of tested proteins inhibiting VERO cell infection with virus was below 3.3 µmol ⋅ L −1 .
The inhibitory effect of α-MMC/MAP30, PEGylated α-MMC/MAP30, and ACV on HSV-1 glycoprotein antigen secretion glycoprotein was tested through the determination of HSV-1 glycoprotein antigen (Table 3) . The results showed a dose-dependent inhibitory effect. IC 50 was 0.67 µmol ⋅ L −1 with α-MMC, 2.94 µmol ⋅ L −1 with PEG-α-MMC, 0.47 µmol ⋅ L −1 with MAP30, 2.39 µmol ⋅ L −1 with PEG-MAP30, and 3.55 µmol ⋅ L −1 with ACV, respectively. Furthermore, IC 50 of α-MMC/MAP30 and PEG-α-MMC/MAP30 was lower than the general drug of ACV. At the same dose, the inhibitory effect on HSV-1 glycoprotein antigen secretion, glycoprotein of MAP30 was higher than that of α-MMC and PEG-MAP30 was higher than PEG-α-MMC. The MTT results showed that the ratio of cell viability can be increased on a dose-dependent feature compared with the control group when protein concentration was higher than 0.03 µmol ⋅ L -1 . But ACV did not display effects on HSV-1 inactivation. At the same dose, the ratio of cell viability with MAP30 was higher than α-MMC and PEG-MAP30 was higher than PEG-α-MMC. This indicated that both α-MMC/MAP30 and PEG-α-MMC/MAP30 had the ability of direct HSV-1 inactivation in a certain range of concentration. Additionally, the effect of inactivation was obvious with the increase of 
A novel preparative strategy of both α-MMC and MAP30 at one time was successfully established. Some properties of these proteins were assessed by SDS-PAGE, ESI-QUAD-MS, MALDI-TOF-MS, and N-terminal sequence as well as Western blotting. This fast, highly efficient methodology enables us to focus more energy on subsequent research. A branched (mPEG) 2 -Lys-NHS(20 kDa) was used to modify these proteins and the serial PEGylated ones were assessed by gradient SDS-PAGE and MALDI-TOF-MS. The α-MMC modifier possessed one-mer, two-mer, and three-mer PEGbound chains, while MAP30 modifier obtained one-mer and two-mer PEG-bound chains. PEGylated conjugates preserved moderate activities on JAR choriocarcinoma cells and HSV-1. Furthermore, both PEGylated proteins showed about 60%-70% antitumor and antivirus activities as well as a 50%-70% immunogenicity decrease when compared with unmodified counterparts. To sum up, PEGylation of α-MMC and MAP30 may offer a possible way for their clinical application as potential therapeutic agents. Nevertheless, to fulfill the requirements of a useful drug, we should meet the challenges, including the reduction of immunogenicity to the greatest extent, the retention of sufficient activity, the extension of the half-life period, and development of various forms of PEG-RIPs. This work is just the beginning for them to be used clinically. After all, therapeutic proteins must fulfill the requirements described previously. PEGylation can reduce immunogenicity and retain certain biological activity of RIPs, but there is lots of work to do for further decreasing their immunogenicity and extending the half-life period. The International Journal of Nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. This journal is indexed on PubMed Central, MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine, Journal Citation Reports/Science Edition, EMBase, Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.
International Journal of Nanomedicine 2012:7
Future work concerns packing RIPs with nanomaterials for targeting drugs and low release. Evaluation of Four Different Systems for Extraction of RNA from Stool Suspensions Using MS-2 Coliphage as an Exogenous Control for RT-PCR Inhibition Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. Diagnosis of enteric viral infections by Real Time reverse transcription -polymerase chain reaction (rRT-PCR) of RNA extracted from stool suspensions is complementing and increasingly replacing diagnosis based on viral isolation and characterization in tissue culture [1, 2] . However, interpretation of results is not always straightforward. The sensitivity of the rRT-PCR is negatively impacted, by compounds present in the clinical sample that may partially or completely inhibit the RT and/or PCR chemistries [3, 4, 5, 6, 7] . Potential inhibitors that might be incompletely removed from stool suspensions during RNA extraction include hemoglobin, immunoglobulins, bilirubin, triglycerides, complex polysaccharides, organic and phenolic compounds, glycogen, fats, and metabolic products especially those from pathological conditions, bacteria, vegetables, medications, anticoagulants, and drugs or alcohol [4, 6, 8, 9, 10, 11] . Adding to the list of endogenous rRT-PCR inhibitors are exogenous inhibitors from extraction protocols such as detergents, chelating compounds and guanidinium HCl [9] .
The presence of inhibitory compounds in the extracted RNA and the extent of inhibition can be determined by semiquantitative rRT-PCR of an RNA template that is present in the sample, added to it prior to extraction, or introduced into the rRT-PCR mix. Natural or modified, encapsulated coliphage MS2 RNA is one source of protected RNA that has been used as a noncompetitive external RNA template control [5, 12, 13, 14] .
The efficiency of removing inhibitors in patient samples may be related to the intrinsic properties of the method used to extract the RNA [15] . In this study we compared four commercial RNA extraction systems efficiencies for RNA isolation and removal of inhibitors in stool samples. The extraction systems were: QIAamp Viral RNA Mini Kit: manual extraction using silica columns (QIAGEN Inc, Valencia, CA, USA); MagNA Pure LC2.0 Automatic extractor with MagNA Pure LC Total Nucleic Acid Isolation Kit-High Performance: automatic RNA extraction using magnetic beads (Roche Diagnostics, IN, USA); KingFisher (Thermo Electron Corporation, Waltham, MA, USA) semi-automatic extraction using magnetic beads of Ambion MagMAX Viral RNA Isolation kit (Ambion, Inc, Austin, Tx, USA); NucliSENS easyMag (bioMerieux, Marcy l'Etoile, France): semi-automatic extractor using magnetic beads of easyMag extraction kit. In addition, the effectiveness of MS2 as an exogenous template control for measuring inhibitors co-extracted with RNA from stool suspen-sions and its relevance for validation of enterovirus diagnostic rRT-PCR suspensions is discussed.
Ninety-three stool suspensions were extracted by the four extraction systems after spiking the lysis buffer of each with MS2 that yielded similar final MS2 concentrations. Any contribution from endogenous MS2 RNA was ruled out since no MS2 RNA was amplified from any of these 93 stool suspensions upon extraction with protocol D when exogenous MS2 was omitted from the lysis buffer. Results for each extraction procedure by assigned levels of inhibition are shown in Table 1 . Individual values are shown in Fig. S1 . The proportion of samples with inhibitors varied among the extraction methods. Of the 93 RNA extracts, 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) extracted by protocols A, B, C, and D, respectively, contained inhibitors of MS2 rRT-PCR. Median interquartile range (IQR) reduction in Cts for protocols A, B, C and D were 2.4 (0 to 34), 1.9 (0 to 33), 0.58 (0 to 29), and 0.26 (0 to 7), respectively. Finally, the number of samples with an inhibition .6 Ct were 6 (6.5%), 3 (6.4%), 2 (2.2%), and 1 (1.1%) for Extraction Protocols A through D, respectively. The Friedman Test indicated that there was a statistically significant difference in co-extraction of inhibitors of MS2 rRT-PCR among the extraction protocols used, P,0.001.
In order to further challenge the performance of the 4 extraction systems to exclude inhibitors of rRT-PCR, 92 stool samples with levels of inhibition ranging from a single cycle to complete inhibition were evaluated. These samples were selected from among archived stool samples previously tested for enterovirus and MS2 after extraction by QIAamp Viral RNA Mini Kit. A sufficient number of samples with high, intermediate, and low levels of inhibitors were chosen for re-analysis to enable comparison between extraction procedures at each of these levels of inhibition. The results of MS2 inhibition for each sample by extraction protocol is shown in Fig. 1 and summarized by levels of inhibition in Table 1 , Experiment 2. The number of samples with inhibitors differed among the four protocols and for some individual samples, the amount of inhibition varied depending on the extraction protocol. Specifically, inhibitors were present in 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the samples prepared by Protocols A, B, C, and D, respectively. Median IQR reduction in Ct levels for protocols A, B, C and D were 11.7 (0 to 34), 7.0 (0 to 30), 4.2 (0 to 29), and 1.3 (0 to 30), respectively. More importantly the number and proportion of samples where inhibition was .6 Ct also varied among the protocols; 44 (47.8%), 29 (31.5%), 14 (15.2%), and 1 (1.1%), for Protocols A, B, C, and D, respectively. The Friedman Test indicated that there was a statistically significant difference in co-extraction of inhibitors of MS2 rRT-PCR among the extraction protocols used, P,0.001.
To differentiate between rRT-PCR inhibition due to the presence of inhibitors in the RNA after extraction and variation due to differences in efficiency of extraction, 23 RNA extracts from samples with inhibitors were spiked with two concentrations of purified CoxB3 enterovirus RNA (Fig. 2 ). The first concentration was equivalent to that of MS2 (R 1 , equivalent) and the second contained 64 fold (6 Cts) more enterovirus RNA (R 2 , high). No significant difference between inhibition of MS2 rRT-PCR and enterovirus rRT-PCR was noted when equivalent amounts of enterovirus RNA was added to extracts prepared by protocols A, B and C (Fig. 2. part 2) . However, when the higher amount of enterovirus RNA was added, there was significantly less inhibition of enterovirus rRT-PCR than MS2 rRT-PCR extracts prepared using protocols B, C, and D, but not A. Thus, the decrease in MS2 rRT-PCR can serve as a measure of the amount of inhibitors in the RNA extracted by each of the extraction protocol.
Analysis of variance, indicated that there were no significant differences between inhibition of Cts for MS2 and enterovirus RNA for Protocols A, B, and C, but not D, when the amount of enterovirus RNA was equivalent to that of MS2 (P.0.5), whereas there was significantly less inhibition when the 64-fold higher amount of enteroviral RNA was tested for RNA extracted by protocols B, C, and D (P,0.05), but not A (P.0.05).
Thirteen stool suspensions with varying amounts of inhibitors were spiked with intact CoxB3 virus at a concentration adjusted to yield a Ct equivalent to that of the MS2. These spiked suspensions were re-extracted in parallel by all four methods. With few exceptions, the extent of inhibition of enterovirus rRT-PCR was similar to that for inhibition of MS2 rRT-PCR (Fig. 3) . The number of samples with $6 Ct inhibition in MS2 rRT-PCR, that also had .6 Ct inhibition of enterovirus rRT-PCR, was 10/10 (100%), 9/9 (100%), 6/7 (85.7%), and 4/4 (100%) for Protocols A, B, C, and D, respectively.
Analysis of variance ( Fig. 3 , part 2), indicated that there was no significant difference (paired t-test, P.0.05) between the inhibition of rRT-PCR of the MS2 external control and the added enterovirus (P.0.05) for protocols A, C, and D. In protocol B the inhibition of the rRT-PCR for enterovirus was actually significantly higher than that for MS2 (P = 0.045). Recovery of enterovirus RNA by the four different protocols was equivalent.
One of the trends in pathogen identification is the increasing reliance on molecular methods to achieve high throughput, high sensitivity and specificity of results in clinically relevant times [2, 16] . However, low pathogen copy number, inefficient extraction and the incomplete removal of inhibitors, reduce the sensitivity and specificity of the molecular assays. Among clinical samples, stool suspensions contain a wide range of materials that have the potential to inhibit RT and/or PCR chemistries [4, 6, 8, 9, 10, 11] . Thus, it is important to develop a reliable method to determine whether inhibitors are present in nucleic acid prepared directly from stool suspensions. An appropriate internal control does not exist because of wide differences in eukaryotic and prokaryotic composition in fecal material between patients.
Four protocols for extracting RNA from stool samples were compared in this study. These four commercial extraction methods are among the most commonly used in the diagnostic laboratories. None of samples 1 to 93 contained MS2 RNA. Similarly Nonove et al (14) did not report any unusual amounts of MS2 RNA that would have indicated prior presence of MS2 in their samples when they added MS2 to 106 stool suspensions at concentrations two to eight-fold less than used in this study. MS2 coliphage was thus used as a non-competitive external control because of its absence from human clinical samples. MS2 is a better external control for rRT-PCR than plasmid or phage DNA since it also measures the efficiency of the RT reaction [5, 12, 13, 14, 17, 18] . Here, the MS2 was added to the lysis buffers to minimize sample manipulation and increase the uniformity between manual and semi-automatic extraction protocols. The presence of rRT-PCR inhibitors was evaluated by extracting equal volumes of stool suspension and eluting with equal volumes of elution buffer. Extractions were performed in parallel to eliminate storage related differences, and RNA was assayed on the same rRT-PCR run to minimize analytical differences [19] . Absence of MS2 in the stool samples 1-93 was confirmed by MS2 rRT-PCR of RNA extracted with the EasyMag RNA extraction system using lysis buffer without exogenous MS2. This extraction protocol, the system that had the least amount of samples with inhibitors, was chosen to increase the chance of finding any endogenous MS2 RNA in the stools.
The number of stool suspension RNA extracts with inhibitors of MS2 rRT-PCR varied between RNA extraction protocols for randomly chosen stool suspensions. The amount of inhibitors in RNA extracts for individual stool suspensions also varied according to extraction procedures. This suggests that more than one type of inhibitor may be present and that different procedures do have different proficiencies in excluding them. Stool samples extracted by the KingFisher and the NucliSENS easyMag had fewer extracts with inhibitors than the QIAamp [18, 20, 21, 22] found that procedures similar to that used by EasyMag outperformed manual extraction procedures similar to QIAgen, and that MagNA Pure and KingFisher-like procedures were intermediate. In contrast fewer did not find major differences between some of these systems [23, 24] . Results presented here further confirm that RNA extracted with magnetic bead-based systems contained fewer inhibitors than column-based systems [21] . The number of samples with inhibitors was low in the first group of samples (samples 1-93) that were used to compare RNA extraction procedures. In order to better compare the four extraction procedures, we selected from among previously tested samples, sufficient numbers of archived stool suspensions that had high, intermediate, and low levels of inhibitors after extraction by QIAamp Viral RNA Mini Kit (samples 101-192). The stool suspensions were re-extracted in parallel by all four protocols and evaluated for inhibition of MS2 rRT-PCR. As before, KingFisher and NucliSENS EasyMag semi were better than the QIAamp Viral RNA Mini Kit and MagNA Pure LC2.0 Automatic extractor.
Differences in extraction efficiency might be at least as important as inhibitors, as Hata et al. [25] showed by adding separate controls for both extraction and amplification. We performed a similar analysis by adding enterovirus RNA to RNA samples after extraction and intact enterovirus to suspensions before extraction. The inhibition of enterovirus rRT-PCR correlated with MS2 rRT-PCR inhibition. It is important to note that all four extraction protocols yielded equivalent amounts of enterovirus RNA from enterovirus added to suspensions where there was no inhibition of MS2 RNA rRT-PCR in the samples for equal starting and elution volumes. The pattern and amount of enterovirus rRT-PCR inhibition was similar for specific suspensions regardless of whether enterovirus was added before extraction or viral RNA added to the reaction mix after extraction. This implies that the apparent decrease in the amount of MS2 RNA in an RNA extract where MS2 RNA was added to the lysis buffer was primarily due to failure to remove inhibitors present in particular stool suspensions.
The standard maximum volumes that could be extracted differ for the procedures: 140 ml for QIAgen; 100 ml for MagNA Pure; 50 ml for KingFisher; and, 200 ml for easyMag. The effect of using these manufacturers' recommended volumes for stool suspensions was not evaluated in the present study, although to have done so would have meant that depending on the procedure used, there would have been a 2 to 4 fold increase in the amount of endogenous inhibitors in the starting volume without a concomitant increase in the extraction, washing and elution buffer volumes. Others have shown that increasing the starting volume increased the co-extraction of inhibitors [21, 25, 26] and that the final rRT-PCR outcome is a balance between specific template and coextracted inhibitors [27] .
The majority of rRT-PCR assays have a lower limit of quantitation of 10 target sequences per reaction with a Ct of approximately 35 cycles. A reaction with a three-fold shift upwards in Ct due the presence of inhibitors would still be positive, i.e. a specific signal would still be detectable below the 40 cycle cap recommended for the majority of rRT-PCR assays. A two Ct difference is within the statistical variation that may occur between repeated analyses of the same RNA. In contrast, a positive signal would no longer be detectible if there were a six-fold shift in Ct, which would produce a false negative outcome. In conclusion, inhibition is a complex process. All four extraction methods were suitable provided that an external control was used to identify problematic samples. rRT-PCR of MS2 RNA recovered from MS2 coliphage added to the lysis buffers of RNA extraction systems is a good predictor of inhibition of enteroviral RNA extracted from stool suspensions. More than one inhibitor may be present in the stool suspensions or added during extraction and their efficiency of removal differs between the extraction protocols. The correlation between the extent of MS2 rRT-PCR inhibition and enteroviral rRT-PCR inhibition increases inversely in relation to the amount of enteroviral RNA in the sample. In agreement with Dreier et al [5] , MS2 rRT-PCR inhibition should be tested for each RNA sample from a stool suspensions each time it is tested since there is no way to predict in advance whether inhibitors have been efficiently removed during extraction or remain active after cycles of frozen storage. Viral rRT-PCR results should not be considered as quantitative results when MS2 rRT-PCR is inhibited by more than 3 Ct. Finally, we recommend that any negative viral rRT-PCR result from a sample with an inhibition of .6 Ct for MS2 rRT-PCR should be considered invalid and alternative methods used to re-assay or re-extract the sample. If the RNA is diluted and re-tested only samples with positive results for enterovirus rRT-PCR should be considered as valid. 
The Ethical Review Board of the Sheba Medical Center, Tel Hashomer approved this study (SMC-8859). The samples and results were stripped of all links to personal details pertaining to, or which could be used to identify individual patients. All data were analyzed anonymously. The Ethical Review Board exempted this study from a requirement for obtaining informed consent.
Stool suspensions (N = 185) prepared for routine analysis of clinical stool samples sent to the Central Virology Laboratory (CVL) at Chaim Sheba Medical Center in Israel were used to evaluate the efficiency of four different RNA extraction systems in excluding inhibitors of rRT-PCR. Ninety-three of the stool samples contained unknown amounts of rRT-PCR inhibitors (Table 1; Fig. S1 ). The remaining ninety-two stools suspensions were selected from among 860 stool suspensions archived between 2009 and 2011 that had been sent for routine enterovirus analysis (Table 1 , Figs. 1, 2, 3 and S2 ). The RNA from these archived suspensions had been extracted manually with the QIAamp Viral RNA Mini Kit and MS2 rRT-PCR and inhibition levels were known. MS2 rRT-PCR inhibitors .5 Ct were found in 93 (10.8%) of these samples. A non-random subset of 92 of these 860 samples (samples 101 to 192) with high, intermediate, and low levels of inhibitors were chosen for re-analysis so that would be a sufficient number of samples for comparison at each of these levels of inhibition.
Small portions of fecal matter were vortexed for 15 seconds in stool suspension buffer, 2 ml 0.9% saline with glass beads (samples 1 to 93) or 5 ml of M199 containing 15.6 mg of dihydrostreptomycin, 15,625 U of Penicillin G, and 156 U of Mycostatin and 0.1 volume (v/v) of chloroform (samples 101-192). Suspensions were clarified by centrifugation at 2,5006g for 10 min and stored at 220uC until use.
A natural E. coli RNA MS2 coliphage [28] (MS2, ATCC 15597-B1) stock was prepared on E. coli Top 10F in NZYCM broth as described by Dreier et al [5] . Aliquots of the stock were frozen at 270uC. The concentrated stock was thawed, serially diluted in dilution buffer [100 mM NaCl, 8 mM MgSO 4 , 50 mM Tris pH 7.5 and 0.01% (w/v) gelatin] and added to the respective extraction lysis buffers used in each of the extraction procedures before use. The amount of external control MS2 template added to the lysis buffers was adjusted to give a Ct of approximately 27 (,10,000 copies/ml) in the rRT-PCR mix upon addition of 5 ml of RNA.
RNA was extracted from clarified fecal suspensions using four different commercial protocols. Samples were extracted in parallel to eliminate storage related differences [19] . Extractions were performed according to manufacturers' instructions except that RNA was extracted from 50 ml of stool suspension for all four protocols with addition of respective suspension buffer to reach the recommended aqueous volumes listed above. The RNA was stored at 270uC pending analysis and between analyses (See Fig. S2 ). Samples 1 to 93 were also extracted by protocol D as above, except that exogenous MS2 was omitted from the lysis buffer.
The ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used for the amplification and detection of the MS2 and Enterovirus RNA by TaqMan technology as previously described [5, 29, 30] . Briefly, for MS2 rRT-PCR, 5 ml of RNA was added to the AgPath Mastermix (Ambion, Applied Biosystems Inc, Foster City, California), which contained the published concentrations of primers and probes and 5-carboxy-X-rhodamine succinimidyl ester (ROX) as an internal reference dye, whereas 8 ml of RNA was added for all enterovirus rRT-PCR assays. rRT-PCR was performed under the following conditions: 30 min at 48uC, 10 min at 95uC, and 60 cycles of 15 s at 95uC and 1 min at 60uC. RNAs extracted from the same stool suspension by the four procedures were assayed on the same rRT-PCR run to minimize analytical differences [19] .
Data were managed and analyzed using Excel (Microsoft) and SPSS (ver. 15) software. For each extraction protocol, the presence and relative amount of rRT-PCR inhibitor(s) was determined by comparing MS2 rRT-PCR Ct results obtained in the absence or presence of stool suspensions. Measured MS2 Cts results were assigned to one of five levels of inhibition: no inhibition, inhibition of 1 to 3 Cts (2 to 8 fold), inhibition of 4 to 6 Cts (16 to 64 fold), inhibition of 7 to 9 Cts (128 to 512 fold) and inhibition of $10 Cts ($1024 fold). Similarly, the presence and relative amount of inhibitors of enterovirus RNA rRT-PCR was determined by comparing enterovirus rRT-PCR Ct results obtained in the absence or presence of stool suspensions for enterovirus added before extraction or enterovirus RNA added after extraction. The inhibition for suspensions that gave Cts for MS2 rRT-PCR below that for MS2 in suspension buffer alone was set to 0. The upper limit for rRT-PCR inhibition was capped at 29 Ct.
The non-parametric Friedman Test was used to determine to determine whether there were significant differences between results among the four different extraction protocols. Post-hoc analysis of pairwise differences between protocols was performed using a Wilcoxon Signed-Rank Test (SPSS) with a Bonferroni correction that set the significance level for the pairwise comparisons to P,0.008. Comparison of MS2 rRT-PCR and enteroviral rRT-PCR Cts was by repeated measurements, analysis of variants. Figure S1 MS2 rRT-PCR inhibition in RNA extracted from stool suspensions using four different RNA extraction protocols. Equal amounts of stool suspensions chosen randomly from among samples sent to the laboratory were extracted by four protocols: (A) QIAgen, (B) magNA Pure, (C) KingFisher, and (D) easyMag as described in Methods. MS2 coliphage calculated to give 27 Ct by rRT-PCR was added to the extraction buffer. rRT-PCR values for MS2 in RNA extracted from buffer controls were subtracted from the values for MS2 in RNA extracted from stool suspensions. This difference, the number of Cts of inhibition, is shown in the box to the right of the sample numbers. Negative values were set to 0 and the maximum values for inhibition ''C'' were capped at 29 Cts. Samples with inhibition .10, 7 to 9, 4 to 6, and 1 to 3 Ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. Blank white boxes indicate no inhibition. The numbers of samples in each category and the significance in differences are shown in Table 1 , Experiment 1. (PDF) Figure S2 MS2 rRT-PCR inhibition for RNA extracted from stool after re-freezing and thawing by extraction protocol. 1. Three repeated measurements of MS2 rRT-PCR were performed for the 23 samples by protocols: (A) QIAgen, (B) magNA Pure, (C) KingFisher, and (D) easyMag as described in Methods. The RNA was stored at 270uC between tests. MS2 coliphage was added to the extraction buffer. rRT-PCR values for MS2 in RNA extracted from buffer alone were subtracted from the values for MS2 in RNA extracted from stool suspensions. This difference, the number of Cts of inhibition, is shown in the boxes to the right of the sample numbers. Negative values were set to 0 and the maximum values for inhibition ''C'' were capped at 29 Cts. Samples with inhibition $10, 7 to 9, 4 to 6, and 1 to 3 Ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. Blank white boxes indicate that the samples were not inhibited. Samples were stored at 270uC between measurements. 2. The means for each of the three repeat tests the square of the means (M Sq), the standard deviation of each run (S.D.), and the significance of differences between the means of the repeated measurements by extraction protocol are presented. There were no significant differences between the repeat measurements for each extraction protocol. 3. Friedman test values for comparison among the 4 extraction methods. There were significant differences among the extraction protocols (shaded yellow boxes). 4. The Wilcoxon Singed-Rank Test (SPSS) with Bonferroni correction (Significance if P,0.008) for the pairwise comparison of the means of the three measurements of the rRT-PCR results from the different extraction protocols. Shaded yellow boxes indicate where pairwise differences between extraction protocols were significant. (PDF) Analysis on the Pathogenesis of Symptomatic Pulmonary Embolism with Human Genomics BACKGROUND: In the present study, the whole human genome oligo microarray was employed to investigate the gene expression profile in symptomatic pulmonary embolism (PE). METHODS: Twenty patients with PE and 20 age and gender matched patients without PE as controls were enrolled into the present study in the same period. The diagnosis of PE was based on the clinical manifestations and findings on imaging examinations. Acute arterial and/or venous thrombosis was excluded in controls. The whole human genome oligo microarray was employed for detection. Statistical analysis was performed with t test following analysis of very small samples of repeated measurements and Gene Ontology (GO) analysis. RESULTS: Genomic data showed no damage to vascular endothelial cells in PE patients. Genomic data only found increased mRNA expression of a small amount of coagulation factors in PE patients. In the PE group, anticoagulant proteins, Fibrinolytic system and proteins related to platelet functions only played partial roles in the pathogenesis of PE. In addition, the mRNA expressions of a fraction of adhesion molecules were markedly up-regulated. Gene Ontology analysis showed the genes with down-regulated expressions mainly explain the compromised T cell immunity. Symptomatic VTE patients have compromised T cell immunity. CONCLUSION: The damage to vascular endothelial cells is not necessary in the pathogenesis of VTE, and only a fraction of factors involved in the shared coagulation cascade are activated. Genomic results may provide a new clue for clinical diagnosis, treatment and prevention of VTE. Thrombus which may result in pulmonary embolism (PE) mostly comes from deep venous thrombosis (DVT). Altogether, both PE and DVT are named venous thromboembolism (VTE). Because of the high incidence of misdiagnosis, morbidity and mortality, VTE has become one of the essential medical problems around world (1, 2) . After 1990's, clinical report of incidence of PE are sharply increased yearly in China (3) . After 1990's, clinical reports of PE are sharply increased yearly in China (3) . In other Asia Ivyspring International Publisher regions, such as Japan, Thailand, Singapore, Taiwan, the number of reported PE cases is also increasing (4) (5) (6) (7) (8) . There are nearly 900,000 new PE cases in USA every year (9) , 78,000 new cases in Canada, 100,000-200,000 cases in Europe including France, Italy, Spain, Britain, Germany, and 47,000 cases in Australia every year (10). According to the statistical data, provided by an epidemiological surveillance in US, PE is the third leading cause of death (11, 12) , only lags behind malignancy and myocardial infarction.
Virchow's classical triad of abnormalities including blood stasis, hypercoagulability and vessel damage has been regarded as the authentic theory for explaining pathogenesis of VTE since 19's century (13) , however, the triad does not fit all clinical profiles.
In 1965, Egeberg et al (14) firstly introduced the concept of thrombophilia based on discovering a family with an autosomal dominant inheritance disease whose members manifested with repeatedly onset of DVT due to depletion of antithrombin III. Since then, many reports have considered that genetic gene mutation was one of the major factors in the pathogenesis of VTE (15) , but there has been a lack of sufficient genetic etiology evidence in many clinical PE patients.
In recent years, some scholars have suggested that DVT-PE is a heterogeneous polygenic and multifactor disease involving the interaction of hereditary factors and environment with many risk factors such as trauma, surgery, advanced age, malignancy, pregnancy, heart failure, stasis, oral contraceptive, and so on (16) . In 2004 and 2008, ACCP proposed risk stratifications in patients receiving surgery, and different strategies should be performed for patients with different risks to prevent the occurrence of VTE (17, 18) . However, the annual number of VTE cases is increasing over year actually. Generally, the traditional concept can merely be verified merely in few patients with VTE, the pathogenesis of majority of other patients is still to be elucidated.
Gene chip analysis provides the advanced tool for the study of gene function (19) (20) (21) . It can be a reliable approach for the study of differential gene expression between healthy people and patients and for the elucidation of molecular etiology of VTE.
Twenty patients enrolled in PE group were those who admitted in hospital during 2007, with 11 males and 9 females, averaged 70(±14) years of age(44-89 years old). ALL patients were diagnosed as PE in accordance with at least 2 of following criteria. 1)Selective pulmonary arteriography showed filling defect or blockage; 2) Pulmonary ventilation perfusion scanning exhibited single or multiple blood flow perfusion defect with normal or abnormal ventilation, mismatched ratio of ventilation/perfusion; 3) Other clinical characteristics including typical manifestation of PE, arterial blood gas analysis, D-dimer test, ultrasoundcardiogram (UCG) and chest computerized tomography (CT) supported the diagnosis and excluded other cardiac and pulmonary disorders. Twenty patients with ischemic heart disease admitted at same period, excluding PE, DVT and other congenital bleeding and thrombosis diseases with comparative clinical presentation (11 males, 9 females, 44-91 years old with mean age 72±14) were enrolled in control group. The study protocol was approved by local ethics committee and informed consent was obtained from all patients in accordance with the declaration of Helsinki.
Total RNA isolation 5 ml of peripheral blood samples anti-coagulated with EDTA were drawn from patients suspected with PE immediately after admitting to the hospital and from those patients without PE, respectively. Mononuclear cells were obtained through density gradient centrifugation with Ficoll solution and remaining red blood cells were destroyed with erythrocyte lysis buffer (Qiagen, Hilden, Germany). Total mononuclear cell RNA was extracted with TRIzol(Invitrogen, Carlsbad, USA) and purified with Qiagen RNeasy column (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Isolated total RNA was testified and quantified by means of Nanodrop ND-1000 spectrophotometer (Nanodrop Technology, Cambrige, UK).
RNA samples of patients with confirmed diagnosis of PE and controls were labeled using indirect labeling method. Briefly, 1μg of total RNA was reverse transcribed. The cDNA then undergoes second strand synthesis and clean-up to become a template for in vitro transcription (IVT) with T7 RNA Polymerase. During IVT, the modified nucleotide, 5-(3-aminoallyl)-UTP (aaUTP) was incorporate into the cDNA. After that, fluorescent Cy3 was chemically coupled to aaUTP which contains a reactive primary amino group on the C5 position of uracil. The dye incorporation rate was assessed with a Nanodrop ND-1000 spectrophotometer and was found to be between 1.2 and 1.4 pmol/μl. Hybridization was carried out using the Agilent oligonucleotide microarray in situ hybridization plus kit (p/n 5184-3568), fol-lowing the manufacturer's instructions. Briefly, 750 ng of Cy3 labeled sample cDNA was subjected to fragmentation (30 min at 60°C) and then hybridization on 44K Human Whole-Genome 60-mer oligo-chips (G4112F, Agilent Technologies) in a rotary oven (10 rpm, 60°C, 17 h). Slides were disassembled and washed in solutions I and II according to the manufacturer's instructions.
Significant differential gene expression analysis Random variance model (RVM) corrected t-test was used for the differential gene expression screening due to the small number of patients (20 cases each group) was far less than the amount of genes and low freedom degree of gene expression signal. P<0.05 is criterion of significantly different genes.
Gene Ontology organizes gene function into hierarchical categories based on biological process, molecular function and cellular component (22) (23) (24) . Fisher's exact test was applied for over representation of selected genes in GO biological categories. In order to assess the significance of a particular category by random chance, false discovery rate (FDR) was estimated for all of categories. After 5,000 re-samplings, FDR was defined as FDR=1-Nk/T, where Nk refers to the subtracted number which was from Fisher's test p-values minus ϰ 2 test p-values in random samples. We specified the threshold of significant GO as p-value<0.05, FDR<0.05 and enrichment parameters. Enrichment represents the degree of gene expression significance. The equation of enrichment is as following Re=(nf/n)/(Nf/N) where nf is the number of significant genes within the particular category, n is the total number of genes within the same category, Nf is the number of significant genes in the entire microarray, N is the number of all genes tested.
Similar to GO analysis, Fisher's exact test was employed for the study of over-representation of selected genes.
According to the traditional theory, the pathogenesis of VTE is associated with abnormalities in blood flow, vessel integrity and blood components. Therefore, we compared gene expressions of these VTE related factors in patients with PE and without PE. Among the expressions of coagulation factor genes, only gene expressions of factor Ⅶ and FIBCD1 were significantly elevated in patients with PE compared with patients without PE. As for genes of other coagulant factors, the expression value of patients with PE was either not significantly different or less than those patients without PE in comparison. There was no significant difference between two groups of patients in gene expression of anticoagulant proteins. And so was in gene expression of fibrinolytic factors except plasminogen Urokinase receptor (PLAUR, Fig.1 ). L-selectin, ITGAL and ICAM-1 are the adhesion molecules originated from different family, mainly distributed on the surface of lymphocytes. ICAM-1 is the ligand of ITGAL which is member of integrin family. Significantly elevated mRNA expression of these adhesion molecules in PE group indicate activated adhesion function of white blood cells. However, mRNA expression of P-selectin (mainly distributed on the surface of ECs and platelets) and E-selection (mainly distributed on the surface of activated ECs) are not elevated in PE group which indicate venous endothelial cells are not impaired in patients with PE (Fig.2) . In expression of platelet aggregation related genes, only 2 genes (GP6 and PAFAH1B2) were significantly elevated in the patients with PE. Compared to patients without PE, the expressions of 3 in 7 genes of platelet adhesion function were significantly increased in patients with PE. As for genes of platelet releasing, the expression of one gene was significantly up and down regulated between 2 groups of patients, respectively (Fig.3 ).
To identify the gene categories with differential expression in patients with or without PE, Gene Ontology analysis was carried out on the experiment data. The union of all differential expression genes resulted from data analysis are 2308 genes in patients with PE compared to patients without PE. Among them, 2238 genes are up-regulated and 70 are down-regulated. The main gene ontology categories impacted by these genes involve the up-regulation of 19 functioning categories against 4 down-regulation categories. Up-regulated genes are those genes whose functions are associated with transformation, phosphorylation, cell survival and cell conjugation, et al. While the function of down-regulated genes are relevant to the function of plasma membrane and activity of receptors, especially to the lymphocyte receptor complex and immunological synapse (Fig.4) . Gene ontology analysis exhibited compromised T cell mediated immune function, and t test indicated associated genes were significantly down-regulated in patients with PE than in control groups.
Two genes with down-regulated expressions are closely related to the T cell mediated immunity according to GO analysis (with high value of Enrichment). These results reveal that T cell mediated immunity has been declined markedly in PE patients. The declined T cell receptor complex displayed as significanlly diminished mRNA expression of CD3G, CD3D, CD247, ZAP-70, T cell granzyme A and B, which will result in loss of functions of cytotoxic T cells.
Additionally, the high mRNA expression of L-selectin, ITGAL and ICAM-1 in PE patients revealed the elevated adhesion of vascular endothelial cells, white blood cells and platelets which indicated that the adhesion molecules play an important role in the pathogenesis of VTE.
The functions of platelets are characterized by aggregation, adhesion and release response (25) . Our results showed the mRNA expressions of 3/7 adhesion molecules were markedly up-regulated in PE patients, which suggests the adhesion of platelets plays an important role in the pathogenesis of PE.
In 2011, we reported the proportions of CD3+T cells and CD8+T cells were markedly reduced and CD4/CD8 significantly increased in a series of CTEPH patients, which suggests the compromised T cell immunity in CTEPH patients and imbalance between CD3+T cells and CD8+T cells (26) . In addition, we also found that the number of CD3+T cells and CD8+T cells was dramatically reduced in a series of acute PE patients and cytological findings also supported the results from genomics studies on PE patients (27) . The occurrence of PE is related to the deficient or compromised T cell mediated immunity. This deficiency of T cell immunity may occur under the following conditions: 1) viral infection; 2) malignant tumors; 3) medication with immunosuppresants; 4) malnutrition. In the present study, most of subjects were old patients and malnutrition was not clinically obvious. Moreover, malignant tumors were not found in these patients and medication with immunosuppressants was absent. Thus, the compromised T cell immunity might be possibly related to viral infection.
In 2010, we reported a patient who died of SARS developed VTE in multiple organs, which implies the viral infection induced systemic VTE and there is correlation between viral infection and occurrence and VTE (28) .
Comparisons between PE patients and controls revealed the mRNA expressions of only a few proteins in the coagulation system, anti-coagulation system and fibrinolysis system were markedly up-regulated and only 3 factors or receptors in the shared coagulation cascade were activated, which was inconsistent with traditional theory that coagulation factors are comprehensively activated.
We reported in our previous study that the main component of thrombus in acute venous thrombosis was fibrinogen and albumin and cytoskeletal proteins accounted for only a minor fraction of the thrombus (29) . The thrombus composed of fibrinogen is unstable, which makes the delayed thrombolysis feasible and also explain the therapeutic effectiveness of transcatheter thrombolysis not long after APE. Findings support that the formation of thrombus in VTE is not due to the comprehensive activation of coagulation factors as in traditional theory.
In the present study, we apply the unitarian theory to explain the pathogenesis of symptomatic VTE: the occurrence of symptomatic VTE is closely related to the compromised immune function as well as the viral infection. This also explains why VTE is frequently found in patients with advanced age, trauma, surgery, malignant tumors, heart failure, immobilization, pregnancy and other risk factors. Our findings provide new knowledge on the etiology and pathophysiology of VTE and novel clue for the clinical diagnosis, treatment and prevention of VTE. Diversity and roles of (t)RNA ligases The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-012-0944-2) contains supplementary material, which is available to authorized users. More recently, split tRNA (Fig. 1A , scheme c) and tri-split archaeal tRNA genes, which encode for parts of the mature domain, have been discovered [17, 18] . This type of tRNA arises from distinct transcription units that are joined by trans-splicing to yield a functional tRNA.
Eukaryal tRNA introns are relatively short and range from 12 to 104 nucleotides in e.g., humans [8] . In eukarya, tRNA introns are less abundant than in archaea-e.g., only 6% of human tRNA genes and 20% of yeast tRNA genes are disrupted by introns [8] -and do not display any conserved structural motifs. Rather, the position of the intron is highly invariable in almost all known eukaryotic pre-tRNA genes (Fig. 1B ). An exception to this rule is exemplified by the non-canonical introns found in the circularly permuted tRNA genes of the red alga Cyanidioschyzon merolae (see scheme accompanying Fig. 1B ) [19] .
Several hypotheses for evolutionary origins of tRNA introns have been put forward. An ''intron-early'' scenario proposes the existence of discontiguous primitive tRNA genes interrupted by introns harboring, for example, genes encoding splicing enzymes or aminoacyl transferases. Throughout evolution, these introns could have lost their initial functions and gradually acquired their current features [20] . The related ''split early'' hypothesis speculates that tRNA introns derive from flanking sequences of a priori split tRNA genes [21, 22] . The contrasting ''intron late'' or ''split late'' scenarios assume that tRNA introns arose later in evolution and propagated themselves by gene conversion or transposition [23] . Recent analyses of archaeal genome sequences suggest that tRNA introns in at least some archaeal species have been inserted into contiguous tRNA genes, e.g., by transposition [24] . A following separation of the sequences encoding the mature domains could have given rise to today's split tRNA genes [25, 26] .
As their phylogenetic origins, the functions of tRNA introns are still being explored. The presence of introns in some particular pre-tRNAs has been demonstrated to be required for enzymatic modification of nucleotides such as methylation [27] and pseudouridylation [28] [29] [30] . Only recently has the function of tRNA introns in vivo begun to be addressed in genetic experiments carried out with Saccharomyces cerevisiae. Here, removal of all introns of a particular tRNA isodecoder family did not affect growth or translation of the obtained mutants at laboratory conditions [11] . A Secondary structure diagram of end-matured, intron-containing archaeal tRNAs. Schemes a and b represent end-matured tRNAs with introns in the D-arm or the T-arm, respectively. Scheme c depicts an end-matured split pre-tRNA assembled from two separate primary transcripts [25] . B Secondary structure diagram of end-matured, intron-containing eukaryotic pre-tRNAs. The accompanying scheme represents non-canonical introns and end processing sites of a permuted pre-tRNA transcript in the red alga C. merolae. The non-canonical intron in the acceptor arm is assumed to be excised by tRNA end processing enzymes rather than pre-tRNA splicing factors [19] . A, adenosine; C, cytosine; G, guanosine; U, uridine; W, pseudouridine; Y, pyrimidine; R, purine; asterisks indicate positions of additional introns, 5 0 -exonic regions are depicted in blue, 3 0 -exonic regions in red and intronic regions in green. Full grey circles indicate nonconserved nucleotides in regions of variable length, full blue circles indicate the position of the anticodon [31] . It is hoped that similar studies will yield further insights into the relevance of tRNA introns.
Yeast mutants accumulating pre-tRNAs [32] initially provided access to substrates suitable for studying tRNA processing in biochemical experiments [33, 34] . These studies led to the conclusion that splicing of pre-tRNAs occurs in two steps. During an endonuclease reaction, the intron is first excised and the resulting tRNA exon halves are then ligated to form a mature tRNA ( Fig. 2A) [35]. The biochemical fractionation of extracts catalyzing pre-tRNA cleavage has led to the identification of tRNA endonuclease proteins in archaea [36] and yeast [37] , facilitating the characterization of the homologous human proteins [38] .
In both archaea and eukarya, the tRNA endonuclease catalyses the cleavage of pre-tRNA at the two exon-intron boundaries and generates tRNA exon halves displaying a 2 0 ,3 0 -cyclic phosphate at the 3 0 -end of the 5 0 -exon and a 5 0hydroxyl at the 5 0 -end of the 3 0 -exon (Fig. 2B , box at the top). In addition, the reaction yields a linear intron with 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini ( Fig. 2A) . Although being mechanistically and evolutionarily related [39] , it has been shown that archaeal and eukaryal tRNA splicing endonucleases differ with respect to substrate recognition [9] .
Studies modifying the substrates used for in vitro processing reactions revealed that splice site recognition by archaeal tRNA splicing endonucleases relies on the presence of the bulge-helix-bulge motif [14] . Archaeal tRNA endonucleases consist of two dimers, each composed of a catalytic and a structural subunit. These dimers combine in an antiparallel fashion and interact with the pseudosymmetric substrate. Each exon-intron boundary is cleaved by one of the diagonally juxtaposed catalytic subunits, which together form the two composite active sites [40] . Despite the conservation of the recognition motif, four types of oligomeric organization of archaeal splicing endonucleases have co-evolved with minor changes of their substrates [41] giving rise to a 4 (homotetramers, found in Euryarchaeota), a 2 (homodimers, found in Euryarchaeota), (ab) 2 (dimers of heterodimers found in some Crenarchaeota such as e.g., Nanoarchaeum equitans), and abcd (heterotetramers, also found in Crenarchaeota) enzymes [39, [42] [43] [44] . In the case of the homotetrameric endonucleases, two of the four identical chains function as structural subunits whereas the two remaining polypeptides serve as catalytic subunits [45] . The monomers of the homodimeric splicing endonucleases might have arisen from an in-frame gene duplication and thus encode for a fusion of a structural and a catalytic domain [42] .
The eukaryal tRNA endonucleases are phylogenetically related to their archaeal counterparts [39] . The yeast endonuclease is of the abcd type and consists of two catalytic subunits, ScSEN2 and ScSEN34, and two structural subunits, ScSEN15 and ScSEN54 [37]. All four yeast subunits have homologous human proteins termed HsTSEN2, HsTSEN34, HsTSEN15, and HsTSEN54 [38] . Although it has been shown that at least one eukaryal tRNA endonuclease retained the ability to process archaeal pre-tRNA substrates [46] , eukaryal endonucleases seem to have a different mode of splice site recognition: Eukaryal tRNA endonucleases cleave pre-tRNA substrates at a conserved distance from a structural feature located in the mature domain [47] . Apart from a strictly conserved A:I base pair (Fig. 1B ) [48] , little sequence constraint seems to exist for the intron itself, which can be extensively mutated without disturbing its proper recognition by the endonuclease [49] .
Proteins catalyzing the final step of tRNA splicing-ligation of tRNA exon halves [35]-have first been identified by biochemical fractionation approaches in yeast [50, 51] and plants [52] . Initial biochemical experiments already indicated that the ligation step is mechanistically not as conserved among the different archaeal and eukaryal organisms as the endonuclease reaction [53] [54] [55] [56] . Interestingly, although not requiring tRNA splicing enzymes, Escherichia coli has been shown to catalyze ligation of tRNA exon halves [57] . Even earlier, an RNA ligase activity had been identified in bacteriophage T4-infected E. coli [58] . These experiments led to the conclusion that, based on the type of phosphodiester bond established, distinct types of RNA ligase activity exist [10] (Fig. 2B ). Bacteriophages on one hand, and fungi and plants on the other, utilize two related but mechanistically distinct multistep reactions to prepare the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini produced by the upstream endonuclease reaction for ligation (Fig. 2B , left box). Both mechanisms require the hydrolysis of the 2 0 ,3 0 -cyclic phosphate and phosphorylation of the 5 0 -hydroxyl prior to ligation and as a result the phosphate at the newly formed phosphodiester linkage originates from the nucleotide triphosphate cofactor used for the kinase reaction [10] . To indicate that a 5 0 -phosphate is joined to a 3 0 -hydroxyl Diversity and roles of (t)RNA ligases 2659 moiety, these two pathways are hereinafter referred to as 5 0 -3 0 RNA ligase mechanisms. In addition, two mechanisms exist that incorporate the cleavage site-derived phosphate into the splice junction (Fig. 2B , central and right boxes). 3 0 -5 0 RNA ligation converts 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini into a 3 0 ,5 0 phosphodiester and can Appp adenosine 5 0 -triphosphate; App adenosine 5 0 -diphosphate; Ap adenosine 5 0 -monophosphate; Gppp guanosine 5 0 -triphosphate; Gpp guanosine 5 0 -diphosphate; Nppp unspecified nucleoside 5 0 -triphosphate; Np unspecified nucleoside 5 0 -monophosphate; pp pyrophosphate; Ap-Lig adenylated ligase protein; NT-domain nucleotidyl transferase domain; Ptase 2 0 -phosphotransferase; NAD ? nicotinamide adenine dinucleotide; Appr[p ADP-ribose-1 00 ,2 00 -cyclic phosphate be detected in archaeal and vertebrate cell extracts. During 2 0 -5 0 RNA ligation, predominantly found in eubacteria and archaea, 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl moieties are joined to give rise to a 2 0 ,5 0 -phosphodiester bond [59, 60] . 5 0 -3 0 RNA ligases in bacteriophages, bacteria, archaea, and kinetoplastids
Several bacterial species have evolved to sacrifice individual members of their populations upon phage infection by activating various suicide response pathways [61] . One of these suicide response mechanisms entails the activation of the latent anticodon nuclease PrrC in phage-infected E. coli CTr5x. The nuclease-kept in an inactive state in absence of phage infection-cleaves the host's tRNA Lys in the anticodon loop to shut down protein synthesis and thus impair phage propagation. Cleavage by PrrC yields 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini resembling the products of tRNA splicing endonuclease reactions. Bacteriophage T4, however, has evolved to cope with this defense mechanism. The phage pnk and rli genes encode for proteins capable of restoring cleaved tRNA Lys [62] . It has been demonstrated that T4 Pnkp (the protein encoded by the pnk gene) acts both as phosphatase [63] and polynucleotide kinase [64] thus converting the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl of tRNA halves into 2 0 ,3 0 -cis diol and 5 0 -phosphate termini (Fig. 2B , left box, left branch). T4 Rnl1 (encoded by the rli gene) subsequently joins these tRNA halves by 5 0 -3 0 RNA ligation thus restores a functional tRNA [65] . In addition to T4 Rnl1, another RNA ligase, T4 Rnl2, has been identified in the genome of bacteriophage T4 [66] . T4 Rnl1 and T4 Rnl2 constitute distinct and only distantly related protein families sharing characteristic features (Fig. 3) . Members of the Rnl2 family have been detected and examined in viral, bacterial, and archaeal genomes, suggesting a common phylogenetic origin and possibly a function in RNA repair [66] [67] [68] [69] [70] . Potentially, this type of ligases might also be involved in archaeal tRNA splicing. In trypanosomatids, 5 0 -3 0 RNA ligases of the Rnl2 type are involved in RNA-guided editing of mitochondrial pre-mRNAs by nucleolytic cleavage and ligation [71] . The finding that tRNA splicing in yeast occurs as a twostep reaction, an ATP-independent endonuclease reaction, and an ATP-dependent ligase reaction constituted the first piece of evidence for the existence of a eukaryotic tRNA ligase [35] . Later, an RNA ligase activity was characterized in wheat germ extracts [55, 72] by using a nucleolytic fragment-obtained by RNase T1 digestion of tobacco mosaic virus RNA [73] -as an artificial ligase substrate.
A thorough characterization of the splice junction revealed that a 2 0 -phosphomonoester-3 0 ,5 0 -phosphodiester linkage is established in this system [55, 72] . The same has been shown to be true for the yeast tRNA ligase [50] . Yeast (ScTRL1) and plant (exemplified by AtRNL from Arabidopsis thaliana) tRNA ligases-both identified by activity-guided chromatographic purification [50] [51] [52] -are multifunctional enzymes harboring all the activities required to join the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini in three functionally independent domains [74, 75] (Fig. 3) . First, the 2 0 ,3 0 -cyclic phosphate is hydrolyzed by a cyclic phosphodiesterase (CPD) activity to yield a 2 0 -phosphate-3 0hydroxyl terminated 5 0 -exon [50, 76] . In a second step, the 5 0 -hydroxyl at the 3 0 -exon is phosphorylated by a kinase activity [50] . The actual ligation is preceded by ATPdependent adenylation of a lysine located within a KxxG consensus motif in the active site of the nucleotidyl transferase domain [50, 76] . Next, the enzyme transfers the adenyl moiety to the 5 0 -phosphate at the 3 0 -exon [50] . The adenylated RNA is then joined to the 2 0 -phosphate-3 0 -hydroxyl group at the 5 0 -exon with the concomitant release of AMP [51, 52] (Fig. 2B , left box, right branch and Fig. 3 ). Although RNA ligases of bacteriophage T4, fungi, and plants share important mechanistic features and key residues required for catalysis (Fig. 2B , left box and Fig. 3) , their overall sequence similarity is low [52] . However, the presence of the conserved nucleotidyl transferase domain with its ligase motifs in bacteriophage T4, fungal and plant RNA ligases suggests a common evolutionary origin for these enzymes [69] . Despite their relatedness, several differences exist between yeast, plant, and bacteriophage T4 ligases. For example, ligase-and kinase/phosphatase activities reside on separate polypeptides in bacteriophage T4 whereas yeast and plant tRNA ligases are multifunctional proteins harboring all activities required for ligation (Fig. 2B , left box, right branch and Fig. 3 ). Hydrolysis of 2 0 ,3 0 -cyclic phosphates by yeast and plant CPD domains yields 2 0 -phosphate-3 0 -hydroxyl products rather than the 2 0 ,3 0 -cis diol termini generated by T4 Pnkp (Fig. 2B , left box) [50, 55, 72, 76] . This arises from the distinct phosphoesterase domains employed by the bacteriophage T4 (aspartic acid based DxDxT phosphoesterase domain [77] ) and yeast/plant (2H phosphoesterase domain, characterized by two conserved histidines [78] ) ligation pathways [77, 79, 80] (Fig. 3) . In contrast to bacteriophage T4 ligases, the yeast and plant enzymes do not accept 3 0 -phosphate RNA substrates and ligation typically depends on the presence of a 2 0 -phosphate at the substrate termini [79, 81] . Yeast and plant RNA ligases generate splice junctions bearing a 2 0 -phosphate [72] . To convert this noncanonical 2 0 -phosphomonoester-3 0 ,5 0 -phosphodiester linkage into a 3 0 ,5 0 -phosphodiester, an NAD ? -dependent phosphotransferase, termed ScTPT1 in yeast, removes the 2 0phosphate generating ADP-ribose-1 00 ,2 00 -cyclic phosphate (Fig. 2B , left box, right branch) [82] [83] [84] [85] . Yeast and plant tRNA ligases also subtly differ with respect to substrate selection. While yeast tRNA ligase prefers tRNA exon halves over artificial substrates such as, e.g., linear introns [51] , the plant enzyme ligates circular introns as efficiently as tRNA exon halves in direct competition experiments [52] . Although a 5 0 -3 0 RNA ligase activity has been detected in biochemical experiments carried out with HeLa cell extracts [86] , no homologues of known 5 0 -3 0 RNA ligase proteins could be identified in animals [52] . It therefore seems likely that the 5 0 -3 0 RNA ligase in animals has diverged too far from the RNA ligases already known in order to be identified by existing algorithms or represents a completely novel type of enzyme. This view is supported by the recent identification of a 5 0 -3 0 RNA ligase in the cephalochordate Branchiostoma floridae (BfRNL) featuring a stand-alone ligase protein with weak homology to yeast and plant RNA ligases [87] . In this system, the cyclic phosphodiesterase and kinase modules (BfKinase/CPD or BfCLP1) do not reside on the ligase polypeptide (Fig. 2B , left box, right branch and Fig. 3 ). Enzymes catalyzing kinase (HsCLP1) [88] , cyclic phosphodiesterase (HsCNP) [89] [90] [91] , and phosphotransferase (HsTRPT1) [92] activities have been identified in humans. The fact that these enzymes can function in tRNA splicing pathways in vivo [93] [94] [95] has triggered speculation that a yet unidentified 5 0 -3 0 RNA ligase protein might also exist in humans [87] . In support of this assumption, the RNA kinase, HsCLP1, is an integral component of the tRNA endonuclease complex, suggesting the occurrence of coupled endonuclease and kinase reactions in humans [38, 88] . In contrast, tRNA exon half phosphorylation is associated with ligation in S. cerevisiae, where ligase and kinase activities both reside on ScTRL1. However, the relevance of the 5 0 -3 0 RNA ligase mechanism for tRNA splicing in vertebrates is under debate since the 2 0 -phosphotransferase is not essential in mice [96] .
To account for the substantial differences in sequence and domain organization, it has been suggested to group 5 0 -3 0 RNA ligases in bacteriophages, yeast, plants, and animals into classes [66, 87] ; however, no uniform nomenclature has become generally accepted to date. Biochemical studies revealed that ligation of tRNA exon halves in vertebrates and archaea is mainly achieved by an alternative mechanism. A 3 0 -5 0 RNA ligase activity resulting in incorporation of the precursor-derived 2 0 ,3 0cyclic phosphate into the splice junction as a 3 0 ,5 0 -phosphodiester was for the first time detected in HeLa cell extracts (Fig. 2B, central box) [53, 56, 97] . The same reaction has later been shown to occur in archaeal cell extracts [54, 98, 99] . Intensive attempts to identify the human tRNA ligase proteins by chromatographic purification were triggered by these initial findings [100, 101] . Recently, chromatographic purification led to the identification of RtcB/HSPC117 proteins as 3 0 -5 0 RNA ligases in the crenarchaeon Pyrobaculum aerophilum and in humans [102, 103] . Concurrently, a candidate approach revealed RtcB in Escherichia coli as a 3 0 -5 0 RNA ligase [104] . The high degree of conservation of HSPC117/RtcB proteins suggested a shared role for this protein family in many organisms already during their initial characterization [102] [103] [104] .
RNA ligase activity of the recombinant RtcB proteins from P. aerophilum and E. coli strictly depends on the presence of bivalent metal ions [102, 104] . Studies probing the active sites of archaeal and bacterial RtcB by mutagenesis [102, 104, 105] could in part confirm early predictions concerning essential residues based on structural analyses of homologous RtcB proteins (pdb files 1UC2 and 2EPG) [106, 107] . However, all crystal structures of RtcB proteins available to date represent apo forms of the enzymes and thus the exact functions of individual amino acids lining the presumed active site cannot be assigned unambiguously. Extended structural studies of RtcB proteins in complex with bivalent metal ions, additional cofactors, and RNA substrates will be required to clarify the active site geometry and the distinct metal ion specificity observed for enzymes from different species [102, 104] .
Initially, HSPC117/RtcB proteins were assumed to catalyze the direct nucleophilic attack of the 2 0 ,3 0 -cyclic phosphate by a 5 0 -hydroxyl group, which seemed likely for two main reasons. First, 2 0 ,3 0 -cyclic phosphates are energyrich substrates with a favorable leaving group [108] and second, HSPC117/RtcB-catalyzed ligation reactions did not seem to be strictly dependent on the addition of nucleotide triphosphate cofactors [102] [103] [104] 108] . However, only enzyme preparations that were rigorously purified in the presence of chelating agents proved to yield RtcB preparations sufficiently pure to demonstrate the real cofactor requirements of RtcB-catalyzed 3 0 -5 0 RNA ligation [108] . Thus, 3 0 -5 0 RNA ligation by RtcB proteins occurs as a sequential reaction involving the stoichiometric hydrolysis of nucleotide triphosphates rather than direct nucleophilic attack of the 2 0 ,3 0 -cyclic phosphate by a 5 0 -hydroxyl group [108] . One advantage of this somewhat counterintuitive strategy might lie in the suppression of the backward reaction-the cleavage of the newly formed phosphodiester-as it is catalyzed by bacterial 2 0 -5 0 -ligases (see below) [59] .
The human tRNA ligase complex 3 0 -5 0 RNA ligation appears to be the prevalent human tRNA splicing pathway [53, 56] and relies on HSPC117 as the essential ligase component as supported by two experimental observations: RNAi-mediated depletion of HSPC117 severely impairs tRNA maturation and mutation of a strictly conserved cysteine residue abolishes ligase activity of the affinity purified protein [103] . Human HSPC117, together with the proteins DDX1, CGI-99, FAM98B, and ASW, forms a stable complex of about 200 kDa (Fig. 4) [103] . Even earlier, the observed co-selection of HSPC117, DDX1, and CGI-99 with cruciform DNA duplexes hinted that these three proteins might interact [109] . In contrast to depletion of HSPC117, RNAimediated silencing of the associated proteins does not severely affect RNA ligase activity in HeLa cell extracts, suggesting further functions of the interacting proteins that remain to be explored [103] . Potential roles of these accessory proteins may include targeting of the complex to appropriate cellular compartments, stabilization of the essential subunit, interaction with substrates or adaptor proteins and prevention of illegitimate, promiscuous ligation. Moreover, individual complex components may mediate a transient association of the ligase complex with the tRNA endonuclease, as it has been suggested for the yeast tRNA endonuclease and ligase [110] . In contrast to its archaeal and bacterial orthologues, recombinant HSPC117 did not act as an RNA ligase in vitro. In addition, HSPC117 per se was incapable of replacing the yeast tRNA ligase TRL1 in complementation experiments (J. Popow, unpublished results).
The human tRNA ligase complex components seem to be constitutively and widely expressed in all human and mouse tissues analyzed so far, indicating that these proteins cooperate in a great variety of cellular contexts [103, 111, 112] . DDX1 is a member of the DEAD-box family of putative RNA helicases characterized by the presence of nine conserved sequence motifs [113] . The DEAD-box helicase domain of DDX1 is interrupted by the insertion of a SPRY domain (Fig. 4 ) detectable in numerous proteins and presumably acting as a protein interaction platform [114, 115] . DDX1 has been associated with many molecular functions ranging from mRNA processing [116, 117] to recognition of DNA double-strand breaks [118] and has been demonstrated to exhibit 3 0 -5 0 RNA unwinding activity [117] . The association of DDX1 with HSPC117 suggests an involvement in tRNA splicing. On the other hand, RNAi-mediated depletion of DDX1 only mildly impaired tRNA maturation activity, suggesting that its function might be dispensable for RNA ligation by HSPC117 in HeLa cell extracts [103] . Alternatively, a lack of DDX1 might also have been compensated for by other proteins present in the assayed extracts. Further experiments assessing the effect of mutations within conserved motifs of DDX1 on ligase activity-carried out with purified RNA ligase complex-might successfully address the function of this protein in tRNA splicing. Co-localization studies and extended analysis of proteins associating with DDX1 could answer the question whether DDX1 mediates its functions in conjunction with HSPC117 or whether it is a shared component of multiple protein complexes acting in distinct biological processes. Furthermore, it might be rewarding to test whether the amplification of DDX1 observed in cancerous cell lines [111, 119, 120] , its function in viral replication [121] [122] [123] , and dsRNA recognition in dendritic cells [124] are linked to RNA ligase activity or tRNA processing. Published information concerning the function of CGI-99 is scarce. Although the protein has been found to interact with human [112, 125] and viral [125] proteins in yeast two-hybrid assays, these studies provided little insight into its molecular function. It remains to be determined whether the interaction of CGI-99 with the PA subunit of influenza virus polymerase [125] or its modulation of transcription by RNA polymerase II [126] is in any respect related to RNA ligase activity.
The identification of FAM98B as a human tRNA ligase complex component is the first piece of information published concerning its molecular function. Within the context of tRNA splicing, the exact role of FAM98B needs to be established. Since its RNAi-mediated depletion has almost no impact on RNA ligase activity in HeLa cell extracts [103] , it is likely that FAM98B acts as an interaction platform recruiting accessory proteins or RNA substrates. On the other hand, it might also be the case that other cellular proteins compensated for a lack of FAM98B in these experiments. Alternatively, FAM98B may mediate completely unrelated, yet-to-be-discovered functions of HSPC117 complexes.
ASW has first been characterized in a genetic screen searching for genes differentially expressed in early neural specification in Xenopus laevis [127] . Although this study could demonstrate that alteration of ASW levels in X. laevis leads to severe developmental defects, it provided limited insights into mechanistic aspects of ASW function. ASW has been speculated to be specific for vertebrates as no homologous genes were detectable in Drosophila melanogaster or Caenorhabditis elegans [127] . Nonetheless, homologues of ASW are detectable in the genomes of at least some arthropods and nematodes (Fig. 5A) . Future studies might answer the question of whether the link between ASW and HSPC117 explains any of the developmental defects observed upon manipulation of ASW levels in X. laevis.
Taken together, apart from HSPC117, which appears to be conserved among all domains of life, the additional components of the HSPC117 complex do not show a comparably broad taxonomic coverage. Orthologues of DDX1, FAM98B, and CGI-99 are spread more widely than ASW, which seems to be restricted to fewer species (Fig. 5A ). Another RNA ligase activity leading to the unusual 2 0 ,5 0 -phosphodiester bond has been shown to act in extracts prepared from various bacterial species (Fig. 2B , right box) [57] . Its biochemical purification led to the identification of the ligase gene ligT, conserved not only in bacteria but also in euryarchaeota and crenarchaeota [59, 78] . The apparent dependence of its activity on the presence of modifications in its tRNA substrates suggests that it is involved in repair or processing of tRNA in its host [10, 59] . LigT from E. coli catalyses an ATP-independent equilibrium reaction between 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini and 2 0 ,5 0 -phosphodiester bonds [59] . More recently, a homologous archaeal 2 0 -5 0 RNA ligase has been characterized in the euryarchaeon Pyrococcus furiosus confirming the broad phyletic occurrence of LigT proteins [60] . Although the high degree of conservation suggests an evolutionarily important role for 2 0 -5 0 ligase genes, the exact biological function of LigT proteins and 2 0 ,5 0 -phosphodiester bond formation are still unknown [59, 60, 78] . Interestingly, a few eukaryotic genomes also encode homologs of bacterial and archaeal LigT proteins (Figs. 5B, 6) . The activities and biological functions of these proteins have not yet been addressed.
Phyletic association of RNA ligases RNA ligase proteins are detectable within all major lines of descent. Considering the different ligation mechanisms and classes of RNA ligase polypeptides, it seems, however, that several species abandoned one or the other mechanism of RNA ligation (Figs. 5B, 6 ). Most representatives of the Basidiomycota and Spermatophyta lineages might have lost RtcB/HSPC117 homologues during the course of evolution. However, there seem to be rare instances (such as the basidiomycote Piriformospora indica, see supplemental information for details) where an RtcB/HSPC117 protein has been retained [52, 102, 103, 128] (Figs. 5B, 6) . None of the 5 0 -3 0 RNA ligases known to date could be allocated to several higher eukaryotes, such as e.g., humans (Fig. 5B) . Nevertheless, this picture might change upon the identification of a currently unknown type of 5 0 -3 0 RNA ligase. The detection of 5 0 -3 0 RNA ligase activity in humans [86] together with the recent discovery of the B. floridae 5 0 -3 0 RNA ligase BfRNL [87] argue in favor of this view.
The subcellular localization of tRNA processing events does not seem to be conserved among all eukaryotes. Many localization studies were carried out in the yeast S. cerevisiae which, however, seems to differ in this respect from many other eukaryotes [129] . tRNA splicing is assumed to occur in the cytoplasm in S. cerevisiae, based on the localization of yeast tRNA endonuclease subunits to the outer mitochondrial membrane [130] . Controversial data have been reported concerning the localization of tRNA splicing in plants. On the one hand, some plant tRNA splicing enzymes at least partly localize to the nucleus [131] . On the other hand, impaired nuclear export of tRNA results in accumulation of unspliced pre-tRNA in Arabidopsis thaliana [132] arguing in favor of cytoplasmic tRNA splicing in plants in analogy to the coupling of nuclear export and cytoplasmic tRNA splicing in yeast rna1-1 mutants [32, 133, 134] . In the vertebrate X. laevis, tRNA splicing-preceded by 3 0 -end formation including CCA addition-has been shown to occur in the nucleus by micro-dissection and micro-injection studies carried out with oocytes [135] [136] [137] . In accord with these observations, subunits of the human tRNA endonuclease have been reported to be nuclear proteins [38] .
The biochemical characterization of identified tRNA ligase enzymes revealed that they accept not only tRNA exon halves but also a broad range of artificial substrates such as nucleolytic RNA fragments, synthetic RNA duplexes, and linear introns generated by tRNA endonucleases [52, 53, 55, 97, [102] [103] [104] [105] . In addition, human cell extracts have been shown to not only ligate 2 0 ,3 0cyclic phosphate bearing but also 3 0 -phosphate-terminated RNA substrates [97, 138] . Biochemical fractionation of HeLa extracts revealed that RNA 3 0 -phosphate terminal cyclase RTCD1 can convert RNA 3 0 -phosphates into 2 0 ,3 0 -cyclic phosphates in an ATP-dependent reaction [139, 140] . Despite the thorough characterization of bacterial and human RNA 3 0 -phosphate terminal cyclase proteins [140, 141] , the physiological function of this enzyme class is still unknown. The recent mechanistic characterization of RtcB from E. coli suggests that 3 0 -phosphate-terminated RNA is per se a substrate for this enzyme [108] , raising questions about the genuine function of RNA terminal cyclase RtcA in E. coli. The potential to prepare a variety of RNA termini for ligation and the relaxed substrate specificity of the ligase itself in many organisms suggests that tRNA splicing enzymes might also act in other biological contexts. As an example of such a case, ScTRL1 has been shown to be involved in stress-induced, non-canonical splicing of the HAC1 transcript in S. cerevisiae [142, 143] . A related pathway acts in human cells, however, the involved RNA ligase has to date not been identified [144] [145] [146] [147] . Furthermore, RNA ligases have been proposed as host factors for the propagation of viruses, viroids, and viroidlike satellite RNAs in humans and plants [148] . In fact, HSPC117 and DDX1 are involved in RNA processing during replication of the hepatitis delta virus [121] , presumably acting as the host RNA ligase factors assumed to be required for cyclization of the viral RNA genome as previously proposed [149] . Furthermore, components of the HSPC117-complex together with the RNA cyclase, RTCD1, have been shown to interact with kinesin-associated RNA transport granules in mouse brain extracts [150] . The exact functions of these RNA metabolic enzymes in the context of RNA transport await further experiments. In addition to processing of RNA transcripts of various origins, RNA ligases are assumed to be involved in RNA repair pathways. Apart from the wellstudied example of tRNA repair by bacteriophage T4 RNA ligase [62] , further examples of RNA repair have been revealed [67, 79, 151] . These studies showed an amazing potential of RNA ligases to function as safeguards against the deleterious effects of cytotoxic nucleases in yeast and bacteria. Similar efforts might unveil examples of RNA repair in unanticipated physiological contexts not only in bacteria and yeast but also in higher organisms. The high degree of conservation of HSPC117/RtcB proteins and preliminary data indicating that HSPC117 seems to be encoded by an essential gene in mice [152] are highly suggestive of universal and important roles for this protein family. Assigning a biological function to the operon harboring the ligase, rtcB, and the cyclase, rtcA, in some bacteria may help to uncover functions of this type of ligase unrelated to tRNA splicing since E. coli does not encode for intron harboring tRNAs. Finally, the identification of genuine substrates interacting with RNA ligase proteins in vivo may yield fascinating insights into the various processes potentially requiring RNA ligases. Diversity and roles of (t)RNA ligases 2667 ESP Abstracts 2012  OFP-01-002 Intraoperative evaluation of breast cancer sentinel lymph node metastasis with an automated molecular detection method as an alternative to standard pathological evaluation F. Beca * , E. Rios, P. Pontes, I. Amendoeira * Centro Hospitalar de São João, Anatomic Pathology, Porto, Portugal Objective: One-step nucleic acid amplification assay (OSNA) from Sysmex® is an automated system to detect sentinel lymph node (SLN) metastasis in breast cancer patients. This assay is based on the detection of cytokeratin 19 mRNA amplification by reverse-transcription loop-mediated isothermal amplification. Traditionally intraoperative evaluation of SLN is accomplished by cytology and/or frozen section, accounting for heavy workload to pathology department. The objective of this study is to compare the OSNA assay with our standard processing for intraoperative SLN evaluation. Method: 82 SLN from 45 patients with CK19+ and invasive primary carcinomas where simultaneously evaluated by cytology and/or frozen section and by OSNA assay. Results where compared with gold-standard definitive H&E evaluation (serially sectioned at 150 μm intervals). Results: The sensitivity and specificity of OSNA assay was 92.3 % and 98.2 %, respectively with a positive predictive value (PPV) and a negative predictive value (NPV) of 96 %. For standard intraoperative evaluation, specificity was 100 % and sensitivity was 73.0 %. Conclusion: OSNA assay showed similar specificity and higher sensitivity than standard pathologic examination. These findings suggest it can be used as an accurate tool for intraoperative evaluation of SLNs metastasis and possibly contribute to reducing the need for re-intervention for axillary lymph node dissection. Objective: Antracycline-based chemotherapy represents a standard of care in breast cancer patients especially who overexpress HER-2. Topoisomerase 2A has been considered a molecular target for antracyclines and its co-amplification with HER-2 genes has been proposed. In our study we investigated if Topoisomerase 2A overexpression can be used as a molecular marker on predicting response to antracycline-based chemotherapy.
Method: Breast cancers tissues from 40 patients underwent to antracycline-based neoadiuvantchemotherapy and 267 treated with adiuvant therapy were collected into 4 Tissues microarrays, and classified according to pathological stage, ER, PR, HER-2 and TOPO2A status. The expression of topoisomerase 2A has been correlated to response to chemotherapy. Results: Our results show that TOPO2A expression is significantly higher in neoplasms with larger size, nodal metastasis, scant ER end PR receptors, and amplification of HER-2 gene (p < 0,00, T-test). Cancers in witch high level of TOPO2A has been found exhibit a better clinical response to treatment with anthracycline (p <0,0000, T-test). Therefore TOPO2A expression could be considered both as predictive and prognostic marker. Conclusion: The employment of TOPO2A as a predictive marker in clinical practice may be useful for the medical therapy of non-endocrine-responsive patients candidate to undergo treatment based on anthracycline, that is no free from adverse effects as cardiomyopathy and leukemia.
OFP-01-004 Stem cells in triple negative breast cancer and associated in situ lesions M. Comanescu * , M. Dobre, G. Bussolati * Institutul Victor Babes, Dept. of Pathology, Bucharest, Romania
Objective: Triple negative breast cancer (TNBC) (negative for expression of estrogen and progesterone receptors (ER, PR) and HER2/neu protein) represent a subtype of breast cancer associated with poor prognosis and highly aggresive behaviour. Genetic characterization of stem cell (CD 44, ....) in this type of carcinoma might provide important data concerning origin and evolution. Data of the literature focused specifically on TNBC but, despite the interest shown to stem cells recently, there are no data concerning the genetic characterization of stem cells in the context of cell biology of TNBC as compared with associated DCIS. Method: We investigated, through immunohistochemistry, the expression and distribution of several stem cell/related antigens, exploring the association of TNBC with DCIS and comparing the presence of stem cells in the invasive and in the in situ component. Results: The multiplicity of parameters to be detected for characterising stem cells and of the diagnostic materials that are available would prevent a straightforward approach and a sequential combination will have to be planned. Conclusion: Optimization of detection, identification and characterisation of tumourigenic breast cancer stem cells might permit further identification of targeted treatment.
Over-and undergrading of Breast Cancer on core biopsies in comparison to surgical specimens T. Decker * , C. Focke, D. Gläser * D. Bonhoeffer Medical Center, Dept. of Pathology, Neubrandenburg, Germany
Objective: To evaluate the relevance of histological grading (HG) based on core biopsies (CB) for clinical decision making in breast cancer (BC) we evaluated the concordance with HG from surgical specimen (SSP) and the reasons for under-or overgrading. Method: CB and related SSP of 398 BCs were prospectively graded according to the Nottingham grading system (SSP: 65 G1, 162 G2 and 171 G3). CB/SSP agreement rates and positive predictive values (PPV) of CB based HG were calculated. Rates of over-and underestimation of glandular differentiation (GD), nuclear pleomorphism (NP) and mitotic activity (MA) were calculated.
Results: CB/SSP agreement rates came out with 95 % for G1, 73 % for G2, and 56 % for G3. The PPVs of CB based HG were 56 % for G1, 61 % for G2 and 100 % for G3. The overgrading and undergrading rates on CB were 1 % and 37 %, respectively. Main causes for undergrading in CB were underestimated MA (40 %) and a combination of underestimated NP and MA (32 %). Conclusion: Whereas overgrading on CB is an exception, undergrading derives largely from underestimation of proliferation. Agreement of HG between CB and SSP ranges from almost perfect for G1, moderate for G2 to slight for G3 tumours.
OFP-01-006 Comparative histopathology of mammary alterations in women and dog V. Deckwirth * , P. Kronqvist, M. Lintunen, A. Sukura * University of Helsinki, Dept. of Veterinary Pathology, Finland
Objective: Companion animals such as dogs share the same environment with people. Mammary tumours are the most prevalent spontaneous neoplasia in intact bitches. More animal models are needed for human mammary tumour research. Our objective was to describe equivalent non-neoplastic and neoplastic mammary gland alterations in woman and dog. Method: Formalin-fixed, paraffin-embedded archived human and canine mammary gland tissue samples. Epidemiological data were collected and analyzed from human (n= 1935) and canine (n=161) patients for the years 2003-2005. Histological comparison was performed with HE-stained tissue sections and immunohistochemistry. Applied antibodies included CK5/6, ERα, PR, Ki67, Her2, p63, SMA, Ecadherin and calponin. Results: Several comparable benign and malignant mammary gland alterations were identified, consisting of lobular, tubular and ductal as well as mesenchymal of origin. Most of these have not been described previously. The epidemiological results show also shared characteristics. Conclusion: Although there are canine specific lesions there also exist equivalent entities. Our results indicate the female canine as a suitable model for translational research.
OFP-01-007 St. Gallen intrinsic subtyping of breast cancer: Influence of proliferation assessment methods C. M. Focke * , D. Gläser, K. Finsterbusch, T. Decker * Dietrich Bonhoeffer Klinikum, Abt. Pathologie, Neubrandenburg, Germany
Objective: The St. Gallen Conference 2011 strongly recommends intrinsic subtyping of breast cancer (BC) for therapeutic decisions. We analyzed the influence of different proliferation assessment methods on the BC subtype distribution. Method: Intrinsic subtyping was performed according to the St. Gallen criteria on 225 BCs. Proliferation was assessed by Mitotic Activity Index (MAI) (area: 1,59 mm², cut-off ≥10) and by several variants of Ki67-Labeling-Index counting any nuclear staining of (1) 100 tumor cells within the hot spot (Ki67-100), (2) 1020 tumor cells in 3 HPF in the tumor periphery, including the hot spot (Ki67-1020periphery), and (3) 1020 tumor cells in 3 HPF, including hot spot, cold spot and an intermediate area (Ki67-1020spectrum) using the recommended cut-off of <14 %. Results: Of 225 carcinomas, 10 % were Triple-negative, 2 % Her2+, 6 % Luminal B Her2+, and 9 % Special Type. The rates of BCs after different proliferation assessment methods were: MAI 49 % vs. 24 %; Ki67-1020spectrum: 34 % vs. 39 %, Ki67-1020 periphery: 24 % vs. 49 % and Ki67-100: 10 % vs. 63 %. Conclusion: Subtyping of Luminal A and Luminal B Her2neg BCs according to St. Gallen 2011 is highly influenced by the method of proliferation assessment used. Its biological relevance seems to be not unequivocal.
OFP-01-008 The utility of cytological smears allied to the OSNA method for intraoperative analysis of sentinel lymph-node metastasis in breast cancer patients S. Foreid * , M. Martins, H. Pereira, M. Oliveira * Loures, Portugal
Objective: One-Step-Nucleic-Acid-Amplification (OSNA) is a recently introduced PCR method to evaluate intraoperative sentinel lymph-node metastasis in breast cancer. It assesses the whole lymph-node in a 20-minutes protocol. Many studies have reported it to be more sensitive than frozen section and/or cytology. Yet, it is more expensive and takes longer to perform. Method: Between 2010 and 2012 at CHLC, we have performed the intraoperative analysis of sentinel lymph-node using both OSNA and cytological smears in a group of patients. Results: 35 lymph-node evaluations were done. 16 cases were OSNA positive (5 micrometastasis and 11 macrometastasis). Of those, 11 were also positive for tumor cells on cytological smears. 19 cases were negative by both methods and 5 cases were detected by OSNA but not on cytological smears. Importantly, all macrometastasis diagnosed by cytology were confirmed by OSNA. Of notice, in this series, 69 % of the positive cases detected by OSNA were also detected by cytological smears. Conclusion: Like previously reported, OSNA showed a greater sensibility than cytology to diagnose sentinel node metastasis. However, the majority of metastasis can be detected by cytological smears alone. Our results suggest that a less expensive and faster cytological analysis should precede OSNA, and if positive, the latter method could be disregarded. Tuesday, 11 September 2012, 17.00-19. Objective: Tumor markers may be concentrated intraductally in early breast cancers. Method: To test this hypothesis, HER2-ECD and CA15-3 of the needle washout fluid from 29 benign lesions, 26 DCIS, and 95 invasive carcinomas of the breast were measured using chemiluminescence. The measuring limit for HER2-ECD (0.5 ng/ml) and that for CA 15-3 (4 U/ml) were used as the cutoff values, respectively. Results: The proportion of patients with any biomarker elevation was 6.9 % in cases of benign lesions, 46.2 % in DCIS, and 15.8 % in invasive carcinomas. Thus, biomarker elevation was most frequent in DCIS, followed by invasive carcinomas (P<0.01). HER2-ECD values over 6 ng/ml or CA 15-3 values over 25 U/ml were s een exclusively in DCIS or invasive ductal carcinomas with an extensive intraductal component, supporting the hypothesis. Conclusion: Our approach may be useful to identify a subset of breast cancer patients who are suitable for intraductal molecular targeted therapy.
OFP-02-002 Biological significance of proliferation and HER2 overexpression in luminal/oestrogen receptor-positive breast cancer D. Jerjees * , A. Green, A. Benhasouna, A. Alshareeda, R. Abduljabbar, F. Barros, C. Nolan, I. Ellis, E. Rakha * University of Nottingham, Dept. of Oncology, United Kingdom
Objective: In this study, we have compared the biological significance of proliferation; assessed using Ki67 labelling index (Ki67-LI), with respect to HER2 expression in oestrogen receptor positive (ER+) breast cancer to assess the impact on growth fraction and biological characteristics of luminal breast cancer. Method: 1562 well-characterised ER + breast cancers were assessed for expression of a large panel of biomarkers (no=30). Results: 53 % of the cases showed high Ki67-LI (>13 %) and 12 % showed HER2 over-expression and both were positively associated with younger age, higher tumour grade, lymph node stage and shorter outcome. Both markers were associated with up-regulation of ER-coactivators (CD71, CARM1, PI3KCA&TK1), P-cadherin, p53 and with lower levels of ER expression and down-regulation of ER-related genes (PgR, AR&GATA3) and BRCA1. Neither of the two markers was associated with expression of basal-associated cytokeratins. High Ki67-LI was associated with down-regulation of luminal enriched markers including luminal cytokeratins, MUC1, GCDFP-15, FHIT, and transcription and differentiation ERrelated genes (FOXA1 and TFF3). In contrast, HER2 expression was associated with up-regulation of luminal cytokeratins, GCDFP-15, FHIT and E-cadherin. Conclusion: Unlike high Ki67-LI, HER2 overexpression is not associated with the down-regulation of luminal enriched genes. Increased growth fraction in ER + tumours may be driven by different mechanisms in HER2+&HER2-disease.
OFP-02-003 Loss of Drosha expression is associated with poor survival in breast cancer patients S. Khoshnaw * , T. Abdel-Fatah, C. Nolan, Z. Hodi, E. Rakha, D. Macmillan, I. Ellis, A. Green * University of Nottingham, Breast Cancer Pathology, United Kingdom
Objective: Drosha is a protein that plays a key role in the biogenesis of microRNAs which are well known to be deranged in human breast cancer. The purpose of this study was to investigate the hypothesis that Drosha is significant in the development and progression of breast cancer and has clinical relevance as a potential predictive and prognostic target. Method: We have examined Drosha protein expression, using microarray and immunohistochemistry, in a well-characterised series of unselected invasive breast cancer patients (n=750) with long-term follow-up and documented expression characteristics for a large range of biomarkers of known relevance in breast cancer. Furthermore, a smaller cohort of selected breast cancer cases (n=24) was also investigated for differential Drosha protein expression in distinct stages of tumour progression, including in situ (DCIS), invasive and metastatic components. Results: Drosha cytoplasmic and nuclear expression was lost with cancer progression, and was associated with loss of BRCA1 expression. Multivariate statistical analyses showed that Drosha cytoplasmic expression was an independent predictor of breast cancer specific survival, tumour recurrence and metastases. Conclusion: Drosha cytoplasmic expression in breast cancer is an independent predictor of patient outcome, tumour recurrence and metastasis. These observations imply that alterations in mechanisms of miRNA regulation can influence tumour behaviour. Objective: Determination of proliferation in breast carcinomas is highly prognostic and can have treatment implications. However, there is no consensus on issues such as counting area or cut-off values. Our aim was to examine and compare Ki-67 counts in various tumor areas, the novel mitosis marker PHH3, and standard mitotic count. Method: We examined a nested case-control series (n= 190) as part of the population-based Norwegian Breast Cancer Screening Program. Mitotic count (mitosis per mm2) was assessed on H&E-sections. The percentage of Ki-67 positive nuclei among 500 tumor cells was recorded for both hot-spots (HS; highest proliferation) and cold-spots (CS; lowest proliferation). PHH3 counts (per mm2) were evaluated by immunohistochemistry. Results: The upper quartiles for Ki-67-HS, Ki-67-CS, PHH3 and mitotic count consistently showed the strongest associations with known unfavorable tumor features (high histologic grade, ER negativity, HER2 positivity, CK5/6+, P-cadherin+). Univariate survival analysis showed comparable prognostic strength of Ki-67-HS, PHH3 count and mitotic count. In multivariate analysis, mitotic count was the strongest prognostic factor among these. Conclusion: By upper quartile, Ki-67-HS, PHH3 and mitotic count all showed strong associations with various tumor features. In multivariate survival analysis, mitotic count was the strongest proliferation marker. Objective: Ki67 LI is a good marker of breast cancer prognosis. We aimed at comparison of performance of 5 Ki67 antibodies. Method: 283 breast cancer cases were retrieved. Immunohistochemical staining for 5 Ki67 clones (30-9, B56, MIB1, polyclonal, SP6) performed in TMA-s in automated immunostainer. Evaluation performed on digital slides utilizing linear 0-10 frequency score. Objective: In breast cancer, classification systems are used to assess pathological response to neoadjuvant chemotherapy. Biomarkers are still needed to better predict the efficiency of primary systemic therapy. Dysregulation of connexin (Cx) channels (gap junctions) is involved in carcinogenesis and tumor progression. Method: We have correlated Cx expression and the pathological response in neoadjuvant treated breast cancers. Fifteen of the 21 human Cx isotypes were pre-screened. Cx26, Cx32, Cx43 and Cx46 were found widespread and tested in tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. Pathological response was characterized according to the EWGBSP, CPS-EG, Miller-Payne, Sataloff, and NSABP classification systems. Results: Only the CPS-EG classification had prognostic relevance by score 1-2 cases presenting better overall survival (p=0.015) than those scoring 3-5. Cx43 levels prechemo correlated with hormone receptor status pre-and post-chemo and had inverse correlation with HER2 levels pre-chemo. Reduced Cx26 expression (<5 %) post-chemo correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20 %) pre-and post-chemo was also associated with better survival in the EWGBSP TR2b (ppre=0.006), Sataloff TB (ppre=0.005; ppost=0, 029) and Miller-Payne G3 (ppre=0.002; ppost=0.012) subgroups. Conclusion: Classifications combined with testing for Cx46 and Cx26 expression can improve prediction of neoadjuvant chemotherapy in breast cancer.
OFP-02-008 Atypical vascular lesions after radiation therapy for breast cancer: A clinicopathologic study and outcomes of 24 cases H. Gobbi * , C. Fraga-Guedes, M. Mastropasqua, M. Kneubil, E. Botteri, G. Viale * UFMG, Dept. of Anatomic Pathology, Belo Horizonte, Brazil
Objective: We assessed clinicopathologic features and outcomes of 24 cases of atypical vascular lesions (AVL) in patients submitted to radiation therapy for breast carcinoma. Method: Data from all patients were retrieved from the files of the European Institute of Oncology (EIO), Milan. Histopathological review of all cases was carried out. Results: All patients were women (median age: 58 year, range: 36-81 year). Median lesion size was 0.5 cm (range: 0.3 to 1.1 cm). Median latency interval from radiation to AVL diagnosis was 49 months (range: 17 to 124 months). 62.5 % of cases presented as an erythematous unique papule confined to superficial/middermis with proliferation of thinwalled, vascular channels, lined by a single layer of bland endothelial cells. No prominent nucleoli, mitoses or blood lakes were seen. Patches of chronic inflammatory infiltrate accompanied all AVL. One patient (4.2 %) developed local recurrence, and two patients (8.3 %) developed angiosarcoma in the previous biopsy site, 19 and 89 months later. Margin involvement was associated to unfavorable outcome. Conclusion: Our data suggest that post-radiation AVL and angiosarcoma may represent a spectrum of the same disease process. The development of local recurrence and subsequent angiosarcoma indicate that AVL must be completely excised, with free surgical margins, and patients must be accompanied by clinical exams and imaging. Objective: Gastric dysplasia is classified as gastric/foveolar and intestinal/adenomatous according to morphological (architectural and cytologic) features. The immunophenotypic classification of dysplasia, based on the expression of mucins and CD10, recognizes three major types: gastric (MUC5AC and/or MUC6); intestinal (MUC2 and/or CD10); hybrid (gastric and intestinal markers). CDX2 is a transcription factor responsible for early intestinal differentiation. ALDH1 is recognized as a stem cell marker in several organs. Method: Nineteen cases of dysplasia were classified as gastric/foveolar (n=8) or intestinal/adenomatous (n=11) and graded as low-grade (n=9) and high-grade (n=10). Immunophenotypic classification showed gastric (n=5; 26 %), intestinal (n=6; 32 %) and hybrid (n=8; 42 %) phenotypes. Results: Cases classified by morphology as gastric/foveolar-type displayed gastric (50 %) or intestinal (50 %) immunophenotype, whereas intestinal/adenomatous-type cases displayed gastric (9 %), intestinal (55 %) or hybrid (36 %) immunophenotype (p=0.023). High-grade dysplasia was identified in cases with gastric (80 %), intestinal (17 %) and hybrid (63 %) immunophenotype (p=0.085). CDX2 expression was significantly lower (p=0.044) in gastric (60 %) than in intestinal and hybrid immunophenotypes (100 %). ALDH1 expression was displayed only in gastric (80 %) and hybrid (50 %) immunophenotypes (p=0.023). Conclusion: Gastric dysplasia is heterogeneous and morphology is insufficient to elucidate cell differentiation. High-grade dysplasia, decreased CDX2 expression and high ALDH1 expression are associated with gastric immunophenotype.
OFP-03-002 Tumor budding in pancreatic cancer: Are we missing important prognostic information? D. Born * , I. Zlobec, M. Montani, B. Gloor, A. Lugli, E. Karamitopoulou-Diamantis * Universität Bern, Inst. für Pathologie, Switzerland
Objective: Tumor budding is defined as single tumor cells or small cell clusters at the invasive front of gastrointestinal (including colorectal, gastric and ampullary) carcinomas and is linked to adverse prognosis. To date it has not been reported in pancreatic ductal adenocarcinomas (PDACs). We assessed the presence and prognostic importance of tumor budding in PDAC. Method: Whole-tissue sections of 120 PDACs with full clinico-pathological and follow-up information were stained with pancytokeratin. Tumor budding was assessed in 10 high-power fields (HPFs) by two pathologists and considered high-grade (HG) when an average of >10 buds was counted. Measurements were correlated to the patient and tumor characteristics. Results: Inter-observer agreement was strong (ICC=0,72). HG-budding was found in 70.3 % of cases and was linked to advanced pT-stage (p=0.0463), lymphatic invasion (p=0.0192) and decreased disease-free and overall survival (p<0,0001 and p=0,0005). There was no association with pN, pM, R-stage or vascular invasion. In multivariate analysis the prognostic effect of HG-budding was independent of lymphatic invasion, pN and R-stage [p<0,0001, HR (95 % CI)=3,65(2,1-6,4)]. Conclusion: Tumor budding occurs frequently in pancreatic cancer, is an indicator of worse outcome and adds independent prognostic information. Routine use of tumor budding would help to better stratify patients into prognostic subgroups.
OFP-03-003 Epstein-Barr virus and p53 in the development of gastric cancer V. Genitsch * , M. Cathomas, L. Terracciano, L. Tornillo, A. Lugli, A. Marx, G. Sauter, F. Carneiro, F. Hofstädter, N. Willi, G. Cathomas * Kantonsspital Baselland, Abt. Pathologie, Liestal, Switzerland
Objective: Epstein-Barr virus (EBV) is associated with a subset of gastric cancers (GC) . In this study, the prevalence of EBV and its presence in the development of GC were assessed. Method: EBV was evaluated in GC by tissue microarrays using in situ hybridization for EBER (n=610). Non-tumorous, dysplasia, intramucosal and invasive carcinomas and metastasis of EBV positive GC were analyzed (n=22). p53 expressen was evaluated in EBV positive and a subset of 84 EBV negative GC. Results: 4.9 % of gastric cancers were positive for EBER. All invasive cancers showed diffuse EBER positivity and only 2 lymph nodes revealed a focal loss of EBER. In 13 GC with foci of atypia/low grade dysplasia, no or only focal EBER positivity was observed. p53 was overexpressed in 24 of 84 EBV negative but not in EBV positive GC (28.6 % vs. 0 %; p=0.007). Conclusion: EBV is diffusely expressed in invasive GC and only occasionally lost in metastasis. In low grade dysplasia, however, EBV is often focal or missing, indicating that EBV infection may be associated with progression from low grade dysplasia to invasive cancer and this progression is p53 independent.
OFP-03-004 P2X7R attenuates colitis associated carcinogenesis P. Hofman * , T. Juhel, M. Ilie, O. Bordonne, O. Boyer, X. Hebuterne, S. Adriouch, V. Vouret-Craviari * CHU de Nice, LPCE, France
Objective: It is now well established that patients with inflammatory bowel disease presented a higher risk to develop a colon cancer. Our previous works characterized the link that exists between bacterial infection and inflammation and highlighted the role of the microenvironment on the onset of chronic inflammation. Here, we wished to understand the role of inflammasome, a complex of proteins that is activated by microbial motifs and the purinergic receptor P2X7 and involved in the maturation of IL-1β. Method: We treated P2X7R knockout mice to induce acute colitis (3 % DSS) or colitis associated colon cancer (AOM and DSS). Results: We showed that P2X7R KO mice are more resistant than wild type mice to a DSS-induced colitis. Further, we demonstrated that P2X7R KO mice are highly susceptible to inflammation driven colon tumorigenesis. In addition, we showed that tumors from P2X7R KO contained more neutrophils (CD11b+/Ly6G + cells) and less macrophage (CD11b+/Ly6G-) than tumors obtained from WT mice. Conclusion: We will present results on the characterization of the pro tumoral tumor associated neutrophils and the molecular mechanisms that are linked to the presence of such immune cells.
OFP-03-005 IEL counts and distribution in normal duodenum, non-GSE IELosis and GSE: Do we need a cut-off? B. A. Karabork * , S. Yuksel, B. Savas, A. Ensari * Ankara University of Medicine, Dept. of Pathology, Turkey
Objective: Intraepithelial lymphocytosis (IELosis) is a characteristic feature of gluten-sensitive enteropathy (GSE), though, the cut-off is still debatable. We aimed to evaluate the number and distribution of IELs in normal duodenum, and in non-GSE IELosis and GSE. Method: The study group comprised of a spectrum of normal duodenal biopsies (n=31), non-GSE IELosis (n= 30), Type 1 (n=22) and Type 3 (n=21) serologically diagnosed GSE cases. The number of IELs/100 enterocytes and distribution on H&E and CD3-immunostained sections, were assessed for each group. Chi square test was used for statistics. Results: IELs increased significantly through the spectrum (15, 55 in normal, 25, 37, 00 in Type 1, 67, 10 in Type 3) on H&E and CD3, respectively (p<0,001). IEL counts ≥20 on H&E and ≥25 on CD3 had a sensitivity of 95,35 % and 100 %, and a spesificity of 40,98 % and 32,79 %, respectively, for GSE. IEL distribution was diffuse in Type 1 (95,5 %), Type 3 (100 %) but focal in non-GSE (80 %) (p<0,001). Conclusion: The current cut-off for IEL counts seems to be sensitive for IELosis, though, not specific for GSE and needs to be accompanied by the diffuse distribution of IELs.
OFP-03-006 Geographic analysis of the protein expression of metastasis suppressor gene RKIP and its clinical relevance in colorectal cancer V. Koelzer * , E. Karamitopoulou, H. Dawson, A. Lugli, I. Zlobec * University of Bern, Inst. of Pathology, Switzerland
Objective: Loss of Raf-1 Kinase Inhibitor Protein (RKIP) expression has been implicated in disease progression in several tumor types. The aim of this study was to evaluate RKIP expression in colorectal cancer stratified by histological "zones" and to determine the zone responsible for its clinical relevance. Method: Immunohistochemical expression of RKIP was assessed in 100 colorectal cancers using whole tissue sections. Four different areas were evaluated using 10 highpower-fields (HPFs) each: normal mucosa, tumor center, invasion front and tumor buds. The average expression for each zone was assessed for its clinical relevance. Results: Expression of RKIP was diffuse in normal mucosa and progressively lost towards the tumor center and front (26 %, 15 % and 8 % RKIP-positivity, respectively; p<0.0001). Only 3 % of tumor buds were RKIP-positive. RKIP loss in the tumor center only corresponded to more frequent lymph node positivity (p=0.0766), distant metastases (p=0.0243), lymphatic invasion (p=0.0533) and more advanced TNM stage (p=0.0278). RKIP loss was highly prognostic (HR (95 % CI): 0.45 (0.2-0.9); p=0.0288) independently of TNM and therapy. Conclusion: The clinical relevance of RKIP is restricted to the tumor center where it acts as an independent prognostic factor. Its absence in tumor buds provides further evidence to support their hypothesized metastatic potential.
OFP-03-007 Outcomes of neoadjuvant chemoradiation for rectal carcinoma G. Ozgun * , F. Oz Atalay, N. Ugras, O. Yerci * Uludag University, Dept. of Pathology, Bursa, Turkey Neoadjuvant radiation and chemoradiation is currently the treatment of choise for patients with locally or advanced carcinoma of the rectum. Patients treated with neoadjuvant chemoradiatheraphy with good clinical response and tumor regression present a controversial management dilemma. To assess the effects of chemoradiation on tumor regression and disease free survival, consecutive series of 27 patients receiving neoadjuvant chemotheraphy or chemoradiation and a control series of 32 patients without pretreatment were studied. The overall survivals and disease free survivals were compared in terms of variables such as tumor regression grade, stromal response in tumor bed, state of lymph node metastasis, and the localization of the tumor in between patients treated with chemoradiation and the control group. Neoadjuvant treatment is one of the treatment modalities in rectal carcinomas. We want to present our experience between 2005 and 2011 in our institution. Key words: Rectal carcinoma, neoadjuvant treatment, pathologic complete response OFP-03-008 Identification of Tissue Microvascular Invasion Biomarkers in Hepatocellular Carcinomas by MALDI Imaging Mass Spectrometry N. Poté * , T. Alexandrov, J. Le Faouder, S. Laouirem, J. Belghiti, J. M. Camadro, V. Paradis, P. Bedossa * Paris, France
Objective: Microvascular invasion (mVI), a major predictive factor of tumoral recurrence and mortality in patients with hepatocellular carcinoma (HCC), is only detectable on pathological examination. So far, there is no reliable tool to identify mVI prior to surgical procedures. MALDI Imaging Mass Spectrometry (IMS) represents a new analytical tool to provide the relative abundance and distribution of the whole proteins expressed in a tissue section. The aim of this study was to compare, using MALDI IMS, the tissue proteome of HCC without and with mVI in order to identify surrogate biomarkers of mVI. Method: A total of 56 HCC samples obtained from surgical specimens, for which frozen samples were available, were retrospectively collected. Two groups of tumors were defined (26 HCC without mVI; 30 HCC with mVI) and were analysed by MALDI IMS. A statistical comparative analysis of acquired mass spectra was then performed in order to identify protein peaks differentially expressed between the two groups. Results: 30 protein peaks were differentially expressed between the two groups, all overexpressed in HCC with mVI. Protein characterization of some of these peaks is in progress. Conclusion: These results highlight the potential of MALDI IMS to uncover new biomarkers in liver carcinogenesis. The identification of mVI biomarkers would be helpful in the therapeutic strategy of patients with HCC.
OFP-03-009 A histopathological scoring system can predict the recurrence of hepatocellular carcinoma in liver transplant patients treated with transarterial chemoembolization F. Rosini * , F. Vasuri, D. Malvi, P. Baldin, W. F. Grigioni, A. D´Errico-Grigioni * University of Bologna, Dept. of Anatomic Pathology, Italy
Objective: Aim of this study is to define the histopathological features predictive of hepatocellular carcinoma (HCC) recurrence in patients treated with orthotopic liver transplantation (OLT) after tumoral transarterial chemoembolization (TACE), in order to establish a predictive "score" applicable by pathologists for recipient risk stratification. Method: We retrospectively enrolled 110 patients (276 total neoplastic or necrotic nodules) who received TACE for HCC downstaging before OLT. The following data were collected: number of neoplastic/necrotic nodules, microscopic thrombosis, the residual neoplastic tissue (RNT), and dysplastic nodules. Sizes, percentage of necrosis, Edmondson's grade, clear cell features were also collected for each nodule. Results: Median follow-up was 1230 days: 14 (13 %) HCC recurrence cases were recorded. Statistically RNT (best cutoff 3 cm3, P=0.032), the total nodule number (best cut-off ≥ 3, P=0.001), and the neoplastic thrombosis (P=0.011) were the only variables predictive of HCC recurrence. Scoring 1+ each parameter, recipients with 0 (n=43) had 0 % recurrence rate; recipients with 1+/2+ (n=49) had 12 %; recipients with 3+ (n=18) had 44 % recurrence rate (P<0.001).
We were able to stratify OLT recipients with TACE-treated HCC in 3 risk groups based only on histopathological analysis.
OFP-03-010 Discovery of a new histopronostic factor in rectal adenocarcinoma treated by radiochemotherapy followed by surgical resection A. Sannier * , J. Lefèvre, F. Bretagnol, D. Cazals-Hatem, Y. Panis, P. Bedossa, N. Guedj * Paris, France
Objective: Neoadjuvant radiochemotherapy (RCT) followed by surgical resection became the treatment for locally advanced rectal cancer. Pathological diagnosis is important for the prediction of prognosis and adjuvant treatment. Study aim was to identify histopronostic factors in a consecutive series of patients treated by RCT and surgery. Method: 113 patients were included, follow-up, tumor morphologic pattern and Modified Rectal Cancer Regression Grade (m-RCRG) were assessed. Univariate and multivariate analysis were used to assess predictors of disease-free survival (DFS). Results: 5-years DFS was 58 %. In univariate analysis, ypT, budding, calcifications, circumferential margin, node involvement, invaded margin, vascular emboles and perineural involvement were pronostic factors (p<0.05). In multivariate analysis, presence of calcification in tumor bed (p=0.027) and small circumferential margin (p=0.037) were the only two independent factor of worse DFS. mRCRG was not correlated to DFS. Among the 50 mRCRG1 tumor, DFS was significantly better in patient with ypT0 than in other ypT stages (p=0.003). Conclusion: Presence of calcification in tumor bed is a new major histopronostic factor described for the first time in rectal cancer. Our results raised the question of whether ypT stage or histological tumor regression is more important in the prediction of patient prognosis.
Tuesday, 11 September 2012, 17.00-19. Objective: The most widely used system for histological grading of colorectal cancer (CRC) is based on the degree of gland formation, despite significant interobserver variability and low prognostic value. Herein we analyzed the prognostic significance of a grading system based on the presence of poorly differentiated clusters in stage I CRC. Method: Poorly differentiated cancer clusters were assessed by two independent pathologists in stage I CRC characterized or not by disease progression. Tumors with <5, 5 to 9, and >10 clusters were classified as G1, G2, and G3, respectively. The prognostic value on disease-free survival and the association with other clinicopathologic characteristics of the conventional and novel grading systems were analyzed. Results: K statistics for inter-observer variability in the assessment of histological grade based on poorly differentiated custers was 0.728 (good). High histological grade assessed with the novel system, but not with the traditional one, represented a negative significant prognostic factor for disease-free survival and it was significantly associated with venous invasion, lymphatic invasion, budding, invasive growth and nodal micrometastases. Conclusion: We suggest that a tumor grading system based on the number of poorly differentiated clusters has a stronger power to stratify stage I CRC patients by prognostic outcome than conventional grading.
OFP-04-003 Cytokeratin 7: A marker for BRAF mutated colorectal carcinomas? S. Gurzu * , Z. Szentirmay, I. Jung * University of Targu Mures, Dept. of Pathology, Romania
Objective: Cytokeratin20+/Cytokeratin7− (CK) is used as the characteristic immunophenotype of colorectal carcinomas (CRC) . Some new studies suggested that aberrant pattern CK20/CK7 can be identified in colorectal cancer with microsatellite instability (MSI). Our aim was to establish which factors may determine changing in this immunophenotype. Method: In 70 CRC, randomly selected, we performed immunohistochemical stains with CK20 and CK7 and analyzed the microsatellite status and BRAF mutations with real time PCR. Results: From the 70 CRC, 15 were MSI and 55 MSS (microsatellite stable) cases. 90 % of MSS cases diffusely expressed CK20 without CK7 expression. From the 15 MSI cases, 6 presented BRAF mutations. In MSI cases with BRAF mutations, CK20 was focally expressed or negative and CK7 was diffusely expressed. Conclusion: Cytokeratin7 positivity may be used to select BRAF mutated MSI colorectal carcinomas. Both CK7 and CK20 should be used for differential diagnosis of colorectal cancer.
OFP-04-004 Intratumoral budding in preoperative biopsies predicts local and distant metastasis in colorectal cancer patients A. Lugli * , M. Hädrich, D. Inderbitzin, M. Borner, I. Zlobec * Medizin. Universität Bern, Inst. für Pathologie, Switzerland
Objective: In 2011, the term "intratumoral budding, ITB" was used to describe the presence of tumor buds within the main tumor body and was correlated to worse clinical outcome in colorectal cancer patients. Here, we further elucidate the potential clinical role of ITB in pre-operative biopsies using pan-cytokeratin stained tissues and a quantitative scoring system. Method: 139 pre-operative biopsies from patients with colorectal cancer underwent immunohistochemistry for pancytokeratin (AE1/AE3). ITB were counted in the area of densest budding (40×) and classified as high-grade when >10 buds/HPF were observed based on receiver operating characteristic (ROC) curve analysis. Results: High-grade ITB occurred in 26.6 % of cases and was associated with right-sided tumor location (p= 0.0356), more advanced pT (p =0.0198) and pN (p< 0.0001) classifications, distant metastasis (p = 0.0164), higher tumor grade (p=0.0037) and lymphatic invasion (p=0.0445). The specificity and positive predictive value for lymph node metastasis was 86.7 % and 75.6 %, respectively. Disease-free survival was significantly worse in patients with high-grade ITB (5-year survival=25 %) in comparison to patients with low-grade ITB (5-year survival= 55 %) (p=0.0157). Conclusion: The assessment of ITB in pre-operative biopsies is predictive of local and distant metastasis in corresponding resections and should be considered in daily management of colorectal cancer patients.
OFP-04-005 Alpha-methylacyl-CoA (AMACR) in gastric cancer -Correlation with clinicopathological data and disease free survival A. Mroz * , M. Kiedrowski, Z. Lewandowski * CMKP, Gastroenterology and Hepatology, Warsaw, Poland
Objective: Diagnostic and prognostic significance of alphamethylacyl-CoA (AMACR) has been established in many human cancers. Its correlation with clinical and pathological data in gastric cancer has not been fully elucidated and its impact on surveillance has not been studied thus far. Method: We analyzed consecutive gastric cancer cases in terms of AMACR immunohistochemical expression and clinical/pathological characteristics and followed patients' postoperative history. Results: AMACR was expressed in 94/164 gastric cancers (57.3 %). We did not find correlation between AMACR expression and gender, age, location, histological type, pTN staging, vascular and nerve sheaths invasion. Overall disease-free survival tended to be worse in AMACR positive patients (p=0.062), and in adenocarcinoma subgroup it was significantly shorter (p=0.021). Conclusion: AMACR expression might represent promising adverse prognostic factor in gastric cancer, particularly in adenocarcinoma histological type.
OFP-04-006 Development of a rabbit monoclonal antibody for determining hENT1 status and predicting response to gemcitabine in pancreatic ductal adenocarcinoma M. Raponi * , J. Isaacson, J. Ranger-Moore, J. Clements, B. Richardson, S. Ormanns, K. Winter, A. Allen, R. Andersson, V. Heinemann, A. P. Dicker * Clovis Oncology, San Francisco, CA, USA Objective: Human equilibrative nucleoside transporter 1 (hENT1) is the primary membrane channel through which gemcitabine (Gem) enters pancreatic tumor cells, and patients whose tumors have low expression of hENT1 may derive little benefit from Gem therapy. Method: An analytically validated rabbit monoclonal antibody immunohistochemistry (IHC) in vitro diagnostic (IVD) for assessing the degree of hENT1 expression in pancreatic adenocarcinoma using an automated IHC platform has been developed. Results: Using primary tumor samples (n=201) from a randomized controlled clinical adjuvant trial (RTOG 97-04), we developed and verified a hENT1 scoring methodology and cut-off for identifying patients least likely to benefit from Gem (hENT1-low). The distribution of hENT1 expression was similar between these samples and an independent set of 130 pancreatic adenocarcinoma specimens from the AIO-PK0104 study of which 77 were confirmed metastatic biopsies. Overall, using this cut-off, approximately two thirds of pancreatic tumors displayed low-hENT1 expression across the different data sets. Conclusion: A robust hENT1 IHC IVD has been developed using a rabbit monoclonal antibody and identifies two thirds of primary and metastatic PDAC cases as having low expression of hENT1.
OFP-04-007 Hypoxia activates pancreatic stellate cells: Development of an organotypic culture model of thick slices of normal human pancreas V. Rebours * , M. Albuquerque, P. Ruszniewski, P. Lévy, A. Sauvanet, V. Paradis, P. Bedossa, A. Couvelard * Beaujon Hospital, Pancreatology Unit, Clichy, France
Objective: Pancreatic stellate cells (PSC) promote oncogenesis by modulating cell proliferation. Aim-To evaluate the early activation of PSC in case of hypoxia in normal human pancreas. Method: An organotypic culture model of thick sections of human pancreas has been developed and validated. Slices of pancreas (300 μm) were prepared from surgical specimens and cultured in hyperoxia conditions. Half of the samples underwent an initial phase of culture in normoxia (21 % O2) to reproduce hypoxia. The total duration of culture was 72 h. Cell viability, hypoxia, apoptosis and activation were monitored. Results: 30 sections per specimen were cultured. Analysis was performed at baseline, 24 h, 48 h and 72 h. Morphological analysis showed gradual appearance of ductal/acinar dedifferentiation. At 72 h, foci of necrosis were identified. Hypoxia was confirmed by the expression of HIF1 and CA9 at 48 h (10 % and 50 % of labeled cells). Apoptosis was limited, acinar cells expressed caspase 3 at 48 and 72 h. Analysis of proliferation using Ki67 index showed significant activation of PSC at 48 h (×5/baseline) and at 72 h (×6/ baseline). Activation of PSC was confirmed by smooth muscle actin immunochemistry. Conclusion: Organotypic culture of normal human pancreas is possible with optimized cell viability at 72 h. Hypoxiainduced activation of PSC occurs very early. Objective: IPN are still poorly characterized precursor lesions of bile duct adenocarcinoma. We present a multicenter study with focus on morphology, subtyping and prognosis and provide a detailed molecular analysis in the stepwise progression from low-grade to high-grade lesions and invasive carcinoma. Method: Fourty-five patients with biliary IPN were included into the cohort, twenty-two patients with conventional adenocarcinoma of the bile duct served as control cohort. Subtyping was performed based on histomorphology and expression profile of mucins and cdx2 expression. Furthermore, low grade lesions (n=14), high-grade lesions (n=38) and invasive components (n=13) of IPN as well as conventional bile duct adenocarcinomas (n=22) were investigated individually for alterations of common oncogenic pathways using immunohistochemistry, real-time polymerase chainreaction and direct sequencing. Results: IPN are mainly detected at preinvasive stage (71 %), pancreato-biliary and intestinal type are the most common subtypes (36 and 29 %, respectively). Detailed molecular analysis showed that p53 overexpression, k-ras mutation, loss of heterozygosity of p16 locus and nuclear ßcatenin expression are early events and loss of smad4 and overexpression of EGFR and HER2 are later events in the tumor progression of IPN. Conclusion: Stepwise alterations of common oncogenic pathways are required for progression of biliary IPN from non-invasive precursors to invasive carcinoma.
OFP-04-009 Influence of BRAF mutations and RAC1b/RAC1 mRNA ratio or protein expression on outcome in patients with metastatic Colorectal Cancer (mCRC) treated with firstline chemotherapy M. Cuatrecasas * , V. Alonso-Espinaco, P. Jares, C. Horndler, A. Castells, J. J. Lozano, J. Maurel * Hospital Clinic, Barcelona, Spain
Objective: Metastatic colorectal cancer (mCRC) patients with BRAF mutation V600E present poor overall survival (OS). RAC1b, a RAC1 spliced variant, is overexpressed in CRC and impairs apoptosis by activation of nuclear-factor-KB. We evaluated if RAC1b was an independent prognostic factor in mCRC. Method: We examined BRAF (V600E) mutational status, RAC1b by immunohistochemistry, RAC1b/RAC1 expression ratio by mRNA RT-PCR, and mismatch repair (MRR) deficiency by microsatellite instability (MSI) analysis, in 186 mCRC patients treated in firstline therapy with FOLFOX or CAPOX from three Spanish institutions. We assessed whether these biomarkers were independently predictive of progression free survival (PFS) or OS. Results: 7 % CRC had BRAF mutations, 5 % MSI-H, 20 % high RAC1b/RAC1 ratio, and 25 % RAC1b protein overexpression. Five of 11 CRC (46 %) with BRAF mutations had high RAC1b/RAC1 ratio/protein vs 25/144 (18 %) without BRAF mutation (p=0.036). All BRAF mutated or RAC1b/RAC1 high were MSS. Low RAC1b/RAC1 ratio and BRAF WT had higher response rate (68 % vs 43 %; p= 0.035). Multivariate regression analysis identified ECOG PS as a significant variable for PFS and LDH levels, PS and high RAC1b/RAC1 ratio for OS. Conclusion: High RAC1b/RAC1 expression ratio or immunoexpression constitutes a marker of poor OS and a potential marker of acquired chemo-resistance in mCRC treated with oxaliplatin-based therapy. Objective: Annually more than 120.000 new cases of papillary thyroid cancer, that is 1 % of malignant tumors, are registered all over the world. Method: There are clinical and morphologiсal methods: immunohistochemistry (MMP-2, MMP-9, TIMP-1, TIMP-2), light, transmission, electron, probe and scanning microscopy with the trace element analysis. It was studied 1541 cases of thyroid cancer since. Results: At the papillary thyroid cancer progression matrix metalloprotease and specific tissue inhibitors of metalloprotease (TIMP-2) increased in the cytoplasm of tumor cells, but the concentration of TIMP-1 decreased. Tumors with low degree of ultrastructural differentiation were presented by cells with electron-dense dark cytoplasm and cells with poor cytoplasm organelles and differently formed nucleus with numerous deep invaginations. High level of some matrix metalloproteaseexpression (ММР-9) and low level of TIMP expression (ТIMP-1) were observed in current cases more (45 % and 58 % respectively), than at more differential papillary thyroid cancer (23 % and 37 %). At performance of atomic force microscopy it was shown that connection between cells had become weak at the decreasing of malignancy degree. It was revealed the significant increasing of oxygen, magnium, potassium, calcium in tumor nodes. Conclusion: Conducted comlex morpho-chemistry analysis display diagnostic patterns of papillary thyroid cancer. Objective: Cytological features of BTN-PH, sometimes interpreted as papillary carcinoma (PC) on fine-needle aspiration biopsies (FNAB), have not been studied extensively. Method: Available material from 48 BTN-PH was reviewed retrospectively and compared with 15 PCs. Results: FNAB diagnoses were: non-diagnostic (n=1), benign (n=18), AUS/FLUS (n=10), suspicious for follicular neoplasm (n=4), suspicious for malignancy (S-PC) (n=13), and malignant (P-PC) (n=2). The extent of papillary hyperplasia on histology was higher in S-PC/P-PC diagnostic categories (50 %; P=0.019; range: 5-90 %). On cytology, papillary structures, present in 53 % of BPN-PH cases, were more frequent and numerous in the S-PC/P-PC group (89 %; P= 0.0093). Focal nuclear atypia was seen in all S-PC/P-PC cases, including rare grooves (87.5 %), enlargement/crowding (75 %), chromatin clearing/rare pseudoinclusions (25 %), but were less than in PCs (P<0.0001). Nuclei were smaller in BTN-PH than in PCs (mean diameter/range (μm): 8.2/6-12 vs 14.2/7-26; P=0.0001). All BTN-PH tested (n=9) were immunonegative for HBME-1, galectin-3, and keratin-19. Conclusion: BTN-PH diagnosed as S-PC/P-PC often show papillary structures and focal nuclear features of PC but they are significantly less than in classical PCs, stressing the need to apply strict criteria for PC diagnosis.
OFP-05-003 Genetic alterations in glucagon cell adenomatosis T. Henopp * , M. Anlauf, S. Biskup, G. Klöppel, B. Sipos * Medizin. Universität Tübingen, Inst. für Pathologie, Germany
Objective: Glucagon cell adenomatosis (GCA) was recently recognized by us as a multifocal neoplastic disease of the endocrine pancreas unrelated to MEN1. Multiple micro-and a few macrotumors are found on the background of a hyperplasia of glucagon cells. The disease may cause unspecific abdominal symptoms and only rarely a glucagonoma syndrome. Recently a mutation in the glucagon receptor (GCGR) gene was described in one GCA patient. Method: The extracted DNA of five patients with GCA was sequenced and the GCGR gene analyzed for mutations. Results: Sequencing of the GCGR gene revealed germline mutations in three out of five patients. One patient shows two different heterozygous point mutations in the hyperplastic alpha cells as well as in the non-tumorous tissue leading to two premature stop codons. One patient harbors a homozygous stop mutation. The third patient shows two homozygous missense mutations of the GCGR gene that most likely also led to a dysfunction of the GCGR. In the two other patients no germ line mutations of the GCGR gene were detected. These variants were not identified in healthy subjects. Conclusion: The finding of germ line and somatic "loss of function" mutations of the GCGR gene in three of five patients with GCA suggests that a change in the signalling function of the GCGR may cause glucagon cell adenomatosis via glucagon cell hyperplasia.
OFP-05-004 TIMP-1 expression and hypoxia in papillary thyroid carcinoma: Relationship to BRAFV600E mutation and clinical behavior M. Ilie * , S. Lassalle, P. Brest, C. Bonnetaud, A. Bozec, N. Guevara, J. Haudebourg, I. Birtwisle-Peyrottes, J. Santini, P. Hofman * CHU de Nice, LPCE, France
Objective: BRAFV600E causes up-regulation of TIMP-1, promoting cell invasion in papillary thyroid carcinoma (PTC). HIF-1α is regulated by hypoxia but also by BRAFV600Emediated signaling pathway in PTC. We assessed the impact on clinical behavior in PTC of TIMP-1, HIF-1α and the hypoxia-inducible CAIX and CAXII. Method: The protein expression was assessed by Western blot in two cell lines, TPC-1/BRAFWT and BCPAP/ BRAFV600E. TMA-immunohistochemistry analysis was used to study protein expression in 114 PTC samples. BRAF status was analyzed by pyrosequencing. Data were correlated with clinicopathological variables of patients. Results: Higher expression of all proteins was detected in BCPAP exposed to hypoxia. TIMP-1 expression displayed 87 % sensitivity and 83 % specificity for identifying BRAF mutation (P<0.001), and was associated with pT-stage (P= 0.001), pN-stage (P= 0.02), and multifocality (P=0.03). HIF-1α expression was correlated to pT-stage (P=0.05). CAIX expression was related to pN-stage (P=0.02), and both CAIX (P=0.004) and CAXII (P=0.05) were highly associated with vascular invasion. Conclusion: TIMP-1 protein expression is reliable surrogate of BRAF mutated status in PTC. TIMP-1 and hypoxiaregulated proteins have potential for future use as predictors of the malignant change in PTC, and warrant further investigation as new therapeutic targets for the treatment of highly aggressive form of PTC. Objective: MTC are usually aggressive tumours which account for 3-5 % of all thyroid carcinomas. Most of MTC are sporadic, the remaining tumours are due to hereditary forms with germline activating mutations of the RET proto-oncogene. The aim of this study was to assess the microRNA profiling expression in both sporadic and hereditary forms of MTC. Method: A total of 40 frozen MTC from patients with welldocumented clinico-pathological parameters were used for microarray analyses (14 HMTC and 26 SMTC) using Human MicroRNA Mircoarray Kit version 2 (Agilent Technologies). After identification of a set of miRNA of interest, validation was done using qPCR. Results: When comparing HMTC harbouring germinal RET mutation with SMTC, seven miRNAwere significantly deregulated (miR-96, −10a, −376c, −15a, −7-2*, −106b, −132). The most highly discriminative miRNA in tumoral samples (>3 fold change) identify miR-375, −129-3p, −136, −376c and −451. Moreover, 5 miRNA could segregate MTC with good prognosis and better clinical outcome (miR-137, −144, −224, −144 and −224) . Conclusion: A specific microRNA signature in HMTC can be identified. Moreover, detection of microRNAs in tissue samples might be of interest for improved clinical management and treatment of patients with MTC.
OFP-05-006 Focus on the follicular variant of papillary thyroid carcinoma: Combination of the immunohistochemical markers CK19, HBME1 and TPO is associated with histopathological diagnosis, better than molecular markers Objective: The follicular variant (FVPTC) is the major variant of papillary thyroid carcinoma (PTC), ranging from 9 % to 22 % of all PTC. It remains however a problematic entity. Some consider that the encapsulated FVPTC (enFVPTC) have an excellent prognosis like follicular adenomas (FA); others showed that enFVPTCs had potential for hematogenous spread like follicular carcinoma. Its place in the spectrum the multivariate Cox regression the FLT3-ITD mutation (p= 0.001) was an independent poor prognostic factor, whereas the goodness-of-fit of the Minkowski fractal dimension an independent favorable prognosticator (p=0.025). The karyotype had no influence on survival. Conclusion: FLT3-ITD and chromatin fractal characteristics were more important as prognostic factors for overall survival than karyotype and gene methylation status. Financial Support: FAPESP and CNPq. Objective: The prevalence of Cyclin D1 (CyD1) positive Bcells with mantle cell lymphoma (MCL) phenotype in the mantle zones of reactive lymph nodes ("in situ" MCL/MCLIS). and related minimal MCL infiltrates in reactive lymphoid tissues of healthy individuals and in MCL patients are unknown. Method: All 1.292 reactive lymph nodes from unselected consecutive surgical specimens of 131 patients without a history of lymphoma obtained over a 3 months´period were stained for CyD1. Additionally, all morphologically reactive lymph nodes and extranodal lymphoid infiltrates of MCL patients from 2000 to 2011 were studied. Samples predating the lymphoma diagnosis for at least 2 months were available from 37/423 (8.7 %) patients. Results: A MCLIS was not identified. However, in four MCL patients, an early manifestation of MCL with mantle zone growth pattern was detected retrospectively, antedating the lymphoma diagnosis for 3-86 months. In six MCL patients, only small groups of CyD1 positive cells in morphologically reactive extranodal infiltrates were detected more than 2 months before the diagnosis of MCL (range 3-59 months). Conclusion: MCL "in situ" is an extremely rare phenomenon in morphologically reactive lymph nodes. In MCL patients, however, immunohistochemically detectable infiltrates of MCL cells antedating the lymphoma diagnosis were found in a significant proportion of cases (10/37=27 %).
OFP-07-003 E2F-1's relationship with tumor growth in Hodgkin's lymphoma is regulated by p53 status E. Georgiadi * , G. Dimtsas, T. P. Vassilakopoulos, V. G. Gorgoulis, I. A. Doussis-Anagnostopoulou * Larisa, Greece
Objective: E2F-1 is a member of the E2F family group of transcriptional factors that play a major role in cell cycle progression and arrest, as well as in apoptosis. In different tumors E2F1 can demonstrate opposing roles, acting either as an oncogene or as a tumor suppressor gene. Method: We have previously established E2F-1's immunohistochemical expression in Hodgkin lymphoma (HL) and its correlation with p53 expression, although no relationship with either proliferation or apoptosis was demonstrated. A further investigation in a larger series of 100 cases of primary HL with the addition of new apoptotic techniques and the use of p21 expression as an indicator of p53 functional status was undertaken. Results: Following stratification of our cases based on p53 functionality, E2F-1 was inversely correlated with the proliferation marker TopoIIa. Independently of p53, high levels of E2F-1 expression showed a linear trend with apoptosis and an inverse trend with proliferation (borderline p value in both cases). Conclusion: Both E2F-1 and p53 activity is upregulated in HL, however it seems that p53 status is a major regulator of the relationship between E2F-1 expression and tumor growth. Alternatively, E2F-1 may use p53-independent pathways to induce apoptosis. Objective: PTLD represent a spectrum of usually EBV-driven lymphoplasmacytic proliferations in the settings of immunodeficiency associated with allograft. EBV infects cells in latent or lytic forms. Although viral oncogenes are expressed in latency programs, lytic EBV replication is required to develop lymphomas in mouse models. The role of intratumoral EBV replication in PTLD has never been addressed before. Method: A series of 35 PTLD was reviewed and EBV latency genes were explored in all cases and included EBER1-2, LMP1 and EBNA2. Moreover, two early lytic genes involved in EBV replication, BZLF1/ZEBRA and EAD11 were also analyzed. All clinical and pathological data were collected. Results: The median age was 50 years (27-77) with a male predominance (26 M: 9 F). EBV infection was observed in 28 cases (80 %). EBV replication was observed in 40 % of the cases. These were more likely to be polymorphic PTLD with latency III in the context of stem cell transplantation. Moreover these cases had atypical clinical presentations such as brain involvement or disseminated disease with aggressive behavior (OS: 11 months vs. 48 months). Conclusion: EBV replication occurs in tumor cells of PTLD-patients associated with poor outcome and may be share common morphological and phenotypical features. In routine praxis, their differential diagnosis might represent a challenge requiring a specific work-up of immunohistochemical (IHC) and fluorescent in situ hybridization (FISH) analyses. Method: Paraffin sections from 17 CD20+/CD5+ blastic B-NHL were examined for cyclin-D1, CD10, bcl-6 and MUM-1 IHC expression together with FISH method using probes for CCND1 rearrangement and/or IGH/CCND1 translocation. The final diagnosis was established according to the criteria of WHO classification. Results: 11/17 cases were diagnosed as bMCL showing cyclin-D1 positivity and CCND1 rearrangement, other analysed IHC markers were negative. Five of these cases were polyploid. 6/17 cases were diagnosed as CD5+ DLBCL in spite of coexpression of cyclin-D1 in 2 of them, as they showed various CD10, bcl-6 and MUM-1 expressions and missed CCND1 gene rearrangement. Conclusion: Cyclin-D1 positivity may appear in other than MCL lymphomas due to changes at transcriptional or posttranscriptional level. In spite of overlaping phenotypes, FISH analysis of CCND1 represents an effective tool to increase the diagnostic accuracy to distinguish CD5+ DLBCL from bMCL. Supported by VEGA grant Nr. 1/ 0378/12 and projects CEPRII (IMTS: 26220120036) and MDCC (IMTS: 26220220113) co-financed by EU sources.
OFP-07-008 Still's Disease associated lymphadenopathy: Report of two cases resembling malignant lymphoma with review of possible morphological findings K. Kamarádová * , M. Nova, M. Kalinova, V. Campr * Prague, Czech Republic Objective: Still's disease (or systemic onset juvenile idiopathic arthritis, SOJIA) is a systemic inflammatory disorder characterized by fever, rash, and arthritis. It has a peak of incidence in early childhood with rare occurence in adulthood (adult-onset Still's disease, AODS). Several other symptoms as hepatosplenomegaly, leukocytosis and lymphadenopathy may be associated possibly mimicking malignant lymphoma. Method: We present two cases of Still's disease associated lymphadenopathy in a child and in an adult simulating morphologically peripheral T-cell lymphoma and classical Hodgkin lymphoma respectively. Results: Histological, immunohistochemical and molecular examination revealed non-neoplastic lymphadenopathy with atypical paracortical T-cell hyperplasia with immunoblastic reaction in the former and burnt-out histiocytic pattern in the latter, both falling into a broad spectrum of reactive lymph node changes associated with Still's disease.
The lymph node lesions in Still's disease can be histologically divided into four major categories: 1) simple atypical paracortical hyperplasia, 2) burnt-out histiocytic pattern, 3) exuberant immunoblastic reaction in paracortical hyperplasia and 4) follicular hyperplasia. The second and the third pattern may closely resemble malignant lymphoma. Extended and careful clinical examination with relevant laboratory findings and possible complex histochemical and molecular analysis are necesarry to rule out the suspition of lymphoma. Objective: Intravascular large B-cell lymphoma (IVLBCL) is a rare form of diffuse large B-cell lymphoma characterized by exclusive or preferential intravascular growth. IVLBCL often goes clinically unrecognized. We report three clinically unsuspected cases of classic variant IVLBCL. Method: Cases diagnosed as IVLBCL were collected from the pathology files of Germans Trias i Pujol Hospital. The diagnosis was reviewed in agreement with WHO 2008 criteria and immunophenotypic and clinical data were gathered. Results: A 71-year-old male and an 87 year-old-male showed neurological symptoms leading to progressive deterioration and death. A 79-year-old female presented with fever of unknown origin and died 2 days after admission. At autopsy there were no gross findings suggestive of a neoplastic process in any instance. Histologic study showed intravascular large B cells with vesicular nuclei involving predominantly the central nervous system in the first two cases and the adrenal glands, liver, and spleen in the third case. Conclusion: The three cases of aggressive, advanced-stage lymphoma herein described illustrate how the clinical diagnosis of IVLBCL remains elusive due to its low frequency and variegated clinical presentation and how the pathologic diagnosis of IVLBCL continues to be highly dependent on autopsy examination.
Objective: The World Health Organisation (WHO) currently recognises four categories of PTLD, which are lymphoid/ plasmacytic proliferations occurring due to immunosuppression after organ transplantation. The aim of this study was to examine the clinicopathologic characteristics of PTLD occurring after renal transplantation in the Republic of Ireland. Method: The National Renal Transplant Registry from 1991 to 2000 (inclusive) was cross referenced with the National Cancer Registry to identify renal transplant recipients who had developed PTLD up to the present day. Review of Pathology and chart review was conducted for each case. Results: Of 2,441 patients (2,667 allografts), 34 cases of PTLD were identified, with pathology review possible in 33 cases. There were 26 (79 %) Monomorphic PTLD (23 Diffuse Large B Cell {DLBCL}, 1 Anaplastic Large Cell, 1 Burkitts-like, 1 Myeloma], 3 Hodgkin-type PTLD, 1 (3 %) Polymorphic PTLD and 1 (3 %) early lesion. In addition, there was 1 Mantle cell lymphoma and 1 Lymphomatoid Granulomatosis. The annual incidence of PTLD was estimated as 0.2 % (95 % CI 0.14-0.28). Median time to diagnosis was 8.7 years (Range 2 to 19.2 years). Conclusion: Similar to other studies, Monomorphic PTLD is the most common type, with DLBCL representing the majority of cases. Of interest, early onset PTLD was very uncommon in this study.
Monday, 10 September 2012, 17.00-19 .00, Forum Hall OFP-08 Oral Free Paper Session Head and Neck Pathology OFP-08-001 Human papilloma virus associated oropharyngeal carcinoma is strongly correlated with cancer of unknown primary S. Ihrler * , G. Assmann, P. Zengel, M. Mollenhauer * Medizin. Universität München, Institut für Pathologie, Germany
Objective: Human papillomavirus (HPV) associated oropharyngeal carcinoma has been identified as a distinct entity within squamous cell carcinoma of the head and neck and is preponderantly restricted to palatinal tonsils and base of tongue. These two primary locations have for long -and HPV-associated carcinomas have recently -been associated with the clinical situation of cancer of unknown primary (CUP). Method: To investigate the relationship between HPV and CUP in detail, we studied 26 consecutive patients who initially presented as CUP and were finally diagnosed with carcinomas of these two locations. Results: Twenty-one carcinomas (81 %) proved to be positive for high-risk HPV (p16 and polymerase chain reaction). They were frequently very small (smallest: 0.3 cm; 6 cases: ≤ 0.6 cm; on average 0.9 cm) and located in a deep, submucosal position. Conclusion: This demonstrates an overrepresentation of HPV-associated carcinoma in patients who were initially diagnosed with CUP, supporting a strong causal relationship between HPV-association and CUP. The frequent manifestation as CUP presumably is caused by the unusual predisposition for small size and submucosal location, combined with frequent and early lymphatic metastization. In order not to miss these small, clinically occult carcinomas, consequent interdisciplinary cooperation and meticulous histological work-up is mandatory.
OFP-08-002 Methylthioadenosine phosphorylase inactivation depends on gene deletion in laryngeal squamous cell carcinoma A. Nadal * , L. Conde, I. Vilaseca, L. Alos, M. Bernal-Sprekelsen, A. Cardesa * Hospital Clínic Barcelona, Dept. of Pathology, Spain
Objective: Methylthioadenosine phosphorylase is an essential enzyme for the methionine and adenosine salvage pathway in normal cells, frequently inactivated in many different human cancers. The MTAP status could be important for tumor cell sensitivity to adjuvant chemotherapy. To date there are no reports on MTAP status in laryngeal carcinoma. Method: A series of 31 laryngeal squamous cell carcinomas was investigated for MTAP mRNA expression through reverse transcription and quantitative PCR (qPCR) as well as for MTAP gene deletion and/or promoter hypermethylation through qPCR and methylation-specific PCR respectively. Results: Low MTAP mRNA expression was found in 32 % of cases, associated with MTAP gene deletion in 70 % (p< 0.001) but not to MTAP promoter hypermethylation, indicating that in this tumors gene deletion is the main mechanism of MTAP inactivation. Neither low mRNA expression nor gene deletion was associated with any of the clinicopathologic parameters investigated. Conclusion: Given the significance of MTAP status for cell sensitivity to different chemotherapeutic regimes, our results suggest that determination of MTAP inactivation could be useful for adjuvant therapy selection in laryngeal squamous cell carcinomas.
OFP-08-003 ERCC1, p16 and ki-67 immunohistochemical expression as predictive and prognostic markers in head and neck squamous cell carcinoma treated with platin-based induction chemotherapy H. Roussel * , M. Housset, H. Tournat, P. Ravel, S. Hans, C. Badoual * HEGP, Dept. de Anatomie-Pathologie, Paris, France Objective: ECC1 enzyme has been associated with resistance to platinum-based chemotherapy. The purpose of this study was to evaluate the role of ERCC1 expression with p16 and Ki-67 as predictive and prognostic markers in the response to platin-based induction chemotherapy in patients with head and neck carcinoma (HNSCC). Method: 208 patients treated from 2000 to 2006 by an induction chemotherapy regimen for HNSCC were included retrospectively. We assessed response to treatment, progression-free survival (PFS) and overall survival (OS). Results: 68 % and 81.5 % of HNSCC showed low expression of 8 F1 and FL297 ERCC1. No correlation was found between the two clones (p=0.1). In the 129 patients treated with cisplatin-5FU chemotherapy, a low expression of 8 F1 ERCC1 was associated with a better response (p=0.027). Over expression of p16, (34.5 % of cancers of the oropharynx) was correlated with a better OS (p=0.0007) and a better PFS (p=0.01). Conclusion: These results suggest that ERCC1 expression might be a useful predictive marker of HNSCC in patients treated by cisplatin-based chemotherapy. The 8 F1 ERCC1 clone appears to be the best for immunohistochemistry. Our study confirms the prognostic value of the over expression of p16 in carcinoma of the oropharynx. Objective: Tumor-stroma ratio or proportion of tumor (PT) has been presented as a prognostic factor in colorectal and breast adenocarcinomas, but there is no information about squamous cell carcinomas (SCC) and laryngeal carcinomas in particular. Method: Eighty-five laryngeal carcinoma cases were included in this series. Five digital images of the tumor sections were obtained (H&Ex20). Percentage of epithelial tumor component was determined by software allowing the pathologist's selection of tiny areas as carcinomatous and stromal region for statistical analysis as a prognostic marker. Results: Median follow up was 48 months (range 3-194). The mean PT was (48,63+18,18) . There was no difference for PT when tumor grade and stage were considered. Although statistically insignificant, the mean PT was the lowest (37,46+12,49) for subglottic carcinomas and cases with perinodal invasion (44,72+20,23), and highest in pN0 cases (50,05+17,34) but there was no statistical significance. The over all and disease free survival analysis did not reveal significance for PT. Only pathological stage was an independent factor for over all survival (p=0,08).
Conclusion: Although there might be an association with adverse prognostic factors and low TP, the findings in this series does not support PT as a prognostic marker in laryngeal carcinomas.
OFP-08-005 Tumoral microvasculature in ameloblastoma subtypes C. H. Siar * , K. H. Ng * University of Malaya, Dept of. Oral Pathology, Kuala Lumpur, Malaysia
Objective: Angiogenesis is essential for tumoral growth and progression. The ameloblastoma is a benign but locallyinvasive odontogenic neoplasm with distinct behavioural characteristics of its subsets. Whether angiogenesis contributes to a more aggressive course in these variants remains unclear. The aim here was to determine and compare the tumoral microvasculature in different ameloblastoma subtypes and to speculate on their significance. Method: Immunohistochemical staining for 4 vascular markers (CD31, CD34, CD105 and VEGF) was performed on archival tissues of 40 cases solid/multicystic (SMA), 20 unicystic (UA), 3 desmoplastic (DA) and 14 recurrent ameloblastoma (RA). Mean microvessel density (MVD) at the tumour advancing front and centres were obtained. Results: VEGF was heterogeneously expressed in the tumoral epithelium in all ameloblastoma subtypes. Protein localization was membranous/cytoplasmic. Mean MVD was slightly higher in SMA compared to UA but the difference was not significant (P>0.05). Mean MVDs were not significantly different between primary and recurrent ameloblastoma; and between tumoral centre and advancing front of each subtype (P>0.05). Conclusion: Although ameloblastoma subtypes are distinctive in their clinicopathologic presentations and behaviour, their tumoral microvasculature is not different. This suggests that angiogenesis is not a major factor influencing the progression of these ameloblastoma subsets.
OFP-08-006 An immunohistochemical study of E-Cadherin and snail expression in laryngeal squamous cell carcinoma A. Tanoglidi * , M. Tsopanomichalou, C. Barbatis, C. Kostopoulos, O. Pantzartzi, V. Gorgoulis * Hellenic Red Cross Hospital, Dept. of Pathology, Athens, Greece
Objective: Snail 1 and 2 (Slug) are zinc-finger transcription factors, repressing the E-Cadherin in epithelial tumors. Published data in Head and Neck Squamous Cell Carcinoma show either inverse relationship or unrelated co-expression, but E-Cadherin reduction/loss and Snail overexpression seem to confer an aggressive phenotype.
Method: Thirty cases of laryngeal squamous cell carcinomas (LSCCs) (gradeI: 11, gradeII: 8, gradeIII: 11) and one large cell neuroendocrine carcinoma (NEC) were studied using double staining immunohistochemical method for E-Cadherin (mAb-DAB) and SNAIL + SLUG (pAb-AP). The colocalization of both molecules was assessed and the expression was compared to the adjacent normal or dysplastic epithelium and the histological grade. Results: Normal squamous and dysplastic epithelium show strong nuclear Snail expression and complete E-Cadherin membranous positivity. Reduction/loss of Snail with or without E-Cadherin membranous expression is observed in 14/30 (47 %) LSCCs with a tendency for inverse relationship with E-Cadherin at the invasive front. 16/30 (53 %) LSCCs strongly express nuclear SNAIL, without absolute relationship to the reduction/loss of E-Cadherin, with a tendency for SNAIL negativity in grade II (75 %). The NEC is nuclear SNAIL negative but cytoplasmic positive. Conclusion: SNAIL and E-Cadherin may be retained, reduced or lost in LSCCs, without a definite inverse relationship. These immunophenotypic combinations need further investigation.
OFP-08-007 Non-sebaceous lymphadenoma of salivary glands: Proposed development from intraparotid lymph nodes and risk of misdiagnosis C. Weiler * , A. Agaimy, P. Zengel, S. Ihrler * Munich, Germany
Objective: Non-sebaceous lymphadenoma (NSLA) is a recently described, rare benign salivary tumour composed of lymphoid and epithelial components, definitionally lacking sebaceous differentiation. Method: Nine cases of NSLA were immunohistochemically stained for CK5/6, CK7, CK14, CK18, p63, and Ki67. Results: All tumours (6 males, 3 females, mean age 50 years) were located in the parotid gland and showed intimate intermingling of lymphoid tissue with islands of epithelium with a wide spectrum of histological differentiation. The immunohistochemical profiles mirrored the epithelial differentiation; tumours with basaloid or lymphoepithelial differentiation strongly expressed CK5/6, CK14, p63, while tumours with ductal differentiation showed strong positivity for CK18/CK7 and CK5/6/CK14/p63 in luminal and basal cell layers, respectively. A hilus structure with salivary inclusions or D2-40 (podoplanin) positive marginal sinus were identifiable in 4 and 9 of the cases, respectively, confirming origin within intra-/periparotid lymph nodes. Six cases were initially misdiagnosed as other benign (n=4) or malignant (n=2) tumours. Conclusion: Our study provides strong evidence that NSLA belongs to the group of salivary tumours that pathogenetically develop from embryonic salivary inclusions in intra-/periparotid lymph nodes. Knowledge of the wide histological spectrum of this rare tumour is important in order to avoid misdiagnosis.
OFP-08-009 CRTC1/MAML2 fusion transcript in central mucoepidermoid carcinoma of mandible: Diagnostic/ Histogenetic implications D. Bell * * MD Anderson Cancer Center, Dept. of Pathology, Houston, TX, USA Objective: MEC typically arises from major/minor salivary glands. Intraosseous salivary carcinomas are extremely rare (2-3 % of all MECs reported). The t(11; 19) and its CRTC1/ MAML1 fusion transcript have been identified in MEC at different sites and are associated with development of a subset of these tumors. We report 9 examples of central MEC of the mandible, including a case with a history of primary retromolar MEC. Method: RT-PCR and DNA sequencing analyses used to study microdissected components of 9 central MEC and 1 noncentral MEC. Of the central MEC tumors, 5 arose from ectopic salivary rests; others appeared to be of glandular odontogenic origins. Results: We identified CRTC1/MAML1 in 5 central MEC arising from ectopic salivary rests. This fusion transcript was not detected in non-central MEC or in another 4 central MEC arising from a glandular odontogenic etiology. Conclusion: Central MEC can manifest the fusion transcript in a subset of central MEC originating from ectopic salivary rests, and may have diagnostic/histogenetic roles in the future analysis of this entity, given the absence of the fusion transcript in MEC with glandular odontogenic precursors. Since the initial clinical and radiological diagnosis in three central low-grade MECs was a benign odontogenic cyst, our findings support a future role for the fusion analysis in initial diagnostic efforts.
OFP-08-010 Salivary duct carcinoma: Morphological and immunohistochemical study of 15 cases M. Vazmitel * , A. Dubrovskij, R. Smoljakova, S. Rjabceva * Minsk, Belarus Objective: An evaluation of morphological and imunohistochemical features of salivary duct carcinoma (SDC). Method: 15 cases SDC were retrieved from tumor archives collected at Belarusian Cancer Centre from 2002 to 2011 years. Immunohistochemistry, FISH. Results: A man to women ratio was 10:5; median age 61,5 years (average . Tumors arose in the parotid (n=13), submandibular (n=2) glands. SDC developed from pleomorphic adenoma (n=3) or de novo (n=12), there were foci of ductal carcinoma in situ (n=4), sialodochodysplasia (n=4) and Pagetoid spread (n=1) additionally to usual features of SDC. As well, tumors demonstrated acinic cell carcinoma-like (n = 2) and micropapillary growth patterns (n=1), oncocytic (n=4), clear cells (n=3), apocrine (n=1) and mucinous (n=1) changes. Immunohistochemically, tumors were characterized by AR-expression (n= 9), Her2/neu overexpression (n=7), of them score 2+ in 2 cases. CK8 and S-100 positivity, STAT5 negativity was detected in one case with acinic cell carcinoma-like pattern. Foci of SDC in situ were highlighted by CK14 and calponin. Amplification of c-erb2-gene was found in 1 case, ETV6/NTRK3 gene was not identified in acinic cell carcinoma-like SDC. Conclusion: Salivary duct carcinoma, like breast cancer, is characterized by wide variability of morphological patterns and immunohistochemical features reflecting possible molecular heterogeneity, which need further investigation. Sunday, 9 September 2012, 17.00-19. Objective: Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. However, routinely used eyeballing scoring systems are time-consuming and subject to significant intra-and interobserver variability. Aim of our study was to explore, whether an image analysis software proves sufficient as an alternative tool to assess protein expression. Method: 630 prostate cancer specimens were stained with one nucleus specific marker (i.e., ERG), one cytoplasmic specific marker (i.e., SLC45A3), and one marker expressed in both compartments (i.e., TMPRSS2). A pathologist visually quantified all stainings, applying a four-step scoring system. For digital quantification, an image analysis software (Tissue Studio v.2.1) obtained a continuous spectrum of staining intensity. Results: For each of the three antibodies we found a strong correlation of the eyeballing protein expression score and the score of the image analysis software (correlation coefficient of 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively, p<0.01). Conclusion: Our data suggest that Tissue Studio is a powerful tool for the quantification of protein expression in IHC stainings. Further, since the digital analysis is precise, it might help to overcome intra-and interobserver variability and increase objectivity of IHC based protein assessment.
OFP-09-002 Is quality of histological and cytological slides a palatable issue?: The TASTE project M. Comanescu * , G. Bussolati, A. Sapino, G. Butur, F. Schmitt, F. Feoli, T. Tot, E. Ovcin * Institutul Victor Babes, Dept. of Pathology, Bucharest, Romania
Objective: The TASTE (technological platform and repository of high quality images and slides) project aims at developing a novel tele-pathology system by means of modern ICTs (Information and Communication Technology) and is addressed to technicians and doctors. Method: Histological and cytological preparations are the basis for pathological diagnosis performed in daily practice throughout Europe in a number of several millions per year and correctness and reproducibility of such diagnosis are heavily dependent on the technical quality. Yet, quality is variable in different places and countries, related to school level, technicians' dedication, standard of apparatuses and reagents. Variation in technical quality of the preparations prevents their open circulation at European level and precludes optimal diagnosis. Results: The TASTE project tackles these problems by building-up an ICT environment TASTE System where professionals from different countries will submit, via Web, "virtual slides" of their own preparations to a panel of internationally recognized experts who will give comments and suggestions on the quality of the preparation. Conclusion: The present approach (unprecedented at world level) will fuel a Web-based community, aimed to a levelling and improvement of histopathological and cytopathological preparations, thus leading to an innovative training and more reproducible diagnosis, a basic requisite for disease treatment.
of the whole atlases. Virtual microscope positions can be stored in the memory and users can navigate quickly back and forward similarly to web pages. All these features are available through any web browser without need to install any additional software. Results: In addition to the current atlases (Dermatopathology, Fetopathology, Pathology of the Newborn and Bone Marrow), an interdisciplinary Atlas of Pathology for pregraduate students of Medicine is under development. It will contain full size annotated virtual slides. The Atlases are free of charge, but registration is required. In order to ease the access, the Atlases are connected to 15 national academic identity federations. The members of institutions connected to identity federations can use theirs home credentials without revealing them to the Atlases. Conclusion: The atlases can serve not only to the students as a source of information, but they can be used by teachers as a source of teaching material.
Proving experiment of international virtual slide telepathology, Japan-Vietnam and Japan-China trial I. Mori * , S. Aida, Y. Osamura * International University of Health, Dept. of Pathology, Tokyo, Japan
Objective: The communications between Asian countries are growing. In the field of medical information and technologies, the necessity of communication is also required. In this work, as communication of pathology field, we tried to prove that international remote pathology diagnosis is possible using virtual microscopy (VS) telepathology. Method: We put VS scanner and server to 1) our Hospital, Tokyo, Japan, 2) Cho-Rai Hospital, Ho-Chi-Minh city, Vietnam, and 3) Chinese Rehabilitation Research Center, Beijing, China. Pathologists go and look the VS through leased optical line circuit and make diagnosis. Discussion was made through teleconference system. Results: The VS image maintained adequate quality for pathology diagnosis even looked cross-border. The diagnoses matched well between hospitals. The response of VS viewer was also good this time. When we use too much band width to the Teleconference system or we sent too many data in the background, the response deteriorate, and was not good enough for diagnosis. Conclusion: It was confirmed that the international VS telepathology diagnosis is now reached to a stage of actual use at least when connected by leased optical line circuit.
Challenges and solutions in the setup of a findings database for a large scale tissue collection R. Reihs * , H. Müller, S. Sauer, K. Zatloukal * Medizin. Universität Graz, Inst. für Pathologie, Austria Objective: Many medical centres have acquired through the last years huge data collections of great relevance for biomedical research. In order to utilize this knowledge it is necessary to analyse this records in a structured way. In our use case the starting point were approximately 1.4 million pathological findings of a non-selected patient group. Method: We developed software tools for the classification of medical records using a phonetic a multi-level spell correction module and an ontology term extraction and decision tree text classification system. With the help of a visual editor a comprehensive decision tree (4174 nodes) was set-up by a team of bioinformaticians and medical experts. Results: With our tool set we achieved in the ICD-10 classification a F-Score of 89,7 % (precession 83,2 % and recall 97,5 %) For the interpretation of the classification results we developed visualization tools and applied the whole software suite at several test cases, e.g. the analysis of a colon data-set with 216.000 findings over 28 years. Conclusion: We developed an automatic classification and visualization system and applied the results in a series of research projects. The software suite is currently used in several research projects and can be easily adjusted to specific phrases used in different institutions and languages.
An image repository for decision support O. Eichhorn * * Aperio, Vista, USA Objective: Pathology is a visual field, and pathologists make decisions based on visual information. An online repository of easily searched, well-categorized pathology images can be of immense use to pathologists, scientists, educators, and students. Method: Individuals and institutions all over the world have accumulated collections with thousands of slides representing a huge variety of pathologies. Some collections are digitized and available online, while others remain libraries of glass slides, with a variety of formats for case information and other metadata. Working with collectors to publish their slides and aggregating them together can yield an amazing decision support resource. Results: Long-term archival storage is a key issue for organizations adopting ePathology. A cloud-based repository can provide an inexpensive and scalable solution. ePathology is increasingly used for remote consultations. Cases managed by online consultation networks can be de-identified and contributed to a common repository, increasing the value of the decision support tool while providing a long-term archive. Conclusion: This talk will present concepts for an Image Repository for Decision Support, and discuss technical and business considerations for making it a reality, including ways to curate image metadata, organizational principles and search tools, relationships with users and contributors, access methods, and security and privacy considerations.
OFP-09-007 Perfect diagnostic accuracy using pathomorphological diagnosis spectra constructed with the help of whole slide imaging K. Furuya * , T. Maeda, K. Kito * Ehime Prefectural Central Hospital, Dept. of Pathology, Matsuyama, Japan
Objective: Pathomorphological diagnosis (PD) is important for estimation of clinical trials and new medical equipment such as elastography. However, interpretation of pathomorphological findings is often subjective, and pathology publications show only a limited number of photographs. Because pathomorphological changes occur in succession, we attempted to construct PD spectra to improve PD accuracy. Method: PD spectra are defined as the series of pathomorphological images arranged in the order of disease progression or cell cycle procession. The images from the whole slide imaging (WSI) server were arranged in order in Power Point files at a fixed magnification, and 4 PD spectra were constructed: chronic hepatitis staging, nuclear grading of hepatocellular carcinoma, nuclear grading of breast cancer, and Ki-67 labelling index (LI). The numbering of all images in the PD spectra corresponded with that of the WSI images so that the whole slides could be viewed alongside. Next, these tools were tried out by 6 people. Results: These tools brought on perfect accuracy with regard to staging and grading, and very high precision of Ki-67 LI for breast cancer. Conclusion: PD spectra are valuable not only for PD but also as learning tools and have a worldwide applicability for measuring PD online.
OFP-09-008 Color correction of immunohistochemistry stained tissue section images by histogram transfer according to control tissues S. Sarioglu * , S. Seyrek, M. Sakar * Dokuz Eylul University, Faculty of Medicine, Izmir, Turkey Objective: For semiquantitative and quantitative analysis, previous normalization attempts of images of tissue sections are not satisfactory. In this study we evaluated, if color correction can be achieved by histogram transfer depending upon control tissue image (CTI) differences. Method: Images from colon and placenta sections stained by anti-CD34 were used as CTI and/or sample tissue images (STI). Thirty-four but one (standard stained slide with images sCTI and sSTI), was stained for different durations and dilutions than the standard procedures. Digital images taken by a CCD camera connected to a light microscope without normalization was stored at a computer. Software was prepared in order to find the histogram difference between two CTIs and transfer the difference to the STI for achieving a corrected STI (corSTI). sSTI (one image) and STI and corSTI (34 images each) were semiquantitatively scored by two observers in blind fashion and the STI and corSTI scores were compared with sSTI score. Results: The wKappa was 0,59 for two observers. The STI semiquantitative score was same as the sSTI in 23,5 % of the STI but this was 76,35 % for corSTI. Conclusion: It seems histogram transfer depending upon CTIs may be a valuable tool for color correction of tissue section images. Sunday, 9 September 2012, 14.15 -16.15 Objective: There is a crucial need for identification of markers to choose the best treatment schedule and monitor the effectiveness of treatment in metastatic melanoma (MM) . The detection of circulating tumor cells (CTC) correlates with prognosis in different cancer subtypes. CTC can be detected by direct and indirect methods such as cytopathological approach, RT-PCR or immunomagnetic separation using the CellSearch (CS) method. Method: From June 2011 to December 2011, blood was drawn from 27 patients with MM and 10 controls patients and processed into 72 h by the CS method. The primary end-point was the overall survival (OS) of MM patients. Results: Enumeration of at least 1 CMC was effective in 61.5 % of patients with MM (n=16) and in 20 % of controls patients (n=2). Nine patients (35 %) had ≥ 2CMC. None of the controls patients had more than one CMC. Mean OS time was 5.6 months among patients with MM and <2 CMC (n=8) and mean OS time was 1.7 months among patients with MM and CMC ≥2 (n=26) (p=0.002). Conclusion: Our results demonstrate that detection of CMC correlates with OS in melanoma. Thus, CMC monitoring should be developed to ensure the best treatment follow-up for patients with MM.
OFP-10-002 Significance of Circulating Tumor Cells (CTCs) detection using the CellSearch system in patients with locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) A. Bozec * , M. Ilie, E. Long, O. Dassonville, G. Poissonnet, J. Santini, E. Chamorey, F. Peyrade, K. Benezery, A. Sudaka, E. Selva, P. Hofman * Centre Antoine Lacassagne, Dept. of Surgery, Nice, France
Objective: The significance of CTCs in patients with HNSCC is debated currently. We evaluated the potential detection of CTCs using the CellSearch (CS) Assay TM (Veridex, NJ, USA) in HNSCC patients and to identify the clinical factors predictive of the presence of CTCs in this population. Method: Forty-nine patients with locally advanced HNSCC were included. The presence and number of CTCs were determined using the CS system in all patients prior to the initiation of therapy, in 21 patients 3 months after treatment, and in 10 healthy individuals. Results: The CS system was able to detect the presence of CTCs in 8 of 49 patients (16 %) before therapy and in 4 of 21 disease-free patients (19 %) after therapy. No CTC was found in the control group. When considering the presence of CTCs before or after therapy, the presence of CTCs was statistically associated with patients' age (p=0.04; t-test) and N-stage (p=0.02; Fisher's exact test). Conclusion: CTCs are identified in a relatively low proportion of patients with locally advanced HNSCC and correlated with initial lymph node involvement. CTC detection could be a future useful prognostic tool to adapt the treatment intensity in HNSCC patients.
The diagnostic utility in identifications of an aneuploid chromosomal (Ch.) pattern by using Urovysion™: A Fluorescence in Situ Hybridization (FISH) commercial test, to improve the efficacy of cytology in peritoneal effusions L. Baron * , M. Postiglione, C. Trombetta, F. Quarto * P.O.S. Leonardo, S.O.C. di Anatomia Patologica, Castellammare di Stabia, Italy
Objective: The challenge in diagnosis of effusion is in differentiation of reactive and neoplastic mesothelial or metastatic cells and identification of primary neoplasia. The difficulties in this differential diagnosis derived from the low sensitivity (~50 %) and specificity of effusion cytology. By identification of tumor associated aneuploidy FISH could enhance the cytodiagnostic yield in effusions. Method: We used Urovysion™ (Abbott), a FISH commercial kit, designed for identifications of Ch. 3, 7, 17 polisomy and 9p21 loss in urothelial tumors. It has been applied to detect chromosomal aneuploidies as a marker of malignancy in 64 effusions with or without known source primary tumors. Also was evaluated reproducibility of molecular alterations with primary tumors and any their specific aneuploidies. Results: FISH positivity was observed in 44 (69 %) of cases, whereas 31 were positive at cytology (48 %). Overall results of cytology and FISH together rised to 75 %. Conclusion: Urovysion™ test could be an useful tool to distinguish malignant cells in inconclusive effusions cytology but it doesn't seem to be used to provide definitive indications about primary neoplasia.
OFP-10-004 Loss of imprinting of isulin-like growth factor 2 (IGF2) in colon carcinomas leads to the cell cycle genes activation -hints to intense proliferation D. Belharazem * , J. Kunanz, A.-K. Henne, P. Kienle, P. Ströbel * Inst. of Pathology, University of Medicine, Mannheim, Germany
Objective: The insulin-like growth factor 2 (IGF2) gene is regulated by imprinting in normal tissues. The loss of imprinting (LOI) results in the bi-allelic expression of IGF2. Its increased activity has been associated with many cancers including colorectal cancer (CRC). To investigate the significance of IGF2 in CRC, normal tissue and tumor biopsies from 400 patients were analyzed and correlated with clinical outcome and the KRAS and PIK3 status. Method: The IGF2 820 G/A gene polymorphism and imprinting status were examined by restriction fragment length polymorphism of DNA derived from normal tissues and tumors then of RNA from heterozygous cases. IGF2 protein analysis as well as an RNA microarray were performed. Results: 41 % of the analysed cases were heterozygous. LOI was detectable in both normal tissues and tumors in 60 % of cases. Tumors with LOI had significantly higher IGF2 protein levels. Gene expression analysis revealed significance of cell cycle progression and mitosis genes in LOI. Conclusion: IGF2 LOI is a common and early change in patients with CRC and in normal tissues, provides possibly a preneoplastic change. However increased IGF2 protein levels as well as a different cell cycle specific gene expression profile were detected in tumors with LOI. Future studies must be performed to find out, whether such subsets of tumors differ in their response to chemotherapy or alternative therapies. Objective: Novel high-throughput technologies has revealed numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHL). This study aimed to detect point mutations involving EZH2 and CD79B in a population of B-cell NHL by applying a multiplex mutation assay to minimal residual material from needle aspirates, and to determine the evolution of their mutational status overtime using all sorts of available samples from these patients. Method: DNA was extracted from residual cytological material stored on FTA® Cards as well as from archived cytological and histological specimens. The presence of point mutations was investigated by a specifically developed assay utilizing MassARRAY spectrometry and confirmed by direct sequencing. Results: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y641) and CD79B (Y196) were detected in 13.2 % and 8 % of the samples, respectively. Mutational status varied in one third of the positive cases, with either gain or loss of mutations over the course of time. Conclusion: Minimal material from lymph node fine needle biopsy is a reliable source for high-throughput multiplex analysis for the detection of point mutation involving EZH2 and CD79B. Whenever possible, the most recent sample should be tested to verify the current status of the disease.
OFP-10-006 MicroRNA expression profile identifies a distinct molecular signature between MYC translocation-positive and negative Burkitt Lymphoma cases G. De Falco * , A. Onnis, F. Morettini, F. Fuligni, C. Bellan, E. Rogena, P. P. Piccaluga, L. Leoncini * University of Siena, Dept. Human Pathology and Oncology, Italy
Objective: The molecular hallmark of Burkitt Lymphoma (BL) is a dysregulation of MYC oncogene, due to one of the three translocations leading to IG-MYC fusion. However, about 10 % of BL cases lack an identifiable MYC rearrangement, although no significant difference of MYC expression among cases has been observed. Additional mechanisms alternative to translocations were explored by microRNA profiling. Method: Ten MYC translocation-positive and ten MYC translocation-negative formalin-fixed and paraffin-embedded BL specimens were used for this study. For microRNA and gene expression profile, the samples were hybridized on the miRCURY™ LNA Array and the Illumina DASL whole genome Assay, respectively. Results: Our results identified a panel of four microRNAs which are differentially expressed between the two groups. Importantly, these microRNAs control relevant biological processes, such as the angiogenesis, apoptosis and cell proliferation, according to Gene Ontology categories. Furthermore, the impact of microRNA dysregulation on the gene expression pattern identified genes which more likely are regulated by the selected microRNA. Conclusion: Using this approach, we showed a clear-cut microRNA and gene signature between MYC translocationpositive and negative BLs. The identification of specific altered microRNAs may represent an alternative molecular mechanism leading to MYC over-expression in the absence of genetic alteration in cancer.
OFP-10-007 Droplet Digital PCR: A highly sensitive assay for B-Raf, K-Ras and EGFR mutation detection J. Frampton * , C. Thorne, V. Spivey, J. Goodall, C. Lowe * Horizon Discovery, Dept. of Diagnostics, Cambridge, United Kingdom
Objective: Droplet Digital™ PCR (ddPCR™) enables the absolute quantitation of nucleic acids in a sample. Horizon Discovery's patented gene editing technology enables it to manufacture reference material that is genetically defined and validated for the allelic frequency of the mutation. These reference standards were used to test the sensitivity of Bio-Rad's QX100™ Droplet Digital™ PCR platform. Method: A panel of defined genomic DNA allelic standards and formalin fixed paraffin embedded (FFPE) cell line samples were used in this study. This panel covered EGFR (G719S, T790M, L858R and L861Q), K-Ras (codon 12) and B-Raf (V600E and V600K) mutations containing allelic frequencies between 0.05 % and 50 %. Droplet Digital™ PCR was performed using mutation specific TaqMan custom SNP assays. Results: There was a strong correlation between the predicted and actual allelic frequencies even down to 0.05 %. The allelic frequency called by the ddPCR™ platform was highly reproducible between experiments particularly in the 1-50 % dilution range.
Conclusion: This study has demonstrated that the Droplet Digital™ PCR QX100™ platform can detect allelic frequencies down to at least 0.05 % using genomic DNA purified from cell lines and down to at least 3.5 % using genomic DNA extracted from FFPE cell lines.
OFP-10-008 Chromothripsis and focal copy number alterations determine poor outcome in malignant melanoma T. Gaiser * , D. Hirsch, R. Kemmerling, J. Camps, T. Ried * Medizin. Universität Mannheim, Inst. für Pathologie, Germany
Objective: Genetic changes during tumorigenesis are usually acquired sequentially. However, a recent study showed that in 2-3 % of all cancers a single catastrophic event, termed chromothripsis, can lead to massive genomic rearrangements confined to one or a few chromosomes. Method: In order to explore whether the degree of genomic instability and chromothripsis would influence prognosis in cancer, we applied high-resolution array comparative genomic hybridization to 20 malignant melanomas (MM) that were, despite comparable conventional clinical and pathological parameters, defined by a profoundly different clinical course. Results: We observed a striking association between both number and structure of chromosomal aberrations and outcome. MM associated with good prognosis showed only few chromosomal imbalances (average 1.6 alterations per case), predominantly presented as whole chromosome or chromosome arm gains and losses. MM with poor prognosis harbored significantly more chromosomal aberrations (13.9 per case; p=0.008). These aberrations were mostly focal events, which culminated in two cases in a pattern consistent with chromothripsis. Conclusion: Here, we describe for the first time the phenomenon of chromothripsis in primary MM and reveal a link between focal copy number alterations and chromothripsis with poor outcome in MM patients (p=0.0002), providing a genetic approach to predict outcome in histopathologically indistinguishable MM.
OFP-10-009 Targeting endometrial stromal sarcoma: Histone deacetylase and PI3K/Akt/mTOR signaling P. Quan * , E. Lederer, I. Halbwedl, H. Denk, K. Zatloukal, J. Haybaeck * Institute of Pathology, Medical University of Graz, Austria
Objective: Endometrial stromal sarcoma (ESS) is a gynecological malignancy with few therapeutic options. Up-regulated histone deacetylase (HDAC) 2 was observed in ESS patients. The HDAC inhibitor SAHA reduced ESS cell growth by inhibiting mTOR. The PI3K/Akt/mTOR signaling cascade, vital to cancer growth is a critical target in cancer therapy. This study aimed at investigating if HDACs interact with the PI3K signaling in ESS pathogenesis and thus might serve as targets for ESS therapy. Method: Tissue microarrays were used to determine HDAC expression in ESS specimens. Western blots revealed protein expression levels of HDACs and PI3K related molecules in two independent ESS and one control cell lines (ESS-1, MES-SA and HESC). Results: Elevated HDAC1 and 2 levels were found in ESS tissues and cell lines. Increased cell growth and Akt/mTOR cascade hyperactivation were detected in ESS cells. SAHA reduced growth of all cells, and induced cell death in ESS cells. SAHA reduced phosphorylated 4EBP1 in all cells, but only inhibited activation of Akt and p70S6k in ESS-1 cells. Conclusion: HDACs are linked to PI3K signaling during ESS pathogenesis. SAHA reduced cell growth via inhibiting PI3K/Akt/mTOR components suggesting that a combination of SAHA with PI3K inhibitors might be effective in treating ESS.
OFP-10-010 Usefulness of linking biobanking field and animal model: High successful rate of human primary Non-small Cell Lung Carcinoma (NSCLC) xenografts in a model system separated by distance and time M. Ilie * , L. Blot, V. Hofman, E. Long, M. Nunes, C. Butori, E. Selva, A. Merino-Trigo, N. Vénissac, J. Mouroux, P. Vrignaud, P. Hofman * CHU de Nice, LPCE, France
Objective: With the ongoing need to improve therapy for NSCLC, there has been an increasing interest in the development of reliable preclinical models to test novel therapeutics. The aim of this study was to evaluate the rate of establishment of patient-derived NSCLC xenografts in the context of a long-distance research network. Method: Fresh surgically resected NSCLC specimens were addressed from the human biobank hospital (Nice) to the animal facilities (Sanofi, Vitry-sur-Seine). Shipment was performed in AQIX medium at room temperature. Within 24 h post surgery, tumour fragments (~63 mm3) were subcutaneously implanted in female SCID mice. The growing tumours were passaged in new mice (≤10 passages). The xenografts were histologically checked to eliminate human or murine lymphoma. Results: Overall, 98 NSCLC samples were implanted leading to 32 (33 %) NSCLC xenografts. The rate of tumour growth was higher in non-adenocarcinoma specimens (23/45; 51 %): 20/38 (53 %) for squamous cell carcinoma, 2/4 (50 %) for large cell carcinoma, and 1/3 (33 %) for pleomorphic carcinoma, when compared to adenocarcinoma samples (8/51; 16 %). Conclusion: We report a high successful rate of xenotransplantation established from patient-derived NSCLC tissues. Our biobanking model system, regardless to extended time and distance, provides a stable and reliable animal model in human lung cancer research.
Monday, 10 September 2012, 14.15 -16.15 Objective: We tried to interpret "the job" of MF in different stages of tumor growth by monitoring the dynamics of their distribution. Method: On a rat model of experimental tumorigenesis using BP6 fibrosarcoma cells injected intraperitoneally, we performed a semiquantitative analysis of the recruitment of MF in the tumor compartment (microenvironment) as well as in peripheral blood. Results: We observed a slow increase of MF count in the initial phase of tumor growth followed by a slow decrease in the late phase within the tumor compartment. Simultaneously peripheral blood showed constant increase throughout the experiment resulting in typical leukocytosis accompanying developed tumors. Conclusion: Increased MF in the initial phase could reflect their enrollment to "prepare a suitable microenvironment for tumor seeding". Their decrease began after the tumor size was macroscopic, thus it was time for them to carry out a "different job". In the mean time their amount in peripheral blood started becoming significant, this is also the time when tumor -host interactions reach a systemic level. In futher experiments we will attempt to verify our assumption: When the tumor flourish in the host organism, it uses informed, transformed and recruited macrophages for growth and building construction of the tumor, and for communication with distant organs on a regulatory level.
OFP-11-002 WWTR1 and CYR61 are early markers of Barrett's esophagus malignant progression M. Mesquita * , J. Cardoso, A. Pereira, S. Braga, M. Bettencourt-Dias, P. Chaves, J. Pereira-Leal * IPOLFG, Dept. of Pathology, Lisboa, Portugal
Objective: Barrett's esophagus (BE) is the major risk factor for esophageal adenocarcinoma (EA). BE has a low risk of progression to EA, being imperative to identify markers to stratify the risk. Our aim was to look for potential early biomarkers of BE malignant progression. Method: Three available microarray datasets were analyzed with R Statistical Computing software complemented with Bioconductor. Biomarker prioritization included: 1) Expression Barcode 2.0; 2) filtering using differential expression analysis. Candidate genes were validated by qRT-PCR on RNA from paraffin embedded samples (RNA-PES) of BE patients with high-grade dysplasia/ADC diagnosed during surveillance and of their index endoscopy dysplasia-free samples. As controls, we used samples from BE patients who have not progressed. Results: Under conservative criteria, we identified 19 upregulated genes that distinguish EA-progressed from nonprogressed BE samples. A second filter, followed by qRT-PCR validation, trimmed the candidates to 2 markers (WWTR1 and CYR61). qRT-PCR on time-series RNA-PES of EA-progressed and EA-free BE samples showed these genes are up-regulated years before the development of EA as compared to patients who did not developed EA. Conclusion: WWTR1 and CYR61 were identified as early risk markers for BE neoplastic progression and may have a potential role in BE risk stratification.
Combined microRNA in situ hybridization and immunohistochemical detection of protein markers B. Nielsen * , T. Møller, K. Holmstrøm * Bioneer A/S, Dept. of Molecular Histology, Hørsholm, Denmark
Objective: MicroRNAs are small noncoding RNAs that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mRNAs. miR-21 is highly prevalent in malignancies. Previous reports on miR-21 in situ hybridization (ISH) in colon and breast cancer identified expression in the stromal cell population that was related to recurrence and cell proliferation (Nielsen et al., Clin Exp Metastasis 2011, 28:27; Rask et al., APMIS 2011, 119:663) . Here, we present a histological assay platform that allows parallel localization of micro-RNAs and protein markers. Method: Routinely processed paraffin embedded breast cancer samples were analyzed. MicroRNA ISH probes were detected with fluorescent tyramine reagents and primary antibodies with compatible immunofluorescence. The reagents were introduced on a semi-automated platform, HistoFlex, which has a precise temperature control unit and utilizes continuous liquid flow. Results: By combination of miR-21 ISH and PDCD4 immunofluorescence we found differential expression in many but not all miR-21 positive cells. Using the HistoFlex, the double fluorescence assay was reduced from 7 to 2 h without loss of sensitivity and specificity. Conclusion: A 3-fold faster assay platform was developed and our in vivo findings support the assumption that PDCD4 is a target of miR-21.
The TNF-superfamily members APRIL and BAFF and their receptors (BAFFR, BCMA, TACI) are differentially expressed in tumors and normal tissues and exert specific effects related to cell fate and differentiation V. Pelekanou * , V.-I. Alexaki, G. Notas, M. Kampa, E. Stathopoulos, A. Tsapis, E. Castanas * University of Crete, Pathology, School of Medicine, Heraklion, Greece
Objective: Classically, tumor immune-related microenvironment was synonym of inflammatory cell infiltrate. Recently, the detection of synthesis and secretion of immune mediators by tumor cells highlighted TNF-superfamily (TNFSF) members as key counterparts of cell fate-related signals. Here, we assayed the synthesis-expression-function of a subset of TNFSF ligands and receptors (BAFF, APRIL, BAFFR, BCMA, TACI) in normal epithelial tissues and tumors and resident adult mesenchymal stem cells. Method: Tissues (breast, CNS, kidney, skin, adipose tissue) have been assayed by means of immunohistochemistry and Real-Time PCR. Cell models were used for signaling, transcriptomics and functional assays. Results: All tumors expressed this TNF-SF subset. BAFFR is absent, while its specific ligand BAFF is ubiquitously expressed; contrairewise, APRIL, BCMA and TACI are expressed in tissue-specific manner. APRIL relays on tumor evolution/grade and is involved in tumor proliferation/differentiation/apoptosis signaling through NFκB-related, or a novel pathway, implicating JNK/FOXO3A/GADD45, triggering transcriptional events. Conclusion: Autocrine/paracrine effects of APRIL and its receptors BCMA and TACI in normal/tumoral parenchyme and the recruitment/attraction of immune-related cells could orchestrate tissue response, regulating cell fate, differentiation, inflammation, tissue remodeling and cancer, with potential tailored-therapy application. Objective: Mutation analysis of KIT and PDGFRA genes is currently used in gastrointestinal stromal tumors (GIST) as an important step in diagnostic protocol. Method: DNA samples obtained from parafin-embedded biopsy material of 278 GIST patients diagnosed in 2004-2011 were screened for mutations in exons 9, 11, 13 and 17 of KIT and 12, 14 and 18 of PDGFRA. Results were tested for associations with clinical parameters of the tumor. Results: Causal mutations (according to in silico analysis with PolyPhen-2 predictor) were identified in 83.8 % of patients, most frequently in KIT exon 11 (62.95 %). The KIT exon 9, PDGRFA exons 18 and 12, KIT exon 13, PDGFRA exon 14 and KIT exon 17 mutations appeared with frequencies 8.3 %, 7.6 %, 2.5 %, 1.4 %, 1.1 % and 0.0 % resp. Genotype-phenotype correlation analysis revealed statistically significant association between intestinal localization of tumors and presence of KIT exon 9 p.503-504_dup2 mutation and gastric localization of tumors and presence of PDGFRA exon 18 p. D842V mutation. Conclusion: Centralized and standardized GIST diagnosis offers clinically and prognostically relevant informations and is necessary for the indication of appropriate targeted therapy of GIST patients. Supported by projects CEPR II (IMTS 26220120036) and MBCC (IMTS 26220220113) at CU JFM co-financed by EU and by grant Novartis Slovakia. Objective: An association between cystic fibrosis transmembrane regulator (CFTR) gene mutations and infertility may occur. This study investigated the frequency of mutations in the CFTR gene, of a group of consecutive patients candidate for assisted reproductive techniques with the aim of identify subjects carriers of the most severe ones. Method: We screened 11208 healthy subjects (5943 females and 5265 males) for 56 CFTR gene mutations and IVS8-poliT polymorphism utilizing the CFTR INNO-LiPA Amplification kit including both general and Italian regional strips. Results: CFTR mutations were detected in 6.2 % of the patients, a percentage similar to that reported in the general population. The most common mutation was ΔF508/N observed in 0.9 % of patients. No difference in the gender distribution was evidenced. In the large group of patients analyzed 87.7 % were wt, 11.8 % carrier of one mutation, and interestingly 0.5 % compound heterozygous. Conclusion: Our data support the relevance of an accurate determination of mutations in the CFTR gene in order to inform the couple of their carrier risk and the possibility on having affected child. Moreover our findings highlight the potential of genetic screening as a tool to identify possible compound heterozygous subjects without CF-like symptoms. Objective: A patient's response to cancer treatment can depend on their biomarker status. Therefore it is important that laboratories perform their biomarker tests well. In 2009 an international external quality assessment scheme for KRAS mutation analysis on FFPE slides was set-up. Method: This scheme was run for 3 years by the coordination center, a medical and technical expert and scheme organizers under the umbrella of ESP. Participants received 10 samples. Genotype results were analyzed and labs with good performance were listed on the ESP website. Written reports of 2011 were evaluated in detail. Results: In 2011, 79 % of participants identified all samples correctly. Results seemed to improve over the years from 9.49/10 to 9.62/10. Genotype results from laboratories that participated for 3 years appeared to improve overtime, although this couldn't be statistically proven. In the reports some important elements weren't always present. For example, the patient's name was only present in 80 %, correct HGVS nomenclature in 90 % and interpretation of results in less than 40 %. Conclusion: KRAS-EQA probably facilitates improvement of laboratory testing. The quality of reports of KRAS tests needs to be improved. A guideline for reporting molecular pathological results would be a good instrument for both participants and assessors. Objective: Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency is reported in a fraction of colorectal cancers (CRCs) (5-10 %) complicating IBDs (IBD-CRCs). Our recent findings argued for the existence of yet unknown mechanisms underlying MMR-deficiency in IBD-CRCs, different from those involved in sporadic and hereditary MSI CRCs. Here we hypothesized that over-expression of miR-155 and miR-21, two inflammation-related miRNAs that target core MMR proteins, could constitute a mechanism underlying MMR deficiency in IBD-CRCs. Method: We compared the expression of both miRs in IBD-CRCs and non-IBD hereditary or sporadic CRCs using nonneoplastic IBD and healthy mucosa samples as controls.
Results: Overall, miR-155 and miR-21 were significantly over-expressed in both non-neoplastic and neoplastic mucosa of IBD patients as compared to healthy controls. MiR-155 alone was preferentially expressed in MSI vs. MSS IBD-CRCs. In contrast, miR-155 was poorly deregulated in non-neoplastic and neoplastic tissues of non-IBD MSI or MSS CRCs. Conclusion: miR-155, alone or synergistically with miR-21, may favor the emergence of MSI IBD-CCRs. Since its deregulation was not limited to neoplastic tissues but extends to non-neoplastic normal mucosa as well, the hypothesis of a miR-155 field defect promoting MMR deficiency and yet MSI-driven transformation can be proposed.
OFP-11-009 PI3K, AKT and PTEN expression in enucleated Brazilian uveal melanoma cases using tissue microarray G. Freeman * , D. Begnami, A. Damascena, J. Neves, S. Nonogaki, F. Soares * São Paulo, Brazil
Objective: In the world literature, little has been published on the molecular pathways in primary uveal melanoma or subsequent development of metastases. The PI3K pathway has been implicated in regulation of apoptosis, cell cycle regulation, transcription and translation. Method: We used immunohistochemistry with TMA to investigate expression of PI3K, AKT and Phos-AKT, and PT in over 200 formalin fixed paraffin embedded blocks of uveal melanoma from the A.C. Camargo Hospital, São Paulo, Brazil from 1988 to 2005 . FISH was performed for PTEN copy numbers.
Results: In this study, neither immunohistochemistry nor FISH results showed a statistically significant difference between the primary tumors and the tumors which metastasized. Statistical analysis of all tumors together (TMA) revealed only one molecular marker (PTEN), which gave results close to statistical significance. Conclusion: This is the first large study of Brazilian patients for PI3K, PTEN AKT expression by Immunohistochemistry and PTEN using FISH. Expression values for molecular markers chosen did not reach statistical significance, although PTEN values were close. Use of FISH to distinguish metastatic from non metastatic cases also came close to statistical significance. These results suggest that the expression of PTEN in uveal melanoma may be a good topic to investigate further. Objective: Chronic antibody-mediated (CAMR) and chronic T-cell-mediated rejection (CTCMR) represent predominant reason for late graft dysfunction. Both categories share similar morphological features. We ask the question whether there are differences between CAMR and CTCMR on the molecular level. Method: Graft biopsies (>3 M) were performed and evaluated according to the Banff classification. Biopsy specimens with CAMR (n=13), CTCMR (n=9) and from protocol biopsies with normal histology (n=10) were stabilized in the RNA-later. Using the Taqman Low Density Array, the intrarenal expressions of 378 genes relating to immune response (B-cell activation, T-cell activation, chemokines, growth factors, immune regulators and apoptosis) were analyzed. Results: Both categories of chronic rejection were associated with up-regulation of many genes in comparison with the control group: chemokines (CCL4, CCL5, CXCL9, CXCL10, CXCL11), growth factor TGFB1, MHC class II (HLA-DMA, HLA-DMB, HLA-DR, UBD), and in T-cell dependent mechanisms (CD3, CD86, LAG3), including cytotoxic T-cell associated transcripts (GBP1, GZMK). In hierarchical clustering, CAMR and CTCMR gene expression profiles were similar. Conclusion: Chronic rejection very probably involves prolonged cooperation of innate immunity and allospecific immune response. Our study showed that CTCMR and CAMR do not differ on the molecular basis.
OFP-12-002 Podocyte loss and glomerulosclerosis in inducible mouse model of podocin mutation-related Nephrotic Syndrome I. Simic * , M. Tabatabaeifar, G. Mollet, C. Antignac, S. Weber, F. Schaefer * Universitätskinderklinik, Nephrologisches Labor, Heidelberg, Germany
Objective: Mutations in NPHS2 gene, encoding podocyte specific protein podocin, cause hereditary nephrotic syndrome. Knock-in mice carrying the R140Q mutation, murine analogue of the most common human mutation R138Q, show developmental arrest of podocytes and renal failure at neonatal age. The aim of this study was to quantify renal histopathological changes in mice with postnatally induced R140Q hemizygosity. Method: C57BL/6 mice with Nphs2Flox/R140Q, Cre + genotype were injected with Tamoxifen for 5 days to induce hemizygosity for R140Q-mutant podocin. Tissue samples were collected at defined intervals after induction. Renal morphology was evaluated by quantitative histology, immunohistochemistry and electron microscopy. Results: Animals developed proteinuria within 1 week that progressed into renal failure and advanced uremia at week 12-16. The number of podocytes per glomerulus started decreasing at week 2 (42±19 vs. 98±31, p= 0.05), whereas glomerular sclerosis index increased from week 4 (1.5±0.15 vs. 0.25±0.08, p<0.00001). Interstitial changes included fibrosis (up to 20 % of section area in end stage renal disease), tubular atrophy and dilatation. Conclusion: Our results implicate that the expression of mutated podocin in induced R140Q-podocin mice leads to podocyte loss that precedes interstitial damage and glomerulosclerosis. Quantification of histological changes will enable better evaluation of the efficacy of different pharmacological approaches directed to improvement of podocyte viability and attenuation of glomerulosclerosis.
OFP-12-003 Heavy chain deposition disease in kidney biopsies A. Vizjak * , J. Mraz, J. Lindic, D. Ferluga * University of Ljubljana, Faculty of Medicine, Slovenia
Objective: Heavy chain deposition disease (HCDD) is a rare and not yet fully explored monoclonal immunoglobulin-related disorder. Method: We studied the histopathology in 4 kidney biopsy cases of HCDD, representing a 0.07 % prevalence among 5481 native kidney biopsies.
Results: HCDD was diagnosed in kidney biopsies of 4 women (mean age 73.0 years). Light microscopy showed diffuse nodular glomerulosclerosis (4/4), associated with mesangial proliferation (3/4) and capillary aneurysms (4/ 4). Immunofluorescence showed abundant mesangial deposits and ribbon-like deposits along glomerular, capsular, tubular and vascular basement membranes, positive for heavy chain IgG3 (3/4) and IgG1 (1/4), with deleted gamma CH1 domain (4/4). Complement C3 and C1q stained positive in all cases. By electron microscopy, punctate and powdery electron-dense deposits were found on the same locations. Conclusion: Immunofluorescence examination of kidney biopsies, including testing for immunoglobulin heavy and light chains, is crucial for diagnosis of HCDD. Our study confirmed that HCDD is peculiar among monoclonal immunoglobulin deposition diseases not only because of its rarity but also because of uniform histomorphologic pattern of nodular glomerulosclerosis with pronounced capillary aneurysms and significant proliferation due to complement activation. Deletion of the heavy chain CH1 domain and its significance in the pathogenesis has to be emphasized.
OFP-12-004 Two cases of ANCA associated vasculitis with geographical necroses in the kidney tissue A. Bartonova * , E. Honsova, R. Rysava * IKEM, Dept. of Pathology, Praha, Czech Republic Objective: ANCA associated vasculitides (AAV) are rare systemic autoimmune diseases affecting small to mediumsized blood vessels. In kidney biopsy samples, AAV usually demonstrate pauci-immune necrotizing crescentic glomerulonephritis (GN). Method: Among 198 cases of AAV evaluated at IKEM during 10 years, 2 had a very unusual pattern of renal involvement. Results: The first patient had been treated for recurrent otitis media with unilateral hearing loss. He also suffered from artralgias and hematuria. An ultrasound study revealed a tumor-like mass in his kidney. During exploratory surgery, the lesion appeared to be infiltrative and a nephrectomy was performed. The second patient presented a granulomatous inflammation in a scar. A month later she developed multiple kidney and spleen "abscesses" with negative hemoculture, and underwent a nephrectomy with splenectomy. In both cases, the final pathological diagnosis was necrotizing granulomatous vasculitis of small and medium-sized vessels, with large geographical necroses of kidney tissue simultaneously with necrotizing crescentic GN. Tests for ANCA antibodies were positive. No microbial pathogens including mycobacteria were detected. Objective: Polyomavirus nephropathy (PVN) is a common complication after renal transplantation. Virus control is achieved by a reduction of immunosuppression allowing an effective T cell-mediated antiviral immune response. The morphology of resolving PVN has not been investigated. Method: 101 protocol biopsies of 34 patients with PV viremia treated by reduction of immunosuppression only were included and scored according to Banff criteria. The extent of interstitial inflammation was estimated as % of cortex. The number of tubular cross sections with SV40+ cells per mm of biopsy length was counted. Findings were grouped as pre-, increasing, decreasing, and post-viremia. Results: During the phase of decreasing viremia, we found a significant increase in the tubulitis score, the extent of tubules with intraepithelial lymphocytes, and interstitial inflammation (p<0.001). These, to a lower extent, persisted after virus clearance. The number of SV40+ tubules correlated with the virus load in the serum, but SV40 immunohistochemistry was frequently negative (33/55 cases), especially if viremia was below 106 geq/ml. Conclusion: Resolving PVN is characterized by a self-limiting acute interstitial nephritis. Our findings are important because the diagnosis of interstitial rejection depends on the same morphological criteria. Therefore, acute interstitial rejection cannot be diagnosed with certainty during PV viremia.
OFP-12-008 Neural cell adhesion molecule and fibroblast growth factor receptor positive interstitial cells increase in interstitial fibrosis in different renal diseases J. Markovic-Lipkovski * , C. Müller, S. Cirovic, S. Tatic, D. Mitrovic, G. Müller * University of Belgrade, Faculty of Medicine, Serbia
Objective: NCAM + cells with dendritic morphology are rarely present in normal renal interstitium. The aim of this study was to evaluate presence of NCAM + cells in kidney biopsies of different renal diseases with and without interstitial fibrosis (IF). Further immunophenotyping of NCAM + renal interstitial cells was performed. Method: 97 kidney biopsies, after routine diagnosis, were stained applying antibodies against NCAM clone123C3.D5 or cloneEric1. For double immunofluorescence, antibodies against NCAM cloneEP2567Y and FGFR1, alpha5beta1 integrin, alphaSMA, cadherin9, cadherin11 were used. By RPCR, different NCAM isoforms (120, 140, 180) were detected using specific primers in 11 renal tissues with and without IF. Results: NCAM + interstitial cells were identified in 48 cases: 69 % lupus nephritis, 64 % focal segmental glomerulosclerosis, 64 % IgA nephropathy, 55 % membranoproliferative glomerulonephritis, 50 % membranous glomerulonephritis. NCAM + cells coexpressed FGFR1 and alpha5beta1 integrin, and were increased in IF. NCAM + cells did not coexpress alphaSMA, cadherin9 and cadherin11, although they were closely colocalised. All normal renal tissues tested for RPCR showed presence of all NCAM isoforms, however NCAM180 lacked in some tissues with IF. Conclusion: NCAM + renal interstitial cells coexpress FGFR1 and alpha5beta1 integrin and are increased in renal diseases with IF, mostly in diffuse proliferative lupus nephritis. In contrast to normal kidneys, in renal tissue with IF NCAM180 is present in a lesser extent.
Reproducibility for C4d immunohistochemistry in renal allografts: Results from the Banff Trial M. Mengel * , S. Chan, J. Climenhaga, P. Randhawa, H. Regele, Y. Kushner, R. Colvin * University of Alberta, Laboratory of Medicine, Edmonton, Canada
Objective: Detection of C4d is crucial for diagnosing antibody mediated rejection, yet formal reproducibility studies are limited. Method: We conducted an international multi-center trial to assess the reproducibility for C4d immunohistochemistry on paraffin-sections. A tissue microarray (TMA) was constructed comprising 44 kidney allograft specimens representing negative, focal, and diffuse C4d positive cases. Participants stained the TMA slides, evaluated their stains and entered their scores online. Stained slides were returned for centralized panel scoring. Weighted kappa statistics were used to determine reproducibility. Results: Inter-institutional reproducibility, i.e. the product from variability between observers and staining methods was low (mean kappa 0.17). Inter-observer reproducibility was fair (kappa 0.39), while inter-laboratory reproducibility was moderate (kappa 0.49). Inter-observer reproducibility could be significantly improved by omitting the Banff C4d grading schema and only considering +/− calls (kappa 0.60). Scoring only C4d+/− inter-laboratory reproducibility improved considerably (kappa 0.78). Higher dilutions of the primary antibody were associated with worse reproducibility. Fixation <1 h or fixation in ethanol had significant negative impact on inter-laboratory reproducibility. Conclusion: C4d results reported from paraffin section are highly variable between institutions. Simplification of the grading schema would improve reproducibility between observers. Technical reproducibility between laboratories is acceptable but could further be improved by standardizing protocols. Objective: Heparanase is a predominant mammalian enzyme that cleaves heparan sulfate, the key polysaccharide found in the basement membranes and at cell surface.
Heparanase is overexpressed in the diabetic kidney; however, its role and mode of action in diabetic kidney disease remains largely unclear. Method: Applying heparanase-null mice we found that deletion of the heparanase gene protects diabetic mice from diabetic nephropathy (DN). Recombinant heparanase enzyme and a specific heparanase inhibitor (SST0001) were used to explore its mode of action in several in vivo and in vitro models. Results: There is essential involvement of heparanase in the pathogenesis of DN. Deleting the heparanase gene protects diabetic mice from DN and administration of specific heparanase inhibitor decreases the extent of albuminuria. In vitro, heparanase enhances macrophage activation by diabetic milieu components, and thus increases the kidneydamaging properties of macrophages. Conclusion: Our results validate the role of heparanase in DN and reveal the mechanism of heparanase action emphasizing its function in coupling chronic inflammation, macrophage activation and diabetic kidney injury. These findings will help in developing effective strategies to disrupt the heparanasedriven sequence of events in diabetic kidney disease, and in designing novel therapeutic interventions in DN. Sunday, 9 September 2012, 17.00-19 .00, Terrace 1 OFP-13 Oral Free Paper Session Neuropathology OFP-13-001 Microvascular angiogenesis occurs in a subset of only the arteriovenous types of vascular malformations and is more abundant in men than in women L. B. Meijer-Jorna * , C. M. van der Horst, C. M. van der Loos, A. C. van der Wal * Medical Center Alkmaar, Symbiant Pathology Center, Netherlands
Objective: Episodic volume expansion may complicate the very slow growth pattern of congenital vascular malformations. We investigated the role of microvascular angiogenesis in this process of sudden growth. Method: 100 resection specimens of symptomatic vascular malformations were screened for presence and extent of sheets or clusters immature microvessels, interpreted as microvascular angiogenesis. Microvessel density (MVD), mast cell density (MCD) and Ki67 labelling index of endothelial cells (EC) were assessed immunohistochemically and quantified. Extent of angiogenesis was correlated with the type of vascular malformation and clinical characteristics. Results: Of 107 cases, 71 were arteriovenous malformations (AVM), 21 were venous (VM), and 8 were lymphatic (LM). Microvascular angiogenesis was observed in 30 % of all vascular malformations, of which 94 % appeared to be AVM. MVD, MCD and Ki67 labelling indexes of EC were significantly higher in immature vessel areas. Moreover, in affected patients these angiogenic responses were far more extensive (in terms of area involvement) in men than in women (p<0.05). Conclusion: Microvascular angiogenesis appears a specific feature of the arteriovenous type of vascular malformations, and is much more extensive in men than in women. We suggest that these microvascular responses may contribute to onset of symptoms due to a mass forming effect.
OFP-13-002 The impact of ventricular assist device prior to transplantation on acute cellular rejection and antibody-mediated rejection in cardiac allografts with due consideration of seasonal behaviour: A prospective study K. Wassilew * , E. Potapov, C. Knosalla, T. Krabatsch, M. Hummel, R. Hetzer * Deutsches Herzzentrum Berlin, Germany
Objective: This study evaluated the impact of bridge-to-transplant ventricular assist device support on development of acute cellular (ACR) and antibody-mediated rejection (AMR) with due consideration of seasonal behaviour in cardiac allografts. Method: We studied 263 consecutive right ventricular endomyocardial biopsies (EMB) between 01/2011 and 03/2012 prospectively. Paraffin-embedded sections were evaluated for acute cellular rejection, endothelial cell swelling and capillary deposition of C4d, C3d and IgA/M/G. The effects of VAD (n= 101) on ACR and AMR, classified according to the ISHLT, were studied for seasonal effects and compared to results of EMB harvested from patients without VAD support (n=162). Results: Our results did not reveal significant differences between the two groups in any given parameter. A positive correlation was found for endothelial cell swelling and capillary C4d deposition; the data failed to show a correlation between C4d and C3d deposition or C3d deposition and endothelial swelling. Complement and immunoglobulin depositions seemed to be more pronounced but without statistical significance in autumn and winter. Conclusion: Our results demonstrate only statistically insignificantly more pronounced capillary complement deposition in autumn and winter. The use of VAD did not predict development of AMR or ACR. The C3d staining does not add to the pathological diagnosis of AMR.
Relevant changes of the molecular profile in the recurrence of glioblastomas with respect to the correspondent primary tumors M. Idoate * , J. Echeveste, R. Diez Valle, M. Montanana, J. Sola, T. Labiano * University of Navarra, Dept. of Pathology, Pamplona, Spain Objective: In the literature, there is not enough information about changes of relevant molecular parameters in the recurrent glioblastoma. Method: The study included a total of 11 grade IV astrocytomas (OMS) and their correspondent recurrences in a series of patients treated by 5-ALA guided surgery. All patients received similar treatment. A comparative histologic and molecular study which included LOH of PTEN region, EGFR amplification SISH, methylation of MGMT by MSP-PCR and sequencing of EGFR variant III mutation on representative tumor samples was obtained. Results: The recurrences appeared adjacent to surgical cavity in seven cases. The mean time between the diagnosis of primary and the recurrence was 500 days (215-1670). The primary glioblastomas showed LOH10q23 in 82 % of cases, hypermethylation of MGMT in 50 %, EGFR amplification in 45 % and EGFRvIII in 37 %. In five cases (45 %) the molecular profile of the recurrences was different to the primaries and most of them (80 %) in the group of vaccines treated glioblastomas. The molecular profile change included one to several of the studied parameters. In one case all of the molecular parameters had changed. Conclusion: The molecular profile change of the recurrences of glioblastomas in respect to the primaries is a frequent event that could be due to the selection of tumor cells due to both heterogeneity and treatment effect. Objective: There is not literature reference on the prognostic significance of the proliferative activity in the border of tumor. A large series of patients with glioblastomas operated by fluorescence guided surgery with uniform treatment and enough follow-up have been studied. Method: A total of 207 samples of different fluorescence quality from 67 glioblastomas, 44 primaries and 23 recurrences, were studied. For each tumor, the maximum value of Ki-67 was determined by a semiquantitative counting by two observers and by an autoanalyzer. These values were compared with relevant oncologic parameters. Results: The results of Ki67 according both counting methods were concordant. The Ki67max values of red (center), pink (intermediate) and blue (border) fluorescence samples obtained by quantitative analysis were 28 % 11 % and 6 %. In the Cox regression analysis for overall survival, the Ki67 value was an independent prognostic factor, stronger than other relevant clinical parameters studied (p=0.002). In the Kaplan-Meier for a Ki67 cut-off of 5 %, the median overall survival was 25.1 months against 13.0 months (p=0.023). For the patients with recurrent glioblastomas the overall survival according to Ki67 values was also significant (p=0.017). Conclusion: The evaluation of the proliferative activity by Ki-67 in the border of tumor defined by 5-ala fluorescence is a relevant independent prognostic marker of primary and recurrent glioblastomas. Objective: According to the levels of estrogen receptor (ER) expression, primary breast carcinoma (BC) cases are divided into two groups: ER-positive and ER-negative (Godhirsch et al. 2011) . In metastatic BC, and especially in brain metastases (BM) of BC, the role of ER expression is not clearly defined. Aim: Investigate ER expression levels in brain metastases of BC and evaluate its prognostic significance. Method: Surgically obtained tumor specimens from sixty BC female patients (median age 52, ranges 29-73) with BM BC were stained with antibodies to ER (DAKO). IHC expression was evaluated according to the semi-quantitative method. Survival was estimated by means of Kaplan-Meyer curves. Statistical analysis was performed using STAITIS-TICA 8.0 software. Results: ER expression was detected in 52 % of patients (31/60), and 48 % (29/60) were ER-negative. Median time period between primary diagnosis and BM development was significantly (p<0.05) longer in ER-positive group compared to ER-negative group (48 and 23 months, respectively). Recurrent BM developed in both groups with a similar frequency (32 % and 34 %, respectively). General survival was significantly (p=0,026) higher in ER-positive patients compared to that of ER-negative group. Conclusion: ER expression is considered to be an important prognostic factor for development of BMBC. Nevertheless, further studies involving more patients with BMBC are required.
OFP-13-006 Dopamine and alpha synuclein interplay in neurodegeneration: A rat animal model G. Stoica * , G. Lungu, N. Bjorklund * Texas A&M University, Veterinary Pathobiology, College Station, USA Objective: A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in alpha synuclein (α-syn) in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia and motor impairments. Method: Histology, immunohistochemistry, transmission electron microscopy were used for morphological analyses. For α-syn assessment real-time PCR and western blot were used. Dopamine measurements were performed by ion mobility mass spectral (IMMS). Results: The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem, and striatum along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a "dying back" type. The disease process is accompanied by gliosis and release of inflammatory cytokines. Conclusion: Decrease dopamine and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease. Sunday, 9 September 2012, 14.15 -16.15 Objective: The mitotic index in thin primary melanomas replaced Clark level in the 2009 staging system of the AJCC. However, the recommended quantification of proliferation by hot spots on HE stains is criticized. An alternative may be the immunohistochemical proliferation marker phosphohistone H3 (PHH3) visualizing all mitotic cells. And, when combined with the melanocytic marker MART1, PHH3 insures quantification of proliferating melanocytes, only. Method: 153 primary melanomas with a median follow-up of 12 years for patients with event-free melanoma were included. PHH3/MART1 stains were performed by an indirect sequential immunoenzymatic technique. The number of PHH3/MART1 positive cells was counted in a fixed 1-mm2 frame in the dermal area with the highest concentration of positive cells (hot spots). Results: In multivariate analysis, PHH3 in hot spots was a strong independent prognostic marker for recurrent disease (HR=3.7, 95 % CI, 1.4 to 9.6; P=.008) and melanoma-specific death (HR=3.4, 95 % CI, 1.3 to 9.0; P=.013), when corrected for primary tumor thickness and ulceration. Conclusion: Cellular proliferation is an independent prognostic marker in primary cutaneous melanoma. However, accurate quantification is crucial for correct clinical staging. For this purpose, proliferation indices of the novel PHH3/MART1 double stains seem very promising. Objective: Some of the more common benign vocal fold lesions are polyps, nodules and polypoid corditis (Reinke's edema). The majority of vocal fold pathology develops in the mucosal layer of the vocal fold, precisely in Reinkes space. Basic science and clinical research over past decades has led to advances in our understanding of benign laryngeal lesions. The aim of our study was compared contact telescopy findings with histological analyzes in a patients with Reinke's edema. Method: The our study included 80 consecutive patients with clinical diagnosis of Reinke's edema. Videoassisted contact telescopy was performed in all cases under general anesthesia and mucosa was stained by methylene blue. Tissue biopsies were taken from areas exhibiting visible pathological changes and sent to routine histopathology and immunohistochemical analyses. We analyzed expression of pancytokeratin antibodies, vimentin and CD 34. Results: In the all case of Reinke's edema, contact telescopic scans showed a change of direction or a disappearance of regular distribution and morphology of blood vessels (irregular shapes, positions, and patterns, apparently running in random directions) in vocal fold mucosa. Marked pathological changes were noted by contact telescopic scans, confirmed with histopathology analyses. Conclusion: Contact telescopy is a useful additional diagnostic tool regarding Reinke's edema.
OFP-14-005 Volunteering in Malawi: A snapshot of surgical pathology in sub-Saharan Africa S. Berezowska * , T. Tomoka, S. Kamiza, D. A. Milner jr., R. Langer * Universität Bern, Institute of Pathology, Switzerland
Objective: The breadth of material found in surgical pathology services in African countries significantly differs from the common spectrum of "the West". We report our experience in the pathology departments of Blantyre and Lilongwe, Malawi. Method: During a six-week period 405 cases were processed (378 histology, 27 cytology). Results: The vast majority of cases showed significant pathological findings (n=369; 91.1 %): 175 (47.4 %) were non-tumoral conditions and 39 (10.6 %) benign tumors or tumor like lesions. The large group of malignancies (n=140; 37.9 %) comprised 11 pediatric tumors (e.g. rhabdomyosarcoma, small blue round cell tumors), and 129 adult tumors. Amongst women (n=76), squamous cell carcinomas (SCC) of the cervix uteri predominated (n=25; 32.9 %), followed by breast carcinomas (n=12; 15.8 %), and esophageal SCC (n=9; 11.8 %). Males (n=53) most often showed SCC of the esophagus (n=9; 17.0 %), and SCC of the urinary bladder (n=7; 13.2 %). Lymphomas (n=7) and Kaposi's sarcomas (n=6) were less frequent. Conclusion: Providing pathology service in a low resource country may be handicapped by lack of personal, inadequate material resources, or insufficient infrastructure. Rotating volunteers offer a bridge for capacity building of both personnel and the local medical service; in addition, the volunteer's horizons are broaden professionally and personally. Objective: Antinuclear antibodies (ANA) are routinely tested by indirect immunofluorescence (IF) and immunoblot. In this paper we aim to retrospectively analyze results of both methods. Method: On two cohort groups: A (a total of 5518 samples), and B (a total of 2615 samples) we performed IF on commercially obtained Hep-2 cell, and immunoblot on (strips Euroline). Results: IF revealed at least 14 different morphological patterns of ANA staining, which we grouped into 5 easily distinguished categories: speckled nuclear fluorescence, homogenous nucleoplasmic fluorescence, multiple nuclear dots, nucleoli staining, and scattered nucleoplasmic dots. We compared these groups according to frequency, type of antibody and correlation with immunoblot resuts.
We summed the characteristics of each IF category, and noticed that both cohorts revealed 7.4 % and 6 %, respectively of sera, which did not show positive IF at an acceptable dilution (only by 1:10) or were fully negative in IF test, but were clearly positive in immunoblot test. This was especially alarming with anti-Scl-70 antibodies, which could be barely recognized in IF test, but were clearly present in 53 of 407 positive stained strips (cohort A) and/ or 14 out of 156 positive stained strips (cohort B).
OFP-14-007 Macrophages and their subtypes in tumorigenesis and growth O. El-Hassoun * , L. Maruscakova, Z. Valaskova, M. Kopani, J. Jakubovsky, I. Hulin * Alphamedical Laboratories, Dept. of Pathological Anatomy, Martin, Slovakia
Objective: It has been established that Macrophages (MF) play a basic role in the tumor microenvironment. Still, the definition of the function of MF with relation to tumors is still vague, mainly when MF are observed acting occasionally as pro tumor and elsewhere as anti tumor. Method: In this paper we attempt to sum up the accumulating observations of MF function with respect to the microenvironment and the tumor entity. Results: Through the different stages of tumorigenesis and growth, macrophages are engaged in various signaling cascades that tame them to perform a certain function in a given microenvironment. This makes MF a multi-program cell with unique adaptivity. We assume that without the macrophages tumors cannot progress. Conclusion: The available MF subtype analysis is still inadequate and doesn't bear into account the dynamics of tumorigenesis and growth. Our presented MF schemes provide an insight on the tumor microenvironment with respect to stage. A complex view on the signaling cascades affecting MF is needed to formulate a novel classification of MF based on their programming. This consequently can be the basis of the development of "anti-MF therapy" or on a larger scale "anti-tumor microenvironment therapy". OFP-14-009 Lodox X-ray is an invaluable asset in autopsy procedures L. Liebenberg * * University of Cape Town, Dept. of Forensic Medicine, South Africa
Objective: The field of autopsy performance has faced numerous challenges internationally. Minimal background information on the deceased, various religious and personal objections regarding cutting a deceased body as well as health hazards face the pathologist regularly. Lodox X-ray is a huge help for the pathologist. Method: Lodox X-ray (low dose X-ray) using the Statscan has proven invaluable in busy forensic pathology laboratories in Cape Town, South Africa. The digitally captured radiology images render a wealth of clear, recognizable, easily documented and user friendly images for use in diagnosis and court evidence. Results: The use of the Statscan has revolutionized the process of forensic autopsy procedures in Cape Town, South Africa. The radiology images obtained in an unusually user friendly and time efficient way have improved efficacy, efficiency, accuracy and pro-active occupational health safety in our forensic laboratories.
Conclusion: Lodox X-ray is a proven invaluable special investigation in forensic, and also academic, autopsy examination procedures. Sunday, 9 September 2012, 17.00-19. Objective: Castration resistant prostate cancer (CR-PCa) is the most aggressive form of prostate cancer (PCa) posing a significant therapeutic challenge. Our aim was to perform whole exome-sequencing on 5 CR-PCa/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform (NGS). We identified two promising genes-YWHAZ and PTK2, that could serve as novel potential therapeutic drug targets in PCa. Method: Genomic DNA was sequenced from 5 CR-PCa/ normal paired FFPE samples. A set of amplified/deleted genes were validated using fluorescence in-situ hybridization (FISH) assays using a PCa progression cohort consisting of 138 cases for localized PCa, 105 patients with primary PCa and corresponding LN metastasis, and 39 samples for CRPCa. Results: Exome-sequencing identified regions of deletions/ amplifications, including NKX3.1, PTEN, CMYC and AR genes, known to play a role in PCa. We identified several amplified genes as druggable targets such as PTK2 and YWHAZ. For YWHAZ we identified 3.5 % amplification in localized PCa, 24.2 % amplification in LN metastasis and 45.5 % amplification in CRPCa. For PTK2 we 4 % amplification for localized PCa and 34 % for both LN metastasis and CR-PCa.
Conclusion: This is the first study to use exome-sequencing approaches on FFPE CR-PCa to understand the biology of disease and its plausible treatment options.
A study of d-Np63 (p40) expression in tumours of stratified epithelium D. Nonaka * * The Christie Hospital, Dept. of Histopathology, Manchester, United Kingdom
Objective: p63 can be expressed by a minority of adenocarcinomas. ΔNp63 (p40), isoform of p63, has recently been reported as more specific in lung squamous cell carcinoma (SCC) than p63, and it appears to be a more reliable marker for SCC. There is no comprehensive study on p40 expression in tumours of different histotypes. Method: 460 tumours of various hisotypes were studied, including tumours of stratified epithelium such as SCC (84 cases) of the skin, head/neck, lung, cervix, and oesophagus, urothelial carcinomas (18), cutaneous basal cell carcinomas (5), thymomas (20) and thymic carcinomas (5), adenocarcinomas (210) from multiple organs, germ cell tumours (25), brain tumours (17), lymphomas (14), and sarcomas (12). p40 immunostains were performed on tissue microarrays and staining extent was evaluated as focal (1-50 %) and diffuse (50-100 %). Results: All but one tumours of stratified epithelium diffusely expressed p40. The negative case represents spindle cell SCC. Other tumours except for basal/squamous component of two teratomas, one breast metaplastic carcinoma, and squamous component of 5 adenosquamous carcinomas were all negative for p40. Conclusion: p40 is specific in tumours of stratified epithelium and its sensitivity appears comparable to p63. p40 can serve as a marker for tumours of stratified epithelium. Objective: Although formaldehyde is the standard fixative used in routine histopathology, it is not considered an "ideal" fixative. Formaldehyde presents carcinogenic properties, a slow fixation and produces cross-links with proteins and nucleic acids. Formaldehyde-free fixatives are commercially available.
Method: From 2002 to 2012 we used Glyo-Fixx (Thermo Scientific, US), a glyoxal-based fixative, as formaldehyde substitute, analysing 54314 surgical pathology specimens and 205 autopsies. All samples were processed and stained routinely. Moreover we performed immunohistochemistry by an automated immunostainer and special stains with standard techniques, where needed. In tumoral cases we fixed 1 cm3 sample in neutral buffered formalin and compared the results with Gyo-Fixx fixation. Results: Glyo-Fixx fixation needed less hours than formaldehyde and not harden the tissues. On gross examination of the adipose tissue, lymph nodes were easily found, due to a more marked whitish appearance. Morphologically we have not found differences between formalin and Glyo-Fixx, except for the eosinophils degranulation. Special stains resulted similar to those with formaldehyde fixations. Almost all antibodies not required pretreatment, but needed adjusts in the standardized protocol for formaldehyde. Conclusion: Our experience demonstrated Glyo-Fixx is a good non-toxic alternative to formaldehyde in routine pathology, capable to preserve morphology and protein integrity of the tissues.
Liposarcoma with solitary fibrous tumor-like dedifferentiated areas: Clues on differential diagnosis M. Aizpurua * * HUVH/ICS, Dept. of Pathology, Barcelona, Spain
Objective: Well-differentiated liposarcoma (WDLS) can undergo dedifferentiation to nonlipogenic sarcomas. Solitary fibrous tumour (SFT) characteristics have not been highlighted as a pattern of dedifferentiated liposarcoma (DLS). However, this does occur and could be the cause of diagnostic pitfalls in atypical locations or partial tumoral resections. The aim of this comparative study is to demonstrate that some DLS's may show morphologic features resembling SFT's, and furthermore to describe the clue features supporting differential diagnosis. Method: Study comprised 11 WDLS-DLS and 12 SFT (4 malignant). Histological features were systematically reviewed, and CD34/CD99/S100/BCL2 immunostains and MDM2 FISH-analysis were evaluated in all cases. Results: There are many overlapping characteristics between DLS and SFT (Table 1 ). Significant differences between both tumor types were observed in lipoblast-like cells (lpc); mature adipocitic cells within tumor (mac); stromal bands surrounding adipose tissue with atypical cells (sac); patternless (pa); keloidal-collagen (kcm) and haemangiopericytoid-pattern (hp). Diagnostic accuracy of different features is shown in table 2.
Conclusion: The dedifferentiated component in some liposarcomas may resemble SFT. In the DLS/SFT differential diagnosis, immunohistochemistry may be confusing. Presence of WDLS areas is the main diagnostic clue and MDM2 FISH amplification is successful in distinguishing DLS from SFT.
Osteoblastoma and diagnostics pitfalls W. Ouahioune * * EHS Douera, Dept. of Pathology, Algiers, Algeria
Objective: Osteoblastoma is a rare benign osteoblastic tumor with a potential for local bone destruction and aggressiveness. The most common site of osteoblastoma is the vertebral column particularly the posterior elements and the sacrum. Method: In this study we present nine cases of osteoblastoma and the principal differential diagnosis. Results: On a period of 10 years, nine cases of osteoblastoma had been diagnosed including seven males and two females. The age of those patients ranged from 5 to 29 year old. The tumor was localized in the spine in five of the nine cases and the other ones in the long bones. The radiological diagnosis of osteoblastoma was made in just two cases. The diagnosis was made by histology in eight cases. All our patients had been treated with curettage. On the nine patients, just one of them had developed two successive recurrences. Conclusion: Osteoblastoma is a rare benign tumor which is rarely diagnosed by radiology alone. The pathologist should always suspect an osteoblastoma in front of a vertebral localization of any tumor. Objective: Congenital hepatic fibrosis (CHF) is a developmental disorder of the portobiliary system. Clinical findings usually include enlarged liver, well-preserved hepatocellular function and portal hypertension. CHF is frequently associated with hepatorenal fibrocystic disease. Method: A 29-year-old woman was referred to our hospital for further evaluation of hepatomegaly, and portal hypertension was found. Laboratory tests were all negative. MRI identified hepatosplenomegaly with hypertrophic left medial segment, high uptake nodular areas and homogeneous boundary liver. Needle biopsy was performed. Results: The biopsy demonstrated slight portal fibrosis and fibrous septa with proliferation of numerous biliary ducts, some of them containing inspissated bile. It was also noticeable the few number of portal vein branches. There was no inflammation or hepatocyte necrosis. Therefore, our diagnosis was congenital hepatic fibrosis. We correlated these results with radiology. Magnetic resonance cholangiopancreatography showed peripherally dilated biliary intrahepatic branches contrasting with preserved caliber of main intrahepatic branches. These findings were also diagnostic for CHF. Conclusion: The low prevalence of CHF makes it hard to think about this entity in patients with portal hypertension and normal laboratory findings. The histological differential diagnosis must be done mainly with cirrhosis, but also with idiopathic portal hypertension and, in small biopsies, with biliary hamartoma. Results: A total of 97 cases of digestive tract lymphoma were reported (64 males, 33 females) with a sex-ratio of 2.0. The patient age ranged between 6 and 82 years with a median age of 48 years. Stomach location was the most frequent with 80 % of cases followed by intestine (15 %) and colon (5 %). Among the 97 digestive tract lymphomas, 48 % were of Marginal zone type (MALT) and 47 % were large B-cell lymphoma. According to the Ann Arbor staging system, 85 % of patients were diagnosed at stage I and II.
Conclusion: Lymphomas of the digestive tract are still frequent in the Centre of Tunisia. The Malt Type lymphoma is main reported histological type. The association with the Helicobacter pylori should be considered.
Collecting duct carcinoma in the west of ireland: A rare experience A. Shalaby * , C. E. Connolly * University Hospital Galway, Dept. of Histopathology, Ireland
Objective: Collecting duct carcinoma is a rare renal neoplasm arising from the epithelium of Bellini's ducts in the distal part of the nephron. Method: We describe our experience of this entity in University Hospital Galway with a series of four cases diagnosed between 2003 and 2011. Results: Three patients aged between 35 and 62 years presented with either lymph node or bone metastases without evident abdominal mass. In each case the biopsy from the metastatic lesion showed adenocarcinoma with an immunohistochemical profile suggestive of renal origin and a renal mass was subsequently confirmed by radiology. The fourth patient was an 81 year old male who presented with haematuria. Abdominal CT scan revealed a 5.6 cm mass in the right kidney extending into the renal vein. Biopsy of the renal mass in all cases confirmed adenocarcinoma with a tubular pattern with positive cytokeratin and Vimentin staining and CD10 negative staining. One patient was treated with nephrectomy with post-neoadjuvant chemotherapy and died 25 months after surgery. The other three patients died few months after their diagnosis. Conclusion: Collecting duct carcinoma is a rare renal neoplasm that can be difficult to diagnose on core biopsies, however, it can be identified based on radiological findings, gross, microscopic, histochemical and immunohistochemical features. Objective: Wilms Tumors (WT) is the most frequent renal tumor of children. Genetic alterations have been suggested as associated factors but the exact pathogenesis of WT is not fully characterized. Tissue factor (TF) is a glycoprotein which happens to be a key receptor for factor VII/VIIa and a primary initiator of coagulation. TF has also been associated with angiogenesis. Recent evidence pointed out an important role for TF in cancer progression and metastasis. Method: In the present investigation the differential expression of TF in WT was assessed by real-time PCR of RNA retrieved from paraffin sections using microdissection. Results: Different histological components of WT were analysed. The results revealed that TF was upregulated in blastema and epithelial components as compared to nonneoplastic tissue (14.38 and 16.02-fold respectively, P< 0.001). Stroma and non-neoplastic tissue presented low levels of TF expression. TF expression in WT metastatic lesions was also significantly upregulated as compared to non-metastatic WT. Microvessel density was positively correlated with TF expression (r=0.721). Conclusion: As described in other tumors, TF seems to play a significant role in the behavior of WT. Further investigations are warranted to better understand the pathways by which TF exerts its effects on tumor progression and its potential as a target for therapy. Objective: Biliary atresia (BA) is the most common neonatal cholestatic disorder and the prime indication for liver transplantation (LTx) in children. Histopathological markers in liver biopsies emerge promise as indicator of early LTx in patients with BA. Method: Ductular proliferation, ductal plate malformations and type I, III, IV and V collagen deposition were evaluated on Kasai portoenterostomy (KPE) and liver transplantation (LTx) biopsies from 36 children with BA. Formalin fixed and paraffin embedded liver biopsies were stained with hematoxylin-eosin, picrosirius-polarization and immunofluorescence methods. There were analyzed liver histoarchitecture, biliary ductus and collagen deposition in hepatic compartments. Pathologic findings were graded according to a 5-point semi-quantitative severity-based scoring system. Impact of these markers was tested on LTx time (<2 year and >2 year). Results: Median age of KPE was 12 weeks (range 6-20) and of LTx was 27 months (range 6-120). In KPE liver biopsies, ductular proliferation, ductal plate malformations and collagen deposition were increased but these parameters presented no association with clinical evolution for early LTx. Furthermore, collagen V prominent deposition was found along of hepatic sinusoids and type I, III and IV were more frequent in portal compartment. Conclusion: These results suggest that histopathological parameters evaluation presented may not determine early LTx in biliary atresia.
The role of Hofbauer cells on villous vasculature in early fetal losses E. Özer * , Y. Arman Karakaya * Dokuz Eylul University, Dept. of Pathology, Izmir, Turkey
Objective: The aim of this retrospective study is investigate the role of Hofbauer cells in early fetal losses. Method: The slides obtained from archieval blocks of missed abortion (MA, n=15) and blighted ovum (BO, n= 15) cases and unwanted pregnancies materials (control group, n=15) were stained by immunuhistochemical methods using CD68 and CD31 antibody to label Hofbauer cells and endotelial cells, respectively. Results: The mean number of vilous Hofbauer cells was found to be significantly higher in both BO and MA in contrast to the control group (p=0.005 and p<<0.001, respectively). However it was not significantly different between BO and MA (P=0.04). Chalkey method revealed no statisticaly significant difference in the control group in comparison with MA and BO in (P=0.29, P=0.09, respectively). Higher microvessel scorring were found in MA in contrast to BO and the control group (p=0.003 and p= 0.003, respectively). However, there was no difference between the control group and BO (p=0.54). Conclusion: We think that Hofbauer cells may be of biological importance in early fetal losses and play a role on defective vasculature formation in MA.
Placental VEGF and its receptors expression patterns in preeclampsia K. Pavlov * , E. Dubova, A. Shchegolev, G. Sukhikh * V.I. Kulakov Scientific Center, Dept. of Pathology, Moscow, Russia
Objective: Placental angiogenesis anomalies play an important role in some complications of pregnancy development, including preeclampsia (PE). VEGF and its receptorsone of the key placental angiogenic factor. Aim: to evaluate patterns of VEGF and its receptors (VEGFRs 1, 2 and 3) expression in placentas from PE complicated pregnancies. Method: We performed complex morphological and immunohistochemical study of 9 term placentas from mild preeclampsia (mPE) cases (1st group), 6 term placentas from severe preeclampsia (sPE) cases (2nd group) and 10 term placentas from uncomplicated pregnancies (control group). Results: We revealed significantly increased syncitial VEGF expression levels in preeclamptic placentas terminal villi and these changes were much prominent in sPE. We also detected insignificantly increased endothelial VEGF expression levels in preeclamptic placentas terminal villi. VEGFR-1 syncitial expression levels in sPE terminal villi were significantly higher in compare to the mPE and control groups. VEGFR-2 endothelial and syncitial expression levels were significantly lower in both PE groups terminal villi and these changes were much prominent in mPE. Patterns of VEGFR-3 expression in preeclamptic and control groups were multidirectional. Conclusion: Revealed patterns of VEGF and its receptors expression point on altered placental angiogenesis in PE and reflect different degree of such alteration in mild and severe PE. Objective: Chorionic villi vascularity disturbances, caused by abnormal expression of angiogenic factors could correlate with maternal and fetal complications in diabetic pregnancies. Method: Aim: to evaluate the patterns of VEGF receptors (VEGFR1, VEGFR2 and VEGFR3) expression in placentas from gestational diabetes (GD) and type 1 diabetes (D1) pregnancies and correlation between them and some clinical parameters in newborn (placental weight (PW), newborn weight (NW), placental/newborn weight index (PNWI),1st day of life blood glucose test results (1BGTR)). 29 term placentas from D1 (n=16) and GD (n=20) and 12 term placentas from normal pregnancy (control group) were studied. Results: In both D1 and GD group we revealed significantly higher level of VEGFR1 (p=0.0001), VEGFR2 (p=0.001) and VEGFR3 (p=0.01) endothelial expression in terminal villi. Difference in VEGFR1 expression among D1 and GD groups was also significant (p=0.0009) as difference in its expression between D1 and control group (p = 0.002). Among clinical parameters we only revealed significant increase in 1BGTR in diabetic groups (p = 0.03) with marked difference between D1 and control group (p = 0.002). We revealed multidirectional correlation between VEGF and VEGFR2 expression and NW in D1 and GD groups.
Conclusion: Revealed changes reflect placental angiogenesis disturbances influence on intrauterine fetal development.
Microvillus inclusion disease (MVID) is a disorder of defective intracellular trafficking and disrupted epithelial cell polarity C. Thoeni * * Division of Cell Biology, Medical University Innsbruck, Austria
Objective: MVID is a congenital enteropathy characterized by loss of microvilli and formation of microvillus inclusions (MI) in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the Myosin Vb (M5B) motor protein involved in intracellular transport and maintenance of epithelial cell polarity. We examined the effects of loss of M5B in enterocytes of MVID patient and in a CaCo-2 cell model. Method: The expression and localization of cell membrane transporters (CD36, Na/K ATPase) and various cell organelle markers : endosomal Rab GTPases [Rab5, 7, 8, 9, 11] ; early (EEA1) and late endosome (LAMP2), and Golgi (Giantin)) were analyzed using multilabel immunoflourescence and confocal microscopy of a duodenal biopsy from MVID patient and in CaCo-2 cells following M5B si RNA knock-down (Hum Mut 31:1-8,2010). Results: Depletion of M5B in both enterocytes and CaCo-2 cells resulted in disruption of epithelial cell polarity with loss of apical microvilli, formation of MI; mislocalization of transporters as well as aggregation of epitopes for different Rab GTP ases, early and late endosomes. Conclusion: M5B plays a critical role in polarized organization of enterocytes in MVID -a disorder characterized by defective intracellular trafficking and altered endosomal sorting. CaCo-2 cells provide an excellent model to study the pathogenesis of MVID.
OFP-16-007 Beta-catenin expression and mutational analysis of CTNNB1 gene in pediatic Adrenocortical Tumors (ACT) R. Alaggio * , P. Dall'Igna, A. Martines, E. Lalli, R. Boldrini, V. D´Onofrio, G. Esposito * Università di Padova, Dipt. di Pathology, Italy
Objective: ACT have an unpredictable clinical behaviour, and no liable histological or molecular parameters are available to predict outcome. The activation of WNT/β-catenin pathway, involved in tumor growth and progression in adult ACT has been explored only in Brasilian pediatric ACT. The aim of this study is the investigation of the possible prognostic role of β-catenin accumulation and/or CTNNB1 (β-catenin gene) mutations also in Italian pediatric ACT.
Method: β-catenin immunostaining and mutational analysis of CTNNB1 gene at exons 3 and 5, when possible, were carried on a series of 19 ACT (7 malignant and 12 benign, according to Wieneke classification), from patients enrolled in the Italian Pediatric Rare Tumor (TREP) Study. Results: Immunostaining for β-catenin showed membrane/ cytoplasmic staining in 9 cases, 5 benign and 4 malignant, and nuclear staining in 1 malignant. No mutations of CTNNB1 gene were found in the 8 tumors analyzed. Conclusion: CTNNB1 gene mutations do not appear to be involved in pathogenesis of pediatric ACT. The accumulation of protein might be related to different mechanisms. Its presence in the majority of malignant ACT suggests a possible role in tumor progression.
OFP-16-008 Neuroblastoma presenting like a Wilms' Tumor with thrombus in inferior vena cava: A case series H. Sartelet * , G. Gaetan, A. Ouimet, C. Lapierre, P. Teira * CHU Sainte Justine, Dept. of Pathology, Montreal, Canada
Objective: Neuroblastomas and Wilms' tumors are frequent pediatric solid tumors. The first is frequently detected in the adrenal gland and the second develops in the kidneys. The extension through the vena cava and the lung metastases are frequent in Wilms' tumors and are rarely in neuroblastoma. We present the cases of three children with abdominal tumors with thrombus in the inferior vena cava and pulmonary metastases were discovered yet demonstrated a stage 4 neuroblastoma. Method: The three male patients were between 23 and 48 months old. They presented an abdominal mass, near the superior pole of the kidney. Thrombus of the vena cava was evoked on imaging studies in all cases and pulmonary metastases were always found. Catecholamine metabolites were present in the first case and negative in the two others. Two out of three patients had a radical nephrectomy. Results: The pathological analysis always found a neuroblastoma poorly differentiated or undifferentiated without MYCN amplification and confirmed the tumoral thrombus in the second case. The evolution of the two first patients was unfavorable and the third is alive. Conclusion: Invasion of the inferior vena cava and pulmonary metastases in children with neuroblastoma is uncommon and can modify the surgical management.
OFP-16-009 EpCAM -A marker for Tufting enteropathy (TE) and a useful tool in the differential diagnosis of congenital enteropathies C. Thoeni * * Division of Cell Biology, Medical University Innsbruck, Austria Objective: Congenital enteropathies (CE) are characterized by villous atrophy and disruption of apical microvilli in duodenal enterocytes. TE is characterized by focal eptithelial tufts and mutations in the EpCAM gene, coding for epithelial cell adhesion molecule, important for cell-cell contacts. Microvillus inclusion disease (MVID), shows loss of apical microvilli and formation of intracytoplasmatic microvillus inclusions. MVID is caused by mutations in the MYO5B gene, important for intracellular transport and organization of epithelial cell polarity. In this study, we identified a new EpCAM mutation in a patient with TE and used anti-EpCAM antibody as a marker for the diagnosis of TE and distinction from MVID. Method: We used Immunohistochemistry (IHC) with monoclonal EpCAM antibody (mouse monoclonal AB for EpCAM, NCL ESA/Leica Microsystems) on routinely processed duodenal biopsies from patients with TE, MVID and age matched controls. Immunostaining for E-Cadherin served as reference. Results: EpCAM expression was completely absent in the biopsy from TE patient homozygous for a novel EpCAM mutation (c227 C>G, Ser 76X). E-Cadherin showed normal expression and distribution pattern in the enterocytes. In MVID, expression and distribution pattern was comparable to controls for both EpCAM and E-Cadherin. Conclusion: Loss of EpCAM is specific and a sensitive marker for confirmation of TE. EpCAM antibody is useful in the differential diagnosis of CE. Objective: In pulmonary adenocarcinoma EGFR mutation analysis has become an important part of the diagnostic work-up. Frequently the tissue of bronchoscopies is of limited diagnostic value. The aim of the present study was to evaluate the impact of ROSE on brochoscopy specimens with regard to EGFR mutation. Method: ROSE was used for adequacy of the specimen and for separating adenocarcinomas and carcinomas NOS from other neoplasms in the lung. If it was not possible to retrieve material for histology, cytology and cellblock were made. If adenocarcinoma or carcinoma NOS was suspected in ROSE, further two biopsies or two cell blocks were obtained. EGFR mutation analysis was performed by PCR (Light Cycler 480; EGFR RGQ PCR Kit; Qiagen). Results: Malignancy was diagnosed in 93 of 354 cases by histology or on cytology with and without cell blocks. Adenocarcinomas (n=30) and carcinomas NOS (n=9), SCC (n =26), SCLC (n = 13), and rare tumors (n=15) were diagnosed, respectively. Among the group of adenocarcinomas and carcinomas NOS (n = 39), EGFR mutation analysis was performed in 29 (74,3 %) cases and showed mutations in 3 (10,3 %) and "wild type" in 26 (89,7 %) tumors. No material was left in 10 (25,6 %) cases. Conclusion: ROSE supports bronchoscopic procedures to retrieve adequate specimens for tumor diagnosis and subsequent EGFR mutation analysis. Objective: Patients with Fibroblast growth factor receptor 1 (FGFR1) amplified squamous cell lung cancers (L-SCC) are treated in phase I clinical trials using small molecule inhibitors (SMI). SCC of the lung share common molecular alterations with squamous cell head and neck cancers (HN-SCC). Aim of our study is to assess if HN-SCCs also harbor FGFR1 amplifications. Furthermore, we aim to inhibit cell proliferation of FGFR1 amplified HN-SCC cell lines using a SMI. Method: The cohort consists of 227 patients suffering from HN-SCC, 97 of these suffering from metastatic disease. Primary tumors and metastatic tumors were assessed for FGFR1 copy number status using fluorescence in-situ hybridization (FISH). We tested cell lines for FGFR1 amplification status and inhibited these with SMIs. Objective: The pulp of pequi has high levels of antioxidants properties. Method: Eighteen male BALB/c mice divided: 14 animals received by gavage 0,5 μL/mg/day of pequi oil (Control + CBCoil=4) during 75 days. After 15 days, 10 of these mices received two doses of 1,5 g/kg intraperitoneal of urethane (Urethane + CBC oil=10). The other 4 animals were only submitted to two doses of 1,5 g/kg intraperitoneal of urethane (Urethane group=4). After 75 days, groups were sacrificed. Antioxidant activity was evaluated in the lung tissues by TBARS (Thiobarbituric acid-reactive substances), CAT (catalase) and SOD (superoxido dismutase) test. DNA damage was estimated by comet test. Results: The lung parenchyma from Urethane groups without oil and with oil showed neoplasic formations induced by the chemical carcinogenesis in contrast with Control + CBC oil group. The results of TBARS test showed a significant decrease of lipid peroxidation in the Urethane + CBC oil, than Urethane group. The CAT and SOD test didn't show a significant difference. Comet assay showed a significant decrease of DNA damage in Urethane + CBC oil when compared with Urethane group. Conclusion: The antioxidant components in the pequi oil diminish the oxidative stress status and DNA damage in chemical carcinogenesis, suggesting that this type of strategies may have a greater impact in lung cancer treatment. Financial Support: FAPESP.
OFP-17-004 An algorithm for gene mutation analysis in lung cancer M. Comanescu * , C. Iosif, M. Dobre, L. Buburuzan, G. Bussolati, F. Vasilescu, C. Ardeleanu * Institutul Victor Babes, Dept. of Pathology, Bucharest, Romania
Objective: Somatic alterations of K-RAS, EGFR and ALK which are increasingly requested in order to predict response to personalized therapies in lung cancer are mutually exclusive and are represented in over 50 % of lung adenocarcinomas. The approach to be followed for planning analyses is not standardized in the literature. Method: We have studied by molecular methods (PCR-RFLP for K-RAS, direct sequencing for EGFR and FISH for ALK) 185 formalin fixed, paraffin embedded cases of lung adenocarcinomas of different subtypes. Results: Based on rational, biological and economic considerations we started with K-RAS analysis by PCR-RFLP, which showed mutations in 38 cases (37 with mutation on codon 12 and one case with mutation on codon 13), The 147 residual cases were all analyzed for EGFR mutations. FISH for ALK translocation followed in EGFR-wild cases. Conclusion: The analysis for K-RAS mutations allows to select the significant percentage (approximately 25 % of adenocarcinomas) when EGFR mutation analysis by direct sequencing can be postponed. K-RAS and EGFR wild cases will then undergo FISH analysis for ALK translocation. ACKNOWLEDGEMENTS Study conducted with the support of the following project: Project PERSOTHER -SMIS-CSNR: 549/12.024; With the support of Sectoral Operational Programme "INCREASE OF ECONOMIC COMPETI-TIVENESS" Priority Axis 2: Research, Technological Development and Innovation for Competitiveness.
OFP-17-005 Gene mutation analysis in adenosquamous carcinomas of the lung M. Comanescu * , G. Bussolati, C. Ardeleanu, L. Daniele, G. Gaina, C. Luca, A. Sapino * Institutul Victor Babes, Dept. of Pathology, Bucharest, Romania
Objective: Adenosquamous carcinomas of the lung constitute a rare and aggressive variant of lung cancers. The pressing interest in evaluating the mutational status in lung carcinomas for predicting responsiveness to targeted therapies is presently focused on adenocarcinomas (of different sub-types), which makes preliminary histological typing a crucial step in order to select cases to be genetically analyzed. Hence the interest in deciding if adenosquamous carcinomas should be included among adenocarcinomas or, viceversa, if they should be interpreted as a variant of squamous cancers and excluded from the process of gene analysis. Method: We have thus collected, from our two Institutions a large number (45 cases) of cancers showing the histological definition of adenosquamous carcinomas according to the WHO criteria and performed gene analysis for k-RAS (codons 12, 13) and EGFR (codons 18, 19 and 21) mutations. The detection of rearrangements of the ALK gene by FISH was also performed.
The results indicate that k-RAS and, specifically, EGFR mutations are detectable in a fraction of these tumors.
In conclusion, adenosquamous carcinomas should not be denied the chance of genetic analysis eventually leading to a targeted therapy. Acknowledgement Project PERSOTHER -SMIS-CSNR: 549/12.024 Romania.
OFP-17-006 Pathology of the lung progenitor cells and their niches in idiopathic interstitial pneumonias and lung sarcoidosis S. Demoura * , E. Kogan, V. Tuong, O. Kichigina * Moscow, Russia
Objective: The aim of the study was to investigate the morphological and the molecular-biological changes of the lung progenitor cells and their niches in idiopathic interstitial pneumonias (IPF) and lung sarcoidosis (LS). Method: We performed an immunohistochemical study on open lung biopsies from 250 patients (usual IPF -87, desqvamative interstitial pneumonia-37, nonspecific IPF -42, Cryptogenic IPF -20, LS -32, control -34 patients). Immunochemistry was done on step paraffin sections with monoclonal and polyclonal antibodies: Apo-Cas, PCNA, 18, 19, PDGF, FGF, IGF II, CD 34, MMP 1, 2, 7, 9, TIMP 4, CD 68, SMA, EMA. Results: Deep injury of Clara cells together with pneumocytes II and their nishes were found in usual IPF, SK with prominent IPF and desqvamative interstitial pneumonia. Myofibroblast proliferation, neoangiogenesis, adenomatosis and fibrosis with high production of TNFα, TGF-β, PDGF, FGF, IGF II, CD 34, MMP 1, 2, 7, 9, TIMP 4 accompanied proliferation of these epithelial cells. While in other variants of IPF pathological changes were localized in the interstitium, vessels and basal bronchiolar epithelial cells. Conclusion: Localization of injury and inflammation in progenitor cells niches results in pathologic reparation, sclerosis and precancer lesions.
OFP-17-007 A small immunohistochemical panel allows for accurate diagnosis of primary and metastatic lung cancer in biopsy specimens D. Felizardo * , R. Henrique, A. L. Cunha * Instituto de Oncologia Porto, Dept. of Pathology, Coimbra, Portugal
Objective: Precise subclassification of lung cancer, mostly performed in biopsy or fine needle-aspiration specimens, is required for appropriate therapy. Moreover, distinction between primary and metastatic carcinoma is critical. Thus, the role of immunohistochemistry (IHC) has been emphasized, although an optimal IHC diagnostic algorithm has not been firmly established. Herein, we evaluated the performance of the IHC panel used at our institution in subclassification of lung cancer and identification of metastatic carcinoma. Method: Cases of non-neuroendocrine lung carcinoma, diagnosed from March 2011 to April 2012 were selected. IHC was performed for CK7, CK20, TTF-1 and p63. Resection specimens were compared with the respective biopsy. Results: Of 184 cases analysed, 73 (39.7 %) were diagnosed as lung adenocarcinoma, 59 (32.1 %) as epidermoid carcinoma, 22 (12 %) as other forms of NSCLC, and 17 (9.2 %) as metastasis. Importantly, 26 % of primary lung adenocarcinomas were initially suspected to be metastasis. In 26 cases submitted to surgical resection, 22 (84.6 %) were correctly diagnosed in the biopsy, revealing a substantial agreement (κ-value=0,757). Conclusion: Our IHC panel allows for reliable subclassification of lung carcinomas in most cases and is decisive for appropriate diagnosis in patients suspected of lung metastasis, which is critical issue in a cancer institute.
OFP-17-008 Rationale for treatment of metastatic squamous cell carcinoma of the lung using FGFR1 Inhibitors F. Göke * , A. Franzen, A. Schroeck, V. Scheble, R. Kirsten, R. Menon, D. Goltz, D. Boehm, W. Vogel, S. Perner * Universitätsklinik Bonn, Inst. für Pathologie, Germany
Objective: We previously identified amplified fibroblast growth factor 1 (FGFR1) as a therapeutic target for small molecule inhibitor (SMI) therapy in squamous cell lung cancer (L-SCC), resulting in currently running clinical trials treating patients with stage III disease. As most patients present with metastatic stage of disease, we attempt to demonstrate FGFR1 amplification in lymph node metastases of amplified primary tumors. Our study aims to give a rational to include these patients in a targeted SMI therapy. Method: We assessed 75 formalin-fixed paraffin-embedded (FFPE) primary L-SCC samples. 46 samples were primary tumours with corresponding FFPE lymph node metastasis. The biotin-labelled FGFR1 target probe (8p11.23 to 8p11.22) was used to determine the FGFR1 amplification status performing fluorescence in situ hybridization (FISH). Results: Of 39 assessable metastatic L-SCC, 7 samples displayed FGFR1 amplification (18 %). All of these primary tumors also harbored FGFR1 amplification in their lymph node metastasis. Non-amplified tumors never displayed FGFR1 amplification in corresponding metastases. Conclusion: We found FGFR1 amplification not only in primary L-SCC, but also in corresponding lymph node metastasis, suggesting that this genetic aberration is a clonal event in tumor genesis. Our study provides data indicating new therapeutic possibilities for patients suffering not only primary, but also metastatic FGFR1 amplified SCC lung cancer disease. KRAS mutation was a G to T transversion in 80 % of the smoker NSCLC population and a G to A transition in 98 % of the non smoker NSCLC population. Mutations were noted at codon 12 (90 %), codon 13 (8 %) and codon 61 (2 %). A KRAS mutation was a negative prognostic factor with a hazard ratio for death of 1.38 (95 % confidence interval, 1.16-1.63). A mucinous histological subtype was observed in more than 75 % of KRAS mutated tumours. Conclusion: KRAS oncogene substitution must be accurately determined in primary lung adenocarcinoma for correlation with tumour behaviour and clinico-pathological parameters. Objective: There is an urgent need for diagnosis of NSCLC at its early-stages and for improving the survival rate of patients. MicroRNAs, small non-coding RNAs, are frequently deregulated in NSCLC. This study aimed to explore plasma microRNAs for diagnostic value, and evaluated the correlation between expression profiles of plasma micro-RNAs and disease-free survival (DFS) in NSCLC patients. Method: We selected eighteen most frequently expressed microRNAs in NSCLC. Total plasma RNA including microRNAs was isolated and reverse-transcribed into cDNAs. The level of microRNAs was determined by quantitative real-time RT-PCR in 42 resectable NSCLC patients and 10 matched cancer-free individuals. The correlation between the expression of microRNAs in plasma and patient DFS were examined by log-rank and Cox analysis. Results: Expression levels of miR-320,−296,−145,−199a, −191,−223,−24,−152,−126, and let-7f in the plasma of NSCLC including stage-I patients were significantly higher compared with controls (P<0.0001). The combination of these microRNAs yielded 87 % sensitivity and 90 % specificity (AUC=0.934) in discriminating NSCLC patients from controls. The levels of miR-155, −152, −20a, −223, −126 and miR-199a were significantly associated with DFS (P<0.05). Conclusion: Our results suggest that high expression of 6plasma miRNAs signature would provide potential noninvasive blood-based biomarker for the prognosis of NSCLC.
Monday, 10 September 2012, 14.15 -16. Objective: Intratumoral disorganized neo-vasculature induces oxygen fluctuations which contribute to tumour growth and metastatic potential. Although the activation by hypoxia of the carbonic anhydrases CAIX and CAXII is well known, responses of these proteins under reoxygenation remain to be elucidated. Method: In this study we evaluated the effects of hypoxiareoxygenation on CAIX and CAXII expression and cell proliferation in A549 and H1975 lung adenocarcinoma cell lines. We further investigated by immunohistochemistry on tissue microarray the value of the combined expression of these proteins to predict outcome in 552 NSCLC patients. Results: CAIX expression was maintained at high level after reoxygenation in contrast of the rapid CAXII downregulation, whereas the cell proliferation rate was significantly increased. Survival analyses showed that high CAIX/ low CAXII was associated with high cumulative incidence of relapse and with poor overall survival of NSCLC patients (P<0.05). Conclusion: Our results provide insight into understanding dynamic responses of CAIX and CAXII expression under tumour cells reoxygenation and demonstrate a critical role for reoxygenation on CAIX and CAXII levels that may select for aggressive lung cancer phenotype. These findings suggest that CAIX and CAXII play selective roles in tumour progression and emphasize their significant prognostic and potential therapeutic value.
OFP-18-002 ALK-gene rearrangement: A comparative analysis on Circulating Tumour Cells (CTCs) and tumour tissue from lung adenocarcinoma patients M. Ilie * , E. Long, C. Butori, V. Hofman, C. Coelle, V. Mauro, K. Zahaf, C.-H. Marquette, J. Mouroux, P. Paterlini-Bréchot, P. Hofman * CHU de Nice, LPCE, France
Objective: Until now the ALK status in CTCs isolated from lung cancer patients has not been characterised. We assessed the ALK status in CTCs detected in lung cancer patients and correlated the results to the ALK status defined in the corresponding tumour tissue. Method: 87 lung adenocarcinoma patients showing CTCs isolated using the Isolation by Size of Epithelial Tumour cell method were screened for their ALK status both in tumour samples and in CTCs. ALK break-apart fluorescence in situ hybridisation (FISH) and immunochemistry using an anti-ALK antibody were done on CTCs and compared with results obtained on corresponding tissue specimens. Results: 5 patients showed ALK-gene rearrangement and strong ALK protein expression in CTCs and corresponding tumour samples. Negative results were found for 82 patients in CTCs and corresponding tumour samples. Conclusion: We demonstrate that the ALK status can be determined in CTCs from lung cancer patients both by immunocytochemistry and FISH analysis. A strong correlation was found for the ALK status obtained from corresponding tissue specimens. These results favour non-invasive, ALK-gene status pre-screening on a routine basis on CTCs isolated form lung cancer patients and open new avenues for real-time monitoring for adapted targeted therapy.
OFP-18-003 Detection of EGFR mutations and EML4-ALK rearrangements in lung adenocarcinomas using archived cytological slides E. Imyanitov * , N. Mitiushkina, A. Iyevleva, A. Poltoratskiy, A. Ivantsov, A. Togo, I. Polyakov, S. Orlov, D. Matsko, V. Novik * N.N. Petrov Institute of Oncology, St. Petersburg, Russia Objective: While the molecular analysis of EGFR and ALK in archival lung cancer tissues is relatively well established, the acceptability of genetic investigation of cytological material in clinical routine remains a subject of debate.
Method: Islets of malignant cells were visually located on the archived cytological slides, lysed in situ by the drop of SDS-containing buffer, and subjected to the standard DNA and RNA extraction. Examination of paraffin-embedded tissue blocks from the same patients was done in parallel. Results: 75 cytological/histological lung adenocarcionoma sample pairs underwent the analysis for EGFR mutation. 2 cytological and 1 morphological samples failed to produce DNA. Concordance for the wild-type and mutation status was observed in 53/72 and 14/72 informative pairs, respectively; 1 pair was non-interpretable; 3 and 1 pairs had mutation only in the cytological or histological material, respectively. RNA extraction followed by RT-PCR analysis for the EML4-ALK translocation was done for the 51 pair; failures were observed for 3 cytological and 8 histological samples. 34/40 informative pairs were concordant for the norm, 3 contained identical translocations, and 2 were noninterpretable. 1 pair demonstrated ALK rearrangement in the tissue block but not in the histological slide. Conclusion: Archived cytological slides appear to be well suitable both for EGFR and ALK analysis. Objective: Non-small cell lung cancer (NSCLC) represents a heterogeneous group of cancers consisting mainly of squamous cell carcinoma (SCC) and adenocarcinoma (AD). Increased sirtuin (SIRT-1) expression leads to deacetylation of p53 that could be important in the pathogenesis of lung cancer. Differences of the molecular mechanisms in NSCLC subtypes may follow subtly different pathways to tumorigenesis. The aim of our study was compare SIRT-1 expression in lung adenocarcinoma, squamous cell carcinoma and control group. Method: 25 patients with stage 2 lung cancer were enrolled in the study. 14 patients had adenocarcinoma, but 11 patients had squamous cell carcinoma. Lung tissue for control group were selected from 15 autopsy cases. Immunohistochemical and western blot methods were used to evaluate SIRT-1 expression in lung tissue. Results: Obtained results showed that patients with lung cancer had increased SIRT-1 expression compared to control group (146±111 vs. 21±16 cells/mm2, p<0.0001). In addition, patients with squamous cell carcinoma had increased SIRT-1 positive cells compared to patients with adenocarcinoma (203±143 vs. 95±49 cells/mm2, p=0.03). Conclusion: Lung cancer is characterized by an increased SIRT-1 expression which ir more prominent in squamous cell carcinoma. Objective: Transplantation is the only treatment for several end-stage lung diseases but limited by chronic allograft dysfunction particularly obliterative bronchiolitis (OB) and its correlate bronchiolitis obliterans syndrome. The development of preclinical models is crucial to better identify immunological/non-immunological mechanisms leading to OB. [group B and C] post-transplant, animals were sacrificed. In animals from group C, cyclosporine was administered at sub-optimal dose. Lung rejection was graded according to the working formulation of ISHLT and the presence of circulating donor-specific (DSA) antibodies determined by flow cytometry. Results: In group A, acute rejection (AR) or OB occurred in 33 % and 17 % of animals, respectively. AR occurred in 33-44 % of animals in group B and C respectively. OB was detected in 33 % and in 44 % of group B and C respectively. High levels of DSA IgG were observed in cases with AR. Conclusion: A novel model of pulmonary OB was developed in the rat. To obtain a reproducible onset of OB, shortterm and sub therapeutic cyclosporin administration appears indispensable, at least in our species combination.
OFP-18-007 Epithelial dysplasia and lung cancer in end-stage idiopathic pulmonary fibrosis: Padova experience N. Nannini * , F. Lunardi, E. Balestro, E. Rossi, M. Loy, M. Saetta, F. Rea, F. Calabrese * University of Padova, Italy
Objective: Idiopathic pulmonary fibrosis (IPF) is associated with increased risk of lung cancer. The prevalence of high grade dysplasia/lung cancer was studied only in a small number of IPF patients. The aim of our study was to investigate the prevalence of precancerous/cancerous changes and their relationship with both metaplastic changes and clinical data. Method: Native lungs from 66 IPF patients were studied. The degree of honeycomb changes and squamous, cuboidal and bronchial cell metaplasia were graded (score: 0-3). The presence or absence of precancerous/cancerous changes were also evaluated. Results: Three patients showed neoplastic transformation (4 %) and nine high grade dysplasia (14 %) ("cancer" group). The "cancer" group had similar smoking history, sex, age and duration of disease than the "no cancer" group. All lungs showed metaplasia, the score of squamous (p= 0.0001), cuboidal (p=0.018) and bronchial cell (p=0.018) metaplasia was significantly higher in the "cancer" than in the "no cancer", while the honeycomb score was similar in the two groups. Conclusion: Advanced IPF patients have a high prevalence of high grade dysplasia/lung cancer, complex epithelial metaplasia, particularly squamous type, is more frequent in "cancer" group, independently from all clinical parameters, including smoking history.
OFP-18-008 Primary pulmonary adenocarcinoma with enteric differentiation, morphologically indistinguishable from metastatic colorectal adenocarcinoma: Case report with a history of metastatic colon adenocarcinoma S. Percinel * , P. Celepli, H. Nalbant, Y. Yuyucu Karabulut * Ankara, Turkey
Objective: According to a computerized medline search in the English literature, the present case is thought to be the first primary pulmonary adenocarcinoma with enteric differentiation, completely resembling metastatic colorectal adenocarcinoma morphologically, with a history of metastatic colon adenocarcinoma. Results: A 62 year old man with a history of colon adenocarcinoma which was detected in 2007 underwent radiologic evaluation. A chest computed tomography scan revealed a 7 mm solitary nodule in the left lower lobe which enlarged in 1 year. The patient underwent left lower lobe wedge resection. Macroscopically, an irregular whitish nodule of 1.5 cm in greatest diameter was detected. Microscopically, the nodule was entirely composed of glandular and papillary structures, some of which had a cribriform pattern, lined by tumor cells that were cuboidal to tall columnar with nuclear pseudostratification, eosinophilic cytoplasm, brush-border, luminal necrosis and nuclear debris. Tumor cells were diffusely positive for cytokeratin (CK)7 and negative for thyroid transcription factor-1, surfactant protein-A, CDX-2 and MUC2. Only a very few tumor cells stained for CK20. Conclusion: Enteric morphology with consistent expression of CK7 and a scattered positivity for CK20 helped in the distinction from metastatic colon adenocarcinoma and favored the diagnosis of primary pulmonary adenocarcinoma with enteric differentiation.
Expression of CXCR4 and CXCL12 in pulmonary carcinoids S. Seiwerth * , L. Brcic, A. Sepac, A. Zanko * Zagreb School of Medicine, Pathology, Croatia
Objective: CXCR4 and its chemochine ligand CXCL12 seem to play an important role in the process of tumor metastasis and, possibly, homing of metastatic tumor cells. The chemochine seems to be responsible for creating microenviromental predispositions for survival of metastatic cells, in a similar way it is done for developing immuno-competent cells. In contrast, expression of CXCL 12 in primary tumor cells, is thought to be associated with lower metastatic potential. CXCR4 is expressed in a wide range of tumors, and is thought to be crucial for the metastatic process and tissue-specific spread firstly of breast and prostate cancer. The role of CXCR4 signaling has been poorly evaluated in carcinoids in general and not at all in pulmonary carcinoids. Method: Immunohistochemically we investigated the expression of CXCR4 and CXCL12 in pulmonary carcinoids. Results: Together 60 tumors (47 typical and 13 atypical) where investigated. In 6 there was a metastatic process (in 4 typical and 2 atypical). Ligand CXCL12 expression was negative in all of the metastatic tumors and in two without known metastasis in contrast to only two non-metastatic tumors showing negative reaction. CXCR4 positivity was found in both metastatic and non-metastatic carcinoids. The other investigated parameters where present in both metastatic and non-metastatic tumors. Objective: To evaluate COL V and decorin expression in pulmonary tissue and to characterize biochemical profile of COLV from lung fibroblasts culture from SSc patients. Method: We evaluated COL V and decorin expression and tridimensional reconstruction (3D) of 6 patients with SSc without pulmonary hypertension that underwent surgical lung biopsy and as control was obtained lung fragments from 6 normal individuals who died from trauma. COL V amount in lung sections was evaluated with immunofluorescence. To biochemical characterization of COL V from lung fibroblasts culture was used quantitative immunoblot. Results: It was found that the structure of COL V fibers was distorted and strongly thickened in lung tissue from SSc patients compared with thin fibers pattern in the healthy controls. Decorin was distributed around COL V fibrils in the bronchovascular interstitium and vascular walls. Histomorphometric analysis of SSc lung demonstrated increased expression of both COL V and decorin when compared to the control (p < 0.01). The semiquantitative imunoblot detected an increased high molecular weight COLV fraction in patients when compared to the control. Conclusion: The over expression and unusual organization of COLV fibers with biochemical changes associated to increased decorin indicates that matrix signalization pathway is involved in COLV fibrillogenesis process in SSc pulmonary fibrosis. Objective: Probe-based confocal laser endomicroscopy (pCLE) is a new method used during bronchoscopy by means of special miniprobe Alveoflex and based on the visualization of intraalveolar structures which possess autofluorescence. Aim: to compare the visual signs of a healthy and pathologically changed lung tissue received at pCLE in patients with infiltrative and local lung nodules with the diagnosis, delivered by light microscopy. Method: An autopsy and surgical material was fixed in 10 % neutral formalin solution and was analyzed by studying with a new method for visualization of structures. Histological specimens were studied at that spaces, where the pCLE was applied. We compared our results by using qualitative method. Results: Normal lung tissue structures include alveolar septum with the high light emission and light-negative spacesthe alveolar spaces. In case of pneumonia alveolar septum were saved, but the light density of alveolar spaces was higher in compare with the normal tissue. In case of alveolar proteinosis we observed unique globules, which had the highest light emission. We found out several authentic signs, which are representative for each kind of pathological feature. Moreover, we revealed some other characteristics, which help us to distinguish some types of lung cancer. Conclusion: pCLE can be used as an additional method of noninvasive diagnostics in vivo.
Increased copy nember of alk gene is not associated with increased immunoreactivity of alk protein in lung adenocarcinomas T. Balharek * , A. Farkasova, Z. Kviatkovska, K. Scheerova, P. Tilandyova, Z. Hutka, L. Plank * Comenius University, Dept. of Pathology, Martin, Slovakia
Objective: Anaplastic lymphoma kinase (ALK) gene rearrangements represent an important predictive marker and promissing therapeutic target in small subset of non-small cell lung carcinomas (NSCLC). Immunohistochemical (IHC) screening of ALK abnormalities in NSCLC was reported to have variable reliability. We analyzed association between ALK immunoreactivity and increased number of ALK gene copies often seen in NSCLC. Method: We examined 10 clinically selected EGFR negative lung adenocarcinomas. ALK protein expression was detected by IHC using antibody clone ALK1 (Dako). ALK gene status was assessed by fluorescent in situ hybridization using LSI ALK Dual Color Rearrangement probe (Abbott) and SPEC ALK/EML4 TriCheck probe (ZytoVision). Results: Increased ALK gene copy number was identified in 7 cases, 3 of them showed cytoplasmic ALK positivity. In 2 of these cases we detected rearrangement of ALK locus, once represented by ALK-EML4 fusion combined with more complex cytogenetic abnormalities. Remaining third ALK + case was negative for ALK rearrangement. Conclusion: IHC seems to be useful method for initial screening of ALK rearrangements in NSCLC. There is no clear association between ALK protein expression and number of ALK gene copies. Prognostic relevance of ALK copy gains or amplification in lung adenocarcinomas remains to be determined, together with role of ALK-inhibitors in those cases.
Expressions of EGFR, ERCC-1, ß Tubulin III, RRM-1 in advanced non-small cell lung carcinoma N. Bassullu * , E. Namal, I. Turkmen, R. Yasar, P. Y. Korkmaz, Z. Akcali, G. Demir, G. B. Dogusoy * Istanbul Bilim University, Dept. of Pathology, Turkey
Objective: EGFR, ERCC1, RRM1 and βTubulinIII predicts sensitivity to therapeutic agents and provide prognostic information in Nonsmall cell lung cancer (NSCLC) which is the most frequent worldwide cause of cancer death. Method: We investigated the expressions of EGFR, ERCC1, βTubulinIII, RRM1 immunohistochemically in 42 advanced NSCLC cases and their correlations with other pathologic features. Results: The age distribution was 30-82 with an average of 63,02. Male/female ratio was 34/8. ERCC1 protein was detected in nuclei of carcinoma cells in 38 patients (90,5 %) (H score >1). ΒTubulinIII expression was detected in cytoplasm of cancer cells in 41 patients (97,6 %) (score ≥ 50). ERCC1 and βTubulinIII expression were not associated with pathological factors. RRM1 was negative (score<9) in 29 (69 %) cases and negativity was significantly correlated with male gender (p<0,032). EGFR was negative (score<200) in 40 (95,2 %) cases and negativity was nearly correlated with absent necrosis (p<0,065). No significant correlations were found between EGFR, ERCC1, βTubuli-nIII, RRM1 and the pathological parameters. Conclusion: In our study, as a first step, EGFR, ERCC1, βTubulinIII, RRM1 showed no significant correlation with pathologic features. We look forward to obtain further results in the next study consisting of correlations with follow ups.
Proliferative markers in idiopathic pulmonary fibrosis: Clinical, radiological and functional significance E. Parra * , M. Cornati, V. Capelozzi * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: Natural course of idiopathic pulmonary fibrosis (IPF) could be predicted by proliferative markers of the fibrotic process, such as myofibroblasts and interleukins (IL)-13 and IL14. Our primary aim was to determine whether these proliferative markers influence the course of IPF course measured by a radiological/functional score. Method: Twenty-eight patients with biopsy-proven IPF disease, who underwent pulmonary evaluation by high-resolution computed tomography (HRCT) fibrosis score and pulmonary function tests were studied. Five normal lung tissues (NLT) were included Biomarkers in lung tissues were detected by immunohistochemistry and quantified by histomorphometry for myofibroblasts alpha-smooth muscle actin (α-SMA), anti-interleukin (IL)-4 and IL-13. Results: Myofibrobalst amount, IL-4 and IL-13 expression were higher in IPF than in NLT (p<0.01). Myofibroblast expression of α-SMA was positively correlated to IL-14 and IL-13 expression. Lung tissue from patients with high HRCT fibrosis scores expressed significantly greater α-SMA+, IL-4 and IL-13 when compared with patients with low HRCT fibrosis scores (p<0.05). Negative correlations were found between myofibroblasts α-SMA + and VC and DLCO. Conclusion: Proliferative markers, detected by immunohistochemistry, in lung tissue allowed recognizing a dichotomous distribution of HRCT fibrosis course and influenced pulmonary function tests, suggesting that they may be promising markers of prognosis in these patients. Objective: Epithelioid mesothelioma is the most common histologic type of the diffuse malignant pleural mesothelioma, also having the best prognosis. Although a great variety of histological patterns within epithelioid type has been described, a clear impact of histological subtyping on clinical outcome is unknown (Kadota et al., 2011.) . Here we compared median survival of six histological subtypes of epithelioid mesothelioma.
We examined hematoxylin and eosin-stained slides of 74 patients diagnosed with epithelioid mesothelioma. According to previously described predominant histological features we grouped them into six subtypes: trabecular, solid, microglandular, tubulopapillary, micropapillary and pleomorphic. Results: The median survival of all 74 patients with epithelioid mesothelioma was 14.5 months. The best median survival was in trabecular and micropapillary subtypes (each 18 months, n=20 and n=5, respectively), followed by tubulopapillary (17 months, n=18), microglandular (16 months, n=7) and solid (11 months, n=19) subtypes. The worst median survival was in pleomorphic subtype (5 months, n=5). Conclusion: Epithelioid type of diffuse malignant pleural mesothelioma shows a great diversity of histological patterns that likely have an impact on the clinical outcome and patient's survival. Further investigations of genetic variations among different subtypes may provide valuable information for better understanding of pathogenesis of these tumors. Objective: Genetic instability resulting in both aneuploidy and polyploidy are discussed to be involved in prostate cancer (PCa) development and progression. Aim of this study was to comprehensively characterize the ploidy status and proliferation levels in PCa with regard to disease progression. Method: Using FISH, we assessed 112 localized PCa, 75 PCa with 125 corresponding lymph node metastases, and 42 hormone-refractory distant metastases for losses and gains of all 24 chromosomes. The proliferation rate was assessed using pHH3 and Ki67 immunohistochemistry. Results: We observed significant increases in aneuploidy with advancing tumor stage (p<0.05). Chromosomes X, 21, Y, 14, 16, and 8 were most frequently numerically altered. Increased levels of proliferation were significantly associated with the extent of aneuploidy and tumor stage (p<0.01). Combining aneusomy of chromosomes 4, 6, 20, and X with PHH3 immunoreactivity resulted in a prediction model for lymph node metastases with a sensitivity of 73.3 % and a specificity of 72.6 %. Conclusion: We present evidence that genomic instability leading to aneuploidy is an important factor in PCa progression. Furthermore, we demonstrate that increased Ki-67 and PHH3 expression are potential indicators of metastatic disease. Lastly, we suggest a new approach to preoperatively determine lymph node metastasized disease in PCa.
OFP-20-002 ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer: A comparative study of two monoclonal antibodies M. Braun * , D. Goltz, Z. Shaikhibrahim, W. Vogel, D. Boehm, A. Dobi, N. Wernert, G. Kristiansen, S. Perner * Universitätsklinik Bonn, Inst. für Pathologie, Germany
Objective: Overexpression of the ERG protein is highly prevalent in prostate cancer (PCa) and most commonly results from gene fusions involving the ERG gene. Recently, an N-terminal epitope targeted mouse and a Cterminal epitope targeted rabbit monoclonal anti-ERG antibody have been introduced for the detection of the ERG protein. Here, we are the first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their concordance on the same PCa cohort. Furthermore, we assessed if the ERG protein expression is conserved in lymph node and distant metastases. Method: We evaluated tissue microarrays of 278 specimens containing 265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. We correlated the ERG protein expression with the ERG rearrangement status using an ERG break-apart fluorescence in-situ hybridization (FISH) assay and IHC of both ERG antibodies. Results: ERG protein expression and ERG rearrangement status were highly concordant regardless of whether the mouse or rabbit ERG-MAb was used (97.8 % versus 98.6 %, respectively). Conclusion: This is the first study to comprehensively compare the two ERG-MAbs. By demonstrating a broad applicability of IHC to study ERG protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application.
OFP-20-003 Improved method of detecting the ERG gene rearrangement in prostate cancer using combined dual-color chromogenic and silver in-situ hybridization M. Braun * , J. Stomper, D. Boehm, W. Vogel, N. Wernert, G. Kristiansen, S. Perner * Universitätsklinik Bonn, Inst. für Pathologie, Germany
Objective: The recently detected ERG rearrangement revealed as a recurrent and prevalent prostate cancer (PCa) specific event. To detect this alteration, FISH is the method of choice. However, FISH harbors disadvantages for widespread adoption in clinical practice. Subsequently, the chromogenic in-situ hybridization (CISH) and the enzymatic metallography silver in-situ hybridization (SISH) emerged as promising bright-field alternatives. We aimed to develop a combined CISH and SISH (CS-ISH) gene break-apart assay on the example of the ERG gene. Method: We assessed and compared 178 PCa and 10 benign specimens for their ERG rearrangement status applying a dual-colour FISH and CS-ISH ERG break-apart assay on consecutive sections. Results: We observed a highly significant concordance (97,7 %) between FISH-based and CS-ISH-based results (Pearson's correlation coefficient 0.955, P<0.001). Conclusion: We demonstrate that the ERG rearrangement status can reliably be assesed by CS-ISH. Further, we confirm that the CS-ISH technique combines the accuracy and precision of FISH with the ease of bright field microscopy. We developed a tool which allows a broad spectrum of applicants to study the biological role and clinical utilization of ERG rearrangements in PCa. Moreoever, our study is the first proof-of-principle for bright-field CS-ISH gene fusion or break-apart assays.
OFP-20-004 Rearrangement of the ETS genes ETV-1, ETV-4, ETV-5 and ELK-1 is a clonal event during prostate cancer progression M. Braun * , Z. Shaikhibrahim, W. Vogel, D. Boehm, R. Menon, N. Wernert, G. Kristiansen, S. Perner * Universitätsklinik Bonn, Inst. für Pathologie, Germany
Objective: ETS gene rearrangements are frequently found in prostate cancer (PCa). Recently, we observed that ERG rearrangement in primary PCa transfers into lymph node metastases, suggesting it to be a clonal expansion event during PCa progression. Here, we investigated whether this also applies to the less frequent ETS genes, ETV-1, ETV-4, ETV-5 and ELK-4. Method: Using break-apart FISH assays, we evaluated the status of above mentioned ETS gene rearrangements on a cohort comprising of primary PCa, corresponding lymph node and distant metastases. Results: ETV-1, ETV-4, ETV-5 and ELK-4 rearrangements were found in 10 %, 6 %, 1 % and 2 % of the primary PCa, respectively, and in 8 %, 6 %, 6 %, 1 % of the corresponding lymph node metastases, respectively. Rearrangements of ETV-1 and ETV-5 were not found in any of the distant metastases cases, whereas ETV-4 and ELK-4 rearrangements were found in 4 % and 4 % of the distant metastases, respectively. Conclusion: Our results suggest that rearrangement of the less frequent ETS genes is a clonal event during prostate cancer progression. Our findings provide insights into potential clonal expansion events during PCa progression and may have significant implications in understanding the molecular basis of the metastatic cascade of PCa.
OFP-20-005 Comparative analysis of two prostate biopsy systems: A study of 120 cases F. E. Costa * , R. Dias, J. R. Vizcaíno * Centro Hospitalar do Porto, Braga, Portugal
Objective: It's expectable that the higher the number of prostate biopsies the greater the probability of prostate cancer detection, presumably improving the diagnostic accuracy and treatment decision. Method: Under this assumption, since May/2010, Santo António's Hospital implemented a prostate mapping system, increasing the number of biopsies performed from 8-12 to 16-18 and processing each core independently. We intend to establish a comparison between the traditional prostate biopsy system (TS) and the prostate mapping system (MS). For this study, all biopsy slides and corresponding surgical specimens from 60 patients subjected to MS and 60 patients subjected to TS were reviewed. Gleason score, percentage of tumor present in prostatic tissue (expressed as small ≤ 5 %, medium 6-19 % and large ≥20 %) and presence of tumor in prostate apex and base were analyzed. Results: Comparing MS with TS, the correlation coefficient was 53,5 % and 31,9 % for Gleason score and 46,9 % and 37,4 % for the percentage of tumor present in prostatic tissue, respectively. Mapped biopsies were able to detect the tumor in the apex and base in 72 % and 80 % of cases. Conclusion: These results suggest that prostate MS improves the diagnostic accuracy of prostate cancer and has a good ability to predict the presence of tumor in prostate apex and base.
OFP-20-006 Frequency and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers A. Fleischmann * , I. Zlobec, T. Visakorpi, G. Thalmann * Universität Bern, Inst. für Pathologie, Switzerland Objective: TMPRSS2-ERG incidence and prognostic significance in lymph node positive prostate cancer are virtually unknown. Method: A tissue-microarray was constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from all Gleason patterns (GP) present in every primary tumor (PT) and corresponding lymph node metastases (MET). TMPRSS2-ERG status was determined by fluorescence in-situ hybridization and correlated with various histomorphological tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, cancer-specific and overall survival. Results: TMPRSS2-ERG fusion was present in 43.5 % (homogeneous 25.2 %, heterogeneous 18.3 %) of the PT and in 29.9 % (homogeneous 28.7 %, heterogeneous 1.2 %) of the MET. Percentage of TMPRSS2-ERG in GP3/4/5 of PT and MET were: 38 %/37 %/24 % and 23 %/30 %/39 %. Concordance in TMPRSS2-ERG status between PT and MET was poor (Kappa 0.39) showing 20.9 % and 8.1 % of cancers with gene fusion solely in the PT and MET, respectively. TMPRSS2-ERG fusion was not correlated with histopathological tumor features and predicted late biochemical recurrence independently (p=0.041) when present in PT. Conclusion: TMPRSS2-ERG fusion in PT is more frequent and its distribution more heterogeneous compared to MET. The gene fusion in primary tumors independently predicts late biochemical recurrence.
OFP-20-007 WNT and SHH pathways activation in penile carcinoma A. Silva * , T. Almeida, F. Soares, M. Buim * Hospital AC Camargo, Investigative Pathology, São Paulo, Brazil
Objective: Penile carcinoma (PC) is rare in developed countries, accounting for less than 1 % of all neoplasms in men, and biological characteristics of this tumor are poorly known. WNT and SHH pathways are important for cell proliferation, differentiation and survival, and therefore, play a role in carcinogenesis of various organs. The goal of this study is to investigate the expression profile of WNT and SHH pathway proteins in PC, characterizing the expression of target proteins from WNT (Wnt-1, Wnt-2, Wnt-2 Gsk3β, β-catenin, D1 Cyclin, MMP7, C-myc, CD44) and SHH (Shh, Smo, Gli, EGFR) pathways. Method: For that, 18 samples of PC were obtained from the files of Anatomic Pathology department from A.C. Camargo Hospital (Brazil) and submitted to immunohistochemistry. Results: We observed that Wnt-1 and Wnt-2 were expressed in 15 and 16 cases, respectively, out of the 18 evaluated samples. Strong Shh expression was detected in 16/18 cases, whereas weak and negative expression was seen only in one case each. Smo and Gli-1 proteins were expressed in almost all cases (17/18), and also D1 Cyclin, b-catenin and EGFR were frequently expressed. Conclusion: These preliminary results suggest that WNT and SHH pathways may be active and participating in the progression of penile cancer.
OFP-20-008 Expression of the multidrug resistance protein 4 correlates with longer PSA relapse free survival and androgen receptor and forkhead box A protein expression in prostate cancer M. Montani * , G. Kristiansen * Medizin. Universität Bern, Inst. für Pathologie, Switzerland
Objective: Multidrug resistance protein 4 (MRP4) a transmembranary transport protein has shown to be expressed in prostate cancer cell lines and cancer cell specimens and turned out to be among the highest upregulated genes in an arraybased transcription analysis. Its expression is regulated in an androgen dependent manner, possibly modulated by forkhead box A (FoxA), an androgen receptor (AR) co-activator. Therefore, we investigated its expression in a large cohort of neoplastic and non-neoplastic prostate tissues (n = 441) and evaluated its prediction of PSA relapse free survival (RFS). Method: TMA (n=441) stained for MRP4, FoxA, AR, ER, PSA, Ki-67: radical prostatectomies (RPE) (n=332), castrate resistant prostate cancer (CRPC) (n=26), metastases (n=39) and non-neoplastic (n=187). PSA-RFS of 258 patients; mean 70 months. Results: MRP4 expression decreases with tumor progression into castrate resistant disease, correlates with PSA, AR and FoxA expression and inversely correlates with Gleason score. Moreover, a strong MRP4 expression is significantly associated with a longer PSA RFS in RPE patients. Normal tissues from the transitional zone show a weak MRP4 expression compared to the peripheral and central zones. Conclusion: MRP4 expression seems to predict PSA relapse free survival in prostate cancer patients. Since its expression is androgen dependent it decreases with tumor progression into CRPC.
OFP-20-009 Synchronous Angiomyolipoma and renal tumors in patients without Tuberous Sclerosis: Clinico-pathological study of 18 cases F. J. Queipo * , R. A. Carías, Án. F. Panizo, M. L. Gómez-Dorronsoro, F. J. Pardo * Clínica Universidad de Navarra, Pathology, Pamplona, Spain Objective: Simultaneous existence of AML and renal neoplasia is frequent in Tuberous Sclerosis (TS) patients, but uncommon in non-TS cases. Method: A total of 18 cases of coexistent renal neoplasia and AML in non-TS patients were identified. Clinico-pathological features were studied. Results: 18 patients: 10 M/8 F (mean age 62,77 year; range: 35-83) . Mean AML size: 0,82 cm (range: 0,2-3,4). The main size of the renal neoplasms was 5,09 cm (range 1,2-15). AML morphology: 6 leiomyomatous, 4 classic-triphasic, 4 lipomatous, and 4 epithelioid. Three cases had multifocal AML. 15 cases had ipsilateral tumors associated with AML: 6 CCRCC (1 sarcomatoid), 4 ChRCC, 1 RO, 1 TFE3 RCC, 1 hybrid (RO-ChRCC) renal-cell tumor, 1 urothelial carcinoma, and 1 case had 2 tumors: MTSC-RCC and concomitant PRCC. 2 cases had contralateral tumours associated with AML: 1 ChRCC and 1 RO. One patient had bilateral tumors associated with AML: an ipsilateral RO and a contralateral CCRCC. The median follow-up was 36,85 mths (range 0,4-199,8) : all patients were alive without disease. Conclusion: The coexistence of renal tumors with AML is a rare event, usually incidental. If AML is found incidentally together with other renal tumors, it is important to exclude TS retrospectively.
OFP-20-010 Primary renal synovial sarcoma: A clinicopathologic, immunohistochemical and molecular genetic study of 16 cases J. Schoolmeester * , J. Cheville, A. Folpe * Mayo Clinic, Dept. of Anatomic Pathology, Rochester, MN, USA Objective: Primary renal synovial sarcoma (R-SS) is a rare malignant neoplasm. Approximately 45 cases have been described, chiefly as case reports or as part of relatively limited small studies. We studied the clinicopathological, immunohistochemical (IHC) and molecular genetic features of 16 well-characterized R-SS. Method: All available slides for 16 institutional and consultation cases of R-SS were reviewed. An IHC panel (TLE1, BCL2, cytokeratins, S100, CD34, INI1) was performed. RT-PCR for SS18-SSX1/2 or FISH for SS18 rearrangement was performed. Follow-up was obtained. Results: The patients (9 M, 7 F) ranged from 17 to 78 years (mean 46). All tumors were of monophasic fibrous type. IHC results: all cases were strongly positive for TLE1 and BCL2; focally positive or negative for cytokeratins; negative for S100 and CD34; and showed retained INI1 expression. Molecular genetic results were: SS18-SSX1 (5 cases), SS18-SSX2 (10 cases), SYT rearrangement (1 case). Follow-up (5 cases): 3 dead of disease, 2 alive without disease. Conclusion: Our data show a striking overrepresentation of the SS18-SSX2 fusion subtype among R-SS, in contrast to other SS, in which the SS18-SSX1 fusion subtype accounts for two-thirds of cases. The reasons for this difference are unclear. The prognosis for R-SS appears similar to that of SS of more common locations. Objective: New data related to the diagnosis and therapy of lung cancer have also affected the position of the pathologist in this area. Principal changes in its classification have appeared in relation to adenocarcinoma and partly also in the large cell type of lung cancer. Method: Presented data represent a five-year retrospective study of a series of 546 cases of lung cancers analyzed in our Institute. Besides routine HE staining the following immunohistochemical reactions were used: antibody against TTF-1, Surfactant A, Chromogranin, Synaptophysin, CK7, AE1/AE3, p63, 34βE12, CD56 and Napsin A. Results: Using the WHO, 2004 Classification of Lung Cancer, an unusual phenomenon of the return of squamous cell cancer (SCC) prevalence over adenocarcinoma has been documented, which in the earlier years has equated the SCC. The difference in obtaining the tissue samples, and changes in the geographical origin of certain patients of Eastern Slovakia may partly explain these differences. Though the new Travis classification (J Thorac Oncol, 2011) was not used, there prevailed mixed forms also in our series. Conclusion: It is important to respect new information regarding the Classification of Lung Cancer, especially in the category of adenocarcinoma. The constructive collaboration of pathologists with clinicians is recommended. Objective: Extranodal marginal zone lymphoma MALT lymphoma and carcinoid are neoplasms occurring most frequently in the gastrointestinal tract and respiratory system. Although each of them occurs relatively frequently and separately, the simultaneous appearance of these two neoplasms is exceptionally rare. Method: We report an exceptional synchronous association of MALT lymphoma and typical carcinoid in the lung. Results: A 52-year-old male with Sjögren's syndrome presented with cough and chest pain with no improvement after antibiotherapy. Bronchial fibroscopy was normal. Computed tomography showed a diffuse interstitial pneumopathy with a persistent left lower lobe nodule suspected of malignancy. A surgical resection of the nodule was achieved. Histopathologically, it measuring 1.2 cm in diameter and was composed of association of malt lymphoma and typical carcinoid tumor which were focally admixed. Immunohistochemical stains were strongly reactive to endocrine marker in carcinomatous component. Tumor cells were diffusely CD20 positive in lymphomatous component. Assessment of extent of lymphoma revealed a gastric location. The patient received chemoptherapy. He's still alive since 8 months. Conclusion: The best of our knowledge, this is the first report of such a collision lung tumor at the same anatomical site. The aim of this study is to describe the pathogenesis and clincicopathological feartures of such exceptional association. Objective: Primary mucoepidermoid carcinoma is rare comprise less than 1 % of all lung tumors. It is characterized by the presence of squamoid cells, mucin-secreting cells and intermediate type. On the basis of morphological and cytological features, it is divided into low and highgrade types. Method: We report a retrospective study of 15 cases diagnosed during a 15-year period. Diagnosis was made by histological examination of specimen obtained from lobectomy in 13 cases and pneumonectomy in 2 cases. No patient had history of salivary mucoepidermoid carcinoma. Results: There were 13 men and 2 women ranging in age from 10 to 76 years. Computed tomography showed a well circumscribed tumor arising in bronchus in all cases. A bronchial fibroscopy showed a main, lobar or segmental mass. Bronchial biopsy revealed a non-small-cell carcinoma in only one case. Histologically, tumors were classified into 7 low grade mucoepidermoid carcinoma and 8 high grade mucoepidermoid carcinoma. Patients with low grade carcinoma remain alive after surgery alone. Patients with high grade carcinoma received chemotherapy, four of them developed distance metastasis. Conclusion: Mucoepidermoid carcinoma is a rare primary malignancy of the tracheobronchial tree which is difficult to diagnose by limited biopsy. The prognosis is variable and depends upon the histological grade. Objective: Primary pulmonary MALT lymphoma is considered to originate from MALT of the bronchus secondary to autoimmune or inflammatory processes. Although, it comprises more than two-thirds of all primary non-Hodgkin's lymphoma of the lungs, it is a rare entity and accounts for less than 1 % of all lymphomas. Method: We report a retrospective study of 6 cases of Malt pulmonary lymphomas diagnosed during an 8-year period. Diagnosis was made on surgical pulmonary biopsy in 2 cases, bronchial biopsy in 3 other cases and on lobectomy in one case. Results: There were 4 women and 2 men ranging in age from 48 to 74 years. Two patients had a history of Sjoren's syndrome and one had a history of previous mammary carcinoma. Computed tomography revealed bilateral nodules in 3 patients, lung mass in 2 patients and an area of consolidation in one patient. Fiberoptic bronchoscopy showed bronchial stenosis in only 3 cases. Morphologically, the neoplasms had features typical of MALT lymphoma. One patient was treated with surgery alone and 5 received chemotherapy. Five patients remained alive while one patient presented with recurrence. Conclusion: Pulmonary MALT lymphomas are characterized by an important dissociation between clinical expression and radiological pattern. Therefore, histological documentation is mandatory to ensure diagnosis. Objective: Malignant mesothelioma (MM) is an aggressive tumour with a poor prognosis. Carbonic anhydrases and their inhibitors offer an opportunity for developing novel anticancer drugs, as well as diagnostic and prognostic tools. Carbonic anhydrase IX (CAIX) is a membranous metalloenzyme involved in cell adhesion and pH homeostasis. It is a direct target of hypoxia-inducible factor and serves as a marker of hypoxia. This study was designed to assess systematically CAIX expression in MM of pleura and peritoneum, and their benign counterparts. Method: 47 MMs of pleura (41 epithelioid, 1 biphasic, 2 sarcomatoid) and peritoneum (3 epithelioid), and 14 normal or reactive pleural samples were analyzed. CAIX expression was determined using immunohistochemistry. Membranous immunoreactivity was evaluated semiquantitatively. Specimens were divided into five subgroups according to the staining pattern and intensity. Results: 95,7 % (45/47) of MMs expressed CAIX. All epithelioid mesotheliomas showed at least a weak focal (8,5 %, 4/47), but predominantly a strong diffuse (70,2 %, 33/47) staining with CAIX antibody, without any perinecrotic pattern. Sarcomatoid mesotheliomas were negative. Normal mesothelial cells were diffusely positive. Conclusion: These data suggest that mechanisms of CAIX overexpression in MM are different than due to hypoxia and appear promising in prospective use of specific therapeutic CAIX targeting in advanced mesothelioma. Objective: EML4-ALK crizotinib therapy needs validation at lower cost and rapid answer in Pathology routine. Method: Histological/WHO 2004 and CK7, TTF1, CK5.6, CD56/chromogranin and vimentin panel classifications with ALK (clone 5A4, Novocastra Laboratories Ltd, Newcastle, United Kingdom) were applied to paraffin sections of 35 bronchial-pulmonary carcinomas: 20 adenocarcinomas, 6 epidermoid carcinomas, 4 pleomorphic carcinomas (mixed type adenocarcinomas with large/giant/fusiform cells), 4 neuroendocrine carcinomas (NEC) (1 combined large cell NEC with adenocarcinoma and 2 with epidermoid carcinomas; 1 SCLC chromogranin positive combined with adenocarcinoma) and 1 adenosquamous carcinoma. Results: The applied antibodies specified bronchial pulmonary carcinomas subtypes clearly. In 3 over 60 years old nonsmoking females mixed type adenocarcinomas ALK expression was over 50 %: acinar, solid, micropapillary and microacinar patterns; one glandular mucinous pattern (mucinous BA pattern) and one BA pattern, all expressing TTF-1. Conclusion: In this study, 3/20 adenocarcinomas of older women had ALK protein expression, only one with a mucinous pattern. As protein positivity cases comprise a lower number, FISH described by S. Lantuejoul seems to be the most appropriate method. It is now necessary to decide whether KRAS and EGFR mutations have to be determined together and/or select TTF-1 positive adenocarcinomas (from terminal respiratory unit) raised by this approach. Objective:
The 5 year survival of bronchial-pulmonary carcinomas remains poor, between 6 % and 14 %/men and 7 % to 18 %/women. Treatment orientation is influenced by clinical staging and morphological classification in biopsies of 70 % of the cases. Method: This study comprised 41 surgical specimens where we compared immunohistochemistry in-between Adenocarcinomas (18), Epidermoid Carcinomas (12) and the heterogeneous groups of Large Cell Neuroendocrine Carcinoma (3), Small Cell Lung Cancer (1), Large Cell Carcinoma (2), Adenosquamous Carcinoma (2) and Pleomorphic Carcinomas (3) with Max 18 F-Fluordesoxiglucose (FDG), a clinical parameter based in PET to preview diagnosis and prognosis. Results: We found significant differences (p=0.028) between TTF-1 positive and negative Adenocarcinomas where the 18 F-FDG capture was lower in TTF-1 positive cases, indicating lower metabolic activity. TTF-1 negative Adenocarcinomas have similar and higher metabolic activity as Epidermoid Carcinomas. The other histological types have FDG capture similar in between the two defined groups. Conclusion: Immunohistochemical and 18 F-FDG analysis correlate with clinical differences between Adenocarcinomas and Epidermoid Carcinomas, where TTF-1 negative Adenocarcinomas are biologically similar to Epidermoid Carcinomas, requiring a different medical approach as well as molecular pathology particular interpretation. These results strain the classification of bronchial TTF-1 negative Adenocarcinomas because they are different from the terminal respiratory unit TTF-1 positive Adenocarcinomas. Objective: According to researchers pneumonias are characterized by increase number of alveolar macrophages population after increased migration of monocytes from bone marrow and blood into the alveolar space. Method: We examined 60 observations of pneumonia with bacteriological, histological and blood monocytes examination in all cases. Bacteriological study was performed at the Institute of antimicrobial chemotherapy. Histological examination was performed in the Smolensk regional institute of pathology. Results: In the foci of pneumonias we discovered mainly mixed gram positive and gram negative microorganisms. Ten observations were characterized by an increase in the absolute number of blood monocytes. Fifty observations did not reveal excess levels of blood monocytes. In all cases with an increase in the number of blood monocytes, gram negative bacteria were isolated. In cases with normal number of blood monocytes some of the microorganisms were gram positive. Conclusion: Gram negative bacteria result in activation of the monocytes and their entry from the bone marrow into the blood more than gram positive bacteria. Objective: Bronchial carcinoids and schwannomas are uncommon, slowly growing, low-grade neoplasms. Carcinoid tumors are thought to arise from neuroendocrine/Kulchitsky's cells of bronchial epithelium. Schwannomas are thought to originate from schwann cells. Clinically they may be asymptomatic or present with non resolving recurrent pneumonia, hemoptysis, respiratory distress. Conclusion: We present two cases of respiratory distress in two teenage patients.
Pulmonary hamartomas mimicking metastatic carcinomas H. Erdem * , L. Yilmaz Aydin, A. K. Uzunlat, M. Oktay, A. N. Annakaya, U. Yilderim, F. Basar * Duzce University, Dept. of Pathology, Turkey Pulmonary hamartomas are usually found incidentally and they mimick metastic tumours. We report a case of a 47 year-old man who admitted to Chest Disease service for cough. He had laryngectomy operation formerly and diagnosed as squamous cell carcinoma. Chest-X-ray and computed tomography revealed pulmonary nodules. It was removed but there was not metastatic carcinomas. This case was presented because it can be difficult to make diagnosis when the patient has malignancy. Objective: Experimental models of pulmonary fibrosis (PF) has been proposed and its later phase tend to the resolution in different degrees depending on the drug/ strain/inflammatory-pattern. Thus the maintenance of these mechanisms may participate in the PF-progression. Our objective was to determine the immune-fibroticpattern in models of pulmonary fibrosis in the late stage (21d). Method: Distinct animal models were used, including Balb/c, C57BL/6 and IL17RA-KO-C57BL/6 mice by Bleomycin and Paraquat. We analyzed the amounts of total peribronchiolar-interstitial collagen (TPC-TIP) by picrosirius and Col3-Col5 by immunofluorescence through the morphometric evaluation. These data were validated by RT-PCR. Results: The TPC did not differ between the treated groups. TIC was higher in the C57BL/6 strain, independent of the absence of IL-17RA. The Col5-immunoexpression was higher in control and BLM-treated IL17RA-KO-C57BL/6 than in wild-mice and lower in BLM-Balb/c mice. The Col3immunoexpression was higher in BLM-Balb/c. Likewise, the Col5 gene expression was higher in the IL17RA-KO mice and lower in the BLM-Balb/c. Conclusion: The perpetuation of fibrosis in PF-susceptiblemice can be modulated by IL-17-dependent Col5-hiperexpression and by Col3-subexpression. This suggests that Col5 is an important component responsible for the development of PF.
The prognostic role of Filamin A protein expression in patients with non-small-cell lung cancer M. Gachechiladze * , J. Skarda * Palacky University, Pathology, Olomouc, Czech Republic Objective: An actin-binding protein Filamin A (FLNA) serves as a scaffold in various signaling pathways. Recently, it has been reported that FLNA interacts with BRCA1 protein and is required for efficient regulation of early stages of DNA repair processes. As DNA repair proteins are important prognostic markers for non-small-cell lung cancer (NSCLC) patients, we aimed to investigate the role of FLNA protein expression in NSCLC, as well as its correlation with BRCA1 protein expression. Method: We performed a preliminary study of FNLA and BRCA1 protein expression in 50 NSCLC patients. Formalin-fixed paraffin-embedded tissue sections were stained by immunohistochemistry using antibodies against FLNA C-terminus (EP2405Y, LSBio), BRCA1 N-termunus (MS110) and against phosphorilated forms of BRCA1 at ser1524 and ser1423 (Abcam). Staining intensity was estimated semi-quantitatively and correlated with all available clinico-pathological factors. Results: FLNA expression was significantly higher in cancer, compared to normal lung tissue. Positive correlation has been revealed between FLNA and BRCA1 phospho-ser1423 expression. Also, 5 year overall survival rate was higher in patients with strong FLNA expression. Conclusion: According to our preliminary study results the prognostic role of FLNA protein expression deserves to be a subject for further studies in patients with NSCLC.
PS-01-014 Pulmonary nodular lymphoid hyperplasia ("pulmonary pseudolymphoma") -The significance of increased numbers of IgG4 positive plasma cells D. Guinee * , A. Gerbino, S. Murakami, M. Koss * Virginia Mason Medical Center, Pathology, Seattle, USA Objective: Pseudolymphomas of the skin, breast, and lacrimal glands show an increase in IgG4 positive plasma cells. We hypothesized that a similar increase in IgG4 positive plasma cells occurs in pulmonary nodular lymphoid hyperplasia (PNLH). Method: Immunohistochemical stains for IgG4 and IgG were performed in 2 cases of PNLH, 7 cases of BALT lymphoma, 8 cases of intraparenchymal lymph nodes (IPL) and 1 case of follicular bronchiolitis (FB). The mean number of IgG4-and IgG-positive plasma cells and the IgG4/IgG ratio was obtained from a manual count of three separate high power fields (hpfs) of areas of highest cellularity. Results: The average number of IgG4 positive plasma cells and the IgG4/IgG ratio was increased in PNLH (case 1: 225/hpf, ratio: 0.39, case 2: 280/hpf, ratio: 0.59). In comparison, average IgG4 positive plasma cells per hpf were much lower in BALT lymphoma (range=0-2.3/hpf, ratio=0-0.03), IPL (range IgG4/ hpf=0-102, ratio=0-0.23) and FB (1/hpf, ratio=0.03). Conclusion: The increase in IgG4 positive plasma cells and IgG4/IgG ratio in PNLH aids in diagnosis and supports our current understanding of PNLH as a distinct form of reactive lymphoid proliferation. The relationship between PNLH and IgG4-related sclerosing disease requires further study.
PS-01-015 Calretinin, CK5/6, TTF-1, CEA, Ber-EP4, and CD15: A useful combination of immunohistochemical markers for differentiating pleural mesothelioma from pulmonary adenocarcinoma N. Gursan * , M. Calik, E. Demirci, S. Sipal, B. Gundogdu, C. Gundogdu * Ataturk University, Medical Faculty, Erzurum, Turkey
Objective: The distinction between pleural epithelial mesothelioma and peripheral lung adenocarcinoma involving the pleura is still an important diagnostic problem for surgical pathologists. The aim of our study was to identify the most specific and sensitive markers for the positive identification of mesothelioma. Method: Paraffin-embedded blocks from surgical material of 19 pleural epithelial mesotheliomas and 22 pulmonary adenocarcinomas were retrieved from the files in our department. The primary antibodies used in each case were the following: antibody anticalretinin, EMA; CEA, BerEP4, TTF-1 and CD15. Results: Of the mesotheliomas, 100 % stained for calretinin, 63.2 % for CK5/6, 78.9 % stained for EMA and AE1/AE3. Of the lung adenocarcinomas, 9.1 % cases showed reactivity for calretinin, 27.3 % for CK5/6, 77.3 % for CD 15, all for TTF-1, 50 % for CEA, 59.1 % for Ber-EP4, 90.1 % for EMA, and all for AE1/AE3. Conclusion: Calretinin were the highly specific positive mesothelial markers, whereas CK 5/6 showed high sensitivity but low specificity. Among negative markers, we advocate the use of TTF-1, CEA and CD15 which were the most specific in differentiating mesotheliomas from adenocarcinomas.
Cyclooxygenase-2 expression in NSCLC I. Kern * , E. Sodja, M. Rot * University Clinic Golnik, Slovenia
Objective: Overexpression of COX-2 correlates with aggressive disease of NSCLC. Aim of our study was to determine the COX-2 expression levels by two methods. Method: Analysis was done on 24 consecutive surgical specimens of NSCLC fixed in Paxgene tissue system. Relative quantification of COX-2 mRNA expression was performed by quantitative RT-PCR using intron-spanning primer-probe set. COX-2 protein expression was assessed by immunohistochemistry (IHC). Scoring was performed using an intensity-extent system, both parameters on the scale 0-3 and multiplied to give IHC index. Results: There were 12 cases of adenocarcinoma, 11 cases of squamous cell carcinoma and one typical carcinoid. COX-2 mRNA expression was detectable in all specimens. The median COX-2 mRNA expression value, normalized against he internal reference gene GADPH, was 0,53 (range 0,08-12,49). We observed cytoplasmic and membranous immunohistochemical reaction patterns. Average IHC index was 3,8. There was positive correlation between mRNA and protein COX-2 expression (R2=0,31). Adenocarcinoma cases had average relative mRNA expression value 2,45 and IHC index 5,08, while squamous cell carcinoma had average relative mRNA expression value 0,67 and IHC index 2,73. Conclusion: COX-2 is expressed in NSCLC with various IHC reaction patterns. Adenocarcinoma and squamous cell carcinoma have different COX-2 expression levels. Objective: The Akt/mammalian target of rapamycin (mTOR) pathway is up-regulated in many human cancers, and agents targeting the mTOR pathway are in various stages of clinical development and application. Method: Expression of pAkt and mTOR was studied by immunohistochemical analysis of 574 surgically resected nonsmall cell lung cancer (NSCLC) specimens on a tissue microarray (TMA). Results: The results were correlated with clinicopathological features. Expression of mTOR showed a strong correlation with the expression of pAkt (p<0.001) and was significantly associated with female gender, tumor size ≤3 cm, adenocarcinoma (ADC), non-smoker status, and lower pathological stage. Expression of pAkt was correlated with older age (≥ 65), ADC, non-smoker status, and lower T stage. Univariate survival analysis revealed that the mTOR and pAkt positive group had a significantly longer cancer-specific survival than the mTOR and pAkt negative group (p=0.038 and p=0.024, respectively). Coexpression of pAkt and mTOR correlated with better prognosis than either single or double negative pAkt and mTOR groups (p=0.016). However, multivariate analysis proved that mTOR and pAkt expression are not independent prognostic factors for cancer-specific survival. Conclusion: Expression of pAkt and mTOR expression is more significantly associated with ADC than squamous cell carcinoma (SCC) and expression of these proteins is associated with better prognosis.
PS-01-018 PAK 6 immunohistochemistry marker in non-small cell lung cancer (NSCLC) A. Kucukosmanoglu * , O. D. İlara Colakkadıoglu, K. Bakir * University of Gaziantep, Pathology, Turkey Objective: PAK 6 is a member of P21-activated kinase family. Many human tumor express and activate this family. Because of their role in cell transformation, they become therapeutic targets. They have important roles in cell survival, cell proliferation and cell migration. They affect cell growth and tumor invasion. Method: 175 cases with NSCLC reported in Gaziantep University Pathology Department between 2000 and 2010, reviewed retrospectively. Immunohistochemically nuclear and cytoplasmic PAK 6 stainings were considered positive and scored according to degree of staining. Results: Our cases included 110 squamous cell carcinoma, 49 adenocarcinoma, 9 adeno squamous carcinoma and 7 large cell carcinoma. There was no significantly associated between tumor types and PAK6 staining. 38 of 175 patients showed recurrence and of 23 cases showed nuclear staining but there were no statistically significance (p:0,267). 31 cases died from the disease and in 21 of these cases nuclear stainig was very intresting, inspite of statistical results. Conclusion: In this study PAK 6 staining patern and score were compared with tumor type, size, recurrence, mortality and lymph node involvement in NSCLC. And we found no statistically significance. 150 of 175 case with NSCLC stained with PAK 6. These results suggest that PAK 6 was expressed in lung like prostat, plasenta and breast.
Pleural Fibroelastosis is a similar spectrum of Histopathology in Chronic Fibrosing Lung Disorders L. Marcal * , E. R. Parra, L. Antonangelo, V. Capelozzi, F. Vargas, E. C. Nascimento, V. Teodoro, K. C. Silva * Lia Junqueira Marçal, São Paulo, Brazil
Objective: Parenchymal fibroelastosis in chronic fibrosing lung disorders has been much investigated, but little attention has been directed at the visceral pleura (VP) participation in these situations. Our aim was to verify whether elastic deposition accompanies collagen deposition in the repairing process of chronic lung injury. In this work we studied the distribution of these fibrous components of VP in bullous disease type I and II, smoking-related interstitial fibrosis and usual interstitial pneumonia (UIP). Method: We employed histochemical methods on conventional histological slides. We measured, by image analysis, the content of fibres of the collagenous and elastic systems of the visceral pleura in histological slides sampled from surgical lung biopsies and bullectomy, using the picrosirius-polarization method and Weigert's resorcin-fuchsin stain, respectively. Results: Four groups were studied: I, 7 patients with spontaneous pneumothorax due to type I bullous disease; II, 12 patients with spontaneous pneumothorax due to type II bullous disease; III, 5 smoking-related interstitial fibrosis; and IV, 5 patients with idiopathic pulmonary fibrosis. The first two groups were used as controls. Patient diagnosed with Idiopathic pulmonary fibrosis (2009), in which experimental transplantation was performed of type II pneumocytes, ingress to the hospital for further study groundmass of the right lung. In the postmortem study a 2340 gr. mass was identified, the mass was formed by sarcomatous cells habit, undifferentiated and pleomorphic, with the presence of multiple implants and adhesions in rib cage, diaphragm, pericardial fat. IHC profile was performed: vimentin (+), EMA focally (+), WT1, CK8, TTF1, CKAE1/AE3, S100, CD34, CD31 and CD45 (−) and low proliferation index, the diagnosis undifferentiated malignant tumor. With inconclusive IHC profile, so blocks were sent to Clinic Barcelona Hospital, for study. The estimated prevalence of idiopathic pulmonary fibrosis is 20 cases per 100,000 population in men, with a mortality of 50 % at 5 years of diagnosis. Cancer rate of in transplanted patients is within the range between 4 % and 18 %. Could the appearance of this tumor be related either to transplanted pneumocytes or it is inmunosupression the main risk factor? Current treatments for idiopathic pulmonary fibrosis are not effective, which requires the study and development of new therapeutic options, using fibroblast proliferation inhibitors, as well as prospective studies, in the long term, the evolution of these patients and the development of secondary entities to it. Objective: Mediastinal hemangiomas are rare tumors accounting for 0,5 % of all mediastinal tumors. These tumors are challenging because of the lack of clinical and radiologic specific signs. Their diagnosis is mainly based on microscopic study. Method: We report a 10-year-experience of a single institution. We describe 5 cases of mediastinal hemangioma. Results: Our study contained 2 men and 3 women with a mean age of 60.4 years. Symptoms consisted mainly in chest pain. Neurologic signs were observed in 2 patients. Surgical treatment was performed in all patients dealing with a total resection in 4 patients. The most used surgical approach was a posterolateral thoracotomy. Video-assisted thoracic excision was tried in one patient and was converted into a median sternotomy. Microscopic examination concluded to a mediastinal hemangioma in all cases. All the patients presented no complications after a follow up periods varying from 9 months to 2 years. Conclusion: Mediastinal hemangiomas are rare tumors whose diagnosis is based on microscopic findings. Their surgical management may be challenging because of their connection to the adjacent structures. Video-assisted thoracic excision is being more frequently used by experienced surgeons. These tumors are benign with a good behavior. Objective: Actinomycosis is an infectious disease caused by Actinomyce israeli in 85 % of the cases. It is mainly observed in alcoholic patients and affects mainly cervicofacial, abdominal and thoracic regions. Pulomnary actinomycosis accounts for 15 % of the localizations and is caused by the inhalation of septic particles causing granulomatous lesions, the extension to the adjacent organs and a cutaneous fistulae. It represents a real pitfall and mimics malignant lesions, tuberculosis or other infectious diseases. Method: We report a retrospective study about 6 cases of pulmonary actinomycosis diagnosed over a 17-year-period. Results: We describe the cases of 6 men aged between 35 and 46 years who presented with respiratory signs. Radiologic investigations showed in all cases parenchymal masses with irregular margins mimicking malignant processes. Positivie diagnosis was based on microscopic examination. The treatment was based on surgical excision associated to a medical treatment without complications after a follow up period ranging from 3 months to 18 months. Conclusion: Pulmonary actinomycosis shares usually the same clinical and radiologic features as malignant lesions.
Positive diagnosis is based on microscopic findings. It inducesusually surroundng organs and may affect, in some cases, the vital prognosis.
Over expression of hyaluronan syntehase-2 activity has impact in the remodeling process and survival evolution in patients with idiopathic pulmonary fibrosis E. Parra * , V. Capelozzi * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: The idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting extracellular matrix (ECM) deposition in the lung. In this regard, the myofibroblasts and the ECM components such as collagen and hyaluronan (HA) have an important role in the fibrosis. We analyzed the expression of HAS1 (HA synthase 1), HAS2, HAS3 and hyaluronic acid receptor (CD44) by epithelial and myofibroblasts cells in patients with IPF and we correlated with a survival. Method: HAS-1, HAS2, HAS3 and CD44 epithelial and myofibroblast expression were evaluated in 27 surgical lung biopsies from patients with IPF in minimal and severe fibrosis by the point-counting technique. Impact of these markers was tested on pulmonary functional tests and follow-up until death from IPF. Results: HAS2 and CD44 expression were significantly increased and directly associated with severe fibrosis. Myofibroblast HAS2 activity was indirectly associated to DLO/VA (r= −0.584; p=0.05). Kaplan Maier curves determined a higher risk of death for patient with high HA2 (>6.83 %) expression than in low expression (Log Rank p=0.05, Figure) . Conclusion: The increased HAS-2 activity in epithelial and myofibroblast cells have impact in the remodeling process and the survival evolution, suggesting that strategies aimed at preventing the effect of this ECM component may have a greater impact in patient's outcome. Financial Support: FAPESP, CNPq.
PS-01-024 Viral antigens are more frequently observed in acute interstitial pneumonia than in other types of idiopathic interstitial pneumonias E. Parra * , G. C. dos Santos, F. Weisshaupt Stegun, C. dos Santos Cirqueira, M. I. Seixas Duarte, V. L. Capelozzi * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: The etiology of idiopathic interstitial pneumonias (IIPs) remains unclear. Many researches assumed that viruses can represent an important factor of aggression to the lung provoking fibrosis. In this regards, we researched the presence of virus infections in patients with IIPs. Method: Biopsy samples from 13 patients with idiopathic interstitial pneumonia (IPF); 8 patients with nonspecific interstitial pneumonia (NSIP); 13 patients with acute interstitial pneumonia (AIP) and 4 patients with idiopathic cetrilobular fibrosis (ICLF) were used to investigate by microarray and immunohistochemistry the presence of measles virus (MV), hepatitis-C virus (HCV), adenovirus (ADV), respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), herpes I and II viruses (HVI and HVII). Results: We detected the epithelial alveolar infection by MV and CMV in 30.8 % and 15.4 % in AIP, respectively. Endothelial CMV infection was observed in 25 % of ICLF group. When we compared with the age of these patients, patients with ≤ of 43 years old had more infection by MV and CMV than the group with ≥ 72 years old. The other viruses were not detected in the different groups. Conclusion: The viral infections observed in AIP and ICLF groups reinforce the possible viral participation in these pulmonary diseases. Financial Support: FAPESP.
Abnormal up regulation of cyclooxygenase-2 is observed in idiopathic pulmonary fibrosis E. Parra * , F. Lin, W. Teodoro, A. P. Velosa, V. L. Capelozzi * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: Several factors modulate fibroblast proliferation and collagen deposition in pulmonary fibrosis diseases, including cyclooxygenase (COX). The main of this study was observed the relationship between COX1 and COX2 in surgical lung biopsies from patients with idiopathic pulmonary fibrosis (IPF). Method: Twenty four patients (64±8.9 year) were characterized with IPF. Immunohistochemistry and histomorphometry were used to evaluate the amount of COX1 and COX2 expression in lung specimens. The expression of these markers was tested with their pulmonary function tests. Results: Significantly a higher amount of COX-2 was observed in IPF patients principally in normal and vascular areas (p=0.05) when compared with control group (Figure) , contrasting with similar amounts of COX-1 in both groups. An important negative correlation was observed between total lung COX-2 expression and DCO/VA (r=0.694, p=0.01) in IPF patients. Conclusion: Higher vascular expressions of COX2 probably mediated the inflammatory reaction in patients with IPF and have an important impact with pulmonary function tests, suggesting that a participation in the pathway of IPF. Financial Support: FAPESP.
Error barr shows the correlations between COX-2 expression in normal and vascular areas of IPF compared with control group PS-01-026 Multiplex Ligation Probe-dependent Amplification (MLPA) as an ancillary method for the diagnosis of malignant pleural effusion E. Parra * , D. Rosolen, L. Kulikowski, R. Dutra, V. l. Capelozzi, F. Vargas, M. Acencio, L. Antonangelo * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: A definitive diagnosis provided by the finding of malignant cells in pleural fluid (PF) can be established in around 50 % of patients with pleural malignancy. However, underdiagnosis risk in cytological suspicious cases is high, which makes the cytological diagnosis quite limited. This is an important clinical problem, especially if we consider that some patients, in bad clinical conditions, can not be submitted to a guided thoracoscopic biopsy. Method: Using multiplex ligation probe-dependent amplification (P315-MRC-Holland) we have studied sequence variations of EGFR gene and amplifications/deletions of chromosomal regions frequently associated to tumors (ATG4B, PAHs, PROS, NSD1, and CDGIF genes). Results: Forty-three malignant PF samples from patients with different cancers were evaluated, even in those cases with scarce pellet cells. Four benign pleural effusions were used as control. Gene sequence changes were observed in 13 (30.2 %) cases, while others copy number abnormalities were found in 19 (44.2 %). Conclusion: The findings suggest that MLPA could be considered an alternative tool to detect molecular genetic changes in malignant pleural effusions, since this technique is relatively low expensive and not time consuming. Our next challenge is to find the best combination of probes capable to recognize malignant cells of any origin in fresh samples of PF.
Multiple primary pulmonary myopericytoma: Case presentation N. Petre * , F. Pop, D. Leonte, S. Bedereag * Medcenter Bucharest, Dept. of Pathology, Romania Objective: Myopericytoma is described as a perivascular myoid tumor which usually develops as a solitary mass in the subcutaneous tissues of the extremities, in adults. Method: The chest radiography of a 52-year-old woman showed multiple, bilateral lung nodules. After the surgical excision of one of them, the histopathologic examination revealed a well circumscribed nodule, composed of fusiform or oval cells, with eosinophilic cytoplasm and ovoid nuclei, without atypia, in a concentric pattern, intimately associated with thin-walled vessels, within a myxoid stroma. Results: The spindle cells were diffusely positive for vimentin, smooth muscle actin (SMA) and occasionally for desmin. CD31 and CD34 were positive only in endothelial cells. Ki 67 was positive in less than 4 % of neoplastic cells. Conclusion: The histological and immuno-histochemical findings led to the diagnosis of multiple primary pulmonary myopericytoma.
PS-01-028 Five cases of lung pneumocytoma: Clinico-pathological, immunohistochemical and ultrastructural study I. Rodriguez * , A. Panizo, B. Larrinaga, I. Amat * C. Hospitalario de Navarra, Dept. of Pathology, Pamplona, Spain
Objective: Lung pneumocytoma (LP), also called sclerosing hemangioma, is an uncommon tumor of uncertain histogenesis. Method: We analyzed the clinical, morphological, immunohistochemical and ultrastructural features of 5 LP. Results: All 5 patients were women, with median age of 48 year (range: 29-73). Mean size was 26 mm (range 15-45 mm). All tumors were located within the right lung (upperlobe: 1; middle-lobe: 2; lower-lobe: 2). All cases showed solid, papillary, sclerotic, and hemorrhagic patterns. The tumors were composed of 2 cells types: pale polygonal and surface cells. EMA, TTF1, and PR were observed in both type cells. The surface cells showed also positivity for CK7, Napsin-A, and surfactant-A, whereas the polygonal cells for vimentin. Surface cells had short microvilli and lamellar bodies in their cytoplasm. Polygonal cells contained abundant microfilaments and rough endoplasmic reticulum. All patients were alive and well without recurrence at the last follow up. Conclusion: LP is likely to be an epithelial tumor with a differentiation toward type II pneumocytes, which exhibits various architectural patterns. The IHC profile provides useful clues for the diagnosis of this lung neoplasm when typical features are absent. All our cases had an excellent prognosis with no evidence of recurrence following surgery.
Rare tracheobroncheal lesions diagnosed by small bronchoscopic biopsies A. Sasmaz * , S. Yuksel, H. Nalbant, P. Celepli, E. Kadan, S. Percinel, S. Dizbay Sak * Ankara, Turkey
Objective: Six different, rarely seen tracheobronchial lesions, taken by bronchoscopy as small fragments were discussed with an emphasis on morphological findings and differential diagnosis. Results: Six patients were men and two patients were women with an age range of 50 to 70 years. Most of the lesions were symptomatic and discovered as small masses by bronchoscopy. The tracheobronchial lesions varied from polypoid, nodular, well-demarcated, fragile masses to nodular thickenings. Microscopically, a polyp composed of a fibrovascular stroma with scattered mononuclear inflammatory cells was compatible with fibroepithelial polyp. Mature adipocyte proliferation in two cases, S-100 and CD68 positive polygonal or ovoid cells with abundant eosinophilic, granular cytoplasm having small, hyperchromatic nuclei, smooth muscle actin and desmin positive spindle cells in interlacing fascicles within a myxoid and collagenous stroma with mononuclear inflammatory cells, CD34 and CD31 positive thin-walled vascular channels, filled with red blood cells and S-100 positive spindle cells having palisading of wavy nuclei in two cases, located in the submucosa were main findings of lipoma, granular cell tumor, inflammatory myofibroblastic tumor, cavernous hemangioma and schwannoma, respectively. Conclusion: Tracheobronchial biopsies, even with small fragments of lesions can allow to render rare specific histopathological diagnoses, some of which need to be confirmed by immunohistochemistry. Objective: Small cell lung carcinomas (SCLC) are aggressive neoplasms that give very early distant metastases. Matrix metalloproteinases (MMPs) are a family of endopeptidases which degrade extracellular matrix and modulate cell adhesion, playing a crucial role in cancer cell invasion and metastasis. The aim of the present study is to investigate the immunohistochemical expression of MMP-2, MMP-9 (type IV collagenases) and MMP-1 (interstitial collagenase) in patients with SCLC. Method: Formalin-fixed, paraffin-embedded lung biopsy specimens from 40 patients with SCLC (M:F=29:11, median age=66.7) were immunostained for MMP-1, MMP-2 and MMP-9 (anti-MMP-1 polyclonal antibody, anti-MMP-2 72 kDa Collagenase IV antibody, anti-MMP-9 92 kDa Collagenase IV antibody, Spring Bioscience). Results: Immunohistochemical expression of MMPs was detected in the cytoplasm of neoplastic cells. Immunoreactivity was designated positive when >10 % of neoplastic cells were stained. Overall, MMPs were expressed in the majority of SCLC. Specifically, MMP-1 was positive in 90 % (36/40), MMP-2 in 87.5 % (35/40) and MMP-9 in 82.5 % (33/40) of SCLC. Non-neoplastic lung parenchyma did not stain for MMPs. Conclusion: The majority of SCLC express immunohistochemically MMP-2, MMP-9 and MMP-1. Given the role of MMPs in cancer progression and metastasis, their expression in SCLC may contribute to their aggressive course. Objective: Number of pulmonary adenocarcinoma (ADC) patients incresease. Treatment of non-small cell pulmonary carcinoma depends of its histological type in era of target molecular therapy particulary of ADC: Diagnosis of ADC is established on small-sized pulmonary biopsies obtained on bronchoscopy and by transthoracic needle pulmonary biopsy. The aim of this study was to evaluate optimal panel of monoclonal antibodies in diagnosis of pulmonary ADC on small-sized biopsy pulmonary samples. Method: Analysis of 50 small-sized biopsy pulmonary samples. Diagnosis of ADC was established on hematoxillyn-eosin stained (H&E) samples and confirmed immunohistochemicaly by Thyreoid Transcription Factor-1 (TTF-1), Napsin-A, Surfactant B and Cytokeratin7 (CK7). Descriptive statistical method (%) was used. Results: TTF-1 specificity was 86 %(43/50), Napsin-A-82 %(41/50), SurfactantB -56 %(28/50) and CK7-90 %(45/50) in ADCs. Two monoclonal antibodies were positive in 24 % (12/50) ADCs, one of them was necessary TTF-1 or Napsin-A. Three monoclonal antibodies were positive in 40 %(20/50) and 4 in 36 %(18/50) ADCs, respectively. There is not statistical significans in number of monoclonal antibodies for diagnosis of ADC. Conclusion: No one monoclonalantibody is specific for one histological type of carcinoma and its origin. TTF-1, Napsin-A, SurfactantB and CK7 belong in optimal panel for diagnosis of pulmonary adenocarcinoma.
PS-01-032 Expression of CD44, E-Cadherin and Bcl-2 in lung cancer I. Strumfa * , A. Abolins, J. Gekis, K. Pavlovs, A. Vanags, J. Grusina-Ujumaza, G. Volanska, J. Gardovskis * Riga Stradins University, Dept. of Pathology, Latvia
Objective: Lung cancer represents major problem in oncology as the incidence and mortality is high (Parkin et al., 2005) . Molecular studies could reveal additional targets for intervention or prognostic evaluation. However, the published data are controversial (Renouf et al.,2009; Leung et al.,2010; Maraz et al.,2011; Ko et al.,2011) . Method: Consecutive lung cancer cases (278) were retrieved by retrospective archive search. The diagnostics has been performed systematically in accordance with WHO classification (Travis et al.,2000) . The expression of CD44, E-Cadherin and Bcl-2 was analysed by immunohistochemistry and evaluated semiquantitatively. Results: The intensity of Bcl-2 expression was highest in small cell cancer, reaching the mean value 2.25. No or very low Bcl-2 expression was found in adenocarcinoma, carcinoid and squamous cell cancer. The membranous expression of E-Cadherin showed significantly higher mean intensity in adenocarcinoma (1.93) and carcinoid (1.90) than in small cell cancer (0.14) and squamous cell cancer (0.85). Cytoplasmic E-cadherin expression was observed in small cell cancer. CD44 expression was widespread and frequent in non-small cell lung cancer. Conclusion: The main histological types of lung cancer show different immunophenotype. Bcl-2 expression is found in small cell cancer. The expression of E-Cadherin is characteristic in adenocarcinoma and carcinoid. CD44 is expressed in non-small cell lung cancer.
Localized nodular pulmonary and thoracic cage amyloidosis I. Strumfa * , A. Abolins, J. Gekis, K. Pavlovs, A. Vanags, B. Strumfs, G. Volanska, J. Gardovskis * Riga Stradins University, Dept. of Pathology, Latvia Objective: Localised amyloidosis represents a rare differential diagnosis of neoplasms. However, in some cases amyloid deposits are manifestation of tumour itself. To increase awareness about these uncommon but important differential diagnostic issues, we show here three well-characterised cases of thoracic cage or pulmonary localised nodular amyloidosis. Method: The cases were identified by systematic retrospective archive search, 2000-2011. Congo red stain, polarisation microscopy and immunohistochemical analysis was applied.
Results: There were 2 males and 1 female among the patients diagnosed with thoracic cage or pulmonary amyloidosis. The patients were 67-84 years old and had no history of previous malignancy. Clinically, either lung cancer or sternal osteochondroma was suspected. Radiologically, 1-2 nodules measuring 1.5-9.0 cm were found in lungs (2 cases, one of these being bifocal) or sternum (1). Surgical treatment was applied in all cases. Amyloid deposits were identified by Congo red stain and polarisation microscopy. AL type of amyloid was confirmed. In case of bone lesion, the plasma cells were monoclonal justifying the diagnosis of multiple myeloma. Conclusion: Localised amyloidosis can involve lung or sternum as tumour-like mass. The lung lesions can be single or multiple. Association with haematologic neoplasm is likely if bone is affected. Clonality analysis of plasma cells surrounding amyloid deposits is mandatory.
Pleural localization of Castleman Disease: A rare entity E. Tastekin * , F. Oz Puyan, E. Isler, U. Usta * Trakya University, Dept. of Pathology, Edirne, Turkey
Objective: Castleman disease (CD) comprises a heterogeneous group of disease with various clinical presentations and prognosis. By definition; CD is a lymphoproliferative disorder of the mediastinal lymph nodes and extranodal tissues with follicular and interfollicular abnormalities. Pleural localization is an unusual presentation of CD. Method: A 27-year-old man applied to the hospital because of cough. Chest x-ray and CT images showed an irregular, 35×56×67 mm sized intrapleural-extrapulmoner mass in the paramediastinal region. Surgical specimen was consisting of a 7×5×3.5 cm sized, irregular-round shaped, gray colored calcified mass. Microscopically, a lymphoid proliferation with distorted follicles and increased dendritic cells (with CD21) was detected. Lymphocyte depleted germinal centers resembling "onion skin" appearance with penetrated sclerosing hyalinized blood vessels were also seen. Interfollicular region was composed of proliferating blood vessels lined by plump endothelial cells and few lymphoplasmositer infiltration. There was heterogeneous positivity with CD3 and CD20. Expanded mantle zones showed diffuse bcl2 positivity. Ki67 proliferation index was low. The mass was diagnosed as "Castleman Disease, hyaline vascular type". Typical appearance of the follicular and interfollicular abnormalities allowed definitive differentiation from mesothelioma and solitary fibrous tumor of the pleura. Conclusion: Pleural CD is a rare lesion but should be considered especially on young patients in the differential diagnosis of pleural masses. Objective: To evaluate FOXp3 expression in bronchus-associated lymphoid tissue (BALT) and correlate with the inflammatory process and collagen content in the lung tissue in an experimental model of scleroderma (SSc) after type V collagen (COL V)-induced nasal tolerance. Method: Female New Zealand rabbits (N=12) were immunized with 1 mg/ml of COL V in Freund's adjuvant (IM). After 150 days, six immunized animals were nasally tolerated with COL V (25 νg/day), during 60 days (IM-TOL). Animals (N = 6) only tolerated served as control (CT). FOXP3 expression in BALT and inflammatory cells in pulmonary interstitium were evaluated by point counting method. Types I, III and V collagen gene expression were evaluated by Real-time PCR. Results: IM-TOL when compared to IM presented decreased lymphocytes, macrophages and monocytes and types I (p=0,002) and V (p=0,009) collagen mRNA expression in pulmonary tissue. T lymphocytes FOXp3 were expressed in 100 % of IM-TOL and 33,3 % of CT (p= 0,03). Additionally, BALT was higher expressed in IM-TOL in relation to CT. Conclusion: COL V-induced nasal tolerance in SSc model induces FOXp3 regulatory T cells in BALT which can trigger an immune regulatory mechanism resulting in decreased inflammation and collagen expression. It suggests that COL V tolerance could be a promising therapeutic for human scleroderma treatment.
IgG4-related lung disease T. Tichy * , H. Hornychova, J. Jankova, J. Skarda, B. Krajsova * University Hospital Olomouc, Inst. of Pathology, Czech Republic
Objective: IgG4 -related lung disease is one of the manifestations of IgG4-related systemic disease. Obliterative vasculitis is considered an organ specific feature of IgG4-related disease in the lung. We report a case of a 78-year-old man with multifocal subpleural consolidations in both lungs. Histologic examination revealed IgG4-related lung disease with obliterative lymphoplasmacytic vasculitis.
Method: Histological sections from paraffin blocks were used for hematoxylin-eosin and special stains (elastica-Masson trichrome, PAS, Geimsa, Grocott), for immunohistochemistry (antibodies against kappa and lambda light chains, CD20, CD3, IgG, IgM, IgA, IgG4, LMP1) as well as for EBER in situ hybridisation and PCR. Results: The lung tissue was irregulary fibrotic with dense lymfoplasmacytic infiltration concentrated in the walls of pulmonary arteries. Some arteries were obliterated by intimal fibrosis. Infiltrating lymphocytes were small and without atypias. The majority of lymphocytes were CD3 positive, nearly all plasmocytes showed IgG positivity. IgG4/IgG ratio was 60 %. EBER in situ hybridisation was negative and monoclonal reaarangement of T cell receptor or immunoglobulin heavy chain genes were not detected. Conclusion: IgG4-related lung disease is rare. Obliterative vasculitis and high percentage of IgG4 positive plasmocytes are main histological features of this entity. Objective: Primary lung carcinoma with heterotopic osteocartilaginous formation is exceedingly rare. To date only 38 cases have been reported in the literature and the pathogenesis is not fully investigated. We report a case of pulmonary adenocarcinoma with osteocartilaginous formation with immunohistochemical staining of bone morphogenetic proteins (BMPs). Method: A 59-year-old man was admitted to our hospital for a lung tumor in his left upper lobe. An open lung biopsy followed a lobectomy was done. The resected tissue was fixed with 10 % formalin and embedded in paraffin. Sections, cut from paraffin blocks, were subjected to hematoxylin and eosin (HE) staining and Masson-Goldner staining, in addition to immunohistochemical staining. Results: The tumor was poorly differentiated adenocarcinoma with heterotopic ossification. Some of the tumor cells were positive for BMP-2, 6, and 7. Some of mesenchymal cells in tumor interstitium were positive for osteoblast (OB)-cadherin or BMP receptor-IA. Conclusion: We report a case of primary lung adenocarcinoma with heterotopic osteocartilaginous formation and reviewed previously published reports. To our best knowledge, this was the first case that not only BMP was expressed in tumor cells but also OB-cadherin or BMP receptor in some mesenchymal cells of tumor interstitium.
Expression of transforming growth factor ß1 and e-cadherin in lung adenocarcinoma J. Yoo * , S. Y. Park * St. Vincent's Hospital, Pathology, Suwon, Republic of Korea Objective: There is evidence supporting the concept of tumor progression from pulmonary adenocarcinoma in situ (formerly bronchioloalveolar carcinoma, BAC) to adenocarcinoma with varying degrees of invasion. The aim of this study was to investigate the role of TGFß1in tumor invasiveness in lung adenocarcinoma, and to determine the potential relationships between its expression and immunophenotypes of cell adhesion molecules. Method: Tumor samples (n=40) from adenocarcinoma in situ (n=13), minimally invasive adenocarcinoma (formerly BAC with ≤5 mm invasion, n=2), and lepidic predominant invasive adenocarcinoma (formerly mixed adenocarcinoma showing non-mucinous BAC features with >5 mm invasion, n=25) were examined for the expression of TGFß1, E-cadherin, N-cadherin, and H-cadherin proteins using immunohistochemistry. Results: Twenty-five tumors (63 %) were positive for TGFß1. The frequency of immunoreactivity in patients with adenocarcinoma in situ, minimally invasive adenocarcinoma, and lepidic predominant invasive adenocarcinoma was 23 % (3/13), 50 % (1/2), and 84 % (21/25), respectively (p=0.001).
Loss of E-cadherin expression was more frequently observed in invasive adenocarcinomas than in adenocarcinomas in situ (p=0.034). TGFß1 expression showed a statistically significant correlation with H-cadherin expression (p=0.040), but not with E-cadherin expression (p=0.752). Conclusion: These results suggest that TGFß1 and E-cadherin may play an important role in invasive progression of lung adenocarcinoma through regulating epithelial-to-mesenchymal transition.
PS-01-040 LIN28A expression as prognostic indicator in lung adenocarcinomas V. Zolota * , V. Tzelepi, G. Psiouri, I. Lilis, N. Panagopoulos, C. Sirinian, C. Scopa * Medical School of Patras, Dept. of Pathology, Greece
Objective: LIN28A is implicated in stem cell pluripotency by blocking let-7miRNAs and Oct-4 is a transcription factor highly expressed in embryonic stem cells. The present study investigates the prognostic significance of both markers in lung adenocarcinomas. Method: We evaluated, by immunohistochemistry, LIN28A and Oct-4A expression in formalin-fixed, paraffin-embedded tissues from 92 lung adenocarcinomas. Immunoreactivity was scored as 1+, <1 % positive tumor cells (ptc); 2+, 1-5 % ptc; 3+, 5-10 % ptc; and 4+, >10 % ptc and was compared with clinicopathologic features and overall survival. Results: Regarding LIN28A expression 23 % of tumors were recorded as 1+, 51 % as 2+, 14 % as 3+ and 12 % as 4+. The stratification for Oct-4 expression was 19 % (1+), 50 % (2+), 23 % (3+) and 8 % (4+). LIN28A and Oct-4 expression was not significantly associated with age, gender and tumor grade or stage. LIN28A 4+ expression predicted a shorter overall survival (P<0.05) on univariate analysis. Oct-4 was not associated with patients' prognosis. Cox multivariate analysis showed that age, TNM and LIN28A 4+ expression were independent prognostic factors of survival. Conclusion: LIN28Α expression appears to be an independent predictor of poor outcome in lung adenocarcinomas. Further studies are warranted in order to investigate its role in stratifying patients at increased risk for poor outcome.
Comparison of the mutation status of EGFR and KRAS on pulmonary adenocarcinoma and corresponding brain metastasis Objective: EGFR and KRAS mutation statuses have been known associated with the sensitivity of tyrosine kinase inhibitor treatment in pulmonary adenocarcinoma. However, the mutation analyses were usually performed on the primary tumors only, due to the availability of the tumor tissue. Method: To compare the KRAS and EGFR mutation statuses between pulmonary adenocarcinomas and corresponding metastases, we performed direct sequencing, followed by Scorpion ARMS method on wild-type cases for EGFR analysis, and allele-specific real-time PCR for KRAS analysis, on the paired samples of pulmonary adenocarcinoma. Results: Thirty-one (63.3 %) pulmonary adenocarcinomas and 30 (61.2 %) brain metastases out of the 49 paired specimens have EGFR mutations, and 30 (61.2 %) paired specimens were concordant for the EGFR mutation status between the primary and metastasis. In addition, 17 (51.5 %) pulmonary adenocarcinomas and 17 (51.5 %) brain metastases out of the 33 paired specimens have KRAS mutations, and only 16 (48.5 %) paired specimens were concordant between the primary and the metastasis. Conclusion: The status of EGFR mutation is relatively consistent between primary and metastasis comparing to that of KRAS mutation in pulmonary adenocarcinomas. However, discordance for the mutation statuses does happen. Accordingly, repeat analysis is recommended if tissue from metastatsis or recurrence is available. Objective: We tried to measure and analyze characteristics of neuroendocrine tumors in lung by image analysis and help to diagnose them. Method: It was analysed that sixteen cases of typical carcinoid tumors, five cases of atypical carcinoid tumors, fifteen of small cell carcinomas, fifty one cases of large cell neuroendocrine carcinomas. We analyzed the nuclear area, perimeter, major axis and minor axis using i-solution image analyzer software package. Results: The mean nuclear area was 488.00 μm2 in the typical carcinoid tumors, 499.30 μm2 in the atypical carcinoid tumors, 481.48 μm2 in the small cell carcinomas, and 684.05 μm2 in the large cell neuroendocrine carcinomas. After the statistical results, every method was effective to distinguish large cell neuroendocrine carcinoma from other tumors and the circumferences of nucleus was the most effective to distinguish among them. Conclusion: Pulmonary neuroendocrine tumors were the nuclear morphologic differences of each tumors. Therefore, diagnosis that considers morphologic differences of pulmonary neuroendocrine tumors contributes to increase reproducibility and accuracy. Sunday, 9 September 2012, 09.30 -10.30 An abundant growth of lactobacilli may result in lysis of vaginal epithelial cells, named as Cytolytic Vaginosis. This cytolytic process may cause the symptoms as seen in candidiasis. We observed Pap smears to evaluate if cytolysis has a relationship with infertility. Method: In the Pathology department of Mardin Maternity Hospital, 2011-2012 period, we examined Pap smear cases suffering from the similar syptoms as candidiasis. Of the 4672 smears, 82 were diagnosed as "Cytolytic Vaginosis". No growth was observed in cultures. Results: The number of the cases suffering from the infertility was 261 (%5.58) among 4672 cases. In the cytolytic vaginosis group this ratio was %32.9 (n=27). The ratio of the infertil cases of cytolytic vaginosis group over general population was significantly higher (p<0.05). Conclusion: Our results are in favour of supporting the hypothesis of the relation between cytolytic vaginosis and infertility. Lactobacilli are thought to have inhibitive role in fertility by changing the vaginal ph and adhering to the epithelial cells so inhibiting the sperm penetration.
Penicillium and aspergillus spp. on pap smears from the surprising origin I. I. Akgun * , B. A. Borsa * Mardin Maternity Hospital, Dept. of Pathology, Turkey
Objective: Fungal organisms are commonly seen on smears. Some unusual species as Aspergillus and Penicillium are so rare and generally seen via contamination. In this study, we investigated the origin of the many extraordinary fungal organisms seen on Pap smears in series in only a few months. Method: In Pathology department of Mardin Maternity Hospital, we observed both smear and vaginal discharge materials, came from the same hospital but different clinicians, for 3 months. Results: 149 smears came from clinician A and 335 smears from clinician B. Curious fungal organisms with huge branching hyphas and macrochonidias were seen in 15 smears among 149 smears (2 Aspergillus and 4 Penicillium species were recognized morphologically). No growth was observed in cultures. Interestingly, among all 335 smears came from clinician B, there was no abnormal funguses. The smear samples with unusual fungal components all came from the same gynecologist. This made us strongly consider the probability of contamination. Conclusion: Penicillium and Aspergillus spp. are extremely rare in vaginal smears. In our study, these funguses are thought to be airborne passed from the thick layer of the mold spreading on the ceiling of the office. Objective: Nearly all cervical cancers are related to human papillomavirus (HPV) infection. Hybrid Capture 2 -HC2 (Qiagen, Hilden, Germany) is the method to determine the presence of high risk HPV (HR HPV) in cytology samples with the best clinical sensitivity. Method: In our study we compared the results of the HPV test in 742 ThinPrep® cervical samples using first HC2 and secondly Cervista (Hologic, Madison). Samples were divided in two groups for the statistical analysis. The first group were HSIL cases confirmed by biopsy (n =65) and the second group cytology samples reported as negative, ASC-US or LSIL (without biopsy or biopsy, not HSIL) (n=677). Results: Overall, concordance between the two techniques was 92 % (683/742). There were 23 cases HC2+ Cervista−, and 36 cases HC2 −Cervista+. The average HC2 viral load in discordant cases was 2.83. In our series the HC2 sensitivity for ≥ CIN2 was 100 % (65/65) and the specificity was 85.4 % (578/677). Cervista results were 98.5 % (64/65) and 83.3 % (564/677), respectively. In the HSIL cases the coincidence was 98.5 % (64/65). Conclusion: Cervista results showed good clinical sensitivity and high concordance with HC2, with a slightly lower specificity. In conclusion, according to our data the determination of HPV with Cervista is comparable with HC2.
Mammary analogue secretory carcinoma of salivary glands: 2 cases including Cytology, Histology, IHC, EM, RT-PCR and FISH P. Farrajota * , E. Tani, C. Carvalho, J. Wejde, G. Elmberger * Centro Hospitalar do Porto, Dipt. do Anatomia Patologica, Portugal
Objective: Mammary analogue secretory carcinoma (MASC) of the salivary glands was described in 2010 by Skálová et al. in a series of 16 cases. We present 2 new molecularly confirmed cases with focus on detailed cytomorphological features and correlation with special studies. Method: A 75 year old man and a 68 year old female presented with history of slow growing parotid and submucosal buccal tumors, respectively. Two fine needle aspiration biopsies (FNAB) were performed in each case. The surgical resection specimens were routinely processed and special studies applied on paraffin tissue section. The FNAB smears were retrospectively studied.
Results: All smears showed similar features of abundant proteinaceous background, moderate cellularity including macrophages and irregular and variable sized groups of cells with slightly enlarged round hypercromatic nuclei and abundant bluish vacuolated cytoplasm frequently containing small reddish granulations. The first FNAB reports confirmed salivary origin and the second ones alert for possible neoplastic nature, advising surgical resection. Electron microscopy performed in the parotid case caracterized the granules as mucigen. Subsquent pathologic evaluation and molecular confirmation was carried out. Conclusion: MASC is a low grade malignant tumor capable of simulating a benign salivary gland process so it is important to know its cytological characteristics.
Adenosquamous lung carcinoma: The importance of cyto-histologic correlation D. Felizardo * , A. L. Cunha, R. Henrique, C. Lobo * Instituto de Oncologia Porto, Dept. of Pathology, Coimbra, Portugal
Objective: Adenosquamous lung carcinomas (ASC) are uncommon and aggressive tumors. Identification of a squamous component within a NSCLC is critical as it excludes anti-VEGF therapy. Likewise, recognition of an adenocarcinoma component is also relevant because it prompts the search for EGFR mutations. Since 70 % of lung cancers are diagnosed in small biopsies/cytological specimens, cytohistological correlation is crucial for correct diagnosis. Thus, we aimed at determining the relevance of combining cytologic and histologic findings in initial diagnostic workup of ASC. Method: We searched for cases of ASC diagnosed at our institution from March 2011 to April 2012. Cytologic and histopathologic findings were evaluated and their accuracy for diagnosing ASC was assessed. Results: Of 184 cases of lung cancer in biopsy, 11 (6 %) corresponded to probable ASC and in 9 cases cytologic examination was also performed. In 7 cases, cytology was not conclusive, but in 2 cases it demonstrated cytomorphological features of probable ASC. We emphasize one case in which only the squamous component was valued by histology, but cytology provided clues that prompted immunohistochemical analysis which led to the diagnosis of probable ASC. Conclusion: Cyto-histological correlation augments the diagnostic accuracy in ASC of the lung, emphasizing the complementarity of both procedures.
The impact of the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC): A retrospective study of 828 aspirates with emphasis on the prior "indeterminate" Objective: The material of this study is consisted of selected FNA biopsies performed in the last 10 mounths period and followed by the Endocrinology Department.
Method: The Bethesda System of reporting thyroid cytopathology was used. Slides were stained with Papanicolaou preparations. Results: Totally 380 cases were examined, 288 of which were female and 92 were male. Age of the patients varied between 5 and 88. 121 cases were diagnosed as nondiagnostic or nonsatisfactory. 211 cases diagnosed as consistent with a benign follicular nodule. Only 4 cases were diagnosed as a consistent with lymphocytic thyroiditis in corelation with the clinical features. Only 1 case diagnosed as a granulomatous thyroiditis. Diagnosis as "Atypia of undetermined significance or follıcular lesion of undetermined significance "was made only in 2 cases whereas "Follıcular neoplasm or suspicious for a follıcular neoplasm" was diagnosed in 1 case. "Suspicious for malignancy" diagnosed in 15 cases. 2 cases were diagnosed as malignant. Conclusion: Although diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions are well established in The Bethesda System, difficulty in categorizing the cases and underestimation or overdiagnosis are quıite frequent issues. To solve this problem, retrospective studies which include surgical and cytopathologic reports, to examine slides by at least two different pathologists in different times will be helpfull.
Metastatic pulmonary adenoid cystic carcinoma: Report of a case diagnosed by fine-needle aspiration cytology P. Karabagli * , S. Ozbek * Selcuklu Medical Faculty, Dept. of Pathology, Konya, Turkey
Objective: A rare case of adenoid cystic carcinoma of ceruminal gland with pulmonary metastasis presented with characteristic cytological and histological findings. Method: The biopsy specimen of the patient, a 45 year-old man with painfull, growing mass in his external auditory canal was interpreted as an adenoid cystic carcinoma. He was treated surgically and received post-operative radiotherapy. After 8 years, a distant metastasis relapse was observed. Computed tomography (CT) of the chest depicted multiple spiculated masses suggestive of metastases throughout both lung fields, the diameter of the largest measuring 2 cm. CT guided percutaneous transthoracic fine needle aspiration biopsy was performed from the largest lesion. Results: Cytological examination of the aspirates revealed large spherical hyaline globules representing basement membrane material surrounded by neoplastic cells. The cells were cohesive, closely packed, and had uniform round to oval hyperchromatic nuclei with scanty cytoplasm. These features were suggestive of an adenoid cystic carcinoma. Conclusion: Patients with adenoid cystic carcinoma could be frequently encountered with disease recurrence confined to the lung. Fine-needle aspiration cytology provided a conclusive diagnosis of adenoid cystic carcinoma. Objective: Often some of a fine needle aspiration (FNA) sample is left behind in the hub of the needle when the specimen is ejected on the slides ( Figure 1A) . Method: We have designed a device that collects this material. It consists of a plastic adaptor that has a core of polyvinyl alcohol foam protruding from its lumen at one end ( Figure 1B) . The other end of the adaptor then fits on to the syringe (Figure 1 C) . As the FNA is being performed the sample material flows up the lumen of the needle's shaft and emerges into the hub of the needle where it is absorbed into the tip of the foam core ( Figure 1D ). Smears are made by ejected the specimen from the needle onto a slide by air pressure from an attached syringe. The air passes through the foam forcing some of the sample in the foam back down the needle but some remains behind. The device is then removed and placed in a formalin specimen pot. Once fixed the core is pulled from the adaptor ( Figure  1E ), processed and sectioned as for routine histology specimens ( Objective: The presence of DCs has been studied histologically in PTC, but their presence and potential diagnostic value in thyroid FNAs has not been evaluated. Method: We assessed the presence of DCs in cytological samples of histologically confirmed PTCs (n=31) and benign thyroid nodules (BTN) (n = 29) using CD1a. Corresponding PTCs (n=11) and BTN (n=10) from surgical excisions were stained with both CD1a and Langerin. Results: CD1a + DCs were identified in 30/31 PTCs on cytology (97 %). They were either isolated in the background or more typically closely associated with tumor cell clusters. The 3 PTC cases with the least DCs corresponded to the follicular variant on histology. In contrast, most BTN (69 %) lacked CD1a + DCs. When DCs were present, they were primarily isolated in the background although 5/29 cases (17 %) contained rare DCs among tumor cells. Tumor-infiltrating DCs and background DCs were both higher in PTCs than in BTN, but only the former was statistically significant (P<0.0001 and P=0.1173 respectively). Similar findings were found on histology where all PTCs contained CD1a + and Langerin + DCs while only 2/10 BTN (20 %) contained rare DCs. Conclusion: DCs are present in FNAs of PTC, typically among tumor cell clusters, while they were absent or rare in FNAs of BTN. Thus DCs may be useful as an additional diagnostic marker for PTC.
Significance of p 16 immunostaining in postmenopausal women with atypical squamous cells A. Repse Fokter * , K. Gornik Kramberger, S. Hutter Čelik, S. Sramek Zatler * Celje General Hospital, Dept. of Pathology and Cytology, Slovenia
Objective: The evaluation of postmenopausal Pap smears can often be challenging. Degeneration associated with atrophic vaginitis, hyperchromatic crowded groups, parabasal cells with organophilic cytoplasm or variations in nuclear size may be falsely interpreted as squamous atypia or even more severe lesion. Method: The study included 25 postmenopausal Papanicolaou patients (26 smears) with the initial cytological diagnosis of ASC-US or ASC-H. The smears were decolorized and immunostaining for p16(Ink4a) was applied. In 21 patients (22 smears) tissue biopsy and histological examination was performed and four ASC-US cases had cytological follow up only. Results: Among cases with histological examination positive p16 reaction was found in 17 patients (18 smears). The histological diagnoses were: CIN1 (3), CIN 1-2 (2), CIN2 (1 case), CIN3 (5 patients/6 smears), AIS (1 case) and invasive squamous carcinoma (2 patients). Two p16 positive patients with three smears and initial negative histology had CIN3 after 2 years. Among p16 negative patients there were two with CIN3 and two with normal histology. Four ASC-US patients without histological examination had normal cytological follow up. Conclusion: P16 is a useful marker in detecting of clinically significant cervical lesions in postmenopausal women.
Endoscopic-ultrasound guided fine needle aspiration cytology of intraductal papillary-mucinous neoplasms of the pancreas: Report of two cases and review of the literature E. Tejerina González * , A. López García, C. González Lois, F. Fernández García, A. Herreros de Tejada, E. Sanz * HU Puerta de Hierro-Majadahonda, Dept. de Anatomía Patológica, Spain
Objective: Intraductal papillary-mucinous neoplasms (IPMN) of the pancreas are mucin-producing tumors recently recognized as a distinct entity among other mucinous pancreatic tumors. Endoscopic-ultrasound guided fine needle aspiration (EUS-FNA) is a sensitive technique which can be very useful in the accurate diagnosis of these neoplasms. Method: We present a case of a 30-year-old female with recurrent episodes of acute pancreatitis and a 49-year-old male with abdominal pain. TC showed a 26 mm-cystic tumor at the uncinated process of the pancreatic head and cystic dilatation of the main pancreatic duct, respectively. In both cases EUS-FNA was performed. Results: Smears showed medium-to-large, cohesive groups and complex papillary clusters set in a clean, mucoid background, composed of cuboidal or columnar epithelial cells with abundant clear or mucinous citoplasms. A honeycomb arrangement was occasionally seen. Although nuclei were predominantly medium-size and uniform, some groups showed some nuclear enlargement and pleomorphism with visible nucleoli. A diagnosis of "mucinous neoplasm with papillary pattern" was rendered in both cases, followed by partial pancreatectomy. The histologic examination was consistent with IPMN. Conclusion: The cytologic features of intraductal mucinous papillary neoplasms of the pancreas, evaluated by EUS-FNA, seems to be quite characteristic of these tumors and allow to suggest a correct preoperative diagnosis. Objective: The human IGH locus at chromosome 14q32 is frequently involved in different translocations of non Hodgkin Lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split signal IGH FISH-CISH DNA probe (IFCD) is a mixture of two fluorochrome-labeled DNAs that binds the telomeric and the centromeric segments, on the IGH breakpoint respectively. We tested the capability of the IFCD to detect IGH translocations and diagnose B-cell NHL on cytological samples. Method: Fifty cytological specimens from lymphoproliferative processes were tested using the IFCD and the results compared to light chain assessment by flow cytometry (FC), IGH status by PCR, and to clinical-histological data. Results: IFCD analysis detected 29 positive, 15 negative and 6 inadequate cases; there were 29 true positive (66 %), 9 true negative (20 %), 6 false negative (14 %) and 0 false positive cases (0 %). Comparing the sensitivity of the IFCD with FC and PCR, the highest sensitivity was obtained by FC followed by IFCD and PCR. Conclusion: The IFCD is effective in detecting any translocation involving the IGH locus; it can be employed on different samples from different B-cell NHL whereas it is not useful to classify the specific entities. Sunday, 9 September 2012, 09.30 -10. Objective: Morphometric analysis of the glands and inflammatory cells proportions in colon mucosa may be crucial for distinction of subtle changes. The most important step is a recognition and proper segmentation of the separate glands, even branched, atrophic or distorted. Method: New sequential algorithm has been proposed, based on mathematical morphology transformations, such as extended regional maxima, hit-or-miss, opening and closing operations. For lumen opened glands snake algorithm with closing has been applied to surrounding elements with sequential elimination of the closed contours under decreasing area criterion. Proposed method has been used to evaluate 47 cases of diversion colitis to compare specimens sampled from functional part of colon and defunctioned distal one. Results: The appropriate results were obtained for specimens with different section planes with efficiency 89 %. In comparison of the B to A was observed decreased participation of epithelium in the glands area measured in deep region (an average 57 % instead 68 %). In addition, the area of the gland was slightly reduced from 51 % to 46 % of the mucosa. Conclusion: The proposed method can be useful to automatic morphometry analysis of the gland shape in colon mucosa. The recognition of the opened glands improves previously designed algorithms. Objective: Quantitative analysis of the immunohistochemical histological samples plays an important role in the diagnosis and prognosis of many cancers. The commercial and non-commercial software's to computerized image analysis of microscopic images based in several approaches. Method: The advance segmentation scheme usually consists of preprocessing, nuclei extraction and classifier blocks. When the preprocessing step includes filtering and conversion to the most discriminative representation and the classifier can be realized by various tools, especially Support Vector Machine as a state-of-art, nuclei extraction is the most crucial part of any algorithms. We compare single thresholding methods such as Otsu, Kurita, minimum error, entropy thresholding, with the sequential thresholding approaches combined with area criteria, local threshold value or extended regional maxima. All of them are accompanied by watershed method and filtration. Results: For Ki-67 stained meningioma specimens in the fields of view without overlapping nuclei the most advance methods are adequate. When increasing the cell number, only sequential thresholding with area criteria and extended regional maxima give acceptable results. The mean relative error is 2.6 ± 2.2 and 5.4 ± 6.4 % respectively. Conclusion: The results confirm the efficiency of using sequential methods for segmentation of cell nuclei. For best results, use sequential thresholding with area criteria.
PS-03-003 NHS improvements: Enhanced IT project for histopathology F. Mayall * * Musgrove Park Hospital, Dept. of Cellular Pathology, Taunton, United Kingdom Objective: Many histopathology laboratories are using antiquated software that hobbles their performance.
We developed open source Filemaker Pro based histopathology reporting software with innovations based on Lean principles to enhance work flow. These include: -Colour coded visual workflow control -"One-click" extrawork requests with order tracking -User defined template reporting -Reporting of complex cases using benchmark profomas -Exportable data-sets using Open Database Connectivity -Easy local customisation and enhancement by the user. Results: The software was used by 15 staff to report more than 7000 histopathology specimens at two histopathology laboratories. Many of these cases were complex cancer resection cases requiring key data capture. The histopathology staff using the software were surveyed on their experience of using the software.
The key steps in Lean IT development: -Start with a small idea -Develop software with multiple Plan, Do, Study, Act (PDSA) cycles -Ask users for more ideas for enhancements -Recruit others into the project by allowing them to use the software and experience its benefits -Standardise new reporting process by achieving agreement between users -Design a project that requires minimal financial investment This open source software is available for download from www.FreeDP.org. Sunday, 9 September 2012, 09.30 -10. Objective: Myxopapillary ependymomas (MPE) often occur at the filum terminale but occasionally occur outside the central nervous system (CNS). Common site for these extraneural tumors is sacrococcygeal area. Method: A cocygeal MPE with radiologic, gross and microscopic features will be presented.
A 39 year old female admitted Neurosurgery Department with painful coccygeal mass. Magnetic resonance imaging revealed a coccygeal, well-demarcated, non-enhancing mass of 4×5 cm in diameter. The mass did not have any connection with the spinal cord. It was completely excised and grossly the cut surface of the encapsulated mass was soft. Microscopically the tumor was comprimised of myxoid stroma with pseudopapillary structures around hyalinized vessels. The tumoral cells showed intracytoplasmic dot-like positivity with EMA. There were no mitosis, and the Ki67 labeling index was low. Conclusion: Extraspinal MPEs are rare and the most common location is sacrococygeal area. These masses are often preoperatively misdiagnosed as pilonidal sinus. Unlike the CNS MPEs, extraspinal MPEs have a potential to metastasize even if they do not show any anaplastic morphological feature. Long term follow-up is recommended as metastases can occur up to 20 years after initial presentation. Objective: Cerebellar liponeurocytoma (CL) is a rare neoplasm with neuronal, astrocytic and lipomatous differentiation arising in adults. The entity is considered as a grade II neoplasm in the last WHO classification of central nervous system tumours. We show two typical cases of this entity stressing the spectrum of histological changes and its differential diagnosis. Method: Patient 1 is a 72-year-old woman with a history of headaches and instability. CT scans and MRI showed a 5 cm in diameter relatively ill-defined, poorly enhanced, round tumour mass in the left hemisphere. Patient 2 is a 56-yearold woman with a history of headaches and instability. MRI showed a 6.5 cm in diameter well-defined triangular lesion in the right hemisphere. Results: Complete surgical resection was achieved in both cases. Both tumours showed highly cellular neoplasms composed by round to polygonal cells with scant cytoplasm and round nuclei with fine chromatin pattern. Mitoses were not seen. Well differentiated lipomatous cells grouped in irregular nests were intermingled with the predominant neuronal component. Conclusion: Cerebellar liponeurocytomas are located in the cerebellar hemispheres and pursue a non aggressive clinical course, with local recurrences detected mainly after incomplete resections. The typical adipous tissue is the result of a lipidization process and not a true metaplasia. Objective: Giant cell tumor of bone is an uncommon neoplasm that accounts for about 5 % of all bone tumors. It usually involves long bones and is very rarely encountered in the skull where it mostly involves the sphenoid and temporal bones. It is a benign neoplasm but can be locally aggressive. Method: We present a case of a 32 year old woman with history of multiple sclerosis whose routine CT scan revealed a mass with corrosive features and calcification situated at the petrous portion of her left temporal bone. A biopsy was sent to our laboratory. Results: Histopathologic examination revealed a neoplasm composed of sheets of oval mononuclear cells, with uniformly distributed nuclear chromatin, evenly intermixed with numerous giant cells that contain a variable number of nuclei with features similar to those of mononuclear cells. Cytoplasm of either cell type was eosinophilic and cell borders were indistinct. Mitoses were scarce and regular. Foamy histiocytes, hemosiderin and microscopic nodules of cartilage formation were also noted. Conclusion: Although giant cell tumor is a benign neoplasm its' localization in this case posses a threat that should be managed surgically and by adjuvant radiotherapy if complete excision is unobtainable.
Leptin and leptin receptor expression in pituitary adenomas G. Ayranci * , T. Avsar, A. Seker, T. Kilic, S. Bozkurt * Marmara University, Dept. of Pathology, Istanbul, Turkey
Objective: Leptin is a regulatory hormone which is mainly synthesized by adipocytes and regulates body fat mass. Leptin also has regulatory function on anterior pituitary. In this study, we have investigated the expression levels of leptin (Ob) and its receptor (Ob-R) in various types of pituitary adenomas and normal anterior pituitaries. Method: 50 pituitary adenoma cases between 2006 and 2011 were selected. Immunohistochemistry for leptin (Ob) was performed for each 10 cases of null cell, GH, ACTH, prolactin and FSH/LH secreting adenomas. Western blot was performed for leptin receptor (Ob-R) in 9 cases consisting of all five subtypes. Results: Immunohistochemical staining showed greater immunoreactivity for leptin in normal pituitaries compared to adenomas. Except for ACTH-secreting pituitary adenomas, all other four subtypes showed widespread and intense staining for leptin (P<0.0001). Null and FSH/LH-secreting subtypes were mainly showed cytoplasmic and nuclear staining. Western blot analysis showed leptin receptor expression in all types of adenomas except null cell. Objective: Primary malignant brain tumors often present local invasive growth. The study aimed to estimate the expression of KAI1 protein, MMP-2, CD44v6 and their correlation in gliomas of different grade of malignancy. Method: Expression of MMP-2, CD44v6 and KAI1 was evaluated on 154 formalin fixed paraffin-embedded tissue blocks divided into: pilocytic astrocytoma (n=15), fibrillary astrocytoma (n=17), anaplastic astrocytomas (n= 27), anaplastic oligodendrogliomas (n=27), glioblastoma multiforme (n=54) and normal brain tissue (n=14) using immunohistrochemistry. Results: MMP-2 and CD44v6 was observed frequently in gliomas with high grade of malignancy. KAI1 immunoreactivity was mainly observed in specimens with low degree of malignancy. MMP-2 and CD44v6 expression was significantly increased when the degree of malignancy of the gliomas increased (p<0.05). Whilst KAI1 expression increased when the degree of gliomas malignancy decreased (p = 0.03). Positive correlations between MMP-2 and CD44v6 (p=0.01) and inverse between KAI1 and MMP-2 expression (p=0.02) were found in gliomas.
These results indicate that association between MMP-2 and CD44v6 expression may increase dissemination of tumor cells. Whereas, high expression of KAI1 protein might suppress the function of MMP-2 in gliomas.
Pediatric glioblastoma: A case report A. Barin * , S. Ekici, C. Altunkaya, S. Yilmaz, G. S. Yalcin, G. Hatipoglu, M. Caydere, H. Ustun * Ankara Education Hospital, Dept. of Pathology, Turkey
Objective: Glioblastoma is the most common malignant tumor of central nervous system in adults which characterized by malignant pleomorphic astrocytic cells with marked nuclear atypia, mitotic activity, necrosis and/or microvascular proliferation. Glioblastoma may manifest at any age but usually affects adults especially older than 50 years. In this case, we present a pediatric glioblastoma case because it has seen rarely under 20 years and it is so important owing to its prognosis. Method: An 11-year-old boy has had headache for 20 days. According to radiological imagings 23×20 mm in diameter infiltrating mass has seen in temporal lobe contains 15× 7 mm in diameter cystic component and contrast enhancement was noted. Clinically grade 3 astrocytoma has thought primarily and he has been operated. Results: In microscopic examination nuclear atypia, mitotic activity and microvascular proliferation has seen without necrosis. Objective: Gliosarcomas are biphasic neoplasms composed of a glioblastoma admixed to a sarcomatous component with different lines of differentiation. Histogenesis of these tumors is still discussed. Our objective is to specify pathological characteristics of this neoplasm its related to or not to glioblastoma. Method: 3 cases of gliosarcomas diagnosed in our department of pathology. Clinical, radiological, therapeutic and follow-up data were reviewed. Histological features and immunohistochemical results were also established. Results: 3 female patients with a median age of 46 years (ranging 30-69 years). intracranial hyperpressure and paralysis are main symptoms. The brain imaging showed frontal, fronto-parietal and intra ventricular expansive process, respectively. Microscopically, a biphasic pattern, with anaplastic astrocytic cells alternated with malignant mesenchymal areas, demonstrating a fibrosarcoma pattern, in two cases and rhabdoïd pattern in one case. Immunohistochemical stain confirmed the diagnosis of gliosarcoma in each case. Patients were treated by surgical excision; One patient was lost on follow-up. One patient died by postoperative complications. Conclusion: Clinical, radiological and follow-up features of gliosarcomas share great similarities with glioblastomas. Histopathological, histochemical and immunohistochemical studies are helpful in accurate diagnosis. Cytogenetic and molecular data support a monoclonal origin for these tumors. Objective: Case of biphasic synovial sarcoma of nerve in a 59 year-old female located between flexor muscles and radial diaphysis and adherent to median nerve and ulnar artery is presented. Method: Tissue was fixed in formalin, embedded in paraffin and stained with Haematoxylin and Eosin. Periodic Acid-Schiff stain without diastase pre-digestion was obtained. Ventana antibodies were employed: TTF-1, ER, EMA, S100, CK 14, CK 7 and MNF 116. SS18-SSX fusion gene transcript was detected with conventional RT-PCR and qRT-PCR. Results: Macroscopically nodule had smooth circumscribed borders, grey-yellowish colour and measured 3,7×3×2,5 cm. Histologically it was delimited by a dense sclero-hyaline capsule and constituted of numerous glands lined by one layer of cuboidal-columnar cells showing eosinophilic cytoplasm and round to ovoid nuclei with a single small nucleolus. They contained dense eosinophilic strongly PAS and EMA positive material. Glands were immersed within tightly packed spindle cells. Mitoses were scanty. Necrosis was absent. EMA strongly stained most of the glandular as well as scanty spindle cell elements. Chromosomal reciprocal translocation t (X; 18) with positive signal for SS18-SSX1 transcript was seen. Conclusion: Synovial sarcoma of nerve is a rare condition that has to be distinguished from histologically similar lesions.
Rabdoid meningioma: Presentation of a rare case I. Dimitriadis * , A. Heva, I. Matzarakis, A. Sekouli, P. Sakellariou, L. Sakkas * Hospital of Thessaloniki, Dept. of Pathology, Greece
Objective: In 1998, Keppes and later Perry suggested the term rhabdoid meningioma which was adopted into the WHO classification (grade III) in 2000.
We present a rare case of a rabdoid meningioma and study the clinicopathological characteristics of the neoplasm. Results: Α 68-year-old woman was submitted in surgical excision of an endocranial tumour (d:3 cm) in our hospital. The histological examination revealed entirely rabdoid morphology. The neoplastic cells expressed the following immunohistochemical phenotype: S100+, Vimentin+, EMA+, GFAP−, PR+, HMB45−, A1/AE3+(focally), CK8/18+(focally), SMA−, Ki-67 (<1)%. Conclusion: Rabdoid phenotype represents an indicator of malignant transformation regardless of the tumor's histogenesis. Expression of Ki-67 antigen correlates with recurrence even in meningiomas with minimal atypical features, which should prompt closer surveillance after excision. Rabdoid meningiomas behave aggressively and have bad prognosis contrary to the classic meningioma. Prognosis is influenced by it's complete excision and the expansion within the intracranial cavity. It has to be distinguished from glioma, melanoma and metastatic carcinoma. The patient after 1 year is in generally good health, and is followed up by oncologists.
Atypical Extraventricular Neurocytoma (Case Report) S. Ersöz * , U. Yazar, A. Reis, K. Kuzeyli * Karadeniz Technical University, Pathology, Trabzon, Turkey
Objective: Central neurocytomas are rare intraventricular tumors with neuronal differentiation, typically located in the ventricles while tumors located outside the ventricles designated as ''extraventricular neurocytomas'' (EVN). Neurocytomas are classified as atypical if they exhibit a MIB-1 labeling index >2 % or atypical features, like focal necrosis, vascular proliferation, and increased mitotic activity. We here report a case of atypical EVN of the left frontal lobe. Method: A 60-year-old woman patient admitted to hospital for headache. CT scan showed a large partially calcified cystic mass. MR imaging showed circumscribed, solid mural nodule within the cystic mass. The patient underwent a left frontal craniotomy. Specimens were fixed in 10 % formalin and stained with hematoxylin and eosin. For immunohistochemistry, sections were incubated with monoclonal antibodies to synaptophysin, neurofilament protein, MAP-2, NSE and GFAP. The proliferative index was assessed with MIB-1. Results: Histopathological examination revealed a neuronal neoplasm composed of uniform cells in solid sheets. Small necrosis areas and mitotic figures were evident. Immunohistochemical examination revealed positive results for synaptophysin, neurofilament protein, MAP-2, and NSE. GFAP staining was negative. MIB-1 index was calculated as 5 %.
Conclusion: In general, central and extraventricular neurocytomas have a good prognosis. Atypical extraventricular neurocytomas are quite rare like this case with malignant histopathological properties.
Meningioma and Schwannoma associated with Neurofibromatosis Type 2: A case report of rare genetic disorder O. E. Gürer * , R. Tuncer * Akdeniz University, Dept. of Pathology, Antalya, Turkey
Objective: Neurofibromatosis is a group of genetic disorders referred as phakomatoses. There are two main forms called NF1 and NF2 which have different incidence, molecular characteristics, and clinical exhibitio. Both have higher risk for the development of certain rare malignant tumors that occur in the brain, nerves or spinal cord. Method: Here, we present a rare case in which multiple meningiomas and vestibular schwannomas diagnosed in the same operation. Results: A 23 year old woman presented with hearing loss clinically. She underwent surgical treatment for tumoral lesions in two different localization in the same session. One of them resected from pontoserebellar location was diagnosed as a schwannoma and the other one located on dura was diagnosed as a meningioma respectively. Conclusion: NF2 is far less common than NF1 and strict diagnostic criteria have been described each of them. Since difficulty to detect genetic abnormalities and no single criteria is pathognomonic, clinical characteristics much more valuable for differentiation. Knowledge of associated clinical features will help to correct diagnosis and to predict much more devastating clinical course for NF2.
Diagnostic confusion: Central neurocytoma N. Gursan * , E. Demirci, M. Calik, F. Daloglu, C. Gundogdu * Ataturk University, Medical Faculty, Erzurum, Turkey
Objective: Central neurocytoma is a rare intraventricular brain tumor that affects young adults and presents with increased intracranial pressure secondary to obstructive hydrocephalus. They are usually located in the supratentorial periventricular region. Because of some clinical and radiological findings CNS neurocytomas were confused with other intraventricular lesions. Method: A 37-year-old male was admitted with chronic headache with 6 months duration on July 21, 2011, when there were no neurological deficits or physical abnormalities. Computed tomographic (CT) scans showed 51×40 mm a mixed density mass with amorphous calcification in the right lateral ventricle, which was irregularly enhanced by contrast medium. Light Microscopy showed sheets of monotonously neoplastic cells with uniform round-to-oval nuclei and inconspicuous nucleoli. The cytoplasm was clear or eosinophilic with indistinct border. Capillary networks were well developed and divided the tumor cells into groups. No nuclear pleomorphism or mitosis was seen. We present a case of intraventricular neurocytomas confirmed by immunohistochemical studies. Conclusion: This rare tumor causing diagnostic confusion, discussed by the literature.
A case of oligodendroglioma with neuronal differentiation T. Hirose * , S. Nobusawa, Y. Nakazato, A. Sasaki * Tokushima Pref. Center Hospital, Dept. of Diagnostic Pathology, Japan
Objective: We report a case of oligodendroglioma showing marked neuronal differentiation. Method: A 46-year-old female visited an emergency room because of an attack of convulsion. Imaging analyses disclosed a calcified, 3.5×3 cm tumor in the right frontal lobe. Results: The resected tumor was composed of a mixture of oligodendroglioma-like (OLG) and gangliocytoma-like (GC) areas. In the former, fried-egg appearance and more cellular nodules were recognized. In the latter, there were numerous small neuron-like cells with basophilic cytoplasm. OLG areas showed immunoreactivity for Olig2, GFAP and mutant IDH1, while GC areas were positive for synaptophysin and NeuN. Ki-67 labeling index was about 10 % in cellular nodules of OLG areas. At least one allelic loss of 1p/19q was detected in 53.4/61.2 % cells in OLG areas and 47.2/40.6 % in GC areas with FISH analyses. Furthermore, an identical mutation of IDH1 (G395A, R132H) was demonstrated in both OLG and GC areas.
Conclusion: Genetic abnormalities including 1p/19q loss and IDH1 mutation indicate that this case is an oligodendroglioma associated with prominent ganglion cell differentiation. Oligodendrogliomas with neuronal differentiation like our case may suggest a close relationship between oligodendroglial progenitor cells and neuronal cells.
Gemistocytic astrocytoma with granulomatous inflammation O. Ipci * , E. Atik Dogan, T. Ozgur, H. Gokce, N. Yilmaz, A. Yilmaz * Mustafa Kemal University, Dept. of Pathology, Hatay, Turkey
Objective: The coexistence of granulomatous inflammation and astrocytoma is extremely rare. Hemostatic agents are used to control intraoperative bleeding in many surgical subspecialties, including neurosurgery. There are occasional reports of granulomatous reaction to biomaterials. Method: We aimed to present a 35 year old female patient, who was operated because of the tumor at left frontoparietal region. Tumor was histopathologically reported as oligoastocytoma. However, no granulomatous inflammation was detected beside the tumor. After 6 months, patient was reoperated because of tumor recurrence, abscess and inflammatory necrotic tissue beside the tumor. Histopathologically, gemistocytic astrocytoma with granulomatous inflammation and abscess formation was reported. No micro-organism had grown in any kind of cultures. At our neurosurgery department surgicel is used after craniotomy as hemostatic agent. It's raw material is cellulose and absorbed in about 3 months. Results: In the literature, germinoma accompanied by granulomatous inflammation have been reported. However, astrocytic tumors don't express such an association. The patient doesn't use any drugs forming granuloma reported in the literature. Conclusion: We encountered that Surgicel causing granulomatous inflammation in very few cases in the literature. The findings might be secondary to surgicel that used in the first operation. Objective: Astrocytic tumors show various molecular and genetic alterations in its development and progression. Glioblastoma multiforme (GM) develops as either de novo or by progression from diffuse astrocytomas (DA) or anaplastic astrocytomas (AA) by genetic alterations. Method: Loss of heterozygosity (LOH) and microsatellite instability (MSI) was investigated in 40 cases of astrocytic tumors (DAs [n=12], AAs [n=15] and GMs [n=13]) with 14 microsatellite markers and 5 microsatellite markers harboring p53 and PTEN, respectively. The patients' age ranged from 27 to 77 years (mean, 50 years). Results: LOH was statistically significant in AAs and GMs, compared to that of DAs (p=0.023). LOH for 17p13 and LOH-H group for 10q23 were statistically significant in AAs and GMs (p=0.045 and p=0.001). MSI rate in DAs, AAs and GMs was detected in 16.7 %, 13.3 %, 23.1 %, but it showed no significant correlation with prognosis factors. MSI and MSI-H rates were correlated with younger (〈50 years) group (p= 0.040, p=0.011, respectively). Conclusion: Alterations on PTEN and p53 may contribute to the development and progression of astrocytic tumors. Especially, LOH-H for 10q23 and 17p13 is considered as clinical application of discriminating the AAs and GMs from DAs. MSI might be involved in the tumorigenesis of relatively young patients. Objective: Charcot-Marie-Tooth disease (CMT) III or Dejerine-Sottas syndrome is a severe hereditary demyelinating motor and sensory neuropathy presenting in infancy or early childhood with delayed motor milestone and extremely slow nerve conduction velocities. Method: Among 530 unrelated Korean CMT patients, mutational screen for well known CMT genes reveals three patients with PMP22 de novo mutation showing DSS. The histopathological findings of distal sural nerves are analyzed in all patients twice; Cases 1 and 2 with 10 years interval and Case 3 with 17 years. Results: On semi-thin transverse sections, all the remaining axons (thinly myelinated or demyelinated) are surrounded by classic or basal lamina onion bulbs (OBs). The first biopsy of Case 1 (2 years-old) shows OBs (4,452/mm2), which consists of 2,295 (51.55 %) MFs with OBs and 2,157 (48.45 %) demyelinated axons with OBs. The second biopsy shows OBs (2,275/mm2), consisting of 425 (18.68 %) MFs with OBs, 489 (21.49 %) demyelinated axons with OBs, and 957 (42.07 %) OBs with no discernible axons. Cases 2 and 3 show similar histopathologic findings with main differences in numbers of OBs. Conclusion: Although the differences noted in two biopsies are probably due to age of the patients, correlations with clinical symptoms will be helpul for management of the patients. Objective: Two mutations of the seipin gene (N88S and S90L) have been reported and known to cause neurodegenerative disorders. We performed whole exome sequencing in two Charcot-Marie-Tooth families and identified three patients with two causative heterozygous mutations (N88S and S90W) and S90W is a novel mutation. Method: The histopathological findings of distal sural nerves are analyzed in two patients (Cases 1 and 2) with S90W novel mutation (FC305) and a patient (Case 3) with N88S mutation (FC51). Results: Transverse semi-thin sections reveal increased numbers of myelinated fibers (MFs) (11,898/mm2; 11,429/ mm2; 9,430/mm2) with increased small MFs forming regenerative axon clusters and loss of large MFs. Histogram shows unimodal distribution pattern. Cases 1 and 2 show the similar ranges and average of diameter of MFs (1.461 3.24 μm, 5.13 μm). Case 3 shows more numbers of small MFs (0.82~10.50 μm, 3.73 μm). Ultra-structural examination reveals MFs with myelin abnormalities, pseudo-onion bulb formation including single axon or axon clusters, and thick MFs. Conclusion: The findings are consistent with axonal neuropathy with features of demyelination. Additional cases will be necessary to know the significance of differences between these cases.
Immunohistochemical subtyping of primary and secondary glioblastomas K.-S. Lee * * Seoul National University Hospital, Republic of Korea
Objective: Glioblastomas (GBLs) may develop de novo (primary GBL; P-GBL) or through progression from lowgrade or anaplastic astrocytomas (secondary GBL; S-GBL).
For subtyping of GBLs, we tried immunohistochemical anaysis. Method: We collected 150 cases of GBLs in SNUBH and SNUH. The mean age at the time of the primary surgery was 58.8 years (range 19-85). Immunohistochemical studies were performed for EGFR, p53 and IDH-1.
Results: According to clinical history, GBLs consisted of 146 primary and four secondary GBLs. However, EGFR(+)/ p53(−) immunohistochemical features of P-GBL consisted 41.3 % and EGFR(−)/p53(+) immunohistochemical features of S-GBL consisted 28.6 % among 150 cases. EGFR(+)/p53 (+) was noted in 20.6 % and EGFR(−)/p53(−) in 9.3 %. Immunohistochemical expression of IDH-1 was noted in 14 cases (9.7 %) out of 144 cases. Expression of IDH-1 and EGFR(−)/p53(+) showed positive correlations with young age. Characteristic features of S-GBL were noted in 3.5 % of clinically P-GBL. Conclusion: We analyzed immunohistochemical subtypes of Korean glioblastomas. Combination of EGFR and p53 was unsatisfactory but combination of EGFR, p53 and IDH-1 can be a good tool for immunohistochemical subtyping of glioblastomas. (also known as erbB-2) is a 185-kD transmembrane glycoprotein with tyrosine kinase activity. HER2 is expressed in some human malignancies and can be a potential target for therapeutic intervention with selective inhibitors. There are only a few studies on the relationship between meningioma and HER2 expression, and the results are different as well. The aim of this study was to determine this relationship. Method: Seventy-two paraffin blocks of meningioma were selected randomly and immunohistochemical staining was then performed for each specimen.
Results: Thirty-one of the 72 meningiomas were HER2-positive. HER2 expression was observed in 11 (55 %) of the 20 grade II/III, and 20 (38.5 %) of the 52 grade I meningiomas.
Conclusion: Consequently, HER2 expression was detected in 43 % of meningiomas. No significant difference was seen between grade I and II/III meningiomas, primary and recurrent tumors, and males and females from the point of view of HER2 expression. Objective: In our previous papers we studied accumulation of iron and proteoglycans in the brain. The aim of this analysis is to build on our previous results alongside other authors to investigate the reason for the presence of excessively accumulated iron observed in Parkinson disease (PD). Method: Postmortem samples taken from the globus pallidus were prepared for light microscopy for iron and proteoglycans detection. Patients had neither iron metabolism disorders nor had clinical signs of neurodegeneration. The samples were analysed using electron paramagnetic resonance (EPR) to investigate the bounds between iron and proteoglycans.
Results: EPR measurements confirm the presence of complex bounds between Fe(III) and proteoglycans. Conclusion: Previous data showed a possible dependance between accumulated iron, proteoglycans, ferrireductive alpha-synuclein and Lewy bodies. We propose a mechanism of toxic iron accumulation in the brain in PD. This cyclic process of amplification is presented by onward steps. It is completed by our own observations into one cycle. The initiator of this vicious circle is probably an impirement of the equilibrium in the presence of useful amount of iron. Understanding these relations could bring new insights to the neurodegenerative disease.
Role of PDGFR overexpression in Schwannoma, and its diagnostic and therapeutic implications T. Neuman * , Y. Fellig * Hadassah Medical Center, Dept. of Pathology, Jerusalem, Israel
Objective: Platelet-Derived-Growth-Factor-Receptors (PDGFR) are transmembrane tyrosine kinase receptors that function as relay points for signaling pathways. They play a key role in numerous processes that affect cell proliferation, tumor genesis, cancer invasion, metastasis, and modulation of apoptosis. Recently, PDGFR has been demonstrated to be overexpressed in Schwannoma in vivo, and in a small series of acoustic neuromas. However, its phenotypic expression in different variants of Schwannoma remains largely unclear.
Method: Immunohistochemical staining was used to detect PDGFR expression in archived formalin-fixed, paraffin-embedded Schwannoma tissue samples (n=24 Objective: Pancreatic ductal carcinoma (PDC) has one of the worst prognoses. Up to 90 % of pancreatic cancers are diagnosed at the locally advanced or metastatic stage. However, brain metastases from pancreatic carcinoma are extremely rare since less than 20 cases have been reported in the literature since 1978.
Method: We report a case of a brain metastasis from pancreatic adenocarcinoma occurred in a 66 years old women.
Results: 62-year-old woman developed severe neurological symptoms and progressively decreased consciousness. CT revealed cystic tumor in cerebral peduncle and medulla oblongata. Stereotactic biopsy revealed metastasis of carcinoma. The patient died from of the twelfth day after STB because of recurrent hemorrhage. Autopsy revealed well-differentiated papillary adenocarcinoma in the head of pancreas and with multiple metastases in liver and two metastases in medulla oblongata and cerebral peduncle. IHC staining for cytokeratins 7, 18 and 19, mucin 1 and 5 AC types was strongly positive. Ki67 labeling index was 14 %. Conclusion: We reported an autopsy case of brain metastases from pancreatic cancer, in which the patient initially developed symptoms of a neurologic disorder without exhibiting any symptoms of pancreatic disease.
The use of Bone Morphogenetic Proteins (BMP2) with nunostrucnure Grey implant in reparation of bone tissue defect T. Pavlova * , L. Pavlova, A. Nesterov, I. Goncharov, D. Kolesnicov * Belgorod State University, Dept. of Pathology, Russia
Objective: The matters of materials development of scull bones defect plastic are obviously necessary this time. The experience in use of nanocostructive titans, composites, bone morphogenetic proteins was accumulated. Method: Implant, made of nanoconstructed titan Grey, covered by 1 layer (gelatin, dextran), 2 layers (1-gelatin, dextran, 2-hydroxyapatite, collagen, dextran) and by 2 layers with use of bone morphogenetic proteins BMP-2. The experiment was conducted on 240 rat-males. In order to study the regeneration processes it were used light, fluorescence, probe and scanning microscopy with the trace element analysis. Results: The most active changes were registered in the experimental group with use of composite from titan Grey with 2 layers of covering and BMP-2. The covering compound increase the rate of regeneration processes due to creation calcium and phosphorus ion depot and perform support function for again-formed tissue. Increase in the concentration of calcium, phosphorus, sodium, magnesium in trabeculae of bones in comparison with matrix bone at term of 12 weeks testify about active regenerative processes; the mature smooth surface with structured relief of bone trabeculae and diploic veins were detected.
Conclusion: Results can be used in neurosurgery, traumatology-orthopedics, dentistry, plastic and cosmetic surgery.
Alpha-synuclein (AS) pathology of the Peripheral Autonomic Nervous System (PANS) in Parkinson Disease (PD) and other Lewy Body Diseases (LBD) T. Ribalta * , E. Tolosa, J. Navarro Otano, E. Gelpi * Hospital Clinic of Barcelona, Dept. of Anatomic Pathology, Spain
Objective: PD and other LBD have been associated with AS aggregates in the central nervous system (CNS). However, autonomic dysfunctions may appear at any time in the course of the disease. Our objective was to investigate the distribution of AS in the PANS in synucleinopathies.
Method: We examined PANS structures obtained at post mortem from 28 subjects (19 female, age range 69-93, mean 81) at our Brain Bank with a diagnosis of PD (10); Lewy body dementia (5); Alzheimer's disease (9); mixed dementia or other (4). A complete neuropathological study of PANS was performed, including dorsal spinal ganglia, vagus nerve, paraspinal sympathetic ganglia, mesentery, adrenals, digestive and genitourinary systems, heart, and skin. Routine and immune stains for AS and tyrosine-hydroxylase were applied. Cases were semiquantitatively assessed.
Results: 71 % of cases showed AS pathology in the CNS. Of them, AS aggregates were additionally present in the PANS in 80 %. A gradient in involvement was observed, being the paraspinal chain (particularly the stellate ganglion) the most constantly involved structure, followed by digestive system, adrenals, and GU system. Conclusion: These findings indicate that AS aggregates are extensively found in PANS in synucleinopathies. The highest expression is found in the paraspinal ganglion chain and decreases in other PANS regions. Our results provide valuable information about potential development of new diagnostic and therapeutic strategies. Objective: Chordoid meningioma is an uncommon histopathological variant of meningioma. Method: We report one case of chordoid meningioma occurring in adult patient. Paraffin embedded tissue was stained with hematoxylin-eosin. We used immunohistochemical markers such as CEA, EMA, vimentin and TLE3.
Results: A 81-year-old woman was hospitalized with clinic of an intracranial hemorrhage. Axial computed tomography (CT) of the brain revealed a 3.1*5.2*4.9 cm lesion with perilesional edema in the left temporoparietal region. Foci of intratumoral hemorrhage were also seen. Compression of the left lateral ventricle and midline shift to right side was seen. The patient underwent a left temporoparietal craniotomy for resection of the tumor and died 3 days after surgery. Histologically, sections revealed sheets, trabeculae and lobules of tumour cells scattered in a pale basophilic myxoid matrix. Some of these cells exhibited characteristic cytoplasmic vacuolization. A typical focal meningiomatous pattern was also observed. The tumor cells were diffusely positive for epithelial membrane antigen, vimentin and a strong nuclear immunoreactivity for TLE3. These cells showed negative immunostaining for CEA.
Conclusion: Chordoid meningioma should be distinguished from chordoma, chondrosarcoma, metastatic mucinous carcinoma and other variants of meningioma.
Morphology of non-specified encephalopathy in cases of polymerase chain reaction proved presence of human herpesvirus-6 S. Roga * , I. Strumfa, S. Kuleznova, S. Chapenko, S. Rasa, M. Murovska * Riga Stradins University, Teaching Department, Latvia
Objective: The morphology of non-specified encephalopathy is a complex medical problem. Human herpesvirus-6 (HHV-6) infection can be discussed as a predisposing factor. The aim of the present study was to investigate the presence of beta HHV-6 in non-specified adult encephalopathy cases. Method: The blood, meninges and brain tissue were obtained in adult (aged 42-74 years) autopsies including 21 cases with encephalopathy and 23 cases in the control group. Tissues were submitted for routine histology including haematoxylin-eosin stain. The presence of HHV-6 genome (DNA) was analysed by nested polymerase chain reaction (nPCR), HHV-6 variants by restriction endonuclease analysis.
Results: The gross and microscopic structure did not reveal any specific changes. In the encephalopathy group, HHV-6 DNA sequence was found in meningeal tissues (16/21 cases; p=0.0036), in brain tissues (15/21 cases; p = 0.0007), and both in brain and meningeal tissues (10/21 cases; p=0.0174). In the control group, the viral DNA was identified in meningeal tissues (7/23 cases), in brain tissues (4/23 cases), both in brain and meningeal tissues (3/23 cases). HHV-6B variant was detected in all cases. Conclusion: On the basis of the present study it can be concluded that HHV-6 is a pathogenic factor that can predispose to encephalopathy.
Pathological variants of brain metastases of breast carcinoma and their prognostic and predictive role in different age groups D. Objective: It is extremely rare that the intramedullary spinal cord metastases (ISCM) are the first manifestation of cancer.
Method: In database of neurosurgery biopsies from 2002 to 2011 two cases of ISCM were found without preoperative signs of primary neoplasm. Surgically removed tissue was stained with hematoxylin-eosin and immunohistochemically using following markers: AE1/ AE3, CK7, CK20, mammaglobin, TTF-1, GFAP, S-100 and vimentin. Results: One patient was male aged 51 and another female aged 44. They presented with neck pain and rapidly progressive upper and lower limb weakness. Imaging analysis showed contrast-enhancing mass at the level C4-C6 in male and C2-C4 in female, reported as an ependymoma. Grossly, tumor tissue was of soft consistency and grey white color with yellowish foci of necrosis. Histopathology revealed moderately differentiated adenocarcinoma with distinct border to surrounded glial tissue. In both cases, tumor cells were immunopositive for AE1/AE3, CK7 and TTF-1 indicating primary lung origin. Postoperatively primary cancer was found in upper lobe of the left lung in both cases, without evidence of lymphadenopathy and other distant metastatic lesions in female.
Conclusion: Diagnosis of ISCM in both cases without preoperative signs of neoplasm in other organs was surprising. Immunohistochemical analysis was essential for determination of cancer origin. Objective: Tetraspanin CD151 is a positive effector of cancer invasion and metastasis in many tumor types. Method: We investigated the protein expression of CD151 in 211 cases of WHO grade I to IV gliomas. Additionally, we performed O6-methylguanin-DNA methyltransferase (MGMT) methylation analysis using real-time methylation-specific PCR in 36 glioblastomas, and the prognostic significance of these biomarkers in glioblastomas was evaluated.
Results: CD151 was overexpressed in a significant proportion (55.6 %) of glioblastomas, while it was not deetected in most of grade I to III glial tumors except for rare overexpression in anaplastic astrocytoma (2/10, 20 %) and oligoastrocytomas (3/23, 13 %). CD151 overexpression was closely associated with MGMT methylation (P= 0.014), and it was a prognostic factor for predicting worse overall survival (OS; P=0.002) and progressionfree survival (PFS; P=0.043). Combination of CD151 overexpression and MGMT methylation better stratified the patients' OS (P=0.001) and PFS (P=0.009). In multivariate analysis, CD151 overexpression was an independent prognostic factor for predicting OS over MGMT methylation (P=0.012). Conclusion: CD151 seems to have a critical role for high grade progression in astroglial tumors. CD151 is a good tissue marker for predicting worse prognosis in glioblastomas. Results: A gross total resection of the tumor was achieved. Histological examination revealed a paucicellular tumor with lobulated architecture and abundant myxoid stroma, containing stellate or spindle cells lacking mitotic activity. Alcian blue and mucicarmin histochemical stains were diffusely and strongly positive. Immunohistochemical analysis showed diffuse reactivity for vimentin and scattered cells positive for CD34. Stains for EMA, GFAP, S100 protein, cytokeratin and smoothmuscle actin were negative.
Conclusion: Primary intracranial myxoma should be distinguished from other myxoid intracranial tumors such as myxomatous meningioma, epithelioid hemangioendothelioma or sarcoma through appropriate pathological and inmunohistochemical analysis. A metastasic cardiac myxoma should also be ruled out through cardiac evaluation including an echocardiography. The distinction between this entity and the reported neurothekeoma of the meninges needs to be reevaluated.
Holocord pylocitic astrocytoma associated to syringomyelia J. Trillo-Tinoco * , T. Garibay-Huarte, E. Gómez-Apo, M. A. Rodríguez-Florido, L. Chávez-Macias, J. E. Olvera-Rabiela * Hospital General de Mexico, Dept. of Surgical Pathology, Mexico City, Mexico
Objective: Spinal intramedullary tumors sometimes extend both superiorly and inferiorly along almost the entire cord, and those diagnosed as pilocytic astrocytoma are rare. The syringomyelia is an ependimary or periependimary cavitation of the spinal cord and is considered a suffering of degenerative and irreversible type, 58 % of the cases is associated with intramedullars tumors.
Method: A three-year-old girl with history of headache, progressive weakness, 3 months before, she was hospitalized for pneumonia, had impairment of neurologic functions and died of cardiac arrest. The autopsy was performed.
Results: The neuropathologic study revealed a intramedullary holocord tumor with secondary syringobulbia, cervical and lumbar syringomyelia. Histopathological examination of all specimen resulted in diagnosis of a pilocytic astrocytoma. Although no signs of atypia were present, an elevated proliferative activity of endothelial vessels was noted. Conclusion: Gross total resection of holocord and longitudinally extensive intramedullary spinal cord tumors can be achieved with preservation of long-term neurological function and also solved the syrinx. Objective: Epithelial cellular adhesion molecule (Ep-CAM) has been studied in many tissues and neoplasm, including thyroid and thyroid tumors. This is a preliminary study to assess the immunohistochemical expression of Ep-CAM in thyroid tumors using Ber-EP4.
We examined the expression of Ep-CAM using the monoclonal antibody Ber-EP4 in 36 cases of thyroid tumors, including 4 adenomas, 11 papillary carcinomas, 5 follicular carcinomas, 8 medullary carcinomas, 2 poorly differentiated carcinomas, and 6 anaplastic carcinomas. We assessed the positivity as a predominantly membranous staining of the cells, and was scored according to the estimated percentage tumor cells in the total tissue section (negative: 0-10 %; positive: ≥10 %).
Results: Ber-EP4 expression was detected in normal thyroid tissue (perilessional), in all the adenomas, follicular carcinomas, and medullary carcinomas. Papillary carcinomas showed in 36 % lacking areas of expression, coinciding histopathologically one case with poorly differentiated component. All of the poorly differentiated and the anaplastic carcinomas were negative. Conclusion: The expression of Ep-CAM, using Ber-EP4, was related to normal thyroid tissue and well differentiated neoplasms. Our study suggest that lost of expression is associated to dedifferentiation. This results match up with the literature. In addition, clinical data and follow up are required to correlate focal areas of lost of expression of Ber-EP4 with dedifferentiated areas of the tumor.
The pathological effects of estradiol valerate on testis tissue: Size and weight in male rat F. Bidhendi * , R. Ahmadi * Imamkhomeini Hospital, Dept. of Radiology, Tehran, Iran
Objective: Studies show that estrogens can influence reproductive system differentiation. The main aim of this study was to determine pathological effects of estradiol valerate on testis histology and morphology in male rats. Method: Adult male albino Wistar rats were divided to control and estradiol valerate (200 μg/kg/day) receiving groups. Estradiol valerate was applied subcutaneously. After 4 weeks, testes were excised and studied morphologically and histologically. Data were statistically analyzed and compared between groups using ANOVA. Results: Our findings revealed that estradiol valerate injection resulted in reducing of testes weight and size (P<0.05). Semeniferous tubules were apparently deformed in estradiol valerate receiving animals and cellular density was also reduced. Number of spermatocytes, spermatids and sperms was decreased in estradiol valerate receving rats compared with control animals (P<0.001).
Conclusion: Estradiol valerate has considerable pathological effects on testes morphology and histology in male rats. Key words: Estradiol, Testes.
Are tumor-dimension, Galectin-3 and CyclinD3 useful to characterize oncocytic adenomas and carcinomas?: mutations in about 45 % of cases. The first clinical manifestation of this tumour is often represented by lymphatic metastases.
Method: Twelve cases of PTC and respective lymph node metastases were retrieved from our archive from 2010 to present. Formalin fixed paraffin embedded tissue sections were stained with routine hematoxilin-eosin and representative tissue areas for both tumour and metastases were microdissected and DNA was extracted. After PCR amplification the mutational status of BRAF and RAs was determined by DNA sequencing.
Results: Seven cases of twelve (58 %) showed a BRAF-V600E mutation. Interestingly, only in three these cases (43 %) there was concordance in the mutational status between primary tumour and metastases. Moreover, all metastase of wild type carcinomas were also wyld type. Conclusion: Even with an overall good prognosis, PTC is characterized by high incidence of lymph node metastases. As BRAF mutational status correlates well with prognosis and tumour progression, a correct molecular assessment is of paramount importance. Our data may indicate that, because the possibility of discrepancy in the mutational status between primary tumour and metastases, molecular analysis should be performed on the primary tumour. Objective: Diabetes may cause chronic and non-healing diabetic foot ulcers (DFU) decreasing the welfare of patients. Some neuropeptides, Substance P and neurotensin (NT), may act as inflammatory modulators and improve wound healing. Natural biopolymers, chitosan derivatives, are receiving great attention as powerful wound dressing materials for wound healing applications due to their favorable properties. The work aim was to use 5-methyl pyrrolidinone chitosan (MPC) as a platform for the delivery of NT. Method: Diabetes was induced by intraperitoneal injection of 200 mg/kg streptozotocin. Mice were anesthetized and two 6 mm excision wounds were created dorsally. MPC alone, NT alone, MPC loaded with NT or PBS were placed daily on wounds. Histological analysis of skin, at days 0, 3 and 10, was done through H&E and Masson´s Trichrome stainnings.
Results: In diabetic mice, the healing process was slower, showing an engulfment of acute inflammatory cells that triggered macrophage activity when compared with controls. At day 3, MPC treatment leaded to a faster healing with retraction of the wound site, NT induced a slower noncontracting healing and combined application delayed inflammatory repair with persistent neutrophils. Conclusion: At day 10, all treatments induced a total healing however, MPC + NT reduced neutrophils infiltration compared with MPC alone.
Acth producing nasal paraganglioma: Case report J. Cassis * , A. Galzerano, M. Chorão * Hospital Egas Moniz, Dept. of Anatomy Pathology, Lisboa, Portugal
Objective: Female patient with chronic sinusitis and hypertension presents with lipothymia, dizziness, polyuria and polydipsia. Laboratory studies showed hyperglicemia, leucocytosis, hypokalemia, hypercortisoluria and elevated serum ACTH levels. Head MRI revealed tumor in the right nasal cavity with implantation at the cribiform plate of the ethmoid bone. Method: Microscopically the tumor had lobular architecture with big, round cells and with eosinophilic granular cytoplasm. These cells were NSE+, synaptophysin+, CD56+, CD57+ and ACTH+. Surrounding the tumor lobules were several spindle cells S-100+ (sustentacular cells).
Results: The lobular growth pattern, the presence of sustentacular cells (S-100 positive) , and the ACTH producing granules favours an ACTH producing nasal paraganglioma diagnosis.
Conclusion: Head and neck paragangliomas account for 0,12 % of the tumors in this region, and are mainly located in the carotid bodies. Paragangliomas arising in the nasal cavity are rare, only 23 cases reported so far. They affect the middle or upper turbinates, and also the etmoid, maxillary and sphenoidal sinuses. We report the third case in literature of a ACTH producing paraganglioma in the nasal cavity.
Sporadic aggressive silent somatotroph pituitary adenoma in the young: Report of two cases L. Chinezu * , A. Vasiljevic, S. Achard Peyregne, G. Raverot, E. Jouanneau, J. Trouillas * Emergency County Hospital, Pathology, Tg. Mures, Romania
Objective: Silent pituitary adenomas are infrequent tumors in adults and exceptional in young.
We describe two cases of 20-year-old girls with pituitary adenoma without genetic history, revealed by visual field defects, with panhypopituitarism in one patient. In both cases, MRI showed giant pituitary adenoma, with invasion of the cavernous and sphenoidal sinuses. Hormone assays revealed normal GH, PRL and IGF1 plasma values. Surgery was incomplete in both cases. Results: Both tumors presented a diffuse pattern. The cells exhibited large nuclei with prominent nucleoli. By immunocytochemistry, focal and low percentages of GH-and PRL-immunoreactive cells were observed respectively in both tumors (GH=1-30 % and PRL=1-5 %). CgA was positive. Cytokeratin and others antibodies against pituitary hormones were negative. Both tumors had high proliferative indexes: Ki-67>3 % (4-10 %) and elevated mitotic number (1-13 mitoses). Detection of p53 was also positive (0.5-5.7 %). The cases were diagnosed as atypical adenomas, according to the WHO classification. Conclusion: The pathological diagnosis of these aggressive GH-PRL tumors has to be taken into account by the clinician to choose the optimal therapeutic strategies. Despite of the important side effects of the radiotherapy, this aggressive treatment might be proposed earlier, even for the young patients, to avoid the tumoral progression.
Intrauterine growth retardation with high fat diet in rats markedly disturbs islet morphology characterized by peri-islet inflammation, fibrosis and haemosiderosis J. Dahlstrom * , V. Delghingaro-Augusto, L. Madad, A. Chandra, E. Bean, C. Simeonovic, C. J. Nolan * ACT Pathology & ANUMS, Dept. of Anatomical Pathology, Garran, Australia
Objective: Pre-and post-natal factors such as intrauterine growth retardation (IUGR) and high fat (HF) diet contribute to type 2 diabetes (T2D). Our aim was to determine if IUGR and HF diets interact in T2D pathogenesis.
Method: A surgical model of IUGR (bilateral uterine artery ligation) in Sprague-Dawley rats with sham (SH) controls was used pups were fed either HF or chow (CH) diets from weaning. Serial measures of body weight and glucose tolerance were made. At 25 weeks, rat pancreases were harvested for histological assessment.
Results: IUGR vs SH pups weights were 17 % lower. HF diet caused excess weight gain, dyslipidaemia, hyperinsulinaemia and mild glucose intolerance not further aggravated by IUGR. Markedly abnormal islet morphology was evident in 0/6 SH-CH, 5/8 SH-HF, 4/8 IUGR-CH and 9/9 IUGR-HF rats (chi-sq, p=0.008). Abnormal islets were characterised by larger size, irregular shape, peri-islet inflammation with CD68 positive cells and marked haemosiderosis. Overall beta-cell mass was not altered by IUGR, with a trend for it to be mildly increased in both HF-fed groups. Conclusion: HF and IUGR independently and together contribute to islet injury. The marked islet haemosiderosis associated with IUGR and HF diets warrants further investigation as iron is toxic to β-cells. Objective: Adreno-hepatic fusion (AHF) is defined as adhesion of the liver and right adrenal gland with or without a fibrous capsule dividing both organs. We report a surgical case of AHF in which a virilising malignant adrenocortical tumour protruded into the liver mimicking a hepatic mass. Method: A 36-years-old woman presented with mid-right abdominal pain and marked hirsutism. Computed tomography (CT) revealed an apparently intrahepatic solid tumour suggestive of a giant hepatic adenoma. The right adrenal gland was not visible on CT and the right kidney showed marked downwards displacement. A radical right adrenohepatectomy was performed, with a satisfactory outcome.
Results: Grossly, a giant, partially encapsulated mass was seen to protrude from the right adrenal gland into the liver. Microscopically, the tumour was constituted by atypical clear cells with nuclear polymorphism, necrosis and moderate mitotic activity. Tumour cells expressed vimentin and Melan-A protein.
Conclusion: To our knowledge, this is the first description of a malignant adrenal tumour in the setting of AHF. As illustrated by this case, awareness of AHF as an entity and attention to its distinctive gross and histologic features are essential to avoid confusion between adrenal and hepatic lesions, especially when imaging studies have provided misleading findings. Objective: Cushing's syndrome (CS) is a rare disease, resulting in the majority of cases from ACTH hypersecretion. 70 % is of pituitary origin, 20 % of adrenal origin, and only 10 % from ectopic ACTH production. Method: We reported a rare cause of ectopic ACTH dependent CS, caused by a pheochromocytoma.
Results: A 54-year-old male was hospitalized for severe high blood pressure, depression and weight loss. Biological investigation revealed serious hypokaliema, severe ACTH dependent hypercortisolism with elevated urinary free cortisol secretion, loss of diurnal variation, and excess plasma ACTH level. CT-scan revealed a nodular lesion in the right adrenal gland with hyperplasia in the left one. The nodular lesion was assumed to be a pheochromocytoma based on the elevated serum and urinary catecholamine and metabolites and local uptake in right adrenal gland in 131 I-MIBG scan. Right adrenalectomy was performed. Macroscopic examination revealed a 4 cm, well-circumscribed, tan-brown tumor, associated with diffuse adrenocortical hyperplasia. Histological examination confirmed the diagnosis of pheochromocytoma, without signs of aggressiveness. The tumor cells were immunopositive for Chromogranin A, Synaptophysin and ACTH. After surgery, catecholamine secretion returned quickly to normal level. Biological and clinical CS regression was noted. Conclusion: Despite the rare association of CS with pheochromocytoma, preoperative diagnosis is required to an appropriate therapy.
A rare case: Extralobar pulmonary sequestration mimicking neuroblastoma G. Emiroglu * , N. Özsan, S. Tiryaki, A. Çelik, Y. Ertan * Ege University, Dept. of Pathology, Izmir, Turkey
Objective: Extralobar intraabdominal pulmonary sequestrations are rare congenital malformations which are characterized by disorganized and nonfunctioning pulmonary parenchyma. These lesions have no communication with the bronchial tree and pulmonary arteries. They receive their blood supply from the systemic circulation. Method: We describe a 5 days old male infant admitted to the hospital with a left subdiaphragmatic, 50×40×35 mm, hiperechogenic and, solid mass that was identified during ultrasonography on the 26th week of gestation.
Results: Abdominal computerized tomography demonstrated a 48× 37× 33 mm, solid, vascularized, encapsulated, mostly cystic suprarenal mass with no calcification reported to be highly suspicious for neuroblastoma. The mass was completely excised. Gross pathologic examination revealed a well circumscribed spongy lesion that mimicked a lung tissue. On microscopic examination, a disorganized lung tissue that was composed of alveoli, alveolar ductus and bronchioles was seen. Normal adrenal tissue was not observed. Later, we learnt that the lesion's arterial blood supply was from the abdominal aort. Based on these findings, our diagnosis was intraabdominal extralobar pulmonary sequestration.
Conclusion: Intraabdominal extralobar pulmonary sequestrations should be kept in mind in cases of the adrenal masses as the surgical resection is the adequate treatment method for these lesions.
Axillary lymph node metastasis of papillary thyroid carcinoma showing anaplastic transformation with cutaneous metastasis S. Erkilic * , A. Ozkur, U. Elboga, F. Celenk, M. Kanlikama * Gaziantep University, Medical Faculty, Turkey
Objective: Papillary thyroid carcinoma (PTC) rarely metastasizes to axillary lymph nodes. Although anaplastic transformation (AT) may occur in the cervical lymph node metastasis from PTC, it is rarely observed in the metastatic axillary lymph nodes. Furthermore, cutaneous metastasis from anaplastic thyroid carcinoma (ATC) is extremely rare. Method: A 65-year-old male patient who had operated for multinodular goiter 8 years ago presented with neck swelling on the right side. Residual thyroid tissue was detected and the lesion was removed surgically in combination with a right neck dissection. Histopathologic examination was consistent with PTC and metastatic lymph nodes. The patient received radioactive iodine therapy. Afterwards, the patient presented with a right axillary mass 14 years after the first operation and underwent surgical excision. Results: Histopathologic examination showed PTC with anaplastic transformation. Histopathologically confirmed multiple skin metastases from ATC emerged in thoracic and abdominal regions 3 months after the last operation. Patient died 3 months after the diagnosis of anaplastic carcinoma.
Conclusion: Although most PTCs show an indolent course and have a favorable prognosis, distant metastasis and anaplastic transformation of the metastatic lymph nodes may occur even more than many years after the primary treatment.
Local "bystander"effect of gene therapy on human tumor cells of medullary thyroid carcinoma in vivo L. Feketeova * , M. Poturnajová, L. Kucerová, P. Babál * Comenius University, Inst. of Pathology, Bratislava, Slovakia
Objective: Gene therapy acts on change of prodrug into cytotoxic drug inside tumor cells transfected by foreign enzyme. It uses also "bystander" effect on surrounding tumor cells without enzyme. We used yeast Cytosine deaminase (yCD) in combination with 5-fluorocytosine (5FC) converted into 5-fluorouracil (5FU) and its metabolites. Medullary thyroid carcinoma (MTC) is a malignant tumor often with therapy resistant distant metastases. Patients with sporadic form have metastases at the time of diagnosis, mostly in bones, lungs and liver. The aim of this study was to evaluate the efficiency of yCD/5FC in MTC treatment using xenotransplants derived from model TT cell line in nude mice. Method: Tumors were immunohistochemically stained with polyclonal antibody anti-EGFP (enhanced green fluorescence protein) and monoclonal antibodies anti-PCNA and anti-Ki67. Positivity was semiquantitatively evaluated. Results: Diffuse positivity for PCNA was seen in untreated and treated tumors, respectively. Positivity for Ki-67 was diffuse in untreated and only sporadic in treated tumors. Conclusion: Diffuse PCNA positivity in treated tumors suggests that the tumor cells stopped in S phase. Scattered positivity of Ki-67 after treatment document suppressed proliferation mediated by yCD/5FC gene therapy, which implies therapeutic application in patients with metastatic MTC. (ITMS 26240220052, VEGA 2/008/11, VEGA 2/0146/10).
PS-05-015 E-cadherin/ß-catenin immunoexpression in thyroid carcinomas K. Ivanova * , E. Onal, J. Ananiev, T. Vlaikova, M. Gulubova * Trakia University, General and Clinical Pathology, Stara Zagora, Bulgaria
Objective: The aberrant activation of Wnt signaling pathway may be a common denominator for the development of thyroid tumorigenesis. It was announced that the loss of Ecadherin rather than β-catenin mutation represents a crucial event in determining the degree of differentiation of thyroid carcinomas. The aim of the study was to evaluate the expression of E-cadherin and β-catenin in the thyroid cancer tissue and to correlate these data with some histological and clinical parameters of the tumors. Method: We investigated 57 patients, having thyroid tumorspapillary, follicular, anaplastic and oncocytic carcinomas immunohistochemically with antibodies against E-cadherin and β-catenin. Survival analyses were done.
Results: E-cadherin expression was focally retained in the tumor cell membranes and in the tumor cell cytoplasm of the papillary, follicular and oncocytic thyroid cancers, weather in anaplastic cancers it was almost lost (p= 0.0042, and p= 0.019, respectively, Fisher's Exact Test). The expression of β-catenin in tumor cytoplasm and membrane in papilary cancers was higher as compared to that in the other tumors (p=0.111, and p=0.0104, respectively). Conclusion: Not surprisingly, the presence of aberrant expression of E-cadherin and/or β-catenin in thyroid cancer has been associated with better patients' prognosis and more well differentiated tumor histology.
Correlation between Estrogen receptor and progesterone receptor with some prognostic factors in papillary thyroid carcinoma M. Jalali Nadoushan * , R. Amirtouri, A. Davati * Shahed University, Dept. of Pathology, Tehran, Iran
Objective: The more prevalence of papillary thyroid cancer in women shows the probability of the role of sex hormones in the cancer. The aim of this study was determination of relation between sex hormones receptors and some prognostic factors. Method: We studied 92 patients with pathology report of papillary thyroid carcinoma after thyroidectomy between 2006 and 2009. The specimens were stained immunohistochemically for ER and PR. The other informations such as sex, age, tumor size and lymph nodes involvement obtained from the patients documents.
Results: The average age of patients was 39.32+16,93. 46.7 % t of samples were ER positive while this was 6.5 % for PR. The percentage of lymph nodes involvement was 23.9 %. The size of tumors was 3.60+2.21 cm. There was a direct relationship between female sex and positivity of ER (p≤0.014). But there was no significant relationship between ER and PR with age, tumor size and lymph nodes involvement.
Conclusion: It seems to be that ER is more prevalent in females but for showing of its role in prognosis, further studies are recommended. Objective: Type 2 diabetes mellitus (T2DM) treatment aims to control metabolic effects, preserve pancreatic function and reduce complications, such as nephropathy. This study intends to evaluate the effects of sitagliptin, a dipeptidyl-peptidase-4 inhibitor, in pancreatic and renal lesions in Zucker Diabetic Fatty (ZDF (fa/fa)) rats, an animal model of T2DM. Method: Male obese diabetic ZDF (fa/fa) rats, 20-weeksold, were treated with vehicle or sitagliptin (10 mg/kgBW/ day) for 6 weeks, and compared with lean control ZDF rats (n=8 each). Biochemical parameters as well as pancreatic and renal lipid peroxidation and histopathology profile were assessed. Specimens we stained with haematoxylin-eosin and periodic acid of Schiff, and examined by light microscopy. Lesions were evaluated by a semiquantitative rating. Endocrine/exocrine pancreas and renal glomerular, tubulointerstitial and vascular lesions were assessed and scored (0 3/0-2). Results are mean ± SEM; ANOVA and Duncan's post-hoc analysis (P≤0.05 was considered as significant). Results: Sitagliptin improved metabolic parameters, reduced lipid peroxidation (p<0.001) in both organs and significantly prevented major diabetic pancreatic and renal lesions in obese diabetic ZDF rats. Conclusion: Sitagliptin seems to comply with the three main objectives of T2DM therapeutic management. Underlying molecular mechanisms deserve further elucidation, but could be related with metabolic improvement and reduction of oxidative stress. Objective: Thyroid tumors of uncertain malignant potential (TT-UMP) include follicular and well-differentiated tumors of UMP (FT-UMP/WDT-UMP), as it refers to the presence of questionable capsular/vascular invasion or incompletely developed papillary thyroid carcinoma (PTC)-type nuclear changes. However, a diagnosis of TT-UMP is difficult in most cases. We aimed to investigate whether immunohistochemistry (HBME-1, Cytokeratin-19, Galectin-3, CD56 and p63) provides additional information concerning such lesions. Method: We performed an immunohistochemical analysis on 14 WDTs-UMP and 4 FTs-UMP.
Results: In the WDT-UMP group, HBME-1 was positive in 6/14 (42.9 %) cases. CD56, a marker whose expression is reduced in thyroid carcinoma, showed a "malignant" profile (no expression) in 9/14 (64.3 %) cases. 6/14 (42.9 %) cases were positive for both antibodies. One case showed the coexpression of HBME-1, CD56, Galectin-3 and Cytokeratin-19. Only one FT-UMP case was positive for HBME-1. The follow-up data revealed no distant metastases or persistent disease.
Conclusion: TT-UMP demonstrated very heterogeneous immunohistochemical profiles. WDTs-UMP revealed a certain tendency toward a PTC profile, suggesting a possible pathogenetic link between these two entities. However, immunohistochemistry is to be regarded more as a supporting factor, while morphological criteria should always prime in the diagnostic decision. Results: There was a positive correlation between TPCFV and HMWCK, CK 19, HBME1, Galectin 3, Fibronectin (p< 0.05), but there was no correlation with TPCFV and RET/ PTC (p>0.05). HBME-1 and CK 19 stained strong and diffuse positive in TPCFVs but weak and focal in FAs.
Our study suggests that morphologic features combined with immunohistochemical panel of HMWCK, CK19, HBME-1, Galectin-3 and fibronectin can help to distinguish benign and malign thyroid neoplasms and TPCFV from follicular adenomas. RET/PTC expression has been nonspecific but its detection can be a useful tool combined with immunohistochemistry for diagnosing TPCFV.
Morphological features of the system mother-placentafetus during pregnancy on diabetes mellitus T. Pavlova * , V. Petrukhin, E. Malutina, A. Nesterov, L. Pavlova, I. Goncharov, D. Kolesnicov * Belgorod State University, Dept. of Pathology, Russia
Objective: Combination of diabetes mellitus and pregnancy has a special concern. Method: It were studied 50 pregnants with diabetes mellitus type 1 and 29 pregnant with gestational diabetes mellitus (GDM). The methods were used: light, transmission, electron, probe and scanning microscopy with microelement analysis.
Results: The pregnancy at diabetes mellitus type 1 and GDM proceeds with complications: treat of pregnancy termination (64,0 % and 50,0 %), hydramnion (27,0 and 62,5 %), preterm birth (36,0 and 12,5 %). It was displayed decreasing of oxygen in mother's erythrocytes. At the diabetes mellitus in uterus of women in birth there are violations of spiral arteries as well as circulatory disorders (stasis, sludge, thrombosis and diapedetic bleedings), that is significantly expressed at gestosis. Tendency to cell form changing is observed in erythrocytes at diabetes mellitus, especially at diabetes mellitus type 1. The frequency of placental insufficiency in pregnant with diabetes mellitus type 1 were 75 %, at GDM-50 %. The gestosis accession were resulted to birth's increasing of children with prenatal injury of central nervous system at diabetes mellitus type 1 in mother and diabetic fetopathy at GDM.
The clinical and morphological parallels of the system mother-placenta-fetus were presiced ad diabetes mellitus type 1 and GDM.
Sclerosing mucoepidermoid carcinoma of the thyroid in a 13 year-old female A. Polónia * , L. Santos, C. Eloy, R. Celestino, P. Soares, M. Jácome, C. Lobo * IPO-Porto, Pathology, Rio Tinto, Portugal
Objective: Sclerosing mucoepidermoid carcinomas (SMECs) are low-grade malignant tumours with both squamous and mucinous differentiation representing less than 1 % of thyroid malignancies. SMECs are usually associated to Hashimoto thyroiditis and classically disclose eosinophilia.
Results: A 13 year-old female with Hashimoto thyroiditis presented a 2.5 cm nodule in the left lobe of the thyroid. The total thyroidectomy specimen disclosed a firm, well circumscribed, non-encapsulated tumour. Microscopically, the tumour was composed by anastomosing clusters of squamoid cells in a sclerotic background without eosinophils. Intra-cytoplasmatic PAS/diastase positive material was focally found. The tumour cells expressed AE1.AE3, TTF-1 and p63 whereas no expression was observed for thyroglobulin, calcitonin and CD5. The Ki-67 labeling index was 5 %. In the molecular analysis no mutations were detected in BRAF or (K-, N-, H-) RAS genes nor RET/PTC or PAX8/PPARgamma rearrangements. The remaining thyroid showed Hashimoto-type thyroiditis. A cervical lymph node metastasis was identified. The patient was treated with radioactive iodine and is alive without signs of disease after 10 months of follow-up. Conclusion: SMECs may not disclose the classical eosinophilia and represent rare low-grade tumours that can give rise to metastases. The most frequent molecular alterations found in thyroid tumours were not detected in this case.
PS-05-024 ALK1 and BMP-9 overexpression as a cause of ossifying papillary thyroid carcinoma
Objective: Ossification is an often encountered finding in papillary thyroid carcinoma (PTC). We hypothesized that osteogenic signaling may be related to osteogenesis of PTC. Bone morphogenic protein (BMP)-9 is the most osteogenic subtypes among BMPs. And as a cellular receptor, activin receptor-like receptor (ALK)1 has been emphasized in BMP-9 induced osteogenic signaling. We investigated the expression of ALK1 and BMP-9 and their correlation with ossification in PTC.
Method: ALK1 and BMP-9 expression were investigated by immunohistochemistry in tumors and adjacent normal follicles of 78 PTCs with bone formation and 64 PTCs without bone formation. ALK1 and BMP-9 expression were further verified by quantitative real time polymerase chain reaction in each group of 15 cases of PTCs with or without bone formation.
Results: ALK1 and BMP-9 immunoreactivity were increased in PTC with bone formation when compared to those without bone formation (P<0.001 and P=0.001).
Both mean values of ALK1 and BMP-9 mRNA expression were elevated in PTCs with bone formation compared with those without bone formation (P=0.037 and P<0.001).
Conclusion: ALK1 and BMP-9 overexpression may be underlying the molecular alteration that accounts for osteogenesis in PTC. Objective: Thyroid is an extremely rare site of metastases. Renal clear cell carcinoma (RCCC) is one of the most frequent primary malignancies causing thyroid metastases as a single nodule (77 %) or multiple nodules (23 %). We report the case of a 62-years-old female patient affected by multinodular goiter, who was diagnosed with RCCC, treated with nephrectomy. Method: Due to compressive symptoms, 6 years after nephrectomy, the patient underwent thyroidectomy and the thyroid was sent for histology.
Results: Histology showed multinodular goiter. In the largest hyperplastic adenomatous nodule (left lobe, diameter mm 53), multiple solide yellow-orange areas were detected grossly. These areas were microscopically composed of sheets of large cells with clear cytoplasm and small hyperchromic nuclei, with a focally infiltrative growth pattern. At immunohistochemistry clear cells were negative for TTF1, HBME1, galectin-3 and positive for CD10.
Conclusion: This is a rare case of multiple RCCC metastases within an adenomatous nodule in goiter. Clinical diagnosis of RCCC metastases to thyroid is extremely difficult, even more if metastases grow in multinodular goiter. It should be always suspected in patients with a clinical history of RCCC. Objective: Follicular carcinoma, oncocytic variant, is a rare type of thyroid carcinoma. We examined a case of oncocytic carcinoma misdiagnosed as medullary carcinoma on fineneedle aspiration (FNA) because it was associated with amyloid material. Method: A 73-year-old female rheumatoid arthritis (RA) patient had a 4.0 cm mass in the right lobe of her thyroid, which showed no enhancement effect on CT scan and was diagnosed as a "cyst". FNA was performed, and she subsequently died of unrelated causes. An autopsy was performed.
Results: Cytology: Large and small round-shaped tumor cells were present with round nuclei with granular chromatin. Double-or triple-nucleated cells and amyloid material were observed. Histology: oncocytic round cells proliferated diffusely in the fibrous capsule. Extra-capsular invasion and vascular infiltration by the tumor cells were recognized. Electron microscopy: the cytoplasm of tumor cells was full of mitochondria. Therefore, we diagnosed this as follicular carcinoma, oncocytic variant. Amyloid deposition was also observed in several other organs. Conclusion: Amyloid deposition was caused by amyloidosis secondary to RA. Narrowing of the tumor feeder artery due to amyloidosis may have prevented early enhancement on the CT image. Objective: Whipple's disease is an infectious disease caused by Tropheryma whipplei, an ubiquitous Gram-positive Actinobacteria. The incidence of the disease is less than 1 per 1 milion. We report five new consecutive cases, four males and one female, diagnosed in our clinic from August 2002 to January 2012. Method: Diagnosis was reached with the help of gastroduodenal endoscopy and histopathological examination of the duodenal biopsies, by lymph node biopsy and by electron microscopy.
Results: The main symptoms were arthralgia, weight loss and diarrhea. The endoscopic aspect of the small bowel mucosa varied from congestion, granularity of the mucosa to whitish plaques. All patients showed PAS positive, diastase resistant, Ziehl-Neelsen negative macrophages in the lamina propria of the duodenal mucosa and, in one patient, suspected for lymphoma, in an abdominal lymph node. The diagnosis was confirmed by electron microscopy in all cases. Classic Whipple's disease was the diagnosis in all five cases, but one patient showed involvement of the endocardium and two patients showed lymphadenopathies. Conclusion: Clinical evolution was favorable under long-term antibiotics (Cephtriaxone/Trimethoprim-Sulfamethoxazole) and follow-up biopsies in three patients showed a normal endoscopical mucosa and a reduced but persistent number of PAS positive macrophages in the duodenal mucosa. Results: Eleven cases of NEN of gastrointestinal tract were identified. They were 6 men and 5 women with a mean age of 53.09 years. The most common localization was the appendix (7 cases). The 4 other cases were localized in the small intestine, the ileo caecal valve, the fundus and the bulb. The mean tumor's size was 13.8 mm. Mitoses were absent in the majority of cases (72,7 %). All the tumors were well differentiated classified as grade 1 in 9 cases and grade 2 in 2 cases. The tumor was aggressive in 2 cases. The small intestine's NET was multifocal infiltrating the subserosa with lymph node metastasis classified pT3 N1. The ileo caecal valve's NET was also aggressive with lymph node and hepatic metastasis classified pT4N1M1. All patients underwent surgical treatment.
Conclusion: Gastrointestinal NENs are complex tumors whose incidence is rising and whose treatment requires precise classification and risk stratification.
Multiple duodenal stromal tumors associated with Neurofibromatosis-1 G. Benkhedda * , Y. Lamouti * CHU Frantz Fanon, Dept. of Pathology, Blida, Algeria
Objective: Gastrointestinal stromal tumors (GIST), most commonly occur sporadically, but there seens to be some increased tendency for these tumors to develop in patients with neurofi-bromatosis1(NF1). There is no histological difference between the NF1 assocaciated cases and the sporadic cases. However, tumors associated with NF1 frequently show multiplicity. Method: A case of multiple duodenal gastrointestinal tumor arising in a 45 year old mal with NF1 is reported. The abdominal exploration revealed multiple solid nodules in the duodenum, and the pancretecticoduodenectomy was performed.
Results: Macroscopy: the resected segment of the duodenal showed seven suberosal solid masses. The largest mass measured 3,5 cm×2 cm×3 cm, and is coupled with the bors lower pancreas but remains limited by a capsule. The cut surface was smooth and white in appearance. Microscopy: the tumors were composed of interlacing fascicles of the uniform spindle cells with elongated cytoplasm. The tumor cells lacked pleomorphism, and mitotic figures were absent. Immunohistochechemistry: the tumors cells were diffusely positive for CD117, CD34, and negative for desmin, AML and pS100. Conclusion: GIST are rarely noted in association with neurofibromatosis-1. Duodenal GISt are most frequently diagnosed in the workup of symptoms not specific to these masses. Duodenal resection is rarely indicated except in the case of duodenal GIST and early-stage adenocarcinoma or if the tumor appeared involve the pancreatic parenchyma on preoperative imagings.
Periampullary adenomyoma: A true trap diagnosis G. Benkhedda * , Y. Lamouti * CHU Frantz Fanon, Dept. of Pathology, Blida, Algeria
Objective: Adenomyoma is a term generally applied to nodular lesions showing proliferation of both epithelial and smooth muscle components. It is usually presented as biliary obstruction. Most cases are misdiagnosed as adenoma or carcinoma by preoperative endoscopic or radiologic. Therefore, it is frequently treated with extensive surgery. Method: We report a case of a 28 year-old man with an adenomyoma located in the ampulla of vater diagnosed by endoscopic piecemeal resection. Results: On histologic examination, the lesion consisted of hyperplastic glandular lobules, mainly located in the muscle layers of the vaterian system. The lobular formations consist of small glands surrounded by myofifroblastic, fibroblastic proliferation, sparse capillaries and inflammatory cells. IHC: Ki67: rare cells with a positive nuclear staining were presenting in the epithelial and mesenchymal components AML: The myofibroblastic of most spindle cells was confirmed by a strong cytoplasmic expression. Conclusion: Real incidence of adenomyoma of the vaterian system is difficult to appreciate as different names (adenomyoma, adenomyomatosis, myoepithelial hamartome) are used to designate the same histological lesion. Adenomyoma was diagnosed only in adult patients -mean age: 63 y). The histogenesis is still a subject of controversy. The most widely accepted hypothesis is that the lesion may represent a form of incomplete heterotopic pancreas.
Adenomyoma is considered as benign and slow growing, but its potential neoplastic nature cannot be excluded. However, the histogenesis of these tumors is subject to further study. Carcinosarcoma of the digestive organs has been reported to exhibit aggressive behavior. Carcinosarcomas in digestive organs have been reportedly associated with a poor prognosis.12,13 However, some cases that have been treated with a curative operation showed long-term survival.
Analysis of HER2 expression level in gastric carcinomas M. Bialas * , A. Sinczak-Kuta, A. Lazar, K. Urbanczyk, K. Galazka, J. Szpor, S. Demczuk, D. Adamek * Jagiellonian University, Dept. of Pathomorphology, Krakow, Poland
Objective: The expression of HER2an oncoprotein belonging to tyrosine kinase family belongs to recently evaluated prognostic and predictive factors in gastric cancer.
Overexpression of HER2 is observed in about 8-27 % gastric cancers. According to many authors, overexpression and/or amplification of HER2 correlates with poor prognosis. The aim of our study was to analyze HER2 expression in gastric cancer in material routinely examined in Pathomorphology Department, JUMC. Method: We have analyzed 50 cases of gastric cancers. The material came from 19 females and 31 males, age: 21-86. Immunohistochemical reactions were performed automatically on BenchMark BMK Classic (Ventana) using PATH-WAY HER-2/neu (4B5) antibody. Scoring system for HER2 expression was ranged from 0 to 3+ where: 0 and 1+ were regarded as a negative, 2+ as equivocal and 3+ as positive.
Results: Most cases of gastric cancers were HER2-negative (45/50): including 0 score (38 patients) and 1+ (7 patients). Three cases were equivocal and two cases showed 3+ expression level.
The lower level of HER2 expression in the analyzed material in comparison with literature could be related to a relatively small group of cases but one cannot exclude that there exist some other factors that stand behind this and surely the investigations should be continued.
PS-06-012 MAPKAP kinase 2 overexpression influences prognosis in gastrointestinal stroma tumors, associates with copy number variations on chromosome 1, and expression of p38 MAP kinase and ETV1 P. Birner * * Medizinische Universität Wien, Institut für Klinische Pathologie, Austria
Objective: ETV1 has been proposed to be activated by KITmutations in gastrointestinal stromal tumors (GISTs). Aim of the study was to evaluate the role of ETV1 and associated proteins in GIST. Method: Expressions of ETV1, MAPKAP kinase 2 (MAP-KAPK2), phosphorylated p38 MAP kinase (pp38), phosphorylated MSK1, phosphorylated RSK1, COP1 and KIT were determined immunohistochemically in 139 GISTs. Sequence analysis of KIT, PDGFRA and MAPKAPK2 and FISH of ETV1 and chromosome 1 was performed.
Results: Prominent ETV1 expression was seen in 50 % of GISTs, but no correlation with clinical outcome was found. Correlation of ETV1 expression and KIT mutation was seen in 60 % of cases. MAPKAPK2 overexpression (n =62/ 44.6 %) correlated with pp38 expression (p=0.021) and alterations of chromosome 1 (p=0.024). In one cases with high MAKAPK2 expression, a MAPKAPK2 gene mutation was found. All relapsing GISTs with very low/low risk showed high MAPKAPK2 and KIT expression. MAP-KAPK2 overexpression was an independent prognostic factor for disease free survival (p=0.006). Conclusion: ETV1 is not universally overexpressed in GIST and seems to be induced also by other pathways than KIT-mutation. MAPKAPK2-overexpression is associated with shorter survival in GIST. Patients with low risk GISTs overexpressing MAPKAPK2 might profit from adjuvant tyrosine kinase inhibitor therapy.
Gastrointestinal stromal tumors: Comparison of two risk stratification systems in a multicenter study of 1963 Turkish cases G. Bulbul Dogusoy * * Gayrettepe Florence Nightingale, Dept. of Pathology, Istanbul, Turkey
Objective: A nationwide database was performed for gastrointestinal stromal tumors (GISTs) in a large series of primary GISTs surgically treated at centers all around Turkey. The aim of this multicenter study was to analyze and compare the performance of the National Institute of Health (NIH) and Armed Forces Institute of Pathology (AFIP) risk criteria to determine the ideal risk stratification system. Method: Statistical analysis of a nationwide database is consisted of age, gender, location, risk groups, histopathologic features and the results of CD117, CD34, Desmin, SM Actin, S-100 protein, and Ki67 immunohistochemistry. Results: In 1965 cases registered in database, male to female ratio was 1.20 and mean age was 57.64 years. Most common location was stomach (47.5 %) followed by small intestine, omentum-peritoneum, large intestine, and esophagus (30.3 %, 12.1 %, 9.1 %, 1.0 % respectively). Comparison of the two risk-stratification systems demonstrated that proposed modified AFIP seems to be better when compared with NIH system. Many histopathologic and immunohistochemical findings showed significant correlation with risk groups of AFIP, even with 'not sufficient data' group (p<0.005).
The results of this multicenter study demonstrates that although follow up results are not provided, AFIP risk criteria seems to be more useful in prognostication for GISTs among the two systems. Objective: HER 2/neu overexpression or amplification is an important biomarker for identifying patients with intestinal-type gastric cancer who respond to therapy with Trastuzumab. Moreover, intestinal type gastric cancer shares many phenotypic and molecular genetic changes with colorectal cancer. In particular a progression from chronic gastritis to intestinal metaplasia, dysplasia, and finally malignant transformation is probably the sequence of gastric carcinogenesis. Somatic mutation of K-ras gene is common in colorectal cancer, being found in more than one-third of cases, but it seems to have, mostly in intestinal-type gastric cancer, a low incidence (7-20 %). The purpose of this study is to assess HER2 gene amplification and K-ras mutational status in a series of intestinal-type gastric carcinoma patients. Method: Twenty paraffin embedded gastric cancer specimens were tested for HER2 amplification by chromogenic in situ hybridization (CISH) and K-ras mutational status (codon 12 e 13) by PCR-RFLP.
Results: Six (30 %) cases were HER2 amplified. Only one case (5 %) was found to have K-ras mutation (codon 12), but it was HER2 not amplified.
Conclusion: The frequency of K-ras mutation and HER2 amplification are in agreement with other studies on this topic. In our study, the two seem mutually exclusive events. Objective: Gastric carcinoma is related with cancer genetic susceptibility that can be investigated as single nucleotide polymorphisms (SNPs) and as cytokine genes are known to predispose to malignant disease, several polymorphisms of Interleukin-6 (IL-6) gene have been reported to in some may be associated with tumour progression including inhibition of malignant epithelial cells apoptosis and stimulation of angiogenesis.
Method: The aim of this study was to clarify the association between IL-6 polymorphisms and the risk of gastric cancer and chronic gastritis development or maintenance. PCR-SSP genotyping for IL-6 -174 C>G polymorphism was performed in 100 biopsies of gastric carcinoma and in 100 biopsies of chronic gastritis.
Results: There was association between IL-6 -174 C allele (p = 0,0466) and -174CC, low producer, genotype (p = 0,0466) and gastric carcinoma, whereas IL-6 G allele (p= 0,0278) and IL-6GG (p<0,0001), high producer, genotype was associated with gastritis.
Conclusion: We conclude that IL-6 -174, low producer genotypes, may have an important role in gastric carcinogenesis and the polymorphism study of this molecule could be a good marker for gastric carcinoma susceptibility when high grade dysplasia is seen in biopsies. Objective: In Helicobacter pylori gastritis, constant antigenic stimulation triggers a sustained B-cell proliferation. Errors made during this continuous DNA replication are corrected by the DNA mismatch repair mechanism. Failure of this mechanism has been described in HNPCC and results in a replication error phenotype. Inherent to their instability during replication, microsatellites are the best markers of this replication error phenotype. We aimed to evaluate the role of defects in the DNA mismatch repair mechanism and microsatellite instability (MSI) in gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Method: We examined 10 microsatellite loci (BAT25, BAT26, D5S346, D17S250, D2S123, TGFB, BAT40, D18S58, D17S787 and D18S69) for instability in 28 patients with MALT lymphomas. In addition, these tumors were also immunostained for MLH1, MSH2 and MSH6, as well as screened for the presence of t(11;198)(q21;q21) by real time polymerase chain reaction (RT-PCR). Results: We found MSI in 5/28 (18 %) lymphomas, with one tumor displaying high levels of instability. MSI occurred in both t(11;18)(q21;q21)-positive and -negative tumors. Conclusion: Our data suggest that a MMR-defect may be involved in the development of gastric MALT lymphomas, and mutations in the MSH6 MMR gene or hypermethylation of the MSH6 promoter might be associated with MSIdriven gastric lymphomas.
Mitosis-specific marker phospho-histone H3 in the diagnosis of gastrointestinal stromal tumors A. Dolzhikov * , A. Tverskoi, K. Hizhnyakov * Regional Pathology Hospital, Dept. of Oncomorphology, Belgorod, Russia
Objective: The assessment of proliferative activity is one of the major parameters in the proper grading of gastrointestinal stromal tumors (GISTs). In the low level of mitotic activity it is difficult to calculate dividing cells correctly. Phospho-histone H3 (PHH3) is a recently introduced immunomarker for mitotic cells.
Method: Immunohistochemical study of 46 cases of GISTs of different malignant potential, statistical analysis. Results: The count of PHH3-immunoreactive cells demonstrates the strong correlation (Gamma 0,756; p<0,05) with malignant potential of GISTs detected by standard parameters. It clearly separates true mitotic cells from apoptotic and piknotic nuclei. In all cases PHH3 count was slightly higher than the mitotic index in H&E stained slides. We have found that PPH3 demonstrated the two types of staining: nucleosomal dot-like type and mitotic homogenous type. The first type, probably, reflects the fraction of cells just before the prophase, which cannot be identified in H&E slides. Conclusion: Immunostaining of PPH3 is a useful additional marker in detection of proliferative activity in GISTs, helping to identify true dividing cells correctly.
PS-06-019 HER2 assessment in gastric carcinoma using IHC and FISH P. Drev * , B. Gazic, J. Contreras * Institute of Oncology, Dept. of Pathology, Ljubljana, Slovenia
Objective: HER2 in gastric cancer (GC) should be assessed following guidelines -recommended testing algorithm employs IHC as a screening tool and is followed by ISH to clarify equivocal cases. Reported incidences of HER2+ GC vary substantially and discordant results are frequent. In a series of GC HER2 was assessed by IHC and FISH to determine incidence of HER2+ GC, concordance of the methods and reevaluate the recommended algorithm.
Method: HER2 was assessed by both IHC (Pathway 4B5™) and FISH (PathVysion™) in 164 GC samples. Reactions were evaluated according to guidelines. HER2 was considered positive in case of strong protein expression (3+) and/or gene amplification (ratio ≥2.0). Frequencies of IHC and FISH scores, incidence of HER2+ GC and concordance (chi-square) of applied methods were analysed. Results: IHC score distribution: neg (0) Objective: Gastric glomus tumors are rare neoplasms originated from modified smooth muscle cells of the glomus bodies. Such lesions present a diagnostic challenge in biopsy material. Herein, we report a case of gastric glomus tumor in a 35-year-old woman. Method: A 35-year-old woman presented with refractory epigastralgy. A gastrointestinal stromal tumor of the gastric antrum was suspected. The diagnosis was made after ultrasound-guided endoscopic biopsy, followed by an endoscopic submucosal resection. Results: The biopsy showed tight convolutes of capillarysized vessels surrounded by collars of small, rounded cells set in a hyalinized, myxoid stroma. Immunohistochemically, these cells were positive for smooth muscle actin and type IV collagen (pericellular pattern), synaptophysin (focal positivity) and negative for CD34, CD117, desmin, S100 and cytokeratins. The diagnosis of gastric glomus tumor was rendered. The resection specimen revealed a submucosal well-circumscribed 2×1,5×1,5 cm nodule with identical histological features. The patient was disease-free 12 months postoperatively.
Conclusion: Visceral glomus tumors are rare neoplasms. When they arise in the gastrointestinal tract, the stomach (antrum) is the most frequent location. The differential diagnosis includes NET, epithelioid GIST and hemangiopericytomas. They usually behave in a benign fashion, although malignant cases have been reported. Surgical excision is the standard treatment.
Immunohistochemical evaluation of replication Protein-A1 in gastric cancer: Clinicopathological associations H. Gakiopoulou * , E. Fourtziala, G. Levidou, A. Stofas, N. Alexakis, P. Korkolopoulou, E. Patsouris * University of Athens, 1st Dept. of Pathology, Greece
Objective: Replication protein A1 (RPA1) is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. In this study, we investigated for the first time the immunohistochemical expression of RPA1 protein in a series of 73 gastric carcinomas in relation with clinicopathological parameters (age, sex, Lauren's histologic classification, histologic grade, lymphovascular invasion, tumor size, depth of invasion (T), lymph node metastasis (N) and stage). Method: A standard immunohistochemical method and a semi-quantitative evaluation for the detection of RPA1 Labeling Index (LI) were applied. Results: Nuclear RPA1 immunoreactivity was seen in all carcinomas with a mean value of 26.5 %. Statistical significant correlations emerged between: 1. RPA1 LI and low Tcategory (p=0.009) 2. RPA1 LI and absence of lymph node metastasis (p=0,014). RPA1 LI was higher in cases without lymphovascular invasion; however this association did not reach statistical significance. Conclusion: The widespread expression of RPA1 in gastric carcinomas suggests that this protein is implicated in gastric cancer growth. The observed significant associations between RPA1 LI and low T as well as N0 tumors could imply that RPA1 might offer a growth advantage in the early stages of gastric cancer progression.
Pigmented histiocytic "Pseudotumoral" reaction due to endoscopic tattooing of the duodenum with India Ink R. Hadhri * , C. Objective: India ink has been used for endoscopic tattooing to facilitate localization of a luminal abnormality at the time of surgery or repeat endoscopic examination. Recognition of this phenomenon is important to prevent misinterpretation of this _nding as other cause of black tissue deposits. Some of them can be easily excluded by microscopic examination; the others, however, necessitate special stains or, as illustrated by our observation, an adequate clinical information! Method: A 76-year-old man was referred for enteroscopy for microcytic anemia. The procedure revealed a 2 cm flat umbilicated lesion of the duodenum. Endoscopic tattooing was performed to guide surgical excision.
Results: Histologic examination of the surgical specimen revealed a tubulovillous adenoma with low-grade dysplasia. The submucosa showed numerous aggregates of large cells containing a heavy black pigmentation. Special histologic stains were inconsistent with melanin or iron. This submucosal cellular infiltration was linked to the preoperative use of India ink when the notion of tattooing was "kindly" provided by the surgeon! Conclusion: Despite a striking and misleading appearance, the microscopic finding of such phenomenon does not represent a pathologic state. Nevertheless, communication between enterologists, surgeons and pathologists are mandatory to assure prompt recognition and avoid unnecessary investigations. Objective: Gastritis cystica polyposa is a unique lesion found on occasion at the stoma of a gastrojejunal anastomosis. However, GCP is rarely found in an unoperated stomach. Method: A 51-year-old woman with abdominal discomfort and vomiting. Physical examination, routine hematological examination and biochemical tests were within normal limits, exept mild anemia.
Results: Upper GI endoscopic examination revealed a pedanculated polyp, in the greater curvature. Endoscopic ultrasonography revealed a polypoid heteroechoic mass with cystic area and 20×22 mm diameter in posterior wall of gastric body. Polypectomy was performed without any complications. Histological examination of the protruding lesion revealed some misplaced cystic glands were entrapped in dense disorganized bundles of smooth muscle of muscularis mucosa surrounded by a rim of lamina propria. These findings were consistent with gastritis cystic polyposa. Objective: Human papillomavirus (HPV) infection is a known risk factor for the development of squamous cell carcinoma (SCC) of the cervix, the oropharynx and the anogenital region. The aim of our investigation was to assess the prevalence of HPV DNA in patients with esophagus cancer in the Western population. Method: Formalin-fixed paraffin-embedded blocks from 37 consecutive patients who underwent esophageal endoscopic mucosal resection or esophagectomy for SCC were tested for the presence of HPV DNA by polymerase chain reaction using consensus primers GP5+/GP6+. Viral genotyping was determined by type-specific primers.
Results: Among the total of 37 cases, no HPV DNA was detected in 33 cases (89.2 %). 4 cases were tested HPV positive (10.8 %) , of which 1 was HPV 16+, 1 was HPV 18+ and 2 were non-high-risk HPV type. The non-high-risk HPVs were detected in patients with previously treated SCC of the mouth (n=1) and the oropharynx (n=1). Patients with high-risk HPVpositive tumors had no history of oropharyngeal cancer. Conclusion: Our study revealed the presence of oncogenic HPV genotypes in a subset of esophageal cancer. The low rates of viral infection detected suggest that HPV unlikely represents a significant etiologic factor in esophageal carcinogenesis. Further studies are needed to confirm these data in larger populations. Objective: Gastric cancer is the second leading cause of cancer mortality in the world. Amplification of HER-2/neu oncogene has become an important biomarker for identifying patients who will respond to HER-2 targeting therapy. The rate of HER2 positivity in gastric cancer is variable, ranging from 6 % to 35 %. Objective: Gastrointestinal stromal tumors (GISTs) are primary mesenchymal tumors that arise in the GI tract. Only 3-5 % of GISTs occur in the duodenum. Here, we report a rare case of duodenal GIST with extramural growth that mimicked a pancreatic tumor. Method: A 57 years old male patient was admitted to our hospital for duodenal ulcer and gastrointestinal bleeding which was diagnosed in an another clinic by endoscopy.
Abdominal MR of the patient showed a 5×5,5 cm necrotic mass in the uncinate process of the pancreas which was excised by whipple operation.
Results: Macroscopically, the resected specimen consisted of solid mass that was connected to the patient's duodenal wall but not to the parenchyma of the pancreas. Microscopic examination of the tumor showed spindle shaped and epithelioid cells with mild nuclear pleiomorphism. Immunohistochemistry revealed that the cells are strongly positive with CD117, with focal expression of CD34 and SMA. Conclusion: GISTs are low-grade malignant mesenchymal tumors of the GI tract and are believed to originate from the neoplastic transformation of the Cajal cells, which are located between the longitudinal and circular layers of the muscularis propria. They most frequently arise from the second part of the duodenum where they push or infiltrate into the pancreas. In our case report, the patient's tumor exhibited extramural growth and mimicked a pancreatic tumor. Mantle cell lymphoma (MCL) is relatively a rare subgroup of non-Hodgkin lymphoma. We have experienced an uncommon case of MCL. A 65-year-old man was admitted to emergency service for upper gastrointestinal bleeding as melena. Gastric corpus wall thickness found increased and splenomegaly determined at whole abdominal ultrasonography. Endoscopic examination revealed subsequently, 1 cm diameter polypoid lesion at corpus anterior wall and 2 cm dimensioned elevated and vascularised lesion at corpus posterior wall. Endoscopic biopsy had reported as active gastritis and mucosal reactive hyperplastic changes with ulcerative background. After than total gastrectomy and splenectomy specimens examined diffuse infiltration of monotonous medium-sized, atypical lymphoid cells with hyperchromatic nuclei. These cells were positive for CD20, CD5, CD43, bcl-2 and CD79a, but negative for CD10, CD23 and bcl-6. Atypical lymphoid cells were present even in splenic hilus and lesser curvature lymph nodes. We reported this case as Primary Gastric Mantle Cell Lyphoma according to morphologic and immunohistochemical staining features. We report a 74-year-old man who had been searched for bicytopenia (anemia and thrombocytopenia). Bone marrow examination revealed extensive bone marrow metastasis of signet ring cell carcinoma. PAS, d-PAS and mucin histochemical markers performed to bone marrow sample which were all positive. Immunohistochemical markers were positive with MUC-1, MUC-5 AC, MUC-2 (focal staining), CK7, CK20, EMA, CEA and negative with vimentin, CD10, chromogranin. According to morphologic and immunohistochemical results we thought primarily gastrointestinal system malignancy, particularly stomach carcinoma. Upper gastrointestinal endoscopy performed subsequently, hemorrhagic ulcerative lesion had seen and taken biopsies. Endoscopic biopsy results showed metastatic signet ring cells origin from stomach. We report this case because bone marrow metastasis can be found commonly in some malignant tumors but diagnosing a nonhematologic malignancy from bone marrow is an unusual event. 
The pathological effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats E. Abdollahi * , R. Ahmadi * Tehran University, Iran
Objective: Studies show that there is association between salvia officinalis extract administration and liver or heart functions. The main aim of this study was to determine the effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats. Method: Male wistar rats were randomly divided into control, normal saline receiving and salvia officinalis extract (100, 150 or 200 mg/kg/day) receiving animals of 5 in each group. After a period of 6 weeks, blood samples were collected using cardiac puncture method. Following serum collection, serum alkaline phosphatase levels were measured by spectrophotometry method. Data were statistically analyzed and compared between groups using ANOVA.
Results: The results indicated that Serum alkaline phosphatase levels were significantly increased in salvia officinalis extract (100, 150 or 200 mg/kg/day) receiving animals compared with control rats. Conclusion: Our findings show that salvia officinalis extract is enhancer of serum alkaline phosphatase according to which, impairing effect of the extract on certain tissues is conceivable.
The pathological effects of waterpipe smoking on serum levels of CEA, alkaline phosphatase or creatine kinase in male and female rats R. Ahmadi * , M. Mafi * Islamic Azad University, Dept. of Physiology, Hamedan, Iran
Objective: Various studies show that smoking can influence serum levels of tumor markers such as carcino embryonic antigen (CEA) and kinase or phosphatase enzymes. The main aim of this study was to determine the pathological effects of waterpipe smoking on serum levels of CEA, alkaline phosphatase (ALP) or creatine kinase (CK) in male and female rats. Method: Male and female Wistar rats were randomly divided into control and waterpipe smoking groups of 5 in each. After a period of 10 weeks, blood samples were collected using cardiac puncture method. Following serum collection, serum CEA, alkaline phosphatase or creatine kinase levels were measured. Intestinal tissue was also examined histologically. Data were statistically analyzed and compared between groups using ANOVA.
Results: The results indicated that serum CEA, ALP or CK levels were significantly increased in male and female waterpipe smoking animals (P<0.01, P<0.01 or P<0.001, respectively). There was not gender effect on serum CEA, ALP or CK levels. There were also histological changes in intestinal tissue including increased tissue plasma cells infiltration and inflammation. Conclusion: Our findings show that waterpipe smoking is an enhancer factor of serum CEA, alkaline phosphatase or creatine kinase levels, according to which, damaging effects of waterpipe smoking on various tissues, particularly intestine should be considered seriously.
The effects of acute or chronic immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats M. Alinavaz * , R. Ahmadi, M. Mafi * Islamic Azad University, Nutrition, Tehran, Iran
Objective: Studies show that immobilization stress has a variety of effects on serum levels of liver enzymes. The main aim of this study was to determine the effects of immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats. Method: 50 male Wistar rats weighing 200±30 g were randomly divided into control, acutely or chronically immobilized animals of 5 in each group. Animals were immobilized for 2 h/day or 8 h/day for a period of 3 weeks or 1 week in chronically or acutely immobilized groups, respectively. Blood samples were collected using cardiac puncture method. Following serum collection, creatine kinase or alkaline phosphatase level was measured by spectrophotometery method. Data were statistically analyzed and compared between groups using ANOVA. Results: The results indicated that serum creatine kinase level was significantly increased in rats enduring acute or chronic immobilization compared with control animals (P< 0.001), however, there was not significant difference between serum alkaline phosphatase levels in immobilized animals compared with control rats. Conclusion: Immobilization stress may leave pathological effects in liver or other organs leading to enhanced serum creatine kinase level.
Immunoexpression of lactoferrin in bone metastases and corresponding primary carcinomas V. Barresi * , A. Ieni, G. Giuffrè, G. Branca, G. Tuccari * University of Messina, Dept. of Human Pathology, Italy
Objective: With reference to primary bone tumors, we previously found lactoferrin (Lf) immunoreactivity in chondroblastomas, chondromyxoid fibromas, giant cell tumours and osteoid osteomas, while no immunoexpression for this protein was detected in chondrosarcomas and osteosarcomas. Herein we aimed to analyze Lf distribution in bone metastases from cancers of different sites. Method: Lf immunohistochemical expression was investigated in 25 formalin-fixed and paraffin-embedded specimens of human bone metastatic lesions as well as in the primitive corresponding carcinomas. The primitive sites of carcinomas were: breast (8), prostate (4), kidney (4), lung (3), colon-rectum (2), uterus (4). A Lf intensity-distribution (ID) score was calculated for each case by multiplying the values of the area staining positivity and the intensity staining.
Results: Lf immunostaining, with variable ID scores, was encountered in 11/25 (44 %) metastatic bone lesions. Immunoreactivity for Lf was found in primary carcinomas with a percentage of neoplastic cells ranging between 50 tand 75 %, although this positivity decreased in breast carcinomas (37.5 %) and was totally absent in lung cancers.
The immunohistochemical concordant evidence of Lf in bone metastases and corresponding primary carcinomas strongly supports the hypothesis of an autoctone production of this protein by the neoplastic elements themselves in order to get a greater availability of iron for their increased turnover. Results: It was observed a negative correlation between IL1/IL6 and IL17/Foxp3 concentration in the SF as measured by FC, before the treatment. In the SM, IL6 and MMP9 were strongly expressed in macrophages, fibroblasts, endothelial cells and the extracellular matrix of control cases, but reduced significantly in the treated cases (p< 0, 05). Il 17 and Foxp3 positive cells remained costantly in a reduced number in the MS. Conclusion: As the proinflammatory cytokines decreased after the therapy, we can conclude that these are more efficiently influenced than some immune cells participating in the autoimmune process.
Histological evaluation of the spleen after acute bleeding followed by blood replacement with two different physiologic solutions M. Cabral * , A. L. Ortiz, C. Venâncio, J. Objective: Chondromyxoid fibroma is a rare benign lesion accounting for less than 0.5 % of all primary neoplasms of bone. A study was conducted to assemble a national study group for rare entities and share the experience of different centers.
Method: The data was collected from the pathology archives of 13 referral hospitals in Turkey.
Results: Among the 61 cases enrolled in the study, 57.4 % (n=35) of them were male. Median age was 35±18 yearsold (Range: 6-67). The most frequent localizations were tibia (28 %, n=17), femur (n=10, 16.4 %) and pelvic bones (n=9, 14.8 %), followed by feet bones (11.5 %), fibula (8.2 %), humerus (4.9 %), hand bones (4.9 %), cranium (3.3 %), costa (3.3 %), radius (1.6 %), scapula (1.6 %), and vertebrae (1.6 %). Although the cases with appendicular skeleton involvement was younger than the others (median age: 29.5±17.03 vs 53±11.5 respectively) no significant correlation was found between age, gender and localization. Conclusion: Cases displayed a wide age-range with a slight male predominance. The most frequent localizations were tibia, femur and pelvic bones. Rare localizations such as temporal frontal bones, vertebrae and scapula were also observed. The study, which may serve as a preliminary work for future studies was presented to share our experience on this rare entity.
Histological changes and granulocytes redistribution in adjuvant arthritis L. Feketeova * , P. Jancová, P. Objective: Surgical treatment options of malignant tumors of the knee includes reconstruction with incorporation megaprosthesis. Inguinal lymphadenopathy due to the lymphatic uptake of metal debris has been described, and may be clinically confused with tumor metastasis. Method: We report the case of a 39-year-old woman with inguinal lymphadenopathy caused by metallic debris from a knee mega prosthesis for Malignant Fibrous Histiocytoma.
Results: The histopathological changes seen in lymph node were metal debris containing sinusoidal macrophages in a background with numerous epithelioid granulomata in the remaining lymph node. Conclusion: The identification of regional lympadenopathy in patients with past history of malignancy usually indicates metastatic disease. Post-prosthesis lymph node histiocytosis resembling metastatic disease is described, and that is why we need resect and examine lymph nodes with the use of polarized light microscopy to identify birefringent particles of prosthetic debris for an accurate histologic diagnosis. Objective: Chordoma is a slow-growing malignant bone tumor that exhibits notochordal differentiation. Nearly 90 % of cases occur in the sacrococcygeal region and in the base of the skull. The remaining cases develop in the mobile spine, predominantly in the cervical and lumbar vertebrae. We report a rare case of paravertebral mediastinal chordoma without bone destruction. Method: Case report.
Results: A 47-year-old Japanese woman was admitted to hospital after a tumor was incidentally detected on a plain chest X-ray image. The tumor was located in the paravertebral region of the mediastinum and did not show any destruction of the thoracic vertebra radiologically. The tumor was clinically diagnosed as a benign neurogenic tumor and the tumor was easily removed surgically. Microscopically, the tumor mainly consisted of tumor cells with extensively vacuolated cytoplasm, arranged in cord-and nest-like fashion in a myxoid matrix background. Immunohistochemically, the tumor cells showed diffuse positivity for pancytokeratin (AE1/AE3), vimentin. The tumor cell nuclei were positive for brachyury, which is a key transcription factor of notochordal development.
Conclusion: These results confirmed the tumor to be an extraosseous chordoma in the paravertebral mediastinal region, which is rather an extremely rare location for usual chordoma.
PS-07-020 Testicular papillary mesothelioma: A case with borderline features S. Mavropoulou * , Z. Tatsiou, P. Nasos * General Hospital, Laboratory of Pathology, Xanthi, Greece
Objective: Well-differentiated papillary mesothelioma occurs rarely in the paratesticular region, with only a few published case reports. Method: We describe a case of a 37-year-old man who initially presented with discomfort in the left testis and underwent resection of a hydrocele in the left testis. A hydrocelectomy was performed, during which a pedunculated mass, 2,5 cm in greatest dimension, was found attached to the testis. Results: Microscopically, the mass was composed of multiple branching papillary structures with fibrovascular cores covered by a single layer of low cuboidal to cuboidal cells with predominantly bland nuclear and cytologic features and rare microscopical necrosis. Immunohistochemical staining for calretinin and cytokeratines 5/6 was positive and proliferative marker Ki67 was <2 %. Accordingly the diagnosis of a well-differentiated papillary mesothelioma was made. The patient has not received additional therapy and is disease free 15 months after diagnosis. Conclusion: In conclusion, we report a rare case of a welldifferentiated papillary mesothelioma of the tunica vaginalis of the testis. The combination of benign and semimalignant characteristics can make the diagnosis of such a lesion problematic and pathologic distinction from malignant mesothelioma is crucial, although it may be difficult because of the variability of associated histologic features. Objective: Lipoma of the bone is a rare benign adipocytic tumor that arises intraosseous and rarely on the cortex or on the surface of the bone (parosteal lipoma). Parosteal lipoma affects the long bones diaphysis of adults over 40 years old. Method: We present the case of a 23 years old female with clinical diagnosis of femoral exostosis. Conventional radiographs shows the presence of a 5 cm length area of lucency on the femoral metaphyseal surface with a periostal reaction at the base of the lesion. Results: Gross examination reveals a 5 cm osseous tumor, whitish on the surface and yellow on the section. On microscopic examination there are mature adipocytes with small foci of bone scattered throughout the adipocytes and hyaline cartilage at the perifery of the lesion. The gross and microscopic examinations correlated with conventional radiographs led us to diagnosis of parosteal lipoma.
Conclusion: This case is interesting being a rare bone tumor, the young age of the patient and the location on the surface of the bone.
Application of Scanning Acoustic Microscope to evaluate lymph node lesions K. Miura * * Hamamatsu University, School of Medicine, Japan
Objective: A scanning acoustic microscope (SAM) is a device that uses ultrasound (frequency, >20 kHz) to image an object or tissue. Because it is known that the harder the tissue, the more the speed of ultrasound, SAM can provide data on the elasticity of cells and tissues. Method: We compared lymph node lesions between acoustic and light microscopic images to evaluate the usefulness of SAM. Results: SAM system discriminated lymph node components and demonstrated distinct acoustic images of the lymph nodes such as cancer metastasis, lymphomas, granulomatous diseases, and deposition diseases such as amyloidosis. Areas with desmoplastic reactions associated with cancer invasion or post-inflammatory fibrosis showed the greater speed of sound than normal lymph nodes. These results corresponded well to those obtained using the conventional microscope.
Conclusion: SAM provides the following benefits: (1) images are acquired in only few minutes without requirement for staining, (2) imaging pattern is similar to that of light microscope, and (3) speed of sound from each lesion is digital and statistical analysis is possible among diseases. Although resolution of SAM is little lower than that of light microscope, the SAM can be an ancillary tool for histological diagnosis and clinical research. Objective: Parkinson's disease (PD) is considered one of the major neurological disorders of the population, and there is increasing data provides enough evidences confirming the involvement of free radicals and other reactive oxygen species (ROS) in a number of physiological and pathological processes. The aim of the present study was to evaluate, the effect of therapy in patients with Parkinson disease on biomarkers of oxidative stress, such as products of lipid peroxidation by two different methods: Electron Paramagnetic Resonance and visible spectrophotometry. Method: The study was performed in blood samples of patients with PD -with therapy, patients with PD -without therapy and healthy volunteers as controls. The products of lipid peroxidation were measured as malondialdehid (MDA), spectrophotometrically by thiobarbituric acid (TBA) method. The levels of lipid radicals were determinated ex vivo at room temperature on an X-band EMX-micro spectrometer, Bruker, Germany.
Results: By the present study we reported higher levels of oxidative stress in PD patients without therapy compared to those with therapy. These results were comfirmed by the EPR method.
Conclusion: The increase of oxidative stress, in PD patients' might be an additional reason for many secondary complications. Objective: Primary Aneurysmal Bone Cyst (ABC) is a histologically complex and mainly cystic lesion that accounts for 2 % of all primary bone tumors. Information regarding its clinical presentation and management in hands and feet remains sparse. Method: The medical records ABC in hands or feet in Hospital La Paz Pathology Department from 1966 to 2011 were retrospectively reviewed and compared with existing data. We also propose pathological criteria for differential diagnosis between ABC and giant cell reparative granuloma (GCRG).
Results: Ten ABC in hands or feet were identified, out of 78 (12.8 %), Five tumors were in metacarpals, 4 in metatarsals and 1 in phalanx. Radiographs showed expansile lytic lesions in metadiaphyseal region, sometimes with aggressive appearance. Histologically, ABC showed a mixture of blood-filled spaces with connective tissue septae containing osteoclast giant cells and foci of osteoid. Scattered areas of so called "blue bone" were present in 5 cases (50 %). There were two solid variants. Five patients underwent resection and 5 curettage, three of which relapsed (30 %). Conclusion: ABC should be considered in the radiologic differential diagnosis of hands and feet tumours because these lesions can even mimic malignancies. Although clinicopathologically some authors consider GCRG is related to the ABC solid variant, we believe they are different entities.
Carb-3 is the superior anti-CD15 monoclonal antibody in optimized protocol settings R. Røge * , S. Nielsen, M. Vyberg * Inst. of Pathology, Aalborg, Denmark
Objective: Immunohistochemical detection of CD15 is important in diagnosis of Hodgkin lymphoma and may be relevant in classification of renal tumours. In four tests with 71-121 participating laboratories conducted by the Nor-diQC external quality assessment scheme only 50-76 % of CD15 stains were sufficient, mainly because of too diluted primary antibody concentration, insufficient HIER and less successful antibody clone. The purpose of this study was to evaluate three anti-CD15 antibodies based on vendor and inhouse optimized protocols.
Method: Multitissue blocks with various malignant lymphomas, renal tumours and normal tissues (n=218) were stained with three concentrated (conc) antibodies (Carb-3, MMA and BY87) according to predetermined in-house optimized protocols on two staining platforms. Ready-to-use (RTU) solutions of Carb-3 and MMA were also examined. Extension and intensity of stains was scored using the H-score method.
Results: Carb-3conc with an in-house optimized protocol gave the highest H-scores in classical Hodgkin lymphoma, renal tumours and normal kidneys. Carb-3RTU and MMAconc gave slightly lower scores, while MMARTU and BY87conc gave the lowest scores and a large proportion of false negative reactions. All in-house optimized protocols gave better staining results than vendor protocols. Conclusion: The importance of antibody selection and protocol optimization in immunohistochemical laboratories is emphasized. Objective: Studies show that there is association between stress and pathophysiological changes in reproductive system. The main aim of this study was to determine the pathological effects of immobilization stress on testes tissue and serum testosterone level in male rats. Method: Wistar rats were randomly divided into control, acutely or chronically immobilized groups of 10 in each. Animals were immobilized for 2 h/day or 8 h/day for a period of 3 weeks or 1 week in chronically or acutely immobilized groups, respectively. After 6 weeks, blood samples were collected using cardiac puncture method. Following serum collection, testosterone level was measured by radioimmunoassay method. The effect of immobilization stress on testes histology was also examined. Data were statistically analyzed and compared between groups using ANOVA.
Results: Serum level of testosterone was decreased in acutely or chronically immobilized rats compared with control animals (P<0.001). In histological study, semeniferous tubules were significantly deformed and cellular concentration was reduced in immobilized rats compared with control animals (P<0.05). The number of spermatocytes, spermatids or sperms was also decreased in immobilized rats (P<0.001) and this pathologic change was more prominent in acutely than chronically immobilized rats. Conclusion: This report underlines the importance of studying the archive material in order to thoroughly comprehend a single museum object. This handling of matters will help to turn anatomical collections into a unique teaching tool for modern medical practice and a noteworthy documentation of scientific, artistic and historical value. Objective: Extramedullary plasmocytoma is an uncommon plasma cell tumor localized preferentially in the upper aerodigestive tract, with no evidence of underlying multiple myeloma. It accounts for less than 1 % of head and neck tumours.
Method: We report a case of a 57-year-old male patient with reccurent left-sided epistaxis and nasal obstruction.
CT-scan showed a left maxillary mass eroding the lateral wall of the nasal cavity. The tumor was surgically removed.
Results: Histologic examination showed a diffuse infiltrate of neoplastic cells in the submucosa, arranged in a scant vascularized stroma. The neoplastic cells were large to medium-sized with amphophilic cytoplasm, irregular nuclei and proeminent, eosinophilic nucleoli. Mitotic figures were frequent. Imunohistochemical stains were performed in order to make a differential diagnosis: the tumor cells were positive for CD38 and CD138 and they expressed cytoplasmic immunoglobulin with kappa light chain restriction. Most of the tumor cells were also CD56 and EMA positive. Conclusion: Even though extramedullary plasmacytoma of the sinonasal tract is rare, it should be included in the differential diagnosis. The cooperation among the otorhinolaryngolists, pathologists and hematologists are required to manage the patients effectively in order to provide optimal treatment. Objective: Sinonasal malign melanoma is a rare antity, mostly arising from nasal cavity and accounting for <5 % of all sinonasal tract neoplasms. Method: We report two patients complaining of nasal obstruction and epistaxis. Endoscopy revealed partially haemorrhagic mass obliterating nasal passage. In one patient diagnosis was done as a plasmacytoma by pathologist in an another hospital. Multiple biopsies were taken. Microscopically, the tumour was consisted of medium sized cells, which had high nuclear to cytoplasmic ratio with pleomorphic nuclei containing eosinophilic nucleoli and intranuclear cytoplasmic inclusions. The cytoplasms contained variable amount of melanin pigment. Mitosesincluding atypical forms-were easily detectable. There were mild inflammatory infiltrate against the tumour and rare tumour necrosis. One was showed pseudopapillary growth pattern and lymphovascular invasion.
Results: Immunhistochemically, tumour cells showed immunreactivity for S-100, Vimentin, HMB-45 and Melan-A(MART-1). Tumour cells were negative for CD-20, CD-3, CD-138, CD-56, SMA, Pan-CK and Desmin. Submandibulary lymph node metastasis was observed in one patient. This patient has undergone chemotherapy of temozolomide and 23 cures of regional radiotherapy. For the other patient, there were no metastasis detected by PET-CT. Conclusion: Sinonasal malign melanoma is a rare malignacy, but this entity could be kept in mind to avoid missdiagnosis with other tumors sharing similar morphology. Objective: Significant changes in the voice occur after 50 years. Vocal quality is dependent on the vocal fold (VF) tissue biomechanics that derive from the extracellular matrix composition and organization. We studied muscularlamina propria interface of human VF in the aging. Method: Two authors evaluated density of vessels and the thickness of the deep and muscle layer by HE and IHC (collagenIV) with semiquantitative score. These data were validated by point-counting morphometric method.
Results: With the aging of the vocal folds we identified increased of vessels density in the muscle and deep layers, increasing the matrix density and thickness of the deep layer, and "dissection" of muscle fibers by dense connective tissue.
The progressive structural changes in musclelamina propria interface play a crucial role in the remodeling and vocal quality. The increased density of vessels and matrix in aging may contribute to the preservation of vocal function by the physiological repairing.
PS-09-012 "Lymphoepithelioma-like" thymic carcinoma in Parotid Gland M. Bugdayci * , G. Atay, B. Sozeri, G. Guler Tezel * Hacettepe University, Pathology, Ankara, Turkey
Objective: We report a 27-year-old woman presented with a slow growing painless mass beneath the right ear. A fineneedle aspiration revealed Whartin tumour. The patient underwent parotidectomy and histopathological examination showed thymic carcinoma with areas of thymoma. Method: Histopathological examination was done according to conventional protocols. Immunohistochemical stainings were performed using LSAB methods. PanCK, CD3, CD20, CK5/6, CK7, Vimentin, Ki-67, CD5, Bcl-2, CD1a, S100, SMA, TdT antibodies were used. Results: Histopathologically, a well circumscribed, nodular, hybride neoplasm finely seperated from parotid gland had two components, uniform spindle cells in basaloid form lobulated by fibrous septae and large epitheloid cells with vesicular nuclei and meganucleoli, suggesting thymoma and thymic carcinoma, respectively. Prominent lymphocytic reaction with germinal centers, high mitotic activity, small necrotic foci and microabcesses were also seen. Immunohistochemisty revealed PanCK, vimentin, CD5 and CD1a positivity. The diagnosis of "lymphoepithelioma-like" thymic carcinoma was made. Conclusion: Thymic carcinoma is a rare tumor most commonly located in the anterior mediastinum. Thymic carcinoma which originates from ectopic rests of thymic tissue caused by defective migration of the embryonic thymus, is extremely rare. In the presented case a thymic carcinoma was found in parotid gland which is an unusual site. Objective: Recent in vitro and in vivo studies have shown that several malignant tumors express an alternatively spliced variant of the receptor of growth hormone -releasing hormone (SV1), which operates by a ligand-dependent and independent manner. Method: Nine (9) adenocarcinomas, 11 pleomorphic adenomas and 11 Warthin tumors were studied by immunohistochemistry for SV1 expression and visualized by diaminobenzidine staining.
Results: SV1 expression was cytoplasmic and was detected in 6/8 malignant (75 %) and 11/11 (100 %) Warthin tumors. However, only 1/11 (9 %) pleomorphic adenomas expressed SV1 (p<0.05, x2-test). Immunoreactivity ranged from mild to intense in all positive specimens, with the exception of Warthin tumors at which only intense immunoreactivity was recorded.
Conclusion: Our study, for the first time reports the presence of anti-SV1 immunoreactivity in tumors of the salivary glands. Furthermore, the high association of SV1 expression with the malignant as opposed to the benign neoplasms implies a role of SV1 in the progression of the disease. A surprising finding of our study was the high positivity for SV1 exhibited by the Warthin tumors that implies biological similarities between these histopathological entities of the salivary glands. These results imply that the use of antagonistic analogs of GHRH merit further investigation. Objective: Sialadenoma papilliferum (SP) is a rare, benign neoplasm of salivary gland origin which manifests as an exophytic papillary excrescence of the mucosa. Indeed, SP is both an exophytic proliferation of papillary stratified squamous epithelium above the mucosal surface and an endophytic salivary ductal proliferation beneath the mucosa. It arises predominantly in minor salivary glands and usually affects patients in the age range of 32-87 years, with reports in young patients being exceedingly rare. Method: We report a case of a previously healthy 20 yearold man diagnosed with a nodular mass in the upper lip buccal mucosa. The tumour was excised and submitted for microscopic examination.
Results: Histologic examination revealed a biphasic proliferation of papillary stratified squamous and salivary ductal epithelia, both underneath the mucosal surface. Conclusion: In this unique case, as the classical SP, the tumour has a biphasic proliferation of squamous and ductal epithelia. However, unlike the classical SP, both epithelia grow under the mucosal surface. As a result, it didn't manifest as an exophytic proliferation, but as a nodule. We excluded squamous papilloma, inverted ductal papilloma, intraductal papilloma and mucoepidermoid carcinoma, the principal entities in the differential diagnosis of SP, and concluded it to be a SP with inverted pattern.
Interest of histopronostic classification in three grades in the therapeutic management of primary epithelial parotid carcinoma V. Results: Microscopic examination showed a biphasic tumor with a prominent myxoid stroma and tumor cells with clear cytoplasm involving pleural tissue. Immunohistochemistry showed tumor cells positive for keratin, S100 protein, smooth-muscle actin, p63 protein, vimentin. Histological and immunohistochemical features confirmed the diagnosis of epithelial-myoepithelial carcinoma. The patient underwent a right parotidectomy 3 years earlier for removal of 3.5 cm mass diagnosed as EMC.
Conclusion: Epithelial-myoepithelial carcinoma of salivary gland is a rare low-grade malignant neoplasm with a potential for local recurrence and metastases. Rare metastases for lungs, kidney and brain were reported. We described an additional case of pleural metastasis. Objective: Salivary defense system recognized as an important protective factor of the child oral environment. Research data reveal that bacterial infection, dental caries and periodontal diseases have a higher incidence in cases of salivary glands dysfunction. The aim of present study was to find out the morphological peculiarities of the fetal parotid gland in cases of restrictions of its intrauterine growth (IUGR) at late gestation.
Method: Parotid gland of twenty human fetuses with IUGR from the late spontaneous abortions material were compared with fifteen fetal glands in cases of induced abortions due to psychological reasons (control group). Tissue samples were stained with hematoxylin and eosin. Stereological examination was done to find out volume fractions of parotid gland structural components. Results: Results have shown the reduction of the area of acini, large collected ducts, striated and intercalated ducts in IUGR group. The volume fractions of vessels were also lower than on controls. The foci of immature secretory lobes in cases of growth restriction occupied wider zones within loose, poorly cellular, fibrous stroma.
Conclusion: Our study demonstrates the delay of the parotid gland structural maturation in pregnancies complicated with IUGR. Impaired growth and secretory gland's dysfunctions may cause pathological changes in oral ecosystem of a child. Objective: Dermal fillers are injectable products commonly used in aesthetic medicine. This type of treatment, which often replaces traditional surgical procedures provides satisfactory cosmetic results. It is known, however, that there is a risk of undesired effects at the site of injection of the product or even at a distance. Literature reports numerous cases of orofacial injuries caused by dermal fillers. The aim of this study is to evaluate two of the most widely used facial dermal fillers for aesthetic purposes in an effort to identify adverse reactions they may produce. Method: Two of the most commonly used materials for aesthetic purposes by dermatologists and plastic surgeons, polymethylmetacrylate and hialuronic Acid were injected in rat tongues: 10 % polymethylmethacrylate (n = 16) and 20 mg/mL hyaluronic acid (n =18), compared to an inert solution for control (n=16). After 7, 60 and 90 days, local clinical and histological alterations were analyzed. Results: The following factors were verified: intensity of the inflammatory response (H&E), amount of newly formed blood vessels (IHC) and macrophages and collagen fibers density (picrosirius). Results showed that both filling materials triggered local inflammatory response to a greater or lesser degree. Objective: The aim of this study was to compare clodronate and zoledronic acid regarding their influence on the repair of surgical wounds in maxillae (soft tissue wound and tooth extraction) and their relation to osteonecrosis.
Method: Thirty-four Wistar rats were allocated into three groups according to the treat-ment received: (i) 12 animals treated with zoledronic acid, (ii) 12 animals treated with clodronate and (iii) 10 animals that were given saline solution. All animals were subjected to tooth extractions and surgically induced soft tissue injury. Histological analysis of the wound sites was per-formed by means of hematoxylineosin (H&E) staining and immunohistochemical staining for receptor activator of nuclear factor-kB ligand (RANKL), osteoprotegerin (OPG), von Willebrand factor, and caspase-3.
Results: The zoledronic acid group showed higher incidence of non-vital bone than did the clodronate group at the tooth extraction site. At the soft tissue wound site, there were no significant differences in non-vital bone between the test groups. RANKL, OPG, von Willebrand factor, and caspase-3 did not show significant differences between the groups for both sites of surgical procedures. Conclusion: Both of the bisphosphonates zoledronic acid and clodronate are capable of inducing maxillary osteonecrosis. Immunohistochemical analysis suggests that the involvement of soft tissues as the initiator of osteonecrosis development is less probable than has been pointed out. Objective: The aim of this study was to evaluate the expression of CD1a, CD4, CD8, CD20, caspase-3, tryptase and basement membrane thickness in oral lesions of lichen planus (LP) and lupus erythematosus (LE). Method: Oral lesions of LP (n=21) and LE (n=23) were biopsied. After confirmation of diagnosis, the specimens were submitted to PAS and immunohistochemistry. Results: CD1a expression was significantly greater in epithelium and connective tissue in LP. Epithelial CD4 and CD20 did not differ between the diseases, but were greater in connective tissue in LP. CD8 expression was greater in both epithelium and connective tissue in LP. Caspase-3 did not differ between the groups, whereas tryptase was greater in LE epithelium. Basement membrane thickness did not differ between LP and LE. Conclusion: CD1a, CD4, CD8, CD20, caspase-3 and tryptase expression and the basement membrane thickness are not definitive criteria for differential diagnosis of oral lichen planus and lupus erythematosus. Objective: Odynophagia may be caused by inflammatory/ infectious conditions and tumors arising within the oropharynx. We present an unusual case of a follicular dendritic cell sarcoma (FDCS) of the tonsil manifesting with odynophagia.
Method: An 80 year-old man presented with odynophagia refractory to medical therapy; a 0.9 cm left tonsillar mass was noted on inspection. Bilateral tonsillectomy was performed. A panel of antibodies was applied on paraffin tissue section.
Results: Grossly, the left tonsil featured a 1.2 cm well circumscribed grey lesion. Histologic examination showed a proliferation of spindle to ovoid cells arranged in fascicles with swirling qualities admixed with scattered lymphocytes. Frequent mitoses and apoptotic bodies were seen but no "tumoral" necrosis was recognized. Tumor nests were separated by thin stromal septi containing a prominent glioblastoma-like microvascular proliferation with focal formation of glomeruloid tufts. The tumor cells were immunoreactive for CD21, CD23 and vimentin. Objective: Salivary gland tumors reveal a broad morphology and immunohistochemistry may be helpful. Method: We examined 12 mucoepidermoid (MEC), 8 adenoid cystic (ADC), 3 asinic cell (ACC) and 4 salivary duct carcinomas (SDC); 2 myoepitheliomas (ME), 5 basal cell (BCA), 31 pleomorphic adenomas (PA) and 18 Warthin tumors (WT) for SMA, Calponin, S100, CD10, GFAP, p63, CEA, GCDFP15, GLUT1, 34BE12, CK14, CK19, CD117 and Galectin3, using tissue microarray.
Results: Tumors expressing myoepithelial markers were ADC (SMA, Calponin), BCA (SMA, Calponin, p63), PA (SMA, Calponin, S100, CD10, GFAP, p63), ME (S100, CD10, GFAP), MEC (p63) and WT (p63); PA being the only tumor expressing all of the markers in the panel. GFAP, S100, CK14, p63, CK5/6 and Galec-tin3 expressions were higher in benign and CK19 was higher in malignant tumors (p < 0.05). Some of our findings helping in differential diagnosis are as follows:
In differentiation of PA from ADC: GFAP, CD10, GCDFP15 positivity; higher expression of S100; lower expression of CK14 and CD117 favors PA. In differentiation of PA from BCA: GFAP and CD10 positivity favors PA. In differentiation of MEC from SDC: CD10, CK14, p63 and CK5/6 positivity favors MEC. Conclusion: We conclude that salivary gland tumors may be well characterized by using distinct markers in each differential diagnosis. Objective: Lobulary capillary hemangioma (LCH) is a benign, rapidly growing vascular lesion of the skin and mucous membrans. It may rarely present as a mass of considerable size and thus entirely fill the nasal cavity. It occasionally appears in the nasal region as a pedunculated or broad base mass. Trauma and hormonal influences are the most common presumed etiologic factors. LCH usually involves the gingiva, lips, tongue, and buccal mucosa. The most common symptoms are epistaxis and nasal obstruction. Method: In this report; a case of 14 years old boy operated surgically of LCD of the nasal septum, which occurred post delivery was described.
Results: On examination, a red pedunculated bloody swelling 3,5*2 cm was noted arising from the posterior part of the nasal septum to nasopharinx. Histopathologically LCH has characteristics consistent with lobular prolifeation of capillaries in a fibromyxoid stroma. Conclusion: We emphasise that rarely seen LCH must be kept in mind in the differantial diagnosis of a rapidly growing mass, who is cured surgically. Follow up 2 months later showed no recurrance. Although the head and neck is not an uncommon region, the nasal cavity is extremely rare sites for LCH in children. Objective: Claudins are integral transmembrane proteins of tight junctions, critical for maintenance of cell adhesion and polarity. Altered claudin expression has been detected in carcinomas and correlates with tumour progression. Actinic cheilits is a pathologic condition that affects the lower lip, caused by chronic exposure to solar radiation; it corresponds to the early phase of squamous cell carcinoma. We investigated claudin patterns in phases of actinic cheilitis, as alterations in adhesion molecules are considered important during the progression of this process. Method: Immunohistochemistry against claudins −1, −3, −5, −7 and −11 was performed in 100 cases of actinic cheilitis/squamous cell carcinoma; results were analysed qualitatively.
Results: Actinic cheilitis (low grade intraepithelial squamous cell carcinoma), and the invasive front of superficially invasive squamous cell carcinoma and in situ squamous cell carcinoma were negative for claudins −1, −3 and −7. Claudin −5 was present on all epithelial layers in most cases evaluated. Claudin −11 was present in all epithelial layers in actinic cheilitis, but negative in cases of superficially invasive and deeply invasive squamous cell carcinoma.
Conclusion: Altered expression of claudins is present in AC from its incipient phase throughout the progression of the disease to invasive squamous cell carcinoma. Objective: Metastases to the jaw bones are uncommon and are most likely to arise from primary lung, breast, prostate or kidney tumours. Jaw bone metastases from a primary oesophageal carcinoma are especially rare, with only seven reports published in the literature.
Method: Here, we describe the case of a 69 year-old male patient where 7 years elapsed between the diagnosis and successful treatment of a poorly differentiated, stage pT2N0 primary oesophageal adenocarcinoma and re-presentation with jaw pain due to a metastatic mandibular deposit.
Results: The morphological appearance of the metastasis and immunohistochemical positivity with CK20, CK7 and CDX2 strongly supported an adenocarcinoma of upper gastrointestinal tract origin.
Conclusion: This case is of particular interest as there is an unusually long time between the detection of the primary oesophageal adenocarcinoma and diagnosis of metastatic disease. The longest period of time we have found for this reported in the literature is 9 months, although it is known that some oral metastases may appear more than 10 years following the primary tumour diagnosis.
Melanotic oncocytic metaplasia of the nasopharynx -A report of three cases and review of the literature J. S. Lee * , J. Y. Na * Chonnam National University, Hwasun Hospital, Pathology, Jeollanam-Do, Republic of Korea
Objective: Melanotic oncocytic metaplasia of the nasopharynx is a rare condition which is characterized by the presence of usually a small, brown to black colored pigmented lesion around the Eustachian tube opening.
Although it is a benign lesion, it may be clinically misdiagnosed as malignant melanoma. Microscopically, melanotic oncocytic metaplasia is a combination of oncocytic metaplasia of the epithelium of the gland and melanin pigmentation in its cytoplasm.
Method: In our present study, we report three cases of melanotic oncocytic metaplasia of the nasopharynx. Results: All the three cases occurred in men and were presented as multiple black pigmented lesions around the torus tubarius. Microscopically, mucous glands with diffuse oncocytic metaplasia and numerous black pigments were observed. No cellular atypia was observed. Immunohistochemically, the scattering of S-100 proteinpositive, and HMB-45-negative dendritic melanocytes was evident.
Conclusion: This is the first report of cases of oncocytic metaplasia of the nasopharynx in Korea. Objective: Angiokeratoma is a rare cutaneous lesion. Mucosal involvement is occasionally found as part of a more generalized cutaneous disease. Isolated angiokeratoma in the oral mucosa is extremely rare with only a few cases reported thus far. Method: We report the case of a 61-year-old female presented with a painless lesion on the left bucal mucosa, of 4 months duration. On clinical examination, a solitary purple lesion of approximately 4 mm in diameter was found.
Results: The lesion was excised and the histological examination revealed a tumour involving lamina propria, composed of large dilated vascular spaces, lined by normal appearing endothelium and filled with blood or with fibrin thrombi. The overlying epithelium showed variable degree of acanthosis and hyperkeratosis. Accordingly the diagnosis of angiokeratoma was made. On clinico-laboratory examination no angiokeratomas were found anywhere on the skin as well as no other malformation or metabolic disorder. The patient received no further treatment and 2 years later remains disease free.
Conclusion: In conclusion we report a case of a solitary angiokeratoma of the oral mucosa. Although rare, it should be included in the differential diagnosis when evaluating any lesion in this location and further investigations should be performed to rule out a metabolic or systemic disease. Results: Histopathologically in macroscopy, the cystic lesion was determined as a small part of tissue in white -grey color, in sizes of 0,6×0,5×0,3 cm. In microscopic evaluation, the epithelial tumor cells were formed mostly tubular, partially in solid form and broadly making pseudopapillary pattern in stromal tissue. In the luminal structures formed by tumor cells, there has been mucin as dark basophilic stained with PAS/Alcian blue (PH: 2, 5). The tumor cells are strong and have common positivity with CK7, and have negativities with CK20 (B-SA method). In the focal area that the tumor cells got the solid pattern, synaptophysin and chromogranin to evaluate the neuroendocrine differentiation wasn't determined.
Conclusion: The lesion in the medial part of the tympanic membrane of the patient was reported as colesteatom and the lesion in the middle ear was reported as middle ear adenoma.
Histopathologic evaluation for Helicobacter Pylori as a possible etiopathogenic factor in chronic tonsillitis E. Özgün * , D. Altinel, A. Albayrak, A. Tan, S. Sayhan, N. Bozlak * Nevehir Devlet Hastanesi, Dept. of Pathology, Turkey
Objective: Helicobacter Pylori is the major gastric pathogen which has an important role in the etiopathogenesis of chronic gastritis. We investigated the presence of Helicobacter Pylori as an extragastric reservoir in the tonsillectomy specimens to display if it is an etiologic factor in the development of chronic tonsillitis.
Method: In the current study, 100 cases with chronic tonsilitis were examined in bilateral tonsillectomy specimens. Objective: Salivary duct carcinoma (SDC) is a rare, aggressive malignancy with poor prognosis. Its histomorphology is distinctly reminiscent of the ductal carcinoma of the breast.
Method: A 48-year-old man was admitted with a mass of the right preauricular area. The mass had been enlarging steadily for the past 7 months. Computed tomography (CT) of the neck revealed 4×3×3 cm contrast enhancing solid mass with irregular borders at the right parotid region. Thorax CT, abdominal and thyroid ultrasonography were unremarkable. As the aspiration cytology was malignant right parotidectomy and right cervical lymphadenectomy was performed. Results: Histopathological examination showed a mixture of ducts, nests and cords of cells often embedded in a desmoplastic stroma with comedonecrosis of some ductal structures. The tumor cells were polygonal with vesicular nuclei, prominent nucleoli and eosinophilic, oncocytic cytoplasm. The tumor margins were lobulated and irregular with perineural invasion. Mucin stains were negative. We noted high proliferative index and cerbB2 overexpression. The tumor was classified as a salivary duct carcinoma, oncocytic variant of parotid gland. The resection margins were negative and the lymph nodes were reactive. Conclusion: As high proliferative index and cerbB2 overexpression are predictive factors, close clinical follow up is recommended for the risk of local recurrences and metastasis.
Immunohistochemical expression of Bcl-2 and Ki-67 in oral lichen planus and leukoplakia with different degrees of dysplasia F. Pigatti * , L. A. de Assis Taveira, C. T. Soares * University of São Paulo, Oral Pathology, Brazil
Objective: The oral lichen planus (OLP) is a chronic inflammatory disease of unknown cause, and its malignant potential is a very controversial issue. Therefore, the aim was to evaluate the immunohistochemical expression of apoptosis-related proteins and cell proliferation in OLP and epithelial dysplasia in order to investigate changes related to carcinogenesis and emphasize the importance of long-term follow-up of patients with OLP. Method: For this purpose, we selected 14 samples of OLP, 14 samples of leukoplakia with epithelial dysplasia, and 09 samples of normal oral mucosa as controls.
The evaluation of the expression of Bcl-2 and Ki-67 was conducted in accordance with the immunoperoxidase technique.
Results: There was also a high expression of Blc-2 protein in inflammatory cells in OLP lesions and leukoplakia with epithelial dysplasia. The expression of Ki-67 marker was higher in all analyzed tissue levels in the lesions of OLP and leukoplakia with epithelial dysplasia when compared with the control group. Objective: Pleomorphic adenoma (PA) is the most common benign salivary gland neoplasm of the major and minor salivary glands. Pleomorphic adenoma was shown sometimes to undergo malignant transformation in its natural course. Carcinoma ex pleomorphic adenoma (CPA) is a rare salivary gland malignancy that may develop from either a long-standing primary or a recurrent PA. The genetic mechanisms involved in the progression of adenoma to a carcinoma is still unclear.
To identify the predictors of disease, more knowledge about their genetic profiles is necessary. This study aimed to characterize alteration in the DNA copy number of PA and CPA. Objective: Leukoplakia is an oral premalignant white lesion. Although its etiology may vary, smoking has been implicated as a possible risk factor. The p27 protein has been shown to inhibit kinases, and it is known that its expression is decreased during carcinogenesis. The reduced expression of p27 has been correlated with poor prognosis in carcinoma. In this study a role for the smoking habit in the expression of this protein was investigated.
Method: Forty cases clinically diagnosed as oral leukoplakias and that presented a mild to intense degree of epithelial dysplasia and could not be diagnosed as any other disease were selected. Twenty cases were from current smokers (more than 20 cigarettes/day for at least 1 year) and neversmokers. Thirty-six cases of leukoplakia without dysplasia were used as controls. Histological sections of each lesion were subjected to the estreptoavidin biotin immunohistochemical method for detection of p27.
Results: A semi quantitative analysis was performed and the results showed that the expression of p27 was independent of the smoking status of the patient (p=0, 5237), using Kruskal-Wallis and Mann-Whitney tests.
Although not statistically significant, due to the small number of cases, the results indicate that the counting of p27 in leukoplakia correlates inversely with degree of epithelial dysplasia.
Low-level laser therapy may influence on the Akt/mTOR signaling pathway in oral cancer cells F. Sperandio * , F. Giudice, L. Correa, D. Pinto jr., S. de Sousa * University of Sao Paulo, Dept. of Oral Pathology, Brazil
Objective: Distinct cells respond differently to low-level laser therapy (LLLT), while the exact molecular mechanisms involved in cell proliferation or growth inhibition, after light stimulation remain poorly understood. Although LLLT has shown promising results in accelerating wound healing and preventing or treating oral mucositis, there is no evidencebased consensus of what this energy could cause in cancer cells. This should be highly pondered when an oral cancer patient is treated with LLLT due to oral mucositis, for example. Method: Two tongue squamous cell carcinoma cell lines (SCC9 and SCC25) were utilized to find the effect of low-level laser irradiation on the Akt/mTOR signaling pathway. Laser irradiation (660 and 780 nm) consisted on 40 mW of power and energy densities of 2, 3 and 6 J/cm2. After a single irradiation the most significant energy densities found with MTS assay were employed to analyze Akt/mTOR signaling pathway related proteins through immunofluorescence and western blotting.
Results: Beyond modifying the growing rates of cancer cells, low-level laser irradiation was able to induce different variations in the studied pathway, however a direct correlation among the proteins was not found.
Conclusion: LLLT may act on Akt/mTOR/Cyclin D1 signaling pathway, which has a widely recognized role in head and neck cancer progression.
Mucins as predictors of recurrent pleomorphic adenoma of salivary glands: An immunohistochemistry analysis of over 60 cases T. Teshima * , R. Ianez, C. Coutinho-Camillo, S. Lourenço * São Paulo, Brazil
Objective: Pleomorphic adenoma (PA) is the most common tumor of salivary glands and, despite its benign behavior, the recurrence after primary surgery is significant. In attempt to find a marker capable to predict the recurrence of this lesion, this study aims to analyze the expression of mucins MUC 1, 2, 4, 5 AC and 6 in 62 cases of PA, considering that mucins have been related to tumour growth of some organs.
Method: This study was performed in 62 cases in PA, which 5 of them presented recurrence after the initial surgery. All the primary tumors were histologically processed and submitted to the immunohistochemistry reaction. The antibodies for MUC 1, 2, 4, 5 AC and 6 were used and then analyzed with conventional optical microscope.
Results: MUC1 was the only mucin significantly expressed in most of all cases (88 %), being present within ductal lumen and cytoplasm. The other mucins showed a focal positivity in few cases, where MUC 2, 5 AC and 6 were cytoplasmic, while MUC4 was expressed in ductal lumen and blood vessels. Objective: Drug resistance remains a major problem in the treatment of NSCLC cancer patients for both, conventional chemotherapeutic and novel biological agents. Intrinsic or acquired resistance is caused by a variety of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that drug resistance mechanisms might also be regulated by microRNAs (miRNAs). Method: We tested 65 lung cancer samples. The total RNA was isolated from individual specimens and then RT-PCR and pre-amplification were performed. We detected levels of miR-21, miR-23a, miR-23b, miR-126, miR-205, miR-335*, miR-3163, miR-491-3p, miR-548p, miR-548x, miR-576-5p, miR-590p, miR-655, miR-656 and miR-944 using Taq-Man® MicroRNA Assays by LightCycler® 480 Real-Time PCR System. We assigned obtained results using statistical methods.
Results: Our results suggest that miR-590-5p and miR-655 are involved in apoptosis. The higher levels of miR-590-5p correlate with Bcl-2 positivity and pro-apoptotic protein BAX seems to be regulated by miR-655. The level of LRP protein responsible for drug resistance in lung cancer patients seems to be regulated by miR-255.
Conclusion: Within our cohort of NSCLC patients we did not find any correlations between the expression profiles of the abovementioned miRNAs and survival.
The 
Modified methacarn fixation as an excellent preservation of histology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens P. Babal * , R. Milcheva, P. Janega, P. Celec * Comenius University, Dept. of Pathology, Bratislava, Slovakia
Objective: Fixation techniques preserving morphological fidelity, immunoreactivity and integrity of nucleic acids may have a high impact on both basic and applied research and diagnostic pathology.
We investigated the effect of formalin, absolute ethanol and methanol; ethanol supplemented with acetic acid and modified methacarn fixative on the tissue morphology and immunoreactivity of different types of tissues and the preservation of RNA fragments of different lengths.
Results: The modified methacarn fixation provided a histomorphological quality comparable to the formalin-fixed tissue specimens. The immunoreactivity was superior in the buffered than in the untreated formalin and the preservation of protein antigenicity in normal and pathologically changed tissues tested with several antibodies was excellent with the use of alcohols with acetic acid. The acidic ethanol and the modified methacarn fixative showed the best preservation of the integrity of RNA in satisfactory quantity and quality of fragments up to 577 bp, which was reliable for relative evaluation of gene expression. Objective: cFLIP prevents the apoptosis by caspase 8 inhibition, its overexpression correlates with the progression of different tumors. Thymomas and thymic carcinomas are thymic epithelial tumors, in which the regulation of apoptosis is still unknown. We investigated the role of cFLIP in regulating the viability of thymus carcinoma cell line 1889c using an RNA Interference. The cell line HaCat was used as a control. Method: 1889c and HaCat cell lines were transfected with an established "short Hairpin" pIRES/PURO cFLIP-shRNA expression vector. CFLIP suppression and its Effect on pro and antiapoptotic molecules were analyzed using q-PCR and western blot. Apoptosis was quantified using the flow cytometry.
Results: The 1889c-shcFlip, but not the shcFlip-HaCat, showed sensitivity to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), accompanied by an overexpression of both the pro-apoptotic protein Noxa (p<10-4) and the anti-apoptotic proteins BIRC2, BIRC3 and XIAP compared to the non-transfected cells (p<10-4). Conclusion: cFLIP shRNA seems to induce apoptosis in the thymus carcinoma cell line. Simultaneously antiapoptotic proteins (AIPs) BIRC2, BIRC3 and XIAP were up regulated in order to protect the cell from apoptosis. Could these AIPs provide the way to escape the cell death? AIPs Selective inhibitions could represent a promising therapeutic approach for malignant thymic carcinomas. Objective: Nucleolin is a multifunctional DNA-, RNA-and protein-binding protein, involved in fundamental aspects of transcription, cell proliferation and growth. It is located in the nuclei/nucleolus, cytoplasm and on the cell surface. The present study aimed at optimizing the histological identification of nucleolin. Method: Mamary invasive ductal carcinoma 3 μm paraffin sections were pretreatead with pronase at room temperature (5-10 min) to epitope exposure. Biotinilated peptide against nucleolin was applied (1/1.5 mM dilutions), incubated at room temperature for 30 min/overnight at 4°C; after phosphate-buffered saline, peptide binding was identified by peroxidase-conjugated streptavidin and 3,3-diaminobenzidine tetrahydrochloride applied according to manufacturer's instructions before haematoxylin counterstaining. Results: Pretreatment for 5 min at 1.5 mM of biotinilated peptide showed only nuclear expression, as 1 mM/10 min pretreatment with overnight peptide incubation; positive cytoplasmic expression was obtained also after 30 min incubation/room temperature. At 1.5 mM concentration for 10 min epitope retrieval, cytoplasmic and nuclear positivity raised over 75 % expression, independently of peptide exposure (overnight/room temperature).
Conclusion: For optimizing peptides/antibodies, we have to be aware that nuclear/cytoplasmic expressions depend on digestion and peptide concentration. Histological morphology is also important and discordant results may be erroneously obtained.
PS-10-013 KRAS testing in clinical laboratory: Optimizing targeted therapy L. Cheng * , L. Miravalle * Indiana University, Dept. of Pathology, Indianapolis, USA
Objective: Activating mutations in the KRAS gene are found in more than 30 % of colorectal tumors, where they are associated with a poor response to anti-epidermal growth factor receptor therapies. Mutation testing techniques have therefore become an urgent concern. Several methods for KRAS mutation detection have been described in the literature. Most of these are laboratory developed tests and only a few commercial assays are currently available. Method: We studied the performance characteristics of a KRAS mutation detection assay on the ABI-3130XL genetic analyzer using a new commercial mutation detection kit. Samples were analyzed in parallel by different reference laboratories using alternative methodologies. Various sample types were used including formalin-fixed paraffin-embedded tissue, fine-needle aspirates, and cyst fluid specimens.
Results: A high level of agreement (100 % correlation for formalin-fixed paraffin-embedded tissue and fine-needle aspirate samples and 93 % correlation for cyst fluid specimens) was obtained despite the use of different methodologies. Conclusion: Shift termination assay is a simple, robust, and sensitive method for the identification of KRAS mutations in wide variety of specimen types. Objective: MicroRNAs (miRs) are small RNAs that modulate protein expression via post-transcriptional regulation of mRNA. They are related to malignancy in several tumors. Deregulation of miRs expression has been described in high grade astrocytomas of adult patients, however there is scarce information in pediatric patients. In this work we quantified the expression profiles of miRs in pediatrics astrocytomas. Method: Total RNA was extracted from 59 astrocytomas and 8 normal brain (NB) tissues, formalin fixed paraffin embedded. The expression levels of miR-124, miR-128-1 and miR-221 were quantified by using miRNA-specific TaqMan miRNA assays.
Results: The miR-128-1 was more abundant in NB against to miR-124 and -221 (p<0.05). MiR-124 and miR-128-1 were significantly down-regulated in all grades compared to NB (p<0.05) but in grade IV was more decreased: 8000-and 1200-fold, respectively. In patients with recurrent tumor, the expression of miR-221 was lower (p<0.002). Live patients expressed high levels of miR-128 (p< 0.02) and miR-221 (p < 0.0026) against deceased.
Conclusion: MicroRNAs are differentially expressed between astrocytomas and NB. The low expression of miR-128 and -221 could be a potential marker in recurrent pediatric astrocytomas, and both are associated to less survival. Embedded tissue is useful to describe and evaluate expression of molecular markers of malignancy. Objective: Isolation of genomic DNA from formalin fixed paraffin embedded (FFPE) tissues is a critical step for molecular diagnostic assays. Horizon Diagnostics has generated FFPE cell line reference standards containing defined mutant allelic frequencies, enabling the quality control of both assay sensitivity and DNA extraction.
Method: A panel of X-Man™ (gene-X, Mutant And Normal) cell lines were developed using our patented gene editing technology (GENESIS™) including; B-Raf V600E, V600K; EGFR ΔE746-A750, T790M, L858R, L861Q; K-Ras G12A, G12C, G12D, G12R, G12S, G12V, G13D; PI3Kα E542K, E545K, H1047R. FFPE blocks containing specific allelic frequencies including: 50 %, 33 %, 25 %, 20 % or 5 % were generated and sections cut. DNA was extracted using five different extraction methods and analysed using Droplet Digital™ PCR. Results: Mutant alleles could be detected using Droplet Digital™ PCR at each defined allelic frequency and the reproducibility of each test was very high. The allelic frequency was consistent throughout each block. The total DNA yield from each section was consistent using the same extraction method but varied between methods.
Conclusion: This study has demonstrated Horizon Diagnostic's FFPE Reference Standards have a highly accurate defined mutation specific allelic frequency together with a consistent DNA quantity.
Transcript variants and isoforms of the phosphatase subunit PPP2CA and its regulatory binding partners in haematological malignancies G. Grech * , C. Saliba, B. Shawn, R. Avellino, P. J. M. Valk, R. Delwel * University of Malta, Dept. of Pathology, Msida, Malta
Objective: The importance of feedback mechanisms involved in suppression of growth factor-induced signals is gaining importance both to understand molecular mechanisms of disease and also as potential therapeutic targets.
Our previous studies show that erythroid differentiation can be blocked by constitutive expression of the pp2a inhibiting subunit, alpha4. The aim of this study was to identify variants and transcript isoforms of PPP2CA and the inhibiting subunits alpha4 and SET, using (1) cell lines derived from haematopoietic disease, and (2) Objective: Epithelial cells grow by proliferation and adhesion, which will form an organ/tissue. STAT3 is involved in cell proliferation and various cellular events. In the present study, we studied the role of STAT3 in cell adhesion in hepatocytes.
Method: STAT3 knockout cells (S3KO cells) and liverspecific STAT3KO (L-S3KO) mice were generated. Cellular adhesion was analyzed by light/electron microscopy. Expression of adhesion molecules was examined by immunohistochemistry and western blot analysis. Protein expression and cellular adhesion were also examined in the pre-/post-hepatectomy liver tissue in control and L-S3KO mice.
Results: mRNA and protein of E-cadherin were not expressed in S3KO cells. IL-6 up-regulated E-cadherin in control cells but not in S3KO cells, and induction of constitutively active mutant of STAT3 restored E-cadherin level in S3KO cells. Interestingly, membrane-bound beta-catenin expression was not affected, but released to cytosol in S3KO cells. S3-KO cells showed almost normal proliferation but did not form cell cluster due to lack of E-cadherin. Electron-micrograph confirmed lack of intercellular adhesion structures (desmosome) in S3KO cells. Also in L-S3KO mice, hepatocytes lack desmosome structure and cell adhesion. Objective: ALK positive anaplastic large cell lymphoma (ALCL) is characterized by anaplastic lymphoma kinase (ALK) expression, most commonly associated with the t (2;5)(p23;q35), fusing the ALK and nucleophosmin (NPM) genes. However, in a significant proportion of cases the ALK gene has a number of other than NPM translocation partners. The aim of the study was to detect the NPM/ ALK fusion gene and the other possible fusion genes. Method: NPM/ALK was detected by RT-PCR, NPM/ ALK negative lymphomas were analyzed by ALK specific Rapid Amplification of 5´cDNA Ends (5´RACE). We prepared Q-RT-PCR assay for quantification of 3Á LK mRNA. Molecular findings were correlated with I-FISH. Results: We analysed 42 ALK positive ALCL. Chromosomal breakpoints affecting the ALK locus were detected by I-FISH in all patients. The NPM/ALK was detected in 32/42 patients. 5´RACE identified ATIC/ALK in 3, CLTC/ALK in 2 patients. Other fusion genes (TPM4/ALK, TPM3/ALK, ALO17/ALK) were found in one patient. In all specimens, overexpression of 3´ALK mRNA suitable for the minimal residual disease (MRD) detection was found. Objective: A 17 year-old girl, presented with an osteolytic epiphyseal lesion of the distal ulna. Based on histopathological examinations, the lesion was classified as giant cell tumor of bone (GCT). Four years later, the girl developed a second lesion at the same site. The histopathological examination revealed aneurysmal bone cyst (ABC) with solid spindle celled and giant celled areas which raised a question of a recurrent GCT or a primary ABC. The aim of this study was to analyze the clonality of these two processes. Determination of maternal and paternal X chromosome activation status is useful in the diagnostic analysis of nonrandom X inactivation patterns. Method: The human androgen receptor (HUMARA) gene polymorphism assay was used to identify the clonality of these two processes. Results: The patient was identified as heterozygous for the HUMARA allele. The GCT and ABC samples exhibited a monoclonal pattern, but one of them was with maternal and the other with paternal X chromosome origin. Conclusion: The findings of this case report demonstrate the clonal behavior of both lesions with different clonal patterns. The above investigation proved to be helpful in distinguishing between recurrent GCT and de novo ABC in the field of previous surgical intervention. . In all specimens gen Her2/neu was quantifed agains reference gen by qRT-PCR method (LyghtCycler® 480 II, Roche). Results: The results of cytogenetic analysis have been showed in 40 % that didn´t corespond with the results of qRT-PCR quantification. On the contrary the results of IHC was correlated with the results of qRT-PCR in 100 %. Methods SISH vs FISH has in 25 % different of results. In regard to the quality of ISH signals the SISH is more preferable for determination Her2/neu. Conclusion: A pilot study conducted on a group of 20 patients showed that among the methods used in the process of determining HER2/neu amplification are significant differences, even in evaluation and interpretation of test results. Regardless of the histological type of tumors (diffuse and intestinal type) as problematic for the evaluation of samples proved to be endoscopic route.
The use of molecular methods in diagnosis of malignant melanoma of biopsy S. Libor * , P. Dundr, S. Lísová, C. Povýšil * General Faculty Hospital, Inst. of Pathology, Prague, Czech Republic
Objective: Distinction between benign and malignant melanocytic lesions commonly represents a big challenge for the pathologist. In this case it appears as a useful auxiliary method of fluorescence in situ hybridization (FISH). The aim of this study was by using fluorescently labeled probes detection of numerical changes occurring in malignant melanoma and thus supply or confirm the diagnosis. Method: The retrospective study included 14 samples tissue with an established histological diagnosis (6x malignant melanoma, benign melanocytic lesions 8x). For detection was used probe Vysis LSI RREB1/LSI Myb/LSI CCND1/ CEP6 (Abbott Molecular, USA), Olympus AX70 fluorescent microscope (immersion lens 1000x). In the area of the tumor were calculated signals of individual probes and evaluated according to the manufacturer's instructions. Results: Evaluable results were obtained in 10/14 Cases (71 %) of the melanomas were 4 and 6 benign melanocytic lesions. In all four cases of malignant melanoma have been burdened with genetic changes, mainly in the CCND1 (in 55 %) gene amplification and PREB1 (in 40 %) and MYB (in 5 %). In contrast, for all 8 benign melanocytic been demonstrated normal findings. Conclusion: DNA abnormalities detected by FISH occurr in the vast majority of malignant melanomas but are not seen in benign nevi. This fact make the FISH test an important extra step in the differential diagnosis melanocytic lesions with ambiguous or borderline histological findings. 
Objective: EGFR is a receptor on the cell membrane with tyrosine-kinase activity and which is a regulator of proliferation, apoptosis, angiogenesis, tumor invasion. He is found to be overexpressed in some lung cancer histological subtypes. MEN1 is a tumor suppressor gene, with a role in cellular growth and differentiation, DNA reparation, and apoptosis. Method: Surgically resected specimen from 99 patients (men n=66; women n=33, age 57±11) with lung cancer were studied: carcinoid tumors (CT)-23, small cell lung carcinomas (SCLC)-13, large cell neuroendocrine carcinomas (LCNEC)-6, adenocarcinomas (AC)-29, and squamous cell carcinomas (SCC)-28. The histological subtype, pTNM stage, and gene expression of MEN1 and EGFR in tumor and normal lung tissue were evaluated. Results: Overexpression of EGFR was observed in 34 %(SCC-61 %, AC-31 %, SCLC-23 %, LCNEC-17 %, CT-13 %). Decreased expression was observed in 22 %(LCNEC-66 %, SCLC-54 %, CT-22 %, AC-14 %, SCC-7 %) (p<0.001). Overexpresssion of MEN1 was observed in 57 % of all tumors (SCLC-69 %, CT-65 %, SCC-57 %, LCNEC-50 %, AC-48 %) (p>0.05). Significant correlation between overexpression of MEN1 and early stage SCC was observed (p=0.03). Conclusion: EGFR is a target for therapy with monoclonal antibodies, so the tumors that overexpress EGFR can be considered for treatment with these drugs. MEN1 can eventually be a marker for good prognosis and a potential target for therapy. Objective: Homeobox genes encode transcription factors controlling cellular proliferation and differentiation. Altered expression of PROX1 homeobox gene is related to many cancers, including breast, esophagus, lymphatic and oral. Method: After overexpression of PROX1 gene in SCC9 cell line, total RNA was extracted from three overexpressing-PROX1 clones (OC) and one control-transfection cell clone (CC). Microarray analyses were performed using the Whole Human Genome 44 K according to the manufacturer's instructions. Genes with a fold change of greater or lower than 2.0 in OC versus CC, were considered as increased or decreased, respectivelly. Gene Ontology (GO) was used to assign biological process related to significantly differentially expressed genes. Results: Down-regulated genes MMP2, TIMP3 and NOTCH1 were further validated by qRT-PCR. Comparative gene analysis of OC and CC revealed 925 up-regulated and 789 down-regulates genes. Pathways induced upon PROX1 overexpression in GO terms included vascular development control, cellular adhesion, regulation of proliferation, among others. MMP2, TIMP3 and NOTCH1 expression by qRT-PCR showed reduced expression leves in OC. These genes are commonly overexpressed in oral squamous cell carcinoma and have been related with metastatic tumors and worse prognosis. Objective: Barrett´s Esophagus is the unique known precursor for esophageal adenocarcinoma (EAC), in a gradual progression to dysplasia. Our objective is to determine the correlation between the hypermethylation in CpG islands of the promoter region of p16 tumoral suppressor gen in epithelial DNA and the histopathological pattern, as possible biomarker for risk of progression. Method: 1. A study was performed about the evolution of precursor lesions, in a group of 55 patients diagnosed following the Vienna classification. 2. p16 hypermethylation is analyzed in paraffin embedding samples, through laser microdissection, DNA extraction, PCR amplification, pyrosequencing and quantification. Results: 1.14 of 20 cases with 2 to 5 biopsies remained as ND, ID or LGD, and 6 cases progressed to HGD and/or EAC. 2. Within the control group the methylation grade is 6.53 % and in the diagnostic groups is 12.04 % (ND), 7 % (ID), 17.05 % (LGD), 8.50 % (HGD) and 23.33 % (EAC).
Conclusion: 1. 70 % remained stable, but those reaching HGD all progressed to ACE (only in 50 % of the latter, HGD is recognized with H&E). 2. Methylation´s grade is higher in all diagnostic groups comparing to the control group, progressively increased as the dysplasia grade found with H&E, so it may be a good biomarker for neoplastic progression. Objective: In High Risk HPVs the expression of oncoprotein E6 is responsible for the degradation of p53, while E7 inactivates pRb and causes the progression to S phase of cell cycle, both sustaining the conversion to and the maintenance of malignancy. The aim of this study is to compare the performance in the detection of the E6 and E7 mRNA expression of HR-HPV using the NucliSENS Easy q test (bioMerieux) or HPV OncoTect (IncellDx) a test based on Flow cytometry-FISH method. Method: We enrolled 50 patients positive for HR-HPV DNA and/or pap smears. All subjects underwent a colposcopy histological evaluation and were tested both for the NucliSENS Easy q test and HPV OncoTect. Results: 31 out of 50 subjects resulted positive for RNA expression. The patients were divided into two cohorts based on the histological diagnosis: low grade lesions (CIN1) and high grade lesion (CIN2+). Patients with CIN1 were 26 with NucliSENS Easy q test and 13 with HPV Onco Tect, those with CIN2+ were 5 with either tests. Conclusion: These preliminary results suggest that HPV OncoTect have a better specificity than NucliSENS Easy q test, while more samples need to assess the difference in sensitivity.
Detection of PIK3CA/AKT mutations in human meningiomas A. Saetta * , G. Tomara, I. Chatziandreou, P. Tsioli, E. El-Habr, I. Sakalidou, G. Vretakos, E. Boviatsis, P. Korkolopoulou, E. Patsouris * University of Athens, 1st Dept. of Pathology, Greece
Objective: The PI3K/AKT pathway is a major signaling pathway frequently activated in human cancer due to PIK3CA and AKT1 gene mutations thus representing a potential therapeutic target and prognostic biomarker. In this study, we examined the mutational status of PIK3CA and AKT1 in meningiomas.
Method: 91 meningiomas were screened for activating mutations in "hot spot" exons 9, 20 of PIK3CA using Real Time PCR and High Resolution Melting Analysis. PIK3CA wild-type samples were analyzed for mutations in exon 4 of AKT1. The mutations were verified by sequencing and/or pyrosequencing Results: Mutations were detected in 2 out of 91 samples (2 %) in exon 9 of PIK3CA and were identified as p.E547K and p.S541F. Regarding exon 20, 7 out of 91 samples (7,5 %) showed the following mutations: p.R1023Q, p.T1025T, p.H1020V, p.M1043I, p.H1047R (2 cases), p.H1046T. Finally, in exon 4 of AKT1, 8 mutant cases were detected (9 %) all identified as p.E17K. In 19 % of the patients the activation of the PI3K/AKT pathway is due to mutations in the two examined genes. Conclusion: Aberrant activation of the PI3K/AKT pathway due to PIK3CA and AKT1 mutations is commonly observed in human meningiomas and these genes could be considered as potential targets in new therapies for cancer.
Study of PI3K/AKT/mTor pathway in urothelial bladder carcinoma A. Saetta * , N. Prekete, E. Trigka, G. Levidou, M. Karlou, P. Pavlopoulos, P. Korkolopoulou, E. Patsouris * University of Athens, 1st Dept. of Pathology, Greece
Objective: Deregulation of the PI3 kinase-AKT/mTOR pathway is a frequent event in tumorigenesis. We examined the possible significance of the components of this pathway in bladder urothelial carcinoma (UC). Method: 108 cases with bladder UC were screened for mutations in exons 9, 20 of PIK3CA gene and exon 4 of AKT1 by PCR-SSCP, HRMA, sequencing and/or Pyrosequencing. The expression of p-mTOR, p-4E-BP1, p-p70S6K, p-AKT, FGFR3 and p-ERK was evaluated by immunohistochemistry. Results: 4,6 % of the cases were mutant in PIK3CA gene and 3 % in AKT1. Cases with wild type AKT1 displayed higher FGFR3 receptor expression (p=0.0521). p-4E-BP1 expression was more frequent in low grade (p=0.018) and in pTa-T1 tumors (p = 0.053). Furthermore, superficial tumors presented higher levels of p-p70S6K expression (p = 0.035) and lower levels of p-AKT expression (p = 0.048). In multivariate survival analysis, p-4E-BP1 immunoexpression emerged as an independent prognostic factor of adverse survival (HR =9.207, p=0.0039), along with tumor grade and T-category. Conclusion: Activation of PI3K/AKT/mTOR pathway in bladder UC is not exclusively related to the presence of AKT1 and PIK3CA mutations. Expression of p-4E-BP1 could serve as an independent prognosticator.
Comparison of DNA extraction methods of formalin fixed, paraffin-embedded archival tissues B. Senguven * , E. Baris, T. Oygur * Gazi University Dental Faculty, Dept. of Oral Pathology, Emek, Turkey
Objective: Formalin fixed, paraffin-embedded (FFPE) archival tissues are valuable resources for many molecular studies. The goal of this study is identify the optimal method for DNA extraction from FFPE tissues. Method: 32 human gingival tissues which has obtained from patients with gingival hyperplasia were used. Serial sections of 10 μm thickness obtained using a standard microtome. Half of the sections were deparaffinized on glass, the other half collected directly to a 1,5 ml tube. In order to identify the optimal method for DNA extraction, we compared phenol-chloroform protocol and DNA Extraction Mini Kit. The duration of proteinase K digestion were also compared. Spectrophotometric evaluation of the yield and purity of DNA was conducted. To deteminate the amplifiablity of extracted DNA, three different bp fragment of the beta-globin gene were amplified using PCR. Results: The phenol-chloroform method had the lowest yield and purity. Deparaffinized specimens on glass, digested for 72 h and isolated using mini kit had the highest yield. Amplification of the 120 bp fragment of beta-globin gene was successful in all samples. Conclusion: According to our results deparaffinization on glass, increasing the time of proteinase K digestion and using commercial kits for isolation seems the best method to obtain amplifiable DNA from archival specimens.
MicroRNA signatures associate with Fallopian tubal implantation in humans R. Shao * * Gothenburg University, Physiology and Endocrinology, Sweden
Objective: MicroRNAs are small non-coding RNA molecules that regulate a large number of cellular pathways and deregulation or altered expression of miRNAs is associated with many disease states. The function of the Fallopian tubes appears to involve orchestrated spatiotemporal alterations in transcriptome profiles, the regulation of tubal gene expression and function by miRNAs may thus be of primary importance in tubal ectopic pregnancy. Method: Both implantation sites and non-implantation sites of Fallopian tubes from women with EP and decidual biopsies from women undergoing therapeutic surgical termination of pregnancy were collected and analyzed by miRNA array. The unique miRNA profiling results were validated by TaqMan qRT-PCR, and bioinformatics' analysis was employed to further predict the miRNA targets. Results: A total of 47 miRNAs were differentially expressed in implantation sites compared with those in non-implantation sites of Fallopian tubes after comparison of decidual miRNAs, among which 19 were up-regulated while 28 are down-regulated. The miR-424 was significantly increased, whereas let-7i, miR-149, and miR-182 were significantly decreased. Differentially expressed miRNAs were predicted to be related with several signaling pathways in normal intrauterine implantation. Conclusion: Our findings establish an miRNA signature associated with tubal implantation and provide the experimental basis for further understanding the molecular and cellular mechanisms of initiation and development of tubal EP in humans. Lung is an organ that can sense any perturbation of the air, due to its constant interaction (long live) of the air. Our hypothesis was that lung could be an organ sensor of climatic change observed today. Method: This preliminary study analyzed the expression of 3 isoforms of the genes of Hsp90. The cases were chosen from archive of autopsies from 1970 to 1979, with different diagnosis, and were matched in sex and age with a same number of cases chosen from 2000 to 2009. The expression was studied by RT-PCR from lung tissue embedded in paraffin, 20 samples of each decade. Results: The expression levels of Hsp90 were normalized to β-actine. We found expression in six cases of 2000-2009: five for Hsp90AA1-2, and 1 for Hsp90AB1, one case expressed both. Their matched cases in 1970-1979 never expressed any isoform. The cause of dead in all cases doesn't fit with any pattern. Conclusion: With these results we propose than the lung could be a sensor of global warming, and the expression of these genes could be molecular markers of this climatic change.
Apoptosis associated genes and their role in predicting responses to neoadjuvant breast cancer therapy D. Tvrdík * , H. Skálová, P. Dundr, L. Stanek, C. Povýšil, L. Petruželka * General University Hospital, Institute of Pathology, Prague, Czech Republic Objective: Neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. This present study is aimed at the investigation of biomarkers that can predict a pathologic response to the therapy. Method: The transcriptional profile of 84 key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific Real-Time PCR assay. Results: On the basis of a hierarchical cluster analysis of 13 significantly changed genes, we divided patients into good and bad prognosis groups, which correlate well with progression-free survival. In the good prognosis group, we found a statistically significant downregulation of the expression of MCL1 and IGF1R genes after neoadjuvant treatment. We also found a statistically significant overexpression of BCL2L10, BCL2AF1, CASP8, CASP10, CASP14, CIDEB, FADD, HRK, TNFRSF25, TNFSF8 and CD70 genes. In contrast, we found upregulation of IGF1R after the treatment in the group with poor prognosis. Conclusion: As we have shown, gene expression profiling after neoadjuvant chemotherapy is a valuable research tool for investigating molecular markers, which may better reflect tumor biology and treatment response than standard prognostic and predictive factors. Objective: Non-Small Cell Lung Carcinoma (NSCLC) is one of the most serious cancers. Identification of genetic changes (mutations, amplifications or rearrangements) within EGFR, KRAS and ALK oncogenes, associated with NSCLC, allows choosing the patients, benefit from biological therapy with tyrosin kinase inhibitors. Method: DNA is isolated from formalin fixed paraffin embedded specimens or cytology specimens. Mutation detection is performed by real-time PCR, fragment analysis, primerextension analysis and mutant-enriched PCR. Wt-EGFR patients (e.g. with no mutation detected) are tested for ALK gene rearrangement and EGFR gene polysomy or amplification using the fluorescence in situ hybridization (FISH) method. Results: Since 10/2010 till 2/2012, 238 DNA samples were analyzed. Out of these, 17 patients (7,14 %) were found to be positive for activating mutations within EGFR gene. Since 07/2011 till 2/2012, 80 patients were analyzed for ALK gene rearrangement, EGFR gene polysomy or amplification. ALK gene rearrangement has been proven in 5 (6,25 %) cases, ALK gene amplification in 2 (2,5 %) cases and EGFR gene polysomy or amplification in 35 (43,75 %) cases. Conclusion: Determination of genetics changes in tumor can provide powerful tool for setting up strategy and therapeutic protocols in NSCLC patients. Objective: A 61 year old gentleman, ex-smoker with a history of asbestos exposure presented to the emergency department with a 1 day attack of severe abdominal pain and bilious vomiting. History of abdominal pain, lethargy, and weight loss over the last 12 months and 4 years of recurrent pleural effusion. Pleural aspirates and a videoassisted thoracic biopsy were negative. Method: X-ray confirmed small bowel obstruction and an emergency laparotomy performed. The terminal ileum was intussuscepted into the caecum causing proximal obstruction. A right hemi-colectomy was performed. Results: On gross examination there was intussusception of the terminal ileum secondary to a polypoid mass at the ileo-caecal valve. Histology showed an oncocytic tumour with neuroendocrine features arising from the serosa of the small bowel. The tumour cells were positive for CK7, calretinin, and vimentin but negative for CK20, chromograninA, CD56 and TTF-1. The features were in keeping with a malignant peritoneal mesothelioma infiltrating the small bowel wall. Conclusion: Peritoneal malignant mesothelioma is an uncommon tumour which rarely causes mechanical small bowel obstruction. To our knowledge this is the first case of localised primary peritoneal mesothelioma presenting with intussusceptions.
Giant mesenteric cystic lymphangioma in adult; rare tumor, unusual location and uncommon age of occurrence: A Tunisian case report S. Attafi * , W. Ajouli, N. Bouchiba, A. Chouchene, M. H. Balti, K. Bellil * FSI Hospital, Dept. of Pathology, Marsa, Tunisia Objective: Cystic lymphangioma is a rare benign neoplasm arising from the lymphatic system. It occurs as a result of congenital malformations of the lymphatics, leading to the obstruction of local lymph flow and the development of lymphangiectasia. Lymphangioma is common in pediatric patients, but it is extremely rare in adults, with only about 100 cases reported in literature. Most lymphangiomas are found in the head and neck; intraabdominal and specially mesenteric locations are very unusual. The aim of this work was to study its clinical, histological and therapeutic features and their diagnostic difficulties. Method: We report the case of a 46 years-old woman, who presented a painful syndrome of the right iliac fossae (RIF). The physical examination found a mass of the RIF. Abdominal ultrasonography and magnetic resonance image showed a mesenteric cystic formation. At laparotomy, a large cystic tumor of the caecal mesentery, measuring 23 cm, was found. Histopathology showed a fibrous and thin walled cyst, lined by flat low lying epithelium with surrounding tissue of scattered lymphoid cells. Results: The diagnosis of cystic lymphangioma was retained. Conclusion: Mesenteric lymphangiomas are very rare, but they can cause acute abdomen that requires an emergent surgery. Therefore, they should be included in the differential diagnosis of cystic intra-abdominal lesions raises several possibilities, including both malignant and benign soft tissue tumours. Objective: The spleen metastases from colon cancer are rare conditions and usually associated with extensive disease. There are only eight reports in English-language literature of isolated splenic metastases from colorectal carcinoma, which generally metastasize to regional lymph nodes, liver and abdominal peritoneum. Method: We report a case of multiple splenic metastases in a 60-year-old woman. Results: In November 2009, the patient had undergone right hemicolectomy, lymphadenectomy and chemotherapy for stage III tubular adenocarcinoma of the ascendant colon with positive pericolic lymph nodes. In December 2011, she was admitted in our hospital for diffuse abdominal pain and intestinal transit disorders. Computed tomography of the abdomen revealed multiple metastases of different sizes in liver and spleen. During the surgery, which was performed with the goal of curing metastatic disease, there were found two nodular hepatic secondary tumors, and numerous spleen metastases, with diameters from 0.2 cm to 1.5 cm. The histopathological evaluation of splenectomy specimen revealed a metastatic tumor deposit, histologically similar to the primary tubular adenocarcinomas of colon. Conclusion: This is the ninth documented case of splenic metastasis from colon cancer. Previously reported cases of this type were isolated tumor, this one being the first reported with multiple splenic metastases.
The role of fatty acid synthase in Inflammatory Bowel Disease (IBS) E. Bas Bozkurtlar * , N. Ozkan, A. E. Kedrah, C. Celikel * Marmara University, Dept. of Pathology, Istanbul, Turkey
Objective: It was demostrated that FAS expression increases not only in mucosa involved by active colitis but also in normal mucosa of ulcerative colitis (UC) patients. Our aims were to evaluate the role of FAS expression in differential diagnosis of IBD, to search for any possible change during the progression of the disease in patients with clinical and endoscopic follow-up. Method: Among 82 colonoscopic biopsy samples of 50 UC cases,13 samples were classified as remission,15 as resolution, and 54 as active period;35 samples of 30 Crohn Disease (CD) cases were classified as active period. Fifteen cases with normal endoscopic and morphologic findings comprised the control group. Results: Between UC and CD active periods and control group, a significant difference was found (p=0,0001). There was a statistical correlation between FAS expression of basal crypt of normal mucosa in UC active period and the disease duration (p=0,036). Conclusion: The increasing in FAS expression in patients with IBD can not be explained only by the inflammation. FAS expression can not aid us in the differential diagnosis of UC and CD, but it seems that it is related to the extent of inflammation in UC. The relation between the disease duration and an increase in FAS expression active UC, might have a significant role in carcinogenesis in UC.
Micropneumatosis -An (un) usual finding in gastrointestinal specimen? K. Blaue * , M. Plauth, J. Knolle * Städtisches Klinikum Dessau, Pathology, Dessau-Roßlau, Germany
Objective: A rather rare disease, micropneumatosis describes the findings of cyst-like cavities devoid of lining epithelium in the gastrointestinal wall. Micropneumatosis is often secondary to intestinal bacterial infection or mechanical factors. This study evaluates the incidence of micropneumatosis in our institute. Method: We analyzed all cases of micropneumatosis obtained in our institute between 2001 and 2011. Available data were retrieved from patients' records. Results: Out of 300.000 specimens obtained between 2001 and 2011, 71 cases (0.02 %) presented with micropneumatosis (37 female, 34 male; mean age 61 years, age range 18 to 85 years). Most common site was the colon (32 cases), followed by stomach (30), small intestine (8) and greater omentum (1). Conclusion: Micropneumatosis represents a rare but harmless diagnosis in gastrointestinal specimen. Due to the histological picture, which may be confused with dilated lymphatic vessels, submucosal lipomas, and, to the untrained eye, even with signet ring cell carcinoma, incidence may even be higher. In unclear cases, where there seems to be a endothelial layer, immunohistochemistry may be necessary to confirm diagnosis of micropneumatosis. This study shows that micropneumatosis should be considered in differential diagnosis in every age and sex.
Predicting lymph node metastasis in pT1 colorectal cancer -A meta-analysis providing rationale for therapy decisions S. Bosch * , I. Nagtegaal * Radboud University Nijmegen, Pathology, Netherlands
Objective: We conducted a meta-analysis of published reports on the predictive value of risk factors for lymph node metastases (LNM) in pT1 colorectal cancer in order to provide a rationale for choosing follow-up or radical surgery after local excision. Method: Local excision is an attractive treatment option for colorectal cancer, but is only safe in the absence of LNM. Several pathological factors have been associated with LNM, however it remains unclear how to integrate these in clinical decision making. Results: A Pubmed search revealed 17 studies totaling 3741 patients. Strong predictors of LNM were lymphatic invasion (RR 5.2 [95 % CI 4.0-6.8]), budding (RR 5.1 [95 % CI 3.6-7.3]), and high grade histology (RR 4.8 [95 % CI 3.3-6.9]). Deep submucosal invasion was also strongly associated with LNM, however in a risk stratification model this factor was of limited added value. Conclusion: The absence of lymphatic invasion, budding and high grade histology may justify withholding radical surgery. The independent role of submucosal invasion depth is probably limited. Models for risk stratification based on these factors need to be validated. Objective: Dendritic S100+ cells in intestines have been described in more studies. Their connection to S100+ fibres of nervous system is lesser of an object of interest. Method: We have processed the samples of intestinal mucosa of people with Crohn's disease and colitis ulcerosa by the means of form-paraffine technique and anti S100 antibodies. Results: In lamina propria and in submucosa at Crohn's disease were S100+ the fibres of various width and orientation. Outside of them the diffusely scattered round S100+ mononuclear cells could be seen. Contrary to colitis ulcerosa the granulation tissue at Crohn's disease contains in the area around abscesses a thick accumulation of S100+ cells and this is similar also in the formed lymphoid tissue. Conclusion: The positive fibres can be seen in submucosa at the edge as well as in LPM between the bases of the glands. In between the plexus/ring and longitudinal muscle there are massive bands of connective tissue which contains bundles S100+ of ganglion cells and nerve fibres. The fibres are present in ring as well as in longitudinal muscle. In this area S100+ fibres can also be seen in vessel walls. Numerous bundles of granulatory tissue do not contain positively reacting substance. In the basal part lamina propria there are visible S100+ cells as well as long, smooth, positively reacting fibres.
PS-11-010 NCF1-deficient mice with impaired oxidative bursthave a more agressive progression of Dextran Sulfate Sodium (DSS) -induced colitis L. Carvalho * , T. Rodrigues-Sousa, A. Alarcão, A. F. Ladeirinha, M. Souto-Carneiro * Amadora, Portugal
Objective: Intestinal Inflammatory Disease (IID) as a primary immunodeficiency depends on mutations in the NADPH oxidase complex, responsible for the production ofreactive oxygen species (ROS). One of the most common clinical patterns in IID is chronic colitis. Ncf1-mutation in mice leads to deficiency in ROS, rendering them susceptible to autoimmunity. Here we studies how ROS-deficiency in Ncf1-mutant mice influenced the immune response to DSS colitis, its recovery and answer to a second induction. Method: Colitis was induced in wild type (WT) and Ncf1mutant (Ncf1) B10.Q mice by administration of 3.5 % DSS in the drinking water for 1 week. After 1 week recovery, DSS was administered for another week. Mice were sacrificed at days 0, 7, 14 and 21, the colon was removed and folded into a Swiss roll. Sections of the colon were stained with HE, and monoclonal antibodies against B cells (B220), CD3+ T cells; and macrophages (Mac1/CD11b) were applied. Results: Colitis was more severe in Ncf1 than WT mice, with epithelial dysplasia, hyperplasia of Peyer's patches and poor epithelium recovery (hyaline scars). At all time-points the amount and location of colonic B cells, T cells and CD11b + cells was distinct between groups. These results suggest that ROS are crucial for leukocyte recruitment and tissue-repair in DSS-induced colitis.
Histopathological pattern of polypoid lesions of colon in Albanian population G. Cekodhima * , A. Cekodhima, A. Beqiri, G. Andrea * University Hospital Maria Teresa, Dept. of Pathology, Tirana, Albania
Objective: Our aim was to study histopathologic pattern of the polypoid lesions in Albania. Method: We studied 216 lesions, 184 polypectomies and 32 colorectal surgery in 139 patients in Tirana. Results: There were observed 216 polypoid lesions of large intestine in 122 males and 91 females.105(48.6 %) were adenomatous polyps, 31(14.35 %) were hyperplastic, 24 (11 %) were inflammatory polyps with ulcerative colitis, 10 (4.6 %) were inflammatory 6(2.77 %) were juvenile polyps, 2(0.92) non Hodgkin's lymphoma and 1 (0.46) was fibrolipoma. Tubular adenomatous polyps were the commonest polyps. They were more common 56 (25.9 %) in male population as compared to female 19(8.79 %). High grade dysplasia was present in 50 (23.14 %) and malignant change in 26 (12 %). The size of the polyps range from 0.5 to 4 cm. Conclusion: Adenomatous polyps were the most frequently found polyps in our study; the approximately 24 % were advanced lesions.
The role of cancer stem cells in biology and prognosis of colon cancers A. F. Çiçek * , Ö. Öngürü, M. Gamsizkan, A. Günal * Gülhane Military Medical Academy, Dept. of Pathology, Ankara, Turkey
Objective: This study was designed as a retrospective clinicopathological observation based on immunohistochemical and statistical findings to show the relationship between the disease progression and the intensity of the stem cell population within the tumor for colorectal cancers.
Method: For this purpose, we investigated 97 colorectal carcinoma cases, retrospectively. Paraffin embedded blocks was obtained from pathology archive and demographical patients' data from the patient's files of Gastroenterological Surgery Department. Immunohistochemically, we used CD133 and Musashi-1 antibody to determine the stem cells within the tumor. We noted the age of the patients, histopathological diagnose, tumor location, grade, TNM status, clinical stage, disease-free-survival and the outcome. Then we statistically compared all prognostic data with the immunohistochemical findings Results: All of the cases were immunoreactive for both antibodies, furthermore we found a significant statistical correlation (p=0.043) between CD133 expression value and patient outcome. When the value of CD133 expression was high, clinical outcome was poor. In addition, there was a relation between high Musashi-1 expression and poor outcome. Conclusion: Based on these findings we reported that overexpression of both CD133 and Musashi-1 antibodies may be a poor prognostic factor in colorectal cancers.
Immunohistochemical evaluation of VEGF expression in colorectal carcinomas D. Crisan * , M. Florea, D. Fodor * University of Medicine and Pharmacy, Pathology, Cluj-Napoca, Romania
Objective: Vascular endothelial growth factor (VEGF) is an important angiogenic glycoprotein secreted by the tumor cells and host cells which proved to be a powerful prognostic factor in various human cancers. The aim of our study was the evaluation of VEGF expression in colorectal carcinomas using immunohistochemistry, in order to identify the relationship between the presence of the protein in the tumor cells and a series of morphological parameters. Method: We analyzed 30 consecutive surgically removed colorectal carcinomas. Immunohistochemistry was done on formalin-fixed and paraffin embeded tissue sections, using the anti-human VEGF-A monoclonal antibody (clone VG1, Dako). The extent and intensity of staining were graded and used to calculate the immunoreactive score for each case. Statistical analysis was performed with SPSS (Statistical Package for the Social Sciences), using nonparametric tests. The level of statistical significance chosen was p <0.05. Results: VEGF was expressed in all tumors, with a heterogeneous distribution. The only parameters that correlated with high VEGF positivity were the extent of the tumor necrosis and the presence of lymph node metastases (p =0,001).
Conclusion: High levels of the VEGF expression may be an indicator of poor prognosis in colorectal carcinomas.
Collision tumour of the appendix: Mucinous cystadenoma and carcinoid -Report of a case C. Dastamani * , A. Paraskeva, E. Carvounis, T. Theodosopoulos, A. Kondi-Pafiti * Aretaieion Hospital, Dept. of Pathology, Athens, Greece
Objective: Appendiceal carcinoids are usually located at the tip of the appendix and occur as incidental findings. Epithelial neoplasms of the appendix are uncommon and consist of mucinous adenomas, carcinomas and neoplasms with features of both carcinoid and adenocarcinomas. Method: A case of dual mucinous cystadenoma and carcinoid of the appendix is reported. Results: The patient, a 57-year old-asymptomatic female presented with a cystic right iliac fossa mass. The excised specimen was appendix cystically dilated measuring up to 8,5 cm in diameter. The luminal content consisted of viscid mucin. The base of the appendix was thickened (up to 1,5 cm) and firm. Microscopically the cystic part of the specimen had features of mucinous cystadenoma with adenomatous epithelium extending as well over the thickened appendiceal wall. At that area the wall was involved by a well differentiated neuroendocrine neoplasm WHO gr I (carcinoid). The neoplastic cells infiltrated the entire thickness of the appendiceal wall, the mesoappendix and reached the serosal surface. The two neoplasms where separate and there was no transitional zone between them. Conclusion: Dual carcinoid/epithelial neoplasia is a rare occurrence in the appendix. The prognosis appears to be no worse than for either of the two components alone.
Gastrointestinal Stromal Tumor (GIST) of the anal canal: A case report L. De Carvalho * , P. A. Teixeira, V. G. Siqueira, M. Pereira, T. Or, E. Pereira * Centro Universitário Lusíada, Dept. of Pathology, Santos, Brazil
Objective: Gastrointestinal stromal tumors (GIST) are mesenchymal tumors derived from interstitial cells of Cajal. They are found more frequently in the stomach, small intestine while colon and rectum represent unusual sites. GIST's of the anal canal are extremely rare. Method: 43-years-old woman who presented bleeding, pain and constipation for several months. The rectal examination revealed a well defined mass located within 10 cm of the anal verge. The magnetic resonance imaging confirmed a well-circumscribed mural mass without adenopathy. The local excision of the tumor was performed for pathological study. Results: Gross examination showed a 4.5×3.0 cm fibrous mass. Histological examination revealed a spindle cell tumor with moderate atypia and mitotic count of 6 mitosis/ 50HPF. Neoplastic cells showed marked positivity for c-Kit and CD34 and negativity for muscular markers. A diagnosis of GIST with intermediate risk of aggressive behavior was made. Conclusion: GISTs of the anal canal are extremely rare with only few cases reported in the literature. We described an additional case of GIST of the anal canal with histological and immunohistochemical study.
Intestinal graft versus host disease with giant cells B. Doganavsargil * , A. Vink, E. J. Petersen, G. J. Arnold Offerhaus * Ege University, School of Medicine, Izmir, Turkey
Objective: Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation in which immune cells in the transplanted marrow attack the immunocompromised host. It can occur in either an acute or in a chronic form and may affect various organs. Intestinal GVHD is particularly important because of its frequency, severity and its effects on the general condition of the patient. Method: Here we report a 68-year-old man who developed GVHD, 8 months after receiving allogeneic non-myeloablative peripheral blood stem cell transplantation for refractory chronic lymphocytic leukemia. Colonic biopsies revealed an edematous and congested lamina propria, dilated crypts lined by flattened epithelium, increased apoptotic figures and vanishing crypts. Notably there were some giant cells in between crypts. However subsequent sections and special stains didn't show any granuloma formation or presence of an infectious agent. Results: To our knowledge presence of giant cells in GVHD hasn't received proper attention before. Although we don't know the actual importance or clinical consequences of the entity yet, we report this case to increase awareness of this feature within the context of GVHD. We have seen more cases with giant cell GVHD afterwards; comparison with other cases of GVHD without giant cells may help clarifying their significance.
Differential mutation patterns of KRAS and BRAF in adenomatous and serrated neoplastic sequences O. Erdogan * , B. Savas, A. Ensari * Ankara University, Faculty of Medicine, Turkey Objective: Colorectal carcinomas develop through adenomatous or serrated neoplastic sequence. Different molecular mechanisms underly these morphologic sequences which may be translated into different therapies. We aimed to assess adenomatous and serrated neoplastic sequences in terms of KRAS and BRAF mutations. Method: The study group comprised of adenomatous polyps (APs, n=20, 13.6 %), conventional adenocarcinomas (ConCA, n = 30, 20.4 %), serrated polyps (SPs, n = 67, 45.6 %) and mucinous adenocarcinoma (MucCA, n=30, 20.4 %). KRAS mutation was assessed for exon 2 and exon 3 using pyrosequencing while BRAF V600E mutation was analysed using allele specific PCR. Chi square test was used for statistics. Results: KRAS mutations were observed in 41.5 % of carcinomas and 25.3 % of polyps. KRAS mutation rate was significantly higher (p<0.05) in ConCA (85 %) than in MucCA (50 %), and in APs (55 %) than in SPs (16.4 %). BRAF mutation was found in one MucCA (1.7 %) while all SPs were mutated. TSAs had the highest KRAS mutation rate (36.8 %) in comparison to SSA/Ps (17.6 %) (p<0.001) whereas SSA/Ps and HPs had significantly higher rates of BRAF mutation (64.7 %, 61.1 %, respectively) than TSAs (26.3 %) (p<0.001). Conclusion: KRAS seems to take part in adenomatous sequence while BRAF, seem to play a significant role in serrated neoplastic progression of the colorectum. Objective: Quiescent ulcerative colitis (Q-UC) is morphologically characterized by typical architectural and cellular mucosal changes that define the process as chronic. It is not clear which of these changes are more ubiquitous in Q-UC. Our aim was to evaluate which classical histological findings of Q-UC are more prevalent in rectal samples. Method: Rectal biopsies were collected from patients with clinical and endoscopical Q-UC. The biopsies were evaluated for mixed inflammation in the lamina propria, crypt architectural abnormalities, basal plasmacytosis, fibrosis, and Paneth cell metaplasia as well as for features of active disease (cryptitis, lamina propria neutrophils and erosion). Results: Forty-five patients (64 % female; median age 56 years) were included. Thirteen (28,9 %) biopsies showed focal activity and four cases (8,9 %) had criteria for active disease. Epithelial distortion, chronic inflammatory infiltrate and basal plasmacytosis emerged as features present in 95, 81 and 73 % of biopsies. Overall, 4 biopsies were histologically normal (no chronic features, no active disease). Conclusion: Rectal mucosa from patients with clinical and endoscopical Q-UC can show microscopic active disease, which illustrates that endoscopy alone may be insufficient to identify quiescent disease. In Q-UC, the most frequent changes (present in over 75 % of the biopsies) were epithelial distortion and chronic inflammatory infiltrate.
Vacuum-based preservation of colorectal cancer specimens: A comparison with formalin fixation J.-F. Fléjou * , S. El-Naderi, P. Cervera * Hôpital Saint-Antoine, Dept. de Anatomie Pathologique, Paris, France Objective: In Pathology, an alternative to immediate fixation in neutral buffered formalin (NBF) is vacuum sealing and cooling (VSC) ("Tissue safe" system). There have been few evaluations of VCS. Method: We assessed MSI, KRAS, BRAF in colorectal cancers, conserved with VCS before fixation (51 cases with surgery in a hospital distant from our centre), or immediately fixed in NBF (56 cases with surgery during the same period in our hospital). DNA was extracted from paraffin embedded tissue. MSI was assessed by MLH1 and MSH2 immunohistochemistry and PCR; KRAS and BRAF were screened by multiwell-plate based real-time PCR (LightCy-cler® 480, Roche), confirmed by Sanger sequencing. Results: There was no difference regarding morphological analysis. Immunohistochemistry was interpretable in all cases, with 4 negative cases in the VCS group (4 MLH1), 11 negative cases in the NBF group (10 MLH1, 1 MSH2), and a 100 % correlation between immunohistochemistry and PCR. DNA extraction was possible in all cases. KRAS and BRAF mutations were detected in 21 and 1 cases in the VCS group and 16 and 7 cases in the NBF group, respectively. Conclusion: We show that analysis of MSI, KRAS and BRAF is feasible in surgical specimens after VCS. This procedure can be an alternative to formalin fixation.
Clinical significance of CD204-positive M2 macrophage in colorectal cancer S. Fushimi * , M. Matsumoto, S. Takahashi, T. Ogino, J. Itakura, T. Ito, A. Matsukawa * Okayama University, Dept. of Pathology, Japan
Objective: Tumor-associated macrophages are divided into two phenotypes, termed M1/M2 macrophages. M1 macrophages promote tumoricidal responses whereas M2 macrophages assist tumor progression and metastasis. In this study, we have analyzed a clinical significance of M2 macrophages in excised sections from patients with colorectal cancer. Method: Patients who had surgical resection for colorectal cancer between 2005 and 2006 were identified from a prospective database. Tissue sections with adenocarcinoma in pT3 category were employed in this study. The sections were stained with anti-CD204, a marker of M2 macrophages. The relation between the staining pattern and tumor budding or patient prognosis was examined. Results: Fifty-two patients (30 male, 22 female) with a median age of 67 years (range 23-86) were studied. The tumors were classified into four subtypes according to the staining patterns of CD204-positive cells, i.e. sparse type (n=9), invading-tip type (n=6), peri-nest type (n=18), and dense diffuse type (n=19). Only a dense diffuse type was related to the high degree of tumor budding. The prognosis with a dense diffuse type demonstrated a poor prognosis as compared to the other types. Conclusion: Even the case with comparable tumor depth, CD204-positive macrophages with dense diffuse distribution was related to the poor prognosis. Objective: The treatment of locally advanced rectal cancer has shown a significant development over the last decade. The total mesorectal excision and the use of pre-operative radio (chemo)-therapy are leading the patients to a gradual improvement of local control of the disease. The aim of this study is to evaluate the pathological tumour response to pre-operative treatment through correlation between the clinical stage (cTNM) and the pathological stage (ypTNM). Method: Since 2009, 35 patients with locally advanced rectal cancer, assessed by high resolution magnetic resonance imaging (HR-MRI) and TNM staging system, have been evaluated by the Colorrectal Multidisciplinary Team. Pre-operative radio (chemo)-therapy was mandatory before undertaking standard surgery. Surgical specimens were evaluated according to the Vikingo Project's protocol implemented by The Spanish Association of Surgeons.
Results: 34 patients completed the pre-operatory treatment. After evaluation of clinical and surgical study specimens, we observed variable degree of downstaging in 27 (79,5 %) patients, 5 of whom showed complete response. The 7 (20,5 %) remainig patients were assessed as stable disease. No local progression of tumours was observed. Conclusion: In this study, approximately 80 % of patients, who were staged by HR-MRI and underwent preoperative radio (chemo)-therapy for locally advanced rectal cancer, showed some degree of downstaging. We remark the fact that nearly 15 % of patients reached complete response.
The density of macrophages in colorectal cancer is inversely correlated to TGF-BETA1 expression and patients' survival M. Gulubova * , I. Manolova, J. Ananiev * Trakia University, General and Clinical Pathology, Stara Zagora, Bulgaria
Objective: The role of macrophages in colorectal cancer tumorogenesis is complex because they can both prevent and promote tumor development. We investigated CD68 infiltration in tumor tissue and its correlations with proteins ot TGF-beta1 signaling pathway. Method: A nonselected panel of 206 primary tumors of colorectal origin was investigated immunohistochemically with antibodies against CD68, TGF-beta1, Smad4, Smad7, TGFRII and levels of TGF-beta1 were measured by ELISA. Results: Lower CD68 infiltration in tumor nests was associated with expression of TGF-beta1 (chi2 = 9.236, р = 0.002) and Smad4 (chi2=2.871, р=0.090) in tumor cells and with TGFRII expression (chi2=5.699, р=0.017) in tumor cells membranes. There was not correlation between CD68 cell numbers in tumor tissue and TGF-beta1 serum levels. We have found higher frequency of liver metastases in patients with lower infiltration with CD68 in invasive margin (chi2=11.364, р=0.001). The survival time was shorter for patients with low CD68 infiltration in tumor nests and invasive margin, compared with the survival time for patients, with higher CD68 infiltration in both tumor compartments (p<0.001). Conclusion: The increased levels of TGF-beta1 in the tumor have an immunosuppressive effect on CD68 infiltration.
Inflammatory myofibroblastic tumor of the colon: The 25th reported case S. Gurzu * , T. Bara, I. Jung * University of Targu Mures, Dept. of Pathology, Romania
Objective: Inflammatory myofibroblastic tumor (IMT) is a very rare tumor of the colon in which the diagnosis is especially based on immunohistochemistry. In the colon, we report the 25th case of IMT. Method: Case presentation Results: We report the case of a 56-year old male who presented with symptoms suggesting colon cancer. A 20 mm protruded tumor was endoscopically described in the ascending colon. Right hemicolectomy was performed. Macroscopically the tumor was well defined, covered by normal mucosa with a central depressed area, suggesting a GIST (Gastrointestinal Stromal Tumor). Microscopically spindle cells with fascicular arrangement admixed with mononuclear leukocytes were observed. The tumor cells expressed Vimentin, SMA, Desmin and ALK and were CD34 and CD117 negative. Intense angiogenesis and CD117 expression in the endothelial cells were also observed. Conclusion: In the IMT of the colon, clinical endoscopic and gross feature can imitate a carcinoma or a GIST. The IHC pattern of IMT can offer informations about its histogenesis. Based on the immunophenotype, we hypothesize that this tumor seems to occur from the the pluripotent stromal cells, CD34 positive which can be either differentiated in the interstitial cells of Cajal (telocytes), which are also CD34 positive or, during differentiation, can loss CD34 positivity. Objective: Determining the fraction of tumor cells in colon carcinoma samples analyzed for KRAS mutation is important for choosing the proper testing modality. However, when asked to determine tumor cell fraction in tissue samples, different pathologists give considerably different estimations, possibly leading to erroneous interpretation of KRAS mutation analysis results and poor treatment choices. Method: We developed a free, easy to use computer program that estimates tumor cell fraction on colon carcinoma slides that are immune-stained with anti-cytokeratin antibody. Sixty samples were evaluated by the program and results were compared to actual measurement of tumor fraction. Results: The tumor cell fraction estimated by the computer program showed highly significant correlation with the actual measurements (R=0.64, p<0.001). Additionally, we found that a short calibration step prior to beginning the computer estimation increased the accuracy of the results. In four cases (7 %) there was some discrepancy between the computer estimation and the actual measurements, however, this was attributed to lower quality immunohistochemical staining. Conclusion: In conclusion, we believe that this program can be used for standardizing the evaluation of tumor cell fraction in colon carcinoma, and that its use might aid in making better diagnosis and treatment choices for these patients.
Magnetic resonance imaging assisted tumour block selection in colorectal cancer C. Hunter * , G. Brown, L. Temple, M. Abulafi, A. Arnaout * Croydon University Hospital, Dept. of Colorectal Surgey, United Kingdom
Objective: Inadequate sampling may result in understaging in colorectal cancer. Fat clearance techniques and whole mount sections are time consuming and costly. We hypothesise that high resolution Magnetic Resonance Imaging (MRI) of the colorectal cancer specimen may aid histological sampling and result in upstaging. Method: Patients undergoing resection are prospectively recruited, and randomised to "conventional histology" or "MRI assisted histology". In addition to routinely selected tumour blocks, 4 additional blocks and any additional lymph nodes are selected with the aid of specimen MRI or visual inspection and palpation according to group. T stage, depth of extramural invasion, distance to resection margin, lymph node (LN) number and involved LN are compared between the two groups. Results: To date, 116 of 218 patients have been recruited. We present the methods and preliminary results. There is a trend towards greater T stage and LN yield. No difference in N stage, extra-mural vascular invasion or involved non-peritonealised resection margin has yet been observed. Conclusion: Early results suggest that specimen MRI may upstage colorectal cancer specimens by aiding tumour block selection. We will complete recruitment of 218 colorectal cancer patients over the next 12 months to determine whether observed differences are significant.
Diverticular disease of transverse colon is rare and perforation may occur in Crohns Disease C. Hunter * , A. Arnaout * Croydon University Hospital, Dept. of Colorectal Surgey, United Kingdom
Objective: Diverticular disease commonly affects the sigmoid colon in the West and the right colon in Asia. Perforation is uncommon affecting 4/100 000 per year. Perforated diverticular disease of the transverse colon is therefore very rare. We present a case of Crohns disease complicating transverse diverticular disease that lead to perforation and unfortunate death. Method: Radiological and histopathological features are presented with a review of the literature Results: To the best of our knowledge, this is the first reported case of Crohns disease complicating diverticular disease of the transverse colon. We suggest that distal stricturing leading to increased intraluminal pressure or increased weakening of the colonic wall due to coincident Crohns fissure and diverticulum are possible pathogenic mechanisms. Conclusion: Crohns disease complicating transverse diverticular disease is uncommon, but may be one pathogenesis leading to the rare event of transverse colon diverticular perforation. Results: There were 12 episodes in 9 patients. They start between day 7 and 499 (178±159 days). Duration ranged from 3 to 87 days (27±22). The three moderate and the three mild AR reversed with treatment. Of the 6 severe AR, 3 caused the failure of the graft. Five episodes de AR began as moderate or severe directly. Severity in the course of each episode was fluctuating. Sixteen indeterminate rejections were seen in 9 grafts (did not reach definitive criteria). They lasted 1-31 days (mean: 5±8 days). Conclusion: 1-Acute rejection is the most common cause of morbidity in the intestinal transplant (56 % of the grafts). 2-It can develop at any time and relapse. 3-The duration and severity of the episodes are fluctuating. 4-In our series, 75 % reversed, but severe rejection often lead to graft loss (50 %).
Histopathological study of 7 intestinal grafts lost in a series of 16 adult transplants. Hospital 12 Octubre. Madrid. Method: We studied 4 explants and 2 autopsy. The autopsy was not authorized in one death. Results: Seven grafts were lost in 6 patients (3 multivisceral). Two types of failure were seen. A) local: 3 severe acute rejections (AR) (days 77, 158, 511) and 1 lymphoproliferative syndrome (LPS) in day 148; B) systemic: 3 sepsis (days 31,88,161). AR showed 3 types of morphology: ulcerative, pseudomembranous and with disappearance of the villi preserving the crypts. LPS was a high-grade B lymphoma and affected the graft and the lymph mesenteric nodes with venous thrombosis that cause ischemic necrosis. The 3 sepsis were due to acinetobacter, to adenovirus in the graft and pulmonary aspergillosis and to pseudomona aeruginosa with necrohemorrhagic pancreatitis. Conclusion: 1-The incidence of IGL was 44 %. 2-The cause of IGL was local (AR and LPS) in 57 % and sepsis in 43 %. 3-Severe AR was the most frequent cause of failure. 4-Histological examination properly determine and document the causes of IGL.
Cap polyposis: Report of two cases R. Ivanova * , D. Kyoseva, G. Trifonov, R. Nikolov * Hospital of Endoclinology, Laboratory of Pathology, Sofia, Bulgaria
Objective: Cap polyposis is a rare colorectal disease characterized by mucoid, bloody diarrhea associated with multiple inflammatory polyps covered by a cap of fibrinopurulent mucous. The disorder was first described in 1985 and up to date a small number of cases has been reported in the literature. Method: We report two cases with multiple polyps of rectum and sigma, which were histologically diagnosed as cap polyposis. Results: The cases were two males on age of 18 years and 64 years. In both cases there was a history of mucoid and bloody diarrhea. On the basis of colonoscopy findings, in the young patient there was a broad differential diagnosis including M. Crohn, inflammatory pseudopolyps and Cronkhite-Canada syndrome. In the second case the clinical diagnosis was polyps of sigma. Endoscopic biopsies and polypectomy were done. In both cases, the histology showed typical histological features of cap polyposis -polypoid lesions containing elongated, tortuous and often distended crypts covered by a cap of inflammatory granulation tissue.
Conclusion: The recognition of this rare disease is of practical significance because its clinical symptoms have some similarity with inflammatory bowel disease or irritable bowel syndrome.
Histopathological findings in 496 consecutive appendectomies: A retrospective analysis J. Jeruc * , Z. Dolenc Stražar, V. Jovic, A. Cerar, N. Zidar * University of Ljubljana, Faculty of Medicine, Slovenia
Objective: Appendectomy is one of the most common surgical procedures. Histology usually confirms the clinical suspicion of acute inflammation, but sometimes other clinically relevant diagnoses are made. The aim of our study was to audit the appendectomies at our institution and summarise atypical pathological findings with emphasis on benign and malignant tumours. Method: We reviewed the histopathology results of 496 consecutive appendectomies received in a 5-year period. Results: Excluding three cases of metastatic process, appendiceal tumour was found in 21 cases (4.2 %), which is a higher rate than reported in the literature. There were four endocrine tumours, all G1, three cases of classical adenomas, 10 cases of low-grade appendiceal mucinous neoplasm (one associated with pseudomyxoma peritonei) and three cases of carcinoma (one mucinous adenocarcinomas, one intestinal type, one undifferentiated carcinoma). We also diagnosed one case of multicystic mesothelioma. Endometriosis was diagnosed in seven cases. In 18 cases appendicitis was associated with diverticula, in 6 cases changes were suggestive for Crohn's disease and in one case for cystic fibrosis. Negative appendectomy rate was 10.3 % that falls within the range reported in the literature. Conclusion: Histopathological examination of appendectomy specimens may reveal many different conditions not previously suspected; therefore, it should be performed in all cases. Results: CDX2, MUC1, MUC2, MUC5AC, and MUC6 expression was observed in 39.7 % (75 cases), 35.4 % (67 cases), 27.5 % (52 cases), 28.6 % (54 cases), and 16.9 % (32 cases), respectively. While SIAC patients with CDX2 expression showed less nodal metastasis (p=0.01), those with MUC1 expression tended to have SIACs with nodular or infiltrative growth (p = 0.003), poor differentiation (p = 0.005), and more lymphatic invasion (p=0.04). MUC5AC expression was associated with SIACs with well differentiation (p=0.02) and frequent pancreatic invasion (p=0.04). Patients with CDX2+/MUC1-had more polypoid (p=0.02) and well differentiated (p=0.006) tumors and a significantly better survival (median, 80.8 months, p<0.0001) than those with other immunophenotypes (CDX2−/MUC1+, CDX2+/ MUC1+, and CDX2−/MUC1−). Patients with CDX2 expression (median, 71.2 months) had a significantly better survival than those without CDX2 expression (23.0 months) by univariate and multivariate analyses (p<0.0001). Conclusion: CDX2 expression is an independent good predictor of survival in surgically resected SIAC patients.
Small neuroendocrine tumor of appendix with metastasis in the ileocecal lymphnode: A case report G. Kalan * , S. Tušar, N. Zidar * General Hospital Jesenice, Dept. of Pathology, Slovenia
Objective: Neuroendocrine tumors (NET) of appendix are believed to behave less aggressively than NETs at other sites. Separate staging criteria have therefore been proposed, and tumor size >2 cm appears to be the dominant criterion for aggressive behavior in appendiceal NETs. However, it is controversial whether radical surgery is indicated in tumors measuring >2 cm. We report a case of a 13-year-old girl with acute appendicitis in whom appendectomy and right-sided hemicolectomy were performed. Method: Surgical specimens were sampled and processed according to standard histological and immunohistochemical (IHK) procedures. Results: In the base of appendix, there was a tumor measuring 1.5 cm in diameter, focally invading mesoappendix, with free resection margins. Microscopically, tumor exhibited characteristic features of NET G1, with positive IHK for chromogranine and synaptophysin, and Ki67 index <2 %. IHK for podoplanin revealed focal lymphovascular invasion. In the right-sided hemicolectomy specimen, there was no residual tumor, metastasis was found in one ilecoecal lymphnode. Conclusion: Our case shows that appendiceal NET smaller than 2 cm can metastasize to regional lymphnodes. IHK against podoplanin might be helpful in searching for lymphovascular invasion, helping to separate it from the retraction clefts, thus providing additional information concerning risk factors for a more aggressive behavior.
Lipomatosis of ileocaecal valve causing small bowel obstruction mimicking Crohn´s Disease M. Kalman * , P. Szépe, J. Marcinek, T. Balhárek, L. Plank * University Hospital Martin, Dept. of Pathology, Slovakia
Objective: Lipomatosis of ileocaecal valve is a rare cause of small bowel obstruction. Method: We describe a 41-year-old male treated for Crohnś disease and clinical and imaging presentation of chronic small bowel stenosis and obstruction. Results: A right hemicolectomy was performed, and hypertrophy of Bauhin´s valve causing intestinal obstruction was found. Microscopical examination of the specimen revealed morphological changes consistent with lipomatosis of the ileocaecal valve. The ileum and colon were macroscopicaly unremarkable and histomorphological features of Crohn´s disease were absent. Conclusion: Many patients with lipomatosis of the ileocaecal valve are asymptomatic, or the lipomatosis gives causes insignificant symptoms only or rarely obstruction may occur. Then, as in the presented case, the surgical resection with ileo-colic anastomosis is the only effective treatment.
Overexpression of CXCR4 in tumor buds is a strong predictor of lymphatic invasion and lymph node metastasis in colorectal cancer E. Karamitopoulou-Diamantis * , D. Kassahn, I. Zlobec, V. Koelzer, H. Dawson, A. Lugli * Universität Bern, Inst. für Pathologie, Switzerland
Objective: CXC chemokines enhance tumor cell survival and proliferation. Especially CXCR4 promotes tumor development by stimulating angiogenesis and favoring metastasis. In contrast, CXCR3 is angiostatic and may exert an anti-tumor effect. Since tumor budding is linked to vascular/lymphatic invasion and lymph node (LN) and/or distant metastasis in colorectal cancer (CRC), here we explored the expression of CXCR3+4 in relation to tumor budding. Method: A multiple-punch tissue microarray of 220 CRCs with full clinicopathological information including therapy underwent immunohistochemistry for CXCR3 and CXCR4. Expression was evaluated in tumor-center, -front and -buds and correlated to clinical data. Results: CXCR3-expression was homogeneous throughout tumor-center, -front and -buds (average 40 %) and was unrelated to clinicopathological features or survival. CXCR4-expression was high in tumor-center and -front but reduced (p<0.001) in buds (76 %, 77 %, and 20 %, respectively). Maintenace of CXCR4-expression within buds was predictive of LN-positivity (p<0.0001) and lymphatic invasion (p<0.0001) but not of venous invasion or distant metastasis. CXCR4-positivity in buds was associated to poor outcome (p=0.0048) in LN-negative CRCs (p=0.0191). Conclusion: Maintenance of CXCR4-expression in buds has a profound effect on tumor aggressiveness and prognosis in LN-negative CRC-patients. This would help in establishing a tumor budding "profile" especially linked to LNmetastasis in CRC. Objective: COX-2 expression was investigated in colorectal cancer and colorectal precancerous lesions. Its expression in adenomas is associated with increasing size and neoplastic potential. Little is known about COX-2 expression in subtypes of serrated colonic polyps, which constitute substantial number of colon cancer precursors. The aim of the study was to assess COX-2 expression in serrated polyps of the colon and to investigate its potential discriminative role. Method: 175 consecutive serrated polyps were analyzed. They included: 26 traditional serrated adenomas (TSA), 36 sessile serrated polyps (SSP) and 113 hyperplastic polyps (HP). COX-2 expression was assessed semi-quantitatively (0-3). Kruskall-Wallis test was used for the comparison of COX-2 expression between serrated categories (α=0.05). Objective: In colorectal cancer (CRC), tumor buds represent a more aggressive tumor cell type at the invasive front with apparently low proliferation. The aim of this study was to determine the proliferation potential of tumor buds by comparing Ki67 staining across different tumor areas and adjacent normal tissue. Method: Whole tissue sections from 197 patients with CRC underwent immunohistochemistry for Ki67. 10 high-powerfields were evaluated for each of the following regions: normal mucosa, tumor center, tumor front and tumor buds. Ki67-positivity was correlated to patient outcome. Results: Average Ki67-positivity was 5.2 % in normal mucosa and significantly higher in the tumor center (38.2 %; p < 0.0001) and invasion front (34.9 %; p < 0.0001). Strikingly, only 0.3 % of all tumor buds showed Ki67-positivity (p<0.0001). Although Ki67-positivity in the tumor center or front was unassociated with clinicopathological features or patient survival, the greater the number of Ki67-positive tumor buds, the worse the prognosis. This effect was also found after adjusting from TNM stage (HR (95 % CI): 2.25 (1.1-4.5); p=0.0193)). Conclusion: A marked absence of Ki67 staining is found in most tumor buds, suggesting a substantially decreased proliferation rate. However, the association of Ki67-positivity with worse prognosis in 15 % of cases points towards a heterogeneous population of tumor buds.
Kayexalate and intestinal necrosis: An underrecognised entity P. Luís * , A. Alves, C. Ferreira * Hospital de Santa Maria, Dept. de Anatomia Patológica, Lisboa, Portugal
Objective: There are only few cases of intestinal necrosis secondary to administration of kayexalate described in the literature, most of them from postoperative patients, having the largest study only 11 patients in 9 years. We report our experience in the last 4 years with a brief review of the pathophysiology and histologic aspects of the lesions induced by the kayexalate. Method: Between 2008 and March 2012 seven patients were diagnosed with ischemic necrosis related to kayexalate administration, five in colon biopsy and two in resection specimens. They're comorbidities and clinical evolution was extracted from the medical records. Results: The patients' age ranged between 47 and 78 years. In one case we had the clinical information of chronic renal insufficiency. In three of them, there was no previous history of renal impairment. One patient developed symptoms after one single administration of kayexalate. The clinical presentation ranged from abdominal pain, gastrointestinal bleeding and three of them had a fatal outcome.
The administration of kayexalate can have associated risks with different clinical impact in any patient. We must be aware of the related complications, as the histological identification of the kayexalate crystals indicates the etiology of the ischemic lesions to the clinicians and alerts them to the dangers of this therapeutic. Objective: EGFR plays an important role in colorectal cancer (CRC) progression and represents a natural target for molecular anticancer drugs. In this study we evaluated 800 consecutive CRC aimed to analyze K-Ras mutational status in relationship to: different clinical-pathological parameters, EGFR immunohistochemical (IHC) expression, response to cetuximab treatment. Method: K-Ras mutations and EGFR expression were determined by direct cycle sequencing and IHC respectively in 565 surgical specimens and 235 biopsies. IHC findings were evaluated using four different score systems. Results: The distribution of K-Ras mutations did not significantly vary between surgical or bioptic specimens or with respect to different anatomic tumor localization. In contrast, K-Ras is more frequently mutated in EGFR negative/low score tumors than in positive ones (p<0.0001). Focusing on the 419 surgical treated CRC patients, we found a higher percentage of K-Ras mutations in T4 CRC (p=0.01) and in younger patients (p=0.002). Finally, we observed that 6 % of primary CRC, concomitantly evaluated with their paired metastases, changed K-Ras mutational status during progression. Conclusion: Our data showed that tumor size, patient age and EGFR IHC expression significantly influenced K-Ras mutations. Interestingly, we observed that cetuximab treated patients, had a better clinical outcome when EGFR presented a high IHC score.
Multiple Immunohistochemical investigation of signaling pathways in colorectal cancer T. Micsik * , L. Kopper, T. Krenács, Z. Nagy, O. Horváth * Semmelweis University Budapest, 1st Department of Pathology, Hungary
Objective: Despite the recent advances in therapeutic armamnet colorectal cancer (CRC) remains one of the leading cancer deaths worlwide. CRC has a heterogenous molecular background with known prognostic and predictive markers, but the whole picture is rather complex with many unexplored connections. Method: We performed multiple immunohistochemical stainings on tissue microarrays (TMA) made of 95 CRC cases to study the correlations between EGFR-RAS, cell cycling, apoptosis, cell adhesion and tumor invasion pathways. We evaluated digitized slides using matrix scores considering both the frequency and positivity of cells and statistical analysis followed. Results: We found strong linear correlation between positivity and (p-1068, p-1173) phosphorylation of the EGFR receptor and p53 positivity detected with different clones. Furthermore, phospho-NF-KB p65 positivity was strongly linked to Survivin-expression. Conclusion: Activation of EGFR-related pathways including NF-kB is a key factor in CRC-growth. The strong link between cancer promoting EGFR signaling and the mutational lack of the p53 driven apoptosis suggests a deadly cooperation, which can be further aggravated by forced tumor survival through survivin. Correlation of these markers potentially predict poor disease outcome but can be specifically targeted by the upcoming new molecules of tailored therapy. Objective: KRAS mutational status is important in colorectal carcinoma (CRC). It is unclear, however, which is the most informative tissue source for study in CRC cases showing more than one tumor mass. To explore this issue we determined KRAS status in a series of primary (PT) and metastatic CRCs.
Method: A total of 68 tumors belonging to 25 patients (17 males and 8 females) with ages ranging between 46 and 80 years (average, 64 years) were studied. KRAS mutation hotspots in codon 12 and 13 were analyzed by polymerase chain reaction and sequentiation. wt-KRAS cases were confirmed with the KRAS StripAssay TM. Objective: Lynch syndrome (LS), the most frequent form of hereditary colorectal cancer (CRC), is caused by germline mutations in the mismatch repair system genes. A recently identified mechanism involving the EPCAM gene is responsible for 6.3 % of MSH2-negative LS cases. We herein explore EPCAM protein expression in LS-associated, MSH2-negative CRCs to evaluate its potential value in the algorithmic approach to LS population screening. Method: We studied a total of 19 MSH2-negative CRCs from 14 different patients in whom we were able to perform a complete germline analysis. Expression of both MSH2 (1:200 dilution, clone G219-1129, Pharmingen) and EPCAM (1:100 dilution, clone Ber-EP4, Dako) was evaluated by immunohistochemistry (IHC). Results: Nine patients showed a deleterious germline mutation that involved the MSH2 gene in three instances and the EPCAM gene exon 9 in six instances. All patients harbouring the EPCAM mutation belonged to the same family. Of the19 CRCs, EPCAM expression loss was seen in only five, all of them were from patients with a germline EPCAM deletion. Conclusion: Due to the high specificity of EPCAM protein loss for identifying LS patients with an EPCAM deletion, we recommend adding EPCAM IHC to the LS diagnostic algorithm in MSH2-negative CRC cases. Objective: Colorectal carcinoma with signet ring cell component (CRC-SRC) is a rare and distinct subtype with little molecular information. We investigated the frequency of BRAF mutation in 28 CRC-SRCs and its relation with clinicopathologic parameters. Method: We categorized tumors into groups 0-9 %, 10-24 %, 25-49 % and >50 % according to signet ring cell component. Genomic DNA was isolated from parafin blocks and analyzed for BRAF V600E mutation by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Results: Eleven of cases showed BRAF mutation (39.3 %). The results were also confirmed with sequence. No statistically significant differences were found in clinicopatologic parameters between BRAF wild-type and mutant CRC-SRC. On the other hand, when we adjusted age, gender, percentage of signet ring compenent and stage, we found a statistically significant increased risk in BRAF mutant group compared to BRAF wild-type in Cox regression analysis (HR=7.68, 95 % CI=1.06-55.78, p=0,044) Conclusion: BRAF mutation is frequent in CRC-SRCs. This finding may support its diverse molecular pathogenesis and could have important therapeutic implications for those patients. To clarify the relation of BRAF mutation with clinicopathologic parameters and prognosis in CRC-SRC, studies with multi-instutional larger series are needed.
Coexisting lipomas and adenocarcinoma of the colon: Case report G. Orgun Sonmez * , U. Bayol, O. Akman, S. Cumurcu, M. Olmez * Tepecik Research and Training Hospital, Izmir, Turkey
Objective: Lipomas of the large bowel are rare, they are invariably submucosal and therefore may intussuscept. Infrequently lipomas may also present in the form of multiple polypoid masses in the colon. We present a 69-years old female with 3 submucosal lipomas and a coexisting adenocarcinoma of transverse colon. Method: 69-years old female patient was admitted to hospital because of rectal bleeding for 3 months. Colonoscopic examination revealed mucosaly intact polypoid tumoral masses and an ulcerated tumoral mass in transverse colon. Biopsy of the ulcerated mass was diagnosed as adenocarcinoma. Extended right hemicolectomy was performed. Results: Grossly, ulcerated tumor was 1,5×1×0,4 cm and there were 3 polypoid tumors in cecal area of the right colon which were 2.5-2.0-1.5 cm in their greatest dimensions. Polypoid tumors were yellow, lipomatous on cut surfaces. Microscopically ulcerated tumor was composed of adenocarcinoma morphology and the other polypoid tumors were composed of submucosal mature adipocytes with a thin capsule around them. The diagnosis was colonic adenocarcinoma (pT1N0M0) and submucosal lipomas of colon. The patient had no additional therapy and she is healthy with no evidence of recurrence. Conclusion: This case is presented to remind that there may be multiple lipomas of colon with coexisting malignant epithelial tumors. Objective: Primary liposarcoma of the small intestine is exceedingly rare. To our knowledge, only 7 cases have been reported so far. We report a case of the primary myxoid liposarcoma of the ileum. Method: The patient was 77-year-old woman. CT and MRI revealed a 6-cm-intrapelvic mass, which was proved to be a tumor of the distal part of the ileum, and, therefore, partial ileectomy was performed. Results: The tumor was 6.7×6.3×3.7 cm in size, relatively well circumscribed and located within the intestinal wall without invasion to the neighboring tissues. Microscopically, the tumor had mixture of cellular and myxoid areas, showing proliferation of short spindle and stellate shaped cells in a delicate capillary vasculature. Univacuolated tumor cells and a small number of lipoblast-like cells were also seen. Immunohistochemically, these proliferating cells were positive for S-100 protein. According to these pathological findings, we diagnosed the tumor as myxoid liposarcoma. FISH methods showed the tumor had chromosomal translocation t(12;16)(q13;p11). Conclusion: The differential diagnosis of this includes other spindle cell tumors such as GIST and myogenic tumors. But we could arrive at the proper pathological diansosis following the above morphological features and the specific chromosomal translocation. We will also discuss the clinico-pathological features of the cases including ours.
Prognostic significance of selected immunohistochemical markers in colorectal cancer E. Paltseva * , O. Samofalova, Y. Gorbacheva, P. Tsarkov * First Moscow State Medical University, Pathology, Russia
Objective: The aim of our study was to identify a combination of markers whose expression is predictive of invasion and metastasis of colorectal carcinoma. Method: The expression of E-cadherin, β-catenin, tenascin C, KAI-1, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) was studied immunohistochemically and correlated clinicopathologically in 72 primary colorectal carcinoma cases (26 with metastatic lymph nodes). 17 patients (24 %) developed haematogenous metastases at median 6,5 months postoperatively (3-11). Results: Expression of following markers significantly correlated with depth of tumour invasion: cytoplasmic accumulation of E-cadherin (p=0,035), nuclear staining for β-catenin (p=0,03), increased tenascin C expression (p=0,001), and loss of EGFR and VEGF expression (p=0,015 and 0,02, respectively). Decreased tenascin C and KAI-1 expression associated with the presence of lymph node metastases (p= 0,027 and 0,046, respectively). Weak cytoplasmic E-cadherin expression correlated with distant metastases (p=0,045). Conclusion: Our results suggest that abnormal expression of proteins involved in cell adhesion and migration (E-cadherin, βcatenin, tenascin C, EGFR) and angiogenesis (VEGF) may be related to the invasion of colorectal carcinoma. Detecting the expression of E-cadherin, tenascin C and KAI-1 probably possesses clinical significance in evaluating lymph node and distant metastasis and predicting the prognosis of colorectal cancer. Objective: In colorectal adenocarcinomas (CRCs) microsatellite instability (MSI) can be documented by immunohistochemical detection of mismatch repair proteins (MMR). The aim of the present study is to identify the percentage of MSI-positive CRCs, in the Epirus region of Greece and compare it to the international data. Method: A total of 50 cases of sporadic CRCs, 25 adenomas and 10 hyperplastic polyps, formalin-fixed paraffinembedded, were examined immunohistochemically using standard methods (En Vision system), with the antibodies MLH1 (Clone ES05), MSH6 (Clone PU29) and PMS2 (Clone M0R4G) (Novocastra-Menarini). Immunostained sections were evaluated semi-quantitatively, taking in account the percentage of neoplastic positive cells and estimating nuclear staining intensity, compared to the internal positive controls (normal epithelium, lymphocytes). Results: All hyperplastic polyps and adenomas expressed the examined MMR. Two out of 50 CRCs were negative for MSH6 and PMS2, while one of them was also negative for MLH1. Interestingly, one of these two CRCs had an adjacent adenoma, which also showed negative staining for MSH6 and PMS2. Conclusion: Immunohistochemical staining for MMR is possible in archival material. Loss of MMR varies, depending on the protein examined. In CRCs from the Epirus region the estimated loss of at least two MMR is 4 %. Objective: Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all extracellular matrix (ECM) components. Genetic alterations in MMP-2 and MMP-9 have been implicated to play an important role in colorectal carcinogenesis (CRC). The aim of this study was to investigate the hypothetic correlations between the MMP-2 and 9 mRNA expression and the clinicopathologic variables of the CRC. Method: The expression of MMP-2 and -9 mRNA was assayed by RT-PCR for 53 Tunisian sporadic colorectal adenocarcinomas. Results: MMP-2 was highly expressed in the 53 cases. MMP-9 was not detected in 18 cases and moderately or highly expressed in 35 cases. Absence of MMP-9 mRNA expression was significantly related to an advanced tumoral invasion stage (pT3) (p=0.003). Conclusion: Our study suggests that the overexpression of MMP-2 could be the result of an imbalance in the system of transcriptional regulation of MMP-2, which would involve the loss of expression or activity of its inhibitor: TIMP-2. A variable expression of transcripts of MMP-9 could be explained by the hypothesis of hypermethylation of the promoter or degradation of its transcript after protein translation.
Relationship between matrix metalloproteinases 2 and 9 promoter polymorphisms and colorectal cancer risk Objective: The carcinogenic schema of colorectal cancer (CRC) follows a multistep process governed by mutational events that affect multiple micro-environmental factors, including matrix metalloproteinases (MMPs). This present work aims to contribute to a better understanding of MMP-2 and -9 promoter polymorphisms involvements in the genesis and progression of sporadic CRC in Tunisian population. Method: The MMP-2 and MMP-9 promoter genotypes were determined by PCR-RFLP in 53 Tunisian patients with sporadic colorectal cancer and 69 healthy control subjects. Results: No significant associations were found regarding MMP-2 (-1306 C/T) polymorphism and CRC susceptibility. There was a connection between the MMP-2-1306 promoter polymorphism and gender (p = 0.012). The MMP-9 (−1562 C/T) was significantly associated with CRC (OR= 2.39 [1.023 to 5.58]). No implication of MMP-9-1562 promoter polymorphism in the clinicopathological parameters of CRC was evident. Conclusion: Our findings suggest that the MMP-2 promoter polymorphism was not a predictive factor for the CRC occurrence while the MMP-9-1562 CT genotype was a risk factor for CRC susceptibility.
Hodgkin's lymphoma in distal rectum in a patient with Crohn´Disease treated by biologic therapy -A case report R. Sampaio * , F. Costa, E. Diane, J. Palla Garcia, J. R. Vizcaino * Anatomia Patológica, Valpaços, Portugal
Objective: Rare cases of Hodgkin`s lymphoma have been reported in the setting of inflammatory bowel disease. Method: We report a case of Hodgkin`s lymphoma in a 37 years old male patient with corticosteroid-dependent Crohn´Disease (CD) who had been medicated with azathioprine for many years. In the last 4 years he began periodical therapy with infliximab, till the time when he complained of worsening of symptoms and the appearance of asthenia, tenesmus and weight loss. Infliximab was withdrawn and replaced by adalimumab. A colonoscopy with several biopsies revealed the presence of a Hodgkin Lymphoma. Results: The histological examination revealed granulation tissues where we identified atypical cells (Reed-Sternberg cells) with the following immunohistological staining: CD30+, CD15+, LMP1 + LCA-, CD20-, CD3-, ALK-. These results in conjunction with the morphological aspects and the clinical history of CD favored the diagnosis of Hodgkin Lymphoma in a patient with preceding CD. The patient was subjected to radical colectomy. Postoperative pathological examination showed CD with involvement of the distal rectum by Hodgkin's lymphoma. Conclusion: The present report may serve as a reminder to clinicians of the possibility of occult lymphoma in patients with colonic CD treated with biological therapy. Objective: DNA mismatch repair (MMR) status in sporadic colonic cancers (CCs) had provided valuable prognostic and predictive information in the appropriate clinical setting; however there are no definitive guidelines for microsatellite instability (MSI) testing. We evaluated the expression of MMR proteins and correlated them with outcome in a cohort of patients with sporadic colon cancer. Method: We reviewed 352 cases of sporadic stage II colonic cancer from Abril 27th 1995 to December 12th 2009. Clinical data including tumor grade, prognostic factors, therapy and outcome were searched for. Tissue microarrays using a manual Beecher system were constructed and stained with antibodies for MLH1, MSH2, PMS2 and MSH6. Results: 8 % (28) of 352 CCs had loss of expression of one or more MMR proteins, including defect in 1 protein 3.7 % (13), 2 proteins 3,7 % (13) and tree proteins 0,6 %(2). The mean overall survival was 144 months with no significant differences in patients between microsatellite stable and unstable tumors. Conclusion: We detected a fairly low prevalence of the loss of immunohistochemical expression of MMR proteins. This result might have influence our mean overall survival between microsatellite stable and unstable tumors.
The pathological effects of orchidectomy on intestine tissue and serum level of carcino embryogenic antigen S. Sattari * , R. Ahmadi * Azad University of Iran, Science Dept., Hamedan, Iran Objective: Carcino Embryogenic Antigen (CEA) is a wellknown tumor marker influenced by carcinogenic changes in intestine tissue. The purpose of this study was to determine the effects of orchidectomy on serum level of CEA and histological changes of intestine in male rats. Method: Male Wistar rats were randomly divided to control, sham, uni-orchidetomised and biorchidectomised groups of 5 in each. 10 weeks after operation, blood samples were collected using cardiac puncture method. Serum CEA level was measured using ELISA method. The effect of orchidectomy on intestine tissue was also histologically studied. The results were statistically analyzed using ANOVA. Results: Serum level of CEA was significantly increased in orchidectomised rats compared with control animals (P< 0.001). The increase in serum CEA level was more in biorchidectomised than uniorchidectomised rats (P<0.001).
There was also an enhancement in tissue lymphocytes, plasma cells and inflammation in intestine of orchidectomised animals.
The results showed that orchidectomy results in pathological changes in intestine tissue leading to increased serum CEA level.
The plasma cells density in pararectal lymph nodes of patients with rectal cancer after neoadjuvant therapy M. Sezak * , N. Ozsan, B. Pehlivanoglu, S. Ozkok, T. Yoldas, B. Daganavsargil * Ege University, Dept. of Pathology, Izmir, Turkey
Objective: Neoadjuvant chemoradiotherapy (NT) is standard procedure for locally advanced rectal cancer. In this study, we compared the morphology of lymph nodes and the amount of plasma cells in the interfollicular region of lymph nodes in rectal tumour surgical excision material, between two groups who had received and not received NT prior to surgery. Method: Fifty cases with NT and forty cases without NT were included. The number and diameter of lymph nodes were recorded. The morphology of the lymph nodes were evaluated, and the percentages of interfollicular plasma cells were demonstrated immunohistochemically (CD138). Tumor regression grade was assessed using Mandard scoring system. Results: Average number and diameter of lymph nodes were greater in patients without NT (p<0,05). Atrophy of germinal centers was noted as 54 % in NT group, versus 5 %. The amount of plasma cells in the interfollicular region was found to be higher than fifty-one percent in 54 % of NT group versus 5 % (p<0,05), as well. Complete response rate (Mandard's: 1) was 18 %. No significant association was found between the amount of plasma cells and tumor regression. Conclusion: NT leads to a decrease in the volume of the lymph nodes and atrophy of germinal centers, conversely, causes stimulation of the proliferation of interfollicular plasma cells.
Adenomyoma of the ileum: Pathohistological features with reference to pathogenesis B. Snezana * , B. Andrejic, N. Solajic * Clinical Center of Vojvodina, Center for Pathology, Novi Sad, Serbia
Objective: Adenomyoma is a rare benign non-neoplastic tumor-like lesion. It originates from abnormal embryonic buds but its pathogenesis has not been fully elucidated. Method: A 63-year old man with severe pain, intussusception and ileus underwent a resection of a part of the intestine containing a tumor mass acting as a lead point. Tissue was analysed using H&E, histochemical and immunohistochemical stains. Results: Lesion located in the mucosa, submucosa and muscularis propria consisted of glandular structures varying in size and morphology, the larger ones containing papillary projections and the smaller ones being similar to Brunner's and peribiliary glands, both of them surrounded by smooth muscle bundles. The lesion was not accompanied by ectopic pancreas, and it indicated a diagnosis of adenomyoma of the ileum. Glandular elements were CK7+ and CK20− and CDX-2−, opposite to intestinal mucosa. Conclusion: Adenomyoma is a mass lesion rarely found distal to the duodenum, which bares close clinical and morfological resemblance to a tumor and some non-neoplastic conditions. Cytokeratin expression favors the heterotopic pancreas theory of pathogenesis, but abnormal interaction between the endoderm-and mesoderm-derived tissues can not be excluded.
Smoothelin in biopsies of colorectal carcinomas as marker of muscularis propria invasion P. Stoemmer * , P. Torres-Galea * Gemeinschaftspraxis Pathologie, Forschungslabor, Augsburg, Germany
Objective: Staging of colorectal carcinomas is of eminent importance for prognostication and treatment; preoperative distinction between high grade dysplasia, invasion of submucosa (with permigration of the muscularis mucosae MM) and infiltration of muscularis propria (MP) is difficult in biopsies, due to similarities of smooth muscle cells in MM and MP. Smoothelin is a robust marker of the MP in normal colon, not expressed in MM.We analysed it value for distinguishing MP and MM near carcinomas. Objective: Tuberculosis can affect any part of the gastrointestinal tract. Anal localization, in particular, has a very low incidence (0,7 %). Method: A 66 year-old male presented, complaining of perianal pain, constipation, weight loss of 5 kilos the last 4 months and night sweating. Physical examination was normal, except for an ulcerated lesion in the anal region. The patient was submitted to computerized tomography (CT) and incisional biopsy of the lesion. Results: The CT of the chest revealed multiple, scattered, calcified nodules in the upper zones of both lungs and histological examination showed confluent, non-necrotizing, epithelioid granulomas with the presence of Langhans' multinucleated giant cells. Acid-fast bacilli were detected both within the anal lesion and in the sputum. Mantoux test was positive. The patient had a history of pulmonary tuberculosis 40 years ago. A diagnosis of anal tuberculosis was made and the patient was put on a four drug anti-tuberculous regimen. In 6 months the symptoms improved and the perianal lesion healed. Conclusion: A tuberculous origin must be considered when the cause of anal and perianal lesions is unclear. Therefore histological and bacteriological confirmation is essential in order to avoid undue delay in diagnosis and treatment.
The relation of BRAF V600E Mutation and microsatellite instability in colorectal carcinomas I. Turkmen * , N. Bassüllü, B. Toptas, T. Öztürk, R. Yasar, P. Korkmaz, N. Saygili, O. Öztürk, G. Demir, G. Dogusoy * Istanbul Bilim University, Dept. of Pathology, Istanbul, Turkey
Objective: Colorectal carcinogenesis is associated with various morphological and molecular pathways. Microsatellite instability (MSI) pathway has familial and sporadic forms.
Among CRC's %15 of the sporadic cases are associated with MSI and these have hypermethylation of the MLH1 promoter associated with BRAF V600E gene mutation. Familial forms do not show BRAF V600E mutation. Method: We investigated the microsatellite instability (MSI) by immunohistochemical study of MLH1, PMS2, MSH2 and MSH6 together with the BRAF mutation presence and their correlation with standard histopathological parameters in 54 sporadic CRC cases. Results: The age distribution was 25-88 with an average of 67,9. Immunohistochemical expression loss of MLH1, PMS2, MSH2 and MSH6 was seen in 18,5 %, 22,2 %, 7,4 % and 11,1 % of the total 54 cases respectively. MSI was seen in 27,8 % of the cases. The BRAF V600E gene mutation was seen in 18 cases and among these 8 cases (44,4 %) showed MSI, while MSI frequency was 19,4 in non-mutated group. And this was statistically significant (p < 0.05). MSI was found significantly correlated with grade and lymph node number (p<0.05). Conclusion: Our study confirmed that sporadic cases of CRC with MSI are associated with the BRAF V600E gene mutation. MSI is correlated with histologic grade and dissected lymph node number.
Colon biopsy diagnostics may reliably be performed using virtual microscopy Objective: Virtual microscopy using whole slide images (WSIs) is a feasible alternative to optical microscopy, offering major advantages for pathology practice. The present study aims to prove reliability of this promising technique for colon biopsy diagnostics. Method: Colon biopsies (n=295) were assessed (into 7 main diagnostic groups) using both glass slides and WSIs by 4 pathologists and 2 residents. Two of the pathologists having ample experience using WSIs, scored the biopsies in a primary diagnostic setting. For each case the criterion standard diagnosis was defined based on glass slide diagnoses. Accuracy was defined as the percentage of concordance with the criterion standard. Kappa statistics were calculated as a measure of observer agreement. Results: The overall concordance rates were 89.5 % for WSIs and 91.4 % for conventional microscopy. The intraobserver (WSIs versus glass slides) agreement was good to excellent, with kappa values ranging from 0.73 to 0.87 (mean 0.78) and was higher than the interobserver agreement for glass slides (mean 0.71). Concordance with the criterion standard varied less between WSIs and glass slides in the diagnoses of pathologists with virtual experience. Conclusion: This study showed good diagnostic accuracy and reproducibility for WSIs, indicating this technology may be used for colon biopsy diagnostics.
Intestinal occlusion due to colonic lipoma: A case report F. Vukmirovic * * Clinical Center of Montenegro, Dept. of Pathology, Podgorica, Montenegro
Objective: Lipomas of the digestive tract are rare benign tumor and most often found incidentally during a colonoscopy, computed tomography scan, surgery or autopsy. Lipomas of the colon were first reported by Bauer in 1757 and are most often located in the ascending colon. The incidence of this lesion is estimated between 0.2 and 4.4 % and represents 1.8 % of the colonic benign lesions. We report a case of patient with symptoms of ileus due to colonic lipoma. Method: A 78-year-old man patient was urgently admitted with symptoms of ileus. Right hemicolectomy was performed, and clinical impression was that it was a malignant tumor. Results: Examination of the cecum we found polypoid whitish yellow tumor, size 4×2,5 cm. On histology the tumor was composed of mature adipose tissue without cellular atypia. The tumor was located into submucosa. Larger parts of the surface mucosa was eroded. The postoperative course was uneventful and intestinal passage was quickly established. Conclusion: Although colonic lipomas seldom cause severe symptoms in patients and are easily removed by endoscope while they are small, severe symptoms like abdominal fullness, intestinal obstruction, intestinal bleeding and intussusceptions may appear as a lipoma grows larger. In some cases lipoma may clinically mimic colonic carcinoma.
Prognostic impact of lymph node ratio outperforms positive lymph nodes and lymph nodes harvested: A time-dependent analysis in mismatch repair-proficient and -deficient colorectal cancers I. Zlobec * , E. Karamitopoulou, L. Terracciano, D. Inderbitzin, A. Lugli * University of Berne, Inst. of Pathology, Bern, Switzerland Objective: We compare the prognostic strength of the lymph node ratio (LNR), positive lymph nodes (+LNs) and collected lymph nodes (LNcoll) using a time-dependent analysis in colorectal cancer patients stratified by mismatch repair (MMR) status. Method: 580 stage III-IV patients were included. Multivariable Cox regression analysis and time-dependent receiver operating characteristic (tROC) curve analysis were performed. The Area under the Curve (AUC) over time was compared for the three features. Results were validated on a second cohort of 105 stage III-IV patients. Results: The AUC for the LNR was 0.71 and outperformed + LNs and LNcoll by 10-15 % in both MMR-proficient and -deficient cancers. LNR and + LNs were both significant (p <0.0001) in multivariable analysis but the effect was considerably stronger for the LNR [LNR: HR=5.18 (95 % CI: 3.5-7.6); +LNs=1.06 (95 % CI: 1.04-1.08)]. Similar results were obtained for patients with >12 LNcoll. An optimal cutoff score for LNR = 0.231 was validated on the second cohort (p<0.001). Conclusion: The LNR outperforms the + LNs and LNcoll even in patients with >12 LNcoll. Its clinical value is not confounded by MMR status. A cut-of score of 0.231 may best stratify patients into prognostic subgroups and could be a basis for the future prospective analysis of the LNR.
Prognostic value of tumor-stroma ratio in rectal adenocarcinomas S. Zoidze * , R. Scheer, J. Klaase, M. Elferink, A. Baidoshvili * Laboratorium Pathologie Oost, Enschede, Netherlands
Objective: Recently, tumor-stroma ratio (TSR) has been identified as a strong predictor for survival in colorectal cancer. Despite an identical biology clinical implications are quite different for colon and rectal cancer regarding to anatomical differences. Method: TSR was estimated on H&E stained histological sections of 154 patients who underwent resection for rectal adenocarcinoma between 1996 and 2006. None of these patients had received neoadjuvant chemo-or radiotherapy. The TSR was determined, by two independent investigators, in different layers of the rectal wall at the point of highest tumor infiltration and at the border of the tumor. TSRs were categorized into three categories: TSR-low, TSR-moderate and TSR-high.
Results: Patients with stage I and II disease (T1-4 N0) and TSR-high showed significantly better 5 year survival rates for overall survival compared to TSR-low and TSR-moderate (p=0.010) and a trend to a better disease specific survival (p=0.067) and disease free survival (0.057). In a multivariate Cox regression analysis the TSR remained an independent prognostic factor for overall survival, when adjusted for age, pT-status and grading. Conclusion: TSR as a prognostic tumor characteristic can be used to identify patients with a good and a poor outcome in lymph node metastasis negative cases. Objective: Goblet cell carcinoid (GCC) of appendix vermiformis is a rare neoplasm that share histological features of both adenocarcinoma and carcinoid tumor. While its malignant potential remains unclear, GCC's particularly show transmural dissemination and are more aggressive than conventional carcinoids. Patients usually present with acute appendicitis. They usually lack the formation of a well-defined tumor mass; thus, it is somewhat difficult to accurately assess their size. Method: Case Presentation: Fifty years old male patient applied to the emergency service with abdominal pain and laparatomy was performed with suspicion of acute appendicitis. Grossly, conjestion and exudation on the distal edge of appendectomy material were seen. A 1×1 cm lesion which was spreading into peripheral adipose tissue was seen on the cut surface. Microscopically, tumor was composed of goblet cell groups resembling "signet ring cells". Tumor was infiltrated into muscularis propria and mesoappendix. Immunohistochemically, chromogranin, synaptophysin, cytokeratin 20, mCEA and p53 were stained positive. Ki67 (MIB 1) proliferation index was 18 %. Conclusion: Adenocarcinoid of the appendix is a rare tumor, which is very difficult to diagnose preoperatively and even macroscopically, making histological examination essential. Immunohistochemical staining is required for definitive and differential diagnosis. Here, we present this rare case with literature reviewed.
Normal colon tissue and colon carcinoma show no difference in heparanase promoter methylation D. Hershkovitz * , Y. Peerless, E. Simon, E. Sabo, O. Ben-Izhak * Rambam Health Care Campus, Dept. of Pathology, Haifa, Israel Objective: Heparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. Accordingly, a large number of studies have demonstrated an association between heparanse expression and tumor stage and patients' prognosis. In colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. One of the regulatory mechanisms of heparanase expression is methylation on its promoter. In the present study we evaluated the role of heparanse promoter methylation in colon carcinoma. Method: Analysis of heparanse promoter methylation was done on 32 samples of colon carcinoma as well as 30 sample of normal colonic mucosa. DNA was extracted from FFPE tissue and subjected to bisulfite conversion. The relative fraction of methylated and unmethylated DNA was evaluated with real-time PCR. Results: The fraction of methylated DNA was 1 %±0.6 in the colon carcinoma group, and 2.4 %±0.6 in the normal colon group (P=0.11). Only one case in the normal group and one case in the tumor group showed more than 10 % methylation in the heparanase promoter. Conclusion: We did not find any difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms. Objective: Among B-cell non-Hodgkin's lymphomas, neural cell adhesion molecule/CD56 expression is exceptional. The aim of this study is to report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). Method: The panel of antibodies included CD3, CD20, CD10, bcl-2, bcl-6, MUM-1 and CD56. Results: A total of 50 cases of DLBCL were identified and one case was also detected positive for CD56. The subject was female, 48 years old. The patient had presented generalized lymphadenopathy. She had not documented involvement of extranodal sites. The case is alive with disease 1 year after diagnosis and chemotherapy treatment. Objective: A case of mediastinal T-cell lymphoblastic lymphoma with indolent clinical course in a 52-yearold man was studied by immunohistology and molecular biology. Method: Formalin-fixed sample. Histology with Ematox-ilin&Eosin and Giemsa; immunohistochemistry by APAAP method. T-cell receptor gamma rearrangement by BIOMED2 protocols. Results: After one fainting episode the patient was found to have pericarditis caused by a XX-cm-diameter mediastinal mass. The 2 first biopsies were unsuccessful and the third diagnostic one was done 6 months later. In this time-period the patient was well without symptoms and the mass stable. After two mediastinal biopsies with unspecific features (Castleman disease?), the diagnostic one showed fibrosis, abundant small reactive lymphocytes (CD20+/CD3+) and areas with diffuse small-medium size lymphocytes positive for CD3, TdT, CD34, CD7, LMO2 and high Ki67. T-cell receptor gamma genes were monoclonally rearranged. Conclusion: Indolent T-LBL is exceedingly rare: for this reason in our case LMO2 was a key marker for the diagnosis which was delayed for the presence of fibrosis and reactive B cells. Objective: Granulocytic sarcoma (GS), also termed myeloid sarcoma or chloroma, is a rare malignant solid tumor resulting from the extramedullary proliferation of myeloblasts or immature myeloid cells. GS most frequently occurs in patients with acute myeloid leukemia, myeloproliferative neoplasms or myelodysplasia. GS rarely presents in the absence of systemic myeloid disease. GS most commonly occurs in the soft tissues of the head and neck, bone, skin and less often in the central nervous system and spinal cord. Results: A 40 year old woman presented with 1 month history of weakness of both legs. MRI of the thoracal spine showed 4 cm diameter epidural mass compressing the dural sac in the spinal canal at the T9-10 levels. Decompressive laminectomy and tumor removal were performed resulting in neurological improvement. Histologically neoplastic cells have round nuclei with finely dispersed chromatin and scant cytoplasm proliferating in a diffuse pattern. Immunohistochemically, tumor cells were positive for MPO, CD117, CD45, CD99. Diagnosis was GS without bone marrow involvement. Conclusion: We report the case of GS due to unusual localization and nonleukemic presentation. Results: There were 5 males and 1 female with a median age of 40 years (22-79 years). Physical examination shows splenomegaly in all cases and hepatomegaly in 2 cases, hematological parameters shows lymphocytosis with peripheral hairy cells in all cases. Flow cytometric immunophenotyping was done in 2 cases, and confirmed the diagnosis of HCL with CD11c, CD25+, CD103+, CD123 +, and CD5-, CD10-, and CD23-. Bone marrow biopsy was done in all cases, showing massive infiltration with villous cytoplasmic projection cells that were CD20 positive. The treatment consists of blood transfusion in all cases, associated with splenectomy in 3 cases, only 1 patient was treated with (2 CDA: cladribine) with a complete remission after a follow up of 24 months. Conclusion: HCL has consistent cytologic histologic and immunologic features that make classification reliable and reproducible; it remains problematic because of the variety of the disorders and the differential diagnosis of splenic lymphoma of the marginal zone. Objective: Primary CNS lymphomas (PCNSL) are uncommon tumors in immunocompetent patients, they represent up to 1 % of non Hodgkin lymphomas (NHL) and 3-5 % of all brain tumors. The majority of PCNSL are diffuse large B cell lymphomas, 2-5 % are T cell lymphomas, in rare instances low grade B cell lymphomas. We determine clinical characteristics, histological findings and treatment outcome of PCNSL. Method: Five cases of PCNSL occurring between 1993 and 2007 were retrospectively reviewed. Results: They were 4 males, 1 female, median age 50 years, range (23-74). Most common symptom was neurological deficits (4cases). MRI of the brain revealed an expansive tumor affecting the parietal bone in 3 cases, the temporal bone in 1 cases and the frontal bone in 1 case. Immunohistological finding showed large B-cell NHL in 2 cases, anaplastic lymphoma in 2 cases, and large-cell immunoblastic lymphoma in 1 case. Treatment consists on exclusive tumoral resection in 3 cases, surgery and chemotherapy (1 case), complicated by death in all these cases. Surgery followed by high dose chemotherapy and radiotherapy (1 case) with complete remission after a follow-up of 7 years. Conclusion: Chemotherapy followed by involved field irradiation appears to be an adapted therapy. Objective: Primary cutaneous non-Hodgkin's lymphoma (PCNHL) is defined as lymphoma limited to skin without extra-cutaneous involvement at presentation. We describe here epidemiological and histo-pathological aspects of PCNHL.
Method: Retrospective review of clinical data of PCNHL patients diagnosed in Hematology Department, Frahet Hached Hospital, Sousse (1993 . Results: Fourty three patients with PCNHL, median age: 48 years (11-84), sex-ratio was 2.3. The site of cutaneous involvement was upper lumb in 4 cases, lower lumb in 7 cases and disseminated in 23 cases. PCNHL was small T cell lymphoma in 13 cases, large B cell lymphoma in 16 cases, anaplastic, CD4+ CD56+ hematodermic neoplasm, and follicular type in 1 case respectively. According to TNM classification 21 cases were T3bNxMx, 12 cases were T1N0M0. After first line therapy, 26 patients in CR, PR was obtained in 1 case and failure in 6 cases, 10 patients relapsed within 1 to 72 months (median 6 months), all of them were disseminated cases. Conclusion: Our study indicate that the extend of cutaneous involvement at the time of diagnosis is a significant prognostic factor in PCNHL. Objective: Follicular lymphomas (FLs) account for one third of non Hodgkin lymphomas (NHLs) in adults. The disease is characterized by a response to initial treatment, followed by relapses, sometimes associated with histologic transformation into high grade NHL. Analyze epidemiological characteristics of patients with FL. Method: Retrospective study of 22 of adult FLs diagnosed in Hematology Department (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . Results: Median age was 59 years (29-77), sex-ratio: 1.7 The most frequent clinical symptom at diagnosis was lymphadenopathy in 13 cases. Involvement of the spleen in 3 cases. B symptoms were present in 10 cases. Performance status was more than 1 in 4 cases. According to the international prognostic index (FLIPI), 7 patients have low index, 5 have intermediate index, and 10 have high index. Grade 2 FL was seen in the majority of cases. Treatment modalities varied over time and according to the disponibility of drugs especially to anti CD20 monoclonal antibody. CR was obtained in 13 cases, PR in 6 cases; 3patients relapsed within 8-48 months. Histologic transformation was seen in 3 cases, we have noted 10 deaths related to the disease in 7 cases. Conclusion: Treatment results in FL might not only be improved by more effective induction regimens but also by maintenance treatment. Objective: CD99 and FLI-1 are widely used for their diagnostic utility in Ewing sarcoma/peripheral neuroectodermal tumor (ES/PNET). CD99 expression has been documented in a variety of tumors, including lymphoid malignancies of T cell origin. Also FLI-1 expression is common in different tumor types. However, few studies have investigated the CD99 and FLI-1 immunoreactivity in diffuse large B-cell lymphoma (DLBCL). We aimed to determine the frequencies of CD99 and FLI-1 immunohistochemical expressions in DLBCL which may lead to a misdiagnosis. Method: CD99 and FLI-1 expressions are retrospectively investigated by immunoperoxidase staining in prechemoterapy primary tumors of 42 DLBCL cases.
Results: Out of 42 cases, CD99 and FLI-1 expressions were observed in 14 (33.3 %) and 7 (16.7 %) tumors, respectively. Concomittant expressions of CD99 and FLI-1 proteins were found in 5/42 (11.9 %) cases.
The present study revealed that CD99 and FLI-1 are frequently expressed in DLBCL, thus DLBCL should be considered in the differential diagnosis of CD99+ and FLI-1+ neoplasms.
Primary cutaneous marginal zone lymphoma with biclonality E. Beretouli * , T. Koletsa, A. Bouzakis, S. Mavropoulou, G. Karkavelas, I. Kostopoulos * AHEPA Hospital, Dept. of Pathology, Thessaloniki, Greece
Objective: Primary cutaneous marginal zone lymphoma (PCMZL) represents a B-cell lymphoma which presents with papules, plaques or nodes. The presence of a monotypical light chain B cell population supports the diagnosis of PCMZL. We describe a case of PCMZL which showed different light chain restriction in two heterochronous lesions. Method: Α 50 year-old male patient presented in 2004 with a cutaneous nodule in the anterior surface of the tibia, which was totally excised. The patient did not show other lesions since 2011, when a new cutaneous nodule located in the right ankle, was appeared. Immunohistochemical analysis was performed in both biopsies. Results: Histological and immunohistochemical findings of both biopsies were consistent with marginal zone lymphoma of the skin with lambda light chain restriction in the biopsy of the first lesion and kappa light chain restriction in the biopsy of the second lesion. Blood laboratory studies, chest and abdominal computerized tomography scans revealed no evidence of systemic involvement by lymphoma. Conclusion: The finding of both kappa and lamda light chain restricted B cell populations in PCMZL is unusual. The monoclonal light chain switching in PCMZL is rare and its pathogenesis is discussed. Objective: Microvessel density (MVD) has prognostic significance in some malignancies. Little information exists about angiogenesis in mantle cell lymphoma (MCL), although antiangiogenic drugs are used experimentally. Prognostic factors of MCL are needed as outcome and therapy are heterogeneous and often unsatisfactory. The aim is to assess MVD in MCL and to evaluate its prognostic significance. Method: 177 MCL specimens (FFPE) were examined by immunohistochemistry with anti-CD34 antibody. MVD was quantified using systematic uniform random sampling and unbiased counting frames. Clinical data were analyzed in Kaplan-Meier survival curves and log-rank test. Results: Median survival (MS) of all patients was 46 months, median progression-free survival (MPFS) 22 months. Mean MVD was 172,7 microvessels/mm2, median 158,2/mm2. Dividing cases into quartiles: <117,4/mm2, <158,2/mm2, <206,6/mm2 and ≥206,6/mm2 (43, 44, 45, 45 cases), MS and MPFS did not differ statistically significantly between these groups (P=0,307; similar for therapy groups). Visible differences are only between the fourth quartile and the rest: Objective: Plasmablastic lymphoma (PBL) is a rare aggressive subtype of diffuse large-B-cell lymphoma which occurs primarily in the oral cavity of HIV-positive patients. It is extremely rare in immunocompetent patients. Method: A total of 5 cases of PB were retrospectively analyzed. All cases had tissue available for immunohistochemistry and in situ hybridization. Results: The age range of the 5 patients was 35-81 years (median 68) and all were men. Three patients were HIV negative. HIV + patients: tumors involved the oral cavity (n =1), and small intestine (n=1). HIV-patients: tumors were located in bone marrow (n=1) and lymph nodes (n=2). All tumors were composed of monomorphic plasmablastic cells. Tumors were negative for CD45 and CD20, while they showed diffuse positivity for CD138, CD38, MUM1 and EMA. By ISH, monoclonal light chain restriction was detected. EBV (EBER) was positive in all cases. The median overall survival was 21,4 months regardless of the intensity of chemotherapy. Only one patient is alive 1 month after diagnosis. Conclusion: PBL may appear in non immunocompromised patients and other locations than oral cavity. Any patient diagnosed with PBL should be tested for HIV. Patients who had HIV-negative PBL have lower rates of oral involvement.
Spinal granulocytic sarcoma preceding clinical manifestation of acute myeloid leukemia V. Cemerikic-Martinovic * , N. Drndarevic, N. Andjelkovic, P. Djordjevic, D. Jovanovic, R. Nikolic, T. Martinovic * Beo-lab, Dept. of Pathology, Belgrade, Serbia
Objective: Granulocytic sarcoma is a rare malignant neoplasm of primitive myeloid cell origin, most commonly found in association with acute myeloid leukemia. Granulocytic sarcomas generally occur in the soft tissues, bone, and skin.
Method: We report a case of a 55-year old man with spinal granulocytic sarcoma manifesting as rapidly progressive paraplegia preceding clinical manifestation of acute myeloid leukemia. Results: Magnetic resonance imaging revealed a tumor in lumbal spine (L1-L3). Baseline laboratory data were normal. He underwent emergent laminectomy and the tumor was totaly resected. Histological examination of lesion demonstrated diffuse proliferation of large atypical cells with round nuclei with delicate chromatin and one or more prominent nucleoli. Immunohistochemical studies revealed positive staining for LCA, CD33, CD43, CD163, Lysozyme and CD15 while myeloperoxidase, lymphoid, neuroendocrine markers, S-100 and CD99 were negative. The final histological diagnosis was granulocitic (monoblastic) sarcoma. No chromosomal aberrations were detected by FISH. A month later, acute myelomonocytic leukemia was diagnosed upon a peripheral blood and bone marrow examination showing an increased number of abnormal monoblasts. Chemotherapy, with multiple regimens for leukemia in combination, did not affect the tumor and the patient died. Conclusion: Granulocytic sarcoma should be considered in the differential diagnosis of spinal tumors. Objective: Survivin, a member of the inhibitor of apoptosis family might play an important role in the pathogenesis of diffuse large B cell lymphoma (DLBCL). We investigate clinical and prognostic significance of survivin expression in nodal DLBCL. Method: Biopsy specimens obtained from 56 patients with newly diagnosed nodal DLBCL treated with immunochemotherapy (R-CHOP) were immunostained for survivin. Results: Survivin immunoexpression (>45 % positive tumor cells) has been found in 22 (39.28 %) patients. Significant difference in immunoexpression was noticed between GCB and non-GCB subtype of DLBCL (p=0.031). There was no significant association with IPI, "bulky"disease, Ki-67 immunoexpression or other clinico-pathological parameters. Univariate analysis showed that survivin expression was unfavorable factor for therapy response and shorter survival in patients with DLBCL (p=0.048 and p=0.034, respectively). Patients with survivin overexpression experienced relapse more often that the patients without expression of this apoptotic protein (27.3 % vs. 11.8 %), but this difference did not reach statistical significance (p=0.131).
The results of this study showed that disregulation of survivin expression had the important role in determination of course of disease in pateints with nodal DLBCL treated with R-CHOP. Therefore, survivin represent potential therapeutic target in DLBCL.
Langerhans cell histiocytosis of the jaws: A report of ten cases with an analysis of the mechanism of eosinophilic infiltration Y. Objective: Langerhans cell histiocytosis (LCH) frequently presents eosinophilic infiltration. Here we report ten cases of LCH that involved the jaws and our investigation of the mechanism underlying eosinophilic infiltration in LCH. Method: We evaluated the CCL-11/eotaxin-1 expression of LCH cells via immunohistochemical staining. Toluidine blue staining was used to inspect the densities of mast cells, with ten periapical granuloma specimens serving as a control group. Results: Every patient was classified as having single-system LCH, even though acute lymphoblastic leukemia occurred in one patient during LCH treatment. The ratio of mandible to maxilla LCH was 5:1. The jaw lesions were the earliest manifestation in seven patients with multifocal LCH. Toluidine blue staining revealed that the number of mast cells in LCH lesions was not significantly higher than in periapical granulomas. However, upon immunohistochemical examination, most of the patients showed diffuse positivity for eotaxin-1 in LCH cells, while few eosinophils/ T cells were immunoreactive. Conclusion: We surmise that the eotaxin-1 expression of LCH cells may be relevant to eosinophilic infiltration in LCH. Further studies of the eotaxin-1 functions including its influence on the immature state of LCH cells may be needed to understand the pathogenesis of LCH as well as the role of tissue eosinophilia in LCH. Method: We report a case of SANT in 53 years-old man presented with abdominal discomfort and pain in a left lumbal region. Abdominal computed tomography scan revealed a massive haematoma extended from spleen to the enlarged left kidney and left retroperitoneal space. The patient also had a spontaneous rupture of right kidney 2 years ago. Results: Macroscopically, the cut surface of spleen showed multiple well circumscribed red brown nodules. Microscopically, SANT consists of multiple well-circumscribed angiomatoid nodules showing plump endothelial cells and extravasated erythrocytes. Nodular formations are surrounded by a variable lymphoplasmacytic infiltrate, spindle cells and collagenous stroma. Immunohistochemical staining displayed endothelial phenotype that resembled splenic capillaries (CD34+/CD31+/CD8−). Expression of CD68 was also present. Conclusion: The differential diagnosis of SANT includes splenic hamartoma, inflammatory myofibroblastic tumor, littoral cell angioma and hemangioendothelioma. It has been postulated that SANT represents a peculiar hamartomatous transformation of splenic red pulp in response to an exaggerated nonneoplastic stromal proliferation. SANT has a benign clinical course and splenectomy has been supposed as curative.
Collision tumor of menningioma and non Hodgin malignant lymphoma of cerebellum H. Erdem * , A. K. Uzunlar, U. Yildirim, A. Sav, M. Dosoglu * Duzce University, Dept. of Pathology, Turkey Primary central nervous system lymphoma (PCNSL) constitutes a rare group of extranodal non-Hodgkin's lymphomas (NHLs), primarily of B cell origin, whose incidence has markedly increased in the last three decades. Immunodeficiency is the main risk factor, but the large majority of patients are immunocompetent. This report presents the case of a 71-year-old woman with a collision tumor of primary malignant lymphoma and meningioma in the cerebellum. Collision tumor of primary malignant lymphoma and meningioma have not been described in the literature. The morphological aspect is interesting with regard to the problem of collision tumors.
Prognostic significance of immunohistochemical expression of the angiogenic molecules VEGF-A, VEGFR-1 and VEGFR-2 in patients with Classical Hodgkin Lymphoma E. Georgiadi * , G. Dimtsas, P. Karakitsos, T. Vassilakopoulos, I. Thymara, P. Korkolopoulou, E. Patsouris, C. Kittas, I. Doussis-Anagnostopoulou * Larisa, Greece
Objective: Classical Hodgkin Lymphoma (cHL) is characterized by the presence of a small percentage of malignant Hodgkin and Reed-Sternberg (HRS) cells amongst a reactive background. The role of angiogenesis in cHL is still unclear. The aim of the study was to evaluate the expression of VEGF-A, VEGFR-1 and VEGFR-2 and their correlation with clinicopathological parameters and prognosis. Method: The immunohistochemical expression of VEGF-A (VG1, Dako), VEGFR-1 (RB-9049, Neomarkers) and VEGFR-2 (sc-6251, Santa Cruz) was studied in a large cohort of 199 patients with cHL and the results were correlated with clinical characteristics and patient outcome. Results: The neoplastic HRS cells expressed VEGF-A, VEGFR-1 and VEGFR-2 in 90.3 %, 97.2 % and 94.1 % of the cases respectively and their expression levels were intercorrelated. Expression of VEGFR-1 and VEGFR-2 was significantly and positively correlated with early disease stage, absence of B-symptoms, WBC <15.000, Alb ≥4, IPS ≤2. VEGFR-2 was additionally positively correlated with male gender and ESR <50. All three molecules were statistically correlated with ramifications of blood vessels but not with microvessel density. Conclusion: Based on our results, we could speculate that, in contrast to solid tumours, the process of angiogenesis is probably an early event in neoplastic progression in the context of Hodgkin's Lymphoma.
PS-12-019 P53 upregulation is associated with proliferation and is not capable of inducing apoptosis in Hodgkin's lymphoma E. Georgiadi * , G. Dimtsas, T. P. Vassilakopoulos, V. G. Gorgoulis, I. A. Doussis-Anagnostopoulou * Larisa, Greece
Objective: P53 is a tumor suppressor protein described as the "guardian of the genome". In a normal cell, p53 is inactivated by its negative regulator, Mdm2. Once activated, p53 induces cell cycle arrest and apoptosis by activating various cell cycle related genes, like WAF1/ CIP1 encoding for p21. Although mutations that deactivate p53 are the most common genetic alteration found in cancer, they are rare in the context of Hodgkin's lymphoma (HL). Method: P53 immunohistochemical expression was investigated in a subset of 81 cases of primary HL. The study of the p53-downstream protein p21 was used as an indirect way of analyzing its functional status. P53 expression was correlated with proliferation, apoptosis, clinicopathological data and prognosis of the patients. Results: P53 showed a median value of 32.4 %, suggesting an upregulation of the p53 gene in HL, and a significant correlation with p21 (p=0.043) and the proliferation marker MIB1 expression (p=0.038).
Conclusion: Although p53 overexpression is a frequent finding in HL and there is indication of its functionality, this upregulation does not lead to apoptosis. It can be speculated that this is possibly due to Mdm2-p53 interactions.
PS-12-020 Simultaneous gastric adenocarcinoma and B cell lymphoma of the stomach F. Gerin * , K. Turkoz, B. Kantarcioglu, C. Ataizi Celikel * Marmara University, Dept. of Pathology, Istanbul, Turkey
Objective: The simultaneous association of gastric carcinoma with gastric lymphoma is a rare event. Method: We describe a 52 years old man, who had been diagnosed as marginal zone lymphoma in a cervical lymph node biopsy 3 years previously.No endoscopic examination was evaluated then. There was no pathological appearance in the gastrointestinal system by CT scanning and patient was considered nodal marginal zone lymphoma. Patient had taken 8 cycles of chemotherapy which consisted of rituximab, cyclophosphamide, vincristine, and prednisolone.2,5 years after completion of chemotherapy, a gastric wall thickening compatible with gastric lymphoma or linitis plastica was detected on CT and gastroscopic examination was indicated. Endoscopic examination revealed an erosive, fragile, hemorrhagic, malignant-looking lesion with a diameter of 4.5 cm in the cardia. Besides, rugae of fundus and corpus were severely rough and hyperemic suggesting a diffuse malignant infiltration. Multiple biopsies were taken from cardia, fundus and corpus of the stomach. Results: Cardiac samples showed pancytokeratin positive signet ring cells which contain neutral and acidic mucine in microscopic examination. Samples of corpus and fundus did not show similar carcinoma cells, instead, there was diffuse infiltration of atipical lymphocytic infiltration consistent with marginal cell lymphoma both morphologically and immunophenotypically. Conclusion: Co-occurrence of carcinoma and marginal zone lymphome is a rare event and dramatically exacerbates prognosis of a patient with such an indolent lymphoma.
Assessment of demographic data, staging and experesion of CD20, CD30, CD15, Bcl-2 in Reed-Sternberg cells of Hodgkin lymphoma cases in their first admission in Urmia Imam Komini Hospital B. Ilkhanizadeh * , P. Mazlomi * Imam Komini Hospital, Dept. of Pathology, Urmia, Iran
Objective: Immunohistochemistry plays an important role in diagnosis of Hodgkin lymphoma. Beyond diagnostic importance of Immunohistochemistry, so far many studies have investigated the correlation between cellular markers and the prognosis of patients with Hodgkin lymphoma. Amongst other markers, CD20 is one of the most studied marker with a lot of controversy around it. Method: Retrospectively 66 eligible patients entered the study. Clinical and laboratory Data and Immunohistochemistry findings analyzed for possible correlation between disease stage and other parameters. Results: In our study 63.6 %, 90.9 % of cases respectively expressed cellular markers CD15 and CD30 while only 18.2 % of cases expressed CD20. Furthermore statistical analysis revealed that CD15 was inversely correlated with disease staging. (P=0.027). In contrast we didn't find any relation between CD20 or CD30 positivity with disease staging. (P=0.482, P= 0.376). However in our study expression of CD20 was not related to stage or other parameters of poor prognosis which proposes that in our patients this marker possibly was not related to disease prognosis. Conclusion: Therefore we suggest that cellular marker CD20 is not beneficial beyond IPS factors and its usage should be confined to diagnostic purposes.
Primary naturall killer cell lymphoblastic leukemia/ lymphoma of central nervous system G. Karagkounis * , T. Argyrakos, E. Ronne, E. Dimitriadis, D. Papadopoulos, G. Stranjzalis, D. Rontogianni * Athens, Greece
Objective: A 25-year old woman with a 2-year history of a multifocal lesion occupying the cerebellum who periodically received cortisol and etoposide as it was considered tumor-forming multiple sclerosis. Method: Two years after initial clinical diagnosis a stereotactic biopsy was performed. Immunohistochemistry and in situ hybridization were performed on the tissue sample. Results: The perivascular (Virchow-Robin) space of microvessels was occupied by middle sized lymphocytes with blastic chromatin and scant cytoplasm. The neoplastic cells were negative for: 1. B-cell markers: CD19, CD22, CD20, CD79a, Pax-5, 2. Myelogenous differentiation antigens: MPO, CD13, CD33, 3. Plasmacytoid dendritic differentiation antigens: CD123, TCL1, 4. Blastic cells markers: CD34, CD117 5. T-cell markers: CD3, CD5 with restricted expression of CD2 and CD7 in a few cells, 6. Cytotoxic enzymes TIA-1, Granzyme-B and perforin. All cells expressed intensely CD56 and TdT and some of them CD16. In situ hybridization for EBV virus (EBERS) was negative. PCR analysis: T-cell receptor and immunoglobulin genes were in germ line. Bone marrow was negative. Objective: Lymphomas constitute 33,65 cases on 100 000 inhabitants annually, its frequency grows. Method: This work presents epidemiological, morphological analysis of nodal/exstranodal lymphomas. Analysis of data from Izhevsk cancer center during 2009-2011 was carried out. Results: Lymphoma appeared in 2.94 % among first diagnosed malignant tumors. The panel of monoclonal antibodies was used for tumor immunophenotype identification. Nodal lymphomas presented 183 cases: 55 -Hodgkin lymphoma (HL), 128non-Hodgkin lymphoma (NHL). Exstranodal lymphomas presented 111 cases, various variants of NHL were more frequent. Small lymphocyte lymphoma -37, diffuse large B-cell lymphoma (DLBCL) -36, follicle center lymphoma -18, mantle cell lymphoma -8 were frequent among nodal lymphomas. T-cellular NHL presented 16 cases (6 -peripheral T-cell lymphoma, 7 -angioimmunoblastic Tcell lymphoma). Among other organs of lymphatic system lymphomas appeared in tonsils (19), thymus (15) and spleen (9). Exstranodal NHL were registered in stomach (25), rarely in other organs -testis (7), central nervous system (3), nose (3), breast (3). As rule DLBCL was more frequent (36). Conclusion: Results coincide with data on lymphoma's frequency and morphological characteristic in other regions.
In situ mantle cell lymphoma in nasopharynx T. Koletsa * , F. Tsiompanou, C. Poulios, G. Karkavelas, I. Kostopoulos * AUTH, Dept. of Pathology, Thessaloniki, Greece
Objective: The nasopharynx normally contains abundant lymphoid tissue and it can be the site of both lymphoid hyperplasia and lymphomas. We present the first case of the recently described in situ mantle cell lymphoma (MCL) in nasopharynx.
A 70 year-old woman presented with nasal obstruction in 2008. A mass located in nasopharynx was found. Biopsy was sent for histological examination. A new biopsy was taken 3 years later. Histological, immunohistochemical and FISH studies were performed. Results: Histological examination of both biopsies revealed lymphoid hyperplasia, characterized by CD5 and cyclinD1 positive cells of mantle zones, findings consistent with in situ MCL. The diagnosis of in situ MCL confirmed by FISH analysis for t(11;14). The patient examined thoroughly and has remained free of an overt lymphoma to the present day.
Conclusion: Pathologists and otorhinolaryngologists should be aware that in situ MCL may be observed in nasopharynx. It may be appropriate to perform cyclinD1 immunostain, even in cases with clinical and histological findings compatible with lymphoid hyperpasia. The patients should be examined thoroughly, since in situ MCL may accompanied by an overt lymphoma in other sites or it may be a precursor of an overt MCL.
Multisystem Langerhans' cell histiocytosis coexisting with metastasizing adenocarcinoma of the lung A. Lovrenski * , M. Panjkovic, Z. Eri * Institute for Lung Diseases, Dept. for Pathology, Sremska Kamenica, Serbia
Objective: Langerhans' cell histiocytosis (LCH) is an uncommon disease of unknown etiology characterized by uncontrolled proliferation and infiltration of various organs by Langerhans' cells. To our knowledge, this is the first case reporting an association of multisystem LCH with metastasizing adenocarcinoma of the lung. Method: We present the case of a 54-year-old man, heavy smoker, with dispnea, cough, hemoptyses, headache and ataxia, who died shortly after admission to our hospital. On the autopsy, we found tumor in the posterior segment of the right upper pulmonary lobe as well as a right-sided occipito-parietal lesion which penetrated into the right ventricle resulting in internal and external hematocephalus. Histologically and immunohistohemically, the diagnosis of primary lung adenocarcinoma with brain metastasis was made (tumor cells showed positivity for CK 7 and TTF-1 which confirmed the diagnosis). Light microscopic examination of the other organs showed LCH involving the pituitary gland, hypothalamus, spleen, mediastinal lymph nodes, and lungs. Immunohistochemical studies revealed CD68, S-100 and CD1a immunoreactivity within the Langerhans' cells.
Conclusion: Multisystem form of LCH with extensive organ involvement was an incidental finding, while the metastatic lung adenocarcinoma to the brain, that led to hematocephalus, was the cause of death.
Langerhans cell histiocytosis associated with extramedullary hematopoiesis in a 65-year-old male patient L. Lungoci * , L. Elena, B. Flavia, M. Rodica * City Hospital, Pathology, Timisoara, Romania
Objective: Langerhans cell histiocytosis is a rare disease histologically characterized by the proliferation of Langerhans cells. Method: A 65-year-old male came to our attention presenting with erythematous-squamous lesions all over the body, axillary and inguinal adenopathies and hepatosplenomegaly. We have taken a cutaneous and an axillary lymph node biopsy. Results: Microscopic examination of the cutaneous fragment revealed the presence of a dermal infiltrate of large cells with an eosinophilic cytoplasm and vesicular, nucleolated nuclei with an irregular nuclear margin, raising suspicion of a cutaneous lymphoma. Immunohistochemical stains for CD45, CD20, CD79a, CD3, CD5, CD15, CD30, and Bcl-2 were negative, and the previous diagnosis was not confirmed. The microscopic examination of the axillary lymph nodes showed foci of extramedullary hematopoiesis, raising suspicion of a lymphoproliferative process, but the immunohistochemical stains and the paraclinical data didn't confirm it. Based on the intense positive reaction for CD68, S100 and Ki-67 showed by the tumoral cells from the prelevated tissues, correlated with the clinical and paraclinical findings, a diagnosis of Langerhans cell histiocytosis was established. Conclusion: The particularity of our case consists in the presence of extramedullary hematopoiesis in the lymph nodes of a patient diagnosed with Langerhans cell histiocytosis.
Unusual primary cutaneous presentation of B-cell chronic lymphocytic leukaemia S. Mavropoulou * , Z. Tatsiou, G. Xanthopoulidis, A. Goutzouvelidis * General Hospital, Laboratory of Pathology, Xanthi, Greece
Objective: B-cell small lymphocytic lymphoma/chronic lymphocytic leukaemia (B-CLL) is a low-grade lymphoproliferative disorder and cutaneous lesions are rarely the presenting findings. Method: We report the case of a 62-year-old woman who presented in winter with erythematous plaque on her nose, of 3 months duration. Laboratory data showed elevated white blood cell count (40.000/mm3) with small mature lymphocytes predominance (60 %). Results: Histopathologic examination of the skin lesion reveals infiltration of the reticular dermis and subcutis, consisting mostly of small lymphoid cells, without epidermotropism. Immunohistochemical examination revealed positivity for CD20, CD79a and CD5 in the neoplastic cells. According to the laboratory and histopathologic findings the diagnosis was consistent with B-CLL with a primary cutaneous presentation. A staging bone marrow aspiration and biopsy showed 80 % infiltration by a clonal B-cell proliferation with typical CLL immunophenotyping. CT-imaging was normal, so stage I according to Binnet classification was confirmed. The patient received 6 cycles of chemotherapy and remained in remission for 1 year, when the skin lesion recurred in winter, at the same location. Conclusion: In conclusion we report an extremely rare case of subclinical B-CLL with cutaneous presentation. It is important to maintain a high index of suspicion for a lymphoproliferative process in skin lesions with atypical lymphocytic infiltration.
Myeloid sarcoma mimicking nasal polyp S. Mungan * , U. Cobanoglu, S. Ersoz, G. Guvendi, M. Yilmaz * Karadeniz Technical University, Pathology, Trabzon, Turkey
Objective: A myeloid sarcoma is a tumour mass consisting of myeloid blasts with or without maturation occuring at an anatomical site other than the bone marrow. Every site of the body can be involved, skin, lymph node, gastrointestinal tract, bone, soft tissue and testis. %10 cases presents at multiple anatomical sites. Method: The current case is 75 year old woman presenting with nasal polyp and underwent polypectomy. The polyp was 2,5 cm in diameter and had a bloody appearance. By microscopic examination there was a diffuse infiltration of neoplastic cells under the cilliated epithelium. The mitotic index was high in neoplastic lymhoid cells. Immunohistochemical study was helpful for the differential diagnosis. Objective: The aim of this study was to describe the epidemiology and anatomo-clinical features of the ear, nose and throat (ENT) lymphomas in the Centre of Tunisia. Method: All ENT lymphoma cases diagnosed in the Pathology Department, Farhet Hached Hospital, Sousse during a 15-year period were analyzed. Results: A total of 34 cases of ENT lymphoma were reported (21 males, 13 females) with a sex-ratio of 1.4. The age at diagnosis varied from 9 to 85 years with a median age of 56.5 years. The majority of ENT lymphoma cases (81 %) were of B cell type including large B-cell lymphoma (58 %) and small-cell B lymphoma (23 %). Nasopharynx and tonsils were the most involved areas with 44 % and 42 % of cases, respectively. According to the Ann Arbor staging system, these lymphomas are diagnosed in the stage II (42 %) followed by stage I (30 %). Conclusion: The most frequent lymphoma developed in ENT region was B cell lymphoma, especially located in nasopharynx and Tonsils. Results: A total of 44 cases were reported (26 males, 18 females) with a sex-ratio of 1.4. The median age was 10 years (range: 3-15 years). Lymph node lymphomas were the most frequent accounting for 63.6 %, followed by ENT, mediastinum, and haematopoietic system (6.8 % each one); cutaneous and digestive location were less frequent (4.5 % each one). Among the 44 patients, 23 (52.3 %) had had Hodgkin's lymphoma and 21 (47.7 %) had had non-Hodgkin's lymphoma (NHL). Diffuse large B-cell lymphoma was the most diagnosed (57 %) followed by Burkitt lymphoma (23.8 %) and T-cell lymphoma (9.5 %). According to the Ann Arbor staging system, 29.6 % of patients were diagnosed in an advanced stage. Method: We present a case of composite AILT and diffuse large B-cell lymphoma (DLBCL) A 45-year-old woman with multiples lymph nodes. Biopsy of inguinal node was performed. Three months later the patient had enlarged cervical node which was removed. Results: Firstly, the inguinal lymph node was diffusely infiltrated by small-medium size lymphocytes with scanty cytoplasm. The nuclei were irregular. Several large cells were observed in a background of prominent arborizing high endothelial venules. The small cells were CD3 positive. The large cells were CD79a, CD30 positive. Theses cells showed nuclear positivity for EBER by ISH. Secondly, the cervical lymph node showed large areas of necrosis admixed with pleomorphic and medium size T lymphocytes. There were a population of neoplastic EBV + pleomorphic B lymphocytes; some of them with RS-like morphology. We performed B and T-cell clonality studies. We detected TCR-gamma T-cell and IgH B-cell gene clonal rearrangement. Conclusion: Occasionally IgH gene rearrangement has been detected but only a few cases have demonstrated, morphologically, composite AILT and EBV-associated diffuse large B-cell lymphoma (DLBCL).
Typhlitis as initial manifestation of granulocytic sarcoma of the appendix: A case report P. Rohani Doost * , B. Ilkhanizadeh, F. Noroozinia, A.
Objective: Myeloid sarcoma is a localized mass of myeloblasts or immature myeloid cells involving any extramedullary site. Reports in the literature documenting leukemic infiltration of appendix are uncommon and extremely rare when symptoms mimicking typhlitis. Method: A 30-year-old man with a history of relapsed acute myelogenous leukemia (FAB classification, M2) admitted for receiving induction chemotherapy. After 10 days of chemotherapy, he presented with fever, nausea, vomiting, abdominal pain and severe neutropenia (0.026×109/L as absolute neutrophil count). This manifestation was attributed to typhlitis and he received antibiotic therapy, but he came back 50 days later with acute abdomen prompting exploratory laparotomy which revealed inflamed appendix. Results: Histologic examination showed diffuse appendiceal wall infiltration of mononuclear cells with medium-to-large vesicular nuclei, conspicuous nucleoli and pale eosinophilic cytoplasm. Immunostains showed the diagnosis of granulocytic sarcoma with myeloperoxidase, leukocyte common antigen, CD15, CD117 and CD68 positivity and epithelial membrane antigen, keratin, CD3, CD5, CD10, CD79a and CD20 negativity. Conclusion: Our case declares that physicians and surgeons should be aware of granulocytic sarcoma in the differential diagnosis of mild tenderness on palpation of the abdomen as a complication of acute myelogenous leukemia. Objective: Angioimmunoblastic T-cell lymphoma (AITL), one of the most frequent entities among peripheral T-cell lymphoma, is characterized by lymphadenopathy, B-symptoms, and an aggressive behaviour. Method: We reported 3 cases of AITL during a period of 4 years (2004) (2005) (2006) (2007) . Results: The age of patients varied between 20 and 73 yearold. They hadn't any pathologic history. Two patients presented with a multiple cervical lymphadenopathy, and weight loss. The other patient had fever and sweat. Physical examination found multiple lymph nodes with hepatosplenomegaly in 2 patients. Abdominal ultrasound and CT-scan showed diffuse lymphadenopathy with features raising the possibility of lymphoma. The diagnosis of AITL was confirmed by a microscopic and an immunhistochemical study of lymph node or liver biopsy. 2 patients received a cure of chemotherapy Conclusion: Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive non-Hodgkin T-cell lymphoma. AITL is frequently associated to immunological and hematologic diseases such as autoimmune hemolytic anemia, vasculitis, rheumatoid arthritis, and autoimmune thyroid disease. Its natural history has been the subject of controversy, considered for many years to be a nonmalignant disorder or a dysimmune disease, until the clonal nature of AITL was proven by molecular studies. Although patients are usually treated with corticosteroids, chemotherapy, and/or plasmapheresis, outcomes were dismal. Objective: Low grade lymphomas (indolent) are commonly located on extranodal sites. nodal location are relatively of low incidence. Our aim is to describe the epidemiological status of NBCL and to assess their pathological features in the center of Tunisia. Method: We report 50 cases of indolent NBCL diagnosed in the department of pathology, during a period of 10 years (2000-2009) . Clinical, pathological and immunohistochemical (large panel of antibodies), treatment and outcome data are collected. Results: The mean age was 63 year old (33 and 89 years). The sex ratio was 1.27. NBCL was 6.5 % of all N H lymphomas and 42.73 % of indolent NBCL of any sites. Nodal enlargement and hepatomegaly were the main symptoms. Small Lymphocytic lymphomas were the most frequent variant (56 %), followed by follicular lymphomas (26 %). Mantle cell lymphoma and marginal zone B cell lymphoma were rare (12 % and 6 %, respectively). They were exclusively of B cell type. 76 % of cases were in stage III-IV. 90 % of patients received chemotherapy. 2 % of recurrence and 48 % of death were found. Conclusion: NBCL in the center of Tunisia are relatively uncommon. Small lymphocytic lymphoma is the most frequent in our population. They are still of relatively worse prognosis. Objective: Bone marrow metastases can be found in some malignant tumors, but diagnosing a nonhematologic malignancy from marrow is an unusual event. Herein we present a case of metastatic signet ring cell carcinoma with an unknown primary site. Method: A 39 year-old woman admitted to the Hospital with weakness and bone pain. Positron emission tomography/computed tomography (PET/CT) revealed disseminated bone lesions showing increased metabolic activity. Bone marrow biopsy and aspiration were performed. Results: Bone marrow biopsy revealed extensive necrosis and nearly total replacement of normal bone marrow elements by atypical tumoral cells. Tumoral cells were arranged individually or in small clusters, having hyperchromatic, eccentric located nuclei and abundant cytoplasm with signet ring cell morphology. Even after thourough investigation of all the systems, a primary site of malignancy could not be detected. She received nine cycles of chemotherapy. Craial MRI revealed multiple metastatic lesions. 9 months after the initial diagnosis, she died of disseminated metastatic lesions. Conclusion: Even after thorough investigations, a primary site of malignancy could not be detected. In the literature, all the presented cases had very short survival varying from days to a few months. Our patient lived for 9 months with multidiciplinary approach. Objective: Approximately 20 % of follicular lymphomas (FLs) are bcl2 negative and 10-15 % of FLs lack a translocation t(14;18). Previously we have found that negative bcl2 staining in cases that do have a t(14;18) is caused by mutations in the epitope for the bcl-2 antibody, but this is never the case in bcl-2 negative, t (14;18) negative cases. Now that translocation detection is more commonly performed we noticed that many of the cases that were referred to us as translocation negative FL are actually often nodal marginal zone lymphomas (NMZLs) with extensive follicular colonization. Method: t(14;18) negative cases that on H&E sections look like FL were analyzed by morphology and immunohistochemistry, including antibodies against CD10, bcl-2, Ki-67, and CD23. Results: Careful observation of the bcl-2 staining pattern revealed that follicles that appeared positive at low power contained negative cells at high magnification. The amount of negative cells varied from follicle to follicle and was associated with the extent of Ki-67 positivity. Conclusion: Based on morphology and immunohistochemistry we reclassified many cases of bcl-2 positive, t(14;18) negative FL into NMZL. Although there is presently no positive marker for NMZL, we believe that such cases are responsible for most t(14;18) negative FL. Objective: Recent studies do not provide a definite answer on the origin and pathogenesis of lichen planus (LP). Histologic findings are the same, regardless of the area involved. Electron microscopy is useful to confirm the diagnosis in atypical cases and monitoring LP development. This study aimed an ultrastructural characterization of keratinocyte degeneration in pemphigoides, hypertrophic, and follicular LP types correlating this with levels of gelatinolytic activity caused by metalloproteinase-9 (MMP-9).
Method: Skin biopsies were processed conventionally and examined in a JEOL 1011 transmission electron microscope. MMP-9 expression was detected immunohistochemically. Results: LP pemphigoides demonstrated intact basal keratinocyte cell membrane, and the lamina densa lining blister cavity correlating with moderate basal MMP-9 expression of weak intensity. Hypertrophic type demonstrated good preservation of keratinocytes and their junctions along with strong basal and suprabasal MMP-9 expression of moderate intensity. Both types demonstrated diffuse and strong MMP-9 immunostaining in dermal lymphohistiocytic infiltrate. A hypertrophic type revealed an increase of expression within dermal sweat glands comparing with LP pemphigoides. A diffuse and strong epidermal and follicular MMP-9 staining was noticed in follicular variant of LP. Conclusion: Ultrastructurally keratinocyte involvement displays deviations in various types of LP, and these changes correlate with levels of gelatinolytic activity caused by MMP-9. Objective: Clevudine is a recently introduced anti-viral agent that shows efficacy against hepatitis B virus related chronic liver diseases. However, it has been reported that certain patients who have received long-term administration of clevudine exhibit myopathy involving mitochondrial abnormalities. To make a differential diagnosis congenital mitochondrial myopathy and drug induced mitochondrial myopathy, we investigate ultrastructural findings of drug induced myopathy. Method: We studied histopathological features of myopathy focused on the various ultrastructural mitochondrial abnormalities found in 18 patients with long-term clevudine therapy. Results: In every case, ragged red fibers and multinucleated fibers with eosinophilic granules were observed. Additionally, type 2 fiber atrophy was found in 6 cases. Mainly concentrated in subsarcolemmal or inter-fibrillar areas, the abnormal mitochondria were enlarged and swollen, showing a variety of morphological types. Most of the abnormal mitochondria indicated structural abnormalities in cristae, including the apparent decrease in the number, concentric lamellar pattern, and structure that is branched or lattice-like. Conclusion: From the overall analysis, clevudine-induced myopathy is characterized by ragged fibers showing proliferation of abnormal mitochondria with various forms of inclusion bodies and abnormal cristae. Another particular feature of interest is the presence of multinucleated fibers, which, in most cases, are filled with abnormal mitochondria.
Ultrastructural features of human mature oocytes subjected to cryopreservation S. A. Nottola * * University La Sapienza, Dipt. die Anatomia Histol., Rome, Italy
Objective: Oocyte cryopreservation protocols have not been fully optimized yet. Our aims were to evaluate and compare the ultrastructure of human mature oocytes frozenthawed (F/T) after slow freezing and vitrification. Method: The oocytes, fixed at sampling (fresh controls) and after freeze/thawing, were processed for light and transmission electron microscopy (LM and TEM) observations. Results: By LM, both fresh and F/T oocytes were rounded cells surrounded by an intact zona pellucida (ZP) and containing uniformly distributed organelles. By TEM, numerous vacuoles were found in F/T oocytes after slow freezing. On the contrary, vacuoles were only occasionally detected in F/T oocytes after vitrification, and in fresh controls as well. Amount and density of cortical granules (CGs) appeared abnormally reduced in F/T oocytes, irrespective of the protocol applied. Conclusion: In conclusion, a) cryopreservation currently ensures a good overall preservation of the oocyte; b) however, vacuolization appears as a recurrent form of cell damage during slow freezing, whereas the quasi absence of vacuoles seems the most relevant marker of quality in vitrified oocytes; c) premature CG exocytosis -and the consequent hardening of the ZP -seems a non-specific, ubiquitous phenomenon occurring during freeze/thawing, suggesting the appropriateness of the use of ICSI as the preferred insemination method after cryopreservation.
Tuesday, 11 September 2012, 09.30 -10.30 Results: There were 3 women and 2 men with a median age of 60 (range 45-80 years). Symptoms were dominated by abdominal pain, jaundice, vomiting, and alteration of the general condition. Radiologic investigation showed multiloculated cystic mass of the pancreas, associated to splenic thrombosis and bone metastasis in one case. In gross, we received multilocular cysts of 3 to 16 cm in wall, their wall was fibrous, and thick, the internal surface showed many papillary projections and mural nodules. There was no communication with the duct system. On histologic examination, cysts had a fibrous wall lined by mucinous atypical cells showing frank anaplasia, wall's cyst was invaded by anaplastic glands; the stroma was of ovarian type in all cases. Conclusion: PMC are uncommon neoplasia, their diagnosis is based on the histopathologic exam, their diagnosis is better than duct carcinoma and depends on the extent of tumour invasion.
Heterotopic pancreas of the gallbladder associated with acute cholecystitis O. Akman * , E. E. Pala, U. Bayol, M. Emiroglu * Tepecik Training Research Center, Dept. of Pathology, Izmir, Turkey
Objective: Pancreatic heterotopia is a rare entity, which is commonly found in the stomach, duodenum, jejunum and Meckel's diverticulum. Heterotopic pancreas is extremely rare in the gallbladder. Despite being a congenital condition, it takes years to become symptomatic. It can be associated with cholecystitis or cholecystolithiasis. Method: A 21-year-old female patient was admitted to emergency service with abdominal pain. On physical examination, right upper abdomen was tender without defense or rebound. Abdominal ultrasonography revealed small sized gallstones and pericholecystic fluid. Magnetic resonance cholangiography showed dilated intrahepatic and extrahepatic bile ducts. Laparoscopic cholecystectomy was performed. Results: Cholecystectomy specimen was 75×35×30 mm. On gross examination we noted a few milimetric yellow colored stones and 12 mm yellow solid intramural nodule. The mucosa was covered with fibrinopurulent exudate. Microscopic examination revealed acute cholecystitis and aberrant pancreatic tissue consisting of acini and ductules. The phlegmonous inflammation of the gallbladder infiltrated the aberrant pancreatic tissue. The patient recovered completely after cholecystectomy. Conclusion: We found this case worth reporting because, pancreatic heterotopia of the gallbladder is a rare, clinically silent entity unless complicated with gallstones and acute cholecystitis.
Expressions of c-erb-B2, EGFR, p27, PTEN, mTOR, PI3K in hepatocellular carcinomas and adjacent liver tissues Method: Fifty HCC cases were stained immunohistochemically with these markers. Correlations between the markers and pathologic characteristics were analyzed. Results: The cases had an average age of 56,72. Male/female ratio was 44/6. HBV is more common in advanced stages and right lobe location (p<0.05). Tumor size is significantly larger in patients older than 50 years (p<0,04). No membranous c erb B2 staining was seen while cytoplasmic positivity was present in 92 % and significant correlation was found with multiplicity (p<0.041) and p27 positivity (p<0,011). EGFR membranous positivity was present in 90 % and significantly correlated with stage (p<0.05). p27 was negative in 92 % and PTEN is reduced or absent in 56 %. All markers were similarly expressed in adjacent noncancerous tissue. No significant correlations were found between PTEN, mTOR, PI3K and the pathological parameters. Conclusion: In our study, as a first step, none of the markers among c-erb-B2, EGFR, p27, PTEN, mTOR, PI3K was found significant in correlation with pathologic features. We look forward to obtain further results in the next study consisting of correlations with follow ups.
The value of echo guided liver biopsy in the positive diagnosis of a rare primary liver tumorpathological approach G. Becheanu * , V. Herlea, V. Serban-Barbu, M. Dumbrava, S. Enache, G. Smira, C. Angelescu, A. Pop, M. Diculescu * Bucharest, Romania
Objective: Hepatic angiomyolipomas are rare mesenchymal hepatic tumour. The most important problem about their diagnosis on biopsy specimens is to exclude a hepatocellular carcinoma. Method: We present two cases of hepatic angiomyolipoma diagnosed in Fundeni Clinical Institute, Bucharest. Both females patients, 39 and 43 years old, were imagistically diagnosed with a 13 cm hepatic left lobe tumor in the first patient, respectively a 10.5 cm hepatic right lobe tumor in the second one. Both tumors were echo guided biopsied. The biopsy specimens measured about 8 mm, respectively 20 mm. The tumors were surgical removed. The biopsy and resection specimens were analyzed in light microscopy, including immunohistochemistry. Results: Initial diagnosis on HE stain was cell variant hepatocellular carcinoma, in the 8 mm biopsy, respectively suspicious for angiomyolipoma in the second case. Further immunohistochemical studies showed positivity for Vimentin, HMB45, MELAN A, S100 and actin, and negativity for OCH1E5, CK7, CK8/18, CEA, CD34, Ki67 and Factor VIII in both biopsy specimens, consistent with hepatic angiomyolipoma diagnosis, confirmed on surgical specimens too. Conclusion: Diagnosing a hepatic angiomyolipoma is not easy to do especially on a biopsy. The size of biopsy specimens plays a very important role in a correct diagnosis. Results: A 62-year-old woman presented with a 2-day history of nausea, fever and diffuse abdominal pain. CT scan revealed a well circumscribed small lesion of the pancreatic body. On arterial phase imaging, the mass was of low density relative to the pancreas without marked contrast enhancement. Based on these findings, the patient underwent surgical resection of this mass and a distal spleno-pancreatectomy was performed. Microscopic examination showed a well circumscribed but non encapsulated soft tissue proliferation consisted of blood vessels lined by a single layer of uniform flattened cells, with dilated lumen filled with red blood cells and inflammatory cells. Immunohistochemical staining confirmed the vascular nature of the lesion. The diagnosis of pancreatic hemangioma was made. Conclusion: Adult pancreatic hemangioma is an extremely rare tumor. The review of all published cases showed that this tumor often do not demonstrate the contrast-enhanced CT features typical of an hemangioma, so a poor arterial phase enhancement cannot rule out pancreatic hemangioma and the histological examination is very important in these cases. Objective: Hepatocholangiocarcinoma (HCC-CC) is an uncommon form of primary liver cancer with features of hepatocellular and biliary epithelial differentiation. It accounts for 0.4-14.2 % of all primary liver carcinomas. Many of the demographic and clinical features of this tumor remain unclear. It was previously suggested that coexisting chronic liver diseases were rarely seen in patients with HCC-CC. Method: We report a case of HCC-CC diagnosed in patient with cirrhosis. Results: A 48 year-old man presented with ascites, jaundice, vomiting and symptoms of portal hypertension. On abdominal CT scan the right hepatic segment was heterogeneous, hypervascular in the late arterial phase and low-attenuated on portal venous phase. Based on a these clinicoradiological informations, diagnosis of infiltrating hepatocellular carcinoma was made and hepatic biopsy was performed. Histopathologic examination showed, in addition to features of liver cirrhosis, the presence of malignant proliferation composed of tubular structures as well as microtrabecular and compact foci. On Immunohistochemical stainings the tumor cells expressed cytokeratins 7 and 20. Cells lining the tubular structures reacted with cytokeratin 19 allowing the diagnosis of hepatocholangiocarcinoma. Conclusion: Hepatocholangiocarcinoma is a rare primary liver cancer. Preoperative noninvasive diagnosis with conventional radiography is often difficult especially when occurring in patients with liver cirrhosis or other hepatic chronic disease. The diagnosis is frequently made only after histological and immunohistochemical examination.
Morphological changes in different zones of the gallbladder in cholelithiasis M. Bokhodir * , T. Vervekina * Tashkent Medical Academy, Dept. of Patological Anatomy, Uzbekistan
Objective: Gallstone disease remains a serious problem of modern surgery. An important aspect of the problem is the question of the urgency of surgical intervention, depending on the condition of the gallbladder. Method: It was carried out the analysis of 1130 case histories and excised gallbladder with different forms of cholelithiasis. 81.3 % were women in the age of 41-50 years. Acute catarrh cholecystitis was found out in 20 % cases, acute phlegmonousin 21 % cases, acute gangrenous -in 11 % cases, chronicin 28 % and chronic relapsingin 21 % cases. Results: All forms of cholecystitis characterized by a maximum thickness of the body wall, minimal at the bottom of the gallbladder. In all forms adipose tissue inflammation in the connective tissue layer was increased and it was found out the atrophy of the mucosa. In the inflammatory infiltrate in acute forms of inflammation were found neutrophilic and basophilic leukocytes: with simple acute cholecystitis -in the bottom, and with acute phlegmonous and gangrenous cholecystitis -in the area of the body of the gallbladder. In the cases with chronic cholecystitis were dominated mononuclear cells in all areas, and in the cases with chronic relapsing -neutrophilic and basophilic leukocytes in the bottom and the body, and mononuclear cells -in the neck of the gallbladder.
PS-14-008 Histological features of chronic hepatitis C in hemodialysis patients I. Delladetsima * , A. Kokkori, V. Sypsa, M. Psichogiou, S. Sakellariou, J. Boletis * Athens University, 1st Dept. of Pathology, Greece
Objective: Chronic hepatitis C (CHC) in hemodialysis patients has not been thoroughly investigated, despite its high frequency. The present comparative study aims to highlight the histological features of CHC and its association with putative pathogenetic parameters in this specific group of patients. Method: Sixty-one biopsies of hemodialysis patients and 326 biopsies from the general population with CHC were comparatively evaluated for the severity and specific histological features of CHC. Results were examined in relation to age, time of dialysis, viral load and genotype. Results: Patients on hemodialysis were older than patients of general population (p=0.031), showing a similar genotype distribution (p=0.328) and lower viral loads (p=0.001). CHC on hemodialysis was significantly milder according to stage (p=0.033), activity and its parameters (p<0.001). Significantly reduced was also the frequency of lymphoid aggregates (p< 0.001), bile duct damage (p<0.001) and steatosis. (p=0.033). Severity of hepatits was not associated with time on hemodialysis. In multivariate analysis the differences were independent of age, which was associated with more severe disease. Steatosis was associated with hemodialysis duration and age. Conclusion: CHC in hemodialysis patients is significantly milder than in general population. Limited necroinflammatory activity and absence of immune mediated lesions are indications of defective immune response although involvement of low viral load cannot be overlooked.
PS-14-009 Inflammatory (myofibroblastic) pseudotumor of the liver: Case report E. Demiralay * , S. Altaner, H. Özdemir * Baskent University, Dept. of Pathology, Istanbul, Turkey
Objective: Inflammatory pseudotumor of the liver (IPT) is a rare, benign tumor-like lesion of which etiology is not fully known. It is confused with primary and metastatic malignant tumors as it seems as an occupying mass on radiologic examinations. Method: Thirtyeight-year-old male patient who had fatigue and dyspepsia was detected to have elevated liver enzymes and hepatomegaly. On computed tomography, two masses of which middle is mildly hypometabolic, measuring 34× 31 mm in right liver lobe and 45×39 mm in left liver lobe were detected. Biochemical tests were normal except elevated AST and ALT levels. Ultrasound-guided needle biopsy was done for two masses. Results: On microscopic examination, a lesion composed of abundant fusiform fibroblastic cells and vascular structures among inflammatory cells composed of lymphocytes, plasmocytes, histiocytes and eosinophillic leucocytes and including sharp margins between regenerated liver tissue was observed. On immunohistochemical examination, SMA was stained focal positive in fibroblastic cells. Staining with desmin, S-100, PanCK, CD34, Ki-67 and CD117 was not seen. Conclusion: IPT does not have a specific radiologic finding and definite diagnosis is made with pathologic examination. This case is presented as it is rarely seen, radiologically resembles to malignant tumors and for review of pathological differential diagnosis.
Enhancer of Zeste Homologue 2 (EZH2) expression in malignant and benign hepatic tumors K. Dezso * , V. Szabó, E. Bugyik, K. Schlachter, S. Paku, Z. Schaff, P. Nagy * Semmelweis University, 1st Dept. of Pathology, Budapest, Hungary Objective: The immunohistochemical demonstration of Enhancer of zeste homologue 2 (EZH2) proved to be a useful marker in several tumor types, including hepatocellular carcinomas.In order to recognize the diagnostic value of this protein in hepatic tumors we have investigated the presence of EZH2 in the most common liver neoplasms. Method: The presence of EZH2 has been studied by standard immunohistochemistry in several formalin-fixed paraffin-embedded tumor samples (hepatocellular adenoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, metastatic liver tumors and primitive childhood tumors). Results: Forty of 44 hepatocellular carcinomas, 22 of 23 cholangiocarcinomas, 29 of 31 hepatoblastomas and 14 of 17 metastatic liver tumors stained positively, but all the investigated hepatocellular adenomas (n = 24) and proliferating biliary structures were negative. The other primitive childhood tumors that were examined all expressed EZH2. Conclusion: Based on these results EZH2 is a sensitive marker of malignancy in hepatic tumors regardless of their histogenesis. In routine surgical pathology EZH2 could be helpful to diagnose hepatocellular carcinomas and it might be the first marker to distinguish transformed and reactive cholangiocytes.
PS-14-011 Nuclear ploidy as a marker of intraductal papillarymucinous pancreatic neoplasms malignancy E. Dubova * , O. Mishnev, A. Shchegolev * V.I. Kulakov Scientific Center, Dept. of Pathology, Moscow, Russia
Objective: Intraductal papillary-mucynous pancreatic neoplasm (IPMN) represents 0,5 to 9.8 % of all tumors of exocrine pancreas. Our aim was to study nuclear ploidy of malignant and benign IPMNs cells. Method: We studied 20 pancreatic surgical specimens from 5 females and 15 males. We performed histological, morphometrical and ploidy analysis of all specimens. Results: IPMN cells nuclear perimeter and size was higher than in normal pancreatic duct cells. This parameters were higher in malignant noninvasive IPMN than in benign tumor. Nuclear size and perimeter of invasive malignant IPMN were in intermediate position between benign and malignant noninvasive tumor. Mean nuclear ploidy (MNP) of normal ductal pancreatic cells was 2.4c. MNP of benign IPMNs was 2.5c, borderline tumors -3.0c. MNP of noninvasive malignant IPMNs was 5.1c, of invasive IPMNs -4.5c. We also revealed that aneuploidy coefficient (AC) of benign IPMNs was 0 (there weren't aneuploid nuclei in its cells). AC of borderline IPMNs was 0.11. Maximal level of AC was in malignant noninvasive IPMNs whereas AC of invasive IPMNs was lesser. Conclusion: Nuclear morphometric parameters and mean nuclear ploidy level as well as aneuploidy coefficient could be used as additional criteria for determining malignant potential of IPMNs.
PS-14-012 An intraductal papillary mucinous neoplasm of the pancreas with concomitant neuroendocrine tumor: A case report J. Garcia * , F. E. Costa, R. Sampaio, R. Dias, A. Duarte, J. Ramon Vizcaíno * Lisbon, Portugal
Objective: Intraductal papillary mucinous neoplasms (IPMNs) are a recently classified pancreatic neoplasm with an increasing incidence. IPMN of the pancreas is a lesion consisting of mucin-producing cells with neoplasic potential, characterized by papillary intraductal growth and varying degrees of cytologic atypia. Neuroendocrine tumors of pancreas (PNET) are rare pancreatic neoplasms comprising 1-2 % of all pancreatic tumors. PNETs express neuroendocrine markers, however the true cell or cells of origin are not fully understood. PNETs are classified as functional or nonfunctional based on the presence or absence of a specific clinical syndrome associated with hormone oversecretion. Intraductal papillary mucinous neoplasm of the pancreas with concomitant Neuroendocrine tumor are a highly rare occurrence. Method: A literature review of IPMNs and PNET was made, as well as a review of the clinical data of a patient with IPMN and concomitant PNET. Results: A 60-years-old Caucasian female, underwent a laparoscopic distal pancreatectomy with splenectomy for IPMN. Macroscopic examination revealed discrete dilatation of pancreatic ducts and nodule in the pancreas tail. Hystologic examination revealed neoplasic lesion of intraductal papillary-mucinous in the body and tail of pancreas with moderate dysplasia (IPMN borderline) and Neuroendocrine Tumor grade 1 (WHO 2010). Conclusion: This case reports a highly rare occurrence.
Glypican 3 and agrin expression in hepatocellular carcinoma and cholangiocellular carcinoma: A immunochemistry study N. Gursan * , H. Balta, B. Gundogdu * Ataturk University, Medical Faculty, Erzurum, Turkey Objective: Glypican 3 (GPC3), which plays a role cellular growth, differentiation and migration is one of members of glypican family. Although GPC3 expression in the liver, glypican upregulation of the vast majority of tumors are available. Agrin is found the surface on the cells and the extracellular matrix, the micro-vessels Method: In this study, formalin-fixed, paraffin-embedded surgical specimens from 20 patients with hepotocellular carcinoma and 20 patients with cholangiocellular carcinoma diagnosed between 2000 and 2011 years in Ataturk University, Faculty of Medicine, Department of Pathology were studied with immunohistochemistry Agrin and GPC3. Results: We detected agrin in around bile ducts and blood vessels within the portal areas in the normal liver. İn the malignant hepatocellular carcinoma (HCC) is seen in a dramatic increase in the quantity of agrin. GPC3 immunpositivity showed in %85 of HCC and %15 of cholengiocellular carcinoma. Agrin immunpositivity showed in %95 HCC and %50 of cholangiocellular carcinoma. Conclusion: GPC3 and Agrin, that is useful in early diagnosis of HCC cases; cholangiocellular carcinoma, or not enough was understood to be very limited.
Value of glypican 3, Hep Par and alpha fetoprotein in diagnosis of hepatocellular carcinoma N. Gursan * , H. Balta, B. Gundogdu * Ataturk University, Medical Faculty, Erzurum, Turkey Objective: Glypican 3 showed high expression of the embryonic liver and intestine, is silenced in normal adult tissues. It is an oncofetal protein. İn general, oncofetal proteins, while having a critical role in tumor progression or immunotherapy is a potent tumor marker used as a target. Alpha fetoprotein, clone C3 localized, normally produced by gestational age, fetal liver and yolc salc, glycoprotein structure emerging marker for patients with HCC. Hep Par 1 antibody was developed in 1993 using fixed liver as immunogen. Method: İn this study formalin-fixed, paraffin-embedded surgical specimens from 20 patients with hepatocellular carcinoma diagnosed between 2000 and 2011 years in Ataturk University, Faculty of Medicine Department of pathology were studied with immunohistochemistry GPC3, Heppar 1, and AFP. GPC3, Heppar 1 and AFP expression was divided into 2 categories negative (negative or weak under %5 tumor cells cytoplasmic staining) and positive (and %5 over of tumour cells) moderate or strong cytoplasmic with membranous accentuation. Results: Immunpositivity at GPC3, Hep Par 1 and AFP were 85 %, 95 %, 75 % respectively. Objective: Fibroblastic growth factor receptor (FGFR) family is known to be related to the development and progression of various types of cancers. The aim of this study is to determine the clinical implication of FGFR expressions in patients with hepatocellular carcinoma (HCC). Method: Immunohistochemical analysis was done in 842 cases of HCC using tissue microarray. Diffuse cytoplasmic staining for FGFR1, 2, 3 and 4 in more than 10 % of tumor cells were designated as positive. The results were analyzed in terms of various clinicopathologic parameters. Results: On univariate analysis, the overall survival rates of patients with FGFR2 expression (152 cases, 18.1 %) were significantly lower than those with no expression (HR 1.838, 95 % CI 1.452-2.328, P<0.001). However, the overall survival rates of patients with FGFR4 expression (446 cases, 59.6 %) were significantly higher than those with no expression (HR 0.636, 95 % CI 0.521-0.776, P<0.001). There was no statistical significance between patients' overall survival rates and expressions of FGFR1 (46 cases, 5.5 %) or FGFR3 (33cases, 4.0 %). On multivariate analysis, only FGFR2 expression is independently associated with reduced OS (FGFR2: HR 1.790, 95 % CI 1.404-2.282, P<0.001). Conclusion: The FGFR2 expression can be used as an independent prognostic factor in patients with surgically resected HCC. Objective: HURP (hepatoma upregulated protein) is a putative oncogene, overexpressed in many human cancers, including hepatocellular carcinoma. It has also been shown recently that high HURP protein levels correlate with resistance to chemotherapeutic agents. In the present study we investigate the expression of HURP and its correlation with pancreatic adenocarcinoma, prognosis and patient survival. Pancreatic adenocarcinoma is one of the most aggressive types of cancer and represents the fourth most common cause of death, either in the male or in the female population of the United States of America. Method: HURP immunoreactivity was assessed by immunohistochemistry in a series of 28 primary pancreatic adenocarcinomas. In parallel, HURP expression was examined in 10 normal pancreatic tissues. Statistical analysis related HURP expression levels with clinicopathological characteristics and survival.
Results: Results showed a positive correlation between HURP overexpression and grade as well as lymphovascular invasion. All non malignant biopsies were negative. Furthermore, positive expression of HURP appeared to be an important independent prognostic factor too, related with poor survival rates. Conclusion: Our results showed that HURP overexpression is associated with poor prognosis in pancreatic adenocarcinoma and indicated a diagnostic potential of this protein. Its role in the carcinogenetic process awaits further elucidation.
Hepatocellular carcinoma in patient with hepatic porphyria: A case report J. Marcinek * , P. Szépe, T. Balhárek, M. Kalman, L. Plank * University Hospital Martin, Dept. of Pathology, Slovakia
Objective: Hepatocellular carcinoma (HCC) is frequently associated with liver cirrhosis or chronic hepatitis of various etiology, less often with other chronic hepatopathies, including hepatic porphyria. We present a case report of HCC diagnosed in patient with clinically unrecognized porphyria cutanea tarda (PCT). Method: A 70-year old male was admitted to hospital complaining of a pressure below low costal margin, anamnesis of a trauma 1 year ago and clinical suspicion of a posttraumatic intrahepatic haematoma. By a surgery, a partialy necrotic and hemorrhagic tumor mass 10 cm in diameter was removed. Results: The mass corresponded histologicaly to moderately differentiated clear cell HCC, resembling renal clear-cell carcinoma. The surrounding parenchyme showed steatohepatitis with fibrosis and distinct intracellular porphyrin crystals. Conclusion: Development of HCC is a well documented complication of PCT, a disorder of porphyrin biosynthesis. PCT can be inherited (autosomal dominant trait), the acquired form (sporadic PCT) is more common. Known etiological factors of PCT include toxins, alcohol abuse, estrogens or chronic viral hepatitis, or association with other hepatic diseases like iron overload (including hereditary haemochromatosis) or chronic hepatitis C, increasing the risk of malignancy. However, in the presented case none of these associations was recognized and its etiology remains obscure. Objective: Signet ring cell carcinoma is an extremely rare type of gallbladder carcinoma composed overwhelmingly (90 %) of signet ring cells. Only a few cases of this histologic type have been published and detailed knowledge of this disease is not available. Method: We report a case of signet-ring cell carcinoma of the gallbladder in a 70-year-old woman who was admitted with epigastric pain. Under the preoperative diagnosis of cholangiocarcinoma, which was based on findings of CT scan and ultrasonography. Cholecystectomy was performed. Results: Microscopic examination revealed a signet ring cells tumor that arose from the sub epithelial layer of mucosa and involved all the layers of gallbladder. Nuclear atypia and mitoses were present. Periodic acid schiff (PAS) stain highlighted the intracellular mucin in the tumor cells. Lympho-vascular emboli were detected in the subserosal layer. The cystic duct surgical margins were invaded. Conclusion: Gallbladder adenocarcinomas are seen frequently, but signet-ring cell carcinoma is a rare entity. Owing to the location of the gallbladder, dissemination of the tumour to the adjacent tissues is usually presented at the time of the diagnosis. It is necessary to exclude a gastric or colonic signet ring cell carcinoma secondarily involving the gallbladder. Objective: Gastrointestinal stromal tumors (GISTs) are mesenchyme neoplasms of the gastrointestinal tract which express CD117. GISTs can occur in the entire length of gastrointestinal tract, but very seldom can also arise in omentum, mesentery and retroperitoneal space, kidney, urinary bladder etc. There they are located adjacent to stomach or intestine, but not originated from the latter. These tumors are designated as "extra-gastrointestinal stromal tumors" (EGISTs). There are only 14 description of the Primary EGISTs reported in the literature. Method: We report a case of a pancreatic EGIST in a 38year-old female patient. Results: CT scan showed a mass in the head of pancreas. Grossly encapsulated tumor, 9 cm in diameter, was found in the head of the pancreas (Figure 1) . Histologically, tumor demonstrated spindle-cell pattern consisted of distinct fascicles and bands. The number of mitoses was 1-2/50 high power fields in "hot-spot" areas. IHC revealed strong positivity for CD117, CD 34 in neoplastic cells and negative for SMA, desmin, S-100. Ki67 labeling index was 3 %. Conclusion: We presented a rare case of pancreatic GIST. The tumor has very good prognosis. Hepatoblastoma is classified as epithelial (56 %) or mixed epithelial/mesenchymal (44 %). Epithelial hepatoblastoma is further divided to pure fetal (31 %), embryonal (19 %), macrotrabecular (3 %) and small cell undifferentiated (3 %). Aim: To increase the awareness regarding possibility of hepatoblastoma in adult age. Method: We review four cases of hepatoblastoma epithelial type diagnosed in Pathology Department of Fundeni Clinical Institute between 2009 and 2012. Immunohistochemistry was done in all cases: CEA, Vimentin, CK 8/18, CK 19, CK 20, Ki 67, TTF1, CD 10, CD 34, CD 56, MUC 5A, alpha-fetoprotein. Results: Two of the patients were males and two females with age ranging between 17 and 27 years. The tumors varying in size from 8 to 12 cm. On microscopic examination the tumor was composed mainly of epithelial elements. The pathological diagnosis was epithelial hepatoblastoma epithelial type. Conclusion: Hepatoblastoma is a rare tumor in adult age and epithelial type is the rarest from all type of hepatoblastoma in adult age. The pathological diagnostic is quite difficult even using immunohistochemistry because none of the markers are not specific for hepatoblastoma.
Submassive hepatic necrosis with regenerative nodules: A series of cases Y. Rodríguez-Gil * , J. Salazar, J. Delgado Sánchez, G. López Alonso, C. Ibarrola de Andrés, F. Colina Ruizdelgado * Hospital Universitar 12 de Octubre, Dept. of Surgical Pathology, Madrid, Spain
Objective: Confluent hepatic necrosis is the morphological correlate of fulminant liver failure. A group of these livers develop a combination of areas of necrosis and regenerative macronodules (RMN). Method: Sequential biochemical and clinical data and tissues from two groups of patients were compared. Group 1: 10 cases of submassive necrosis associated with RMN; Group 2: 19 cases with total confluent necrosis without nodular regeneration. Results: Group 1: mean age was 33,6 years (ranged 10 months-63 years); group 2 mean age was 42,6 years (ranged 18 months-63 years) (p>0,05). A longer course (42,8±34,6 days vs 12,6±6,0, p<0.05) and higher maximum serum bilirubin levels (23,5 mg/dl ±13,08 vs 14,9 mg/dl± 10,9, p<0 .05) were observed in group 1. Serum AST, ALT and gamma-GT were lower in group 1 (p>0.05). Morphologically livers from group 1 showed well defined regenerative green-yellow nodules with large regenerative hepatocytes in acinar pattern without surrounding fibrosis. The remaining parenchyma showed confluent necrosis, haemorrhage, mixed inflammatory infiltrate and ductular proliferation. Conclusion: A special anatomoclinical form of subacute liver failure was characterized by regenerative macronodules in a background of extensive confluent liver necrosis. Illness duration was longer and had higher maximum bilirubin levels than those without RMN.
Immunohistochemical study of MUC gene family in pancreatic cancer G. Setdikova * , O. Paklina * Moscow, Russia
Objective: Investigation of the MUC gene in pancreatic ductal adenocarcinomas (PDA). Method: In the present study, we examined the expression of MUC1, 2 and 5 AC types by immunohistochemical analyses in PDA from 74 patients. Overall survival curves were drawn by the Kaplan-Meier method. For all analyses, p<0.05 was considered to be statistically significant. Results: In our cases most of the PDA was presented as MUC1+/MUC5AC + phenotype -42 % cases (31/74). The group with intestinal phenotype mucin, which characterized by positive expression of MUC2 and CDX2, was only 7 % (5/74). The group which characterized by positive expression only MUC5AC (gastric phenotype), was 15 % (11/74). The survival rate of patient better in group with MUC2 expression. Cumulative survival at 12 months after surgery was 1.0 and the median postoperative follow-up period was 17 months. The most aggressive behavior was PDA with expression only MUC1 ("true pancreatobilliary type"). Cumulative survival at 12 months after surgery was 0.25 and the median postoperative follow-up period was 7 months. Conclusion: The mucin profile as a prognostic factor is important not only for intraductal pancreatic mucinous neoplasm, but and for ductal pancreatic adenocarcinoma.
PS-14-026 Hepatocellular carcinoma microvessels density depends on tumor differentiation: Radiologic-pathologic correlations A. Shchegolev * , E. Dubova, U. Tumanova * V.I. Kulakov Scientific Center, Dept. of Pathology, Moscow, Russia Objective: Prognosis and recurrence level in hepatocellular carcinoma (HCC) depends on microvessels density. Method: We performed CT and pathological correlations in 22 cases of HC (5 cases of highly (h-HCC), 11 cases of moderate (m-HCC) and 4 cases of low differentiated HCC (l-HCC)). Results: Vessels number in w-HCC in compare to m-HCC was higher by 26.1 % (p<0.05). Total vessel area was higher in m-HCC and l-HCC than in w-HCC by 11.7 % and 20.4 % accordingly. We observed positive correlation between CT density of h-HCC and number and total vessel area. Correlation between h-HCC CT density and mean vessel area was negative. We also revealed modest negative correlation between m-HCC density and the number vessels in all phases of CT. Negative correlation between total and mean vessel area and m-HCC density was revealed in native and arterial CT phases in compare with venous and delayed phases where positive correlation was revealed. In all phases of CT we revealed strong negative correlation between l-HCC density and the number of vessels whereas correlation between l-HCC density and mean vessel area was strongly negative. Conclusion: We revealed decreasing in angiogenic activity in HCCs neoplastic progression and growth. CT signs correlate with HCC histological differentiation.
PS-14-027 Primary sporadic liver schwannoma: An extremely rare diagnosis R. Silva * , C. Eloy, J. M. Lopes * Centro Hospitalar de São Joâo, Serviço de Anatomia Patológica, Porto, Portugal
Objective: Primary liver schwannoma (PLS) is extremely rare. Results: Case Report: A 69-year-old female with previous history of cholecystectomy, appendectomy, arterial hypertension and diabetes mellitus was admitted due to an intrahepatic nodular lesion identified in routine ultrasound examination. She was submitted to partial hepatectomy with postoperative uneventful evolution. Macroscopically, the specimen disclosed a 2.1 cm nodular, well circumscribed, yellowish tumour. Histologically, the tumour displayed an expansive growth pattern, comprising short bundles of spindle cells with mild atypia, without mitotic figures or necrosis. Immunohistochemically, the tumor cells expressed diffusely vimentin, S-100 and GFAP, in the absence of AE1/AE3, CAM5.2, CD117, and HMB45; Ki-67 index was 2 %. Staging procedures did not disclose evidence of any other tumor. There was no personal or family evidence of neurofibromatosis. These features are consistent with sporadic PLS.
Conclusion: Schwannoma is a rare benign tumour in the gastrointestinal tract with few cases reported in the liver. The clinical presentation is usually an upper abdominal pain but they can be asymptomatic. Secondary cystic degeneration and hemorrhage are common in large tumours. Differential diagnosis by imaging evaluation includes several benign and malignant, namely metastatic, tumours. Therefore, the pathological examination is crucial for the diagnosis of primary liver schwannoma.
Metastases of hepatocellular carcinoma to the costa and soft tissue: A very rare entity E. Tastekin * , T. D. Yalta, O. Yalcin, T. Ciftci * Trakya University, Dept. of Pathology, Edirne, Turkey
Objective: Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver with a potential of metastasis primarily to the lung, abdominal lymph nodes and bone. However, metastases to the costa, soft tissue are rare. Method: An 82-year-old man was admitted to hospital with weakness, anorexia. Chest x-ray and CT-scan demonstrated a 5×5 cm sized, intrathoracic mass on the lateral-side of the left 2.costa and also infiltrating the axillary soft tissues.A secondary mass with the identical radiological features was also detected within the C7-vertebra. Whole-body CT-scan was planned.A 2×2 cm sized nodular, hipodense mass was detected in the upper segment of the liver. Interestingly, none of these masses demonstrated a significant pathological FDG-uptake in the PET.A trucut biopsy was taken from the axillar. Microscopically, large tumoral nests were observed in connective tissues. Polygonal shaped tumoral cells were found to have granular cytoplasm, pleomorphic nuclei. Immunohistochemistry results: Positive cytoplasmic reaction with Heppar, TTF-1; canalicular staining with Polyclonal-CEA, CD10; weak cytoplasmic staining with pankeratin, B72.3; Negative reaction with Vimentin, AFP, CK7, CK20. Results: These histomorphological, immunohistochemical findings strongly supported to the diagnosis of HCC metastasis. Conclusion: HCC that is, in general, morphologically similar to mesenchymal and epithelial tumors, should be considered among the differential diagnosis of unknown primary tumors.
Morphological justfication of using pulsed electric discharge in surgical treatment of echinococcosis M. Tussupbekova * , D. Kaliyeva, E. Turgunov * Medical University Karaganda, Dept. of Pathological Anatomy, Kazakhstan Objective: Echinococcosis is characterized by lesions of internal organs, severe complications, often lead to disability and death. Method: New method of processing of the residual cavity with impulsive electric discharge after echinococcectomy was experimentally developed and introduced into clinical practice. The effectiveness of the method is confirmed by morphological examination of operational bioptic of the liver tissue. Results: 20 patients underwent surgical treatment with the new method (Patent of the Republic of Kazakhstan ¹ 63558). Parasitic cysts with an inner germinal layer and protoskoleks, outdoor layer -chitinous shell with a productive reaction with lymphocytes infiltration, single eosinophils, separate groups of hydatid bubbles with their invasion in the liver tissue were detected in the histological examination of material taken intraoperatively prior processing of residual cavity with electropulse discharge. Fibrinoid necrosis, destruction of germinal elements, single lymphoid infiltration, hyperemia of blood vessels, absence o inflammatory reaction were marked in the histological material after processing by impulsive electric discharge.
The results showed that echinococcectomy with processing of the residual cavity with impulsive electric discharge is an alternative for pericystectomy and liver resection. Objective: Congenital peribronchial myofibroblastic tumor is a rare, solid mesenchymal tumor of the fetus and neonate, usually associated with nonimmune hydrops fetalis. We report the case with clinical, radiological and pathologic findings and review the other cases in the English language literature. Method: We present a case of CPMT, whom a right lung mass was detected in intrauterine life. 12 days after delivery by cesarean section, right lobectomy was performed. Results: The tumor was limited to lung, and composed of spindle cells, proliferating around bronchial unit. Central necrosis and 4-5/10 hpf was present. Patient is well 15 months after surgery. Conclusion: Congenital peribronchial myofibroblastic tumor is a rare solid pediatric tumor of lung which was named differently in the past, such as congenital fibrosarcoma, congenital leiomyosarcoma, congenital mesenchymal malformation of the lung, neonatal pulmonary hamartoma. albeit it resembles sarcoma with high cellularity, mitosis and necrosis features microscopically, no additional therapy to tumor resection is needed. So it is important to keep in mind this scary looking, but innocent tumor both in prenatal and postnatal evaluation.
Morphologic alteration of metastatic neuroblastoma in bone marrow after chemotherapy G.-E. Bae * * Samsung Medical Center, Dept. of Pathology, Seoul, Republic of Korea
Objective: The aims of our study are to evaluate the histologic features of metastatic neuroblastoma in bone marrow (BM) in comparison with those of primary neuroblastoma, and to compare the histologic characteristics of metastatic neuroblastoma in BM before vs. after chemotherapy. Method: Total 92 biopsies from 19 children diagnosed as stage 4 neuroblastoma with BM metastasis were examined histologically; 19 primary neoplasm biopsies, 19 BM biopsies before chemotherapy, 19 primary neoplasm excision biopsies after chemotherapy, and 35 BM biopsies after chemotherapy. Results: 1) Primary neoplasms were classified as neuroblastoma poorly differentiated (n=10), neuroblastoma differentiating (n=5), ganglioneuroblastoma intermixed (n=1) and neuroblastoma type unclassifiable (n=3). 2) Metastatic foci in BM before chemotherapy were composed of undifferentiated and/or differentiating neuroblasts but not ganglion cells, in neuropil but not schwannian stroma. 3) Metastatic foci of BM after chemotherapy showed differentiation such as ganglion cells and schwannian stroma, which was more prominent after more cycles of chemotherapy. 4) Metastatic neuroblastomas in BM after chemotherapy were as mature as or less mature than those in primary neuroblastomas after chemotherapy. Conclusion: Metastatic neuroblastomas in BM initially consist of more immature components than primary neuroblastomas, whereas they become differentiated as primary neuroblastomas after multi-cycle chemotherapy.
Morphometric evaluation and clinical correlations in malignant small round cell tumors C. Bansal * , A. Gupta, A. Kumar, A. Srivastava * CSMMU, Dept. of Pathology, Lucknow, India
Objective: Nuclear size increases in malignant tumors and reflects DNA content, ploidy and proliferation index. Present study investigated; could nuclear morphometery differentiate histomorphologically similar paediatric malignant small round cell tumors in H & E stained sections for using in a poor resource country? Method: Morphometric analysis was done in 26 confirmed but difficult to differentiate round cell tumors histomorphologically and were analyzed by cell images from 6 different areas in each section, using Leica Q win 500 images software. Results: Nuclear measurements were obtained for retinoblastoma (9), Hodgkin lymphoma (6), Wilms tumor (3), medulloblastoma (2), Ewing's sarcoma (2), alveolar rhabdomyosarcoma (1), malignant hemangiopericytoma (1), non-Hodgkin lymphoma (1) and neuroblastoma (1). Amongst the retinoblastomas, maximum mean nuclear area percent (24.93) was seen in cases with nerve involvement and metastasis, followed by cases with only nerve involvement (21.60) and smallest area (16.57) was in non-nerve involving, non metastatic cases. Wilms tumor cases with metastasis had higher nuclear area (21.25) than non metastatic (19.47). Non-Hodgkin lymphoma nuclear area (20.03) was more than Hodgkin's (18.60). Amongst all tumors, minimum value (14.93) was seen in malignant hemangiopericytoma. Conclusion: Morphometric evaluation in paediatric malignant round cell tumors have generated useful data, and needs further multicentric confirmation for implementation. Objective: Survivin, a bifunctional protein that regulates cell division and suppresses apoptosis, may play an important role in tumorigenesis. The aim of this study was to determine survivin expression patterns in Wilms tumor (WT) and to analyze it in relation to stage, prognostic category and histological type. Method: Immunohistochemical expression of survivin was analysed in 59 cases of primary WT and in 10 normal kidney specimens uninvolved by the tumor. Results: Fifty one out of 59 cases of WT (86.44 %) showed decreased cytoplasmic survivin expression compared to the expression in normal kidney tissue. Decreased cytoplasmic expression (in all components of WT) of survivin was found significantly more often in low stage compared to high stage WTs (86.7 % vs. 27.3 %; p=0.002). Tumors of intermediate risk group showed more often decreased cytoplasmic expression of survivin in comparison to high risk group, but the difference was not significant. Decreased survivin expression was found more frequent in WTs with diffuse anaplasia and in epithelial WTs compared to other histological types, but without statistically significant difference. Conclusion: Decreased survivin cytoplasmic expression may be associated with the favorable prognosis WT. Objective: Hepatic tumors accounted 5 % of congenital neoplasms. Mesenchymal hamartoma of the liver is a rare benign tumor of children. We report a case of hepatic mesenchymal hamartoma in a premature male neonate. Method: A premature neonate born at 33 gestational weeks from 34 year-old mother, G3P3 with non-consanguineous marriage; prenatal ultrasonography showed a fetal macrosomia, a highly vascularized abdominal mass occupying two-third of the abdomen and displacing the bowel loops associated with poly-hydramnios. The newborn examination showed abdominal firm lump. Ultrasound found and anterior highly vascularized mass displacing the left lobe of liver. The infant died few hours later. A complete autopsy was performed. Results: External examination showed a male neonate anatomically of 34-35 week having an increased periombilical diameter and a macrosomia, ascitis, pulmonary hypoplasia, cardiomegaly, pleuropericardial effusion, liver tumor developed in left lobe and dilation of renal vessels. Histologically the tumor showed a mixture of normal liver tissues with blood or lymphatic vessels, bile ducts within an abundant edematous and myxoid stroma. Conclusion: In neonate and fetus, prenatal diagnosis is possible by ultrasonography. Large tumors can affect the viability of the newborn. Adequate excision is curative in most of cases.
PS-15-007 NCAM polysialylation as potential initiator of differentiation and proliferation of renal progenitors in human fetal tissue S. Cirovic * , J. Tadic, N. Radunovic, C. Müller, G. Müller, J. Markovic-Lipkovski * Inst. for Pathology, Nephropathology, Belgrade, Serbia Objective: Objective: Neural cell adhesion molecule (NCAM) is widely expressed on mesenchymal and early tubular epithelial cells during kidney development although with still undefined function. NCAM can be polysialylated and as PSA-NCAM has been shown to be involved in proliferation and migration of neuronal cells during brain development. The aim of this study was to evaluate the presence of PSA-NCAM in nephron precursors in relation to the expression of renal progenitor and proliferation markers. Method: Human fetal and neonatal kidneys were analyzed using double-immunofluorescence (dIF) staining and Western Blot (WB) analysis. Specific antibodies against NCAM, PSA-NCAM, EpCAM, CD24 and Ki-67 were applied. Results: On WB only fetal tissue samples have bands with NCAM at 140 to 250 kDa which suggest that NCAM molecule is polysialylated; dIF analysis of fetal tissue show PSA-NCAM + Ki-67+ cells in all structures known as nephron precursors. While in neonatal tissue PSA-NCAM and Ki-67 were positive only on rare single cells in intersticium. Conclusion: PSA-NCAM expression appears to characterize a very early stage of induced nephron progenitors differentiating from NCAM + EpCAM-mesenchymal cells. According to PSA-NCAM localization and coexpression with Ki-67 during development and its practically absence in neonatal tissue, suggest that PSA-NCAM present potential initiator of proliferation and differentiation of renal progenitor. Objective: Cystic hygroma (CH) typically develops in utero, late in the first trimester to early in the second trimester of gestation. Many of the fetuses with CH present additional malformations commonly associated with chromosomal anomalies. Method: A review of 548 fetal autopsies performed over a 10 year period revealed 19 cases of CH (3,4 %). The results of cytogenetic analysis and the prenatal ultrasound findings were retrieved and compared to the autopsy findings. Results: Fetal death was due to therapeutic abortion in 9/19 cases, intrauterine death in 8/19 cases, and spontaneous abortion in 2/19 cases. Cytogenetic analysis was available in 12 cases. The results showed an abnormal karyotype in 7 cases (5 cases of Turner syndrome and 2 cases of trisomy 21). The mean size of CH was 5,4 cm. Other findings suggestive of the cause of fetal death were diagnosed in 10/19 cases (52,6 %). The most common autopsy findings were hydrops and central nervous system anomalies. The autopsy findings were in agreement with the prenatal ultrasound findings in 14/19 cases, while in 5 cases (26,3 %) additional findings were detected during autopsy. Conclusion: Our study is confirms the strong correlation between CH and chromosomal anomalies of the fetus.
Immunohistochemical expression of E-cadherin in primary and metastatic nephroblastoma cases I. Franckevicha * , R. Kleina * University Children´s Hospital, Dept. of Pathology, Riga, Latvia
Objective: In nephroblastoma cases association between decreased E-cadherin expression and higher stage of tumor (Safford SD, 2005) and lower expression in metastatic tumors (Alami J, 2003) was described. Some authors considered that E cadherin is not likely to play tumor suppressor role in nephroblastoma ( Shulz S, 2000) . Purpose of this study was to compare the expression of E-cadherin in metastatic and primary tumor cases in Latvia. Method: 26 cases of primary tumors, 8 metastases and 1 case of relapse were analyzed immunohistochemically using visualization system EnVision. The number of E-cadherin positive structures per field was assessed (magnification× 100). Comparison between primary tumors and metastatic/ relapse tumors groups was made using Mann-Whitney test. Results: In primary tumor group the number of positive structures in the field ranged from 0 to 86, average 27.19, SD 24.13. In the metastatic tumor group, the number of positive structures varied from 0 to 40, average 9.89, SD 14.87. Comparison of expression observed in both primary and metastatic tumors groups showed the decreased E-cadherin expression in metastatic tumor group (Z= −2.33, p=0.046). Conclusion: Expression of E-cadherin is lower in metastatic tumor group that may suggest about negative correlation between it and higher tumor grade.
PS-15-010 Testicular fibrous hamartoma: A case report A. Kilitci * , F. Yilmaz, S. Yanik, H. Ozturk * Abant Izzet Baysal University, Dept. of Pathology, Bolu, Turkey
Objective: Fibrous Hamartoma (FH) of childhood is uncommon benign tumor. They are generally seen in the head and neck region, gastrointestinal system and lung. As in the case we report, they may also occur in other unusual sites such as groin and testis. The clinical presentation is almost always a mass or swelling, however our case was admitted to our hospital because of a left testicular atrophy. We report a FH in the testis, which has a rarely location and clinical presentation. Method: A 7-year-old male presented to department of pediatric surgery because of left testicular atrophy. Left orchiectomy was performed. Results: In macroscopic examination, testis dimension was 1.5×1.3×0.4 cm. Its cut surface was smooth and dirtywhite. In microscopic examination, the tumor showed disorganized matur tissue that composed of fibrocollagen stroma, vessels, muscular and adipose tissue. By this findings, it was diagnosed as testicular fibrous hamartoma (TFH). Conclusion: In conclusion, TFH should be always kept in mind with testicular atrophy not only testicular mass or swelling. And knowledge of this particular type lesion is important to distinguish the FH of childhood from other situations in testis such as testicular torsion, incarcerated hernia, malign neoplasm, etc. in order to allow a correct diagnosis and avoid inadequate treatment.
Placental pathologic features in diabetes and hypertension P. Luís * , A. Costa-Silva, A. Alves * Hospital de Santa Maria, Dept. de Anatomia Patológica, Lisboa, Portugal
Objective: The aim of this study was to evaluate the most frequent placental findings in diabetes and hypertension and their main differences. Method: Retrospective study of 229 selected placentas from 19 to 41 weeks gestation, in a universe of 3231 placentas examined over the last 10 years (6 % associated with hypertension, 1,5 % with diabetes and 0,4 % with both diabetes and hypertension (DwH). Results: In diabetes the most frequently found abnormalities were immature villi (35 %) and infarction (27 %). In DwH the most frequent lesions were the immature villi (29 %) and inflammatory lesions (either acute or chronic) (29 %). In the hypertension group the most frequently found lesions were infarction (54 %) and accelerated maturation of the villi (23 %). In 35 % of placentas in diabetes, 21 % in DwH and only 15 % in hypertension, no lesions were found. The incidence of fetal death was 8 % in diabetes, 0 % in DwH and 5,5 % in hypertension. When evaluating placental weight, small placentas were more frequent in the hypertension group (37 %), and large placentas were more frequent in diabetes (27 %). Conclusion: Our findings may contribute to evaluate the consequences of diabetes and hypertension in fetal outcome.
As inflammatory conditions are usually not directly attributed to diabetes or hypertension, placental examination may help in diagnosis of associated pathology like infection.
Placental villi morphometry in preeclampsia K. Pavlov * , E. Dubova, R. Shmakov, A. Shchegolev * V.I. Kulakov Scientific Center, Dept. of Pathology, Moscow, Russia
Objective: Terminal villi structure abnormalities could play an important role in preeclampsia (PE) and its complications development. Our aim was to perform comparative morphometric study of the placentas from mild and severe PE pregnancies. Method: Complex morphological and morphometric study of 9 term placentas from mild PE (mPE) cases (1st group), 6 term placentas from severe PE (sPE) cases (2nd group) and 10 term placentas from uncomplicated pregnancies (control group) was performed. Results: We revealed significant decreasing in terminal villi size in both preeclamptic groups in compare to control. This decreasing was much prominent in sPE group. We also observed significant terminal villi perimeter decreasing in both PE groups without any difference between mPE and sPE groups. Morphometry of CD31 stained specimens revealed significant decreasing in mean capillary number in both PE groups. Single capillary area and perimeter were significantly lower in mPE and sPE groups and these changes were much prominent in sPE group. Total villous capillary area and perimeter were significantly lower in both PE groups with minimal values in sPE group. Degree of villous capillarisation was significantly lower in both PE groups. Conclusion: Revealed features reflect villous structure changes in preplacental hypoxia, caused by mild and severe preeclampsia.
Sudden intrauterine death: The usefulness of autopsy F. Portelli * , E. Orlando, E. Di Stefano, E. Maresi * University of Palermo, Section of Pathology, Italy
Objective: We investigated in 1006 autopsies of stillbirths the usefulness of autopsy even in the absence of risk factors and/or apparent anatomical/clinical causes which could explain the death. Method: From January 1990 to December 2010 (Institute of Pathology, Paolo Giaccone-Palermo) 2643 autopsies on dead uterine foetuses were performed. 1637 cases were abortions (<25 weeks), 1006 were stillbirths (≥25 weeks). In all cases the autopsy included a macroscopic and microscopic examination both of the foetus and the placenta. The final diagnosis of death was based on both the morphological and clinical data. Of the 1006 stillbirths, 715 cases were "risk factor pregnancies", 291 non. Results: The stillbirth mortality was classified as follows: -Sudden Intrauterine Unexplained Death (SIUD,"sine materia" and absence of risk factor autopsies),4 cases; -Explained Intrauterine Death ("cum materia" with or without risk factor autopsies),987 cases; -Borderline Intrauterine Death ("sine materia" and presence of risk factor autopsies),15 cases. Conclusion: In the absence of autopsy in 715/1006 cases it was possible to establish the cause of death based on the presence of risk factors. In the absence of risk factors the autopsy showed a certain anatomical cause in 287/1006, only in 4/1006 it was "sine materia".
The role of "traditional" and "tomography" autopsy in foetal Congenital Heart Disease (CHD) F. Portelli * , F. P. Busardò, L. Gutsul, L. Averna, E. Orlando, E. Maresi * University of Palermo, Section of Pathology, Italy
Objective: The role of systematic autopsy in foetal CHD is to identify the morphology of cardiac and possible associated extracardiac malformations (ECM) and the correlation between morphology, suspected clinical etiology and genetics. We evaluated the role of autopsy in 643 foetal CHDs with/without prenatal diagnosis. Method: From January 1990 to December 2010 (Institute of Pathology, Paolo Giaccone -Palermo), 2371 foetal autopsies were performed. The autopsy protocol used was "tomography" for abortions, the "traditional" technique was adopted for stillbirths. Results: In 643 cases CHDs were identified. In 196 cases CHD diagnosis was made only through autopsy, in 447 after a certain (244) or suspect (203) clinical/genetic diagnosis. The etiology of CHD associated to ECM (621) was: chromosomic type (271), syndromic/sequence type (233), association type (117). The etiology of CHD without ECM (22) was never syndromic. Conclusion: "Traditional" and "tomography" autopsy plays a key role in the diagnosis and counselling of CHD, either when it represents the only diagnostic tool (30,48 %) or when it is preceded by a clinical/genetic study. In the latter cases, its value depends on the detection of ECM, useful to "consolidate" a suspect clinical diagnosis (31,57 %) and to "complete" a malformative picture etiologically known thanks to a clinical/genetic analysis (37,9 %). Objective: Deregulation of cell cycle control is a hallmark of cancer. We have examined protein expression and gene amplification of cyclin A in Wilms tumor (WT) and to analyze it in relation to tumor stage, prognostic group and histological type. Method: Real-Time Quantitative PCR was used to detect gene amplification of cyclin A in tumor tissue from 36 patients with WT, while immunohistochemistry was applied to detect protein expression of the same cyclin. Results: Cyclin A gene amplification was found in 4 out of the 36 (11.8 %) cases of WT. Cyclin A protein overexpression was detected in all four cases, but was also found in 84.7 % of cases without detectable gene amplification. So, there was no significant correlation between cyclin A gene amplification and protein overexpression. All cases with amplification of cyclin A were of favorable histological type, intermediate risk group and three out of four cases were low stage WTs. On the other hand, overexpression of cyclin A was found significantly more often in high stage WTs compared to low stage WTs (p=0.04). Conclusion: Cyclin A gene amplification might be associated with the favorable prognosis of WT (low stage, intermediate risk group and non-anaplastic tumors). The gold standard to diagnose is lung biopsy or necropsy. Up to 40 % of cases have mutations or deletions in the gene FOXF1 (cr16q24.1) who plays a crucial role in the development of the lung vasculature. Method: We report a case of a term newborn female affected of ACD/MPV who was diagnosed by a lung biopsy. She died at 38 days of life. Autopsy and molecular diagnosis were also performed Results: In the biopsy and autopsy specimens a decline in the number of capillaries in the alveolar septa and detachment of the epithelial lining was observed. Secondary proximal plexiform arteriopathy, muscularization of arterioles and venous-venular dilatation and proliferation were evident. A pathogenic mutation in the gene FOXF1 (frame shift mutation in the first exon) confirmed our diagnosis. Conclusion: ACD is defined by a decrease in capillaries with alveolar septal thickening and hypertrophy of the middle muscular layer of arterioles. MPV suggests an imbalance of angiogenesis. The FOXF1 mutation helps to prenatal diagnoses of high risk families and to give the diagnosis to patients who fail to perform biopsy or autopsy. Objective: Prognosis of Rhabdomyosarcoma (RMS) has improved significantly over the last 20 years. Overall survival (OS) is>80 % in the majority of patients; even though, children with metastatic tumors have a dismal prognosis with an OS <30 %. Method: We analyzed in 18 cases, the possible correlation of Notch activation with histology, presence of metastasis and outcome. Immunohistochemistry (IHC) was performed for the Notch downstream effectors Hes1 and Hey1. Results: Hes 1 was strong or moderate in 95 % of the cases. Hey1 was positive in 50 %. 3 out of 4 alveolar RMS (aRMS) were positive for Hes1 and 1 was positive for Hey1. Both stains were negative in the fusion-negative aRMS. In embryonal RMS (eRMS), eight out the 9 were Hey1 positive and 14 of the 17 were Hes1 positive. The 2 patients whit metastases had staining for Hes1 and Hey1. 3 patients who died showed Hes1(+). 14 of the 15 patients in remission showed positivity for Hes1 and 7 for Hey1. Conclusion: Hes1 expression was found in the majority of RMS while Hey1 was more eRMS specific. Is no correlation between pathway activation, metastasis or outcome. The blockage of the pathway with specific inhibitors could offer a new therapeutic option for this patients.
A renal epithelioid angiomyolipoma in a young woman with tuberous sclerosis complex, cortical tubers by neuroimaging, facial angiofribromas and lung lymphangioleiomyomatosis J. Trillo-Tinoco * * Hospital General de Mexico, Dept. of Surgical Pathology, Mexico City, Mexico
Objective: Tuberous sclerosis (TS) is a genetic disorder affecting cellular differentiation and proliferation, which results in hamartoma formation in many organs like skin, brain, lung, kidney and heart. Method: A seventeen-year-old female with history of facial angiofibromas a 10 years before, right renal tumor diagnosed a year before as theratoid-rhabdoid tumor, with chemoradiation as adjuvant treatment, cortical tubers by neuroimaging a few months before, and diagnosis of lung lymphangioleiomyomatosis recently. After last diagnostics, we reviewed again the renal tumor, new orientation of tissue, with new histological sections were performed. (Fig) Results: At histological examination, the kidney showed an infiltrating tumor, very cellular, consisting mainly of polygonal cells with eosinophilic cytoplasm, other multinucleated similar to the ganglion cells, also small hamartomatous areas with smooth muscle, fat and blood vessels proliferation. The tumor cells expressed HMB45, MART-1, SMA, VIM and CD10, CKAE1/3 were negative. (Fig)  Conclusion: The majority of renal angiomyolipomas is sporadic and 50 to 80 % occurs as part of TS, and their partnership is more close with epithelioid variant, recently, a rare entity with aggressive behavior, difficult histological characterization and poor prognosis.
Pulmonary mast cells in sudden infant death syndrome (SIDS) C. Zaharia * , C. Loddo, P. Schmidt, R. M. Bohle * University of Saarland, Inst. of Pathology, Homburg, Germany
Objective: Several theories of the underlying mechanisms of SIDS have been proposed, one of them is focusing on shock including anaphylaxis. Increased concentrations of mast cell tryptase in post mortem blood have been observed without increased mast cell numbers in lung tissue. The aim was to evaluate the age-related distribution of pulmonary mast cells in infants dying of sudden infant death syndrome and controls. Method: 6 infants (up to 1 year of age) who died of SIDS and 23 controls who died of other non-pulmonary causes were examined. Peribronchial mast cells exhibiting tryptase immunoreactivity were evaluated and quantified in high power fields in lung sections. Results: The number of mast cells in peribronchial regions amounted to 18,9 (± 6,6)/mm2 in children aged 1 month up to 13 months. Mast cells in SIDS cases were 21,2 (± 4,5)/mm2. The difference was not significant (P=0,113, Student'st test). Conclusion: It is unlikely that increased pulmonary mast cells are indicators of SIDS. The role of mast cells in SIDS remains controversial.
Trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia S. Zekic Tomas * , I. Kuzmic Prusac, D. Roje, I. Tadin * Clinical Hospital Centre Split, Pathology, Croatia Objective: To assess trophoblast apoptosis separately in cytotrophoblast, syncytiotrophoblast, total villous trophoblast and syncytial knots, as well as to investigate the expression of apoptotic factors Fas ligand (FasL), Bcl-2 and proliferation marker Ki-67 in trophoblast of placentas from preeclamptic patients. Method: The study included placental samples from 25 preeclamptic and 25 normal pregnancies. For the detection of apoptosis and proliferation antibody M30 and antibody against Ki-67 antigen were used. Expression of Fas ligand and Bcl-2 was assessed using semi quantitative HSCORE method. Syncytial knots were expressed as the number of syncytial knots per individual villus and as the total number of syncytial knots in each placental sample.
Results: apoptosis in all stages of trophoblast differentiation, number of syncytial knots per individual villus and the total number of syncytial knots were significantly higher in preeclamptic placentas than in control group placentas. Fas ligand expression was significantly less, and Bcl-2 expression significantly greater in the villus trophoblast among the study subjects compared with controls. There was no difference in the trophoblast proliferation between groups. Conclusion: our findings might suggests that increased apoptosis and syncytial knots formation combined with reduced Fas ligand expression could be involved in pathophysiological mechanisms of preeclampsia.
Contribution of fetal autopsy for diagnosis of Meckel-Gruber Syndrome M. Jo * , I. Guerra, G. Perez de Nanclares, P. Morales, J. J. Aguirre, Z. S. Quintero, C. Gomez, N. T. Villagra * Hospital Txagorritxu, Pathology, Vitoria, Spain
Objective: Meckel-Gruber Syndrome (MGS) is a lethal rare autosomal recessive malformation. The six implicated genes encode proteins involved in primary cilia function. Groups of families in Finland, India and North of Africa have been identified. Method: A fetus,46 XY, karyotype, therapeutically aborted at 21 weeks with alobar holoprosencephaly in ultrasound. Fetal autopsy was performed. Genetic counseling was proposed to the family revealing Moroccan origin and consanguinity, the parents were first cousins. Results: A male fetus showing cyclopia, proboscis and a single opening with two rudimentary eyes was the external morphology. Histological examination confirmed holoprosencephaly and also showed bilateral corticomedullary renal multicystic and periportal hepatic fibrosis with bile duct dilatation. The main diagnosis was MGS although other ciliophaties and non-ciliopathies conditions were considered, such as Bardet-Bield, Joubert, Smith-Lemli-Opitz syndrome and trisomy 13. However, the pathological characterization, parent's consanguinity and their North African origin makes MGS the likely diagnosis.A genetic study on paraffin-embedded material was requested, the poor quality of DNA stopped definitive genetic diagnosis. Conclusion: The pathologist may encounter atypical cases that require morphologic diagnosis to determine the type of underlying mutation and provide genetic counseling to parents.A meticulous autopsy is necessary to establish the diagnosis of MGS. Fresh material would have to be frozen in order to make current diagnostic techniques of molecular pathology.
Mesenteric cysts in the pediatric age group R. Jankovic * , J. Sopta, M. Stojanovic, B. Lekic, B. Jovanovic, Z. Stojsic * University of Belgrade, Faculty of Medicine, Serbia
Objective: Mesenteric cysts are extremely rare lesions arising with an incidence of 1/20,000 admissions in children. Clinically, mesenteric cysts are generally comprehended as a unique diagnostic entity, although they exhibit histological diversity. The objective of this study was to determine the incidence and the histology of the mesenteric cysts in the pediatric age. Method: All cases of mesenteric cysts operated at the University Children's Hospital Belgrade over the 10-year period of 2002 to 2011 were reviewed using pathology reports from the files of the Institute of Pathology. Histological slides were re-examined and immunohistochemistry was applied, when necessary. Results: A total of 27 cases of mesenteric cysts were identified. Cysts of lymphatic origin (cystic lymphangiomas) were recorded in 12 patients (44 %), cysts of enteric origin -in 14 patients (52 %): 13 duplication cysts and one isolated enteric cyst of the mesenterium. Only one example of the cyst of mesothelial origin, i.e. benign cystic mesothelioma was diagnosed (4 %). The most frequent site was mesenterium and mesocolon (86 %), followed by omentum (7 %) and the retroperitoneum (7 %). Conclusion: Cysts of enteric origin are easily recognized. It is important to differentiate between cystic lymphangioma and cystic mesothelioma due to their different natural history. Objective: Myxoid liposarcoma belongs to the group of soft tissue sarcomas with lipomatous differentiation. Breast is a rarely affected (only 0.3 % of breast sarcomas), and often misdiagnosed. Method: A 67-years old woman presented with a painful timorous lump of the left breast. Mammography showed oval, lobulated lesion between medial quadrants, while ultrasonography revealed hipoechogenic, inhomogeneous nodule. Patient underwent core biopsy which was histologicaly inconclusive and followed by quadrantectomy with excision of both medial quadrants.
Results: A lump (6 cm) was visible on the skin surface. Serial sectioning revealed solid gray-white tumor with cystic and prominent necrotic areas. The tumor was located in the deep mammary tissue an it infiltrated the overlying dermis without involving the epidermis. It consisted of atypical stelate and spindle cells with infrequent mitoses and low Ki-67 proliferative index. On the periphery mature adipocytes and lipoblasts were present. Stroma was abundant, myxoid with plexiform capillary pattern. The myxoid substance stained slightly Alcian-blue positive and tumor cells showed cytoplasmic immunopositivity for S100. Phylodes tumor was excluded because of absence of epithelial component. Conclusion: Myxoid liposarcoma has a distinct morphology, rarely confused with other soft tissue tumors, although on cytological smears or biopsy samples it may be unrecognized. Objective: Synovial sarcoma poses a difficult diagnostic challenge since it can be confused with other benign or malignant entities. Method: Four biphasic and one monophasic synovial sarcomas were studied. We used the break apart/split signal kit (VYSIS) to detect the t(X;18) translocation and we performed immunohistochemistry for TLE-1, INI-1, D2-40, CD56, CD99, bcl-2, EMA, CK7, CD34, S-100, Desmin and Claudin-1. Results: Immunohistochemically we observed: TLE-1+(5/ 5), INI-1 (4/5) with reduced nuclear and (1/5) with no expression, D2-40+(1/5), CD56+(5/5), CD99+(5/5), bcl-2 +(5/5), EMA focal expression (5/5), CK7−(5/5), CD34−(5/ 5), S-100−(5/5), Desmin−(5/5), Claudin-1 + (5/5). FISH revealed the split signal between the centromeric and telomeric end of the SYT gene. Conclusion: t(X;18) translocation remains the diagnostic hallmark of synovial sarcoma. INI-1 which is typically negative in atypical teratoid/rhabdoid tumor, epithelioid sarcoma and myoepithelial carcinoma seems to be reduced or even negative in synovial sarcomas. The focal expression of D2-40 must be considered when the differential diagnosis includes mesothelioma. Finally TLE-1 is a very sensitive marker for synovial sarcoma.
PS-16-004 "Mixed aneurysmal bone cyst" and "simple bone cyst", represent a different group of cystic lesion E. Ayhan Cinar * , B. Doganavsargil, M. Sezak, F. Oztop * EGE University, Pathology, Izmir, Turkey
Objective: There are some mixed cysts (MC) with overlapping histological features of aneurysmal bone cyst (ABC) and simple bone cysts (SBC). Method: We reviewed 69 pure ABC, 40 SBC and 23 "Mixed Cysts (MC)", and compared them by nonparametric tests. Results: MCs, mainly showed two paterns as "Type 1: SBC with secondary ABC foci (n=18)"and "Type 2: Cysts with fully developed SBC and ABC areas (n=5)". The median age for ABC, SBC, and MC were 16±18.2 (range: 3-53), 21±13.7 (range: 3-54) and 9±11.47 years old (range: 3-50) respectively. MCs were more frequent in males than ABC and SBC (p=0.017) and the most frequently involved bones were humerus-femur-pelvic bones in descending order (p= 0,021). "Type 1 MC" showed more frequent cementum-like amorph material (p<0.001), ectatic venules (p=0.040), calcifications (p=0.044), less cholesterol deposition, necrosis and no different fracture and osteoid matrix when compared with Type 2 MCs and SBCs. Conclusion: Cysts with overlapping features of ABC and SBC are not uncommon. Though they usually represent "SBCs with secondary ABC component", they have dissimilarites with ordinary SBCs which needs to be further clarified with larger series. Objective: Percent of tumor necrosis after neoadjuvant chemotherapy, determined by detailed specimen mapping, has a high prognostic value. However both the mapping and grading systems has some practical problems. Method: 128 cases were reviewed for potential pitfalls in handling and reporting processes and correlated with radiological findings by nonparametric-tests. Results: Most of the tumors were osteosarcoma (74.2 %) and Ewing Sarcoma (16.4 %), located in femur (53 %), tibia (22,7 %) and humerus (10.2 %). Cortex, soft tissue and joint invasion was observed in 94 %, 77.6 % and 30.3 % of the cases, respectively. A median of 27.5 tumor blocks (9-74± 14,64) were submitted for histology. Tumors were totally necrotic in 30 cases (24 %) (Huvos Grade-HG-IV), HG III in 20.3 %, HG II in 28.1 % and HG I in 24.2 % of the cases. Overall good (HG III-IV) and poor responders (HG I-II) correlated well with radiological findings (p<0.001). Discrepant cases showed extensive oedema and congestion enhancing contrast medium, patchy necrosis unabling accurate histologic evalation, or failure to prove tibiofibuler joint involvement because of sagittal sectioning (p<0.05).
Conclusion: Evaluating chemotherapy responce is a laborious work. Radiologic orientation prior to grossing and applying morphometric technics may enhance more accurate evaluation.
Atypical Ewing sarcoma/Primitive neuroectodermal tumor with unusual melanocytic differentiation -A case report P. Buzrla * , J. Dvorackova, I. Urbanovska, H. Bielnikova * Inst. of Pathology, Ostrava-Marianske Hory, Czech Republic
Objective: Atypical Ewing sarcoma/primitive neuroectodermal tumor with the melanocytic differentiation is a very rare, malignant tumor which occurs in infants and adolescents. It is localized mainly in soft tissues. Its biological behavior is aggressive, but its response to the chemotherapy is prognostically good. Method: A 4-month old male is presented with the 17×11 cm tumor of the soft tissue, localized in the right front over the cranial margin of the orbit. The tumor was surgically exstirpated and then sent to the histological investigation with the additional immunohistochemistry and the FISH. Results: Microscopically, the tumor is hypercelullar and consisted of oval to polygonal cells with the vesicular nuclei, the prominent nuclei and the amphophilic cytoplasm. In other parts of the tumor, there are smaller neoplastic cells with hyperchromatic nuclei. Some neoplastic cells contain the melanin pigment in the cytoplasm, which was positive in Fontana-Masson method. The immunohistochemical stains for vimentin, AE1/3, CK7, CD99, HMB-45, FLI-1 were positive. The FISH investigation demonstrated the translocations (t 11;22, q24;q12) in 95 % of neoplastic cells and (t 21;22, q12;12) of 5 % ones.
Conclusion: In literature, the Ewing sarcoma/PNET showing the myogennic differentiation is usual, but the melanocytic differentiation is rare and can be confirmed not only immunohistochemically, but also by FISH investigation. Objective: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in the repair of DNA single-strand breaks. PARP-1 inhibitors may be efficient in therapy of malignancies. This report evaluates the expression of PARP-1 in primary testicular germ cell tumors and correlates expression patterns with histological subtypes and patient/ tumor characteristics. Method: Group of 124 patients with testicular germ cell tumors were investigated for PARP-1 expression by immunohistochemistry, scored by the multiplicative quickscore (QS) method and compared to PARP-1 expression in normal testis. Results: We observed higher expression of PARP1 in testicular tumors compared to normal testis (mean QS=10.04 vs. 3.60, p< 0.0000001). The PARP-1 overexpression (QS>9) was most often detected in intratubular germ cell neoplasia ITGCN (100 % of specimen with PARP-1 overexpression), compared to 1.9 % of normal testicular tissue specimen. There was no association between PARP-1 expression and clinical variables. Conclusion: PARP-1 expression is higher in tumor tissue than in normal testis. PARP-1 could represent a novel treatment target in TGCTs and the assessment of PARP-1 expression in tumor samples may lead to the consideration of TGCTs patients for PARP inhibitor therapy. Supported by 2007/30-NOU-01, VEGA 1/0724/11 and ITMS: 26240220052 cofinanced by European Regional Development Fund. Objective: Gardner fibroma (GAF), a rare lesion, typically occurs in infants, children and young adults, with predilection for the trunk (particularly paraspinal region) and no gender predominance. GAF is an ill-defined plaque-like mass, with rubbery consistence, usually asymptomatic, ranging from 0.3 to 12 cm. Although benign, 70-90 % of the cases may be associated with APC mutations, familial adenomatous polyposis (FAP) and/or Gardner Syndrome (GS). We report 2 cases of GAF, which allowed the identification of 2 families with GS. Results: CASE 1: 7-month-old boy, with 2 nodules in the left paraspinal region, the largest with 2.5 cm. CASE 2: 5month-old boy, presenting a 1.5 cm ill-defined deep right scapular mass. Both lesions were infiltrative, paucicelular densely collagenized proliferations of bland spindle cells. A GAF diagnosis was made and clinical/genetic investigation advised. APC gene sequentiation revealed, in both cases, a frameshift germline mutation in exon 15. Additionally, case 1 presented a del(5)(q11q35). Heredogram showed typical manifestations of GS: CASE 1: Mandibular osteomas, epidermoid cysts, "soft-tissue tumours", colo-rectal carcinoma. CASE 2: Hepatoblastoma, colo-rectal carcinoma. Conclusion: Given the high association with GS and the fact that it can be its first manifestation, GAF is considered a sentinel lesion for this syndrome. Therefore, an accurate diagnosis is of the utmost importance.
Prognostic significance of bcl-2, c-myc and survivin in synovial sarcoma D. Demir * , B. Yaman, Y. Anacak, B. Keçeci, G. Kandiloğlu, T. Akalın * Ege University, Pathology, Izmir, Turkey
Objective: In this study, we evaluated 81 synovial sarcoma cases, who had been referred to our tertiary tumor center during the last 20 years. We applied bcl-2, c-myc and survivin as immunohistochemical markers and evaluated the relation with conventional prognostic findings and prognosis for those 64 patients who have follow-up. Method: In this study; ten-year tumor free survival rate was 38 % reflecting the agressive behaviour of synovial sarcoma. Tumor grade was the most valuable prognostic input. Progression free survival (PFS) was 159 months for gradeII cases (40 cases) and 36 months for gradeIII cases (24 cases) (p=0,000). Results: Immunohistochemically, there was weak relation between bcl-2 staining intensity with prognosis. Overall survival was 211 months for weak or negative cases (9cases), 132 months for focally intense cases (21cases) and 101 months for diffuse and intense cases (34 cases) (p= 0,042). There was also weak relation with c-myc staining pattern with prognosis. Overall survival was 193 months for c-myc negative cases (25cases), 114 months for cytoplasmic positive cases (23cases) and 68 months for nuclear positive cases (16 cases) (p=0,043). There was no relation between survivin and prognosis. Conclusion: In conclusion; tumor grade is the most valuable prognostic parameter in synovial sarcomas. Immunohistochemically c-myc and bcl-2 staining have weak relation with synovial sarcoma prognosis.
Immunohistichemical profile of primary and recurrent desmoids E. Dubova * , T. Sidorenko, A. Shchegolev, A. Adamyan * V.I. Kulakov Scientific Center, Dept. of Pathology, Moscow, Russia
Objective: Desmoids is a locally recurrent and invasive but not metastatic tumor. Our aim was to study immunohistochemical profile of primary and recurrent desmoids. Method: Complex morphological study of 16 abdominal desmoids. All the tumors were divided into 3 groups: primary, first recurrent and second and more recurrent cases. We used antibodies against β-cathenin, COX-2, APC, survivin and Ki-67 for immunohistochemical study. Results: Nuclear and cytoplasmic β-cathenin expression levels were significantly higher in recurrent than in primary desmoids. APC cytoplasmic expression level was also significantly higher in recurrent tumors. We revealed only cytoplasmic COX-2 expression in desmoids, and its level was significantly higher in recurrent tumors with the activity increasing accordingly to the number of recurrences. Survivin expressed both in nuclei and cytoplasm of the tumor cells and its expression levels were significantly lower in recurrent desmoids. Conclusion: Revealed immunohistochemical properties of primary and recurrent desmoids reflect tumor transformation and progression and could be used as additional prognostic markers in this disease. Objective: Many bone and soft tissue sarcomas (BSTS) are aggressive tumors with fatal prognosis. The importance of angiogenesis for the growth and progression of solid BSTS is now well recognized. A variety of chemokines have been described that either promote (angiogenic) or inhibit (angiostatic) angiogenesis. The aim of this study is to characterize the expression profile of some chemokines in a series of xenotransplanted human BSTS. Method: One Ewing sarcoma (ES), 1 grade 3 chondrosarcoma (Chs), 1 osteosarcoma (Os), 1 synovial sarcoma (SS), 1 fibrosarcoma and 1 gastrointestinal stromal tumor (GIST) were xenotransplanted into the backs of nude mice (athymic Balb-c nude mice). When the tumor size reached the 3 cm, animals were sacrificed and tumors analyzed for the expression of CXCL1/2/3, CXCL9 and CXCL10, using two-color staining fluorescence on each slide under a confocal microscope (Olympus FV1000). Results: We observed that angiostatic chemokines (CXCL9 and CXCL10) presented higher expression than angiogenic (CXCL1/2/3) chemokines in ES, grade 3 Chs, Os and GIST whereas fibrosarcoma and SS were more positive for angiogenic chemokines. Conclusion: The expression profile of angiostatic and angiogenic chemokines depends on the type of BSTS and could be related to their different biological behaviour. Other elements such as angiogenic or pro-inflammatory markers should also be considered. Objective: The wide range of differential diagnostic possibilities of rhabdomyosarcomas (RMS) shows the need for more specific and sensitive markers. Low survival rates of high risk RMS patients require new prospective therapeutic targets. Method: Archival material FFPE samples of 15 RMS (12 embryonal -eRMS, 3 alveolar -aRMS) and non-RMS soft tissue tumors were evaluated by immunohistochemistry for myogenin, MyoD1, EGFR, VEGF, COX-2, p-Akt and p-mTOR expression. The presence of PAX3/7-FKHR forming translocations, myogenin, MyoD1, gamma subunit of fetal acetylcholine receptor (AChR) and K-RAS mutational state were determined by RT-PCR. Results: 10/12 eRMS and 3/3 aRMS showed expression of myogenin, MyoD1 and gamma AChR. Non-RMS tumors were negative. Translocations were only found in aRMS. EGFR expression was characteristic for eRMS, without the presence of activating K-RAS mutation. Strong expression of VEGF was detected in all samples. p-AKT and p-mTOR showed overlapping expressions in 86,7 % RMS. In most of the samples weak COX-2 positivity was demonstrated. Conclusion: Myogenin, MyoD1 and the fetal AChR are specific and sensitive diagnostic markers of RMS. RMS subtypes are identifiable by PAX3/7-FKHR detection and probably EGFR expression. The results indicate VEGF, EGFR, COX-2 and Akt-mTOR pathway directed therapy to be considered in RMS. Supported by ITMS 26240220052.
Immunohistochemical analysis of potential targets in desmoid tumor therapy A. Janegova * , Z. Hlavata, P. Babal, P. Janega * Comenius University, Dept. of Pathology, Bratislava, Slovakia
Objective: Desmoid tumors (DTs) are clonal fibroblastic/ myofibroblastic proliferations. Although histologically benign, desmoids are locally invasive and often have an unpredictable clinical course. DTs are infrequent lesions, but they have a high incidence in patients with familial adenomatous polyposis (FAP). The treatment of DTs needs to be individualized. Method: To explore the molecular bases of potential pharmacologic targets in DTs we evaluated the immunohistochemical expression of steroid hormone receptors (ERa, ERb, PR) and COX-2 protein in sporadic (n=6) and FAPassociated desmoid tumors (n=9) together with GI adenomas of FAP patients (n=5). Results: Nuclear ERb expression was found in 14/15 DTs. All adenoma samples showed nuclear ERb positivity, which was weaker than in the surrounding normal epithelial cell. ERa and PR expression were lacking in all samples. COX-2 was found in 14/15 DTs. Adenomatous polyps showed intense COX-2 expression compared to surrounding normal mucosa. Conclusion: High incidence of ERb positivity in DTs supports the usage of hormonal therapy in these lesions. Open question is the effect of anti-estrogen therapy in DT patients with adenomatous polyps, as in adenomas estrogen seems to have preventive potential. COX-2 expression suggests the benefit of anti-inflammatory treatment in DTs adenomatous polyps. Supported by ITMS: 26240220052.
Retroperitoneal sarcomas: Clinicopathological features in a series of 68 cases E. Kairi-Vasilatou * , A. Tsagkas, A. Melloy, A. Paraskeva, A. Kondi-Pafiti * Aretaieio Nosokomeio University, Dept. of Histopathology, Athens, Greece
Objective: Retroperitoneum is the less common site of origin accounting for approximately 10 % of soft tissue sarcomas. Method: Between January 1990 and December 2010, our hospital's records of 68 patients with retroperitoneal sarcomas were retrospectively studied.
Results: The patient median age was 57 years and there was no sex predominance. Median tumor size was 18.5 cm (ranging from 6 to 55 cm) with 60 % of them being larger than 10 cm. The most common histological type was liposarcoma (36/68-52,9 %), followed by leiomyosarcoma (16/68-23,5 %) and undifferentiated soft tissue sarcoma (7/68-10,2 %). The remaining 9 tumors (13,2 %) included 2 chondrosarcomas, 2 well-differentiated fibrosarcomas, 1 PEComa, 1 hemangiopericytoma, 1 Ewing sarcoma and 1 malignant peripheral nerve sheath tumor. 44 of the sarcomas were high grade (65 %) and 24 (35 %) low grade. 1-year recurrence rate was 34,3 %. The 3-and 5-year overall survival rates were 56,2 % and 53,1 % respectively. Seven patients received adjuvant chemotherapy. Conclusion: The most commonly encountered histologic subtypes are liposarcoma and leiomyosarcoma, which are consistent with the results of the present study. Complete tumor resection at first operation is the only treatment factor that consistently predicts improved survival.
Abdominal desmoid tumor Y. Lorenzo Mahia * , M. San Martin Alonso, B. Iglesias Rodriguez * Hospital Maixoeiro, Anatomia Patologica, Vigo, Spain
Objective: Abdominal desmoid tumours are rare benign neoplasms. They are commonly found in the mesentery, while they are rarely found in the intestinal wall. Most cases are sporadic, although there is a link with colonic polyposis, trauma, and oestrogen. They predominate in 25-35 year-old women. This present case is notable due to its location in the jejunal wall, possible relation with previous surgery, and the age and sex of the patient. Method: We present a 51-year-old male patient previously operated for umbilical hernia years before. He came to the consultation on noticing an abdominal mass. Since the initial suspicion was of jejunal wall GIST tumour, surgery was performed. Results: The histopathological and immunohistochemical findings, support the diagnosis of desmoid tumour. Conclusion: Desmoid tumours consist of fibroblastic monoclonal proliferation developed from aponeurotic muscle structures. Some authors consider them non-neoplastic processes given their limited aggressiveness while others classify them within distinct low-grade sarcomas. Their origin is not well established, although there are known factors involved such as mutations in the APC gene or beta-catenin and trisomy 20 and 8. The originating cell, the myofibroblast, is involved in post-traumatic cellular regeneration. This explains why we find these tumours associated with previous surgery.
Treatment of advanced dermatofibrosarcoma protuberans with imatinib mesylate with or without surgical resection W. Michej * * Cancer Centre Institute of Warsaw, Dept. of Pathology, Poland
Objective: Dermatofibrosarcoma protuberans (DFSP) is a rare soft tissue sarcoma of the skin characterized by the presence of specific COL1A1-PDGFB fusion protein, which appears as a consequence of the t(17;22) (q22;q13) translocation. Method: The aim of the study was to perform an analysis of patients with advanced DFSP treated with imatinib, with or without surgery. We examined 15 patients (6 male, 9 female; median age 56 years) with locally advanced/initially inoperable and/or metastatic DFSP treated with imatinib 400-800 mg daily between 12/2004 and 06/2009. All diagnoses were ascertained cytogenetically (fluorescent in situ hybridization). Median follow-up time was 16 months (range: 4-81). Results: Metastases were present in 8 cases (two lungs, two soft tissue, two lymph nodes). Fibrosarcomatous transformation was confirmed in 7 patients. After treatment with imatinib overall responses were: 10 partial responses, 2 stable diseases (13 %) and 3 progressive diseases (20 %). Seven patients (47 %) after resection had residual disease confirmed by pathologic examination and remained free of disease. Conclusion: We proved that anti-tumour effect of imatinib in DFSP with presence t(17;22) had in most cases good responses. Imatinib therapy may in some cases leads to tumour resection because of lesser size. Objective: At an estimated incidence of 2 cases per million per year, osteosarcoma is the most common malignant primary bone tumor. Method: We conducted a retrospective study to identify the osteosarcoma cases diagnosed at major tertiary care hospitals in Turkey. Results: Our study group was made up of 745 cases: 440 men, 305 women, aged 3-82 years (mean 23.7 year). All patients had been diagnosed with skeletal osteosarcoma between 2001 and 2011 at one of the 10 tertiary care referral center. Tumor was most frequently located in femur (50.4 %) followed by tibia. Apart from the long bones, pelvic and gnatic bones were the next in location, 4.4 % and 1.6 % respectively. There were 16 (2.1 %) secondary osteosarcomas, related to previous irradiation and various underlying diseases. For the histological types conventional osteblastic intramedullary tumors were most prevalent, making 71.8.% of the cases. Chondroblastic and telengiectatic osteosarcoma are the next common histologic types. Surface tumors were detected in 51 (6.8 %) cases. Rare histological types like small cell, epitheloid, chondroblastoma like and fibrous dysplasia like were also reported. Conclusion: This study is conducted as a preliminary work to form the basis of a pathologic database for the osteosarcoma cases diagnosed in our country. Objective: Kaposi's sarcoma is a rare disease likely associated with human herpes virus 8 infection, and occurs predominantly in Jewish, Mediterranean and middle eastern men. Since there is a paucity of reports on the pattern of its occurrence in Tunisia, we here analysed the epidemiological pattern and anatomoclinical features. Method: We retrospectively studied 71 consecutive cases of Kaposi's sarcoma diagnosed in the Pathology Department, Farhet Hached Hospital, Sousse during a 15-year period.
Results: Kaposi's sarcoma represented 21 % of soft tissue sarcomas. There were 23 (32.4 %) females and 48 (67.6 %) males (male-to-female ratio: 2.1:1). Median age at diagnosis was 69 (range: 10-98 years). The age distribution showed that elderly (≥60 years) were the most affected patients with a frequency of 66.2 %, followed by patients aged 31-59 years (25.3 %), and patients under 30 years (8.4 %). The most common location was the lower limbs, particularly the distal lower extremity (73.3 %), followed by contiguous location (7 %), and soft tissue, NOS (7 %). Conclusion: Kaposi's sarcomas were more frequently diagnosed in elderly. The distal lower extremities were more involved. Objective: Rhabdomyosarcoma is the most common soft tissue sarcoma in the first two decades of life. In this study, we analysed the epidemiological pattern and antomoclinical features of rhabdomyosarcoma in Central Tunisia. Method: We retrospectively studied all cases of rhabdomyosarcoma diagnosed in the Pathology Department, Farhet Hached Hospital, Sousse during a 15-year period. Results: There were 15 (31.9 %) females and 32 (68.1 %) males (male-to-female ratio: 2.1:1). Median age at diagnosis was 9 (range: 0-85 years). Rhabdomyosarcoma was more frequently diagnosed in childhood (63 %) than in adults (37 %). In children, the tumour size was higher than 5 cm in 73 % of cases, the embryonal subtype was the most frequent (60 %) and the two most common sites of disease were the head and neck (50 %) and genito-urinary tract (23.3 %). In adults, the tumour size was higher than 5 cm in 90 % of cases, the pleomorphic subtype was the most diagnosed (41 %), and limbs were the most involved sites (41 %). Conclusion: Rhabdomyosarcomas is more frequently diagnosed in children than in adult. Head and neck locations were the most involved and embryologic type was the most diagnosed. In adults, rhabdomyosarcomas were more frequently localized in limbs and diagnosed as pleomorphic type.
Microarray-based DNA methylation study in Ewing sarcoma of bone H.-R. Park * , Y.-K. Park * Hallym University, Sacred Heart Hospital, Anyang, Republic of Korea Objective: Alterations in the DNA methylation pattern are a hallmark of malignancy and also of Ewing sarcomas. However, most epigenetic studies in Ewing sarcoma have focused on the analysis of few candidate genes and comprehensive studies are required. Method: Here, we report for the first time a microarraybased DNA methylation study of 1505 CpG sites of cancerrelated 807 genes in 69 Ewing sarcomas. We used Illumina's GoldenGate Methylation Cancer Panel I microarray.
Results: Using appropriate controls (n=14), we identified a total of 104 CpG sites hypermethylated in Ewing sarcoma. Most of hypermethylated genes are related with cell adhesion, cell regulation, development, and signal transduction. We compared the methylation mean of each tumor according to the survival data. The methylation mean was significantly higher in the alive patient group (0.25±0.03) compared to the dead patient group (0.22±0.05) (p=0.0322). However, the methylation mean was not significantly correlated with age, sex, or tumor location. We selected the most popular hypermethylated genes, GDF10, OSM, APC, and HOXA11, but, their methylation levels were not significantly correlated with the survival data. Conclusion: We have characterized the DNA methylation profile of Ewing sarcomas and detected 104 CpG sites that were significantly hypermethylated in Ewing sarcomas. These might therefore play an important role in the development of Ewing sarcomas.
Pleomorphic and dedifferentiated leiomyosarcoma associated with Lynch syndrome: A case report H. Quiceno * , F. J. Queipo, R. Carías, J. J. Sola, F. J. Pardo * Clinic University of Navarra, Anatomical Pathology Lab, Pamplona, Spain
Objective: The Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC) is a hereditary syndrome that predisposes to different types of colonic and extracolonic cancer, mainly in endometrium, stomach, ovary, and hepatobiliary and urinary tract. Occasional sarcomas have been reported in HNPCC patients. Method: We describe a pleomorphic and dedifferentiated leiomyosarcoma of the gluteus in a patient with Lynch syndrome. We study her clinical, pathology, immunochemistry and molecular alterations.
Results: We present a 71 year old woman with previous colorectal, endometrium and breast cancers in a Lynch syndrome with mutations in exons 1 and 2 of MLH2, which appeared with a right gluteus mass. Grossly, it was encapsulated, whitish-yellowish with necrotic areas and measured 7,2 cm. Microscopically, the tumor was composed by a diffuse and polymorph spindle cells with fascicular pattern, focally myxoid and zones of necrosis and large hyalinization. The cells had large nuclei, sometimes giant multinucleated, with a brisk atypical mitosis activity. There was IHQ variability: positivity to desmine, MSA, calponin and negative to caldesmon and SM. There was loss of MSH2 and MSH6 repair proteins. Conclusion: We must suspect a Lynch Syndrome relational sarcomas if we found one in a patient with HNPCC in order to test to mismatch repair proteins.
Immunohistochemical review of 42 synovial sarcomas, including expression of TLE1: A "new" marker, with molecular confirmation in 21 cases B. Rekhi * , R. Basak, S. Desai, N. Jambhekar * Tata Memorial Hospital, Dept. of Pathology, Mumbai, India
Objective: Synovial sarcoma displays a wide clinicopathological spectrum and a specific translocation t(X; 18)(SYT-SSX)(p11.2; q11.2). Cost constraints limit molecular confirmation in every case. Lately, TLE1 has been recognized as a useful IHC marker. Herein, we present IHC review of 42 synovial sarcomas, including TLE1 expression. Method: Forty-two synovial sarcomas included monophasic spindle-cell type (26) (61.9 %), biphasic 13 (30.9 %), calcifying (2) (4.7 %) and poorly-differentiated type (1) (2.3 %). 21 tumors were confirmed with molecular analysis. TLE1 immunostaining was graded from 0, 1+, 2+, 3+, with 2+ or 3+ grades interpreted as positive staining. Results: On IHC, various synovial sarcomas were positive for EMA (26/34)(76.4 %), CK7 (6/10) (60 %), CK/MNF116 (6/21)(28.6 %), BCL2 (36/37)(97.3 %), MIC2 (23/ 31)(74.1 %) and TLE1 (40/42)(95.2 %), while negative for CD34 (0/21). Among 70 other tumors, TLE1 was positive in schwannomas (5/5) (100 %), neurofibromas (2/2)(100 %), MPNSTs (2/12)(17 %) and PNETs (4/10)(40 %). Sensitivity and specificity of TLE1 for synovial sarcoma was 95.2 % and 72 %. 50 % of PNST (75 % of schwanomas, 50 % of MPNST and 25 % of neurofibromas). In contrast, HER3 positivity was observed in 20 % of other mesenchymal tumors (28 % uterine and 9 % non-uterine leiomiomas, 18 % uterine and 25 % nonuterine leiomiosarcomas, 29 % UPS). Conclusion: HER3 overexpression is frequently found in PNST, including 50 % of MPNST. Due to the crucial role of HER3 receptor in cell signalling as a main activator of the PI3K pathway, these results support the rationale of developing new therapeutic approaches in MPNST. Objective: Nonneural granular cell tumour (NNGCT) is a rare neoplasm described in 1991 by Leboit et al. Till now, no more than 35 cases have been presented in the literature, nearly all were limited to the skin. We report the first NNGCT of the breast and present its morphological and immunohistochemical characteristics. Method: A 39 year-old female underwent lumpectomy because of tumour of the breast. After routine histological examination, the immunohistochemical studies were performed using commercially available antibodies against cytokeratins (AE1/AE3), CD68, CD1A, S100, CD34, smooth muscle actin, desmin, miogenin, inhibin, and Ki67 antigen (MiB1). Results: Microscopically, the tumour was composed of ovoid and polygonal cells with abundant granular, eosinophilic cytoplasm. The cells presented mild or moderate polymorphism and trace mitotic activity. They were arranged in diffuse sheets with no evidence of nesting or fasciculation. The histological texture of the tumour resembled that of classical granular cell tumour of Abrikosoff. However, the tumour cells were strongly immunoreactive for CD68 and inhibin, whereas the reactions against S100 protein as well as other antigens studied gave negative results. The value of Ki67 index did not exceed 2-3 %. Conclusion: The differential diagnosis of granular cell myoblastoma (Abrikossoff tumour) should include the nonneural granular cell tumour.
Low grade fibromyxoid sarcoma: A study of 10 cases K. Shelekhova * , A. Konstantinova * Petrov´s Research Institute for Oncology, St. Petersburg, Russia
Objective: Low grade fibromyxoid sarcoma (LGFMS) is a specific type of fibrosarcoma with deceptively banal appearance and malignant behavior. Method: A 5 year retrospective study revealed 10 LGFMS cases. Morphological and immunohistochemical analysis was performed. Follow-up information was obtained for 5 cases. Results: All tumors occurred in adults (mean 40 year). They developed in neck (1), extremities (6) and trunk (3). Beside classical morphology of LGFMS following features were observed: necrosis (3), rosettes (3), areas of increased cellularity (5), foci of epithelioid cells (5), marked nuclear pleomorphism (1), prominent myxoid change (3), invasive border (6), bone formation (1), focal retiform pattern (1) and sclerosing epithelioid fibrosarcoma-like areas (1). Immunoprofile was the follow: vimentin (100 %), EMA (30 %), CD34 (10 % focally), S100 (0), SMA(0), desmin (0), AE1/AE3 (0). Two patients were without evidence of disease, one developed lung metastasis after 1 year. Two patients developed secondary tumours in 6 and 12 years after initial diagnosis. There were differences in the location and histologic features compared with primary ones. Secondary tumors were less cellular, without necrosis or epithelioid cells. Behavior of secondary tumors did not fit to the classical concept of tumor progression that implies increase of malignancy. Conclusion:
LGFMS is a distinctive but unusual type of soft tissue sarcoma demonstrating a wide histologic spectrum and behavioral range.
Immunohistochemical and mutational study of synovial sarcomas K. Specht * , M. Bettstetter, G. Keller, H. Rechl, R. von Eisenhart-Rothe, H. Höfler, M. Straub * Technische Universität München, Inst. für Pathologie, Germany
Objective: Synovial sarcomas are mesenchymal tumors of unknown histogenesis. Their molecular signature is a specific t(X; 18)(p11.2;q11.2) translocation. No effective targeted therapies are currently available. The aim of this study was to evaluate the expression and mutational status of potential molecular therapeutic targets. Method: 38 molecularly confirmed cases of synovial sarcomas were included in this study. Immunohistochemical stainings of the EGF-R family (EGF-R, HER2/neu, HER3), and signaling molecules implicated in the mTOR pathway (AKT, mTOR, PTEN), as well as E-Cadherin and snail was performed. In addition, cases were screend for mutations in the EGFR, PIK3C, B-RAF, K-RAS, and N-RAS genes. Results: EGF-receptor family members as well as E-Cadherin and snail are important for defining the tumor phenotype by determining epithelial-mesenchymal transition of synovial sarcomas. Activation oft the mTOR pathway is seen in a significant number of cases. Mutations of the genes studied are an overall rare event in synovial sarcomas and other types of sarcomas studied. Conclusion: EGF-R expression is found in many synovial sarcomas, however, mutations of EGFR or downstream molecules appear to be rare. Activation of mTOR pathway is frequently seen in synovial sarcomas. The benefit of targeted therapy against these genes in synovial sarcomas remains to be determined.
Collagen V induces differentiation of rabbit adipose tissue-derived stem cells in chondrocyte-like phenotype W. Teodoro * , I. Brindo da Cruz, A. P. Velosa, S. Carrasco, C. Goldenstein-Schainberg, R. Fuller, E. Parra, V. Capelozzi * Faculdade de Medicina da USP, Disciplina de Reumatologia, São Paulo, Brazil
Objective: Stimulated mesenchymal stem cells (MSCs) have capacity of differentiation in many cell types. It is being used in degenerative diseases treatment protocols. We evaluated the collagen V (COL V) and collagen XI (COL XI) influence in the differentiation of rabbits adipose tissue-derived MSCs in a chondrocyte-like cell phenotype. Method: MSCs isolated of New Zealand rabbits adiposetissue were maintained in culture by 4 weeks. COLV, COLXI and COLV/XI (10 μg/ml) were added to culture during 72 h. The cells aggregates were stained with Toluidine blue, Alcian blue and Picrosirius. Chondrocyte-like phenotype was confirmed by immunofluorescence to CD34, vimentin and collagens I, II and III. Results: MSCs stimulated with COLV expressed proteoglicans and collagen, when compared with COLXI and COLV/ XI and control. In the presence of COLV, MSCs was capable to increase collagen II expression confirming its chondrocyte-like cell phenotype. In contrast, MSCs cultured with COLXI and COLV/XI express collagen I and III. Conclusion: The data suggest that COLV may facilitate the differentiation of rabbit adipose tissue-derived stem cells into a chondrocyte-like phenotype. Further studies are urged in order to evaluate the influence of COLV in the ability of chondrocytes to remodel osteoarthritic joint surface at ultra structural and molecular levels. Objective: XRCC1 is essential for DNA base excision repair, single strand break repair and nucleotide excision repair. Method: We evaluated XRCC1 immunohistochemically in early stage breast (n=2046), ovarian (n=157), gastric (n= 140), colorectal (n=250) and pancreaticobiliary cancers (n= 240). Pre-clinically, we evaluated a panel of XRCC1 deficient and proficient Chinese hamster ovary and human cancer cell lines. Double strand break repair (DSB) inhibitors targeting ATM (KU55933), DNA-PKcs (NU7441) and ATR (NU6027) were evaluated for synthetic lethality and cisplatin alone or in combination with DSB inhibitors for chemopotentiation. Results: In breast cancer, XRCC1 loss (16 %) was associated with a 2-fold increase in risk of death and metastasis (p< 0.0001). In ovarian cancer, XRCC1 positive tumours (44 %) were more resistant to platinum chemotherapy (p=0.0001). XRCC1 positivity conferred a 2 fold increase of risk of death (p=0.002) and independently associated with poor survival (p= 0.002). Pre-clinically, KU55933, NU7441 and NU6027 were synthetically lethal in XRCC1 deficient compared to proficient cells as evidenced by DSB accumulation, G2/M cell cycle arrest and apoptosis. XRCC1 deficient cells were hypersensitive to cisplatin which was enhanced by DSB repair inhibitors compared to in proficient cells. Conclusion: Conclusions: XRCC1 deficiency in human tumours may be suitable for synthetic lethality application and exploited for cisplatin chemotherapy potentiation.
Analysis of BCL2 oncoprotein expressing breast cancer by molecular subtype A. Abolins * , I. Strumfa, Z. Simtniece, A. Vanags, G. Trofimovics, J. Gardovskis * Riga Stradinš University, Inst. of Oncology, Latvia
Objective: Breast cancer is the most common malignancy in Western women. Despite the progress in morphological investigation, active research is devoted to potentially important targets for prognosis and intervention. BCL2 oncoprotein represents such factor. Method: Consecutive breast cancer cases were examined by routine protocol approach. The BCL2 oncoprotein expression was detected immunohistochemically. Expression was considered positive if it was in more than 25 % of tumour cells. The molecular subtype ( Objective: Breast cancer is the most common malignant tumour of Latvian women (www.csb.lv). Correct diagnosis, including identification of tumour histogenesis, is the prerequisite for appropriate treatment.
Method: Consecutive breast tumour cases were selected by systematic retrospective archive search and were examined by breast cancer panel. If expression of oestrogen and progesterone receptors and HER2 protein was negative, mammaglobin, cytokeratin AE1/AE3, vimentin, CD45, CDX2, cytokeratin 20, TTF-1 and melanosome protein HMB-45 were detected. Results: Five patients (0.89 %, 95 % CI=0.38-2.10 %) with secondary breast tumours were identified among 559 cases. Breast was affected by metastatic small cell lung cancer (1), malignant neuroendocrine tumour of small intestine (1) as well as by epithelioid melanoma metastasis in breast tissues (2) or intramammary lymph node (1). Analysing medical records, multiple synchronous and/or metachronous metastasis, involving brain, kidneys and ovaries, were found in all patients with haematogenous tumour metastases in the breast. In contrast, the lymphogenic tumour spread was isolated. Conclusion: 1. Lymphogenic or haematogenous metastasis rarely (0.89 % of malignant breast tumours by morphology) can develop in breast tissues. It should be taking into account when planning the differential diagnostic approach, especially immunohistochemistry. 2. Haematogenous metastases in breast are associated with a wide synchronous or metachronous extramammary tumour spread.
The prognostic significance of tumor-associated stroma in invasive breast carcinoma Objective: Fibroblasts in the stromal component of a tumor may influence tumor progression in various organs. The prognostic significance of tumor-infiltrating lymphocytes is also frequently reported. However, the prognostic significance of the stromal component in breast cancers, particularly those of high grade, has not been established. Method: In this study, we analyzed surgically resected specimens from 545 patients with breast carcinoma, including 193 high grade tumors, for tumor-stroma ratio, dominant stroma type (collagen (C), fibroblast (F) or lymphocyte (L) dominant type), and central fibrosis on hematoxylin-eosin stained histological sections. We correlated these features with clinical prognosis. Results: Among the 533 specimens examined, 127 (23.3 %) were of C type, 292 (53.6 %) of F type, and 114 (20.9 %) of L type. Central fibrosis was found in 99 tumors (18 %). The dominant stroma type was a significant prognostic factor on univariate and multivariate analyses, together with T classification, nodal status and Bloom-Richardson grade. Tumorstroma ratio and central fibrosis did not predict survival on multivariate analysis. Even in high-grade tumors, relapse-free intervals differed significantly according to dominant stroma type. Conclusion: Conventional hematoxylin-eosin stained tumor slides may contain more prognostic information than previously thought; in particular, the dominant stroma type in invasive breast cancer may potentially be used to predict outcome.
PS-17-007 Pathology in breast implants substitution L. Alfaro * , J. Serra, J. M. Ibañez * Valencia, Spain
Objective: Health alert concerning breast implants brand PIP led to a review of patients harboring these prosthesis and in many cases replacement by new ones. Anaplastic lymphoma described in these patients has been an additional problem to be faced by plastic-surgeons and pathologists. Method: Ninety two cases of women with breast implants were studied. Most of them had PIP implants (although no information of prosthesis type was available in all cases). Eighty of them presented with ruptures of different size. After implant replacement, histopathologic analysis of fibrous capsules and liquid from seromas in periprosthetic cavities was carry out. Following FDA recommendations, immunohistochemical studies to rule out lymphoma was performed in 85 cases. Results: Fibrous capsules showed synovial metaplasia in all cases. Morphology was practically identical that in real articular synovial cells, and occasionally intracavitary nodules were seen detached from the surface in a process similar to synovial chondromatosis. No cases of lymphoma were seen and no expression of CD30 or ALK markers occurred. A case of pericapsular ductal invasive carcinoma was discovered. Conclusion: Synovial metaplasia seems to be very common in capsules around breast implants. Development of conventional breast carcinoma is probably much more frequent than lymphomas independently of the possible implant influence.
Post-radiation angiosarcoma of the breast: Report of a case A. Apostolaki * , M. Sofopoulos, S. Tsitsiou, E. Pigadioti, N. Mylona, N. Arnogiannaki * Agios Savvas Hospital, Dept. of Anatomical Pathology, Athens, Greece
Objective: Post-irradiation angiosarcoma generally occurs after breast conservation and radiation therapy. It affects the dermis of the breast within the radiation field. The incidence of post-radiational angiosarcoma is about 0.14 %.
Method: An 82-year-old woman who had undergone conservative surgery and radiotherapy for breast cancer 10 years ago presented with multiple red-purplish papules on the skin of her right breast. Excisional biopsy followed by simple mastectomy was performed. Results: Excisional biopsy revealed a neoplasm composed of highly pleomorphic cells with prominent nucleoli forming solid areas and neoplastic slit-like vascular channels. Many mitotic figures and some individual apoptotic cells were also present. The tumor cells were positive for endotelial cell markers (factor VIII, CD31, CD34) and negative for CK7. The gross inspection of the mastectomy specimen showed multiple reddish-purple papule-like lesions on the skin, spreaded in an area of 12×10 cm. Sectioning showed numerous homogenous slightly hemorrhagic white nodules measuring from 2 to 12 mm located in the dermis. Histopatology and immunohistochemistry findings were consistent to those of the excisional biopsy. Infiltration of the subdermis was noted. Conclusion: Diagnosis was high grade post-irradiation angiosarcoma. Simple mastectomy is the treatment of choice. Adjuvant chemotherapy should be considered in high grade neoplasms like this one.
Prognostic factors in invasive lobular carcinoma of the breast G. Askan * , G. Ayranci, N. Özkan, H. Kaya, U. Ugurlu * Marmara University, Dept. of Pathology, Istanbul, Turkey
Objective: Determination of molecular features in breast carcinomas, such as hormone receptor expression, can guide clinicians to the optimal choice of therapy. In this study, the relationship between the histologic grade, pathologic stage and the prognosis of invasive lobular carcinoma of the breast, and the tissue expression levels of ER, PR, HER-2/neu, p53, bcl-2, Ki-67 and E-cadherin was investigated. Method: 31 cases of invasive lobular carcinoma of the breast, from 2003 to 2011, were included in this study. A single best representative paraffin block was selected for each case and H&E staining and immunohistochemistry procedures were performed. Ki-kare test was used for statistical method. Results: 24 of 31 cases had classical lobular carcinoma, 7 cases had tubulolobular, pleomorphic, signet ring cell, or apocrine features. The median age of patients was 54 years. ER and PR were positive in 28 cases. All cases were negative for E-cadherin. Ki-67 was greater than %15 in 2 cases with pleomorphic lobular carcinoma. p53 positivity increased with grade. In 14 cases p53 was negative and bcl-2 was positive. Bcl-2 and p53 were positive in 29 and 17 cases respectively.
Conclusion: In contrast to the literature, there was no correlation between bcl-2 status and other molecular markers, including p53.
Rapid immunohistochemistry in intraoperative sentinel axillary lymph node evaluation P. Baldin * , M. C. Cucchi, Y. Ishikawa, V. Eusebi, M. P. Foschini * University of Bologna, Dept. of Anatomic Pathology, Italy
Objective: Sentinel lymph node (SN) examination is the current procedure to establish the status of axillary lymph nodes in breast cancer. To avoid a two step delayed surgical procedure, a reliable and quick method of SN evaluation is advocated. Rapid immunohistochemical technique (UICH) for keratin has been only recently proposed. Aim of this study is to apply keratin UICH in frozen sections (FSs) of SNs. Method: A consecutive series of 231(Series A) SN cases was studied at FS level followed by two sections stained with keratin UIHC. All procedure requires 23 min. For comparison 131 consecutive cases (series B) of SN were studied with FS only. All residual tissue from both series was paraffin embedded (PS). Results: Series A: SNs showed tumour involvement in 41 cases (17.7 %). In only 8 cases (3.75 %), PS sections evidenced additional neoplastic cells (micometastases) not seen with keratin UIHC, that led to a delayed axillary dissection. Series B: PS sections revealed metastatic deposits (micrometastases) not seen in FS in 6.1 % of cases. Conclusion: FS coupled with keratin UHIC of all the entire lymph node accurately evidences carcinoma cells in SNs.
High concordance of 6 HER2 in situ hybridization methods with Abbott FISH J. Boers * , L. Krol, C. Netjes, H. Meeuwissen, C. Prinsen, C. van Krimpen, E. van der Logt, J. Bart, E. Schuuring * Isala Klinieken, Dept. of Pathlogy, Zwolle, Netherlands
Objective: We conducted a comprehensive concordance study of 6 ISH methods with Abbott FISH in a large series of breast carcinomas. Method: Tissue Micro Arrays (TMA) were constructed by taking 3 tissue-cores from praffin blocks of 402 primary breast carcinomas. Up to 384 cases were analyzable in 7 ISH assays. Scoring was performed by two independent observers without knowledge of the other ISH data according the ASCO-guidelines for HER2-testing. Cases were considered positive when the ratio was ≥2.0. Discordant cases were reviewed and scores were reassigned on consensus of opinion. Concordance and Cohen's kappa score were calculated in relation to FISH, Abbott. Results: In 372 cases analyzable with Abbott HER2 FISH, 12.1 % were HER2-positive. Concordances (kappa-scores) of the 6 other assays were: DAKO FISH 98.1 % (0.90), DAKO duoCISH 97.2 % (10.4 %), Zytovision FISH 99.1 % (0.96), Zytovision duoCISH 99.1 % (0.96), single probe SISH Ventana 98.9 % (0.95), dual probe SISH Ventana 99.4 % (0.97). Conclusion: Conclusion: Concordance of 6 HER2 ISH assays with Abbott FISH were shown to be 97.2 % or higher. In this study, DAKO assays had a lower kappa score with Abbott FISH than Ventana or Zytovision assays.
Which is the best method to measure multiple breast cancer? M. Boros * , C. Marian, O. Pop, S. Stolnicu * UMPh Targu Mures, Pathology, Romania
The size of the breast tumor is relevant in a patient s management also affecting the prognosis. For unifocal lesions, tumor staging depends on the maximum diameter of the tumor, whereas in multiple lesions, this issue is not standardized. The aim of this paper is to study which is the best method in the assessment of the tumor size in multiple invazive carcinomas (multifocal and multicentric) in correlation with the lymph node metastases developement. Two different assessments of the tumor size (diameter of the largest focus=LD, and combined, aggregate diameter of all the foci=AD) were used in 418 primary invasive breast lesions (91 multiple, 327 unifocal) and compared with the nodal status (CHI-test). The use of combined tumor focus upstaged 23 (25.27 %) patients with multiple tumors (16 upstaged from pT1 to pT2 and 7 from pT2 to pT3). There was no difference in nodal positivity based on pT status between LD and AD. We observed a statistically significant difference in the mean diameter of the largest focus between the unifocal and multifocal group (31,47 vs 39,67 mm) (p=0,0013). Aggregate diameter in multiple breast cancer is not correlated with an increase of axillary metasases and should not be used for staging.
Central nervous system metastases in women with invasive breast carcinoma, not otherwise specified, are associated with estrogen receptor status E. Cambruzzi * , A. G. Reginatto, C. G. Zettler, K. L. Pêgas, V. Grings, J. M. Venites, C. A. Coelho * ULBRA and UFRGS, Dept. of Pathology, Porto Alegre, Brazil
Objective: Central nervous system metastases (CNSM) from breast cancer (BC) are relatively common and can present as the first site of disease progression. Lymph node status and tumor size are regarded as important prognostic indicators for disease-free and overall survival in BC. The aim of this study was to investigate prognostic/predictive pathological data in BC that could define a high-risk group to develop CNSM. Method: The authors evaluated 97 female patients with invasive breast carcinoma, not otherwise specified, previously submitted to setorectomy/mastectomy, in order to determine lymph node status, tumor size, histologic grade, estrogen receptor status (ER) and immunoexpression of HER2/neu. Of these cases, 10 patients developed CNSM. Results: The patients who developed CNSM were younger (median age 54.7±8.152 years/p=0.251) and more likely to have T2N2 disease than patients with no CNSM. The presence of encephalic disease was associated with ER (p= 0.031). Lymph node status (p = 0.84), tumor size (p = 0.339), histologic grade (p=0.933), and HER2/neu expression (p=0.31) were not significant risk factors. Conclusion: Although the literature data discriminate that HER2/neu overexpression in BC is related with CNSM, the authors suggests that these lesions can be related to ER too. Efforts to determine other risk factor for development of CNSM may be warranted.
Androgen receptors and sex hormone serum levels in breast carcinoma: Study of the ORDET cohort L. Cimetti * , S. Sieri, A. M. Chiaravalli, N. Sahnane, F. Sessa, C. Riva, C. Capella * University of Insubria and Ospedale di Circolo, Varese, Italy
Objective: Androgens and androgen receptors (AR) are involved in breast cancer (BC) pathogenesis. High testosterone serum levels increase the risk of developing mainly ER + BC, especially after menopause, although androgen role in tumor progression is not clearly elucidated. Method: Correlations between serum sex hormones and clinico-pathological features of 131 BC arisen among 10,786 women previously recruited for ORDET study were investigated. Prediagnostic estradiol, testosterone (free/total) and SHBG serum levels were available. Immunohistochemistry for AR was evaluated along with ER, PR, HER-2 and MIB-1. Results: AR + was found in 90.8 % of BC. Higher estradiol (p=0.0001), free testosterone (p=0.02) and SHBG (p=0.02) were seen in premenopausal patients. In dead patients higher free testosterone was observed (p=0.0003). No correlation was found for hormone levels vs stage, histotype and grade. Higher SHBG was seen in PR-rich tumours (p=0.02). HER-2+/ER-cases showed a trend for a higher total testosterone (p=0.18) and SHBG (p=0.2). Testosterone, estradiol and SHBG were similar in AR + and AR-tumors. Among triple negative, AR + tumours showed higher free testosterone (p =0.04). BC with few AR + cells showed higher total testosterone (p=0.0004) and a worse outcome (p=0.29). Conclusion: Our results confirm the role of AR in BC and suggest the androgen involvement in tumour progression.
Mitotic count in metastatic breast carcinoma to lymph nodes: Preoperative study A. Córdoba * , L. Gomez, F. Vicente, I. Amat, C. Llanos, D. Guerrero * Hospital Navarra, AP, Pamplona, Spain
Objective: Axillary lymph node metastasis (ALNM) is one of the most important prognostic factors in breast cancer. The reasons why tumours are capable to result in axillary metastasis remain unclear. The evaluation risk of ALNM would improve the treatment planning. We study the metastatic breast carcinoma to the lymph node to obtain information about the metastatic risk. Method: We study 65 patients with metastatic breast carcinoma to the lymph node diagnosed preoperatively by needle biopsy. We study the mitotic average (×10 HPF), metastasis size, positive lymph nodes, total lymph node studied, lymph node ratio (nº of positive node/total nodes nº), primary tumour size and grade. Results: All the cases with high mitotic count were associated with macrometastasis. We didn't find any relation between the mitotic rate or the metastatic size with the number of lymph node affected, and the lymph node ratio. Breast carcinomas G1, T1, <6 mitosis/HPF lymph nodes showed 25-50 % of positive lymph nodes. Conclusion: We tried to obtain information from the metastatic breast carcinoma to the lymph node to predict the axillary status. In our cases we couldn't predict the lymph node involvement based in the tumour size, grade, metastatic size, and metastatic mitotic rate. The preoperative study of a breast tumour and their lymph node metastasis don't allow predicting the lymph node status in our series.
PS-17-020 RASSF1A hypermethylation is associated to the presence of tumoral cells detected by One-step Nucleic Acid Amplification (OSNA) A. Córdoba * , F. Vicente, N. Perez Janices, J. Perez Vizcaino, E. Gochi, N. Torrea, D. Guerrero * Hospital Navarra, AP, Pamplona, Spain
Objective: One-step nucleic acid amplification (OSNA) is used in routine clinical use for sentinel lymph node biopsy (SLNB). It consists of the molecular quantitation of a tumoral marker (cytokeratin-CK 19 mRNA). Gene hypermethylation is one of the most common mechanisms of inactivation of suppressor genes. RASSF1A gene, is a region frequently hypermethylated in breast cancer. Method: 51 patients with breast cancer were included in the study, and a total of 87 lymph nodes were analysed. Results: 41 %, 33.3 % and 21.6 % of the tumours were of low, intermediate and high-grade, respectively. In 40.8 % of the cases there are not tumoral cells, in contrast to 12.2 %, 4.1 %, 14.3 %, 28.6 % that showed low number of cells/ITC (<250 copies/sample), Micro-(<5000 copies/sample) and Macro-Mtx, respectively. There is a very clear association between RASSF1A hypermethylation and the presence of tumoral cells (p= 0.004), being more frequent in Macro-mtx compared to the rest of groups (P=0.007). RASSF1A hypermethylation is also correlated to unfavorable histologic grade in the tumour (P=0.025) and lymph node involvement at the diagnosis (0.001). Conclusion: The analysis of RASSF1A hypermethylation could provide additional information to OSNA to detect tumoral cells in lymph nodes. The spare tissue material derived from OSNA could be good material to consider new molecular studies. Results: Of these, 15 patients (88,23 %) had palpable tumor at presentation, 12 (70,58 %) with palpable axillary adenopathy. In 10 cases (58,82 %) the tumor diameter was over 2 cm at the time of diagnosis. In 15 cases (88,23 %) the histological type was infiltrative ductal carcinoma, with 13 cases having high grade. Most of the tumors expressed ER and PR and were Her-2 negative (none was triple negative). However, in 11 cases (64,70 %) the ki-67 index was more than 50 %. Conclusion: Young women with breast cancer have been shown to have a poorer prognosis because of the high grade and hormonal status. Our study shows some similar aspects, but the hormonal status is totally different from the dates in the literature, revealing a possible better response to hormonal treatment and a better survival.
Immunohistochemical predictive markers for trastuzumab resistence A. Cuesta Diaz de Rada * , E. Honrado Franco, M. Baltasar Moreira, F. M. Izquierdo Garcia * Hospital de León, Anatomía Patológica, Spain
Objective: HER2 positivity defines a clinically challenging subgroup of patients with breast cancer with variable prognosis and response to therapy. The main aim of this study is to identify immunohistochemical markers to predict trastuzumab resistence. Method: Tumours from 57 patients with Invasive Ductal Carcinoma, who were previously treated with trastuzumab, were included in a tissue microarray and stained for ER, Ki67, p53, cyclin D1, p16 and for HER2 and SISH (silver in situ hybridization). Results: After 5 years of follow up, 80 % of patients with p53 negative tumours were disease free (p 0.029) and 90 % were alive (p 0,014) and the patients cyclin D1 positive, 90 % were alive (p 0.031) and 84 % were disease free (p 0.005). Among HER2 3+, 10 % did not amplify (polysomy 17). Moreover, 12 % of treated patients were HER2 0−2+ and did not amplify by SISH. Conclusion: The best immunohistochemical markers to predict a good response to trastuzumab treatment were P53 negative and/or cyclin D1 positive. All HER2 positive 3+ tumours should be confirmed by hybridization, since polysomy 17 is found in 10 %, and these tumours should not be treated with trastuzumab. Variations in the immunostaining techniques could induce trastuzumab treatment to patients with HER2 non amplified tumours.
PS-17-023 CD99 expression in breast carcinoma P. Czapiewski * , J. Szade, A. Zaczek, M. Welnicka-Jaskiewicz, W. Biernat * Medical University of Gdansk, Dept. of Pathology, Poland
Objective: CD99 is a membraneous protein that is expressed widely among various soft tissue tumors. There is growing evidence that its expression in some carcinomas correlates with epithelial to mesenchymal transition (EMT) and is a poor prognostic factor. Its importance in the breast carcinoma remains unsettled. Method: The analysis was performed on breast cancer samples from 122 patients. Tumors were graded histologically according to the Nottingham system. CD99 expression was scored by grading system used for HER-2. Only cases showing grade (3+) were regarded as positive. Additionally, expression of estrogen (ER) and progesterone receptor (PR), HER-2, E-cadherin, Vimentin, Twist, Ki-67, c-myc, cyclin D1 and Topoisomerase 2alfa was performed. Expression of the CD99 was correlated with all these markers and with clinical outcome. Results: Expression of CD99 was observed in 11 patients and correlated significantly with negative PR status (p=0,01), higher histological grade (p=0,05), positive Twist (p=0,04) and Topoisomerase2alfa expression (p=0,04). There was also a trend toward higher frequency of cyclin D1 positivity (p= 0.09). No impact on prognosis for CD99 expression was found. Conclusion: Expression of CD99 correlates with high histological grade, negative progesterone receptor status and expression of certain EMT and proliferation markers, but it does not influence prognosis in breast carcinoma.
Primary plasmacytoma of the breast: A case report L. De Carvalho * , G. G. Monteiro, L. Casagrande, T. Ricci, T. Lebrão, V. Tarricone, P. Dinamarco * Centro Universitário Lusíada, Dept. of Pathology, Santos, Brazil
Objective: extramedullary plasmacytomas of the breast are extremely rare, especially those that are not associated with multiple myeloma and they can mimic mammary carcinoma. Method: case report:69-year-old woman presented a palpable mass in the left breast previously diagnosed as a lobular carcinoma by core biopsy performed in other service. In our institution a conservative surgery with lymph node dissection was proposed. Results: Frozen section was required and the gross examination showed a 4.0×4.0×3.0 cm firm-elastic nodule with surgical margins free of tumor and the microscopic examination showed tumor cells with abundant cytoplasm with hyaline appearance. The paraffin sections showed a proliferation of plasma cells with moderate atypia. The immunohistochemical study confirmed the diagnosis of plasmacytoma. There was no axillary lymph node involvement. There were no bone lesions in the additional investigation. Conclusion: primary plasmacytomas of the breast are extremely rare and have to be included in the differential diagnosis with breast carcinomas especially in material of core biopsy.
Sentinel lymph node in breast cancer: Form morphology to molecular examination L. Di Tommaso * , B. Fernandes, B. Fiamengo, C. Navligu, P. Spaggiari, S. Manara, C. A. Garcia Etienne, G. Masci, A. Testori, C. Tinterri, M. Roncalli * IRCCS Istituto Clinico Humanitas, Dept. of Pathology, Rozzano, Italy Objective: Sentinel lymph node (SLN) examination is a standard in breast cancer treatment. It can be performed on formalin-fixed paraffin-embedded material (FFPE) or on frozen sections (FS). FFPE or FS suffer two drawbacks: 1) partial examination; 2) operator's dependence. To avoid these limitations, a molecular technique (OSNA) targeted to quantify a tumoral fingerprint (CK19), has been introduced. Our aim is to compare the performance of FFPE, FS and OSNA. PS-17-026 HER2 assessment in invasive breast cancer using IHC and FISH: Results from 7479 consecutive cases P. Drev * , B. Gazic, J. Contreras * Inst. of Oncology, Dept. of Pathology, Ljubljana, Slovenia Objective: HER2 in invasive breast cancer (IBC) should be assessed according to recommended algorithms employing IHC as screening tool and ISH only in equivocal cases. Therefore there is little data on possible missdiagnosis in discordant cases. Method: HER2 was assessed by both IHC and FISH in 7479 consecutive IBC. Distribution of IHC and FISH scores, incidence of HER2+ IBC, concordance and level of amplification in discordant cases were analysed. Results: IHC distribution: neg(0) 49.6 %, neg(1+) 27.0 %, equivocal(2+) 11.3 %, pos(3+) 12.1 %. FISH distribution: nonamplified 84.6 %, amplified 13.6 %, equivocal 1.8 % of which in 42.1 % ratio was ≥2.0. 15.0 % IBC were HER2+ and 0.7 % were double-equivocal. 14.4 % of equivocal(2+) were amplified. Discordance was infrequent (1.2 %) (p< 0.0001): among neg(0) and neg(1+) 0,3 % and 1,6 % were amplified, while among pos(3+) 4.7 % were nonamplified. 64 IHC negative tumors, representing 5.7 % of all HER2+, were amplified, however in 89.1 % ratio was ≤4.0.
Conclusion: 15 % IBC are HER2+. Application of two standardised methods results in excellent concordance and enables detection of all HER2+ IBC, while recommended algorithms lead to missdiagnosis in 6 % of HER2+ IBC, but in these amplification is low-level.
Case report: Unusual breast cystic lesion in a 46-yearold female P. Farrajota * , P. Cusati, D. Esteves, C. Dias, C. Carvalho, G. Falconieri * Centro Hospitalar do Porto, Dipt. do Anatomia Patologica, Portugal
Objective: Papillary carcinoma of the female breast may exhibit a broad phenotype. We present an unusual case of a pseudoencapsulated invasive papillary carcinoma featuring transitional cell features. Method: A specimen of internal right female breast biquadrantectomy was routinely processed and a panel of antibodies applied on paraffin tissue section. Results: Grossly, the specimen showed a 12.5 cm cystic lesion with fibrino-hematic material and multiple pinkish-grey papillary structures, the largest measuring 4 cm. The histological examination revealed a malignant epithelial papillary proliferation reminiscent of transitional cell carcinoma, with high nuclear grade, frequent mitotic figures, multiple areas of invasion and rare necrotic foci. Tumor cells were immunoreactive for p63, 34βE12 and Ki-67 (90 %) and negative for other stains including HER2/neu, estrogen and progesterone receptors. Our differential diagnosis included a metastasis from a primary urothelial carcinoma, an intraductal papillary lesion, a metaplastic carcinoma and a papillary adnexal neoplasm, all reasonably excluded after careful clinico-pathologic evaluation. Conclusion: Metastatic involvement of the breast is uncommon but it should be considered if tumor phenotype is inconsistent within usual or "special type" breast carcinoma. Yet, some rare primary misleading lesions are difficult to recognize.
PS-17-028 Volume measurement of female Sprague-Dawley mammary tumors induced by N-methyl-N-nitrosourea: Comparing ultrasonography and caliper A. Faustino-Rocha * , C. Teixeira-Guedes, J. Pinho-Oliveira, R. Soares-Maia, R. Arantes-Rodrigues, B. Colaço, R. Ferreira, P. Oliveira, M. Ginja * UTAD, Dept. of Veterinary Sciences, Vila Real, Portugal
Objective: N-methyl-N-nitrosourea (MNU) is a chemical carcinogen frequently used to induce mammary tumors in female rats, which experimental evaluation requires the monitoring of tumor's volume. For this purpose several methods are described, namely: caliper and ultrasonography measurement. The aim of this work was to compare data obtained by caliper and ultrasonography. Method: Twelve female Sprague-Dawley rats with 187.7± 14.3 g body weight were intraperitoneally injected with MNU (50 mg/Kg) at 50 days of age. Thirty-six weeks after MNU administration forty-one tumors volume was determined by caliper (Vito®) and ultrasonography (LOGIQP6®, General Electric Healthcare) measurement. The tumor volume (V) was calculated according to the following formula V=π.〖S_1^2〗^.S_2⁄12, being S_(1) and S_2 the tumor diameters (S_(1)<S_(2)). Results: The means tumor caliper and ultrasonography measures were 4.116±5.801 cm3 and 2.578±4.302 cm3, respectively. The ICC for scoring reproducibility was 0.75 (95 % Confidence Interval, 0.57 to 0.86) and Paired-Sample T-Test was significant (p<0.01). Conclusion: Usually mammary tumors in rat are measured by caliper. However, our results showed that there are volume differences between both methods studied. Ultrasonographic method is more accurate and more precise than caliper. Therefore, we recommend the use of ultrasonography to rat tumor evaluation. Objective: There is a significant percentage of patients with breast cancer and HER2 receptor overexpression that do not respond to trastuzumab-directed therapy. The aim of this paper is to study the alterations of one of the HER2 signalling pathways related to trastuzumab response in breast cancer. Method: The study comprised 127 cases of breast carcinoma in different stages of evolution with HER2 overexpression (HercepTest 3+) or gene amplification (FISH). All patients were treated with trastuzumab. The assessment of PTEN and pAkt expression was based on staining intensity and percentage of stained cells. We also determined the molecular alterations in PIK3CA. Results: Our series showed a statistically significant association between a positive expression of PTEN and the absence of PIK3CA mutations with better overall survival from treatment onset with trastuzumab (p=0.022 and p= 0.003, respectively). The association with the expression of pAkt was statistically significant if survival is evaluated from the pathological diagnosis of the tumor (p=0.043), pAkt positive tumors had worse survival. Objective: Expression of mammaglobin A (MG-A) in breast cancer has been reported to be of prognostic value and to be associated to hormone receptor-positivity. MG-A is also useful in the diagnosis of metastatic breast cancer. The present study evaluates the differential MG-A expression in two subsets of primary breast cancer (metastasizing vs. non-metastasizing) and in their correspondent metastases. Method: Immunohistochemical analysis of MG-A (clone 304-1A5) was performed in 55 non-metastasizing primary breast tumors and 19 paired primary and metastatic breast cancers. A percentage of 1 % of stained tumor cells was considered positive. The expression of MG-A in the different groups, together with other clinicopathologic factors (grade, size, hormone receptors, HER2, etc.) was analyzed by t-test. Results: Expression of MG-A was significantly higher in estrogen receptor-positive (ER+) tumors (p<0.003). A trend was noted for ER + metastasizing primary breast cancers to show a higher expression of MG-A, compared to those nonmetastasizing (p=0.064). No significant differences were noted in the expression of MG-A between metastasizing primary tumors and their correspondent metastases, with a good correlation (p=0.033). Conclusion: These results suggest that MG-A might be of prognostic value in determining a subset of ER + breast cancers prone to progression. Larger series are needed to confirm these observations.
Concordance of St. Gallen intrinsic subtypes in core biopsies and related surgical specimens of breast cancer C. M. Focke * , T. Decker * Dietrich Bonhoeffer Klinikum, Abt. Pathologie, Neubrandenburg, Germany
Objective: To evaluate the relevance of intrinsic breast cancer subtyping (IBCS) on core biopsies (CB). Results: 39384 samples were tested in 2009. Score 0 was found in 45.43 % cases, 1+ in 28.86 %, 2+ in 12.97 %, 3+ in 14.73 %. The amplification was revealed in 32.12 % of 2+ cases, 15.82 % tumors were 3+/FISH+. 43485 cases were tested in 2011: score 0 −38.42 %, 1+ 30.61 %, 2+ 15.65 %, 3+ 15.32 %. The amplification was seen in 28.17 % 2+ cases and 18.68 % tumors became 3+/FISH+. There was no significant distinction using 10 % and 30 % cells score system for 3+ tumors, whereas for groups 1+ and 2+ it was. Conclusion: ASCO/CAP scoring criteria developed in 2007 have no effect on HER2-positive tumors detection but increases number of cases with equivocal result, what require retesting with ISH.
Microvessel density in triple-negative breast cancer: Characteristics and prognostic implications H. Gobbi * , M. De Brot, R. Rocha, F. Soares * UFMG, Dept. of Anatomic Pathology, Belo Horizonte, Brazil
Objective: We assessed topography and characteristics of blood vessels in triple-negative breast cancer (TNC) and examined their association(s) with clinicopathological features, basal-like phenotype and influence on patient prognosis. Method: Blood vessels were assessed in tissue sections from 133 TNCs stained for CD34. Slides were scanned using ScanScope® XT and analysed with Aperio Image-ScopeTM, Microvascular Density v1 algorithm. Microvessel density (MVD) was assessed in three areas: intratumoural (IT), peripheral tumoural (PP) and peritumoural (PT), and correlated with clinicopathological features and prognosis. Results: The majority of blood vessels were located largely at the peripheral tumoural and peritumoural areas. Tumours with higher total MVD and PT-MVD were significantly associated with the presence of recurrence (p = 0.05; 0.01), distant metastasis (p = 0.01; 0.005), hematogenous metastasis (p= 0.006; 0.01); and shorter overall survival (OS; p= 0.006; 0.01) and disease free interval (DFI; p=0.04; 0.09). High PT-MVD correlated with a higher stage at diagnosis (III and IV; p= 0.02), lymph node (LN) metastasis (p=0.04), basallike phenotype (p = 0.01), high histologic grade (p = 0.05), presence of distant metastasis (p = 0.02), and shorter OS (p =0.01). Conclusion: Blood vessels are mainly located at the peritumoural and peripheral areas in triple negative breast cancers and seem to have a major role in tumour progression by representing means for disease dissemination.
Expression of CD44 and CD24 in primary ductal invasive carcinoma of breast and metastatic lymph nodes M. Gudadze * , K. Kankava, T. Javakhishvili, M. Sarishvili, G. Burkadze * Central University Clinic, Dept. of Pathological Anatomy, Tbilisi, Georgian, Republic of Georgia Objective: Cancer stem cells are believed to have certain role in progression, metastatic potential and outcome of ductal invasive carcinoma of breast. Method: The archived materials of 2008-2012 years from the Department of Pathological Anatomy, N. Kipshidze Central University Clinic were used for the research. 196 cases of ductal invasive carcinoma were chosen for the study. The CD44, CD24 immunohistochemistry (Biocare Medical) was used for staining. Evaluation was performed using SPSS software. Results: The results showed: 68.5 % of grade II cases and 90.5 % of grade III cases were lymph node positive. CD24 positivity in grade II tumors was seen in 29.6 % of primary site and 45.9 % of metastasis; in grade III tumors-50 % of primary site and 55.2 % of metastasis. CD44 positive cases in grade II-48.1 % for primary tumor and 27 % in metastasis, for grade III-73.8 % for primary tumor and 39.4 % for metastasis.
The results show that CD44 positivity is significantly less frequently seen in metastasis than in primary tumor; CD24 positivity is more frequently seen in metastasis for grade II cases. This indicates, that cells with stem cell phenotype decrease in metastasis reflecting heterogeneity between primary and metastatic sites, which should be taken into consideration for assessing progression and treatment.
PS-17-035 Solid neuroendocrine breast carcinoma: Three case reports B. Ince Alkan * , S. Yuksel, E. Kadan, S. Percinel, S. Dizbay Sak * Ankara University, Turkey
Objective: It seems that neuroendocrine breast carcinoma (NEBC) is underrecognized in routine practice. 2003 World Health Organization classification of tumors of the breast defined NEBC as a subtype of invasive mammary carcinoma in which >50 % of the tumor cells express neuroendocrine markers. Three cases compatible with solid NEBC are discussed with an emphasis on identifying features useful in recognition of this tumor type. Results: The patients were 35, 55 and 57 years old women. All patients presented with a palpable mass, two in the right and one in the left breast. Microscopically, infiltrating, solid cohesive nesting pattern with delicate sinusoidal vasculature or peliosis and papillary pattern were observed. The tumor cells were round or polygonal with abundant granular, eosinophilic cytoplasm. The nuclei had hyperchromatic/vesicular or "salt and pepper" chromatin. Two cases had ductal carcinoma in situ component. All cases were positive for synaptophysin and chromogranin A in >50 % of tumor cells. While a positive status for estrogen receptor was detected in all cases, progesterone receptor was observed in two cases. None of the tumors displayed HER2 overexpression. Conclusion: Morphological clues suggestive of NEBC must be recognized for immunohistochemical confirmation for appropriate classification of this clinically distinct subtype of invasive breast carcinoma. Objective: Automation of HER2 FISH may improve HER2 gene testing. The aim of our study was to evaluate an automated HER2 FISH assay for assessing the HER2 genomic status. Method: Core biopsies of 100 invasive breast carcinomas were analysed in parallel using the manual PathVysion™ HER-2 DNA Probe Kit and the automated Leica HER2 FISH System for the BOND™ instrument. To assess intermethod agreement, concordance analysis was performed for various numerical and categorical parameters. Results: Carcinomas with all HER2 immunohistochemical scores were included (0+: 20; 1+: 20; 2+: 30; 3+: 30). Using either HER2/CEP 17 ratio >2.2 or ≥2.0 as criterion for HER2 amplification, high levels of concordance were observed between automated and manual FISH (concordance rate 96 %, K coefficient 0.92). High levels of inter-method agreement were also found for HER2 copy number, CEP17 copy number, HER2/CEP17 ratio, the percentage of carcinoma cells with HER2/CEP17 ratio >2.2, and the presence of HER2 genetic heterogeneity, HER2 clusters and CEP17 polyploidy. Conclusion: HER2 testing using automated FISH is feasible on breast carcinoma core biopsies. Automated HER2 FISH using the Leica HER2 FISH System for BOND is an alternative to manual HER2 FISH in evaluating the HER2 status of primary invasive breast carcinomas.
Myofibroblastoma of the breast: Presentation of three cases E. Kairi-Vasilatou * , C. Dastamani, A. Tsagkas, A. Paraskeva, A. Kondi-Pafiti * Aretaieio Nosokomeio University, Dept. of Histopathology, Athens, Greece Objective: Myofibroblastoma is a rare, usually solitary, benign spindle cell tumour composed of myofibroblasts. It affects both genders equally. Method: Three cases diagnosed in our laboratory are presented. Results: Two patients were postmenopausal females (53 and 75 years old) and one was male (55 years old). The tumour was 1,6 cm in greatest diameter in the first case, 7 cm in the second and 2 cm in the third. Microscopically, all neoplasms were circumscribed and consisted of uniform, bland spindle cells separated by broad bands of hyalinized collagen. The neoplasmatic cells had abundant eosinophilic cytoplasm, oval nuclei and were arranged in fascicles. Cellular atypia was found only in one case and mitoses were scarce. Immunohistochemically, all neoplasms were strongly positive for vimentin, CD34 and Bcl-2, while the expression of desmin and SMA varied. S-100 protein was negative. The neoplasms of the female patients were strongly positive for ER and negative for PgR. The stains for estrogen and androgen receptors were not carried out in the case of the male patient. Conclusion: Differential diagnosis can be complex. Fibromatosis of the breast, nodular fasciitis, myoepithelioma, myofibrosarcoma, solitary fibrous tumor, inflammatory myofibroblastic tumour should be excluded based on the histological and immunohistochemical findings. Method: Using light-and transmission electron microscopy fragments of mammary gland tumors, received intraoperative, from 58 patients 26-82 years with infiltrative ductal breast cancer were studied. Results: Most of the mast cells were localized in the areas of infiltrative growth, at a background of lymphohistiocyte infiltration and in peripheral part of the tumor. Intratumourous mast cells were totally degranulated. The mast cells with intact structure at a background of cells and tissue destruction were also in mammary gland. In destructive mast cells the nucleus with electron-dense circle and chain of granules were observed. On transmission electron microscopy their deformations and the decrease of density were revealed. The mast cells were of elongated of rounded shape with large nucleus and invaginations of karyolemma. The nucleus occupies almost completely the cell's area. It has a ring of large but pale granules. Small protuberances were observed on the surface of the cell membrane. Their number decreased in direct proportion to the size of must cells and their remoteness from microvessels. Such protuberances are necessary for mast cells in moving. Destructively changed cells with the signs of swelling, vacuolization and clasmatosis were frequent in breast cancer. Objective: Determination of eligibility of breast cancer patients for treatment with anti-angiogenic drugs has been always considered as a challenge for oncologists. Each mutation in the genes of proliferation phase enhances the angiogenesis of tumor. We aimed to determine the effect of concurrent mutations of HER-2 and TP53 on angiogenesis. Method: 32 Women affected by invasive ductal carcinoma (IDC) sporadic breast cancer were included. Immunohistochemical study was performed with HER-2, TP53, Ki-67 and AnnexinV markers. Angiogenesis index was semiquantitatively calculated by MVD-CD34 technique. Statistical associations between parameters were evaluated. Results: Prevalence of HER-2 positive and TP53 positive cases were 21.4 % and 20.0 %, respectively. 6.5 % of patients showed concurrent mutation of these genes. Concurrent mutation led to significant increases in both angiogenesis and proliferation and a significant decrease in apoptosis. There was no statistically significant association between concurrent mutation and tumor grade. Conclusion: This study demonstrates that most of the tissue prognostic factors are poor in concurrent mutation of both genes. Also, our study illustrates that the concurrent mutation correlates with a higher angiogenesis and thus an increased risk of recurrence. We can conclude that in priority setting for administration of anti-angiogenic agents, patients with concurrent mutations are more eligible.
Adenoid cystic carcinoma in male breast: A case report E. Kimiloglu Sahan * , U. Karinoglu, A. Akyildiz Igdem, N. Erdogan * Taksim´s Hospital, Dept. of Pathology, Istanbul, Turkey
Objective: Adenoid cystic carcinoma of the breast is a rare variant of breast cancer that accounts for 0.1 % of all breast carcinomas and occurs commonly in women between the ages of 25 to 80. In the literature, only a few examples have been reported in men. It is well-differentiated tumour with favorable prognosis and generally presents as a painful breast mass. Method: Here, we are presenting a 60 years old male patient with a 13×8 mm diameter solid mass on left breast retroareolar region. On fine needle aspiration biopsy, there were 'atipical proliferating ductal epitelial groups'. Then, hookwire localization and excision have been performed. Microscopically, there were tubulary and cribriform islands composed of basaloid type cells with eosinophilic cytoplasm and myxoid material in the cribriform spaces at the center of the islands. We used immunohistochemical markers such as P 63, S 100, CK 7, CD 117, CK 14 and Smooth Muscle Actin for differential diagnosis. Results: The diagnosis was 'Adenoid Cystic Carcinoma'. The margins of the tumor were positive, so radical mastectomy was performed for treatment. Conclusion: Because adenoid cystic carcinoma of the breast in male is a very rare example, we present our case here.
Comparison between the Bond Oracle HER2 immunohistochemical system, the polyclonal HER2 Dako antibody and Chromogenic In Situ Hybridization in breast carcinoma H. Kourea * , V. TzelepiI, I. Nikolatou, P. Ravazoula, V. Zolota * University of Patras, Dept. of Pathology, Greece
Objective: The sensitivity and specificity of immunohistochemical (IHC) methods for HER2 testing are very important given the therapeutic implications. This study compares the concordance between Oracle and the HER2 Dako polyclonal antibody (HER2), in breast carcinomas (BCs) that were equivocal (2+ or not evaluable/NE), by HER2 staining, considering as gold standard the chromogenic in situ hybridization (CISH). Method: BCs (n=34), problematic by HER2 staining (1−2/ 2+ or NE), and studied by CISH in our institution, and 24 additional consecutive BCs were stained with HER2 and Oracle, and scored separately by three pathologists. Consensus scoring for each IHC method and CISH results were recorded. Descriptive statistics and measurement of the Cohen's Kappa coefficient were performed. Results: The overall agreement between the 2 tests in a 3×3 analysis shows a concordance in 62 % of cases (κ=0.405). Among the cases studied by CISH, equivocal were 26 and 11 cases, for HER2 and Oracle, respectively. Seventeen HER2 equivocal cases were negative with Oracle and CISH. In problematic cases, using CISH as gold standard, the sensitivity, specificity, positive and negative predictive values for HER2 and Oracle were 100, 20.7, 17.9, 100 and 100, 72.4, 38.5, 100, respectively. Conclusion: In problematic cases, Oracle testing shows higher specificity and positive predictive value.
Stromal P53 and Ki67 expressions of the mammary phyllodes tumors: Are they the clues in determination of tumor grade? U. Kucuk * , U. Bayol, E. E. Pala, S. Cumurcu * Tepecik Training Hospital, Dept. of Pathology, Izmir, Turkey
Objective: Conventionally growth pattern, stromal overgrowth, stromal cellularity, stromal mitotic activity are the main parameters in grading of phyllodes tumors. Recent studies revealed that P53 and Ki67 expressions are both correlated with grade of phyllodes tumors of the breast. Method: We searched for p53 and Ki67 expression rates of benign and malignant phyllodes tumors in our archival data and correlated them with conventional parameters such as stromal cellularity and mitotic activity rates. 17 benign, 9 malignant phyllodes tumors were reevaluated as regards stromal cellularity (low/moderate and high), mitotic activity (low and high), p53 expression (low, moderate, high), Ki67 expression (low and high) rates. Statistical correlation amongst the whole parameters were searched with Chi-Square test. Results: Stromal cellularity, mitotic rate, p53 and Ki67 expression rates were all closely correlated (p = 0.000-0.001) for benign and malignant histologic subgroups. Ki67 expression was significantly correlated with histologic subgroups, stromal cellularity and mitotic rate (p=0.000-0.001). Similarly P53 expression was correlated with histologic subgroups, stromal cellularity and mitotic rate (p= 0.000-0.002). Conclusion: Both Ki67 and p53 expression rates are statistically significantly correlated with grade of mammary phyllodes tumors, so they can be used in determination of tumor grade, especially for differential diagnosis of benign and malignant ones.
Quantitative measure proliferative markers by image analysis of invasive ductal carcinoma A. Kudaybergenova * , S. Kalantarli * RSCRCT, Dept. of Immunohistochemistry, St. Petersburg, Russia
Objective: We analyzed a total number of tumor cells) in invasive ductal breast carcinoma, proliferative activity (% Ki67-positive cells) and mitotic index (% phh3positive cells) to establish absolute quantity tumor cells per sq.mm of histological slide and relations this measure with proliferation and mitosis.
Method: The study included 46 patients diagnosed with breast carcinoma from Baku Oncology Hospital during the 2001-05. After whole slide scanning by Mirax scanner (3DHistech, Budapest) of HE, Ki67 and phh3 stained slides we juxtaposed all three slides in one screen to the found area with maximal ki67 level in tumor and the corresponding area in other slides. Morphometric analysis was performed using the Pannoramic Viewer software (3DHistech, Budapest). For each case we analyzed a total number of tumor cells in1 mm2, number of Ki67 and phh3 positive cells. Results: Mean tumor cells in 1 mm2 of histology slide was 4180+\\−251 cells, median -3976 cells, 1185+\\−166 (31 %) were positive for Ki67 and 124+\\−23 (3 %) were in mitosis. There was moderate correlation between cell density and Ki67 r=0,44 (p=0,0032) and phh3 r=0,42 (p=0,0018). Conclusion: By analysis of breast cancer, was established a total tumor cell per mm2 and main proliferative characteristics for invasive ductal carcinoma. Objective: Adenomyoepithelioma of the breast is a very rare benign tumor with biphasic proliferations of epithelial and myoepithelial elements. It is morphologically and immunohistochemically identical to epithelial-myoepithelial cell carcinoma of the salivary gland. The histologic criteria of malignant AME is not well-established because of the rarity of AME. We report a case of malignant adenomyoepithelioma in a 65-year-old woman. Method: On ultrasonography, a well-marginated and lobulated solid mass was found at LIQ of the right breast.. An ultrasono-guided core biopsy was performed. The diagnosis of core biopsy was myoepithelial lesion. She subsequently underwent a wide local excision of the lesion. Results: Microscopically the tumor mass was composed of biphasic patterns which showed formation of tubules lined by an inner layer of ductal epithelial cells surrounded by proliferation of myoepithelial cells that also formed solid nests. But this tumor had foci of infiltrating margins and proliferation of spindle cell components. Also noted are numerous mitotic figures and increased mylepithelial Ki-67 positivity (10-15 % of tumor cells). Conclusion: Breast lesions which have predominently myoepithelial cells can be divided into myoepithelial hyperplasia, adenomyoepithelioma and malignant adenomyoepithelioma. Tavassoli divied AME into tubular, papillary, and solid subtypes. The criteria of malignant AME is not wellestablished but some criterias can apply to make a diagnosis of malignant AME. Objective: Neoadjuvant chemotherapy (NACT) is available for patients with breast carcinoma. However, resistance to chemotherapy is still a main cause of mortality. Method: Differentially expressed genes were identified from previously published studies that examined chemoresistant and chemoresponsive cell lines or patients with breast carcinoma. The expression of 14 selected gene products was assessed in tissue microarray slides comprising 75 post-NACT resection specimens from breast carcinoma patients using immunohistochemistry, and analyzed according to the molecular subtype and Residual Cancer Burden (RCB) grade. Results: Most cases were positive for ABCB1 (97.7 %) and MYC (96.0 %), but negative for TOP2A (100 %). RCB-II cases expressed much higher levels of MUC1 (p=0.011) and CLU (p=0.021) than RCB-III cases. Positive expression for CALR (p=0.002) and LGALS3 (p=0.018) was observed more often in triple negative types than in luminal types, and cytoplasmic CDKN1B expression was observed more often in luminal types (p=0.014). Conclusion: Positive expression of ABCB1 and MYC and negative expression of TOP2A in the residual carcinoma after NACT implies general resistance to NACT. Expression of CALR, LGLAS3 and cytoplasmic CDKN1B may be associated with resistance depending on the subtype. Expression of MUC1 and CLU can be used to predict the RCB grade or response to NACT.
Primary and metastatic melanoma of the breast-review of 4 cases I. Liepniece-Karele * , L. Osipova, M. Sperga, S. Isajevs, A. Grjunbergs, J. Eglitis * Riga ECUH, Dept. of Pathology, Latvia
Objective: Primary breast melanoma is a very rare tumour accounting for <5 % of all malignant melanomas. The malignant melanoma can be with different manifestation in the breast (primary breast tissue or primary breast skin melanoma as well as metastatic melanoma). Method: In this study 4 cases of breast melanoma were identified from our records of the past 3 years (0.41 % of all breast cancer cases). A histological and an immunohistochemical (IHC) study was performed using antibodies against CKAE1/3, HMB-45 and Melan-A on both the biopsy and operation material. Results: Obtained results showed that in 3 cases pigmented, epitheloid and spindle cell melanoma, but in one case epitheloid cell amelanotic melanoma was found. By IHC the melanoma cells expressed HMB45 in three cases, melan-A and S100 in all cases. No expression of CKAE1/3 was observed. Conclusion: Careful histological and immunohistochemical examination of malignant tumour is essential for adequate diagnosis, follow-up and treatment of breast melanoma.
Stem cell expansion in ductal carcinoma in situ of breast C. Lopes * , A. Paula, O. Marques, A. Rosa, A. Rema, F. Carvalho * ICBAS, Dept. of Pathology, Porto, Portugal Objective: The aim is to study the ability of a cell marker panel -ALDH1, CD44 and Ki67to identify breast stem cells in no malignant and ductal carcinoma in situ. Method: Double-color triple-immunohistochemistry -to ALDH1, CD44 and Ki 67 -was done in 169 paraffin embedded tissue specimens from 111 patients arrayed in tissue microarray blocks. Statistical was done using Chisquare probability test: differences were considered significant when p<0.05. Results: Significantly higher immunoreactivity was seen in DCIS than in benign lesions of breast (p<0.01) with used markers. In a total of 169 specimens, CD44+/ALDH1+/ Ki67-cells were identied in 110 cases. The distribution was as follows: DCIS (79/57); fibroadenoma (45/29), Atypical hyperplasia (23/13); other benign lesions ( Objective: Data from in vitro and clinical studies suggest that CaSR expression can be associated with the development of bone metastases. Most probably CaSR stimulates production and secretion of PTHRP via EGFR pathway. The aim of our study was to assess expression of CaSR in the primary breast cancer and correlate it with the risk of bone metastases. Method: We have analysed 170 patients with the breast cancer. Bone metastases were diagnosed in 102 cases, 68 patients died without skeletal involvement (control group). CaSR expression was assessed in primary tumors using tissue microarray (TMA) and immunohistochemical technique (polyclonal antibody Pierce Bio. PA1-37213). To evaluate cytoplasmatic CaSR expression we have used 0-1 point scale in which 1 was defined as uniformly strong or medium staining in more than 50 % of tumor cells. Results: Strong or medium staining in more than 50 % was identified in most studied cases, however it was more predominant in patients diagnosed with bone metastases than in the control group (93,14 % vs 83,82 %), p=0,053. Conclusion: Expression of CaSR is common in primary tumors of patients with disseminated breast cancer irrespectively of the metastatic site. However, the patients with bone involvement have higher rate of expression, that is borderline significant statistically. As a result, a group of patients with very high risk of bone dissemination might be separated.
Bcl2 expression is associated with centromere 17 alterations in luminal B breast cancer A. Matsionis * , I. Pavlenko, A. Petrov * Rostov Refional Institute, Dept. of Experimental Pathology, Rostov Regional Institute Objective: Bcl2 is an important established prognostic parameter in human breast cancer (BC) and chromosome 17 centromere (CEP17) copy number is proposed to be the same too. We evaluated Bcl2 expression in different BC's molecular subtypes in relation to increased CEP17 level (CEP17>3 per nucleus). Method: Immunohistochemistry for Bcl2 assessment and fluorescence in situ hybridization for detection of CEP17 alterations were used. Statistical analysis was performed with Fisher's exact test. Results: A total of 226 cases of female invasive BC's were analyzed (2010) (2011) . The tumour subtypes were as follows: luminal A (ER/PR+, Her2/neu-, Ki67<14 %) -118; luminal B (ER/PR+, Her2/neu-, Ki67>14 % or ER/PR+, Her2/neu+) -62; Her2/neu+(ER/PR-, Her2/neu+) -24; triple-negative -20. In our study group, Bcl2-cases were preferentially ER-in agreement with previous reports. However, 20,5 % tumours of luminal B subtype were Bcl2-too and all of them had increased CEP17 copy number (p= 0,0011, rφ=0,57). We therefore hypothesized that not only ER had influenced Bcl2 in luminal B BC but CEP17 alterations also. Conclusion: Centromere 17 alterations are associated to bcl2 downregulation in luminal B BC's. The mechanisms responsible for that remain to be established but the underlying cause could be promoter methylation or transcriptional repression.
Outcome of excision of radial scar diagnosed on core biopsy: A single centre analysis F. Menezes * , M. Caldas, N. Coimbra, C. Leal * IPO Porto, Dept. de Anatomia Patologica, Portugal
Objective: Radial scar (RS) is a sclerosing lesion of the breast which may be associated with a spectrum of epithelial proliferative lesions and carcinoma. It is frequently subject to biopsy when presenting as a mammographic abnormality. The need to excise RS diagnosed on core biopsy (CB) remains controversial. The aim of our study is to determine the frequency of upgrade in diagnosis after excision of RS. Method: A retrospective study of RS diagnosed on CB in our department between 01/01/2000 and 31/12/2011 was performed.
Results: 117 cases were retrieved, all women, with a median age of 53 years. Of these, 101 pairs of CB/resection specimens were obtained. CB diagnosis was RS without atypia in 79 cases, RS with atypia in 19 cases and RS with in situ carcinoma in 3 cases. After excision, 21/79 (26,6 %) cases diagnosed as RS without atypia were upgraded: 17 to atypia, 3 to in situ carcinoma and 1 to invasive carcinoma. Of those cases diagnosed on CB as RS with atypia, 9/19 (47, 4 %) were upgraded: 8 to in situ carcinoma and 1 to invasive carcinoma. Conclusion: Our results support the excision of all lesions diagnosed on biopsy as RS. *FM and MC are joint first authors.
Outcome of excision of papillary lesions diagnosed on core biopsy: A single centre analysis F. Menezes * , M. Caldas, N. Coimbra, C. Leal * IPO Porto, Dept. de Anatomia Patologica, Portugal
Objective: Papillary lesions of the breast (PLB) comprehend a spectrum of entities with different morphologies and malignancy risk, which present a diagnosis challenge on core biopsy (CB). Although diagnostic accuracy has improved with immunohistochemistry, the need to excise benign PLB remains controversial. The aim of our study is to determine CB diagnosis accuracy in PLB, and subsequent need to excise all lesions. Method: A retrospective study of PLB diagnosed by CB in our department between 01/01/2000 and 31/12/2011 was performed. Results: 96 cases were identified, with a median age of 59 years. CB diagnosis was benign in 66 cases, some kind of atypia found in 10, and malignant lesions ('in situ' and encysted/invasive types of carcinoma) in 20. Surgical excision was performed on 70 cases, including all cases with carcinoma. Following excision, 6/42 and 4/42 benign CB results were respectively upgraded to atypical and malignant; 3/8 with atypia were upgraded to malignant. Overall, 14 % (7/50) of benign and atypical PLB CB diagnosis therefore missed malignancy. Conclusion: Our results show that, despite good correlation between CB and excision diagnosis, some cases of carcinoma are missed on CB, so the excision of all PLB remains advisable. *FM and MC are joint first authors.
Myoepithelial carcinoma of the breast: A case report I. Michalopoulou Manoloutsiou * , B. Christoforidou, P. Xirou, V. Bostani, E. Goupou, I. Themeli, F. Patakiouta * Theagenion Hospital Thessaloniki, Dept. of Pathology, Greece
Objective: Myoepithelial carcinoma of the breast is an extremely rare tumor, composed purely of myoepithelial cells, predominantly spindle, with identifiable mitotic activity. Method: We report a case of a 50 year-old female patient with a palpable, well demarcated lump in her left breast, measuring 3,5 cm in its maximal diameter. Surgical excision and axillary lymph node dissection were performed. Results: Histologically, the tumor displayed an infiltrating growth pattern and consisted of spindle cells, that appear to emanate from myoepithelial cells of ductules entrapped in the center of the lesion. Mitotic activity did not exceed 5 mitotic figures/10 HPF. Immunohistochemically, the tumor cells revealed positivity for p63, cytokeratin 5/6, cytokeratin 34βΕ12, smooth muscle actin, CD10 and S100 protein, whereas they were negative for desmin, cytokeratin 7, CD34, HMB45, estrogen, progesterone receptors and Her-2 oncoprotein. Approximatelly 25 % of tumor cells showed nuclear positivity for MIB-1/Ki-67. All axillary lymph nodes were free of metastases. Conclusion: Myoepithelial carcinoma of the breast is a potentially highly aggressive neoplasm and its differential diagnosis is fairly broad, including metaplastic spindle cell carcinoma and a variety of myofibroblastic lesions. Objective: We investigated the differential expression of several biological markers between primary invasive breast carcinomas and their paired lymph node metastasis analyzing separately epithelial and stromal components. Method: Representative samples of 42 IDC and paired compromised lymph nodes were arrayed in a TMA and 15 selected markers: hormonal receptors, HER-1, proliferation (p53, pAKT, pmTOR, TGFβ1) , motility (CD9 and CXCR) and basal markers (CK5, CK14, c-kit) were evaluated by IC. Results: In the primary tumor, p53, MIB-1, TGFβ1, CD9 and CXCR4 were more expressed in epithelial cells (p< 0.05), while pAKT, pmTOR, c-myc and c-kit showed a similar frequency in both components. Hormone receptors, HER-1/HER-2 and cytokeratins were not expressed in stromal cells. The proliferative biomarkers were concordant in the epithelial component. CD9 frequency was similar but cytoplasmic CXCR4 as opposite to nuclear was predominantly expressed in lymph nodes (p=0.008). Stromal cells from lymph node showed a reduced frequency of CD9 (p= 0.029) and c-myc (p=0.003) when compared to the stromal component of primary tumors. Conclusion: Epithelial CXCR4 expression may facilitate lymph node metastasis whereas the low frequency of cmyc and CD9 in the lymph node stromal component indicated decreased proliferation enhanced motility of stromal cells in this site.
Expression of hypoxia-inducible factor-1a and associations with vascular endothelial growth factor expression, high microvessel density and features of aggressive tumors in African breast cancer H. Nalwoga * , J. B. Arnes, H. Wabinga, L. A. Akslen * University of Bergen, The Gade Institute, Norway
Objective: Breast cancer in Africans is reported to have poor clinical outcome. Whereas hypoxia-inducible factor-1α (HIF-1α) expression has been linked to treatment failure and poor prognosis in breast cancer, there is a lack of reports about HIF-1α expression in Africans. The aim of this study was to evaluate HIF-1α expression in relation to vascular endothelial growth factor (VEGF) expression, angiogenesis, and other tumor characteristics in an African population. Method: In total, we analyzed 192 breast cancers by immunohistochemical staining. We determined microvessel density (MVD), proliferating microvessel density (pMVD), and vascular proliferation index (VPI) in the most vascularized areas as well as expression of HIF-1α and other biomarkers using tissue microarrays. Results: Expression of HIF-1α (in 128/182 tumors; 70 %) was associated with VEGF expression (p<0.0005), MVD (p =0.037), high tumor grade (p=0.001), high Ki-67 proliferative rate (p<0.0005), and p53 expression (p=0.032). Conclusion: There is a high expression of HIF-1α in this series of breast cancer which is strongly associated with VEGF expression and increased MVD. More studies are required to assess the therapeutic implications of HIF-1α expression in this population. The patient underwent lumpectomy. The gross specimen had a tan grey firm nodule of 25×15×15 mm. Histological examination revealed a proliferation made of signet ring cell and glandular structures, with islands of goblet cells. Immunohistochemistry revealed strong positive staining with CK7 and CK20, and a sparse positive staining with synaptophysin and chromogranin.ER, PR and HER2 were negative. The patient underwent appendicectomy with a final diagnosis of breast localization of a primary occult appendiceal GCC. Conclusion: The differential diagnosis between primary carcinoid tumor of the breast and signet ring cell carcinoma metastatic to the breast is often controversial in surgical pathology. Diagnoses need to be made correlating clinical and histological examination in difficult cases in which there is not a diagnosis of carcinoid tumor elsewhere. Their histological appearance may mimic ductal adenocarcinoma of the breast. The distinction is important due to differences in management and prognosis.
Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in breast carcinoma M. Neagu * , C. Ardeleanu, G. Butur, A. Florin * National Institute of Pathology, Bucharest, Romania
Objective: Detection of Her2neu amplification is an integral part of breast carcinoma diagnostics to decide therapy. Method: We study 55 cases of breast carcinoma embended paraffin tissues, both CISH and FISH were performed on each case using (SPoT-Light HER2) for CISH and both Her-2 and chromosome 17 probes for FISH (Vysis). Sixty tumor cells were evaluated in each case. The scoring system and interpretation of CISH -INVITROGEN. Results: Concordance between CISH and FISH was found in 94,8 % cases, considering FISH as gold standard, sensitivity of CISH was 97.5 % and specificity 94 %. CISH is more practical alternative due to lower cost, no requirement of fluorescence microscope, use of existing bright-field microscopy and techniques it s similar to IHC, archivable and quantitative results, it s easy to observe both the tissue morphology and the gene amplification evaluation Of the 55 cases analyzed, 53 showed similar results for both methods. Two cases were discordant. In these cases, low-level amplification was suggested by CISH but nonamplification by FISH. Conclusion: our results, suggest that CISH is a useful technique to determine Her-2/neu oncogene status, in breast carcinoma for paraffin embendded tissues, is a highly accurate, reproducible and practical technique, with a high sensitivity.
Usual ductal hyperplasia with central necrosis, microcalcifications and multiple foci of pseudoinvasion arising in a radial scar: A potential diagnostic pitfall A. Nechifor-Boila * , S. Stolnicu * University of Medicine and Pharmacy, Tirgu-Mures, Romania
Objective: The diagnosis of radial scar (RS) is often difficult, especially when associated with proliferative changes, pseudoinvasion and necrosis. Method: A 41-year-old woman was referred to the Surgery Department for a palpable mass in her left breast, regarded as a possible fibroadenoma on ultrasonography. Results: Lumpectomy was performed and the macroscopy revealed a tan-white 11 mm diameter lesion, with irregular margins and firm consistency. On light microscopy, a sclerosing lesion, with a stellate arrangement of ducts surrounding a central fibro-collagenous zone was identified. Haphazardly arranged, distorted ducts associated with massive intraductal proliferation, highly suspicious of infiltrative were also present in the center of the lesion. The proliferating ducts showed slit-like, irregular, secondary lumens, several containing central necrosis and a heterogenous cell population. The presence of central necrosis and pseudoinvasion were, however, worrisome. Immunohistochemistry for p63 demonstrated the presence of myoepithelial cells surrounding all the areas with pseudoinfiltrating features, while CK 5/6 displayed a heterogenous, mosaic-like positivity, characteristic for usual ductal hyperplasia (UDH). Conclusion: Because pseudoinvasion in benign RS can easily be misinterpreted as invasive carcinoma, immunohistochemistry is mandatory to establish the presence of myoepithelial cells. Although rare, central necrosis may occur in UDH and should not be used as a single diagnostic criterion of malignancy.
Phenotypical and morphological heterogenity of breast cancer: Our experience J. Nieslanik * , J. Dvorácková, M. Uvírová, Š. Laciok, R. Ondruššek, D. Žiak * CGB Laboratory A.S., Dept. of Pathology, Ostrava, Czech Republic Objective: Breast cancer is the most frequent malignant tumor in women with a rising incidence. About 4 500 new cases are diagnosed in Czeck republic every year, up to 43 % die on it´s account.
In CGB laboratory are investigated 500 of malignant breast tumors every year. We evaluate the morphology, grade of differentiation and phenotype properties-hormone receptors expression, overexpression and amplification of HER-2/neu gene. 2 % of carcinomas posses significantly morphologically and phenotypically heterogenic tumor population in one and the same tumor leasion. Results: The most often sign of phenotype heterogenity found is hormone receptors expression or overexpression of HER-2/neu gene. Often we see two morphologically different tumor populations coexisting. The most interesting cases from our practice are on the poster. Conclusion: As for now, studies about the origin of heterogenity inside the same leasions, did not find a single theory to resolve it. A possible answer gives the theory of tumor stem cells and the model of clonal evolution. Our observations demonstrate and may explain the practical reason of different phenotype properties found in core cut biopsy where one population may be held, against the heterogenic properties of the tumor shown when resected as a whole.
High grade infiltrating carcinoma with squamous features -A case report D. R. Novac * , C. Ardeleanu, S. Taban, F. Cadariu, A. Dema * Municipal Clinical Hospital, Anatomical Pathology, Timisoara, Romania
Objective: Apocrine carcinoma is a rare and histologically distinct type of invasive breast carcinoma. Method: The patient, a 73 years-old woman was admitted to the hospital for a tumor mass in the upper internal quadrant of the left breast. Tissue fragments from the quadrantectomy specimen were routinely processed by paraffin embedding. For the immunohistochemical (IHC) study we used the following antibodies: CK7, androgen receptors (AR), estrogen receptors (ER), progesterone receptors (PR), high molecular weight cytokeratin (HMWCK), p63, gross cystic disease fluid protein-15 (GCDFP-15), En Vision system, visualization with diaminobenzidine. Results: The gross examination revealed a nodular, well delineated mass of 2,5/2,7/3 cm, firm, gray-beige with areas of necrosis on cut section. Microscopically, the tumor had a predominantly solid, partially cystic and less tubular growth pattern. The tumor cells had abundant eosinophilic granular cytoplasm and pleomorphic high-grade nuclei. A small tumor contingent presented squamoid features. IHC stains showed the following profile: CK7+, AR+, GCDFP-15+, ER-, PR-, p63/HMWCK + (focal/zonal). A diagnosis of high-grade infiltrating apocrine carcinoma with squamous differentiation was established. Conclusion: There are only a few cases of apocrine carcinoma on record and our case is even more special because of his peculiar aspect, the squamous differentiation.
The effects of treadmill exercise in the number and weight of mammary tumors chemically induced in female Sprague-Dawley rats: Preliminary results J. Oliveira * , C. Teixeira-Guedes, A. Faustino-Rocha, R. Soares-Maia, R. Arantes-Rodrigues, M. J. Pires, M. Ginja, P. Oliveira, R. Ferreira * UTAD, Dept. of Veterinary Sciences, Vila Real, Portugal
Objective: We hypothesized that moderate exercise in Treadmill may affect the mammary tumor development. N-methyl-N-nitrosourea (MNU) is a commonly used carcinogen to induce mammary carcinomas. The aim of this study was to evaluate the influence of Treadmill exercise in the development (number and weight) of female rat mammary tumors. Method: In this experimental protocol were used 21 female Sprague-Dawley rats. MNU was intraperitoneally administered at 50 days of age in a dose of 50 mg/Kg. Animals were randomly divided in two groups: sedentary (n=11) and exercised (n=10). The exercise program was started after carcinogen administration. Animals were exercised in a Treadmill Control LE8710® after an initial period of familiarization. Thirty-six weeks after MNU administration animals were sacrificed and tumors were counted and weighted. Results: Sedentary and exercised group presented 25 and 20 tumors, respectively. Pearson Chi-Square value was not significant (p>0.05). The mean tumors weight of sedentary group (5.34±10.58 g) was lower than exercised group (8.64 ±13.01 g). The difference between groups was not significant (p>0.05). Conclusion: We observed that exercised group showed minor number of tumors, however the lesions presented higher volume. Future morphological and biochemical analysis of tumors will allow a better understanding of the relation between mammary cancer and physical exercise. Objective: Primary non-Hodgkin's lymphomas is an uncommon disease representing approximately 0.15 % of all reported malignant mammary neoplasms. Clinically they are mainly observed as solitary lesions but may also be seen as multiple foci. Herein, we present a very rare case with the diagnosis of multifocal malignant lymphoma of the breast with a detailed clinicopathologic evaluation. Method: A 56-year-old-female patient with a right palpable breast lump admitted to the hospital. Mammography and ultrasonography findings showed 2 different foci of hypoechoic solid mass forming lesion in the right breast. Excisional biopsy has been performed. Results: In the histopathological evaluation, both lesions demonstrated diffuse infiltration of mammarian tissue with foci of necrosis; mitotically active tumor cells with large nucleus and prominent nucleoli. The immunohistochemical analysis revealed diffuse and strong LCA, CD20, CD43, focal CD68 positivity while pancytokeratin, EMA, CD34, SMA were negative. The case was diagnosed as Diffuse Large B-cell Lymphoma. Conclusion: Breast is an uncommon site for primary malignant lymphomas. We report a very rare case with a diagnosis of multifocal primary non-Hodgkin's lymphoma of the breast.
PS-17-065 Cytological, histopathological and clinical correlation at differential diagnosis of granulomatous mastitismalignancy T. Ozgur * , E. Atik, S. Toprak, H. Gokce, N. Sengul, M. Temiz * Mustafa Kemal University, Medical Faculty, Antakya, Turkey
Objective: Idiopathic Granulomatous Mastitis (IGM) is a rare disease that is difficult to diagnose by only radiological methods and clinical findings. Method: First case; a 30 years old woman with a well circumscribed mass of 5 cm diameter at right breast applied to our hospital general surgery outpatient clinic. In the examination of fine needle aspiration biopsy (FNAB), palisaded epitheloid histiocytes and scattered atypical cells have been seen on a bloody and inflammatory background. We suggested excisional biopsy. Second case; a 39 years old woman applied to our hospital general surgery outpatient clinic with a sore mass in her left breast. The mass has been excised for malignancy suspect. Foci of abscesses, active chronical inflammation, areas of hemorrhage and numerous granulomas containing epitheloid histiocytes are observed microscopically. Results: In microscobic evaluation of the excisional biopsy of the first case prediagnosed as granulomatous mastitis invasive ductal carcinoma has been our diagnosis. In the second case there have been numerous granulomas containing epitheloid histiocytes, microscopically. Our diagnosis was Granulomatous mastitis in this patient prediagnosed as malignancy.
Conclusion: Especially FNAB findings are easy to confuse with malignancy. We wanted to emphasize to clinician and pathologist to be more alert on distinction of granulomatous mastitis and malignancy. Objective: Triple-negative-breast-cancer (TNBC) that accounts for 10-20 % of all breast carcinomas is defined by the lack of estrogen receptor, progesterone receptor, HER2 expression with agressive clinical behavior. TNBC is categorized into basal like subtype which is characterized by the expression of basal cell markers and normal breast subtypes. Method: We studied on 41 immunohistochemically TNBC patients to determine EGFR, Cytokeratine5/6 (CK5/6), p53, Ki67, GCDFP15 expression patterns by immunohistochemistry, HER2/Chromosome 17 gene status by FISH. Results: Most of the tumors (90,2 %) were invasive ductal carcinomas. p53, Ki67, GCDFP15 mean positivity rates were 55,6 %; 51,7 %; 3,2 % respectively. GCDFP15 positivity was noted in 8 cases of which 6 were CK5/6(−). The cut-off value for CK5/6 positivity was 5 %. EGFR immunoreactivity was grouped into 0, 1+ as negative; 2+, 3+ as positive categories. CK5/6 was positive in 56,1 %, EGFR was positive in 51,2 % of the patients. The relation between CK5/6 and EGFR expression was statistically significant (p <0.01). HER2 FISH was negative in all cases. Conclusion: As a result GCDFP15 alone is not a useful marker to detect the metastasis of basaloid type breast cancers. CK5/6 and EGFR coexpression can be used to diagnose basaloid tumors with 5 % cut-off value. Objective: Lobular neoplasia (LNs) of the breast include atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS). Because LNs do not present typical clinical or radiologic findings, the diagnosis is incidental. Many reports suggest that LN is not only a risk factor of invasive lobular carcinoma but also is as a precursor. It is important to know about the incidence of LNs and associated disease in Korea. Method: A total 1551 cases of breast biopsy or excision from five major hospitals in Daegu were reviewed independently by 5 pathologists of the Daegu Breast Pathology Study Group. The incidence of ALH and LCIS, associated disease, the presence of microcalcification and necrosis were reviewed. All suspected cases of LNs were confirmed on E-cadherin immunohistochemical staining. Results: Only 46 cases out of 1551 cases (2.9 %) revealed LNs. Associated disease were 17cases of invasive ductal carcinoma, 10cases of in situ ductal carcinoma, 6 cases of invasive lobular carcinoma, 5 cases o f other benign disease, and 3 cases of florid ductal hyperplasia or columnar cell change.
Conclusion: The incidence of LNs was similar to western women. Incidental LNs were frequently associated with invasive ductal carcinoma and further excision is suggested if it was present on the needle biopsy specimen.
Silver-enhanced in Situ Hybridization (SISH) detection assay for HER2 gene status determination in breast carcinoma: A four year experience in our laboratory F. Patakiouta * , G. C. Balis, V. Theodorou, E. Triantafillidou, C. Fotiou, I. Boukovinas, C. Andreadis, G. Sibilidis * "Theagenion" Cancer Hospital, Dept. of Pathology, Thessaloniki, Greece
Objective: Assessment of HER2 status in breast cancer is important in the clinical management of patients and can be identified by a number of methods. In this study we present the results of the SISH technique used in our laboratory in the last 4 years. Method: We analyzed 398 cases of invasive breast carcinoma, including 41 core biopsies, which were previously characterized immunohistochemically (CB11) as equivocal on a protein level (HER2 2+). All cases were evaluated by bright-field SISH using the automated Ventana Benchmark XT machine. Evaluation was independently performed by two pathologists (FP, GB) based upon the algorithms of the manufacturers and ASCO/CAP guidelines. Results: Out of 398 cases the HER2 gene was amplified in 82 cases (20.6 %), while in 302 cases (75.9 %) there was no gene amplification. Fourteen of the cases (3.5 %) were characterized as equivocal. The concordance between the two pathologists was high (97.6 %). Conclusion: The SISH method is new, fully automated and very rapid. Furthermore the bright-field SISH signal does not decay and can be visualized by any standard bright-field microscope even after long periods of time, making it particularly suitable for routine application in surgical pathology. Objective: Breast fibromatosis (BF) represents 0.2 % of breast tumours that simulate carcinoma. The aim of this study is to characterize the cases of BF diagnosed in our institution. Method: BF cases diagnosed from 1999 to 2009 were evaluated with clinicopathological and immunohistochemical parameters. We also studied mutations in exon 3 of the gene for β-catenin in 4 cases. Ultrastructural study (US) was performed in 1 case. Results: There were 5 women between 33 and 69 years old. Radiologically they showed deeply located hypoechoic irregular lesions. Size ranged between 1 and 23 cm. Histologically they were characterized by an ill-defined spindle cell proliferation without epithelial elements or atypia. Immunohistochemical study was negative for AE1/AE3, CK5/ 6, P63, CD34 and BCL2. Actin was diffusely positive in 4/ 5, and S100 and Desmin focally positive in 4/5. β-catenin showed cytoplasmic(5/5) and nuclear(1/5) expression. The US showed double smooth muscle and myofibroblastic differentiation. Three patients showed mutations in exon 3 of the β-catenin gene (2 with substitution at codon 41 (T41A) and 1 deletion of 33 base pairs (A43_E65del)). Conclusion: Focal desmin expression and US suggest smooth muscle differentiation in some BF. All cases showed cytoplasmatic positivity for β-catenin but nuclear expression was seen in only 20 % of cases. 75 % of the valuable cases showed genetic alterations of β-catenin. Objective: Breast cancer is a heterogenous disease consisting of distinct entities characterized by different gene expression patterns. Gene expression profiling of breast cancer revealed 4 major subtypes, including hormone receptor (HR) positive luminal A and B, HER-2 positive and basallike breast cancer. Most of the basal-like carcinomas are triple negative (HR and HER-2 negative). As most triple negative breast cancers express EGFR, mutations in the signal transducing cascade of RAS/RAF/MAP kinase might play a role in tumor progression. The aim of this study was to determine the incidence of KRAS and BRAF V600 mutations in triple negative breast carcinomas. Method: Total genomic DNA was obtained from 37 formalin-fixed, paraffin embedded, triple negative breast tumors. KRAS was analyzed by pyrosequencing of codon 12, 13, 61. BRAF (exon 15, V600) mutations were analyzed with AutoGenomics INFINITI BRAF assay on the Autogenomics INFINITI analyzer.
Results: We found KRAS mutation in only one case (2.7 %), apocrine carcinoma which was Gly12Asp (GGT> GAT) mutation. The BRAF V600 mutations were not detected in all analyzed tumors. Conclusion: We concluded that KRAS mutation was rare in triple negative breast carcinomas and high frequency of EGFR overexpression in this subtype might be related to other pathways in EGFR signaling.
The pre-lymphatic system and the lymphatic network of the breast in menstruating and in menopausal women: A comparative study S. Popovska * , I. Ivanov, T. Betova, T. Dineva, Z. Ivanova * Medical University Hospital, Dept. of Clinical Pathology, Pleven, Bulgaria
Objective: The pre-lymphatic system was initially described in the 1960s by Casley-Smith and Florey. It consists of CD34 positive fibroblasts lined stromal spaces communicating with the lymphatic capillaries. The aim of the following investigation was to compare the lympho-vascular density and pre-lymphatic network density of breast tissues in the context of the menstrual status of the patients. Method: Formalin-fixed, paraffin embedded tissue specimens from 105 patients with primary breast cancer were studied. Tumor-free tissue materials were selected. Tissue processing and immunohistochemical staining with D2-40 and CD34 antibody was performed in accordance to standard laboratory protocols. Quantitative evaluation of the lymphatic and pre-lymphatic system was performed. The Kruskal -Wallies test was used to compare differences between the median values of the studied variables. Statistical significance of the differences was considered if p<0.05. Results: The pre-lymphatic network was found to be significantly more dense in menstruating, compared to menopausal women K-W=61.04; p<0.0001 n=105. Lymphatic vascular density was higher in menstruating compared to menopausal women K-W=7.60882; p=0.006; n=105. Conclusion: Involution changes in the pre-lymphatic and lymphatic system of the breast were found to appear in postmenopausal women. This may be the explanation for the slow local progression of breast cancer observed often in elder patients.
Comperative study of Herceptest and fluorescence in situ hybridization results in breast carcinoma: Indeterminate (2+) group must not be a wide range uncertainty category Y. Saglican * , U. Ince * Acibadem Maslak Hospital, Dept. of Pathology, Istanbul, Turkey Objective: HercepTest is an exclusively used immunohistochemistry method for testing Her2/neu overexpression in breast carcinomas. The purpose of this study is to select the proper patients candidate to in situ hybridization, with an indeterminate Her2 score. Method: A retrospective analysis of 163 cases with invasive breast carcinoma were evaluated for Her2 status using immunohistochemistry and fluorescence in situ hybridization. İmmunohistochemistry of cases that had been previously evaluated by nine different pathologists in our department, were reviewed by two experienced pathologists. Interpretation of the immunohistochemistry results was made without knowing the first results. Results: Compared to the first evaluation; cases with score 0, 1+ and 3+correlated with the review of immunhistochemistry and none of score 0 and 1+ tumors were positive with in situ hybridization. Gene amplification was detected in 43 cases; 23 of them with 2+score. Three of 3+ tumors and 106 of 2+ cases were negative with in situ hybridization. Discordance was detected in 21 cases in 2+ score; 17 of them were reclassified as negative and 4 were technically unsatisfactory. Conclusion: Using a different cut-off value for indeterminate Her2 results and training of pathologists to minimaze the interpretational differences, will maximize the accuracy, while not submitting to unnecessary molecular tests. Objective: The excision repair cross-complementation 1 (ERCC1) enzyme plays an essential role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in different types of cancer. The aim of the present study was to evaluate the clinicopathological significance of ERCC1 expression in breast cancer patients. Method: We used immunohistochemical to analyze ERCC1 expression in a tissue microarray from 135 breast carcinomas. ERCC1 expression analysis was available for 109 cases and was correlated with clinicopathologic factors and outcome data. Results: ERCC1-positive was observed in 58 (53.2 %) cases and was correlated with smaller tumor size (P= 0.007) and with positivity for estrogen receptor (P=0.040). ERCC1 expression did not correlate with overall and disease-free survival rates. The majority (72.7 %) of special histological types of invasive breast carcinomas was positive for ERCC1 compared to invasive ductal carcinomas (ERCC1-positive in 51.1 % of the cases). Similarly, triple negative breast cancers (TNBC) were more frequently negative for ERCC1 (61.5 % of the cases) compared to the non-TNBC (41.5 %). Conclusion: ERCC1 expression correlated significantly with favorable prognostic factors, such as smaller tumor size and ER-positivity, suggesting a possible role for ERCC1 as a predictive and/or prognostic marker in breast cancer.
A study of CD10 positive basal/myoepithelial cells in a consecutive series of in situ lobular neoplasia of the breast S. Shousha * , G. Forbes * Charing Cross Hospital, Dept. of Histopathology, London, United Kingdom
Objective: We have previously reported a few sporadic cases of in situ lobular neoplasia (ILN) associated with marked proliferation of CD10 positive basal/myoepithelial cells. Method: Twenty consecutive cases of pure ILN and 9 DCIS were studied. New sections were cut and stained for E-Cadherin and CD10. Results: One out of the 20 cases showed foci of invasive lobular carcinoma in the new cut sections. All 20 ILN cases showed increased proliferation of CD10 positive basal cells, varying from a focal mild increase to a marked proliferation surrounding or mingling with the lobular cells. CD10 positive cell proliferation was least observed in a case of ILN with central necrosis and in foci of ILN seen adjacent to the invasive carcinoma. The invasive tumour cells were CD10 negative. All cases of DCIS had a thin layer of CD10 positive myoepithelial cells, that was incomplete or absent in some foci. Conclusion: Our findings confirm the presence of a unique relationship between excess proliferation of CD10 positive basal cells and ILN; an association which is not seen in DCIS or invasive carcinoma. This is particularly interesting as recent evidence indicates that CD10 is involved in the regulation of mammary stem and 'sphere forming' cells. Objective: Primary neuroendocrine carcinomas (NEC) of the breast are defined by the WHO classification as a group of neoplasms that express neuroendocrine markers in more than 50 % of the cells. These tumors can exhibit different morphologic appearances including solid, nested or alveolar pattern, papillary or mucinous differentiation or conventional ductal structures. The aim of this study is to identify histomorphological features useful in the recognition of this tumors. Method: 160 invasive breast carcinomas were reviewed, none of them previously typified as NEC. 154 were ductal carcinomas (26 grade I, 61 grade II and 66 grade III) and 6 lobullilar carcinomas. In all cases immunohistochemical staining for the neuroendocrine marker synaptophysin was performed. Results: Eight of the 160 cases showed intense immunoreactivity for synaptophysin. All of them were predominantly composed of solid, confluent nests and cords of cells with medium-to high grade nuclear atypia. Two cases showed intense desmoplastic or sclerotic stromal response. A colloid component was identified in another two cases. Conclusion: Due to its morphologic variability, mammary NEC is frequently underrecognized. A predominantly solid pattern of confluent nests and trabeculae associated with a prominent sclerotic stromal response or colloid differentiation should prompt to perform additional immunohistochemical staining in order to exclude neuroendocrine differentiation. Objective: Association between centrosome abnormalities and response to chemotherapy has not been fully elucidated in breast carcinomas (BC). Here we analysed the association between Aurora kinase A (AURKA) and Gamma-tubulin (GT) and response to neoadjuvant chemotherapy (PST) in patients treated with BC. Method: The immunohistochemical expression of AURKA and GT was analysed in 44 core biopsies of BCs taken before administration of PST. AURKA expression was analysed using a modified Allred-like scoring system (intensity and percentage of staining combined). Cells containing one or two centrosomes as determined by GT were considered negative, whereas cells containing more than two centrosomes were regarded as positive. Centrosome amplification was graded in each specimen as follows: negative (0-2 % of cells); weak (2-10 %); moderate (11-20 %); and strong (21 %<). Pathological response rates were assessed using Chevallier's classification. Results: Centrosome amplification was significantly higher in patients achieving complete response when compared with the cases where partial remission or no response was defined (p=0.03), whereas we found no significant correlation between the expression of AURKA protein and response rate to neoadjuvant chemotherapy (p=0.24). Conclusion: BCs showing centrosomal amplification as determined by GT present higher response rates to neoadjuvant chemotherapy, but need further validation.
Breast granulocytic sarcoma with aleukemic presentation: A case report O. Tzaida * , P. Giagkazogloy, P. Repousis, M. Kotsopoulou, I. Kasselaki, C. Valavanis, I. Iacovidou * Anticancer Hospital Metaxa, Peiraias, Greece
Objective: Granulocytic Sarcoma (GS), a rare solid tumor of immature myeloid cells in exrtamedullary sites, usually occurs during the natural course of acute myelogenic leukemia (AML). Rarely is presented without overt hematological disease. The breast is an uncommon site of localization. Method: We report a case of breast GS with aleukemic presentation, condition that is extremely rare and requires a high index of suspicion for diagnosis. A 46 year old woman, presented with a solitary, non-tender, palpable left breast mass. A lumpectomy was done. Results: Histopathology revealed breast parenchyma diffuse infiltration, in a targetoid pattern, by immature small cells intermingled with eosinophils. Lobular carcinoma and lymphoma were considered in differential diagnosis. The myeloid origin of the neoplasm was established by immunohistochemical analysis that revealed LCA, MPO, CD34, HLADR, CD68 (Kp1) positivity. There was no evidence of leukemia in the peripheral blood and bone marrow. The patient was treated as AML with systemic chemotherapy and 5 months after the diagnosis she is without evidence of disease. Conclusion: Breast GS is a challenging situation both for pathologists and hematologists. Available evidence strongly favors the application of systemic chemotherapy despite the appeared localized nature of the disease. Method: Paraffin blocks of tumor tissue of 152 breast cancer cases collected at our Cancer Centre for 3 years. The immunohistochemical study was performed by standard techniques using following antibodies (Dako): ER, PR, HER-2, EGFR, CK5/6, CK17, Ki67. Results: All cases were classified into the 5 subtypes: luminal A (n = 91; 59.8 %); luminal B (n =13; 8.6 %); HER-2-positive (n=19; 12.5 %); basal (n=19; 12.5 %); 5negative (n=10; 6.6 %). Conclusion: Luminal breast cancer phenotype predominates in our collection, predictive/prognostic value of molecular genetic subtypes is planned to be investigated. We recognized statistically significant differences (P<0,001) in five-year survival rates between tumors of luminal (A, B) and basal subtypes.
Proliferating trichilemmal cyst with atypical cytological features R. G. Wright * , C. Gallivan, T. Molden-Hauer, R. Liang * Gold Coast Hospital, Pathology Queensland, Australia
Objective: Proliferating trichilemmal cysts are relatively common occurring within the scalp of elderly women. Proliferating trichilemmal cysts of the breast, however, are rare. These cysts are benign with very few demonstrating malignant transformation. Method: We report a case of proliferating trichilemmal cyst of the breast with atypical cytology which prompted excisional biopsy. Cytological assessment was useful in this case as it indicated excisional biopsy rather than a more extensive excision. Results: Smears produced from the FNA showed sheets of atypical epithelial cells with foamy histiocytes, multinucleate cells and fibrotic material with evidence suggestive of fat necrosis. A cell block was acellular. A specific diagnosis was not made. An excision specimen was advised and a proliferating trichilemmal cyst was demonstrated. Objective: A large clinical value has determination of molecular subtype of breast cancer (BC). To clarify morphological and molecular biological similarities and differences between BC depending on age, we compared features between tumors of younger and older women. Method: 573 patients with BC are included in research: from 18 till 86 years, among them 254 patients were till 35 years. ER, PR, Her-2/neu, p53, p63, Ki67, CK5/14, p21, Bag1, Mcl1, pS2, VEGFR, Her-1 were analyzed in all cases by immunohistochemistry. Results: The younger patients with BC had higher expression of p53, p63, p21, Bag1, Mcl1, Ki-67, VEGFR, HER-1 (p<0,001) than older patients. There was a basal-like molecular subtype of BC 3-fold more frequent compared with older patients (21,6 % versus 7,2 %; p<0,0001). Threeyears overall survival in patients till 35 years was by 11,5 % and a five-year overall survival was by 15,5 % lower than in patients over 35 years (p<0,001). Objective: Breast HER2 ISH relies on correct enumeration of HER2/CEP 17 signal. However, as with immunohistochemistry, there are technical pitfalls which may render ISH difficult/unreliable to interpret. The UK NEQAS has established a 'technical' ISH module to provide feedback to laboratories using both chromogen and fluorescence based tehcniques. Method: Unstained slides consisting of 4 breast cancer cell lines were distributed. Laboratories were asked to demonstrate HER2 gene amplification using their routine assay, and then return the slides for assessment. Chromogen based methods were assessed around a multi-header microscope with 4 assessors scoring each slides, whilst the fluorescent ISH method was assessed by a single individual. Assessors scored the 'readability' of each slide, without counting the probes, and provided feedback where the hybridisation technique could hinder interpretation. Results: CISH based methods showed acceptable staining in 50 % (N=32) of cases. Staining problems ranged from loss/poor HER2/CEP17 signal, non-specific staining and morphological damage. The FISH pilot assessment showed an acceptable rate of 32 % (n=21), with the low pass rate being attributed to poor preservation/quenching of fluorescence signal.
Conclusion: Greater emphasis needs to be placed on the 'readability' of an ISH slide, prior to carrying out the process of enumeration.
Tuesday, 11 September 2012, 09.30 -10.30 Objective: Intravenous leiomyomatosis is a very rare growth pattern variant of leiomyoma in which nodular masses of tumor grow within venous channels. Occasionally the tumor can extend to vena cava and the right heart. Method: We present a case of a 45-year-old woman, who was admitted in the emergency room with rapidly evolving exertional dyspnea. Cardiac ultrasonography revealed a "big mass in the right chambers". She was submitted to a right atriotomy with resection of part of the tumor, which was sent for intraoperative consultation. Results: Grossly, the tumor was polypoid, firm, with a smooth surface. The frozen section showed a lesion composed of tortuous vessels and in some areas a fibrillar eosinophil extracellular matrix and others with spindle cells. No significant atypic or pleomorphic cells, mitosis or necrosis were observed. The diagnosis was deferred for definitive paraffin sections. In the definitive H&E and with immunohistochemical stains, the case was diagnosed as an intravenous leiomyomatosis, and the diagnosis was confirmed in the hysterectomy specimen. Conclusion: Intravenous leiomyomatosis with cardiac involvement is an extremely rare condition. Clinical information is essential for the correct diagnosis in frozen section.
Characterization of a specific mechanism for late loss of cardiac allograft: The antibody mediated rejection (AMR) as a major factor of cardiac allograft vasculopathy (CAV) P. Bruneval * , C. Toquet, A. Loupy, P. Pouvier, A. Cazes, J.-P. Duong van Huyen * France
Objective: The rational of this study was to investigate explanted failing grafts in the light of new concepts in rejection mechanisms, namely AMR. Method: This retrospective multicentric study collected 31 explanted cardiac grafts failing ≥1 year posttransplantation. The vasculature of the grafts was assessed from epicardial coronary arteries to myocardial microcirculation. Immunohistochemistry was performed for C4d complement fraction deposition and CD68-positive macrophages in the explanted grafts and previous endomyocardial biopsies. Donor specific antibodies (DSA) were retrospectively assessed using Luminex SA technique. Results: A pure classical coronary atherosclerosis pattern was observed in 6/31 (19 %). A pure pattern of CAV was present in 12/31 (39 %) and a mixed pattern associating CAV and atherosclerosis features in 9/31 (29 %). Interestingly the CAV pure and mixed patterns were associated with vascular inflammation within the arteries and/or the microcirculation with C4d deposits and macrophages (15/21) in the explanted grafts versus nil (0/6) in pure atherosclerosis pattern (p<0.002). Furthermore they were associated with previous AMR episodes on endomyocardial biopsies (11/ 21) and by positive DSA (10/14) versus nil in pure atherosclerosis pattern (p<0.05 and p=0.05, respectively). Conclusion: In failing cardiac grafts CAV lesions are associated with makers of AMR. CAV should be the consequence in coronary arteries of an ongoing AMR process. Results: In our cases increase in fibrous and adipose tissue concordant with age, indicating an age related nature, were detected. Fibrous and fatty tissue infiltration appeared at the age of 35. Fatty infiltration started at the age between 20 and 34 at sinoatrial node. In 4 cases calcification and in 19 cases inflammation was observed. Amyloid accumulation was not present. In 7 cases myocardial infarction not involving CCS was seen. In 1 case fibroelastoma was detected. Conclusion: In Turkish population age related fibrosis and fatty infiltration in CCS appeared at the age of 35 and increased with age. Fatty infiltration in sinoatrial node started at a younger age than that of reported in the literature. In cases whom the cause of death could not be determined we could not detect lethal pathologic features. However we think that examination of the CCS will improve the quality of autopsy diagnosis.
Periadventitial adipose tissue in human coronary atherosclerosis: A neurotrophin study P. Ghenev * , P. Panayotov, G. Chaldakov, L. Aloe * Medical University Varna, Dept. of Pathology, Bulgaria
Objective: Recent evidence demonstrates that epicardial adipose tissue, including coronary periadventitial adipose tissue (tunica adiposa), are paracrine sources of bioactive mediators (adipokines, NO, H2S) which may be involved in coronary atherogenesis. Because of the increasing interest for extra-neuronal effects (inflammation, wound healing, lipid and glucose metabolism) of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), including in human coronary atherosclerosis in autopsy samples, the aim of the present study is to evaluate the expression of NGF and BDNF and their receptors (TrkA and TrkB, respectively) in cardiosurgery biopsy samples of pericoronary adipose tissue. Method: Immunohistochemistry of NGF, BDNF and their Trk receptors. Results: NGF, BDNF, Trk A and TrkB expression was lower in periadventitial adipose tissue of atherosclerotic coronary arteries as compared to non-affected coronaries. Conclusion: We provide the first evidence for a possible role of neurotrophins in the molecular remodeling of tunica adiposa in human coronary atherosclerosis. This study is dedicated to 103rd Anniversary of Rita Levi-Montalcini, the Nobel laureate for the discovery of NGF. Objective: In normal myocardium, apoptotic myocytes are usually absent, or if affected, with most of 28 positive cells per million. Method: The apoptosis was measured by the TUNEL method in 60 patients with dilated cardiomyopathy (DCM) and myocarditis (MC), and expresed by apoptotic index (AI) in 30 patients within both groups, and to correlated with ejection fraction (EF) and with different morphological stages of these entities. Statistical analisys was performed, and p values ≤0.05 were considered statistically significant. Results: AI in MC cases was 4,23+12,16, and in DCM cases the value was significantly higher 5,41+9,33 (p= 0.005). In the group MC patients, we didn't found statistically significant correlation between AI and different values of EF (p=0.701). The analysis of the value of AI between patients with different morphological stages of MC didn't show statistically significant correlation (p=0.535), as well as in DCM (p=0.312). Conclusion: Apoptosis play a significant role in DCM and MC but its significance in the progression to heart failure has still to be established. Objective: Cardiac lesions following blunt chest trauma are multiple and of various degrees of severity. "Cardiac Contusion" is a difficult diagnosis, especially antemortem and if signs of thoracic trauma are not evident on external habitus and on the underlying internal plans of cardiac area. Is the contusion a cause of death per se? Method: The authors present two cases of road accident male victims, aged 43 and 50 year-old, submited to complete postmortem examination with ancillary diagnostic methods. Results: No traumatic lesions or underlying pathology was found at the autopsy. Epicardial and myocardial haemorrhagic foci were histologicaly documented. Toxicology was negative. Conclusion: Cardiac Contusions are caused by one of the following mechanisms: 1) heart compression between bone structures, 2) sudden acceleration-deceleration movements and 3) sudden thoracic/abdominal pressure increase. They may be asymptomatic or lead to rhythm disturbances responsible for unexpected death (10 %) . They seem to be underdiagnosed, thus suspecting the entity "Contusio Cordis" is crucial, not only antemortem for correct surveillance and treatment, but also postmortem to adequately establish causality nexus in cases of post-thoracic trauma deaths.
The influence of bone marrow-derived multipotent stromal cells on myocardial scar healing in experimental myocardial infarction L. Kakturskiy * , T. Fatkhudinov * Institute of Human Morphology, Central Laboratory of Pathology, Moscow, Russia Objective: Previous studies have shown the benefits of intracoronary infusion of bone marrow-derived multipotent stromal cells (BM-MSCs) in heart disease. Our aim was to study engraftment, differentiation of BM-MSCs and its role in myocardial reparation. Method: Acute myocardial ischemia was produced by transient occlusion (total of 20 min) of the proximal left anterior descending coronary artery and followed by reperfusion. BM-MSCs were isolated, expanded by standard and labeled with PKH26 (Sigma). Cells were CD90 and CD105 positive. 30 days after occlusion during cross-clamping of aorta the cells were administered into the cavity of the left ventricle at the concentration 5×106 in saline solution. In 1 day, 2, 4 and 6 weeks after transplantation the labeled cells were detected in the cryosections and heart morphometry was performed. Results: BM-MSCs were detected only in the scar tissue and had a fibroblast-like phenotype. They neither differentiated into cardiomyocytes nor into the cells of blood vessels. In 4 weeks after transplantation the scar thickness was higher in the group with cell transplantation (p<0.001). Conclusion: Engrafted BM-MSCs promote myocardial fibrosis only in the scar, but not in the perifocal myocardium, provided strengthening of the scar, remodelling of outflow tract, and improvements the myocardial function.
Objective: From January 2000 to December 2009 (Institute of Pathology and Legal Medicine, Paolo Giaccone -Palermo) 57 autopsies were performed on 37 males and 20 females who died of a massive PTE. Method: Systematic autopsy. Results: Saddle pulmonary thrombosis in 33 cases, in 24 isolated in both the main pulmonary arteries. In all cases the transverse heart diameter was 1,5-2 cm superior to the longitudinal one, the interventricular septum became rectilinear with a right ventricular dilation. The coronary artery anatomy was: critical stenosis through fibroatheromatous plaque of the dominant right coronary artery (RCA, 2), non dominant left coronary artery (LCA, 3); non critical stenosis of RCA (14), LCA (8); absence of stenosis (30). Histological ventricular examination showed hypoxic-ischaemic and/or reperfusion and overload lesions. In all cases, the lungs showed plurifocal occlusive thrombosis of intramural pulmonary arterial (IPA) vessels associated with adjacent infarctual necrosis (49); acute partially haemorrhagic basal oedema (35) and desquamative macrophagic alveolitis (28). Conclusion: In all cases of massive PTE the thrombosis of IPA vessels associated with an adjacent infarction (86 %) is constant. This suggests that death is nearly always preceded or triggered by one or more episodes (clinically silent or with non specific symptoms) of thromboembolism of IPA vessels.
Cardiac findings in routine fetal autopsies: More than it meets the eye? E. Rios * , C. Bartosch, C. Ramalho, J. Monterroso, O. Brandão * Centro Hospitalar S João, Pathology, Porto, Portugal Objective: Congenital heart disease (CHD) is the most common malformation in newborns. Our aim was to evaluate the spectrum of CHD in consecutively performed fetal autopsies and to correlate prenatal and postmortem diagnoses. Method: A retrospective study of 726 fetal autopsies was performed in a tertiary referral hospital. CHD was classified in seven categories: left ventricular outflow tract obstruction-LVOTO, right ventricular outflow tract obstruction-RVOTO, septal defects, connection anomalies, conotruncal anomalies, complex anomalies and others. Cardiac defects were also classified as isolated or associated with others anomalies.
Results: CHD was identified in 99 (13.6 %) fetuses. Most common categories were septal defects and complex anomalies. Associated anomalies were found in 67 fetuses (67.7 %). In this group, septal defects were the most common CHD, frequency being significantly higher than in the group of isolated cardiac anomalies (p=0.012). Comparison of prenatal and postmortem diagnoses (50 cases) showed complete or partial agreement in 36 and 10 cases (72 % and 20 %, respectively) and complete disagreement in 4 cases (8 %). In the latter group, prenatal diagnosis had not been done by a pediatric cardiologist. Conclusion: The high prevalence of CHD in lost pregnancies highlights the importance of systematic fetal autopsy performed by a specialized pediatric pathologist. Objective: The appearance of pathological calcification, which leads to significant changes of vascular wall, which underlie the development of atherosclerotic complications, has a great importance in the morphology of atherosclerosis development. Purpose of the work is the study of pathological biomineralization in heart valves affected by atherosclerosis. Method: The study was conducted on sectional material of mitral and aortic valves obtained during autopsy. The tissue of heart valves was studied by methods of histology, electron microscopy, X-ray diffraction, infrared spectroscopy. Results: Macroscopically mitral and aortic valves are thickened, opaque, dull whitish, sometimes with deformity and ulceration. Histology observed the formation of atherosclerotic plaques, fibrous changes, and inflammatory infiltration. According to X-ray phase analysis biominerals in heart valves are represented by apatite crystalline phase. Sizes of crystals have distinct age dependence. The results of infrared spectroscopy revealed absorption bands of carbonate apatite replacement; in all instances they correspond to the type of substitution B (CO3 2-replaces PO4 3-). Conclusion: The study of pathological mineral formations on human heart valves by the range of morphological and physicochemical methods show that they are aggregates of interacting organic and mineral components, their ratio changes with "maturation": the organic component decreases and the mineral component, represented by carbonate containing hydroxyapatite doped with chlorine grows while improving, so the process is dynamic.
Relationship between myocardial injury, oxidative stress mechanism and sepsis/septic shock in infants submitted to surgery for congenital heart defects M. Silva de Oliveira * , E. Medeiros Floriano, P. Henrique Manso, R. Nilsson Sgabieri, J. Guilherme Klamt, E. Zangiacomi Martinez, W. Vilella de Andrade Vicente, S. Gusmão Ramos * University of São Paulo, Dept. of Pathology, Ribeirão Preto, Brazil
Objective: A progressive ventricular dysfunction caused by ischemic myocardial injuries remains one of the leading causes of death during the postoperative course in congenital heart disease (CHD). The aim of this study was to investigate the role of oxidative stress in these myocardial injuries. Method: Myocardial injuries and oxidative stress mechanisms were assessed by histopathology and immunohistochemistry and quantified by morphometrical analyses. Results: Myocardial injury was observed in pediatric patients submitted to surgery for CHD with cardiopulmonary bypass, followed by lethal exit. Oxidative stress mechanisms were directly related to these particular types of myocardial injuries. Importantly, 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation, is strongly expressed, especially in irreversible myocardial lesions. Although morphologically similar, myocardial injuries observed in patients who evolved with sepsis in the peri-operatory period exhibited a completely different set of oxidative stress mechanisms. Increased concentrations of nitrotyrosine protein adducts were observed in these patients, suggesting that peroxynitrite-mediated protein nitration may be the predominant oxidative stress mechanism found in these situations. Conclusion: The underlying mechanisms of these lesions seem to be related to the development of ischemia or ischemia/reperfusion followed by oxidative stress mechanisms that vary depending on whether sepsis was present. While the exact mechanism is not fully understood, it has been suggested that endogenous catecholamine release could have a role in this process. Objective: A significant decrease of the mean cardiomyocyte DNA content and increased numbers of diploid cardiomyocytes after ventricular unloading was demonstrated, suggesting a numerical increase of cardiomyocytes. The heart harbours several stem cells populations including c-kit (CD 117) + cells and side population cells (SPC), that might proliferate after unloading and generate diploid cardiomyocytes. It was tested, whether there is an increase of ABCG2+ SPC and CD117+ cells after unloading. Method: In paired myocardial samples (before and after LVAD), the number of cells with immunoexpression of ABCG-2, c-kit/CD 117 and MEF-2 was assessed by immunohistochemistry and morphometrically determined. Results: A significant increase of SPC and cells with coexpression of c-kit and MEF-2 after unloading was observed (p=0.001). A significant positive correlation between both SPC and cells with coexpression of c-kit and MEF-2 expression was observed (p=0.007 and 0.01). No correlation was found between the number of SPC and the mean cardiomyocyte DNA content. Conclusion: SPC are significantly increased in the myocardium after ventricular unloading, suggesting a role of stem cell proliferation during "reverse cardiac remodelling". These cells might proliferate and commit to different cell lineages such as cardiomyocytes or endothelium, and thus ameliorate cardiac function.
Role of elastin gene polymorphism in ascending aorta aneurysm development in patients with essential hypertension V. Zakharova * , V. Dosenko, M. Kostiv, E. Rudenko * Cardiovascular Surgery Institute, Pathology, Kiyv, Ukraine
Objective: There is supposition about genetic propensity in the ascending aorta aneurysm (AAA) development. Aim: to investigate role of elastin gene polymorphism (rs2071307) in AAA development in essential hypertension (EH). Method: DNA samples from persons with AAA and EH (1 group), with EH only (2 group) and healthy subjects (3 group) were examined. Polymorphism rs2071307 was studied with real-time PCR. Histological examination of AAA walls was performed in 1 group. Results: Distribution on genotypes A/A, A/G, G/G was the following: 1 group -42.1 %, 42.1 %, 15.8 %; 2 group -68 %, 24 %, 8 %; 3 group -18.5 %, 45.6 %, 35.9 %. Fragmentation of elastic membranes and media collagen component strengthening were revealed in persons with A/A, A/G, G/ G genotypes in 54.2 %, 70.8 %, 11.1 % respectively. Persons with G/G genotype have more frequently (66.6 %) than others (A/A -16.6 %, AG -12.5 %) signs of inflammation, which promote AAA development parallel to EH. Conclusion: We obtained evidence that elastin gene polymorphism (rs2071307) plays important role in EH and AAA development. In persons with allele A in rs2071307 risk of EH and AAA development is higher than in G/G genotype. Mostly allele A in rs2071307 is accompanied with elastic membranes fragmentation and their substitution by collagen that decreases arterial wall elasticity.
Tuesday, 11 September 2012, 09.30 -10.30 Objective: Our aim is to inform on the frequency, clinical aspects, and histopathological features of the tumor metastatic to the orbit. In adults, the most common tumor metastatic to the orbit are carcinomas, breast carcinoma the most frequent. Less common are the metastases from gastrointestinal tract, kidneys, and prostate. Cutaneous malignant melanoma is the most common nonepithelial tumor metastatic to the orbit. Method: Orbital metastatic tumors may occur at any age, but most often between 40 and 70 years of age. Most reported statistics reveal a slight predominance of women, accounted for by the fact that breast carcinoma is causing metastases most frequently. A frequent finding in the cases of metastatic carcinoma is enophthalmos that is associated with scirrhous carcinomas. We present two cases of orbital metastatic tumors in which the primary tumors were a basal cell carcinoma and a cutaneous malignant melanoma. Results: As to the metastasis from a basal cell carcinoma, the primary tumor was located in the external angle of the eyeball and metastasized the external 1/3 of the orbit. In the case of malignant melanoma, the primary tumor was located in the upper lid and invaded the orbital floor and fat. In both patients eyeball enucleation was performed. Conclusion: We chose to present these cases given the differential diagnostic challenge and rarity of their location.
Uveal melanoma A. Dumitriu * , S. Dumitriu * UMF Iasi, Dept. of Neurology, Romania
Objective: Uveal melanoma which arises from melanocytes residing in the stroma is the most common primary intraocular cancer in adults.
We report a case of uveal melanoma presenting as complicated cataract in a 65 years-old male. Results: The right eye was blind for 6 months. The patient underwent extended enucleation and histopathology was consistent with uveal melanoma. Conclusion: We present this case in order to provide the medical community a basic reference that would help to make further progress in this rare disease, which remains difficult to treat. Results: There were more women than men (52 % versus 48 %). Metastases developed in 39 % (total group mean follow up of 7.14 year). Mixed tumors were most common overall (43.5 %): for non metastasizing tumors there were 41.1 % mixed, 36.1 % fusiform and 17.2 % epithelioid types. For metastatic tumors there were 46.7 % mixed, 21.5 % epithelioid and 21.5 % fusiform types. The difference between cell type and metastatic development was significant in all but epithelioid compared with mixed tumors. Epithelioid type had a relative risk of mortality of 3.51 and mixed had 2.18 compared to fusiform cell type. Conclusion: Our patients were younger, with larger tumors and different histological proportions from those of the Collaborative Ocular Melanoma Study. Five year survival times were similar to those for Europe.
Uveal melanoma between preserving vision and prolonging survival. Pathological contributions to a better clinical management M. Mera * , L. Blaga * UMF Cluj-Napoca, Anatomic Pathology, Romania
Objective: Uveal melanoma has a high metastatic potential which is invariably fatal. There is an ongoing debate on the impact of ocular treatment on metastatic disease and survival. Improved prognostication is needed to identify cases that could avoid unnecessary loss of vision while avoiding unethical care. Our study is aimed at contributing to the developing of reliable tools to assess the metastatic risk.
Method: An accurate prediction should include both clinical features and histologic parameters as biopsy techniques continuously advance. 120 cases of uveal melanoma, either reviewed or prospectively managed in our Pathology Department, were included in a Log-logistic multivariate survival model that we have considered would best accomodate our data. The performance of the model was assessed by bootstrap re-sampling. Results: Extravascular matrix patterns, optic nerve invasion, tumorinfiltrating lymphocytes, tumor cytomorphology, nucleolar size and the mitotic count together with initial visual acuity have been found to be the most important predictors, ahead of the traditional TNM stage. The results are provisional but encouraging. Conclusion: There is still need for multidisciplinary approach and multicenter collaboration in an effort to elucidate the uncertainty related to the best management of one of the most traumatising types of cancer.
Orbital hemangiopericytoma: A case report E. Omoscharka * , K. Lankachandra * University of Missouri, Dept. of Pathology, Kansas City, USA Objective: Hemangiopericytomas are rare vascular tumors arising from Zimmerman's pericytes, which ubiquitously surround blood capillaries and post-capillary venules. They rarely occur in the orbit, accounting for only 1 % of all orbital neoplasms. In most cases the primary presentation is progressive proptosis. Method: A 26-year-old female was first seen for the evaluation of a right orbital mass which has been causing proptosis. Associated symptoms included peripheral vision loss in the right eye, persistent headache with occasional balance problems. A palpable, 3.2 × 2.5 cm well circumscribed mass with a smooth surface, resting on the inferior orbital wall was completely removed through right anterior orbitotomy. H&E stained histologic sections were studied. In addition, tissue sections were subjected to immunohistochemical stains for CD34, CD99, BCL2, SMA, desmin, EMA and S-100. Results: The mass was composed of spindle cells with very low mitotic activity. Staghorn vascular channels were evident, and in several areas the tumor cells invaded the pseudocapsule. The tumor cells were positive for CD34, CD99 and bcl-2 supporting the histopathologic diagnosis of benign hemangiopericytoma.
Conclusion: Orbital hemangiopericytoma is a slow growing tumor with a potentially malignant behavior, and a high local recurrence rate if incompletely excised. Complete, intact removal prevents tumor recurrence and provides a good outcome.
Expression of SOD family in rat lacrimal gland after intermittent light exposure C. L. Zamfir * , F. E. Zugun, E. Cojocaru, R. O. Temneanu, R. Folescu, M. Spataru * University of Medicine Iasi, Dept. of Histology, Romania
Objective: Any disturbance of chronobiological rhytms represents a potential stressor, acting like a strong oxidant trigger. SOD family (CuZn-SOD, Mn-SOD and EC-SOD), is one of the most significant enzymatic complexes involved in antioxidant defense. Monitoring local SOD distribution in lacrimal gland after intermittent light exposure can be able to provide insights into the distinct links of antioxidant adaptative mechanisms.So long as lacrimal gland morphology and functions are dependent to a normal light exposure, mobilisation of its own antioxidant resources, especially SOD, is an usefull starting point in antioxidant ophtalmic therapy. Method: Immunohistochemistry was performed in microscopic analysis of glandular fragments prelevated from 30 male Wistar rats, randomized in control/intermittent light exposed groups for 45 days. There have been used specific antibodies for each enzymatic member of SOD family.
Blood samples were prelevated to control SOD levels. The study respected all imposed ethical criteria. Results: Lacrimal glandular tissue exhibits a strong CuZn-SOD immunoreactivity, a moderate one for Mn-SOD and only a small EC-SOD reactivity, while the level of sanguin SOD is high, as a marker of oxidative aggression. Conclusion: Oxidative stress may be engagged in a deep alteration of lacrimal gland. Mobilisation of SOD, depending on their specific glandular location and tissular receptivity, should be regarded as a promising alternative for antioxidant therapy.
Tuesday, 11 September 2012, 09.30 -10.30 Objective: Liposarcoma is the most commonly diagnosed soft tissue sarcoma in adults and occurs predominantly in the lower limbs and retroperitoneum. Primary mediastinal liposarcomas are rare accounting for less than 1 % of all mediastinal tumors. Method: We report a retrospective study of 4 cases of mediastinal liposarcoma during an 8-year period. Diagnosis was made on mediastinal biopsy in 2 cases and on resected specimen in the 2 other cases. Results: There were 3 men and a women ranging in age from 28 to 81 years, explored for respiratory symptoms. Radiological findings showed an anterior inhomogeneous mediastinal mass in all cases with extension to left hemithorax in one case. A surgical resection was achieved in only 2 cases but was incomplete. Histopathologically, tumors were classified into 2 well differentiated lipoma-like liposarcoma, one myxoid liposarcoma and one mixed liposarcoma (sclerosing liposarcoma with myxoid liposarcoma). All patients received adjuvant radio or chemotherapy. One of them presented lung metastasis during the follow up and another died from respiratory failure. The two other are still alive. Conclusion: Mediastinal liposarcomas include a heterogeneous group of bulky tumours, the progression of which depends on the histological type. Histopathologic examination is always necessary as much for diagnosis as prognosis.
Mediastinal epithelioid hemangioendothelioma with a highgrade clinical course A. Konstantinova * , K. Shelekhova, V. F. Klimashevsky * St. Petersburg State University, Russia
Objective: Epithelioid hemangioendothelioma (EH) is a rare neoplasm usually presenting in soft tissues. About twenty cases of EH have been reported in the mediastinum. Most of these mediastinal EH exhibited an indolent course. Results: A 21-year-old woman presented with focally encapsulated mass, 5.5×4 cm in size, in the anterior mediastinum. The tumour of bone density was adhered to the lung tissue and occluded left subclavian vein. Microscopically, the tumor consisted of foci of so-called blister cells typical for EH, anastomosing cords of small epithelioid cells embedded in myxohyaline matrix with haphazardly distributed metaplastic bone and hemorrhagic cellular stroma. Spindling of the tumor cells was prominent. The tumor cells demonstrated moderate atypia, but mitotic figures were not found. Immunohistochemically, cells of primary tumor were positive for Fli1, vimentin, CD31 and CD34. Focal staining with EMA was observed. Multiple metastases were subsequently found in the liver and lungs. Metastatic deposits had the same morphology as a primary tumor.
We present the case of rare mediastinum epithelioid hemangioendothelioma with peculiar histological features such as prominent spindling of neoplastic cells and abundant metaplastic bone formation in aggregate with aggressive clinical behavior that exhibited a profound metastatic poteintial. Objective: Thymic carcinomas comprise rare malignant epithelial neoplasms, which exhibit a disproportionately large variety of growth patterns. We present three cases of primary thymic carcinomas, including two squamous cell carcinomas and one papillary adenocarcinoma. Method: All three patients were males, aged 51, 61 and 77 years, and presented with mediastinal masses measuring 17 cm, 6.5 cm, 3.7 cm in maximal diameter respectively. Results: Microscopically the first two tumors were poorly differentiated squamous cell carcinomas, immunohistochemically positive for CD5 and CD117, whereas the third tumor was a papillary adenocarcinoma, positive for CD5 and negative for CD117 and TTF-1. Immunohistochemical features of the tumors were supportive of thymic derivation. Conclusion: Primary thymic carcinomas are rare malignant tumors with clear-cut atypia, largely lack of organotypic features and a very diverse differentiation. Squamous cell carcinoma is the most frequent subtype. They are often a diagnosis of exclusion, since metastases to thymus and anterior mediastinum, mainly from the lung, are far more common. Immunoreactivity to CD5, CD70 and CD117 may support the thymic origin of neoplastic squamous cells. Treatment options include surgical excision, radiation and/ or chemotherapy depending on tumor stage and patient's condition. The prognosis is generally poor with squamous cell carcinomas having a slightly more favorable outcome. Wednesday, 12 September 2012, 09.30 -10.30 Objective: Surgical blood loss can lead to ineffective tissue perfusion of vital organs. Physiologic solution chosen for blood volume replacement may be determinant for preserving renal integrity. Our aim is to study kidney histopathological changes in a hemorrhagic model, followed by intravascular volume replacement with Ringer's lactate or Hydroxyethylstarch (HES) 130/0.4 solutions. Method: Thirty one pigs under general anaesthesia with propofol and remifentanil underwent haemorrhage at a volume of 30 ml kg-1, over 20 min. After a waiting period of 20 min, intravascular volume was replaced using HES 130/ 0.4 (G1) and Ringer's lactate (G2). One hour after, pigs were euthanized with IV potassium chloride and sixty two renal samples were taken for histopathological examination, using PAS staining. Renal damage was assessed for glomerular, tubulointerstitial and vascular lesions. Results: Mean arterial pressure reached 40 mmHg after bleeding, and recovered for values above 60 mmHg in both groups after volume replacement. Histopathological lesions observed in G2 were more frequent than those in G1. Conclusion: HES 130/0.4 may reduce the incidence of histopathological lesions secondary to renal hypoperfusion after severe bleeding when compared with Ringer Lactate.
Results suggest that the reestablishment of intravascular volume with HES 130/0.4 may preserve renal integrity secondary to blood loss.
Expression of AEG-1, P53 and its clinicopathological significances in the malignant lesions of renal cell carcinomas H. Erdem * , M. Oktay, U. Yildirim, A. K. Uzunlar, M. A. Kayikci * Duzce University, Dept. of Pathology, Turkey
Objective: Astrocyte elevated gene-1 (AEG-1 also known as Metadherin) is associated with various aspects of tumor malignancy. The aim of this study was to investigate P53 relationship between AEG-1 and prognostic parameters. Method: This study was made 50 paraffin blocks (tumoral samples), which were histopathologically diagnosed at department of pathology from 2005 to 2011. Subtypes of the cases were 24 (48 %) clear cell renal cell carcinomas (RCC) and 26 (52 %), non-clear RCC respectively. By immunohistochemical analysis, we investigated AEG-1, P53 expression in carcinomas of kidney and we determined its relationship with clinicopathological parameters. Results: There were significant relationship between increased AEG staining score and tumor capsule (P=0.01), lymphovascular invasion (p=0.015) and significant relationship between the increased diameter with the increase of p53 (p=0.028). There were significant correlation between increased diameter of tumor and degree of increase the Fuhrman (p=0.02). Conclusion: High AEG-1, P53 expression correlates with prognostic parameters in the RCC. In addition, AEG-1, P53 expression in RCC may be associated with tumor progression.
Relationship of CD95, COX-2 and P53 in renal cell carcinomas with survival and other prognostic parameters: A tissue microarray study H. Erdem * , U. Yildirim, A. K. Uzunlar, M. A. Kayikci * Duzce University, Dept. of Pathology, Turkey
Objective: Renal cell carcinomas (RCC) is the seventh most common human malignancy. RCC is now recognized to be a complex neoplasm consisting of several different tumor subtypes, each with distinct genetic and clinical features. The aim of this study was to investigate the expressions of cyclooxygenase-2 (COX-2), P53 and CD95 in RCC that has different clinicopathologic characteristics. Method: This study was conducted on a total of 49 paraffin embedded kidney samples (tumoral samples), which were histopathologically diagnosed at department of pathology from 2005 to 2011. IHC stains for COX-2, P53 and CD95 were performed on tissue microarray using standard procedures. Results: There were significant correlations between COX-2 and subtype (p=0,044), COX-2 and diameter (p=0,026). Significant relationships were found between p53 and age (p=0,050), P53 and diameter (p=0.050). Besides, there were significant correlations between CD95 and furhman grade (p=0,050). Conclusion: CD95, COX-2, P53 expression correlates with prognostic parameters in the RCC patients. In addition, COX-2, P53, CD95 expression in RCC may be associated with tumor progression. Objective: EBV related malignancies (skin cancers, lymphomas, Kaposi sarcomas) complicates organ transplantation EBV-associated smooth-muscle tumors are rare. Method: Among 1500 kidney grafted patients, three developed EBV-SMT. Case 1. Female 51 y, with EBV-SMT (Generalized) Case 2. Female 46 y, breast phylodes sarcoma, reviewed diagnosis: EBV-SMT. Case 3. Male 23 year, severe abdominal pain, 2 gut tumor-like nodules were excised suspection of TBC. EBV-SMT was microscopic diagnosis. Results: Progressive weight-loss all 3patients, chronic cough in patient 1, chest X ray multiple pulmonary lesions, on CT-scan paravertebral lesions. Transthoracic biopsy: spindle cells proliferation with mixed cellularity eosinophilic cells intermingled fascicles resembling smooth muscle, mononuclear inflamatory cells and capillarie evoqued myofibroblastic tumor. No mitosis, mild nuclear atypia. Negative immunohistochemestry: CD21, CD34, CD99, EMA, cytokeratin, S100, CD117. Alpha-smooth muscle actin diffusely positive, EVB-LMP negative. In situ hybridization : EBV + nuclei. EBV-SMT was established. Conclusion: SMT in immunocompromised are EBV associated. Primary target of EBV are B lymphocytes, may infect smooth-muscle through receptor for EBV CD21 Mechanisms of EBV related-tumor genesis is integration of EBV-DNA within ALK locus in tumor cells. ALKgene rearrangement and expression; associated with inflammatory myofibroblastic tumors, anaplastic large-cell lymphomas etc. Clonally of multifocal EBV-SMT using southern blot showed, multiple tumors constitute independent primary lesions. Method: The degree of glomerulosclerosis was scored into 0-3, mesangial proliferation was scored into 0 (absent) and 1 (present), and the degree of crescents was scored into 0-2. Glomerular injury score (GIS) was obtained by adding the above 3 scores and grouped into 3 categories (group 1, score 0-1; group 2, 2-4; group 3, 5-7). Results: Serum creatinine level was significantly increased (1.3±0.4, 1.8±0.9, 2.2±0.8 mg/dL, p=0.007), estimated glomerular filtration rate (eGFR) was significantly decreased (68.5±17.6, 52.1±22.5, 40 .9±20.4 mL/min, p= 0.001) and proteinuria was significantly increased as GIS increased (1.3±2.7, 1.9±1.6, 4.6±7.1 g/24 h, p=0.030). Interstitial fibrosis of more than 25 % of cortical area increased as GIS increased (2.3, 26.0, and 57.1 %, p<0.001). When a multivarate analysis was done, GIS group 3 was the most important predictive factor of eGFR (p=0.009) and proteinuria (p=0.014). Objective: The aim of the study is to evaluate the histopathological finding and the C4d staining patterns of the patients who were biopsied due to graft dysfunction (GD) after initial well-function within 1 month of deceased donor kidney transplantation (DDKT). Method: Histological analysis and C4d immunostaining were performed 34 on needle core biopsies. Results: Thirty-four patients (mean age: 37±8 years, male: 59 %) were included. Histological analysis revealed acute rejection (AR) (n:7), acute tubular injury (ATI) (n:8), allograft infection (n:1), borderline changes (n:8), normal morphology (n:3), and donor-related changes (n:7). C4d staining was detected in 44 % (15 of 34) biopsies; staining patterns were diffuse (n:5), focal (n:5) and minimal (n:5). Diffuse (n:3) and focal (n:1) C4d positivity accompanied 4 of 7 cases of AR. In remaining 27 patients having non-AR histological picture, diffuse or focal C4d positivity were detected in 2 and 4 cases, respectively. Peritubular capillaritis was detected in 41.1 % (14/34) of biopsies, of which four had C4d diffuse, one had C4d focal positive. Conclusion: Beyond cold ischemia time-induced ATI, immunological causes including antibody mediated process may play an important role in early impairment of graft function after DDKT.
Immunohistochemical analysis of the renal interstitial fibroblasts S. Kostadinova-Kunovska * , R. Jovanovic, M. Bogdanovska, V. Janevska, L. Grchevska, G. Petrushevska * Faculty of Medicine, Inst. of Pathology, Skopje, Macedonia
Objective: Renal fibrogenesis is a process common to all progressive kidney diseases. The main executive cell of this process is the fibroblast, by secreting and remodeling the extracellular matrix. The number of fibroblasts is minor in healthy kidney interstitium, but it increases during the process of fibrosis. Their morphology and immunophenotype vary due to different intrinsic and extrinsic factors, thus making their identification and visualization, as well as determination of their origin, very difficult. Method: We performed morphological and immunohistochemical analyses on kidney biopsies with primary glomerulopathy and interstitial fibrosis, using the following antibodies: Vimentin, α-SMA, S100A4, Cadherin 9 and CD34. Results: Interstitial fibrosis with focal, rather than diffuse distribution, was present in all analyzed cases. The total interstitial fibroblast population was positive for Vimentin, majority of the cells were positive for S100A4, and a smaller proportion of cells were positive for α-SMA, Cadherin 9 and CD34. Furthermore, different cells in the fibroblastic population showed positivity for different markers. Conclusion: The above stated observations contribute to the theory that different subpopulations of fibroblasts, with different origin, take part in the renal fibrogenesis.
Soluble epoxide hydrolase inhibition reduce blood pressure and organ damage independently of Nitric Oxide (NO) in mice with Goldblatt two kidney, one clip model (2K1C) P. Kujal * , L. Kopkan, L. Cervenka, Z. Vernerova * Charles University, Third Faculty of Medicine, Prague, Czech Republic
Objective: Investigate the role of NO in the blood pressure (BP)-lowering effects of soluble epoxide hydrolase (sEH) inhibition in 2K1C model. Method: The endothelial NO synthase gene knockout mice and their wild-type controls were used. Renal concentrations of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) were measured in nonclipped kidney. Renal NO synthase activity was determined by measuring the rate of formation of L-[14 C]citruline. Results: Treatment with the sEH inhibitor caused the same BP decrease that was associated with increase in daily sodium excretion in both types of mice. The ratio of EETs/DHETEs in the nonclipped kidney was increased and did not alter renal NOS activity. sEH inhibition reduced significantly glomerular and tubulointestitial injury. Conclusion: BP-lowering effects of chronic sEH inhibition in 2K1C mice are associated with normalization of the reduced availability of biologically active EETs in the nonclipped kidney and their direct natriuretic actions.
Total inflammation in 6-month surveillance renal transplant biopsies is associated with decreased renal function and de novo class 2 donor specific antibody Z. Laszik * , S. Chandran, F. Vincenti * UCSF, Dept. of Pathology, San Francisco, USA Objective: The goal was to correlate inflammation and acute rejection (AR) with renal function and antibody status in 6-month renal transplant surveillance biopsies. Method: Relative risks of inflammation and AR in 380 6month biopsies was calculated by Multivariable Poisson regression. Results: AR was seen in 7.3 % (n=28), borderline change in 19.7 % (n=75), and C4d positivity in 2.1 % (n=8) of all biopsies. Total cortical inflammation (ti), present in 32.6 % (n=124) cases, was associated with 7 ml/min/1.73 m2 lower eGFR at 6 months (95 % CI= −10.8, −3.2). De novo HLA Class 1 was identified in 17.4 % (n=44) and Class 2 DSA in 26.1 % (n=66), with most having MFI values <1000. Increased risk of AR was seen with higher levels of de novo Class 1 (RR 1.49, 95 % CI=1.08, 2.0) or Class 2 DSA (RR 1.34, 95 % CI=1.04, 1.72). Class 2 (but not class 1) DSA was associated with a higher risk of ti (RR 1.40 for ti-score ≥1, 95 % CI=1.13, 1.72) at 6 months. Conclusion: Both ti and AR in 6-month surveillance kidney transplant biopsies are strongly associated with de novo HLA Class 2 DSA. Total inflammation at 6 months correlates with decreased kidney function. Objective: Tenofovir disoproxil fumarate has been used in the treatment of HIV patients producing occasional renal dysfunction and Fanconi Syndrome. Method: A 38-year-old patient was referred with severe acute renal failure and important metabolic acidosis and glycosuria with normoglycemia in urine analysis, altogether with urinary alkaline pH and proteinuria with a protein/ creatinine ratio of 4. He was HIV and B positive diagnosed 17 years earlier. His medication regimen consisted of lopinavir, ritonavir, tenofovir and lamivudine. The patient had a history of nausea, vomiting and some diarrhea with low fluid intake for a week. Besides slight dehydration, the physical examination was unremarkable. Renal ultrasound showed normal kidneys. With adequate fluid restoration, the renal function improved but other metabolic alterations like nephrogenic diabetes remained. Results: Renal biopsy revealed toxic acute tubular necrosis predominantly involving proximal tubules with prominent eosinophilic inclusions within proximal tubular cell cytoplasm, which represented giant, abnormal mitochondria, consistent with tenofovir toxicity. Some histochemichal studies (COX and SDH) were performed. Conclusion: Although prospective clinical trials have demonstrated a low incidence of renal toxicity with tenofovir, there are several such reports in the literature. Current recommendations suggest close monitoring of renal function after initiation of therapy with tenofovir, specially at the beginning and when used in combination with certain antiretroviral agents. Objective: Nutcracker syndrome (NS) is caused by compression of the left renal vein between the aorta and superior mesenteric artery. The main presenting symptom of this rare entity is haematuria, with various degrees of proteinuria. The diagnosis of NS syndrome is primarily by imaging and can sometimes be challenging.
Method: A 21-year-old male was referred to nephrology with a 3 months history of intermittent gross haematuria. All blood test including blood cell count, biochemistry, immunoglobulin electrophoresis, antinuclear antibody were normal. Urinary analysis revealed numerous red blood cells and a protein/creatinine ratio of 0.3. A previous cystoscopic examination was normal. Renal ultrasound revealed a diffuse increased echogenicity. Results: Renal biopsy showed a normal histology with no immunofluorescence deposits. CT scan revealed dilatation of the distal left renal vein with narrowing between the superior mesenteric artery and the aorta. Conclusion: NS is a rare entity causing haematuria. There are few reported cases with histology, although it usually shows no abnormalities. The proposed mechanism for the haematuria is an abnormal communication between the submucosal venous plexus and the calyceal system presumably induced by renal venous hypertension.
Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility for BKV immunohistochemistry in renal allografts M. Mengel * , S. Chan, J. Climenhaga, H. Regele, B. Colvin, P. Randhawa * University of Alberta, Laboratory of Medicine, Edmonton, Canada
Objective: Detection of BK virus associated large T-antigen is crucial for the diagnosis of polyomavirus nephropathy. Method: In an international multi-centre trial we assessed the inter-observer and inter-laboratory variability for BK immunohistochemistry. A tissue microarray was constructed comprising 23 specimens representative of the whole analytical spectrum from negative over mild to strong SV40 positive cases. 81 participants at 60 centers stained the TMA slides using local protocols. Participants evaluated their slides following a provisional Banff grading schema. Details regarding local staining protocols and evaluation scores were collected online. Stained slides were returned for centralized panel re-evaluation. Weighted kappa statistics were used to determine the variability. Results: The BK inter-observer reproducibility was substantial (mean kappa 0.64), but inter-laboratory reproducibility was below chance (kappa −0.22). Separating components of BK evaluation schema into stain intensity and stain percentage showed no significant improvements in reproducibility. However, collapsing the proposed BK scoring schema into a simple positive/negative call improved BK inter-laboratory variability to 0.77. Conclusion: These results indicate a significant variability between laboratories for detecting the SV40 large-T antigen by immunohistochemistry in paraffin sections. Any proposed grading schema for BK nephropathy, which is dependent on percentage and intensity of nuclear staining, will essentially not be reproducible between laboratories. Objective: Cryopyrin-Associated Periodic Syndrome (CAPS) is a rare hereditary inflammatory disorder with three differents phenotypes: familial cold autoinflamatory syndrome, Muckle-Wells syndrome and neonatal-onset multisysten inflamtory disease CAPS results from a mutation of the NLRP3 gene (1q44) coding for cryopyrin, which forms intracellular protein complexes (inflammasomes). Results: Case report: 41 year old women with chronic renal disease stage 3 (creatinin 2,2 mg/dl and proteinuria 180 mg/ 24 h) who developed urticarial episodies since 6 months old related to cold exposure. After 11 years old patient describe associated to these episodios bilateral arthritis (knes, ankles and elbows), conjunctival inflammation, shivers, and asthenia, more frequently in winter and precipitated by cold exposure, air conditioning, stress and menstruatión. Aditionaly the patient developed in last years bilateral hypoacusis A kidney biopsy showed a amorphous, acellular and acidophilic material Congo red positive deposits at interstition, arteries and arterioles and less frequent at glomeruli. This material displays apple green birefringence by polarizad light microscopy. Diagnosis: SECONDARY RENAL AMYLOIDOSIS Conclusion: The clinico-Pathologial findings of this case are compatible with Muckle-Wells syndrome. Secondary amyloidosis is a severe complication which occurs in 25 % of Muckle-Wells cases. Amyloidosis is cause by increase of C-protein and A amyloid during the episodies before described and deposition of the A amyloid in differents tissues.
Glomerulocystic disease associated with thrombotic microangiopathy in two kidney allografts J. M. Mosquera Reboredo * , E. Vazquez Martul * Complexo Hospitalario Universitario, Dept. of Pathology, La Coruña, Spain
Objective: Glomerulocystic kidney disease (GCKD) is a rare condition usually congenital and reported in infants and young children. Only few cases of adquired GCKD had been reported often following Hemolytic Uremic Syndrome (HUS). Method: Histological study of two kidney graft explants. Results: We present two cases of kidney transplant who developed HUS in allografts. Both cases showed at the histological examination typical vascular and glomerular changes of Thrombotic Microangyopathy (TMA). A cystic transformation with increase of urinary space and retraction of glomerular tuft was frequently observed. Chronic transplant vasculopathy was found in the two cases. Humoral active rejection was also demonstrate in one case with moderate peritubular capillaritis and glomerulitis and diffuse C4d deposition at peritubular capillaries. Conclusion: In a few cases GCKD appear to develop after another kidney disease includes single case reports of GCKD associated with mesangial glomerulonephritis, Wegener's granulomatosis, progressive systemic sclerosis, after HUS (include some case in adult patient). Our two cases represent a initial stage of adquired GCKD. The ethiopathogenetic relationship is not clear but some authors propous that cystic dilatation of the Bowman's capsule associated to TMA/ HUS may be secondary to ischemic mechanism.
Case report: Cytomegalovirus gastritis in renal transplanted man F. Noroozinia * , K. Makhdoomi, A. Esmaeili, A. Saffarifard * Emam Khomeini Hospital, Dept. of Pathology, Urmia, Iran
Objective: CMV is an important pathogen in immunocompromised hosts, including patients with AIDS, neonates and transplant recipients. This infection develops in 70-90 % of transplant patients. Upper GI symptoms in solid organ recipient are common (20 %) and clinical signs are more serious in 10 % of cases.
Method: We report a 30 year old man with end stage renal disease underwent kidney transplantation from a CMV negative donor on June 2011. After 1.5 months he admitted with fever, generalized body pain, oral aphtous ulcers and epigastric pain accompanied by malaise. Endoscopic examination revealed multiple antral erosions with surrounding erythema. Clinicopathological investigations revealed CMV viremia with a Ig-M antibody titer and CMV gastritis confirmed by histopathological examination. Results: The patient was started on intravenous (IV) ganciclovir 5 mg/kg per day every 12 h initially for 3 weeks, afterwards the fever decreased; cell blood counts throwback to normal ranges and general condition of the patients improved.
Conclusion: CMV infection develops in 70-90 % of the transplant patients. The colon and stomach are the most common sites of gastrointestinal infection. Though the rate of GI affliction by CMV is high, localization to the gastric antrum is not common.
Immunohistochemistry study of C-Kit expression in renal cell carcinoma F. Noroozinia * , F. Abbasi, Z. Yekta, F. Meisami, A. Saffarifard * Emam Khomeini Hospital, Dept. of Pathology, Urmia, Iran
Objective: Renal Cell Carcinomas include about 2-3 % of adults neoplasms and 90-95 % of all renal tumors. In many cases, it is possible to distinguish. RCC subtypes on the basis of hematoxylin-eosin staining alone. However, overlapping morphologic features pose some difficulties in making a proper diagnosis. To render an accurate diagnosis, additional methods like immunohistochemichal staining against C-Kit have been recommended. Method: We reviewed 65 cases of RCC diagnosed during 9 years. Formalin fixed, paraffin embedded specimens was available in 65 cases. The expression of C-kit was evaluated using immunohitochemistry. Results: Six cases of 39 clear cell type (15.4 %), 8 of 13 papillary type (61.5 %), and 11 of 11 chromophobe type (100 %) were positive for C-kit that considering Chi-Square test there is significant relevation between RCC's subtypes and C-Kit expression (P: 0.001). From 8 cases with renal vein invasion, 3 showed positive expression of C-Kit (37.5 %) and in 55 cases with no venous invasion, C-kit expression was detected in 22 (38.6), so no significant relation was found between renal vein invasion and C-kit expression. Conclusion: The expression of C-Kit in RCC may have diagnostic significance. Objective: IgG4-RSD shows abundant IgG4-positive plasma cells, diffuse fibrosis, and increased serum IgG4 levels. Although IgG4-RSD typically results in autoimmune pancreatitis (AIP), any organ may be involved. Thus, IgG4-RSD TIN often goes unrecognized in the absence of AIP. This is an IgG4-RSD TIN case with mediastinal lymph node and pulmonary involvement. Method: A 73-year-old male presented with rapidly progressive renal failure, hypergammaglobulinemia, hypocomplementemia, and enlarged mediastinal lymph nodes and bilateral pulmonary nodules on CT. Renal and mediastinal lymph node biopsies were performed and IgG4 IHC was done on both after standard techniques. Results: The renal cortex and medulla showed diffuse interstitial fibrosis with tubular atrophy and abundant plasma cells (25 IgG4-positive cells/HPF), numerous lymphocytes and some eosinophils. IF showed IgG, C3, kappa, and lambda granular deposits in tubular basal membranes and Bowmann capsules. Lymph node follicular and paracortical hyperplasia with abundance of mostly IgG4positive plasma cells was seen. An IgG4-RSD diagnosis was rendered and high serum IgG4 levels were demonstrated. Steroid therapy resulted in lymph node and lung nodule reduction. Conclusion: TIN with abundant plasma cells and diffuse interstitial fibrosis, especially if accompanied by hypergammaglobulinemia, hypocomplementemia, or extrarenal involvement, should suggest IgG4-RSD and prompt serum IgG4 level determination and renal IgG4 IHC. Objective: A 39-year-old woman was admitted for chronic renal failure. Clinical examination was normal. Biological explorations showed creatinin clearance around 55 ml/min, tubular proteinuria with Bence Jones κ protein. Serum immunoelectrophoresis identified abnormal monoclonal immunoglobulin G and κ-light chains (LC). Bone marrow histology was normal. Method: Kidney biopsy revealed diffuse intracytoplasmic vacuoles in the proximal tubules resembling osmotic nephrosis. Distal tubules, glomeruli, vessels and interstitial compartment were normal. Immunofluorescence (including anti-κ and -λ staining) was negative. Electronic microscopy (EM) revealed intracytoplasmic immunoglobulinic crystals containing κ LC inside the vacuoles, leading to the diagnosis of Light Chain Proximal Tubulopathy (LCPT). Results: LCPT is a rare complication of dysglobulinemia. It may be associated with crystals within the cytoplasm of proximal, less frequently distal, tubular cells, consisting more frequently in κ LC. Rarely, diffuse tubular vacuolization is present, often indistinguishable from osmotic nephrosis. In our case, there was no proximal tubular dysfunction and immunofluorescence was negative. The first evocated diagnosis by light microscopy was "osmotic nephrosis". However, we failed to identify any causal factor. Finally, the diagnosis was performed by immunoEM. Conclusion: In conclusion, before a picture of osmotic nephrosis without obvious cause, EM and immunoEM may be helpful for the diagnosis of LCPT, often revealing a dysglobulinemia.
Opportunistic infections in renal transplantation -A case series R. Sampaio * , R. Dias, P. Farrajota, A. Coelho, T. Almeida, A. Duarte, J. R. Vizcaino * Anatomia Patológica, Valpaços, Portugal
Objective: Oporto's Hospital Centre is one of the portuguese hospitals with more renal transplantation activity (performed since 1982). Although increasingly rare, opportunistic infections (OI) in transplanted patients remain a major diagnostic challenge and are associated with high mortality rate. Method: In order to evaluate the incidence of OI in renal transplant patients and to identify the location of infection, the respective techniques of diagnosis used and the survival time after infection, we conducted a retrospective study using the Nephrology Department's database on renal transplants. We consulted the registries from 2004 to 2012. Results: We investigated 2041 cases and found 82 cases of OI caused by Herpes virus (n=7), Cytomegalovirus (n=27), Polyomavirus (n=16), Aspergillosis (n=4), Alternaria (n= 2), Mucormycosis (n=1), Candidiasis (n=12), Tuberculosis (n=9), Cryptococcus (n=2) and Pneumocystis (n=2). The lung and urogenital system were the most affected systems. Kidneys were affected in 3 cases respectively by mucormycosis, tuberculosis and cryptococcus. Of all the cases of OI, 39 were diagnosed by the Department of Anatomic Pathology (21 by biopsy; 16 by cytology; 2 by biopsy and cytology). Conclusion: In many cases the diagnosis could only be performed through histologic/cytologic examination. Prompt diagnosis and treatment are necessary to avoid life threatening complications and may greatly improve prognosis.
Papillary renal cell carcinoma with osseous metaplasia and bone marrow elements: A case report L. L. Santos * , A. Polónia, R. Henrique, C. Lobo * IPO Porto, Dept. of Pathology, Viana do Castelo, Portugal
Objective: Renal cell carcinomas might display foci of calcification and even ossification. This rare event has been reported mostly in the clear cell variant. Herein, we present the case of a 69 year-old man, previously diagnosed with colon cancer, incidentally found to have a calcified mass in the kidney, which was interpreted as non-characteristic for renal cell origin. Results: Radical nephrectomy was performed. A 5.5×5.0× 4.0 cm tumor was found, with a heterogeneous brown cut surface, containing areas of necrotic tissue and extensive calcifications. Histopathologic examination disclosed papillary structures covered by small cells with scant cytoplasm, dispersed among mature bone tissue enclosing marrow elements. Epithelial tumor cells were imunorreactive for cytokeratins, CD10 and PAX2, and negative with HMB-45. Hale`s colloidal iron was negative. Trisomy of 3p, 7p and 17p was detected by FISH. A diagnosis of PRCC type 1, Fuhrman grade 2, with extensive osseous metaplasia was rendered. Conclusion: Ossification of renal cell tumors is rare, occurring mostly in clear cell type and the underlying mechanism is unclear. These tumors show atypical radiological features and might be confused with non-renal cell tumors. To the best of our knowledge, this is the first report of PRCC with osseous metaplasia and bone marrow elements.
The effect of doxycycline on glomerulosclerosis in 5/6 renal ablation S. Sarioglu * , D. Sonmez, A. Celik, F. Saglam, O. Yilmaz, E. Koraltan, Z. Cavdar, G. Oktay * Dokuz Eylul University, Faculty of Medicine, Izmir, Turkey
Objective: The effect of matrix metalloproteinase (MMP) inhibitors in segmental sclerosis is unknown. The aim of this study is to investigate the effect of a MMP inhibitor, doxycycline, on glomerulosclerosis (GS) in renal ablation nephropathy. Method: Fourteen of the 32 female Wistar albinos were 5/6 nephrectomised. Doxycyline was given to half of each group (40 mg/kg/day total 28 days). After sacrification, the GS, MMP-2, MMP-9, TIMP-2 expressions were analyzed histopathologically. Pro and active MMP-2 and -9 were analyzed by gelatin zymography. TIMP-1 and TIMP-2 were measured with ELISA assay. Results: Doxycycline administration to the 5/6 nephrectomy group improved GS, but did not inhibit glomerular MMP-9 or cortical pro-and active-MMP-2 and pro-MMP9 but increased TIMP-1 and TIMP-2 expression in all groups in cortical tissue. MMP-9 expression and GS were increased in all groups receiving doxycycline. Conclusion: We have demonstrated improved GS in renal ablation model by doxycycline administration but also doxycycline has an unexpected adverse effect. The effect of doxycycline on the expression of MMP-2 and -9 cannot explain the improvement in GS, but increased cortical TIPM-1 and -2 may be an important contributing factor for inhibition of MMPs.
Digitally reinforced hematoxylin-eosine polarization in diagnosis of renal amyloidosis S. Sen * , B. Sarsik * Ege University, Faculty of Medicine, Izmir, Turkey
Objective: Systemic amyloidosis is a rare disorder, characterized by extracellular accumulation of Congo-red (CR) positive fibrillar amyloid protein deposits. The kidney is the most commonly affected organ by systemic amyloidosis. CR staining which increases the positive birefringence of the weakly birefringent unstained amyloid. In this study we investigated potential power of digitally reinforced birefringence of routine Hematoxylin-eosine (HE) slides on the renal biopsies.
Method: We reviewed 130 HE stained slides for polarization. Sixty five amyloidosis cases were diagnosed by renal biopsy from 2008 to 2012 at our laboratory. All biopsies were evaluated by light and immunofluorescence microscopy. Slides were reevaluated blindly using a microscope (Olympus BX51) attached polarization filter and connected to a digital camera (Olympus DP21, SAL). Depositions which show green birefringence on HE with digitalized microscopy were considered as positive and results were confirmed using CR.
Results: Of the 65 CR confirmed amyloid positive biopsies, 61 showed green birefringence with HE. Of the 65 CR confirmed amyloid negative biopsies, two were considered as false positive. The sensitivity, specificity, positive and negative predictive values were estimated as 94 %, 97 %, 97 % and 94 %, respectively. Conclusion: We concluded digitally polarized HE sections can be used as a fast and first step diagnostic method for renal amyloidosis.
Sirolimus ameliorates cyclosporin-induced nephrotoxicity in a rat modelfocus on renal lesions, oxidative stress, inflammation, proliferation and angiogenesis J. Sereno * , A. M. Romão, M. Teixeira, B. Parada, C. Mega, H. Vala, E. T. lemos, F. Teixeir, F. Reis * IBILI, Medicine Faculty, Laboratory of Pharmacology and Experimental Therapeutics, Coimbra, Portugal
Objective: Sirolimus (SRL) have been pointed as a feasible option for minimize the use of Cyclosporin A (CsA), especially because of putatively less nephrotoxicity. This study aimed to characterize the histological lesions and the molecular pathways implicated in CsA-induced nephropathy and prevention when converted to SRL. Method: The following 4 groups (n=6) were tested during 9 weeks: Vehicle, CsA, SRL and Conversion (CsA 3 weeks + SRL 6 weeks). BP and HR were monitored. Blood was collected and kidney gene expression of markers of inflammation, proliferation, angiogenesis and oxidative stress were assessed. Histology: H&E, PAS and Gordon & Sweets staining. Statistics: ANOVA and Post´hoc tests (p<0.05).
Results: After 9 week of CsA treatment, there was important kidney lesions, including glomerular, tubulointerstitial and vascular: mesangial expansion, atrophy, bowman capsule enlargement, hyaline cylinders formation, tubular calcification and vascular congestion, as well as arteriolar vacuolization and arteriolosclerosis. Conversion to SRL, HT and tachycardia were reduced, accompanied by amelioration of kidney dysfunction and lesions (glomerulosclerosis and tubulointerstitial fibrosis), together with reduction of oxidative stress, proliferation and angiogenesis. Conclusion: In conclusion, SRL ameliorates CsA-induced nephrotoxicity in a rat model, which might be due to protection against oxidative stress, proliferation and angiogenesis, but these mechanisms deserve better exploitation. Acknowledgements: FCT(SFRH/BD/63962/2009).
Non-lupus "full-house" nephropathy in Serbian population in last six years J. Vjestica * , S. Cirovic, S. Tatic, R. Naumovic, S. Simic-Ogrizovic, J. Markovic-Lipkovski * Inst. for Pathology, Medical Faculty, Belgrade, Serbia Diffuse glomerular and sometimes focal mesangial staining of immune complex depositas (IgA, IgG, IgM, C3, and C1q), also known as "full-house" pattern commonly indicates lupus nephritis. However, some non-lupus nephropathy also can be present with a "full-house" immunofluorescence pattern, mimicking lupus nephritis. The aim of this study was to define the clinicopathological spectrum of originally non-lupus "fullhouse" nephropathy. Biopsies from January 2005 till December 2011 were analyzed in order to identify all renal biopsies cases showing "full-house" pattern. The study included 192 "full-house" renal biopsy diagnosiss. From all analyzed cases 117 (60 %) had lupus nephritis expressing "full-house" pattern and 75 (40 %) cases had non-lupus "full-house" nephropathy. In the absence of clinical and/or serological evidence of systemic lupus erythematosus (SLE), at the time of renal biopsy, in 36 cases diagnose was membranous glomerulonephritis (GN), than 11 mesangioproliferative GN, 9 membranoproliferative GN, 7 IgA nephropathy, 6 rapidly-progressive GN and 6 membranous/membranoproliferative GN cases. Nonlupus "full-house" nephropathy is present in broad spectrum of different types of GN, predominantly in cases of membranous GN. The possibility of "full-house" nephropathy preceding the emergence of overt SLE remained to be clarified. Objective: Although the renoprotective effects of prostacyclin have been demonstrated in many studies, the protection mechanisms of prostacyclin in chronic kidney disease, especially at the terminal stage, are still remained unclear. In the present study, we performed pathological and pathphysiological analyses of prostacyclin renoprotective effects using a stable prostacyclin analogue, beraprost sodium, in the disease kidney of anti-GBM glomerulonephritis (GN) rats. Method: Beraprost was administrated from 2 weeks after induction of GN. The condition of renal microvascular network and localization of apoptotic cells were examined using renal vascular corrosion casts, immunostainings and TUNEL-staining. The intracellular apoptotic signaling pathway was analyzed by Western blot and qPCR. Results: In the kidney of beraprost-treated rat, significantly high density of renal microvascular network was maintained, and apoptosis of vascular endothelial cells was suppressed even at the terminal stage of anti-GBM GN. Pathophysiological analyses revealed that transcriptional and post-translational modifications of Bcl2 and XIAP, which were anti-apoptotic proteins in mitochondria dependent apoptotic pathway, were occurred in the kidney of beraprost-treated rat. Conclusion: These results suggested that prostacyclin protects renal vascular network by inhibiting mitochondria dependent endothelial apoptosis, and it play an important role for preservation of renal function in the chronic kidney disease. Wednesday, 12 September 2012, 09.30 -10.30 Objective: Although villous adenomas commonly occur in the gastrointestinal tract villous adenomas of the urinary tract, including the bladder, are infrequently encountered. Method: We report a case of urinary bladder villous adenoma in a 72-year-old man. The patient was undergone cystoprostatectomy because of diagnosis infiltrative urothelial carcinoma in the transurethral resection of bladder. Results: Histopathological examination of cystoprostatectomy specimen there was no urothelial carcinoma. A 2,5 cm polypoid mass was seen at the dome of the urinary bladder. Histopathology confirmed that this tumor was a villous adenoma with a polypoid growth of the glandular epithelium consisting of small tubular glands, dilated cystic glands or papillary fronds lined by a columnar epithelium. The glandular epithelial cells displayed mild nuclear atypia and nuclear pseudostratification with some mucus cells admixed. The patient was diagnosed with a rare case of villous adenoma of the bladder. Over the past 5 months of follow up, the patient is alive and no metastasis. Conclusion: Patients with isolated villous adenomas in the urinary bladder have an excellent prognosis and surgical resection is curative. However, it is uncertain whether an untreated lesion might eventually develop into an adenocarcinoma. Therefore, close follow up is recommended because of the possibility that this condition might be premalignant.
Caprin 1 overexpression in urothelial carcinomas of bladder B. Akkaya * , Z. Cetin, S. Berker-Karauzum, M. Baykara * Akdeniz University, School of Medicine, Antalya, Turkey Objective: Caprin1 encoded by Cytoplasmic Activation/ Protein-1 gene located in 11p13 chromosome region. It has been reported that Caprin 1 is associated with cell proliferation in various types of cell lineages. Method: We researched whether Caprin 1 might be overexpressed or not in urothelial carcinoma of bladder and its overexpression could be correlated with clinicopathologic parameter (age, sex, invasion). Fifty urothelial carcinoma of bladder (29 infiltrative; 21 non-infiltrative) were stained by immunohistochemically in tissue microarrays. Results: The expression of Caprin 1 was observed in 24infiltrative urotelial carcinoma cases (%82) and 18 non-invasive urothelial carcinoma cases (%85). Age range was 32-85. Eight patients were female, 42patients were male. Conclusion: In the literature Caprin 1 overexpression was reported in different types of tumors including esophageal, stomach, prostate, lung, liver. Caprin 1 overexpression might be correlated with the cellular proliferation potential.
To determine of importance of Caprin -1 overexpression new studies are necessary. Objective: Pseudohyperplastic squamous cell carcinoma of the penis (PSCC) is a low grade tumor with specific clinical and pathological features. This very uncommon tumor occurs in association with lichen sclerosus, the main location is foreskin, and the high degree of differentiation may difficult its discrimination with pseudoepitheliomatous hyperplasia. Method: A 66-year-old male presented a penis lesion involving glans and foreskin, clinically suspicious of malignancy. Size was 1,6×1,4 cm and a conservative resection was done. Pathological study was performed, and HPV detection with a commercially available kit: PCR amplification and reverse hybridization with probes to 35 HPV types. Results: The lesion showed a non-verrucous well differentiated squamous cell tumor. Upper layers lacked any atypia but infiltrative growth was evident in basal layers, with atypical cells and mitosis. Lichen slerosus changes were evident bordering the tumor. HPV was negative for all types studied. No further treatment was employed and after a 18 months follow-up no recurrence has been observed. Conclusion: PSCC should be taken in mind when handling penis tumors. A correct differentiation from benign lesions and a knowledge of its low grade to avoid overtreatment will benefit patients. Lichen sclerosus and not HPV seems to play a precancerous role.
Evaluation of sunitinib malate and meloxicam as single agents or in combination in bladder cancer cell lines R. Arantes * , R. Pinto-Leite, C. Lopes, L. Santos, A. Colaço, P. Oliveira * UTAD, Dept. of Veterinary Sciences, Vila Real, Portugal
Objective: Currently accepted for the treatment of advanced renal cancer, sunitinib malate is a small molecule inhibitor of the VEGFR family, with ability to regulate tumor growth, progression, angiogenesis and metastasis. Several reports have suggested that encouraging effects can be achieved by combining COX-2 inhibitors with anticancer agents. The goal of this work was to evaluate the effects of sunitinib malate and meloxicam isolated and combined on three human bladder cancer cell lines. Method: T24, 5637 and HT1376 cells were treated with several concentrations of sunitinib malate and meloxicam, as single agents or in combined schedule. Their influence on cell proliferation was determined by MTT method after 72 h of treatment. Control samples were processed in the same way as treated samples but in drug-free medium. Absorbance values of each well were read at 492 nm using an ELISA plate reader. Results: A reduction in cell proliferation rate was observed when all cell lines were treated either with sunitinib malate or meloxicam isolated. Simultaneous exposure to both agents enhances the inhibition of cell proliferation. Statistical significances were obtained when treatment groups were compared with control group. Conclusion: These results suggest a potential clinical application of sunitinib malate in combination with meloxicam on bladder cancer.
Bcl-2 expression in prostate carcinomas B. Balinisteanu * , A. Dema, S. Taban, C. Lazureanu, D. Herman, S. Ursoniu, A. Loghin, A. Vaduva * Municipal Hospital, Pathology, Timisoara, Romania
Objective: The disturbance of apoptosis represents an important event in the genesis of tumors with different localization. The study of anti-apoptotic protein Bcl-2 expression from the perspectives of the prognostic and predictive value in prostate cancer has led to inconsistent results, even contradictory. Method: Expression of Bcl-2 was analyzed in 3 groups of prostatic carcinoma: localized, locally advanced and with distant metastases. For histological grading of carcinomas we used Gleason score. Classification of the tumors into prognostic subgroups was made according to NCCN Guidelines. For the immunohistochemical study we used anti-Bcl-2 antibody (clone 174), EnVision system, visualization with diaminobenzidine. The results of immunohistochemical reaction were assessed by evaluating the extent and intensity of immunostaining. Statistical analysis was performed using STATA 9.2. Results: 17 of the 59 analyzed cases of prostate carcinomas showed Bcl-2 over-expression: 5,9 % localized carcinomas, 23,5 % locally advanced carcinomas and 70,6 % carcinomas with distant metastases (p<0.001). Although most of the Bcl-2 positive tumors were poorly and moderately differentiated, the correlation between Bcl-2 over-expression and tumor grade did not show statistical significance (p=0,085). Conclusion: Bcl-2 overexpression in advanced prostate carcinomas suggests involvement of this marker in the progression of tumors in this location.
Urothelial carcinoma of the bladder: A clinicopathologic study of 92 cases G. Benkhedda * , S. Khalifa, Y. Lamouti * CHU Frantz Fanon, Dept. of Pathology, Blida, Algeria
Objective: Urothélial carcinoma (UC) accounts for nearly 90 % of urinary bladder tumors.A variety of histological variant of UC have been recently recognosed. Some variants have prognostic and therapeutic implications. The aim to this study is to assess the pathological features from our series and to compare our results that of the literature. Method: We retrospectively studied 92 patients who were diagnosed histopthologically with urothelial carcinoma using the WHO classification system. Results: The mean age of patients at diagnosis was 60(range, 30-70 years). 86,67 % were male (80 H/12 F). All tumors were classified as urothelial carcinomas: 2,1 % urothelial neoplasm with squamous differentiation,2,1 % with glandular differentiation,2,1 % urothelial tumors nested and 1 % sarcomatoid. In this study most tumors were grade 2 (67 cases) and stage pT1. Conclusion: Adult urothelium has the capacity to undergo several pathways of phenotypic cellular and structural differentiation as a result of the embryological origin of the bladder from the multipotent tissues of the cloacal endoderm and the mesodermal wolffian ducts. The clinical course of bladder cancer varies depending on the histological type of neoplasm, grade and stage of the tumor. Hight-grade muscle-invasive urothelial cancers and tumors schowing variant microscopic morphology have in general hight mortality and poor prognosis.
Comparison of insignificant cancer detection rates in prostatectomies performed following 6 and 12-core biopsy schemes U. Berber * , A. Haholu, I. Yilmaz, Z. Kucukodaci, D. Demirel * GATA HEH, Dept. of Pathology, Istanbul, Turkey Objective: Widespread use of extended biopsy protocols have increased the prostate cancer detection rates. Besides this improvement, whether detection of clinically insignificant cancer detection rates are increased by extended biopsy protocols is not well documented. In the study, we aimed to compare the rates of insignificant cancers found in prostatectomy specimens performed following 6 and 12-core biopsy protocols. Method: Retrospectively, we investigated the low volume/ low grade (LV/LG) prostate cancers in 160 prostatectomy specimens. Tumors volumes were calculated digitally as multiplying total tumor areas by 3 mm for average block thickness, and corrected for tissue shrinkage by multiplying a factor of 1.25. Results: Of the 160 prostatectomies, 54 were performed following sextant technique, and 106 were performed after 12-core protocol. Review of the H&E stained sections revealed 21 insignificant cancers. Number of LV/LG tumors found in 6 and 12-core groups were 3(5.6 %) and 18 (16.9 %), respectively. Conclusion: When compared to sextant technique, detection of LV/LG tumors were significantly raised in prostatectomies performed following 12-core protocol, and this increase points out the need for new approaches in patient management to avoid overtreatment after extended biopsy protocols.
PS-22-008 Impact of total core length for cancer detection in a lateral zone targetted 12-core prostate biopsy scheme U. Berber * , A. Haholu, Z. Kucukodaci, I. Yilmaz, D. Demirel * GATA HEH, Dept. of Pathology, Istanbul, Turkey Conclusion: Total core length is significantly associated with cancer detection rates and may be used as a reliable adjunctive tool in deciding repeat biopsies for patients with negative biopsy result. Objective: Primitive neuroectodermal tumors (PNETs) are highly malignant tumors of neuroectodermal origin. We report a case of renal PNET in a 52-year-old male with a 6-month history of intermittent hematuria.. He underwent a right radical nephrectomy. Macroscopically, the inferior pole was replaced by a multinodular, grey, glistening tumour measuring 8.7/7/6 cm, with foci of necrosis and hemorrhage. Method: Serial histological sections have been assessed using hematoxylin-eosin and Van Gieson stain and the indirect immunohistochemical analysis for antibodies: MNF 116, VIM, CD56, NSE, MIC2/CD99. Results: Histological examination revealed a uniform population of undifferentiated, small-to medium-sized tumor cells, arranged in alveolar-insular patterns, with round to oval nuclei, small nucleoli, numerous signs of mitosis and scattered apoptosis, geographic zones of necrosis. Dispersed cells showed cytoplasmic vimentin positivity favouring the diagnosis of PNET. Few tumoral cells appeared positive for MNF116. Expression of CD56 was positive in a large number of tumoral cells and an area of the tumour exhibit a milder reaction of positive NSE. MIC2 was positive with moderate staining in almost all tumoral cells. Conclusion: Diagnosis is based on histology and immunohistochemistry but pathological evaluation can be challenging because of the differential diagnosis with other small round cell tumors.
Nonamyloid fibrillary glomerulonephritis: Presentation of two cases E. Beretouli * , G. Dimas, G. Karayannopoulou, T. Koletsa, D. Grekas, G. Karkavelas * AHEPA Hospital, Dept. of Pathology, Thessaloniki, Greece
Objective: Fibrillary glomerulonephritis (FGN) is a rare disease, characterized by fibrillar deposits in the mesangium and the glomerular capillary loops. These deposits do not have an amyloidlike cross-β structure and are readily distinguishable from amyloid by the larger thickness of fibrils and lack of Congo red staining.
Method: We report two cases of a 52-and 64-year-old women, who presented with severe nephrotic syndrome, rapidly progressive chronic kidney disease and lymphoproliferative disorders. Glomerular crescents were present in about 30 % of both renal biopsy specimens (18 of 54 and 9 of 27 glomeruli, respectively). Immunohistochemical analysis, immunofluorescence and an electron microscopy (EM) studies were performed. Results: Renal biopsy showed a deposition of an amyloidlike extraneous substance in the mesangium, as well as within the glomerular basement membranes. Congo red staining was negative. The EM examination revealed FGN. Conclusion: FGN must be included in the differential diagnosis of rapidly progressive chronic renal disease. EM confirm the diagnosis of FGN, which suggests a poor outcome. Objective: Urachal carcinomas represent less than 1 % of bladder-related cancers. Most are adenocarcinomas, but urothelial, squamous and small cell carcinomas may occur. There are pathological criteria for assessing an urachal origin. A specific staging system is lacking for these tumors. Method: A mass of the bladder dome was found in two 56 and 58 year-old men. A 76 year-old woman presented an urachal cyst. CT-scan revealed no other tumors and partial cystecomy was performed. Pathological examination showed partially cystic adenocarcinomas, one enteric type G2, and two mucinous type G1. Two tumors extended into the bladder mucosa and one was limited to the urachal cyst. Surgical margins were negative. Immunostainings were positive for CK7, CK20, CDX2, cytoplasmic beta-catenin, and negative for p63. Results: With a follow-up of 1, 6 months and 6 years, all patients are free of disease. Conclusion: No TNM classification exists for urachal carcinomas. Specific staging systems have been proposed by Sheldon, and more recently by the Mayo Clinic. Two cases are Sheldon IIIA/Mayo Clinic II, and one Sheldon II/Mayo Clinic I. Since stage, grade and surgical margins are the main prognostic factors, a clear and relevant staging system is needed for these rare carcinomas. Objective: Renal cell carcinoma (RCC) with two different histologies must be included in the unclassified group (WHO classification). Whether these cases should be included in this histological subgroup or be considered hybrid RCC is a matter of debate. We report one of such cases in which papillary and chromophobe phenotypes meet. Method: A 5 cm in diameter asymptomatic left renal mass was discovered incidentally in the radiological follow-up of a breast carcinoma diagnosed 12 years before in a 62 yearold woman. Follow-up showed right adrenal gland metastasis. Patient died 12 months later. Results: Grossly, tumor well circumscribed, tan-yellow, with haemorrhagic and necrotic areas. The neoplasm showed two different histologies clearly defined: One showed solid nests of polyhedral cells, with eosinofilic cytoplasm, central and hyperchromatic nuclei, occasional mitosis recapitulating chromophobe RCC. The other component presented a well defined tubulopapillary growth pattern typical of papillary RCC. Focally sarcomatoid transformation, with tumor necrosis and chronic inflammation. By immunohistochemistry, chromophobe and papillary areas retained their specific phenotypes. Conclusion: Hybrid renal carcinomas do exist, but they are most probably hidden in the unclassified group of renal tumors. However, the exact histological context for which a renal neoplasm deserves the name "hybrid" remains to be defined. Objective: Renal cell carcinoma with thyroid-like follicular pattern is a rare histological subtype of renal carcinomas that has been very recently described. Method: A 3.3 cm in diameter asymptomatic left renal tumor was discovered during the study of a macroscopic hematuria in a 32 years old man. The lesion was organconfined. Results: Grossly, the tumor was a well circumscribed, solid, brown and homogenous intraparenchymatous nodule. Proliferating cells were arranged in a microfollicular pattern with colloid-like material resembling thyroid adenoma. Cells displayed low grade nuclear features and had eosinophilic cytoplasm. Some areas showed a solid pattern of growth resembling an oncocytoid neoplasm. By inmunohistochemistry, the tumor was negative for thyroglobulin and TTF1, and positive for EMA, CK7, AE1/AE3, and ecadherin.
Conclusion: Renal cell carcinoma with microfollicular thyroid-like features has been very recently identified in the literature. There is no agreement on the exact nature of this neoplasm so far, and the 2004 WHO classification of renal tumors still does not consider this phenotype as a distinct histological subtype. Anyway, the tumor must be distinguished from metastatic thryroid carcinoma, another quite unusual condition.
Evidence for clonal fibroblast proliferation and autoimmune process in idiopathic retroperitoneal fibrosis L. Cheng * , J. Clevenger, A. Lopez-Beltran * Indiana University, Dept. of Pathology, Indianapolis, USA Objective: We sought to determine if idiopathic retroperitoneal fibrosis is clonal process and if it is an autoimmune, or IgG4-driven, process. Method: Thirty cases of idiopathic retroperitoneal fibrosis, in whom known causes of retroperitoneal fibrosis were excluded and those for which paraffin blocks were available, were included in this study. We performed clonality analysis in 16 female patients. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection.
Results: Eight of 15 information cases (53 %) showed nonrandom X-chromosome inactivation, or a clonal process. Of the 26 patients for which IgG4 analysis was performed, 14 (54 %) were positive for IgG4-positive plasma cells and all were negative for ALK. Of the 12 patients, for which both clonality analysis and IgG4 analysis were performed, 4 were clonal and IgG4 negative (33 %), 2 were clonal and IgG4 positive (17 %), 2 were nonclonal and IgG4 positive (17 %), and 4 were nonclonal and IgG4 negative (33 %). Conclusion: Our data indicate that a significant proportion (53 %) of idiopathic retroperitoneal fibrosis cases in females is associated with a clonal expansion of fibroblasts. In addition, a subset of idiopathic retroperitoneal fibrosis cases could be classified in the IgG4-related sclerosing disease spectrum. Although a conclusive association with malignancy, urologic disorder, or systemic disease has not been established, often the lesion carries a challenging clinical differential diagnosis that includes malignancy.
We examined clinical and histopathologic characteristics in 41 patients. Medical records were assessed for presentation, clinical diagnosis, associated urothelial carcinoma, radiation treatment, tobacco use, immunologic/urologic disorder, and treatment strategy/outcome. Results: The mean age was 68 (range 28-87 years). Presenting symptoms were: pain (37 %), hematuria (27 %), and dysuria (20 %), in contrast to asymptomatic (32 %). Clinical diagnosis favored malignancy in 10 % of cases. Concurrent or subsequent urothelial carcinoma was present for five patients (12 %), though none developed urethral carcinoma. Histologic features included mixed hyperplastic urothelial and squamous lining, overlying a variably fibrotic, edematous, inflamed, and vascular stroma. Invaginations of urothelium extending into the stroma were common (68 %), showing rounded nests with cystic or glandular luminal spaces, similar to urethritis cystica/glandularis, without intestinal metaplasia. Two lesions included an organizing thrombus, one with intravascular papillary endothelial hyperplasia. Twenty patients were treated with topical medications without resolution. Three lesions recurred (7 %) after excision. Conclusion: Urethral caruncle is an uncommon lesion that may clinically mimic benign and malignant conditions, making tissue diagnosis critically important.
Human Papillomavirus (HPV) is not involved in urothelial tumorigenesis L. Cheng * , R. Alexander, A. Lopez-Beltran * Indiana University, Dept. of Pathology, Indianapolis, USA Objective: The purposes of this study were to investigate the possible role of human papillomavirus in the development of squamous cell carcinoma of the urinary bladder and to determine if p16 expression could serve as a surrogate marker for human papillomavirus in this malignancy. Method: Forty-two cases of squamous cell carcinoma of the urinary bladder and 27 cases of urothelial carcinoma with squamous differentiation were investigated. HPV infection was analyzed by both in situ hybridization at the DNA level and immunohistochemistry at the protein level. p16 protein expression was analyzed by immunohistochemistry. Results: Human papillomavirus DNA and protein were not detected in 42 cases of squamous cell carcinoma (0 %, 0/42) or 27 cases of urothelial carcinoma with squamous differentiation (0 %, 0/15). p16 expression was detected in ten cases (31 %, 13/42) of squamous cell carcinoma and nine cases (33 %, 9/27) Method: A review of clinical data from an ADPKD patient with TCC admitted in our hospital and a review of current literature regarding ADPKD and TCC were made. Results: The patient is a 77 year-old woman with chronic renal failure due to ADPKD, on hemodialysis for 2 years. She was admitted with left lumbar pain, hematuria and fever. Radiological exams revealed various complex cysts with dense material that did not enhance with contrast, compatible with hematic cysts in the context of ADPKD. The whole clinical findings suggested cystic infection complicated with sepsis, so nefrectomy was performed. Macroscopic examination of the ressected kidney revealed a white granulous nodular formation with 4.5 cm diameter in the renal pelvis. Histological examination confirmed a highgrade papillary TCC with parenchymal infiltration (pT3) and extense scamous differentiation. Conclusion: The present case illustrates that TCC can occur in ADPKD, despite its rarity. It can be difficult to successfully diagnose TCC on ADPKD based on clinical-radiological findings. Objective: Carcinoid tumor is a very uncommon neoplasm in the kidney. We report the histopathologic and immunohistochemical (IHQ) study of two new cases. Method: Case 1: 48 year-old female with a 4 cm asymptom-atic¨cystic¨mass discovered in a routine exam. The patient underwent tumorectomy. Case 2: 77 year-old female with a 3 cm renal mass who underwent left nephrectomy. Two months later a needle biopsy confirmed carcinoid tumor metastasis in the liver. Both patients currently free of disease. Results: Both tumors showed similar histologic features. Neoplastic cells were grouped in nests, ribbons, and pseudoglands with rosette-like appearance and showed eosinophilic granular cytoplasm and chromatic nuclei. Low mitotic index. IHC: diffuse/intense cytoplasmic staining for CD56, CD57, AE1/AE3, chromogranin and synaptophysin. Conclusion: Primary renal carcinoid tumor is rare in the clinical practice. The histological findings correspond to a well differentiated neoplasm and are similar to carcinoid tumors in other locations, which makes the diagnosis feasible even without previous personal experience. IHC confirms the diagnosis. It usually behaves as a low grade neoplasm, but recurrences and metastases do occur. Only single cases and short series have been published so far. There is no accumulated experience to establish long term prognosis.
Prostatic adenocarcinoma occurring simultaneously with large cell neuroendocrine carcinoma of the urinary bladder: An extraordinary collision tumor P. Czapiewski * , M. Sieczkowski, M. Matuszewski, K. Krajka, W. Biernat * Medical University of Gdansk, Dept. of Pathology, Poland
Objective: Radical cystoprostatectomy is a standard surgical procedure for male patients with muscle-invasive urinary bladder (UB) carcinoma. Vast majority of these tumors are urothelial carcinomas, while large cell neuroendocrine carcinoma (LCNEC) is a very uncommon tumor with less than 20 reported cases. Invasive prostate carcinoma is incidentally detected in up to 20 % of cystoprostatectomy specimens. It is usually well differentiated and shows low propensity for dissemination and local recurrence. Method: Clinical and pathological description of an extremely rare collision tumor composed of LCNEC of the urinary bladder and a high grade acinar prostate carcinoma. Results: A 79-year-old male patient was admitted to the urology department due to severe gross haematuria. Cystoscopy revealed large tumor of the posteriorinferior wall of the UB with involvement of the left uretheral orifice. In the radical cystoprostatectomy specimen LCNEC of the UB, involving bilaterally prostate and spermatic vesicle (pT4a), was diagnosed. Additionally both lobes of the prostate were involved by the acinar prostatic carcinoma (pT2c, Gleason score 5+4=9). Conclusion: High-grade prostate carcinoma may rarely coexist with UB tumors of uncommon histology. Objective: Despite of numerous researches in the field, data regarding immunohistochemical (IHC) expression of androgen receptor (AR) in prostate carcinomas, in terms of prognosis and therapy, are quite controversial. Method: The IHC expression of AR was analyzed on 3 groups, each of 20 primary diagnosed prostate carcinomas: localized, locally advanced and distant metastasized. The anti-AR antibody (clone AR441) was used. For each tumor was determined the percentage of AR-positive nuclei, assigning a staining score (0 to 4). A value of 64 % was considered to be discriminat o r y b e t w e e n t u m o r s w i t h h i g h a n d l o w A R expression. Results: All the analyzed carcinomas showed AR. 54.2 % of tumors had high AR expression and 45.8 % showed low AR expression. 7/19 localized tumors (one tumor vanished during processing), 15/20 locally advanced and 10/20 distant metastasized tumors showed high AR expression (p=0.051). 5/18 well-differentiated, 16 of 23 moderately differentiated and 11/18 poorly differentiated tumors showed high expression of AR (p=0.022). Conclusion: Although not reaching statistical significance, the AR expression tended to correlate with the stage of disease and with the degree of differentiation in terms of an increased expression of AR in advanced, moderately and poorly differentiated tumors. Objective: Prostate lesions with atypical stromal component are rare and poorly characterized in terms of evolution and prognosis. Method: We retrospectively analyzed 3 cases of unusual prostate tumors diagnosed on prostate needle biopsy. The biopsies were performed due to an elevated serum PSA and/ or phenomena of bladder outlet obstruction. For the immunohistochemical (IHC) diagnosis there were used the following antibodies: CK AE1/AE3, PSA, PSMA, vimentin, SMA, ER, Desmin, PgR, CD34. Results: Two of the three patients, one 68 and the other 66 years-old, with serum PSA level of 34 ng/ml and 14.58 ng/ml respectively, were diagnosed with sarcomatoid carcinoma, the former presenting with liver metastases at diagnosis. In evolution, the death of the first patient occurred 3 months after the diagnosis and the second patient refused surgery. The third patient, 75 years-old, with a nodular prostatic mass of 3.3 cm, was clinically suspected to have an unusual malignant prostatic tumor considering that, despite his 7 ng/ml of serum PSA, he presented multiple bone metastases. The histopathological diagnosis was Stromal Tumor of Uncertain Malignant Potential. Conclusion: The prostate lesions with atypical mesenchymal component are rarities in urologic pathology. An accurate diagnosis relies on meticulous pathological examination techniques and supplemented, for rare cases, with other clinical and laboratory data.
Immunohistochemistery study in a case of nephrogenic bladder adenoma M. R. Farzaneh * , A. Safaei, A. Amin Shareefi * Shiraz, Iran
Objective: Nephrogenic adenoma is a rare benign lesion of bladder that may be confused with malignant lesions. There is strong relation with urinary tract irritation and intravesicl instrumentations. Nephrogenic adenoma was initially thought to originate from urothelial metaplasia however no solid proof is available.
Method: We present this 55 years old lady with urinary problem. Cystocopic examination show a sessile mass and biopsy show circumscribed proliferation of tubules, cysts, and papillae lined by cells with low cuboidal to columnar epithelial cell. Nephrogenic adenoma can be a significant diagnostic pitfall as certain histological features, such as the presence of enlarged nuclei with prominent nucleoli Results: Immunohistochemistery study showed strong reactivity to CK7, P504S, CD10, and EMA but negative for CK20, PSA, and P63. Conclusion: We recommend that any lesion in cystoscopic examination should be followed and immunohistochmistery examination some times is mandatory to differentiate them from malignant tumors.
Regulators of apoptosis and the cell cycle are overexpressed in bladder cancer metastases and may predict survival A. Fleischmann * , R. Seiler, A. Perren, T. George * Universität Bern, Institut für Pathologie, Switzerland
Objective: Expression of biomarkers and their prognostic relevance may differ between primary tumours (PT) and its metastases (MET). There is little information about this phenomenon concerning apoptosis and cellcycle associated biomarkers in urothelial bladder cancer (UCB). Method: Nodal positive patients (n=152) with UCB underwent cystectomy and lymphadenectomy. Immunohistochemical expression of bcl-2, bcl-6, MDM-2, p53 and CyclinD1 was quantified in tissue microarrays constructed from PT and corresponding nodal MET. Results: Frequency of MDM-2 positivity increased from PT (17 %) to MET (37 %, p < 0.001). This trend was not significant for bcl-2 (PT: 7 %; MET: 10 %, p=0.4) and bcl-6 (PT: 8 %; MET: 12 %, p=0.08). Median percentage of p53 and CyclinD1 immunostained cells increased significantly (p<0.05) from PT (15 %/30 %) to MET (40 %/50 %). MDM-2 and CyclinD1 expression were positively correlated in PT (p<0.004) and MET (p<0.002). p53 and MDM-2 positivity were inversely correlated in PT (p<0.03). Only high CyclinD1 expression in the metastases predicted early death significantly and independently (p=0.017). Conclusion: Biomarkers of apoptosis and cell-cycle associated are up-regulated in the metastases indicating differences in survival and proliferation of cancer cell compared to primary tumours. Both tumor components may harbor different prognostic information and are not necessarily surrogates for each other.
Evaluation of histopathologic and histomorphometric changes of testicular tissue and gonadotropins levels following consumption of Methylphenidate in male mice Z. Ghahri Saremi * , S. Fazelipour, Z. Tootian, M. Shafii * Tehran, Iran
Objective: One of the most common psychiatric disorders in children is ADHD (Attention Deficit Hyperactivity Disorder), which is treated extensively by Methylphenidate. This study investigates the assessment of the effects of methylfenidate on Histopathologic and Histomorphometric changes of testes and serum level of gonadotropin in adulthood which produces gametes and has Importance for future generations. Method: In this study 36 adult male mice (Balb/C) were used. After determining the body weight, the animals were divided randomly into two experimental groups of and one control group. The Experimental groups received Ritalin via gavage as follow: The group 1 received 2 mg/kg/day and the group 2 received 10 mg/kg/ day for a period of 40 days. After evaluation of body weight, general anesthesia was used for taking blood samples from the heart in order to measure gonadothropins levels in serum. Then for the purpose of body weighing and measuring of diameter of germinal epithelium the testes were removed and the possibility of any pathologic changes was considered. Results: The results showed that Methylphenidate with different doses could decrease germinal epithelium and also body weight significantly. Besides some significant changes in serum gonadotropins, without any pathological changes were observed. Conclusion: Our findings demonstrated that Methylphenidate administration in adulthood due to Influence of enhanced computed tomography and magnetic resonance imaging was characterized as 8×8×7 cm sized welldemarcated left renal middle-superior polar mass with central necrosis and cystic changes. The contralateral kidney was normal. He underwent left radical nephrectomy by thoracoabdominal approach. Gross examination of the specimen revealed yellowish white 9×9×7 cm sized mass, with multifocal necrozis and cystic changes. Histopathology of these resection specimens; scattered spindle cells and foam cells, fine vascular network and necrosis. Immunohistochemistry revealed CD68-positive xanthoma cells. The tumor also stained positive for vimentin, CD34, but negative for cytokeratin, EMA, desmin, SMA and myoD1. A diagnosis of MFH was made. Conclusion: MFH is a primitive mesenchymal tumor with some histiosytic and fibroblastic differentiation Primary renal MFH is an extremely rare lesion. Because this malign mesenchymal tumor is indistinguishable clinically and radiologically from renal cell carcinoma diagnosis and histopathology of this rare lesion are discussed.
Hyaline ring granulomas in the urinary bladder: A case report N. Hammer * , N. Gatt, J. De Gaetano * Swatar, Malta
Objective: Hyaline ring granulomas, or pulse granulomas, are rare inflammatory responses to vegetable matter, characterised by aggregates of hyaline rings and other inflammatory components around vegetable matter. The vast majority occur in the oral cavity and only a few specific extra-oral cases have been reported. We present a case of pulse granulomas in the wall of the urinary bladder, occurring in a 50 year-old gentleman with a concurrent history of diverticulitis and colo-vesical fistula. Pulse granulomas were recently described in the bladder in association with interstitial cystitis, in mesocolonic fat and mesocolonic lymph nodes in association with inflammatory bowel disease and in enterocutaneous and recto-salpingeal fistulas in association with diverticulitis. To our knowledge this is the first case reporting the direct association of the occurrence of bladder pulse granulomas in relation to diverticulitis. Method: The tissue sections were fixed with 10 % buffered formalin and stained with H&E. Results: Histopathological examination revealed several hyaline ring granulomas within the outer muscle layer and serosa of the urinary bladder, together with hyaline ring granulomata within the outer wall of the sigmoid colon associated with diverticula. Conclusion: It is important to differentiate pulse granulomas from parasites, hyaline ring vasculopathy, granulomatous inflammatory disorders and even malignancy. Objective: Micropapillary carcinoma (MPC) is associated with poor prognosis, and may lead to immediate cystectomy irrespective of stage. We study MPC, with "classical" histologic features, to "stage-matched urothelial carcinoma (UCa), not otherwise specified (NOS)", and other UCa with divergent differentiation to compare outcome.
Method: 89 cases from 80 patients with MPC, UCa with squamous (SQ) or glandular (GL) differentiation, small cell carcinoma (SmCa), and nested variant (NeCa) variant of UCa were identified. Histologic and clinical data including stage and outcome were collected. Results: All groups show male predominance and similar age range. MPC showed the highest rate of nodal metastases (Table 1) . Patients with MPC tumor volume of >50 % showed the highest rate of nodal metastases of all other subgroups. The majority of patients with MPC died, a large percentage from unknown causes. Compared to MPC, SQ showed similar presentation at higher stages and similar survival, but a lower propensity for nodal metastases. In our cohort, GL presented at lower stages, showed slightly better survival, and a lower rate of nodal metastases, when compared to MPC. Our cohort included fewer cases of SmCa and NeCa. However, both patients with NeCa died of disease. Conclusion: MPC, particularly when it represents >50 % of tumor volume, shows higher rate of nodal metastases than remaining subgroups, and is associated with a larger percentage of patient deaths. Objective: Müllerian-like stroma with ER/PR expression is typical of renal mixed epithelial and stromal tumor and has been described in kidneys with obstructive pathology. WT1 overexpression is not reported in this setting. Method: Immunohistochemical study for ER, PR and WT1 in autosomal dominant polycystic kidney disease (ADPKD) (n=5), acquired cystic kidney disease (ACKD) (n=5), xanthogranulomatous pyelonephritis (XP) (n=5), and renal lithiasis (RL) (n=5). Controls: fetal (<24 w, n=4; >24 w, n= 4) and adult autopsy kidneys (n=5). Results: Stromal ER was found in 100 % of ADPKD, ACKD, XP and RL; PR expression in 100 % of ADPKD, ACKD and RL and 80 % of XP, and nuclear WT1 in 20 % of XP and RL. ER were negative in 100 % of controls. We found PR expression in 100 % of fetal controls of second trimester. WT1 was negative in the stroma of adult controls (100 %). 80 % of fetal controls showed WT1 expression in the peripheral cortex. Conclusion: ER and PR expression is frequent in kidneys with chronic obstructive and inflammatory pathology. WT1 is expressed occasionally. More studies are needed to determine whether these findings are a consequence of renal obstruction or they are involved in its pathogenesis, as well as their potential therapeutic implications.
Telomerase expression in urothelial carcinomas of the urinary bladder: Does it make sense for carcinogenesis or prognosis? D. Kankaya * , S. Kiremitci, K. Zengin, C. Tuygun, A. Sertcelik * Ankara University Medical School, Dept. of Pathology, Turkey
Objective: Human telomerase reverse transcriptase (hTERT) has been reported as poor prognostic marker in several cancers. In the present study, we examined hTERT expression in urothelial carcinoma (UC) to investigate whether it has a role on the carcinogenesis or prognosis. Method: Immunohistochemistry was performed to detect hTERT protein expression in tisse microarray blocks consisting of cores of papillary UC (n = 63) and infiltrating UC (n = 13) cases. Nucleolar staining was considered and staining scores (intensity × distribution) were determined. Tumors were grouped as low or high in terms of histological grade, and as early (Ta, T1), or late stage (T2-T4) in terms of pathological stage. Results: hTERT expression was significantly higher in the papillary UC group (p=0.028). Tumors in late stage were more likely to show low staining scores of hTERT (p= 0.013). No correlation was found with tumor grade and recurrence rate. No effect on survival.was detected. Conclusion: These findings indicated an association of hTERT protein with early stage UCs as its expression significantly decreased with muscularis propria invasion.
Incidental multifocal renal cell carcinoma in graft nephrectomy with cellular rejection findings: Concurrence of papillary renal cell carcinoma and renal carcinoma associated with Xp11.2 translocation D. Kankaya * , S. Kiremitci, A. Ensari, A. Sertcelik * Ankara University Medical School, Dept. of Pathology, Turkey
Objective: Malignancy represents the second main cause of death in renal transplant patients and increase markedly by 20 years after transplantation. Method: A 41 year old male who has been on hemodialysis for 8 months for chronic renal failure, underwent living kidney transplantation from his brother. He received immunosupressive therapy of siclosporin and prednisolon. Twelve years later, due to the impairment of renal functions hemodialysis was started again and ultrasonography revealed chronic renal parenchymal disease. Graft nephrectomy was performed with the pre-diagnosis of chronic rejection. Results: Macroscopic examination revealed two well-circumscribed tumor nodules with 12 mm and 6 mm in largest diameter. Microscopically, both tumor nodules showed tubulopapillary organization. Larger tumor consisted of clear cells with TFE3 positivity, whereas the small one showed eosinophilic cells with diffuse cytokeratin7 and AMACR positivity, without any TFE3 expression. Concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with Xp11.2 translocation was reported. Non-tumoral renal paranchyme revealed interstitial inflammation, tubulitis and transmural arteritis with fibrinoid necrosis compatible with type III cellular rejection. Conclusion: As far as we know this is the first case in the English literature demonstrating concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with Xp11.2 translocation in a graft nephrectomy.
The effect of postoperative intravesical BCG and Mitomycin C therapy on recurrence in superficial bladder cancer D. Kankaya * , K. Zengin, A. Sertcelik, C. Tuygun, N. Sertcelik * Ankara University Medical School, Dept. of Pathology, Turkey
Objective: To evaluate the efficacy of two mostly used intravesical agent, BCG and mitomycin c, in case of tumor recurrence. Method: Between 2002 and 2008, we performed TUR-BT to 127 patients whom pathology results were superficial bladder cancer. 41 patients were treated with intravesical BCG once for week 6 weeks then monthly up to one year beginning from 14. day postoperatively. 26 patients were treated with intravesical mitomycin c, beginning from first 6 h postoperatively and once a week for 8 weeks. 60 patients didn't get any further treatment, and excluded from study. Results: For BCG group, expected disease free interval was 58.1 months, and 34.6 months for mitomycin c group. When two groups compared for expected disease free interval, statistically significant difference observed (p=0.017). For BCG and mitomycin c group, recurrence was seen in 13 and 9 patients, respectively. For patients treated with BCG, 1 and 3 years disease free survival rate was %92.7 and %75.7, respectively. In mitomycin c group 1 and 3 years disease free survival rate was %76.9 and %62.9. Conclusion: Intravesical adjuvant BCG maintenance therapy is more effective for tumor reccurence than intravesical mitomycin c therapy.
Papillary cystadenomas of the epididymis: Case presentation P. Katafygiotis * , L. Abou-Asabeh, A. Nomikos, S. Chranioti * Hospital of Voula, Dept. of Pathology, Athens, Greece
Objective: Papillary cystadenomas of the epididymis are rare, first described by Sherrick in 1956. They are considered hamartomatous lesions rather than neoplastic and may be seen as intrascrotal swellings in a wide age range. We report here the case of a 32-year-old male who presented with a swelling of the right testis over the past year. His past medical history was otherwise unremarkable. Method: Ultrasonography revealed the presence of an epididymal cyst. Grossly the cyst had a 2 cm diameter with a cyst wall of 0.2 cm thick attached to a part of epididymis measuring 2×1 cm filled with clear fluid. Results: Histologically the cyst showed pseudopapillary structures lined by monotonous clear cells. There was mild nuclear pleomorphism, no mitotic activity and no necrosis.
Immunostaining for AE1/AE3, EMA, were positive, as well as focally for CK5/6 whereas calretinin and CEA was negative. Similar pattern of staining was seen in the epididymal parenchyma. Conclusion: Papillary cystadenomas are benign neoplasms and there have been no reports of recurrence or metastasis in the recent literature. Bilateral disease is often associated with von Hippel-Lindau syndrome. Our patient received no further treatment and remains asymptomatic and well 10 months after the intervention.
Effect of neoadjuvant sorafenib treatment on histology of clear cell renal cell carcinoma and occurrence of circulating tumor fragments G. Kats-Ugurlu * * UMC St. Radboud, Dept. of Pathologie, Nijmegen, Netherlands
Objective: Clear cell renal cell carcinoma (ccRCC) generally presents with a micronodular phenotype (MP) due to high expression levels of vascular endothelial growth factor (VEGF-A). Earlier we have shown that MP is associated with shedding of multicellular tumor fragments (MTF) into the circulation and pulmonary metastasis. We hypothesized that VEGF inhibition will destroy MP resulting in less MTF. Method: 8 ccRCC patients were treated for 4 weeks by daily administration of Sorafenib (400 mg bid). Three days after therapy, nephrectomy was performed and kidneys were perfused via the arteria renalis. Venous perfundate was filtered and processed to AgarCytoblocks for MTF. Treatment effects were studied using immunohistochemistry. Results: All tumors were ccRCC as demonstrated by high VEGF-A expression. None of the tumors showed MP after Sorafenib treatment. Tumors showed large areas of necrosis and fibrinoid necrosis of the blood vessels, concomitant with profound perivascular inflammation. 6/8 ccRCC patients (75 %) had MFT vs 33 % in a control group (p= 0.03 Fisher's exact test). Individual tumor cells in the MTFs showed increased mitotic activity. Conclusion: Sorafenib destroys MP in ccRCC, attacks tumor vasculature, causes extensive necrosis and inflammation. Post-treatment MTF are increased in venous perfundate. Care should therefore be taken with neo-adjuvant sorafenib treatment of ccRCC. Objective: Claudins are major transmembrane proteins of tight junctions. As the disruption of their function have important impact on tumorogenesis, invasion and metastasis. Claudins became a focus of interest for targeting therapies. Although their expression profiles have been studied in many organs, researches on Claudin expression in bladder are in limited number. The aim of this study is to present the differential expression of Claudins in invasive urothelial cell carcinoma (IUCC), noninvasive papillary urothelial carcinoma (NPUC), carcinoma in-situ (CIS), papillary urothelial neoplasm of low malignant potentiall (PUNLMP) and control group (CG). Results: 83 cases (31 IUCC -further divided into:15 muscle invasive UCC, 16 UCC with lamina propria invasion-, 17 NPUC, 13 PUNLMP, 7 CIS and 15 CG) were evaluated with Claudin-1, 3, 4. Interestingly, high Claudin-1 and -3 score in CG (100 %, 80 %), decreased significantly in all non-invasive lesions (mean 18 %, 22 %). Claudin-4 expression appeared to decrease in IUCC vs. Others (%52 vs mean %82). Conclusion: Higher expression of Claudin-4 in low-grade and non-invasive lesions may be used as a diagnostic tool. In terms of Claudin-1 and -3, their decreased expression in non-invasive lesions when compared to CG and their trend to show more increased expression in IUCC needs to be studied further in larger studies.
Can we rely on alternative sampling method of radical prostatectomy specimens? T. D. Kokenek-Unal * , A. S. Oguz-Erdogan, M. Alper * YB Research and Training Hospital, Dept. of Pathology, Ankara, Turkey
Objective: Prostatic adenocarcinoma is the most common cancer and second leading cause of cancer death in men. The incidence of prostate cancer has been increasing because of efficiency of modern cancer scanning programs and residual awareness of the patients. For that reason, there is a considerable increase in the number of prostatectomy specimens in the university and research hospitals.It is costly and time-consuming procedure and causes in turn increased workload. The aim of this study is to compare the results of total and alternative sampling methods and to delineate the differences if there are any. Method: Totally embedded 50 radical prostatectomy specimens were randomly selected and evaluated for key pathologic parameters. These cases then were reevaluated with limited sampling. The limited sampling method was built to include similarly embedded apical margins, bladder neck margins and seminal vesicles. In addition to that new slices were selected by skipping every other slice as differently from totally sampling protocol.
Results: The %37 reduction was achieved in number of blocks. The concordance rates between two sampling methods were %40, %50.4, %70.3, and %60 respectively for Gleason scores, perineural invasion, extraprostatic extension and pathologic stages. Conclusion: Although the limited sampling protocol provides statistically significant results, because of critical role of pathological assessment in treatment of prostatic adenocarcinoma, they can be found unsatisfactory for many pathologists. Objective: Initially described in embryonic neuroepithelium, nestin is an intermediate filament involved in cell differentiation and transiently related to vimentin, keratin and glial fibrillary acidic protein (GFAP). In tumoral and vascular proliferation, nestin is recognized in stem/progenitor cells. The octamer-binding transcription factor 4 (OCT-4) is a biomarker of this lineage. Since there are few data concerning the presence of nestin in Leydig cells and testicular tumors, our aim was to investigate a series of Leydig cell hyperplasia and tumors. Method: 31 cases (13 hyperplasia, 14 adenomas, 2 malignant Leydig cell tumors and 2 lymph node metastases) and controls were immunostained with anti-nestin, vimentin, keratin, GFAP and OCT-4 antibodies. Results: Twenty-six (84 %) cases (tumors and hyperplasia) were nestin positive with mostly weak, finely granular cytoplasmic staining. Four (13 %) were negative, 1 (3 %) not interpretable. Vimentin was expressed in twenty-seven (87 %) cases. Seventeen (55 %) were focally keratin positive. Only three (10 %) showed OCT-4 positivity. All were GAFP negative. Conclusion: In Leydig cell hyperplasia, adenomas and malignant tumors nestin and vimentin are expressed frequently, sometimes with keratin. This would be a sign of epithelialmesenchymal transition. Further investigations are needed to understand the relationship with the malignant potential of these tumors.
Morphological changes of testis in atherosclerosis N. Krupnov * , A. Astrakhantsev * Bureau of Forensic Medical Exp., Ryazan, Russia
Objective: Up to now in the field of morphology of reproductive system of men the problems of testis involution and morphogenesis at atherosclerosis haven't been thoroughly investigated.
We have investigated histologically testis of 63 died patients aged 36-89 with general atherosclerosis and testis of 38 men aged 36-89 with minimal manifestations of atherosclerosis died in an accident. Results: The absolute volume rete testis of patients being ill with atherosclerosis decreases by 65-80 %. Absolute volume of convoluted seminiferous tubules and interstitial tissue decrease by 24-27 %. In testis there are zones of focal sclerosis of seminiferous tubules, whose area goes as far as 12.7 % of shear section area. It was revealed that on the periphery of sclerosis zones there is a reduction of transaction area of seminiferous tubules by 20-33 % with Sertolli cells quantity reduction by 17-24 %. It has been stated the downward changes in index of spermatogenesis in convoluted seminiferous tubules by 63-65.6 %, accounted for the quantity reduction of all kinds of cells. Conclusion: Morphological changes of testis at general atherosclerosis characterize atherosclerotic testiculopatie, caused by chronic ischemia of testis.
A Leydig-cell tumor in a cryptorchid testis: Report of a case E. Lambropoulou * , A. Datsis, P. Morfaki, G. Charalambopoulou * General Hospital of Messologhi, Dept. of Pathology, Greece
Objective: Leydig cell tumor is a rare form of testicular neoplasm, representing only 1-3 % of all testicular tumors. According to accepted medical knowledge, this type of tumor isn't linked to cryptorchidism, unlike germ cell tumors of the testicles. Herein, a case of Leydig cell tumor in a cryptorchid testis is described. Method: A 59-year-old, unmarried man was admitted for surgical repair of inguinal hernia. His past medical history included untreated bilateral cryptorchidism first noted in childhood, as well as poliomyelitis that presented at age two. The past surgical history and review of systems were noncontributory. A CT scan of the abdomen and pelvis revealed two 3×3 cm testicles at the level of the inguinal canal. He underwent bilateral inguinal orchiectomy combined with surgical repair of his inguinal hernia. Results: The right testis contained a solid, well-circumscribed, round, tan-colored tumor 1.5 cm in diameter. The histological appearance was that of a Leydig cell tumor exhibiting no mitotic figure. There was no clinical or radiological evidence of metastatic spread. Conclusion: Although cryptorchidism is considered to be a risk factor for developing germ cell tumors, there have been a few reported cases of Leydig cell tumors with a history of cryptorchidism. Our case adds to the evidence that there may be a link between the two conditions. Objective: CD44, E-cadherin, β-catenin are "cell adhesion molecules" and appear to influence development, inflammation, cancer invasion and metastasis. We studied the expression of these CAMs in prostatic adenocarcinoma (PCa), high grade prostatic intraepithelial neoplasia (HGPIN) and nodular adenomatous hyperplasia (NH). Method: 135 specimens of radical prostatectomies were assessed. These CAMs were determined by immunohistochemistry. All sections included PCa, HGPIN, and NH. The expression of these markers was evaluated with three scores. The correlation of immunopositivity with Gleason score and TNM stage was investigated. Results: CD44 was strongly expressed in 41.5 %, 46.7 % and 37.8 % of NH, HGPIN and PCa, respectively. E-cadherin immunostaining was higly detected in 71.1 %, 78.5 % and 63.0 % of NH, HGPIN and PCa areas while β-catenin immunostaining was exclusively membranous in 80.7 % of NH and nuclear/cytoplasmic in 70.4 % and 48.9 % of HGPIN and PCa areas. All markers were unrelated to Gleason score (p=0.352). CD44 and E-cadherin immunopositivities were inversely associated with TNM stage (p=0.021 and p=0.042 respectively); such an association was not observed in β-catenin (p=0.556). Conclusion: CD44 and E-cadherin decreased expression is probably associated with invasive potential of prostate cancer. β-catenin staining pattern in neoplastic lesions differs from that in non-neoplastic prostate lesions. Results: The loss of ECAD expression was significantly higher at the TIF when comparing with TSCP and NNM. The loss of ECAD was correlated with histological grade, infiltrative pattern, lymph node metastasis, perineural and vascular invasion. The vimentin expression showed association with histological grade, infiltrative pattern, T stage, lymph node metastasis, perineural and vascular invasion. Conclusion: The loss of ECAD and the gain of vimentin expressions occur more frequently at the tumor invasion front and are associated with classic factors of poor prognosis and low survival rates.
Prognostic value of immunohistochemical markers in bladder cancer I. M´Sakni * , A. Chaabane, F. Bougrine, B. Laabidi, A. Bouziani * Rades Meliane, Tunisia
Objective: The tumor stage and grade of bladder tumors are the major elements to define the prognosis. However, it is sometimes difficult to identify an infiltration of chorion or detrusor muscle. Moreover, the evaluation of tumor grade is subjective and not reproducible. Method: Our objective is to study the prognostic value of the expression of proliferating cell nuclear antigen (PCNA), Ki67 antigen, the tumor suppressor gene p53, the protooncogene c-erb B2, the receptor for Epidermal Growth Factor (EGF-R), the apoptosis suppressor gene bcl2, carcinoembryonic antigen (CEA) and Epithelial Membrane Antigen (EMA). Results: The study showed that the PCNA expression was significantly associated with the early recurrence (p=0.010) and the tumor stage (p=0.003). The Mib1 expression was correlated to the early recurrence (p=0.010), and tumor progression in stage and/or grade (p=0.007). The c-erbB2 expression showed significant association with the tumor grade (p=0.007). The prognostic value of other markers has not been proven. Conclusion: These findings may be useful providing better classification of bladder tumors thus the better management of patients. The c-erbB2 expression contributes to refine the tumor grading. PCNA and Mib1 can predict the early tumor recurrence; they could be relevant for the determination of endoscopic controls rhythms of patients. Objective: Primary urinary bladder neuroendocrine carcinoma (PUBNEC) is a rare tumor characterised by an aggressive behaviour and poor prognosis. Method: We report five cases of PUBNEC diagnosed in the departement pathology of Farhat Hached Hospital between 1990 and 2011. Results: Our population is composed by four mens and a woman. All patient are heavy smokers. The most common presenting symptom is hematuria and dysuria in all cases. One patient presented a complete urinary retention. A cystoscopic examination with transurethral resection was performed in all cases. The pathological examination with use of immunohistochemical markers of neuroendocrine differentiation were consistent with a large cell neuroendocrine carcinoma in four cases and a small cell neuroendocrine carcinoma in a case. A cystoprostatectomy was made in two cases followed by chemotherapy. Conclusion: The clinical presentation of PUBNEC is similar to other bladder cancers and is characterized by advanced stage at diagnosis and rapidly progressive disease. The diagnosis of poses several problems: a vesical metastasis has to be excluded and such lesions have to be differentiated from transitional cell carcinoma, lymphoma, paraganglioma and peripheral nerve neuroblastoma. There is no gold standard for the management of patients affected due to low disease frequency PS-22-052 IgG4-associated Inflammatory Pseudo-tumor (IPT) of the ureter: A case report A. Marando * , G. D´Ambrosio, F. Catanzaro, F. Sessa * University of Insubria, Dept. of Surgical, Varese, Italy
Objective: IgG4-associated inflammatory pseudo-tumor (IPT) is a novel clinico-pathologic entity characterized by intensive infiltration of IgG4-positive plasma cells, associated with systemic IgG4-related sclerosing disease. Many reports described IgG4-related IPT in various locations such as pancreas, salivary glands, liver, breast, lung and recently also ureter. Method: It is described a case of ureteral IPT with pathologic and immunohistochemical features of IgG4-related IPT, fibrohistiocytic type. Results: The study case is a 82-year-old female with severe stenosis of the left ureter and hydronephrosis, who underwent to nephroureterectomy and endoscopic resection of multiple lesions in the bladder. The histological examination showed transmural fibrosing inflammatory lesion of the affected ureteral wall, with abundant plasma cells intermixed with many histiocytes, lymphocytes, fibroblasts and scattered eosinophils. The majority of infiltrating plasma cells were positive for IgG4. Bladder lesions showed similar histological features. The diagnosis was IgG4-related IPT, fibrohistiocytic type. Conclusion: IgG4-related IPT of ureter is extremely rare, with only few cases reported in literature. Recognition of this entity is clinically relevant because this type of IPT can be treated with steroid therapy and may be associated with sclerosing autoimmune disease in other organs.
A rare case of malignant fibrous histiocytoma of the urinary bladder S. Mavropoulou * , Z. Tatsiou, I. Amplianitis, P. Nasos * General Hospital, Laboratory of Pathology, Xanthi, Greece
Objective: Malignant fibrous histiocytoma (MFH) is an extremely rare malignant mesenchymal neoplasm of the urinary bladder with only a few well-documented cases reported in the English literature. Method: We report the case of an 88-year-old man who was brought to our hospital due to sudden massive haematuria. Catheterization failed to obtain haemostasis so, a suprapubic incision and direct exploration of the bladder was performed. A large solid tumor was found with a shaggy haemorrhagic surface and biopsies were taken. Results: Histological examination revealed an infiltrating malignant neoplasm composed of variably pleomorphic ovoid neoplastic cells with eosinophilic cytoplasm, bizarre tumor giant cells and prominent stromal osteoclastic giant cell reaction. Immunohistochemical examination was negative for cytokeratin, desmin, smooth muscle actin, PSAP, NSE, S100protein and HMB45 whereas it was positive for CD68 in a large number of tumor cells. Accordingly, the diagnosis of undifferentiated pleomorphic sarcoma with histological features compatible with giant cell MFH of the urinary bladder was made. The patient died 4 days after the diagnosis. Conclusion: In conclusion, MFH of the bladder should be kept in mind when facing with an undifferentiated malignant tumor. Despite the poor prognosis, early diagnosis and aggressive salvage therapy might offer the chance of long-term survival in selected cases.
Partial nephrectomy experience at a single tertiary-care oncology centre: A clinicopathologic study of 60 cases S. Menon * , G. Bakshi, H. Tongaonkar, V. Noronha, A. Joshi, K. Prabhash, S. Desai * Tata Memorial Hospital, Dept. of Pathology, Mumbai, India
Objective: Partial nephrectomy (PN) is replacing radical surgery as a gold standard in the treatment of small renal masses. Intra-operative frozen consultation for margin status is aimed at achieving a disease free state in order to reduce chances of recurrence. Method: A retrospective clinicopathologic analysis of consecutive PNs performed at our institute from 2004 to 2011 was undertaken. Results: Sixty cases of PN were analysed. Male to female ratio was 1.8:1. Median age was 51 years. Six cases were benign: oncocytomas (3), angiomyolipoma (3); while 54 cases were malignant: renal cell carcinoma (RCC) -conventional (43), papillary (8), chromophobe (1), mucinous tubular spindle cell carcinoma (1) and one case of primitive neuroectodermal tumour. In 4 patients, the renal tumour was a second malignancy. Median tumour size was 3.5 cm with 42 cases of stage pT1. Intra-operative margin was positive in 16 cases. Mean margin for all cases was 2.3 mm. Median follow-up was of 24 months. None of the tumours recurred or metastasized during follow-up. Conclusion: Conventional RCC is the commonest histology in PN cases. Frozen section analysis has a definite role in achieving margin free status. PN is not associated with increased risk of local recurrence in small renal tumours.
Clear cell tubulopapillary renal cell carcinoma: A clinicopathologic study of two cases G. Muñiz * , A. Corominas, N. Cerda, A. Perez, V. Caamaño, M. Gonzalez, L. Etxegarai, J. I. López * Hospital Universitario Cruces, Dept. de Anatomía Patológica, Barakaldo, Spain
Objective: Cleal cell tubulopapillary renal cell carcinoma has been recently identified as a low grade renal cell tumor with distinct histological features. Method: Patient 1: 65 year old female with a history of fibromyalgia and persistent loin pain. A right renal mass, 2.8 cm in diameter, was discovered in the rheumatologist's follow up. Right nephrectomy was performed and the patient is free of disease 7 months later. Patient 2; 55 year-old female with a history of diabetes mellitus type 2, hypertension and renal failure grade 3 with a 2.5 cm in diameter renal tumor in the routine studies. She underwent tumorectomy. Results: Grossly, both were cystic tumors with gelatinous fluid and white-yellowish solid areas. Tumors showed a tubular and papillary architecture. Proliferating cells displayed clear cell cytoplasm and hyperchromatic nuclei placed in the luminal side. IHC showed positive staining with e-cadherin, EMA and CK7. Conversely, CD10, CD117 and AMACR were negative. Conclusion: Clear cell tubulopapillary renal cell carcinoma should be considered a distinct subtype of renal cell carcinoma according to its unique morphologic and inmunohistochemical features. The few cases reported so far behave in an indolent course.
Survivin expression in renal epithelial tumors: Its usage in the differential diagnosis of eosinophilic renal epithelial tumors A. Ozcan * , N. Yigit, O. Onguru, B. A. Firat, S. Ozaydin * Gulhane Military Medical Academy, Dept. of Pathology, Ankara, Turkey Objective: The differential diagnosis of renal tumors can be problematic due to overlapping morphologic features. The purpose of this study was to assess the potential contribution of survivin expression in the differential diagnosis and determination of therapy modalities of these tumors. Method: This study consisted of 15 chromophobe (ChRCC), 15 clear cell (CCRCC) and 9 papillary (PRCC) renal cell carcinomas, and 13 oncocytomas. Sections were stained against survivin antibody. Results: PRCCs and CCRCCs showed diffuse and strong survivin expression. Survivin expression was strikingly prominent in type1 PRCCs and cystic CCRCCs. In CCRCCs, survivin expression was more pronounced in low grade areas than high grade and sarcomatoid areas. In ChRCC, survivin expression was more limited and weaker than that of oncocytomas and other malignant renal tumors. In non-neoplastic renal tissue, survivin expression was more pronounced in podocytes and atrophic tubules than other nephron parts. Conclusion: Our findings suggest that survivin may contribute to the differential diagnosis of renal tumors because of the partially unique staining patterns. It was purposed that knockdown of survivin reduced growth, induce apoptosis and enhance in vitro radiosensitivity of RCC cells. Taken together, to be known different survivin expression patterns in renal tumors may help to determine new therapeutic strategies for RCCs. Objective: There are several tumor-like lesions and miscellaneous neoplasms of rete testis. We present a case with adenomatous hyperplasia of rete testis (AHRT). Method: The patient was 24 years old with undescended testis and referred to our hospital. There was no clinical or endocrin abnormalities. Cryptoorchidism was unilateral and the other testis was normal. Right orciectomy was performed and sent to pathology labarotory for examination. Results: There was no tumoral lesion in gross examination but in microscobic examination there was gland like tubular structures. Some of these were back to back position with little intervening stroma and mild to moderate atypia. EMA and pancytokeratin immunohistochemistry findings with morphology confirmed the diagnosis of AHRT in this case. Conclusion: AHRT is a rare proliferatif lesion and can be confused with malignancy. It is incidentally realised in microscobic investigation. It may present as a very small lesion detected in microscobic examination or solid-cystic mass lesion which is macroscobically evident. Clinic history, localization, histologic features and immunohistochemistry are criterias for differentiating these lesions.We present this case for both surgeons and pathologists with its importance to be confused with malignancy. Objective: Prostate cancer is the second leading cause of death in men. The localized disease often responds to conventional therapies like androgen ablation via castration and/or administration of chemical inhibitors but advanced disease resistant to any curative therapies is still challenge for investigators. There are increasing efforts to enhance the possibility of finding positive and sensitive immune markers for diagnosing and treating prostate cancer. Method: We applied immunohistochemical markers; AMACR and iNOS. Formalin-fixed parafin embedded tissues of 64 prostate needle biopsy specimens diagnosed as prostate adenocarcinoma between 2005 and 2010 years were enrolled in the study. Results: AMACR expression has been found in 58 (90.6 %) and iNOS expression in 54 (84.4 %) of 64 prostate adenocarcinomas.No significant relationship of AMACR and iNOS has been obtained (p>0.05). There was no significant correlation of histopathologic grade of the tumors with AMACR and iNOS expression (p>0.05). Conclusion: The expression of AMACR and iNOS might be important diagnostic immune markers for prostate adenocarcinomas especially in needle biopsies when the quantity and quality of tissue are limited.
A tissue microarray study of Napsin-A expression in renal tumors A. Panizo * , F. J. Queipo, J. J. Sola, J. Pardo * Hospital de Navarra, Anatomia Patologica, Pamplona, Spain
Objective: Napsin-A is an aspartic protease present lung, renal, and thyroid cells. There are few studies evaluating Napsin-A in renal neoplasms. Therefore, we studied IHC expression of Napsin-A in a wide spectrum of renal tumors. Method: IHC for Napsin-A (rabbit polyclonal antibody) was performed in a series of 334 cases of primary and metastatic renal tumors on TMA. Cytoplasmic immunoreactivity was scored: intensity (0-3+) and extent (% of tumor cells: 0-3). The 2 scores were added: positive case: combined IHC score>2; negative if combined score of 2 or less. Objective: The diagnosis of prostatic carcinoma can be challenging on needle core biopsies. The aim of this study was to assess the utility of alpha-methylacyl-CoA racemase (AMACR/P63) antibody cocktail for prostate cancer diagnosis. A prospective analysis of 50 consecutive radical prostatectomy specimens and 50 prostate needle biopsy semples was performed to select histological sections showing foci of minimal prostatic carcinoma, high grade prostatic intraepithelial neoplasia (HGPIN) and benign mimickers of prostatic carcinoma (atrophy, adenosis). Method: Serial histological sections were stained with hematoxylin and eosin, Van Gieson and immunomarkers: AMACR and P63 using a prediluted antibody cocktail. Results: The cocktail was very useful in highlighting prostatic carcinoma associated with HGPIN (flat or cribriform) and distorted foci of minimal carcinoma. AMACR was positive with moderate and strong staining in almost all cases for which the immunohistochemical result converted the atypical diagnosis to a final cancer diagnosis. The cases whose diagnosis was changed from "atypical" to cancer were all highly suspicious for cancer based on HE histology and negative basal cell markers. Conclusion: This cocktail would be of diagnosis utility when limited tissue is available for histopathological evaluation of small diagnostically difficult foci (prostate needle biopsy and surgical specimens).
Overexpression of Cytokeratin 20, Ki-67 and Topoisomerase-II-a can significantly stratify the recurrence risk in patients with bladder cancer after transurethral resection S. Petrov * , K. Malkhasyan, R. Khasanov * Kazan Cancer Center, Dept. of Pathology, Russia
Objective: The current predictive models based on main clinical tumor features are not accurate for the biggest bladder cancer patient group, who underwent the transurethral resection (TUR). Method: Overall 103 patients with primary urocarcinoma after TUR were included in this retrospective study. The follow up plan in all cases included cystoscopy and biopsy every 3 months in the first 2 years. The recurrence criteria were cystoscopical and pathological confirmation of the tumor growth. In all cases using TMA technique (TMA Master, 3DHistech) there was done the IHC expression study of p53, p63, CK20, E-cadherine, B-catenin, CD44v6, Ki-67 (10 % cut-off), Topo-II-a (10 % cut-off) and Her2, as well c-erb-B2 amplification study (CISH). Results: There were no association found between the Her2 expression and c-erb-B2 amplification in urocarcinoma patients. In multivariate regression analysis only CK20, Ki-67 and Topo-II-a showed the significant prognostic power in recurrence prediction. These markers were used to develop the powerful predictive index for bladder cancer patients after TUR. Conclusion: Although the Her2 overexpression is relatively common event in bladder cancer, c-erb-B2 gene amplification isn't main mechanism of its realization. The CK20, Ki-67 and Topo-II-a are promising prognostic markers for recurrence and should be validated in further prospective study.
Solitary fibrous tumor of the urinary bladder associated with a high-grade urothelial invasive carcinoma. A case report A. Pitino * , S. Squillaci, C. Spairani, M. Ferrari, M. F. Cosimi, W. Fusco, C. Rossi, F. Montefiore, G. L. Bigatti, V. La Paglia * San Giacomo Hospital, Division of Anatomic Pathology, Novi Ligure, Italy
Objective: Solitary fibrous tumor (SFT) is an unusual spindle cell neoplasm, which can exceptionally occur in the urinary bladder. Method: We present a case of urinary bladder SFT in a 60 year-old man who complained of pelvic pain. Cystoscopy revealed a large protruding, fleshy mass at the anterior wall of the bladder. A biopsy was first misclassified as inflammatory myofibroblastic tumor (IMT). Subsequent complete transurethral resection was performed. Results: Macroscopically, a 9×7×5 cm greyish lobulated firm polypoid tumor was seen. Microscopically, it consisted of uniform spindle cells with elongated tapered ends nuclei forming a patternless growth in a collagenous background. Mitotic figures were rare. Immunohistochemically, the neoplastic cells were positive for CD34 and bcl-2, and negative for α-SMA, desmin, CK AE1/ AE3 and Alk-1. The bladder urothelium showed foci of high-grade transitional carcinoma with lamina propria invasion. Conclusion: Initially, SFTs were thought to be of mesothelial origin. Then, these tumors have also been observed in extrapleural and extraserosal sites, which suggests a mesenchymal cell origin. The differential diagnosis should always include other spindle cells lesions such as sarcomatoid carcinoma, leiomyosarcoma and IMT. To date, this is the first reported case of association of urinary bladder SFT and high-grade urothelial invasive carcinoma.
Renal oncocytomas with unusual features: Clinicopathological study of 9 cases F. J. Queipo * , H. D. Quiceno, F. J. Pardo, Án. F. Panizo, M. L. Gómez-Dorronsoro, G. Aisa, F. J. Monzón, E. Mejía, C. Del Agua, J. Alfaro * Clínica Universidad de Navarra, Pathology, Pamplona, Spain
Objective: Oncocytoma (RO) is a benign renal neoplasm, with a wide morphologic spectrum and excellent prognosis. Recently, it has been described worrisome morphological features.
Method: RO treated in our centres were reviewed, and we focused on identifying the worrisome and the atypical features. Results: We identified 9 RO with at least one of the worrisome feature. Patients: 5 M/4 F (mean age 72,89 year; range: 63-88). Mean tumor size: 4,24 cm (range: 1,6-11,5 cm); right kidney: 5, and left: 4 cases. Invasion into the perinephric or renal sinus fat was the most frequent worrisome feature: 7 tumors (77,8 %) and focal chromophobe carcinoma-like areas in 3 cases (33,3 %). Lymphovascular invasion, entrapped renal tubules-glomeruli, necrosis and mitosis were found in 2 (22,2 %) respectively. All but two ROs had at least 2 worrisome features. Followup was available for all cases (median 11 months; range 3-108): all patients were alive without recurrence or metastasis. Conclusion: RO is a tumor which often can show worrisome and atypical morphology. It is necessary to know and recognize the worrisome features to prevent diagnostic errors, if otherwise typical oncocytoma morphology is present. Despite these atypical morphological data, the prognosis is excellent.
Ki67 and p53 expression in urothelial carcinomas and clinicopathologic correlation A. Ribeiro * * CHLC, EPE, Serviço de Anatomia Patológica, Lisboa, Portugal
Objective: The most important predictive parameter for the biological behaviour of urothelial carcinoma is histological grade, except for depth of invasion. The aim of this study was to investigate the expression of p53 oncoprotein and Ki67 antigen in a series of transitional cell bladder carcinoma with papillary morphology (pTapapillary carcinoma and pT1) with histological grade and recurrence. Method: This study included 59 cases diagnosed with urothelial carcinoma with papillary morphology (pTa, pT1). Immunohistochemical expression of Ki67 and p53 were examined in each case, and were graded accordingly to the percentage of cells stained, in low, moderate and high-expression groups. Results: As described previously in other publications the expression of p53 and Ki67 has a relationship with histological grade. We also noted that of the 13 recurrences, 11 were associated with moderate to high expression of either p53 or Ki67, or both. Conclusion: We concluded that p53 and Ki67 expression combined with histological grade and pathological stage may be helpful in assessing more accurately the biological behaviour of urothelial carcinoma. And the overexpression of p53 and Ki67 are related with an unfavourable prognosis. Objective: Prostatic stromal hyperplasia with atypia is a rare lesion with fewer than 200 cases reported worldwide. It can be mistaken for sarcoma because of the presence of atypical, bizarre cells. Its malignant potential is uncertain. The follow up study of every case is important. Method: A 71-year-old man with previously diagnosed atypical prostatic adenostromal hyperplasia with atypia of stromal cells was hospitalized with urinary obstruction after transurethral resection of prostate made 6 years ago. Repeat TURP was performed and tissue specimens were investigated and compared with the initial biopsy. Results: The second biopsy showed the benign hyperplastic prostatic glands with atypical, bizarre, frequently multinucleated giant stromal cells between them. They displayed intense immunoreactivity for actin, vimentin and androgen receptors. The microscopical picture was identical to that seen in the first specimens. The nuclear abnormalities looked like in an atypical symplastic leiomyoma of myometrium. No evidence of sarcomatous or carcinomatous transformation, mitotic index evaluation was noted. Conclusion: This case maintains the viewpoint that the prostatic adenostromal hyperplasia with stromal cell atypia is a benign lesion. But it can recur and requires the repeat TURP or radical surgery.
Cancer risk in patients with precancerous lesions of the prostate Y. Rogov * , V. Zakharava, T. Liatkouskaya, E. Cherstvoy * Belarusian Medical Academy, Dept. of Pathology, Minsk, Belarus
Objective: Prostatic intraepitelial neoplasia (PIN) and atypical small acinar proliferation (ASAP) has a high predictive value as markers for prostate cancer (PCa). Method: PCa-risk in patients with precancerous lesions has been assessed on biopsy material in 172 patients having morphological suspicious to PCa. Suspicious foci were estimated with use of cocktail AMACR + HWC + p63. Results: According to our results revealing of precancerous lesions in biopsy specimens has been associated with PCa identification in re-biopsies (F=0,04). Within the first-5years the overall incidence of PCa in re-biopsies made 27 % in the group of precancerous lesions and 36 %in the ASAP group. Life-time-without-PCa median made up 5-years with no reliable difference between PIN and ASAP groups (WW=2,35; p=0,06). Thus, the cumulative share of patients without PCa in the ASAP group formed 86 %-82 %-82 %-73 %-60 % at the end of the first-second-thirdfourth-fifth year of supervision respectively. Conclusion: Within the first 5 years PCa risk makes 27 % in the general group of precancerous lesions of the prostate and 36 % in ASAP group with no difference between ASAP and PIN groups. Сumulative share of patients without PCa in rebiopsy specimens decreased from 86 % to 60 % during these 5 years.
Benign prostatic hyperplasia and prostate carcinogenesis after the Chernobyl accident in Ukraine A. Romanenko * , A. Chekalova, A. Yurakh, P. Harkonen, S. Vozianov * Institute of Urology, Kiev, Ukraine
Objective: The prevalence as well as immunohistochemical (IHC) study of latent, incidentally found prostate cancer (LPC) as well as precancer lesions, in patients, who underwent surgery for BPH were studied. Method: BPH samples were obtained by prostatectomy from 120 Ukrainian patients consisting of 30 patients from areas without radio-contamination (control group 1) and of 90 patients living in 137Cs contaminated areas of Ukraine (group 2). Ki-67, p53, p27Kip-1, p63 and Bcl-2 proteins were IHC investigated in BPH from all patients. Results: The incidences of LPC (Gleason score 4), chronic prostatitis, PIA and PIN were 16.67, 53.34, 20, and 26.67 % in group 1; 12.23, 64.45, 43.45 and 36 .67 % in group 2, respectively. Greatly elevated levels of p53, Ki-67, Bcl-2 associated with decreased levels of p27Kip-1and p63 in areas of PIA and less LPC and PIN in group 2 to compare with group 1 patients were obtained with statistically significant differences. Conclusion: Our study suggests that chronic long-term lowdose radiation exposure might result in the increase of chronic inflammation and it is now found to be associated with increased incidences of PIA and PIN in BPH accompanied by p53, p27KIP-1 and Bcl-2 alteration which in turn could lead to prostate carcinogenesis.
Eosinophilic globules in rete testis mimicking yolk sac tumor in a testicle with seminoma E. Ronne * , T. Argyrakos, D. Rontogianni * Evangelismos Hospital, Dept. of Pathology, Athens, Greece
Objective: The recognition of a non-seminomatous component in an otherwise typical testicular seminoma changes the choice of adjuvant chemotherapy.
Method: A 37-year old male underwent orchiectomy for a testicular mass. Serum tumor markers levels (β-hCG/AFP/ LDH) were normal. Results: Macroscopical examination revealed a 2.1 cm white-yellow tumor. The tumor was composed of the characteristic for seminoma homogenous, clear cells arranged in nests and islands separated by septa and infiltrated by lymphocytes. Immunohistochemically the tumor cells were positive for PLAP, OCT3/4, CD117 and D2-40 and negative for CD30 and CK8.18. There was also a regular pattern of tubular structures infiltrated by the seminoma cells while retaining a low columnar type epithelium. The presence of sphaerical eosinophilic globules (PAS+/dPAS+/AFP−) within the tubular lumina was strongly reminiscent of the hyaline globules of yolk sac tumor. The tubular structures were positive for CK8.18 and negative for AFP, glypican-3 and OCT3/4. Conclusion: The presence of tubular structures with sphaerical eosinophilic globules creates a suspicion for a yolk sac tumor component. The absence of AFP and glypican-3 expression, the regular pattern of the tubular structures and their continuity with rete testis excluded this suspicion. Hyaline eosinophilic globules in rete testis should not be confused with the globules produced by yolk sac tumors.
Beta-catenin expression and CTNNB1 mutations in a series of Wilms tumours R. Santi * , P. Pinzani, F. Salvianti, G. Baroni, M. Pepi, G. Nesi * University of Florence, Pathological Anatomy Section, Italy
Objective: CTNNB1 mutations have been found in 15-30 % of Wilms tumour (WT) cases. Nuclear beta-catenin protein has been detected by immunohistochemistry in a higher proportion of WTs, thus suggesting alternative genetic pathways leading to beta-catenin activation in these neoplasms. Method: Sixteen renal WTs and 7 secondary WT localizations were retrospectively investigated. The series included 17 paediatric patients, 10 females and 7 males, with a mean age of 4.5 years and a 34 year-old female patient. Immunohistochemical analysis of beta-catenin was performed and findings were reported for each neoplastic component (i.e. epithelium, stroma and blastema). Tumour DNA was extracted for direct sequencing analysis. Results: The majority of WTs showed moderate to strong membranous staining for beta-catenin in the epithelial (76.5 %) and the blastemal (60 %) components. Nuclear betacatenin expression was observed in combination with cytoplasmic staining in the mesenchyma and/or the blastema of two primary renal tumours. In these cases the deletion of codon 45 p.S45del and the missense substitution p.T41A were detected. Conclusion: Preliminary results of this ongoing study highlight beta-catenin cytoplasmic and membranous expression in the tumour cells of primary and metastatic WTs, with few cases demonstrating nuclear expression. In our series, betacatenin nuclear expression was invariably associated to CTNNB1 mutation.
A preliminary study on O6-methylguanine-DNA methyltransferase and Type 2 transglutaminase expression profile of renal cell carcinomas B. Sarsik * , B. Pehlivanoglu, D. Tunali, A. Simsir, E. Gokmen, S. Sen * Ege University, Faculty of Medicine, Izmir, Turkey
Objective: O6-methylguanine-DNA methyltransferase (MGMT) repairs O6-methylguanine in DNA, therefore provides a tumor supressor effect. Type 2 transglutaminase (TGase-2) is a multifunctional enzyme involved in many biological processes. Few data are available in the literature on their expression in kidney cancers. In this preliminary study, we evaluated immunohistochemical expression of MGMT and TGase-2 in renal carcinoma cases. Method: Forty cases of renal carcinoma including ten clear cell, ten chromophobe cell, ten papillary and ten urothelial carcinoma were randomly selected. Thirty-one patients were male and average age was 61,5. Staining intensity and percentage of staining tumor cells were scored. Total score was categorized as weak, moderate and strong. Results: Strong MGMT positivity was demonstrated in 15 cases (38 %). Eight cases (20 %) showed strong TGase-2 expression. Fifty percent of clear cell carcinomas strongly expressed MGMT and TGase-2. No correlation was found between MGMT and/or TGase-2 expression, tumor size and grade. Eighty percent of urothelial carcinomas strongly expressed MGMT. Conclusion: The prognostic value of MGMT and TGase-2 as well as their potential role in treatment response have been investigated recently. MGMT and TGase-2 may be prognostic factors in renal carcinomas. Further investigation is required to verify our findings.
Three dimensional topographic analysis of 904 cases of radical prostatectomy A. N. Seo * , K. S. Lee, G. Choe * Seoul National Univ. Bundang, Pathology, Seongnam-Si,
Objective: Prostate cancer is typically mutifocal, and there has been no report about the exact number of prostatic carcinomas in each case. We have performed topographic analysis using three dimensional mapping technique. Method: We established data base including 904 cases of radical prostatectomy consisting of 2717 individual adenocarcinomas, and performed comprehensive pathologic analysis. Objective: TMPRSS2-ERG gene fusion is the most common genetic alternation in prostate cancer. It is associated with the expression of oncogene ERG protein. Recently, the immunohistochemical staining method that using anti-ERG antibody was verified strong correlation with ERG protein which is the product of genetic alteration. Aim of this study is to declare that the relationship between ERG expression and clinicopathological factor. Method: otal 307 cases of radical prostatectomy specimen were assessed. All cases were constructed tissue microarray and immunohistochemical staining was performed.
Results: ERG-positive rate was 24.1 % (74/307) and significantly higher expression of ERG expression was observed in the subgroup that has lower Gleason score. (p<0.05) Analysis with the histologic pattern of prostate adenocarcinoma, tumors with discrete glandular unit (Gleason pattern 3) is shown higher frequency of ERG expression (p=0.007). Conclusion: ERG-positive case was smaller than that of western population (about 50 %) and other factors including age, tumor volume, initial PSA level, pathological stage and margin status were not significantly related with ERG expression. In conclusion, positive rate of ERG immunohistochemical staining is meaningful higher in the tumors with wellformed gland that is represented by lower Gleason score.
Solitary fibrous tumour of the kidney mimicking renal cystic neoplasm T. Tichy * , J. Skarda * University Hospital Olomouc, Institute of Pathology, Czech Republic
Objective: Solitary fibrous tumour (SFT) can develop at any anatomic site, but in the kidney is described rarely. In general, SFT forms an unencapsulated solid mass. We report a case of a 57-year old woman with benign SFT of the left kidney. The tumour showed extensive pseudocystic change and mimicked renal cystic neoplasm. Method: A nephrectomy specimen showed cystic tumour beneath renal capsule and in peripelvic adipose tisssue. Histological sections were used for hematoxylin-eosin and for immunohistochemistry (antibodies against vimentin, smooth muscle actin, desmin, S-100 protein, CD117, CD99, bcl-2, CD34, CK18, AE1-AE3, EMA). Results: Microscopically the tumour showed uniform spindle cell proliferation with expansive tumour margins, without necrosis or hemorrhagies. Mitotic activity was 1 per 10 high power fields. Cystic spaces without epithelial lining contained eosinophilic proteinaceous fluid. Imunohistochemically tumour showed difuse positivity for CD34, CD99, bcl-2, vimentin and stained negatively for S-100 protein, cytokeratins, EMA, smooth muscle actin, desmin and CD117. Conclusion: SFT of the kidney is infrequent. Some renal SFT can undergo pseudocystic transformation and mimic renal cystic neoplasm both clinically and macroscopically. Objective: Renal angiomyolipomas are mesenchymal tumors that comprised of adipose tissue, smooth muscle like cells and abnormal thick walled blood vessels admixed in various proportions. Epithelial renal angiomyolipomas are rare variants and may exhibit atypia. In the literature epithelioid renal angiomyolipomas with atypia are reported to have malignant potential. Method: Left radical nephrectomy was performed due to renal mass with flank pain and hematuria. Results: In gross examination; 6.5×3.5×3 cm gray white mass with occasionally necrotic areas at the middle portion of left kidney renal sinus and perinephritic fatty tissue, macroscopically. Microscopic eveluation reveals; tumoral lesion comprised of adipose tissue, smooth muscle and blood vessels, showing areas of epitheloid morphology and moderate to severe degree of nuclear atypia. In addition to histomorphology positivity HMB-45 immunohistocemistry elaborated to the diagnosis of "epitheloid renal angiomyolipoma with atypia". Conclusion: This case has been presented due to its infrequent occurrence and malignancy potential.
Correlation of minute focus of prostate adenocarcinoma on random multifocal needle biopsy with radical prostatectomy specimen A. Urbanskiy * * Russian Research Centre for Ra, Pathology, St. Petersburg, Russia
Objective: This work attempts to determine the importance of small foci of prostatic cancer in random multifocal needle biopsy specimens. Method: 64 patients with a microscopic focus confined to a single core specimen (which defined as tumor less than 1 mm with a Gleason score of 6 or less) were identified from a retrospective review of 1206 needle biopsies of the prostate. Twelve of these 64 subsequently under went radical retropubic prostatectomy at our centre. Clinically significant tumors were defined as those with volume greater than 0.5 cc. Results: Average tumor volume was 0,9±0,6 cc (range 0,068-2,9 cc). In 41,66 % (5) of the cases was less 0,5 cc (range 0,068-0,458 cc, mean volume -0,22±0,15 cc). There was 1 (8,4 %) of patient with extraprostatic extension. Conclusion: Our data have shown high frequency of revealing insignificant tumours (mean 42±28 %; Р=0,95). However, about 60 % of patients had clinically significant tumors warranting definitive therapy. The smallest focus of cancer on needle biopsy is not a guarantee of a clinically insignificant tumor.
PS-22-085 E-cadherin in mice: Expression in normal urothelium, pre-neoplastic and neoplastic urothelial lesions C. Vasconcelos Nóbrega * , C. Costa, R. Arantes-Rodrigues, A. Henriques, H. Vala, A. Colaço, L. Santos, C. Lopes, P. Oliveira * Escola Sup. Agrária Viseu, DZERV, Portugal
Objective: E-cadherin is an adhesion molecule that promotes the integrity and stability of the urothelium. A decrease in its expression is associated with more aggressive tumour phenotypes, with the ability to invade and metastasize. Our aim was to describe the expression of E-cadherin in normal urothelium and in urothelium with pre-neoplastic and neoplastic lesions of ICR male mice. Method: Urothelial lesions were chemically induced by Nbutyl-N-(4-hydroxybutyl) nitrosamine in ICR mice, and evaluated by immunohistochemistry in order to determine the staining pattern of E-cadherin. Results: In normal urothelium, 87.5 % of E-cadherin expression was at the cellular membrane level. In simple hyperplasia, the same pattern was observed in 66.67 % of lesions. Nodular hyperplasia exhibited a mixed pattern (50 % membrane and 50 % cytoplasmic pattern). In 86.67 % of dysplasia, a cytoplasmic pattern was seen. On invasive carcinoma the majority of invasive urothelial cells exhibited a pattern of membrane and cytoplasmic staining. On squamous metaplasia it was observed a membrane pattern on basal and intermediate layers, and a loss of immunoreactivity in the most superficial ones.
Conclusion: E-cadherin is a valuable tool for investigating the cellular adhesion status of the urothelium in mice. Neoplastic lesions exhibited an abnormal, heterogeneous staining pattern.
New potential prognostic and predictive factors in conventional clear cell renal carcinoma P. Latalova * , P. Flodr * FMD PU and FH Olomouc, Dept. of Clinical and Molecular Pathology, Czech Republic
Objective: Biological behavior of conventional clear cell renal carcinoma (CCRC) is associated with tumour stage and grade. Cancer progression with metastasis is part of a process epithelial-mesenchymal transition (EMT) and is joined with invasiveness due to adhesion and cytoskeleton change. The central role of the EMT mechanism is attributed to Snail factor. The intermediate filament expression is changed through the progression of many types of neoplasms. These changes are organ or tissue specific and depend also on the degree of malignant transformation (genome instability acceleration). The expression changes of cytokeratin 18 (CK18) are signs of aggressive biological behaviour in some tumour types -e.g. colorectal and breast carcinoma (downregulation of expression) or CCRC (upregulation of expression). The expression of Snail and CK18 molecules and mRNA CK18 levels correlate to stage and grade of CCRC (according Messai et al. 2010) . Objective: Study was based on the expression of immunohistochemical markers (CK18, CK7, vimentin, Snail, CD10) in 15 cases with evaluation of the primary tumour and its metastasis. Results: The difference and similarity of intensity and percentage of positive neoplastic population between the primary and the secondary carcinoma will be presented. Conclusion: Snail and CK18 seem to be potential tumour progression factors and may be included in so called personalised medicine. Objective: Sarcomatoid carcinoma (SC) of the prostate is a rare variant of prostatic cancer representing less than 1 % of prostate tumors. Tumors are most commonly composed of an admixture of both malignant glandular and spindle cell elements. The sarcomatoid component can vary from 5 % to 99 %. Method: We report the case of a 63-years-old patient with an invasive tumor of the prostate for which he had a radical prostatectomy. Histopathological examination showed that the tumor was responding to an undifferentiated proliferation made of beaches and clusters of pleomorphic cells usually spindle-shaped, with eosinophilic cytoplasm, and elongated or oval nuclei, which are very irregular, sometimes monstrous, multinucleated, basophils with numerous mitotic figures. In some places, there were areas of glandular differentiation and foci of comedocarcinoma. Immunohistochemical study showed immunoreactivity of spindle and pleomorphic cells with cytokeratin and vimentin. Results: The diagnosis of sarcomatoid carcinoma of the prostate-grade Gleason 5+5 was confirmed. Conclusion: SC of the prostate is an exceedingly rare tumor. Retrospective analyses render prostate SC as one of the most aggressive prostate malignancies. The prognosis is dismal regardless of other histologic or clinical findings. extra-uterine extension (FIGO stage II-IV) were significant parameters of poor prognosis. No multivariate analyses were performed due series limitation. Conclusion: Detailed pathology evaluation, including histological subtype, and FIGO stage are essential in the adequate management decision/prognosis evaluation of U-LMS. Objective: Over 90 % of women with endometrial cancer show signs of uterine bleeding, so most cases are diagnosed at an initial stage. This study covers two patients who presented the first signs of this disease in an exceptional manner. Method: Case-1 A 71-year-old woman was afflicted with a six-month toxic syndrome. An omentum tumor was identified in the x-ray. Eleven days after the excision, she demonstrated oliguria, dysuria, and scant uterine bleeding. The pelvic ultrasound revealed a uterine mass. Case-2 A 56-year-old woman demonstrated disabling right inguinal pain. After an x-ray examination of her pelvis, a metastasis was suspected. A biopsy confirmed it. The immunochemistry pointed to a gynecologic, pancreatic or intestinal tumor. The computer tomography scan revealed a uterine mass. Results: The first case only suggested an estromal gastrointestinal tumor or a mesothelioma, misdiagnosing and missing the endometrial carcinoma. The second case was uncommon as bone metastasis is found with solid tumors but seldom occurs with endometrial cancer and even less so as a first sign. Conclusion: We must consider endometrial cancer at signs of uterine bleeding, however, further investigation is required in order to consider the 10 % who do not show the common signs.
Extrapelvic endometriosis presenting as a retroperitoneal tumor A. Alves * , P. Luís, A. Ribeiro, M. Ferreira * Hospital Santa Maria, Servico de Anatomia Patologica, Lisboa, Portugal Objective: Endometriosis is a benign disease characterized by the presence of functional endometrial tissue in ectopic locations. Retroperitoneal, liver or kidney involvement are extremely rare. Method: We present a case of a 52-year-old woman, which was referred to surgical consultation because of a retroperitoneal mass that was found on a routine abdominal ultrasonography (US). A subsequent CT scan showed a 12 cm tumor involving the liver, right kidney and adrenal gland. The gynecological US revealed ovaries with normal size and two probable uterine leiomyomas. She was submitted to a tumorectomy with segmental hepatectomy and nephrectomy. Results: Grossly, the tumor was 12×8×6 cm, invaded the kidney, the liver and a segment of diaphragm. It was white, fasciculate and with hemorrhagic focus. Histologically the tumor was composed of a biphasic proliferation of endometrial glands and endometrial stroma, both without atypia. The case was diagnosed as deep infiltrating extrapelvic endometriosis. With a 5 years follow-up, the patient is doing well and without evidence of disease. Conclusion: Despite being an extremely rare presentation, endometriosis can involve retroperitoneal organs and simulate a malignant neoplasm, even without a previous history of pelvic involvement. Objective: Malignant melanoma of the female genital tract is a multifocal disease resulting from a disorder of melanocytes Cotyledonoid dissecting leiomyoma is a benign smooth muscle tumor which can mimic a malignant lesion due to its alarming aspect. There are some variant forms of leiomyomas with an unusual infiltrative growth pattern. Due to its worrying appearance of the gross specimen, it is often mistaken for malignant lesion and it is important to be aware of this entity not to over treat this benign smooth-muscle neoplasm. We report a case of a 55 years old woman who underwent hysterectomy because of the suspect of a tumor growing from the right lateral uterine wall. On gross study the tumor measured 10 mm in diameter and was nodular, brown in color and irregular on cut section. Histological examination showed a nodular tumor composed of smooth muscle cells with a storiform pattern which dissects the uterine wall. The tumor was highly vascularized, included a myxoid component and showed some areas with invasion of vascular components. Cellular atypia, mitosis and necrosis were absent. The patient has had a good clinical course, without relapses until the date, what supports the fact that these tumors have a benign clinical behavior, even though they may have a malignant appearance.
Uterine leiomyoma with lymphoid infiltration A. N. Deger * , C. Kocak * S.B. DPU. Kec. EA Hastanesi, Patoloji Bolumu, Kutahya, Turkey
Objective: Although leimyomas of the uterus are common, lymphoid infiltration of leimyomas is a rare occurrence. We presented a case of a 45-year-old woman whose leiomyoma was diffusely infiltrated by lymphocytes. Lymphoid infiltration was analyzed with immunohistochemical methods and infiltration was found to be polyclonal type. Method: A 45-year-old woman with abnormal uterine bleeding was prediagnosed uterine leiomyoma and she was applied total abdominal hysterectomy and bilateral salphingo-oopherectomy. The analysis of the sample sent to pathology lab showed two leiomyomas. Hematoxylin eosin -stained slides were prepared after sampling. ımmunohistochemical stains were applied. Results: Microscopic examination of leiomyoma in greater diameter revealed a lesion with well-circumscribed borders composed of interlacing fascicles of bland monomorphic spindle cells diffusely infiltrated by lymphoid cells. The infiltrate not extended into the surrounding myometrium. Immunohistochemical analysis showed positive staining with CD 20, CD3, CD68. It was seen that inflammatory infiltration was polyclonal and involved T cells, B cells and histiocytes. Leiomyoma with lymphoid infiltration was diagnosed. The postoperative course was uneventful within a 6 month follow up period. Conclusion: Leiomyoma with lymphoid infiltration first described in 1989 by Ferry et all. From that date on, the literature involves few cases. Because it is rare and differential diagnose with malignant lymphoma is crucial, we presented the case and reviewed the literature. Objective: Vulvar intraepithelial neoplasia is divided into two groups: usual type and differentiated type. The differentiated vulvar intraepithelial neoplasia, which is frequently seen with invasive squamous cell carcinoma, can be confused with some benign lesions. The aim of this study is to analyse p16, p53, and Ki-67 expression characteristics of different histological types of vulvar intraepithelial neoplasia, invasive squamous cell carcinoma, and benign lesions of the vulva. Method: In this study, immunohistochemical analysis of 18 vulvectomy cases with p16, p53, and Ki-67 was performed. Results: Of 18 patients who underwent vulvectomy, nine had invasive squamous cell carcinoma and nine had vulvar intraepithelial neoplasia. Four additional vulvar intraepithelial neoplasia lesions were found accompanying the invasive squamous cell carcinomas. Nine benign lesions were found accompanying the invasive squamous cell carcinomas and vulvar intraepithelial neoplasia. Mean Ki-67 proliferation index was 32.3 % in the usual type of vulvar intraepithelial neoplasia cases and 26.4 % in the differentiated vulvar intraepithelial neoplasia cases. No p53 expression was present in benign lesions. Conclusion: Ki-67 PI does not recognize the usual type or differentiated type of vulvar intraepithelial neoplasia. p53 positivity can be of value in distinguishing differentiated type vulvar intraepithelial neoplasia from benign lesions.
An audit of surgical pathology reports of endometrial carcinoma: Experience from a referral centre in India K. Deodhar * , B. Rekhi, S. Menon, B. Ganesh * Tata Memorial Hospital, Dept. of Pathology, Mumbai, India
Objective: The aim was to see, compliance to minimum data information in carcinoma endometrium reports, in a team of 13 pathologists; and also to analyze these parameters e.g. tumor size, type, grade, depth of myometrial invasion, lymph node yield, pTNM stage etc. Method: During the period of 2008-2010, from the files of Pathology department of our hospital, reports of operated 114 carcinoma endometrium cases were retrieved and analyzed for various, above mentioned, parameters. Results: The median age was 58.04 years and median tumor size was 4 cm. Endometrioid adenocarcinoma was the commonest type (82.5 %); followed by MMMT (6.1 %) and Serous carcinoma (3.5 %). Grade 2 was the commonest tumor grade (42.1 %). Less than half of myometrial invasion was seen in 50 % of the cases, =/> half myometrial invasion was seen in 46.5 % of cases. (Information-not available in 4 cases). Parametrial involvement was seen in 5.3 % cases. The pTNM stage was not mentioned in 71.9 % reports. The median lymph node yield was 15. Conclusion: The compliance to adhere to minimum data information in carcinoma endometrium reports is generally good. Lymph node yield is reasonable. Parametrial involvement and mentioning of pTNM staging is to be done more meticulously. Use of proformas/checklists is recommended.
Extraovarian granulosa cell tumor: A case report I. Efstratiou * , S. Pervana, E. Pazarli, D. Alataki * Papageorgiou Hospital, Dept. of Pathology, Thessaloniki, Greece
Objective: Extraovarian granulosa cell tumor is a very rare neoplasm. We report a case of a tumor located in mesocolon. Method: A 58-years-old woman presented with abdominal pain and fever. Abdomen CT showed a mesocolic mass adherent to the left kidney and the spleen. She underwent resection of a 22 cm long segment of the left colon with a circumscribed mesocolic tumor measuring 7×10 cm. The tumor was infiltrating the subserosa of the large intestine. Intraoperatively normal ovaries have been identified. Results: Histologically the tumor is composed by monomorphous cells in solid nests developing cavities which contain serous or hemmorhagic fluid. The tumor cells have scanty cytoplasm and often grooved nuclei. Mitoses are very rare. The tumor cells were immunoreactive for vimentin, inhibin and progesterone receptors. The postoperative course was uncomplicated and the patient is free of disease 18 months later. Conclusion: Light microscopic and immunohistochemical features of the tumor are similar to those of ovarian granulosa cell tumors (GCT). Since the tumor was resected in toto and the mitotic activity of the cells is very low, we expect a favorable prognosis. Extraovarian GCT are very rare and only 7 cases have been published during the last 50 years. Probably these tumors arise from residual tissue of the genital ridge (so-called secondary mullerian system).
Detection of amplification of TERC and TERT genes in cytology samples from cervical neoplasias by fluorescent in situ hybridization A. Farkasova * , E. Kudela, T. Balharek, P. Zubor, J. Danko, L. Plank * JFM CU, Dept. of Pathology, Martin, Slovakia Objective: Increased telomerase activity represents an early event in cervical carcinogenesis allowing cell immortality by recovering chromosomal telomeres. Thus, detection of TERC and TERT gene amplification might represent a diagnostic and valuable prognostic biomarker of cervical neoplasias. Method: Cervical smears from 13 patients classified according to Bethesda as NILM (n=3), ASC-US (n=2), L-SIL (n=2), H-SIL (n=4) and SCC (n=2) were analysed for TERC (3q26) and TERT (5p15) gene amplification by fluorescent in situ hybridization (FISH) in 100 cells per slide using a four-color FISH probe (FHACT™). Results: The numbers of TERC and TERT copies, average ratio of gene copies and average number of cells with ≥4 TERC gene copies were highest in SCC, followed by H-SIL, L-SIL, ASC-US and NILM cases. Correlation between TERC/TERT amplification intensity and oncocytological findings was statistically significant (p<0.0001). Conclusion: FISH analysis of TERC and TERT genes could be effective tool for the diagnosis of cervical neoplastic lesions. Using together with cytology and HPV DNA testing it can achieve higher sensitivity and specificity to discriminate H-SIL and invasive carcinomas from L-SIL lesions. Objective: Ovarian stromal hyperplasia (OSH) is characterised by non-neoplastic overgrowth of the ovarian cortical. Mild hyperplasia of the cortical and medullary stroma is found in the ovaries of about one-third of perimenopausal and postmenopausal women. It is nearly always diffusely bilateral. We report a case of OSH associated with endometrial carcinoma (EC). Method: A 40-years-old caucasian woman, obese, smoker, with fatty liver, Gilbert´s syndrome and cholelithiasis. She had endometrial curettage biopsy for dysfunctional uterine bleeding for 5 month, which was diagnosed of complex atypical endometrial hiperplasia. A total abdominal hysterectomy whith bilateral salpingo-oophorectomy were performed. Results: The diagnosis was Well differentiated endometrioid EC and bilateral OSH with focal stromal hypertecosis. Conclusion: OSH of moderate to severe degree may be found in women with disorders associated with androgenic and estrogenic manifestations including EC, obesity, hypertension, and glucose intolerance, but these findings are less frequent and less obtrusive than in stromal hyperthecosis. Obese woman are at risk for developing endometrioid EC as a result of the increased capacity in adipose tissue to convert androstenedione to oestrone, and testosterone to oestradiol. A relationship in the origin of hormone-dependent endometrial pathology may exist between OSH, blood androgen levels and EC.
Detection of micrometastases in para-aortic lymph nodes in patients with carcinoma of the uterine cervix after negative frozen section analysis L.-C. Horn * , C. Kellner, R. Scherling, M. Höckel, J. Einenkel * Medizin. Universität Leipzig, Institut für Pathologie, Germany
Objective: Previous studies considered the presence of micrometastases (MM) in pelvic lymph nodes as clinically relevant prognostic indicator. Method: Frozen section analysis was performed in all cases. After FS-examination nodes were examined by one H&E-stained slide. All nodes without metastatic disease after frozen section and permanent section examination were subject of the present study. 43 patients and 418 PAN were enrolled and immunohistochemical staining using two cytokeratin-cocktail antibodies (AE 1/AE 3 and KL-) was performed.
Results: In one case, one single node showed micrometastasis, representing an incidence of 2.3 % of the studied cases and 0.23 % of the examined lymph nodes. In three cases benign endosalpingiosis was seen. The patient with MM is alive without evidence of disease 96 months after surgery. ITC were not observed. Conclusion: The frequency of MM in PAN is very low. There are only limited data regarding their prognostic impact within the literature. After careful examination of all removed PAN using H&E-staining (and step sectioning), immunohistochmeical ultrastaging cannot be recommend for routine use.
Serous tubal in situ carcinoma (STIC) in tubal and primary peritoneal carcinomas L.-C. Horn * , K. Leonhardt, S. Kafkova, J. Einenkel * Medizin. Universität Leipzig, Institut für Pathologie, Germany
Objective: Serous tubal in situ carcinoma (STIC) has been defined as one important precursor of pelvic serous cancer. Morphologically, STIC is defined by are cytologic atypia, high proliferative index and strong staining for p53. Method: The present study evaluates the presence of STIC and p53-signature in consecutive cases of 12 prophylactic salpingo-oophorectomy in women with BRCA-1-mutaion (BSO), 11 macroscopically inconspicuous tubes of patients with primary tubal cancer (TC) and 9 cases of primary peritoneal cancer (PPC) using immunohistochemistry against Ki-67 and p53 (clone DO-7). Results: The frequency of p53-signature and STIC was 8 % and 0 % in cases of prophylactic surgery, 9 % and 18 % in TC and 0 % and 33 % in PPC.
Conclusion: STIC and p53-signature as precursor lesions of pelvic serous cancer is seen in macroscopically inconspicuous Fallopian tubes in unilateral TC in patients with elective BSO and patients affected by PPC. We propose that the sectioning and extensively examining the fimbria protocol be applied to all cases with PPC, TC and in women with prophylactic BSO. Objective: Primary adenocarcinoma of the vulva is rare, and enteric differentiation is excepcional. Only a few cases of neoplasms of pure intestinal-type in the low genital tract have been reported. They are considered to arise from cloacal remants or from intestinal heterotopia. Method: We present the case of a 54 year-old woman, with a histerectomy performed 10 years before. On clinical examination a polipoid lesion in the vulvar vestibule was noticed. Biopsy revealed a tubular adenoma of intestinal type with a focus of adenocarcinoma. The first histologic interpretation was a metastatic intestinal tumor. Clinical examination, recto-colonoscopy and magnetic resonance imaging of the abdomen excluded this possibility. Results: The immunohistochemical study showed reactivity with citokeratin 20 but didn't with citokeratin 7. All this led to conclude that it was a primary intestinaltype adenocarcinoma of the vulva arisen from an adenoma. Conclusion: On our knowledge there are less than 100 reported cases of intestinal adenoma in the genital tract. They have been described also in the vagina and cervix but only a few developed an adenocarcinoma. It is important to be aware of this tumor type and to distinguish it from metastatic colorectal adenocarcinoma in order to plan appropriate treatment.
Ligneous cervicovaginitis and endometritis: Case report M. Koyuncuoglu * , E. Dogan * Dokuz Eylul University, Dept. of Pathology, Izmir, Turkey
Objective: Ligneous (wood-like) disease is a rare chronic pseudomembraneous inflammation of the mucous membranes which may also affect genital tract. The underlying pathogenesis is still unclear and an effective method of treatment has not yet emerged. Method: Here we present a 32 years old female patient presented to our clinic with 7 years of unexplained infertility. Her diagnostic infertility work-up revealed no abnormality. However, at gynecologic examination there was a thick and hard granulation tissue at the cervix extending to the posterior vaginal wall. Transvaginal ultrasound was unremarkable except for increased endometrial thickness. Results: Cervicovaginal and endometrial biopsy with diagnostic hysteroscopy was performed which demonstrated ligneous inflammation of both cervix and vagina. Endometrial biopsy was reported as chronic non-specific endometritis with dense fibrin deposition. Conclusion:. The disease may also affect other organs including oral cavity and eyes. This unusual condition is difficult to treat and lack of awareness makes the diagnosis also problematic. Objective: Differential diagnosis LM vs STUMP may be controversial, especially regarding necrosis evaluation; few studies address the relation between different treatments and their morphological effects. We aim to characterize a series LMs and putative STUMPs harboring necrosis. Method: Consecutive putative STUMPs (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) and a series of LMs with necrosis diagnosed at CHSJ & IPOP-Porto. Clinical files, particularly previous hormonal treatment (HT), gross specimen and histology features. Results: 18 putative STUMPs and 35 LMs with necrosis. LMs mean age at diagnosis: 43 years; mean size: 9.0 cm (2.3-18); 45 % with other LMs; treatment: myomectomy (49 %) and hysterectomy (51 %); 23 % peri-partum, 8.5 % post-uterine artery embolization; 17 (49 %) previous HT: progestin (17 %), oral contraceptive-OC (20 %), hormonal-IUD (8.6 %), and medical assisted reproduction (8.6 %). STUMPs mean age at diagnosis: 45 years; mean size: 10 cm (2.0-20); 44 % with other LMs; low mitotic index-MI (mean 3.16; median 1/10HPF); slight cell atypia; variable ischemic/ tumor-type necrosis. Treatment: myomectomy (22 %), hysterectomy (83 %) and radiotherapy (11 %); 12 (67 %) previous HT: progestin (22 %), OC (39 %), hormonal-IUD (11 %); no recurrence in remaining cases (n=6): median follow-up: 97.5 months. Conclusion: LMs with necrosis and STUMP may display overlapping features. Diagnosis of STUMP should consider previous HT, detailed morphology (necrosis type/atypia/MI), to prevent overdiagnosis of STUMP. positivity. PLAP and Chromogranin were negative. Ki-67 nuclear positivity was found in less than 1 % of Sertoli cells and in about 1 % of Leydig cells in hyperplastic areas. Karyotype was 46xy. Conclusion: Although, surgical pathologists encounter TFS rare in a clinical practice, they should be aware of this condition, especially in lack of relevant clinical data, when it could be interpreted as a tumor. Objective: Frozen section (FS) diagnosis of ovarian mucinous tumors can be difficult due to the size of these tumors, heterogeneity and potential risk of metastasis from gastrointestinal (GI) neoplasms. Method: We reported 79 ovarian mucinous tumors submitted for (FS) evaluation between January 2007 and Avril 2012 was conducted. Results: FS and final pathology results were collected. The average tumor size was 22,1 cm (1-55 cm). The FS and final pathology diagnosis were concordant in 84,8 % (67/79) of the cases. Of the 12(15,1 %) discordant cases, one (1,2 %) was downgraded and 8 cases (13,9 %) were upgraded. Of the 30 tumors interpreted as borderline mucinous tumors (BMT) on FS, 8(26,6 %) were malignant at final diagnosis (4 ovarian, 4 GI), 21 (70 %) remain as BMT and 1 (3,3 %) was benign. Of the 18 benign tumors on FS, 3 cases (17, 6 %) were upgraded to BMT at final diagnosis. Tumors with a malignant diagnosis on FS (30 cases) were 100 % concordant with final diagnosis. Conclusion: Our study showed a 15,1 % rate of discordance between FS and final diagnosis. Given that GI origin is a possible finding, intraoperative assessment of the appendix should be performed in all mucinous ovarian tumors.
Uterine müllerian adenosarcoma: A clinicopathologic study of 31 cases A. Nasfi * , L. Charfi, K. Mrad, S. Sassi, R. Sellami-Dhouib, R. Doghri, M. Driss, I. Abbes, S. Nechi, W. Jomaa, K. Ben Romdhane * Salah Azaiez Institute, Dept. of Pathology, Tunis, Tunisia Objective: Müllerian adenosarcoma (MA) is a distinctive type of uterine tumor, traditionally regarded as a low-grade variant of mixed müllerian tumors. Method: Thirty one cases of adenosarcoma were examined during a period of 19 years (May 1993 -February 2012 . Results: Thirty tumors were of the uterine corpus and one of the cervix. The mean patient age was 54,2 years (range: 16 to 73 year). The main clinical manifestations were vaginal bleeding and pelvic pain. Physical examination showed cervical/vaginal mass or pelvic mass. Treatment was known in 25 cases: Patients underwent hysterectomy with bilateral salpingo-oophorectomy in 17 cases and lymphadenectomy in 6 cases. Tumor size ranged from 2,5 to 12 cm (mean: 7.1 cm). Microscopically, sarcomatous overgrowth was found in 4 cases (13 %), heterologous elements in 5 (16 %). Eleven cases (35 %) had myometrial invasion involving the internal half of the myometrial thickness in 9 cases, and more than 50 % in 3 cases (9 %). The cervical tumor presented as an endocervical polyp without invasion of the cervical wall. Conclusion: Uterine MA are low-grade neoplasms capable of local recurrence and much less commonly distant metastasis. Surgical excision is the main treatment strategy with a good prognosis in the early stage disease. The 2 most important adverse prognostic factors are deep myometrial invasion and sarcomatous overgrowth.
Spectrum of epithelium changes in adenomyosis N. Nizyaeva * , E. Kogan, T. Demura * Scientific Center of Obstetric, Dept. of Pathology, Moscow, Russia
Objective: Adenomyosis (AM) is a very common gynecological disorder. Despite high incidence of the disease epithelial changes precise developmental events leading to the condition remain controversial. The aim of the study was to investigate and compare epithelium morphologicaly and markers expression of proliferation, apoptosis, invasion and neoangigogenesis in AM foci. Method: This study was done on biopsy samples of uterus taken from 70 women with adenomyosis. Immunohistochemical staining of tissues was performed with antibodies to ApoCas, Ki-67, MMP-2, TIMP-1, claudins 3, 5 (CL3,5), E-cadherin, COX-2, EGFR, VEGF. Results: Four variants of epithelium changes were found in AM foci such as proliferative type, hyperplastic type with and without atypia, and atrophic type. times during 3 years and investigated using routine light microscopy and immunohistochemistry (IHC) to control the treatment response. Results: In the course of progestin treatment the complex endometrial hyperplasia with intraepithelial neoplasia became histologically less complicated and than normal. IHC profiles and Ki-67 expression had the same dynamics. In the youngest patient the unusual gland cell atypia with focal calcification was observed one time and hysterectomy was considered. Conclusion: The study confirms that the repeated curettages with conservative treatment and histological studying can be successfully used in some cases of complex endometrial hyperplasia with intraepithelial neoplasia.
Value of Ki67, P16 and CK17 markers in differentiating cervical intraepithelial neoplasia and benign lesions A. Safaei * , M. Pourjabali, F. Sari Aslani, M. Momtahan * Shiraz University of Medical Sciences, Iran
Objective: The cervical cancer is one of the most common cancers among women worldwide. Diagnosis of CIN affected by high rates of discordance among pathologists. Therefore, we need to other adjunct methods for accurate diagnosis of CIN versus benign lesions in equivocal cases. The aim of this study was evaluation of Ki-67 (MIB-1), CK17 and P16 INK 4a (P16) markers by immunohistochemical method in differentiating CIN from benign cervical lesions. Method: Seventy-seven cervical biopsies that originally diagnosed as non-CIN (n = 31) and CIN (n = 46), were reviewed by three pathologists and re-classified as non-CIN (n = 54) and CIN (n = 23), based on agreement between at least two of three, to obtain a consensus diagnosis. Consensus diagnosis was defined as the "Gold Standard". Then immunostaining for Ki67, P16 and CK17 was performed on all cases and their results were compared with original and consensus diagnosis. Results: The overall agreement between original and consensus diagnosis was 67.5 % (Kappa = 0.39, P-value < 0.001). The sensitivity and specificity of Ki67 immunostaining were 95.6 % and 85.1 % and for P16 were 91.3 and 98.1 %, respectively. The sensitivity and specificity of CK17 negative staining for CIN detection were 39.1 % and 40.7 % respectively. Conclusion: We recommended using Ki67 and P16 markers as complementary tests for differentiation between dysplastic and non-dysplastic lesions.
PS-23-038 WT1 expression in ovarian borderline and malignant surface epithelial tumors M. Tahamtan * , F. Sari Aslani, A. Safaee * Shiraz, Iran
Objective: Wilms tumor gene product, a tumor suppressor gene, now is considered to have oncogenic functions. There seems to be differences in WT1 expression among surface epithelial ovarian tumor subtypes. Method: Immunohistochemistry for WT1 was done on 35 serous & 3 mucinous cystadenocarcinomas, 9 borderline serous & 10 borderline mucinous tumors, 7 endometrioid ovarian carcinomas, 3 clear cell carcinomas, 1 malignant Brenner tumor, 2 metastatic adenocarcinomas and 6 endometrial adenocarcinomas. A tumor was considered negative if<1 % of tumor cells were stained. Positive reactions were graded :1+,1-24 %; 2+,25-49 %; 3+,50-74 %; 4+,75-100 %. Results: Of serous cystadenocarcinomaes, 30(85.7 %) were positive,4 showed reactivity of<50 % of the tumor cells and one were negative. All borderline serous tumors were positive. All mucinous tumors, endometrioid carcinomas, clear cell carcinomas, metastatic adenocarcinomas and primary endometrial carcinomas were negative. The single malignant Brenner tumor were positive for WT1. Conclusion: WT1 is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. Degree of expression is not an indicator to separate high grade borderline serous tumors from low grade ones. Objective: FATWO is a rare neoplasm originating from the mesonephric duct remnants that occurs predominantly in the broad ligament but also in the ovary. It is considered a lowmalignant potential tumor. However local recurrences and metastases have been reported. Method: A 45 year-old female, on routine gynecological physical examination, followed by pelvic ultrasonography and computer tomography was diagnosed with a mass at the right ovary. A salpingo-oopherectomy was performed. On gross examination a well-circumscribed, solid and focally cystic tumor measuring 10,5×7×5,5 cm arose in the ovarian hilus. Results: Microscopically the tumor consisted of mediumsized, ovoid to polygonal cells arranged in a solid, tubular and sieve-like pattern. Some cystic spaces were lined by low-cuboidal cells and contained amorphous, eosinophilic material. Cellular atypia and mitoses were rare. The tumor cells were positive for Vimentin, Inhibin-a (focally), CD10, cytokeratins 8/18, 19 and 7 (focally) and negative for cytokeratin 20, epithelial membrane antigen, carcinoembryonic antigen and a-fetoprotein. Conclusion: Histopathological and imunohistochemical findings consistent with an ovarian FATWO. Differential diagnosis includes endometrioid carcinoma, clear-cell carcinoma, Sertoli-Leydig cell tumors (retiform variant) and rete ovarii adenoma. Surgical excision is the optimal treatment. Radiation therapy, chemotherapy or even targeting molecular therapy is questionable. Müllerian adenosarcoma is a rare mixed tumor of low malignant potential. Usually presented as a large endometrial polyp in postmenopausal women. They are associated with tamoxifen or radiation therapy. Microscopy shows a mixture of benign glandular epithelium and low-grade endometrial sarcoma is typically concentrated around the glands. The differential diagnosis is made with the adenofibroma, but now is doubted the existence of this tumor and is considered more of a distinct adenosarcoma. The treatment is total hysterectomy. Sisters of 24 and 16 years with a history of dysmenorrhea. Hysteroscopy was performed to the eldest one, in which multiple polyps are observed and partially resected. The pathological diagnosis was Mixed Mullerian tumor, adenofibroma/adenosarcoma. Total abdominal hysterectomy was performed. Histological examination showed a mixed tumor with glandular component and a low-grade sarcomatous, scarce mitosis, mild atypia and no necrosis. The diagnosis was: mullerian adenosarcoma. A year later, a hysteroscopy and endometrial biopsy was performed to the youngest sister, that showed endometrial polyps with stromal predominance, and no significant atypia. With the diagnosis of mullerian adenosarcoma, and taking into account family history, total hysterectomy was performed. Adenosarcoma is a rare tumor whose family presentation is not described in the literature.
Gliomatosis peritonei is associated with frequent relapse but not affects overall survival in patients with ovarian immature teratoma N.-R. Yoon * * Samsung Medical Center, Dept. of Pathology, Seoul, Republic of Korea Objective: Gliomatosis peritonei (GP) associated with ovarian teratoma has known to have no adverse prognostic effect. We investigated the clinicopathological features of ovarian teratoma associated with GP, and compared immature teratomas (ITs) with GP to ITs without GP. Method: We investigated 16 patients with ovarian teratoma with GP and 27 patients with IT without GP, who were diagnosed at Samsung Medical Center (seoul) from January 1995 to August 2010. Results: Six patients with IT with GP (37.5 % of 16 patients) had recurrence. When IT with GP (n=15) was compared to IT without GP (n=27), patient of IT with GP showed larger tumor size (median, 19 cm vs. median 13 cm) (P<0.001), more frequent relapse (40 %, 6/15 vs. 3.7 %, 1/ 27) (P= 0.005), and frequently elevated pre-operative CA125 level (100 %, 12/12 vs. 50 %, 10/20) (P=0.004). Survival curves showed significantly shorter relapse-free survival in patients of IT with GP (P=0.002). Two-year relapse-free survival rates were 59.3 % and 96.3 % in IT with GP and IT without GP, respectively. However, all patients except one case of IT with GP alive. Conclusion: Ovarian IT with GP was characterized by larger tumor size and frequent elevation of preoperative CA125 level, and GP was associated with frequent relapse in patients with ovarian IT.
Corelation between histopathologic diagnosis with p16 and Ki67 immunostaining, cytologic features in cervical neoplasia C. Altunkaya * , S. Yilmaz, A. Barin, S. Ekici, G. S. Yalcin, M. Caydere, H. Ustun * S.B Ankara E. A.H, Pathology, Turkey Objective: Frequency of cervical carcinoma decreases nowadays since cytologic screening methods have been using extensively. The incidance of cervical intraepithelial neoplasia increases. Cytologic diagnosis og these lesions are substantially important. So we aim to identify the role of p16 and Ki67 immunostainings for predictive factors of cervical neoplasia. Method: The study was conducted January 2007 to January 2012. Ninety-three cases (mean age 43,9; range 22-71 years) diagnosed as squamous intraepithelial lesions were included, Objective: There are several prognostic factors in melanoma. It was suggested that HER-3 expression may influence the tumor behavior. The aim of the study was to investigate the relationship of HER-3 expression with various prognostic factors. Method: HER-3 expression was evaluated in 52 melanomas, 26 without metastases and 19 with lymph node and 7 with distant metastases. Membranous, cytoplasmic and nuclear HER-3 expression was separately analysed. The staining intensity and percentage of positive tumor cells were evaluated. Results: The cytoplasmic staininig was seen in all cases, with average intensity of 2 and percentage of positive cells ranging from 5 % to 90 % (mean 48.8 %). The percentage of cytoplasmic HER-3 positive cells was inversely correlated with Clark and Breslow stage (R=−0.29 and R=−0.43). Cytoplasmic HER-3 reaction was significantly stronger in cases with lymph node metastases (2.3 vs. 1.8 p<0.03). In 22 cases a dot-like cytoplasmic reaction was seen. Membranous positivity was seen in 28 cases (54 %). Conclusion: HER-3 may play a role in melanoma progression. It may be involved in lymphatic dissemination. Further studies of prognostic significance of HER-expression in melanoma are needed.
Hypopigmented mycosis fungoides with unusual vitiligolike presentation in child H. Erdem * , N. Buyukbabani, H. Turan * Duzce University, Dept. of Pathology, Turkey
Objective: Mycosis fungoides (MF) is the cutaneous T cell lymphoma. Classical, clinical and histopathological findings of MF are detected in most of the patients. However, some of the patients have defined atypical MF. hypopigmented MF (HMF) is one of the atypical forms. HMF could be misdiagnosed with clinical and histopathological examination. HMF is considered mistakenly vitiligo. Therefore, it should be considered differential diagnosis. MF is usually seen in the middle aged and elderly. The occurrence of mycosis fungoides in children is very rare. Method: An 7-year-old girl child attended dermatology clinic with complaints of pruritus and hipopigmented patches. Lesions was performed punch biopsy and reported HMF. Conclusion: Herein, this case presented because it was considered vitiligo as clinically and diagnosed HMFas histopathologically.
Reducing block sampling in wide local excisions for melanoma C. Fives * , C.C.B.B. Heffron * Cork University Hospital, Dept. of Histopathology, Ireland Objective: It is established practice that wide local excision (WLE) is undertaken for the further management of cutaneous melanoma, however, definitive guidelines for macroscopic sampling have not been established. Our aim was to determine whether our sampling of WLE specimens was adequate, inadequate or excessive and to establish guidelines for these specimens. Method: 128 cases which underwent initial biopsy and subsequent WLE in 2010 were identified. We recorded the specimen size, macroscopic appearance, number of blocks sampled and margins of the original biopsy. Results: There was wide variation in the number of blocks sampled (range 1-27). Residual melanoma was identified in 7 of the cases (9.4 %) which had clear margins on the original biopsy. Of these, 4 had evidence of a pigmented lesion on macroscopic examination. The remaining 3 cases had margins of 1 mm or less on the original excision. No subsequent surgery was performed on these cases. Conclusion: Our study has shown that in WLE specimens with no evidence of a macroscopic lesion and in which margins of the original biopsy were clear by greater than 1 mm, little is to be gained from extensive sampling. Reduced sampling would result in saving laboratory resources with a predicted 49 % block reduction in our laboratory alone.
Functioning oxyphil adenoma of parathyroid gland: A case report M. Genadieva -Yordanova * , S. Hristova, A. Vlahova, G. Todorov, B. Miserliovska, S. Yordanov * Alexandrovska Hospital, Dept. of Pathology, Sofia, Bulgaria
Objective: The most frequent cause of primary hyperparathyroidism are adenomas of the parathyroid gland, the majority of which are composed of chief cells. Oxyphil adenomas are uncommon and account for 3 % of functioning parathyroid adenomas. Up until 1978 year they have been considered non-functional. Method: Laboratory findings revealed calcium levels of 2.86 prior to operation. The serum parathyroid hormone level was 151.40 pg/ml. A CT scan of the neck showed normal size thyroid lobes with heterodense structure with an oval hypodense lesion in the lower right pole of the thyroid. Results: At surgery a mahagony-brown mass measuring 10 mm in diameter was removed and reported as oxyphil adenoma on frozen section. Histopathological examination revealed encapsulated adenoma composed of oxyphil cells with abundant, granular pink cytoplasm and a rim of normal parathyroid tissue. Mitotic figures were absent. Postoperatively, a decrease of the serum calcium down to 2.31 mmol/l was declared. The followup period was unremarkable. Conclusion: There appear to be a growing evidence that a big part of oxyphil adenomas of parathyroid gland can produce parathyroid hormone and contribute to the cases of primary hyperthyroidism.
Effectiveness of UVB phototherapy on mycosis fungoides using histologic guitart criteria in Iranian patients A. Ghanadan * , A.-H. Ehsani, H. Seirafi, M. Khiabani * Tehran University, Razi Hospital, Iran
Objective: Mycosis fungoides (MF) is the most common primary cutaneous lymphoma. Mycosis fungoides often develops slowly over many years, presenting with a generalized erythroderma, skin patches or plaques. The diagnosis of MF requires the integration of clinical and histopathologic findings. Narrowband UVB (NBUVB) is widely used to treat MF. To evaluate the effectiveness of therapy the histopathologic findings before and after NBUVB reviewed using GUITART criteria. Method: We enrolled 20 patients (12 women, 8 men; age range, 10-80 years; mean age, 45.5 years) with clinically and histologically proven MF. The patients received NBUVB phototherapy three times a week. A biopsy was performed 3 months after onset of the treatment and GUITART criteria used to scoring MF before and after therapy. Results: Phototherapy was reduced the primary intraepidermal atypical lymphocytes (P value=0.008), dermal atypical lymphocytes (P value=0.008), epidermotropism (P value= 0.001), density of infiltration (P Value=0.002) and lymphocytic infiltrate without inflammatory features (P Value = 0.046) in all patient. But the relationship between the reticular fibroplasia of papillary dermis and phototherapy was not proved (P value=0.18). Overall score of patients was significantly lower after phototherapy (P value=0.000). Conclusion: Our data suggest that NBUVB therapy is reduces histiologic score of MF and is effective for the treatment of Iranian patients.
Nodular colloid degeneration of the skin: Report of three cases A. Ghanadan * , K. Kamyab-Hesari, M. Daneshpajouh, K. Balighi, M. Khosravi * Tehran University, Razi Hospital, Iran
Objective: Nodular colloid degeneration (NCD) is a rare dermatological disorder and also a rare type of colloid milium. The degeneration may be related to sun exposure.
Method: Three cases of NCD were enrolled from the archive of dermatopathology department of Razi hospital during 2009-2011. Results: In this report, three cases, all presented with multiple plaques and nodules in the nose and the face, are depicted. Histologically, these nodular masses were homogeneous, with eosinophilic clefted materials expanding the papillary dermis and extending into the deep dermis. Histochemical review showed the reactivity of the colloid materials via the PAS, crystal violet and methyl violet staining. All the three cases were finally diagnosed as nodular colloid degeneration. Conclusion: NCD is a rare disease but it should be considered in any cases with a history of long exposure to the light. We suggest the long term exposure to the sun as an etiologic factor thus, sun protection would be the most preventive and available treatment.
Glypican-3 protein expression in melanoma: A immunocytochemistry study N. Gursan * , H. Balta, B. Gundogdu * Ataturk University, Medical Faculty, Erzurum, Turkey Objective: Glypican-3 (GPC3) is a cell surface heparan sulfate proteoglycan. Serum GPC3 was shown to be expressed in 40 % of melanomas (Ms) but GPC3 expression in Melanoma tissues had not been investigated.In this study, immunohistochemical analysis of GPC3 protein expression was investigated in histologic sections from Melanoma tissues. Method: 60 melanoma patients, twenty patients with insitu melanoma, twenty patients stage 0 and stage I melanoma, twenty patients stage II and stage III melanoma. All cases were stained with anti-GPC3 antibody. GPC3 expression was divided into 2 categories: negative (negative or weak cytoplasmic staining) and positive (moderate or strong cytoplasmic with membranous accentuation). Results: GPC3 immunopositivity. showed in 40,3 % of melanomas. GPC3 expression at stage 0, I, II, III were 44.4 %, 40.0 %, 41.6 %, 35 % respectively. Method: We report the case of a 77 year old man presenting with an erythematous plaque of the right hemithorax. Results: Macroscopically the lesion had irregular borders and was mildly infiltrative with a violet hue. Microscopic examination revealed tumor cells within lymphatic spaces, without infiltration of the adjacent stroma. The histologic and immunophenotypic characteristics of these cells were compatible with a carcinoma of lung origin. CT scan revealed a lung mass as well as multiple liver and chest wall metastases. Conclusion: Malignancies originating from the breasts, lungs and large bowl are the most common to involve the lymphatic net of the skin. Melanoma on the other hand is more frequently presenting with lymphangitic invasion, whereas inflammatory carcinomas may also affect lymphatic vessels and may be confused with erysipelas. The revelation of the primary origin is not always easy and differential diagnosis should also include gynecological malignancies, kidney and urinary bladder carcinomas. In our patient the diagnosis was established on the grounds of histology and immunohistochemistry, and was supported by the clinical and radiological findings. Objective: Inverted follicular keratosis (IFK) is almost always a solitary lesion occuring mainly in adult life. Men are affected twice as often as women. Pathologically, They can be confused with a variety of lesions, both benign and malignant. Squamous carcinoma is the most serious differential diagnosis. Method: We decribe a retrospective study about 7 cases of IFK diagnosed over a 18-year-period (1994-2012) . Results: We collected 7 lesions, all treated by surgical excision. Haemalum eosin sections were studied in all cases. All lesions were single. Five, of them, were situated on the face and two on the scalp. All the patients were adults with a mean age of 34 years, average between 19 and 66 years. They presented mostly as asymptomatic papules and all were small lesions. The different sections of the lesion showed skin well delineated endophytic epithelial proliferations with inverted papillomatous and acanthotic components containing several circumscribed squamous eddies. They was no atypia, mitotic activity necrosis or stromal invasion. Conclusion: IFK pose very real diagnostic problems unless one is aware of this entity. In fact, they may mimic malignant lesions especially squamous cell carcinoma, both clinically and pathologically.
Primary cutaneous follicular lymphoma with prominent spindle cell areas. So called spindle cell follicular lymphoma. Case report G. Ivády -Szabó * , T. Strausz, E. Tóth * National Institute of Oncology, Dept. of Surgical Oncology, Budapest, Hungary
Objective: Spindle cell type variant of cutaneous follicular lymphoma is a rare histologic variant of primary cutaneous follicular lymphomas. It is caracterised by the presence of spindle and bizarr cells. We present a case of a 25 years old man who had a nodule on his scalp and two smaller nodules on his frontal region. We examined an excisional biopsy specimen of the forehead. Method: HE, Stains all and Immunohistochemical stains were used. Results: Histology revealed dense lymphoid infiltrate showing nodular pattern in the dermis and in the subcutis. The lymphoid infiltrate predominantly composed of large centrocytes. There were areas where the neoplastic cells show spindle cell morphology with bizarre nuclei and in these areas the stroma were myxoid, mucinous. With immunohistocemical stains the neoplastic cells were positive for CD20, Bcl-6, and negative for CD5, CD10, vimentin, CD34, S100, actin, desmin, AE1/AE3. CD21 showed residual network of follicular dendritic cells in the background. Conclusion: Spindle cell lymphoma is a very rare variant of cutaneous follicle centre cell lymphoma and the presence of the spindled bizarre cells can cause differential diagnostic problems. The main differential diagnostic entities are primary or metastatic spindle cell sarcoma and spindle cell melanoma. Careful morphological and immunohistochemical analysis are required to the correct diagnosis.
The histopathologic and immunohistochemical features of mycosis fungoides B. A. Karabork * , A. Okcu Heper, S. Yuksel, I. Kuzu * Ankara University of Medicine, Dept. of Pathology, Turkey
Objective: The histopathologic diagnosis of early mycosis fungoides (MF) is often difficult. The lesions can mimic a variety of inflamatory dermatitis. We aimed to establish and draw attention to the most frequent histopathologic and immunohistochemical features of MF. Method: We reviewed 115 skin biopsies of clinicopathologically diagnosed MF cases at Medical University of Ankara. We looked for a) epidermotropism, atypical lymphocytes, morphologic features in the epidermis, dermoepidermal junction and dermis b) immunohistochemical staining ratios of CD3, CD4, CD8, CD20, CD30 and expression loss of CD5, CD7. Results: Atypical lymphocytes) (96 %), dermal fibrosis (90 %), epidermotropism (86 % single cells, 44 % linear arrangement, 15 % Pautrier's microabscesses, 83 % 'haloed' lymphocytes), epidermal acantosis (72 %), basal vacuolar degeneration (focally 43 %, marked 15 %) and a perivascular lymphocytic infiltrate were the most common and important features. Dermal edema (46 %) extravasated erythrocytes (34 %), spongiosis (27 %), eosinophils (17 %) and necrotic keratinocytes (14 %) were the less seen and non-specific ones. Immunohistochemical results correlated with a ratio of 78 % CD4, 22 % CD8 positive MF. Expression loss of CD7 was seen in 73 %, CD5 in 31 % of cases. Conclusion: Diagnosis of early MF requires the correlation of morphologic, immunohistochemical and clinical features. Also due to the disease's heterogenity, biopsies from different locations and rebiopsies will enhance the diagnosis.
Connexins of cutaneous melanocytic tumours G. Kiszner * , Z. Buday, I. Teleki, E. Varga, I. B. Nemeth, I. Korom, T. Krenacs * Semmelweis University, 1st Dept. of Pathology, Budapest, Hungary
Objective: Connexins (Cx) form transmembrane channels that can transport ions and small regulatory molecules between adjacent cells. They also function as hemichannels and through protein interactions and are involved in the control of cell replication and maintenance of multicellular homeostasis. Method: We have tested the expression of connexins in melanocytic tumours using immunohistochemistry in tissue microarrays of 21 common and 73 dysplastic nevi, and 61 primary and 23 metastatic malignant melanomas. Results: Cx23 was not found in melanocytic tumours despite expressed in the basal epidermis. Cx30.3 reaction showed punctate cell membrane staining in 71 % of naevi in their superficial regions including atypical nests, and displayed cytoplasmic staining in 28 % of melanomas. Low levels of Cx32 were revealed in the cytoplasm of >80 % of naevi and melanomas but only 23 % and 8 % showed membranous positivity, respectively. Cx36 perinuclear/cytoplasmic immunostaining was observed in 57 % of naevi and 24 % of melanomas and as cell membrane reaction in 24 % and 12 %, respectively. Punctate Cx43 reaction was detected in vertical tumour nests in 69 % of naevi, while only 11 % of melanomas proved positive and showed cytoplasmic Cx43 delocalization.
Conclusion: Therefore, most tested connexins were significantly down-regulated in malignant vs. benign melanocytic tumours that possibly contribute to their malignant phenotype.
Quantitative follow up study of CD 1a, CD 8 and CD 68 positive cells in multiple basal cell carcinoma cases after combined treatment R. Kleina * , I. Truksane, J. Kisis, I. Franckevica * Riga Stradins University, Dept. of Pathology, Latvia
Objective: In last years appear more cases with multiple basal cell carcinomas (BCC). Skin immune system reaction is especially important in nonsurgically treated cases. Aim of study is to evaluate the dynamics of CD 1a, CD 68, CD8 marked cells in BCC and in skin adjacent to tumour (5 and 10 mm) before and after the treatment with cryotherapy and imiquimodum. Method: From 68 BCC cases with multiple tumours investigated immunohistochemically, in dynamics we have characterized 8 patients. Antibodies for CD1a, CD8, CD68+ cells in derma and epidermis were used. They were evaluated in 3 fields of vision (400×) before and after treatment. 5 % imiquimod cream and double freezing was used. Control group: three normal skin samples. Statistical analyses of results were done. Results: After treatment were erythema, then crust and exfoliation. Microscopically expressed fibrosis instead BCC was found. Amount of immune cells before and after treatment were: CD8 25±3,1/34±4,8 (also in epidermis), Langerhans cells 7±2,3/14±3,4, CD 68 19±3,8/10±2,9. Conclusion: All immune cells of skin react to the combined treatment of BCC in radius of 5 mm. There is significant statistical difference between the numbers of Langerhans cells in normal epidermis and in BCC cases treated with cryomethod and imiquimodum.
Regulatory T-cells in invasive and in situ squamous cell carcinoma of the skin and actinic keratosis H. Kourea * , A. Stravodimou, V. Tzelepi, H. Papaioannou, H. Papadaki, C. Scopa * University of Patras, Dept. of Pathology, Greece
Objective: Regulatory Τ-cells (Τregs) participate in tumor tolerance and facilitate tumor growth and Foxp3 transcription factor is necessary and sufficient for their development and function. We investigated the presence of T-regs in invasive (IN) and in situ (IS) squamous cell carcinoma (SCC) of the skin and actinic keratosis (AK). Method: Tregs were identified using immunohistochemistry for Foxp3 and recorded using image analysis in 55 cases of INSCC (and their adjacent IS, AK or benign tissue (BN), when present), in 18 cases of IS and 46 cases of AK (and their adjacent BN tissue). Statistical analysis was performed using the paired T-test. p-values <0.05 were considered statistically significant. Results: In INSCC cases, Τregs in the tumor were more numerous than the adjacent ΒN (n=46) (mean 155 vs. 79, p <0,001). Additionally, adjacent IS had more Tregs than adjacent AK (n=6) (160 vs. 86, p=0,045). Significant differences between INSCC, and IS or AK were not observed. In IS and AK cases, Τregs were more numerous than the adjacent BN (206 vs. 42, p<0,001 and 148 vs. 47, p< 0,001), respectively. Conclusion: Treg infiltration of the skin increases early from the precancerous AK, indicating early involvement of Tregs in the development of SCC. Objective: Mastocytosis is a rare disorder and its true incidence is unknown. Skin is most commonly involved, followed by the bones and the gastrointestinal tract. Method: Five men presented with many brown lesions in the trunk and one woman with multiple redish brown nonpruritic macules in the extremities. We used immunohistochemistry for c-kit and tryptase for the definite diagnosis. The expression of CD25 and CD2 were also evaluated. Results: Histology showed five cases of urticaria pigmentosa with severe diffuse infiltration of the papillary dermis by mast cells and one case of telangiectasia macularis eruptiva perstans (TMEP) with scattered mast cells around dilated capillaries and venules of the papillary dermis. The mast cells were positive to c-kit and tryptase and only in one case there was a coexpression of CD25 and CD2 >50 % of the mast cells. In that case involvement of bone marrow was observed and the diagnosis of indolent systemic mastocytosis was established according to WHO 2008 criteria. Conclusion: Urticaria pigmentosa is the most common form seen in adults while TMEP is an uncommon form that occurs exclusively in adults. The skin involvement in indolent systemic mastocytosis is a part of the spectrum of the disease.
PS-24-025 Subvisual nuclear characteristics are different between keratoacanthoma and keratoacanthoma-like squamous cell carcinoma K. Metze * , M. Tabai, R. Adam, I. Watanabe, A. de Moraes, M. Cintra * University of Campinas, Pathology, Brazil Objective: The differential diagnosis between keratoacanthorna and keratoacantoma-like squamous cell carcinoma may be extremely diffcult. Our objective was to study whether computerized image analysis could be helpful. Method: In 34 patients biopsies of keratoacantoma-like lesions were taken at admission. One month later surgical excision was performed in growing lesions, whereas regressing lesions were left untreated. A final diagnosis was established combining clinical and histological evaluation, Digitalized images from KI-67 immunostained and hematoxylin counterstained sections of first biopsies were obtained. Tumor nuclei were marked interactively. An inhouse computer program analyzed the geometric relations between the nuclei. Nuclear gray values and their histogram entropy were calculated. Results: 27 keratoacnthomas and 7 keratoacantomalike squamous cell carcinomas entered this investigation. Basic variables of the geometric analysis did not differ between the two entities regardless whether all nuclei or only the Ki67 positive ones had been examined. Chromatin gray values were significantly lower and their histogram entropy higher in the keratoacantoma-like squamous cell carcinomas. Conclusion: Basic geometric variables do not seem to be different beteen both lesions, but a shift in the gray value histogram to lower values with increased entropy in carcinomas indicated important differences of the chromatin structure, Supported by FAPESP and CNPq.
Non-infectious erythematous papular and squamous lesions of the skin in our institute, with clinicopathologic correlation T. Ozgur * , A. C. Dogramaci, E. Atik, S. Hakverdi, M. Yaldiz, Z. A. Tas * Mustafa Kemal University, Medical Faculty, Antakya, Turkey
Objective: Non-infectious erythematous, papular and squamous lesions of the skin are basic lesions that pathologists differentiate in routine laboratory examinations. Our aim has been to analyse these lesions by pathologic and clinical findings in our institute with determining clinicopathologic correlation. Method: In our study 420 cases prediagnosed as erythematous, papular and squamous lesion by dermatologists and evaluated in pathology laboratory between 2004 and 2010 have been reviewed. Results: The lesions comprised %14.3 of the total load of surgical pathology and 9.1 % of total number of skin biopsies. The highest percentage was in the 41-50 year age group (39.1 %) with a female predominance of 51.2 %. The limbs were most frequently involved (36.9 %). Psoriatic lesions were the commonest (50.7 %), classic generalized plaque variant psoriasis (89 %) being the most frequent. Correlation with the histopathologic diagnosis was positive in 75.3 % cases and negative in 24.7 % cases.
Conclusion: The contribution of histopathology to the final diagnosis was significant. It confirmed the diagnosis in 75.3 % cases and gave the diagnosis in 7.3 % cases.
PS-24-027 Vulvar lichen sclerosus: A misnomer for an entity with decreased fibrillar components and increased amorphous components in extracellular matrix remodeling E. Parra * , C. A. Pires de Godoy, V. L. Correia Feitosa, W. Teodoro, A. P. Velosa, V. L. Capelozzi * FMUSP, Dept. of Pathology, São Paulo, Brazil
Objective: The hyalinization of subepidermal skin is one of the histopathological characteristics of the vulvar lichen sclerosus (VLS). It was found that patients with VLS present autoantibodies against the extracellular matrix protein 1 (ECM-1) and the deficiency of this protein is responsible for the development of a different disease, the Lipoid Proteinosis. This disease shows a similar histology with VLS and has better characterized morphology. Method: We analyzed 20 VLS patients biopsies and the control group was composed by 20 vulva samples from authopsy. The biopsies and control samples were analysed by immunofluorescence for collagen I, III and V and th total collagen fibers by Picrosirius staining. The elastic fibers were stained with Verhoeff and the proteoglycans and glycosaminoglycans with Periodic Acid-Schiff and Alcian Blue. Collagen quantification was performed through image analysis.
Results: It was observed a significant reduction in all studied collagens as well as in the elastic fibers. On the other hand, the proteoglycans were increased in the VLS biopsies. Conclusion: This study did not found an increase in collagen sclerosis that would justify the term used for the LS. It was observed a predominance of the edema area probably caused by the increase of glycoproteins as in lipoid proteinosis.
Combined high-grade basosquamous carcinoma and malignant melanoma of the scalp (malignant basomelanocytic tumor) metastasizing to the breast. A case report A. Pitino * , S. Squillaci, C. Spairani, F. Ottelli Zoletti, M. F. Cosimi, M. Ferrari, A. Tropiano, P. C. Rassu, F. Tuo, P. Maiocchi * San Giacomo Hospital, Division of Anatomic Pathology, Novi Ligure, Italy Objective: Background. Basal cell carcinoma (BCC) is a very low grade, usually not metastasizing skin malignancy, which needs to be radically excised.
Methods. An 80 year-old woman was treated for a 3×2,2 cm multinodular mass in the scalp. One month later an ultrasonography of the left breast showed a 5,5 cm mass which was excised with clean margins. Axillary lymph nodes were free of metastasis. Results: Results. The dermal tumor presented well demarcated basaloid epithelial nests with squamous differentiation focally connected with the epidermis. They showed peripheral palisading and high nuclear grade with pleomorphism, brisk mitotic activity and multiple prominent nucleoli. Atypical cells, arranged as strips, nests or isolated elements, were observed at the tumor edge, separated by a grey-zone from the overlying malpighian epithelium. Immunostains were positive for cytokeratins, melanocytic (S-100/HMB-45/ MART-1) and neuroendocrine (CD56/NSE/Cromogranin) markers. The 4,8 cm breast tumor corresponded to a high grade metastatic BCC with reactivity for epithelial markers, S-100 and CD56. Objective: Pyoderma gangrenosum (PG) was described at first by Brocq and named by Brunsting et al. in 1930. Superficial granulomatous pyoderma (SGP) was described as a variant of PG in 1988. We present three cases of this rare variant. Method: There were two women and one man with age ranging from 40 to 78 years. All of them had a long history of a slowly enlarging, eritematous and ulcerative plaque in different areas including leg and breast skin. A biopsy was performed in all cases. Results: The superficial dermis showed neutrophilic inflammation with an admixture of granulomatous inflammation and sinus formation. There wasn't vasculitis and fat tissue was not affected. Conclusion: SGP is a rare variant of PG. Diagnosis of this entity generally is made on the basis of skin biopsy results with all features mentioned previously and a typical clinical appearance. Most of times, these lesions are diagnosed as granulomatous dermatitis, thinking in infectious pathology. This is the most important differential diagnosis, because SGP is responsive to corticosteroids. The pathogenesis of PG is unknown, though is now believed to altered neutrophil chemotaxis and some authors suggest that SGP may be a delayed-type hypersensitivity to an unknown antigen. Pathergy is the inciting factor in several patients.
High concordance in BRAF status between native-BRAF malignant melanoma and matched lymph node metastases A. Santos Briz * , E. Godoy, L. Arango, P. Antúnez, M. Yuste, C. Roman, E. Fernández, M. D. Ludena * Hospital Universitario Salamanca, Dept. de Anatomía Patológica, Spain
Objective: The discovery of selective v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600 mutation as an oncogenic mutation in cutaneous malignant melanoma (MM) has changed the treatment paradigm for melanoma. Selective BRAF inhibitors have demonstrated response rates far higher than standard chemotherapeutic options. BRAF mutation analysis is usually performed on primary tumour tissue; however, no conclusive data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of BRAF mutation status in a study of 16 primary BRAFnative tumours and their corresponding lymph node metastases. Method: 16 patients with histologically confirmed nonmutated BRAF MM who underwent surgical resection of the primary tumour and positive selective lymph node biopsy or lymphadenectomy were included. Mutation status was determined by means of a realtime PCR assay (Cobas 4800 BRAF V600 Mutation Test, Roche Molecular Systems). Results: There was a 100 % concordance of the BRAF results in primary tumor and corresponding metastases in 16 analyzed pairs. Conclusion: Our findings show total concordance in BRAF status between primary BRAF native tumors and their paired metastases, and suggest that acquisition of BRAF V600 mutation in metastases from primary native tumors is a rare event.
Dermatoscopic and histologic score correlation in atypical Spitz/Reed nevi M. Saravia * , Z. Pellicer, J. M. Martin, C. Monteagudo * Hospital Clinic University Valencia, Dept. of Pathology, Spain
Objective: Different dermatoscopic scores of pigmented skin lesions have provided a valuable tool in daily routine practice to differentiate malignant from benign lesions. However, these semiquatitative methods have a high rate of false positives in Spitz/Reed nevi. We have compared semiquantitative histopathologic and dermatoscopic findings in a series of Spitz/Reed nevi. Method: We collected 19 cases (15 female and 4 men) of atypical Spitz and/or Reed nevi. All cases were microscopically confirmed. A histologic index (HI) was constructed scoring the following findings: symmetry, sharp demarcation, architectural dysplasia, melanocytic atipia and nest size, and compared with validated dermatoscopic scores (ABCD score, 7-point checklist and Menzies score). Distributions of these scores for the different dermatoscopic patterns were also analyzed. Results: Median age at presentation was 15 years (range 1 to 36). The HI varied from 0 to 5 points. We found a strong positive correlation between the HI and Menzies score (r=0,539, p=0,01). In addition, HI values differed significantly when the multicomponent dermatoscopic pattern group was compared with the other patterns (U =22, 5 Z =−2,25, p =0.01). Conclusion: Menzies is the only dermatoscopic score which strongly correlates with histologic findings in Spitz/Reed nevi and may be useful in the diagnosis of atypical lesions.
Correlation of nonmolecular and molecular subtyping of inherited epidermolysis bullosa K. Veselý * , H. Bucková, L. Fajkusová, M. Veselá, B. Jerábková * St. Anne's Hospital, Dept. of Pathology, Brno, Czech Republic Objective: Inherited epidermolysis bullosa (EB) is a heterogeneous group of hereditary mechanobullous diseases with skin blister formation of variable severity and numerous extracutaneous manifestations. EB comprises three main groups and many subtypes and their recognition is important for prognosis and genetic counselling. Method: We examined skin biopsy samples of 42 patients with EB using transmission electron microscopy, immunofluorescence antingen mapping (AM) and their peripheral blood samples by mutational analysis. Results: In the total number of 42 cases, concordant results of molecular and nonmolecular methods were found in 35 cases (83 %). In 7 patients (17 %) mutational analysis did not confirm the results of ultrastructural and AM analysis. Conclusion: Strong correlation in the results obtained by different methods was observed. Using combination of nonmolecular and molecular diagnostics methods high diagnostic accuracy can be achieved.
Granulomatous Slack Skin (GSS): A case report with evidence of T cell clonality I. Yilmaz * , H. Baloglu, U. Berber, Z. Kucukodaci, M. Arcila * GATA HEH, Dept. of Pathology, Istanbul, Turkey
Objective: GSS is a very rare variant of MF. We describe an additional case of this rare disorder in a 42 yo female that has been approved by TCRB&G Tcell Clonality Assay. Method: A 42 yo female with a single bulky skin lesion in the inguinal fold which had first appeared 6 y before and enlarged up to 20 cm, referred to plastic surgery for esthetics. H&E section of the deep skin biopsy of lesion showed a granulomatous infiltrate in the dermis. Nuclear atypia and epidermotropism is not significant. But there is numerous multinucleated histiocytic giant cells. Elastophagocytosis are present. TCRB&GT cell Clonality Assay showed a clonal rearrangement in TCR gamma gene. She was referred to dermatology for treatment and now she is getting UVA1 phototherapy. Results: The diagnosis of GSS is difficult in early stages of the disease. Clonal rearrangement of TCR genes can be a useful diagnostic tool in early stages of the disease. Conclusion: 20-50 % of patients with GSS carry a risk for the development of a second malignancy. GSS may be associated with lymphoproliferative disorders. Therefore, follow up of the patient is important. This case report supports that GSS is an indolent variant of MF due to clinical, histological and T-cell gene rearrangement results.
Unusual occurrence of multicentric reticulohistiocytosis with systemic involvement in childhood S. Zurac * , C. Dobrea, A. Diaconeasa, A. Colita, C. Solovan, C. Arion, F. Staniceanu * Colentina University Hospital, Dept. of Pathology, Bucharest, Romania
Objective: Multicentric reticulohistiocytosis (MR) with systemic involvement is an extremely rare disease afflicting mainly adults. Method: We report a case of SR arising in a 3 year 7 month old boy with concomitant celiac disease. Results: Since 6-7 months, he developed several brown to dark-red skin nodules (up to 0.8 cm) located mainly on face and arms; current episode include hemorrhagic syndrome, severe pancytopenia and massive hepato-splenomegaly. Bone marrow aspirate ruled out acute leukemia and tezaurismoses; bone marrow biopsy and skin biopsy showed important infiltration with numerous large cells with abundant fine granular/ground glass eosinophilic cytoplasms in a reactive inflammatory stroma (lymphocytes, few plasma cells and few eosinophils); bone marrow cells were mononucleated, cutaneous cells were both mono&multinucleated; tumor cells immunophenotype was consistent with histiocytes (CD68+, S-100 protein + (faint), CD1a-, langerin-) both in bone marrow and skin biopsies; no hemophagocytosis was seen. MR with systemic involvement of skin, bone marrow, liver and spleen was diagnosed. Cyclophosphamide therapy was instituted with initial diminishing of splenomegaly; latter on, despite adding cyclosporine and etoposide, no further impact on pancytopenia and hepatosplenomegaly was recorded. Conclusion: Complete hematologic examination is mandatory for patients diagnosed with cutaneous reticulohistiocitosis, irrespective of their age, in order to identify MR and institute proper treatment.
Primary cutaneous follicle centre lymphoma: A case report and review of literature K. Diamantopoulou * , C. Zorzos, C. Eftychiadis, I. Famellos, G. Piagkos, G. Karamanis, I. Babalis, S. Binder, H. Mahera * General Hospital Kat, Pathology, Athens, Greece
Objective: Primary cutaneous follicle centre lymphoma (PCFCL) is an indolent, primary cutaneous B-lymphoma with an excellent prognosis but a high incidence of reccurence. Apart from standard surgery and local radiotherapy, targeted strategies with anti-CD20 (rituximab) has introduced new treatment modalities. Method: Woman, aged 82, with solitary skin lesion on her face. After surgical excision, histo-and immunohistochemistry with the ABC system on paraffin embedded tissue was performed. Results: Microscopically, a non-epidermotropic perivascular and periadnexal neoplastic infiltrate with a follicular growth pattern was observed. The lesion consisted of predominantly medium-sized centrocytes and several centroblasts enmeshed in a network of CD23(+) dendritic cells. Immunohistochemically, the neoplastic lymphoid cells were CD20, CD79a, BCL2 and BCL6 positive. CD30, MUM1 and Ig were all negative. The patient received one cycle of rituximab and 6 months later is free of disease. Conclusion: Primary cutaneous B-cell lymphomas (CBCL) account for 25 % of cutaneous lymphomas. PCFCL is one of the three major categories of CBCL, recently classified by WHO, (the other two: PCMZL and PCDLBCL, leg-type). BCL-2, except for systemic follicular lymphoma, it can also occur in PCFCL, as in our case. PCFCL, usually locally treated with surgery and radiotherapy, could possibly offer a candidate for anti-CD20 targeted therapy, especially in multifocal lesions. Objective: Thirteen and ten years old male patients admitted with 4 months interval, having diffuse hemorrhagic papules on extremities and trunk. The first has not responded to methorexate previously. The second had varicella infection 3 months before and, his rushes spreaded out recently. Method: Multiple punch biopsies of the first patient revealed epidermal necrosis, basal vacuolisation and subepidermal diffuse infiltrate composed of CD3 and mostly CD8 positive T cells showing a striking angiocentric distribution. There were CD30 positive large cells with vesicular nuclei both in epidermis and dermis. Some of the vessels exhibited lymphocytic vasculitis with fibrinoid necrosis. Punch biopsy of the second patient revealed subcorneal pustule, dyskeratosis and basal vacuolisation, papillary dermal edema, erythrocyte extravasation and severe lymphocyte-rich cellular reaction infiltrating the interface. Lymphocytes were CD3 and mostly CD8 positive. In between, there were large cells with vesicular nuclei exhibiting CD30 positivity. Results: Diagnoses were PLEVA based on the vascular and interface changes in accordance with clinical findings. Both patients responded to oral antibiotics. Conclusion: PLEVA is among the reactive conditions that can simulate CD30 positive lymphoproliferative diseases. Although CD8 positive lymphomatoid papulosis is defined in pediatric patients, no response was seen to oral antibiotics.
Are HMB-45 and MIB1 results reliable for safe diagnosis of nevi? Z. Yusifli * , O. Kurtulan, Ç. Aydin, M. Bugdayci, Ö. Gököz * Hacettepe University, Pathology, Ankara, Turkey
Objective: Melanocytic lesions having subtle features suspicious for malignancy create considerable difficulty in decision-making.process. Lack of deep HMB-45 positivity and low dermal MIB-1 index can be reliable tools in association with morphology. Method: Twenty-seven acquired melanocytic lesions in which HMB-45 and MIB-1 have been performed are reviewed to determine the importance of these markers in routine diagnostic practice. Asymmetry, focal loss of maturation, presence of mitoses, epidermal consumption and lymphocytic inflammation were the morphological features that have created difficulty especially when seen alone in a bland appearing nevus.
Results: All cases with epidermal consumption and/or mitoses and nearly half of cases with inflammation, asymmetry and focal loss of maturation was associated with.focal deep dermal HMB-45 positivity. Thirteen cases had an MIB-1 index of ≤1 %. Two of 3 cases with MIB-1 index of >1 % had also HMB-45 positivity and/or inflammation. The case with a MIB-1 index of 8 % was an acral nevus bearing 4 of the evaluated morphological features. Conclusion: Focal deep dermal HMB-45 positivity goes parallel with epidermal consumption and dermal mitoses, mostly used prevailing criteria of malignancy in melanocytic lesions but it's expression is not adequate for diagnosis of melanoma. Low MIB1 index can be a reliable marker of benignity. Objective: Leptospirosis in humans usually courses with hypokalemia and hypomagnesemia, and the putative mechanism may be related to nitric oxide production. Methylene blue is a known inhibitor of inducible nitric oxide synthase, and have beneficial effects on clinical and experimental sepsis. Method: Serum creatinine and ionic changes were evaluated at various time points (4, 8, 16 and 28 days) in hamsters. We also determined the effect of methylene-blue treatment when used as adjuvant therapy combined with a late start of standard antibiotic (ampicillin) treatment. Results: Rather than K and Mg depletion, hyperkalemia and hypermagnesemia were observed during acute infection. These findings are probably associated with an accelerated progression to renal failure, since this model is not feasible to mirror the supportive therapy (including fluid expansion) that retards progression to the oliguric/hyperkalemic state. Infected hamsters at day 8 presented diffuse tubular cell swelling with mild or no nephritis. At days 16 and 28 they showed variable degrees of acute tubular changes, regeneration of tubular epithelia and nephritis. Survival and renal pathology did not differ among different treatment groups. Conclusion: Adjuvant methylene blue had no effect on the survival, renal pathology or Mg and K serum levels during acute-phase leptospirosis in hamsters.
Occupation of the striated muscle fibre by Trichinella spiralis is associated with increased intracellular sialylation P. Babal * , R. Milcheva, D. Ivanov, S. Petkova * Comenius University, Dept. of Pathology, Bratislava, Slovakia
Objective: The knowledge about glycoproteome in skeletal muscle is limited and most of the information come from studies on aberrant glycosylation in inherited muscle diseases. This work describes the intracellular changes in sialylation of skeletal muscle fiber during the process of its transformation into a nurse cell after occupation by the nematode Trichinella spiralis. Results: The study was performed at defined time points post infection. Lectin histochemistry with TML, MAL and SNA detected increased production of sialylated glycoproteins within the affected fibers, and it was evaluated by acidic ninhydrin reaction in muscle homogenates. Increase of total sialyltransferase activity was estimated by measurement of incorporated CMP-N-[14 C]-acetylneuraminic acid, and immunohistochemistry showed higher expression of α-2,3-sialyltransferases II and IV within the affected fibers. SNA lectin affino-blots showed at least four protein bands with approximate molecular weight between 126 and 159 kDa, which were more reactive to SNA compared to their counterparts from the control samples. Conclusion: It is evident that skeletal muscle injury induced by Trichinella activates biosynthesis of glycoproteins bearing sialic acids, which are not present in the normal muscle fiber. However, the protein identity, function and the biological significance of these sialoglycoproteins remain to be elucidated. BG051PO001 Objective: Protozoal and helminthic infestations of the central nervous system (CNS) are rare and their incidence is less than 1 %. They are more common among children, the elderly and immunocompromised individuals and tend to follow a fatal course. Method: We report 2 cases of cerebral amebic abscess occurring in 2 immunocompetent men. Results: They were 2 men aged of 43 and 18 years old. Headache; altered mental status and fever were common presenting symptoms. In the 2 cases the diagnosis of cerebral abscess was suspected on cerebral CT scan. Histopathologic analysis revealed extensive areas of necrosis and hemorrhage with granulomatous lymphoplasmacytic inflammatory infiltrate. other conditions such as malignancy and tuberculosis. It occurs normally in the mouth and tonsils, however, it can sometimes occur in the chest, abdomen, pelvis or other areas of the body. Method: We describe a retrospective study about 56 cases of actinomycosis diagnosed over a 21-year-period (1990-2011) . Results: The 56 cases were divided in 20 cervicofacial actinomycosis, 18 genito-urinary tract actinomycosis, 8 cutaneous actinomycosis, 7 abdominal actinomycosis and 3 cases occur in central nervous system. All patients were symptomatic. The most frequent symptom consisted in fever. The diagnosis was based on histologic study in all cases. Tissue histologic studies show suppurative and granulomatous inflammatory changes, connective proliferation, in which bacteria form typical granular colonies composed of radiating gram positive filaments. Conclusion: Diagnosis of actinomycosis is usually made retrospectively by means of histologic examination of surgically obtained specimens, but rarely preoperatively. It is generally treated with long term antibiotics.
Expression of angiogenesis factors in placentas with intrauterine transmission of HIV A. Kolobov * , E. Musatova, V. Karev, V. Zinserling, D. Niauri * St. Petersburg, Russia
Objective: Placenta plays an important role in the prevention of the mother-to-child transmission of HIV during pregnancy. The purpose of this investigation was to study expression CD31, bFGF and TGFβ in human placentas in cases of intrauterine HIV infection. Method: Group A -11 placentas from women and children with HIV infection. In control -Group B, were 16 placentas from women without HIV infection. On paraffin slices expression of CD31, bFGF, TGFβ and p24 was evaluated. We counted the area of positive cells related to the square of the slice. Results: In all placentas with HIV infection p24 has been found in placental macrophages and villi's vessels. In group A expression of CD31 in endothelial cells was 4,22±0,85 % of the slice's square (in control -3,69± 0,53 %) (p>0,05). Expression of bFGF in villi's stroma was 5,49±1,48 % (in control -4,01±0,6 %) (p>0,05). Expression of TGFβ in villi's vessels was 11,2±3,6 % (in control -2,71±0,63 %) (p<0,05). Conclusion: Thus, placentas in cases with intrauterine transmission of HIV are characterized by increased expression of TGFβ the significance of this fact is still to be evaluated. Objective: Unilocular cystic echinococcosis which Echinococcus granulosis causes is seen frequently in the animal husbandry practised underdeveloped countries. Method: The 1709 unilocular cystic echinococcosis cases which were seen in the eastern part of Turkey, in 30 years between 01.01.1981 and 01.01.2010, except Faculty of Medicine of Atatürk University are presented. Results: Organ distribution: Female: Liver 557 (SD:15.614), lung 259 (SD 15.928), spleen 42 (SD 13.108), abdomen 25 (SD 11.55), kidney 21 (SD 11.92); Male: Liver 331(SD 15.406), lung 233 (SD 18.027), spleen 25 (SD 10.12), brain 23 (SD 11.55), soft tissue 15 (SD 14.197) . Conclusion: The frequently seen organs in both gender are liver and lung. The rarely seen organs were spleen 67, brain 40, abdomen 39, kidney 36, vertebrae 29, soft tissue 27, pleura 26, bone 25, breast22. The others were very rare: eye 4, gallbladder 4, thyroid 4, neck 4, ovary 1, testis 1.
Relation of the length of appendix and parasitosis: Preliminary report A. Kurt * , S. A. Özmen, I. Erdem * Bölge Egitim ve Arastirma Hastanesi, Dept. of Pathology, Erzurum, Turkey Objective: Is there an effect of the length of appendix vermiformis on the settlement of the bowel parasites? We have decided to research. Method: It was studied retrospectively for not to be under the influence. Randomly chosen from the archives, 65 parasitic appendix (41 women + 24 men) and 65 appendix vermiformis (41 women and 24 men) lengths were compared. The last 65 appendix materials of the appendixes which include parasites (56 E.vermicularis, 8 A.lumbricoides,1 T.saginata) and operated any causes were examined. Placeholder conditions such as fibrous obliteration and tumour were excluded. The premeasured length of the appendix in the series (65+65=130) were listed with the gender. Results: Appendix length in the control series: The length of appendix in men:7.15 The length of appendix in women:6.85 The length of appendix in both gender:6.95 cm. The length of parasitic appendix: The length of appendix in men:7.30 The length of appendix in women:6.76 The length of appendix in both gender:6.94. Objective: On the basis of recently detected receptors for erythropoietin on the surface of MSCs we hypothesized that introduction of EPO together with MSCs may enhance their effect and improve the results of sepsis treatment. Method: 50 Wistar male rats were randomized into 5 groups with 10 animals in each: Group1 -the healthy controls, 2-5 Groups were intraperitoneally introduced bacterial LPS 20 mg/kg. Group3-4 got × 105 allogeneic MSCs, Group4 -8.5 μg of recombinant EPO-beta, Group 5 -MSCs and EPO in the same doses. The morphological study of liver, spleen, thymus, lung and kidney was performed. Histological specimens were evaluated by qualitative, semiquantitative and quantitative methods. Results: In lung tissue the thickness of alveolar septums progressivly reduced from Group 3 to 5.In kidney and liver tissue the dystrophy of hepatocytes and nefrocytes and a blood vessel's dilatation in studied groups also reduced.In lien tissue we observed hyperplasia of white pulp in groups 4 and 5. Structural features of thymus were connected with patterns of T-lymphocyte proliferation and differentiation. Conclusion: Combined treatment with EPO and MSCs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of sepsis in rats.
Mycobacterial spindle cell pseudotumor of the liver A. Alves * , A. Costa, A. Fernandes, G. do Carmo, I. Tavora * Hospital Santa Maria, Servico de Anatomia Patologica, Lisboa, Portugal
Objective: Spindle cell pseudotumors are rare manifestations of mycobacterial infections. We describe a case in the liver in an immunocompromised patient with tuberculosis. Method: A 28-year-old man, with active substance dependency, HIV-1 infection known for 7 years and acquired immunodeficiency syndrome for 3 years, was receiving tuberculostatic medication for disseminated tuberculosis and investigated for persistent fever. A liver ultrasonography confirmed a moderate hepatomegaly and showed multiple dispersed nodules in both lobes, with 1 to 3 cm of greater diameter. Results: A needle biopsy from one of the nodules showed a proliferation of spindle cells without atypia, grouped in intersecting bundles, with immunoreactivity to vimentin, CD68 and lysozyme and negative for CD34, pS100, SMA and desmin; a Ziehl-Neelsen stain revealed several acid fast bacilli in the spindle cells. They were PAS negative. Conclusion: Pseudotumoral lesions in hepatic tuberculosis usually correspond to granulomatous inflammation. A spindle cell proliferation, like that seen in other organs are more frequently associated with non tuberculous mycobacteria, is very rare. We found only another case in the literature of liver spindle cell pseudotumor associated with tuberculosis. This lesion must be differentiated from mesenchymal neoplasms.
Sixteen months follow-up in a treated case of Whipple Disease (WD) C. Popp * , M. Petre, G. Micu, A. Bastian, R. Chirculescu, R. Mateescu, F. Staniceanu * Colentina University Hospital, Dept. of Pathology, Bucharest, Romania
Objective: WD is a rare, multi-systemic infection caused by a ubiquitous environmental bacteria -Tropheryma whippleithat affects the duodenum and small intestine but also the brain, endocardium, skin, lungs and joints. Method: We are presenting a 49 years old man with a 2 years history of diarrhea, malabsorption and weight lost (about 30 kg). Endoscopical examination revealed an unspecific, yet unusual pattern of lesions: small, yellow, multiple, slightly elevated patches involving duodenum and jejunum. Histological examination revealed numerous foamy macrophages in lamina propria with PAS (+), Ziehl-Neelsen (−) material in cytoplasm. Results: Treatment with trimethoprim/sulfamethoxazole was initiated with a spectacular response: after 2 days of therapy the patient had normal stool and in 6 months he already gained back 20 kg. After 1 year of treatment the biopsy confirmed a partial remission with fewer macrophages persisting in lamina propria of duodenum. After 16 months of treatment (March 2012), very few macrophages with PAS (+) material are still identified on duodenal biopsy. Conclusion: WD is a potentially fatal disease with unspecific clinical and endoscopical signs that can be diagnosed on duodenal biopsies. Histological follow-up is a useful tool of managing the treatment and histopathological confirmation of the absence of foamy macrophages is mandatory in discontinuing therapy. Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [(18)F]-2-deoxy-2-fluoro-D-glucose ([(18)F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([(18)F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [(18)F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time. In March of 2009, an outbreak of a novel variant of H1N1 influenza A virus was reported in cases of influenza illness in Mexico [1] . By June 11, the World Health Organization raised the pandemic alert level to its highest level, declaring the first influenza pandemic in over 40 years [1] . Unlike seasonal influenza viruses, this novel H1N1 pandemic strain (H1N1pdm) tended to affect younger healthier populations and had an increased risk of morbidity and mortality [2] [3] [4] with 12-30% of the population developing clinical influenza, 4% of those requiring hospital admission, and 1 in 5 requiring critical care [5] . In general, however, infection of the H1N1pdm was relatively mild in most persons, although a fatal viral pneumonia with acute respiratory distress syndrome occurred in approximately 18,000 cases.
In contrast to seasonal influenza in human cases, H1N1pdm infections showed a tropism for the lung similar to H5N1 [6] . The ability of H1N1pdm viruses to infect the lower respiratory track has been attributed to a broader specificity in the binding of the viral hemagglutinin (HA) to a2-3in addition to a2-6-linked sialic acid (SA) receptors [7, 8] . It is reasonable that the lung tropism of the H1N1pdm contributed to the severity of disease in those individuals with preexisting complications such as asthma and chronic obstructive pulmonary disease (COPD) [6, [9] [10] [11] [12] . Data from limited human autopsies and animal studies of various pandemic strains also suggest contribution of the host innate immune response and the virus in the progression of disease [13] [14] [15] [16] .
Molecular imaging can potentially play a strong role in basic infectious disease research and clinical response by providing a noninvasive, spatiotemporal measurement of viral infection and host inflammation [17, 18] . To explore the potential utility of molecular imaging in influenza infection, we chose the ferret (Mustela putorius furo) model. Ferrets have been used as an animal model of influenza infection and pathogenesis since 1934, when they were reported to develop an acute respiratory tract illness when exposed to influenza viruses from humans and swine [19] . In contrast to mice, the ferret can be infected by human isolates without adaptation and display signs and symptoms of infection such as sneezing and nasal secretions that are similar to what is seen in humans [20] [21] [22] [23] . The ferret is an attractive model for imaging influenza pulmonary infections given the ferret's long trachea, large lung capacity, and bronchiolar branching. These anatomical features can potentially bridge imaging with histopathologic evaluation. Finally, the ferret more closely mimics humans in distribution of sialic acid (SA) receptors in the respiratory tract with higher a-2-6 SA in the upper respiratory tract and SA with a-2-3in the lower [24] .
Historically, plain film (x-ray) radiography and computed tomography (CT) have been useful for clinical assessments of influenza disease severity in clinical cases [25, 26] . These imaging modalities are limited by characterizing only anatomic changes in the lung parenchyma, such as ground-glass opacity (GGO) and consolidations, which represent different degrees of interstitial and alveolar filling by cells, edema, and inflammatory exudate [27] . In contrast, positron emission tomography (PET) imaging with the radiotracer, [ 18 F]-2-fluoro-2-deoxy-D-glucose ([ 18 F]-FDG), can provide data on metabolic activity of cells by measuring sites of increased glycolysis from leukocyte chemotaxis and accumulation, and provide increased sensitivity in detection of cells during inflammation. [ 18 F]-FDG, an analog of glucose that is moved into cells via facilitated transport, is commonly used in PET imaging as a radiotracer in clinical and basic science research. Recent studies have demonstrated the utility of the [ 18 F]-FDG in assessment of infectious disease burden in animal models of schistosomiasis and tuberculosis [28] [29] [30] . PET/CT has been used in the assessment of one hospitalized H1N1pdm patient and revealed an intense inflammatory response [31] . The uptake of [ 18 F]-FDG in humans and animals suggests the predominant presence of activated neutrophils [32] [33] [34] [35] . In studies of mice infected with influenza A virus, neutrophils play a critical role in protection and recovery from infection, and participate in the process of adaptive immunity to the virus [36] . Coupled with molecular virology and pathology, molecular imaging with important probes of disease has enormous potential to reveal early critical factors that contribute to the clinical progression of illness as well as accelerate screening, the efficacy and mechanistic studies of vaccines and antiviral therapies [17, 18] .
To test the hypothesis that FDG-PET/CT imaging could reveal the spatiotemporal nature of H1N1pdm inflammation and disease progression, we chose the ferret model of H1N1pdm influenza infection. Further, for these studies we chose a low passage clinical isolate, A/Kentucky/180/2010 or KY/180, herein, which has a change in the HA1 gene, D222G, which correlates with increased severity of disease in patient cases from several countries [37] [38] [39] [40] . In mice, infection with H1N1pdm engineered to include this change show increased viral titers and pathology, however, in ferrets there do not seem to be any major differences in clinical signs, transmissibility or pathogenicity [41, 42] . Our results show for the first time, the spatiotemporal progression of inflammation with CT and PET using [ 18 F]-FDG in ferrets infected with H1N1pdm in conjunction with histopathology and viral titers over a seven day period. Importantly, the extensive areas of patchy GGO and consolidation seen in the ferret model at 6 days post-infection (DPI) are similar to that reported for human H1N1pdm infections [31] . In vivo imaging with these modalities for anatomic (CT) and molecular (PET) data suggests increased pulmonary inflammation as the amount of circulating virus becomes undetectable. These results suggest that molecular imaging will be a great asset in gaining insight into the temporal and spatial progression of the inflammatory process caused by influenza virus infection.
Due to the size of the Siemens Trimodal gantry for PET/CT imaging we chose four month old females rather than male ferrets. The pandemic H1N1 isolate KY/180 employed in these studies was isolated from nasal swab sample provided by the Severe Influenza Pneumonia Surveillance project, a clinical study of hospitalized patients with influenza pneumonia in Kentucky. The patient had a severe course of influenza disease and died after 19 days. Sequencing of the HA1 gene from this isolate revealed the D222G mutation, which has been associated with severe disease in human cases [37] [38] [39] [40] . The second passage of the KY/180 seed was employed in the characterization of infection in the female ferrets. The 50% infectious dose in female ferrets was determined to be 10 0.07 TCID 50 (data not shown). In group 1, six ferrets were mock infected with PBS. In group 2, six ferrets were infected intranasally (i.n.) with KY/180 with a 0.5 ml dose of 0.5610 5.7 TCID 50 per naris. Ferrets were monitored for temperature for 10 DPI, and for body weight and clinical symptoms for 28 DPI. Two animals in each group were euthanized on days 2, 14 and 18 to determine virus and HI titers in blood, lung and several additional organs at 2 DPI.
In figure 1A , the body weight changes are shown for mock-and KY/180-infected ferrets. Body weight showed a drop on day 2 where the weight remained for the remainder of the study. Figure 1B shows the average temperatures of the mock-and KY/ 180-infected ferrets for the first 10 DPI. Temperature peaked on days 1 and 5 for KY/180 infected animals with a mean temperature of 103.4uF (SD = 1.71uF) and 103.2uF (SD = 0.52uF), respectively. The average hemagglutinin inhibition (HI) serum antibody titer in the blood on day 14 was 540 (reciprocal dilution) and the average total serum influenza-specific IgG by ELISA was 7610 (reciprocal dilution) (Fig. 1C) . In studies to determine the infectious dose, additional tissues were taken from animals infected at with KY/180 with a 0.5 ml dose of 0.5610 4.7 TCID 50 per naris. On day 2, viral titers were the highest in the nasal turbinates (10 6.25 TCID 50 /g) followed by the caudal lung (10 6.0 TCID 50 /g) and trachea (10 3.75 TCID 50 /g). The lowest levels of virus were observed in the cranial lobe of the lung (10 3.2 TCID 50 /g). Viral titers were measured by TCID 50 in nasal turbinates, trachea, right cranial lobe of the lung, right caudal lobe, brain, liver, spleen, kidney, duodenum, jejunoilieum, colon and rectum. KY/180 was detected in jejunum in one animal (10 2.0 TCID 50 /g), but was not detected in any other tissues (data not shown).
Female Fitch ferrets were divided into five groups, with two per group of animals that were mock-infected with PBS (group 1) or intranasally infected with KY/180 (groups 2-5), in a 0.5 ml dose of 0.5610 6.0 TCID 50 per naris, on day 0 (Table 1) . Group 5 was the only group that was imaged each day; while groups 2-4 were imaged and sacrificed on days 1, 2 and 3 post-infection. This study design permitted evaluation of the progression of imaging with infection and pathology in two animals each day as well as continuous imaging of the lungs in one cohort over the seven-day time-period.
FDG-PET and CT images of the H1N1pdm-infected and mock-infected ferrets were successfully obtained and fused for two ferrets on days 1, 2, 3 and 6 ( Fig. 2 and 3 ). Volumes of interest (VOI) and corresponding maximum standardized uptake values (SUVMax) were generated for any metabolically active lesions in the lung as well as background activity in the lungs, liver, heart, thymus, and thoracic lymph nodes. Baseline imaging prior to infection showed no focal areas of lung consolidation on CT and background standard uptake values (SUVMax) of the [ 18 F]-FDG levels ranged from 0.7-1.0 for PET ( Fig. 2A and Fig. 3A ). Each figure shows the one two-dimensional coronal plane that were standardized across days to provide a similar orientation and do not necessarily represent the SUVMax as that plane may be out of view.
By 1 DPI, an area of consolidation was identified on CT in the right caudal lobe with corresponding radiotracer uptake on PET (Fig. 2B , SUVMax of 4.7). Consolidative areas in the right caudal lobe increased by day 2, with a persistently elevated SUVMax of 3.1(data not shown). By day 3, the consolidation increased in the right caudal lobe ( Fig. 2C and Fig. 3C , SUVMax of 3.7 and 4.4, respectively) and also appeared in the left caudal lobe (SUVMax of 3.2) of ferret 2214 (Fig. 3C ). By 6 DPI, there were widespread areas of patchy consolidation on CT with multiple areas of increased radiotracer uptake in both ferrets in caudal and cranial lobes ( Fig. 2D and Fig. 3D , SUVMax of 6.0 and 7.6 on the right, 4.2 and 4.6 on the left, respectively). These results suggest that inflammation progresses into the lower respiratory airways after infection into the upper part of the lower respiratory system. A ferret from the uninfected cohort was also imaged on day 6, with no focal appearance of consolidation on CT and no evidence of increased [ 18 F]-FDG uptake on PET (image not shown, background SUV of 0.6).
To measure viral shedding, each day each ferret was swabbed in the nasal, throat and fecal passages and the viral titer was measured by TCID 50 ( Table 2 ). The highest levels of viral shedding were measured in the throat swabs. Nasal swabs also showed viral shedding for most animals, while the presence of virus in rectal swabs was low although detectable in a few animals. Replication of H1N1pdm in nasal turbinates and lungs were determined post-mortem from the right caudal lobe of the lung taken on euthanasia (Table 3) . Four sections were taken per lobe to provide greater insight into the spread of the virus in the tissue (Fig. 4) . High levels of virus were detected in all nasal turbinate samples at 1, 2, and 3 DPI (95% C.I. = 5.43+/21.00 TCID 50 / mL). Virus was also detected in a majority of lung sections from 1, 2, and 3 DPI. It was absent in the lung sections from one animal on 2 DPI, although it was present in the ferret's nasal turbinates, suggesting that the timing of infection in this animal was slower than the others. Of note, this same animal had a focus of consolidation on CT and radiotracer uptake on PET. This observation also suggests that, while the virus may have been undetectable by TCID 50 , low levels of virus had entered the lower respiratory system. No animals on day 7 post-infection had detectable virus in the nasal turbinates or the sampled lung tissue. Virus was not detected in nasal, throat and fecal passages or the sampled lung tissue from ferrets in any of the controls.
Upon necropsy, all but the right caudal lobe of the ferret lung was fixed with paraformaldehyde. Following fixation, sections were taken for histopathology from the right and left cranial lobes, left caudal lobe and the middle accessory lobe. Representative photographs from slides of the left caudal lobe are shown in figure 5 . The ferrets in the control group had intact bronchiolar walls with very minimal infiltration by neutrophils with the exception of the left caudal lobe from control animal 2206 sacrificed on Day 1. Possible causes of this pattern of change may be an underlying systemic vasculopathy which is typically confirmed by evaluation of other organs that were not collected (e.g., kidney, spleen, liver).
In general pulmonary lesions associated with influenza infection were roughly comparable at Days 1 and 2 and consisted of variable suppurative or necrosuppurative bronchiolitis and mixed cell alveolitis at minimal to moderate severity levels. By 1 DPI, there were some small foci of inflammation without much infiltration of the bronchi or bronchioles. There was an increased severity of inflammatory findings in lung lobes from infected ferrets on day 3. Specifically, more extensive infiltration of neutrophils can be seen within the bronchiolar lumen, along with necrosupprative bronchiolitis and mixed cell alveolitis. At Day 7, lesions observed in the lung lobes continued to exhibit an increased severity compared to the majority of lung lesions seen at 1 and 2 DPI. Bronchiolar epithelial hyperplasia and cytokaryomegaly were noted in addition to bronchiolitis.
To evaluate potential correlations between PET/CT with histopathology, the SUVMax of lesions in the right and left lung of each ferret were compared with the cumulative histopathology scores assigned by the veterinary pathologist (Fig. 6 ). On average, the SUVMax was higher in the right lung than the left lung but the slopes and Spearman's correlation coefficients (r) were similar between the two sides. The highest correlation was seen between the cumulative bronchiolitis score and SUVMax (r of 0.71 and 0.75 on the right and left, respectively). The next highest was between the cumulative bronchitis score and SUVMax (r of 0.69 on the right and 0.67 on the left). A weaker positive correlation was seen between the cumulative alveolitis score and SUVMax (r of 0.47 on the right and 0.57 on the left).
Herein, we show for the first time the feasibility of utilizing [ 18 F]-FDG PET coupled with CT imaging of H1N1pdm in ferret to track the progression of pulmonary disease in real-time. We chose a low passage clinical isolate, KY/180, which has a change in the HA1 gene, D222G. The D222G change in H1N1pdm correlates with increased severity of disease in patient cases from several countries [37] [38] [39] [40] . The patient from which we obtained the KY/180 isolate also had a severe course of influenza illness over a period of 19 days that resulted in death. Recently, studies in mice and ferrets infected with pandemic influenza viruses A/California/04/2009 and A/Netherlands/602/2009 engineered with the D222G mutation have shown that the D222G mutation are lethal in mice, but not ferret [41, 42] . The lethality in mice, but not ferrets, has been attributed to the greater abundance of a2-3-SA in the mouse model [42, 43] . All of these viruses have an affinity for a2,6-SAs associated with attachment to and replication in cells of the upper respiratory tract as shown by the high levels of viral replication in the nasal turbinates. Thus, infection of ferrets with these H1N1pdm isolates engineered with D222G and our clinical isolate have not correlated with clinical findings in patients. These results in ferrets are not surprising given that 80% of the fatal cases of H1N1pdm had underlying medical conditions and bacterial infections [6] . Discovery of the molecular components of the host response that may promote pathogenesis will be critical for defining new treatments.
Noninvasive imaging can provide real-time in vivo monitoring of the progression of infection, inflammation and disease that may give insight into the mechanisms that modulate disease progression. Recently, Veldhuis Kroeze et al, presented data on the monitoring of pulmonary lesions of H1N1pdm influenza virusinfected ferrets with CT scanning which correlated with disease progression and severity [44] . As those studies demonstrate, CT is a powerful tool, but it will not give the molecular details that can be provided by PET or SPECT imaging of probes that target critical host responses such as neutrophil invasion. In our study we coupled CT scanning with the [ 18 F]-FDG radiotracer and show infection and inflammation of influenza infection in the lower respiratory system with foci of increased [ 18 F]-FDG uptake corresponding to areas of lung opacity on CT, with underlying inflammation on necropsy. In comparison to human CT imaging studies of influenza, the molecular images in the ferret show strong similarity. CT findings in patients with confirmed influenza infection show patchy ground-glass opacities in segmental multifocal distributions, mixed with areas of consolidation in the lung [12, 25, 45] . Moreover, the few case reports of human influenza in which lungs were imaged by [ 18 F]-FDG PET demonstrate areas of high uptake in these ground-glass opacities and consolidation [31] . Our study similarly demonstrates this pattern in the ferret model, also showing patchy opacities on CT with high uptake of radiotracer on PET, with necroscopy-based confirmation of inflammation in the left caudal lobe. Specifically, we show the ferret lung demonstrated progressive consolidation on CT and FDG uptake on PET predominantly in the right caudal lobe, which progressed to the left caudal lobe by day 3 p.i. By day 6, the diffuse metabolically active lesions seen on PET/CT were similar to what has been reported in the human literature during the 2009 H1N1 pandemic [31] .
Histopathologic evaluation of the lungs confirmed the progressive nature of the pulmonary lesions and corroborated the radiologic data. Suppurative and necrosuppurative bronchiolitis seen on days 1 and 2 became progressively worse by days 3 and 7 post-infection. The inflammation tended to be patchy or multifocal and an entire lung lobe was never uniformly affected, corresponding to the multiple patchy lesions seen on PET/CT by the end of the study. This also agreed with the analyses of the viral titers in various sections of the right caudal lobe and the PET/CT imaging. Our analyses of viral titers in the four representative sections suggest different levels of infiltration of the lobe. The histopathologic scoring for bronchiolitis correlated the best with the SUVMax of the lesions seen in the right and left lungs on PET.
In general, the severity of infection and inflammation on imaging can be represented by (1) the volume of affected lung (i.e. the percentage of diseased lung relative to total lung capacity), and (2) the extent of parenchymal destruction (disruption of pulmonary architecture) and inflammatory cell migration. Our study first aims to correlate FDG uptake measurements with histology, thereby analyzing the extent of parenchymal destruction and cellular infiltrates.
It should be considered that there can be variation in matching FDG uptake with histologic severity because more severe architectural distortion can lead to necrosis with more dead cells, therefore showing less uptake of radiotracer among metabolically inactive dead cells and nonviable tissue. Our study, however, shows that progressing inflammatory infiltrates on histology in the studied time period after acute influenza infection corresponds to radiologic trends. Additionally, our study demonstrates spatial progression with increased size and number of abnormal foci in the lung parenchyma during acute infection.
Ultimately, utilizing these new imaging tools, we envision a number of future experiments to delineate potential differences in the course of H1N1pdm and H5N1 infection in ferrets. We also plan to explore additional radiotracers that might reveal potential differences in host responses in the immune system and the process of acute injury in the lung. Future studies will assess differences in presentation of those who recover from infection versus those who eventually succumb to infection such as with more lethal isolates such as H5N1. This model should be valuable in rapid assessment of the effect of various treatments on pulmonary inflammation and damage. Finally, these first PET/CT imaging approaches could be extended to a number of other important pulmonary infections caused by pathogens such as hantaviruses, respiratory syncytial virus, and SARS CoV, to gain further insight into the spatiotemporal in vivo dynamics of disease progression [18] .
The influenza H1N1pdm virus, A/Kentucky/180/2010, (KY/ 180; GenBank CY99332 and CY99333) was isolated from the nasal swab of a severe hospitalized case (hospitalized in March 2010) provided by the Severe Influenza Pneumonia Surveillance project, an ongoing clinical study of hospitalized patients with influenza pneumonia in Kentucky (courtesy of Dr. Julio Ramirez). The virus was isolated and passaged in the allantoic cavity of tenday-old embryonated hens' eggs at 37uC. The allantoic fluid was harvested 72 h after inoculation, pooled and stored in stored in aliquots at 280C until use. The infectious virus titer of the resulting seed stock was determined by TCID 50 (50% tissue culture infectious dose) and the titer calculated by Reed and Muench [46] and confirmed by plaque assay on MDCK cells. Passage E2 was used for the studies reported herein.
Ferret studies were approved by the University of Louisville Institutional Animal Care and Use Committee. University of Louisville has Veterinary Medicine tasked to monitor and support all animal experiments. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research will be conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
All female Fitch ferrets were obtained from Triple F Farms (Sayre, PA). Ferrets were selected after screening blood samples for the presence of influenza antibodies using a hemagglutination inhibition assay (HI). Ferrets that were seronegative for seasonal and pandemic viruses were shipped directly to the University of Louisville Regional Biocontainment Laboratory and acclimated for seven days prior to initiation of the studies. Animals were fed Teklad Laboratory Diet #2072 (Harlan/Teklad, Madison, WI) and water ad libitum.
For the characterization of the progression of infection of the KY/180 clinical isolate we utilized four month old, female ferrets. Prior to infection with virus, ferrets were anesthetized with 0.05 mg/kg atropine, 5.0 mg/kg ketamine, and 0.08 mg/kg dexmedetomidine intramuscularly. Subsequently, six animals were inoculated intransally (i.n.) with 0.5 mL of infectious virus per naris as a bolus, which was diluted to 10 5.7 TCID 50 /mL in phosphate buffered saline (PBS). Six additional animals were inoculated by i.n. with 0.5 mL of infectious virus per naris as a bolus with PBS (mock). Anesthesia was reversed with 0.4 mg/kg atipamezole. Ferrets were monitored daily for temperature and clinical symptoms. On day 2 post-infection, two ferrets were taken to measure viral titers in blood, lung, brain, trachea, nasal turbinates, spleen, kidney, thymus, liver, duodenum, jejuno-ileum, large intestine, and rectum. Gross pathology was defined during necropsy for the lung. At 14 and 28 days post-infection two additional ferrets from each group were analyzed for viral titers and pathology in the lung.
For molecular imaging studies, twelve, four-month-old female Fitch ferrets were utilized. Animals were fed food and water ad libitum except 4 h prior to and during CT/PET imaging. On day 0, ferrets were anesthetized prior to inoculation with virus or PBS with ketamine, dexmedetomidine and atropine. Eight animals were inoculated i.n. with 0.5 mL of infectious virus per naris as a bolus, which was diluted to 10 6.0 TCID 50 /mL in phosphate buffered saline (PBS). Four ferrets were inoculated i.n. with 0.5 mL PBS per naris as a mock-infected control. For imaging, on days 0, 1, 2, 3 and 6, anesthetic induction and maintenance were achieved with 1-3% isoflurane. Blood glucose levels were checked prior to administration of the radiolabeled tracer to ensure that they were within normal limits, which typically range from 62-134 mg/dL in ferrets [47] . Glucose was provided to animals to compensate for body fluids lost during imaging. Typically 30 mls of Lactated Ringer's solution (Hospira) was administered subcutaneously (s.q.) following completion of the imaging. Animals were monitored for body temperature and vital signs during imaging.
Imaging was performed on 0, 1, 2, 3, and 6 days post-infection (DPI). Each day, four ferrets were imaged with CT and PET on hardware designed for preclinical animal studies, including microCT and microPET, respectively. Two ferrets were euthanized the each day and necropsied to obtain tissue samples for virologic and histopathologic analyses (please see study design Table 1 ). Image acquisition was conducted with a Siemens Inveon Trimodal Scanner (Siemens Preclinical, Knoxville, TN), which is a small animal imaging platform that combines microPET, microCT, and microSPECT modalities within one unit. This combination facilitated co-registration of PET and CT images as the study subject was kept in a uniform position on the scanner bed, minimizing potentially large motion artifacts as a result of repositioning the animal between each scan. The Inveon microCT scanner features a variable-focus tungsten X-ray source with an achievable resolution of 20 mm and a detector with a maximum field of view (FOV) of 8.4 cm65.5 cm. The source-to-object distance was 263.24 mm and the source-to-detector distance was 335.67 mm. The Inveon PET detector provided an axial field of view (FOV) of 12.7 cm with a spatial resolution of 1.44 mm. PET images were reconstructed using a 2D-filtered backprojection algorithm with attenuation correction provided by microCT imaging. For the microCT scan, the following imaging settings were used: two bed positions, 80 kVp, 500 mA, 500 ms exposure time, and 464 binning. After each ferret underwent microCT imaging, the bed position was reset and microPET imaging with 18F-FDG (PETNET, Louisville, KY) began immediately. For each ferret, 2 mCi of 18F-FDG was administered (i.p.) with a 60-90 min uptake period. Radioactive dose was confirmed with an Atomlab 500 Dose Calibrator (Biodex Medical Systems Inc., NY).
All imaging data were processed with PMOD software (v3.1; PMOD Technologies Ltd., Zurich, Switzerland). MicroCT data were received from the Inveon platform as DICOM files and PET data as microPET files. Scans were imported into the program's local SQL database with the units for the PET radiotracer in kBq/ cc. PET images were co-registered with the CT images with reslicing done as necessary to facilitate later calculations. For analysis of 18F-FDG levels, the standardized uptake value (SUV) was used. SUV is a widely used semi-quantitative measure that normalizes radiotracer uptake in a given region of interest based on body weight, and calculated for this study as follows: 
For all calculations, animal weights were expressed in kilograms and FDG activity in megabecquerels. For each image series, SUVs for each voxel were calculated using an external filter in PMOD, with the radionuclide half-life set at 6586.2 sec for 18F-FDG. For each pulmonary lesion, an ellipsoid volume of interest (VOI) was generated that encompassed the structure. Then, automatic isocontour detection was used to refit the VOI by setting a threshold of 50-60% of the difference between the maximum and minimum intensity SUVs in the ellipsoid VOI such that 0.5*(SUV max 2SUV min ). In cases where the automated threshold included contiguous structures in the VOI, manual refitting in conjunction with the co-registered CT scan was used to exclude those surrounding structures. For all VOIs, maximum SUV (SUV Max ) and average and standard deviation of all pixels in the volume (SUV Mean 6 SD) were calculated.
For CT analysis, image interpretation was performed by a radiologist (in consultation with the scientific team) having more than ten years of diagnostic experience along with formal certifications by the American Board of Radiology (ABR) and the American Board of Nuclear Medicine (ABNM). Lesions on CT were identified using conventional criteria and terminology; Ground-glass opacity (GGO) is defined in this study as hazy increased lung opacity, with discernible underlying lung architecture such as visible bronchial and vascular structures, representing partial displacement of air in interstitial and alveolar airspaces; Consolidation is defined in this study as high density lung lesions (more dense than GGO) in which vascular and bronchial margins are obscured, representing complete displacement of alveolar air [27] .
On days 1, 2, 3 and 6 and prior to euthanasia, swabs were taken from each ferret from nasal, throat and rectal regions. Following scheduled euthanasia, the nasal turbinates and the right caudal lobe of the lung from each ferret, which was divided laterally into four segments, were isolated. All swab and tissue samples were snap-frozen in liquid nitrogen and stored at 280uC until analyzed for virus titer by TCID 50 . Frozen tissues were weighed and diluted 10% weight per volume into cold DMEM with 1% penicillin/ streptomycin and 0.2% BSA before being homogenized and centrifuged to remove debris. Tissue homogenate and swabs were serially diluted 10-fold in DMEM with 2 mg/mL TPCK-Trypsin, 0.2% BSA, 4.5 g/L glucose, 1% penicillin/streptomycin, 2 mM L-glutamine, and 25 mM HEPES. Each sample was analyzed in quadruplicate following incubation in 96-well plates with Madin-Darby canine kidney (MDCK) cell monolayers at 37uC in 5% CO 2 for three days. Supernates were collected from each well were assayed for hemagglutination activity using 0.5% turkey red blood cells as an indicator of infection. Viral titers were expressed as log 10 TCID 50 /mL and were calculated using the Reed-Muench method [48] .
The HI test quantitates serum antibody to influenza virus which can prevent agglutination of turkey RBCs (Fitzgerald Industries International Inc., MA). Heat-inactivated serum samples were treated with receptor-destroying enzyme (Sigma-Aldrich) for removing nonspecific inhibitors (followed by RBC adsorption) and were diluted 2-fold serially from initial dilution of 1:10. HA antigen (8 HA units in 25 mL) were added onto each well and incubated for 1 h at RT. Following antigen-antibody reaction, 50 mL of 0.5% turkey RBC were added to each well and incubated for 1 h at RT. HI negative wells were scored based upon a diffuse sheet of agglutinated RBCs covering the bottom. HI positive wells were scored if they showed a well circumscribed button of nonagglutinated RBCs.
Lungs were inflated and stored in 10% neutral-buffered formalin. Three lung sections were placed into cassettes per lung section (right cranial, left cranial, left caudal, and right middle lobe) until they were trimmed, paraffin-embedded, and sectioned. Sections were mounted on glass slides and stained with hematoxylin and eosin for microscopic evaluation at Experimental Pathology Laboratories, Inc. by a veterinary pathologist. Sections were examined for the presence of abnormal findings including supprative and necrosupprative inflammation; epithelial hyperplasia and cytokaryomegaly; and fibrinous and exudative changes. Changes were graded with a standardized scale of 0-5, with 0 classified as ''not present'', 1 as ''minimal'', 2 as ''slight/mild'', 3 as ''moderate'', 4 as ''moderately severe'', and 5 as ''severe/high.'' For each ferret, a composite score for pathological changes was generated based on the locations in the respiratory tract (alveoli, bronchioli, bronchi) for statistical evaluation.
All statistics were performed using R version 2.13.0 and GraphPad Prism 5. For each image, mean SUVMax and standard deviations were obtained. For each ferret, SUVMax values were correlated with histopathologic scoring using Spearman's Rho (r). The Dynamics, Causes and Possible Prevention of Hepatitis E Outbreaks Rapidly spreading infectious diseases are a serious risk to public health. The dynamics and the factors causing outbreaks of these diseases can be better understood using mathematical models, which are fit to data. Here we investigate the dynamics of a Hepatitis E outbreak in the Kitgum region of northern Uganda during 2007 to 2009. First, we use the data to determine that [Image: see text] is approximately 2.25 for the outbreak. Secondly, we use a model to estimate that the critical level of latrine and bore hole coverages needed to eradicate the epidemic is at least [Image: see text] and [Image: see text] respectively. Lastly, we further investigate the relationship between the co-infection factor for malaria and Hepatitis E on the value of [Image: see text] for Hepatitis E. Taken together, these results provide us with a better understanding of the dynamics and possible causes of Hepatitis E outbreaks. Outbreaks of diseases such as avian influenza, SARS and West Nile Virus have alerted us to the potentially grave public health threat from emerging and re-emerging pathogens [1] [2] [3] . Many important infectious diseases persist on a knife-edge: rapid rates of transmission coupled with brief infectious periods. Such violent epidemic behavior has been observed in plague [4] , cholera [5] , pertussis [6] and more recently Hepatitis E. The recent outbreak of Hepatitis E in northern Uganda, has left many dead and a number of infectives that continue to spread the infection [7] . Hepatitis E is caused by infection with the Hepatitis E virus (HEV) which has a fecal-oral transmission route. It is a self-limiting disease but occasionally develops into an acute severe liver disease. As emerging and re-emerging infectious diseases increase in outbreak frequency, there is a compelling interest in understanding their dynamics [8] [9] [10] .
The Kitgum outbreak, which we study here, has been linked to contaminated water or food supplies [11] . An assessment conducted by the Uganda Red Cross and district representatives in Agoro revealed that for a population of about 28,045 with 6,039 households mainly living in camps for internally displaced people in Potika as well as Agoro and Oboko satellite camps, the latrine coverage was as low as 3.7%. This means that there is one latrine for every 27 people. Further, only 23 boreholes were functional implying that the bore hole coverage is 0:38%, or one bore hole per 263 households.
Another possible factor that could be implicated in the outbreak of Hepatitis E is its possible relationship with malaria. Malaria has been shown to disarm the immune system and increase susceptibility to viral infections such as HIV [11] . Recently, in a 3-month follow-up study the pattern of co-infection of Plasmodium falciparum malaria and acute Hepatitis A (HAV), in 222 Kenyan children under the age of 5 years was observed [12] . The incidence of HAV infections during P. falciparum malaria was found to be 6.3 times higher than the cumulative incidence of HAV, suggesting that co-infection of the two pathogens may result from changes in host susceptibility. There is also evidence both for [13, 14] and against [15, 16] an association between Hepatitis B viruses and malaria. HEV transmission route is similar to the Hepatitis A virus and thus for HEV it is important to consider possible links to co-infection with malaria. This can be done using mathematical models of multiple pathogens [17] [18] [19] [20] [21] [22] .
In this paper, mathematical models are used to study the effects of both environmental conditions and malaria on Hepatitis E infections. The models designed are fit to data from the Kitgum outbreak, to estimate the basic reproduction number and to relate them to the level of contamination of the environment. We assume that the small number of latrines [23] , leads to contamination of environment. This in turn leads to contaminated water. Owing to the few number of bore holes in the region, lack of access to clean water gives rise to the viral infection of Hepatitis E.
We formulate two mathematical models: one for Hepatitis Eonly and another for the co-infection with malaria, based on prior work as in [4, 24, 25] . In their framework, an individual is categorized according to their infection status and passes sequentially through the series of non infectious, infectious and recovered classes. A system of ordinary differential equations are then designed, analyzed and later fit to data from the Hepatitis E outbreak in Kitgum district to estimate desired parameters.
First we model the epidemiology of Hepatitis E, an environmentally transmitted viral infection. The dynamics of the disease are an SEIR framework, i.e. Susceptible, Exposed, Infectious and Recovered. Hepatitis E virus is mainly spread by the fecal-oral route. This results either from directly touching the contaminated environment and eat without washing hands, or drinking contaminated water. In Kitgum district Uganda, most people live in internally displaced camps. The number of latrines in the area are not enough for the entire population, [11] , and people use the local environment for this purpose. When rain falls, it washes the faeces into water bodies. In the Kitgum region, few people have access to clean bore hole water [11] , and therefore collect water from the contaminated water sources. To model this phenomenon we use l, to denote the proportion of households in Kitgum with access to latrines. Therefore, the rate of change of contamination c of the environment is given by
where r is the transmission rate of HEV from the infected human, I, to the environment.
In the human population, susceptibles, S, are recruited at a rate m that equals to the per capita natural mortality rate for each group. This assumption is made to keep the population constant, while keeping a turnover of individuals in the population. We assume that a fraction b of the population has access to clean bore hole water and cannot become infected. Susceptible individuals without bore hole access become infected with the Hepatitis E virus at a rate bc, where b is the transmission rate of HEV from the contaminated environment c, to the human. This gives
After successful infection, the individual is now exposed to HEV and moves to the exposed class E. The incubation period takes a mean period of 1 s days. The equation for this group is given by
At the end of the incubation period 1 s , the individual becomes infectious and moves to group I. At this point, they display signs and symptoms that include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, jaundice, dark urine, clay-colored stool and joint pain [26] . The infected individual may recover at a rate c. These dynamics are given by dI dt~s E{(mzc)I:
Of the total infected individuals, a fraction p of them die due to the infection, and (1{p) recover to join the immune group R. This implies that
Equations (1) to (5) provide a system of equations defining the transmission of HEV between a contaminated environment and humans. Assuming that the dynamics of the environment are fast. This means that the environment reaches a steady-state before the humans. The quasi-stationary-state (QSS) for c can be obtained from equation (1) to give
This steady state is then substituted in the human equations to give a reduced system of equations as follows:
dR dt~( 1{p)cI{mR:
As in [27, 28] , in this system, 1~SzEzIzR. Thus, the last equation in (7) is redundant. This system of equations will be analyzed and fit to data. The endemic stationary state is given by
where
is the basic reproduction number for HEV. The term s mzs is the proportion of the exposed humans that survive the incubation period. The other fraction, br(1{l)(1{b) mzc is transmission rate of HEV during the infectious period of the human. The disease-free equilibrium point is stable if R 0 v1 (see Supporting Information S1) When R 0 w1 the endemic equilibrium point in equation (8) exists and is stable. This equilibrium is attained via oscillatory dynamics, with period T*2p
is the mean age at infection, and G~1 mzs z 1 mzc is the ecological generation length of the infection. Figure 1a is a plot of the data for the outbreak from 2007 through 2009. To estimate model parameters and determine the critical level of control needed to eradicate the epidemic, the model described by the equations in (7) is fit to the data collected during the Kitgum outbreak ( Figure 1 ) During the invasion phase of HEV, the prevalence is approximately
Taking the log of both sides of equation (10) and performing linear regression (details in Supporting Information S1) on this equation, (Figure 1b gives an estimated value R 0~2 :15.
To determine R 0 when natural mortality is not equal to zero, (i.e. mw0), we use a non-linear differential equation fitting tool, called the PottersWheel Toolbox [29] . In this fitting technique, the c 2 value of the sum of the squares of the differences between the observed and fitted values is minimized by searching through different parameter values. We set m~0:0004, p~0:0169, b~0:0038, l~0:0037, and fit the free parameters, s,c, and the force of transmission br. We repeat this process 50 times to produce a range of best fits. The basic reproduction number for each run is then calculated using the expression in equation (9) The fitted parameters estimate the basic reproduction number R 0 between 2.08-2.39 with average 2.25. This value is similar to that found from the linear regression fitting. Figure 1c is a plot of the model outcome using the parameters generated from the fitting to the Kitgum outbreak.
In addition to Hepatitis E, individuals in the Kitgum region were at a risk of acquiring malaria which is endemic to Uganda. To model possible co-infection we adopt the model to include a susceptible group which comprises both those with and without malaria. That is, the total susceptible population S9 = S+M where M is the proportion of individuals infected with malaria. The malaria dynamics will not be modelled in detail here but an assumption is made that malaria continuously invades the population, and individuals move back and forth between infection and recovery from the disease. This implies that
The equilibrium state for this model is given by 
Clearly, this assumption provides a much simplified model when compared to a full model of vector-borne malaria [3, 12, 27] . Our concern here, however, is how background levels of malaria effect transmission dynamics of HEV. In Kitgum, at its lowest point during March 2009, 2,316 cases of malaria were reported out of a total population of 28,045 [7] . Thus 8.3% of the population are infected with malaria at any time, M Ã~0 :083. Recovery rate for malaria is r~0:1429 per week, and thus we set f~0:0129.
Under the above assumption, equations (7) are rewritten to incorporate the malaria dynamics in equation (11) where j is a parameter that models change the increase (or decrease) in susceptibility to Hepatitis E of malaria infected individuals [12] . The other parameters are as defined in equations (7) and remain as defined there. We assume here that after exposure to HEV, both the susceptible and malaria infected groups join the exposed, E and subsequently the I group. In other words, individuals that harbor both infections are assumed to develop HEV symptoms at the same speed as those with only HEV. The dynamics of this model for standard parameter values are shown in Figure 2 .
Using the next generation method as in van den Driessche and Watmough (2002), [30] , the basic reproduction number for Hepatitis E in presence of malaria is given by
where R 0 is as defined in equation (9) When R C v1 infected individuals will have more chances of recovery than of transmitting the disease further hence the epidemic will die out. When R C w1, there exists an endemic equilibrium point as shown in Supporting Information S2 given by p . This implies that the endemic stationary point is attained via damped oscillations. The stability of this point would depend on the sign of the real part, a. If a.0, then the steady state is an unstable spiral, otherwise, it is a stable spiral. If ½j(mzf)z(mzr) 2 4jm(mzfzr) w1, then we have real roots, and stability of this equilibrium state will depend on the signs of these roots. If both are positive, the steady state is an unstable node; if both are negative, it is a stable node. If one of them is positive and the other negative, the steady state is a saddle point. Figure 2a shows the evolution of the malaria infected, M, the exposed, E, and the infected, I with time, while Figure 2b is a phase space portrait in the SI plane. From equation (14) it can be seen that the value for R C is determined by the proportions of susceptibles and malaria infectives in the population. Rearranging and assuming that SzM~1 this equation gives a criteria for an epidemic of
This criteria is plotted in Figure 3a . As expected, if jw1 then presence of malaria increases the probability of an outbreak of Hepatitis E, while if jv1 the presence of malaria inhibits Hepatitis E. Assuming that malaria is at equilibrium in the population. (For example week of March 2009, with 2,316 malaria cases), gives M Ã~0 :083, then equation (16) gives a direct relation between j and R 0 . In fitting the model we note that the transmission rates br and j are not independent. Indeed, using PottersWheel to fit the co-infection model shows that the range of values for br is between 1.28-4.69, c between 1.01-1.75 and j values are between 0.02-13.27 (Figure 3b ) All of these values fall on line corresponding to R C between 2.19-2.48. This relationship follows the same curve as the analytical results in Figure 3a .
To test potential interaction between Hepatitis E and malaria empirically, we now assume that in the absence of malaria, Hepatitis E has R 0~1 and does not spread. Thus malaria is required for the spread of Hepatitis and jw1. Since R C~2 :3 from the data and M Ã~0 :083, then substituting these values in to equation 14 gives j~16:9. This implies that, under the assumption of co-infection as the factor which promotes Hepatitis E, malaria infected individuals were infected with Hepatitis E up to 16.9 times more than those not infected with malaria. As we gain more information about the role of co-infection, this relationship can be used to improve estimation of br.
As in [25, 27] , the criterion under which Hepatitis E will invade the population when malaria is endemic is derived in the Supporting Information S3. Thus, Hepatitis E virus invades if
and the co-infection persists if j fzr(1{R 0 ) fR 0 : ð18Þ
As in the Hepatitis E-only model, PottersWheel Toolbox is used to investigate the basic reproduction number R C , when natural mortality is not equal to zero, (i.e. mw0) A sequence of parameter estimates are generated, this time setting the fits in sequence to 20. The process is repeated until a set of 50 readings is obtained. The parameters are chosen in such a way that 0:1077vsv0:4777 (i.e. 15v1=sv64 days, the Hepatitis E incubation period, [26, 31, 32] ), and x 2 value is less than 65. The basic reproduction number for each run is calculated using the expression in equation (14) .
The Global Burden of Disease (GBD) concept, first published in 1996, constituted the most comprehensive and consistent set of estimates of mortality and morbidity yet produced [33] . A GBD study aims to quantify the burden of premature mortality and disability for major diseases or disease groups, and uses a summary measure of population health, the DALY (Disability-Adjusted Life Years), to combine estimates of the years of life lost and years lived with disabilities. A DALY is defined as an indicator to quantify the burden of the disease and the functional limitation and premature mortality [34] . It can be used across cultures to measure health gaps as opposed to health expectancies, and the difference between a current and an ideal situation where everyone lives up to the age of the standard life expectancy, and in perfect health. In developing the DALY indicator, Murray and Lopez (1996) , [33] 
Since theDALY combines in one measure the time lived with disability, YLD, and the time lost due to premature mortality, YLL, then
The YLL metric essentially corresponds to the number of deaths, P, multiplied by the standard life expectancy, L, at the age at which death occurs. Therefore,
To estimate YLD on a population basis, the number of disability cases is multiplied by the average duration of the disease and a weight factor that reflects the severity of the disease on a scale from 0 (perfect health) to 1 (dead) The basic formula (without applying social preferences) for one disabling event is given by
where I is the number of incidence cases, DW is the disability weight, and D is the average duration of disability.
Since the reported cases are not specified according to age, the estimate will be done on a population basis. Typical symptoms of Hepatitis E include jaundice (yellow discoloration of the skin and sclera of the eyes, dark urine and pale stools), anorexia (loss of appetite), an enlarged, tender liver (hepatomegaly), abdominal pain and tenderness, nausea and vomiting, and fever and the disease may range in severity from sub-clinical to fulminant [35] . To calculate the YLD, we will set the disability weight to that for a diarrhea disease episode, (equal to 0.11, [33] ), in untreated or treated form.
In developing the DALY indicator, additional social choices are taken into account. For example, is a year of healthy life gained now worth more to society than a year of healthy life gained sometime in the future? The DALY is an incidence-based measure, rather than a prevalence-based measure. Therefore, to estimate the net present value of years of life lost, a time discount rate to years of life lost in the future is applied, to adjust both costs and health outcomes [36] . Discounting health with time reflects the social preference of a healthy year now, rather than in the future. To do this, the value of a year of life is generally decreased annually by a fixed percentage, d. Therefore, equations (20) and (21) are respectively transformed to
YLD~I
According the WHO [35] , the life expectancy for a Ugandan male is 51 and 48 for a female. An average of 50 years will be used. 
The number of latrines and boreholes that would have prevented the Hepatitis E outbreak in Kitgum are calculated using our results in preceding sections. First, it is assumed that if the people had the necessary and sufficient number of latrines in addition to safe drinking, then the outbreak would not have occurred. Then, the costs of constructing the required latrines and boreholes are computed. From the results, the cost of saving one life from Hepatitis E, for one year is determined.
The current number of latrines in Kitgum is 1,038 [11] . This implies one latrine per 27 people. According to the rules and regulations of Kampala City Council Authority, Building Inspection department, 1 latrine should be shared by a maximum of 5 people. We can use our estimated values of the basic reproduction number R 0 to determine the level of l and b that make R 0 v1. First, we use equation (9), the parameters in Table 2 , and the value of R 0~2 :11, (linear regression) Assume that contamination is due to insufficient latrines. Then, for R 0~1 , this method estimates that the latrine coverage should be increased to at least 17.1%. This translates into increasing the number of latrines from 1,038 to 4,796 (i.e. 3,756 extra latrines): 1 latrine per 7 people. Similarly, the boreholes should be increased from 23 to 230, that is, 17.7%, or 1 bore hole per 26 households. Similar results are obtained using R 0~2 :25 found from the non-linear fitting tool. In this case of Hepatitis E-only, the latrines should be increased to 16.1% and boreholes must cover 16.6% of the population. From the coinfection model, latrines should be increased to 17.5%: 4,908, (3,870 extra), 1 for 6 people. Boreholes should be increased to 18.1%, a total of 234, or 1 bore hole per 26 households.
Our model suggests that to eradicate the epidemic, the minimum number of additional latrines required is 3,47. The average cost of digging and constructing a basic pit latrine is approximately USD 250.00 (quotation from city council official) Therefore, 3,477 would cost a total of USD 869,250.00. Thus, the cost per disability adjusted life year averted in Kitgum, in the case of Hepatitis E is 869,250/7,066 = USD 123.00. In addition to improving hygiene we should consider education. Let us now consider the case of education to the camp dwellers. Taking the simplest and cheapest scenario of hiring twenty (20) guidance and counseling officials to educate the dwellers about hepatitis E for about a month (that is 30) days, moving around the camp. Let us assign each counselor, 10 households per day. We then calculate the total amount in USD that would facilitate such an exercise as shown in Table 3 . From the calculations, it is seen that 104,000 USD would be required. Assuming the success of such an operation this translates into 104,000/7,066 = USD 14.71 cost per disability adjusted life year.
The epidemic of HEV in Kitgum lasted a period of over two years [7] . Within this period, 160 individuals have lost their lives. As a result, the disease burden, the functional limitation and premature mortality have equaled a disability adjusted life years equal to 7,066, even allowing for the relatively low life expectancy in this part of Uganda. This paper provides a case study of how a simple epidemic model can be fit to such an outbreak disease. Two fitting methods have been used; the first, an analytical method and the other based on a freely available fitting tool. Using these methods, a reliable estimate of R 0 &2:2 has been provided.
We then use the model to find the measures to keep R 0 v1. The necessary levels of latrine and bore hole coverages needed to eradicate the epidemic are both around 16 to 18%. Although the cost of construction of the required number of latrines is a one off cost, the benefits are large. Here we show what the benefits would have been in terms of protection against Hepatitis E. However, other diseases due to poor sanitation that have been reported in Uganda, such as cholera and dysentery, could be prevented in the same way [7] . [35] c Per capita rate of recovery from HEV 0.0238-0.1429/day [11] p
The proportion that died during the outbreak 160 9449 [23] b The latent period of HEV 15v 1 s v64/day [26, 32] l
The proportion of humans with latrines 3.7% [11] b
The proportion of humans with bore hole water 0.38% [11] t  Human Bocaviruses Are Not Significantly Associated with Gastroenteritis: Results of Retesting Archive DNA from a Case Control Study in the UK Gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. Despite improvements in detection methods, a significant diagnostic gap still remains. Human bocavirus (HBoV)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between HBoVs, and in particular type-2 HBoVs, and gastroenteritis has previously been made. The aim of this study was to determine the role of HBoVs in gastroenteritis, using archived DNA samples from the case-control Infectious Intestinal Disease Study (IID). DNA extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of HBoV DNA. All samples were screened in a real time PCR pan-HBoV assay, and positive samples were then tested in genotype 1 to 3-specific assays. HBoV was detected in 7.4% but no significantly different prevalence was observed between cases and controls. In the genotype-specific assays 106 of the 324 HBoV-positive samples were genotyped, with HBoV-1 predominantly found in controls whilst HBoV-2 was more frequently associated with cases of gastroenteritis (p<0.01). A significant proportion of HBoV positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. However, the distribution of the untyped HBoV strains was no different between cases and controls. In conclusion, HBoVs, including HBoV-2 do not appear to be a significant cause of gastroenteritis in the UK population. In 2005 a novel parvovirus was discovered in respiratory secretions of young children and was termed Human Bocavirus (HBoV-1) [1] . Other important members of the parvoviridae family include B19 which causes fith disease and human parvovirus 4 (Parv 4) which has not yet been associated with a disease [2] . Parvoviruses in animals are generally associated with systemic disease but also with respiratory and enteric symptoms [3, 4] . Since the discovery of HBoV-1 three other HBoV genotypes have been described, HBoV-2, HBoV-3 and HBoV-4. The association between HBoV-1 and respiratory disease has previously been well established [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19] . Although all HBoVs have also been detected in stool samples with prevalences ranging from ,1% to 20%, only HBoV-2 has been reported to be associated with symptoms of gastroenteritis [20] . Nevertheless, the role of HBoV2 as an aetiological agent of gastroenteritis has not been clearly confirmed, furthermore, to date, no clear association between the presence of HBoV-3 and HBoV-4 and disease has been established [21, 22] .
Recent seroepidemiological studies indicate that exposure to HBoVs occurs early in life and 90% of the population are seropositive by the age of 5, although differences were reported in the seroprevalence of type-specific antibodies to the different HBoVs, which suggested that HBoV-1 infections are more prevalent [23] .
The Infectious Intestinal Disease Study (IID Study) was a large case control study of gastroenteritis carried out in the UK between 1993-1996 [24] with the aim to determine the burden and aetiology of sporadic cases IID in the UK population. Initially, the use of classical microbiology diagnostic methods and electron microscopy (for virus detection) failed to detect a potential aetiological agent or toxin in 49% of the cases [25] . Retesting of the archived samples from this study using molecular methods for the detection of enteric viruses, bacteria and protozoa revealed viruses to be the most common aetiological agents of gastroenteritis,the diagnostic gap for IID was reduced to 25% from 49% [26] . The aim of the present study was to evaluate the role of HBoVs in IID in the UK population, using archived DNA samples from the matched case-control IID-1 study [26] . In addition, the presence specifically of HBoV-1, 2 or 3 was investigated in order to determine any possible associations between specific HBoV genotypes and IID.
A total of 4,380 archived DNA from the IID study [26, 27] were tested for the presence of HBoV DNA. This archive comprised DNA extracted from stool samples from 2,256 cases and 2,124 controls.
The qPCR assay targeted the NS1 gene (Ratcliff et al., unpublished method, personal communication) and was performed using an ABI Taqman7500. Oligonucleotide primer and probe sequences and positions are described in table 1.
The reaction consisted of 0.1 M DDT (Invitrogen), 1X Platinum Quantitative PCR Supermix-UDG (Invitrogen), Pan-HBoV-F and Pan-HBoV-R primers each at a concentration of 100 mm, Pan-HBoV-NS1 probe at 10 mm concentration, ROX 25 mm (Invitrogen) 2.5 ml of template DNA and RNase free water to a final reaction volume of 25 ml. The amplification consisted of an initial denaturation at 95uC for 10 min, followed by 40 cycles with denaturation at 95uC for 15 sec, annealing at 55uC for 30 sec and extension at 60uC for 45 sec.
HBoV-1, 2 and 3-specific primer pair and probes were designed in house through alignment of sequence data available in GenBank. The reaction conditions are as follows; 1X Platinum Quantitative PCR Supermix-UDG (Invitrogen), HBoV-NS1-1F, HBoV-1R, HBoV2-R and HBoV3R primers each at a concentration of 20 mm, the HBoV1,2 and 3 probe at 10 mm concentration, ROX 25 mm (Invitrogen), 2.5 ml of template and RNase free water was added to a final reaction volume of 25 ml. The amplification conditions for the typing assay are the same as those described in the HBoV NS1 detection assay above.
Plasmids containing a 1773 bp and a 1737 bp region of the NS1 encoding gene of HBoV-1 and HBoV-3 respectively were used for assay optimisation and as controls. Control material was kindly provided by R. Ratcliff, Adelaide, Australia.
The controls were also used in order to generate a standard curve for use with the pan-HBoV assay in order to allow for normalisation of the data generated including the comparison of relative sensitivities of the different assays and for quantitation of DNA present in each of the positive samples. The standard curve was generated using the plasmid containing a genome segment of the HBoV-1 and consisted of a series of 10 fold dilution containing from 300,000 copies/ml down to 3 copies/ml. Inter-and intraassay reproducibility was analysed by performing replicate testing of the standards in a single run (X11) and repeated runs (X2), and the standard curve was also included in each assay run for quality control and normalisation of results.
A subset of 17 samples positive in the Pan-HBoV assay but which failed to amplify in the type-specific assays were confirmed using an alternative method published elsewhere [28, 29] , and 6 were further confirmed though direct sequencing of the amplicons obtained after purification either from solution or agarose gels using Agencourt AMPure (Beckman Coulter, USA) and GeneClean Spin kit (QBiogene), respectively, following manufactures protocols.
The chi-squared test was used in order to evaluate the significance of differences observed between groups. For comparison of median values (analysis of CT values) the Mann Witney Utest was used. Prevalence Odds Ratio (POR = Pcases/(1-Pcases)/ Pcontrols/(1-Pcontrols)) was calculated in the total cohorts and by age group. Table 1 . HBoV-specific oligonucleotide primers and probes (all located at the NS gene).
Sequence ( 
A total of 7.4% of the samples tested were positive for HBoV. No statistically significant differences were seen in the prevalence of HBoV between cases and asymptomatic controls, POR = 0.79 (Table 2) . Peak HBoV infection was observed in children under the age of 5, both in cases and controls, with significantly higher HBoV incidence in children between 1 and 4 in asymptomatic controls than in the cases of gastroenteritis (POR = 0.6; p,0.02). The number of HBoV positives in older age groups was too small for meaningful statistical analysis.
The average CT values were 34.5 and 34.8, and the median CT values were 36.3 and 37.4 in cases and controls respectively (see distribution in Figure 1 ). The majority of HBoV-positives in both cases and controls had copy numbers ranging between 30 and 299 copies/reaction (or between 4.5610 3 and 4.5610 4 copies/ml of feaces). The distribution of HBoV viral loads between cases and controls was comparable and the median CT values between cases and controls were not significantly different (U-test; z = 0.458139, p.0.05).
HBoV DNA was found in 149 (46%) samples in the absence of other co-pathogens (Table 3) . No statistically significant differences were observed in the proportion of cases or controls in which HBoV was found as a single organism or in the presence of one or more pathogens in the cohort as a whole, however in the 1-4 years of age group, HBoV in the absence of any other enteric pathogens was found in 29% of the cases, but in 56% of the controls (p,0.05).
HBoV infections were detected year round although a peak was observed in the spring/early summer months, between April and June 1994 (Figure 2 ).
HBoV DNA was found in 48.8% and 45.7% of female cases and controls, respectively. The distribution of HBoV among females and males was not significantly different from the distribution of females and males in the entire cohort which was 53% and 47%, respectively.
A total of 106 (32.7%) HBoV positives were genotyped, whilst 218 (67.3%) remained untyped after testing in the HBoV types 1, 2 or 3 specific assays (Table 4 ). HBoV-1 detection was found predominantly in controls, (p,0.001) and HBoV-2 was predominantly associated with cases (p,0.01). The prevalence of HBoV-3 was not significantly different between cases and controls. HBoV-1 and -3 were predominantly found in children (Table 4) . HBoV-2 in the absence of any other pathogen was detected in 17 (81.3%) of the cases, compared to 9 (47.4%) of the controls. In cases, HBoV-2 was found across the age groups, although more frequently in children ,5, whereas in controls they were found predominantly in children ,5 with only 1 example in an adult ( Table 4 ). The prevalence of HBoV-2 in children ,5 years old was however not significantly different between cases and controls, 2.8% and 2.6%, respectively. A subset of HBoV that were negative in the type 1,2 or 3specific assays were confirmed in an alternative pan-HBoV PCR, and sequencing of a small number confirmed them as types 1, 2 or 3. The majority of the untyped samples (70%) had a CT value of .37 in the screening pan-HBoV PCR, indicative of low viral loads being present in the samples.
This represents the largest study to date investigating the role and distribution of HBoVs infections in community acquired sporadic gastroenteritis and in asymptomatic controls. The prevalence of HBoV infection in the UK population was found to be 7.4% across all ages, with a higher percentage of the infections occurring in children ,5 years of age (19%). However, the prevalence of HBoV infections was comparable in cases of gastroenteritis and in age-matched asymptomatic controls. Although the presence of enteric pathogens, eg norovirus or rotavirus, in asymptomatic individuals is well documented, a significantly higher prevalence of the pathogen is seen in cases than in the controls [26] . Therefore, our data suggests that HBoV are not causally associated with gastrointestinal disease in the UK population as a whole, nor in children. The prevalence of detection of HBoV in stool samples in previous studies varies widely (see summary in Table 5 ), but most coincide in reporting the highest prevalence in children.
HBoV infections were detected all year round in the UK although a tentative peak was observed in the spring/early summer months in 1994 (between April and June). Different seasonal patterns in the peak prevalence of HBoV have been reported in different countries (see Table 5 ),
Of the 324 HBoV positive samples, 106 (32.7%) were genotyped in the type-specific assays. HBoV-1 was found predominantly in controls (p,0.001) and the prevalence of HBoV-3 was similar in cases and controls. Both HBoV-1 and -3 were predominantly found in children. HBoV-2 was predominantly associated with gastroenteritis cases (p,0.01). The overall prevalence in cases was 1.4% and 0.8% in controls, however, in children ,5 year of age, the prevalence in cases and controls was similar, 2.8% and 2.6%, respectively. The prevalence of HBoV-2 in children in the UK was significantly lower than that reported in a study in Australia, in which HBoV-2 was detected in 17.2% and 8.1% of the cases and controls, respectively [22] . The findings of the study in Australia lead to the proposal of HBoV-2 as an important aetiological agent of infantile gastroenteritis. It is noteworthy however, that in the Australian study, the association of HBoV-2 with gastroenteritis was only significant when cases with a bacterial co-pathogen were included in the analysis. Although in the present study HBoV-2 in the absence of other enteric pathogens was found more frequently in cases than in controls, the small numbers found in such large study suggest that the role of these viruses in IID, if any, is likely to be small. A lack of correlation between HBoVs or HBoV-2 and paediatric gastroenteritis was also reported in several smaller studies published elsewhere [30, 31, 32] . A total of 67% of the HBoV-positive samples could not be genotyped using the genotype-specific PCR assays. The majority of these untyped samples (70%) had CT values .37. This suggests that failure to type may be associated with low viral loads and differences in the relative sensitivities of the genotyping assays compared to the detection assay. Although under experimental conditions and using plasmid controls the sensitivities of all assays were comparable, it is likely that when applied to true clinical samples the sensitivity of the type-specific assays was inferior, possibly due to as yet not identified strain variability within genotypes. Also, a HBoV type 4-specific assay was not included in this study, therefore, any possible HBoV4 infections would not have been typed. Of the panel of samples that were tested in an alternative pan-HBoV PCR, the strains typed through sequencing were HBoV-1 (2 samples), HBoV-2 (1 sample) and HBoV-3 (3 samples). Furthermore, the distribution of untyped HBoVs was not significantly different in cases and controls.
HBoVs in the absence of other enteric pathogens were seen in 46% of the HBoV-positive samples, and more frequently in the controls, 50.3% vs 40.9% in cases. No significant difference in HBoV load was observed between cases and controls, or between the samples positive for HBoV alone or in the presence of other pathogens. Previous studies have investigated the relationships between viral load and disease severity [33, 34, 35, 36, 37] . In respiratory infections significantly higher HBoV loads were seen in samples collected from children positive for HBoV alone than in those from children with co-infections. In respiratory infections also, viral loads .10 4 were associated with disease, whereas loads ,10 4 were associated with asymptomatic children [33, 38] . This lead to the suggestion that higher viral loads are indicative of a causative role of HBoV in respiratory infections [33, 38] . However, Brieu et al [38] found no significant correlation between viral load and clinical symptoms or disease severity.
In conclusion, the results obtained from investigating for the presence of HBoV DNA in archived DNA samples from a large and previously well described case-control study of IID suggest that HBoV, including HBoV-2,do not appear to be a significant cause of gastroenteritis in the UK population, and particularly in the paediatric population. Although HBoVs are relatively frequent across all ages, and in particular in preschool age children, they are found just as frequently among children and adults without symptoms of gastroenteritis. Structural Origins for the Loss of Catalytic Activities of Bifunctional Human LTA4H Revealed through Molecular Dynamics Simulations Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents. Leukotriene cascade is associated with the biosynthesis of variety of leukotrienes (LT) from the phospholipids of the nuclear membrane of the leukocytes [1] . The LTs are a group of lipid mediators associated with acute and chronic inflammatory diseases such as asthma, rhinitis, psoriasis, chronic obstructive pulmonary disease, and atherosclerosis [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . Cytosolic phospholipase A2 (cPLA2) hydrolyzes the ester bond present in sn-2 position of phospholipids and yields lysophospholipids and free fatty acid, arachidonic acid (AA) [1, 14] . This increases the level of free AA available for the synthesis of inflammatory leukotrienes upon the action of more enzymes. The enzyme 5-lipoxygenase (5-LO) assisted by file-lipoxygenase-activating protein (FLAP) converts the AA into the highly unstable allylic epoxide, leukotriene A4 (LTA4) [15] [16] [17] [18] [19] [20] [21] . This unstable intermediate is converted into two different products LTB4 and LTC4 by the action of two different enzymes LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), respectively [1, [22] [23] [24] [25] . The LTC4 is subsequently converted to LTD4 and LTE4 substances by the action of different enzymes. All of these LTB4, LTC4, LTD4, and LTE4 are powerful proinflammatory mediators [1, 26] . The LTA4H, which catalyzes the conversion of LTA4 to the chemotactic agent LTB4, was identified as a bi-functional enzyme capable of processing two highly diverse substrates such as LTA4 (a fatty acid) and peptide through its epoxide hydrolase and aminopeptidase activities [27, 28] . This enzyme was first discovered for its epoxide hydrolase activity and later for its aminopeptidase activity based on the presence of consensus Zn binding motif (HEXXH-X 18 -E), which was found in M1 family of Zn containing aminopeptidases [29] [30] [31] [32] . The natural peptide substrate for this enzyme is still not known but preference is shown over arginyl di-and tripeptide and can selectively be blocked by the mutation of either E296 or Y383 residues [33] [34] [35] [36] . Upon the determination of LTA4H crystal structures it was revealed that this enzyme is composed of three domains, a fully beta N-terminal domain, a mixed alpha/beta catalytic domain, and a fully alpha-helical C-terminal domain ( Figure 1 ) [37] [38] [39] [40] [41] [42] . In terms of the hydrolase activity of the enzyme, D375 from a narrow hydrophobic pocket is specifically required as it is involved in the nucleophilic attack targeting C12 atom of LTA4 [43] . In addition, this residue belongs to the peptide K21 (L365-K385) segment identified by Lys-specific peptide mapping of suicide inactivated LTA4H. The carboxylate moiety of LTA4 was observed to form direct electrostatic interactions with the two positively charged conserved R563 and K565 residues present at the entrance of the active center [28, 44] . These interactions are very much essential in aligning LTA4 along with the catalytic elements of the active site. Based on the mutagenic experiments, E271 residue from another conserved GXMEN motif in the family of zinc peptidases was found to be important for both the functions of the enzyme [14] as the mutagenic replacements abrogated both the activities. A crystal structure of LTA4H with E271Q mutation has revealed only minimal conformational changes and did not explain the loss of enzyme function [14] . It was also suggested that the carboxylate of E271 participates in an acid-induced opening of the epoxide moiety of LTA4 and as N-terminal recognition site in terms of peptide substrates [14, 26, 45] . Some mutagenic experiments have also reported the critical role of R563 residue in epoxide hydrolase reaction by positioning the carboxylate tail along the catalytic elements of the active site [28, 34] . In aminopeptidase reaction, both R563 and K565 residues co-operate each other to ensure the necessary binding strength and productive alignment of the substrate. Altogether, R563 plays important roles in both the functions of the enzyme whereas K565 residue assists R563 in catalyzing the peptide substrate but not in hydrolase reaction, which catalyzes the fatty acid substrate [27] . These residues were reported to be the common carboxylate recognition site for both lipid and peptide substrates in the active site of LTA4H [27] .
Mutations of R563 to any other amino acid including a conservative replacement of R by K preserving the positive charge abolished the enzyme function but exhibited a significant residual aminopeptidase activity [27] . Crystal structure with R563A mutant could not reveal any structural changes explaining the complete loss of catalytic activity. It was also reported that esterified LTA4 cannot be the substrate of the enzyme and this phenomenon was explained with the steric hindrance [46] , which also proved that a free carboxylate group of LTA4 is critical for the hydrolase function. The other carboxylate recognition residue K565 located in a way that it can also involve in carboxylate recognition but its mutagenic replacements have not decreased the epoxide hydrolase activity [27] . The difference in observed aminopeptidase activities between the wild type and K565 mutants have suggested that K565 is a carboxylate binding site for peptide substrates also [27] . The information of a binding pocket for its ligand is very important for drug design, particularly for conducting mutagenesis studies [47] . In the literature, the binding pocket of a protein receptor to a ligand is usually defined by those residues that have at least one heavy atom (i.e., an atom other than hydrogen) with a distance from a heavy atom of the ligand. Such a criterion was originally used to define the binding pocket of ATP in the Cdk5-Nck5a* complex [48] that has later proved quite useful in identifying functional domains and stimulating the relevant truncation experiments [49] . The similar approach has also been used to define the binding pockets of many other receptor-ligand interactions important for drug design [50] [51] [52] [53] [54] .
This study has focused on structural changes and binding mode differences between wild types and mutant forms of hLTA4H-fatty acid and -tripeptide substrate complexes. Molecular dynamics (MD) simulations of two wild types of the enzyme-substrate complexes and three mutations including E272Q, R563A, and K565A were studied with LTA4 (fatty acid) and Arg-Ala-Arg (RAR, tripeptide) substrate ( Figure 2 ). The binding mode comparison of enzyme-substrate complexes revealed essential structural differences, which were not shown in X-ray structures, explaining the loss of catalytic functions of the enzyme. The overlay of binding modes of LTA4 and RAR substrates has proved the previous assumption of similar but overlapped active site is used in both the functions of the enzyme. The results from this study provide valuable information over the way the two functions of the enzyme are exerted over two substrates of diverse nature. In addition, the structural information obtained from this study can be utilized in structure-based hLTA4H inhibitor design as inhibition of the hydrolase and aminopeptidase functions will lead to the development of anticancer and anti-inflammatory drugs, respectively.
As this study was aimed at investigating the binding of fatty acid and peptide substrates of the enzyme, independent hLTA4H-LTA4 (L-LTA4) and hLTA4H-RAR (L-RAR) complexes were prepared. The LTA4 of AA cascade is the natural substrate of the enzyme but no 3D structural information was available for its binding from X-ray studies. Some previous studies have reported the proposed binding of this fatty acid substrate at the active site of the enzyme. In this study, molecular docking methodology using GOLD 5.0.1 program was employed to obtain the reliable binding mode of LTA4. The GOLD program from Cambridge Crystallographic Data Centre, UK uses a genetic algorithm to dock the small molecules into the protein active site [46, 55] . The GOLD allows for a full range of flexibility of the ligands and partial flexibility of the protein. One of the crystal structures of hLTA4H bound with most active inhibitor (PDB ID: 3FUN) solved at high resolution was used in molecular docking. The bound inhibitor of this crystal structure was removed and the apoform of this enzyme was subjected to MD simulations with the parameters discussed below. The representative structure with a RMSD value close to the average structure of last 3 ns of the 5 ns MD simulations was selected and utilized in molecular docking experiments. The active site was defined with a 10 Å radius around the bound inhibitor. The ten top-scoring conformations of every ligand were saved at the end of the calculation. Early termination option was used to skip the genetic optimization calculation when any five conformations of a particular compound were predicted within an RMS deviation value of 1.5 Å . The GOLD fitness score is calculated from the contributions of hydrogen bond and van der Waals interactions between the protein and ligand, intramolecular hydrogen bonds and strains of the ligand [56] . Protein-ligand interactions were analyzed using DS and Molegro Molecule Viewer [57] . The best pose was selected based on the molecular interactions and the distance between epoxy group of LTA4 and metal ion (Zn 2+ ) present in the active site as well as the location of its carboxylate group which interacts with the carboxylate recognition residues R563 and K565. Finally, the enzyme-LTA4 complex was prepared to be used in further steps in this study.
In terms of preparing the enzyme-peptide complex to investigate the aminopeptidase function of LTA4H enzyme, 3D coordinates of the bound tripeptide Arg-Ala-Arg (RAR) in a solved X-ray structure of hLTA4H with a mutation E271Q (PDB ID: 3B7T) was utilized. The Superimpose Structures protocol as available in Accelrys Discovery Studio 3.0 (DS) was employed to copy the 3D coordinates of this tripeptide into the representative structure of LTA4H picked from the 5 ns MD simulation by superimposition. This complex was subjected to energy minimization using Energy Minimization protocol of DS before considered further in this study.
Many marvelous biological functions in proteins and DNA and their profound dynamic mechanisms, such as switch between active and inactive states [58, 59] , cooperative effects [60] , allosteric transition [61, 62] , intercalation of drugs into DNA [63] , and assembly of microtubules [64] , can be revealed by studying their internal motions [65] . Likewise, to really in-depth understand the action mechanism of receptor-ligand binding, we should consider not only the static structures concerned but also the dynamical information obtained by simulating their internal motions or dynamic process. To realize this, the MD simulation is one of the feasible tools. Initial coordinates for the protein atoms were taken from the wild type (WT) and mutant forms of both L-LTA4, L-RAR complex structures. Mutations were introduced at E271Q, R563A, and K565A of the enzyme based on the previous experimental reports to investigate the single active site that catalyzes two different functions upon diverse substrates [14, 45] . The protonation states of all ionizable residues were set to their normal states at pH 7. Eight MD simulations were performed for systems including WT and mutant forms of L-LTA4 and L-RAR complexes ( Table 1 ). All MD simulations were performed with GROMOS96 forcefield using GROMACS 4.5.3 package running on a high performance linux cluster computer [66, 67] . During the MD simulations, all the protein atoms including divalent metal ion (Zn 2+ ) were surrounded by a cubic water box of SPC3 water molecules that extended 10 Å from the protein and periodic boundary conditions were applied in all directions. The systems were neutralized with Na + and Cl 2 counter ions replacing the water molecules and energy minimization was performed using steepest descent algorithm for 10,000 steps. A 100 ps position restrained MD simulations were performed for every system followed by 5 ns production MD simulations with a time step of 2 fs at constant pressure (1 atm), temperature (300 K). The electrostatic interactions were calculated by the PME algorithm and all bonds were constrained using LINCS algorithm. A twin range cutoff was used for long-range interactions including 0.9 nm for van der Waals and 1.4 nm for electrostatic interactions. The snapshots were collected at every 1 ps and stored for further analyses of MD simulations. The system stability and behavior of the catalytic structural components present in every system were analyzed using the tools available with GROMACS 4.0.5 and PyMol programs.
Surprisingly, the LTA4H enzyme catalyzes both hydrolase and aminopeptidase functions over fatty acid and peptide substrates utilizing the same active site. In order to obtain deeper insight upon this unique characteristic of the enzyme, a set of MD simulations were performed with WT and mutated enzymesubstrate complex structures. The natural epoxy substrate LTA4 of arachidonic acid pathway, which is converted to LTB4 upon the action of the enzyme, was selected as fatty acid substrate to investigate the hydrolase function of the enzyme. In the other hand, RAR tripeptide that is reported to be the preferred peptide substrate of the enzyme [14] was selected to investigate the aminopeptidase function of the enzyme. The L-LTA4 complex was prepared through the molecular docking methodology whereas L-RAR complex was prepared by copying the 3D coordinates of RAR from the X-ray crystal structure of LTA4 ( Figure 3 ). The representative structure obtained from the MD simulation of LTA4H-apoform was used in preparing both the complex structures to compare the structural changes effectively with no artifacts. From the reported site-directed mutagenesis experiments, three amino acid residues from the catalytic active site of the enzyme were predicted to be very important for the enzymatic activities of the enzyme. These residues include one negatively charged E271 residue from the central catalytic domain and two positively charged R563 and K565 residues from the Cterminal domain. Studies mentioned that mutation of this negatively charged residue to a neutral glutamine (E271Q) has completely abrogated both the catalytic activities of the enzyme. But the crystal structure solved with this mutation (PDB ID: 1H19) could not report any structural or conformational changes causing this drastic change in the catalytic activity [14] . It was also proposed that metal (Zn 2+ ) ion present close to the epoxide moiety of LTA4 acts as a weak Lewis acid to activate and open the epoxide ring. It continued to explain that the E271 located in proximity to Zn 2+ also participates in this acid-induced opening of the epoxide ring of LTA4. It was also reported that E271 acts as the N-terminal recognition point in stabilizing the peptide substrates in terms of aminopeptidase reaction of the enzyme. The positively charged residues R563 and K565 were predicted as the carboxylate recognition sites for both the substrates of the enzyme. All mutations of R563 including R563K, which preserved the positive charge, have resulted in complete loss of catalytic functions of the enzyme. The R563K mutant has shown a significant aminopeptidase activity. All these mutations of R563 leading to tremendous change in enzyme functions did not reveal any structural changes explaining the loss of activities. Especially in epoxide hydrolase reaction, the role of R563 was presumed to position the carboxylate tail of the substrates along the catalytic components of the active site [45] . In terms of aminopeptidase reaction, both the positively charged (R563 and K565) residues help each other in aligning the substrate with the catalytic elements and maintaining the binding strength. The mutations K565A and K565M lacking the positive charge have reduced the aminopeptidase and revealed that this positively charged residue assists R563 in carboxylate recognition in aminopeptidase reaction [27] . Despite of this information over the importance of E271, R563, and R565, the structural changes explaining the catalytic activities of the enzyme are lacking. The results of MD simulations of WT and mutant enzyme-substrate complexes discussed in this study will provide a deeper insight from the structural perspective.
Overall structural stability of the systems The overall stability analyses are considered important to note that the systems did not undergo any unusual changes during the time scale of simulation because of erratic system preparation. In this study, root mean square deviation (rmsd), root mean square fluctuation (rmsf), and intra-molecular hydrogen bonds were used in analyzing and comparing the stability of the systems under study (Figure 4) .
In terms of L-LTA4 systems, the calculated average rmsd value of K565A system was 0.158 nm which is lower than the rmsd values of WT (0.189 nm), E272Q (0.186 nm), and R563A (0.202 nm) systems ( Figure 4A ). The R563A system has shown the higher rmsd value and thereby indicating the additional effect of this particular mutation. Despite of these small differences in the rmsd values between the L-LTA4 systems, the systems were stabilized well throughout the timescale of simulation. As another method to investigate the stability of the systems, rmsf values of all systems were calculated during the simulation and plotted. From the plot, it was observed that none of the active site residues were fluctuating higher than 0.2 nm and explained the stable nature of the systems over the time scale of simulation. In addition, the number of intramolecular hydrogen bonds were calculated for all the systems and plotted. The average number of hydrogen bonds revealed that R563A system has formed more number of hydrogen bonds (466.9) compared to WT (452.8), E272Q (451.7) and R565A (450.6) systems which displayed the reduced number of hydrogen bonds. This result also has confirmed the stability of the systems despite of small differences between systems ( Figure 4A ).
In L-RAR systems, the average rmsd value of K565A (0.212 nm) was higher than other systems, which is completely contrasted to the rmsd value of equivalent L-LTA4 system whereas the R563A system has shown the average lower rmsd value (0.168 nm). The other two systems, WT and E272Q, have shown the same average rmsd value of 0.189 nm from last 3 ns of the simulation time ( Figure 4B ). In terms of rmsf calculations, the rmsf plot has shown that except D375 and Y378 residues of the active site all other important active site components were stable throughout the simulation. This high fluctuation of these two residues was mainly observed in K565A system. The average number of intramolecular hydrogen bonds during last 3 ns of the simulation was very similar in all the systems. At the end of the simulation time, the R563A and K565A systems started losing their hydrogen bonds and thereby became less intact compared to other systems. All the systems were investigated for the stability and found to be well stabilized during the simulation. Thus the representative structures close to the least rmsd value of each system was obtained and used in structural comparison.
The distance between the most important metal (Zn 2+ ) ion and oxygen atom of the epoxy ring in case of L-LTA4 systems is very important for the hydrolase function of the enzyme. The average distance value observed in WT system (0.56 nm) was lower compared to the mutant systems. Among mutant systems R563A system has shown the higher average distance value of 0.78 nm whereas E271Q and K565A systems have shown similar average distance value of 0.69 nm and 0.63 nm, respectively. During the end of the simulations, this distance in E271Q and K565A systems has reduced close to the distance of WT but R563A has maintained higher distance until the end ( Figure 5A ). In terms of L-RAR systems, the distance between the same metal ion and carbonyl oxygen atom of N-terminal peptide bond was measured and compared between the L-RAR WT and mutant systems. As observed in L-LTA4 systems, WT of L-RAR systems has maintained the lower average distance value of 0.59 nm whereas Figure 5B ). The hydrogen bonds formed between the protein and bound ligands were also calculated for all the systems under study to investigate the molecular interactions that are lost during the mutations. In L-LTA4 systems, WT system has formed high number of average hydrogen bonds (5.7) compared to the mutant systems. The K565A system has shown an average number of hydrogen bonds of 5.0 whereas E271Q and R563A systems have formed only 1.7 and 1.8 average number of hydrogen bonds with the bound ligand ( Figure 6A ). This reduced number of hydrogen bonds correlate well with previously reported loss of hydrolase activity of the enzyme in E271Q and R563A mutated systems [14, 45] . In terms of L-RAR systems, the E271Q system has formed more number of average hydrogen bonds (8.7) with the bound RAR. Whereas the WT system has shown an average hydrogen bond value of 8.0, the other mutant systems R563A and K565A have shown the average hydrogen bond values of 4.3 and 3.9, respectively ( Figure 6B ). This result of number of hydrogen bond values observed between protein and RAR has proved the importance of both R564 and K565 to maintain the substrate alignment in the active site and the binding strength of the substrate. But the high number of observed hydrogen bonds in E271Q system does not correlate with the observed loss of catalytic activity due to the mutation of E271. This indicated that other structural disturbances script the loss of catalytic function of the enzyme.
L-LTA4 systems. In L-LTA4 systems, the binding mode of LTA4 in WT system has formed hydrogen bonds with Y383 through the oxygen atom of epoxy ring and the distance between this oxygen atom and the metal ion was maintained in a closer distance compared to that of mutant systems. The carboxylate group of LTA4 in WT system was well recognized by both the positively charged residues R563 and K565 which are reported to be the carboxylate recognition sites. Because of this recognition the carboxylate group has formed strong hydrogen bond interactions with R563 and K565. The distance between the carboxylate of E271 and metal (Zn 2+ ) ion was maintained in close vicinity for the reported acid-induced catalytic reaction. The other residue Y378 has also formed a hydrogen bond interaction with the carboxylate group of LTA4 ( Figure 7A ). In E271Q system, the binding mode of LTA4 is so different from that of WT system. Because of the uncharged nature of the mutation (E271Q) the metal ion has slightly moved towards the carboxylate group of LTA4, which has also mutually moved towards the metal ion. This movement of carboxylate group of LTA4 has moved the central epoxy group of LTA4 further down and made it inaccessible by the catalytic metal ion ( Figure 7B ). This change in the E271 and distance between Zn 2+ and epoxy group (0.69 nm) can be directly correlated with the loss of activity ( Figure 5A ). The hydrogen bonds formed with R563 and K565 residues were completely lost because of this mutation. In R563A system, though E271 residue has maintained its hold on the metal ion because of the absence of R563, the important carboxylate recognition site, the LTA4 has moved backwards into the hydrophobic cavity formed by hydrophobic residues such as W311, F362, K364, L365, V366, V367, and V381 ( Figure 7C ). This change has not only brought the distance between Zn 2+ and the epoxy ring of LTA4 higher (0.78 nm) compared to any other L-LTA4 systems but also made hydrogen bonding with K565 impossible. In terms of K565A system, the binding mode of LTA4 was similar to that of WT system. Regardless of mutated K565 the hydrogen bonds were maintained with R563 but still the distance between Zn 2+ and epoxy ring of LTA4 was higher (0.68 nm) in this mutant system as well. The hydrogen bond between LTA4 and Y378 was also maintained as observed in WT system ( Figure 7D ). This observation also correlated the experimental observation that K565A mutation reduces the activity but does not abolish it.
L-RAR systems. The binding modes of the tripeptide RAR in all systems were observed and compared to investigate the changes due to the mutated residues. More number of hydrogen bonds was observed between the protein and bound substrate compared to the L-LTA4 systems because of the high number of polar hydrogen in the peptide substrate. In the WT system, strong molecular interactions were observed through the hydrogen bonds and p-cation interactions formed between protein and substrate ( Figure 8A ). The C-terminal carboxylate which is equivalent to the carboxylate of LTA4 has formed strong hydrogen bond interactions with both positively charged residues R563 and K565, the carboxylate recognition sites. These interactions mainly hold the RAR at the active site and improve its binding strength. As reported, the N-terminal amino group interacted with the E271 which is the N-terminal recognition site for the peptide substrates.
These interactions altogether brings the carbonyl oxygen atom of N-terminal peptide bond close to the catalytic metal ion. A pcation interaction was formed between the side chain of Cterminal Arg residue and Y378, which was found highly fluctuating in rmsf analysis. This p-cation interaction between the same atoms was observed in R563A and K565A systems as well whereas it was between Y383 and C-terminal Arg residue in E271Q system (Figure 8 ). The binding mode of the substrate in E271Q system was different to that of WT system. The strength of the hydrogen bonds with R563 and K565 has become weak in this system because of the conformational changes of both carboxylate of substrate and R565 residue ( Figure 8B ). The metal ion located in the active center was thrown away from its initial position because of the absence of negatively charged E271. This behavior observed in this system clearly reveals that E271 acts as a hook to hold the Zn 2+ ion in the active site. In terms of R563A system, the observed binding mode of this system is similar to that of E271Q system ( Figure 8C ). The metal ion was hooked by the presence of E271 residue but still the distance between Zn 2+ and the carbonyl oxygen atom of N-terminal peptide bond was high compared to that of WT. The hydrogen bonds were formed between Y378, Y383, and G269 residues. Surprisingly, two hydrogen bonds were formed with K565 residue in absence of R563. This is different compared to that of the equivalent L-LTA4 system where the hydrogen bonds with both the positively charge residues were completely lost. This observation indicates the importance of K565 in assisting R563 in carboxylate recognition during peptide binding. In K565A system, the binding mode of the substrate is folded and completely different to the other systems. The hydrogen bonds were observed only with Y383 and A565 residues along with an additional p-s interaction between Y378 and C- terminal Arg residue ( Figure 8D ). No hydrogen bonds were formed with R563 residue, which is one of the positively charged carboxylate recognition site residues. The molecular interactions observed in R563A and K565A systems revealed that both the residues are important for recognizing the carboxylate group and aligning the peptide substrate along the catalytic elements as reported.
Active site structural changes L-LTA4 system. The overlay of each mutant system with the WT system has allowed observing the structural changes occurred because of the mutations (Figure 9 ). In E271Q system, the loop of G269 was fluctuating and moved into the active site when compared to other systems. This change observed in this loop could be because of the newly formed hydrogen bond between G269 and the tilted carboxylate group of LTA4, which was found to be a response to the metal ion that lost the interaction with mutated E271. The carboxylate group of LTA4 and terminal NH 2 of R563 moved away from each other (2.7 to 8.4 Å ) because of the missing E271. A short beta sheet formed by the residues V306-N308 of HEXXH-(X) 18 -E motif that possess two catalytically conserved histidine residues coordinating with Zn 2+ ion disappeared in E271Q system. Another helix followed by this short beta sheet was extended by four amino acids W311-F314 making the important F314 slightly backward from the active center ( Figure 9A ). Two tyrosine residues Y378 and Y383 located opposite to each other in the active site were highly fluctuating in this system to adjust the binding of the LTA4. The K565 residue present in the loop has become a part of the long helix originally formed by T567-A575 residues during the simulation of E271Q system. This change slightly drew back the K565 residue from the active site. The other important positively charged residue did not show any structural changes during the simulation. The R563A system has shown different structural changes compared to E271Q system. The loop containing G269 has shown slight fluctuation only at the location of G269 because of the hydrogen bonds formed between the carboxylate of LTA4 and G269 but the lower part of the loop was stable unlike E271Q system ( Figure 9B ). The short helix formed by V306-N308 residues was maintained in this system and the helix containing F314 was extended but kept for the same length during the simulation. The region (W311-F314) that turned an extended helix was highly fluctuating in this system because of the moving alkyl part of LTA4. This backward movement buried the alkyl part into the hydrophobic pocket, formed by a mixture of aliphatic and aromatic hydrophobic residues (W311, F362, K364, L365, V366, V367, and V381), was observed because of the missing interactions with R563 residue (not shown in figure). Unlike E271Q system, Y378 residue has shown only slight side chain movement as a response to the moving LTA4 whereas Y383 did not show any fluctuations from its initial position. The same helix extension was observed as in E271Q system and thus K565 was included in helix formed by T567-A575 residues. The missing R563 led to the loss of correct alignment of LTA4 along the catalytic elements and severe instability of the binding mode of LTA4. In the final mutant (K565A) system, the G269 loop was completely stable and no hydrogen bond interaction was observed between LTA4 and G269 residue. The short helix of V306-N308 disappeared during the simulation of K565A system as observed in E271Q system ( Figure 9C ). The helix of F314 was extended as seen in R563A system and thus has shown the mixed characteristics of E271Q and R563A systems. The Y378, one of the oppositely located pair of tyrosine residues, has fluctuated highly in this system. The binding mode of LTA4 was quite similar to that of WT except its carboxylate group, which moved back because of the missing hydrogen bonds from K565 residue. But the hydrogen bonds with R563 were maintained and thus kept the alignment of LTA4 along with the catalytic elements. The overlay of active sites of all the systems have made clear about the structural changes where Y378 was observed to be fluctuating differently in each system maintaining a close distance with the substrate ( Figure 9D ). Thus Y378 residue, along with the carboxylate recognition site residues R563 and K565, can play a key role in aligning the substrate at the active site.
L-RAR systems. Comparison of active site residues of WT and E271Q systems using the representative structures obtained from the simulations revealed the structural changes led to the differences in the catalytic activity of the enzyme ( Figure 10 ). The overlaid WT and E271Q structures have shown the difference in the locations of catalytically important Zn 2+ ion in the catalytic center ( Figure 10A ). The uncharged nature of the mutant residue Q271 the metal ion has lost the important interaction and moved far away from its original location. The distance between the Zn 2+ ion and the oxygen atom of the N-terminal peptide bond was so high compared to the WT system. Thus the catalytic aminopeptidase reaction becomes impossible in E271Q system. The helix containing Y383 was extended during the simulation E271Q system moving Y383 backward from the active site. This movement of Y383 has formed p-cation and a hydrogen bond interactions with the C-terminal part of RAR whereas in WT system this residue has formed a hydrogen bond interaction with the N-terminal amino group. The other tyrosine residue Y378, which was found to be guiding the substrate along with the carboxylate recognition site residues, has formed hydrogen bond with the carboxylate of RAR. The binding mode of RAR observed in E271Q system has shown only weak hydrogen bonding interactions with R563 as it was moving away from it. The helix formed by K565-A575 residues was shortened slightly leaving K565, one of the carboxylate recognition residues, as a part of loop making it more flexible. But this residue has maintained hydrogen bond interactions with the peptide substrate through its carboxylate group. This change observed in helix containing K565 is different from that of L-LTA4 systems. The K565 is the key residue that assists the other positively charged residue R563 to maintain the proper alignment of RAR in the active site whereas in LTA4 binding K565 is not required to assist R563 residue [27] . Moreover, the absence of negatively charged E271 residue also played a major role in observed loss of catalytic activity. The R563A mutation has caused a high fluctuation of E271 that moved far from the N-terminal amino group of RAR and makes it impossible to act as N-terminal recognition site ( Figure 10B ). The interacting distance between Zn 2+ and E271 was maintained in this mutation. The helix extension was observed near Y383 as displayed in E271Q system and this changed the flexibility of Y383 in the active site. The other tyrosine residue Y378 was highly fluctuating in this mutant system compared to any other systems and formed strong p-cation interaction than it is in WT system. The C-terminal part of RAR substrate has slightly went back as it missed the strong interactions from R563 but the alignment was almost maintained as observed in WT system except the side Cterminal side chain of RAR. Interestingly, K565 has taken the location of R563 in this mutant system to maintain the hydrogen bonds with the carboxylate of RAR and there was no change observed in the K565-A575 helix as observed in E271Q system. The K565A system also has shown some structural changes that were not observed in other L-RAR systems ( Figure 10C ). The short helix (V306-N308) that has shown structural changes in L-LTA4 system disappeared in K565A system and the F314 helix was extended including W311-F314 residues. These changes were observed in other L-RAR systems. The extension of helix has drawn F314 residue back from the active site center. A folded binding mode of RAR was observed in K565 system much different from that of WT and other mutant L-RAR systems. Though R563 is present, the carboxylate group of RAR has moved back from its original position and formed p-interactions with Y378 and completely lost interactions with R563. This observation completely correlates with the observed activity and the reported statement that K565 assists R563 to act as carboxylate recognition site in aligning the substrate along the catalytic elements of the enzyme. The overlay of all L-RAR systems revealed that along with E271, R563, K565 residues, Y378 and Y383 were also important in keeping the peptide substrate aligned within the active site ( Figure 10D ). As observed in L-LTA4 systems, Y378 residue has acted as a baffle to control the binding modes of the substrates. This part of the study has documented various structural changes explaining the differences in activities between WT and mutated forms of the enzyme bound to its two different substrates which were not determined by the X-ray crystallography so far (Table S1 ).
The binding modes of fatty acid and peptide substrates that are catalyzed by hydrolase and aminopeptidase functions of the enzyme using same active site were compared. The overlay of two WT systems has given the overview of which parts of the active site were occupied by these two highly diverse substrates. Both the substrates bind perpendicular to each other occupying majorly the different portion of the active site and sharing the carboxylate recognition sites in common ( Figure 11A ). The long alkyl part of LTA4 was snuggly bound into the hydrophobic pocket formed by a mixture of aliphatic and aromatic residues including W311, F314, K364, L365, V367, Y378, and V381. The side chain of Cterminal Arg residue of RAR was fit into the small cavity formed by F356, Y378, S379, M564, K565, and R568 residues. The overlay of active site residues has shown very few structural changes between the WT systems of L-LTA4 and L-RAR systems including the side chain movements of Y378, Y383, and K565 residues ( Figure 11B ). The K565 residue was present in the loop in WT of L-LTA4 system and in the extended helix in WT of L-RAR system and thereby changing the flexibility and interacting behavior of K565. This adds explanation to the importance of K565 residue in assisting R563 residue in aligning the peptide substrate whereas this residue is not required in aligning fatty acid substrate.
In this study MD simulation methodology was used to simulate hLTA4H enzyme complexed with its two diverse substrates along with mutated key residues. The aim of this study was to investigate the structural and conformational changes in the bi-functional active site of the enzyme reflecting on the catalytic activity. This was considered very important and necessary to script the reasons for the observed loss of activity due to particular mutations as the solved X-ray structures failed to show the structural changes. Eight systems including two WT, enzyme-LTA4 and enzyme-RAR complexes along with three independent mutations (E271Q, R563A, and K565A) in each complex were simulated in this study. The observed hydrogen bond interaction network and distance between the catalytically important atoms have correlated well with the experimental results. The E271 residue which is considered very important for both functions of the enzyme and E271Q systems have revealed from our study that this residue acts as a hook to hold the catalytic metal ion at its location and also plays a role of N-terminal recognition point for the aminopeptidase function. Both the E271Q systems have lost the expected binding mode of the substrates for the successful catalysis. The other mutant R563A and K565A systems have also revealed the structural changes and binding mode differences explaining the loss of activity in mutant systems. In L-LTA4 systems, the substrate binding mode in R563A system has changed completely that the long alkyl chain of LTA4 was completely buried into the hydrophobic pocket. This difference in binding mode of LTA4 was completely because of the loss of hydrogen bond interaction with R563 residue. In terms of L-RAR systems, the same mutation R563A has affected the binding mode of RAR and N-terminal recognition through E271 residue in peptide binding. Because of this missing N-terminal recognition the catalytic distance between the metal ion and the carbonyl group of the N-terminal peptide bond was high in this system. The K565A systems in both the substrate complexes have shown different structural changes. In L-LTA4 system the binding mode of the substrate was very much similar to the WT explaining the less importance of K565 in LTA4 binding whereas in L-RAR system the binding mode has lost both the N-terminal and C-terminal recognitions leading to the loss of activity. These results obtained from this study can be effectively used in designing future hLTA4H inhibitors as anti-inflammatory and anti-cancer therapeutics. Table S1 The structural changes which were not seen in experimental studies observed through the MD simulation studies.  Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses. Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix. Bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects [1] [2] . In addition, there is also a possible release of biowarfare agents in a man-made bio-terror event. A number of studies demonstrated that the respiratory tract can be colonized with disease organisms [3] [4] [5] . Through talking, coughing, sneezing or singing, the potential virulent organisms can be exhaled and spread into the ambient environment [6] , which accordingly causes air contamination. For example, SARS in 2003 and H1N1 in 2009 outbreaks were shown to be attributed to the airborne route of disease transmission [7] [8] [9] [10] .
Among many other diseases, respiratory infection accounts for 23.3-42.1% of the total hospital infections [11] , and is listed as the third leading killer [12] . However, present diagnosis procedures using nasal swabs, bronchoalveolar lavages, nasopharyngeal aspirates or sputum samples, appear to cause unpleasant experiences in addition to long detection time. During flu outbreaks, body temperature or isolation procedures are often used to control and prevent further spread, however such methods are lacking scientific evidence and not always effective with those patients infected but in latent period. On another front, exhaled breath condensate (EBC) as a simple and noninvasive method is increasingly being utilized in early disease screening and infectious aerosols measurements, e.g., lung cancer [13, 14] , asthma [15, 16] , and other respiratory problems [17, 18] . In previous studies, human influenza A viruses were detected in exhaled breath using EBC [19, 20] as well as filter [21] , mask [22, 23] and a liquid sampler [24] . In another study, foot-and-mouth disease viruses were also found in the exhaled air from experimentally infected cattle [25] . In addition, high levels of bacterial concentrations in EBC were also observed in other studies [26] [27] [28] [29] . It was recently shown that exhaled breath could be also analyzed for fungal infection by relevant biomarker, e.g., 2-Pentyl furan (2PF) for aspergillosis [30] . Overall, EBC has demonstrated great potential and advantages in early disease screening and diagnosis [31] , opening a new arena for studying airway inflammation and chemistry [32] . Recently, Vereb et al (2011) suggested that exhaled breath can be also used for assessing a variety of environmental exposures [33] .
For EBC related studies, the first key step is the collection of exhaled breath. Over the years, a variety of devices (Table S1 , Supporting Information) were developed including Rtube collection system (Respiratory Research, Inc, Charlottesville, VA) and EcoScreenH condenser (Erich Jaeger Gmbh, Wurzbur, Germany). Typically, these devices would be able to collect 1000 ml of EBC samples within about 10 min, however the collection often comes with a lengthy procedure and a higher cost. For example, use of the EcoScreen involves 7 steps: 1) turn on to cool, 2) clean collection tube, 3) clean condensation chamber insert, 4) retrieve cooling sleeve from freezer, 5) sample collection, 6) sample storage and transport, 7) removal of sample (Respiratory Research, Inc, Charlottesville, VA). The RTube eliminates the first 3 steps, but each collection still requires 10 min and costs $23.25 (Respiratory Research, Inc, Charlottesville, VA) compared to 31 min and $47.17 per collection for the EcoScreen. These collection devices are generally expensive, e.g., the EcoScreen costs around $9000. A recent study compared the sampling efficiency of the Rtube (widely used EBC collection device) with that of throat swab method, showing detection rates of 7% and 46.8% for the Rtube and the throat swab method, respectively [20] . It was suggested that the RTube is not applicable for viral detection in exhaled breath [20] . In addition, condenser coatings [34] , sampling temperature [35] and sampling times [36] were shown to affect physical collection efficiencies of available EBC collectors. Among others, the noted problems with these available EBC collection devices are the device availability, reusability and possible cross contamination [37] , which would negatively impact their wide applications. In addition, EBC collection is strictly limited to the method condensation only in most studies [38] . To fully utilize EBC in early disease screening, diagnosis and environmental exposure assessment, simple yet efficient EBC collection device using different methods and biological characterization of the EBC sample are needed.
In this study, a novel EBC collection method was developed by using hydrophobic surface, a layer of ice, and a droplet scavenging procedure. The physical collection efficiency (amount of EBC collected per unit of time) of the device was evaluated. In addition, biological analysis and characterization of EBC samples collected from human subjects were conducted using culturing, DNA stain, SEM, qPCR and species identification tool VITEK 2. This work contributes to the effort in applying EBC together with molecular tools as a non-invasive method in rapid disease diagnosis.
The collection method and device developed and experiential set up for collecting EBC are shown in Figure 1 and Figure 2 , respectively. As observed in Figure 1 , a simple EBC collection device was developed here. The EBC collection device is composed of four major parts as shown in Figure 1 : collection device cover, collection device base, a layer of ice, and a hydrophobic film (treated by ultralow temperature 270uC). The collection device cover and base were made of Teflon TM polytetrafluoroethylene (PTFE) material, and a parafilm (Parafilm Co. Menasha, WI) used as the hydrophobic surface. The dimensions of the collection device are measured as 80640640 (mm) (length6width6height). In the collection device cover, there is a hole with a diameter of 6 mm as the exhaled breath inlet. The thickness of the collection device cover and base was about 3 mm, and the whole collection device weighs around 105 g. The layer of ice is used to keep the treated hydrophobic film cool.
For EBC collection, sterile water (DNA and RNA free) was first added into the collection base of the device up to a depth of 5 mm as observed in Figure 2 . And then, the device base together with the cover was placed in an ultralow temperature (270uC) refrigerator (Thermo Fisher Scientific Co. Marietta, OH) to form a layer of ice. Following this step, a sterile hydrophobic parafilm measured as 8064060.3 (mm)(length6width6thickness) was placed onto the surface of the ice suited in the collection base. To collect EBC samples, a disposable sterile straw with a diameter of 5 mm (16 cm long) is inserted through the exhaled breath inlet shown in Figure 2 , with its end 2 mm above the hydrophobic parafilm. The human subjects are then advised to mouth-breathe without wearing a nose clip through the exhaled breath inlet shown in Figure 2 toward the hydrophobic film for a selected time (1-4 min). Due to the low temperature and hydrophobic nature of the parafilm surface, exhaled breath quickly condenses into tiny liquid droplets on the hydrophobic surface. Assuming an average breathing rate of 12 L/min for an adult, the particle speed from the exhaled breath would be around 10 m/s given the size of the straw (5 mm in diameter). Therefore, during the exhaled breath collection, the bacteria or virus particles would impact onto the hydrophobic surface at a speed of 10 m/s. In addition to condensing used for other EBC collection procedures, the method developed here also rely on the impaction to collect the bacterial and viral particles. Given such a speed, there might be possible particle bounce problems, however the bacterial or viral particles in the exhaled breath usually come with water droplets, which thus minimizes the potential particle bounce problem.
After the collection, about 10 ml of DNA and RNA free DI water was pipetted onto the hydrophobic film as observed in Figure 1 . To collect breath samples on the hydrophobic film, one only needs to use the pipette to touch the DI water droplet, and then drag the DI water droplet to scroll over the entire surface. The DI water droplet would move with the pipette without an extra step. The materials collected on the surface would be subsequently scavenged into the water droplet. After this operation, the collected EBC samples in the form of bigger liquid droplet as shown in Figure 1D were transferred to a sterile tube by a pipette for subsequent analysis. The samples collected without the exhaled breath from human subjects are used as the negative controls. The EBC collection efficiency and biological analysis of collected samples were performed as outlined in the experimental procedure shown in Figure S1 (Supporting Information).
To investigate the amount of variability in EBC collected by the method developed, six student volunteers were recruited to exhale through the device for 1, 2, 3 and 4 min. The volume of collected EBC was measured by a calibrated pipette (Eppendorf, Hauppauge, NY). The amount of EBC per unit time collected by the device was determined using averages of EBC samples obtained by the volunteers under each of specific collection time tested. For each EBC collection, a different hydrophobic film and a different exhalation straw were used. In addition, the particle size distributions in the exhaled breath through mouth-breathing were also measured in a particle free bio-safety hood using an Optical Particle Counter (OPC) (GRIMM Co. Ltd., Ainring, Germany) at a flow rate of 1.2 L/min. To ensure air stream balance, the OPC was connected to a two-way tubing, which connects to clean air (Biological SafetyHood) and the breathing straw, respectively.
In this work, seven patients with onset flu symptoms (their medical information is listed in Table S2 , Supporting Information) were also recruited from the respiratory clinic of Peking University Third Hospital in Beijing. About 40 ml of exhaled breath condensate collected from each of 7 patients was diluted by 10 times and then plated on Trypticase Soy Agar (TSA) (Becton, Dickson and Company, Sparks, MD) plates at 26uC for 2-3 days, and colony forming units (CFUs) were manually counted. The total culturable bacterial aerosol concentration was calculated as CFU/m 3 (exhaled breath) by considering the collection time and an average breathing rate of 12 L/min for an adult. Besides, the culturable bacterial species were identified using VITEKH 2 (BioMérieux, Inc,100 Rodolphe Street, Durham, NC). In addition, molecular detection of bacteria and virus using qPCR and RT-qPCR, respectively, were performed according to the procedures described in Supporting Information S1. To further confirm the bacterial presence DNA stain of EBC sample by Acridine Orange (AO) was also conducted.
The differences in collected EBC volumes and culturable bacterial aerosol concentrations obtained by the EBC collection device were analyzed by Analysis of Variance (ANOVA). A pvalue of less than 0.05 indicates a statistically significant difference at a confidence level of 95%. Collection of EBC from human subjects was approved by Peking University Ethnics Committee.
Here, a novel EBC collection method and device was developed and evaluated in collecting EBC samples from human subjects using culturing and molecular methods. Compared to those currently available devices shown in Table S1 , our device is lightweight with simplicity, reusability, and lower cost. The developed collection device itself costs less than $10, with about $0.5 for consumables (straw and hydrophobic film) per collection. The time needed for 100 ml EBC including sample collection and removal was around 2 min. The physical collection efficiency of the device is shown in Figure 3 . The data points shown in the figure were averages of the EBC samples collected from six volunteers under each of the collection times (1, 2, 3 and 4 min) tested. In general, the amount of EBC sample collected was observed to increase with increasing collection time were observed among subjects. As also observed in Figure 3 , the method has a good reproducibility (small variations). ANOVA analysis indicated that the collection time had a statistically significant effect on the amount of EBC sample collected per unit of time (p-val- Table S2 ; F and M indicate Female and Male, respectively, 1-7 indicate the subject ID corresponding to those listed in Table  S2 ; EBC collection time was 3 min; data points represent averages and standard deviations from at least three replicates. doi:10.1371/journal.pone.0041137.g005 ue = 0.0026). For the 4 min collection, the volume of collected EBC (168.7 mL) was 1.8 times of that (60.0 mL in average) by 1 min. In our study, when no EBC was collected about 1 mL of liquid was obtained from the hydrophobic surface in an environment with a temperature of 17.9-19.3uC and a relative humidity level of 46-52%. In addition, during the breath sample collection, the collection device had a higher air pressure due to the exhaling, thus it is less likely that environmental air would come into the device. This suggests that environmental water vapor had limited impact on the collection method given the total amount of EBC collected. A recent study indicated that the minimum required volume of EBC was 50 mL for follow-up biological and chemical analysis, such as multiplexed cytokine analysis [35] . This on the other hand implies that the EBC device developed in this study can provide adequate amount of EBC sample for rapid analysis. Here, only one type of hydrophobic surface (parafilm) was tested, and in the future different Table S2 ; DI water was used as the negative control. Table S2 ; Bacillus subtilis species was used as the positive control and DI water was used as the negative control; the curves shown here include two duplicates for each EBC sample. doi:10.1371/journal.pone.0041137.g007 hydrophobic materials should be also explored to improve the overall efficiency.
As listed in Table S1 , currently available EBC collection devices, e.g., the Rtube and the EcoScreen, are comparable to ours with respect to rate of EBC collection. However, our EBC device has advantages in size, weight, and simplicity. In our study, we used a 16 cm long straw for exhaling toward to the super hydrophobic surface without any control of saliva for the possible contamination. However, our collection time was only 1-4 min, and during such short sampling period the sample contamination by saliva is very limited given the length of the straw. Another advantage of our developed device is the one time use of the hydrophobic parafilm (disposable) and exhalation straw with an easy collection of EBC, which thus prevents the possible cross contamination and facilitates the collection of EBC samples from a large number of subjects. This is particularly useful during an influenza outbreak or a man-made bio-terrorism attack in which a rapid screening of exposed persons needs to be conducted immediately.
Here, the EBC samples collected by the developed device from seven human subjects recruited from a respiratory unit of Peking University Third Hospital in Beijing were studied using culturing, DNA stain, SEM and molecular methods. In this study, the particle size distributions trends in a typical exhaled breath were also measured and are shown in Figure 4 . As observed in the figure, the number concentration decreased with increasing particle diameter. For bacterial size ranges (0.65-2.2 mm), a concentration level of 329 to 25819 particles/L was observed, while for larger particles of 2.2-4 mm a concentration level of 60 to 400 particles/L was obtained. In previous studies, similar particle size distribution trend in exhaled breath was also found using the OPC, although the droplet concentrations for respective size ranges were slightly different [21, 39] . Nonetheless, due to its rapid evaporation water droplet itself or those adsorbing on bacterial particles in the exhale breath will certainly affect the results obtained here. The results from OPC indicated that particles of larger than 2.5 mm only accounted for 0.4% of the total particles exhaled. According to ICRP (1994), the total lung deposition efficiency for particles larger than 2 mm is more than 80%, while for smaller particles of less than 1 mm, the deposition efficiency is less than 40%, i.e., 60% exhaled out [44] . In addition, larger particles could stick to the straw wall. Therefore, in the exhaled breath as well as those collected into DI water droplet smaller particles would dominate. Figure 5 shows the concentrations of culturable bacterial aerosols in EBC samples collected from seven human subjects. As shown in the figure, bacterial concentration levels ranged from 693 to 6,293 CFU/m 3 . ANOVA tests indicated that there were statistically significant differences in culturable bacterial aerosol concentrations for EBC samples collected from different subjects (p-value = .0001). In a recent study, human occupants are also identified as the significant contributors for indoor bacteria, i.e., the emission rate is about 37 million gene copies per person per hour, and a distinct indoor air signature of bacteria was demonstrated to be associated with human skin, hair, and nostrils [40] . During human breathing, the bacterial particles from environmental air are continuously inhaled, some of which, i.e., smaller ones, can be exhaled out again by the lung and reside with nostrils. Here, bacterial species Sphingomonas paucimobilis and Kocuria rosea were detected using Vitek2 in six EBC samples as shown in Table S2 . Because of limitation of Vitek 2, certain bacterial species were not identified in our study. Among the subjects, subject #6 had substantially higher culturable bacterial concentrations than other subjects. From his medical conditions shown in Table S2 , it was likely that his fever was caused by the bacterial infections. In his EBC sample, we found Kocuria variants which were thought to cause catheter-related bacteremia [41] . For other human subjects, the culturable bacterial aerosol concentra- tion levels ranged from 700 to 3000 CFU/m 3 and Sphingomonas paucimobilis, a non-fermenting Gram-negative bacillus, were detected. In a previous study, S. paucimobilis was found to cause nosocomia bacteremia outbreak [42] . For negative control samples, we did not observe the bacterial growth, indicating no contamination during the EBC collection. Ideally, bacterial particles in EBC should be collected using a suitable size-selective sampling tool to investigate the bacterial counts for different size range. However, such device is currently not available yet. Compared to the environmental culturable bioaerosol concentrations, those in EBC samples collected had relatively higher levels, thus representing an important source of bioaerosols particularly in a high human occupancy environment. In addition to viruses, Rhodococcus equi, a bacterium causing pyogranulomatous bronchopneumonia, were detected in the exhaled air from foals in a recent study [43] . When pathogenic bacteria are breathed out, they could pose a serious public health threat. Figure 6 shows the qPCR amplification plot from EBC samples collected from seven human subjects in a respiratory clinic. As observed from the figure, bacterial samples were successfully amplified (Ct values were [16] [17] [18] [19] , while the positive sample (B. subtilis) had a Ct value of 15 and the negative control had a value of 28. Based on the DNA standards used, the concentrations of bacterial DNA in the EBC samples (Sample 1-7) were in the range of 0.32 mg/mL-3.15 mg/mL. Detection of the bacterial DNA in EBC samples was also confirmed by the melting curve of qPCR amplification as shown in Figure 7 . As observed in the figure, most EBC samples had a peak at 68uC, the same as that of the positive control B. subtilis. For a few different peaks observed, they might be the possible primer dimer (PD) from the PCR non-specific amplification process. In addition to the qPCR amplification of bacteria in EBC samples collected, DNA stain (AO method) was also performed and the results are shown in Figure S2 . As observed in the figure, both viable (green) and dead (yellow) were found in the EBC samples collected and the positive control B. subtilis samples, while no cells were detected in the negative control. SEM images with different resolutions and agar plate culturing shown in Figure 8 also indicated that EBC samples (cultured) had various types of bacteria based on their morphologies and colony color. From SEM images, it can be estimated that most bacteria are in the range of 0.5-1.0 mm. According to total particle deposition curve developed by ICRP (1994) [44] , more than 60% of bacterial particles of below 1 mm could be exhaled out. These smaller bacterial particles could remain airborne for a prolonged time period, thus playing an important role in airborne transmission of potential diseases. Results shown in Figures 5, 6 , 7, and 8 indicate that high levels of bacterial aerosols were detected in the EBC samples collected, and the results on the other hand also implied that the developed device was efficient in collecting bacterial particles in the exhaled breath. These experimental data further confirm that exhaled breath is an important source of bacterial aerosols in the built environments.
In this study, qPCR was also applied to detecting influenza A H3N2 viruses in EBC samples collected by the device. As observed in Figure S3 , H3N2 viruses were detected in the EBC sample collected from subject #3 with a Ct value of 28, while those for subject #1, #2 were shown below the detection limits. In addition, spiking viruses into the samples in general enhanced the overall qPCR signal as observed in Figure S3 . This on the other hand suggests no inhibition or amplification occurred when amplifying H3N2 viruses in EBC samples using qPCR. According to information shown in Table S2 , subject #3 had a fever, but no other information was available at the time of the experiment. In a previous study, it was indicated that use of the RTube for EBC collection had a very low viral detection rate (7%) compared to nasal swabs (46.8%) [20] . Recently, a mask-like sampler was also tested and proved to be useful in detecting viruses using PCR in exhaled breath [23] . It was indicated that airborne virus detection is difficult due to their low concentration and the presence of a wide range of inhibitors, thus optimized molecular biology should be performed to enhance their detection [45] . Although the number of the subjects tested is limited here, the developed method, i.e., EBC collection and qPCR application, was demonstrated successful in detecting viruses from human exhaled breath. This would offer a non-invasive method for diagnosis of respiratory infections by using EBC. In the future, more patients should be tested with the EBC collection device developed here for viral detections.
Exhaled breath holds great promise for monitoring human health and for the diagnosis of various lung and systemic diseases, but analysis challenges remain due to the complex matrix of the breath [46, 47] . In this study, different from available devices restricted solely to condensation a simple and low cost EBC collection method using impaction and condensing was developed here for collecting bacteria and virus particles. An important advantage is the reusability of the collection device with a disposable hydrophobic film and an exhalation straw yet with a rapid EBC collection. This would offer the opportunity to collect EBC samples from a large number of subjects, especially during an influenza outbreak or a man-made bioterrorism event, within a shorter time frame. The developed EBC collection method was shown highly successful in detecting bacteria in EBC samples in a clinical setting. The developed EBC collection method was also shown applicable in detecting influenza viruses too. Experimental data here also suggest that exhaled breath, which was shown to contain smaller bacterial particles, could play an important role in airborne transmission of potential diseases. The collection efficiency of other substances including bio-markers (NO,CO, 8isoprostane, hydrogen peroxide, nitrite, volatile organic compounds) using the developed method here is subject to further investigations. In addition, different exhalation modes should be also investigated with the method in collecting EBC. Besides, the dynamics of the air flow, mixing, and effects of temperatures and humidity, condensation, evaporation, growth of particles during the collection as well as the optimal straw length should be also investigated for improving the developed technique. Overall, our developed method here could be easily made available to a laboratory, and have impacts on current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of those influencing factors described. Figure S1 Experimental procedures used in this study include physical characterization and molecular analysis of the EBC collection efficiencies of the device and its pilot application in a respiratory clinic. (TIF) Figure S2 Optical images of EBC samples stained by Acridine Orange (AO): Bacillus subtilis species were used as the positive control and DI water was used as the negative control. (TIF) Figure S3 Detection of H3N2 influenza viruses in EBC samples collected from three human subjects with ID: 1, 2, 3 corresponding to those listed in Table S2 ; In addition, spiked H3N2 virus samples were also amplified; H3N2 viruses were used as the positive control and DI water was used as the negative control. (TIF)  Seasonal influenza vaccination knowledge, risk perception, health beliefs and vaccination behaviours of nurses The relationship between knowledge, risk perceptions, health belief towards seasonal influenza and vaccination and the vaccination behaviours of nurses was explored. Qualified nurses attending continuing professional education courses at a large London university between 18 April and 18 October 2010 were surveyed (522/672; response rate 77·7%). Of these, 82·6% worked in hospitals; 37·0% reported receiving seasonal influenza vaccination in the previous season and 44·9% reported never being vaccinated during the last 5 years. All respondents were categorized using two-step cluster analyses into never, occasionally, and continuously vaccinated groups. Nurses vaccinated the season before had higher scores of knowledge and risk perception compared to the unvaccinated (P<0·001). Nurses never vaccinated had the lowest scores of knowledge and risk perception compared to other groups (P<0·001). Nurses' seasonal influenza vaccination behaviours are complex. Knowledge and risk perception predict uptake of vaccination in nurses. Annual epidemics of seasonal influenza result in about 3-5 million cases of severe illness and 250 000-500 000 deaths worldwide [1] . Healthcare workers (HCWs) can be a key source for influenza transmission in communities and hospitals as they are exposed to both infected patients and high-risk groups [2, 3] . Vaccination is the most effective way to prevent infection and severe outcomes [1] and the principal measure to reduce the impact of epidemics, such as hospitalization, mortality and morbidity [2, [3] [4] [5] . Moreover, studies suggest that the vaccination of HCWs has substantial economic benefits as well as health-related benefits, including reduced absenteeism from work and the extra costs of sick leave and staff replacement [4, 6, 7] .
For the above reasons, the World Health Organization (WHO), United Kingdom Department of Health (DoH) [8] , United States Centers for Disease Control and Prevention (CDC), other healthcare professional organizations and many countries' government agencies [1, 9, 10] strongly recommend the annual seasonal influenza vaccination of HCWs. However, studies suggest that influenza vaccine uptake in HCWs is often low worldwide [11] [12] [13] [14] . For example, the overall seasonal vaccination rate in England for HCWs was 26 . 4% for the 2009/2010 season [15] . Nurses, as the group having the most patient contact, are more reluctant to be vaccinated than other HCWs [16] [17] [18] [19] [20] [21] [22] [23] .
Although predictors influencing nurses' vaccination practices have been identified to some extent regarding knowledge and risk perception [16] [17] [18] [19] [23] [24] [25] [26] [27] , further studies are needed to explore the influences on nurses' attitudes and practices regarding influenza vaccination and to identify the major influencing factors for their vaccination behaviours. This study aimed to examine the relationship between knowledge, risk perceptions, health beliefs towards seasonal influenza and vaccination and the vaccination behaviours of nurses.
A cross-sectional survey was conducted of qualified nurses between 18 April and 18 October, 2010. Qualified nurses attending continuing professional education courses at a large university in central London were invited to participate in the study. Potential respondents were given a study information sheet and a questionnaire by the investigator. Completed questionnaires were collected immediately by the investigator or returned by mail to the research team using Freepost addressed envelopes. Questionnaire completion was anonymous so that it was not possible to follow up non-response. Ethical approval was obtained from the University Ethics Committee.
The questionnaire collected the following data : (1) knowledge about seasonal influenza and vaccination (22 items requiring true, false or unsure responses) included five dimensions to assess general information, severity of influenza, influenza vaccination, high-risk groups and vaccination-recommended groups; (2) risk perception (12 items with a 4-point Likert scale) towards influenza and pandemic with three dimensions (i.e. personal vulnerability to illness, negative consequences of contracting influenza and severity of influenza) ; (3) health locus of control including internal, chance and powerful others dimensions assessed by the Multidimensional Health Locus of Control (MHLC) scales [28] (18 items) ; (4) vaccination behaviours (nine items) including vaccination status (whether respondents had been vaccinated in the previous season), vaccination intent (whether respondents intended to be vaccinated next season) and vaccination history (how many times respondents had been vaccinated in the last 5 years) ; (5) reasons for accepting or refusing vaccination using two open questions; and (6) demographic characteristics (10 items) including gender, age group, highest educational qualification, place of work, clinical speciality, year of qualification as a nurse and whether or not respondents had direct patient contact. The Cronbach's a-coefficients for the three newly developed scales (sections 1, 2, 4) ranged from 0 . 701 to 0 . 763 and principal components analysis produced a good fit and confirmed the internal design of the instrument.
Statistical analysis was performed using SPSS version 15.0 (SPSS Inc., USA). The x 2 test or Fisher's exact test was used to explore the statistical differences between categorical variables. The independentsamples t test was used to compare statistical difference between continuous variables in two groups. The one-way between-groups analysis of variance (ANOVA) was used to explore the differences between more than two groups. Logistic regression was performed to explore the impact of the variables on vaccination status. The two-step cluster analysis procedure was performed to explore the natural groupings (i.e. clusters) within the respondents. The clustering criterion was that the solution had smaller values of Schwarz's Bayesian Information Criterion (BIC), a reasonably large ratio of BIC changes and a large ratio of distance measures. A P value <0 . 05 was considered to denote statistical significance.
In total, 672 questionnaires were distributed and 522 were returned representing a response rate of 77 . 7%. The characteristics of the respondents are summarized in Table 1 . Overall 188/508 respondents (37 . 0%) reported receiving a vaccination in the previous season with 44 . 9% never receiving a vaccination during the last 5 years. There was no difference in the demographic characteristics of the vaccinated or unvaccinated respondents in the previous season. The number of years qualified as a nurse for the two groups were 11 . 99¡9 . 085 years and 11 . 89¡8 . 624 years (P=0 . 898), respectively.
Comparison of knowledge and risk perception scores and sub-scores of MHLC are summarized in Table 2 . There were significant differences in knowledge scores and risk perception between the vaccinated and unvaccinated nurses and between those with vaccination intent, no intent or unsure. There was no significant difference in the sub-scores of MHLC between the vaccinated and unvaccinated (data not shown in table) but there was a significant difference for the sub-score of powerful others between those groups with different vaccination intent.
Direct logistic regression was performed to assess the impact of a number of factors on the likelihood that respondents had been vaccinated in the previous season. The model contained five independent Table 3 , only two of the independent variables made a unique statistically significant contribution to the model (knowledge score and risk perception score). The strongest predictor of vaccination status was the risk perception score, recording an odds ratio of 1 . 76, indicating that respondents who had higher risk perception scores were >1 . 76 times more likely to have been vaccinated in the last 12 months than those with lower scores, controlling for all other factors in the model. Knowledge score with an odds ratio of 1 . 05 indicated that knowledgeable respondents were more likely to be vaccinated than the unknowledgeable, controlling for other factors in the model.
The two-step cluster analysis procedure was used to explore the natural groupings within the respondents. First, the auto-clustering exploratory analysis was performed using the categorical variables of vaccination status, vaccination intent, vaccination history and the continuous variables of knowledge score and risk perception score. Of the 522 respondents, 64 were automatically excluded from the analysis due to missing values on one or more of the variables. Of the 458 respondents assigned to clusters, 195 (42 . 6%) were assigned to the first cluster, 143 (31 . Subsequently the analysis was performed using the combined categorical variables of vaccination status in the previous season (=yes) and vaccination history and the continuous variables of knowledge and risk perception scores. The results were auto-clustered into four groups but not explainable. The procedure was repeated with the cluster number fixed to 2 due to the values of BIC, ratio of BIC changes and ratio of distance measures. Of the total 188 vaccinated respondents, 12 were excluded due to missing values. Of the remaining 176 respondents, 107 (60 . 8%) were assigned to cluster 1 and 69 (39 . 2 %) to cluster 2. Vaccinated cluster 1 comprised those vaccinated only in the previous season, i.e. the newly vaccinated group and vaccinated cluster 2 contained those vaccinated in the previous season who had more than one previous vaccination, i.e. the continuously vaccinated group. Then, the same analysis was repeated for the unvaccinated respondents and two clusters emerged, i.e. unvaccinated cluster 1 (never vaccinated) and unvaccinated cluster 2 (used to be vaccinated).
The analysis had therefore separated the respondents into reasonable categories. A comparison of variables across all clusters revealed that the never vaccinated had the lowest knowledge score, risk perception score and powerful others sub-score of MHLC compared to the other clusters (P<0 . 001, P<0 . 001, P=0 . 020, respectively) and this difference was statistically significant. For the vaccinated, there were no significant differences across any variable for the newly vaccinated and continuously vaccinated clusters although there was a trend of higher average scores for knowledge and risk perception in the newly vaccinated cluster compared to those of the other clusters (P=0 . 652, P=0 . 288, respectively). For the unvaccinated, there were no statistically significant differences across the variables except for the MHLC 'powerful others ' sub-score (P=0 . 008).
Further comparisons were performed to explore whether there were differences across the different items of knowledge and risk perception in the clusters.
In the clusters of never vaccinated, other vaccination history and vaccinated with intent, there were significant differences in knowledge related to general information, high-risk groups and vaccination of recommended groups with P values of <0 . 001, <0 . 003 and <0 . 006, respectively. On average those never vaccinated had the lowest score while those vaccinated with intent had the highest scores across all knowledge items. For only one item of risk perception, i.e. personal vulnerability to illness, was there a significant difference between the clusters of never vaccinated and other vaccination history and between never vaccinated and vaccinated with intent (P<0 . 000 respectively). Those never vaccinated had the lowest average score. There was no statistically significant difference in the knowledge and risk perception item scores between the two vaccinated clusters. However, the newly vaccinated usually had higher scores than those of the continuously vaccinated except for one item, i.e. the vaccination of recommended groups. Similarly, for the two unvaccinated clusters there was no difference for knowledge scores, but there was a significant difference in one risk perception item, i.e. personal vulnerability to illness (P=0 . 001). Those never vaccinated had a lower score for this item than those who used to be vaccinated and they were also less knowledgeable compared to the other group. Tables  4 and 5 .
In this study, the seasonal influenza vaccination rate in nurses was 37 . 0 % which is higher than previous reports of vaccination coverage ranging from 14 . 3-26 . 4% in HCWs in UK [12, 29, 30] and 16% in nurses reported by Chalmers [27] and similar to O'Reilly et al.'s reported vaccination coverage of nurses in elderly care units [19] . This higher vaccination rate might be explained to some extent by the UK media reports of the risk of seasonal influenza and H1N1 pandemics in 2009 which may have increased the sample nurses' risk perception towards influenza and consequently changed their vaccination decisions as noted in a previous study [31] .
This study found that vaccination behaviours in nurses were more complex requiring an analysis of both vaccinated and unvaccinated nurses' behaviours. More levels of vaccination behaviours existed in the sample with the two-step cluster analysis revealing three whole population clusters, i.e. those never vaccinated, those vaccinated this season with intent next year, and those with other vaccination history. Two clusters, the newly vaccinated and continuously vaccinated, were identified for the vaccinated group and another two clusters, never vaccinated and used to be vaccinated, were identified in the unvaccinated group. To improve the influenza vaccination rates in nurses, it may be helpful to develop different strategies which target the nurse groups of the never vaccinated and the occasionally vaccinated.
We found that a lack of knowledge about influenza and vaccination was a strong predictor of nurses' vaccination behaviours, especially for those never vaccinated. This cluster had the lowest knowledge score, suggesting that increasing their knowledge might improve their vaccination behaviours. However, it seems there are 'persistent decliners ' who are in the 'habit ' of not having a vaccination. This suggests that future educational campaigns need to be persistent, durative, and intensive if their vaccination behaviours are to be modified. For those who had been vaccinated in the past but not in the current season, knowledge was also a predictor for their vaccination behaviours, which suggests that current vaccination campaigns have failed to address their misgivings about vaccination to maintain their compliance with the annual vaccination recommendation for HCWs. Between those occasionally vaccinated and continuously vaccinated, knowledge levels were not significantly different but the newly vaccinated in 2009 had on average higher knowledge scores than those continuously vaccinated. This may reflect an increase in their risk perceptions towards influenza due to widespread reporting of the risks in the media encouraging them to be vaccinated for the first time in their lives. This suggests that timing may be crucial to the success of vaccination campaigns making behaviour modification easier. Future studies are required to explore the relationship between the content and timings of vaccination campaigns and nurses' first vaccination uptake.
This study showed that the perception of personal vulnerability to illness was important in nurses making vaccination decisions. But perceptions of the negative consequences of contracting influenza and severity of influenza were not major factors, a finding which is consistent with findings of previous studies [16] . This suggests that future educational campaigns might be more effective if they focus on the negative personal consequences of contracting influenza and its sequelae rather than nurses' professional duty to protect patients or other vulnerable groups. Additionally, the reasons which nurses gave for having vaccination focused upon their personal health motivation rather than a professional responsibility regardless of whether they were vaccinated or unvaccinated. Concerns about the vaccine's side-effects and effectiveness or safety were the two most frequent reasons for not having a vaccination indicating continuing misconceptions about influenza vaccine in nurses. Future educational campaigns may wish to consider providing targeted information to change these widespread myths in nurses. However, these concerns did not seem to influence vaccination decisions because both vaccinated as well as unvaccinated nurses noted these reasons against vaccination. It may be the case that 2 days of minor discomfort postvaccination is tolerable when set against a year's influenza protection. Unvaccinated nurses reported 'no need ' as their reason not having a vaccination which is consistent with their low-risk perception of contracting influenza. The convenience of the vaccination programme was identified as an organizational reason highlighting the importance of easy access to vaccination to increase its coverage in nurses.
Our analysis of health locus of control data found that those never vaccinated had a lowest 'powerful others ' locus of control for their vaccination behaviours, indicating that they did not believe their health was something over which they had no control [32] . This pattern of health beliefs towards influenza vaccination is consistent with their low-risk perception of personal vulnerability to illness and 'no need ' as their reason refusing vaccination and may be an important factor for never vaccinated nurses. Further studies are needed to explore what may influence this pattern of health locus of control in order to modify nurses' vaccination behaviours.
Some organizations have recently required mandatory seasonal influenza vaccination for HCWs as a professional and ethical obligation to protect their patients' health [33, 34] . However, ethical issues have been raised with mandatory vaccination because, while promoting the interests of patients and employers, it challenges HCWs' personal autonomy and freedom of choice [35, 36] . Moreover, it has been suggested that vaccination is not the only avenue of influenza prevention and there are several other important measures that healthcare organizations may take to protect both patients and HCWs [37] . Further previous studies have also suggested that not all HCWs support mandatory vaccination [38] . Until mandatory influenza vaccination for HCWs is accepted worldwide, continued efforts to improve nurses' vaccination behaviours will be required.
This study has some limitations. First, there is possible selection bias of a convenience sample ; however, the broad range of qualified nurses together with a high response rate strengthen the results. The extent of bias is unknown especially regarding nurses not working in London or in different care settings. Second, the survey relied on self-report vaccination data ; however, Zimmerman et al. [39] found that selfreport data were reliable in comparison with medical records. Third, the three factors explored relating to nurses' vaccination behaviours explained only 8 . 7-11 . 9% of the variance according to the logistic regression analysis (although it was statistically significant) and therefore our results cannot fully explain nurses' vaccination behaviours. Additional predictors will need to be introduced into the model in future studies to fully explain nurses' vaccination behaviours.
In conclusion, this study revealed that nurses' influenza vaccination behaviours are complex. Knowledge and risk perception were identified as two predictors influencing nurses' vaccination decisions with the health belief pattern of 'less powerful others ' being an important predictor in the never vaccinated ; however, there are other influential factors which need to be identified in future studies. IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors. CNS resident cells are targets of autoimmune mediated damage but also active participants in disease development, progression and control [1, 2] . However, their contributions to neuroprotection and regulation by inflammatory mediators are not well defined. CNS insults, including autoimmune disease, initiate rapid astrocyte activation characterized by cellular hypertrophy and increased intermediate filament glial fibrillary acidic protein (GFAP) expression [3] [4] [5] . Astrocytes form a physical barrier surrounding areas of inflammation initially limiting bystander tissue damage [6, 7] . However, this barrier subsequently impedes axonal regeneration contributing to sustained disability [3, 5, 8] . Innate and adaptive pro-inflammatory astrocyte functions include production of pro-inflammatory cytokines, reactive oxygen species, chemokines, and matrix metalloproteinases [3] [4] [5] . By contrast, secretion of anti-inflammatory cytokines and scavengers of reactive oxygen species, as well as inhibition of both microglial activation and tumor necrosis factor (TNF) secretion, all support an astrocyte mediated anti-inflammatory function [3] [4] [5] . Therefore, astrocyte activation constitutes a ubiquitous, yet heteroge-neous response associated with both promoting and inhibiting CNS repair [3] [4] [5] .
MS and EAE are both associated with T cells secreting IFN-c (Th1 cells) and IL-17 (Th17 cells) which play complex, not fully understood roles in disease initiation and progression [2, 9] . In vivo and in vitro evidence indicates that suppression of encephalitogenic T cell proliferation within the CNS during EAE and activation of anti-inflammatory programs are in part mediated via astrocytes [4, 5] . Similar to astrocytes, IFN-c mediates both proand anti-inflammatory functions during autoimmune disease [10] . Early IFN-c induced effects are pro-inflammatory; IFN-c facilitates inflammatory cell access, shapes their composition, increases major histocompatibility complex (MHC) expression, contributes to macrophage and microglia activation, and initiates oligodendrocyte death [11] . Similarly, IFN-c mediated protection during EAE is also multifaceted [12] [13] [14] [15] . It acts as a negative regulator of neutrophil accumulation, Th17 cell activation, IL-1R signaling, protease secretion, and chemokine activity. It also inhibits proinflammatory cytokine secretion via induction of suppressor of cytokine secretion (SOCS) proteins, facilitates T cell apoptosis and protects oligodendrocytes via inducing endoplasmic reticulum (ER) stress responses [10, 11, 16, 17] . This highlights the critical role of a single mediator in both promoting disease but also limiting inflammatory mediated damage required to initiate repair cascades. Based on the gatekeeper functions of astrocytes and the diverse biological effects of IFN-c, we set out to determine how IFN-c signaling specifically to astrocytes influences CNS autoimmune disease. The results demonstrate that IFN-c signaling to astrocytes had no profound effects on initial disease progression, but played an essential protective role during the transition from acute to chronic disease. Clinical remission induced by IFN-c signaling to astrocytes coincided with reduced demyelination, axonal degeneration, and astrocyte activation. The IFN-c receptor is expressed ubiquitously; however, these data reveal astrocytes as the primary target and mediator of the well established antiinflammatory activity of IFN-c within the CNS.
To understand the role of IFN-c signaling to astrocytes during the pathogenesis of CNS autoimmune disease, EAE was induced in transgenic mice expressing a signaling deficient dominant negative IFN-c receptor 1 specifically on astrocytes (GFAPcR1D mice) [18] . Peripheral activation of self reactive T cells in GFAPcR1D mice was similar to wt mice (data not shown). This is consistent with both the CNS restricted transgene expression in the GFAPcR1D mice as well as the similar T cell activation following peripheral immunization with a non-self antigen [18] . Following immunization the incidence of disease, initiation of clinical symptoms, and initial symptom progression were unaltered by the inability of astrocytes to respond to IFN-c ( Fig. 1A ; Table 1 ). In addition, neither immunized group exhibited clinical symptoms of atypical EAE associated with the absence of IFN-c [19] . However, clinical symptoms in GFAPcR1D mice began to diverge from wt mice at , day 12 post immunization (p.i.) prior to the peak of clinical disease. In both groups clinical disease peaked at , day 14 p.i., but severity was increased from a score of 3.1 in wt mice to 4.0 in the GFAPcR1D group ( Fig. 1A ; Table 1 ).
GFAPcR1D and wt mice were compared at the peak of acute disease to determine if IFN-c signaling altered astrocyte activation or CNS inflammation. Astrocyte hypertrophy and GFAP expression ( Fig. 1B) were similar in both groups, indicating no overt effects of IFN-c on initial astrocyte reactivity. Furthermore, neither the extent of inflammation nor the anatomic distribution of inflammatory cells was altered (Fig. 1B) . Flow cytometry confirmed no difference in the overall extent of CD45 hi inflammatory cells recruited into the CNS ( Fig. 2A) . Furthermore, percentages of CD4 + T cells ( Fig. 2A) , CD8 + T cells and macrophages within the infiltrates were also similar (Fig. 3) . In contrast to the association between increased EAE severity and neutrophil accumulation in the global absence of IFN-c [14, 15] , only a small percentage of neutrophils were identified in the CNS of both groups by flow cytometry (Fig. 3) ; their low presence was confirmed by the inability to identify cells with the characteristic morphology of neutrophils in the brain by histopathology (Fig. 1B) . The absence of increased neutrophils in the CNS of GFAPcR1D mice during acute disease suggested that clinical disease was aggravated by a mechanism distinct from global IFN-c deficiency. In addition to the similar frequency of CD4 + T cells in the brains of the two groups ( Fig. 2A) , myelin oligodendrocyte glycoprotein (MOG) reactive CD4 + T cells secreting IFN-c were also similar ( Fig. 2B ). By contrast, MOG specific CD4 + T cells secreting IL-17 ( Fig. 2B ) and Foxp3 + regulatory CD4 + T cells (Tregs) were decreased in GFAPcR1D mice compared to wt mice (Fig. 2C) . Reduced Th17 and Tregs may be attributed to increased IFN-c [10, 16] . Alternatively, as IFN-c is protective in EAE [12] [13] [14] [15] , increased disease severity may reflect reduced bioavailable IFN-c due to sequestration of IFN-c binding to the dominant negative receptor. However, measurement of IFN-c in cell free supernatants derived from dissociated brains at the peak of acute disease demonstrated protein levels of 2.160.2 ng/brain in wt mice versus 3.660.5 ng/brain in GFAPcR1D mice (n = 3; p,0.05). As overall frequencies of MOG reactive CD4 + T cells secreting IFN-c were similar, increased IFN-c in the brains of GFAPcR1D mice suggested enhanced secretion at the cellular level. Although a contribution of NK or CD8 T cells could not be excluded, these potential sources of IFN-c were unlikely due to their low frequencies (NK ,5% and CD8 + T cells , 5%, see Fig. 3 ) and their equivalent frequencies in the wt and GFAPcR1D mice. Furthermore, reduced Th17 cell and Treg frequencies were consistent with suppression of these populations due to elevated IFN-c [10, 16, 20] .
Inflammation in spinal cords from the GFAPcR1D mice was also similar to wt controls at the peak of clinical symptoms (Fig. 4) . Although numbers and distribution of CD4 + T cells were also similar (4.661.3/mm 2 in GFAPcR1D mice vs. 3.860.1/ mm 2 in wt mice; Fig. S1 ), spinal cords of GFAPcR1D mice exhibited a ,2-fold increase in demyelination (Fig. 4) . Areas of demyelination encompassed 7.361.0% of spinal cord white matter in GFAPcR1D mice versus 3.860.9% in wt mice (p#0.01). Furthermore, the increase in demyelination was associated with a prominent loss of axons in GFAPcR1D mice compared to controls (Fig. 4) . Despite elevated demyelination and axonal loss in the absence of IFN-c signaling to astrocytes, spinal cords showed no evidence of differential astrocyte activation by either immunohistochemistry (Fig. 4 ), or differences in GFAP mRNA expression during the peak of acute disease (Fig. 5 ). Although CCL5, IL-1 and TNF mRNA expression were increased in the spinal cords of the GFAPcR1D mice, no differences in IFN-c, iNOS, CXCL10 or IL-6 mRNA were consistent with similar inflammation (Fig. 5) . These data suggest that the initial disease pathogenesis, reflected by an increased demyelination in spinal cords, but not brain, during ascending paralysis is dampened by IFN-c signaling to astrocytes.
Subsequent to peak disease severity the clinical symptoms in wt mice began a modest decline by day 16 p.i. (Fig. 1A ). By contrast, GFAPcR1D mice not only exhibited increased severity of clinical symptoms following day 14 p.i., but the continued disease escalation was associated with increased mortality ( Fig. 1A ; Table 1 ). Sustained morbidity and significant mortality in GFAPcR1D mice after day 30 p.i. implied a critical role for IFN-c induced astrocyte signaling in neuroprotection and limiting disability. A similar absence of clinical remission was found in a preliminary experiment comparing EAE SJL mice carrying the GFAPcR1D gene (data not shown). These data suggest that astrocyte responses to IFN-c are protective, irrespective of genetic background.
Escalating disease in GFAPcR1D mice coincided with focal areas of intense inflammation in spinal cords, which contrasted with the more diffuse inflammation in wt mice (Fig. 6 ). Demyelination was also increased with myelin loss encompassing 5.761.8% of spinal cord area in GFAPcR1D mice versus 2.361.4% in wt mice (p#0.05) at day 35 p.i. (Fig. 6 ). The demyelinated areas further exhibited enhanced axonal damage in GFAPcR1D mice (Fig. 6) , supporting a correlation between enhanced tissue damage, sustained morbidity and increased mortality ( Fig. 1A ; Table 1 ). Astrocyte activation associated with areas of myelin loss is a prominent finding in the CNS of both patients with MS and rodents with EAE. Although demyelination was increased in the CNS of GFAPcR1D mice, the extent of astrocyte activation associated with spinal cord white matter lesions was similar in both groups ( Fig. 6 ; ,60 GFAP + cells/mm 2 ). By contrast, the frequency of activated astrocytes in spinal cord grey matter areas that were not associated with demyelinated lesions, was increased in GFAPcR1D mice with 101.5623.0 GFAP + activated astrocytes/mm 2 versus 25.5615.5 in wt mice (p,0.001) (Fig. 7) . A similar increase in astrocyte activation within grey matter distal to white matter lesions was also detected in GFAPcR1D SJL mice during chronic EAE (data not shown).
Flow cytometric analysis during chronic disease revealed an ,4fold increase in CD45 hi inflammatory cells confirming increased inflammation in the absence of IFN-c signaling to astrocytes (Fig. 8A ). Similar to the acute disease, relative percentages of neutrophils, macrophages, CD4 + and CD8 + T cells were all similar (Fig. 3) . Furthermore, no differences in expression of activation markers on CNS derived CD4 + T cells, including CD69, CD122, CD127, Fas, FasL, ICOS and CD152 were evident between the groups (data not shown). The percentages of MOG reactive Th1 cells were also not altered in the CNS of GFAPcR1D relative to wt mice (Fig. 8B ) and the decreased percentage of potentially destructive Th17 cells identified during acute disease (Fig 2) , was also sustained during chronic disease (Fig. 8B ). Equivalent expression of IL-7, IL-23 and TGF-b mRNA (Fig. 8C ) suggested that the decrease in Th17 cells was dependent upon an increase in IFN-c and not related to a defect in activation or maintenance signals [20] . Increased inflammation and sustained MHC class II expression on microglia (Fig. 8A ) further suggested that IFN-c signaling to astrocytes down regulates inflammation universally without altering the composition of the CNS infiltrates. This concept is supported by sustained composition of CNS infiltrates during inflammation induced astrocyte apoptosis [21] .
The inability to restrain ongoing inflammation during EAE in GFAPcR1D mice was evident at multiple levels. Astrocyte activation was sustained consistent with increased expression of GFAP mRNA. CCL2, CCL5 and CXCL10 mRNA levels were increased (Fig. 8C ). The expression of mRNA encoding potentially destructive immune mediators including IL-1, IL-6, iNOS, and TNF were all increased (Fig. 8C ). IFN-c was ,2-fold higher in the CNS of GFAPcR1D mice (Fig. 9) . Lastly, the level of the antiinflammatory cytokine IL-10 was reduced ( Fig. 9 ), suggesting limited availability of IL-10 may be a key in prolonging astrocyte activation and inflammation [22] . A possible link between reduced IL-10 and the inability of IFN-c signaling to astrocytes is provided by the capacity of IFN-c to induce IL-27 in astrocytes [23] , thereby promoting IL-10 production. Indeed IL-27 in the CNS of the GFAPcR1D mice was significantly reduced compared to wt mice during chronic disease (Fig. 9 ). By contrast, IL-12 ( Fig. 9) , an indirect suppressor of CNS inflammation by promoting IFN-c production [24] , was similar in the CNS of both GFAPcR1D and wt mice. Astrocytes derived from GFAPcR1D and wt mice were stimulated with IFN-c to confirm IFN-c dependent IL-27 secretion by astrocytes [25] . While IFN-c induced IL-27 secretion by astrocytes from wt mice, IL-27 secretion was significantly reduced in cultures derived from GFAPcR1D mice (Fig. 10) . These data support the possibility that IFN-c mediated IL-27 secretion by astrocytes regulates EAE effector T cell function, inflammation and tissue destruction via induction of IL-10; however, this does not exclude the possibility that the inability of astrocytes to respond to IFN-c could directly or indirectly dysregulate a variety of other immunomodulatory functions [4, 5, 17] .
In the EAE model of MS, IFN-c functions as a proinflammatory cytokine in the early stages of disease [10, 11, 26 ], yet it also assumes a prominent anti-inflammatory role during the transition to remission [10, [12] [13] [14] [15] . Protection has been attributed to a variety of potentially interrelated mechanisms including limiting neutrophil accumulation, Th17 cell activation, IL-1R signaling, matrix metalloproteinase secretion, pro-inflammatory cytokine secretion and chemokine activity; in addition IFN-c facilitates T cell apoptosis and protects oligodendrocytes from death via an ER stress response [10, 11, 17] . The activities of astrocytes during autoimmunity also range from pro-to antiinflammatory [3] [4] [5] . However, to what extent a direct action of IFN-c on astrocytes contributes to inhibitory mechanism is unclear. The data herein demonstrate that among the multiple Early functions of astrocytes imposed by innate responses are largely pro-inflammatory during EAE [3, 6, 7, 26] . For example, astrocytes contribute to the loss of BBB integrity via secretion of reactive oxygen species, chemokines and pro-inflammatory cytokines [3] [4] [5] . This pro-inflammatory milieu is associated with initial axonal damage prior to accumulation of self reactive T cells [27] , which further promotes immune mediated damage. Blocking the pro-inflammatory transcription factor NF-kB in astrocytes improves recovery during chronic EAE [28, 29] . Supporting an initial pro-inflammatory role of astrocytes dependent on innate, not IFN-c responsiveness, inhibition of IFN-c signaling to astrocytes did not influence EAE onset or incidence, initial disease progression, astrocyte activation, or BBB integrity as indicated by similar entry of inflammatory cells into the brain. By contrast, an inflammation dampening effect of IFN-c became evident during the onset of disease remission. Astrocytes limit ongoing inflammation and pathogenic processes at several levels. They not only facilitate repair by inhibiting inflammatory cell entry into the CNS parenchyma [6, 7, 30] , but also down regulate T cell effector function and proliferation [4, 5, 21] . For example, EAE in GFAP deficient mice results in more severe and widespread inflammation [31] . In addition, T cell-astrocyte interactions as well as astrocyte secretion of an unidentified soluble product, distinct from nitric oxide, prostaglandins, or tryptophan metabolism, suppress T cell proliferation [4, 5] . T cell proliferation was also not inhibited via defective IL-2 release, despite the suggestion that T cell-astrocyte interactions facilitate secretion of anti-inflammatory cytokines [4, 5] . Lastly, astrocytes may limit inflammation by triggering apoptosis in T cells [32] . Nevertheless, the role of IFN-c signaling in these potentially anti-inflammatory, protective mechanisms is not clear.
Elevated IFN-c in the CNS of GFAPcR1D mice during both acute and chronic disease excluded limited IFN-c due to sequestration by the transgenic receptor as a mediator of increased clinical severity and mortality. Indeed, enhanced IFN-c production may reflect an attempt to compensate for the loss of IFN-c dependent astrocyte mediated control of inflammation [33] . Sustained expression of pro-inflammatory cytokines, particularly IL-6 and TNF, thus represents a primary mechanism underlying ongoing tissue destruction in GFAPcR1D mice. IL-6 is known to mediate neurological dysfunction [34] and astrocytes are the predominant source of IL-6 during CNS autoimmune disease. Furthermore, its secretion is down regulated by IFN-c induced SOCS activity [17] . On the other hand, sustained TNF implicated dysregulated activation of microglia or macrophages via increased IFN-c or IL-6, as TNF is predominantly secreted by activated CNS macrophages and microglia during EAE [35, 36] . However, the down regulation of microglia activation, including TNF secretion, following interaction with activated astrocytes [37] questions this notion. While both IL-6 and TNF may thus contribute to sustained pathological changes, the source of TNF remains unclear. Similarly, astrocytes are a potential source of the IFN-c induced chemokine CXCL10 during EAE, one of the chemokines controlling T cell recruitment [38] . However, the increased expression of CXCL10 coupled with the inability of the astrocytes in the GFAPcR1D mice to respond to IFN-c suggests altered microglia activation and secretion of CXCL10 [38] or activation of CXCL10 transcription via an independent signaling pathway mediated by TNF or Type 1 interferons [39, 40] .
Importantly, pathology further correlated with decreases in both Tregs and IL-10, but not with increased antigen specific Th17 cells, although astrocytes are critical targets of IL-17 [41] . Although it is possible that the increased IFN-c in the CNS influenced peripheral activation of antigen specific Th17 cells, previous data demonstrated no evidence for expression of the transgene in peripheral organs, including lymphoid organs [18] . IL-10, secreted by a variety of cells types including CD4 + T cells, CD8 + T cells and Tregs [42] , inhibits both acute and chronic EAE [43, 44] . The decrease in this anti-inflammatory cytokine suggests that the induction of IL-27 secretion is a critical aspect of astrocyte responses to IFN-c, thus promoting IL-10 secretion by T cells [23, 25] . However, our data is unable to distinguish the contribution of paracrine versus autocrine IFN-c induced signals to astrocytes on IL-10 production, as IL-10 also regulates astrocyte activation [22] , and can itself be secreted by astrocytes to exert autologous functions [45] .
In addition to mediating an imbalance of protective versus detrimental cytokines, our results demonstrate that IFN-c signaling to astrocytes limits the extent of astrocyte activation during chronic EAE, specifically in grey matter. Activated astrocytes, especially within and adjacent to areas of demyelination are a prominent feature of white matter plaques associated with both chronic MS and EAE [3] [4] [5] . Astrocytes protect neurons and oligodendroglia, but also form glial scars which inhibit regeneration after neuronal injury [3, 5, 8, 20] . The absence of reactive astrocytes increases axonal regeneration after injury [46] , consistent with the concept that limiting astrogliosis is critical for axonal regeneration after neuronal injury. While the significance of sustained astrocyte activation in grey matter tracks is unclear in our model, it is consistent with increased axonal damage, and suggests possible damage to axons outside the lesions, which may not be manifested by histological analysis.
Limiting inflammation following either infection or during an autoimmune attack is a prerequisite for the initiation of repair. This is critically important within the CNS which has limited regenerative capacity. In summary, our data identify astrocytes as prominent targets underlying IFN-c mediated suppression of chronic CNS inflammation [19] . The data further provide a link between sustained inflammatory responses, enhanced demyelination and axonal degeneration associated with loss of neurological function during EAE and chronic progressive MS. Although the inability of astrocytes to respond to IFN-c did not alter disease in the brain, engagement of the IFN-c receptor on astrocytes in spinal cord limits demyelination and functions in a neuroprotective capacity. Current therapies for MS are primarily focused on antiinflammatory and immunomodulatory approaches and have been partially successful in treating acute episodes. The identification of astrocytes as critical responders and mediators of IFN-c signaling in limiting CNS autoimmune disease may provide insights into new approaches to limit long term progression to disability. 
Brains and spinal cords from perfused mice were homogenized separately as previously described [47, 48] . Homogenates were centrifuged at 4506g for 7 min at 4uC. Supernatants were stored at -80uC for cytokine determination (see below). Cells were resuspended in 30% Percoll (Amersham Biosciences, Piscataway, NJ) and isolated by centrifugation (8006g for 30 min at 4uC) onto 70% Percoll cushions. Non-specific binding was inhibited by incubation with anti-CD16/CD32 (2.4G2; BD Biosciences, San Diego, CA) and a 10% mixture of normal goat, human, mouse and rat serum for 10 min on ice. FITC, PE, PerCP, and APC conjugates with monoclonal antibodies (mAb) used to identify and quantify microglia and inflammatory cells were: CD4 (GK1.5), CD8a (53-6.7), CD45 (30-F11), MHC class II (2G9), Ly6G (1A8) (BD Biosciences), and F4/80 (Serotec, Raleigh, NC). Microglia and inflammatory cells were distinguished based on differential CD45 expression. CD4 + T cells were identified as CD45 hi CD4 + , CD8 + T cells as CD45 hi CD8 + , macrophages as CD45 hi F4/80 + and neutrophils as CD45 hi Ly6G + MHC class II -. MOG specific induction of cytokines was determined by stimulation of 1610 6 CNS cells with 20 mg/ml peptide for 6 h at 37uC with GolgiStop (BD Biosciences) added for the last 4 h. Intracellular cytokines were detected with FITC-anti-IFN-c (clone XMG1.2; BD Biosciences) and PE-anti-IL-17 (clone TC11-18H10; BD Biosciences). Intracellular Foxp3 was detected by staining for cell surface markers, followed by permeabilization with Fixation/ Permeabilization Reagent (eBioscience, San Diego, CA) and incubation with PE-labeled anti-Foxp3 (FJK-16s; eBioscience). Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest Pro software (BD Biosciences). Data was analyzed using FlowJo (7.6.1) software (Tree Star Inc., Ashland, OR).
Cytokines were determined by ELISA using antibody pairs and recombinant cytokine standards from BD Bioscience. IL-27 was measured using QUANTIKINE Mouse IL-27p28 Immunoassays (R&D Systems Inc., Minneapolis, MN).
Following anesthesia, mice were perfused with PBS (pH 7.2). Brains and spinal cords were fixed with Clark's solution (75% ethanol and 25% glacial acetic acid), and embedded in paraffin. Spinal cords were divided into 6 sections prior to embedding, corresponding to cervical, thoracic and lumbar levels. Cross sections (6 mm), were stained with either hematoxylin and eosin (H&E) or luxol fast blue (LFB). Immunoperoxidase staining was used to identify activated astrocytes with anti-GFAP (AbCam, Cambridge, MA) and axonal integrity with SMI-31 and SMI-32 mAb (Sternberger Monoclonals Inc., Lutherville, MD) followed by visualization using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and 3,39-diaminobenzidine (Sigma-Aldrich).
Sections from at least 3 separate experiments containing at least 3 mice per group were reviewed in a blinded manner. Numbers of GFAP + cells in spinal cord were determined in 15 non-overlapping 406 fields (0.2 mm 2 ) in white matter and gray matter. Stained spinal cord sections of all 6 levels on individual glass slides were scanned (406) and digitally imaged at high resolution with an Aperio ScanScope (Vista, CA). Aperio software was used to quantify areas of demyelination within the white matter tracks of each of the 6 sections per individual mouse. For analysis of CD4 + T cells spinal cords were embedded in Tissue-Tek O.C.T. (Andwin Scientific, Tryon, NC), flash frozen in liquid nitrogen and stored at 280uC. Blocks were warmed to 220uC prior to cutting 10 mm sections by cryostat. Following fixation with acetone for 2 min at 4uC, non-specific antibody binding was blocked with Cyto Q Background Buster (Innovex Biosciences, Richmond, CA) for 15 min. Sections were stained with anti-CD4 (L3T4) antibody (Vector Laboratories) diluted in Cyto Q Immuno Diluent (Innovex Biosciences) followed by biotinylated rabbit anti-rat, peroxidase ABC reagent and visualized with NovaRED substrate (all from Vector Laboratories). Sections were counter stained with hematoxylin to visualize the nuclei. Spinal cord sections were scanned (406) and digitally imaged at high resolution with an Aperio ScanScope.
Mixed glial cultures (,70% astrocytes) were established from neonatal GFAPcR1D and wt mice as previously described [47] . IL-27 secretion was determined 48 h after rIFN-c (100 ng/ml) stimulation.
Frozen tissues were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and cDNA prepared as described [47, 48] . Quantitative real-time PCR was performed using 4 ml of cDNA and SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in duplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression levels were normalized to ubiquitin or GAPDH using the following formula: 
Statistical significance was determined by two-tailed Student's t test. A value of p,0.05 was considered statistically significant. Figure S1 CD4 + T cell recruitment into the spinal cord during acute EAE. CD4 + T cells were visualized in 10 mm frozen sections of spinal cords from wt and GFAPcR1D tg mice at day 18 p.i. Immunoperoxidase stain (NovaRED chromogen with hematoxylin counterstain). Scale bars = 50 microns. (TIF)  Influence of Mabs on PrP(Sc) Formation Using In Vitro and Cell-Free Systems PrP(Sc) is believed to serve as a template for the conversion of PrP(C) to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(c)or PrP(Sc) would hinder or prevent the formation of the PrP(C)–PrP(Sc) complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc) formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP(Sc) as a template or seed, and a series of incubations and sonications, to convert PrP(C) to PrP(Sc). The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP(C)–PrP(Sc) conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C) conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy). Prion diseases are a group of fatal neurodegenerative disorders that are associated with conformational conversion of the cellular prion protein, PrP C , which is mainly a-helical with very few beta sheets, into a b-sheet-rich form, PrP Sc [1] [2] [3] [4] [5] . The mechanism by which PrP C is converted to the abnormal isoform is still not clear, but it is presumed to involve a PrP C -PrP Sc complex, with the latter serving as a conformational template [6] . In this model, PrP Sc serves as a template that binds to PrP C and produces a conformational conversion into the abnormal isoform. This raises the issue of whether there are critical contact sites that mediate conversion. If this is the case, interfering with or blocking complex formation should prevent the PrP C to PrP Sc conversion process. Previous reports have described anti-PrP antibodies that can stop or hinder the conversion process add reference 44 and renumber [7] [8] [9] [10] [11] [12] [13] [14] .
Protein misfolding cyclic amplification (PMCA) is an assay that mimics the PrP Sc propagation process under cell-free conditions. In this method PrP Sc is amplified by converting PrP C to a PrP Sc seed during incubation with periodic sonication [15] . PrP Sc generated by PMCA is infectious in wild-type animals [16] and can be indefinitely propagated while preserving the properties of the original PrP Sc strain [16] [17] [18] . Furthermore, PMCA has been quite useful in studying the cofactors that influence PrP conversion [19] [20] [21] [22] [23] [24] [25] [26] , and in detecting PrP Sc from biological samples of humans and animals [17, [27] [28] [29] [30] [31] [32] [33] [34] .
We hypothesized that antibodies binding to PrP c and/or PrP Sc might hinder or prevent the formation of the PrP C -PrP Sc complex and thus prevent the conversion process. We compared the effect of individual PrP-specific monoclonal antibodies (Mabs) on the PrP C -PrP Sc conversion process using both an N2a/22L cell culture model and the test-tube PMCA system. Our results demonstrate that the Mabs have a range of inhibitory effects on the PrP C -PrP Sc conversion process. The degree of inhibition is Mab specific and more dependent on the antibody targeting region than on the specific epitope being recognized. Furthermore, since the PMCA-based method is dose-dependent and rapid, it may serve as an ideal screening assay for potential inhibitors of both PrP Sc accumulation and the progression of prion diseases.
All procedures involving animals and their care were conducted in accordance with the United States Department of Agriculture Animal Welfare Act and the National Institute of Health policy on Humane Care and Use of Laboratory Animals. Tissue samples from uninfected and prion agent-infected mice and hamsters were obtained using protocols approved by the Institutional Animal Care and Use Committee of the SUNY Downstate Medical Center (protocol #'s 07-250-09 and 07-251-09).
A 10% normal hamster brain homogenate (NBH) was prepared in phosphate buffered saline (PBS) containing 1% Triton X-100, 4mM EDTA and 1% protease inhibitor cocktail (Abcam). PrPspecific Mabs were generated against recombinant (murine or hamster) PrP or brain-derived proteinase K (PK)-resistant purified PrP Sc [35] from brains of clinical mice infected with the ME7 mouse-adapted scrapie strain or clinical hamsters infected with the 263K hamster-adapted scrapie strain. The Mabs used in this study were purified (Montage Antibody Purification kit; Millipore, Billerica, CA), isotyped (ELISA Mouse Antibody Isotyping kit; Thermo Fisher, Rockford, IL), and epitope mapped ( Table 1) . The immunoreactivity of all the Mabs were analyzed on western blots against denatured, PK-digested and undigested PrP derived from uninfected and infected brain homogenates as well as by ELISA against recombinant PrP. With the exception of Mab 3F4, each of the individual Mabs had equivalent immunoreactivity against murine and hamster PrP Sc on an immunoglobulin concentration basis. All of the Mabs were highly reactive against both hamster PrP C and PrP Sc isoforms and, for the PMCA studies, were individually added to the 10% NBH at a final concentrations of 50 mg/ml. A 10% 263K brain homogenate was prepared in PBS only and diluted to a final concentration of 10 24 . A 100 ml aliquot of this homogenate was initially combined with 10 ml of 10% NBH (with or without added Mab). Each sample was sonicated (QSONIC at 480W power, 60 Amplitude, 40,000 J energy, 90 sec process time, 3 sec pulse on21 sec pulse off), then incubated at 37uC for 1 hr. This was defined as one cycle of serial PMCA (sPMCA). At the completion of each cycle, an additional 10 ml of 10% NBH (with or without Mab) was added. At the end of every five cycles, 100 ml of the total volume was transferred to a new tube containing an equal volume of 10% NBH (with or without Mab) and the cycling reactions continued. At the completion of 40 cycles (sPMCA 40 ), 500 ml from each sample was PK-treated (100 mg/ml final concentration, 50uC, 30 min), followed by the addition of protease inhibitor cocktail. The sample was heated (100uC, 10 min) and then centrifuged at 16,0006g for 2 min at room temperature. The supernatant was combined with 6X Laemmli sample buffer, and 50 ml was electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose membrane. The membrane was blocked for 1 hr in PBS containing 0.1% Tween 20 (PBST) with 5% non-fat dry milk and incubated with 2 mg/ml biotinylated Mab 08-6/2F11. The membrane was washed 3 times (10 min each) with PBST, incubated for 60 min in HRP-conjugated streptavidin (Invitrogen) (1:5000 in PBST containing 5% non-fat dry milk) followed by 3 additional PBST washes and detection of proteins with ECL Supersignal West Dura kit (Thermo Fisher). Quantification of PrP Sc was performed by densitometric analysis using NIH Image J software.
Cellulose membranes spotted with 99 overlapping 13-mer PrP peptides were produced as previously described [36] . The membranes were blocked with 5% non-fat dry milk/tris-buffered saline containing 0.1% Tween 20 (TBST) probed with antibody diluted 1:5000 in 1% normal goat serum/TBS at 4uC overnight, followed by horseradish peroxidase (HRP)-conjugated goat antimouse secondary (Cappel 55570) for 2 hours at room temperature, and detected using Millipore Immobilon Western chemiluminescent HRP substrate (Cat WBKLS0500). Membranes were regenerated for re-use by shaking with dimethylformamide for 30 minutes, then 8M urea/50mM Tris-HCl pH 8.0/1% b-mercaptoethanol (b-MC)/1%SDS overnight at 37uC, followed by a 30 min wash in the same buffer, and then twice for 30 minutes each in 50% methanol/glacial acetic acid, and finally three times for 5 minutes each in methanol. After air drying membranes were stored in a sealed container at room temperature.
Murine neuroblastoma N2a cells (ATCC line CCL 131) were grown in the Minimal Essential Medium supplemented with 10% FBS, penicillin and streptomycin and infected with 2% 22L brain homogenate as described previously [12] . Following infection, the amount of PrP Sc in 200 mg cell lysate aliquots of the N2a/22L cells was determined by PK digestion (1 mg/ml PK for 30 min at 37uC), SDS-PAGE on 12.5% Tris-tricine gels [37] and western blot analysis as previously described [12] .
For treatment of cells with Mabs, N2a/22L cells (from the fifth passage after infection and higher) were plated in six-well plates and once the cells were 70-80% confluent, Mabs were added at a final concentration of 10 mg/ml and incubation was continued for 96 hr. Each Mab was tested in three independent experiments using independently infected cell lines. Each experiment included both a positive control (untreated N2a/22L cells) and a negative control (N2a cells), which were subjected to PK digestion. The level of PK-resistant PrP Sc was measured in western blots using HRP-conjugated sheep anti-mouse IgG as the secondary reagent and ECL Supersignal West Dura kit. Membranes were exposed to X-ray film (X-Omat Blue XB-1; Kodak, New Haven, CT,) with a constant exposure time of 30 sec. The films were converted into eight-bit grayscale digital files. Quantification of PrP Sc was performed by densitometric analysis using NIH Image J software v. 1.34. Areas under the curves for three PrP bands representing non-, mono-and diglycosylated isoforms of the protein were summarized from each sample to calculate the total amount of PrP and expressed as percentages of the average value from a positive control (untreated N2a/22L), whereas the optic density of the background was taken from negative control lanes (N2a cells).
The PrP-specific Mabs that were evaluated for their ability to prevent PrP C to PrP Sc conversion have linear epitopes that span the entire prion protein from the amino to the carboxy terminus ( Table 1 , Fig. 1 ). We used N2a cells persistently infected with the 22L mouseadapted scrapie strain (N2a/22L) to evaluate the affect of each Mab on PrP Sc formation ( Fig. 2A and 2B) . Treatment with the Mabs did not result in any cytotoxicity to the N2a/22L cells throughout the incubation period. Further, incubation of the. N2a/22L cells with 10 mg/ml purified, irrelevant mouse IgG had no effect on PrP Sc formation compared to untreated N2a/22L cultures (Figs. 2A and 2B). Mab 3F4 did not reduce PrP Sc formation compared to control N2a/22L cultures lacking Mab. Mab 3F4 does not react with mouse prion protein so this was not surprising [38] . The ability of a singly added Mab to inhibit PrP Sc formation was not related to a specific epitope since all of the remaining singly added Mabs inhibited PrP Sc formation to varying degrees. Of the individually added Mabs, 5D6 was the most effective at inhibiting PrP Sc formation (95% inhibition) while 3A2 was the least effective (38% inhibition). Targeting the amino terminus with Mab 7E4 was effective at inhibiting 73% PrP Sc formation as was targeting the octapeptide repeat region using Mab 10E4 which resulted in almost 90% inhibition. Strangely, although their epitopes overlap, Mab 11F12 was less effective than Mab 5D6 at inhibiting PrP Sc formation (53% vs 95% inhibition). This is in contrast to Mabs 8E9 and 1B11, which have overlapping epitopes with 8E9 being more expansive, and resulted in 52% and 42% inhibition, respectively. The combination of 5D6 and 11F12 did not result in an additive inhibitory effect and, in fact, resulted in less inhibition than either one alone. This was confirmed in studies where the addition of 8E9 to 5D6 and 11F12 caused a 45% PrP Sc inhibition, which was slightly better than 8E9 alone, although the predicted additive inhibitory effect of 63% for the three Mab combination (48% for 8E9 plus 15% for the 5D6 and 11F12 combination) was not observed.
Studies were performed with PMCA to determine whether a cell-free system can recapitulate the effect of Mabs on PrP C conversion observed in infected cells. This system also allowed us to evaluate whether accessibility of Mab to membrane associated PrP C in the living cells influences the PrP C to PrP Sc conversion process. Mabs (12-50 mg/ml final concentration) were added throughout the sPMCA 40 protocol along with the 10% NBH spiked with a 10 24 dilution of 263K infected brain homogenate as described in the Methods section. This dilution of infected brain homogenate does not result in detectable PK resistant PrP Sc immunostaining (Fig. 3A) and, therefore, did not interfere with the detection of newly formed PrP Sc . At the completion of sPMCA 40 , the samples were digested with PK (100 mg/ml) and analyzed on immunoblots using biotinylated Mab 2F11 which reacts equally with both hamster PrP C and PrP Sc . It is interesting to note that although the 263K-infected brain homogenate displayed the 3 band pattern typical for the multiple glycosylated forms of PrP C and PrP Sc (Fig. 3A) , the sPMCA 40 products in the positive controls and Mab-treated reactions consisted of only a single diglycosylated 30 kDa PrP Sc band observed after PK digestion at the higher levels of inhibition, .50%, but had two bands or a smear when there was less inhibition (Fig. 3B) .
PMCA in the presence of Mabs was also used to study the importance of binding site specificity in the PrP C to PrP Sc conversion process (Fig. 3) . We performed sPMCA with different Mab concentrations to determine the minimum amount of Mab necessary to inhibit the conversion process. Using a 10 24 dilution of 263K-infected hamster brain homogenate as the PrP Sc seed and a 10% normal brain homogenate (NBH) as the source of PrP C , we tested the ability of Mabs to inhibit the conversion of PrP C to PrP Sc . For each Mab, final concentrations of 12 mg/ml (lanes 3, 6, 9, 12, 15, 18, 21 and 24), 25 mg/ml (lanes 4, 7, 10, 13, 16, 19, 22 and 25) and 50 mg/ml (lanes 5, 8, 11, 14, 17, 20, 23, and 26) were prepared in hamster NBH and used in the sPMCA reactions. Compared to sPMCA 40 , which contained no Mab (lane 1) and with the exception of 02-3/3A2, the majority of the PrP-specific Mabs inhibited the conversion process in a dose-related manner although some were more effective than others. Mabs 7E4, 10E4, 11F12, 8E11, and 8E9 completely inhibited the conversion process at 50 mg/ml while Mabs 1B11 and 2F7 inhibited the conversion process to a lesser degree. The inhibition caused by the Mabs was a specific response since sPMCA 40 studies replacing Mabs with purified normal mouse IgG (at 12-50 mg/ml) in the 10% NBH did not cause any inhibition of PrP Sc formation (data not shown). It is interesting to note that, with the exception of only 8E9, the epitopes for all the Mabs that caused complete inhibition are located in the amino half of the PrP while those that caused incomplete inhibition are located in the carboxy half of PrP. There was good correlation between the extent of PrP Sc inhibition when 10 mg/ml Mab in cell culture was compared to 12 mg/ml Mab with sPMCA 40 .
A separate study using sPMCA 40 demonstrated that Mabs 3F4 and 5D6 caused complete inhibition of PrP Sc formation at 12-50 mg/ml (Fig. 4) . Therefore we extended those studies and evaluated the effects of Mabs 3F4 and 5D6 using a wider range of Mab concentrations (1.5-50 mg/ml). Compared to the other antibodies in this study, Mabs 3F4 and 5D6 had the most pronounced effects on PrP Sc formation as demonstrated by the low concentrations of 3 and 6 mg/ml, respectively, causing complete inhibition (Fig. 4) . The potent inhibitory effect of 5D6 on PrP Sc observed using sPMCA 40 coincides with its dramatic effect in the N2a/22L culture model. Furthermore, the poor PrP Sc inhibition by 3A2 with sPMCA 40 (Fig. 3B) corresponded well with the poor inhibition (only 32% reduction compared to negative control) observed in the cell culture system ( Fig. 2A  and 2B ). 
Currently, there is no effective treatment for prion diseases. To date, hundreds of chemical compounds have been identified that antagonize prion propagation in vitro in cell culture-based assays and/or in vivo in animal studies [39] [40] [41] [42] [43] . Unfortunately, many compounds efficient in in vitro studies were only effective in animal models if treatment was begun before or close to the time of inoculation with the infectious agent [44] . Furthermore, many of the candidate compounds have limited usefulness clinically due to toxicity or their inability to cross the bloodbrain barrier [e.g. Congo red [45] , iododoxorubicin, b-sheet breakers].
Additional therapeutic and/or prophylactic strategies have been and continue to be pursued. Vaccination with recombinant mouse PrP delays the onset of prion disease in mice [46] . Passive immunization with anti-PrP antibodies was shown not only to inhibit formation of PrP Sc in a cell-free system [47] , but was also shown to prevent infection of susceptible N2a cells [7] and to inhibit prion replication in infected cells [8, 47, 48] . The effectiveness of these treatments were also dependent on when they were administered relative to the time of infection.
In an initial passive immunization study using wild-type CD1 mice, Mabs 8B4 (to mouse PrP residues 34-52) and 8H4 (to mouse PrP residues 175-185) given immediately after challenge with 139A scrapie by intraperitoneal (IP) injection (50 mg/week), resulted in a significant prolongation of the incubation period with 10% of the 8B4 treated animals remaining disease free in the group challenged with a lower dose of PrP Sc [10] . In another study using higher antibody doses (4000 mg/week IP) of either ICSM 18 (to mouse PrP residues 146-158) or ICSM 35 (to mouse residues 95 to 105), prion infection from a peripheral source was completely prevented if treatment was continued for 7 or 30 days immediately following PrP Sc challenge [9] . Furthermore, a transgenic mouse model that expresses Mab 6H4 is resistant to prion infection via IP injection by a mechanism that involves either perturbation of cellular PrP trafficking/PrP C degradation or disruption of the PrP C -PrP Sc interaction [49] .
Previous studies have reported that the 132-140 portion of PrP C [8] or the 132-156 region of PrP [50] [51] [52] [53] are important for the generation of PrP Sc . Rigter et al. [54] found two high affinity binding regions for protein-protein interactions using ovine peptide-arrays: (i) sheep-PrP peptides 43-102, including the amino-terminal octarepeats, and (ii) sheep-PrP peptides 134-177 Figure 2B for representative western blots). PrP Sc western blots were quantitated and the amount of inhibition was determined relative to N2a/22L control cultures. The controls consisted of cells both in the absence of Mab and in the presence of normal mouse IgG. The % PrP Sc inhibition plotted represents the mean 6 SD from three independent experiments as described in Methods. doi:10.1371/journal.pone.0041626.g002 which encompasses most of the scrapie susceptibility-associated polymorphisms in sheep. Moroncini et al. [55] found that residues within the 89-112 and 136-158 segments of PrP C are key components of the PrP C -PrP Sc complex. Beringue et al. [11] reported that antibodies exclusively binding PrP C were relatively inefficient inhibitors of PrP Sc accumulation compared with antibodies that additionally recognize disease-associated PrP isoforms. Féraudet et al. [56] screened 145 anti-PrP Mabs for their capacity to inhibit PrP Sc replication in infected N2a or Rov9 cells. They identified four different linear epitopes that hindered the PrP C to PrP Sc conversion: the amino terminal region 26-35, the octarepeat region 59-89, the intermediate region 97-102, and the central region 130-160. The observation that antibodies that bind to the amino terminus of the prion protein are capable of inhibiting conversion suggests that the Figure 3 . A. Western blot of 263K brain homogenate that was used as the seed for PMCA. Dilutions of the brain homogenate was prepared and either untreated (lanes 1 and 2) or PK-treated (lanes 3 and 4) prior to SDS-PAGE and western blotting. A 1023 dilution prior to and after PK demonstrates the three protein banding pattern typical for 263K brain homogenate whereas no bands are visible at a 1024 dilution of the same homogenate. B. Western blotting of the PMCA products following sPMCA40 in the absence and presence of PrP-Specific Mabs. Fourty cycles of serial PMCA was carried out in the absence or presence of Mabs as described in the text. Each Mab was added at a final concentration of 12, 25, and 50 mg/ ml. Following PK treatment, the PMCA products were subjected to SDS-PAGE, western blotted and immunostained for PrPSc. The protein bands were quantitated and the level of PrPSc inhibition, relative to the no Mab and normal mouse IgG controls, were determined. doi:10.1371/journal.pone.0041626.g003 Figure 4 . Influence of Mabs 3F4 and 5D6 on PrP Sc formation following sPMCA 40 . Mabs 3F4 and 5D6 were added to sPMCA 40 at final concentrations of 0-50 mg/ml. The PMCA products were PK treated and western blotted. The PrP Sc was quantitated and the level of PrP Sc inhibition was determined relative to control reactions. doi:10.1371/journal.pone.0041626.g004 endogenous proteolytic cleavage occurs after the site of conversion.
To more completely explore the possible therapeutic effect of anti-PrP antibodies, and to establish another system to analyze the influence of Abs on the conversion process, we screened Mabs produced in our laboratory for their capacity to inhibit PrP Sc formation. This screening was performed using N2a/22L cells and cell-free sPMCA. In N2a/22L cultures, all Mabs that react with mouse PrP reduce PrP Sc formation although with varying efficiency. Thus, similar to previous results [55] , we found that the ability to inhibit PrP C to PrP Sc conversion was not restricted to a single epitope or limited to a specific region of the protein. However, the greatest inhibition was observed with Mabs that targeted epitopes in the amino terminal, unstructured region of the PrP. The greatest inhibition in the N2a/22L cells was with Mab 5D6. This is consistent with a prior study using Mab 6D11 (anti-PrP residues 95-105) which in a screen of multiple Mabs, only one produced the greatest inhibition (,100%) [12] . Mab 6D11 has also been shown to have some efficacy in vivo prolonging the presymptomatic incubation period [57] . The Mab inhibition results obtained using PMCA were similar to that found in the cell culture system. PMCA has the advantages over the cell culture model of being cost-effective, simple, rapid, sensitive, and more amenable to studies of dose dependence. For identification of potential candidate Mabs that might have in vivo activity it is likely that such Mabs would have to produce 90 to 100% inhibition in the much simpler in vitro systems.
The interaction of PrP C to PrP Sc is critically dependent on the structural compatibility of the molecules as supported by the existence of a species barrier for prion infection, related to minor differences in the primary sequence of PrP C in different species. Therefore, it is not surprising that antibodies that may alter or mask the critical epitopes on PrP C and/or PrP Sc , involved during the mutual conformational complementarity required in prion propagation, will be inhibitory for prion replication. Although many anti-PrP antibodies targeting different regions of PrP may have some therapeutic effect in vitro, it is not clear how this relates to their efficacy in vivo. On the one hand, it is tempting to speculate that only the antibodies exhibiting near complete inhibition in vitro would be effective in vivo given the obstacle of the blood brain barrier and access to PrP in cells. However, it is also possible that only partial inhibition of conversion is required in vivo allowing the cells to ''recover''. In either case, it would be advantageous for these therapeutic antibodies to have high affinities of binding to PrP C and/or PrP Sc , as well as targeting specific critical PrP domains. One can hypothesize that the simultaneous targeting of more than one critical epitope will lead to greater benefits. However, co-treatment experiments performed with a mixture of two antibodies compatibly binding cell-surface PrP C did not show any benefit with compared to treatment involving a single Mab in our current experiments. In a previous study [58] , we demonstrated synergistic binding with one of our antibody pairs. Synergistic binding of inhibitory Mabs, i.e. reaction with an antibody that increases the binding of the second antibody, would be predicted to enhance the inhibitory effect. Further studies with antibody pairs fitting this description will be required to test this hypothesis. In addition, determining the significance of the Mab's ability to bind both PrP C and PrP Sc may provide further insight into the conversion process.
Conceived and designed the experiments: RR TW. Performed the experiments: BC RP TW RR. Analyzed the data: BC RP TW RR. Contributed reagents/materials/analysis tools: RP TW RR. Wrote the paper: RR. Predictors and outcome of patients with acute respiratory distress syndrome caused by miliary tuberculosis: a retrospective study in Chongqing, China BACKGROUND: Miliary tuberculosis (TB) is an uncommon cause of acute respiratory distress syndrome (ARDS) with a high mortality. The aim of the present study was to evaluate the clinical characteristics, predictors and outcome of patients with ARDS caused by miliary TB. METHODS: A retrospective study was conducted among patients with a diagnosis of ARDS with miliary TB in four hospitals from 2006 to 2010. Medical records and laboratory examinations of these patients were taken during the first 24 h of admission. RESULTS: Eighty-five patients with miliary TB developed ARDS, 45 of whom survived (52.9%). The median age was 36.6 ± 12.5 years with 38 males (44.7%). Diabetes mellitus (DM) was the most common underlying disease (18.8%).ICU mortality was 47.1%. The time from admission to anti-tuberculosis therapy was 4.5 ± 2.0 days. Mean duration of mechanical ventilation was 8.5 ± 3.0 days in all patients. Duration of time to diagnosis, time from diagnosis to mechanical ventilation, and time to anti-tuberculosis therapy were significantly shorter in survivors than those in non-survivors. Diabetes mellitus (OR 5.431, 95%CI 1.471-20.049; P = 0.005), ALT (70-100U/L, OR 10.029, 95%CI 2.764-36.389; P = 0.001), AST (>94U/L,OR 8.034, 95%CI 2.200-29.341; P = 0.002), D-dimer (>1.6mg/L, OR 3.167, 95%CI 0.896-11.187; P = 0.042), hemoglobin (<90g/L, OR 14.824, 95%CI 3.713-59.179; P = 0.001), albumin (<25g/L, OR 15.896, 95%CI 3.975-63.566; P = 0.001) were independent predictors of ARDS development in the setting of miliary TB. CONCLUSIONS: Accurate diagnosis, early initiation of anti-tuberculosis therapy and mechanical ventilation are important for the outcome of patients with ARDS caused by miliary TB. DM, ALT, AST, D-dimer, hemoglobin, and albumin are independent predictors of ARDS development in patients with miliary TB. Tuberculosis(TB) remains a major and global health disease [1, 2] . Recent studies have shown the link between acute respiratory distress syndrome (ARDS) and pulmonary TB [3, 4] . Pulmonary TB complicated by ARDS is often found in the setting of miliary TB [3] [4] [5] [6] . Most reports on ARDS caused by miliary TB are small numbers of patients in the case reports. Compared with miliary TB alone, miliary TB with ARDS portends a higher mortality of 33-90% [7] [8] [9] . Duration of miliary TB beyond 20 days tends to markedly increase the risk of ARDS [10] . It is very important for recognition of ARDS caused by miliary TB.
Despite being a well-documented entity, miliary TB complicated by ARDS remains a challenging diagnosis due to its variable clinical manifestations and low morbidity. Some predictors such as AST, and ALT [4, 10] in miliary TB with ARDS have drawn our attention. Numerous case reports have mentioned that TB with ARDS is more common than TB complicated by anemia [4, 11] and hypoproteinemia [6, 11] . Thus, identifying the predictors of miliary TB associated with ARDS can play an important role in diagnosis and therapy. The aim of the present study was to determine the predictors and their impact on outcome based on a retrospective analysis of patients with ARDS caused by miliary TB in four hospitals of Chongqing in China, with the hope that this will contribute to a better understanding and improved management of the disease.
Over a 5-year period (2006.03. Our research protocol was approved by the institutional review boards of all participating institutions.
The original case records including clinical profiles and laboratory parameters at admission were gathered from the registration departments. Age, sex, past medical history, underlying diseases, PaO2/FiO2, time from diagnosis to mechanical ventilation, time from admission to antituberculosis therapy, duration of time to diagnosis, and lengths of stay in the ICU and in the hospital were collected. Acute Physiology and Chronic Health Evaluation (APACHE) III score were calculated on the day of diagnosis with ARDS. The results of acid-fast bacilli(AFB) smears, culture of respiratory specimens including sputum, tracheal aspirate or bronchoalveolarlavage (BAL)fluid and histopathological examination were recorded. Laboratory data including aspartate aminotransferase (AST), alanine aminotransferase (ALT), erythrocyte sedimentation rate (ESR), D-dimer, hemoglobin, and albumin were taken during the first 24 h of admission.
The diagnosis of miliary TB was made based on: equality of the size, distribution, density miliary-like nodules bilaterally diffused on chest radiography by at least 2 independent of radiologists. Pulmonary TB was confirmed by at least one of the three following criteria:1)positive AFB smear and/or culture for M. tuberculosis from respiratory specimes;2) histopathological identification of TB granulomain in biopsied tissues of lung, and/or pleura;3)clinical and radiographic improvement after anti-tuberculosis treatment [4] . The diagnosis of ARDS was made in accordance to the diagnostic criteria of European-American Consensus Conference on ARDS [12] :acute in onset with PaO 2 /FiO 2 ≤ 200mmHg, bilateral infiltrates seen on chest radiograph, and pulmonary artery wedge pressure ≤ 18 mm Hg. Survivors were defined as patients who survived to discharge from hospital. Patients with human immunodeficiency virus (HIV), H1N1 Influenza A,and procalcitonin (PCT) positive were excluded.
Continuous variables were presented as mean ± standard deviation(SD) or median and compared using an unpaired t-test and the Mann-Whitney U test. Categorical variables were compared using the Chi-squared test. Multivariate logistic regression analysis was performed to determine the predictors. All statistical analysis were performed using SPSS 13.0. P <0.05 was considered significant.
Patients' clinical characteristics and outcome are presented in Table 1 . All patients experience typical symptoms of miliary TB. Diabetes mellitus (DM) was the most common underlying disease (18.8%). Thirty-three (38.8%) patients had been initially misdiagnosed with viral pneumonia, hypersensitivity pneumonitis, acute interstitial pneumonia, fungal pneumonia, alveolar cell carcinoma, or meningitis for a median of 7.2 ± 3.4 days from admission. The diagnosis of miliary TB was established by AFB smear and/or culture of respiratory specimens (including sputum,tracheal aspirate or BAL fluid) in 61 patients(71.8%), by histopathological examination of tissue biopsy in 11 patients(12.9%), and by clinicoradiological diagnosis in 13 patients(15.3%). Bacterial isolate drug sensitivity data were available from 43 patients (50.6%), 3(3.5%) demonstrated at least single drug resistance.
The time from admission to anti-tuberculosis therapy was 4.5 ± 2.0 days. All 85 patients with ARDS were prescribed anti-tuberculosis medication consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. Mechanical ventilation was necessary in all 85 patients. Thirty-eight patients(44.7%) required invasive mechanical ventilation while the rest were given non-invasive mechanical ventilation with BiPAP. Mean duration of mechanical ventilation was 8.5 ± 3.0 days with ICU mortality of 47.1%. Thirty-five patients (41.2%) received glucocorticoid therapy (methylprednisolone:80 mg/day) intravenously for a maximum of 5 days when anti-tuberculosis therapy was started. The use of glucocorticoids was associated with a mortality of 22.9% (8/35) compared with 76.0% (38/50) in those who were not treated with glucocorticoids. Comparison between patients with miliary TB developing ARDS and patients with miliary TB alone are shown in Table 2 . Comparison between survivors and non-survivors of ARDS patients are shown in Table 3 . Duration of time to diagnosis, time from diagnosis to mechanical ventilation, and time from admission to anti-tuberculosis therapy were significantly shorter in survivors than non-survivors. Also, DM, ALT, AST, Ddimer, hemoglobin, and albumin showed significant difference between the survivor group and non-survivor group. The 3 pregnant patients underwent termination of pregnancy, one of whom died of respiratory failure.
Positive likelihood ratio were performed to analyze sensitivity and specificity of the predictors level(the greater the ratio the greater probability of true positive in a positive result) ( were independent predictors of ARDS development caused by military TB.
In the present study,our results demonstrated accurate diagnosis and early therapeutic management were crucial to optimizing outcome and DM, ALT, AST, D-dimer, hemoglobin, and albumin are independent predictors of ARDS development in patients with miliary TB. Tuberculosis remains a major cause of morbidity and mortality around the world, especially in developing countries [13, 14] . According to the 13th annual tuberculosis report of the World Health Organization (WHO), there were an estimated 9.27 million new cases worldwide in 2007, an increase from 9.24 million in 2006 [1] . Further, miliary TB is an uncommon cause of ARDS with a high mortality [4, 15] . The current study was performed in Chongqing, the fourth central municipality of China, which has a prevalence rate of 0.54%, higher than the national average. In our study, the average age of patients with ARDS caused by miliary TB was younger than that previously reported due to the possible reason of host factors including region, race, and environment [3, 4] . Mechanical ventilation is an important treatment for miliary TB complicated by ARDS. Prompt mechanical ventilation from the day of diagnosis can effectively improve outcome, which benefits for the management of ARDS caused by miliary TB. Also, accurate diagnosis and early anti-tuberculosis therapy are crucial to the treatment of miliary TB with ARDS. However, the high overall mortality is attributed to case mix, misdiagnosis, and severity of illness, which ultimately lead to the delay in the initiation of anti-tuberculosis therapy or mechanical ventilation.
Albumin plays an important role in regulating plasma osmolality. Hypoproteinemia accelerates fluid exudation, promotes alveolar edema, and contributes to ventilation-perfusion imbalance. Also, in infected mycobacterium tuberculosis, inflammatory cells accumulate in the alveolar spaces, releasing granular enzymes and oxidants which participate in local inflammation and overlapping reactions and damaging the alveolar basement which allows increase in cellular permeability that aggravates oxygen dysfunction and consequently causes ARDS [16, 17] . If the process continues, cytokines activate an inflammatory cascade reaction and lead to other organs dysfunction, resulting in increases in AST and ALT. The changes in AST, ALT, and serum albumin were significantly different between survivor and non-survivor groups. The results showed that AST, ALT, and serum albumin could be independent predictors of ARDS development in miliary TB.
A number of studies have documented anemia associated with TB [18] [19] [20] . It is well established that erythropoiesis is suppressed by inflammatory mediators in anemia of chronic infection or inflammation and anemia caused by chronic infections including TB results from the effects of cytokines that mediate the inflammatory response,which may provides the development for ARDS [21] [22] [23] . As we know,the severity of anemia is mainly determined by hemoglobin level. The decrease in hemoglobin is assumed as a reflection of inflammation, which can explain the relationship between ARDS and hemoglobin in our study. This relationship is supported by our finding of hemoglobin being an independent predictor of ARDS development in miliary TB.
D-dimer is a fibrin degradation product formed during fibrinolysis, the process of breaking down a blood/fibrin clot. Our study showed that D-dimer was an independent predictor of ARDS development and mortality. Hyperglycemia is known to have a proinflammatory effect, and may be correlated with poor outcome in hospitalized/critically ill patients. Consistent with this correlation, odds ratio analysis after ARDS development identified DM as a risk factor. To further understand the accurate levels of the predictors for predicting ARDS, we used stratified analysis for each index. Positive likelihood ratio was included in the study due to a combination of sensitivity and specificity to reflect the reality of the indicators. ALT (>70-100U/L), AST (> 94U/L), D-dimer (>1.6mg/L), hemoglobin (<90g/ L), and albumin (<25g/L) at the time of admission were independent predictors for ARDS development in the setting of miliary TB. Simple miliary TB can damage organs, inducing mild increases in ALT and AST, although these incremental changes are too small to have any accuracy in predicting the occurrence of ARDS. However, when one develops ARDS, inflammatory mediators will seriously damage the organs, resulting in significantly higher increases in the indexes than those in simple miliary TB, which may translate into higher accuracy for predicting ARDS. Though ESR was significantly elevated in miliary TB, it did not reach statistical significance in association with ARDS. Our findings suggested that ESR has minimal value in predicting ARDS.
In addition, for patients with miliary TB who developed ARDS during pregnancy, there are few case reports with favorable outcome. Our data suggested that pregnancy was a risk factor for ARDS, but this clinical observation was limited by the small sample size. The efficacy of systemic corticosteroids is well-documented for several extrapulmonary complications of tuberculosis such as tuberculous meningitis and tuberculous pericarditis [24] [25] [26] [27] [28] , as well as ARDS [29] [30] [31] [32] . The role of glucocorticoids remains controversial in the management of miliary TB complicated by ARDS [33, 34] . In our study, patients treated with glucocorticoids had a lower mortality than those who did not, which might suggest that methylprednisolone at a dose of 80 mg/day was given intravenously at the time when anti-tuberculosis therapy was started might be benefit for ARDS associated with miliary TB. The limitations of this study are studies with large numbers of patients may be required to validate the observations due to a relatively small sample size in the present study.
In conclusion, the mortality of ARDS caused by miliary TB remains high. Accurate diagnosis and early therapeutic management of therapy including anti-tuberculosis agents and mechanical ventilation are crucial to optimizing outcome. DM, ALT, AST, D-dimer, hemoglobin, and albumin are independent predictors of ARDS development in patients with miliary TB. Symptomatic Venous Thromboembolism Is a Disease Related to Infection and Immune Dysfunction The characteristics of human genomics and cellular immune function between clinically symptomatic venous thromboembolism (VTE) and controls were systematically compared to explore the immunologic pathogenesis of VTE. Microarray assay showed the mRNA expressions of genes related to non-specific cellarer immune and cytokines were significantly down-regulated. Abnormal expressions of CD3+, CD4+, CD8+, NK marker CD16+56+, CD19 and aberrant CD4+/CD8+ ratio were detected in 54 among 56 patients. In PE patients, microarray assay revealed the imbalance in the expressions of genes related to the immune system. The expressions of genes related to non-specific immune cells and cytokines were markedly up-regulated and those associated with cellular immune were dramatically down-regulated. In VTE patients, cytological examination indicated the functions of NK cells were significantly compromised, and the antigen recognition and killing function of T cells markedly decreased. The consistence between genomic and cytological examination suggests the symptomatic VTE is closely associated with the infection and immune dysfunction. Venous thromboembolism (VTE) including acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH) and deep venous thrombosis (DVT) is a global disease. The high morbidity, high misdiagnosis rate and high mortality render PE as a worldwide health problem (1) . In 1965, Egeberg et al first described a parentage with inherited antithrombin deficiency, a member of which repeatedly developed DVT. The antithrombin deficiency is an autosomal dominant genetic disease and they first proposed the concept of thrombophilia (2) . However, evidence on the genetic pathogenesis of VTE is rarely identified in a majority of VTE patients (3) . According to the pathogenesis, VTE is classified into genetic VTE and acquired VTE which is frequently found in clinical practice. The symptomatic VTE is an entity of hereditary VTE and acquired VTE.
The American College of Chest Physicians (ACCP) has recommended the guidelines for the diagnosis and prevention of VTE since 1995. A total of 9 issues have been published by the end of 2012 (4) , and the contents have also been continuously renewed. ACCP proposed that Trauma, surgery, old age, malignancies, pregnancy, heart failure and oral administration of contraceptives are the main risk factors of VTE and ACCP also proposed the concept of risk stratification for prophylaxis of VTE by which differ-Ivyspring International Publisher ent managements were used for prevention from VTE in different risk patients (5, 6) . The incidence of symptomatic VTE is not reduced but gradually increased, the reason of which may be related to the unclear pathogenesis of VTE (7) .
In 2006, Smeeth et al reported the occurrence of VTE was associated with infection, and VTE was frequently observed within 2 weeks after infection (8) .
In 2010, we reported VTE in multiple organs of a patient who died of SARS, suggesting viral infection is a cause of systemic VTE (9) . In addition, in 2010, we detected virus-like microorganisms in the lymphocytes of a young pulmonary hypertension patient with increased D-Dimer, which morphologically confirmed the attack of T cells by virus, and peripheral decreased CD3 + and CD8 + level also indicated virus infection caused significantly compromised function of T cells (10) . In 2011, we reported the decreased CD3 + and CD8 + level with an increased CD4 + /CD8 + ratio in a group of CTEPH patients, suggesting T cellular immune dysfunction and ratio imbalance in CTEPH patients (11) . In the present study, the whole human genome microarray and Gene Ontology (GO) analysis were employed to detect the targeting of symptomatic pulmonary embolism (PE). In addition, flow cytometry was performed to investigate the changes in immune cells in VTE patients, which aimed to validate the results from genome analysis. Based on the findings above, the relationship between immune dysfunction and clinical symptomatic VTE was analyzed. The CPR level in part of VTE inpatients was determined.
Genomic study 20 PE inpatients and 20 controls were randomly selected in Cardiology Department, Tongji Hospital of Tongji University. In the PE group, there were 11 males and 9 females, with a mean age of 70±14 years (44~89 years). There were 13 patients with acute PE and 7 with CTEPH. The pulmonary artery pressure was 50-108 mmHg in CTEPH patients. In the control group, 20 patients (11 males and 9 females) with a mean age of 72±14 years (44~91 years) were enrolled during the same period. No significant difference in age was found between PE patients and control patients (P>0.05). Malignancies, use of immunosuppresants or autoimmune diseases were excluded in all patients.
A total of 56 clinically proven VTE patients (34 with APE, 9 with DVT and 13 with CTEPH) aged 64±18 years (13~88 years) were recruited from Tongji Hospital, including 26 males and 30 females. The immunological parameters of these patients were all compared with the detection intervals of normal population.
The diagnostic criteria of acute PE were the same as the above and the diagnosis of DVT was based on the criteria previously reported (12) . The criteria for CTEPH developed by American AHA were used for the diagnosis of CTEPH (13) . In the present study, 13 patients had the mean pulmonary artery systolic pressure of 54-106 mmHg. Malignancies, use of immunosuppresants or autoimmune diseases were excluded in all patients. All patients were from Shanghai, China. The present study was approved by the Ethics Committee of Tongji Hospital and informed consent was obtained before study.
A total of 5 ml of venous EDTA anti-coagulated blood was obtained from patients of both groups and mononuclear cells were isolated by density gradient centrifugation. Red blood cell lysis buffer (Qiagen, Hilden, Germany) was used to isolate mononuclear cells and total RNA was extracted from mononuclear cells with TRIzol (Invitrogen, Carlsbad, USA) followed by purification with RNeasy column (QIAGEN). Treatment with DNase was performed to avoid the influence by genomic DNA. Quantification of extracted RNA was performed with Nanodrop ND-1000 spectrophotometer (Nanodrop Technology, Cambridge, UK).
Agilent G4112A Whole Human Genome Oligo Microarrays were purchased from Agilent (USA). A microarray is composed of 44,290 spots including 41675 genes or transcripts, 314 negative control spots, 1924 positive control spots and 359 blank spots. The functions of more than 70% of genes in the microarray have been known. All patients of both groups were subjected to microarray analysis.
Indirect approach was applied to mark samples. About 1 μg of total RNA was reversely transcribed into double strand cDNA. After purification, in vitro amplification was performed with Agilent Low RNA Input Linear Amplification Kit (Agilent, Pal alto, USA) and modified UTP [aaUTP, 5-(3-aminoally1)-UTP] was used to replace UTP. The integrated aaUTP can interact with Cy3 NHS ester forming fluorescent products which are then used for hybridization. The integration rate of fluorescence can be determined with a NanodropND-1000 spectrophotometer. Then, hybridization mixture was prepared with Agilent oligonucleotide microarray in situ hybridization plus kit. About 750 ng of fluorescent products were fragmented at 60℃ and hybridization was conducted in Human Whole-Genome 60-mer oligo-chips (G4112F, Agilent Technologies) at 60℃ for 17 h at 10 rpm. After hybridization, the chips were washed with Agilent Gene Expression Wash Buffer according to manufacturer's instructions. Original signals were obtained Agilent scanner and Feature Extraction software. The standardization of original signals was carried out with RMA standardized method and standardized signal values were used for screening of differentially expressed genes.
The spots in the microarray were randomly selected and their expressions were confirmed by RT-PCR. Among genes with differential expressions, 3 genes were randomly selected and these genes and house keeping gene (GAPDH) were subjected to RT-PCR. The relative expressions were expressed as the expressions of target genes normalized by that of GAPDH (2 -△△ Ct ). Melting curve and 2 -△△ Ct method were used to compare the difference in the expressions between control group and PE group. Results from RT-PCR were consistent with microarray analysis.
Gene Ontology organizes gene function into hierarchical categories based on biological process, molecular function and cellular component. Fisher's exact test was applied for over representation of selected genes in GO biological categories. In order to assess the significance of a particular category by random chance, false discovery rate (FDR) was estimated for all of categories. After 5,000 re-samplings, FDR was defined as FDR=1-Nk/T, where Nk refers to the subtracted number which was from Fisher's test in random samples. We specified the threshold of significant GO as p-value<0.05, FDR<0.05 and enrichment parameters. Enrichment represents the degree of gene expression significance. The equation of enrichment is as following: Re=(nf/n)/(Nf/N) (14) , where nf is the number of significant genes within the particular category, n is the total number of genes within the same category, Nf is the number of significant genes in the entire microarray, N is the number of all genes tested.
Agilent Feature extraction software was used to collect original data from microarray followed by analysis with robust multichip average (RMA). Gene intensity data between PE group and control group were compared with t test after calibration with a stochastic variance model. Differentially expressed genes were identified from whole genomes. Independent-Samples T Test was used to compare mRNA levels in samples from PE patients and controls. Statistical tests were performed using SPSS 17.0, and p values <0.05 were considered significant. Before t test, test for equality of variances was performed, if variances were not equal, t test result would be corrected.
Sample collection: the fasting venous blood (2 ml) was collected in the morning and added to the ET tube. Flow cytometry was performed to detect the differentiation antigens on immune cells with BECKMANCOULTER EPICS XL-II flow cytometer. In 56 patients, the CD3 + , CD4 + , CD8 + , and CD19 were detected, and the NK cell marker CD16 + +56 + was detected in 50 patients.
The CRP level in VTE patients was detected by scintillation turbidimetry.
GO analysis targets the compromised immune functions of T cells and the decreased expression of immune receptor complex in PE patients.
Among 12 genes related to the activation and chemotaxis of neutrophils, the mRNA expressions of 9 genes were significantly up-regulated in PE group (Figure 1 ).
In the PE patients, the mRNA expression of CD14, a mononuclear cell surface antigen, was markedly up-regulated and that of CD74, a macrophage activating factor, was also significantly up-regulated. In addition, the mRNA expressions related to Fc fragment of surface receptors (FCGR2A, FCGR2B, FCGR2C, ITGAL and SCARB1) were largely increased significantly ( Figure 2 ). 
The mRNA expressions of C1 and C3 remain unchanged. In PE group, the mRNA expressions of C4b, C5, C5b as well as their receptors and complement integrins were markedly up-regulated but those of membrane attack complex component C6, C7, and C9 were significantly down-regulated when compared with the control group ( Figure 3 ).
In the PE group, the mRNA expressions of IFN regulatory factors, TNF and IL-10 were markedly up-regulated. IL-2 and IL-23A mRNA expressions were significantly down-regulated when compared with control group (Figure 4 ).
In the PE group, the expression of killer lectin-like receptor (KLR) was markedly down-regulated when compared with control group, and the NCR1 mRNA expression was also markedly down-regulated ( Figure 5 ).
Only CD86 mRNA expression was significantly up-regulated in PE group ( Figure 6 ).
In the PE group, the mRNA expressions of T cell mediated cellular immunity, protein kinases related to transmembrane signal transduction (protein tyro-sine kinase-ZAP70), T cell surface antigens (especially CD3), membrane surface immune receptor complex and T cell granzymes were markedly down-regulated when compared with the control group (Figure 7) . 
A total of 6 parameters(CD3 + , CD4 + , CD8 + , CD4 + /CD8 + , CD19 and CD16 + +56 + ) were measured in the 56 PE patients, and 53 had abnormalities in one or more parameters: 27 had abnormal CD3 + expression (decreased in 25 and increased in 2) ( Figure 8A ); 18 had aberrant CD4 + expression (decreased in 10 and increased in 8) ( Figure 8B) ; 26 had abnormal CD8 + expression(decreased in 25 and increased in 1) ( Figure  8C ); 30 had aberrant CD4 + /CD8 + ratio (decreased in 7 and increased in 23) ( Figure 8D ); 23 had abnormal NK CD16 + +56 + expression in 50 patients (decreased in 14 and increased in 9) ( Figure 8E ) and 17 had aberrant CD19 expression in 56 patients (decreased in 15 and increased in 2)( Figure 8F ); The CD8 + expression was decreased in 25 out of 56 patients(44.6%), the CD8 + and CD16 + +56 + expressions were decreased in 34 out of 56 patients(60.7%), and the CD8 + , CD16 + +56 + and CD19 expressions were decreased in 39 out of 56 patients(69.8%)( Figure 8G ). 
In 56 VTE patients, 44 patients received CRP determination. The CRP level in 35 out of 44 patients (79.5%) was higher than normal range (Figure 9 ).
The Go analysis of the genomic study targeted the decreased immune function of T cells and immune receptor complex in PE patients, suggesting the occurrence of PE is closely related to the immune dysfunction. Statistical analysis revealed the mRNA expressions of genes associated with innate immunity and cytokines were markedly up-regulated and those related to the cellular immunity of T cells and NK cells significantly down-regulated. In addition, cytological experiment indicated 6 parameters related to immune function were abnormal in 53 of 56 VTE patients. The expressions of CD3 + and CD8 + were markedly reduced and the CD4 + /CD8 + ratio significantly increased. The number of CD16 + +56 + and CD19 cells was reduced. The results from cytological examination and genome analysis were consistent.
Among the 56 patients with VTE, 25 (44.6%) had decreased CD3 + T cells, 25 had reduced CD8 + T cells and 23 (41%) had increased CD4 + /CD8 + ratio. These findings indicated the ability of T cells to recognize antigen and transmit activation signals was significantly compromised, and the capability of T cells to kill the pathogen infected cells decreased. The compromised T cell immune function is often identified in patients with malignancy, use of immunosuppressant, viral infection or malnutrition (15, 16) . In the present study, none of patients had malignancies or were treated with immunosuppressants. Therefore, the pathogenesis of VTE might be closely related to viral infection or malnutrition. In our report, the syn-thesis and release of virus-like micro-organisms were noted in the lymphocytes under an electron microscope in a young pulmonary hypertension patient with increased D-Dimer (10) .
Among 56 patients with VTE, 14 had decrease of CD16 + +56 + NK cells, which suggests the ability of NK cells to kill intracellular pathogens including virus is impaired. CD19 is only expressed on the B lymphocytes of normal hemopoietic system and the follicular dendritic cells (FDC) of germinal center. The expression of CD19 is detectable back to progenitor B cells and present during the maturation of B lymphocytes. Once the B cells differentiate into plasma cells, the CD19 expression is absent. CD19 involves in the flux of Ca 2+ in the B lymphocytes, and can regulate the activation and proliferation of B cells (17) . Among 56 VTE patients, 15 had decreased CD19 expression, which suggests the activity and proliferation of B cells are compromised.
In 53 of 56 VTE patients, the expressions of CD3 + , CD8 + , CD16 + +56 + and CD19 were separately or combinedly down-regulated or the CD4 + /CD8 + ratio was abnormal. These results imply the occurrence of VTE is closely related to the immune function. In the present study, CD16 + +56 + T cells and/or CD8 + T cells were decreased in 34 out of 56 VTE patients (60.7%). Down-regulation of CD16 + +56 + , CD8 + and/or CD19 was found in 39 out of 56 patients (69.64%). These findings indicate the symptomatic VTE is associated with the decrease of innate immunity and adaptive immunity in more than 2/3 of patients. Moreover, 53 VTE patients (94.6%) had one or more immune dysfunctions.
Among 56 VTE patients, only 3 had normal immune function. Two patients were a 31-year-old male acute PE patient and 62-year-old female acute PE patient who did not receive genetic testing. The remaining one patient was a 66-year-old female patient who was diagnosed as CTEPH and underwent splenectomy 20 years ago. For patients with abnormal immune function on admission, it was difficult to confirm when the immune function became abnormal. However, we found that more than 10 patients with down-regulation of CD8 + and CD16 + +56 + developed symptomatic VTE sequentially.
Among 56 VTE patients, more than 50% of patients had symptoms of respiratory infection or a history of respiratory infection recently. Among 56 VTE patients, CRP was measured in 44 patients and 35 (79.5%) had increase of CRP, which implies inflammation is related to the occurrence of VTE. In response to the invasion of foreign pathogens to human body, instantaneous innate immune response occurs within 0-4 hours after infection, and early innate immune response occurs 4-96 hours after infection (18, 19) . DVT and PE often occurred after 2-10 days postoperatively, which coincided with the infection process of innate and adaptive immune function (20) .
During the 3-year follow up period, 21 VTE patients (40%; including those died) were lost to follow up. Among patients receiving follow up, all were treated with warfarin for anti-coagulation. In addition, immunological examination and detection of D-Dimer were also carried at designed time points. Our results showed about 30% of VTE patients receiving follow up did not have increase of D-Dimer level any more at 0.1~1 year after warfarin discontinuation when the immunological examination and detection of D-D dimer showed normal.
Of 56 patients with symptomatic VTE, the relationship between VTE and immune dysfunction was found in 53 (94.6%). Nevertheless, patients with immune dysfunction do not develop symptomatic VTE in a short time. The compromised or disorganized immune function may be the internal cause of susceptibility to acquired VTE, and infection acts as a triggering factor of acquired VTE. When the pathogens invade the subjects with immune dysfunction, the pathogens can not be completely removed by the immune system. Thus, patients with compromised or disorganized immune function are susceptible to acquired VTE. Cedar Virus: A Novel Henipavirus Isolated from Australian Bats The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV. Henipaviruses were first discovered in the 1990s following investigation of serious disease outbreaks in horses, pigs and humans in Australia and Malaysia [1, 2] and comprise the only known Biosafety Level 4 (BSL4) agents in the family Paramyxoviridae [3] . Depending upon the geographic locations of outbreaks, and the virus and animal species involved, case mortality is between 40% to 100% in both humans and animals [4, 5] , making them one of the most deadly group of viruses known to infect humans. The genus Henipavirus in the subfamily Paramyxovirinae currently contains two members, Hendra virus (HeV) and Nipah virus (NiV) [6] . Fruit bats in the genus Pteropus, commonly known as flying foxes, have been identified as the main natural reservoir of both viruses although serological evidence suggests that henipaviruses also circulate in non-pteropid bats [7, 8, 9, 10] .
The discovery of henipaviruses had a significant impact on our understanding of genetic diversity, virus evolution and host range of paramyxoviruses. Paramyxoviruses, such as measles virus and canine distemper virus, were traditionally considered to have a narrow host range and to be genetically stable with a close to uniform genome size shared by all members of Paramyxovirinae [3] .
Henipaviruses shifted this paradigm on both counts having a much wider host range and a significantly larger genome [6] . Identification of bats as the natural reservoir of henipaviruses also played an important role in significantly increasing international scientific attention on bats as an important reservoir of zoonotic viruses, including Ebola, Marburg, SARS and Melaka viruses [11, 12, 13, 14] .
Since the discovery of the first henipavirus in 1994, much progress has been made in henipavirus research, from identification of functional cellular receptors to the development of novel diagnostics, vaccine and therapeutics [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25] . By contrast, there is little understanding of the pathogenesis of these highly lethal viruses. This is due in part to the requirement of a high security BSL4 facility for any live infection studies and in part to the limited range of research tools and reagents for the current small animal models. Research into the mechanisms of henipavirus pathogenesis is also hampered by the lack of related, but non-pathogenic or less pathogenic viruses, thus preventing targeted comparative pathogenetic studies.
Early serological investigations in Australia and more recent studies in other regions (e.g., China) indicated the presence of cross-reactive, but not cross-neutralizing, antibodies to henipaviruses in bats of different species [8] . These findings were further supported by the detection of henipavirus-like genomic sequences in African bats [26] . Discovery and isolation of these related viruses will be highly important to our further understanding of henipavirus evolution, mechanism of cross-species transmission, and pathogenesis in different animal species.
Here we report the isolation and characterization of a new bat henipavirus which, based on preliminary infection studies, is nonpathogenic in two of the small animal infection models currently used in henipavirus research. We believe that this new virus will provide a powerful tool to facilitate our future study into different aspects of henipaviruses, especially in the less advanced area of pathogenesis.
As part of our on-going field studies on HeV genetic diversity and infection dynamics in the Queensland flying fox populations, urine samples were collected on a regular basis for PCR and virus isolation. Since the establishment of the Pteropus alecto primary cell lines in our group [27] , we have intensified our effort to isolate live virus from these urine samples by routinely inoculating separate primary cell lines derived from kidney, spleen, brain, and placenta, as well as Vero cells. Syncytial CPE was observed in kidney cell (PaKi) monolayers 5 days post inoculation (dpi) with two different urine samples (Fig. S1 ) collected in September 2009 from a flying fox colony in Cedar Grove, South East Queensland (see Fig. S2 for map location). No CPE was observed in any of the four other cell lines. Supernatant harvested 6 dpi was used to inoculate fresh PaKi cell monolayers. After two passages in PaKi cells, the virus was able to infect and cause CPE in Vero cells. However, the CPE morphology of CedPV infection in Vero cells was different from that of HeV infection. Further analysis using HeV-specific PCR primers indicated that the new bat virus was not an isolate of HeV.
Considering the formation of syncytial CPE by this new virus and the previous success in isolating paramyxoviruses from bat urine [28, 29, 30] , paramyxovirus family-specific and genus-specific primers were used to determine whether this new virus was a member of the family Paramyxoviridae. Positive PCR fragments of the expected sizes were obtained from the Paramyxovirinae and Respirovirus/Morbillivirus/Henipavirus primer sets developed by Tong et al [31] . Sequencing of the PCR products indicated that it was a new paramyxovirus most closely related to HeV and NiV. Based on these preliminary data, the virus was named Cedar virus (CedPV) after the location of the bat colony sampled.
Full length genome sequence was determined by a combination of three different approaches, random deep sequencing using 454 technology, sequencing of PCR products obtained using degenerate primers designed based on known henipaviruses, and RACE to determine the precise genome terminal sequences. As shown in Fig. 1 , the genome of CedPV is 18,162 nt in length most similar to that of HeV in the family. The full genome sequence has been deposited to GenBank (Accession No. JQ001776). The genome size is a multiple of 6, hence abiding by the Rule-of-Six observed for all known members of the subfamily Paramyxovirinae [3] . It has a 3-nt intergenic sequence of CTT absolutely conserved at all seven positions and highly conserved gene start and stop signals similar to those present in HeV and NiV (Fig. S3) . Also similar to the HeV genome is the presence of relatively large non-coding regions in the CedPV genome ( Fig. 1 and Table 1 ). The overall proteincoding capacity of the CedPV genome is 87.41% which is significantly lower than the average of 92.00% for other family members but higher than HeV at 82.12%. As the genome size of CedPV and HeV is very similar, the increased coding capacity of CedPV is attributed to an increase in protein sizes for five of the six major proteins, with the L protein being 257-aa larger (Table 1) . At 2,501 aa, the CedPV L protein is the largest, not only in the family Paramyxoviridae but also for all known viruses in the order Mononegavirale.
Phylogenetic analysis based on the full length genome sequence and the deduced amino acid sequences of each structural protein confirmed the initial observation that CedPV is most closely related to henipaviruses in the family. A phylogenetic tree based on the deduced sequences of the nucleocapsid protein (N) is presented in Fig. 2 . Phylogenetic tree based on whole genome sequences gave similar results (Fig. S4 ). CedPV is more closely related to HeV and NiV than henipavirus-like sequences detected in African bats [26, 32] as shown in a phylogenetic tree based on the only sequences available of a 550-nt L gene fragment (Fig. S5) .
A phosphoprotein (P) gene lacking RNA editing and coding capacity for the V protein First discovered for the parainfluenza virus 5 (PIV5, previously known as simian virus 5), almost all members of Paramyxovirinae have a P gene which produces multiple proteins through an RNA editing mechanism by addition of non-templated G residues leading to production of N-terminal co-linear proteins from different reading frames downstream from the editing site [3, 33] . These multiple gene products are known to play a key role in antagonizing the innate response of susceptible hosts [3] . A search of CedPV for open reading frames (ORF) in the P gene revealed a 737-aa P protein and a 177-aa C protein, but failed to find the highly conserved, cysteine-rich V ORF present in most other paramyxoviruses. The RNA editing site with the sequence of AAAAGGG, which is absolutely conserved in all known HeV and NiV isolates discovered to date, is also missing from the CedPV P gene sequence. To further verify that there are no multiple mRNAs produced from the CedPV P gene, direct sequencing of P gene transcripts was conducted from CedPV-infected Vero cells using multiple sets of primers generating overlapping fragments covering the entire coding region of the P gene. Each produced
Hendra and Nipah viruses are 2 highly pathogenic paramyxoviruses that have emerged from bats within the last two decades. Both are capable of causing fatal disease in both humans and many mammal species. Serological and molecular evidence for henipa-like viruses have been reported from numerous locations including Asia and Africa, however, until now no successful isolation of these viruses have been reported. This paper reports the isolation of a novel paramyxovirus, named Cedar virus, from fruit bats in Australia. Full genome sequencing of this virus suggests a close relationship with the henipaviruses. Antibodies to Cedar virus were shown to cross react with, but not cross neutralize Hendra or Nipah virus. Despite this close relationship, when Cedar virus was tested in experimental challenge models in ferrets and guinea pigs, we identified virus replication and generation of neutralizing antibodies, but no clinical disease was observed. As such, this virus provides a useful reference for future reverse genetics experiments to determine the molecular basis of the pathogenicity of the henipaviruses. uniform trace files indicating a lack of RNA editing activities, which is very different from the mixed peaks generated by HeV and NiV immediately after the editing site (Fig. S7 ). To our knowledge, CedPV is the first member of Paramyxovirinae that lacks both RNA editing and any V-related coding sequence in its P gene. Further investigation is required to exclude the possibility that the P-gene editing in CedPV is cell-or tissue-specific and not present or present at an extremely low level in the current viruscell system.
The striking similarity in genome size and organization and the presence of highly conserved protein domains among the N, M and L proteins between CedPV and henipaviruses prompted us to investigate the antigenic relatedness of these viruses. Staining of CedPV-infected Vero cells using rabbit anti-henipavirus antibodies indicated the presence of cross-reactivity. This cross-reactivity was further confirmed in reverse by staining of HeV-infected Vero cells using a rabbit serum raised against a recombinant CedPV N protein (Fig. 3) . However, analysis by virus neutralization test using either polyclonal or monoclonal antibodies found that henipavirus-neutralizing antibodies were unable to neutralize CedPV. Similarly, CedPV-neutralizing antibodies obtained in our infection studies (see below) also failed to neutralize either HeV or NiV. It can therefore be concluded that CedPV and henipaviruses share cross-reactive antigenic regions, but not crossneutralizing epitopes.
To further investigate the relationship between CedPV and recognized henipaviruses, we investigated the use of the henipavirus receptors, the ephrin-B2 and -B3 host cell proteins, as potential receptors for CedPV infection. Our previous studies have demonstrated that the ephrin-B2 and -B3 expression negative HeLa-USU cell line could support henipavirus infection and formation of syncytial CPE only when either the ephrin-B2 or -B3 gene was transiently expressed in the cells [22, 34] . For CedPV, similar observations were made with respect to the ephrin-B2 receptor. As shown in Fig. 4 , CedPV failed to infect HeLa-USU, but was able to infect and cause syncytial CPE when the human ephrin-B2 gene was expressed. In contrast, when ephrin-B3 molecule was introduced, there was no evidence of infection.
Ferrets, guinea pigs, and mice exhibit differing responses to the previously described henipaviruses HeV and NiV, with ferrets and guinea pigs, but not mice developing severe disease characterized by systemic vasculitis [20, 35, 36, 37, 38] . In contrast, ferrets and guinea pigs exposed to CedPV by, respectively, oronasal and intraperitoneal routes remained clinically well although neutralizing antibody was detected in serum between 10 to 21 days pi ( Table 2) . Balb-C mice exposed to CedPV by the oronasal route remained clinically well and did not develop neutralizing antibody in serum by day 21 pi. In ferrets electively euthanized at earlier time-points, there was reactive hyperplasia of tonsillar lymphoid tissue, retropharyngeal and bronchial lymph nodes, accompanied by edema and erythrophagocytosis. CedPV antigen was detected in bronchial lymph node of one animal euthanized on day 6 pi, consistent with viral replication in that tissue; cross-reactive immunostaining against anti-NiV N protein antibodies was also noted (Fig. 5) . No other significant histological lesions were identified. Viral RNA was detected in selected lymphoid tissues of 3 (of 4) ferrets sampled day 6 to 8 pi, including pharynx, spleen, and retropharyngeal and bronchial lymph nodes, as well as the submandibular lymph node of the ferret euthanized on day 20 pi. This pattern of lymphoid involvement suggests that there may be transient replication in the upper and lower respiratory tracts although CedPV genome was not recovered from nasal washes, oral swabs, pharynx or lung tissue of affected animals. Virus isolation was unsuccessful for all PCR positive tissues.
As a first step towards the understanding of the pathogenicity difference between CedPV and HeV, we examined the IFN responses in human HeLa cells upon virus infection. As shown in Fig. 6 , while the induction of IFN-a was similar in cells infected with HeV or CedPV, there was a significant difference of IFN-b production upon infection by HeV or CedPV, with CedPVinfected cell producing a much higher level of IFN-b.
To investigate the CedPV exposure status of pteropid bats in Queensland and potential co-infection (either concurrent or consecutive) of CedPV with HeV, we tested 100 flying fox sera collected previously for other studies for antibody against the two viruses. Due to the cross-reactivity observed above, virus neutralization tests were conducted to obtain more accurate infection data for each virus. Overall, 23% of the sera were CedPV-positive and 37% HeV-positive (Table S1 ). Co-infection was reflected in 8% of the sera tested.
The emergence of bat-borne zoonotic viruses (including HeV, NiV, Ebola, Marburg, and SARS) has had a significant impact on public health and the global economy during the past few decades. With the rapidly expanding knowledge of virus diversity in bat populations around the world, it is predicted that more bat-borne zoonotic viruses are likely to emerge in the future. The discovery of a novel ebolavirus-like filovirus in Spanish microbats demonstrates that the potential for such spill over events is not limited to Africa or Asia [39] . It is therefore important to enhance our preparedness to counter future outbreaks by conducting active pre-emergence research into surveillance, triggers for cross-species transmission, and the science of identification of potential pathogens.
Henipaviruses represent one of the most important bat-borne pathogens to be discovered in recent history. Although CedPV displays some differences from existing members of the genus Henipavirus, we propose that CedPV be classified as a new henipavirus based on the following shared features with known henipaviruses: 1) it is antigenically related to current henipaviruses; 2) its genome size and organization is almost identical to those of HeV and NiV; 3) it has a similar prevalence in flying foxes; and 4) it uses ephrin-B2 as the cell entry receptor.
The lack of cross-neutralization between CedPV and HeV or NiV was not unexpected from the comparative sequence analysis of all the deduced proteins, especially the G protein (see Table 1 ). It is clear that the genetic relatedness of CedPV with HeV or NiV is much lower than between HeV and NiV. However, the percentage sequence identities of the major viral proteins between CedPV and HeV/NiV are on average at least 10% higher than that between HeV/NiV and any other known paramyxoviruses. Also, there was no antigenic cross-reactivity observed between CedPV and representative viruses of the other paramyxovirus genera in the subfamily Paramyxovirinae (Fig. S6) .
Like other paramyxoviruses, the P gene of henipaviruses produces multiple proteins which play a key role in viral evasion of host innate immune responses [4, 40, 41] . One of these is the Cys-rich V protein: all members of the subfamily Paramyxovirinae produce the V protein with the exception of the human parainfluenza virus 1 (hPIV1). Although a putative RNA editing sequence (AAGAGGG) is present at the expected editing site of the P gene, the hPIV1 RNA polymerase does not produce an edited mRNA of the P gene [42] . There are remnants of the V ORF easily detectable in the hPIV1 P gene although the predicted 68-aa ORF region is interrupted by multiple in-frame stop codons. Of the 7 Cys residues conserved between bovine parainfluenza virus 3 and Sendai virus, four are still present in the non-functional V ORF of hPIV1 [42] . In contrast, an extensive ORF and sequence homology search of the CedPV P gene only identified one aa coding region with minimal sequence identity to the V ORFs of HeV and NiV (see Fig. S8 ). In this region, out of the 9 Cys residues conserved between HeV and NiV V proteins, only 2 are present in the CedPV P gene. Furthermore, the sequence (AGATGAG) upstream from this putative ORF V coding region does not match the consensus RNA editing site. It can therefore be concluded that CedPV is the only member of Paramyxovirinae which lacks both the functional V mRNA/protein and the coding capacity for the RNA editing site and ORF V. The evolutionary significance of this finding needs further investigation.
Our in vitro study indicated that ephrin B2, but not ephrin B3, was able to restore CedPV infection in the ephrin B2-deficient HeLa cells. While this is highly suggestive that ephrin B2 is the functional entry receptor for CedPV, it should be emphasized that this was not a direct proof that ephrin B2 is the receptor. Further investigation is required to confirm this.
In our preliminary studies, it was shown that CedPV was able to replicate in guinea pigs and ferrets, but failed to cause significant clinical diseases, unlike that of the closely related HeV and NiV. These first infection experiments were conducted with a high dose if virus to establish whether the CedPV could replicate in these animals and determine the degree of any clinical disease. A second experiment was then carried out in ferrets to determine the site of replication and tissue tropism in sequentially sacrificed animals. A lower dose was used to gain better comparison with similar infection experiments using HeV and NiV [18, 35] . Although these initial experimental infection studies indicate that CedPV is less or non-pathogenic in these species, it is possible that CedPV may be pathogenic in other hosts, such as horses. We hypothesize that the lack of a V protein may impact on the pathogenicity. In this regard, it was encouraging to observe that infection of human cells by CedPV induced a much more robust IFN-b response than HeV. Further study is required to dissect the exact molecular mechanism of this observed difference. Due to the close relationship between CedPV and HeV, it was important to investigate the possibility of co-infection by these two viruses in the Australian bat population. Based on the detection of neutralizing antibodies at 23% for CedPV, 37% for HeV and 8% for both, it can be concluded that the co-infection rate is very close to the theoretical rate of 8.5% (the product of the two independent infection rates). Based on this limited preliminary analysis, it appears that infection of bats by one henipavirus neither prevents nor enhances the likelihood of infection by the other.
In summary, the discovery of another henipavirus in Australian flying foxes highlights the importance of bats as a significant reservoir of potential zoonotic agents and the need to expand our understanding of virus-bat relationships in general. Our future research will be directed at determining whether spill-over of CedPV into other hosts has occurred in the past in Australia, whether CedPV is pathogenic in certain mammalian hosts, and whether CedPV exists in bat populations in geographically diverse regions.
All animal studies were approved by the CSIRO Australian Animal Health Laboratory's Animal Ethics Committee and conducted following the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes guidelines for housing and care of laboratory animals.
Cell lines used this study were Vero (ATCC), HeLa-USU [22] , and the P. alecto primary cell lines derived from kidney (PaKi), brain (PaBr), (spleen) PaSp and placenta (PaPl) recently established in our group [27] . Cells were grown in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 Ham supplemented with double strength antibiotic-antimycotic (Invitrogen), 10 mg/ml ciprofloxacin (MP Biomedicals) and 10% fetal calf serum at 37uC in the presence of 5% CO 2 .
Urine (approximately 0.5-1 ml) was collected off plastic sheets placed underneath a colony of flying foxes (predominantly Pteropus alecto with some P. Poliocephalus in the mixed population) in Cedar Grove, South East Queensland, Australia and pooled into 2-ml tubes containing 0.5 ml of viral transport medium (SPGA: a mix of sucrose, phosphate, glutamate and albumin plus penicillin, streptomycin and fungizone). The tubes were temporarily stored on ice after collection and transported to a laboratory in Queensland, frozen at 280uC, and then shipped on dry ice to the CSIRO Australian Animal Health Laboratory (AAHL) in Geelong, Victoria for virus isolation. The samples were thawed at 4uC and centrifuged at 16,0006g for 1 min to pellet debris. Urine in the supernatant (approximately 0.5-1 ml) was diluted 1:10 in cell culture media. The diluted urine was then centrifuged at 1,2006g for 5 min and split evenly over Vero, PaKi, PaBr, PaSp and PaPl cell monolayers in 75-cm 2 tissue culture flasks. The flasks were rocked for 2 h at 37uC, 14 ml of fresh cell culture media was added and then incubated for 7 d at 37uC. The flasks were observed daily for toxicity, contamination, or viral cytopathic effect (CPE).
Cells showing syncytial CPE were screened using published broadly reactive primers [31] for all known paramyxoviruses and a subset of paramyxoviruses. PCR products were gel extracted and cloned into pGEM T-Easy (Promega) to facilitate sequencing using M13 primers. Sequences were obtained and aligned with known paramyxovirus sequences allowing for initial classification.
Whole genome sequence was determined using a combination of 454 sequencing [43] and conventional Sanger sequencing. Virions from tissue culture supernatant were collected by centrifugation at 30,0006g for 60 min and resuspended in 140 ml of PBS and mixed with 560 ml of freshly made AVL for RNA extraction using QIAamp Viral RNA mini kit (Qiagen). Synthesis of cDNA and random amplification was conducted using a modification of a published procedure [44] . Briefly, cDNA synthesis was performed using a random octomer-linked to a 17-mer defined primer sequence: (59-GTTTCCCAGTAGGTCTCNNN NNNNN-39) and SuperScript III Reverse Transcriptase (Invitrogen). 8 ml of ds-cDNA was amplified in 200 ml PCR reactions with hot-start Taq polymerase enzyme (Promega) and 59-A*G*C*A*C TGTAGGTTTCCCAG-TAGGTCTC-39 (where * denotes thiol modifications) as amplification primers for 40 cycles of 95uC/1 min, 48uC/1 min, 72uC/ 1 min after an initial denaturation step of 5 min at 95uC and followed by purification with the QIAquick PCR purification kit (Qiagen). Sample preparation for Roche 454 sequencing (454 Life Sciences Branford, CT, USA) was according to their Titanium series manuals, Rapid Library Preparation and emPCR Lib-L SV.
To obtain an accurate CedPV genome sequence, 454 generated data (after removing low quality, ambiguous and adapter sequences) was analysed by both de novo assembly and read mapping of raw reads onto the CedPV draft genome sequence derived from Sanger sequencing. For 454 read mapping, SNPs and DIPs generated with the CLC software were manually assessed for accuracy by visualising the mapped raw reads (random PCR errors are obvious compared to real SNPs and DIPs especially when read coverage is deep). Consensus sequences for both 454 de novo and read mapping assembly methods were then compared to the Sanger sequence with the latter used to resolve conflicts within the low coverage regions as well as to resolve 454 homopolymer errors.
Sequences of genome termini were determined by 39-and 59-RACE using a protocol previously published by our group [45] . Briefly, approximately 100 ng of RNA was ligated with adaptor DT88 (see reference for sequence information) using T4 RNA ligase (Promega) followed by cDNA synthesis using the Super-Script III RT kit (Invitrogen) and an adaptor-specific primer, DT89. PCR amplification was then carried out using DT89 and one or more genome-specific primers. PCR products were sequenced directly using either DT89 or genome specific primers by an in-house service group on the ABI Sequencer 3100.
The CLC Genomics Workbench v4.5.1 (CLC Inc, Aarhus, Denmark) was used to trim 454 adapter and cDNA/PCR primer sequences, to remove low quality, ambiguous and small reads ,15 bp and to perform de novo and read mapping assemblies all with default parameters. Clone Manager Professional ver 9.11 (Scientific and Educational Software, Cary, NC, USA) was used to join overlapping contigs generated by de novo assembly. Phylogenetic trees were constructed by using the neighbor-joining algorithm with bootstrap values determined by 1,000 replicates in the MEGA4 software package [46] .
Quantitative PCR assays (qPCR) were established based on CedPV-specific sequences obtained from the high throughput sequencing. A TaqMan assay on the P gene was developed and used for all subsequent studies. The sequences of the primer/probe are as follows: forward primer, 59-TGCAT TGAGC GAACC CATAT AC; reverse primer, 59-GCACG CTTCT TGACA GAGTT GT; probe, 59-TCCCG AGAAA CCCTC TGTGT TTGA-MGB.
The coding region for the CedPV N protein was amplified by PCR with a pair of primers flanked by AscI (59 end) and NotI (39 end) sites for cloning into our previously described GST-fusion expression vector [47] . The expression and purification by gel elution was conducted as previously described [48] . For antibody production, purified protein was injected subcutaneously into 4 different sites of 2 adult (at a dose of 100 mg per animal) New Zealand white female rabbits at days 0 and 27. The CSIRO's triple adjuvant [49] was used for the immunization. Animals were checked for specific antibodies after days 5 and 42 and euthanized at day 69 for the final blood collection.
For immunofluorescence antibody test, Vero cell monolayers were prepared in 8-well chamber slides by seeding at a concentration of 30,000 cells/well in 300 ml of cell media and incubating over night at 37uC. The cell monolayers were infected with an MOI of 0.01 of CedPV, HeV or NiV and fixed with 100% ice-cold methanol at 24 h post-infection. The chamber slides were blocked with 100 ml/well of 1%BSA in PBS for 30 min at 37uC before adding 50 ml/well of rabbit sera against CedPV N or NiV N diluted 1:1000. After incubation at 37uC for 30 min, the slides were washed three times in PBS-T and incubated with 50 ml/well of anti-rabbit 488 Alexafluore conjugate (Invitrogen) diluted 1:1000 at 37uC for 30 min. The slides were then washed three times in PBS-T and mounted in 50% glycerol/PBS for observation under a fluorescence microscope.
For virus neutralization test, serial two-fold dilutions of sera were prepared in duplicate in a 96-well tissue culture plate in 50 ml cell media (Minimal Essential Medium containing Earle's salts and supplemented with 2 mM glutamine, antibiotic-antimycotic and 10% fetal calf serum). An equal volume containing 200 TCID 50 of target virus was added and the virus-sera mix incubated for 30 min at 37uC in a humidified 5% CO 2 incubator. 100 ml of Vero cell suspension containing 2610 5 cells/ml was added and the plate incubated at 37uC in a humidified 5% CO 2 incubator. After 4 days, the plate was examined for viral CPE. The highest serum dilution generating complete inhibition of CPE is defined as the final neutralizing titer.
Human ephrin B2 and B3 genes were cloned into pQCXIH (Clontech) and the resulting plasmids packaged into retrovirus particles in the GP2-293 packaging cell line (Clontech) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G) following the manufacturer's instructions. HeLa-USU cell line [22] was infected with the VSV-G pseudotyped retrovirus particles in the presence of 1 mg/ml polybrene (Sigma). 8 h post infection, the medium was changed and the cells were allowed to recover for 24 h, allowing time for the retroviral insert to be incorporated into the cell genome and for expression of the hygromycin resistance gene. 24 h post infection, cells transformed by the retrovirus were selected for by the addition of 200 mg/ml hygromycin in the media. Stocks of cells that were resistant to hygromycin were prepared and frozen. HeLa-USU and ephrin-expressing HeLa-USU cells were seeded in 6-well tissue culture plates at a density of 250,000 cells/well overnight. The viruses (HeV and CedPV) were diluted to give an MOI of 0.01 and inoculated into the wells. The cell monolayers were examined daily for syncytial CPE.
Animal studies were carried out in the BSL4 animal facility at AAHL. Ferrets, guinea pigs and mice were used on the basis of their known and varying responses to exposure to other henipaviruses.
Firstly, 2610 6 TCID 50 /ml CedPV passaged twice in bat PaKi cells was administered to 2 male ferrets (1 ml oronasally); 4 female guinea pigs (1 ml intraperitoneally); and 5 female Balb-C mice (50 ml oronasally). Guinea pigs and mice were implanted with temperature sensing microchips (LifeChip Bio-thermo, Destron Fearing) and weighed daily. Ferret rectal temperature and weight was recorded at sampling times. Animals were observed daily for clinical signs of illness and were euthanized at 21 d postinoculation. Sera were collected on days 10, 15 and 21 to test for neutralizing antibody against CedPV.
Secondly, on the basis of asymptomatic seroconversion to CedPV noted in ferrets in the first study, 7 further female ferrets were exposed by the oronasal route to a lower dose of 3610 3 TCID 50 . Two animals were euthanized on each of days 6, 8 and 10 post-inoculation and one on day 20. Nasal washes, oral swabs, and rectal swabs were collected on days 2, 4, 6, 8 and 10 and urine was sampled on the day of euthanazia; each specimen was assessed for CedPV genome. A wide range of tissue samples were collected at post mortem examination and assessed by routine histology, immunohistochemistry (using rabbit antibodies raised against recombinant CedPV and NiV N proteins, respectively), qPCR (see above) and virus isolation using reagents and procedures previously established in our group [16] .
HeLa cells were infected with Hendra and Cedar viruses at an MOI 0.5 for 24 hours, at which time total cellular RNA was extracted and IFN-a and IFN-b mRNA levels were quantified by real-time PCR using Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems). Primers were as previously described [50] .
Sera from 100 flying foxes collected during 2003-2005 from Queensland, Australia were screened for neutralizing antibodies to CedPV. Virus neutralization test was conducted as described above (antibody tests). All serum samples were tested at a dilution of 1:20.  Host Modulators of H1N1 Cytopathogenicity Influenza A virus infects 5–20% of the population annually, resulting in ∼35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors. The Orthomyxoviridae family member influenza A virus is the causal agent of acute respiratory tract infections suffered annually by 5-20% of the human population. There is a significant impact on morbidity, concentrated in people younger than 20 years, with economic consequences running into the billions of dollars during large epidemics [1] . In addition, viral infections are associated with development of chronic asthma and disease exacerbation in both children and adults. In particular, acute influenza infection can amplify airway inflammation in asthmatic patients and induce alterations in epithelial and stromal cell physiology contributing to allergen sensitization, exaggerated bronchoconstriction, and remodeling of airway epithelia [2] . Mortality rates associated with seasonal flu are low, but the aging population is at risk for development of severe congestive pneumonia which kills ,35,000 people each year in the U.S. [1] . Of continual concern is the threat of emergent high virulence strains such as the Spanish flu (H1N1), Asian flu (H2N2) and Hong Kong flu (H3N2) pandemics which claimed millions of lives world-wide. Current treatments are focused on vaccines and drugs that target viral proteins. However, both of these approaches have limitations as vaccines require yearly development and lag detection of new strains, while viral proteins have a stunning capacity to evolve resistance to targeted agents [3] . The genome of the influenza A virus consists of 8 negative single-strand RNA segments that encode 11 functional peptides necessary for viral replication and virulence [1] . Thus the viral-autonomous repertoire of gene products is extremely limited and influenza A replication is dependent upon hijacking host-cell biological systems to facilitate viral entry, replication, assembly, and budding. The recognition that a suit of human host proteins are required for IVA infection and replication presents additional targeting strategies that may be less prone to deflection by the highly plastic viral genome.
Here we have employed the cytopathic effects of H1N1 infection in bronchial epithelial cells as a mechanism to isolate host genes that represent intervention target opportunities by virtue of their contribution to H1N1 infection and replication, or by virtue of their contribution to viral virulence factor-dependent evasion of innate immune responses. A primary whole-genome arrayed siRNA screen identified gene depletions that either deflected or promoted bronchial epithelial cell death upon exposure to the H1N1 A/WSN/33 influenza virus and were not cytotoxic to mock infected cells. Integration with orthogonal data sets, describing host gene function [4] [5] [6] [7] [8] , parsed collective 'targets' into four functional classes. 1) Targets that, when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This class presumably represents host factors that facilitate viral infection and/or are required to support viral replication. 2) Targets that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This important and initially unanticipated class, likely represents proviral host factors that deflect cell death checkpoint responses that would otherwise engage upon detection of viral infection. 3) Targets that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication relative to controls. Recently discovered innate immune pathway components, such as IFITM3 that are responsive to H1N1 infection, are members of this class, which presumably represent antiviral restriction factors that normally oppose infection. 4) Targets, that when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication as compared to controls. These host factors are likely responsible for influenza virus-mediated cytopathic effects. Chemical inhibition of gene products from two classes, RABGGTASE and CHEK1, indicated these targets might be pharmacologically addressable for H1N1 intervention in an epithelial cell autonomous context.
Influenza A infection is associated with pathological changes throughout the respiratory tract, however the major site of impact appears to be the respiratory epithelia. Bronchoscopy of patients with uncomplicated influenza infections reveals alterations in the ciliated epithelia of the larynx, trachea, and bronchi that includes vacuolization, loss of cilia, and desquamation of columnar epithelial cells and goblet cells down to the basal cell layer. Importantly, viral antigen is found predominantly in the epithelial cells and mononuclear cells [1] . Therefore, for the studies described here, we employed telomerase-immortalized human bronchial epithelial cells (HBEC30) that retain the capacity to differentiate into a polarized ciliated epithelial sheet [9] . In undifferentiated cell culture, we found that 100% of HBEC30 in culture display viral protein production after a 24-hour exposure to mouse-adapted virus at an MOI of 5 ( Figure 1A , B). This leads to an approximately 50% decrease in cell viability 48-hours post infection ( Figure 1C ). Given these observations, we adopted a whole genome siRNA screening strategy that involved a 48-hour incubation post siRNA transfection, followed by a 48 hour exposure to influenza A/WSN/1933 or carrier, with cell viability as the endpoint assay. Raw viability values were converted to viability Z-scores with a metric that normalized for both position and batch effects ( Figure 1D , see methods). The dynamic range of viability scores observed under screening conditions potentially affords the opportunity to identify both enhancers and resistors of viral pathogenicity ( Table 1 in Supporting Information S1). Considering siRNA pools associated with Z-scores that were equal to or greater than 3 standard deviations above (resistors) or below (sensitizers) the mean of the population, 53 candidate resistors and 182 candidate sensitizers were identified ( Table 2 in Supporting Information S1). A representative sample of targets was further tested for consequences on viral protein accumulation and viral replication. As might be expected, the majority of the siRNA pools that deflect a viral cytopathic response resulted in reduced viral protein accumulation, as detected by quantitation of viral proteins at single cell resolution, and reduced production of infectious particles ( Figure 2A ). Among these, IVNS1ABP and the splicing factor SFPQ directly interact with the viral pathogenicity factor NS1, presumably reflecting a positive role in support of viral corruption of host machinery for viral protein production [10] . Of interest in this class is RRAGD, a small G-protein that supports the amino-acid responsiveness of mTOR as a component of the ''ragulator'' [11] . Several reports have highlighted the importance of viral induction of mTOR for viral replication, but the mechanism is not fully elaborated [6, 12] . Given the participation of endosomes as a viral entry mechanism [13] , it is tempting to speculate that RRAGD is a limiting host factor for viral corruption of mTOR regulation. Additional factors in this group are involved with the host defense response, p53-mediated cell death and vesicle maturation and trafficking. To test for false positives arising from off-target effects of siRNA treatment, we retested 88 siRNA pools as four individual oligos. Approximately 60% of siRNAs retested with two or more oligos reproducing the original phenotype ( Figure S1 ).
Among the most potent members of the sensitizer class were the previously described proviral host factor IFITM3 and its homolog IFITM1 ( Table 7 in Supporting Information S1). IFITM3 has been reported to be required for restriction of viral infection and is thought to inhibit viral entry [4, 14] . These gene products are interferon responsive, and depletion was associated with enhanced viral pathogenicity and enhanced viral protein production at limiting MOIs (1 and 0.1) as compared to controls (Figure 2A , B, C). Unexpectedly, cells depleted of IFITM3 produced fewer infection competent viral particles as determined by secondary infection of MDCK cells with cell culture supernatants (Figure 2 A, E). For these assays, HBEC30 cell cultures were infected with an MOI of 5 for 48 hours post transfection with siRNA pools. Supernatants were collected 24 hours post infection and used to infect confluent MDCK cell cultures. Notably, we observed enhanced frequency as well as enhanced amplitude of viral protein accumulation in IFITM3 depleted cells during primary infection. Reduced production of infectious particles, in the face of enhance viral protein production, may therefore be a consequence of either limiting host factors or disruption of viral protein/host factor stoichiometry required for assembly of viable viral particles. Of interest, the viral cytophathic effect was greatly enhanced upon IFITM3 depletion in the presence or absence of the virulence factor NS1, a viral protein known to block many of the innate immunity responses [15] [16] [17] [18] [19] (figure 2F, G). However, deletion of NS1 results in complete failure of infectious particle production even upon IFITM3 depletion ( Figure 2H ). These observations would place IFITM3 function early in the viral life cycle and independent of NS1 function, consistent with reports that indicate IFITM3's antiviral activity is at the level of viral entry [14] .
Depletion of the cell cycle/DNA damage checkpoint proteins CDC2 and CHEK1, like IFITM3, appeared to promote viral protein production and cytopathic response, while impairing assembly of infection-competent viral particles. A global comparison of the candidate modulators of H1N1 pathogenicity identified here with two whole-genome siRNA screens for modulators of cell cycle progression revealed a significant intersection ( Figure 3A ). However, CDC2 and CHEK1 depletion show quite distinct consequences on G1 versus G2 arrest suggesting their contribution to H1N1 infection may be independent of cell cycle control. CHEK1 has not been previously isolated in viral pathogenicity or viral replication screens, including those performed with the same siRNA library employed here ( Figure 3B , C, Tables 3 and 4 in Supporting Information S1). To investigate additional biological processes that may be associated with CHEK1 modulation of viral infection, we assembled a context-specific protein-protein interaction sub-network defined by the genomic Z-score distribution of the primary screen ( Figure S7 ). This subnetwork revealed the circadian gene Timeless, recently defined as a master regulator of the host defense response [20] , within the first-degree neighborhood of CHEK1 ( Figure 3D ). Given this association, we investigated the consequence of chemical inhibition of CHEK1 on H1N1 infection.
We employed SB218078, an investigational CHEK1 inhibitor similar to one currently in clinical trials as an anti-neoplastic agent, with an in vitro IC50 of 0.015 mM and a K i,app. of 1564 [21, 22] . Pretreatment of cultures with 1 uM or 100 nM SB218078 for 12 hours resulted in significant inhibition of viral protein accumulation together with a marked virus-specific death response by 24 hours (Figure 4A , B). While some viral infection was detected at the 100 nM dose, viral protein production was severely limited at single cell resolution ( Figure 4B , C). These observations suggest that SB218078 is releasing a cell death response to viral detection that would otherwise be suppressed during the viral replication cycle. Viral-induced cell death was also observed upon siRNA-mediated CHEK1 depletion (Figure 2A ). The seemingly contradictory increase in infection frequency upon CHEK1 depletion may therefore be an indirect consequence of infection of low density residual cell populations with hypomorphic CHEK1 activity. Remarkably, SB218078 had no consequence on H1N1 replication in A549 cells, a cancer cell line often employed to test for modulators of viral replication and host responses [5, 23, 24] ( Figure 4E , F). However, a nontransformed, telomerase-immortalized bronchial epithelial cell line, derived from a different patient, HBEC3 [25] , was identical to HBEC30 in its re- Figure 4G ). These observations indicate intervention targets may be available in non-tumorigenic cells that are uncoupled from host regulatory networks in cancer cells, and potentially explain why CHEK1 was not identified in other efforts to date that have universally relied on cancer lines as screen hosts [4] [5] [6] [7] 26] . We next queried the behavior of gene depletions identified here that modulate H1N1 cytopathic effects to those in 4 wholegenome siRNA screens which measured influenza virus replication as the end-point assay [4] [5] [6] [7] . This allowed us to parse collective 'hits' into four functional classes ( Figure 3C , Table 5 in Supporting Information S1). Class 1: genes that, when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This class presumably represents host factors that facilitate viral infection and/or are required to support viral replication. Class 2: genes that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure, and are required for viral replication. This, initially unanticipated but very intriguing class, likely represents host factors that deflect cell death checkpoint responses that would otherwise engage upon detection of viral infection. Class 3: genes that, when depleted, reduce bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication relative to controls. This class presumably represents antiviral restriction factors that normally oppose infection. Class 4: genes, that when depleted, enhance bronchial epithelial cell survival upon H1N1 exposure and enhance viral replication as compared to controls. Of note, Class 2, which may represent novel intervention target opportunities, includes TRRAP, a large multidomain protein of the phosphoinositide 3-kinase-related kinases (PIKK) family that is a component of many histone acetyltransferase (HAT) complexes. TRRAP was recently identified as a bona fide oncogene in melanoma through cancer genome resequencing efforts, however, its transforming mechanism is unknown [27] .
By nature, a challenge to siRNA-screening efforts is false negatives that derive from weak phenotypes due to suboptimal depletion of what are otherwise key factors in the biological process under investigation. One opportunity to help meet this challenge is to employ coherent behavior of gene sets to identify key biological processes supporting a phenotype rather than relying solely on an arbitrary scoring threshold for each individual gene. We employed Netwalk [28] here to facilitate identification of such gene sets based on overrepresentation of functionally coherent subnetworks within the graph (Figures S1, S2, S3, S4, S5, S6, S7, S8 and S9). One such subnetwork implicated prenylation of Rab-family GTPases in support of H1N1 replication ( Figure S4 and Figure 3D ). To test this we employed 3-IPEHPC, a specific inhibitor of the type II Geranylgeranyltransferases (IC50 of 1.27 mM and a K i of 0.211 mM for Rab1a modification [29] ). As such, 3-IPEHPC specifically inhibits modification of Rab-family proteins with a carboxy-terminal CC motif as opposed to the carboxy-terminal CAAX motif [29] . HBEC-30 cells pretreated with 3-IPEHPC for 24 hours were significantly refractory to infection by H1N1 (Figure 5A, B) . Inhibitory activity was observed at concentrations as low as 125 nM ( Figure 5C ). Unlike SB218078, A549 cells were also responsive to 3-IPEHPC ( Figure 5D, E) . While the use of a mouseadapted virus facilitates large-scale screening and allows comparisons with other published screening efforts, the extent to which results translate to seasonal or highly pathogenic strains is not established. Importantly, 3-IPEHPC was protective against infection with the avian strain H5/N1 and the recent pandemic swine flu strain H1/N1 ( Figure 5D, E) .
A stark limitation of arrayed siRNA screens is the requirement for ''single gene'' phenotypic penetrance. This can limit sensitivity of detection of relevant molecular entities due to insufficient protein depletion and/or the presence of functionally redundant gene products. As a mechanism to potentially reveal combinatorial contributions of gene function to viral replication and cytopathic effects, we repeated the original screen using a library of 426 human microRNA mimics. These reagents have the advantage of inducing multigenic perturbations, though accurate assignment of target space is a significant challenge. This effort identified a small cohort of miRNA mimics that either enhanced or deflected H1N1induced cell death ( Figure 6A , B, Table 6 in Supporting Information S1). 11 of these were further examined for consequences on H1N1 viral protein production in HBEC30 cells, which identified both sensitizers and resistors that enhanced or repressed viral replication ( Figure 6C ). Of note, a test for ''hits'' that also have activity against the recent pandemic strain Cal/04/ 09 identified two miRNA mimics that impair Cal/04/09 protein production in A549 cells (hsa-miR-495 and hsa-miR-519a, Figure 6D ). To infer biological processes that may be engaged by the miRNAs that can impair H1N1 replication, we examined the intersection of predicted miRNA targets and single-gene perturbations that behaved similarly to the subject miRNA. Candidate miRNA target genes were selected based on seed sequence presence in 39 UTRs as defined by Target Scan context scores. These predictions were intersected with siRNA data from this study and those of the 4 whole-genome siRNA screens that measured influenza virus replication [4] [5] [6] [7] . When considered as a heuristic, this analysis produced three subnetworks that may correspond to the miRNA mode of action, namely the glycosylphosphatidylinositol transamidase, viral and host protein ubiquitylation [30, 31] and alternative mRNA splicing ( Figure 6E ).
Here we have focused on isolation of H1N1 pathogenicity response modifiers in human bronchial airway epithelial cells (HBEC). This cell type was selected as tissue culture model that may be enriched for conservation of cell autonomous biological features representative of the viral target tissue. These cells resist plaque formation, but are highly sensitive to single cycle infection. From whole-genome siRNA and miRNA mimic screening, both candidate sensitizer and resistor response modifiers were identified. A key deliverable from this analysis was the identification of gene products that apparently serve to restrain cell death responses that would otherwise engage upon detection of viral infection. Though not required to support cell viability in the absence of viral challenge, depletion of genes in this class enhanced the death response to H1N1 infection concomitant with restraining H1N1 protein production. As such, this class may represent targets for interventions that restrain propagation of multi-cycle infection by facilitating suicide of infected cells prior to production of new infectious particles. A chemically addressable member of this class, CHEK1, showed strong activity in multiple HBEC lines but not in A549, a non-small cell lung tumor derived line commonly employed to model influenza virus infection. This suggests that intervention targets may be available in normal epithelial cells that are uncoupled from host regulatory networks in cancer cells. 
Racemic 3-IPHPC (2-hydroxy-3-imidazo[1,2-a]pyridine-3-yl-2phosphonopropionic acid) was prepared and characterized as described previously [32, 33] and stored at ,0uC and pH $7 [32, 33] . The purity was $98%by 1 H NMR. The inhibitor was tested in this work as the racemate [32, 33] . It was subsequently demonstrated that the individual enantiomers have markedly different IC 50 and Ki values for inhibition of Rab1a prenylation, thus the racemate value obtained here probably represents an upper limit with respect to the potency of the more active stereoisomer.
HBEC30-KT cells were cultured in KSFM (Invitrogen Cat#17005) with 1% Pen/strep antibiotics as previously described [34] . MDCK and A549 cells [from ATCC] were grown in DMEM with 10% FBS.
Plaque Assay 5610 5 MDCK [from ATCC] and HBEC-30KT cells [34] were plated in 6 well plates and grown to confluence overnight. Cells were infected with WSN virus at 10 fold dilutions with a starting concentration of 10 8 pfu/ml. Infected cells were allowed to incubate at 37uC with tilting every 10 minutes. After incubation liquid was aspirated and 2 ml of agar solution was added to wells and allowed to solidify for 1 min. Plates were incubated for 48 hrs at 37uC. Following incubation plates were fixed with formaldehyde for 1 hr. Fixative and agar was removed and cells were stained with crystal violet.
HBEC30-KT cells [34] were plated in 96 well plates and incubated overnight. Cells were infected with WSN virus at an MOI 5. Whole cell lysates were collected at the indicated time point and separated by 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Cultures for immunofluorescence were fixed with 4% formaldehyde at indicated time point. Viral protein was detected in both cases with antibodies for pan influenza A (1:200), M2 (1:500) or NP (1:500) proteins{a-tubulin (cellsignalling rabbit mAb, cat#2125S), anti-NS1 [35] , anti-NP (Abcam, cat# ab20343)} followed by detection with either HRP conjugated secondary or staining with Alexa 498 (1:5,000) or Alexa 594 (1:5,000) conjugated secondary antibodies(from Invitrogen). Wells were imaged with a 20x lens on a BD Pathway 855 microscope. Imaged cells were segmented using Hoescht staining and distance from nucleus, aIVA fluorescence intensity was measured, with Attovision software.
HBEC30-KT cells were infected with WSN virus and supernatants were collected at 24 hours post infection. Supernatants were then added to MDCK cells at 1% final concentration and MDCKs were fixed 14 hours after supernatant addition and viral production in MDCK cells was detected immunostaining. 
The siRNA screen was performed using the Dharmacon library targeting 21,125 genes HBEC30 were plated into 96 well plates at 10,000 cells per well and siRNAs were reverse transfected. Each siRNA pool was transfected in two sets of triplicates for a total of 6 wells for each siRNA, three wells for infection with IVA and three wells for mock infection, with a concentration of 50 nM for oligos and 0.1% DharmaFECT 3 reagent. Cells were incubated for 48 hrs after transfection and infected with influenza A/WSN/33 (H1N1) virus at an MOI of 5. Forty-eight hours after infection cell viability was assayed using CellTiter-Glo, 15 ml of Promega's CellTiter-Glo was added to wells on a 96 well plate for a final concentration of 7.5%. Plates were rocked for two minutes followed by 10 minutes incubation. Luciferase activity was measured with a PerkinElmer EnVision reader. The miRNA mimic screen was performed with the Dharmacon miRNA mimic library corresponding to 426 human miRNA's. Screening conditions were identical to those described above with the exception of a 72-hour incubation between transfection and infection.
To remove position effects, raw values from each well were normalized to the median well of their respective row using the siMacro found at (http://sourceforge.net). To control for contamination and technical issues the top 5% of outliers with the highest coefficient of variation among triplicates were removed. Outliers were defined as wells with the largest distance among triplicate values. Normalized data was log2 transformed for proper distribution of sensitizers and resistors and a ratio of infected over mock infected was obtained. To control for batch effects, Z-Scores were calculated using batch specific variance where for each siRNA pool i Zi = xi-mbatch/sbatch, where x is the raw data to be normalized, m is the mean of the batch population, and s is the standard deviation of the batch population.
Published data sets were obtained from four siRNA screens for influenza A modulators that used viral replication as an end point assay [4] [5] [6] [7] . Candidate hits in our screen were queried for behavior as regards viral replication. Hits that modulated viral replication greater than 1.5 standard deviations were assigned to functional classes. In cases were hits showed multiple phenotypes the strongest phenotype was used for classification.
Screening data was compared for overlap with published hit lists for cell cycle regulators [36, 37] , host regulators of HIV infection [38] [39] [40] , and interferon stimulated genes (ISGs) [41] . Published hits that correlated with a change in cell viability greater than two standard deviations were considered as positive hits.
Predicted targets of miRNAs based on seed sequence were obtained from TargetScan (http://targetscan.org). Network analysis of predicted hits was completed using Ingenuity IPA (http:// ingenuity.com) and queried for behavior in siRNA screens for regulators of influenza A infection.
Z-scores were used as weights for NetWalk analysis [28] . Interactions with 350 highest and 350 lowest Edge Flux values were used to construct the networks with high and low z-scores, respectively. Analyses and graphics were done in the NetWalker desktop application (Komurov et al, manuscript submitted, http://research.cchmc.org/netwalker).
HBEC30 or A549 cells were plated on 96 well plates overnight. Media was removed and replaced with media containing SB218078 (1mM, 100nM 10nM) 3-IPEHPC (12.5mM, 1.25mM, 125nM) DMSO (0.06%) or plain media. Cells were incubated overnight and then infected with WSN at an MOI of 5. Cells were fixed with 4% formaldehyde at 8 hours, 12 hours and 24 hours post infection and stained as described previously. SB218078 was purchased from Tocris biosciences cat # 2560 and dissolved in DMSO. 3-IPEHPC was dissolved in PBS. Figure S1 Individual siRNA oligo assays. HBEC30 cells were transfected in triplicate with four individual siRNA oligos and infected with WSN. Cell viability was measured 48 hours post infection and a two-tailed Student's t-test was performed to determine significance. Green boxes are oligos with a p value less than 0.05. (PDF) Figure S2 Network analysis of siRNA screen results. Data from siRNA screen results was used for NetWalk analysis. Nodes are colored based on Z-Score with red for positive and green for negative, edges are colored based on interactions, PPI: protein-protein interaction, TF-Target: gene regulation, GO: GO similarity. Networks analysis was performed with entire data set. (PDF)  Association of Fcγ Receptor IIB Polymorphism with Cryptococcal Meningitis in HIV-Uninfected Chinese Patients BACKGROUND: As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis. METHODOLOGY/PRINCIPAL FINDINGS: In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02–2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33–0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05–3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24–0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls. CONCLUSION/SIGNIFICANCE: We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals. Cryptococcal meningitis is the most common opportunistic fungal infection of the central nervous system in AIDS patients. Among HIV-uninfected patients, several predisposing factors for cryptococcal meningitis such as corticosteroid medication, solid organ transplantation and malignancy, etc, have been indentified. Yet cryptococcal infections in apparently healthy individuals are also increasingly being reported, especially from Asian data [1] [2] [3] . Our previous study has demonstrated an association between mannose-binding lectin (MBL) genetic deficiency and cryptococcal meningitis in HIV-uninfected patients [4] . However, MBL deficiency was present in only 21% of the cases, and for the remaining 79% of patients the underlying mechanism for susceptibility remained unclear.
Fc gamma receptors (FccRs) mediate a variety of immune responses after binding to IgG-opsonized pathogens or immune complexes, and therefore act as immune regulators in both autoimmune and infectious diseases [5] [6] [7] [8] [9] . According to their affinity to IgG, FccRs are categorized into high-affinity and lowaffinity receptors. FccRI is the only known high-affinity receptor.
Low-affinity FccRs which include FccRIIA, FccRIIB, FccRIIC, FccRIIIA, and FccRIIIB, are encoded by FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes, respectively.
FCGR polymorphisms had been associated with the susceptibility and severity of various infections. FCGR2A 131R/R had been reported to attribute to the susceptibility of meningococcal infection, community-acquired pneumonia (CAP) caused by Haemophilus influenza, and the development of severe malaria [10] [11] [12] . FCGR2A 131H/H was reported to contribute to higher risk of bacteremia in pneumococcal CAP patients [13] . Another study showed that HIV-infected patients with FCGR2A 131R/R genotype progressed to a low CD4 + cell count at a faster rate, but conversely in individuals carried FCGR2A 131H/H there was an increased risk of Pneumocystis jiroveci pneumonia [14] . FCGR3A 158F/V gene polymorphism was not associated with progression of HIV infection, but predicted the risk of Kaposi's sarcoma [14] . A study on infections during induction chemotherapy found that FCGR2A 131H/H was associated with a decreased risk of pneumonia, FCGR3B NA1/NA1 associated with infections, and FCGR3A polymorphisms not associated with infections [15] . Sadki et al. investigated the influence of FCGR3A 158V/F and FCGR2A 131H/R polymorphisms on susceptibility to pulmonary tuberculosis in the Moroccan population but no association was found [16] . A study in East Africa found that the FCGR2B 232T/T genotype provided protectiveness for children against severe malaria [17] .
A previous study by Meletiadis et al. investigated FCGR polymorphisms in patients with cryptococcosis, and found that FCGR2A 131R/R and FCGR3A 158V/V were over-presented, and FCGR3B NA2/NA2 was under-presented in patients with cryptococcosis [18] . The purpose of this study was to investigate FCGR polymorphisms in our series of patients to further verify the association between FCGR and cryptococcal meningitis.
A total of 117 HIV-uninfected patients with cryptococcal meningitis were included. Subjects from both the patient and control groups were of Chinese Han ethnicity. Clinical information and predisposing factors of the patients are summarized in Table 1 . Of the 190 healthy control subjects, 111 were male (58.4%). The median age of the control subjects was 44 years (range, 12-79 years).
Two samples failed genotyping of FCGR3A and 2 samples failed in genotyping of FCGR2B. Allele distributions of the tested FCGR genes in the control group were in Hardy-Weinberg equilibrium. The frequencies of FCGR2A, FCGR3A, FCGR3B and FCGR2B genotypes were shown in Table 2 . An association was found between FCGR2B 232I/T genotypes and cryptococcal meningitis based on dominant and over-dominant model. The FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02-2.67]; P = 0.039) and the FCGR2B 232I/T genotype was underpresented (OR = 0.542, 95% CI [0.33-0.90]; P = 0.016) in patients with cryptococcal meningitis in comparison with controls. No significant difference was found in the distribution of FCGR2A 131H/R, FCGR3A 158 F/V and FCGR3B NA1/NA2 genotypes.
We further compared the genotype distribution of FCGR2A, FCGR3A, FCGR3B and FCGR2B between the 58 patients without predisposing condition and controls. Similar to results from the overall patient group, associations were also found between FCGR2B 232I/T genotypes and cryptococcal meningitis based on dominant and over-dominant model. Specifically, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05-3.66]; P = 0.033), and FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24-0.91]; P = 0.023) in patients without predisposing factor than in controls. For the genotype distribution of other polymorphisms (FCGR2A 131H/R, FCGR3A 158 F/V and FCGR3B NA1/NA2), there was also no significant difference between patients and controls.
The distribution of FCGR polymorphisms has been reported to exhibit substantial inter-ethnic variation. According to our population, frequencies of FCGR2A 131R/R, FCGR3B NA2/ NA2, and FCGR2B 232T/T in all subjects were 16%, 11%, and 7% respectively, similar to those reported among other Asian populations (which ranged 9-14%, 11-21%, and 5-11%) [19] [20] [21] [22] [23] [24] [25] [26] . Frequencies of these genotypes in Caucasian population were reported to be 19-34%, 38-43%, and 1-3% [18, 23, [27] [28] [29] [30] , which were different from our data. There seems no marked difference in the distribution of FCGR3A 158F/V genotypes between the Asian and Caucasian populations [18, 21, 23, 31, 32] .
The four polymorphisms of low-affinity receptors genotyped in our study have each been demonstrated to affect functions of their encoded receptors. In FCGR2A, the G.A SNP at amino acid position 131 results in a histidine (H) to arginine (R) change (FCGR2A 131H/R), resulting in reduced affinity of the correspondent receptor to IgG2 [33, 34] . The T.G SNP at position 158 of FCGR3A causes a phenylalanine (F) to valine (V) substitution (FCGR23A 131F/V) and FCGR3A 158V/V encoded receptors show higher affinity to IgG1 and IgG3 [35, 36] . In the FCGR3B gene, five nucleotides (141,147,227,277 and 349) are combined to form two main haplotypes termed FCGR3B NA1 and FCGR3B NA2, and receptor encoded by FCG3B NA1 haplotype binds to IgG1 and IgG3 more easily [37] . Finally, FCGR2B 232I/T causes an isoleucine (I) to threonine (T) substitution in the transmembrane domain [22, 38] and receptors encoded by FCGR2B 232T are unable to interact with activating receptors [39] .
Although FCGR polymorphisms have been demonstrated to be associated with susceptibility and severity of numerous infections, there has only been one previous genetic association study on the relationship of FCGR genotypes and cryptococcosis [18] . Meletiadis and colleagues genotyped FCGR2A 131H/R, FCGR3A 158F/V and FCGR3B NA1/NA2 in 103 cryptococcosis patients and 395 healthy controls. They found that in patients with cryptococcosis FCGR2A 131R/R and FCGR3A 158V/V were over-presented (Pvalues were 0.04 and 0.04), while FCGR3B NA2/NA2 was underpresented (P-value was 0.04).
In our study, we found for the first time that cryptococcal meningitis was associated with the FCGR2B 232I/T genotypes, which was not reported in Metediatis' study. As the only known inhibitory FccR, FccRIIB has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, and thus it plays an important role in regulating the immune system [40] . FCGR2B 232I/T is located in the transmembrane domain, and the FccRIIB receptors encoded by FCGR2B 232T are unable to interact with activating receptors and exert inhibitory activity [38] . Published data have suggested the mutation genotype FCGR2B 232T/T to be a susceptible genotype for systemic lupus erythematosus [17, 22, 32] , and this genotype also provided protective effect for severe malaria in East African children [17] . The role of FccRIIB in cryptococcal infection is still not very clear. Like the activatory FccRs, FccRIIB can also recognize the major component of the capsule of C. neoformans, glucuronoxylomannan (GXM). In a previous study by Monari et al., the immunosuppressive effect of GXM was demonstrated to be dependent on FcRcIIB, based on the evidences that FccRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-a secretion, and that the addition of monoclonal antibody to GXM reverses GXM-induced immunosuppression by shifting recognition from FccRIIB to FccRIIA [41] . Another study subsequently demonstrated that GXM triggered NO-induced macrophage apoptosis, which was dependent on FccRII [42] . These data support that FccRIIB plays a critical role in the pathogenesis of cryptococcal infection. In our study, it is the FCGR2B 232I/T heterozygote instead of the minor homozygote 232T/T that is under-presented in patient group. One study on children with idiopathic thrombocytopenia (ITP) also showed a similar pattern, that the FCGR2B 232I/T was less frequently detected in acute ITP in comparison with chronic ITP [27] . The reason for the heterozygotes 232I/T rather than 232T/T under-presenting in our patients and those acute ITP children has not been clarified.
Unlike results from Meletiadis' study, no association among FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA2/NA2 and cryptococcal meningitis was found in our study. The cause for discrepant results may be multifactorial. One was the ethnic differences between the two studies. Subjects in our study were of Chinese Han ethnicity, while the majority of subjects in Meletiadis' study were Caucasians (60%). As a matter of fact, the FCGR3A 158V allele was significantly increased only in patients who were Caucasian in Meletiadis' study. Secondly, all the cases in our study were diagnosed with cryptococcal meningitis, while some patients from Meletiadis' study were cryptococcosis without central nervous system involvement. Furthermore, both studies had relatively small sample sizes, which could be underpowered to generate positive results.
In conclusion, our study suggested that FccRIIB genetic polymorphism may contribute to the susceptibility of cryptococcal meningitis. The overall association is relatively weak, which warrants validation in larger population.
This study was reviewed and approved by the Ethic Committee/Institutional Review Board (HIRB) of Huashan Hospital, Fudan University, and informed written consent was obtained from each participant.
A total of 200 volunteers and 117 unrelated patients with proven or probably diagnosed cryptococcal meningitis who were referred to Huashan Hospital, Fudan University, China, from 2001 through 2011 were recruited for the present study. Patients who met at least one of the following criteria were considered as proven cryptococcal meningitis: (1) Isolation of C. neoformans from cerebrospinal fluid (CSF) by culture or positive India ink smear, and (2) compatible histopathological findings, which are 5-10 mm encapsulated yeasts observed in brain tissue. Patients who had no microbiological or pathological documentation but present with positive cryptococcal antigen titer ($1:10) in CSF and met at least one of the following criteria were regarded as probable cryptococcal meningitis: (1) abnormal laboratory tests or an increased open pressure ($200 mmH 2 O) of CSF, (2) abnormalities of cranial imaging (Computerized Tomography or Magnetic Resonance Imaging) which could not be explained by other factors, and (3) comorbidities that compromise the host immune system. Cryptococcal antigen was determined using diluted CSF with the Latex-Cryptococcus antigen detection system (Immuno-Mycologics). Patients and volunteers were assessed for predisposing factors as follow, immunocompromising diseases (liver cirrhosis, chronic kidney diseases, autoimmune diseases, malignancies, solid organ transplantation) [2, 3, 43] , and corticosteroid (at prednisone equivalent dose of .0.3 mg/kg/day of for .3 weeks) or immunosuppressive medications (within 90 days before onset of cryptococcal meningitis) [44] , and idiopathic CD4 + T lymphocytopenia (unexplained CD4 + T lymphocytopenia with CD4 + T lymphocyte count ,300 cells/mm 3 ) [45] . Diabetes mellitus was also included, although this common condition is a controversial predisposing factor [3, 46] . Patients without any of the above mentioned predisposing factors were considered as apparently healthy hosts. Ten volunteers were excluded because of disclosed predisposing conditions, and the remaining 190 healthy volunteers were included in the control group.
Four functional FCGR polymorphisms including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T were selected for genotyping after literature review of previous studies on association between FCGR polymorphisms and infectious diseases [11] [12] [13] [14] [15] [16] [17] .
Venous blood was obtained by venepuncture from each subject. Genomic DNA was extracted using the QIAamp DNA kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. Genotyping of 8 SNPs in FCGRs (Table 3 ) was performed by multiplex SNaPshot technology using an ABI fluorescence-based assay discrimination method (Applied Biosystems, Foster city, CA, USA), which has been described in detail in previous studies [47, 48] . The multiplex SNaPshot detection of single-base extended probe primers was based on fluorescence and extended length detected by capillary electrophoresis on ABI3130XL Sequencer (Applied Biosystems, Foster City, CA, USA).
Four pairs of primers for PCR amplification including 5 fragments of 587-2394 bp and 8 primers for SNaPshot extension reactions were designed by Primer3 online software (v.0.4.0) (http://frodo.wi.mit.edu/primer3/) according to the reference sequences from dbSNP (http://www.ncbi.nlm.nih.gov/SNP). There were homologous sequences between FCGRs, the specificity sequences were checked with the sequence databases using BLAST (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). These sequences were also verified by SNPmasker1.1 (http://bioinfo.ebc. ee/snpmasker) to make sure that the different bases were caused by SNP [49] . And each primer pair was tested for potential primer-dimer and hairpin structures using the AutoDimer software (http://www.cstl.nist.gov/biotech/strbase/ AutoDimerHomepage/AutoDimerProgramHomepage.htm). The primers used in this study were listed in Tables 3.
The PCR reactions were performed with 1 mL of DNA and 1 mL multiple PCR primers (the concentration was 1 mM) in a total volume of 20 mL containing 16 HotStarTaq buffer, 2.0 mM Mg 2+ , 0.3 mM dNTP, and 1 U HotStarTaq polymerase (Qiagen, Hilden, Germany). The cycling conditions for FCGR2A and FCGR3A were 95uC for 2 min, 35 cycles using 96uC for 20 s, 62uC for 2 min, and 72uC for 3 min, then 72uC for 10 min, and finally kept at 4uC. The cycling conditions for FCGR2B and FCGR3B were 95uC for 2 min, 7 cycles using 96uC for 20 s, 55uC for 2 min, and 72uC for 3 min, then 72uC for 10 min, and finally kept at 4uC. PCR products were then purified (add 1U SAP enzyme to 10 mL PCR products, incubate at 37uC for 1 hour, then, inactivate at 75uC for 15 min).
The extension reaction to identify single nucleotide polymorphisms in the PCR products was performed in a total volume of 10 mL containing 2 mL purified PCR product, 1 mL primer (the concentration was 0.8 mM), 5 mL SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA, USA), and 2 mL ultrapure water. The cycling conditions for extension were 96uC for 1 min, 28 cycles of 96uC for 10 s, 52uC for 5 s, and 60uC for 30 s, and kept at 4uC. Then each extended product was added to 1 U shrimp alkaline phosphatase, incubated at 37uC for 1 hour, and the enzyme inactivated at 75uC for 15 min. Then, 0.5 mL was added to 0.5 mL Liz120 SIZE STANDARD (Applied Biosystems, Foster City, CA, USA), 9 mL Hi-Di (Applied Biosystems, Foster City, CA, USA), and sequenced by ABI3130XL Sequencer (Applied Biosystems, Foster City, CA, USA). Finally, the primary data was analyzed by GeneMapper 4.0 (Applied Biosystems, Foster City, CA, USA). Genotypes were determined by the type of nucleotide presented at SNP site, which was visualized by one or two different color peaks on the figures.
For quality control, a random sample of 5% of the cases and controls was genotyped twice by different researchers, with a reproducibility of 100%. The minor allele counts were compared with database (http://www.ncbi.nlm.nih.gov/projects/SNP), and the data were matched well. Genotyping was performed blind to group status.
Dominant, over-dominant, recessive and allelic models were applied for the analysis of genotype distribution. Hardy-Weinberg equilibrium, differences in gene polymorphism distributions between patients and controls were analyzed with 262 x 2 tests or Fisher's exact test where appropriate. P-values, odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by SPSS 16.0 for Windows (SPSS, Inc, Chicago, IL). P-values ,0.05 were considered statistically significant. Infectious Disease Transmission during Organ and Tissue Transplantation Infectious disease transmission through organ and tissue transplantation has been associated with severe complications in recipients. Determination of donor-derived infectious risk associated with organ and tissue transplantation is challenging and limited by availability and performance characteristics of current donor epidemiologic screening (e.g., questionnaire) and laboratory testing tools. Common methods and standards for evaluating potential donors of organs and tissues are needed to facilitate effective data collection for assessing the risk for infectious disease transmission. Research programs can use advanced microbiological technologies to define infectious risks posed by pathogens that are known to be transplant transmissible and provide insights into transmission potential of emerging infectious diseases for which transmission characteristics are unknown. Key research needs are explored. Stakeholder collaboration for surveillance and research infrastructure is required to enhance transplant safety.  Non-HIV Pneumocystis pneumonia: do conventional community-acquired pneumonia guidelines under estimate its severity? BACKGROUND: Non-HIV Pneumocystis pneumonia (PCP) can occur in immunosuppressed patients having malignancy or on immunosuppressive agents. To classify severity, the A-DROP scale proposed by the Japanese Respiratory Society (JRS), the CURB-65 score of the British Respiratory Society (BTS) and the Pneumonia Severity Index (PSI) of the Infectious Diseases Society of America (IDSA) are widely used in patients with community-acquired pneumonia (CAP) in Japan. To evaluate how correctly these conventional prognostic guidelines for CAP reflect the severity of non-HIV PCP, we retrospectively analyzed 21 patients with non-HIV PCP. METHODS: A total of 21 patients were diagnosed by conventional staining and polymerase chain reaction (PCR) for respiratory samples with chest x-ray and computed tomography (CT) findings. We compared the severity of 21 patients with PCP classified by A-DROP, CURB-65, and PSI. Also, patients’ characteristics, clinical pictures, laboratory results at first visit or admission and intervals from diagnosis to start of specific-PCP therapy were evaluated in both survivor and non-survivor groups. RESULTS: Based on A-DROP, 18 patients were classified as mild or moderate; respiratory failure developed in 15 of these 18 (83.3%), and 7/15 (46.7%) died. Based on CURB-65, 19 patients were classified as mild or moderate; respiratory failure developed in 16/19 (84.2%), and 8 of the 16 (50%) died. In contrast, PSI classified 14 as severe or extremely severe; all of the 14 (100%) developed respiratory failure and 8/14 (57.1%) died. There were no significant differences in laboratory results in these groups. The time between the initial visit and diagnosis, and the time between the initial visit and starting of specific-PCP therapy were statistically shorter in the survivor group than in the non-survivor group. CONCLUSIONS: Conventional prognostic guidelines for CAP could underestimate the severity of non-HIV PCP, resulting in a therapeutic delay resulting in high mortality. The most important factor to improve the mortality of non-HIV PCP is early diagnosis and starting of specific-PCP therapy as soon as possible. Pneumocystis pneumonia (PCP) not related to human immunodeficiency virus (HIV) can occur in immunosuppressed patients having malignancy or on immunosuppressive agents [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . The mortality of patients with PCP without HIV infections ranges from 0 to 70% [1, [3] [4] [5] [6] [7] [8] [9] [10] , compared to that of HIV-infected PCP patients, which ranges from 10 to 20% [1, 4, 9] . Besides, mortality rates as high as 60-75% have been reported in PCP patients without AIDS who required mechanical ventilation [11, 12] . The higher mortality among non-HIV PCP patients has been attributed to severe lung inflammation [1, 4, 10] , although the exact etiology accounting for these large differences in mortality has not yet been determined.
The A-DROP system proposed by the Japanese Respiratory Society (JRS), the CURB-65 score proposed by British Respiratory Society (BTS) and the Pneumonia Severity Index (PSI) proposed by the Infectious of Disease Society of America (IDSA) are widely used in classifying patients with community-acquired pneumonia (CAP) [13] [14] [15] in Japan. For the purpose of evaluating how correctly the conventional prognostic guidelines of CAP reflect the severity of non-HIV PCP, we retrospectively analyzed 21 patients with non-HIV PCP. It has never previously been reported that the severity of non-HIV PCP may be underestimated by these prognostic guidelines. This is the first report focusing on the limit of conventional prognostic guidelines of CAP for non-HIV PCP.
From the end of 2009 to the beginning of 2010 we retrospectively reviewed all the cases of PCP diagnosed as CAP in the Kameda Medical Center, Chiba, Japan. All the patients had undergone HIV testing and were negative. Patients with hospital associated pneumonia (HAP) were excluded. PCP was diagnosed based on polymerase chain reaction (PCR) and conventional PCP staining with Grocott methenamine silver stain or Diff-Quick™ staining in respiratory samples such as induced sputum (IS) or bronchoalveolar lavage (BAL) fluid associated with radiographic infiltration on chest X-ray and computed tomography (CT) findings on admission. All radiographic pictures showed infiltration confirmed by the pulmonologist and radiologist in our hospital. The decision to perform IS or BAL examination depended on the patient's general condition at the discretion of attending physicians. PCP diagnosis was based not solely on the positive PCR respiratory specimen but also on the clinical and radiological findings consistent with the diagnosis of PCP as well as complete recovery with anti-Pneumocystis jirovecii treatment alone. No biopsy was performed during this study. We compared the severity of 21 patients with PCP classified by A-DROP, CURB-65, and PSI, and analyzed the background and laboratory data of each.
Comparisons of group means were made by unpaired or paired t-tests or Mann-Whitney U-test. Contingency tables were evaluated by Fisher's exact probability test. p values < 0.05 were considered significant.
The characteristics of the 21 patients with PCP are shown in Tables 1 and 2. The mean age was 71.5 years (range 57-88). Twelve patients (57.1%) had rheumatic or autoimmune disease which was the most common underlying disease, followed by malignancy (n = 10, 47.6%). Seventeen patients (77.8%) were receiving steroid or immunosuppressants for the underlying disease. Prophylactic therapy consisting of trimethoprim-sulfamethoxazole (TMP/SMX) was used by only 1 patient (4.8%).
All the patients received oral or parenteral (TMP/SMX and adjuvant steroid therapy. Eighteen (85.7%) of the 21 patients had acute respiratory failure. Eight patients (38.1%) in the study died.
The A-DROP scale was one of the prognostic guidelines for CAP proposed by JRS in 2005. It is a scoring system using age, degree of dehydration (serum blood urea nitrogen (BUN)), SpO 2 < 90%(PaO 2 < 60 mm Hg), orientation, and Table 3) .
CURB-65 is also a prognostic guideline for CAP, proposed by BTS in 1996. It seems to be similar to the A-DROP system, the greatest difference being the age value used in the scoring. While JRS defines males > 70 years and females > 75 years as high risk elderly, BTS classifies high risk elderly age as > 65 years for both sexes. In our study 19 patients (90.5%) were classified as mild or moderate (class 0-2); 16 of the 19 patients (84.2%) developed acute respiratory failure, and 8 (42.1%) of the 16 died (Table 3) .
The PSI was proposed by the Infectious Diseases Society of America/American Thoracic Society (IDSA/ATS). It is considered to be more complicated and less convenient to use than the other scoring systems. It consists of 19 items such as age, underlying disease, gender, vital signs, etc. In our study, 14 patients (66.7%) were classified as severe or extremely severe by PSI. All the 14 patients developed respiratory failure and 7 patients (50%) died ( Table 3) .
The majority of the PCP patients were categorized in risk classes 2-3 by A-DROP and CURB-65, and III/IV by PSI. Of note are the very high patient mortalities in risk classes 2 by A-DROP and CURB-65, and class III/IV by PSI ( Table 3) .
The comparison of the prognostic accuracy of each guideline for CAP is shown in Table 4 . It can be seen that the positive predictive values and negative predictive values for mortality in each system were low.
The lactate dehydrogenase (LDH) value tended to be higher and Alb/BUN tended to be lower in the non survivor group compared with the survival group, but this was not statistically significant. There were no significant differences in serum β-D-glucan (β-DG), Krebs von den Lungen 6 (KL-6), body mass index (BMI) between the two groups. However, both the time between the initial visit and establishment of a diagnosis and the time between the initial visit and starting PCP therapy were significant much shorter in the survivor than non survivor group (Table 5 ).
There are some widely used prognostic guidelines for CAP. These systems appear to be useful in assisting physicians to make more rational decisions regarding the need for admission [13] [14] [15] . Patient mortalities in the risk groups 3-5 on A-DROP and CURB-65, and IV-V on PSI have previously been reported as 11.5-23.3%, 11.6-21.0% and 12.5-29.2%, respectively [16] [17] [18] [19] . A striking fact is that the majority of the PCP patients were categorized as mild to moderate by these guidelines and resulted in respiratory failure, and poor outcomes. We emphasize that mortality prediction in PCP is not correct when these conventional guidelines for CAP are applied, even when PCP develops in the setting of CAP. Also, these guidelines definitely underestimate the severity of PCP as CAP. The important issue is why these guidelines cannot correctly estimate PCP severity. PCP without HIV infection shows quite different clinical pictures compared to PCP with HIV infection. PCP with HIV occurs slowly and gradually [20] . On the other hand, PCP without HIV is typically more acute and severe than when associated with AIDS [10] , often resulting in acute respiratory failure requiring a need for mechanical ventilation. We suppose that this results from the differences of pathologic mechanisms between PCP with and without HIV. It is evident that PCP without HIV is an allergic reaction originating from Pneumocystis jirovecii. Pneumocystis elicits many kinds of immune responses, including those by lymphocytes, macrophages, neutrophils, dendritic cells, and epithelial cells [1, 21] .
There is now a considerable body of evidence showing that immune and inflammatory responses to Pneumocystis can have harmful as well as beneficial effects on host lungs. Another reason why the mortality rate of PCP without HIV remains high is presumably that it is difficult to diagnose PCP according to nonspecific signs, symptoms and/or no reliable culture. Bollée et al. documented that the leading symptoms of PCP in HIV-uninfected cancer patients were fever (85.7%), dyspnea (78.6%), cough (57.1%), and all three symptoms (44.6%) on diagnosis [5] , and 14.3% of the patients showed only one symptom. In our study, 4 out of 21 patients (19%) were asymptomatic. In addition, 6/21 patients (28.6%) showed abnormality in chest X-ray on admission. It is possible that steroids and immunosuppressive drugs could mask fever and general fatigue on the initial visit. We strongly believe that clinicians are unable to diagnose non-HIV PCP by clinical picture or chest X-ray alone. The association with P. jirovecii cysts has been reported in HIV-uninfected PCP to be one tenth of that in HIV-PCP [4] . Therefore, the sensitivity of conventional staining methods for diagnosis of HIV-uninfected PCP is lower than that for PCP with HIV. Our study demonstrated the sensitivity of conventional staining to be 23.8%. While Diff-Quik staining is highly sensitive, it requires considerable technical expertise [22] . It is likely that physicians are unable to diagnose PCP without HIV soon enough due to the reasons mentioned above.
A clue for making the early diagnosis of PCP is serum β-D-gulcan (β-DG) and chest CT findings. Tasaka et al. reported the β-DG could be a serum indicator for the diagnosis of PCP with the cut-off value of 31 pg/ml [23, 24] . In our study, the sensitivity of β-DG in diagnosing PCP was 10/21 (47.6%) setting the cut-off value at 31 pg/ml. We suggest that testing β-DG is effective for diagnosis of PCP. In testing, BAL is also well known to be more sensitive than IS, as many physicians previously reported [23, 25] . In terms of a radiological approach, high resolution computed tomography (HRCT) should be performed if PCP is suspected. It is commonly known that chest CT shows ground glass appearance with a panlobular pattern or so-called crazy paving appearance in PCP patients [26, 27] . These findings are also found in viral pneumonias, mycoplasmal pneumonia, alveolar hemorrhage, methotrexate pneumonia, and others. However, where the patient's background and characteristics are conducive, the presence of PCP should be suspected. Conventional guidelines for CAP have recommended that clinical outcomes should be evaluated three days after initial therapy has been started [13, [28] [29] [30] [31] [32] [33] . In our study, 12 of the 13 (92.3%) patients who received accurate anti-PCP therapy within 3 days from initial visit were cured. Ten of the 12 (83.3%) patients received empiric therapy for PCP based on patient characteristics, laboratory data and radiological findings on HRCT. On the other hand, 7 of the 8 (87.5%) patients who received anti-PCP therapy that was initiated after day 4 died. PCP without HIV tends to develop acute respiratory failure and results in a more severe, acute form of acute respiratory distress syndrome (ARDS) than PCP with HIV. Thus, only three days of doctor's delay in starting PCP therapy could be fatal as our study showed.
The limitation of our study is that it is a retrospective analysis in a very small population. Retrospective studies may be less reliable in terms of the data collected, particularly for data such as physical examination. A prospective study should be carried out and with more cases.
In conclusion, we suggest that conventional prognostic guidelines for CAP might underestimate the severity of HIV-uninfected PCP. Physicians should be aware of the possibility that PCP may occur in non-HIV patients having malignancy or rheumatic disease, receiving steroid and/or immunosuppressive therapy. The most important factor for improving the mortality of PCP without HIV could be the time when anti-PCP therapy is started. Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates the potential of this analytical approach for the detection of malaria as well as other human diseases. The burden of malaria continues to worsen globally with a devastating impact on human health and corresponding impediment to economic improvement [1] . Despite worldwide initiatives, emerging drug resistance in different species of Plasmodium and paucity of information about the exact underlying mechanism of the disease pathogenesis are creating challenges for the management and eradication of the disease. Plasmodium falciparum (Pf) infection represents the major cause of malaria associated morbidity and mortality worldwide. Falciparum malaria (FM) accounts for approximately 247 million cases and one million deaths annually, particularly in sub-Saharan Africa [2] , while outside the African continents, Plasmodium vivax (Pv) is responsible for more than 50% of all malaria cases [3] . In order to survive within the host cells and ensure their reproduction, intracellular parasites like Plasmodium develop versatile mechanisms to exploit their host cells and induce new permeability pathways to permit the uptake of nutrients and the removal of waste products, resulting into activation of multiple host immune cascades and inflammatory responses [4] . Plasmodium infection also affects blood coagulation by diverse pathobiological mechanisms, which results into development of fatal hemorrhagic complication [5, 6] . Investigation of the parasite induced alterations in host proteome and modulation of different vital physiological processes have great clinical relevance in the light of diagnosis and prognosis. Recently, proteomic studies have contributed substantially to our understanding of the clinical proteome of human malaria parasites [7] , profiling humoral immune responses to Plasmodium infection [8] and the malaria parasite infection-induced changes in host erythrocyte membrane proteins [9] . The findings obtained from such studies have provided better understanding of the disease pathogenesis, host-pathogen interactions and host immune response.
Analysis of human serum proteome is found to be very useful for the identification of potential disease-related markers, understanding disease pathogenesis and host immune response since various serum proteins exhibit rapid alteration in expression pattern in response to diseased conditions and show direct correlation with disease progression [10] . In recent years, a number of proteomic studies have been carried out to investigate the pathogen induced alterations in human serum/plasma proteome in different infectious diseases including dengue [11] , SARS [12] , leishmaniasis [13] , and leptospirosis [14] . In this study we have investigated the alterations in human serum proteome due to the P. falciparum infection for obtaining mechanistic insight about the disease pathogenesis and host immune response in the most virulent form of human malaria. Additionally, serum proteome changes in FM were compared with vivax malaria (VM); another widely distributed human malaria to study the similarities and differences in host responses against these two major plasmodial infections. To achieve this comparative analysis we have utilized selected dataset of our previous serum proteomics study on VM [15] , while additional proteomics and immune-assay-based experiments were performed using a bigger (compared to our previous report) clinical cohort of VM patients. The comparative study on FM and VM revealed that quite a few serum proteins associated with diverse essential physiological pathways, including acute phase response signaling, cytokine and chemokine signaling, complement cascades and blood coagulation are commonly altered in both of the plasmodial infections, while some uniquely modulated candidates such as calcium binding protein 39, calpain 10, regulator of G-protein signaling 7, serum paraoxonase/arylesterase, transthyretin in FM and ceruloplasmin, vitamin D-binding protein, serum amyloid P, alpha-2-macroglobulin, fibrinogen beta chain precursor in VM, were also identified. Recently, we performed serum proteomic alterations in another clinically relevant infectious disease, leptospirosis [14] . To evaluate the specificity of the identified protein targets and eliminate the generic febrile responses; expression level of the serum proteins differentially expressed in plasmodial infections (compared to the healthy subjects) was analyzed in leptospirosis patients from our previous study [14] . Another major intention of this present study was identification of the characteristics marker proteins, which can readily discriminate malaria patients (FM from VM as well) from healthy population as well as closely related infectious diseases with high accuracy. The potential serum protein biomarkers identified in our study were used to build statistical models, which successfully classified and predicted the clinical phenotypes of controls (healthy and febrile), FM and VM in a blinded study.
Stringent inclusion criteria were employed during the selection of malaria patients and controls (HC and FC) to reduce preanalytical variations. Malaria patients (FM and VM) selected for this proteomic analysis were suffering from uncomplicated, nonsevere plasmodial infections with comparable range of parasitemia. Blood samples were collected from the malaria patients before administration of any antimalarial drugs. Majority of the patients were suffering their first episode of malaria, while some of the subjects had a past history of this disease (relapse or recurrent). The average age of the FM and VM patients included in this proteomic analysis was 34.2 years (SD = 10.93; range 20-53; median 35) and 32.9 years (SD = 10.76; range 20-52; median 32), respectively (Table 1) . To maintain uniform population profiles of test (FM and VM) and controls (HC and FC) for differential protein expression analysis, healthy and febrile control (leptospirosis patients) populations with comparable age distribution; mean values 33.4 years (SD = 8.69; range 20-44; median 31.6) and 30.5 years (SD = 8.31; range 23-42; median 26.5), respectively for HC and FC, were selected ( Table 1) .
In this proteomics study we have performed two levels of gelbased proteomic analysis using classical two-dimensional gel In classical 2DE analysis, patients suffering their first episode of malaria as well as few patients with a past history of malaria (relapse or recurrent) and higher level of parasitemia (.5000 infected RBCs/mL blood) were included since it was difficult to get bigger cohort of malaria patients with similar parameters. In gel-based proteomic analysis samples were studied individually (n = 63 for classical 2DE and n = 30 for 2D-DIGE) rather than sample pooling to achieve better insights about biological variability from individual samples. In proteomic analysis two major high-abundance serum proteins; albumin and IgG were removed using Albumin & IgG Depletion SpinTrap (GE Healthcare) to reduce the dynamic range of the serum proteome. Depletion of these top two highabundance proteins removes more than 60% of the total protein content in human plasma or serum allowing detection of more proteins by increasing the effective concentration of the lowabundance proteins. Depletion of albumin and IgG effectively increased the overall spot number in 2D gels ( Figure S1A ). The efficiency of albumin and IgG depletion from human serum was evaluated by densitometric analysis of SDS-PAGE gels containing resolved serum proteins before and after depletion ( Figure S1B ). The densitometric analysis revealed around 85% and 80% depletion of albumin and IgG, respectively ( Figure S1C ). Serum proteome analysis of FM patients and healthy controls by 2DE identified 22 statistically significant (p,0.05) differentially expressed (with changes from 24.28 to +78.73-fold) protein spots (Table S1 .1). After staining with GelCode Blue Safe Protein Stain, over 700 protein spots were detected reproducibly in each gel by IMP7 software. Representative 2DE images of serum proteome profile of FM subjects and healthy individuals, and bar-diagrammatic representation of the fold change and 3D views of few selected spots are illustrated in Figure 1A and B. In MS analysis 12 different proteins were identified from the 22 differentially expressed protein spots, since in few cases MS analysis revealed similar identity for multiple protein spots appearing as different entities in 2D gels. The similar identity of multiple spots indicates the possibility of presence of various isoforms of those particular proteins probably due to the complex combinations of posttranslational modifications. Among the 12 identified proteins; 7 proteins were up-regulated (serum amyloid A, hemopexin precursor, apolipoprotein E, a-1-antitrypsin precursor, leucinerich a-2-glycoprotein, a-1-BN glycoprotein and a-1-antichymotrypsin precursor) and 5 proteins were down-regulated (haptoglobin, ficolin 3 precursor, apolipoprotein A-I, clusterin precursor and serum albumin) ( Figure S2 ; Table 2 and S2.1). Interestingly, serum amyloid A (spot U13 and U14) was found to be highly over expressed (.25-fold) in all the FM patients.
Around 1300 protein spots were detected on each 2D-DIGE gels in DeCyder 2D software analysis. In 2D-DIGE analysis of FM and HC, total of 121 (around 9.3% of the entire detected spots) differentially expressed spots satisfied the statistical parameters (t-test; p,0.05), among which, 70 protein spots were up-regulated (with changes from 1.02 to 50.9-fold) and the remaining 51 were down-regulated (range from 1.2 to 24-fold) (Table S1 .2). Out of 121 spots, 36 and 9 spots were found to be statistically significant after performing false discovery rate (FDR) correction (Benjamini-Hochberg) and Bonferroni correction, respectively (Table S1 .2). All of the 121 differentially expressed spots (in FM compared to HC) identified in 2D-DIGE analysis were excised and subjected to MALDI-TOF/TOF MS analysis. We obtained reliable MS IDs for 63 protein spots out of 121 (Table S2. 2); while remaining 58 spots remained unidentified and produced virtually empty spectra, most likely owing to the presence of extremely diminutive quantity of proteins as indicated by the retrospective scrutiny of the spot volumes. The 63 protein spots identified by MS represented 30 (14 up-regulated and 16 down-regulated) differentially expressed proteins in FM patients ( Table 2 ; Figure 1C and S3). Proteins identified in 2DE experiment were also obtained in 2D-DIGE; additionally, new candidates were also identified by 2D-DIGE due to the higher sensitivity and reproducibility. 3D views and graphical representation of selected protein spots are shown in Figure 1D .
A comprehensive comparative analysis of host responses in FM with that of VM (from the findings of our previous study on VM [15] ) was carried out to categorize the common and unique proteomic alterations in human serum in Pf and Pv infections. Almost half (46.2%) of the total identified proteins were commonly modulated in both plasmodial infections; however, the magnitude of proteomic alteration was different in these two types of malaria (Figure 2A and D). Compared to healthy controls, quite a few serum proteins such as calcium binding protein 39, calpain 10, regulator of G-protein signaling 7, serum paraoxonase/arylesterase and transthyretin precursor were found to be differentially expressed in FM but not VM. In contrary, some proteins like ceruloplasmin, vitamin D-binding protein, serum amyloid P, alpha-2-macroglobulin, fibrinogen beta chain precursor exhibited altered expressions only in VM patients (Table S3 ). Among the 19 proteins, which were differentially expressed (compared to HC) in both of the malaria, only alpha-2-HS-glycoprotein and serotransferrin precursor (transferrin) exhibited opposite trends in Pf and Pv infections. Rest of the 17 proteins exhibited similar trend of differential expression in FM and VM; although, fold-change values were different ( Figure 2D ).
Compared to VM, 84 protein spots were found to be differentially expressed in FM (t-test; p,0.05). After FDR (Benjamini-Hochberg) and Bonferroni correction 10 and 3 protein spots (out of 84) remained significant, respectively (Table  S1 .3). Out of 84, MS IDs for 43 protein spots were obtained in MALDI-TOF/TOF MS analysis, which indicated differential expressions of 13 proteins in FM compared to VM. Among those 13 differentially expressed proteins, 5 proteins (interleukin-17E precursor, serum amyloid A, ficolin 3 precursor, alpha-1antitrypsin and Ig kappa chain C region) were up-regulated while the remaining 8 proteins (alpha-2-HS-glycoprotein, apolipoprotein E, serotransferrin precursor, alpha-1-antichymotrypsin, leucinerich alpha-2-glycoprotein, AMBP protein, vitamin D-binding protein and haptoglobin) exhibited reduced expression level in Pf infected patients (Table S3 .4).
Principal component analysis (PCA) using the extended data analysis (EDA) module of the DeCyder software v7 revealed distinct clustering of the three experimental groups (FM, VM and HC) ( Figure 2B ). Proteins present in at least 80% of the spot maps, which passed the filter of one-way ANOVA (p,0.01) test were included in this multivariate analysis. Additionally, a hierarchical cluster analysis was performed using the same protein selection Figure 2C ). 85 protein spots were found to be significantly differentially expressed in the malaria subjects compared to the healthy individuals.
Further comparative analysis was performed keeping leptospiral infection as a febrile control to appraise the specificity of the identified malaria related serum markers. Although, some of the identified proteins exhibited similar trends of differential expressions in malaria and febrile controls, interestingly, expression levels of quite a few candidates including serum amyloid A, haptoglobin precursor, ficolin 3 precursor, hemopexin precursor, interleukin-17E precursor, retinol-binding protein, serotransferrin precursor, and vitronectin precursor were found to be altered in malaria patients (both FM and VM) but not in leptospiral infection (Table  S4 ). Altered expression levels of identified serum proteins in falciparum and vivax malaria and leptospirosis (FC) has been illustrated bar-diagrammatically in Figure S4 .
Validation of a few differentially expressed proteins was performed using different immunoassay-based methods including immunoturbidimetric assay, ELISA and western blotting to confirm the results of proteomic analysis. Haptoglobin and apolipoprotein A-I (Apo A-I) concentrations were directly quantified turbidimetrically in the serum samples of malaria patients (n = 37), healthy subjects (n = 20) and febrile controls (n = 6). Compared to the healthy controls, both FM and VM patients found to have lower serum level of haptoglobin and Apo A-I (p,0.0001 in a Mann-Whitney test) ( Figure 3A and B). The mean haptoglobin concentration was found to be 0.20860.048 and 0.33360.06 g/L in FM and VM patients respectively, while the healthy and leptospirosis (FC) populations exhibited a mean values of 0.91860.1 g/L and 0.88860.056 g/L (mean 6 SE). Likewise, Apo A-I exhibited more than three times lower mean value in both the malaria patients compared to the healthy subjects (39.3965.43, 43.1964 .96 and 137.0565.33 mg/dL in FM, VM and HC respectively). While the febrile controls shown a mean value of 76.5264.12 mg/dL, which is around 2-fold higher than the malaria patients. Serum retinol-binding protein (RBP) concentration was measured by sandwich ELISA. The serum levels of RBP was found to be significantly (p,0.01) lower in malaria patients ( Figure 3C ). Western blot analyses of four differentially expressed targets proteins; haptoglobin, serum amyloid A, clusterin and retinol-binding protein were performed on a subset of control [HC (n = 12) and FC (n = 6)] and diseased samples [FM and VM (n = 12 each)] ( Figure 3D ). CBB staining of the SDS-PAGE gels and Ponceau staining of the transferred blots containing the resolved proteins indicated equal loading of the samples in each lane ( Figure S5 ). Western blot analysis showed up-regulation of serum amyloid A and downregulation of haptoglobin, clusterin and retinol-binding protein in FM and VM patients (p,0.01) compared to the healthy and febrile controls. These results confirmed our findings from the proteomic analysis, and are illustrated graphically in Figure 3E .
Thirty differentially expressed serum proteins identified in FM patients (compared to HC) were subjected to functional pathway analysis using Ingenuity Pathway Analysis (IPA). Out of those 30 candidates, 27 were eligible for network analysis (focus molecule) based on the IPA Knowledge Base criteria. Two overlapping interaction networks were identified where the highest scoring network included 14 out of the 27 focus molecules, while the second network included 10 focus molecules ( Figure S6 ; Table  S5 .1). The most significant related functions derived from these overlapping networks included, lipid metabolism (14 proteins, p = 2.92E 209 -5.48E 203 ), inflammatory response (18 proteins, p = 1.07E 211 -5.48E 203 ), molecular transport (15 proteins, p = 2.92E 209 -5.48E 203 ), immune cell trafficking (10 proteins, p = 9.32E 208 -4.12E 203 ) and humoral immune response (7 proteins, p = 1.10E 205 -5.48E 203 ). According to this functional pathway analysis, Pf infection leads to the alteration of multiple serum proteins involved in diverse essential physiological pathways, including acute phase response (Ratio = 0.067, p = 1.11E 218 ) and primary immunodeficiency signaling (ratio = 0.048, p = 5.1E 205 ) (Table S5. 2). Functional analysis of differentially expressed proteins was also performed using Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) databases. In PANTHER analysis blood coagulation system (p = 4.88E 205 ) was again identified. Moreover, the heterotrimeric G-protein signaling, interleukin signaling pathway and inflammation mediated by chemokine and cytokine signaling pathways were identified as related physiological pathways with statistical significance (p,0.05) (Table S5. 3). Further, DAVID analysis also confirmed modulation of complement and coagulation cascades (p = 1.28E 204 ) in FM (Table S5. 
According to the molecular function analysis by GeneSpring, most of the differentially expressed proteins identified in FM are related to binding (59.5%) and enzyme regulation activity (24%). A small fraction is involved in transport (9.5%) and antioxidant activity (7%) ( Figure S7A ; Table S6 ). Majority of the proteins reside in the extracellular region (61%), while some are located in cell (15%), organelle (11%), macromolecular complex (9%), and lumen (4%) as depicted in Figure S7B by cellular component analysis (Table S6 ). Biological processes analysis by GeneSpring indicated that identified proteins are involved in following biological process: response to stimulus (20%), biological regulation (20%), localization (14.5%), cellular process (11.5%), metabolic process (9%), immune system (9%), multi-cellular organismal process (8.5%), biogenesis (3%), signaling and development process (,4%) ( Figure S7C ; Table S6 ).
Further comparative analysis with VM [15] indicates that both Pf and Pv infection lead to alteration of multiple serum proteins involved in diverse essential physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades, lipid transport and metabolism, and blood coagulation ( Figure 4 ). Table S7 and Figure S8 summarize different biological pathways and physiological functions associated with the differentially expressed serum proteins identified in FM and VM using multiple analytical tools. serum proteome of HC and FM patients. FM and HC samples were labeled with Cy3 and Cy5 respectively, while the protein reference pool (internal standard) was labeled with Cy2. (D) Graphical and 3D fluorescence intensity representations of few selected statistically significant (p,0.05) differentially expressed proteins in FM patients identified in biological variation analysis (BVA) using DeCyder 2D software. Graphs showing the decrease/increase in the standardized log abundance of spot intensity in FM compared to the HC cohort of the study (n = 8 Initially, the fidelity of a potential biomarker subset containing 5 proteins identified in 2DE (Table S8 .1A) was evaluated for discrimination of FM and HC. As shown in Figure S9A , the two groups (FM and HC) could be clearly classified by phenotype, thereby providing an additional, unbiased estimate of class prediction. Secondly, we applied the class prediction model based on initial cohort (n = 10) to independently predict (assign) the phenotypic class to either FM or HC group on an independent blind group of 16 subjects (8 newly recruited FM patients and 8 HC). The model provided accurate phenotypic classification; and 100% of the FM (n = 19) and 94.74% of the HC (n = 19) subjects were accurately classified into correct phenotypes (Table  S8 .1C). We achieved 97.37% overall prediction accuracy on independent prediction [HC (n = 19) and FM (n = 19)] using partial least squares discriminant analysis (PLS-DA). For the final validation phase, we compared the performance of the biomarker subset using three well-known machine-learning methods: Decision Trees, Naïve Bayes and support vector machines (SVM). Table S8 .1B summarizes the percentage of samples classified during model training, cross-validation and independent prediction respectively, using the three different classifiers. We achieved, 97.37% overall prediction accuracy with SVM, Decision Trees and Naïve Bayes as well, on blinded prediction using the biomarker dataset for FM and HC (n = 19 each).
Further, 7 differentially expressed proteins (Table S8 .2A) identified in 2D-DIGE were implicated as potential classifiers for the discrimination of FM and VM patients and healthy subjects employing similar type of analysis ( Figure S9B ). We achieved 95.83% overall prediction accuracy on blinded prediction (n = 23) using PLS-DA. Table S8 .2B summarizes the percentage of the samples classified during model training, cross-validation and independent prediction respectively. In the final validation phase; we achieved 100 and 95.83% overall prediction accuracy with Decision Trees and SVM respectively, followed by Naïve Bayes (91.66%) on blinded prediction using the biomarker dataset for FM (n = 8), VM (n = 7) and HC (n = 8).
Next round of multivariate statistical analysis was performed to evaluate the efficiency of the identified serum proteins to discriminate the FM, VM and FC (patients suffering from leptospiral infection) ( Figure 5A ). 6 differentially expressed proteins (Table S8 .3A) identified in 2D-DIGE were implicated as potential classifiers. Table S8 .3B summarizes the percentage of the samples correctly classified during model training, cross-validation and independent prediction respectively using different classifiers. We achieved, 100% overall prediction accuracy with Decision Trees; 95.83% with SVM and Naïve Bayes and 91.67% with PLS-DA on independent prediction [FM (n = 8), VM (n = 8) and Lep (n = 6)]. Table S8 .1C, S8.2C and S8.3C, provides additional details on the confidence measure obtained on blinded prediction for each subject using a given statistical method. The confidence measure defines the strength of the prediction belonging to the particular class.
Receiver operating characteristic (ROC) curve analysis was carried out to evaluate the individual performance of 3 classifier proteins; apolipoprotein A-I, haptoglobin and retinol-binding protein in malaria prediction. These 3 serum proteins were used as potential classifiers (along with other 3 candidates) to build statistical sample class prediction models employing PLS-DA and For proteins with multiple spots in the 2D gels, representative spot detail is provided. Exact values for each spot are provided in (Table S2) . other classification methods for FM, VM, FC and HC discrimination. The area under the ROC curve (AUC) indicates the accuracy of different classifier proteins to distinguish FM, VM and leptospirosis from HC ( Figure 5 ). ROC curves demonstrate apolipoprotein A-I (AUC = 0.957) and haptoglobin (AUC = 0.936) as efficient predictor proteins for falciparum malaria detection. A cut off value .112.1 mg/dL for Apo A-I revealed the specificity and sensitivity of 90% and 95%, respectively; while haptoglobin at a cut off value .0.465 g/L provided 95% specificity and 90% sensitivity in predicting Pf infection. Retinolbinding protein (AUC = 0.879) exhibited moderate sensitivity (66.6%) and specificity (80%) for FM at a cut off value .30.88 mg/mL ( Figure S9C ; Table S9 ). Precondition efficiency of the classifier proteins for vivax malaria was also evaluated and found to be highly appreciable for Apo A-I (AUC = 0.979; 94.12% sensitivity and 95% specificity at a threshold value .96.59 mg/ dL), haptoglobin (AUC = 0.888; 76.47% sensitivity and 95% specificity at a threshold value .0.45 g/L) and retinol-binding protein (AUC = 0.875; 76% sensitivity and 90% specificity at a threshold value .28.61 mg/mL) as well ( Figure S9D ; Table S9 ).
Accuracy of classifier serum proteins in prediction of leptospirosis (FC) was also tested ( Figure 5 ). Although Apo A-I (AUC = 0.783; 66.6% sensitivity and 90% specificity at a threshold value .111.1 mg/dL) exhibited fine proficiency in detection of leptospiral infection; performance of haptoglobin (AUC = 0.508; 66.6% sensitivity and 50% specificity at a threshold value .0.845 g/L) and retinol-binding protein (AUC = 0.558; 50% sensitivity and 65% specificity at a threshold value .34.99 mg/ mL) were poor (Table S9 ). ROC analysis revealed that the serum levels of the classifier proteins, particularly Apo A-I and haptoglobin exhibited good correlation with plasmodial infections and could further be investigated as potential surrogate protein markers for malaria.
Among the four different species of Plasmodia, which cause malaria in human, Pf and Pv account for over 90% of the total malaria cases worldwide. In this study, we used proteomics to decipher the alteration in human serum proteome due to the Pf infection to gain insight into the disease pathogenesis and host immune response. We also performed a comparative analysis of host response in Pf and Pv infection. The comparative proteomic analysis of plasmodial and leptospiral infection (febrile control) was performed to appraise the generic febrile responses and specify the malaria related serum markers. The ultimate aspiration of this study was to identify potential marker proteins that can distinguish the malaria patients from the healthy or febrile controls as well as discriminate between the Pf and Pv infections with high accuracy. Although a number of earlier proteomic and immunoassay-based studies have demonstrated the alteration of limited set of serum proteins in malaria [16] [17] [18] hitherto, there was no attempt for a comprehensive analysis to establish a panel of classifier proteins that can readily discriminate the FM and VM groups from the controls.
Our results indicate that various vital physiological pathways, including acute phase response signaling, cytokine and chemokine signaling, complement cascades, lipid transport and metabolism, and blood coagulation are modulated in Pf and Pv infections (Figure 4) . Alteration of the levels of several acute phase proteins (APPs) and multiple members of serum complement cascade as well as complement regulatory proteins (Table 2) due to the plasmodial infections is consistent with earlier findings [19] [20] [21] . Increased expression levels of circulating acute-phase amyloid proteins like serum amyloid A and P provide non-specific resistance against the pre-erythrocytic stages of Plasmodium, limit tissue damage and promote a rapid return to homeostasis [22, 23] . Interestingly, human serum paraoxonase (PON1) an HDLassociated esterase which protects lipoproteins against oxidation, found to be down-regulated in falciparum malaria patients. Acute inflammatory stimuli lead to reciprocal regulation of SAA and PON-1 [24] . Decreased serum PON-1 activity in context with falciparum malaria may in part be attributable to higher SAA level.
In course of the disease progression, malarial parasites growing in the erythrocytes degrade hemoglobin and generate reactive oxygen species (ROS), which lead to increased oxidative stress within the erythrocytes and outside the parasitized cells. As a result, to circumvent the situation, enhanced plasma levels of antioxidant defense associated enzymes/proteins such as superoxide dismutase-1 (SOD-1) are observed in acute malaria patients [25] . In both FM and VM patients we have identified elevated serum level of hemopexin, a heme-binding protein, which provides the second line of defense against hemoglobin-mediated oxidative damage during intravascular hemolysis [26] . Increased production of this acute phase protein by the host defense system could be helpful to circumvent the pro-inflammatory response with oxidative stress generated in patients with Pf or Pv infection. In contrast, haptoglobin (Hp) found to be significantly downregulated in FM and VM patients. Hp removes free hemoglobin (Hb) released during parasite induced hemolysis, and disappears as the Hp-Hb complexes leading to the malaria associated hypo-or ahaptoglobinemia and is a promising inflammatory marker to evaluate the severity of the Plasmodium infection [21] . Earlier reports have demonstrated the possible role of this APP as an epidemiological marker for malaria [21, 27] .
Erythrocyte invasion is an essential gateway to malaria disease and a key target for disease intervention. Signaling via the erythrocyte beta 2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Gas) regulates the entry of the human malaria parasite P. falciparum. Disruption of the interaction between the G-alpha-s subunit of the Gs protein and the receptor results in a reduced erythrocyte invasion by the parasite and subsequent low level of parasitemia [28] . Down-regulation in regulator of G-protein signaling in FM patients might be due to some host response to combat this parasitic infection. Conversely, up-regulation of apolipoprotein E was observed in the malaria patients. This apolipoprotein also inhibits Plasmodium invasion, since it shares the cell entry mediators (heparan sulphate proteoglycans and/or low density lipoprotein receptor) with the parasite [29] . The pathway analyses and densely connected networks based on our results provide an insight into the underlying molecular mechanisms of malaria.
Early and accurate diagnosis is critical for the effective treatment and management of malaria. In recent years, multivariate projection methods are being successfully applied to analyze biological data obtained through genomic, transcriptomic or proteomic approaches to study various human diseases, with implications for diagnostics and clinical management [30, 31] . A sub-set of the proteins identified in our proteomic analysis was used to build statistical sample class prediction models to identify the classifier marker proteins for FM, VM and HC discrimination. Interestingly, two key classifier proteins: serum amyloid A and haptoglobin differentially expressed consistently in all of the malaria patients (FM and VM) compared to the control subjects (HC and FC) and remained statistically significant after FDR (Benjamini-Hochberg) and Bonferroni correction of the p-values obtained in t-test; indicating very strong correlation between the expression levels of these two serum proteins and plasmodial infections. The recognition ability of the prediction models for FM, VM and HC discrimination and cross-validation was almost 100% (Table S8) . We controlled for the statistical false discovery rate using three distinct, iterative validation steps: (i) k-fold crossvalidation algorithm for the original cohort, (ii) application of the marker subset identified in the original cohort to classify newly recruited patients, and (iii) performance of the marker subset validated using three well-known machine learning methods. In our study, biological replicates were investigated, i.e. each proteome profile was representative of a different human subject, and hence the data-sets are characterized by low homogeneity, conferring to the protocol a very high level of variability and complexity. Indeed the extreme heterogeneity or large biological variations including gender, age, genetic factors, dietary considerations, environmental factors and drug treatment affects the detection, validation and establishment of ''gold standard'' serological biomarkers [10, 32] . Nonetheless, the accurate discrimination among the FM, VM and control groups obtained by various prediction models on the basis of differentially expressed candidate proteins testifies to the excellent potential of this analytical approach for the detection and discrimination of VM and FM ( Figure 5 ; Figure S9 ). It should also be noted that uncomplicated FM and VM patients with diverse range of parasitemia; mainly low and moderate parasitemic (,5000 parasites/mL blood), were used for the validation of the prediction models (Table 1) . Even so, the discrimination accuracy of the study is very appreciable indicating the capability of our analytical approach for the detection of very low-level of parasitemia, which is highly promising from a diagnostic point of view. Although, diagnosis of malaria on the basis of microscopic examination of thin or thick smears of peripheral blood is the most commonly used and well-accepted method, but it requires highly trained personnel for smear interpretation, frequently fails to distinguish mixed-species infections or diagnose patients with ''sub-microscopic'' parasitemia below the detectable limit of blood smears, and in many areas of endemicity the operating characteristics of microscopy are poor [33, 34] . In quest of an early and accurate diagnosis of malaria and discrimination of Pf, Pv or mixed infection, establishment of serum protein markers can be an attractive approach apart from clinical symptoms and conventional microscopic examination of blood smears. To this end, some of the classifier candidate proteins identified in this study; such as serum amyloid A, paraoxonase, apolipoprotein A-I and E, haptoglobin, hemopexin, and complement C4 are very important due to their functional relevance in malaria pathogenesis and could further be investigated as potential surrogate protein markers for clinical implications. Various rapid diagnostic tests (RDTs) are in practice for malaria diagnosis, which diagnose the infection on the basis of detection of parasite proteins/antigens e.g. histidine-rich protein II (HRP-II) or lactate dehydrogenase (LDH) [35] , whereas for the first time we have demonstrated the discrimination between FM and VM patients based on protein expression in human host. Malaria RDTs are used regularly in clinics due to the low cost, sensitivity and less detection time. However, analysis of frozen specimens of blood from parasitaemic patients using existing RDTs is bit challenging. Another limiting factor is the shelf-life of RDTs, since most of the existing RDTs deteriorate rapidly on exposure to moisture (humidity) and high temperature. Moreover, significant variations may appear between technicians in both RDT preparation and result interpretation process depending on experience of the performer, manual proficiency and visual perception [36] . To this end, serum protein markers can be potential candidates for development of an alternative sensitive diagnostic approach for malaria. Development of highly sensitive biosensors for the identified surrogate proteins might be attractive from a diagnostic point of view.
In summary, the present study demonstrates the application of diagnostic proteomics to decipher host responses against the human malaria parasites Pf and Pv, and identifies potential candidate biomarkers for these two plasmodial infections. In this comprehensive proteomic analysis we have identified multiple differentially expressed serum proteins with versatile biological functions, indicating the modulation of multiple vital physiological pathways in FM and VM patients. We anticipate that information obtained from this study will provide valuable insight into the underlying molecular mechanisms of malaria and may help to establish early detection surrogates for these infectious parasitic diseases to meet the need for better diagnostics and effective therapy. Some of our identified classifier proteins such as serum amyloid A, apolipoprotein A-I and E and haptoglobin, which successfully discriminated FM from VM might be prognostic host markers for disease severity. To this end, it would be interesting to elucidate the fate of the identified serum proteins in severe malaria patients and could be a future continuation of this study. Diagnostic impact of the identified serum biomarkers in clinics and specificity for malaria prediction can only be established after investigation of the disease patterns in large clinical cohorts.
This proteomic analysis was performed with the approval of the institutional ethics committee of Seth GS Medical College and King Edward Memorial hospital, Parel, Mumbai, India. Patients suffering from uncomplicated Pf or Pv infection with asexual parasite count more than 1000 per mL of blood were selected for this study. A total of 37 patients, with uncomplicated FM (n = 20) or VM (n = 17) confirmed through microscopic examination of a thin peripheral blood smear followed by RDT were enrolled for this proteomic study. In addition, blood specimens were collected from age and sex matched leptospirosis patients (n = 6) as febrile controls, and healthy subjects (n = 20) to perform comparative proteomic analysis. Written informed consent was taken from each participant (malaria patients and controls) prior to the sample collection process. Demographic, epidemiological and clinical details of all malaria patients and febrile controls (FC) selected for this proteomic study are provided in Table 1 . Blood samples (5.0 mL) were collected from the antecubital vein of the subjects using serum separation tubes (BD VacutainerH; BD Biosciences). Immediately after blood collection the tubes were kept in ice for 30 mins for clotting. Serum separation was performed as described previously [15] . In brief, after clotting, the samples were centrifuged at 2500 rpm at 20uC for 10 mins and serum was collected carefully from the upper surface. Collected serum was divided into multiple aliquots and stored at 280uC until time of analysis to prevent protein degradation. Prior to proteomic analysis, maximum 2-3 freeze/thaw cycles were allowed for any serum sample to reduce pre-analytical variations.
Crude serum was diluted five times with phosphate buffer (pH 7.4) and subjected to mild sonication in a Vibra cell sonicator using the following settings: 6 cycles of 5 sec pulse; 30 sec gap in between; at 20% amplitude. After sonication, the top two highabundance serum proteins (albumin and IgG) were removed using Albumin & IgG Depletion SpinTrap (GE Healthcare) following the manufacturer's instructions. Extraction of protein from depleted serum samples was performed employing TCA/acetone precipitation method as described by Chen et al., with slight modifications [37] . In brief, depleted serum samples were diluted (1:4 ratio) with ice-cold acetone containing 10% (w/v) TCA. Uniform mixing was performed using mild vortexing for 15 sec and the mixture was allowed to incubate at 220uC for 2 hrs for protein precipitation. After completion of the incubation period, tubes were centrifuged at 1000 g for 15 min at 4uC. Supernatants were separated and kept in fresh microcentrifuge tubes, and the pellets were dissolved in rehydration buffer [8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer (pH 4-7; Linear), 40 mM DTT and traces of bromophenol blue]. In order to precipitate the remaining amount of proteins present in the collected 10% TCA/acetone-containing supernatants, 1 mL icecold acetone was added to each tube and the samples were subjected one additional round of precipitation and extraction process. In all cases, prior to re-suspension in rehydration buffer, the pellet was briefly air-dried. Prior to proteomic analyses, protein concentration in the samples was quantified using the 2D-Quant kit (GE Healthcare) following the manufacturer's instructions. A total of 600 mg of depleted serum protein extract dissolved in 350 mL of rehydration buffer was loaded on 4-7 pH range IPG strips (18 cm) and underwent passive rehydration for 14-16 hrs. Isoelectric focusing (IEF) was performed on an Ettan IPGphore 3 isoelectric focusing unit (GE Healthcare) for overall approximately 78 kVh using the following voltage settings: 200 V for 4 h (step and hold), 500 V for 1 h (step and hold), 1000 V for 1 h (step and hold), 8000 V for 3 h (gradient), and 8000 V for 7:30 h (step and hold). After completion of IEF, the focused IPG strips were stored at 220uC until the second dimensional analysis was performed. Preceding to the second dimensional separation, each strip was equilibrated to reduce and alkylate the proteins (for 15 min each) using equilibration buffer containing 6 M Urea, 75 mM Tris-HCl pH 8.8, 29.3% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue. Just prior to use, 1% (w/v) DTT or 2.5% (w/v) IAA was added in the first (reducing) and second (alkylating) equilibration buffer, respectively. The second dimension was performed on 12.5% SDS polyacrylamide gels using an Ettan DALTsix electrophoresis unit (GE Healthcare). After electrophoresis GelCode Blue Safe Protein Stain (Thermo Scientific, USA) was utilized for visualization of the protein spots. Proteins extracted from each of the subjects were run in duplicate to verify the reproducibility and curtail technical artifacts.
Each CyDye (Cy3, Cy5 and Cy2) was resuspended in anhydrous N, N-dimethylformamide (DMF) to prepare a stock dye concentration of 1 mM. A working solution of 400 pmol of each CyDye was made by further dilution of the stock with DMF. Samples (test and control) were labeled with Cy3 and Cy5, while a mixture of equal amounts of all samples to be analyzed in the experiment, regarded as internal standard, was labeled with the third fluorescent dye; Cy2 according to the manufacturer's instructions (GE Healthcare). In brief, the pH of each sample was adjusted to 8.5 using 100 mM NaOH. 50 mg of each protein sample [malaria, controls (HC/FC) and internal standard] were separately labeled with 400 pmol of CyDyes. After addition of CyDyes, samples were incubated on ice for 30 in the dark. Labeling reaction was stopped by addition of 10 mM lysine followed by incubation on ice for additional 10 min. Dyeswapping was performed while labeling the test and control samples for eliminating any type of dye effects. After labeling, samples labeled with Cy3, Cy5 and Cy2 were mixed, diluted with the rehydration buffer and loaded on 18 cm, 4-7 pH IPG strips. Subsequent IEF and SDS-PAGE separation were performed following the same protocol as previously described in the 2DE section.
Image acquisition and data analysis was performed as described previously [15] . In brief, after staining, the 2D gels were scanned by using LabScan software version 6.0 (GE Healthcare) and analyzed by using ImageMaster 2D Platinum 7.0 software (GE Healthcare). Comparative analysis of FM samples was performed by creating different ''match sets'' and using the HC samples as reference. Spot detection parameters were specified as: Smooth: 7, Saliency: 100 and Min Area: 5. After automatic detection of the spots through IMP7, manual refinement was performed to eliminate any contaminating artifacts, such as streaks or dust particles. Spot quantification was performed in % vol value using ImageMaster algorithm. It provided normalized value that remains relatively independent of variations due to staining or protein loading. The gel analysis tables, histograms and 3D images generated by the software were used for further analysis.
2D-DIGE gels were scanned using Typhoon 9400 variable mode imager (GE Healthcare) at a 100 mm resolution employing suitable excitation/emission wavelengths for each of the CyDye [Cy3 (523/580 nm), Cy5 (633/670 nm) and Cy2 (488/520 nm)]. After scanning, gel images were cropped properly using Im-ageQuant software; version 5.0 (GE Healthcare) prior to importing in DeCyder 2D software; version 7.0 (GE Healthcare) for comparative analysis and relative protein quantification across the FM and control samples. Comparative analysis was performed using two different modules, differential in-gel analysis (DIA) and biological variation analysis (BVA) of the DeCyder software. Preliminary analysis was performed using DIA module to detect spots on a cumulative image derived from merging up to three individual images from an in-gel linked image set (malaria, controls and internal standard). It permits the pair-wise comparisons of each normal and malaria samples to the mixed standard present in each gel and offers spot-wise protein abundance as ratios. Further analysis was performed using BVA module to get the variation in protein expression levels between any of the two experimental groups (FM vs. VM, FM vs. HC and VM vs HC) across all the sets. Statistical significance of the average ratio of expressions was analyzed by Student's t-test. Protein spots exhibiting differential expression with reproducibly and statistical significance (p,0.05) were considered for further analysis. Bonferroni correction (for reducing Type I errors) of the p-values obtained from Student's t-test was performed using standard Bonferroni procedure to recognize those marker proteins which have very strong connection with the diseased state (remains significant after Bonferroni correction). Since Bonferroni correction is extremely conservative; comparatively less stringent false discovery rate (FDR) correction was also performed as detailed in Benjamini and Hochberg [38] .
We also performed a comparative analysis of FM data-set obtained in this study with our previously published VM data [15] . Clustering of the three experimental groups (FM, VM and HC) was performed by principal component analysis (PCA) using an algorithm included in the extended data analysis (EDA) module of the DeCyder software. Proteins present in at least 80% of the spot maps and passed the filter of the one-way ANOVA (p,0.01) test were included in this multivariate analysis. Additionally, a hierarchical cluster analysis was performed using the same protein selection criteria.
Statistically significant (t-test, p,0.05) differentially expressed proteins spots identified in regular 2DE and 2D-DIGE experiments were selected for further MS analysis to establish protein identity. GelCode Blue stained preparative gels containing much higher amount of protein (1 mg) were used for excision of the spots of interest specified in the 2D-DIGE experiment. Spot excision was performed manually. In-gel digestion of the proteins separated by 2D gel electrophoresis was performed as described by Shevchenko et al., with slight modifications [39] . In short, gel slices were cut into small cubes (,161 mm) and washed with 50 mL of stain removal solution (25 mM ammonium bicarbonate buffer) for removal of CBB stain. After washing, 50 mL of 25 mM ammonium bicarbonate/acetonitrile (1:1 v/v) was added, followed by 5 min incubation with occasional vortexing at room temperature. After incubation, the solutions were removed. These two steps are repeated for three times. Then, 50 mL reduction solution (10 mM DTT in 100 mM ammonium bicarbonate) was added and the gel pieces were incubated for 60 mins at 56uC in an air thermostat. Tubes were allowed to cool to room temperature after incubation, and 50 mL of 25 mM ammonium bicarbonate buffer was added to wash the gel pieces followed by dehydration with 25 mM ammonium bicarbonate/acetonitrile (1:1, v/v). After this step, alkylation solution (50 mM IAA in 100 mM ammonium bicarbonate) was added and the tubes containing the gel pieces were incubated for 30 mins at room temperature in dark. Rehydration and dehydration steps were performed twice and gel pieces were allowed to dry. Once the gel slices were properly dried, trypsin solution (Trypsin Gold; Promega, Madison, Wisconsin, United States) was added to the gel pieces keeping the ratio of trypsin: protein around 1:10 (w/w) and incubated at ice for 30 mins for absorption of the solution. After this step, the tubes were incubated overnight at 37uC. Adequate amount of ammonium bicarbonate buffer was added to cover the gel pieces. Extraction of the digested peptides from the gel matrix was performed using 100 mL of extraction buffer (0.2% formic acid in 66% acetonitrile) after completion of the enzymatic reaction. Extraction step was repeated thrice to ensure maximum recovery of the digested peptides. The collected supernatants were pooled in a single tube and concentrated using speed vac. After extraction trypsin digested samples were further processed using Zip-Tip C18 pipette tips (Millipore, USA) according to the manufacturer's protocol for removal of salts and enrichment of the peptides.
Subsequent to enrichment and purification through the Zip-Tip pipette tips, peptide mixtures were dissolved in 0.5 mL of CHCA matrix solution (5 mg/mL CHCA in 50% ACN/0.1% TFA) and spotted onto a freshly cleaned MALDI target plate. Spots were allowed to dry for 30 mins at room temperature. After air drying, the crystallized spots were analysed using a 4800 MALDI-TOF/ TOF mass spectrometer (AB Sciex, Framingham, MA) linked to 4000 series explorer software (version 3.5.3). All mass spectra were recorded in a reflector mode within a mass range from 800 to 4000 Da, using a Nd:YAG 355 nm laser. The acceleration voltage and extraction voltage were kept at 20 kV and 18 kV respectively. Six point calibration of the instrument was automatically performed by a peptide standard Kit (AB Sciex) that included des-Arg1-bradykinin (m/z 904.468), Angiotensin I (m/z 1296.685), Glu1-fibrinopeptide B (m/z 1570.677), ACTH (18-39, m/z 2465.199), ACTH (1-17, m/z 2903.087), and ACTH (7-38, m/z 3657.923). All the MS spectra were obtained from accumulation of 900 shots. MS/MS spectra were acquired for the 15 most abundant precursor ions, with a total accumulation of 1500 laser shots and collision energy of 1 kV. Once the MS survey scans were completed, the data were processed to generate a list of precursor ions for interrogation by MS/MS. The combined MS and MS/ MS peak lists were searched using the GPS TM Explorer software version 3.6 (AB Sciex). Protein identification was performed by MS/MS ion search using MASCOT version 2.1 (http://www. martixscience.com) search engine against the Swiss-Prot database. Searches were carried out with the following parameters; all entries taxonomy, trypsin digestion with one missed cleavage, fixed modifications: carbamidomethylation of cysteine residues, variable modifications: oxidation of methionine residues, mass tolerance 150 ppm for MS and 0.4 Da for MS/MS. Identified proteins having at least two unique matched peptides were selected for further analysis. We have reported only those proteins with a protein identification confidence interval of $95%.
Quantitative immunological measurement of two of the differentially expressed proteins identified in this study; haptoglobin and apolipoprotein A-I, in serum samples of healthy controls (n = 20), falciparum malaria (n = 20) and leptospirosis patients (n = 6) were performed using COBAS INTEGRA 400 PLUS system (ROCHE). The serum concentration of those two target proteins in vivax malaria was taken in account for a comparative analysis from our previous report [15] . Crude individual serum samples were subjected directly to immunoturbidimetric quantification using the Tina-quant ver.2 kits (Roche Diagnostics) according to the manufacturer's instructions. Samples and controls were automatically prediluted 1:21 with NaCl solution by the instrument. In this immunological assay the target proteins form precipitates with the specific antiserum which are determined turbidimetrically at 340 nm. Anti-human haptoglobin (rabbit) and Apo A-I (sheep) antibodies were applied for the immunoturbidimetric quantification of haptoglobin and Apo A-I respectively. The instrument was monitored at absorbance measuring mode where the absorbance increase was directly proportional to the concentration of the target proteins.
Quantification of another interesting target; retinol-binding protein (RBP) was performed using ELISA. Concentrations of RBP4 in serum samples of HC (n = 20), FC (n = 6), FM (n = 12) and VM (n = 12) patients were measured using AssayMax Human Retinol-Binding Protein-4 (RBP4) ELISA kit (Cat# ER3005-1) from AssayPro (USA) following the manufacturer's instructions. Briefly, quantitative sandwich enzyme assay was employed where RBP4 standard and serum samples (HC, FC, FM and VM) at a dilution of 1:100 were subjected to a microplate pre-coated with a polyclonal antibody specific for RBP4. Samples were sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for RBP4, which was recognized by a streptavidinperoxidase complex. Color development was performed through the addition of a peroxidase enzyme substrate and optical densities were measured at 450 nm and 570 nm using a SpectraMax M2 e (Molecular Devices, USA).
Prior to the western blotting experiment protein concentration in each sample [malaria patients (n = 24), FC (n = 6) and HC (n = 12)] was accurately estimated using the 2D-Quant kit (GE Healthcare) and BCA Protein Assay (Thermo Fisher Scientific). Western blot analysis was performed as described previously [40] . Briefly, serum proteins were separated by 12% SDS-PAGE (50 mg per track) and then transferred onto PVDF membranes under semidry conditions by using ECL semi-dry transfer unit (GE Healthcare). Western blot was performed by using monoclonal/ polyclonal antibody against serum amyloid A (Santacruz Biotechnology, sc-20651), haptoglobin (Santacruz Biotechnology, sc-71207), clusterin (Santacruz biotechnology, sc-8354) and retinolbinding protein (RBP) (Santacruz Biotechnology, sc-69795) and appropriate secondary antibody conjugated with HRP (GeNei (MERCK)-621140380011730 or 621140680011730). Candidate proteins for validation were selected on the basis of fold changes, possible association of the proteins with malaria pathobiology and accessibility of the required antibodies. ImageQuant software; version 5.0 (GE Healthcare) was applied for quantitation of signal intensity of the bands in western blots.
Differentially expressed serum proteins in FM were subjected to functional pathway analysis using IPAversion 9.0 (IngenuityH Systems, www.ingenuity.com) to determine association of the identified proteins with various physiological pathways. The significance of association between our dataset and identified networks/pathways was considered on basis of two parameters, ratio and p-values. Differentially expressed proteins in FM patients were also analyzed using PANTHER system; version 7 (http:// www. pantherdb.org) [41] and DAVID database version 6.7 (http://david.abcc.ncifcrf.gov/home.jsp) [42, 43] . The list of Uni-Prot Accession from each dataset was uploaded in tab delimited text format at once, which was mapped against the reference Homo sapiens dataset to extract and summarize functional annotations associated with individual or group of genes and proteins.
The gene ontology (GO) categories for 30 proteins was assigned using GeneSpring software package (version 11.5; Agilent Technologies, Santa Clara, USA). Since, GO vocabulary is organized in a hierarchical fashion, the second level of GO terms were presented as a balance between GO term for specificity and maximal coverage. GO terms that were enriched in two or more proteins were considered. In addition, a significance p-value of the enrichment was computed using the hypergeometric probability distribution, which identifies GO categories represented by the 30 proteins relative to their representation on the Biological Genome for Human created using information available at NCBI (ftp://ftp. ncbi.nlm.nih.gov/gene/DATA).
To determine the biological pathways with significant enrichment of the input proteins, algorithms in GeneSpring software package performs a standard hyper-geometric calculation to obtain p-value, which signifies the enrichment. Prior to analysis, manually curated biological pathways from Reactome, Biocarta, NCI and PathwayCommons (in Biopax level 2 format) were populated in GeneSpring's database. Pathways with significance pvalue (p,0.05) were chosen for subsequent analysis and interpretation. We used GeneSpring's pathway database to create Shortest Connect network from the selected pathways. The Expand Selection algorithm was performed on the above network to include first and second degree neighbors. Expand Selection uses the GeneSpring pathway database for finding expansion on entities and takes a series of expansions to connect processes/ functions/other biomolecules to the given entities (proteins). This algorithm allows listing all processes and functions in which the given entities participate.
We applied proteomics data obtained from 2DE and 2D-DIGE analyses to discriminate among FM, VM, FC and HC groups using multivariate statistical analysis. 5 proteins (haptoglobin, apolipoprotein A-I, hemopexin, apolipoprotein E and serum amyloid A) identified by 2DE (Table S8 .1A) and 7 proteins (haptoglobin, apolipoprotein A-I, hemopexin, apolipoprotein E serum amyloid A, serum amyloid P and serum paraoxonase/ arylesterase 1) identified by 2D-DIGE (Table S8 .2A) were used to develop statistical classifier designed to categorize and predict clinical phenotypes (i.e., FM, VM and HC). Discrimination of malaria (FM and VM) from FC (leptospirosis) and statistical sample class prediction was performed on the basis of differential expression levels of 6 candidate proteins (serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I) (Table S8 .3A). Selection of the candidate proteins was executed on the basis of their level of differential expression and ability to discriminate between the clinical phenotypes. For multivariate statistical analysis and machine learning, the data were mean centered; scaled and logarithmic transformation was performed in order to lower relatively large differences among the respective spot abundances. 2DE data was additionally normalized using Quantile method to correct for batch difference. 3 levels of validation were used to establish the reliability of identified differentially expressed proteins to detect correct phenotypic classes using Mass Profiler Professional (MPP).
We used PLS-DA, SVM, Decision Trees and Naïve Bayes implemented in MPP software package (version 2.2, Agilent Technologies, Santa Clara, USA) for all multivariate and machine learning analysis in this study. Partial least squares is a regression method using the information contained in X data matrix (predictor variables) to predict the behavior of Y data matrix (response variables). PLS method models both X and Y variables simultaneously to find the latent variables in X that will predict the latent variables in Y [44] . The application of PLS as a classification method is indicated as PLS-DA [45, 46] . SVM separates two classes by generating the hyperplane (in a highdimensional feature space) which maximizes the distance from the hyperplane to the closest training examples [47] . In Decision Trees a sample gets classified by following the appropriate path down the decision tree. The Naive Bayesian model is built based on the probability distribution function of the training data along each feature. Since Decision Trees and Naïve Bayes directly handle multi-class problems, we have used the default parameters for these techniques. The SVMs are trained using sequential minimal optimization with a linear kernel. Validation of the obtained predictive models was performed using a standard K-fold cross-validation procedure: observations in input data were randomly divided into three equal parts, two parts were used for model training, and the remaining samples were classified using the constructed model. The whole process was repeated for 10 times.
Efficiency of 3 classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein for prediction of malaria (FM and VM) and leptospirosis (febrile control) was analyzed using receiver operating characteristic (ROC) curves [plot of true positives (sensitivity) vs false positives (1-specificity) for each possible cutoff] using GraphPad Prism software package (version 5.02). ROC curve analysis was performed for only those 3 classifier proteins (out of 6) for which absolute serum concentration values (immunoturbidimetric assay/ELISA) were measured. Sensitivity and specificity values for the marker proteins were calculated at different threshold points. Two-sided p-values less than 0.05 were considered statistically significant. Figure S1 Evaluation of the depletion efficiency for albumin and IgG from human serum. Two major highabundance serum proteins; albumin and IgG were removed using Albumin & IgG Depletion SpinTrap (GE Healthcare) to reduce the dynamic range of serum protein concentration. (A) Levels of albumin and IgG in CBB stained 2D gel before and after depletion. 600 mg of total serum proteins were focused on linear pH 4-7 IPG strips (18 cm) and then separated on 12.5% polyacrylamide gels. Depletion of the top two high-abundance proteins (albumin and IgG) introduced nearly two-fold increase in overall spot number in 2D gels. (B) Levels of albumin and IgG in CBB stained 1D-SDS-PAGE gel before and after depletion showing the efficiency of the depletion process. 10 mg of total crude [C] and depleted [D] serum proteins were loaded onto each lane and separated on 10% polyacrylamide gels. (C) Densitometric analysis of the 1D-SDS-PAGE gels revealed around 85% and 80% depletion of albumin and IgG respectively. (PDF) Figure S2 Trends of differentially expressed proteins in falciparum malaria patients visualized in 2DE gels. (A) Representative 2D gels of serum from healthy controls and FM patients. 600 mg of total serum proteins were focused on linear pH 4-7 IPG strips (18 cm) and then separated on 12.5% polyacrylamide gels, which were stained with Gel Code Blue Stain. Protein spots exhibiting significantly altered expression levels are marked on the gels. Down (B) and up (C) -regulation of protein expression levels in FM patients. The 3D images of statistically significant (p,0.05) differentially expressed spots were analyzed using IMP7 software. Data is represented as mean 6 SEM (where n = 20). (PDF) Figure S6 IPA defined interaction networks associated with the differentially expressed proteins in falciparum malaria. Differentially expressed serum proteins identified in FM patients were entered as focus molecules in the analytical software to generate biological processes, pathways and molecular networks associated with the identified proteins. (A) The top-scoring network (score 35); cell signaling, molecular transport, vitamin and mineral metabolism. This network incorporated 14 out of the 27 differentially expressed proteins (focus molecules), (B) The second net-work; lipid metabolism, molecular transport, small molecule biochemistry (score 23). This network incorporated 10 focus molecules. Green and red symbols represent proteins that were down and up-regulated in falciparum malaria, respectively (identified in this study). White symbols represent associated proteins identified in the functional analysis for which the difference in expression level did not achieve statistical significance in our study. (PDF) Figure S7 Gene Ontology (GO) terms for molecular functions, cellular components and biological processes associated with the differentially expressed serum proteins identified in falciparum malaria. A total of 1394 Gene Ontology (GO) terms were identified, of which the distribution of second level of GO terms that were enriched in two or more proteins is shown as molecular functions (A) and cellular components (B) and biological processes (C). (PDF) Figure S8 Biological process regulated by differentially expressed serum proteins identified in falciparum and vivax malaria patients. Regulations were based on Natural Language Processing performed on MEDLINE abstracts as available in GeneSpring software package (version 11.5, Agilent Technologies). Identified process (A) common in both the plasmodial infections (B) specific for P. falciparum (C) specific for P. vivax infection. Red triangles and blue squares represent positive and negative regulations, respectively. (PDF) Figure S9 Discrimination of falciparum and vivax malaria from healthy controls on the basis of differential expressions of selected serum proteins. PLS-DA scores plot for (A) FM (red spheres, n = 10) and HC (green spheres, n = 10) samples, based on 5 differentially expressed proteins (Table  S8 .1A) identified using 2DE, (B) FM (red spheres, n = 6), VM (blue spheres, n = 5) and HC (green spheres, n = 5) samples based on 7 differentially expressed proteins (Table S8.  Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(−)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models. Human lungs are composed of three functional and morphological compartments: proximal and distal airways and the alveolar compartment. Proximal airways are lined by a pseudostratified epithelium with a number of cell types with important protective functions such as ciliated cells, goblet cells, and basal cells. More distally, the lining is a simplified columnar epithelium largely made up of non-ciliated secretory cells called Clara cells, and a few ciliated and basal cells. [1, 2] . Further down, the respiratory bronchioles are lined by cuboidal epithelium comprised entirely of ciliated and Clara cells, whereas, the epithelium of the alveolar compartment is comprised of type I and type II cells. In mouse, the pseudostratified epithelium is limited to trachea and extrapulmonary main bronchi while Clara cells make up over 80% of the epithelium, with few interspersed ciliated cells, that line intrapulmonary conducting airways [3] . These features make mouse an excellent tool for studying the functions of Clara cells.
Clara cells have several protective properties. They detoxify xenobiotics and oxidant gasses, control inflammation, participate in mucociliary clearance of environmental agents, and proliferate/ differentiate to maintain the ciliated and non-ciliated cell population. Clara cells are a source of cytochrome P450 enzymes that contribute to the metabolism of a variety of substances [4] . In addition to the major Clara cell secretory protein (CCSP), also known as CC10, CC16, Clara cell antigen, secretoglobin 1A1 (SCGB1A1) or uteroglobin, Clara cells also contribute surfactant apoproteins A, B and D, proteases, anti-microbial peptides, several cytokines and chemokines, and mucins in the extracellular fluid lining airspaces. CCSP is the most abundant secretory protein found in the airway surface fluid, expressed exclusively in nonciliated Clara cells and widely used as a marker of the cells [5, 6, 7, 8] .Changes in CCSP levels have a profound impact on not only the composition of airway surface fluid but also the airway epithelial response to environmental stimuli [9, 10] . Another important property of Clara cells is their ability to serve as progenitors for airway lining cells in response to injury. Moreover, subpopulations of CCSP-expressing cells may function as true stem cells of adult airways. Presently it is not known whether the groups overlap or represent distinct cells such as variant Clara cells [11] , type A cells [12] , OCT4-expressing stem cells [13] and bronchioalveolar stem cells (BASCs) [14] .
Due to the lack of simple methods for the isolation of primary Clara cells from the lung, the majority of studies have been carried out in vivo or using lung cancer cells for in vitro tests. The major disadvantage of such approaches is the difficulty in performing mechanistic studies in non-neoplastic primary cells. Recently, Wong et al. developed a method for isolating CCSP + cells from bone marrow by flow cytometry sorting [15] . We speculated that this method may also be used to isolate CCSP + (Clara) cells from the lung. In this study we established a simple method for the isolation of CCSP + cells from mouse lung and applied several different means to identify stem cell-like characteristics of CCSP + cell in vitro and in vivo. We propose that this new procedure method for CCSP + cell isolation provides a useful instrument for Clara cell research, for instance in the field of stem cell biology.
Mice FVB mice were purchased from the Frederick National Lab, Maryland. Mice were housed under specific pathogen-free conditions under a 12-h light/dark cycle with access to food and water ad libitum. All the procedures used in this study were approved by the NIH Animal Care and Use Committee.
The heart, lungs and trachea were removed en bloc from mice following euthanasia by carbon dioxide inhalation. Lungs were separated and lobes minced on ice and incubated with collagenase type I (Invitrogen, Grand Island, NY) at 3 mg/ml in PBS in a volume of 2 ml per lung for 1 hour at 37uC with continuous agitation in an incubator. The suspension was further disaggregated by trituration through a 19 gauge needle (Sherwood Medical Co, St. Louis, MO), diluted in PBS. The crude cell suspension was filtered through a 40 mm cell strainer (BD Biosciences, Sparks, MD) and centrifuged at 700 rpm for 5 min. After discarding supernatant, cells were resuspended in 2 ml of red blood cell lysis buffer (eBioscience, San Diego, CA) for 4 min. Neutralization was performed with 10 ml of Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% FBS (Invitrogen) and cells were centrifuged at 700 rpm for 5 min. Cells were resuspended in DMEM/10% FBS with 20 ng/ml gentamycin/0.5 ng/ml amphotericin B (Cascade Biologicas tm , Portland, Oregon), plated in 100 mm dishes and placed to recover in an incubator at 37uC and 5% CO 2 for 18 hours ( Figure 1A ).
Recovered cells were trypsinized in 0.05% Trypsin-EDTA (Invitrogen) and resuspended at a concentration of 1610 7 cells in 100 ml PBS with 3% FBS. Two microliters of the rabbit anti-CCSP antibody (Millipore, Billerica, MA) was added, followed by a 30 min incubation on ice. Cells were washed twice in PBS with 3% FBS, then 2 mL goat anti-rabbit-FITC secondary antibody was added and incubated on ice for 30 min. After two washes in PBS with 3% FBS, cells were resuspended in the same but fresh media. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control. CCSP positive (CCSP + ) and negative (CCSP 2 ) fractions were obtained by fluorescence-activated cell sorting (FACS) using Vantage SEH cell sorter (BD, Bedford, MA). They were examined by immunfluorescence, adherent or 2D and sphere cell (3D) cultures and qRT-PCR.
Single and dual labeling of cells and tissue sections by immunofluorescence (IF) or immunohistochemistry (IHC) was performed according to previously described methods [16] [17] . The primary antibodies were: goat polyclonal anti-CCSP(T18) (1:50, Santa Cruz Biotech, Santa Cruz, CA), rabbit antiuteroglobin-related protein 1 (UGRP1) (1:100, a kind gift from Dr. Shioko Kimura, NCI/NIH, Bethesda, MD), rabbit anti-pan-cadherin (, 1:50, Abcam, Cambridge, MA), mouse anti-b-catenin (BD). rabbit anti-pan-cytokeratin (1:100, Dako, Carpinteria, CA), rabbit anti-pro-SPC (1:200, Millipore), rat anti-BrdU (1:100, Accurate Chemical & Scientific Corp, Westbury, NY), rabbit anti-sox2(1:2000, Seven Hill, Cincinnati, OH) and rabbit anti-ALDH1(1:500. Abcam). Approximately 1610 5 FACS sorted cells, were washed twice in PBS with 1% FBS and resuspended in 30 ml of Cell Adherence Solution (Crystalgen, Commack, NY). After standing for 2 minutes, 3 ml of the cell mixture was mounted on glass slides, dried for 2 minutes and fixed with 4% paraformaldehyde in PBS for 15 minutes. Both fixed cells and tissue sections were blocked with 1% goat or rabbit normal serum for 1 hour. The blocking solution was removed and 75 ml of primary antibody was added to cells. After 1 hour of incubation at room temperature, slides were washed in PBS three times. Secondary antibodies were added. For dual-labeling IF, additional primary antibodies were added after the third PBS wash, followed by incubation with secondary antibodies conjugated with Alexa fluor 488 or 594 (Invitrogen). All incubations were performed at room temperature and slides were washed in PBS (365 min) between each step and mounted with an anti-fading reagent with 49,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Control slides were included in each analysis in which non-immune serum was substituted for primary antibodies and secondary antibodies individually. All IF images were taken with a Zeiss LSM 510 Meta Mk4 Confocal Microscope (Zeiss, Thornwood, New York). For IHC, signals were developed using 3,39diaminobenzidine (DAB).
Total RNA from sorted cells was isolated using an RNeasy minikit (Qiagen, Valencia, CA) by following the manufacturer's protocol. One microgram of RNA was reverse transcribed in a total volume of 20 ml using the QuantiTect RT kit (Qiagen). PCR was performed in triplicate in a MyiQ single color real time PCR detection system (Bio-Rad, Hercules, CA) using SYBR Green PCR kit (Qiagen) according to the manufacturer's protocol. Amplification was confirmed by ethidium bromide staining of the PCR products on an agarose gel. The expression of each target gene was normalized to the expression of 18 S RNA and presented as the ratio of the target gene to 18 S RNA, expressed as 2 2DCt , where Ct is the threshold cycle and DCt = Ct Target 
For continuous labeling in vivo, BrdU (50 mg/ml) was administered to mice throughout a 7-day period via a subcutaneous miniosmotic pump (Alzet model 2001, Durect Corporation, Cupertino, CA). Alzet pumps were implanted in mice and removed after one week. Mice were sacrificed 4 weeks after removal of the Alzet pumps. Lungs were fixed overnight via tracheal instillation of fresh 4% paraformaldehyde and embedded in paraffin prior to sectioning. Label-retaining cells were identified by BrdU immunofluorescence. BrdU and CCSP double staining was performed and cells exhibiting a nucleus and attachment to basement membrane were counted. Bronchiolioles (BLs) were defined as intrapulmonary airways in which smooth muscle, but neither cartilage nor glands, could be seen. Terminal bronchioles (TBs) contained an intact bronchioalveolar duct junction (BADJ) and visible alveolar duct [17] . In TBs quantification of staining included all cells within 200 mm of the BADJ. A total 49 TBs and 26 BL structures were analyzed in lung sections of five mice. Figure 1 . Schema for purification of primary CCSP positive cells from mouse lung. A) Two month old FVB mice were euthanized by CO 2, lungs removed and lobes collected. After washing in PBS, lobes were minced on ice and incubated in a small cell culture dish with 3 mg/ml collagenase in PBS (total 5 ml) in a shaking platform for 1 hour at 37uC. The suspension was further disaggregated by trituration through a 19 gauge needle, with 5 ml of PBS, filtered through a 40 mm cell strainer and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, cells resuspended in red blood cell lysis buffer for 4 min re-plated into 10 cm culture dishes for recovery overnight (18 hrs). Surviving cells were adhering to the dish. After trypsinization and neutralization by 10% FBS media, cells were resuspended in PBS with 3% FBS, and stained with rabbit anti-CCSP antibody and FITC conjugated anti-rabbit secondary antibody. CCSP + and CCSP 2 cells were sorted with FACS Vantage SE cell sorter. B) Rabbit IgG was used as an isotype -matched negative control; CCSP + population sorted with FACS Vantage SE cell sorter from dissociated lung tissue was 25.37%. doi:10.1371/journal.pone.0043008.g001
It is well established that CCSP is expressed in non-ciliated Clara cells in the airways. CCSP which is widely used as a Clara cell marker is a cytoplasmic secretory protein [3] . Figure 2A revealed intense immunorectivity along the lining of mouse TB. Recently, Wong AP et al. was able to isolate CCSP + cells from bone marrow using flow cytometry [15] . Therefore, we postulated that CCSP may be expressed not only in the cytoplasm, but also in the cell membrane of Clara cells. To obtain evidence for this, we used the well-known cell membrane marker pan-Cadherin [18] . Indeed, in high magnification photomicrographs we were able to demonstrate co-expression with pan-Cadherin in the cell membrane using confocal microscope ( Figure 2B) . These data suggested a possibility for isolating living Clara cells by flow cytometry sorting and lead us to develop the protocol outlined in this study.
To test the possibility of CCSP + cell isolation by flow cytometry from mouse lung, we established a simple method to make single cell suspensions from lung tissues. Using a combination of mincing by scissors and incubation in a high concentration of collagenase (3 mg/ml) for digestion, single cells were obtained within 2 hours from euthanasia. After an overnight recovery in DMEM/10%FBS cell culture media, cells adherent to culture dishes were trypsinized and sorted using a flow cytometry sorter. In FVB mice, about 25% of the lung cells sorted from one whole lung single cell suspension were CCSP + (Figure 1 ). Typical yields of sorted cells per mouse were about ,2.5610 5 of CCSP + cells and ,4610 5 of CCSP 2 cells. Sorted cells were plated into 100 mm cell culture dish for overnight in an incubator prior to further studies. Unattached dead cells were removed with the media.
Sorted cells were used for RNA isolation and RT-PCR following an overnight recovery. CCSP mRNA expression was detected in CCSP + cell fraction, but not in CCSP 2 cells ( Figure 3A) . We also mounted cells on slides using Cell Adherence Media for immunofluorescence (IF). We found that 98% (225/ 230) of the cells in CCSP + sorted fraction were positive for CCSP IF. We also found that 97% of the cells in the CCSP + fraction revealed the expression of another Clara cell maker UGRP1 by IF. All of the cells in CCSP + sorted fraction expressed pan-keratin. Rare CCSP + sorted cells revealed the presence of pro-SPC ( Figure 3) . These data demonstrated that sorting by flow cytometry is a useful and simple method for harvesting purified CCSPcontaining Clara cells. 
One of the features of stem/progenitor cells is the expression of stem cell markers. Therefore we performed qRT-PCR for several common stem cell markers including CD44, CD133, Sca-1 and SOX2 in the sorted cells. Interestingly, CD44 was expressed in both CCSP + and CCSP 2 populations at similar levels. In contrast, CD133, Sca-1 and Sox2 demonstrated much lower but detectable levels in CCSP + cells than the levels in CCSP 2 cells. These data indicate that CCSP + cells express stem cell markers, although at low levels ( Figure 4 ).
Spheroid culture is a common method to detect stem cell features in vitro [19] . Spheroid colony formation was tested by serial dilution technique. Both CCSP + and CCSP 2 cellular fractions were able to form sphere clones. However, following 10 days of culture ( Figure 5 ) CCSPcells demonstrated a larger colony size and higher efficiency of colony formation than CCSP + cells. Dissociation of spheroid colonies into single cells resulted in reformation of the spheroid colonies, indicating that this phenotype was stable (data not shown).
Quiescent or slow-cycling stem cells in adult tissues can retain BrdU over long periods by either segregating chromosomes asymmetrically or dividing slowly. Label-retaining cells can be used to identify populations that contain stem cells [20] . In fact, many such studies have been used to determine putative stem cell locations in mammalian tissues [21, 22] . Using CCSP and BrdU double staining by IF, we found that 1.59% (39/2450) of cells in TBs and only 0.39% (12/4138) of them in BLs were BrdU + / CCSP + (Figure 6 ). The results suggest that the majority of mouse airway CCSP + stem/progenitor cells may reside in TBs.
A subpopulation of CCSP + /pro-SPC + cells known as bronchioalveolar stem cells (BASCs) are capable of differentiating into Clara cells and alveolar type II cells and are considered to be adult lung stem cells [14] . In the current study, a rare portion of sorted CCSP + cells were also found to express the type II cell marker pro-SPC ( Figure 3D ). In order to confirm the existence of BASCs in vivo, we performed CCSP/pro-SPC double staining by IF in mouse lungs. Our results showed that 1.1% of TB epithelial cells contained BASCs while no CCSP + /pro-SPC + double positive epitheliums were detected in BLs ( Figure 6 ). In addition, a number of stem cell markers such as CD44, Sox2 and ALDH1 were detected by IF or IHC along the TB epithelium ( Figure 6C-E) . We also found that CD133, CD44, Sca-1 and Sox2 mRNAs were expressed at variable levels in mouse lung tissues ( Figure 6F, 6G) . This provides further evidence for the progenitor role that Clara cells may have in the mouse lung. 
In this study, we isolated and characterized significantly purified CCSP-expressing cell populations from mouse lung by using high concentrations of collagenase and a flow cytometric sorting method. In addition, we showed that CCSP + cells expressed stem cell markers and form three dimensional spheroid colonies in culture. Furthermore, we confirmed that CCSP + cells may also express stemness characteristic in vivo as evidenced by label retention, the presence of CCSP/pro-SPC double positive BASCs and expression of stem cell markers in the epithelial lining of TBs of mice. Accordingly, the novel method described herein is a significant step in the progress of isolating and characterizing highly purified Clara cells in primary cultures.
Based on previous publications, the distribution of CCSP expression in non-ciliated Clara cells is described as cytoplasmic [23, 24, 25] . We made the surprising and novel discovery of CCSP immunoreactivity along cellular membranes of bronchiolar Clara cells. Using pan-cadherin as a cell membrane marker in normal airway epithelium [18, 26, 27] we found CCSP was expressed not only in the cytoplasm, but also in the membrane. These findings gave rise to the possibility that living Clara cells can be isolated by flow cytometry using fluorescing tags. Our successful CCSP + cell sorting further confirmed the distribution of CCSP membranous expression. One explanation is that bronchiolar Clara cells secrete such large quantities of CCSP that part of it remains stuck to the outer surfaces of cell membranes, allowing sorting of CCSPcontaining cells from suspension. Clara cell isolation from rabbit was first reported in the early 1980s by Devereux et al. [28] . After that, several groups were able to isolate pulmonary Clara cells from mouse [29, 30, 31, 32, 33] . The studies have been instrumental in establishing the many functions of Clara cells. However, the majority of the methods are quite complex and rely on protease digestion followed by centrifugal elutriation and/or Percoll density gradient centrifugation. Only one group used FACS for Clara cell isolation from rat based on the reaction of their glutathione content with monochlorobimine to a fluorescent product [34] . The techniques typically resulted in a Clara cell enrichment of 55,90%. A reproducible source of considerably purified Clara cells is necessary for airway stem/ progenitor cell research. Using high concentrations of collagenase for lung tissue digestion followed by flow cytometry sorting, we were able to achieve 98% pure CCSP + (Clara) cell population, providing a very useful and reliable method for Clara cell function and stem cell research. A notable application will be to directly address molecular mechanisms of genes that have been expressed in Clara cells by using CCSP as a lung specific promoter in transgenic mice.
To further characterize sorted CCSP + cells, we evaluated the expression of pan-keratin protein in CCSP + cells. All cells expressed pan-keratin indicating that all the CCSP + cells were epithelial. We also found that a few cells expressed pro-SPC. This suggests that CCSP + cells contain rare populations of BASCs (CCSP/SPC double positive cells).
In this study, the expression of well documented stem cell markers such as CD44 [35] , CD133 [36, 37] , Sca1 [14, 38] and Sox2 [39, 40] was detectable by qRT-PCR in CCSP + cells. However, the level of CD133, Sca-1 and Sox2 expression was lower in CCSP + cells than that in CCSP 2 cells. One possible explanation is that CCSP is a Clara cell differentiation marker, so a CCSP + population of cells may contain more mature Clara cells, but few stem/progenitor cells, while CCSP 2 cells fraction is a mixture of many cells, such as type I, type II, ciliated cells, basal cells, smooth muscle cells and fibroblast cells and so on. Many of the cells have been shown to have stem cell features [19, 41] . Sphere culture showed that CCSP + cells were able to form spheroid colonies. The sphere colony size and efficiency of colony formation were lower in CCSP + cells compared to CCSP 2 cells. This data further suggests that CCSP + cells do have stem cell features, but stem cell activities are lower than in CCSP 2 cells. Using tissue sections, we found that 1.59% of CCSP positive cells in TBs were label-retaining cells and 1.1% were CCSP/SPC double positive BASCs. These data provide in vivo validation for the in vitro results that the CCSP + cell population contains a small subset of stem cells in the airway.
In summary, we discovered that CCSP was not only expressed in the cytoplasm but there was also marked immunoreactivity along in the cell membranes of airway Clara cells. This provided the basis of flow cytometry sorting technology for the isolation of CCSP expressing Clara cells from murine lung. We also found that in vitro, CCSP + cells demonstrated stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Moreover, a subset of labelretaining cells and BASCs were detectable in the CCSP + population in vivo located in the TBs We conclude that Clara cell isolation by FACS is a useful method for investigating Clara cell function and overall pulmonary stem cell research biology. Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development. Rapid progress and improvement in molecular technologies have allowed researchers to switch from the traditional approaches of virus detection in clinical samples to multiplexing for simultaneous detection of multiple pathogens in a single assay. A number of different PCR based assays for detection and discovery of multiple pathogens have been developed [1] [2] [3] [4] . Detection microarrays are proven to be useful in the identification and discovery of viruses homologous to known species. They have been used to guide the selection of samples for further analysis by sequencing [1] [2] [3] 5] . However, microarrays based on nucleic acid hybridization are too complex in design and performance for the routine donors testing, and exhibit a comparatively low sensitivity of detection, usually around 10021,000 genome copies of target virus per analyzed sample [1] [2] . Several PCR based assays coupled with oligonucleotide microarray technology (so called resequencing arrays) have been designed to allow simultaneous detection or genotyping of a target group of viruses, such as some critical blood-borne pathogens (3 viruses) [6] , respiratory viruses (16-21 viruses) [7] [8] , and respiratory adenoviruses (6 different serotypes) [9] . Such PCR based approach allows increasing the sensitivity of detection down to 10-100 copies of the target RNA or DNA in a sample.
PCR multiplexing should be highly useful when both the volume of the samples and the time of testing are critical, as in the donor eligibility (DE) testing for tissue or organ transplantation [10] . Current regulation requires that DE testing be performed using assays approved and licensed by the U.S. Food and Drug Administration (FDA). However, the automated assay systems that are designed to screen large numbers of samples, without the strict limitation of sample volumes, may not be completely suitable or ideal for the needs of DE testing for tissue or organ transplantation.
The main goal of the study presented here was to evaluate the feasibility of developing a sensitive and specific assay for rapid detection and identification of a group of target viral pathogens.
The following viral pathogens were included in our array: human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia virus-1 and -2 (HTLV-1 and HTLV-2), hepatitis C virus (HCV) and West Nile virus (WNV), all with singlestranded RNA genome; vaccinia virus (VACV) and hepatitis B virus (HBV), both with double-stranded DNA genome, HBV also has single-stranded RNA stage. Some of the listed viruses are included to the required DE testing for tissue transplantation. Besides, historically, some of the targeted viruses have been found to be allograft-transmitted to recipients [11] [12] . In the present study, a real-time PCR array with SYBR-Green chemistry targeting these eight viral pathogens listed above was developed and evaluated with analytical and clinical panels. The array demonstrated acceptable performance in the testing with both analytical panels and donors' clinical samples.
The research study conducted at FDA using previously frozen blood samples was reviewed by Department of Health and Human Services, Food and Drug Administration, Research Involving Human Subjects Committee (RISHC Protocol #10-008B entitled ''Detection of Infectious Agents in Previously frozen blood Samples from Patients with Various Illnesses and Healthy Blood Donors''). The 17 clinical plasma samples positive for HBV, HCV or HIV-1 used in this study were existing clinical diagnostic samples kept in NIH Blood Bank. Information of these left over samples had been recorded in such a manner that subjects can not be identified, directly or through identifiers. The written informed consent from the participants was waived under 45 CFR 46.101 (b) (4) . The six plasma samples positive for HIV-1 were estimated to contain 50 to ,90,000 genome copy numbers per ml; six plasma samples positive for HCV were estimated to contain 780 to ,123,000 genome copy number/ml and five plasma samples positive for HBV were estimated to contain ,150 to 16,000 copy number/ml. The copy numbers of viruses in these samples were provided by the NIH Blood Bank. No information about the subtypes or genotypes of these viruses was available. The amount of each clinical sample was sufficient to be tested only once by the PCR array in the study. All positive PCR products obtained in the testing using the PCR array were confirmed for validity by sequencing in the Facility for Biotechnology Resources of FDA/ CBER.
We used the ''Insignia'' program (http://insignia.cbcb.umd. edu/query.php), a bioinformatics on line tool developed in the Center for Bioinformatics and Computational Biology, University of Maryland [13] to choose a specific DNA or RNA ''signature'' (a sequence, with customized length and G/C content) for targeted viruses. Comparative sequence analysis of the complete genomes was performed using mVISTA (http://genome.lbl.gov/vista/ mvista/submit.shtml). Multiple nucleotide sequence alignments (NSAs) were then created to visualize the most conserved genome areas using MEGA4 (http://www.megasoftware.net).
Specific criteria for the primers and amplicon selection for the SYBR Green based PCR array were: 1) the same range of annealing temperature (T) -57-60uC -for all primers, 2) high G/C content for primers, allowing higher specificity of annealing, and 3) an amplicon size in the range of 100-200 b.p. in order to have a high PCR amplification efficiency and to sufficiently distinguish the products from primer dimers based on melting T peak (Tm). All primers were checked for potential dimer formation using ''Primer Express'' software (version 3.0, Applied Biosystems). After design, all primers were again checked using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to avoid any cross-reactivity with other species. Newly designed and previously published primer sets adapted for the final version of the real-time PCR array are listed in Table 1 . In addition, primers specific for the human beta-globin gene were included in the array as an internal control for the quality of DNA/RNA preparation as well as for estimation of viral copy number per host cell if needed (the last row of Table 1 ).
Total cellular DNA of the chronically infected cell cultures (for HIV-1, HIV-2; HTLV-1, HTLV-2 and VACV), viral genome cDNA copy spiked into human DNA (for HCV and WNV), and DNA isolated from human plasma of the infected individual (for HBV) were used as the positive templates in the initial testing and are listed in the last column of Table S1 . DNA or RNA panels were created by cloning of specific synthetic templates for each virus into the pGEM-T-Easy vector (Promega) by TA cloning, following by in vitro transcription to obtain RNA standards for HCV and WNV. All created plasmids are listed in Table S1 . Nucleotide numbers in Table S1 refer to the location of the partial viral genome cloned into pGEM-T-Easy vector according to the following complete genome sequences available in GenBank: HTLV-1 -L03562.2, HTLV-2 -M10060.1, HIV-1 -K02083.1, HIV-2 -J03654.1, HBV -AF462041.1, HCV -AF271632.1, VACV -AY243312.1, WNV -HQ596519.1. To establish the real-time PCR standard curve the copy number was calculated for each plasmid carrying one copy of the specific viral gene. The size of each plasmid (X b.p.) was used to determine the molecular weight in Daltons (g/mol): W (g/mol) = X b.p. (330 Da 6 2). The copy number of the target viral gene (molecules/ml) was determined from the plasmid concentration (C DNA ) and the molecular weight of each plasmid molecule:
Copy number = C DNA (ng/ml) 6 6.02 6 10 23 (Avogadro's number)/W.
Knowing the number of plasmid molecules with the target viral gene in a ml, a series of dilutions was made to generate a PCR standard curve.
The developed analytical standards were used to calculate the intra and inter-assay reproducibility of quantification for each virus-specific primer set. Mean C(t) values, standard deviation (SD) and coefficient of variation (CV) were calculated from the data obtained in three replicates of each standard dilution for the intraassay reproducibility, and in three real-time PCR assays consisted of three replicates each (nine total) for the inter-assay reproducibility. CV was calculated as SD/Mean C (t) * 100%.
Preparation of Viral RNA/DNA for PCR Array Analysis 0.5-1 ml of human plasma was used for the total viral RNA/ DNA extraction using ''QIAamp Viral RNA Mini-Kit'' (Qiagen) and tRNA (SIGMA) was used as a carrier RNA during the preparation. After the final elution step RNA/DNA in 160 ml of buffer AVE was precipitated with 100% ethanol and 3 mM NaCl at 220uC overnight. An RNA/DNA pellet was washed with 70% ethanol, dissolved in 10 ml of DEPC-treated water and then immediately used for cDNA synthesis with SuperScript II RT (Invitrogen) and random hexamers (Invitrogen) in a total reaction volume of 20 ml. The volume of cDNA/DNA sample was then adjusted up to 30 ml with DEPC-treated water and the whole 
PCR was performed using ''Bio-Rad CFX96 Real-Time System'' with ''Power SYBR Green PCR master mix'' (Applied Biosystems). One reaction (25 ml total) contained: 12.5 ml of PCR master mix, 0.5 mM of each primer and 1.25 ml of DNA/cDNA template. In the single virus testing (sections 3 and 4 of the ''Results'') we used 2.5 ml of DNA/cDNA template from 20 ml of sample after cDNA synthesis. ''Universal'' PCR conditions for all primer sets included to the array were: 95uC for 8 min (one cycle), then 50 cycles of: denaturation at 95uC for 15 s, annealing and extension at 60uC for 1 min, followed by melting curve read from 65uC to 95uC with increment 0.2uC for 5 s. Real-time PCR data were downloaded in 96-well plate format from ''Bio-Rad CFX Manager 2.1'' to MS Excel and analyzed manually.
Two types of samples served as the background control for determination of the C(t) cut off. The 1 st type of negative control was 50 ng of human cellular DNA. The 2 nd type of negative control was negative donors' plasma. Data were collected in separate experiments from 8 human cellular DNA controls and 3 negative donors' plasma. To standardized the C(t) cut off for all primers the threshold was set at the PCR machine default setting. Based on a false positive rate of less than 5% the following method [14] was used to estimate the C(t) cut off from the range of C(t) obtained with negative samples for all 24 primer sets in the array:
1. Calculate the margin of error of the confidence interval (CI), W = t* 6 SD/!n, where: n -number of obtained C(t) values, SD-standard deviation, df = n21, and t* (for 95% confidence) is a ''critical value of the T distribution'' [14] . 2. One side CI covers this range: M-W, where M is sample mean.
The C(t) cut off calculated from the range (n = 50) of the 1 st type of negative control data was C(t)#41.03. The C(t) cut off calculated from the range (n = 50) of the 2 nd type of negative control data was C(t)#42.7. Even some overlap between the C(t) measurements of truly positive and truly negative samples was detected in PCR array data, the Tm parameter was used to define if the obtained PCR product is specific by comparison to the expected Tm peak range.
The analytical sensitivity of each primer set was determined in the single virus testing using FDA/CBER panels (kindly provided by Dr. Stephen Kerby, FDA/CBER) consisting of various amounts of the viruses (0-1,000 genome copies/ml) spiked into the ''normal'' human plasma. Panels for the following viruses were used in testing: HIV-1 (three different panels), HIV-2, HBV (based on genotype A), HCV (genotype 1b), and WNV (based on strain HU2002). These panels have been used for testing of commercially licensed NAT assays and their development was described previously [15] . Three separate HIV-1 RNA panels were used for testing. The first consisted of various amounts of an HIV-1 group M subtype B isolate: 0, 5, 10, 25, 50, 100, and 500 copies/ml. The second consisted of 25 samples representing various concentrations of HIV-1 groups O and N, and group M subtypes A, C, D, E, F and G: 10, 100 and 1,000 copies/ml for each virus. The third panel consisted of HIV-1 circulating recombinant forms (CRFs) AE and AG: 100, 1,000, and 10,000 copies/ml for each CRF. NP24 CAACTTCATCCACGTTTCACC a -to simplify the process of evaluation we used our primer names with sequential numbers, however some of the primers have been designed previously with their original names in the articles listed in the last column of this [25] [26] . HIV-1 groups and subtypes are based on designations reported by the NIH ARRRP. Low passage virus stocks were prepared and median tissue culture infective dose (TCID 50 )/ml of virus containing supernatant determined in fresh human PBMCs isolated as previously described [27] . Infectious unit (IU) of the virus stocks in TCID 50 s were titrated for each isolate and the number of IU used for viral RNA isolation and PCR was calculated based on the dilution factor (1:100) and ranged from ,10 to 2,500 or 1 to 3.4 log 10 TCID 50 per PCR reaction.
PCR approach based on SYBR Green chemistry, allowing simultaneous detection of multiple targets, was chosen to be applied for the array performance. Virus-specific primer sets targeting at least three different genomic sites for each viral pathogen were designed for the real-time PCR array. We used the ''Insignia'' program, a bioinformatics tool that helps to choose a specific DNA or RNA ''signature'' for different bacteria and viral pathogens that are included in the pre-built ''Insignia'' database [13] . Sequences of the highly conserved regions, such as coding viral polymerase or structural proteins were selected to design the candidate primers. In addition, we performed nucleotide sequence alignments (NSA) of the complete viral genomes and of the most conserved genome areas for different subtypes/genotypes or different isolates of all targeted viruses. Some previously published primers designed for PCR detection of the target viruses were also adapted and evaluated. Overall, a total of 120 primers were initially designed using specific criteria for the current real-time PCR array (see Materials and Methods) to cover the eight targeted viruses: HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV -2, WNV, and VACV. The primers were designed and tested for their effectiveness and specificity of amplifying the respective target under uniform PCR conditions (using the same annealing temperature for all primers).
Each candidate primer pair was first tested for its specificity and sensitivity of PCR amplification. This was assessed in a real-time PCR cross-testing against human PBMC DNA (50 ng/PCR reaction) from a healthy donor, DNA from human cell cultures infected by various target viruses, or human DNA spiked with a known amount of genome copies of various viruses. It was important to ensure that the selected primers targeting a specific virus would not non-specifically amplify any DNA in a sample. The melting temperature (Tm) peak of the product amplified from the target virus should be clearly differentiable from the Tm peak of primer dimers or any non-specific products produced in PCR. DNA or RNA panels were created from the cloned synthetic templates (listed in Table S1 ) by spiking of 2-10 4 genome copies of each virus into 50 ng of human background DNA. The sensitivity of each candidate primer set for each target virus was assessed using these panels.
Example of the experimental testing of HIV-1 specific primer set targeting gag gene (NP3/4) for its sensitivity with DNA analytical standards is shown in Figure S1 . Serial dilutions of a plasmid DNA corresponding to 5-10 4 HIV-1 genome copies spiked into 50 ng of normal human DNA, HIV-1 infected cells (H9/IIIB) (''positive'' control DNA) and uninfected human PBMC (''negative'' control DNA) are used in the experiment. Only a single melting peak Tm = 72.5uC corresponding to HIV-1 specific product was observed and no unspecific amplification was registered. Figure S1B revealed a standard curve showing the correlation between copy number of the target gene and Ct values with a slope = 23.77. The limit of sensitivity was determined in this assay to be 10 viral genome copies/PCR.
After evaluation, a total of 24 primer pairs targeting the eight different viruses were chosen for the real-time PCR array based on their specificity and sensitivity (Table 1) . Among them, five of the primer sets were previously published and the other 19 primer sets were originally designed in the current study. Table 1 shows the sequences of the previously published primer sets selected for the real-time PCR array with the reference to the original source. Analytical sensitivity expressed in genome copy/PCR for each primer set (Table 1 ) was estimated using DNA/RNA analytical panels, as described above. Coverage of variants (i.e., different subtypes or genotypes) for each virus (Table 1 ) was estimated using the NSAs. Degenerative nucleotides were introduced into some of the primer sets based on the NSAs performed in-house to obtain a broader coverage. The Tm peak range of the product stated in Table 1 for each primer set was established during further testing of selected primers with analytical and clinical panels.
In addition, the intra and inter-assay reproducibility of quantification for all primer sets was evaluated using three replicates of each standard dilution (of DNA or RNA analytical standards) in each of three real-time PCR assay runs. The coefficient of variation (CV) for the C (t) values was #3.3% and #6.7% for intra-and inter-assay, respectively. All the data depicting mean C (t), standard deviation (SD), and CV for each primer set selected for the real-time PCR array with each standard concentration are shown in Table S2 .
To further evaluate the sensitivity of the selected primers, we used FDA/CBER panels (kindly provided by Dr. Stephen Kerby, FDA), consisting of various amounts of viruses spiked into ''normal'' human plasma, that are specifically developed and used for the evaluation of commercially licensed NAT assays. Table 2 summarizes the testing results for the selected primers against the target viruses. One out of four primer sets targeting HIV-1, NP3/4, could detect the subtype B HIV-1 RNA at the concentration of 50 copies/ml of human plasma. Two other HIV-1 specific primer sets (NP51/52 and NP170/171) detected the HIV-1 RNA at 100 copies/ml of plasma. The 4th primer set, NP175/174, (targeting the conserved pol region and containing degenerative nucleotides to support broader variant coverage) could detect the virus only at 500 viral genome copies/ml of plasma. HIV-2 RNA was detected with primer set NP86/87 at the concentration of 10 copies/ml and with the primer set NP76/77 at the concentration of 50 copies/ml. Another primer set (NP84/ 85) detected HIV-2 at 100 copies/ml. HBV (genotype A) DNA was detected with the primer set NP11/97 at 50 copies/ml and with NP94/100 at 100 copies/ml. The third HBV specific primer set (NP11/97-mod) detected HBV at 500 copies/ml of plasma. Both HCV specific primer sets targeting the viral 59NTR detected HCV RNA at the concentration of 10 copies/ml of plasma. Two of the WNV-specific primer sets (NP21/22, targeting E protein gene, and NP176/177, targeting NS5) detected WNV at 50 copies/ml and the third primer set (NP178/179, also targeting NS5) gave a positive signal at 100 copies/ml of plasma.
The four HIV-1 specific primer sets were further evaluated by testing them against another FDA/CBER analytical panel containing a broad spectrum of HIV-1 subtypes. As summarized in Table 3 , the HIV-1 subtype F (group M) and group O isolates in Table 2 . The results of sensitivity testing of the real-time PCR array primer sets specific for HIV-1, HIV-2, HBV, HCV, and WNV the with FDA/CBER analytical plasma panels. Table 3 . Sensitivity of four HIV-1 specific primer sets selected for the real-time PCR array in testing with FDA/CBER analytical HIV-1 broad spectrum panel. Detection limit (copy number/ml of plasma) was evaluated using FDA/CBER analytical panel, containing pre-set copy number of HIV-1 spiked into 1 ml of ''normal'' human plasma. RNA from 1 ml of plasma was converted to cDNA and divided into eight PCR reactions; two PCR repeats were performed for each primer set. The estimated copy number per PCR reaction is shown in parentheses. the panel could not be successfully amplified by any of the four selected primer sets (detection limit is .1,000 RNA copy/ml of plasma). All other subtypes and CRFs of group M, and the group N isolate could be amplified with at least one out of the four primer sets at 50 to 1,000 RNA copies/ml of plasma (Table 3) . The Tm peaks of the PCR products amplified from different subtypes of HIV-1 varied within 1.5uC ( Table 3 ). All of the four selected HIV-1 specific primer sets amplified HIV-1 subtype B (group M) with the highest sensitivity (50-500 copies/ml of plasma).
To examine the specificity and the ability of the array to detect different isolates within subtypes of HIV-1 and different isolates of HIV-2 by the array, we additionally tested our primers with another panel, developed by the Southern Research Institute. This panel contained three different isolates from each subtype (A to G) of HIV-1 group M, three isolates from HIV-1 group O, one isolate from HIV-1 group N and three different isolates of HIV-2. The infectious dose of each HIV isolate in the panel was determined by TCID 50 (median tissue culture infective dose) titration and the dose of virus used in each PCR reaction was calculated. The results of testing of HIV-1 specific primers are shown in Figure 1 . In this experiment we evaluated the coverage of HIV-1 variants and estimated the relative sensitivity of the four HIV-1 specific primer sets based on cycle threshold (C(t)) values obtained with each isolate tested. Two primer sets targeting the conserved pol regions (NP170/171 and NP175/174) were able to detect most of the HIV-1 subtypes with a high sensitivity (C(t) = 15-25). Only one primer set (NP175/174) detected both the group N isolate and all three isolates of group O with Ct = 20-35. We did not detect crossreactivity of HIV-1 or HIV-2 specific primers with the other type of HIV. All three HIV-2 isolates studied (1-3 log 10 TCID 50 /PCR input) were detected with all HIV-2 specific primer sets with a low Ct value: 12-20 (data not shown).
After completion of sensitivity testing of primer sets with analytical panels we finalized the expected Tm peaks range for each amplicon and arranged a working array in 96-well plate format ( Figure S2 ). To evaluate the specificity and possible crossreactivity of primers in the array, 20-100 genome copies of each virus were spiked into 50 ng of human DNA to be used as positive templates and the same amount of human DNA was used as a negative control in each experiment. The array was tested with all targeted viruses using DNA standards (listed in Table S1 ). Each positive template was tested in duplicate; one human DNA negative control and one no-template control (NTC) were included in every testing. In these experiments the definition of ''positive'' signal was set up as the threshold cycle cut-off of C(t)#41 (see Materials and Methods) and all Tm peaks are within the expected range ( Figure S2) . No cross-reactivity was detected for any primer sets in the array using DNA standards with this setting (data not shown).
Seventeen (17) clinical plasma samples (obtained from NIH Blood Bank) from donors who tested positive for HIV-1, HBV, or HCV were used to evaluate the array sensitivity and specificity. The genome copy of each virus in virus-positive plasma samples was previously determined by the NIH Blood Bank using commercial assays approved for donor screening. Representative results from the testing of three HBV-positive plasma samples (pt.#13-pt.#15) using our array are shown on Figure 2 . The threshold cycle cut off of the assay performed with plasma samples was set up as C(t)#43 (see Materials and Methods). The wells with C(t) lower than the cut-off and the obtained Tm peaks within the expected Tm range indicate positive amplification of HBV target genes and are highlighted with blue circles (Figure 2 , green color code for HBV). For samples #13 and #14 Tm peaks of the products, obtained with only two HBV specific primer sets (wells G2, G3-pt.#13 and wells G5, G6-pt.#14), were within the expected range, with C(t) values of 36.7-42.9 and 36.5-36.6 respectively. For sample #15, all three HBV specific primer sets gave the Tm peaks within the expected range (wells G7, G8 and G9), and the C(t) values range was 33.8-34.2. The genome copy number of HBV in plasma samples #13-15 was 151-518 copies/ ml, corresponding to 6-21 copies/PCR. The wells (white color code, red circles) with the human beta-globin gene specific primer set, serving as the internal control, produced positive signals from all three plasma samples tested, with C(t) values of 25.5-26.3 (wells H3, H6 and H9 - Figure 2 ), which shows that the quality of the RNA/DNA sample preparation was equally good for the plasma of these three patients.
The final Tm range for each primer set was adjusted after testing of this clinical panel. Based on the results from the testing of clinical samples and according to NSA performed during the design, two primer sets targeting the S gene region of HBV (NP11/97 and NP11/97-mod) have two different Tm ranges depending on the HBV genotype ( Figure 2 -wells G1 and G3). The lower Tm range (74.5-75.5uC) was detected for genotype A and the higher Tm range (76.8-77.4uC) -for genotypes B, C, and D. The difference in Tm ranges occurred due to single nucleotide polymorphism (SNP) in the amplicon sequence leading to a difference in the G/C nucleotide content of the products. Table 4 summarizes the results from testing the 17 virus-positive clinical samples using the developed array. At least two different specific primer sets could detect a target virus in all cases, with one exception: one HIV-1 positive sample (#4) with ,4 viral genome copies per PCR reaction was amplified with only one primer set (NP175/174). HIV-1 positive clinical samples (#1-#3) with 51-80 viral genome copies/ml of plasma or 2-3 copies/PCR reaction were detected with at least two primer sets. All six HCV-positive samples (#7 to #12) with 16-2,570 viral genome copies/ml of plasma tested positive with both HCV-specific primer sets. Three HBV-positive samples (#15, #16, #17) at 21-66 copies/PCR tested positive by the three HBV-specific primer sets and two HBV-positive samples (#13, #14) with 6-10 copies/PCR tested positive with two out of three HBV-specific primer sets. It is important to note that none of the primer sets showed any crossreactivity with other viruses in the panel of clinical samples tested.
In the study presented here we applied a real-time PCR array approach in a 96-well plate platform for detection of a group of target viral pathogens. In contrast to TaqMan PCR, which is commonly used for viral diagnostics, this platform based on PCR with SYBR Green chemistry supports simultaneous detection and identification of 24 different targets corresponding to eight different viruses. PCR based on SYBR Green staining of the double stranded DNA is economically affordable and allows for the detection of mutants with SNPs within the amplicon sequence [34] . The strategy of primer selection for the array included the sequential use of different bioinformatics programs to identify highly-specific primer sets with a maximal variants' coverage of the targeted viruses, while working under ''universal'' PCR conditions. Experimental selection process using a panel of DNA or RNA analytical standards allowed choosing two to four most sensitive and specific primer sets for each targeted virus. The sensitivity (in copy number per PCR reaction) and the intra and inter-assay reproducibility of quantification were characterized for each primer set selected for the array.
FDA/CBER analytical plasma panels were used to assess the detection sensitivity of the primer sets selected for the working array. The HIV-2 and HCV RNA was detected at as low as 10 genome copies/ml of human plasma by one out of three and two out two primer sets, respectively. Similarly, WNV RNA and HBV DNA were detected at 50 genome copies/ml of plasma by two out of three and one out of three selected primer sets respectively.
All four of the selected HIV-1 specific primer sets detected group M, subtype B, the most common subtype of HIV-1 in the Americas and Western Europe [35] , at 50-00 genome copies/ml of plasma. However, the array was able to amplify all other subtypes, excluding subtype F, of HIV-1 group M with only one primer set (NP170/171), targeting the conserved pol region, at 100-1,000 genome copies/ml of plasma. In addition, the array amplified the group N isolate at 1,000 copies/ml with another primer set (NP175/174) targeting the conserved pol region. All the selected HIV-1 specific primer sets of the array failed to amplify the group O isolate (detection limit is .1,000 copies/ml of plasma). In comparison, the limit of detection (LOD) of recently improved commercial multiplex NAT assays for the broad spectrum of HIV-1 group M isolates is 40 copies/ml, and the LOD for group O is 200 copies/ml [36] [37] [38] . Thus, specific primer sets targeting group O isolates of HIV-1, as well as subtype F (group M) will need to be included in the array to increase the coverage of all existing subtypes/groups of the HIV-1. No crossreactivity was shown for HIV-1 or HIV-2 specific primers with the other type of HIV in a testing with medically relevant levels of viruses in a diversity panel containing 25 different HIV-1 isolates and three natural HIV-2 isolates collected worldwide.
There is presently no US Food and Drug Administration (FDA) approved PCR-based NAT testing for blood donors' screening for HTLV-1 and HTLV-2. There is no official FDA (or World Health Organization (WHO)) viral panel released for these two viruses. It is also difficult to obtain HTLV-1 or HTLV-2 positive blood donors' samples in the United States. In the absence of the analytical panels and clinical samples we tested HTLV-1, HTLV-2 and VACV specific primers only with cell culture derived DNA and with DNA standards. The minimum detection limit of HTLV-1 and HTLV-2 specific primer sets estimated with analytical DNA standards was 5-10 genome copies/PCR reaction, which is in the same range as for other primers included to the array. The coverage of the viral variants for the primers targeting these viruses was estimated in silico using multiple sequence alignments performed with complete genome sequences available in Gen Bank.
The working array arranged in 96-well plate format was subsequently tested for specificity and potential cross-reactivity with human DNA and with each of the targeted viruses. None of the primer sets selected for a particular target virus in the array produced non-specific cross-reaction toward the other viruses. Comparative performance of the array was also evaluated through the testing of 17 clinical specimens from the United States patient population. All 17 samples were correctly identified in our PCR array with a high sensitivity to contain HIV-1, HCV, or HBV. We found that a combination of several primer sets targeting each virus in the array allows for the detection of different variants of the virus; however, it makes the absolute quantification with uniform DNA/RNA standards a challenge. Quantification by the assay is not always possible when the genetic group of the viral isolate being tested is different than the assay standards. This is one of the reasons why in certain cases the commercial assays underestimate viral loads by up to 1-10 log 10 copies per ml [37] [38] [39] . Nevertheless, relative quantification can be done using all primer sets selected for the array, and the absolute quantification can be performed with DNA/RNA standards using the primers targeting the most conserved genome areas. There are several commercial qualitative multiplex NAT assays now available on the market simultaneously targeting three most important blood-borne viral pathogens (HIV-1, HCV and HBV) [40] . One of them was recently approved by U.S. FDA for screening of blood and organ donors (http://www.fda.gov/BiologicsBlood Vaccines/BloodBloodProducts/ApprovedProducts/LicensedProduc tsBLAs/BloodDonorScreening/InfectiousDisease/ucm306073.htm).
We compared the LOD of these multiplex NAT assays [40] with the results of our working PCR array in sensitivity of testing against these important viruses. The LOD for HBV are 38.1-195 geq/ml by ''Procleix Ultrio Tigris'' and 9.2-37. Other PCR-coupled techniques have been developed previously for highly-sensitive pathogens' detection that could reach the sensitivity of the assay up to 1 genome copy per PCR reaction. For example the bioactive amplification with probing (BAP), utilizing a nested PCR and magnetic bead-based hybridization with the specific probe, has been developed for the detection of bovine and avian viruses [41] [42] [43] . In spite of the exceeding sensitivity of such assays targeting a single virus, it may be difficult to adapt the approach or method to meet the main objective of simultaneous detection of multiple target viral pathogens by an array using universal PCR conditions.
It is important to note that this array was developed to be adapted by any laboratory. Comparison of experimentally obtained Tm peaks to the range of expected specific Tm peaks allows rapid identification or exclusion of the viral pathogen in a sample. This is an initial study to examine the suitability of using PCR arrays for the detection of a group of target viruses. The current array was developed utilizing five previously published and Table 4 . Tm and C(t) values obtained with primer sets specific for HIV-1, HCV, or HBV in testing of 17 human clinical samples in the format of PCR array targeting eight different viruses. 19 originally designed primers sets. However, it can be expanded to a larger number of targets for the same virus. Targeting of several genome areas increases the detection sensitivity of the target virus and provides an intra-assay confirmation of positive signals. Additionally, any new virus of interest can be added to the list of targeted pathogens. Efforts are underway to test the utility of this real-time PCR array using samples with more diverse biological origin and pathogen content.  TcdC Does Not Significantly Repress Toxin Expression in Clostridium difficile 630ΔErm In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested. Clostridium difficile is an anaerobic, Gram-positive, spore forming rod shaped bacterium that can cause disease with a wide variety of symptoms, ranging from mild diarrhea to severe forms of pseudomembranous colitis [1] [2] [3] . Since 2004, numerous countries have reported outbreaks in health-care facilities caused by hypervirulent C. difficile PCR Ribotype (Type) 027 [1] [2] [3] [4] [5] [6] . Clostridium difficile infection (CDI) caused by Type 027 is associated with a more severe course of the disease and a higher mortality rate than other ribotypes [1, 3, 6] . Recently, increasing numbers of the hypervirulent Type 078 are reported [7] . C. difficile Type 078 is more frequently associated with community acquired CDI and affects a younger population than Type 027 [6] [7] [8] [9] . Furthermore, CDI caused by Type 078 is associated with an increased morbidity compared to other ribotypes [8] .
The main virulence factors of the enteropathogenic C. difficile are the two large clostridial toxins, toxin A (TcdA) and toxin B (TcdB). These toxins are glycosyltransferases that inactivate Rho, Rac and Cdc42, thereby disrupting the cytoskeleton and tight junctions of the cells, resulting in apoptosis [10] . This induces an inflammatory response and degradation of the intestinal epithelial cell layer. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC) [11] [12] [13] . In between the toxin genes the tcdE gene is situated, which encodes a putative holin protein [14] . Interestingly, both hypervirulent Types 027 and 078 have been shown to contain mutations in the tcdC gene, encoding the putative negative regulator of toxin gene transcription, and this has been proposed as a possible explanation for their increased virulence [8, 15] .
The exponential growth phase of C. difficile has been reported to be associated with a high transcription level of the tcdC gene and low transcription levels of tcdR and the toxin genes, whereas the stationary growth phase is associated with a low transcription level of the tcdC gene and high transcription levels of tcdR and the toxin genes in strain VPI10463 [16] . The synthesis and secretion of the toxins is increased upon entry into the stationary growth phase [16] [17] [18] [19] . The decreasing transcription of tcdC correlates with diminishing TcdC protein levels in stationary growth phase [16, 20] .
TcdR is an alternative sigma factor that positively regulates toxin production [11, 12] . The direct interaction of TcdR and the RNA polymerase core enzyme mediates recognition of the toxin promoters and the tcdR promoter [11, 12, 21] . TcdC has been reported to act like an anti-sigma factor for toxin production by destabilizing the TcdR-RNA polymerase core enzyme complex in a way that is not yet fully understood [12] .
The reported inverse correlation between the transcription of tcdC and the toxin genes and the expression patterns of the corresponding proteins, together with the biochemical data, has led to the prevailing model that TcdC is an important repressor of toxin expression [12, 16, 17, 20] . This model seems to be supported by the finding that the absence of a functional TcdC caused by a frame shift mutation (D117 bp) in the tcdC gene is linked to a supposed increased toxin production in certain (hyper) virulent strains [15, 22] .
Recently, some doubts were raised about the importance of TcdC for regulation of toxin expression on the basis of two findings. First, two studies have found increasing levels of tcdC transcription in time that coincide with increasing transcription of the toxin genes and increasing amounts of toxin production [18, 19] . Second, there is a great variability in toxin expression levels among (hyper) virulent strains, even though these generally carry mutations in tcdC [15, 18, 19] . Therefore, a minor (or modulatory) role for TcdC in the regulation of toxin expression was proposed [18, 19] .
Here, we sought to clarify the role of TcdC in regulation of the toxin production by generating an isogenic tcdC mutant (CT::tcdC) using the ClosTron technology. We find only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and the expressed total toxin levels did not significantly differ, suggesting that the role of TcdC in toxin regulation is not of significance under the conditions tested in C. difficile strain 630DErm.
The importance of TcdC for regulation of toxin expression was recently challenged by two studies [18, 19] . It was proposed, based on the increasing transcription levels of the PaLoc genes in time and the variability in toxin expression levels among virulent strains, that TcdC has a minor or modulatory role on toxin expression rather than a major role as previously assumed. In this study we sought to clarify the role of TcdC for toxin expression by generating an isogenic tcdC mutant. As toxin gene expression is subject to complex regulation influenced by glucose and cysteine, we performed our experiments in a trypton-yeast (TY) based broth [17, 23] . TY broth does not contain glucose and no cysteine was added. We verified that in TY broth earlier and higher expression of toxins was achieved in comparison to the commonly used Brain Heart Infusion (BHI) broth (data not shown).
TcdC consists of three domains: a hydrophobic domain, a proposed dimerization domain and a proposed C-terminal repressor domain ( Figure 1A ) [12] . We successfully disrupted the tcdC gene in the region coding for the repressor domain using ClosTron technology. Disruption of genes using the ClosTron technology results in stable mutants and no or non-functional proteins [24] [25] [26] . The genotype of the disruption was confirmed with conventional PCRs using the tcdC2 primer and the EBS universal primer and with a primer pair (tcdC1 and tcdC2) flanking the ClosTron insertion site ( Figure B ). Sequence analysis confirmed that the disruption was in the proposed repressor domain of the tcdC gene at the expected site (data not shown). In addition, Southern blot analysis using intron-, ermB and tcdCspecific probes clearly confirmed a specific single insertion of the Group II intron in the genome ( Figure 1C ).
Western blot analysis, using antibodies against TcdC, confirmed that the isogenic tcdC mutant no longer expressed TcdC ( Figure 1D ). A control blot using antibodies against F 0 F 1 ATPase indicated that the lack of signal in the TcdC Western blot was not a result of lower amounts of proteins loaded in the lanes of PCR ribotype 035 (a PaLoc negative strain) and the tcdC mutant.
The growth kinetics of the wild type and CT::tcdC strains showed no significant differences in various media tested ( Figure 1E and data not shown). In TY broth, which does not contain glucose or added cysteine, the wild type strain and the CT::tcdC strains showed an exponential growth phase in the first 8 hours post inoculation and after 12 hours post inoculation both strains entered into the stationary growth phase ( Figure 1E ). Conventional control PCRs confirmed that the disruption of the tcdC gene had remained intact during our growth curves experiments (data not shown).
Comparable Relative Transcription Levels of PaLoc Genes in Wild Type and CT::tcdC
In order to determine the influence of TcdC on the transcription levels of the PaLoc genes we compared the relative transcription levels of the PaLoc genes of wild type and CT::tcdC strains by reverse transcriptase quantitative real-time PCR (RT-qPCR). We found comparable transcription levels of all PaLoc genes in wild type and CT::tcdC strains.
Overall, the logarithmic growth phase was associated with lower transcription levels of the PaLoc genes and by entering into the stationary phase increasing transcription levels of PaLoc genes were found, as previously described for tcdR, tcdE, tcdB and tcdA [16, 18, 19] and tcdC [18, 19] (Figure 2 ). The transcription levels of tcdR in wild type and CT::tcdC strains increased approximately 100-fold between 6 and 24 hours post inoculation ( Figure 2A ). Though the expression of tcdR was, on average, 3-fold higher at the various time points in the CT::tcdC strains compared to the wild type, this difference was not statistically significant ( Figure 2A , all p values $0.088). Similarly, we observed a 10-to 100-fold increase in the transcription levels of tcdB ( Figure 2B ), tcdE ( Figure 2C ), tcdA ( Figure 2D ) and tcdC ( Figure 2E ) when comparing values from the logarithmic growth phase with those observed in the stationary growth phase. The expression levels of tcdB, tcdE, tcdA and tcdC were, on average, 1.5-fold, 2.5-fold, 1.4-fold and 1.7fold higher, respectively, in the CT::tcdC strains compared to the wild type. With one exception, these differences were not found to be significant. The transcription level of tcdB in the CT::tcdC1 strain is significantly (P = 0.046) higher compared to wild type level at 8 hours post inoculation ( Figure 2D ). However, no significant differences are found between the wild type and CT::tcdC strains at any of the other time points.
Therefore, we conclude that the disruption of the tcdC gene does not result in a consistently and significantly increased transcription level of the PaLoc genes.
Considering the small increase in PaLoc gene expression in the CT::tcdC mutants observed in the RT-qPCR experiments, we were interested to see if this difference translated into higher toxin levels. We determined toxin levels using two independent assays, but found no consistent difference between wild type and mutant cells.
First, filter sterilized bacterial supernatants were incubated on a Vero cell (a kind gift of Dr. E.J. Snijder [27] ) monolayer and cytotoxic effects were quantified after 24 hours by determining the end-point titer ( Figure 3A ) [26] . In the exponential growth phase (5 and 8 hours post inoculation) no cytotoxic effects were detectable (data not shown). In the stationary growth phase (12, 24 and 48 hours post inoculation) we observed increasing cytotoxic effects, indicative of the presence of toxin. Importantly, the observed cytotoxic effects were specific for C. difficile toxin A and B, as a pre-incubation of the filter sterilized bacterial supernatants with anti-toxin, a polyclonal antibody against toxin A and toxin B (Techlab), resulted in complete neutralization of cytotoxic effects on the Vero cells at all time points (data not shown). The tcdC mutant strains showed no significant differences in toxin levels compared to the wild type strain (Fig. 3A) . Next, we used an enzyme immunoassay (Ridascreen, Biopharma) for the direct detection and relative quantification of the secreted toxins. In the exponential growth phase (5 and 8 hours post inoculation) no toxins were detectable (data not shown), consistent with the lack of toxicity towards Vero cells described above. In the stationary growth phase (12, 24 and 48 hours post inoculation) increasing toxin levels were detectable. When we compared the toxin levels at various time points, there were equal amounts of toxins in the wild type and tcdC mutant strains.
We conclude that the disruption of the tcdC gene does not result in consistently and significantly increased toxin levels.
C. difficile infections caused by the (hyper-)virulent Type 027 (NAP1/REA B1) and Type 078 (NAP7/REA BK) are associated with an increased morbidity and severity of disease compared to other types [1, 3] . This increase is suggested to be linked to toxin hyper production [3, 22, 28] . A potential mechanism by which this could occur is through inactivation of a negative regulator of the toxin gene transcription. TcdC has been identified as a negative regulator of toxin production [12] .
In the currently prevailing model, a major role for TcdC in the repression of toxin genes has been proposed on the basis of three lines of evidence. First, in C. difficile VPI10463 (a high toxin producing strain that also expresses high levels of TcdC [18, 29] ), an inverse correlation between the transcription of tcdC and the genes encoding the toxins is found [16, 18, 29] . This correlation for TcdC is also observed in protein levels [20] . Second, elegant in vitro experiments have established that heterologously produced and purified TcdC protein can interfere with TcdR-mediated transcription of toxin genes in a way that is not yet fully understood [12] . Finally, a frame shift mutation (D117 bp) in tcdC, that results in a non-functional protein, is associated with increased toxin production in certain (hyper)virulent strains [15, 22] .
Recently, it was reported that the introduction of a functional tcdC gene from a high toxin-producing strain that lacks any of the hyper virulence associated tcdC mutations (VPI10463, PCR ribotype 087) into an epidemic strain carrying a non-functional tcdC (M7404, PCR ribotype 027/NAP1/REA B1) can reduce toxin expression levels and moderately attenuate virulence [30] . This data seems consistent with the model discussed above. However, it is unclear how the levels of TcdC in the complemented strain relate to the physiological levels of the protein prior to the inactivation of TcdC in this strain background. The introduced tcdC gene, including its transcription signals, was derived from a different genetic background (VPI10463, Type 087) and was introduced on a multicopy plasmid. In addition, the reintroduction of TcdC in a strain lacking a functional TcdC, may affect processes that are not normally affected. Finally, the experiments were not corrected for the additional copies of the tcdC promoter that could result in the titration of regulators binding to those sequences.
In an alternative approach that addresses many of the issues above, the role of tcdC in toxin expression could be addressed by removing it from a background in which it is normally functional. To this end, we generated two independent isogenic ClosTronbased tcdC mutants strain that could be directly compared to its wild type counterpart, in which the TcdC protein was expected to be functional. Our data obtained with these mutant strains show that TcdC does not exert a major or even significant effect on the transcription of the PaLoc genes or the expression levels of the toxins under the conditions tested.
Our experiments were performed in a glucose free TY broth medium, since glucose is a known repressor of toxin production [17] . Indeed, we observed earlier and higher levels of toxin production in TY broth than in the commonly used Brain-Heat-Infusion broth (BHIS) based media, that does contain low amounts of glucose (0.2%) and to which frequently cysteine is added. However, also in BHIS we did not observe a significant effect of a tcdC deletion on toxin expression (data not shown).
We controlled critical parameters in our experiments by performing conventional PCRs which confirmed that the disruption of tcdC remained intact throughout the growth curve. Western blot analysis with antibodies raised against a TcdC epitope confirmed that the disruption of the tcdC gene resulted in the absence of TcdC protein ( Figure 1D ). The disruption of the tcdC gene did not affect the growth kinetics compared to the wild type strain ( Figure 1E ).
In the RT-qPCR experiments, sample to sample variation was corrected by normalizing to the reference gene rpsJ [31] . The rpsJ gene was selected for normalization, since rpsJ was overall the highest ranked reference gene regarding gene expression stability [31] .Reverse transcription was carried out using random hexamers, to prevent gene specific biases [32] . PCR efficiency in the qPCR was determined using a standard curve for each gene, enabling post run correction [33] . To obtain objective data concerning the quantification of the secreted toxins, we used an end point titer assay and an enzyme immunoassay rather than a manual (subjective) cell scoring system [26] .
The trends observed in the transcription of the PaLoc genes and the expression of the toxins generally conform to previously reported data [18, 19] . It should be noted that the up-regulation in time of tcdC transcription was not observed in earlier studies on C. difficile VPI10463 [16] but is consistent with more recent reports [18, 19] . We observed an increase in transcription of the PaLoc genes in time, and a concomitant increase in toxicity of culture supernatant in stationary phase that can be attributed to the toxins as it is fully neutralized by anti-toxin against toxin A and B.
The disruption of the tcdC gene resulted in an on average 1.7 fold higher transcription level of tcdC in time compared to the wild type strain, although this difference was not found to be statistically significant. It should be noted that we detect these differences because the real time PCR probe detects a region of the gene upstream of the ClosTron insertion site ( Figure 1A ). This finding might indicate some kind of feedback mechanism on TcdC expression. Similar to tcdC gene expression, the disruption of tcdC resulted in a slightly higher transcription level of the other PaLoc genes, although this was generally not significant. Moreover, the increased transcription level of the toxin genes did not result in generated a PCR product of 302 bp for the CT::tcdC strains. Primers tcdC1 and tcdC2 generated a 699 bp PCR product for the wild type and for the CT::tcdC strain a PCR product of circa 2800 bp. (C) Southern blot analysis of EcoRV digested genomic DNA of wild type and CT::tcdC strains with a Group II intron, ermB gene and tcdC specific probes. Note that probing with the ermB probe results in 2 bands for the CT::tcdC strains, since wild type already carries a copy of the ermB gene in the genome [35] . (D) Western blot analysis of TcdC production in wild type and CT::tcdC strain 8 hours post inoculation. The arrow indicates the location of TcdC protein based on MW and absence of the protein in the PaLoc negative Type 035 strain. Note that cross-reaction of TcdC antibody with a protein of similar MW was also observed in Carter et al. [30] . (E) Growth curves of C. difficile 630DErm and C. difficile CT::tcdC mutant strains. The absorbance (OD 600 ) was measured over 48 hrs of growth in TY medium. The error bars indicate the standard error of the mean of six experiments. doi:10.1371/journal.pone.0043247.g001 a detectable increase in toxin levels as measured with two independent assays.
Based on the paradigm that TcdC is a major suppressor of toxin production we expected precocious and significantly elevated transcription levels of tcdA, tcdB, tcdE and tcdR in the CT::tcdC strains compared to wild type. However, our data indicate that TcdC exerts a moderate, if any, effect on the transcriptional levels of the PaLoc genes and the expression of toxins in C. difficile 630Derm under the conditions tested.
Clostridium difficile strain 630DErm is a derivative of the clinical isolate 630 [34, 35] , a PCR ribotype 012 strain. PCR ribotypes 012 strains constitute 4% of the clinically isolated toxinogenic isolates in Europe [7] . Clostridium difficile 630 (PCR ribotype 012)-derived strains are commonly used to investigate virulence of mutants [26, 36, 37] .
An independent study, published during the preparation of this manuscript, reached a similar conclusion with respect to the role of TcdC in toxin regulation in C. difficile 630Derm using an allelic exchange technique [38] . In that paper reintroduction of a single functional copy of tcdC at its native locus did not affect toxin production in strain R20291 either [38] . R20291 is a strain from problematic PCR ribotype 027 (NAP1/REA B1) that was isolated following an outbreak in Stoke Mandeville, UK.
Our work and that of Cartman and coworkers [38] seem at odds with the previous reports that clearly demonstrate that TcdC can act as a repressor for toxin gene expression [12, 30] . However, we cannot exclude the possibility that TcdC exerts a more profound effect under specific conditions, or in other strains of C. difficile than 630Derm and R20291. It should be clear though that in vivo relevance of TcdC for toxin regulation in these two strains is limited.
In conclusion, we suggest that TcdC might have a modulatory role in regulating toxin expression, and that TcdC functionality is therefore not a major determinant of the (hyper)virulence of C. difficile. This is supported by the lack of correlation between virulence, toxin production and tcdC gene variants that was noted by several other studies [18, 19, 30, 39] .
The Clostridium difficile and Escherichia coli (E. coli) strains and plasmids used in this study are described in Table 1 . E. coli strains were grown in Luria Bertani (LB, USB cooperation) medium supplemented with appropriate antibiotics when required. C. difficile strains were grown anaerobically in a microaerobic cabinet (Don Whitley VA1000) at 37uC in pre-reduced 3% Bacto Tryptose (Difco), 2% Yeast extract (Difco) and 0.1% thioglycolate (pH 7.4) medium (TY) or Brain Heart Infusion broth (Oxoid) supplemented with 0.5% yeast extract and 0.01% L-cysteine (Sigma) (BHIS) [40, 41] . When required, the broths were supplemented with appropriated antibiotics. For RNA extraction and toxin quantification, C. difficile 630DErm (wild type) and two independent isogenic tcdC mutant strains (CT::tcdC) were serially diluted and pre-cultured (overnight) in pre-reduced TY broth. Mid-logarithmic growth phase pre-cultures (OD 600 0.4-0.8) were used to inoculate pre-reduced TY broth to a starting OD 600 of 0.05 (60.01). Optical density readings and samples for total toxin quantification were taken at 2, 3, 4, 5, 6 and 8 hours post inoculation in the exponential growth phase and at 12, 24 and 48 hours post inoculation in the stationary phase. Samples for RNA extraction were taken at 6, 8, 12, and 24 hours post inoculation. Samples for Western blot detection of TcdC were taken at 8 hours post inoculation. We routinely monitored the purity of the 
We generated two independent isogenic tcdC mutants by insertional inactivation of the tcdC gene in the wild type strain 630Derm using ClosTron technology [24, 25] . Briefly, the Perutka algorithm on the ClosTron website (http://www.clostron.com) was used to design primers ( Table 2) for retargeting the Group II intron (Sigma; Targetron). The retargeted intron was cloned using the restriction enzymes BsrGI and HindIII into plasmids pMTL007C-E2 and the constructs were verified by sequencing [25] . The verified plasmid (pDB001) was transformed to E. coli CA434 and transferred to the wild type strain 630Derm via conjugation [34, 41] . The selection of C. difficile transconjugants was done by subculturing on pre-reduced BHIS agar supplemented with thiamphenicol (Sigma; 10 mg/ml) and C. difficile selective supplement (Oxoid). This was followed by several rounds of subculturing on pre-reduced BHIS agar supplemented with lincomycin (Sigma; 20 mg/ml) and C. difficile selective supplement to promote integration of the GroupII intron into the gene of interest. Chromosomal DNA isolated from the transconjugants using a QIAamp blood kit (Qiagen) was used in conventional PCRs and sequence runs to confirm the disruption of tcdC and the nucleotide position of the insertion in the tcdC gene. Primers used for cloning and sequencing are listed in Table 2 .
Complementation can be a valuable control for knockout studies. However, as our tcdC mutant strains have no clearly detectable phenotype regarding toxin production, complemented mutant strains are expected to be comparable to wild type and tcdC mutant strains, as also reported recently in an independent study [38] . Therefore, a complementation study would not add to the message of this manuscript.
Southern blot analysis was performed to verify a specific single integration into the genome. Genomic DNA was extracted using a Phenol-chloroform extraction [42] . Four mg of genomic DNA was digested with EcoRV enzyme and separated on a 0.8% agarose/0.5xTris-acetate-EDTA gel by electrophoresis. DNA was transferred onto a Hybond N+ filter (Amersham) in 10x saline sodium citrate (SSC) solution. The filter was washed in 2X SSC and baked at 80uC for 2 hours. Prehybridization of the filter was done for 2 hrs at 60uC in 5x SCC, 5x Denhart and 100 mg/ml of yeast tRNA. Probes specific for the group II intron (EBS2-tcdC623as/Sal-R1), ermB gene (oWKS1131/oWKS1132) and tcdC gene (tcdC5-tcdC6) were generated. Primers are listed in Table 2 . The generated probes (100 ng) were radiolabeled ( 32 P dATP) using Klenow enzyme (Roche) and overnight hybridized in 10 ml fresh pre-hybridization buffer at 60uC. The filter was washed for 30 min in 2x SCC, 0.5% SDS, 30 min in 1X SSC, 0.5% SDS and 30 min in 0.5X SSC, 0.5% SDS and analyzed using phosphorimage screen and a Typhoon 9410 scanner (GE healthcare).
Antibodies against TcdC were generated by immunizing rabbits with a synthetic peptide (CQLARTPDDYKYKKV) representing a specific TcdC epitope (Genscript). Note that this epitope is located before the Clostron insertion site, and would therefore also be expected to detect truncated TcdC protein, would this be produced. Western blots were performed as follows. C. difficile (2 mL) cultures were harvested by centrifugation (2 min, 11.0006g, 4uC) and washed with Phosphate Buffered Saline (PBS). The bacterial pellets were resuspended in PBS containing protease inhibitor cocktail (Complete, Roche) and lysed by sonification. The bacterial lysates were centrifuged at low speed (3 min, 10006g, 4uC) to remove unbroken bacterial cells [20] . To separate the cytosolic proteins from the membrane associated proteins the bacterial supernatant was centrifuged at 200.0006g, 4uC for 1 hr [20] . The pelleted membrane associated proteins were resuspended in 10 mM Tris-HCl (pH 7.4), 5 mM EDTA with 2% Triton X-100 for 30 min at room temperature. Equal amounts of the resuspended membrane associated proteins were separated on 15% SDS PAGE gel and transferred onto polyvinyl difluoride (PVDF) membranes. Similarly generated membranes with the transferred membrane associated proteins of a Type 035 (PaLoc negative) strain were used for pre incubation of the TcdC antibodies. The membranes were probed with the pre incubated TcdC antibody and an antibody against the b subunit of the E. coli F 0 F 1 ATPase that cross reacts with the homologous protein in C. difficile [20, 43] . The probed membranes were analyzed using secondary anti-mouse horse radish peroxidase conjugated antibodies (Dako), a chemiluminescence detection kit (Amersham) and a Typhoon 9410 scanner (GE Healthcare).
Five mL of the C. difficile cultures were 1:1 diluted with ice cold methanol and stored overnight at 280uC. Bacterial pellets, obtained by centrifugation (20 min, 30006g, 4uC), were resuspended into 200 ml lysisbuffer (100 mM EDTA, 200 mM Tris-HCl pH 7.0, 50 mg/ml lysozyme) and incubated for 1 hr at 37uC. Tri-pure reagent (Roche) was used for the extraction of RNA according to the manufacturer's instruction with minor modifications. Briefly, 1 ml Tri-pure was added to the lysed bacterial pellets and incubated for 5 min at room temperature. Per 1 ml Tri-pure, 200 ml chloroform was added and carefully shaken by hand for 3 min, followed by an incubation of 2-5 min at room temperature. The aqueous phase was collected after centrifugation (12,0006g for 15 min at 4uC) and transferred to a fresh tube. RNA was precipitated by mixing the aqueous phase with 500 ml isopropanol, followed by an incubation of 10 min at room temperature. The precipitated RNA was collected by centrifugation (12,0006g, 10 min, 4uC) and resuspended in 100 ml DNase/ RNase free water. The RNA was re-precipitated overnight at 280uC with ammonium acetate (Fluka; 10 mM) and 3 volumes of absolute ethanol. The re-precipitated RNA was washed once with 80% ethanol and dissolved in 50 ml DNase/RNase free water. The RNA was treated twice with a TurboDNase (Ambion) according to the manufacturer's instruction followed by another Tri-pure RNA isolation. The quality and purity of the extracted RNA was assessed using a RNA nano chip on an Agilent Bioanalyzer.
A RevertAid TM H Minus Reverse Transcriptase kit (Fermentas) was used to synthesize cDNA according to the manufacturer's instruction. Random hexamers were used to convert 750 ng RNA into cDNA. The synthesized cDNA was treated with RNase (Qiagen) for 1 hour at 37uC and stored at 220uC. The software program Molecular Beacon (Premier Biosoft) was used to design primer pairs and probes (Table 2) for the 2 multiplex quantitative PCRs (qPCR), based on the available genome of C. difficile strain 630 [35] . All primer pairs were first tested by conventional PCR and multiplex PCR to confirm specificity and amplicon sizes. The primer pair and the probe for the amplification of the tcdC gene are in front of the insertion site in the tcdC gene ( Figure 1A ), allowing detection of tcdC transcription levels in wild type and CT::tcdC strains. The real-time multiplex qPCR amplification of the PaLoc genes and the reference gene encoding for a ribosomal protein (rpsJ) was performed on a CFX96 real-time PCR detection system (Biorad) [31] . The amplification efficiencies of the PaLoc and reference genes were determined using serially diluted genomic DNA (standard curve). The manually calculated efficiencies and the reference gene rpsJ were used to normalize the expression levels of the PaLoc genes. The amplification was performed in a 25 ml final volume. The first real-time multiplex qPCR (target genes: tcdA, tcdA and tcdC) contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 2.5 mM MgCl 2 , 100 nM of each probe and 2 ml synthesized cDNA. The second multiplex real-time multiplex Q-PCR (target genes: tcdR, tcdE) contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 3.5 mM MgCl 2 , 100 nM of each probe and 2 ml synthesized cDNA. The real-time qPCR to quantify the reference gene rpsJ contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 2.5 mM MgCl 2 , 0.06% SYBRgreen (Sigma) and 2 ml synthesized cDNA. The real-time qPCR protocol included an enzyme activation step for 15 min at 95uC, followed by 50 cycles of amplification; 95uC for 30 sec, 52uC for 30 sec and 72uC for 30 sec.
Total toxin amounts were quantified using 2 assays; a toxin end point titer assay and a commercial available ELISA (Ridascreen, Biopharma). The supernatants of culture samples (1 mL) were collected after centrifugation (30 min, 30006g, 4uC), filter sterilized (0.45 mM cellulose acetate membrane) and stored at 4uC.
For the toxin end point titer assay, Vero cells were seeded into a 96 wells plate at a density of 1610 4 cells per well and incubated overnight at 37uC and 5% CO 2 . The filter sterilized supernatants of 5, 8, 12, 24 and 48 hours post inoculation (hpi) were diluted 2, 10 1 , 10 2 , 10 3 , 10 4 and 10 5 fold in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with penicillin 100 u/mL, streptomycin 100 U/mL, fetal calf serum(10%). Fifty ml of the dilutions were added onto the Vero cell monolayers and incubated for 1 hr at 37uC and 5% CO 2 . For the neutralization assay a 2-fold dilution of each tested time point (5, 8, 12, 24 and 48 hpi) was pre-incubated with a 1/100 diluted anti-toxin (Techlab) for 1 hr at 37uC and 5% CO 2 . After the pre-incubation, 50 ml was added onto the Vero cell monolayers. The incubated bacterial supernatants were aspirated off after one hour and replaced with 100 ml cell culture medium. After 24 hrs of incubation the end-point titer was determined of each diluted time point [26] . The end-point titer was defined as the first dilution at which the Vero Cell morphology was indistinguishable from the neutralized 2-fold diluted supernatants [26] . The enzyme immunoassay (Ridascreen, Biopharma) was performed according manufacture's protocol.
Statistical analysis was performed using the software package SPSS 18 (IBM). An independent sample t-test was employed to compare the strains at different time points. Childhood Tuberculosis Presenting with Haemophagocytic Syndrome Haemophagocytic syndrome is a life threatening complication of systemic infection resulting from an exaggerated immune response to a triggering agent. Prompt recognition and treatment of this disorder can abrogate otherwise high fatality associated with this disorder. A 2 year old girl presented with acute enteritis, developed prolonged fever and organomegaly complicated by multi-organ failure. She fulfilled the diagnostic criteria for haemophagocytic lymphohistiocytosis including bone marrow evidence of haemophagocytosis. In addition she had serological evidence of tubercular infection as well as a positive family history of tuberculosis. She responded rapidly to immunosuppressive therapy and anti-tubercular therapy. Our case illustrates the association of haemophagocytic syndrome with tuberculosis as well as the favourable response obtained with prompt diagnosis and treatment. Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder caused by unrestrained proliferation and activity of the monocyte-macrophage system with phagocytosis of the mature and immature formed blood cells, release of inflammatory mediators, coagulopathy and often multiorgan failure. It has been described in all age groups, especially in the paediatric-adolescent population. Management usually consists of immunosuppressive agents along with treatment of the underlying condition. The HLH 2004 protocol consists of repeated cycles of cyclosporine-etoposidedexamethasone; however, sustained responses are rare, especially in familial HLH, and most patients eventually relapse [1] . Bone marrow transplant remains the only effective therapy for refractory cases but entails high procedure related mortality.
Various studies have reported 5 year survival rates of 50-60% for children with HLH, including familial and acquired forms [2, 3] . The diagnosis of familial HLH is often based on the age of onset, family history including a history of consanguinity, the clinical profile and/or co-existence of inherited immune deficiencies. Frequent relapses are common and these patients are usually candidates for BMT [4] . However, differentiation from early onset acquired HLH can be difficult. Absence of markers of immune deficiency (CHS, GS or XLP) or genetic perforin-granyzyme mutations does not rule out familial HLH. Acquired HLH has been described in association with collagen vascular disease (macrophage activation syndrome), post-transplant, malignancies especially T-cell lymphomas (lymphoma associated HLH) and infections (infection associated HLH). [5] . Both familial and secondary HLH are usually precipitated by an immunological trigger which may be an infectious agent or a drug. Among the infectious agents viruses especially Ebstein-Barr virus and Cytomegalovirus (virus associated HLH) are most commonly implicated, but bacterial, fungal and parasitic infections have also been described [6, 7] . With the possible exception of visceral leishmaniasis, immunomodulation is indicated in most cases [8] . Mycobacterium tuberculosis has been related to haemophagocytic syndrome in case reports from the Indian subcontinent, often with high mortality despite aggressive immunosuppressive therapy [1, [9] [10] [11] . We report a case of haemophagocytic syndrome related to mycobacterial infection which was managed with steroids and IVIG with complete clinical and haematological response.
The patient was a 2-year-old female with an unremarkable past, perinatal or family history. She was admitted with fever and diarrhoea of 2 days duration. She was managed with broad spectrum antibiotics, hydration and other supportive measures. High grade fever persisted along with progressive hepatosplenomegaly; on the 10th day of admission she developed ascites, respiratory distress and bilateral ptosis. Chest X-ray revealed bilateral pulmonary infiltrates suggestive of Acute respiratory distress syndrome. Peripheral blood counts revealed anaemia (7.6 gm/dl) and thrombocytopenia (87 9 10 3 /ll). Leucopenia (total leucocyte count 2.4 9 10 3 /ll, absolute neutrophil count 1.1 9 10 3 /ll) developed 4-5 days later. The coagulation profile was deranged with prolonged PT (32 s, INR 3.02) and APTT (39 s) in the absence of overt bleeding. D-dimer was positive. Serum triglycerides were 457 mg/dl, serum ferritin was 1,331 ng/ml and LDH was 1,889 IU/l. Bone marrow aspiration and biopsy revealed prominence of macrophages and histiocytes and phagocytosis of mature myeloid and lymphoid elements (Fig. 1 ). In addition, ELISA (IgM) for M tuberculosis was unequivocally positive at 1.08 U/ml (normal \ 0.90 U/ml) while IgG (0.18 U/ml, normal \ 0.90) and IgA (45.53 U/ml, normal \ 300) were negative, suggestive of acute Tubercular infection. Mantoux test was negative; tests for HBV, HCV and HIV were negative.
Transaminases showed a twofold increase (AST 74 IU/l, ALT 87 IU/l) with normal bilirubin levels and normal renal function tests. Based on the fulfilment of 6/8 HLH-2004 criteria, namely fever, splenomegaly, cytopenias, hypertriglyceridemia, hyperferritinemia and bone marrow findings, a diagnosis of Haemophagocytic syndrome was made (Infection Associated HLH) [1] .
Immunosuppressive therapy was initiated immediately after bone marrow studies. Methylprednisoslone (30 mg/ kg/day 9 3 days) followed by IVIG (1 gm/kg/day 9 2days) were used initially. HLH protocol was held in abeyance in the event of relapse of cytopenia or persistent fever. The patient was also exhibited anti-tubercular therapy consiting of isoniazid, rifampin, ethambutol and pyrazinamide. With the above treatment the patient responded rapidly; respiratory distress resolved within 24-48 h with resolution of radiological findings on follow-up X-ray chest. High grade fever settled within 24 h, organomegaly resolved over 7-10 days. Cytopenias also resolved over 4-5 days as did biological markers of Haemophagocytic Syndrome. The child was discharged on the 16th day of methylprednisolone and is on regular follow-up with no recurrence of symptoms and normal blood counts.
HLH is a distinct clinical entity characterised by fever, pancytopenia, splenomegaly and haemophagocytosis in bone marrow, spleen, liver or lymph nodes. Laboratory investigations usually reveal high triglyceride and ferritin levels, impaired NK and cytotoxic T-cell function and low fibrinogen. It is a syndrome of macrophage activation, usually secondary to an immunological trigger, resulting in phagocytosis of mature and immature red cells, myeloid elements and platelets. In addition there is intense immune system activation causing release of inflammatory mediators IFNc, TNFa, IL-6, IL-10; Th-1 responses and organ system damage.
The case described could well have been familial HLH, especially in view of age of onset. However, absence of a history of consanguinity, demonstration of recent mycobacterial infection and prompt response to treatment suggest infection associated HLH. The patient presented with prolonged fever complicated by organomegaly, cytopenias and ARDS, was investigated and treated promptly with good response to treatment. In a study of HLH in children a median age of onset of 17.4 months was described with average duration of fever ranging from 6 to 14 days. Our patient had onset at 24 months of age with fever duration of 10 days before developing symptoms. The patient was diagnosed and treated early at the 11th day of admission as against a median of 19 days described in Western literature [12] . Haemophagocytic syndrome related to childhood tuberculosis has been reported previously, in this patient the diagnosis remained presumptive based on the ELISA, positive family history and rapidity of response to ATT and immunosuppression [10] .
Neurological signs described in HLH are encephalopathy, meningism, hypotonia, hemiplegia and seizures [2, 13] . Our patient developed bilateral ptosis which eventually resolved over 2-3 weeks. Phagocytosis, reportedly, most affects the red cells and platelets, however, in our case the majority of the ingested cells were of the myeloid and lymphoid lineages [14] .
The classical picture of florid haemophagocytosis is usually not seen in the initial bone marrow and develops over the course of the illness. In this case bone marrow biopsy was not repeated as the parents were unwilling and blood counts rapidly normalised along with signs and symptoms. EBV infection markers were not available at this centre and hence not performed.
Our patient had a favourable clinical outcome possibly due to early diagnosis and prompt initiation of specific treatment. A high index of suspicion is required for such cases as it may be an important cause of FUO [12] . Infection associated HLH related to tuberculosis is a treatable disorder with early immunosuppressive therapy. Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures. As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury. Mammalian lungs are designed to optimize exposure of blood to oxygen. To achieve this, two intertwined and highly branched tree-like tubular systems, one conducting air and the other conducting blood must develop in a coordinated way to generate the millions of functional alveolar gas-exchange units [1, 2, 3] .
Lung development in the mouse begins on embryonic day 9.5 (E9.5) when the lung primordium appears as a ventral bud in the primitive foregut [4] . Airway branching begins around E9. [5] [6] [7] [8] [9] [10] [11] [12] and continues through the ''pseudoglandular'' [E12-E16.5] and ''canalicular'' [E16.5-E17.5] stages. Thereafter, during the ''saccular'' stage [E17.5 to postnatal day 4 (P4)] the distal airways form the saccular units which are further subdivided by secondary septae formed during the alveolar stage (P4-P28 in mice) to form mature alveoli. This sequence of events is tightly controlled by the concerted action of growth factors, transcription factors, and mechanical forces [5, 6, 7] .
Prominent role in the regulation of lung development and homeostasis is played by members of the Bone Morphogenetic Protein (BMP) family [8] . BMPs, like all other members of the TGFb superfamily, signal via specific membrane receptors that have serine-threonine kinase catalytic activity [9] . Functional BMP receptor units are composed of two Type-I and two Type-II receptor polypeptides. Four different Type-I BMP receptors (ALK2, ALK3/BMPRIa, ALK6/BMPRIb and ALK1), and three Type-II receptors (BMPRII, ActRIIA and ActRIIB) have been identified [10] . Upon ligand binding, the constitutively active Type-II receptors phosphorylate and thus activate their Type-I partners, which in turn phosphorylate their intracellular targets, the receptor-regulated Smad proteins 1, 5 and 8. Phosphorylated Smads form complexes with the ''common'' Smad4 and translocate to the nucleus where they regulate expression of their target genes, synergistically with other transcription factors [8, 11] . BMPs can also signal via Smad-independent intracellular pathways that involve mitogen-activated protein (MAP) kinases [12, 13] .
Several studies using transgenic and conventional or conditional knock-out mice have clearly demonstrated the key role played by BMPs during early lung development [14, 15, 16, 17, 18, 19, 20, 21] . Disruption of BMP signaling by ectopically expressing the BMP antagonists noggin or gremlin in the lung epithelium [15, 22] , inactivating BMP receptors [16] or expressing a dominant negative form of the BMP Type-I receptor (dnALK6) result in abnormal distal lung architecture. Remarkably, over-activation of the BMP pathway is also incompatible with normal lung development. Ectopic over-expression of Bmp4 in the epithelium leads to smaller lungs and to substantially reduced epithelial cell proliferation [14] and mice with deletion of the BMP antagonist Follistatin-Like 1 (Fstl1) gene die at birth from respiratory distress and show multiple defects in lung development [23, 24] .
Moreover, increasing evidence supports a key role for BMP signaling angiogenesis and vasculogenesis in the lung [25, 26] . A large number of genetically modified mice with lesions in genes encoding either BMP ligands, receptors or antagonists exhibit defective angiogenesis. The importance of BMP signaling for the vascular system is demonstrated by the association of mutations in genes encoding for BMPRII and ALK1 with the development of two genetic vascular diseases, namely, pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia.
The BMP signaling pathway has also been implicated in the regulation of adult lung homeostasis and tissue repair following injury [27, 28, 29, 30, 31] . Several BMP ligands have been found upregulated in allergen challenged lungs [27] and notably, ectopic expression of gremlin by adenovirus mediated gene transfer in the lung of adult rats causes severe pulmonary fibrosis [32] illustrating the importance of the BMP pathway for lung homeostasis as well. Consistently, over-expression of gremlin has been observed in humans suffering from Idiopathic Pulmonary Fibrosis (IPF) [28, 29] .
Despite the significant progress, the precise mechanism(s) by which BMPs regulate lung development have not been fully delineated [5] . Moreover, characterization of the mechanisms by which BMPs affect adult lung homeostasis and tissue repair is still rudimentary. The current study was based on the premise that identification of the actual cellular targets of BMP mediated signaling during lung development, homeostasis and adult lung tissue repair will facilitate interpretation of earlier genetic studies, guide rational targeting of BMP signaling components in future experiments and contribute to the clarification of the mechanisms of action of this signaling system. Therefore, utilizing a mouse transgenic line carrying the eGFP reporter gene under the control of a canonical BMP pathway responsive regulatory element [33] we have constructed a detailed spatiotemporal map of canonical BMP signaling in the lung and identified putative cellular targets of this signaling pathway during lung development and adult lung tissue-injury repair. Our studies suggest that canonical BMP pathway plays key roles during early development of the pulmonary vasculature and the management of lung epithelial cell progenitors during late lung development and repair of adult lung tissue injury.
Animals were housed in individually ventilated cages under specific pathogen free conditions, in full compliance with FELASA (Federation of Laboratory Animal Science Associations) recommendations, at the Animal House Facility of the Foundation for Biomedical Research of the Academy of Athens (Athens, Greece). All procedures for care and treatment of animals were approved by the Institutional Committee on Ethics of Animal Experiments and the Greek Ministry of Agriculture (Permit Number: K/1054).
BMP-responsive eGFP expressing (BRE-eGFP) mice [33] kept in the C57BL/6 background maintained on a 12:12 hours light:dark cycle at the Animal Facility of the Foundation for Biomedical Research of the Academy of Athens.
Collection of fetal lung tissues was carried out at the embryonic (E11 and E12) pseudoglandular (E13.5 and E14.5), canalicular (E17) and saccular (E19.5) stages. Noon of the day of the vaginal plug was considered embryonic day (E) 0.5. Additionally, tissues were collected on post-natal days 1 (P1), 15 (P15) and 2 months after birth (adult). All embryonic and P1 lungs were removed surgically, cleaned from the surrounding tissues and placed in 4% paraformaldehyde (Merck, 104004) in PBS for 24 hours at 4uC. For analysis of P15 and adult BRE mice the right lung-lobes were used for RNA/Protein isolation, whereas the left lungs were gently perfused with a mixture of 4% PFA:OCT (2:1) using a 20G catheter (Abbocath G717-A01, 4535-20), the bronchus was ligated and placed in 4% PFA for 24 hours at 4uC. Thereafter, the tissues were placed in PBS with 30% sucrose for 24 hours at 4uC, washed with PBS, embedded in OCT (Shandon Cryomatrix) and kept at 280uC until use. Cryostat sections 6-10 mm (Leica CM3050S) were placed on polylysine or SuperFrost Plus slides (Menzel Glaser), kept at room temperature for 3 hours with silica gel (Merck, 101969) and then stored at 280uC. The primary antibodies used for immunostaining were: goat anti-CC10 (Santa Cruz, SC9773), rat anti-PECAM1 (CD31) (BD-Biosciences, 550274), rat anti-VEGFR2 (eBiosiences 14-5821), rabbit anti-SpC (Chemicon, AB3786), rabbit anti-CGRP (Sigma-Aldrich, C8198), mouse anti-a smooth muscle actin-Cy3 (Sigma-Aldrich, C6198), Syrian hamster anti-T1a (DSHB 8.1.1), Sheep anti-BrdU (Fitzgerald, 20-BS17), mouse anti-Foxj1 (generous gift of Dr. S. Brody), mouse biotinylated anti-Muc5ac (Abcam, ab79082), anti-SM22alpha (Abcam, ab14106) and chicken anti-GFP (Abcam, ab13970). The secondary antibodies used were: donkey anti-goat Texas Red (Jackson Immuno-research, 705-076-147), donkey antichicken FITC (Jackson Immuno-research, 703-096-155), goat anti-rat AlexaFluor594 (Molecular Probes, A11007), goat anti-rat AlexaFluor647 (Molecular Probes, A21247), goat anti-Syrian hamster AlexaFluor568 (Molecular Probes, A21112), goat anti-Syrian hamster AlexaFluor647 (Molecular Probes, A21541), donkey anti-rabbit Texas-Red (Jackson Immuno-research, 711-076-152), donkey anti-sheep AlexaFluor568 (Molecular Probes, A21099) and donkey anti-rabbit Cy5 (Jackson Immuno-research, 711-176-152). The slides were mounted using the ProLong Gold Antifade Reagent with DAPI (Molecular Probes, P36931). Images were captured with a Leica DMRA2 fluorescent microscope equipped with a Leica DFC320 and DFC350 FX digital cameras and a Leica TCS-SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Image analysis was performed using Adobe Photoshop CS3, ImageJ 1.41 and Volocity LE. pSmad1/5/8 immuno-staining Tissue sections prepared as described in the previous section were treated 1.5% H 2 O 2 in MetOH for 30 min at RT in the dark to inactivate endogenous peroxidise. After blocking with 2% normal donkey serum (Sigma D9663) in TBS-Tween 0.3% for 2 hours at room temperature, the sections were incubated with rabbit anti-pSmad1/5/8 (Chemicon, ab3848) antibody overnight at 4uC in TBS-Tween 0.3% and then incubated for 1 hour at room temperature with the secondary anti-Rabbit HRP (Santa Cruz, SC2301). The slides were developed using DAB chromogen (DAKO K3468) development according to the manufacturer's instructions. Normal rabbit IgG fraction was used as control primary antibody (isotype control). Nuclei were counterstained with Mayer's Hematoxylene.
Total RNA from mouse lungs was isolated using the Tri-Reagent protocol (Sigma, St. Louis, MO) and the yield and purity of RNA was determined electrophoretically and spectrophotometrically. After DNase treatment with RQ1 RNase-Free DNase (Promega, M160A), 2 mg of RNA were reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, M170B) and random primers (Invitrogen, 58875) according to the manufacturer's instructions. The primer pairs for real-time PCR were designed using Beacon Designer v7.01 software (Premier Biosoft International, Palo Alto, CA). Sequences for the primer pairs used are given in table S1. The PCR reactions contained SYBRH Green ER TM qPCR Super Mix Universal (Invitrogen, 11762-500), 200 nM of each primer (Invitrogen, Carlsbad), and 0.2 ml of cDNA template in a 20 ml reaction volume. RT-qPCR cycling parameters were initial incubation at 50uC for 2 minutes, denaturation at 95uC for 10 minutes followed by 50 cycles of 15 seconds at 95uC and 40 seconds at 60uC. Data collection was carried out using a Chromo4 Real-Time PCR detector (BioRad) and analyzed with Opticon Monitor 3 expression analysis software (BioRad). Relative levels of mRNA expression were calculated according to the DDCt method [34] .
For protein extraction cells were homogenized in lysis buffer containing 50 mM Tris-Cl pH7.6, 150 mM NaCl, 1% (v/v) Triton 6100, 5 mM EDTA pH 8.0, 1 mM PMSF, 1:25 (v/v), Protease Inhibitor (Roche Applied Science, 11836153001), 2 mM Na 3 VO 4 , 5 mM NaF, 2 mM Sodium Pyrophosphate, 13.3 mM b-Glycerophosphate disodium salt. Homogenized cells were centrifuged at 13.000 rpm for 15 min at 4uC and protein samples were stored at 220uC until use. For detection of pSmad1/5/8 separated proteins were transferred to Immobilon-P membrane (Millipore, IPVH00010) and analyzed using rabbit anti-pSmad1/ 5/8 (Chemicon, ab3848, 1:1000) coupled with the anti-rabbit HRP (Santa Cruz SC2301, 1:5.000) and anti-b actin (Sigma-Aldrich, A5316, 1:5.000) combined with the anti-mouse HRP (Sigma-Aldrich, A4416, 1:40.000). Densitometry was done using the ImageJ 1.41 software.
In-vivo lung tissue-injury/repair models Female BRE-eGFP animals, between 12-16 weeks of age were treated with naphthalene or bleomycin. For naphthalene treatment, 300 mg/kg naphthalene in corn oil (vehicle) or vehicle alone was administered by intra-peritoneal injection. Lung tissues were collected on days 2, 3, 6, 9 post naphthalene-treatment. For bleomycin treatment, bleomycin (0.033 Units/mouse, Nippon Kayaku co ltd, Bleocin) diluted in PBS or PBS alone (vehicle) was administered intra-tracheally in 30 ml total volume. Lungs were collected on days 2, 4 and 12 post-bleomycin-treatment.
Ex-vivo culture of fetal lung explants Transgenic E12.5 lung explants were isolated and cultured, for 8 or 24 hours, in a 1:1 mixture of DMEM: F12 (GiBCO, 11039), supplemented with Insulin-Transferrin-Selenium (Gibco, 51300-044) and antibiotics. Explants were placed on Nuclepore Track-Etched Membranes (Whatman, 110414) and they were incubated in 4-well plates (Thermo Scientific, 144444). LDN193189 (Axon MedChem, Axon 1509) and SB431542 (Sigma-Aldrich, S4317) were used at a concentration of 10 mM. Untreated and vehicle (DMSO, Sigma-Aldrich, D2650) treated lungs were used as controls. In some experiments, E12.5 lung explants were treated with 100 ng/ml BMP4 (Peprotech, 120-05ET), or 250 ng/ml FGF10 (Peprotech, 100-26) or 500 nM Sonic Hedgehog (Shh) agonist-Purmorphamin (Calbiochem, 540220) for 8 hours.
Endodermal buds and distal lung mesenchyme were isolated from E12.5 lungs as previously described [35, 36] . Briefly, isolated distal lung tips were treated for approximately 5 min with Trypsin 0.5% Pancreatin 2.5% in HBSS 16 (Gibco, 14170) on ice. Mesenchyme was removed using 26G needles and the epithelial buds as well as the isolated mesenchyme were placed in a (1:1) mixture of Growth Factor Reduced Matrigel (BD Biosciences, 356230) and DMEM: F12 (GiBCO, 11039) 10% FCS (Gibco, 10500), Pen Strep (Gibco, 15070, 1:100) and incubated for 8 hours at 37uC, 5% CO 2 .
Adult primary lung cells were isolated as described in [37] with small modifications. Briefly, lungs were isolated, cut in small pieces and digested over-night at 4uC with 0.1% protease type-XIV (Sigma-Aldrich, P5149) in Joklik's MEM (Sigma Aldrich, 8028) containing antibiotics (Gibco, 15070). Lung pieces were transferred in Joklik's MEM supplemented with 10% FBS (Gibco 10500), pipetted gently several times to release cells and passed through 70 mm cell strainer (BD Biosciences, 352350). Cells were washed with MCDB-201 (Sigma-Aldrich, M6770) containing Insulin-Transferrin-Selenium (Gibco, 51300-044) and viability was measured using Trypan Blue. 10 6 cells (10 6 /ml) were cultured in 12-well plates (Nunc, 150628) coated with Bovine collagen (Nutacon, 5409) for 24 hours. Coating was done with 100 mg/cm 2 collagen, for 1 hour at 37uC. Thereafter, cultures were rinsed with MCDB-201 to remove detached cells and 500 ml MCDB-201 with Insulin-Transferrin-Selenium and 1 ng/ml EGF (Peprotech, 315-09) was added. After 1 week, colonies of epithelial cells were formed and infected with recombinant adenoviruses expressing either constitutively-activated ALK3 (caALK3), caALK5, Smad1 or Smad3. After 48 hours incubation the treated cells were used either for cytochemistry or for RNA extraction.
P3 mice were anesthetized, the abdominal cavity was opened and ventricular vein and artery were cut. The chest was opened and the right atrium nicked. Lungs were perfused through the right ventricle of the heart with 1 ml ice-cold 16PBS using a 26G syringe, cut in small pieces using a razor blade and digested with the Enzyme mix [Elastase (Sigma-Aldrich, 45124): 3 U/ml, Collagenase: 0.1 U/ml, Dispase: 0.8 U/ml (Roche Applied Science, 11097113001) and DnaseI 50 mg/ml (Sigma-Aldrich, DN25] in PBS, at 37uC for 1 hour with rotation. Equal volume of HBSS ++ [HBSS 16 (Gibco, 14170) , supplemented with 2% FCS (Gibco, 10500), 0.1 M HEPES (Sigma-Aldrich, H0887) and 15% Cell Dissociation Buffer (Gibco, 1315-016) or EGTA 2 mM] was added. Cells were gently agitated several times, passed through a 40 mm cell strainer (BD Biosciences, 352340) and centrifuged 10 minutes at 1200 rpm, at 4uC. The supernatant was discarded and cells were re-suspended in HBSS ++ and counted with a hemocytometer. Specific subpopulations of lung cells were isolated using a BD FACS-Aria IIu cell sorter by staining the isolated lung cells as previously described by Teisanu et al. [38] with biotinylated anti-CD31 (BD Biosciences, 553371), anti-CD45 (Biolegend, 103103) and anti-CD34 (eBioscience, 13-0341-85) combined with Streptavidin APCCy7 (Biolegend, 405208), with anti-Sca1-Alexa Fluor647 (Biolegend, 108118) andanti-EpCam-PECy7 (Biolegend, 118216). DAPI was included for dead cell exclusion. The EpCam pos -CD31 neg -CD45 neg -CD34 neg cells were separated into Sca1 neg and Sca1 low and each of these two populations were separated by sorting into eGFP neg , eGFP low and eGFP high . Isolated cells were used either for further in-vitro cultivation, RNA expression or Western Blot analysis.
In vitro cultivation of sorted epithelial cells in Matrigel was done as previously described [38] with minor modifications. Selenium (Gibco, 51300, 1:100), Pen Strep (Gibco, 15070, 1:100), amphotericin B (Gibco, 15290, 1:1000) and 10 mM SB431542 (Sigma-Aldrich, S4317). Culture medium was changed every other day until day 8. SB431542 was then removed and the cultures were maintained for an additional four days before fixing overnight at 4uC with 4% PFA in PBS. The Matrigel was removed by freezing-thawing and rinsing with fresh PBS. The content of the Transwell inserts was embedded in OCT (Shandon Cryomatrix) and 10 mm sections were prepared by cryostat (Leica CM1950).
Although epithelial colonies developed in Matrigel even in the absence of SB431542 its presence resulted in higher number of larger colonies and therefore this culture system was preferred. Treatment of the Mlg cells with SB431542 for 24 hours led to substantial increase in FGF10 mRNA synthesis (data not shown) suggesting that stroma cell derived FGF10 may be part of the underlying mechanism.
One way analysis of variance (One-way ANOVA) combined with ''Bonferoni's Multiple Comparison Test'', was done using GraphPad Prism. (*/ # ) P,0.05, (**/ ## ) P,0.01, (***/ ### ) P,0.001.
Spatiotemporal pattern of expression of the BRE-eGFP reporter during lung development Previous studies utilizing two independent canonical BMP pathway reporter mouse lines, one harboring a BRE-eGFP [33] and another harboring a BRE-lacZ allele [39] have demonstrated expression of the BRE-eGFP and BRE-lacZ reporters respectively in the epithelium of trachea and primary-bronchi already at around E10-E12.5. To extend these findings and map in detail the spatiotemporal pattern of canonical BMP signaling in the lungs, we collected lung tissues from the BRE-eGFP reporter mice at embryonic days E11, E12, E13.5, E14.5, E16, E17.5, E19.5 and postnatal days 1, 15 and 2 months and analyzed tissue sections for eGFP expression. Our analysis demonstrated that the BRE-eGFP reporter was active already at E11, reached maximal levels around birth, thereafter declining to minimal levels in the adult ( Figure 1 and Figure S1 ).
Around E11, eGFP expression was confined to the endothelial cells of the large pulmonary vessels, and the developing vascular network in the parenchyma. A very small fraction of sub-epithelial smooth muscle cells (SMCs) expressed very low levels of eGFP at E12 ( Figure S1 ). However, from E13.5 onwards robust eGFP expression was detected in sub-epithelial SMCs located at the proximal regions of the developing airways (Figures 1 and Figures  S1 and S2) .
From E16 onwards, expression of eGFP was detected in both proximal and distal epithelial compartments.In the proximal epithelium (i.e. the developing airways) eGFP expression exhibited a proximal-distal axis pattern, with high eGFP expression in bronchi and large airways and surprisingly no expression at the distal portion of the growing airway tree. Interestingly, isolated clusters of eGFP pos cells were observed at the tips of airway branch-points. In the distal lung compartment, eGFP expression was detected in cuboidal epithelial cells of the developing saccules ( Figure 1B ). As lung morphogenesis approached completion, the eGFP signal was reduced and in adult mice,(,2 months old), few eGFP pos cells were detected scattered in the epithelium of bronchi and large airways, in the alveolar compartment and in a subpopulation of cardiac cells in the outer layer of the tunica media of the pulmonary veins ( Figure 1B ) [40] .
Although the capacity of BRE element to monitor BMP mediated transcriptional activation in-vivo has been demonstrated previously [33, 39, 41] , additional studies were undertaken to further increase confidence with respect to transgene expression in the lung. The kinetics of mRNA expression for eGFP and the BMP target gene Id1 were analyzed by quantitative PCR. EGFP and Id1 mRNA levels followed similar kinetics to eGFP protein expression, reaching maximum levels around birth ( Figure 2A ). P1 lungs were therefore enzymatically digested and lung cells were separated by cell sorting on the basis of eGFP expression into eGFP negative (eGFP neg ), eGFPlow (eGFP low ) and eGFP high (eGFP high ) cells ( Figure 2B ). Western blot analysis of protein extracts from the isolated population demonstrated good correlation between expression of eGFP and levels of pSmad1/5/8 ( Figure 2C , D). Analysis of the mRNA levels for eGFP and BMPregulated target genes such as Smad6, Id1 and Id3 demonstrated very good correlation between the level of eGFP expression and the activation of known BMP targets ( Figure 2E ). Interestingly, expression of Id2 mRNA did not correlate with levels of eGFP expression. Furthermore, treatment of embryonic lung explants exvivo with either BMP-or TGFb-receptor inhibitors resulted in either suppression or augmentation of BRE-eGFP transgene expression, respectively (see below Figure 3B ). Moreover, treatment of embryonic lung explants with FGF10, a known inducer of BMP4 production by distal endodermal cells, or recombinant BMP4 resulted in substantial up-regulation of eGFP expression ( Figure S3 ). Treatment of the lung explants with purmorphamin, a Shh agonist, resulted in redistribution of the eGFP staining pattern that involved a small increase of eGFP expression in a narrow zone of sub-mesothelial cells and a reduction of eGFP expression in the more central parts of the mesenchyme. This pattern of expression could stem from a Shh mediated reduction in FGF10 synthesis by mesenchymal cells [35] that in turn leads to a more confined BMP expression zone.
Too further validate the BRE-eGFP reporter animals and assess its sensitivity in comparison to staining tissues for nuclear pSmad1/5/8 by immuno-histochemistry, we stained adjacent tissue sections of P1 lungs, the developmental stage demonstrating maximal BRE-eGFP reporter activity, with anti-eGFP and anti-pSmad1/5/8 antibodies. As shown in Figure S4 , nuclear pSmad1/5/8 was detected in regions with intense eGFP immuno-staining demonstrating consistency between BRE-eGFP reporter activity and pSmad1/5/8 immuno-staining ( figure S4 ). Unfortunately, in our hands the anti-pSmad1,5,8 reagents we have analyzed so far were unable to detect pSmad1/5/8 in lung tissues from embryos before stage E19.5 and thus we were not able to confirm correlation of eGFP and pSmad1/5/8 at earlier developmental stages. A number of studies have indicated that the value of measuring pSmad1/5/8 as the sole indicator for canonical BMP pathway activation is questionable since presence of pSmad1/5/8 does not necessarily lead to transcriptional activity of BMP target genes. Canonical BMP pathway activity is known to be regulated by cell type-specific Smad-interacting transcription factors, co-activators and co-repressors. For example Tbx1 can bind to Smad1, interfere with Smad1/Smad4 interaction and decrease BMP transcriptional activity without affecting the amount of pSmad1/5/8 in the responding cell [42] . Furthermore, Leeuwis et al. [41] demonstrated that in the kidney of BRE-eGFP animals, whereas the expression pattern of BRE-eGFP exhibited excellent correlation with the expression of the BMP7 and the BMP target genes Id1, and Smad6, in contrast pSmad1/5/8 exhibited a brought expression pattern that did not correlate with the expression of BRE-eGFP, BMP7, Id1 or Smad6. Therefore, a safer assessment of canonical BMP pathway should combine analysis of several known BMP target genes and pSmad1/5/8 immuno-stainings when this is technically possible. Collectively, the analysis by Monteiro et al. [33] , Leeuwis et al. [41] and the results described herein indicate that the BRE-eGFP transgenic line reports quite faithfully activation of the canonical BMP pathway in-vivo and ex-vivo and thus it provides an additional method for assessing potential canonical BMP pathway activation.
Canonical BMP-pathway is activated in the developing vascular and sub-epithelial smooth muscle networks during early lung development
To identify the putative cellular targets of canonical BMP signaling, tissue sections collected from lungs at different developmental stages were stained with antibodies specific for appropriate cellular markers. Double immunostaining for eGFP and CD31 (PECAM-1), a surface marker of mature endothelium, or VEGFR2, a surface marker of vascular progenitor cells ( Figure  S1 ), demonstrated that the majority of CD31 pos or VEGFR2 pos cells at E12, E14.5 and E17 were also eGFP pos ( Figure 3A ), confirming that the network of eGFP pos cells in the parenchyma is composed of vascular cells and that the canonical BMP pathway is activated during the establishment of the vascular network in the developing lung. At P1, ,40% of the CD31 pos cells were eGFP pos , from P15 onwards the number of eGFP pos -CD31 pos cells declined and in the adult they were undetectable. EGFP expression with similar kinetics was observed in the endothelial cells lining the large blood vessels ( Figure 3A ). Very few eGFP pos alpha smooth muscle actin (aSMA) double positive cells were found in the walls of the vessels ( Figure 3A , white arrow in P1 vessel).
To validate the functional significance of BMP signaling for the development of the vascular network of the developing lung, ex-vivo cultures of E12 lung explants were treated with the respective BMP-or TGFb/Activin-receptor inhibitors LDN193189 and SB431542. Treatment of lung explants for 24 hours with SB431542 led to a substantial increase in eGFP, Id1, and FGF10 mRNA expression, whereas treatment with LDN193189 led to a decrease ineGFP, Id1 and FGF10 mRNA expression and an increase, probably compensatory, in BMP4 and VEGFa mRNA expression ( Figure 3B and D). Remarkably, even 8 hours of treatment with the SB431542 led to an increase in the density of the CD31 pos network and a more pronounced increase in the eGFP pos network, whereas, similar treatment with LDN193189 led to reduction in the density and connectivity of the eGFP pos and CD31 pos cells ( Figure 3C) .
Collectively, these findings indicate that during the pseudoglandular stage, in the course of which most of the stereotypical branching morphogenesis is thought to take place, an important target of canonical BMP signaling in the lung is the developing pulmonary vasculature.
Strong eGFP expression was detected in the developingsubepithelial SMC layer of the proximal airways from E14.5 to P1 ( Figure 3A and Figures S1 & S2) . The eGFP expression, which interestingly involved only a portion of the sub-epithelial SMCs, was undetectable by P15. It has been proposed that sub-epithelial SMCs originate from a pool of sub-mesothelial, FGF10-expressing, mesenchymal cells. Under the influence of several signalling pathways, such as those mediated by FGF, BMP, Shh, and Wnt, these cells proliferate and passively translocate to surround the proximal portion of the developing airway tree [35, 43, 44, 45, 46] . http://www.sciencedirect.com/science/article/pii/S0012160611 010189 -bb0125To map more precisely the temporal activation of the BRE-eGFP reporter during development of the subepithelial SMC compartment we isolated P11, P12 and P13.5 lungs from the BRE-eGFP reporter animals and analysed coexpression of eGFP and SM22a, a marker of both primitive and mature SMCs [47] , or aSMA a marker of more differentiated SMCs. As shown in Figure S1B , SM22a expression was observed in a sub-epithelial population of cells around the developing proximal airways already at embryonic stage E11, aSMA expression was evident only in the most proximal portion of the SM22a pos zone and eGFP expression was clearly detected in the aSMA pos zone only after embryonic stage E13.5 (very few aSMA pos cells expressed very low levels of eGFP at E12). These findings are consistent with the notion that canonical BMP signalling does not involve primitive SMCs but rather mature aSMA expressing sub-epithelial SMCs.
Canonical BMP pathway activation in developing airway epithelial cells Although strong BRE activity was detected in the trachea and primary bronchi as early as E10-E12.5 [33] , consistent with the demonstrated role of BMP signaling in trachea development [48, 49] , BMP-driven eGFP-expression was not detected in the epithelial compartments of the interlobular airways before the canalicular stage of lung development. As shown in Figures 1 and  4A , minimal eGFP expression was detected around E14.5 in the large airways, and only after E17 strong eGFP signal was detected in cartilaginous and large airways following a proximal-distal axis pattern, with more intense signal in the proximal part of the bronchial tree. Immunostaining of BRE-eGFP lung tissue sections with an antibody specific for the Clara cell specific differentiation marker Scgb1a1/CCSP/CC10 (hereafter referred to as CC10), revealed expression of the eGFP reporter from E17.5 and onwards, in luminal cells of the developing airways. Strong CC10 expression was detected around E19.5, approximately two days after the onset of eGFP expression in the airway epithelium ( Figure 4A ) CC10 and eGFP exhibited partially overlapping zones of expression, with eGFP being highly expressed in the proximal portion and CC10 highly expressed in the distal portion of the developing airway tree. A zone of eGFP pos -CC10 pos cells appeared to separate the CC10 pos epithelial population from the eGFP pos airway regions. Postnatal expansion of CC10 pos regions in the conducting airways was accompanied by a gradual reduction of the eGFP pos regions ( Figure 4A ).
Interestingly, regions of airways exhibiting enhanced eGFP expression were detected close to the tips of airway branch-points in close proximity to neuro-epithelial bodies (NEBs). Analysis of the eGFP expression in relation to CC10 and CGRP, a differentiation marker of neuro-epithelial cells, revealed that eGFP pos -CC10 low cells formed ''caps'' over clusters of CGRP pos cells. From E19.5, the eGFP pos -CC10 low ''cap'' cells (green-arrows in Figure 4B ) were separated from the eGFP neg -CC10 pos cells by a zone of double positive epithelial cells (yellow-arrows in Figure 4B) . Notably, the zone of eGFP pos -CC10 pos cells coincided with zones richin NEBs ( Figure S5 ). From P15 and beyond, the only NEBassociated eGFP pos cells were CGRP pos neuro-epithelial cells (blue-arrows in Figure 4B ).
As the majority of eGFP pos cells did not express the marker for secretory airway epithelium (CC10), their possible relationship to ciliated airway epithelial cells was investigated by staining with antibodies for Foxj1 a ciliated cell specific differentiation marker. As shown in Figure S6 , a remarkably small portion of eGFP pos cells were FoxJ1 pos (yellow-arrows in Figure S6 ).
Two alternative models have been proposed regarding the role of BMP signalling during lung branching morphogenesis. One model suggests that mesenchymal derived FGF10 induces Bmp4 expression by the distal bud epithelium to limit, in an autocrine manner, Fgf10-mediated bud outgrowth [45, 50] . The other model, based on the differential response of the distal epithelial buds to BMP4 in the presence or absence of mesenchyme, proposes that Bmp4 produced by the distal epithelium acts in an autocrine manner to limit proliferation of distal epithelial buds, and in a paracrine manner, on the adjacent mesenchyme, to induce production of a mesenchymal signal that enhances proliferation of distal epithelial buds [51] . Thus, according to the latter model the negative or positive effect of BMP signalling on branching is the outcome of a dynamic balance between negative autocrine and positive paracrine mechanism.
The absence of eGFP reporter activation in the distal buds of the branching airway tree (Figures 1 and 4) prompted us to investigate whether this is the result of an intrinsic inability of the distal epithelium to activate the canonical BMP pathway or, in accordance to the second model, the result of a dynamic interplay with the adjacent mesenchyme. Therefore, mesenchyme free distal epithelial buds and epithelial free mesenchyme were isolated as previously described [35, 36] and cultured in Matrigel. Remarkably, within 8 hours of incubation, the BRE-eGFP reporter was strongly up-regulated in the mesenchyme-free epithelial buds and conversely it exhibited a tendency for decline in the isolated mesenchyme ( Figure 5 ). These findings are consistent with the model of Bragg et al. [51] and moreover suggest that the elusive, mesenchyme derived, epithelial growth promoting signal could simply act by negatively regulating activation of the canonical BMP pathway in the distal epithelium buds.
Collectively, the above findings demonstrate that activation of the canonical BMP pathway in airway epithelial cells coincides with the beginning of the canalicular stage, when the developmental plan of the lung shifts from branching morphogenesis to the development of distinct respiratory epithelial cell compartments [52] . Keeping in mind that gas conducting airways and blood conducting vessels need to develop in a coordinated manner and be properly juxtaposed to support optimal gas-exchange in the adult, it is understandable that the tips of developing airways should reciprocally exchange growth, differentiation or guidance signals with the developing vasculature and other mesenchymal cells to ensure coordinated development and spatial integration of these two branching systems. The finding that disruption of the vascular network in growing lung explants affects branching morphogenesis by reducing primarily ''orthogonal bifurcations'' of the epithelial tubules [53, 54] and the findings presented herein demonstrating the influence of the adjacent mesenchyme on the activation of the canonical BMP pathway reporter ( Figure 5 ) illustrates the importance of the crosstalk between epithelial and vascular cells during early lung development.
It should be pointed out that due to our failure to successfully apply anti-pSmad1/5/8 immuno-histochemistry in tissues from embryos earlier than stage E19.5, we cannot conclusively rule out for the moment activation of the canonical BMP pathway at the tips of the early branching endoderm. Further studies will be required to assess whether the BRE-eGFP reporter is not active in the distal endoderm because: a) non-canonical BMP pathways are primarily involved; b) the canonical BMP pathway is active however, does not reach the activation threshold required to activate the BRE-eGFP reporter; or c) that the dynamic balance between the mesenchyme and endoderm which keeps the BRE-eGFP reporter inactive most of the time (Figure 5 ), allows at critical points along the branching process transient spikes of canonical BMP pathway signaling to reach the nucleus and regulate expression of target genes.
Over-expression of BMP ligands or inactivation of BMP receptors in the distal part of the developing lung leads to dramatic defects in alveolar development [14, 16, 18, 19] . Since eGFP expression was highly increased in lung parenchyma of the BRE-eGFP mice during the saccular and alveolar stages, the period during which functional alveoli are formed, we characterized the nature of the eGFP pos cells in this part of the developing lung. Tissue sections from different developmental stages were analyzed by co-staining for eGFP and pro-surfactant protein C (pro-SpC), a marker of type-II pneumocytes. As shown in figure 6 and Figure S2 , eGFP staining during the pseudoglandular stage was confined to endothelial cells and peri-bronchial smooth muscle cells of the proximal airways. Pro-SpC pos epithelial cells in the distal airways were uniformly eGFP neg at this developmental stage ( Figure S2 ).In contrast, starting at the canalicular stage (E17) and persisting until approximately P15, a population of eGFP pospro-SpC pos cells emerged in the distal lung compartments ( Figure 6A ).The SpC pos cells at this stage of development are thought to be immature type-II pneumocytes [55] . Morphometric analysis demonstrated that during the saccular and alveolar stages, approximately half of the pro-SpC pos alveolar cells were eGFP pos . Adult tissues, in which alveolarization was complete, contained a very small number of eGFP pos -pro-SpC pos alveolar cells (,0.5-1%) ( Figure 6B ). High resolution confocal microscopy analysis revealed that at all the developmental stages analyzed only a very small portion of type-I pneumocytes were eGFP pos ( Figure S7 ). Interestingly, ectopic over-expression of a constitutive ALK3 in cultures of adult primary pulmonary cells resulted in increased pro-SpC (,6-fold) and Id1 (,15 fold) mRNA levels, in comparison to the untreated or ALK5ca treated groups ( Figure 6C ) further implicating canonical BMP-signaling in the physiology of type-II pneumocytes.
The sequential appearance of eGFP neg -SpC pos (immature type-II pneumocytes), eGFP pos -SpC pos and eGFP neg -SpC pos (mature type-II pneumocytes) may suggest that the eGFP pos -SpC pos population is an intermediate step through which immature type-II pneumocytes must pass before developing into mature functionally competent type-II pneumocytes. This conclusion is compatible with the finding that mice with canonical BMP pathway disrupted due to deletion of the Smad1 gene accumulate in their developing lungs high numbers of periodic acid-Schiff (PAS) positive immature type-II pneumocytes [19] .
The above findings indicate that during the cannalicular stage, an important target of canonical BMP signaling in the distal lung compartment is the developing type-II pneumocyte population. This could explain the dramatic distal lung phenotype of animals with disrupted BMP signaling in the distal lung epithelium [15, 16, 18, 19] .
It is very intriguing that with the beginning of the canalicular stage, when the developmental plan of the lung shifts from branching morphogenesis to the development of distinct respiratory epithelial cell compartments [52] , the BMP-responsive eGFP reporter is activated in two epithelial domains, namely, the proximal airways and the distal developing alveolar sacs. The conclusion that canonical BMP signaling becomes crucial for lung epithelial cell development at the cannalicular stage is consistent with earlier genetic studies demonstrating that deletion of Bmpr1 [16, 18] or Smad1 [19] and ectopic expression of negative regulators of BMP signaling such as XNoggin and dominantnegative ALK6 [15] by SpC-promoter-driven expression, resulted in defects in lung development visible only around E15-E16, i.e. after completion of the branching morphogenesis.
The localization of eGFP pos cells in the vicinity of NEBs and the minimal overlap between cells expressing eGFP and markers for specialized secretory (CC10) or ciliated cells (FoxJ1) prompted us to investigate whether active canonical BMP signaling may define cells with an immature progenitor phenotype. Thus, a single two hour pulse of BrdU was given to pregnant mothers at E17 to label cycling epithelial cells which were subsequently detected by staining with anti-BrdU antibodies. BrdU pos cells accounted for ,6.7% of the surface airway epithelium in conducting airways and were uniformly distributed throughout the airway tree ( Figure 7A ). This pattern is compatible with the findings of Giangreco et al. [56] and Rawlins et al. [57] demonstrating that randomly distributed progenitor cells contribute to normal epithelial homeostasis. Of the BrdU pos cells ,77.8% were eGFP neg and ,22.2% were eGFP pos , indicating that both populations contribute to the expansion of the airway epithelial compartment ( Figure 7B ). Despite the dominance of the eGFP neg -BrdU pos cells (in absolute numbers), ,13.4% of the eGFP pos were BrdU pos, compared to ,5.5% for the eGFP neg cells ( Figure 7C ).
To further compare the proliferative potential of the eGFP pos and eGFP neg cells different epithelial populations were isolated from dissociated lung tissue by cell sorting and analyzedin-vitro for colony-forming ability. Following a previously described protocol [38] , EpCAM pos , Lineage negative (i.e. CD45 neg -CD31 neg -CD34 neg ) cells from BRE-eGFP transgenic animals were subdivided into Sca1 neg and Sca1 low cells. Finally the Sca1 neg and Sca1 low cells were separated by sorting into eGFP neg , eGFP low and eGFP high cells respectively ( Figure 7D ). Isolated cells were cocultured with MLg feeder cells in a 3-dimensional Matrigel culture system [38] with a modification developed by some of us (HC and BRS) that involved co-culture of the cells in Matrigel for eight days in the presence of the TGFb receptor inhibitor SB431542 (10 mM), followed by an additional four days in the absence of inhibitor.
Lin neg -EpCAM pos -Sca1 low cells cultured in this way, in agreement with previous reports [58] , gave rise primarily to large cystic epithelial colonies and Lin neg -EpCAM pos -Sca1 neg cells gave rise almost exclusively to smaller compact epithelial colonies ( Figure 7E ). The clonogenic capacity of both Sca1 low and Sca1 neg cells correlated with the expression levels of the BRE-eGFP reporter with the corresponding eGFP high populations exhibiting highest clonogenic capacity ( Figure 7F ). Previous studies have suggested that cystic colonies derived from Lin neg -EpCAM pos -Sca1 low cells contain progenitors of airway epithelial cells (Muc5Ac pos ), whereas, the compact colonies derived from Lin neg -EpCAM pos -Sca1 neg cells contain primarily SpC pos alveolar progenitors [58] . Consistently, as shown in Figure 7G , whereas all compact colonies derived from the Sca1 neg subpopulations exhibited a similar SpC pos -Muc5Ac neg staining pattern, the large cystic colonies derived from the Sca1 low subpopulations exhibited a more heterogeneous pattern of staining with approximately half of them expressing Muc5Ac and half of them expressing SpC.
It is worth noting that adult Lin neg -EpCAM pos -Sca1 neg epithelial cells have very low clonogenic capacity in Matrigel [38] . The development of colonies fromLin neg -EpCAM pos -Sca1 neg cells in the current study is most probably due to the fact that the analysis was done with P3 lungs which were still undergoing intense alveolar development.
Previous studies have provided evidence for two types of airway epithelial progenitors, one randomly distributed in the airways that maintains normal epithelial homeostasis and responds to minor injuries of the epithelium, and a second, associated with previously described progenitor cell niches that can renew depleted Clara cells upon severe epithelial injury [38, 56] . The randomly distributed eGFP neg -BrdU pos cells detected in the developing lungs ( Figure 7A ) could represent the former type whereas the eGFP pos epithelial cells could represent the latter type of progenitors. Once lung development is completed, the homeostatic, low turnover, maintenance of the epithelial population can be supported by the distributed Clara cells [38, 56] and thus active BMP signaling may not be required any longer. The decline in eGFP expression in the lungs of the adult BRE-eGFP transgenics may reflect just that. Reactivation of the BRE-eGFP reporter in adult lung after tissue injury suggests an active role of BMP-signaling in adult lung repair after injury
The dramatic decline of eGFP expression in the adult lung of the BRE-eGFP reporter mice and the association of eGFP expression with the epithelial progenitor pool prompted us to investigate whether the reporter was reactivated in adult lung during injury and repair. Adult BRE-eGFP animals were analysed in two established models of lung tissue injury and repair, namely the naphthalene and bleomycin induce lung injury models. Naphthalene treatment causes selective depletion of Clara cells in the airways [59] . In contrast, bleomycin causes epithelial cell death, acute inflammation and fibro-proliferative remodeling of the peripheral lung [60] . Immuno-staining for CC10 and CGRP demonstrated that by day 3, naphthalene treatment had caused a large reduction in CC10 pos cells in the airways, an increase in the number of CGRP pos cells per NEB, and a substantial increase in BRE-eGFP reporter activity at airway branch-points and terminal bronchioles ( Figure 8A ).This pattern of CC10 localization and eGFP reporter expression corresponds with previously determined anatomic locations that maintain chemically resistant epithelial progenitor cells [61, 62, 63, 64] .
Analysis at different time points after naphthalene-treatment demonstrated a gradual increase in the number of eGFP pos bronchial epithelial cells that was evident on day 2 and maximum increase of 15-fold over control levels by day-9 post-naphthalene treatment ( Figure 8B ). Up to day-2, the increase in eGFP pos airway epithelial cell numbers involved CGRP pos and CGRP neg -CC10 lowcells ( Figures 8C and D) . Thereafter, although the total number of eGFP pos -CGRP pos cells remained stable from days 2-9, a wave of eGFP pos -CC10 low -CGRP neg cells that reached maximum around day 3 was followed by a wave of eGFP pos -CC10 pos cells that reached plateau around day-9 ( Figure 8D) .
Interestingly, the distribution of eGFP and CC10 expression recapitulated the pattern observed during early lung development i.e. eGFP pos -CC10 pos cells separating eGFP pos -CC10 low from apparently newly formed eGFP neg -CC10 pos cells. The kinetics of appearance and relative localization of these cells within the tissues are consistent with the notion that they may represent sequential stages of development from the eGFP pos -CC10 low to the eGFP neg -CC10 pos phenotype.
Bleomycin is an anti-cancer drug, which induces lung injury and fibrosis [60] . As shown in Figures 9A and 9B , treatment of adult BRE-eGFP animals by intra-tracheal administration of bleomycin resulted in substantial reactivation of the eGFP reporter in type-II pneumocytes (Pro-SpC pos ). During the first days of treatment the eGFP pos cells were primarily cuboidal SpC pos cells. From day-4 onwards both cuboidal and squamous eGFP pos cells were found in the alveolar regions. Interestingly, the squamous eGFP pos cells were T1a-expressing type-I pneumocytes and were always localized in the borders between affected and apparently normal tissue ( Figure 9C ). We postulate that the cuboidal eGFP pos cells observed in the early stages of the bleomycin induced response represent a reparatory progenitor population that expands and differentiates into the eGFP pos -T1a low cells observed in the later stages to repair the damaged alveolar epithelium. Further studies will be required, however, to validate this hypothesis.
Collectively, our findings demonstrate that the canonical BMP pathway is re-activated during adult lung injury in a manner that bears resemblance to the activation of this pathway during early lung development, strongly supporting its importance during adult lung tissue injury repair.
Utilizing a transgenic reporter mouse line harboring a BMPresponsive eGFP reporter allele we were able to construct a detailed spatiotemporal map of canonical BMP signalling during early lung development and adult lung tissue injury repair. Our studies demonstrated that during the pseudoglandular stage, when branching morphogenesis characterises lung development, canonical BMP pathway is active mainly in the vascular network and the airway smooth muscle layer. Only after the completion of branching morphogenesis and the initiation of epithelial cell differentiation canonical BMP pathway activation commences in airway and alveolar epithelial cell that are enriched in progenitors that can form epithelial colonies in Matrigel in-vitro. The BRE-eGFP reporter is reactivated in mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that the low reporter activity in intact lungs is the result of a dynamic interplay between the endoderm and the mesenchyme and moreover suggest that the elusive, mesenchyme derived, epithelial growth promoting signal postulated by Bragg et al. [51] may act by regulating negatively activation of the canonical BMP pathway in the distal epithelium buds.
BRE-eGFP expression, in agreement with earlier reports describing the temporal expression pattern of some BMP pathway components during late lung development [65] , peaks around birth and returns to very low levels upon completion of lung development. Remarkably, severe depletion of Clara cells in the adult lung by naphthalene treatment, leads to re-expression of the eGFP reporter around NEBs and terminal bronchioles, areas known to harbour airway progenitor cells. Likewise, injury of the alveolar epithelium in the adult lung by bleomycin treatment, leads to re-expression of eGFP initially in SpC pos cuboidal epithelial cells, which we postulated to be alveolar epithelial progenitors and subsequently in squamous T1a low cells which we postulated to be derived from the former population and on the way to differentiate to Type-I pneumonocytes. Our findings are compatible with the notion that during the late stages of lung development the role of canonical BMP signalling pathway is to maintain the undifferentiated state of the airway and alveolar epithelial progenitors preventing premature exhaustion of their pools and securing the continuous supply of the differentiated epithelial cells that are required for a fully developed lung. The reactivation of the BMP responsive reporter in adult animals undergoing repair of severe epithelial injury, where the same requirements regarding proper management of the undifferentiated epithelial progenitor pools apply, is also compatible with this conjecture. An analogous function has been previously proposed for BMP signaling regarding the maintenance of the undifferentiated state of pluripotent mouse embryonic stem (ES) cells [66, 67] and the maintenance of the undifferentiated, multipotent state of distal lung progenitor cells [45, 68] .
The construction of a spatiotemporal map of canonical BMP signalling during lung development and adult lung tissue injury repair (summarised in Figure 10 and table S2) will facilitate interpretation of earlier genetic studies and guide rational targeting of BMP signaling components in future experiments. Moreover, the ease isolation of living eGFP pos i.e. BMP responding, subpopulations of lung cells by cell sorting will greatly facilitate definitive clarification of the mechanisms of action of this signaling system during early lung development and repair of lung tissue injury in the adult. Figure S3 FGF10, BMP4 and a Sonic Hedgehog agonist affect expression of the BRE-eGFP reporter. A) Whole E12 lung-explants were cultured on Nuclepore membranes for eight hours in the presence of vehicle, FGF10 (250 ng/ml), BMP4 (100 ng/ml) or Sonic Hedgehog agonist Purmorphamin (500 nM). Upper panel shows bright field images of representative explants. Lower panel shows eGFP expression in the same explants. B) Confocal images of tissue sections prepared from the explants described above stained for eGFP (green stain). Nuclei were counterstained with DAPI. Note the strong upregulation of eGFP expression in the FGF10 and BMP4 treated explants, and the Figure S4 Correlation between pSmad1/5/8 and BRE-eGFP immune-staining in P1 lung tissue sections. Adjacent tissue section of lungs from BRE-eGFP reporter animals were stained with anti-GFP or anti-pSmad1/5/8 antibodies as described in materials and methods. A) Staining of adjacent sections of a large airway with anti-GFP and anti pSmad1/5/ 8antibodies or normal rabbit IgG fraction as isotype control. B) Representative images from the indicated tissue regions demonstrating that tissue areas with intense BRE-eGFP expression coincide with regions exhibiting pSma1/5/8 immuno-staining. The scale bar corresponds to 25 mm. (TIF) Figure S5 The zone of eGFP pos CC10 pos cells coincides with the NEB-rich portion of the airway tree. A) Confocal image of a lung tissue section derived from a P1 BRE-eGFP animal stained for eGFP (green staining), CC10 (red staining) and CGRP (white staining). The images illustrate the zone in the airway-tree where co-expression of eGFP and CC10 occurs. The lower images, showing the CGRP and DAPI channels of the upper images, illustrate the coincidence of eGFP pos -CC10 pos zone with the NEB-rich regions of the airway tree. B) Confocal images of the transitional zone between the eGFP pos -CC10 neg and eGFP neg -CC10pos domains of the airways demonstrating the preferential association of eGFP pos -CC10 low cells with NEBs. The image depicting NEBs is obtained from a section sequential to the ones depicting CC10 and eGFP expression. Nuclei were counterstained with DAPI (blue staining). (TIF) Figure S6 Minimal eGFP expression in the Foxj-1 pos airway ciliated cells. Representative confocal images demonstrating minimal activation of the BRE-eGFP transgene in FoxJ1 pos ciliated airway epithelial cells. Tissue section collected from E17.5, E19.5, P1, P15 and adult BRE-eGFP transgenic lungs were stained for eGFP (green staining), FoxJ1 (red staining) and CGRP (white staining). Nuclei were counterstained with DAPI (blue staining). The images demonstrate the presence of remark-ably low number of eGFP pos -FoxJ1 pos epithelial cell (depicted with yellow arrows). (TIF) Figure S7 A small, however, detectable number of BRE-eGFP developing type-I pneumocytes (T1a pos ) express eGFP. Representative confocal images of lung sections of E14.5, E17.5, E19.5, P1, P15 and adult BRE-eGFP transgenic lungs stained for eGFP (green staining), CD31 (red staining) and T1a (white staining). Nuclei were counterstained with DAPI (blue staining). The Scale bars are 20 mm. The images demonstrate that whereas the majority of the developing endothelial cells express eGFP, only a small number of type-I pneumocytes, shown in the inserts of the P1 and P15 image, are eGFP pos . (TIF)  Pulmonary sequelae in a patient recovered from swine flu The pandemic of swine flu (H1N1) influenza spread to involve the whole world rapidly. Many patients manifested a mild clinical illness but some developed pneumonia and respiratory failure. High mortality was observed in patients with severe disease. Among survivors, studies are limited. Ground-glass opacities on a high-resolution computerized tomography scan and reduced diffusion capacity were noted after 3 months in a study. But long-term complications in patients with swine flu pneumonia have not been studied well. We are presenting an unusual case of swine flu pneumonia who developed interstitial lung disease after recovery.  Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production BACKGROUND: Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection. CONCLUSIONS/SIGNIFICANCE: To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production. Dengue virus (serotypes DENV-1, -2, -3 and -4) is a positivestrand RNA virus belonging to the family Flaviviridae, genus Flavivirus. Mosquitoes transmitting DENV in humans has been a major cause of dengue diseases in tropical and subtropical counties; approximately one-third of the world's population is at risk of the infection. People infected with DENV typically show self-limited febrile dengue fever and dengue hemorrhagic fever (DHF). Life-threatening dengue shock syndrome (DSS) is more likely to occur after a second DENV infection [1, 2] . Other than supportive treatments, no specific therapy is available for denguerelated diseases. Several tetravalent DENV vaccine candidates are under development, but an effective, safe and affordable dengue vaccine remains elusive [3] .
DENV infects multiple organs and cell types in humans. Particularly, the mononuclear phagocyte lineage of macrophages, monocyte-derived dendritic cells (DCs) and skin Langerhans cells are the primary cell targets [4, 5, 6] . Similar cellular tropism of macrophages and DCs was observed in the experimental DENV mouse infection model [7] . Macrophages and DCs are the most crucial cell types in innate immunity and rapidly produce type I interferons (IFNs) and cytokines to fight against microbe invasion. Type I IFNs are potent inhibitors of virus replication. Therefore, many pathogenic viruses have developed strategies to escape the IFN-triggered anti-viral effects. More than 170 different virusencoded IFN antagonists from 93 distinct viruses have been described [8] . For example, hepatitis C virus (HCV), a member of Flaviviridae, evades innate immunity by cleaving mitochondrial antiviral signaling protein, an IFN stimulator, with its protease NS3/4A [9] . The nonstructural proteins NS4B of DENV-2, West Nile virus (WNV), and yellow fever virus (YFV) block the activation of STAT1 in cells stimulated with type I IFN [10, 11] .
We previously found that two flaviviruses, Japanese encephalitis virus and DENV-2, trigger type I IFN transcription through an RIG-I-dependent signaling cascade to activate interferon regulatory factor (IRF) and NF-kB. However, JEV induced higher activation of IRF3 and NF-kB than DENV-2 in human A549 cells [12] . Furthermore, type I IFN production triggered by doublestranded RNA (dsRNA) stimulation was blocked in DENV-2infected human DCs [13, 14] . Microarray results from rhesus macaques also indicated that type I IFN, interleukin 10 (IL-10), IL-8, IL-6 and tumor necrosis factor a (TNFa) were not upregulated with DENV-1 infection [15] . Therefore, DENV-2 might modulate the induction pathway of type I IFN and other cytokines.
The Toll-like receptor (TLR) family is one of the best-studied pattern-recognition receptor families and is responsible for sensing invading pathogens [16] . Different TLRs recognize the different molecular patterns of microorganisms; for example, TLR4 recognizes lipopolysaccharide (LPS) and TLR3 recognizes dsRNA. Engagement of TLRs with their ligands triggers signal cascades to activate IRFs and NF-kB, thus leading to production of cytokines and type I IFN [17] . NF-kB activation is crucial for cytokine induction, and many viruses evolve various strategies to manipulate NF-kB signaling [18, 19] . Several RNA virus-encoded proteins, such as HCV NS5B, SARS CoV M protein, measles virus V protein, and enterovirus 71 2C, inhibit NF-kB activation directly or indirectly [20, 21, 22, 23] .
In this study, we investigated whether DENV-2 could block type I IFN and cytokine induction triggered by TLR signaling. We studied the influence of DENV-2 infection on activation of NF-kB and extracellular signal-regulated protein kinase (ERK).
The DENV-2 PL046 strain (Genbank accession: AJ968413.1) was isolated from a Taiwanese DF patient. The DENV-2 prototype New Guinea C (NGC) strain was kindly provided by D. J. Gubler of the Centers for Disease Control and Prevention, USA. These viruses were propagated in the mosquito cell line C6/ 36 (ATCC: CRL-1660) grown in RPMI 1640 medium containing 5% fetal bovine serum (FBS) [24] . The J774A.1 mouse macrophage cell line (ATCC: TIB-67), A549 human lung epithelial carcinoma cell line (ATCC: CCL-185), and African green monkey kidney epithelial cell line Vero (ATCC: CCL-81) were cultured in DMEM medium supplemented with 10% FBS (Invitrogen). The TLR3 ligand polyinosine-polycytidylic acid (polyI:C) and TLR9 ligand CpG oligodeoxynucleotides 1826 (CpG ODN 1826; hereafter CpG) were from InvivoGen. The TLR4 ligand LPS (Sigma-Aldrich), anti-ERK antibody, anti-phospho-ERK antibody (Cell Signaling, catalog# 9102 and 9101S) and anti-NF-kB p65 antibody (sc-372, Santa Cruz Biotechnology) were used.
Female C57BL/6 mice at 6-8 weeks old were used in accordance with the guidelines of Kaohsiung Veterans General Hospital animal care and use committee under the approved animal study protocol (VGHKS-99-A028). BMDCs were generated by culturing bone-marrow hematopoietic cells with FMS-like tyrosine kinase 3 ligand (Flt3L) for 8 days [25, 26] . For DENV-2 infection, 10 6 DCs were adsorbed with DENV-2 at a multiplicity of infection (MOI) of 5 for 1 h. After removing the virus inoculant, cells were incubated with complete medium. For stimulation with TLR ligands, DCs were incubated with 100 mg/ml polyI:C or 1 mg/ml CpG.
TRIzol reagent (Invitrogen) was used for total RNA extraction, and cDNA was synthesized from 0.5 mg total RNA by Superscript III reverse transcriptase (Invitrogen). qPCR amplification was done with 4 ng cDNA in 10 ml SYBR Green PCR master mix (Applied Biosystems) with 3 mM of primers in the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Transcript levels were normalized to that of hypoxanthine phosphoribosyltransferase ( Table S1 .
Cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min. After 2 washes with PBS, cells were blocked with 10% skim milk in PBS. NF-kB p65 subcellular location was determined by immunostaining with rabbit anti-NF-kB p65, then Alexa Fluor-568-conjugated goat antirabbit IgG antibody (Invitrogen). DENV-2 NS3 was detected by a specific monoclonal antibody against NS3 (#YH3304, 1:500 dilution, Yao-Hong Biotechnology) plus Alexa Fluor-488-conjugated goat anti-mouse IgG antibody (Invitrogen). Fluorescence signals were observed under a fluorescence microscope (Olympus BX51).
TurboFect transfection reagent (Fermentas) was used for transient transfection following the manufacturer's protocol. Cells cultured in 12-well plate were transfected with NF-kB-or IFN-b-Luc reporter plasmids [12, 25] . pRL-TK (Promega), encoding Renilla luciferase under an HSV thymidine kinase promoter, was used as an internal control. After transfection for 24 h, cells were infected with DENV-2; in some experiments, cells were further stimulated with LPS or polyI:C (both 1 mg/ml). Cell lysates were collected at the indicated times for dual-luciferase assays (Promega). Relative firefly luciferase activity was normalized to Renilla luciferase activity.
Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) containing protease inhibitor and phosphatase inhibitor cocktails (Roche). Harvested cell extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes, which were reacted with primary antibody, and then horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory) and visualized with an enhance chemiluminescence system (Thermo). Images were acquired by a digital image system (UVP or Fujifilm).
To study the effect of DENV-2 on modulating innate immune response, we determined the levels of type I IFN and cytokines in DENV-2-infected mouse BMDCs by quantitative RT-PCR. The TLR3 ligand polyI:C and the TLR9 ligand CpG greatly stimulated the mRNA expression of IFN-b (124-fold induction at 2 h by polyI:C and 531-fold induction at 6 h by CpG), IL-10 (77-fold at 12 h by polyI:C and 74-fold at 36 h by CpG), IL-12p40 (381-fold at 24 h by polyI:C and 1551-fold at 6 h by CpG), and TNFa (64-fold at 2 h by polyI:C and 180-fold at 6 h by CpG) ( Figure 1A -D, left panels). However, levels of IFN-b and these cytokines were much lower in cells with DENV-2 infection (Figure 1 , left and right panels): especially, IL-10 was not induced by DENV-2 infection. The kinetic of DENV-2 replication in BMDCs was measured by qPCR with primers specific for DENV-2 59-UTR ( Figure 1E ) and by immunofluorescence staining with antibody specific against DENV-2 NS3 ( Figure 1F ). DENV-2 replication peaked around 12-24 h post infection, in consistence with cytokine induction peaked around 12-36 h post infection. Therefore, infection with DENV-2 inefficiently triggered type I IFN expression and that of other cytokines. Since DENV-2 infection would produce intracellular viral RNA to turn on RLR and TLR signaling cascades for cytokine production, weak induction of DENV-2 for these cytokine genes ( Figure 1 ) implies that DENV-2 may interfere with a common signaling pathway for inducing type I IFN and other inflammatory cytokines.
To determine whether DENV infection could suppress the cytokine induction triggered by TLR signaling, we investigated infection with a murine macrophage cell line J774A.1. Consistent with previous report that J774A.1 is susceptible to DENV infection [27] , DENV-2 infection in J774A.1 was shown by immunofluorescence staining with antibody specific against DENV-2 NS3 (Figure S1A) , and by qPCR with DENV-2 59 UTR primers ( Figure S1B ). J774A.1 cells were mock-infected or infected with DENV-2, then stimulated with LPS or polyI:C. LPS and polyI:C readily promoted the expression of IFN-b (Figure 2A and B; left panels) and IL-10 ( Figure 2A and B right panels), but with DENV-2, the expression was diminished. Similar results were noticed in DENV-2-infected BMDCs that polyI:C-activated IFN-b and IL-10 were reduced with DENV-2 infection ( Figure 2C ). Thus, DENV-2 appears to interfere with IFN-b and cytokine production triggered by TLRs signaling cascade.
NF-kB is an essential molecule in the TLR signaling pathway for inducing type I IFN-b and cytokines [16, 28, 29] . In our infection system, two strains of DENV-2, PL046 and NGC, triggered low degree of NFkB p65 nuclear translocation. Nuclear staining of p65 was noted in 7% and 13% of PL046-and NGCinfected cells, respectively ( Figure S2A panels b and c) ; in contrast, about 80% of the control virus infected cells showed nuclear staining of NF-kB p65 ( Figure S2A, panel d) . Thus, we checked whether DENV-2 could suppress NF-kB activation triggered by TLRs engagement. With use of a NF-kB-dependent luciferase reporter, NF-kB activation stimulated by the TLR ligands LPS and polyI:C was readily blocked in DENV-2-infected cells at 12 and 24 h post stimulation ( Figure 3A ). Immunoblotting result of DENV-2 NS3 indicated that the virus was replicating in A549 cells and was not affected by LPS and polyI:C posttreatment ( Figure 3B ). As well, DENV-2 infection blocked LPS-triggered NF-kB activation in Vero cells as measured by nuclear translocation of NF-kB p65 ( Figure 4A) ; similar results were also observed in J774A.1 macrophages ( Figure S2B ). Nuclear translocation of NF-kB p65 was slightly increased in A549 cells up to 60 h post DENV-2 infection, but the polyI:C-triggered p65 nuclear translocation was greatly blocked in DENV-2 infected cells ( Figure  S2C ). We also tested the influence of TLR singling in DENV-2 replication, polyI:C pretreatment blocked DENV-2 replication; whereas, the antiviral effect of polyI:C was not seen in cells with established DENV-2 infection ( Figure 4B ). This data is consistent with previous report [30] and suggest that DENV-2 inhibits TLR signaling to benefit its replication. DENV-2 replication in these two cell lines, A549 and Vero, was confirmed by qPCR with viral 59 UTR primers and plaque assay for virion production ( Figure  S3 ). Thus, DENV-2 downregulates TLR-activated NF-kB, which leads to reduced cytokine expression.
The activity of ERK is associated with the expression of type I IFN and cytokines, particularly IL-10 [31, 32, 33] . We thus further checked whether DENV-2 targets ERK activation by examining the phosphorylated ERK (p-ERK). DENV-2 infection did not activate ERK1/2 phosphorylation and even impaired the basal level of p-ERK1/2 ( Figure 5A ). Furthermore, polyI:C-and LPS-stimulated phosphorylation of ERK1/2 was decreased with DENV-2 infection ( Figure 5B and 5C), so DENV blocks TLR-mediated ERK activation to modulate both arms of the innate immunity response to infection: type I IFN and cytokines.
The pathogenesis mechanism of severe DHF/DSS with DENV infection is not well understood, but evidence suggests that the magnitude of DENV replication and its regulation of innate and adaptive immunity may both contribute [34] . The incidence of DHF/DSS is higher in people with previous exposure to different serotypes of DENV, and antibody-dependent enhancement (ADE) may be a mechanism for DHF/DSS [1] . Enhancing antibodies may increase viral entry and increase the number of infected cells. DENV infection via the ADE route has also been shown to downregulate several genes of the innate immunity system, resulting in suppression of the innate response and increase of DENV replication [35, 36, 37] . In this study, we further demonstrate that DENV per se is a weak cytokine inducer because IL-10, IL-12, and TNFa were induced to a lower extent in DENV-2infected BMDCs. Furthermore, even in the absence of enhancing antibody, DENV could block NF-kB activation and cytokine induction triggered by TLR signaling. Downregulation of the cytokine system may provide a growth advantage for DENV to propagate in host macrophages and DCs.
IL-10 is a potent immunosuppressor produced by several immune cells [38] , and its expression can be triggered by TLR [31, 32] . IL-10 is critical in suppressing excessive inflammation and immunopathologic conditions caused by the host immune system responding to infections [39, 40] . DHF patients showed high serum levels of IL-10, which may be involved in the pathogenesis of severe dengue disease [41, 42] . In a WNV animal infection model, IL-10 downregulated T cell-mediated immunity and had a negative role in antiviral immunity [43] . Thus, our findings that DENV-2 failed to induce IL-10 expression in BMDCs (Figure 1 ) are unexpected but are consistent with previous reports that IL-10 is not produced from mature human CD1a+ DCs infected with DENV-2 [44, 45] and that microarray data from DENV-1-infected rhesus macaques showed no transcription of IL-10 or other cytokine genes [15] . In addition, we found that DENV-2 could inhibit TLR-triggered IL-10 expression. Therefore, the high IL-10 expression found in patients with dengue-related diseases might not be simply stimulated by the DENV itself. Instead, it may be resulted from uncontrolled DENV replication, which then triggers increased levels of immune activation and increased IL-10 production. DENV infection through the ADE route often induces IL-10 expression, which worsens the host anti-viral system and results in increased DENV production [35, 37, 45] . Thus, DENV infection via ADE or non-ADE routes might have different effects in the immune system.
TLR signaling cascades are mainly controlled by the MyD88dependent and TRIF-dependent pathways, which both lead to activation of NF-kB and mitogen activated protein kinases (MAPKs) [17, 46] . NF-kB and MAPKs have been suggested to have critical roles in cytokine induction [47, 48] . A recent study showed that constitutive intestinal NF-kB activation does not lead to destructive inflammation unless accompanied by activation of MAPKs such as p38 and ERK [49] . Our results that DENV-2 blocked activation of NF-kB, as well as ERK1/2, support our findings of cytokine production hampered in DENV-2-infected cells. Because both NFkB and ERK1/2 were affected, DENV may suppress an upstream molecular event such as TLR gene expression, as that has been reported for DENV infection through an ADE route [37] . We also detected TLR genes expression in DENV-2 infected J774A.1 macrophages and found that polyI:Ctriggered TLR4, TLR5 and TLR13 expression was significantly downregulated by DENV-2 ( Figure S4 , panels a-c), but not for that of TRL6, TLR7, TLR8 and TLR2 ( Figure S4 , panels d-g). In contrast, DENV-2 enhanced the gene expression of TLR1 and TLR3 triggered by polyI:C stimulation ( Figure S4 , panels h-i), suggesting that DENV-2 may modulate certain TLR genes expression in macrophage. Other possibilities, such as whether DENV protease, found to block IFN-b promoter activation [13] , may target common molecules involved in type I IFN and cytokine production in TLR signaling, or whether ISG15 that is induced by DENV-2 and functions as an inhibitor of type I IFN production [50] may also contribute to immune evasion, remain to be further studied.
Taiwanese DENV-2 strain PL046 used in this study was isolated from patient with DF, and this virus has been used in the studies of viral pathogenesis and host responses mechanism in vitro and in vivo [7, 12, 51, 52, 53] . Other groups have reported that DENV-2 strains MON601 (a laboratory strain of NGC) and 16681 downregulate the activation of NF-kB and production of type I IFN and TNFa in human DC, macrophage, and Huh7 cells [14, 54] . A recent report by Chase A. J. et al. [55] also revealed that human DCs infected with several endemic DENV-2 strains failed to polarize the naïve CD4 + T cells to effectors, suggesting a defect on T cell priming for DENV-infected DCs [55] . However, one of the strain ARA6894 did not show such kind of inhibition effect, as ARA6894-infected DCs triggered CD4 + Th1 polarization with high expression of IFNc and TNFa. Interestingly, the polyprotein sequences of strain ARA6894 contain nonsynonymous amino acids that are not present in other DENV-2 strains such as PL046, 16681 and NGC [55] , suggesting that different impacts on DC's function between these DENV-2 strains might be due to viral genome diversity.
Dengue may be the most important arboviral disease potentially affecting 2 to 3 billion people living in tropical and subtropical areas. DENV mainly infects monocytes, macrophages and DCs that are also the most important innate immune cells. The balance between the protective and pathological immune responses likely contributes to the DENV infection outcomes. Our results demonstrating that DENV can modulate the signaling events triggered by several TLRs are of interest and provide an explanation for how DENV may skew the host immune system. Immunofluorescence analysis showed the subcellular localization of NF-kB p65 (red, panels a-d) and the detection of viral proteins DENV-2 NS3 or JEV NS1 (green, panels e-h). (B) J774A.1 macrophages were infected with DENV-2 PL046 for 48 h before stimulation with LPS (1 mg/ml). After 6 h of LPS treatment, the localization of NF-kB p65 was determined by immunostaining with anti-NF-kB p65 antibody (red fluorescence, panels a-d). DENV-2 infection was determined by anti-DENV-2 NS3 antibody (green fluorescence, panels e-h), and the DAPI presents the nuclear counter stain (blue fluorescence, panels i-l). Representative cells from the same field are shown for each experimental group. (C) A549 cells were infected with DENV-2 PL046 (MOI of 5) for the indicated times. For polyI:C stimulation, DENV-2-infected cells at 36 h p.i. were transfected with polyI:C (2 mg) and incubated for another 24 h, meaning DENV-2 infection for a total of 60 h. For the mock control group, polyI:C stimulation was conducted by polyI:C (2 mg) transfection for 24 h. The immunofluorescence staining of NF-kB P65 was performed as described above, and the DENV-2 infected cells with nuclear p65 were counted. Data are mean6SD from 3 determinations. Table S1 . (TIF)  High Influenza A Virus Infection Rates in Mallards Bred for Hunting in the Camargue, South of France During the last decade, the role of wildlife in emerging pathogen transmission to domestic animals has often been pointed out. Conversely, far less attention has been paid to pathogen transmission from domestic animals to wildlife. Here, we focus on the case of game restocking, which implies the release of millions of animals worldwide each year. We conducted a 2-year study in the Camargue (Southern France) to investigate the influence of hand-reared Mallard releases on avian influenza virus dynamics in surrounding wildlife. We sampled Mallards (cloacal swabs) from several game duck facilities in 2009 and 2010 before their release. A very high (99%) infection rate caused by an H10N7 strain was detected in the game bird facility we sampled in 2009. We did not detect this strain in shot ducks we sampled, neither during the 2008/2009 nor the 2009/2010 hunting seasons. In 2010 infection rates ranged from 0 to 24% in hand-reared ducks. The 2009 H10N7 strain was fully sequenced. It results from multiple reassortment events between Eurasian low pathogenic strains. Interestingly, H10N7 strains had previously caused human infections in Egypt and Australia. The H10 and N7 segments we sequenced were clearly distinct from the Australian ones but they belonged to the same large cluster as the Egyptian ones. We did not observe any mutation linked to increased virulence, transmission to mammals, or antiviral resistance in the H10N7 strain we identified. Our results indicate that the potential role of hand-reared Mallards in influenza virus epizootics must be taken into account given the likely risk of viral exchange between game bird facilities and wild habitats, owing to duck rearing conditions. Measures implemented to limit transmission from wildlife to domestic animals as well as measures to control transmission from domestic animals to wild ones need to be equally reinforced. During the last decade, awareness concerning the intimate links between human and animal health has rapidly increased in the context of disease emergence [1] . Indeed, approximately 80% of the infectious diseases that recently emerged were zoonotic [2] . The role of wildlife in emerging pathogen transmission to humans and domestic animals has in many cases been pointed out [3±5] . Conversely, pathogen transmission from domestic animals to wildlife has received far less attention, although the importance of this issue was often mentioned [6, 7] . Indeed, contacts between wildlife and livestock or their environment sometimes result in wildlife diseases with conservation issues [8, 9] . Besides, handreared animal releases into the wild for either conservation or exploitation purposes represent a particular case in which handreared individuals eventually share natural habitats with their wild congeners. In both cases such releases can dramatically influence disease dynamics in the surrounding wild animal populations [ 10± 14] .
In the present study we focused on the case of game restocking, which implies the release of millions of individuals worldwide each year [15] . Birds are the most frequently involved, with millions of individuals being released annually in Europe only. For example more than 3 millions red-legged partridges are released annually in Spain [15] , and ca. 1.4 million Mallards are being so in France [16] . The Camargue region, a complex network of wetlands situated in the Rhone delta, is a major duck winter quarter [17] and a central place for wildfowl hunting in France [18] . Hunting is also among the most important economic activities in the area, which is one of the reasons for the massive Mallard releases in the Camargue [19] . At least 30 000 hand-reared individuals are released annually in the region [20] . Maximum Mallard numbers in the wild are reached in September after the beginning of the hunting season, with 56 500 individuals on average over the last seven years (Gauthier-Clerc, unpubl. data), these numbers certainly include a mixing of wild and released Mallards. Given its central position on the flyway of many European migratory species, the Camargue is also a potential hotspot for the introduction and transmission of bird-borne pathogens [21] .
For this reason, avian influenza viruses (AIV) have been studied since 2004 in the area. These negative-sense single stranded RNA viruses belonging to the Orthomyxoviridae family are commonly characterized by the combination of their surface proteins: hemagglutinin (HA) and neuraminidase (NA) [22, 23] . AIVs are highly variable and undergo continuous genetic evolution via two mechanisms: i) accumulation of point mutations at each replication cycle, ii) reassortment involving gene segment exchanges that occur when a cell is co-infected by different viruses [24] . These mechanisms contribute to the emergence of new variants with the ability to transmit to new hosts and/or with epidemic or even pandemic potential. Aquatic birds, particularly Anseriforms (ducks, geese and swans) and Charadriiforms (gulls, terns and shorebirds) constitute their major natural reservoir [23, 25] . The AIV circulating in wild birds are usually low pathogenic ones (LPAIV). LPAIV generally have little impact on their host [26, 27] , although some studies have reported a possible influence on migration capacities [28] . Besides, when LPAIV of H5 or H7 subtypes are transmitted from wild birds to domestic ones reared in artificial environments, their virulence can evolve to high pathogenicity [29, 30] . Highly pathogenic avian influenza viruses (HPAIV), such as HP H5N1 strains currently circulating in Asia and Africa, are still of great economic concern, notably due to the cost of preventive actions including vaccination and massive birds culling [31] . Moreover, HPAIV infections represent a threat for human health since 603 HPAIV H5N1 human infections including 356 fatal cases have been reported worldwide since 2003 [32] .
Relatively high prevalence of AIV was regularly detected in the wintering Mallard population of the Camargue (e.g. 5.4% prevalence during the 2006±2007 hunting season) [33] . Moreover a seasonal infection pattern was identified in Mallards during autumn and winter, with higher infection rates in early fall [33] . Mallards hence represent a focal study species in AIV research. Indeed wild Mallards are one of the main low pathogenic AIV natural reservoir host [34] , and have proven to be healthy carriers of some of the H5N1 HPAIV strains [35] . However, to our knowledge no study ever aimed at investigating the potential role of hand-reared Mallards released for hunting in the epidemiology of AIV, despite the very large number of ducks being released in the wild annually.
To clear this gap we conducted a 2-year study in the Camargue to investigate the potential influence of hand-reared Mallard releases on AIV dynamics in surrounding wildlife. We first hypothesized that, owing to high density rearing conditions and to their genetic uniformity, hand-reared Mallards should be highly susceptible to AIV infections and could play an amplification role in AIV dynamics. This phenomenon has already been pointed out in red-legged partridge (Alectoris rufa) reared for hunting in Spain, where Escherichia coli prevalence was much higher in hand-reared populations before their release than in the wild ones [36] . To test this assumption we collected cloacal swabs from Mallards reared for hunting in several game bird facilities (GBF) in 2009 and 2010, and analysed these samples to measure AIV prevalence. Second, we hypothesized that AIV exchange occurs between wild and hand-reared Mallards, potentially leading either to the circulation of new strains in wild populations or to the amplification and dispersal of wild strains. Indeed, no barrier prevents AIV exchange between wild birds and hand-reared ducks in the GBF since water flows exist between pens and ponds used by wild birds. Moreover, as the GBF roofs are made of nets wild birds can deposit feces in the pens. Finally, hand-reared ducks are in direct contact with wild ones after their release. To investigate these issues, we tested shot waterfowl before and after handreared Mallards were sampled. Noteworthy, genetic analyses suggest that 76% of hunted Mallards in the Camargue have a captive origin [37] . Considering the low annual survival of released Mallards (0.8±15.9% depending on the release site) [37] , the individuals we tested from the Camargue hunting bags certainly included a large proportion of ducks released some months before being shot. These released ducks cannot be differentiated morphologically from the wild ones [38] . Here we hence analyzed shot ducks as a whole since they are a representative sample of the Mallard population wintering in the Camargue, which is composed of individuals of both wild and captive origin that share habitats and can thus be considered as a single epidemiological unit.
Our third hypothesis was that any AIV strain potentially found in captive reared Mallards might present genetic characteristics linked to its circulation in domestic populations. We therefore performed a molecular study of the identified strains in order to look for such characteristics, and in particular to test for the presence of mutations known to be associated with increased virulence, since it has been highlighted that artificial environments are favorable to the appearance of such mutations in birds [30] . We also searched for mutations linked with transmission to other species including humans, since some studies proved that they can be acquired during their transmission among birds [39] . Thirdly, we tested for the presence of mutations conferring resistance to common antiviral drugs, since such resistance has recently been recorded in wild birds, notably in Sweden [40] . Lastly, full sequence analysis of some strains was performed, so as to get insight into their geographic origin through a phylogenetic study, and to determine their relatedness with strains, which have caused human infections in the past.
The infection rates in the different GBF were determined by real-time RT-PCR targeting the conserved M gene of AIV (Table 1) . A very high infection rate (99%) was observed in the single GBF sampled in 2009. In 2010 the infection rates ranged between 0 and 24%, being of 8% in the farm infected in 2009. Initial subtyping, searching for H5, H7, H9, N1, and N7 by realtime RT-PCR performed on positive samples led to the identification of an H10N7 strain in the GBF in 2009 and further testing for H10 by real-time RT-PCR showed that the outbreak involved a single H10N7 virus (named H10N7 Camargue below). The viral strains involved in the infections observed in 2010 were LPAIV. H5, H7, H9, H10, and N7 subtypes were searched among them but none was detected.
In shot ducks, mean AIV infection rates were 15% and 5% during the 2008/2009 and the 2009/2010 hunting seasons, respectively ( Table 2) . During these two periods the maximum monthly infection rates (respectively 20% and 16%) were observed in September (Table 2 ). All the involved strains were LPAIV but an important proportion of these were of the H5 subtype (2008/ 2009:17%; 2009/2010:30%). No H7 or H10N7 subtype was detected in AIV found in shot ducks.
Phylogeny. The H10N7 Camargue strain was isolated in embryonated chicken eggs and amplified. Determination of the sequence of the whole genome was performed in order to gain insights into its phylogeny and molecular characteristics. The phylogenetic analysis of four H10N7 Camargue isolates confirmed that they were very closely related to each other and formed a cluster (Figures 1 and S1 ). Analysis of each of the sequences of the 8 viral segments highlighted some shared characteristics (Figures 1 and S1). First, the sequences of all segments were clearly distinct from those of LPAIV strains from the North-American lineage. Second, none were closely related to highly pathogenic viruses (H5N1 or H7N7) that circulated in Europe and Asia. However, the PB2 segment was more closely related to the Asian HP H5N1 cluster than to most of the European LPAIV strains we included in our phylogenetic analysis. This finding suggests an ancient Asian origin of this segment. Third, the sequences of all segments were closely related to Eurasian strains. Interestingly, the most closelyrelated Eurasian strains differed between segments. As an example the LPAIV strain A/mute swan/Hungary/5973/2007 (H7N7) was very closely related to the H10N7 Camargue virus for the N7 segment, but less so for the M segment and belonged to a distant phylogenetic group for the PB2 segment. In the same way, the LPAIV strain A/mallard/Netherlands/9/2005 (H7N7) was closely related to the H10N7 Camargue virus for the M segment, whereas its PB2 segment belonged to a distinct phylogenetic group. African strains that we included in our analysis were part of Eurasian clusters and clearly distinct from American strains. For one of them, A/Pekin duck/South Africa/AI1642/2009 (H10N7) the NS, N7 and H10 sequences were closely related to those of the H10N7 Camargue virus but available sequences of two other segments (NP and M) were clearly distinct. Finally, it is important to note that based on the analysis of partial sequences of the H10 (data not shown) the H10N7 strains that caused human infections in Australia (Genebank accession numbers ADG58106 and ADG58107; Ratnamohan,V.M. and Dwyer, D.E. unpublished) are clearly distinct from the H10N7 Camargue strain. On the contrary Egyptian H10N7 strains that circulated in birds in 2004, i.e. at the time of the detection of two human infections [41] , are relatively closely related to the H10N7 Camargue strain. However, this relatedness is difficult to interpret since the involved nodes are not strongly supported statistically (Figure 1) .
Study of the extremities of the viral segments. We also determined the sequences of the extremities including the full noncoding sequences of all segments of the H10N7 Camargue strain, which to our knowledge was not performed before for any H10N7 strain. The extremities of the N7 segment revealed two characteristics that confirmed the phylogenetic distinction between the H10N7 Camargue strain and those of the American lineage. First, in the non-coding region of the 59 extremity, an A was found at position 1440, as commonly seen in Eurasian strains, while strains of the American lineage possess a T at this position. Second, at position 145±147 of the 39 extremity that belongs to the coding region the Camargue strain, like other Eurasian ones, has a supplementary amino acid compared to strains of the American lineage.
Molecular characterization. The molecular analysis of the H10N7 Camargue strain is detailed in Table 3 . The focus strain is a low pathogenic one since the HA segment possesses a monobasic cleavage site. HA, NA and PB2 sequences all presented some typical avian characteristics (see Table 3 ). No known determinants of adaptation to mammals (humans or mice) were detected. In addition, we did not identify any mutation known to be linked with increased virulence in mammals. Yet, we detected the V149A mutation in PB2, which is associated with high virulence in chickens. However, 149A was also observed in low pathogenic H7 strains sampled from wild and domestic ducks in China. This suggests that this mutation alone does not increase the virulence in ducks [42] . Furthermore, no mutation was detected in the H10N7 Camargue strain at positions known to be linked with reduced susceptibility or resistance to neuraminidase inhibitors or M2blockers. 
This study confirms our first hypothesis that hand-reared Mallards are highly sensitive to AIV infections. Indeed, we detected high infection rates in the GBF we sampled in 2009 (99%) and in 2010 (up to 24%), whereas the prevalence usually observed in duck populations very rarely exceeds 20% in the wild [33, 43, 44] . This shows that massive outbreaks can occasionally affect GBF, potentially impacting on AIV dynamics in the wild populations with which hand-reared Mallards are in contact before and after their release into the wild. This pattern has already been highlighted in Spain where rabbit (Oryctolagus cuniculus) restocking caused sarcoptic mange dispersion in the wild rabbit population, leading to massive decrease in numbers [11] . The possible outcomes of GBF infection onto wild duck populations should here depend on the origin of the strain involved. Indeed, low pathogenic AIV strains that naturally circulate in wild ducks have little impact on their health [26] . Our data did not permit to determine the origin of the strain we identified in 2009. Indeed, we did not detect the H10N7 Camargue strain among the samples collected on shot Mallards, neither during the hunting season that preceded the release of the infected individuals, nor during the following one.
Our sample size for hunted Mallards was reasonably large (n = 299 in 2008/2009 and 555 in 2009/2010). Moreover, the sites where we sampled hand-reared and shot Mallards were close to each other (see Figure 2 ; for instance one of the hunting estates we sampled was within 4 km of the GBF where we detected an AIV outbreak). Nevertheless, our sampling effort was limited in time to the hunting seasons: we do not have any data from February to August 2009. We therefore cannot exclude that the H10N7 strain originated from wild birds. Population density during the breeding season is low in wild Mallard [45] , and thus, even if the H10N7 Camargue virus originated from wild birds, hand-reared Mallards would then have played an amplifying role. In addition, commercial trade of Mallard chicks is common and most of the juveniles sampled in this study traveled 600 km from the producer when one-day old. It is also possible that the H10N7 Camargue strain originated from the birthplace of the chicks. If so, domestic ducks would have both played an amplifying role and potentially a dispersal one, since they could have spread a new strain in wild duck populations.
The lack of samples during the weeks that directly followed the release of the positive Mallards (because the hunting season had not yet started) could explain why we did not detect the H10N7 Camargue virus in wild individuals. Furthermore, the lack of large-scale dispersal of that H10N7 strain could be due to different parameters. First, we sampled the hand-reared ducks of the GBF1 21 days before their release. It is possible that only few birds were still excreting viruses when released into the wild, since excretion time in birds can be as short as 2 days (although it can also last up to 30 days in some cases) [46±48]. Second, a parallel capturerecapture demographic study ran in the Camargue has shown that hand-reared Mallards exhibit low monthly survival before the hunting season (only 44% survive from release until the onset of the hunting period on average) [49] . Moreover, their dispersal capacities appear to be very low [49] . The same pattern has been observed for red-legged partridges released in Spain [13] . Although intestinal parasites were much more numerous in the individuals living in the hunting estates where domestic birds were released, this was interestingly not observed in the neighboring estates [13] . It is possible that the poor survival and low dispersal of the hand-reared Mallards after release limited the potential spread of the H10N7 Camargue virus within the Mallard population in the wild.
Besides, we did not detect any H5 subtype circulating in the GBF whereas 23% of all the AIV we isolated from samples collected on shot Mallards in the Camargue belonged to this subtype. Again we could not highlight any AIV exchange between ducks living in captivity and in the wild. Yet, hand-reared ducks are exposed to wild bird feces and GBF are connected to wild bird habitats through common use of water. Thus, AIV exchanges are very likely to exist. Our results highlighted that: i) GBF represent an epidemiological compartment into which important AIV outbreaks can occur; ii) a significant proportion of Mallards wintering in the Camargue are infected by LPAIV, including H5 strains that are known to be able to evolve to HPAIV in domestic birds [30] . Knowing that rearing conditions in the GBF may favor AIV exchanges between wild and hand-reared Mallards, these findings outline the need for influenza surveillance in GBF to prevent HPAIV outbreaks in both wild and domestic birds in the region.
We used the phylogenetical analysis of whole-genome sequence of the H10N7 Camargue strain as a supplementary tool to get insights into its evolutionary and geographical origins. The four isolates studied clearly belonged to a cluster that did not include other strains. The infections we detected were therefore certainly due to a single introduction event. All the viral segments of the H10N7 Camargue strain belong to the Eurasiatic lineage also including African strains. The sequences of the extremities of the N7 segment, that exhibit features characteristic of the Eurasian lineage viruses, confirmed this finding. The Eurasian lineage is clearly distinct from the American lineage and our observations further support the fact that inter-hemispheric AIV exchanges are rare. The Camargue strain was also distinct from the H10N7 viruses that previously caused human infections in Australia, but belonged to the same large cluster as the Egyptian H10N7 strains that circulated in birds when H10N7 human infections occurred in this country. These Egyptian strains did not cause severe disease. Nevertheless, this information raises concern about a possible transmission to humans of strains such as the Camargue one, in particular to the GBF owners and hunters who live in close contact with ducks. Interestingly, the strains that are genetically closest relatives differ for each segment of the H10N7 Camargue strain. The Camargue strain thus seems to result from multiple reassortments between different Eurasiatic strains, a common phenomenon which has been illustrated by many studies [50, 51] . Regrettably, we could not determine if the H10N7 Camargue strain was more closely related to strains observed in the wild or in GBF. Indeed, the data associated with viral sequences from AIV infected Mallards in GenBank are often too scarce to determine if the sample was taken on a wild or a domestic individual. This underlines the need for more detailed information on AIV sequences included in common databases.
The molecular analysis of the H10N7 Camargue strain allowed us to question whether it may exhibit some characteristics related to its circulation in an artificial environment, namely some mutations linked to increased virulence, resistance to antiviral drugs or transmission to humans. Our results do not support this assumption. The sequences of the studied segments presented typical avian characteristics. No mutation known to contribute to transmission to humans was detected. Moreover, the Camargue strain is a low pathogenic one like all viruses isolated during our research program in the Camargue since 2005 [33, 52] . No mutation conferring antiviral resistance was detected. This analysis therefore failed to highlight any risk factor linked to this particular strain. Yet, one must keep in mind that we only looked for mutations previously described in the literature, while many others may remain undiscovered so far. It is also important to stress that most experimental studies focusing on mutations modifying host specificity and virulence are carried out on chicken, although it has been demonstrated that phenotypic outcomes of a given mutation can greatly differ from one host species to another [42] .
In conclusion, our results point out the important role that could be played by hand-reared ducks released for hunting in AIV dynamics. As we did not detect similar AIV strains in shot and hand-reared ducks we could not prove that AIV exchanges exist between these two epidemiological compartments. Yet, due to rearing conditions in GBF, AIV exchange risks seem to be high enough to urge for sanitary control of hand-reared animals prior to their release into the wild, which appears to be highly insufficient so far. Such surveillance would also prevent HPAIV circulation that may arise from the evolution in GBF of H5 LPAIV that proved to be commonly infecting free-living ducks in the Camargue. The world organization for animal health (OIE) stresses that surveillance of AIV infection should be applied to all domesticated birds including those used``for restocking supplies of game'' [53] , but control measures generally appear to be poor in game bird rearing estates. This surveillance gap has recently been highlighted in the USA, where game bird holders reported very variable sanitary practices [54] . The problem appears similar in Europe, including France. Additionally, according to French law, game birds should be ringed to allow for the differentiation of wild and released individuals [55] . Most of GBF owners ignore this obligation. If released birds were clearly identified the specific role they may have in AIV dynamics could be addressed both before and after their release. Our study illustrates the reality of the epidemiological consequences that can result from surveillance gaps and the knowledge that could be gained if released individuals were recognizable. Sharing knowledge and strictly controlling viral exchanges between wild birds and all kinds of domestic ones represent major steps to anticipate and face HPAIV epizootics.
This study has been submitted for approval and the results reviewed by the Scientific Council of the Tour du Valat Foundation. Birds were handled, ringed and sampled under the supervision of a veterinarian (Michel Gauthier-Clerc) and a registered duck ringer of the « Museum National d'Histoire Naturelle » of Paris (Matthieu Guillemain). All procedures were carried out in accordance with the permit delivered by the prefect of Paris to the « Office National de la Chasse et de la Faune Sauvage » (permit n 2009±014). (Table 1) . Samples were stored in 3 ml of universal transport medium (Biolys kit) and frozen at 280uC until molecular analysis was performed.
RNA isolation from 140 ml of each sample was carried out using a Macherey-Nagel NucleoSpin 96 virus system with RNA elution into a final volume of 60 ml. Influenza A virus was detected by real-time RT-PCR targeting the conserved matrix as describe previously [56] . All real-time RT-PCR assays were performed on a LightCycler 480 (Roche Diagnostic) in a final volume of 10 ml with 2.5 ml RNA, 0.5 mM of each primer, 0.2 mM probe and 0.4 ml enzyme mix using a Superscript III Platinum One-Step quantitative RT-PCR system (Invitrogen). The reaction was carried out with the following temperature profile: 15 min at 45uC, 3 min at 95 C, 50 cycles of 10 s at 95uC, 10 s at 55uC, 20 s at 72uC, and finally 30 s at 40uC. Positive samples were further tested for H5, H7, H9, N1 and N7 subtypes using the same real-time RT-PCR technique (primers available upon request).
For 10 RT-PCR positive samples, 200 ml of specimen was inoculated in the allantoic cavity of 10-days-old embryonated hen's eggs. The allantoic fluid was harvested after 72 h at 37uC and influenza A virus was detected by hemagglutination assay with hen erythrocytes. Viral RNA was purified from allantoic fluid using a QIAamp viral RNA mini kit (Qiagen) following manufacturer's instructions. HA subtype of virus isolates was determined by RT-PCR and sequencing of the HA 0 cleavage site region using a universal set of primers as described previously [57] . HA sequence was analyzed with the basic local alignment search tool available from NCBI (data not shown) and confirmed by sequencing of the whole HA gene using a set of H10-specific primers (primer sequences available upon request). The NA subtype was deduced from this analysis and confirmed by RT-PCR and sequencing of 172 nt of the NA using a set of H10specific primers (primer sequences available upon request).
Amplification of viral RNA extracted from 4 virus isolates was carried out using a Superscript Platinum One-step RT-PCR system (Invitrogen) and primers specific of each segment. Sequencing was done using a Big Dye Terminator V1.1 kit and a sequencer ABI DNA Analyzer 3730XL (Applied Biosystems). Sequences of all primers are available upon request. Sequences of the whole viral genome were analyzed with CLC Main Workbench 5.6.1. We performed alignments for the 8 segments using sequences available from the Influenza Sequence Database with CLC Main Workbench 5.6.1. Phylogenetic trees were constructed using maximum parsimony (MP) methods with the dnapars program of the PHYLIP 3.68 package and the maximum likelihood (ML) with the software PhyML 2.4.4. Evolutionary model was selected using Model Generator 0.85 [58] . Nodal supports were assessed with 100 bootstrap replicates generated for each method.
Determination of 39 and 59 End Non Coding Sequences of the 8 Segments of the H10N7 Virus Viral genomic RNA was extracted using the QIAamp Viral RNA Mini kit (Qiagen) from 140 ml of allantoic fluid according to the manufacturer's recommendations. The RNA was eluted in 60 ml of RNase-free water and the 39 and 59 NC regions were amplified as previously described [59] using an anchored (dT) 14 oligonucleotide and primers specific for the coding sequences of both segments for reverse transcription and amplification. After purification, the PCR products were sequenced with internal oligonucleotides using a Big Dye terminator sequencing kit and an automated sequencer (Perkin Elmer). All sequences of the primers are available from the authors upon request.
One-step real-time RT-PCR assays were developed to be specific of the virus identified. All influenza A positive samples detected between August 2008 and July 2010 on wild and domestic Mallards were tested for H10 and N7 subtypes respectively. Detection was performed in the same conditions as described above except annealing temperature was decreased from 55uC to 50uC using the following primers and probes: H10-780Fw  Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis. Human bocavirus 1 (HBoV1) was initially identified in 2005, in nasopharyngeal aspirates of patients with acute respiratory-tract infections (ARTI) [1] . It was found to be associated with ARTI in children, at a detection rate of 2-19% [2] [3] [4] [5] . Three additional human bocaviruses, HBoV2, 3 and 4, discovered in human stool samples, have since been phylogenetically and serologically characterized [6] [7] [8] [9] . However, whether these are associated with any diseases is currently unknown. HBoV1 is commonly detected in association with other respiratory viruses, and is the fourth most common respiratory virus (after respiratory syncytial virus (RSV), adenovirus and rhinovirus) in infants less than 2 years of age who are hospitalized for the treatment of acute wheezing [2, [10] [11] [12] . Indeed, ARTI is one of the leading causes of hospitalization of young children in developed countries [13, 14] . Acute HBoV1 infection, diagnosed by a virus load of .10 4 genome copies (gc)/ ml in respiratory samples, viraemia, or by detection of HBoV1specific IgM or of an increase in the levels of IgG antibodies, results in respiratory illness [2, [15] [16] [17] [18] [19] [20] . Recent descriptions of lifethreatening HBoV1 infections in pediatric patients in association with high virus loads or diagnostic HBoV1-specific antibodies [21] [22] [23] , in addition to a recent longitudinal study of children from infants to puberty, documenting a clear association of acute primary HBoV1 infection with respiratory symptoms [24] , strongly support that HBoV1 is an etiological agent of both upper and lower ARTI.
HBoV1 has been classified as a new member of the genus Bocavirus of the family Parvoviridae [25] , of which bovine parvovirus (BPV1) and minute virus of canines (MVC) are the prototypes [26, 27] . In comparison with the BPV1 and MVC genomes, the HBoV1 genome sequences obtained previously appeared to exclude the two termini, and therefore, were incomplete [28] . However, sequencing of the head-to-tail junctions of HBoV1 and HBoV3 ''episomes,'' which had been amplified in DNA samples extracted from HBoV1-infected differentiated human epithelial cells and from intestinal biopsies of HBoV3-infected patients, respectively, revealed portions of the HBoV termini [29, 30] . Notably, these sequences were conserved with the terminal sequences of BPV1 and MVC [28] .
In vitro HBoV1 infection has been reported only once in welldifferentiated human airway epithelia (HAE) [31] . That study provided only minimal information on virus replication, and did not include observations of pathophysiology. Obviously, the lack of a sustainable and highly reproducible system that enables highyield virus production, as well as the ability to conduct reverse genetics is a significant barrier to further elucidation of HBoV1 replication and pathogenesis. In the current study, we have successfully sequenced the full-length HBoV1 genome and cloned it in a plasmid referred to as pIHBoV1. Furthermore, we have demonstrated that transfection of human embryonic kidney 293 (HEK293) cells with pIHBoV1 results in efficient production of HBoV1 virions at a high titer, and that these virions are able to productively infect both primary and conditionally transformed polarized HAE.
The terminal hairpins of the HBoV1 genome are typical of those of the genus Bocavirus A head-to-tail junction of an HBoV1 episome identified in an HBoV1-infected HAE [28, 29] was found to possess two sequences (39-CGCGCGTA-59 and 39-GATTAG-59) identical to parts of the BPV1 left-end hairpin (LEH) [27, 32] . This finding suggested that the head sequence is part of the HBoV1 LEH (nucleotides in blue; Figure 1A ). We therefore used the head sequence as the 39 end of a reverse primer (RHBoV1_LEH). Together with a forward primer (FHBoV1_nt1), which anchors the 39 end of the HBoV1 genome predicted from the BPV1 LEH, we amplified the hairpin of the LEH from a viral DNA extract (1.2610 8 gc/ml) prepared from a nasopharyngeal aspirate taken from an HBoV1-infected patient (HBoV1 Salvador1 isolate) [17] . Only one specific DNA band was detected at approximately (,)150-bp ( Figure 1D , lane 1). Sequencing of this DNA revealed a novel sequence of the HBoV1 LEH (nucleotides in red between the two arrows; Figures 1A and S1A). Because the LEHs of the prototype bocaviruses BPV1 and MVC are asymmetric [27, 32] , we set up another PCR reaction with a forward primer located in the hairpin (FHBoV1_LEH) and a reverse primer targeting a sequence downstream of the LEH at nt 576 (RHBoV1_nt576; Figure 1B ). Sequencing of a DNA fragment ( Figure S1B ), detected as expected as a ,600-bp band ( Figure 1D, lane 3) , confirmed the presence of the novel joint sequence and the LEH ( Figure 1B) .
The tail of the HBoV1 head-to-tail junction [28, 29] was found to contain a sequence (59- GCG CCT TAG TTA TAT ATA ACA T -39) identical to that of the right-end hairpin (REH) of the other prototypic bocavirus MVC [27] . We thus speculated that the entire HBoV1 REH is similar in structure to its MVC counterpart. Using a reverse primer targeted to this sequence (RHBoV1_nt5464) and a forward primer located upstream of the REH (FHBoV1_nt5201), we were able to amplify a specific ,300-bp-long DNA fragment ( Figure 1D , lane 5). Sequencing confirmed the presence of the palindromic hairpin of the predicted REH (nucleotides in red; Figures 1C and S1C), and revealed two novel nucleotides at the end of the hairpin (GC in red; Figure 1C ).
These results indicate that we have identified, for the first time, both the LEH and REH of the HBoV1 genome from a clinical specimen, and confirm that the HBoV1 genome structure is typical of the genus Bocavirus.
A full-length HBoV1 clone (pIHBoV1) is capable of replicating and producing progeny virus in HEK293 cells
We also cloned and sequenced the non-structural (NS) and capsid (VP) protein-coding (NSVP) genes of the HBoV1 Salvador1 isolate from the patient-extracted viral DNA. We then ligated the LEH, NSVP genes and REH into pBBSmaI using strategies diagramed in Figure S2 , and refer to this full-length clone as pIHBoV1. We have deposited the sequence of the full-length genome of the isolate in GenBank (JQ923422).
As we previously showed that HEK293 cells support replication of the DNA of an autonomous human parvovirus (B19V) in the presence of adenovirus helper genes or adenovirus [33] , we first investigated whether the adenovirus helper function is necessary for pIHBoV1 replication in HEK293 cells. Specifically, we transfected pIHBoV1 into HEK293 cells (untreated or infected with adenovirus), alone or with pHelper. Interestingly, we found that pIHBoV1 replicated well in the absence of helper virus. Indeed, all the three representative forms of replicated bocavirus DNA [27, 34] (DpnI digestion-resistant dRF DNA, mRF DNA and ssDNA) were detected in each test case, and at similar levels ( Figure 2A ). DpnI digestion-resistant DNA bands are newly replicated DNA in cells as DpnI digestion only cleaves plasmid DNA prepared from prokaryotic cells, which is methylated at the dam site [35] . In contrast, these DNA forms of the viral genome were absent in pIHBoV1-transfected primary airway epithelial cells (NHBE; Figure 2B , lanes 7&8) and present at very low levels (over 20 times lower than in pIHBoV1-transfected HEK293 cells) in pIHBoV1-transfected human airway epithelial cell lines BEAS-2B ( Figure 2B , lanes 5&6), A549 and 16HBE14o-( Figure 2C ), even in the presence of adenovirus. Thus, replication in these cells appears to be non-existent or poor in these contexts.
To confirm the specificity of DNA replication and the identity of the DpnI-resistant DNA bands, we disrupted the ORFs encoding viral proteins NS1, NP1, VP1 and VP2 in pIHBoV1; knockout of expression of the corresponding viral protein was confirmed by Western blot analysis. When the NS1 ORF was disrupted, no DpnI digestion-resistant DNA was detected ( Figure 2D , lane 4), confirming that replication of this DNA requires NS1. Notably, when the NP1 ORF was disrupted, an RF DNA band was detected but it was very weak ( Figure 2D , lane 6), suggesting that NP1 is also involved. When the VP2 ORF was knocked out, the ssDNA band disappeared, but this was not the case when VP1 was disrupted (VP2 was still expressed; Figure 2D , compare lanes 7 to 9), these findings are consistent with a role for
Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. HBoV1 productively infects polarized primary human airway epithelia. However, no cell lines permissive to HBoV1 infection have yet been established. More importantly, the sequences at both ends of the HBoV1 genome have remained unknown. We have resolved both of these issues in this study. We have sequenced a full-length HBoV1 genome and cloned it into a plasmid. We further demonstrated that this HBoV1 plasmid replicated and produced viruses in human embryonic kidney 293 cells. Infection of these HBoV1 progeny virions produced obvious cytopathogenic effects in polarized human airway epithelia, which were represented by disruption of the epithelial barrier. Moreover, we identified an airway epithelial cell line supporting HBoV1 infection, when it was polarized. This is the first study to obtain the full-length HBoV1 genome, to demonstrate pathogenesis of HBoV1 infection in human airway epithelia, and to identify the first cell line to support productive HBoV1 infection.
the capsid formation in packaging of the parvoviral ssDNA genome [36] [37] [38] .
The presence of the ssDNA band in pIHBoV1-transfected HEK293 cells suggested that progeny virions were produced. To prove this, we carried out large-scale pIHBoV1 transfection and CsCl equilibrium centrifugation to purify the virus that was produced. We fractionated the CsCl gradient, and found the highest HBoV1 gc (1-5610 8 gc/ml) at a density of 1.40 mg/ml, which is typical of the parvovirus virion. Electron microscopy analysis revealed that purified virus displayed a typical icosahedral structure, with a diameter of ,26 nm ( Figure 2E ).
Collectively, these findings confirm that we have generated a full-length clone of HBoV1 capable of replicating and producing progeny virus in transfected HEK293 cells.
The infectivity of the HBoV1 virions purified from pIHBoV1transfected HEK293 cells was examined in polarized primary HAE, the in vitro culture model known to be permissive to HBoV1 infection [31] . Three sets (different donors, culture lots #B29-11, B31-11 and B33-11) of B-HAE were generated, and these were Figure 1 . Sequencing the terminal hairpins of the HBoV1 Salvador1 isolate. Sequence and predicted structure of the left-end, LEH (A&B), and right-end, REH (C), hairpins are shown and diagramed, with PCR primers indicated by arrowed lines. PCR products were analyzed by electrophoresis on 2% agarose gels; the expected DNA bands are indicated by arrowheads (D). In both the LEH and REH, nucleotides in red represent new sequences identified in this study, nucleotides in blue represent sequences identified from the head-tail junction of an HBoV1 episome [28, 29] , and nucleotides in black are the 59end and 39end sequences of the incomplete HBoV1 genome (GenBank: JQ411251). doi:10.1371/journal.ppat.1002899.g001 infected with HBoV1 from the apical side. Initially the B-HAE cultures were infected with various amounts of virus, and when a multiplicity of infection (MOI) of ,750 gc/cell was used, most of the cells (,80%) were positive for anti-NS1 staining (indicating that the viral genome had replicated and that genes encoded by it had been expressed) at 5 days post-infection (p.i.). This MOI was subsequently used for apical infection. Notably, B29-11, B31-11 and B33-11 HAE each supported productive HBoV1 infection ( Figures 3 and S3 ). Immunofluorescence (IF) analysis of infected B31-11 HAE at 12 days p.i. showed that virtually all the cells expressed NS1 and NP1 ( Figures 3A and 3B ), and that a good portion of the infected cells expressed capsid proteins (VP1/2; Figure 3C ).
The production of progeny virus following HBoV1 infection was monitored daily by collecting samples from both the apical and basolateral chambers of the HAE culture and carrying out (2) been infected with Ad as indicated. Lanes 1-8 in panel B were analyzed on the same gel, and the gels shown in panels B&C were transferred and blotted together. Ten ng of the HBoV1 dsDNA genome (,5.6-kb), excised from pIHBoV1 using the SalI and XhoI sites, was used as a control (Ctrl) for DpnI digestion in panels A-C. (D) HEK293 cells were transfected with pIHBoV1 and its various mutants as indicated. At 48 h post-transfection, Hirt DNA was extracted and digested with (+) or without (2) DpnI, followed by Southern blotting using the HBoV1 dsDNA genome as a probe. dRF DNA, double replicative form DNA; mRF DNA, monomer replicative form DNA. (E) Negative staining electron micrograph. Purified HBoV1 particles were negatively stained and examined by a transmission electron microscopy. Bar indicates 100-nm. doi:10.1371/journal.ppat.1002899.g002
HBoV1-specific quantitative PCR (qPCR; Figures 4A and S3B ). In the case of B33-11 B-HAE, apical release was obviously initiated at 3 days p.i., then continued to increase to a peak of ,10 8 gc/ml at 5-7 days p.i., then decreased slightly through day 10 p.i. and was maintained at a level of ,10 7 gc/ml through day 22 p.i. ( Figure 4A ). The total virus yield from one Millicell insert of 0.6 cm 2 over a 24-h interval was greater than 2610 10 gc. This result suggested that productive HBoV1 infection of primary B-HAE is persistent. Notably, in the B-HAE cultures from both donors, virus was also continuously released from the basolateral side, keeping pace with apical secretion throughout, though at levels about one log lower than the release from the apical surface ( Figures 4A and S3B ). The genomes of the progeny virions released from infected B-HAE were amplified and sequenced using the primers listed in Figure 1 and primers spanning the NSVP genes between the termini. The result showed an identical sequence with that of the HBoV1 Salvador isolate (Genbank JQ923422). Additionally, no virus was detected in mock-infected B-HAE (data not shown).
Taken together, these results demonstrate that the HBoV1 virions produced by pIHBoV1 transfection is capable of infecting polarized primary HAE cultures from cells derived from various donors and releasing identical progeny virions from infected primary HAE. More importantly, we found that productive HBoV1 infection was persistent.
Although no gross cytopathic effects were observed in HBoV1infected B-HAE, histology analysis of mock-vs. HBoV1-infected epithelia (B33-11) revealed morphological differences: infected B-HAE did not feature obvious cilia at 7 days p.i., and was significantly thinner than the mock-infected one on average at 22 days p.i. ( Figure 4B ). We further monitored the transepithelial electrical resistance (TEER) during infection of B-HAE, and found that at 6 days p.i., it was reduced from a value of ,1,200 to ,400 V.cm 2 , while the mock-infected B-HAE maintained the initial TEER ( Figure 4C) . Notably, the decrease in TEER in the infected B-HAE was accompanied by an increase in HBoV1 secretion ( Figure 4A ).
To confirm a role for HBoV1 infection in disruption of the barrier function of the epithelium, we examined the distribution of the tight junction protein Zona occludens-1 (ZO-1) [39] . Infected B-HAE showed dissociation of ZO-1 from the periphery of cells started from 7 days p.i., compared with mock-infected B-HAE ( Figure 5A ), which likely plays a role in reducing TEER. Cumulatively, these results demonstrate that HBoV1 infection disrupts the integrity of HAE and that this may involve breakdown of polarity and redistribution of the tight junction protein ZO-1. To confirm a role for HBoV1 infection in the loss of cilia, we examined expression of the b-tubulin IV, which is a marker of cilia [40, 41] . In HBoV1-infected B-HAE, expression of b-tubulin IV was drastically decreased at 7 days p.i., and was not detected at 22 days p.i., in contrast to that in mock-infected B-HAE ( Figure 5B ). These results confirmed that HBoV1 infection caused the loss of cilia in infected B-HAE. Notably, infected B-HAE showed changes of nuclear enlargement, which became obvious at 22 days p.i. (Figure 5 , DAPI), indicating airway epithelial cell hypertrophy.
Collectively, we found that productive HBoV1 infection disrupted the tight junction barrier, lead to the loss of cilia and airway epithelial cell hypertrophy. These are hallmarks of respiratory tract injury when a loss of epithelial cell polarity occurs.
Although primary HAE cultures support HBoV1 infection, their usefulness is limited by the variability between donors, tissue availability and high cost. We thus explored alternative cell culture models for their abilities to support HBoV1 infection. Using the purified HBoV1, we examined HEK293 cells, other common epithelial cell lines permissive to common respiratory viruses [42] , including HeLa, MDCK, MRC-5, LLC-MK2 and Vero-E6, and several transformed or immortalized human airway epithelial cell lines (A549, BEAS-2B, 16HBE14o- [43] , NuLi-1 and CuFi-8 [44] ), as well as primary NHBE cells for the ability to support infection in conventional monolayer culture. All were negative for HBoV1 infection as determined by IF analysis (data not shown). We next speculated that since some respiratory viruses infect polarized HAE but not undifferentiated cells [45] , some characteristics of the polarized epithelia may be critical for HBoV1 infection. We thus polarized immortalized cells (NuLi-1 and CuFi- 8) at an air-liquid interface (ALI) for one month. Once polarization was confirmed by detection of a TEER of .500 V.cm 2 , the cultures were infected with HBoV1, under the same conditions as used for primary B-HAE cultures. Notably, IF analysis revealed that at 10 days p.i., HBoV1-infected CuFi-HAE (differentiated from CuFi-8 cells) was uniformly positive for NS1 ( Figure 6A ), whereas the HBoV1-infected NuLi-HAE (differentiated from NuLi-1 cells) was not ( Figure S4) . Moreover, the CuFi-HAE did express HBoV1 NS1, NP1 and VP1/VP2 proteins (Figures 6B and  6C ). The kinetics of virus release from the apical surface was similar to that of a primary B-HAE infected with virus at a similar titer (maximally 2610 7 gc/ml), although virus release from the basolateral surface was undetectable ( Figure 6D ). HBoV1 infection also resulted in a decrease in the thickness of the epithelium ( Figure 6E ), and dissociation of the tight junction protein ZO-1 from the epithelial cell peripheries ( Figure 6F) .
Collectively, these findings demonstrate that the immortalized cell line CuFi-8 [44] , when cultured and polarized at an ALI, supports HBoV1 infection, and recapitulates the infection phenotypes observed in primary HAE, including destruction of the airway epithelial structure.
In this study, we have cloned the full-length HBoV1 genome and identified its terminal hairpins. Virions produced from transfection of this clone into HEK293 cells are capable of infecting polarized HAE cultures. Thus, we have established a reverse genetics system that overcomes the critical barriers to studying the molecular biology and pathogenesis of HBoV1, using an in vitro culture model system of HAE.
It is notable that the HBoV1 terminal hairpins appear to be hybrid relicts of the prototype bocavirus BPV1 at the LEH, but of MVC at the REH [28] . Replication of HBoV1 DNA in HEK293 cells revealed typical replicative intermediates of parvoviral DNA. Although the head-tail junctions are unexpected in the replication of autonomous parvoviruses, they were likely generated during the cycle of rolling hairpin-dependent DNA replication [46] . Therefore, we believe that the replication of HBoV1 DNA basically follows the model of rolling hairpin-dependent DNA replication of autonomous parvoviruses, with terminal and junction resolutions at the REH and LEH, respectively [46] . The replication of parvoviral DNA depends on entry into S phase of the cell cycle or the presence of helper viruses [46, 47] . In this regard, it is puzzling that mature, uninjured airway epithelia are mitotically quiescent (,1% of cells dividing) [48] [49] [50] , as are the majority of the cells in polarized HAE (in the G0 phase of the cell cycle). However, recombinant adeno-associated virus (AAV; in genus Dependovirus of the family of Parvoviridae) infects HAE apically and expresses reporter genes [51] [52] [53] . Gene expression by recombinant AAV requires a conversion of the ssDNA viral genome to a doublestranded DNA form that is capable to be transcribed [54] . This conversion involves DNA synthesis. Hence, we hypothesize that HBoV1 employs a similar approach to synthesize its replicative form DNA. Notably, wild type AAV infected primary HAE apically and replicated when adenovirus was co-infected [55] . The exact mechanism of how HBoV1 replicates in normal HAE will be an interesting topic for further investigation.
The airway epithelium, a ciliated pseudo-stratified columnar epithelium, represents the first barrier against inhaled microbes and actively prevents the entry of respiratory pathogens. It consists of ciliated cells, basal cells and secretory goblet cells that together with the mucosal immune system, provide local defense mechanisms for the mucociliary clearance of inhaled microorganisms [56] . The polarized ciliated primary HAE, which is generated by growing isolated tracheobronchial epithelial cells at an ALI for on average one month, forms a pseudo-stratified, mucociliary epithelium and displays morphologic and phenotypic characteristics resembling those of the in vivo human cartilaginous airway epithelium of the lung [57, 58] . Recent studies have revealed that this model system recapitulates important characteristics of interactions between respiratory viruses and their host cells [41, 45, [59] [60] [61] [62] .
In the current study, we have examined primary B-HAE cultures obtained from three different donors. HBoV1 infection of primary B-HAE was persistent and caused morphological changes of the epithelia, i.e. disruption of the tight barrier junctions, loss of cilia and epithelial cell hypertrophy. The loss of the former, plasma membrane structures that seal the perimeters of the polarized epithelial cells of the monolayer, is known to damage the cell barrier necessary to maintain vectorial secretion, absorption and transport. ZO-1, which we monitored here, is specifically associated with the tight junctions and remains the standard marker for these structures. Similarly, cilia play important roles in airway epithelia, in that they drive inhaled particles that adhere to mucus secreted by goblet cells outward [63] . HBoV1 infection compromises barrier function, and thus potentially increases permeability of the airway epithelia to allergens and susceptibility to secondary infections by microbes. The observed shedding of virus from the basolateral surface of infected primary HAE, albeit at a lower level (,1 log lower than that from the apical surface), is consistent with the facts that HBoV1 infection disrupted the polarity of the pseudo-stratified epithelial barrier and resulted in the leakage to the basolateral chamber. This explanation is also supported by HBoV1 infection of CuFi-HAE, where disruption of the tight junction structure was less severe and virus was released only from the apical membrane. The induction of leakage by HBoV1 also suggests a mechanism that accounts for the viraemia observed in HBoV1-infected patients [5] . Further disease pathology could be accounted for by infection-induced loss of cilia of the airway epithelia; a lack of cilia is often responsible for bronchiolitis [64] [65] [66] . Therefore, our study provides direct evidence that HBoV1 is pathogenic to polarized HAE, which serves as in vitro model of the lung [57, 58] . Since HBoV1 is frequently detected with other respiratory viruses in infants hospitalized for acute wheezing [2, [10] [11] [12] , the apparent pathological changes observed in HBoV1-infected HAE suggest that prior-infection of HBoV1 likely facilitates the progression of co-infection-driven pathogenesis in the patient.
The kinetics of virus release from the apical chamber of HAE infected with the progeny virus of pIHBoV1 (cloned from the clinical Salvador1 isolate) was similar to that following infection with the HBoV1 Bonn1 isolate, a clinical specimen [31] . We believe that our study of HBoV1 infection of primary HAE reproduces infection of the virus from clinical specimens. In addition, we generated virus from a pIHBoV1-b clone, which contains the NSVP genes from the prototype HBoV1 st2 isolate [1] . Infection of primary B-HAE with this st2 virus resulted in a level of virus production similar to that observed here using the Salvador1 isolate (data not shown). We believe that our study with the laboratory-produced HBoV1 Salvador1 represents infection of HBoV1 of clinical specimens in HAE. The MOI used for infection in the current study was high. However, it should be noted that this titer is based on the physical numbers of virion particles as there are no practical methods for determining the infectious titer of HBoV1 preparations. It should also be taken into consideration that extensive parvovirus inactivation occurs during the purification process, i.e. during CsCl equilibrium ultracentrifugation [67] . Virus infection of HAE most likely reflects HBoV1 infection of the lung airways in patients with a high virus load in respiratory secretions [5] .
The fact that pIHBoV1 did not replicate well in undifferentiated human airway epithelial cells (Figures 2B and 2C) indicates that polarization and differentiation of the HAE is critical for HBoV1 DNA replication. Nevertheless, polarized NuLi-HAE, which is derived from normal human airway epithelial cells, did not support HBoV1 infection, but the CuFi-HAE derived from airway epithelial cells isolated from a cystic fibrosis patient did. The CuFi-HAE is unique relative to the others in that it retains the capacity to develop epithelia that actively transport in Na + but not Cl 2 because of the mutation in the cystic fibrosis gene [44] . Given the high complexity of the airway epithelium, we speculate that the permissiveness of HBoV1 infection is dependent on various steps of virus infection, e.g. attachment, entry, intracellular trafficking, and DNA replication of the virus. Nevertheless, a polarized CuFi-HAE model derived from the CuFi-8 cell line represents a novel stable cell culture model that is providing unexpected insights into the infection characteristics of HBoV1. Although HBoV1 infection of CuFi-HAE reproduced disruption of the barrier tight junctions like that seen also in primary B-HAE, the absence of virus on the basolateral side implies that in HAE the secretion of HBoV1 is apically polarized. We speculate that the milder damage of tight junctions in these cells might prevent virus release from the basolateral side of infected CuFi-HAE. Further studies will focus on understanding the permissiveness of CuFi-HAE to HBoV1 infection and on the reason for the ease of infection of an HAE with a cystic fibrosis phenotype.
It has been shown that HBoV1 remains detectable in the upper airways of patients for weeks and months, even up to half a year [68] [69] [70] [71] . However, the mechanism behind this persistence, i.e. whether it is due to persistent replication and shedding, passive persistence after primary infection, or recurrent mucosal surface contamination, has remained unknown. Our results in in vitro HAE cultures showed that HBoV1 is able to replicate and shed from both the apical and basolateral surfaces at least for three weeks, supporting the notion that shedding of the virus from the airways is a long-lasting process. This may further explain why a high rate of co-infection, or co-detection, between HBoV1 and other respiratory viruses has been reported [5] . Since recombinant AAV persists as an episome in transduced tissues, which prolongs gene expression [72, 73] , it is possible that also the HBoV1 genome can be presented as an episome [29, 30] for long term expression and replication. Apparently, the mechanism underlying this feature of HBoV1 infection warrants further investigation. However, in contrast to the other human-pathogenic B19V, HBoV1 does not seem to persist in human tissues for many years [74] .
In conclusion, our findings indicate that the innovative reverse genetics system for studying HBoV1 infection that we describe here will enable us to elucidate the mechanism of HBoV1 replication and pathogenesis in a polarized HAE. Our system mimics natural HBoV1 infection of the in vivo human cartilaginous airway epithelia. The pathogenesis of HBoV1 in co-infection with other respiratory viruses and in conditions of lung diseases is a focus of future study. [43] ), as well as NuLi-1 and CuFi-8 (Tissue and Cell Culture Core, Center for Gene Therapy, University of Iowa). NuLi-1 and CuFi-8 were immortalized from normal and cystic fibrosis human primary airway cells, respectively, by expressing hTERT and HPV E6/E7 genes [44] . Primary Clonetics normal human bronchial/tracheal epithelial cells (NHBE) were purchased from Lonza (Walkersville, MD). Cells were cultured in media following instructions provided by the supplier.
Human airway epithelium cultures. Polarized primary HAE, termed as primary B-HAE, was generated by growing isolated human airway (tracheobronchial) epithelial cells (three HAE cultures were generated from different donors) on collagencoated, semipermeable membrane inserts (0.6 cm 2 , Millicell-PCF; Millipore, Billerica, MA), and then allowing them to differentiate at an air-liquid interface (ALI); this was carried out at the Tissue and Cell Culture Core of the Center for Gene Therapy, University of Iowa [44, 58, 75, 76] . After 3-4 weeks of culture at an ALI, the polarity of the HAE was determined based on the transepithelial electrical resistance (TEER) using an epithelial Volt-Ohm Meter (Millipore) and the relationship to infectability was assessed; a value of over 1,000 V.cm 2 was required for HBoV1 infection. CuFi-and NuLi-HAE were generated following the same method as above, but using the immortalized airway epithelial cell lines, CuFi-8 and NuLi-1, respectively. The primary B-, CuFi-, and NuLi-HAE were cultured, differentiated and maintained in (50%:50%) DMEM:F12 medium containing 2% Ultroser G (Pall BioSepra, Cergy-Staint-Christophe, France).
A nasopharyngeal aspirate was obtained from a child with community-acquired pneumonia in Salvador, Brazil, who had an acute HBoV1 infection (seroconversion, viraemia, and over 10 4 gc of HBoV1 per ml of aspirate) [17] . Viral DNA was extracted according to a method described previously [77] .
The sequence of the head-to-tail junction of the HBoV1 episome suggests that HBoV LEH and REH share similarities both in structure and sequence with that of the BPV LEH and MVC REH, respectively [27, 29] . Based on this information [28] , we designed primers to amplify the HBoV1 termini, which are shown in Table 1 and Figure 1 . The Phusion high fidelity PCR kit (NEB, Ipswich, MA) was used following the manufactures' instructions, to amplify the left-end hairpin (LEH) and the rightend hairpin (REH) of HBoV1. Briefly, the DNA denaturation at 98uC for 30 s was followed by 35 cycles of: denaturing at 98uC for 10 s; annealing at 55uC for 15 s; and extension at 72uC for 30 s. Following the final cycle, extension was continued at 72uC for 10 min. The PCR products were analyzed by electrophoresis in a 2% agarose gel. DNA bands were extracted using the QIAquick gel extraction kit (Qiagen, Valencia, CA), and the extracted DNA was directly sequenced at MCLAB (South San Francisco, CA), using primers complementary to the extended sequences on the forward and reverse amplification primers. PCR-generated DNA was cloned in pGEM-T vector (Promega, Madison, WI), and DNAs isolated from cultures of individual clones were subsequently sequenced.
Construction of the pBB vector. We first constructed a pBBSmaI vector by inserting a linker of 59-SalI-SacII-KpnI-SmaI-ApaI-SphI-KpnI-HindIII-XhoI-39 into a vector backbone (pProEX HTb vector; Invitrogen) generated from the B19V infectious clone pM20 [78] by removing all of the B19V sequence (SalI-digestion). All cloning work was carried out in the Escherichia coli strain of Sure 2 (Agilent, La Jolla, CA). All the nucleotide numbers of HBoV1 refer to the HBoV1 full-length genome (GenBank accession no.:JQ923422).
Cloning of the left-end hairpin ( Figure S2B ). The DNA fragment SalI-BglII-nt93-518(BspEI)-576-XhoI-HindIII (containing the HBoV1 sequence nt 93-576), was amplified from the viral DNA and inserted into SaII/HindIII-digested pBBSmaI, to produce pBB2.1. Another DNA, SalI-nt1-86-BclI (containing HBoV1 nt 1-86 sequence), was synthesized according to the LEH sequence obtained in Figures 1A and 1B , and placed between the SalI and BglII sites in pBB2.1, with ligation of the BclI and BglII sites reproducing the HBoV1 sequence nt 87-92. The resultant plasmid harboring the 59 HBoV1 nt 1-576 sequence with an intact LEH is designated pBB-LEH.
Cloning of the right-end hairpin ( Figure S2C ). The DNA fragment SalI-nt4097-4139(BglII)-5427(KasI)-ApaI (containing the HBoV1 nt 4097-5427 sequence) was amplified from viral DNA and inserted into SaII/ApaI-digested pBBSmaI, resulting in pBB2.2. Another DNA fragment, ApaI-nt5460(KasI)-5543-XhoI (containing HBoV1 nt 5460-5543 sequence) was synthesized based on the REH sequence ( Figure 1C ) and placed between the ApaI and HindIII sequences in pBB2.2, resulting in pBB-REH(D5428-5459). The missing short fragment between the two KasI sites encompassing nt 5428-5459 was recovered by a synthesized KasI linker based on the REH sequence ( Figure 1C ) and inserted into KasI-digested pBB-REH(D5428-5459). The resultant plasmid harboring the 39 HBoV1 nt 4097-5543 sequence with an intact REH is designated pBB-REH.
Cloning of the pIHBoV1 ( Figure S2D ). The HBoV1 DNA fragment SalI-nt1-518(BspEI)-576-XhoI, which was obtained from SalI/XhoI-digestion of pBB-LEH, was ligated into SalI-digested pBB-REH, resulting in pBB-LEH(BspEI/BglII)REH. The larger fragment produced by digestion of this plasmid with BspEI/BglII was ligated to the HBoV1 DNA fragment nt 518(BspEI)-4139(BglII), which was amplified from the viral DNA. The final construct containing the full-length HBoV1 (nt 1-5543) was designated pIHBoV1.
Construction of pIHBoV1 mutants. pIHBoV1NS1(2) and pIHBoV1NP1 (2) were constructed by mutating HBoV1 nt 542 from T to A, and nt 2588 from G to A, resulting in stop codons that lead to early termination of the NS1 and NP1 ORFs, respectively. Similarly, pIHBoV1VP1(2) and pIHBoV1VP2 (2) were generated by mutating HBoV1 nt 3205 from T to A, and nt 3540 from T to G, disrupting VP1 and VP2 ORFs, respectively.
Cells grown in 60-mm dishes were transfected with 2 mg of plasmid as indicated in Figure 2 ; the Lipofectamine and Plus reagents (Invitrogen/Life Technologies, Carlsbad, CA) were used as previously described [79] . For some of the transfection experiments, HEK293 cells were cotransfected with 2 mg of pHelper plasmid (Agilent), which contains the adenovirus 5 (Ad5) E2a, E4orf6, and VA genes, or infected with adenovirus type 5 (Ad) at an MOI of 5 as previously described [79] .
Low molecular weight (Hirt) DNA was extracted from transfected cells, digested with DpnI (or left undigested) and analyzed by Southern blotting as previously described [80] .
Cells were lysed, separated by SDS-8% polyacrylamide gel electrophoresis (PAGE), and blotted with antibodies as indicated as previously described [81] .
HEK293 cells were cultured on fifteen 150-mm plates in DMEM-10%FCS, and transfected with 15 mg of pIHBoV1 per dish using LipoD293 (SignaGen, Gaithersburg, MD). After being maintained for 48 h at 5% CO 2 and 37uC, the cells were collected, resuspended in 10 ml of phosphate buffered saline, pH7.4 (PBS), and lysed by subjecting them to four freezing (2196uC) and thawing (37uC) cycles. The cell lysate was then spun at 10,000 rpm for 30 min. The supernatant was collected and assessed on a continuous CsCl gradient. In brief, the density was adjusted to 1.40 g/ml by adding CsCl, and the sample was loaded into an 11ml centrifuge tube and spun in a Sorvall TH641 rotor at 36,000 rpm, for 36 h at 20uC.
Fractions of 550 ml (20 fractions) were collected with a Piston Gradient Fractionator (BioComp, Fredericton, NB, Canada), and the density of each was determined by an Abbe's Refractometer. Viral DNA was extracted from each fraction and quantified with respect to the number of HBoV1 gc, using HBoV1-specific qPCR as described below. Those fractions containing the highest numbers of HBoV1 gc were dialyzed against PBS, and then viewed by electron microscope and used to infect HAE cultures. Observation by electron microscopy (EM)
The final purified virus preparation was concentrated by ,5fold, and adsorbed for 1 min on a 300-mesh copper EM grid coated with a carbon film, followed by washing with deionized water for 5 s and staining with 1% uranyl acetate for 1 min. The grid was air dried, and was inspected on a 200 kV Tecnai F20 G2 transmission electron microscope equipped with a field emission gun.
Fully differentiated primary B-(each of the three distinct subtypes), CuFi-and NuLi-HAE were cultured in Millicell inserts (0.6 cm 2 ; Millipore) and inoculated with 150 ml of purified HBoV1 (1610 7 gc/ml in phosphate buffered saline, pH7.4; PBS) from the apical surface (at a multiplicity of infection, MOI, of ,750 gc/cell; an average of 2610 6 cells per insert). For each of the HAE, a 2-h incubation was followed by aspiration of the virus from the apical chamber and by three washes of the cells with 200 ml of PBS to remove unbound virus. The HAEs were then further cultured at an ALI.
For conventional monolayer cells, cells cultured in chamber slides (Lab-Tek II; Nalge Nunc) were infected with purified HBoV1 at an MOI of 1,000 gc/cell.
After HBoV1 infection, ALI membranes were fixed with 3.7% paraformaldehyde in PBS at room temperature for 15 min. The fixed membranes were cut into several small pieces, washed in PBS three times for 5 min, and permeabilized with 0.2% Triton X-100 for 15 min at room temperature. The membranes were then incubated with primary antibody at a dilution of 1:100 in PBS with 2% FCS for 1 h at 37uC. This was followed by incubation with a fluorescein isothiocyanate-or rhodamine-conjugated secondary antibody. Confocal images were taken with an Eclipse C1 Plus confocal microscope (Nikon, Melville, NY) controlled by Nikon EZ-C1 software. Primary antibodies used were anti-(HBoV1) NS1, NP1 and VP1/2 antibodies, as reported previously [82] .
For infected cells cultured in chamber slides, IF analysis was carried out as previously described [83] .
Virus samples were collected from both the apical and basolateral surfaces at multiple time points. Apical washing and harvesting was performed by adding 200 ml of PBS to the apical chamber, incubating the samples for 10 min at 37uC and 5% CO 2 , and removing and storing the 200 ml of PBS from the apical chamber. Thereafter, 50 ml of medium were collected from each basolateral chamber.
Aliquots (100 ml apical or 50 ml basolateral) of the samples were incubated with 25 units of Benzonase (Sigma, St Louis, MO) for 2 h at 37uC, and then digested with 20 ml of proteinase K (15 mg/ ml) at 56uC for 10 min. Viral DNA was extracted using QIAamp blood mini kit (Qiagen), and eluted in 100 ml or 50 ml of deionized H 2 O. The extracted DNA was quantified with respect to the number of HBoV1 gc, by a qPCR method that has been used previously [84] . Briefly, the pskHBoV1 plasmid [82] , which contains the HBoV1 sequence (nt 1-5299), was used as a control (1 gc = 5.4610 212 mg) to establish a standard curve for absolute quantification with an Applied Biosystems 7500 Fast system (Foster City, CA). The amplicon primers and the PrimeTime duallabeled probe were designed by Primer Express (version 2.0.0; Applied Biosystems/Life Technologies) and synthesized at IDT Inc. (Coralville, Iowa). Their sequences are as follows (GenBank: JQ411251): forward primers, 59-GCA CAG CCA CGT GAC GAA-39 (nt 2391 to 2408); reverse primer, 59-TGG ACT CCC TTT TCT TTT GTA GGA-39 (nt 2466 to 2443); and PrimeTime probe, 59 6FAM-TGA GCT CAG GGA ATA TGA AAG ACA AGC ATC G-39 Iowa Black FQ (nt 2411 to 2441). Premix Ex Taq (Takara Bio USA, Madison, WI) was used for qPCR following a standard protocol. 2.5 ml of extracted DNA was used in a reaction volume of 25 ml.
On the last day of infection, the HAE on the Millicell inserts were washed with PBS and fixed in 4% paraformaldehyde for ,30 min. The fixed membranes were cut into several small pieces, and washed with PBS three times. Each membrane fragment was transferred to 20% sucrose in a 15-ml conical tube and allowed to drop to the bottom; it was then embedded vertically in cryoprotectant OCT in an orientation that enabled sectioning of the membrane perpendicular to the blade. Cryostat sections were cut at a thickness of 10 mm, placed onto slides, and stained with hematoxylin and eosin (H&E). Images were taken with a Nikon 80i fluorescence microscope at a magnification of 660. Figure S1 Sequencing of PCR DNA fragments. PCR DNA fragments indicated by arrowheads in Figure 1D were extracted and sequenced. A representative result of sequencing is shown in each chromatogram. The sequences between the arrows in the chromatograms (A-C) show the sequences which are complementary to those sequences between the arrows in the hairpin drawings in Figure 1A -C, respectively. (TIF) Figure S2 Construction of a full-length pIHBoV1 clone. (A) HBoV1 genome. The full-length genome of HBoV1 is diagramed with structures of the left-end hairpin (LEH) and rightend hairpin (REH) in a form of negative ssDNA from 39end to 59end. Restriction enzyme sites of BspEI and BglII in the replicative form (RF) DNA are shown. (B) Cloning of the LEH. PCR-amplified DNA fragments from the LEH, shown in red, were ligated into pBBSmaI or its derivative. (C) Cloning of the REH. PCR-amplified or synthesized HBoV1 DNA fragment from the REH, shown in blue, were ligated into pBBSmaI or its derivatives. (D) Cloning of the pIHBoV1. The pIHBoV1 was constructed by ligating HBoV1 DNA nt 1-517 digested from pBB-LEH and nt 518-4139 amplified from viral DNA extract (HBoV1 Salvador isolate) into the pBB-REH that contains HBoV1 REH (nt 4140-5543). All the numbers are nucleotide numbers of the HBoV1 genome (Genbank JQ923422). (TIF) Figure S3 Kinetics of virus release from HBoV1 infection of primary B31-11 and B29-11 HAE. Primary B-HAE (donor B31-11 or B29-11) was infected with purified HBoV1 at an MOI of ,750 genome copy numbers (gc)/cell. Virus was collected from the apical chamber (A), or from both the apical and basolateral chambers (B) for detection of nuclease-resistant viral gc. Averages and standard deviations are shown. ND, not determined. (TIF) Figure S4 Immunofluorescence analysis of HBoV1infected HAE polarized from NuLi-1 cells (NuLi-HAE). NuLi-1 cells were polarized by growth at an ALI for 4 weeks on Millicell inserts of 0.6 cm 2 , until a transepithelial electrical resistance (TEER) of .500 V.cm 2 was detected. Polarized HAE was infected with purified HBoV1 at an MOI of ,750 gc/cell. At 10 days p.i., infected NuLi-HAE was fixed and stained with an anti-(HBoV1)NS1 antibody. Nuclei were stained with DAPI and cells were visualized by confocal microscopy at a magnification of 640.
(TIF) Personal, Occupational, and Public Health Perspectives on Dealing with the First Case of Influenza A (H1N1) in the United Arab Emirates New epidemics of infectious diseases often involve health care workers. In this short communication we present a case report of a health care professional who became the first case of influenza H1N1 virus to be notified in the United Arab Emirates. There are several issues related to workplace considerations and general public health, including preventive measures, the need for isolation of the patient, dealing with contacts, return to work, and communication with the workforce. In recent years influenza viruses have circulated in seasonal (H3N2, H1N1) and avian (including H5N1) forms. There has been concern that Influenza A (H5N1), a worldwide cause of large poultry outbreaks, which by December 2009 had affected 467 persons (282 deaths), would drift or shift to become the next pandemic strain [1] . However in April 2009 'Swine flu' caused by a new strain of influenza A, Pandemic (H1N1) 2009 emerged.
This has now become the dominant strain producing an illness that is transmitted in the same way as seasonal influen-
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. za, which in most cases is mild, which can be effectively treated with antiviral drugs and for which a vaccine is now available. By the end of 2009 many countries were still reporting disease activity and an impact on health-care services [2] .
In the early days of the H1N1 pandemic, when there was uncertainty about the infectivity and virulence of the new virus, a more precautionary approach to management was advocated. This included laboratory testing of suspected cases, contact tracing, isolation of cases and contacts, anti-viral medication for treatment and prophylaxis, and clinical surveillance and follow-up.
In this short case report we describe the personal experience and management of the first case of H1N1 reported in the United Arab Emirates (UAE).
The patient was a 48 year-old male academic public health physician who had just returned to the Middle East after
www.e-shaw.org spending a week with his family in Saskatoon in Canada. His journey to the UAE was via Calgary and Heathrow airport in London, UK. He started feeling lethargic, and developed a sore throat, with cough and high fever for around 10 hours since the night of his arrival in Dubai, UAE. This led him to consult the on-duty infectious disease consultant at the Emergency Department of a local hospital at around 8:00 am the following day.
The consultation included a discussion of any possible exposure to H1N1 since Canada was recognized then as experiencing a large number of cases of the infection. A combined influenza A&B antigen screen on a nasopharyngeal swab was positive, and an additional swab and a blood sample were then sent for further confirmatory testing. He was prescribed Oseltamivir 75 mg orally twice daily for five days, azithromycin 500 mg daily and paracetamol 500 mg three times daily for three days, and advised to remain at home until the confirmatory test results were available.
By the next morning, the patient's fever and sore throat had subsided and he was feeling better. Despite the very low, but nevertheless real risk of having 'swine flu', the patient had to make some important difficult decisions regarding his state of health and his work deadlines. His work place was a university campus and as he had no lectures that day, he had no need to be in contact with any students. All scheduled appointments on his calendar for the day were cancelled, but he decided to proceed with a ten-minute scheduled presentation to six of his peers regarding a large research grant proposal. A mask was not worn during the presentation, and he returned home immediately after the event. The patient was alone at home but one of his relatives came to visit him unannounced, accompanied by his wife and a ten year old child from a neighboring town. They stayed at his home for that night, as the distance for return travel was considerable.
On the next day the patient received a call from the Health Authority confirming Influenza A (H1N1) infection and he was therefore in the unenviable although historical position of being the first reported case of H1N1 infection in the UAE. The patient was admitted to hospital with airborne and contact isolation, where he completed the rest of the maximum recommended 10 days quarantine period. The visiting couple and child also had to stay at the patient's home for 10 days of quarantine and all also received prophylactic medicine (Oseltamivir). No lab tests were advised.
As a public health physician, the index case had considered the H1N1 situation before commencing his travels to Canada. At that time (May 8 2009), the World Health Organization (WHO) did not recommend restricting travel, although some individual national authorities were advising against non-essential travel. The advice on the various websites seemed very pragmatic: observe basic hygiene, hand-washing and cough etiquette; do not travel when ill and seek medical advice if you become ill after your return. The patient's route to Canada took him through London (34 cases reported in the UK at that time) and Toronto (15 cases in Ontario) to Saskatoon (2 cases). By the time he was due to return to the UAE from Saskatoon via Calgary, the number of cases in Canada had increased from 242 to 496 with 19 in Saskatchewan and 67 in Alberta. During his stay in Saskatoon he did not recall meeting anyone with respiratory symptoms and he was quite well on his journey back to Dubai. He was therefore not certain where and from whom he caught the infection.
This case raised several issues related to workplace and general public health. Measures taken by the UAE government to prevent an influenza epidemic include the installation of thermal scanners at Dubai, Sharjah and Abu Dhabi airports (three major international airports in the United Arab Emirates). The individual was afebrile and symptom-free on arrival at the airport, and so was not detained for further enquiry. The thermal scanners will detect individuals with fever from whatever cause, but will not necessarily detect those with early H1N1 infection, especially if they are afebrile [3] .
Effective and timely communication is essential to allay unwarranted concerns from the public and at the workplace. Queries from the media were channeled to a senior member of the administration from the office of the Dean -to ensure consistency in the information provided. He was briefed by public health physicians, occupational health physicians and hospital clinicians dealing directly with the case. A central news release was provided to staff and students on H1N1 reiterating the importance of hygiene in regards to limitation of transmission. The workplace was a university campus. This case did not have any lectures or meetings with students. Contact with a few coworkers was transient (not more than 15 minutes in the same area). These contacts were counselled on the low likelihood of acquiring the infection. They were informed about seeking medical advice if they had any other reasons for concern or if they developed H1N1 symptoms. Doctors, nurses and ancillary healthcare workers looking after the case while in hospital were briefed on hygiene and infection control procedures. N-95 masks, gloves and gowns were provided to health-care staff.
The Health department took prompt action. Family members with close contact were quarantined at home. They were given a prophylactic course of Oseltamivir. Adequate supplies of food and provisions and maintenance of phone communiwww.e-shaw.org cation was confirmed. The public health department dealt with general queries from the public. Official release of information and contact with the WHO was through the federal Ministry of Health. The airline that transported the case from Canada to the UAE sought to contact passengers in the rows adjacent to the passenger's allocated seat. None of those who were traced developed any flu-like illness within the incubation period following the timing of the flight.
Where new epidemics of infectious diseases appear, history has shown that the cases have often included healthcare workers, and their family members [4] . The index case for Ebola infection was a hospital laboratory worker, and secondary cases occurred in other healthcare workers and within the family. Two-thirds of the deaths from the early outbreaks of Ebola infection occurred in healthcare staff. The early cases of SARS and H5N1 infection included doctors and nurses [5] [6] [7] . The likelihood of healthcare staff being affected in such infections is high, especially in the absence of adequate preventive measures, or if there is poor compliance with recommended precautions. In this particular first reported case of H1N1 infection in the UAE, prompt and appropriate action resulted in the individual being treated, the risk of transmission being reduced, and the provision of information being timely and adequate. None of the known contacts developed signs and symptoms of the disease. It was not possible to contact the taxi driver who shared the same vehicle with the case during the hour long journey from the airport home, but there were no reports of infection in any Dubai taxi driver in the 2 weeks following the journey.
We now believe that even if they are infectious, clinicians who practice good respiratory and hand hygiene will limit the risk of transmission to others. Standard and droplet precautions should be in place [8] . Standard Precautions minimize exposure to potentially infected blood and body fluids and include hand hygiene and the use of appropriate personal protective equipment. Droplet precautions require that a medical mask is worn when working within one meter of the patient and that when performing aerosol-generating procedures, further measures are taken including the use of eye protection, N-95 masks or other equivalent or more effective respirators and other personal protective equipment. In addition, respiratory or cough etiquette should be observed so that all persons cover their mouth and nose with a disposable tissue when coughing or sneezing, and then disposing the used tissue promptly. Within the healthcare setting, administrative, environmental and engineering controls such as frequent cleaning of work areas should also be in place.
Generally it will not be appropriate to conduct contact tracing of patients or to provide anti-viral prophylaxis. However if there has been a particular type of contact between a healthcare worker and a patient (for example intubation) or a patient is at high risk of severe or complicated infection, then further risk assessment is indicated with a view to offering prophylaxis. An alternative approach, if practical, is to monitor exposed persons and administer antiviral treatment when symptoms develop. When a vaccine becomes available the first priority should be to immunize healthcare staff.
When pandemic influenza is widespread in a community it will inevitably have consequences for the workplace not least because that is a setting where transmission can occur. In these circumstances occupational health practitioners should be prepared to lead a consistent and proportionate response. Staff with influenza will be diagnosed on the basis of symptoms. The clinical diagnostic criteria are fever (≥ 38 o C) or a history of fever and two or more symptoms of an influenza-like illness i.e. cough, sore throat, headache etc. Those who satisfy this case definition should be sent home and advised not to work until fully recovered. A risk assessment should be carried out and the risk of transmission to other staff members should be considered in terms of the excess risk compared to acquiring the infection from other community sources.
Stories about the new H1N1 case in town appeared daily and reflected public anxiety. The media can play an important role in allaying the fears of the community by providing adequate and accurate information. The installation of thermal scanners at points of entry has their limitations, and is not recommended by the WHO. Studies indicate many of its drawbacks, including a low positive predictive value of 3.5% [2] .
An unpublished population study carried out in the UAE during October 2009 by medical students investigated the impact of the recent H1N1 pandemic on the parents of primary school children. They found that while the majority of parents had good knowledge of H1N1 and its mode of transmission, many had mistaken beliefs about the origin of the virus, for example thinking that it had been genetically engineered. Parents reported changing their behaviour because of H1N1, taking measures such as cancelling travel plans and restricting socializing. Also, while most had confidence in the way in which the authorities had managed the pandemic, they continued to worry that their families were at risk of infection and were not persuaded of the safety of available vaccines.
In conclusions, as for many epidemics, dealing with initial cases is often a key to successful subsequent management of further outbreaks. This case documents the experience of a public health physician as a patient in an infectious disease epidemic, with lessons for occupational and public health management. The lack of further transmission from this first case in the UAE may be a combination of good and effective public health intervention, or serendipity. Even though H1N1 has high infectivity with low case fatality rates, the number of cases globally declined, and WHO declared the end of the pandemic on 10 th August 2010. Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma BACKGROUND: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). METHODOLOGY/PRINCIPAL FINDINGS: In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4(+) tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G(+) FcγRIIIa(CD16A)(+) murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4(+) tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A(+) CD56(+) NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. CONCLUSIONS/SIGNIFICANCE: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A(+) immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL. Cutaneous T cell lymphomas (CTCLs) are a clinically heterogeneous group of lymphoproliferative malignancies characterized by the clonal accumulation of mature and skin-homing memory T cells. Mycosis fungoides (MF), which is the most common and indolent form of CTCL, accounts for 50%-60% of CTCL cases [1] ; primary cutaneous CD30 + lymphoproliferative disorders, more specifically primary cutaneous anaplastic large cell lymphoma (PC-ALCL) -the second most common CTCL, accounts for circa 30%; and Sézary syndrome, which is an aggressive leukemic variant of CTCL, affects approximately 5% of patients. These patients exhibit significant immune dysfunction [2, 3] because of the global dysregulation of T cells, which is due to an unknown etiology [4, 5] . Bacterial sepsis is the terminal event in most patients with advanced cancer. Current therapies for patients with advanced CTCL, including its leukemic variant, are only palliative, and extensive long-term remissions are rare. The poor 5-year survival rate of these patients receiving existing therapies clearly emphasizes the need for the development of new targeted therapies in this fatal disease [6] .
Over the past few years, several studies have described the expression of chemokine receptors in the skin and blood of CTCL patients, including the uniformly high expression of CC chemokine receptor 4 (CCR4) [7, 8, 9, 10] . CCR4 is highly expressed in both leukemic CTCL including Sézary syndrome and in MF, both in the very early stages (patch and plaque stages) of the disease and in large cell transformations [7, 8, 10, 11, 12] . It is also expressed on circa 60% of PC-ATCL cells [1] . In a recently published consensus article regarding the classification of CTCL, it is clear that CCR4 is expressed in the vast majority of CTCL cells, regardless of their histological subtype [1] . On the other hand, expression of CCR4 is limited amongst non-malignant cells [13] . It is not present on neutrophils, monocytes, or B cells [14] . It is absent on naïve T cells, and present on fewer than half of all memory T cells [15] . expression of CCR4 by tumor cells is associated with their skin involvement, CCR4 also has an important role in normal and tumor immunity [13, 14] . CCR4 is expressed at high levels on T regulatory cells (Tregs) that can migrate to tumor cells that secrete the CCR4 chemokines CCL17 and CCL21 to facilitate evasion from immune surveillance [16, 17] . High expression of the these CCR4 ligands has been detected in CTCL lesions [11] , breast cancer [16] , ovarian cancer [17] and oral squamous cell carcinoma [18] . Thus, targeted therapy against CCR4 may be an attractive treatment option for these malignancies, not only to directly kill the CCR4 + tumor cells, but also to overcome the suppressive effect of CCR4 + Tregs on the host antitumor immune response.
Monoclonal antibody (mAb)-based immunotherapies have become the standard therapy in an increasing number of human cancers [19, 20] . Tumor targeting with a human mAb directed against tumor-associated markers, such as CCR4, might provide a powerful therapeutic strategy against CTCL. In this study, we used recombinant adeno-associated viral (AAV) vector-mediated antibody gene transfer into SCID-BEIGE mice to evaluate the effectiveness of h1567, a novel humanized anti-CCR4 mAb to inhibit CCR4 + tumor cell growth and increase survival. The CCR4-specific antibody gene was packaged into an AAV vector and then delivered by a single direct intravenous (i.v.) injection which leads to the endogenous synthesis and durable expression of therapeutic antibody levels for months. Intravenous delivery of this h1567 minibody-encoding AAV vector allowed for rapid and accurate assessment of its therapeutic potential, thereby avoiding ex vivo manipulations involved in the production and purification of therapeutic mAbs.
In vivo studies using therapeutic mAb gene transfer after CCR4+ tumor cell implantation demonstrated the potent antitumor activity of the mAb h1567. In addition, the in vivo effector cells that mediate tumor cell killing through h1567 Fc binding to Fcc receptors, namely FccRIIIa (CD16A), were delineated. These studies suggest that mAb 1567 can serve as an effective antibodydirected therapy for immunodepleting malignant CTCL cells and may minimize collateral damage to the already compromised immune system. Furthermore, in the context of anti-cancer mAb therapies that require frequent and repeated administration, this AAV-based therapeutic antibody gene transfer strategy might serve as an alternative platform for their delivery.
In vitro and in vivo Expression of AAV8-encoding Anti-CCR4 h1567 A modified scFvFc minibody format was used as the antibody moiety in the AAV8 vector, in which the V domains of heavy (VH) and light (VL) chains of the humanized scFv h1567 were fused to the coding region of the hinge and constant domains 2 and 3 (CH2 and CH3) of the human IgG1 heavy chain, to yield bivalent binding to the target molecule hCCR4 (Figure 1a ) (DK. Chang et al., in press). The resulting recombinant AAV8 vector was used for both in vitro protein synthesis and virus production for in vivo antibody gene delivery. In a pilot dosing study, nude mice received a single injection of two different concentrations of AAV8-h1567 via intravenous tail vein injection. Serum h1567 minibody levels were followed for 15 weeks. H1567 minibody levels rose for the first 2-3 weeks, reaching levels of circa 65 and 96 ug/ml for the low (0.8610 11 vg/mouse) and high (2.0610 11 vg/mouse) vector doses, respectively and then through the remaining weeks of the study leveled off at near peak levels for the high dosed vector and circa 1/3rd that level (,35 ug/ml) for the low dosed vector ( Figure S1 ). Because 2610 11 vg per mouse gave higher serum levels of h1567, this vector concentration was used in the subsequent in vivo studies.
CCR4 + Mac-1 tumor cells grow well in SCID-BEIGE mice and therefore we established a SCID-BEIGE/Mac-1 xenograft tumor model to evaluate the efficacy of AAV8-h1567 therapeutic minibody gene transfer. In SCID-BEIGE mice treated with a single intravenous tail vein injection of the AAV8 vectors, a timedependent increase in serum concentrations of the control 11A and h1567 minibodies, reaching steady state levels of circa 50 ug/ ml after 7-14 days and remaining at those peak levels through day 28, the last day of the study ( Figure 1b) . The control 11A is a irrelevant minibody that is directed against SARS Spike protein [21] . To determine whether the AAV8-minibody transduction in vivo could result in production of properly folded scFvFc, protein A-purified minibodies recovered from serum of SCID-BEIGE mice three weeks following intravenous delivery of AAV8 vectors were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. As shown in Figure 1c , when examined under reducing conditions, the 11A and h1567 minibodies recovered from both in vitro and in vivo sources showed bands at the expected size for scFvFc, circa 60 kD. Analysis under non-reducing conditions showed dimer formation (mol wt circa 120 kD), thereby confirming that the minibodies were divalent in vitro and in vivo ( Figure 1c ). In addition, the ease of recovery of the AAV8-derived minibodies from serum using affinity purification on protein A, their reactivity on Western blot with the anti-human Fc antibody, and their stable dimer formation confirms the proper folding and structural integrity of their CH2-CH3 domains (Figure 1c and 1d ).
Binding Activity of h1567 Minibody in Serum Following AAV8-mediated Gene Transfer
To determine the functional integrity of the AAV8-derived scFvFc minibodies, sera obtained from mice 14 days after in vivo AAV8 transduction were examined for the level of binding to CCR4 by flow cytometry. As shown in Figure 1e , the secreted h1567 minibody in the mouse serum could specifically bind to the CCR4 + Mac-1 cells and CCR4 + 293T cells but not to parental 293T cells, indicating that the scFv domain was correctly folded and that it retained full antigen-binding activity. Irrelevant 11A minibody, which served as a negative control, did not bind to CCR4-expressing cells.
The therapeutic effects of AAV8-h1567 gene transfer were next evaluated in vivo in SCID-BEIGE mice that carried subcutaneously implanted Mac-1 tumor xenografts. Groups of 4 mice were given a single intravenous injection of AAV8-h1567 or control AAV8-11A vector on day 7 after tumor inoculation and tumor volume was assessed twice weekly. As shown in Figure 2a , a single injection of AAV8-h1567 resulted in significantly reduced tumor growth compared with AAV8-11A treated mice or PBS control treated mice (P,0.01 at day 18, P,0.0005 at day 21). Mouse survival was monitored for up to 2 months. Tumor-bearing mice treated with AAV8-h1567 significantly outlived (P,0.005) mice treated with AAV8-11A or untreated mice ( Figure 2b ).
Since SCID-BEIGE mice lack T and B lymphocytes as well as functional natural killer (NK) cells, it is possible that the CCR4 + Mac-1 tumor cells were eliminated by h1567 through neutrophildependent ADCC as neutrophils are intact in SCID-BEIGE mice and they express FccRIIIA receptors which have been shown to mediated ADCC [22, 23] . Tumor sections were excised 21 days after AAV8 gene transfer and analyzed histologically for expression of Ly6G, a member of the Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored proteins expressed on murine neutrophils [24, 25] . Immunostaining of tumors sections with neutrophil-specific Ly-6G mAb confirmed infiltration of neutrophils into tumors treated with AAV8-h1567 (Figure 2c , upper-left and middle panels) but not with AAV8-11A (Figure 2c , lower-left and right panels). Quantification of the neutrophil infiltration demonstrated a marked accumulation of Ly-6G+ staining cells only in the h1567 treated mice (Figure 2d) .
To further assess the h1567-mediated, mouse neutrophildependent tumor cell killing, in vitro ADCC assay was carried out using purified SCID-BEIGE mouse neutrophils and h1567 minibody. Coculturing Mac-1 cells with mouse neutrophils in the presence of h1567 at the effector to target ratio of 80:1 resulted in significant neutrophil-mediated ADCC as measured by lactate dehydrogenase (LDH) release from Mac-1 cell (Figure 2e ). Control 11A minibody was not able to induce neutrophilmediated cytotoxicity. These in vitro results correlate with the observed anti-tumor activity in vivo and suggest that the antitumor activity of the h1567 minibody in this CTCL murine model is mediated, at least in part, through Fcc receptor IIIA (CD16A) engagement on mouse neutrophils to induce ADCC effector functions.
The therapeutic CTCL model was further extended to evaluate the role of human effector cells in tumor cell killing using bioluminescence imaging (BLI) of luciferase expressing CCR4 + Mac-1 cells established by retroviral transduction. Ten SCID-BEIGE mice that were grafted with 1610 6 CCR4 + Mac-1 cells and developed equivalent sized tumors as detected on day 7 by BLI were divided into two groups. Eleven days after initial tumor cell inoculation, the AAV8-minibody vectors were administered intravenously. Next, human PBMCs (hPBMCs) were given by intraperitoneal injection 7 days after AAV vector administration. As shown in Figure 3a , treatment with AAV8-h1567 and hPBMCs resulted in substantial tumor growth inhibition compared to AAV8-11A plus hPBMC treated mice. Quantitative monitoring of tumor growth by in vivo BLI correlated with visible tumor growth, further confirming the tumor growth inhibition effect of AAV8-h1567 compared with control group (Figure 3b) . A significant difference was observed between the control AAV8-11A and therapeutic AAV8-h1567 groups on days 40, 42, and 45 after tumor inoculation by caliper measurement and by days 25 and 38 by BLI (Figure 3a and b) . Real-time whole-body BLI of a representative mouse showed that tumor growth was considerably inhibited in mice treated with AAV8-h1567 compared with control mice over the treatment period ( Figure 3c) . Analysis of micro-computed tomography/positron emission tomography (mCT/PET) images also revealed tumor growth inhibition with AAV8-h1567 treatment compared with the control group. While both AAV8-h1567 and AAV8-11A showed primary tumor growth 28 days after tumor inoculation, the tumor cells became much more locally invasive in the AAV8-11A treated group and showed increased metabolic activity as indicated by the accumulation of the PET tracer 18 F-fluorodeoxyglucose (FDG) in whole-body images of mice (Figure 3d) .
To further assess the in vivo mechanisms of tumor cell killing in the AAV8-h1567 plus human PBMC treated group, the role of human NK cells, which also express FccRIIIA receptors, was evaluated. In the AAV8-h1567 treatment group, a substantial increase in tumor-infiltrating human NK cells was observed, as shown by the intense CD56 immunostaining compared with control 11A treated mice (Figure 4a) . Quantitative color deconvolution analysis showed a significantly increased staining in the mouse group treated with AAV8-h1567 compared with the control group treated with AAV8-11A (P,0.01; Figure 4b ).
Human NK cell-mediated ADCC activity was also evaluated in vitro using purified human NK cells as effector cells. As shown in Figure 4c , human NK cells were able to kill Mac-1 target cells in the presence of h1567 in a dose dependent fashion. Control 11A minibody showed only very low levels of killing. As both mouse neutrophils and human NK cells express FccRIIIA receptors (CD16A) on their surface that can bind h1567, these in vitro and in vivo data strongly support that h1567 mediated killing occurs, at least in part, through FccRIIIA engagement and activation of immune cell effector functions.
In this study, an AAV8-based therapeutic antibody gene transfer model was developed to evaluate a novel humanized anti-CCR4 monoclonal antibody h1567 as a therapeutic drug candidate against CTCL. The SCID-BEIGE mice that were used to establish this CTCL model lack T and B cells and functional NK cells [26] . High level, durable expression of the h1567 minibody was achieved after a single intravenous injection and significant anti-tumor activity against CCR4 + Mac-1 cells was seen in two animal treatment studies. These results provide the first in vivo evidence that mAb h1567 may be clinically active against CTCL cells and suggest that further studies should be undertaken to investigate its clinical efficacy.
Remarkable among the findings of this study is that a single intravenous tail vein treatment with AAV8-h1567 minibody resulted in a dose dependent increase in serum minibody levels that steadily increased over a two week period and remained at near peak levels through the end of this 15 week study ( Figure S1 , Figure 1b ). The integrity of the minibodies was demonstrated biochemically in vitro and in vivo by several parameters including their CCR4 + binding activity, stable dimer formation and ease of recovery by protein A chromatography ( Figure 1 c and 1d) . This scFvFc minibody format may be ideal for experimental AAV8 delivery since conventional mAb expression is derived from heavy and light chain genes, and it can be difficult to achieve the balanced expression of two genes within a single AAV vector that has a small packaging capacity (less than 5 kb), although a 2A selfprocessing peptide and furin cleavage have been successfully used to drive the expression of full-length rat IgG [27, 28] . For cancer immunotherapy, scFvFc minibodies appear to be promising because they have been shown to be functionally comparable with full-length IgG and have been successfully used to treat various tumors in preclinical studies [29, 30, 31] . As an added benefit, along with their bivalent antigen-binding avidity and intact antibody effector functions, they comprise a single polypeptide chain that does not require balanced heavy and light antibody chain heterocomplex associations and have a smaller molecular weight for better tissue penetration compared with whole IgG molecules [32] .
The functional integrity of the h1567 minibody was also shown by its potent in vivo anti-tumor and in vitro killing activities. In the first treatment study (model 1), marked inhibition of Mac-1 tumor cell growth and increased survival was seen (Figure 2a and b ) even though these SCID-BEIGE mice have profound immune cell defects including lack of T and B cells as well as impaired macrophage and NK cell effector functions [26] . Further IHC staining of the paraffin-embedded tumor tissues revealed a predominant infiltration of Ly-6G + CD16A + neutrophils only in the h1567 minibody but not control 11A minibody treated mice (Figure 2c) . Furthermore, in vitro ADCC assay using purified mouse neutrophils as an effector cells demonstrated that h1567 induced significant lysis of CCR4 + Mac-1 cells, while no lysis was seen with 11A ( Figure 2e) . These results support the view that neutrophil-mediated ADCC is involved in anti-tumor activities following AAV8-h1567 gene delivery in the SCID-BEIGE CTCL mouse model.
The therapeutic SCID-BEIGE CTCL model was further extended to evaluate the role of human effector cells in tumor cell killing. In the second treatment study (model 2), AAV8-h1567 gene delivery together with human PBMCs was evaluated and a significant inhibition of CCR4 + Mac-1 tumor cell growth was again seen (Figure 3) . The therapeutic effect of the h1567 was monitored using tumor size measurements and BLI. In comparison with the measurement of tumor volume, BLI analysis enabled earlier tumor detection and revealed extensive cell death in response to h1567 treatment (Figure 3c ). The addition of microPET and CT images provided three-dimensional analysis of the primary tumor and further evaluation of the effectiveness of the AAV8-h1567 treatment in vivo. PET imaging indicated invasive tumor cell infiltration into surrounding tissues which was not seen in the h1567 treated mice (Figure 3d) .
The FccRIIIA receptor (CD16A) is the dominant FccR involved in human NK cell-mediated ADCC. Treatment of CCR4 + Mac-1 tumor bearing mice with AAV8-h1567 and human PBMCs resulted in a marked increase in the number of tumorinfiltrating human CD56 + NK cells, suggesting that CD16A which is expressed on human NK cells is involved in this tumor cell killing through it's interaction with the Fc portion of h1567, a finding that has been experimentally confirmed through Fc mutagenesis studies (data not shown). Moreover, in vitro ADCC studies with purified human NK cells demonstrated a concentration dependent killing by h1567 (Figure 4c) . Thus the unifying observations from both treatment studies strongly suggest that the in vivo anti-tumor activity of h1567 is mediated, at least in large part, by ADCC through engagement of FccRIIIA on mouse neutrophils (model 1) and human NK cells (model 2). Since Fc gamma receptors (FccRs) of different types are present on a variety of effector cell populations, including NK cells, dendritic cells, macrophages, monocytes and neutrophils [33, 34] , it is possible that FccR engagement on other immune effector cells, not investigated in this study could also be involved.
MAb therapy for advanced CTCL has been proposed [3] and numerous trials with alemtuzumab (anti-CD52) have shown modest to moderate clinical effects [35, 36, 37] . A recent trial with low dose alemtuzumab has shown complete remission in 50% of patients with refractory leukemic forms of the disease and without infectious disease complications although it was found completely ineffective in the treatment of MF [6] . A mAb to CD4 (GenMab) has been designated an orphan drug for the treatment of MF by the FDA [38] . While both approaches are designed to eliminate CTCL cells, there can be significant adverse effects from either treatment. CD52 is expressed by virtually all T and B cells, and the elimination of all CD4-positive cells has well-known negative consequences [39] . Clonal malignant T cells in these CTCL patients express uniformly high levels of CCR4, but variable to low levels of other skin homing addressins, including CLA, CCR10 and CCR6. CCR7, which is also uniformly highly expressed on leukemic variants of CTCL with T CM phenotype, is not expressed on the phenotypic T EM cells that are found in MF skin lesions [7] . Thus, only CCR4 is uniformly expressed on all forms of CTCL and has a restricted expression pattern on normal T cells, including Tregs [40] . Indeed, a subset of malignant T cells in some CTCL have been shown to act as CCR4 + Tregs to suppress antitumor responses and may fuel disease progression [41] . A therapeutic mAb that could preferentially target all forms of the disease and reverse Treg mediated immune suppression would be a major advance in the effective therapy of CTCL. The activity of mAb1567 in abrogating Treg mediated suppression of T effector cell function is described elsewhere (DK. Chang et al., in press). MAb KM0761, is another humanized anti-CCR4 mAb that has shown promising results in CTCL animal studies [42] and in clinical trials for refractory Adult T-cell leukemia (ATLL) and peripheral T cell lymphoma (PLCL) where good clinical activity without severe adverse side effects was seen [43, 44] . Our data support further exploration of the clinical potential of therapeutic mAbs that target CCR4 in CTCL.
In summary, the results of the present study have validated the utility of an AAV8-based therapeutic minibody gene transfer platform for the rapid experimental evaluation of mAbs for the treatment of human cancer. Furthermore, this study showed that the AAV8-h1567 minibody inhibited the primary CCR4 + tumor burden, suppressed local metastasis and prolonged the survival time in tumor-bearing SCID-BEIGE mice. We remain hopeful that additional studies will support this humanized mAb1567 moving from bench to bedside. 
The human skin-tropic Anaplastic large-cell lymphoma (ALCL) cell line Mac-1, which was originally isolated in the laboratory of Marshall E. Kadin at Harvard Medical School [45] , was cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 0.06 mM 2-mercaptoethanol, and 500 mg/ml G418. Immunophenotyping of the Mac-1 cell line showed the expression of all known tumor-specific chemokine receptors, including high levels of CCR4, CCR7, and CXCR4. This MAC-1 cell line was stably transduced with a luciferase encoding retrovirus. HEK 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). All cells and cultures were maintained at 37uC in a 5% CO 2 humidified incubator. Human PBMCs obtained from the Dana-Farber Blood Center were purified by a Ficoll-Hypaque density gradient centrifugation as described in the general protocol of Miltenyi Biotec Inc. (Auburn, CA). Mouse neutrophils were isolated from SCID-BEIGE mouse blood by Percoll density gradient centrifugation, as described [46] . Human NK cells were isolated from human PBMC using the NK cell isolation kit, according to the manufacturer's protocol (Miltenyi Biotec, CA).
To construct the scFvFc h1567 minibody expression cassette, the scFvFc h1567 gene was PCR-amplified from a plasmid coding for the humanized anti-human CCR4 antibody that is derived from heavy and light antibody chains of mAb 1567 (R&D Systems, Inc) previously cloned in our laboratory (DK. Chang et al., in press) and inserted into the AAV-cloning vector pTRUF (obtained from the University of Iowa Viral Vector Core) at the restriction sites of Sfi1 and Not1. Consequently, to efficiently direct the expression and secretion of the single chain mAb, the pTRUF vector was modified by inserting the human IgG VH4 leader sequence and the Fc sequence (hinge, CH2 and CH3 domains) of the human IgG1 flanked by 145-bp and AAV2-inverted terminal repeats (ITRs) (Figure 1a ).
Recombinant AAV8 viral vectors were produced using a helper virus-free system with some modifications [47] . Low-passage human HEK 293 cells were cotransfected by linear polyethylenimine (Polysciences) with three plasmids: the AAV cis-plasmid pTRUF encoding the human mAb gene expression cassette flanked with ITRs; the AAV-packaging plasmid p5e18 (2/8) containing AAV2 rep and AV8 cap genes; and the Ad helper plasmid pXX6-80 containing the VA RNA, E2, and E4 genes required for AAV propagation (obtained from Dr. Jim Wilson, University of Pennsylvania) [48] . At 48 h post-transfection, the cells were harvested, and the AAV virus extracted by freezing and thawing the cells. Subsequently, AAV was purified by two sequential iodixanol density gradients, concentrated, then desalted by centrifugation through Biomax 100-K filters (Millipore) according to the manufacturer's instructions. Viral titers were determined as genome copy titers (vg), by quantitative real-time PCR using primers and probe speicific for AAV vector pTRUF [49] . Forward primer (59-TCTGAGTAGGTGTCATTC-TATTCTGGG-39) is located at the end of the 39-poly(A), and reverse primer (59-CACTAGGGGTTCCTAGATCTCTCCC-39) is at the beginning of the 39 inverted terminal repeat (ITR). The probe (59-TCTTCCCAATCCTCCCCCTTGCTGTC-3; FAM/TAMRA) is located in between.
Larger quantity of the AAV serotype 8 vectors encoding scFvFc 11A, control minibody specific for SARS [21] , and scFvFc h1567 were produced at Harvard Gene Therapy Initiative (Harvard Institute of Medicine, Boston, MA) and used in the animal studies. 
Mice were monitored for tumor development and progression by both caliber measurement and Xenogen BLI. The latter was initiated for the monitoring of tumor growth 7 days after tumor implantation, which was repeated once a week. Mice were anesthetized with 3.5% isoflurane in an induction chamber, which was followed by the intraperitoneal administration of 50 mg/ml D-luciferin. For imaging, mice were maintained under 1.5% isoflurane anesthesia that was delivered through a nose cone. Whole body images were repeatedly acquired until the maximum peak of photon number was confirmed during various exposure times (10 s-1 min). Data were quantified using the time point that gave the highest photon number during the scanning time and analyzed using the Living Imaging software (Caliper Life Sciences, Hopkinton, MA).
PET/CT scans were performed at the Harvard Medical School Imaging Core Facility. Mice were fasted for 12 h before the 18 F-FDG injections, but provided water ad libitum. For 18 F-FDG injection and imaging, mice were anesthetized using 2% isoflurane. The animals were then intraperitoneally injected with 7.4 MBq (200 mCi) of 18 F-FDG, allowed to regain consciousness, and then kept at 37uC until imaging. Imaging was started 30 min after the intraperitoneal injection. Mice were imaged in a chamber that minimized positioning errors between PET and CT to less than 1 mm. Image acquisition time was 10 min. Images were analyzed using AMIDE software [50] . All regions of interest were defined on fused PET/CT images to ensure reproducible positioning.
HEK 293T cells (ATCC, Manassas, VA) were transfected with the AAV-coding plasmid containing the minibody-expressing constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Three days after transfection, the minibodies were purified from the supernatants with protein A sepharose affinity chromatography. The in vivo production of AAV8-minibodies was generated by i.v. injections into SCID-BEIGE mice as described above. Levels of minibodies in the serum were measured in duplicate using a human IgG ELISA quantitation kit according to the manufacturer's protocol (Bethyl Laboratories, Inc., Montgomery, TX).
Western immunoblotting was performed on protein A column purified samples containing in vitro synthesized minibodies and in vivo AAV8-derived minibodies. The proteins were separated by SDS-PAGE under reducing or nonreducing conditions and electrophoretically transferred onto a nitrocellulose membrane using the iBLot dry blotting system (Invitrogen). After blocking with 5% skim milk overnight, the blot was probed with an APconjugated human IgG-Fc antibody that was diluted 1:30,000 in blocking buffer for 1 h at room temperature. Excess conjugate was removed by five washes with Phosphate buffered saline containing 0.1% Tween 20 (PBS-T). The detection of protein was performed by incubating the membrane with BCIP/NBT alkaline phosphatase substrate (KPL).
The biological activity of the in vivo AAV8-derived h1567 minbodies was analyzed by fluorescence-activated cell sorting (FACS) for binding activity. Mac-1 cells or 293T-CCR4 cells were washed with PBS supplemented with 0.5% bovine serum albumin (PBS-B) and then incubated with in vivo produced h1567 for 1 h at room temperature, which was followed by incubation with antihuman IgG-Fc conjugated to fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed using BD FacsCalibur (BD Biosciences, San Jose, CA) and FlowJo data analysis software (Tree Star, Inc., Ashland, OR).
Immunohistochemical staining was performed at DFCI/Harvard Cancer Center Research Pathology Core. For qualitative and quantitative immunohistochemical analysis, formalin-fixed and paraffin-embedded tissue sections were stained with antibodies directed against Ly-6G on the surface of mouse neutrophils and human CD56 antigen on human NK cells. The stained slides were then scanned using the Aperio ImageScope (Aperio Technologies, Inc., Vista, CA), and full tumor sections were selected for quantitative analyses. The percentage of positively stained cells in the entire tumor sections was calculated using a color deconvolution algorithm.
In vitro Antibody-dependent Cell Cytotoxicity Assay ADCC was performed using the lactate dehydrogenase (LDH) release assay method, according to the CytoTox96 non-radioactive cytotoxicity assay procedure specified by the manufacturer (Promega, Madison, WI). Mouse neutrophils purified from SCID-BEIGE mouse or purified human NK cells from PBMC was used as effector cells and CCR4+ Mac1 tumor cells were used as target cells. Briefly, purified SCID-BEIGE mouse neutrophils or NK cells were plated at a density of 1610 4 cells per well in a roundbottom 96-well plate in the presence of h1567 or 11A minibodies.
After 1 h of incubation, freshly prepared effector cells were added at an effector-target cell ratio (E:T) of 80:1 (mouse neutrophils) or 2:1 (human NK cells). After 2 h incubation at 37uC, supernatants of each well were recovered by centrifugation at 3006g for 5 min. LDH activity in the supernatant was determined by measuring absorbance at a wavelength of 490 nm. The cytotoxicity (%) was calculated according to the following formula:
where E is the LDH release by effector-target coculture, SE the spontaneous release of the LDH from the effector cells, ST the spontaneous release of the LDH from the target cells and M the maximum release of the LDH from the target cells incubated with lysis solution (10% Triton-X). All measurements were done in triplicate.
Statistical analyses were performed using 2-way ANOVA with Bonferroni post hoc tests and unpaired 2-tailed t-tests using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). P values less than 0.05 were considered statistically significant. Figure S1 Dose dependent expression of h1567 anti-CCR4 minibody. Nude mice (4 mice per group) were treated one time by tail vein injection with AAV8-h1567 viral vectors at the two concentrations shows. PBS buffer treated mice served as controls. Mice were bled at the indicated time points over 15 weeks and their h1567 scFv-Fc levels were determined by ELISA on anti-human Ig capture and detection. (TIF) The VNTR Polymorphism of the DC-SIGNR Gene and Susceptibility to HIV-1 Infection: A Meta-Analysis BACKGROUND: Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin related (DC-SIGNR) can bind to the human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein and is thus important for the host-pathogen interaction in HIV-1 infection. Studies of the association between the variable number tandem repeat (VNTR) polymorphism of the DC-SIGNR gene and HIV-1 susceptibility have produced controversial results. METHODS AND FINDINGS: We conducted a meta-analysis of the data contained in the literature to clarify these findings. In total, 10 studies consisting of 2683 HIV-1 patients and 3263 controls (2130 healthy controls and 1133 HIV-1 exposed but seronegative (HESN) controls) were included. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were assessed in the main analyses. Further stratified analyses by ethnicity and sample size were performed. By dividing the controls into two groups, healthy controls and HIV-1 exposed but seronegative (HESN) controls, we explored different genetic models to detect any association between the VNTR polymorphism and predisposition to HIV-1 infection. The results showed that the 5-repeat allele carriers (OR = 0.84, 95% CI = 0.73–0.96) and the 5/5 homozygous (OR = 0.68, 95% CI = 0.50–0.93) had significantly reduced risk when using the HIV-1 exposed but seronegative (HESN) as controls. The stratified analyses by ethnicity and sample size confirmed these findings. However, a low to moderate degree of heterogeneity was also found across studies. CONCLUSIONS: Our findings demonstrate that the VNTR polymorphism of the DC-SIGNR gene is associated with a moderate effect on host susceptibility to HIV-1 infection. Similar to the 32-bp deletion in the chemokine receptor-5 gene (CCR5Δ32), the DC-SIGNR VNTR 5-repeat allele might have a role in resistance to HIV infection, particularly in Asian populations. The incidence of acquired immunodeficiency syndrome (AIDS) has increased over the past few decades. Up to now, nearly 34 million people suffered from human immunodeficiency virus-1 (HIV-1) infection, and an estimated 2.7 million people were newly infected with the virus in 2010 (http://www.who.int/features/ factfiles/hiv/facts/en/index3.html). However, the natural course of HIV-1 infection and the susceptibility to infection after exposure are highly heterogeneous among individuals [1, 2] . Despite of high-risk behavior and/or multiple exposures to HIV-1, some individuals remained seronegative, or uninfected. These individuals may have a different course of progression to AIDS and may have different clinical outcomes. It is a common observation that some infected individuals became symptomatic within 2-3 years while others remained asymptomatic for more than 10-15 years [3] . Last year, NIH published a conference report about definition of HIV-exposed seronegative (HESN) individuals. It is now a consensus to define HESN from several group of individuals who are at high risk of exposure which include : (1) the commercial sex workers, (2) people with hemophilia, (3) discordant couples, (4) intravenous drug users, and (5) mother-to-child transmission [4] .
In the past few years, many studies revealed that host immunogenetics, including genetic polymorphisms, play important roles in host resistance to HIV-1 infection and predict different progressions to AIDS after infection [5] [6] [7] . Polymorphisms of certain chemokines and chemokine receptors have been reported to play important roles in individual variability in response to HIV-1/AIDS. The most investigated variation is the 32-bp deletion in the chemokine receptor-5 (CCR5 D32) gene, which was shown to confer resistance to HIV-1 infection in homozygous carriers, and its role has been investigated in a clinical context [8, 9] . Similar to CCR5, DC-SIGNR is potentially an important gene affecting host susceptibility to HIV-1 infection and disease progression. DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) is able to bind the HIV-1 gp120 surface glycoprotein with high affinity and is able to enhance trans-infection of T cells by HIV-1 [10] . DC-SIGNR is a homolog of DC-SIGN with 77% amino acid identity and preferential expression in liver and lymph node epithelial cells. DC-SIGNR functions similarly to DC-SIGN in capturing HIV-1 and enhancing HIV-1 infection of T cells [11] .
Both DC-SIGN and DC-SIGNR are organized into 3 domains: (1) an N-terminal cytoplasmic region followed by a transmembrane domain, (2) a neck-region containing a variable number tandem repeat (VNTR) of a conserved 23 amino acid sequence, and (3) a C-terminal extracellular domain with a C-type carbohydrate recognition domain (CRD) involved in pathogen binding [12] . While the CRD forms complicated carbohydrates with high mannose-containing ligands, the neck-region is essential for lectin tetramerization and influences the capability of CRD to interact with pathogens such as HIV-1. Because the VNTR of the neck-region in DC-SIGNR is highly polymorphic, ranging from 3 to 9 repeats in worldwide populations, the polymorphism has been widely studied with regard to host genetic predisposition to HIV-1 infection [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . However, the results are controversial and inconclusive, most likely because of the different ethnic populations used in the different studies or lacking the statistical power in any individual study to produce a reliable conclusion. Thus, a comprehensive analysis is critical.
In this study, a meta-analysis was performed to investigate the association between the VNTR polymorphism of the DC-SIGNR gene and host susceptibility to HIV-1 infection. We also performed a stratified analysis by ethnicity and sample size to explore the variation in the relationship between VNTR polymorphism and HIV-1 infection risk among different ethnic populations.
We performed a literature search in PubMed, MEDLINE, Embase, Wanfang Database and Weipu Database for studies published through April 2011 to use in this meta-analysis. The key terms were ''DC-SIGNR or CLEC4M or CD209L or L-SIGN or CD299 or DC-SIGN2'', ''HIV-1'', and ''polymorphism'' in various combinations. The search was limited to articles published in English and Chinese. We also manually searched the references matching the above criteria to identify additional studies.
The eligible investigations met the following criteria: (1) the studies were case-control designed to explore the association between VNTR polymorphism in the DC-SIGNR gene and HIV-1 susceptibility; (2) the studies provided data on the distribution of VNTR polymorphism in the case-control population; (3) the studies were published in the English or Chinese languages. Studies were excluded if they did not contain enough data for meta-analysis, if they were abstracts or reviews, or if they were duplicated within other included studies.
Two authors perused the papers and extracted the data according to the above inclusion and exclusion criteria. The following characteristics were collected from the eligible studies: first author, year of publication, country of studied population, ethnicity, sample size of the cohorts, number of HIV-1 patients, number of normal healthy controls, number of HIV-1 exposed but seronegative (HESN) controls, and the distribution of the DC-SIGNR VNTR genotype among case and control groups.
ORs with 95% CI were calculated to assess the association between VNTR polymorphism in the DC-SIGNR gene and HIV-1 susceptibility. In the primary analysis, an allelic association test was performed for the prevalent alleles, which were (1) the 7repeat allele, (2) the 5-repeat allele, (3) the 6-repeat allele and (4) the 9-repeat allele. These alleles represented the four most prevalent alleles in human population. To correct for multiple testing for these 4 alleles, a Bonferroni correction was also applied to determine the corrected p-value (p-value corrected). After that it was found that 5-repeat allele had a protective effect. An exhaustive round of post-hoc analysis was carried out to identify if any particular genetic model was in operation. In this post-hoc analysis, the pooled ORs were performed between HIV-1 patients and control groups in the following genetic models: (1) the homozygote proportion [24, 25] , (2) the 7/7 genotype vs. the other genotypes, (3) the 5/5 genotype vs. the other genotypes, (4) the 5/7 genotype vs. the other genotypes, (5) the 6/7 genotype vs. the other genotypes, (6) the 7/9 genotype vs. the other genotypes. As it is an explorative attempt to delineate the mode of operation account for the protective effect in the genetic test, no Bonferroni correction was carried out in this stage. In all the genetic models, the types of controls were used: (1) healthy normal or population controls and (2) HIV-1 exposed but seronegative (HESN) controls (see the Table S1 for detailed breakdown). Because the remaining rare genotypes and alleles of the VNTR locus have negligible frequencies in the cohorts, their comparisons were not conducted in this analysis. Stratified analyses were performed by ethnicity and sample size.
Heterogeneity across the publications was assessed with the Cochran's ,chi. 2 test (Q-test) [26] , and p,0.10 was considered statistically significant. The I 2 test was also conducted to evaluate heterogeneity between studies. A high heterogeneity is considered present when I 2 .50% and much higher when I 2 .75% [27] . The pooled OR was calculated by a fixed effect model (using the Mantel-Haenszel method) or a random effect model (using the DerSimonian-Laird method) according to the heterogeneity among studies [28] . The statistical significance of OR was analyzed by the Z test, and p,0.05 was considered statistically significant. Publication bias was evaluated by Egger's and Begg's tests and was considered significant if p,0.05 [29, 30] . Sensitivity analysis was performed by sequentially excluding individual studies to assess the stability of the results.
Statistical analyses were performed using the Revman 5.1 software and STATA 10.0 software. The p value was two-tailed and was considered to be statistically significant if its value was ,0.05.
As shown in Figure 1 , a total of 115 results were identified after an initial search from the selected electronic databases. After screening the titles and abstracts, 40 articles were selected for further review. Among them, five reviews were excluded; 22 studies were excluded for not referring to the association between the DC-SIGNR VNTR polymorphism and HIV-1 susceptibility.
Finally, one study was excluded because it lacked extractable data; and two studies were excluded because the data or sample sets overlapped with another one. Thus, a total of 10 studies were suitable for meta-analysis [13] [14] [15] [16] [17] [18] [20] [21] [22] [23] . Among them, seven studies were conducted with Asians [13, [16] [17] [18] 20, 21, 23] , two were conducted with European-Americans [14, 15] and one was conducted with Africans [22] . Two studies only included subjects of HIV-1 patients and HIV-1 exposed but seronegative (HESN) controls [21, 22] . Three studies included subjects of HIV-1 patients and healthy controls [15, 18, 20] , while the remaining five studies included subjects of HIV-1 patients, HIV-1 exposed but seronegative (HESN) controls, and healthy controls [13, 14, 16, 17, 23] . The total number of samples involved in the 10 eligible studies was 5946, which included 2683 HIV-1 patients and 3263 controls (2130 healthy controls and 1133 HIV-1 exposed but seronegative (HESN) controls). The characteristics of each study are listed in Table 1 .
After pooling the data from the 10 studies for meta-analysis, the results were calculated according to different genetic models. For each genetic model, two comparisons were carried out separately using either the healthy samples or the HIV-1 exposed but seronegative samples (HESN) as controls.
In the primary analysis for allelic risk model of the 5-repeat allele vs. the other alleles, using the healthy individuals as controls, the value of X 2 was 9.52 with 7 degrees of freedom, and p was 0.22 in a fixed effect model (Figure 2A ). The I 2 was 27%, suggesting a low to moderate heterogeneity. The fixed effect model was applied to synthesize the data. Overall, the OR was 1.03 (95% CI = 0.93-1.15) and was not significant (Figure 2A ). On the other hand, using the HIV-1 exposed but seronegative (HESN) samples as controls, the value of X 2 was 8.84 with 6 degrees of freedom, and p was 0.18 ( Figure 2B ). The I 2 was 32%, suggesting a moderate heterogeneity.
Then the fixed effect model was applied to synthesize the data and the pooled OR was calculated. The pooled OR value was significant at 0.84 (95% CI = 0.73-0.96, p = 0.01, Figure 2B ). The Bonferroni corrected P-value was 0.05. These results suggested that the 5-repeat allele carriers tended to be associated with resistance to HIV-1 infection in HIV-1 exposed seronegative (HESN) individuals.
In the genetic models, we analyzed the heterogeneity of 5/5 vs. the other genotypes by using the healthy subjects as controls. The results indicated a low heterogeneity (X 2 = 9.30, I 2 = 25%, p = 0.23) ( Figure 3A ). Next, we pooled the 8 studies under the fixed effect model. The pooled OR was 0.90 (95% CI = 0.71-1.44, p = 0.38) ( Figure 3A) . Then, we analyzed the heterogeneity of 5/5 vs. the other genotypes by using the HIV-1 exposed but seronegative samples subjects (HESN) as controls ( Figure 3B) . The results indicated a low to moderate heterogeneity (X 2 = 7.09, I 2 = 29%, p = 0.21). The fixed effect model was then used to synthesize the data. Overall, the pooled OR was significant at 0.68 (95% CI = 0.50-0.93, p = 0.01) ( Figure 3B ). These results suggested that the 5/5 homozygous genotype showed a significantly reduced risk of HIV-1 infection in HIV-1 exposed seronegative individuals (HESN) but not in healthy individuals. A summary of the results of all the comparisons with different genetic models was listed in Table S1 .
We also performed stratified analyses by ethnicity and sample size to explore potential sources of heterogeneity and examine the relationship between the DC-SIGNR VNTR polymorphism and susceptibility to HIV-1 infection. The results are also summarized in Table S2 . Most of the results of the stratified analyses were consistent with the main analysis. For ethnicity, the studies were divided into two subgroups, one subgroup of Asian descendants (7 studies) and the other subgroup of European-American descendants (2 studies). The resistance to HIV-1 infection found among 5/5 homozygous subjects was predominantly due to the HIV-1 exposed seronegative (HESN) subjects of the Asian population (OR = 0.58, 95% CI = 0.39-0.85, p = 0.006, Pheterogeneity = 0.34 and I 2 = 12%) ( Figure 4A ). The 5-repeat allele presented the trend of having the protective effect among Asian (OR = 0.84, 95% CI = 0.71-1.00, p = 0.05, Pheterogeneity = 0.20 and I 2 = 33%), though the p value was marginal ( Figure 4B ). This result also showed that the sample size of the Asian population in present study was not large enough to achieve enough statistical power to obtain significant observations. As there was only one study performed with African and European-Americans involving HIV-1 patients and HIV-1 exposed seronegative (HESN) controls, the stratified analyses in these populations were not performed.
For sample size, the studies were stratified into two subgroups, one comprising studies with more than 200 subjects and one with fewer than 200 subjects. In these subgroups, significantly reduced HIV-1 infection of 5/5 homozygous (OR = 0.69, 95% CI = 0.50-0.94, and p = 0.02) and 5-repeat allele carriers (OR = 0.82, 95% CI = 0.68-0.98, and p = 0.03) was found in the subgroup with a sample size of more than 200 subjects, but not in the subgroup with fewer than 200 subjects (Table S2 ).
Publication bias was assessed by Begg's test and Egger's test (data not shown) in the total population and all the subgroups. For the genotypic risk model, 5/5 vs. other genotypes, in which the HIV-1 exposed but seronegative samples (HESN) subjects were used as controls, both the Begg's test (p = 0.462) and the Egger's test (p = 0.215) did not present any significantly statistical evidence of publication bias. For the allelic risk model of 5-repeat allele vs. other alleles, in which the HIV-1 exposed but seronegative 
Sensitivity analysis was performed by deleting one study at one time to assess the stability of the pooled ORs. None of the corresponding pooled ORs was statistically changed, implying the stability of the results. By using the healthy samples as controls, the comparison was carried out between the HIV-1 patients and healthy samples; (B) By using the HIV-1 exposed but seronegative (HESN) samples as controls, the comparison was carried out between the HIV-1 patients and the HIV-1 exposed but seronegative (HESN) samples. Note: A Bonferroni correction for multiple testing of 4 was applied to get a corrected P value in this primary analysis. The association between 5-repeat allele had a corrected P value of 0.05. doi:10.1371/journal.pone.0042972.g002
A potential role of host genetic factors in the predisposition to HIV-1 infection has been suggested by different reports [8, 31, 32] . DC-SIGNR serves as an HIV-1 ligand to facilitate HIV-1 virion infection into adjacent CD4+T cells in trans and has been the subject of many recent studies. The VNTR polymorphism in its neck-region was found to be associated with host susceptibility to HIV-1 infection, but the conclusions were controversial.
In the present study, we performed a meta-analysis of 10 eligible studies with 2683 HIV-1 patients and 3263 controls to elucidate the relationship between the VNTR polymorphism and HIV-1 infection risk. The strength of the present analysis is based on the accumulation of published data that provides enough information to generate a more precise conclusion. By using different genetic models, we could preliminarily estimate the effect of the allele frequency, genotype frequency, and homozygous proportion. The results indicated that the 5/5 homozygous genotype was associated with resistance to HIV-1 infection. The protective effect was most predominant in HIV-1 exposed seronegative (HESN) individuals. The stratified analyses by ethnicity and sample size confirmed these findings. Thus, it is important to emphasize that in studies concerning HIV-1 susceptibility, taking samples from HIV-1 exposed seronegative (HESN) individuals as controls may be more powerful than taking random healthy individuals as controls. In future studies, collecting both HIV-1 exposed seronegative individuals (HESN) and random healthy individuals as controls would be helpful for clarifying the relationship between candidate genes and host susceptibility to HIV-1 infection. But under the definition and criteria of HIV-1 exposed seronegative individuals (HESN), it is still a heterogeneous group. A workshop sponsored by NIH was conducted in 2011 and a consistent definition and criteria were drawn in the term of HIV-exposed seronegative (HESN) individuals [4] . In the following studies, the collection of the HESN as controls should strictly obey these definitions, which would be helpful in explaining natural HIV-1 protection in individuals exposed to HIV-1 who remain seronegative or demonstrate resistance to infection.
Pooled data analysis by ethnicity was only performed in Asians since only one study involving HIV-1 patients and HIV-1 exposed seronegative controls (HESN) was separately performed in European-Americans and Africans. More studies on these populations are needed in future works, which would help us better understand the relationship between the VNTR polymorphism and HIV-1 infection risk in different ethnic populations.
Our findings are consistent with the study by Rathore et al. [16] but are different from other reports [14, 15, 17] . The potential explanation for the discrepancy may be the limited sample number included in the single study and the relative impact of this polymorphism in the different populations. As shown, the associations were only confirmed in stratified analyses by combining studies with a sample size greater than 200 subjects, which emphasized the importance of having sufficient power with a large enough sample size and the requirement of further largescale analysis.
Although the function of the association between the DC-SIGNR VNTR polymorphism and host susceptibility to HIV-1 infection has not been fully explored, outcomes of this metaanalysis suggest that the DC-SIGNR VNTR polymorphism had an impact on host susceptibility to HIV-1 infection. A study by Xu By using the healthy samples as controls, the comparison was carried out between the HIV-1 patients and healthy samples; (B) By using the HIV-1 exposed but seronegative (HESN) samples as controls, the comparison was carried out between the HIV-1 patients and the HIV-1 exposed but seronegative (HESN) samples. doi:10.1371/journal.pone.0042972.g003 et al. [18] showed that patients carrying the 5-repeat DC-SIGNR allele had significantly lower HIV-RNA levels compared with those observed in patients with the 7-and 9-repeat DC-SIGNR alleles, though they did not observe that the different genotypes/ alleles were associated with CD4+ T cell numbers. In vitro studies also demonstrated that DC-SIGNR with equal to or less than 5repeat alleles displayed unstable homozygous or heterozygous DC-SIGNR aggregates [33] accompanied by changes in affinity for HIV-1gp120 glycoprotein [34] . Data from these in vivo/vitro functional studies gave some support to the hypothesis that the 5/5 homozygous genotype might confer more resistance to HIV-1 infection. However, these associations were far from conclusive. More functional in vivo studies are needed.
Although meta-analysis is a powerful statistical method, inherent limitations of this study should be addressed. First, we only included the studies written in English and Chinese, and the related reports in other languages were not included, which might bias our conclusion in this study. Second, publication bias could not be excluded though the test showed negative results. The studies reporting significant associations between certain genotypes and reduced susceptibility to HIV infection would be more readily published while the studies with nonsignificant associations would be more difficult to publish. Third, most of the studies were conducted using Asian population groups. In the stratified analysis by ethnicity, there was only one study performed with Africans and two studies with European Americans, each of which had a sample size too small to achieve enough statistical power to obtain significant observations. In fact, many association studies showed different results for different populations. Thus, further studies are warranted in other ethnic populations to evaluate the possible ethnic differences of the VNTR polymorphism and HIV-1 susceptibility. Fourth, gene-gene and gene-environment interactions may influence host susceptibility to HIV-1 infection. In fact, many genes have been proven to influence HIV-1 infection risk, but we did not have enough data to eliminate these interfering factors. Fifth, as most studies did not mentioned about potential population stratification of patients and controls samples, we cannot rule out a role of population structure in the observed association. Finally, further stratified analyses of patients and HESN individuals by infection exposure routes (sexual contact, intravenous drug use, etc.) could not be performed because the data detailing the infection route for the HIV-1 patients were lacking. Because DC-SIGNR is expressed at relatively lower levels in tissues in vivo [35] , the association between the VNTR polymorphism and HIV-1 infection risk by different infection routes might be different.
Humans show remarkable variation in vulnerability to infection by HIV-1 and especially in the clinical outcomes after infection. Understanding why some people establish and maintain effective control of HIV-1 and others do not is a priority in the effort to (A) By using the HIV-1 exposed but seronegative (HESN) samples as controls, the comparison was carried out between the HIV-1 patients and the HIV-1 exposed but seronegative (HESN) samples under the genotypic risk model of 5/5 vs. other genotypes. (B) By using the HIV-1 exposed but seronegative (HESN) samples as controls, the comparison was carried out between the HIV-1 patients and the HIV-1 exposed but seronegative (HESN) samples under the allelic risk model of 5-repeat allele vs. other alleles. doi:10.1371/journal.pone.0042972.g004 develop new treatments for HIV/AIDS. To our knowledge, this is the first meta-analysis to assess the relationship between the DC-SIGNR VNTR polymorphism and HIV-1 infection risk. Our results showed that the 5/5 homozygous genotype was associated with resistance to HIV-1 in HIV-1 exposed seronegative subjects (HESN), especially in the Asian population. Future studies in different ethnic populations and with clear infection routes should be performed to evaluate these associations. Table S1 The breakdown of HESN controls in the included studies of this meta-analysis according the routine of exposure. (DOC)  Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice Brucella spp. cause undulant fever in humans and brucellosis in variety of other animals. Both innate and adaptive immunity have been shown to be important in controlling Brucella infection. Toll-like receptors (TLRs) represent a group of pattern recognition receptors (PRRs) that play critical roles in the host innate immune response, as well as development of adaptive immunity. In the current report, we investigated the role of TLR signaling in the clearance of Brucella and development of adaptive immunity in TLR2(−/−), TLR4(−/−), or MyD88(−/−) mice following aerosol exposure to B. melitensis 16 M. Consistent with previous reports, MyD88 is required for efficient clearance of Brucella from all three organs (lung, spleen, and liver). The results reveal Th2-skewed immune responses in TLR2(−/−) mice late in infection and support a TLR2 requirement for efficient clearance of Brucella from the lungs, but not from the spleen or liver. Similarly, TLR4 is required for efficient clearance of Brucella from the lung, but exhibits a minor contribution to clearance from the spleen and no demonstrable contribution to clearance from the liver. Lymphocyte proliferation assays suggest that the TLRs are not involved in the development of cell-mediated memory response to Brucella antigen. Antibody detection reveals that TLR2 and 4 are required to generate early antigen-specific IgG, but not during the late stages of infection. TLR2 and 4 are only transiently required for IgM production and not at all for IgA production. In contrast, MyD88 is essential for antigen specific IgG production late in infection, but is not required for IgM generation over the course of infection. Surprisingly, despite the prominent role for MyD88 in clearance from all tissues, MyD88-knockout mice express significantly higher levels of serum IgA. These results confirm the important role of MyD88 in controlling infection in the spleen while providing evidence of a prominent contribution to protection in other tissues. In addition, although TLR4 and TLR2 contribute little to control of spleen infection, a significant contribution to clearance of lung infection is described. The genus Brucella is a group of Gram-negative, facultative intracellular bacteria that cause brucellosis, a reproductive disease in ruminants, and undulating fever in humans. Brucellosis is one of the most important worldwide zoonotic diseases. Ten species have been identified to date, three of which, including B. melitensis, B. abortus, and B. suis are virulent in humans and represent a significant threat to public health (Atluri et al., 2011) . Humans often become infected following inhalation of particles carrying the bacteria or consumption of dairy products contaminated with the organism. Although vaccination is used to successfully reduce the spread of disease, the risk remains high in underdeveloped nations. There are currently no vaccines available that are safe for use in humans, and although generally effective, antibiotic treatments do not always prevent disease recrudescence. As a result of these factors and concern over their potential weaponization, NIH and the CDC/USDA have classified these three species as category B agents.
Both innate and adaptive immunity have been described as contributing to the control of Brucella infection (Baldwin and Parent, 2002; Dornand et al., 2002; Baldwin and Goenka, 2006) . The role of innate immunity against infection by this pathogen has drawn recent attention as a result of awareness of the role of innate immunity in the establishment of infection and the development of adaptive immunity (Weiss et al., 2005; Oliveira et al., 2011) . In contrast, adaptive immunity, including cell-mediated and humoral responses, has been the prominent focus of Brucella research over the past few decades. The innate immune system is composed of a variety of cellular and humoral components, which are the first line of the host defense against invading pathogens. Recognition relies on pattern recognition receptors (PRRs) expressed on/in the cellular components of the innate immune system. Toll-like receptors (TLRs) are the best characterized PRRs. Receptorligand interaction via TLRs induces the production of antimicrobial peptides and proinflammatory cytokines through NF-κB, mitogen-activated protein kinase (MAPK) and other signaling pathways (Kawai and Akira, 2006) . As a result, TLR signaling is critical to development of the host innate immune response, including recruitment of dendritic cells (DCs) and T effector cells, and upregulation of MHC I and II on antigen presenting cells (APCs) and by extension adaptive immunity against infection. 10 TLRs in human and 13 in the mouse have been identified to date (Kawai and Akira, 2006) . TLR2, TLR4, TLR5, and TLR9 recognizing lipopeptide, lipopolysaccharides, flagellin and CpG DNA, respectively, are known to be important in controlling bacterial infections. With the exception of TLR3, the TLRs require the adapter molecule myeloid differentiation factor 88 (MyD88) for signal transduction (Kawai and Akira, 2007) . As expected, MyD88 have been shown to be essential for clearance of Brucella infection from mice (Weiss et al., 2005; Copin et al., 2007; Macedo et al., 2008) .
Several groups have investigated the contribution of TLR signaling to innate immunity against Brucella infection in the mouse model. The consensus opinion is that TLR2 is not required to control Brucella infection in the mouse (Campos et al., 2004; Copin et al., 2007) . However, TLR2 has been shown to be important for cytokine production (Huang et al., 2003; Giambartolomei et al., 2004; Weiss et al., 2005; Macedo et al., 2008; Zwerdling et al., 2008) , MHC-II expression and down regulation of the type I receptor for the Fc portion of IgG (FcγRI, CD64) (Barrionuevo et al., 2011) in tissue culture.
The role of TLR4 in Brucella infection remains controversial. Some studies suggest that TLR4 is required to control Brucella replication in the mouse (Campos et al., 2004; Copin et al., 2007; Macedo et al., 2008) ; others indicate that TLR4 is not involved (Weiss et al., 2005; Barquero-Calvo et al., 2007) . The use of different Brucella strains/species in these experiments may account for the observed differences. The influence of the route of infection on the role of TLRs in studies with other pathogens suggests a need to do so with brucellosis, as is the role of TLR-mediated innate immunity in the development of adaptive immune response. These studies employed intraperitoneal (i.p.) inoculation, which is not a typical route for Brucella infection. As a result of these reports, the consensus of scientific opinion is that MyD88 contributes significantly to the control of Brucella infection while TLR-based signaling plays a lesser role at best. The absence of any role for TLR signaling is consistent with results indicating that the Brucella protein TcpB interrupts TLR-based signaling by promoting degradation of MyD88 adaptor like protein, MAL (Chaudhary et al., 2012) , and modification of Brucella LPS reduces agonist activity (Duenas et al., 2004) . However, these studies have been restricted to an atypical route of exposure, and the potential for differential TLR expression associated with different tissues has not been considered (Juarez et al., 2010) . Since inhalation represents a major concern to public health, experiments were undertaken to determine the role of TLR signaling in the control of Brucella infection following aerosol exposure. The current study, investigates the roles of TLR2, TLR4, and MyD88 in clearance of Brucella following respiratory exposure and development of adaptive immune response against Brucella. Since the mucosal/respiratory system is the primary portal of entry for human infection documented in many laboratory incidents and relevant to biothreats, it is important to understand the pathogenesis and immune responses resulting from aerosol exposure.
Since TLR signaling is an important component bridging innate and adaptive immunity (Iwasaki and Medzhitov, 2004) , failure to clear Brucella from organs by TLR signaling deficient mice could be due to defects in the development of adaptive immunity. To test this hypothesis, we determined cellular-and humoral-mediated adaptive immunity in these TLR knockout mice following Brucella infection. The results reveal that TLR signaling exhibits significant differences in control and clearance of Brucella from selected tissues and in the associated development of an adaptive immune response.
The bacterial strain used in these experiments, B. melitensis 16 M was obtained from ATCC (#23456) and recovered from an aborted goat fetus. Bacterial cultures were prepared as previously described (Pei and Ficht, 2004) . Briefly, B. melitensis 16 M was cultured in TSB for 24 h, pelleted by centrifugation at 20,000 ×g, washed with and resuspended in PBS (pH 7.4) at a density of approximately 5 × 10 9 CFU/ml (Kahl-McDonagh et al., 2007) .
Breeding pairs of TLR2 −/− , TLR4 −/− , and MyD88 −/− mice were obtained from Dr. S. Akira (Osaka University, Osaka, Japan) via Dr. Michael Berton (University of Texas at San Antonio, San Antonio, TX) and colonies maintained by Comparative Medicine Program (CMP) personnel at TAMU. C57BL/6 control mice were purchased from Jackson Laboratory. All mice were housed in BSL-3 suite in the CMP at Texas A&M University. Mice of both sexes between 8 and 12 weeks old were used in the experiments. Euthanasia was performed using carbon dioxide inhalation. All personnel working with animals received training in rodent handling and euthanasia via the CMP. All animal work was performed in compliance with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals as described by the National Research Council's Institute for Laboratory Animal Research (ILAR) Guide for the Care and Use of Laboratory Animals.
Aerosols were generated via nebulization of a Brucella suspension (∼ 5 × 10 9 CFU/ml in PBS) into a Madison Chamber (Madison, Wisconsin) according to the manufacturer's instructions (Kahl-McDonagh et al., 2007) . The aerosol chamber was located in a biosafety level 3 (BSL3) facility in the Laboratory Animal Research and Resources (LARR) building staffed and managed by the CMP at Texas A&M University. To assure personal safety, the principal investigator implemented a strict safety protocol, and all procedures were approved by the IBC and IACUC. Following aerosol exposure, one group of mice (n = 5) were immediately euthanized via carbon dioxide inhalation and the lungs collected to determine the infecting dose (Kahl-McDonagh et al., 2007) . At 1, 2, 4, 6, 8 , and 10 weeks after exposure, mice were euthanized and lung, spleen and liver were collected and homogenized in 1 ml each of sterile water containing 0.5% (v/v) Tween-20. One hundred microliters of appropriate dilution of the homogenized tissue were plated on Farrell's selective medium plates followed by incubation at 37 • C for at least 3 days. The experimental limit of detection was determined to be 10 CFU per lung, spleen or gram of live, and the results described represent the accumulated data from 4 independent aerosol exposures.
At 8-week post infection (p.i.), one-half of the spleen from each euthanized mouse was used to produce single cell suspensions as previously described. The cell density was adjusted to 2 × 10 6 per ml using complete RPMI-1640 containing 10% (v/v) heatinactivated FBS, 1 mM non-essential amino acid, 100 μg/ml of penicillin and 100 U/ml streptomycin. The cell suspension was dispensed to 24-well plates with 1 ml/well and stimulated with heat killed Brucella (HKB) or ConA (2 μg/ml) for three days. Following stimulation, supernatants were collected and cytokine synthesis characterized. Cells collected from 100 μl of the suspension were lysed using 1% (v/v) Triton X-100, and the LDH released from live cells was determined using CytoTox 96™ Nonradioactive Cytotoxicity Assay kit (Promega) following the manufacturer's instructions.
IFN-γ levels in the culture supernatants were determined 72 h post stimulation using sandwich ELISA kits (PeproTech Inc., Rocky Hill, NJ) according to the manufacturer's instructions (Pei et al., 2008) .
Blood samples were collected just prior to euthanization from mice at 1, 2, 4, 6, 8, and 10 weeks post exposure. Sera were separated and anti-Brucella antibodies, including IgG, IgG 1 , IgG 2a , IgM, and IgA were evaluated using ELISA. ELISA plates (Nunc) were coated with B. melitensis 16 M cell lysate. Briefly, 100 μl of 1 μg/ml lysate in bicarbonate/carbonate buffer (2.93 g NaHCO 3 , 1.59 g Na 2 CO 3 , 0.203 g MgCl 2 in 1 L of distilled water, pH 9.6) was added to microtiter wells (96 wells/plate) and incubated overnight at 4 • C (Pei and Collisson, 2005) . Non-specific binding was blocked via incubation with 5% (w/v) non-fat milk in PBS for 1 h at room temperature. The antigen-coated wells were incubated 2 h at room temperature (25 • C) with mouse sera diluted in PBS (pH 7.4) containing 0.05% (v/v) Tween-20 (PBS-T) 200 times for IgG1, IgG 2a , IgM, IgA detection or 1000 times for IgG detection. The plates were then incubated with HRP-conjugated goat anti-mouse immunoglobulins (IgG 1 , IgG 2a , IgM, IgA, and IgG from Kirkegard-Perry Labs (KPL) for 1 h at room temperature. The wells were washed with PBS-T between steps to remove any unbound materials. Color development followed the addition of 100 μl of TMB substrate KPL. The reaction was terminated by the addition of 50 μl of stop buffer (1 M H 2 SO 4 ) and OD 450 was determined using an ELISA reader (Bio-Rad).
Statistical significance was determined using one-way ANOVA or Student's t-test. P-values <0.05 * and <0.01 * * were considered to be significant.
In these experiments, the infecting dose determined using lungs collected from control C57BL/6 mice (n = 5) immediately following challenge is 4.26 ± 0.28 log CFU/mouse, which is consistent with a previous report (Kahl-McDonagh et al., 2007) . Bacterial load recovered from the lungs of TLR2 −/− , TLR4 −/− , and MyD88 −/− mice during the first two weeks following exposure is not significantly different from the C57BL/6 control mice (Figure 1 , panels W1-2), indicating that TLR2, TLR4, and MyD88 are not required to clear lung infection early. A significant difference in bacterial burden is detectable by week 4 and extends through week 10, indicating that the contribution of TLR2, TLR4, and MyD88 to the control of Brucella infection in the lungs occurs via adaptive immunity (Figure 1 ). By week 4 p.i., there was a significant difference in the clearance of the organism from each of the knockouts. This pattern persisted through week 10, although significance is lost due to the reduction in the number of animals available at this time point. The absence of MyD88 appears to have a greater impact on the clearance of infection from the lungs over this period, as the bacterial burden in the lungs of MyD88 −/− mice is significantly higher than that in TLR2 −/− and TLR4 −/− mice by week 8 p.i. (Figure 1, panel W8 ). The kinetics of Brucella clearance from the lungs is summarized in Figure 1K , and taken together, these results indicate that TLR2, TLR4, and MyD88 contribute to Brucella clearance from the lungs following aerosol challenge.
Infection and the kinetics of clearance of Brucella from the spleens of mice is notably different from that observed in the lung. Initially, the organism exhibits a delay in systemic distribution to the spleen that requires up to 4 weeks to achieve maximum burden. The delay in systemic spread of infection is evident in the spleen and less dramatically in the liver (see below), with bacterial burden in 9 of 13 spleens from TLR4 −/− and 10 of 13 from MyD88 −/− mice below the detection limit (10 cfu/spleen). This compares with a delay in only 1 of 17 TLR2 −/− mice and 5 of 20 C57BL/6 mice. The MyD88 −/− knockout appears to have a reduced ability to control the infection as evidenced by the significant difference in spleen colonization by week 4 (Figure 2 , panel W4). Once established infection proceeds similarly, although the MyD88 −/− and TLR4 −/− mice exhibit a delay in clearance relative to the TLR2 −/− and C57BL6 mice ( week, which is accompanied by a delay in clearance at later times. Again, the exaggerated decline observed between weeks 8 and 10 in TLR4 −/− mice may be an artifact of the reduced animals numbers.
Similar to the results described for the spleen, systemic spread of the organism to the liver is delayed in TLR4 −/− and MyD88 −/− mice, and is associated with a corresponding delay in the clearance of infection. Here again, TLR2 −/− mice are indistinguishable from control mice in the ability to restrict systemic infection (Figures 2 and 3) . As observed in the spleen, the capacity to control infection is most dramatically affected in the MyD88 −/− mice (Figure 3 , panels W1-10). From week 2 and onward, TLR2 −/− and TLR4 −/− mice controlled infection and cleared the organisms from the livers indistinguishably from the C57BL/6 mice (Figure 3, panel W10 ). Taken together, these results reveal that, in the mouse liver; (1) TLR2 and TLR4 are not required to control infection or clearance;
(2) the absence of TLR4 reduces accumulation of Brucella in the liver, but has little effect on late stage clearance;
(3) MyD88 is critical in early control and late clearance of the organism.
To determine the development of cell-mediated immunity, the splenocytes collected at 8 weeks p.i. from knockout and control mice are stimulated with Brucella antigen ex vivo. Using an LDH release assay to measure lymphocyte proliferation, no significant differences in proliferation was detected for TLR2 −/− , TLR4 −/− , MyD88 −/− and control C57BL/6 mice ( Figure 4A ). This result indicates that cell-mediated adaptive immunity against Brucella is unimpaired in these knockout mice. Consistent with the results of the lymphocyte proliferation assay, IFN-γ levels detected in splenocyte supernatants collected from the knockout mice are not significantly different from C57BL/6 mice ( Figure 4B ).
To determine the role of TLR signaling in antibody production and isotype switching, anti-Brucella IgM, IgG, IgG 1 , IgG 2a , and IgA responses were determined following aerosol infection. The results presented reveal detectable levels of IgM by 2 weeks p.i. that peaks by 4 weeks p.i. (Figure 5 , panel A). IgM levels begin to decline in the MyD88 −/− and C57BL/6 mice at 4 weeks p.i. while, IgM levels in the TLR2 −/− and TLR4 −/− mice plateau at 4 weeks with a gradual decline by 10 weeks p.i. Any requirement for TLR2 and TLR4 in IgM production is transient (only at 4 weeks p.i.). and IgM levels were never significantly different between MyD88 −/− and C57BL/6 mice throughout the experiment. Total Brucella-specific IgG in wild-type mice is detectable starting 4 weeks post-infection (Figure 5, panel B) , and increases continuously over the next 6 weeks. In contrast, elevation of the Brucella specific IgG levels is delayed in all of the knockout mice. Comparison among the groups revealed that, between weeks 4 and 8 p.i., IgG levels in TLR2 −/− and TLR4 −/− mice are significantly lower than that observed in the C57BL/6 mice. IgG levels increase in TLR4 −/− and TLR2 −/− mice by 10 weeks p.i. to levels that are not significantly different from the control C57BL/6 mice. In contrast, IgG levels in MyD88 −/− mice exhibit a significant reduction in titer over the course of infection. Serum IgA is not detected in the TLR2 −/− and TLR4 −/− mice, nor in C57BL/6 mice out to week 10 ( Figure 5 , panel C). Interestingly, serum IgA is prominent in the sera of MyD88 −/− mice and continuously increases over the course of the experiment.
Recent studies have shown that TLRs play a critical role in determining the fate of naive T cells, directing them toward either Th1 or Th2 responses (Dabbagh and Lewis, 2003; Xu et al., 2004) . To characterize the role of TLR signaling in determining a Th1 or Th2 response, IgG 1 (Th2) and IgG 2a (Th1) levels and the IgG 1 /IgG 2a ratio are calculated following Brucella aerosol exposure. IgG 1 and IgG 2a production are above background levels only 8 weeks p.i. (Figure 5 , panels D and E). By week 10, TLR2 and C57BL/6 mice produce significantly elevated levels of IgG 1 (Figure 5 , panel D) indicating TLR2 is not required for IgG 1 production. In contrast, IgG 1 levels in TLR4 and MyD88 knockout mice are significantly reduced, indicating TLR4 and MyD88 are essential for IgG 1 production (Figure 5, panel D) . Relatively low levels of IgG 2a are present at 10 weeks p.i. in all groups, but IgG 2a levels in TLR2 and MyD88 knockout mice are significantly reduced relative to control mice. IgG 1 /IgG 2a 
The kinetics of Brucella spp. clearance from infected mice differs depending on the genetic background of the strain. In C57BL/6 mice, infection is at or below the limit of detection in the liver as early as 6 weeks p.i. or by 8 weeks in the lung (Figures 1 and 3) .
In contrast, these same organs in BALB/c mice contain persistent levels of organism 8 weeks p.i. and beyond (Kahl-McDonagh et al., 2007) . These results are consistent with previous reports describing enhanced sensitivity of BALB/c mice to Brucella infection following i.p. inoculation (Copin et al., 2007) , confirming the expected immunological outcome despite the use of different routes of inoculation. One explanation for the improved clearance from C57BL6 mice is generation of a TH1-skewed immune response as opposed to a TH2-skewed response in BALB/c mice (Watanabe et al., 2004) . TLR activation induces the maturation of APCs, enhances antigen presentation, up-regulation of co-stimulatory molecules and cytokine production. Cytokine profiles produced by APCs control CD4 + T cell differentiation into either TH1 or TH2 cells. Engagement of different TLRs is expected to affect cytokine production, with significant effect on CD4 + T cell differentiation (Dabbagh and Lewis, 2003) . Previous studies have shown that Brucella infection induces mainly a TH1 response during acute disease (Agranovich et al., 1999; Pasquali et al., 2001; Giambartolomei et al., 2002; Rafiei et al., 2006; Khatun et al., 2009) .
It is clear that TLR signaling plays a critical role in the activation of the host innate immune response, including cytokine and chemokine secretion and up-regulation of co-stimulatory molecules in APCs (Kawai and Akira, 2006) . Recent studies have shown that these activations indirectly affect the development of adaptive immunity (Iwasaki and Medzhitov, 2010) . In addition, it has been demonstrated that functional TLRs are expressed on various T and B cell subsets (Bekeredjian-Ding and Jego, 2009; Booth et al., 2011; Kulkarni et al., 2011) . For example, functional expression of TLRs has been extensively investigated on γδ-T cells (Wesch et al., 2011) that have been shown to be important in controlling Brucella infection (Bertotto et al., 1993; Ottones et al., 2000) . Interaction of TLRs with their respective microbial ligands provides a third signal for B cell activation, which is essential for optimal antigen-specific antibody production and class switch recombination (Bekeredjian-Ding and Jego, 2009; Booth et al., 2011) .
Brucella are adept at inhibiting the host immune response. Brucella virulence derives from expression of an LPS with reduced agonist activity that limits activation of innate immunity and development of an effective adaptive response that promotes invasion and establishment of a replicative niche. In addition, Brucella express a protein TcpB (or Btp1) that restricts the proinflammatory response by interfering with MyD88 function. The result is enhanced degradation of MAL (MyD88-adapter-like protein) and restricted expression of NFκB. However, our current results indicate infection varies in different tissues, and that this variation is attributable in part to various PRRs, including TLR2, TLR4, and downstream signaling partners, like MyD88. Interference with TLR signaling by the organism has been described in numerous studies, as has the prominent contribution of MyD88 in limiting infection. The results reported here confirm the importance of MyD88 in the control of infection and extend those findings to include three tissues; spleen, liver and lung. In addition, the experiments performed confirm previous findings concerning the lack of significant contribution of TLR4 and TLR2 with regard to spleen (and liver) infection, but reveal their significant contribution preventing lung infection.
In an effort to explain the control exerted by each of these host factors, an evaluation was made of the corresponding immune response. Since cellular immunity is critical in controlling Brucella infection, splenocytes were used to characterize the role of TLR2, TLR4 and MyD88 in the development of cellmediated adaptive immunity against Brucella. Finding that the loss of TLR2, TLR4 and MyD88 had no demonstrable effect on the development of memory T cell response against Brucella infection was initially a surprise (Figure 4) . However, recent experiments have revealed a disruption of TLR signaling resulting from enhanced degradation of MyD88-adaptor like protein (MAL) that is consistent with previous reports documenting a minimal role for TLR signaling in memory T cell development against other viral and bacterial infections (Way et al., 2003; Fremond et al., 2004; Heer et al., 2007; Seibert et al., 2010; McBride et al., 2011) . These results are also in agreement with a recent study showing that IRAK4 (interleukin-1 receptor associated kinase 4), one of the kinases recruited by MyD88 upon stimulation, is not required for generation of CD4+ and CD8+ T cells producing IFN-γ in the late stage of Brucella infection (Oliveira et al., 2011) . These results suggest that TLR-independent signaling is likely involved in the development of cellular adaptive immunity against Brucella.
The contribution of humoral immunity to the outcome of Brucella infection is controversial. IgM may enhance opsonic uptake or the activity of complement. However, the differences in IgM production between knockouts are only transitory and have no correlation with differences in immune clearance. In general, IgG levels are significantly reduced in each of the knockout mice. However, the effect in the TLR4 and TLR2 knockouts is transient, while reduced IgG levels are consistently observed in the absence of MyD88 function. A second marker associated with the loss of MyD88 is the significant level of circulating IgA observed in the MyD88 knockout mice. Surprisingly, serum IgA levels were elevated in MyD88 −/− mice, but not in TLR2 −/− and TLR4 −/− mice following Brucella infection, suggesting that the IgA class switch recombination or secretion is enhanced in MyD88 knockout mice. Alternatively, higher Brucella burden in the lung constantly stimulates IgA secretion. However, this hypothesis is not supported by the fact that serum IgA is not detected in the TLR2 and TLR4 knockout mice despite similar levels of bacterial burden in the lung. In fact, this phenomenon has been previously reported in MyD88 −/− mice orally vaccinated with attenuated Salmonella expressing a Streptococcus pneumoniae surface antigen (Park et al., 2008) . It could prove useful to understand the biological mechanism involved and its potential application to vaccine design. The contribution of either elevated IgA or suppressed IgG to the inability of MyD88 knockouts to control infection is under investigation.
Since a reduction in the IgG1/IgG2a ratio is associated with the development of a T H 1 response and protection against Brucella infection, IgG, IgG1 and IgG2a levels were characterized in TLR2, TLR4 and MyD88 following aerosol exposure (Figure 5 , panel F). The results reveal a significant contribution of TLR4 and MyD88 to IgG1 production and the importance of TLR2 and MyD88 to IgG2a production. Examination of the IgG1/IgG2a ratio suggests the dependence of a protective T H 1 response on expression of MyD88 and TLR2, but not TLR4. This is not a totally unexpected result since the Brucella LPS, and/or TcpB primarily exert their influence on TLR4-based signaling. The contribution of MyD88 and TLR2 to protective immune response as determined by the reduced bacterial burden in selected tissues is borne out in previous discussion. To reiterate, MyD88 −/− exhibits significant delays in clearance from all three tissues examined; TLR2 and TLR4 contribute to clearance from the lung alone.
The importance of TLR signaling in the induction of antibody production remains controversial. In one such study, it was shown that TLR is not required for mice to generate robust antibody response (Gavin et al., 2006) . Other researchers report that TLR signaling is critical for antibody production and isotype switching (Pasare and Medzhitov, 2005; Heer et al., 2007) . A recent study by Weiss et al. show that TLR2, TLR4, TLR2/TLR4, and MyD88 are not required for anti-Brucella IgG production (Weiss et al., 2005) . In contrast, our results reveal that production of Brucella specific IgG is delayed in TLR2 and TLR4 knockout mice and impaired in MyD88 knockout mice. Possible reasons for the discrepancies include the use of different Brucella species, B. melitensis 16 M vs B. abortus S19, differences in virulence and different routes of inoculation (aerosol exposure vs. intraperitoneal injection). Since IgM production is not affected by MyD88 deficiency and only transiently affected by TLR2 and TLR4 absence, we conclude that TLR2, TLR4 and MyD88 are critical for IgG class switch recombination, which might be critical to the control of Brucella infection, and should be considered when Brucella designing vaccines.
In our current report, we have clearly demonstrated that TLR2 and TLR4 are critical to clearance of Brucella infection from the lung, although less prominently than MyD88. This contrasts significantly with the clearance profile for the spleen and liver and may arise as a result of differences in the TLR expression pattern or levels reported for different tissues (Nishimura and Naito, 2005) . It should be noted that based on results of previous studies Frontiers in Cellular and Infection Microbiology www.frontiersin.org September 2012 | Volume 2 | Article 115 | 8 following i.p. inoculation in which only the spleens were examined, it was concluded that Brucella infection was unaffected by the loss of TLR2, however, bacterial load in the lungs and livers were not determined in these studies. (Campos et al., 2004; Weiss et al., 2005; Copin et al., 2007; Macedo et al., 2008) . In agreement with previous studies, the results confirm MyD88 as a critical factor in the control of Brucella infection in the spleen (Campos et al., 2004; Weiss et al., 2005; Barquero-Calvo et al., 2007; Copin et al., 2007; Macedo et al., 2008) . However, caution is warranted in interpretation of the significance of host factors based on the analysis of a single tissue. Recent results in our laboratory indicate that Brucella may be recovered from the lung following i.p. inoculation, indicating a need to consider persistence in this tissue irrespective of the route of infection.
Another unexpected observation in this study was the delay in infection of the spleens and livers of TLR4 −/− and MyD88 −/− mice 1-2 weeks post-inoculation. These results suggest that TLR4 and MyD88 play significant roles in mediating Brucella dissemination from the lungs to other tissues, which is consistent with a previous observation indicating a role for TLR4 in the uptake of smooth Brucella by macrophage (Pei et al., 2008) , and that translocation of bacteria across mucosal or intestinal barriers is mediated by TLR4 (Neal et al., 2006) . Evaluation of Brucella infection dynamics derived from different routes of entry during the first week post aerosol exposure may be expected to confirm these results and potentially suggest post-exposure treatments.
Over millions of year's co-evolution between host and pathogen have resulted in strategies to survive in the host and cause disease presumably without eliminating the host entirely. Recent studies reveal that bacteria exploit the host TLR pathway, resulting either in enhanced virulence or immune suppression (Arpaia et al., 2011; Round et al., 2011) . Our previous study has shown that Brucella can use TLR4 to gain entry into the host cells (Pei et al., 2008) . It is possible that Brucella inhibit IgA class switch recombination or secretion via MyD88 by an unknown mechanism in order to initiate infection through mucosal surfaces.
Overall, our data demonstrate for the first time that TLR2, TLR4, MyD88 are essential for clearance of Brucella from the lung following aerosol exposure. Although TLR2, TLR4 and MyD88 are not required for the development of cell-mediated adaptive immunity, they play diverse roles in Brucella antigen specific antibody production and antibody class switching. The information obtained from this study will greatly facilitate efforts to understand immunity to Brucella and the rational design of novel vaccines against Brucella infection. Diagnostic Devices for Isothermal Nucleic Acid Amplification Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. Nucleic acid amplification is one of the most valuable tools in nucleic acid detection because it can amplify fewer than 10 target copies, significantly improving assay sensitivity. The polymerase chain reaction (PCR) was introduced by Mullis [1] and has since become an indispensable tool in numerous OPEN ACCESS molecular research and diagnostic applications. Related advanced technologies, such as multiplex PCR, nested PCR, real-time PCR, and reverse transcription PCR (RT-PCR), have been used for bimolecular analysis. However, there are numerous features confining the applicability of PCR. The approach requires thermal cycling instrumentation, considerable expertise, and a substantial amount of space in routine diagnostic laboratories, thus limiting its use to highly sophisticated facilities. These limitations in current PCR-based techniques have spurred the development of a new molecular-biological technique known as isothermal nucleic acid amplification. The major difference between PCR and isothermal amplification are the temperature reaction condition requirements. Stringent reaction conditions, including thermal cycling steps at specific temperatures, are employed in PCR, whereas only a single optimal reaction temperature is required for the entire isothermal amplification reaction, thus providing simpler and more effective reaction conditions without expensive equipment. Additionally, isothermal DNA amplification produces longer DNA fragments than the conventional PCR technique. Overall, isothermal nucleic acid amplifications have greater amplification efficiency and produce higher DNA yields than PCR owing to their undisrupted and sustained enzyme activity.
With the advent of microfabrication technology, one of the directions taken to address the future needs of bioanalysis and clinical diagnosis is the development of micro total analysis systems (µTAS) or labs-on-a-chip (LOC). This scaling down capability supports an exceptional ability to miniaturize various functional units such as pumps and reactors, making it possible to integrate and automate processes into a microsystem. Additionally, it offers important advantages over bulk or large-scale analysis including rapid assay results, high-throughput screening, and low consumption of reagents. Further, the energy required for microfabrication and operation is remarkably reduced. Most importantly, these benefits make microchip systems amenable to near-patient and point-of-care testing. The development of DNA amplification microinstruments began in the 1990s, when the concepts of integrated microfluidic devices were introduced to take advantage of microfabrication technology for biological and chemical analyses [2] . To establish such a system, it was desirable to create a totally integrated device performing a series of specific molecular functions such as nucleic acid extraction and purification, nucleic acid amplification and detection, and other supporting analysis techniques, with minimal dead volumes.
Owing to the overwhelming quantity of literature available on isothermal DNA amplification devices, we will describe the strategies of five major isothermal techniques. Because several reviews have previously focused on isothermal methods in bioanalysis applications [3] [4] [5] , we focus mainly on recent advances in the rational design and fabrication of integrated DNA microchips. The measurements of amplified DNA using different approaches will also be reviewed. Finally, future challenges and perspectives on diagnostic device construction are described.
Isothermal approaches can facilitate rapid target amplification through single-temperature incubation, reducing system complexity compared to PCR-based methods. Established isothermal amplification methods differ in terms of complexity (multiple enzymes or primers), attainable sensitivity, and specificity. In this section, we introduce the main isothermal methods used in diagnostic systems, including nucleic acid sequence-based amplification, strand displacement from the molecular beacon, and the fluorescence provides a real-time monitoring of NASBA progress [5, 14, 15] . Recent effort has shown that an automated NASBA system, NucliSENS EasyQ, can perform simultaneous amplification and detection using fluorescence quantification. The detection of amplification products takes place in a single closed tube to significantly reduce contamination risks. This platform also helps decrease the hands-on time and provides rapid results (within 4 h), thus becoming a potentially suitable device for diagnostic applications [16] [17] [18] [19] .
Although the NucliSENS EasyQ platform can obtain measurements simply and rapidly at central laboratories, the system has had limited application outside of this context. With the goal of bedside monitoring, many researchers have reported on integrated analysis systems that make it possible to shift NASBA applications from high-cost, tabletop systems to low-cost, portable devices. Esch et al. developed a NASBA assay in conjunction with fluorescence detection on a microfluidic device [20] . This device consisted of a polydimethylsiloxane (PDMS) block with a single channel, placed on a gold-coated glass slide at the device's center to immobilize the probe. Detection was accomplished using a sandwich hybridization of the NASBA products between capture probes and reporter probes tagged with carboxyfluorescein-filled liposomes. This technique had a detection limit of 5 fmol/L for a sample size of 12.5 μL. A later publication by Dimov et al. reported a microfluidic diagnostic device that integrated solid-phase extraction, real-time fluorescence detection, and a NASBA assay [21] . The integrated microfluidic NASBA chip consisted of two reaction chambers: a silica bead-bed chamber for sample purification and concentration, and a NASBA chamber for RNA amplification. To improve the efficiency of the NASBA reaction, all chambers were incubated with bovine serum albumin overnight before the reaction was started. Adequate amounts of the NASBA product were obtained after a reaction time of 30 min. Earlier this year, Zhao et al. introduced the concept of an integrated microfluidic chip-based system to monitor pathogens in a water environment with femtomolar sensitivity. The system, called immuno-NASBA, combined the versatility of enzyme-linked immunosorbent assay (ELISA) with the amplification power of NASBA [22] . The device was modeled on a 96-well ELISA microplate with 43 reaction chambers so that it would be fully compatible with a conventional reader. Moreover, the chip contained six parallel reaction channels to perform the simultaneous detection of six targets. Immuno-NASBA diagnostic devices have powerful potential to be applied for the diagnosis of various infectious diseases.
Strand displacement amplification (SDA) was described in 1992 [23] and was improved in the same year [24] . There are four sequence-specific primers used in this isothermal amplification. The first set of primers (S 1 and S 2 ) is designed to have single-stranded restriction enzyme recognition site overhangs, and the second set of the primers (B 1 and B 2 ) represent the bumper primers. The DNA target is first denatured by heat and each strand is allowed to hybridize with two primers (S 1 and B 1 ), which are annealed to the DNA template. The B 1 extended product displaces the extension from the S 1 primer, which can hybridize to the opposite strand primers (B 2 and S 2 ). Thus, newly synthesized DNA that has been extended from the primers is cleaved by a corresponding restriction endonuclease, and the amplification is repeated by the polymerase, thus generating the newly synthesized strands rolling-circle and circle-to-circle amplification and the subsequent microchip electrophoretic analysis of bacterial genes (Figure 3 (B)) [60, 61] . A clinical sample was detectable in less than 65 min after the reaction was initiated.
In addition to single-target detection, RCA is also desirable for multiple-analyte sensing assays because amplified products are considered to be localized at the array spot [62] . An array of real-time RCA in combination with the parallelism of arrays was developed by Yang et al. for protein quantitation down to the low nanomolar range [53] . Konry et al. constructed a two-layer sandwich assay on microbead surfaces for the combined detection of DNA and protein molecules in a single approach [63] . This array chip achieved detection limits of 1 pM and 10 fM for target DNA and proteins, respectively.
Loop-mediated isothermal amplification (LAMP) is one of the DNA amplification technologies that employ a constant temperature [64] . The Bst polymerase plays a key role in the LAMP reaction process. The Bst polymerase, which is derived from Bacillus stearothermophilus living in hot springs with temperature around 70 °C, has polymerize activity, 5'-3' exonuclease activity, and strand displacement ability. At a suitable temperature, Bst polymerase with strand displacement activity can separate the non-template strand from the template DNA without the thermal cycles of the PCR process, which uses Taq polymerase to synthesize new DNA strands. Subtle primer design is also necessary for a successful LAMP reaction. In the first stage of the reaction, the so-called outer and inner primer pairs can make dumbbell-like loop DNA strands from the target DNA templates, and the dumbbell-like DNA strands become the new template DNA for the next step ( Figure 4) . The dumbbell-like DNA strands then continue replicating to become a flower-like long-chain DNA product [65] . In addition to these two primer pairs, a third pair known as loop primers has been designed and proven to be beneficial in accelerating the amplification process. A good primer design not only ensures successful execution of LAMP, but also increases the sensitivity and specificity of the reaction result [66] . Thus, the LAMP reaction is carried out by three pairs of primers in an isothermal condition. Compared to the PCR, the reaction time of LAMP is shorter while the sensitivity and specificity are almost the same or even better. For fixed temperature heating, the heater component of the device can be simpler relative to traditional DNA amplification instruments. These features afford LAMP strong potential as a disease screening method based on the economic benefits of clinical point-of-care devices with simpler designs. Because of convenience, high efficiency, and the specificity of LAMP, it has been applied to many DNA screening tests, especially virus detection.
Microfluidic chips have been applied to the detection of LAMP reactions in recent years. Some chips are used only for guiding the reaction buffer and DNA solution to the reaction chamber, whereas others are combined with additional technologies such as nanostructures for sample concentrating, electrophoresis, magnetics beads, etc. A microfluidics chip made of PMMA has been used for the turbidity detection of the hepatitis B virus (HBV) LAMP reaction by our group [67, 68] . With a disposable LAMP microreactor and optical fiber-based turbidimetry device, as shown in Figure 4 , the lowest limitation for detection of the HBV DNA template was 50 copies/25 μL with the critical detecting time set at 30 min. 
Helicase-dependent amplification (HDA) is based on natural DNA replication mechanisms. Initially, the coordinated action of helicases unwinds and separates the template DNA duplex. The primer can hybridize with the free single-stranded templates, and the subsequent extension by a DNA polymerase will result in DNA amplification (Figure 6(A) ). The original reaction reported in the literature is performed at 37 °C for the entire process, and more than a million-fold amplification of DNA fragments can be achieved from nanogram quantities of genomic DNA [79] . Unlike the PCR, HDA uses helicases instead of heat, thus eliminating the need for any denaturation steps. Nevertheless, two additional accessory proteins are required in this approach: MutL to stimulate helicase unwinding activity and a single-strand binding (SSB) protein to prevent premature re-association of the separated ssDNA. A thermostable helicase may be also advantageous for HDA. Recently, a new helicase was developed from Thermoanaerobacter tengcongensis, which can be operated at temperatures from 45 °C to 65 °C [80] , so HDA reactions are now generally performed at the higher temperature of 65 °C. The use of thermostable helicase led researchers to abandon both the MutL and SSB proteins, while simultaneously improving the DNA yield of the reaction [81] . This simple thermal management option makes HDA very attractive for the development of simple portable DNA diagnostic devices and point-of-care testing.
Recently, electrochemical methods for the detection of DNA in combination with HDA have been developed. A DNA-based sensor for the detection of M. tuberculosis using the electrochemical detection of gold nanoparticles was developed [82] . The dextrin-coated gold nanoparticles (AuNPs) used as a reporter can be electrochemically detected on a screen-printed carbon electrode chip ( Figure 6(B) ). Kivlehan et al. developed a real-time electrochemical method for HDA using the monitoring of intercalating redox probes [83] . The binding of redox probes to the HDA products (amplified double-stranded DNA) led to less electrochemically detectability, compared with the probes' free counterpart. This method of electrochemical HDA detection does not require the immobilization of the probe on the electrode; real-time isothermal HDA reactions with 48-electrochemical microwells can be performed in 1 h. Therefore, it has the potential to be a reliable method for sequence-specific DNA detection.
Lateral flow test strips provide a promising tool for the development of point-of-care nucleic acid biosensors. Consequently, HDA has been employed with an embedded lateral-flow DNA detection strip for end-point assay to detect HIV-1 in human plasma [84] . The principle of this approach is based on a sandwich immunoassay using two probes: a fluorescein isothiocyanate (FITC)-labeled capture probe and a biotin-labeled detection probe. The HDA products hybridize with the capture probes and detection probes to form the complex. The hybrids are bound to streptavidin-conjugated color particles and are captured on the test zone by the interaction between the target DNA-FITC capture probe and an anti-FITC antibody. The accumulation of color beads in the test zone of the fiberglass paper is visualized as a characteristic red band. This assay provides the satisfactory detection of HIV-1 RNA at 50 copies/assay. This disposable amplicon detection device based on HDA has also been applied to the herpes simplex virus [85] and Mycobacterium tuberculosis diagnosis [86] and shows a performance comparable with conventional detection assay. Nevertheless, sample preparation, target amplification, and nucleic acid testing are conducted as distinct steps. 
Trau's group has proposed a beacon-assisted detection amplification (BAD-AMP) by DNA polymerization in conjunction with the nicking event [90, 91] . Two enzymes are used in BAD-AMP: the DNA polymerase that replicates the DNA target on the beacon and the nicking endonuclease that cuts the replicated single strand at the recognition position. Initially, the reaction can be activated by the addition of target DNA to switch the conformation of the beacon. When a new DNA is synthesized, the target is displaced by the polymerase with strand-displacement activity. This polymerization eventually leads to the newly synthesized DNA strand with a recognition sequence for the DNA endonuclease. This allows an enzyme to nick the DNA strand, such that the polymerase can also displace the nicked strand. BAD-AMP leads to exponential amplification by repeating cycles of polymerase and endonuclease activity. Because this strategy is a relatively simple technique, BAD-AMP has also been applied for the construction of molecular logic gates [92] .
Hybridization chain reaction (HCR) is a short DNA amplification technique that is based on hybridization and strand-exchange reactions for selective and specific extension [93] . Two complementary, kinetically trapped DNA hairpins coexist in solution until the introduction of target strands initiates a cascade of hybridization events. Because there is no requirement for enzyme amplification of the signal, HCR can be performed at room temperature. The major drawback of HCR is that it provides linear amplification only, compared to the PCR, which produces exponential amplification. Various approaches with labeled hairpin probes have been reported to improve the sensitivity of targets [94] [95] [96] [97] . Although HCR is the simplest method among the isothermal nucleic acid amplifications, there are no reports on the development of an integrated HCR chip.
The aim of this review was to briefly describe the current state of the art of diagnostic devices for isothermal nucleic acid amplification. The isothermal strategy has been a versatile and powerful technique applied in the detection of microbial and viral pathogens, among many other uses in the diagnostic laboratory. The combination of the properties derived from isothermal amplification and biosensing platforms proved a valuable strategy for simplifying the analytical science of nucleic acid detection. In reviewing the various detection configurations, we observed that integrated microchip systems are particularly desirable because these systems provide significant advantages in convenience and cost-effectiveness, simultaneously simplifying operational procedures and shortening analysis times.
To date, the development of chip-based isothermal assay systems has received great attention, whereas achieving a higher degree of portability remains a challenge. No device reported thus far is clearly superior, resulting in the possibility that sensing platforms based on different isothermal amplifications may find their way to market. Commercialization requires further improvement in on-chip sample pretreatment, miniaturization of detectors, decrease in power consumption, and the establishment of quality control. We can expect the full integration of all components on disposable credit-card-sized systems for isothermal nucleic acid amplification and detection in the near future. Given the great effort being invested in isothermal DNA microchip systems, there is no doubt that they will provide significant contributions to point-of-care diagnostics and decentralized testing. Clinical characteristics of pediatric hospitalizations associated with 2009 pandemic influenza A (H1N1) in Northern Bavaria, Germany BACKGROUND: The 2009 pandemic influenza A (H1N1) (PIA) virus infected large parts of the pediatric population with a wide clinical spectrum and an initially unknown complication rate. The aims of our study were to define clinical characteristics and outcome of pandemic influenza A (H1N1) 2009-associated hospitalizations (PIAH) in children <18 years of age. All hospitalized cases of children <18 years of age with laboratory-confirmed pandemic influenza A (H1N1) 2009 in the region of Wuerzburg (Northern Bavaria, Germany) between July 2009 and March 2010 were identified. For these children a medical chart review was performed to determine their clinical characteristics and complications. RESULTS: Between July 2009 and March 2010, 94 PIAH (62% males) occurred in children <18 years of age, with a median age of 7 years (IQR: 3–12 years). Underlying diseases and predisposing factors were documented in 40 (43%) children; obesity (n = 12, 30%), asthma (n = 10, 25%) and neurologic disorders (n = 8, 20%) were most frequently reported. Sixteen (17%) children received oxygen supplementation; three (3%) children required mechanical ventilation. Six (6%) children were admitted to an intensive care unit, four of them with underlying chronic diseases. CONCLUSIONS: Most PIAH demonstrated a benign course of disease. However, six children (6%) needed treatment at an intensive care unit for severe complications. Influenza is a common cause of illness in children, predominantly treated as outpatients. For seasonal influenza, annual incidences for influenza-associated hospitalizations were estimated as 90 (CI 95%: 80-110) /100,000 in children <5 years old in the USA [1] , and as 123/100,000 in children <6 years of age in a German region [2] .
In 2009, an influenza pandemic was caused by a new influenza A/H1N1 virus (PIA). In more than 214 countries laboratory-confirmed PIA cases were reported, including over 18,097 deaths [3] . During this pandemic especially children appeared to be affected. In the USA, about 87,000 PIA hospitalizations (PIAH) and 1,280 fatalities occurred in children <18 years of age from April 2009 to April 2010, representing 32% of all PIAH and 10% of all PIA-associated fatalities in the US population [4] .
In Germany, first cases of PIA were reported in April 2009; the first wave of the pandemic lasted from calendar week 42/2009 to 02/2010 [5] . The sentinel surveillance system for laboratory-confirmed PIA reported 226,137 cases and 253 fatalities for the total population until April 2010 [6] . A nationwide surveillance of critically ill children admitted to pediatric intensive care units (PICU) and fatalities associated with PIA estimated an incidence rate of 2.8 cases/100,000 children in infants * Correspondence: Liese_J@kinderklinik.uni-wuerzburg.de † Equal contributors 1 <1 year of age and 0.8 cases/100,000 in children <15 years of age [7] . Thus far, there is only limited data on clinical characteristics and outcome of PIAH in Germany [8] . In the current study, we therefore investigated clinical characteristics of all pediatric PIAH in a defined geographical region.
All children and adolescents <18 years of age with laboratory-confirmed PIA admitted to one of the three hospitals covering pediatric in-patients in the area of Wuerzburg (Northern Bavaria / Germany) from July 2009 to March 2010, covering the influenza season 2009/2010, were included. The study population size in our defined catchment area corresponded to approximately 60,300 children younger than 18 years of age (Bavarian State Office for Statistics and Data Processing 2009, https://www.statistik.bayern.de).
Respiratory samples (nasopharyngeal aspirate or nasopharyngeal swab or tracheal secretion) from all three hospitals were routinely sent to the Institute of Virology and Immunobiology of the University of Wuerzburg and analyzed using reverse transcriptase-polymerase-chain -reaction (RT-PCR) or direct immunofluorescence (DIF) testing for influenza A and B. All specimens tested positive for influenza by DIF were determined by PCR for subtype determination.
Laboratory-confirmed PIA-infections were reported by the Institute of Virology and Immunobiology. For all identified PIAH patients <18 years of age a medical chart review was conducted by a representative of the study coordination centre (University Children's Hospital, Wuerzburg). Date of admission, number of days in hospital, data on treatment, demographic, clinical and epidemiological data were obtained using a standardized and anonymous data collection form. All data were collected for the duration of hospital stay.
For analysis of clinical parameters, all identified PIAH patients with symptoms starting before hospitalization or less than 72 h after hospitalization were included. Nosocomial infections with symptoms starting >72 h after hospitalization (n = 10 cases, including one fatality) were excluded from the present analysis and will be described elsewhere. Data were analyzed descriptively using SPSS (version 18.0 and 19.0, Chicago, IL). Continuous data were reported as median with interquartile range (IQR) and categorical variables as percentage of patients.
The study was approved by the Institutional Ethics Review Board of the University of Wuerzburg. Figure 2 ). Of all 94 patients, 88 (94%; 60% males, median 7 (2-12) years of age) were admitted to a general ward and stayed for a median of four (3-6) days in hospital. Six children (6%; 83% males, age range 0.1-16 years) were admitted to an intensive care unit (ICU), with a median stay at ICU of three (2-6) days and of seven (3-18) days in hospital. For 48 patients (51%), a contact with a person with suspected (n = 35) or confirmed (n = 29) PIA was reported as potential source of infection; most frequently, contacts to siblings (n = 30) or the child's mother (n = 15) had been documented.
The onset of symptoms, reported for 92 (98%) patients, occurred at a median of two (1-3) days before admission to hospital. The most frequently reported symptoms at admission were cough in 75 (80% of the 94 children), fever in 73 (78%), rhinorrhea in 48 (51%), and refusal of food or drink in 36 (38%) cases ( Table 1 ). The most frequent diagnoses were upper respiratory tract infection in 84 (89%) children, bronchitis in 20 (21%; including nine (10%) cases of obstructive bronchitis), and pneumonia in 16 (17%) children, including one (1%) case of secondary bacterial pneumonia. Other reported diagnoses were fever convulsion in seven (7%), laryngitis/croup in six (6%), and otitis media in three (3%) children (multiple diseases per patient possible).
At least one underlying medical condition was documented for 40 (43%) of the 94 children; the most frequent condition was obesity defined as Body Mass Index >90th percentile (n = 12, 30% of all 40 children with predisposing factors), asthma (n = 10, 25%), neurologic disorders (n = 8, 20%), preterm birth, allergic diseases and other chronic diseases (each n = 6, 15%).
Oseltamivir was administered in 23 (25%) children for a median of three (2-5) days. A total of 28 (30%) children were treated with antibiotics, administered orally for a median of four (1-6) days and intravenously for a median of five (3-6) days. A total of 16 (17%) children received oxygen supplementation; three (3%) children required mechanical ventilation for a median of four (range 2-5) days.
Patients admitted to an intensive care unit ICU treatment was reported for six (6%) of the 94 patients. Four of them had known underlying chronic diseases or predisposing factors: i) A two-month-old former preterm (male) with congenital heart defect was admitted due to desaturation and pneumonia. After treatment with oxygen and antibiotics, he was discharged after three days on PICU and 31 days in hospital. ii) A ten-year-old boy with chronic neurologic and lung diseases was treated at PICU for one day due to fever convulsion. iii) A ten-year-old boy with tuberous sclerosis as chronic disease was admitted to PICU due to fever convulsion and was intubated and mechanically ventilated at the PICU for five days because of hypopnoea. iiii) A sixteen-year-old girl with obesity (BMI >30) and nicotine abuse as risk factors was treated at PICU due to pneumonia and acute respiratory distress syndrome. She was mechanically ventilated at the PICU for four days due to respiratory failure.
Three of 94 hospitalized children (3%), two with underlying diseases, had been vaccinated against PIA five days (n = 2) or about two months before hospitalization (n = 1). One child without underlying diseases had been vaccinated against seasonal influenza before hospitalization (no information about date of vaccination). Only two (5%) of 40 children with underlying chronic conditions for whom influenza vaccination is generally recommended in Germany had received PIA vaccination, compared to one child (2%) of the 54 children without underlying chronic conditions. None of the six children with severe complications had received any influenza vaccination.
During the influenza A/H1N1 epidemic a total of 94 children with PCR-confirmed PIA were hospitalized in the three hospitals in Wuerzburg, Northern Bavaria, which cover the pediatric population of the city of Wuerzburg and its surroundings. The clinical course was mostly benign with cough (80%), fever (78%), and rhinorrhea (51%) as predominant symptoms. Only six percent of PIAH were admitted to an ICU; which is about three times lower than reported from Argentina, Canada and USA (17-19%) [9] [10] [11] but similar to 8% found in a UK hospital [12] and identical to 6% of ICU admissions reported from a large hospital in Hamburg (Northern Germany) [8] . In Germany, both in our study and in Hamburg, a clearly lower percentage of patients with PIA received treatment with oseltamivir (25% and 28%, respectively) and antibiotics (30% and 25%, respectively) [8] , when compared to antiviral (46-99%) [9] [10] [11] and antibiotic (74-86%) treatment in other PIAH studies [9] [10] [11] . In Argentina, oxygen was supplemented five times more often (82%) than was to be observed in our study (17%) [11] . Mechanical ventilation was required six times more often (17%) than documented in our study (3%), whereas data from USA and Canada revealed a similar frequency (6%) for hospitalized patients [9, 10] . The low rates of severe influenza cases in the present study correspond with the results of an earlier study on severe seasonal influenza in Germany [13] . Hence, on the one hand, the higher primary and secondary complication rates among hospitalized patients in other countries may reflect a real increase in the complication rate, due to delayed treatment with a limited access to primary health care. On the other hand, observed heterogeneity in the severity of hospitalized patients may result from differences in hospitalization access, with a higher threshold for hospitalization in countries with lower socio-economic status or limited health insurance (as in the USA) compared to Germany [11] .
Underlying diseases were documented in 43% of PIAH, with asthma (25% of all children with predisposing factors) reported as one of the most frequent conditions. These results are comparable to the 32-40% of cases with underlying diseases (predominantly led by asthma) reported by most other surveys on pediatric PIAH [8, 11, 12, 14] . Of 40 patients with underlying diseases, 10% received ICU treatment, in contrast to only 4% of 54 previously healthy patients, indicating a more severe course of disease in risk group children. However, the majority of children with underlying disease had an uncomplicated course of disease. It may be assumed that at least in part they were hospitalized for pre-emptive treatment and monitoring of possible complications. In contrast, a recent study from seven Austrian hospitals on PIA patients seeking emergency medical care reported underlying chronic conditions only in 13% of PIA patients <18 years of age [15] .
In October 2009, the German Advisory Board on Immunization (STIKO) recommended vaccination against PIA for selected risk groups. For children, it was recommended that primarily children above six months of age with underlying diseases, such as chronic diseases of the air ways, cardiovascular system, liver or kidneys, should be vaccinated. Secondarily, healthy children should be vaccinated as well [16] . The first PIA vaccine was available in Germany at the end of October 2009. In our study, 40 (43%) children suffered from underlying diseases and, hence, ideally should have been vaccinated against influenza. Of these 40 children, two children were younger than six months and 16 children became ill before the PIA vaccine was available. Of the remaining 22 children with underlying diseases only two (9%) had received a PIA vaccination. Only for one child (2%) out of 51 children without predisposing factors aged above six months a PIA vaccination was reported. The low PIA vaccination coverage found in our study is confirmed by results from cross-sectional surveys in children <14 years of age in Germany (8% coverage) [17] , and from a German surveillance study on severe PIA cases <15 years of age admitted to intensive care units (9% coverage) [7] .
Potential limitations may result from the differences in criteria for inpatient treatment and use of diagnostic methods depending on the individual decision by the admitting physician. The number of hospitalizations corresponded to a conservative incidence estimate of at least 118 PIAH per 100,000 children <18 years of age. However, this may considerably underestimate the true pediatric PIAH incidence as only patients with laboratory-confirmed PIA were included; children hospitalized with respiratory symptoms or influenza-like illness without being tested for influenza were not captured in this study.
The course of PIAH was predominantly benign; 43% occurred in children with chronic underlying diseases. Severe complications implying treatment at ICU occurred in six (6%) of the children, including four children with chronic underlying diseases. Better acceptance and higher vaccination coverage of risk group children with the recommended and available PIA vaccine could have prevented a considerable number of pediatric PIAH in Germany. Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD. Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by a disruption of the dystrophin mRNA reading frame resulting in an out-of-frame transcript and a non-functional dystrophin protein. 1 In the past decade, a number of new treatments for DMD have been investigated, of which antisense oligonucleotide (AO)-mediated splice correction is one of the most promising approaches. [2] [3] [4] AOs modulate dystrophin pre-mRNA splicing, by specifically restoring the reading frame of the dystrophin gene via exon skipping, and therefore generate truncated but semi-functional dystrophin protein isoforms.
In vivo studies in the DMD mouse model, mdx, have shown that systemic delivery of naked phosphorodiamidate morpholino oligomers (PMO) 5 and 2′-O-methyl (2′OMe) 6 AOs are not capable of restoring significant dystrophin protein in cardiac muscle. Notably, even direct intra-cardiac injections of naked AOs resulted in very low exon skipping. 7 And although clinical trials with 2′OMe 8, 9 and PMO 10,11 chemistries have shown great promise, there is still a need for further optimization to improve AO delivery to all skeletal muscles and to the heart. This is critical as respiratory complications 12 and cardiac dysfunction 13 are the major causes of premature death in DMD patients. In particular the cardiomyopathy becomes clinically apparent at ~10 years of age, and is exhibited in all DMD patients by the age of 18. 13, 14 While some studies have shown that the restoration of dystrophin in respiratory muscles can improve cardiac function in the absence of restored dystrophin protein in the heart, 15, 16 there is concern that any improvement in skeletal muscle function in the absence of cardiac correction may worsen the cardiac disease progression due to increases in cardiac work load. [17] [18] [19] It is therefore highly desirable that future therapies endeavor to restore dystrophin in cardiac as well as in skeletal muscles.
Cell-penetrating peptides (CPPs), which may be readily conjugated to charge neutral AOs such as PMO, have shown potential for improved systemic delivery. CPPs that contain cationic amino acids, particularly multiple arginines, are highly effective in enhancing AO delivery due to their unique ability to deliver associated cargoes across the plasma and endosomal membranes. 20 Various arginine-rich peptides have been found to be particularly effective for delivery of such charge neutral AOs, [21] [22] [23] leading to systemic dystrophin production. However, dystrophin protein restoration in heart has typically required repeated 24 and/or very high dose administrations. 25
Corinne Betts 1 , Amer F Saleh 2 , Andrey A Arzumanov 2 , Suzan M Hammond 1 , Caroline Godfrey 1 , Thibault Coursindel 2 , Michael J Gait 2 and Matthew JA Wood 1 Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. 26 has also been investigated, namely R6-Penetratin which contains six additional arginines. 27 Employing R6-Penetratin as the lead peptide, a series of peptide nucleic acids/PMO internalization peptides (Pips) were derived that were found to be much more stable to serum proteolysis. 28 Two such Pip peptides, Pip2a and Pip2b, conjugated to a dystrophin exon 23-specific (peptide nucleic acids) AO, were shown to be capable of inducing strong exon skipping and dystrophin positive fibres following intramuscular injection into the tibialis anterior (TA) muscle of the mdx mouse. Further optimization of this peptide series was carried out as conjugates to PMO, and Pip5e-PMO was identified as the most efficient peptide-PMO conjugate capable of inducing high levels of exon skipping and dystrophin restoration body wide, including in the heart, following a single dose intravenous administration. 29 The Pip5e structure comprises a hydrophobic core region flanked on each side by arginine-rich domains containing aminohexanoyl (X) and β-alanine (B) spacers. By analogy with the previous arginine-rich B peptide, 22 it was thought that the high arginine content of Pip5e contributed to overall delivery efficiency into all muscle tissues, whereas the hydrophobic region might be important for heart muscle delivery.
We now report the results of a series of mutations to the hydrophobic core region of the Pip5e peptide, where this central core region amino acid sequence is reversed, scrambled, or partially deleted. These changes affect the levels of exon skipping and dystrophin restoration in multiple muscle groups, including the heart, following a single, low dose intravenous injection of the corresponding Pip6-PMO conjugates. The results show that a core length of 5 amino acids (5-aa) appears to be essential for heart dystrophin production, since reductions in core length reduced cardiac activity. Unexpectedly, an arginine residue was tolerated in one position of the hydrophobic core, but two arginine residues were not tolerated, nor an arginine in a different position. Surprisingly, skeletal dystrophin production was also reduced in these two latter cases.
Our previous lead Pip series CPP, Pip5e, 29 contains two arginine-rich flanking regions and a central hydrophobic core. To further probe the composition requirements of the hydrophobic core for maintenance of good heart dystrophin production, we synthesized a range of Pip5e derivative peptides (Pip6 a-f) (Figure 1a) where mutations were made only to the hydrophobic core region, for example scrambled and partially deleted core region peptides. All peptides contained the same number of arginine residues (10) in the flanking sequences as in Pip5e, with the exception of Pip6e. These peptides were conjugated to a 25-mer PMO complementary to dystrophin exon 23, 30,31 previously validated for exon skipping in mdx mice. In contrast to the method of conjugation to the 5′ end of PMO that we utilised previously, 29 Pip6-PMO conjugates were prepared by conjugation of the 3′ end of the PMO to the C-terminal carboxylic acid moiety of the Pip peptide (Figure 1b) . We reported that there was no significant difference between the in vivo dystrophin production or exon skipping activity for Pip5e-PMO conjugated to the 3′ end of the PMO or to the 5′ end and therefore chose to utilise 3′ end conjugation for these experiments. 32
The exon skipping potential of Pip6-PMO conjugates was evaluated in differentiated mouse H2K mdx myotubes in the absence of any transfection agent (Figure 2 ) at concentrations ranging from 0.125 to 1 µmol/l. This showed that exon skipping activity in cultured muscle cells was very similar for all these constructs, including Pip5e-PMO. These results differ from the previous Pip5 series, 29 where the flanking argininerich sequences mostly contained a fixed number of arginine residues (10) but where spacings were varied through alternative placement of aminohexanoyl and β-alanine units. This resulted in small variations in exon skipping activity that correlated well with in vivo activity. In the case of Pip6 sequences, the flanking arginine-rich sequences are identical (with the exception of Pip6e, which is identical except for one arginine immediately preceding the core which is displaced into the second position of the core). The results demonstrate that cellular exon skipping activity does not depend on the sequence or length of the hydrophobic core. Note that we have previously shown that major changes in in vitro exon skipping activities are correlated instead with the total numbers of arginine residues. 33 Alterations to the Pip5e hydrophobic sequence improve splicing activity in vivo Given the potency of Pip5e-PMO in heart tissue, the aim of altering the sequence of the hydrophobic core (whilst maintaining the 5-aa length) was to identify peptides that might be more efficient at lower doses. These modifications included inversion of the hydrophobic region (Pip6a), substitution of tyrosine by isoleucine (Pip6b), substitution of glutamine in the Pip6a sequence by displacement of the arginine immediately flanking the core in the first arginine-rich flanking region (Pip6e), and a scrambled hydrophobic core sequence (Pip6f). All Pip6 peptide-PMO conjugates were administered to mdx mice as single 12.5 mg/kg intravenous injections via the tail vein and tissues were harvested 2 weeks later and assessed for activity at both RNA and protein levels.
Immunohistochemical staining of dystrophin expression for all 5-aa core Pip6-PMOs revealed high levels of dystrophin production in skeletal muscles including the TA, diaphragm, and the heart (Figure 3 ). Immunohistochemical staining quantification (Figure 4a , b) was performed as previously described 16, 34 and was achieved by taking four representative frames of the dystrophin staining and correlating this with laminin staining for each section (n = 3) of the quadriceps, diaphragm and heart for each peptide-PMO treatment. Untreated mdx and treated mdx mice were normalized to C57BL10 mice. This method allows comparison of the staining intensity of dystrophin at the sarcolemma relative to laminin for each treatment group. Intensity ratios are normalized to C57BL10 samples and each region of interest at the sarcolemma (120 regions for each treatment group) is plotted on a scatter graph. The relative intensity values obtained for all four of the 5-aa core Pip6-PMO conjugates were significantly different to those of untreated mdx mice for the quadriceps and diaphragm ( Figure 4b and Table 1 ). There were very similar dystrophin restoration levels in the quadriceps (percent recovery score-%RS-range between 21.10 and 33.44%; Figure 4b ) and in the diaphragm for all treatments, with the exception of Pip6b which had a higher recovery score in the diaphragm (%RS range between 38.87 and 48.43%, Pip6b 56.72%; Figure 4b ).
All 5-aa core Pip6-PMO-treated mice exhibited high dystrophin intensity values in the heart with the exception of Pip6e (other Pip6-PMOs were statistically significant compared to mdx = P < 0.0001; Table 1 ). Pip6a-and Pip6b-PMO conjugates displayed the highest recovery scores, as observed in Figure 4b (%RS 37.66% and 34.22%, respectively) closely followed by Pip6f-PMO (%RS 26.24%) and then Pip5e-PMO (%RS 17.32%). When directly compared to Pip5e-PMO treatment, only Pip6a-PMO was significantly better in the heart ( Table 2 ). These 5-aa core Pip6-PMOs were also shown to restore other dystrophin complex proteins, namely neuronal nitric oxide synthase, α-sarcoglycan, and β-sarcoglycan as illustrated by immunohistochemical staining in the TA muscle (Supplementary Figure S4) .
The PCR and western blot analyses exhibited similar results to the immunostaining. The reverse transcription-PCR (RT-PCR) representative images (Figure 4d ) illustrate high exon skipping efficiency in all tissues analyzed. This is better shown by quantitative real-time PCR (qRT-PCR) results for the quadriceps, diaphragm and heart (Figure 4c) . The delta 23 transcript is normalized against "total dystrophin" for each muscle group (n = 3). Quantification of this data revealed similar levels of ∆23 skipping in the quadriceps of all 5-aa Pip6-PMO-treated mice. The data trends suggest that Pip6f-and Pip5e-PMO show the highest exon skipping in the diaphragm, and Pip6f-PMO the highest in heart (for splicing mean values, see Supplementary Figure S2a ). Western blots (Figure 4e) were performed on the tissues of each mouse and were quantified against a 50 and 10% C57BL10 control. These results were averaged and are presented in Supplementary Figure S2b. Pip6a-, Pip6b-, Pip6e-, and Pip6f-PMO conjugates exhibited the highest dystrophin protein restoration in the TA and quadriceps muscles. The levels of dystrophin restoration in the diaphragm were uniform across all of these treatments, whereas in the case of the heart, Pip6b-and Pip6f-PMO conjugates showed the highest dystrophin restoration.
Protein restoration as measured by immunohistochemical staining is consistently higher than protein restoration 4 calculated by western blot analysis. These differences may be attributed to the differing "housing proteins" used i.e., dystrophin restoration quantified by immunohistochemical staining is normalized against laminin, whereas western blot analysis uses α-actinin for normalisation. Quantification of western blots has only recently been reported for dystrophin and currently uses chemiluminescence methods. It may therefore be judicious to give greater weight to the trends in dystrophin protein levels revealed by western blot rather than to the absolute values. Therefore, considering the results overall, mdx mice treated with each of the four 5-aa core Pip6-PMOs (Pip6a-, Pip6b-, Pip6e-, and Pip6f-PMO) appear to demonstrate improved dystrophin production and exon skipping in TA, quadriceps, and heart muscles compared with the previous lead candidate, Pip5e-PMO. In addition, these 5-aa core Pip6-PMOs do not exhibit evidence of toxicity, as assessed by plasma levels of relevant toxicity biomarkers, alanine aminotransferase, aspartate aminotransferase, and creatine kinase (see Supplementary Figure S5a ). Blood urea nitrogen and creatinine levels were similar to untreated mdx levels (see Supplementary Figure S5b ). All Pip6-PMO treatment groups exhibit similar biomarker levels to untreated C57BL10 controls.
In addition to the need to identify Pip-PMOs with high efficiency and cardiac delivery, it was a further aim to better define those elements of the hydrophobic core of Pip peptides that are important for heart delivery. To this end, Pip peptides containing partial deletions of the hydrophobic core by 1 aa (removal of tyrosine; Pip6c) and by 2 aa (removal of isoleucine and tyrosine; Pip6d) were synthesised as PMO conjugates (Figure 1a) .
Following treatment of mdx mice, immunohistochemical staining was performed and this revealed some dystrophin expression in skeletal muscles such as the TA and diaphragm for both these deletion Pip6-PMOs ( Figure 5) . Quantification of the immunohistochemical staining revealed the lowest dystrophin restoration in the quadriceps with Pip6d-treatment, closely followed by Pip6c-PMO when compared to the other Pip6-PMOs (Figure 6a,b and Table 1 ). Similarly, Pip6d-PMO displayed the lowest dystrophin restoration in the diaphragm. The recovery scores for Pip6c-and Pip6d-PMO conjugates in the heart were very low, indicating their poor efficiency (Pip6c %RS -1.06% and Pip6d 2.50%; Figure 6b) .
These results are corroborated by the PCR and western blot analyses. The RT-PCR representative images ( Figure  6d ) and the qRT-PCR exon skipping results (Figure 6c ) both indicate reduced exon skipping in mdx mice treated with Pip6c-and Pip6d-PMO conjugates in quadriceps and diaphragm and negligible exon skipping in the heart. Western blot analysis revealed inefficient dystrophin protein production in the TA and quadriceps muscles and negligible dystrophin restoration in the heart (Figure 6e and Supplementary Figure S2b) .
These results show that the length of the hydrophobic core is crucial not only for good heart dystrophin production but also for activity in some other muscle groups. Therefore, the arginine content of the CPP alone is not the sole predictor of dystrophin production and exon skipping efficiency for this class of peptides.
Altering the position of arginine in the hydrophobic core or adding a second arginine is detrimental to dystrophin production The repositioning of an arginine from a flanking region into the core was unexpectedly well-tolerated (Pip6e-PMO) Two further Pip6-PMO conjugates were thus synthesized (Figure 1a) as derivatives of Pip6e-PMO. Pip6g-PMO contained a second arginine residue, which was moved from the second flanking region into the central hydrophobic core, and Pip6h-PMO contains an inversion of the Pip6e hydrophobic region, such that the single arginine location is altered within the core. (e) Representative western blot images for each treatment. Ten micrograms of total protein was loaded (TA, quadriceps, gastrocnemius, diaphragm, heart, and abdomen muscles) relative to 50% (5 µg protein) and 10% (1 µg) C57BL10 controls, and normalized to α-actinin loading control (for quantification see Supplementary Figure S2a) . PMO, phosphorodiamidate morpholino oligonucleotide.
Surprisingly, these changes to the hydrophobic core resulted in further reductions in dystrophin expression not only in heart but also in all other tissues, as observed in the immunohistochemical staining (Figure 7a ) and in the quantifications thereof (Figure 7b,c) . The immunohistochemical staining representative images revealed very few dystrophin positive fibers in all tissues with the exception of the TA and quadriceps (Figure 7a) . With reference to the quantifications, Pip6g-PMO was not significantly different to untreated mdx in the quadriceps or diaphragm (Supplementary Figure S3a) . Statistical significance tables for immunohistochemical staining of quadriceps, diaphragm, and heart muscles for Pip6a-f-treated mdx mice relative to untreated mdx mice. Statistical significance was determined using a repeated measures, multilevel statistical model (****P < 0.0001, ***P < 0.001, **P < 0.01*P <0.05, N/S, not significant). Statistical significance tables for immunohistochemical staining of quadriceps, diaphragm, and heart muscles for Pip6a-f-treated mdx mice relative to Pip5e-treated mice. Statistical significance was determined using a repeated measures, multilevel statistical model (****P < 0.0001, ***P < 0.001, **P < 0.01*P <0.05, N/S, not significant). Both Pip6e-PMO derivatives were not significantly different to untreated mdx in heart muscles, illustrating the general inefficiency of these two peptides. Similarly, these Pip6e-PMO derivatives showed reduced efficiency in exon skipping, as illustrated in representative RT-PCR images (Figure 7e ) and in qRT-PCR analyses (Figure 7d and Supplementary Figure S3c ) in all tissues. Western blots revealed negligible dystrophin protein restoration (Figure 7f and Supplementary Figure S3d ) in all tissues with the exception of the TA muscle. These data show that an increase in the number of arginines or alteration in the location of the single arginine in the hydrophobic region of Pip6e are detrimental to both heart as well as skeletal muscle dystrophin production.
The most promising therapy to date for the severely debilitating neuromuscular disorder DMD is treatment with AOs, which restores the reading frame of the dystrophin pre-mRNA by exon skipping. Two AOs, a PMO 10,11 and a 2′OMe oligonucleotide, 8, 9 are currently in clinical trials and the early Ten micrograms of total protein was loaded (TA, quadriceps, gastrocnemius, diaphragm, heart, and abdomen muscles) relative to 50% (5 µg protein) and 10% (1 µg) C57BL10 controls, and normalized to α-actinin loading control (for quantification see Supplementary Figure S2a) . PMO, phosphorodiamidate morpholino oligonucleotide.
promising results have increased hope for DMD patients. However, studies involving the administration of very high doses of naked PMO into mdx mice have shown only partial restoration of dystrophin in body-wide skeletal muscles and negligible correction in heart. 16, 35 The necessity to correct dystrophin in the heart is ever more apparent following studies whereby the correction of the skeletal phenotype resulted in an increase in the cardiac workload and thus further progression of the cardiomyopathy. 17, 18 The discovery that CPP-conjugated PMOs can achieve much more effective dystrophin correction in mdx mice than naked PMOs has brought renewed promise for enhanced AO efficacy by improving cellular and in vivo delivery. We previously reported a promising peptide-PMO candidate, Pip5e-PMO, capable of restoring dystrophin protein to high levels in all muscle types, including heart, following a single 25 mg/kg administration. 29 In addition to arginine-rich sequences, Pip peptides contain a 5-aa hydrophobic section not present in the previous B-peptide lead, 26 which seemed likely to be responsible for the improved heart activity. The Pip6 series was developed as derivatives of Pip5e-PMO in an attempt to cast light on aspects of the hydrophobic core required for heart dystrophin production and also to identify even more active Pip-PMO conjugates. Our study using a moderate, single dose administration regimen has produced some interesting and sometimes surprising results.
A key finding is that maintenance of the 5-aa length of the hydrophobic core region is imperative for good heart dystrophin production. One might imagine that diminished efficiency of dystrophin restoration in the heart for Pip6c-PMO and Pip6d-PMO, with sequential amino acid deletions in the core, might be correlated with the resultant lower hydrophobicity and hence a reduced capacity to enter the cell. 36 However, the in vitro results would suggest that all of these constructs are capable of entering the cells as they are all fully capable of exon skipping in muscle cells (Figure 2) . Thus, the length of the 5-aa hydrophobic core must affect a different parameter essential for in vivo heart delivery. Enhanced uptake into whole heart slices of fluorescently labeled Pip5e-PMO, compared to B-PMO, suggested instead that crossing of another barrier (for example, the endothelial lining to the heart) is improved. 30 Further heart studies are continuing with a Pip6-PMO that may help to address this issue. More surprising perhaps is that for Pip6c-PMO and Pip6d-PMO there was also some loss of dystrophin production in other muscle types. This suggests that the hydrophobic/cationic balance and/or the precise spacings of hydrophobic and cationic residues in the CPP impose more subtle effects on in vivo delivery parameters.
Another clear conclusion arising from the Pip6-PMO analogues is that a specific order of hydrophobic residues within the hydrophobic core is less important at maintaining the heart dystrophin production, since an inverted sequence (Pip6a), a single substitution of an equally hydrophobic residue (Pip6b), and a scrambled sequence (Pip6f) were at least as active as Pip5e-PMO, and more efficient in heart and some muscle groups (Figure 4 and Supplementary Figure  S2) . These results provide evidence that the hydrophobic core of Pip peptides is unlikely to contain a particular amino acid sequence that recognizes a specific receptor in a membrane barrier required to penetrate heart tissue, but instead the core acts as a hydrophobic spacer of some kind.
Most surprising however is that Pip6e-PMO did induce some dystrophin splicing and protein restoration in heart muscle as indicated by the western and qRT-PCR results (note: not significantly different in immunohistochemical staining quantification). In the Pip-6e peptide, one arginine residue is moved into the hydrophobic core, which also results in alignment of a hydrophobic X residue adjacent to the core (X-YRFLI). One might have expected heart dystrophin production to have been completely lost in this conjugate, since a cationic amino acid (arginine) is now included in the core. By contrast, such heart activity was lost for Pip6h-PMO (X-ILFRY core) and the double arginine core conjugate Pip6g-PMO (X-YRFRLI-X core). Unexpectedly, dystrophin production was also lost in quadriceps and diaphragm for both Pip6g-and Pip6h-PMO. The unanticipated inconsistencies within the activity results for Pip6e, Pip6g, and Pip6h, and the losses of activities for Pip6c-and Pip6d-PMO, are perhaps best explained by the realization that precise spacings of the arginine residue within the Pip peptides with respect to both the outer hydrophobic amino acid spacers (X and B) and the inner hydrophobic core residues may drastically alter the pharmacological properties of each conjugate. This might occur not only through alteration in cationic/hydrophobic balance but alternatively due to secondary or tertiary structure changes of the Pip-PMOs, which could in turn affect serum protein binding or another parameter that alters the circulatory half-life, or which could affect the ability to traverse barriers required to penetrate muscle tissues. Such more subtle effects will require a more wide-ranging pharmacological and biophysical study, upon which we are currently embarking.
For a complete understanding of the role of the hydrophobic core within Pip peptides, one would ideally wish to study the activities, pharmacology and biophysical parameters of a much larger range of sequence-variant Pip-PMO conjugates. The need for multi-mg synthesis of each conjugate as well as the availability of a large number of mdx 37 ). This is greatly promising for the Pip6-PMOs, as it would not be considered the optimal peptide yet still demonstrated the significant therapeutic effect of this group of peptides. These new leads provide a good basis for identification of a Pip-PMO candidate suitable for detailed physiological studies of muscle and heart function, as well as thorough toxicity profiling including dose escalation studies, in anticipation that one such Pip-PMO will proceed to clinical trial.
Synthesis of peptide-PMO conjugates. Peptides were synthesized by standard Fmoc chemistry and purified by highperformance liquid chromatography. The PMO sequence (5 ′-GGCCAAACCTCGGCTTACCTGAAAT-3′) was purchased from Gene Tools LLC (Corvallis, OR). Peptides were conjugated to PMO through an amide linkage at the 3′ end of the PMO, followed by purification by high-performance liquid chromatography and analyzed by MALDI-TOF MS as previously described in preliminary communication. 32 Full details of synthesis including improvements to the experimental procedures are described in detail in the Supplementary Materials and Methods. Peptide-PMO conjugates were dissolved in sterile water and filtered through a 0.22-µm cellulose acetate membrane before use.
Conjugates of PMO of Pip6a, Pip6b, Pip6e, and Pip6f were found to be predominantly stable and of similar stability to Pip5e-PMO in 100% serum for 2 hours at 37 °C, as seen by high-performance liquid chromatography and MALDI-TOF mass spectral analysis. The conjugates all showed similar degradation patterns, and intact conjugates were still observed up to 4 hours (data not shown).
In vitro assays: exon skipping in mdx mouse myotubes. H2K mdx myotubes were prepared and incubated with peptide-PMO conjugates in the absence of any transfection agent at concentrations of 0.125, 0.25, 0.5, and 1.0 µmol/l by the method described previously. 29 The products of nested RT-PCR from total isolated RNA were examined by electrophoresis on a 2% agarose gel. Quantification of ∆23 transcript levels was calculated using densitometry. The MTS cell viability test (Promega, Madison, WI) showed 100% survival at the highest concentrations of peptide-PMO conjugates used in the study (data not shown).
Animals and intravenous injections. Four and a half month old to 5½-month-old mdx mice were used in these experiments (n = 3). The experiments were carried out in the Biomedical Sciences Unit, University of Oxford according to procedures authorized by the UK Home Office. Pip6-PMO conjugates were prepared in 0.9% saline solution at a final dose of 12.5 mg/kg. The 160 µl total volume was administered via the tail vein of anaesthetized mice. Two weeks later mice were sacrificed by CO 2 inhalation, and muscles and other tissues harvested and snap-frozen in cooled isopentane before storage at -80 °C.
Immunohistochemistry and quantification of dystrophin expression. Transverse sections of tissue samples were cut (8-µm thick) for the examination of dystrophin expression. For dystrophin visualisation and quantification, sections were costained with rabbit-anti-dystrophin (Abcam, Cambridge, MA) and rat anti-laminin (Sigma, St Louis, MO), and detected by goat-anti-rabbit immunoglobulin G Alexa 594 and goat-antirat immunoglobulin G 488 secondary antibodies, respectively (Invitrogen, Carlsbad, CA). Images were captured using a Leica DM IRB microscope and Axiovision software (Carl Zeiss, Cambridge, UK). Quantitative immunohistochemistry was performed as previously described. 16, 34 A representative image for each treatment was taken. For quantification, four representative frames of the dystrophin and correlating laminin were taken for each section (n = 3) of the quadriceps, diaphragm and heart for each treatment. Using ImagePro software, 10 regions of interest were randomly placed on the laminin image which was overlaid on the corresponding dystrophin image. The minimum and maximum fluorescence intensity for 120 regions were recorded for each treatment. The intensity difference was calculated for each region to correct for background fluorescence and untreated mdx and treated mdx were normalized to C57BL10. These values were plotted on a scatter graph. The "relative intensity means" were calculated using a multilevel statistics model. Using these values, the percentage recovery score was calculated by implementing the following equation, as described on the TREAT-NMD website (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/ DMD_M.1.1_001.pdf): (dystrophin recovery of treated mdx mice-dystrophin recovery of untreated mdx mice)/(dystrophin recovery of C57BL10 mice-dystrophin recovery of untreated mdx mice). Staining of dystrophin associated proteins was performed as previously described 29 using a MOM blocking kit (Vector Labs, Burlingame, CA) and α-sarcoglycan and α-dystroglycan (Novocastra, Newcastle-Upon-Tyne, UK) antibodies (1:100 dilution). Neuronal nitric oxide synthase staining was performed using a goat anti-rabbit antibody (Abcam).
Exon skipping in mdx mouse tissues. Total RNA was extracted from control and treated mouse tissues using TRIzol reagent (Invitrogen) following manufacturer's instructions.
RT-PCR: Four hundred nanograms of RNA template was used in a 50 µl reverse transcription reaction using One Step RT-PCR Kit (Qiagen, Hilden, Germany) and gene-specific primers (Ex 20-26, Fwd: 5′-CAG AAT TCT GCC AAT TGC TGA G-3′, Rev: 5′-TTC TTC AGC TTG TGT CAT CC-3′). Cycle conditions: 50 °C for 30 minutes, followed by 30 cycles of 30 seconds at 94 °C, 1 minute at 58 °C, and 2 minutes at 72 °C. Two microliters of cDNA was further amplified in a 50 µl nested PCR (QIAGEN PCR kit) using the following cycle control, mdx untreated and Pip6g-and Pip6h-PMO-treated mice. Dystrophin immunostaining in TA, quadriceps, gastrocnemius, diaphragm, heart, and abdomen muscle groups for C57BL10, mdx-untreated and mdx-treated mice are shown. (b) Quantification of dystrophin immunohistochemical staining relative to laminin counter-stain in quadriceps, diaphragm, and heart muscles of C57BL10, mdx-untreated and mdx-treated mice. Relative intensity values for each region of interest (120 regions) are plotted and the model estimate averages calculated (presented in c) from the repeated measures, multilevel statistical model. For statistical significance tables see Supplementary Figure S3a ,b. Percentage recovery score is represented below. (d) Percentage ∆23 exon skipping as determined by quantitative real time (q-RT)-PCR in quadriceps, diaphragm and heart muscles. (e) Representative real-time (RT)-PCR images demonstrating exon skipping (skipped) in TA, quadriceps, gastrocnemius, diaphragm, heart, and abdomen muscles. The top band indicates full-length (FL) or unskipped transcript. (f) Representative western blot images for each treatment. Ten micrograms of total protein was loaded (TA, quadriceps, gastrocnemius, diaphragm, heart, and abdomen muscles) relative to 50% (5 µg protein) and 10% (1 µg) C57BL10 controls, and normalized to α-actinin loading protein (for quantification, see Supplementary Figure S3c) . PMO, phosphorodiamidate morpholino oligonucleotide.
conditions: 94 °C for 30 seconds, 58 °C for 1 minute, and 72 °C for 1 minute for 24 cycles (Ex 20-26: Fwd: CCC AGT CTA CCA CCC TAT CAG AGC, Rev: CCT GCC TTT AAG GCT TCC TT). PCR products were examined by electrophoresis on a 2% agarose gel.
Quantitative real time PCR: Two micrograms of RNA was reverse transcribed using a High Capacity cDNA Synthesis kit (Applied Biosystems, Branchburg, NJ). Exon skipping qPCR was performed using Syber green Kits (Applied Biosystems), primer sets (IDT) and the StepOne Plus Real-Time PCR system (Applied Biosystems). Primer sets used were as follows: total dystrophin transcripts, ex19-20: Fwd: GCCATAG-CACGAGAAAAAGC, Rev: GCATTAACACCCTCATTTGC; Delta23 dmd transcript, Fwd: GCG CTA TCA GGA GAC AAT GAG, Rev: GTT TTT ATG TGA TTC TGT AAT TTC CC.
Plasmids (total dystrophin and delta 23 skipped) were used for the standard curve.
Protein extraction and western blot. Control and treated muscle samples were homogenised in lysis buffer comprising 75 mmol/l Tris-HCl (pH 6.5) and 10% sodium dodecyl sulphate complemented with 5% 2-mercaptoethanol. Samples were heated at 100 °C for 3 minutes before centrifugation and removal of supernatant. Protein levels were measured by Bradford assay (Sigma) and quantified using BSA standards. Ten to fifteen micrograms of protein of untreated and treated mdx sample, and 50% and 10% of these concentrations of C57BL10 protein (positive control) were loaded onto 3-8% Tris-Acetate gels. Proteins were blotted onto polyvinylidene fluoride membrane and probed for dystrophin using DYS1 (Novocastra) and loading control, α-actinin (Sigma), antibodies. Primary antibody was detected by binding of horseradish peroxidase-conjugated anti-mouse immunoglobulin G with lumigen. Western blots were imaged (LiCOR Biosciences, Lincoln, NE) and analyzed using the Odyssey imaging system.
Clinical biochemistry. Plasma samples were taken from the jugular vein of mdx mice immediately following sacrifice by CO 2 inhalation. Analysis of toxicity biomarkers was performed by a clinical pathology lab, Mary Lyon Centre, MRC, Harwell, UK.
Statistical analysis. All data reported mean values ± SEM. A multilevel, repeated measures model was implemented for this study. The multilevel statistical approach builds upon traditional statistical methods and is being increasingly implemented in the social, medical and biological sciences. [38] [39] [40] [41] The model used for this study takes into account the multiple "relative intensity units" (level 1) for each mouse (level 2) for each treatment (level 3) as performed in the immunohistochemical staining quantification. In this example mdx untreated mice and Pip5e-PMO-treated mice were applied as the constant/ fixed parameter, to which the other treatments and wild-type control were compared. This was following a Box-Cox power transformation which was performed to ensure a normal distribution. Statistical analysis was performed using MLwIN version 2.25. Figure S1 . HPLC chromatogram and MALDI-TOF data for Pip6e-PMO. Figure S2 . qRT-PCR mean values table and quantification of western blots for C57BL10 control, mdx-untreated and Pip6-PMO-treated mdx mice, following a single 12.5 mg/kg, i.v. injection. Figure S3 . Statistical tables for quantitative immunohistochemical staining, qRT-PCR mean values table and quantification of western blots for C57BL10 control, mdx untreated and Pip6e-PMO derivative (Pip6g and Pip6h) treated mdx mice, following a single 12.5 mg/kg, i.v. injection. Figure S4 . Immunohistochemical staining of dystrophin complex proteins in C57BL10 control, mdx untreated, the 5-aa hydrophobic core Pip6-PMO-treated mice. Figure S5 . Toxicity assays assessed in blood samples of C57BL10 control, mdx-untreated, Pip6-PMO-and Pip5e-PMO-treated mdx mice, following a single 12.5 mg/kg, i.v. injection. Clinical characteristics and outcome of Penicillium marneffei infection among HIV-infected patients in northern Vietnam OBJECTIVE: This study reports the clinical characteristics and outcome of HIV-associated Penicilliummarneffei infection in northern Vietnam. METHODS: We conducted a retrospective chart review of all patients with laboratory confirmed Penicilliummarneffei infection admitted to the National Hospital for Tropical Diseases in Hanoi, Vietnam, between July 2006 and September 2009. RESULTS: 127 patients with P. marneffei infection were identified. All were HIV-infected; median CD4+ T-cell count was 24 cells/μl (IQR:12-48); 76% were men. Common clinical features were fever (92.9%), skin lesions (82.6%), hepatomegaly (61.4%), lymphadenopathy (40.2%), weight loss (59.1%) and cough (49.6%). Concurrent opportunistic infections were present in 22.0%; half of those had tuberculosis. Initial treatment regimens were: itraconazole or ketoconazole capsule (77.2%), amphotericin B (20.5%), and fluconazole (1.6%). In-hospital mortality was 12.6% and showed no significant difference in patients treated with itraconazole (or ketoconazole) and amphotericin B (p = 0.43). Dyspnea, ascites, and increased LDH level were independent predictors of mortality. No seasonality was observed. CONCLUSION: The clinical features, treatments and outcomes of HIV-associated P. marneffei infection in northern Vietnam are similar to those reported in other endemic regions. Dyspnea was an important predictor of mortality. More patients were treated with itraconazole than amphotericin B and no significant difference in treatment outcome was observed. It would be of clinical value to compare the efficacy of oral itraconazole and amphotericin B in a clinical trial. Penicillium marneffei can cause a fatal systemic mycosis in immunosuppressed patients and is one of theleading causesof mortality in people living with Human Immunodeficiency Virus (HIV) in South-East Asia [1] [2] [3] . Penicilliosis presents primarily as a disseminated disease in HIVinfected patients with CD4+ T-cell count <100 cells/μL, involving the blood stream, skin, liver, spleen, lymph nodes, bone marrow, lung and gastrointestinal tract [2, 4] . Typical umbilicated skin lesions are present in 70% of patients, facilitating early empirical antifungal therapy and resulting in better outcomes [4] . Laboratory diagnosis is made by microscopy and culture of skin lesions, blood, lymph node, or other body fluids [3] .The majority of patients respond well to either amphotericin B or itraconazole treatment [2, [4] [5] [6] ; however no randomized controlled trials have been conducted to evaluate treatment choices for penicilliosis.
We conducted a retrospective patient chart review of all patients with a compatible clinical syndrome and culture confirmed P. marneffei infection admitted to the National Hospital for Tropical Diseases (NHTD) in Hanoi between July 2006 and September 2009. Data collected included: demographics, admission date, clinical characteristics, HIV status, CD4+ T-cell count, laboratory investigations, concurrent opportunistic infections (OIs), treatment, and hospital outcome. P. marneffei was cultured from clinical specimens according to standard culture techniques [7] . For seasonality analysis, we assessed the number of penicilliosis admissions during dry versus rainy months in relation to all HIV-related admissions identified from hospital records. For outcome analysis three variables were used: 1. Survival including clinical improvement, defined as regression of symptoms such as fever, skin lesions, lymphadenopathy, hepatomegaly and splenomegaly, or resolution; 2. death at hospital discharge; 3. Lost to follow up due to that the patient were taken home or referred to another health facility and could not be contacted. We performed univariate and multivariate logistic regressions using forward stepwise selection of predictor variables. Statistically significant results (p < 0.05) from the univariate analysis were entered into a multivariate logistic regression model where continuous variables were dichotomized based on the mean values and after exclusion of correlated variables. The analysis was performed using Statistics Package of Social Sciences (SPSS version 19, USA).
P. marneffei infection was diagnosed in 127 patients, 42.5% by both blood and skin lesion culture, 29.1% by blood culture alone, and 28.3% by skin lesion culture alone. The average age was 32 years (range 21-50); the majority was male (75.6%), and 81.9% came from provinces outside of Hanoi in northern Vietnam. The reported route of HIV infection was intravenous drug use (IDU) (37.0%), commercial sex (29.9%), husband to wife (15.7%), combination of IDU and commercial sex (7.9%) and unknown (9.0%). 29.9% had been on antiretroviral therapy (ART) prior to the diagnosis of penicilliosis; the mean duration of ART was 10.8 weeks (SD 18.4).
The clinical and laboratory features are shown in Table 1 and Table 2 . The median CD4+ T-cell count on admission was 24 cells/μl (IQR: 12-48). Other OIs were diagnosed (n = 28, 22%) patients; including tuberculosis (n = 14, 11.0%), Pneumocystis jirovecii pneumonia (PCP) (n = 6, 4.7%), varicella-zoster virus (n = 4, 3.1%) as well as toxoplasmosis, cytomegalovirus retinitis and herpes simplex virus type 1 infection all (n = 1, 0.8%). Five patients also had bacteremia with the following pathogens: Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes and Salmonella spp. Six patients were previously diagnosed and received treatment for penicilliosis; however, none had started ART. In addition to blood and skin lesion cultures, there were 9 CSF cultures performed of which one was positive for P. marneffei and 11 lymph node aspirate cultures were all negative.
The initial choices of antifungal treatments are listed in Table 3 . Outcome at discharge was clinical improvement or resolution in 107 (84.2%) patients and death in 16 (12.6%). Nine patients were discharged early due to request from the family members, or referred to other hospital. Five of them were assessed within a month after discharge and four patients were lost to follow-up. The mean duration of treatment in the hospital was 15.5 days (SD 11). For diseased patients deaths occurred early in the course of treatment after an average of 6.6 days (SD 10), compared to survivors, who were treated in average 16.7 days (SD 10)(p < 0.001). Among 98 patients receiving itraconazole or ketoconazole, 85 (86.7%) had an improvement; 13 (13.3%) died. By comparison, among 26 patients receiving amphotericin B, 24 (92.3%) had an improvement; and two (7.7%) patients died. The mortality differences between the two treatment groups did not reach statistical significance (p = 0.43, χ 2test).
Results of the logistic regression are shown in Table 1 for clinical variables and Table 2 for laboratory variables. Univariate analysis showed significantly higher risk of death among patients with dyspnea, defined as a combination of subjective sensation of difficulty breathing and observed tachypnea, ascites, jaundice, splenomegaly (Table 1) , increased levels of alanine transaminase (AST), bilirubin, lactate dehydrogenase (LDH), white blood cell count, blood urea, thrombocytopenia and prolonged prothromb in time (Table 2 ). However, in the multivariate analysis, only dyspnea, ascites and increased LDH levels remained independent predictors of mortality. Of the 11 patients that died 6 had dyspnea, none of them had tuberculosis or Pneumocystis jiroveci. However, two were diagnosed with sepsis, one Escherichia coli and one Streptococcus pyogenes. In total 23 patients had X-ray confirmed lesions of the lungs, 5 of these also had dyspnea (p = 0.015), 4 had pulmonary tuberculosis and 2 had Pneumocystis jiroveci, 4 died, of those 2 were diagnosed with tuberculosis. Of the 5 patients with dyspnea and lung lesions one was diagnosed with Pneumocystis jiroveci and none with tuberculosis.
Assessment of seasonality was performed for the year 2007 and 2008. During these two years P.marneffei accounted for 87 of 793 (11.0%) of all HIV related admissions. The number of penicilliosis admissions was 43 during the hot rainy months (May to October) and 44 during the cooler dry months (November to April). The number of all HIV-related admissions was 463 during the rainy months and 330 during the dry months. The proportion of penicilliosis admissions in relation to all HIV-related admissions comparing dry versus rainy season did not show statistically significant difference (p = 0.07, χ 2test).
Penicilliosis accounted for 11% of all HIV-related admissions at NHTD in Hanoi during 2007 and 2008 which is higher than the 4.4% reported from the major referral hospital for infectious diseases in Ho Chi Minh City, southern Vietnam [4] . However as NHTD is a specialized tertiary hospital, and most (82%) of the cases were referred, and because other epidemiological data were lacking, it cannot be concluded that there is a difference in disease prevalence between northern and southern Vietnam. The clinical features of disseminated penicillosis are consistent with other studies including profound immunosuppression (median CD4+ T-cell count: 24 cells/μl) and high rate of co-infections with other opportunistic pathogens [2, 4, 6] . One third of the patients were already on ART for in average 10 days, this indicates that many patients had an ongoing P. marneffei infection that was not revealed before initiation of ART, but was probably unmasked by immune reconstitution inflammatory syndrome (IRIS) after initiation of ART, this has earlier been reported in a few case studies [8, 9] . In our study the presence of dyspnea, ascites, and high LDH levels on admission independently predicted hospital mortality. Of the six patients that had dyspnea and died no one had tuberculosis or Pneumocystis jiroveci diagnosis, however two had septicemia. This may indicate that pulmonary involvement (i.e. P. marneffei pneumonia) drives disease severity or that the disease severity result in pulmonary lesions. This is consistent with a prior study showing that high respiratory rates and dyspnea among penicilliosis patients predicted poor hospital outcome [4] . Of the 11 patients with dyspnea 5 also had lung lesions, of these one was diagnosed with Pneumocystis jiroveci and non with tuberculosis. Lung involvement of P. marneffei has been shown in Taiwan where it was found to be the most common cause of cavities in the lungs of immunosuppressed HIV infected patients [10] . The majority of patients with dyspnea did not have lung lesions, hence the dyspnea might be due to the severe condition with multi-organ involvement and acute respiratory distress syndrome (ARDS). It should be noted that there could be some under diagnosis of tuberculosis due to the poor sensitivity for sputum microcopy and culture in immunosuppressed individuals as well as for Pneumocystis jiroveci as microscopy of sputum, obtained by nebulizer or bronchoscopy, is not routinely performed.
Typical skin lesions were present in 80% of patients in this study. The pathogenesis and prognosis of skin involvement is poorly understood. Although a previous study showed that presence of typical skin lesions facilitates early initiation of empirical antifungal treatment and results in better outcome [4] , it is unclear whether skin involvement itself is a prognostic marker. In the absence of skin lesions, the differential diagnoses for an AIDS-associated febrile illness with reticuloendothelial system involvement are broad and include: Mycobacterium tuberculosis, Mycobacterium Avium Complex, histoplasmosis and cryptococcosis among others [11] . This poses a major challenge in diagnosis and treatment, especially in areas with poor access to blood culture and other diagnostic laboratory. Penicilliosis should be considered in all severely immunosuppressed HIV patients with one or more of the common presentations, skin lesions, lymphadenopathy, hepatomegaly, splenomegaly, ascites, jaundice and dyspnoea, who have been in P. marneffei endemic areas, and empirical antifungal treatment in very ill patients may be indicated.
In this study 22% of the patients had a concurrent OI; of whom half had tuberculosis. Hence, multiple OIs, particularly pulmonary tuberculosis, need to be considered. Tuberculosis and penicilliosis co-infection poses a major therapeutic dilemma in resource-poor settings as rifampicin is a potent P450 inducer and markedly reduces itraconazole concentrations [12] Amphotericin B is recommended for patients with concurrent tuberculosis treatment. An alternative tuberculosis drug rifabutin is not available in most penicilliosis endemic areas.
The WHO recommended treatment for severe penicilliosis, amphotericin B, was only given to 20.5% of patients. The majority (77.2%) was treated with either itraconazole or ketoconazole. Although itraconazole is a recommended alternative treatment for mild to moderate disease or when amphotericin B is unavailable [13] , in Vietnam both mild and severe cases are commonly treated with oral itraconazole because amphotericin B is often not available or is too expensive. In our study there was no significant difference between itraconazole and amphotericin B in treatment outcome. So far no randomized, prospective treatment trials have been conducted to compare the efficacy of different antifungal treatment regimens for penicilliosis. Compared to northern Thailand and southern Vietnam where cases peak in the rainy season [4] , seasonality was not observed in our cohort. It should be noted that our small sample size and short time frame does not enable any conclusive results about seasonality. However, the cool and dry season in northern Vietnam is often damp with high humidity which might have an impact on the reservoirs of P. marneffei.
This study shows that penicilliosis in northern Vietnam presents with similar clinical characteristics as in other endemic areas, and that dyspnea is an important predictor of mortality. It is common practice to treat patients with oral itraconazole rather than amphotericin B, and no significant difference in treatment outcome was observed. It would be of clinical value to compare the efficacy of oral itraconazole and amphotericin B in a clinical trial to develop evidenced based guidelines.
Abbreviations HIV: Human Immunodeficiency Virus; NHTD: National Hospital for Tropical Diseases; IDU: Intravenous drug use.
The author declares that they have no competing interests Authors' contributions NTLH -conception and design, acquisition of data, analysis and interpretation of data, has been involved in drafting the manuscript and have given final approval of the version to be published. ML -Analysis and interpretation of data, has been involved in drafting the manuscript and have given final approval of the version to be published. HFLW -conception and design, acquisition of data, interpretation of data, revising it critically for important intellectual content and have given final approval of the version to be published. DTT -Laboratory analysis and have given final approval of the version to be published. WT -conception and design, acquisition of data, revising it critically for important intellectual content and have given final approval of the version to be published. PH -conception and design, acquisition of data, interpretation of data, revising it critically for important intellectual content and have given final approval of the version to be published. NVT-Laboratory analysis and have given final approval of the version to be published. NTMH -Acquisition of data and have given final approval of the version to be published. TL -Revising it critically for important intellectual content and have given final approval of the version to be published. NVK -conception and design, acquisition of data and have given final approval of the version to be published. All authors read and approved the final manuscript. Prospective application of clinician-performed lung ultrasonography during the 2009 H1N1 influenza A pandemic: distinguishing viral from bacterial pneumonia BACKGROUND: Emergency department visits quadrupled with the initial onset and surge during the 2009 H1N1 influenza pandemic in New York City from April to June 2009. This time period was unique in that >90% of the circulating virus was surveyed to be the novel 2009 H1N1 influenza A according to the New York City Department of Health. We describe our experience using lung ultrasound in a case series of patients with respiratory symptoms requiring chest X-ray during the initial onset and surge of the 2009 H1N1 influenza pandemic. METHODS: We describe a case series of patients from a prospective observational cohort study of lung ultrasound, enrolling patients requiring chest X-ray for suspected pneumonia that coincided with the onset and surge of the 2009 H1N1 influenza pandemic. RESULTS: Twenty pandemic 2009 H1N1 influenza patients requiring chest X-ray were enrolled during this time period. Median age was 6.7 years. Lung ultrasound via modified Bedside Lung Ultrasound in Emergency protocol assisted in the identification of viral pneumonia (n = 15; 75%), viral pneumonia with superimposed bacterial pneumonia (n = 7; 35%), isolated bacterial pneumonia only (n = 1; 5%), and no findings of viral or bacterial pneumonia (n = 4; 20%) in this cohort of patients. Based on 54 observations, interobserver agreement for distinguishing viral from bacterial pneumonia using lung ultrasound was ĸ = 0.82 (0.63 to 0.99). CONCLUSIONS: Lung ultrasound may be used to distinguish viral from bacterial pneumonia. Lung ultrasound may be useful during epidemics or pandemics of acute respiratory illnesses for rapid point-of-care triage and management of patients. Emergency department visits quadrupled with the initial onset and surge during the 2009 H1N1 influenza pandemic in New York City (NYC) from April to June 2009 (Figures 1 and 2) [1, 2] . This time period was unique in that >90% of the circulating virus was surveyed to be the novel 2009 H1N1 influenza A according to the New York City Department of Health. Five-hundred sixtyseven patients requiring hospitalization were confirmed with the 2009 H1N1 influenza A in NYC [1] . In NYC, there were 16 deaths, 46% of admitted patients were <18 years old and 20% were <5 years old [2] . Eighty percent of confirmed cases had a known underlying risk condition, most commonly asthma (40% of confirmed cases) [1] .
This fourfold increase in patient volume presented logistical challenges for emergency departments [1] . In response to mass casualty incident-type conditions and overcrowding, emergency departments in New York City added staffing and created alternate sites of care to accommodate the increased patient volume. Increased demand for chest radiography for those patients with more severe disease led to increased delays and length of stay for those patients with suspected, but non-severe pneumonia.
Clinicians are challenged by the diagnostic dilemma that influenza cannot reliably be distinguished from other acute respiratory illnesses on the basis of clinical presentation alone [3] . Rapid viral antigen testing for diagnosis, which under ideal situations can yield results within 30 min, is not practical nor cost-effective in pandemic conditions [3] . Point-of-care ultrasound has been demonstrated to identify, in real-time, various pathologies of the lung, such as pneumonia, viral pneumonia, and acute respiratory distress syndrome (ARDS) [4] [5] [6] [7] [8] [9] [10] An algorithm for differentiating between various respiratory pathologies has been described ( Figure 3 ) [4] , and evidence-based recommendations regarding the use of point-of-care lung ultrasound have recently been published [11] . The use of lung ultrasound during the 2009 H1N1 influenza pandemic in adults has also been recently described [12] . We describe a prospective case series of children in whom clinicianperformed lung ultrasonography was used to differentiate between different respiratory pathologies and assessed interobserver agreement of these ultrasound findings during the initial onset and surge of the 2009 H1N1 pandemic (April to June 2009).
We describe a subcohort of patients who required chest X-ray for suspected pneumonia and were enrolled into a prospective study of lung ultrasound for diagnosing pneumonia that coincided with the onset and surge of the 2009 H1N1 influenza pandemic from April to June 2009 [1, 2, 13] . We also describe the application of a modified Bedside Lung Ultrasound in Emergency (BLUE) protocol [4] with posterior thorax scanning ( Figure 3 ) during the This study was approved by our institutional review board. The study population consisted of a convenience sample of patients who met predetermined inclusion criteria and in whom informed consent had been obtained and documented from the patient or guardian for enrollment into the study.
Inclusion criteria consisted of patients < 21 years of age presenting to the emergency department with clinical suspicion of pneumonia requiring chest X-ray for eva-luationWe excluded those patients who presented the following: (1) arrival in the emergency department with a chest X-ray, (2) a confirmed diagnosis of pneumonia by diagnostic imaging, or (3) hemodynamic instability.
Enrolled patients had a screening history and physical examination performed at the time of triage to determine eligibility into the study. After informed consent was obtained, enrolled patients had clinical exam findings documented on a standardized form and underwent point-of-care lung ultrasound examination. An ultrasound machine with a linear array transducer at 7.5 to 10 MHz (Sonosite Micromaxx, Bothell, WA, USA) was used to image the lungs in perpendicular planes (transverse, parasagittal, and coronal) in the midclavicular line anteriorly and posteriorly on the chest and the midaxillary line from the axillae to diaphragm (Figure 4 ).
Using a six-zone lung ultrasound scanning protocol similar to that described by Copetti et al. [7] , we defined and classified patients as positive or negative for viral pneumonia based on the presence of small subpleural consolidations usually <0.5 cm ( Figure 5 and Additional file 1) and/or individual B-lines or confluent B-lines (echogenic vertical lines arising from the pleural line to the bottom of the ultrasound screen; Figure 6 and Additional file 2) [7] . These ultrasound findings are similar to those described in interstitial syndrome which is defined as three or more B-lines in a given lung region [10, 14, 15] . A-lines (horizontal, reverberation artifacts of the pleural line; Figure 7 left) which indicate areas of the normal lung were also noted when present [10, 14] . Patients were classified as positive or negative for bacterial pneumonia based on the presence or absence of lung consolidation with air bronchograms [6, 7, 16] seen on ultrasound (Figures 7 right, 8, and Additional file 3). A clinical course with follow-up after 2 weeks (via electronic medical record and telephone interview) was used to determine disposition and outcomes of enrolled patients. Clinicians performing and interpreting ultrasound were blinded to chest X-ray results, and when performed per hospital protocol for possible admission, viral antigen testing results. Bacterial pneumonia on chest X-ray (posterior-anterior and lateral views) was classified based on the attending pediatric radiologist reading for 'consolidation, 'infiltrate, or 'pneumonia. For analysis purposes only, viral pneumonia on chest X-ray was defined as 'peri-bronchial cuffing, 'peri-bronchial thickening, or 'increased interstitial markings identified by the pediatric radiologist. Pediatric radiologists were blinded to the lung ultrasound results.
Ultrasound images and videos were reviewed between two blinded investigator sonologists (enrolling sonologist and reviewing sonologist) to determine interobserver agreement by unweighted Cohens Kappa for viral pneumonia (small subpleural consolidation and/or B-lines), normal lung ultrasound pattern (A-lines), and bacterial pneumonia (lung consolidation with sonographic air bronchograms).
Patient demographic and study characteristics are presented in Table 1 . Twenty pandemic 2009 H1N1 influenza patients requiring chest X-ray (CXR) were enrolled during this time period. 
Distribution of diagnoses based on lung ultrasound findings, chest X-ray findings, and clinical outcomes using a modified BLUE protocol [4] is presented in Table 2 . Interobserver agreement for ultrasound findings of lung consolidation with air bronchograms, B-lines or small subpleural consolidations, and A-lines by Cohen's Kappa was 0.82 (95% confidence interval (CI), 0.63 to 0.99) ( Table 3) .
Ultrasound findings of lung consolidation with sonographic air bronchograms [6, 7, 16] correlated 100% with chest X-ray findings of bacterial pneumonia (reported as consolidation or infiltrate) in eight patients. All of these patients were confirmed to have pneumonia based on the clinical course at 2-week follow-up. This represented a doubling (40% vs. 20%) in the prevalence rate of bacterial pneumonia in our study during the H1N1influenza A onset and surge time period compared to the time period prior to the onset of H1N1 influenza A. The prevalence of viral lung ultrasound findings increased from approximately 50% for the overall study [13] to 75% during the surge of H1N1 influenza. Chest X-ray findings for viral pneumonia (most commonly described as peri-bronchial thickening or peri-bronchial cuffing) were present in 8 of 15 (53%) patients identified as having viral pneumonia on ultrasound. Seven of these 15 patients with viral pneumonia based on ultrasound had superimposed bacterial pneumonia also identified by ultrasound (Figure 7 and Additional file 4). All four patients in our series that required hospitalization had viral and bacterial pneumonia based on ultrasound.
All patients in our series were recovering or recovered from their influenza illness on follow-up after 2 weeks. All admitted patients were subsequently confirmed with the 2009 H1N1 influenza A by the New York City Department of Health. Per hospital protocol for possible hospital admission, four of nine patients tested positive for influenza A by viral antigen testing, despite the New York City Department of Health reporting >90% of the circulating virus during this pandemic time period was the novel influenza A H1N1 [1] . One infant in the cohort was co-infected with respiratory syncytial virus based on viral antigen testing. Three patients, all <5 years of age requiring hospital admission had evidence of both bacterial and viral pneumonia on ultrasound. The only patient requiring ICU admission, a 20-year-old female, was intubated after deteriorating during her ED stay with persistent hypotension and septic shock from a left lower lobe bacterial pneumonia. This patient initially presented with an influenza-like illness and acute abdominal pain.
To our knowledge, this is the first prospective series describing the use of lung ultrasound in children as a potential real-time diagnostic triage tool during a mass casualty-type incident due to an acute respiratory illness pandemic surge [17, 18] . Testa et al. have reported on similar lung ultrasound findings in adults during the 2009 H1N1 influenza A pandemic [12] . Single case reports of clinician-performed lung ultrasound to monitor the progression of H1N1 influenza-associated ARDS [19] and point-of-care echocardiography to diagnose H1N1 influenza myocarditis [20] have been described.
Retrospective reports of the role of ultrasound in mass casualty incidents during disasters such as earthquakes have also been described [21, 22] . Lichtenstein et al. described an algorithm using lung ultrasonography to distinguish between various respiratory pathologies of the lung [4] . We modified Lichtenstein's BLUE protocol [4] to recognize basic lung ultrasound patterns to distinguish between the normal unaffected lung, viral pneumonia pattern, and bacterial pneumonia (Figure 3 ). Scanning the posterior thorax was added to increase the sensitivity of the protocol [23] . Point-of-care lung ultrasound was able to identify, in real-time, four groups of pandemic patients: viral pneumonia only (subpleural consolidations and/or B-lines or confluent B-lines), bacterial pneumonia only (lung consolidation with sonographic air bronchograms), both viral and bacterial pneumonia (Figure 7) , and normal lungs (A-lines only). Our calculated Kappa was 0.82, which means that the interobserver agreement in distinguishing between these ultrasound findings was excellent. These ultrasound findings facilitated triage and immediate decision making regarding the need for respiratory isolation in a negative pressure room without waiting for chest X-ray. Our median time to chest X-ray tripled (Table 1 ) during the pandemic compared to a time period prior to the pandemic. Our time to chest X-ray interpretation during the pandemic was longer than the median of 98 min reported by Zanobetti et al. in the study of emergency department lung ultrasound in nonpandemic conditions [5] .
When lung consolidation with sonographic air bronchograms was visualized, point-of-care ultrasound facilitated the immediate decision to treat with antibiotics, without waiting for chest X-ray. Visualization of viral pneumonia on ultrasound may be useful to assist in the decision to initiate immediate empiric treatment with antiviral medication for future pandemic or epidemic influenza patients. In a large cohort of hospitalized H1N1 influenza A pandemic patients, only 73% of patients with radiographic evidence of pneumonia received antiviral drugs, whereas 97% received antibiotics [24] . Better recognition of viral pneumonia by ultrasound may impact outcomes, as available data have shown treatment with antiviral medication reduces mortality in hospitalized patients with influenza, even when therapy is initiated after 48 h of illness onset [24] .
Our sample size was limited by the inability to enroll during the surge of pandemic patients due to time and resource constraints. Selection bias from convenience sampling may have occurred because patients were more likely to have been enrolled at less busier or better staffed times. In general, the patients in this series had illnesses severe enough to warrant investigation with chest X-ray. Thus, information about less ill or asymptomatic pandemic patients is lacking.
Although our calculated interobserver agreement for lung ultrasound to distinguish between viral and bacterial pneumonia is high, the number of total observations was limited, and this is reflected in our wide 95% confidence intervals. However, it is notable that our point estimate Kappa for ultrasound is higher than the reported interobserver agreement for chest X-ray for pneumonia by pediatric radiologists, 0.51 (0.39 to 0.64) [25] .
Due to the large numbers of patients presenting to our emergency department during the pandemic, only hospitalized patients (four patients in our series) were confirmed with 2009 H1N1 influenza A [1] . Finding small subpleural consolidations and/or B-lines on ultrasound allows the recognition of viral pneumonia from bacterial pneumonia (lung consolidation with sonographic air bronchograms), but it is unknown if different viruses have unique lung ultrasound patterns (e.g., influenza A from RSV). We could not report test performance characteristics, such as sensitivity and specificity, as there was no practical reference gold standard for viral pneumonia at the time our study was conducted. Additionally, chest X-ray cannot be used as a gold standard for viral pneumonia. However, according to the New York City Department of Health, >90% of the circulating virus during this pandemic time period was the novel influenza A H1N1 [1] . Serum Levels of Gelatinase Associated Lipocalin as Indicator of the Inflammatory Status in Coronary Artery Disease Background. Atherosclerosis is a chronic inflammatory disease and the acute clinical manifestations represent acute on chronic inflammation. Neutrophil gelatinase-associated lipocalin (NGAL) is found in the granules of human neutrophils, with many diverse functions. The aim of this study was to evaluate the hypothesis that levels NGAL in blood may reflect the inflammatory process in various stages of coronary artery disease. Methods. We studied 140 patients, with SA 40, UA 35, NSTEMI 40, and STEMI 25, and 20 healthy controls. Serum NGAL was measured upon admission and before coronary angiography. Results. Significant differences were observed in median serum-NGAL(ng/mL) between patients with SA (79.23 (IQR, 37.50–100.32)), when compared with UA (108.00 (68.34–177.59)), NSTEMI (166.49 (109.24–247.20)), and STEMI (178.63 (111.18–305.92)) patients and controls (50.31 (44.30–69.78)) with significant incremental value from SA to STEMI. We observed a positive and significant correlation between serum-NGAL and hs-CRP (spearman coefficient rho = 0.685, P < 0.0001) as well as with neutrophil counts (r = 0.511, P < 0.0001). Conclusions. In patients with coronary artery disease serum levels of NGAL increase and reflect the degree of inflammatory process. In patients with acute coronary syndromes, serum levels of NGAL have high negative predictive value and reflecting the inflammatory status could show the severity of coronary clinical syndrome. Systemic inflammation participates in atherosclerosis evolution from the early development of endothelial dysfunction, to formation of mature atheromatic plaques, to the ultimate endpoint, rupture, and thrombotic complications [1] . Plaque rupture with the formation of an occlusive thrombus is the cause of acute coronary syndromes (ACS) [2] . Inflammatory cells, involving activated neutrophils, are more frequently found in plaques vulnerable to rupture [3] . Neutrophil activation has been reported in unstable angina (UA) and acute myocardial infarction (AMI) but not in patients with stable angina (SA) [4] [5] [6] [7] [8] [9] [10] . This activation seems to precede myocardial injury in patients with AMI [11] . Therefore biomarkers of neutrophil activation could be of prognostic and even diagnostic importance.
Recent studies have shown that gelatinase B also known as matrix metalloproteinase-9 (MMP-9), an endopeptidase capable of degrading the extracellular matrix, is thought to be associated with atherosclerosis, and plaque rupture [12, 13] . Therefore, MMP-9 is considered to be an important mediator of vascular remodeling and plaque instability. The MMP-9 action is enhanced b neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin-2, a 25 kDa glycoprotein, that is, found in the granules of human neutrophils, with many diverse functions, such as scavenger of bacterial products, modulator of inflammation, iron trafficking, and apoptosis [14] .
The formation of a complex with NGAL and MMP-9 is crucial for atherosclerotic plaque erosion and thrombus formation [15] . NGAL is also produced by kidney tubular cells in response to various ischemic or toxic insults and has 2 International Journal of Inflammation been proposed as an early biomarker for the diagnosis of acute kidney injury [16, 17] .
In this study, we hypothesized that levels NGAL in blood may reflect the extent of neutrophil activation in various stages of ACS and could discriminate various types of ACS (UA, NSTEMI, and STEMI) and stable from unstable coronary syndromes.
One hundred and seventy consecutive patients programmed for coronary angiography to the Invasive Cardiology Department of the KAT General Hospital Athens, Greece, were recruited for this study, from June 2010 to October 2010.
The study was performed according to the principles of the Declaration of Helsinki and was approved by the hospital's ethics committee. Written informed consent was obtained from all participating patients.
Thirty patients were excluded from the study. Exclusion criteria included a negative coronary angiography in patients with a typical chest pain which was considered as angina or had a false positive single photon emission computed tomography (SPECT), any surgery in the previous six months, liver disease, end stage renal disease, renal cardiac or liver transplantation, neoplasia, and infection since all these can affect serum-NGAL levels.
The 140 patients who fulfilled the study criteria after the clinical assessment and final diagnosis were divided into the following 4 groups: SA (n = 40), UA (n = 35), NSTEMI (n = 40), and STEMI (n = 25). Twenty (20) healthy amateur athletes without risk factors served as control group (Figure 1 ). The demographics and clinical characteristics of patients and controls are shown on Table 1 .
All patients, upon presentation in emergency room, underwent an initial clinical assessment that included clinical history, physical examination, 12-lead ECG, continuous ECG monitoring, and standard blood tests (including white blood cell, polymorphonuclear neutrophil counts, and troponin-I). These tests were repeated at 6 at 12 and 24 hours as long as clinically indicated. To determine the final diagnosis for each patient 2 cardiologists blinded to NGAL results reviewed all patients available records (including patient history, laboratory results, radiologic testing, ECG, echocardiography, and coronary angiography) at the completion of their hospital stay.
The SA group consisted of patients with angiographically documented organic coronary stenosis >70% by quantitative coronary angiography in major arteries who had chronic symptoms of angina or a positive SPECT test. UA was diagnosed in patients with typical angina at rest, or a sudden increase in episodes of a previously stable angina.
AMI was diagnosed when there was evidence of myocardial necrosis in a clinical setting consistent with myocardial ischemia. Necrosis was diagnosed by a rising and/or falling pattern of troponin-I with at least one value above the cutoff value (defined as the 99th percentile of a normal population where the assay shows an imprecision <10%). Our troponin-I assay fulfills the imprecision criteria for concentration >0.2 ng/mL.
Serum and K 2 EDTA-plasma samples were collected from all SA patients in the morning before the coronary angiography. In all ACS patients PCI was performed within 24 hours from admission and the blood samples were collected on admission. From all healthy subjects, samples were collected in the morning and before training. Serum samples were kept frozen at −80 • C until tested.
Total white blood cell count (WBC), and peripheral polymorphonuclear neutrophil count (PMN) were assessed using the Cell-Dyn Sapphire haematology analyzer (Abbott, Chicago, Il, USA). Serum creatinine was measured with a modified jaffe method on Architect ci16200 analyzer (Abbott, Chicago, Il, USA). High-sensitivity CRP (hs-CRP) was measured with a turbidimetric assay on the same analyzer. Troponin-I was measured with a chemiluminescent immunoassay on the same analyzer. Serum-NGAL was measured with an ELISA (Bioporto, Gentofte, Denmark).
This was performed with NCSS statistical program. Normality of distributions for quantitative data was tested with the Shapiro-Wilk test. For normally 
The mean age and the mean BMI of the patients did not differ significantly among the four groups whereas the controls were significantly younger and their BMI was significantly lower (ANOVA-test). The proportion of diabetic patients did not differ significantly among four patient groups (chisquare = 1.69, P = 0.639) as well as the proportion of patients with hypertension and dyslipidemia (chi-square = 1.63, P = 0.652). Finally smoking habits did not differ significantly in the first three patient groups while it was significantly higher in group 4. Those risk factors were absent from our controls. (50.31 ng/mL) ( Table 2 and Figure 2 ). Also significant were the differences observed between healthy controls and SA patients, between UA patients and patients with AMI (NSTEMI or STEMI). Patients with STEMI had higher levels of NGAL than patients with NSTEMI but the difference was nonsignificant ( Table 2) .
Markers. The median plasma levels of hs-CRP were similar in patients with SA (0.40 mg/dL) and those with UA (0.69 mg/dL) and were significantly higher than the levels in the control group (0.12 mg/dL). Hs-CRP levels were significantly increased in patients with NSTEMI (1.20 mg/dL) and STEMI (6.76 mg/dL). In order to further investigate the relationship between serum-NGAL and hs-CRP, we performed regression analysis between serum-NGAL and hs-CRP (Figure 3 ). This analysis revealed that there is a linear and positive correlation between hs-CRP and serum NGAL (spearman rank correlation coefficient rho = 0.685, P < 0.0001). The differences that were observed among the four patient groups in WBC and PMN counts were statistically significant (P < 0.001, ANOVA-test). There was a positive and significant correlation between serum-NGAL and WBC (r = 0.510, P < 0.0001) and PMN (r = 0.511, P < 0.0001) counts (Figure 4) .
In a multivariate regression analysis model entering as independent parameters age, serum creatinine, hs-CRP, and PMN count, we identified only hs-CRP (P < 0.005) and PMN count (P < 0.0001) as independent predictors of serum-NGAL levels. curve for serum-NGAL hs-CRP and PMN counts ( Figures  5 and 6 ). The diagnostic value for serum-NGAL in discriminating patients with UA, from those with SA is high (AUC = 0.852) and better than of hs-CRP (AUC = 0.735) or PMN count (AUC = 0.761). If we use as cutoff for serum-NGAL 83.74 ng/mL, we can predict an UA event with sensitivity and specificity, 82.8% and 75%, respectively. The negative predictive value of this cutoff is high (97.28%).
The diagnostic value for serum-NGAL in discriminating ACS patients, from patients with SA is high (AUC = 0.929) and better than of hs-CRP (AUC = 0.794) and PMN count (AUC = 0.830). If we use as cut-off for serum-NGAL 89.29 ng/mL, we can discriminate an ACS patient from a stable patient with sensitivity and specificity, 89.3% and 81.6%, respectively. The negative predictive value of this cutoff is high (98.65%).
In this study, we demonstrated that serum levels of NGAL are higher in patients with CAD than in healthy controls patients. Among ACS patients, these levels are gradually elevated according to the severity of the coronary clinical syndrome (UA, NSTEMI, and STEMI). Also serum levels of NGAL are higher in patients with ACS than in patients with SA and could be used, with high negative value, to discriminate patients with stable or unstable coronary syndromes. The relevance of NGAL to cardiovascular disease (CVD) remains primarily unknown. Elevated plasma NGAL levels were associated with atherosclerosis and were implicated as a predictor for cardiovascular mortality after cerebrovascular ischemia, possibly because of activation of blood leukocytes [18] [19] [20] . Although in recent reports has been shown that NGAL is present in atherosclerotic plaques and in human abdominal aortic aneurisms, raising the possibility that expression of NGAL can be induced in vascular cells during atherogenesis, the underlying mechanism for the induction of NGAL in vascular cells remains unknown [15, 21] . In further analysis the main source of NGAL was found to be neutrophils, probably recruited in the vascular wall by platelet activation [21] .
NGAL is considered to have a protective effect on MMP-9 and enhancing its proteolytic activity, could be considered as an important factor indirectly contributing to the progression of aneurism as well as involved in the physiologic and pathologic remodeling of vessel walls. This view is further supported by the observation that similar neutrophil NGAL/MMP-9 overexpression can be found in atherosclerotic plaques, particularly those with intramural haemorrhagic debris and central necrosis [15, 22] . The above evidence supports the clinical observations that highcirculating leucocyte (particularly neutrophil) counts are independent predictors of recurrent ischaemic attacks. This may be explained by their presence in the necrotic core of unstable plaques and by their proteolytic activity towards atherosclerotic tissue and secondary mobilization of thromboembolic fragments [23] . The evidence derived from these experimental studies, showing the close link between neutrophils, their products and the natural history of atherosclerosis, and its complications, generated clinical studies that investigated the clinical utility of serum-NGAL measurements.
In two recent studies it was found that serum levels of NGAL were significantly elevated in patients with angiographically confirmed CAD compared to those with normal arteries or controls [24, 25] . Our data agree with these reports since we found that levels of serum-NGAL are significantly higher in patients with all clinical syndromes of CAD than in healthy controls, reinforcing the utility of NGAL as biomarker of detection and the extent of CAD. The expression of NGAL from vascular cells during atherogenesis can also explain the differences between patients with SA and control subjects with no risk factors observed in our study. In addition to its induction in the vessels after mechanistic injury, previous studies suggest that NGAL is strongly upregulated in atherosclerotic lesions and also in the heart after ischemic injury [15] . It is possible that NGAL produced by vascular cells could also be secreted into the systemic circulation.
Inflammation plays a critical role not only in development and progression of atherosclerosis but also in pathogenesis of the destabilization of atherosclerotic plaque that leads to ACS [1, 26] . Activation and degranulation of polymorphonuclear neutrophils and probably an underestimated critical components of an acute coronary inflammation event. Infiltrating macrophages and neutrophils participate in the transformation of stable coronary artery plaques to unstable lesions with a thin fibrous cap [27] . It has been repeatedly reported that thrombosed plaques were densely infiltrated by neutrophils and macrophages [28, 29] . Macrophages and neutrophils and some other types of leukocytes produce various proteolytic enzymes which facilitate the rupture of plaques by thinning and weakening their normally thick and firm cap [30, 31] . NGAL is one protein, that is, produced not only by the distressed kidney but also by activated neutrophils and by the vascular wall cells. Recent studies have shown that neutrophils are the main source of NGAL in blood [32, 33] .
Increase in serum NGAL resulting from activation of neutrophils may reflect an acute systemic inflammatory response to events such as stroke, renal failure, or infection [18, [34] [35] [36] but are also linked with the presence of chronic inflammatory diseases such as atherosclerosis [18] whose acute clinical manifestations represent acute on chronic inflammation. Besides neutrophils, NGAL is also expressed by epithelial cells, renal tubular cells, and hepatocytes during inflammation or injury [37] [38] [39] . Our data agree with the above studies since we found a positive correlation between levels of serum-NGAL and systemic inflammation (expressed by the serum hs-CRP levels and neutrophil count), and also serum levels of NGAL were higher in patients with ACS than with SA. The higher levels of serum-NGAL observed in patients with ACS compared to SA could be explained by International Journal of Inflammation 7 the fact that neutrophil activation is present only in patients with acute coronary events (10, 11) . Also, our results, as far as patients with SA and AMI, are similar with the findings of a recent published study which showed that the plasma level of NGAL is higher in patients with AMI compared with the patients with stable CAD [40] .
In clinical practice, levels of serum-NGAL have a high negative predictive value, 97.28% and 98.65% for patients with UA and ACS, respectively. So, serum-NGAL could be used in discriminating of patients with ACS or especially UA from whom with SA or without CAD, giving the possibility to exclude patients with symptoms similar to angina but not having true ACS. As far as the gradual increase of serum-NGAL, according to the seriousness of unstable coronary clinical syndrome, this could reflect the intensity of the inflammatory reaction, as it is expressed by the incremental increase of hs-CRP and neutrophil count and their combination with serum NGAL. Especially between serum-NGAL and hs-CRP, the correlation is linear and positive.
In conclusion, our study shows that serum levels of NGAL increase in patients with CAD with every coronary clinical syndrome and reflect the inflammatory status in the same population. Having high negative predictive value could be used as a marker for the discrimination of SA or chest pain without CAD from those with ACS. Also in patients with ACS, serum levels of NGAL reflecting the inflammatory status could show the severity of coronary clinical syndrome (UA, NSTEMI, and STEMI).
Acute coronary syndrome CAD:
Coronary artery disease SA:
Stable angina UA:
Unstable angina AMI:
Acute myocardial infarction NGAL: Neutrophil gelatinase associated lipocalin NSTEMI: Non-ST-elevation myocardial infarction STEMI: ST-elevation myocardial infarction PCI:
Percutaneous coronary intervention. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used recently to investigate host–virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host–virus interaction. Most enzymes are tightly regulated post-translationally. Many enzymes are synthesized as zymogens, which are functionally inactive. Moreover, enzyme functions can be changed by alterations in pH and binding to inhibitors. Thus, methods that allow direct quantification of protein activities rather than simply protein abundance are required to delineate distinct protein functions in physiological and pathological events. Activity-based protein profiling (ABPP) is a chemoproteomic platform for monitoring active proteins or enzymes. ABPP utilizes chemical probes to interrogate the functional state of large numbers of enzymes in complex proteomes in vitro or in vivo biological systems. ABPP probes consist of two key elements: (1) a reactive group/warhead (e.g., small molecule inhibitors, substrate-based scaffolds, or protein-reactive molecules) for binding and covalently labeling the active sites of many members of a given enzyme class (or classes), and (2) a reporter tag for the detection, enrichment, and/or identification of labeled enzymes from proteomes. A variety of reporter tags are used in ABPP, such as fluorophores (e.g., rhodamine) for visualization, biotin for enrichment as well as "clickable" handles, such as azides and acetylenes for in vivo or in situ labeling of proteins. The linker region is a flexible chain of varying length and hydrophobicity that connects and acts as a spacer between the warhead and the reporter tag.
Serine hydrolases represent one of the largest and most diverse classes of enzymes in higher eukaryotes, collectively composing about 3% of the predicted Drosophila proteome (Rubin et al., 2000) and about 1% of all predicted expressed human genes (Lander et al., 2001) . Serine hydrolases are involved in a variety of physiological and pathological processes including blood coagulation (Kalafatis et al., 1997) , T cell cytotoxicity (Smyth et al., 1996) , inflammation (Bonventre et al., 1997) , neural plasticity (Yoshida and Shiosaka, 1999) , neurotransmitter catabolism (Taylor, 1991; Cravatt et al., 1996) , peptide/protein processing (Steiner, 1998) , protein/lipid digestion (Lowe, 1997) , angiogenesis (Mignatti and Rifkin, 1996) , emphysema (Kato, 1999) , and cancer (DeClerck et al., 1997) . Serine hydrolases also perform crucial functions in bacteria and viruses, where they contribute to pathogen life cycle (Steuber and Hilgenfeld, 2010) , virulence (White et al., 2011) , and drug resistance (Damblon et al., 1996) . Most enzymes hydrolyze metabolites, peptides or post-translational ester and thioester modifications on proteins. Because of the biological importance of serine hydrolases, clinically approved drugs target members of this enzyme class to treat diseases such as obesity (Henness and Perry, 2006) , diabetes (Thornberry and Weber, 2007) , microbial infections (Kluge and Petter, 2010) , and Alzheimer's disease (Racchi et al., 2004) .
Proteolytic cleavages of viral proteins by cellular or viral proteases are necessary for host cell attachment, invasion, and reproduction of viral progeny. Host serine proteases are essential for the influenza virus life cycle because the viral hemagglutinin is synthesized as a precursor which requires proteolytic maturation (Garten and Klenk, 2008) . Recently, the non-structural 3 protease (NS3 -is a chymotrypsin-like serine protease which requires a polypeptide cofactor NS2B for activation) has been shown to be responsible for cleavage of the viral polyprotein precursor and to play a pivotal role in the replication of flaviviruses (Falgout et al., 1991; Mukhopadhyay et al., 2005; Chappell et al., 2006) including Hepatitis C (HCV), West Nile virus, and Dengue virus. NS3 also facilitates viral pathogenicity by cleaving host proteins and down-regulating the innate immune response of the cell (Failla et al., 1994; Meylan et al., 2005) . In fact, site-directed mutagenesis www.frontiersin.org that focused on the NS3 cleavage sites in the polyprotein precursor abolishes viral infectivity (Chappell et al., 2006) . Cell culture models provided important clues about potential inhibition of several protease inhibitors against NS3 for Dengue virus and West Nile virus (Cregar-Hernandez et al., 2011; Steuer et al., 2011) . Clinical trials of NS3 serine protease inhibitors showed good success rates (Lee et al., 2012) as anti-HCV. Therefore, NS3 is one of the most promising targets for drug development against Flaviviridae infections (Kolykhalov et al., 2000; Chappell et al., 2008) . Other serine proteases involved in the pathogenesis and virus life cycles are being considered as targets for chemotherapy. The catalytic activity of the herpes simplex virus type 1 serine protease is essential for viral nucleocapsid formation and for viral replication (Gao et al., 1994) . A trypsin-like serine protease is involved in pseudorabies viral penetration of the basement membrane during mucosal invasion (Glorieux et al., 2011) . Serine protease inhibitors inhibit pseudorabies virus invasion in basal membranes. A vaccinia virus serine protease inhibitor prevents virus induced cell fusion (Law and Smith, 1992) . Serine protease inhibitor AEBSF and pAB significantly reduce influenza A virus replication in mouse models (Bahgat et al., 2011) . However, identification of active SHS and their functional characterization are necessary for better understanding the molecular pathogenesis and development of antiviral strategies.
All serine hydrolases possess a common catalytic mechanism that involves activation of a conserved serine nucleophile for attack on a substrate ester/thioester/amide bond to form an acylenzyme intermediate, followed by water-catalyzed hydrolysis of this intermediate to liberate the product. The greatly enhanced nucleophilicity of the catalytic serine renders it susceptible to covalent modification by many types of electrophiles, including fluorophosphonates (FPs) and aryl phosphonates, sulfonyl fluorides, and carbamates (Alexander and Cravatt, 2005; Jessani et al., 2005; Okerberg et al., 2005) . FPs are highly reactive and provide broad coverage, with the capacity to react with nearly all essential serine hydrolases (Bachovchin et al., 2010) . Therefore, they are ideal reagents to use for ABPP of serine hydrolases (Liu et al., 1999; Patricelli et al., 2001) . However, certain serine proteases displayed restricted substrate selectivities that reduce their labeling with FPs. To address this limitation of FPs, selective inhibitors (e.g., carbamates, triazole ureas) have been introduced to probe the function of individual serine hydrolase in biological systems (Bachovchin et al., 2010; Adibekian et al., 2011) .
Microarray technologies in the field of genomics (transcriptomics), and mass spectrometry and bioinformatics technologies in proteomics, have facilitated the specific and global analyses of genes and their expression, and this has accelerated understanding the molecular basis of disease. These technologies, coupled with two-dimensional gel electrophoresis, mass spectrometry enhanced with chromatographic separations such as MudPIT (Shaw et al., 2008) , or isotope coding-ICAT (Yan et al., 2004) , iTRAQ (Lu et al., 2012) , and SILAC (Coombs et al., 2010) , have provided valuable insight into the quantitative differences in protein abundance during virus infections. However, these methods lack the inherent ability to profile and distinguish proteins according to their actual biological activities or functional state, which has more important bearings on understanding the implications of these macromolecules in vivo (Barglow and Cravatt, 2007) . The lack of functional assessment of these other omic methods has prompted the development of alternative strategies such as ABPP, for the discovery and characterization of enzyme activities within highly complex biological samples.
A typical target discovery experiment would comparatively analyze two or more proteomes by ABPP to identify enzymes with differing levels of activity (Figure 1) . The differentially expressed serine hydrolases in healthy and diseased samples can be hypothesized to regulate the host-virus interaction. The testing of such hypotheses, of course, requires further experimentation for validation (e.g., functional interference of the target enzyme). ABPP has been used to profile a number of enzyme classes including proteases, hydrolases, oxidoreductases, and isomerases in the process of host-virus interaction Schlieker et al., 2005; Wang et al., 2006; Gredmark et al., 2007; Jarosinski et al., 2007; Shah et al., 2010) . Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme which has an important role in HCV propagation (Blais et al., 2010) . We have examined the activity of serine hydrolases during reovirus, Influenza A, and Sindbis virus replication in cell culture in different cell lines. Differential serine hydrolase activities were induced by different viruses and alterations of serine hydrolases were dependent on the time course of viral infection. Several of these differentially active serine hydrolases represent possible virus-host interactions that could be targeted for development of antivirals.
ABPP can also be used as a competitive screen to identify both reversible and irreversible enzyme inhibitors and also to confirm target inhibition because inhibitors have the ability to block probe labeling of enzymes (Kidd et al., 2001; Greenbaum et al., 2002; Leung et al., 2003; Adibekian et al., 2011) . Competitive ABPP has already led to the discovery of selective serine inhibitors (e.g., carbamates, trizole ureas) for several enzymes (e.g., peptidase, lipases), which have in turn been used to test the function of these proteins in living systems (Bachovchin et al., 2010; Adibekian et al., 2011 ). An alternative omic strategy would be to examine libraries of commercially available protease inhibitors for their ability to inhibit a virus' pathological process; this would potentially lead to development of novel therapeutic options.
Quantification of differentially expressed active proteins after virus infection is essential for better analysis of results, particularly when examining enzymes. It is difficult to compare the altered serine hydrolases between healthy and infected samples by simply visualizing gel images or merely by mass spectrometry. To address this problem an advanced quantitative mass spectrometry-based method called ABPP-SILAC (stable isotope labeling with amino acid in cell culture) has been used to identify alterations in the levels of active enzyme targets (Everley et al., 2007) and in small molecule-binding proteins in cell lysates (Ong et al., 2009) . Comparative ABPP -SILAC can be used to quantify more accurately the intricate changes in host proteins caused by viral infection. Similarly, competitive ABPP-SILAC is valuable to identify inhibited enzymes during global screening of inhibitors.
Many enzymes and metabolites display difficult physicochemical properties that complicate their analysis in biological samples, and many metabolic pathways that enzymes regulate in a disease-specific context are not understood. These challenges can be addressed by applying innovative metabolomics and ABPP approaches to mapping biochemical pathways that support disease. Using selective inhibitors developed through competitive ABPP efforts or RNA interference technology, the function of an enzyme of interest can be specifically blocked, and then the metabolites that the enzyme regulates can be profiled. In this manner, not only can the substrates and products of an enzyme in specific (patho)physiological contexts be examined, but also the metabolic networks that the enzyme regulates can be identified and annotated. Collectively, this platform will allow identification of novel biochemical roles of already characterized enzymes, or may allow the identification of metabolic roles of completely uncharacterized enzymes.
Understanding the mechanisms by which viruses develop resistance is a vital component of the fight against viral diseases, and can lengthen the lifespan of existing antivirals. Potentially any antivirus molecule could be transformed into an activity-based or affinity-based probe, allowing isolation and characterization of enzymes that detoxify the antiviral drug.
ABPP with live cell imaging may provide additional insight into understanding the pathogenesis due to viral infection (Furman et al., 2009) . Identification and functional characterization of serine hydrolases involved in pathogenesis and virulence of viruses would be a novel approach to uncover molecular processes at the basis of viral diseases.
Natural products represent an important treasure box of biologically active molecules, from which many drug candidates have been developed (Newman and Cragg, 2007) . Since a large number of the proteome remains functionally uncharacterized and is therefore difficult to assemble into larger biochemical networks, competitive ABPP will inevitably accelerate the development of novel inhibitors from natural products.
This mini review describes briefly a limited number of approaches involved in profiling serine hydrolases during viral infection and assigning catalytic functions to previously uncharacterized serine hydrolases. Visualization of the altered active serine hydrolase in situ during viral disease progression, trying to fully understand mechanisms of resistance and developing new antiviral therapeutics and viral diagnostics will make the ABPP application more worthwhile for the field of virology. www.frontiersin.org Murphy's law—if anything can go wrong, it will: Problems in phage electron microscopy The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles. Legend has it that Murphy's law was formulated in 1949 by a Captain Edward A. Murphy, then at Edwards Air Force Base in California. 15 There are several variants, all meant to express a perverse outcome. Murphy's law certainly applies to bacteriophage electron microscopy.
This type of investigation is a multistep procedure that depends on expensive and complicated instruments, refined techniques, bacteriophages, and, not least, the investigator. Problems and errors beset every step. Artifacts in particular have received little attention and will be the focus of this article.
Negative staining of viruses, arguably the technically simplest and most important single method in virology, was introduced in 1956. Hall and later Brenner and Horne used phosphotungstic acid for contrasting plant viruses. 8, 12 This technique was extended in 1959 to coliphage T2. 9 Viruses and their components stood out as white on a dark background with unprecedented clarity. Negative staining by phosphotungstates rapidly superseeded shadowing for virus visualization. Uranyl salts and ammonium molybdate were introduced later. The standards of viral electron microscopy were set in the 1970s. 11, 20, 21 Today, negative staining is done almost exclusively with uranyl acetate (UA) and phosphotungstate (PT) salts (Figs. 1 and 2) and has been applied to thousands of viruses. At present, at least 6,300 bacterial and archaeal viruses have been examined in the electron microscope after negative staining. 6 Transmission electron microsocpes (TEMs) fall into three categories. (1) Conventional or "manual," also called "analogue" microscopes using mechanical devices for stage drive and aperture alignment and analog potentiometers or variable resistors for electronic controls. Images are recorded on photographic film or plates. (2) "Digital" microscopes using far fewer controls due to a computer-based system with digital potentiometers, electronic stage drives, and a digital alignment via a centralized computer interface. Objects are generally visualized on a monitor screen and images are acquired via a "chargedcouple device" or CCD sensor, replacing film or plates as the recording media.
(3) Hybrids or conventional microscopes equipped with a digital camera.
Since about 1985, "manual" electron microscopes were gradually replaced by "digital" instruments. These instruments were marketed almost simultanously by three major competing companies that did so with meager or no instructions relating to contrast and image quality. An additional modification by microscope manufacturers was to change the focal length of the objective pole piece (zeta or Z angle) to increase fidelity at the cost of reduced contrast. All these factors could explain a wave of poor electron microscopical images 1 which, seemingly, has not abated. Unfortunately, this wave coincided with the death of several famous and highly skilled electron microscopists, such as E. Kellenberger in Switzerland, D.E.
Bradley in Canada, and A.S. Tikhonenko in Russia, who could have stemmed the tide.
To assess this problem, one of us (HWA) analyzed 155 publications with phage micrographs originating in 28 countries, published between 2007 and January 2012 in over 50 journals. Twothirds (109) were of poor quality, namely contrastless, unsharp, astigmatic, of small size and low magnification (below 150,000¾). Only 46 could be considered as good or acceptable. The adjectives "poor and good" are somewhat subjective and we apologize for this. The micrographs had been obtained with a wide selection of about 50 types or models of electron microscopes, most of which were "digital." The vast majority of articles presented several pictures. "Manual" microscopes were apparently disappearing fast. Clearly, poor electron microscopy is a global problem.
Electron microscopes. The most frequent types of transmission electron microscopes (TEMs) used in phage research are produced by JEOL (Japanese Electron Optical Laboratories), Hitachi and FEI, a successor of Philips (Eindhoven, The Netherlands), followed distantly by Carl Zeiss, Germany. One also finds a few old AEM (England) and Tesla (Czechia) instruments. The prevalence of JEOL and Hitachi instruments may be attributed to their relatively low cost. The most popular types are the JEOL 1200 EX, the Hitachi H-1700, and three instruments of the Philips family (FEI CM 100, Technai Spirit or Morgagni). A few "manual" microscopes, for example the Philips EM 300, persist and seem to be, because of their high quality, the objects of a cult among inconditionals of electron microscopy. FEI-Tecnai electron microscopes appear to be the most highly evolved TEMs and have indeed produced excellent micrographs, whereas, for example, the JEOL 1200 is often associated with poor pictures. This impression is superficial. Indeed, good and poor pictures have been produced with JEOL, Hitachi and Philips-FEI instruments. In conclusion, electron microscopes are above all "operator-sensitive." Poor pictures must generally be attributed to poor maintenance and untrained users. There is no cure but know-how.
The dirty picture. Quite often, specimens are not purified in any way and investigators examine crude lysates. This is almost certain to go wrong. Crude lysates contain huge amounts of impurities, notably proteins (Figs. S4 and S19). This results in contrastless, flat images and the presence of bacterial debris and even complete bacteria. The debris may mimic certain types of bacteriophages ( Table 2) . Images of nonpurified specimens should not be accepted for publication. Purification can be achieved by centrifugation in sucrose or CsCl density gradients; unfortunately, this technique is limited to a small number of specialized laboratories. Instead, we recommend to sediment phages in Eppendorf tubes followed by two washes in buffer or even tap water. Using fixed-angle rotors, phages can be sedimented in as little as 1 h at 25,000 g. It is not necessary to stain phages for long minutes or to fix them. Staining is almost instantaneous and fixation is merely a complication. While purification is mandatory for any real investigation, crude lysates can be examined for quick checks of phage presence and identity. It is also possible to examine phages extracted from a lysed agar surface. 23 For this, a drop of staining solution is deposited on the agar and the Petri dish is agitated gently. The drop is then touched with a grid and excess liquid is drawn off.
The technically deficient picture. The root causes of unsharp, "fuzzy" images (Figs. S5 and S6) are poor microscope maintenance and misalignment of column components. Both focusing and astigmatism correction can go wrong and produce unsharp images. Much has been said in manuals of electron microscopy (e.g., in ref. 7) about focusing and astigmatism and this does not need to be repeated. Digital microscopes allow the investigator to use the FFT (Fast fourier transform) function as a measure of astigmatism (Fig. 3) . This function is available in FEI, Hitachi, and JEOL instruments and, for example, the widespread AMT, Gatan, and OlympusSis cameras. If fuzziness and astigmatism persist, the electron microscope should be checked by the installer. Test specimens for resolution checks and conventional astigmatism correction ("holey" grids) are commercially available and a must for the serious microscopist. Unsharp images are generated by:
(1) Misalignment of the whole EM column and/or the objective aperture.
(2) Object drift due to a specimen support film that breaks, does not adhere to the grid, or has not been stabilized with carbon, thus causing the the substrate to charge and float.
(3) Discharges in the electron gun altering the focus.
(4) Over-or under-focusing by the user (Fig. S7) .
(5) Astigmatism, generally caused by a dirty object holder or objective lens diaphragm, resulting in fuzziness and fine parallel lines instead the normal grain of micrographs (Fig. S8) .
Contrast. Poor contrast (Figs. S5 and S6) seems to be a pervasive, if not the main problem of "digital" electron microscopy (EM). Indeed, many "digital" micrographs are lamentably dark and poorly contrasted. This is not an intrinsic limitation of "digital" microscopes or cameras, but rather due to inappropriate parameters or complete misunderstanding of the dynamic signal range during image acquisition.
In "manual" instruments, poor contrast can be overcome by apertures, highcontrast films, or elongating the focal distance of the objective lens, for example in the old Siemens Elmiskop I. Finally, contrast can be improved in the darkroom by means of fast developers, polycontrast papers and optical filters. Unfortunately, this is very difficult with "digital" electron microscopes where contrast must be adjusted before or after image acquisition. CCD digital technology offers a wide range of gray scale values. They can be selected to favor the mid-tonal range, thus reducing the extreme dark and white values. For example, a 12-bit image comprises 4,096 levels from black to white. If the software is set to adjust the predominant intensity to a middle tonality, all values will be adjusted accordingly. The mid-tones typically yield lowcontrast images. Contrast levels should therefore be set to exclude a given percentage of black and white outliers. After image acquisition, the histogram camera software allows for adjustments of gamma (a function of luminance), contrast, and brightness. 19 The observer is left to find his or her best combination by trial and error. Contrast may also be improved by third-party software (e.g., Adobe Photoshop or Image J; http// rsbweb.nih.gov/ij/). This seems often to be misunderstood or ignored. Poorly contrasted digital images should be a thing of the past.
Farming out. The high cost of electron microscopes and the specialized knowledge needed to operate them generated a questionable, even damnable development: the "farming out" of investigations to central laboratories or institutions carrying out examinations for a fee. Anything might go wrong during this procedure. It might be acceptable if phage researchers themselves have access to the electron microscope used or the technician that carries our investigations, is properly qualified or backed by an experienced phage researcher. Unfortunately, this seems to be rarely the case and has resulted in situations when examinations are performed by incompetents without instructions or personal interest in the subject, or simply unused to magnifications above 200.000¾. The investigating laboratories sometimes charge outlandish fees for essentially worthless data. This should be resisted. We propose fees of $50 US for access to an electron microscope and $75 US for an investigation aided by a technician or a scientist.
The journals. Murphy's law also rules the final step of publication. Indeed, a good micrograph can be ruined by a journal that reduces it to postage stamp size or darkens it beyond recognition. Part of this may be attributed to the now frequent procedure of outsourcing manuscript assembly to distant offices, e.g., in South East Asia. Only protests will help here and sometimes do.
Positive staining. This is the most frequent artifact in virus electron microscopy. Uranyl salts cause both negative and positive staining, while phosphotungstates and molybdates cause negative staining only ( Table 1) . The principal incriminated stain is uranyl acetate (UA). In positive staining (Figs. S9 and S13), virus particles themselves are stained and then may appear black on a white background. This is due to the strong affinity of uranyl ions to dsDNA 13 and seen in any viruses with compact masses of DNA, e.g., phage heads and adeno-or herpes-viruses. It never occurs in filamentous or ssRNA phages. Positively stained phage heads are invariably shrunken by about 10-15% 5, 16 and show neither capsomers nor transverse edges. Phage tails are not stained and appear as shadows. Consequently, these viruses can rarely be identified by EM. Their dimensions are perfectly useless and should not be published. If positive staining is generally an undesirable artifact, it has however two important applications. It allows (A) the visualization of phage heads in sectioned bacteria and (B) facilitates the quantification of aquatic viruses. 4 The reasons for positive staining are unknown. Both negative and positive staining may occur in adjacent areas of the same EM grid (Figs. S10, S12 and S13). The addition of protein to UA solutions seems to alleviate the incidence of positive staining.
Halo formation. UA positive staining is often accompanied a gray halo around the virus capsid that increases with the time of irradiation (Figs. S12 and S13). The halo has unsharp margins and resembles an envelope. Its origin and significance are unknown.
UA crystals and precipitates. UA tends to crystallize on the grid, often starting with viruses as crystallization origin. Crystals come in many varieties. Some are small (Fig. S14) and feathery or appear as long black needles. Others are flat, large sheets (Figs. S15 and S16). One also observes, albeit rarely, membranaceous precipitates around phage particles. Viruses within crystals often appear as brilliant white structures of abnormally large size (Fig. S17) .
Swollen proteins. As an acidic stain (pH 4 to 4.5), UA causes proteinic structures to swell. 5 Compared with PT, the walls of UA-stained empty phage capsids and tails appear as relatively thick and less sharply defined. Typically, contractile tails are 2 nm larger in UA than in PT. Tail length is not affected. On the other hand, UA acts as a fixative and preservative, so that UA-stained specimens can be kept over years. Phage heads are better preserved in UA than in PT.
Artifacts caused by phosphotungstate. Surprisingly, these are few. Compared with UA, and depending on the phage, heads tend to be rounded and are sometimes broken and empty.
From the medium. Unwashed preparations contain proteins, sugars, and cell debris (below). They yield dirty, poorly contrasted, even opaque images with little structural details (Fig. S4) . PEG (polyethylene glycol), which is frequently used to concentrate phages, produces similar effects. PEG will collect anything: DNA, proteins, cell debris, phages and phage debris. Fortunately, PEG is is easily removed by washing. In one recent case, four washes were needed to obtain a clean preparation. CsCl, used for phage purification, may persist after incomplete dialysis. CsCl forms flat crystals, but does not interfere with staining. Arborescent or flat NaCl crystals are frequent in lysates from halophilic bacteria which require NaCl for growth, e.g., Vibrio and Halobacterium. Again, bacteriophages may act as nuclei of crystallization (Fig. S18) . NaCl crystals may obscure phage particles, but do not alter the quality of staining. In lysates from bacteria prepared with meatbased media, e.g., of clostridia, one may observe occasional fibers of striated muscle and bundles of double-walled rings which resemble phage tails (Fig. S41) and may be be attributed to self-assembly under the action of phospholipase C. 3 From bacteria (Table 2 ). Bacteria contribute proteins, DNA, ribosomes, cytoplasm, capsular material, pili, flagella, and fragments of plasma membrane and cell wall. Proteins and cytoplasm may interfere with the staining process, but are not a source of error. However, this is the case with the other cellular constituents that one may find in a lysate. Their presence depends very much on the phage host. For example, slime and capsular material are present in huge amounts in lysates of Acinetobacter and Xanthomonas and cell wall debris abound in those of Pseudomonas.
Slime and capsular material normally appear as rounded elements, but can be distorted by centrifugation and then superficially resemble filamentous viruses (Inoviridae) (Figs. S20 and S21) or even tailed phages (Fig. S27) . Similarly, pili and debris of flagella may be mistaken as filamentous phages and short debris of flagella, of 100 to 200 nm length, may be confused with contractile phage tails. Occasionally, a fragment of a pilus still attached to a piece of plasma membrane may suggest the presence of a tailed phage. These elements should not be much of a problem as they are easily identified at magnifications above 150,000¾.
Chloroform, sometimes used for sterilization of lysates, is dangerous as it may produce a thick smear of particles thought to be lipopolysaccharide (LPS). This goo interferes with staining and can make observations impossible. The amount of smear probably depends on the bacterium and seems to be particularly high in lysates of Brucella spp and Cronobacter sakazakii (Fig. S22) .
Cell wall and plasma membrane fragments (Figs. S23 and S26) may be misdiagnosed as enveloped pleomorphic viruses, novel viruses, or tailless phages. This error is easily explained. Upon bacteriophage lysis, bacteria fall into pieces, many of which are round and have indeed the size of plasmaviruses or cystoviruses (70-80 nm). The latter are characterized by an envelope surrounding an isometric capsid. When superposed over each other, some cellular elements exhibit what seems to be external and internal (Fig. S25) . Cell wall fragments carrying an adsorbed phage tail may be taken at low magnification for tailed phages (Fig. S26) . These membrane fragments are much more than an inconsequential nuisance because phage counts in seawater and freshwater are increasingly often based on fluorescent microscopy. 19 As it happens, bacterial DNA can associate with membrane fragments during lysis 18 and will stain with fluorescent dies. The fluorescent membrane fragments are approximately of the size of phage heads and appear as tiny green dots simulating phages. So far, investigations of phage prevalence in the environment seem to have neglected this potentially serious source of errors. 18 From Bacteriophages (Tables 2 and 3) Bacterial viruses produce a wide variety of abnormal particles ( Table 3) . Nearly all of them have been observed in tailed phages, although the filamentous inoviruses sometimes give rise to doublelength particles. Tailed phages are classified into three families according to tail structure: Myoviridae with contractile tails, Siphoviridae with long, noncontractile tails and Podoviridae with short tails. Nature and frequency of "freaks" or "monsters" vary with the complexity of phages. Some aberrations have a genetic basis, others reflect errors of assembly, and still others result from propagating phages and their hosts on the wrong substrate, e.g., a medium containing amino acid analogs. 10 The subject has been reviewed elsewhere in some detail. 2 Phage T4 and its relatives have a particular propensity to produce aberrant particles. Some sources of error (and Mr Murphy's and the electron microscopist's delight) are:
(1) Abnormally long tails (Fig. S28 ). They are found in very numerous siphoviruses, are extremely rare in myoviruses, and have never been reported in podoviruses. Normal tails may appear short when the head partly covers the tail (Figs. S29 and S30).
(2) Isolated contracted tails, which have been interpreted by inexperienced observers as complete tailed phages and even novel species of viruses. (4) Proheads, recognizable by their small size and wavy outline (Fig. S33) , may be mistaken as complete isometric viruses or phage debris.
(5) Mottled heads and polyheads (Figs. S34 and S35); they seem products of faulty phage synthesis.
(6) Freak particles with two tails, two tail sheaths (Figs. S36 and S37) or even two heads. The latter were observed in Lactococcus lactis phages of the c2 species (not shown).
(7) Myoviruses mimicking as siphoviruses after loss of their tail sheath (Fig. S38) .
(8) Head-size variants (Fig. S40) , notably in phages with prolate heads www.landesbioscience.com Bacteriophage producing particles with short or isometric capsids. (9) Broken tails, suggesting the presence of podoviruses or isometric phages instead of sipho-or myo-viruses. 22 (10) Virus-like particles (Figs. S41 and S43) and true, but deformed phages (Fig. S44) .
The presence of abnormal or damaged particles indicates a contamination by other phages or the presence of lysogenic phages produced by the phage host. The observer has to decide whether there are different phage populations or aberrant particles, e.g., a spectrum of phage tails of different length. The latter indicates the presence of a malformation. The matter requires prolonged observation at magnifications above 150,000¾.
Exact magnification depends first on the adjustments made on installing an electron microscope. It must be controlled later by means of test specimens (e.g., beef liver catalase crystals 13 or T4 phage tails) because magnification may change over time and at every repair. Latex crystals and diffraction grating replicas are for low magnification only and to be rejected. In "manual" TEMs, magnification is easily and rapidly adjusted in the darkroom by means of a photographical enlarger. In "digital" TEMs, magnification can be controlled, if necessary, by calculation of correction factors. This potentially tedious procedure seems to be very unpopular with today's phage electron microscopists. Indeed, magnification control is seldom mentioned in recent phage descriptions and one suspects that it is rarely done. As a result, particle dimensions from "digital" TEMs sometimes appear as products of fantasy.
Lastly, electron microscopy depends heavily on the quality of observations and interpretations. 19 For example, a common error is to call every phage head with a hexagonal outline an icosahedron although, geometrically speaking, it could also be an octahedron or a dodecahedron ( Table 2 ). The past five years have generated scores of strange publications. Some investigators of soil phages saw novel viruses in every round or oval particle or isolated tail sheath, others interpreted particles with contracted and extended tails of the same Bacillus myovirus as members of different species, and still others confused negative and positive staining or myo-, sipho-and podo-viruses. This denotes a decline in the general knowledge of viruses and of bacteriophages in particular. Misdiagnosis is particularly frequent in the interpretation of natural samples (water, soil and feces) because these may contain almost anything: algal, plant and vertebrate viruses, muscle fibers, abiogenic material, and of course the omnipresent bacterial debris of any kind. Ultimately, the human factor is the root cause of most problems: failure to maintain, repair and align an electron microscope; failure to focus properly and to correct astigmatism; improper or no specimen preparation; failure to recognize artifacts and fake viruses; absence of magnification control; finally failure to consult the now very abundant literature on electron microscopy in general and that on phages in particular. This translates as "everything that can go wrong, will." Fortunately, there are reasons to be optimistic as any man-made problems can be corrected by humans.
Supplemental materials may be found here: www.landesbioscience.com/journals/ bacteriophage/article/20693 Herbal Products: Benefits, Limits, and Applications in Chronic Liver Disease Complementary and alternative medicine soughts and encompasses a wide range of approaches; its use begun in ancient China at the time of Xia dynasty and in India during the Vedic period, but thanks to its long-lasting curative effect, easy availability, natural way of healing, and poor side-effects it is gaining importance throughout the world in clinical practice. We conducted a review describing the effects and the limits of using herbal products in chronic liver disease, focusing our attention on those most known, such as quercetin or curcumin. We tried to describe their pharmacokinetics, biological properties, and their beneficial effects (as antioxidant role) in metabolic, alcoholic, and viral hepatitis (considering that oxidative stress is the common pathway of chronic liver diseases of different etiology). The main limit of applicability of CAM comes from the lacking of randomized, placebo-controlled clinical trials giving a real proof of efficacy of those products, so that anecdotal success and personal experience are frequently the driving force for acceptance of CAM in the population. Complementary and alternative medicine (CAM) therapies sought and encompass a wide range of approaches, including two broad categories: exogenous chemicals such as herbal supplements, vitamins, or plant extract, and natural or selftherapies (NST) techniques including relaxation, meditation, prayer, hypnosis, biofeedback, or physical strengthening [1] .
The use of herbal medicine began in ancient China at the time of Xia dynasty and in India during the Vedic period [2] . With the revolution of the natural sciences and evidence-based medicine, the divide between Western and Eastern medicines appeared to widen, with CAM reaching an increasing popularity in western countries through years (from 34% of the population in 1990 to 48% in 2004) [3] .
The age-old system of herbal medicine is being revived by day-to-day practice for its long-lasting curative effect, easy availability, natural way of healing, and less side-effects, so that today herbal medicines are gaining importance and expanding throughout the world [4] .
The widespread use of CAM is emphasized among people with chronic disease, since it promotes greater personal control over health decision, empowers people to manage their chronic condition, and helps to avoid dissatisfaction often associated with conventional health care [5] .
CAM is believed to be safer and better than standard medical practice because they are "natural" or are based on a religious, philosophical or a strongly felt concept of "wellness" and health. Treatments with herbal medicine concentrate on reestablishing or reinforcing natural healing processes and wellness [6] .
Despite increasing popularity, communication about the use of CAM between physicians and patients is limited: most physicians know little about CAM and patients avoid discussing CAM because they fear being received with indifference [7] .
Moreover, physicians used to focus attention on potential toxicities, even though identification of toxicity from herbal preparations is often difficult, because patients generally selfmedicate with these and may withhold this information. 
Toxic hepatitis is the most common adverse reaction resulting from the use of CAM [8] , often associated with the concomitant consumption of hepatotoxic ingredients such as acetaminophen and nonsteroidal anti-inflammatory agents or with hepatotoxicity of herbal ingredients themselves [9] .
Physicians and health care providers need to become familiar with these products and to recognize potential interaction between conventional drugs and herbals, considering their actual diffusion [10] .
Botanical medicines have been used traditionally by herbalists and indigenous healers worldwide for the prevention and treatment of liver disease. Clinical research in this century has confirmed the efficacy of several plants in the treatment of liver disease, so the fact that the patients with chronic liver disease seek primary or adjunctive herbal treatment is not surprising.
Particularly, silymarin (an extract of milk thistle) is the most popular product taken by subjects with liver disease and especially by those with hepatitis C virus infection [11] . Seeff et al. [12] found that 41% of outpatients with diagnosis of liver disease had used some form of CAM. Herbal products are often used to improve well-being and quality of life [13] and to ameliorate side effects in patients on antiviral treatment, as fatigue, irritability, and depression: lessening of these symptoms might permit a higher compliance and avoid the need to limit the dose and finally withdraw inter-feron.
A systematic review about the use of CAM in chronic hepatitis C has been conducted by Coon and Ernst [14] , The authors cited fourteen randomized clinical trials considering the combined use of herbal products and interferon-alfa during antiviral treatment. Although difficulty in extrapolation and interpretation of results because of different methodological limits of the considered studies, the authors found that several herbal products and supplements (vitamin E, thymic extract, zinc, traditional Chinese medicine, Glycyrrhiza glabra, and oxymatrine) could exert potential virological and biochemical effects in the treatment of chronic hepatitis C infection, as a greater clearance of HCV-RNA and normalization of liver enzymes.
As shown in various studies [15, 16] , the use of CAM could be predicted by social, cultural, and geographic factors: sex, age, higher education level, or marriage status of patients are associated with a different use of herbal products.
The aim of this study is to describe the potential role, benefits, and limits of some of known widespread herbal products in chronic liver disease. We conducted an updated research on Pubmed and Medline in order to refer to more recent articles about this issue.
Quercetin is one of the major flavonoids, which represent a class of naturally occurring polyphenolic compounds, ubiquitously present in photosynthesising cells. The intake of flavones and flavonols is determined as 23-24 mg/day and quercetin, the main flavonol present in our diet, represents 70% of this intake. Quercetin is found in fruits (apple) and vegetables, especially onions [17] .
Various ways of supplementing quercetin are possible, including a pure supplement or a diet intervention using a food component with a high quercetin content. Supplement usually contains only the aglycon form of quercetin, whereas a food component normally comprises high amounts of various quercetin derivatives that might have a better biological availability than the aglycon itself. Another advantage of a dietary supplementation versus a "conventional" supplement might be a better compliance, especially in long-term use [18] .
The absorption of quercetin is considerably enhanced by its conjugation with a sugar group. After their facilitated uptake by means of carrier-mediated transport, quercetin glycosides often become hydrolysed by intracellular βglucosidases. After absorption, quercetin becomes metabolized in various organs including the small intestine, colon, liver, and kidney. Metabolites formed in the small intestine and liver are mainly the result of phase II metabolism by biotransformation enzymes and, therefore, include the methylated, sulphated, and glucuronidated forms. Moreover, bacterial ring fission of the aglycon occurs in both small intestine and colon, resulting in the breakdown of the backbone structure of quercetin and the subsequent formation of smaller phenolics [19] .
Quercetin appears to have many beneficial effects on human health, including cardiovascular protection, anticancer activity, antiulcer and antiallergy activity, cataract prevention, and anti-inflammatory effects. Quercetin has been shown to be an excellent in vitro antioxidant. Within the flavonoid family, it is the most potent scavenger of ROS (reactive oxygen species), including O 2 [20] .
The pathogenesis and progression of ALD are associated with free radical injury and oxidative stress, which could be partially attenuated by antioxidants and free radical scavengers. Lipid metabolism disorder and oxidative stress play an important role on the development and progression of ALD, and mitochondria compartment is presumed to be the main source and susceptible target of intracellular ROS. The hypothesis that quercetin could prevent ethanolinduced oxidative damage in hepatocytes has been investigated.
In animal studies, prophylaxis with quercetinameliorated ethanol-stimulated mitochondrial dysfunction manifested by decreased membrane potential and by induced permeability transition though suppressing glutathione depletion, enzymatic inactivation of manganese superoxide dismutase, and glutathione peroxidase, ROS overgeneration, and lipid peroxidation in mitochondria. Quercetin, thus, may protect rat, especially hepatic mitochondria, from chronic ethanol toxicity through its hypolipidemic effect and antioxidative role, highlighting a promising preventive strategy for ALD by naturally occurring phytochemicals [21, 22] .
Evidence-Based Complementary and Alternative Medicine 3 Quercetin tends to downregulate the ethanol-induced expression of glutathionine peroxidase 4 (GPX4). Furthermore, it tends to reduce the expression of SOD2 induced by ethanol, to downregulate the expression of Gadd45b at the presence of ethanol, which could permit to explain DNA demethylation associated with the upregulation of gene expression in experimental ALD [23] .
Another study evaluated the effect of quercetin on the parameters classically associated with alcohol liver injury, as lactate dehydrogenase (LDH), aspartate transaminase (AST), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in order to address the alterations of cell damage and antioxidant state after quercetin intervention; the ethanol-intoxicated (100 mM for 8 h) rat primary hepatocytes were simultaneously treated, pretreated (2 h) and posttreated (2 h) with quercetin. The toxic insult of ethanol on hepatocytes was challenged by quercetin and biochemical parameters almost returned to the level of control group when hepatocytes were treated with quercetin at the dose of 50muM for 2-4 h before ethanol exposure [24] .
A recent study elucidates also a neuroprotective effect of quercetin in alcohol-induced neuropathy through modulation of membrane-bound inorganic phosphate enzyme and inhibition of release of oxidoinflammatory mediators, such as malondialdehyde (MDA), myeloperoxidase (MPO) MPO, and nitric oxide (NO) [25] .
In conclusion, pretreatment with quercetin provided protection against ethanol-induced oxidative stress in hepatocytes and may be used as a new natural drug for the prevention and/or treatment of ALD. Antioxidants significantly reduced the oxidative stress induced by ethanol intoxication, increased membrane integrity, and also increased organ regeneration [26] .
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States. It represents the hepatic manifestation of metabolic derangements, known as metabolic syndrome, with insulin resistance playing the major role. Nonalcoholic fatty liver disease includes a variety of histological conditions (ranging from liver steatosis and steatohepatitis, to fibrosis and hepatocarcinoma), all characterized by an increased accumulation/deposition of fat within the liver and associated with alterations in hepatic and systemic inflammatory state. Hepatocytes of a primary cell culture that are exposed to high glucose, insulin, and linoleic acid concentration respond with lipid accumulation, oxidative stress up to cell death.
Regarding the role of quercetin in NAFLD, it has been show that in mice fed with a Western diet chronic dietary intake of quercetin reduces liver fat accumulation and improves systemic parameters related to metabolic syndrome, probably mainly through decreasing oxidative stress and reducing expression of genes related to steatosis (as PPARα) [27] .
Another study was aimed to examine the hypoglycemic and insulin-sensitizing capacity of onion peel extract (OPE, containing a high content of quercetin) in high fat diet/streptozotocin-induced diabetic rats and to elucidate the mechanism of its insulin-sensitizing effect. OPE might improve glucose response and insulin resistance associated with type-2 diabetes by alleviating metabolic dysregulation of free fatty acids, by suppressing oxidative stress, upregulating glucose uptake at peripheral tissues, and/or downregulating inflammatory gene expression in liver. Moreover, in most cases, OPE showed greater potency than pure quercetin equivalent. These findings provide a basis for the use of onion peel to improve insulin insensitivity in type-2 diabetes [28] .
In hepatocytes from normal rats, the decrease in de novo fatty acid and TAG synthesis induced by quercetin represent a potential mechanism contributing to the reported hypotriacylglycerolemic effect of this agent [29] .
The hepatic response to chronic noxious stimuli may lead to liver fibrosis and to preneoplastic cirrhotic liver. Fibrogenic cells activate in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin is an epidermal growth factor receptor (EGFR) ligand, specifically induced upon liver injury. Recent study investigated the effects of quercetin on the amphiregulin/EGFR signal and on the activation of downstream pathways leading to cell growth: quercetin-ameliorated activation of survival pathways and downregulated the expression of genes related to inflammation and precancerous conditions. Suppression of amphiregulin/EGFR signals may contribute to this effect [30] .
Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection: these aspects were investigated by several studies.
Park et al. [31] investigated about the antiviral activity in HCV-infected patients of derivates of 7-O-arylmethylquercetins. Only five quercetin derivatives showed selective antiviral activity in HCV replicon cell-based assay.
Recent study studied the quercetin as a potential nontoxic anti-HCV agent in reducing viral production by inhibiting both NS3 and heat shock proteins (that are essential for HCV replication). It was found that quercetin inhibit NS3 activity in a specific dose-dependent manner in vitro catalysis assay. Moreover, as analyzed in the subgenomic HCV RNA replicon system, quercetin seemed to exert adjunctive effect: to inhibit HCV RNA replication and production in the HCV infectious cell culture system (HCVcc), as analyzed by the focusforming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3-engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to NS3 and does not involve other HCV proteins. This work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease [32] . (9) showed anti-HBV activity in vitro. Anti-HBV activity was closely related to the parent structure of these compounds (agigenin > luteolin > quercetin) as well as to the number of glucoside (flavone monoglucoside > flavone diglucoside). The structure of these agent also influences their cytotoxicity (flavone > flavone monoglucoside > flavone diglucoside). In addition, the substitution of acyl group on glucoside may be important to keep their anti-HBV activities (galloyl > feruloyl > coumaroyl) [33] . Quercetin did not show activities against HBeAg secretion: this limit might be due to the absence of the saccharide group in their structures [34] .
Curcumin is a low-molecular-weight polyphenol derived from the rhizomes of turmeric (curcuma longa). It represents a yellow pigment widely used as a coloring agent and spice in many foods. It has various beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic, hypocholesterolemic, antibacterial, antispasmodic, anticoagulant, and hepatoprotective activities [35] .
Phase I clinical trials have shown that curcumin is safe even at high doses (12 g/day) in humans but exhibit poor bioavailability. Despite the promising biological effects of curcumin, low plasma and tissue levels of curcumin due to its poor oral bioavailability and absorption, rapid metabolism and systemic elimination in both rodents and humans [36] may be responsible for the unfavorable pharmacokinetic of this molecule. To improve the bioavailability of curcumin, numerous approaches have been undertaken. These approaches involve, first, the use of adjuvant like piperine that interferes with glucuronidation; second, the use of liposomal curcumin; third, curcumin nanoparticles; fourth, the use of curcumin phospholipid complex; fifth, the use of structural analogues of curcumin (e.g., EF-24) [35, 36] .
Liver fi-brosis can be explained with an increased deposition of extracellular matrix (ECM). Chronic alcohol abuse is one of the main causes of liver fibrosis. Ingestion of polyunsaturated fatty acids (PUFAs) together with alcohol can aggravate the toxicity of alcohol. The degree of abnormal ECM degradation depends on the ratio of active matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). A recent work studied the influence of bis-desmethoxy curcumin analog (BDMC-A) on the expression of MMPs and TIMPs during alcohol and DeltaPUFA-induced liver toxicity. Administration of BDMC-A significantly decreased the levels of collagen and TIMPs and positively modulated the expression of MMPs. From this study, we can conclude that BDMC-A influences MMPs, TIMPs expression and that it acts as an efficient anti-fibrotic agent [37] .
It has been demonstrated the potential protective effect of curcumin pretreatment against ethanol-induced hepatocytes oxidative damage, with emphasis on heme oxygenase-1 (HO-1) induction. Ethanol exposure resulted in a sustained malondialdehyde (MDA) elevation, glutathione (GSH) depletion, and evident release of cellular lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), which was significantly ameliorated by curcumin pretreatment. In addition, dose-and time-dependent induction of HO-1 was involved in such hepatoprotective effects by curcumin. Curcumin exerts hepatoprotective properties against ethanol involving HO-1 induction, which provide new insights into the pharmacological targets of curcumin in the prevention of alcoholic liver disease [38] .
To study the mechanism of curcumin-attenuated inflammation and liver pathology in early stage of alcoholic liver disease, female Sprague-Dawley rats were divided into four groups and treated with ethanol or curcumin via an intragastric tube for 4 weeks. A control group was treated with distilled water and an ethanol group was treated with ethanol (7.5 g/kg bw). Treatment groups were fed with ethanol supplemented with curcumin (400 or 1 200 mg/kg bw). The liver histopathology in ethanol group revealed mild-to-moderate steatosis and mild necroinflammation. Hepatic MDA, hepatocyte apoptosis, and NF-kappaB activation increased significantly in ethanol-treated group when compared with control. Curcumin treatments resulted in improving of liver pathology, decreasing the elevation of hepatic MDA, and inhibition of NF-kappaB activation. The 400 mg/kg bw of curcumin treatment also revealed a trend of decreased hepatocyte apoptosis. However, the results of SOD activity, PPARgamma protein expression showed no difference among the groups. In conclusion, curcumin improved liver histopathology in early stage of ethanol-induced liver injury by reduction of oxidative stress and inhibition of NF-kappaB activation [39] .
In animal study, it has been demonstrated the capacity of curcumin to improve liver histology of NAFLD. The NASH model was induced by highfat diet combined with carbon tetrachloride. These rats were successively treated with curcumin and curcumin derivative. The results showed a remarkable reduction in serum ALT (U/L), AST (U/L) in rats treated with curcumin derivatives. The degrees of fibrosis were also significantly alleviated. The reduction of the gene transcriptions of TNF-alpha, NF-kappaB, and HMG-CoA was the mechanism proposed by wich curcumin exercise its beneficial effects in NASH.
The results of this study indicate, moreover, that the watersoluble curcumin derivative displays superior bioavailability to the parent curcumin, which can effectively improve the lipid metabolism and delay the progression of hepatic fibrosis in rats with steatohepatitis [40] .
Interestingly, it also demonstrates that the activating effect of LDL can be reversed by curcumin. The hypocholesterolemic action of curcumin was reported by numerous studies: curcumin seems to reduce serum cholesterol concentrations by increasing hepatic expression of LDL receptors, by blocking LDL oxidation, increasing bile acid secretion, and faecal excretion of cholesterol, repressing the expression of genes involved in cholesterol biosynthesis and protecting against liver injury and fibrogenesis [41, 42] . Curcumin induces apoptosis and blocks proliferation of hepatic stellate cells (HSCs), and, via activation of PPARγ, inhibits extracellular matrix formation, downregulates the expression of LDL receptors, induces SREBP-1c,and increases the fat-storing capacity of HSC, and it may thereby restore this "protective" functionality, proving of therapeutic usage in preventing liver steatosis and fibrosis [43] .
An Italian study tested whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. They demonstrated that curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I, and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of alpha-smooth muscle actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. This study so proposed that curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells [44] .
High consumption of dietary fructose is an important contributory factor in the development of hepatic steatosis in insulin or leptin resistance. The effects of curcumin on fructose-induced hypertriglyceridemia and liver steatosis and its preventive mechanisms in rats have been investigated. Curcumin reduced serum insulin and leptin levels in fructose-fed rats and it protects against fructose-induced hypertriglyceridemia and hepatic steatosis by inhibition of PTP1B (hepatic protein tyrosine phosphatase 1B) and subsequent improvement of insulin and leptin sensitivity in the liver of rats. This PTP1B inhibitory property may represent a promising role for curcumin to treat fructoseinduced hepatic steatosis induced by hepatic insulin and leptin resistance [45, 46] .
Curcumin is known to exert antiviral activity against influenza virus, adenovirus, coxsackievirus, and the human immunodeficiency virus [47, 48] . However, it remains to be determined whether curcumin can inhibit the replication of hepatitis C virus (HCV). A study showed that curcumin decreases HCV gene expression via suppression of the Akt-SREBP-1 activation, not by NF-kappaB pathway. The combination of curcumin and IFN-alpha exerted profound inhibitory effects on HCV replication. These results indicate that curcumin can suppress HCV replication in vitro and may be potentially useful as novel anti-HCV reagents [49] .
Hepatitis B virus (HBV) infects the liver and uses its cell host for gene expression and propagation. Therefore, since targeting host factors is essential for HBV gene expression, it could represent a potential anti-viral strategy.
Curcumin treatment could complement the antiviral activity of the nucleotide/nucleoside analogues, which are considered as the gold standards for anti-HBV therapy. The combination of Lamivudine and curcumin treatments resulted in an enhanced suppression of HBV expression by up to 75%, as compared to nontreated cells. These results suggest that curcumin may work synergistically with the current anti-HBV nucleotide/nucleoside analogous, and that this combination may result in a better suppression of HBV. Moreover, curcumin inhibits HBV gene expression and replication, by downregulating PGC-1alpha, a starvationinduced protein that initiates the gluconeogenesis cascade and that has been shown to robustly coactivate HBV transcription [50, 51] .
Silybum marianum, also known as milk thistle, is a member of Asteraceae family and is well recognized as a hepatoprotective herbal medicine. Silymarin is a lipophilic extract of the milk thistle seeds. It is composed of three isomers of flavonolignans (silybin, silydianin, and silychristin), and two flavonoids (taxifolin and quercetin).
Silymarin has revealed poor absorption, rapid metabolism, and ultimately poor oral bioavailability. For optimum silymarin bioavailability, issues of solubility, permeability, metabolism, and excretion must be addressed. An array of methods have been described in recent years that can improve its bioavailability, including complexation with βcyclodextrins, solid dispersion method, formation of microparticles and nanoparticles, self-microemulsifying drug delivery systems, micelles, liposomes, and phytosomes [52] .
Silymarin possesses various pharmacological activities, including hepatoprotective, antioxidant, anti-inflammatory, anticancer, and cardioprotective effects. Silybum marianum is the most well-researched plant in the treatment of liver diseases. Silymarin has been shown to have a variety of anti-inflammatory effects on liver, including mast cell stabilization, inhibition of neutrophil migration, and Kupffer cell inhibition [53] . Silymarin is commonly prescribed in cases of cirrhosis or viral hepatitis.
Hepatocyte models were proposed as a platform for screening of herbal 6 Evidence-Based Complementary and Alternative Medicine component against ethanol hepatotoxicity. Nanosilibinin, for the first time, found to perform significant protection against ethanol-induced hepatotoxicity while silibinin in normal particles could not inhibit such toxicity. This protection of nanosilibinin might be related to its high bioavailability compared to normal insoluble silibinin and could act as an antioxidative and antisteatosis agent against ethanol-induced hepatotoxicity [54] .
The affect of silymarin on the levels of serum ALT and GGT in ethanol-induced hepatotoxicity in albino rats has also been tested. Eighteen male albino rats aged 6-8 weeks, weighing 150-200 gm, were divided into 3 groups of 6 rats each: group A as control, group B as rats taking ethanol at a dose of 0.6 mL (0.5 gm)/100 gm/day, and group C taking ethanol and silymarin at a dose of 0.5 gm/100 gm/day and 20 mg/100 gm/day, respectively, for 8 weeks. Silymarin tends to normalize liver function test in alcoholic liver disease [55] .
Acute ethanol administration caused prominent hepatic microvesicular steatosis with mild necrosis and an elevation of serum ALT activity, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation (oxidative stress) and increased hepatic TNF (necrosis factor-alpha) production. Supplementation with a standardized silymarin with its both antioxidant and antiinflammation properties decreases TNF production and attenuated these adverse changes induced by acute ethanol administration. In view of its nontoxic nature, it may be developed as an effective therapeutic agent for alcoholinduced liver disease by its antioxidative stress and antiinflammatory features [56] .
Silymarin showed a significant hypocholesterolemic effect compared to the diet model with high fat-diet (HFD). Moreover, silymarin significantly reduces TG levels compared to HFD group.
The elevation of transaminases usually reflects necrosis of hepatocytes: with silymarin, ALT levels (a specific index of hepatic necrosis) were particularly reduced [57] .
Assuming that oxidative stress leads to chronic liver damage, Loguercio et al. conducted a study about the antioxidant activity of silybin conjugated with vitamin E and phospholipids. Eighty-five patients were divided into 2 groups: those affected by nonalcoholic fatty liver disease (group A) and those with HCV-related chronic hepatitis associated with nonalcoholic fatty liver disease (group B), nonresponders to antiviral treatment. The treatment consisted of silybin/vitamin E/phospholipids. After treatment, group A showed a significant reduction in ultrasonographic scores for liver steatosis. Liver enzyme levels, hyperinsulinemia, and indexes of liver fibrosis showed an improvement in treated individuals. A significant correlation among indexes of fibrosis, body mass index, insulinemia, plasma levels of transforming growth factorbeta, tumor necrosis factor-alpha, degree of steatosis, and gamma-glutamyl transpeptidase was observed. Our data suggest that silybin conjugated with vitamin E and phospholipids could be used as a complementary approach in the treatment of patients with chronic liver damage [58] .
Silymarin showed antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin also blocked cell-to-cell spread of virus [59] .
Pegylated interferon (PEG-IFN) plus Ribavirin therapy is the current treatment for the patient with chronic hepatitis C. The main goal of therapy is to achieve a sustained virological response (SVR is defined as undetectable HCV-RNA in peripheral blood determined with the most sensitive polymerase chain reaction technique 24 weeks after the end of treatment). This goal is practically equivalent with eradication of HCV infection and cure of the underlying HCV-induced liver disease. This therapy is effective only in half of patients, because of important side-effects, resistance, and high cost related to therapy. Silymarin inhibits both HCV RNA (in a dose-dependent manner) and HCV core expression thanks to its direct effect against HCV 3a core or activation of JAK/STAT pathways, resulting in inhibition of HCV core gene, by phosphorylation of Stat1 on tyrosine and serine [60] .
Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b. Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission by targeting the host cell [60, 61] .
In another study, patients with chronic hepatitis C performing silymarin (650 mg/day) for 6 months improved serum HCV-RNA titer, serum aminotransferases (ALT, AST), hepatic fibrosis, and patient's quality of life. After the treatment, nine patients were found with negative HCV-RNA and statistically significant improvement in results of liver fibrosis markers was found only in fibrosis group.
So, for its antioxidant and anti-inflammatory actions, sylimarin could result useful in reducing hepatic inflammation in chronic liver disease, including HCV-related damage. It has been hypothesized that decreased hepatic inflammation-due to both direct and indirect effects of silymarin in decreasing viral replication has the potential to induce long-term benefit to the infected liver [62] .
Since oxidative stress may play a pathogenetic role in chronic hepatitis C, and sustained virological response to antiviral therapy is limited in HCV1 genotype infection, a double-blind study was performed in HCV1 patients treated with pegylated interferon + ribavirin in order to assess the Evidence-Based Complementary and Alternative Medicine 7 efficacy of supplementation with the antioxidant flavonoid silymarin. In the silymarin group, a more rapid decrease in the malondialdehyde level as well as a marked decrease in superoxide dismutase and an increase in myeloperoxidase activity after twelve months of treatment were found. In particular, alanine aminotransferase normalized in 6/16 (versus control 9/16) cases and sustained virological response occurred in 3/16 (versus 7/16) patients [63] .
Few recent data discuss about the role of silymarin in the hepatitis B. We only reported silymarin beneficial effects in early stages of liver pathogenesis, in preventing and delaying liver carcinogenesis.
This drug should be considered as a potential chemopreventive agent for HBV-related hepatocarcinogenesis [64] .
Betaine is a naturally occurring dietary compound that is also synthesized in vivo from choline. In vivo, betaine acts as a methyl donor for the conversion of homocysteine to methionine and its also functions as an osmolyte.
Chronic ethanol exposure has been shown both to decrease hepatic concentrations of Sadenosylmethionine (SAM) and plasma concentrations of folate in animal and human studies and to increase plasma concentrations of homocysteine and hepatic levels of Sadenosylhomocysteine (SAH) [65] .
In the liver, betaine can transfer its methyl group to homocysteine in order to form methionine. This can result in decreased concentrations of homocysteine and increased concentrations of methionine in the liver, resulting in decreased hepatic concentrations of SAH, whereas the latter can increase hepatic SAM concentrations, which leads to an increased SAM : SAH. An elevated SAM : SAH can trigger a cascade of events leading to formation of proper VLDL, to export of triacylglycerol and attenuation of fatty liver. Increased hepatic concentrations of SAM can activate cystathionine-synthase and lead to upregulation of the transsulfuration pathway, to increase synthesis of glutathione and attenuate oxidative stress. Thus, betaine can ameliorate ALD by attenuating fatty liver, inflammation, and fibrosis [66] .
The role of mitochondrial dysfunction in the pathogenesis of alcoholic liver disease has been long documented by multiple laboratories. The dietary supplementation with betaine protects against ethanol-induced loss in oxidative phosphorylation system proteins. Even if the exact mechanism for this protection at the organelle level is not known, betaine shows to preserve the function of the electron transport chain, to maintain the integrity of the liver, and to protect against the development of alcoholic liver injury by preventing NOS 2 induction and NO generation [67] . Moreover, specific changes are associated with the normalization of hepatic SAM : SAH ratio and maintenance of methylation potential in response to betaine supplementation during chronic ethanol ingestion.
Chronic alcohol administration increases gut-derived endotoxin in the portal circulation, activating Kupffer cells to produce several proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1.
Ethanol administration can also lead to the synthesis of Toll-like receptor 4 (TLR4) protein and its gene expression in Kupffer cells, indicating that TLR4 may play a major role in the development of alcohol-induced liver injury. The intragastric ethanol-fed rat model, which reproduces the pathological features of early alcohol-induced liver injury, was used to observe the changes of TLR4 expression and the effect of betaine in alcohol-induced liver injury animal models.
It has been suggested that betaine can prevent alcoholinduced liver injury effectively and improve liver function. The hepatoprotective mechanism of betaine is probably related to the inhibition of endotoxin/TLR4 signaling pathways. In rats with alcohol-induced liver injury, betaine feeding can decrease the levels of serum ALT, AST, endotoxin, TNF-α, IFN-γ, and IL-18, reduce the expressions of TLR4, and improve the degree of hepatic steatosis and inflammation in liver tissues.
In summary, the results of this study show that the expression of TLR4 increased significantly in ethanol-fed rats. Betaine administration can inhibit TLR4 expression, which may be the mechanism of protection from alcoholic liver injury exerted by betaine [68] .
The role of betaine in the treatment of NASH has been evaluated in human studies. Oral administration of betaine glucuronate in NASH patients for 8 weeks reduced both hepatic steatosis by 25% and hepatomegaly by 8%, and it significantly attenuated serum concentrations of AST, ALT, and glutamyl transferase. Similarly, a marked improvement in the degree of steatosis, necroinflammatory grade, and stage of fibrosis was obtained after treatment with betaine [69] .
Nonalcoholic fatty liver disease (NAFLD) is a common liver disease, associated with insulin resistance. Betaine treatment would prevent or treat NAFLD in mice. Betaine reduces fasting glucose, insulin, triglycerides, and hepatic fat in mice submitted to a moderate high-fat diet (MHF). Betaine significantly improved insulin resistance and hepatic steatosis. Betaine treatment reversed the inhibition of hepatic insulin signaling in mHF and in insulin-resistant HepG2 cells, including normalization of insulin receptor substrate 1 (IRS1) phosphorylation and of signaling pathways for gluconeogenesis and glycogen synthesis. We can conclude that betaine treatment prevents and treats fatty liver in a moderate high-dietary-fat model of NAFL D in mice [70] .
Moreover, betaine supplementation alleviated hepatic pathological changes, which were concomitant with attenuated insulin resistance (as shown by improved homeostasis model assessment of basal insulin resistance values and glucose tolerance test) and corrected abnormal adipokine productions (adiponectin, resistin, and leptin). Specifically, 8 Evidence-Based Complementary and Alternative Medicine betaine supplementation enhanced insulin sensitivity in adipose tissue as shown by improved extracellular signalregulated kinases 1/2 and protein kinase B activations. In adipocytes, freshly isolated from mice fed a high-fat diet, pretreatment of betaine enhanced the insulin signaling pathway and improved adipokine productions. Further investigation using whole liver tissues revealed that betaine supplementation alleviated high-fat diet-induced endoplasmic reticulum stress response in adipose tissue as shown by attenuated glucose-regulated protein 78/C/EBP homologous protein (CHOP) protein abundance and c-Jun NH2-terminal kinase activation [71] .
Song et al. showed that betaine significantly attenuated hepatic steatosis induced by high-sucrose diet (animal model), and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver [72] .
The cause of failure of antiviral treatment with standard therapy, that is, pegylated interferon alpha (pegIFNalpha) combined with ribavirin, in half patients, is unknown, but viral interference with IFNalpha signal transduction through the Jak-STAT pathway could be considered. The expression of HCV proteins leads to an impairment of Jak-STAT signaling because of the inhibition of STAT1 methylation. Unmethylated STAT1 is less active since it can be bound and inactivated by its inhibitor, protein inhibitor of activated STAT1 (PIAS1). The treatment of cells with S-adenosyl-L-methionine (AdoMet) and betaine could restore STAT1 methylation and improve IFN alpha signaling. Furthermore, the antiviral effect of IFNalpha in cell culture could be significantly enhanced by the addition of AdoMet and betaine. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells expressing hepatitis C virus (HCV) proteins and enhance the inhibitory effect of IFNα on HCV replicons. SAMe and betaine were found to be safe when used with pegIFNα/ribavirin [73] .
In conclusion, the addition of these drugs to antiviral standard therapy for patients with chronic hepatitis C could overcome the problem of drug resistance [74] .
Homocysteine, a sulfuric amino acid involved in methionine metabolism, belongs to the group of intracellular thiols. Hyperhomocysteinemia is frequent in the Caucasian and its role in vascular pathology has been clearly established. In hepatology, experimental data in transgenic mice deficient in homocysteine metabolism enzymes have shown the presence of severe liver steatosis with occasional steatohepatitis. In chronic hepatitis C, preliminary data have shown that hyperhomocysteinemia is an independent risk factor for steatosis or even fibrosis. The physiopathological mechanism has now begun to be better understood. On one hand, there is a strong correlation between homocysteine and insulin resistance whatever its etiology. On the other hand, homocysteine has a direct effect on the liver, resulting in overexpression of SREBP-1 and favoring steatosis. It stimulates proinflammatory cytokine secretion such as NF kappa B increasing the risk of NASH. Finally, homocysteine could increase the risk of fibrosis by stimulating TIMP 1. Moreover, hepatitis C virus induces hypomethylation of STAT 1 and could decrease the antiviral activity of interferon. Results from in vitro studies have shown that the normalization of STAT 1 methylation by bringing betaine and S Adenosyl Methionine (which belongs to homocysteine cycle) restores the antiviral activity of interferon. Finally, treatment of hyperhomocysteinemia could have favorable consequences in steatohepatites and HCV infection [75, 76] .
Only few data exist about the role of betaine in histological or clinical effects of hepatitis-B-virusinfected patients.
Glycyrrhiza glabra (licorice root), a perennial herb cultivated in temperate and subtropical regions of the world and native to Mediterranean region as well as to central and South-Western Asia, belongs to the Leguminosae family, genus Glycyrrhiza [77] . The aqueous extract of this plant contains Glycyrrizin (GL), a conjugate of two molecules of glucuronic acid and one of 18β-glycyrrhetic acid (GA) [78] and other substances as flavonoids, hydroxycoumarins and beta-sitosterols [79] .
Stronger neominophagen C (SNMC) is a product used in Japan for the treatment of acute and chronic hepatitis. This solution is administered intravenously, 80-200 mg/day, for variable periods of time, and contains 0.2% glycyrrhizin, 0.1% cysteine, and 2% glycine in physiological solution. In the United States, glycyrrhizin is available in a multiplicity of nonstandardized oral formulations found over the country [80, 81] .
GL injected i.v., is partially metabolized to 3-monoglucuronyl-glycyrrhetinic acid (3MGA) in the liver by lysosomal β-D-glucuronidase, and GL and 3MGA could be excreted with bile. The biliary-excreted GL and 3MGA are hydrolyzed by intestinal bacteria into GA, which is reabsorbed into the bloodstream. Orally administered GL is enzymatically hydrolyzed to GA by intestinal bacterial flora before absorption into the bloodstream. The circulated GA is further metabolized to 3MGA in the liver by UDPglucuronyl transferase and then excreted with bile into the intestine [82] . The pharmacokinetic characteristics of i.v. administration of GL in patients with liver disease was studied in a Japanese and European report: SNMC has linear pharmacokinetics up to 200 mg, and steady state in achieved after two weeks of 200 mg doses administrated six times per week [83] .
Potential interactions may occur with drugs metabolized by CYP450 3A4, although those have not been reported to date [80, 81] .
Decreases of potassium, sodium retention, worsening of ascites, and hypertension are possible adverse effects due to the 11-hydroxy-steroid dehydrogenase inhibitory activity of GL and GA [84] . However, published data show no increased rate of these side-effects during treatment although documentation of toxicity is poor in most reports [77] .
The use of GL in acute and chronic hepatitis is due to its hepatoprotective, immunomodulatory, and antiinflammatory effect. It reduced ischemia/reperfusion (I/R-) induced liver injury [85] and inhibited hight-mobility group box 1 (HMGB1), an inflammatory cytokine that acted in inflammation and organ damage in hepatic I/R-injury [85, 86] .
Many studies shown that GL attenuated inflammatory responses due to decreased activation of nuclear factor kB (NF-kB) and mitogen-activated protein kinase (MAPK) pathways [87] , moreover, inhibited the production of LPSinduced nitric oxide (NO) and tumor necrosis factor-α (TNF-α), prostaglandin E2, intracellular reactive oxygen species (ROS), proinflammatory interleukins as IL-4, IL-5, IL-6, IL-18, IL-1β [88] [89] [90] [91] [92] , and increased the production of anti-infiammatory interleukins as IL-12 and IL-10 [92] .
In animal studies, GL inhibits CD4+ T-cell and tumor necrosis factor-(TNF-) mediated cytotoxicity [93] , activated NK cells and extrathymic T lymphocyte differentiation [94, 95] , and promoted maturation of dendritic cells [92] .
GL inhibited serum AST and ALT levels and histologically inhibited the infiltration of inflammatory cells and the spreading of degenerative areas of hepatocytes in an animal model of concanavalin A-induced liver injury [96] .
Many studies showed GL have antiviral activity (reviewed in [97] ). A mechanism proposed for explain this propriety is the membrane stabilizing effect, as demonstrated in rat hepatocytes incubated with antibody raised against rat liver cell membranes: rat hepatocytes released AST after incubation with antiliver cell antibody in the presence of complement, and the endogenous phospholipase A2 activity was increased, but glycyrrhizin suppressed phospholipase A2 activity and reduced transaminase level [98] . A more recent study confirms this propriety in HIV and Influenza A virus [99] .
Despite a precedent review [100] evidenced that SNMC acts as an antiinflammatory or cytoprotective drug but does not have antiviral properties, a recent in vitro study found that GL inhibit HCV full-length viral particles and HCV core gene expression or function in a dose-dependent manner and had synergistic effect with interferon [101] and a European randomized trial showed biochemical effects of 26-week treatment with SNMC in patients with chronic hepatitis C [102] .
Glycyrrhizin-modified glycosylation and blocked sialylation of hepatitis B surface antigen (HBsAg) [103] . An in vitro study, measuring the release of surface protein (HBsAg) and HBV-DNA in transfected HepG2 2.2.15 cells, showed that this compound had a moderate ability in reducing viral production [104] .
Long-term clinical trials in Japan and The Netherlands demonstrate that interferon nonresponder patients with chronic hepatitis C and fibrosis stage 3 or 4 have a reduced incidence rate of HCC after glycyrrhizin therapy normalizes ALT levels [105, 106] . Other well-diagnosed studies are needed to better define the role of GL in HBV and HCVrelated liver disease.
Steatosis. 18 beta-glycyrrhetinic acid (18α-GL) can suppress the activation of hepatic stellate cells (HCSs) and induce their apoptosis by blocking the translocation of NF-kappaB into the nucleus, furthermore, it promoted the proliferation of hepatocytes in rats with CCl4-induced liver fibrosis [107] . GA inhibited type I collagen synthesis and progression of liver fibrosis probably through the suppression of collagen gene (COL1A2) promoter [108] . In transgenic mice expressing the HCV polyprotein fed an excess iron diet, SNMC prevented hepatic steatosis: this product attenuated ultrastructural alterations of mitochondria of the liver, activated mitochondrial β-oxidation with increased expression of carnitine palmitoyl transferase I and decreased the production of reactive oxygen species.
Wu et al. found that 18α-GL, the biologically active metabolite of GL, prevented FFA-induced lipid accumulation and cell apoptosis in in vitro HepG2 NAFLD models and also prevented high-fat-diet-induced hepatic lipotoxicity and liver injury in vivo rat NAFLD models. GA stabilized lysosomal membranes, inhibited cathepsin B expression and enzyme activity, inhibited mitochondrial cytochrome c release, and reduced FFA-induced oxidative stress [109] .
A recent review revealed that triterpenoids, which are also found in Glycyzirra glabra extract, had antitumor activities. Triterpenoids could induce apoptosis in various cancer cells by activating various proapoptotic signaling cascades. The molecular mechanisms involved include inhibition of various oncogenic and antiapoptotic signaling pathways and suppression or nuclear translocation of transcription factors including NF-κB [110] . In a human hepatoma cell line, the expression of junB mRNA, a tumor suppressor gene, and JUNG protein is highly increased by GL treatment [111] . Zhao et al. studied the β-Cyclodextrin/glycyrrhizic acid functionalized quantum dots (β-CD/GA-functionalized QDs [112] , and found that this drug has proapoptotic effects in hepatocarcinoma cells. β-CD/GA-functionalized QDs triggered G0/G1 phase arrest and induced apoptosis through an reactive oxygen species mediated mitochondrial dysfunction pathway.
7.1. Definition, Pharmacokinetics, and Biological Aspects. The genus Phyllantus (Euphorbiaceae) consist of about 6500 species in 300 genera, of which 200 are American, 100 African, 70 from Madagascar, and the remaining are Asian and Australian [113] . Many species are used in traditional medicine mainly in India and China to treat several diseases [114] and a morphological analysis of samples of Phyllanthus used in raw drug trade in southern India shown that 76% of the market samples contained P. amarus as the predominant species (>95%) and other five different species, namely, P. debilis, P. fraternus, P. urinaria, P. maderaspatensis, and P. kozhikodianus, were found in the remaining 24% of the shops [115] .
P. amarus has been widely studied because it is the most commonly used in the Indian Ayurvedic medicine in the treatment of gastrointestinal and genitourinary diseases. Its main active components are ligans, as phyllanthin and hypophyllanthin, and flavonoids, alkaloids, hydrolysable tannins, polyphenols, triterperens, sterols, and volatile oil [113] .
Many animal studies evidenced the hepatoprotective activity of P. amarus. The extract enhanced liver and serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), acid phosphatase (ACP), glutathione-S transferase (GST). Furthermore lipid peroxidation level was significantly reduced in ethanol and CCl4-induced liver disease animal models [116] [117] [118] [119] . The administration of P. amarus extract significantly decreased the levels of collagen and tissue inhibitors of matrix metalloproteinases (TIMPs) and positively modulated the expression of matrix metalloproteinases (MMPs) in rats with alcohol and thermally oxidized polyunsaturated fatty acid-(PUFA-) induced hepatic fibrosis [120] . In mice with aflatoxin B1-induced liver damage, P. amarus extract lowered down the content of thiobarbituric acid reactive substances (TBARSs) and enhanced the reduced glutathione level and the activities of antioxidant enzymes, glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) [121] .
In a recent animal study, it was found a synergistic effect between silymarin and P. amarus, especially with ethanoic extract of P. amarus, due to its higher concentration of phyllanthin in comparison to aqueous extract against CClinduced nepatotoxicity [122] .
The hepatoprotective action was studied also in other Phyllanthus species, as P. simplex [123] , P. atropurpureus [124] , P. acidus [125, 126] , P. fraternus [127] , P. emblica [128] , P. urinaria, and P. maderaspatensis [129, 130] , and significant antioxidant and anti-inflammatory activities were found especially in P. simplex extracts because of its high phenolic content [123] . Finally, the antioxidant activity was compared between P. virgatus and P. amarus and was found that the first had higher cytotoxicity, higher free radical scavenging activity, and more inhibition of peroxidation capacity [131] .
Preclinical studies have shown that Emblica officinalis (P. emblica) protect against ethanol-induced hepatotoxicity (reviewed in [132] ). Animal studies showed that the fruit extract improved plasma enzymes level, reduced lipid peroxidation, and restored the enzymatic and nonenzymatic antioxidants level in alcohol-induced liver disease, this action is probably due to tannoid, flavonoid and NO scavenging compounds present in the extract [133] [134] [135] . Phyllanthin restored the antioxidant capability of rat hepatocytes including level of total glutathione, and activities of superoxide dismutase (SOD) and glutathione reductase (GR) which were reduced by ethanol [136] and P. amarus-modified alcohol and thermally oxidized PUFA-induced fibrosis in rats because decreased the levels of collagen and TIMPs and positively modulated the expression of MMPs [120] .
7.3. Phyllanthus in HCV and HBV Infection. P. amarus extract was used, in 1988, in a preliminary study involving 37 patients with chronic hepatitis B. 22 of 37 treated patients had cleared the virus 2-3 weeks after the end of the treatment period and only one of 23 placebo treated became HBsAg negative [137] . The mechanism of action appears to be related to the suppressive effect of Phyllanthus extract on HBsAg secretion and HBsAg mRNA expression [138] and the inhibition of hepatitis B virus polymerase activity [139] . A recent study isolated a polyphenolic compound, 1,2,4,6tetra-O-galloyl-β-D-glucose (1246TGG), from P. emblica, and found that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant of HepG2.2.15 cells [140] . The role of Phyllanthus spp. in the treatment of chronic hepatitis B was studied in several reports that were evaluated in a recent Cochrane review. The authors included a total of 16 randomized trials but only one compared Phyllanthus with placebo and found no significant difference in HBeAg seroconversion after the end of treatment or followup. Fifteen trials compared Phyllanthus plus an antiviral drug like interferon alpha, lamivudine, adefovir dipivoxil, thymosin, vidarabine, or conventional treatment with the same antiviral drug alone and found that the combined treatment affect serum HBV DNA, serum HBeAg, and HBeAg seroconversion. The authors conclude that Phyllanthus in combination with an antiviral drug may be better than the same antiviral drug alone but clinical trials with large sample size and low risk of bias are needed to confirm these findings [141] .
The metanoic extracts of P. amarus root and leaf are also recently studied for the treatment of chronic hepatitis C and the root extract showed significant inhibition of HCV-NS3 protease enzyme; whereas the leaf extract showed considerable inhibition of NS5B in the in vitro assays. Both extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. Furthermore, addition of root extract together with IFN-α showed additive effect in the inhibition of HCV RNA replication [142] .
P. emblica and P. urinaria are Phyllanthus species most studied for cancer treatment.
Water extract of P. urinaria induces apoptosis by DNA fragmentation and increased caspase-3 activity, reduces the viability of numerous cancer cells lines probably by telomerase suppression activity, and reduces the angiogenesis as suppressing MMP-2 secretion and inhibiting MMP-2 activity through zinc chelation [143] .
P. emblica and some of its phytochemicals such as gallic acid, ellagic acid, pyrogallol, some norsesquiterpenoids, corilagin, geraniin, elaeocarpusin, and prodelphinidins B1 and B2 possess antineoplastic effects. It also possess other properties that are efficacious in the treatment and prevention of cancer as radiomodulatory, chemomodulatory, chemopreventive effects, free radical scavenging, antioxidant, anti-inflammatory, antimutagenic, and immunomodulatory activities [144] .
In liver, P. emblica and P. urinaria inhibited HepG2 cell growth and five other cancer cell lines [145, 146] . Progallin A isolated from the acetic ether part of the leaves inhibited the proliferation of BEL-7404 cells, upregulated Bax, and downregulated Bcl-2 expression [147] . Defatted methanolic fruit extract of P. emblica suppressed carcinogen-induced response in rat liver with diethylnitrosamine-induced hepatocarcinoma [148] .
Liv.52 is an Ayurvedic medicine that was used for 50 years in in the prevention and treatment of viral hepatitis, alcoholic liver disease, early cirrhosis, and a variety of conditions as protein energy malnutrition, loss of appetite, and others. It is composed by Capparis spinosa, Cichorium intybus, Mandur bhamsa, Solarium nigrum, Terminalia arjuna, Cassia occidentalis, Achillea millefolium, and Tamarix gallica [149] .
The potential cytoprotective effect of Liv.52 was studied in vitro studies: it improved copper [150] and tert-butyl hydroperoxide (t-BHP) [151] toxicity in HepG2 cells by inhibition of lipid peroxidation, and increase of GSH content and antioxidant enzyme activity. Another recent study found that Liv.52 abrogated the ethanol-induced PPARγ suppression and ethanol-induced TNFα gene expression, it also upregulated PPARγ mRNA [152] . Pretreatment with low (2.6 mL/kg/day) and higher doses (5.2 mL/kg/day) of Liv.52 reversed paracetamol-induced liver toxicity in mice [153, 154] .
Few randomized controlled clinical trials were made and results were conflicting [155] , recently, a double-blind, placebo-controlled study reported that cirrhotic patients treated for 6 months with Liv.52 had significantly better Child-Pugh score, decreased ascites, and decreased serum ALT and AST levels compared with placebo group [156] .
Hepatocellular carcinoma (HCC) is one of the most frequent cancers in the world and its incidence has been increasing recently in countries including the United States of America, western Europe, and eastern Asia [157] . Systemic chemotherapy plays a palliative role while yields unsatisfactory response rates, which is partly due to the poor selectivity and low uptake efficiency of chemotherapeutic drugs in tumor [158] . Since the prognosis of cirrhotic patients seems to be largely influenced by the development of HCC, every attempt should be performed to prevent HCC in such a highrisk group. Oka et al. reported in a randomized controlled trial that a kind of medicinal herb, "Sho-saiko-to" could significantly decrease hepatic carcinogenesis rate in patients wiith cirrhosis [159] . Moreover, a number of clinical and laboratory studies have been done in the past decades in order to provide the scientific basis for the effectiveness of traditional Chinese medicine against cancer. However, actually, there are a number of contradictory reports due to various factors, as inconsistency in treatment schemes, limited sampling sizes and lack of quality assurance of the herbal products well-designed randomized controlled trials (RCT). In general, most of the published clinical studies are trials without rigorous randomization or they involved single group pre-post, cohort, time series, or matched case-control studies [160] . Herbs are generally used in combination as "formulas," in the belief that in this way their benefits were enhanced and side effects reduced. Moreover, practitioners can adjust or customize the formulas to suit individual cancer patients. Through synergistic interactions between different effective ingredients, the herbal preparation can exert its effects in several ways: (i) they can protect the noncancerous cells and tissues in the body from the possible damage caused by chemo/radiotherapy; (ii) they can enhance the potency of chemo/radiotherapy; (iii) they can reduce inflammatory and infectious complications in the tissues surrounding the carcinoma; (iv) they can enhance immunity and body resistance; (v) they can improve general condition and quality of life; (vi) they can prolong the life span of the patients in the late stages of cancer [161] .
The anticancer herbal drugs can be divided into three categories based on their target: (i) drugs that uniquely target topoisomerases (Topos) and perturb DNA replication; (ii) drugs that kill tumor cells through apoptotic pathways; (iii) drugs that alter signaling pathway(s) required for the maintenance of transforming phenotypes of the tumor cells. The cellular and animal studies have provided strong molecular evidences for the anticancer activities of the herbal medicine; however, several important questions remain to be answered. Specifically, three specific issues that will require focused attention: (i) more well-designed clinical trials to support the effectiveness and the safety of TCM in the management of cancers; (ii) new parameters based on the unique properties and theory of TCM to assess the clinical efficacy of TCM in clinical trials; (iii) new approaches to research, given the nature of TCM herbs as being fundamentally different from drugs. Undoubtedly, a clinical study of TCM treatment is more difficult and complicated than the study of single compound drugs. In addition, the effects, as well as the toxicity, of individual herbs or single compounds derived from the herb cannot completely reflect the benefits and toxicity of the herbal combination [162] . As a goal, to develop TCM into rational cancer therapy, more well-designed intensive clinical evaluations and translational laboratory studies are absolutely needed. Also, close collaboration between TCM and conventional Western medicine professions and a combination of TCM with modern multidisciplinary cutting-edge technologies, such as omic methodology on systems biology [163] , would provide us with an attractive and effective strategy to achieve this goal. Although there are many therapeutic strategies including chemotherapy to treat cancer, high systemic toxicity and drug resistance limit the success full outcomes in most cases. Accordingly, several new strategies are being developed to control and treat cancer. One approach could be a combination of and effective phytochemicals with chemotherapeutic agent, which, when combined, would enhance efficacy and reduce toxicity to tissues [164] .
Several herbs and plants with different pharmacological properties are known to be rich of sources of chemical constituents that may have a potential for the prevention and the treatment of several human cancers.
9.1. Curcumin. Curcumin has been shown with chemopreventive and chemotherapeutic properties against tumors in animal models and clinical trials [165] [166] [167] . The anticancer effects of curcumin have been documented in many cancers; it induces apoptosis through the death receptor mediated pathway and mitochondrial dysfunction and also induces DNA damage response by cleaving caspase-3. In addition, curcumin induces cell cycle arrest by downregulating the protein expression on cdc2 and inhibits the proliferation of human hepatocellular carcinoma J5 cells in a time-and dosedependent manner.
Glycyrrhizin has been shown to successfully prevent the occurrence of primary HCC in patients with HCV-related chronic liver disease by unknown mechanisms [168] . One of the principal roles of long-term administration of glycyrrhizin in decreasing the carcinogenesis rate seemed to be anti-inflammatory ones, which would retrieve an active carcinogenic process.
It has been shown that quercetin inhibits the growth of hepatoma cells in dose-and time-dependent manners. Particularly, in a recent study, quercetin treatment of hepatoma cells resulted in changes of cell cycle, reducing HCC progression [169] .
Most of studies involving quercetin and HCC analyzed cotreatment with different chemotherapeutics.
A study showed that BB-102 (a recombinant adenovirus vector expressing the human p53, GM-CSF and B7-1 genes) and quercetin synergetically suppress HCC cell proliferation and induce HCC cell apoptosis, suggesting a possible use as a combined anticancer agent [170] .
In a different study, the authors explored the effect of combination treatment of quercetin in combination with roscovitine in hepatoma cells. Results showed that roscovitine in combination with quercetin can be considered as a potential therapeutic target for treatment of HCC [171] .
Furthermore, it has been demonstrate that reactive oxygen species production is involved in quercetin-induced apoptosis in human HCC cell lines so quercetin induces favorable changes in the antioxidant defense system of hepatoma cells that prevent or delay conditions which favor cellular oxidative stress [172] .
Otherwise, quercetin, by inducing oxidative stress, potentiates the apoptotic action of 2-methoxyestradiol in human hepatoma cells [173] .
The chemopreventive effect of silymarin on HCC has been established in several studies using in vitro and in vivo methods; it can exert a beneficial effect on the balance of cell survival and apoptosis by interfering cytokines. In addition, anti-inflammatory activity and inhibitory effect of silymarin on the development of metastases have also been detected. In some neoplastic diseases, silymarin can similarly be administered as adjuvant therapy. 9.5. Phyllanthus. Phyllantus Emblica exhibits a variety of pharmacological effects including antiinflammatory, antipyretic, antioxidant and anti-mutagenic effects [174] . The active principles of extracts of P. emblica have demonstrated anti-proliferative effects in several cancer cell lines both in vivo and in vivo, thanks to their ability to interfere with cell cycle regulation via the inhibition of cdc 25 phosphatase and partial inhibition of cdc 2 kinase activity [175] .
A study examined the growth inhibitory effect of P. emblica on human hepatocellular carcinoma (HepG2) and its synergistic effect with doxorubicin and cisplatin: the effect of chemotherapeutic agents may be modified by combination of P. emblica and be synergistically enhanced in some cases [176] . Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be reduced. The mechanism involved in this interaction between chemotherapeutic drugs and plant extracts remains unclear and should be further evaluated.
Although research on complementary and alternative medicine (CAM) therapies is still limited, this systematic review has revealed sufficient evidence to conclude that CAM, particularly the herbal products examined can be effective for certain conditions. There are reliable evidences of potential therapeutic benefit. At the same time, the more limited state of knowledge regarding the side effects of this herbal products are studied in this issue.
These "natural products" have multiple pharmacological actions on various human physiological systems that would support the treatment of chronic disease like cancer. Moreover, the use of herbal medicines is safe compared with synthetic drugs. Further studies are required to determine the molecular mechanisms of their active ingredients.
The limitations of available clinical trials with regard to establishing safety are the same as those for establishing efficacy.
Several studies remark the importance of their protective effects for their principle antioxidants effects useful because it may help to prevent carcinogenicity-associated proliferative processes, but there are not recent publication about their toxicity or their side effects derived by their cronic or acute use. Anyway, if it presents, the side effects are poor (i.e., Glycyrrizin can induce hypokalemia, sodium retention, increase in body weight, and elevated blood pressure) [177] . Finally, hepatic damage from conventional drugs is widely acknowledged and most physicians are well aware of them. It is important to remember that acute and/or chronic liver damage occurred after ingestion of some Chinese herbs, herbals that contain pyrrolizidine alkaloids, germander, greater celandine, kava, atractylis gummifera, callilepsis laureola, senna alkaloids, chaparral, and many others. Several herbals have been identified as a cause of acute and chronic hepatitis, cholestasis, drug-induced autoimmunity, vascular lesions, and evenhepatic failure [79] ( Table 1 ).
Oxidative stress is the common pathway of chronic liver diseases of different etiology (both viral and alcoholic). CAM seems to exert an antioxidant and antifibrotic effect on liver (even if histological proof of these actions is not provided in all studies), so its use alone or in association with etiologic and causal standard therapies is actually common.
For the majority of herbal products, proof of efficacy by randomized, placebo-controlled clinical trials is often lacking. Anecdotal success and personal experience are frequently the driving force for acceptance of CAM in the population [178] .
In contrast to pharmaceuticals, CAM are usually distributed as "food supplements" and not evaluated formally for safety and efficacy; variations in methods of harvesting, preparing, and extracting the herb, which can result in dramatically different levels of certain alkaloids. The biologically active substances have been structurally defined and standardized for only a few of the herb: in most countries, their use is neither regulated nor controlled [179] .
It has been clearly shown that herbal products can protect the liver from oxidative injury, promote virus elimination, block fibrogenesis, or inhibit tumor growth, but the active molecules must be isolated and tested in suitable culture and animal experiments and finally in randomized, placebocontrolled studies to enable rational clinical use of the agents [180] . Identification of pneumonia and influenza deaths using the death certificate pipeline BACKGROUND: Death records are a rich source of data, which can be used to assist with public surveillance and/or decision support. However, to use this type of data for such purposes it has to be transformed into a coded format to make it computable. Because the cause of death in the certificates is reported as free text, encoding the data is currently the single largest barrier of using death certificates for surveillance. Therefore, the purpose of this study was to demonstrate the feasibility of using a pipeline, composed of a detection rule and a natural language processor, for the real time encoding of death certificates using the identification of pneumonia and influenza cases as an example and demonstrating that its accuracy is comparable to existing methods. RESULTS: A Death Certificates Pipeline (DCP) was developed to automatically code death certificates and identify pneumonia and influenza cases. The pipeline used MetaMap to code death certificates from the Utah Department of Health for the year 2008. The output of MetaMap was then accessed by detection rules which flagged pneumonia and influenza cases based on the Centers of Disease and Control and Prevention (CDC) case definition. The output from the DCP was compared with the current method used by the CDC and with a keyword search. Recall, precision, positive predictive value and F-measure with respect to the CDC method were calculated for the two other methods considered here. The two different techniques compared here with the CDC method showed the following recall/ precision results: DCP: 0.998/0.98 and keyword searching: 0.96/0.96. The F-measure were 0.99 and 0.96 respectively (DCP and keyword searching). Both the keyword and the DCP can run in interactive form with modest computer resources, but DCP showed superior performance. CONCLUSION: The pipeline proposed here for coding death certificates and the detection of cases is feasible and can be extended to other conditions. This method provides an alternative that allows for coding free-text death certificates in real time that may increase its utilization not only in the public health domain but also for biomedical researchers and developers. TRIAL REGISTRATION: This study did not involved any clinical trials. The ongoing monitoring of mortality is crucial to detect and estimate the magnitude of deaths during epidemics, emergence of new diseases (for example, seasonal or pandemic influenza, AIDS, SARS), and the impact of extreme environmental conditions on a population such as heat waves or other relevant public health events or threats [1, 2] . The surveillance of vital statistics is not a novel idea; mortality surveillance has played an integral part in public health since the London Bills of Mortality were devised in the seventeenth century [3] . The Bills served as an early warning tool against bubonic plague by monitoring deaths from the 1635 to the 1830s. Today, mortality surveillance continues to be a critical activity for public health agencies throughout the world [4] [5] [6] [7] .
Pneumonia and influenza are serious public health threats and are a cause of substantial morbidity and mortality worldwide; for instance, the World Health Organization (WHO) estimates seasonal influenza causes between 250,000 to 500,000 deaths worldwide each year [8] while pneumonia kills more than 4 million people worldwide every year [9] . Worldwide, the morbidity and mortality of influenza and pneumonia have a considerable economic impact in the form of hospital and other health care costs. Each year in the United States approximately 3 million persons acquire pneumonia and, depending on the severity of the influenza season, 15 to 61 million people in the US contract influenza [9] . These numbers contribute to approximately 1.3 million hospitalizations, of which 1.1 million are pneumonia cases [10] and the remainder for influenza [11] . Moreover, pneumonia cases and influenza together cost the American economy 40.2 billion dollars in 2005 [12] . In The Netherlands it has been estimated that influenza accounts for 3713 and 744 days of hospitalization per 100,000 highrisk and low-risk elderly, respectively [13] . Due to the public health burden and the unpredictability of an influenza season, strong pneumonia and influenza surveillance systems are a priority for health authorities.
Mortality monitoring is an important tool for the surveillance of pneumonia and influenza which can aid in the rapid detection and estimates of excess deaths and inform and evaluate the effect of vaccination and control programs. Traditionally, influenza mortality surveillance often uses the category of "pneumonia and influenza" (P-I) on death certificates as an indicator of the severity of an influenza season or to identify trends within a season; however, only a small proportion of these deaths are influenza related. It has been reported that only 8. [5] [6] [7] [8] [9] .8% of all pneumonia and influenza deaths are influenza related [14, 15] . The non-influenza-related pneumonia deaths tend to be stable from year to year and fluctuations in this category are largely driven by the prevalence and severity of seasonal influenza. As a result, the P-I category is an important sentinel indicator.
In the US, death certificates are the primary data source for mortality surveillance whose findings are widely used to exemplify epidemics and measure the severity of influenza seasons [16] . Currently, there are three systems to monitor influenza-related mortality; one system in particular, the 122 Cities Mortality Reporting System, provides a rapid assessment of pneumonia and influenza mortality [6] . Each week, this system summarizes the total number of death certificates filed in 122 US cities, as well as the number of deaths due to pneumonia and influenza. However, even these data can be delayed by approximately 2-3 weeks from the times of death. This delay can be attributed to one of the following reasons: 1) timeliness of death registration and 2) reviewing of the death certificates to identify pneumonia and influenza deaths [6, 16, 17] . The registration and reviewing of death certificates varies by states and, as a result, there is variability in length of time to report a death to CDC. For instance, states with paper-based death registration system typically perform manual reviews of the death certificates which can take up to 3 weeks; however states with electronic death registration systems (EDRS) may perform automatic reviews which can decrease this time significantly.
The current 122 Cities Mortality Reporting System surveillance system also lacks flexibility for expanding the number of conditions and/or the geographic distribution. Moreover, the unavailability of coded death records due to the complexity of the National Center of Health Statistics (NCHS) coding process results in multiple strategies to identify common outbreaks such as pneumonia and influenza deaths, which greatly vary by jurisdiction. To bypass the lengthy NCHS process, a variety of approaches have been attempted that are close to 'realtime' but less than optimal. For instance, in Utah keyword searching is used to identify pneumonia and influenza deaths; although this method is fast and easy to implement, it can easily result in the over or under estimation of cases. This can occur by missing cases due to misspelled terms, synonyms, variations, or the selection of strings containing the search term.
Other research groups [18, 19] have demonstrated the feasibility of using mortality data for real time surveillance but all used "free text" search for the string "pneumonia", "flu" or "influenza." As noted earlier, although this method can provide the semi quantitative measurements for disease surveillance purposes, keyword searches can also result in an array of problems that result from complexities of human language such as causal relationships and synonyms [20] . Therefore, the lack of coded death data that may not be available for months [21] seriously limits the use of death records in automated systems. At this time, there is little published on the automatic assignment of codes to death certificates for automatic case detection.
Currently the coding of death certificates is a complex process which involves many entities. In the US, where we are focusing this study, the codes on death certificates that are generated by the National Center for Health Statistics (NCHS) depend on information reported on the death certificate by the medical examiner, coroner, or another certifier, and there is substantial variation in how certifiers interpret and adhere to causeof-death definitions [22] . The cause of death literals are coded into International Classification of Diseases Tenth Revision (ICD-10) [23] and the underlying and multiplecause-of-death codes are selected based on the World Health Organization coding rules. These coding rules have been automated by CDC with the development the Mortality Medical Data System (MMDS) which consists of four programs: Super Mortality Medical Indexing Classification and Retrieval (SuperMICAR) Data Entry; Mortality Medical Indexing Classification and Retrieval (MICAR); Automated Classification of Medical Entities (ACME) and TRANSAX (Translation Axes). SuperMI-CAR was designed to facilitate the entry of literal text of causes of death in death certificates and convert them into standardized expressions acceptable by MICAR. It contains a dictionary which assigns an entity reference number (ERN) to statements on the death certificate. These ERNs are fed into MICAR200 which transforms the ERNS into ICD-10 codes by using specific mortality coding rules; the rules require look-up files and a dictionary. ACME and TRANSAX then selects the underlying and multiple causes of death respectively. ICD-10 codes from MICAR200 are fed into ACME which assigns the underlying cause of death using decision tables. The decision table contains all possible pairings of diseases for which the first disease can cause the second. In the latest version of the system, ACME is comprised of eight decision tables including three tables of valid and invalid codes, causal relationships (General Principle and Rule 1), and direct sequel (Rule 3), and three other tables needed by modification rules. Figure 1 provides the workflow for the MMDS system.
Of the 2.3 million deaths that occur each year 80-85 percent are automatically coded through Super-MICAR, and the remaining records are then manually coded by nosologists, a medical classification specialist [24] ; this is a tedious and lengthy process lasting up to 3 months. Although the automation process has decreased the time required for coding death data to 1-2 weeks, the national vital statistics data is not available for at least two years. Therefore, local health department still manually code records or perform basic process techniques to quickly characterize disease patterns [25] .
Records that were processed through Super-MICAR or were manually coded are then processed through the remaining components (MICAR200, ACME and TRANSAX) of MMDS. In 1999, MICAR200 had a throughput rate of 95-97%, while ACME rate was 98 percent. Moreover, based on a reliability study, ACME error rate for selecting the underlying cause is at onehalf percent, while TRANSAX, the multiple cause codes had a one-half percent error rate [26] . Due to the high processing rates and low error rates, MMDS is considered by practitioners as the gold standard for the processing and coding of death certificates in the US and other countries (such as Canada, the United Kingdom (UK) and Australia). Therefore, we used the codes produced by this system as the "gold standard" when comparing with the methods developed here.
In 1997, the US Steering Committee to Reengineer the Death Registration Process (a task force representing federal agencies, the National Center for Health Statistics and the Social Security Administration, and professional organizations representing funeral directors, physicians, medical examiners, coroners, hospitals, medical records professionals, and vital records and statistics officials (NAPHSIS) published the report "Toward an Electronic Death Registration System in the United States: Report of the Steering Committee to Reengineer the Death Registration Process." This report explained the feasibility of developing electronic death registration in the United States [27] and argued that these electronic death records have the potential to be an effective source of information for nation-wide tracking and detecting of disease outbreaks. However, little actions have been taken to implement such recommendations in a comprehensive manner. As of July 2011, electronic death registration systems were operating in 36 states, the District of Colombia, and in development or planning stage in a dozen others [28] .
Information representing the 'cause of death' field on the death certificates is free text. One major goal of natural language processing (NLP) is to extract and encode data from free-texts. There have been many research groups developing NLP systems to aid in clinical research, decision support, quality assurance, the automation of encoding free text data and disease surveillance [29] [30] [31] . Although, there have been a few NLP applications to the public health domain [32, 33] , little is known about its capability to automatically code death certificates for outbreak and disease surveillance. Recently, Medical Match Master (MMM) [25] , developed by Riedl et al at the University of California Davis, was used to match unstructured cause of death phrases to concepts and semantic types within the Unified Medical Language System (UMLS). The system annotates each death phrase input with two types of information, the Concept Unique Identifier, CUI, and a semantic type both assigned by the UMLS. MMM was able to identify an exact concept identifier (CUI) from the UMLS for over 50% of 'cause of death' phrases. Although, the focus of this study was to use NLP techniques to process death certificates, the description of this system reported in the literature did not show how well coded data from an NLP tool along with predefined rules can detect countable cases for a specific disease or condition.
The purpose of our project is to create a pipeline which automatically encodes death certificates using a NLP tool and identify deaths related to pneumonia and influenza which provides daily and/or weekly counts. We compared the new technique developed here with keyword searching and MMDS as exemplars of the easiest possible approach and the current "gold standard", respectively. The comparison of the techniques was done by calculating recall, precision, F-measure, positive predictive value and agreement (Cohen's Kappa).
We obtained 14,440 de-identified electronic death records all with multiple-cause-of-death from the Utah Department of Health (UDOH) for the period 1 January 2008 to 31 December 2008. The records included a section describing the disease or condition directly leading to death, and any antecedent causes, co-morbid conditions and other significant contributing conditions. An example of a paper and electronic death certificate are shown in Figures 2 and 3 respectively. All death certificates used in this study have been processed using the Mortality Medical Data System (MMDS) and the record axis codes were received from UDOH. For our study we randomly selected 6,450 (45%) records. All death records included in the study were previously also coded by NCHS into ICD-10, but this information was not used for our coding, it was only used as posteriori to assess to quality of the automatic coding.
We chose to apply the Centers of Disease Control and Prevention case definition of pneumonia and influenza deaths defined by CDC's epidemiologist staff through personal communication. Therefore, the operational definition for deaths from influenza includes deaths from all types of influenza with the exception of deaths from HAEMOPHILUS INFLUENZAE infection and deaths from PARAINFLUENZAE VIRUS infection. Pneumonia deaths include deaths from all types of pneumonia including pneumonia due to H. influenza and pneumonia due to parainfluenzae virus. The exceptions include aspiration pneumonia (O74.0, O29, O89.0, J69.-and P24.-)1, pneumonitis (J84.1, J67-J70), and pneumonia due to pneumococcal meningitis (J13, G00.1) 1. Pneumonia and influenza related deaths were defined as one of the diagnoses listed in Table 1 which were reported in any cause of death field. These codes were selected through manual review of the ICD-10 version 2007 manual [23] .
The Death Certificates Pipeline, DCP, was developed to identify pneumonia and influenza cases. The pipeline consisted of two components. The first component of the system was the natural language processor, for which we used MetaMap [34] , and the second component was the definitional rules that were applied to the output generated by MetaMap. The study procedures for this pipeline included: preprocessing, NLP, extraction of coded data and the detection of pneumonia and influenza cases (Figure 4 ).
Spelling errors are common on death certificates; therefore, the death records were first processed through a spell checker to identify misspellings. Although the UMLS SL has a spell suggestion tool called GSPELL [35] [36] [37] , we decided not to use it and chose to utilize ASPELL [38] . Our motivation for this decision was based upon an evaluation which showed ASPELL outperforming GSPELL; ASPELL performed better on three areas of performance which were (2) whether the correct word was ranked in the top ten; and (3) whether the correct word was found at all [35] . PERL (www.perl.org), a high-level computer programming language that aids in the manipulation and processing of large volume of text data was then used to prepare the cause of death free text for NLP. The preprocessing also involved the removal of non-ASCII characters; this was a required technical step for MetaMap processing.
Step 2: Natural language processing
MetaMap was used to convert the electronic death records to coded descriptions appropriate for the rule based system. MetaMap [34] , developed by the National Library of Medicine (NLM), is useful in identifying biomedical concepts from free-form textual input and maps them into concepts from the Unified Medical Language System (UMLS) Metathesaurus [34, 39] . MetaMap works by breaking the inputted text into words or phrases, map them to standard terms, and then match the terms to concepts in the Unified Medical Language System (UMLS) [40] . For each matched phrase, MetaMap classifies it into a semantic type then returns the concept unique identifier (CUI) and the mapping options which are ranked according to the strength of the mapping. output from MetaMap. Text bolded in the output from NLP represent the code and its corresponding phrase.
Step 3: Extraction of coded data
The data produced by MetaMap (XML format) was processed through a PERL script to extract the inputted text and its corresponding meta-mapped CUIs. This extracted data was outputted to a text document.
Step 4: Identification of P-I deaths
The identification of pneumonia and influenza cases involved two steps: 1) identifying CUIs relating to pneumonia and influenza and 2) use of the CUIs to create a rules based algorithm to identify cases. Details of each step are explained in the following paragraphs.
To determine which CUI codes were relevant for identifying pneumonia and influenza deaths it was necessary to create a "CUI code list" that represents all the ICD-10 codes of interest (see Table 1 ). To create this list, we generated a subset of the UMLS 2010 AB database [41] using the Metamorphosys [40] tool provided by the National Library of Medicine, NLM. The UMLS database includes many vocabularies, therefore, to determine which vocabularies are relevant to our aims we used the procedure used by Riedl Three queries were performed on the subset described above to map pneumonia and influenza ICD-10 codes to CUIs and identify related pneumonia and influenza concepts. Each query was then placed in a separate database, all duplicates were removed and a sub-query was run to ensure that only the ICD-10 codes in Table 1 were included in this list. This produced 241 distinct concept identifiers (CUIs) relating to pneumonia or influenza. These codes were used to develop the rules to identify the cases of interest.
The coded data produced by MetaMap was accessed by rules, aimed at identifying the presence of pneumonia and influenza based on the coded data. The rules for identifying these deaths used the CUI code list described above. The rule looks at each cause of death field (Underlying Cause, Additional Causes, etc.) to flag records with relevant codes. These rules used boolean operators (And, Or, Not) and if-then statements to create a chain of rules ( Figure 5 ).
The list of cases identified by our automated detection system was compared with those identified by two other methods: a) keyword searching and b) the reference standard: the ICD-10 codes given by the CDC MMDS method. For key-word searching we followed the process To evaluate the performance of both techniques against the reference standard, we needed to specify what constituted a match. Each death record is associated to a unique number; therefore, we considered a match if the unique identifier was identified by the comparator and also found by the reference standard.
Three standard measures were used to evaluate the performance of one method in relation to the reference standard used in this study: precision (equivalent to positive predictive value; recall (equivalent to sensitivity or true positive rate), and F-measure. Kappa statistics were used to assess agreement and McNemar's test was used to analyze the significance between the two methods. All calculations were performed in R [42] .
To calculate these values, pneumonia and influenza related deaths were examined by comparing the reference standard output vs. the two comparators: DCP and keyword search. For both comparators, the deaths were counted and categorized as TRUE POSI-TIVES (cases found by the comparator-pneumonia deaths being correctly classified); FALSE POSITIVES (incorrect cases found by the comparator-the number of pneumonia and influenza deaths incorrectly identified by the comparator); FALSE NEGATIVES (correct cases not found by the comparator-the number of pneumonia deaths not identified by the comparator). Precision, recall and F-score were calculated as follows: Precision = True Positives/(True Positives + False Positives) (1) Recall = True Positives/(True Positives + False Negatives) (2) F-measure = 2 *(P R/ P + R) (3) McNemar's test was also calculated to evaluate the significance of the difference between the two comparators. To calculate this value a confusion matrix was created where A is the number of times both methods have correct predictions; B is the number of times method 1 has a correct prediction and method 2 has a wrong prediction; C is the number of times method 2 has a correct prediction and method 1 has a wrong prediction; D is the number of times both methods have incorrect predictions.
Ethics approval was not required for this study. Identifying variables that could be used for re-identifying individuals were excluded from the study data.
The records were processed and analyzed on a server with two Opteron Dual-Core 2.8 GHz processors and 16 GB RAM at the Center of High Performance Computing at the University of Utah. Using keyword searching the CPU processing time to identify pneumonia and influenza cases was 0.21 seconds and the wall time was 0.37 seconds. For the DCP, the total CPU processing time was 881.83 seconds. The NLP portion of the pipeline attributed to 99.4 percent of the processing time (NLP-877 seconds). While the DCP execution time is much longer, still it is well within the "in real time" realm. For instance, it would take 6,364.3 seconds CPU time seconds for DCP to code and flag all the weekly death records of the US ( 46,523).
Recall and precision were calculated at a 0.95 confidence intervals; the F-measure was also calculated. The performance of each method is described below.
Of the 6,450 records analyzed keyword search identified 473 records as pneumonia and influenza deaths, 21 being identified as false positives. Precision for keyword searching was calculated at 96%. Of the 21 false positives, 6 records correctly mentioned pneumonia in the cause of death text but their corresponding ICD-10 codes failed to provide any code related to pneumonia, while 2 records were flagged because it included the sub-string "pneumonia" in the additional cause of death field. The death literal for these two records were "bacteremia due to Streptococcus pneumonia" and "Streptococcal Pneumoniae Septicemia", The remaining 13 errors were due to the entry of the death literals; in all cases the negation of 'aspiration pneumonia' either due to: 1) 'pneumonia' being in a separate cause of death field to 'aspiration' or 2) 'pneumonia' not being directly followed by 'aspiration' in the death text (example "pneumonia due to secondary aspiration"). A total of 20 false negatives were recorded, yielding a recall of 96%. The false negatives could be generalized into two categories: 1) misspellings of pneumonia on the death certificated (n = 8) and 2) appropriate pneumonia or influenza ICD-10 code was coded but the death literals did not mention an appropriate scanned phrase (n = 12). F-measure was also calculated at 96%. A high level of agreement was seen among keyword searching and the reference standard (kappa 0.95).
Utilizing the Death Certificates Pipeline (DCP), we identified 481 records as pneumonia and influenza deaths, 9 of which were false positives. The precision for this method was calculated at 98%. Like the keyword searching method, of the 9 false positives, 6 records mentioned pneumonia in the cause of death field but their corresponding ICD-10 codes failed to provide any code related to pneumonia and the remaining errors were due to the reporting of aspiration pneumonia on the death certificate. This method had only 1 false negative for the death literal stating "recurrent aspiration with pneumonia", thus yielding a recall at 99.8%, being less than keyword searching. F-measure was calculated at 99%. The level of agreement between the pipeline and the gold standard was almost perfect with a Cohen's kappa of 0.988. The precision and recall scores that are reported above suggest that the DCP is a better method for identifying pneumonia and influenza deaths than keywordsearching. Therefore, we investigated if this observation is supported by statistical analysis. Performing a Fisher's exact test at α = 0.05, significant difference was seen for both recall (p = 1.742e-05) and precision (p = 0.026). The McNemar's test result also showed DCP to be a better method with a p-value = 2.152e-05.
For the 472 pneumonia and influenza cases found by the reference standard, DCP correctly identified 471 cases, missed one case and incorrectly flagged nine cases. Most failures were due to discrepancies between the death literal and its respective ICD-10 code. For the only case which the pipeline did not match, the phrase 'recurrent aspiration with pneumonia' was present in the death literal. MetaMap coded this literal as aspiration pneumonia which was excluded from the CUI code list, but its respective ICD-10 included J189. For the 9 additional cases which were not present in the reference standard, we noticed two categories of errors: 1)cases where the string 'pneumonia' is present in the death literal but not coded into ICD-10 and 2) the reporting of aspiration pneumonia on the death certificate. The first category of errors was not due to MetaMap or the rule algorithm, but perhaps due to the coding process. As described earlier, MMDS produces entity axis and record axis codes. The entity axis codes would be a more appropriate reference standard for they provide the ICD 10 codes for the conditions or events reported as listed by the death certifier and maintains the order as written on the death certificate [43] ; but as noted earlier only the record axis codes were made available for this study. The algorithm used to produce record axis codes from the entity axis data removes duplicate codes and contradictory diagnoses within the entity axis data to produce the more standardized record axis [44] . For example, if a medical examiner reports pneumonia with chronic obstructive pulmonary disease both conditions will be shown in entity axis code data. However, in record axis code data, they will be replaced with a single condition: Chronic obstructive pulmonary disease with acute lower respiratory infection (J44.0). We were unable to verify that codes related to pneumonia were present in the entity axis codes for the six cases; therefore, we can only speculate the reason for this failure.
The second category of errors was due to the reporting of aspiration pneumonia on the death certificate. In cases where the string "aspiration" and pneumonia" were not reported in the same text field MetaMap processed the string separately thus yielding two codes: one for aspiration and the other pneumonia, instead of one code for "aspiration pneumonia" [C0032290]. In an initial review of MetaMap we found MetaMap had difficulties processing the phrase "pneumonia secondary to acute aspiration", therefore, our rule detection algorithm excluded cases where the code for pneumonia and aspiration were present in the same text field.
To our knowledge, this is the first published report on using a natural language processing tool and the UMLS to identify pneumonia and influenza deaths from death certificates. We found that automated coding and identification of pneumonia and influenza deaths is possible and computationally efficient. The Death Certificates Pipeline developed here was statistically different to keyword searching and has higher recall and precision when compared to the current semi-automatic methods in use by the CDC. A good recall is required to help capture the 'true' P-I deaths and a good precision is needed to avoidoverestimating the number of P-I deaths. This study also indicated that keyword searching underestimated pneumonia and influenza deaths in Utah. The simple keyword search method not only decreased recall and precision but also reduced the level of agreement. When reporting counts for surveillance purposes it's best to be as accurate as possible; however, there's a trade-off between recall and precision. For disease surveillance, increased precision enables public health officials to more accurately focus resources for control and prevention, therefore, although both methods had good precision the pipeline developed would be more advantageous to utilize.
MetaMap did an excellent job at extracting cause of deaths from free-form text which is consistent with the results of Reid et Al [25] . Most of the concepts were present in the UMLS which attributed good recall. Both recall and precision depended on the comprehensiveness of the CUI code list. The performance of this system is determined largely by the coverage of terms and sources in the UMLS. Both keyword searching and the system's weakest point is its lack of precision. Most of the concepts the system did not identify had either the aspiration text in another field or pneumonia was mentioned in the cause of death text but not coded (9 cases fit these criteria). The sample size was sufficient to show difference between the two methods. It is important to note that utilizing trained nosologists, who would manually code the death certificates, would have developed an absolute gold standard which may or may not be a better reference standard than ICD-10 codes. However, our motivation for utilizing ICD codes was influenced due to the fact that the use of ICD codes to identify all-cause pneumonia has been examined and has showed to be a valid tool for the identification of these cases [45, 46] .
In terms of timing, while keyword searching is faster than the DCP, our method is also sub 1/10 second range, which implies that it is possible to process the daily Utah deaths (~40) in approximately 5.47 seconds and all deaths in the US (~6646) in approximately 909.17 seconds using current hardware. This timing would be much faster than the minimum of two weeks to receive the coded data from the current CDC process. Moreover, these timings make it apparent that this system can be integrated in a real time surveillance system without introducing any additional bottlenecks.
There are several potential limitations with this analysis. First, the generalizability of the findings is limited because the death records were only from one institution. Although death certificates have a standardized format, the death registration process and the reviewing of death records differ by institutions. UDOH utilizes keyword searching to identify pneumonia and influenza cases, other institutions may use more accurate (manual review) or less accurate methods for finding cases. Second, a separate evaluation of the NLP component of the DCP was not performed. Further research is needed to examine the use of NLP on electronic death records across institutions and countries which may have different documentation procedures.
This study shows that it is feasible to achieve high levels of accuracy when using NLP tools to identify cases of pneumonia and influenza cases from electronic death records while still providing a system that can be used for real time coding of death certificates. Identification of concept identifiers related to the CDC's case definition of pneumonia and influenza was very important in producing a highly accurate rule for the identification of these cases. Future work will aim to improve the preprocessing phase of the pipeline by providing the inclusion of the spellchecker used by the CDC's Mortality Medical Data System. Future work will also involve evaluating the flexibility (e.g. identification of different diseases) of the system to deploy the pipeline tool, along with other public health related analytical tools, as a grid service to provide to real time public health surveillance tool that uses data and services under the control of different administrative domains.
We have shown that it is feasible to automate the coding of electronic death records for real-time surveillance of deaths of public health concern. The performance of the Pipeline outperformed the performance of current methods, keyword searching, in the identification of pneumonia and influenza related deaths from death certificates. Therefore, the Pipeline has the potential to aid in the encoding of death certificates and is flexible to identify deaths due to other conditions of interest as the need arises. Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation Rift Valley fever virus (RVFV), belongs to genus Phlebovirus of the family Bunyaviridae, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-β mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210–230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-β promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6–30, 31–55, 56–80, 81–105, 106–130, 131–155, 156–180, 181–205, 206–230, 231–248 or 249–265 lack functions of IFN–β mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81–105, 106–130, 131–155, 156–180, 181–205, 206–230 or 231–248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-β suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype. Rift Valley fever virus (RVFV) belongs to genus Phlebovirus of the family Bunyaviridae, and is a mosquito-borne zoonotic pathogen which causes Rift Valley fever (RVF). RVF is characterized by an acute febrile illness, hemorrhagic fever, neurological disorder or blindness in humans [1, 2, 3, 4] . In ruminants, RVFV induces a high rate of abortion or fetal malformation as well as lethal hepatitis in newborn lambs [5] . The first recognized outbreak of RVF occurred in Kenya in 1930 [6] , and RVFV has spread from endemic region in sub-Saharan Africa into Egypt [7] , Madagascar and the Arabian Peninsula [8, 9, 10, 11, 12] . The potential threat of RVFV introduction into non-endemic countries raises concern of agriculture and public health [13, 14, 15] . RVFV is a risk group 3 pathogen, Category A pathogen and an overlap select agent by the CDC/USDA [16] . The handling of wild-type (wt) RVFV within the U.S. requires BSL3+ or BSL4 facilities. Live-attenuated MP-12 vaccine strain is excluded from select agent rule, and handled at BSL2 level. MP-12 encodes for functional NSs protein, which is useful for the analyses of various NSs functions at BSL2 level [17, 18, 19] .
RVFV has a tripartite negative-stranded RNA genome, referred to as Small (S)-, Medium (M)-and Large (L)-segment. The Ssegment encodes for N and NSs genes in an ambi-sense manner, M-segments encodes for NSm, 78-kD protein, NSm-Gn, Gn, and Gc proteins, and L-segment encodes for RNA-dependent RNA polymerase [20, 21, 22, 23] . NSs is a major virulence factor of RVFV and inhibits host general transcription through sequestration of TFIIH p44 [24] or promotion of TFIIH p62 subunits degradation [19] . NSs also inhibits host antiviral response by inhibiting the activation of interferon (IFN)-b promoter through interaction with Sin3A-associated protein (SAP30) at aa.210-230 [25, 26] , and promotion of dsRNA-dependent protein kinase (PKR) degradation [17, 27, 28] .
Developing countermeasures against RVFV is important for the prevention of RVF outbreaks or decreasing impact of RVFV introduction. A number of candidate vaccines are under development including live-attenuated vaccine [29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40] , formalin-inactivated vaccine [41, 42] , subunit vaccine [43] , virus-like particle [44, 45, 46, 47] , nonspreading RVFV replicon [48, 49] , viral vectors [50, 51, 52, 53, 54, 55, 56] , and DNA vaccines [57, 58, 59] . For treatment of ongoing outbreaks, several antivirals have been tested for RVFV infection. Liposome-encapsulated ribavirin is effective to treat RVFV infection in mice [60] , while a potent IFN inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-Lysine and carboxymethyl cellulose [Poly(LCIC)] are effective in combination with ribavirin [61] . Therapeutic administration of IFN-a into rhesus monkeys infected with RVFV also limits RVFV replication [62] . The 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is shown to be more effective to inhibit RVFV replication than ribavirin [63] , while antiviral small molecule, LJ001 was shown to be effective to numerous enveloped viruses including RVFV [64] . These studies suggest that increased innate immune responses could inhibit RVFV replication in addition to antivirals specific to viral proteins.
Since the major virulence factor, NSs protein, is an antagonist of IFN responses, the direct attenuation of NSs function may increase host innate immune responses in cells infected with RVFV potentially limiting RVFV replication. Thus, we aimed to attenuate RVFV NSs function(s) by co-expressing nonfunctional NSs. It has been shown that recombinant MP-12 virus encoding truncated NSs at aa.210 to 230 (SAP30-binding domain) does not inhibit IFN-b mRNA synthesis [26] . Thus, we employed a similar strategy to abolish a part of NSs functions by truncating each 17 to 25 aa. Co-expression of such truncated NSs may exhibit dominant-negative phenotype by self-association through the Cterminus at the self-association domain (aa.249 to 265) [65] . In this study, we generated NSs encoding deletions of 17 to 25 amino acids, characterized the functions of truncated NSs, and analyzed the dominant-negative effects of co-expressed truncated NSs in cells infected with RVFV.
Generation of recombinant MP-12 encoding NSs encoding a 17 to 25 aa. truncation Using reverse genetics for the RVFV MP-12 strain, we recovered 11 recombinant MP-12 viruses which encode NSs protein with a 17 to 25 aa. truncation (Fig. 1 ). These NSs mutants exhibited different plaque phenotypes in plaque assay (Fig. S1 ) suggesting possible variation of attenuation by each NSs mutant. The plaques of NSD6-30 and NSD56-80 were clear in neutral red stain, while other mutants made turbid plaques. To test the functions of each NSs protein, VeroE6 cells (type-I IFNincompetent) were mock-infected or infected with MP-12, rMP12-C13type (a control lacking NSs functions) [18] , and NSs mutants using an moi of 3. After 16 hours, cells were collected and the abundance of PKR was measured ( Fig. 2A) as described previously [17] . As expected, PKR was not detectable in cells infected with MP-12 by post-translational downregulation [17] , but was detected in cells mock-infected or infected with rMP12-C13type. However, cells infected with the MP-12 encoding partially truncated NSs did not decrease PKR abundance.
Next, we tested if partial deletions within the NSs gene would affect the inhibition of IFN-b mRNA synthesis. Type-I IFNcompetent A549 cells were mock-infected or infected with MP-12, rMP12-C13type or NSs mutants at an moi of 3, and then total RNA was extracted at 7 hpi. Northern blot was performed using RNA probe specific to human IFN-b, ISG56 or RVFV anti-viralsense S RNA/N mRNA as described previously [66, 67] . We tested ISG56 gene, one of the genes controlled under IFN-stimulated response element (ISRE), to confirm the inhibition of host transcription suppression including IFN-b mRNA by NSs. As expected, cells infected with MP-12 inhibited the synthesis of IFNb and ISG56 mRNA, while those infected with rMP12-C13type induced both IFN-b and ISG56 mRNA (Fig. 2B) . Interestingly, none of NSs mutants, including NSD210-230 lacking SAP30binding domain, had inhibited IFN-b mRNA synthesis. Viral replication of those mutants was significantly decreased in type-I IFN-competent MRC-5 cells (Fig. S2) . Therefore, it was concluded that a series of MP-12 encoding partially truncated NSs gene does not degrade PKR and inhibit IFN-b mRNA synthesis.
In Western blot as shown in Fig. 2A , all NSs mutants, except for NSD249-265, could be detectable by using anti-RVFV mouse polyclonal antibody. It was possible the anti-RVFV polyclonal antibody does not sufficiently contain antibodies reactive to linear epitopes except for the C-terminus. Thus, we next tested the accumulation of NSD249-265 by using indirect immunofluorescent assay to know if the same anti-RVFV polyclonal antibody can recognize conformational epitopes on NSD249-265. 293 cells were transfected with in vitro synthesized RNA encoding NSs of MP-12, NSD249-265 or chloramphenicol acetyltransferase (CAT) (control), and the cells were fixed with methanol at 16 hours post transfection. Nuclear filamentous inclusion was observed in cells expressing NSs of MP-12 or NSD249-265, while the specific signals of NSs accumulation were weaker in cells expressing NSD249-265 than those expressing MP-12 NSs (Fig. 3) . NSD249-265 was also accumulated in cytoplasm.
Next, we tested the cellular localization of truncated NSs other than NSD249-265 by Western blot (Fig. 4) . We did not include NSD249-265 for the experiment as no antibodies were available to detect this NSs in Western blot. 293 cells were mock-infected or infected with MP-12 or NSs truncation mutants at an moi of 3. Cells were collected at 16 hpi, and nuclear and cellular fractions were analyzed for the presence of NSs proteins. MP-12 NSs were accumulated both at cytoplasm and nucleus, while N proteins were exclusively localized at cytoplasm, which is consistent with previous study [65] . Abundant accumulation of NSs in nucleus was only observed in cells infected with NSsD6-30, NSsD 31-55 and NSsD56-80, while other mutants, NSsD 81-105, NSsD106-130, NSsD131-155, NSsD156-180, NSsD181-205 and NSsD206-230, NSsD231-248 poorly accumulated NSs in nucleus.
To find if co-expression of nonfunctional NSs is able to attenuate PKR degradation function of MP-12 NSs, VeroE6 cells were infected with MP-12 and then co-infected with rMP12-C13type or one of the NSs truncation mutants using an moi of 3. Cells were collected at 16 hpi and Western blot was used to measure abundance of PKR and RVFV NSs. However, it was found that levels of MP-12 NSs accumulation were not identical to those expressing truncated NSs (Fig. S3) . Only cells co-infected with NSD6-30 and NSD56-80 allowed an efficient accumulation of MP-12 NSs. As a result, PKR was abundantly detected in cells infected with MP-12 and NSs mutants.
We attempted to allow accumulation of MP-12 NSs by using 293 cells. Cells were mock-infected or infected with rMP12-NSs-Flag, which encode Flag-tagged NSs in place of intact NSs, at an moi of 3, and subsequently mock-transfected (a control) or immediately transfected with in vitro synthesized RNA encoding CAT (a control), or NSs mutants, as described previously [17] . Cells were collected at 16 hpi, and the abundance of PKR and RVFV proteins were analyzed by Western blot. All cells infected with rMP12-NSs-Flag accumulated abundant levels of NSs (Fig. 5A) . PKR was degraded in infected cells mock-transfected or transfected with CAT RNA, while PKR was also degraded in infected cells transfected with RNA encoding NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230 or NSD231-248. On the other hand, the expression of NSD249-265 lacking the Cterminus self-association domain slightly increased the abundance of PKR in cells infected with rMP12-NSs-Flag.
We next tested the effect of co-expression of NSs mutants in the inhibition of IFN-b mRNA synthesis by MP-12 NSs. A549 cells were mock-infected or infected with rMP12-NSs-Flag using moi of 3 and then either mock-transfected or immediately transfected with in vitro synthesized RNA encoding CAT or NSs mutants. Total RNA was extracted at 7 hpi, and Northern blot was performed as described above. None of cells transfected with RNA encoding NSs mutants increased the synthesis of IFN-b mRNA (Fig. 5B) . On the other hand, cells transfected with RNA encoding NSD249-265 slightly increased ISG56 mRNA abundance (Fig. 5B) . These results suggest that nonfunctional NSs encoding the C-terminus self-association domain do not have dominant-negative function, while those lacking the C-terminus domain slightly inhibit PKR degradation as well as ISG56 mRNA synthesis.
Co-affinity precipitation studies were conducted with use of Strep-tagged protein purification to know if over-expressed truncated NSs can interact with MP-12 NSs in infected cells. 293 cells were infected with moi 3 of rMP-12-NSs-SF (recombinant virus tagged with tandem Strep-Tag and Flag) [19] and were then transfected using the in vitro synthesized capped RNA encoding each of the truncated NSs mutants. After 6 hours, newly synthesized host and viral proteins were labeled with [ 35 S] methionine/cysteine for 4 hours. Whole cell lysates were mixed with Strep-Tactin beads, and SF-tagged MP-12 NSs and bound host and viral proteins were precipitated. Presence of NSs bands were visualized with autoradiography ( Fig. 6 ). As expected, MP-12 NSs was co-precipitated with NSs-SF, and MP-12 NSs was migrating slightly faster than that of NSs-SF. The expression of truncated NSs was lower than that of MP-12 NSs (see input), while the expression of NSsD206-230 was not detectable. The same phenomenon was observed in a repeated experiment, suggesting the instability of NSsD206-230 expression in this experiment. On the other hand, we could not detect co-precipitation of any truncated NSs with NSs-SF. Collectively, our results suggest that those truncated NSs accumulates in cells at low level, mislocalizes, and do not interrupt the MP-12 NSs functions by co-expression.
As NSs lacking C-terminus exhibited slight dominant-negative effect on PKR degradation, we hypothesized that an intact sequence at aa.1 to 248 is required for the dominant-negative effect. We next tested the effect of the C-terminus on the dominant-negative effect. We substituted two sequential acidic amino acids triplets located at the C-terminus with alanines as shown in Fig. 7A; i.e., NSs-E253-255A/D257-259A, NSsD257-259A or NSs-E253-255A. We found that NSs-E253-255A could form filamentous inclusion bodies (data not shown), and accumulation was equivalent to that of MP-12 NSs (Fig. 7B) . On the other hand, NSs-E253-255A/D257-259A or NSsD257-259A did not accumulate in cells efficiently, and the NSs could not be detected with IFA (data not shown). We then tested PKR degradation function and IFN-b mRNA suppression function of those mutants using VeroE6 cells and A549 cells using the same method as described above. Cells infected with NSs-E253-255A degraded PKR (Fig. 7B) , and inhibited the synthesis of IFN-b mRNA (Fig. 7C) , while those infected with NSs-E253-255A/ D257-259A or NSsD257-259A did not degrade PKR and did not inhibit IFN-b mRNA synthesis. The results suggest that the glutamic acid at aa.253 to 255 can be replaced without affecting [26] and C-terminus selfassociation domain (aa.249-265) [65] . The rMP12-C13type (C13type) encodes an in-frame truncation of aa. 16-198, NSs functions, while aspartic acid at aa.257 to 259 is important for NSs stability.
We next tested the effect of co-expression of NSs-E253-255A/ D257-259A, NSsD257-259A or NSs-E253-255A in infected cells, as described above. 293 cells were mock-infected or infected with rMP12-NSs-Flag using a moi of 3, then cells were mocktransfected or transfected with in vitro synthesized RNA encoding CAT, NSD249-265, NSs-E253-255A/D257-259A, NSsD257-259A or NSs-E253-255A. Cells were collected at 16 hpi for Western blot analysis. As shown in Fig. 8A , co-expression of NSs-E253-255A/D257-259A or NSs-D257-259A or NSs-E253-255A did not inhibit PKR degradation by rMP12-NSs-Flag, while that of NSD249-265 very slightly increased the PKR abundance, which is consistent with the result in Fig. 5A .
To test the co-expression effect of those NSs mutants in IFN-b mRNA synthesis, A549 cells were mock-infected or infected with rMP12-NSs-Flag at a moi of 3, and immediately transfected with in vitro synthesized RNA encoding NSs-E253-255A/D257-259A, NSsD257-259A or NSs-E253-255A. Total RNA was extracted at 7 hpi, and Northern blot was performed to detect IFN-b, ISG56 or RVFV S-RNA/N mRNA. None of those mutants attenuated NSsmediated IFN-b mRNA synthesis suppression (Fig. 8B) . These results suggest that attenuation of PKR degradation function might occur due to the accumulation of nonfunctional truncated NSs with some stability (Fig. 3) by the lack of C-terminus 17 amino acids residues.
Next, we tested whether the co-expression of NSD249-265 can inhibit MP-12 replication. A549 cells were infected with MP-12 using an moi of 0.01, and then were either mock-transfected or immediately transfected with in vitro synthesized RNA encoding CAT (RNA transfection control), NSD249-265 or NSs-E253-255A (a control with functional NSs). Culture supernatants were harvested at 72 hpi for viral titration using plaque assay. RVFV titer was significantly decreased by the CAT RNA transfection when compared to mock-transfection control. Transfection with RNA encoding NSD249-265 did not further decrease RVFV titer compared to CAT RNA transfected control, while RNA encoding NSs-E253-255A increased RVFV titer significantly. These results suggest that co-expression of NSD249-265 NSs does not significantly decrease viral replication, while that of NSs-E253-255A facilitates RVFV replication by inhibiting IFN-b and PKR in transfected cells. Overall, co-expression of truncated NSs inhibited neither NSs functions nor RVFV replication efficiently. Even though NSs encode self-association domain at the Cterminus domain, the expressed protein mislocalizes in cells, and does not maintain the stability of intact NSs, which minimizes the dominant-negative effect on MP-12 NSs. Discussion Dominant-negative suppression of viral replication has been characterized in a number of different viral proteins [68, 69, 70, 71, 72, 73] . For RVFV, L proteins form oligomer, and exhibit dominant-negative function [74] . In this study, we used NSs protein as a target protein of dominant-negative suppression, because a lack of NSs dramatically attenuates RVFV [75, 76] .
Since NSs can self-associate and form filamentous inclusion bodies in infected cells [65, 77] , we hypothesized that co-expression of nonfunctional NSs with the C-terminal self-association domain in cells infected with RVFV allows incorporation of such nonfunctional NSs into the NSs filament, and attenuates a part of NSs functions. However, our results were not as expected; 1) most of truncated NSs localized at cytoplasm, 2) all of truncated NSs did Our results suggest truncation of NSs causes mis-folding and/or mis-localization of protein, which might abolish the ability to interact with authentic MP-12 NSs through the C-terminus selfassociation domain. Unexpectedly, only the co-expression of NSs lacking C-terminus self-association domain (NSD249-265) slightly inhibited PKR degradation by MP-12 NSs. The NSD249-265 could accumulate both in cytoplasm and nucleus, which is consistent with previous study [65] . We speculate that NSD249-265 could compete with host factors required for PKR degradation with intact NSs. In the meantime, co-expression of NSD249-265 NSs did not result in a significant decrease of RVFV replication when compared to CAT RNA transfection control. Therefore, the co-expression of NSs fragments in infected cells might not be an effective strategy to inhibit RVFV replication in vivo.
Another novel finding is that all of truncation mutants; i.e., NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230, NSD231-248 and NSD249-265, had lacked both PKR degradation and IFN-b suppression functions. This suggests that conformation structure might be important for those NSs functions rather than the presence of linear domain. Our results suggest that those truncated NSs decrease accumulation level, and change the localization pattern in cells. The stability and cellular localization of NSs, which are probably controlled by conformational domain, might be important for biological functions of NSs. Although the C-terminus 17 amino acids were determined as a self-association domain important for filament formation [65] , our result suggests that filament formation does not occur by NSs encoding an in-frame truncation with 25 amino acids. It is possible that the C-terminus 17 amino acids are the prerequisite of NSs self-association, and other structural domains play a role in the filament formation through the C-terminus domain.
Our result showed that NSD206-230 dominantly localized at cytoplasm. Previous study showed that rec-ZHD210-230 (recombinant ZH548 encoding an in-frame truncation in NSs at 210-230) could induce IFN-b mRNA due to a lack of SAP30-binding domain in infected cells [26] . It was also shown that NSs binding to SAP30 is required for NSs filaments to target pericentromeric DNA and induce nuclear anomalies [78] . They described that rec-ZHD210-230 expresses a stable NSs protein located in the nucleus in the discussion [78] . Thus, it might be possible that NSs encoding 20 aa. does not change the nuclear localization. On the other hand, NSD6-30, NSD31-55, NSD56-80 and NSD249-265, all of which encode SAP30-binding domain at aa.210-130, could be accumulated in nucleus, whereas they did not inhibit IFN-b gene, suggesting that NSs has another structural requirement to inhibit IFN-b gene activation in addition to SAP30 binding. The requirement of NSs localization and IFN-b gene suppression should be further studied to understand the detailed mechanism of IFN-b gene regulation by RVFV NSs.
We also characterized the role of C-terminus acidic residues in NSs functions. We found that both NSs-E253-255A/D257-259A and NSsD257-259A were not abundantly accumulated in infected cells. On the other hand, the NSs-E253-255A accumulated efficiently in cells, and showed a similar phenotype with authentic NSs. Thus, the aspartic acids at 257-259 but not glutamic acid at 253-255 must be important for the stability of NSs. Since the accumulation of NSs-E253-255A/D257-259A and NSsD257-259A were very low, we could not determine whether those mutants are lacking the functions to degrade PKR or inhibit IFNb mRNA synthesis.
We noted that the co-infection of recombinant MP-12 encoding truncated NSs with MP-12, except for NSD6-30 and NSD56-80, had resulted in dominant accumulation of truncated NSs. This effect may possibly occur at the transcription or translation level rather than post-translation level, since MP-12 NSs can also accumulate with truncated NSs when RNA transfection was used for truncated NSs expression. If a selective viral transcription of truncated NSs mRNA, or a selective translation of truncated NSs proteins could occur in co-infected cells, then a virus exhibiting these traits may be useful for post-exposure vaccination. However, new studies will be required to detail this mechanism.
In summary, short in-frame truncations of RVFV NSs affect the expression level and cellular localization, which lessen or abolish biological functions of NSs most probably due to the lack of functional conformation domains. Thus, co-expression of truncated nonfunctional NSs in RVFV-infected cells does not attenuate NSs functions of RVFV.
VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573) and A549 cells (ATCC CCL-185) were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS). BHK/T7-9 cells that stably express T7 RNA polymerase [79] were maintained in MEM-alpha containing 10% FCS. Penicilin (100 U/ml) and streptomycin (100 g/ml) were added to the culture media.
The plasmid encoding anti-viral-sense of MP-12 S-segment at the downstream of the T7 promoter, pProT7-S(+), was described previously [18] . Serial deletion of 75 bp (25 aa.) was introduced into the NSs open reading frame (ORF) of pProT7-S(+) by sitedirected mutagenesis with Pfu Turbo DNA polymerase (Stratagene), designated as pProT7-S(+)-NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230 or NSD231-248, respectively. For C-terminus mutant, the PCR fragment encoding NSs ORF with C-terminus 51 bp (17 aa.) deletion was amplified, and cloned between HpaI and SpeI of pProT7-S(+) [18] , designated as NSD249-265. The alanine substitutions of NSs-E253-255A/ D257-259A, NSs-E253-255A or NSsD257-259A were made onto pProT7-S(+) plasmid by site-directed mutagenesis with Pfu Ultra (Agilent Technologies), and designated as pProT7-NSs-E253-255A/D257-259A, NSs-E253-255A or NSsD257-259A, respectively. NSs ORF of those NSs mutants were amplified by PCR with Phusion High Fidelity DNA polymerase (New England Biolab), and cloned into pcDNA3.1mycHisA (Invitrogen) between KpnI and XhoI, and designated as pcDNA3.1mycHisA-NSD6- were transfected with those plasmids as described previously [18] .
Total RNA was extracted from infected or mock-infected cells using TRIzol reagent. Denatured RNA was separated on 1% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane (Roche Applied Science). Northern blot analysis was performed as described previously with strand-specific RNA probes to detect RVFV anti-sense S-segment/N mRNA, human IFN-b mRNA, or human ISG56 mRNA [66, 67] .
Western blot analysis was performed as described previously [17] . The membranes were incubated with anti-PKR monoclonal 
A549 cells were infected with rMP12-NSs-Flag at an moi of 0.01, and mock-transfected or immediately transfected with in vitro synthesized RNA encoding CAT, NSD249-265 or NSs-E253-255A. At 72 hpi, culture supernatants were collected, and plaque assay was performed as described previously [18, 80] .
The pcDNA3.1mycHisA plasmids encoding CAT [17] , NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230, NSD231-248, NSD249-265, NSs-E253-255A/D257-259A, NSs-E253-255A or NSsD257-259A were linearized, and in vitro transcribed by using mMESSAGE mMACHINE T7 Ultra kit (Ambion) according to manufacturer's instruction. The linearized CAT DNA contained myc-His tag at the 39end.
Transfection of in vitro synthesized RNA was performed by using TransIT-mRNA Transfection Kit (Mirus) according to manufacturer's instruction. Figure 8 . Co-expression of truncated NSs in RVFV-infected cells. 293 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT (control) or NSs with indicated NSs mutants. Cells were collected at 16 hpi, and NSs-Flag/NSs (a-RVFV antibody), NSs-Flag (a-Flag antibody), PKR (anti-PKR antibody), CAT-myc (anti-myc antibody) and b-actin (anti-actin antibody) were detected by Western blot. (B) A549 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs with indicated NSs mutants. As a control to induce IFN-b and ISG56 mRNA synthesis, A549 cells were infected with rMP12-C13type (C13type) at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-b mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown. (C) A549 cells were infected with MP-12 at moi of 0.01, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs of NSD249-265 or NSs-E253-255A. Culture supernatants were harvested at 72 hpi, and virus titers were measured by plaque assay. P-values of unpaired Student's t-test are shown (*; p,0.05, ns; not significant). doi:10.1371/journal.pone.0045730.g008
VeroE6 cells were mock-infected or infected with MP-12 or recombinant MP-12 encoding partially truncated NSs at an moi of 4 in 6-well plate. At 16 hpi, cells were collected, and washed once in PBS. Then, cytoplasmic fraction was lyzed with PBS containing 1% TritonX-100 on ice for 5 min. After centrifugation at 10,000 xg at 4 u C for 5 min, nuclear fraction was washed once with cold PBS, and resuspended in PBS containing 1% TritonX-100. Both cytoplasmic and nuclear fractions were mixed with 26 SDS sample buffer, and subjected to SDS-PAGE and Western blot analysis.
Co-affinity precipitation SF (Strep-Flag)-tagged protein was precipitated with Strep-Tactin magnetic beads (Qiagen). 293 cells were first infected with rMP-12 tagged with SF-tag (moi 3) and were then transfected with in vitro synthesized capped RNA (encoding NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230, NSD231-248, or NSD249-265). After incubation for 6 hours, newly synthesized proteins were then labeled with [ 35 S] methionine/cysteine (MP Biomedicals). Using cell lysates, SF-tagged proteins were precipitated with Strep-Tactin beads according to manufacturer's instructions. Then, co-precipitated proteins were analyzed by separating on 10% SDS-PAGE gel and followed by autoradiography.
Unpaired Student's t-test was performed by using the Graphpad Prism 5.03 program (Graphpad Software Inc.) for the comparison of two groups,
All the recombinant DNA and RVFV were created upon the approval of the Notification of Use by the Institutional Biosafety Committee at UTMB. Figure S1 Plaque phenotypes of MP-12 encoding NSs mutants. VeroE6 cells were infected with indicated virus as 10-fold dilution, and overlaid with 0.6% noble agar containing 5% FBS and 5% Tryptose phosphate broth in MEM as described previously [80] . Second overlay of agar containing 0.011% of neutral red was performed at 3 dpi. Plaques at 4 dpi are shown. (TIF) Figure S2 Titer of MP-12 NSs mutants in MRC-5 cells. Human lung diploid MRC-5 cells were infected with MP-12, rMP12-C13type (C13type) or NSs mutants encoding indicated truncations at an moi of 0.01. At 72 hpi, culture supernatants were collected, and virus titers were measured by plaque assay using VeroE6 cells. Means and standard deviations of 3 independent experiments are shown. **p,0.01, Student's unpaired t-test compared to MP-12. (TIF) Figure S3 Co-infection of MP-12 and MP-12 encoding truncated NSs. VeroE6 cells were mock-infected or infected with a mixture of MP-12 (an moi of 3) and either of rMP12-C13type (C13type) or indicated NSs truncation mutants (an moi of 3). Cells were collected at 16 hpi, and PKR (anti-PKR antibody), NSs and N (anti-RVFV antibody) and b-actin (anti-actin antibody) were detected by Western blot. (TIF) Predictive and Reactive Distribution of Vaccines and Antivirals during Cross-Regional Pandemic Outbreaks As recently pointed out by the Institute of Medicine, the existing pandemic mitigation models lack the dynamic decision support capability. We develop a large-scale simulation-driven optimization model for generating dynamic predictive distribution of vaccines and antivirals over a network of regional pandemic outbreaks. The model incorporates measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. The performance of the strategy is compared to that of the reactive myopic policy, using a sample outbreak in Fla, USA, with an affected population of over four millions. The comparison is implemented at different levels of vaccine and antiviral availability and administration capacity. Sensitivity analysis is performed to assess the impact of variability of some critical factors on policy performance. The model is intended to support public health policy making for effective distribution of limited mitigation resources. As of July 2010, WHO has reported 501 confirmed human cases of avian influenza A/(H5N1) which resulted in 287 deaths worldwide [1] . At the same time, the statistics for the H1N1 2009 outbreak has so far included 214 countries with a total reported number of infections and deaths of 419,289 and 18,239, respectively [2] . Today, an ominous expectation exists that the next pandemic will be triggered by a highly pathogenic virus, to which there is little or no pre-existing immunity in humans [3] .
The nation's ability to mitigate a pandemic influenza depends on the available emergency response resources and infrastructure, and, at present, challenges abound. Predicting the exact virus subtype remains a difficult task, and even when identified, reaching an adequate vaccine supply can currently take up to nine months [4, 5] . Even if the existing vaccines prove to be potent, their availability will be limited by high production and inventory costs [6, 7] and also will be constrained by the supply of antiviral drugs, healthcare providers, hospital beds, medical supplies, and logistics. Hence, pandemic mitigation will have to be done amidst limited availability of resources and supporting infrastructure. This challenge has been acknowledged by WHO [7] and echoed by the HHS and CDC [8, 9] .
The existing models on pandemic influenza (PI) containment and mitigation aims to address various complex aspects of the pandemic evolution process including: (i) the mechanism of disease progression, from the initial contact and infection transmission to the asymptomatic phase, manifestation of symptoms, and the final health outcome [10] [11] [12] , (ii) the population dynamics, including individual susceptibility [13, 14] and transmissibility [10, [15] [16] [17] , and behavioral factors affecting infection generation and effectiveness of interventions [18] [19] [20] , (iii) the impact of pharmaceutical and nonpharmaceutical measures, including vaccination [21] [22] [23] , antiviral therapy [24] [25] [26] , social distancing [27] [28] [29] [30] [31] and travel restrictions, and the use of low-cost measures, such as face masks and hand washing [26, [32] [33] [34] .
Recently, the modeling efforts have focused on combining pharmaceutical and nonpharmaceutical interventions in search for synergistic strategies, aimed at better resource utilization. Most of such approaches attempt implementing a form of social distancing followed by application of pharmaceutical measures. For significant contributions in this area see [33, [35] [36] [37] [38] [39] [40] [41] . One of the most notable among these efforts is a 2006-07 initiative by MIDAS [42] , which cross-examined independent simulation models of PI spread in rural areas of Asia [43, 44] , USA and UK [45, 46] , and the city of Chicago [47] , respectively. MIDAS crossvalidated the models by simulating the city of Chicago, with 8.6M inhabitants and implementing a targeted layered containment [48, 49] . The research findings of MIDAS and some other groups [12, 33] were used in a recent "Modeling Community Containment for Pandemic Influenza" report by IOM, to formulate a set of recommendations for PI mitigation [50] . These findings were also used in a pandemic preparedness guidance developed by CDC [51] .
At the same time, The IOM report [50] points out several limitations of the MIDAS models, observing that "because of the significant constraints placed on the models . . . the scope of models should be expanded." The IOM recommends "to adapt or develop decision-aid models that can . . . provide real-time feedback . . . and include the costs and benefits of intervention strategies." Our literature review yields a similar observation that most existing approaches focus on assessment of a priori defined strategies, and virtually none of the models are capable of "learning," that is, adapting to changes in the pandemic progress, or even predicting them, to generate dynamic strategies. Such a strategy has the advantage of being developed dynamically, as the pandemic spreads, by selecting a mix of available mitigation options at each decision epoch, based on both the present state of the pandemic and its predicted evolution.
In an attempt to address the IOM recommendations, we present a simulation optimization model for developing predictive resource distribution over a network of regional outbreaks. The underlying simulation model mimics the disease and population dynamics of each of the affected regions (Sections 2.1 and 2.2). As the pandemic spreads from region to region, the optimization model distributes mitigation resources, including stockpiles of vaccines and antiviral and administration capacities (Section 2.3). The model seeks to minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks, using measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. The methodology is calibrated and implemented on a sample outbreak in Fla, USA with over 4M inhabitants (Section 3). The strategy is compared to the reactive myopic policy, which allocates resources from one actual outbreak region to the next, each time trying to cover the entire regional population at risk, regardless of the resource availability. The comparison is done at different levels of vaccine and antiviral availability and administration capacity. We also present a sensitivity analysis for assessing the impact of variability of some critical factors, including: (i) antiviral efficacy, (ii) social distancing conformance, and (iii) CDC response delay.
The objective of our methodology is to generate a progressive allocation of the total resource availability over a network of regional outbreaks. The methodology incorporates (i) a cross-regional simulation model, (ii) a set of single-region simulation models, and (iii) an embedded optimization model.
We consider a network of regions with each of which classified as either unaffected, ongoing outbreak, or contained outbreak ( Figure 1) . The cross-regional simulation model connects the regions by air and land travel. The single-region simulation models mimic the population and disease dynamics of each ongoing region, impacted by intervention measures. The pandemic can spread from ongoing to unaffected regions by infectious travelers who pass through regional border control. At every new regional outbreak epoch, the optimization model allocates available resources to the new outbreak region (actual distribution) and unaffected regions (virtual distribution). Daily statistics is collected for each ongoing region, including the number of infected, deceased, and quarantined cases, for different age groups. As a regional outbreak is contained, its societal and economic costs are calculated.
In Sections 2.1-2.3, we present the details of the simulation and optimization models. A testbed illustration and a comparison of our strategy to the myopic policy is given in Section 3.
A schematic of the cross-regional simulation model is shown in Figure 2 . The model is initialized by creating population entities and mixing groups, for each region. A pandemic is started by an infectious case injected into a randomly chosen region. The details of the resulting regional contact dynamics and infection transmission are given in Section 2.2. As the infected cases start seeking medical help, a new regional outbreak is detected. A resource distribution is then determined and returned to the single-region model. The outbreak can Begin cross-regional simulation 
The single-region model subsumes the following components (see Figure 3 ): (i) population dynamics (mixing groups and schedules), (ii) contact and infection process, (iii) disease natural history, and (iv) mitigation strategies, including social distancing, vaccination, and antiviral application. The model collects detailed statistics, including number of infected, recovered, deceased, and quarantined cases, for different age groups. For a contained outbreak, its societal and economic costs are calculated. The societal cost includes the cost of lost lifetime productivity of the deceased; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined [52] .
Each region is modeled as a set of population centers formed by mixing groups or places where individuals come into contact with each other during the course of their social interaction. Examples of mixing groups include households, offices, schools, universities, shopping centers, entertainment centers, and so forth, [53] . Each individual is assigned a set of attributes such as age, gender, parenthood, workplace, infection susceptibility, and probability of travel, among others. Each person is also assigned Δt time-discrete (e.g., Δt = 1 hour) weekday and weekend schedules, which depend on: (i) person's age, parenthood, and employment status, (ii) disease status, (iii) travel status, and (iv) person's compliance to social distancing decrees [54] . As their schedules advance, the individuals circulate throughout the mixing groups and come into contact with each other (see Section 2.2.2).
It is assumed that at any point of time, an individual belongs to one of the following compartments (see Figure 4 ): susceptible, contacted (by an infectious individual), infected (asymptomatic or symptomatic), and recovered/deceased. In what follows, we present the infection transmission and disease natural history model, which delineates the transitions between the above compartments.
Process. Infection transmission occurs during contact events between susceptible and infectious cases, which take place in the mixing groups. At the beginning of every Δt period (e.g., one hour), for each mixing group g, the simulation tracks the total number of infectious cases, n g , present in the group. It is assumed that each infectious case generates r g per Δt unit of time new contacts [46] , chosen randomly (uniformly) from the pool of susceptibles present in the group. We also assume the following: (i) during Δt period, a susceptible may come into contact with at most one infectious case and (ii) each contact exposure lasts Δt units of time. Once a susceptible has started her contact exposure at time t, she will develop infection at time t + Δt with a certain probability that is calculated as shown below.
Let L i (t) be a nonnegative continuous random variable that represents the duration of contact exposure, starting at time t, required for susceptible i to become infected. We assume that L i (t) is distributed exponentially with mean 1/λ i (t), where λ i (t) represents the instantaneous force of infection applied to susceptible i at time t [55] [56] [57] . The probability that susceptible i, whose contact exposure has started at time t, will develop infection at time t + Δt is then given as
A schematic of the disease natural history is shown in Figure 5 . During the incubation phase, the infected case stays asymptomatic. At the end of the latency phase, she enters the infectious phase [44, 46, 48] . She becomes symptomatic at the end of the incubation period. At the end of the infectious phase, she enters the period leading to a health outcome, which culminates in her recovery or death.
Mortality for influenza-like diseases is a complex process affected by many factors and variables, most of which have limited accurate data support available from past pandemics. Furthermore, the time of death can sometimes be weeks following the disease episode (which is often attributable to pneumonia-related complications [58] ). Because of the uncertainty underlying the mortality process, we adopted an age-based form of the mortality probability of infected i, as follows:
where μ i is the age-dependent base mortality probability of infected i, ρ i is her status of antiviral therapy (0 or 1), and τ is the antiviral efficacy measured as the relative decrease in the base probability [44] . We assume that a recovered case develops full immunity but continues circulating in the region.
Mitigation is initiated upon detection of a critical number of confirmed infected cases [59] , which triggers resource distribution and deployment. The model incorporates a certain delay for deploying field responders. Pharmaceutical intervention (PHI) includes vaccination and antiviral application. Vaccination is targeted at individuals at risk [60] to reduce their infection susceptibility. The vaccine takes a certain period to become effective [61] . Vaccination is constrained by the allocated stockpile and administration capacity, measured in terms of the immunizer-hours. We assume that as some symptomatic cases seek medical help [62, 63] , those at risk of them will receive an antiviral. The process is constrained by the allocated stockpile and administration capacity, measured in terms of the number of certified providers.
Both vaccination and antiviral application are affected by a number of sociobehavioral factors, including conformance of the target population, degree of risk perception, and compliance of healthcare personnel [64] [65] [66] . The conformance level of the population at risk can be affected, among other factors, by the demographics and income level [67] [68] [69] [70] [71] as well as by the quality of public information available [54] . The degree of risk perception can be influenced by the negative experience developed during previous pharmaceutical campaigns [72, 73] , as well as by public fear and rumors [74, 75] .
Nonpharmaceutical intervention (NPI) includes social distancing and travel restrictions. We adopted a CDC guidance [51] , which establishes five categories of pandemic severity and recommends quarantine and closure options according to the category. The categories are determined based on the value of the case fatality ratio (CFR), the proportion of fatalities in the total infected population. For CFR values lower than 0.1% (Category 1), voluntary at-home isolation of infected cases is implemented. For CFR values Influenza Research and Treatment 5 between 0.1% and 1.0% (Categories 2 and 3), in addition to at-home isolation, the following measures are recommended: (i) voluntary quarantine of household members of infected cases and (ii) child and adult social distancing. For CFR values exceeding 1.0% (Categories 4 and 5), all the above measures are implemented. As the effectiveness of social distancing is affected by some of the behavioral factors listed above [54] , we assume a certain social distancing conformance level. Travel restrictions considered in the model included regional air and land border control for infected travelers. Figure 2 , the optimization model is invoked at the beginning of every nth new regional outbreak epoch (n = 1, 2, . . .), starting from the initial outbreak region (n = 1). The objective of the model is to allocate some of the available mitigation resources to the new outbreak region (actual distribution) while reserving the rest of the quantities for potential outbreak regions (virtual distribution). By doing so, the model seeks to progressively minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks, spreading from the ongoing locations. Mitigation resources can include stockpiles of vaccines and antivirals, administration capacity, hospital beds, medical supplies, and social distancing enforcement resources, among others. The predictive mechanism of the optimization model is based on a set of regression equations obtained using single-region simulation models. In what follows, we present the construction of the optimization model and explain the solution algorithm for the overall simulation-based optimization methodology.
We introduce the following general terminology and notation: The optimization criterion (objective function) of the model incorporates measures of expected societal and economic costs of the pandemic: the societal cost includes the cost of lost lifetime productivity of the deceased; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined. To compute these costs, the following impact measures of morbidity, mortality, and quarantine are used, for each region k: To estimate these measures, we use the following regression models obtained using a single-region simulation of each region k:
where δ i ·· denotes the regression coefficient associated with resource i and δ im ·· is the regression coefficient for the interaction between resources i and m. Similar models are used for Y hk , D hk , and V hk .
The above relationships between the impact measures and the resource distributions ought to be determined a priori of implementing a cross-regional scenario (see Section 3). Here, we consider each region k as the initial outbreak region. We assume, however, that as the pandemic evolves, the disease infectivity will naturally subside. Hence, the regression equations need to be re-estimated at every new outbreak epoch, for each region k ∈ C n , using the singleregion simulation models, where each simulation must be initialized to the current outbreak status in region k in the cross-regional simulation. As an alternative to using a computationally burdensome approach of re-estimating the regression equations, a modeler may choose to use a certain decay factor α n [76] to adjust the estimates of the regional impact measures at every nth outbreak epoch, in the following way:
In addition, we use the following regression model to estimate the probability of pandemic spread from affected region l to unaffected region k, as a function of resources allocated to region l, which, in turn, impact the number of outgoing infectious travelers from the region:
where γ i ·· denotes the regression coefficient associated with resource i, γ im ·· is the regression coefficient associated with interaction between resources i and m, and γ 0 ·· represents the intercept. Consequently, the total outbreak probability for unaffected region k can be found as p k = l∈B n p lk . As in the case of the impact measures, the estimates of the regional outbreak probabilities need to be progressively re-estimated or adjusted using a scheme similar to (4), as follows:
6
Finally, we calculate the total cost of an outbreak in region k at the nth decision epoch as follows:
where m h is total medical cost of an infected case in age group h over his/her disease period, w h is total cost of lost wages of an infected case in age group h over his/her disease period, w h is cost of lost lifetime wages of a deceased case in age group h, and w h is daily cost of lost wages of a non-infected case in age group h who complies with quarantine.
The model. The optimization model has the following form.
Minimize TC n j q 1 j , q 2 j , . . . , q r j + s∈C n TC n s q 1s , q 2s , . . . , q rs · p n s subject to
The first term of the objective function represents the total cost of the new outbreak j, estimated at the nth outbreak epoch, based on the actual resource distribution {q 1 j , q 2 j , . . . , q r j } (see (7)). The second term represents the total expected cost of outbreaks in currently unaffected regions, based on the virtual distributions {q 1s , q 2s , . . . , q rs } (7) and the regional outbreak probabilities p n s (6) . The set of constraints assures that for each resource i, the total quantity allocated (current and virtual, both nonnegative) does not exceed the total resource availability at the nth decision epoch. Note that both the objective function and the availability constraints are nonlinear in the decision variables.
(1) Estimate regression equations for each region using the single-region simulation model.
(2) Begin the cross-regional simulation model. (4) Select randomly the initial outbreak region j. Set n = 1. (c) Re-estimate regression equations for each region k ∈ B n ∪ C n using the single-region simulations, where each simulation is initialized to the current outbreak status in the region (alternatively, use (4) and (6)). (d) Solve the resource distribution model for region j. (e) Update the total resource availabilities.
(10) Calculate the total cost for each contained region and update the overall pandemic cost.
To illustrate the use of our methodology, we present a sample H5N1 outbreak scenario including four counties in Fla, USA: Hillsborough, Miami Dade, Duval, and Leon, with populations of 1.0, 2.2, 0.8, and 0.25 million people, respectively. A basic unit of time for population and disease dynamics models was taken to be Δt = 1 hour. Regional simulations were run for a period (up to 180 days) until the daily infection rate approached near zero (see Section 3.3). Below, we present the details on selecting model parameter values. Most of the testbed data can be found in the supplement [77] .
Models. Demographic and social dynamics data for each region [77] were extracted from the U.S. Census [78] and the National Household Travel Survey [79] . Daily (hourly) schedules [77] were adopted from [53] . Each infected person was assigned a daily travel probability of 0.24% [79] , of which 7% was by air and 93% by land. The probabilities of travel among the four regions were calculated using traffic volume data [80] [81] [82] [83] , see Table 1 . Infection detection probabilities for border control for symptomatic cases were assumed to be 95% and 90%, for air and land, respectively [84] .
The instantaneous force of infection applied to contact i at time t ((1), [57] ) was modeled as
Influenza Research and Treatment 7 where α i is the age-dependent base instantaneous infection probability of contact i, θ i (t) is her status of vaccination at time t (0 or 1), and δ is the vaccine efficacy, measured as the reduction in the base instantaneous infection probability (achieved after 10 days [61] ). The values of age-dependent base instantaneous infection probabilities were adopted from [46] (see Table 2 ). The disease natural history included a latent period of 29 hours (1.21 days), an incubation period of 46 hours (1.92 days), an infectiousness period from 29 to 127 hours (1.21 to 5.29 days), and a period leading to health outcome from 127 to 240 hours (5.29 to 10 days) [85] .
Base mortality probabilities (μ i in (2)) were found using the statistics recommended by the Working Group on Pandemic Preparedness and Influenza Response [52] . This data shows the percentage of mortality for age-based high-risk cases (HRC) ( Table 3 , columns 1-3). Mortality probabilities (column 4) were estimated under the assumption that highrisk cases are expected to account for 85% of the total number of fatalities, for each age group [52] .
Single-region simulation models were calibrated using two common measures of pandemic severity [35, 45, 46] : the basic reproduction number (R 0 ) and the infection attack rate (IAR). R 0 is defined as the average number of secondary infections produced by a typical infected case in a totally susceptible population. IAR is defined as the ratio of the total number of infections over the pandemic period to the size of the initial susceptible population. To determine R 0 , all infected cases inside the simulation were classified by generation of infection, as in [33, 43] . The value of R 0 was calculated as the average reproduction number of a typical generation in the early stage of the pandemic, with no interventions implemented (the baseline scenario) [33] . Historically, R 0 values for PI ranged between 1.4 and 3.9 [37, 43] . To attain similar values, we calibrated the hourly contact rates of mixing groups [77] (original rates were adopted from [46] ). For the four regions, the average baseline value of R 0 was 2.54, which represented a high transmissibility scenario. The values of regional baseline IAR averaged 0.538.
Mitigation resources included stockpiles of vaccines and antiviral and administration capacities (Section 3.4). A 24-hour delay was assumed for deployment of resources and filed responders [59] .
PHI. The vaccination risk group included healthcare providers [66] , and individuals younger than 5 years (excluding younger than 12 months old) and older than 65 years [60] . The risk group for antiviral included symptomatic individuals below 15 years and above 55 years [60, 86] . The efficacy levels for the vaccine (δ in (9)) and antiviral (τ in (2)) were assumed to be 40% [44, 87] and 70%, respectively. We did not consider the use of antiviral for a mass prophylactic reduction of infection susceptibility due to the limited antiviral availability [9] and the risk of emergence of antiviral resistant transmissible virus strains [26] . We assumed a 90% target population conformance for both vaccination and antiviral treatment [64] . The immunity development period for the vaccine was taken as 10 days [61] .
A version of the CDC guidance for quarantine and isolation for Category 5 was implemented (Section 2.2.4, [51] ). Once the reported CFR value had reached 1.0%, the following policy was declared and remained in effect for 14 days [51] : (i) individuals below a certain age ξ (22 years) stayed at home during the entire policy duration, (ii) of the remaining population, a certain proportion φ [88] stayed at home and was allowed a one-hour leave, every three days, to buy essential supplies, and (iii) the remaining (1 − φ) noncompliant proportion followed a regular schedule. All testbed scenarios considered the quarantine conformance level φ equal to 80% [54] .
An outbreak was considered contained, if the daily infection rate did not exceed five cases, for seven consecutive days. Once contained, a region was simulated for an additional 10 days for accurate estimation of the pandemic statistics. A 2 5 statistical design of experiment [89] was used to estimate the regression coefficient values of the significant decision factors and their interactions (see Section 2.3; the values of adjusted R 2 ranged from 96.36% to 99.97%).
The simulation code was developed using C++. The running time for a cross-regional simulation replicate involving over four million inhabitants was between 17 and 26 minutes (depending on the initial outbreak region, with a total of 150 replicates) on a Pentium 3.40 GHz with 4.0 GB of RAM.
The performance of the DPO and myopic policies is compared at different levels of resource availability. Table 4 summarizes the total vaccine and antiviral requirements for each region, based on the composition of 8 Influenza Research and Treatment Average daily cost of lost productivity of a non-infected quarantined case (20-99) $432.54 theregional risk groups (see Section 3.3). Table 5 shows the per capita costs of lost productivity and medical expenses, which were adopted from [52] and adjusted for inflation for the year of 2010 [90] . Comparison of the two strategies is done at the levels of 20%, 50%, and 80% of the total resource requirement shown in Table 4 . Figures 6(a) and 6(b) show the policy comparison in the form of the 95% confidence intervals (CI) for the average number of infected and deceased, respectively. Figure 7 also shows the policy comparison using the 95% CI for the average total pandemic cost, calculated using the pandemic statistics, and the per capita costs from Table 5 . For illustrative purposes, we also show the average number of regional outbreaks, for each policy, at different levels of resource availability, in the testbed scenario involving four regions, with the Hillsborough as the initial outbreak region ( Table 6) .
It can be observed that the values of all impact measures exhibit a downward trend, for both DPO and myopic policies, as the total resource availability increases from 20% to 80%.
An increased total resource availability not only helps alleviating the pandemic impact inside the ongoing regions but also reduces the probability of spread to the unaffected regions. For both policies, as the total resource availability approaches the total resource requirement (starting from approximately 60%), the impact numbers show a converging behavior, whereby the marginal utility of additional resource availability diminishes. This behavior can be explained by noting that the total resource requirements were determined assuming the worst case scenario when all (four) regions would be affected and ought to provided with enough resources to cover their respective regional populations at risk. It can also be seen that on average, the DPO policy outperforms the myopic approach at all levels, which can attest to a more efficient resource utilization achieved by the DPO policy (see also Table 6 ). The difference in the policy performance is particularly noticeable at the lower levels of resource availability, and it gradually diminishes, as the resource availability increases and becomes closer to be sufficient to cover the entire populations at risk in all regions. It can also be noted that the variability in the performance of the DPO strategy is generally smaller than that of the myopic policy. In general, for both strategies, the performance variability decreases with higher availability of resources.
In this section, we assess the marginal impact of variability of some of the critical factors. The impact was measured separately by the change in the total pandemic cost and the number of deaths (averaged over multiple replicates), resulting from a unit change in a decision factor value, one factor at a time. Factors under consideration included: (i) antiviral efficacy, (ii) social distancing conformance, and (iii) CDC response delay. We have used all four regions, separately, as initial outbreak regions for each type of sensitivity analysis. The results (patterns) were rather similar. Due to limited space, we have opted to show the results for only one initial region, chosen arbitrarily, for each of the three types of sensitivity studies. While Duval County was selected as the initial outbreak region to show the sensitivity results on antiviral efficacy, Hillsborough and Miami Dade were used as the initial regions to show the results on, respectively, social distancing conformance and CDC response delay. Figure 8 depicts the sensitivity of the average total cost and average total deaths to antiviral efficacy values between 0% and 80%. As expected, for both policies, the curves for the average number of deaths exhibit a decreasing trend which is almost linear for the values of τ between 0% and 40%. As the value of τ approaches 70%, the curves start exhibit a converging behavior. The curves for the average total pandemic cost exhibit a similar pattern for both policies.
It can be noted that the performance of both policies is somewhat identical for low antiviral efficacy (between 0% and 30%). However, the performance of the DPO policy improves consistently as τ increases which can be attributed to a more discretionary allocation of the antiviral stockpile by the DPO policy.
Reduction of the contact intensity through quarantine and social distancing has proven to be one of the most effective containment measures, especially in the early stages of the pandemic [27, 30, 31, 41] . Figure 9 shows the sensitivity of the average total cost and average total deaths to the social distancing conformance ranging between 60% and 80%. We observed that for both impact measures, the DPO policy demonstrated a better performance with the difference ranging from $3B to $26B in the total cost and from 1,400 to 20,000 in the number of fatalities. The biggest difference in performance was achieved at the lower-to-medium levels of conformance (between 65% and 72%). As the conformance level approached 80%, the dominating impact of social distancing masked the effect of better utilization of vaccines and antivirals achieved by the DPO strategy.
The CDC response delay corresponds to the interval of time from the moment an outbreak is detected to a complete deployment of mitigation resources. Depending on the disease infectivity, CDC response delay may represent one of the most critical factors in the mitigation process. Figure 10 shows how the performance of both policies was significantly impacted by this factor. The DPO policy showed a uniformly better performance with the difference ranging between $3B to $4B in the average total cost, and between 800 to 1,800 in the average number of mortalities, over the range (24- Figure 10 : Sensitivity analysis for CDC response delay.
As recently pointed by the IOM, the existing models for PI mitigation fall short of providing dynamic decision support which would incorporate "the costs and benefits of intervention" [50] . In this paper, we present a large-scale simulation optimization model which is attempted at filling this gap. The model supports dynamic predictive resource distribution over a network of regions exposed to the pandemic. The model aims to balance both the ongoing and potential outbreak impact, which is measured in terms of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. The model was calibrated using historic pandemic data and compared to the myopic strategy, using a sample outbreak in Fla, USA, with over 4 million inhabitants.
Summary of the main results. In the testbed scenario, for both strategies, the marginal utility of additional resource availability was found to be diminishing, as the total resource availability approached the total requirement.
In the testbed scenario, the DPO strategy on average outperformed the myopic policy. As opposed to the DPO strategy, the myopic policy is reactive, rather than predictive, as it allocates resources regardless of the remaining availability and the overall cross-regional pandemic status. In contrast, the DPO model distributes resources trying to balance the impact of actual outbreaks and the expected impact of potential outbreaks. It does so by exploiting regionspecific effectiveness of mitigation resources and dynamic reassessment of pandemic spread probabilities, using a set of regression submodels. Hence, we believe that in scenarios involving regions with a more heterogeneous demographics, the DPO policy will likely to perform even better and with less variability than the myopic strategy. We also note that the difference in the model performance was particularly noticeable at lower levels of resource availability, which is in accordance with a higher marginal utility of additional availability at that levels. We thus believe that the DPO model can be particularly useful in scenarios with very limited resources.
Contributions of the paper. The simulation optimization methodology presented in this paper is one of the first attempts to offer dynamic predictive decision support for pandemic mitigation, which incorporates measures of societal and economic costs. Our comparison study of the DPO versus myopic cross-regional resource distribution is also novel. Additionally, our simulation model represents one of the first of its kind in considering a broader range of social behavioral aspects, including vaccination and antiviral treatment conformance. The simulation features a flexible design which can be particularized to a broader range of PHI and NPI and even more granular mixing groups.
We also developed a decision-aid simulator which is made available to the general public through our web site at http://imse.eng.usf.edu/pandemics.aspx. The tool is intended to assist public health decision makers in implementing what-if analysis for assessment of mitigation options and development of policy guidelines. Examples of such guidelines include vaccine and antiviral risk groups, social distancing policies (e.g., thresholds for declaration/lifting and closure options), and travel restrictions.
Limitations of the model. Lack of reliable data prevented us from considering geo-spatial aspects of mixing group formation. We also did not consider the impact of public education and the use of personal protective measures (e.g., face masks) on transmission, again due to a lack of effectiveness data [91] . We did not study the marginal effectiveness of individual resources due to a considerable uncertainty about the transmissibility of an emerging pandemic virus and efficacy of vaccine and antiviral. For the same reason, the vaccine and antiviral risk groups considered in the testbed can be adjusted, as different prioritization schemes have been suggested. The form of social distancing implemented in the testbed can also be modified as a variety of schemes can be found in the literature, including those based on geographical and social targeting. Effectiveness of these approaches is substantially influenced by the compliance factor, for which limited accurate data support exists. It will thus be vital to gather the most detailed data on the epidemiology of a new virus and the population dynamics early in the evolution of a pandemic, and expeditiously analyze the data to adjust the interventions accordingly. A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI. Avian influenza (AI) is a devastating poultry disease with serious economic consequences to the commercial poultry industry. AI is also a significant public health concern because of recent highly pathogenic H5N1 avian influenza outbreaks causing also human deaths in Asia, Europe, and North Africa. According to the world health organization (WHO) update, 2011, since 2003, 520 confirmed cases of human infection with H5N1 have been reported, of which 307 died due to disease complications. However, other avian influenza viruses including low-pathogenic avian influenza (LPAI) can also be a risk to public health. For instance, the LPAI subtype H9N2 infection in chickens is mild to asymptomatic and easily overlooked. However, it shares similar receptor binding epitopes with human influenza viruses and can infect humans [1] . There is a risk for LPAI subtypes H5 and H7 to become high-pathogenic avian influenza (HPAI) viruses in chickens due to constant virus shedding and transmission to new birds within the flock or neighboring flocks [2, 3] . Vaccination is an effective way for prevention of viral diseases in poultry. However, routine vaccination against AI has not been widely practiced throughout the world mainly for surveillance reasons [1, 2] . When there is the desire for routine vaccination, constant 2 Influenza Research and Treatment reformulation of AI vaccines is required according to the circulating field virus, which can be cumbersome in the case of an immediate outbreak. Current vaccines against AI viruses can reduce mortality, clinical signs, shedding, and transmission of the virus in poultry, but they are not capable of preventing infection and virus replication [4] .
The design of a universal influenza vaccine has been the major focus of researchers in the influenza vaccinology field. The external domain of matrix protein 2 (M2e) has been one of the main interests for the generation of a universal AI vaccine. The M2e is encoded by a separate open reading frame of segment 7 of the influenza virus genome, is located in the viral envelope, and projects from the surface of the virus as tetramers [5, 6] . The M2 is composed of 97 amino acids which forms 3 domains: the external domain, the transmembrane domain, and the internal domain. The external domain of M2 (M2e) is recognized by the host's immune system [7] [8] [9] . Initially, vaccination of ferrets with whole M-or M2-expressing recombinant vaccinia virus showed no evidence of protection [10] . However, later vaccine constructs using plasmid and recombinant salmonella expressing M or M2 induced significant protection in terms of reduction in virus growth and mortality in mice and chickens, respectively [11] [12] [13] . A multiple antigenic peptide construct containing M2e (M2e-MAP) induced strong M2especific antibody titers in the serum of mice and resulted in significant protection against influenza virus challenge [13] . Liang et al., 1994 [14] showed the importance of CD4 + T cells for nasal resistance and protection against the virus. It is assumed that M2e-specific memory T h cells also may have an important role in protection against the virus in the nose and trachea of mice [13] . De Filette et al., 2005 [15] used the hepatitis B virus core particle (HBc) as a carrier and fused M2e (conserved region of human influenza A virus) to either the C-terminus of HBc or inserted it in the immune-dominant loop of HBc. Immunization of mice with this M2e-HBc vaccine was 100% protective against lethal challenge [15] [16] [17] .
Antigenic epitopes of pathogens are peptides that are capable of inducing an immune response. However, their small size limits their immunogenicity. Therefore, usually a larger carrier protein, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or a virus-like particle (VLP), is required for optimal immunogenicity [18] . Structural organization of the epitope on the carrier is critical for inducing stronger immune responses. Denis et al., 2007 [19] demonstrated that a monomeric form of M2e peptide was not immunogenic and Huleatt et al., 2008 [20] tried to solve that problem by adding 4 copies of the M2e peptide in their platform. Here, we used peptide nanoparticles as a platform to display the M2e peptide to the host's immune system. These nanoparticles represent a novel type of repetitive antigen display system which allows presenting the M2e peptide in high density in both, either in its monomeric or its tetrameric form.
This idea was first presented in Raman et al., 2006 [21] ; the monomeric peptide is composed of two coiled coils connected by a short linker region. The association between the coiled coils induces self-assembly of the monomers into Figure 1 : Computer model (a): the pentameric-trimeric architecture of Mono-M2e and the fully assembled icosahedral nanoparticle. (b) Tetra-M2e, with tetrameric-trimeric architecture, and the resulting octahedral nanoparticle. In both images, green: pentameric coiled coil, turquoise: tetrameric coiled coil, and blue: trimeric coiled coil. Red represents M2e in either its monomeric or tetrameric state. spherical nanoparticles with either icosahedral or octahedral symmetry ( Figure 1 ) according to our computer models. The potential for these nanoparticles to serve as platforms for vaccines is apparent. As opposed to live attenuated vaccines, there is no risk of infection within the vaccinated population [21] . Furthermore, the ease and speed of protein expression, purification, and self-assembly into nanoparticles reduce the cost and time of large-scale production. The concept has been successfully used for the design of malaria [22] and SARS [23] vaccines prototypes.
Here, we present the biophysical characterization of the nanoparticles and an immunological profiling using chickens as test animals. The results suggest that the selfassembling polypeptide nanoparticle shows promise as a potential vaccine against AI.
2.1. Nanoparticle Synthesis. The DNA coding for the nanoparticle constructs was prepared using standard molecular biology procedures. Shortly, plasmids containing the peptide monomers (Table 1) were constructed by cloning complementary oligonucleotides (CCCGGGGGGGCAGCGGC AGCCTGCTGACCGAAGTGGAAACCCCGACCCGCAAC-GGCTGGGAATAATGAATTC) encoding the avian M2e epitope with flanking residues (ARGGSGSLLTEVETPTRNGW-E * * E) into the XmaI/EcoRI restriction sites of the basic SAPN expression construct to yield Mono-M2e. To Table 1 : Summary of self-assembling nanoparticle peptide sequences.
Peptide sequence
The peptide Mono-M2e is composed of a pentameric coiled coil (green) and a trimeric coiled coil (blue). Tetra-M2e uses the same trimer but has a tetrameric coiled coil (turquoise). In both sequences, the M2e epitope is shown in red. Other amino acid residues, such as linkers and his-tags, are shown in black. M2eN-GCN4 consists of M2e attached to the tetrameric GCN4 coiled coil, shown in brown. Monomeric M2e, used for ELISA, coating is shown in red.
generate Tetra-M2e, we first cloned the tetrameric oligomerization domain of tetrabrachion into the BamHI/BssHII restriction sites of pPEP-T ( Figure 5 ), before cloning complementary oligonucleotides (ATGCATCCCTGGTTCC GCGTGGAAGCCTGCTGACCGAAGTGGAAACCCCGAC-CCGCAACGGCTGGGAATGCAAATGCAGCGATAGCAGC GGATCC) coding for the slightly longer avian M2e sequence (HASLLTEVETPTRNGWECKCSDSSGS) including flanking residues into the N-terminal NsiI/BamHI restriction sites. The M2e-GCN4 construct was made by replacing the nanoparticle fragment of Tetra-M2e with the GCN4 sequence. The plasmids were transformed into Escherichia coli BL21 (DE3) cells, which were grown in Luria broth with ampicillin at 37 • C. Expression was induced with isopropyl β-D-thiogalactopyranoside. Four hours after induction, cells were removed from 37 • C and harvested by centrifugation at 4,000 ×g for 15 min. The cell pellet was stored at −20 • C. The pellet was thawed on ice and suspended in a lysis buffer consisting of 9 M urea, 100 mM NaH 2 PO 4 , 10 mM Tris pH 8, 20 mM imidazole, and 0.2 mM Tris-2-carboxyethl phosphine (TCEP). Cells were lysed by sonication and the lysate was cleared by centrifuging at 30.500 ×g for 45 min. The cleared lysate was incubated with Ni-NTA Agarose Beads (Qiagen, Valencia, CA, USA) for at least 1 hour. The column was washed with lysis buffer and then a buffer containing 9 M urea, 500 mM NaH 2 PO 4 , 10 mM tris pH 8, 20 mM imidazole, and 0.2 mM TCEP. Protein was eluted with a pH gradient: 9 M urea, 100 mM NaH 2 PO 4 , 20 mM citrate, 20 mM imidazole, and 0.2 mM TCEP. Subsequent washes were done at pH 6.3, 5.9, and 4.3. Following the pH gradient, a gradient of lysis buffer with increasing imidazole strength was used to further elute the protein. Purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as shown in Figure 6 .
The protein solution was filtered with a 0.1 μm polyvinylidene fluoride membrane filter (Millipore Billerica, MA, USA). Nanoparticle self-assembly was performed by dialysis into buffer containing 8 M urea, 20 mM Tris pH 7.5, 150 mM NaCl, and 5% glycerol, at a protein concentration of 0.1 mg/mL. This was followed by dialysis into the same buffer containing decreasing concentrations of urea: 6 M, 4 M, 2 M, 1 M, and two changes of the same buffer without urea. Following self-assembly, the nanoparticle solution was again filtered with a 0.1 μm filter.
Scattering. Dynamic light scattering experiments were carried out on a Zetasizer Nano S Instrument (Malvern, Worcestershire, UK), with a 633 nm He-Ne laser. All measurements were carried out at 25 • C in a buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5% glycerol.
Samples were negatively stained with 1% uranyl acetate (SPI Supplies, Westchester, PA, USA) and observed with a FEI Tecnai T12 S/TEM at an accelerating voltage of 80 kV (FEI, Hillsboro, Oregon). The peptide concentration of the constructs was about 0.05 mg/mL.
Samples were dialyzed into 20 mM sodium phosphate pH 7.5, 150 mM NaCl, and 5% glycerol and concentrated or diluted to a peptide concentration of about 0.13 mg/mL for Mono-M2e and about 0.05 mg/mL for Tetra-M2e. Circular dichroism measurements were performed at room temperature using an Applied Photophysics (Surrey, UK) Pi Star 180 spectropolarimeter, taking measurements from 200 to 250 nm.
The influenza virus used in the direct challenge AI study was A/Turkey/CA/D0208651-C/02 H5N2 low pathogenic. Influenza A/Turkey/Wisconsin/1/1966 H9N2 low pathogenic was used for hyperimmune serum production provided by Charles River Avian Vaccine Services (Storrs, CT). Viruses were grown and titered in 9-to 11-dayold embryonated specific pathogen-free (SPF) chicken eggs as previously described [24] .
Groups. SPF P2a line (B19/ B19) white Leghorn chickens eggs were obtained from Cornell University, Ithaca, NY. The eggs were hatched in the University of Connecticut Poultry Farm and after the hatch, the chickens were moved to the Office of Animal Research Services (OARS) facilities. After 2 weeks in the brooders with free access to water and a standard starter diet, the chickens were divided into groups, bled for baseline serology, transferred to isolators equipped with high-efficiency particulate air (HEPA) filters, and were provided commercial diets and water ad libitum.
A previously described plaque reduction assay was modified and used to evaluate the virus neutralization activity of collected sera after vaccination [25] . Briefly, serum samples from each treatment group were pooled. An equal volume of a 1 : 10 dilution of pooled serum and LPAI subtype H5N2 was mixed and incubated for 30 min at 37 • C. A commercially available anti-M2 antibody (ProSci-Inc, Poway, CA) was used in a 1 : 1000 dilution as a control for antibody activity. Chicken embryo kidney cell (CEKC) monolayers in 6-well plates were washed twice with prewarmed PBS and 400 μL of the above mixture was added to the CEKC monolayer. The plates were incubated for 60 min at 37 • C. Then, the inoculums were removed and after 2 washes with prewarmed PBS, they were overlaid with 0.8% agar (University of Connecticut Cell Culture Facility) in Minimum Essential Medium Eagle (MEM). After 72 h, the plates were checked for plaque formation and for further evaluation were fixed with 99% methanol and stained with crystal violet for plaque counting.
ELISA. The M2e epitopes, including the nanoparticle platforms with M2e epitopes (Tetra-M2e and Mono-M2e) and M2e linked to GCN4, (M2eN-GCN4), were used for coating of the ELISA plates. Briefly, individual wells of the flat-bottom 96-well Immulon 1B plates (NUNC/Thermo Fisher Scientific, Rochester, NY) were coated with 5 μg/mL of tetrameric M2e (M2eN-GCN4) or the nanoparticle of interest. Antigen adhesion was allowed to proceed at 4 • C overnight. Plates were rinsed with 2% Tween 20 in phosphate-buffered saline (PBS) (PBS/Tween 20 Ther-moFisher) and blocked with a 3% BSA in PBS solution.
Plates were incubated at 37 • C for 3-4 h or 4 • C overnight (preliminary studies showed that there was no difference in the result). After incubation, plates were rinsed 4 times with PBS/Tween 20 and incubated for 1 h at room temperature with the previously collected sera. Briefly, 2-fold serial dilutions of each serum sample were prepared in a PBS solution containing 0.2 to 0.5% BSA. Hyperimmune serum from previously infected birds with the LPAI subtype H9N2 or commercial anti-M2e antibody were used as positive controls; and sera from healthy, unvaccinated birds were used as a negative control. After appropriate washes, peroxidaseconjugated goat anti-chicken IgY (Sigma Aldrich,) was prepared in a 1 : 10,000 dilution in PBS and was added to each well and plates were incubated for an additional hour at room temperature. After subsequent rinsing, the plates were developed using 3,3 ,5,5 tetramethylbenzidine (TMB) peroxidase substrate (Thermo Fisher Scientific Inc., Rockford, IL) followed by a room temperature incubation period of 15 to 30 min. The absorbance was read in a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.
In order to generate a standard curve for realtime PCR, we transcribed standard RNA in vitro using T7 RiboMAX Express Large-Scale RNA Production System (Promega, Madison, WI). Briefly, RNA extraction was done by using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer. The coding region of the M gene from the LPAI subtype H5N2 was amplified using previously described universal primers [26] . RT-PCR was performed using a Qiagen One-Step RT-PCR kit (Qiagen, Valencia, CA) according to the standard manufacturer's protocol. PCR products were visualized by electrophoresis through ethidium-bromide-stained (0.5 μg/mL) 1.5% (40 mM Tris-Acetate pH 7.8, 0.1 mM EDTA) agarose gels under UV light.
The amplified fragment was excised from the gel and cDNA was recovered from agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol and the purified DNA product was ligated with a pCR 2.1 vector (Invitrogen, Carlsland, CA) according to the manufacturer's protocol to generate the pCR-M5 plasmid. For further confirmation, PCR positive plasmids were sequenced in the DNA Biotechnology Facility of the University of Connecticut. Six micrograms of plasmid DNA was linearized using 6 units of the restriction enzyme Bam HI for 4 h at 37 • C. Then, linearized DNA was used as a template in an in vitro transcription reaction with the T7 RiboMAX Express Large-Scale RNA Production System (Promega, Madison, WI) according to the manufacturer's recommendation. After the in vitro transcription reaction at 37 • C for 1 h, the possible remaining plasmid DNA was digested by DNase I and purified RNA was quantified with a spectrophotometer NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE). The copy numbers of purified RNA were determined using a previously described method [27] and was used for the generation of a real time standard curve.
In this study, real-time RT-PCR was performed using the previously published primers M+25: AGA TGA GTC TTC TAA CCG AGG TCG and M-124: TGC AAA AAC ATC TTC AAG TCT CTG for quantification of viral load [28] . RNA extraction was done on each swab sample followed by PCR in duplicate or triplicate using 5 μL of RNA per each PCR reaction. Briefly, the Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystem, Foster City, CA) was used with a 20 μL reaction mixture. For each PCR run, standards were designated for the plate and viral loads were calculated using fluorescence data acquired at the end of each annealing step. The amount of unknown sample was extrapolated based on the standard curve and was reported as viral copy number.
Prior to vaccination and challenge study, a pilot study was performed to evaluate pathogenicity Influenza Research and Treatment 5 Upon determination of the peak of virus shedding and appropriate infectious dose in the pilot study, the vaccination and challenge trial was initiated. Briefly, 42 SPF chickens were divided into six groups of seven and received their first inoculation at 2 weeks of age followed by two boosters, two weeks apart, at 10 weeks and 12 weeks after hatch as described in Table 2 . Preceding injection, nanoparticles were concentrated using Amicon Centrifugal Filter units with a 100 kDa MWCO (Millipore, Billerica, MA). Concentration was determined by absorbance at 280 nm and nanoparticle quality was assured by DLS. The nanoparticle vaccine constructs were emulsified with either Freund's complete adjuvant (prime) or Freund's incomplete adjuvant (boosters) and injected into the pectoral muscle of each chicken. Two weeks after the second booster, the birds, except for those in the negative control group, were challenged with 10 7.2 EID 50 LPAI subtype H5N2. Briefly, each bird received 1 mL allantoic fluid containing 10 7.2 EID 50 LPAI subtype H5N2 divided among the eyes, nasal cavity, and oropharynx, while temporarily blocking the fresh air delivery to the isolator. Fresh air was resumed after 5-10 min the following challenge of the last bird in the isolator. Although the clinical signs associated with LPAI viruses are rare, they were observed for possible clinical symptoms daily; and the presence of the symptoms and their severity was recorded.
Tracheal and cloacal swabs were taken from each bird at days 2, 4, 6, and 8 after challenge and they were placed in a 3.0 mL UVT tube (Becton, Dickinson, NJ). Blood samples from each bird were collected before each booster as well as two weeks after the second booster prior to challenge. Each blood sample was collected in a separating blood tube and serum was separated by placing the tubes at 37 • C for 1 h then at room temperature overnight followed by a 5 to 10 min centrifugation at 1000 rpm at 4 • C. Then, the collected serum samples were stored at −20 • C until analysis.
Design. An obvious model for a selfassembling protein particle is a viral capsid. The capsids of spherical viruses often have icosahedral symmetry, due to their need to build a large encapsulating structure from many copies of the same, or only few, capsid proteins. An icosahedron is the most efficient way to accomplish this. By utilizing pentameric and trimeric coiled coils, we have built a self-assembling nanoparticle which uses the threefold and fivefold symmetry of an icosahedrons [21] . The pentameric coiledcoil motif of the monomer is taken from Cartilage Oligomeric Matrix Protein (COMP) and the trimer is a de novo designed coiled coil. Self-assembly occurs when the coiled-coil domains of different monomers associate, forming the icosahedral nanoparticle ( Figure 1 ). A nanoparticle with this sort of architecture can then be used as a vaccine platform by extending the ends of the monomer with an epitope sequence. The Mono-M2e species of nanoparticle follows this plan ( Table 1) . As a result, it repetitively displays a monomeric form of M2e on the surface of the nanoparticles. The M2e peptide on the icosahedral nanoparticles lacks its C-terminal five residues to avoid problems with disulfide crosslinking that presumably require the native tetrameric conformation for proper formation. Although the simplest icosahedral particle with T1 icosahedral symmetry is made from 60 polypeptide chains, it may also be possible that Mono-M2e particles possess higher triangulation numbers, resulting in particles with an even greater molecular mass.
On the other hand, the native conformation of M2 is a tetramer. Hence, to elicit conformationally specific antibodies, the M2e antigen displayed by a vaccine particle should ideally be tetrameric. With that in mind, we designed the Tetra-M2e peptide ( Table 1) . Instead of a pentameric coiled coil, this polypeptide uses the tetrameric coiledcoil motif from the protein tetrabrachion [29] . Self-assembly using this peptide would result in a nanoparticle with threefold and fourfold symmetry axes or octahedral symmetry. As opposed to the larger icosahedral Mono-M2e, this octahedral particle would only have 24 polypeptide chains. In addition, the epitope is now constrained to its native tetrameric conformation. The full-length M2e contains two cysteine residues. The formation of disulfide bridges between the cysteines of adjacent chains under oxidizing conditions is thought to stabilize their tetrameric conformation. The speed and ease of protein expression and purification, as well as of the self-assembly process, contribute to the overall viability of this technology as a vaccine platform. To facilitate purification, we have included polyhistidine tags at the N-terminal ends of the peptides.
To enable detection of antibodies against tetrameric M2e, the peptide M2eN-GCN4 was designed (Table 1 ). M2e is linked to a GCN4, a coiled coil whose oligomerization state can be determined by the identity of amino acid residues in key a and d positions of the coiled coil. In this case, the tetrameric version of GCN4 was used [30] . By affixing M2e to a tetrameric protein, we can constrain it in its tetrameric conformation. The effect is similar to that experienced by the ends of the tetrameric coiled coil from tetrabrachion of the Tetra-M2e nanoparticle. However, the coiled coil sequence is different. This guarantees that any antibodies bound to M2e-GCN4 are specific for the tetrameric version of M2e and not against the coiled coil or other parts of the nanoparticle.
Mono-M2e formed particles whose hydrodynamic diameters have a distribution which peaks at 34.5 nm, while the distribution of Tetra-M2e peaks at 22.9 nm (Figure 2 ). It is also noteworthy that the size distribution peak of Mono-M2e is broader than that of Tetra-M2e, suggesting that the former has a higher degree of polydispersity.
The results were confirmed by transmission electron microscopy ( Figure 3) . We can see that nanoparticles were formed and that their diameters are comparable with those measured by dynamic light scattering. It can be seen from the micrographs that neither Mono-M2e nor Tetra-M2e form nanoparticles with perfectly spherical morphology. This may in some way explain the polydispersity observed by dynamic light scattering.
Structure. The double minima found by circular dichroism confirm the alpha helical structure of the nanoparticles (Figure 4 ). It appears that Tetra-M2e exhibits this behavior much less than Mono-M2e. This may be partly due to the larger M2e epitope sequence in the Tetra-M2e peptide as compared to that used in Mono-M2e.
The plaque reduction assay performed by using pooled serum from chickens inoculated with Tetra-M2e did not show a significant (P > 0.05) difference compared to control nonvaccinated chicken serum and commercial anti-M2e antibody.
The anti-M2e immune response was monitored by determining the titer of the M2e-specific IgY at three different time points (2 weeks after each inoculation). Chickens after each inoculation developed high levels of antibody against the injected construct and anamnestic response clearly was seen when the plates were coated with Mono-M2e and Tetra-M2e nanoparticles and M2e-GCN4 (tetrameric M2e), respectively (Table 1, Figures 7 and 8) . For further investigation of the antibodies, the level of M2e specific antibody was measured using plates coated with tetrameric M2e-GNC4 peptide to evaluate the specific antibody against tetrameric M2e rather than the whole particle. The result of this study indicated that in chickens, after the second booster, the antibody levels are not at the same level as our previous results in mice with the same backbone but a different (malaria) epitope had been shown [23] . The dose level was also higher than what was shown to be required in mice. This could be because of the lower haplotype-specific immunogenicity of the particles in chickens, the route of administration in mice (intraperitoneal and intranasally), the body weight of the mice compared with chickens, and different immune system repertoires of mammalian and avian species. In future studies, changing the administration route can be another approach to reducing the dose of vaccine construct. We also coated the plate with inactivated purified virus to observe seroconversion of the chickens after challenge with the virus at 2 weeks after the last boost. Results indicated that whole virus response was higher as expected with hyperimmune serum (Figure 9 (a)), however, ELISA response from chicken vaccinated with tetra-M2e and with whole virus reacted similarly on GCN-M2e coated plate (Figure 9(b) ). The protective efficacy of the anti-M2e antibody responses induced by different constructs was assessed by evaluation of viral shedding post challenge. To determine the peak of shedding, viral copy number was Figure 10 . We determined viral loads in tracheal and cloacal swabs samples on day 8 following challenge with 10 7.2 EID 50 LPAI subtype H5N2. Reduction of cloacal and oropharyngeal shedding in vaccinated birds was significant in chickens vaccinated with Tetra-M2e with Freund's adjuvant. Virus shedding was evaluated at day 4 and day 6 after challenge; the swabs were tested for virus load ( Figure 11 ). It is seen that virus shedding reduction starts at day 4 post infection with a significant decrease at day 8 post infection.
Currently available vaccines induce antibodies against specific field strains or closely related avian influenza strains.
Most of these vaccines are killed virus vaccines that induce short-lived immunity and are lacking a broad cross-reactive humoral immune response. Recently, the generation of a universal influenza vaccine using conserved peptide regions among several influenza virus strains has been an area of interest in the human influenza vaccine field. M2e is a highly conserved region among influenza viruses and it has been studied as a possible universal vaccine candidate against human influenza virus infection [16, 17] .
In the present study, protection efficiency of two different nanoparticle constructs harboring M2e was studied as possible vaccine candidates for low-pathogenicity avian influenza infection. Biophysical analysis confirms that they are of relatively regular shape and size, but there is some degree of heterogeneity. Though molecular weight measurements still remain to be carried out and we have no high resolution Influenza Research and Treatment structural data, we assumed that nanoparticles assembled in a state close to what was expected, that is, icosahedral and octahedral nanoparticles, respectively. This will repetitively display M2e in both, either in its monomeric or its tetrameric form.
There is speculation as to how the polyhistidine tag at the N-terminal end of the monomers may affect the selfassembly process, the final nanoparticle structure, or the immunogenicity of the vaccine, but attempts at producing his-tag free versions of the nanoparticle constructs either did not reliably provide pure protein or never adequately self-assembled. Similarly, we attempted to include CD4 T cell epitopes to increase the immune response, but this also interfered with nanoparticle formation.
Since many variables can affect the host's virus shedding and the course of disease [31, 32] , prior to evaluation of vaccine constructs, the pathogenicity after challenge with the LPAI subtype H5N2 virus was evaluated. A biphasic virus shedding was observed in this study. For the LPAI subtype H5N2, the peaks for tracheal and cloacal shedding were at days 4 and 8 postinfection.
The Tetra-M2e vaccine construct provided a significant viral load reduction at the peak of viral shedding in immunized chickens. Chickens immunized with Tetra-M2e that harbors the tetrameric M2e with Freund's adjuvant showed a clear reduction in cloacal and tracheal excretion of LPAI compared to challenge control groups. The results of immunization with Mono-M2e with adjuvant and Tetra-M2e without adjuvant were also promising and by improving both B-and T-cell epitopes of those constructs, desirable results may be obtained. In vaccine design, repetitive Bcell epitope display is considered a strategy for improving the humoral immune response [33, 34] . In addition to repetitive antigen display on the nanoparticle, we were able to present M2e in its native tetrameric conformation. The correlation between high protection and antibody response specific for tetrameric M2e elicited by Tetra-M2e supports our assumption of tetrameric M2e presentation. The results of our studies show that tetrameric M2e stimulates a more specific immune response compared to the monomeric presentation and induces a significant protection against homologous virus challenge. The fact that a large portion of the antibody response is directed against the carrier and not only against the epitope(s) (Figures 7 and 8) can be explained by the fact that significant portions of the core of the nanoparticles are also exposed to the immune system (compare Figure 1) and hence, these portions will also induce a significant immune response.
In this study, we showed that anti-M2e antibodies are not neutralizing antibodies; however they are capable of binding to the M2 proteins that are abundantly presented on the surface of the infected cells (data not shown). These can describe an efficient delayed clearance of the virus in M2e vaccinated chickens based on the previously described NK cell involvement in ADCC [35] . Significant improvement of virus clearance in vaccinated chickens with tetrameric M2e may be considered in a new vaccination strategy by vaccinating chickens with both a killed vaccine and a nanoparticle vaccine in order to provide robust protection, cross-reactive immunity, and clearance in case of emerging new strains of the virus.
However, there remains the risk that such vaccination may cause a long-term persistence of HPAI in poultry flocks, because the vaccine could not prevent the viral infection but rather suppresses the symptoms of HPAI virus-infected chickens by reducing the virus shedding in chicken. Thereby, especially in the case of HPAI infection, the vaccination may make the infection less visible and the eradication of virus more difficult, and consequently it may provide a good opportunity for HPAI virus to survive and persist in poultry flocks for a long time. For this reason, we plan to design new nanoparticle constructs that also contain fragments of hemagglutinin in addition to the M2e domain. Immunization would then result in the generation of neutralizing hemagglutinin-specific antibodies in addition to the disease modulating M2e-specific antibodies.
In this study, we evaluated a new approach to immunizing chickens against AI that uses a nanoparticle platform to carry an antigenic epitope. Further designing and testing of new nanoparticle vaccines should demonstrate that they are effective tools for stimulation of an immune response against M2e and other B-or T-cell epitopes. Therefore, application of the nanoparticle platform facilitates the development of a new generation of vaccines that harbor conserved epitopes of avian influenza viruses and would not be rendered ineffective by viral mutations such as antigenic shifts and drifts. The nanotechnology described here offers the opportunity to rapidly produce new vaccines according to the emergence of new strains of influenza virus without going through the time-consuming steps of production currently used in manufacturing commercial influenza vaccines. For future studies, the chicken's LPAI infection model needs to be improved to evaluate clinical signs and higher virus shedding. This may help to better evaluate virus shedding, specifically cloacal virus shedding. Also, vaccination and HPAI challenge may be used to evaluate the vaccine efficiency in protection against high-pathogenicity AI viruses.
Avian influenza M2e: Ectodomain of matrix protein 2 LPAI: Low pathogenicity avian influenza HPAI: High pathogenicity avian influenza SPF: Specific pathogen free. Factors Associated with Increased Risk Perception of Pandemic Influenza in Australia The aim of this study was to assess factors associated with increased risk perception of pandemic influenza in Australia. The sample consisted of 2081 Australian adults aged 16 years and older who completed a short three item pandemic influenza question module which was incorporated into the NSW Health Adult Population Health Survey during the first quarter of 2007. After adjusting for covariates, multivariate analysis indicated that those living in rural regions were significantly more likely to perceive a high risk that a pandemic influenza would occur, while those with poor self-rated health perceived both a high likelihood of pandemic and high concern that self/family would be directly affected were such an event to occur. Those who spoke a language other than English at home and those on low incomes and younger people (16–24 years) were significantly more likely to have changed the way they lived their lives due to the possibility of pandemic influenza, compared to those who spoke only English at home, middle-high income earners, and older age groups, respectively. This data provides an Australian population baseline against which the risk perceptions of demographic subgroups regarding the current, and potential future pandemics, can be compared and monitored. The pattern of recurrence of pandemics since the mideighteenth century indicates that pandemics occur about every 30 years [1] . Prior to 2009, expert consensus was that another pandemic influenza was almost inevitable [2] [3] [4] [5] [6] [7] , and although the H5N1 avian viruses were the most likely candidate for an influenza outbreak, the unexpected H1N1 swine influenza reached pandemic in June, 2009. With previous influenza pandemics and the current H1N1 influenza pandemic arriving with little to no warning, we are afforded a unique opportunity to prepare for the next pandemic threat, which has the potential to be more severe than the current pandemic. Important for preparation is knowledge about the public's response to such a threat, and a key component to the public's response is their perception of risk.
Knowing how a risk is perceived is essential for preparing an effective plan for risk communication, and may be predictive of the public's response. In a study of the NSW population, Barr et al. [8] found that respondents with higher levels of risk perception reported more willingness to comply with public health behaviours in the event of an outbreak of influenza. Similar results were found in Hong Kong [9] and Italy [10] , where respondents in both studies with an increased perception of risk were more likely to be engaged in risk-reducing behaviours.
In 2007, 14.9% of the NSW population reported that they thought pandemic influenza was very or extremely likely to occur and 45.5% were very or extremely concerned that they or their family would be affected by an influenza pandemic should it occur [8] . What may be of particular importance however, is how risk perception varies within the population. Risk perception may be affected by factors such as awareness of a hazard, cultural and social factors or the experience or memory of a prior similar hazard, all of which may result in variation in risk perception among individuals. Lau et al. [11] found in a Hong Kong sample that the odds of females reporting worry about themselves or their families contracting an outbreak of avian influenza if it is to occurr were 1.6 times higher than the odds of males reporting such worry. De Zwart et al. [12] similarly found that women and older respondents scored significantly higher on a composite measure of risk perception (combining perceived seriousness of threat and vulnerability to threat) than men and younger respondents, respectively. In an Italian population, Di Giuseppe et al. [10] found that risk perception was higher for respondents with lower socioeconomic status and lower education.
In preparation for a pandemic influenza outbreak, the Australian Government recommends a number of measures the general public could take, such as having enough food, water, and essential items to enable a household to be confined at home for up to 14 days [13] , ensuring such food is rotated and use by dates are checked regularly [13] ; practicing good personal hygiene, and teaching children about hand washing and cough etiquette [14] .The World Health Organisation has also recommended seasonal influenza vaccinations for health care workers to reduce the risk of genetic shifts in the influenza virus [15] .The preparation of the general public for an outbreak of influenza may be a key strategy in preventing the spread of the disease in the event of a pandemic. Thus it is important to identify subpopulations in Australia who are more and less likely to have changed their life in response to the possibility of pandemic influenza.
The aim of the current study was to obtain baseline Australian data on factors associated with perceptions of the likelihood of pandemic influenza, concern for self and family in the event of an influenza pandemic and broad changes in living as a result of the threat of pandemic influenza.
A short three item pandemic influenza question module was developed as the first part of a larger module of questions on potential threats. These questions were field tested and inserted into the New South Wales Population Health Survey, administered between 22 January and 31 March, 2007 [8] . The New South Wales Population Health Survey is a continuous telephone survey including questions on health behaviours, health status, and access to health services of the state population using the in-house CATI facility of the New South Wales Department of Health [16] . Households were contacted using random digit dialing. Up to 7 calls were made to establish initial contact with a household, and 5 calls were made in order to contact a selected respondent. Only residential phone numbers were used in the sample, as residential phone coverage in Australia still remains high [17] and results from persons who only have mobile phones has been shown to be comparable in the United States [18, 19] . Interviews were conducted in English, Arabic, Chinese, Greek, Italian, or Vietnamese, depending on respondent preference. More details of the sampling approach can be found in the 2007 NSW Health survey report [20] .
A three item pandemic influenza question module was developed which addressed pandemic influenza threat perceptions. The wording of the questions was as follows:
(1) How likely do you think it is that pandemic influenza will occur in Australia?
(2) If a pandemic influenza were to occur in Australia, how concerned would you be that you or your family would be affected by it?
(3) How much have you changed the way you live your life because of the possibility of an influenza pandemic?
All responses were coded on a five-point Likert scale. Response options for all questions were "not at all", "a little", "moderately", "very", and "extremely". In addition, "do not know" and "refused" responses were coded as missing.
Analysis. Data analysis was performed using the "SVY" commands of Stata version 9.2 (Stata Corp, College Station, TX, USA), which allowed for adjustments for sampling weights.
The five-point Likert-scale responses were dichotomised. The definitions of the variables used are as follows:
(1) Pandemic influenza likely to occur: the proportion of households aged 16 years and older who rated pandemic influenza as very or extremely likely to occur.
(2) Concern for self/family: the proportion of households aged 16 years and older who were very or extremely concerned that self/family would be directly affected if pandemic influenza were to occur. To determine factors associated with risk perception, the dichotomized risk question indicators and the "composite" indicators were used as outcome measures and these were investigated using the following set of independent variables: age, gender, marital status, children in household, location (urban/rural) as defined by respondents' area health region, born in Australia, speaking a language other than English at home, highest level of formal education, household income, living alone, self-rated health status, and psychological distress. Self-rated health status was assessed with the question "Overall, how would you rate your health during the past 4 weeks?" with possible responses being "excellent", "very good", "good", "fair", "poor", and "very poor". Responses of "very good" and "good" were combined and reported as "good" self-rated health, and responses of "poor" and Influenza Research and Treatment 3 "very poor" were combined and reported as "poor" selfrated health. Psychological distress was assessed using the Kessler 10 measure (K10). The K10 provides a measure of nonspecific psychological distress. Questions in the K10 include "In the past 4 weeks about how often did you feel . . ." "tired out for no good reason", "nervous", "so nervous that nothing could calm you down", "hopeless", "restless of fidgety", "so restless you could not sit still", "depressed", "that everything was an effort", "so sad that nothing could cheer you up", and "worthless". Possible responses were "all of the time", "most of the time", "some of the time", "a little of the time", and "none of the time". The K10 provides a score ranging from 10-50. For the current analysis a score below 22 was considered as low-psychological distress, and a score of 22 or above was considered as high-psychological distress.
Multiple survey logistic regression using stepwise backwards model was used in order to identify the factors significantly associated with risk perception. All variables with statistical significance of P ≤ .05 were retained in the final model.
In total, 2081 state residents aged 16 and over completed the module on pandemic influenza. The overall response rate was 65%. The key demographics of the weighted survey were comparable to Australian Bureau of Statistics (ABS) 2006 Australian population census data [21] .
Multiple survey logistic regression analyses using a backward stepwise method were performed for the nine outcome variables. Table 1 shows that Australian households who lived in rural areas were significantly more likely to think that pandemic influenza was very or extremely likely to occur than those in urban region (AOR = 1.59 (95% CI: 1.02-2.49, P = .041)). Respondents with poor self-rated health were also significantly more likely to think that pandemic influenza was very or extremely likely to occur, compared to those with good self-rated health (AOR = 1.92 (95% CI: 1.12-3.31, P = .018)) and were also more likely to report being very or extremely concerned that self or family would be directly affected if a pandemic was to occur (AOR = 1.64 (95% CI: 1.09-2.47, P = .017). Those from low income households, those who spoke a language other than English, and young people (16-24 years) were more likely to have changed the way they lived their lives because of the possibility of pandemic influenza, compared to their respective reference groups. Table 1 also shows that respondents who lived in rural areas and respondents who reported poor self-rated health were significantly more likely to report combined indicator (1) than those who lived in urban areas and those with good self-rated health. The odds of respondents with highpsychological distress reporting combined indicator (2) were 3.03 (AOR = 3.03) times higher than the odds of respondents with low-psychological distress levels reporting combined indicator (2).
The aim of this study was to assess factors associated with increased risk perception of pandemic influenza in Australia, increased concern for self and family if a pandemic influenza were to occur in Australia and associated changes in living due to the threat of such an event. Particular strengths of this study are the population-based sampling method and appropriate adjustment for sampling weight to reflect the population of interest.
Generally, pandemic influenza was not regarded as a high threat by NSW residents, with only 14.9% reporting that they felt pandemic influenza was very or extremely likely to occur. Those living in rural areas and those with poor self-rated health were more likely to report pandemic influenza very or extremely likely to occur compared with those living in urban areas and those with good self-rated health, respectively. Although not regarded as a high threat by Australians, 45.5% of respondents said they would be very or extremely concerned for self and family in the event of a pandemic influenza. Respondents with poor self-rated health were more likely to report more concern for self and family if an influenza pandemic occurred as compared to respondents with good self-rated health.
These results are dissimilar to those in prior studies. Although the studies of Lau et al. [11] and De Zwart et al. [12] found that females scored higher on risk perception than males, gender was not a significant risk factor for high-perceived pandemic likelihood or concern for self and family in this study. Similarly, although in prior studies older respondents [12] , those with lower socioeconomic status [10] and lower education [10] reported significantly higher risk perception, in the current study none of these were risk factors for high-perceived pandemic likelihood or concern for self and family. What is common to all these groups is that they represent the groups typically most vulnerable to concern due to a focal threat. The prior studies were conducted on populations responding to a tangible threat as they were conducted around either the time of the avian influenza outbreaks or on populations which were most affected by the avian influenza or the SARS outbreaks. As this study was conducted on an Australian population which was not directly affected by avian influenza or SARS and where influenza was not a media focus, the null findings in this study may indicate that the threat of pandemic influenza was so general and distal that it did not have the capacity to concern even the portion of the population normally most sensitive to threat.
It is not surprising that individuals with poor self-rated health reported greater risk perception and concern for self and others than individuals with good self-rated health. The health concerns of these individuals may lead them to believe they are more susceptible to infection or complications which may occur with an outbreak of pandemic influenza. Also, since poor self-rated health has been associated with increased levels of anxiety [22, 23] and distress [24] , there may be a heightened focus on, concern about, and belief in the likelihood of major external threats such as a pandemic influenza would represent. Such anxiety and distress may 4 Influenza Research and Treatment also lead these individuals to be more concerned about others as well as themselves in the event of a pandemic influenza. The potential link between self-rated health and heightened risk perception and concern for self and other in the event of a risky external event such as pandemic influenza warrants further examination. We can only speculate as to why individuals living in rural areas believed pandemic influenza was more likely to occur than those living in urban areas. Perhaps individuals living in rural areas are more broadly aware of disease transmission and its health and economic consequences, including the possibility of influenza transmission from animals to human. However, though individuals living in rural areas believed that pandemic influenza was more likely to occur than individuals living in urban areas, they did not display more concern for self and family should a pandemic influenza occur. This is not unexpected given that the influenza virus is more easily transmitted from person to person in crowded environments, and that rural environments are typically not densely populated. It might be expected however that individuals living in urban environments may be more concerned for self and family in the case of a pandemic influenza, as urban environments are typically crowded. This, however, was not the case in this study. Similarly, it is of particular note that in this study concern for self and family did not increase when there were children or elderly in the household, despite individuals in these age groups being particularly vulnerable to influenza morbidity and mortality. As suggested above, these null results might reflect that pandemic influenza is too distal a threat for concern for the whole Australian population. Perhaps higher levels of perceived likelihood and concern for self and family in the context of a specific imminent threat (e.g., swine flu) are required for significant group differentiation to emerge.
Generally, a minority of people had changed the way they live their life because of the possibility of pandemic influenza, with only 23.8% reporting they had changed their life at all. This is not surprising as the current data also indicate that few Australians believed pandemic influenza was likely to occur. Households which had a lower income, households which spoke a language other than English and those respondents aged between 16 and 24 were more likely to have changed the way they lived their life because of the possibility of a pandemic influenza than those households with middle-high income, those who only spoke English and those older than 24 years, respectively. Further investigation into specific actions people take to change their lives in response to the threat of a pandemic influenza may provide useful information.
Interestingly, all of the factors associated with living changes in the case of a pandemic influenza are independent of the factors associated with perceived likelihood and concern for self and family in the case of pandemic influenza. That is, these groups are reporting living changes in the absence of heightened perceived threat or concern relative to the remainder of the population. This suggests that these groups may not be changing their way of life because they feel pandemic influenza is more likely than the remainder of the population, or because they feel themselves or their families are particularly vulnerable should pandemic influenza occur, but for some other reason. It is possible that these results may be due to methodological issues. These groups (lower income, language other than English, and younger respondents) are somewhat marginalized groups, that we would expect to have a higher threat perception and concern. The results therefore may be due to the broad nature of the question, which may have tapped into a more general and pervasive sense of threat vulnerability within the community, such as upswings in terrorism, war, and climate change, which may have been felt more strongly in more exposed or vulnerable groups. Similarly, the response set for this question was extremely broad compared to the remaining questions. A respondent was considered to have changed his way of life if he reported to have done so a "little", "moderately", "very", or "extremely". This is in contrast to the perception and concern questions where only responses of "very" and "extremely" were included in analyses. Changed life a "little" could be interpreted by some respondents as an increased feeling of threat which represents a change in effect rather than in behaviour. Even though individuals with poor self-rated health believed that pandemic influenza was more likely to occur and felt more concern for self and family in the case of a pandemic influenza than those with good self-rated health, they were not more likely to report changing their life as a result of the possibility of pandemic influenza. Again, these populations may be responding to a more distal than proximal threat. Though they report some concern, it may not have been of the extent to prompt actual changes in way of living. More practically, neither the government nor the media were concerned at this time with promoting that the population makes life changes as a result of the threat of pandemic influenza, nor what these actions should be.
In a previous study, the role of concern for self and family was a key factor associated with likely compliance with protective health behaviours [25] . This suggests the benefit of risk communication messages that strategically heighten and then utilise public concern when a pandemic has or is likely to occur to increase compliance behaviours. For example, risk communication strategies could selectively target sub-population for whom risk beliefs are particularly low; in the current study these groups are urban populations and populations with good self-rated health. However, some authors have cautioned that increasing the risk perception of the population through such strategies risks societal estrangement and may frighten health care workers, first responders, and those who would have contact with the public in the event of a pandemic [26] . When consensus is reached regarding the optimal level of risk perception required for specific populations to elicit appropriate protective responses, the results of this study may be useful to guide which population groups these artificially inflating or deflating risk communication messages should be targeted.
It is likely that due to the recent H1N1 swine influenza pandemic that the current risk perceptions of the population are significantly different to those reported in this paper. Further research could examine changes in risk perceptions following this current pandemic for the whole population as well as the subpopulations examined in this paper. As such, the results of this paper provide a baseline measure for which future studies on risk perceptions can be compared. The population may have also recently made changes in daily living as a result of the H1N1 pandemic, as information on preventative measures such as personal hygiene have featured prominently in social marketing messages since its outbreak. This represents a response to a pandemic rather than a preventative measure for a potential pandemic. However, it would be important to determine which subpopulations maintain key behaviours (e.g., sneeze etiquette) following the end of the current pandemic, as this information can assist in the prevention of future pandemic threats.
With the outbreak of the current H1N1 swine influenza, research has emerged which has reexamined pandemic influenza attitudes and reactions in the Australian population. In research conducted during the World Health Organization (WHO) Phase 5 of the swine flu pandemic (between 2 May and 29 May, 2009) [27] , 21% of a Sydney-based sample ranked their risk of catching pandemic influenza as high. The same authors conducted a similar survey also on a Sydney-based population during the WHO Phase 6 of the swine flu pandemic (between September and October, 2009) and found that 17.4% believed they had a high to very high risk of acquiring H1N1 influenza [28] . In a CATI survey conducted between August and September, 2009, in an Australian nationally representative sample, Eastwood et al. [29] found that of the respondents, 5% were extremely concerned and 17% were quite concerned that they or a member of their family would contract swine influenza. Although in the latter study consideration was given to the reasons for concern (e.g., close family member/friend in high-risk group, having an underlying illness, being employed in a position with high public contact), none of these studies examined factors associated with increased perceived risk for acquiring swine H1N1 influenza. These results provide interesting comparison to the results of the current study. When the likelihood of pandemic influenza occurring was generally considered to be low, 45.5% of the population reported they would be concerned for themselves and their family should it occur. However, in the midst of the current pandemic, individuals perceived less risk to the self which has decreased as the influenza pandemic has progressed [27, 28] , and with as few as 22% [29] of the population reporting concern that they or a family member would contract the virus.
Lastly, consideration should be given to the limitations of the current study. The main limitation is that the study was conducted using telephone interviews which may have introduced selection bias. However, residential phone coverage in Australia remains high [17] , and a large number of studies on SARS and avian influenza have utilized this method. Also, risk perception and protective behaviours are likely to be mediated by a number of factors in addition to those identified in this study. Factors such as anxiety, risk perception of others, media, and recent events such as the current swine flu pandemic are all factors likely to affect risk perception. However, despite these limitations the results of this study suggest that it may be appropriate to direct risk communication strategies to individuals living in urban populations and individuals with good self-rated health, which may result in an increased likelihood of appropriate protective responses if an influenza pandemic was to occur. Data in this study further suggest that in contexts where pandemic influenza is generally not regarded as a high threat by the population, messages highlighting actions individuals can take to prepare for a pandemic influenza should be directed to households with higher incomes, households which do not speak a language other than English, and individuals above the age of 24 years. Finally, data from this study provide an Australian population baseline against which factors associated with risk perception related to outbreaks of pandemic influenza, both current and future, can be compared. Blow Flies Were One of the Possible Candidates for Transmission of Highly Pathogenic H5N1 Avian Influenza Virus during the 2004 Outbreaks in Japan The 2003-2004 H5N1 highly pathogenic avian influenza (HPAI) outbreaks in Japan were the first such outbreaks in 79 years in Japan. Epidemic outbreaks have been occurring in Southeast Asia, with the most recent in 2010. Knowledge of the transmission route responsible for the HPAI outbreaks in these countries remains elusive. Our studies strongly suggested that field and laboratory studies focusing on mechanical transmission by blow flies should be considered to control H5N1 avian influenza outbreaks, in particular in epidemic areas, where there are high densities of different fly species throughout the year. In this paper, we review these field and laboratory entomological studies and discuss the possibility of blow flies transmitting H5N1 viruses. The H5N1 subtype of highly pathogenic avian influenza (HPAI) A virus has frequently infected wild and domestic ducks in Asia, causing huge economic damage to both poultry farms and governments in the affected countries. Most avian influenza viruses do not infect humans, but the 1997 outbreak of the H5N1 virus in Hong Kong [1, 2] alerted the medical community that some subtypes of avian influenza viruses include highly pathogenic strains that can affect humans. In this influenza virus outbreak, there were 6 deaths in the 18 human cases caused by the H5N1 subtype [3] . As of August 2, 2010, WHO has identified 502 human cases of H5N1 influenza around the world, and 298 of these were fatal [4] . In particular, H5N1 outbreaks have occurred recently in Egypt, Indonesia, and Vietnam. Therefore, H5N1 influenza virus can cause serious public health problems in birds and humans and is one of the most infectious avian diseases transmissible to humans.
From January 2004 to March 2004, there were outbreaks of acute, highly transmissible, lethal diseases in chickens at four poultry farms in Japan: one in Oita, one in Yamaguchi, and two in Kyoto Prefecture ( Figure 1 ). Virus isolates from infected chickens were all identified as influenza A virus of the H5N1 subtype [5] . Such highly pathogenic avian influenza (HPAI) epidemics had not been reported in Japan for 79 years. Two avian influenza outbreaks at poultry farms in Tamba Town, Kyoto Prefecture, were the last two outbreaks of the 2004 avian epidemics in Japan. Since then, there were outbreaks of H5N1 avian influenza in Okayama and Miyazaki Prefectures in 2007. The H5N1 virus was also isolated from dead Whooper swans, Cygnus cygnus, in 2008 in Towada Lake, Akita Prefecture [6] . In addition, outbreaks of other subtypes of avian influenza virus have frequently occurred in Japan. The H5N2 avian influenza was reported in Ibaraki and Saitama Prefectures in 2005 and 2006, the H3 subtype was reported in Saitama Prefecture in 2009, and the H7 subtype was in Aichi Prefecture in 2009. We know of no report suggesting that H5N1 virus could be transmitted efficaciously from person to person, but the possibility remains that such transmission could evolve [7] .
Tamba Town (35 • 9 42 N and 135 • 26 31 E) is located in a hilly area 150-300 m above sea level, 50 km northwest of Kyoto City, Japan ( Figure 2 
Knowledge of the transmission route responsible for the HPAI outbreaks in Southeast and East Asian countries still remains elusive [5, 9] . Four hypotheses have been suggested for transmission of H5N1 in the HPAI outbreaks in Japan [6] : (1) H5N1-virus-infected chickens may have been imported from other countries, (2) materials (e.g., vehicles and egg containers) from infected area may have been used, (3) viruses may have been carried on clothes, boots, hands, and so forth, and (4) infected wild birds may have carried H5N1 virus into poultry farms to infect chickens. In particular, it has been suggested that migratory birds carried the viruses and subsequently infected domestic and/or wild ducks [9] . HPAI outbreaks in Japan. Therefore, it is very likely that the 2004 epidemics H5N1 virus was transmitted to Japan from East Asian countries. The important question is how could the H5N1 virus be transmitted from virus-positive migratory wild birds to domestic poultry in Japan? We have noted that it is unlikely that wild birds directly transmitted influenza viruses to poultry in Japan, because all of the Japanese poultry farms, where H5N1 virus outbreaks occurred, had fowling nets in place to prevent entry of wild birds. However, flying insects (e.g., flies) can easily get through the nets and invade a poultry farm. We have shown that a chicken can eat all 31 blow flies put inside its cage in just 7 min [10] . A chicken can catch and break down the body of a fly and even catch and swallow a fly in flight. Therefore, we have been interested in whether, if a chicken eats blow flies carrying H5N1 virus, the chickens might become infected and develop symptoms of H5N1 influenza.
Although the spring season in Japan is cold, some fly species are present. However, no studies have been reported on the possible role of flies in transmission of H5N1 influenza virus. Therefore, an entomological survey was conducted in March 2004 to investigate the possibility of blow flies transmitting H5N1 virus, using flies collected from around the infected poultry farm in Tamba Town for virus detection and isolation. Blow fly collection was carried out on 10-11 March 2004, just after the H5N1 outbreak at poultry farm B [11] . A sunny place protected from strong wind was selected, and rotten fish bait was placed on the ground. A total of 926 flies were collected within a 2.3 km radius of poultry farm A in Tamba Town ( Figure 2 ), representing eight fly species with >80% of the collected flies identified as either Calliphora nigribarbis Vollenhoven or Aldrichina grahami (Aldrich) (Figure 3 ). Influenza A virus matrix protein (M) and hemagglutinin (HA) genes were detected in the intestinal organs, crop, and gut of C. nigribarbis and A. grahami by reverse transcriptionpolymerase chain reaction (RT-PCR) [11] . The prevalence of H5 subtype virus (20-30%) was higher in flies of both species collected 600-700 m from poultry farm A and lower (10%) in flies collected >2 km from poultry farm A. We found that nearly 5% of C. nigribarbis collected around the affected areas contained infectious H5N1 viruses. viral M, HA, and NA genes were amplified by PCR with universal primers, full-length sequences were analyzed, and sizes were found to be 991, 1,707, and 1,362 bp, respectively. These sequences showed high similarity to those of strains from chickens (A/chicken/Kyoto/3/2004) and crows (A/crows/Kyoto/53/2004) isolated during the 2004 outbreaks in Kyoto, with >99.9% identity for all three genes. The virus from C. nigribarbis (A/blow fly/Kyoto/93/2004) was characterized as an H5N1 subtype influenza A virus based on neuraminidase gene (NA) sequences. In addition, the HA1-HA2 connecting peptide sequence in the HA gene segment was RERRRKKR↓G. Finally, virus isolated from C. nigribarbis was characterized as a highly pathogenic H5N1 subtype influenza A virus.
To investigate whether H5N1 virus could survive in the blow fly C. nigribarbis, we monitored the titer of infectious virus in flies after they were exposed to the H5N1 avian influenza A virus (A/duck/Hyogo/35/2001) [10] . Fifty female blow flies (Kyoto strain C. nigribarbis), approximately 14 days old, were put into a 20 cm 3 fly cage for 3 h at 20 • C with a piece of cotton impregnated with 10 8 EID 50 /mL allantoic fluid from an H5N1 virus (A/duck/Hyogo/35/2001 [10] )-infected egg diluted with MEM diluents [11] . Following the 3 h virus exposure, the blow flies were individually reared at 20 • C or 10 • C until tested. Incubation at 10 • C was chosen because the average temperature around Tamba Town was 3.6 • C in February and 6.4 • C in March, with average daytime highs of 11.1 • C in February and 13.1 • C in March [12] .
Crops and intestines dissected from flies' bodies at various times after virus exposure were used for virus isolation and titration. Virus was isolated from fly crops and intestines up to 24 h after exposure and from feces and vomit matter of 1 of 3 blow flies at 48 h after exposure (Table 1) 
Two species of the blow fly, C. nigribarbis and A. graham, are categorized as larger-sized fly species in particular in comparison to the house fly Musca domestica (L.); its body size is 5-8 mm (Figure 2 ). The body length of female C. nigribarbis is 11-15 mm and approximately 1.5 times larger than that of female A. grahami (8-13 mm) . The capacity of the crop of female C. nigribarbis (average = 23 mL) is approximately five times greater than that of female A. grahami (average = 4.4 mL) [11] . The consumption rate of both C. nigribarbis and A. grahami might have been high because of their large body size. In fact, virus genes were found more often in C. nigribarbis than in A. grahami [11] . Stable flies, Muscina stabulans (Falle'n) and M. angustifrons (Loew), collected at the same collection sites and the same time as the fly surveillance in Kyoto, showed much smaller body size than C. nigribarbis and A. grahami, and no virus was detected in these smaller-sized flies [11] .
Blow flies prefer to lick animal carcasses and droppings. If food for blow flies is contaminated by pathogens, the flies might ingest significant numbers of pathogens. One possible mechanism for mechanical transmission of pathogens by blow flies is regurgitation and the feces on the food source [13, 14] . The effectiveness of mechanical transmission through regurgitation may depend on the viability and titer of pathogens in the fly's body. The accumulated droppings at a poultry farm should be a good breeding site for blow flies. If the flies reproduced at a poultry farm, they should have many opportunities for contact with viruses in the feces of infected chickens and/or their dead bodies.
Calliphora nigribarbis has a characteristic temperate-zone life cycle. For example, in Japan, they become more active between winter and spring for migration and reproduction [15, 16] . It is well known that C. nigribarbis has excellent flight capacity and high dispersal ability. They have been identified by weather ships at stations located on the Pacific Ocean and East China Sea, 300-450 km from Kyushu Island, Japan [17] . It was also suggested that the number of flies found in autumn in the Kyushu District appears to increase due to their transoceanic migration [18] . Female blow flies can survive for about one year in Japan [17] , in comparison to the house fly Musca domestica which has a mean longevity of 34.2 days [19] . The longevity and high dispersal ability of blow flies may also result in wide dispersion of viruses that they carry. Mark-release-recapture experiments conducted at Tamba Town in 2005 suggested that C. nigribarbis generally could migrate up to 2-3 km in 24 h [20] . The distance between the two poultry farms in Kyoto prefecture, where the two H5N1 virus outbreaks took place in 2004, was approximately 4 km. In fact, 10% of all C. nigribarbis flies collected at a site intermediate between the affected farms expressed H5N1 virus genes [11] . Viable titers of H5N1 influenza virus, but not virus replication, were detected for up to 24 h in the crop and intestine of virus-exposed C. nigribarbis [10] . The presence of infectious virus in blow flies for 24 h could have a strong implication for virus dispersion since blow flies, with their excellent flight capacity, could transport the H5N1 virus over significant distances. In addition, H5N1 virus has been isolated from feces and vomit matter of blow flies at the 48 h postexposure, but virus titers in flies at 48 h were lower than that of the virus-containing cotton used in these experiments. This suggested that the viability of influenza virus decreases steadily in the blow fly crop and intestine, although some infectious virus remains for longer than 24 h. Therefore, C. nigribarbis could transport H5N1 virus to poultry farms 2-3 km apart.
How often do H5 influenza viruses migrate to Japan? Which subtypes of H5 influenza virus migrate to Japan? To following previous studies [10, 11] . No H5 influenza virus gene was detected from a total number of 96 fly pools examined ( 
It is well known that the domestic house fly, Musca domestica spp., and some other fly species can transmit many kinds of pathogens mechanically [21] [22] [23] [24] [25] . In particular, M. domestica spp. are the most important fly species at poultry farms [26] with regard to mechanical transmission of >30 different pathogens [13] , for example, bacteria, protozoa, viruses, and parasite oocysts and eggs. Some viruses can be transported to animals by contact with contaminated body surfaces of flies. In the case of the house fly, it has been shown that rotavirus can be mechanically transported by contaminated fly surfaces [21] . House flies frequently defecate while feeding and resting on food surfaces [27] . However, in studies of C. nigribarbis, neither defecation nor vomiting was observed within 24 h after feeding (data not shown). The body surface of the house fly could be contaminated by viruses easier than that of blow flies. This would suggest that the mechanisms of virus transmission by blow flies could be different from those of house flies. Therefore, to evaluate virus transmission mechanisms that are more complex than contact with a contaminated fly surface, blow fly intestinal organs, crop, and gut were analyzed for their possible role in transmission of avian influenza virus. A seasonal consideration is that M. domestica vicina populations are generally highest in the summer in Japan. In fact, no house fly was found around any poultry farm or pigpen in Tamba Town during our survey in March. Therefore, it seems reasonable that winter blow flies may be involved in transmission of winter pathogens, like influenza virus, by maintaining minimum infectious titers.
We have suggested here that blow flies are likely candidates for mechanical transmission of HPAI because of their ecological and physiological characteristics as reviewed here. In fact, blow flies have already been recognized as important vectors for mechanical transmission of several serious infectious diseases, that is, poxvirus [28] , rabbit hemorrhagic disease [29] , and paratuberculosis [30] . Recently, it has been reported that the H5N1 viral gene was detected in house flies [31] and engorged mosquitoes [32] . We suggest that mechanical transmission by flies may also be involved in the outbreak and pandemic of infectious diseases other than HPAI. However, although there are high densities of a variety of fly species during all seasons in Southeast Asia, their ability to transmit viruses has not been evaluated. The prevalence of H5N1 avian influenza is still a public health problem for birds and humans. Therefore, field and laboratory studies on mechanical transmission of pathogens by flies would be very important for controlling H5N1 avian influenza outbreaks, at least in epidemic Southeast Asian countries. Recently, the H5N1 virus surveillance conducted in Indonesia suggested that pigs are at risk of infection during outbreaks and pigs can serve as intermediate hosts in which this avian virus can adapt to mammals [33] . They also found the evidence of pig-to-pig transmission of this virus without any significant influenza-like signs. The transmission mechanism of this virus became more complicated and serious. As we introduced in the previous section, blow flies prefer to lick carcasses and droppings of not only chickens but also pigs. Furthermore, we can assume that the flies can access the pigpen easier than the poultry farm. This finding from Indonesia [33] strongly suggest that it is important to pay attention to pigpens as well as poultry farms within 2-3 km, where viable H5N1 viruses are transmitted by blow flies. Examining the knowledge, attitudes and practices of domestic and international university students towards seasonal and pandemic influenza BACKGROUND: Prior to the availability of the specific pandemic vaccine, strategies to mitigate the impact of the disease typically involved antiviral treatment and “non-pharmaceutical” community interventions. However, compliance with these strategies is linked to risk perceptions, perceived severity and perceived effectiveness of the strategies. In 2010, we undertook a study to examine the knowledge, attitudes, risk perceptions, practices and barriers towards influenza and infection control strategies amongst domestic and international university students. METHODS: A study using qualitative methods that incorporated 20 semi-structured interviews was undertaken with domestic and international undergraduate and postgraduate university students based at one university in Sydney, Australia. Participants were invited to discuss their perceptions of influenza (seasonal vs. pandemic) in terms of perceived severity and impact, and attitudes towards infection control measures including hand-washing and the use of social distancing, isolation or cough etiquette. RESULTS: While participants were generally knowledgeable about influenza transmission, they were unable to accurately define what ‘pandemic influenza’ meant. While avian flu or SARS were mistaken as examples of past pandemics, almost all participants were able to associate the recent “swine flu” situation as an example of a pandemic event. Not surprisingly, it was uncommon for participants to identify university students as being at risk of catching pandemic influenza. Amongst those interviewed, it was felt that ‘students’ were capable of fighting off any illness. The participant’s nominated hand washing as the most feasible and acceptable compared with social distancing and mask use. CONCLUSIONS: Given the high levels of interaction that occurs in a university setting, it is really important that students are informed about disease transmission and about risk of infection. It may be necessary to emphasize that pandemic influenza could pose a real threat to them, that it is important to protect oneself from infection and that infection control measures can be effective. Public cooperation in complying with infection control measures is required to minimize the spread of infectious diseases. Previous studies have demonstrated the positive correlation between willingness to adhere to the recommendations around infection control practices and perceived infectiousness and severity of the disease [1] [2] [3] [4] , perceptions about the effectiveness of control measures [5] and trust in the information being provided by national and international public health authorities [1] .
From the literature published to date on the general public's risk perceptions and behaviour changes during the 2009 influenza A/H1N1 pandemic [1, 6, 7] , higher risk perception scores were reported from Asian countries than from Western countries. For example, participants from studies conducted in India [8] , Saudi Arabia [6] and Hong Kong [9] expressed higher concern and perceived susceptibility levels than the respondents from studies conducted in the UK [1] and Australia [3] . While these variations may be correlated with methodological issues or the time period during the 2009 pandemic in which the study was conducted (i.e. May 2009 versus November/December 2009), if the trends are accurate it has the potential to affect the speed and extent to which infection control measures are accepted.
During the height of the pandemic, we undertook a study which aimed to measure the perceptions and responses of staff and students at our University [10] . While a large proportion of the sample reported either "no anxiety" or "disinterest", Asian respondents were significantly (p < 0.001) more likely to believe that the pandemic was serious compared to their counterparts from other regions. Although, most participants reported not adopting any specific behaviour changes, those who did were significantly more likely to be of Asian origin.
In order to further explore these trends amongst our domestic and international university students, we used qualitative methods to explore their attitudes, risk perceptions and adoption of health behaviour interventions against seasonal and pandemic influenza.
This study was carried out from May to August 2010. Qualitative semi-structured interviews were undertaken at the University of New South Wales (UNSW) in Sydney, Australia. The relevant Human Research Ethics Committee located at the university approved this study.
Students attending the main campus of the university were approached to participate in the study. Two methods were used to identify potential participants. Firstly, the interviewer (JP) directly approached a convenience sample of students who were located in the food halls and recreation areas of the university campus and invited them to participate. In the latter half of the study, a snowball approach was used. The snowball approach was adopted due to problems with identifying and recruiting postgraduate students. They constitute a considerably smaller percentage of the total student body, often are enrolled externally and attend classes in the late afternoon/evening. Students were classified on the basis of their enrolment status: undergraduate vs. postgraduate, and domestic vs. international. We aimed to recruit a sample of students from each classification and hence we firstly screened the student to identify their enrolment status. Students enrolled in the bachelor of medicine were excluded as it was assumed they would not be representative of the general student body, and would have had more exposure to issues surrounding disease spread and control. Participation was voluntary and written consent was obtained. During the study period, pandemic influenza H1N1 activity remained low and sporadic cases of pandemic influenza continued to be reported without evidence of sustained community transmission [11] .
The study researchers collaboratively developed an interview guide. Questions were shaped to cover the key areas of interest that included: knowledge, perceived severity, risk perceptions and concerns towards seasonal and pandemic influenza and personal health seeking behaviours and practices. Small variations in the questions were used to provide relevance for the overseas students. For example, we explored whether the international students believed their personal risk varied when located at home versus while residing in Australia and whether they had adopted or discontinued any health related behaviours whilst studying in Australia. We were not prescriptive around the term 'pandemic influenza'; instead we left it up to the student to interpret what they felt it meant to them. Pre-designed prompts were employed throughout the interview to trigger interviewees' thought. An interview face sheet was used to collect demographic information (sex, age, enrolment status etc.) from the participants. All interviews were conducted by JM and lasted up to one hour in length.
The interviews were recorded and transcribed verbatim. Two investigators (HS and JM) developed a list of themes after the analysis of one-quarter of the transcripts. An agreed framework was then applied to another subsample of transcripts and further modified. Using this final framework, all of the transcripts were analysed and coded. Text was organized within the identified themes of the developed framework. No software was used in the process.
A total of 20 university students' aged ≥18 years completed the interview (RR: 35 %). The participants ranged in age between 21 to 30 years and 70 % were born overseas (14/20). International students were over-represented in our sample (50 %, 10/20) compared to the actual proportion enrolled at UNSW (25 %).
There was a reasonably level of knowledge amongst the participants about the transmission modes and common symptoms of influenza and the common cold. A number of international students associated the occurrence of seasonal influenza with the temperature drop in winter; however they did not elaborated on the mechanisms of the connection.
"....people tend to get sick, the flu during winter because of the change in temperature, it gets colder. . .." (International postgraduate student) Many of the participants were unable to accurately define what pandemic influenza was. While avian flu or SARS were mistaken as examples of past pandemics, almost all participants were able to associate the recent "swine flu" situation as an example of a pandemic event. In comparison to seasonal influenza and colds, participants generally perceived pandemic influenza as being more serious. Pandemic influenza was associated with increased numbers of medical consultations/hospitalizations and a higher mortality rate. However, there were a few sceptical participants who were doubtful about the actual disease impact and felt that it was only "promoted" as being serious by the government and the media.
Young children and the elderly were nominated as being the most vulnerable groups during a pandemic outbreak due to their 'sub-optimal immune systems'. Participants believed that children were less conscious about hygiene and were therefore more likely to be exposed to other infected children or contaminated objects in a school environment. On the other hand, teenagers and young adults (20s-30s), the 'physically and socially healthy' and the 'well educated' , were considered to be at lowest risk of contracting pandemic influenza.
In regards to differences in risk between racially or culturally diverse groups, one participant commented that people or cultures that have frequent proximate interaction with each other were at risk of contracting the disease during pandemic. While another suggested that in countries where there are higher levels of respect for traditional medicines over western medicines, people may also be at risk. Amongst the international students it was suggested that the risk of contracting pandemic influenza is higher for people in their 'home towns' because of differences in their health care system, population density, personal hygiene practices and environmental quality.
". . .culture. . ...where they interact with a lot of people. . . such as Italians, they're very outgoing. . ., lots of interaction, proximate to each other, perhaps they're more prone," (Domestic postgraduate student) ". . ..some cultures where they have let say more respect for traditional medicine than modern medicine. . .. . ..are also going to be a problem. . .that's why lots of pandemic in say Asia and Africa, and not so much in Europe or America" (International postgraduate student)
Not surprisingly, only a few postgraduate participants stated that university students were at risk of catching pandemic influenza. When participants were required to rate their self-perceived risk of contracting pandemic influenza in Australia during an outbreak, almost all of them rated themselves at the low end of the scale. Being young and leading a healthy lifestyle were the major reasons provided to justify the low self-perceived risks levels. Only two overseas participants considered themselves at the 'relatively high risk' end. However, they presented very different justifications for this ranking, as one thought that their 'adventurous lifestyle' put them at risk and the other because of the dynamic nature of the university environment.
". . .may be I travel a lot more than the other people, and I go to polluted environments, institutions. . .. and I meet people I don't know. "(International undergraduate student)
Five of the international students perceived themselves to be at a higher risk of catching pandemic influenza in their 'home town' than in Australia. Differences in population density, quality of transport, connectivity to other countries, hygiene levels, accessibility to and quality of health care were the main reasons given for the differences.
Regular hand washing, cough etiquette (covering mouth and nose when coughing or sneezing), and avoiding the sick were suggested as good strategies to prevent becoming infected with pandemic influenza. The use of social distancing/isolation or masks/respirators was not very popular. Social avoidance was considered to be the most difficult intervention to comply with and impractical due to the vast amount of human interaction existing in the society. One international student also felt that it was impolite to maintain a distance to a sick acquaintance or relative. The use of masks was dismissed, as they were considered uncomfortable, inconvenient and unnecessary. Moreover, participants believed that wearing mask would cause embarrassment and social stigma. 
Outbreaks of seasonal influenza amongst student and university populations have been previously reported [12, 13] . These outbreaks have resulted in increased absenteeism, impaired school performance, and increased health care utilization [14] . The first reported university outbreak of 2009 pandemic H1N1 occurred at the University of Delaware (UD), affecting an estimated 10 % of the student population. It spread rapidly through the University of Delaware community with a surge in illness over a 2-week period. Although severe illness was rare in this instance, the authors documented that the outbreak caused a substantial burden and challenge to the university health care system [15] . In Japan, Uchida et al. reported that the infection rate among university students they surveyed ranged from 4.3 % to 15.5 % during the 2009 pandemic [16] . The authors suggested that continued exposure to sick individuals and disease transmission occurred during the pandemic, mainly through university club activities.
While our participants were knowledgeable about the modes of transmission of influenza, very few were able to accurately describe what 'pandemic influenza' actually meant. The participants had heard of 'swine flu' , however only a few demonstrated a high level of knowledge around how it originated. There was a lot of confusion around the role that animals play in regard to 'pandemics'. Unconfirmed beliefs and misconceptions regarding pandemic influenza H1N1 2009 have been previously documented [17, 18] .
In accordance with most of the pre-and post pandemic general public studies conducted worldwide [1, 2, 8, 19, 20] , our participants held a common belief that they were not at risk of acquiring the disease. Amongst our participants, it was felt that they were protected against the infection because they were 'fit and young'. This sense of non-vulnerability has also been previously documented in our previous university study [10] and amongst dormitory housed university students (aged18-23 years) in the USA [21] . However, given the low level of comprehension about pandemic influenza, the general public may be over or underestimating their level of risk towards acquiring the disease and the health consequences if infected (serious illness, need for hospitalization, mortality risk).
As highlighted through the interviews, our students believed in the classic picture of morbidity attributable to the flu, such that only the very young, the elderly, those with co-morbidities and those with weakened immunity are at risk. This result is consistent with the risk groups identified by participants in previous studies [4] . Given this low level of anxiety towards the pandemic, it is perhaps not surprising that the students did not undertake any behavioural changes in response to the H1N1 pandemic, as highlighted here and in our previous study [10] .
During the 2009 H1N1 pandemic, posters developed by the Commonwealth Department of Health and Ageing and UNSW were placed in high traffic areas. They focused on: (1) encouraging faculty, staff and students to stay at home if symptomatic (i.e. with a fever, cough, and runny nose) and to protect each other; (2) cough/sneeze etiquette (i.e. "cover your mouth and nose when you cough and sneeze" and "dispose of used tissues in the bin) (3) hand hygiene (i.e. "Wash your hands properly and regularly"). Our participants considered regular hand washing, cough etiquette (covering mouth and nose when coughing or sneezing), and avoiding the sick as good strategies to prevent infection. During the early and peak pandemic periods, hand washing was found to be the most accepted intervention among university students in Hong Kong [22] , Korea [23] , United States [24] and Australia [10] . Young people such as our university students may be more amenable to hand hygiene as a strategy because of a number of reasons. Firstly, these practices are community learnt and represent actions that the person has been encouraged to carry out from a young age. Secondly, hygiene-based measures pose minimal disruptions to daily routine. However, this is just a hypothesis and was not explored in depth in the study.
Amongst our participants, mask use, as an infection control strategy was extremely unpopular. In many western settings, where medical mask/respirator use is generally restricted to the hospital setting, it is not unanticipated that people would associate embarrassment and social stigma with the use of these products. At the University, it is extremely rare to see a student wearing a medical mask. This maybe because students believe that masks are uncomfortable, inconvenient and unnecessary. Habit is an important influence on routine behaviour [25] , including hygiene behaviour [26] , such that despite their best intentions people may find it difficult to implement new hygiene measures during a pandemic if they have not previously made these a habit.
The implementation of infection control behaviours appears to depend on a number of environmental (e.g. time, energy, availability of facilities, social norms), and motivational (e.g. social responsibility, social relationships, selfishness) factors. In the future however, the level of adoption of measures such as masks will fluctuate with changes in perceptions of risks and the perceived infectiousness and severity of the disease.
The use of voluntary home quarantine, social distancing, and school dismissal to prevent the transmission of pandemic influenza is a standard inclusion in most countries pandemic plans [27] [28] [29] . However, lower acceptance of isolation and social distancing, which can disrupt routine and enjoyable activities, has been observed in prior studies [30, 31] . When participants were asked to comment on how they felt about the use of these interventions they stated that they were not in favour of adopting these actions and would find them extremely difficult to comply with. During an outbreak of pandemic H1N1 virus infection at a large public university in April 2009, Mitchell et al. undertook an online survey of students, faculty, and staff to assess knowledge of and adherence to university recommended non-pharmaceutical interventions [24] . They found that amongst the students with an acute respiratory infection (ARI), 44 % reported leaving campus for >1 day while sick, 35 % had visitors and only 34 % reported missing days of class. Most students attended class or work, went out in public, and participated in purely social activities (including having visitors) while having an ARI. Aside from not wanting to miss these important events, it could be suggested that low risk perceptions and mixed messages about the severity of the 2009 influenza H1N1 pandemic and about the actual need for isolation and social distancing would probably have contributed to a low acceptance rate.
There are a number of logistical issues that universities and other institutions need to contend with instigating measures such as isolation. For example, universities may have large numbers of students living on or around the campus. While some of these facilities are self-contained, others have large common dining, entertainment and study rooms. The difficulty of introducing home quarantine in this setting is that many of these students (especially international and interstate students) may be unable to leave the campus facilities and would end up having to care and cater for themselves. Given the inevitability of future disease outbreaks or pandemic, universities must undertake efforts ensure that the needs of the students are catered for in these situations. Students, their parents, and other members of the university community must be involved with planning for these events so that feasible action plans are developed. These plans must ensure that there is continuity for the student.
A strength of this study was using interviews that allowed to uncover in greater depth the attitudes and perceptions of the students. However, there are several limitations in this study. These include: (1) over reporting: as the study was conducted through face-to-face interviews with our interviewer, it may have resulted in an overreporting of infection control behaviours to avoid embarrassment or judgement; (2) recall bias: some questions in the interview guide required participants to recall their past experiences during the 2009 H1N1 pandemic, therefore recalling errors may have occurred; (3) representation: as the study was undertaken one year after the pandemic, the responses received may not represent the attitudes that participants held during the pandemic; (4) participation rate was low.
Communicating to students effectively about the spread of influenza and the need to adopt preventative measures on a large campus presents a challenge. University officers need to find a balance between promoting and educating, while trying not to incite unnecessary fear. In the event of prolonged public health threats, such as infectious disease disasters, online messaging and regularly updated web sites have been shown to be timely and effective in providing risk communication and health messages [32] . However, it has also been demonstrated that pandemic influenza-specific web sites are among the least accessible and most difficult to understand compared with web sites addressing other types of disasters [33] . Poor accessibility can significantly undermine the effectiveness of university pandemic preparedness efforts and limit the ability of individuals to make well informed decisions during pandemics [34] . Other mediums favoured by young adults such as popular internet sites (Facebook or twitter) should be considered as a possible means of information provision to this susceptible cohort and in increasing uptake of preventative health advice. Education campaigns targeting young adults could also utilise the university networks and information gateways, or distribute information through universitywide emails and newsletters.
Given the high levels of interaction that occurs in a university setting, it is really important that students are informed about disease transmission and about risk of infection. It may be necessary to emphasize that pandemic influenza could pose a real threat to them, that it is important to protect oneself from infection and that infection control measures can be effective. Our participants believed that it would be extremely difficult to comply with infection control measures such as social distancing. In this university setting, practical measures may also be needed to support implementation, such as education, reminders and provision of hand gel.
Raina MacIntyre receives funding from influenza vaccine manufacturers GSK and CSL Biotherapies for investigator-driven research. These payments were not associated with this study. The remaining authors have no competing interests. Use of functional gene arrays for elucidating in situ biodegradation Microarrays have revolutionized the study of microbiology by providing a high-throughput method for examining thousands of genes with a single test and overcome the limitations of many culture-independent approaches. Functional gene arrays (FGA) probe a wide range of genes involved in a variety of functions of interest to microbial ecology (e.g., carbon degradation, N fixation, metal resistance) from many different microorganisms, cultured and uncultured. The most comprehensive FGA to date is the GeoChip array, which targets tens of thousands of genes involved in the geochemical cycling of carbon, nitrogen, phosphorus, and sulfur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). Since the development of GeoChips, many studies have been performed using this FGA and have shown it to be a powerful tool for rapid, sensitive, and specific examination of microbial communities in a high-throughput manner. As such, the GeoChip is well-suited for linking geochemical processes with microbial community function and structure. This technology has been used successfully to examine microbial communities before, during, and after in situ bioremediation at a variety of contaminated sites. These studies have expanded our understanding of biodegradation and bioremediation processes and the associated microorganisms and environmental conditions responsible. This review provides an overview of FGA development with a focus on the GeoChip and highlights specific GeoChip studies involving in situ bioremediation. As the most phylogenetically and functionally diverse group of organisms on the planet (estimated 2000-50,000 microbial species per gram of soil; Torsvik et al., 1990; Hong et al., 2006; Schloss and Handelsman, 2006; Roesch et al., 2007) , microorganisms are critical to ecosystem functioning and are involved in the biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus, and metals, as well as degradation or stabilization of contaminants in the environment. However, because a vast majority (>99%) of microorganisms remain uncultured (Amann et al., 1995; Fuhrman and Campbell, 1998; Whitman et al., 1998) , culture-independent approaches must be used to gain a comprehensive picture of microbial communities. However, many of the culture-independent methods, such as 16S rRNA genebased cloning or quantitative PCR, require a PCR amplification step, which introduces well-known biases (Suzuki and Giovannoni, 1996; Warnecke et al., 1997; Lueders and Friedrich, 2003) . In addition, since many functional genes have too much variance or too few sequences available, conserved PCR primers cannot be designed for many functional genes. Even if primers could be designed for many functional genes, performing PCR with many different primer sets would be cost-and time-prohibitive.
Microarrays allow the examination of thousands of genes at one time without the need for PCR amplification of each gene. Since microarrays were first shown to be valuable for the study of microbial communities (Guschin et al., 1997) , several types have been designed to examine microbial communities. These include (i) phylogenetic oligonucleotide arrays (POA), designed to examine phylogenetic relatedness or community composition using 16S rRNA or other conserved phylogenetic genes (Small et al., 2001; Loy et al., 2002; Wilson et al., 2002; Brodie et al., 2006) ; (ii) community genome arrays (CGA), designed to examine the relatedness of microbial species or strains or to identify community members using whole-genomic DNA probes Zhang et al., 2004) ; (iii) metagenomic arrays (MGA), designed as a highthroughput screening method using environmental clone library inserts as probes (Sebat et al., 2003; Mockler and Ecker, 2005; Gresham et al., 2008) ; (iv) whole-genome ORF arrays (WGA), designed to examine gene expression of individual microorganisms using probes for all ORFs in one or more genomes (Wilson et al., 1999) , but can also be used for comparative genomics (Murray et al., 2001) ; and (v) functional gene arrays (FGAs), designed to examine multiple functional genes at one time using probes for key genes involved in microbial functional processes of interest He et al., 2007 He et al., , 2010a . This review will focus on FGAs.
for examining and monitoring microbial communities and is even being used for metatranscriptome analysis (van Vliet, 2010) . However, while many of the technical challenges of microarrays and high-throughput sequencing have been overcome, each still has some distinct advantages and disadvantages, which make them ideal as complementary approaches: (i) Random sampling errors. In most sequencing studies, only a small proportion of the microbial community is actually sampled (McKenna et al., 2008) and while theoretically with true random sampling the probability of sampling the same fraction of the community multiple times is low , one would expect that dominant populations would have a greater chance of being sampled multiple times. These sampling errors can result in low reproducibility between technical replicates (17.2 ± 2.3% for two replicates; 8.2 ± 2.3% for three; Zhou et al., 2011) . Microarrays, in contrast, interrogate all samples against the same set of sequences (probes), so that the same population is sampled each time. (ii) Relative abundance. Abundance of individual species will vary greatly within microbial communities. With sequencing-based approaches there will be a bias toward the most abundant sequences in the environment so that many of the obtained sequences will represent the most abundant species/sequences while possibly missing lesser abundant species/sequences. Microarrays are not affected in the same way since lesser abundance sequences will still hybridize to their corresponding probe and as long as it is above the detection limit, it will be detected. (iii) New sequence detection. One of the greatest advantages of sequencing is that new sequences are easily detected since any sequences in the sample can be sequenced (open system). Microarrays, in contrast, can detect only the limited number of sequences covered by the probe set on the array (closed system), as such, it is not able to detect new sequences. Some new microarray techniques have been developed to allow the discovery of new sequences. Capture microarrays have been developed, which use lower stringency conditions to hybridize or "capture" sequence variants Okou et al., 2007) . These captured sequences are then washed off and sequenced. An array with probes specific for viral families has been developed that uses the hybridization pattern to classify novel viruses (Wang et al., 2002; Ksiazek et al., 2003) . As such, microarray and sequencing approaches could be used to maximize the benefits and minimize the deficiencies of each.
The first FGA developed used PCR-amplicon probes and targeted four N-cycling genes (nirS, nirK, amoA, and pmoA; Wu et al., 2001) . However, since PCR-amplicons were used, only a limited number of genes could be included because conserved primers can only be designed for a few functional genes. In addition, it would be cost-and time-prohibitive to amplify genes from hundreds of microorganisms or clones in order to achieve a truly diverse probe set. Most microarrays now use oligonucleotide probes, which are more specific (Zhou, 2003) , can be easily customized (Denef et al., 2003; Zhou, 2003; Gentry et al., 2006) , and are relatively inexpensive.
Since then several different FGAs have been developed, although most of these cover only a limited number of genes and focus on specific functional groups or locations. For example, FGAs have been designed to examine methanotrophs (Bodrossy et al., 2003 (Bodrossy et al., , 2006 Stralis-Pavese et al., 2004) , N-cycling genes (Taroncher-Oldenburg et al., 2003; Jenkins et al., 2004; Steward et al., 2004; Zhang et al., 2007a) , pathogens and virulence factors (Call et al., 2003; Kostić et al., 2005; Cleven et al., 2006; Miller et al., 2008; Palka-Santini et al., 2009) , rhizobial isolates (Bontemps et al., 2005) , and acid mine drainage (AMD) and bioleaching systems (Yin et al., 2007) .
The most comprehensive FGAs reported to date are the GeoChip arrays. The GeoChip 1.0 had 2006 oligonucleotide probes (50-mers) for genes involved in nitrification, denitrification, nitrogen fixation, methane oxidation, sulfate reduction (Tiquia et al., 2004) , organic contaminant degradation, and metal resistance (Rhee et al., 2004) . This array was used in several studies examining microbial communities at uranium (U)-contaminated sites (Wu et al., 2006a; Waldron et al., 2009) , in the Gulf of Mexico (Wu et al., 2008) , and under different land use strategies (Zhang et al., 2007b) and showed FGAs to be useful for microbial community studies. GeoChip 2.0 was developed to provide a truly comprehensive probe set for multiple functional gene categories and to provide increased specificity for highly homologous gene variants . GeoChip 2.0 contains 24,243 (50-mer) oligonucleotide probes targeting ∼10,000 functional genes from 150 gene families involved in the geochemical cycling of C, N, and P cycling, sulfate reduction, metal reduction and resistance, and organic contaminant degradation. This array has been used in numerous studies to examine microbial communities at metals contaminated sites (Gao et al., 2007; Van Nostrand et al., 2009 , oil or diesel-contaminated sites (Rodríguez-Martínez et al., 2006; Liang et al., 2009a,b) , coral mucus (Kimes et al., 2009) , lake or river samples (Taş et al., 2009; Parnell et al., 2010) , deep sea samples (Mason et al., 2009; Wang et al., 2009) , Antarctic soils (Yergeau et al., 2007) and to examine the taxa-area relationship .
GeoChip 3.0 covers 56,990 sequences from 292 gene families, greatly increasing the number of genes and categories covered compared to GeoChip 2.0 and added new control features (He et al., 2010a) . New gene categories include antibiotic resistance, energy processing, and phylogenetic markers (i.e., gyrB). A set of 16S rRNA gene probes were added as positive controls, human, plant, or hyperthermophile gene probes were added as negative controls, and a common oligo reference standard (CORS) was added for data normalization and comparison. The CORS is composed of an artificial sequence probe that is co-spotted with each gene probe and the complementary CORS target, labeled with a contrasting fluorescent dye to the sample, which is then spiked into each sample prior to hybridization (Liang et al., 2010) . The signal intensity of the CORS probe can then used to normalize the signal intensity of the sample and allows comparison of samples hybridized at different times. In addition, a computational pipeline has been developed for GeoChip probe design and data analysis. The GeoChip 3.0 has been used to examine microbial communities associated with elevated CO 2 (He et al., 2010b) , to examine communities within coal formation production waters (Wawrik et al., 2012b) or rhizosphere communities in As-contaminated sites (Xiong et al., 2010) . GeoChip 4.0, the newest version, is synthesized by Nimblegen (Madison, WI, USA) in their 12-plex format and contains 83,992 probes targeting 152,414 genes in 410 gene categories (Lu et al., 2012a) . In addition to added genes in most categories, new categories added include stress response, antibiotic resistance, and bacteriophage genes. It has been used to examine microbial communities during the 2010 Gulf oil spill (Lu et al., 2012a) ,
GeoChip covers a wide range of functional genes and currently includes sequences from bacteria, Achaea, fungi, and viruses. The first step in designing new probes for the array is deciding which processes should be included. Then genes for enzymes or proteins that are key to the process of interest are selected. These could be catalytic subunits or proteins with recognition sites or that provide functional specificity. Next, keywords are selected to search public sequence databases (e.g., GenBank). The keywords should be as broad as possible since proteins from different microorganisms may be annotated differently or have more general or specific annotations. Once the sequences are downloaded, they are confirmed by HMMER alignment 1 with preselected seed sequences. The seed sequences are those sequences for which the protein identity and function have been experimentally confirmed. This is a critical step in the design process and these sequences should be selected with care. The HMMER confirmed sequences are then used to design gene-or group-specific 50-mer oligonucleotide probes using new versions of the CommOligo software (Li et al., 2005) and experimentally determined criteria based on sequence homology (≤90% identity for gene-specific probes, and ≥96% for group-specific probes), continuous stretch length (≤20 bases for gene-specific probes, and ≥35 for group-specific probes), and free energy (≥35 kJ mol −1 for gene-specific probes, and ≤60 kJ mol −1 for group-specific probes; He et al., 2005b; Liebich et al., 2006) . The probes are then BLASTed against the GenBank database to confirm specificity. Keywords, downloaded sequences, seed sequences, HMMER confirmed sequences, and designed probes are stored in corresponding databases for use in future array updates.
The newly designed probe sets can then be commercially synthesized. Several options are available for producing arrays. Synthesized oligonucleotide probes can be spotted onto nylon membranes or glass slides (Taroncher-Oldenburg et al., 2003; Rhee et al., 2004; Tiquia et al., 2004) . Glass slides are more frequently used since they have less background fluorescence (Schena et al., 1995 (Schena et al., , 1996 and allow higher probe density (Ehrenreich, 2006) . Probes can also be added to slides using bubble Jet printing (Okamoto et al., 2000) , laser-induced forward transfer (Serra et al., 2004) , or photolithography (Chen et al., 2009 ). In addition, a few companies, such as Agilent or Affymetrix, synthesize custom microarrays using a customer's probe set.
GeoChip can be hybridized with either DNA or RNA. Most DNA samples used for GeoChip analysis are extracted using a 1 http://hmmer.wustl.edu/ well-established freeze-grind method with detergent lysis (Zhou et al., 1996; Hurt et al., 2001) since it provides high molecular weight DNA, important for later amplification steps. The use of RNA presents some challenges as mRNA is unstable and has a low abundance in environmental samples. Several papers have described methods for extracting environmental RNA, including a protocol for the dual extraction of both DNA and RNA Burgmann et al., 2003) or RNA alone (McGrath et al., 2008; Poretsky et al., 2009a) . Methods for mRNA enrichment include size separation by gel electrophoresis (McGrath et al., 2008) or use of commercial kits [MICROBExpress (Ambion) and/or mRNA-ONLY (Epicentre Biotechnologies); Poretsky et al., 2009b; Mettel et al., 2010] . Size separation obtained 115-155 ng mRNA from 4.6-5.3 μg total RNA (McGrath et al., 2008) . Using commercial kits, Mettel et al. (2010) were able to obtain 140-530 ng of mRNA from 0.4-2.0 μg total RNA per 0.5 g soil.
Nucleic acid quality is of great importance for microarray analysis. DNA and RNA should have an A 260 to A 280 ratio ∼1.8 and >1.9, respectively and an A 260 to A 320 ≥ 1.7. The A 260 to A 320 ratio is most important in determining microarray success (Ning et al., 2009) . Some environmental samples, especially those with high humics, can be difficult to purify up to the necessary level. A gel purification strategy followed by a phenol-chloroform-butanol extraction (Xie et al., 2007; Liang et al., 2011) has been successful with a wide range of soil and sediment samples.
Large amounts of DNA (e.g., 1 μg) or RNA (e.g., 5 μg) are needed for GeoChip hybridization. However, it can be difficult to get sufficient quantities of nucleic acid from some types of samples (e.g., water) or the sample is too difficult or impossible to replace to use such large quantities of nucleic acid. In this case, amplification of DNA or RNA can be done using either whole community genome amplification (WCGA; Wu et al., 2006a) or whole community RNA amplification (WCRA; Gao et al., 2007) . WCGA uses the Templiphi 500 amplification kit (phi 29 DNA polymerase, GE Healthcare, Piscataway, NJ, USA) with a modified amplification buffer and using 1-100 ng DNA provides a sensitive (10 fg detection limit) and representative amplification (<0.5% of amplified genes showed >2-fold difference from unamplified; Wu et al., 2006a) . WCRA provides a representative amplification with 50-100 ng of starting material.
There are commercial kits available for microbial RNA amplification such as the MessageAmp TM II-Bacteria RNA Amplification Kit (Life Technologies, Grand Island, NY, USA). There are also other commercially available methods for WCGA. Wang et al. (2011) compared two of these (Bacillus stearothermophilus DNA polymerase (Bst) and REPLI-g; Qiagen, Valencia, CA, USA) with the modified Templiphi kit (Wu et al., 2006a) . The amplification bias for all methods was relatively low (<3-fold). Less bias was observed with REPLI-g and Templiphi for pure culture DNA and with REPLI-g for community DNA while Bst showed the least inhibition by lesser quality DNA.
The amplified (or unamplified) nucleic acids are directly labeled with a fluorescent dye (Cy3 or Cy5) using random priming with the Klenow fragment of DNA polymerase for DNA (Wu et al., 2006a) or Superscript TM II/III RNase H-reverse transcriptase for RNA (He et al., 2005b) . The labeled DNA/RNA is then purified and dried for hybridization.
The labeled nucleic acids are then hybridized to the microarray at 42-50 • C with 40-50% formamide (He et al., , 2010a Lu et al., 2012a) . Hybridization specificity can be adjusted by varying the temperature or the formamide concentration (the effective hybridization temperature increases by 0.6 • C for every 1% of formamide). Hybridized slides are then scanned and analyzed by quantifying the pixel density (intensity) of each spot using image analysis software. Commercial manufacturers often have their own analysis software or other microarray analysis software can be used, such as GenePix Pro (Molecular Devices, Sunnyvale, CA, USA), GeneSpotter (MicroDiscovery, San Diego, CA, USA), or ImaGene (BioDiscovery, El Segundo, CA, USA). For GeoChip data, there is a data analysis pipeline 2 for rapid preprocessing and data analysis. Poor and low quality spots and outliers, based on Grubbs' test of outliers (Grubbs, 1969) , are removed and then the signal intensities of all spots are normalized. Positive spots can be determined using signal-to-noise ratio [SNR = (signal mean − background mean)/background standard deviation], signal-to-both-standard-deviations ratio [SSDR = (signal mean − background mean)/(signal standard deviation − background standard deviation)] (He and Zhou, 2008) , or signalto-background ratio (SBR = signal mean/background mean) (Loy et al., 2002) .
Due to the large volume of data obtained from GeoChip, data analysis can be very challenging. The data has a multivariate structure and the number of variables is much larger than the number of observations (p n). To assist users with data analysis steps, a pipeline is available which performs many of the common analyses 3 . Some common descriptive statistics used include relative abundance of gene categories or subcategories, richness and diversity (α and β) indices, and percentages of gene overlap between samples. To compare the overall community structure, unconstrained ordination [principal component analysis (PCA) and correspondence analysis (CA)] to reduce the dimensionality of variables in order to maximize the visible variability of the data or hierarchical cluster analysis (HCA), which groups communities based on the similarity of their gene profiles, can be used. To compare communities, response ratios, which compare the signal intensity of genes between conditions (Luo et al., 2006; Liang et al., 2009a) , t-tests, ANOVA, and dissimilarity tests can be used. Several methods can be used to examine the relationship between communities and environmental parameters. These include constrained ordination, such as canonical correspondence analysis (CCA; ter Braak, 1986), distance-based redundancy analysis (db-RDA; Legendre and Anderson, 1999), variation partitioning analysis (VPA; Økland and Eilertsen, 1994; Ramette and Tiedje, 2007) , and Mantel test. A relatively new analysis method is the random matrix theorybased (Mehta, 1990) neural network analysis (NNA) used to examine gene relationships within microbial ecological networks .
Having high-quality nucleic acids (non-degraded, large fragments to improve amplification yields, absence of inhibitors or contaminants which may impede subsequent amplification and labeling steps) is the most important criterion for successful microarray experiments. Nucleic acids can be purified using commercial kits although the presence of humic acids and other contaminants can still be a problem. If large amounts of DNA are present, an agarose gel purification followed by phenol-chloroform-butanol extraction (Xie et al., 2007; Liang et al., 2009b) can be used, but large amounts of DNA are lost with this method so it is not practical for low abundance samples. So, better purification methods with high recovery yields are needed.
One of the main objectives in developing FGAs was to provide a truly comprehensive probe set . Each new GeoChip version has expanded the coverage of gene variants and expands the number of genes covered (He et al., , 2010a Lu et al., 2012a) . This continued expansion is challenging as the number of gene sequences available is constantly increasing as new sequences are being submitted to public databases. While the GeoChip design pipeline 2 has an automated update feature which uses the previously selected key words and seed sequences to search the NCBI database, downloading new sequences and designing probes is still time consuming due to the sheer volume of sequences available. As such, better and faster computation systems are needed. In addition, available microarray probe density limits are rapidly being approached as the number of GeoChip probes increases. So, new methods of array construction to increase probe density are needed.
Two key issues for microarray hybridization of microbial communities are specificity and sensitivity since environmental communities can have such vast diversities. Both of these conditions can be improved at various stages of microarray design, construction, target preparation, or hybridization. During probe design, determining the best criteria for probe design, such as using experimentally determined design criteria (He et al., 2005b; Liebich et al., 2006) can improve specificity, thus decreasing the number of false positives . Probe length also affects specificity and sensitivity; longer probes are more sensitive, but less specific (Denef et al., 2003; He et al., 2005a) .
The method of array synthesis can also affect sensitivity and specificity. Increasing the probe concentration per spot can increase sensitivity (Cho and Tiedje, 2002; Relógio et al., 2002; Zhou and Thompson, 2002) . However, this may also decrease specificity by decreasing the overall probe signal intensity (Denef et al., 2003) . The choice of array surface can also be important as use of unmodified array slides can decrease background fluorescence thus requiring a lower signal fluorescence for detection (Kumar et al., 2000; Gudnason et al., 2008) .
Target preparation strategies can also affect these parameters. Amplification of community DNA can increase sensitivity. WCGA was able to representatively amplify 1-250 ng of community DNA (Wu et al., 2006a) , increasing the detection limit from 25 ng to 10 pg (2 bacterial cells); however, using such small quantities of starting material greatly increases the amplification bias compared to the bias observed with 1 ng of DNA. Labeling methods can also affect sensitivity. For example, cyanine dye-doped nanoparticles or tyramide signal amplification labeling are able to increase sensitivity 10-fold (Denef et al., 2003; Zhou and Zhou, 2004) .
Hybridization conditions can also be used to increase specificity and sensitivity. Temperature and formamide concentration can be modified to adjust stringency thus altering specificity . A lower hybridization solution volume and mixing during hybridization (Adey et al., 2002) have both been shown to increase sensitivity. Decreasing ozone levels, which can degrade cy-dye signal (Branham et al., 2007) , can also improve sensitivity.
Most GeoChip analysis has involved the use of DNA, so that only gene abundance can be determined. These changes can be used to infer microbial activity, but cannot provide direct proof of activity. mRNA can be used for FGA analysis to monitor activity (Dennis et al., 2003; Bodrossy et al., 2006; Gao et al., 2007; Wawrik et al., 2012a) , although as mentioned above, working with environmental RNA can be challenging. Stable isotope probing (SIP) has also been used with GeoChip to monitor microbial activity (Leigh et al., 2007) . Gao et al. (2007) used amplified community mRNA from a denitrifying fluidized bed reactor to examine microbial activity. Genes for nitrate and nitrite reduction, organic contaminant degradation, sulfite reduction, and polyphosphate kinase were detected, consistent with reactor operation (Gao et al., 2007) . Another study used amplified community mRNA to examine nitrate utilization in marine bacterial communities (Wawrik et al., 2012a) . Hybridization results indicated activity by ureC, nirS, nirK, narG, nosZ, napA, nrfA, amoA, and nifH genes, indicating that urea cycling, denitrification, dissimilatory nitrate, nitrite reduction, and N fixation were occurring (Wawrik et al., 2012a) .
Another method of monitoring microbial activity with GeoChip is to combine it with SIP (Leigh et al., 2007) . Microcosms were set up from soil samples collected from the root zone of a tree growing in a PCB-contaminated site and fed 13 C-labeled or unlabeled biphenyl. Genes involved in biphenyl degradation were detected as were other organic contaminant degradation genes including those for degradation of benzoate, catechol, naphthalene, and phenol.
Several GeoChip-related studies have examined microbial communities from U-contaminated groundwater at the U.S. Department of Energy (DOE) Oak Ridge Integrated Field Research Challenge (OR-IFRC) site. Groundwater samples covering a range of contamination levels and an uncontaminated background sample were compared using GeoChip 1.0 (Wu et al., 2006a) . Samples from the uncontaminated site and those with lower levels of contaminants had higher functional gene diversity and gene numbers. In addition, as expected based on the contaminants present at this site, genes for denitrification, organic contaminant degradation, metal resistance, and sulfite reduction (dsr) were detected. A similar sample set using the same array was examined in greater detail in a later study (Waldron et al., 2009) . In this study, sulfate, pH, U, and Tc were found to be the most important drivers in determining the microbial community structure, with pH and the combination of U and Tc explaining ∼21% of the variance observed or 29-40% when all four variables were included.
Another study at this site examined a pilot-scale field bioremediation system which used ethanol as an electron donor to stimulate microbial communities and immobilize U(VI) by reduction to U(IV) (Luo et al., 2006; Wu et al., 2006b,c) . GeoChip 2.0 was used to examine the microbial communities during different phases of operation. A period of active U(VI) reduction occurred after initial start-up (days 137-304). During this period U(VI) reduction was relatively rapid and genes associated with denitrification, sulfate reduction, and Fe(III) reduction increased in abundance, suggesting that these populations were involved in U(VI) reduction . This active reduction was followed by a maintenance period during which the low level of U(VI) was maintained, and the denitrifying, sulfateand Fe(III)-reducing communities remained in higher abundance. Next, the stability of the bioreduced U(IV) was examined by allowing the system to enter periods of starvation (ethanol injections were halted) and reoxidation (dissolved O 2 entered the system). The functional communities showed distinct clustering patterns based on whether the system received ethanol or not, indicating a shift in community structure with the return of ethanol injections . While total gene numbers increased once ethanol injection was restarted, the relative abundance of each gene group changed little during and after starvation, indicating a functionally diverse community which could be stimulated after adverse conditions. Chemical oxygen demand (COD, i.e., ethanol) was the most important driver in determining community structure, but temperature, sulfate, and U(VI) were also important.
In this same remediation system, the sediment microbial community was examined with GeoChip 2.0 after 2 years of operation (Xu et al., 2010) . Sediment samples were collected from 11 wells, 5 from the outer loop and 6 from the inner loop. Results revealed significant differences between the microbial communities in the inner and outer loops. The inner loop communities had higher gene numbers and greater diversity than those in the outer loop and inner and outer loop samples were grouped separately based on hierarchical clustering and principle component analysis, indicating that the ethanol injections stimulated the microbial communities in the inner loop. In addition, genes important for U(VI) reduction such as cytochrome c, dsr, and denitrification as well as genes involved in metal resistance and organic contaminant degradation were enriched in the inner loop where electron donor was added. This study demonstrated the importance of U(VI)reducing populations for the maintenance of reducing U(IV) in this bioremediation system.
Another GeoChip 2.0 study examined groundwater microbial communities at a field site examining the use of acetate to stimulate U(VI)-reducing microorganisms in the subsurface at the Old Rifle site, a former U ore processing facility in Rifle CO (Liang www.frontiersin.org et al., 2012) . The study compared communities taken during a shift from sulfate to Fe(III)-reducing conditions. The overall community structure changed with the switch from Fe(III)to sulfate-reducing conditions and were reflective of the redox conditions at the site. Sulfate-reducing and methane-generating microorganisms increased in abundance under sulfate-reducing conditions. Acetate, U(VI) and redox potential were important environmental variables in determining the microbial community structure. Xie et al. (2011) examined five AMD sites in China using GeoChip 2.0 to determine the functional diversity and metabolic potential of microbial communities in these sites and to determine how the communities responded to environmental conditions. The sites showed a great deal of variability in regards to the microbial communities with ∼150-1000 functional genes detected in each sample. Most of the genes represented on the GeoChip that were involved in C, N, S cycling and metal resistance were detected in all of the AMD sites. Results indicated that the immediate environmental conditions were important in forming the variations in the functional structure of microbial communities as opposed to spatial distance. There was a positive correlation between Zn resistance gene abundance and Zn concentration but not for other metals. However, the concentrations of B, Co, Cu, La, Mg, and S were significantly correlated with the community structure in these communities. Overall, results suggested that AMD microbial communities may not be as simple as previously thought.
GeoChip 2.0 has also been used to probe pure culture isolates for the presence of specific genes. Four Ni-resistant Gram-positive actinomycetes were hybridized to GeoChip to get a better idea of what metal resistance genes were present (Van Nostrand et al., 2007) . Genes associated with resistance to Al, As, Cd, Cr, Cu, Hg, Ni, Te, and Zn were detected.
Microbial communities from the rhizosphere of the arsenichyperaccumulating plant Pteris vittata and non-rhizosphere samples were examined using GeoChip 3.0 (Xiong et al., 2010) . The functional gene diversity was significantly correlated with As concentration. Interestingly, As contaminated rhizosphere samples had higher functional gene diversity than non-rhizosphere samples even though the non-rhizosphere samples had a lower level of As. In addition, greater numbers of As resistance genes, with higher signal intensities, were detected in rhizosphere samples compared to non-rhizosphere samples and very few genes were detected in both environments, suggesting that the rhizosphere and non-rhizosphere microbial communities were distinct. Results suggested that the P. vittata rhizosphere may protect the microbial communities from As contamination.
Another study used GeoChip 2.0 to examine microbial communities in Zn-and Cd-contaminated soil microcosms with or without Thlaspi caerulescens, a Cd and Zn hyperaccumulator plant (Epelde et al., 2010) . Higher numbers of functional genes were detected in the contaminated samples than in uncontaminated samples and in planted samples compared to unplanted. Thirty-five to forty-seven percent of the variation in community structure observed was explained by metal concentrations. All of the Cd and/or Zn resistance genes (12) were detected in the contaminated, planted samples while only 7 were detected in the contaminated/unplanted samples. Substrate-induced respiration, K concentration, and nitrate concentration were the most important environmental variables in determining the functional community structure.
The microbial community associated with a bioremediation system comprised of a fluidized bed reactor to clean dieselcontaminated groundwater in Vega Baja, Puerto Rico was examined with the GeoChip 1.0 (Rodríguez-Martínez et al., 2006) . Genes involved in the degradation of diesel fuel and other organic contaminants (acetylene, aniline, benzoate, biphenyl, cyclohexanol, methyl tert-butyl ether, naphthalene, phthalate, protocatechuate, and toluene) were detected. Increased signal intensities for genes involved in anaerobic benzoate degradation indicated a shift toward anaerobiosis over time, a conclusion supported by other experimental evidence. Liang et al. (2009b) examined the effect of different bioremediation treatments on microbial communities using laboratory scale bioremediation systems with sediment from contaminated oil fields and inoculated with oil degrading enrichment cultures. The systems were incubated 242 days, treated with ozone, and incubated an additional 125 days. Many oil degradation genes (benzene, benzoate, catechol, polyaromatic hydrocarbon aromatics, protocatechuate, phthalate) were detected with GeoChip 2.0. Ozonation treatment resulted in an almost 50% reduction in the number of functional genes detected. Gene numbers increased again after a recovery period and the community retained the ability to degrade oil.
Another study used GeoChip 2.0 to characterize microbial communities along an oil contaminant gradient and found a decreased number of functional genes as the contaminant levels increased although genes involved in the degradation of biphenyl, catechol, and protocatechuate increased in the more contaminated samples (Liang et al., 2009a) . The most important environmental factors in determining the microbial community structure were oil concentration and soil available nitrogen. Liang et al. (2011) collected contaminated and uncontaminated soils from five oil fields across China in order to determine whether oil contamination or geographic location played a larger role in determining the microbial community structure. Results from GeoChip 2.0 indicated that communities from uncontaminated sites had higher functional gene diversity than those from contaminated sites in the same geographical area. Overall, the microbial communities clustered based on geographic location; however, when only organic contaminant degradation genes were examined, the contaminated samples clustered together. Geographic location was able to explain ∼33% of the microbial community variation observed, oil explained ∼10% of the variation, and soil geochemistry explained another 12%, while the remainder (∼41%) was unexplained.
GeoChip 4.0 was used to compare microbial communities in oil-contaminated water to those from uncontaminated water in order to understand the effects of the 2010 Gulf of Mexico oil spill Lu et al., 2012a) . Results indicated that after only 40 days the presence of the hydrocarbon plume Frontiers in Microbiology | Microbiotechnology, Ecotoxicology and Bioremediation (1100 m depth) caused a significant shift in the microbial community functional structure and composition and that indigenous microorganisms, similar to known petroleum degraders, were stimulated by the hydrocarbon plume. Many genes associated with hydrocarbon degradation were significantly enriched in plume samples Lu et al., 2012a) . Genes that were enriched in plume samples included those for naphthalene 1,2-dioxygenase, β-oxidation of benzylsuccinate, cyclohexanone 1,2-monooxygenase, and alkene monooxygenase (Lu et al., 2012a) . These findings suggest that the microbial communities in the Gulf of Mexico were capable of intrinsic bioremediation and that the presence of the oil stimulated the oil-degrading community members and were important in determining the fate of the deep-sea oil spill.
In a study using GeoChip 2.0 to examine three atrazinecontaminated aquifers and a background site, Liebich et al. (2009) detected more genes in the background site compared to the contaminated sites. The aquifer with the highest level of contamination had the highest number of genes, most involved in contaminant degradation, compared to the other contaminated samples. Atrazine-degradation genes were detected in all contaminated samples and verified by PCR. These results indicated that even small amounts of contaminant were enough to select for specific degrading populations.
River sediments from industrial pollutant and pesticidecontaminated sites were examined with GeoChip 2.0 and the results indicated that contaminant level was not a major driver in these systems (Taş et al., 2009) . Instead, C/N ratio, depth, total Kjeldahl N, and location were the strongest drivers in determining the community structure. Most of the reductive dehalogenation genes detected were from Dehalococcoides spp., suggesting that this microorganism may play an important role in contaminant degradation in this system.
GeoChip 2.0 was used to examine phenanthrene-spiked soil microcosms to examine the effect of phenanthrene on microbial communities (Ding et al., 2012) . Communities were examined after a 21-day incubation and compared with communities from day 0. A larger number of genes were detected in spiked soils compared to the control soils. Genes showing an increase in the spiked soils included dioxygenases involved in aromatic compound degradation, genes involved in the degradation of PAHs (nahA, rhda, nahQ, narR) , and genes involved in the degradation of one-ring aromatic compounds. In addition, an overall shift in community composition and structure was noted in spiked soils as determined by non-metric multidimensional scaling.
Another study examined microbial communities associated with a leachate-contaminated landfill using GeoChip 3.0 (Lu et al., 2012b) . Groundwater samples were collected from wells along a flowpath of the landfill. Communities directly under the landfill and in the closest well had significantly lower functional gene diversity and richness. Genes involved in the anaerobic degradation of organic contaminates such as aromatic acids (bclA, bbs, tutFDG), phenoxyacetic acid herbicides (ftdA) atrazine (atzABC, trzN, trzA, trzE) were detected in all wells. Based on canonical correspondence analysis, the environmental variables (pH, sulfate, ammonia, and dissolved organic carbon) had significant effects on the community structure.
The GeoChip arrays have been shown to be powerful tools in linking microbial function to ecosystem processes and are able to provide sensitive, specific, and potentially quantitative information. Use of this array in bioremediation studies have expanded our understanding of the microbial processes and communities at work in these sites and provide information necessary for the successful improvement and application of bioremediation strategies. Over the past decade, great improvements have been made in regards to microarray technology, design, and application. However, there are still technical hurdles that need to be overcome to further improve sensitivity and specificity in addition to better methods of nucleic acid extraction and purification. Improved bioinformatics tools are also needed to assist with data processing and analysis.
The effort for preparing this review was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231 through ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory and the Oklahoma Applied Research Support (OARS), Oklahoma Center for the Advancement of Science and Technology (OCAST), the State of Oklahoma through the Project AR062-034. Functional Studies of ssDNA Binding Ability of MarR Family Protein TcaR from Staphylococcus epidermidis The negative transcription regulator of the ica locus, TcaR, regulates proteins involved in the biosynthesis of poly-N-acetylglucosamine (PNAG). Absence of TcaR increases PNAG production and promotes biofilm formation in Staphylococci. Previously, the 3D structure of TcaR in its apo form and its complex structure with several antibiotics have been analyzed. However, the detailed mechanism of multiple antibiotic resistance regulator (MarR) family proteins such as TcaR is unclear and only restricted on the binding ability of double-strand DNA (dsDNA). Here we show by electrophoretic mobility shift assay (EMSA), electron microscopy (EM), circular dichroism (CD), and Biacore analysis that TcaR can interact strongly with single-stranded DNA (ssDNA), thereby identifying a new role in MarR family proteins. Moreover, we show that TcaR preferentially binds 33-mer ssDNA over double-stranded DNA and inhibits viral ssDNA replication. In contrast, such ssDNA binding properties were not observed for other MarR family protein and TetR family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Overall, these results suggest a novel role for TcaR in regulation of DNA replication. We anticipate that the results of this work will extend our understanding of MarR family protein and broaden the development of new therapeutic strategies for Staphylococci. Staphylococci are among the most common causes of bacterial infection in the community and pose a major danger to human health. S. aureus is the most well-known of the species which produce hospital-and community-acquired infections, with methicillin-resistant S. aureus presenting a serious public health threat [1] . S. epidermidis is the sister species of S. aureus which often causes infection in immunocompromised individuals or those following damage to the epithelium. Both of them produce biofilm to protect themselves from host immune system and enhance their resistance to antibiotic chemotherapy [2] . The key component of the biofilm extracellular matrix in Staphylococci is polysaccharide intercellular adhesin (PIA) [3] , an essential factor in biofilm formation composed of homopolymer of b-1,6-linked N-acetylglucosamine (GlcNAc). The production of PIA depends on the expression of the icaADBC operon, and TcaR and IcaR are a weak and a strong negative regulator of transcription of the ica locus, respectively [4] .
The transcription regulator TcaR is a member of the MarR family, and is involved in teicoplanin and methicillin resistance in Staphylococci [5] . The MarR family proteins function as regulators of protein expression and these regulated proteins confer resistance to multiple antibiotics, household disinfectants, organic solvent virulence factors, and oxidative stress agents [6, 7, 8, 9, 10, 11] . The crystal structures of a number of MarR family proteins have been reported, including MarR from Escherichia coli [12] , OhrR from Bacillis subtilis [13] , MexR from Pseudomonas aeruginosa [14] , MarR from Xanthomonas campestris [15] , SlyA from Salmonella typhimurium [16] , AdcR from Streptococcus pneumonia [10] and TcaR, which is studied in this work, from S. epidermidis [17] . These structures revealed that MarR family proteins are all homodimers. The overall structure of each monomer could be divided into two functional domains, one is the dimerization domain and the other is the winged helix-turn-helix (wHTH) DNA binding domain. The N and the C-terminal a-helices (a1, 5, 6) of one monomer interdigitate with those of the other monomer to produce dimerization interaction. In addition, the wHTH DNA binding domain is composed of a2-a3-a4-bA-W1-bB which adopts the winged-helix-fold, and the amino acid sequences of this domain are highly conserved.
As the MarR-type proteins can act as positive, negative, or bifunctional regulators, TcaR also acts as a multi-functional regulator. It is not only as a regulatory factor to affect the transcription of icaADBC [4] , the first regulator reported for cell wall-anchored proteins (SpA and sasF), but also as the regulator of sarS [18, 19] . We previously described the 3D structures of TcaR in its apo form and in complex with salicylate as well as several aminoglycoside and b-lactam antibiotics [17] . In this research, comparison of the native TcaR structures and those in complexes indicated that the regulation mechanism involves a large conformational shift in the DNA binding lobe. Several antimicrobial compounds inhibited TcaR-DNA interaction and further induced biofilm formation in S. epidermidis. In the present study, we found that TcaR could interact with ssDNA and the result demonstrated that TcaR shows a stronger preference toward GCrich ssDNA than to dsDNA by using EMSA, CD, and Biacore experiments. However, the detailed mechanism of the interaction between TcaR and ssDNA still remains to be elucidated. In order to investigate the regulation mechanism of the ssDNA binding ability of TcaR, we applied electron microscopy (EM) technique to reveal TcaR-ssDNA complex. Furthermore, we clarified the role of TcaR-ssDNA interaction by in vitro replication assay and in vivo plaque assay. Taken together, these results provide an in-depth investigation on the multiple functions of TcaR in S. epidermidis.
Strong TcaR Binding to ssDNA Oligomers Revealed by EMSA TcaR is known to bind and regulate the ica promoter [4] . We previously identified that TcaR most strongly interacts with IcaR DNA1 (a 33-mer pseudo-palindromic sequence containing consensus sequence TTNNAA) compared with other designed IcaR DNA fragments [17] . However, when using the sense strand of IcaR DNA1 (IcaR DNA1S) and the antisense strand of IcaR DNA1 (IcaR DNA1A) ( Figure 1A ) as controls in electrophoretic mobility shift assays (EMSA), the result demonstrated that TcaR shows a stronger preferences toward ssDNA fragments (IcaR DNA1S and DNA1A) ( Figure 1B) . To determine the type and length of the TcaR-binding site on ssDNA, a series of GC-rich and AT-rich ssDNA segments were designed ( Figure 1A ) [20, 21] . Their TcaR binding ability was tested using EMSA. As shown in Figure 1C , TcaR does not significantly interact with 17-mer GCrich (GC17) and AT-rich (AT17) ssDNA oligomers, but shows strong interaction with 33-mer GC-rich (GC33) and AT-rich (AT33) ssDNA sequences with a preference toward the 33-mer ssDNA sequence with a molar ratio of 1:1. Thus, we suggest that TcaR prefers binding to the 33-mer ssDNA.
In order to evaluate the minimal DNA binding length of TcaR, GC-rich fragments of different lengths were designed. As seen in Figure 1D , GC-rich fragments with 33, 29, and 25 bases showed similar binding strength to TcaR; with TcaR forming a large, apparently multimeric complex with GC33, a small complex with GC25, and both small and large complexes with GC29 in EMSA. These results indicated that the minimal observed ssDNA fragment size to allow TcaR binding ranges between 17 to 25mer; providing useful information for the design of a DNA fragment with precise length suitable for crystal packing. Up to now, only three MarR family protein complex structures have been reported, and the first one is complexed with dsDNA [13, 16, 22] . The second one is complexed with salicylate [12, 17, 22, 23] and we discovered the third case which is complexed with antibiotics [17] . We have already obtained TcaR-ssDNA crystals and collected X-ray diffraction data to 3.6 Å resolution at SPring-8 (Hyogo, Japan), beamline BL12B2. However, the phase problem is still the main challenge and the works are currently under progress.
Moreover, to investigate whether TcaR preferentially binds to ssDNA or dsDNA, the ability of ssDNA to compete with the TcaR-dsDNA complex was evaluated. For the competition assay, the IcaR DNA1 probe was preincubated with TcaR (dimer) protein to allow formation of the dsDNA-TcaR complex prior to mixing with increasing amounts of single-stranded GC33 DNA. It has been known that ssDNA products have lower migration velocity compared to its dsDNA counterparts in polyacrylamide electrophoresis [24, 25] . As shown in Figure 1E , ssDNA, as a competitor, interfered the binding of TcaR to the dsDNA, suggesting a binding preference for ssDNA. To further confirm this result, IcaR DNA1 and GC33 ssDNA oligomers were mixed, and their interaction strengths with TcaR were compared using EMSA ( Figure 1F ). Findings indicated that increasing the concentration of TcaR produces a ssDNA band shift greater than that for dsDNA, confirming a stronger interaction between TcaR and ssDNA. Moreover, to investigate possible pH effect of ssDNA binding activity of TcaR, a series of buffers with increasing pH were tested for their potential interfere in TcaR-ssDNA binding. As shown in Figure 1G , the EMSA results showed that TcaR had a strongest affinity for GC33 at pH 8.0 and the affinity was reduced by decreasing pH. Consequently, the result indicates that the ssDNA binding activity of TcaR is pH-dependent.
To clarify whether the ssDNA binding site of TcaR is close, or identical, to the dsDNA binding site, a TcaR quadruple mutant (4 positively charged amino acids responsible for DNA binding mutated to alanines to produce R71A/K73A/R93A/K95A) [17] was designed and its ssDNA and dsDNA binding ability tested. As seen in Figure 1H , the mutant failed to interact with either dsDNA or ssDNA. This indicated that these amino acids are essential for binding in both ssDNA and dsDNA.
The MarR protein of E. coli is a multidrug binding transcription regulator. A wide range of compounds, including 2,4-dinitrophenol, plumbagin, menadione, and salicylate, attenuate and inhibit its association with cognate DNA [26] . In our previous study, salicylate and multiple antibiotics interfered with the transcriptional repressor activity of TcaR [17] . These findings prompted the current investigation into the possible effects of antibiotics on the ssDNA binding ability of TcaR. Here, to investigate the possible effect of some drugs on TcaR, four compounds were tested for their potential inhibition on TcaR-ssDNA interaction. These include one beta-lactam antibiotics (ampicillin) that contain a b-lactam nucleus in their molecular structure and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls, two aminoglycoside antibiotics (kanamycin and gentamicin) that composed of several sugar groups and amino groups, and bacteriostatic antimicrobial (chloramphenicol) which is considered as a prototypical broad-spectrum antibiotic.
As shown in Figure 1I , kanamycin, chloramphenicol and gentamycin interfered with the ssDNA (GC33) binding activity of TcaR at a concentration of 2 mM and this effect was more pronounced at a higher concentration, suggesting that antibiotics inhibit formation of the TcaR-ssDNA complex. Results indicated that ampicillin also antagonized the ssDNA binding activity of TcaR at a concentration of 20 mM. This result is consistent with the observation seen in SPR, as discussed below. Taken together, we believe that diverse kinds of antibiotics may interact with TcaR to regulate its ssDNA binding ability.
The binding affinity of GC33 and TcaR was determined quantitatively using surface plasmon resonance. Increasing concentrations of TcaR were passed across a flow cell coated with ssDNA GC33 and the binding response was recorded as changes in response units (RU) after subtraction of the binding response for the reference flow cell. Figure 2A shows a representative sensorgram. Analysis of the sensorgram data indicates k a for the interaction of TcaR with the ssDNA GC33 is 8.8610 5 M 21 s 21 ; k d for the interaction of TcaR with the ssDNA GC33 is 9.5610 23 s 21 (Table 1) . To investigate whether TcaR binds to different types and different lengths of DNA molecules, Biacore experiment was used to test the binding of TcaR to DNA fragments of biotin-labeled ssDNA and hairpin DNA duplex (IcaR DNA1). Consistent with previous observations in Fig. 1B , TcaR binds to ssDNA GC33 and ssDNA AT33 with the higher affinity and the association rates of TcaR to IcaR DNA1, and ssDNA GC17, ranging from 4.3610 5 M 21 s 21 to 1.2610 5 M 21 s 21 , are much lower (shown in Figure 2B ). However, when look at a wider range of DNA sizes, TcaR showed no detectable binding ability to ssDNA 8-mer GC8 but the highest binding ability to ssDNA GC99. The association rate with ssDNA GC99 is 36-fold higher than with ssDNA GC33, along with a 20-fold higher off-rate, suggested that cooperative binding of TcaR may contribute significantly in its ssDNA binding activity (Table 1) . Interestingly, the association rate of GC33 ssDNA is two-times higher than IcaR DNA1, but the dissociation rate of IcaR DNA1 is the lowest compared to the DNA fragments tested in this study, suggesting different modes of interaction occurs in ssDNA-TcaR and dsDNA-TcaR complex ( Figure 2B ).
Furthermore, in order to determine whether other MarR family and TetR family proteins such as SAR2349 from S. aureus and . IcaR DNA1 probe duplex of 1 mM was pre-incubated with 2 mM TcaR (dimer) at room temperature for 15 min before mixing with increasing concentration of GC33 ssDNA, followed by the same procedure as described in the legend to Figure 1B . (F) Competition experiment was carried out to compare the binding strength of TcaR to ssDNA and to dsDNA. In the EMSA analysis, 1 mM IcaR DNA1 probe duplex was pre-incubated with 1 mM GC33 ssDNA fragment for 15 min at room temperature before mixing with TcaR protein of increasing concentration. (G) The effects of pH to the TcaR binding to ssDNA GC33 in EMSA experiment. Molar ratio of GC33:TcaR was 1:2. (H) EMSA analysis of mutant TcaR protein binding to ssDNA (GC33) and dsDNA (IcaR DNA1) fragments with different protein-DNA ratio. (I) EMSA analysis of the binding of TcaR protein to ssDNA(GC33) in the present of different types of antibiotics. GC33 ssDNA probe of 1 mM was preincubated with 2 mM TcaR (dimer) at room temperature for 15 min before mixing with 2 mM or 20 mM antibiotics, followed by the same procedure as described in the legend to Figure 1B IcaR from S. epidermidis have the ssDNA binding ability, we conducted a series of SPR experiments to analyze the binding ability of SAR2349 and IcaR proteins to GC33 ssDNA ( Figure 2C ). The result shows that not all MarR family proteins have this ssDNA binding ability, thus pointing to the specific ssDNA-binding feature of TcaR. In addition, we have previously demonstrated that antibiotics appear to antagonize the ssDNA binding activity of TcaR ( Figure 1I ). Therefore, a measurement for the effect of kanamycin and ampicillin to the GC33 ssDNA binding affinity of TcaR is conducted using surface plasmon resonance to confirm the result. As seen in Figure 2D , the affinity between TcaR and GC33 ssDNA is shown by a decrease in RU values in the presence of antibiotics. This was especially apparent with kanamycin, which yield the lower binding capacity. Taken together, we demonstrate that TcaR shows a higher binding affinity to ssDNA than to dsDNA, and several antibiotics could regulate the ssDNA binding activity of TcaR.
Conformational changes of TcaR in response to ssDNA were monitored using CD spectroscopy [27] . As shown in Figure 3 , the CD spectra of TcaR protein were scanned from 200 to 250 nm in the presence of GC33. With increasing concentrations of GC33 ssDNA, the CD spectrum shows a concomitant decrease in the intensity of the negative peak at 222 and 210 nm, revealing the conformational change of TcaR.
Furthermore, in order to examine whether TcaR shows a binding ability towards much longer ssDNA fragments such as viral wx174-ssDNA, the interaction between them was also examined with increasing concentration (0, 2.5, 10 mM) of viral 
In order to further confirm our finding that TcaR forms complex with viral ssDNA, the M13 and wX174 phage ssDNA circles were used as probes in EMSA to evaluate TcaR binding. As shown in Figure 4A , TcaR reduced the mobility of the M13 and wX174 ssDNA, but S. epidermidis IcaR and S. aureus MarR family protein SAR2349 had no specific interactions with ssDNA. This indicated that TcaR has strong viral ssDNA-binding ability. It is also worth noting that other MarR family protein and TetR family protein do not have such ssDNA binding properties with high affinity, suggesting that the results from TcaR studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Furthermore, since the attempt to obtain the TcaR-ssDNA complex structure was not successful, we resorted to EM analysis to image TcaR-wx174 complex and to pursue its 3-D reconstruction. After staining for 4 min with 2.5% uranyl acetate, EM analysis was performed with a Tecnai tm G 2 Spirit Bio TWIN (FEI CO., The Netherlands) using 120kV acceleration voltage. As seen in Figure 4B -D, EM imaging revealed that no complex was found in the negative control sections, whereas TcaR form a nucleoprotein filament with a circular viral wX174-ssDNA fully covered with proteins, suggesting strong and cooperatively interaction between viral ssDNA and TcaR. This is consistent with the EMSA results we observed. We are now testing another EM method as described by Lurz R et al. [28] to confirm the cooperative binding between the TcaR and viral wX174-ssDNA.
A distinct group of DNA-binding proteins called the ssDNAbinding proteins (SBP) could specifically bind ssDNA and be used in processes where the double helix is separated, including DNA replication, transcription, and recombination. Because TcaR is known as a MarR family transcription regulator that binds to specific dsDNA sequence with the winged helix-turn-helix (wHTH) DNA binding domain, the ssDNA binding ability of TcaR may not be involved in transcription. In order to clarify the role of TcaR-viral ssDNA interaction, our approach is to examine it with in vitro replication. We used single-primed M13 replication assay to measure the ability of purified TcaR protein to convert a primed single-strand M13 template to the duplex form in a manner that requires processive DNA synthesis. As seen in Figure 5A , M13-based in vitro DNA replication assay showed that the addition of TcaR protein to the reaction mixture, and incubation for up to 30 min, resulted in almost no DNA replication activity compared to controls. This indicated that TcaR markedly inhibits DNA replication and that the mechanism of inhibition, at least in part, involves interaction with viral ssDNA. These results suggest a possible role for TcaR in bacteriophage resistance.
Since 1980, investigators have developed an increasing number of bacteriophage therapies for the treatment, or prophylaxis, of bacterial infectious diseases [29, 30, 31] . Reports have described that appropriately administered phages can treat lethal infectious diseases caused by gram-negative and gram-positive bacteria, such as Pseudomonas aeruginae, Klebsiella pneumoniae, Enterococcus faecium, and S. aureus [32, 33, 34, 35] . Antibiotic resistance has become a global public health concern; thus investigators are extensively reevalu-ating phage therapies to fully exploit their antimicrobial potential [36, 37] . However, phages encounter a variety of different antiviral mechanisms during their infection of bacterial cells, such as prevention of phage adsorption and DNA entry, cutting of phage nucleic acids, and abortive infection systems [38] . Most reported antiphage systems have been shown to be relevant to the dsDNA phage, but not ssDNA, ssRNA, or dsRNA phages. To further confirm and clarify the first relationship between the TcaR protein and ssDNA phage resistance, the standard plaque assay was performed in E. coli as a model system since little is known about the ssDNA phage infecting Staphylococci. As seen in Figure 5B , induction of the TcaR protein in E. coli conferred increased host resistance to ssDNA phage (M13 and wX174) infection. However, a TcaR-expressing strain did not demonstrate reduced sensitivity to dsDNA phage Lambda (l) infection, suggesting that the phage resistance was caused by TcaR-viral ssDNA complex. The observed biological differences point to a remarkable plasticity of TcaR. These findings, thus, may support a hypothesis that TcaR might interfere with viral ssDNA replication and establish a link between TcaR and ssDNA phage resistance.
The MarR family transcriptional regulators serve as sensors of changing environments, allowing pathogenic bacteria to survive and persist in a dynamic environment [39] . However, up to now, the knowledge of MarR family protein-nucleic acid interaction has been focused on dsDNA and the MarR family protein-ssDNA interaction ability as well as their contribution to the multiple functions of TcaR is yet to be discovered. Better understanding of these interactions not only will benefit the understanding of many biological mechanisms but also is expected to provide a concept for designing a new therapy for Streptococci. In this work, we present the first attempt to investigate the TcaR-ssDNA interaction. The information of TcaR-ssDNA binding mode and the minimal binding length that we obtained from EMSA analysis and Biacore will be helpful for us to obtain TcaR-ssDNA complexed structure successfully. Moreover, we used in vitro replication assay and plaque assay to elucidate the specific biological role of the ssDNA binding ability of TcaR. Such observations may help us understand the mechanism of antibiotic resistance in the MarR family regulators.
The IcaR and TcaR proteins were expressed in E. coli BL21 (DE3) and purified as already described [17, 40] . The MarR homologous gene, SAR2349, was amplified directly from the S. aureus MRSA252 genome by polymerase chain reaction (PCR) and subsequently cloned into expression vector pET-32. This construct was transferred into E. coli of Arctic Express TM (DE3) RIL strain. The His 6 -tagged wild-type protein was over-expressed in Difco Luria-Bertani (LB) broth containing 50 mg/l ampicillin to an optical density at 600 nm of 0.5-0.6 and then induced with 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside). Cells were grown for 2 days at 16uC. The cells were then harvested by centrifugation at 12,000 g for 30 min and disrupted by Constant Cell Disruption System (CONSTANT SYSTEM Ltd, UK) with lysis buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 20 mM imidazole. The homogenate was centrifuged at 27,000g for 30 min and the cell-free extract was loaded onto a Ni 2+ -NTA column, which had been previously equilibrated with lysis buffer. The column was washed with lysis buffer, and the His 6 -tagged SAR2349 was subsequently eluted by a linear gradient of imidazole from 10 mM to 500 mM. His-tagged SAR2349 eluted was dialyzed twice against 5 liters of buffer (20 mM Tris-HCl, pH 8.0, and 500 mM NaCl) and then subjected to Thrombin digestion to remove the tag. The mixture was then passed through another Ni 2+ -NTA column, and subsequently untagged SAR2349 protein was dialyzed twice against 3 liters of buffer (20 mM Tris-HCl, pH 8.0) and then passed through a Q-Sepharose anion-exchange column for further purification, and subsequently SAR2349 was eluted by a linear gradient of 10 mM to 500 mM NaCl-containing buffer and then dialyzed twice against 5 liters of buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM DTT) for storage. The purified SAR2349 protein was finally concentrated by 3 kDa cut-off size membrane of Amicon ultra-15 centrifugal filter units (Millipore, MA, USA) for storage at -80uC.
The six oligonucleotide probes used in EMSA experiments were purchased from MDBio Inc. (Taiwan) ( Figure 1A ). The viron wX174 and M13 ssDNA were purchased from New England Biolabd (USA). For the preparation of double-stranded IcaR DNA1, equimolar amounts (100 mM each) of complementary oligonucleotides were mixed, fully denatured by heating at 95uC for 5 min in 10 mM Tris-HCl pH 8.0, 20 mM NaCl and allowed to cool gradually to room temperature. Gel shift assays were performed by incubating 1 mM of ssDNA or dsDNA with 1-4 mM purified recombinant proteins under binding conditions (20 mM Tris-HCl, pH 8.0, 150 mM KCl, 0.1 mM MgCl 2 , 0.05 mM EDTA, 12.5% Glycerol, 0.1 mM DTT and 1 mg/ml BSA) for 15 min at room temperature with gentle vortex. After incubation, 15 ml of the reaction solution was mixed with 3 ml of the sample loading dye and subsequently electrophoresed on 6% preequilibrated polyacrylamide gels in 1/2 Tris/acetate/EDTA (TAE) at 100 V for 30 min and visualized using SYBR Green I nucleic acid gel stain (Invitrogen). For competition assay, IcaR DNA1 probe duplex of 1 mM was pre-incubated with 2 mM TcaR (dimer) at room temperature for 15 min, then mixed with increasing concentration of GC33 ssDNA. In the assay for analyzing the effect of different antibiotics on the interaction between TcaR and ssDNA, 1 mM GC33 probe was pre-incubated with 2 mM TcaR (dimer) at room temperature for 15 min before mixing with 2 mM or 20 mM antibiotics.
The affinity, association and dissociation between the drug and the DNA duplexes were measured using a BIAcore 3000A surface plasmon resonance (SPR) instrument (Pharmacia, Uppsala, Sweden) with a SensorChip SA5 from Pharmacia by monitoring the refractive index change of the sensor chip surface [41, 42, 43] . These changes are proportional to the amount of bound analyte. The SPR angle change is reported as resonance units (RU). Five fragments of 59 biotinylated oligonucleotides probes purified by PAGE were purchased from MDBio Inc. (Taiwan). Activation buffer (100 mM NaCl, 50 mM NaOH) was injected for 1 min (20 ml) to remove any unbound streptavidin from the sensor chip. To control the amount of the DNA bound to the SA chip surface, 200 nM of the biotinated oligonucleotides were immobilized manually onto the surface of a streptavidin chip until 120 RU was reached in the first cell. The chip surface was then washed with 10 ml of 10 mM HCl to eliminate non-specific binding. The second flow cell was unmodified and served as a control. Different concentration of TcaR, IcaR, and SAR2349 proteins were injected at a flow rate of 30 ml/min in 50 mM Tris, 150 mM NaCl, pH 7.5 for 170 s to reach equilibrium. Blank buffer solution was then passed over the chip to initiate the dissociation reaction. At the end of each cycle, the surface was recovered with two 30 s injections of 0.025% SDS. SPR-binding constant is analyzed as described previously [44] . Sensorgrams for the interactions between DNA and TcaR were analyzed using BIA evaluation software to determine the association and dissociation rate constant (k a /k d ). In the assay analyzing the effect of antibiotics on the interaction between TcaR and ssDNA, TcaR protein with 640 nM kanamycin or ampicillin in 50 mM Tris, 150 mM NaCl, pH 7.5 was injected on to the sensor chip.
Circular dichroism (CD) spectra were obtained using a JASCO-815 CD spectropolarimeter. Temperature was controlled by circulating water at the desired temperature in the cell jacket. TcaR and DNA samples were prepared under the conditions identical to those prepared for EMSA assay. The CD spectra were collected between 250 and 200 nm with 1 nm bandwidth at 1 nm intervals. All spectra were obtained from an average of five scans. The photomultiplier absorbance did not exceed 600 V during the analysis. CD spectra were normalized by subtraction of the background scan with buffer alone. The mean residue ellipticity, [h], was calculated based on the equation, [h] = MRW6hl/ 106l6c, where MRW is the mean residue weight, hl is the measured ellipticity in milidegrees at wavelength l, l is the cuvette pathlength (0.1 cm), and c is the protein concentration in g/mL.
The TcaR proteins (0.3 mM) were first incubated at 30uC for 15 min in reaction buffer [20 mM Tris-HCl, pH 8.0, 150 mM KCl, 0.1 mM MgCl 2 , 0.05 mM EDTA, 12.5% glycerol, 10 mM DTT, 12 mM circular viron wX174 ssDNA (5386 nucleotides in length), 0.2 M ammonium acetate], and then chilled on ice to stop the reaction. The reaction product was diluted 100-fold with EM sample dilution buffer (2 mM MgCl 2 , 0.5 mM DTT, 10 mM HEPES pH 7.0). A droplet (4 ml) was placed for 1 min at room temperature on a copper grid (300 mesh, Pelco, USA) coated with fresh carbon. The excess buffer was then carefully blotted away from the edge of the grid with Whatman #1 filter paper (Whatman Inc., USA). After staining for 1 min with 2% uranyl acetate, excess liquid was removed and samples were dried at room temperature. Bio-transmission EM was performed with a Tecnai F20 Bio TWIN (FEI Co., Netherlands) using an acceleration voltage of 200 kV. Images were recorded with a slow scan CCD camera (Gatan MultiScan TM 600, USA) at a resolution of at least 4k64k pixels.
For M13 replication assay, 250 mM of single primed M13mp18 ssDNA was incubated with/without 2 mM TcaR protein in the reaction mixture (20 ml) containing 25 unit Klenow fragment, 25 mM NaCl, 7 mM MgCl 2 , 1 mM EDTA, and 0.5 mM DTT for 3 min at 30uC to allow replication complexes to assemble at the primer template junction [45, 46] . Replication was allowed to proceed by addition of 60 mM dNTP. After incubation at 30uC for 30 min, the reactions were terminated by addition of 10 mM Tris-HCl, 5 mM EDTA, 0.5% SDS, and 50 mg of proteinase K (with total volume of 20 ml) and incubated at 50uC for 1 h. Conventional electrophoresis was then performed to verify the result of DNA replication (20-cm 0.8% agarose gel, 15 mA, 0.5X TBE buffer). The bands are visualized using SYBR Green I nucleic acid gel stain (Invitrogen) and quantified by Quantify One (BIO-RAD, USA).
Host sensitivity to phages was tested using a virulent variant of phage (M13, wX174 and c) and E. coli BL21 (DE3) RIL transformed with engineered pET-16b-TcaR plasmid containing lacI gene and lac operator as host [47, 48, 49] . Cells were grown in LB media until the optical density (OD 600 ) reached 0.6. TcaR protein was then induced by adding a final concentration of 0.1 mM IPTG and used in plaque assays as previously described [50, 51] . Plaque assays were performed in triplicate. Plates and topagar contained LB and above mentioned concentrations of inducers. The sensitivity of the host to phage infection was calculated as the efficiency of plaquing, which is the plaque count ratio of a non-IPTG set to the IPTG set [52] . Error-bars were calculated as one standard deviation.
The atomic coordinates and structure factors for the TcaR-RNA complex have been deposited in the wwPDB with accession numbers of 4EJT. Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics The use of bioinformatics to integrate phenotypic and genomic data from mammalian models is well established as a means of understanding human biology and disease. Beyond direct biomedical applications of these approaches in predicting structure–function relationships between coding sequences and protein activities, comparative studies also promote understanding of molecular evolution and the relationship between genomic sequence and morphological and physiological specialization. Recently recognized is the potential of comparative studies to identify functionally significant regulatory regions and to generate experimentally testable hypotheses that contribute to understanding mechanisms that regulate gene expression, including transcriptional activity, alternative splicing and transcript stability. Functional tests of hypotheses generated by computational approaches require experimentally tractable in vitro systems, including cell cultures. Comparative sequence analysis strategies that use genomic sequences from a variety of evolutionarily diverse organisms are critical for identifying conserved regulatory motifs in the 5′-upstream, 3′-downstream and introns of genes. Genomic sequences and gene orthologues in the first aquatic vertebrate and protovertebrate organisms to be fully sequenced (Fugu rubripes, Ciona intestinalis, Tetraodon nigroviridis, Danio rerio) as well as in the elasmobranchs, spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea), and marine invertebrate models such as the sea urchin (Strongylocentrotus purpuratus) are valuable in the prediction of putative genomic regulatory regions. Cell cultures have been derived for these and other model species. Data and tools resulting from these kinds of studies will contribute to understanding transcriptional regulation of biomedically important genes and provide new avenues for medical therapeutics and disease prevention. the species and provided additional insights about genomic evolution. Another area of interest that stands to benefit greatly from genomic data is that of gene regulation. Understanding in this field is fragmented and, as with any scientific discipline, the concepts and questions that can be conceived and addressed experimentally are dependent on available technological approaches.
Mechanisms by which expression of a single gene is regulated can be extremely complicated. Multiple phosphorylation-or ligand-dependent nuclear receptors that homo-or heterodimerize may be required to achieve activity. Each of these receptors may have different activation specificity or duration, even when acting via the same regulatory DNA sequence such as classical proximal promoter elements. These receptors may also work in combination with other transcription factors that function at sites more distal from the proximal promoter or in introns. Alternatively spliced transcripts represent another complex aspect of gene expression regulation that is influenced by extracellular and intracellular signaling but is not well understood (Stamm et al. 2005) . Furthermore, individual genes often are part of a broader, coordinately regulated network of genes that function to elicit a set of cellular responses (Wagner 1999) . Through such mechanisms, ligands may, for instance, induce their own metabolism or export, a process that further complicates understanding of gene regulation and that also has critical implications for models of pharmacokinetics and drug efficacy.
Experimental identification of functional genomic sequences depends heavily on cell culture and other techniques of in vitro cell biology. Traditionally, identification of gene regulatory regions has been limited by the labor-intensiveness of the requisite strategies. Generally only regions close to the transcriptional start sites have been experimentally tractable for detailed examination, despite evidence that important regulatory regions exist more than 10 kb upstream or downstream from the coding region or in introns of genes (Rowntree et al. 2001) .
Identification of specific functional sequences through the generation of deletion constructs is limited in the sequence size that can be analyzed and is restricted to examination of single genes. Transfection of cells with reporter constructs containing putative proximal promoters may elicit strong activation when treated with receptor-specific ligands in culture, while in vivo studies may yield inconsistent results, suggesting that these receptors and transcription factors are a subset of a larger complex of regulators of gene expression. Techniques such as DNASE I hypersensitivity studies and gel shifts are excellent methods for testing functionality of putative regulatory elements; however, they are not efficient for screening candidate sequences. By contrast, computational analyses allow the examination of a significantly larger region when predicting conserved regulatory regions and signals. Computational techniques lend themselves to the identification of patterns or clusters of regulatory motifs and prediction of coregulated genes, and generate targeted predictions of candidate regulatory sequences and signaling molecules that can then be directly tested in functional and mutagenesis studies (Hughes et al. 2000; Loots et al. 2000; Pennacchio and Rubin 2003; Ovcharenko et al. 2005) .
Understanding causative relationships between specific regulatory elements and expression patterns would greatly enhance the ability to predict disease-associated genes (Pennacchio and Rubin 2003) . However, even in computational approaches the ability to identify and predict the regulatory functions of non-coding sequences has been limited (Pennacchio and Rubin 2003) . Pairwise comparisons have helped to predict functionally conserved regions, however the statistical accuracy of these predictions is increased when more than two sequences are used (Dubchak et al. 2000) . Several studies suggest that comparative analysis of multiple evolutionarily diverse organisms facilitate the prediction of functionally important non-coding regions (Dubchak et al. 2000; Matys et al. 2003; Thomas et al. 2003) . Comparisons of genome sequences from evolutionarily diverse organisms also elucidate regulatory regions that are specific to a particular species or group of species. Recent additions of the African tree frog (Xenopus tropicalis), chicken (Gallus gallus) and dog (Canis familiaris) to the sequencing pipeline will provide important complementary perspectives for tetrapods, but data from more divergent vertebrate genomes is still needed. Different rates of molecular evolution, including gene duplication, underscore the importance of the examination of genomic sequences from an evolutionary range of organisms for comparative sequence analyses. When such an approach is taken, the contrast of divergent sequences differentiates between functionally conserved regions and generally conserved regions that simply reflect a lack of divergence time (Dubchak et al. 2000) . Using sequences from evolutionarily diverse organisms may provide the necessary divergence to identify functionally conserved regions in genes that have evolved slowly.
The increasing availability of genomic data for non-mammalian organisms and similarities to the human genome underscore the value of these organisms as models of a variety of diseases in humans (Aparicio et al. 2002; Dehal et al. 2002; Ballatori et al. 2003) . The identification of conserved coding and regulatory regions is enhanced by including divergent sequences in comparative studies (Thomas et al. 2003 ) because these sequences provide more stringent filters for detecting conserved, and presumably functionally important elements (Dubchak et al. 2000; Thomas et al. 2003; Ahituv et al. 2004) . Consequently, sequences from evolutionarily distant marine vertebrates and protovertebrates are being used in comparative studies with increasing frequency. The pufferfish (Fugu rubripes) genome sequencing project supported this approach as it led to the discovery of nearly 1000 human genes not previously described (Aparicio et al. 2002) . Sequence comparisons of the Hoxb-4 gene in mouse and Fugu identified novel regulatory elements that directed subsets of the full Hoxb-4 expression pattern in transgenic mice (Aparicio et al. 1995; Amores et al. 2004) . In a recent comparative analysis of the HoxA cluster in human, horned shark and zebrafish (Chiu et al. 2002) , extensive conservation of non-coding sequence motifs was found between the human and shark sequences, whereas zebrafish sequences exhibited significant loss of conservation. The majority of newly identified regulatory elements for this cluster of genes were identical to known binding sites for regulatory proteins as defined in the transcription factor database, TRANSFAC (http://www.biobase.de; Matys et al. 2003) , demonstrating the accuracy of this approach (Matys et al. 2003; Santini et al. 2003) .
Special physiological attributes exhibited by many evolutionarily diverse aquatic organisms have led to the increased appreciation of their importance as models of human disease. These characteristics have been exploited experimentally to further understanding of human immunology, genomics, stem cell and cancer biology, pharmacology, toxicology and neurobiology. Historically, marine vertebrates provided critical insights into fundamental mechanisms of physiological processes, and the value of these organisms has not been diminished by the advent of molecular approaches. Membrane transporters that are the sites of action of diuretic drugs were first cloned from specialized organs in marine species (Gamba et al. 1993; Xu et al. 1994) . Mutagenesis studies in bony fish generated a spectrum of biologically relevant and distinct phenotypes (Naruse et al. 2004; Walter et al. 2004 ). Large-scale genetic screens produced more than 500 zebrafish mutants, many with phenotypes similar to human disorders (Dooley and Zon 2000) . Immunogenetic studies in carp, salmonids and other species are contributing to the growing database of marine vertebrate genomics Fujiki et al. 2001 Fujiki et al. , 2003 Tomana et al. 2002; Ishikawa et al. 2004) .
Although increasing numbers of vertebrate genomes are being sequenced, there are still stretches of evolutionary history without representation (Thomas and Touchman 2002) . Among aquatic organisms, teleosts are represented by significant EST and genomic sequencing, such as those in zebrafish and pufferfish. Until recently, chondrichthyes, which include elasmobranchs (sharks, rays and skates) were the only major line of gnathostomes, or jawed vertebrates, for which there are no major initiatives to generate genomic sequences. Sequence data from elasmobranchs have provided unique insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, G protein coupled receptors, natriuretic peptide receptors, and other biomedically relevant genes (Valentich and Forrest 1991; Henson et al. 1997; Aller et al. 1999; Silva et al. 1999; Greger et al. 1999; Waldegger et al. 1999; Ke et al. 2002; Wang et al. 2002; Yang et al. 2002; Cai et al. 2001 Cai et al. , 2003 Mattingly et al. 2004b) .
In March, 2005 the National Human Genome Research Institute of the National Institutes of Health (NIH), USA, announced that it will fund the whole genome sequencing of Raja erinacea. In the announcement of the Skate Genome Sequencing Initiative (http://www.genome.gov/ 13014443), NIH states: 'The skate (related to many species of shark and cartilaginous fish) was chosen because it belongs to the first group of primitive vertebrates that developed jaws, an important step in vertebrate evolution. Other innovations in this group of animals include an adaptive immune system similar to that of humans, a closed and pressurized circulatory system, and myelination of the nervous system. Understanding these systems of the skate at a genetic level will help scientists identify the minimum set of genes that create a nervous system or develop a jaw, possibly illustrating how these systems have evolved in humans, and how they sometimes go wrong.' Chondrichthyes (cartilaginous) fish appeared approximately 450 million years ago. Elasmobranchs comprise most chondrichthyan organisms. They exhibit fundamental vertebrate characteristics, including a recombinatorial immune system (Hinds and Litman 1986; Adelman et al. 2004) , and are also the oldest existing vertebrates with circulatory system-related signaling molecules and receptors such as platelet-derived growth factor and adenosine receptors. The specialized rectal gland of Squalus acanthias, the spiny dogfish shark, has greatly facilitated the study of cystic fibrosis, sodium and chloride secretion (Devor et al. 1995; Lehrich et al. 1995; Forrest 1996; Henson et al. 1997; Lehrich et al. 1998; Silva et al. 1999; Aller et al. 1999; Greger et al. 1999; Waldegger et al. 1999; Ke et al. 2002; Yang et al. 2002) . Unlike many primary cell cultures that dedifferentiate and lose transport polarity immediately after isolation, primary cultures of shark rectal gland tubular cells maintain fully differentiated function and expression of all known receptors, transporters, ion channels and signal transduction pathways in vitro (Valentich and Forrest 1991; Devor et al. 1995; Lehrich et al. 1995; Aller et al. 1999; Greger et al. 1999; Waldegger et al. 1999; Ke et al. 2002; Yang et al. 2002; Mattingly et al. 2004b) . A comparison of the properties of the cloned shark and human cystic fibrosis transmembrane regulator (CFTR) has provided insights into structural domains related to functional differences in the normal and mutant proteins (Marshall et al. 1991) . The spiny dogfish shark CFTR protein is 72% identical to the human ortholog and comparison of the human and shark CFTR sequences revealed conservation of five cyclic AMP-dependent kinase phosphorylation sites and three residues that, when mutated in the human protein, are associated with cystic fibrosis.
The coding sequences and functions of a number of medically relevant genes are conserved in the spiny dogfish shark and little skate (Cai et al. 2001 (Cai et al. , 2003 Wang et al. 2002; Yang et al. 2002; Mattingly et al. 2004b ). Primary hepatocytes from little skate retain hepatobiliary polarity for at least 8 h and possibly up to several days in culture, offering particular advantages for studies of liverspecific functions . Genomic information applied to existing physiological data in these systems, along with the further development of in vitro cell culture systems, will allow the testing of molecular hypotheses and understanding of regulatory mechanisms that are directly applicable to human biology. Targeted sequencing of well-defined genomic regions generated from BAC clones has been extremely useful in providing supporting genomic information in species for which complete genome data are not available . In addition to constructing four-fold coverage bacterial artificial chromosome libraries from sperm DNA of dogfish shark and little skate (http://www.mdibl.org/research/ skategenome.shtml), an expressed sequence tag (EST) sequencing project is underway to substantially increase the availability of sequence data for these model organisms (Mattingly et al. 2004b ). Over 10,000 sequences are publicly available through the EST database at the National Center for Biotechnology Information (dbEST; Boguski et al. 1993 ) and http://www.mdibl.org/decypher. These data sets are updated as new sequences become available.
One of the remarkable findings of the human genome sequencing project was the discovery that coding regions account for only 5% of the genome (Venter et al. 2001) . The remaining sequence consists of repetitive DNA ($40-45%) and extensive non-coding regions for which there is very little functional information. It is within these regions that significant regulatory information presumably is concealed. The major challenge in the postgenomic age is uncovering important functional regions within this non-coding DNA. The rapid development of sequence analysis software tools, increasing availability of genomic data, and recognition of the importance of comparing data from diverse organisms are allowing scientists to make fruitful inroads to understanding genomic structure and gene expression regulation. A brief summary of software tools that are valuable for identifying regulatory information from crossspecies comparative analyses follows.
The University of California Santa Cruz Genomic Browser (http://genome.ucsc.edu/; Karolchik et al. 2003 ) is among the most popular databases for querying and retrieving genomic sequences of interest. It currently provides access to genomic assemblies from 23 organisms, including 10 vertebrates, 8 insects, 2 nematodes, a sea squirt, baker's yeast, and the SARS virus. Whether genomic regions are retrieved from an existing database or sequenced locally, there are an increasing number of options for analysis and annotation. Several computational tools allow identification of genes and exon boundaries in genomic sequences. GENSCAN (Burge and Karlin 1997) , TWINSCAN (Korf et al. 2001) , MZEF (Zhang 1997) , and Gene Recognition and Analysis Internet Link (GRAIL; Uberbacher and Mural 1991) are among the most popular. Most gene finding programs were optimized to predict genes in sequences from mammalian models; as a result accuracy is sometimes reduced when using sequences from more divergent organisms or nonvertebrates. These programs use different strategies and are based on current, but incomplete understanding of gene structure. Therefore, predictions from multiple programs should be combined computationally to enhance accuracy and confidence (Rogic et al. 2002) .
Quality control strategies can be used to increase accuracy of and confidence in results from gene finding tools. First, the abundance of repetitive elements in genomic sequences can distort predictions of genes and exon locations. To counter this problem, masking genomic sequences is recommended, before gene analysis, using a program like RepeatMasker (A.F. A. Smit et al. 1996, unpublished) . The effectiveness of Repeat-Masker with marine and other evolutionarily divergent organisms is not yet clear because interspersed repeats are specific to a species or group of species. Sequences from such organisms should be evaluated under masked and unmasked conditions. Second, aligning ESTs or cDNAs with genomic sequences using programs like Spidey (http://www.ncbi.nlm.nih.gov/spidey/; Wheelan et al. 2001 ) refines predictions of exon and intron boundaries, promoter regions, and splice sites. This approach presents challenges for transcripts that are expressed at very low levels, have significant tissue-or age-specific requirements, or are from species for which minimal sequencing has been done (Schwartz et al. 2000) . A new, publicly available resource, the Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org; Mattingly et al. 2003 Mattingly et al. , 2004a , provides multiple alignment and phylogenetic analysis results with sequences from diverse organisms for biomedically significant genes and proteins. CTD provides access to data valuable for identifying homologous genomic sequences and confirming gene and gene feature predictions. Identification of gene features greatly improves interpretation of subsequent multiple sequence analysis results.
Aligning multiple genomic sequences is becoming widely accepted as a powerful mechanism for identifying important functional regions such as regulatory elements. MultiPipMaker (http:// pipmaker.bx.psu.edu/pipmaker/; Schwartz et al. 2003 ) and mVISTA (http://gsd.lbl.gov/vista/index.shtml; Bray et al. 2003; Frazer et al. 2004 ) are two of the most popular web servers that conveniently combine alignment engines with visualization capabilities. MultiPipMaker, and a new server zPicture (http://zpicture.dcode.org/; Ovcharenko et al. 2004) , use the local alignment program BLASTZ as their alignment engine (Schwartz et al. 2000) ; VISTA uses AVID, a global alignment program (Bray et al. 2003; Frazer et al. 2004 ). Local alignment tools find high-scoring, short matching segments and extend these regions based on a scoring threshold. Local alignments may permit greater diversity between sequences by finding regional similarities. Segments of similarity need not be conserved in order or orientation. This feature may be advantageous for finding conserved transcription factor binding sites, which are very short and prone to reordering (Bray et al. 2003) .
High similarity of short regions, however, does not necessarily imply homology (a gene derived from a common ancestral gene) and can lead to false implications of relatedness among sequences that are not homologs. By contrast, global alignments do require that the order and orientation of similar regions is conserved, because similar architecture is often observed in homologous sequences (Bray et al. 2003) . MultiPipMaker and VISTA provide visualization options for alignments that include percent similarity and usersubmitted annotations (e.g., exon locations, repetitive elements). It is important to note that local and global alignment tools are being refined so rapidly that it is becoming difficult to distinguish between them (Frazer et al. 2004) . Readers are referred to two recent reviews of alignment programs for detailed comparisons (Frazer et al. 2003; Pollard et al. 2004) .
A major challenge to identifying transcription factor binding sites (TFBSs) is that they tend to be short, degenerate, and occur frequently throughout the genome. Analysis of a single sequence usually leads to an abundance of false positive predictions of TFBSs. Several programs have been developed to respond to this challenge based on two principles. First, functional regulatory elements are often conserved evolutionarily; therefore, identifying TFBSs that are conserved or aligned in multiple sequences may effectively filter false positive predictions (Ovcharenko et al. 2005) . Second, gene expression often results from coordinate activation of multiple, proximal regulatory elements; therefore identifying TFBSs in clusters, rather than isolation, may enhance confidence in the functionality of predicted TFBSs. These principles have been leveraged, albeit differently, by rVISTA (http:// gsd.lbl.gov/vista/index.shtml; Loots et al. 2002; Loots and Ovcharenko 2004) , which is a member of the VISTA suite of tools and is also integrated with zPicture, and the newly launched Mulan (http://mulan.dcode.org/; Ovcharenko et al. 2005) . Both servers use profiles of transcription factor binding sites from the TRANSFAC database (http://www.biobase.de; Matys et al. 2003) . Because TRANSFAC and other similar resources only contain information for known transcription factor binding sites, they are inherently incomplete. Furthermore, existing tools do not address other important regulatory features such as properties related to protein-protein interactions and chromatin structure, and clusters of binding sites that may have been reshuffled between organisms over evolutionary time (Loots et al. 2002) .
Cell culture of marine genomic model species and experimental verification of predictions from comparative analysis of genomic sequences Using in vitro cell culture systems, the functional significance of conserved, putative regulatory sequences predicted through comparative computational analysis in, for instance, the 5¢upstream region of select genes can be tested experimentally. The availability of sufficient genomic information facilitates targeted studies to evaluate such potential functional regulatory regions. Comparative experimental studies can be designed employing cell lines derived from any species, though mammalian cell lines have thus far been favored (Mather and Barnes 1997; Barnes and Sato 2000) . Reporter constructs containing regulatory regions of select genes can be generated using up to 5 kb upstream of the transcriptional start site of the relevant genes; these are inserted upstream of a reporter gene such as that for an enhanced fluorescent protein.
In the last decade, significant progress has been made in development of marine and freshwater organism cell lines with utility for genomic studies. A variety of zebrafish cell lines have been developed, some of which maintain a normal karyotype for extended periods in vitro (Barnes and Collodi 2005) . Zebrafish cells in culture can be transfected with plasmid DNA using adaptations of approaches common for mammalian cells in vitro, and transient transfection methods have identified expression of genes under control of a number of mammalian and fish promoters. In addition, cell cultures from pufferfish (genera Fugu and Tetraodon) provide a biological complement to the genomic libraries derived to study the molecular biology of these animals, allowing the extension of this model to experiments in functional genomics. Multipassage cell cultures have been established from embryo and adult tissues of species of both of these pufferfish genera (Barnes and Collodi 2005) . One of the Fugu rubripes cultures has been maintained for more than 200 population doublings, and flow cytometry showed that the relative amount of DNA present in cultured cells was approximately 15% of that in human cells, as predicted by biochemical analysis. Telomerase, an enzyme associated with indefinite proliferation in mammalian cell cultures, was easily detectable in these cells, suggesting that the cultures are capable of indefinite growth.
Until recently, the lack of in vitro culture systems for elasmobranch models has been a major limitation for the use of these species in comparative functional genomics. Cold-water marine animals such as the little skate and dogfish shark are useful models for physiology and genomics, but homologous in vitro systems with which investigators can test hypotheses or confirm predictions at a molecular level are essential for widespread use of these models. Expression of elasmobranch genes in heterologous systems such as mammalian cell cultures or Xenopus oocytes may be compromised by differences in membrane lipid composition and missing or interfering accessory and messenger proteins. Heterologous systems are also not adaptable to mechanistic studies using genetically altered, dominant negative molecules. Another advantage derived from cell cultures from species such as shark and skate (that may be only seasonably available) is that they make biological material available yearround in any laboratory. Studies on regulation of gene expression in dogfish shark and little skate are beginning to benefit from the expanding genomic databases for these animals. Skate embryo-derived cell cultures also provide new avenues for elasmobranch research in embryology and organogenesis, toxicology, neurobiology, genome regulation and comparative stem cell biology.
Cells have been cultured from the rectal gland, eye, brain, kidney and early embryo of Squalus acanthias. Medium is either LDF, developed for zebrafish cells (Barnes and Collodi 2005) or VCM (Valentich and Forrest 1991) , a urea-timethylamine oxide (TMAO)-containing medium that supports the short-term culture of shark rectal gland cells. In some cases cells are plated onto dishes pretreated with collagen. Medium is further supplemented with a variety of cell typespecific peptide growth factors, nutrients and purified proteins at a range of concentrations. These include insulin, transferrin, epidermal growth factor (EGF), basic fibroblast growth factor (FGF), transforming growth factor-beta, vitamins A and E, selenium, mercaptoethanol, dexamethasone, fetal calf serum, shark serum and shark yolk extract. For example shark brain cells can be grown in primary culture in VCM containing EGF, FGF, insulin, transferrin, selenium, a chemically-defined lipid supplement and vitamin E on a fibronectin substratum (Figure 1 ). Expression of genes from plasmids has been achieved in primary dogfish shark rectal gland cell cultures by lipofection using the CMV promoter to direct expression of the gene of interest ( Figure 2 ). Differentiated function also is maintained, as evidenced by expression of CFTR and vasoactive intestinal peptide receptor (VIPR) mRNA detected by reverse-transcription polymerase chain reaction (RT-PCR) assay (Figures 3  and 4) .
Assay for telomerase on cultures of a variety of dogfish shark and little skate cell types showed that all cultures tested were positive, although specific activity varied almost 100-fold among different cell types. Assay for cell proliferation by in situ bromo-deoxyuridine (BrdU) incorporation also was carried out on cultures from shark brain, kidney and rectal gland. The assay involves an overnight incubation with BrdU, followed by an immunoassay identifying cells synthesizing DNA during the time of incubation and incorporating the nucleoside analogue. The results showed that cells in the cultures were synthesizing DNA. The most active synthesis was seen in cultures from early shark embryos. Medium was supplemented with insulin, tranferrin, selenous acid, EGF, FGF, Lglutamine, chemically defined lipids, non-essential amino acids, heat-inactivated fetal bovine serum (heat treated for 30 min at 56°C) and shark yolk extract. Conditioned medium from the cells was stimulatory for other shark cell cultures, including the shark rectal gland cells ( Figure 5) . A normalized c-DNA library has been made from these cells and attempts will be made to identify the cell type by extensive EST analysis.
In addition to the scarcity of cell lines from marine vertebrates, a persistent absence of cell lines from non-arthropod invertebrates has stymied research on many valuable species useful in comparative genomics and a variety of other disciplines (Bayne 1998; Rinkevich 1999) . Selection of the echinoderm Strongylocentrotus purpuratus (purple sea urchin) as a model species for genomic sequencing has enhanced the need for cell lines from this species in particular. We recently have explored cell culture using both this species and the closely related Strongylocentrotus droebachi- ensis (green sea urchin). Cells from Polian vesicles and axial organ (Figures 6 and 7) appear to have proliferated in vitro for more than 8 months (Parton and Bayne 2004, 2005) . Other cultures yielded thraustochytrid protists that are common parasites of marine invertebrates worldwide (Rinkevich 1999) . Basal nutrient culture medium was LDF diluted with 4 volumes of filtered sea water (Kawamura and Fujiwara 1995) with antibiotics (penicillin 200 U/ml, streptomycin 200 lg/ ml, ampicillin 25 lg/ml), sodium bicarbonate at 0.18 g/l and HEPES at 15 mM. Culture vessels were held at 20°C in ambient air. Additives included fetal calf serum (heat inactivated) at 1-3%; insulin at 10 lg/ml; transferrin at 10 lg/ml; FGF at 50 ng/ml; EGF at 50 ng/ml; b-mercapto-etha-nol at 55 lM; chemically defined lipid supplement at 1 ll/ml; selenium at 10 nM, and L-glutamine at 200 lM.
Transcriptional regulation involves complex interactions of diverse proteins, or transcription factors, with specific DNA sequences in the noncoding regions of target genes. Furthermore, cells respond to environmental stimuli and to developmental signals by altering expression of gene networks. The limited number of transcription factors suggests that few, if any of these proteins exert their activity exclusively on a single gene; rather, they bind to conserved sites in several genes to coordinate their expression (Wagner 1999; Pennacchio and Rubin 2003) . In situations in which there are no clinically acceptable inhibitors or other modulators of clinically relevant proteins, elucidating mechanisms by which these genes are regulated and identifying other coordinately regulated genes may reveal novel strategies by which disease processes may be disrupted or controlled.
Comparative studies provide insight into the possible common ancestor of a gene, trace the accumulation of mutations over time and suggest selective pressures that influence the expression and functions of genes (Pennacchio and Rubin 2003) . Comparisons have provided useful predictions about which sequences are minimally essential for function as well as those that may be important on a species-specific level (Aparicio et al. 1995) . Comparative genomic computational approaches continue to identify conserved regions in non-coding sequences. The challenges will be to determine which sequences are functionally significant and to identify coordinately regulated genes and common regulatory pathways. Understanding such networks, the transcription factor binding sites and genes involved in disease states may reveal alternative points of intervention and contribute to a more predictive approach to molecular medicine.
Significant progress has been made in the development of powerful sequence analysis tools. Although often optimized for sequences from more traditional model organisms, they offer great value for comparative studies that include evolutionarily divergent sequences. They should be used cautiously, however, with awareness of their limitations, which are largely a reflection of current scientific knowledge and understanding of genome structure. As the diversity of available sequence data continues to increase, it will drive refinements and development of new tools and provide important insights into the evolution and essential mechanisms of gene expression regulation.  Regulated multicistronic expression technology for mammalian metabolic engineering Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering. There are two general levels of genetic engineering in which a suitable production cell line is generated; (i) stable introduction of the genetic information for the product protein and (ii) an optional metabolic engineering step to improve cellular activities by the manipulation of enzymatic, transport, and regulatory functions of the cell (Bailey, 1991) . Metabolic engineering of animal cells has already been proven useful for improving diverse key characteristics of cultured cells including cell viability (apoptosis engineering: Cotter and Al-Rubeai, 1995; Mastrangelo and Betenbaugh, 1998) , product quality (glycosylation engineering: Bailey et al., 1998; Jenkins et al., 1996) , product yield (controlled proliferation technology: Fussenegger et al., 1997a; Fussenegger et al., 1998a; Fussenegger et al., 1998b; Papoutsakis, 1998) and growth in protein-free medium (cell-cycle engi- * Author for all correspondence.
neering: Renner et al., 1995; Lee et al., 1996; Rivard et al., 1996; Greulich and Erikson, 1998) . Most of these successes have been realised by the addition of a single gene to the host cell's genome. However, just as single-gene interpretations of human disease have limited scope (Lander and Schork, 1994) , one-gene metabolic engineering cultured cells cannot access anything approaching the full potential set of useful engineered phenotypes (Papoutsakis, 1998) .
Owing to the genetic complexity of higher eukaryotic cells and the absence of sophisticated genetic tools (compared to those for several microbial hosts), introduction of heterologous genetic information into mammalian hosts is usually achieved by cotransfection of a selection marker and the gene of interest with subsequent selection for clones containing the marker, and as empirical experience has shown, often also include the cotransfected gene (Kaufman and Sharp, 1982) . Many undesired phenomena accompany this haphazard genetic engineering of mammalian cells because of the undefined, mechanistically obscure selection of random integration sites in different stable clones, giving rise to variability in product expression levels, genetic stability, and second order effects on growth, viability, and productivity resulting from disruption of host genes (or regulatory loci) at the integration site. Recently, chromosomal locations of some industrially relevant mammalian cells lines have been found which show high transcription and stability for integration of transgenes (Karreman et al., 1996) . Screening for such sites is a time-consuming process that involves establishment of a genetic platform for subsequent targeted integration. However, unlike the situation in mouse ES stem cells (Hicks et al., 1997) gene targeting is difficult to achieve in most industrially relevant cell lines because they seem to lack necessary basic recombination machinery, and therefore they require installation of complex heterologous site-specific recombination systems (Fukushige and Sauer, 1992; Karreman et al., 1996) .
Regardless of the method of integration and the chosen combination of product and metabolic engineering genes, it is desirable to manipulate the cell in a minimal number of steps. This goal is addressed by technology for simultaneous cloning and subsequent expression of multiple genes in a desired host. Besides providing a platform for future metabolic engineering breakthroughs, multicistronic expression technology should speed basic functional genomic research and new applications in tissue engineering and gene therapy.
Here we review recent developments in multicistronic expression technology and their use to enable one-step multigene metabolic engineering, positive feedback regulation circuits and auto-selective expression systems.
Bacteria have evolved expression units called operons which unite functionally related genes under the control of a single promoter, thus enabling coordinated, simultaneous and rapid expression of metabolically coordinated genes in response to specific environmental signals or physiological constraints (the classic example is the lactose operon; Dickson et al., 1975) . Individual genes in a bacterial operon are preceded by characteristic sequences, socalled ribosomal binding sites (RBS), for translation-initiation at appropriate points within a single mRNA molecule. In contrast to bacterial multigene transcripts, most eukaryotic mRNAs are monocistronic, and optimal translation of the encoded gene relies on a post-transcriptional 5 modification (capping) for ribosome binding and subsequent AUG-scanning (Shatkin, 1985; Kozak, 1989) . However, other capindependent modes of translation-initiation such as leaky scanning, termination-reinitiation, and internal initiation are used in rare cases (Jackson et al., 1995;  Table 1; Figure 1 ).
As part of their pathogenic life cycle, picornaviruses have evolved specific genetic elements (internal ribosomal entry sites; IRES) in their 5 nontranslated leader regions (ntr) which adopt a particular secondary structure able to attract eukaryotic ribosomes and to allow internal translation-initiation (Belsham and Sonenberg, 1996 ; Table 1 ). The pivotal role of IRES in picornaviral pathogenesis is based on the expression of a viral protease which cleaves the host cap-binding translation-initiation factor eIF4G and allows redirection of host translation machinery for exclusive translation-initiation of IRES-containing viral mRNAs (Etchison et al., 1982; Pelletier and Sonenberg, 1988; Jackson et al., 1990; Belsham and Sonenberg, 1996; Rueckert, 1996) .
IRES-like elements are present in other viral systems and were recently discovered in eukaryotic cells which give certain mRNA molecules cap-independent translation ability in response to viral infection or stress conditions, as was shown for immunoglobulin heavy chain binding protein (Bip) and the cap-binding protein eIF4G (Macejak and Sarnow, 1991; Gan and Rhoads, 1996) (Table 1) . Cap-independent translation can also enforce an alternative translation start site, resulting in translation of different proteins from the same mRNA, such as that mediated by the human fibroblast growth factor 2 (FGF2) (Vagner et al., 1995) . Recently, IRES elements were also identified in the translation regulation of developmentally regulated genes such as the homeotic gene Antennapedia or Ultrabithorax of Drosophila (Oh et al., 1992; Ye et al., 1997) , the genes for human insulinlike growth factor (IGF-II) (Teerink et al., 1995) , and the platelet-derived growth factor B (developmental IRES or D-IRES; Bernstein et al., 1997) . The potential for cap-independent translation-initiation has also been found in yeast and Xenopus oocytes (Iizuka et al., 1994; Kneiper and Rhoads, 1997) . Furthermore, the finding of an internal ribosomal entry site in the 5 untranslated region of c-myc suggests that IRES- Figure 1 . Strategies for simultaneous and in some cases regulated expression of more than one gene in mammalian cells. Key genetic elements for expression in mammalian cells such as the promoter (P), internal ribosomal entry sites of polioviralorigin (IRES) or derived from the encephalomyocarditis virus (CITE), the splice donor (SD) and acceptor (SA) and the polyadenylation site (PA) as well as for regulated gene expression including the tetracycline-responsive transactivator (tTA) and the tet-responsive promoter (P hCMV * −1 ) are indicated. In some cases translation is shown below the genetic configuration (mRNA, ribosome, proteins). Iizuka et al., 1994 mediated translational control may be vital for higher organisms as aberrant translational regulation of cmyc is likely to play a role in tumorigenesis (Stoneley et al., 1998) .
Despite the potential of IRES as key elements of operon-like multicistronic expression units in mammalian genomes, such genetic configurations seem to have rarely evolved in a natural context, perhaps because most complex and fine-tuned regulatory cir-cuits in mammalian cells are best configured with independent regulation of individual genes.
Since the transcription and translation of separate cotransfected genes is not strictly correlated, the reliability of product expression based on selection of the cotransfected marker gene can be very low. Further- more, even under high selection pressure, the genetic stability of the expressed product can not be assured in long term cultivations. The combination of product and marker genes on the same vector does not completely alleviate these complications. For these reasons, dicistronic genetic configurations were developed. The first dicistronic constructs used IRES elements of picornaviral origin or from encephalomyocarditis virus (EMCV) for cap-independent translation of the second cistron while the first cistron relied on classical cap-dependent translation-initiation (Pelletier and Sonnenberg, 1988; Kaufman et al., 1991) . Although the two IRES elements differ completely at the sequence level, their secondary structure is very similar and typical for such internal translation initiators. A large number of dicistronic product-marker configurations have since been developed for many different applications. Table 2 gives an overview of recent dicistronic expression vectors. Although genetic combinations used for dicistronic expression vary among different applications, EMCV and picornaviral IRES remained the most popular cap-independent translation-initiators for dicistronic configurations because these elements function in a wide variety of cell lines including the industrially relevant CHO and BHK cell lines (Borman et al., 1997) . However, recent reports of varying translation-initiation capabilities of IRES in different host cell environments and discovery of new IRES elements is stimulating new development to apply these IRES elements for multicistronic expression (Bernstein et al., 1997; Schumacher and Wirth, 1997) . Dicistronic genetic configurations which contain the marker gene in the second cistron enable autoselective expression in addition to simultaneous and coordinated gene expression. Resistance to the marker gene or expression of the reporter gene is only possible if all 5 -encoded genetic elements are intact. This intrinsic self-selective program was found to be very reliable, with nearly all of the resistant cells also expressing the desired product gene (Gurtu et al., 1996; Rees et al., 1996) . Furthermore, product-marker configurations can also be used for efficient generation and screening of high producing cell clones: IRES-based translation-initiation of the second cistron is usually less efficient compared to cap-dependent translation, and can be decreased further by loss-infunction mutations of the IRES or the marker gene itself. The overall lower translation efficiency or activity is then compensated under high selective pressure by integration of the dicistronic expression unit into chromosomal sites with high transcriptional activity (Kaufman et al., 1991; Gurtu et al., 1996; Rees et al., 1996) . Certainly, simultaneous expression of two genetic traits can also be achieved by gene fusions (Krömer et al., 1997 ; pTracer plasmids of Clontech) or recently developed splicing expression technology (Lucas et al., 1996;  Figure 1 ), but gene fusion strategies are limited in functional applications or may lead to fusion products with altered physiologic specificities, and splicing-based two-gene expression leads to unequal expression levels of both proteins. Only IRESbased dicistronic expression guarantees simultaneous and coordinated expression of both transgenes at comparable levels for multi-subunit proteins (for example antibodies) which enables genetic configurations for a wide variety of contemporary research and development applications which are also listed in Table 2 (Dirks et al., 1993; Dirks et al., 1994; Fussenegger et al., 1997a) . Furthermore, IRES-mediated expression systems can be extended beyond the dicistronic level to tri-or even quattrocistronic artificial eukaryotic operons (Fussenegger et al., 1997b; Fussenegger et al., 1998c) .
Despite the numerous expression vectors available containing dicistronic expression units ( Table 2) , most of these expression systems express a marker or reporter gene in a fixed configuration, leaving only one cistron free for heterologous gene expression. However, for one-step transfection of a product protein, metabolic engineering, and a selection marker in a single expression unit, multicistronic artificial mammalian operons with 3 or even 4 cistrons are desirable. We recently reported the construction of a novel vector family, pTRIDENT, for tricistronic gene expression in mammalian cells (Fussenegger et al., 1998c; Fig-ure 2) . A single promoter allows high level expression and, in some vectors, adjustable transcription of all three genes. Whereas the first cistron is translated in a classical cap-dependent manner, translation-initiation of the subsequent two cistrons rely on IRES elements of picornaviral (IRES; pTRIDENT1) and EMCV origin (denoted here CITE, cap-independent translation enhancer; third cistron; pTRIDENT3; Fussenegger et al., 1998c) .
Tricistronic pTRIDENT1-and pTRIDENT3-derived test vectors encoding the model product gene SEAP (secreted alkaline phosphatase; first cistron), a metabolic engineering determinant (the cyclindependent kinase inhibitor p21 (CDI) second cistron), and the reporter gene GFP (green fluorescent protein; third cistron) were transfected into a CHO cell derivative which allows tetracycline-responsive gene expression (Fussenegger et al., 1998c) . Both tricistronic configurations were stable in CHO cells and showed strict simultaneous, coordinated as well as regulated expression of all three cistrons. The expression levels of individual cistrons were assessed by comparison to respective values of isogenic monocistronic expression vectors. Although expression levels of genes encoded on different cistrons are largely dependent on the overall stability of the polycistronic mRNA and therefore a direct function of the genetic configuration of encoded genes, our test vectors showed similar expression levels to those provided by the monocistronic vector on the first two cistrons and approximately 35% (CITE) to 50% (IRES II) lower expression levels on the third cistron. For enhanced translation-initiation of the third cistron, CITE was specially mutated (CITE * ) to avoid erroneous translation-initiation at upstream ATG start codons (Jackson et al., 1990; Kaufman et al., 1991; Davies and Kaufman, 1992; Rees et al., 1996; Fussenegger et al., 1998c) . Initially, the use of pTRIDENT3 derivatives (IRES-CITE) was preferred over double IREScontaining counterparts because pTRIDENT3-based vectors show a slightly higher translation efficiency of the third cistron, and they contain no duplicated sequence elements (IRESI-IRESII; pTRIDENT1) which bear the risk of recombination-mediated deletion of the second cistron. However, genetic rearrangements or deletions in double IRES-containing pTRIDENT1 derivatives were never observed during cloning steps in recA − E.coli nor in mammalian cells (Fussenegger et al., 1998a and 1998c) .
pTRIDENT vector backbones encode a bacterial ampicillin resistance and origin of replication (ori) Figure 2 . Examples for multicistronic expression in mammalian cells: pTRIDENT1 and pQuattro-tTA. Both vectors allow tetracycline-regulated expression of all transgenes. Since pQuattro-tTA encodes all genetic elements for regulated gene expression in its multicistronic expression unit, pQuattro-tTA allows autoregulation and can be used to achieve one-step regulated expression the genes of interest in any cell line where the internal ribosomal entry sites of polioviral origin function (IRES, CITE).
for high copy number amplification of these plasmids (Figure 2 ). High copy number amplifications in bacterial hosts is a prerequisite for large-scale transient transfection protocols which are becoming increasingly popular for industrial R&D applications (Fussenegger et al., 1997a) . The tricistronic expression unit contains three multiple cloning sites (MCS) with up to 18 unique restriction sites, many for 8 bptargeting, rare-cutting enzymes to allow sequential, complication-free cloning of all three transgenes into pTRIDENT. The general modular set-up of the key genetic elements in the pTRIDENT series, including, promoter, IRES elements, polyadenylation site, and vector backbone with their well selected flanking (or sometimes internal) restriction sites or MCS allows straightforward elimination or exchange of cistrons between existing conventional monocistronic or pTRI-DENT expression vectors. Also, the modular set-up enables rapid adaptation of the pTRIDENT vector concept for special applications and stimulates future developments in expression vector design. Based on the compatibility of pTRIDENT to existing vector families, for example the one presented by Dirks et al. (1993 and , recent developments of the growing pTRIDENT family resulted in tricistronic vector derivatives with various constitutive (P SV 40 , P MP SV ), tetracycline-and ecdysone-regulated promoters (P hCMV * −1 ; P EC ) and in construction of auto-regulated, self-selective, one-step transfection systems described below.
Pioneering reports by Suzuki and Ollis (1990) and Al-Rubeai et al. (1992) showing increased specific productivity of growth-inhibited hybridoma cells stimulated research on chemical culture additives to arrest cell growth and initiated efforts to control cell growth by controlled overexpression or inhibition of selected genes. Three successful one-gene metabolic engineering strategies have been developed to reversibly control mammalian cell growth: (i) estrogen-regulated overexpression of the interferon-responsive factor (IRF-1), a transcription factor which is upregulated by interferons as response to viral cell invasion, in BHK cells (Koester et al., 1995) ; (ii) dexamethasoneinducible suppression of the key transcription factor c-jun by antisense technology in Friend murine erythroleukemia cells (F-MEL) (Kim et al., 1998) ; and (iii) tetracycline-regulated overexpression of negative key regulators of the cell-cycle including the tumor suppressor p53 and the CDIs p21 and p27 in CHO cells (Fussenegger et al., 1997a; Fussenegger et al., 1998a and 1998b) . Overexpression of IRF-1 resulted in cell-cycle-independent growth arrest, but heterologous gene expression was not enhanced unless the exogenous genes were placed under control of IRF1-responsive promoters. Furthermore, IRF-1overexpressing BHK cells rapidly die, probably by an apoptosis-independent pathway (Koester et al., 1995; Müller et al., 1998) . On the contrary, c-jun suppression leads to sustained G0-phase arrest of F-MEL cells for over two weeks and protects these cells against apoptosis (Kim et al., 1998) . Unfortunately, this promising antisense technology remains to be assessed in an industrially relevant cell line and in connection with cloned protein production. However, G0-arrested cells have previously shown to produce exogenous protein at a lower rate (Kim et al., 1998) .
In another strategy, transient tetracycline-responsive overexpression of p53, p21 or p27 in a dicistronic configuration (SEAP-p53; SEAP-p21; SEAP-p27) led to G1-specific cell-cycle arrest, and in each case was accompanied by an up to 4-fold increase in SEAP production compared to proliferation-competent control cells (Fussenegger et al., 1997a) . These results compare favourably with those from G1-arrested, temperature-sensitive CHO cells generated by random mutagenesis, which also showed a 3-4-fold higher heterologous protein production upon growth arrest but retained low cell viability at elevated permissive temperatures (Jenkins and Hovey, 1993) .
However, in a stable genetic configuration in CHO cells, only SEAP-p27 overexpression lead to a significant increase in productivity, with specific SEAP productivity increasing by 15-fold compared to control cells (Fussenegger et al., 1998b) . Intracellular p21 levels were probably insufficiently high to cause significant growth inhibition, and p53-based cell-cycle arrest led to rapid decrease in cell viability accompanied by cell morphologies indicative of apoptosis, even when achieved by overexpression of the apoptosis-deficient mutant p53175P (Rowan et al., 1996) , a phenomenon which could not be observed with p27-induced G1-arrest (Fussenegger et al., 1998b) .
The failure to produce stable growth-controllable CHO cells by p21-mediated overexpression exemplifies current limitations of one-gene metabolic engineering strategies. Although global regulatory proteins certainly exist, such key metabolic effectors are rare, difficult to find and their overexpression may imbalance fine-tuned interconnected cellular circuits, as seems the case with overexpression of p53.
Using tricistronic expression technology we extended the SEAP-p21-encoding dicistronic configu-ration by an additional cistron harbouring the differentiation factor, CCAAT/enhancer binding protein α (C/EBPα) (pSS5; Figure 3 ). C/EBPα has been shown to stabilise p21 at the protein level and also to induce endogenous p21 alleles (Timchenko et al., 1996) . Using this tricistronic set-up for metabolic engineering, the induction of conditional growth arrest of CHO cells was successful, and the sustained cell-cycle arrest achieved was accompanied by an up to 15-fold higher specific SEAP productivity compared to proliferationcompetent control cells, similar to that achieved by p27-based one-gene metabolic engineering (Fussenegger et al., 1998a and 1998c) .
In a further preventive measure against possible apoptosis, which was strongly suggested by morphologies of p53 overexpressing cells, we linked SEAP-p27 expression with the expression of the survival gene bcl-x L in a tricistronic configuration (pDD6; Figure 3 ). bcl-x L belongs to the family of bcl-2 anti-apoptosis genes which have been successfully used to suppress apoptosis in production cell lines (Cotter and Al-Rubeai, 1995; Mastrangelo and Betenbaugh, 1998) . Although overexpression of SEAP-p27-bcl-x L induced sustained growth arrest in CHO cells like its dicistronic counterpart, the specific SEAP productivity of arrested cells was increased by an additional factor of three, which corresponds to 30-times higher specific SEAP productivity than respective proliferation-competent control cell lines (Fussenegger et al., 1998a) . This unexpected effect of bcl-x L expression cannot be explained based on the current knowledge of cell-cycle and apoptosis regulatory pathways, and further investigations are needed to reveal the mechanism of this new, apparently apoptosis-unrelated effect of bcl-x L .
Thus, using controlled proliferation technology as an example, multigene metabolic engineering has proven to be useful for achieving difficult-to-attain cell culture states, and the combinatorial expression of an intuitively unrelated gene revealed previously unknown functions and potential molecular links of complex cellular pathways.
There is much current interest in the development of regulatable expression systems in basic functional genomic research, since externally regulated transcription enables the effects of a particular gene product Figure 3 . Tricistronic expression vectors enabling multigene metabolic engineering. Both vectors express the model product gene, the secreted alkaline phosphatase (SEAP) and one of the cell-cycle inhibitors p21 (pSS5) and p27 (pDD6). While the expression of p27 is sufficient to cause cell-cycle arrest and result in enhanced specific SEAP productivity which is additionally increased by coexpression of the anti-apoptosis gene bcl-x L , cell-cycle arrest using p21 is only effective when the differentiation factor c/ebpα is coexpressed and stabilizes p21.
to be assessed in an identical genetic background. Regulated gene expression is also gaining increasing importance for biotechnological applications since it allows conditional metabolic engineering and achievement of specific cell culture states in a timely manner (Fussenegger et al., 1997a; Fussenegger et al., 1998a) . For example, regulated metabolic engineering in a multicistronic configuration allows differentiation of a cell culture process into two stages: a nonproductive growth phase in which the cells are rapidly expanded to the desired cell density, and a subsequent non-proliferating production phase where the cells can devote all of their metabolic capabilities to the production of protein instead of biomass.
Several in vivo regulated eukaryotic promoters have been described (Schweinfest et al., 1988; Israel and Kaufman, 1989; Ko et al., 1989; Hu and Davidson; Mattioni et al., 1994) and used for regulated gene expression. However, as most of these regulated promoters are derived from regulatory circuits which mediate metabolic responses, the corresponding regulating external stimuli may lead to undesired pleiotropic effects. More successful transcriptional regulation circuits rely on basic regulatory machineries of heterologous origin which are genetically adapted for use in mammalian cells. Besides the lac switch (Fieck et al., 1992) and the ecdysoneresponsive system , the tetracyclineregulatable expression system (tet system; Gossen and Bujard, 1992) is by far the most popular. The tet system consists of two separate genetic entities, the tet-responsive transactivator (tTA) and the tTA responsive promoter, P hCMV * −1 each of which represents a chimeric genetic configuration composed, respectively, of a protein fusion between the bacterial tet repressor and the VP16 domain of the herpes simplex virus (tTA) and a genetic fusion which places a tet operator adjacent to a minimal cmV promoter (P hCMV * −1 ). While the bacterial parts are responsible for promoter recognition and integrate responsiveness to the external stimulus tetracycline, the viral parts initiate transcription (Gossen and Bujard, 1992) .
Despite its success story, the tet system has two major limitations. First, prior to introduction of the regulated transgene, each host cell line must be engineered to express tTA in a fashion which affords efficient tetracycline-mediated control of P hCMV * −1initiated transcription. Screening for this phenotype is tedious and time-consuming. Cotransfection of the tTA-expression plasmid and the vector encoding the gene of interest or the transfection of a single vector with a combination of both genes, are not recommended since, in either case, the close proximity of the two genes in the host chromosome may cause the enhancer of tTA-driving promoter expression to interfere with P hCMV * −1 thus leading to hardly regulatable configurations (Gossen and Bujard, 1992) . Second, a transcriptional 'squelching' effect by the VP16 transactivator domain may be lethal for the host cell, even at moderate expression levels (Gill and Ptashne, 1988) . Consequently, since the activity of P hCMV * −1 is proportional to the intracellular tTA levels, moderate tTA expression may lead to apparently low expression levels of regulated transgenes (Furth et al., 1994) . Several improvements have been made to alleviate these complications including (i) fusion of the tTA to the ligand-binding domain of the estrogen receptor to control the transfer of tTA into the nucleus (Iida et al., 1996) , (ii) fusion of tTA to a nuclear localisation signal enabling tight regulation and high-level induction (Yoshida and Hamada, 1997) , (iii) construction of a regulatory cascade by controlling tTA expression by another higher-order control system, for example, by the lac switch (Aubrecht et al., 1996) , (iv) construction of P hCMV * −1 derivatives harbouring minimal promoters of various sources which show altered regulatory features, promoter strength and tTA responsiveness (Hoffmann et al., 1997) , and (v) placing tTA expression itself under control of P hCMV * −1 to prevent accumulation of toxic tTA levels prior to induction (Shocket et al., 1995) . However, all of these improvements still require two rounds of transfection for their implementation.
We recently reported one-step, auto-regulated and auto-selective multicistronic mammalian expression systems which included the tTA in a multicistronic, pTRIDENT-based or quattrocistronic configuration (pQuattro-tTA; Fussenegger et al., 1997b ; Figure 2 ). Since the tTA gene is encoded on the multicistronic expression unit itself, little or no tTA is expressed under repressive conditions. This genetic configuration alleviates intracellular accumulation of toxic tTA levels. However, when the auto-regulated system is induced, the few tTA molecules originating from the leakiness of P hCMV * −1 activate this tet-responsive promoter ( Figure 1 ). Since tTA is itself encoded on the artificial operon, every round of transcription generates also an additional tTA message resulting in a positive feedback regulation system with high tTA levels and consequently high level expression of all cocistronically expressed transgenes. Since all genetic elements essential for regulated gene expression are united in a single vector, these autoregulated pTRIDENT derivatives mediate one-step regulated gene expression in various cell lines including CHO, BHK and HeLa cells (Fussenegger et al., 1997b) . Previously, HeLa cells have been reported to be very sensitive to squelching, and prolonged screening procedures are usually necessary to select HeLa clones with moderate tTA expression to avoid this problem (Gossen and Bujard, 1992) . To our knowledge, no convincing tet-regulation gene expression has previously been established in BHK cells apart from a recent report by Sekigushi and Hunter (1998) which shows very high background under repressed conditions and only 10-fold induction. On the contrary, our positive feedback regulation system showed both tight regulation as well as high levels of tet-responsive gene expression in all these cell lines with no signs of squelching. The lack of squelching is rather surprising, considering that the positive feedback circuit is expected to produce high intracellular levels of tTA. However, recent experiments with a monocistronic positive feedback configuration in transgenic animals also showed no detrimental effects (Shocket et al., 1995) .
Positive feedback configurations with tTA in the last cistron consist of both essential and interdependent elements for regulated expression, with P hCMV * −1 and tTA at the perimeters of the multicistronic expression unit. This set-up harbours an intrinsic, auto-selective program which guarantees full length transcripts and maintains the functional integrity of all genetic elements encoded on this autoregulatory operon. Recently, a similar autoregulated dicistronic expression system was reported (Shocket et al., 1995; Hofmann et al., 1996; Zhang et al., 1997) , but only pTRIDENT-based or pQuattro-tTA systems allow one-step, autoregulated and auto-selective multigene metabolic engineering in industrially-relevant cell lines (Fussenegger et al., 1997b) .
Since one-gene metabolic engineering will necessarily reach its limits when coping with today's increasingly complex challenges, the recent development of artificial eukaryotic operons enables effective multigene metabolic engineering of mammalian cells. This greatly expands possibilities to reprogram interconnected cellular networks in desired ways to improve key characteristics of mammalian cells. Besides use in next-generation multigene metabolic engineering, multicistronic expression units are expected to have great impact on very specific applications including (i) straightforward combinatorial evaluation of gene functions and metabolic networks, (ii) one-step transfection, selection and maintenance of difficult-toexpress (multi-subunit) proteins, (iii) selection of high producer cell lines, and (iv) genetic immunisation and gene therapy in combination using sense, antisense or ribozyme technology. Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. The incidence of genital herpes is high and increasing world-wide (Corey, 1993 ). An important aim in combating the disease is achieving an effective vaccine which can act against both the primary and recurrent disease caused by herpes simplex virus. The company strategy has therefore been to adapt live viruses by genetic manipulation to introduce an acceptable margin of safety through the development of DISC (Disabled Infectious Single Cycle) virus vaccines. These are viruses which lack an essential gene, and are therefore unable to undergo multi-cycle replication in a vaccinated host. They can, however be prepared in a way that allows them to go through a single cycle of replication in cells of the vaccinee, leading to an effective immune response. It has been shown that herpes simplex viruses lacking the essential glycoprotein H (gH) gene can be used as effective vaccines (Farrell et al., 1994; McLean et al., 1994) . The virus is therefore genetically inactivated so that it is unable to spread within the host. * Part of this work was presented as a poster at the ESACT meeting in Tours, France. September 1997.
Currently the DISC-HSV is propagated in a complementing cell line derived from a Vero (African Green monkey kidney) cell line approved by the World Health Organisation (WHO) for use in vaccine manufacture (WHO, 1989) . The Vero cells were modified to contain the HSV-2 gH gene under the control of the HSV-1 glycoprotein D (gD) gene promoter (Boursnell et al., 1997) . The complementing cell line was designated CR2. Because the gD promoter requires additional HSV proteins for its induction, gH is regulated so that it is only produced in the cell following an infection with virus. Virus produced from CR2 cells can infect normal cells, but can only perform one cycle of replication. The progeny virus from this replication cycle lack the gH protein and are therefore non-infectious ( Figure 1 ).
Previously it has been shown that a DISC gHdeleted HSV-1 can protect against HSV-1 challenge in the mouse ear model (Farrell et al., 1994) . The DISC virus, by virtue of its capacity for a single round of replication in normal cells, is more potent than a non-replicating, inactivated virus preparation. A gHdeleted HSV-2 virus has also been tested as a vaccine in a guinea pig model. Animals vaccinated with DISC HSV-2 showed complete protection against primary HSV-2 induced disease, even when challenged six months after vaccination (Boursnell et al., 1997) . The animals were also almost completely protected against recurrent disease.
For the vaccine to become a viable option as a commercial product, it must be manufactured on a large scale with appropriate processing to meet the demands of the regulatory requirements of safety and efficacy. Currently the DISC-HSV is propagated in the CR2 cell line. The CR2 cell line is an adherent cell and therefore routine culture was performed in roller bottle cultures. Whilst this method of cell growth and virus production is suitable for development work it is not a desirable system for the manufacture of the virus on a larger scale. The use of microcarriers as a support for anchorage-dependent cells has been reported previously (Hu et al., 1985; Van Wezel, 1973) . Anchorage-dependant cell lines have been cultured to produce a variety of viruses on a large scale (Talbot et al., 1989; Lesko et al., 1993; Baijot et al., 1987; Meignier, 1978) . Some cell lines such as Vero and MRC5 have been propagated on microcarriers to produce human viral vaccines (Fabry et al., 1989; Griffiths et al., 1980; Montagnon et al., 1981) . Griffiths et al. (1982) have demonstrated HSV-2 production from cells cultured on low (2 g l −1 ) concentrations of Cytodex microcarriers. The yields obtained were ten-fold lower when compared to those achieved from a roller bottle system. It was hoped that our current microcarrier system would produce equivalent productivity when compared to the standard roller bottle process.
The aim of this work was to evaluate the production of DISC-HSV in a microcarrier based culture system in comparison to a conventional roller bottle culture process. The work set out to determine the conditions for the optimum growth of cells and ultimately, production of high titre virus. Production of the virus was initially determined in small-scale (1 l) cultures. Scale-up of the production system was then demonstrated at the 15 l scale.
This paper details the way in which we have approached this issue of manufacturing DISC-HSV only as far as the initial upstream bulk harvest product and does not discuss the subsequent virus purification.
Viruses: The DISC-HSV virus, grown in gHexpressing CR2 cells was constructed at Cantab Pharmaceuticals, Cambridge, U.K.
Cells: Two cell lines are used in our study. One has been designated CR1 and the other CR2. The CR1 cell line is a line used for the assay of the virus only. It is a Vero derived cell line that has been modified to express the glycoprotein H (gH) gene derived from HSV-1 this gene is under the control of the HSV-1 glycoprotein D (gD) promoter. CR2 cells are a modified Vero cell from the WHO Vero accredited bank (No. 88020401; European Collection of Animal Cell Cultures ECACC, Porton Down, U.K.) that has been modified to express the glycoprotein H (gH) gene derived from HSV-2. This gene is under the control of the HSV-1 glycoprotein D (gD) promoter. The CR1 cells are solely used to assay the virus in the TCID 50 assay whereas the CR2 cells are the production cells for manufacture of the DISC-HSV. Cells were cultivated in Dulbecco's Modified Eagles Medium (DMEM with high glucose, Life Technologies, U.K.) supplemented with 5% Foetal Bovine Serum (PABCO, New Zealand origin).
The bottles used had a total surface area of 850 cm 2 and were obtained from Corning.
Cytodex 1 microcarriers: (Pharmacia Biotech, U.K.): The microcarriers were used at a density of 5 g l −1 and were prepared according to the manufacturers instructions. Before use they were pre-conditioned in DMEM (5%FBS).
Hypotonic saline: The solution comprised of Na 2 HPO 4 2.29 g l −1 , NaH 2 PO 4 .2H 2 O 0.599 g l −1 NaCl 0.58 g l −1 . The reagents were sourced from BDH.
Roller bottle cultures were seeded with a total of 2 × 10 7 CR2 cells per roller bottle. The cultures contained 100 ml of DMEM (5% FBS) per bottle culture. Cultures had a five day cell growth period at 37 • C prior to infection with DISC-HSV.
At infection the cell monolayers were washed with Dulbecco's PBS to decrease the level of any contaminating BSA from the FBS. Serum-free DMEM was added to the cells as a maintenance medium throughout the virus production phase. Cultures were infected with a working seed preparation of DISC-HSV at a multiplicity of infection (MOI) of 0.01 pfu cell −1 . The temperature of the cultures during the virus production phase was decreased and maintained at 34 • C.
Approximately 64-68 h post infection cell monolayers have between 90-100% CPE. This observed level of CPE is ideal for harvesting the DISC-HSV. The harvest method involved pouring off the medium and adding 10 ml of hypotonic saline. The cultures were then incubated at 34 • C for 5-10 min. The cells and virus were collected by scraping the surface with a plastic scraper.
Production of DISC-HSV in microcarrier culture 1 l and 15 l scale
The microcarriers were prepared as according to instructions from Pharmacia. They were prepared in a siliconised Schott bottle of suitable size. All microcarriers were conditioned by washing with DMEM (5% FBS) prior to addition to the vessel. The microcarriers for the 1 l cultures were washed twice with 250 ml of complete medium whilst the microcarriers for the 15 l cultures were washed twice with 1500 ml of medium.
All vessels were obtained from FT Applikon Ltd. For the 1 l cultures a 2 l total volume jacketed glass vessel was used. A BioBench 20 l total volume, jacketed stainless steel vessel was used to culture the 15 l cultures. Both vessels had spin filters (76 µm mesh size) and used reverse marine impellers. The 2 l vessels had an H:D ratio of 1.5 and the 20 l vessel had an H:D ratio of 2.2.
The growth of our CR2 cells was examined on several commercially available microcarriers. The range used included the following: -Cytodex 1 (Pharmacia), Cytodex 2 (Pharmacia), Cytodex 3 (Pharmacia), Cultisphere (Cellon Sarl), Cytopore 1 (Pharmacia), Cytopore 2 (Pharmacia), Cytoline 2 (Pharmacia) and FACT (Solohill).
The microcarrier Cytodex 1 was chosen as the production carrier because the cell density obtained using this carrier was the greatest from the least amount of microcarriers used. Cytodex 3 microcarriers gave a similar cell number as expected but this microcarrier has a pig skin collagen layer on the bead. Cytodex 1 was selected in preference to the other microcarriers because of good cell growth and to remove the issue of animal products in the manufacturing process. We currently use the Cytodex 1 at a level of 5 g l −1 . The growth medium used was Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% Foetal Approximately 75% of the final volume of cultivation medium and 5 g l −1 of microcarriers were pre-incubated in the culture vessel overnight. The culture parameter set points were controlled by using the Applikon 1030 controlling system for both vessels. The culture pH was maintained by controlling at a set point of 7.2±0.2 by sparging air/CO 2 (90:10%) into the spin filter. Dissolved oxygen concentrations were kept above a minimum set point of 30% saturation by sparging when necessary into a spin filter with air for the 1 l cultures and with pure oxygen in the 15 l cultures. The culture temperature was maintained at 37 • C throughout the cell growth period by use of a recirculating thermocirculator attached to the jacket of the 2 l vessels and in the case of the 20 l vessels the Applikon controlling system used a similar integral recirculating thermocirculator.
The cell inoculum for cultivation on microcarriers was prepared from late exponential CR2 cultures in 850 cm 2 roller bottles. Cultures were inoculated at a density of approximately ten CR2 cells per microcarrier and 5 g of Cytodex 1 l −1 (Figure 2 ). Cell growth was monitored by estimating nuclei released from a sample of microcarrier culture taken from the reactor after incubation in 0.1 M citric acid containing 0.1% (w/v) crystal violet (Sanford et al., 1951) . Glucose and lactate concentrations were determined using an off-line YSI 2700 glucose/lactate analyser (YSI, U.K. Ltd). Glucose and lactate concentrations were controlled by partial media changes on days 2 and 3 for the 1 l culture and days 2, 3, 4 and 5 for the 15 l culture. The 15 l culture was maintained for an additional 48 h period compared to the small scale 1 l cultures in Figure 5 . The bionebuliser is a low pressure shear disruption method which uses nitrogen as a carrier gas. The disrupter is depicted here schematically indicating how the process disrupts cells. Using a carrier gas pressure of 50 psi cells are drawn up and nebulised. This cell mixture is directed to a ceramic ball bearing where they impact. The cells are disrupted to release the virus and the whole disrupted cell/virus solution is collected. order to provide a maximal cell density at the point of infection.
Confluent microcarriers (Figure 3) , approximately 100 h post inoculation in the 1 l cultures and approximately 140-160 h in the 15 l cultures were infected with DISC-HSV at an MOI of 0.01 pfu cell −1 . The agitation was stopped and the microcarriers were allowed to settle out. The medium was then removed to waste. The 1 l cultures were washed by addition of 1 l of Dulbecco's PBS with agitation. The PBS wash was removed and this step repeated a further two times. The 15 l cultures were washed by addition of 10 l of Dulbecco's PBS with agitation. The PBS wash was removed to waste and this step was repeated a further two times. After washing, the original culture volume was replenished with serum free DMEM. The temperature during the infection period was decreased and maintained at 34 • C.
Infected cells (Figure 4) were harvested from the microcarriers 60-72 h post infection when the cells on the majority of the microcarriers showed 100% CPE. Harvesting was accomplished by removal of the media from the culture, addition of hypotonic saline at a volume equal to 10 ml hypotonic saline per roller bottle equivalent of surface area of microcarriers used in the culture. The detached cells were separated from Cytodex 1 by filtration through a sterilisable 70µm mesh in the bottom of the vessel. The resulting cell/virus supernatant was subjected to low pressure disruption (Degouys et al., 1997) , (Figure 5) , to release the DISC-HSV virus from the cells and cellular debris. Virus titres were calculated using an in-house TCID 50 assay with subsequent conversion to pfu ml −1 .
The cell and virus harvests from either pooled roller bottle cultures or microcarrier cultures were passed through a bionebuliser (Glas-Col) at a pressure of 50 psi using sterile filtered nitrogen as the carrier gas. The carrier gas causes a vacuum over the hole leading to the cell suspension. This vacuum draws the cell suspension up the tubing and allows the cell suspension to become mixed with the carrier gas. The mixture then flows through the orifice to impact on the target a ceramic ball to disrupt the cells in the suspension. The flow rate of the gas determines the density of cells in the mixture and the target ceramic ball can be adjusted to maintain a good bionebulisation of the cells. The process is depicted in Figure 5 . This low-pressure shear system has been used previously to disrupt cells (Degouys et al., 1997) .
The DISC virus infectious titre (TCID 50 ml −1 ) was estimated in CR1 cells on 96-well plates infected with serial tenfold dilution's of virus material. The CPE was read 3-4 days post infection using a TCID 50 ml −1 method (Reed and Muench, 1938) . The TCID 50 ml −1 assay is used with a subsequent conversion to pfu ml −1 as described by Dougherty (1964) . The TCID 50 dose can be measured by a number of means and the method of Reed and Muench (1938) was employed in this case. This TCID 50 is the usual end point for many virus assays. The conversion to pfu ml −1 takes into account the distribution of virus in the suspension according to Poisson's Law. According to Poisson distribution the TCID 50 would contain 0.69 infectious particles per unit volume. Therefore this correction factor can only be applied when chance distribution of virus in suspension occurs (Dougherty, 1964) .
Roller bottle process CR2 cells were cultured in roller bottles for 5 days until a cell density of 1 × 10 8 cells was typically achieved. At this density 100% cell confluency was observed. Roller bottle cultures infected with DISC-HSV at an MOI of 0.01, exhibited 100% CPE after approximately 65 h. Harvesting the roller bottles with hypotonic saline it was possible to achieve 10 9 pfu per bottle. Figure 6 . Typical growth of CR2 cells and DISC-HSV production at 1 l scale. The total DISC-HSV virus titre produced in a 1 l vessel is approximately 1.5 × 10 10 pfu. Figure 7 . Typical growth of CR2 cells and DISC-HSV production at 15 l scale. The amount of DISC-HSV virus titre able to be produced from a 15 l culture is 7 × 10 11 pfu. This demonstrates a considerable scale up potential for a microcarrier production system.
Based on available surface area for cell attachment, a 1 l microcarrier culture is equivalent to approximately 25 × 850 cm 2 roller bottles in terms of potential virus production capability (Table 1) . A typical growth curve for CR2 cells on Cytodex 1 microcarriers is shown in Figure 6 . Cells multiplied exponentially during the first 4 days before reaching stationary phase. A cell density of 1.5-2.0 × 10 9 total cells was typically obtained after approximately 100 h growth. At this density a confluent monolayer of CR2 cells covered the microcarriers (Figure 3 ). At 100% confluency the cells appeared elongated, resembling the surface of a golf ball. Cells ready for harvest exhibited complete CPE (Figure 4 ) 60-72 h after infection. The cultures were harvested before they became detached from the microcarriers. The morphological appearance of the cells at both critical stages was identical to the appearance observed in roller bottle cultures. Infectious DISC-HSV released from the CR2 cells was quantified using a TCID 50 assay and compared to the amount obtained from the roller bottle culture harvests. The results are shown in Table 1 . Typically 1.5-2.0 × 10 10 total pfu from a 1 l culture was regularly achieved, with approximately a 20 hour period during which the culture could be harvested without significant decrease of the titre. The total surface area available in the culture is equivalent to 25 roller bottles. This enables a comparison in terms of productivity to be made. We routinely achieve 2.0-2.5 × 10 10 total pfu from 25 roller bottle cultures. Therefore, the two production systems yield similar total amounts of DISC-HSV.
Based on available surface area for cell attachment, a 15 l culture is equivalent to approximately 388 × 850 cm 2 roller bottles in terms of potential virus production (Table 1) . A typical growth curve for CR2 cells on Cytodex 1 microcarriers grown at the 15 l scale is shown in Figure 7 . Cells multiplied exponentially during the first 6 days before reaching stationary phase. Typically a cell density of 4.0-5.0 × 10 10 total cells was obtained after approximately 150 h. After this growth period a confluent monolayer of CR2 cells (Figure 3 ) covered the microcarriers. When the cells exhibited complete CPE (Figure 4 ) 60-72 h after infection, the cultures were harvested. Infectious DISC-HSV released from the CR2 cells was quantified using a TCID 50 assay and was again compared to the titre achieved from the equivalent roller bottle culture harvests. The results are shown in Table 1 . Typically 4-7.0 × 10 11 total pfu from a 15 l culture was achieved, with a period of approximately 9 h during which this high titre was sustained. From an equivalent number of 388 roller bottles we would expect to achieve approximately 3.88 × 10 11 total pfu. Our results, therefore, compare very favourably with the expectations from roller bottle cultures and the results achieved from the 1 l cultures.
Cytodex 1 microcarriers can be used as a Vero cell culture support for the production of DISC-HSV virus. It could be envisaged that the microcarrier washing and conditioning procedure at a large manufacturing scale could be extremely time consuming. It is expected that the microcarriers will be sterilised in-situ inside the production vessels. Preliminary work at 35 l scale employs the use of an internal sieve to aid the washing and conditioning process. At present at the 35 l scale this step takes approximately 2 h to complete. This washing and conditioning step may be an extensive time constraint at a manufacturing scale. Tackling the engineering problem in terms of pipe work and increasing flow rates could reduce this time. This is an issue that is under review and will require suitable development time to reduce it satisfactorily. Yields achieved from the microcarrier cultures were comparable to those obtained in the standard roller bottle culture systems (Table 1) .
It has been demonstrated that a 15 l microcarrier culture can produce an equivalent amount of virus as that achieved from 700 × 850 cm 2 roller bottles despite having the surface area of only 400 roller bottles. This maintenance of the cell productivity has not always been observed when virus production systems have been scaled up. Griffiths et al. (1982) had a tenfold decrease in cell productivity of HSV per cell when switching from roller bottle cultures to a microcarrier system using Vero cells. This may have been an artefact of poor virus release from the cells. In comparison, when culturing the HSV-2 on MRC-5 cells, a significant but smaller decrease in production levels was observed when changing from roller bottle cultures to microcarrier cultures. This observation reinforces the issues made in some early virus production work reported by Giard et al., 1977. They proposed that there are specific virus/cell line requirements coupled with a specific optimisation of the conditions not only for the growth of the cells but also for the virus production phase. The production of hepatitis A in microcarrier culture was reported to be 8-fold lower than when cultured in conventional flask cultures (Widell et al., 1984) . The maintenance of productivity seen in this study is very encouraging in moving the culture system forward to a larger scale which may be suitable for the production of Phase III material and for the supply of commercial requirements. Due to the productivity's shown here, production at a 15 l scale would be large enough to supply material for Phase I and Phase II studies.
Additional benefits of this system arise in the need for decreased culture medium. The very large culture surface area to volume ratio offered by the microcarrier system provides high cell yields in a minimal volume. When compared with the roller bottle system approximately half the volume of medium is required to produce an equivalent cell density (Van Wezel, 1972) . This leads to savings on the cost for culture medium and in particular for costly serum additions.
The number of direct manipulations is also reduced when switching to a microcarrier based production system. This decreases the labour intensity of the process, minimising costs of materials and the overall process time. The virus is produced from a single batch culture with one set of aseptic manipulations throughout the process rather than multiple manipulations that are required when using a roller bottle system.
If the system can be operated at a larger scale (e.g. >50 l) and still maintain DISC-HSV productivity then it would become a viable production method. Passaging Vero cells to a large production vessel (500 l) has been suggested (Baijot et al., 1987) , but it likely that the trypsinisation method will be all important in such a process.
The high productivity cultures using perfusion techniques, such as the production system used for the poliovirus production at 20 g l −1 of microcarriers (Fabry et al., 1989) , may also be employed for high titre virus production. However, further development work would be needed to adopt a similar production system for our DISC-HSV vaccine.
In our laboratory we are considering serial passaging and perfusion techniques to improve our production capabilities further. From the results reported in this present study we are confident that we have a productive scaleable microcarrier system for our DISC-HSV vaccine that will allow us to produce adequate material for both Phase III studies and commercial product. Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance. The current outbreak of swine infl uenza that originated in Mexico in March 2009 has spread to more than 80 countries causing more than 3,99,232 laboratory confi rmed cases of pandemic infl uenza H1N1 globally and over 4735 deaths reported to World Health Organization (WHO)as of 11 October 2009 [1] . The WHO declared pandemic alert stage 6 on 11 June 2009, indicating an ongoing infl uenza pandemic [2] . The 2009 swine fl u virus designated H1N1 A/swine/California/04/2009 is not zoonotic swine fl u and is not transmitted from pigs to humans, but rather from person to person and has higher transmissibility than seasonal infl uenza viruses [3] . In humans, H1N1 swine fl u presents as an infl uenza-like illness (ILI) with symptoms similar to seasonal infl uenza, i.e. fever, cough, sore throat, runny nose, muscle pains, severe headache, however, a considerable proportion of patients reported vomiting or diarrhea which is unusual in seasonal infl uenza [4, 5] . Since these symptoms are not specifi c to swine fl u, early in the pandemic physicians were advised to consider swine infl uenza in the differential diagnosis of patients with acute febrile respiratory illness who had returned from Mexico or been in contact with persons with confi rmed swine fl u [6] . This new strain of H1N1 swine infl uenza has a unique combination of genes from both North American and Eurasian swine lineages that has not been identifi ed previously in either swine or human populations [7] . The virus appears to be a result of reassortment of two swine infl uenza viruses, one from North America and one from Europe with the North American virus itself the product of previous re-assortments, carrying an avian PB2 gene for at least 10 years and a human PB1 gene since 1993. The virus also has genome segments of avian origin. Hence scientists call this novel strain as a "quadruple reassortant" virus. The hemagglutinin (HA) gene is similar to that of swine fl u viruses present in pigs in United States since 1999, where as neuraminidase (NA) and matrix (M) genes resemble viruses present in European pigs. Viruses with this genetic makeup have not previously been found in humans or pigs.
In India the novel H1N1 virus infection has been reported from all over the country. The most affected states are Maharshtra, Delhi, Tamil Nadu, Karnataka, Andhra Pradesh, Haryana, Kerala, Uttar Pradesh and Gujarat. As on 21 October 2009, a total of 68,919 samples from clinically suspected persons have been tested for infl uenza A H1N1 in government laboratories and a few private laboratories across the country and 13,330 (18.9%) of them have been found positive with 427 deaths [8] .
Genomic analysis of the 2009 infl uenza A (H1N1) virus in humans indicates that it is closely related to reassortant swine infl uenza A viruses isolated in North America, Europe and Asia [ Fig. 1 ] [9] [10] [11] . The segments coding for the polymerase complex, hemagglutinin, nuclear protein, and non-structural proteins show high similarity with the swine H1N2 infl uenza A viruses isolated in North America in the late 1990s. The segments coding for the neuraminidase and the matrix proteins of the new human H1N1 virus are, however, distantly related to swine viruses isolated in Europe in the early 1990s. In particular, the closest isolated relatives of the neuraminidase segment have 94.4% similarity at the nucleotide level with European swine infl uenza A virus strains from 1992 [11] .
The incubation period for novel H1N1 2009 infection appears to range from 2 to 7 days; however, additional information is needed. On the basis of data regarding viral shedding from studies of seasonal infl uenza, most patients with novel H1N1 2009 infection might shed virus from 1 day before the onset of symptoms through 5 to 7 days after the onset of symptoms or until symptoms resolve; in young children and in immunocompromised or severely ill patients, the infectious period might be longer [12] . Patients who are at highest risk for severe complications of novel H1N1 2009 infection are likely to include but may not be limited to groups at highest risk for severe seasonal infl uenza: children under the age of 5 years, adults 65 years of age or older, children and adults of any age with underlying chronic medical conditions and pregnant women [13] .
Two classes of antiviral medication are available for the treatment of seasonal human infl uenza: neuraminidase inhibitors (oseltamivir and zanamivir) and adamantanes (rimantadine and amantadine). During the 2008-2009 infl uenza season, almost all circulating human infl uenza A (H1N1) viruses in the United States were resistant to oseltamivir [14] . However, genetic and phenotypic analyses indicate that novel H1N1 2009 is susceptible to oseltamivir and zanamivir but resistant to the adamantanes [15] . The Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA has recommended that given the severity of illness observed among some patients with novel H1N1 2009 infection, therapy with neuraminidase inhibitors should be prioritized for hospitalized patients with suspected or confi rmed novel H1N1 2009 infection and for patients who are at high risk for complications from seasonal infl uenza.
A number of different laboratory diagnostic tests can be used for detecting the presence of novel H1N1 infl uenza virus in respiratory specimens, including direct antigen detection tests, virus isolation in cell culture, or detection of infl uenza-specifi c RNA by real-time reverse transcriptasepolymerase chain reaction (Real-time RT-PCR).
During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines, antiviral treatment and prophylaxis where available and other public health-based nonpharmaceutical measures. Appropriate treatment of patients with respiratory illness depends on accurate and timely diagnosis and early diagnosis of infl uenza can reduce the inappropriate use of antibiotics and provide the option of using antiviral therapy.
Preferred respiratory samples for infl uenza testing include nasopharyngeal or nasal swab, throat swab and nasal wash or aspirate, depending on which type of test is used. Samples should be collected within the fi rst 4 days of illness. Routine serological testing for infl uenza requires paired acute and convalescent sera, does not provide results to help with clinical decision-making. Serological testing results for human infl uenza on a single serum specimen is not interpretable and is not recommended. All respiratory specimens should be kept at 4°C for no longer than 72 hours before testing and ideally should be tested within 24 hours of collection. If storage longer than 72 hours is necessary, clinical specimens should be stored at -70°C [16].
Antigen detection tests; also known as rapid infl uenza diagnostic tests (RIDTs) detect infl uenza viral antigens in clinical specimens. These rapid infl uenza diagnostic tests can provide results within 30 min or less. Hence the results are available in a clinically relevant time period. Diagnostic tests for detection of novel H1N1 infl uenza virus antigen may be of two main types: direct fl uorescent antibody (DFA) tests and rapid enzyme/optical immunoassays or assay for NA enzymatic activity. Direct fl uorescent antibody (DFA) staining of clinical specimens using specifi c monoclonal antibodies against novel H1N1 infl uenza virus antigen can be a reliable and relatively rapid technique for the pandemic novel H1N1 infl uenza virus detection. Studies of DFA detection of infl uenza viruses have shown highly variable results with sensitivities ranging from 40% to 100%. Recent analytical studies indicate that commercially available RIDTs can detect novel infl uenza A (H1N1) virus [17] . In a study Chan et al. showed that the rapid antigen tests they evaluated in their study have comparable sensitivity for detection of novel H1N1 infl uenza and seasonal infl uenza viruses [18] . Data on analytical sensitivity for detection of different viruses does not directly refl ect clinical sensitivity on patient specimens. However, only limited data have been published on the performance of RIDTs compared with RT-PCR for detecting the presence of novel infl uenza A (H1N1) virus in clinical specimens [19] . Compared to RT-PCR, the sensitivity of RIDTs for detecting novel infl uenza A (H1N1) virus infections ranged from 10% to 70%. The sensitivity of RIDTs to detect novel infl uenza A (H1N1) virus is equal to or lower than the sensitivity to detect seasonal infl uenza viruses [17] . Although these rapid tests do not differentiate between novel H1N1 2009 infl uenza virus and seasonal infl uenza A or even between subtypes H1 and H3 but they may provide useful information that might impact patient care. Understanding the limitations of rapid tests is very important to appropriately interpret results for clinical management of the disease [20] .
Novel H1N1 infl uenza virus detection can also be achieved by inoculating the clinical specimen on MDCK cells for virus isolation with subsequent characterization by hemagglutination inhibition (HI) and neuraminidase inhibition tests using monospecifi c antiserum. Although the cell culture method is sensitive, it requires viable virus, needs expertise and at least 6-8 days to grow the virus to a level where cells are examined for cytopathic effect (CPE). Virus isolation is not only labor-intensive it is timeconsuming also and requires a week for declaring a sample positive or negative hence not appropriate for an epidemic situation.
Although the extreme genetic variability of infl uenza viruses is a challenge for design of molecular-based diagnostic tests. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a widely used molecular tool that has been applied to both infl uenza virus detection and subtype characterization of virus isolates. Most infl uenza A PCR assays in use target conserved regions of the M gene and therefore should detect infl uenza A from all established subtypes, including the newly emergent novel H1N1 infl uenza. However, such methods need to be complemented with a rapid subtyping test to distinguish seasonal infl uenza A from novel H1N1 2009 infl uenza virus. Multiplex PCR testing for the detection of respiratory viruses has seen major advances over the past decade resulting in the development of several commercially available tests. These tests can amplify one or more genes from a number of respiratory viruses and detect amplifi ed products using microgene arrays. One such assay the xTAGTM RVP test was developed in 2005 immediately following SARS and H5N1 infl uenza and was designed to detect and type the three infl uenza A subtypes circulating at that time viz. H1, H3 and H5 [21, 22] .
A limitation of PCR methods is that false-negative results may occur due to sequence variation in primer and probe targets and is particularly relevant for the detection of emerging viruses. However the use of multiple targets can reduce such limitations, and may serve as a means of confi rming positive results. Mahony 
The effi ciency and performance of nucleic acid amplifi cationbased assays depends on the amount and quality of sample template. Nucleic acid amplifi cation assays, including reverse transcriptase RT-PCR (rRT-PCR), and real-time RT-PCR are the most sensitive and specifi c infl uenza virus diagnostic assays. Real-time RT-PCR remains the method of choice for clinical diagnosis of novel H1N1 2009 virus in respiratory specimens and for differentiating it from seasonal infl uenza viruses [25] . Laboratory tests, such as real-time RT-PCR should be prioritized for hospitalized patients to diagnose 2009 H1N1 infl uenza and immunocompromised persons with suspected infl uenza where RIDT or DFA testing is negative or to determine infl uenza A virus subtype in patients who have died from suspected or confi rmed infl uenza A virus infection.
The CDC has developed and recommended a realtime RT-PCR asaay for detection and characterization of novel H1N1 infl uenza. The assay includes a panel of oligonucleotide primers and dual-labeled hydrolysis (Taqman®) probes. The assay can be used to detect and characterize the novel H1N1 virus (swine infl uenza) in respiratory specimens and viral cultures. The assay has InfA primer and probe set designed for universal detection of type A infl uenza viruses and swInfA primer and probe set to specifi cally detect all swine infl uenza A viruses. The assay also includes a set of specifi c primer and probes for HA gene to specifi cally detect swine H1 infl uenza virus in specimens positive with SwInfA primers and probes. The assay can be applied on a wide range of specimens such as broncheoalveolar lavage, tracheal aspirates, sputum, nasopharyngeal or oropharyngeal aspirates or washes, and nasopharyngeal or oropharyngeal swabs taken from suspect swine infl uenza A infected patients. Recently Carr et al. developed an M gene-based real-time reverse transcriptase polymerase chain reaction (rtRT-PCR) assay for the detection of novel H1N1 2009 infl uenza virus that does not cross-react with human seasonal infl uenza A viruses (subtypes H1N1 and H3N2) [26] .
An internal control should be included for each and every clinical sample tested for novel H1N1 2009 virus. The inclusion of internal control ensures proper specimen collection, processing and RNA extraction. The CDC realtime RT-PCR protocol uses Human RNaseP gene (RNP) as internal control for human nucleic acids. No template controls and positive template controls should also be included in each run. A human specimen control provides a secondary negative control that further validates the nucleic extraction procedure and reagent integrity.
The no template control reactions should not exhibit fl uorescence growth curves that cross the threshold line. Although the real-time RT-PCR is highly sensitive and specifi c assay for novel H1N1 virus detection, the limitations include need of trained personnel for assay set up and result interpretation, false negative results which may occur if inadequate numbers of organisms are present in the specimen due to improper collection, transport, handling or excess of DNA/RNA template in the reaction and initial cost of machine. (Table 1) (Fig. 2) .
There is no perfect test for the diagnosis of infl uenza. Virus culture, the present 'gold-standard test' is not 100% sensitive and does not provide results in a time-frame that allows optimal use of potentially effective antiviral treatment. Although rapid diagnostic tests provide results in less than 30 minutes, they are signifi cantly less sensitive and do not differentiate between different subtypes of infl uenza A virus. Rapid testing is only offered after the fi rst culture-confi rmed cases of infl uenza are reported from the community. Molecular assays; reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR targeting conserved regions of infl uenza virus genome have advantages over other methods and provide sensitive, highly specifi c and rapid diagnosis. The realtime RT-PCR should be the method of choice and both in-house developed and CDC-developed real-time PCR assays can be used for the specifi c detection of novel H1N1 2009 infl uenza virus. Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics. Infl uenza is an acute respiratory disease which affects the upper and/or lower respiratory tracts. Infl uenza outbreaks occur every year and globally account for about 3-5 million severe cases with 250,000 to 500,000 deaths annually. It is caused by infl uenza viruses which are of three types: A, B and C and they can all affect humans.
Infl uenza A viruses have 2 main surface glycoproteins -hemagglutinin (HA) and neuraminidase (NA) -which have 16 and 9 subtypes respectively. The infl uenza A virus subtypes are classifi ed on the basis of the different HA and NA glycoprotein subtype combinations. All the subtypes can affect birds which are the natural hosts. Only a few subtypes are capable of infecting humans. The past few years have seen the emergence of two new infl uenza A subtypes which have become a cause of concern for the global community. These are the highly pathogenic H5N1 infl uenza A virus (H5N1) and the pandemic 2009 infl uenza H1N1 virus. The H5N1 virus emerged initially in 1997 and then in 2003. Since 2003 this virus has caused widespread disease and death in poultry. In humans the number of cases infected with this virus is few but the mortality is very high. The second infl uenza virus, the pandemic H1N1 or the swine origin infl uenza A (H1N1) virus emerged this year and within a few months quickly spread throughout the world. The virus acquired the ability for effi cient human-tohuman spread which resulted in a large number of infected individuals. Fortunately the mortality has been low.
Infl uenza A viruses are dynamic and can evolve by two processes, antigenic drift and antigenic shift. This capability of antigenic variation is responsible for the severe outbreaks of disease. Antigenic drift occurs by point mutations in the two genes coding for HA and NA and this causes minor changes in surface proteins. This leads to a new strain which is not recognized by antibodies to previous infl uenza strains. Antigenic shift is a major change through genetic reassortment which produces a novel infl uenza A subtype in humans. This occurs through mixing of human and animal infl uenza A virus genes or by animal to human transmission. A pandemic occurs when a new type of infl uenza A virus is introduced in humans that can cause a serious illness and is capable of sustained human to human transmission.
H5N1 is a highly pathogenic avian infl uenza virus which has caused a widespread epizootic illness among birds. Although the virus is widely present in birds in various parts of the world, human disease from H5N1 has been uncommon as bird to human transmission is ineffi cient and human to human transmission is rare. The virus could cause an infl uenza pandemic if it attains the ability for effi cient and sustained human to human transmission. This is of great concern as there is little natural immunity to H5N1 virus in humans and the disease in humans is severe with a high mortality.
The emergence of H5N1 infl uenza in humans for the fi rst time occurred in 1997 in Hong Kong. Eighteen people admitted with a respiratory illness were found to be H5N1 positive. Six of them (33%) died. This outbreak was epidemiologically linked to H5N1 infection in a live bird market in Hong Kong. At this time 1.4 million poultry was culled in Hong Kong, the market was disinfected and import of poultry from mainland China was halted. This outbreak was successfully controlled with these measures [1].
In 2003, 2 members of a 5 member family from Hong Kong were infected with H5N1 virus after travelling to China. The source of the infection could not be confi rmed. One of these two persons died while the other, a young boy recovered. Subsequently an outbreak of H5N1 infection in poultry and humans occurred in south East Asia. In 2003-2004, fatal and severe respiratory infection, mostly pneumonia and respiratory failure, were seen in China, Thailand and Vietnam. All of these were associated with an outbreak of H5N1 in poultry. A total of 50 cases with 36 deaths were reported with the mode of disease transmission being from sick or dead poultry to humans. Only one case of human-to-human transmission from a sick child to mother was reported in Thailand [1].
In 2005, 98 human cases with 43 mortalities were reported from Cambodia, China, Indonesia, Thailand and Vietnam. Again all these outbreaks were associated with an ongoing H5N1 infection in poultry. The next year the cases and geographical area increased and 115 cases with 79 deaths were reported from China, Cambodia, Azerbaijan, Djibouti, Egypt, Indonesia, Iraq, Thailand and Turkey. Most of these were again from contact with dead or live infected poultry. The only evidence of human to human transmission was in a family cluster of 8 in Indonesia in which 7 individuals died [1].
In 2007, 86 human cases with 59 mortalities were reported from nine countries: Cambodia, China, Egypt, Indonesia, Laos, Myanmar, Nigeria, Pakistan and Vietnam [1]. A total of 442 confi rmed cases of avian fl u (H5N1) have been reported till 24 September 2009 from the above-mentioned 15 countries with 262 deaths [2].The mortality rate is around 60% which is very high. Infection with H5N1 in humans has so far remained confi ned to individuals with close contact with infected birds or surfaces or objects heavily contaminated with their droppings. This virus has not acquired the ability for sustained human to human spread and therefore has not been able to infect a large number of individuals and cause a pandemic.
90% of patients infected with H5N1 are less than 40 years old with a median age of 18 years [3] . The mortality is highest among the 10-19 years age group and lower in people more than 50 years old. The reason for lower infection rate and mortality in older people has not been ascertained.
The route of transmission is usually from birds to humans. There is usually a history of exposure to dead or sick poultry/wild birds, their secretions or excretions during the week prior to illness. Activities involving close contact such as defeathering, preparing poultry for cooking, holding or playing with sick poultry, handling fi ghting cocks and eating raw or undercooked poultry products have been implicated [4] [5] [6] [7] .
Human-to-human transmission has only been documented in 1 case of transmission from sick child to mother and possibly 1 case of a cluster of 8 patients in a family in Indonesia in 2006 [7, 8] .
The incubation period of H5N1 infection in humans has usually ranged from 2-5 days, though clinical features have appeared even up to 8-17 days after exposure . Almost all patients present with high grade fever (>38°C). Cough and dyspnea are seen commonly (about 90%). Sore throat is seen in around half the patients with rhinitis and upper respiratory symptoms being less common. Headache, myalgia and weakness have also been reported. Gastrointestinal symptoms such as diarrhea, vomiting and abdominal pain may also be present. Watery diarrhea may precede respiratory symptoms by a week [9] [10] [11] . One case with fever, diarrhea, seizure and coma has been reported from Vietnam leading to a clinical diagnosis of encephalitis. H5N1 was detected from CSF, serum, throat and fecal samples [12] . Conjunctivitis is also described occasionally [13, 14] .
Most patients develop lower respiratory features early during the illness. Dyspnea usually develops after a median of 5 days of initial symptoms [15] . Respiratory distress, tachypnea and inspiratory crackles are commonly seen. Most patients have clinical and radiological features of pneumonia which is seen to rapidly progress to respiratory failure with manifestations of adult respiratory distress syndrome (ARDS). In a report from Thailand, the median time for progression to ARDS was 6 days (range 4 to 13 days) [15] .
Complications such as multiorgan failure, cardiac dilatation, arrhythmias, ventilator-associated pneumonia, pulmonary hemorrhage, pneumothorax, sepsis syndrome, Reye's syndrome and pancytopenia have been described. A very high mortality of more than 60% has been reported though the risk factors for severe disease are not clear. In 1997, old age, delayed hospitalization and lower respiratory infection were found to be associated with severe disease with children less than 6 years having milder disease. However, recent H5N1 infections have caused high mortality rates in infants and young children. Knowledge related to epidemiology and clinical features remains incomplete and coordination between affected countries is needed to fully understand the profi le of this new viral infection.
Novel strains of infl uenza virus arise due to antigenic shifts and drifts. These strains have very different surface glycoproteins which did not exist in human strains before. A pandemic occurs when such a virus emerges in humans with effi cient human to human transmission. As there is very little or no immunity against it, the virus quickly infects a large number of individuals in all age groups. A pandemic has been expected for long and it was feared that H5N1 avian infl uenza virus which caused severe disease in clusters of humans was the most likely candidate virus to cause a pandemic. For any pandemic to start three conditions need to be met:
A infl uenza virus subtype not seen in humans for at least a generation should emerge; 2.
The new virus should have the ability to infect and replicate effi ciently in humans; and 3.
The new virus should have developed the ability for easy and sustained human-to-human spread. H5N1 virus was unable to cause a pandemic due to its ineffective human to human transmission. In March 2009, a novel strain of H1N1 infl uenza virus evolved from a reassortant between triple re-assortant swine infl uenza viruses in North American pigs and infl uenza A virus circulating in Eurasian pigs [16, 17] . This combination had not been seen previously. By the end of April 2009, WHO declared the emergence of human cases of H1N1 swine infl uenza virus. On 11 June 2009, the WHO raised the pandemic alert from phase 5 to phase 6 and announced that the world was at the beginning of an infl uenza pandemic [18] . 
The Centers for Disease Control, Atlanta, USA compiled and analyzed data collected from the beginning of the outbreak till 24 July 2009 and this gives a fair idea of the demographic characteristics of H1N1 infl uenza. During this period, 43,771 cases were reported in the USA with the majority being in people under 24 years of age [21] . Few cases were reported in people older than 60 years. The age distribution of the number of cases per 100,000 according to this analysis is given in table 1 [22] .
In India, all ages have been affected, with the age group 15-34 years being the worst affected. The incidence has been low in children below 3 and people above 60 years of age [23] . The hospitalization rate was highest in children less than 4 years followed by the 5-24 year age group. Hospitalization was less common in 25-65 years age group and increased thereafter. This is unlike seasonal fl u infection where the elderly and young children are found to be at higher risk for fl u related complications [24] .
Complications were also higher in people with underlying diseases such as asthma, cardiac diseases, renal diseases and in pregnancy. Obesity has also found to predispose to severe disease [25] .
Human-to-human transmission is through inhalation of respiratory droplets which are expelled when an infected person sneezes or coughs or by contact with surfaces that have been contaminated by respiratory secretions and then by touching the mouth or nose. The rate of secondary transmission has been found to be 22-33% [26] . In a study in Kenya the secondary household transmission was found to be 26% [27] . The transmissibility in schools has been found to be around 20% [28] . The incubation period is between 1 and 7 days and an infected person can transmit the infection from a day prior to onset of symptoms to a day after symptoms have subsided.
The clinical picture of H1N1 infl uenza encompasses a wide spectrum ranging from the mild self-limiting upper respiratory illness to lower respiratory infection including ARDS, cardiac involvement, neurological involvement, multiorgan failure, septicemia and death.
H1N1 most commonly causes a mild respiratory illness with fever, cough, sore throat, dyspnea, rhinorrhea, myalgias, chills, headache and fatigue. Diarrhea and vomiting are more commonly seen than with seasonal fl u. In a study of patients from April to June 2009 in the USA, gastrointestinal symptoms were seen in 39% patients [29] . Fever and cough are the most common features seen in 93% and 83% respectively [30].
It is usually a self limiting, mild illness but may occasionally present as a serious illness needing admission.
The hospitalization rate in the USA between 15 April and 24 June 2009 was 2.21% with the highest rate of hospitalization being seen in people below 24 years. Most patients who required admission did so within 1-7 days, with a median of 4 days, from the onset of illness [31] . 73% of these patients had some underlying disease which predisposed them to a severe disease and complications. The most common underlying disorders were asthma (28%), neurological disorders (21%), diabetes (15%), immunosuppression (15%) and cardiovascular disorders (13%), with the others being chronic renal disorder, chronic obstructive pulmonary disorder and pregnancy. Obesity was found in 29% of the hospitalized patients with or without other risk factors. 25% of the hospitalized patients were critically ill [29] . Patients who required ICU admission, vasopressors, inhaled oxygen at FiO2 more than 60% or required mechanical ventilation were said to be critically ill [31] .
Younger people are at higher risk of being critically ill with infants and people between 26 and 64 years being the worst affected, the mean age being between 30 and 40 years of age. Around one-third of these patients were young or middle aged adults and were not pregnant and had no underlying disorder [32] .
Most patients who were critically ill presented with fever, cough, dyspnea, myalgias, malaise, weakness, tachypnoea, tachycardia, hypotension, cyanosis and low oxygen saturation. The most common presentation was with adult respiratory distress syndrome or an acute lung injury picture. 31% of these patients had superadded bacterial pneumonia. About 60-80% of these patients required mechanical ventilation [31] .
Other complications that have been reported include myocarditis, pericarditis, encephalitis, seizures, myositis, multiorgan failure and toxic shock syndrome [33] . The mortality in this group has been found to be around 18% with older age, requirement of mechanical ventilation and co-morbid conditions being major risk factors. A large number of deaths are seen in young to middle aged adults due to the higher incidence in this group [31, 32] .
Till date, the pandemic 2009 H1N1 infl uenza virus has spread rapidly and caused a massive burden of disease around the world. It affects the younger population more frequently and can cause severe illness in a small proportion of people. The mortality rate is low and similar to seasonal infl uenza. How the virus will behave in subsequent months in terms of virulence and morbidity is unclear. A better understanding of the disease will help us prepare for the future.
The last decade or so has seen the emergence of two new infl uenza A viruses which have a different epidemiolgical and behavior pattern. The H5N1 virus has remained mainly an avian virus with human spread being limited and occuring only in persons coming in close contact with infected or dead poultry. The virus is however lethal causing a very high mortality. The pandemic 2009 infl uenza A (H1N1) virus on the other hand has emerged as a new human virus with the ability for effi cient human to human spread. Within a span of less than 6 months this virus has spread to more than 191 countries and emerged as the fi rst pandemic virus of this century. Fortunately, this virus causes a low mortality and hopefully this pandemic will be as mild as the last two infl uenza pandemic that occurred in 1957 and 1968. The fear that this virus may become more virulent and lead to a more severe pandemic as occurred in 1918 still exists. We therefore need to be vigilant and prepared if this happens in subsequent waves. Lessons learned from the 1918–1919 influenza pandemic The 1918 influenza pandemic was one of the most virulent strains of influenza in history. Phylogenic evidence of the novel H1N1 strain of influenza discovered in Mexico last spring (2009) links it to the 1918 influenza strain. With information gained from analyzing viral genetics, public health records and advances in medical science we can confront the 2009 H1N1 influenza on a global scale. The paper analyses the causes and characteristics of a pandemic, and major issues in controlling the spread of the disease. Wide public vaccination and open communication between government and health sciences professionals will be an essential and vital component in managing the 2009 H1N1 pandemic and any future pandemics. The infl uenza pandemic of 1918 is generally ranked second only to the 14th century "Black Death" plaque in terms of relativity and absolute mortality. [1] In fact, the death toll of the First World War failed to infl ict the human casualty rate that the 1918-1919 Infl uenza Pandemic did. Little progress has been made toward understanding the condition responsible for the extreme virulence of the "1918 type," and/or the conditions necessary to prevent the reappearance of this infl uenza. Unlike the typical "fl u" that strikes the very young, chronically ill and the elderly, this fl u would attack and kill healthy young adults. Taubenberger [2] reported that deaths resulting from the infl uenza and pneumonia for the 15-34-year-old cohort were 20 times higher in 1918 than any previous time, and 99% of excess deaths among people under 65 years of age [3] This strain of infl uenza killed so many people that it reduced the life expectancy of the United States with ten years during its course.
The present H1N1 strain discovered in the spring of 2009, almost 90 years from the onset of the 1918 pandemic, is resurrecting this specter from the past. This novel H1N1 infl uenza strain emerged from a quiet village in Mexico. The Mexican government responded on 24 April 2009, closing schools, canceling public gatherings in Mexico City and surrounding states until 6 May 2009 [4] . This drastic step may have slowed down the regional spread of H1N1 in Mexico, but it had already left the country through international trade and travel. As in past quarantines, this quarantine would fail as well [5] .
The early focus on slowing this new strain by the health community and governmental agencies would be depended on non-pharmaceutical interventions focusing on measures to:
Limit international spread of the virus (e.g. travel screening and restrictions); 2.
Reduce the spread within national and local populations; 3.
Reduce an individual person's risk for infection; and 4.
Communicate risk to the public.
Infl uenza pandemics occur as a result of two different mechanisms: novel emergence of an avian descendent virus (as in the 1918 virus) or a modifi cation of a human adapted virus by genetic mixing as in the reassortment of novel hemagglutinin (HA) with or without non-committent neuraminidase, (NA) as in the 1968 H3N2 strain [19] . The pandemics of 1948, 1957 and 1968 were caused by variations of the infl uenza virus resulting from this shuffl ing of the eight gene pairs within the virus. Infl uenza pandemics occur in three waves. The typical fi rst wave or initial outbreak, as in the spring of 1918, was relatively mild, starting from the Midwest and spreading along the rail lines with soldiers from Ft. Funston, Kansas, modern day Ft. Riley [2, 3, 14, 16] . The novel H1N1 discovered in Mexico also had such a humble origin. Both patient zeros reported little problems and made complete recoveries. Unlike the Mexican outbreak, the 1918 spring outbreak was not even noted in the index in the 1918 volumes of the Journals of the American Medical Association. Infl uenza was not a reportable disease until 1925 in the United States: the only evidence of the early occurrence was the registration of deaths reported as uncomplicated cases of pneumonia by physicians to various public health departments [3, 8, 9] .
Those who had suffered from the earlier spring infl uenza generally suffered less discomfort in the second wave which would occur in the early summer of 1918 in Europe, affecting the outcome of the war. The third and most deadly wave of the infl uenza would occur later that year in the late fall. Despite the obvious differences between the strains in each wave, it is suggested that the more virulent form of infl uenza was genetically derived from the spring infl uenza [3, 10, 12] . The antigenic composition of the 1918 virus is related to the H1N1 viral group. Phylogenetic studies indicate that the virus responsible for the 1918 infl uenza and viruses that provided gene segments for the Asian/1957 and Hong Kong/1968 pandemics are still circulating in wild birds, with few or no mutations [10,11,16,] . The extreme virulence of fall 1918 infl uenza strain has been blamed on severe pathology with acute pulmonary edema, as well as hemorrhage with acute bronchiolitis, aveolitis and bronchopneumonia [20] it is believed that the severe infl ammation of the lungs initiated high levels of cytokines resulting in a depletion of neutrophils and alveolar macrophages, causing death. The stronger the host's immune system, the stronger response to the infl uenza infection, and greater release of cytokines to counter the virus [19, 20] . This is what resulted in the much higher than average mortality among the 15-40-year-old cohort. Patterson and Pyle [12] , Crosby [8] and many other researchers believe that a strain of pneumonia bacteria accompanied the virus [3, 12] . Noyes [3, 14, 17] noted that the nation's people were stricken and died from the illness at differing rates, just as the cities were hit at differing rates. There was no correlation between populations, or even geographical demographics. Sex and age both played a major factor in determining the susceptibility to the disease of the individual. Females were stricken in rates greater than males, and young adults were sickened in greater numbers that other age cohorts [3, 13 and 14] .
The current international strain of novel H1N1 virus discovered in Mexico is derived from two unrelated swine viruses, one associated with a fourth generation of the 1918 human infl uenza virus with which acts to recombine the viruses and its progeny [4, 18] . Essentially the virus continues by shuffl ing its eight genes in the avian reservoir to eventually be passed to swine and other mammals before the encounter with humans. Seldom are there transfer of infl uenza between humans and avian. Pigs act as the "transformers or converters" for the various infl uenza viruses and let loose the world new "strains" of the infl uenza virus. The intervening passage continues to be through the domesticated pig, as they have the mechanism to convert the arrangement of the sialic acids to become receptive to human cells [21, 22] . The arrangement of the genes, determine the protein sheath structure and the interaction between the antibody defenses of the host. A mere change of one amino acid can change the impact of the infection, from a mild discomfort to a killer virus [14] . One of the major concerns of the 2009 H1N1 is this very issue; will it change between the waves of outbreaks?
In the 1918 pandemic control was sought after, as each wave of the infl uenza outbreak proved deadlier than the previous. Communication between the medical community, the government, media and the public was non-existent. Because of the war, information concerning the infl uenza was blocked, except for neutral Spain, which won the honor of being the name sake for this deadly virus: "The Spanish Infl uenza". In Britain, the battle between preventive and curative medicine raged. Previously these medical approaches were championed by the Medical Offi cers of Health in their efforts to prevent illness, and a therapeutic practice by private physicians [1] . The curative physicians, primarily general practitioners, were overwhelmed by the cases of infl uenza and relied on traditional methods to cope with the illness ranging from aspirin, quinine, opium, ammonia, alcohol, camphor, eucalyptus and iodine to musk, wet packs, blood serum, creosote, turpentine, cinnamon and turtle soup [1, 2] . At best, these prescriptions offered symptomatic relief, and some actually harmed the patients. In the United States, the 1918 pandemic was met with local quarantines, and large public gatherings were discouraged, and like Great Britain folk remedies were widely used [2, 8, 22] . There was no coordinated information from medical authorities to the communities on how to cope with this outbreak. The best advice that was offered then was common sense: bed rest and careful nursing to avoid complications, which is still issued today.
The issue of vaccination was not available until 1931, when a viral growth in embryonated hens' eggs was discovered, and in the 1940s, the US military developed the fi rst approved inactivated vaccines for infl uenza, which were used in the Second World War with limited success. Vaccination is the best defense against the infl uenza if the right strain is predicted, and if there are no mutations after the administration of the vaccine. In the 1957 and 1968, infl uenza vaccination programs were credited with the reduction of the severity of both pandemics. The 1976 swine fl u scare provided the general public a forum to exam the role of vaccination, when the "predicted" swine fl u pandemic failed to materialize after hundreds of thousands of Americans were vaccinated [15] . As shared by many authorities, the directions of pandemics are diffi cult to predict. Successive pandemics and pandemiclike episodes have been decreasing in severity over time. This is due to advances in medicine, public health and understanding the genetics of the disease, but this may also refl ect the evolutionary course of the virus, to that favors transmissibility with minimal pathogenicity. A virus that kills its host, or causes its host to remain at home, will not be transmitted [10] . The swine fl u scare did not result in a pandemic: was it because it lost its pathogenicity or because of the massive immunization clinics which would result in a "herd immunization"?
Harvey Fineberg from the Institute of Medicine, reviewed the1976 swine fl u scare, and shared these fi ve principles facts for preparing the public for a pandemic: (i) Build a base for decision-making; (ii) Think thoroughly each decision point; (iii) Consider and maintain good ties to the media; (iv) Maintain long-term credibility; and (v) Think twice about medical knowledge [15] . These fi ve lessons have been applied to several situations since 1976, i.e. the foot and mouth disease outbreak in UK, and the recent SARS outbreak and the bird fl u threat of only a few years ago. The application of these fi ve procedures has prevented panic, allowed professionals to do their jobs, and provided the media sound and correct information.
Current health and government offi cials have been implementing these fi ve principles, and the 2009 H1N1 pandemic preparedness has been successful thus far.
Steps have been implemented at the various levels of government in the United States, and most institutions where people gather have procedures in place to cope with a pandemic (see Table 1 ). Infl uenza pandemics will not be prevented, but with lessons learned from previous pandemics, the effect of infl uenza pandemics can be reduced. Governments and health professionals must continue to maintain surveillance against all diseases, share information and work together in developing vaccines. As learned in 1918-1919, infl uenza respects no politics, nor borders. Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R Complex interactions between effector T cells and Foxp3(+) regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4(+)Foxp3(−) T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression. Natural regulatory T cells (Tregs), identified by the expression of Foxp3, play an important role in down-regulating immune responses and an imbalance between numbers of Tregs and conventional CD4 (Tconv) and CD8 T cells contributes to outcomes in cancer and autoimmune and infectious diseases [1] [2] [3] [4] . Interactions between Tregs and non-Tregs result in modifications of the function of both cell types, with the goal of minimizing detrimental side effects of pro-inflammatory immune responses while maximizing the efficacy of processes such as pathogen clearance. Further, the function of both cell types and the balance between the pro-and anti-inflammatory immune responses may be affected by cytokines and other molecules produced by cells such as dendritic cells, in the inflammatory milieu.
While Tregs are immunosuppressive at sites of inflammation, Tregs may express transcription factors and cytokines that parallel those of Tconv cells. Thus, Tregs express T-bet and IRF4 at sites of Th1 and Th2 inflammation, respectively [5] [6] [7] . Additionally, we and others have shown that Foxp3 + Tregs express IFN-c during infections [5, 8] . Unfractionated populations of Tregs harvested from inflammatory sites, which include cells expressing IFN-c, remain immunosuppressive. Suppression can be demonstrated in T cell proliferation assays after stimulation with either anti-CD3 mAb or virus-specific peptides [8] . In contrast, other studies suggest that a pro-inflammatory milieu may result in diminished Treg suppressive function [9] [10] [11] . In a model of experimental autoimmune encephalomyelitis (EAE), CNS-derived epitope MOG -specific Tregs showed reduced ability to inhibit proliferation of Tconv of the same specificity isolated from the inflamed CNS [9] . This reduction in inhibitory function was IL-6 and TNF-dependent.
IL-12 has also been shown to enhance activation and proliferation of Tconvs even if Tregs are present, possibly reflecting an IL-12-mediated reduction in Treg suppressive function [12] . In other studies, IL-12 was shown to induce IFNc production by Tregs in vitro and in vivo [5, 13] ; IFN-c expression by Tregs may indicate that these cells are transiting to Th1 effector cells [13] . Thus, in apparent conflict with the results using suppression assays, these data suggest that IFN-c expression is a marker for reduced immunosuppressive ability. However whether IFN-c expression actually affects Treg suppressive function has not been assessed experimentally. Collectively these results suggest that IL-12 has independent effects on Tconv and CD8 T cells as opposed to Tregs. Consequently, the effect of IL-12 (or other cytokines) on the T cell response in a total lymphocyte culture will reflect the summation of effects on the different cell types.
In order to delineate the roles of these cytokines, particularly IL-12, on Treg function, we treated lymphocytes harvested from spleens and lymph nodes of naïve mice with a panel of cytokines. Treatment with IL-12 induced IFN-c expression by both Tconv and Tregs and reduced Treg frequency and Foxp3 expression. We also demonstrated that IFN-c + Tregs are as immunosuppressive as IFN-c -Tregs. Our results showed that IL-12 functioned, in part, by diminishing IL-2 production by Tconv and CD8 T cells in the mixed cultures and by down-regulating IL-2 receptor expression on Tregs but up-regulating it on non-Treg T cells.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in the Animal Care Facility at the University of Iowa. The protocol was approved by the University of Iowa Animal Care and Use Committee (Protocol Number: 1007161). All efforts were made to minimize animal suffering.
Specific pathogen-free C57BL/6 (B6) and B6/Thy1.1 mice were purchased from the National Cancer Institute, Bethesda, MD. Foxp3 gfp mice on a B6 background were kindly provided by Dr. A. Rudensky (Sloan-Kettering Institute) and were bred to a Thy1.1 background. IL-12Rb2 2/2 mice on a B6 background were obtained from Dr. J. Harty (University of Iowa). 
Lymphocytes prepared from lymph nodes and/or spleens of mice were stimulated with soluble anti-CD3 mAb (0.5 mg/ml) in the presence of various cytokines. Unless indicated, cells were cultured at 1610 6 cells/ml in 24-well tissue culture plates (2 ml/well) and IL-12 was used at 1 ng/ml. To analyze cell proliferation, cells were labeled with CFSE (2 mM, Invitrogen) before culture. To detect intracellular IFN-c or IL-2 production by T cell subpopulations, PMA (50 ng/ml, Sigma), ionomycin (1 mg/ml, Sigma) and Golgi plug (1 ml/ml, BD Biosciences) were added for the last 4 hr of culture.
Treatment with TLR Agonists 5x10 6 B16-Flt3L cells [14] (obtained from Dr. J. Harty) were inoculated subcutaneously into 12-wk old B6 mice. Twelve to fourteen days post inoculation, spleens were harvested, digested with collagenase and DCs were isolated using anti-CD11c microbeads (Miltenyi Biotec, Auburn, CA). T cells were enriched from spleens of B6 or IL-12Rb2 2/2 mice using a Pan T Cell Isolation Kit II (Miltenyi Biotec). 1610 5 T cells were cultured with 1610 4 DCs in the presence of anti-CD3 mAb and medium or IL-12 or LPS (1 mg/ ml) or CpG (1 mM) for 72 hr in a 96-well round-bottom plate. In parallel wells, 2610 5 unfractionated lymphocytes were treated under the same conditions. Samples were analyzed in triplicate.
To evaluate the function of IFN-c producing Tregs, lymphocytes were prepared from Foxp3 gfp /Thy1.1 mice and stimulated with anti-CD3 mAb in the presence of IL-12 or medium as control. After 66 hr, cells were harvested, washed and enriched for CD4 + T cells using a CD4 T Cell Isolation Kit II (Miltenyi Biotech). GFP + Tregs were then sorted. Approximately .90% and ,3% of Tregs expressed IFN-c in the IL-12 and medium groups, respectively. Responder Tconvs were isolated from naïve Foxp3 gfp (Thy1.2) mice. For suppression assays, Tregs were co-cultured with CFSE-labeled (2.5 mM) responder Tconvs at the indicated ratios (Tregs plus responders = 5610 4 cells/well) in 96-well round bottom plates in the absence of IL-12. Wells also contained 2610 5 T-cell depleted splenocytes (irradiated at 2500 rad) and anti-CD3 mAb. After 66 hr, Thy1.1 2 Thy1.2 + Tconv cells were analyzed for CFSE dilution by flow cytometry.
To evaluate the function of Tregs in the presence of IL-12, responder T cells were enriched from naïve B6/Thy1.1 mice using a Pan T cell Isolation Kit II, and Tregs were isolated from B6 mice using a CD4 + CD25 + Regulatory T cell Isolation Kit (Miltenyi Biotec). For suppression assays, Tregs were mixed with CFSElabeled (2.5 mM) responder T cells at the indicated ratios (Tregs plus responders = 5610 4 cells/well). Cells were cultured in the presence of 2610 5 irradiated splenocytes and anti-CD3 mAb with or without IL-12 in 96-well round bottom plates. After 66 hr, Thy1.1 + Tconvs and Thy1.1 + CD8 T cells were analyzed for CFSE dilution by flow cytometry. The Division Index (DI) was obtained using FlowJo software (Tree Star, Inc., Ashland, OR). A normalized DI was calculated as follows: % normalized DI = 100% 6(DI of responders plus Tregs/DI of responders only).
A Foxp3 Staining Buffer Set (eBioscience) was used for Foxp3 or T-bet staining or when cells were analyzed for Foxp3 and cytokine expression simultaneously; otherwise, BD Cytofix/Cytoperm and Perm/Wash buffers (BD Biosciences) were used in intracellular cytokine staining assays. Cell sorting was performed with a FACSDiva or FACSAria and cell analysis with a FACSCalibur or LSRII (BD Biosciences).
Lymphocytes from lymph nodes of B6 mice were stimulated with anti-CD3 mAb in the presence of IL-12 (1 ng/ml) or IFN-c (100 ng/ml) in 24-well plates as described above. Supernatants were collected at 48 hr after culture. IL-2 ELISAs were performed using reagents and protocols provided by the manufacturer (eBioscience, Mouse IL-2 ELISA Ready-SET-Go kit). Samples were plated in duplicate.
Data are presented as means 6 standard errors of the means (SEM). Differences between two groups were determined by Student's two-tailed unpaired t tests or paired t tests (when normalized DI were compared), using GraphPad Prism 5.03 software. Differences with values of P,0.05 were considered significant. *, P,0.05; **, P,0.01; ***, P,0.001.
We and others showed that Tregs express IFN-c at sites of inflammation and can be induced to express IFN-c in vitro [5, 8, 13] .
In specific, IFN-c is expressed by virus-specific Tregs in the CNS of mice infected with the rJ2.2 strain of mouse hepatitis virus (MHV) [8] ; we used information obtained from this experimental system as the basis for the approach described here. To determine whether cytokines had a role in IFN-c expression by Tregs, we initially focused on cytokines that are up-regulated in the CNS of MHV-infected mice and remain elevated as the infection resolves [15] . Among these cytokines, IL-12, IL-6 and IFN-c have roles in T cell polarization and Treg development [16] [17] [18] [19] [20] , so we first evaluated their participation in Treg-mediated IFN-c expression. Lymphocytes were prepared from the lymph nodes of naïve B6 mice, labeled with CFSE and treated with each cytokine and anti-CD3 mAb for 66 hours. Cells were then stimulated for 4 hours with PMA and ionomycin and evaluated for T cell proliferation and IFN-c expression.
As shown in Fig. 1B , IL-12 but not IL-6 or IFN-c induced IFNc production by Tregs and Tconvs although IL-12 induced lower levels of IFN-c in Tregs than in Tconvs. After 42 hours, the majority of Tconv and Tregs expressed IFN-c; by 66 hrs nearly all of the cells were IFN-c + (Fig. 1C) . Further, IL-12 treatment increased proliferation of Tconvs (Fig. 1B, D) and CD8 T cells (data not shown), but, in contrast, diminished frequency and proliferation of Tregs (Fig. 1A, D, E) . IL-12 also decreased levels of Foxp3 in Tregs (Fig. 1F ). The net result was a decreased proportion of Tregs expressing reduced levels of Foxp3 in IL-12treated cell cultures (Fig. 1A, E) . IL-6 reduced both Tconv and Treg proliferation although the effects on Treg proliferation were greater (Fig. 1B, D) .
IL-12 treatment increases expression of T-bet, a transcription factor associated with Th1 development [19, 20] ; we and others previously showed that T-bet was up-regulated in Tregs localized to sites of Th1-type inflammation [5, 7, 8] . Consistent with these results, T-bet was also increased in Foxp3 + Tregs as well as in Tconvs after IL-12 treatment (Fig. 2) .
To examine the role of IL-12 in IFN-c induction in Tregs when inflammation is induced by a more physiological mechanism, we treated samples with TLR agonists, LPS or CpG. T cells (B6 or IL-12Rb2 2/2 ) and splenic DCs (B6) were isolated and incubated together in the presence of anti-CD3 mAb and LPS or CpG. The fraction of Tregs expressing IFN-c increased after LPS or CpG exposure and was similar to levels obtained in cultures treated with IL-12 (Fig. 3 ). LPS and CpG likely functioned via effects on DCs because no IFN-c was induced when unfractionated lymph nodederived lymphocytes were treated with LPS or CpG in the absence of added DCs (data not shown). CpG or LPS-mediated augmentation in the proportion of cells expressing IFN-c was largely dependent on IL-12, since the fraction of IFN-c + Tregs increased only slightly in cultures containing IL-12Rb2 2/2 T cells (Fig. 3) . Of note, IL-12Rb2 2/2 mice are deficient in IL-12 but not IL-23 signaling [21] .
Mixed populations of IFN-c + and IFN-c -Tregs are able to suppress proliferation of both anti-CD3 mAb and MHV peptidestimulated T cells [8] . However, it was not possible from these experiments to determine whether IFN-c expression attenuated suppressive function. To address this question, we treated spleen and lymph node-derived lymphocytes isolated from Foxp3 gfp mice with anti-CD3 mAb in the presence or absence of IL-12. GFP + Tregs were then sorted from the mixed cultures. In the presence of IL-12, .90% of all GFP + cells expressed IFN-c while 90-98% IFN-c -Tregs were detected in cultures treated with anti-CD3 mAb alone. Both sets of Tregs (IFN-c + or IFN-c -) were then mixed with CFSE-labeled Tconvs. Cells were then stimulated with anti-CD3 antibody in the absence of IL-12. As shown in Fig. 4 , IFN-c + and IFN-c -Tregs inhibited the proliferation of CFSE-labeled Tconv to the same extent.
In these assays, we compared equal numbers of IFN-c + and IFN-c -Tregs for suppressive function. However, results in Fig. 1 showed that the numbers of Tregs and their relative expression of Foxp3 were diminished in IL-12-treated cultures. Further, a previous report showed that Treg suppressive function was alleviated by IL-12 [12] . To reconcile these disparate results, we assessed Treg suppression in the presence of IL-12 using Tregs and responder T cells enriched from lymph nodes and spleens. Responder T cells were labeled with CFSE and co-cultured with unlabeled Tregs at several cell ratios. We observed diminished suppression of CFSE-labeled CD4 T cells in the presence of IL-12, in agreement with published results (Fig. 5) . Suppression of CD8 T cell proliferation was almost completely abrogated if IL-12 was present in the culture.
Treg proliferation was diminished in the presence of IL-12, resulting in an effective change in ratio of the two cell types within a single culture (Fig. 1C, D) . However, it is also possible that IL-12 augmented Treg cell death or induced conversion of Tregs to Tconvs, which would have the same effect. To address the role of apoptosis, we measured AnnexinV expression on Tconvs and Tregs in lymph nodes cultures. IL-12 treatment did not increase the percentage of either AnnexinV + Tregs or Tconvs in the cultures, demonstrating that there was no preferential Treg death (Fig. 6) .
Loss of Foxp3 expression by Tregs has been demonstrated in vivo, although whether this represents Treg instability is controversial [22] [23] [24] . IL-12 induces IFN-c expression by Tregs (Fig. 1B) ; if IL-12 also mediated Treg conversion, this would contribute to a decrease in Treg, and an increase in Tconv numbers. To examine this possibility, Foxp3 gfp /Thy1.1 Tregs were purified from naïve spleens and incubated with unfractionated lymphocytes from Foxp3 gfp /Thy1.2 mice in the presence of anti-CD3 mAb and IL-12 or media. As shown in Fig. 7 , IL-12 did not induce an appreciable level of Treg (Foxp3 + ) to Tconv (Foxp3 2 ) conversion when compared to samples exposed to medium alone.
Treg development, homeostasis and suppressive function are dependent upon IL-2 [25] [26] [27] [28] [29] . Further, IL-12 diminishes IL-2 expression by CD4 T cells [30] . Initially, we showed that IL-2 expression by Tconvs was modestly reduced after 48 hr with more substantial effects noted after 72 hr of IL-12 treatment (Fig. 8A ). More strikingly, IL-2 production by CD8 T cells was greatly reduced after both 48 and 72 hr (Fig. 8A ). In agreement with this diminished expression, levels of IL-2, measured by ELISA, were reduced in IL-12-treated cultures at 48 hr (Fig. 8B) . T cell proliferation is augmented by IL-2, especially in tissues at sites of inflammation [31] [32] [33] , yet the results in Fig. 1 show that Tconv proliferation is increased by IL-12 treatment. One possible explanation for these results is that IL-12 preferentially induces Effects of IL-12 on IL-2 and IL-2R Expression PLOS ONE | www.plosone.org IL-2R expression on Tconvs; IL-12 was previously shown to enhance IL-2R expression on CD8 T cells [34] . As shown in Fig. 9 (A-B, D-E), this is indeed the case. Treatment with IL-12 in the presence of anti-CD3 mAb induced up-regulation of both the a (CD25) and b (CD122) chains of the IL-2R on Tconv and CD8 T cells in a dose-dependent manner. In contrast, IL-12 treatment diminished CD25 expression on Tregs while not affecting CD122 expression, reducing the ability of Tregs to compete for IL-2 in the culture. Of note, even though IL-12 induced increased IFN-c expression by Tconv and CD8 T cells, IFN-c, by itself, was unable to induce diminished expression of IL-2 (Fig. 8B) or IL-2R upregulation on non-Treg T cells (Fig. 9C) .
As shown in Fig. 1 , one of the effects of IL-12 treatment was decreased Foxp3 expression by Tregs. Previous studies showed that IL-2 is required for maximal Foxp3 expression [35] but whether IL-12-mediated reduction in Foxp3 expression occurred through effects on IL-2 signaling was not examined. To address the role for IL-2 signaling in IL-12-mediated effects, we treated cells with exogenous IL-2. IL-2 treatment partially reversed the effect of IL-12, resulting in higher levels of Foxp3 in Tregs (Fig. 9F ). [30] . To determine whether IL-27 has similar effects as IL-12, we treated lymphocyte cultures with anti-CD3 mAb and IL-27 or medium. Similarly to IL-12, IL-27 diminished IL-2 production by Tconvs and CD8 T cells (Fig. 10A ) and down-regulated Foxp3 expression by Tregs (Fig. 10B) . However, unlike IL-12, IL-27 decreased proliferation of Tconvs and Tregs and had no effect on CD8 T cell expansion (Fig. 10C , D) resulting in a relatively slight decrease in Treg frequency (Fig. 10E) . The effects of IL-12 were mediated both by diminished IL-2 expression and changes in the relative amounts of IL-2R on Tregs and Tconvs and CD8 T cells. IL-27 reduced CD25 and CD122 expression on Tregs but only marginally affected expression on non-Tregs (Fig. 10F) . Collectively, these results suggest that Tregs are better able to compete for IL-2 in IL-27 compared to IL-12-treated cultures.
A previous report, using co-cultures of purified Tconvs and Tregs, showed that IL-12 could relieve Treg-mediated suppression of Tconv proliferation and activation [12] . Here, we extend these observations to analyze the effects of IL-12 on T cells in unfractionated lymphoid cultures and identify a basis for at least some of the effects of IL-12. We show that IL-12 decreases IL-2 production by Tconv and CD8 T cells and differentially modulates IL-2R expression on Tregs and non-Tregs. IL-2R is up-regulated on Tconv and CD8 T cells and down-regulated on Tregs. When coupled with decreased IL-2 production, this results in a milieu that favors non-Treg proliferation. The effect of IL-12 on CD25 expression by CD8 T cells is especially notable. In the absence of IL-12, levels of CD25 are much greater on Tregs than CD8 T cells, but treatment with IL-12 at 1 ng/ml results in levels of CD25 on CD8 T cells that exceed those on Tregs in the same culture (Fig. 8B, C) . Levels on CD8 T cells are substantially higher than those on Tconv cells, explaining why IL-12 more readily relieves Treg-mediated suppression of CD8 T cell proliferation. These results are consistent with previous reports showing that IL-2R is down-regulated on Tregs in inflamed tissues in the setting of infectious or autoimmune diseases and that IL-2 levels are reduced at these sites, resulting in the outgrowth of non-Treg T cells [5, 36, 37] .
One probable consequence of these changes in IL-2R expression, in conjunction with IL-12-mediated decreases in IL-2 production, is that Tregs are less able to compete for the smaller amount of IL-2 present in the culture. These changes result in diminished Treg proliferation and perhaps decreased Treg Effects of IL-12 on IL-2 and IL-2R Expression PLOS ONE | www.plosone.org suppressive function on a per cell basis (as measured by decreased Foxp3 expression (Fig. 1E) ), resulting in decreased total Treg suppressive capacity in the culture.
The importance of IL-2 for Treg function is well established [29, 35, 36] and has been confirmed in clinical studies. In patients with either hepatitis C virus-induced vasculitis or graft versus host disease, treatment with low doses of IL-2 therapy increased Treg numbers and decreased clinical disease [38, 39] . These results are also consistent with others showing that Treg numbers were increased when cancer patients were treated with low dose IL-2 [40, 41] . Exogenously administered IL-2 also corrects IL-12mediated decreases in Foxp3 levels in Tregs (Fig. 8B) ; whether low level IL-2 treatment also increases Foxp3 levels in Tregs in patients, in addition to increasing Treg numbers, has not yet been addressed.
Further support for an important role for IL-2R levels on Tconv and CD8 T cells comes from studies of IL-27. IL-27 is a cytokine with pro-and anti-inflammatory properties [42] [43] [44] [45] . Mice transgenic for the expression of IL-27 exhibit diminished IL-2 production, which results in decreased numbers of Tregs and subsequently, a systemic inflammatory disease [46] . We showed that IL-27 diminished IL-2 expression, consistent with these results, but IL-27 only modestly decreased Treg frequency (Fig. 9D ). Consistent with a role for IL-2R expression on non-Tregs, there were minimal changes in IL-2R levels on these cells after IL-27 treatment (Fig. 9E) . Thus, under these conditions, Tregs were presumably able to compete for IL-2 even in the presence of reduced amounts of IL-2.
Another consequence of IL-12 treatment is the induction of IFN-c expression by Tregs. Tregs express IFN-c at sites of inflammation under conditions of Th1-type lethal and non-lethal infections [5, 8] . It has been difficult to determine whether IFN-c affects Treg suppressive function, since Tregs in these settings include both IFN-c + and IFN-ccells. Here we showed that IFN-c expression did not diminish Treg suppressive function. These results are in agreement with others showing that in vitro-generated IFN-c + and IFN-c -Tregs were equally suppressive in a mouse model of colitis [13] . In other experimental settings, loss of Foxp3 and gain of inflammatory cytokine expression suggested that Tregs converted into effector T cells [22, 47] . If Tregs at sites of inflammation can generally express IFN-c and still remain suppressive, these results raise the possibility that IFN-c + Tregs, instead of entering the effector T cell pool, could lose IFN-c and revert to a classic Treg phenotype. Whether these cells would preferentially re-express IFN-c on repeat exposure to antigen remains to be determined.
Several studies including this one demonstrate an important role for IL-12 in IFN-c expression by Tconv and CD8 T cells and Tregs in vitro [5, 13] . Further, IL-12 is critical for IFN-c production by Tregs in mice with autoimmune colitis [13] . However, the requirement for IL-12 in IFN-c expression by Tconv and CD8 T cells or Tregs in virus-infected animals is less clear. To investigate whether IL-12 signaling is critical for IFN-c production by Tregs in virus-infected mice, we infected IL-12Rb2 2/2 mice with the mildly neurovirulent rJ2.2 strain of MHV [48] . We detected slightly lower frequencies of virus-specific IFN-c expressing Tregs and Tconv in IL-12Rb2 2/2 mice, consistent with a previous report showing that IL-12 modestly decreased the frequencies of virus-specific T cells in MHV-infected IL-12p35 2/2 mice [49] . The absence of IL-12 signalling may not have had a substantial effect because we detected no differences in CD25 expression on Tregs and virus-specific non-Treg T cells when cells harvested from the brains of IL-12Rb2 2/2 mice and B6 mice were compared (data not shown). Our results are also consistent with others showing IFN-c production was relatively normal in IL-12 2/2 or IL-12R 2/2 mice infected with viruses such as lymphocytic choriomeningitis virus, influenza A virus, adenovirus or hepatotropic strains of MHV. In contrast, IL-12 was essential for IFN-c expression in mice infected with some intracellular nonviral pathogens, such as Leishmania species and Toxoplasma gondii [50] .
In summary, our results show that Treg-mediated suppression of T cell proliferation, especially that of CD8 T cells is nearly ablated in the presence of IL-12. IL-12 induces IFN-c expression by Tregs, but this does not affect Treg immunosuppressive function. Rather, the ability of IL-12 to reverse Treg immunosuppression results, at least in part, from effects on IL-2 and IL-2R expression and by extension, differential IL-2 signaling in mixed cultures of lymphocytes. Genetic Variation in the TNF Gene Is Associated with Susceptibility to Severe Sepsis, but Not with Mortality BACKGROUND: Tumor necrosis factor (TNF) and TNF receptor superfamily (TNFR)-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA) single nucleotide polymorphisms (SNPs) with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS: Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432), sepsis (n = 384) and healthy controls (n = 624). Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (P(adj) = 0.00046, odds ratio (OR)(adj) = 1.92) and sepsis patients (P(adj) = 0.002, OR(adj) = 1.56). Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs) of healthy volunteers exposed to lipopolysaccharides (LPS) ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007). Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (P(adj) = 0.02). However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis. CONCLUSIONS/SIGNIFICANCE: Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with susceptibility to severe sepsis, but not with lethality in Chinese Han population. Sepsis is an infection-initiated and inflammation-induced syndrome. Despite progress in the development of antibiotics and other supportive care therapies, severe sepsis remains an unconquered challenge for the clinicians with an unacceptable high mortality rate of 30%-50% [1] . The response to infection is diverse among different individuals. Given the same therapies, most sepsis patients will recover and do well, while a small, but significant portion, will develop severe sepsis and multiple organ system failure, refractory hypotension and death [2, 3] . Currently, more and more evidence showed that genetic factors played an important role in the development and severity of sepsis [4, 5, 6, 7, 8, 9, 10, 11] . Common sequence variants within genes involved in pro-inflammatory response have received particular attention [12, 13] .
Although the pathogenesis of sepsis remains incompletely understood, an excessive pro-inflammatory response has been established as a fundamental component of severe sepsis [14] . The proinflammatory cytokine TNF-a is an essential component in the host immune response to infection and has been widely reported to be an important mediator in severe sepsis and septic shock. High circulating levels of TNF-a were correlated with poor outcomes in sepsis patients [15] . TNF-a and lymphotoxin-a (LT-a) share the same receptors as well as many biological activities, and they are central mediators of immune responses [16] . TNF-a and LT-a are encoded by adjacent gene loci in the central or class III region of the human major histocompatibility complex (MHC), between the HLA class I and II genes on the short arm of chromosome 6 [17] . Several SNPs within the promoter region of TNF (2238, 2308, 2857, 2863, 21031) and the first intron of LTA (+252) were thought to influence TNF-a and LT-a production, and have therefore been identified as candidate variants that might influence susceptibility to and/or outcomes from severe sepsis and infectious diseases [18, 19, 20, 21, 22, 23, 24, 25] . In particular, rs1800629 (TNF 2308) and rs909253 (LTA +252) have been the focus of many investigations on sepsis. Although several studies have identified associations for rs1800629 and rs909253 with sepsis risk or outcomes [18, 19, 20, 23, 24] , other studies have not replicated the associations [21, 26, 27, 28] . This inconsistency may be due to small samples size studied and ethnic differences [29] .
TNF-a and LT-a exert their pleiotropic functions by activating intracellular signaling cascades via binding to two types of receptors, TNFR-1 (encoded by the TNFRSF1A gene) and TNFR-2 (encoded by the TNFRSF1B gene) [30] . TNFR1deficient mice are resistant to endotoxic shock and have prolonged survival with less hypothermia [31] . TNFR2 influences the biological activity of TNF-a and LT-a both in a membranebound and a soluble form. Membrane-bound TNFR2 facilitates activation of nuclear factor (NF)-kB and mitogen-activated protein kinase signaling cascades upon binding with TNF-a and LT-a, whereas soluble TNFR2 is capable of binding and inactivating circulating TNF-a and LT-a [16, 30] . Moreover, animal studies showed that TNFR2 mediated protective effects in the development of severe sepsis [31] . Recent studies proposed that genetic variation in TNFRSF1A and TNFRSF1B was associated with susceptibility to inflammatory and autoimmune diseases, such as tuberculosis, systemic lupus erythematosus, rheumatoid arthritis and Crohn's disease [32, 33, 34, 35] . However, to date, only one study investigated the role of TNFRSF1A and TNFRSF1B polymorphisms in sepsis susceptibility and mortality [36] .
Considering the important role of TNF-a, LT-a, TNFR1 and TNFR2 in the pathogenesis of severe sepsis, we hypothesized that genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B might be associated with susceptibility to and outcomes from severe sepsis in Chinese Han population. To test this hypothesis, we conducted a relatively large-scale case-control study enrolling 432 severe sepsis patients, 384 sepsis patients and 624 healthy individuals to investigate the association of genetic variants in TNF, LTA, TNFRSF1A and TNFRSF1B with severe sepsis susceptibility and prognosis in Chinese Han population. Furthermore, we investigated the association between the genotypes of the TNF gene SNP rs1800629 and TNF-a concentration in culture supernatants of LPS simulated PBMCs obtained from healthy donors and in serum from severe sepsis patients.
A total of 432 severe sepsis patients, 384 sepsis patients and 624 health volunteers were enrolled in this case-control study. According to the mortality within 30 days, severe sepsis patients were divided into survivor and non-survivor groups. The baseline characteristics and clinical data of all subjects are shown in Table 1 . The average age and proportion of male among the severe sepsis, sepsis and healthy control groups did not show significant difference. The primary source of infection in severe sepsis patients was the lungs (69.9%), followed by abdomen (21.8%), blood stream (3.5%), urinary tract (2.5%) and others (2.3%). The overall 30-day mortality rate of severe sepsis patients was 36.1%. The mean APACHE II and SOFA score in nonsurvivor group was higher than that in survivor group (P,0.05).
Association Analyses of TNF, LTA, TNFRSF1A and TNFRSF1B SNPs with Susceptibility to Severe Sepsis
The genotyping success rates of all tested SNPs ranged from 95% to 99% and none of the ten SNPs diverged significantly from Hardy-Weinberg equilibrium (P.0.05) ( Table 2 ). The allele and genotype distributions of all tested SNPs in severe sepsis patients, sepsis patients and healthy controls are listed in Table 3 . Our results showed that rs1800629 (2308G/A), located in the promoter region of TNF, was associated with significantly increased risk for severe sepsis. The frequency of rs1800629A in severe sepsis patients was significantly higher than that in both the healthy control subjects (P = 0.00028, OR = 2.08) and the sepsis patients (P = 0.00035, OR = 2.39), and the difference remained significant after Bonferroni correction. Moreover, in multivariate analyses after adjustment for covariates, rs1800629A was still significantly associated with the development of severe sepsis when compared with healthy control group (P adj = 0.00046, ORadj = 1.92) and sepsis group (P adj = 0.002, OR adj = 1.56). The genotype distribution of rs1800629 in the severe sepsis group was also significantly different from that in the healthy control group (P adj = 0.003) and the sepsis group (P adj = 0.004), and the significance remained after Bonferroni correction. However, the difference of the allele and genotype frequencies of rs1800629 between subjects with sepsis and healthy controls were not statistically significant (P.0.05). When we analyzed the allele and genotype distributions of the other nine SNPs (rs361525, rs1799724, rs1799964, rs767455, rs4149570, rs1061622, rs3397, rs1800630 and rs909253), no significant difference was found between the severe sepsis, sepsis and healthy control groups (Table 3) .
Association Analyses of TNF, LTA, TNFRSF1A and TNFRSF1B SNPs with Severe Sepsis Outcomes We next investigated the association between all tested SNPs and 30-day mortality. The overall 30-day mortality rate among severe sepsis patients was 36.1%. We compared the allele and genotype distributions of all tested SNPs between survivors and non survivors of severe sepsis patients. No association was observed between TNF, LTA, TNFRSF1A and TNFRSF1B variants and 30-day mortality in the severe sepsis cohort in either the unadjusted or adjusted models (Table 4 ).
To determine whether rs1800629 genotypes influenced TNF-a production, we investigated TNF-a levels in culture supernatants of PBMCs obtained from 24 healthy volunteers. We observed a significant association between TNF-a levels and rs1800629 genotypes under the LPS-stimulated condition. AA+AG genotypes were associated with higher levels of TNF-a compared with GG genotype after LPS stimulation (P = 0.007) ( Figure 1 ). However, no significant association was observed under the unstimulated condition.
Furthermore, we measured TNF-a serum concentrations in 120 severe sepsis patients, including 104 patients with rs1800629GG genotype, 14 patients with GA genotype and 2 patients with AA genotype. Our results showed that rs1800629A allele was associated with higher TNF-a serum concentrations on the first day of severe sepsis. As shown in Figure 2 , the serum concentration of TNF-a in severe sepsis patients with AA+AG genotypes was significantly higher than that of patients with GG genotype (550.4673.6 pg/mL vs. 488.0668.5 pg/mL, P = 0.001). To control confounding variables, we used the possible confound- ing factors (age, gender and APACHE II scores) as covariates in a linear regression model and found that the rs1800629 genotypes remained associated with TNF serum concentration (P adj = 0.02).
Several genetic variants within genes involved in pro-inflammatory response have been associated with morbidity and mortality in patients with severe sepsis or septic shock, which is a complex and multifactorial syndrome [2] . TNF-a is an important pro-inflammatory cytokine involved in sepsis; several functional SNPs in TNF and LTA have been extensively studied in sepsis [29, 37] . However, results from previous studies were inconsistent. Discrepancies among previous studies may have resulted from differences in the populations studied, sepsis phenotype or imprecise definition of phenotype and limited sample size [38] . Considering these factors that might affect the results, we designed the present study with large samples size to achieve greater statistical power. Furthermore, all the samples were recruited from central Chinese Han population, thus the ethnic heterogeneity could be eliminated.
To our knowledge, this was the first relatively large-scale study investigating associations of genetic variants within TNF, LTA, TNFRSF1A and TNFRSF1B with severe sepsis in Chinese Han population. Our results provided evidence that rs1800629, a functional SNP in the promoter region of TNF, was significantly associated with susceptibility to severe sepsis in Chinese Han population. The association between rs1800629 and severe sepsis risk may be explained by its influence on the expression of TNF-a. In this study, we found that the risk allele (rs1800629A) is associated with increased TNF-a production in PBMCs from healthy subjects after stimulation with LPS. Moreover, we found that TNF-a serum levels in severe sepsis patients with AA+AG genotypes for rs1800629 were significantly higher than those in the individuals with GG genotype. Previous studies also showed that rs1800629A allele was associated with a six fold higher expression of both basal and induced TNF mRNA [39, 40] . Menges et al. found that the plasma TNF-a concentrations in patients with sepsis secondary to severe traumatic injury were significantly elevated in rs1800629A carriers on the first day after admission and for the following 14 days [41] . As TNF-a plays a pivotal role in the pathogenesis of severe sepsis in response to infection, it is reasonable to assume that patients with rs1800629A allele might produce a higher amount of TNF-a, and therefore become more susceptible to severe sepsis. In contrast to the TNF 2308G/A polymorphism, the LTA +252A/G was not associated with the development of severe sepsis in our study. Our data showed that 2308G/A of TNF and +252A/G of LTA were in weak linkage disequilibrium (LD) (D9 = 0.118) in Chinese Han population. The LD pattern is quite dissimilar to Caucasian population, which might result from the racial difference [24] .
Rs1800629 was not associated with mortality among subjects with severe sepsis in our study. This was consistent with the study by Stuber et al., which demonstrated that the rs1800629 genotypes were not associated with poorer prognosis in severe sepsis. However, they did not find an association between rs1800629 genotypes and plasma TNF-a levels [26] . Recently, Teuffel et al. conducted a systematic review and meta-analysis, which also concluded that rs1800629 (TNF 2308 AA/AG, TNF2) was associated with susceptibility to sepsis, but not with sepsis mortality [29] .
Several studies have proposed that genetic variation in TNFRSF1A and TNFRSF1B was associated with susceptibility to inflammatory and autoimmune diseases, such as tuberculosis, systemic lupus erythematosus, rheumatoid arthritis and Crohn's disease [32, 33, 34, 35] . However, up to now, only one case control study investigated associations between TNFRSF1A and TNFRSF1B polymorphisms and sepsis susceptibility [36] . Four potentially functional SNPs in TNFRSF1A and TNFRSF1B were genotyped in our study. However, none showed association with susceptibility to or death from severe sepsis in Chinese Han population. Our findings were consistent with the results of Gordon et al. that five functional SNPs in TNFRSF1A and TNFRSF1B were not associated with susceptibility to or outcomes from sepsis in Caucasian population [36] .
Potential limitations of this study should be addressed. First, although we knew that different pathogens had different impact on severity and outcomes of sepsis, we did not perform stratification analysis by different pathogens due to small number of cases with a definite microbiologic diagnosis. Second, we did not resequence these genes or select tag SNPs for genotyping. Instead, only ten potentially functional SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were included in our study, which was far from comprehensive. Indeed, these four genes are highly polymorphic. Therefore, it was possible that some important SNPs might be missed or the observed association might be due to other polymorphisms in LD with the studied ones. Additionally, assuming the prevalence of 0.01 for severe sepsis and using a significance level of 0.05, our study with 432 severe sepsis patients and 624 healthy controls had about 80% power to detect a 5% risk allele with an odds ratio of 1.63. Variant with an effect size smaller than this cannot convincingly be excluded based on these results. Therefore, our results cannot exclude variant associations with weaker effects between severe sepsis and the other three candidate genes (LTA, TNFRSF1A, TNFRSF1B). A more highly powered study involving thousands of subjects may yet exclude the role of these variants in severe sepsis susceptibility and outcomes.
In conclusion, our relatively large scale association study demonstrated that individuals with a functional variant in the promoter region of TNF may confer susceptibility to severe sepsis. However, common functional genetic variants in TNF, LTA, TNFRSF1A and TNFRSF1B were not associated with severe sepsis mortality in Chinese Han population.
This study was approved by the Ethics Study Board of Zhongshan Hospital, Fudan University, Shanghai, China (Record no: 2006-23). Written informed consent was obtained from patients or the next of kin, carers or guardians on the behalf of the participants before enrollment.
From May 2005 to March 2011, a total of 432 severe sepsis patients, 384 sepsis patients and 624 ethnic-matched healthy controls were enrolled in this study ( Table 1) . The severe sepsis patients were those admitted to the Emergency, Surgical and Respiratory ICU at Zhongshan Hospital. The sepsis patients were those admitted to Zhongshan Hospital, but did not develop severe sepsis during hospital stay. The sepsis patients were considered as at risk controls for severe sepsis. Of 384 sepsis patients, 174 patients overlapped with that from our previous study [42] . Another 210 sepsis patients were collected between May 2008 and March 2011, and these patients were not included in our previous study. Sepsis patients recruited in the current study included multitrauma subjects and patients with a history of chronic heart, renal, liver or pulmonary failure, thus they spent a long time (more than 8 days on average) on ICU (Table 1) . Sex-and age-matched controls were selected from healthy blood donors. To reduce the potential confounding from ethnic backgrounds, only central Han Chinese individuals were recruited in this study.
The diagnosis of sepsis was based on the criteria presented at the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference in 1992 [43] . Severe sepsis was defined as sepsis in combination with infection-induced acute organ dysfunction in at least one organ. Acute organ dysfunction was defined as Sequential Organ Failure Assessment (SOFA) scores more than 2 for the organ in question. Baseline characteristics (age, gender and previous health status), as well as clinical data including Acute Physiology and Chronic Health Evaluation II (APACHE II) and SOFA scores, source of infection, microbiology and ICU mortality were obtained after the patient met sepsis criteria. The APACHE II and SOFA scores were calculated in the first 24 hours after the diagnosis of sepsis and severe sepsis. All patients included in the protocol were followed up for 30 days or hospital discharge. When cultures were absent or negative, the source of infection was determined by two senior physicians. Exclusion criteria included age below 18 years, pregnancy, severe chronic respiratory disease, severe chronic liver disease (defined as a Child-Pugh score of .10), malignancy, using of high-dose immunosuppressive therapy and AIDS diagnosis. Questionnaires were obtained from all control subjects to document smoking status, and history of chronic illness or severe sepsis. Healthy controls were defined as individuals without any recent acute illness, chronic illness or history of sepsis and severe sepsis.
Previous studies found that several functional SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were associated with inflammatory and autoimmune diseases. In our study, SNPs in TNF, LTA, and TNFRSF1A and TNFRSF1B were selected based on the following criteria: (1) location within the gene region (promoter, intron, exon, 39UTR and 59UTR); (2) association with inflammatory and autoimmune diseases such as sepsis, asthma, tuberculosis, systemic lupus erythematosus, rheumatoid arthritis and Crohn's disease in more than two studies. A total of ten SNPs were selected and genotyped in our study. Location and characterization of all selected SNPs were listed in Table 2 .
Genomic DNA was extracted from whole blood with a FlexiGene DNA Kit (Qiagen, Hilden, Germany) in accordance with the protocol of the manufacturer. Six SNPs (rs1800629, rs1799724, rs361525, rs1800630, rs1799964 and rs909253) in TNF and LTA were selected and genotyped by direct sequencing. The sequencing reactions were performed using Applied Biosystems BigDye (version 3.1) chemistry (Applied Biosystem, Foster City, CA, USA), and the sequences were resolved using an ABI 3730 Genetic Analyzer. Analyses of the sequence traces were performed using the Staden package and were double scored by a second operator. The primers and PCR protocols used were shown in Table S1 . Four SNPs in TNFRSF1A (rs767455, rs4149570) and TNFRSF1B (rs1061622, rs3397) were selected and genotyped on the GenomeLab SNPstream high-throughput 12-plex genotyping platform (Beckman Coulter, Fullerton, CA) following the manufacturer's instructions. The primers for PCR and single base extension were performed with Beckman Coulter Autoprimer software and shown in Table S2 .
To determine the associations between rs1800629 genotypes and TNF-a levels in PBMCs, we investigated 15 subjects with rs1800629GG genotype, 8 subjects with GA genotype and 1 subject with AA genotype. PBMCs were derived by using Ficoll gradient density centrifugation method. Isolated PBMCs were plated at a density of 1610 6 cells/ml in 24-well plates and cultured in RPMI 1640 medium with 10% FBS at 37 uC with 5% CO 2 . The cells were then incubated for 6 hours in presence or absence of 100 ng/ml Escherichia coli 0111:B4 LPS (Sigma, USA). After incubation, supernatants were harvested and stored at 280uC until use.
Blood samples (5 mL) were collected within 24 hours of meeting criteria for severe sepsis. Samples were centrifuged at 4uC for 10 min at 3200 rpm within 60 min after collection. Then the serum was stored at 280uC until use. TNF-a level was determined by human ELISA kit (R&D Systems, USA) according to the manufacturer's protocol.
The genotype data of cases and controls was analyzed for deviations from Hardy-Weinberg equilibrium by the Haploview v4.1 software [44] . The differences in allele and genotype distributions between severe sepsis and control groups were compared using x 2 -test or Fisher's exact test when appropriate. The test for association with genotypes used the global genotype test in the 362 contingency table. Allele frequencies of cases and controls were used to calculate the OR and the 95% CI. Multivariate logistic regression was used to adjust for potential confounding factors. When comparing severe sepsis group to sepsis group, we entered the genotypes or alleles in the multivariate models controlling for the confounding variables including age, gender, history of diseases, source of infection, APACHE II and SOFA scores. When comparing severe sepsis patients to healthy controls, age and gender were included in the multivariate models. The Bonferroni method was used to correct for multiple comparisons where applicable. The power analysis was performed using the Genetic Power Calculator web tool [45] . A two tailed Pvalue of ,0.05 was considered statistically significant, whereas a value of corrected P,(0.05/number of tests) was considered significant after Bonferroni correction. Continuous variables were described as either a mean 6 standard deviation, or as a median with interquartile range. TNF-a serum levels between individuals with different rs1800629 genotypes (AA+GA vs. GG) were compared by Student's t-test. To determine whether an association with rs1800629 genotypes might depend on other potential confounding factors for TNF-a serum levels, we investigated the association of rs1800629 genotypes by adding the polymorphisms to a linear regression model controlling for age, gender and APACHE II scores. The software used for statistical calculations was SPSS 15.0 (SPSS Inc., Chicago, IL, USA) unless specified.
Table S1 Primers and PCR protocols for six SNPs in TNF and LTA.
(DOC)  Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2 BACKGROUND: Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. CONCLUSION/SIGNIFICANCE: In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2. Anterior gradient homolog 2 (AGR2) was identified initially as a secretory factor expressed in the anterior region of the dorsal ectoderm in Xenopuslaevis embryos, where it was postulated to mediate the specification of dorsoanterior ectodermal fate, particularly in the formation of the cement gland [1] . Clinical studies have further shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung [2] [3] [4] [5] [6] . More biological studies in these cancer cell lines have indicated a significant role for AGR2 in tumor-associated pathways, including tumor growth, cellular transformation, cell migration, limb regeneration, and metastasis [5, [7] [8] [9] . However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated [10] . Therefore, the development of molecular ligands specifically recognizing AGR2 is of great significance to early diagnosis and prognosis of cancer and to fundamental research for the elucidation of the biochemical functions of AGR2.
Various ligands have been developed for specific molecular recognition, such as small molecules, antibodies, and peptides [11] [12] [13] . More recently, another type of molecular ligand, named aptamer, has drawn significant attention. Aptamers, singlestranded modified or unmodified oligonucleotides (RNA or DNA), are generated through in vitro selection process or SELEX (Systematic Evolution of Ligands by EXponential enrichment) with high binding affinity and specificity towards defined targets [14, 15] . The selected aptamers can recognize a wide variety of targets, including small molecules, proteins, cells and tissues relying on their diverse tertiary structures. Compared to antibodies, aptamers have low molecular weight, fast tissue penetration rate, high stability and low immunogenesis [16] . They can be chemically synthesized with low cost and modified easily with various reporters [17] . Furthermore, they can be ligated and/or amplified by enzymes in vitro [18] . These advantages make aptamers promising ligands for medical and pharmaceutical research, such as drug development, disease diagnosis, and targeted therapy [19] .
The possibilities provided by aptamers are enormous, and some aptamers have already shown many important applications in bioanalysisand biomedicine [20] [21] [22] [23] . Particularly, several aptamers have been generated against cancer-related proteins, such as PDGF, VEGF, HER3, NFkB, tenascin-C, or PMSA [24] [25] [26] . Many aptameric sensors, probes and assays have been developed to allow sensitive and selective detection of these cancer biomarker proteins [27] . For instance, Yang et al has reported a lightswitching excimer aptamer probes for sensitive quantitative detection of PDGF in cell media [28] . Kwon et al have developed a functionalized polypyrrole nanotube with aptamer to build a VEGF biosensor [29] . Aptamers have also been applied for molecular imaging to in vivo characterize the complex pathogenic activities that accompany tumor growth for disease early diagnosis and pathogenesis measurement [30] [31] [32] [33] . Since the targets for aptamers could be intracellular, extracellular or cell-surface biomolecules, various therapeutic methods have been developed using the aptamers as targeting reagents [34] [35] [36] [37] , which greatly broaden the range of targeted therapy. In addition, some therapeutically useful aptamers have been found to inhibit protein-protein interactions, such as receptor-ligand interactions, and thereby function as antagonists [38] .
In this study, using the bead-based and flow cytometry monitored SELEX technology, we aimed to obtain specific aptamers to AGR2 and study theirs structure and potential function. Beads-based SELEX allowed the use of simple, yet effective, flow cytometry analysis to monitor the progress of the selection, avoiding the tedious, time consuming and radioactive EMSA process [39] [40] [41] [42] [43] . After 14 rounds of selection, we have identified a group of DNA aptamers that specifically bound to AGR2 with high affinities. Structural studies on one of the aptamer sequences, C14B, revealed an intramolecular parallel Gquadruplex, and its structure and binding affinity to AGR2 depend on K + ion intensively. Furthermore, we designed an allosteric molecule beacon AGR2-aMB based on the identified aptamer, which enables simple, sensitive and selective detection AGR2. The aptamer sequences and AGR2-aMB reported in this study are potentially useful tools for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.
To identify aptamers against AGR2, recombinant AGR2 was fused with glutathione-S-transferase (GST) to facilitate the attachment of the protein to solid supports (Sepharose GSHbeads). The resulting AGR2-GST-beads were used as the positive target in SELEX while the GST-beads as negative control to remove non-specific surface binding sequences. The process of in vitro sepharose-bead-based SELEX is schematically illustrated in Figure 1 .
An 87-nucleotide (87-nt) single-stranded DNA (ssDNA) library with 45 random bases flanked by two primer sequences (22-nt and 20-nt) was subjected to the SELEX procedure. The library was first allowed to interact with excess negative control beads, and only the DNA sequences that did not bound to the GST-beads were collected. The collected sequences were then incubated with AGR2-GST-beads. After rigorous washing, those sequences that either did not bind, or only weakly bound to the target were discarded. Only the sequences that bound strongly enough were retained on beads, and the bead-ssDNA complexes were collected and amplified by PCR for the next round of selection. After multiple rounds of selection, the subtraction process efficiently reduced the DNA sequences that bound to the GST beads, while those AGR2-specific aptamer candidates were gradually enriched. The progress of the selection process was monitored by flow cytometry. The stronger binding of DNA library to AGR2, the more FAM labeled sequences bound to the beads, thus the higher fluorescence intensity the beads would emit. With the increasing number of selection cycles, steady increases in fluorescence intensity on the target beads were observed (Figure 2a) . The binding affinity of the enriched library after 14 rounds of selection was determined to be in the nanomolar range (K d = 64.165.4 nM), while there was no observable binding of the library to control beads ( Figure 2b ). These results suggested that the DNA aptamers specifically recognizing AGR2 were enriched during the selection process.
After 14 rounds of selection, the enriched DNA pool was cloned and sequenced. The sequencing data for clones were analyzed by using sequencing analysis software Clustal W 6.0 [44] . The sequences were grouped based on the homology similarity of the DNA sequences from individual clone. Among the sequences from 62 clones, there were two subfamilies each containing multiple sequences with common features. One subfamily is guanosine-rich sequences (22 clones) , and the other is thymine-rich sequences (40 clones) ( Figure S1 ). Four sequences were chosen and synthesized for further characterization (Table S1 ): three sequences from Grich subfamily, C14A (appeared 2 times), C14B (appeared 3 times) and C14C (appeared 5 times), and one sequence C14D (appeared 2 times) from T-rich subfamily. Only one T-rich sequence was chosen because of T-rich sequences tend to from non-rigid tertiary structures and the chance of being aptamer was thought to be very low. As shown in Figure 2c , C14B can bind AGR2 with the highest affinity (K d = 13.167.2 nM). Titration of C14B to GSTbeads revealed no observable binding, establishing that the binding target of C14B was indeed AGR2. Other three sequences have similar binding constants to AGR2 but a little weaker than C14B, in which the K d of C14A is 20.965.2 nM, C14C is 44.667.0 nM and C14D is 48.4615.6 nM. ( Figure S2 ).
Since C14B is the best aptamer we obtained from the four sequences tested, it was choses for future optimization and characterization. The length of C14B is 87mer, which is disadvantageous for future applications because it is inconvenient and expensive to synthesize such a long sequence. To identify the binding region of the aptamer, the marginal sequences of C14Bwas gradually truncated. Two truncated sequencesC14B0 and C14B1 from C14B were shown in Table 1 . Subsequent binding affinity experiments revealed that both C14B0 (8.563.6 nM) and C14B1 (19.165.1 nM) have similar K d to AGR2 as that of original C14B (13.167.2 nM). The majority of eliminated sequences were primer sequences, implying that the binding region of the aptamer was the middle of random sequences and the primer sequences do not or contribute little to the binding affinity of aptamer. C14B1 has five portions of poly-G and we designed five truncated sequences by removing one of poly-G portion each (Table S2) . Removal of any poly-G portion would destroy its binding affinity to certain extend ( Figure S3) , suggesting the whole G-motif is required for aptamer binding. Taking together, these results indicate that C14B1, which was only 33 mer, was the essential binding region to AGR2. Thus, C14B1 was applied for further characterization and probe design.
In order to demonstrate the specific interaction between AGR2 and C14B1, three control proteins BSA, thrombin and trypsin were coupled with the NHS-sepharose-beads and then were incubated with C14B1. As shown in Figure 3 , C14B1 could bind to target AGR2 strongly, while there were no or little binding affinity towards thrombin, BSA and trypsin, demonstrating the high selectivity of the aptamer.
Further examination revealed that C14B1 has multiple stretches of guanines (CGGGTGGGAGTTGTGGGGGGGGGTGG-GAGGGT). It is well-known that guanine-rich sequences can fold into four-stranded secondary structures called quadruplexes. According to G-Quadruplex prediction formula d(G 3+ N 1-7 G 3+ N 1- Figure 1 . Schematics of systematic evolution of DNA aptamers against AGR2. In sepharose-beads-based SELEX process for protein AGR2, the GST-beads were incubated with the ssDNA library for counter-selection to remove nonspecific sequences. The unbound DNAs were then incubated with AGR2-GST-beads for target-selection. After harshly washing, the AGR2-specific DNA sequences were subsequently amplified by PCR for the next round of selection, or for cloning and sequencing to identify individual aptamers after flow cytometry analysis. doi:10.1371/journal.pone.0046393.g001 single stranded G-quadruplex structure, the dependence of melting temperature on the concentration of C14B1 was studied [48] . The melting temperature of C14B1 was found to be 59uC, which was independent of oligonucleotide concentration ( Figure  S4 ), indicating that the aptamer forms an intramolecular Gquadruplex.
Binding Affinity and Structure Stability of C14B1 is K + Dependent Many DNA aptamers have been found to form G-quadruplex structures [49] [50] [51] . It is well established that the existence of a monovalent cation (especially potassium) in the center of these tetrads can significantly stabilize G-quadruplexes [52] [53] [54] . Thus, we investigated how a cation would affect the structural stability of C14B1 and its binding activity towards to AGR2. As shown in Figure 4a , the stability of the C14B1 G-quadruplex structure is strongly dependent on the presence of the monovalent ion. With 60 mM of KCl, strong CD peaks were observed, suggesting the formation of stable G-quadruplex structure. Replacing KCl with the same concentration of LiCl, NaCl, and MgCl 2 led to a dramatic intensity decrease in CD.
We further studied the effect of K + concentration on the stability of G-quadruplex. In Figure 4b , addition of 0.1 mM K + in phosphate buffer dramatically increased the CD intensity at 240 and 260 nm. CD absorption intensity enhanced with the increase of K + concentration and reached a plateau when K + concentration was higher than 20 mM. The binding affinity of C14B1 at different concentrations of K + was also investigated. As the flow cytometry results shown in Figure 4c , very weak binding of C14B1 towards AGR2-beads was observed when there was no K + in the buffer, and the binding affinity kept increasing with the addition of K + . The binding constants of C14B1 were measured and compared in the presence or absence of K + (Figure 4d ). The K d value in the presence of K + was determined to be 6.261.9 nM. The results demonstrated that formation of G-quadruplex is important for the binding capability of aptamer C14B1 to its target, which is highly K + dependent.
Clinical studies have already shown that the AGR2 protein is overexpressed in a wide range of human cancers. The sensitive and selective detection of AGR2 is thus of great importance to early cancer diagnostics. Herein, by applying the selected and optimized aptamer C14B1, we designed and developed an allosteric molecular beacon against AGR2, named AGR2-aMB, which converts the molecule recognition property of aptamer to fluorescence flow cytometry signal for AGR2 sensing [55] . Figure 5 illustrates the working principle of AGR2-aMB. An AGR2-aMB is a ssDNA consisting of an streptavidin (SA) aptamer sequence [56] , a C14B1 sequence, a short sequence complimentary to a small part of the SA aptamer sequence, and a fluorophore. A stable hairpin structure is formed by intramolecular hybridization between the SA aptamer sequence and the complementary sequence, temporarily disabling the probe's ability to bind with SA beads. Consequently, when incubated with SA beads, no probe can bind to SA beads, and the beads display very low fluorescence. In the presence of AGR2, however, the C14B1 sequence in the loop of aMB binds to the target sequence, which in turn disrupts the hairpin structure to free the SA binding sequence, thereby activating the probe's binding affinity to SA beads. Thus, the AGR2-bound probe will bind to SA beads, which will fluoresce strongly because the probe is FAM labeled. Target molecules can be detected and quantified by reading the fluorescence intensity of SA beads through flow cytometer.
We designed AGR2-aMB, with the following sequence: CGACGCACCGATCGCAGGTTCGGGATTTTCGGGTGG-GAGTTGTGGGGGGGGGTGGGAGGGTT-FAM, where the AGR2 binding region is underlined and the SA aptamer sequence is in bold. Stable hairpin structure is formed by intramolecular hybridization ( Figure S5 ). In our experiment, when only AGR2-aMB incubated with SA beads, no probe can bind to SA beads, and the beads fluoresced very weakly. In the presence of AGR2, however, the fluorescence intensity of SA beads increase significantly, suggesting the binding of AGR2-bound probe to SA beads. With the increase of AGR2 concentration, steady increases in fluorescence intensity on the target beads were observed. AGR2 at 100 nM concentration can be easily detected (Figure 6a) . A series of proteins including BSA, trypsin, thrombin, and IgG were employed as controls (500 nM), and no distinct fluorescent signals were observed (Figure 6b) , indicating an excellent specificity of the AGR2-aMB towards target molecule. The results demonstrated the sensitivity and specificity of the allosteric molecular beacon probe, implying its potential for application in real sample analysis, such as protein function study and disease diagnosis.
In conclusion, we have developed new aptamer probes for specific recognition of the AGR2. In the SELEX process, AGR2-GST fused protein was used as the target protein, and linked to sepharose beads through the mild, yet specific, noncovalent GST-Glutathione interaction. Beads-based SELEX allowed the use of simple, yet effective, flow cytometry analysis to monitor the progress of the selection, avoiding the tedious, time consuming and radioactive EMSA process. Through multiple rounds of selection with GST as a control, we have identified aptamers that selectively recognize AGR2 with nanomolar K d values. CD measurements and melting-temperature assays demonstrated that the optimal aptamer C14B1 forms an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected The aptamer sequences and sensors reported here has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.
The HPLC-purified library containing a central randomized sequence of 45 nucleotides flanked by two 20-nt and 22-nt primer hybridization sites. Initial library was: 59-TCT CGG ACG CGT GTG GTC GG-N45-C TCG CTG CCT GGC CCT AGA GTG-39, forward primer: 59-FAM-TCT CGG ACG CGT GTG 
The plasmid pGEX-GST-AGR2 was transformed into the engineering strain BL-21 and the GST-tagged AGR2 protein was expressed. After purification with glutathione sepharose beads (GE healthcare) by affinity chromatography, the GST-AGR2 fused protein was linked to sepharose beads for positive selection. After being washed several times with W1 buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.07% b-mercaptoethanol, 1% Triton X-100), the final purified AGR2-GST-beads were stored in sterilized PBS buffer. Flow cytometry analysis with TAMRA-labeled anti-AGR2 monoclonal antibody (Santa Cruz) and SDS-PAGE indicated that the GST-AGR2 fused protein was successfully linked to the sepharose beads.
The procedures of selection were as follows. The ssDNA pool (200 pmol) dissolved in binding buffer (200 mL, 16PBS buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 and 2.0 mM KH 2 PO 4 , pH 7.4) was denatured by heating at 95uC for 5 min, then quickly cooled on ice for 10 min, and subsequently incubated for another 10 min at room temperature before binding. The ssDNA pool was then incubated with negative GST beads (1.0610 5 beads) for counter selection to remove sequences non-specifically binding to GST beads. After filtering with a homemade filter column, the filtrate was incubated with positive AGR2-GST-beads (1.0610 5 beads) at 37uC for 45 min. The unbound or nonspecifically bound oligoes were removed by filtration. The sequences bound to the target-coated beads were then amplified by PCR with FAM and biotin-labeled primers (5-15 cycles of 0.5 min at 94uC, 0.5 min at 53uC, and 0.5 min at 72uC, followed by 5 min at 72uC; the Easytaq plus polymerase and dNTPs were obtained from Transgen Beijing). After denaturation in NaOH (0.1 M), the selected sense ssDNA was separated from the biotinylated antisense ssDNA strand on streptavidin-coated sepharose beads (GE healthcare) and used for next round selection. For the first-round selection, the amount of initial ssDNA pool was 5 nmol, dissolved in 500 mL binding buffer, and the counter-selection step was eliminated. To acquire aptamers with high affinity and specificity, the selection strength was enhanced gradually by increasing the number of washes (from three to ten times with 200 mL 16PBS buffer each) and decreasing the amount of the ssDNA library per round (from 200 to 150 pmol).
The resulting pool from the 14 th round was PCR amplified, cloned and sequenced (Shanghai Sangon sequencing facility). The resulting 62 sequences were subjected to multiple sequence alignment analysis by using Clustal W 6.0 software to discover highly conserved motifs in groups of selected DNA sequences. The discovered consensus sequences with high repeats among selected pools were then chemically synthesized for further testing.
To monitor the enrichment of aptamers after selection, the FAM-labeled ssDNA pool was incubated with 5610 4 AGR2-GST-beads or GST-beads in binding buffer (200 mL) at 37uC for 45 min. Beads were washed three times with 200 mL binding buffer by means of filtration, and suspended in binding buffer (250 mL). The fluorescence intensity of the resulting beads was monitored with a FACSAria cytometer (Becton Dickinson Immuno cytometry systems) by counting 10000 events. The binding affinities of aptamers were determined by incubating AGR2-GST-beads (5610 4 ) with various concentrations of FAMlabeled aptamers (pre-heat-treated) in binding buffer (200 mL) at 37uC for 45 min in the dark. Beads were then washed three times with the binding buffer, then resuspended in binding buffer (250 mL) and subjected to flow cytometry analysis. The FAMlabeled unselected ssDNA library was used as negative control for the nonspecific binding evaluation. All binding experiments were repeated two to four times. The mean fluorescence intensity of target protein labeled by aptamers was used to evaluate binding affinity by subtracting the mean fluorescence intensity of nonspecific binding produced by unselected ssDNA library. The dissociation constants (K d ) of the fluorescent ligands were obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the ligands to the equation (1): Y = B max X/(K d +X) using SigmaPlot software.
CD measurements were carried out on a Jasco J-810 spectropolarimeter equipped with a programmable temperature-control unit (Julabo HP-4). The concentration of DNA samples were 2 mM. Before the CD spectrum measurement, the DNA samples were annealed by heating to 95uC for 5 min, then rapidly cooled on ice for 10 min, and incubated for another 10 min at room temperature. The spectra from 400 to 200 nm were obtained by using 1 nm slit width and 0.1 nm scanning resolution. Each CD spectrum was an average of 8 scans with the buffer background subtracted.
UV absorbance and melting studies were carried out on an Agilent 8453 spectrophotometer equipped with a programmable temperature-control unit (Agilent 89090A). Melting temperatures (T m ) were taken as the temperature of half-dissociation of the quadruplex and were obtained from the maximum of the first derivative dA/dT plots at 295 nm. The heat-treated DNA solutions at several concentrations were introduced into a quartz cuvette and overlaid with a thin layer of silicone oil to prevent evaporation. The optical path length was 1 cm. Absorbance and temperature were recorded every 2uC.
Evaluation of AGR2-aMB AGR2-aMB (40 nM) was annealed by heating to 95uC for 5 minutes in 200 mL Tris-HCl buffer (25 mM Tris-HCl, 120 mM NaCl, 0.5 mM KCl, 2 mM MgCl 2 at pH 7.4), and then rapidly cooled on ice for 10 min, and subsequently waiting for another 10 min at room temperature before use. To the solution of AGR2-aMB, various concentrations of AGR2 were added and the resulting solutions were allowed to incubate at 37uC for 30 min. To the mixture, 1 mL of streptavidin beads (about 5610 4 beads) were added and the mixture was allowed to set for 45 min in dark. After washing twice with Tris-HCl buffer, SA beads were filtered to eliminate unbound AGR2-aMB and resuspended in Tris-HCl buffer solution 250 mL before flow cytometry analysis. In selectivity tests, BSA (Tagene Biotechnology Xiamen), trypsin (Dingguo Beijing), thrombin (alfa) and IgG (Dingguo Beijing) were used. Figure S1 The sequences of 62 clones. One subfamily is guanosine-rich sequences (22 clones) , and the other is thymine-rich sequences (40 clones). (TIF) Figure S2 The dissociation constant measurement of C14A, C14B, C14C and C14D against AGR2 GST and GSH. (TIF) Figure S3 Flow cytometry assay to monitor the binding of C14B1 and its five truncated sequences with a) AGR2 (target protein) and b) GST (control protein). (TIF) Figure S4 UV thermal-denaturation experiment of C14B1. Denaturation profiles obtained at 295 nm for the aptamer at three different concentrations (2 mM, 4 mM, 8 mM). The T m (59uC) at 295 nm is independent of oligonucleotide concentration, indicating that the aptamer forms an intramolecular G-quartet. (TIF) Figure S5 The secondary structure of AGR-aMB. Stable hairpin structure is formed by intramolecular hybridization between the SA aptamer sequence and the complementary sequence of C14B 1 .
(TIF)  Flt3L Combined with Rapamycin Promotes Cardiac Allograft Tolerance by Inducing Regulatory Dendritic Cells and Allograft Autophagy in Mice The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25(+) Foxp3(+) regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation. Organ transplantation has become the primary treatment for patients with end-stage organ failure [1] . Although the application of immunosuppressive drugs has contributed significantly to the success of allograft survival, side effects resulted from immunosuppression or drug toxicity also markedly impact the quality of life of recipients [2, 3] . Therefore, establishment of strategies aimed at inducing allograft tolerance is wanting.
In the setting of transplantation, DCs actively mediate graft rejection by presenting donor-derived alloantigens to naïve T cells. However, there is emerging evidence indicating that other than mediation of allograft rejection, DCs also possess the capability to induce allograft tolerance [4] . Therefore, DCs are capable of inducing either immune response or tolerance depending on their activation and maturation status [5, 6] .
Flt3, a member of the tyrosine-kinase receptor family, was initially cloned from fetal liver of cells with hematopoietic stem cell activity [7] . Flt3L is the ligand for Flt3, which is a key regulator for DC commitment and development. Mice administered with Flt3L displayed the expansion of certain subtypes of DCs such as the plasmacytoid DCs (pDCs) and conventional DCs (cDCs) in the spleen [8] . Studies have shown that pDCs play an important role in the induction of Tregs in vivo manifested by that their precursors are able to prolong graft survival [9] . Studies in hematopoietic cell transplantation also revealed that Flt3L is a potent mobilizer to induce murine hematopoietic chimerism, while long-term persistence of donor hematopoietic cells in peripheral lymphoid and non-lymphoid tissues of organ allograft recipients is postulated to be an essential prerequisite for the induction of donor-specific tolerance [10, 11] .
Autophagy in eukaryotic cells is a cellular quality control process to deliver cytoplasmic constituents for lysosomal degradation, which enables cells to recycle nutrients for survival during nutrient starvation [12] . Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) has been used to stimulate autophagy. In the steady state, autophagy-mediated antigen processing in thymic epithelial cells could have a crucial role in the induction and maintenance of CD4 + T-cell tolerance [12, 13] .
In this study, we examined the effects of Flt3L in combination with Rapamycin in a BALB/c-to-C57BL/6 cardiac allograft model. Our results demonstrate that Flt3L combined with lowdose of Rapamycin prevented acute allograft rejection and prolonged allograft survival time. The enhanced allograft survival is associated with the induction of tolerogenic DCs along with the production of CD25 + Foxp3 + regulatory T cells and increased allograft autophagy. Our data suggest that Flt3L combined with low-dose of Rapa could be a promising therapeutic strategy in clinical transplantation.
We first sought to investigate the regulatory effect of Flt3L combined with a short-term of low-dose Rapa treatment on allograft acute rejection. A murine cardiac allograft transplantation model was employed to address this question. As shown in Figure 1A , untreated recipients rapidly rejected their grafts (7.660.516 days) along with typical pathological features of acute rejection (Fig. 1B) . Similar pathological features were observed in recipients treated with either PBS or rGST (controls, Figure 1B ). As expected, administration of Rapa significantly increased graft mean survival time (MST) (16.962 .767 days, Fig. 1A) . Similarly, recipients treated with Flt3L doubled allograft MST (13.161.287 days, Fig. 1A ). In sharp contrast, a significant enhanced graft survival was observed in mice treated with Flt3L and low-dose of Rapa (36.4618.704 days, Fig. 1A) . Remarkably, about 20% of recipients showed long-term graft survival (MST.100 days, Fig. 1A ). In consistent with these results, a significant reduction for inflammatory infiltration was observed in the allograft sections (Fig. 1B ).
We next examined DC phenotypes in the recipient mice. It was noted that a significant increase in the number of pDCs in mice treated with Flt3L or Flt3L with low-dose of Rapa as compared with that of control mice ( Fig. 2A) . Similarly, we also detected a significantly higher proportion of splenic CD8a + DCs in Flt3L or Flt3L/Rapa treated mice ( Fig. 2A) . Since both CD8a + DCs and pDCs have regulatory functions [14] , Our data suggest that Flt3Lcombined with Rapa could induce the expansion of regulatory DCs in the recipient mice.
Rapamycin has been shown to inhibit the up-regulation of maturation markers expressed by DCs [15] , while immature DCs have been demonstrated with capability to prolong allograft survival [16, 17] . In line with this notion, Flt3L combined with Rapa significantly blocked CD11c + DC maturation as manifested by the decreased CD40, CD80 expression (Fig. 2B ). Since CCR9 + pDCs had previously been reported to be potent inducers for Tregs [18] , we then investigated whether CCR9 + pDCs are implicated in the induction of allograft tolerance. Indeed, recipients treated with Flt3L and low-dose of Rapa displayed a significant increase for CCR9 + pDCs as compared with those recipients treated with Flt3L or Rapa alone or other groups (Fig. 2C ).
Flt3L Combined with Rapa Promotes the Production of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + T Cells
The above results prompted us to examine the proportion of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + Tregs. To this end, splenic T cells isolated from each group of recipient mice were stained for intracellular Foxp3 after co-staining with anti-CD4/ CD25 antibodies or anti-CD3/CD8/CD25 antibodies, and the cells were next analyzed by flow cytometry. A significant increase of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + Tregs was observed in recipients treated with Flt3L and Rapa as compared with recipients in other groups (Fig. 3 ).
We then tested the reactivity of bulk T cells isolated from the spleens of the recipients against donor-derived DCs. As shown in Figure 4A , T cells derived from Flt3L/Rapa treated mice exhibited a significantly lower proliferative response against donor BALB/c DCs in MLR when compared with that of T cells derived from untreated, rGST-treated and PBS-treated recipients. Unexpectedly, a similar proliferation rate was noted in recipients treated with either Flt3L or Rapa alone as compared with that of recipients treated with Flt3L/Rapa. This result promoted us to further examine cytokine production in the culture supernatants by ELISA. Of Interestingly note, the production of IL-10 was significantly higher in the Flt3L/Rapa treated mice. More importantly, although T cells derived from Flt3L or Rapa treated mice showed similar potency for proliferation (Fig. 4A ), but their capacity for secretion of IL-10 was similar to those T cells from control mice (Fig. 4B ). Collectively, our data suggest that Flt3L/ Rapa treatment enhances IL-10 secretion which could favor the induction of T anergy.
Given that Flt3L/Rapa therapy resulted in reproducible longterm allograft survival in about 20% of recipients without immunosuppression ( Fig. 1A) , we thus employed adoptive transfer studies to characterize the cell subpopulations for sustained allograft survival. For this purpose, CD4 + T cells, CD8 + T cells, pDCs and total splenocytes were first isolated from long-term survival recipients, and then adoptively transferred into naive recipients. The mice were next transplanted with cardiac allografts as described. As shown in Figure 5 , transferred CD8 + T cells and pDCs exhibited the ability to prolong allograft survival. Unexpectedly, compared with CD8 + T cells and pDCs, adoptive transfer of CD4 + T cells failed to provide protection against allograft rejection.
To determine whether the prolonged allograft survival was associated with enhanced autophagy, we examined the levels of autophagy-related protein beclin-1, microtubule associated pro-tein1 light chain 3 (LC3) I and II by Western blot analysis. As shown in Fig. 6A , only low levels of Beclin-1 and LC3 II were detected in control mice (rGST, untreated and PBS groups), but higher levels of Beclin-1 and LC3 II were observed in mice treated with Flt3L or Rapa alone. Importantly, the highest Beclin-1 and LC3 II expressions were noted in Flt3L/Rapa treated mice. To further confirm these results, we did immunohistochemical analysis, and similar results were obtained (Fig. 6A ). To examine the functional relevance for the enhanced autophagy in allograft survival, we performed similar transplantation along with the administration of 3-MA, an autophagy inhibitor [19, 20] . As expected, administration of 3-MA significantly repressed autophagy as determined by Western blot and immunohitochemical analysis (Fig. 6B) . Remarkably, repression of autophagy significantly attenuated Flt3L/Rapa therapy mediated protection against allograft rejection (Fig. 6C ). In consistent with this result, we also observed a decrease for the number of regulatory T cells and regulatory DCs (Fig. 6D) . Taken together, these data indicate that autophagy plays an essential role in Flt3L/Rapa therapy mediated protection against allograft rejection.
Despite the benefit of Flt3L and Rapa for anti-rejection in setting of transplantation, the effect for Flt3L or Rapa based mono-therapy on allograft survival remains poor [21, 22, 23, 24] . In the present study, we have demonstrated that Flt3Lcombined with low-dose of Rapa could markedly increase graft survival time as compared with that of Flt3L or Rapa alone. The enhanced protective effect was achieved by promoting the generation of tolerogenic DCs and Tregs along with increased autophagy in the allograft.
It has been reported that DCs expanded by Flt3L only express low levels of costimulatory molecules, and therefore, they are able to delay allograft rejection in transplantation settings [25, 26] . In contrast to Flt3L or Rapa alone, our data show that Flt3L combined with low-dose of Rapa are more potent to expand DCs with tolerogenic characteristics. Moreover, short-term of low-dose Rapa exerts the effect to expand Tregs while to avoid side effect due to high-dose of Rapa.
Several DC subsets have been reported to promote tolerance induction [4, 27] . The CD11c + CD8a + DC subset could promote peripheral self-tolerance and prolong the survival of cardiac allografts in rodents [28, 29] . More recently, pDC precursors have been identified and are implicated in the induction of allogeneic Tcell hyporesponsiveness [9, 30] . In line with these findings, our phenotypic analysis of splenic DCs in the recipient mice revealed that Flt3L/Rapa treatment increased the proportion of DC subsets with regulatory effect such as CD11c + CD8a + DCs and pDCs. It is known that immature DCs not only fail to prime T cells effectively, but also serve to promote tolerance induction [31] . Our studies show that Flt3L combined with Rapa could block DC maturation and increase the ratio of CCR9 + pDCs, while CCR9 + pDCs are immature pDCs with potency to suppress antigen-specific immune responses both in vitro and in vivo [18] .
Regulatory T cells are a subpopulation of T cells that have the ability to suppress immune reactions and maintain tolerance. They were believed to play a key role in transplant tolerance induction. The most important representative of regulatory T cells is the naturally occurring regulatory T cells (Tregs), with a CD4 + CD25 + Foxp3 + phenotype. They were found to mediate donor-specific Figure 2 . DCs from recipients treated with Flt3L/Rapa display a tolerogenic phenotype. Spleen cells isolated from mice treated with Flt3L/ Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were stained with PE-cy7-labeled anti-mouse CD11c mAb, Per-cy5.5-labeled anti-mouse B220 mAb, APC-cy7-labeled anti-mouse CD8a mAb, FITC-labeled anti-mouse CCR9 mAb, APC-labeled anti-mouse CD80 mAb and APC-labeled anti-mouse CD40 mAb. CD11c + and CD11c + B220 + gated DCs were analyzed by flow cytometry for expression of the various DC markers. (A) The bar graph was a summary of percentages of pDC and CD8a + DC in the recipients. (B) CD11c + -gated DCs were analyzed for expression of costimulatory markers CD80 and CD40 to assess their maturation state. (C) The percentage of CCR9 + cell of gated pDC subsets was assessed by FACS analysis. The results are representative of three independently performed experiments (* P,0.05). doi:10.1371/journal.pone.0046230.g002 Figure 3 . Flt3L combined with Rapa promotes the production of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + T cells. Spleen cells isolated from mice treated with Flt3L/Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were analyzed to determine the proportion of CD4 + CD25 + Foxp3 + and CD8 + CD25 + Foxp3 + T cells. (A, B) The expression of Foxp3 by CD4 + CD25 + and CD8 + CD25 + T cells was analyzed by flow cytometry after intracellular staining. The bar graph was a summary of percentages of CD4 + CD25 + Foxp3 + T cells and CD8 + CD25 + Foxp3 + T cells in the recipients. The data shown are representative of three independent experiments that yielded comparable results (* P,0.05). doi:10.1371/journal.pone.0046230.g003 unresponsiveness in some stable renal [32, 33, 34, 35, 36] and liver [37] transplant patients. In addition, adoptive transfers of Tregs could prevent allograft rejection and prolong graft survival in the mouse models [38] . Previously, addition of rapamycin to human and murine T cell cultures preferentially expanded regulatory T cells [39, 40] . Our data indicated that infusion of Flt3L with Rapa and Rapa alone enhances the production of CD4 + CD25 + Foxp3 + Tregs. In addition, we found a significant increase of splenic CD8 + CD25 + Foxp3 + T cell populations in the recipient mice treated with Flt3L/Rapa, while CD8 + CD25 + Foxp3 + T cell have been found being able to inhibit antigen-specific CD4 + and CD8 + T cell responses by cell contact inhibition, secretion of cytokines and other ways [41] .
Our adoptive transfer studies showed that CD8 + T cells are responsible for induction of long-lasting tolerance toward donor alloantigen. We therefore proposed that Flt3L-mobilized recipient DCs might process indirect antigen presentation of allopeptides to induce tolerance for host CD8 + alloreactive T cells, which are the main effector mechanism of cellular rejection across an MHC class I barrier. This notion is well supported by a recent study, in which Rapa-conditioned, alloAg-pulsed DC can present acquired MHCpeptide complexes from donor cells and modulate directly-reactive CD8 + T cell function, and through which, these DCs enhance Agspecific CD8 + T cell undergoing apoptosis [42] . Furthermore, CD8 + CD28 -T cells may also play an important role in tolerance induction. These T cells represent a transient state of effector CD8 + T cells, which promotes the production of the immunosuppressive cytokine IL-10 [43, 44] . Emerging data suggest that the appearance of these T cells in transplant recipients is associated with better graft acceptance along with stable function [45, 46, 47] . Finally, our data indicate that autophagy plays an important role in the protective effect of Flt3L in combination with Rapa on cardiac allograft survival. The possible mechanisms is that autophagy delivers self-proteins for MHC class II loading, and their peptidic fragments are essential for the deletion of selfreactive T cells in the thymus. Recent studies have also suggested that the possible link between autophagy and tolerance is that autophagy plays a role in the removal of apoptotic cell corpses [48] . Regarding to the effect of autophagy on allograft survival seems controversial. In a rat liver transplantation study, it has been shown that autophagy-associated hepatocyte death triggers liver graft dysfunction, and suppression of autophagy prevent cold ischemia-warm reperfusion injury associated with liver transplantation [49] . Thus, further relevant investigations are needed.
In summary, Flt3L combined with Rapamycin is able to promote the prolongation of allograft survival. This protective effect is associated with the generation of tolerogenic DC subsets, CD25 + Foxp3 + regulatory T cells (Tregs), and enhanced allograft autophagy.
Six to eight week-old female C57BL/6 (H-2 b ), BALB/c (H-2 d ) mice were obtained from the animal facilities at Tongji Medical College. All mice were maintained under specific pathogen-free conditions and the studies were carried out in compliance with the institutional animal care and use guidelines. 
DNA sequence encoding Flt3L (696 bp) was amplified from the C57BL/6 mouse spleen cDNA using the following oligonucleotides: 59-CGG GAT CCA TGA CAG TGC TGG CGC C-39 and 59-CGG AAT TCA TCC TAG GGA TGG GAG-39. The resulting products were subsequently cloned into a pGEX-4T3 vector with a glutathione S-transferase tag. The plasmid was transformed into Escherichia coli strain BL21. Recombinant Flt3L expression was induced by addition of 1 mM IPTG and then purified using the glutathione sepharose 4B resin columns as instructed by the manufacturer. A GST vector protein was also expressed and purified to homogeneity. The proteins were passed over polymyxin B columns (PIERCE) to remove any contaminated LPS and further concentrated by 3KD Micropore Filters. The purified recombinant Flt3L and rGST were stored in aliquots at 280uC until use. 
Heterotopic cardiac transplantation was performed as previously described [50] . In this study, transplant surgery involved the transfer of fully MHC-mismatched hearts from BALB/c (H-2 d ) donors to C57BL/6 (H-2 b ) recipients. Heart beat of the grafts was monitored and evaluated daily by direct abdominal palpation to detect the state of cardiac health/rejection.
Heart transplant recipients were randomly assigned to six groups (n = 10): Group 1, rGST alone; Group 2, untreated control; Group 3, PBS alone; Group 4, Flt3L alone (10 mg/day, i.v) for 10 consecutive days before heart transplantation; Group 5, Rapa alone (2 mg/kg/day; p.o, POD0-15); Group 6, combination treatment of Flt3L and Rapa.
Allograft samples were fixed in 4% paraformaldehyde. Serial sections (5 mm in thickness) were prepared using a microtome and stained with hematoxylin/eosin for the analysis of pathological changes.
Spleen cells isolated from mice treated with Flt3L/Rapa and other control mice were incubated with anti-CD16/CD32 monoclonal antibody to block FcR, and then stained with PE-cy7-labeled anti-mouse CD11c mAb, Per-cy5.5-labeled antimouse B220 mAb, APC-cy7-labeled anti-CD8a mAb, FITClabeled anti-mouse CCR9 mAb, APC-labeled anti-mouse CD80 mAb and APC-labeled anti-mouse CD40 mAb. CD11c + and CD11c + B220 + gated DCs were analyzed by flow cytometry for expression of the various DC markers. For analysis of regulatory T cells, splenocytes were isolated from the recipient mice and subsequently co-stained with CD4, CD25 or CD3, CD8a CD25 antibodies. The cells were further stained with Foxp3 using established techniques [51] . Cells were then analyzed using FACS flow cytometer as previously reported [52] .
Splenic T cells (purified using nylon wool columns) isolated from naive or grafted C57BL/6 mice serving as responders (2610 6 / well) were incubated for 7 days in the presence of Mitomycin Ctreated (50 mg/ml, Sigma-Aldrich) DCs used as stimulators derived from naive BALB/c mice (4610 5 /well, DC: T ratio of 1:5) in 12-well flat bottom plates (Nunc, Roskilde, Denmark). Cultures were kept at 37uC in 5% CO 2 atmosphere. Supernatants were recovered after 7 days for determination of IL-10 production. Cytokine level was quantified using Mouse IL-10 ELISA MAX TM Deluxe. T-cell proliferation was assessed by serial dilution of the intracellular CFSE dye as described previously [53] .
Proteins (50 mg) extracted from cardiac allograft were subjected to electrophoresis on a 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were then incubated with primary antibodies for Beclin-1, LC3B and GAPDH at 37uC for 2 h, respectively. The blots were visualized using enhanced chemiluminescence (Amersham).
After deparaffin and rehydration, the paraffin-embedded heart sections (5 mm) were treated with 3% H 2 O 2 for 5 min. The nonspecific proteins were blocked with 5-10% goat serum for 10 min. For Beclin-1 staining, specimens were incubated with a rabbit antimouse Bcelin-1 Monoclonal antibody (1:200) at 4uC overnight, followed by incubation with a HRP conjugated goat anti-rabbit secondary antibody. The sections were finally incubated with DAB chromogenic substrate and counterstained with hematoxylin.
Total T cells were first enriched with T cells using nylon wool columns, and then subjected to isolation of mouse CD4 + T cells using the (L3T4) MicroBeads, and CD8 + T cells using the mouse CD8a (Ly-2) MicroBeads as instructed (Miltenyi Biotec, CA)., pDCs were isolated using a mouse Plasmacytoid Dendritic Cell Isolation Kit II as instructed (Miltenyi Biotec, CA). The purified CD4 + T cells (5610 6 ), CD8 + T cells (5610 6 ), pDCs (5610 5 ) and total splenocytes (5610 6 ) originated from recipients with long-term survival allograft were then adoptively transferred into naive recipients, respectively. One day after the adoptive transfer, the mice were transplanted with cardiac allografts and monitored for allograft survival as described above.
The data are presented as means 6 SD. Statistical differences were determined by one-way ANOVA. Allograft survival differences between groups were determined using the log-rank test. P values less than 0.05 were considered with statistical significant. Renalase's Expression and Distribution in Renal Tissue and Cells To study renalase's expression and distribution in renal tissues and cells, renalase coded DNA vaccine was constructed, and anti-renalase monoclonal antibodies were produced using DNA immunization and hybridoma technique, followed by further investigation with immunological testing and western blotting to detect the expression and distribution of renalase among the renal tissue and cells. Anti-renalase monoclonal antibodies were successfully prepared by using DNA immunization technique. Further studies with anti-renalase monoclonal antibody showed that renalase expressed in glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells and its cells supernatant. Renalase is wildly expressed in kidney, including glomeruli, tubule, mesangial cells, podocytes and tubule epithelial cells, and may be secreted by tubule epithelial cells primarily. Renalase is a newly discovered monoamine oxidase enzyme originating from renal tissues [1] . It degrades circulating catecholamines, regulates blood pressure and cardiac function, and is closely associated with cardiovascular diseases and chronic kidney disease (CKD) [2] [3] [4] . Renalase is a protein made of 342 amino acids with a molecular weight of 37.8 KDa approximately. The N-terminate of renalase contains one signal peptide, one flavin adenine dinucleotide binding site and one monoamine oxidase domain, and 13.2% of its amino acid sequence is similar to the monoamine oxidase A.
Obtaining recombinant renalase protein and preparation of monoclonal antibodies are the essential steps for the study of renalase's function, expression and distribution in renal tissues and cells. Recently we have been using recombinant renalase protein produced by prokaryotic expression system to develop monoclonal antibodies, and we also have tried to use recombinant protein produced by eukaryotic expression systems such as baculovirus etc, to develop monoclonal antibodies. However, obtaining large volume of eukaryotic expressed recombinant renalase protein has not been such an easy task [5, 6] .
DNA immunization technique is a vaccination method, which is a fast and effective way of stimulating body to generate immune reaction against target protein [7] [8] . After DNA vaccine's uptake and processing by muscle cells, the natural structure of target protein remains well preserved. In recent years, DNA vaccine techniques have been used to obtain monoclonal antibodies, especially when it is difficult to get protein antigens. Recently we have successfully utilized such techniques to prepare monoclonal antibodies for retinol binding protein (RBP4) [9] , and established immunological testing methods as well as explored renalase related DNA vaccine techniques [10] . On this basis, we utilized DNA immunization technique to prepare anti-renalase monoclonal antibodies and those antibodies were used to analyze renalase expression in renal tissues and cells.
Primer for renalase gene was designed as Sp 59-ATAA-GAATGCGGCCGCATGGCGCAGGT GCTGATC -39, As 59-GGAAGATCTCTAAATATAATTCTTTAAAGCTT -39, and then renalase gene was replicated using RT-PCR technique. Renalase gene was then inserted in pBudCE4.1 (Invitrogen, Carlsbad, CA, USA) vector plasmid, after which the immunization plasmid was completed.
Renalase protein was obtained using prokaryotic expression system. After being purified and measured for concentration, it then was stored at 220uC [5] .
HEK293T cells purchased from ATCC (ATCC No. CRL-11268 TM , USA) were cultured in Dulbecco's Modified Eagle Medium (Invitrogen, USA) containing 10% fetal bovine serum. Plasmids were transfected into HEK 293T cells using Lipofectamine 2000 Reagent (Invitrogen, USA), and this process was carried out according to the manual guidelines. After 48 hours, culture media was removed and rinsed once with PBS (pH 7.4), and then dissolved (0.2 ml/hole). After the collection, the sample was heated for 5 min at 100uC, and then stored at 220uC.
Six weeks old BALB/c mice (Shanghai Experimental Animal Center of Chinese Academy of Sciences) were kept in SPF grade animal room. Animals were used according to the feeding and utilization guidelines prescribed by Chinese Academy of Science. Animal immunization was done as per the recommended methods in the literature [11] : 100 mg plasmids (100 ml sterile PBS, pH 7.4) was given intramuscularly in the quadriceps. Immediately after the injection, electric impulse stimulation was given with ECM830 (BTX, Holliston, USA). Stimulation parameters were square wave, 100 V/50 ms, positive and negative impulses given three times each, with an interval of 1 second. A total of three injections were given with an interval of 3 weeks. Three weeks after the last injection, cell fusion was carried out. A shot of intra-abdominal booster of recombinant renalase protein was given three days before the cell fusion. 10 days after the third injection, blood was withdrawn from the orbit, kept for 1 hour at the temperature of 37uC, followed by 24 hours at 4uC, and then centrifuged. Upper layer of colorless or pale yellow serum was collected and stored at 220uC for further use.
The classic method of monoclonal antibody preparation was used [12, 13] . In brief, selected one mouse that had high sensitive activation to renalase protein, harvested the spleen, then extracted spleen cells and myeloma cell line SP2/0, and then performed cell fusion with 50%(w/v)polyethylene glycol in a 50 ml centrifuge tube (Corning, New York, USA). Following the cell fusion, cells were subjected to centrifugation and transferred to 96-well plate, and then with HAT added, cell selection was performed. Ten days after the cell-fusion, cells selection was performed with selecting ELISA positive holes.
Anti-renalase monoclonal antibodies were used as primary antibodies and SABC testing kit (Boster, Wuhan, China) was used to detect renalase's expression in renal tissues. Each testing step was performed according to the testing manual guidelines. The renal tissues subjected to testing were from a post-traumatic kidney.
With self-prepared anti-renalase antibodies used as primary antibodies and HRP labeled goat-anti-mouse IgG (Shanghai Immune Biotech, China) used as secondary antibodies, western blotting was carried out to detect renalase's expression in 
After the renal tissues were obtained, 3 mm thickness frozen sections or renal cell climbing slices were made and fixed with acetone, and self-prepared anti-renalase monoclonal antibodies were added as primary antibodies. Then, FITC labeled goat-antimouse IgG (Santa Cruz, USA) were added as secondary antibody, slide sealed with glycerol and observed under fluorescence microscope.
Renalase coded gene was inserted into the expression vector pBudCE4.1 (contains one His Tag gene after the sequence of multiple cloning sites), and the correct sequencing was verified. After transfection in vitro expression of pBudCE4.1-Renalase was verified by western blotting demonstrated in Fig. 1 , renalase was detected by anti-His Tag monoclonal antibody. No response was observed in empty vector, indicating that renalase can be expressed in mammalian cells.
The titer of antibody from mice serum was tested before the cell fusion. One mouse with high titer was selected as spleen cells donor and cell fusion was done with cell line SP2/0. The serum titer of anti-renalase was 1:32000 and the titer of normal mouse was below 1:1000. After 3 cycles of cloning, two hybridoma cell lines were obtained. Culture supernatant was collected and ascites was prepared. Analysis of culture supernatant showed an antibody titer of 1:128000, and an antibody sub type IgG1/k. Titration ELISA of purified anti-renalase monoclonal antibody was over 1:512000, higher than that of poly-serum titer 1:128000. Western blotting was performed by using prokaryotic expressed renalase protein as the sample and the prepared anti-renalase monoclonal antibodies as primary antibodies. The results showed the recombinant renalase protein can be recognized by the monoclonal antibody (Fig. 2) . Renalase's expression in renal tissues can be detected by immunofluorescence Indirect immunofluorescence testing was conducted by using anti-renalase monoclonal antibody. The results showed that renalase expressed in cytoplasm of glomerular mesangial cells (Fig. 3A) . And renalase can also be detected for its expression in cytoplasm of tubule epithelial cells (Fig. 3C ).
Immunohistochemical testing was carried out with renalase monoclonal antibodies as the primary antibodies. The results showed that renalase expressed in renal glomeruli (Fig. 4A) and tubule (Fig. 4C ).
Indirect immunofluorescence testing was conducted with antirenalase monoclonal antibodies as the primary antibodies. The results showed that renalase expressed in cytoplasm of mesangial cells (Fig. 5A), podocytes (Fig. 5C ) and renal tubular epithelial cells (Fig. 5E ).
Western blotting was done with anti-renalase monoclonal antibodies as the primary antibodies. The results showed that mesangial cells (Fig. 6A) , podocytes (Fig. 6C ) and renal tubular epithelial cells (Fig. 6E ) cultured in vitro also could express renalase. But only tubular epithelial cells' supernatant could be detected the expression of renalase, as shown in Fig. 6F .
Recent studies showed that elevated levels of catecholamine in renal failure patients compared to normal individuals may be associated with hypertension and cardiovascular complications of CKD. But the exact mechanism of it is still unclear [14] . Renalase was discovered in 2005, it is an enzyme expressed in kidneys that can degrade circulating catecholamines. This finding has changed the previous understanding about renal physiology and neurophysiology [15] [16] [17] . In patients with CKD, renalase production in serum and tissues decreases. Current studies indicate that the decreased renalase production is associated with hypertension, as well as increased levels of circulating catecholamines [18] . In 2007, renalase-like substance was successfully cloned from mice by Wang [19] .
Renalase is a highly conserved protein, of which 95% of amino acids are similar to that of primates. This is probably because renalase coded genes have evolved from a same ancestor gene [4] . Studies have shown that kidneys are the main source of renalase production. In vitro study has shown that renalase can degrade catecholamines and has strongest enzymatic hydrolysis action against dopamine, followed by adrenaline and noradrenaline. In vivo studies indicate that catecholamine activates renalase precursor which promotes deactivation of catecholamines and regulation of cardiovascular function [20, 21] .
As of today, renalase is the only enzyme known to be secreted into blood that can degrade circulating catecholamine and may have a great value in prevention of kidney diseases and cardiovascular diseases. Discovery of renalase has drawn the attention of the scientific world [22] [23] [24] . Even though there have been some debates (Boomsma F), much importance is given to its significance as further studies are carried out [25] . It may have a great value in further understanding increased sympathetic activity and mechanism of cardiovascular complications in CKD patients as well as a prevention of CKD and cardiovascular diseases [26] [27] [28] . Both in vitro and animal studies have demonstrated the possible effects of renalase in chronic renal failure and cardiovascular diseases [29] . It can be speculated that replacement or supplementation of renalase may bring up a promising treatment.
At present, there are many ongoing studies on genetic background of renalase. Zhao et al have found that renalase gene polymorphysim is associated with primary hypertension, indicat-ing this gene may become the novel marker of genetic susceptibility in essential hypertension [30] . Farzaneh-Far discovered that renalase gene polymorphism also is associated with ventricular hypertrophy [31]. Buraczynska found that an association between renalase gene polymorphism and hypertension in type 2 diabetes [32] . Stec et al investigated patients with end stage renal disease and found that renalase gene polymorphism is associated with hypertension among these patients [33] .
Despite the role of renalase in regulating serum catecholamines and blood pressure, recent studies demonstrated that extra-renal renalase is also important. In renalase gene knocked-out mice, serum urea nitrogen, creatinine and aldosterone levels were unaffected, and cardiac systolic function was intact. However, mild ventricular hypertrophy was presented with decreased tolerance to ischemia, and increased risk of myocardial infarction, as much as 3 times higher than that of wild mice. Recombinant renalase replacement therapy could abort such abnormal changes [34] . Gu et al recently reported that renalase's expression is influenced by renal perfusion and abnormality of the renal perfusion is probably implicated in elevated serum catecholamine levels in cardiac failure [35] . Both renalase gene and protein are also observed in central and peripheral nerve systems [26] . Renalase is found in hypothalamus, pons, medulla oblongata and spinal cord, where the presympathetic and preganglionic neurons are located. It is highly plausible that renalase is also involved in regulating the central sympathetic output. Other extra-renal sources of renalase (i.e. skeletal muscles, small intestine and vasculature) are yet to be determined. The regulation of renalase's expression is another enigma that requires further studies. As of today, there have been some animal experiments that showed decreased renalase production associated with high intake of sodium and phosphate [36] . Renal denervation, a potential anti-hypertension treatment [37] , was reported to increase plasma renalase content and renalase expression in the kidneys in spontaneously hypertensive rats [38] . Data from clinical studies has shown that renalase level significantly decrease in patients with stroke and chronic kidney diseases, especially those on renal replacement therapy [39] [40] [41] , and is normalized in kidney and heart transplant recipients [42, 43] .
However what parts of kidney and which renal cell renalase is expressed in are not clear yet. In order to obtain high sensitive anti-renalase antibody, this study used DNA immunization which is an established laboratory technique that has been done successfully. Study was conducted using self-made anti-renalase monoclonal antibody, and findings were consistent with the literature, that is, renalase was expressed in both glomeruli and renal tubule [1] . Further in vitro testing showed renalase expressed in mesangial cells, podocytes and tubular epithelial cells, especially only tubular epithelial cells can secrete renalase to the supernatant. Therefore, tubular epithelial cells may be the major cells that can secrete renalase. Such observation has not been reported. These study findings indicate that anti-renalase monoclonal antobodies used in this study could identify natural renalase protein and it is a good experiment tool.
Renalase is widely expressed in glomeruli, tubules, mesangial cells, podocytes and tubular epithelial cells, but its physiological function is not clear yet. Further studies about this novel protein's molecular structure as well as function, carry a great value. At present, a lot more works need to be done on renalase's expression, regulation, function and replacement therapy etc.  Virus contaminations of cell cultures – A biotechnological view In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect)which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. Since the development of viral vaccines, animal cell technology has been used for the production of biologicals for prophylaxis and therapy of humans and animals. As many of these products are injectables, the microbiological safety is of particular importance and is a permanent concern. Whereas microbial contaminations (fungi, yeasts, and bacteria) can be rather easily detected via cultivation methods, the detection of mycoplasma and viruses is more difficult because they are not observable by routine light microscopy. However, fluorescence microscopy, mycoplasma amplification and culture techniques, ELISA and PCR are well developed for determining the extent of mycoplasma contamination. Viral contamination, on the other hand, represents a greater concern because viruses require more complex and frequently need highly sophisticated detection methods (see later). In addition the potential for viruses to cause silent infection of cell culture needs to be addressed, negative results do not always signify that there is no virus contamination. Viral infection can originate from contaminated cell lines, contaminated raw materials, or from a GMP breakdown in the production and purification process (Minor, 1996) leading to a virus-contaminated final product. In addition, although downstream processing is able to eliminate or inactivate certain viruses, not all viruses can be eliminated in such a way because they can be resistant to elimination and/or inactivation steps. Viral infection can be highly pathogenic, and in contrast to microbial infection, there are frequently no effective treatments available, this requires serious consideration to be given to the prevention of contamination. As a result of this everything possible has to be done in order to maintain the entire manufacturing process, and thus the final product, virus-free.
In this review, the problem of viral contaminations in animal cell culture will be presented with special emphasis on animal cell technology used for the production of biologicals for prophylaxis and therapy. In addition, this article will suggest actions which can be taken, in order to assure the absence of viral contaminations. These may include the use of production media devoid of animal derived substances, validation of viral clearance in downstream processing or analytics for detecting adventitious viruses in cell culture and final biological product.
At the end of this article, the implications for the more basic research laboratory will be discussed.
Viral contamination of cultured cells is associated with several problems:
-In contrast to bacterial and fungal contamination, viral contamination cannnot be easily detected, because they cannot be observed by normal light microscopy. It is only when a viral contamination leads to a morphological modification of the cultured cells, such as a cytopathic effect, that a contamination by a virus can be suspected. Silent infection by viruses with no observable morphological modification of the infected cell are clearly of greater concern. -A restricted number of viruses can infect cells and can integrate as a provirus, as in the case of Adeno-Associated virus (AAV), for instance. In this case, the provirus is present, but cannot replicate without the assistance of a helper virus, when one is present both virus species will be produced, together (Mayor and Melnick, 1966; Mayor et al., 1967; Hoggan et al., 1972; Berns et al., 1975) . -Virus contaminated cell cultures represent a risk for the operators, collaborators, patients, as well as for non-contaminated cell cultures. -Cells contaminated with some viruses can show a change to their susceptibility to infection by other viruses. For example some safety testing proto-cols require indicator cells to be used to show the presence of virus, if these are chronically infected by viruses this reduces their susceptibility to other virus species, this in turn, can lead to false negative results, because the virus to be detected can no longer infect the indicator cells. -As a general statement, cell lines contaminated by viruses cannot be treated to become virus free. The result of this is that potentially valuable cell lines will have to be discarded and replaced by new, non-contaminated cells. One of the few exceptions to this rule is the case of Lactate Dehydrogenase virus (LDV). Cultures of cells recovered from a passage in infected animals contain this virus; however, as this virus cannot infect the cells, it will be diluted during subsequent in vitro passaging and thus will be lost (Nicklas et al., 1993; Nakai et al., 2000) .
In order to avoid viral contamination or reduce the incidence it is helpful to know the source of the contamination. Viruses can be introduced by a limited number of different routes and knowing this provides the possibility to avoid infection. The identified routes of infection are: (i) the cells used to produce the production cells are already contaminated by exogenous virus because the cells were already contaminated at source, e.g., the donor animal from which the cells were explanted (see 'Contamination via the cell source'), the pre-culture (in vitro), or the in vivo passage in an animal led to the virus contamination (see 'Passages via virus infected animals'). (ii) Endogenous viruses, such as retroviruses are a particular concern. Several cell lines of biotechnological importance, such as murine hybridomas or Chinese Hamster Ovary (CHO) cells, contain endogenous retroviruses and can produce retroviral particles during production. In the case of murine retroviruses these can be capable of replication, as observed with hybridoma cells, or which are incapable of replication, as in the case of CHO-cells (see 'Cell lines of biotechnological interest-endogenous retroviruses and other cell associated/latent viruses'). (iii) The cell cultures can be contaminated by viruses which were present in the animal derived materials used in the manipulation or for the growth of the cells. These types of materials include serum or trypsin (see 'Use of contaminated raw materials'). Animal derived raw materials are of particular concern as many different animal viruses can potentially be present originating from the use of infected source animals. Non-animal derived raw materials can also be contaminated by viruses due to contact with virus shed from animals or man from production until its eventual use in the medium of which they are a component. (iv) Finally, errors made by the operator can also result in viral contamination of animal cells (see 'Handling errors of the operator').
In the following paragraph, these issues will be described in more detail.
Primary cells derived from explants or continuous cell lines immortalised/transformed by viruses can be contaminated by adventitious viruses. Primary cell cultures derived from animal tissues are seldom used for the production of vaccines for humans but are more frequently applied for veterinary use. When the donor animals are already latently infected with viruses the subsequent in vitro cultures of cells derived from these animals may be infected. If such primary cells are then used for the production of viral vaccines, these vaccines are likely to be contaminated with the adventitious virus. In the period between 1954 and 1961, when primary kidney cells from macaque or rhesus monkeys were used for the production of poliovirus vaccine, this vaccine was frequently contaminated by SV40. The source of this virus was from the kidney cells of infected monkeys (Sweet and Hilleman, 1960; Shah and Nathanson, 1976 ) (for details, see 'Passages via viruses infected animals'). Young immunocompetent rhesus or macaque monkeys can readily be infected with SV40 by the oral, intranasal, and subcutaneous routes, and viremia and viruria occur in these animals (Shah et al., 1969) . The use of such animals as source for kidney cells may lead automatically to a SV40 contaminated primary kidney cell culture because the virus may persist in the kidneys in a latent form (Shah et al., 1969) . Similar observations were made with secondary lamb kidney cells, which are widely used for the production of veterinary vaccines. In the case described, an attenuated Aujeczky's Disease viral vaccine was produced on lamb kidney cells. The master virus stock used for the production had become contaminated with the Border Disease virus due to the contamination of the cell culture used for its production. The vaccination of the sows with this vaccine during the first third of their pregnancy led to the infection of the fetuses which led to a disease similar to classical swine fever (Vannier et al., 1988) . Leiter et al. (1978) reported on the establishment of a mouse epithelial pancreatic cell line which was persistently infected by a polyoma virus. The origin of this infection was not completely clear, but it seemed probable that the mouse which was the source of these cells was also the source of the virus.
Finally, all cell lines which have been established by using a virus transformation (e.g. EBV-transformed B-lymphocytes, such as the Namalva cell line (Klein et al., 1972; Butler, 1991) ) are potentially able to produce the virus used for the transformation and therefore also represent a potential infection risk for the operators, the cell culture lab, and the patients receiving a biological produced with such cell lines.
The in vitro production of viral vaccines began with the demonstration that explanted embryonic tissue could be used for the production of poliovirus (Enders et al., 1949) . Subsequently primary cells, in particular primary monkey kidney cells, were used for the production of this virus. Although the use of such cells was very convenient and the first accepted way to produce a viral vaccine, the source of these cells (the primary monkey kidney cells) was associated with a number of problems particularly with the introduction of a number of viral contaminats. The advantage of the use of primary cells for the vaccine production system was that it was the first in vitro system for the production of viral vaccines, that high titers of viruses could be obtained, and that the system could be scaled up to a reactor stage by using microcarriers . However, it has been recognised that, this system could be prone to contamination originating from the donor animals. The frequency of contaminants may have been due initially because, the animals were caught in the wild with no control of the disease risks with these animals, leading to a high incidence of contaminations by adventitious viruses. Stones (1977) reported that 40-80% of the cultures of kidney cells from Vervet monkeys were positive for adventitious agents. As mentioned, many of the viruses which infect primates are pathogenic for humans, the species barrier is being able to be crossed (Eloit, 1997) .
The notion of the species barrier is sometimes seen as the ultimate rampart that will protect humans against animal viruses, and this can be true for a number of animal viruses which are not able to intitate infection in human cells. However, the species barrier relies on a number of inate features of the immune system which are bypassed in the case of medicinal products (injectables). In addition, the barrier may only be a quantitative issue (quantity of active virus, e.g. the LD 50 of rabies virus in mice is 10 6 times higher by the oral than by the intracerebral route) and is a question of the route of administration (e.g. the mucous membrane is a rather efficient barrier, however, when a virus is inoculated parenterally, the species barrier is no longer valid). Finally, even in the case of an abortive replication cycle, cell transformation via the expression of early viral genes is still possible, eventually leading to a transformed phenotype of non-permissive cells (e.g., mouse cells are transformed by SV40, which in monkey cells leads only to a lytic cycle, or non-permissive cells transformed by bovine polyomavirus) (Eloit, 1997 (Eloit, , 1999 .
Zoonotic infection where humans are infected by animal pathogens is frequently observed in nature.
Only two examples are described here: Minor (1996) reported on 28 cases of infections occuring between 1932 and 1987 in which individuals who had close contacts with macaque monkeys infected with a highly pathogenic virus (Herpes simiae or B virus) fell ill. This virus is latent in these monkeys, but causes fatal disease in man. Twenty cases were fatal and seven had severe sequelae. The second example concerns the contamination by the Marburg virus. Smith et al. (1967) reported that the contamination of monkeys with this filovirus led to an infection of 31 operators/monkey handlers of which 7 died. These monkeys had been imported into Germany for vaccine production by using their kidney cells. More details can be found in Peters et al. (1992) .
A further relevant example from the production of poliovirus vaccine is provided by SV40, a polyomavirus which is an extremely common infection of macaques and rhesus monkeys (Shah and Nathanson, 1976) where it persists in the kidneys in a latent form without causing a cytopathic effect (Sweet and Hilleman, 1960) . This virus grows much more slowly than poliovirus and thus an infection might not be observed during the vaccine production. It is estimated that almost everybody who was vaccinated against poliovirus between 1954 and 1961 also recieved viable SV40 together with the poliovirus vaccine. This is true for the live attenuated as well as for the inactivated poliovaccine, because, in the first case, no inactivation step was applied and, in the second case, the formalin inactivation step was insufficient to inactivate SV40 (Sweet and Hilleman, 1960) . Whereas in the case of the live vaccine, the route by mouth is a poor route for infection with SV40 (Sweet and Hilleman, 1960) , others were injected with inactivated poliovaccine together with infectious SV40. A long-term follow-up study with a small number of individuals, as well as the observation that, in spite of the large number of vaccinees (10-30 millions of 98 millions who were vaccinated, or almost 90% of individuals under 20 yr in 1961 (Shah and Nathanson, 1976) ) which are believed to have received infectious SV40, showed no corresponding increase in related cancers (Shah and Nathanson, 1976) . It should be mentioned, however, that (i) DNA-sequences of SV40 have been detected in association with different human tumors and at an higher incidence in mesotheliomas (Horaud, 1997) , (ii) that SV40 isolated from primary monkey kidney cells by Sweet and Hilleman (1960) induced sarcomas in newborn hamsters (Eddy et al., 1961) , and (iii) that SV40 is oncogenic for laboratory rodents (Magrath, 1991) .
In 1970, Hoggan reported the detection of latent infections of AAV in human and monkey kidney cells. He and his coworkers screened cell lines intended for vaccine production and found that approximately 1% of human embryonic kidney cells and 20% of African green monkey kidney cells produced AAV when infected with helper adenovirus. These results suggested that AAV stayed in an integrated form in the absence of helper virus and that this inapparent infection was a rather frequent natural occurrence. Hoggan et al. (1972) could prove by using Detroit 6 cells that a deliberate infection with AAV in the absence of helper virus led to a latent infection. A superinfection with helper adenovirus led to the induction of AAV replication.
It is evident that steps can be taken to reduce the risk of contamination risk in primary cell cultures. In practice, different actions are possible: use of virus free animals, use of kidney cells from animals which are less susceptible to virus infections, establishment of veterinary examination and quarantine of animals intended for use, and/or use of diploid or continuous cell lines (use of the cell bank concept/seed stock concept); all reduce the frequency of contamination, Use of captive-bred monkeys versus use of imported wild-caught animals. Besides the advantage of independence from the diminishing wild populations and the sparing of these, it is evident that breeding under controlled conditions led to the production of better quality animals especially in respect of virus infections (Van Steenis et al., 1980) . Van Wezel et al. (1978) could show, by performing serological tests on 18 captive-bred cynomolgous monkeys and 40 imported wild caught parent animals that most of the wild caught animals were positive for antibodies against herpes simplex B, parainfluenza 3, or measles virus, whereas two thirds of the captive bred animals were only positive for antibodies against rotaviruses. Twenty out of 36 imported animals were positive for foamy virus 1 antibodies whereas these antibodies were not observed in the animals bred in captivity (Table I) .
During production, control cultures are generally established in parallel to the cultures used for the production of polio vaccine. Van Steenis et al. (1980) compared the frequency of contaminated primary, secondary, tertiary, and quaternary cultures (flasks as well as microcarrier cultures) and stated that all cultures derived from captive-bred animals were free of virusinduced cytopathic effects or hemadsorption, whereas 30 out of 45 kidney cultures from single imported wild caught animals and all of 71 cultures derived from multiple animals showed cytopathology (Table II) .
Reduction of the frequency of contaminations. Statistically, the use of fewer animals (kidneys) will increase the probability to establish virus free cultures. As primary kidney cells from monkeys can be amplified to reactor scale cultures by using microcarriers, the number of animals (kidneys) per batch could be considerably reduced (Van Wezel et al., 1978 , 1980 . The calculations done by Van Wezel et al. (1978) in- dicated very clearly that this approach could reduce the number of animals necessary for production purposes of Polio virus vaccine by 5-7 fold. Shah and Nathanson (1976) calculated the probability to obtain kidney cultures free from SV40 with respect to the number of animals used per production batch. By using one animal the frequency of SV40 contamination was about 20%. However, the frequency increased to 70% when the kidneys of two to three animals were pooled and was 100% when the kidneys of more than 10 animals were pooled, indicating very clearly that the increase of the number of animals per batch increased considerably the probability of the presence of virus contamination.
Use of kidney cells from animals, which are less susceptible for virus infections. One way to reduce the contamination by human pathogenic virus is to change the species of the animal as donor of the primary cells. Shah and Nathanson (1976) proposed that new world spider monkeys should be used instead of the rhesus monkeys or macaques because SV40 does not readily multiply in cells from spider monkeys. On the other hand, macaque monkeys can be infected with Herpes simiae or B viruses, which are highly pathogenic for humans. The replacement of the macaques as donors by African Green Monkeys, which are not susceptible to infection by herpes simiae virus, would be the best precaution in this case (Minor, 1996) .
Other means. In addition to the above mentioned measures, there are some other measures which can be performed in order to increase biological safety. The animals intended for use should be examined for their health status and must pass through a quarantine regime. For safety reasons, there has to be routine use of in vitro and in vivo culture systems for detection of viruses in any case.
However, the best means to increase the biological safety of the produced viral vaccines is the use of diploid or continuous cell lines, because it can be determined that such cells are free of animal derived viruses: This can be achieved by establishing master (seed stock) and working (distribution and user stocks) cell banks which have been rigorously tested and validated for the absence of microbial as well as viral contaminants (see chapter by Freshney and the section on 'Testing-virus screening in cell banks' of this article). By this means producers of viral vaccines and all other biotech products can make use of a homogeneous pool of characterized cells from which each production run will be started, in the knowledge that they are free of any contaminant (because they have been tested) (Berthold et al., 1996) . In addition, by using the seed stock/working stock concept for the viral inoculum the manufacturer of viral vaccines can use a tested and validated stock of virus inocula of which one aliquot is used for the infection of each production run.
Many contaminating murine viruses, such as Minute virus of mice (MVM), K virus, Mouse Encephalomyelitis virus, and Mouse Adenovirus have been isolated from contaminated virus pools. Viruses, such as A similar study was published by Nicklas et al. (1993) , however, revealing a lower rate of contamina- (Nicklas et al., 1993) Origin tions probably due to the improvement of the microbiological quality of the laboratory rodents. Of 297 tumors examinated, 75 (25.3%) were contaminated. Considerable differences were observed for in vivo (36.6% positive of 186 tumors) and in vitro (6.3% positive of 111 tumors) passaged transplantable tumors.
Mouse transplantable tumors showed the highest frequency of contamination, whereas tumors of other species showed much lower frequencies (Table IV) . Contamination with reovirus 3 and MVM was found in 4 (3.7%) of 109 cell lines tested, and in 2 of 60 monoclonal antibody bulk preparations. With respect to LCMV, Bhatt et al. (1986) reported its isolation from transplantable tumor cell lines. The testing of tumor cell lines revealed that 16 out of 55 in vivo tumor samples and one out of eight in vitro samples were positive. A similar situation was found in a New Jersey research institute, where human cell lines and tumor cell lines were passaged via nude mice for the development of monoclonal immunodiagnostics and immunotherapeutic agents. This LCMV contamination led to the outbreak of laboratory-acquired human LCMV infection (Mahy et al., 1991) . LCMV contaminated hamster tumor cell lines have also been responsible for an outbreak of infections occurred in medical center personal at the University of Rochester (Hay, 1991) .
Mouse hybridomas are of particular concern because, first, these cells have been created by fusion of mouse spleenocytes with mouse myeloma cells, second, many hybridoma cells have been cultivated in animals, and third they are used for the produc-tion of injectables. This signifies that mouse viruses are potential contaminants of these cell lines and their products. These viral contaminants can be divided into two groups; group 1 contains viruses which are also known to cause human diseases or to be able to infect human cells, while group 2 contains other mouse viruses (Table V) (Minor, 1996) . Although Ectromelia virus is listed in group 2, cultures infected with this virus are only processed in the special P4 unit available at the NIH (Hay, 1991) . Ectromelia, a member of the orthopoxviurs group, is a natural pathogen in mice, and is able to replicate in all mouse lymphoma lines, in some hybridoma cell lines, and in BS-C-1 cells (Buller et al., 1987) . Consequently, the ATCC has screened its collection of murine cell lines but no characteristic cytopathic effects have been observed (Hay, 1991) . Moore (1992) listed the mouse viruses which had been detected in production cell banks of hybridomas: LMCV, MVM, Sendai, LDH, and epizootic diarrhea virus of infant mice. The present view is that hybridoma cell lines should be tested for the viruses indicated in Table V , and only those should be used for biotechnological applications which are free of these viruses. The only acceptable viral particles in bulk supernatants from hybridoma cell lines are those of endogenous origin.
The passage of human tumor cells in nude mice can also lead to the infection of these cells by murine endogenous retroviruses. Crawford et al. (1979) reported the contamination of a human nasopharyngeal carcinoma with murine endogenous xenotropic retroviruses after a passage in a nude mouse. As these Table V . Viruses potentially infecting rodent cells (Minor, 1996) Group contaminations are of animal origin, it is necessary to verify the contamination status of laboratory animals. Minor (1996) indicated that all mouse strains, which were received at NIBSC from breeders of laboratory animals, were tested positive for MVM and Sendai virus.
Finally working with rat cell lines, which have been passaged via rats, is also a concern because they can be contaminated by different rat viruses, in general, and by Hantaan virus, in particular. Hantaan virus has been isolated in cell culture from rat immunocytomas. Transplantation into LOU/M/Wsl rats and storage of passaged immunocytomas at -70 • C over a period of 8-10 yr did not eliminate the virus. Lloyd and Jones (1986) also showed that the passage of rat immunocytomas in infected LOU/Wsl rats led to a contamination of these cell lines.
Hantaan virus is a silent pathogen in rats and mice, but causes disease in humans (hemorrhagic fever with renal syndrome) after infection (Leduc et al., 1985) . Laboratory animal care workers working with infected animals as well as persons handling contaminated rat immunocytoma cell lines have been infected with this virus (Leduc et al., 1985; Lloyd and Jones, 1986; Mahy et al., 1991) . For this reason, all ATCC certified cell lines of rat origin have been screened for the presence of hantavirus. The following rat cell lines and rat hybridomas appeared free of hantavirus infection: CCL 38, 43, 45, 47, 82, 82.1, 97, 107, 144, 149, 165, 192, 216; CRL 1213 CRL , 1278 CRL , 1439 CRL , 1442 CRL , 1444 CRL , 1446 CRL , 1458 CRL , 1468 CRL , 1476 CRL , 1492 CRL , 1548 CRL , 1569 CRL , 1570 CRL , 1571 CRL , 1578 CRL , 1589 CRL , 1592 CRL , 1600 CRL , 1601 CRL , 1602 CRL , 1603 CRL , 1604 CRL , 1607 CRL , 1631 CRL , 1655 CRL , 1662 CRL , 1674 HB 58, 88, 90, 92, 100, 132; and TIB 104, 105, 106, 107, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 145, 146, 164, 166, 168, 175, 183, 184, 207, 210, 211, 213 (Leduc et al., 1985) . It should also be mentioned that cell lines of other species can also be contaminated by hantavirus. This virus had been detected in a human lung carcinoma cell line (A549) (French et al., 1981) and in Vero E6 cells (McCormick et al., 1982) .
Precautions: The precautions which should be taken to reduce the risk of contamination of cells during passage in animals: (i) Cells which have been once passaged in animals have to be screened for the absence of microbial and viral contaminants normally found in the animal species; (ii) Animal passages should be avoided as much as possible, the contamination risk by mouse pathogens is reduced 6-fold when these cells are cultivated in vitro (Nicklas et al., 1993) ; and (iii) only tested laboratory-bred animals (virus-defined, specific pathogen free), and no wild caught animals, should be used for animal passages of mammalian cells.
The most important cell lines of biotechnological interest, mouse hybridomas and CHO cells, are known to contain endogenous retroviruses (ERVs) and are known to produce retroviral particles. ERVs exist in 2 forms, a viral form and a proviral form. Retroviral proviruses are transmitted through germ cells and are present in the genomes of almost all vertebrates thus far studied. Humans possess many ERV genomes related to mammalian C type retroviruses and to A, B and D types of this family of viruses. The presence of ERV-like sequences in a cell line to be used in the production of a biological is a potential cause for concern because of the possibility that the endogenous retrovirus may be activated and result in infectious virus being present. particles (IAP) as well as budding C-type particles (Table VI) . With respect to IAPs, Anderson et al. (1990) demonstrated, that the CHO cells' genome contained approximately 300 copies of viral sequence per haploid genome. No intact open reading frame for gag, pol, or env could be detected in clones of either family (Anderson et al., 1990) . In addition, no infectivity has been associated with A-type retroviruses from CHO cells (Kuff and Lueders, 1988; Adamson, 1998) . CHO cells also produce C-type particles at concentrations of <10 3 -10 6 ml −1 . The presence of virus particles was correlated with detectable reversed transcriptase activity (a retrovirus specific enzyme) (Dinowitz et al., 1992) (Table VII) . As for the IAPs, each CHO cell contains between 100 and 300 copies/genome (Dinowitz et al., 1992) . No evidence of infectivity could be detected (Dinowitz et al., 1992; Adamson, 1998) , this may be due to the lack of functional open reading frames, rendering the retroviruses incapable of encoding an intact endonuclease (Dinowitz et al., 1992) .
Hybridoma and murine plasmacytoma cell lines. As for CHO cells, plasmacytoma and hybridoma cells produce IAPs and C-type particles (Table VI) (Spriggs and Krueger, 1979; Weiss, 1982) . As for CHO IAPs, hybridoma IAPs stay inside the cells and are noninfectious because they are devoid of intact open reading frames. However, unlike CHO C-type retroviral particles, hybridoma C-type particles have the ability to replicate in several different cell lines including a small number of human cell lines (lung fibroblasts, RD cells (Weiss, 1982; Levy, 1983; Adamson, 1998) ).
By using electron microscopy, the retroviruses present in cell culture supernatant have been as high as 10 9 particles per ml (Moore, 1992) . About one in 10 4 -10 6 particles is infectious. In this context, Froud et al. (1997) presented data from Lonza Biologics (former Celltech Biologics), indicating that all hybridoma cell lines processed by the company produced retroviral particles. Five to six percent of the cells produced infectious mouse ecotropic retrovirus, whereas almost all (about 85%) mouse cell lines tested produced low levels of infectious mouse xenotropic retrovirus (X-MLV) when the cell banks were tested.
All mouse cell lines commonly used for antibody or recombinant protein production are derived from the MOPC21 tumor of a female BALB/c mouse. This indicates that all cell lines, clones, and subclones which are derived from the MOPC21 tumor (the plasmacytomas P3X63.Ag8.653, NS1, NS0, and the hybridoma SP2/0.Ag14) produce X-MLV (Froud et al., 1997) .
Infectious retrovirus has also been found in mouse/human hybridomas. In co-cultivation studies it could be established that these retroviruses were of the X-MLV type, and no human retroviruses have been detected in any mouse/human hybridoma or genetically engineered human cell line (Moore, 1992) . Although not detected so far, the possibility of molecular recombinations leading to pseudotyped particles is a concern.
Other cell associated viruses and safety considerations Mouse/human hybridomas can be established by fusing human EBV transformed lymphoblastoid cells with mouse plasmacytomas. As these cells were EBV transformed, the hybridoma cells are potential EBV producers. Cells transformed by EBV are potential EBV producers (e.g. Namalva), and the downstream processing protocol as well as the safety testing have to take this fact into account (Cartwright, 1994; Robertson, 1996) (see Section on 'Process validation -downstream -processing -viral clearance').
BHK cells are also transformed rodent cells and it was possible to induce production of R-type particles in these cells (Moore, 1992) .
Table VIII presents a short résumé on cell lines of biotechnological interest which contain endogenous viruses or latent proviruses and are therefore producers or potential producers of these viruses. Whereas plasmacytomas, hybridomas, CHO and BHK cells contain endogenous viruses which integrated into the genome Table VII . Characteristics of C-type particles isolated from CHO cells (Dinowitz et al., 1992) Reversed transcriptase activity Detected in highly concentrated (4000-7000-fold) culture fluids from some cell lines; Mn 2+ preferring.
Similar to other C-type particles in sucrose density gradients (about 1.13-1.16 g ml −1 ).
Nucleotide homology to other C-type Endnuclease region contains significant homology particles with mammalian C-type retroviruses. No intact open reading frames detected in cloned cDNA sequences. About 100-300 copies per CHO genome.
Proteins P30 core protein is related to murine and other C-type retroviruses.
No infectivity detected by direct inoculation of reverse transcriptase-containing concentrates or cocultivation of CHO cells with a battery of cell lines. of the respective species after an infection millions of years ago, the presence of EBV (due to EBV transformation of human B-lymphocytes) or sequences of parvoviruses (e.g. AAV (latent infection leading to integration) due to natural contamination of human embryonic kidney cells and African Green Monkey kidney cells (Hoggan, 1970) , or Procine Parvovirus (latent infection) (Fikrig and Tattersall, 1992 ) (see Section on 'Trypsin')) are due to recent events and can eventually be avoided by using tested cell lines (absence of the respective sequences).
It is evident that all biological products derived from cell lines containing endogenous retroviruses or other latent viruses have to be characterized for the presence of virus. In addition, in order to increase their biological safety, first, all biotech products derived from such cell cultures have to be rigorously tested for the absence of retroviral activities/viruses or latent viruses, and second, the purification protocols for biotech products derived from these cells have to be validated for their capacity to eliminate or inactivate retroviruses/latent viruses.
An important potential source of viral contaminations are raw materials used for the preparation of culture media in animal cell technology. Although any component of the culture medium can theoretically by contaminated by viruses, the materials with the highest probability of viral contamination are those derived from animal origin. The classical animal cell culture technology currently makes use of several raw materials of animal or human origin. This is true for the production of viral vaccines for human or veterinary use or many other biological therapeutic. Animal sera as medium additive is the most widespread animal derived material used today. Fetal, new born or adult bovine sera and in some cases also horse sera are used. Trypsin, mainly from pig pancreas, is a very important detachment agent for all adherent cells. Other animal or human derived substances are often used in association with the replacement of serum by serumfree media, but which can eventually also be found as excipients: human albumin, protein hydrolysates (casein, gelatin, etc.), and human transferrin. Other substances of animal origin are some amino acids, which are derived from complete hydrolysis of proteins. However, because of the chemical conditions used in their production, there is less risk than that of materials prepared without this process. Although the use of entirely chemically defined media devoid of any animal derived substance reduces the risk of viral contaminations, it is important to mention that this risk will never be zero.
Several zoonotic viruses are known and can be transmitted from animal sources (Eloit, 1999) . Because of the recognised risks from these agents careful sourcing and screening can easily prevent the risk of their transmission. However, it should be noted that other viruses not known to be harmful for humans might be infectious and might lead to severe disease.
Contamination problems associated with the use of serum Bovine serum might be contaminated by many different bovine viruses. Although theoretically qualitycontrolled serum should have been tested for all possible viruses, this is not possible for economic reasons and may not even be necessary. It is evident that each serum batch has to be tested for those viruses which are ubiquitous and known risks, such as the Bovine Viral Diarrhoea Virus (BVDV). However, there is also the question of the geographical origin of the serum which may indicate the need for additional virus tests. It is only necessary to test for those viruses, which are present in the geographical region from which the serum is coming. Table IX presents bovine viruses for which tests have to be performed depending on the geographical origin of the serum. The testing Table IX . Specific tests applicable to the screening of calf serum and porcine trypsin used for the production of medicinal products for human use (Eloit, 1999) Calf serum Trypsin (Table X) . In the following, the most important bovine viruses are presented in more detail.
BVDV. Bovine viral diarrhoea/mucosal disease is one of the most important viral diseases of cattle. The natural prevalence is very high with approximately 80% of cattle being seropositive and 1-2% of these animals being persistently viremic animals due to immune tolerance which occurs after infection of the fetus (Kniazeff, 1973) . The infection rate has been increased by the uncontrolled use of live vaccines and by heterologous vaccines fortituously contaminated with BVDV virus (Kreeft et al., 1990) . Together with Hog Cholera and Border Disease virus of sheep, BVDV constitutes the pestivirus group. Bolin et al. (1991) have studied the frequency of contamination of fetal calf serum with BVDV and reported that 332 of 1608 raw fetal serum samples (20.6%) derived from the abbatoirs were positive for this virus, 224 of these samples (13.9%) contained antibodies against BVDV and 3.1% of the samples (50/1608) were positive for both, BVDV and antibodies against BVDV (Table XI ). They have also tested commercial fetal calf serum for cell culture and detec-ted BVDV in 47% of the samples (90/190): 88 contained non-cytolytic and only two contained cytolytic BVDV isolates. Two percent of these samples (3/190) were positive for Infectious Bovine Rhinothracheitis virus isolates. Wessman and Levings (1999) have reported similar results, indicating that 32 to 68% of fetal bovine serum samples (pooled one liter lots from two bovine fetuses) were rejected for presence of BVDV or antibodies against BVDV, in the period of 1990-1997.
These studies indicate the importance of the problem of BVDV contamination in fetal calf serum and several conclusions can be made: first, veterinary diagnostic laboratories should avoid the use of fetal calf serum in diagnostic procedures for pestivirus infections, second, there is a significant risk that adventitious BVDV from fetal calf serum may lead to contaminations in the veterinary biologicals industry (see Section on 'Other substances of animal/human origin and non-animal derived substances'), and third, the results indicate a very high rate of fetal infection with BVDV, possibly reflecting a failure in hygiene issues or in control measures (Bolin et al., 1991) .
Finally the most important question concerns the contamination status of cell lines in culture collections, because many cell lines have existed for many years in these collections and might have been contaminated in periods when no or fewer tests were performed for proving the absence of BVDV. The question most relevant to the use of fetal calf serum for animal cell culture and animal cell culture technology is: which cells are/were contaminated by BVDV and are the cells from different species as easily contaminated as bovine cells or is there any species barrier. In this context, Bolin et al. (1994) performed a survey of cell lines from the American Type Culture Collection and observed the following contamination status: Using immunocytochemical procedures and PCR amplification, 13 of 41 ATCC cell lines were tested BVDV positive: these cell lines were derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat. Attemps to experimentally infect 14 different cell lines from animals, which were not found positive in the survey of the ATCC cell lines, led to the result that all swine cell lines (3/3: MPK, ESK-4, and one other) and most rabbit (3 out of 4: Sf 1 Ep, R9AB, RAB-9) and cat cell lines (3 out of 4: CRFK, AK-D, NCE-F161) became infected with BVDV, whereas hamster (BHK-21), human (IMR-90), dog (MDCK), rabbit (SIRC) and cat (Fc3Tg) cells were refractory to BVDV infections. The results concerning monkey cells (LLC MK2) were variable -no clear answer was obtained. Wessman and Levings (1999) reported that the following cell lines could be infected with BVDV: bovine cells (EBK, MDBK, BoTur, primary and continuous kidney cell lines, lung, trachea, and aortic endothe-lium), sheep choroid plexus and lamb kidney cells, monkey kidney cells (Vero and others), mosquito cells, porcine cells (PK-15 and others, testis, minipig kidney cells), goat cells (kidney and oesophagus), cat cells (lung, CRFK, tongue, feline embryo), rabbit kidney cells (RK-13), and others. Harasawa and Mizusawa (1995) published a study on the pestivirus contamination of cell stocks of the Japanese Cancer Research Resources Bank. Fifteen out of 20 cell lines (75%) were positive using RT-PCR. Whereas bovine cell lines (HH, MDBK, CPA, CPAE, EBTr, Ch1Es) were contaminated with genotypes I, II, and III, cell lines of dog, cat, and primate origin were contaminated with genotype II of BVDV (HeLa, MOLT-4, U937, WI-38, WiDr, CV-1, Vero, MDCK, CRFK). Roehe and Edwards (1994) assessed the ability of 11 pestiviruses from pig, eight from cattle, and five from sheep to replicate in cells of porcine (PK-15), bovine (BT) and ovine (SCP) origin. The pattern of replication in different cell types varied between different isolates of the same virus species.
These results indicate that the virus suceptibilities of a species are not completely predictible and that many cells derived from other species than cattle can be infected by BVDV.
of importance. Viral infections lead to respiratory syndrome in cattle (shipping fever) and abortion in bovines. In 68% of calves significant antibody levels against PI-3 have been detected. The virus can be easily replicated in primary (bovine kidney cells) and established bovine cells (EBTr, MDBK) (Kniazeff, 1973) .
tracheitis -a very common infection of cattle -, abortion, pustular vulvovaginitis, meningoencephalitis of calves, and conjunctivitis. The incidence of infection is high with viremia a common feature. It replicates in leukocytes and can stay there latently. It is never found free in the bloodstream. It replicates in bovine cells, but also in cell cultures from: elk, mule deer, sheep, L cells, chick embryo cells, pig, human (amnion (probably also a clon of HeLa cells (see chapter by Masters)), HeLa), and primary monkey kidney cells (Kniazeff, 1973) .
BPyV. Bovine polyomavirus belongs to the family of the polyomaviruses. These viruses have been isolated by several laboratories (e.g., from monkey kidney and other cells cultured in the presence of bovine serum. By infecting permissive cells, this virus leads to a cytopathic effect, whereas non-permissive cells are transformed and they acquire certain properties of a malignant cell.
As for the other bovine derived viruses, BPyV is an ubiquitous virus and about 40% of the calves are seropositive in the first month after birth. In the subsequent months this seropositivity decreases to about 11% at an age of one year, however, the older the animals become seropositive again and the final percentage of seropositivity is beyond 80%. It was also shown that bovine fetuses were infected in utero, leading to the presence of antibodies against this virus in fetal bovine serum batches (in about 12% of the tested batches). Despite this rather high incidence of infection in fetuses, no known disease is associated with this virus, neither for cattle nor for humans. Using PCR, 14/20 European serum batches (70%) contained BPyV DNA sequences . A similar frequency of contamination was observed in North American serum batches (Kappler et al., 1996; Van der Noordaa et al., 1999) . There was no correlation between the PCR results and the presence/absence of antibodies against BPyV, however, could show that all PCR-positive sera contained infectious BPyV. IBR or BHV-1. BPyV can infect calf kidney cells, and monkey kidney cells (Vero, BSC-1, CV-1, RITA) but also human embryonic kidney cell cultures (Waldeck and Sauer, 1977; Wognum et al., 1984) . The virus does not seem to replicate in mouse 3T3 cells, nor in the human embryonic lung cell WI-38 (Waldeck and Sauer, 1977) . The BPy virus is known to lead to cell transformation and tumorigenesis, which is induced by the expression of the large-T antigen .
All adherently growing cells have to be detached for passaging from time to time. To facilitate this the enzyme trypsin is frequently used. As for serum, trypsin is an animal derived product, generally from porcine pancreas. Therefore, similar safety criteria as for serum have to be applied for trypsin. A special concern is Porcine Parvovirus (PPV). Latent parvovirus contamination has been found in many permanent human cell lines. The first contamination was observed by Hallauer and Kronauer (1960) when they observed that some control cultures (non-infected with Yellow Fever virus) yielded a different hemagglutinating agent, unrelated to Yellow Fever virus, when subjected to their isotonic, high pH glycine extraction buffer (= physiological stress). Further studies identified this infectious agent as a member of the parvovirus group. Following this, Hallauer et al. (1971) isolated 38 parvoviruses in 43 permanent human cell lines obtained from 19 laboratories. Some cell lines showed signs of degenerescence when arriving into the laboratory of Hallauer, others appeared completely normal. Three different serotypes of parvovirus could be identified, the origin of them is not really known. However, the recovery of the same serotypes from different laboratories suggests a common source, such as a reagent used in cell cultivation. One of the serotypes had been identified as PPV, indicating that the use of contaminated trypsin lots was the probable source of contamination (Fikrig and Tattersall, 1992) . The definite proof that porcine trypsin was the source for cell culture contaminations by PPV was apported by Croghan et al. (1973) , because they detected the same serotype in commercial trypsin lots.
It could be shown that various cell lines from different species can be infected by PPV, such as human continuous cell lines (Lu 106, HeLa, and the following HeLa clones (see chapter by Masters): KB, Amnion, and Hep-2) or swine kidney cells (PK 15) (Hallauer et al., 1971) .
Recently, a new group of viruses, the circoviridae, was described. The circoviridae are very small viruses (non-enveloped, circular single stranded DNA, diameter of 17 nm) and are very resistant against most of the inactivation methods currently used. This group of viruses was found in Japanese patients suffering from non A to G hepatitis, described as TT virus (Nishizawa et al. 1997) , and also found in chickens, where it is described as Chicken Anemia virus (Yuasa et al., 1979) . This virus seems to be ubiquitous in humans because DNA of the TT virus was identified in plasma of 76% of French blood donors (Biagini et al., 2000) . It is evident that such a virus might be a problem when human derived proteins are used, because this it is very resistant against most inactivation methods. Tischer et al. (1982) described a circovirus in pigs, and it has been reported that the swine cell line PK-15 was chronically contaminated by this type of virus. A serological study showed that 20 out of 22 randomly collected pig sera contained specific antibodies against the virus, whereas no specific antibodies could be detected in sera from rabbits, mice, calves, and man, including the laboratory staff working with this virus (Tischer et al., 1982) . The virus exists as 2 subtypes, type 2 porcine circovirus replicates actively in porcine fetuses (Sanchez et al., 2001) and is associated with abortions, reproductive failure and postweaning multisystemic wasting disease in swine (O'Connor et al., 2001; Ellis et al., 2001) , however, it seems that this virus is very porcine specific .
Although there is no record of Porcine Circovirus being able to infect man, precautions should be taken when porcine derived substances, such as trypsin, are used in animal cell technology. As this type of virus is very resistant it is preferable to avoid contaminations of the cell culture, and thus of the biotech product, than to try to separate the product from the virus (due to stability reasons autoclaving the final product is not possible).
For the moment, these viruses are most frequently detected by PCR, presently there is no good culture method available.
The highest risk is associated with the use of human or animal derived substances. With respect to material of human origin, it is evident that there exists an important risk of viral transmission, because of the absence of any species barrier to infection. Human sourced raw materials should be checked for the absence of viruses, like Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human Immunodeficiency virus (HIV), and EBV, or CMV (Committee for Proprietary Medicinal Products: Ad Hoc Working Party on Biotechnology/Pharmacy and Working Party on Safety Medicines, 1992). The testing for these viruses is most frequently accomplished by PCR assay. The extent of the use of human derived materials is limited with only human transferrin and human serum albumin still in use. The development of serum-and protein-free media leads more and more to media devoid of any animal/human derived substance.
Other substances of animal origin, such as certain amino acids, lipids, or other protein additives (peptones) are also potentially contaminated by viruses and have to be rigorously tested for virus absence or should be replaced by non-animal derived compounds (Merten, 1999; Jayme, 1999) .
Finally it should be mentioned that adventitious viruses can also be introduced via contaminated nonanimal derived substances (medium components), as observed by the contamination of a manufactured material by viruses of extraneous origin. The most widely reported case was that occuring with the manufacturer Genentech (Garnick, 1996) (see next section).
The scientific literature gives few descriptions of viral contamination in biotechnological productions of recombinant proteins or viral vaccines (Table X) . Nine cases have been published indicating that viral contamination can be acquired via the serum source (in 7 out of 9 cases) or the culture medium (in 1 out of 9 cases). Biotechnological products were contaminated by different viruses, by those leading to a cytopathic effect (epizootic haemorrhagic disease virus) but in most cases by those which have no effect on the morphology of the contaminated cells (Minute Virus of Mice (MVM), BVDV, BPyV, bluetongue virus, reovirus). In cases where the viral infection leads to observable morphological effects, the contamination is easily detectable. However, those viruses which do not lead to a modification of the morphology and/or growth characteristics of the cells require methods such as RT-PCR, PCR, bulk in vitro testing. Or alternatively the onset of diseases in animals administered with the test material. These virus positive products were not delivered in the case of the products destined for human use, because the virus detection was performed before product release (Garnick, 1996; Rabenau et al., 1993; Harasawa and Sasaki, 1995) , however, with respect to the live attenuated veterinary vaccines, several incidents of disease in vaccinated animals were reported (Kreeft et al., 1990; Wilbur et al., 1994; Falcone et al., 2000) .
It should be mentioned here, that the case of MVM contamination of CHO cultures for the production of TPA, did not lead to a cytopathic effect and could not be detected without specific virus tests (Garnick, 1996) . In contrast, Nettleton and Rweyemamu (1980) and Hughes (1996) reported on a MVM contamination of BHK-21 cells for veterinary vaccine production, which was detected via persistent cell deaths of these cells. It could be shown that the serum batch was the origin of this contamination. It is evident that rigourous testing of raw materials is necessary because of the following: -In the case of the production of recombinant proteins for human use, virus contaminations can only be eliminated with difficulty from the bulk product. Should a virus present in the bulk product be able to be eliminated during downstream processing, the FDA will generally not accept the final product after purification (Burstyn, 1996) . -The problem associated with live attenuated virus vaccines is that these products cannot be treated for virus inactivation because the active ingredient would be inactivated at the same time. Such products require a more extended quality control testing for the raw materials. For instance, new serum batches should be tested for a more extensive range of bovine viruses and in particular for those viruses, which are of relevance for the final application of the product. For instance, in the case of the contamination of a canine vaccine by bluetongue virus which lead to the death of some bitches (Table X) (Wilbur et al., 1994) , the application of a specific test would have avoided this incidence. -In conclusion, the best solution for reducing the risks of viral contaminations is the use of raw materials which are not of animal or human origin, but of plant or microbial origin or produced by chemical synthesis. It is evident that such an approach will not eliminate the risk of viral contaminations, but represents an important step towards risk minimization.
Sourcing. This approach is clearly limited to agents for which there is a well-documented specific geographical distribution. Such examples are quite rare and only the case of TSE agents will be mentioned here, where sourcing of bovine serum from diseasefree countries (a geographical choice) is possible. This approach, however, can also be used for viruses such as bluetongue virus.
Screening. In principle, all raw materials, of animal origine or produced by chemical synthesis, have to be rigourously tested and should fulfill certain quality attributes, when used for the production of biological injectables (GMP-guidelines). The characteristics which are frequently required to be described in raw materials are identity (testing, tracability, labels), purity (testing, inspection, vendor certificate of analysis), suitability for intended use (process validation, vendor audit programme, performance testing if needed), tracability (vendor audit programme, vendor certification, certificates of analysis, contractual obligations under change control, labelling, control). For more details, consult Lubiniecki and Shadle (1997) .
Although it would be desirable that all raw materials should be tested for the absence of adventitious agents, in order to be sure that they are safe, this is often impracticable. Therefore there are two approaches: first, tests are employed which are based on the detection of general characteristics of viruses (cytopathic effects, haemadsorption) and, second, specific tests using imunological and/or PCR methods are employed for detecting virus antigens or specific viral sequences after amplification in permissive cells (see Table IX for testing of bovine serum and porcine trypsin, see Section on 'Testing -virus screening in cell banks').
However, such a screening gives only a limited guarantee of safety because of the following:
-Complete testing can be impracticable on a batch to batch basis. In most cases, screening will only be done for certain viruses, e.g. for BVDV, IBR, and PI-3 in the case of bovine serum, for porcine parvovirus in the case of trypsin, because these are the most probable viral contaminants. However, depending on the geographical origin of the serum or the trypsin, additional tests for viruses which are present in that geographical area from where the raw material is coming may have to be performed (Table IX) . If a raw material is of animal origin, screening tests should also include the use of cells of the species of origin. With respect to the use of serum for the GMP production of biologicals for human use, the EMEA proposes in a draft that more viral screening tests should be performed for proving the absence of BVDV, IBR, PI-3, Bovine adenovirus, Bovine Parvovirus, Bovine Respiratory Syncytial virus, Bovine Re- virus tests, otherwise they pass undetected. -Due to sampling size, low titers of some adventitious viruses can remain undetected but may be amplified during the manufactureing process. In this context it should be remarked that screening methods are not always sufficient because contaminated serum batches which had passed as uncontaminated have been detected by and Yanagi et al., (1996) ; contamination of serum samples with BPyV and BVDV, respectively.
Recommendations for fetal bovine serum quality (Hansen and Foster, 1997) . Although the best choice would be a serum-free cell culture process which is devoid of any animal or human derived substances, this is not always possible. Where serum supplementation is necessary, the serum should be of high quality. In addition to the absence of viruses the hemoglobin level should be <10 mg, the endotoxin level should be below 10 Eµ ml −1 , and there should be a reliable tracability to countries without BSE nor foot and mouth disease. A serial filtration using 40 nm poresize filters should be used and the serum should be γ irradiated with >25 kGy using validated procedures. For veterinary use, the radiation dose should be 35 kGy.
More on the quality control of bovine serum used for the production of viral vaccines for human use can be found in Mareschal (1999) .
Other precautions: The screening of animal derived raw materials for the presence of adventitious viruses is of utmost importance, however, as already mentioned, screening has its limits, because it is impractical to screen for all theoretical viruses, and other new viruses might emerge for which no tests are available. Because of this supplementary precautions have to be undertaken for reducing the risk of viral contamination, by inactivating or eliminating at least viruses of families, which are susceptible for inactivation and/or elimination. With respect to the treatment of fetal bovine serum for animal cell technology, γ irradition, and UVC irradiation are used. Heat treatment as well as treatment with peracetic acid are possible, however, they are not really used.
Some treatments are presented in the following: (a) Nanofiltration (Troccoli et al., 1998; Aranha-Creado et al., 1997; Graf et al., 1999) . If the size difference (molecular weight) between the raw material and the virus is large enough, viruses can be removed by nanofiltration, which makes use of pore cut offs of 50, 35 nm, and even 15 nm. Filters with a pore cut offs of 50 nm can be used to eliminate viruses which have a diameter larger than 50 nm, such as retroviruses or influenza A virus (80-120 nm) (typical log titer reduction in a validation study: >6.3). However, such filters only partly reduce the quantity of poliovirus (28-30 nm), and viruses of a size of 25 nm (model particle: Bacteriophage PP7) pass without any significant retention (log titer reduction: <1-8.5, depending on the buffer system used) (Graf et al., 1999) . It should be mentioned that the composition of the medium/buffer system in which the virus is placed, has an effect on the log titer reduction of viruses which are below the molecular weight cut-off of the membrane used. An example of typical virus retention data for a commercial hydrophilic PVDF membrane filter is shown in Table XII .
Improved virus retention can be obtained by using pore cut offs of 35 nm. Using a 35 nm membrane in line with two prefilters (one 75 nm filter followed by a first 35 nm filter) led to log titer reductions of >4.3 for Hepatitis A Virus (HAV) and Encephalomyocarditis Virus, although both viruses are smaller (28-30 nm) than the cut-off of the filters (Troccoli et al., 1998) . All viruses larger than 35 nm were completely removed.
Finally the use of a 35 nm membrane filter followed by a 15 nm pore size membrane filter assures a log reduction factor of >6.7 and >5.8 for HAV and BVDV, respectively, signifying that in principle all potential adventitious viruses (also the small ones) can be removed from the product (Johnston et al., 2000) .
Nanofiltration is mainly used as a final step in the production of biologicals purified from human plasma and of recombinant DNA-derived products. Fetal calf serum is often filtered three times using a cut-off of 100 nm (removal of, for instance, IBR and PI-3, but not of BVDV). Only some companies provide fetal bovine serum which is serially filtered through 40 nm pore size filters (Hanson and Foster, 1997) , because this cut-off allows also the elimination of, for instance, BVDV, which has a size of 45-55 nm (see Table XIII ).
(b) γ Irradition (Plavsic et al., 1999) . γ irradition (using a 60 Co source) is a very efficient and straightforward means for inactivating many different virus types. As animal derived substances such as serum can be contaminated by adventitious viruses, γ irradition is, after routine quality control for virus detection, the best method to increase the safety of using serum in the production of animal cell culture derived biologicals. Using PETG (polyethylene terephthalate G copolymer) bottles with 500 ml of frozen serum (-40 • C) inoculated with model virus (Hanson and Foster, 1997) , validation experiments have been performed to determine the optimal radiation dose to inactivate relevant bovine and porcine viruses, and in parallel to assure that the irradiated serum has still a sufficient growth supporting ability. Plavsic et al. (1999) could show that at a radiation dose of 25 kGy, all tested viruses (Bovine Reovirus, Porcine Parvovirus, Canine Adenovirus, IBR, and BVDV) showed a significant decline in titer. An exposure of 35 kGy led to titers of all viruses tested falling below the detection level (≤0.5 TCID 50 ml −1 ). Even very resistant viruses, such as the Porcine Parvovirus, could be reduced to below the detection level. For all viruses tested the log reduction factor was at least 6.78 (Table XIV) . Willkommen et al. (1999) reported an overview on virus inactivation and removal from serum and serum substitutes. With respect to the efficiency of PPV inactivation by γ irradition they indicated that even after application of a radiation dose of 40 kGy, a TCID 50 of 5.3 per ml was observed, indicating that the log reduction was only about 2. This difference with data published by Plavsic et al. (1999) might be due to differences in the design of the respective studies. However, with respect to the other viruses tested (BVDV, IBR, PI-3, reovirus 3), no differences in the inactivation doses were observed. It should be mentioned here, that sera are normally irradiated using a dose of 20 to 25 kGy. For veterinary use, the radiation dose has to be 35 kGy.
A very important consideration is the capacity of the irradiated serum to support cell growth. By performing long-term standard cultures (three passages in a medium supplemented with 5% of the irradiated sera), Plavsic et al. (1999) were able to show that in principle all tested cell lines could be cultivated, but also that different cells reacted relatively differently on the radiation doses used. Whereas low passage BHK cells, Vero (only slightly), and CHO cells displayed an inverse relationship between growth and radiation dose, high passage BHK cells and the human diploid fibroblasts, WI-38 and MRC-5 -the latter are of special interest for vaccine production and virology -did not display growth decline as a function of radiation dose (Table XV ). None of the tested cell lines showed a modifed morphology.
The advantages of irradiation is that it is easy and safe and does not leave residual molecules in the final product as when chemical inactivation methods are used. Today γ irradition is mandatory in Europe for fetal bovine serum. Validation experiments of the γ irradiation (dose: 25-35 kGy) of porcine pancreatic trypsin powder indicated a 6.7 log 10 reduction of the median tissue culture infective dose (TCID 50 ) (Erickson et al., 1989) .
(c) UVC irradiation (Kurth et al., 1999) : A second rather easy and safe method is UVC irradiation for inactivating adventitious agents. As distinct from γ irradition, the UVC irradiation has to be performed by using a continuous flow through irradiator. An irradiation time of 8±1 s and a fluence of 0.1 J cm −2 are used normally. The principle of this type of irradiation is a DNA excitation leading to electron transfer (→ 8-hydroxoy-guanine), photohydration (→ cytosin hydrates), photoaddition and dimerization (→ Pyr -Pyr, Thy -Ade, Pyrimidine (6-4) Pyrimidone). UVC irradiation is very effective for selected virus groups, especially for those with single-stranded nucleic acids. The data shown in Table XIII indicate that all tested single-stranded viruses were rather efficiently inactivated with a log clearance ranging from >5.5 to 8. Only the reovirus 3 which has a double stranded RNA shows a lower log reduction (4). Long term growth assays did not reveal any reduction in the ability of the UVC irradiated sera (used at 1%) to support cell growth.
(d) Other treatments: Substances of animal origin, such as serum or trypsin, or final biotech products can also be treated by other methods for reducing the eventual viral burden. These treatments can be of chemical nature, such as treatment with peracetic acid (Hughes, 1996) , solvents (e.g. 1% Tween 80 and 0.3% tri-n-butyl-phosphate at 25 • C for 8.5 h for the treatment of plasma derived Factor IX, works only for lipid enveloped viruses) (Johnston et al., 2000) , or imines (Brown et al., 1999) , or physical methods, such as heating (Hughes, 1996) or the reduction of the pH to 4.5. With respect to heat treatment, it is less effective than irradiation methods (Willkommen et al., 1999) and the serum composition is too much altered (Hanson and Foster, 1997) , leading to a rather weak growth promotion. The addition of a chemical substance, such as peracetic acid, is not ideal because a chemically reactive substance is added which might also lead to an inactivation of some medium compounds. In spite of this, virus inactivation based on the treatment with peracetic acid is rather effective for inactivating resistant virus, such as poliovirus Table XV . The effect of increasing doses of gamma radiation on the ability of fetal bovine serum to support the long term growth of selected cell lines in culture (expressed as percent of growth of control cultures) (Plavsic et al., 1999) (Sprössig and Mücke, 1967) , as well as for maintaining the growth supporting ability of the serum. Other adventitous agents like mycoplasmas and bacteria are also efficiently inactivated (Schweizer et al., 1972) .
(e) Replacement of animal derived substances by non-animal/non-human derived substances: Although viral screening tests are efficient for detecting adventitious viruses, the best remedy for avoiding the presence of these viruses is the use of non-animal/nonhuman derived raw materials. This is not a complete assurance for the absence of virus, but a considerable risk reduction, since adventitious viruses might also be introduced via non animal derived raw materials, such as medium components as shown by Garnick (1996) . In this context it should be mentioned that most of the recent biologicals based on the use of animal cell technology are produced in serum-free or protein-free media (Froud, 1999; Merten, 1999) . With respect to the production of viral vaccines, the first serum-free viral vaccines were developed and tested in clinical studies (Brands et al., 1999; Kistner et al., 1999) , and are going to be put on the market.
Recently, a study concerning a veterinary live virus vaccine, which was produced under protein-free conditions (devoid of any animal or human derived substances), showed that such a vaccine was as efficacious and safe as a classically produced vaccine (under serum-conditions) (Makoschey et al., 2002) . This indicates very clearly that the use of serum for the production of viral vaccines, in particular, and of biologicals, in general, is an anachronism and that the efficient replacement of non-animal derived serum-supplements is feasible.
Operator induced biological contaminations in cell culture is a multifaceted problem involving the unex-pected introduction of other animal cells (see chapter by Masters), microbial (see chapter by Drexler and Uphoff), and viral contaminants. There are few reports on operator induced viral contaminations. The potential exists, however, as reports have appeared documenting the considerable stability of Rhinoviruses, Respiratory Syncytial virus, Rotaviruses, and others, in aerosols on worker's hand and safety hood surface (for more details, see Hay, 1991) .
In general, viral contaminations of cell lines cannot be treated and contaminated cultures should be discarded, with the exception of LDV. This virus causes a life long viremia in infected mice without any clinical signs, and each sample of these animals is virus contaminated. Because LDV requires primary mouse macrophages for replication it cannot survive repeated in vitro subcultivations, leading to a loss of this virus in infected in vitro cultures. Another elimination method is the passage of the contaminated cell line/tumor in another species, for example nude rats (Nicklas et al., 1993; Nakai et al., 2000) .
The absence of virus can only be assured by performing a rigorous testing programme, which includes all steps in a bioprocess: master cell bank, working cell bank, the raw materials, the unprocessed bulk harvest, late expanded cells, and the final product. A summary on the tests to perform is presented in Table XVI .
Whereas research cell banks are mostly tested for sterility and absence of mycoplasmas, GMP cell banks a Used for preparation of WCB or in production runs starting from MCB or WCB. b Cells at the end of a typical production run are tested to determine the virus load in case of retrovirus like particle bearing production cells.
Only few harvests need to be tested (validation). c Old cells may be from production runs (as post production cells) or from a separate culture kept in continuous culture for a long period and prepared for this analysis of 'limit of cell age' only (as late expanded cells). Extensive testing performed as part of the qualification of the MCB regarding absence of latent virus, inducible by cultivation on production conditions. d A very large sample volume for testing would be required for statistics of a sufficiently sensitive detection of low virus titers.
for the production of biologicals for parenteral applications have to be tested much more rigorously. ICH Topic Q5A (1997) suggests the following virus tests for different cell banks: 'A master cell bank has to be extensively screened for both endogeneous and non-endogeneous viral contaminants. For heterohybridoma cell lines in which one or more partners are human or non-human primate in origin, tests should be performed in order to detect viruses of human or nonhuman primate origin as viral contaminants arising from these cells may pose a particular hazard. Testing for non-endogeneous viruses should include in vitro and in vivo inoculation tests and any other specific tests, including species-specific tests such as mouse antibody (MAP) test, that are appropriate, based on the passage history of the cell line, to detect possible contaminating viruses.'
'The working cell bank as a starting cell substrate for drug production should be tested for adventitious viruses either by direct testing or by analysis of cells at the limit of in vitro cell age, initiated from the WCB. When appropriate non-endogenous virus tests have been performed on the MCB and cells cultured up to or beyond the limit of in vitro age have been derived from the WCB and used for testing for the presence of adventitious viruses, similar tests need not to be performed on the initial WCB. Antibody production tests (MAP, RAP, or HAP) are usually not necessary for the WCB. An alternative approach in which full tests are carried out on the WCB rather than on the MCB would also be acceptable. ' 'The limit of in vitro cell age used for production should be based on data derived from production cells expanded under pilot-plant scale or commercial-scale conditions to the proposed in vitro cell age or beyond. Generally, the production cells are obtained by expansion of the WCB; the MCB could also be used to prepare the production cells. Cells at the limit of in vitro cell age should be evaluated once for those endogenous viruses that may have been undetected in the MCB and WCB. The performance of suitable tests (e.g. in vitro and in vivo) at least once on cells at the limit of in vitro cell age used for production would provide further assurance that the production process is not prone to contamination by adventitious virus. If any adventitious viruses are detected at this level, the process should be carefully checked in order to determine the cause of the contamination, and completely redesigned if necessary.'
The detection of adventitious viruses in cell banks has to follow two principles -use of detection methods for specific viruses such as MAP, HAP, RAP (mouse, hamster, rat antibody production tests -examination of serum antibody levels against specific viruses or enzyme activity after a specified period), and different specific PCRs, as well as the use of general tests which may indicate the presence of one or more of a large variety of different viruses. General tests include the in vitro test for adventitous virus. This test involves the inoculation of different cell lines -a human, a primate and a bovine (if bovine material was used for the production, otherwise a cell line from the species of origin of the cell substrate), and the production cell line (co-cultivation test). The RTase assay is a general test which will detect the presence of all viruses which contain reverse transcriptase enzyme. The last general test which is normally applied is the, in vivo tests where animals are used to identify the presence of virus. The animals are treated using different inoculation routes (the health of these animals should be monitored and any abnormality should be investigated to establish the cause of the illness). Finally, electron microscopic examination is also a general test which can be used for detecting adventitious viruses in the case of rather high virus loads. More details can be found in the articles by Poiley (1990) , by Werz et al. (1997) , and in the ICH Topic Q5A (1997).
When a producer cell line of murine origin is used, the consensus opinion among regulators is that all known murine viruses should be tested for. If a cell of human origin is involved in production, then there should be tests for human viruses, such as HIV, HTLV, EBV, CMV, HHV6 and HHV7. Human-mouse heterohybridoma cell lines have to be tested for both human and murine viruses (Robertson, 1996) .
In general, the unprocessed bulk material (pool of harvests of cells and culture media) should be tested for viruses after the end of production and before any downstream processing. The scope, extent, and frequency of virus testing on the unprocessed bulk should be determined by taking several points into consideration including the nature of the cell lines used to produce the desired product, the results and extent of virus tests performed during the qualification of the cell line, the cultivation method, raw material sources and results of viral clearance studies. In vitro screening tests, using several cell lines, are generally employed for testing. If appropriate, a PCR test or other suitable methods may be used. The presence of an adventitious virus will block the further use and processing of the harvest material.
For screening of raw materials, mainly serum, see Section on 'Sourcing, screening and other precautions'.
In summary general virus tests are vital because past incidents of viral contaminations have derived from viruses not known to be present within the production systems. Therefore the approach involving a variety of both general and specific tests applied at more than one stage of manufacture in combination with viral elimination steps during subsequent processing to assure the safety of the product is of utmost importance.
The use of modern biotechnology for the production of biopharmaceuticals allows the treatment of diseases, which could not be treated previously. However, virus infection and replication is an inherent risk during cultivation of mammalian cells. Raw material testing, rigorous characterization of the master and the working cell bank as well as testing of the final bulk product (before downstream processing and virus inactivation steps have been performed) lead to a considerable increase in the viral safety. Although viral contamination might happen during production only preventive measures can be taken in fermentation and product biosynthesis. Thus the downstream processing is an integral part of the manufacturing process and has to be validated in order to assess its potential to eliminate, clear, or inactivate viruses. The downstream processing has two, sometimes contradictory aims: (i) purify the product to the required purity at high recovery using the lowest number of steps possible, and (ii) eventually increase the number of purification steps in order to assure the elimination of potential viral contaminants. The general difficulty resides in the physico-chemical properties of the product. The properties that maintain the beneficial effects of a product are often very similar to those carried by all viruses, in particular when the bioproduct is a live virus (for vaccination or for gene therapy purposes). Therefore only a limited spectrum of techniques can be used for virus inactivation/elimination. To assess the capability of individual process steps to remove viruses these steps have to be tested with live model viruses for which clearing factors can then be calculated. These, so called, viral clearance studies have the objective to demonstrate the capacity of different steps of the purification process to eliminate or inactivate adventitious agents acquired during the production process (contaminated cells, raw materials, process failure, etc.). They are performed via spiking experiments. As viruses vary greatly in properties such as size, resistance to inactivation, type of envelope, type and structure of their genome, the model viruses used for these spiking experiments have to be selected in order to cover the whole spectrum of potentially present viruses. However, in order to assure absence of viral contaminations derived from the producer cell line in the product, the downstream protocol has also to be validated for its capacity to inactivate or eliminte these specific viruses (e.g. retroviruses derived from hybridoma and CHO cells, or EBV derived from human lymphoblastoid cell lines). In general, it is difficult to show more than a five log removal on any given step due to the titers of the model virus used. More details can be found in the following references: Fritsch (1992) , Cartwright (1994) , Werz et al. (1997) , ICH Topic Q5A (1997), and Larzul (1999) .
It is evident that a research laboratory cannot afford all tests, which have to be performed by the biotech industry in the case of GMP production. In addition, there is no need for such exhaustive testing unless the materials are to be used in the treatment of human patients. However, most of the issues described in the chapters on 'Problems associated with viral contaminations' and on 'Origin of viral contaminations' are valid for everyone working in the field of animal cell culture. The use of validated cell lines, shown to be 'virus-free', is the best choice because cell collections, such as the ATCC (www.atcc.org), perform entrance tests for new cell lines for assuring the absence of mycoplasma, bacteria, fungi, protozoa, and cytopathic viruses and can guarantee a certain microbial quality for the delivered cell lines. The German Cell Line Bank (www.dsmz.de) provides cells, most of them have been tested for the absence of HIV-I, HTLV-I and II, EBV, HBV, HCV, and HHV-8 by using PCR or RT-PCR. The absence of retroviruses is proven by performing a reverse transcriptase assay.
However, cell lines obtained from such collections, can still be contaminated by viruses because viruses, which do not lead to a cytopathic effect, are not detected by the tests commonly used, on one side (ATCC), or only tests for detecting human pathogenviruses have been performed on the other side (DSMZ). If a very important cell line of a research laboratory has to be more rigorously screened, commercial screening services are needed and their use is recommended.
The use of controlled animals, free of microbial contaminants, for animal passages of cells and of controlled raw materials derived from accredited dealers, who are performing viral screens (e.g. for virus absence in serum and trypsin preparations), for the preparation of culture media are steps in the right direction for reducing the risk for viral contaminations. The rules concerning the general cell culture operation procedures for avoiding viral contaminations are largely the same as for preventing mycoplasmal contaminations (see chapter by Drexler and Uphoff).
Viral contaminations are a serious threat for animal cell cultures and may lead to false results in research, development, and virus screening, to viral contaminations in the biologicals derived from the contaminated cultures and finally to an infection of the treated patient. Fortunately due to rigorous testing of the animals used as the source of explants, the production of ascites or the passage of cells, of raw materials, of the cell strains and cell lines in use, and finally of bulk and the final product can prevent potentially dangerous viral contaminations. Existing data demonstrate that contamination of cells and harvests by viruses can occur for products of biotechnology, and while the frequency may be low it is not zero. For instance, routine testing of cell lines of biotech interest revealed a contamination frequency with adventitious viruses of less than 1% (Moore, 1992) . However, the possibility of new emerging viruses and the permanently existing risk of contaminations by adventitious agents and viruses leads to the conclusion that the user of animal cells as well as the producer of biotech products by using animal cells have to be attentive to this possible threat and that they have to assure the absence of adventitious agents/viruses by any mean. Only then, animal cell technology biotech products can be used for the benefit of everyone. DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice. Viral infections have a vast impact on human health, resulting in hundreds of thousands of deaths yearly. To replicate and spread, these intracellular pathogens subvert the host cellular defense machinery. Dengue virus (DENV) is the most prevalent arbovirus in humans, and productively infects cells that are involved in the immune response, such as monocytes, B cells, macrophages and dendritic cells (DCs) among others [1, 2, 3, 4, 5] . Like most viruses, DENV has evolved in order to inhibit or evade different aspects of the innate immune system, the first line of human defense against microbes. DCs are antigen presenting cells (APCs) and some of the first cells that interact with the virus after the bite of an infected mosquito. Infection of these cells induces their activation, which results in their migration to the lymph nodes where the virus can infect other susceptible cells. The kinetics of infection of different cells in the immune system is not well documented, due to the lack of immune-competent mouse models for dengue disease. Nevertheless, in mice defective for type I IFN signaling, one of the most accepted current models for dengue disease, it has been shown that DCs and macrophages are productively infected by DENV [3, 4, 5, 6] reviewed in [7] .
DENV is a single stranded RNA virus of positive polarity that, after entering the cytoplasm of the host cell, releases its genome and synthesizes a polyprotein using the cellular machinery, as a first event of the viral cycle. The DENV polyprotein is cleaved by the viral protease complex (NS2B3) and cellular proteases, including furin [8] . After this processing, some of the viral proteins have the ability to rearrange the ER membrane and create the micro-environment necessary for the production of de novo synthesized viral genomic RNA. During this event, DENV accumulates products with conserved molecular structures, like RNA with 59-triphosphate moiety or double stranded RNA, also referred to as pathogen associated molecular patterns (PAMPs). These foreign molecules are ligands of different cellular receptors engaged in their recognition, known as pattern recognition receptors (PRRs). PRRs are mainly divided into two different classes depending on their localization, associated with either the membrane or the cytoplasm. The Toll-like receptor (TLR) family is composed of membrane proteins with domains that are designed to detect extracellular PAMPs. On the other hand, the cytosolic DExD/Hbox RNA helicase proteins that contain caspase-recruiting domains (CARDs), referred to as RIG-I and MDA-5, can detect specific PAMPs present in the cytoplasm. These last two cytoplasmic sensors together with the TLR family members (TLR3/TLR7/TLR8) have been described so far as the most relevant DENV sensors [9, 10, 11] . After recognition of the mentioned PAMPs by the C-terminal helicase domain of RIG-I and MDA-5, these undergo a conformational change that exposes their CARD domains and promote the interaction with different down-stream molecules. One of the most well studied downstream molecules, referred as IPS-1 (also known as, MAVS, CARDIF or VISA), is located in the outer membrane of the mitochondria and transmits the signal via different molecules, including the tumor necrosis factor receptor associated factors 6 and 3 (TRAF6 and TRAF3) and the IkB kinase (IKK) family members (TBK1, IKKa, IKKb and IKKe) among other cellular factors [reviewed in [12] ]. Recently three different groups, using cDNA library screening of genes that induced the IFNb promoter, described an adaptor protein that localizes in the endoplasmic reticulum (ER). This protein was named as stimulator of the interferon gene (STING) [13] , mediator of IRF3 activation (MITA) [14] and endoplasmic reticulum IFN stimulator (ERIS) [15] . Also the same protein, referred to as MYPS, was previously identified as a mediator of anti-major histocompatibility complex II monoclonal antibody-induced apoptosis in B-lymphoma cells [16] . STING is highly expressed in several immune cells, including macrophages and DCs, as well as endothelial and epithelial cells [13] . This protein can interact with RIG-I and IPS-1, but not with MDA-5, and the signaling mediated by this adaptor is independent of the sensing by the TLR family members [17] . In two recent reports, it was documented that STING is involved in the pathway that mediates the detection of pathogens with DNA genomes [18] and has a role as a direct sensor of cyclic dinucleotides, a signaling molecule produced exclusively by bacteria and archea [19] . Activation of STING by some of these stimuli leads to its relocalization with TBK1 from the ER to perinuclear vesicles containing the subunit of the exocyst complex 5 (Sec5) followed by the phosphorylation of TBK-1 and the subsequent activation of the transcription factors IRF3/7 and NFkB, which translocate to the nucleus and complex with ATF2/c-Jun to induce the expression of type I IFN and pro-inflammatory cytokines [17] .
A remarkable hallmark of highly virulent human pathogens is the ability, acquired through evolution, to inhibit this innate immune response by the expression of viral factors that affect one or several steps of the above described signaling cascade. Some of the most notorious examples are the influenza virus NS1 protein, that targets RIG-I for degradation, minimizing the sensing of influenza virus PAMPs by this PRR [20] or the Hepatitis C virus NS34A protease complex that cleaves the adaptor IPS-1 to interrupt the signaling that ends with the activation of IRF3, NFkB and the subsequent production of type I IFN in human hepatocytes [21] .
Our group has documented that DENV is a weak inducer of type I interferon in human DCs, in particular when compared with other viruses that competently produce these cytokines in large amounts, such as Newcastle disease virus (NDV) [22] and Semliki Forest virus (SFV) [23] . This lack of type I IFN production by DCs infected with DENV results in an impaired ability of those DCs to prime T cells toward Th1 immunity, an effect that can be reversed by the addition of IFNb [24] . Nevertheless, DENV is able to induce the expression of some pro-inflammatory cytokines at early times post infection, which we hypothesize is a strategy used by this virus to attract more cells to the site of infection by allowing the expression of some chemo-attractants by infected cells. Our group described that the infection by DENV does not induce the phosphorylation of IRF3 in human primary cells, resulting in an inhibition of type I IFN production [24] . In a subsequent report, we examined the ability of DENV-infected DCs to respond to a variety of type I IFN-triggering signals using potent stimulators such as NDV, SeV, SFV, or TLR-3 ligand poly(I:C) [25] . This effect is viral dose dependent and takes place as early as 2 hours after DENV infection. We also showed that the inhibition of IFNa/b production after NDV infection in DENV-infected DCs is not a bystander effect, implying an active role of the DENVinfected DC population in the inhibition of IFNa/b. By using an NDV vector strategy to express the individual DENV nonstructural proteins (NS2A, NS2B3, NS4A and NS4B), we showed that only the recombinant NDV expressing the protease complex NS2B3 inhibited IFNa expression in infected MDDCs, as compared to NDV alone. Similar results were obtained using an IFNb promoter activity assay in 293T cells. Catalytically inactive NS2B3 mutants showed a diminished inhibition of this phenotype, which highlighted the important role for the protease activity of the NS2B3 protein as inhibitor of the type I IFN production. Interestingly, the proteolytic core of NS2B3, consisting of the last 40 amino acids of NS2B and the first 180 amino acids of NS3, was enough to reduce the activation of the IFNb promoter by a strong stimulus, such as Sendai virus (SeV) infection.
DENV has also been shown to express inhibitors of the type I IFN signaling cascade [26] and has been shown to encode for at least four non-structural proteins NS2A, NS4A, NS4B and NS5 that target different components of this pathway. The most remarkable example is the proteasomal degradation of human STAT2 by the NS5, a phenomenon that does not occur in mouse cells, which makes mouse STAT2 a restriction factor for DENV replication in these animals [27, 28, 29, 30, 31] . In summary, DENV can successfully inhibit two fundamental steps of the innate immune system, both the inhibition of the type I IFN production and the signaling. In this way, DENV reduces the expression of hundreds of interferon inducible genes that would otherwise establish the antiviral state and control the spread of the infection in the host.
In the present report, we describe the mechanism of inhibition of type I IFN production by DENV in primary human and mouse cells and identify the human adaptor molecule STING as a target
Dengue virus (DENV) is a pathogen with a high impact in human health that replicates in a wide range of cells of the immune system. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response (IFN). Thus, DENV can inhibit type I IFN signaling (described by several groups), and type I IFN production (described by our group). We documented the inhibition of type I IFN production in human monocyte derived dendritic cells (MDDCs) with an otherwise strong cytokine and chemokine profile in those cells and that the NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this event. Here we identify the human adaptor molecule STING as a target of the NS2B3 protease complex and characterize the mechanism of inhibition of the type I IFN production in primary human MDDCs mediated by this viral factor. We also describe that DENV NS2B3 cannot degrade the mouse version of STING, a phenomenon that strictly restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.
of the DENV NS2B3 protease complex. We demonstrate that the proteolytic activity of this viral factor is crucial for the cleavage and degradation of STING and this phenomenon impairs the production of type I IFN in DENV infected cells. Furthermore, we show that DENV NS2B3 is not able to cleave the mouse version of STING. Using STING double knockout mouse embryonic fibroblast (MEFs) and human dendritic cells, we demonstrate the relevant role of this host factor in the restriction of DENV replication in mouse cells. This is the first report showing STING as a target for cleavage and degradation by a viral protein to inhibit innate immune responses and as a host restriction factor for virus infection in primary cells.
Previous results from our laboratory showed that dengue virus inhibits type I IFN production in human primary dendritic cells and that this inhibition requires a proteolytically active NS2B3 protease complex [24, 25] .
In order to identify potential NS2B3 targets we performed a bioinformatic search for potential DENV protease cleavage sites contained within members of the type I IFN pathway [32] . We identified putative cleavage sites in several known members of the type I IFN pathway (see table 1 ). After testing the factors shown in table 1 for their susceptibility to be cleaved by the DENV protease NS2B3, we observed that only STING was cleaved in our experimental set up (data not shown and figure 1). We have generated a wild type DENV-NS2B3, and a proteolytically inactive version (NS2B3-S135A), ( Figure 1A ) by direct mutagenesis [25] that were used to analyze the potential cleavage of STING by the DENV protease complex. When we compared the human amino acid sequence of human STING to its mouse counterpart we noticed that the putative NS2B3 cleavage site in hSTING which is situated at the beginning of transmembrane domain 3 ( Figure 1B ), is absent in mouse STING. In order to test the susceptibility of human and mouse STING proteins to proteolytic cleavage or degradation, we co-expressed a Cterminally HA-tagged STING alongside a wild type or catalytically active DENV-NS2B3, and a proteolytically inactive version (NS2B3-S135A) in 293T cells, ( Figure 1A ) and analyzed them by western blot (Figures 1C, 1D ). In the presence of WT NS2B3 we observed the full-length 42 kDa human STING and an additional band of about 32 KD, which is consistent with a C-terminal region product of a cleavage occurring within the first 96 aa of STING ( Figure 1C ). The additional band was not visible when the mouse version of STING or a catalytically inactive NS2B3 was used ( Figure 1C and 1D ). This putative cleavage site for the DENV NS2B3 lies very close to the conserved cysteine motif C88xxC91, or redox motif, recently described to be required for dimerization of STING and subsequent signaling in the type I IFN production pathway [33] . Regardless of their susceptibility to cleavage by the DENV NS2B3 complex, both the human and mouse versions of STING co-immunoprecipitated with the WT DENV NS2B3 complex and the proteolytically inactive mutant NS2B3 S135A ( Figure 1E , lanes 2, 3, 6 and 7). To map the putative cleavage site of human STING for the DENV NS2B3 complex, we mutated the sequence corresponding to the first three amino acids of the human site, RRG (shown in figure 1B as hSTING in red) with the sequence corresponding to the amino acids HCM found in the mouse version of STING (shown in figure 1B as mSTING). These recombinant versions of STING were co-transfected into 293T cells with the WT and mutant version of the NS2B3 protease and the ability of the DENV protease to cleave STING ( Figure 1F , lane 5) was drastically reduced when the mouse sequence was present in hSTING ( Figure 1F , lane 2). These data confirm the requirement for amino acids RRG for efficient cleavage of STING by the DENV NS2B3. However, replacement of the corresponding amino acid sequence of mouse STING (IHCM) by the human putative cleavage sequence (LRRG) does not render mouse STING susceptible to cleavage by the DENV protease ( Figure 1G , lane 2), suggesting that additional flanking amino acids are required for this cleavage. Altogether, these results strongly suggest that STING is a target for NS2B3 in human cells and possibly a restriction factor for DENV infection in the mouse.
To test whether endogenous STING undergoes the same NS2B3-dependent processing as in overexpression experiments in 293T cells, we infected human MDDCs with DENV-2 (16681 strain) and analyzed the cell lysates by western blot at different time points ( Figure 1H ). Infection of MDDCs by DENV resulted in the degradation of STING that could be detected at 24 and 48 hours post infection (hpi) ( Figure 1H , lanes 9 and 10) which correlate with peak expression levels of the NS2B3 (as detected with NS3 specific antibodies). As expected, this degradation of STING was not observed in MDDCs treated with UV-inactivated DENV or mock treated cells ( Figure 1H , lanes1-5 and 11-15). These data demonstrate that DENV NS2B3-dependent cleavage of endogenous human STING occurs in cells relevant to DENV infection (MDDCs), and therefore has the potential to play a crucial role in inhibition of type I IFN production.
We next investigated whether STING cleavage by DENV NS2B3 had an impact on its ability to mediate the signaling necessary for type I IFN production. We transfected 293T cells with either hSTING (Figures 2A and 2B ) or mSTING (Figures 2C and 2D) and the three different versions of the DENV protease: wild type, the proteolytically inactive version (NS2B3-S135A) and the proteolytic core (NS2Bh-NS3pro) alongside luciferase reporter constructs driven by either an IFNb promoter (IFNb-Luc) or by three IRF3/7 binding sites (p55-C1B-Luc) (kindly provided by Dr. Megan Shaw and shown in figure 2E schematically) [34] . As shown in figures 2A and 2B, cleavage of hSTING by the DENV NS2B3-WT and NS2Bh-NS3pro greatly inhibited its ability to activate both reporter constructs, while transfection of the mutant version of the protease did not. Conversely, the DENV NS2B3 had minimal or no impact on the ability of mSTING to induce activation of either of the reporter constructs used. We did not observe any DENV NS2B3-dependent inhibition when the human adaptor TBK1 was used as a positive control to induce the IFNb promoter, demonstrating that the inhibition during dengue infection occurs upstream of this adaptor (data not shown). Taking together, these data demonstrate that cleavage of hSTING by the DENV NS2B3 precludes the induction of type I IFN responses. Moreover, to validate the observed results in human primary cells, we used E. coli DNA, a known inducer of STING signaling [35] to treat human MDDCs previously infected with DENV, UV-inactivated DENV or mock treated (as shown schematically in figure 2F ). Figures 2G, 2H and 2I show that only live DENV but not UV inactivated DENV was able to inhibit the induction of IFNb, IFNa or ISG15 by this ligand in human MDDCs.
To validate the results described using p55-C1B and IFNb promoter assays in a primary cell model, we measured IFNa/b production upon infection of MDDCs either with DENV or a Semliki forest virus (SFV) expressing the DENV NS2B3 protease complex or the mutant version of the protease (NS2B3-S135A) as a control [23, 25] . Consistent with our earlier report [24] , human MDDCs infected with DENV-2 (16681 strain) were unable to produce IFNa/b. Furthermore, SFV-NS2B3 induced significantly lower levels of IFNa/b mRNA than the SFV-NS2B3-S135A ( Figures 3A and 3B ). Interestingly, SFV-NS2B3-WT induced significantly higher expression of TNFa at early times postinfection compared to SFV-NS2B3-S135A control ( Figure 3C ). As shown previously, this would suggest an involvement of the NS2B3 protease complex in the expression of this pro-inflammatory cytokine [24] . As expected, the infection of MDDCs by DENV upregulated the expression of STING in these cells ( Figure 3D ). Figure 3E shows the kinetics of infection by DENV in MDDCs, with the peak of viral RNA at 48 hpi. In contrast, the SFV vectors used in these studies show low levels of viral RNA at late times after treatment ( Figure 3F ), since these vectors are replication deficient [36] . Then we infected mouse bone marrow-DCs (BM-DCs) using the same viruses, and analyzed the gene induction profile in those cells. As expected, infection of BM-DCs by DENV was rapidly controlled, consistent with the inability of DENV to infect mouse cells, and showed an opposite kinetic of viral RNA synthesis compared to the observed pattern in human DCs, with a modest induction of cytokines ( Figure 3G to 3K). SFV is an alphavirus that can replicate in mouse cells, and DCs in particular. In this context, the SFV-NS2B3 exhibited a higher induction of both IFNa/b genes compared to the SFV-NS2B3-S135A control, showing an opposite profile than that observed in human DCs, in which SFV-NS2B3-S135A induced higher levels of IFNa/b genes ( Figure 3G and 3H) . Again, the kinetics of infection of the SFV vectors in mouse DCs ( Figure 3L) show very low levels of viral RNA, consistent with their lack of productive infection in these cells [36] . The observed phenomenon agrees with the inability of recombinant DENV-NS2B3 to cleave mouse STING and decrease the activity of the IFNb and p55-C1B promoters induced by this adaptor protein ( Figure 2C and 2D).
To explore STING's impact on the DENV replication in mouse cells, we used WT (Sting +/+ ) and STING double knockout (Sting 2/2 ) mouse embryonic fibroblasts (MEFs). First, we infected WT and Sting 2/2 MEFs with two different DENV-2 strains, 16681 and NGC (a strain that was obtained after several passages in mouse brain) [37] , with an MOI of 5. Then, we measured the ability of the two DENV-2 strains to induce IFNb production, to replicate in these cells and to release infectious particles from those cells ( Figure 4A-4F ). Both DENV-2 strains induced significantly higher levels of IFNb in WT MEFs as compared to the Sting 2/2 MEFs ( Figure 4A and 4D) , underlining the relevance of STING in the signaling of type I IFN upon the infection with DENV. Consistent with the observed low induction of IFNb, Sting 2/2 MEFs were permissive to DENV replication while, despite the high MOI used, replication of DENV 16681 and NGC was rapidly controlled in WT MEFs ( Figure 4B and 4E) . The production of infectious particles by the two DENV-2 strains in WT and Sting 2/2 MEFs was measured by plaque assay and shows that the KO MEFs were more permissive to DENV infection than the WT MEFs and have very different peaks of infection ( Figure 2C and 2F). To test whether our observation was independent from the high MOI and viral strains used, we repeated the infection using different DENV serotypes (DENV-2 16681 strain, DENV-3 PR-6 strain and DENV-4 H-241 strain) and an MOI of 1, with similar results (data not shown). These results are likely due to the inability of DENV to inhibit the type I IFN signaling in mouse cells and the establishment of the antiviral state [31] Mutation of cleavage site for DENV-NS2B3 restores the ability of STING to induce type I IFN production upon DENV infection
To confirm the relevance of STING cleavage by the DENV-NS2B3 on the inhibition of type I IFN production upon DENV infection, we transduced Sting 2/2 MEFs with lentiviruses expressing either WT human STING (STING-WT) or a mutant (uncleavable) version, that harbors the mouse STING sequence at the NS2B3-cleavage site (STING-MUT) ( Figure 1B and 1F) . Twenty-four hours after transduction MEFs were infected with DENV-2 at an MOI of 1 (strains 16681 and NGC) or mock treated and the levels of IFNb, IFNa and viral RNA were measured by qRT-PCR after total RNA extraction from the cells at different times post infection ( Figure 5A-5F) . A schematic representation of the lentiviruses used is shown in figure 5G . For these experiments, as shown in figure 5A and 5D, MEFs expressing the uncleavable version of STING (STING-MUT) expressed significantly higher levels of IFNb mRNA when compared to MEFs expressing WT STING (STING-WT) or to the control MEFs (GFP) confirming that the cleavage of STING by DENV-NS2B3 is necessary for the inhibition of IFNb production in infected cells. The induction of IFNb was detected as early as 2 hpi. Infection with the two DENV-2 strains also induced higher levels of IFNa mRNA in MEFs expressing STING-MUT than in the MEFs expressing STING-WT and the GFP control ( Figure 5B and 5E ). Under these experimental conditions, the replication of the mouse adapted DENV-2 (NGC) was increased in Sting 2/2 MEFs expressing wild type STING as compared with STING-MUT ( Figure 5F ). In the case of 16681 strain, a significant increase of replication was observed only with Sting 2/2 MEFs (GFP) at 48 hpi., and no significant difference was observed in MEFs expressing the two versions of STING, presumably due to a high level of lentiviral-expressed STING that could overwhelm the ability of this non mouse-adapted DENV strain to replicate in this system ( Figure 5C ).
As shown in figure 5 , the lack of STING cleavage was sufficient to increase the expression of type I IFN in MEFs infected with DENV. To validate these results in primary human cells, we transduced MDDCs from three different donors with the STINGexpressing lentiviruses and the GFP-only control ( Figure 5G ). STING transduced DCs were then infected with DENV-2 at an MOI of 5 and we assessed the production of IFNb in those cells. As shown in Figure 6A (showing one representative donor out of three), there were no significant differences between the levels of STING-WT and STING-MUT mRNA. This demonstrates that any difference in antiviral effect observed with the two different versions of STING is independent of the expression levels for these proteins. Upon DENV-2 infection, DCs expressing STING-MUT produced higher levels of IFNb when compared with STING-WT or GFP controls (showing statistical significance at 2 and 12 hpi). The induction of IFNb messenger RNA was detected as early as 2 hpi, which is in agreement with the results described with MEFs ( Figures 4, 5 and 6B ). These data confirm that detection of DENV infection by DCs takes place at early times post infection and that STING cleavage by DENV NS2B3 is fundamental to inhibit the signaling mediated by this adaptor in human cells. We next measured DENV replication kinetics and we found that viral RNA levels were significantly lower in DCs over-expressing STING-MUT when compared with STING-WT and GFP control ( Figure 6C ). Suggesting that the inability of DENV to cleave mutant STING and inhibit the induction of type I IFN has a direct impact on its replication kinetic and the accumulation of viral RNA in those cells.
To determine STING's impact on DENV replication in primary human MDDCs, we used RNA interference (RNAi) to silence its endogenous expression. A decrease of STING mRNA level was observed when specific siRNAs were used compared to two scrambled control siRNAs ( Figure 7A ). As expected, the previously observed upregulation of STING after DENV infection was controlled by the STING siRNAs ( Figure 7A ). As a consequence, the reduction in STING expression resulted in an increase of DENV replication, illustrated in Figure 7B . When the viral progeny released by those infected MDDCs was quantified by plaque assay, the six donors treated with STING specific siRNA, showed viral production under this experimental conditions, however when scrambled siRNA was used, only three out of the six donors released detectable viral progeny in the supernatant ( Figure 7C ). Data shown in figures 7A and 7B correspond to donor 4 in figure 7C . Taken together, these data confirm that STING is a crucial restriction factor of DENV replication in human dendritic cells, since its silencing increases the levels of DENV replication in those cells.
Different populations of cells were isolated from human blood and subsequently infected with DENV-2 at MOI of 1 and 12 h after infection supernatants were collected and RNA was extracted from cells. DENV-2 RNA was detected in all cells tested including plasmacytoid DCs (pDCs), B cells, blood circulating DCs (cDCs), monocytes as well as in monocyte-derived DCs (MDDCs) ( Figure 8A ). We also analyzed the cytokine and chemokine expression profile in all those cells by qRT-PCR (data not shown) and by multiplex ELISA (Figure 8B ) in the supernatants at 12 hpi. We observed a marked chemokine response (IL-8 and MIP1b) in monocytes, MDDCs, B cells and cDCs at this early time point, ( Figure 8B ). However pDCs did not show any significant chemokine profile after DENV-2 infection at this time point ( Figure 8B ). More interestingly, there was no significant type I IFN production observed in any of the cells tested by qRT-PCR (data not shown) or ELISA ( Figure 8B ). These data suggest that there is a coordinated and distinct kinetic of infection of DENV-2 in different cell populations in blood and there is a lack of type I IFN production in those cells after infection with this virus, at least at this early time point. The early time point of 12 hpi was chosen to obtain sufficient numbers of pDCs, since these cells have short half-lives and downregulate their specific cell surface markers in a rapid fashion. Nevertheless, we have previously reported that as early as 8 hpi, pDCs are able to produce type I IFN after infection with other viruses, such as NDV [24] .
To rule out that the lack of type I IFN production resulted from lack of cell to cell interactions, we infected whole PBMCs with DENV-2 (MOI of 1) and 18 h after infection cell supernatants were collected. Figure 8C shows multiplex ELISA data of cell supernatants from those cultures. While there is a clear IL-8 response to DENV-2 infection in PBMCs, consistent with the strong IL-8 signature observed in sera from patients [38] , there was no detectable IFNa secretion from infected PBMCs ( Figure 8C ). This suggests that DENV-2 may inhibit type I IFN production in susceptible cells within those cultures.
Macrophages have been shown to support DENV infection in animal models, and have been proposed to play an important role during early phases of dengue virus infection [39, 40] . We tested if monocyte-derived macrophages (MDMs) when infected with DENV were able to produce type I IFN. Figure 8D shows that macrophages are efficiently infected with DENV, with an early peak of replication and produce TNFa and IL-6 after DENV infection and after SFV expressing the WT and mutant versions of the NS2B3 DENV protease complex (Figures 8E, 8F ). Under these experimental conditions we were unable to detect IFNa released by macrophages after DENV infection. Interestingly, when compared to MDDCs infected with the same viruses ( Figure 8H) , macrophages produce at least 10-fold lower levels of IFNa after SFV infection, and the inhibitory effect of the DENV protease in this system was less apparent (8G and 8H).
Activation of innate immunity due to the detection of viral replication products in the cell leads to the expression of hundreds of antiviral genes that controls the spread of the infection [12] . The inhibition of different steps implicated in these molecular pathways by viruses has been a matter of extensive study for several years. It has been demonstrated by others and by our group that DENV can inhibit both the production and signaling of type I IFN by the expression of viral proteins. In this way, DENV can mitigate the immune response induced by the host upon infection [24, 25, 29] . Here we have identified the human adaptor molecule STING as a protein with a predominant role in the recognition of DENV by the innate immune system. This adaptor protein was described to reside in the ER, a cellular organelle intimately related to the DENV replication process. Also, STING has been described as part of the TRAP (translocon associated protein) complex that can associate with RIG-I and IPS-1, two proteins with relevant roles in viral detection [41] . Ishikawa et al. also described an inhibition of the STING mediated IFNb production by the yellow fever virus (YFV) NS4B [42] . However, when we tried to replicate these results using the DENV NS4B, this viral protein was unable to decrease the induction of luciferase mediated by STING in an IFNb promoter assay (data not shown).
By co-expression experiments of human STING with the DENV NS2B3 protease complex we observed a specific cleavage at (94-RRGA-99), a site described as a putative target for DENV NS2B3 [32] that generated a cleaved band of approximately 32 KDa (Figure 1C and 1F) . Interestingly, by analysis of the sequence alignment between human STING and its mouse version we, observed a drastic difference in the amino acid sequence in this region (94-HCMA-99) (shown in figure 1B ) and the inability to cleave the mouse STING by the DENV NS2B3 was confirmed by co-expression experiments ( Figure 1D and 1G) . Furthermore, the impact that the STING cleavage by NS2B3 had on the signaling of IFNb production pathway was subsequently demonstrated using IFNb and p55-C1B promoter systems ( Figure 2 ). In these experiments, a reduction in luciferase induction was only observed for human STING, suggesting that the cleavage confirmed by WB (showed in Figures 1C and 1F ) impaired the ability of this adaptor to induce IFNb. Recently, Jin et al. described a series of mutations in hSTING that were implicated in the activation/dimerization and subsequent induction of interferon. Interestingly, two cysteines located at C88XXC91 were fundamental for the proper induction of type I IFN after stimulation [33] . It could be interesting to investigate the presence of mutations at the cleavage site of STING for DENV NS2B3 in the human population, to identify a natural resistance to DENV infection.
While this manuscript was under review, Yu and colleagues reported by overexpression experiments that the DENV protease can cleave the adaptor molecule MITA, [43] . In the present report we provide important data on the role of this adaptor molecule in primary human and mouse cells and during the context of DENV infection. We also confirmed the cleavage and degradation of STING by the DENV NS2B3 protease in human MDDCs in the context of DENV infection, since it is important to validate these findings in a relevant primary cell system and during virus infection ( Figure 1H ). In primary human cells as well as in mouse cells, such as MEFs and DCs, we also found that the presence of human STING allowed for greater DENV replication and the presence of mouse STING seemed to restrict DENV replication (Figure 3, figure 4 and figure 7) . We also show that the NS2B3 protease of DENV has specificity for the human STING and not for the mouse homologue of this protein ( Figure 5 and figure 6 ), suggesting that STING may be an important restriction factor in mice. Several viral proteases have been described as proteins that modulate cellular pathways, allowing many viruses to modify the intra and extracellular environment to promote optimal conditions for replication and spread. Some of the most remarkable characteristics observed at early times after infection by DENV are the lack of IFNa/b induction and a robust induction of proinflammatory cytokines like TNFa [24, 25] . As it was described in our previous work, DENV infection can abrogate IRF3 phosphorylation, but has no impact on NF-kB activity [25] . Using recombinant viruses expressing DENV-NS2B3 we observed a clear effect on the induction of TNFa in human DCs, similar to that observed with DENV infection (Figure 3 and figure 8) . Also, the inhibition of luciferase activity driven by p55-C1B promoter was considerably more efficient when compared with IFNbpromoter, since p55-C1B only harbors sites for IRF3/7 transcription factors, and IFNb-Luc has also has response elements for NFkb and AP-1 transcription factors (Figures 2B and 2D ). Furthermore, Ishikawa et al. overexpressed STING in 293T cells in the presence of different promoters driving the luciferase gene. Interestingly, STING stimulated IFNb promoter up to 400-fold, IRF3 response element (PRDIII-I-Luc) up to 1,000-fold, and NFkb responsive promoter (NF-kb-Luc) only up to 12-fold [41] . This observation suggested that STING is fundamentally involved in phosphorylation of IRF3, and under these experimental conditions showed a 100 fold less influence on NF-kb induction. Taken together, these observations suggest that DENV NS2B3 protease inhibits IFNb production by cleavage of the adaptor STING without modifying the observed NF-kb activity induced after infection by DENV. Further work exploring the impact that DENV-NS2B3 has on the induction of NF-kb activity in infected cells would confirm a putative role of this viral factor in the modulation of innate immune response by the induction of proinflammatory cytokines, a hallmark phenomenon observed during infection by DENV [44] .
It is becoming increasingly clear that STING is a crucial adaptor in immune cells after infection with different viruses, such as HIV and DENV, among others [35, 45, 46] . These viruses require activation of their target cells in order to establish infection, or in the case of DENV to induce viremia in the host. Nevertheless, all viruses need to limit or inhibit the production of type I IFN in infected cells to avoid the establishment of an antiviral state in those cells. STING could be instrumental in these types of virus infections, since it can discriminate between the induction of type I IFN and the activation of the NFkB pathway. Along those lines, this report shows a novel mechanism of inhibition of IFN production by an RNA virus, namely DENV targeting STING. By inhibiting only type I IFN but not the NF-kB pathway, DENV induces a specific profile in infected human MDDCs and other susceptible primary cells that allow the virus to efficiently reach the lymph nodes and spread in the infected host, culminating in the production of viremia. All our experiments were performed in the context of primary infections with DENV, since we believe that the early events in primary infections dictate the quality of adaptive immune responses and the outcome of the infection. By targeting DCs and inhibiting the production of type I IFN in those cells, DENV may be able to efficiently modulate the generation of adaptive immune responses and establish infection in the host [24, 25] .
It has been proposed that during DENV infection IPS-1 may be responsible for controlling early viral replication and type I IFN production [47] , while the IFN signaling pathway (JAK/STAT) may control late viral replication and type I IFN production in DENV infected cells [48] . It is possible that interactions between STING and IPS-1 [46] may be disrupted by the DENV NS2B3 targeting of STING. This mayinhibit type I IFN production early during DENV infection in susceptible cells, although the NS2B3 has not been shown to directly interact with IPS-1. Further experiments are required to understand these complex interplays between different signaling molecules in human primary cells infected with DENV. Our experiments demonstrate that primary human cells implicated in dengue virus infection, such as dendritic cells, macrophages, monocytes and B cells can support DENV replication, although at different levels. Interestingly, DENV infection did not induce type I IFN production in any of those human primary cells tested (Figure 8) . These different blood cells may play different roles during DENV infection in humans, such as being involved in the initial infection or in the final stage of viremia. Also, since the mouse models that support DENV replication and recapitulate dengue symptoms are deficient in type I IFN responses or are reconstituted with human immune cells [6, 49] , the inhibition of type I IFN in infected cells seems to be crucial for the establishment of infection by DENV. Our data on different cells from blood also show that the inhibition of type I IFN production by DENV is not a DC specific phenomenon ( Figure 8) .
The inability of DENV to replicate in wild type mouse cells is well documented, and many attempts have been made to develop a competent animal model to study DENV infection [50] . The data presented in this manuscript, showing the ability of DENV to replicate in Sting 2/2 MEF (Figure 4) , open new approaches to develop a mouse model to study DENV infection and also highlights the requirement that type I IFN production has on the innate immune system and for the control of invading pathogens. Ashour et al. described the adaptor STAT2 as a restriction factor for DENV replication in mouse cells [31] . Based on our combined data, an interesting approach would be the development of a transgenic mouse model with humanized STING and STAT2. This approach could provide an immune competent mouse model for DENV that eliminates two of the potential bottlenecks that exist for DENV replication in mice.
The animal protocol used in this study was reviewed and approved by the University of Miami Institutional Animal Care and Use Committee (IACUC) under IACUC protocol 11-181 ''Host Defense and the Regulation of Interferon Production: STING.''
The University of Miami has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. The assurance number is #A-3224-01, effective July 11, 2007 . Additionally, as of July 20, 2010, the Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) has continued the University of Miami's full accreditation.
Vero, 293T and mouse embryonic fibroblast (MEFs), were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Baby hamster kidney cells (BHK) were grown in Glasgow minimal essential medium (MEM) supplemented with 10% FBS, and 20 mM HEPES. Mosquito cells derived from Aedes albopictus, clone C6/36, were expanded at 33uC in RPMI medium with 10% FBS. All media were supplemented with 100 U/ml of L-glutamine and 100 mg/ml of penicillin-streptomycin. All tissue culture reagents were purchased from Invitrogen.
Dengue virus serotype 2 (DENV-2) strains 16681 and New Guinea C were used in this study. DENV was grown in C6/36 insect cells for 6 days as described elsewhere [51] . Briefly, C6/36 cells were infected at a multiplicity of infection (MOI) of 0.01, and 6 days after infection, cell supernatants were collected, clarified, and stored at 80uC. The titers of DENV stocks were determined by limiting-dilution plaque assay on BHK cells [52] . Semliki Forest virus (SFV) expressing GFP and DENV-NS2B3 were generated as described previously [36] and titrated in BHK cells by immunofluorescence [53] .
Lentiviral vector constructs were built using conventional molecular biology techniques. Briefly, human STING cDNA was PCR amplified from pcDNA3.1 hSTING [13] and cloned into a lentiviral vector derived from pHR SIN CSGW [54] ( Figure 5G ). Mutations in the NS2B3 cleavage site at positions 94-96 of hSTING were obtained by overlap PCR. Residues RRG were changed to the corresponding murine sequence HCM.
Lentiviral vector derived viruses were obtained by transfection of HEK 293T with 3 plasmids encoding STING, HIV-1 Gag-Pol, and VSV-G respectively [55] . Viral supernatants were harvested 48 and 72 hours post-transfection, 0.45 mm filtered, concentrated at 14,000 g for 6 hours over a 20% sucrose cushion and frozen at 280uC until used.
Monocytes, pDCs, B cells and circulating CD11c + DCs (cDCs) were isolated from blood of healthy donors (New York Blood Center) using Miltenyi isolation kits. CD14 + clinimacs, for monocytes, CD123/BDCA4 kit for pDCs and BDCA1 kit for cDCs. B cells were isolated as part of the BDCA1 kit for isolation of cDCs according to manufacturers' instructions. The purity of each cell population was tested by flow cytometry as described below and was routinely 85-95% for CD14 + cells, 87-90% for pDCs, and 95-99% for both MDDCs and cDCs.
Samples of 5610 5 isolated cell populations were infected with DENV-2, 16681 at the indicated MOI in a total volume of 100 ml of DC media for 1 hour at 37 C. Then, DC media supplemented with 4% HS was added up to a final concentration of 10 6 cells/ml and cells were incubated for the remainder of the infections at 37 C. At the indicated times, cell supernatants were collected and cell pellets were used for RNA extractions. Whole PBMCs were used after ficoll centrifugation and samples of 60610 6 PBMCs were infected with DENV-2 at MOI of 1 or left uninfected. After 1 hour DC media 4% HS was added. Eighteen hpi supernatants were collected and isolation of the different cell populations after DENV-2 infection was carried out as described above.
Human MDDCs were obtained from healthy human blood donors (New York Blood Center), following a standard protocol as previously described [24] and described above. Briefly, after Ficoll-Hypaque gradient centrifugation, CD14+ cells were isolated from the mononuclear fraction using a MACS CD14 isolation kit (Milteny Biotec) according to the manufacturer's directions. CD14+ cells were then differentiated to naïve DCs by incubation during 5 to 6 days in DC medium (RPMI supplemented with 100 U/ml L-glutamine, 100 g/ml penicillin-streptomycin, and 1 mM sodium pyruvate) with the presence of 500 U/ml human granulocyte-macrophage colony-stimulated factor (GM-CSF) (Pe-proTech), 1,000 U/ml human interleukin 4 (IL-4) (PeproTech), and 10% FBS (Hyclone). To generate MDMs, monocytes were cultured in the presence of 2000 U/ml human granulocytemacrophage colony-stimulated factor (GM-CSF) for 10 days, and media was replenished (with same concentration of GMCSF) at days 2, 5 and 8. The purity of each cell population was confirmed by flow cytometry analysis and was at least 99% for MDDCs and 95% for MDMs.
Femurs and tibia of wild-type C57BL/6 mice (Jackson) were soaked in 70% ethanol, washed with RPMI (Invitrogen), and epiphyses were cut to expose the bone marrow. The bones were flushed with RPMI supplemented with 10% fetal bovine serum (FBS) to extract the bone marrow. Cells were pelleted by centrifugation, washed once with RPMI and resuspended in ammonium chloride red blood cell lysis buffer. RBC lysis was performed for 1 minute at room temperature, stopped with RPMI-FBS and cells were collected by centrifugation. Bone marrow cells were seeded in 6-well dishes in RPMI containing 10% FBS, 50 U/ml Penicillin (Invitrogen), 50 mg/ml Streptomycin (Invitrogen), 20 ng/ml GM-CSF (Peprotech), 10 ng/ml IL-4 (eBioscience), and 40 mM beta-mercaptoethanol (BIO-RAD). Cells were cultured for 5 days at 37 degrees C, 5% CO2 and fresh media was added every 2-3 days.
Human and mouse DCs were obtained as described above, and at day 5 of culture, samples of 1610 6 cells were resuspended in 100 ml of DC-medium and were infected for 45 min at 37uC with the indicated MOI of virus (diluted in DC media) or with DC medium (mock group) in a total volume of 200 ml. After the adsorption period, DC medium supplemented with 10% FBS was added up to a final volume of 1 ml, and cells were incubated for the appropriate time at 37uC. siRNA transfection and Dengue infection of human MDDCs 2.5610 4 MDDCs were seeded per well in 96 well plates and transfected with the corresponding siRNA using the StemFect RNA transfection kit (Stemgent), according to the manufacturer instructions. Chemically synthesized 27mer siRNA duplexes were obtained from OriGene Technologies, Inc. The sequences of the STING siRNA oligonucleotides used in this study are as follows:
siSTING -1:59-rGrGrCrArUrGrGrUrCrArUrArUrUrArCrAr-UrCrGrGrArUAT-39.
siSTING-2:59-rArCrCrUrGrUrGrArArArUrGrGrGrArUrCr-ArUrArArUrCAC-39.
siSTING-3:59-rGrGrArUrUrCrGrArArCrUrUrArCrArArUr-CrArGrCrArUTA-39.
Two random non-coding control siRNA were used: Sc-1 (59-rUrArCrGrUrArCrUrArUrCrGrCrGrCrGrGAT-3) from Qiagen, and Sc-2 (universal scrambled negative control siRNA duplex SR30004) from OriGene Technologies, INC. 48 h after transfection, cells were infected with Dengue virus at an MOI of 1. Briefly, cells were centrifuged (4006g, 10 min), the media was removed and 25 ml of RPMI containing the appropriate amount of virus was added and the plates were incubated for 45 min at 37uC. Then, 75 ml of RPMI with 10% FBS were added and cells were incubated at 37uC for the indicated hours. Subsequently, cells were recovered by centrifugation for 10 min at 4006g, and the cell pellets were lysed for RNA isolation.
Plated monocytes were transduced as previously described [56] . In brief, freshly isolated monocytes were transduced with VSV-G pseudo-typed SIV VLPs and each lentiviral vector construct for 3 h by spinoculation, in the presence of 2 mg/mL polybrene (Sigma). Subsequently, cells were washed, resuspended in regular growth medium described before for the generation of monocytes derived human dendritic cells and incubated for 5 days at 37uC until stimulation. At day 5 post-transduction, MDDCs were infected with DENV as described before, and cell pellets were collected at the indicated time points, and lysed for RNA isolation.
293T cells were transfected by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. A type I IFN production antagonist assay was performed as described previously [25] using IFNb-Luc and p55-C1B-Luc [34, 57] . 293T cells seeded on 24-well plates were transiently transfected with 50 ng of the luciferase reporter plasmid together with a total of 400 ng of various expression plasmids or empty control plasmids. As an internal control, 50 ng pRL-TK was transfected simultaneously. Then, 24 or 48 h later, the luciferase activity in the total cell lysate was measured by using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's directions.
Transfection of 293T cells and infection of human DCs was performed as described above. Cell lysates were obtained after incubation of cells with RIPA lysis buffer (Sigma Aldrich) supplemented with complete protease inhibitor (Roche) and resuspended in a total of 50 ml of Laemmli sample buffer (Bio-Rad). Crude lysates were either boiled for 10 min or incubated at 42uC for 20 min and then kept on ice. Each sample was loaded in a polyacrylamide-SDS gel, and the proteins were electrophoretically separated by conventional methods. Proteins were transferred to nitrocellulose, and blots were incubated with anti-HA, anti-FLAG, anti-Actin anti-GAPDH (Sigma Aldrich) and rabbit polyclonal antibodies anti-hSTING [41] and anti-DENV NS3 (kind gift of Dr. Andrea Gamarnik), and developed using SNAP ID detection system (Millipore), following the manufacturer's instructions. Antibody-protein complexes were detected using a Western Lighting chemiluminescence system (Perkin Elmer).
RNA from different cells was extracted using Trizol (Invitrogen), followed by a treatment with DNase using DNA-free Ambion. The concentration was evaluated in a spectrophotometer at 260 nm, and 500 ng of RNA were reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions.
Evaluation of the expression of human and mouse cytokines from different cell types and viral RNA was carried out using iQ SYBR green Supermix (Bio-Rad) according to the manufacturer's instructions. The PCR temperature profile was 95uC for 10 min, followed by 40 cycles of 95uC for 10 s and 60uC for 60 s. Expression levels for individual mRNAs were calculated based on their CT values using two different housekeeping genes (human: rps11 and a-tubulin genes) and (mouse: S18 and b-Actin) to normalize the data.
One paired two tailed Student's t-test was used to analyze data. Data considered significant demonstrated p values less than 0.05. Acute Reactogenicity after Intramuscular Immunization with Recombinant Vesicular Stomatitis Virus Is Linked to Production of IL-1β Vaccines based on live viruses are attractive because they are immunogenic, cost-effective, and can be delivered by multiple routes. However, live virus vaccines also cause reactogenic side effects such as fever, myalgia, and injection site pain that have reduced their acceptance in the clinic. Several recent studies have linked vaccine-induced reactogenic side effects to production of the pro-inflammatory cytokine interleukin-1β (IL-1β) in humans. Our objective was therefore to determine whether IL-1β contributed to pathology after immunization with recombinant vesicular stomatitis virus (rVSV) vaccine vectors, and if so, to identify strategies by which IL-1β mediated pathology might be reduced without compromising immunogenicity. We found that an rVSV vaccine induced local and systemic production of IL-1β in vivo, and that accumulation of IL-1β correlated with acute pathology after rVSV immunization. rVSV-induced pathology was reduced in mice deficient in the IL-1 receptor Type I, but the IL-1R−/− mice were fully protected from lethal rechallenge with a high dose of VSV. This result demonstrated that IL-1 contributed to reactogenicity of the rVSV, but was dispensable for induction of protective immunity. The amount of IL-1β detected in mice deficient in either caspase-1 or the inflammasome adaptor molecule ASC after rVSV immunization was not significantly different than that produced by wild type animals, and caspase-1−/− and ASC−/− mice were only partially protected from rVSV-induced pathology. Those data support the idea that some of the IL-1β expressed in vivo in response to VSV may be activated by a caspase-1 and ASC-independent mechanism. Together these results suggest that rVSV vectors engineered to suppress the induction of IL-1β, or signaling through the IL-1R would be less reactogenic in vivo, but would retain their immunogenicity and protective capacity. Such rVSV would be highly desirable as either vaccine vectors or oncolytic therapies, and would likely be better tolerated in human vaccinees. Vaccines based on attenuated viral vectors (e.g. poxviruses, adenovirus) are highly immunogenic, relatively inexpensive to produce, and can be delivered by multiple routes. These advantages have accelerated the development of virus-vectored vaccines, especially as concerns about emerging infectious diseases such as avian influenza and multi-drug resistant tuberculosis increase. However, pre-existing anti-vector immunity can significantly impair the ability to raise immune responses to vaccination in the pre-immune host. That suggests that it will be advantageous to prioritize development of viral vaccine vectors to which preexisting immunity is rare in the human population. Vaccine vectors based on recombinant vesicular stomatitis virus (rVSV) are highly immunogenic and can protect small animals and nonhuman primates against a range of infectious diseases [1, 2, 3, 4, 5] . VSV is also a potent oncolytic agent, and has been shown to be superior to nine other viruses tested against multifocal glioma [6, 7] and other cancers [8, 9] . Natural infection of humans with VSV is extremely uncommon, and therefore pre-existing immunity to rVSV vectors is almost non-existent [10, 11, 12] . Despite these advantages, development of rVSV for human clinical use continues to be delayed because of concerns about vectorassociated pathology. Small laboratory animals immunized with rVSV vaccines display clinical signs of acute illness and lose up to 20% of their pre-immunization body weight in the first four days after immunization [13, 14] . Although most animals recover, undesirable side effects such as these would be unacceptable in human vaccinees. Weight loss is less pronounced in non-human primates immunized with rVSVs, but the one human to date therapeutically immunized with a single cycle non-replicating experimental rVSV vaccine against Ebola virus developed a high fever and transient viremia in the first 24 hours after vector administration [15] . That result demonstrated that even single cycle vectors can induce significant adverse side effects, and suggested that alternate strategies to reduce residual viral vector reactogenicity are required.
For rVSV-based vaccines to move forward to clinical use it is important to identify ways to eliminate rVSV vector-associated pathology that do not at the same time compromise vector immunogenicity or oncolytic properties. It has been shown previously that TNF-a produced in response to intranasal immunization with rVSV vaccine vectors contributes to, but is not the only factor responsible for, rapid weight loss after rVSV immunization [14] . In those studies, TNF-a deficient mice were partially protected from acute pathology after VSV challenge, and TNF-a was not required for the generation of humoral immune responses to rVSV [14] . We undertook the present study to expand on those observations, focusing on the importance of IL-1. We addressed intramuscular immunization, because that is the most likely route of administration for rVSV vectors in humans.
Mice deficient in the interleukin-1 receptor (IL-1R2/2) are protected from weight loss after immunization with VSV vaccine vectors Mice immunized with recombinant VSV (rVSV) vaccine vectors lose weight rapidly after immunization. Weight loss is usually maximal by the second day after immunization but most animals recover, with weight returning to normal by five to seven days after immunization. Interleukin-1b (IL-1b) is a pro-inflammatory cytokine that causes acute weight loss and fever in mice and humans [16, 17, 18] , and which stimulates the production of other pro-inflammatory mediators such as IL-6 [17, 19] and prostaglandin E2 (PGE 2 ) [20, 21] . To determine whether IL-1 can contribute to the induction of acute pathology after administration of rVSV vaccine vectors, we immunized groups of C57BL/6 wild type (WT, n = 4) or IL-1 receptor deficient (IL-1R2/2, n = 3) mice with a single intramuscular injection of 5610 8 PFU live replication-competent rVSV. All animals survived the immunization, but wild type mice lost approximately 10% of their preimmunization body weight by the first day after immunization ( Figure 1 ) and did not fully regain their pre-immunization weight until eight days after immunization. In contrast, IL-1R2/2 mice had minimal pathology, losing only a small amount (,5%) of weight on the first day after immunization and returning to their starting weight by the second day after immunization. IL-1R2/2 mice lost significantly less weight than wild type control animals after challenge (P = 0.03 from day 1-day 4 after challenge, Mann-Whitney test). Because mice deficient in the IL-1R cannot respond to IL-1, this result demonstrated that either IL-1a or IL-1b, both of which bind to the IL-1R, contributed to acute pathology after rVSV immunization.
Mice immunized with VSV vaccine vectors produce IL-1b systemically and at the injection site Both IL-1a and IL-1b are regarded as pro-inflammatory cytokines, but IL-1b rather than IL-1a has been more commonly associated with symptoms such as weight loss and fever that are induced by infection with a live virus or other immune stimulus [16, 17] . To determine whether intramuscular immunization with VSV vaccine vectors induced production of IL-1b in vivo we challenged wild type C57BL/6 mice with rVSV and used an ELISA to quantitate the amount of IL-1b produced locally (at the injection site and in the draining lymph node) and systemically (in the blood). As shown in Figure 2A -C, mice immunized intramuscularly with rVSV had accumulations of IL-1b in the blood, in the quadriceps muscle, and in the popliteal lymph node that drains the quadriceps at 12 and 24 hours after immunization with VSV rwt. These results confirmed that IL-1b was produced in vivo both locally and systemically after intramuscular rVSV challenge, and was consistent with the results of Poeck et al. who found that wild-type mice injected intravenously with rVSV had detectable levels of IL-1b in the serum at six hours after challenge [22] . Our results were also consistent with the hypothesis that IL-1b caused the acute pathology that occurs after immunization with rVSV vaccine vectors, and suggested that reducing the production of IL-1b after rVSV immunization might correspondingly reduce reactogenicity and other undesirable side effects of vaccination.
Mice deficient in the IL-1R control rVSV replication, have normal cellular and humoral immune responses, and are immune to high dose re-challenge IL-1b causes some deleterious effects such as fever and anorexia, but has also been shown to contribute positively to the generation of adaptive immune responses induced by live virus infection [23] . The role of IL-1 in control of VSV replication has not been examined previously, and reports describing the role of IL-1 in control of other viruses in vivo are not in agreement. Because our ultimate goal is to be able to generate less reactogenic rVSV vectors by attenuating the host inflammatory response, it was important to determine whether IL-1 was required for the control of vaccine vector replication, generation of humoral and cellular immune responses, or to generate protection against rechallenge. To investigate these questions we intramuscularly immunized mice deficient in the IL-1 receptor (IL-1R2/2) with 5610 8 PFU of rVSV and measured viral loads, induction of antibody and T cell responses, and protection from rechallenge. As shown in Figure 3A , viral loads in the quadriceps muscle (injection site) were not significantly different between wild type and IL-1R2/2 mice (n = 3 per group) at 24 hours after infection, which is the peak of VSV replication in vivo. Also, virus was not detected 2) are protected from acute weight loss after intramuscular immunization with rVSV. Eight to ten week old female C57BL/6 mice were immunized with a single intramuscular injection to the left rear quadriceps of 5610 8 PFU of live replicating rVSV. Graph shows average percent initial weight by day for each group of mice beginning on the day of immunization (Day 0). IL-1R2/2 mice (n = 3) lost less weight and recovered more quickly than wild type (WT, n = 4) mice after immunization. The difference in weight loss was significant from day 1-day 4 after challenge (P = 0.03, Mann-Whitney test). This experiment has been performed two times with consistent results. doi:10.1371/journal.pone.0046516.g001
in the blood of any infected animal. Those results demonstrated that IL-1 is not required for the control of VSV replication in vivo. As shown in Figure 3B , serum neutralizing antibody titers against VSV were not significantly different in IL-1R2/2 (n = 4 per group per timepoint) and wild type mice (n = 5 per group per timepoint) at any time after immunization. When we used an MHC Class I tetramer to measure CD8 T cell responses to an immunodominant H-2 K b -restricted epitope (N-RGYVYQGL-C) in the VSV N protein [24] , IL-1R2/2 mice had slightly fewer anti-VSV N specific CD8 T cells in the blood than did wild type animals ( Figure 3C ) at 14 and 28 days after immunization, although the difference was only statistically significant at 14 days after immunization (P = 0.03, Two-tailed T test). Finally, to determine whether these immune responses were sufficient to protect wild type and IL-1R2/2 mice from re-challenge with rVSV, we challenged all animals intranasally with 1610 8 PFU of rVSV at eight weeks after the primary infection. As shown in Figure 3D , pre-immune wild type and pre-immune IL-1R2/2 mice were fully protected from rechallenge, while naïve wild type animals (n = 7, open triangles, Figure 3D ), lost up to 20% of their pre-infection body weight. Two out of the seven naïve animals succumbed to infection. Together, these results indicated that IL-1 was not required either for the control of rVSV replication in vivo, or for the generation of protective anti-VSV immune responses. The results also supported the idea that suppressing the production of or response to IL-1b in response to rVSV vector administration would not render rVSV vectors unsafe or non-immunogenic.
Mice deficient in inflammasome adaptor molecule ASC are partially protected from acute weight loss after immunization with rVSV vaccine vectors Because the ultimate goal of these studies was to devise strategies by which we could suppress IL-1b production and thereby reduce the pathology of rVSV vectors in vivo, we sought to determine the mechanism by which IL-1b was being produced in response to VSV. IL-1b is synthesized as an inactive precursor molecule (pro-IL-1b), which must be cleaved either intracellularly by endogenous protease caspase-1 [25, 26, 27] , or extracellularly by matrix metalloprotease 9 [28] or other neutrophil [29] and mast cell-associated proteases [30] to become biologically active. It was shown recently that murine bone marrow derived dendritic cells (BMDC) infected with VSV in vitro produce IL-1b via formation of an inflammasome composed of RNA helicase RIG-I, adaptor molecule ASC, and caspase-1 [22] . Because the authors did not test whether RIG-I, ASC, and caspase-1 were required to produce IL-1b in response to VSV in vivo we used caspase-1-deficient and ASC-deficient mice to determine the effects of the absence of these molecules on acute pathology after rVSV immunization. It was not practical to test RIG-I deficient mice for IL-1b induction, because RIG-I deficient mice do not produce IFN in response to VSV and therefore rapidly succumb to infection [31] .
We challenged ASC-deficient mice (ASC2/2) and wild type C57BL/6 mice intramuscularly with 5610 8 PFU rVSV as in Figures 1, 2 , and 3 and measured production of IL-1b and acute pathology after immunization. As shown in Figure 4A , the systemic and local production of IL-1b was not significantly reduced in ASC2/2 mice relative wild type control mice immunized in parallel. Consistent with that result, and with our prediction that IL-1b induces acute weight loss after rVSV infection, ASC2/2 (n = 5) mice lost slightly less weight after rVSV challenge than did wild type C57BL/6 (n = 6) mice infected in parallel, with the difference only reaching statistical significance only on the second day after challenge (P = 0.004, Mann Whitney Test, Figure 4B ). A second experiment replicated these findings almost exactly, with ASC2/2 mice (n = 6) showing slightly enhanced protection relative wild type animals (n = 10, Figure S1 ). The difference in weight loss between wild type and ASC2/2 mice was small but highly reproducible, and ASC2/2 mice were never protected to the same extent as IL-1R2/2 mice. To confirm that observation, we infected the three groups in parallel (5610 8 PFU rVSV intramuscular). As shown in Figure 4C , IL-1R2/2 mice (n = 4) lost significantly less weight than wild type (n = 5) mice (days 1-4 after challenge P,0.05 via one way ANOVA with Bonferroni multiple comparison test) and recovered their pre-immunization body weight more quickly than either wild type or ASC2/2 mice. The difference in weight loss between IL-1R2/2 and ASC2/2 mice was significant on the first and second day after challenge (P,0.05 via one way ANOVA with Bonferroni multiple comparison test). Similar to the results obtained in IL-1R2/2 mice, ASC2/2 mice made equivalent humoral responses and slightly reduced cellular responses to VSV, and were fully protected from high dose rechallenge ( Figure 4D -F). Taken together, these results demonstrated that production of IL-1b in vivo after intramuscular rVSV immunization occurred independent of inflammasome adaptor molecule ASC. Because ASC deficient mice were not protected fully from acute pathology after rVSV immunization, strategies that suppress the function of ASC would be predicted to partially but not completely abrogate acute pathology after rVSV immunization. Mice deficient in caspase-1 are partially protected from acute weight loss after immunization with rVSV vaccine vectors Similarly, when we immunized mice deficient in caspase-1 (caspase-12/2) or wild type mice with 1610 9 PFU of rVSV, caspase-1 deficient mice did not have significantly reduced levels of IL-1b relative wild type mice ( Figure 5A ) when we measured IL-1b production at the injection site and in the draining lymph node by ELISA. Consistent with those data, caspase-1 deficient mice immunized intramuscularly with 5610 8 PFU of rVSV as in Figure 4B -C (n = 5 per group), were partially but not completely protected from acute weight loss ( Figure 5B ) relative wild type mice (n = 5), with the difference in weight loss between wild type and caspase-12/2 mice being significant only on days 2 and 3 after challenge (P,0.05, Mann Whitney test). Caspase-1 deficient mice controlled viral replication as well as wild type mice ( Figure 5C , n = 6 per group), with no significant difference in viral loads in the quadriceps muscle of infected animals at 24 hours after infection. As with IL-1R2/2 mice, no virus was recovered from the blood of infected mice. Caspase-1 deficient mice made robust humoral ( Figure 5D ) and cellular ( Figure 5E ) responses to VSV, which were not significantly different than those of wild type mice.
We undertook this study with the goal of determining which cytokines induced by rVSV contribute to acute pathology after intramuscular immunization. This is important because while VSV is a highly promising vaccine vector and oncolytic agent, its clinical development has lagged behind that of other live viruses because of concerns about vector-associated pathology. Fever, myalgia, and the ''sickness response'' are induced by many live viral or bacterial vaccines [32, 33, 34, 35, 36] , and these vaccineinduced side effects are among the leading reasons why some individuals elect not to receive protective vaccines [37, 38, 39] . Consistent with our results obtained in the mouse model and presented here, several recent studies have correlated increased levels of IL-1 or other pro-inflammatory cytokines with the induction of high fevers or other adverse events in response to live There was no significant difference in viral loads between the two groups. In Panels B-D, adult female wild type (n = 5) or IL-1R2/2 (n = 4) mice were immunized intramuscularly with a single injection of 5610 8 PFU rVSV in the rear quadriceps. At the indicated timepoints after infection mice were bled and humoral and cellular immune responses were assayed. Panel B shows average anti-VSV neutralizing antibody responses by group as measured by microneutralization assay. Error bars represent the upper and lower limits of the 95% confidence interval. Panel C shows average percent CD8 T cells specific for the VSV N1 epitope as measured by MHC Class I tetramer. At 14 days after immunization, WT mice had significantly more (P = 0.03, Twotailed T test) VSV N specific CD8 T cells than IL-1R2/2 mice, but by day 28 the difference was no longer significant (P = 0.13). At eight weeks after the primary infection all mice were challenged intranasally with a semi-lethal dose of rVSV (1610 8 PFU). A cohort of naïve wild type mice (n = 7) was challenged at the same time. All pre-immune mice had robust immunity to rechallenge (Panel D) and did not lose weight or exhibit other signs of pathology. Two of the naïve mice succumbed to infection. Days on which naïve animals succumbed are indicated with an asterisk on the graph. doi:10.1371/journal.pone.0046516.g003 Figure 4 . Mice deficient in the inflammasome adaptor ASC (ASC2/2) are partially protected from acute weight loss after intramuscular immunization with rVSV. Groups of adult female C57BL/6 wild type or ASC2/2 mice were immunized with two injections (5610 8 PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 12 and 24 hours after infection mice (n = at least 4 per timepoint), were sacrificed and IL-1b in the blood (Panel A left), draining popliteal LN (Panel A middle), and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1b produced by wild type and ASC2/2 mice was not significantly different at any time or in any organ. The comparison of IL-1b production by wild type and ASC2/2 mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type (n = 6) and ASC2/2 (n = 5) mice after intramuscular challenge with 5610 8 PFU of rVSV. The comparison of WT and ASC2/2 mice has been performed four times with consistent results. Panel C shows average percent initial weight for wild type (n = 5), ASC2/2 (n = 5), and IL-1R2/2 (n = 4) mice infected with rVSV. IL-1R2/2 mice lost significantly less weight than wild type (days 2-4) or ASC2/2 mice (days 1-2) (P,0.05 via one way ANOVA with Bonferroni test). The comparison of WT, IL-1R2/2, and ASC2/2 mice has been performed twice with consistent results. Panel D shows average serum neutralizing titers for WT (n = ) and ASC2/2 (n = ) mice after primary immunization with VSV. There were no significant differences in neutralizing titer between the two groups. Panel E shows average percent CD8 T cells specific for the VSV N1 epitope as measured by MHC Class I tetramer. At 14 days after immunization, WT mice (n = 9) had significantly more (P = 0.001, Two-tailed T test) VSV N specific CD8 T cells than ASC2/2 mice (n = 9), but by day 28 the difference was no longer significant (P = 0.07). At eight weeks after the primary infection preimmune WT (n = 10) and ASC2/2 (n = 6) mice were challenged intranasally with a semi-lethal dose of rVSV (1610 8 PFU). A cohort of naïve wild type mice (n = 5) was challenged at the same time. All pre-immune mice had robust immunity to rechallenge (Panel F) and did not lose weight or exhibit other signs of pathology. One of the naïve mice succumbed to infection. doi:10.1371/journal.pone.0046516.g004 Figure 5 . Mice deficient in caspase-1 are partially protected from acute weight loss after intramuscular immunization. Groups of adult C57BL/6 wild type or caspase 12/2 mice were immunized with two injections (5610 8 PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 24 hours after infection mice (n = 2-3 mice per timepoint), were sacrificed and IL-1b in the draining popliteal LN (Panel A left) and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1b produced by wild type and caspase 12/2 mice was not significantly different in either organ. The comparison of IL-1b production by wild type and caspase 12/2 mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type and caspase 12/2 mice (n = 5 per group) after intramuscular challenge with 5610 8 PFU of rVSV. The comparison of WT and caspase 12/2 mice has been performed twice with consistent results. Caspase 12/2 mice lost significantly less weight than wild type controls on days 2 and 3 after challenge (P,0.05 via Mann Whitney test). Panel C shows average viral loads in the quadriceps muscle of infected mice (n = 6 per group). Data is compiled from two identically performed experiments. Viral loads in WT and caspase 12/2 mice were not significantly different. Panel D shows average serum neutralizing antibody titers for wild type and caspase 12/2 mice immunized with rVSV (n = 5 per group per timepoint). Error bars represent the upper and lower limits of the 95% confidence interval. Panel E shows average percent 6 SEM of CD8 T cells in the blood binding to an MHC Class I tetramer recognizing an immunodominant epitope within VSV N. There were no significant differences in the antibody or CD8 T cell responses between the two groups at any time. The comparison of humoral and cellular immune responses in WT and caspase 12/2 mice has been performed twice with consistent results. doi:10.1371/journal.pone.0046516.g005 virus vaccination in humans [36, 40] , or have identified genetic polymorphisms in the IL-1 gene which predispose individuals to severe adverse events after receiving live virus vaccines [41] . For these reasons it is important to determine the ways in which proinflammatory cytokines contribute to reactogenicity of VSV vectors in particular, not only because those findings could advance development of VSV-based therapeutics, but also because our findings might help to inform the development of other live virus vaccines and oncotherapies.
Previous studies in which VSV vectors were delivered to mice intranasally showed that intranasal immunization with VSV induces the pro-inflammatory cytokine TNF-a, and that TNF-a production directly correlated with weight loss and acute pathology [14] . Although intranasal immunization has many advantages (needle free delivery, induction of mucosal immunity, etc), a significant drawback to intranasal immunization with rVSV is the risk of neurotropic spread of the virus. VSV instilled in the nose rapidly colonizes the olfactory neurons, and migrates into the brain [42] . The neurovirulence of VSV vectors has been significantly reduced by attenuating the capacity of the VSV vector to replicate [43] , but even a remote chance of neurotropic spread will likely prevent use of the intranasal route for human inoculations. Therefore, we decided to determine which cytokines were responsible for acute pathology after intramuscular immunization, which is regarded as a safer route by which to administer potentially neurotropic agents.
We show here that mice deficient in the interleukin-1 receptor (IL-1R) are significantly protected from weight loss after intramuscular challenge with VSV. Although these results do not preclude the contribution of other inflammatory processes to pathology, they do positively identify IL-1 as an important target for intervention.
The IL-1 receptor binds IL-1a and IL-1b. Therefore because IL-1R2/2 mice were protected from pathology, it was possible that IL-1a, IL-1b, or both cytokines contributed to VSVassociated pathology in vivo. IL-1b has been well characterized as a mediator of acute inflammatory responses in mice and humans, namely the induction of fever and cachexia, the acute phase response, and upregulation of other inflammatory mediators such as IL-6 in response to infectious or non-infectious stimuli [44, 45] , reviewed in [46] . IL-1b is more commonly associated with these pathologies than is IL-1a. For example, IL-1b deficient mice, but not IL-1a deficient mice, are protected from fever after injection of turpentine [16, 17] . Similarly, IL-1b, but not IL-1a, is the etiologic agent of hereditary periodic fever syndromes and other ''autoinflammatory'' diseases in humans [47, 48] . IL-1a is more commonly associated with ''sterile'' inflammation that accompanies apoptotic cell death [49] , and with the exception of adenovirus mediated inflammation [50] has not been associated with pathology arising from viral infection. For these reasons, we predict that IL-1b is the primary mediator of pathology in our system, but further experiments will be required to formally exclude a role for IL-1a.
Once we had established that IL-1 contributed to acute pathology after intramuscular VSV inoculation, the most important question to pursue was whether IL-1 would be required for control of VSV replication in vivo, and for the induction of immune responses to the VSV vector. If IL-1 were not required for control of virus replication, or for generation of protective immune responses, then that would suggest that engineering rVSV vectors to suppress IL-1 production and/or signaling would be a rational approach to reducing rVSV associated pathology.
Although three separate reports have now described the induction of IL-1 in response to VSV challenge in vivo [22, 51, 52] , none of these had examined whether or not the induced IL-1 contributed positively to or was required for generation of immune responses or protection. In other viral infection models where this has been investigated, published reports are not in agreement. For example, Schmitz et al found that IL-1R2/2 mice infected with influenza had a significantly higher rate of mortality than did wild type control animals, but the increase in mortality did not correlate with a higher virus burden in the IL-1R2/2 mice [53] . Three additional reports examined the role of IL-1b, as well as components of the Nlrp3 inflammasome, in control of influenza infection [23, 54, 55] . In these, two out of three found transiently elevated viral loads in mice in which IL-1b production was decreased [23, 54] , the other report found no difference in virus burden [55] . Similarly, only one of the three reports [23] found that a reduction in IL-1b correlated with a reduction in cellular immune responses-the others either did not examine immune responses or did not find a correlation.
In this study, we found that IL-1R2/2 mice controlled viral loads as well as wild type control animals. Viral loads at the injection site were not significantly different between groups, and neither group had a detectable viremia after challenge. This result highlights the relative safety of the intramuscular immunization route. Equally important was that IL-1R2/2 mice challenged with VSV made robust humoral and cellular responses to VSV antigens, and were fully protected from a high dose intranasal rechallenge with VSV. Interestingly, we did observe levels of anti-VSV CD8 T cells in the IL-1R2/2 mice and in ASC2/2 mice were transiently but significantly decreased, which is consistent with the report of Iwasaki et al, who found that anti-influenza CD8 T cell responses were slightly decreased in mice with reduced levels of IL-1b production [23] . In our studies, the number of anti-VSV specific CD8 T cells was significantly lower in IL-1R2/2 and ASC2/2 mice at 14 days after infection, but was not significantly different at either 7 or 28 days after infection. This finding warrants further investigation, and follow-up studies will focus on determining whether CD8 T cells primed in the absence of IL-1 signaling are functionally different (e.g. in cytotoxic capacity, or in acquisition of central memory phenotype) from those primed in IL-1 intact animals.
In summary, these results support the idea that IL-1 production is not required either for control of VSV replication in vivo, or for the induction of protective immune responses to VSV antigens after intramuscular immunization. Because the relative magnitude of immune response to foreign antigens expressed by VSV is generally similar to the relative magnitude of the immune response to VSVs own antigens [56] , we predict that IL-1 would also not be required for the induction of immune responses to vaccine antigens expressed by VSV. Nonetheless, determining whether IL-1 is important for the response to foreign (vaccine) antigens expressed by an rVSV will be an important direction for further study.
The second question arising from our results is how the VSVinduced production of IL-1 might be suppressed. One means of suppressing the biological response to IL-1 in humans is via injection of recombinant IL-1 receptor antagonists. Currently marketed as the drugs Anakinra or Kineret, these agents effect a systemic suppression of IL-1a and IL-1b. Although systemic suppression of IL-1 is an effective treatment for some conditions (severe rheumatoid arthritis, for example) [57] , systemic suppression of IL-1 also carries the risk of enhanced susceptibility to infection [57, 58] . Therefore we decided to determine whether there might be a way of suppressing IL-1 only in the cells directly infected with VSV, or within the focus of VSV-infected cells. To determine the appropriate target molecule(s) to effect local suppression of IL-1, it was necessary to determine the mechanism by which IL-1 was being produced in our model. Several years ago, Muruve et al reported that adenovirus activated human monocytes (THP-1 cells) to produce IL-1b in vitro, and that IL-1b production was dependent upon NLRP3 and caspase-1 [51] . In the supplement to that manuscript, the authors reported that infection of THP-1 cells with VSV did not induce IL-1b production, although detailed methods for that study were not provided. In contrast to that, two more recent studies reported that VSV-infected THP-1 cells do produce IL-1b in vitro. In the first study, Poeck et al found that VSV induced production of IL-1b was dependent upon caspase-1, but not upon NLRP3 [22] . In contrast to that, Rajan et al found VSV mediated induction of IL-1b from THP-1 cells to be dependent on NLRP3, and dependent on caspase-1 [52] . It is likely that the discrepancies in these reports were due to subtle differences in experimental procedures, but the different outcomes reported by each of these groups highlight the complexity of elucidating the precise mechanism(s) by which VSV may induce IL-1.
In our studies we found that VSV induced robust production of IL-1b in vivo and that IL-1b was still produced in mice lacking caspase-1 or ASC. One caveat to those findings is that ELISA assays such as the one used here do not rigorously discriminate between detection of pro-IL-1b and detection of the cleaved active IL-1b. Therefore it is possible that the some of the IL-1b detected in caspase2/2 mice was not biologically active. Precise determination of the amount of active IL-1b present in caspase2/2 mice will require the development of better detection reagents. Despite this limitation, our results support a model in which there are multiple pathways by which mature IL-1b is produced in vivo in response to VSV. The data are also consistent with the idea that the manner in which mature IL-1b is produced (via caspase-1 or not) may vary with the cell in which the IL-1b is induced. We further observed that mice lacking caspase-1 or ASC were not protected from acute pathology to the same extent that IL-1R2/ 2 mice were. That meant that VSV vectors engineered to suppress components of the RIG-I/ASC/caspase-1 inflammasome, or the Nlrp3/ASC/caspase-1 inflammasome, would be unlikely to be significantly less reactogenic than the parent vector. On the other hand, rVSV designed to suppress the biological response to IL-1b (for example via inclusion of a soluble IL-1b trap), or to IL-1a and IL-1b (via inclusion of a soluble IL-1 receptor antagonist) might reduce the biological response to IL-1 in vivo and therefore be less reactogenic.
In summary, we have shown here that IL-1 contributes to acute pathology after intramuscular immunization with VSV. IL-1 was not required for control of viral replication, for the induction of cellular or humoral immune responses, or for development of protective immunity to rechallenge. These results add to our understanding of the role for IL-1 in promoting immunity to viral challenge, and support the notion that the requirement for IL-1b in promoting adaptive immunity may vary according to the type and dose of pathogen encountered as well as the route of exposure. Finally, by identifying IL-1b as a major source of reactogenicity for rVSV vaccines, we are able to propose a novel strategy to ameliorate side effects without compromising immunogenicity.
All animal studies were reviewed and approved by the Duke University Institutional Animal Care and Use Committee.
Vesicular stomatitis virus (Indiana strain) was originally obtained from Dr. John Rose (Yale University). Virus was propagated on BHK-21 cells (ATCC CCL-10) and titered using a standard plaque assay.
Eight to ten-week-old C57BL/6 wildtype and IL-1 receptor type 1 deficient (IL-1R2/2, strain name B6.129S7-Il1r1 tm1Imx/J ) mice were obtained from Jackson Laboratories. Caspase12/2 on the C57BL/6 background were generously provided by Dr. Fayyaz Sutterwala and Dr. Richard Flavell. ASC2/2 and Nlrp32/2 mice on the C57BL/6 background were generously provided from Genentech Inc. (San Francisco, CA). Mice obtained commercially were housed for at least 1 week before experiments were initiated. Mice were housed in microisolator cages in a biosafety level 2-equipped animal facility. Viral stocks were diluted to appropriate titers in serum-free DMEM. For intramuscular immunization (i.m.), mice were injected with the indicated amount of virus(es) in 50 ml total volume. For intranasal (i.n.) vaccination, mice were lightly anesthetized with isoflurane using a vaporizer and administered the indicated amount of virus in 30 ml total volume. The Institutional Animal Care and Use Committee of Duke University approved all animal experiments.
Mice were sacrificed via anesthetic overdose and organs removed aseptically. After dissection organs were weighed, and homogenized in sterile buffer (100 ml buffer per 0.1 g organ weight). Homogenates were titered by standard plaque assay on BHK-21 cells (ATCC CCL-10) using a semi-solid overlay to detect infectious VSV. After 48 hours the overlay was removed and the cell layer stained with crystal violet to visualize plaques.
Mice were bled and then sacrificed via anesthetic overdose and organs removed aseptically. Organs were homogenized in 500 mL (100 mL for lymph nodes) of buffer containing 137 mM NaCl, 20 mM Tris-Cl pH 8.0, 5 mM EDTA, 0.05% Triton-X 100, and protease inhibitor cocktail (Roche). Serum and organ homogenate supernatants were assayed for IL-1b by ELISA (R&D Systems). Organ IL-1b amounts were normalized to the amount of protein in the samples, as determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific).
Blood was obtained from mice on days 7, 14, and 28 after vaccination via cheek bleed. Heat inactivated serum was diluted in serum-free DMEM such that the final dilution in the first well of a 96-well plate was 1:10 for day 7 samples and 1:100 for day 14 and 28 samples, with subsequent two-fold dilutions. Samples were assayed in duplicate. 100 PFU of rVSV diluted in serum-free DMEM was added to each well and incubated for one hour at 37uC-5%CO 2 , after which 4,000 BHK-21 cells (ATCC CCL-10) diluted in 5%FBS-DMEM were added to each well. Plates were incubated at 37uC-5%CO 2 for three days, and cytopathic effect was observed. The neutralizing titer was defined as the highest dilution of serum that gave 100% neutralization of rVSV.
To obtain peripheral blood lymphocytes blood was collected into serum free medium (DMEM) containing heparin. Blood was layered onto a Ficoll gradient and spun, after which lymphocytes were collected from the interface. Cells were washed and resuspended in DMEM containing 5% FCS.
Staining was performed on freshly isolated lymphocytes as previously described [4] . Briefly, approximately 5610 6 cells were added to the wells of a 96-well V-bottom plate and were blocked with unconjugated streptavidin (Molecular Probes) and F c block (Pharmingen) for 15 min at room temperature (RT). Following a 5-min centrifugation at 5006g, lymphocytes were labeled with a FITC-conjugated anti-CD62L antibody, (Pharmingen), an allophycocyanin-conjugated anti-CD8 antibody (Pharmingen), and tetramer for 30 min at RT. The tetramer was a PE-conjugated major histocompatibility complex (MHC) class I K b tetramer (NIH Tetramer Facility) containing the H-2K b restricted peptide VSV N 53-59 (N-RGYVYQGL-C). Sham-inoculated control animals were used to determine background levels of tetramer binding. Background was routinely less than 0.1% and was subtracted from all reported percentages.
Statistical comparisons were made using GraphPad Prism software. Results were considered significant when P,0.05. Figure S1 Mice deficient in the inflammasome adaptor ASC (ASC2/2) are partially protected from acute weight loss after intramuscular immunization with rVSV. Average percent initial weight for wild type (n = 10) and ASC2/2 (n = 6) mice after intramuscular challenge with 5610 8 PFU of rVSV. The difference in weight loss between wild type and ASC2/2 mice was significant on the first and second day after challenge (P,0.05, Mann Whitney test).
Author Contributions Competition between Influenza A Virus Genome Segments Influenza A virus (IAV) contains a segmented negative-strand RNA genome. How IAV balances the replication and transcription of its multiple genome segments is not understood. We developed a dual competition assay based on the co-transfection of firefly or Gaussia luciferase-encoding genome segments together with plasmids encoding IAV polymerase subunits and nucleoprotein. At limiting amounts of polymerase subunits, expression of the firefly luciferase segment was negatively affected by the presence of its Gaussia luciferase counterpart, indicative of competition between reporter genome segments. This competition could be relieved by increasing or decreasing the relative amounts of firefly or Gaussia reporter segment, respectively. The balance between the luciferase expression levels was also affected by the identity of the untranslated regions (UTRs) as well as segment length. In general it appeared that genome segments displaying inherent higher expression levels were more efficient competitors of another segment. When natural genome segments were tested for their ability to suppress reporter gene expression, shorter genome segments generally reduced firefly luciferase expression to a larger extent, with the M and NS segments having the largest effect. The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells. Our data indicate that IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription. The mechanism of replication and transcription varies greatly among viruses depending on the nature and structure of their viral genomes. Negative-strand RNA viruses replicate their viral genome via the synthesis of full length positive-strand complementary RNA (cRNA) molecules that in turn serve as templates for the synthesis of negative-strand virion RNA (vRNA) genomes. The negative-strand genomes also function as templates for the production of mRNAs [1, 2] . In non-segmented negative-strand RNA viruses, sequential transcription of successive genes results in a gradient of transcript abundance that steadily decreases towards the end of the template. Thus, the expression level of each gene is governed by the gene order [3] . This does, however, not apply to all negative-strand viruses as some of them acquired segmented genomes during their evolution. Each genome segment of these viruses is individually replicated and transcribed, necessitating careful regulation of these distinctive processes to generate sufficient vRNAs and proteins for the production of progeny virions [2] .
Influenza A virus (IAV) of the family Orthomyxoviridae is an enveloped, negative-strand RNA virus. The IAV genome is composed of eight different vRNA segments that altogether encode up to 13 proteins [4] [5] [6] [7] . Each vRNA and cRNA possesses untranslated regions (UTRs) of varying length at the 39 and 59 ends. The first 12 and 13 nucleotides at the 39 and 59 UTRs of the vRNAs and cRNAs are highly conserved among different RNA segments. These highly conserved partly complementary UTRs, which form a ''panhandle'' or ''corkscrew'' conformation by alternative modes of base-pairing, constitute the promoter structure for RNA synthesis [8, 9] . The panhandle conformation results from base-pairing between 59 and 39 terminal ends of the viral RNA segment with a small internal loop [10, 11] , while the corkscrew structure consists of a six base-pair RNA rod in the distal element in conjunction with two stem-loop structures of two short-range base-pairs [12] .
The IAV vRNA and cRNA segments form ribonucleoprotein (RNP) complexes by association to the polymerase and to multiple copies of the nucleoprotein (NP). These RNPs may be regarded as independent molecular machines responsible for transcription and replication of each segment. The viral RNA polymerase, which consists of the PA, PB1 and PB2 subunits, recognizes the RNA promoter, and stabilizes a supercoiled conformation of the RNPs. Different models have been proposed for the regulation of transcription and replication. One model suggests that the RNA polymerase switches from a transcriptase, used for mRNA synthesis, to a replicase, used for cRNA and vRNA synthesis, which is triggered by newly synthesized NP protein [13] . Another model suggests that cRNAs can be directly synthesized from incoming vRNAs, but require newly synthesized polymerase and NP to be stabilized in RNPs [14] . More recently, Jorba and colleagues proposed a model, in which a template RNP is replicated in trans by a soluble polymerase complex, whereas transcription of the vRNA occurs in cis and the resident polymerase complex is responsible for mRNA synthesis [15] .
Early studies, in which semi-quantitative hybridization techniques were used, described differential expression rates and levels of the different vRNAs. In general it appeared that the mRNAs for NS1 and NP are synthesized preferentially at the early times post infection, while the synthesis of matrix (M1) mRNA is delayed [16] [17] [18] . More recently, Vester and coworkers showed, by using quantitative RT-PCR that the vRNAs are synthesized in equimolar amounts and with similar kinetics, whereas early in infection preferential synthesis of NS1 mRNA and a delay in that of M1 mRNA was found [19] . However, how IAV temporally regulates the replication and transcription levels of its multiple genome segments is not known.
Several reporter assays have been described to study and quantify IAV transcription/replication in vivo. These reporter systems usually consist of a reporter protein-encoding cDNA, flanked by 39 and 59 UTRs, inserted in an antisense orientation between a PolI promoter and a transcription terminator or ribozyme sequence. After introduction of the reporter construct into a cell, reporter gene expression is induced by co-transfection of plasmids encoding NP, PA, PB1 and PB2 (transfection assay) or by subsequent infection with a helper IAV (infection assay). Such reporter assays are very helpful to quantify virus replication or virus production, and to assess the antiviral activity of compounds including antibodies [20] [21] [22] . These assays have also been used for the mutational analysis of IAV promoter elements in vivo [12, 23, 24] .
To get more insight in the mechanism by which IAV regulates and balances the replication and transcription of its genome segments, we converted the IAV single reporter assay into a dual reporter assay, by which the expression of two different luciferase genes can be monitored simultaneously. This assay more closely resembles the multiple segment transcription and replication conditions that occur in IAV infected cells than the single reporter assay. Our results indicate that different vRNA segments compete with each other, as transcription/replication of one vRNA Figure 1 . Schematic representations of the dual luciferase reporter constructs, and of transfection and infection assays. A) Schematic outline of the firefly and Gaussia luciferase reporter constructs. The firefly and Gaussia luciferase genes, flanked by 39 and 59 UTR of the NP segment, were inserted in antisense orientation between a PolI promoter and a ribozyme sequence, resulting in FNP and GNP, respectively. The extended Gaussia luciferase reporter construct (GFsNP) additionally contains the 39 terminal half of the firefly luciferase gene (indicated as Fs) behind the stop codon of the Gaussia gene. B) HEK 293T cells were transfected with one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases (PB1, PB2, PA) and NP either by simultaneous co-transfection of expression plasmids (transfection assay) or by infection with IAV at an MOI 1 at 24 h post-transfection (infection assay). The firefly and Gaussia luciferase expression levels can be measured consecutively using a dual luciferase assay system (Promega) 24 h post-transfection or post-infection. doi:10.1371/journal.pone.0047529.g001 segment can affect that of another. Using this multiple segment reporter assay we subsequently assessed the contribution of vRNA segment length, UTRs sequence and coding sequence to the competition between the different segments.
A schematic overview of the dual luciferase reporter constructs and the assays is shown in Figure 1 . The firefly and Gaussia luciferase genes, in this example flanked by 39 and 59 UTRs of the NP segment (referred to as FNP and GNP, respectively), are inserted in an antisense orientation between a PolI promoter and a ribozyme sequence. Cells are transfected with either one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases and NP either by co-transfection of expression plasmids (transfection assay) or by virus infection (infection assay). The expression levels of the firefly and Gaussia luciferase reporter constructs are determined consecutively using a single tube, dual luciferase assay system.
First we determined the luciferase expression levels of the firefly (FNP) and Gaussia (GNP) luciferase reporter constructs when transfected alone or in combination by using the transfection assay. As shown in Figure 2 , single transfection of each reporter gene resulted in high expression levels of both the firefly ( Fig. 2A) and Gaussia (Fig. 2B ) luciferase genes. However, when the reporter constructs were co-transfected, the firefly luciferase expression level was dramatically reduced ( Fig. 2A) , while the Gaussia luciferase expression level in the same cells was not affected when compared to the single-transfected cells (Fig. 2B) . The differential expression of firefly and Gaussia reporter plasmids when transfected alone or together can also be illustrated by plotting the normalized ratio of firefly to Gaussia luciferase activity (normFluc/ Gluc; the normalized ratio's were calculated as indicated in the Materials and Methods section). As shown in Figure 2C , this ratio is significantly decreased upon co-transfection of the two reporter constructs, when compared to the ratio of the luciferase expression levels in the single-transfected cells. Similar results were obtained at earlier and later time points post transfection (data not shown). This indicates that the balance of firefly and Gaussia luciferase expression is strongly in favor of Gaussia luciferase, when both reporter constructs are present within the same cell. Thus, the results indicate that expression of the firefly luciferase gene is Figure 2 . Competition between firefly and Gaussia luciferase reporter genome segments. Plasmids encoding firefly (FNP) or Gaussia (GNP) luciferase reporter constructs were transfected alone (Single) or in combination (Co). Luciferase expression was induced by simultaneous cotransfection of polymerase and NP expression plasmids (transfection assay). A) Firefly luciferase activity after transfection of FNP or FNP together with GNP. B) Gaussia luciferase activity after transfection of GNP or GNP together with FNP. C) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/ Gluc) when FNP and GNP were transfected singly or in combination. D) Quantitative RT-PCR analysis of mRNA levels derived from FNP and GNP after single or co-transfection of these constructs. RNAs were extracted 24 h post-transfection and subjected to quantitative RT-PCR. The comparative Ct method was used to determine the relative mRNA levels using the housekeeping gene GAPDH as a reference. The mRNA levels were normalized relative to the samples in which a single reporter construct was transfected. E) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) when reporter gene constructs FNP and GNP were transfected singly or in combination. The amounts of reporter gene constructs transfected are indicated. Significant differences in A-D are indicated (**; P,0,01). doi:10.1371/journal.pone.0047529.g002 negatively affected by co-transfection of the Gaussia luciferase reporter plasmid.
Very similar results were obtained when an empty plasmid (pUC18) was included in the transfection mixture when only one reporter construct was transfected ( Fig. S1A-C) . Thus, the observed differences in firefly luciferase expression do not result from a lower transfection efficiency of the firefly luciferase, but not of the Gaussia luciferase reporter construct, when an additional plasmid was included in the transfection mixture.
Next, to analyze whether the observed difference in luciferase protein levels results from differences at the RNA level, we performed a quantitative RT-PCR analysis of the mRNA levels [19] . The results are shown in Figure 2D . The mRNA levels of the Gaussia reporter gene were not affected by co-transfection of the other reporter construct. However, co-transfection of the Gaussia luciferase construct significantly affected mRNA levels of the firefly luciferase gene. From these results we conclude that the observed inhibitory effect of co-transfection of the Gaussia luciferase construct on the firefly luciferase activity is a reflection of lower firefly luciferase mRNA levels.
The results indicate that replication and transcription of the Gaussia and firefly luciferase genome segments are in competition with each other. If so the observed inhibitory effect of cotransfection of the Gaussia luciferase construct on the firefly luciferase expression level is expected to depend on the ratio of the transfected reporter constructs. As shown in Figure 2E , this is indeed the case. Lowering the amount of co-transfected Gaussia as well as increasing the amount of co-transfected firefly luciferase reporter plasmid shifted the balance in the competition between the firefly and Gaussia luciferase genome segments as judged from the increased normFluc/Gluc ratio (Fig. 2E ). This increased ratio resulted from altered firefly rather than Gaussia luciferase expression levels ( Fig. S2A and B ). From these results we conclude that the Gaussia luciferase genome segment is much more efficiently replicated and transcribed than its firefly luciferase counterpart, the latter of which is outcompeted by the presence of the former.
The firefly and Gaussia luciferase genome segments are most likely competing for host and/or viral factors that are necessary for transcription and/or replication. To analyze whether a limiting availability of viral proteins is an important factor in the competition between firefly and Gaussia luciferase genome segments, we increased the amount of plasmids encoding the RNA polymerase subunits and NP in the transfection assay. Empty plasmid (pUC18) was included in the transfection mixture when needed to achieve the same total amount plasmid DNA for each transfection condition. Upon increasing the amounts of transfected plasmids encoding PB1/PB2/PA/NP, the normFluc/ Gluc ratio increased ,10-fold (Fig. 3A ) as a result of increased firefly luciferase expression levels ( Fig. S3A and B ). This result indicates that increased amounts of polymerase subunits and NP can alleviate the competition between the firefly and Gaussia luciferase genome segments. Increasing the amount of transfected NP-encoding plasmid alone did not affect the normFluc/Gluc ratio ( Fig. 3B ) or the absolute Fluc and Gluc levels ( Fig. S3C and D). Increasing the amount of polymerase subunit-encoding plasmids, but not of the NP-encoding plasmid, appeared to alleviate the competition between the two segments ( Fig. 3C ). However, it also negatively affected the reporter gene expression levels per se, with most dramatic effects being observed for the firefly luciferase reporter segment ( Fig. S3E and F). We speculate that this negative effect correlates with the requirement for NP for replication, which appears less stringent for short RNA templates [25, 26] . Although we did not analyze the NP and polymerase protein levels directly, our results indicate that the luciferase genome segments compete for RNA polymerase subunits and/or NP, with a limiting amount of polymerase subunits being the most likely explanation for the observed competition. However, we cannot exclude that the observed competition between reporter segments is partly caused by limiting amounts of host factors.
Next we analyzed to what extent the competition between the luciferase genome segments is affected by characteristics of the genome segments themselves. An obvious difference between the firefly and Gaussia genome segments is their gene length as the firefly and Gaussia luciferase genes consist of 1653 and 558 nucleotides, respectively. Small genome segments are likely to be replicated faster than long ones. To test this hypothesis, we generated an extended Gaussia luciferase gene construct, in which part of the firefly luciferase gene (39-terminal half) was inserted immediately behind the stop codon of the Gaussia luciferase gene in the GNP plasmid (referred to as GFsNP, Fig. 1A) to produce a genome segment with exactly the same length as the firefly luciferase genome segment. The extended and the normal Gaussia luciferase reporter segments were compared for their ability to compete with the firefly luciferase reporter segment. The absolute Gaussia luciferase expression level from the GFsNP segment was lower than that from the GNP segment (Fig. S4B ), probably resulting from its extended length, for which is corrected by plotting normalized Fluc/Gluc ratios in Figure 4 . These norm-Fluc/Gluc ratios (Fig. 4A) indicate that the extended GFsNP segment is still a strong competitor of the FNP segment, although much less efficient than the smaller GNP segment. We conclude that segment size is an important factor in the competition between vRNA segments. However, our results also suggest that coding regions are important as FNP and GFsNP segments do not differ in size, while they contain identical 59 and 39UTRs.
Next, we analyzed to what extent the competition between different reporter constructs is affected by the identity of the 39 and 59 UTRs. The genome segment UTRs provide signals for viral RNA transcription and replication, as well as for packaging of vRNP into virus particles [27] . The first 12 and 13 nucleotides at the 39 and 59 UTRs, which are highly conserved among the eight viral RNA segments, constitute the promoter structure for RNA synthesis [8, 9] . Also the non-conserved regions of the different segments have been implicated in viral RNA replication [28] . We inserted the 39 and 59 UTRs of the eight IAV-WSN segments (Table 1) into the extended Gaussia reporter plasmid and tested them in the competition assay with the firefly luciferase reporter construct FNP. The extended Gaussia construct was chosen instead of the short version as the former construct affects expression of the firefly luciferase construct less than the latter, resulting in a more balanced system in which it will be easier to detect UTRdependent up and down effects. Introduction of the UTRs of different segments into the extended Gaussia reporter construct affected the balance between the Gaussia and firefly luciferase expression to different extents (Fig. 4B) . Introduction of PB1, NA or NS segment UTRs resulted in balanced firefly and Gaussia luciferase expression levels as similar ratio's were observed when expressed alone or in combination (normFluc/Gluc ,1; Fig. 4B ). When the extended Gaussia construct was provided with the PB2, PA, HA and M segment UTRs, the normalized ratio's observed after co-transfection with FNP were similar to those observed when the construct containing the NP segment UTRs was used (normFluc/Gluc ,1), indicating that the balance had shifted in favor of Gaussia luciferase expression. Subsequently, we analyzed whether the absolute expression levels of the eight different Gaussia luciferase segments (Fig. S5A ) correlated with their ability to inhibit expression of firefly luciferase (Fig. S5B) . Correlation between inhibition of firefly luciferase expression and the expression of Gaussia luciferase with different UTRs resulted in an R 2 value of 0.8 (Fig. 4C) , which indicates that 80% of the variance in firefly luciferase inhibition correlates with variability in Gaussia luciferase expression levels. A similar R 2 value was obtained when the results obtained with the short GNP reporter segment were also taken into account (Fig. S6) . The results indicate that not only the expression of reporter genome segments, but also the balance between different reporter genome segments is affected by 39 and 59 UTRs, and that these two phenomena are largely correlated. Thus, Gaussia luciferase genome segments that are expressed to a higher extent are more efficient inhibitors of firefly luciferase expression driven by another genome segment.
Subsequently, we analyzed to what extent reporter gene expression was affected by the presence of the natural viral genome segments. To this end, the reporter constructs were cotransfected with plasmids encoding each of the IAV genome segments under the same control of human RNA polymerase I promoter. As a control, empty plasmid (pUC18) was cotransfected. The different IAV segments significantly affected the firefly luciferase levels (Fig. 5A) . In general, the shorter segments gave stronger competition on the firefly expression compared to longer segments, with the M and NS segments having the largest effect. Correlation between inhibition on firefly luciferase expression and the gene length resulted in an R 2 value of 0.7 (Fig. 5C) , which indicates that 70% of the variance in firefly luciferase inhibition correlates with variability in genome segment length. In contrast, expression of Gaussia luciferase was hardly affected by the presence of the viral RNA segments (Fig. 5B) , while no correlation was observed between the modest decrease/increase of Gaussia luciferase expression and the genome segment length (Fig. S7) . We speculate that the lack of inhibition of Gaussia luciferase expression correlates with the very efficient replication/transcription of this reporter segment.
Mutations in the 39UTR of the NP segment that increase gene expression have been described [23] . The nucleotide changes were predicted to improve base pairing of the 39 and 59 UTRs and thus to stabilize the panhandle structure. Considering the results described above, we expected that these mutations would affect competition between different reporter genome segments. The results show that reporter constructs containing these panhandlestabilizing UTRs (referred to as NPph; Fig. 6A ) indeed displayed 3-5 fold higher luciferase expression levels in the transfection assay than their wild-type UTR-containing counterparts (Fig. S8) . Remarkably, however, the normalized ratio between firefly and Gaussia luciferase was much more affected by the presence of the NPph UTRs. Introducing the NPph UTR in the background of the extended Gaussia construct (GFsNPph) resulted in a much decreased normFluc/Gluc when compared to its counterpart with the wild type NP UTRs (GFsNP; Fig. 6B) . A similar level of competition was not observed when the mutations that increase the number of base-pairs were introduced in the 59 UTR of the NP segment (referred to as NPphR; Fig. 6A ) instead of in the 39UTR. In this case, the Gaussia, rather than the firefly luciferase expression was affected by the co-transfection of both reporter plasmids (normFluc/Gluc .1; Fig. 6B and Fig. S8B ). Similar results were obtained when NPph UTR was introduced in the firefly luciferase genome segment (referred to as FNPph; Fig. 6C ). In general, normFluc/Gluc was increased when FNPph rather than FNP was used, except for the combination with GNPph ( Fig. 6C & Fig. S8C and D) . Thus, the balance between different segments is dramatically affected by the introduction of panhandle structure-stabilizing mutations in the 39UTR. Infection assay Neumann and Hobom (1995) previously reported increased reporter gene expression upon the introduction of panhandlestabilizing mutations in the 39 UTR. In their experimental system, however, the differences in reporter gene expression appeared much larger than ours. We hypothesized that this difference might be explained by Neuman and Hobom using virus infection to drive reporter gene expression, while we used transfection of polymerase subunit-and NP-encoding plasmids. Infection with IAV will not only provide viral RNA polymerase and NP, but will also introduce natural vRNPs that may compete with reporter genome segments for replication and/or transcription. Thus, in virusinfected cells, the natural virus genome segments might be preferentially replicated and transcribed over the reporter genome segments, unless the panhandle-stabilizing mutations in the 39 UTR are present. To test this hypothesis we compared reporter gene expression driven by co-transfection of expression plasmids (transfection assay) with expression driven by virus infection (infection assay). Reporter genes flanked either by natural NP UTRs or by the mutant NPph UTRs were used. Both firefly and Gaussia luciferase genes were expressed at high levels in the transfection assay, with the reporter constructs containing the NPph UTRs again displaying somewhat higher luciferase levels than their counterparts with the natural NP UTRs (Fig. 7A) . However, when using the infection assay, dramatic differences in reporter gene expression levels were observed (Fig. 7B) . Thus, while the reporter genes flanked by the NPph UTRs reached 1 to 2 fold higher expression levels than those flanked by the natural NP UTR in the transfection assay, this fold difference was much increased (130 to 160 fold) in the infection assay (Fig. 7C) . Quantitative RT PCR confirmed that mRNAs levels of the Gaussia luciferase RNAs were very similar, regardless of the presence of the natural NP UTRs or the mutant NPph UTRs in the transfection assay, but not in the infection assay (Fig. 7D) . These results are in agreement with our model, in which IAV genome segments compete for available resources, likely the viral proteins, to maximize their replication and/or transcription. Only reporter genome segments carrying panhandle-stabilizing mutations in their 39 UTR are able to efficiently compete with the natural genome segments in the infection assay. 
The molecular mechanisms by which IAV replicates and transcribes its genome segments have generally been well studied. However, the way by which IAV regulates and balances the replication/transcription of its 8 genome segments is much less understood. In order to study and manipulate these processes, we developed a dual reporter genome segment assay that enabled us to analyze whether the replication/transcription of one genome segment is affected by that of another. Our results indicate that this is indeed the case as luciferase expression driven from a reporter genome segment was shown to be affected by the presence of other genome segments, both in the context of virus infection and in the presence of polymerase and NP proteins provided by transfection of the expression plasmids. Furthermore, our results indicate that genome segments are likely to compete with each other for the available viral proteins and that the balance between different genome segments is affected by reporter genome segment length, by the identity of 39 and 59 UTRs, and probably also by their coding regions.
Our results indicate that replication/transcription of a genome segment can be negatively affected by the presence of another genome segment. This interference became less pronounced when the length of the smaller segment was extended, indicating that genome segment length plays a role in the competition between different segments. This ''length effect'' was also observed when natural genome segments were present in addition to the reporter construct, with the shortest segments, M and NS, giving the strongest inhibition of the reporter gene expression. In agreement herewith, IAV defective interfering (DI) RNAs, which are formed by internal deletion of progenitor RNA segments, interfere with vRNA synthesis, probably because of the competitive advantage of the smaller DI RNA molecules (reviewed by Nayak [29] ). In addition, our data indicate that the coding region of the vRNA segment may also be of importance, as the extended version of the Gaussia reporter segment was still able to outcompete its firefly luciferase counterpart, albeit less efficiently than its shorter version.
The segment UTRs are known to contain signals for transcription, replication and packaging of vRNP [24, 27, 28, 30] . We now show that the identity of the 39 and 59 UTRs also influences the competition between different segments. Relatively minor differences were observed when reporter genome segments with different natural UTRs were compared in the competition assay. This result is in agreement with the observation that non- conserved regions of the UTRs contribute to some but limited extent to viral RNA replication [28, 31] . However, introducing three nucleotide changes in the 39 UTR (G3A/U5C/C8U) of the NP segment, which is predicted to stabilize the UTR panhandle structure and is known to lead to increased reporter gene expression in infected cells [23] , dramatically increased the competitive ability of the reporter segment, both when replication/transcription was driven by transfection of polymerase-and NP-encoding segments and when mediated by IAV infection. Thus, while reporter segments carrying the natural NP UTRs or the mutant NPph UTRs were both efficiently expressed in the absence of competitor segments, large differences in luciferase expression were observed in favor of the luciferase segment carrying the panhandle-stabilizing mutations when other reporter segments were co-transfected or in IAV infected cells. In agreement herewith, recombinant viruses carrying two nucleotide changes (G3A/C8U) in the UTR of either the PB1 or PA segment displayed enhanced replication/transcription of the mutated segments in detriment of the wild-type UTR-bearing segments [32] .
The most likely scenario suggested by our observations is that replication/transcription of one reporter segment interferes with that of another by sequestering UTR-binding proteins, probably polymerases, required for RNA synthesis. Several observations by us and others support this hypothesis: 1) increasing the amount of polymerase and NP proteins, but not of NP protein alone, alleviated the competition between different segments, 2) the polymerase proteins have been shown to bind to 59 and 39 UTRs of vRNAs, with most strong binding observed to the 59 UTR [33] , 3) introduction of mutations in the 39 UTR (NPph) that stabilize the panhandle structure and are predicted to result in increased polymerase binding [23] result in increased ability of the reporter segment to be replicated/transcribed in the presence of competitor segments ( [23] and this study), 4) introduction of similar mutations in the 59 UTR (NPphR) that are likely to interfere with polymerase binding [33] , had a negative effect on the competitive ability of the reporter construct, and 5) panhandle-stabilizing mutations in the 39 UTR (NPph), that increased the competitive ability of the reporter construct, partly compensated for replication-debilitating mutations in PB2 (R142A or E361A) [34, 35] , but not in NP (M331K or F488G) [36] (Fig. S9) , suggesting a link between the interaction of the UTR with polymerase and the ability to compete with other segments. Although our experimental system (i.e. the transfection assay) does not approach the complexity of the IAV infected cells with respect to number of vRNA segments and viral proteins present, our data suggest that IAV RNA segments compete with each other for available polymerases. This competition is expected to be most apparent early during infection, when only low amounts of polymerase are present. It is conceivable that at this stage of the infection the low level of RNA polymerase plays a critical role in the regulation of segment-specific replication and/or transcription. At later times during infection competition between vRNA segments is expected to be alleviated by the increased levels of the polymerase subunits, thereby ensuring the efficient replication/transcription of all genome segments. 
HEK 293T and MDCK cells were maintained in complete Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 10% (v/v) Fetal Calf Serum (FCS; Bodinco B. V.), 100 U/ml Penicillin and 100 mg/ml Streptomycin. Influenza A/WSN/33 (H1N1) (IAV-WSN) was grown on MDCK cells. Briefly ,70% confluent MDCK cells were infected with IAV-WSN at a multiplicity of infection (MOI) of 0.02 50% tissue culture infectious dose (TCID50) per cell. Supernatant was harvested after 48 h of incubation at 37uC and cell debris was removed by centrifugation at 2,000 rpm for 10 min. Virus was stored at 280uC and TCID50 on MDCK cells was determined.
Precursor firefly and Gaussia luciferase reporter gene constructs were generated by GenScript. These constructs have the following schematic make up: hepatitis delta virus (HDV) ribozyme -AloI restriction site -firefly or Gaussia luciferase gene -BaeI restriction site -human PolI promoter. These constructs did not yet contain IAV 59 and 39 UTR sequences. 39 UTR sequences were introduced by ligation of primer dimers into the AloI-digested constructs. Subsequently, primer dimers corresponding to 59 UTR sequences were ligated into the resulting BaeI-digested plasmid. The AloI and BaeI restriction recognition sites are completely removed by this procedure and the corresponding PolI transcripts are similar to genuine vRNA segments with the exception of the coding region. In addition, we generated an extended version of the Gaussia luciferase reporter gene construct by introduction of a firefly luciferase gene fragment. As a result, the length of the extended Gaussia luciferase vRNA is identical to that of the corresponding firefly luciferase-encoding vRNA. To generate this construct, a NotI-digested PCR fragment, corresponding to part of the firefly luciferase gene (nucleotide 559 -stop codon) and containing flanking NotI restriction sites, was ligated into the NotIrestricted precursor Gaussia luciferase plasmid, immediately downstream of the Gaussia luciferase gene stop codon. The IAV segment UTRs were inserted in the extended Gaussia luciferase plasmid in the same manner as described above. For a schematic overview of the reporter constructs, see Figure 1A .
The protein expression plasmids encoding PB2, PB1, PA and NP (pcDNA-PB2, pcDNA-PB1, pcDNA-PA and pcDNA-NP) and transcription plasmids encoding eight IAV-WSN vRNA segments (pPOLI-PB2, pPOLI-PB1, pPOLI-PA, pPOLI-HA, pPOLI-NP, pPOLI-NA, pPOLI-M, and pPOLI-NS) were a kind gift of Dr. Ervin Fodor [37] .
HEK 293T cells were seeded in 96-wells plates at a density of 10,000 cells per well and incubated overnight. For the transfection assay, cells were transfected with reporter plasmids encoding firefly or Gaussia luciferase along with expression plasmids encoding PB2, PB1, PA, or NP using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. Fifty nanogram of each plasmid was used in the transfection unless mentioned otherwise. For the infection assay, cells were transfected with reporter plasmids encoding firefly or Gaussia luciferase. The next day, cells were infected with IAV-WSN at a multiplicity of infection (MOI) of 1 TCID50 units per cells.
Twenty-four h post-transfection or -infection, cells were lysed by incubation with Passive Lysis Buffer (Promega) for 15 min at room temperature. Cell lysates were assayed for luciferase activity using the Dual-Luciferase assay system (Promega) according to the manufacturer's protocols, and the relative light units (RLU) were determined using a Centro LB 960 Luminometer (Berthold Technologies). The ratio of firefly luciferase/Gaussia luciferase activity after co-transfection of both reporter constructs (norm-Fluc/Gluc) was normalized to the ratio of firefly luciferase/Gaussia luciferase activity after single transfection of reporter constructs, which was set at 1.
Quantitative RT-PCR to determine the amount of mRNA synthesized for the reporter genes during the transfection and infection assays was performed according to Vester et al. [19] . Briefly, following the removal of the cell culture medium, cells were washed with PBS and lysed by incubation with TriZol reagent (Invitrogen) for 3 min at room temperature. The lysates were mixed with chloroform and centrifuged at 14,000 rpm for 20 min at 4uC. The water phase was collected and mixed with 70% (v/v) ethanol. Subsequent RNA purification was performed using the RNAeasy Kit (Qiagen) according to the manufacturer's protocols. The concentration of total RNA was determined using NanoDrop 1000 Spectrophotometer (Thermo Scientific). The total RNA was treated with amplification grade DNase (Invitrogen) according to the manufacturer's protocols to digest the plasmid DNA. Reverse transcription from total RNA was performed using mRNA-specific primer ( Table 2) . Reverse transcription was carried out using Superscript II reverse transcriptase (Invitrogen). Briefly, 100 ng of DNase-treated total RNA was mixed with 2 pmol of primer and 1 ml of 25 mM dNTP in the total volume of 12 ml. The mixture was incubated at 65uC for 5 min. After the cooling step to 4uC, 4 ml of 56 first strand buffer, 2 ml of 0.1 M DTT, 1 ml of RNase Inhibitor (40 U/ml) were added and the mixture was incubated at 42uC for 2 min. Reverse transcription was carried out at 42uC for 50 min after addition of 1 ml superscript II reverse transcriptase (50 U/ml) and was terminated by heating at 70uC for 15 min. Real time quantitative PCR was performed using qPCR MasterMix Plus for SYBR Green (Eurogentech) on a LightCycler 480 II (Roche). qPCR forward and reverse primers ( Table 2 ) that primed at the coding sequence of corresponding reporter gene were used to amplify cDNA. Quantitative PCR reactions were set up in triplicates according to the manufacturer's instruction by mixing 20 pmol of forward and reverse primers and 1 ml of cDNA products. The PCR mixture was incubated at 95uC for 10 min, followed by 40 cycles of 15 sec and 1 min incubations at 95uC and 60uC, respectively. To check the specificity of PCR product, melting curve analysis was performed at the end of the PCR. The comparative Ct method was used to determine the relative mRNA levels using the housekeeping gene GAPDH as a reference [38, 39] . The mRNA levels were normalized relative to the samples in which a single reporter construct was transfected.
The means of multiple experiments are shown. All experiments were performed 2-4 times, with each experiment containing 4 replicates. Differences between means were determined using Student's t-test. Differences were considered significant if P,0.05. Significant differences are indicated by symbols in the figures where appropriate. Figure S1 Competition between firefly and Gaussia luciferase reporter genome segments. Plasmids encoding firefly (FNP) or Gaussia (GNP) luciferase reporter constructs were transfected alone (Single) or in combination (Co). Luciferase expression was induced by simultaneous co-transfection of polymerase and NP expression plasmids (transfection assay). A) Firefly luciferase activity after transfection of FNP with empty plasmid (pUC18) or FNP together with GNP. B) Gaussia luciferase activity after transfection of GNP with empty plasmid (pUC18) or GNP together with FNP. C) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) when FNP and GNP were transfected singly or in combination. (TIF)  Oral health in China – trends and challenges For a long time, oral disease is one of the major problems of the public health for its high prevalence and incidence throughout the world, which is especially true for low-income populations. Since China's economic reform in 1978, great changes have taken place in China. These changes have significant impact on and have been reflected in oral disease trends in China. This paper provides an overview and assessment of the oral health status in China. It focuses on changes in the nation's demographic profile, in the marketplace, the oral disease status and trends. The paper also suggests some possible measures and strategies for bettering oral health in future China. In recent years, chronic noncontagious diseases have become a major health problem in the world. Besides social and environmental factors, the changes in lifestyle worldwide have great effects on the changes in the disease status and trends. Oral disease is one of the major problems of the public health for its high prevalence and incidence, especially in low-income populations. Oral diseases have become one of the major maladies. This paper provides an overview of the oral health status in China, focusing on changes in the nation's demographic profile, the marketplace, and trends of Chinese dentistry. The paper also suggests some related measures and strategies for improving oral health in future China.
China's population is expected to grow from 1. cing aging of its population and the senescence process develops rapidly. Since 1990, the aged population (people over 60 years) has increased at an average annual rate of 3.32%. In the 21st century, China has entered the aging society. In 2005, 11% of Chinese population, i.e. 144 million people, are older than 60 years of age. The number of Chinese senior citizens over 65 years old has amounted to 100 millions, accounting for 7.7% of the total population. By 2040, the number will have increased to about 374 million, accounting for about 25%, thus making China the largest aging society in history (Tables 1, 2 ). The change of population will have major effects on oral health [1] . In the same time, many rural people have rushed into the city, altering the rural to urban population proportion a great deal (Table 3) . The rural population has decreased from 73.6% of the total population in 1990 to 57.0% in 2005. A few years later, this proportion will further decrease to about a half. Meanwhile, the great changes in lifestyle will affect their general and oral health. These demographic changes will alter disease patterns, cultural attitudes, health behaviors and the health care delivery systems and services. [2] . Economic development has instigated the development of general medical health care and oral health care. However, despite the high growth rate of GDP, the spending on public health care, especially the oral health care, remains low, as seen by the average spending on health care per person [3] [4] [5] (Tables 4, 5). The data are calculated at current prices. 
Recently, China's gini coefficient (a measurement of wealth distribution of a society, the greater the value, the deeper the gap between rich and poor) keeps growing. According to the World Bank assessment, China's gini coefficient was 0.447 in 2001. In 2002, the Chinese Academy of Social Sciences calculated the gini coefficient at 0.454. In both years, China has exceeded the alerting threshold of 0.4, indicating that the income gap in China has been too wide [6] . For the difference in oral health status between rich and poor, it is the same story. Low-income populations and people in need of special care (aged people, children, disabled people) experience illness more often and need more treatment. Besides lack of money and ignorance of oral health knowledge, they do not have easy access to oral health care.
According to the statistics released by the Chinese Ministry of Civil Affairs, there are more than 60 million victims of natural disasters each year, over 22.82 million low-income people who receive subsistence allowances in city, 40 million rural people who have very low incomes and live in absolute poverty, 60 million disabled people and 100 million elder people over 65 years old who need all kinds of assistance [7] . Meanwhile, the government budgetary funding only accounts for 0.3% of the national GDP.
In the past two decades, both the general medical and oral health conditions of the Chinese people have been improving. However, oral diseases remain prevalent.
Many factors, such as area, race, age and gender, affect the severity of oral diseases. Oral health conditions differ greatly under different influences.
Caries of the primary dentition still calls for attention, as the number of children, although accounting for a decreased percentage of the total population, remains large. In some areas, caries of the permanent dentition has increased in number. The elderly with their relative and absolute number increasing have complicated medical conditions and an increased need for treatment.
According to the third national epidemiological investigation on oral diseases conducted in 2005, the caries prevalence rate of children aged between 5-6 years old remains high, people of 35-44 and 65-74 years experience high caries prevalence rate and low filling rate [8] [9] (Table 6 ). With more teeth remaining in the oral cavity, the elderly are apt to develop caries and other oral disorders. Investigations conducted in Bejing and Shanghai have revealed that people aged between 65-70 years exhibit a root caries prevalence rate of 57%, a root caries index of 4.0-5.8, DFS index of 5.9-6.3 while 60%-70% of the fillings need replacement or have secondary caries underneath [8] [9] [10] [11] .
The caries status in China exhibits characteristics typically found in developing countries. About 97% carious teeth of children aged 5 are left untreated, while for children aged 12, this percentage is 89%. About 78.9%-91.7% of carious teeth (including third molars) of the middle-aged and elderly people have been left untreated. This situation has not been much improved with China's economic development, challenging China's oral health care resources [8] [9] .
The third national epidemiological investigation on oral diseases conducted in 2005 revealed that, gingival bleeding and calculus occur in over a half of 12-year-olds (57.7% and 59.1% respectively), most middle-aged people (77.3%, 97.3%) and many elderly people (68.0%, 88.7%) ( Table 7) . Periodontal conditions of all age groups were found to be better in cities, females and eastern areas than in rural areas, males, middle and western areas. The findings of this investigation did not differ much from the previous one [8] [9] . In all factors contributing to periodontal diseases, smoking and diabetes should not be ignored. Tobacco consumption related to oral disorders is one of the major sources of the global oral disease load. Over a half of adult periodontitis cases are triggered by it. China has become the biggest tobacco consuming country in the world. Its tobacco consumption increases at an annual rate of 5.3%. China's tobacco yield is equal to the sum production of the other 7 biggest tobacco producing countries. About 160 thousand billion cigarettes are sold in China each year, accounting for over 30% of the world total tobacco consumption [12] [13] . On the other hand, in recent years, the incidence rate of diabetes has been growing in China. According to a survey of the Chinese Academy of Endocrinology, the prevalence of diabetes is reaching 11% in urban and suburban areas. The sufferers of diabetes in China are ranked second in the world. According to the WHO assessment, there will be about 50 million diabetes patients in China by the year of 2025.
According to summary of the Chinese Academy of Orthodontics, the prevalence rate of malocclusion has increased by 20% from 1950-60s [14] (Table 8 ). 
According to the third national epidemiological survey on oral diseases conducted in 2005, the prevalence of edentulism in 65-74 age group was 7%, lower than the finding of 1995 investigation (11%), indicating that the elderly have more remaining teeth than before. However, considering the criteria of this survey (any remaining tooth was excluded regardless of the treatment need for extraction), the actual rate should be higher [8] [9] .
The prevalence of dental erosion is relatively high and appears to be growing. Statistics show that the prevalence for the 65-74 age-group is 30.2%-87.6%. With the increased consumption of sugar-containing soft drinks, the incidence for children aged 3-5 has even reached 9.3% [15] [16] .
Only regional surveys contain information about oral cancer [17] [18] [19] (Table 9 ). Calculated at a prevalence rate of 81-41.2 per 100 thousand, there are 100 thousand oral cancer victims in China, with 10 thousand patients dying each year. Besides, oral soft tissue diseases, oral and facial injuries and trauma, tooth abrasion, mucosal infections and diseases, developmental disorders, craniofacial anomalies are very common. 
Suffering from multiple chronic diseases and more other health problems, having complicated medical and dental problems, adults especially elderly people present complicated medical problems. More than 70% elderly people take medicine, which will affect doctor's diagnosis and treatment. The treatment for old people is particularly complex. Dental therapies are frequently intricate, and the elderly also have chronic medical conditions, which complicate patient management. Dentists will face more complicated treatment and medically complicated or compromised patients.
With the economical development, the dental service need will increase for the people and patient.
As people retain more teeth into old age, the need for all kinds of oral health services will continue to increase. Dentists will be faced with more difficult cases in the future. At the same time, patients' expectations keep growing, their need for oral care, oral health products and medicals expand quickly. These special needs and trends will affect China's oral product market for a considerable time.
The oral health care system is an arrangement that combines the patients with the service provider. So far, health care services in China are mainly paid through the ways listed in Table 10 [3 -5] . Although there are no statistics about oral health care expenses, it's believed that over 85% of the total expense is paid by the patients themselves.
In recent years, the number of dentists and that of oral health education institutions have grown rapidly (Table  11) , however, the utilization efficiency of oral health resources remains low. Difficulty in visiting the doctor and paying for medication remains to be serious problems that concern people's livelihood. 
According to Qiang Gao, the former minister of Ministry of Health of P. R. China, at present, the development of China's health care system is faced with important opportunities as well as problems. It is an important historical mission for us to strengthen health care system reform, increase the governmental input for more preventive services, products and programs, to establish examples of successful prevention programs.
Comparing the findings of the national epidemiological investigations in 1995 and in 2005, it is indicated that the oral health status of the whole population will not be improved by only increasing the number of dentists and oral health institutions. Prevention as a public policy measure should be underlined in the oral health care system. New policies must have the necessary resources to translate need, currently not met, into effective demand. Policy should be developed to emphasize dental prevention and insurance reimbursement for preventive services. It should be emphasized that the reform start with innovating the structure of oral health care system, improve public health, rural health, and enhance the construction of the medical care system in urban communities. Meanwhile, it is important to pro-mote dental care professionals to pinpoint prevention service and to gear people to participate in preventive services. Insurance should cover the field of oral preventive services. Successful experiences of preventive model should be explored. In recent years, Beijing and Shanghai municipal governments have increased the inputs in oral health care for students, making a good start.
Nowadays, the effectiveness of present oral preventive methods has been proved by the evidence-based medicine. Therefore, the use of fluorides, pit and fissure sealing, oral health products and medicines should be advocated. After early diagnosis is made, the process of oral diseases can be monitored and specific advice can be given to patients. The significance of technology in decision making should not be overlooked in continuing education. Furthermore, the real challenge lies in the use of innovations rather than the conventional surgeries. Recently, the new concept of minimal invasive dentistry (MID) has become the theme in the clinical practice of dentistry.
The former U.S. Secretary of Health and Human Services Dr. Louis Sullivan said: getting patients to practice better health habits is the greatest challenge in medicine. The emphasis is on promoting health, rather than preventing diseases. Oral health education and promotion through community and clinical practice will play a leading role in the future public health initiatives.
Changed demographics, disease trends and scientific advances are stimulating the need of pharmaceuticals and oral health products. More patients require dental medicine rather than traditional dental treatment. Oral pharmaceuticals and dental health products are increasingly used for diagnosing, treating and preventing oral disease. Therefore, pharmaceutical solutions will play a greater role in oral care because they are passive, economic and preventive rather than surgical care. Medical agents can reach people of high caries risk who fall outside the dental delivery system and using these products require less contact with dental care providers. Medical interventions have a potential market in both urban and rural areas. Dentists will be able to save time and patient care costs. Toffler et al. [20] argued in their book, Revolutionary Wealth, that novel technologies are fortifying the fusion of producers and consumers, creating a new class, the prosumers. They pointed out, consumers can take advantage of new treatments brought about by nanotechnology and other new technology to accomplish the task that used to be executed by doctors, namely diagnosis and treatment. Such revolution will change the functioning manner of the entire business in the arena of oral health area in the future.
Just as the SARS virus and the bird flu virus can be transported around the world in hours, health care information can be transmitted from one corner of the globe to another in seconds. Health care is a global concern that breaks down national boundaries. New scientific findings and technologies can arise anywhere in the world. The globalization of the health system will surely affect the area of oral health. Success in preventing and controlling oral disease in China is increasingly dependent on the ability to share knowledge and expertise with others around the world in a free and open manner. Dentistry in China must be fully involved in international organizations and activities for research, education, clinical practice, product development and distribution, and health promotion. In the same time, China will benefit from international cooperation and collaboration. National intensive care unit bed capacity and ICU patient characteristics in a low income country BACKGROUND: Primary health care delivery in the developing world faces many challenges. Priority setting favours HIV, TB and malaria interventions. Little is known about the challenges faced in this setting with regard to critical care medicine. The aim of this study was to analyse and categorise the diagnosis and outcomes of 1,774 patients admitted to a hospital intensive care unit (ICU) in a low-income country over a 7-year period. We also assessed the country’s ICU bed capacity and described the challenges faced in dealing with critically ill patients in this setting. FINDINGS: A retrospective audit was conducted in a general ICU in a university hospital in Uganda. Demographic data, admission diagnosis, and ICU length of stay were recorded for the 1,774 patients who presented to the ICU in the period January 2003 to December 2009. Their mean age was 35.5 years. Males accounted for 56.5% of the study population; 92.8% were indigenous, and 42.9% were referrals from upcountry units. The average mortality rate over the study period was 40.1% (n = 715). The highest mortality rate (44%) was recorded in 2004 and the lowest (33.2%) in 2005. Children accounted for 11.6% of admissions (40.1% mortality). Sepsis, ARDS, traumatic brain injuries and HIV related conditions were the most frequent admission diagnoses. A telephonic survey determined that there are 33 adult ICU beds in the whole country. CONCLUSIONS: Mortality was 40.1%, with sepsis, head injury, acute lung injury and HIV/AIDS the most common admission diagnoses. The country has a very low ICU bed capacity. Prioritising infectious diseases poses a challenge to ensuring that critical care is an essential part of the health care package in Uganda. The prevalence of critical illness in developing countries is disproportionately high in view of the disproportionate burden of diseases such as HIV/AIDS, malaria, tuberculosis and trauma. Sub-Saharan Africa bears 25% of the global burden of disease [1] . Management of critically ill patients requires significant human, infrastructural, and financial resources. These resources are typically limited in low-income countries. Major intensive care units (ICUs) are mostly found in large hospitals in urban or metropolitan areas [2] . The most common admission criteria to these units are post-operative treatment, infectious diseases, trauma and obstetric complications [2, 3] .
A recent review highlighted the paucity of knowledge regarding critical care in the developing world [4] . Knowledge of the characteristics and outcomes of critically ill patients admitted to ICUs in low-income countries may help with the identification of priorities and the resources required for improvement of the care of critically ill patients in resource-limited regions of the world.
The aim of this study was to determine the admission diagnoses and outcome of patients admitted to the Mulago Hospital ICU from 2003 until 2009 and to highlight the country's ICU bed capacity. It is hoped that the findings will be a useful addition to the increasing body of evidence highlighting the disparities between critical care in high-and low-income countries.
This study was a retrospective audit. The study protocol was approved by the hospital Research and Ethics Committee. Medical charts were reviewed and anonymity was preserved for each case record.
The study intensive care unit is a 12-bed unit with the ability to ventilate only six patients at any one time. It provides level II ICU services to all kinds of critically ill patients. Level II care includes mechanical ventilation for longer than 24 h, and specific organ support like dialysis and inotropic infusions. The ICU can provide mechanical ventilation, post-operative care, intermittent haemodialysis, peritoneal dialysis, and basic neurocritical care. The ICU serves Mulago Hospital, which is a 1,500 bed national referral hospital, and Makerere University teaching hospital. The unit was started in the late 90s with a foreign donation and was initially run by a UK trained anaesthesiologist who has since retired. The ICU is currently staffed by three full-time ICU doctors (two internists and an anaesthesiologist) who have undergone further training in higher income countries, and 20 nurses. It receives technical support from the Department of Anaesthesia.
Apart from the study ICU, Mulago hospital also has a four-bed cardiac ICU, a four-bed coronary care unit (the heart institute is a semi-autonomous unit within the hospital that caters for paying patients and open-heart surgery patients), a six-bed paediatric high dependency unit, a new five-bed obstetric high dependency unit and a neonatal special care unit that can only provide nasal CPAP ventilation. No unit in the hospital can ventilate infants or neonates. Currently, the study ICU uses early warning score criteria to admit patients, together with a first come first served basis system, due to the limited number of beds. It is estimated that about ten critically ill patients are deprived of ICU admission daily, and as a result succumb to their illnesses. An ongoing study is being conducted in the hospital to identify missed opportunities for saving such patients.
The audit included all patients admitted to the study ICU from January 1, 2003, until December 31, 2009. No patient was excluded from the study. The following information was recorded for each study patient: basic demographic data (including age and gender), admission criterion, duration of stay in the ICU, and survival to ICU discharge. We also conducted a telephonic survey to establish the ICU bed capacity in the whole country.
Basic descriptive statistics were used to analyse demographics data and other study variables. Logistic regression analysis was used to determine the association between different durations of ICU stay and survival to discharge. P-values <0.05 were considered statistically significance. Data are presented as mean values, with standard deviations, unless otherwise indicated.
For the purposes of the telephonic survey, an ICU bed was defined as comprising a bed, a pulse oximeter, a mechanical ventilator, a suction machine and an anaesthesia provider in the vicinity. We determined that, based on our definition, there were 33 ICU beds in the whole country for a population of 33 million people (Table 1) .
During the study period, 1,774 patients were admitted to the study ICU ( Table 2 ). The mean age of the study patients was 35.5 ears. The majority of the patients (56.5%) were male. Indigenous Ugandans accounted for the majority (92.8%) of the patients. Upcountry referrals constituted 42.9%, and the remaining patients were from within and around Kampala, the capital city of Uganda. The mean mortality rate over the 7-year period was 40.1% (n = 715) ( Table 3 ). The highest mortality rate (44%) was observed in 2004; the lowest mortality rate (33.2%) was observed in 2005. Children (age <18 years) accounted for 11.6% of admissions, and their mortality rate was 40.1%, with paediatric post-operative admissions being higher than paediatric medical admissions.
Sepsis, ARDS, traumatic head injury, and HIV/AIDS were the most frequent admission diagnoses during the study period (Table 2) . Neurosurgical conditions accounted for the ICU admission diagnosis with the highest mortality. Patients who stayed in the hospital for 6 to 10 days were three times more likely to survive compared with patients who stayed for 1 to 5 days. Patients who stayed for 11-20 days were at twice as likely to surviveas likely to die compared with patients who stayed for 1 to 5 days ( Figure 1 ) (Table 4 ). Patients who stayed for >20 days were almost twice as likely to survive compared with patients staying for 1 to 5 days.
In this retrospective audit, we aimed to determine admission patterns in our ICU during a 7-year observation period. We found that the two most common admission diagnoses were identical to those reported by ICUs located in other parts of the world [5] [6] [7] . The overall mortality rate of 40.1% is comparable to reports from other African country ICUs [5] , but much higher than that reported by ICUs in high-income regions of the world (at between 10-20.9%) [6] [7] [8] .
Head injuries were a common reason for ICU admission and associated with the highest mortality rates in this audit. This is not surprising, considering that the study ICU is a general ICU and does not have specialised neurocritical care resources (e.g. facilities to measure intracranial pressure or arterial blood gases). This is despite the ICU being served by four neurosurgeons; therefore the limitations are related to infrastructure rather than skills or personnel. It was difficult to determine what proportion of deaths was preventable because reliable data for this was not available.
A paper by Mock et al. estimated that improved trauma systems can avert between one and two million deaths a year in severely injured patients in low-and middle-income countries [9] . The lack of neurocritical monitoring equipment is coupled with the fact that Uganda does not have a functional emergency medical response system. This leads to inadequate transportation of trauma victims to health care facilities and delays in definitive care.
There are a limited number of ICU beds in Uganda as a whole -only one ICU bed for every one million Ugandans or 0.1 ICU beds/100,000 (Table 1) . This compares poorly with South Africa (8.9/100,000), Sri Lanka (1.6/ 100,000), and the United States of America (20/100,000) [1] . This also explains the high number of referrals to Mulago hospital from upcountry centres. This limitation is further compounded by a well-documented dearth of anaesthesiologists-a critical human resource for intensive care units [10, 11] .
Adequate emergency care at a crash scene (e.g. airway management, positioning, oxygen and fluid resuscitation) is known to improve trauma outcome [12] . The high number of non-helmet wearing motorcycle riders in Uganda, and in Kampala in particular, also contributes to the high injury severity and mortality rate of neurotrauma observed in this study [13] .
Sepsis was also a common cause of mortality, with mortality rates higher than those reported from industrialised countries [6] [7] [8] . Although our study data cannot explain the high mortality rates associated with sepsis, it is likely that insufficient early sepsis care may have contributed. Delayed presentation of sepsis patients to the hospital, and subsequently to the ICU, is common [14] . The paucity of resources to manage patients with sepsis (e.g. insufficient amounts of fluids, unavailability of intravenous broad-spectrum antibiotics and unavailability/unreliability of microbiological diagnostics) may have prevented adequate sepsis management at the study ICU. Recently, an expert group published guidelines to help resource poor settings manage critically ill patients with sepsis [15] . The recommendations have been well received in a number of resource limited countries. The patient population included in this study is younger compared with patients admitted to ICUs in industrialised countries [16] . However, our findings are similar to those reported from surveys of critically ill patients treated in other African countries, where life expectancy is comparably low to that of Uganda [5] . Similarly, the mean length of ICU stay in this study resembled that in other parts of Africa. The finding that patients who stayed 6 to 15 days in the ICU experienced better survival to discharge than those treated for less than 5 days or longer than 2 weeks indicates that patients in the study ICU typically die early (within a few days) or relatively late (after 2 weeks). Early deaths can most likely be explained by the lack of trained staff and resources to provide adequate care for critically ill patients with a high disease severity (e.g. those with brain trauma, shock or sepsis).
Children accounted for 11% of all ICU admissions with a mortality rate of 40%. This is similar to other African country ICUs [17, 18] , but considerably higher than in industrialised countries [19] . The lack of ventilators and accompanying resources in the paediatric high dependency unit at the Mulago hospital is one of the main reasons why children are admitted to the study ICU. Although our study cannot prove a causal relationship, it is likely that delayed initiation of mechanical ventilation and aggressive resuscitation could explain the high death rate in the paediatric patients in the study population There was a higher mortality in the paediatric medical group than in the surgical group, and we hypothesise that this is because a lot of the post-operative patients were elective surgical patients who were admitted for observation. Most paediatric referrals were, and continue to be, children with acute respiratory failure who are transferred from the paediatric high dependency unit because they are in need of mechanical ventilation. The relatively younger population in LICs and the fact that respiratory illness is the leading cause of deaths in under-5-year olds in such countries [20] , implies that more emphasis should be placed on strengthening paediatric critical care resources in LICs. Previous studies have suggested the need to estimate the cost effectiveness of critical care in this setting, given the relatively younger and economically active population. [1, 2] The fact that HIV is endemic in Uganda explains why HIV/AIDS was one of the most common reasons for admission in the study population. Due to the advent of easily accessible highly active anti-retroviral therapy, together with septrin prophylaxis, the incidence of HIVrelated diseases (such as pulmonary infection with Pneumocystis jiroveci, which usually presents as acute respiratory failure) has markedly decreased [21, 22] . In this survey, it was difficult to retrospectively determine from the medical records whether acute respiratory failure was due to infection or other causes. We could, however, determine that chronic obstructive pulmonary disease was a very rare cause of acute respiratory failure in our setting. Other rare HIV-related causes of ICU admission were viral encephalitis and liver failure.
Obstetric admissions in our study were largely due to perioperative cardiac arrest occurring as a consequence of peripartum haemorrhage, eclampsia and/or sepsis. Following the introduction of protocolised care for peripartum emergencies and the establishment of the obstetric high dependency unit (patient monitors and more intense nursing and protocols without mechanical ventilation) at the Mulago hospital, the number of obstetric critically ill patients admitted to the study ICU dropped substantially.
Limitations of this study include its retrospective nature with the consequence that it could not provide the same level of evidence as a prospective survey. Furthermore, due to the concise format of medical records, only limited data could be retrieved for this audit. For example, information on whether patients received mechanical ventilation; the volume of fluids; and drugs was not available. According to anecdotal evidence, 99% of all admissions are mechanically ventilated; however, the lack of data to support this precludes us stating this as a fact. Other ICU-relevant data would have allowed better description of the study population. More detailed data may have allowed for examination of other variables associated with mortality. It is hoped that advances in health information technology in low-income countries will result in improved reporting ability in the future.
This is the largest study to date of critically ill patients in a low-income setting in sub-Saharan Africa. Our ICU study population is a young one and, even though we have limited data for comparison, high-income countries may have an older ICU population. We had a mortality rate of 40.1%, with sepsis, head injury, acute lung injury and HIV/AIDS the most common admission diagnoses. The mortality rate stayed the same over time, possibly because the admitting doctors stuck to their prognoses, and there are limitations in resources and a paucity of use of evidence-based practice. The fact that half the patients came from outside of the capital city is explained by the dearth of ICU beds in the country as a whole. Critical care remains a neglected area of health service delivery in this setting, with large numbers of patients with potentially treatable conditions not having access to such services. Further research needs to be carried out in ICUs in other resource limited settings, including a prospective study to estimate the resources required to design resource appropriate units in such settings and the impact on morbidity and mortality, especially for the most common conditions. Influenza Virus Infection in Nonhuman Primates To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza. W orldwide, infections with infl uenza A viruses are associated with substantial illness and death among mammals and birds. Public health and research have placed major focus on understanding the pathogenicity of different infl uenza virus strains and characterizing new infl uenza vaccines. Nonhuman primates (NHPs), including macaques, have become popular experimental models for studying the pathogenesis and immunology of seasonal and emerging infl uenza viruses. NHPs readily seroconvert after experimental inoculation with seasonal infl uenza virus and have been used to test candidate vaccines for strains of human and avian origin. Like humans, macaques infected with infl uenza virus exhibit fever, malaise, nasal discharge, and nonproductive cough; virus replication can be detected in the nasal passages and respiratory tract (1, 2) . However, whether NHPs are infected with infl uenza viruses in nature remains unknown.
Over the past decade, we have focused on the role of pet and performing monkeys in disease transmission throughout Asia. Commonly trapped in the wild, these monkeys might be sold at wet markets, the putative source of several zoonotic outbreaks (3), where they might be caged next to any number of animal species (Figure 1 , panel A) (4). Pet and performing monkeys are likely conduits for cross-species transmission of respiratory pathogens like infl uenza viruses because of their close and long-term contact with their owners, audiences, domestic animals, wild animals, and birds ( Figure 1, panel B) (5) . However, the breadth and diversity of this interface presents a challenge for monitoring the emergence of infectious diseases. We have approached this challenge by conducting longitudinal studies at several sites and collecting biological samples and behavioral data representing various contexts of human-NHP contact (4-7). We used these historical and newly acquired samples, representing various countries and contexts of human-macaque contact, to determine whether NHPs are infected with infl uenza viruses in nature.
As part of our decade-long longitudinal studies, ≈200 serum samples were collected from macaques. These included pet macaques (Macaca nigra, M. nigrescens, M. hecki) from Sulawesi, Indonesia; performing macaques from Java, Indonesia (M. fascicularis) and from Bangladesh (M. mulatta); M. fascicularis macaques from the Bukit Timah and Central Catchment Nature Reserves in Singapore, where they freely interact with wild avian fauna and visitors (occasionally entering residential areas) (7); M. sylvanus macaques from the Upper Rock Nature Reserve in Gibraltar, where international tourists frequently use food to entice the macaques to climb about their heads and shoulders (6) ; and free-ranging macaques (M. fascicularis and M. nemestrina) at temple shrines or M. fascicularis macaques that range throughout a wildlife rescue center and nearby villages in Cambodia ( Figure 2 ). Serum was collected and stored as described (8) . All samples were stored on cold packs in the fi eld and transferred to dry ice for shipment to the United States, where they were then stored at −80°C.
For initial screening for antibodies against infl uenza virus, serum samples were treated with receptor-destroying enzyme as described (9) and tested by using a multispecies Infl uenza A Virus NP Antibody Inhibition Test (Virusys Corporation, Taneytown, MD, USA) according to manufacturer's instructions. ELISA results indicated nucleocapsid protein antibodies against infl uenza in samples from macaques from Cambodia (29.2%), Singapore (16.7%), Sulawesi (16.1%), Bangladesh (13.3%), and Java (6.0%) ( Table 1) . Antibodies were detected in animals 1-10 years of age at the time of sampling. No infl uenza virusspecifi c antibodies were detected from the 73 total samples from Gibraltar, perhaps because persons with infl uenza virus infection infrequently travel to the Upper Rock Reserve (healthy-visitor effect) (10) or perhaps because monkeys from Gibraltar are less susceptible to infection. Seroprevalence of antibodies against infl uenza A virus, by site and collection year, human and NHP population densities, and prevalence of avian infl uenza viruses are shown in Figure 2 .
Serum samples that were positive by ELISA were also screened by hemagglutination-inhibition assay as described (9) . Based on the year and location of NHP sample collection, the estimated ages of the NHPs at the time of sample collection, and the presence of avian H5 and H9 infl uenza viruses in many of these countries during the sampling period (11) (12) (13) , a panel of human vaccine strains and avian infl uenza virus strains was used in the hemagglutination-inhibition assay. Although not all ELISA-positive serum samples could be subtyped, antibodies against seasonal subtype H1N1 and H3N2 infl uenza A strains were detected from macaques in Bangladesh, Singapore, Java, and Sulawesi (Table 2) . Of the performing macaques in Bangladesh, 2 had antibodies against A/chicken/Bangladesh/5473/2010, a strain of G1 clade subtype H9N2 avian infl uenza virus. Subtype H9N2 infl uenza viruses are prevalent in poultry in Bangladesh (14) and have been detected in humans (12) . We did not detect antibodies against highly pathogenic avian infl uenza subtype H5 viruses, which might not be surprising given our relatively small sample size (Table 2) . Also given the small sample size, we were unable to perform microneutralization studies, which would be useful to perform with future samples.
In 2011, to determine whether any macaques were actively infected with infl uenza virus, we collected oral swabs from 48 monkeys in Cambodia to test for infl uenza virus by real-time reverse transcription PCR as described (8) . In brief, the inside of the anesthetized and immobilized monkeys' mouths (cheeks, tongue, and gums) were swabbed. Swabs were immediately placed into viral transport media, stored, and shipped as previously described. RNA was isolated by using an Ambion MagMAX-96 AI/ND Viral RNA Isolation Kit (Life Technologies Corporation, Grand Island, NY, USA) on a Kingfi sher Flex system (Thermo Fisher Scientifi c, Waltham, MA, USA). Viral RNA was analyzed in a Bio-Rad CFX96 Real-Time PCR Detection System and a C1000 Thermocycler (Bio-Rad, Hercules, CA, USA) with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Foster City, CA, USA) and the InfA primer/ probe sets as described (15) . Of the 48 respiratory samples, 1 (2.1%) was positive for infl uenza virus; cycle threshold value was 38 (limit of detection is 40). Attempts to amplify longer PCR fragments of matrix, hemagglutinin, or neuraminidase genes or to isolate the virus by blind passage in embryonated chicken eggs or MDCK cells were unsuccessful.
Our results indicate that NHPs that have contact with humans can be naturally infected with seasonal endemic human infl uenza viruses and with emerging pandemicrisk avian infl uenza viruses. We found serologic evidence of infection in several countries, contexts, and macaque species. Preliminary real-time reverse transcription PCR results also pointed to the presence of virus in a buccal swab from an adult macaque from Cambodia, indicating active infection at the time of sampling. On the basis of results from this study, it seems that pet, performing, and free-ranging macaques are susceptible to infl uenza virus infection. Given the close relationship between humans and NHPs in areas of the world where avian and human infl uenza viruses cocirculate, further surveillance of these populations is warranted. The ability to detect and eventually isolate strains of infl uenza virus currently infecting NHPs and humans at the animal-human interface is paramount to understanding how NHP-human interactions can affect the genetics, transmission, and public health risk for infection with infl uenza A viruses. Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg. In recent years, various types of biosensors have been increasingly becoming practical and useful tools in a wide variety of analytical devices [1, 2] . The immobilization of biological elements to realize the biosensor is an essential step for the successful construction of a diagnostic system. In order to allow the detection of a small amount of target sample and improve detection performance, bioreceptor proteins must be immobilized onto biosensor chip surfaces with high density and nonspecific adsorption avoided or at least minimized. Moreover, orientation control with retention of protein conformation and activity is a required task to be established [3] . One method involves the physical adsorption via van der Waals forces, ionic binding or hydrophobic and polar forces on an insoluble matrix. This is a simple process which causes little disruption of the proteins, while it is unstable during the binding procedure due to the highly dependency against environmental conditions in maintaining its functional characteristics. Thus, the resulting receptor layer seems to form heterogeneous and random orientation. Another method can also be constructed by crosslinking functional reagents by a certain number of functional groups due to its simple procedure and strong chemical bond of proteins. This is widely used for stabilization of proteins that are covalently bound onto the support platform generated by chemical treatment. However, this method has also disadvantages as follows: the difficulty in controlling the crosslinking reaction, the gelatinous nature of the proteins and the relatively low activity of the proteins due to the specific structural features [3, 4] . In each of these methods used for the favorable performance of biosensors, a number of factors deserve careful consideration for strong binding forces between the solid surface and recognition elements, exposure of active sites on the target sample, conservation of biological activity after immobilization process, and simple protocol [3, 4] .
Many researchers have studied in vivo combinatorial biotechnology, e.g., either phage or cell-surface display techniques, and developed polypeptide sequences, which can specifically bind to metals [5] [6] [7] , oxides [8, 9] , and semiconductors [10] [11] [12] . Among them, gold-binding polypeptide (GBP) is one of the genetically engineered proteins for a strong binding onto the gold surface [7, 13, 14] . Whereas many proteins and chemicals bind to the gold surface with thiol linkage, GBP does not contain a cysteine residue having thiol group. Although the definite mechanism is not clear yet, it is estimated that these polar groups in GBPs seem to coordinately interact with the gold surface within a monolayer [15, 16] . In addition, the kinetics and thermodynamics of biomimetic interactions between the GBP, and the gold surface were investigated by surface plasmon resonance (SPR) [14, 17, 18] . Compared with other thiol-based systems, the GBP binds tightly to the gold surface due to the lower standard Gibbs free energy for the bond, and the binding process is fast under aqueous conditions compatible with biological environments [14, 17, 19] . These characteristics suggest its potential applications in nano-and bio-technologies as novel agents for surface functionalization [13] .
Moreover, immobilization with correct orientation of biological material is a problem of prime importance in biosensors. We employed GBP-fusion proteins in the construction of biosensor for the detection of hepatitis B viral surface antigen (HBsAg) as a model (Figure 1 ), which is a biomarker for diagnosing the hepatitis B virus (HBV). The strong affinity between the GBP and the gold surface guarantees the stability of this sensor system and orients the sensing parts outward from the solid surface, exposing them directly to the target sample [13, 16] . Furthermore, electrochemical detection has attracted considerable interest recently for miniaturized analytical systems [20, 21] , including remarkable sensitivity (approaching that of fluorescence), inherent miniaturization of both the detector and control instrumentation, independence of optical path length or sample turbidity, low cost, low-power requirements and high compatibility [22, 23] . Besides, one of the most attractive points of this method is its potential for portable assays in a variety of point-of-care testing (POCT) environments. We here developed a simple platform biosensor technology by mediating the recognition parts and the solid surface on the gold substrate. SPR analyses were used for optimization of sample concentrations and verification of target sensing. Electrical signal-based detection methods for HBV such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were developed on the gold electrode surface, which has been a very versatile material in the field of biosensors. 
Restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). Agarose was from Cambrex BioScience Rockland (Rockland, ME, USA). 30% (w/v) acrylamide/bis solution and protein assay were purchased from Bio-Rad (Hercules, CA, USA). HBsAg PreS2 peptide (H 2 N-NSTTFHQALLDPRVRGLYFPAGG-COOH) was synthesized at Peptron (Daejeon, Korea). Ni-NTA affinity kit was from Qiagen (Hilden, Germany). Other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. All oligonucleotides were synthesized at Bioneer (Daejeon, Korea).
Polymerase chain reaction (PCR) experiments were performed with a PCR Thermal Cycler (Bio-Rad) using High-Fidelity PCR System (Boehringer Mannheim, Mannheim, Germany). DNA sequences were confirmed by automatic DNA sequencer (ABI Prism model 377, Perkin Elmer, Grove, IL, USA). Cell growth was monitored by measuring the absorbance at 600 nm (OD 600 ; DU ® 650 spectrophotometer, Beckman, Fullerton, CA, USA). Cells were disrupted by using ultra-sonicator (Braun Ultrasonics, Danbury, CT, USA). SPR experiments were performed by using SPRLAB™ system (K-MAC, Daejeon, Korea) and BIAcore3000 (GE Healthcare, Uppsala, Sweden). Electrochemical detection analysis was carried out using CHI 660C Electrochemical Analyzer/Workstation (CH Instruments, Austin, TX, USA).
The flow-chart for the construction of the fusion protein between the GBP and its single-chain antibody (ScFv) is shown in Figure S1 (Supplementary material). The 6HGBP-ScFv fusion protein was prepared by genetically fusing the GBP and ScFv, allowing two specific interactions between GBP and gold substrates, and the capture of HBsAg and its ScFv, respectively. For easy purification of the fusion protein by metal affinity chromatography, the coding sequence of a six-histidine (6H) was introduced at the N-terminus of the GBP. For the cloning of the fusion gene, the DNA fragments encoding 6histidine-fused GBP (6HGBP) were obtained by PCR amplification using plasmid pTacFadLGBP-1 [16] as a template, and P1 (5'-AAAATACCATATGGGCCACCATCACCATCACCACGG-3') and P2 (5'-TTCCCCATGGAGACGAATGGTACCGCTCGT-3') as primers. The PCR product was digested with NdeI and NcoI, and ligated into the same sites of pET-22b(+) (Novagen, San Diego, CA, USA) to make pET-6HGBP. For the cloning and expression of the 6HGBP-ScFv fusion gene, the DNA sequence encoding ScFv fragment was amplified by PCR using plasmid pET-ScFv-SBD [18] as a template, and P3 (5'-CAAGACCATGGGTGTCGACTGAGGAGTCTGGA-3') and P4 (5'-TCCGCTCGAGACGTTTTATTTCCAGGTAGGT-3') as primers. This PCR product was digested with NcoI and XhoI, and ligated into the same sites of pET-6HGBP to make pET-6HGBP-ScFv.
) gal dcm (DE3)] was used as a host strain for the expression of GBP-fused ScFv fragment (GBP-ScFv). Recombinant E. coli BL21(DE3) strain harboring pET-6HGBP-ScFv was cultivated in 250 mL flasks containing 100 mL Luria-Bertani medium supplemented with 2% (w/v) glucose and 100 μg/mL of ampicillin at 37 °C in a rotary shaking incubator. Cell growth was monitored by measuring the absorbance at 600 nm. At an OD 600 of 0.4, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce the gene expression. Then, cells were further cultivated for 6 h and harvested by centrifugation. Cells were disrupted by sonication for 1 min at 40% output in cell lysis solution (Tris-NaCl buffer containing 6 M GuHCl and 5 mM imidazole, pH 8.0) and centrifuged at 16,000× g for 10 min at 4 °C. The pellet containing insoluble proteins was denatured, purified and underwent dialysis for further experiments. Since the 6HGBP-ScFv fusion protein contains 6H tag at N-terminal, they could be simply purified using Ni-chelating resin (Qiagen, Valencia, CA, USA) without further purification step. Protein concentration was determined by Bradford's method using bovine serum albumin (BSA) as a standard.
The SPR experiments were carried out at 25 °C by using a repeated angle mode and a fixed angle mode. The repeated mode is the method for measuring the changes of the minimum resonance angle in specially fixed angle range by repetitive angle scanning and fitting, which is a plot of change in the reflectance intensity as a function of time (Resonance vs. Time). SPRLAB™ (K-MAC, Korea) system detects a refractive index and thickness change containing semiconductor laser source with 635 nm wavelength and dielectric silicon photodiode detector, which has incident angle range of 30-80 degree.
For the HBsAg detection, the concentration of 6HGBP-ScFv fusion protein was roughly optimized at first. Sequential injection of solutions was as follows: For equilibration, phosphate-buffered saline (PBS) solution was flowed over the bare gold surface to wash away any potential contaminants. Then, several different concentrations of 6HGBP-ScFv fusion protein were injected. The optimal concentration of 6HGBP-ScFv fusion protein, determined in this process, was used for detecting various concentrations of target HBsAg in the following experiments. In the process of HBsAg detection, BSA instead of the target was injected as a negative control. Prior to the target binding, 0.5 mg/mL of BSA was injected with a flow rate at 20 μL/min for an effective blocking of nonspecific binding sites. In order to remove nonspecifically bound molecules and unbound samples, washing step was applied intermittently with PBS for about 5 min. For the binding of targets, a fixed flow rate at 5 μL/min and binding time of 5 min was applied, consuming a total sample volume of 100 μL.
Electrochemical measurements including EIS and CV were performed on a conventional electrochemical cell equipped with Ag/AgCl with 3 M KCl as a reference electrode, platinum wire as a counter electrode and bare gold electrode as a working electrode. All potentials were referred to the Ag/AgCl reference electrode.
Immediately prior to use, working electrodes were cleaned by five cycles of CV in a potential window of −0.5 to 0.5 V to remove any potential contaminants. Then, the clean gold surface was serially modified by sequential immersion in 50 μg/mL 6HGBP-ScFv and 0.5 mg/mL BSA as a negative control for 1 h at room temperature, respectively. In the last step, the modified electrodes were incubated with various concentrations of HBsAg solution for about 2 h. The washing step with DI water and nitrogen gas was performed after each binding event.
The electrode fabrication process, namely, confirmation of binding of recognition elements onto the gold electrode was characterized by EIS and CV, while EIS was used for target detection. Electrochemical measurements including EIS and CV were performed in 1 mM ferricyanide in 0.1 M KCl (Nyquist plot). CV experiments were carried out in unstirred solutions at a scan rate of 0.1 V/s and at a fixed potential window of −0.5 to 0.5 V vs. Ag/AgCl. Impedance measurements were carried out in the frequency range from 10 4 down to 0.1 Hz with AC amplitude of 5 mV and bias potential of 0.22 V.
To verify the expression level of the 6HGBP-ScFv fusion proteins, the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 6HGBP-ScFv fusion protein was carried out as shown in Figure S2 (Supplementary material). Compared with the wild-type E. coli BL21(DE3) strain as a negative control, E. coli BL21(DE3) harboring pET-6HGBP-ScFv expresses a thick band between marker bands of size 27.5 kDa and 37 kDa. This band size is in agreement with the size of 6HGBP-ScFv fusion protein, which was calculated as approximately 31.9 kDa. Since this fusion protein is expressed as an insoluble fraction, it was purified in denaturing condition and sequentially subjected to dialysis. According to the result, the most right band shows the 6HGBP-ScFv fusion protein purified using a Ni-chelating column resulting with good purity and quantity for further use in several sensing platforms.
SPR is a label-free and sensitive spectroscopic technique used to study bioaffinity interactions on gold thin films by measuring changes of the refractive index under conditions of total internal reflection [24, 25] . To prove that GBP-fusion proteins could be functionally immobilized on the gold surface, the 6HGBP-ScFv fusion protein was flown over the gold sensor chip to monitor its binding affinity by combining the GBP-fusion approach with SPR. The dynamic and specific binding of fusion proteins onto the gold sensor chip could be directly monitored in real time by Biacore ® SPR spectroscopy ( Figure S3 in Supplementary material). A sharp increase in the SPR signal was observed upon introducing the 6HGBP-ScFv fusion protein solution onto the chip surface. About 93% of the injected total 6HGBP-ScFv fusion proteins were bound onto the gold sensor chip surface. Most 6HGBP-ScFv proteins remained bound to the gold surface after washing with PBS solution. Whereas a little decline in SPR signal during washing is due to the removal of unbound 6HGBP-ScFv proteins from the gold surface, the SPR signal sharply decreased after washing with the PBS solution when ScFv instead of 6HGBP-ScFv was flown over the gold sensor chip. About 23% of ScFv protein was nonspecifically bound to the gold surface compared with 6HGBP-ScFv fusion protein. The binding affinities were calculated by assuming a single binding site model as shown in Table S1 . Strong binding of GBP-fusion protein onto the gold chip surface suggests that various protein-protein interactions can be performed by using this system.
To confirm the successful binding of a series of molecules, the detection of HBV was first performed using commercial SPR system. At first, 6HGBP-ScFv concentration was optimized as follows: 100, 50 and 10 µg/mL of 6HGBP-ScFv fusion protein was flowed over the gold surface, respectively. The fusion protein 6HGBP-ScFv was immobilized onto the gold surface via its intrinsic affinity with the gold as shown in Figure 2 . Interestingly, coverage of 6HGBP-ScFv on the gold surface didn't always increase with increasing protein concentrations, a maximal RU change appearing at concentrations of 50 µg/mL. From this result, 6HGBP-ScFv concentration of 50 µg/mL was chosen for subsequent experiments. Next, we checked the specificity of this platform with BSA as a negative control. First, 50 µg/mL of 6HGBP-ScFv was injected into the fluid phase, followed by 0.5 mg/mL BSA blocking of the binding sites on the fusion protein. Then, 10 µg/mL of control peptide (HOOC-CGPTGPTGPTGPTGPT-NH 2 ) as a negative control, 50, 10, 5, 2.5 and 1 µg/mL of target HBsAg were injected into respective channels, respectively. Below concentrations of 50 µg/mL for HBsAg, RU levels dropped rapidly while target concentrations as low as 2.5 µg/mL can be detected compared with the control sample. For 50, 10, 5, 2.5 and 1 µg/mL of target HBsAg, and 0.5 mg/mL of BSA, RU increase is 542.2 RU, 55.7 RU, 48.5 RU, 17.4 RU, 4.9 RU and 5.6 RU, respectively, showing an increasing trend of SPR signals with increasing concentration of target within this scope.
To detect HBsAg, electrochemical assay was also carried out, which have traditionally received the major share of the attention in biosensor development for their various advantages such as high sensitivity, specificity and simplicity, and inherent miniaturization of modern electrical bioassays permits them to rival the most advanced optical protocols [26] . Such miniaturization allows packing of numerous microscopic electrode transducers onto a small footprint of the biosensor device, and hence the design of high-density arrays was required. In this paper, EIS and CV were performed in order to characterize the electrode fabrication process, and EIS was applied to detect HBsAg. For the determination of optimal 6HGBP-ScFv coverage, its concentration and volume were carefully calculated in regard to that used in SPR analysis.
For the characterization of the electrode fabrication process, EIS and CV were performed each time after binding of each reagent. As shown in Figure 3(a) , the dotted line is the impedance spectrum obtained on the bare gold electrode. Frame circle line is an impedance spectrum of 50 µg/mL 6HGBP-ScFv fusion protein modified electrode, and solid circle line is that of after BSA blocking. Finally, solid line represents an impedance spectrum obtained after HBsAg binding. The result reveals the resistance of the electron transfer at the electrode surface increasing step by step because of the insulating effect of the binding proteins. This electron transfer resistance at the interface on the electrode surface, and solution can be determined by the diameter of the semi-circle of the curves in EIS spectra. After the gold electrodes were immobilized with GBP-ScFv, the peak current decreased dramatically with an increase of the peak-to-peak potential separation (ΔEp) for the bare gold electrode. When HBsAg was bound on the gold chip surface, the peak current more decreased and ΔEp (approximately 110 mV) increased comparing with those of the gold electrode and the GBP-ScFv immobilized chip, resulting from the electron transfer resistance of bound HBsAg molecules. BSA was used as a negative control. It can also be observed that the current responses in CV spectra (Figure 3 (b)) are decreasing in the process of electrode fabrication, which coincides with a conclusion drawn from impedance assay. These results were due to the insulating characteristics of the protein. As shown in Figure 4 , 50 µg/mL, 10 µg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg were detected by using this method, respectively, and they presented a rough trend of increasing electron transfer resistance with increasing target concentrations. As a negative control, BSA of 10 µg/mL was detected at the same time. Numerical data were drawn by fitting with a circuit model as shown in an inset of Figure 4 . Correspondingly, binding of 50 µg/mL, 10 µg/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL of HBsAg and 10 µg/mL BSA caused electron transfer resistance increase of 8,493 Ω, 6,473 Ω, 4,047 Ω, 3,097 Ω, 2,143 Ω, 1,513 Ω and 777 Ω, respectively. Though linearity is not very strict as a function of HBsAg concentration, it demonstrated a rough linear trend into log scale. Target concentrations as low as 0.14 ng/mL of HBsAg calculated via 3-sigma rule was successfully detected compared with the negative control of 10 µg/mL of BSA ( Figure 4 ). Furthermore, a lower limit of detection (LOD) can be further expected if experimental conditions, such as 6HGBP-ScFv concentration, binding time, temperature and washing condition, were optimized. In order to check the nonspecific binding of real blood sample, we tested a fetal bovine serum of 10 µg/mL instead of HBsAg for clinical trials [27, 28] and confirmed no effect. Several procedures for HBV diagnosis with GBP-fusion protein were developed in this study. First, 6HGBP-ScFv fusion protein was prepared from simple cultivation of recombinant E. coli for HBsAg detection. This GBP-fusion protein allowed for the direct and easy immobilization onto the gold surface. Successful binding of the proteins was demonstrated by SPR optical analysis due to versatile use of gold in the sensing area. Second, this one-step immobilization process onto the solid surface is very simple. Strong affinity between the GBP and the gold surface guarantees the stability preventing possible leakage of sensing proteins. Binding of GBP onto the gold surface also might help to expose sensing molecules outward to react with their targets by arrangement for correct orientation of bioreceptor. Additionally, GBP prevents direct contact between proteins and the gold surface, which is advantageous for protein activity conservation. Furthermore, it is easy to conclude that a device capable of detecting multiple targets can be designed since the bio-recognition elements against targets can be easily fused with GBP via recombinant DNA technology or linker chemistry. Coupling with microfluidics, it can serve to minimize sample volume required as well as to decrease diagnosis time.
In summary, we have developed a label-free electrochemical method for HBV detection based on a gold-specific immobilization strategy. The results showed that optimal concentration of bioreceptor was 50 μg/mL in SPR analysis, and the LOD for HBsAg was about 0.14 ng/mL in the electrochemical analysis. This EIS analysis successfully presents the effectiveness of this sensing platform. Regarding intrinsic advantageous property of the electrochemical assay, such as high sensitivity, simplicity and especially its inherent miniaturization characteristics, and a popular trend of development of electricity-based sensors in everyday life, the proposed method presented in this study has a huge potential in commercialization of a POCT device for viral diagnosis. We have to be conducted with clinical trials in the near future to determine its performance. Development of a Plastic-Based Microfluidic Immunosensor Chip for Detection of H1N1 Influenza Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device. Swine-origin influenza virus, a high-risk human influenza A virus (H1N1), is a serious health threat and potential leading cause of death all around the World [1] . The World Health Organization (WHO) has reported that more than 16,000 cumulative deaths were reported from 213 countries due to H1N1 in February 2010 [2] . Several laboratory diagnostic methods have been developed to monitor the outbreaks of the virus as follows: (1) specific real-time polymerase chain reaction (PCR)-based detection method, (2) isolation of H1N1 influenza virus, (3) detection of 4-fold rise of neutralization antibodies to the virus [3, 4] . However, these methods require highly skilled-personnel and expensive laboratory instruments. In addition, they are not suitable for undeveloped countries because of the limited access to central laboratories and expensive costs.
To overcome these issues, microfluidic immunoassay systems have been introduced because of their various advantages, including high throughput, high-efficiency, low-cost and minimized consumption of samples and reagents [5] . After the development of soft lithography techniques using poly(dimethylsiloxane) (PDMS), PDMS has become the most popular microfluidic device materials and offers several advantages such as easy handling, good sealing properties and high optical transparency [6] . However, the poor chemical stability in different types of organic solvents, difficulty in surface modification and mass production have limited the use of PDMS in the various applications [7] .
Recently, because of the material issues, some researchers have been attempted to use plastic materials as an alternative solution. Among the various types of polymers, cyclic olefin copolymer (COC) is one of the most popular polymeric materials in the fabrication of microfluidic chips. COC is a well-known polymeric material with various advantages, including high clarity and light transmission, excellent mechanical properties and great biocompatibility [8] .
Furthermore, effective immobilization of proteins is essential and important in microfluidic chips to be used as immunosensors. Several methods to immobilize antibodies on the sensor chip surface have been developed, including physical adsorption, covalent binding, and specific interaction between avidin and biotin [9, 10] . However, these previous methods have limitations in terms of denaturization, extra chemical modification and random orientation. In order to overcome these issues, Brown et al. and Park et al. developed specific gold-binding polypeptide (GBP) that endows the orientation of proteins in their functional state [11, 12] . GBP shows a strong binding affinity to the gold surface without any surface modifications [13] [14] [15] . Therefore, GBP-fusion proteins could be selectively and functionally immobilized onto the gold surface.
In this study, we carefully designed microfluidic devices, and the surface of a detection chamber was coated with gold for the direct assembly of proteins. A microfluidic-based immunosensor to detect human H1N1 influenza was developed into a low-cost immunosensor based on the exploration of fluorescence signals. The detection of a specific antibody among serological assays in blood samples was performed in the microfluidic biosensor chip by immunoreactions between the GBP-recombinant influenza hemagglutinin antigen (GBP-H1a) fusion protein and its specific antibody (Ab). The GBP-H1a fusion protein as a bioreceptor and the fluorescence-labeled Ab as a marker were used to provide an excellent detection signal. In addition, the chip fabrication and sensing characteristics are reported in detail.
COC was purchased from TOPAS Advanced Polymers (Frankfurt-Höchst, Germany). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification steps. Rabbit anti-H1 polyclonal Ab was obtained from AbFrontier (Seoul, Korea).
The microfluidic immunosensor device is fabricated by the injection molding method. The master stamp was fabricated using a micromilling process. The size and thickness of the stamp were 95 × 95 mm and 1.2 mm, respectively. We used a plastic microinjection mold machine (A270C 400-100, ARBURG, Lossburg, Germany) to produce the microfluidic device in order to achieve cost-effectiveness and facilitate mass production. Once the chip was fabricated, chromium (50 Å) and gold (100 Å) were coated on the detection area for further immobilization of GBP-H1a. In this work, the ultrasonic bonding method with an ultrasonic bonder (2000X, Branson, Danbury, CT, USA) was employed to bond the COC chips. For this purpose a melting line with a height of 20 μm was made around microchannels. Once the ultrasonic energy was applied to the COC plates, the sonic energy was intensively localized on the top of the melting line. After the lines were immediately melted and then cooled, both COC plates were tightly bonded with each other to fabricate the plastic-based microfluidic device.
Bifunctional fusion protein was created by genetically fusing GBP and H1a, allowing specific interactions between GBP and the gold substrates as well as the capture of H1a and its antibodies. As described in the previous report [16] , the DNA fragments encoding the H1N1 viral surface antigen (H1a) were amplified by PCR with forward primer (5′-CCATGGCATATGGG CCACCATCACCATCACCACGGCAA-3′) and reverse primer (5′-CCGCTCGAGCTGGCTACG CACTTTTTCATACAGGTTTTTAACGTTGCTATCGTGATAGCCGCAAGCTTGTCGACA-3′) for the construction of 6His-GBP-H1a fusion gene. Then, the PCR product was cloned into the NdeI-XhoI fragment of pET-6HGBP to make pET-6HGBP-H1a.
Recombinant E. coli BL21 (DE3) strain harboring pET-6HGBP-H1a was cultivated in Luria-Bertani (LB) medium (10 g/L bacto-tryptone, 5 g/L yeast extract, and 5 g/L NaCl) supplemented with 100 µg/mL of ampicillin at 37 °C and 250 rpm. At OD 600 (DU600 ® Spectrophotometer, Beckman Coulter, Brea, CA, USA) of 0.4, cells were induced with 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) for the production of the fusion protein. After induction, cells were further cultured for 4 h. The cells were then harvested and disrupted by sonication (Braun Ultrasonics, Orlando, FL, USA) for 1 min at 20% output power. After centrifugation at 16,000× g for 10 min at 4 °C, the pellet containing the soluble protein fraction with high-level expressed target fusion-protein was collected for purification of the fusion protein. Because of the 6His tags, a HisTrap TM column (GE Healthcare, Chalfont St. Giles, UK) was used to purify the fusion protein without further purification steps. The protein concentration was determined by the Bradford assay with bovine serum albumin (BSA) as a standard.
The binding of the GBP-H1a fusion protein onto the surface of a SPR bare gold chip was characterized by SPR measurement using a BIAcore3000 TM with an automatic flow injection system (Biacore AB, Uppsala, Sweden). All experiments were performed in phosphate-buffered saline (PBS, pH 7.4) at room temperature. A fresh SPR sensor chip was attached to a separate chip carrier for easy assembly in the SPR system. After docking and priming of the SPR chip, PBS was used to flush the activated surface and thereby minimize non-specific binding and any unbound sites by removing loosely bound material and dust. All samples were injected onto the gold chip surface at a flow rate of 5 μL/min for 10 min at room temperature using a liquid-handling micropipette in the SPR system. The surface was then washed and equilibrated using PBS. The GBP-H1a fusion protein (50 μL of a 25 μg/mL solution) was injected onto the SPR chip surface. Before binding anti-H1 Ab, 50 μL BSA of 100 μg/mL was injected to block non-specific binding for 10 min. All SPR sensorgrams were fitted globally using BIA evaluation software (Biacore).
The plastic-based microfluidic devices were fabricated by an injection molding technique. The microchannels-embedded master mold was obtained by a milling technique, and it was used for casing the microfluidic devices. In this case, COC was chosen as a substrate material to produce the device due to its great advantages over the other thermoplastic polymers in terms of optical, physical and chemical properties and biocompatibility. The total cycle time of the microinjection molding process took less than 1 min to replicate the COC substrate. After location of the master mold in the injection mold machine, COC was injected through the nozzle, and the microfluidic device was released from the mold. The production process and conditions are similar to the previous research which demonstrated various microinjection conditions to form microstructures [17] . Holes of 1 mm in diameter for an inlet and outlet ports were punched to load the sample and buffer solutions into the microchannels. As shown in Figure 1 , the channel depths are 200 and 500 μm with 450 μm in width, respectively. The diameter of the detection chamber is 1 mm, and it was coated with chromium and gold by sputtering for the immobilization of GBP-H1a fusion protein. The design and fabrication process of microfluidic device are described in Figure 1(A) . The key features of microstructures including welding lines, gold deposition on detection chamber, and backflow prevention structures are schematically demonstrated in Figure 1B . Previously, thermal bonding method has been popular to bond plastic-based microfluidic device. However, this method requires high temperature and takes too much time to bond it. Recently, an ultrasonic bonding method has been developed as an alternative solution to the thermal bonding. However, welding line is essential for bonding between plastic chips in this technique. In this experiment, we carefully designed the welding lines as shown in red color in Figure 1 (B), and these lines were melted during the ultrasonic bonding process for the sealing of the chips. After coating of gold, two COC plates were placed on the ultrasonic bonder (2,000X, Branson) and set the bonder in time mode with weld frequency of 20 kHz. After the setting of the mode, the COC was bonded as following conditions: (1) 800 Pa weld pressure, (2) 0.2 s of weld time, (3) 75% of amplitude, (4) 10 s of hold time, (5) 1.5 kPa of hold pressure. Occasionally, backflow of the buffer or target solutions occurs in microfluidic channels during the injection to cause a contamination problem. Therefore, there would be required a unique microstructure in a microchannel to prevent backflow of solution and contamination of microchannel. Among various methods, two different channel depths were applied into this device as shown in Figure 1(B) . This method was worked properly due to the pressure difference in the microchannel to prevent the backflow. The integrated microfluidic immunosensor chips can reduce the analytical time compared to the conventional methods. In addition, usage of plastic facilitates low-cost mass production of disposable and easy-to-use microfluidic chips. Previously reported plastic-based immunosensors required a surface modification step for the immobilization of biomolecules. Among these methods, silanization using 3-aminopropyltriethyoxysilane (APTES) is a widely used method for coupling of biomolecules onto the inorganic substrate [18] . However, silane is difficult to react with organic materials without any hydroxyl groups present on the surface by using UV/ozone treatment [5] . In order to overcome these issues, a specially engineered peptide was developed and used to immobilize onto the specific targeted surface, especially gold. There is no requirement of complicated steps for coupling of antibody or antigen. The GBP-H1a fusion protein can be simply and selectively immobilized in the microchannel on the gold surface as described in Figure 1(C) .
In this device, Y shaped inlet channels with backflow-prevented microstructure and detection chambers were designed for an efficient immunoassay as shown in Figure 2 . The key features of microstructures in microfluidic device were successfully replicated using the microinjection molding, and they were confirmed through top and tilted scanning electron microscopy (SEM, Hitachi S4800, Ibaraki, Japan) images as shown in Figure 2(C-F) . Due to the height differences between inlet channels and main channels (Figure 2(C,D) ), this could prevent the backflow of the solutions. This device is composed of three detection chambers with 1 mm in diameter for the further immobilization of GBP-H1a fusion protein as shown in Figure 2 , and these chambers may reduce the errors during analysis by averaging signals. All protuberant microstructures near to the microchannels were specially designed as welding lines. During the ultrasonic bonding process to bond the top and bottom of the microfluidic chip, these lines would be melted by concentrating the ultrasonic energies on the top of welding lines. To investigate the sensing window of the fusion protein, different concentrations (6.25, 12.5, 25, 50, 100 and 200 μg/mL, respectively) of GBP-H1a fusion protein were immobilized onto the gold chip surface by surface plasmon resonance (SPR) microfluidics. A greater shift in resonance unit (RU) was observed by SPR analysis with increasing concentration of immobilized GBP-H1a fusion protein bound on the planar surface at various concentrations as shown in Figure 3(A) . These results suggest that the SPR sensor with GBP-fusion protein implemented on the gold surface can be an effective system for biomolecular immobilization. Furthermore, the concentration of GBP-H1a fusion protein was fixed to 100 μg/mL due to its best immobilization concentration.
For the subsequent binding of anti-H1 Ab, different concentrations (1.5 to 400 μg/mL) of specific antibody were bound to the GBP-H1a fusion protein on the gold sensor chip. The saturated 4,000 RU value obtained with SRP experiments implies that about 4 ng of anti-H1 Ab was immobilized onto the gold surface area of 1 mm 2 . One RU is determined as 0.0001° of resonance angle shift and equivalent to a mass change of the 1 pg/mm 2 on the SPR sensor chip surface [19, 20] . Specific anti-H1 Ab against the H1 influenza surface antigen, which is a hemagglutinin subunit having a high immunogenicity and surface probability, was applied to the GBP-H1a fusion protein-layered surface to monitor specific binding between GBP-H1a fusion protein and anti-H1 Ab by SPR biosensor as shown in Figure 3(B) . To further investigate whether this microfluidic device can be used in immunoassay, we employed GBP-H1a fusion protein, BSA as a blocking agent and Cy3-labeled anti-H1 Ab. Cy3-labeled anti-H1 Ab is a strongly fluorescent molecule, and the fluorescence-based immunoassay is more sensitive compared to the most colorimetric assays in most of the cases [5] . The whole immunosensing process was carried out by using COC microfluidic chips at room temperature. The three detection chambers are included in one microchannel to verify the sensing results which may reduce the error of the signal. In addition, one chip is composed of three different detection zones to test different concentrations of the target Abs. First of all, 100 μg/mL of GBP-H1a fusion protein was injected through the microchannel for the immobilization on the surface of gold surface. After immobilization for 1 h, all the chips were washed with PBS solution and BSA solution (1 mg/mL) was injected to the channels to prevent the non-specific binding then washed with PBS solution. After the blocking and washing process, five different concentrations of Cy3-labeled-Ab (100, 50, 10, 5, and 1 μg/mL, respectively) were injected through the microchannel and left them for 1 h. After incubation for the further reaction, all immunosensors were rinsed with PBS solution three times, and the microchannels were blown off by air. All microfluidic immunosensor chips were examined under same conditions of confocal microscopy (Carl Zeiss LSM510 Meta NLO, Göttingen, Germany) as shown in Figure 4 . At the low concentration of Cy3-labeled Ab applied, small fluorescent signals were observed in the detection chambers. As increasing the concentration of Ab, the whole detection chamber was covered with red fluorescence, and the signal intensities were also increased. In order to compare the signal intensity, the intensity profiles were also recorded because the fluorescence intensity is directly proportional to the amount of Cy3-labeled Ab attached to the surface of detection chamber. The fluorescence intensity changes at the center of chamber were measured, and their fluorescent images with same scale in Y-axis were showed. The fluorescent intensity graphs which correspond to each inserted white line also showed similar signal changing patterns compared with the fluorescent image. From these results, the specific binding of GBP-H1a was successfully immobilized on the gold surface, and the fluorescent images and emission profiles were subsequently increased due to the effective binding of Cy3-labeled anti-H1 Ab, which could be applicable in immunoassay onto the microfluidic chip surface. As a result, the fluorescent intensity depends on the number of immobilized Cy3-labeled-Ab in the detection chamber under the same incubation period. In addition, all detection chambers were analyzed using the line profiles. As increasing the amount of Ab, the intensity of line profiles also increases over the time, and the intensity line is getting increased, which is properly matching with fluorescence images. From the analyzed fluorescence results, the calibration curve, which represents the relationship between the fluorescence intensity and Cy3-labeled-Ab, is shown in Figure 4 (G). The regression equation could be expressed as:
where I is the fluorescence intensity and C is the concentration (μg/mL) of Cy3-labeled-Ab with a correlation coefficient of 0.983.
In this study, we report a development of a microfluidic device for the detection of human influenza by antigen-antibody interaction based on a highly transparent and inexpensive polymer. The significant fluorescence intensity changes over the different concentrations to the serological antibodies and three different chambers in one microchannel provide more accurate information to detect the H1N1 flu virus. In addition, the immunosensor chips were successfully applied for the detection of the H1N1 without any surface modification of microfluidic chip. The proposed integrated plastic-based microfluidic chip could provide a significant improvement in the miniaturization and a cost-effective way for bio-analysis systems. Therefore, this platform offers perspective for point-of-care testing diagnosis in various infectious disease areas. Hepatitis C VLPs Delivered to Dendritic Cells by a TLR2 Targeting Lipopeptide Results in Enhanced Antibody and Cell-Mediated Responses Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund’s adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV. Hepatitis C virus (HCV) infection affects an estimated 200 million individuals worldwide and contributes to significant morbidity and mortality rates associated with liver cirrhosis and hepatocellular carcinoma. Approximately 80% of infected individuals do not clear the virus following acute infection and will develop chronic infection that can lead to end-stage liver disease and complications. Although treatment options using a combination of pegylated interferon-a and ribavirin are available, sustained clearance of the virus is only achieved in approximately 40% of individuals infected with HCV genotype 1 and 60-70% of those who are infected with genotypes 2 or 3 [1] . Recent advances in the treatment of HCV using directly acting antiviral agents (DAAs) such as boceprevir and telaprevir have improved SVR rates in both treatment naïve and experienced patients (reviewed in [2] ). However, treatment can be prolonged, expensive and also associated with substantial side effects. The development of an effective vaccine that can significantly reduce the number of new infections and improve sustained virological response rates could therefore be a useful adjunct to current therapeutic approaches and reduce the impact of infection on global health care systems.
Whilst the immune correlates mediating the clearance of virus are still not entirely clear or defined, there is substantial evidence demonstrating that the development of a broad multifunctional T cell response against an array of key viral proteins such as core, E1, NS3, NS4 and NS5 during acute HCV infection is associated with disease resolution [3, 4] and may also provide a level of protection against reinfection [5] . It is also becoming increasingly apparent that such responses alone are not enough [6] and that neutralising antibodies also play an integral role in conferring protection [7, 8] and facilitating viral clearance by mechanisms including antibodydependent cellular cytotoxic mechanisms [9] . An effective HCV vaccine will need to induce antibody and cell-mediated responses and also provide cross protection against different viral genotypes and quasispecies. Neutralising antibodies induced against conserved, conformational epitopes in the viral envelope E1 and E2 glycoproteins [10] [11] [12] , notably antigenic region 3 (AR3) [13] of E2, including the critical neutralisation contact residues contained within domain I of E2 [14] and amino acids 313-327 of E1 [15] , can be broadly cross-neutralising. The fact that these antibodies neutralise different HCV genotypes highlights the importance of including epitopes from both envelope proteins for a vaccine strategy to be effective.
Virus-like particles (VLPs) possess features which make them ideal vehicles for the delivery of viral antigens to the immune system; (i) antibody epitopes are presented in the native conformation for induction of potentially neutralising antibodies (ii) multiple T cell, CD4 + and CD8 + , epitopes are packaged in VLPs (iii) VLPs lack regulatory proteins as well as genetic material that could pose a risk of reversion or mutation (iv) encouraging results have been obtained using insect cell-derived recombinant VLPs expressing HCV antigens which induce virus-specific humoral and cellular responses [16] [17] [18] (v) HCV VLPs appear to possess properties favourable for dendritic cell uptake [19] and (vi) they exhibit superior immunogenicity and antigenicity over recombinant protein and DNA-based vaccine approaches [16, 17] .
An important consideration in the manufacture of HCV-based VLPs is the cell-type used for their manufacture. For example, it has been shown that vaccination with recombinant HCV envelope proteins expressed in mammalian cells, but not in yeast or insect cells, protect chimpanzees from primary infection by an homologous HCV isolate [20] . Similarly, Rosa et al have demonstrated that mammalian cell-derived recombinant envelope proteins bind to human cells with higher affinity than those produced in yeast or insect cells and appear to be antigenically and functionally similar to the viral proteins produced in an infected host cell [21] . More recently, vaccination of macaques using VLPs in a prime-boost regime has been reported to induce broadly neutralising antibody responses against different HCV genotypes [22] . Although all of these studies provide encouraging results supporting the development of HCV VLP-based vaccines, the fact that heterologous viral vectors and/or unrealistic dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity.
In this study we evaluate the immunogenicity of mammalian cell-derived VLPs containing structural proteins (core, E1 and E2) of HCV genotype 1a when delivered directly to dendritic cells (DCs) using a Toll-like receptor 2 (TLR2) targeting lipopeptide. This lipopeptide contains the TLR2 agonist dipalmitoyl-Sglyceryl-cysteine (Pam 2 Cys) and associates electrostatically with protein antigens significantly improving their ability to induce both humoral and cell-mediated responses [23] . The ability of lipopeptide-VLP complexes to facilitate DC uptake, induce antibody capable of inhibiting VLP entry into target cells and to elicit cell-mediated antigen-specific responses were each determined.
HCV virus-like particles were constructed using a recombinant adenovirus containing encoding the HCV structural proteins (core, E1 and E2) of HCV 77H, genotype 1a. Briefly, the core/E1 genes were amplified from pBRTM_HCV 1-3011 plasmid containing the genome of HCV H77 genotype 1a (a gift from Prof C Rice). The core/E1 genes were amplified from pBRTM_HCV 1-3011 plasmid containing the genome of HCV H77 genotype 1a (a gift from Prof C Rice). The forward Core/E1 primer (59gCCTCTAgAgCCACCATgCATCACCATCACCAT-CACACAAgCACgAATCCTAAACTCAAAgAAAAACC 39) was designed to introduce an XbaI enzyme restriction site followed by a Kozak sequence, a start codon and a His(6) tag at amino terminal end of the core protein. The reverse primer of core/E1 (59 ggCTTAAgCCCggTgACgTgggTTTCC gCgTCgAC 39) was designed to amplify from the sequence downstream of the region corresponding to E1/E2 cleavage site (amino acids 383/384) and to introduce an Afl II restriction site at the 39 end. Next, the E2 genome was amplified using a forward primer (59CgACTAgT-gAAACCCACgTCACCgggggAAgTgCCggCCgC 39) that also introduced a SpeI site at the 59 end. The reverse E2 primer (59CgGATATCTCATCAC gCCTCCgCTTgggATATgAgTAA-CATCATCC 39) was designed to introduce a double stop codon and an EcoRV restriction site at the 39 end of the amplicon.
The core/E1 and E2 amplicons were cloned into pGEMEasy (Promega) and subsequently subcloned and ligated to produce a construct which was verified by DNA sequencing. This construct was subsequently subcloned into pAdTrack-CMV (provided by B Vogelstein, Howard Hughes Medical Centre, Baltimore), digested with PmeI and transformed into AdEasier-1 cells by electroporation (Bio-Rad Gene Pulser) as previously described [24] .
High titres of recombinant adenovirions encoding the HCV proteins (rAdHCV-CE1E2) were produced in 293T cells by serial passaging and the equivalent multiplicity of infection (MOI) was determined as described previously [24] . To produce HCV VLPs, Huh7 cells were infected with rAdHCV-CE1E2 at a MOI of 1. At 72 hours post-infection, cells were collected and disrupted using a dounce homogeniser and centrifuged at 17,000 g for 5 min. The supernatant was further centrifuged through a 30% sucrose cushion (containing 20 mM Tris pH 7.4 and 150 mM NaCl) at 178,000 g for 4 hours at 4uC. The resulting pellet was resuspended in 50 mM Tris pH 7.and 100 mM NaCl and purified through a 33% caesium chloride gradient by ultracentrifugation at 14uC at 143,000 g) for 72 hours. Twelve 1 ml gradients were recovered and dialysed against sterile PBS at 4uC overnight. Fluorescent labelling of VLPs was achieved by adding VLPs (6 mg/ml) to 2 mg/ml of fluorescein isothiocyanate (FITC; Sigma Aldrich) in 50 ml of DMSO. The suspension was vortexed vigorously and incubated overnight at 4uC before dialysis against PBS the next day. All VLP preparations were stored in aliquots at 270uC until use. Huh7 and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen USA) supplemented with 10% fetal calf serum (FCS) and streptomycin 50 mg/ml at 37 C in 5% CO 2 .
The syntheses of the branched anionic peptide construct containing eight N-terminal glutamic acid residues (E 8 ) using traditional Fmoc chemistry has been described previously [23] . Briefly, synthesis was carried out manually using PEG-S RAM solid support (Rapp Polymere, Tübingen, Germany; substitution factor 0.27 mmol/g). Fmoc-lysine(Mtt)-OH (Novabiochem, Läufelfingen, Switzerland) was first coupled to the support and the Fmoc protecting group present on the a-amino group then removed and Fmoc-lysine(Fmoc)-OH was then coupled to the exposed N-terminal amino group. Subsequent de-protection and acylation of another two rounds of Fmoc-lysine(Fmoc)-OH yielded eight branch points to which glutamic acid residues were coupled. The primary amino groups of the glutamic acid residues were then acetylated using a 20-fold excess of acetic anhydride and a 40-fold excess of diisopropylethylamine (DIPEA; Sigma, Australia) to generate E 8 which has an overall charge of 28. Lipidation of E 8 was then carried out by removing the Mtt protective group present on the e-amino group of the C-terminal lysine followed by acylation of the exposed e-amino group with two serially added serine residues. The Pam 2 Cys lipid moiety was then coupled according to Zeng et al [25] to generate E 8 (Pam 2 Cys) ( Figure 1A ).
Following assembly, lipopeptides were cleaved from the solid phase support and all side-chain protecting groups removed with 88% TFA, 5% phenol, 2% TIPS, 5% water for 3 hours at room temperature. Lipopeptides were analysed by reversed phase highpressure liquid chromatography (RP-HPLC) using a Vydac C4 column (4.66300 mm) installed in a Waters HPLC system. The chromatogram was developed at a flow rate of 1 ml/min using 0.1% TFA in H 2 O and 0.1% TFA in acetonitrile as the limit solvent. Lipopeptides were purified if necessary. All products presented as a single major peak on analytical RP-HPLC and had the expected mass when analysed using an Agilent series 1100 ion trap mass spectrometer.
A line of murine BALB/c-derived DCs (D1 cells) was prepared and propagated according to the method described by Chua et al [26] . After a minimum of 21 days in culture, cells were stained for Class II MHC using FITC conjugated anti-IA/IE antibody (Clone M5/114.15.2; Becton Dickinson, USA) and PE-conjugated CD11c (Clone 2G9; Becton Dickinson, USA) prior to use. Cells were verified to be CD11c + MHC Class II + by flow cytometry using a FACSCaliber (Becton Dickinson, USA). D1 cells (2610 5 ) were seeded onto a petri dish in 1 ml of fresh D1 media [26] and incubated at 37uC and 5% CO 2 in the presence or absence of 5 mg FITC-labelled VLPs alone or with VLPs mixed with E 8 Pam 2 Cys (0.2 pmole/ml).
Cells were harvested 24 hours later and washed with FACs wash (1% FCS/5 mM EDTA in PBS) before fixation in 1% paraformaldehyde in PBS. To examine cellular association of VLPs, cell fluorescence was analysed by flow cytometry (FACSCaliber, Becton Dickinson, USA). For examination of intracellular uptake of VLPs, extracellular fluorescence was quenched by addition of an equal volume of 0.1 M citrate buffer (pH 4.0) containing 250 mg/ml trypan blue (Merck, Damstadt, Germany) prior to analysis [27] . Data were analysed using FlowJo software (Tree Star, San Carlos, CA).
To assess the degree of DC maturation resulting from exposure to different adjuvants, cells were exposed to varying concentrations of aluminium hydroxide gel, Alhydrogel (Sigma Aldrich, Missouri, USA), E 8 Pam 2 Cys or lipopolysaccharide (5 mg/ml) as a positive control (LPS; Sigma Aldrich, Milwaukee, USA). In some experiments, cells were incubated with VLPs alone (5 mg/ml) or in the presence of E 8 Pam 2 Cys (0.032 mg/ml). After 16 hours, cells were harvested, washed and analysed for expression of surface Class II MHC antigen.
All experimental procedures involving animals were approved by the University of Melbourne's animal ethics committee under the AEC numbers 0707207 and 061061. HLA-A2k b transgenic mice (HHD mice) were obtained from the Queensland Institute for Medical Research and bred in the Animal House Facility of the Department of Microbiology and Immunology at The University of Melbourne under specific pathogen free conditions. These mice do not express H-2D b but instead express the chimeric monochain of the a1 & a2 domains of HLA-A2.1 and the a3 cytoplasmic and transmembrane domains of H-2D b linked at its N-terminus to the C terminus of human b2 microglobulin [28] .
Female HHD mice (3 per group) were inoculated subcutaneously on each side of the base of tail (50 ml per dose) with VLPs (30 mg) either alone, emulsified in an equal volume of complete Freund's adjuvant (CFA) or with an equal amount of E 8 Pam 2 Cys on days 0 and 14. Spleens were removed 28 days after the second dose and splenocytes restimulated in vitro at a concentration of 3610 6 cells/ml in RF-10 medium consisting of RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (CSL, Parkville, Australia) 7.5 mM HEPES, 2 mM L-glutamine, 76 mM 2-mercaptoethanol, 150 U/ml penicillin, 150 mg/ml streptomycin, 150 mM non-essential amino acids and 10 U/ml of recombinant IL-2 (Roche, Indianapolis, USA) at 37uC in an atmosphere of 5% CO 2 . Restimulation was carried out in the presence of 10 mg of VLPs or 10 mM of an irrelevant HCVderived HLA-A2-restricted epitope (NS5B2594-2602) that is not contained in the VLP construct.
Cells were harvested 5 days later, washed and serial dilutions commencing at 5610 5 cells/ml performed in polyvinylidene fluoride (PVDF) membrane-lined 96-well plates (Millipore, Ireland) previously coated with 5 mg/ml anti-IFN-c capture antibody (clone R4-6A2-BD Pharmingen, San Diego, USA). Cells were then cultured in the presence of 3.75610 5 irradiated autologous VLP-pulsed (10 mg) splenocytes for 40 hours at 37uC and 5% CO 2 . After washing with PBST (PBS containing 0.05% Tween 20), biotinylated anti-IFN-c detection antibody (clone XMG1.2; Becton Dickinson, USA) was added and incubated for 2 hours at room temperature in a humidified atmosphere. Plates were then washed and streptavidin-alkaline phosphatase (Becton Dickinson, USA) added and incubated for a further 2 hours. Spots representative of IFN-c-producing cells were developed by the addition of 100 ml of 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich, USA) for 30 minutes. Individual spots were counted using an AID iSpot EliSpot Reader (GmbH, Strassberg, Germany). Analysis of variance in all experiments and all p values in this study were conducted and obtained using one-way ANOVA nonparametric statistical analysis and Tukey's post-hoc range tests performed with Prism 5 (GraphPad Software, La Jolla, California USA). Flat bottom 96-well polyvinyl plates were coated with either purified HCV VLPs (10 mg/ml) or recombinant E2 protein (5 mg/ ml) in PBSN 3 overnight at 4uC. Prior to coating plates with HCV VLPs, wells were pre-incubated with Galanthus nivalis lectin (2 mg/ml; Sigma Aldrich Australia) in carbonate buffer (15 mM NaCO 3 , 35 mM NaHCO 3 , 0.21 mM NaCl) for 30 minutes at room temperature Following removal of antigen, 100 ml of BSA (10 mg/ml) in PBS was added and plates incubated for 1 hour at room temperature before washing four times with PBST (PBS containing v/v 0.05% Tween-20 [Sigma Aldrich, Milwaukee, USA]). Serial dilutions of sera obtained from immunised mice were added to wells and incubated in a humidified atmosphere overnight. After washing, bound antibody was detected using horseradish peroxidase-conjugated rabbit antimouse IgG antibodies (Dako, Glostrup, Denmark) in conjunction with enzyme substrate (0.2 mM 2,2_-azino-bis 3-ethylbenzthiazoline-sulfonic acid in 50 mM citric acid containing 0.004% hydrogen peroxide). The reaction was stopped by addition of 50 ml of 0.05 M NaF. Titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2.
For the detection of specific antibody secreting cells by ELISPOT, PVDF membrane-lined 96-well plates (Mabtech, Nacka Strand, Sweden) were coated with 100 ml of PBS containing VLPs (10 mg/ml), recombinant E2 (10 mg/ml) or anti IgG antibody (10 mg/ml) overnight at 4uC. Plates were washed 5 times with PBS and blocked for 2 hours using RPMI 1640 medium (Gibco, USA) supplemented with 20% BSA (Sigma, Australia). Wells were emptied before 5610 5 splenocytes in 200 ml of RF-10 medium was added and incubated for 36 hours at 37uC in an atmosphere of 5% CO 2 . Spot forming units representative of specific antibody-producing cells were developed as previously described for the detection of IFN-c-secreting cells except that biotinylated anti-IgG antibody and streptavidin-conjugated horseradish peroxidase (both from Mabtech, Nacka Strand, Sweden) were used as detecting reagents.
In order to determine any inhibition of cell entry by VLPs using antibodies present in sera of vaccinated animals, Huh7 cells were Enhancement of HCV-VLP Immunogenicity PLOS ONE | www.plosone.org first incubated with PBS (10% FCS) for 15 min at 4uC to reduce non-specific binding of antibodies subsequently added. Cells were washed twice, resuspended in PBS (0.1% FCS) and incubated with FITC-labelled VLPs at 4uC for 1 hr. Serial dilutions of sera from vaccinated or non-vaccinated mice were then added and incubated for a further 1 hour at 37uC. For each reaction, 5610 5 Huh7 cells and 200 ng of FITC-labelled VLPs were used in a total volume of 500 ml. At the end of this incubation period, cells were washed with PBS (0.1% FCS) and then fixed in BD Cytofix (Becton Dickinson, USA). Inhibition of VLP entry was determined by flow cytometry and analysed using WEASEL 2.0 software (Walter and Eliza Hall Institute, Melbourne, Australia). Sera from vaccinated mice that demonstrated a decrease in specific cellular binding of 50% or more compared to sera from naïve mice were considered to contain neutralising antibodies [18] .
HCV genotype 1a VLPs were produced by transducing a human hepatocyte-derived cell line with recombinant adenovirus containing encoding the HCV structural proteins (core, E1 and E2) of genotype 1a to produce particles that harbour antigenic resemblance to virions produced in an infected host cell. Because of the essential role that DCs play in the induction of both humoral and cell-mediated responses, we first examined the ability of a spleen-derived DC line (D1 cells) to take up fluorescein isothiocyanate-labelled HCV VLPs (FITC-VLPs). Flow cytometric analysis revealed that DCs incubated with FITC-VLPs exhibited higher whole cell fluorescence intensities compared to untreated DCs indicating the presence of cell-associated VLPs ( Figure 1B) . Exposure of DCs to VLPs pre-mixed with the lipopeptide E 8 Pam 2 Cys also resulted in higher levels of cell fluorescence compared to untreated DCs. The percentage of fluorescenated cells in these cultures was similar to cultures that contained FITC-VLPs alone ( Figure 1C) .
To determine the magnitude of VLP cell uptake, intracellular fluorescence was measured by quenching extracellular fluorescence after exposure of cells to trypan blue prior to flow cytometric analysis [27] . Although the resulting fluorescence intensities of DCs incubated with FITC-VLPs were now lower following this treatment, the levels were still notably higher than untreated cells confirming the presence of intracellular FITC-VLPs ( Figure 1D ). Equivalent fluorescence cell intensities were also observed in DCs that were incubated with FITC-VLPs pre-mixed with E 8 Pam 2 Cys. However, a higher percentage of fluorescenated cells were detected in those cultures compared to those exposed to FITC-VLPs alone ( Figure 1E ) indicating that an increase in uptake of these constructs is facilitated using the lipopeptide.
To investigate the ability of HCV VLPs and E 8 Pam 2 Cys to cause activation of DCs, we measured the expression of surface MHC class II molecules following incubation with the various antigens. The results (Figure 2A) indicate that untreated DCs contained two populations of cells which were MHC class II low and MHC class II high , the latter comprising ,24% of the population analysed. While the distribution of these populations was not affected by exposure to HCV VLPs alone, incubation with HCV VLPs mixed with E 8 Pam 2 Cys caused a dramatic shift in the distribution of MHC class II expressing cells such that 83% of cells were MHC class II high . The upregulation of MHC class II expression on these cells were comparable to those cultured in the presence of LPS which is a potent DC maturation stimulus.
Further dosing experiments showed that increasing the concentrations of HCV VLPs to 20 mg/ml, did not induce DC activation because the percentage of MHC Class II high DCs in cultures containing escalating doses of VLPs remained similar to those containing untreated DCs ( Figure 2B ). In contrast, exposure to as little as 0.1 nmoles of E 8 Pam 2 Cys was sufficient at inducing a greater than two-fold increase in activation of DC compared to untreated cells ( Figure 2C) and was similar to the levels of activation observed with LPS. No DC activation was observed in the presence of Alhydrogel ( Figure 2D ). The presence of E 8 Pam 2 Cys in HCV VLP-containing formulations however, not only promotes uptake of HCV VLPs by DCs but also considerably increases the level of DC activation.
To determine if the DC activating properties of VLP formulations containing E 8 Pam 2 Cys translate to an improvement in HCV VLP immunogenicity, BALB/c mice were inoculated with VLPs alone or with VLPs mixed with E 8 Pam 2 Cys. HCV VLP-specific antibody titres in sera obtained after one, two or three doses of each formulation were then determined by ELISA.
Administration of VLPs alone in saline was able to elicit detectable titres of specific antibody that were marginally increased after each dose of antigen ( Figure 3A ). In animals that received HCV VLPs mixed with E 8 Pam 2 Cys, however, antibody levels were significantly higher, in some cases by up to ten-fold more than those from animals that received the same dose of HCV VLPs alone. In fact the titre of specific antibody induced following administration of 3 doses of HCV VLPs alone was achieved using a single dose only of HCV VLP mixed with E 8 Pam 2 Cys. When compared to animals that were inoculated with HCV VLPs formulated with Alhydrogel, an adjuvant widely used to induce antibody responses to both human and veterinary vaccines [29] , lower antibody titres were observed in these animals than in those that received the VLP-lipopeptide formulation. In examining levels of E2 specific antibodies elicited by vaccination, higher titres were once again demonstrated in animals that received 3 doses of VLPs mixed with E 8 Pam 2 Cys compared to those that were inoculated with VLPs alone or with Alhydrogel ( Figure 3B ).
The hierarchical pattern of antibody responses induced by E 8 Pam 2 Cys and Alhydrogel was also confirmed by the numbers of specific antibody secreting cells that were detected in the spleens of vaccinated mice. Once again significantly higher numbers of cells secreting both HCV VLP ( Figure 4A ) or E2-specific antibodies ( Figure 4B ) were detected in animals that received HCV VLPs mixed with E 8 Pam 2 Cys than those that were inoculated with HCV VLPs alone or VLPs formulated with Alhydrogel.
To assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if VLP entry into human hepatocyte cell line Huh7 could be inhibited. Pre-incubation of FITC-labelled VLPs (VLP-FITC) with PBS or naïve serum resulted in minimal inhibition of VLP entry ( Figure 5A ). However, the presence of an antibody against CD81, a cell surface molecule implicated in HCV entry into hepatocytes [30] , was able to prevent VLP entry into these cells by .90% confirming that these VLPs also utilise this molecule to facilitate cell entry. We next analysed the ability of sera obtained from mice inoculated with VLPs to inhibit the binding and entry of VLPs into Huh 7 cells ( Figure 5B ). Neutralisation of binding of VLPs to Huh7 cells was significantly greater in sera obtained from mice inoculated with VLPs in E 8 Pam 2 Cys (,50%) compared to sera obtained from mice inoculated with VLPs administered in Alhydrogel (,30%) or in saline (,20%).
The ability of VLP formulations containing E 8 Pam 2 Cys to induce a cell-meditated immune response was examined by inoculating transgenic mice expressing the MHC class I (HLA-A2) allele but not endogenous H-2D b molecules [28] . Control transgenic animals were inoculated with VLPs alone or VLPs emulsified with an equal amount of complete Freund's adjuvant (CFA). Splenocytes from vaccinated animals were obtained 28 days post-inoculation and re-stimulated with antigen in vitro. The results ( Figure 6 ) of an ELISPOT assay carried out revealed significantly higher numbers of HCV VLP-specific IFN-c producing cells in the spleens of mice inoculated with VLPs in the presence of E 8 Pam 2 Cys or VLPs emulsified in CFA than in those that received VLPs alone.
The development of novel, effective anti-viral vaccine strategies in recent times has seen a notable shift away from the use of traditional formulations which utilize whole inactivated or live attenuated viruses towards approaches based on recombinant subunit protein antigens which are more easily characterised and defined. VLPs offer features that make them a useful platform for delivering viral antigens in a single vaccine construct which not only minimises the risks that may be associated with preparations containing or requiring the use of a replicating pathogen but will also closely resemble native viral antigens from which they are derived. The most convincing demonstration of VLPs efficacy is the quadrivalent VLP-based vaccine Gardasil which prevents persistent infection and associated disease caused by human papillomavirus [31] . Other studies of VLP-based vaccination strategies have also shown promising results and led to Phase I testing against a number of disease indications including seasonal and pandemic influenza, Hepatitis B, malaria and HIV (reviewed in [32] ).
Depending on the type of virus used to manufacture a VLP construct, studies have shown that protective responses induced by VLPs can be elicited without co-administration of adjuvant [33] [34] [35] . In many cases, however, the induction of useful immune responses may require multiple doses [36, 37] , a regime that may be impractical to implement in the field or involve a viral vector to provide an initial priming dose followed by a boost using VLPs [22, 38] . Of relevance to the present study, the use of adjuvants to enhance VLP immunogenicity has been shown to induce strong antibody responses using dose-sparing amounts of HIV [39] or Norwalk virus-derived VLPs [40] and also elicits cell-mediated were incubated with VLPs (5 mg) alone or formulated with E 8 Pam 2 Cys (0.01 nmoles/ml) or Alhydrogel (5 mg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 mg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PEconjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class II high expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E 8 Pam 2 Cys, (D) Alhydrogel or VLPs (5 mg) formulated with increasing amounts of (E) E 8 Pam 2 Cys, or (F) Alhydrogel. doi:10.1371/journal.pone.0047492.g002 responses that culminate in improved protection against lethal influenza viral [41, 42] and tumorigenic challenge [43] .
Our previous studies have shown peptide epitope and proteinbased antigens can be made far more immunogenic when covalently attached to Pam 2 Cys in order to target their delivery via TLR2 to dendritic cells (DCs) [44] . This results in the induction of robust antibody and CD8 + T cell-mediated immune responses and has been shown for multiple indications [44] [45] [46] [47] .
Each of these vaccine candidates demonstrated the ability of this simple lipid structure to dramatically enhance the immunogenicity of antigens that are otherwise immunologically inert. Nevertheless, the approach introduces complexities into the vaccine manufacturing process due to the requirement for covalent attachment of Pam 2 Cys to an antigen. The use of the anionic lipopeptide E 8 Pam 2 Cys overcomes many of the technical complexities related to this process, especially the use of covalent chemistries, by making use of electrostatic association with antigen [23] .
The ability of Pam2Cys to dramatically enhance the immunogenicity of HCV VLPs was demonstrated in the improved overall antibody responses that we observed. Not only are greater antibody titres induced following vaccination with VLPs formulated with E 8 Pam 2 Cys compared to the use of VLPs alone or when co-administered with Alhydrogel but up to 3 doses of nonadjuvanted or traditionally adjuvanted antigen were required to match the titres obtained with a single dose using lipopeptide. Most importantly in the context of HCV the trend translates to improved E2-specific antibody responses and the use of lower doses of VLPs to achieve this while maintaining efficacy has major advantages by providing cost benefits to vaccine manufacturers.
Our studies examining the interaction of VLPs with DCs indicate that while improvements in VLP uptake mediated by this lipopeptide is minimal, its ability to concomitantly induce the maturation of DCs at very small doses is a feature not observed using VLPs alone or VLPs administered in the presence of alum. Our previous work also demonstrated that association of antigen with charged lipopeptide facilitates trafficking of antigen to lymph Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5610 5 ) in a total volume of (500 ml) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice. doi:10.1371/journal.pone.0047492.g005 Figure 6 . Cell-mediated responses elicited by vaccination. HLA-A2k b transgenic mice (n = 3 per group) were inoculated (100 ml) subcutaneously at the base of the tail on days 0 and 14 with 30 mg of VLPs alone, emulsified with an equal amount of complete freund's adjuvant (CFA) or pre-mixed with 30 mg of E 8 Pam 2 Cys in saline. Splenocytes were obtained 28 days later and restimulated for 7 days in the presence of 10 mg VLPs or an irrelevant HCV-derived HLA-A2restricted epitope not part of the VLP construct. The frequency of peptide-specific T cells producing IFN-c was determined in an ELISPOT assay. Each bar represents the average number of IFN-c producing T cells and standard deviation in each group after subtracting nonspecific responses from corresponding samples stimulated with the irrelevant peptide. doi:10.1371/journal.pone.0047492.g006
Enhancement of HCV-VLP Immunogenicity PLOS ONE | www.plosone.org nodes draining from the vaccination site [23] and together with the results presented in this study provide an explanation for the dose-sparing neutralising antibody responses that we observe. Coadministration of VLPs using charged lipopeptide has the added benefit of eliciting VLP-specific cell-mediated responses in HLA-A2 transgenic mice, a fact that may also be attributed to DC targeting and activation. Given the results of the work described in this study, we conclude that the use of this branched anionic lipopeptide together with VLPs containing HCV antigens in order to provide a broad spectrum of conformational epitopes can provide benefits in terms of inducing improved levels of neutralising antibody titres and eliciting cell-mediated responses. This strategy could therefore constitute a valuable addition to the armamentarium of current VLP-based vaccine developments against HCV.  Viral metagenomic analysis of bushpigs (Potamochoerus larvatus) in Uganda identifies novel variants of Porcine parvovirus 4 and Torque teno sus virus 1 and 2 BACKGROUND: As a result of rapidly growing human populations, intensification of livestock production and increasing exploitation of wildlife habitats for animal agriculture, the interface between wildlife, livestock and humans is expanding, with potential impacts on both domestic animal and human health. Wild animals serve as reservoirs for many viruses, which may occasionally result in novel infections of domestic animals and/or the human population. Given this background, we used metagenomics to investigate the presence of viral pathogens in sera collected from bushpigs (Potamochoerus larvatus), a nocturnal species of wild Suid known to move between national parks and farmland, in Uganda. RESULTS: Application of 454 pyrosequencing demonstrated the presence of Torque teno sus virus (TTSuV), porcine parvovirus 4 (PPV4), porcine endogenous retrovirus (PERV), a GB Hepatitis C–like virus, and a Sclerotinia hypovirulence-associated-like virus in sera from the bushpigs. PCR assays for each specific virus combined with Sanger sequencing revealed two TTSuV-1 variants, one TTSuV-2 variant as well as PPV4 in the serum samples and thereby confirming the findings from the 454 sequencing. CONCLUSIONS: Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs. Conclusions: Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs.
Wild animals are carriers of a number of pathogens that have the potential to infect the human population and/ or domestic animals. The intensity of contact between wildlife, livestock and human population is increasing due to a number of factors, primarily human and livestock population growth leading to encroachment onto wildlife habitat [1, 2] . During recent decades a number of pathogen crossovers from wildlife to humans and livestock have occurred resulting in emerging diseases, such as SARS, Hantavirus Pulmonary syndrome 1, Nipah virus disease 1, and Hendra virus-induced diseases among others. However, it is also evident that transmission can occur both ways i.e. pathogens may spill-over to wildlife from humans and/or from livestock. One example of this is the spill-over of Canine distemper virus from domestic dogs (Canis familiaris) to African wild dogs (Lycaon pictus) in Serengeti in 1991 leading to local extinction of wild dogs in the area [2] . Apart from being carriers of novel viruses with the potential to cause disease in naïve domestic hosts, wildlife may also act as reservoirs for known viral pathogensfor example there are a number of wildlife reservoirs for foot and mouth disease virus such as African buffalo (Syncerus caffer), reindeer (Rangifer tarandus) and wild boar (Sus scrofa) [3] . However, information on the viral flora in wildlife is typically scarce or non-existent.
Traditional viral detection methods, such as virus isolation, are often hindered by the inability to grow virus in cell culture. The divergence of many viruses and absence of a common viral marker gene makes detection difficult using standard molecular techniques including PCR and microarray as they are frequently targetspecific through the use of specific primers, probes and/ or antibodies. Viral metagenomics is a sequence, and culture-independent approach that has proven to be a valuable tool for the investigation not only of diseases of unknown etiology but also of the complete viral flora of different reservoirs and vectors. By providing insights into a wide range of unknown potential pathogens and revealing novel aspects of biodiversity, metagenomics is able to detect and characterise novel pathogens [4] [5] [6] .
In many rural parts of the developing world, domestic livestock are kept in free-range systems, potentially allowing contact with wild animals. In some parts of Uganda, free-range scavenging by pigs is frequent. At the same time wild species of suidae such as bushpigs (Potamochoerus larvatus), with a wide distribution in Eastern and Southern Africa, live and move at the interface between the national parks and the farmland where there is an opportunity for interaction and sharing of pathogens with domestic relatives. Bushpigs are considered to be possible natural reservoirs for African swine fever virus [7] , but less is known about what other viruses bushpigs might carry. Therefore, to investigate whether bushpigs are carriers of known and or unknown porcine viruses we have in this study investigated the viral flora of bushpig sera.
A total of 171,466 reads were obtained from the 454sequencing run, and after assembly 4,441 contigs were created while 32,863 reads remained unmatched (singletons). Although blastn and blastx analysis showed that a majority of the sequences were non-viral, 35 contigs and singletons sequences were identified as viral sequences ( Table 1 ). The high percentage of host genetic sequences found despite of the nuclease treatment prior to sequencing demonstrates the difficulties of completely reducing the background of the host as discussed in the review by Blomström A-L 2011 [4] . Most viral hits showed closest similarity to known pig viruses such as, Torque teno virus (TTSuV) and porcine parvovirus 4 (PPV4). By design of primers based on the sequences obtained from the 454-sequencing run, PCR assays were established to verify the presence of these viruses. These PCR assays confirm the presence of TTSuV1, TTSuV2, PPV4, and the porcine endogenous retrovirus (PERV). The GB Hepatitis C-like, and the Sclerotinia hypovirulence associated like virus, on the other hand, were not detected by the PCR approach, possibly due to the low concentration of these viruses in the samples as only one sequencing read was found for each among the total 171,466 reads obtained from the 454-sequencing run. Also it is possible that in some cases sequencing errors led to primer mismatches.
Parvoviruses are small single-stranded linear DNA viruses with a genome of approximately 5000 nucleotides, which have been found in a number of species such as human, swine, cattle and gorilla [8] . In swine, porcine parvovirus (PPV) is a known agent that causes reproductive failure [9] . However, in recent years a number of new parvovirusesporcine hokovirus (PHoV) [10] , porcine bocavirus (PBoV) [11] and porcine parvovirus 4 (PPV4) [12] -have been discovered in pigs, with their potential involvement in disease currently unknown.
The parvovirus sequences discovered in the investigated bushpigs showed closest similarity to PPV4. Porcine parvovirus 4 was originally discovered in 2010 in samples collected from pigs in North Carolina in 2005 after an outbreak of acute-onset of disease with high mortality [12] . Subsequently, PPV4 was reported in pigs in China where 1.84% of the investigated pigs were PPV4 positive [13] . The genome of parvoviruses consists of two major open reading frames (ORF) encoding the non-structural and the capsid proteins. However, the genomes of PPV4 as well as of PBoV contain an additional third ORF [12] . The parvovirus sequences obtained from this study could be found in the capsid and in the non-structural ORF. All reads classified as parvovirus gave significant similarity to PPV4 through Blastx searches. A PCR with primers designed to amplify PPV4-like sequences from the original extracted genetic material showed the presence of this virus in one of the three bushpig sera. Sequencing of the PCR product (GenBank accession number: JQ277337) confirmed correct amplification and sequence analysis showed that at protein level (84 amino acids), the product displayed a 75.9-77.1% sequence similarity to the PPV4 sequences available in GenBank. In addition, the phylogenetic tree generated from these data ( Figure 1 ) grouped the sequence with PPV4 when analysed together with sequences from all the different parvovirus genera (Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus) and PPV4. However, the tree also confirms the divergence of the bushpig PPV4 from the other PPV4. PPV4 has previously been reported in domestic pigs only in USA and China [12, 13] . However with this study we show the presence of a PPV4 variant in a wild suid in Uganda. Figure 1 Neighbour-joining phylogenetic tree of Parvovirus. The Neighbour-joining tree shows the phylogenetic relationship between the bushpig PPV4 sequence (84 amino acid long) and 49 sequences available from GenBank. The bushpig PPV4 is indicated with ♦.
Torque teno virus was discovered 1997 in a serum sample from a patient with posttransfusion hepatitis of unknown etiology using representational difference analysis [14] . Since then the virus has been detected and characterised in a number of species such as primates, cats, dogs and pigs [15] , but the role of these viruses in disease development is still controversial. These viruses have small (approximately 2.8 kb) circular DNA genomes. Torque teno virus in pigs is divided into two different species, Torque teno sus virus 1 and 2, and prevalence studies have shown that TTSuV is widely spread in pig populations across the world [16, 17] . In a previous study [18] , we have found that 51.6% of a sample population of domestic pigs in Uganda were carrying one or both these TTSuV variants. Although, most studies have targeted domestic pigs, TTSuV have also been found in wild boar in Europe [19] .
As shown in Table 1 , our data indicated the presence of both TTSuV-1 and 2 in the investigated serum samples. Both the TTSuV-1 and 2 sequence reads were located in the major open reading frame (ORF1) and all reads showed significant similarity to the respective virus in both blastn and blastx analyses.
Two of the TTSuV-1 sequence reads partially overlapped and the sequence analysis indicated a significant variation between the two and therefore two different TTSuV-1 PCR assays were designed. The results from the specific PCR assays showed that one of the TTSuV-1 variants (here named TTSuV-1a) could be found in two of the three bushpig sera while the other one (here named TTSuV-1b) was found in all three. The sequence analysis of the PCR amplified TTSuV-1 products (Gen-Bank accession number JQ277338 -42) showed a sequence similarity between TTSuV-1a and b on protein level at 53-54.5% in the analysed 66 amino acid region. Compared to sequences from other studies, TTSuV-1a showed a 60.6-84.8% protein sequence similarity while TTSuV-1b was more divergent (50-56% similarity). These protein sequence identity values were similar to those seen when comparing the sequences retrieved from the GenBank with each other (59-100% similarity). The phylogenetic tree (Figure 2 ) also indicated that TTSuV-1b was more divergent than the other sequences. Sequences from different parts of the world such as China, Germany, Spain, US etc. was used in the phylogenetic study however no clear geographical clustering was seen.
TTSuV-2 was confirmed in one of the bushpig sera and the amplified product GenBank accession number JQ277343) showed a protein sequence identity of 66.6-79.4% to the other TTSuV-2 sequences used in the phylogenetic study using a 313 amino acid region. This sequence identity was in the range of the similarity seen between the different TTSuV-2 sequences from other studies used for comparison (64.2-100%). In the phylogenetic study ( Figure 3 ) the sequenced TTSuV-2 grouped with the other TTSuVs but in its own clade.
TTSuV-1 and 2 have previously, as mentioned, been detected both in domestic pigs across the world [16] [17] [18] and wild suidae in Europe [19] and now for the first time in bushpigs on the African continent. Studies on the genetic variability of TTSuV-1 and 2 have shown a higher genetic diversity in the coding regions compared to the untranslated region [20] . The sequence analysis of both the bushpig-derived TTSuV and those from GenBank shows a high genetic variability among the different TTSuV-1 and TTSuV-2 and also shows the co-infection of two different TTSuV-1 variants and one TTSuV-2.
Endogenous retroviruses are integrated in the host genome and all vertebrates investigated have been shown to carry retroviral sequences. It is estimated that up to 10% of animal genomes are retroviral elements [21] . Also the bushpig genome has been investigated and confirmed to contain PERV [22] [23] [24] . By running the specific PERV PCR on both DNA and on DNase treated RNA we could as expected see that all the bushpigs had both integrated proviral DNA and expressed PERV RNA, indicating active viral transcription and replication. The sequenced PERV products (GenBank accession number JX566717-719) showed a high similarity (85 -89%) to those available in GenBank.
Through investigating sera collected from bushpigs in Uganda by viral metagenomics, we have for the first time showed the presence of PPV4 in a wild Suid on the African continent. The region of PPV4 investigated indicates a sequence divergence relative to previously described PPV4. In addition, novel TTSuV-1 and 2 variants were found. Further sequence analysis and prevalence studies can be used to define the genetic relationships of these viruses and their distribution in both domestic pigs and in wildlife.
The sera from three bushpigs collected from Lake Mburo National Park, Uganda in March 2010, as part of a research project on African swine fever epidemiology were used in this study. The animals were captured using game capture nets (50x3m, 150 mm square mesh, 3.5 mm nylon braid khaki, ALNET Ltd, South Africa) with assistance from Uganda Wildlife Authority (UWA) staff, and sedated with zolazepam and tiletamine (Zoletil forte vet 50 mg/ml + 50 mg/ml, Virbac Laboratories, France) before blood sampling from the saphenous vein. The Ministry of Agriculture Animal Industry and Fisheries and Uganda Wildlife Authority, together with Makerere University are mandated to carry out animal disease investigations in livestock and wildlife in the country. This is done by veterinarians who handle the animals under internationally recognized guidelines.
Fifty microliters of serum was aliquoted for the RNA and DNA extraction respectively. One hundred and fifty microliter of 1x DNase buffer (Roche, Mannheim, Germany) was added to each aliquot of serum after which the sample was treated with nucleases -100 U DNase (Roche, Mannheim, Germany) and 2 μg RNase (Invitrogen, Carlsbad, CA, USA) for two hours at 37°C in order to degrade the host nucleic acid. Trizol was added to one of the two aliquots and RNA was extracted using a combination of Trizol and Qiagen RNAeasy kit. DNA was extracted using the Qiagen DNAeasy mini extraction kit according to the manufacturer's instructions and eluted in 50 μl elution buffer (EB). The DNA and RNA were amplified by random PCR as described earlier [25] . Before sequencing, the primers were cleaved using EcoRV (NEB, Ipswich, MA, USA) and the cleaved product was purified using the Qiagen PCR purification kit (Qiagen, Hilden, Germany) and eluted in 30 μl EB. 
The purified product was sequenced on 1/8 th of a pico titre plate using the 454 technology by Roche at Inqaba Biotech (South Africa). The sequences were analysed through quality check and removal of very short sequences before being assembled using CLC genomic workbench v4.6 (http://www.clcbio.com/index.php). Blastn and blastx searches were performed through the Camera 2.0 portal [26, 27] and searches through NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The viral blast hits with an e-value of 10 -4 or lower were further analysed.
Confirmation PCRs, sequencing and phylogenetic studies PCR primers were designed based on the reads from the 454-sequencing run ( Table 2 ) and PCR assays were set up with the aim to look for each individual virus in each bushpig sera. For the RNA viruses cDNA synthesis was performed prior to PCR using Superscript III (Invitrogen, Carlsbad, CA, USA) and random primers according to the manufacturer's instructions. The PERV RNA was treated with DNase prior to the cDNA synthesis and both a "+" and a "-" RT cDNA synthesis reaction was performed. The PCR using each specific primer pair (Table 1) was performed according to the following procedure: 1x PCR buffer, 2.5 mM MgCl 2 , 1.0 mM dNTP, 0.4 μM forward primer and reverse primer each, and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). For each reaction, two μl DNA or cDNA from each respective bushpig was used. The amplification was performed with the following reaction conditions: a 12 minute enzyme activation step at 95°C followed by 39 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 90 seconds, finishing with one cycle for 10 minutes at 72°C. The PCR products were visualized on a 1.5% agarose gel. The PCR-positive products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and eluted in 25 μl EB. The purified products were sequenced with standard Sanger sequencing using Big Dye Termination kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The obtained chromatograms were edited using SeqMan (Lasergene 9, DNASTAR Inc., Madison, USA). Sequence identity plots were performed using the Bioedit software [28] . ClustalW as well as the phylogenetic analyses were carried out using Mega 5 [29] . The phylogenetic trees were constructed using the Neighbour-joining algorithm with p-distances and with a bootstrap value of 1000.  A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection. Vector-borne flaviviruses, such as Dengue (DENV), Yellow Fever, Japanese Encephalitis virus (JEV), and tick-borne encephalitis (TBEV) viruses are recognized as significant human pathogens and cause considerable mortality and morbidity worldwide. TBEV, a tick-borne flavivirus (TBFV), is responsible for 14,000 infections per year [1] and has a fatality rate of up to 40% [2] . Symptoms of TBEV infection can include fever, malaise, meningitis, and encephalitis. TBEV and other TBFV, such as Omsk Hemorrhagic Fever virus, are classified as NIAID Category C pathogens and are treated as biosafety level 4 agents in the United States. One TBFV, Langat virus (LGTV), is naturally attenuated [3, 4] , making it suitable for biosafety level 2 work and ideal for use in laboratory studies as a model for higher pathogenicity viruses.
In nature, LGTV and other TBFV maintain a complex cycle between ticks and vertebrate hosts. Historically, Ixodidae ticks (hard ticks) have been considered to be the arthropod vector, but, some findings with Alkhurma virus [5] and Kyasanur Forest virus [6] suggest that the soft-bodied Ornithodoros ticks can transmit TBFV. Thus, the arthropod host-range for TBFV may be greater than assumed. The TBFV present a unique situation because the viruses persistently infect ticks and are maintained by vertical transmission across the developmental instars (larval, nymph, and adult). Horizontal transmission (from tick to vertebrate host) then allows amplification of the frequency of the virus within the tick population, as uninfected ticks feeding upon infected vertebrates can acquire the virus [7, 8] . The primary vertebrate hosts are generally small rodents; however, infection of larger mammals also occurs in endemic areas. Humans are an inadvertent, dead-end host, contracting TBFV via tick bite or less frequently by consumption of milk from infected animals [1] . The impact of TBFV infection on vertebrates can vary considerably; some reports describe persistent infection of vertebrates and vertebrate cell lines [9] [10] [11] while other laboratory studies show severe disease development in infected animals [12] [13] [14] .
Like other members of the family Flaviviridae, LGTV is a singlestranded, positive-sense RNA virus. Upon infecting a cell, the virions are thought to traffic to the endosome, where they undergo structural transformation and fuse with host cell membranes, releasing the 11-kb viral RNA genome into the cytoplasm [15] . The genomic RNA, which can function as mRNA, is translated into an approximately 400 kDa polyprotein [16] that is subsequently cleaved into at least ten proteins by both viral and cellular host proteases [17] . The currently defined complement consists of three structural proteins (Capsid [C] , membrane [M] , and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [17, 18] . Precise functions for all of the nonstructural proteins are not fully elucidated, but some roles have been determined. NS1 can be found on the cell surface but is known to be secreted [17] and is also detected in perinuclear areas where it is believed to play a part in viral replication [19] ; however, its role has not yet been clearly defined. NS3 is a RNA helicase and also acts as a viral protease using NS2B as a co-factor [20, 21] . NS4A, a small hydrophobic protein, has been implicated in the cellular changes that accompany virus reproduction [22] and has been shown to localize to foci within the perinuclear region of infected cells [23] . NS5 is the RNA-dependent RNA polymerase and methyltransferase [17] ; however, recent implications of LGTV NS5 as an innate immune response antagonist confirms a broader role for this protein beyond genome replication [24] . This broader role is also seen in NS59s wider distribution, being located in cell nuclei, perinuclear regions, and diffusely throughout the cytoplasm [25] . Following synthesis of the viral proteins, the single stranded genome is converted into a doublestranded RNA (dsRNA), which serves as a transcriptional template as well as the viral replicative form. Together with the replication complex, made up of NS1, NS2A, NS3, NS4A, and NS5, dsRNA is found in cytoplasmic, perinuclear foci throughout LGTV infected cells. This dsRNA species is an additional obligatory replicative intermediate.
Dramatic cellular changes accompany flavivirus replication and have been the subject of considerable study, although most of the studies have been done in mammalian cells using the mosquitoborne flaviviruses. Replication has been linked to areas of the cell containing large amounts of virus-induced structures, including convoluted membranes, dilated cisternae, vesicles, tubules, and paracrystalline arrays [19, [26] [27] [28] . These structures are localized in a perinuclear distribution of vastly proliferated membranes which, in the case of cells infected with mosquito-borne flaviviruses, are derived from endoplasmic reticulum (ER) [29] [30] [31] . The vesicles are typically 70-150 nm in diameter [28] with a pore-like connection either between vesicles and the cytoplasm or between individual vesicles [30, 31] .
Vector-borne flaviviruses, like DENV, JEV, and TBEV, must replicate in both mammalian and arthropod cells, and a limited number of comparative studies have been published [32] [33] [34] [35] [36] . The findings, generally, show agreement between mammalian and arthropod cells in virus induced structures, such as cytoplasmic membrane proliferation and vesicle formation. Some changes were noted via TEM by Senigl et al. [35] four to seven days post TBEV infection; viral particles were observed inside rough ER in mammalian cells but were visualized within vacuoles or within the cytosol in tick cells. Consequently, we have undertaken a direct comparison of LGTV infection in both mammalian and arthropod cells, and extended the results to include 3D reconstructions. In addition, we have shown for the first time the 3D structure of acute and persistent flavivirus infection in an arthropod cell line.
African green monkey kidney cells (Vero, ATCC) were maintained in Dulbecco's minimal essential media (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS) and 50 ug/ml gentamicin (complete DMEM) at 37uC in 5% CO 2 . ISE6 cells derived from I. scapularis embryonated eggs (kindly provided by Dr. Timothy Kurtti, University of Minnesota) were cultured at 32uC as previously described [37] with the addition of 10 ug/ml gentamicin to the L-15B media.
A virus stock of Langat virus strain TP21 (LGTV) (kindly provided by Dr. Alexander Pletnev, NIH/NIAID) was prepared by infection of Vero cell cultures at a multiplicity of infection (MOI) of 0.005 [38, 39] . For virus titration by immunofocus assay, 1610 5 Vero cells were seeded in complete DMEM into wells of twelve-well plates. Ten-fold dilutions of LGTV were adsorbed for 60 minutes at 37uC, 5% CO 2 with constant rocking. Infected cells were then washed twice with Dulbecco's phosphate buffered saline (DPBS, Invitrogen) and fresh complete DMEM added. After three days incubation, cells were washed twice with DPBS and fixed with 100% methanol for 30 minutes. Cells were rinsed with DPBS, blocked with OptiMEM supplemented with 2% FCS for 10 minutes, and incubated with Russian Spring-Summer Encephalitis immune ascites fluid (ATCC) diluted 1:1000 in OptiMEM/2% FCS. Following two DPBS washes, cells were incubated with 1:1000 dilution of anti-mouse horseradish peroxidase antibody (Dako) in OptiMEM/2% FCS. Foci were visualized using peroxidase substrate containing 0.4 mg/ml 3, 39 diaminobenzidine (Sigma Aldrich) and 0.0135% H 2 O 2 (JT Baker) in PBS. For all experiments, Vero and ISE6 cells were infected at an MOI of 10.
To establish a persistently infected culture, 1610 7 ISE6 cells were plated in a 150 cm 2 flask and incubated for 24 hours at 32uC. Cells were then infected at an MOI of 10 with LGTV TP21. Cells were passaged every two to three weeks as needed and supernatants collected for viral titer via immunofocus assay to confirm productive viral infection. Samples for immunofluorescence and electron microscopy were prepared at multiple time points post-infection. No differences were noted between samples collected at various time points. Representative images for figures depicted were acquired between 90 and 260 days post infection.
The primary antibodies against viral proteins used were: mouse monoclonal anti-prM 13A10, IgG2a; mouse monoclonal anti-E 11H12, IgG2a; mouse monoclonal anti-NS1 6E11, IgG2a (all kind gifts from Dr. Connie Schmaljohn, USAMRID, Fort Detrick, Frederick, MD), chicken poly-clonal anti-NS3 (sequence: CZRDIREFVSYASGRR) and chicken polyclonal anti-NS5 (sequence: CZDRHDLHWELKLESSIF) (custom prepared by Aves Labs), mouse anti-dsRNA clone J2 (purchased from English & Scientific Consulting, Szirak, Hungary). Markers against cellular organelles used were: Alexa Fluor 594-conjugated wheat germ agglutinin (WGA, Invitrogen), and mouse monoclonal Dylight 488-conjugated protein disulfide isomerase 1D3 (PDI, Enzo Life Sciences). Secondary antibodies used were: Alexa Fluor 488-and 594-conjugated anti-mouse-and anti-rabbitspecific IgG and Alexa Fluor 647-conjugated anti-chickenspecific IgG (Invitrogen).
Vero and ISE6 cells were seeded at 3610 4 cells/well in 8 well Labtek dishes (Nunc), infected with LGTV (MOI 10), and incubated for 48 hours at 37uC and 32uC, respectively. The cells were washed twice with PBS, fixed for 10 minutes in 4% paraformaldehyde (PFA)/5% sucrose in PBS, permeabilized with 0.1% Triton X-100/4% PFA for 10 minutes, washed with 50 mM glycine for 5 minutes, and blocked for 60 minutes with 2% bovine serum albumin (BSA)/PBS. Primary and secondary antibodies were incubated at a 1:1000 dilution in 2% BSA/PBS for 60 minutes each, with three five-minute washes with PBS between incubation steps. After washes, slides were dried and coverslips mounted using Prolong Gold Antifade with DAPI (49, 6-diamidino-2-phenylindole, Invitrogen). Confocal images were acquired using a Carl Zeiss LSM 710 confocal laser scanning microscope. TIFF files of individual channels and merge images were made using Bitplane Imaris 7.2 and images assembled for publication using Adobe Photoshop software.
For transmission electron microscopy (TEM), Vero or ISE6 cells were seeded on 13-mm Thermanox coverslips (Nunc) in 12 well plates at 1610 5 cells/well. After 24 hours, cells were infected with LGTV TP21 at an MOI of 10 and incubated for 48 hours at 37uC and 32uC, respectively. Cells were then washed twice with DPBS and fixed with 2.5% glutaraldehyde in cacodylate buffer (100 mM sodium cacodylate, 50 mM KCl, 2.5 mM CaCl 2 ) for 30 minutes at room temperature prior to overnight incubation at 4uC.
Samples were processed further in a Biowave model laboratory microwave oven, equipped with a Coldspot water circulator (Ted Pella, Inc.) as follows: washed twice in 0.1 M sodium phosphate buffer, pH 7.2, for 1 minute each; post-fixed in 1% osmium tetroxide in phosphate buffer for two cycles of 2 minutes on, 2 minutes off, and 2 minutes on; washed once in phosphate buffer for 1 minute and twice in water for 1 minute each; contrasted with 1% uranyl acetate in water for two cycles of 2 minutes on, 2 minutes off, and 2 minutes on; dehydrated in three changes of ethanol for 1 minutes each; and embedded in Spurr's resin using steps of 50%, 75%, and two changes of 100% resin in ethanol for two periods of 5 minutes each. The power output of the oven was set at 250 watts for dehydration and embedding. All other steps were performed at a setting of 167 watts. The cover slips were placed cell-side down onto resin block molds, polymerized overnight at 65uC, and removed from hardened blocks after a 5 second immersion in liquid nitrogen.
Thin and semi-thick sections of approximately 70 and 250 nm, respectively, were cut using a diamond knife and a model EM UC6 microtome (Leica Microsystems). Thin sections were collected on uncoated 200 mesh copper grids. Once thoroughly dried, the sections were stained with fresh 1% lead citrate using 1 minute of microwave irradiation at 167 watts, followed by a 1 minute water wash at 167 watts and standard drop-wise washing.
For electron tomography (ET), semi-thick sections were collected on carbon coated 200 mesh copper grids (Ted Pella, Inc.), dried, and stained and washed similarly, except that the microwave irradiation steps were extended to 2 minutes each. Tomography grids were then immersed briefly in goat anti-mouse IgG 10 nm gold conjugate (Ted Pella, Inc.) to add fiducial markers on both sides of the sections and then thoroughly washed with water.
Thin sections were examined at 80 kV using a model H7500 transmission electron microscope (Hitachi High Technologies, Inc.). Digital images were captured using a model HR-100 camera system (Advanced Microscopy Techniques, Inc.) and processed with PhotoShop (Adobe Systems, Inc.). Semi-thick sections were examined at 120 kV using a G2 Spirit BioTwin model TEM (FEI, Inc.) equipped with a tilt stage, Xplore3D acquisition system, and model Ultrascan 1000 CCD system (Gatan, Inc.). Tilt series images were collected at 0 and +/268 degrees with 1 degree intervals. Images were aligned and reconstructed into tomogram volumes using Inspect3D software (FEI, Inc.) and rendered using Amira (Visage Imaging, Inc.).
Vector-borne flaviviruses replicate in both mammalian and arthropod cells. However, the three-dimensional (3D) fine structure of TBFV replication in mammalian cells has not yet been compared to that in acutely or persistently infected tick cells. In order to undertake this comparison, baseline characterizations of acutely infected mammalian (African green monkey kidney, Vero) and tick (Ixodes scapularis embryo derived, ISE6) cells were performed by light microscopy and virus titer was assayed from culture supernatants. Uninfected Vero cell cultures formed a monolayer of uniform elongated, fibroblast-like cells that were firmly adherent to the culture vessel. Infected Vero cells first showed cytopathic effect (CPE) by 24 hpi, consisting of rounded cells and syncytia (Fig. 1A) . The CPE was progressive and by 96 hpi, Vero cells showed extensive evidence of cell death (shape irregularity, cytoplasmic condensation, blebbing, and an inability to exclude trypan blue). After 96 hours, very few cells remained attached to the culture vessel.
ISE6 cell cultures, on the other hand, are a mixed population of cells consisting of clumps of loosely adherent, round cells as well as more firmly adherent, stellate cells with branching pseudopodia. In marked contrast to infected Vero cell cultures, there was no obvious evidence of CPE following acute infection of the ISE6 cells (Fig. 1A) .
LGTV replicated well in both cell lines, but the titer was 2-3 logs lower in acutely infected ISE6 cells than in Vero cells (Fig. 1B) . Thus, there was a prominent difference in the cellular response to acute LGTV infection between mammalian and tick cells. For subsequent experiments, we selected a time point of 48 hpi as a compromise between virus production and integrity of cellular morphology.
In nature, TBFV establish and maintain a persistent infection across the various life-stages of the arthropod host [8] . In an effort to model and study this aspect of TBFV infection in vitro, ISE6 cells were infected with LGTV, serially passaged at confluence, and examined at intervals for the induction and maintenance of a persistent infection. Interestingly, cultures of infected ISE6 cells appeared normal for a period of at least one year following initial infection.
LGTV infection was maintained in the ISE6 cells, and the viral titer oscillated from a low of 2610 3 ffu/ml to a high of 2610 4 ffu/ml (Fig. 1C) . After roughly one year, approximately 50% of the cells were positive for viral proteins, and the culture supernatant at this point had a viral titer of 2610 4 ffu/ml (Fig. 1C) . Thus, a persistent infection had been established in the ISE6 cells and that infection was still productive a year later.
The findings described in the previous section provided us with a model system in which to examine the distribution of several structural and non-structural proteins in infected mammalian and tick cells (Fig. 2) . The analysis is complicated by the fact that the basic cellular morphology differs substantially between the mammalian and tick cell lines. Nevertheless, in acute infections in Vero and ISE6 cells, as well as in the persistently infected ISE6 cells, M, E, and NS3 proteins were localized to the perinuclear region, although some staining was seen throughout the cytoplasm (Fig. 2B) . NS1 exhibited a more punctate cytoplasmic staining pattern and NS5 showed a diffuse cytoplasmic staining pattern with occasional evidence of nuclear staining. Similar patterns were seen for all the viral proteins in both cells lines.
To more precisely evaluate the relative distribution of the proteins during infection, we co-stained specimens for multiple viral proteins. In each case, the bulk of NS3 staining was colocalized with both the viral membrane (prM) (data not shown) and envelope (E) proteins (Fig. 3A) ; although the overlap appeared less complete in the ISE6 cells. NS5 was diffusely distributed in the cytoplasm, but there was clear evidence that the staining for E overlapped (Fig. 3B) . The punctate staining pattern for NS1 also exhibited coincidence with prM, E, and NS3 but with fewer areas of colocalization with NS5 (data not shown) in both Vero and ISE6 cells. There were no appreciable differences between the acute and persistently infected ISE6 cells (Fig. 3) .
Flavivirus replication and assembly takes place on membranes derived from ER and that maturation of virus particles occurs within the trans-Golgi [40] [41] [42] [43] . Thus, we next looked at LGTV protein localization in relationship to markers for ER (Dylight 488conjugated protein disulfide isomerase, PDI) or Golgi membranes (Alexa Fluor 594-conjugated wheat germ agglutinin, WGA). In both infected mammalian and tick cells (Fig. 4C) , we observed a notable increase in the amount of ER staining in comparison to mock infected cells (Fig. 4A & B) , while the amount of Golgi staining remained relatively constant (date not shown). In infected Vero cells, the bulk of structural protein E and the nonstructural protein NS3 colocalized in large concentrated areas of ER (Fig. 4C ) but a small amount of signal from E overlapped with the Golgi marker (WGA) (data not shown). Similar results were observed for both the acutely and persistently infected ISE6 cell cultures (Fig. 4C) . Thus, LGTV infection of mammalian and tick cells was accompanied by an increase in ER related structures, and furthermore, both structural and nonstructural LGTV proteins are concentrated in those areas.
An obligate marker for flavivirus replication is the presence of the replicative form, double-stranded RNA (dsRNA). Therefore, to further demonstrate that LGTV replication was occurring in an ER derived compartment [44] , we co-stained infected Vero and ISE6 cells for dsRNA and either viral proteins or cellular markers. Uninfected cells showed no dsRNA staining (Fig. 5A) . In infected Vero cells, labeling for dsRNA was largely confined to areas staining for NS3 (Fig. 5B) and NS5 (Fig. 5C) . The same general pattern was seen in acutely ( Fig. 5B and 5C ) and persistently (data not shown) infected ISE6 cells, but the dsRNA signal was not as prominent as in the Vero cells. The majority of dsRNA labeling was coincident with ER staining (Fig. 6 ), but not with Golgi staining (data not shown) in acutely infected Vero or in ISE6 cells, both acutely and persistently infected. Taken together, these findings indicated that in both mammalian and 
In the previous sections, we showed that LGTV replication was occurring in part of the cytoplasm associated with expanded ER and that there was no obvious difference between mammalian and tick cells. However, immunofluorescence does not provide sufficient resolution to clearly define the cellular compartments associated with replication. Therefore, to compare TBFV infection in mammalian and tick cells at higher resolution, we used TEM of fixed, resin-embedded specimens to examine mock-infected Vero and ISE6 cells (Fig. 7A) , LGTV infected Vero, and either acutely or persistently infected ISE6 cells. In Vero cells, extensive areas of altered membrane proliferation (Fig. 7B-D) could be found in the majority of cells, consistent with the expansion of the ER system evident by fluorescence microscopy [27, 29, 30] . In some sections, these regions of proliferation encompassed nearly half of the cytoplasmic area, thus explaining our immunofluorescence results. Single-membrane bound vesicles, ranging in diameter from 60-100 nm, were frequently found within these proliferated ER areas, often occurring in large groups contained within dilated ER cisternae. Single, small groups, and large paracrystalline arrays of virions were readily observed (Fig. 7A) . Infrequently, tubular structures or elongated vesicles were seen (Fig. 7D) . Tubules have been seen in other reports on ultrastructure of mosquito-borne flavivirus mammalian cell infection [30, 45] and mosquito cell infection [26, 46, 47] ; however, no function has been assigned to these tubules.
In acutely infected ISE6 cells, membrane proliferation and vesicles were also observed ( Fig. 7B & C) , but the extent was notably less widespread than in mammalian cells, consistent with the findings of previously published work [35] . The vesicles were also found in dilated ER cisternae and were the same diameter as those seen in Vero cells (Fig. 7C) . Virions were not detected until 96 hpi (data not shown), likely a consequence of the lower viral titer and slower replication observed in the tick cells (Fig. 1B) . The membranous tubules or vesicles were present at a slightly higher frequency than in acutely infected Vero cells.
In persistently infected ISE6 cells, membrane proliferation was more extensive than in acutely infected tick cells; but, again, this was not as pronounced as in infected Vero cells (Fig. 7B) . Vesicles were again seen within proliferated, dilated ER and remained within the same size range as in acute infection. Virions were not noted in the samples we examined. One difference in the persistently infected ISE6 cells was particularly striking. Tubules were seen in almost all persistently infected ISE6 cells and often occurred in fascicle-like bundles of multiple tubules cloaked by a single membranous sheath (Fig. 7D ).
The recent application of electron tomography (ET) to the study of virus infection has allowed 3D evaluation of virus replication [48] [49] [50] [51] [52] [53] including in mosquito-borne flavivirus infection [30, 31] . We employed ET of 250 nm thick, fixed, and resin-embedded specimens to enhance the findings described in the earlier sections. Tilt series were acquired, aligned using gold particles, and tomograms assembled to reveal 3D structures. In Vero cells acutely infected with LGTV, we found the vesicles and virions were contained within an anastomosing network of dilated membranes. The 60-100 nm diameter vesicles were round in shape ( Fig. 8A and Movie S1) and there were pore-like openings connecting vesicles to the cytoplasm (Fig. 9A ) and to other vesicles (Fig. 9D) .
In tick cells, acute LGTV infection induced 3D rearrangements similar to those seen in Vero cells (Fig. 8B and Movie S2). The membrane proliferation, while not as marked as in Vero cells, still resulted in a continuous network that surrounded the vesicles. The majority of vesicles observed in the tick cells were round (Fig. 7C &  D) ; however, slightly elongated vesicles were seen more frequently than in Vero cells. Communicating pores or openings between the vesicles and the cytoplasm (Fig. 9B ) and between individual vesicles (Fig. 9E) were also seen in the tick cells.
The persistently infected tick cells also contained round vesicles enclosed within dilated membrane structures (Fig. 8C and Movie S3). The 3D analysis confirmed that the elongated profiles were in fact tubular, and that they were clustered in fascicle-like groups surrounded by a single membrane. These structures were cylindrical in shape with closed ends, varying in width from 60 to 100 nm and in length from 100 to 800 nm. Pores were seen between tubules and vesicles ( Fig. 9C & F) ; however, in our examinations of numerous tubular structures, we were unable to identify pores linking tubules with other tubules or with the cytoplasm.
structure. Similar to other positive strand viruses, such as Semliki Forest virus [54] , rubella virus [52] , and poliovirus [51] , the flaviviruses induce remarkable alterations in the cytoplasmic membrane system. In our study, Vero cells acutely infected with LGTV show the ER proliferation, vesicle accumulation, and membrane network (Fig. 7B-D) described in earlier studies with mosquito-borne flaviviruses [30, 31] . A number of functions have been ascribed to the alterations, including concentration of viral replication machinery, provision of a solid state platform for viral protein synthesis and replication, and sequestration of viral dsRNA replicative form from innate immune sensors [27, 55, 56] . In general, viral protein distribution in acutely infected tick cells was similar; however, some differences were apparent. Vero cells had higher levels of ER proliferation and rearrangement as well as significantly more viral particles. This may have been the result of the greater level of replication seen in Vero cells but, it is also possible that the differences are the result of a fundamental difference between the two host cell lines.
In our study, an absence of observed viral particles in LGTVinfected ISE6 cells did not allow for confirmation of previously reported differences seen with TBEV [35] between virus particle location in mammalian versus arthropod cells. However, we have augmented this earlier published work by reporting the 3D structure of acutely infected tick cells. By ET, the proliferating membranes are revealed to be a complex anastomosing system of membranes almost certainly derived from ER. In the Semliki Forest virus replicon system containing Kunjin virus proteins, expression of a single flavivirus protein, NS4A, can induce the membrane rearrangements [22] , and similar results have been reported for Dengue virus NS4A [57] , but it is uncertain at this time if this role can be assigned to NS4A in the TBFV. Future investigation is in progress to determine which TBFV protein or combination of proteins cause membrane rearrangement in mammalian and tick cells.
The circular profiles seen in TEM were clearly demonstrated by ET to be spherical vesicles bound by a single membrane and to have pore-like connections to the cytoplasm or to other vesicles in Vero and ISE6 cells. The function of the vesicles is thought to be to minimize exposure of the replicative form dsRNA to innate immune sensors, such as RIG I [27, 58, 59] , while the pores are thought to provide a conduit for nucleotides, amino acids, and other components required for replication and gene expression [30] . In brief, our findings suggest marked similarities between acute replication in ISE6 and Vero cells, and further demonstrate the similarity between cellular features of acute tick-borne and mosquito-borne [30, 31] flavivirus replication.
Our work also allowed a detailed examination of cellular features of persistent LGTV infection in ISE6 cells and an opportunity to compare acute and persistent infection in tick cells. Over a yearlong period, the persistently infected ISE6 cells maintained a grossly normal morphology. Furthermore, the distribution of viral proteins and their co-localization with cellular markers mirrored the acute infection.
However, study by EM and 3D reconstruction revealed differences between the persistently infected and the acutely infected ISE6 cells. The persistently infected tick cells had greater changes in ER structure and ER abundance (Fig. 7B-D) than the acutely infected cells, although viral titers (Fig. 1B & C) were similar in both settings. However, the most remarkable difference between acute and persistent infection was the number of tubular structures. These structures have been noted infrequently in infected cells [26, 30, 34] , and their relevance in flavivirus replication is uncertain. In our acute mammalian and tick cell experiments, we confirmed these observations and saw tubules only occasionally (Fig. 7D, 8 , and Movies S1, S2, and S3). However, in the persistently infected tick cells, the number of tubules increased dramatically (Fig. 7D, 8C , & Movie S3). While the diameter was similar to that seen in round vesicles (60-100 nm), the length was up to eight times as long. The tubules were often arranged in membrane-bound, fascicle-like bundles ( Fig. 8C and Movie S3). The 3D reconstructions demonstrated that the tubules were closed on each end and, although closely juxtaposed, were not connected by pores to other tubules or to the cytoplasm, unlike the round vesicles. The function of the tubules is obscure at this time, and it is unclear if they represent bona fide features of replication, aberrant structures, or the result of a cellular process to restrict infection. It is possible the tubules may play a role in initiation or maintenance of the persistent infections or if the apparent lack of pores in the tubules is consequential. It Figure 6 . Localization of LGTV TP21 replication complex to endoplasmic reticulum. Cells were infected at a MOI of 10, fixed, and costained for double-stranded RNA (dsRNA, red) and endoplasmic reticulum (protein disulfide isomerase (PDI), green). Nuclei counterstaining (DAPI, blue) is only shown in the merge panel. Scale bars, 10 um. doi:10.1371/journal.pone.0047912.g006 3D LGTV Infection in Mammalian and Tick Cells PLOS ONE | www.plosone.org may be that the increase in the number of tubules is the result of the higher number of defective virus particles, which are known to exist in persistently infected cells [60] [61] [62] . Tubules may be formed as a result of a failure to correctly gather the membrane to form the round vesicles and pores that are associated with the flavivirus replication complex. Perhaps the lack of pores prevents proper replication or packaging of the viral genome. Additional cell biology or biochemistry studies may shed light on the role of the tubules.
In summary, our experiments have provided the first analysis of the 3D structure of tick-borne flavivirus infection in both mammalian and arthropod host cell systems. We observed vesicles with pores connecting to other vesicles or opening to the cytosol in tick-borne flavivirus infection, similar to those seen in mosquito-borne flavivirus infection. We have shown for the first time the 3D ultrastructure of acutely and persistently, flavivirusinfected arthropod cells, facilitating the observation of the shift that occurs from round vesicles during acute TBFV-infection to the elongated tubules that dominate persistent infection. Future experiments are needed to better understand the increasing presence of tubules at the site of membrane rearrangement during persistent infection. 
Movie S1 LGTV TP21-induced structures in acutely infected Vero cells. Animation through a z-series and 3D surface rendering of a semi-thick section of an acutely infected Vero cell. ER is depicted in green, vesicles in blue, and virions in red. Both virions and vesicles are contained within a network of proliferated ER. The images were aligned using Inspect3D software (FEI, Inc.) and rendered using Amira (Visage Imaging, Inc., San Diego, CA). (MP4)
LGTV TP21-induced structures in acutely infected ISE6 cells. Animation through a z-series and 3D surface rendering of a semi-thick section of an acutely infected ISE6 cell. ER is depicted in green and vesicles & tubules in blue. Vesicles and tubules are contained within proliferated ER. Tubules are short in length, only reaching approximately twice the diameter length. The images were aligned using Inspect3D software (FEI, Inc.) and rendered using Amira (Visage Imaging, Inc., San Diego, CA).
Movie S3 LGTV TP21-induced structures in persistently infected ISE6 cells. Animation through a z-series and 3D surface rendering of a semi-thick section of a persistently infected ISE6 cell. ER is depicted in green and vesicles & tubules in blue. Numerous long tubules are seen in a large bundle; however, smaller tubules and round vesicles are also seen. The images were aligned using Inspect3D software (FEI, Inc.) and rendered using Amira (Visage Imaging, Inc., San Diego, CA). (MP4)  SEED Servers: High-Performance Access to the SEED Genomes, Annotations, and Metabolic Models The remarkable advance in sequencing technology and the rising interest in medical and environmental microbiology, biotechnology, and synthetic biology resulted in a deluge of published microbial genomes. Yet, genome annotation, comparison, and modeling remain a major bottleneck to the translation of sequence information into biological knowledge, hence computational analysis tools are continuously being developed for rapid genome annotation and interpretation. Among the earliest, most comprehensive resources for prokaryotic genome analysis, the SEED project, initiated in 2003 as an integration of genomic data and analysis tools, now contains >5,000 complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, a derived set of protein families, and hundreds of genome-scale metabolic models. Until recently, however, maintaining current copies of the SEED code and data at remote locations has been a pressing issue. To allow high-performance remote access to the SEED database, we developed the SEED Servers (http://www.theseed.org/servers): four network-based servers intended to expose the data in the underlying relational database, support basic annotation services, offer programmatic access to the capabilities of the RAST annotation server, and provide access to a growing collection of metabolic models that support flux balance analysis. The SEED servers offer open access to regularly updated data, the ability to annotate prokaryotic genomes, the ability to create metabolic reconstructions and detailed models of metabolism, and access to hundreds of existing metabolic models. This work offers and supports a framework upon which other groups can build independent research efforts. Large integrations of genomic data represent one of the major intellectual resources driving research in biology, and programmatic access to the SEED data will provide significant utility to a broad collection of potential users. The growth in the number of sequenced genomes has exceeded the most ambitious past estimations [1, 2] . Today, tens of thousands of genomes are being sequenced by public and private institutions around the globe [3, 4] , and whole-genome sequencing/re-sequencing has become a routine step of virtually any analysis of microbial behavior. In some fields, researchers are sequencing hundreds of genomes from distant taxa [5] to explore microbial diversity, while elsewhere, hundreds of closely related genomes are being sequenced to understand environmental adaptation, to support pangenome reconstruction [6, 7, 8] , and to build maps of microbial variomes [9] . Sequencing is also being applied to aid in solving urgent biological problems, including the development of strategies to combat emerging or reemerging biothreats, such as the severe acute respiratory syndrome (SARS) virus [10] , the 2009 H1N1 influenza virus [11, 12] , and the 2011 German Escherichia coli outbreak [13, 14] .
With the vast speed and minimal costs of modern sequencing technology, new demands are being placed on the bioinformatics pipelines used to analyze sequence data [1] . Modern bioinformatics must be high throughput and high volume with a strong emphasis on comparative genomics, and the SEED family of resources for genome interpretation and analysis were specifically constructed to embody these characteristics [15] . The SEED resources are built upon the organization of biology into a modular set of subsystems, each describing a specific biological functionality (e.g., flagellar motility or histidine biosynthesis) [16] . Subsystems-based technologies were developed in the SEED with the view that the interpretation of one genome can be made more efficient and consistent if hundreds of genomes are simultaneously annotated in one subsystem at a time [16, 17] . The SEED Project, a multi-institutional effort coordinated by the Fellowship for Interpretation of Genomes (FIG) and Argonne National Laboratory, began with the ambitious goal of consistently annotating 1,000 genomes (http://theseed.org/wiki/ Annotating_1000_genomes); today, over 50,000 viral and prokaryotic genomes have been annotated by the subsystems technology. More recently, the SEED Project has been expanded to include the capacity to automatically generate genome-scale metabolic models based on SEED annotations of genome sequences, and the Model SEED system developed from this pipeline has now been applied to construct metabolic models for thousands of genomes [18, 19, 20] . The SEED family of resources now collectively serves as a repository for almost 5,000 distinct complete prokaryotic genomes, associated with approximately 30,000 annotations, 11,000 metabolic models, 178,000 protein families, 10,250 functional roles, and 1,060 subsystems.
While a web interface is available for visualizing the data within the SEED (http://pubseed.theseed.org), this interface is inadequate to make full effective use of the massive quantities of data now embodied within the SEED. To truly expose the SEED data for efficient large-scale use by the global scientific community, we initially developed a Simple Object Access Protocol (SOAP) server to allow the needed programmatic access to the SEED data [21] ; however, the SOAP server could not scale up with the increased volume of data. To cope with the increasing demands placed on the SEED web services, as well as the increasing volumes of data included within the SEED resources, we developed a secondgeneration of web services to improve throughput, response time, and access to SEED functionality. Here, we describe in detail the four primary servers now available for remotely accessing SEED data and we describe the client-side distribution of server scripts that we provide as examples of how the servers may be applied to perform complex analyses of genome data.
The Servers and the Services The four servers described here, collectively referred to as the SEED servers, currently support 181 methods that can be invoked to extract data and services (Fig. 1) . The server code resides at the location of the SEED data. Users download a distribution with their choice of runtime environment that they may use to write programs to access SEED data or perform a number of common bioinformatics tasks using a supplied set of preprogrammed scripts. Currently, Perl and Java integrations are supported. The following subsections describe the four servers in more detail.
The Sapling Server offers access to the underlying integration of genomic data-including genomes, genes, proteins, annotations, subsystems, FIGfams, and co-occurrence data. On the server side, the Sapling Server accesses a database implemented by an entity-relationship data model (ERDB). The ERDB model is defined by a set of XML metadata describing the entities, relationships, and attributes in a form that can be used to generate queries as well as the documentation and an up-to-date database diagram may be viewed at http://servers.nmpdr.org/ figdisk/FIG/ErdbDocWidget.cgi?database = Sapling. Thus, the public description of the database always remains synchronized with the internal data structures-an important benefit in a database designed for public use.
The Sapling Server is architected such that new features can be added quickly. New data tables may be added as updates to the XML metadata, which is processed by a special load program to build the initial database tables. The list of services offered is maintained on the server, so that client software does not need to be updated in order for users to access new features. A web application that converts general database queries to Perl code helps speed implementation of new functions.
A database query is specified by naming the entities and relationships along a path through the ERDB diagram ( Fig. 2) along with a list of the data items to be returned and a filter clause that limits the results to the desired data objects (e.g., a particular genome or identifier). The Sapling Server allows direct queries against the database; however, a set of common data requests is implemented as direct server functions. Sapling Server functions typically accept multiple input values within a single call, allowing a client to minimize the number of requests that must be made to the server. Additional input parameters allow a client to modify the query, for example, to request that the output be in FASTA format or to ask for protein rather than DNA sequences.
In a sample ids_to_sequences request (Fig. 3A) , the user specifies four identifiers, and the server returns them as a table (actually a Perl hash) with the associated DNA sequences attached.
The Sapling Server currently supports 127 access functions. These functions are listed on a web page generated automatically from the latest version of the code, ensuring that the documentation remains up to date. A sample showing the web page description of ids_to_sequences is shown (Fig. 3B ).
The Annotation Support Server supports two distinct capabilities relating to the annotation of genomes: de novo annotation of protein or DNA sequences, and aggregation of annotations into subsystems. The Annotation Support Server accepts either DNA or protein sequence as input and, depending on the user options, can either use existing gene calls or invoke standard gene callers (e.g., GLIMMER-3 for protein-encoding genes). The server also houses newly developed high-performance methods to assign function to protein sequences or regions of genomic DNA sequences, based on FIGfams [22] . Below is an example application using these methods that produces a relatively accurate annotation of most microbial genomes within a few minutes. To evaluate the technology, users are encouraged to simply submit a known prokaryotic genome to the server for annotation.
Sequences can be submitted to the server in three ways:
1. Programs can directly access the services needed to call genes and assign functions to the proteins encoded within the genome. 2. If the protein-encoding genes have already been identified, the program can assign functions to these sequences. An example program is provided in the download library and is described at http://servers.theseed.org/sapling/server.cgi?pod=svr_assign_ using_figfams.pl.
A program can take as input fragments of DNA (e.g., from a metagenomic sample) and use the services to detect pieces of protein-encoding genes. Again, an example program is provided in the download library and is described at http://servers.theseed. org/sapling/server.cgi?pod=svr_assign_to_dna_using_figfams.pl).
The Annotation Support Server also provides the ability to take as input a set of functional roles (in the controlled vocabulary established by the subsystem collection) and to produce a detailed estimate of which subsystems are represented by those functional roles. That is, one can also use the server to develop a metabolic reconstruction based on the functional roles that have been assigned to the protein-encoding genes.
The RAST (Rapid Annotations using Subsystems Technology) [23] service provides access to high-throughput, high-quality annotation of prokaryotic genomes, and routinely processes ,25-75 genomes a day, with peak throughputs that have exceeded 200 genomes per day. This service is accessible for individual genome submission via a graphical web interface, and the next generation SEED servers provide new access for programmatic batch submission of genomes via the RAST submission and retrieval server. This server supports programmatic submission of genomes to the RAST Server, retrieval of job status, and retrieval of the final set of annotations. These scripts and the underlying application programming interface (API) enable users to submit genomes to the RAST Server, test the status of submitted jobs, and retrieve the output (i.e., annotated genomes).
Three types of input are supported:
1. A FASTA file of contigs that make up the genome to be annotated 2. A file of GenBank-formatted entries (with the option to retain the gene calls as given in the uploaded files)
3. An Entrez ID or a Genome Project ID.
In the case of the first type of input (FASTA file), the RAST Server will perform de novo annotation, i.e., will start by calling RNA-and protein-coding genes, and then will proceed to functional annotation, subsystems assignment, and subsequent metabolic reconstruction. In the second case (GenBank-formatted files), gene calling is optional, since GenBank-formatted files already include the coordinates for open-reading frames and RNA genes. In the last case (Entrez ID or Genome Project ID), the tools provided within the RAST Submission/Retrieval Server will query NCBI using the query ID(s) and will retrieve the sequence (whether that sequence consists of one or a set of contigs that make up the genome project); that sequence then becomes the input to the RAST Server.
The Metabolic Modeling and Flux Balance Analysis (FBA) Server provides programmatic remote access to the Model SEED biochemistry and genome-scale metabolic model database, as well as some model analysis algorithms. The Model SEED biochemistry database integrates into a single, nonredundant set all the reactions and compounds found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [24, 25, 26] , together with additional curated reactions and compounds [18] , and a continuously growing number of published genome-scale metabolic models.
Currently this database consists of 16,279 compounds and 13,272 reactions. For compounds, the database also includes database IDs from KEGG compounds and SEED models, names/ synonyms, molecular masses, molecular formulas, molecular charge, and estimated Gibbs free energy of formation [27] . For reactions, the database includes database IDs from KEGG reactions and SEED models, names/synonyms, stoichiometry, EC numbers, pathways, and estimated Gibbs free energy change of reactions [27] . Compound charge, formula, formation energies and reaction stoichiometry are all calculated for aqueous conditions at neutral pH. All API functions used to access the Metabolic Modeling and FBA Server capabilities are documented in detail at http://servers.theseed.org/.
The SEED database also contains a large number of genomescale metabolic models, including 14 published models [28] and approximately 11,000 models generated from the annotated genomes stored in the SEED [29] . The Metabolic Modeling and FBA Server also provides the user with an API to remotely obtain a list of the models in the SEED and to download data on the compounds and reactions in each SEED model. The server returns the following data for each reaction in a specified model: (i) all data from the SEED biochemistry database, (ii) a list of the genes associated with each reaction in the model in a format that captures how the protein products encoded by the genes function to catalyze the reaction (as either independent enzymes or multienzyme complexes), and (iii) a list of compartments in the model where the reaction takes place and the directionality/ reversibility of the reaction in each compartment. For the model compounds, the server returns the data from the SEED Figure 2 . Entities and relationships in the SEED. The entities (boxes) are connected to each other by a series of relationships (diamonds) that describe how the two entities relate. To move from one entity (e.g., ''Identifier'') to another (e.g., ''DNA Sequence''), the series of connections shown by the gray arrow is made. This way, any entity can be connected, either directly or indirectly, to any other entity. doi:10.1371/journal.pone.0048053.g002 biochemistry database. As with the biochemistry data, all the model data in the server are accessible either via Perl programs or the API.
The Metabolic Modeling and FBA Server also enables users to run various FBA studies on any of the genome-scale metabolic models stored in the SEED database. These studies can be performed while simulating any of 525 distinct media conditions currently encoded in the SEED database (which includes all BiologH [30] media conditions and a variety of complex media formulations). Both the Perl program and the API enable users to obtain a list of the media conditions currently stored in the SEED and details on the compounds included in each formulation. Once a model and media condition have been selected for simulation, the server provides an interface for running three types of FBA simulation: (i) simple growth simulation to predict maximum growth rate of the organism in the selected media, (ii) flux variability analysis (FVA) [31] to classify the reactions and compounds in the model according to their behavior during growth in the selected media, and (iii) single gene knockout analysis to predict the genes essential for growth in the selected media.
The simple growth simulation returns the maximum predicted growth rate of the model given the input parameters, the predicted flux through the model reactions during maximum growth, and the predicted uptake and production of nutrients from and to the environment during maximum growth.
The FVA simulation returns the predicted class of every reaction and compound in the model during growth given the input parameters. Reactions in the model are classified as forward essential or reverse essential if they are required for growth to occur, with forward and reverse referring to the direction in which the reactions must proceed. Reactions that are not essential for growth, but still active, are classified as forward variable, reverse variable, and variable, with forward and reverse indicating when reactions proceed only in a single direction. Reactions are classified as blocked if they cannot carry flux under the conditions specified by the user. Metabolites in the model are classified as essential nutrients or essential products if their uptake or secretion is required for growth in the input conditions, and they are classified as transported if they can be taken up or secreted but are not essential for growth. In addition to classifying the reactions and compounds in the model, the FVA simulation returns the maximum and minimum values for the flux through each reactions and the uptake/secretion of each metabolite.
The single gene knockout analysis rapidly simulates the individual knockout of every gene represented in the model during growth in the input conditions. Based on these simulations, the analysis produces a list of the predicted essential genes and the predicted nonessential genes in the model. Both the Perl program and API allow the user to run any of the three simulation types from the command line.
All three simulation types accept the same user input: the name of the model to be run, the name of the media formulation that growth should be simulated in, a list of genes in the model that should be knocked out during the simulation, and a list of the reactions in the model that should be knocked out during the simulation. See http://servers.theseed.org/for detailed documentation on all Metabolic Modeling and FBA Server functions.
The SEED servers can have limitless applications. Below, we demonstrate various applications of SEED servers through a small set of potential applications and coding examples.
Dealing with IDs of genes or the proteins they encode is often nontrivial. In the SEED database, we use IDs that specify proteinencoding genes in a rapidly growing set of genomes, and we support correspondences between these IDs and those used by other annotation efforts. The SEED has two notions of equivalence: (i) two IDs that represent either protein-encoding genes or protein sequences are said to be sequence equivalent if the protein sequences are identical and (ii) two IDs that represent either exactly the same protein-encoding gene or the precise protein encoded by the gene (that is, ''the protein sequence of gene X in genome Y'') are said to be precisely equivalent. Unfortunately, in the presence of multiple versions of thousands of genomes, perfect maintenance of the ''precisely equivalent'' correspondence is virtually impossible.
Our first example script takes a command-line argument containing a single ID and produces a table for all assertions of functions for sequence equivalent IDs. Each ID in the input is associated with the name of the genome containing it, the function for that ID, the source of the functional assignment assertion, and an indication of whether the source of the assertion provided a confidence for their estimate. The code is available at http://servers.theseed.org/sapling/server. cgi?code = server_paper_example1.pl (also provided as Supporting Information S1).
Given a set of functional roles, one often wishes to understand which subsystems can be inferred from the set. The following example script reads as input a set of functional roles and constructs a table of subsystems that can be identified, along with their variation codes. The data displayed in this simple example could form the start of a research project to gather the functional roles not connected to subsystems to determine whether they were not connected because a small set of functional roles were not present in the input, and to seek candidates for such "missing functional roles." The ability to easily map functional roles into subsystems will improve as the SEED annotation effort improves its collection of encoded subsystems [32] . The code for this example is shown at http://servers.theseed.org/sapling/server. cgi?code=server_paper_example2.pl. (also provided as Supporting Information S1).
The SEED provides the ability to graphically display the chromosomal regions around a set of genes (normally from distinct genomes); for example, see http://seed-viewer.theseed.org/ seedviewer.cgi?page=Annotation&feature=fig|83333.1.peg.4. The SEED also offers an alternative for creating custom interfaces, moreover, one that does not require the user to know appropriate SEED IDs. This approach exploits the conversion capabilities of the SEED for creating a program to accept arbitrary protein IDs. It also exploits the ability of SEED to map functional roles into subsystems as described in the preceding example. The result is a tool that enables the user to take a SEED ID and a region size, and extract the genes that are found within a region centered on the designated gene. The code for this example is shown at. http://servers.theseed.org/ sapling/server.cgi?code=server_paper_example3.pl (also provided as Supporting Information S1).
A great deal has been learned from studying genes that tend to occur close to one another in diverse genomes [33, 34, 35, 36, 37, 38] . In particular, the co-occurrence of hypothetical and non-hypothetical proteins can be exploited to suggest the function of the former based on the function of the latter.
The following program at http://servers.theseed.org/sapling/ server.cgi?code=server_paper_example4.pl illustrates the potential for constructing custom tools by going through all of the protein-encoding genes in all of the complete prokaryotic genomes maintained within the SEED looking for ''hypothetical proteins'' that tend to co-occur with genes encoding functions that can be connected to subsystems. The program constructs a table showing the following: This table can therefore be used to suggest functions for hypothetical proteins that could be tested experimentally. A copy of the code is provided (Supporting Information S1).
The SEED can be used to assign functions to a file of protein sequences. The code for this example is at http://servers.theseed. org/sapling/server.cgi?code=server_paper_example6.pl (also provided as Supporting Information S1).
This program reads a FASTA file of protein sequences and attempts to assign function to those sequences using a K-merbased algorithm. When a function is proposed, the program will produce a ''score'' (the number of distinct K-mers that were matched) and an estimate of phylogenetic neighborhood-a representative genome that is ''phylogenetically close'' to the genome containing the protein, if an estimate can reasonably be given.
A similar approach has been adopted for rapid, real-time analysis of metagenomics samples, which might elsewhere take days or months for BLAST-based analysis. This real-time metagenomics analysis (URL: http://edwards.sdsu.edu/rtmg) can be performed on computers or cellular phones [39] .
Here, we demonstrate how to run a variety of FBA algorithms on the SEED model of E. coli and how to print all data from the E. coli model and the results of the FBA into an output table. For the code, see http://servers.theseed.org/sapling/server. cgi?code=server_paper_example7.pl (also provided as Supporting Information S1).
The program starts by obtaining a list of all compounds and reactions in the SEED E. coli model (Seed83333.1) using the ''get_compound_id_list'' and ''get_reaction_id_list'' functions, respectively. The program then uses these lists to obtain detailed data on all the E. coli compounds and reactions (using the ''get_compound_-data'' and ''get_reaction_data'' functions, respectively). These data are stored in two tables: one for compounds and one for reactions. Next the ''classify_model_entities'' function is used to run a FVA on the SEED E. coli model. In this particular FVA, the reactions and compounds in the E. coli model are classified while simulating growth in LB media (called ArgonneLBMedia in the SEED model). At this point, the data returned by the ''classify_model_entities'' function is added onto the compound and reaction tables prepared previously. In the next step, the code uses the ''simulate_model_growth'' function to run a standard FBA on the SEED E. coli model, maximizing the model growth rate in simulated glucose minimal media (called Carbon-D-Glucose in the Model SEED). The data returned by this function are also added to the reaction and compound tables. In the final call to the server, the program uses the ''simulate_all_single_gene_knockout'' function to simulate the single knockout of all E. coli genes, and the results of this study are stored in a gene table. The remainder of the program handles the printing of the compound, reaction, and gene tables to the files CompoundTbl.txt, ReactionTbl.txt, and GeneTbl.txt, respectively.
The Gene Ontology (GO) project aims to unify biology by providing a controlled vocabulary of terms for all genes and gene products [40] , but has long had a focus on eukaryotes with less emphasis on prokaryotes and their viruses. On the other hand, the SEED database contains high quality annotations for hundreds of microbial and viral genomes, using subsystems-based controlled vocabulary. Mapping SEED functional roles to GO annotations for a given set of genes or gene products can be achieved via SEED servers. A workflow, detailed elsewhere (Short URL: http://bit.ly/server_paper_example8 ), uses two SEED serversbased programs to update SEED to GO comparisons through the use of UniProt [41, 42] protein identifiers.
The workflow consists of the following steps: 
The SEED project [15] focuses on developing technology to support rapid, high-volume, accurate annotation of genomes, and has so far achieved four advances of central importance:
1. The subsystems strategy, adopted as the guiding principle of the effort [16] , centers on leveraging expert annotations to define a small set of functional roles in all genomes rather than all the functional roles in a small number of genomes. 2. The subsystem effort provided a convenient framework for the curation of a set of protein families that became known as FIGfams [22, 23] , intended to contain only isofunctional homologsthat is, each family was intended to contain only homologous proteins playing the same functional role. When errors in FiGfams are detected, the underlying subsystems are updated and then the FIGfams are regenerated to correct those errors. The rapid evolution of the FIGfam collection has made possible a number of the services described in this article. 3. Using subsystems and FIGfams as the underlying technology, the RAST server was developed and made available in 2007 [23] . Thousands of viral and prokaryotic genomes have been annotated with the RAST system, and hundreds more are being annotated each week. 4. The high-quality annotations generated by RAST together with the ability to manually modify those annotations by human experts allowed for the automatic generation of genome-scale metabolic models in the Model SEED [18, 19, 20] .
As indicated above, the improvement in sequencing speed and efficiency led to a rapid accumulation of genome sequences and necessitated more efficient methods for large-scale access to genomes, annotations, subsystems, and genome-scale models within the SEED database. A SOAP server was first developed to allow programmatic access to the SEED data [21] , but several performance issues prevented the service from scaling with the volume of data contained within the SEED database. The server abstraction layer required the loading of numerous large modules on each invocation of server functionality, resulting in a noticeable delay in response to each server request. The encapsulation of the results in SOAP XML conferred significant overhead on the data being transferred. Finally, each operation of the SOAP server was atomic, accepting a single argument and returning a single datum. Trivial requests such as retrieving all the functions for all of the proteins in a genome took unacceptably long to complete, requiring a separate call for each protein and instantiating many threads on the server. The four SEED servers described here provide programmatic access to the SEED data and methods. They expose the current data in a form that is conveniently accessed computationally. The installation and maintenance of the client-side software require minimal effort. We have constructed the underlying methods to support relatively large-grained data transfers, allowing the construction of relatively efficient programs. In comparison to the SOAP server [21] , the new web services provide access to larger amounts of data in less time, and they have been engineered to respond to server requests with little or no server-side delay. Furthermore, the new web services provide a more efficient and flexible computing approach because they are designed to process batches of requests at a time, streaming the responses as they complete. These services provide access to the integrated genomic data, subsystems, FIGfams, co-occurrence data, annotation services, RAST annotation submission and job retrieval (thereby offering access to our continuing improvements in microbial annotation), and metabolic modeling. All client modules, code examples and documentation are available online at http:// servers.theseed.org, and we are continually expanding these services and improving the underlying documentation.
In conclusion, the SEED integration of genomic data now contains over 5,000 complete or nearly complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, and a derived set of protein families. The client-server code discussed in this article gives users easy programmatic access to the data in the SEED. It has already been successfully been used for building other applications, such as finding prophages in microbial genomes [43] , desktop-based RAST genome annotation and comparison (manuscript in preparation), and real-time annotation of metagenomes (RTMG, URL: http://edwards.sdsu.edu/rtmg [39] ). We encourage researchers to use this software to retrieve and analyze data from the SEED database and we believe that the underlying implementation of these new servers is efficient enough to address the needs of most users and we will continue providing occasional stand-alone versions of the SEED to users who need more performance or privacy.
The latest documentation and downloads are available at the following web addresses: http://servers.theseed.org and http:// blog.theseed.org/servers/ N Project name: SEED Servers N Project home page: http://www.theseed.org/ N Operating system(s): Mac OS, Linux N Programming language: Perl, JAVA N License: SEED Toolkit Public License N Any restrictions to use by non-academics: no limitations
The SEED servers project is documented and can be downloaded from the servers' web site, http://servers.theseed.org.
The Perl distribution contains the following: Client Packages.
1. The Sapling server -SAPserver.pm 2. The MODEL server -MODELserver.pm 3. The Annotation Support Server -ANNOserver.pm 4. The RAST server -RASTserver.pm
Programming using the servers. The SEED servers provide all necessary network operations in a client package that can be used to access the server functions. One uses these like any other Perl package. For instance, to find all genomes in the SEED, one does the following:
#!/usr/bin/perl w use strict; use SAPserver; my $sapObject = SAPserver-.new(); my $genomes = $sapObject-.all_genomes(); foreach my $g (sort { $genomes-.{$a} cmp $genomes-.{$b} } keys(%$genomes)) { print "$g\t$genomes-.{$g}\n"; } The function call $sapObject-.all_genomes() marshals the correct server-side function call and arguments into a network package, transmits that package to the server, waits for and retrieves the answer, processes any returned error codes, decodes the return package into a Perl data structure, and returns the result. All function calls in all the client packages perform these basic services.
The Java Distribution Contains the Following Client Packages. The org.theseed.servers.serverConnections package handles connecting to the server, transmitting and receiving the data, and converting data structures from the server into Java data structures. The classes in org.theseed.servers.servers packages handle connecting to each of the servers and making the appropriate calls.
Programming using the servers. We recommend that the code be accessed in eclipse (http://www.eclipse.org/), netbeans (http://www.netbeans.org/), or a similar graphical integrated development environment (IDE). These are used like any other class. For instance, to find all genomes in the SEED, one does the following import java.util.HashMap; import org.theseed.servers.SAPserver; public class AllGenomes { public static void main(String [35] args) { SAPserver sapling = new SAPserver(); HashMap,String, String.genomes = sapling. allGenomes(); for (String id : genomes.keySet()) System.out.
println(id + "\t"+ genomes.get(id)); } } Future directions include expanding the applications and releasing packages for use by other programming languages such as Python.
Supporting Information S1 Examples of programming using the SEED servers (coded in Perl). Eight application examples are provided, seven of which (1-4 and 6-8) are described in detail in the text. (PDF) ECR 2012 Book of Abstracts - A - Postergraduate Educational Programme  The rapid technological development of medical imaging over the past few decades has transformed radiology into an essential part of modern medical practice. In parallel with this, the rapid pace of biologic advances has made this the age of molecular medicine. Thanks to the emerging field of molecular imaging, the radiology community has the opportunity to help lead a revolution in modern medicine towards optimisd diagnosis and therapy of individual patients. Molecular imaging is the in vivo characteristion and measurement of biological processes that occur at the cellular and molecular level at a macroscopic level of resolution. This is in contrast to the current conventional, anatomically based radiology. The most widely used molecular imaging modalities include optical (fluorescence, bioluminescence); radionuclide (PET, SPECT); ultrasound; and MRI (spectroscopy, diffusion-weighed imaging). The interaction of several disciplines, including molecular cell biology and chemistry, has demonstrated the advantages of multi-modality, multi-disciplinary approach to molecular imaging. There are three currently used imaging strategies to noninvasively monitor and measure molecular events. They have been broadly defined as "direct", "biomarker" and "indirect" imaging. Although most of these imaging techniques are in the preclinical or early clinical phases, radiology will benefit greatly from these methods in the effort to detect and diagnose these diseases, to target therapies and to monitor results at the molecular level. Molecular imaging is the key technology by which to accomplish these goals. Imaging practitioners should think of themselves primarily as biologists whose task is to image what happens functionally and structurally at the molecular level. Learning Objectives:
1. To learn about basic concepts, principles and strategies of molecular imaging. 2. To understand the multimodality and multidisciplinary approach of molecular imaging. 3. To become familiar with task of imaging functional and structural events at the molecular level.
A-002 12 Imaging scientists are similar to the fly floating in a swimming pool described by Richard Feynman: they imagine the real world from the few waves that reach their small niche. Biological time scales range from fractions of seconds to evolutionary ages; biological spatial dimensions from atomic scales to tens of centimeters. Not one single technique can produce images spanning more than three temporal or spatial orders of magnitude. To reach further precision, either a priori information must be introduced in the imaging method, or several imaging techniques must be combined through co-registration. Here we give one example for each approach.
Optical reporter genes can be exquisitely addressed to sub-cellular organelles through targeted genetic encoding. A protein emitting light upon abrupt rises of local calcium concentrations is genetically encoded into the mitochondria of a mouse. Providing that the sampling of data acquisition is fast enough, the localistion of the signal is attributed to the mitochondria, roughly one micrometre in size each, even though the detection sensors have a millimetric spatial resolution. In neuroscience research, it is a classical paradigm to superimpose molecular images from PET with anatomical images from MRI. New experimental techniques allow co-registering multimodal images with widely differing spatial resolutions, by rebuilding 3D sets of in vivo PET images of the rat brain with post mortem histological stains of the same brains. Extension of such techniques beyond the artisanal knowhow is bound to become a major experimental tool for translation of molecular information into in vivo clinical applications. Learning Objectives: 1. To understand the temporal and spatial scales within living animal systems. 2. To become familiar with the temporal and spatial scales imaged by in vivo and in vitro imaging methods. 3. To learn how to bridge temporal and spatial scales between the different imaging methods.
In the era of intravenous thrombolysis and intra-arterial thrombolysis/ thrombectomy a fast and accurate diagnosis of acute cerebral ischaemia is mandatory. Several requirements for imaging are relevant: 1) All acute stroke patients should be imaged and treated as fast as emergency trauma patients. 2) Exclusion of other causes of acute stroke. 3) Detection of early signs of acute ischaemic stroke. 4) Assessment of the type of ischaemic stroke. 5) Assessment of the severity and extent of cerebral blood flow impairment. 6) Evaluation of the vasculature for causes of ischaemic stroke. 7) Optimal logistics with continuous interaction between nurses, technicians, radiologists and neurologists. Both structural and functional imaging have to be performed. Both CT and MRI are adequate techniques to deal with the imaging requirements, although CT with CT perfusion and CT angiography has become the standard technique in most hospitals. Nevertheless, MRI has several advantages over CT and should be available as back-up or substitute for CT. Important questions are 1) can CT detect all non-ischaemic causes of stroke? 2) Can CT detect ischaemic stroke in the early phase and make a distinction between the different types of ischaemic stroke? 3) How to interpret CT-and MRI-perfusion images? 4) How to detect distal occlusions? 5) How to differentiate between real and pseudo-occlusions of the internal carotid artery? Finally, a systematic approach in the analysis of the acquired images will facilitate a fast and accurate contribution of radiology to the optimal treatment of patients with acute ischaemic stroke. Stroke has gained acceptance as a treatable disease due to new developments both in diagnostic and therapeutic tools. The aims of therapy in stroke are to reperfuse hypoperfused areas and to open occluded vessels in the presence of a neurological deficit. Initially after the occlusion there is a sudden decrease in local blood flow. After a sufficient decrease, there is at first a reduction in function that is still associated with viability: this area where the thresholds for irreversible ischemia have not yet been attained is called the penumbra. While initially this was an area with reduced electric function but retained viability and membrane integrity, this area has been defined by diminished blood flow. Additionally a "core" infarct can be detected that is constituted of irreversibly ischaemic tissue. On MRI the penumbra was first delineated as the so-called diffusion-perfusion mismatch. CTbased algorithms also allow defining areas of reduced flow that are not yet necrotic. The ischaemic core can be both seen on DWI MR images or on unenhanced CT images: the addition of perfusion CT and perfusion MR techniques allows us to refine the visualisation of what should be the therapeutic target: the penumbra, since its reversibility should restore function.
Learning Objectives: 1. To understand the physiology of cerebral blood flow. 2. To be familiar with the concept of 'core infarct' and 'penumbra'. 3 . To learn how we can identify the penumbra on CT perfusion imaging. 4 . To learn how we can identify the penumbra on MR perfusion imaging.
A-011 16 At initiation, tumours in a pre-vascular phase are supplied with oxygen and nutrients that diffuse from pre-existing normal vessels. When the tumour reaches a critical size of approximately 1-4 mm diameter, the resultant ischaemia leads to the secretion of angiogenic factors. These factors, such as vascular endothelial growth factor (VEGF), recruit and maintain tumour vessels. "New" vessels (neovasculature) exhibit increased blood volume and permeability compared with normal vessels.
Various new specific therapies target tumour vasculature or tumour neoangiogenesis. It is not uncommon that these targeted therapies have pronounced cytostatic and not predominantly cytotoxic effects. This limits the usefulness of size-based morphological tumour response assessments. Of newer CT or magnetic resonance imaging (MRI) modalities, perfusion has emerged as valid marker of tumour-induced blood vessels and their function. Perfusion measures the vascularity within a tumour, as well as its component heterogeneous parts. Of parameters which can be measured to date, blood volume and permeability are commonly applied in patient studies. Blood volume measures the aggregate size of the vascular space, while the permeability function informs about the integrity of vessels and their ‚leakiness' to contrast agents. We will describe the use of MR perfusion to monitor new targeted therapies and discuss its advantages and disadvantages in comparison with CT perfusion protocols. Ultrasound as an alternate modality for perfusion assessment will be briefly presented. PET strategies for treatment-monitoring will be mentioned, with prospect on the role of combined vascular and metabolic imaging to further optimise non-invasive response assessment in specific anticancer therapies.
Learning Objectives:
1. To know about perfusion imaging protocols, specific advantages and technical challenges. Apart from morphology and perfusion characterisitcs, the evaluation of tumor agressiveness is closely related to other components of the tumor biology. Some of these "tumor variables" can be measured by the use of imaging biomarkers of tumour activity and phenotype. Imaging biomarkers are objectively measured characteristics that can be used as indicators of tumour biopathological changes. By using the qualitative and quantitative information of the different imaging modalities (US, CT, MR, PET) imaging specialists may be able to predict tumor prognosis and define the most appropriate therapeutic strategies. In this talk the most relevant components (cell density, cell and vascular heterogeneity, tissular entropy, tumor metabolites, cellular receptors, intracellular and interstitial physiological changes, pH, oxygen and iron concentration, elastic properties, physical quantities as the T1 and T2 relaxation times and biochemical molecular expresions) will be commented together with the different ways to explore them thought imaging parametric and multiparametric maps. These graphic maps characterice imaging biomarkers in two ways. First, they represent quantitative variables that characterize and measure different parameters obtained from medical images that are relevant to the tumour grading. Second, parametric images allow us to analyze the spatial distribution of the biomarker in the tumor through its visual representation. These images are generated to provide a graphical representation of the values of the calculated parameters on the basis of original image by post-processing computing. The use of imaging biomarkers in information-driven decision-making in clinical trials and oncologic clinical care will be simplified and supported through computer-aided diagnosis/quantitative image analysis. Imaging investigation of cholesteatoma is required before surgery. If no surgery has been performed previously, CT will provide information about the location of the lesion (epi, pro, meso, retro, hypotympanum), the partial or total destruction of the ossicles and possible extension to the inner ear. If there is no doubt about any of these factors CT is sufficient. In doubtful cases an MRI examination is performed to confirm or refute the presence of cholesteatoma using T1 sequences without IV contrast medium, and diffusion-weighted imaging with or without high-resolution T2, depending on the age of the patient. In postoperative recurrent cholesteatoma, MRI is becoming the first choice modality for detecting cholesteatomas, appearing low in signal on T1 sequences and high in signal on diffusion-weighted imaging. However, care is required since performing diffusion-weighted imaging without T1 may lead to false positives. A granuloma with a slightly or markedly increased T1 signal is often associated with a high signal on diffusion. Measurement of ADC is useful for detecting cholesteatomas, infected cholesteatomas or abscess. Finally, whilst MRI is the first examination in the follow-up of postoperative patients, the use of contrast medium is not necessary in most of the cases. Inner ear enhancement can only be seen in a reliable way on gadolinium-enhanced MR images. High field MR even allows inner ear screening with sub-millimetric contrast-enhanced T1-weighted images. On these images one can distinguish whether the enhancement is in the scala tympani or scala vestibule of the cochlea. The most frequent causes of inner ear enhancement are intralabyrinthine schwannoma (ILS) and labyrinthitis. Labyrinthitis can be viral, bacterial, autoimmune and luetic. Moderate to strong enhancement will only be seen in the acute phase and when the labyrinthitis is causing an important enough blood-labyrinth barrier rupture. Most often both the cochlea and the vestibular system are involved and the edges of the enhancement are unsharp. Under treatment this enhancement gets weaker and will finally disappear. Schwannomas have a stronger enhancement, the enhancement is better delineated, the lesions are most often restricted to the cochlea or the vestibular labyrinth and they can grow over time. When they grow they follow the pre-existing intralabyrinthine anatomical pathways. Many other less frequent lesions like meningiomas, granulomatous diseases (e.g. tuberculosis), trauma-surgery, cholesteatoma, etc., can also affect the inner ear and cause inner ear enhancement. The most frequent causes of inner ear enhancement, their MR characteristics and their natural behaviour will be discussed in this lecture. This session will provide the participants with a practical update on the principal indications to pediatric neuroimaging with a discussion of the main study techniques. The first presentation deals with the indications for ultrasound in neonates, where the advantages compared to MRI will be elucidated. The second presentation focuses on the applications of MRI in the study of normal brain development both before and after birth with an emphasis on how to correctly report an MRI study and the main pitfalls in children. The third presentation focuses on advanced MRI A-015 17 Acute stroke is increasingly being treated by neurointerventional techniques. Intra-arterial recanalisation provides better recanalisation rates and potentially better outcome for large vessel occlusion as compared with systemic thrombolysis.
The key to success is to select patients with salvageable brain tissue at the time of intervention. Besides the time window, this largely depends on the collateral circulation provided to the involved tissue. Diffusion-perfusion weighted MRI and perfusion weighted CT scan are being used to demonstrate viable penumbra around the infarct core. Direct injection of thrombolytics (rTPA) provides recanalisation in 50-60% of cases treated with this technique. This is associated with a 9-10% rate of symptomatic parenchymal haemorrhage. Today, mechanical thrombectomy or thrombaspiration is the first choice of interventional treatment. Stent thrombectomy is capable of providing a recanalisation rate of 80-90% on the TICI scale and good clinical outcome (0-2 on the modified Rankin scale) in 45-60% of patients. Results might be improved applying the bridging technique, that is a combination of iv-thrombolysis and local thrombectomy. Faster and more effective recanalisation as well as reduced usage of thrombolytics results in a lower symptomatic haemorrhage rate (4-10%).
Learning Objectives: 1. To learn how to select stroke patients for interventional neuroradiological treatment. 2. To be familiar with the techniques for intra-arterial thrombolysis. 3 . To be familiar with the techniques for mechanical thrombectomy. 4 . To understand what the advantages, disadvantages, risks and limitations of these methods are.
Quite often radiologists consider the anatomy of the temporal bone (t.b). as difficult. Therefore, the first part of the lecture will give a compressed presentation of radiologically relevant t.b. structures. Optimal findings require the knowledge of normal morphology. This is especially true concerning malformations, which are in the focus of the second part. Malformations are characterised by a deviation from the normal morphology combined with functional disturbance (in t.b. predominantly congenital hearing loss). They count among rare diseases. Most of them are diagnosed in early childhood, but in cases of unilateral hearing loss of lesser degree without deformed auricle the diagnosis may be delayed until adulthood. Due to different tissue of origin and different times of development, typical and less typical combinations of malformed parts of the ear result. Combined external and middle ear malformations (congenital aural atresia) are more common than combined malformations of the middle and inner ear. While in suspicion of middle ear malformations a correlate on images can be found in a high percentage, presumed inner ear malformations show a normal radiological finding in about four-fifths of the cases. Nowadays, middle ear malformations are diagnosed by CT or Cone Beam CT. Both methods can also detect most radiologically discernible inner ear malformations, but MRI delivers more details at this. The radiological appearance of malformations of the external auditory canal, middle and inner ear will be demonstrated by giving examples. Therapeutically relevant points are briefly mentioned at the end.
Learning Objectives: 1. To review the normal anatomy of the temporal bone. 2. To become familiar with congenital malformations of the external and middle ear. 3. To recognise and differentiate the most frequent congenital malformations of the inner ear.
so-called normal appearing white matter. Dynamic susceptibility-weighted contrastenhanced perfusion MR imaging provides haemodynamic information that complements traditional structural MR imaging and is being increasingly used in clinical practice to diagnose, manage and understand brain tumours in the paediatric patient group. Clinical studies of today involve multidimensional high-resolution images containing a substantial amount of structural and functional information. The role of quantitative imaging and the parameters measured have become more and more important to evaluate severity of a disease, disease progression and response to therapy, to mention a few examples. Increasing number of different quantitative image analysis techniques that can be used both in adult and children have been described in the recent years. Different imaging analyses like region-of-interest analysis, intra-individual voxel-based analysis and inter-voxel based analysis are a few examples all with different limitations and drawbacks as will be discussed in this lecture. The role of different imaging analyses such as DTI tractography and perfusion MR and the use of quantitative measures of different parameters and their application in children will be presented. Pancreatic neoplasms have a very different prognosis and treatment. In order to illustrate the experiences of a multidisciplinary group at the University of Verona, the participants (surgeon, pathologist and radiologist) will discuss some of the issues that are critical in the diagnosis, differential diagnosis and management of pancreatic cancer patients. Session Objectives: 1. To understand the role of a multidisciplinary approach.
2. To learn about the goal of each medical specialty in pancreatic tumours.
What the surgeon needs to know C. Bassi; Verona/IT (claudio.bassi@univr.it)
The third issue that will be collegially discussed is as to whether there are any liver metastases. The surgeon will illustrate that the presence of liver metastases will contraindicate surgical treatment, especially depicting small liver metastases may avoid unnecessary laparotomies that are always associated with a different level of morbidity and mortality. The pathologist will show us the criteria to diagnose a liver metastases and the importance of histological subtyping (i.e. adenocarcinoma vs. endocrine neoplasm).The radiologist will illustrate the sensitivity of different imaging modalities in detecting liver metastases and the improvement in sensitivity after the administration of liver-specific contrast agents. Furthermore, the diagnostic imaging criteria for the differential diagnosis of focal liver lesions will be illustrated. The fourth issue that will be collegially discussed is whether there are there positive lymph nodes. The surgeon will illustrate that the lymph nodes are a marginal surgical problem. The pathologist will illustrate the lymph nodes more frequently involved in pancreatic cancer and the criteria for a pathological diagnosis. The radiologist will illustrate the strength and especially the limitation in characterising lymph nodes. The fifth issue that will be collegially discussed is whether the tumor is locally advanced. The surgeon will illustrate the criteria that may contraindicate the surgical procedure, namely which are the vessel of interest in planning a surgical procedure that a radiologist should take care of. The pathologist will illustrate the criteria for vessel wall infiltration and the pattern of peri-vessel infiltration. The radiologist will illustrate which are the diagnostic imaging criteria to define vessel wall infiltration and their relative accuracies.
applications, and the role of different imaging analyses such as DTI tractography and perfusion MR and the use of quantitative measures of different parameters and their application in children will be presented. To stress the role of DTI and tractography in the more detailed evaluation of brain pathology. 3. To discuss the role of voxel-based methods in detecting abnormalities that can be missed by even the most experienced radiological eye.
What is the potential and role of brain ultrasound in the MRI era? M.I. Argyropoulou; Ioannina/GR (margyrop@cc.uoi.gr)
Brain ultrasound (US) represents the first line imaging modality for the evaluation of the neonatal brain. The main advantages of the technique are safety, performance at bed side, possibility for serial imaging without sedation, real-time information, coupling with colour Doppler and low cost. Anterior fontanel is the classic acoustic window but mastoid and posterior fontanel approaches are increasingly being used for better evaluation of the occipital lobes and the posterior fossa structures. Tranducers of different frequencies are routinely used to optimise imaging and increase lesion detectability. Intubated ventilated preterm babies are examined in the incubator and ischaemic-haemorrhagic brain disease can be detected at very early stages. Early periventricular leukomalacia (PVL) appearing as periventricular hyperchogenicity with sharp or hazy speculated margins and early periventricular venous infarct appearing as fan-shaped paraventricular echogenicity associated with flow disappearance in the terminal vein are important US and colour Doppler findings appearing before detection of any abnormality on MRI. Infectious haemorrhagic infarcts appearing initially as punctuate hyperechogenic lesions and progressing into microcystic lesions are almost never detected by MRI. Increased echogenicity is detected at the early stages of less common acquired lesions such as gangliothalamic ischaemia and cortical necrosis. Intraventricular haemorrhage can be easily detected by US. Colour Doppler by depicting moving echoes in the aqueduct of Sylvius is very helpful in the detection of haemorrhage at its very early stages and in the differential diagnosis between intraventricular and subependymal haemmorhage. Brain maturation-myelination and long-term follow-up of perinatal lesions should be performed with MRI. Learning Objectives:
1. To learn about the role of US in diagnosing the encephalopathy of prematurity. 2. To become familiar with US patterns of periventricular leukomalacia and brain haemorrhagic disease. 3. To understand the role of US in diagnosing hypoxic-ischaemic disease in full term babies.
When is MRI of the brain indicated? P.D. Griffiths; Sheffield/UK (p.griffiths@sheffield.ac.uk)
In this presentation I will concentrate on normal development of the white matter in the cerebral hemispheres. I will use images of tissue sections and foetal brain MR examinations to highlight the important changes occurring in the cerebral hemispheres of the second and third trimester foetus and their relationship with developmental abnormalities. I will then move onto the normal post-natal development of the hemispheric myelin detailing the minimum knowledge required in order to report MR brain examinations of the neonate and infant. A number of potential interpretive pitfalls will be discussed. Learning Objectives: 1. To understand the changes in MR signal characteristics found in the foetal, neonatal and paediatric brain as myelination progresses. 2. To understand why different approaches need to be made when performing MR imaging of the brain in those groups. 3. To appreciate the effects that myelination has on the ability to detect the more common brain malformations.
Although all pancreatic neoplasia, including ductal carcinoma (PDC), may clinically appear as a cystic lesion due to degenerative changes in a solid tumour, the most typical cystic neoplasms of the pancreas are cysts lined by different epithelium. The cystic neoplasms are classified into benign, borderline and malignant. Nonneoplastic mimickers of PDC mostly fall into the category of inflammatory and fibrotic conditions correlated in various ways with chronic pancreatitis and autoimmune pancreatitis. PDC must be differentiated from other malignancies such as acinar or endocrine carcinomas. It is essential to identify mucinous-cystic tumours and intraductal-papillary tumours because of their incomparably better prognosis. The 'cystic' variant of ductal adenocarcinoma, due either to degeneration changes or to ectatic changes of the duct system, can mimic the former two neoplasms. The ideal diagnostic test for liver metastasis should be the tissue diagnosis with a trucut biopsy, since the tissue can be also used for ancillary studies. The pathological criteria and the importance of histological subtyping will be presented. The surgically removed lymph nodes are classified and numbered according to their topography, considering that part of them are dissected with a radical pancreaticoduodenectomy and others are dissected by the surgeon and submitted separately. Because the most frequent site of local recurrence of PDCA involves the retroperitoneal posterior bed of the pancreas, the most important tissue to study is the peripancreatic fibrous and adipose tissue that runs behind the head of the pancreas and dorsally and laterally to the superior mesenteric artery.
Learning Objectives: 1. To understand the pathologic features of different pancreatic neoplasms.
2. To learn about distinct histo-pathologic findings that enable tumour characterisation. 3 . To be aware of histo-pathologic findings that show prognostic relevance.
Imaging of pancreatic tumours R. Manfredi; Verona/IT (riccardo.manfredi@univr.it)
The first issue that we will discuss as a group is whether it is a solid or a cystic lesion of the pancreas that we are dealing with. In this topic the surgeon will underline the importance in this differential diagnosis, since a solid lesion must be resected, if possible, where some cystic lesion could be followed up. In this settings, the pathologist will illustrate us the different pathological patterns of pancreatic neoplasms, cystic and solid, their immunohistochemistry and the possible progression from benign to malignant pancreatic tumour. According to the different pathological patterns, the radiologist will illustrate the appearance on the different pancreatic tumours on the different diagnostic imaging modalities. The second issue that we will collegially discuss is whether we are sure that it is a neoplasm. The surgeon will illustrate the different surgical procedures that he/she is able to perform and the relative indication and the different questions that each procedure need to be answered by the pathologist and the radiologist. The pathologist will show the different pathological patterns that are useful for the differential diagnosis among different pancreatic tumours and especially the criteria useful for the differential diagnosis with tumour-like conditions that do not require surgery but medical therapy instead. The radiologist will illustrate the diagnostic imaging signs that are helpful to make a differential diagnosis in patients with a mass in the pancreas. Learning Objectives:
1. To know the diagnostic imaging findings in different pancreatic neoplasms.
2. To learn about the role of imaging in surgical planning. 3 . To be familiar with the strengths and weaknesses of imaging in patient management.
Dynamic contrast-enhanced MR-mammography is the most sensitive method for detection of breast cancer. Diagnostic results using this technique may vary due to reader experience as image interpretation is to some degree a subjective task.
In the past years, further, more or less quantitative MRI techniques have been investigated. While pharmacokinetic modelling of high temporal resolution dynamic contrast-enhanced imaging (perfusion imaging) promises further, quantitative insights into the pathological characteristics of neoplastic vasculature, diffusionweighted imaging (DWI) and MR-spectroscopy (MRS) are based on entirely different concepts. While MRS is a molecular imaging technique able to quantify biochemical tissue properties, DWI is influenced by microstructural tissue changes. This talk aims to outline the concepts of perfusion, DWI and MRS, provide knowledge to implement these techniques into clinical practice and critically discusses the possible diagnostic benefit of doing so. Acute pelvic pain in pregnant women may be the manifestation of various gynaecological and non-gynaecological conditions. The correct diagnosis of the causes of acute pain during pregnancy is critical to minimise maternal-foetal morbidity and mortality. Although ultrasound (US) is the primary imaging investigation in the diagnostic evaluation of the pregnant patient, the role of magnetic resonance (MR) imaging in the evaluation of foetal and maternal diseases in pregnant patients continues to expand. MR imaging offers different potential advantages in comparison with US for evaluating acute abdominal and pelvic pain; these include multiplanar imaging capabilities, a higher soft tissue contrast and the ability to detect and distinguish blood from other fluid collections. When US is equivocal or nondiagnostic, MR imaging is a valuable complement to determine the exact aetiology of acute abdominal pain. The intrinsic safety and the accuracy of MRI in diagnosing abdominal and pelvic diseases make it an excellent choice for triage of pregnant patients with acute pelvic pain. MRI provides important information that influences the appropriate management and it is important for the radiologists to recognise the MR findings of the common causes of acute abdominal and pelvic pain. This lecture will discuss the use of MR imaging for maternal diseases that cause acute pelvic pain during pregnancy. Moreover, this lecture will discuss the different MR imaging techniques to use, and will show how to detect and to differentiate the gynaecological and non-gynaecological causes of acute pelvic pain during pregnancy. Learning Objectives: 1. To become familiar with the most common causes of pelvic pain in pregnancy. 2. To understand how to diagnose non-gynaecological causes of pain in pregnancy.
glass opacity, air trapping and reticular pattern) which have been recognised in asymptomatic healthy subjects. The appearances in normal older individuals (such as large airway abnormalities, a subpleural reticular pattern and cysts) will be highlighted. Learning Objectives: 1. To become familiar with the particular HRCT findings that occupy the gray area between unequivocal health and definite disease. 2. To become familiar with these findings, with a particular focus on the effects of cigarette smoking and ageing. 3. To understand the dilemma between the increased sensitivity of HRCT to detect preclinical disease and the potential risk of overdiagnosis. Breast clinical examination provides information of tissue elasticity Young modulus E = σ / ε, where σ is the compression (stress) and ε is the deformation (strain) of the tissue. Cancers tend to be stiffer than benign lesions. Breast ultrasound elastography provides a mapping of lesion stiffness, independent of its morphologic features, displayed in real-time as a color overlay of the lesion and surrounding tissue. Two techniques are available: 1. Strain imaging, where an external compression is gradually applied to the lesion by means of the ultrasound probe, thus deforming the lesion. Results are qualitative or semi-quantitative. 2. Shear wave imaging (no external compression applied) where the ultrasound system induces mechanical vibration by using shear wave imaging. Quantitative values of Young modulus are provided. Combining elastography with B-mode can improve specificity and accuracy for BIRADS four lesions and potentially reduce breast biopsy rates. For BIRADS 3 lesions, elastography can help identifying complicated cysts and reduce FNA rates. It can also indicate a biopsy instead of follow-up in case of BIRADS 3 lesions with suspicious elastography findings. Elastography can improve delimitation of iso-echoic lesions such as infiltrating lobular carcinomas. Further applications include lymph node characterisation and monitoring neoadjuvant treatment. 3D elastography is actually in progress.
In recent years, technical advances and improvements in cardiac computed tomography (CT) have provoked increasing interest in the potential clinical role of this technique in the non-invasive work-up of patients with various cardiac diseasesmost importantly in patients with suspected coronary artery disease (CAD) -and have initiated discussions on correct patient selection for this emerging imaging technology. A huge number of publications have abounded evaluating the diagnostic performance of CT coronary angiography in detecting significant coronary artery disease. With continuous development in CT system generations, the diagnostic accuracy increased steadily. During the first years of cardiac CT, technical improvements focused on the increase of detector rows, enabling faster scans with less contrast agent volume and improved robustness of the technique. In recent years, with the introduction of wide detectors with 256 or even 320 detector rows, as well as with the advent of Dual-Source CT scanners, scanning of the heart within one or two heart beats became feasible, as well as new options such as dual energy imaging of the heart or highly radiation dose-efficient protocols, e.g. applying highpitch scanning protocols. These options can broaden the clinical applications of cardiac CT, such as the depiction of myocardial changes, e.g. myocardial infarctions or perfusion defects, or -due to scanning options with less than 1mSv equivalent dose -including younger patients, even infants, e.g. with congenital heart disease. Learning Objectives:
1. To learn about the latest technical developments in cardiac CT. 2. To understand how the technical characteristics of CT scanners influence results of cardiac CTA. 3 . To know about the optimal combination of scanning parameters to get the most out of CT scanners in cardiac examinations.
A-039 17:00 C. Cardiac hybrid imaging P.A. Kaufmann; Zurich/CH Recent data using SPECT and CT suggest that hybrid imaging provides added diagnostic clinical value beyond that of either technique alone or that of side-byside analysis. The value of non-invasive hybrid imaging lies far beyond the simple addition of a further diagnostic test as it allows accurate spatial association of perfusion defects to their subtending coronary stenoses providing key information for evidence-driven intervention targeting relevant lesion only. While a negative coronary computerised tomography angiography (CCTA) has high negative predictive value to rule out relevant coronary artery disease (CAD), an abnormal CCTA -like an abnormal conventional angiography study -is a poor predictor of ischaemia, and further perfusion imaging testing is mandatory for decision-making towards revascularisation. Conversely, a normal MPI result does not exclude the presence of subclinical CAD requiring risk modification. The incongruence of CCTA and MPI is inherent to the duality of morphologic versus functional testing. However, no matter how accurate CCTA will possibly get with future advances in technology, the two pieces of information obtained with MPI versus morphology are difficult to compare, as they are complementary. Hybrid images offer superior diagnostic information with regard to identification of the culprit vessel with the haemodynamic relevant lesion providing added diagnostic information not obtained on side-by-side analysis. However, it remains uncertain at this point whether hybrid scanners offer advantages over software fusion of data sets obtained from different scanners, as by either way one can obtain hybrid images.
A-035 16 In this lecture, the role of imaging in the evaluation of gynaecological emergencies will be presented. A combined approach using both clinical findings and imaging features is necessary. Accurate evaluation is important as failure to make a diagnosis may lead to serious consequences. Presenting symptoms, such as pelvic pain or vaginal bleeding or discharge, may overlap with pregnancy-related emergencies and with non-gynaecological abdominal emergencies. The range of conditions to be considered includes ovarian cyst emergencies (cyst rupture, haemorrhage or torsion), infective conditions (Bartholins' or vulval abcess, pelvic inflammatory disease or tubo-ovarian abcess) and acute bleeding (from inflammation, neoplasm, or trauma). Pain may be related to the menstrual cycle, as in endometriosis or ruptured corpus luteum, or may be unrelated, such as in fibroid or ovarian torsion or pelvic inflammatory disease. The imaging features of these acute abnormalities will be reviewed and discussed in the context of the differential diagnoses.
Learning Objectives: 1. To recognise the causes of common gynaecological emergencies. 2. To understand the diagnostic imaging of common gynaecological emergencies.
A-036 17:00 C. Imaging for non-gynaecological emergencies R.F. El Sayed; Cairo/EG (rania 729 @hotmail.com)
Acute pelvic pain in women is a routine situation in an emergency unit. It can be secondary to a variety of disorders. Clinical setting often indicates an obstetric or gynaecological origin of acute pelvic pain, but sometimes it is not easy to distinguish if the primary cause is related to genitourinary system or gastrointestinal tract or to other unusual conditions. Diagnostic imaging can be very valuable in this situation. Ultrasonography is usually the first choice imaging modality for diagnosis of acute pelvic pain-and, for the most part, the only one needed for the diagnosis. MRI is the preferred technique in young women, also in pregnancy in second or third trimester. CT is more valuable for assessing non-gynaecological disorders or post-operative infections. The radiologist should know how to investigate the patient with regards to history and clinical findings as certain contexts can encourage specific diagnoses. The purpose of this lecture is to highlight the contribution of different imaging modalities in emergency radiology as a useful diagnostic tool in management of acute female pelvic disease of non-gynaecological origin and to describe key radiologic signs to improve differential diagnosis. Learning Objectives: 1. To become familiar with the causes and diagnostic findings of common nongynaecological emergencies. 2. To know how to investigate patients with reference to history and clinical findings. 3. To learn how to approach pelvic pain using multimodality imaging techniques in different clinical contexts.
A-043 16 Vascular grafts replace occluded vessels to maintain patency. Although rare (1-6%), graft infections are severe surgical complications, with poor prognosis. Optimal treatment of infected grafts is surgical removal. The challenging question affecting management is whether the graft or only adjacent soft tissue wound is infected. False positives lead to unnecessary surgery while false negatives are associated with high morbidity. SPECT and PET radiopharmaceuticals are used for diagnosis of vascular graft infection. Labelled leucocytes (Tc-99m, In-111) are SPECT agents of choice, accumulate by diapedesis, chemotaxis and vascular permeability. False-negative studies are related to antibiotherapy or duration of symptoms, and false positives occur with lymphocelle, haematoma, thrombosis, and physiologic uptake in recent grafts. FDG, the main PET tracer, is taken up by infections with high cellular metabolism. Advantages of PET over SPECT imaging include lower background activity, shorter duration of studies and no handling of blood of potentially infected patients. False positives due to increased uptake in native vessels, early grafts or healing scars can be differentiated by pattern and intensity of uptake. Size and proximity of structures and positional changes can lead to inaccurate localisation of tracer-avid foci and faulty diagnosis. Combining anatomic CT landmarks with functional SPECT changes or increased metabolism on PET decrease the false-positive rate. Uptake foci are accurately localised to graft or soft tissues improving specificity and diagnostic accuracy. SPECT/CT provided accurate data for diagnosis and localisation in 67% of patients and accuracy of FDG-PET/CT was above 95% for diagnosis of vascular graft infection. Learning Objectives: 1. To understand the potential value of nuclear medicine procedures in suspected vascular graft infection. 2. To understand the specific incremental role of hybrid imaging in suspected vascular graft infection. 3 . To recognise patterns of true and false positive findings in infected vascular grafts and to know the referral criteria for the best utilisation of hybrid imaging in vascular graft infection. 
A-040 16 :00
Chairmen's introduction P. Bourguet 1 , A. Palkó 2 ; 1 Rennes/FR, 2 Szeged/HU
The session is aimed at clarifying the role of radiological imaging diagnostics and the contribution of nuclear medicine methods in the diagnosis and differential diagnosis of inflammatory conditions. Obviously the number of these conditions is endless, therefore we decided to focus on two areas where both specialties contribute to the evaluation of the clinical situation: vascular graft infections and inflammation and inflammatory bowel diseases. Speakers from both disciplines reveal the most up-to-date information, including the role of hybrid imaging techniques in order to give a comprehensive overview of the morphological and functional diagnostic algorithm and define the most appropriate examination protocols. The panel discussion following the presentations will provide the opportunity to further deepen consensual knowledge leading to an integrated diagnostic approach to these clinically challenging conditions. Session Objectives: 1. To understand the importance of functional and morphological imaging evaluation of inflammatory conditions in certain inflammatory conditions. 2. To evaluate the specific role and the proper diagnostic algorithm of nuclear medicine and radiological techniques. 3. To highlight potential avenues of future development for functional and morphological imaging in the diagnostics of these conditions.
Imaging inflammatory bowel disease: the nuclear medicine perspective A. Signore; Rome/IT (alberto.signore@uniroma 1 .it)
Nuclear Medicine offers several possibilities for the imaging of inflammatory bowel diseases (IBD). Most data available in the literature concern the use of radiolabelled autologous white blood cells (WBC). With this method it is possible to evaluate with a single scan the whole body and, therefore, depict the extent of pathological gut wall infiltration by leucocytes: a sign of disease activity. The method of WBC has been criticised for its long preparation time and lack of anatomical accuracy. However, there are now available several kits for easy labelling of WBC with 99mTc and the possibility to use hybrid SPECT/CT cameras that improve accuracy and anatomical localisation of lesions. These kits can also use 18 F and 64Cu for WBC labelling, two positron-emitting isotopes, that allow PET/CT imaging with further improvement of image detail and possibility to quantify uptake in lesions for early therapy follow-up. But the availability of new SPECT and PET radiopharmaceutical for molecular imaging and for in vivo histological characterisation of lesions has recently opened a new indication for IBD that is therapy decision making. We can quantify the presence of activated T-cells or lymphokines (such as TNFa) in gut wall lesions, thus providing the clinician with important information for starting the appropriate treatment and to early assess (within weeks) the efficacy of the treatment. Since MRI and Nuclear Medicine are complementary for the study of IBD, we aim at clarifying what is the role of both methods and how to integrate them in the diagnostic flow-chart of patients. The future will probably be a single scan with PET/MRI hybrid cameras, recently available. Learning Objectives: 1. To highlight available imaging methods, imaging techniques and diagnostic algorithms for the evaluation of inflammatory bowel lesions. 2. To learn more about the specificity of nuclear medicine techniques and their clinical role. 3. To understand the role of nuclear medicine imaging in the evaluation of disease activity and extent, therapy decision making and early therapy follow-up.
The arterial trauma occurs in different scenarios such as politrauma, stab and gunshot wounds and iatrogenic. Surgical vessels ligation was the common method of bleeding control in World War II with an amputation rate of 50%. Surgical vessels reconstruction is currently considered the first option; however, this is no longer necessary in many cases. There are different mechanisms of arterial injury ranging between intimal flap and spasm to AV fistula and complete transection with or without arterial destruction. The amount and speed of bleeding after arterial trauma are the main factors to decide if an invasive treatment is necessary and when and how to treat it. Embolisation is a smarter, less invasive and safer method of ligation with similar results than surgery in terms of bleeding control. Vessel reconstruction is now also possible with endovascular devices. In general we will choose embolisation for small and peripheral arteries and vessel reconstruction with stent grafts for injuries in central and large arteries. In pseudoaneurysm and AV fistula there is no active bleeding and endovascular treatment can be done either by embolisation or stent graft. Coils are widespread as embolisation material but sometimes do not achieve a complete bleeding control mainly if patients suffer coagulopathy. Liquid embolics like cyanoacrylate and onix are less used but have some advantages over the coils. Many aortic injuries can be treated with stent graft and with less morbidity and mortality than surgery. The follow-up is mainly clinic with Doppler ultrasound and CTA as ancillary tools.
Learning Objectives: 1. To understand potential treatment options and when to treat and when not to treat. 2. To be familiar with different embolisations and other treatments. 3 . To learn about the results and appropriate follow-up strategies.
A-048 16 Trauma is a serious global health problem, accounting for approximately one in ten deaths worldwide. Injuries of CNS and great vessels are responsible for death at the site of accident while visceral haemorrhage is the principal cause of mortality during the first following hours after a trauma. The role of the interventional radiologist (IR) has changed dramatically during the past decades. It is now possible to temporise or definitively treat polytrauma with endovascular techniques. Imaging in the evaluation of patients with blunt abdominal trauma is fundamental in order to "Control the Uncontrollable bleedings" which is the main role of interventional radiology in damage control. Based on the spectrum of injury, interventional techniques are a solid alternative not only in spleen, liver, kidneys, pancreas and adrenal injuries, but also in pelvic fractures. Patient selection for treatment and follow-up is mainly governed by vascular imaging focused on MDCT, especially 3-D CT reconstructions. CT is a good predictor of the need for endovascular management or conservative treatment, based on key findings. Key images that may alter management will be discussed. The role of IR depends on local practice and carries broad applications where getting the patient to IR-angiosuite/hybrid-room is the first challenge. Finally, IR can control most haemorrhages and this is done in a multidisciplinary approach with proactive management to reduce haemorrhage and allow appropriate surgery. Various angiographic techniques have the potential of definite treatment even in case of marginal haemodynamic instability. Learning Objectives:
1. To know about the causes and the imaging appearances of solid organ trauma. 2. To understand various methods of IR treatment. 3 . To learn about the results and appropriate follow-up strategies.
Panel discussion: Do we need IR in the ER?
A-044 16 :59
Vascular graft infection (VGI) is a serious complication, prognosis and treatment depend on early diagnosis. Current imaging methods have different applications and value in VGI. Plain film does not have a major role for diagnosis of VGI. It is suspected when perigraft gas is present. Ultrasound (US) is used for basic diagnostic due to availability, costs and rapid use. Evaluates graft morphology, anastomosis, native vessels, surrounding tissues and fluid collections. DopplerUS determines flow disturbances. Computed Tomography (CT) is currently the choice imaging method. Examines all anatomic regions. Grafts are visualised to full extension. Detects perigraft fluid, perigraft soft-tissue attenuation, ectopic gas and pseudoaneurysms. In low-grade infection, sensitivity decreases as morphological changes are difficult to distinguish from postoperatory changes. CT examines vascular and perivascular masses, ectopic gas and fluid collections, native adjacent arteries, arterial grafts and their lumen, venous system and other pathologies that can explain graft infection. CT detects aortoenteric fistulas, can distinguish between enteric loops and interenteric abscess and bleeding after graft infection. Provides image guidance for aspiration of fluid collections and percutaneous drainage placement. Magnetic Resonance Imaging (MRI) has proven accuracy in diagnosis of aortic VGI. Some disadvantages compared with CT are higher costs, less availability and more time spending procedure. It Is superior to CT for detection of perivascular inflammatory changes and small amounts of periprosthetic fluid MRI evaluation. Angiography has limited value for VGI diagnosis but is used for complications of VGI as pseudoaneurysms, rupture or graft thrombosis in order to perform endovascular treatment.
Learning Objectives: 1. To understand the importance and role of radiological imaging in the detection, evaluation and therapy planning of vascular graft infections and inflammations. 2. To know more about available imaging methods, imaging techniques and diagnostic algorithms for the evaluation of vascular graft complications. 3 . To learn more about the specific imaging symptoms and differential diagnostic considerations in vascular graft infections and inflammation.
Generally, using precise semantics is fundamental to guarantee the reliability of automated data processing, as well as the consistency of information recorded in databases, and their meaningful statistical exploitation. In medicine, this issue has been addressed for many years using standard terminologies such as MeSH or SNOMED. In medical imaging, and especially in the field of Computer Assisted Detection (CAD), information semantics is defined through DICOM Structured Reports that organise relevant information in reference to terminological resources specified in the DICOM standard (DICOM Part 16). This course explains the limitations of this approach, as well as the added value that could be brought in the future by semantic web technologies and especially ontologies. This added value concerns primarily: (1) more effective decision support, thanks to the more precise definition of involved information, e.g. the quantitative markers extracted from the images, (2) enhanced possibilities regarding integration of data from various institutions, e.g. in the context of large clinical trials, or for data mining in the context of translational research and (3) enhanced possibilities in terms of database querying, thanks to the use of the knowledge embedded in referred ontologies (e.g. properties of "is-a" and "part-of" relationships). The presentation will introduce the basic ideas There have been considerable advances in the imaging tools available for imaging the supra aortic vessels, specifically the carotid arteries. Developments ranging from rotational DSA (allowing for accurate delineation of vascular disease) to improvements in CTA and MRA (particularly Gadolinium enhanced studies) have greatly improved our diagnostic armamentarium. In addition there is increased understanding that atheromatous disease localised to the carotid bifurcation is only part of the problem with cerebrovascular disease. Increased coverage of the arch and intracranial circulation are becoming increasingly important requiring greater spatial coverage. In the future imaging of the wall components may become important. For the moment, however, management remains dependent on the accurate assessment of luminal stenosis. The greatest evidence suggests that contrast-enhanced MRA is the investigation of choice. However, CTA is extremely popular especially with clinicians, particularly as it allows algorithms for including or excluding calcification. There is a lesser evidence base, although the relentless progress of technology such as dual beam techniques makes it difficult to provide timely evidence. There are pros and cons for all methodologies, which will be addressed.
Learning Objectives:
1. To learn about structured reporting in angiographic studies of supra-aortic arteries. 2. To understand the role of post-processing techniques and quantitative analysis of arterial stenosis. 3. To be able to answer specific clinical questions in supra-aortic arterial occlusive diseases.
A-050 16 This presentation focuses on technical backgrounds and requirements for successful acquisiton of angiographic MR and CT data to a minor extent. Apart from conventional techniques also functional imaging approaches for quantifying disease severity will be presented such as MR-flow measurements and time-resolved phase-contrast MRA. The main focus of this presentation is the display of typical disease entities that can be encountered in the thoracic and abdominal aorta. The topics covered will include atherosclerosis, congenital vessel diseases and inflammatory vessel diseases such as Takaysu's diseaes or pseudoaneursyms. Special emphasis will be put on the implications for therapy that can be deducted from the MRA and CTA data. Finally, MRA and CTA will be compared with regard to their diagnostic capabilities and their optimal applications. Learning Objectives:
1. To learn about structured reporting of aneurysmal and obstructive diseases. the most stunning kind of new natural artefact. In 1562, Arcimboldo left Milan for Vienna to become court painter to Maximilian II, who like his father, Ferdinand I, was particularly interested in medicine and the natural sciences and had made his court a major centre of scientific exchange. The renowned physician Pietro Andrea Mattioli from Siena had already worked for the court of Ferdinand I, and both of his sons, Maximilian II and Ferdinand II, and had earned fame for his commentaries on the materia medica of Dioscurides, which were published in many languages. Arcimboldo became engaged in the illustration of various species of plants and animals, which the Emperor housed in his botanical and zoological gardens, most of which were particularly rare or came from the New World. As a result of his interaction with Italian physicians at royal courts in both Vienna and Bohemia his drawings were sent to a renowned natural scientist in Bologna, Ulisse Aldrovandi. He had Arcimboldo's drawings carved into wooden blocks in order to use them as illustrations in his books on quadrupeds and birds. Thus, Arcimboldo's artistic drawings came to be used as scientific illustrations.
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advanced segmentation tools, every tissue may be highlighted or displayed in the background, thus providing additional information and helping to analyse liver anatomy. Virtual models can be ideally used in interventional procedures. Radiologists have to know the size and volume of the lesion to treat, as well as its precise location and relationship with the surrounding tissues. For a percutaneous approach such as radiofrequency ablation, the intervention can be planned and simulated by introducing, on screen, a "virtual" needle in the liver to reduce potential damage to the vessels and other organs when the "real" procedure will take place. These models may help not only interventional radiologists but also surgeons, who need to know the size and precise location of the lesion before operating on a patient. Laparoscopy is a typical example of how imaging can help to guide surgery. With this approach, the problem is that the field of view surgeons have is very limited because of the small size of the laparoscopic camera. A very good solution is to have a combination of both the live laparoscopic view and the preoperative or intraoperative processed images and models. Hepatic surgery requires detailed pre-operative evaluation of anatomical and structural analysis of each patient anatomical variation and topological distribution of anomalous structures and tumours. This includes a three-dimensional assessment of the liver segments based on vascular and biliary tree distribution. Furthermore, endoscopic and robotic surgeries rely on pre-operative planning and modelling of minimally invasive procedures that do not provide visual navigation that open surgery provides. Advanced image rendering and visualisation techniques are now available for surgeons to prepare their surgical interventions by extracting appropriate anatomical features from cross-section imaging techniques such as CT and MRI. These software programs that were initially expensive and only accessible to computer-savvy experts are becoming more widely accessible on personal computers and portable devices. Example of such software distributed for free under Open-Source licensing is the OsiriX software that allows 3D and 4D image manipulation and visualisation on standard personal computers. This software will be used to demonstrate the feasibility and practical usefulness of 3D rendering techniques for planning and modelling of surgical interventions. In addition to illustrating the different segmentation techniques, we will also review the different technical solutions available for accessing and manipulating three-dimensional images in the operating rooms. Different settings are applicable for different types of surgical interventions from endoscopic imaging to virtual reality rendering of images that can be used in the OR to the latest generation of "augmented-reality" techniques allowing real-time fusion of 3D rendered images with patient's physical anatomy. In the past decades x-ray fluoroscopy, ultrasound and endoscopic/laparoscopic imaging systems have been successfully used for intra-operative 2D and rarely 3D anatomical imaging. However, in the functional field only gamma probes were used as "1D" tools within surgical suites. In this presentation, the focus is on a novel imaging approach which allows the surgeon to acquire required functional 3D imaging information within the operating room with minimum interruption of the surgical workflow. We present the first success of this approach in terms of 3D intra-operative imaging using navigated low-energy gamma probes called freehand SPECT. We provide many examples of its successful deployment for different clinical applications based on diverse SPECT tracers. We will then discuss the possible application of similar technologies for intra-operative liver imaging. In this case, higher energy probes are needed as most liver tracers are PET tracers. We present the challenges that need to be overcome to finalise such solutions and discuss a few possible solutions. We will also demonstrate the possibility of then complementing such functional information with real-time 2D or 3D ultrasound imaging, and intra-operative 4D ultrasound-driven models of liver. In conjunction with advance As imaging modalities and computers have reached unmatched levels of precision, radiologists can now acquire very detailed information that they can use in 3D models within minutes after the examination, speeding up treatment planning. The co-called virtual imaging is proving to improve therapy outcome manifold, and its role might only increase in the future. This session will review all the progresses made from the detection and characterisation of liver disease to treatment planning. Knowing the latest advances in detecting and characterising liver disease has thus become mandatory to all radiologists, since an increasing number of molecular tools are currently being developed, and contrast media products targeting specific cells are multiplying. Biomarkers to assess liver functions or tumour activity/response have also become available. These spectacular computer science contributions to medicine will not require that the radiologist or the surgeon will eventually have to become computer experts themselves. The future will rather be based on a multidisciplinary integration which will allow surgery to be increasingly assisted by advanced imaging and computer technology. To develop virtual models of the liver that can serve as clinically useful imaging biomarkers, both the magnitude and the spatial distribution of the different objective measurements must be displayed. To be properly done, it is necessary to validate the relationship of the biomarkers with the reality of the organ and to check its technical and clinical usefulness. This process includes defining tests for the concepts and mechanisms; obtaining standardised and optimised anatomical, functional, and molecular images; analysing the data with computer models; displaying data appropriately; obtaining the appropriate statistic measures; and conducting tests on the principle, efficacy, and effectiveness. The development, standardisation, and optimisation of the different imaging acquisition protocols (for US, CT, MR and PET) and data analyses (physiological and biochemical computing, fusion of modalities, adequate measurements) will change some clinical research and patient care scenarios. In multimodality imaging, the need to combine morphofunctional and biological information can be approached by either acquiring images at different times (asynchronous) and fusing them through digital image manipulation techniques or simultaneously acquiring or computing images (synchronous) and merging them automatically. MR diffusion-weighted, MR and US contrast-enhanced pharmacokinetic imaging, cellular imaging, chemical quantitative imaging and PET tracers will be co-registered and merged to further increase molecular and functional imaging as a relevant medical discipline in hepatology. Multimodality imaging models will be displayed as parametric or multiparametric graphs of the liver reality associated with the DICOM Structured Reporting initiative as the standardisation of structured data and clinical observations in the liver imaging environment. Osmolality, iconicity and viscosity are important physicochemical factors which influence the safety of these agents. Gd-CM are either linear or macrocyclic and available as ionic or non-ionic preparations. The stability of the Gd-CA is an important safety feature of these agents particularly in patients with reduced renal function. The macrocyclic agents have high kinetic stability, whereas the non-ionic linear preparations have low kinetic and thermodynamic stability. Contrast media adverse effects can be divided into acute and delayed reactions. Acute reactions are allergic like and usually develop within one hour of CM administration. Delayed reactions develop after an hour and within a week of CM administration. Acute reduction in renal function referred to as contrast-induced nephropathy (CIN) may develop after contrast administration in patients with pre-existing risk factors. Patients with advanced reduction in renal function may develop the complication of nephrogenic systemic fibrosis (NSF) after administration of low-stability Gd-CM. In this session contrast-induced nephropathy and nephrogenic systemic fibrosis will be discussed. Measures to reduce the incidence these adverse effects will also be presented. Session Objectives: 1. To provide an understanding of the bio-distribution of extra cellular water soluble contrast agents after intravenous administration. 2. To highlight the physico-chemical properties that influence the safety of iodinebased and gadolinium-based contrast agents. 3. To provide a summary of the different adverse effects that might occur with the use of contrast agents including an approach to minimising the risk of these adverse events. The pharmacokinetics of iodinated contrast media are quite simple. They distribute evenly in the extravascular space and are excreted by glomerular filtration. No metabolisation occurs. The pathophysiology of CIN is complex and not well understood. Basically, a misbalance between vasodilatation and vasoconstriction takes place inside the kidney after intra arterial or intravenous CM administration. Furthermore, increased oxygen demand of tubular cells due to increased reabsorbtion of sodium and water is a second mechanism, leading to transient medullar ischaemia. The third mechanism is the increased production of reactive oxygen species (ROS) due to the developed ischaemia. ROS lead to more cellular injury and increased misbalance between vasodilatative and vasoconstrictive mediators. Identifying the patient at risk is the first step in prevention. Knowledge of the patient's medical record and a recent basic kidney function are mandatory. High-risk patients should receive prevention. Two major topics in CIN prevention are the questions whether iso-osmolar CM cause significantly less CIN than low-osmolar CM and whether hydration schedules with NaHCO3 give significantly less CIN than hydration schedules with NaCl 0.9%. The radiologist has three roles in the evaluation of pulmonary infection: 1) detection and description; 2) differential diagnosis; and 3) evaluation of complications. We will approach these through unknown cases and audience participation. Detection and description: In everyday practice, if the chest X-ray is negative, it is assumed, clinically, that the patient has no pneumonia. Unfortunately, this is not the case for immunocompromised patients, such as AIDS patients, bone marrow patients, etc. If there is strong clinical suspicion of pneumonia, a CT is often revealing when the chest X-ray was negative. Descriptors of pneumonia patterns are often confusing, because some descriptors are clinical, some are pathological, and many are based on early X-ray descriptions. These confusing, and often conflicting terminology issues will be addressed. Differential diagnosis: Imaging is not a substitute for microbiology. However, certain patterns are characteristic of certain types of infection and are often helpful in the initial clinical management of patients until cultures are available. The imaging appearance, combined with basic clinical information (white blood count, fever, and duration of illness) can often lead to the correct or narrowed differential diagnosis. Complications: The majority of pneumonias show imaging and clinical improvement within several days and complete resolution within four to six weeks. Deviation from this pattern suggests a complication, such as abscess, empyema, resistant organisms, a structural problem in the lung, or noninfectious diagnosis. These complications and the role of CT will be emphasised. Non-pulmonary infections may involve mediastinum, heart and vessels, chest wall and pleura. This course will focus on mediastinal, pleural and chest wall disease. Acute mediastinitis is a potentially life-threatening condition that is rare but requires prompt diagnosis and treatment. Spontaneous or iatrogenic esophageal rupture is the by far most common cause. Other causes include post-surgical mediastinitis and extension of infection from adjacent spaces (neck, pharynx, pleura or retroperitoneum). This course uses typical cases to discuss important differential diagnoses and the sensitivity and specificity of various radiologic findings for diagnosis of acute mediastinitis. The distinction between a parapneumonic pleural effusion and an empyema based on radiologic findings may be impossible. Features will be discussed hat suggest a "complicated" course that may requires interventional or even surgical treatment. Features that differentiate empyema from lung abscess at CT will be presented. An empyema necessitatis describes a chronic empyema that attempts to decompress through the chest wall. Underlying infectious agents include tuberculosis, actinomyces, staphylococcus and various types of fungi. It has to be differentiated from other mostly neoplastic diseases that tend to cross fascial planes such as lymphoma or Pancoast tumor. Currently, up to 50% of all MRI examinations worldwide are performed using contrast agents, either an extracellular agent or an organ-specific agent. The extracellular MRI contrast agents are chelates that contain the paramagnetic ion gadolinium which strongly affects the relaxation properties of water protons, leading to changes in tissue contrast. Gd-DTPA was the first extracellular agent to be introduced in clinical practice. Since the introduction of Gd-DTPA in 1988, various gadolinium chelates with different chemical properties became available for clinical use. For many years, it was believed that gadolinium-based contrast agents (GBCA) were nearly a 100% safe which led to a liberal and off label use of these agents. The condition that came to be known as nephrogenic systemic fibrosis (NSF) was first identified in 1997, but reported in the peer-reviewed literature in 2000. Since then, NSF has been the subject of a wide-ranging multidisciplinary medical investigation that has proven an indisputable link to renal disease and a compelling association with the increasing use of GBCA in the renally impaired. NSF is a severely disabling complication of renal disease with some features that overlap with those of other conditions. Accurate diagnosis of NSF requires careful clinicopathological correlation. US-and European authorities have confirmed the categorisation of GBCAs in three risk categories in view of NSF. The number of new NSF cases has been dramatically reduced since the introduction of these risk categories. In this talk also an update of NSF reports will be given. Both ultrasound (US) and magnetic resonance imaging (MRI) are recognised as generally safe imaging modalities and thus appropriate for use in the diagnostics of pregnant females and foetuses. However, to some extent both techniques may carry some risks when used during pregnancy which every radiologist must be aware of. Ultrasound may induce adverse effects by either thermal or non-thermal means. The risk of inducing thermal effects is greater in the second and third trimesters, when foetal bone is intercepted by the ultrasound beam. Non-thermal mechanisms include acoustic cavitation, radiation force and acoustic streaming and may be more significant in early gestation, when the relatively loosely tethered embryonic tissues are exposed to an ultrasound beam in a liquid path. The likelihood of producing cavitation-type non-thermal effects is enhanced by the presence in the sound field of gas-encapsulated echo-contrast media. There is no scientific evidence in humans to suggest that the risk to the foetus from a routine MRI imaging examination is significantly increased during pregnancy. The radiofrequency radiation pulses used in MRI imaging although nonionizing, result in energy deposition and can potentially lead to tissue heating. However, the use of single-shot echo train spin-echo sequences is commonplace in foetal imaging and unlikely to result in significant temperature changes. Another potential risk of MRI to the foetus is connected to intravenous application of gadolinium-based contrast agents, which are not recommended during pregnancy. Yet, available evidence suggests it is unlikely that these compounds have an adverse effect on the developing foetus. Patients suffering polytrauma require to be handled with a special approach and attitude of the medical personnel from the on-site primary care through the emergency room, diagnostic and life support measures to the operation theatre and the intensive care unit, because of the complexity and the life-threatening nature of the multiple injuries causing unique clinical consequences. The survival of these patients is greatly dependent on the duration of time elapsing between the injury and the start of definitive therapy. The role of imaging diagnostics is, on the one hand, essential to explore the lesions of key importance and, on the other hand, is limited by time constraint and urgency. A clear understanding of the task and an ability to work under pressure are required of the imaging diagnostic team. Suffering polytrauma in pregnancy is a very uncommon situation, which typically may occur as a result of motor vehicle accident or interpersonal (usually domestic) violence. In these cases, the absolute priority is the preservation of the life of the mother; therefore, the usual concerns regarding the use of ionising radiation and contrast media are of less importance. At the same time, the radiologist has to be aware of the special risks caused by potential injuries and their consequences on the pregnant uterus, placenta and foetus. The altered anatomical situation may also hinder the proper imaging evaluation of the situation. Pregnancy is a specific time during which special care must be taken of a mother and foetus whenever diagnostic imaging is necessary. Ultrasonography (US) and magnetic resonance imaging (MRI) are the safest imaging methods as they do not involve ionising radiation. They are not completely adverse effect free, i.e. both can cause tissue heating, which is potentially harmful to the foetus. Nevertheless, they should be considered as the methods of choice when maternal problems require diagnostic imaging, both preferentially without the use of contrast media. There are however clinical problems which cannot be solved without the use of ionising radiation. Two main clinical settings will be discussed during this session: polytrauma and pulmonary embolism (PE), both requiring computed tomography (CT) in most cases. In cases of polytrauma, saving the mother's life is a priority, and radiation protection of the foetus is a secondary problem. When CT angiography is performed to confirm or rule out PE, the protocols are optimised as far as the radiation dose and contrast medium volume are concerned (i.e. 100 or even 80 kV instead of 140 kV). The use of CT is considered to be quite safe in terms of foetus safety if the examination covers the region of the head, neck and thorax of the mother. However even with the use of CT in the region of the abdomen and pelvis, the threshold of 100 mGy is rarely exceeded. Session Objectives: 1. To become familiar with the risk of using radiological modalities in pregnant woman. 2. To consolidate knowledge of the pros and cons of the different imaging techniques. 3 . To learn about the indications requiring the use of imaging in pregnancy.
What are the real risks of radiation and contrast media to the mother and the foetus? D. Prayer; Vienna/AT (daniela.prayer@meduniwien.ac.at)
Contrast media (CM) that might be considered for use during pregnancy comprise mainly iodinated CM and gadolinium derivates. Iodinated contrast media should be avoided during pregnancy, as the iodine will be stored in the foetal thyroid gland, but a single dosage is unlikely to have a negative effect. Systemic cystic fibrosis has not been described in pregnant women or foetuses; however, no human data proving the safety of gadolinium during pregnancy are available. Gadolinium may be used in maternal conditions unrelated to pregnancy, such as suspect appendicitis or pregnancy-related problems of the placenta, and foetal indications. The latter ones have not been studied yet in larger groups. The greatest risk for radiationassociated damage of developing tissues lies in the embryonic period: at a dosage of > 100 mSV, an embryo might die during the preimplantation phase, and the threshold dose that puts the embryo at risk for malformations or mental retardation lies from 100 mSV onwards. CT may be used during pregnancy for diagnosis of skeletal malformations. Low-dose fast protocols, for instance using a dual source system, are currently under investigation. In conclusion, MRI without contrast administration should be the first choice in case of insufficient ultrasound information. Gadolinium. compounds may provide additional information in selected indications. Low-dose CT may be helpful in the management of skeletal malformations. The extent of patient's injuries depends mostly on the mechanisms of injury and magnitude of energy which is absorbed by human body. Knowledge of Mechanism's details provides valuable information for appropriate management of patients. Although there is still controversy on direct whole-body multi detector computed tomography versus stepwise imaging starting with plain x-rays of the cervical spine and chest, followed by FAST Ultrasound and regional CT, MDCT is an accepted routine method in evaluation of trauma patients. However, there is no existing one and the only standard trauma protocol of examination. A lot of factors must be taken into account by choosing the optimal way of conducting CT examination: patient's state of consciousness, penetrating wounds, stable/unstable haemodynamics, presence of visible instabilities, general medical condition, etc. One of the first "Musts" is to clear the emergency head and cervical spine injuries; MDCT protocols without contrast media are sufficient in such cases. Thoracic injuries like tension pneumothorax, open pneumothorax, flail chest or massive haemothorax must be excluded/confirm almost immediately. Intra-thoracic and intra-abdominal bleeding must be considered in every hypotensive patient; this requires the use of contrast media MDCT protocol. In evaluation of liver and spleen injuries, 5-minute delay in scanning after contrast media administration can be very helpful. Arterial and late venous phases are of extreme importance in appropriate diagnosis of the extent pelvic injuries. One must not forget about the possible fatal consequences of undiagnosed injuries of extremities, resulting in huge loss of blood. A radiology department (RD) providing emergency services remains a core part of hospital emergency care. Modern imaging has become the key diagnostic tool that determines whether a patient will be discharged home or will have to be admitted for further treatment from the accident and emergency department (ED). The most important factor remains the professionalism of the medical and nursing staff in the radiology department. Prompt image interpretation of any imaging can be achieved by the use of Teleradiology. The interpretation of images used outside receiving hospitals in emergency transfers can be very useful. It can help bridge the gap in communication between transferring and receiving institutions and to reduce interpretational discrepancies. The next important issue is the location of the RD. The RD should be built adjacent to or within the ED. It is worthwhile to mention that the benefit-risk ratio of each individual procedure should be considered; the responsibility of this decision lies not only with the radiologist but on the medical community as a whole. Organisation of radiology department in the ED is most important issue. Last but no means the least, we should always remember that one day we might find ourselves having an accident -what kind of service would we like to get? Familiarity with the normal anatomic appearance of the airways is the essential pre-condition to fully exploit the potential of modern technology and especially of volumetric CT. Besides, the importance of correctly phenotyping the chronic obstructive pulmonary disease (COPD), both from a prognostic and therapeutic viewpoint, is now well established. From the trachea to the alveolar spaces, derangement of normal anatomy can give us the explanation of a large number of CT signs and patterns commonly found in the different phenotypes and sub-phenotypes of COPD, such as saber sheath trachea, tracheo-bronchial dynamic instability (tracheobronchomalacia) and bronchial outpouchings diverticula. Moving further along the bronchial tree towards the periphery, a sound knowledge of the borderlands between normal and abnormal anatomy is also of paramount importance to detect early bronchiectasis, bronchial remodelling and the signs of infectious bronchiolitis (centrilobular nodules, tree in bud opacities), more rare inflammatory conditions (Respiratory bronchiolitis, for example), areas of mosaic oligoemia with expiratory air trapping (another possible manifestation of small airways disease), and, finally, the irreversible destruction of the respiratory spaces (particularly, centrilobular emphysema). COPD is a very heterogeneous disease. Different patterns can overlap and the same patient can present with a number of different findings, co-morbidities and complications. A little stroll through the airway anatomy, from the trachea to the alveolar sacs, can help us to understand and remind what COPD is, what we know and the large amount of things that remain to be discovered and clarified. Learning Objectives: 1. To understand the imaging characteristics of large airways, small airways, and alveoli. 2. To understand the imaging characteristics of the pathological entities contributing to the development of the clinical COPD. 3. To be able to describe the potential continuum between these components, as clinically manifested in the spectrum of chronic obstructive pulmonary disease (COPD).
to move to 3D and 4D imaging, to deal with contrast-enhanced techniques and to consider ultrafast imaging, high-resolution imaging technology and elastographic imaging, The integration of ultrasound in a multimodality approach, with modalities such as fusion, will be exposed mainly for interventional applications. Questions regarding safety in these new developments will finally be discussed. The choice of the US equipment is always a dilemma. The precise definition of the true needs is a difficult task, including ergonomics, cleaning, B-mode, basic functions, Doppler imaging, overall imaging quality, networking, advanced capabilities and general and specific transducers. None of the existing system provides an optimal image quality for all applications, so it is necessary to prioritise the most important ones. The ideal situation is to evaluate the candidate systems in your routine practice and eventually compare the systems with each other with side by side evaluations. Of course, the price of the acquisition is playing an important role, but it is necessary to take into account the additional cost of the set of transducers, the advanced imaging options and the maintenance (that can include the transducers and the delay of intervention). It is necessary to distinguish the truly portable systems and the transportable systems (for dedicated applications, general-purpose applications or high-end imaging) from the larger and heavier systems. The mammographic image of the breast reflects the tissue composition of the breast, i.e. epithelial, connective and adipose tissue. Different methods are used to assess the mammographic density: Wolfe (1976) described four densities (N1, P1, P2, DY) and the Breast Imaging Reporting and Data System described four categories. As risk calculation is related with the percentage of mammographic density (PMD) rather than with "density", more adequate measurements are necessary. Attempts on computer-assisted methods based on interactive thresholding were developed. Due to the introduction of digital mammography, information of the digital data can be used for the quantification of the density of the breast. Several studies concluded that PMD is associated with a higher risk of breast cancer: 6 to 8 times higher than for women with primarily adipose breasts. This association was stronger in studies in the general population than in symptomatic patients and stronger for quantitative measurements than for qualitative measurements. Although high density is associated with an increased risk of breast cancer, it also makes the detection of breast cancer with mammography more difficult. The DIMST trial already showed a higher accuracy of digital mammography for dense breasts. Additional breast ultrasound in women with mammographically dense breasts permits detection of small, occult breast cancers, but is also correlated with a significantly higher biopsy rate. Recent reports on tomosynthesis showed improved lesion conspicuity of masses and stellate lesions, which are often masked in dense breasts. Implementation of newer techniques such as dual energy mammography has just started. Breast MRI represents the most sensitive modality in the identification of breast cancer. Even if its use is increasing, the main concern on a wider use of this examination in clinical practice still remains its sub-optimal specificity and falsepositive rate. Several systematic reviews evaluating the diagnostic performance of contrast-enhanced MR imaging in patients with breast lesions showed a specificity ranging between 67% and 72%. The reported false-positive rate of the examination ranged, in Literature, between 3.5% and 7%. However, any parameter of diagnostic performance should consider the clinical context. Radiologists need to have a basic knowledge of the radiological anatomy of the organs imaged within the thorax, among which the pulmonary circulation plays an important role as it can provide morphologic but also functional information useful for the management of numerous thoracic disorders. This new trends in evaluating the pulmonary circulation is driven by the technological developments of multidetector-row CT (MDCT) technology which offers the possibility not only to search for morphological abnormalities at the level of central and peripheral pulmonary vessels with single-source MDCT but also to integrate the evaluation of the capillary level by means of dual-energy CT. Detection of morphological abnormalities at the level of central and peripheral pulmonary vessels in congenital and acquired chest disorders requires the knowledge of the prevailing arterial and venous patterns whose analysis can now benefit from the development of computer-aided-diagnosis tools. The analysis of the most distal portion of the pulmonary circulation, sometime referred to as "pulmonary perfusion", is now available in routine clinical practice and requires, for proper interpretation of images, a basic knowledge of the anatomical relationships between the pulmonary and systemic circulations of the chest. CT can also participate in the noninvasive approach of pulmonary hypertension by means of the analysis of the degree of distensibility of central pulmonary arteries. All these pieces of information are accessible to radiologists providing the selection of the adequate scanning protocol. The purpose of this lecture is to review these anatomical and physiological perspectives offered by MDCT in 2012. While the three controversies covered in this session are apparently disparate they do have a clear common theme relating to the risks (in particular the false-positive test) and benefits of breast imaging and our perceptions of these. Perceptions of risk (and benefits) are complex and are determined by an interaction of our personal past experience, information provided and our current point of observation. A woman with a newly diagnosed breast cancer will have a very different perspective to a public health doctor, or for that matter a politician, commissioning a service for their population in financially difficult times. This is compounded by the general population's woefully inadequate grasp of statistics used to provide evidence which is not helped by our own presentation of the facts which some claim is based entirely on vested interest. In our drive to find every cancer (or perhaps our anxiety not to miss any) regardless of its biological potential it is easy to forget the need for specificity. The false-positive result (abnormal test in a normal patient) can have both short-and long-term physical and psycho-social consequences for the individual and definitely has significant financial costs to the health economy. The three talks start with populations, move seamlessly to individuals and finally provide help and advice on reducing the number of false-positive results. Conventional MR imaging (T2-weighted and contrast-enhanced T1-weighted sequences) is the standard modality for the initial visualisation and characterisation of CNS lesions, with superior sensitivity compared with alternative imaging modalities. However, advanced MR techniques such as perfusion-weighted imaging (PWI), diffusion-weighted imaging (DWI) and MR spectroscopy (MRS) are increasingly incorporated into imaging protocols to provide not only better specificity, but also physiological information that complements the morphological detail of conventional MR studies. DWI provides useful information for providing an accurate differential diagnosis between pyogenic abscesses and necrotic tumoural lesions (both metastasis and high-grade gliomas), although it cannot distinguish between necrotic CNS tumours from inflammatory pseudotumoural cystic lesions. Data on the literature regarding the diagnostic value of MRS for differentiating pseudotumoural lesions from brain tumours have yielded conflicting results. Some authors have shown that there are not enough spectral differences that allow a precise diagnosis in individual cases, while others demonstrated that this discrimination can be achieved based on metabolite ratios obtained at short TE and long TE. PWI can also be helpful in differentiating pseudotumoural inflammatory lesions and high-grade gliomas, as only the latter show a significantly increase in cerebral blood volume. Accurate characterisation of brain lesions with conventional and advanced MRI techniques is feasible in clinical practice and might be helpful in distinguishing between tumoural from pseudotumoural lesions. This will have a clear impact on patient management as unnecessary aggressive diagnostic or therapeutic procedures could be avoided. Learning Objectives: 1. To become familiar with the most common causes of multiple brain lesions caused by primary and secondary brain tumours. 2. To learn about the most common causes of multiple non-neoplastic brain lesions that mimic tumours. 3. To learn the characteristic neuroimaging findings that may be useful in establishing differential diagnoses. 4. To be aware of the importance of advanced neuroimaging techniques for evaluation of multiple brain lesions. Oncologic imaging examinations are often complex, and specialised knowledge is required to interpret them in a clinically relevant manner. The radiologist needs to be aware of the details of the staging system and pattern of spread for a given tumour; the strengths and limitations of available imaging modalities in specific oncologic applications, especially in assessing tumour response to different conventional and newer therapies; and various pitfalls in the overall approach to interpretation and reporting of oncologic imaging examinations. Because a radiologic study is only a "snapshot" taken during a brief moment of a patient's medical timeline, meaningful interpretation (the radiologist's "added value") requires integration of current The anatomy of the brain is often perceived as being complicated. Especially the cortex is seen as an irregular arrangement of variable structures, which are difficult to differentiate and to identify. We will review the overall subdivision of the brain into lobes and describe their boundaries and their major gyri and sulci. We will then describe the location of areas specific to language in terms of their cortical anatomy as well as their cytoarchitectonic structure: 1. areas of speech production, most importantly Broca's area, which is centred on the pars opercularis of the 3rd frontal convolution; we will discuss the identification of this area and its variability; 2. areas of language comprehension, with special attention to Wernicke's area, its definition and variability; and 3. areas related to hearing, mainly the primary auditory cortex (A1), centred on the postero-medial part of Heschl's gyrus (HG), we will present simple landmarks in each of the three planes: a) axial: adhaesio interthalamica, b) sagittal: omega/heart shape of HG and c) coronal: omega shape of HG. We will discuss the most important tracts connecting these areas, mainly the uncinate and the arcuate fascicles. At the end of this lecture, you will know the general subdivision of the cortex and will have a basic understanding of the functional anatomy of language. Learning Objectives:
1. To learn about the general brain cortical anatomy. 2. To be able to identify the most important cortical anatomy involved in the language system and its imaging representation. 3. To be able to recognise the most import white matter tracts involved in the language system and its imaging representation. 4. To get a glimpse into the relevant anatomical and clinical insights coming from MR microscopy. MRI is an imaging technique that is particularly sensitive to haemorrhages in the brain. Apart from large haematomas and recent subarachnoid haemorrhages, that are also visible on CT, using MRI minute haemorrhages (microbleeds) can be detected within the brain and subtle remote haemorrhages in the subarachnoid space (superficial siderosis) can be detected. In addition, MRI allows for assessment of the age of cerebral haemorrhages, based on signal characteristics. The location of cerebral haemorrhages allows for an estimation of the underlying pathology. For example, haematomas with a lobar distribution are suggestive of cerebral amyloid angiopathy, those located in the basal ganglia in a patient with hypertension are suggestive of hypertensive microangiopathy, and blood in the subarachnoid space is suggestive of a ruptured aneurysm. A comprehensive MRI protocol (comprising T2*-weighted images, post-gadolinium images, and MRA techniques) also allows Complexity and heterogeneity of the lymphomas and especially the DLBCL (Diffuse large B-cell lymphoma) have been demonstrated over the past 10 years, first by the most recent WHO classification that includes no less than 50 different subentities, and second by the gene expression profiling analyses leading to a molecular classification in the most common form of adult non Hodgkin's lymphoma, the DLBCL entity. The prognosis has been demonstrated to be variable according to the morphological, immunophenotypic and molecular classification. The therapeutic approach is different according to the classification. So the importance of the material obtained by different techniques is crucial: excisional lymph node biopsy or core needle biopsy (which is increasingly replacing chirurgical techniques) must be properly used in order to obtain a good morphology and frozen material dedicated to molecular analysis. Learning Objectives:
1. To learn about the evolution of the successive classifications of lymphomas according to new molecular prognostic markers. 2. To understand the limitations of image-guided biopsies in some peculiar entities. 3. To appreciate to what extent molecular analyses can predict outcome.
What the haematologist needs to know P. Brice; Paris/FR (pauline.brice@sls.aphp.fr)
Lymphomas are a heterogenous group of malignant disease ranging from very aggressive clinical course like Burkitt lymphoma to indolent diseases which do not need to be treated. Histological diagnosis is crucial to define treatment and to target it (Rituximab is only given to B-cell lymphoma expressing CD20 antigen). Unless refractory or very early relapses, patients with recurrent disease should have a radiological non-invasive biopsy to detect histological modifications: a) histological transformation from low-to high-grade B-cell lymphoma, b) Hodgkin lymphoma recurring with non-Hodgkin lymphomas (NHL), c) loss of CD20 antigen expression in relapsing B-cell lymphoma and d) second solid tumour. Before treatment radiological evaluation must be planned with CT or PET-CT and criteria of response must be defined if PET-Ct is used as an interim response and not only at the end of treatment (Juweid JCO 2007). Physicians must define their therapeutic goals: a) complete remission leading to long-term cure, b) prolongation of survival and c) quality of life in elderly or incurable disease. Response with radiological evaluation may guide therapeutic options. 1. good responders: limit the treatment and avoid long-term toxicities (mostly for Hodgkin lymphoma), 2. slow responders: give the optimal amount of treatment and radiation therapy in some localised cases and 3. bad responders: increase the treatment and proceed to high-dose therapy. In conclusion, the management of lymphoma patients needs non-invasive biopsies and multiple (PET)-CT evaluation for optimal care. imaging findings with results from various prior radiologic studies and pertinent clinical information. The frequently numerous findings visible on an imaging study need to be distilled into a focused, clinically relevant report; otherwise, the radiologist functions simply as a "film reader" (rather than as a true consultant), and the resultant radiology report may be technically accurate but clinically unhelpful---or even misleading. Better reports can be produced by using standardised report templates, integrated imaging summaries and consistent lexicons. The most important parameter for the developing of the subgroups was overall survival based on disease stage. The major changes involve the T and M categories, resulting in subgroups that would more accurately be associated with prognosis of a patient with defined descriptors. In this course, the new TNM staging classification will be demonstrated, and case examples will be used to interactively enhance the learning experience of attendees. Also, the role of imaging methods in evaluating standard and innovative therapy regimen will be discussed, and typical findings and pitfalls will be presented. Over the past decade, the treatment of colorectal cancer has become increasingly individualised with therapeutic options ranging from neoadjuvant therapy with FOLFOX and radiation therapy, to transanal mucosal resection, to robotic surgery with low anterior resection and surgical extirpation including APR. This lecture reviews the role of CT, MR, transrectal ultrasound and PET-CT in guiding the increasingly sophisticated process of staging and managing patients with colorectal cancer. Guidelines concerning the most efficacious use of the DWI MR imaging and perfusion and diffusion CT and MR with a view towards optimising patient management are also presented. Over the past years, the number of coronary CTA studies performed has been steadily growing. To perform a coronary CTA study using appropriate protocols in a correct manner is mandatory in order to obtain a diagnostic image quality. Beyond that, there is a lot more to a coronary CTA study than acquiring the data. Correct interpretation required knowledge of the advantages and disadvantages of various post-processing methods in order to avoid false-positive and false-negative findings regarding coronary artery disease. In addition, a standardised approach for interpreting the CT data and for reporting the imaging findings to the referring clinician is reasonable. The presentation illustrates a practical approach to the post-processing and interpretation of data acquired during a coronary CTA study, including the reporting of the relevant imaging findings to the referring clinician. Learning Objectives:
1. To know the scope of information needed by a referring physician from a coronary CTA examination. 2. To be familiar with possible sources of mistakes in the interpretation of cardiac CT. 3. To learn how to write a report on coronary CTA.
C. Cardiac MRI in ischaemic heart disease J. Bogaert; Leuven/BE (Jan.Bogaert@uz.kuleuven.ac.be)
The reporting part is probably the most important step in a radiological exam because the radiologist's expertise comes into play. Radiologists in training learn the pecularities of reporting during their training. For novel domains in medical imaging, such cardiac MRI (eg in patients of with ischemic heart disease), experience is often limited. Moreover, reporting of these exams differs from 'traditional' radiological exams, since it necessitates (extensive) image analysis (eg, contouring of endo-and epicardial myocardial contours), interpretation of dynamic sequences (myocardial contractility, myocardial perfusion), and multisequence imaging (e.g. imaging of acute myocardial infarct patients). All the information needs to be interpreted together, to achieve an idea about the status of the heart. In some patients these studies are (or need to be) performed during stress conditions to evaluate the impact on stress on myocardial perfusion and function. To streamline the interpretation of MRI study results with other information obtained with other modalities (eg echocardiography, nuclear medicine), standardized reporting is required, otherwise the referring clinician risks to get lost in the abundance of information. In this lecture, the focus is how to practically approach and report a cardiac MRI exam in ischemic heart disease patients starting from several clinical cases. Imaging plays now a major role at every step in the management of lymphomas: initial staging, response assessment, follow-up and investigation of suspected relapse. Beyond conventional cross-sectional imaging, molecular imaging has changed dramatically the management of routinely FDG-avid lymphomas in the past decade. Recommendations for the use of PET-CT have been published in 2007 for standardisation of the technique, optimal timing and a better understanding of false positives and negatives, providing uniform endpoints for clinical trials. Depending on the type and course of lymphoma, and on protocol inclusion, CT, PET of both will be done at any step in the course of the disease. This exhaustive workup better assesses therapeutic response but raises new questions. Occult lesions or new events are early detected, prompting image-guided biopsy for histological confirmation. The aim of this presentation is to consolidate knowledge of the imaging workup in lymphoma and illustrate how imaging strongly influences patients' management. Proper imaging strategies are of paramount importance for selecting patients for interventional radiological treatment in the setting of abdominal trauma. Contrastenhanced CT should be performed whenever possible in the severely injured trauma patient as it accurately grades injury to parenchymal abdominal organs, identifies active bleeding or the presence of other vascular injuries that require treatment and also assesses the presence of concomitant injuries such as pelvic fracture with bleeding. Angiography and embolisation is performed to treat ongoing bleeding or to prevent delayed bleeding from false aneurysms or traumatically dissected vessels, thus rendering non-operative management safer. Depending on the location and the nature of the vascular abnormalities coils and /or gelfoam are the most commonly used embolisation materials. The most frequently involved organs / vessels amenable to embolisation are the spleen, the liver, the internal iliac arteries due to pelvic fracture and the kidneys. Rarely a covered stent can be used to treat the vascular injury while maintaining patency of the treated vessel.
Results are good with haemostasis being obtained in more than 90% of cases and non-operative management succeeding in more than 80-90% of cases treated with embolisation. Embolisation may also be used as an adjunct to operative treatment.
Complications include contrast-enhanced nefropathy, splenic infarct, renal infarct, liver abscess and buttock claudication (after bilateral internal iliac artery occlusion). A proper trauma management infrastructure with rapid access to CT-scanning and a 24/7 interventional radiology service should be present in all centres dealing with severely injured trauma patients. Currently is the largest medical society in Italy with over nine thousand members and provides the main framework for presenting and exchanging the scientific experience of different hospital-and university-based working group. At present SIRM is the national member of the European Society of Radiology with the largest number of both institutional and individual affiliated radiologist. Our Society manages its activity through two organising structures: study sections and regional groups that work together, representing the real scientific and professional core of the society. Each section has one president and four councillors and its aim is to promote the radiological advancement and also to organise the Continual Medical Education (ECM) for our members. Regional groups are based on the geographical division of our Country. The Society has two main seats: the first one, the Headquarters in Milan, and the other one House of Radiologic Area in Rome where E-learning, research and development take place. This venue hosts also inter-society meetings
Acute arterial and venous ischaemia-presentation, management and outcome L. Boyer; Clermont-Ferrand/FR (lboyer@chu-clermontferrand.fr)
Acute mesenteric ischemia (AMI) is a life threatening diagnosis and a therapeutic emergency with high mortality rates (50-95%). Its diagnosis and management are challenging issues. It is due to arterial thrombosis in 20-30% of cases, emboli in 40-50%, and venous obstruction in 5-20%; non occlusive mesenteric ischemia accounts for 20-30% of cases. Clinical presentation includes severe abdominal pain which contrasts with poor clinical findings, and depends also on the cause. Quick AngioMDCT is the cornerstone of diagnosis, with a reliability of 95.6%: it detects vascular obstruction as well as signs of intestinal ischemia (reduced wall enhancement) or necrosis (pneumatosis intestinalis and/or portal gas) and peritoneal irritation signs. There are no randomized or controlled trials of AMI therapy. Therapeutic options include surgery, with an important morbi-mortality, but also increasingly endovascular procedures such as: -administration of vasodilatators to treat NOMI, -local thromboaspiration and thrombolysis for arterial emboli, -balloon angioplasty ± stenting ± thrombolysis to treat arterial thrombosis and mesenteric complications of aortic dissection, -in situ thrombolysis ± mechanical disobliteration of mesenteric venous thrombosis. Percutaneous endovascular techniques may be valuable alternatives to surgery in selected patients, and can be helpful in limiting bowel resection. Endoscopy is the primary diagnostic and treatment modality in non-variceal upper gastrointestinal haemorrhage (NVUGH). When endoscopy fails to control bleeding embolisation or surgery are the haemostatic options. Placing endoscopic clips at the site of endoscopically uncontrollable haemorrhage facilitates successful embolisation. CT angiography sensitivity in acute bleeding is 90% but this depends on careful case selection. CT is particularly useful when endoscopy fails to identify the site haemorrhage or there is a history of aortic reconstruction or pancreaticobiliary procedure or disease (ACR Appropriateness Criteria 2010). In transpapillary (bile or pancreatic duct) haemorrhage embolisation is the first line intervention. In known site life-threatening post-surgical haemorrhage (e.g. surgical drain bleeding) proceeding directly to angiography expedites haemostasis. The decision making process when endoscopy fails to control NVUGH depends on the available evidence but also on the 24/7 availability of services and/or the speed of response. A recent systematic analysis of embolisation in NVGUH reported pooled mean technical and clinical success rates in primary UGI haemorrhage only, trans-papillary haemorrhage only, and mixed studies were 84% and 67%, 93% and 89%, and 93% and 64%, respectively. The evidence comparing surgery and embolisation is limited to six single-centre cohort studies. Despite decision making bias, with more elderly patients, coagulopathy and other adverse co-morbidities in the embolisation group: mortality, clinical success and re-bleeding were equivalent. Large-volume resuscitation, particularly when clotting derangement or multi-organ failure results, is associated with increased mortality. This reinforces the need for rapid control haemorrhage and has implications for service organisation and delivery. Acute Mesenteric ischaemia (AMI) is a life-threatening vascular emergency due to acute decrease of small bowel blood supply that may present occlusive (arterial/venous) or non-occlusive ethiopathogenesis. It has been estimated that the majority of cases (65%) are caused by arterial embolism or thrombosis, 25% by non-occlusive aetiology and the remaining 10% from venous thrombotic aetiology. Despite the advances in imaging techniques and, consequently, in therapeutic approaches, the overall mortality rate of this condition still ranges from 60 to 100%. An early diagnosis is important for a correct therapeutic approach able to reduce the mortality. Till now the best imaging method in diagnosis of mesenteric ischaemia/ infarction is enhanced CT even if some authors are considering a role for MRI in the evaluation of these disorders. Moreover, in available literature, there exists no systematic evaluation of the radiological findings related to different ethiopathogenesis of AMI and their chronological evolution. The aim of our studies was to identify the macroscopic, microscopic and μ-MRI findings of each aetiology of AMI and to document their chronological evolution in rat models. The progression of the ischaemic disease, observed in these experimental models, appears in line with the human pathology and so it could represent a useful tool to evaluate the role of MRI as an alternative technique in the diagnosis of intestinal infarction. Learning Objectives:
1. To understand the chronological evolution of findings in mesenteric ischemia and infarction studying the damage either with a 7T-micro MR or by macromicroscopic observation. 2. To be able to attribute each finding to one or more of the three etiological types of mesenteric ischaemia (arterial, venous, ischaemia/reperfusion). 3. To appreciate the efficacy of MRI, as an alternative tool in the early detection of this pathology.
Interlude: Reasons to come to the 45th SIRM National Congress C. Faletti; Turin/IT (carlo.faletti@cto.to.it) From 1st to 5th June, 2012, Torino, will be the venue for the 45th National Congress of the "Società italiana di Radiologia Medica" (Italian Society for Medical Radiology). The society will unite 12,000 Radiologists. In fact, all those involved in the field of diagnostic imaging consider the radiologist a protagonist in the numerous pathological fields. This congress will offer not only the possibility of aggregation, but also the chance to compare oneself with others and the state-of-the-art-technology at our disposal nowadays both as to research and technological aspects, all of which enriched with numerous dedicated workshops, symposiums and monothematic courses. Although this year's congress will have a central theme of vascular alterations, no pathological field will be left out. The venue is one of International prestige, i.e. the "Centro Congressi Lingotto", a multi-complex of easy reach with all facilities which include an underground railway, surface buses and taxis as well as the most comfortable and modern of hotels. It is easily reached by road through a network of motorways and by airport. Torino is a city rich in history and culture, with monuments that include the Mole Antonelliana (housing the Cinema Museum), The Royal Palace and The Palace in Venaria, the famous Egyptian Museum and the automobile museum. Torino is at the foot of the mountains (Piedmont), situated between two hills, rich in enogastronomic culture, surrounded by some of the most famous wine countries in Italy, adding a "taste" for tourism to that of scientific culture.
MR contrast agents for liver imaging A. Giovagnoni; Ancona/IT (a.giovagnoni@univpm.it)
Hepatobiliary-specific Gd-based contrast agents (Gadobenate dimeglumine : Gd-BOPTA and Gadoxetic acid: Gd-EOB-DTPA) are one of several classes of contrast agents presently available for MRI of the liver. These agents are taken up by functioning hepatocytes and excreted in the bile, and their paramagnetic properties cause a selected enhancement of the liver and biliary tree. This results in an increased contrast-to-noise ratio for nonhepatocellular lesions compared with that of the background liver, thereby increasing lesion conspicuity on delayed T1weighted images. However, due to their pharmaco-dynamic properties, Gd-BOPTA and Gd-EOB-DTPA are sometimes described as "combination agents" because of their dual capability for imaging in the dynamic and delayed hepatocyte-specific phases of enhancement. These two agents potentially allow comprehensive noninvasive imaging assessment of the liver parenchyma, intrahepatic lesions, hepatic vessels, and the biliary tree in one examination. These MR contrast agents vary as Italian Association of Neuroradiology (AINR), Italian Association of Oncologic Radiotherapy (AIRO) and National Radiological Union (SNR). The great scientific activity of SIRM is proved by the Official Scientific Journal, "La Radiologia Medica", with an impact factor of 1.698. In the past decade Multislice CT Coronary Angiography (MSCT-CA) achieved excellent results in the diagnosis of coronary artery disease (CAD) with improving spatial and temporal resolution. High sensitivity and negative predictive value were elicited for the detection/exclusion of coronary artery stenoses in patients with suspected CAD. MSCT-CA may be employed in the emergency setting to perform the work-up of acute chest pain, including a one-stop-shop imaging of main thoracic killers: coronary syndrome without ST elevation at the ECG, pulmonary embolism and aorta dissection. Another relevant application is coronary plaque imaging which provides findings and clues of plaque vulnerability (i.e. composition and wall remodeling). MSCT-CA may address a paradigm shift from demonstration of ischaemia to the assessment of pure atherosclerosis. Recent studies pointed out the excellent prognostic value of the technique. Other potential applications of MSCT-CA are the assessment of revascularised patients (with stenting or coronary artery by-pass grafts), and the depiction of coronary anomalies. Radiation dose concerns triggered technical innovations such as higher spatial resolution with larger and novel detectors, faster data acquisition, modulation dose systems, and prospectively ECG-triggered high-pitch acquisition. New scanners may provide excellent image quality at a dose below 1 mSv, to levels lower than conventional angiography. MSCT-CA may also provide useful information of cardiac functional assessment (left and right ventricular function, evaluation of valves). In this context, the fusion imaging of hybrid scanners (CT and SPECT/PET) could represent a promising technological solution able to assess morphology and function at the same time. The winter Olympic Games held in Torino in 2006 were testimonial a very hard challenge which took on the form of the following motto: Passion Lives Here. This was also held dear in the organisation and set up of the diagnostic imaging, which was developed in three separate sites, at varying altitudes, reaching 2,000 metres high up in the Alps, in Sestriere. The most complete of diagnostic equipment was to be found there, including Ultrasound Diagnostic Equipment, C.T. Scans and Magnetic Resonance. Moreover, for the first time, all of these diagnostic techniques were pooled into a network that allowed for real-time visualisation/consultation between one site and another. The results gave credit to the organisational efforts, allowing for examinations and diagnosis to be made in the shortest possible time lapses. The pathologies diagnosed in the field of musculoskeletal imaging enabled the most suitable of surgical and/or therapeutic solutions to be chosen, as well as the possibility to programme the athletes' participation in the forthcoming competitions. Ultrasound examinations were carried out "on the field" for real-time interventions and the CT scans were done in dedicated containers at an altitude of 2,000 metres. It was for the first time that high field resolution Magnetic Resonance, both total body and those dedicated to new concepts, were executed at such an altitude, but, above all, the technology available was applied to this specific sports sector. Thanks to the excellent results obtained, satisfaction rates ran high for the athlete and medical teams alike. The evolution of ultrasound technology has allowed the wide application of colourflow duplex scanning (CFDS) in the imaging of abdominal arteries. Duplex criteria have been set to identify renal artery stenoses (RAS) that are haemodynamically significant. Threshold values for PSV consistent with a 60% or greater diameter reduction of the renal artery obtained in most studies are 180 to 190 cm/sec. In some cases it is useful to use the ratio between the velocity of the renal artery and the aorta. Threshold values of this ratio diagnostic of a 60% or greater RAS vary in different studies from 3 to 3.5. Renal hilar duplex scanning from a flank or translumbar approach has been suggested as a simplified alternative for screening, especially in those patients in whom conventional renal duplex examination is difficult to perform. Contrast-enhanced US (CEUS) seems promising in diminishing the rate of technically inadequate renal artery examinations. Regarding the mesenteric arteries, with adequate patient preparation, the CA and SMA can be identified and in mode of administration and dose, mechanism of cellular up-take, degree of excretion through the biliary pathway, and imaging characteristics. In the liver, hepatobiliary-specific agents can be used to improve lesion detection, to characterise lesions as hepatocellular or nonhepatocellular, and to specifically characterise some hepatocellular lesions, notably focal nodular hyperplasia. Biliary excretion of these agents can be used to evaluate the anatomic structure and function of the biliary tree. Hepatobiliary-specific contrast agents may have wider applications, such as grading of cirrhosis and quantification of liver function in patients with liver trasplantation. Clinical applications, advantages, pitfalls, and problems with different contrast agents will be described and discussed. Acute abdomen refers to any clinical condition characterised by severe abdominal pain that develops over a period of hours. This is a great challenge to the radiologist because differential diagnoses of acute abdomen include a wide spectrum of disorders, ranging from life-threatening diseases to benign self-limiting conditions. Rapid, accurate diagnosis is essential if morbidity and mortality have to be significantly decreased. The diagnostic work-up of patients admitted with acute abdomen is based on various imaging modalities such as abdominal plain film, ultrasound, CT and MRI: the topographic classification of acute abdominal pain (pain in one of the four abdominal quadrants, diffuse abdominal pain, flank or epigastric pain) facilitates the choice of the imaging technique and allows to narrow the range of possible diagnoses. A practical approach to acute abdomen is to confirm or to exclude the most common disease and to look for general signs of pathology such as inflamed fat, bowel wall thickening, ileus, free fluid, free air, etc: the role of US, CT and MRI in achieving these goals will be discussed. The common and more unusual causes of acute abdomen, with reference to the site of pain, will be also discussed in an interactive fashion. Findings useful for differential diagnoses will be presented in order to obtain the correct diagnosis beginning from the imaging sign. 58,240 Americans were diagnosed with renal cancer in 2010 and an estimated 13,040 will die from renal carcinoma in 2010. Deaths worldwide are over 100,000 annually making renal cancer one of the leading causes of cancer death. With the advent of cross sectional imaging modalities like CT and ultrasound over two thirds of cases of renal cell carcinoma are detected as incidental findings. Classic presentations like hematuria only occur in up to 40% of cases and so many of these patients are diagnosed with larger tumors. In this presentation we will focus on the role of state of the art CT for the detection, staging and management of the patient with known or suspected renal carcinoma. We will look at some of the challenges in diagnosis focusing on optimising scan protocols as well as the role of 3D mapping in staging and pre-operative planning. The role of imaging in patient management be it partial nephrectomy, ablation therapy or nephrectomy will be discussed. Potential pitfalls and limitations of study technique will be addressed. Finally we will also focus on some of the newer discoveries showing that enhancement patterns on CT can not only differentiate in most cases between papillary and clear cell RCC but enhancement patterns can also predict genetic errors which may impact on therapy for drug selection. Trends for future innovation will also be discussed. Global epidemiological statistics demonstrate that mortality from ovarian cancer has not changed in half a century. Advances in molecular medicine and biomedical imaging are poised to make a difference. Early ovarian cancer is treated with comprehensive staging laparotomy. For advanced disease deemed nonresectable, neoadjuvant chemotherapy followed by surgical debulking is recommended. In the management of ovarian cancer, cross-sectional imaging is now essential in (1) tumour detection and characterisation; (2) treatment selection and planning (identifying difficult-to-reach tumour deposits or inoperable disease for which neoadjuvant chemotherapy is indicated); (3) monitoring treatment response; (4) detecting recurrent disease, and, depending on tumour size and location, choosing between secondary cytoreduction and chemotherapy. Ultrasound is the primary modality for detecting and characterising adnexal masses. MRI is useful for characterising sonographically indeterminate adnexal masses, and contrast-enhanced CT is the modality of choice for preoperative staging. FDG PET-CT is valuable for detecting recurrent disease, particularly in the mesentery, bowel serosa and normal-sized lymph nodes. We are witnessing a paradigm shift in cancer care, as imaging permeates all facets of cancer diagnosis, treatment and follow-up. The opportunities for biomedical imaging have never been greater. Our role is to understand the key clinical questions and, using imaging, serve as essential physician consultants. Abnormalities of the abdominal aorta and the visceral vessels may represent a diagnostic challenge in patients with acute or chronic clinical symptoms. In addition to the primary diagnosis in b-scan, colour-coded duplex ultrasound, contrast-enhanced ultrasound (CEUS) with low mechanical index (low MI) is a new promising method in the diagnosis and follow-up of pathological aortic lesions. These pathology findings were compared with b-scan, colour-coded duplex ultrasound, CEUS and multislice computed tomography angiography (MS-CTA). Pathologies of the abdominal aorta will be often treated with an endovascular aneurysm repair (EVAR). Endoleaks following endovascular aneurysm repair (EVAR) are common and present a diagnostic challenge in the follow-up after EVAR. CEUS with SonoVue® allows a more rapid and noninvasive diagnosis in the follow-up after EVAR. This course will describe the aetiology, classification and importance of pathologies of the abdominal aorta and the different types of endoleaks allowing the participants to appreciate the usefulness of CEUS in this clinical situation. Despite advances in cross-sectional techniques imaging still has difficulties on assessing pancreatic tumours and in particular adenocarcinoma. The main problems remain the early detection of small tumours and the clearcut definition of patients that are amenable to curative surgery. After reviewing current concepts in the classification of pancreatic tumours including the role of molecular biomarkers, the lecture will address the strategies that may be used to maximise tumour conspicuity in a multi modality perspective that also encompasses uprising techniques such as dual energy CT, perfusion CT/MR and diffusion-weighted MRI. The concept of borderline resectable pancreatic cancer will be explained as well the key points for image interpretation in the setting of clinical decisions for patient management. Also, the current role of adjuvant or neoadjuvant therapy will be shortly addressed especially concerning its imaging implications, as well as the new concepts on pancreatic adenocarcinoma oncogenesis and possible imaging strategies that may be used for earlier detection. Optical imaging is unequivocally the most versatile and widespread visualisation modality in the life sciences. Yet it is significantly limited by photon scattering, which complicates imaging beyond a few hundred microns. Recently, however, there has been an emergence of powerful new optical imaging methods that offer high-resolution imaging beyond the penetration limits of microscopic procedures. Of particular importance is the development of multi-spectral opto-acoustic tomography (MSOT) methods that bring unprecedented imaging performance in visualising anatomical, physiological and molecular imaging biomarkers through several millimetres to centimetres of tissue. Its attractive features include the ability to offer 10-100 micron resolution and real-time imaging. In parallel, we have achieved the first in-human clinical translation of targeted fluorescent probes which opens the way for advanced surgical and endoscopy procedures and personalised theranostics and screening. MSOT can enable exceptional insights to cellular and sub-cellular processes through entire small animals, embryos, fish and insects. This talk describes current progress with instruments, methods and applications for in-vivo optical-and opto-acoustic tomography of whole intact animals and model biological organisms. We show how new opto-acoustic and fluorescence imaging concepts are necessary for accurate and quantitative molecular investigations in tissues and why it could be a valuable tool for accelerated research of therapeutic efficacy and outcome. We further demonstrate that cellular functions and bio-chemical changes can be detected in-vivo, through intact tissues at high sensitivity and molecular specificity. Pre-clinical and clinical results are presented and advantages and limitations of these methods along with future directions are discussed. Pulmonary nodules are one of the classic challenges in radiology: detection and lesion characterisation are constant sources of trouble, be it in the form of unnecessary workup or missed lesions and ensuing lawsuits. This presentation will review the progress that has been made on these issues and also discuss current limitations of radiological techniques. Human observers are notoriously inaccurate when it comes to detection and characterisation of nodules. Like the development in chess computers, computer programs (computer-aided diagnosis, CAD) now start to outperform humans. Rib suppression or temporal subtraction techniques on chest radiographs aid in lesion detection. New CAD techniques combine algorithms and can detect, quantify and characterise pulmonary lesions. However, while radiologists are still able to detect lesions missed by CAD, they also dismiss true-positive and accept false-positive CAD markers. CAD development has been stimulated by the large image databases from lung cancer screening trials. These trials provide new information about prevalence and prognosis of various nodule phenotypes: small solid nodules are extremely common but have a low cancer risk, persistent part solid and ground glass nodules (GGN) have a high malignancy rate and GNN grow slowly. Volume and mass doubling times are more accurate measures than nodule diameter. Form and location allow for identifying benign perifissural nodules. Dynamic-contrast-enhanced imaging with CT or MR as well as PET so far only plays a role in larger lesions. Still, however, the role of CT screening is not fully defined: false positives and radiation dose remain important issues. The possibility of carrying out Molecular Imaging protocols by means of MRI is very attractive for the superb anatomical resolution that is attainable by this technique. However, MRI suffers from an intrinsic insensitivity with respect to the competing imaging modalities that has to be overcome by designing suitable amplification procedures based on the development of reporting units endowed with an enhanced sensitivity and on the identification of efficient routes of accumulation of the imaging probes at the sites of interest. Now, the need of targeting molecules that are present at very low concentrations requires the development of novel classes of contrast agents characterised by enhanced contrasting ability and improved targeting capabilities. The possibility of delivering a high number of imaging agents at the target of interest appears the solution of choice to overcome the drawback associated with the low sensitivity of the MRI approach. Currently much attention is devoted to the design and use of self-assembled systems based on lipophilic molecules, where the imaging reporters are represented by highly stable paramagnetic Gd (III) complexes. Moreover, nano-sized carriers for Gd-complexes based on naturally occurring systems (e.g. lipoproteins) have also been considered for targeting specific epitopes on diseased cells. Besides paramagnetic agents much attention is currently devoted to the new class of frequency-encoding probes, the CEST agents (CEST= Chemical Exchange Saturation Transfer). The use of frequency-encoding agents opens the interesting perspective of detecting more than one agent in the same anatomical region. Ultrasound is one of the workhorses in clinical diagnosis. In this context, the diagnostic power of ultrasound can be increased when using microbubbles as contrast agents providing information about tissue vascularisation and perfusion. By conjugating biomolecules to the microbubble surface even intravascular molecular ultrasound imaging becomes feasible. In this context, for the discrimination of low-and high-aggressive breast cancers it has been shown that the assessment of the VEGFR2-expression by molecular ultrasound is superior to the functional information about tumour vascularisation. First targeted ultrasound contrast agents are currently evaluated in clinical trials. Potential clinical indications are characterisation of tumours, improvement of ultrasound guided biopsy, personalised tumour therapy, the characterisation of atherosclerotic plaques and the diagnosis of other diseases that go along with vascular remodelling and inflammation. Unfortunately, microbubble-based imaging is restricted to the endovascular compartment. Here, photoacoustic imaging may be helpful, which bases on the ultrasound acquisition of the thermoelastic expansion of nanoparticles after laser excitation. Haemoglobin, gold nanoparticles, dyes, fluorescent proteins and other light-absorbing materials can sensitively be imaged by this technique. In this talk, the composition of molecular ultrasound contrast agents and their measurement techniques will be explained and illustrated. In addition, the mechanistic principle of photoacoustic imaging will be explained intending to exemplify both emerging diagnostic technologies to the clinical radiologists. Friday and long-term assessment of therapeutic result is usually achieved by contrastenhanced CT (CECT) or MRI (CEMRI) which can be performed only at the end of the treatment session. In addition, the possibility of achieving local tumour control with percutaneous ablation is largely related to tumour size. Consequently, there is an increased need to detect and treat small tumours that are often clearly visualised on CT or MRI, but not on US. Contrast-enhanced sonography (CEUS) is helpful for guiding electrode insertions into targets hardly visible with US. However, the most important role of CEUS is the immediate post-ablation control. The sensitivity of CEUS for the detection of residual tumour is almost equivalent to that of CECT and CEMRI. Cost-effectiveness and reduction of patients' discomfort related to the need of re-treatment are the two main advantages of CEUS in RFA of liver malignancies. The use of CEUS during liver surgical resections is gaining more and more importance since it can allow to detect small additional neoplastic lesions not visualised in the pre-surgical staging with imaging modalities and to depict with great accuracy liver vascularity, thus allowing to perform precise liver-sparing anatomical resections. The routine practice of oncologic imaging requires standardisation, which means that we need to harmonise technical protocols and agree on the meaning of selected words for the radiological report. The words "Response, "Progression" and "Stable disease" are precisely defined according to internationally accepted thresholds and criteria. Although the rules are quite simple and rather easy to apply, they are very efficient in the classification of the response to treatment, and therefore for the medical decisions. However, the role of the radiologist is not limited to measurements and calculation. The detection of new lesions may be challenging and requires experience. The differential between cancer progression and complications of the treatment might be very difficult and requires an adequate communication with the referring clinician. Overall, most of the decisions taken by the clinician will be related to imaging results, stressing the importance of adequate protocols and reports. In the past decade, contrast-enhanced ultrasound (CEUS) of the liver has been firmly established as an excellent imaging modality for focal lesions. Its strong points include high spatial and contrast resolution as well as its unparalleled temporal resolution, enabling dynamic imaging after contrast injection in real time.
Disadvantages are poor sonographic access in some patients (mainly obese) and operator dependence. All common liver lesions display characteristic dynamic enhancement patterns on CEUS in the arterial, portal venous and delayed phase. This is used for lesion classification and characterisation. Furthermore, the fact that the vast majority of malignant lesions appear hypoenhancing on delayed phase imaging is exploited to improve detection of malignant lesions. Typical features of common benign and malignant lesions as well as clinical indications for CEUS of the liver will be discussed. Detection of focal renal lesions is very common in the clinical practice. Most renal lesions are non specific on baseline ultrasound (US) and require further examination with imaging modalities after the intravenous administration of a contrast agent. US contrast agents are strictly intravascular and unlike CT and MR contrast agents, US contrast agents are not eliminated by the urinary system and can be used in patients with renal failure. The main indication of contrast-enhanced ultrasound (CEUS) in the kidney is the characterisation of complex cysts. The detection of enhancing thickened irregular wall or septa or the presence of soft-tissue enhancing mass independent of the wall is the most specific sign suggesting malignancy. Characterisation of complex cysts with CEUS is also very useful when complex masses do not show a conclusive CT/MR diagnosis. CEUS may also help in the characterisation of pseudotumours such as prominent Bertin columns and dromedary hungs. Regarding solid renal tumours, there is an overlap of the enhancement pattern between benign and malignant lesions. For this reason, CEUS is not routinely recommended to detect and characterise solid masses. However, it can help in the differentiation of subtypes of renal cell carcinomas based on tumour vascularity since non clear-cell carcinomas, such as the papillary type, are usually more hypovascular than clear-cell carcinomas. CEUS has also a role in the evaluation of renal perfusion including ischaemia, trauma and complicated pyelonephritis that may in some cases show a focal appearance that mimics a renal tumour. Ultrasound is used for visualisation of vascular pathology in various areas. It is used to evaluate peripheral arterial disease, carotid and vertebral arteries disease, diseases of aorta and its visceral branches and pathology of deep and superficial veins. Although ultrasound is often neglected in imaging of blood vessels accuracy of ultrasound in most areas is comparable with CTA and MRA. Ultrasound has numerous advantages to CTA and MRA, since it is cheap, widely and easily available, non-invasive, safe, with no exposure to ionising radiation, mostly does not require administration of contrast media and can be performed at the patient's bedside. It demonstrates not only morphology of the vessel but also provides important haemodynamic information in real-time. Role of vascular ultrasound is very important in planning of surgical and/or endovascular therapy, in evaluation of immediate success and long-term follow-up after the treatment. Ultrasound is very useful in angio-suite during endovascular procedures, and several cases will be presented to demonstrate usefulness of ultrasound in vascular interventions. Thorough understanding of haemodynamic changes and Doppler physics is needed for adequate interpretation of Doppler findings. Basic technique of vascular US exam will be presented in the lecture, as well as factors that influence morphology of Doppler spectra. Doppler artefacts will be discussed. Various types of normal and pathological Doppler waveforms will be presented. Criteria to establish the diagnosis of stenosis in various arteries will be discussed. Use of ultrasound to diagnose deep venous thrombosis and alternative findings that mimic DVT will be presented. 
Chest x-ray in children
Interpretation of the chest radiograph in children is quite different to the adult CXR. It requires knowledge of the normal appearance of the paediatric CXR and an understanding of the diseases that affect children. I shall first present the normal appearance with an emphasis on the appearance of the thymus in the paediatric CXR. Four patterns of disease will be described: airway obstruction, interstitial pattern, cardiomegaly and atelectasis. I shall then present two main diagnoses in each of four categories: neonatal medical chest disease, congenital lung abnormalities, cyanotic and non-cyanotic heart disease, and common and less common respiratory infection in children. Four miscellaneous conditions, diaphragmatic hernias, cystic fibrosis, non-accidental injury and foreign body aspiration will complete this whirlwind review of a vast and very important topic. has a varied imaging appearance based on the underlying histologic stroma (from glandular mucin producing to dense fibrous). The contrast enhancement characteristics that vary with the stroma are key to detecting and characterising these lesions. There is no effective chemotherapy or interventional cure, and treatment options are limited to aggressive surgical approaches and the imaging assessment of extent of disease is critical in planning. Metastatic liver disease most often has very nonspecific imaging features that preclude diagnosis on imaging characteristics alone. Certain contrast enhancement characteristics, however, allow for characterisation and more importantly, assessment of response to treatment, particularly in vascular metastatic lesions such as GIST tumours. Detection of small lesions that can be critical for staging can be optimised with MR liver-specific imaging agents and with diffusion-weighted imaging. This presentation provides guidelines for how to be successful in prostate MRI and convince urologists to use MRI. It is important that radiologists performing prostate MRI speak the same language as their referring physicians. They should know as to what is important for the patient. Therefore, this presentation provides guidelines for prostate MRI, assessed by prostate MRI experts from the European Society of Urogenital Radiology (ESUR). The proposed MRI protocols for "detection", "staging" and "node and bone" will be shown. The use of endorectal coil versus pelvic phased array coil and 1.5 versus 3 T will be discussed. And most importantly, clinical indications are provided. Finally, the ESUR PiRADS classification and a reporting system will be presented. Molecular imaging (MI) appears as a great challenge for the future of imaging, because it is able to characterise cellular and molecular processes and will serve as a guide for new targeted or personalised therapies. MI is already part of clinical practice using PET and targeted probes, due to the very low doses of tracers used. Introduction of MI into radiological techniques will take more time because our techniques are lacking sensitivity. The main issues of MI are targeting of cells, receptors and gene expression. Cell targeting has already been used in patients using MRI and iron oxide particles: these are phagocytized by macrophages in deep tissues making it possible to target inflammation into brain, bone, kidney and arterial wall. Ex vivo labelling of cell progenitors is also promising to monitor cell therapies with imaging. Targeting receptors has a great potential to identify molecular processes involved during cancer development, inflammation, apoptosis, extracellular matrix changes¼ but it requires to validate the specificity of targets, the stability of the agent and to obtain enough sensitivity with our clinical imaging techniques. Imaging gene expression is more challenging. Friday CT is the technique of choice because of its accessibility and excellent diagnostic results. The aim of this presentation is to show the key radiological features for an adequate diagnosis and staging of pancreatic cancer. In addition specific guidelines for the radiological report in these patients will be discussed in order to convey all the necessary information to the referring physician. Unresectability criteria with CT include liver metastases, distant locoregional lymph nodes, peritoneal carcinomatosis or tumour invasion of superior mesenteric artery, celiac axis or hepatic artery. The presence of direct contiguity between the tumour and arterial vessel, regardless of the degree of contact between them is a sign of tumour invasion. In general arterial invasion is always considered a criterion of unresectability and no patients with arterial involvement should be operated on. Venous invasion is generally not considered a contraindication for radical resection. Recently a small subgroup of patients with marginally resectable adenocarcinoma of the pancreas has been described. These are patients with arterial tumoural infiltration less than 50% who respond to aggressive neoadjuvant chemotherapy and radiotherapy. Neurological emergencies, i.e., the sudden loss of motor, sensory, or cognitive functions up to coma, have numerous causes. The probably most important neurological emergency, both in absolute number of affected patients as in socio-economic impact, is stroke. With recent advances in devices and techniques for interventional neuroradiology, such as microcatheters and 3D DSA, neuroendovascular surgery is playing an increasing role in the treatment of acute stroke. Early diagnosis with a complete imaging work-up, either by CT or MRI, is mandatory before these therapies can be applied, and although the time span for a successful therapy has increased to 4.5 hours for i.v.-thrombolysis and up to eight hours for intraarterial therapy, an efficient time-saving algorithm in the management of stroke patients remains crucial. Other causes besides stroke, however, must not be forgotten. This talk focuses on CNS pathology; conditions like coma due to metabolic causes or intoxication will not be covered. A wide range of possible etiologies remains, ranging from vascular pathology (e.g., cervical artery dissection or venous sinus thrombosis) to epilepsy, metastatic disease, and inflammation. As in stroke, imaging studies today are an essential component of early diagnosis. MRI is the method of choice for nearly all of the above mentioned conditions; at least in tertiary care centers, indications for MR in a neurological emergency should be seen generously -around the clock (24/7). In an emergency situation, structural imaging should routinely be complemented by special techniques like diffusion-and susceptibility-weighted imaging and perfusion studies. Examples will be shown and recommendations for optimized emergency study protocols are given. Traumatic brain injury (TBI) encompasses a wide, heterogeneous group of intracranial injuries that includes acute primary insults that occurred at the time of impact and secondary ones such as cerebral swelling or herniation. Imaging is critical for diagnosis and proper management in all patients with TBI. Noncontrast CT is still the "gold standard" imaging modality in acute setting, because it identifies extravasation immediately. Epidural, subdural and subarachnoid haemorrhage (extra-axial) MRI provides critical information for preoperative decision making in rectal cancer. High-resolution T2 sequences covering the whole tumour, inclined to the tumour axis are required in axial, coronal and sagittal planes. An accurate assessment allows the surgical and oncology team to decide whether the patients should proceed straight to surgery or if they require neoadjuvant radiotherapy alone (short course) or chemoradiotherapy (long course). The radiologist must therefore report the following tumour features that predict adverse outcome or suitability for radiotherapy: precise tumour location in the rectum and relationship to the peritoneal reflection; presence and extent of tumour penetration thorough the rectal wall (T3 or more); presence of tumour within 1 mm of the Circumferential Resection Margin (CRM) (corresponding to the mesorectal fascia); invasion of the peritoneum; invasion of the anal sphincters; presence of vascular invasion; malignant lymphadenopathy; mucinous differentiation. The role of diffusion-weighted imaging is yet to be defined in clinical practice in terms of initial tumour assessment and predicting response to therapy. In addition to MRI, accurate staging requires MDCT evaluation for distant metastasis to liver, lung, retroperitoneal nodes and peritoneum. The optimal imaging report provides a comprehensive radiological staging to compare with final histology (Tumour, Nodes, Vascular Invasion, Involvement of CRM and distant Metastasis). This is essential for audit of clinical practice and service development. There is a potential role for structured clinical reports to ensure that all of this information is provided in a reproducible way to facilitate multidisciplinary team decision making. The questions regarding the CT diagnosis of bowel obstructions are as follows: is there an obstruction? What is the level and the cause? Are there findings of closed loop obstruction and of strangulation? What is the treatment recommended: surgery or follow-up, laparoscopy or laporotomy? To answer these questions, the CT semiology is based on findings validated in the literature that will be described in this lecture, by underlining the potential pitfalls in interpretating a CT exam in bowel obstruction. The technical modalities of the CT will be detailed: thickness of the slices, role of acquisition without contrast and reformatting Learning Objectives:
1. To understand a simple classification for the causes of bowel obstruction. Radiological staging of adenocarcinoma of the pancreas is critical for adequate management of these patients because treatment depends on the stage of the disease. If pancreatic cancer is limited radical surgery can be performed whereas only palliative treatment with chemotherapy and radiotherapy can be offered in patients with locoregional or distant extension of the disease. There are many diagnostic techniques for studying the pancreas, but most authors agree that multidetector
ence or absence of specific features predicts the risks for local recurrence, distant metastatic disease or both. This information thus determines the best of use of the many preoperative treatment strategies available. The initial preoperative assessment enables selection of patients for local therapies including non surgical and less radical therapy options when appropriate and identification of patients that require preoperative downsizing and downstaging in order to achieve the best results. Defining a robust preoperative strategy for patients with rectal cancer relies on consistent and reliable definitions of the risk factors by detailed imaging assessment that incorporates an understanding of surgical and oncological therapeutic limits.
Learning Objectives: 1. To understand precise definitions of a resection margin at risk.
2. To learn about the prognostic relevance of nodal stage, vascular invasion and depth of extramural spread, and pelvic sidewall lymphadenopathy. 3. To appreciate strategies for low rectal cancers and tumours beyond the TME plane.
Treatment tailored according to staging K. Haustermans; Leuven/BE (karin.haustermans@uzleuven.be)
A patient presenting with an adenocarcinoma of the rectum should be staged according the TNM staging system using different imaging modalities. Next to these classical clinical factors also circumferential margin involvement, size of the primary tumour and location of the primary tumour in relation to the anal sphincter are important factors in the treatment decision. The goals of preoperative treatment in rectal cancer are to reduce the risk of local relapse, to enable R0-resection and to preserve the anal sphincter in low-lying tumours. Radiotherapy can be administered either as a short course (5 fractions of 5 Gy, one fraction a day, overall treatment time of one week) or as a long course (25 fractions of 1.8 Gy, one fraction a day, overall treatment time of five weeks combined with chemotherapy eventually followed by an extra dose of radiation to the macroscopic tumor). In case of a short course of radiation total mesorectal excision (TME) is performed in the week following the end of radiation. In case of a long course of chemoradiation TME surgery is performed after an interval of 6 to 8 weeks. When immediate surgery is performed no tumour downstaging or downsizing can occur while after a long course of chemoradiation followed by a long interval before surgery up to 20% of patients have no tumour left in the resection specimen. Currently it is not known whether aiming for sphincter preservation after a good response to preoperative chemoradiation increases the risk of local recurrence. Learning Objectives: 1. To understand that the initial staging of rectal cancer determines the treatment approach (patient tailored treatment). 2. To understand the difference in approach between a short course of radiation and a long course of chemoradiation. 3. To become familiar with the concept of "the good, the bad and the ugly" in rectal cancer.
Response evaluation by imaging R.G.H. Beets-Tan; Maastricht/NL (r.beets.tan@mumc.nl)
The standard treatment for advanced rectal cancer is preoperative chemoradiotherapy (CRT) followed by standard resection of the rectum. Neoadjuvant CRT allows downsizing and downstaging of the tumour, leading to improved resectability and local control. The clinical question is what to do with good response after CRT. Most surgeons advocate performing a resection on the basis of the status before CRT disregarding the response. However, major pelvic surgery is associated with a high postoperative morbidity rate of 40%-50%. The paradigm shift in treatment is further tailoring of treatment for the good responders after CRT aiming at organ saving treatment options with less morbidity, such as local excision. Although still controversial, a wait-and-see-policy (omission of surgery under close monitoring) is being advocated for the complete responders. This shift in treatment has consequences for radiologists. Our major challenge will be to provide tools that can help in precise selection of these patients. The challenge will not only restrict to an accurate evaluation of the local tumour response but also extend to an accurate evaluation of the nodal response. This lecture will focus on the evidence-based role of EUS and MRI for response evaluation after CRT. Accuracy and limitations of each method will be discussed. The lecture will dwell on potential new techniques (diffusion and perfusion MRI, lymph node-specific contrast enhanced MRI, PET/ CT) that can overcome the limitations of standard imaging. Learning Objectives: 1. To understand the evidence based role of imaging for re-staging locally advanced rectal cancer after chemoradiotherapy.
as well as cortical contusion, intraparenchymal haematoma and diffuse axonal injury (intra-axial hemorrhage) will be discussed. Conventional MRI sequences are less sensitive than CT in detection of hyperacute intracranial bleeding. However, FLAIR techniques are capable to detect even small amount of extravasated blood. Susceptibility-weighted imaging (SWI) is mandatory in evaluation of microhaemorrhages; together with DWI they play an important role in diagnosis of axonal injury. Traumatic injury of spinal cord (SCI) in majority of cases results with devastating medical and social consequences. MR examination is the imaging modality of choice in such patients. It enables the clear assessment of lesion morphology, extent and severity of trauma. To interpret MR images properly, we have to be familiar with mechanisms of spinal cord injury which are presented in this lecture. For optimal characterisation of SCI one has to start with estimation of canal compromise and the degree of spinal cord compression. Then qualitative intramedullary changes like cord swelling, oedema, contusive haemorrhage, haematomyelia, partial or complete laceration should be evaluated. Protocols for routine MRI of the patients SCI are proposed and discussed. This Session, jointly organised with ESTRO (European Society of Radiotherapy and Oncology), aims at promoting an integrated approach between specialists involved in multidisciplinary tumour boards to tailor the best treatment for each individual patient by exploiting the use of imaging. New advanced imaging technology provides not only morphological information on tumour extension, but also information on tumour function and biology. It allows not only a good evaluation of tumour response during and after treatment, but also an early detection of tumour recurrence. Radiation oncologists increasingly use hybrid equipment in which diagnostic imaging technology is incorporated within the radiation treatment machines to allow continuous adaptation of radiation treatment according to the daily response of the tumour, the surrounding organs and their movement. Thus, it is our interest to enhance the collaboration between imaging specialists and to optimise and adapt the different use of imaging to the comprehensive clinical management of the oncological patient. By dedicating a combination of Radiology and Radiotherapy lectures to rectal cancer model, the Session aims at offering a programme focusing on the use of imaging in a truly multidisciplinary environment, in order to support understanding between specialists belonging to different disciplines, yet using a common language. Session Objectives: 1. To become familiar with an integrated approach between radiologists and radiation oncologists in multidisciplinary tumour boards to tailor the best treatment for each individual patient with rectal cancer. 2. To understand how to evaluate the tumour response during and after treatment, as well as the early detection of tumour recurrence. 3. To appreciate the benefit for radiation oncologists in using hybrid equipment to allow continuous adaptation of radiation treatment according to the daily response of tumour, the surrounding organs and their movement. The common cold is one of the most frequent illnesses in Europe and the United States. Although most cold are mild and resolve within a short time period, colds cost billions of dollars per year, mostly due to lost time at work and school. The common cold is a group of symptoms caused by one of a large number of viruses. Rhinoviruses cause the greatest number of colds; there are more than 100 different varieties of rhinovirus. The average adult experiences two to three colds per year, while children average 8 to 12 colds per year. In most cases, colds do not cause serious illness. Most colds last for three to seven days, although many people continue to have symptoms (coughing, sneezing, congestion) for up to two weeks. Some viruses that cause the common cold can also depress the immune system or cause swelling in the lining of the nose or airways; this can lead to bacterial infection. One of the more common complications is sinusitis, which is usually caused by viruses and rarely (about 2 per cent of the time) by bacteria. However, it can be difficult to distinguish bacterial sinusitis from sinusitis caused by a cold because the signs and symptoms can be similar. However, due to the fact that a runny nose can also result from inflammation, trauma, foreign body and other abnormal processes, including tumors, an excellent diagnostic workup is necessary. Session Objectives: 1. To discuss the epidemiology. 2. To discuss the pathophysiology. 3. To become familiar with facts concerning economic aspects.
It is important for the radiologist to understand the anatomy of the drainage pathways and the frequent anatomical variants in this region in order to guide the surgeon. These variants may impair the functional drainage pathways, increase the risk of endoscopic surgery and make access to sites of disease extremely diffcult. This lecture will highlight the clinical relevant sinonasal anatomy (osteometal unit (OMU), frontal and sphenoethmoidal recesses) and variants (frontal ethmoidal, infraorbital and sphenoethmoidal air cells, ethmoid roof and anterior skull base). By the end of the lecture I hope that you will have learnt a logical approach to assessing the sinonasal anatomy and variants and an ability to review and understand the sinonasal region in all three orthogonal planes in order to accurately assess this region. For locally advanced rectal cancer (LARC) an early accurate prediction of tumour response, more specifically pathological complete response (pCR), after preoperative chemoradiotherapy (CRT) is valuable to tailor treatment. Accurate prediction could enable more individualised surgical approaches, including less extensive resection or even a wait-and-see policy on one hand and possibly an intensification of pre-operative treatment in selected patients on the other hand. In our research groups several response prediction models for LARC have been developed mainly based on longitudinal PET-imaging, and specifically SUVmax on MR imaging, and on multifactorial nomograms including clinical parameters; see also (www. predictcancer.org). An innovative method consists in adding CT-based features for pre-treatment response prediction in LARC, the so-called "radiomics" approach. We conclude that imaging-based models and the nomogram developed based on clinical and sequential imaging data can accurately predict pCR and can be used as a decision support tool for surgery after prospective validation. MRI, a problem-solving imaging modality a few years ago, has become a first-line imaging technique in the diagnosis of focal liver lesions. Technological improvements lead to high-quality images and fast sequences improving its diagnostic accuracy. The contribution of the new cellular-specific contrast agents has been crucial in selected situations. Diffusion-weighted images are nowadays routinely included in the liver study protocol and its role in the detection and characterisation of the focal liver lesions will be discussed. 3D dynamic fat saturation protocols allow to obtain multiphasic high-quality studies and contrast bolus timing can be carefully assessed with the development of techniques such as fluorotriggered contrastenhanced MRI. The newer imaging protocols and the typical imaging features of the focal liver lesions using different MRI contrast agents will be discussed.
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should be evaluated for defining a differential diagnosis, such as multiplicity, location, origin (e.g. subcutaneous, intramuscular), size, shape, margins, peritumoral oedema, bone involvement and rate of growth. It is not uncommon for a reporting radiologist to come across vertebral body collapse in day to day practice. A number of imaging options are available to the radiologist to assess the nature of the vertebral body collapse. Vertebral body collapses are broadly divided into benign and malignant depending on the aetiology. Benign collapses are most often due to metabolic diseases such as osteoporosis and trauma. It is vital to be able to differentiate these two categories of vertebral involvement to initiate appropriate therapy. Radiographs have a low sensitivity and specificity in differentiating these categories of vertebral body collapse. MRI on the other hand is excellent at differentiating between benign and malignant lesions on standard imaging sequences. A number of features including retropulsion, T1 signal characteristics, clefts, soft tissue abnormalities, posterior element involvement and contrast enhancement help in this differentiation. Advanced imaging protocols including diffusion and in/out of phase imaging are rarely needed. In some clinical circumstances where the differentiation is not possible despite all these measures, CT scan, follow-up imaging and/or a biopsy may be necessary. Static instabilities are defined by the absence of classic symptoms of instability and are associated with rotator cuff or degenerative joint disease. Dynamic instabilities are subdivided in two main categories. The first category is traumatic unidirectional Bankart surgery (TUBS), which is characterised by a history of trauma resulting in unidirectional anteroinferior instability, commonly associated with a fibrous or osseous Bankart lesion that requires surgical repair. The second category is known as atraumatic multidirectional bilateral rehabilitation inferior capsular shift (AMBRI). This pattern of multidirectional instability is believed to be the result of atraumatic ligamentous and capsular laxity. Treatment is rehabilitation initially, followed by inferior capsular shift if indicated. The anterior instability is characterised by avulsion of the labroligamentous complex from the anteroinferior aspect of the glenoid, which, with complete disruption of the scapular periosteum, is termed a fibrous Bankart lesion. The presence of an associated adjacent glenoid rim fracture is referred to as an osseous Bankart lesion. Osseous Bankart lesions can easily be missed on MR images; therefore, CT arthrography is preferred by some authorities. In posttraumatic posterior dislocation, tears occurring in the posterior labrum are referred to as a reverse Bankart lesion and impaction of the anterosuperior humeral head are called reverse Hill-Sachs defect. Recurrent (atraumatic) posterior shoulder instability has to be distinguished from acute and chronic (locked) posterior dislocation. Recurrent (atraumatic) posterior instability is not related to trauma, but rather to laxity of supporting capsular and muscular structures and/or to the shape of the bony glenoid or the labrum. Learning Objectives:
1. To learn about the specific imaging findings of instability. 2. To be familiar with the different types of shoulder instability.
rhinosinusitis, though only when sign and symptoms suggest complications. In this scenario orbits, skull base, anterior and central skull base require even more meticulous assessment than sinus cavities; contrast administration is mandatory, particularly to detect intracranial lesions. On the other hand, the role of CT is much more extensive in chronic rhinosinusitis, both to indicate the need for endoscopic sinus surgery and to plan the most proper strategy. Thorough knowledge of the anatomy of sinuses and mucus drainage pathways is the key to interpret CT findings. A centrifugal approach is normally used in reporting, assessing nasal fossae and drainage conduits first, and only ultimately moving to the periphery, i.e. sinus cavities, the involvement of which is quite often only a secondary effect. After surgery, CT scan may be indicated if major signs and symptoms (nasal obstruction, pus drainage, facial pain) persist. Sinus anatomy may be significantly altered, althoughas a general rule -it is simplified; however, interpretation of CT images is relatively easy if the main (and most commonly used) procedures performed are known by the radiologist. The main goals of imaging are the detection of any residual sign of mucosal inflammation along the surgically widened mucus drainage pathways, as well as the identification of retention mucocele or infectious complications. There is a great variety of sinonasal tumours due to the many different tissue types present within the maxillofacial complex. Benign lesions may be of epithelial origin, but also fibro-osseous or vascular lesions do occur. Squamous cell carcinoma is the most common malignancy of the sinonasal area, but other tumours may originate from glandular tissue (e.g. adenocarcinoma) or neural crest cells (e.g. aesthesioneuroblastoma or melanoma). Only few of them show typical imaging features. The main goal of imaging is to evaluate tumour extent and guide treatment planning. In this lecture, imaging patterns of sinonasal tumours will be shown. The current TNM classification for malignant tumours will be discussed and illustrated. Ultrasonography (US) is a readily available non-invasive technique that can be used for the detection of a soft tissue mass. It gives a first impression of the size, location in relation to the fascia and consistency of soft tissue lesions. An important ability of US is the potential to differentiate between cystic and solid lesions; however, specificity in further characterising a soft tissue mass is low. MR is the next imaging modality to perform for the evaluation of soft tissue lesions. MR is accurate in determining size, confinement to or extension beyond the anatomic compartment of origin and the relationship to the neurovascular bundle and adjacent bone and joints. Although histopathological examination remains the gold standard, differentiation between malignant and benign soft tissue masses, indication of the grade of malignancy and prediction of the histological diagnosis are challenging goals of MR imaging. As such, characterisation of soft tissue masses starts with interpretation of signal characteristics on T1-and T2-weighted sequences. Combinations of signal intensities reveal the different tumour components (e.g. fat, water, and blood), and thus provide indirect information about the nature of soft tissue masses. Additional imaging parameters combined with clinical parameters such as age, gender, etc.
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Friday the radiology unit. Ultrasound is a rapid, portable and reliable method for screening of patients with abdominal trauma. Emergency sonography is performed bedside and simultaneously with physical assessment, resuscitation and stabilisation within the first minutes of the patient's arrival. Focused assessment with sonography for trauma (FAST) is now part of the Advanced Trauma Life Support (ATLS) protocol, while becoming the standard of care in the management of adult and children trauma patient for the detection of intraperitoneal, intrathoraic and pericardial fluid. The primary goal of this examination is to determine the need for immediate laparotomy. Recently, FAST has been extended to evaluation of pneumothorax and vascular filling (extended FAST or EFAST). Limitations of FAST include its inability to detect the injuries, and the possibility that the initial bedside ultrasound results negative because fluids are too less to be identified or they have not yet accumulated in the peritoneal pouches. Interpreting the results of imaging studies is more and more challenging and time consuming due to the large volume of data to evaluate, compare and post-process. Radiological errors are inevitable, affect all radiologists and may be defined as a mistake that has management implications for the patient. Errors can be broadly classified into technical errors, active errors (errors in perception, judgment or knowledge) and errors of communication. The majority of errors are false-negative interpretations and occur during interpretation of CT examinations. Good communication between the referring physician and the radiologist is essential. Unfortunately, only a small minority of radiologists keep a personal record of their errors. Patient safety should benefit from the repeat organisation of "error meetings" through the act of collective learning. Radiologists and radiology departments must continue to improve the process of recording and addressing errors. Session Objectives: 1. To understand the potential pitfalls that may be encountered in interpreting imaging exams with emphasis on oncologic and emergency cases. 2. To describe the different types of errors: technical or active errors. 3 . To learn about methods to facilitate identification of errors in order to minimise their occurrence.
Breast
Breast cancer remains a major cause of cancer death among women worldwide. Imaging plays a key role in the early detection of breast cancer. Mammography is an accepted screening modality. Ultrasound and MR imaging of the breast can significantly improve cancer detection and characterisation and are used as an adjunct to mammography. Misinterpretation due to technical and human errors has a significant impact on mortality and morbidity of breast tumour/ cancer patients. The potential harm caused by breast imaging includes the creation of unnecessary anxiety, morbidity, costs and the use of ionising radiation. It is for this reason that the strongest possible emphasis on quality control and assurance is required. These start with the identification of eligible women, with the commitment that breast imaging of the highest possible standard is performed, that films are read by proper trained radiologists, that prompt and further effective investigations are provided, that outcomes are monitored and evaluated regularly, that regular audits are performed, and that training of the staff is ensured. During this session the auditorium will learn about the spectrum and factors that contribute to errors in the interpretation by breast imaging exams highlighted by case studies. Although CT is commonly regarded as the best imaging method in patients with acute abdominal symptoms, the rapid growth of its use in emergency medicine has raised concern about increasing radiation exposure to population. There is a need for strategies using a non-ionising radiation technique such as US as first approach in this clinical setting, with CT used in equivocal or negative US results. Furthermore, US may be the preferred choice to approach patients with renal impairment, since it does not need the use of potentially nephrotoxic contrast media. In addition, it must be stressed that US is clearly the first imaging approach to the pregnant patient with acute symptoms. This presentation deals with the comparison of the results of these two imaging techniques in emergency patients with acute abdomen. Their sensitivity and specificity is compared, and special attention is given to the need of a standardised complete protocol of US examination of the whole abdomen. Since the results of US are considered to be highly dependent on the level of expertise of the examiner, such protocol could possibly allow a more standardised level of results. In some cases, US may be also requested as integration to emergency CT. For instance, presence of thin septations or small echoes within fluid collections can provide clues to their nature, while small parenchymal lesions can be easily recognised as either cystic or solid without the need of dedicated CT scanning protocols. Examples are provided of the many possibilities of US in this field. In paediatric radiology abdominal ultrasonography plays an important role in dayto-day clinical practice. The well-known advantages of ultrasonography in children are the lack of ionising radiation, the bed-side applicability, the potential to perform the study without sedation, and the relative low costs of the examinations. The body composition of most children, in contrast to adults, yields an exquisite depiction of anatomical structures which makes US the paediatric abdominal imaging modality of first choice. However, CT is being increasingly used for the diagnosis of patients presenting with an acute abdomen. Overall the number of CT exams, more so in adults compared with children, has shown a steady increase over the past decades. In this session the application of both US and CT, based on clinical paediatric cases, will be discussed and compared. Both gastrointestinal as well as non gastrointestinal causes of the paediatric acute abdomen will be presented. MR mammography has become an important imaging technique, but because of low specificity, controversies remain, especially in the field of preoperative staging and screening. Some multicentre studies showed disappointing results due to inexperienced participating centres. Lack of standardisation also can be a reason of inferior results. To have the best results, MR units ≥ 1 T are necessary, with double dedicated breast coils, preferably multichannel. Regular checks using standardised quality control are recommended. Gentle pressure of the breasts must be performed to avoid motion, but this should not result in compression of vascular structures. A T2 weighted series must be done, followed by a dynamic contrastenhanced examination with slice thickness ≤ 3 mm, spatial in-plane resolution ≤ 1.5 mm2 and temporal resolution ≤ 120 s. Reporting must contain description of lesion morphology, per cent enhancement versus time curves, number and location of lesions and extent of disease. The use of a standardised interpretation system, such as the BI-RADS lexicon, is recommended. Clinical examination, mammography, ultrasound and earlier MR must be included in the interpretation. To reduce false positives, MR mammography must be performed between day 7 and 12 of the menstrual cycle or at least 4 weeks after stop of hormone substitution therapy, except in urgent cases. To optimise specificity, techniques as diffusion, spectroscopy and ultra fast scanning have been studied and new signs are described. But of utmost importance to make MR mammography a useful examination is to reserve it only for good indications. Contrast-enhanced MRI has proven to be superior in the detection and staging of breast cancer. Besides the primary tumour stage the lymph node (LN) status and the presence of distant metastases have significant impact on treatment planning and overall prognosis. Even small LN metastases can already have a negative impact on the disease-free and overall survival of patients. Sentinel LN biopsy or complete axillary LN dissection remains the standard of care in spite of the associated morbidity. Diagnostic imaging strategies using ultrasound (with FNA), MRI or FDG/PET can be used in the preoperative LN evaluation. Due to the limited accuracy of these strategies they are so far not able to overcome the surgical standard and reduce the patient's morbidity. The presence of distant metastases results in a stage IV breast cancer and is considered non curable with a 10-year survival of about 10%. For the detection of distant metastases a chest x-ray, liver ultrasound and bone scintigraphy (BS) are commonly used. PET/CT or whole body Learning Objectives:
1. To learn about the potential pitfalls that may be encountered in interpreting breast imaging exams. 2. To understand the technical errors in the realisation of breast imaging exams that contribute to misinterpretation. 3. To be aware of the spectrum of factors that contribute to active errors (detection, characterisation) in interpretation of breast imaging exams.
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Imaging of the musculoskeletal system differs from that of other organs by many features: limited specificity of presenting symptoms (pain), variable qualification of referring physicians, high frequency of trauma-related lesions and relative rarity of non-traumatic life-threatening conditions, frequent anatomic variants, possibility of successive imaging procedures with unsatisfactory communication of previous imaging or clinical findings. These features may contribute to numerous pitfalls and errors at MSK imaging. During this lecture, we will focus on common causes of errors. For lesion detection, we will emphasise the limitations of ultrasound, the inappropriate use of which may lead to significant difficulties. For lesion characterisation, we will focus on a several causes of misinterpretation at MR imaging including the lack of recognition of air, gas and calcium at MR imaging and the inappropriate use of fat-saturation. We will also remind the audience of the underestimation of septic disorders with inappropriate management of percutaneous biopsies. Finally, we will also draw the attention to mistakes that are related to the patients, including unconscious patients, relatives or VIP patients.
Learning Objectives:
1. To learn about the potential pitfalls that may be encountered in interpreting MSK imaging exams. 2. To understand the technical errors in the realisation of MSK imaging exams that contribute to misinterpretation. 3. To be aware of the spectrum of factors that contribute to active errors (detection, characterisation) in interpretation of MSK imaging exams.
Neuro P.C. Maly Sundgren; Lund/SE (Pia.Sundgren@med.lu.se)
The reasons for mistakes in the interpretation of neuro-imaging examinations are similar to those in radiology in general, i.e. poor technique, artefacts, failure in perception, lack of knowledge and misjudgment. The reader may commit so-called scanning errors (overlook the primary or additional lesion), recognition errors or decision-making errors. Factors like misleading-or even correct clinical history, presence or absence of previous studies, knowledge and experience of the reader, etc, may lie behind an incorrect interpretation. Although experience increases the accuracy, marked differences have been reported between the most experienced readers. In neuro-imaging there is an increased risk of errors due to the large (and increasing) number of images to be reviewed per case, the complexity of some of the images and of the analysis. The risk of errors is increased also due to artefacts, especially in the interpretation of MRI, or due to poor imaging quality, for example in MRA or CTA. In the present lecture different causes for mistakes, such as artefacts, misdiagnosis due to lack of-or comparison to wrong previous studies, poor imaging technique and wrong clinical information will be addressed. Learning Objectives:
1. To learn about the potential pitfalls that may be encountered in interpreting neuro imaging exams. 2. To understand the technical errors in the realisation of neuro imaging exams that contribute to misinterpretation. 3. To be aware of the spectrum of factors that contribute to active errors (detection, characterisation) in interpretation of neuro exams. Friday the type and extent of parenchymal injuries. It can help detect active extravasation of contrast and is of great help in guiding transcatheter embolisation. It may also demonstrate urine leakage, preexisting abormalities with increased risk of injury from blunt trauma, and associated abdominal and retroperitoneal injuries. The volumetric data acquired can be used to obtain high-resolution MPR, MIP and 3D images that help display complex injuries. The wide availability of MDCT in major trauma patients has reduced the use of other imaging modalities: IVU in unstable patients already in the operative room, and ultrasonography. Learning Objectives:
1. To learn the indications, advantages and disadvantages of imaging modalities in evaluating the kidney and ureter after trauma. 2. To learn the appropriate diagnostic imaging studies and imaging findings of different types of kidney and ureteral trauma. 3. To be able to identify a kidney that is in danger after trauma.
B. Imaging the bladder and urethra U.G. Mueller-Lisse; Munich/DE Trauma to the bladder and urethra is seldom an isolated injury, such as in selfinserted urethral foreign objects in men. Often, the lower urinary tract (LUT) is one site of a more complex trauma injury process involving the pelvis and abdomen. Trauma scan (TS) protocols for computed tomography (CT) have been widely applied in emergency radiology since the advent of multidetector-row CT (MDCT). Acute LUT injury has thus become subject to TS as the primary imaging modality, with intravenous contrast media being used in various phases of its distribution, potentially including arterial, venous and excretory, as in CT-urography (CTU). Delayed diagnosis appears to rarely affect LUT in TS patients. Cystography and urethrography may play a complimentary role and remain useful modalities in the initial evaluation and follow-up of LUT trauma that may determine urological management. Complications of LUT trauma, such as urine leaks, urinomas and fistulas, are increasingly being diagnosed by means of MDCT. However, particularly in LUT fistulas, voiding cystourethrography and urethrography are still important. Complications of LUT injury may require imaging-guided interventional procedures, whether by means of fluoroscopy, ultrasonography or CT. Learning Objectives:
1. To be able to identify patients at risk of bladder and urethral trauma and those requiring urgent urethrography. 2. To recognise the imaging findings identifying bladder and urethral injury. 3. To learn the imaging techniques necessary for accurate initial evaluation of the urethra in cases of complicated pelvic trauma.
C. Interventional radiology for GU trauma B. Peynircioglu; Ankara/TR (borapeynir@gmail.com)
GU trauma can easily be missed when associated with other abdominal/pelvic injuries. The type/mechanism of the trauma is the key for both imaging and treatment. Iatrogenic injuries of the GU tract are getting more and more common as a result of increasing numbers of percutaneous procedures (e.g. nephrolithotomies, biopsies). In general, renals are the most common injured part of the tract; however, by increasing numbers of renal transplantation, ureteral injuries are now common as well. Timing of the intervention is as important as taking the decision of percutaneous approach for optimal management of injury. Multiphasic CT imaging with contrast injection via both IV line and trans-urethral catheter (if possible) can demonstrate most of the injured sites with high sensitivity. CT is also useful in predicting which haemodynamically stable patients may benefit from percutaneous (non-operative) management. Vascular injury of the GU tract almost always involves renals, and bleeding and/or ischaemia is the problem. Unless the patient is at unresponsive haemodynamic shock, or has complete vascular avulsion, endovascular treatment may always be the choice. The American Association for the Surgery of Trauma system for grading injury to the kidney is also helpful in making a decision whether surgical or endovascular intervention is used. Embolisation of a bleeding artery/ pseudoaneurysm or stenting of an intimal dissection are the most common cases.
Although the vascular injuries of the GU results in retroperitoneal haemorrhages, non-vascular injuries leading urine leaks may present with intra or extra peritoneal urinomas. Nonvascular percutaneous intervention may be applied to urinoma, urine leak, ureteral laceration and transection injuries. These interventions include percutaneous nephrostomy for urine diversion, ureteral stent placement for ureteral injuries and drainage tube placement for urinoma formation.
Learning Objectives: 1. To be able to determine which cases deserve management by interventional radiology.
MRI can also be used for the detection of distant metastases. Earlier detection of for instance bone metastases is needed to optimise treatment and reduce or delay skeletal-related events. Diffusion weighted whole body imaging with background suppression (DWIBS) was found to be equally sensitive with respect to staging. However, DWIBS was able to detect more lesions in cases with many bone lesions. In this presentation imaging techniques that can be used in the evaluation of axillary lymph nodes and distant metastases are discussed. Multimodal cancer therapy, including chemotherapy, biologicals and radiotherapy, is frequently associated with adverse, toxic effects. Depending on the specific agent different organs, such as brain, lung, liver, bowel, bone and heart might be affected. Inflammatory " reactions" will occur. These will be detected on imaging as white matter lesions in the brain, reticular changes and consolidations in the lung (fibrosis, cryptogenic organizing pneumonia), diffuse liver disease, colitis, bone necrosis, or cardiomyopathy. These direct toxic effects have to be differentiated from infectious complications due to chemotherapy-induced neutropenia. These infections mainly affect the lung and are caused by fungi or viruses. Angioinvasive aspergillosis of the lung is the most frequent, but sinusitis, abscesses in brain and liver as well as sepsis with haematogeneous foci are also encountered. Toxic effects of radiotherapy will mainly occur within the planned target volume and result from application of high doses to radiosensitive normal tissue leading to inflammation, fibrosis and necrosis. As imaging is routinely performed for therapy response monitoring or surveillance, in patients suffering from cancer toxic effects have to be differentiated from infectious complications, postsurgical or postradiation scar tissue or tumour recurrence. Specific patterns of toxic effects of cancer therapy in brain, lung, liver, pelvis and their differential diagnoses will be reviewed. PET/CT with FDG is useful for staging, response assessment and follow-up of Hodgkin and Non Hodgkin lymphoma (HL, NHL). Most HL and NHL show FDG uptake higher than local background; disease sites are therefore easily recognised. Some entities (e.g., follicular lymphoma I) have low FDG uptake, reflecting their less aggressive nature. For staging, combined CT and PET define the disease better than either test alone. For instance, PET can better show involvement of spleen and bone marrow, but CT remains necessary for anatomic detail, detection of disease with low FDG uptake and size measurements. FDG PET is more accurate than morphologic criteria in assessing response early (decline in FDG uptake correlates with rapidity of response and often predicts ultimate outcome) and also at the end of treatment (lack of FDG in residual masses reliably indicates lack of residual viable tumour). A negative PET after 2 or 4 cycles of chemotherapy predicts excellent outcome. In contrast, up to 50% of positive interim scans are false-positive, and hence cure may still be possible. Persistent FDG uptake after end of treatment indicates a worse prognosis, even when treated with radiotherapy. Recurrent disease is well detected on CT and PET, but there is no proof that prolonged routine imaging after end of therapy is necessary or advantageous (possible exception: poor prognosis aggressive NHL). Follow-up imaging can probably be avoided entirely in patients with early-stage HL and negative end of treatment FDG PET/CT. Recent data with the agent 18 F fluorothymidine will be discussed. Varicose veins of the lower extremity are a common problem. Besides the unsightly look, venous reflux can lead to swelling, heaviness and itching, and eventually to ulcerations. In many cases, the underlying cause is an insufficient saphenous vein. Duplex ultrasound is essential for the diagnosis of reflux and treatment planning. Endovenous thermal ablation has largely replaced surgery (vein stripping) in the United States because of equal results, outpatient set-up and an overall much quicker and easier recovery. The endovenous treatment is well tolerated. Postinterventional pain and tenderness is low (VAS < 2). It seems that the discomfort is slightly lower for RFA (closure fast) than laser (bare fibre). Severe complications such as deep vein thrombosis or skin burn are extremely rare. Minor complications such as hyperpigmentation, erythema and paraesthesia occur in < 5%. Thermal ablation can be done with RFA or laser, including different wavelength and bare or covered fibre tip). The results seem similar between the different endovenous treatments. In the short term (1 month), an occlusion rate of close to 100% can be expected. Mid-and long-term results (up to 48 months) maintain an elimination of reflux > 90%. Learning Objectives:
1. To learn about imaging and clinical evaluation of varicose veins.
To know more about the minimaly invasive treatment modalities. 3. To become familiar with the short-term and long-term results.
The impact of radiographers on performance and quality issues arising with novel technology F. Girard; Pont de Roide/FR (franck.girard@opteamage.com)
The emergence of new technologies in the medical field is putting pressure on the technologist's job. Among other questions, training, performance of these technologies and image quality are especially under scrutiny. This presentation will cover these aspects, always from a technologist's point of view and will give an outlook on the implications expected from the emergence of these new technologies in the radiographers' world. The influence of the technicians' role will be demonstrated through MR and CT images acquired following standard procedures. A longitudinal follow-up study will show not only that regular refresher courses are one part of the solution to be applied for a better image quality, but also that the technologists should be part of the front line of developments, through regular readings of specialised papers, participation to technical congresses and to professionals forums. This is very often not compatible with a daily work and it is one topic of concern to be addressed by the radiology management. The radiographer is instrumental in today's and tomorrow's medicine, but he/she needs to be constantly challenged, helped and brought to the highest possible level of education, always ultimately to the benefit of the patient. Continuous education of radiographers is needed to keep up with state-of-the-art techniques. To maintain a high quality standard of radiological examinations we introduced a continuous education programme for radiographers. The Academic Medical Center Amsterdam started to make an inventory of the actual expertise and preferences for education of radiographers in 2008. The main outcome of the initial inventory was that radiographers appeared less competent on new technology, such as emergency CT. Also, the prior level of education appeared to be a mediating factor in the competence of the radiographers. Subsequently, continuous education was implemented on a per modality basis and at two levels for some modalities. The attendance to education was registered by means of Edumanager. Also, radiographers were able to maintain a digital education portfolio by themselves. The effectiveness of the education was evaluated after two years (2010). The radiographers valued the education highly. Higher expertise of the radiographers was found after two years regarding emergency CT. The effectiveness of the education varied with the prior education level of the radiographers. Additionally, radiologists were asked to evaluate the quality of the radiological examinations. The radiologists valued about 5% of the radiological examinations as insufficient and approximately 75% as good-excellent. We did not find a significant trend over time. Continuous education remains urgently needed to keep up to date with current radiological technique. Our continuous education will be adapted based on the outcomes of the questionnaires. The difference in prior education is a matter of concern and needs special attention. Radiographers working within cross-sectional imaging need to understand their role in respect to the pathology they are imaging and the significance of the images they produce in respect to patient care-pathways. Adaptation and implementation of new and 'novel' techniques is fundamental in achieving the best possible information for the clinician and thus improved patient treatment. The modern cross-sectional radiographer needs to be knowledgeable, flexible and be able to operate in an MDT environment in order to deliver a 'first class' service. Only then can the profession advance to become an integral part in the patient care delivery. Besides drug therapy, the mainstays in the treatment of coronary heart disease (CHD) are interventional therapy and coronary bypass surgery. The need to image coronary bypass grafts is based on their limited lifetime: the occlusion rate at 10 years ranges between 5 and 15 % for arterial, and between 40 and 50% for venous grafts, respectively. Graft sclerosis develops in 38% of nonoccluded venous bypass vessels after 5 years and in 75% after 10 years. This sclerosis causes more than 50% luminal narrowing in approximately half of the affected vessels. Non-invasive imaging of coronary bypass grafts by MD-CT requires information about the operative procedure. With the increasing implementation of 64slice CT scanners and beyond, it is possible to scan the heart and the full anatomic extent of grafts with sub-millimeter slice-thickness within a single breath-hold. When analysing the grafts, three graft segments should be assessed: the origin or proximal anastomosis, the body of the graft and the cardiac anastomosis, either single or sequential. Recent studies have shown that graft patency and the presence of significant graft stenosis can be assessed with an accuracy of 100% using most recent MD-CT technology (256/320 slice CT or dual source CT). The assessment of native coronaries with respect to the progression of CAD may still be problematic in cases with severe calcifications of the native coronary arteries. In these cases, MR perfusion imaging in combination with Cine and late gadolinium enhancement (LGE) imaging may be helpful in detecting newly developed, stress-induced myocardial ischaemia. The metabolic syndrome refers to the clustering of cardiovascular risk factors that include diabetes, obesity, dyslipidaemia and hypertension. The association between metabolic syndrome and cardiovascular diseases raises important questions about the underlying pathological processes, especially for designing targeted therapeutic interventions. Insulin resistance and visceral obesity have been recognised as the most important pathogenic factors. Metabolic syndrome generally precedes and is often associated with type 2 diabetes. Cardiovascular risk reduction in individuals with metabolic syndrome should include 1) control of obesity, unhealthy diet and lack of physical activity; 2) control of the individual components of metabolic syndrome, especially atherogenic dyslipidaemia, hypertension, hyperglycaemia and prothrombotic state; and 3) control of insulin resistance, a defect closely linked to metabolic syndrome. Appropriate management of metabolic syndrome should be able to prevent the progression from impaired glucose tolerance to frank diabetes and thus to prevent the increasing prevalence of type 2 diabetes and vascular diseases. Each 1% increase in HbA1c is associated with a 28% increase in risk of incident PAD. Diabetes is also highly associated with progression of PAD and especially with development of critical limb ischaemia. Rigorous control of blood The testicular varicocele is a pathological dilatation and tortuosity of the veins of the pampiniform plexus of unknown aetiology. Clinical presentation can include scrotal swelling and pain; the varicocele is more often seen in the subfertile or infertile man. The primary diagnostic imaging tool is ultrasound. Right-sided varicoceles can be symptomatic; a profound abdominal diagnostics is necessary. Therapeutic options include minimally invasive surgical and open microsurgical treatment, as well as interventional radiological therapy. The IR treatment by percutaneous venography includes the selective catheterisation of the spermatic vein and subsequent embolisation or sclerotherapy. Technical and clinical success rates are encouraging and complications are rare. In this lecture, diagnostic modalities, clinical and imaging features plus the indications for therapy will be discussed. In addition, the different therapy options and its results will be elucidated; furthermore, the management of the endovascular occlusion in treatment of varicoceles will be demonstrated in a step by step fashion. Myocardial function and coronary anatomy from one scan? This "science"-fiction has become true now for computed tomography (CT). Over years, we have often worried whether a lesion found on CTA is "significant" (= causes myocardial ischaemia indicating the need for coronary revascularisation) or not. Now, CT-perfusion (CTP) provides that information. Different CTP-techniques will be discussed (e.g. the "one-shot" approach and "dynamic"). New CT technology providing low radiation dose will be highlighted in this course. CT is also established technique for imaging of global and regional cardiac function. Beyond, imaging of myocardial viablity using delayed enhancement will be discussed. The aim of this course is to understand basic principles of functional imaging by CT, to learn "how-to" perform these scans, to review current scientific evidence and to discuss potential clinical applications. Most commonly MRI myocardial perfusion images are analysed by visual inspection, noting regions of hypointensity (ischaemia) during the first pass of a contrast medium bolus of a gadolinium chelate. High temporal resolution is required to capture transient ischaemia in the myocardium under pharmacological stress and to distinguish artefacts (e.g. dark rim artefacts during a few frames at peak enhancement). Various modifications of T1-weigthed, ECG gated, multislice acquisitions have been proposed to assess first-pass myocardial signal intensity changes in cine mode. The time-versus-intensity curves allow quantitative analysis by deriving parameter values from the time series of intensity values (e.g. upslope, peak, absolute myocardial blood flow). Epi/endocardial contour tracing is required for segmental analysis, by pixels or to construct perfusion maps. Time intensity curves are deconvoluted for arterial input function using a Fermi function for estimating Peripheral arterial disease (PAD) is a common cardiovascular complication in patients with diabetes. In contrast to non-diabetic PAD, it is more prevalent and, because of the distal territory of vessel involvement and its association with peripheral neuropathy, it is more commonly asymptomatic. Diabetic PAD may present later with more severe disease and have a greater risk of amputation. The pervasive influence of diabetes on the atherothrombotic milieu of the peripheral vasculature is unique. The abnormal metabolic state accompanying diabetes results in changes in the arterial structure and function. The proatherogenic changes include increases in vascular inflammation and derangements in the vascular cellular components, alterations in blood cells and haemostatic factors. These changes are associated with an increased risk for accelerated atherogenesis as well as poor outcomes. In contrast to the focal and proximal atherosclerotic lesions of non-diabetic PAD, in diabetic patients the lesions are more likely to be more heavily calcified, diffuse and distal, sparing the proximal vessels and mainly affecting the more distal arteries in the calf and the foot. By identifying a patient with subclinical disease and instituting preventative measures, it may be possible to avoid acute, limb-threatening ischaemia. The primary imaging modality to be used should be duplex ultrasound, due to its non-invasive nature, lower risks and costs. CT-angiography and MR-angiography are now replacing DSA as standard imaging methods, providing a non-invasive assessment of the localisation and extension of a vascular lesion and allowing an accurate planning of endovascular and/or surgical treatment. The choice of the appropriate modality for imaging of the peripheral vascular system strongly depends on the type of disease. In the acute setting of lower leg ischaemia caused by embolism or thrombosis on top of a pre-existing high-grade stenosis, CT angiography frequently is the imaging modality of choice due to its 24-hour availability and robustness with reasonable accuracy in the upper leg exceeding 90%. With the introduction of dual energy CT, visualisation of lower leg arteries and highly calcified areas has improved. For elective patients, contrast-enhanced MR-angiography (CE-MRA) is the technique of choice, in patients with renal insufficiency preferably at 3 Tesla with a single dose of macrocyclic contrast agents. Recent continuous table-movement techniques have substantially reduced the complexity and scan time and extended the spectrum to the emergency stetting. Time-resolved CE-MRA improves the detection of small arteries in the lower leg and reduces artefacts from venous overlay. Accuracy of state-of-the art CE-MRA exceeds 95% compared to digital subtraction angiography (DSA). New fast-spin echo and steady-state free precession MRA techniques without contrast media demonstrate a high negative predictive value in areas without motion. DSA should be restricted to the display of the target lesion, inflow and run-off during the interventional procedure, as well as to the in-detail visualisation of the anastomosis site prior to surgical revascularisation, particularly in the lower calf. Learning Objectives:
1. To learn about the appropriate imaging protocols for each modality.
2. To understand quantitative analysis of occlusive disease in peripheral arteries in order to plan revascularisation. 3 . To learn about the specific pros and cons of ultrasound, MRA, CTA and DSA in diabetic foot syndrome prior to revascularisation. RF is also currently used within the multidisciplinary approach of patients with liver metastases, either as a complementary treatment during a hepatectomy or as a unique method to increase the local control of a liver nodule. Finally, in recent years several series have demonstrated excellent results with the application of RF in other non-liver tumours such as renal or lung carcinomas. In spite of being a relatively safe procedure the use of RF has some limitations and is not exempt from complications. In general they include the need of the introduction of a needle, the decrease in its efficacy when the tumour is located close to a blood vessel and the impossibility to obtain a complete necrosis, with a safety margin, in tumours bigger than 3.5 cm in its greatest diameter. To overcome some of these limitations, new locally ablative methods have been developed. Some may not need the insertion of a needle and can be applied in many different tumoural locations, among them "high intensity focused ultrasound" (HIFU) or "stereotactic body radiation therapy" (SBRT). Others like "microwave ablation" or "irreversible electroporation" may be applied to tumours bigger than 4 cm or situated close to blood vessels. Radiofrequency ablation (RFA) provides complete ablation of most of the tumours less than 3 cm and away from vessels larger than 3 mm. For tumours larger than 3 cm success rate of RFA decreases due to limited volume of ablation that can be obtained with one RF delivery and impossibility to deliver treatment through several RFA electrodes at the same time. Success rate of RFA also decreases when tumours are close to large vessels. Microwave (MWA) might be a valid answer to these difficulties in size and vascular cooling due to the possibility to use several microwave antennas at the same time and better thermal profile, namely by being able to reach rapidly temperature superior to the ones used for RFA. High MWA energy can be nowadays delivered through microwave antennas inserted percutaneously, especially due to needle shaft cooling. Synergy has been demonstrated when multiple antennas are used at the same time. Consequently, MWA has been applied in lung, liver and kidney ablation where large ablation volumes were obtained with promising success rate of complete ablation when treating large tumour. Great care should be taken to avoid complications that can occur when very large volumes of ablation are performed. Peritoneal carcinomatosis is usually associated with a poor overall survival rate.
Recently, introduction of more aggressive surgical treatment and intraperitoneal chemotherapy appears to significantly increase the overall survival rate for these patients. A detailed preoperative assessment of peritoneal carcinomatosis could be very challenging in the field of imaging, but a new aggressive surgical approach requires an accurate preoperative assessment of the disease. Cross-imaging using CT and MRI with diffusion-weighted (DW) sequences is important for appropriate management of patients with peritoneal carcinomatosis. Appreciation of the spectrum of diagnostic patterns and pitfalls as well as different sites of involvement of peritoneal carcinomatosis using CT and DWI is crucial for appropriate surgical treatment. The aims of the lecture are to underline the role of 64 MDCT and DWI 3 T MRI in the evaluation of Peritoneal Metastases (PMs) in patients undergoing peritonectomy and hypertermic intraperitoneal chemotherapy (HIPEC), to understand advantages and drawbacks of each diagnostic imaging technique and to address which particular abdominal areas are the most important and crucial to evaluate in order to exclude the presence of PMs for a right selection of patients that must be surgically treated by HIPEC. 
What is the added value of PET/CT?
Diagnostic potentials -as well as limitations -associated with morphological cross-sectional imaging on the one hand, and functional imaging on the other, are increasingly well understood. It has become obvious that in many cases both kinds of imaging complement one another. So hybrid PET/CT imaging must be considered one of the most promising new developments in medical imaging. The most recently launched line of PET/CT scanners combines high-definition PET with high-end multislice CT. These imaging systems do not only provide a higher diagnostic accuracy based on detection of smaller lesions with CT and PET, but also offer integration of complex CT protocols into the PET/CT scan. Detection of peritoneal carcinomatosis has been challenging with all imaging procedures. A "peritoneal cake" can be detected easily; however, in many cases peritoneal disease will be rather diffuse compromising its detection with cross-sectional imaging techniques. This also applies to FDG-PET/CT where low FDG-uptake in low-grade tumours and the anatomical resolution of PET may hamper detection of small lesions. Additional contrast-enhanced CT as part of the PET/CT scan may aid lesion detection in this situation. The aim of this talk is to give an overview concerning PET/CT in detection of peritoneal carcinomatosis. The specific advantages of US over CT are that US has an image definition in the close range which is much higher. US is more interactive: patient's history as well as painful area or palpable mass can be correlated with the US findings. US shows peristalsis, pulsations and blood flow. US shows the effects of respiration, Valsalva manoeuvre, gravity and compression with the probe, allowing to assess whether organs as bowel and gallbladder are soft or rigid. US allows easy puncture of intraperitoneal fluid and drainage of pus. US in acute abdomen is performed with graded compression. Compression is necessary to displace or compress bowel, eliminating the disturbing influence of bowel gas and to approach the pathological structure closely. This allows using a high-frequency transducer with a better image quality. The compression should be graded to avoid unnecessary pain and to avoid pushing organs out of the US plane. US examination should be symptom-directed and requires communication with the patient. In patients with an acute abdomen the entire abdomen should be examined, i.e. from the axilla to the groin. The final US report should be integrated with the clinical findings, laboratory data, CT-scan and possible other radiological examinations. The US images of appendicitis, diverticulitis, intussusceptions, acute biliary, urological and gynaecological conditions, infectious ileocecitis, perforated peptic ulcer, small bowel obstruction, ruptured aneurysm, pancreatitis, Crohn's disease, epiploic appendagitis, omental infarction and perforating malignancy will be demonstrated using illustrative case histories, with emphasis on the specific advantages of US over CT. Multidetector computed tomography (CT) has emerged as the modality of choice for evaluation of patients with most of the traumatic and nontraumatic conditions causing acute abdominal pain. Operator dependency and technical inadequacy secondary to the body habitués or intestinal gas of the patient are not so rare limitations of the ultrasound exam. Although radiation and use of contrast material are the main drawbacks, CT is the fastest imaging modality for scanning patients. Moreover, multidetector CT scanners allow performing multiplanar reformation without loss of image resolution, a technique that has been shown to improve physician confidence in either confirming or excluding the diagnosis of abdominal emergencies. Increased certainty improves treatment planning and can reduce inappropriate utilisation of hospital resources. Also in trauma patients with multiorgan injuries additional post processing techniques, such as maximum intensity projection and volume rendering, multiplanar reformation may also improve identification and characterisation of especially vessel and bone injuries in the same setting. MDCT is the primary imaging technique used in evaluating patients suspected with most common causes of abdominal pain; acute pancreatitis, acute appendicitis, abdominal abscess or renal stone. It has a high sensitivity and specificity and high confidence level to diagnose or rule out the pathology. CT also has an indispensable role to differentiate nonsurgical rare conditions of acute abdominal pain such as epiploic appendages, omental infarct, mesenteric panniculitis or diverticulitis. The most common viral infection of the brain is herpes encephalitis (HSV1). It is a necrotising encephalitis with a mortality rate of more than 50%. On imaging studies lesions will be seen in the "limbic system", i.e. the temporal lobes, hippocampi, insular cortex and cingulate gyrus. They appear as hypodensity on CT, T2-and FLAIR hyperintensity on MRI, possibly with haemorrhagic transformation. Diffusion is restricted in the early phase. Enhancement occurs at a later stage. Human immunodeficiency virus (HIV) infection is a multifocal giant-cell encephalitis eventually leading to a progressive leuco-encephalopathy. On MR atypical focal or diffuse symmetrical signal abnormalities are seen neither with mass-effect nor with enhancement and typically sparing the U-fibres. Spectroscopy can show an increase of myo-inositol. Progressive multifocal leuco-encephalopathy (PML) is due to reactivation of the JC polyomavirus in immunocompromised patients, 10% of which are HIV positive. On MRI "scalloped" multifocal asymmetrical lesions are seen with minimal mass effect and without enhancement. New treatments of HIV, especially highly active antiretroviral therapy (HAART) can lead to a paradoxical worsening of patients due to the immune reconstitution inflammatory syndrome (IRIS). On MRI mass lesions are seen with diffuse patchy enhancement. Cerebral toxoplamosis appears as multiple enhancing lesions with marked perilesional oedema. Calcification is possible. Prion diseases are caused by a proteinaceous infectious particle leading to Creutzfeld-Jacob disease in humans. Diffusion weighted MR images show high signal intensities in the cortex and the basal ganglia. Abnormalities on T2-weighted images and FLAIR occur at a later stage where atrophy is mostly prominent. Adrenal tumours are a wide group of masses where imaging plays an important role in diagnosis. Most of them are incidental findings during radiological CT or MRI studies, performed for oncological or non-oncological indications. A list of all possibilities will be presented with special consideration on the embryological origin of the tumours. A rationale for technical protocols in CT and MRI is presented in particular to apply to clinical settings. The differential diagnosis between adenoma and mets is the more frequent dilemma in oncologic patients. In non-neoplastic cases the tool is to distinguish between functioning and non-functioning masses, with all the endocrinological implications. More rarely, larger lesions may represent the diagnostic challenge for radiology: to recognise benign and malignant findings is crucial to orient towards different histotypes. Relations with neighbouring structures are also necessary to correctly stage the disease. CT and MRI have some potential diagnostic possibilities to be utilised in the characterisation attempt: density, enhancement, de-enhancement, variations of signal intensity, in particular in FS and IN-OUT of false sequences. Particular considerations will be given to some particular tumours as angioma, myelolipoma, adrenal carcinoma and collision tumours.
Learning Objectives: 1. To understand the role of different radiological techniques used for demonstration and characterisation of adrenal tissue and masses. 2. To know the major problems encountered with radiological imaging in the differential diagnosis of adrenal nodules in oncologic and non-oncologic patients. 3. To know the appearance of primary benign and malignant tumours of adrenals, either originating from the medulla or from the adrenal cortex, using different radiological techniques. The term bone marrow oedema was introduced to describe ill-defined bone marrow hypointensity on T1Wi and hyperintensity on T2Wi and water sensitive sequences. Bone marrow edema can be found in many similar unrelated disorders, such as bone contusions, osteonecrosis, inflammatory or degenerative disease, being a non-specific MRI abnormality representing a diagnostic challenge for radiologist.
Recently has been demonstrated that bone marrow oedema might be a prognosis marker for OA (osteoarthritis) and inflammatory disease, and could be used as a powerful predictive tool for treatment options. Therefore our role as radiologist is to try to increase specificity to help patient management and decrease progression. The aim of this refresher course is to describe MR imaging features of bone marrow edema in different important and frequent disorders: osteoarthritis, inflammatory diseases and trauma.
A. BME and osteoarthritis F.
The objective is to discuss the terminology, differential diagnoses, and clinical and structural significance of MRI-detected subchondral bone marrow lesions (BML) of osteoarthritic joints. Subchondral BMLs are a hallmark of knee osteoarthritis (OA) on MRI, but are also regularly seen in hip OA and in non-weight bearing de- The staging of kidney tumours includes a systematic approach to recognise and describe the local, regional, and distant extent of tumour burden. Staging results determine therapeutic approach, success of individual treatments, and prognosis of individual patients. Evolving diagnostic, therapeutic, and prognostic experience continues to re-define staging categories. In renal cell carcinoma (RCC), the Robson classification has been replaced by the TNM classification. Since 2002, the TNM classification includes RCC of 4 cm or less in stage T1a, since recent surgical experience suggests that partial nephrectomy (nephron-sparing surgery) can be curative, while it preserves excretory capacity. Since surgery is currently the only means of curative treatment, lymph node (N-) or distant metastatic (M-) extent of RCC is associated with unfavourable prognosis. However, new therapeutic concepts involving tumour anti-angioneogenesis factors or multi-kinase inhibitors demonstrate promising effects on tumour volume or tumour viability. Staging and follow-up of RCC may involve various imaging modalities. However, the increase in speed and range of both CT and MRI suggests that these modalities may soon limit or obviate the need for other imaging modalities in the staging of renal tumours.
PET-CT appears to be promising for the follow-up on new therapies in patients with metastatic RCC. Transitional cell carcinoma (TCC) of the kidney is a differential diagnosis in patients with solid renal lesions who do not have other primary tumours.
Learning Objectives: 1. To know the different imaging methods for the detection and staging of renal cell carcinoma. CT urography is defined as CT examination of the kidneys, ureters and bladder with at least one imaging series acquired during the excretory phase of contrast enhancement. The principal reason for the existence of CT urography is for diagnosing UUT-UCC. In adults, CT urography is now the preferred initial examination for patients with haematuria at high risk for upper urinary tract urothelial cell cancer (UUT-UCC). CT urography is evaluated for diagnosing UUT-UCC by analysis of results from haematuria clinics as well as by comparison studies using ultrasound, excretory urography and retrograde ureteropyelography. Technical aspects of image acquisition and processing will be explored and technical tips relating to protocol design given to optimise CT urography for diagnosing UUT-UCC.The principal problems with using CT urography for diagnosing UUT-UCC are reader error and false-positive diagnoses. Solutions to the problems will be proposed. Use of a formative teaching programme with the aim of reducing reader error will be put forward. A radiologically guided method for upper tract lesion biopsy will be The addition of 3D and 4D techniques to ultrasound in the area of obstetrical and gynaecologic ultrasound has expanded the diagnostic capabilities of the modality and has provided a means to improve scanning efficiency. For gynaecologic images, 4D acquisitions of the uterus permit real-time visualisation of the coronal plane of the uterus, a plane that cannot be imaged with conventional ultrasound. This coronal plane is ideal for assessing the uterine shape and contour to diagnose duplication anomalies, for presence and position of masses such as fibroids and polyps, and for IUD placement. In obstetrical imaging, anomalies of the face and extremities, including facial clefts, limb reduction defects, and abnormal posturing of the hands and feet, are best visualised using 3D imaging with surface rendering. Anomalies of the spine and extremities can be assessed with 3D imaging in skeletal mode to provide an image similar to an x-ray, for evaluation of such abnormalities as hemivertebrae and skeletal dysplasias. With respect to scan efficiency, 3D sonography permits acquisition of a volume or several volumes of data in just a few minutes. After the patient has left, these volumes can be rendered into sectional planes for interpretation on the workstation, or they can be manipulated into the usual scanning planes for measurements and diagnosis. Storing 3D volumes for subsequent interpretation can diminish scanning time significantly with no loss of diagnostic accuracy. Users are discovering that this is not just an additional way to image, but with innovative thinking, volume acquisition ultrasound can change the way we practice or deliver a service; often this is has the double benefit of greater efficiency and greater accuracy. This presentation will review the latest volume ultrasound technology, outline the basic concepts and techniques and provide evidence of how this can change our approach to general imaging with ultrasound.
generative joints. BMLs in OA are understood as non-cystic subchondral areas of ill-defined hyperintensity on T2w images and of hypointensity on T1w MR images. BMLs are observed regularly in conjunction with adjacent cartilage alterations. As the disease progresses, an increase in BML volume is seen in the same region subchondrally in many patients, which is positively correlated with an increase in cartilage loss and radiographic joint space narrowing. Subchondral cysts are strongly associated with BMLs in the same subregion and develop within non-cystic BMLs. BMLs fluctuate in size and the majority of subchondral BMLs may regress or resolve completely. Fluctuation of joint pain is positively correlated with fluctuation of BMLs in the same direction. Several validated semiquantitative and quantitative methods exist to assess BMLs in a research setting. Histologic correlation studies showed that the lesions consisted of a mixture of different tissue patterns with only little oedema. Specific changes in bone mineralisation and remodelling in areas of BMLs have further been reported. Differential diagnoses of OA-related BMLs include traumatic bone contusions and fractures with or without disruption of the articular surface. Osteonecrosis, inflammation, idiopathic BMLs, red marrow and post-surgical alterations should also be considered.
Learning Objectives:
1. To learn about the basic physiopathology of OA and its relation to BME.
To be informed about the distribution and natural history of BME in OA.
3. To understand the differential diagnosis and relevance of BME in staging OA and as a marker of prognosis.
B. BME and early inflammatory disease A.J. Grainger; Leeds/UK (andrew.grainger@leedsth.nhs.uk)
Marrow oedema is identified as a feature of many forms of inflammatory and mechanical arthritis, but has been most studied in inflammatory arthritides and particularly in rheumatoid arthritis. It was first reported as a feature of RA as far back as 1986. Studies have been undertaken both using human specimens and specimens from animal models, which suggest that marrow oedema seen on MRI in RA corresponds to areas of inflammation associated with invading pannus, lymphocytic aggregates and hypervascularity. There is also evidence that the marrow lesions seen on MRI in ankylosing spondylitis correspond to histopathological inflammatory change. Marrow oedema has been shown to be an important predictor of future joint damage in patients with rheumatoid arthritis. In addition to predicting bone destruction for erosion, marrow oedema is independently predictive of joint space loss and therefore cartilage destruction. It also correlates well with other measures of disease activity. We have applied dynamic contrast enhancement techniques to show that treatment with anti-TNF therapy brings about a reduction in contrast uptake in areas of marrow oedema in patients with RA. In the seronegative arthritides, marrow oedema in the spine in ankylosing spondylitis has been shown to be predictive of future changes and of response to treatment. Diffusionweighted imaging of marrow lesions in ankylosing spondylitis can also be used to show a treatment response, seen as a change in the apparent diffusion coefficient. Learning Objectives: 1. To become familiar with the imaging pattern of seronegative and rheumatoid arthritis distribution. 2. To understand the relationship between BME and early diagnosis of inflammatory disease. 3. To understand whether BME helps in patient management, diagnosis and follow-up.
C. BME and trauma S. Dzelzite, P. Likums; Riga/LV (dzelzitis@apollo.lv)
Most of the acute traumatic skeletal injuries are evaluated and treated on the basis of plain radiographs. Increased use of magnetic resonance imaging in investigation of acute musculoskeletal injuries during the last few years highlighted the value of recognition of the different bone marrow oedema patterns for more precise diagnosis of possible obscured soft tissue injuries. BME-like lesions in cases of traumatic injuries presented as focal bone marrow changes with decreased signal on T1W images and increased signal on T2W and STIR images; this appearance is thought to represent areas of haemorrhage, oedema or infarction secondary to trabecular microfractures. The osseous injuries may be the result of a direct blow or of compressive forces of adjacent bones impacting on one another. These injuries produce extensive BME. The indirect injury is the result of traction forces, which displays as avulsion injuries with localised BME. Sanders and Hayes in two different articles in "Radiographics" in the year 2000 identified distribution patterns of BME with different soft tissue and ligamentous injuries in the knee. Subsequently, the term "footprint" was introduced. In the knee, 5 different contusion patterns such as Saturday ing, especially for assessing lung and liver metastases. Despite inherent low tissue contrast, the excellent spatial resolution due to isotropic and multiplanar capacities has nowadays also improved the accuracy for local staging, provided that watery rectal enema is performed. Thanks to excellent tissue contrast, MRI has evolved as a powerful tool for regional staging, in particular to distinguish between T2 and T3 tumors and to assess the relationship of the tumour with the mesorectal fascia. The prerequisites are data acquisition in high-resolution mode and adequate slice positioning. However, perirectal desmoplastic reaction, inherent in rectal cancers, may mimic tumour infiltration, thus overestimating the T-stage. This drawback does not seem to change significantly with high-field MRI (3 T). For accurate lymph node staging, neither EUS, neither CT, nor MRI has proven excellent accuracy. Thus, alternative imaging modalities are steadily being evaluated, without any international consensus so far. Furthermore, state-of-the-art requirements and advantages of the most important recently developed imaging techniques, such as perfusion-CT, diffusion-MRI and USPIO-enhanced MRI for lymph node staging, as well as the indication for 18F-FDG PET/CT, will briefly be discussed. Patients identified at initial MRI staging as having locally advanced rectal cancer undergo neoadjuvant chemoradiation therapy (CRT) for their tumour to be downsized and downstaged, especially in low rectal cancers so that sphincter sparing surgery may be performed. In 15-30% of patients, complete pathological response is achieved. Reimaging with MRI at 6 weeks post-treatment is of great importance for assessing tumour response. However, referral depends greatly on the strategy that is to be followed by the surgeons in case of complete imaging response (no deviation from initial surgical plan, "wait and see" policy). Conventional MRI has a reported moderate accuracy for prediction of mesorectal fascia (MRF) involvement after CRT therapy, mainly due to its inability to differentiate between fibrosis with/ without the presence of tumour cells. Morphological MR imaging criteria that correspond to free of disease/ involved MRF have been reported, as well as criteria indicating disease being confined to the rectal wall (ypT0-T2). High-resolution MR images combined with radiologists' rising level of familiarity regarding the assessment of reactive changes post-CRT have increased local staging accuracy of MRI, including both T and N stage reporting. Introduction of imaging techniques such as diffusion weighted imaging, especially when combined with morphological T2w sequences and to a lesser degree PET/CT, for early and late tumour assessment response have shown promising results, especially when quantitative analysis is performed by means of post-CRT or % change of ADC values and SUV Response Index measurements. The increasing role of radiologists in the multidisciplinary management team of patients with rectal cancer is recognised and the role of MR imaging for stratifying these patients into a differentiated treatment is consolidated. Nowadays, with preoperative chemoradiotherapy (CRT), advanced rectal cancer shows phenomenal response with downstaging to small tumours (ypT0-2) confined to the wall. In 15-20%, no residual tumour is found in the resection specimen (complete response, ypT0N0). The paradigm shift in treatment has been further tailoring the treatment for the good responders after CRT, aiming at organ saving treatment options with less morbidity, such as local excision. Although still controversial, a wait-and-see policy (omission of surgery under close monitoring) is being advocated for the complete responders. This shift in treatment has consequences for radiologists.
Our major challenge will be to provide tools that can help in precise selection of these patients. The challenge will not only be restricted to an accurate evaluation Fusion imaging is not new. However when combined with volume imaging and navigation, it becomes a very strong tool broadening ultrasound as well as CT clinical applications for both diagnostic and interventional practices and in particular in guiding therapy without or reduced ionising radiation. Currently available systems have been shown to be useful in a) the US tracking of liver and renal lesions for detection, characterisation of difficult/occult lesions and follow-up following therapy, b) in the guidance of biopsy and ablation of occult liver or renal tumours as well as c) a much needed training tool for interventional clinicians. A practical work-flow through various fusion and navigation systems for the clinical applications as well as their current benefits, limitations and future developments will be reviewed. Preoperative staging of rectal cancer implies detailed recognition of tumour extent, providing the basis for multidisciplinary team (MDT) treatment planning. The role of diagnostic imaging continues to increase in this setting. There are technological advances in all imaging modalities, and treatment is based on a synthesis of information in each patient. Diagnostic imaging discussed in MDT conferences is recognised as a quality assurance parameter in rectal cancer, ensuring that the available prognostic information is adequately used all the way to treatment decision. The multidisciplinary approach also has continuous educational advantages. For radiologists, recognition and reporting of tumour relation to anatomical structures such as the mesorectal fascia, the peritoneal reflection, the levators and anal sphincters, as well as identification of extramural venous invasion, lymph node involvement and evaluation of tumour response to neoadjuvant treatment, are all important parts of this educational process. MDT-based imaging discussions continue to result in new surgical and oncological treatment strategies to be considered both in randomised clinical trials or in single patients. Being able to stratify patients into prognostic groups based on imaging may help to identify not only those in whom neoadjuvant treatment is beneficial, but also those where primary surgery can be performed without need for radio-or chemotherapy. In this session, the present role of different imaging techniques for local and distant staging of rectal cancer will be presented, including their advantages and limitations. New treatment strategies and challenges based on assessment of response to treatment will also be presented and discussed. Coronary computed tomography angiography (CCTA) using 64-detector rows or greater has been recently introduced as a novel non-invasive anatomic method for evaluation of CAD, demonstrating high diagnostic accuracy for detection and exclusion of obstructive CAD. Several recent reports have also examined the prognostic value of CCTA findings for prediction of future adverse CAD events, but have been generally limited to single centres and by small patient cohorts. Further, prior studies examining risk stratification by CCTA findings have been primarily restricted to measures of obstructive CAD by maximal luminal diameter stenosis severity on a per-patient or per-vessel basis, with other plaque characteristics visualised by CCTA, including location, distribution, extent, and composition and non-coronary cardiac findings including left ventricular systolic function, volume, and regional wall motion, largely neglected within development of prognostic models. Several preliminary studies showed that CCTA has an incremental value over traditional risk factors, coronary calcium score and other imaging tests for the prediction of cardiovascular events. The interesting value of CCTA is also related to the capability of stratification in the area of sub-clinical atherosclerosis and in the definition of plaque type. With the implementation of low-dose CCTA the prognostic potential of CCTA can be imagined as a reliable clinical information. At the moment there is one large international multicentre registry that started to explore the predictive potential of CCTA in large number of patients (> 20.000). Coronary artery disease (CAD) is the leading cause of morbidity and mortality in the industrialised countries. In order to determine which patients are at risk to develop sudden cardiac death or non-fatal future cardiac events (e.g. myocardial infarction/ischaemia-related cardiac arrhythmias), traditional risk factors have been established. These are helpful to triage patients in high/intermediate/low risk groups. However, many patients in the high-risk group will never experience a cardiac event, while patients in the other risk groups can be falsely reassured. This has urged clinicians and scientists to assess novel prognostic factors better predicting future cardiac events. MRI, amongst other techniques, seems promising to improve prediction of patient prognosis. MRI is the reference technique to assess cardiac volumes and function. It allows to reliably assess the impact of coronary artery stenoses on myocardial perfusion and function in resting and stressed conditions, and this technique enables to noninvasively depict myocardial necrosis/ fibrosis and to fully characterise the jeopardised myocardium in the acute setting. These applications are not only useful in the diagnosis but also to predict clinical outcome and to assess patient prognosis as increasingly shown in literature. In this presentation, the added value of MRI in the different CAD subgroups (stable CAD, acute ischemic event, chronic ischemic cardiomyopathy) will be discussed in detail.
of the local tumour response, but also be extended to an accurate evaluation of the nodal response. The objective of this lecture is to understand the increasingly important role of radiologists for stratifying rectal cancer patients, not only before but also after preoperative treatment. It will discuss the evidence for organ saving new treatment options and how this evolution will influence our diagnostic decisionmaking process. It will elucidate the (limited) role of present imaging and point at potential future techniques (diffusion and perfusion MRI, lymph node-specific contrast-enhanced MRI, PET/CT) for the precise selection of patients. Identification of (a)symptomatic individuals at high risk of myocardial infarction is important to optimise treatment and prevent a coronary event. However, optimal patient selection remains a challenge, even after evaluation of cardiovascular risk factors. The coronary calcium score, derived from newer CT generations (at least 16-MDCT), accurately reflects the amount of coronary atherosclerosis and can improve cardiovascular risk assessment. In this presentation, results from recent prospective population-based studies will be discussed, including the Heinz Nixdorf Study, Multi-Ethnic Study of Atherosclerosis and Rotterdam study. These asymptomatic, general populations consistently show strong predictive value of calcium However, there are caveats as it uses the strong static magnetic field, transmitted radiofrequency and time-varying gradients fields. These can affect instrumentation including projectile attraction and/or twisting of tools to align with magnetic field and operational interference. Hence the MRI interventional suite requires care and the use of specialised non-magnetic instruments (e.g. Titanium tools). Interventional MRI also requires staff to stay in the scanning room during imaging which presents a range of safety issues that will be discussed. Access to the patient is also a consideration; options include using an open bore scanner or having a surgical suite where the patient can be moved on a sliding patient table between the MRI scanner and another one for access. Does improved technology lead to better outcome in vascular interventions? In some cases it does, but most of the time we just have no idea and we all follow the delusion of the day. Maybe the European notified bodies should start to handle registration of new medical devices and implants in the same way as they do with new drugs. This would be a great stimulus for good research, better patient care and most likely a reduction in medical treatment related costs. More than 90% of the new devices will never reach a more than a 3-4 level of evidence. Single arm and single centre studies, often underpowered and with a dubious methodology, presented at medical meetings or published in second class journals, that is main stream in new technologies for vascular medical devices, and probably this is also true for other specialities in medicine. Who are the main stakeholders in this game? The patients, who will be treated with a device without any proof of efficacy or superiority compared to standard treatment. The doctors who like new technology (gadgets). The health care providers, who will have to pay for expensive non-proven technology. The manufacturers who want a good return of investment for their shareholders. The weakness of the current regulatory will be discussed. Traditionally, interventional procedures, such as coronary interventions or orthopedic procedures, were guided using simple x-ray fluoroscopy, which delivers two-dimensional radiographic images as a function of time. During the past decade these c-arm systems became capable of acquiring tomographic information. This so-called rotational angiography or interventional cone-beam CT has found rapid application in the catheterisation laboratories and in the operating rooms. Today, many interventional procedures rely on the tomographic information taken before, during or after the intervention. While standard tomographic c-arm devices are equipped with cost-efficient image intensifiers the high-end systems use the flat detector technology, which is more compact and not prone to distortions. Compared with clinical CT rotational angiography allows for a far better patient access, provides images of higher spatial resolution, but suffers from the lack of low-contrast resolution and from low temporal resolution. The main reason for these differences is the During the presentation, technical aspects and clinical indications for all imaging modalities will be reviewed. The development of imaging modalities now allows scanning large body volumes in a very short time, necessary to obtain pure vascular phases. In particular, scanning of the extracranial and intracranial carotid arteries should be performed in less than 10 seconds, to avoid filling of jugular veins and image degradation. Either with computed tomography or magnetic resonance, such a short scanning is feasible, maintaining an excellent spatial resolution that allows detection of even minimal parietal irregularities. By performing such exams, it is possible not only to assess the patency of the vessels and demonstrate the degree of stenosis or the presence of aneurysms, but also to evaluate the characteristics of atherosclerotic plaque, which is also important for treatment planning. In this setting, magnetic resonance may provide some advantages over computed tomography, either exploiting its high intrinsic contrast or by using intravascular or high relaxivity contrast agents, which allow acquisition not only during the first pass, but also in the steady state. In particular during the steady state, it is possible to acquire high-resolution images with the result of increasing the detectability of small parietal ulcerations and wall irregularities. Magnetic resonance is generally preferred also when dynamic imaging is required, especially when malformations and/or fistulas need to be evaluated. Interpretation of bedside chest radiograph is particularly difficult, because only one antero-posterior view is available in patients with severe illness and sub-optimal breath-holding. The storage phosphore-based computed radiography system is frequently used in this clinical setting and provides several advantages: for example, compensation for variations in exposure and decreased radiation to the patient with a better image quality. Technical requirements are important to know because the bedside chest radiography has to be reported using optimal conditions: patient upright or in a semi-erect position at deep inspiration with a target-to-film distance of approximately 130 cm. In ICU, technical parameters are often limited with the patient supine and a shorter focus-film distance. Consistency in technique and positioning is critical for optimal evaluation of the patient on serial examinations, and technical parameters have to be transmitted between technicians in order to reproduce the same conditions. Careful plain film analysis begins with systematic control of the technical quality, followed by a careful analysis of external devices, chest tubes and catheters. Potential kinking and aberrant positions must be reported. Systematic comparison between both lungs will help the radiologist to discover abnormal findings. Repartition of air and fluid is different on bedside chest radiography and knowledge of their appearance is essential. Differential diagnosis of radiological patterns is often difficult and will be confronted with the clinical information. In many situations, a side-by-side comparison between standard radiography and CT will be performed to better understand the imaging findings, discover the main pitfalls and help the radiologist for eventual reinterpretation. Current multi-slice spiral computed tomography (MSCT) allows for a robust examination of the entire chest with submillimeter collimated protocols in a single breathhold. This is the basis for dedicated analysis of the thoracic vasculature, especially in an interactive setting, for instance using curved multiplanar reformats (cMPR). CT angiography (CTA) is mainly used for clinical suspicion of acute pulmonary embolism (PE), and for the assessment of acute and chronic aortic disease. With ECG synchronisation of the data set, a complete assessment of the cardiothoracic vasculature can be achieved, including the coronary arteries. Iodine mapping (depicting the segmental defects in iodine distribution in locations corresponding to embolic vessel occlusions) has become technically feasible in PE, e.g. using dual energy acquisition protocols. This might further improve detection of emboli and give insights into the effects on perfusion deficits in the adjacent lung parenchyma. In addition, an overview on the current status on computer-aided diagnosis software for the semi-automated assessment of PE will be provided. Image-guided percutaneous abscess drainage is one of the most common interventional procedures performed today. It can provide definitive treatment of most collections in the chest, abdomen, pelvis and, selectively, other body parts, thus obviating surgical exploration. The most critical step in performing percutaneous abscess drainage is selection of the imaging modality and selecting the appropriate access route. Uncorrectable coagulopathy and lack of a safe route for catheter placement, most commonly due to interposed bowel loops, are contraindications to the procedure. Local anaesthesia or light sedation is usually employed. Children and uncooperative patients may need general anaesthesia. The shortest pathway between the skin entry point and the abscess is usually chosen. Tandem-trocar technique, Seldinger technique and direct trocar catheter placement are the most common catheter insertion methods. The catheter sizes used range from 8 to 24 Fr and most have an inner retention mechanism. The drainage catheter is secured to the skin after placement and the abscess is completely evacuated while the patient is in the x-ray department. The catheter is then left to gravity drainage connected to a collection bag. Occasionally, if catheter placement is not feasible, needle aspiration may be safe and effective. The catheter is checked on a daily basis and irrigated with 5-10 mL of normal saline to maintain patency. Intracranial atherosclerosis is a systemic and multifactorial disease, associated with atherosclerosis of carotids, coronaries, aorta, renal and iliofemoral arteries. Over one third of ischemic strokes occur in the posterior circulation, a leading cause of which is atherosclerotic vertebrobasilar (VB) disease. Symptomatic VB disease carries a high annual risk of recurrent stroke, averaging 10-15% per year, despite medical therapy. VB stroke is particularly prone to devastating consequences due to the eloquence of the regional brain tissue and is associated with high rates of death and disability. More common in older age and in Western countries, without differences of gender. CTA and/or MRA are excellent screening tool, but DSA is the gold standard to show focal stenosis, luminal irregularities, thrombosis, occlusion, arteries ectasia and elongation, serpentine aneurysms. Transient ischemic attack, along with severe stenosis and progressive occlusion, are most common symptoms. An important stroke mechanism in VB atherostenosis is regional hypoperfusion. Furthermore, both embolic and flow processes can synergize to increase stroke risk, reducing the wash-out of emboli from the distal circulation in hypoperfused regions. Posterior circulation collateral channels may maintain adequate distal flow. The existence and extent of these compensatory blood flow pathways, evaluated by DSA, may influence the risk of stroke. Percutaneous angioplasty and stenting for symptomatic atherosclerotic VB stenosis is a feasible and effective therapeutic method, but good experience and full understanding of neurology and hemodynamics are required. Cross sectional MRI demonstrates the vessel wall and intramural hematoma. MR and CT perfusion imaging are useful to assess the hemodynamic effects of dissections. Cerebral vasculitis can affect the large (proximal intracranial) vessels, medium-sized vessels (distal to the MCA bifurcation) and small vessels < 300 μm. Cerebral vasculitis can occur as primary angiitis of the CNS, as complication of a systemic vasculitis (Takayashu's, Poloyartitis nodosa), as secondary vasculitis in connective tissue disorders (SLE, rheumatoid arthritis and Behçet's), infections (tuberculosis, aspergillosis, varicella, HIV, borreliosis), and drug use (cocaine, heroin). CTA and MRA can demonstrate involvement of large and medium sized vessels. Invasive cerebral angiography has the best spatial resolution but carries a procedural risk and is still unable to detect involvement of small vessels (< 300 μm). The latter is diagnosed by the effects on the brain parenchyma on MRI (infarcts, microhaemorrhages) and local hypoperfusion on MR perfusion studies. Whilst MRI is highly sensitive to detect changes in cerebral vasculitis, its specificity is relatively low and brain biopsy is frequently required to make a definitive diagnosis of cerebral vasculitis. neuroimaging studies. Despite these advantages, operating at such field intensity raises technical challenges. First, pulse sequence design and implementation must take into account specific characteristics of MR physics at high-field strength (increased T1 and decreased T2 relaxation times, increased chemical shift or B0and B1-related inhomogeneities). For this reason, merely transposing imaging sequence protocols that are effective at lower fields is not sufficient, because there are particular theoretical issues. Second, there are several artefacts and drawbacks resulting from some of these physical events, such as magnetic susceptibilityinduced artefacts (resulting in signal misregistration and intravoxel dephasing), compromised fat suppression (specially on air-tissue or air-bone interfaces) or image B1-induced dielectric effects on abdominal imaging. Other types of artefacts such as patient (in)voluntary motion, CSF pulsation, blood flow or Gibbs are also present at lower-field strength; there are nevertheless variations that are more misleading and appear frequently and in a more severe way at high-field strength.
By understanding the cause of such particularities, the radiographer should be able to identify and deal with most of the high-field strength-related difficulties, despite some hardware-related situations which cannot be dismissed. This presentation will revise the physical explanation for most of the artefacts present in high-field strength MRI, on both body and neuroimaging studies, as well as ways to avoid or compensate for them without impairment of diagnostic quality. MRI is a safe technique for imaging the foetus due to the lack of ionising radiation. It is considered an adjunct to ultrasound for studying the foetal brain in vivo and additionally offers improved soft tissue differentiation and increased field of view compared to ultrasound. The use of MRI in this field of diagnosis is increasing steadily following hardware and software advancements as well as clinical needs. Perhaps, the single most important problem in foetal MRI after low signal-to-noise ratio (SNR) is foetal motion, which can induce severe motion artefacts and turn images non-diagnostic. Foetal motion is unpredictable and it is currently one of the most challenging areas for motion correction for image post-processing. However, there are many different ways to compensate for foetal motion from a patient preparation/ protocol planning point of view (sequence optimisation, scanning time minimisation, motion-resistant MRI protocols) and the radiographer's knowledge and contribution are essential to this end. This paper will review different motion compensation techniques in foetal MRI from a radiographer's perspective. High-field magnetic resonance (MR) imaging may provide better SNR compared to low field. Many are excited about the opportunity not only to use the increased SNR for clearer images, but also to exchange it for better resolution or faster scans. The benefits of 3 T MR imaging in the depiction of CNS were initially established. In addition, it has been recently noticed that also body and cardiac high-field imaging combined with parallel imaging techniques decreases scan time exam and resultant motion artefacts, resulting in higher image quality compared to low field. Specifically, high-field MRI has increased susceptibility effect and prolonged T1 relaxation time. The first improves the sensitivity to the presence of haemorrhages and produces better perfusion and functional images, while the second has been exploited to succeed superior time-of-flight MRA angiographies and T1 contrast-enhanced imaging. Chemical fat suppression can be optimised due to the processional frequency difference between fat protons and water protons, which is double at 3.0 T compared to 1.5 T. Combined with the ability to increase resolution, high-field MRI is superior in the depiction of breast, musculoskeletal, adrenal, in mrcp and in spectacular. Recently, 3-dimensional contrast-enhanced magnetic resonance venography in high-field MRI has been able to more clearly visualize intracranial venous, vena cava and peripheral veins. MRI at high field combined with phased array coil, modern sequences and techniques is promising for the future and in the edge of technology. Therefore, every radiographer should be familiar with it and apply it properly. Learning Objectives: 1. To be able to identify human anatomical areas where the use of high field MRI is advantageous compared to low field MRI. 2. To be able to identify human body and brain pathologies whose depiction is facilitated by high field MRI compared to low field MRI. The use of high-field magnetic resonance imaging (MRI) is known to provide better signal-to-noise ratio, spatial resolution and fat suppression efficacy, enabling increased quality on morphological images, as well as on spectroscopy and functional Saturday urge for competing at the highest level leads to intensive training programmes in young children and adolescents. The growing lumbar spine is an area of well-known overuse-related injuries. Within the range of stress-induced bone marrow oedemalike patterns of abnormality, the devastating end stage is a stress fracture of the pars intervertebralis. The potential role of imaging, both in detecting as well as prognosis, is discussed. Imaging strategy both in the acute as well as the overuse type of injury will finalise the presentation. The Egyptian Society of Radiology and Nuclear Medicine is invited to meet the European Society of Radiology and to be given the opportunity to present some of the main topics which are of special interest to our community. Egypt is the largest country in the Middle East with a population exceeding 80 millions. Imhotep the great Egyptian pharaoh is recognised as the founder of medicine and the world's first doctor in 27th century BC (2655-2600 BC). The number of radiologists in Egypt is approximately 3425, working in more than 565 public and university radiology departments as well as 258 specialised private radiology centers. In this session we have chosen to give an overview of imaging of the more frequent cancers in Egypt namely urinary bladder, liver and breast. The place of imaging of bladder carcinoma and its relation to schistosomiasis will be highlighted. The Egyptian experience in interventional management of hepato-cellular carcinoma shall be presented. An update of the Egyptian National Screening program for early detection of breast cancer among Egyptian women will also be demonstrated. We have also thought of Paleoradiology in imaging Egyptian mummies as an interesting topic with the use of Multi Detector CT to unveil their secrets. Three short interludes, between presentations, will tackle urinary diversion and ancient Egyptian medicine and present Egypt's charm. Intramedullary and intradural-extramedullary tumours are less common than intracranial tumours. Spinal cord enlargement and heterogeneous appearance with solid and cystic components represent the main characteristics of intramedullary tumours. Low grade astrocytomas (pilocytic) are the most common, followed by gangliogliomas. Extensive involvement of the spinal cord is common. Holocord involvement and calcifications are most frequently seen in gangliogliomas. Paediatric intramedullary ependymomas are almost never seen outside the context of NF2. Metastatic disease due to CSF seeding of intracranial tumours (medulloblastomas) represents the most common intradural-extramedullary tumours. Multiple intradural neurinomas and meningiomas are found in NF2. Inflammatory demyelinating disorders my affect the cord and the spinal nerve roots in childhood. Guillain-Barre syndrome is an autoimmune acute demyelinating poly-radiculoneuropathy characterised by either diffuse or ventral nerve root enhancement. Spinal cord lesions in multiple sclerosis (MS) are mainly located dorsolaterally, extend over less than two vertebral segments in length and affect less than half the cross-sectional area of the cord. Spinal cord lesions in acute disseminated encephalomyelitis (ADEM) extend over more than three vertebral segments in length and occupy more than two-thirds of the cross-sectional area of the cord. Differences in brain and spinal cord imaging findings are useful in the differential diagnosis between MS and ADEM. Spinal cord involvement similar to that found in ADEM may be found in idiopathic acute transverse myelitis (ATM) and in neuromyelitis optica (NMO). Lack of brain involvement in ATM and the presence of the NMO-specific IgG autoantibody are useful in the differential diagnosis with ADEM. Learning Objectives:
1. To learn about the etiologies and the imaging findings of infectious, parainfectious and autoimmune disorders. The presentation will focus on acute trauma in the cervical spine and more chronic, sports-related overuse kind of trauma in the lumbar spine of paediatric patients. Although it is thought that cervical spine injuries in paediatric patients following acute trauma are extremely rare, the clearance of the paediatric cervical spine on admission to the emergency room remains a challenge for both clinicians as well as attending radiologists. For adults, the use of The National Emergency X-Radiography Utilization Study (NEXUS) guidelines is validated and thus useful in daily practice. The number of imaging cervical spine in adults, especially using high-dose multi-detector computed tomography (MDCT) has increased rapidly. The literature on the use of Nexus criteria and MDCT in the paediatric population will be critically debated. In clearing C-spine, the mainstay is conventional radiography. Guidelines for interpretation of these views will be given and examples of its use are illustrated. Also, the imaging findings when using MDCT will be enhanced. The Egypt's MOHP has identified breast cancer as a priority health area and is determined to improve awareness, services and outcomes. However, as Egyptians, we need to ask as to why breast cancer should be a priority health area. The Egyptian Women's National Council announced that breast cancer represents 33% of all female cancers, with 10% of cases metastatic at presentation; average size at presentation is 5 cm, and the average age of presentation is 10 years less than that in the west. Consequently, launching of "WHOP" was announced in October 2007. It is governmentally funded offering free screening for breast cancer, diabetes, hypertension and obesity for all women above 45 years. Double reading Digital mammography interpretation is performed using a specially designed BIRADS interpretation report. Positive cases are offered treatment free of charge. Implementation of this project, with well-constructed policies and procedures is sketched over a 5-year plan to reach all 27 Egyptian governorates through mobile and fixed digital mammography units. To date we have served 17 governorates and screened over 75,000 eligible ladies, out of whom 1545 were suspicious by mammography, of which 383 cases were operated upon. Learning Objectives:
1. To learn about the basic requirements for implementation of a screening programme for breast cancer in limited/medium resource countries using the maximum available resources. 2. To learn how to make use of the available resources to achieve international standards. 3. To identify the challenges and hear about solutions for them. 
Imaging of urinary bladder cancer T. El-Diasty; Mansoura/EG (teldiasty@hotmail.com)
The urinary bladder is the most common site of cancer in the urinary tract. Squamous cell carcinoma (SCC) accounts for only 1% of bladder cancer in England and around 5% in US. In an Egyptian series SCC represented 59% of 1026 cystectomy specimens. Most of SCCs in Egypt are associated with Schistosomiasis. The aim of imaging is to assess the extent of the local tumour and to detect tumour spread to lymph nodes and other organs. Anatomical and functional information to help in making therapeutic decisions can be obtained using different imaging methods. The first treatment decision is whether the patient has a superficial or muscle-invasive tumour. If it is superficial, patients should undergo MR imaging to confirm lack of invasion prior to undergoing trans-urethral resection (TUR). Further imaging modalities, such as CT, bone scan, PET and certain MRI techniques, are reserved for patients with muscle-invasive bladder cancer. The second treatment decision, based on staging, is to identify patients with organ-confined from patients with non-organ confined cancer and in particular those with locally advanced or metastatic disease. Computed tomography (CT) cannot differentiate accurately between organ-confined and extravesical extension. The correlation between CT findings and tumour extent in cystectomy specimens is 65-80%. MDCT urography is helpful for monitoring patients undergoing neo-adjuvant chemotherapy or bladdersparing treatment modalities. Magnetic resonance imaging (MRI) cannot detect microscopic extension into the perivesical fat. However, the overall accuracy of MR staging remains better than CT by 10-30%. Learning Objectives:
1. To understand the relation between schistosomiasis and carcinoma of the urinary bladder in Egyptian patients. Paleoradiology (paleo means ancient) involves the use of x-rays and advanced medical imaging modalities to evaluate ancient human and animal skeletons as well as biological materials from archaeological sites. Paleoradiological studies have been performed on mummies, skeletal remains and fossils to determine their sex and age at death as well as to detect ancient diseases. The great advantage of the roentgenographic examination is that it allows examination of mummies without either destroying or unwrapping them. Mummification is the preservation of human bodies after death. It symbolises the fear ancient Egyptians had of death and answers their belief and eager desire for immortality. The main principle was to dry the bodies so that bacteria could not live on its tissues. The mummification process secrets and technique will be presented. The Egyptian Supreme Council of Antiquities has initiated a large ongoing project, started in 2005, for scanning all the royal mummies that belong to the 18th and 19th dynasties. Among the great Kings and Queens scanned are Tut Ankh Amun and his family, KV55 (presumed Akhenaton), Amenhotep 3rd, Ramses 2nd and 3rd, Tuthmosis family, KV 60 (presumed Queen Hatchepsut), KV 35 (presumed Nefertiti) and many others. The CT scanning of all these great pharaohs revealed intense amount of information that helped to unveil their secrets. The most important of these discoveries will be presented some of which for the first time. Real-time elastography (RTE) of the breast may easily and quickly integrate conventional breast imaging. A mechanical force is applied to the tissue and sophisticated algorithms are used to estimate the tissue stiffness. Qualitative scores are usually derived from the estimate of the strain and help to differentiate soft benign lesions from malignancies. These are usually stiffer due to the secretion of collagen and fibronectin, and the surrounding oedema. Fluid lesions almost always show a typical three-layered pattern. Strain scores may be integrated by semi-quantitative data (fat-to-lesion ratio); unfortunately, cutoff values vary for different companies. Dedicated RTE technologies as an alternative track the shear wave propagation through tissues to obtain a true quantitative evaluation of the acoustic modulus and promise to be the gold standard for the future applications. Clinical reports show a high diagnostic accuracy: increased specificity for atypical carcinomas and a very high specificity in benign lesions, including BI-RADS category 3 lesions. With the best cutoff point between elasticity scores 3 and 4, the true negative predictive value is over 90%. In invasive carcinomas RTE clearly shows the peripheral infiltration improving the volume measurement; 3D elastography and tomographic imaging may help in this respect. RTE works better with small lesions, less than 10 mm in diameter. RTE is almost insensitive to breast thickness and echogenicity. RTE scores are well reproducible. Indexes of intra-observer and inter-observer agreement are very good. Still RTE score is only a complementary descriptor that requires global experience in breast imaging and strict guidelines. Ultrasonography is the widest accessible and most cost-effective means to assess thyroid morphology and neck masses. Diffuse thyroid disease evaluation implies not only thyroid volume measurement but also parenchymal echogenicity and vascularisation assessment as this may offer clues to different types of hyperthyroidism and thyroiditis. In thyroid nodule management, whether single or nodular goiter, the main role of US is to point, for further fine needle aspiration biopsy assessment, to suspicious nodules that might represent papillary carcinoma. Sonographic criteria pointing to nodular malignancy are marked hypoechogenicity, interrupted halo, irregular /invasive contour, punctate microcalcification or central or interrupted annular macrocalcifications, taller than wide appearance, increased central vascularisation and hypo / avascular rim and accelerated volume increase on follow-up. Malignant lymphadenopathy and extrathyroid spread are late signs. Evocative signs of benignity are also discussed. The current role of elastography and contrast imaging is presented briefly. The peculiar appearance of rare thyroid malignancies is presented. Clues on how to identify abnormal parathyroid glands and differentiate them from thyroid nodules are pointed out. The most common differentials of neck masses are illustrated: lymph nodes, branchial and thyroglossal duct cysts, carotid body and salivary gland tumours and strap muscle haematomas. The lecture concludes with a brief overview on US-guided diagnostic and therapeutic procedures in the neck. It is important to identify malignant nodules because they are potentially curable. The first step in assessing a pulmonary nodule on a chest radiograph is to determine that it is indeed a lung nodule rather than a pleural or chest wall abnormality. It is essential to review images from previous examinations, because a solid nodule that remains stable for at least 2 years is probably benign. Topics discussed in this talk include the importance of nodule size, growth rate, margin morphology, density (solid, ground-glass, part solid), calcifications or fatty components within the nodules, the significance of cavitations or bubble-like densities, enhancement patterns at dynamic contrast-enhanced CT, and findings on positron emission tomography (PET). The talk also covers the current guidelines for the management of incidentally detected nodules (solid and subsolid). The mediastinum is a region of the thorax that separates both lungs and communicates with the neck and the abdomen. These two anatomic features are very important to understand the behaviour of some diseases and their radiological manifestations. Most asymptomatic mediastinal masses are benign, while clinical symptoms may raise the possibility of a malignant lesion. Imaging plays a very important role, especially CT and MRI. In the presence of a mediastinal mass we must ask ourselves two questions: 1) where is the mass located? The classic divisions of the mediastinum in compartments remain very useful, because it narrows the differential diagnosis. 2) Is the lesion cystic or solid? Pure mediastinal cysts are benign and their characterisation depends on their location: Thymic cyst (anterior mediastinum), bronchogenic and duplication cysts (middle mediastinum) and menyngoceles (posterior mediastinum). Solid lesions may be benign or malignant while some lesions may have a cystic component. Solid lesions of the anterior mediastinum are usually thymomas, germ cell tumours or lymphomas. In the middle mediastinum most masses are of lymphatic origin but we should also include aortic or oesophageal pathology. Intrathoracic thyroid usually follows the trachea and thus is situated in the upper-middle mediastinum although posterior and anterior extensions may occur. In the posterior mediastinum most masses are of neural origin. There are some locations that will typically indicate specific diagnosis or a narrow differential. Such is the case of the cardiophrenic angle masses, juxtadiaphragmatic lesions and thoracic inlet pathology. Different medical specialties are using fluoroscopy-guided procedures due to many clinical benefits. X-ray systems are becoming more complex and many imaging protocols are available including rotational and cone beam CT. The potential of high-radiation doses for patients and staff are the reasons to implement specific radiation safety and quality assurance programs. Radiation quantities and units used to describe the radiation risks for patients need to be understood by clinicians and dose values considered as part of the optimisation strategies. Electronic dosimeters to inform in real-time on the level of scatter dose may be used as part of the occupational radiation protection program. Patient dose reports and the future integration in DICOM standards will be presented together with the need to promote automatic software tools to process all the dosimetric information and to help in the optimisation of the clinical procedures including a clinical follow-up programme for high-dose procedures. Strategies of radiation protection (RP) of patients and staff will be addressed. The new ICRP recommendations on the use of diagnostic reference levels for interventional procedures and the new suggested annual limit for the equivalent dose to the lens of the eye of 20 mSv for occupational exposure need to be implemented in the angiography and interventional radiology units. Practical recommendations for RP of patients and staff will be summarised together with the international recommendations and standards, dealing with interventional radiology, including the need of a dedicated training and certification in RP for professionals using these techniques.
Learning Objectives: 1. To comprehend the complexity of the modern interventional x-ray systems, the many imaging protocols and the involved radiation doses. 2. To understand the need to include the radiation protection aspects as part of the quality assurance program.
Panel discussion with angiographic equipment manufacturers L. Desponds 1 , B. Hoornaert 2 , M. Lendl 3 ; 1 Buc/FR, 2 Eindhoven/NL, 3 Structured reporting uses standardised language and predefined formats to create clinical radiology reports. In this presentation, we will define structured reporting, describe its advantages and disadvantages and identify the motivations for its adoption. This session will discuss how structured reporting can make it easier to retrieve reported information, evaluate the appropriateness of imaging procedures and aggregate data across healthcare enterprises. Structured reporting can support radiology quality improvement, research, and education, and has the potential to improve the quality of communication between radiologists and their referring colleagues. The use of standardised terminologies, such as RadLex® and SNOMED Clinical Terms®, allows interoperability across a variety of languages and information systems. We will explore how structured reporting might advance the quality and effectiveness of radiology worldwide.
Ultrasound (US) has gained widespread acceptance as an accurate, dynamic, non invasive and inexpensive modality to assess joints, tendons, muscles, nerves and vessels of the extremities. The couple US-standard radiographs, obtained after a good clinical examination, allow a correct diagnosis in most patients and orient towards more expensive imaging technique in the remaining patients. In this lecture we will recall the fundamental practical rules of musculoskeletal US examination. Then we will present the basic normal and pathologic US appearance and the role of US in the clinical workup of the main disorders of the musculoskeletal system. In the head and neck, a great number of anatomical structures and functions are concentrated within a very confined area. Some of these structures allow us to explore the surrounding environment through "senses" like sight, hearing, smell and taste. Others, as the larynx and pharynx, perfectly organise and coordinate breathing and swallowing. Also, the combination of voice and hearing permits a sophisticated interaction with the other humans. Surprisingly, all these functions are provided by miniaturised structures. Hence, miniaturisation is the least common denominator of the head and neck field. To image these structures and to identify subtle abnormalities requires the combination of two main factors. The first is the availability of high spatial and high-contrast resolution imaging techniques. Today, "high" goes beyond MSCT, high field MR, CBCT and PET-CT, getting to high-resolution hybrid imaging like MR-PET. The second factor is an adequate knowledge of anatomy, functions and physiopathology. In fact, if not properly trained, the radiologist might not be able either to correctly perform the examination -though using advanced imaging equipment -or to detect key findings for diagnosis and treatment planning. So far, parallel to technical progress, a whole community of radiologists has been walking, sharing a true enthusiasm for the fascinating depths of the head and neck imaging field, like small -very small indeed -structures and complex lesions. And today, the real challenge is to transmit and share this interest, enticing those young radiologists willing to voyage into the future of this wonderful "inner space". The tumour microenvironment plays a major role in determining tumour response to radiation therapy or chemotherapy. As a key factor, tumour hypoxia is considered to be a therapeutic problem, as it makes solid tumours resistant to ionising radiation and some forms of chemotherapy. Overall, it has been shown that modification of tumour hypoxia significantly improved the effect of radiotherapy for the outcome of loco-regional control and with an associated significant overall survival benefit. Therefore, the predictive value of imaging markers of response to treatment is of crucial importance in the management of cancer patients in order to improve the therapeutic index by allowing better individualisation of treatment. In parallel, innovative therapies able to modulate the microenvironment such as flow and oxygenation are currently being developed in order to improve treatment outcome. In this lecture, we will discuss how it is possible to get parametric oxygen images from tumours or surrogate imaging markers of tumour hypoxia (MRI and PET). We will also discuss Learning Objectives: 1. To define structured reporting and its role in radiology. 2. To become familiar with current efforts for structured reporting of imaging procedures. 3. To identify how report consistency will promote the quality of radiology services.
Teleradiology: more disadvantages than advantages R. FitzGerald; Wolverhampton/UK (richardfitzgerald@nhs.net) Teleradiology benefits many patients: those caring for them, and healthcare systems. Many radiologists choose to practice/use teleradiology and derive substantial economic and/or work-life benefits. Large-scale teleradiology is less than 10 years old and we have yet to see the full extent of its negative, often unintended, consequences. Commoditisation of radiology by teleradiology, and increasing pressure on healthcare budgets in this age of austerity may lead to hospital departments losing contracts for less complex reporting, the funding from which is needed to cross-subsidise complex imaging. Local departments may have to curtail services and cut staff. The cut-throat marketplace of radiology reporting facilitated by teleradiology may encourage faster reporting and may discourage comparison with relevant previous imaging and reports, interrogation of electronic patient records, direct discussion with clinicians and service development. This could have adverse consequences for patients. We have yet to see the long-term occupational health and accuracy implications of call centre productivity applied to teleradiology reporting by companies having to secure contracts with ever tighter margins. Radiologists' pay, pensionability, tenure and working conditions could be at risk. Medical regulation of teleradiologists is patchy and this poses risks to patients. Legislative regulatory solutions and their implementation are years away, despite lobbying since 2004. Learning Objectives:
1. To understand the risks to hospital radiology services and their staff from teleradiology companies securing reporting contracts. 2. To explore the possible long term occupational health and reporting accuracy implications in the ever more competitive commoditised radiology reporting market. 3. To discuss the potential implications for patients from current inadequate medical regulation of teleradiologists.
Teleradiology: more advantages than disadvantages L. Donoso; Barcelona/ES (ldonoso@clinic.ub.es)
Teleradiology services, initially limited to consultations between smaller hospitals and tertiary care centres for second opinions and patient transfer, have developed substantially to include international reporting services. Although teleradiology confers many advantages in many circumstances, it also has a number of inherent limitations regarding the proper provision of imaging services and may therefore increase risks for the patient. Teleradiology involves much more than just technology. It is also important to optimize the workflow and communication among radiologists and other medical specialists. It is time we were aware that teleradiology procedures constitute medical acts with implications that go beyond mere reporting. Teleradiology requires a technological infrastructure adapted to distributed environments enabling collaborative networks to improve the efficiency of radiology procedures and radiologists' work/life balance. This presentation will highlight the advantages of using teleradiology and key parts of the guidelines developed for the benefit of patient care. Emergency radiology is one of the fastest growing fields in modern radiology and the delivery of advanced radiological diagnosis and interventional procedures for patients after polytrauma in a 24-hour x 7-day setting remains a very challenging task. Presently, many emergency departments rely much on height quality radiological diagnosis; an exponential increase in the use of MDCT could be observed in recent years with an impressive annual growth rate of 15-20% and a doubling time of only 4.7 years in many emergency departments. Trauma remains the leading cause of death in people below the age of 45 years. Radiology became a key player in the trauma teams worldwide and is today an integrated part of emergency medicine services. Emergency radiology developed into a new subspecialty in modern radiology and only recently a European Society of Emergency Radiology (ESER) was founded. This state-of-the-art session summarises today's high-end use of diagnostic imaging and radiological interventional therapy, as well as all major aspects of patient handling after polytrauma. It is challenging to keep up with technological developments and advanced use of imaging techniques and to provide a well-trained staff for these critically injured patients. Session Objectives: 1. To understand the key role of emergency radiologists in the management of patients with polytrauma. 2. To become familiar with current state-of-the-art emergency radiology services and future trends. 3. To learn about state-of-the-art logistics and the interdisciplinary approach of modern polytrauma patient care.
Ultrasound: why, when, how and by whom? P.-A. Poletti; Geneva/CH (pierre-alexandre.poletti@hcuge.ch)
The term «FAST» (Focused Assessment with Sonography for Trauma) refers to real-time sonographic scanning of four regions for depiction of intra-peritoneal blood, which is generally performed at patient's admission. This method of examination, which does not include direct visualisation of parenchymal injuries, gives an immediate overview about a possible major intra-abdominal injury, especially in haemodynamically unstable patients who may require immediate treatment. The value of FAST in haemodynamically stable patients is more controversial since free intra (or retro-) peritoneal fluid is not always found in patients with blunt abdominal organ injuries, including potentially life-threatening injuries. Besides, the adequate training schedule of FAST operators (radiologist or non-radiologists) is subject to controversy; there is no consensus with regard to the number of examinations an operator performs under supervision before being entitled to perform a FAST examination alone. In spite of the fact that ultrasound achieves a poor sensitivity (40 to 55%) for the direct depiction of parenchymal injuries, a full abdominal sonographic examination, including solid organ analysis, may be useful for the triage of patients towards CT or clinical observation, when time constraint is not a major issue and CT not immediately available. Contrast-enhanced sonography can be useful to improve the detection of solid organ lesions and for the detection of vascular injuries, such as delayed splenic pseudoaneurysms. how the delineation of hypoxic target volumes within solid tumours could be used to optimise the treatment efficacy either by delivering optimal radiation doses into the resistant areas (dose painting) or by delivering an associated treatment for potentiating the efficacy of radiation treatments (by alleviation of tumour hypoxia). Imaging targets for tumour characterisation range from simple size measurements of the tumour to more specific imaging biomarkers on functional, cellular, metabolic and molecular levels. The biodistribution of these specific biomarkers should complement the morphological information acquired using traditional techniques. This lecture will discuss current and emerging imaging techniques for tumour characterisation: (1) Functional measures of tumour blood flow and vascular permeability can be made using MRI, CT and ultrasound. (2) Cellular imaging targets include measurements of water and fat content using MRI. Diffusion-weighted imaging can be used to estimate tumour cellularity, which may decrease following successful chemotherapy or radiotherapy. Emerging techniques allow tumour necrosis to be specifically probed. (3) Changes in the extracellular space can be used to characterise tumours; for example the majority of tumours are acidic when compared with the surrounding normal tissue and recent advances in PET and MRI have been used to detect tumour pH. (4) On the molecular level, cell surface expression of proteins and enzyme activity within the cell have been exploited as imaging biomarkers. PET-and MRI-labelled probes have been developed which can bind to these proteins or mimic endogenous tumour metabolites; for example, fluorodeoxyglucose is a PET-labelled analogue of glucose that is widely used in oncological imaging. In the future, functional and molecular imaging techniques will be used in conjunction with anatomical imaging methods to aid cancer diagnosis, as well as to detect early response to chemotherapy and radiotherapy. The most common pancreatic malignancy is ductal adenocarcinoma, with neuroendocrine tumours (NET), lymphoma and metastases being important differential diagnoses. Non-neoplastic pitfalls include focal pancreatitis and focal fatty infiltration. Multi-phasic hydro-MDCT is very effective in detection of hypoattenuating adenocarcinoma, with a sensitivity of up to 90%. In 5-11% of patients, isoattenuating cancers will only show indirect tumour signs (duct dilatation, contour deformity, or mass effect). MRI is a problem-solving tool in equivocal CT to depict small cancers. Best pulse sequences for tumour detection are T1w 2D-GRE fatsat, dynamic gadolinium-enhanced 3D-GRE fatsat and DWI. Important issues in cancer staging are vascular involvement (celiac trunk, SMA, SMV, portal vein), lymph nodes and liver metastases. 3D MDCT reformations (MIP, VRT, CPR) are helpful to demonstrate vascular invasion, although minimal invasion may elapse CT or MR imaging. NET tend to be hypervascular, which is best seen in the arterial phase. RCC metastases to the pancreas are not uncommon. They are typically hypervascular, mimicking NET at imaging. The most important non-neoplastic solid mass is focal pancreatitis. The duct-penetrating sign at MRCP helps to differentiate focal inflammation from cancer, although multimodality imaging including biopsy is often required to make a definitive diagnosis. Focal steatosis or lipoma is easily diagnosed by chemical shift imaging (T1w GRE in-and opposed-phase).
In conclusion, multi-phasic hydro-MDCT is excellent for pancreatic cancer detection. 3D reformations are helpful for demonstration of vascular involvement during staging. For differentiation between cancer and tumour-simulating disease (focal pancreatitis, steatosis), MRI is complementary to MDCT. Cystic tumours of the pancreas are less frequent than solid lesions, and quite often they are occasionally recognised, as many of these lesions are small and asymptomatic, but they may be associated with pancreatitis or have malignant potential. An accurate differentiation between different cystic tumours is important because they require a different treatment according to their histological type and differentiation, but due to the frequent lack of specific clinical and laboratoristic signs, the overlap of imaging findings between different cystic tumours and between non neoplastic and neoplastic cistic lesions of the pancreas, the management of these patients is complex, and knowledge of symptoms of the patients, natural history and predictors of malignancy for pancreatic cysts are important. When dealing with pancreatic cysts, the aim of the imaging is to differentiate cystic tumour from tumour-like lesions and to characterise cystic tumour, distinguishing benign tumour, which usually does not require surgical excision, from border-line or malignant ones, which must be resected whenever possible. On the basis of imaging criteria alone, it can be very difficult to differentiate non tumoural cystic lesions from neoplastic ones; in order to achieve a correct diagnosis it is important to correlate the imaging findings with the clinical history of the patient, the presence or absence of symptoms and their type. US, CT and MRI are excellent tools which permit to accurately evaluate these lesions, making thus possible their correct management. Diagnosis of trauma-related injuries is a key task in modern radiology. Early, thorough and accurate detection of potentially life-threatening injuries is crucial for fast and targeted initiation of treatment. Conventional radiography (CR) and ultrasound (US) are well established and still represent the basic diagnostic tools for trauma imaging. However, a number of studies have shown a lower detection rate of injuries for radiography and ultrasound compared with computed tomography (CT). Multi-detector CT (MDCT) with its shorter scan time and increased accuracy has become the gold standard for most indications in trauma imaging. As MDCT has a higher radiation dose, its use should be restricted and carefully indicated especially when dealing with a younger patient population. Careful optimisation of imaging parameters has to be performed to minimise exposure and maximise diagnostic safety. Modern MDCT examinations produce a large number of images, which have to be limited to a reasonable number for interpretation. This review article focuses on optimisation of examination protocols and on how to handle the flood of images for viewing and archiving. Arterial haemorrhage in polytrauma remains the leading cause of death. The aim of this presentation is to evaluate interventional radiology as a life-saving procedure. Contrast-enhanced CT has been reported to be an accurate technique for identifying ongoing arterial haemorrhage in polytraumatised patients. TAE may be a rapid, safe and effective technique for treatment of haemorrhage. To increase the efficacy of the method and to reduce complications, the trend is to combine several materials for embolisation. Several embolisation devices are available: a fibrin sponge is used for temporary embolisation (PVA) and microspheres are commonly used for permanent embolisation, whereas detachable balloons, glue, coils and plugs are used for definitive embolisation. Stent grafting permits the exclusion of bleeding and obtaining the patency of the vessel. Different types and sizes of stent grafts are available. The success rate, expressed in terms of haemorrhage control and reduction in transfusion requirement, for pelvic haemorrhage, liver, kidney and spleen is high. Stent grafting has different rates of success related to the injury site. TAE should be performed as early as possible, because effective embolisation must be achieved before severe systemic coagulopathy and multiple organ failure develop. CT allows locating and characterising the haemorrhages, which will reduce the diagnosis time and, above all, improve the embolisation procedures. These features make it very useful in this setting, where the clinical course and prognosis are related to the achievement of haemodynamic stability. The patient presenting with an abdominal mass represents a common clinical problem. Clearly, the differential diagnosis is large and obviously will depend on the age and sex of the patient as well as location of the mass. Of course history and clinical examination are mandatory, but formal diagnosis will usually rest on radiological interpretation. Contrast examinations have now been replaced by cross-sectional imaging and endoscopy and it behoves the clinical radiologist to be aware of the advantages and limitations of these methods in order to reach a diagnosis. Particular difficulty may be encountered when the mass is so large that it is difficult to determine the organ of origin. The purpose of this interactive case discussion is to explore the relative merits of ultrasound, CT, MR and Endoscopy in establishing a diagnosis in two different cases. The cases concerned are 1. a 21-year-old man who presents with a right iliac fossa mass, but is otherwise asymptomatic. 2. A 54-year-old woman who presents with anaemia and a large upper abdominal mass. Active audience participation will be encouraged by means of key pads in order to respond to issues raised during the debate. Clinical presentation: patients with spontaneous (SAH) most often present with severe, so-called thunderclap headache. However, the clinical picture can vary from mild headache to abrupt death. Rupture of an intracranial arterial aneurysm (AA) is the most common source; other non-aneurysmatic causes may be perimesencepahic haemorrhage or SAH secondary to cerebral vasculitis. Arteriovenous malformations (AVM) and dural arteriovenous fistulae (DAVF) may also present with SAH. Diagnosis of SAH is typically by non contrast CT, less often by lumbar puncture or MRI. Intracranial AA that has ruptured carries a 50% chance of re-rupture the first year, with 20% mortality. The risk for re-bleeding from an AVM or DAVF is 5-10%. Imaging strategies to detect aneurysms: The aim of the neuroradiological investigation is to identify AA or other causes for the SAH. CT and CT angiography are effective in finding AA and excluding some pathologies, such as cerebral vasculitis. The sensitivity of MRI or MRA is less well documented. An arterial dissection, a microAVM or a DAVF may not be visible on CTA or MRI/MRA. Thus further investigation with conventional catheter angiography is warranted in all cases where there are doubts about the source of the bleeding and for further anatomical assessment. Different forms of aneurysms: Berry aneurysms comprise the majority of the AA, the rest being partially thrombosed AA or dissecting AA. Screening: Non-invasive screening is with MRI/MRA or CT/CTA and may be warranted in patients with two relatives having suffered from aneurysmal SAH or a family history of AA. Finding of a palpable mass in the abdomen always raises the possibility of an important clinical problem. A potentially life-threatening process, especially malignancy is the major concern. The list of differential diagnosis of "abdominal mass" is a very long one. In the process of differential diagnosis the most common approach is to evaluate the patient according to the gender, age, patient history and coexisting clinical and laboratory findings. The accompanying symptoms and signs and the location of the abdominal mass are the key indicators in the clinician's way of thinking in the process of differential diagnosis. For instance, a palpable mass with acute abdomen or intestinal obstruction will be assessed differently than a mass found incidentally. Imaging is commonly required to confirm or ascertain the diagnosis. Cross-sectional imaging is required to accurately evaluate a palpable abdominal mass in most situations. Ultrasound and computed tomography have each been used successfully in evaluating patients with palpable abdominal mass. Although each modality is appropriate in most situations, the advantages and disadvantages of each modality in certain situations will be addressed and the appropriateness criteria will be reviewed in this lecture. Knowledge of a detailed clinical history is as important to the radiologist as to the clinician. Its impact on the diagnostic accuracy in the interpretation of the images will also be addressed. A palpable abdominal mass has a long list of benign and malignant differential diagnoses. These diagnoses may be as different as a hydatid cyst of the liver, a volvolus, an aneurysm, or a giant renal cell carcinoma. The diagnostic approach is based on two major steps: first, the affected organ must be identified. Second, the differential diagnosis must be established based on imaging characteristics. The choice of ultrasound, CT or MRI should be based on location and size of the mass. Despite the fact that ultrasound is frequently used as a first step, the overview and anatomic orientation in large masses may be hampered, making ultrasound a better technique for image-guided biopsy than for primary diagnosis. In the vast majority of cases, multidetector CT is the first technique of choice. A thin-section protocol should be used to allow for high-quality multiplanar imaging. A pre-contrast scan is usually not required but can be helpful in suspected haemorrhage. For most upper and middle abdominal masses, biphasic imaging in the arterial and portal phase is recommended in order to establish the relation of the mass to the vasculature and to assess vascularity. In the small pelvis, MR is the superior imaging technique. Otherwise, MRI is mainly used for problem-solving. This course will discuss how to use the various imaging tools efficiently to narrow the differential diagnosis, decide about the need for biopsy and establish a suitable therapy. Normal genital anatomy varies with the age and the hormonal status of the paediatric patient. Knowledge of embryology is essential to understand congenital anomalies of the male and female developing genital systems. Common congenital anomalies include cryptorchidism in males and Müllerian duct anomalies and/or obstructing vaginal anomalies in females. Abnormalities of sexual differentiation result from non-accord of chromosomal, gonadal and genital sex. Dysplastic gonads carry a high risk of gonadal malignancy. Acquired disorders include a variety of acute and non-acute conditions such as testicular or ovary torsion, inflammatory and infectious scrotal or pelvic disorders, ovarian cysts and genital neoplasms, ectopic pregnancy and genital (scrotal) trauma. Ultrasound, including Doppler tools, is the primary modality for imaging both male and female genitalia. A proper and dedicated ultrasound technique examination is essential to obtain maximum diagnostic information. Further genital system imaging, when needed, is preferably accomplished with magnetic resonance such as in cases of complex urogenital (female) malformations and oncologic conditions. Except for tumour spread assessment, computed tomography has a limited role in paediatric genital disorders and should be avoided due to the associated high radiation burden. Plain films are obtained to assess bone age in pubertal disorders, and genitography is usually restricted to the evaluation of rare complex genital malformation. Genitourinary tumours in paediatrics usually present as a painless abdominal mass, the patient's age being an important clue in the differential diagnosis. Neonatal renal tumours are rare and mesoblastic nephroma is the most common tumour at this age. In children older than one year, the most common intra-abdominal masses in children are Wilms tumour and neuroblastoma. Wilms tumour represents approximately 95% of paediatric renal tumours (peak incidence of 2-3 years); mainly sporadic, it may occur in a small number of genetically predisposed children; it may be unilateral or bilateral. Abdominal color Doppler-US and chest radiograph are first-line tests; abdominal contrast-enhanced CT or MRI further delineates tumour extension and excludes bilateral disease; the role of CT versus chest radiograph to detect pulmonary lesion is still controversial. US surveillance is recommended in predisposed children. Non-Wilms tumours may represent a significant proportion of renal tumours in children (especially in children aged 12 years); preoperative imaging, however, is of limited value in the differential diagnosis. The differential diagnosis for a painless scrotal mass in a child includes primary testicular tumours, rabdomyosarcoma, and leukaemia. Scrotal ultrasound remains the imaging modality of choice. The presentation of adnexal masses in childhood differs from that in adult women: children may present with poorly localised symptoms, precocious puberty or with an acute abdomen due to torsion or haemorrhage within an adnexal mass. Both imaging studies and the assessment of the patient's hormonal and pubertal status should be considered in the differential diagnosis. Intracranial aneurysms are in most cases the cause of a subarachnoid haemorrhage (SAH). Ideally, ruptured aneurysms should be excluded from the circulation as soon as possible to prevent rebleeding and allow aggressive treatment of SAH complications such as vasospasm. Endovascular treatment of intracranial aneurysms has evolved in the twentieth century from parent vessel occlusion to selective occlusion of the aneurysm sac with detachable microcoils ("coiling"). The first coiling was performed twenty years ago in 1992. The endovascular approach to the coiling of aneurysms has become an alternative to surgical clipping both due to the low procedural morbidity and mortality, and to the possibility to treat aneurysms with complex vascular anatomy and at locations not accessible to clipping. Compared with clipping the disadvantage of coiling is the incomplete occlusion of aneurysms (neck remnants). Therefore, coiled aneurysms need to be regularly followed by angiography or magnetic resonance imaging. Recently, supportive techniques to coiling have been optimised to help occlude the aneurysm and preserve the parent artery. Compliant balloons can be used to temporarily support the introduction of coils in the aneurysm sac. In wide-necked or dissecting aneurysms intracranial self-expanding stents can be placed. Anticoagulants need to be taken after stent placement complicating the post procedural care of recently ruptured aneurysms. The most recent development is the use of flow-diverting stents that alter the haemodynamics inside the aneurysm sac. Based on various patient and aneurysm characteristics the treatment options need to be discussed with a neurovascular surgeon, neurologist and interventional neuroradiologist. At the subacute phase after surgical or endovascular aneurysm treatment, aneurysm rebleeding or intracranial vasospasm must be clinically suspected when the neurological status suddenly worsens including increased headaches, neurological deficit or loss of consciousness. Doppler sonography and brain CT scan must be performed in emergency to confirm the diagnosis and to discuss an additional treatment in selective cases. In the mid-and long-term follow-up, non-invasive imaging is needed to detect aneurysm recurrence, neck remnant regrowing or de novo aneurysm. After endovascular treatment, the rate of aneurysm recurrence may be estimated between 10 and 30% according to the aneurismal size with a risk of rebleeding from 1 to 2% per year. Most of recurrences are detected during the first 6 months after embolisation leading to an additional treatment in about half of cases. MR angiography is the technique of choice including time-of-flight technique and/or Gd-enhanced MR angiography at 6 months after the initial event and then every year during the following 5 years and finally every 3 years in the long-term follow-up. After surgical treatment, the rate of aneurysm recurrence and the risk of rebleeding are much lower when the aneurismal neck has been clipped in good technical conditions. CT angiography using 64-detector rows or more is definitely the best technique to detect post-surgical aneurysm recurrence. CT is usually performed at 3 years after the initial event and then every 5 years.
Learning Objectives: 1. To become familiar with the signs of vasospasm or re-bleeding in the subacute phase.
To learn what is the best technique for follow-up of these patients after treatment. 3. To understand the advantages and disadvantages of CT versus MR angiography after aneurysm treatment (endovascular coiling or surgical clipping).
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equipment available which differ both as to conception and field intensity and the various sequences must respond to precise technical indications. Basically, the structures that are to be evidenced are represented by fibrous-connective tissue, cartilage, synovial tissue and bone, and the modifications that may be caused on these by the varying pathologies must be taken into consideration. Sensitivity, specificity and multiplanarity are all fundamental elements to be considered when evaluating a magnetic resonance of the articulation. The morphology of the structure is best defined with the use of spin-echo sequences, in particular that of T1 weighted images. The sequences that exploit the suppression of fat, or fat--water separation, do seem to be the most suitable for a panoramic and comprehensive evaluation of the joint under study, as the various components may be observed in such a way so as to make a primary diagnostic codification on the underlying problem. Depending on the individual requirements, specific sequences may be added, such as those that give more detailed information/evaluation of cartilage damage, or a better view of flogistic synovial processes. With modern multidetector CT scanners CT arthrography with multiplanar reconstruction in high resolution is possible. CT arthrography is performed with intra articular injection of non diluted iodinated contrast material. CT arthrography of virtually any joint from head to toe is possible. There are several indications and advantages for CT arthrography: 1. Imaging time: imaging time with CT arthrography is much faster than MR. This is especially advantageous for anxious patients. Also for patients with claustrophobia, CT is often a better option. 2. Cartilage: CT arthrography has the advantage of high contrast between cartilage and contrast material. Therefore, CT arthrography may be superior in defining cartilage defects.
3. Postoperative Imaging: in the postoperative patient, artefacts from metal may be present in or near the joint. With CT arthrography these artefacts are often less pronounced, compared with MR imaging. 4. Bone and calcifications: assessment of bony structures may be easier with CT arthrography compared with MR arthrography. Small osseous fragments, such as glenoid rim fractures may be difficult to see with MR arthrography. With CT these fragment or calcifications may be easily seen. 5. MR contraindications: CT arthrography is a valuable alternative in patients that have a contraindication for MR imaging. For example, the diagnosis of a meniscal tear is possible in the same way using CT arthrography as with MR imaging. In the shoulder, CT arthrography is well suited for the assessment of labral lesions, cartilage damage and rotator cuff tendons using the same diagnostic criteria as with MR arthrography. MR arthrography is an outpatient procedure, requires no anaesthesia and is essentially devoid of complications. It is relatively easy to perform, and is a definite aid in patients when conventional MR exams cannot provide for exact information which may be needed for sufficient therapy. MR arthrography, by virtue of its ability to demonstrate accurately intra-articular structures and abnormalities of these structures (e.g. labral lesions -shoulder, hip; partial articular-sided cuff tears), adds an important component to the radiologist`s armamentarium. Although not appropriate for all patients, MR arthrography plays an important role in the evaluation of patients with suspected intra-articular pathology who have equivocal clinical and conventional MR imaging findings. However, even nowadays, clinical use of MR arthrography is limited due to three main reasons: the conversion of a non-invasive procedure into an invasive, albeit minimally invasive, procedure; increased cost and time required to perform MR arthrography compared with conventional MR imaging; and the need to obtain patient`s consent for performing a MR arthrography exam since the use of intra-articular gadolinium compounds has not yet been approved generally. MR arthrography is an outpatient procedure, requires no anaesthesia and is essentially devoid of complications. It is relatively easy to perform, and is a definite aid in patients when conventional MR exams cannot provide for exact information which may be needed for sufficient therapy. MR arthrography, by virtue of its ability to demonstrate accurately intra-articular structures and abnormalities A-275 17:00
The imaging algorithm and imaging examples for most common conditions in neonatal obstructive uropathy (ureteropelvic junction obstruction, obstructive megaureter, posterior urethral valve and stone disease) will be presented and discussed. Imaging urinary tract obstruction is a common query in paediatric uroradiology. The submitted infants have increased by fetal screening ultrasound (US). With the advent of more conservative treatment approaches the task of imaging is not only to establish the diagnosis, but also to identify those kidneys as early as possible which will deteriorate and lose function and/or growth potential and thus benefit from surgery. For these, however, there is at present no reliable a-priori pro-futuro assessment in spite of all modern techniques such as diuretic MR-Urography (MRU). Thus, often repeated follow-up imaging is necessary to monitor the development. Imaging usually starts with US, additionally complemented by diuretic renal scintigraphy and/or MRU; voiding cystourethrography (VCUG) is used for detection of vesicoureteral reflux (VUR). The frequency and timing of investigations as well as the detailed protocol vary within institutions, partially also because of different criteria used for indicating surgery. Intravenous urography (IVU) has practically vanished for this query; apart from few exceptions, the "Whitaker test" is today also seldom performed only for complicated cases (e.g., peri-operatively).
Obstructive uropathy is usually detected foetally and primarily assessed by US.
Once VUR is ruled out, further follow-up will include MAG3-scintigraphy or MRU. CT and IVU are used only exceptionally, with the most common indication in the work-up of complicated childhood urolithiasis. Breast surgery has significantly changed in recent years---from mastectomy to breast conservation as a standard, from single discipline to inter-disciplinarity and from mechanistic to biology-driven strategy. By virtue of all these changes, breast surgeons today offer breast-conserving approaches for almost every breast cancer patient, sentinel-node dissection for most and cure for the majority. Breast cancer is the leading cause of cancer death among women worldwide. Imaging plays a key role in the early detection of breast cancer. Beside imaging accurate treatment is pivotal. It has been shown that cooperation between different disciplines is the base for accurate patient management and improved treatment.
The audience will learn about the importance of multidisciplinary breast centres and their effect on improved detection and treatment of breast cancer. We look forward to welcome you all to get an updated information that could directly be utilised in your clinical services. Breast cancer is the leading cause of cancer death among women worldwide. Imaging plays a key role in the early detection of breast cancer. Mammography is an accepted screening modality. It has been shown that additional imaging modalities like ultrasound and MR imaging of the breast can significantly improve cancer detection and characterisation. An international classification system (BIRADS®) has been introduced and offers a global language for breast imager. In case of BIRADS® 4 and 5 lesions imaging guided biopsies are recommended before definitive surgery. The interpretation of imaging findings and pathologic results (b-classification) is essential and should be performed in multidisciplinary team conferences. It has been shown that such conferences have a strong impact on overall survival because mismanagement and failures can be reduced significantly. During this session the assembly will learn basics as well as about the importance of guidelines and risk management (failure conferences), illustrated by case studies. Clinical management of patients with HCC is a decision-making process that should be approached following evidence-based data. Staging is a relevant topic in these patients because it determines the therapeutic approach. Tumoural extension based on MRI or CT findings and prognosis assessment are critical steps in the management of patients with HCC to indicate treatment, to predict response to treatment and to predict survival. For optimal results any proposal has to take into account liver function, tumour stage and physical status. Up to now, the only proposal linking staging to prognostic prediction and treatment indication is the BCLC approach. Updated BCLC staging in 2012, first presented in 1999 in Seminars in Liver disease will be discussed and also the recent imaging features supporting the tumoural extension. Genomic studies attempted to provide the basis to identify the abnormalities that reflect a higher risk of cancer and the biological profile will be discussed. In cirrhotic liver disease those unfit for surgery but with paucinodular (usually less than 3 nodules of < 3 cm) or intermediate disease (more advanced) are increasingly amenable to locoregional therapies. Treatment in situ is, however, difficult to assess for the purposes of treatment response and continued surveillance. Modified RECIST criteria have been proposed more recently and these aim to quantify and validate in situ tumour necrosis in the late arterial phase as a valid surrogate endpoint, increasingly justified by clear survival benefit after ablation, TACE and anti-angiogenic drugs. Optimised late arterial phase imaging is critical in the diligent assessment of treatment effect and to ensure that residual disease is not left untreated. There are, however, still typical patterns of residual and recurrent disease that the radiologist must recognise whilst becoming familiar with benign post-treatment features seen after ablation and radioembolisation, for example. These can include marginal hyperaemia, portal tract oedema, arterioportal shunting, etc. Post-treatment assessment can be complex but recent work suggests that diffusion-weighted MR and increased, mapped ADC values may be useful in confirming treatment response. Careful subtraction MR (late arterial phase -baseline) has also been shown to be a robust guide to residual disease. PET/CT assessment has proved problematic due to the highly variable degree of FDG-avidity in HCC. Changes in perfusion CT parameters have been observed in different treatment response groups. With regard to ablation semi-automatic volume segmentation processing is now permitting much more accurate assessment of treatment adequacy. Medicolegal consequence is now recognised to be a major aspect of modern radiological practice. Sins of omission and commission are the building bricks of proper imaging technique in a multimodality perspective. Cirrhosis is a fertile soil for the development of different focal liver lesions, and vascular abnormalities can be now currently recognised by morphological and functional imaging including the use of cell-specific MR contrast agents. Presently, imaging acts as the common denominator in clinical decision-making, being instrumental in confirming the presence and staging of HCC and heavily influencing patient management. Apart from its diagnostic capabilities, radiology has special responsibilities in providing minimally invasive treatment options and in assessing tumour response. The current session aims to provide an overall integrated approach of the modern role of radiology in this clinical scenario, especially for all of those involved in providing consultation in a multidisciplinary environment. Although some authors still consider that fine-needle biopsy is safe, accurate, enables final diagnosis of focal liver lesions and is essential for deciding the therapeutic strategy, recent cohort studies have raised suspicions of its utility. The diagnosis with fine-needle biopsy has several limitations: location and size of tumour, clotting disorders or ascites interfere with needle insertion. False-negative results caused by sampling error may occur at a higher rate. Moreover, needle biopsy of HCC may carry a 2% to 5% risk of seeding tumour cells. Typical HCC is supplied mainly via the hepatic artery and the portal flow is lacking or reduced within the tumour. The characteristic HCC profile, including intense enhancement in AP (wash-in) and followed by contrast washout in PVP or EP (washout) on CEUS, MDCT and dynamic MR images using ordinary extracellular agents, has been established. A prospective study indicated a high PPV (> 95%) using these noninvasive imaging criteria (wash-in and washout) for diagnosis of HCC (> 1 cm), which was comparable with that of a liver biopsy. Identification of washout finding in PVP or EP is also crucial for reliable diagnosis of HCC, in particular differentiation between non-neoplastic early arterial enhancement (A-P shunt) and HCC. Based on these recent results, AASLD and EASL recommended that the diagnosis of HCC in cirrhotic patients can be made if these noninvasive imaging criteria are identified on contrast-enhanced US, MDCT or MR imaging without the use of liver biopsy. Cirrhosis is a chronic liver disease defined by the presence of fibrosis and regenerative nodules. These regenerative nodules may either remain stable or enlarge, or evolve to dysplastic nodules and hepatocellular carcinoma (HCC). This multistep carcinogenesis explains why the diagnosis of small HCC remains challenging. Other lesions such as perfusion disorders and confluent fibrosis are also often observed in cirrhosis. Last, the prevalence of peripheral cholangiocarcinoma has been shown higher than in normal livers. Therefore the goal of imaging is to provide clues which enable lesion characterisation. The classical imaging findings are mostly based on lesion size and lesion enhancement on arterial-dominant, portal venous and delayed phases. Yet, both HCCs and other lesions may show atypical enhancement. This lecture will detail the input of functional tools. For instance, combination of classical sequences and diffusion-weighted MR imaging increases distinction between HCC and other lesions developed on cirrhosis. The role and limitations of hepatospecific MR contrast agents will also be stressed.
Learning Objectives: 1. To be aware of the differential diagnosis of focal liver lesions in the cirrhotic liver in a multimodality perspective. Saturday is handicapped by these factors, the emergency radiologist responding to such a patient is at an even greater disadvantage. In case of unconscious or irresponsible patient, having a kind manner, offering adequate information to patients' relatives, checking on the patient's comfort and trying to reassure the patients' relatives are as important for the radiologist as for the physician more directly involved in patient care. It is very important that family members or friends of the patients are politely treated. Moreover, before providing information to the family of a patient with cerebral death, clear communication between the emergency department physician and the radiologist is crucial. Learning Objectives:
1. To evaluate the difficult balance between emergency, safety of diagnostic procedures and patient information. 2. To learn how to inform the patients' relatives in case of unconscious or irresponsible patient. 3. To learn the criteria for cerebral death and the necessity of appropriate communication with the clinician before information is provided to the family. In this presentation the new classification of posterior fossa malformations will be presented. This classification is based on embryology, making it easier to understand and remember. Embryology and radiological features will play together to teach the audience how to differentiate and diagnose this malformations. Knowing the features of these malformations is important, making it possible to look for them in MR scans, done on the paediatric population, suffering from developmental delay, and will lighten the way to the correct diagnosis. Developmental and genetic classification of mid-hindbrain malformations: 1. Malformations secondary to early anteroposterior and dorsoventral patterning defects, or to misspecification of mid-hindbrain germinal zone. 2. Malformations associated with later generalised developmental disorders that significantly affect the brainstem and cerebellum. 3. Localised brain malformations that significantly affect the brainstem and cerebellum. 4. Combined hypoplasia and atrophy in putative prenatal onset degenerative disorders.
an ever expanding industry of regulation, legislation and litigation. It is important for radiologists to recognise the implications of this development not only as an ongoing consideration in daily practice, but also in an attempt to protect themselves from unforeseen legal consequences. The personal impact of medicolegal litigation on practicing radiologists cannot be under estimated, and though this is at its infancy in certain countries, in many others it has gained significant momentum. The presenters in this Session will address and advise an approach to medicolegal avoidance in three clinical scenarios: when the radiologist has made a mistake, specific challenges within paediatric practice and emergency radiology. Session Objectives: 1. To learn appropriate responses following a clinical radiological mistake (miss, inadvertant outcome). 2. To learn potential medico-legal dangers and responsibilities specific to paediatric radiology practice. 3. To learn appropriate responses to medico-legal issues specific to emergency radiology practice.
The correct conduct when you have just made a mistake L. Berlin; Skokie, IL/US (lberlin@live.com)
Ethical standards, moral values and mandates of virtually all professional societies call for full disclosure of medical errors to patients affected by them. However, many radiologists are reluctant to divulge errors because of potential repercussions ranging from simple embarrassment, loss of face or professional standing, to malpractice litigation or restriction of clinical privileges. Nevertheless, divulging the mistake and offering an explanation and apology for it may diminish the patient's pain, assuage the radiologist's guilt and recrimination and strengthen the physicianpatient relationship. All radiologists and non-radiological physicians must always keep in mind that it is their fundamental obligation to act in the best interest of the patient, not in their own personal interest.
Learning Objectives: 1. To understand why facing the patient and complications is always better than hiding. 2. To learn the appropriate words, recognising facts, but not responsibility. 3. To understand the difference between 'normal' complications and malpractice.
Medico-legal issues within paediatric practice: the history, the challenges, and the future C. Owens; London/UK (owensc@gosh.nhs.uk)
Aim and objective of this lecure is to introduce the attendee to the historical, clinical and legal challenges which faced (and continued to face) health professionals dealing with child protection, with special reference and focus upon the unique issues which occur in the paediatric environment. The attendee will have the opportunity to hear a summary gleaned from opinions from those learned health professionals involved with the difficult challenges in non accidental injury and all of the issues arising and pursuing the medicolegal expert dealing in child protection. Specific individual examples from high profile UK legal cases will be highlighted to allow the listener to understand the harsh climate facing those involved in child protection from the legal perspective. 
Case-based review of medico-legal aspects in emergency radiology A. Pinto; Naples/IT (antopin 1968 @libero.it)
Legal considerations relevant to emergency department radiology are significant. Most patients seeking emergency care are acutely ill. Imaging examination must be done promptly and interpreted without delay. Misdiagnosis is the most common allegation lodged against emergency radiologists as a basis for liability. Missing an abnormality on a radiograph performed in the emergency setting is never easy to explain: it can occur for a variety of reasons. Trauma care creates a "perfect storm" for medical and radiological errors: unstable patients, incomplete histories, timecritical decisions, concurrent tasks and often junior personnel working after-hours in busy emergency departments. Although good medical practice helps ensure against claims, the value of effective communication is also important. The emergency department regularly receives inebriated and uncooperative patients with whom the physician has had no prior contact. If the emergency department physician
that intravenous contrast administration (presumably because of a relative but temporary increase in capillary blood volume causing partial displacement of air) can unpredictably increase lung density. Disease processes which lead to partial filling of the air spaces, thickening of the interstitium, partial collapse of alveoli and/or an increased capillary blood volume will also manifest as a pattern of ground-glass opacification. In clinical practice, the recognised causes of ground-glass opacities on HRCT include pulmonary oedema (cardiogenic or otherwise), infections (e.g. pneumocystis jiroveci pneumonia) and some of the idiopathic interstitial pneumonias (e.g. non-specific interstitial pneumonia and respiratory bronchiolitis-associated interstitial lung disease). The presentation will review and revise the causes of physiological and disease-related ground-glass opacification on HRCT. Ground-glass opacity (GGO) is defined as increased attenuation of the lung parenchyma without obscuration of the pulmonary vascular markings on CT images. GGO may be the result of a variety of interstitial and alveolar infectious and noninfectious inflammatory diseases. As an imaging finding alone GGO does usually not allow a specific diagnosis. GGO in inflammatory disorders often will be present in the company of other interstitial or alveolar findings. However, the number of diseases that cause diffuse isolated GGO or GGO as the predominant finding is relatively small and can be prioritised with clinical information. The most common cause of diffuse isolated GGO in immunocompromised hosts are a variety of diffuse, opportunistic pneumonias, e.g. Pneumocystis jiroveci pneumonia (PCP), cytomegalovirus pneumonia (CMV) or herpes simplex pneumonia (HSV), that constitute the first differential. Chronic onset disorders in immunocompetent patients include cellular nonspecific interstitial pneumonia (NSIP), subacute hypersensitivity pneumonitis (HP), organising pneumonia, air-space sarcoid and drug-induced lung disease. In these disorders, ancillary findings such as an associated reticular pattern with traction bronchiectasis/bronchiolectasis (NSIP), mediastinal lymphadenopathy (sarcoidosis), superimposed nodularity or cysts may help to refine the diagnosis.
In patients with collagen vascular disorders, e.g. scleroderma, GGO secondary to pulmonary involvement needs to be differentiated from drug-induced lung disease. This refresher course will put GGO in the context of outpatients versus inpatients, the acuity of clinical symptoms, e.g. fever, cough and dyspnoea, signs of massive systemic inflammation, and the clinical situation such as inhalational history, pneumotoxic drug administration, immunocompromisation, or bone marrow-suppression. This lecture will introduce the neuroradiological findings of the three commonest phakomatoses referred for neuroimaging in paediatric practice, namely Sturge-Weber syndrome, Tuberous sclerosis complex and Neurofibromatosis type 1. I will present the findings on the background of the suspected pathogenesis of the conditions with the aim of making the imaging findings understandable. Appropriate imaging protocols will be discussed for each of the conditions. Ground-glass opacity (GGO) is characterised on HRCT by the presence of a hazy increase in lung opacity that does not cause obscuration of underlying bronchial and vascular margins. Although a very common finding, it also constitutes a very nonspecific term since it can be seen in a variety of different intraalveolar and interstitial processes with different histology including inflammatory, infectious and neoplastic diseases that have a common physiologic mechanism: partial displacement of air. Ground-glass opacity may even be seen in normal processes such as poor ventilation in dependant lung areas and in expiration. Moreover, GGO can represent either an ongoing, active and potentially treatable disease or an irreversible process. In order to interprete correctly this highly nonspecific but very significant finding it is crucial to attempt to further classify the different large main entities in which this radiological finding appears. Are there specific radiological and HRCT findings that can help us differentiate GGO in autoimmune-inflammatory conditions from infectious and neoplastic processes? Are there associated findings other than GGO -such as nodules, reticulation or focal disease and distribution of findings that can narrow the differential diagnosis? Systematic evaluation of GGO and associated findings as well as integration with clinical information (acute, subacute or chronic symptoms) is essential in defining GGO subtypes in order to improve radiological diagnosis. Radiologists who regularly review high-resolution CT (HRCT) should be aware of the range of patterns and, more importantly, their potential pathological meaning. A pattern of ground-glass opacification is one of the more common HRCT findings but, to the unwary, its interpretation can be problematic. An important underlying principle is that a ground-glass pattern may be caused by any process -physiological or pathological -which partially displaces air. Physiological (i.e. non-diseaserelated) ground-glass opacification is perhaps most commonly seen in subjects who, for whatever reason (e.g. breathlessness, obesity), are unable maintain or achieve a satisfactory inspiratory effort during scanning. A generally increased lung density (in contrast to adults) is also a feature in infants and young children simply because there are fewer alveoli in the developing lung. Finally, it is worth noting The number of 7T magnetic resonance (MR) scanners is growing rapidly, and 7T has proved beneficial for MR imaging and spectroscopy. 7T provides the principal advantages of higher image signal-to-noise ratio, changes in T1, T2 and T2* contrast and increased blood oxygenation level-dependent (BOLD) contrast for use in functional MRI. These gains can be exploited to improve the spatial resolution and/or temporal resolution of anatomical and functional images. Changes in contrast can be exploited to enhance depiction of pathology such as lesions in multiple sclerosis, and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Whilst for MR spectroscopy, 7T provides the advantage of increased spectral resolution. 7T has opened up novel contrast mechanisms including susceptibility-weighted imaging (SWI) which provides information on the local variation in magnetic susceptibility highlighting differences in iron concentration and myelin. Ultra-high field provides increased longitudinal relaxation times, providing increased signal changes for Arterial Spin Labelling (ASL). This presentation will discuss the advantages of 7T and the applications which benefit from 7T over lower field MR. The limitations of 7T in the clinical setting will be discussed, including the issue of specific absorption rate (SAR), and B1 and B0 inhomogeneity. Technical developments to overcome these limitations and enable the wider clinical use of 7T MR will be discussed. Over the recent years the clinical MRI field strengths have gradually been increased to 3 Tesla; however, whole body MRI systems with higher fields of up to 9.4 Tesla have become available in experimental settings. Compared with clinical field strengths, MRI at very high magnetic fields has several advantages but also some unique challenges. With increasing field strength the signal-to-noise ratio increases, which can be used to either increase the spatial resolution in the images, or to acquire the images more rapidly. Unfortunately, the energy deposited in the human body via the RF excitation scales quadratically with the field strength. Thus, the specific absorption rate (SAR) is a critical factor in all rapid imaging protocols and requires the design of RF pulses with low SAR, (e.g. VERSE pulses). Inhomogeneities of the RF field which are induced by standing wave phenomena have to be compensated and make the design of efficient spin echo pulse sequences very difficult. At higher field also the field inhomogeneities become larger and stronger imaging gradients are required to overcome the susceptibility-induced image distortion. Stronger gradient systems are difficult to manufacture, and the usable gradient slew rate is limited by peripheral nerve stimulation thresholds. Furthermore, stronger and faster gradients become very loud at high fields, and special measures for sound protection are required. Despite these limitations, high field MRI offers image with very high resolution, it provides unique contrasts, a better spectral separation of the resonance lines and high signal for non-proton applications. MRI has become a vital tool for clinical diagnosis and research. A major current trend is the introduction of more powerful static magnetic fields, in particular human research magnets > 3 T. Advantages of higher magnetic fields include higher SNR and unique tissue contrasts due to enhanced sensitivity to tissue susceptibility differences. As field strength increases, the frequency of the RF fields used for tissue excitation increases linearly according to the Larmor relation, leading to correspondingly shorter wavelengths inside the tissue. Interference and penetration effects make it quite difficult to achieve RF excitation uniformity, particularly in body applications when a large cross-section is imaged, and lead to subjectdependent signal dropouts according to body habitus. The inherent advantages of higher magnetic fields can thus often not be leveraged to realise expected imaging benefits. Preliminary research indicates that these effects can be addressed by use of parallel transmission strategies such as RF shimming and Transmit SENSE: in RF shimming a multi-element transmit coil array is driven with amplitude and phase weightings optimised for a particular body region; Transmit SENSE is a further generalisation in which different RF pulse waveforms are applied to each element. These novel excitation strategies result in alterations of the RF energy distribution in the tissue, making new demands on the prediction and monitoring of SAR and concomitant tissue heating. Once these challenges are mastered, it Most biliary intervention is performed endoscopically but endoscopy may fail and the antegrade placement of a catheter into the bowel assists endoscopic access. Previously, the primary management of malignant bile duct obstruction was with the endoscopic placement of 10 F plastic stents. When ERCP failed, PTC using a 7 F catheter allowed the placement of an endoscopic 10 F stent but kept complications low because of the avoidance of a 10 F trans-hepatic track. Now, with the advent of 6 Fr trans-hepatic delivery systems for 10 mm diameter metal stents this combined procedure has fallen into disuse. Endoscopic access for the management of stones can fail, most commonly because of a peri-ampullary diverticulum.
In this situation, a trans-hepatic approach can allow wire-guided sphincterotomy leading to successful endoscopic management and the avoidance of open bile duct surgery. This presentation will describe the technical aspects of this approach together with illustrative case examples. The technique has a very high success rate with complications of haemorrhage, bile leak and cholangitis in approximately 10% of patients. Polya-gastrectomy is declining in its incidence and experienced endoscopists who can deal with these patients are becoming uncommon. A transhepatic approach may allow a guide wire to be passed along the afferent loop for endoscopic access. There is a small but growing experience of EUS-guided biliary drainage for the management of biliary obstruction. Gastroenterologists favour this approach because it appears to generate fewer complications than a trans-hepatic approach and is certainly less uncomfortable for the patient. These newer techniques will be illustrated and discussed. The concept of reference dose levels was introduced in the UK to act as an aid to radiation protection. Initially, DRL were calculated for common diagnostic x-ray examinations and in more recent years the concept has been extended into interventional studies. The aim of DRL was to indicate abnormally high doses for common x-ray examinations. Recent literature states that DRLs are still very variable across Europe and Asia for common examinations using similar x-ray equipment. Hence, patients are consistently receiving higher radiation doses in different countries. It has also been reported that poor image quality results in unnecessary radiation exposure to these patients. Simple measures can be put in place by the radiographer to ensure that image quality is maintained and the ALARA principle is adhered to at an international level. The use of anti-scatter grids, standardised techniques, DRLs and the impact of digital imaging will be explored and recommendations for best practice made. These simple modifications to technique can be easily implemented into daily practice in the imaging department and have achieved dose reductions of up to 49% with minimal loss of image quality during simulated experimental conditions. The session will conclude with a discussion on the concept of producing a diagnostic image which is adequate for the purpose with a lower radiation dose, rather than optimum image quality with a high radiation dose.
Benign biliary stenoses may be due to postoperative injury (laparoscopic cholecystectomy, gastric/hepatic resection and bilio-enteric anastomosis), post-liver transplantation, post traumatic (blunt or penetrating trauma), inflammation associated with lithiasis, congenital (usually in paediatric population), parasitic and post radiation. Internal biliary fistulas occur due to acute inflammation with obstruction that leads to adhesions, erosion of the bile duct or gallbladder wall and eventual erosion with formation of a fistula. The most common causes of benign fistulas are cholelithiasis, peptic ulcerartion and Crohn's disease of the duodenum. Diagnosis of benign biliary strictures is usually performed with Ultrasound, Computed Tomography, Magnetic Resonance Cholangiopancreatography or Endocopic Retrograde Cholangiopancreatography (ERCP), whereas biliary fistulas are difficult to diagnose with non invasive methods and the diagnosis is usually made either with Percutaneous Transhepatic Cholagiography, ERCP or even intraoperatively. Percutaneous approach is an established method of treatment of benign stenosis, particularly when they are a result of bilio-digestive anastomosis, either with the use of multiple and prolonged balloon dilatations, or with the use of plastic or metallic (bare or covered) stents. Biliary fistulas can be percutaneously treated by correcting the reason keeping the fistula active (patent). A biliary stricture or stone may be such a reason and can be dilated or removed, respectively, either through the existing fistula or through a new transhepatic access. Embolisation of the fistula tract or use of a metallic stent can be also discussed. In case of portalbiliary fistula, embolisation of the portal branches or positioning of a covered stent might be the solution. Bile tract complications occur in 10-35% of liver transplants, with major incidence in partial LT. The incidence is highest in the first few month after LT and 80% are diagnosed within 6 months. Type of surgical anastomosis, cold and warm ischaemic liver injury and pre-existing biliary diseases are all factors influencing the frequency, type and severity of complications which include biliary strictures, bile leakage, stone formation and bilomas. Screening sonography (38% sensitivity) may be misleading due to false-negative results. Therefore, systematic PTC at 6 month after LT is recommended in order to disclose biliary complications at early stage. Main indications for percutaneous approach include 1. Early anastomotic strictures unaccessible to endoscopy (segmental graft or hepaticojejunostomy anastomosis The screen-film system has been the primary tool in radiology for over a century and the radiation dose was a minor consideration during the early days. The technology evolution for digital radiography provided a lot of advantages, like a better workflow, a high image quality and when properly used can decrease the dose per exam. It is mandatory for radiographer follow the whole process of transition from screen-film to digital system and has thorough knowledge of new installed technology. Internationally, medical radiation accounts for the majority of radiation exposure to the public. With the increasing international focus on radiation protection in clinical practice, there has never been a more appropriate time to promote education in this area amongst the radiography community. With the advent of new technologies and changes in working role, the radiographer's role is ever developing. Because radiographers are directly involved with the technology and patients on a daily basis, their role in quality assurance (QA) and radiation protection is pivotal. This presentation will focus on the current role of the radiographer in QA and radiation protection from a European perspective and will explore the increasing role of QA in the clinical department. The importance of appropriate education and training and the types of education currently available for radiographers will be discussed alongside current literature and guidelines available in this field. The experience of an online radiation safety course and its impact on the role of the radiographer will be reviewed. The future of the radiographer's role in this field and deficiencies in current practice will be examined and conclusions will be drawn. Meta-analyses of studies (mostly nonconsecutive patient series) showed that breast MRI detects otherwise occult ipsilateral and contralateral disease in 11% and 4% of women who had the test, respectively. The ongoing controversy regarding preoperative breast MRI and the balance between potential advantages (reduction in re-operation rate for positive margins and ipsi-/contralateral recurrence) and drawbacks (overdiagnosis/overtreatment) persists given limited evidence. Association of increasing use of preoperative MRI and increased rate of mastectomies has been suggested. Two recent randomized controlled trials did not show advantages from preoperative MRI but have been criticized due to various limitations. Thus, EuroAIM in cooperation with EUSOBI, designed an observational multicenter study which will analyses individual women data: "Preoperative breast MRI multicenter international prospective analysis (MIPA) study". Two concurrent consecutive cohorts of newly diagnosed breast cancer patients defined by receiving/not receiving preoperative breast MRI will be compared matched for age/breast density. Analytical adjustment will be performed for relevant covariates. All involved centers will be high-volume breast imaging/care facilities. We plan to enroll about 2,600 women from 18 to 80 years of age with newly diagnosed breast cancer: 1,300 for the MRI group and 1,300 for the no-MRI group. Rate and type of changed surgical planning in the MRI group will be assessed. Primary outcomes will be rate of primary upfront mastectomy and re-excision rate for positive margins. Secondary outcome will be ipsilateral recurrence rate, contralateral cancer rate, and distant metastasis occurrence during 5-year follow-up. Recent results show that CTC is an accurate test to detect colonic polyps and masses. Although the CMS has declined reimbursement for screening CTC in the United States in 2009, CTC is being used increasingly in the radiologic community. Indications for CTC include examination of elderly and symptomatic patients as well as examination of asymptomatic adults who select CTC as their screening option for colorectal cancer. In the light of a lack of reimbursement certain steps have to be taken to set up a successful CTC service. In many countries, ionizing radiation cannot be used for screening examinations other than mammography. Therefore, radiologists have to be well familiar with radiation exposure considerations in CTC. Also, specialists have to know about indications, contraindications, and pitfalls that have to be followed for succesful CTC. Furthermore, training strategies for CTC interpretation will be discussed; in the beginning, expert double reading is recommended. In this presentation it will be outlined which steps have to be undertaken to set up a succesful CTC program. Learning Objectives:
1. To appreciate the need for training prior to CTC and understand the role of training courses and double reporting. 2. To become familiar with ways of maximising service efficiency. 3. To appreciate the differences in approach between setting up a service for older symptomatic patients and setting up colorectal cancer screening. 4. To learn how to formulate local polyp reporting guidelines and how best to integrate the service with the needs of local clinicians. 5. To learn a basic audit framework. CT colonography is widely considered as the radiological examination of choice for the large bowel. It has a variety of indications including investigation of symptomatic patients and potentially in screening of asymptomatic individuals. Radiologists undertaking CTC must be well versed in the technical protocol requirements as well as how best to interpret the examination. Furthermore, they must have a thorough understanding of the epidemiology of colorectal neoplasia, so reporting practices are evidence based. This session will guide participants in a stepwise fashion through the pre-requisites to high-quality CTC in 2012. Lectures will discuss how to achieve a high-quality examination, how to interpret the examination and finally consider the requirements for setting up and running a successful CTC service.
A.
Step 1: bowel prep and distension A. Laghi; Latina/IT (andrea.laghi@uniroma 1 .it)
Bowel preparation and colon distention represent two critical steps of CT colonography (CTC) examination. The ideal colonic preparation is still under debate. It is general consensus that patients should undergo a low-residue diet before the examination, and to ingest a cathartic agent the day before. Different cathartic agents are available. In order to improve sensitivity and specificity, "tagging" of residual fluids, in combination with the administration of a cathartic agent, is now routinely used. It consists in the administration, prior to CTC, of water-soluble iodinated contrast medium or alternatively diluted barium sulfate suspension. Since bowel cleansing is consistently the most unpleasant part of colonic examination, minimally-invasive preparation schemes (using mild or no laxatives at all), more comfortable and less demanding for patients, are now available. For reduced bowel preparations the combination with faecal/fluid tagging is mandatory. Gaseous colonic distention is another critical step since collapsed bowel is a frequent cause of missed lesions at CTC. The use of a rectal balloon catheter has been replaced by a thin rubber tube in order to improve patient compliance and to minimise risks of perforation. To further improve Patient comfort, automatic CO2 insufflation represents a possible alternative to room air. The administration of a spasmolytic agent is particularly useful in the case of colonic spasm, typically in sigmoid colon, and in patients with severe diverticular disease. The use of a MDCT scanner is mandatory and dedicated scanning protocols need to take into account radiation exposure, especially in individuals undergoing screening CTC. Learning Objectives: 1. To become familiar with the options available for bowel preparation, including the use of tagging agents, and to learn three examples of a bowel preparation regimen that 'works'. 2. To learn a step-by-step evidence-based approach to distending the colon (including spasmolytic agents, patient positioning, insufflation technique). 3. To appreciate optimum CTC acquisition parameters. 4. To become familiar with described complications (notably perforation) and how the risk may be minimised.
A-316 08:58 B.
Step 2: analysis and how to avoid pitfalls T. Mang; Vienna/AT (thomas.mang@meduniwien.ac.at)
The evaluation of CT colonograpy (CTC) studies is based on detection, interpretation and reporting of colonic findings. It is performed on a workstation equipped with dedicated CTC software by a primary 2D or a primary 3D approach. In either case, the alternative viewing technique must be available for rapid correlation and characterisation of suspicious findings. Primary 2D evaluation is based on "lumen tracking" by interactively scrolling through the axial slices and multiplanar reformatted images, focusing only on the air-distended colonic lumen from one end to the other. Primary 2D evaluation provides information about the attenuation of findings during the search process and is time-efficient. Primary 3D evaluation is based on 3D virtual endoscopy in an antegrade and retrograde direction and increases both, the conspicuity, especially of small-and medium-sized polyps, and the duration of Given the vast selection of possible imaging modalities and techniques, it is practically impossible to detail all the imaging techniques and typical findings of Neuroradiology in 25 minutes. It has become clear lately, that except for politrauma patients -where CT is still the first method of choice -MRI is the gold standard for Neuroradiology examinations and thus my talk will focus on MRI. In the first part of the talk I will give a short introduction to the MRI techniques used in the evaluation of focal neurological disorders and discuss their utility and possible pitfalls.
In the second part I will highlight some hot topics of diagnostic Neuroradiology, starting with recent changes in diagnostic guidelines, e.g. in acute stroke, where MRI become the first modality of choice. Then I will continue with lesser-known but nevertheless important diseases: the acute disseminated encephalomyelitis (ADEM), the posterior reversible encephalopathy syndrome (PRES) and the neonatal asphyxia. I will detail the up-to-date MRI protocols, the differential diagnostic questions and the possible pitfalls. Original clinical cases illustrating the importance of appropriate imaging modalities in the right interpretation of neurological symptoms and evaluation of correct diagnosis are presented. The role of clinical history and disease course are accented. The spectrum of pathologies includes vascular, inflammation, metabolic, degenerative diseases and tumours of the central nervous system. Each case story is described shortly in a standard form followed by demonstration of typical CT and/or MRI images. In some cases conventional MRI is supplemented by MR spectroscopy, DTI and MR tractography images. Follow-up images are presented where appropriate. Several diagnostic options are offered for attendants. The audience will be asked to participate in the diagnostic process by the use of voting pads. After highlighting of final diagnosis the basic differential diagnostic considerations will be summarised and emphasised from the clinical and imaging point of view for each presented case. Learning Objectives:
1. To review typical cases illustrating the role of imaging modalities in the differential diagnosis of focal neurological symptoms. 2. To get involved in the diagnostic process by the use of electronic voting pads. 3. To understand the conclusion that may be drawn on the basis of the discussed cases.
characterising incidentally detected lesions in patients with cancer. The performance of the established and new techniques in CT, MRI and to a lesser extent PET, that can be used to distinguish benign adenomas and malignant lesions of the adrenal gland will be reviewed. With the increasing use of imaging, incidentally detected renal masses are very common. While masses detected by CT or MR usually can be properly classified, renal masses detected by ultrasound frequently require further workup. The following considerations determine the diagnostic workup: simple cysts are very common but may present atypically. Renal cell carcinomas may be cystic but usually display at least a small solid component. Renal cell carcinomas have a bad prognosis when metastasized but metastases hardly every develop before the tumour has reached 3 cm in diameter. Differentiation between solid tumours by imaging alone is exceedingly difficult, save for the identification of angiomyolipomas in adults. This course will discuss suitable diagnostic algorithms based on the initial presentation of the mass. Typical imaging findings of various benign and malignant renal masses will be presented. The role of the Bosniak classification will be illustrated. Newer developments such as a wait-and-see approach or primary biopsy for small solid renal masses will be discussed. When dealing with "focal neurological disorders", what kind of neuroimaging should we apply and what should be the optimal diagnostic work-up? What pathological condition must be searched for in function of the focal neurological signs? When is imaging playing a vital role? The radiologist must and should remain first of all a Medical Doctor: he/she should keep close contact with the clinical world and always keep an active dialogue with the referring clinician. As medical imaging has become more and more sophisticated and also more expensive, it should not just be a "screening procedure" for brain pathology but should be performed in order to confirm a clinical, suspected diagnosis. The radiologist must answer precise questions about the patient's suspected pathological condition and if necessary, discuss the imaging findings with the clinician in order to narrow the differential diagnosis. Each MRI or CT must be performed with a clear knowledge of the clinical question and the suspected pathology. Symptoms and clinical history may already strongly orient towards lesion location and even the nature of the pathology. Still, similar symptoms may be present in multiple and very diverse pathological conditions such as infectious, neoplastic, haemorrhagic, vascular diseases. Therefore, the radiologist should make the proper choices of imaging techniques, especially with MRI, where imaging sequences have become numerous: by knowing the clinical history, and what the focal neurological disorders are, a good, "clinically conscious" radiologist will undoubtedly be of greater "added value".
S80 C B D E F G H A
Learning Objectives: 1. To become familiar with the different types of traumatic haemorrhage (epidural, subdural, intracerebral, subarachnoid). 2. To understand the difference between primary and secondary traumatic brain lesions. 3. To understand how the brain can be severely damaged in closed head injuries (deceleration trauma, diffuse axonal injuries).
C. Spine trauma A. Cianfoni; Charleston, SC/US (acianfoni@hotmail.com)
The role of imaging in acute c-spine trauma is to assess the spinal injury, determine the stability and instability of the injury, evaluate integrity of neural elements, to direct appropriate management, and to predict neurological outcome. A set of clinical and/or amnestic criteria can be very useful in identifying patients who need acute spinal imaging. Underdiagnosis should absolutely be avoided, due to potentially devastating sequelae. Awareness of the strengths and limitations of plain radiography, MDCT and MRI in the diagnosis of spinal injuries is fundamental. Traumatic spinal lesions are represented by fractures, dislocations, ligamentous disruption, disc injury, acute disc herniations, and epidural haematomas, often combined, with variable involvement of neural structures. Fractures and dislocations usually occur based upon a single predominant mechanism of injury. The six major mechanisms of injuries are hyperflexion, simultaneous hyperflexion and rotation, hyperextension, simultaneous hyperextension and rotation, vertical compression or lateral flexion. The cranio-vertebral junction and the mid-lower cervical spine have different anatomy and different pattern of injuries. This lecture will review the role of different imaging modalities clinically used to assess the cervical spine in acute trauma patients and the various injury patterns. Elements of imaging diagnosis in paediatric patients will be presented. Imaging pitfalls will be also discussed. Endometriosis is defined by ectopic endometrial glands and stroma and sensitive to changes in estrogen leading to peri-menstruel bleeding causing pain and adhesions. Ultrasound is the first imaging method to diagnose endometriosis of the ovary and the bladder. Posterior localisations of endometriosis pelvis include utero-sacral ligaments, the retrocervical region, the posterior vaginal fornix and the recto-sigmoid wall. Those lesions can be diagnosed by trans vaginal ultrasound but include difficulties due to a limited field of view, pain during mobilisation of the probe and absent bowel or vaginal distension. MRI of the pelvis has an excellent sensitivity for all pelvic localisation of endometriosis. Endometrioma is typically hyper-intense at T1 fat-suppressed sequences and of variable signal intensity at T2-weighted imaging. Other localisations are displayed by focal thickening of ligaments/walls or a T2 hypo-intense nodule. Vaginal or bowel wall distention due to ultrasound gel filling, thin slice sections and slice orientation perpendicular to the main direction of the organ helps identification and diagnosis of extent of deep localisations of endometriosis. Intra-venous contrast injection details depths of wall invasion and adnexal mass characterisation. Rare lesion sites such as the abdominal wall, the round ligament, the sciatic nerve or the parametrium can also be detected with MRI. A delayed contrast-enhanced URO-MRI might be useful to display ureteral lesions and the urinary tree. According to lesion localisation, extent and clinical symptoms additional imaging methods might include CT of the abdomen for small bowel endometriosis and hysterosalpingography to check tubal patency in infertility patients. Maxillofacial injuries may occur in isolation or in conjunction with cranio-cerebral trauma. The goal of imaging is to depict the presence, location, and displacement of fractures, to delineate compromise of airways, ocular motility, and dental occlusion and to recognise concomitant skull base and intracranial injury. A concise imaging strategy employs MDCT with thin multiplanar reformations to delineate and classify fractures, to assess stability and to provide a basis for surgical midface restoration. 3D techniques add information in complex fractures and in perception of fracture displacement. In isolated mandibular trauma, panoramex and pa radiographs maintained a certain role, which is challenged by superiority of CT or Digital volume tomography (DVT) for condylar fractures as well. MR in maxillofacial trauma serves as an adjunct to CT in case of injury to cranial nerves, dura, vessels, orbital contents and brain parenchyma. Late complications of maxillofacial trauma related to sinus drainage, ocular motility and dural integrity require a combination of MR and CT as well. Maxillofacial fractures are classified into mandibular, zygomatico-maxillary fractures including LeFort I and II fractures, centrolateral Lefort III fractures and nasoethmoid fractures. Mandibular fractures comprise dento-alveolar, subcondylar, condylar neck and condylar head fractures. Orbital fractures are divided into inferior and medial blowout fractures, orbital trap door and roof fractures. Orbital penetrating injuries are an indication for both CT and MR to recognise the type_organic versus nonorganic_and the location of a foreign body and to depict potential retro-orbital, optic nerve and intracranial brain and vascular involvement. Neuroimaging techniques constitute an essential part of the diagnostic work-up of patients with traumatic brain injury (TBI). In the acute setting, imaging findings determine patient management and influence clinical course. CT remains the first choice technique to determine the presence and extent of injury, and to guide surgical planning. Multi-detector CT allows simultaneous assessment of head and cervical spine, obviating the need for plain radiographs. From a clinical point of view, it is important to understand the difference between primary and secondary TBI. Primary injuries occur as a direct result of the impact with damage to brain tissue. Examples include fractures, different types of traumatic haemorrhage (epidural, subdural, intracerebral, subarachnoid), cerebral contusion, diffuse axonal injury (DAI). CT-angiography is useful to document traumatic blood vessel injury. Whenever there is a discrepancy between the patient's clinical status and imaging findings, MRI is indicated. Secondary brain injuries are caused by systemic factors such as increased intracranial pressure, edema, brain herniation, decreased cerebral blood flow, excitotoxic damage. These lesions can be documented with multiparametric MRI including diffusion, perfusion, and susceptibility-weighted imaging. Diffusion tensor imaging with fractional anisotropy mapping may show microstructural abnormalities in patients with mild TBI, even when traditional MRI sequences appear normal. Neuroimaging also plays a role in the chronic stage of TBI, identifying sequelae, determining prognosis, and guiding rehabilitation. In conclusion, advances in neuroimaging improve our understanding of the pathophysiology of craniocerebral trauma and allow us to detect abnormalities, even in patients with mild TBI, when routine imaging studies appear normal. US and MR are excellent imaging techniques for studying tendons and muscles. During this integrated session we will review the advantages and disadvantages of US and MR for the diagnosis and follow-up of sports injuries. Tricks of both techniques will be addressed, together with some useful guidelines for specific sports injuries. Ultrasound is being used at the pitch side and in sports medicine practice as an adjunct to clinical practice. With this in mind there are a number of questions that will be answered in these talks: 1. when are US and MRI the primary imaging and when are they complimentary? 2. What advances have there been in US and MRI imaging to help advance our use of these techniques in tendon and muscle injury? 3. Should we be aspirating haematomas and using autologous blood injections or PRP to treat tendon or muscle disease? 4. Can we predict the athletes return to sport?
Muscle imaging is inherently complex and presents unique morphological challenges and continuing integration of dynamic, physiological and functional capabilities as imaging technology progresses. In sports medicine, high-resolution ultrasound (US), which is now available not only in high-end equipments but also in portable machines, has proved to be an excellent tool to evaluate muscle strain and contusion injuries in athletes, providing good correlation with clinical findings. In the acute phase, US has nearly equal sensitivity to MR imaging to diagnose muscle strains, except in the first few hours after the injury, when fresh haemorrhage and oedema have similar echogenicity to normal muscle and strains may go unnoticed. Later in the process, US has been shown to be a useful tool in assessing the sequential stages of muscle repair. Local complications, such as vein thrombosis, irritation of adjacent neurovascular bundles, chronic haematoma and myositic ossificans can be demonstrated with this technique as well. However, US tends to underestimate the extent of injury and the abnormalities seen disappear more quickly when compared with MR imaging. At least in elite athletes, MR imaging seems, therefore, to play a more significant role in management of muscle injury, particularly when decisions regarding the time at which the patient can return to play are needed. By contrast, US is quicker, more accessible and cheaper than MR imaging. In most clinical settings, US should be regarded as the first-line imaging choice for assessing skeletal muscle injury. Learning Objectives: 1. To understand the mechanism of injury of muscles in athletes. Sports activity can affect tendons due to chronic overuse or acute injury. Both can result in complete tendon rupture. US is helpful not only in precise assessment of rupture severity and extent, but also in assessment of tendon degeneration, where rupture of individual collagen fibres stimulates a chronic cycle of reparative response caused due to repetitive microtrauma. In chronic tendinopathy histopathological changes, such as hypoxic, mucoid, calcifying, or lipoid degenerations are present. US enables differentiation of partial tears, tendinosis, tenosynovitis or paratendinosis because of active and passive dynamic examination possibilities and high-resolution capability when using high-frequency probes. US developments as power Doppler US, sonoelastography and contrast-enhanced US allow further new insights into tendinopathy. With the use of US, tendon changes can be Learning Objectives: 1. To appreciate the proposed mechanisms of pathogenesis of endometriosis and the relationship with the distribution of disease. 2. To be able to identify the most common imaging findings of endometriosis and to discuss the differential diagnosis.
A-327 09:00 B. The acute female pelvis E. Sala; Cambridge/UK (es 220 @radiol.cam.ac.uk)
Imaging plays a crucial role in diagnosis and management of female patients presenting with acute pelvic pain. The combination of transabdominal and transvaginal US is the study of choice for initial evaluation of acute female pelvis. Colour, power and spectral Doppler provide additional information regarding associated vascularity which is of particular importance when evaluating for presence of ovarian torsion. US is very accurate in determining the specific cause of acute pelvic pain; common causes include ovarian cyst rupture, persistent corpus luteum cysts, ectopic pregnancy and endometriosis. US can reliably demonstrate the presence of a pyosalpinx or tubo-ovarian abscess complicating pelvic inflammatory disease. US is the initial modality for evaluating patients with a pelvic mass with or without suspected torsion. It can confirm the presence of the mass, establish its organ of origin and demonstrate its internal consistency (endometrioma versus dermoid versus complex/solid mass) as well as complications such as torsion, rupture, etc. US is also the first imaging modality for assessment of lost intra-uterine coil devices which can present with acute pelvic pain. CT may be performed after US to visualise the full extent of disease in severe cases of tubo-ovarian abscess. It is also indicated if clinical symptoms mimic appendicitis and is very useful in detection of post-operative complications. MRI is a problem-solving modality. It helps differentiate benign processes that necessitate specific immediate therapy from those that can be managed expectantly. This triage is particularly important in pregnant patients presenting with acute pelvic pain or indeterminate pelvic mass. Learning Objectives:
1. To learn about the role of US as the primary imaging modality in the evaluation of the acute female pelvis. 2. To be able to recognise the added value of CT and MRI in the assessment of acute female pelvis. Uterine artery embolisation (UAE) is a non-surgical intervention for treating symptomatic uterine leiomyomas and represents an alternative to surgical removal (hysterectomy, moymectomy, hysteroscopic resection). The indication for uterine artery embolisation crucially relies on the pre-interventional assessment of symptomatology and burden of disease. Especially the location, size, and number of leiomyomas are important to determine the treatment options of the patient. As a rule, both single and multiple fibroids can be treated by UAE. However, the clinical outcome of UAE does not primarily depend on the number and location of the individual tumours (subserosal, intramural, transmural, submucosal) but the infraction rate of fibroids. MR guided high-intensity focused ultrasound (HIFUS) is a non-invasive treatment option for symptomatic leiomyomata. In contrast to UAE and surgery it lacks the invasiveness of these procedures since the targeted leiomyoma is ablated by energy transmitted through the skin by focused ultrasound while exact delivery is monitored online by MR Imaging. Size, location and number of fibroids are limiting factors for the application of HIFUS. Magnetic resonance imaging (MRI) is superior compared to ultrasound in delineating the extent of fibroid disease and excluding other pathologies or disease processes that may mimic fibroid-related complaints. Further advantages of MRI result from the use of MR angiography and contrast-enhanced imaging in assessing outcome and complications following UAE. This presentation summarises the current role of UAE and ultrasound ablation (HIFUS) for uterine leiomyomata as well as the role of imaging before and after the procedure. of normal of 10 mm is universally applied. However, size criteria alone are unreliable: CT for lung cancer staging has a pooled sensitivity of 51% (i.e., false-negative diagnoses of metastatic deposits in nodes < 10 mm), and a specificity of 86% (i.e., false-positive diagnoses due to enlarged benign nodes). With MRI the same size criteria apply. However, additionally, imaging features such as central necrosis on T2w fatsat or gadolinium-enhanced images are suggestive of metastasis (or suppurative infection). Lymph node-specific USPIO MR agents can depict tumour deposits in subcentimeter pelvic nodes. Unfortunately, they did not receive market approval. DWI is helpful in identifying in lymph nodes as they exhibit high SI with higher b-values. However, diffusion pattern of benign and malignant nodes overlap, so that measurement of ADC values do not aid in characterisation. Despite the use of modern MDCT and MRI techniques, lymph node characterisation needs further improvement. Up to date lymph node staging is based on size and shape criteria only; however, micrometastases can also be present in normal sized lymph nodes and nodes can be enlarged due to inflammatory changes. New contrast agents in MRI such as ultrasmall particles of iron oxide have substantially improved the diagnostic accuracy of lymph node staging compared with conventional MRI. Unfortunately, USPIO is not commercially available and therefore new approaches to differentiate benign from malignant lymph nodes are required. Diffusion-weighted MRI (DWI) is a noninvasive method that provides microstructural information on the underlying tissue. Up to date several studies mainly in the pelvis have shown promising results to detect lymph nodes and also to allow differentiation between benign and malignant nodes with reported sensitivities of 79-87% and specificities of 74-93% based on the underlying mean apparent diffusion coefficient (ADC) value. In these studies any size of lymph nodes has been included with the smallest short axis diameter of 5 mm. There is an overlap between ADC values of benign and malignant nodes; therefore, further studies with histopathological correlation are needed to reduce the high rate of false-positive nodes. Combination of USPIO and DWI might facilitate and improve lymph node staging in the future provided that USPIO will be available. Currently, nuclear medicine uses sentinel node scintigraphy and PET with the glucose analogue fluorodeoxyglucose (FDG-PET) to detect lymph node metastases. Sentinel node scintigraphy images the lymphatic drainage of a tumour. A radiolabelled colloid is injected at the site of the tumour and the drainage of the colloid to the regional lymph node is imaged with a gamma camera. Intraoperatively, lymph nodes are identified with a handheld gamma probe. Lymph nodes that have accumulated the radiolabelled colloid are resected and analysed histopathologically. Randomised studies have shown that in patient with breast cancer sentinel node biopsy significantly reduces the rate of complications as compared with systematic axillary dissection. Conversely, the risk for axillary recurrence is not increased. FDG-PET imaging can detect metastases in lymph nodes that are not pathologically enlarged. Furthermore, many unspecifically enlarged lymph nodes do not demonstrate increased FDG uptake. However, it is important to note that false-negative cases can occur in very small lymph node metastases, in mostly necrotic lymph node metastases or in tumour types with low metabolic activity. False-positive findings occur in active inflammation. Despite these limitations, studies have clearly documented that FDG PET significantly improves the accuracy of lymph node staging in several malignant tumours. In non-small cell lung cancer randomised trials have shown that FDG-PET significantly improves patient management by avoiding unnecessary surgery. Ongoing preclinical and initial clinical studies evaluate radiolabelled amino acid analogues that are potentially diagnosed before they become symptomatic and a reduction of tendon load and initiation of treatment before the condition becomes chronic seem to gain important place in therapeutic regimes. Furthermore, US-guided therapies are advisable over blinded-guided injections to minimise side effects and to allow an accurate targeted therapeutic approach. MRI is an excellent tool in depicting sports-related muscle and tendon injuries. Most sports-related muscle injuries are located in the lower extremities and some are quite deep-seated, thereby somewhat harder for US to show. Another advantage of MRI over US in this setting is that concomitant bone injury is readily displayed. US is admittedly a high-resolution, well-tolerated and readily available tool for musculoskeletal soft tissue injuries. On the other hand, considerable expertise is needed for musculoskeletal US, which is also quite time consuming. During the time needed for a single musculoskeletal US to be performed and reported by an expert musculoskeletal radiologist, several MRI studies may well be reported. Overall, considering reimbursement constraints, "fee-for-service" healthcare systems favour MRI over US for evaluating sports-related musculoskeletal injuries. Myositis ossificans, which is usually readily displayed by plain radiographs, is a caveat in MRI of the muscles, may be overlooked or mistaken for more sinister conditions, and is a case in point highlighting the need for evaluating musculoskeletal MRI exams along with plain radiographs. Muscle herniations are better depicted by US, given dynamic imaging capabilities of this modality. Positive lymph node status is an important predictor for a poor prognosis. Accurate nodal staging often influences the choice of treatment and outcome of these patients. However, detection of nodal infiltration by non-invasive imaging has been challenging and to date FNA or biopsy has remained important to dictate treatment. Nowadays, the combination of morphological and functional or metabolic information with modern imaging technology offers us new perspectives. In this session we will learn about the present status of lymph node imaging using new generation CT, MRI or PET and become familiar with the strength and weaknesses of each.
A. The current criteria for nodal involvement on CT/MRI W. Schima; Vienna/AT (Wolfgang.Schima@khgh.at)
Lymph node involvement in metastatic spreading and benign lymph node enlargement are common in a variety of diseases. Thus, lymph node characterisation is an important issue. It is based on size (short axis diameter) and morphologic criteria such as shape, homogeneity and contrast enhancement. For abdominal nodes, location-specific size criteria apply (upper limit of normal: lower paraaortic 11 mm, upper paraaortic 9 mm, gastrohepatic ligament 8 mm, portocaval space 10 mm, retrocrural space 6 mm; pelvic nodes 10 mm). In chest CT, an upper limit Mobile devices such as smart phones and touch screen tablets have taken the market by storm and are becoming major players in medical informatics providing convenient solution for physicians on the move. The resolution and processing power of these devices allow nowadays displaying medical images with sufficient resolution for image review and analysis in clinical practice. While they may not be adequate for routine diagnostic tasks they provide a convenient mobile solution for on-call and remote consultations. There are, however, different types of software solutions that can be implemented for such tasks. Two major different design are (1) online web-based applications where the device serves as a "thin-client" to display images rendered and manipulated on a remote computer and (2) local applications that reside on the mobile device and can run independently after images have been downloaded on the device. The first solution requires the user to be constantly connected to the network to be able to display and manipulate images, while the second solution can continue to function after disconnecting from For MR imaging optimisation for ultimate reporting, the radiologist needs to not only understand pelvic MR imaging but also be aware of what the surgeons / oncologists / radiotherapists need to know to plan the correct therapy. The MR imaging report should start with clinical information and requirements, followed by the technique used. Patients with uterine and cervical cancer undergo MR imaging after diagnosis for staging purposes. MR imaging may detect accurate findings regarding the T and the N stage. The report should be focused on this findings and the radiologist should give an estimation of stage. Patients with adnexal masses undergo MR imaging for characterisation purposes. MR imaging findings need to be correlated with clinical information and blood tests to be as accurate as possible. The report should be focused on the features of the adnexal mass and offer a possible diagnosis to the clinician. In this presentation a standardised reporting system for multiparametric prostate MRI examinations will be presented. Furthermore, the imaging assessment of prostate cancer, with emphasis on information useful for surgical and focal treatment planning, will be discussed. Emphasis will be placed on functional MR imaging techniques in conjunction with clinical staging nomograms and tumour localisation. The major teaching points of this exhibit are knowledge of the role of multi-parametric MR imaging in the detection, localisation, and characterisation of prostate cancer. Knowledge of standardised reports will enable us to overcome current limitations in communication with the referring physicians.
Learning Objectives: 1. To learn tips for MR imaging optimisation for ultimate reporting. 2. To learn the most essential points and details to be reported in prostate cancer patients. 3. To understand the major weaknesses of a prostate MR report.
A-346 09:30 C. CT urography N.C. Cowan; Oxford/UK Technological advances in computed tomography (CT) have improved the diagnostic imaging of the urinary tract, surpassing ultrasound and the intravenous urogram. CT urography is defined as CT examination of the kidneys, ureters and bladder with at least one imaging series acquired during the excretory phase of contrast enhancement. In adults, CT urography is now the preferred initial examination for patients with haematuria at high risk for upper urinary tract urothelial cell cancer (UUT-UCC). A practical method for risk stratification will be discussed. Technical aspects of image acquisition and processing will be explored and technical tips relating to protocol design given to optimise CT urography for ultimate reporting. The principal reason for the existence of CT urography is for diagnosing UUT-UCC. Examples of the typical and atypical upper urinary tract urothelial tumours and bladder cancers will be demonstrated. A method for reporting CT urography will be demonstrated. For patients with haematuria, early and accurate diagnosis helps optimise prognosis but conventional investigative pathways are complicated and lengthy, utilising multiple imaging tests and many diagnostic algorithms exist without rigorous evaluation. CT urography offers a single imaging test of high diagnostic accuracy with the potential to replace multiple alternative imaging tests in the diagnostic pathway, improve patient experience, improve diagnostic performance and accelerate diagnosis. A system for imaging haematuria involving use of shown that large core needle biopsy (LCNB) is less costly and better tolerated by the patient than open surgical biopsy, while still achieving high diagnostic accuracy, i.e. 98%. A disadvantage of minimally invasive breast biopsy is malignancy underestimation, defined as less severe pathology in the biopsy specimen than in the subsequent surgical excision specimen. Consequently, these patients might receive insufficient treatment or need to undergo additional surgery. Improvement of breast biopsy methods to reduce malignancy underestimation is ongoing. Vacuum-assisted large core needle biopsy (VACNB) enables removal of larger tissue volumes from the target region with a single needle insertion. A recently introduced biopsy method, the Breast Lesion Excision System (BLES), allows image-guided, percutaneous removal of an intact lump of the target lesion. Malignancy underestimation in breast biopsy specimens is addressed in two recent meta-analyses. Brennan et al. report pooled DCIS underestimation rates of 30.3% for LCNB and 18.9% for VACNB. Specifically for stereotactic breast biopsy, Bruening et al. report pooled DCIS underestimation rates of 24.4% (LCNB) and 13.0% (VACNB), and pooled high-risk underestimation rates of 43.5% (LCNB) and 21.7% (VACNB). Reported DCIS underestimation rates for BLES vary from 3.2% to 21.3%, and high-risk underestimation rates from 0% to 9.4%. In conclusion, malignancy underestimation is an important issue in minimally invasive breast biopsy. With BLES, further reduction of underestimation rates may be realised. Learning Objectives:
1. To realise the risk of a false negative result in needle biopsies. 2. To understand the performance standards needed to minimise the risk of underestimation. 3. To be aware of the importance of radiologic-pathologic correlation prior to definite diagnosis.
C. New developments: therapeutic interventional procedures G. Manenti; Rome/IT (guggi@tiscali.it)
Breast cancer management has been evolving toward minimally invasive approaches. With the improvements in imaging techniques that have allowed the earlier detection of smaller breast cancers and the desire for improvements in cosmetic outcome, a number of minimally invasive techniques for the treatment of early stage breast cancers are being investigated. Breast conservation therapy has become the treatment standard for early-stage breast cancer. The next challenge is to treat primary tumors without surgery. For this purpose, several new minimally invasive procedures, including radiofrequency ablation, cryotherapy, interstitial laser ablation, microwave ablation, focused ultrasound ablation and percutaneous tumor excision, are currently under development and may offer effective tumor management and provide treatment options that are psychologically and cosmetically more acceptable to the patients than are traditional surgical therapies. In this course, we give an overview of minimally invasive approaches for the therapeutic management of benign breast lumps and early-stage breast cancer. It is cautiously optimistic that these therapies can be used as a routine adjunct in the treatment of selected breast cancers. The challenge will lie in the ability to identify multifocal disease and in situ carcinoma as well as to ensure complete and effective eradication of the breast cancer. Actually, breast conserving surgery remains the standard of care for breast malignancies and additional research is needed to determine the efficacy of these techniques when they are used as the sole therapy and to determine the long-term local recurrence rates and survival associated with these treatment strategies. Sunday Several cardiomyopathies will be reviewed such as ischaemic cardiomyopathy and hypertropic cardiomyopathy, including their appearance on regular chest CT. Several valve disease entities such as mitral valve stenosis and prolapse, heavily calcified aortic valve disease/stenosis, bicuspid aortic valves and infective endocarditis will also be reviewed on regular chest CT. Several different types of cardiac shunts such as ASD, VSD, PAPVR-with sinus venosus ASD and interatrial septal aneurysm will also be discussed and their appearance evaluated on routine chest CT. A spectrum of coronary anomalies/fistulas that may be detected on standard chest CT will be discussed. The talk will also review the typical pulmonary changes seen in heart failure, both in the lung parenchyma and the heart and its vessels. Finally, the cardiopulmonary manifestations of smoking will be discussed including the appearances and implications of coronary artery disease. Learning Objectives:
1. To learn about incidental cardiac findings to be reported on a regular chest CT. 2. To review typical pulmonary findings in heart failure. 3. To review typical cardiopulmonary findings associated with smoking. The causes of pulmonary hypertension (PH) are diverse and include a wide variety of diseases. There is increasing recognition that PH is---besides acute and chronic thromboembolism---associated with diseases such as connective tissue disease, COPD and interstitial lung disease. Emphysema is visualised by a decrease in mean lung density; the characteristic findings of fibrotic changes include honeycombing, reticular opacities, ground-glass attenuation, and traction bronchiectasis and architectural distortion. Both changes go along with chronic, progressive hypoxia leading to hypoxic vasoconstriction of the peripheral pulmonary arteries as well as parenchymal destruction. The introduction of fast rotation speed and dedicated cardiac reconstruction algorithms has opened new possibilities for thoracic imaging: besides the better delineation of peripheral pulmonary arteries; there is the possibility of integrating cardiac functional information into a diagnostic CT scan of the chest. Assessment of right ventricular function and analysis of the distensibility of the right pulmonary artery provide insights not only of right heart imapairment but also enable recognition of patients with elevated pulmonary arterial pressures. Furthermore, it is possible to assess the severity of coronary artery disease to determine left vetricular function parameters as well as to depict the sequelae of ischaemic heart disease such as postinfarct aneurysms or wall-thinning due to myocardial infarction. Last but not least, imaging of lung perfusion either by using colour-coded maps of lung density without or without subtraction technique or using dual energy imaging will further widen the clinical applications of thoracic CT. Learning Objectives:
1. To learn about the different etiologies of pulmonary hypertension and their specific imaging findings. 2. To learn about a comprehensive concept for imaging and reporting pulmonary hypertension. 3. To become familiar with the dedicated evaluation of right heart function. Whenever a diagnostic x-ray examination of a pregnant patient is considered to be necessary, conceptus dose estimation is an important step in assessing the risks to the unborn child. Accurate estimation of conceptus dose is also needed after inadvertent irradiation of a pregnant patient from a diagnostic x-ray procedure.
CT urography, unenhanced CT of the kidneys, ureters and bladder, urinary tract ultrasound and cystoscopy will be proposed. Before the advent of fast-scanning multidetector-row CT (MDCT) technology, thoracic CT studies were exclusively used for the morphological assessment of thoracic organs but concurrent examination of the heart has traditionally been hampered by image degradation from cardiac motion artefact. The introduction of fast rotation speed and dedicated cardiac reconstruction algorithms exploiting the multislice acquisition scheme of the data has opened new possibilities for chest imaging, starting with the possibility to integrate cardiac functional information into a diagnostic CT scan of the chest. Initiated with 16-slice MDCT, this concept of integrating morphology and function has been further simplified with 64-slice and dual-source CT scanners, thus allowing the radiologists to provide vital information in the management of patients with a wide variety of acute or chronic respiratory disorders. Because this CT technology offers the possibility of generating highresolution and motion-free images of the coronary arteries, evaluation of the coronary arteries during CT examinations of the chest should further widen the clinical applications of CT for respiratory patients, keeping in mind that cigarette smoking is a shared risk factor for both impaired lung function and cardiovascular events. The purpose of this session is to review practical aspects of this new concept in thoracic imaging.
Increasing temporal, contrast and spatial resolution of modern imaging techniques is paralleled by improved conspicuity of cardiac structures, even when examinations are not focused to the heart. This requires thorough knowledge of cardiac anatomy which is crucial for all Radiologists in order to identify relevant pathology and to avoid overcalling normal anatomy or normal variants resembling pathology. Systematic review of cardiac structures is required on all plain or cross-sectional images of the thorax. Detailed assessment may require reconstruction in specific planes similar to echocardiography or along coronary arteries for further analysis. Knowledge of cardiac anatomy enables identification of pathology such as thrombi, infarcts, aberrant coronary arteries, masses and ventricular enlargement or hypertrophy. On the other hand such knowledge is required to avoid overcalling of normal structures such as the crista terminalis as mass or pericardial recessus as lymphadenopathy. Moreover, it is important to identify variants of coronary anatomy and to distinguish benign from malignant variants. Thorough knowledge of cardiac anatomy and systematic review of all cardiac structures on plain and cross-sectional images of the thorax is relevant to identify pathology and to distinguish pathology from normal anatomy and normal variants. The main outline of this talk will cover cardiac incidental findings, pulmonary findings in heart failure and the cardiopulmonary findings associated with smoking.
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exposure in MRI will also be discussed including the following situations: the patient may not be aware that she is pregnant, likely to be in the first trimester; the mother is referred for direct foetal imaging after ultrasound (normally second or third trimester); the expectant mother may need diagnosis; research on pregnant volunteers. The exposure for pregnant staff working in MRI is also an essential consideration. Finally, how to minimise the exposure for the foetus during MR imaging will be discussed. Although surgery, including hepatic segmentectomy, hemihepatectomy and orthotopic liver transplantation, remains the only curative treatment option for patients suffering from hepatocellular carcinoma (HCC), locoregional interventional procedures increasingly play an important role in the preoperative and palliative management (and potentially even in the curative management) of HCC. First, the technique and clinical value of portal vein embolisation prior to hemihepatectomy will be discussed. Next, the role of radiofrequency (RF) ablation and other, new ablative interventional technologies will be highlighted for the palliative and potentially also for the curative treatment of HCC. Finally, an overview of different transcatheter procedures will be given, including bland and chemo-embolisation with or without drug-eluting beads as well as Y90-radioembolisation, to target HCC lesions. Results of technical and clinical outcome will be discussed as well as the potential of combination therapies.
A. RF ablation V. Válek; Brno/CZ (vlvalek@med.muni.cz)
RFA is recommended as a technique for the treatment of early stage (Child A or B, solitary HCC or up to 3 nodules < 3 cm in size) HCC. The best outcomes have been reported in Child-Pugh A patients with small single tumour, commonly less than 2 cm in diameter. When the patient is considered inoperable, RFA can be indicated also in huge tumours, even in combination with other procedures. RF is coagulation induction from electromagnetic energy sources with frequencies less than 30 MHz For tumour ablation purposes the frequency is usually in the 375-500 kHz range. Even if there exist optimistic data concerning survival of the patients with HCC treated by RFA, RFA is "only" palliation with therapeutic potential. After correctly indicating and performing RFA we can expect 5-yr survival in 40-70% and curative treatments in 30% patients. Major complications after percutaneous ablation such as severe pain, neoplastic seeding, intrahepatic abscesses, intestinal perforation, pleural effusion and peritoneal bleeding after RFA have been reported in up to 6% of patients and mortality related to RFA up to 0.3%. The most common complications are abdominal haemorrhage, abscess, biliary tract damage, liver failure, pulmonary complications and ground pad burns. Follow-up imaging studies should be aimed at detecting local tumour progression, the development of new hepatic lesions, or the emergence of extra hepatic disease. A recommended follow-up protocol includes CT or MR examinations at 3-, 6-, 9-, and 12 months after the treatment and at 6-month intervals thereafter for the subsequent 3 years. Several methods have been developed to estimate conceptus dose from radiologic examinations. When the uterus is remote from the directly exposed tissues, the conceptus is exposed to scattered radiation and its dose is negligible (< 1 mGy). Examinations involving the abdomen-pelvis may deliver higher dose to the child. Variations in maternal body size and uterus position should be taken into account to obtain accurate conceptus dose estimation. Multidetector CT (MDCT) scanners have replaced conventional CT technology. Conceptus doses from abdominal MDCT range from about 13 to about 31 mGy during the first post-conception weeks for a scan acquired at 120 kVp, 200 mAs with a pitch of 1.0, depending on maternal body size and uterine position. Multi-phase abdominal CT examinations may deliver relatively high doses to the unborn child. Doses to the conceptus below 100 mGy should not be considered a reason for termination of pregnancy. The risk to the embryo/fetus for stochastic effects is assessed on the basis of dose using appropriate risk factors. Although these risks from a single diagnostic procedure are low for the majority of diagnostic x-ray examinations, it is important to ensure that doses are kept as low as reasonably achievable.
Learning Objectives: 1. To learn how to manage and counsel pregnant patients in case of (a) intentional and (b) accidental exposure. 2. To learn how to estimate conceptus radiation dose from diagnostic x-ray examinations. 3. To learn how to assess the radiogenic risks to the embryo/foetus from diagnostic
x-ray examinations.
B. X-ray imaging and pregnancy: justification and optimisation of exposure P. Vock; Berne/CH (peter.vock@insel.ch)
As outside pregnancy, justification and optimisation are the main steps to be done when an imaging examination using ionising radiation is considered during pregnancy. However, the risk concerns the embryo/foetus in addition to the mother which means that justification has to be more critical whenever the uterine dose is not neglectable. The practical approach to an examination in any woman of child-bearing age starts by ruling out pregnancy, whether by taking history or by a laboratory test. When pregnancy cannot be ruled out, further steps will depend on the type of examination needed and the urgency of diagnostic clarification. Ultrasound is the alternative to be preferred when it can answer the clinical question. But even among x-ray examinations, the uterine dose is varying widely which asks for a careful selection, optimisation and, maybe, for postponing the test. Once pregnancy is confirmed, the major question is whether the specific type of diagnostic examination will include the uterus in the primary radiation field. Examinations not involving the uterus by direct radiation -despite a potentially significant exposure by scattered radiation -can usually be performed without a relevant risk to the embryo/foetus. The situation is more critical when the uterus is within the examination field and when therapeutic interventions are considered. The presentation will discuss the practical approach to these different situations, the influence of the stage of pregnancy, optimisation methods and the choice between alternative methods in some frequent clinical situations. This paper explores the risks to the foetus when magnetic resonance imaging (MRI) is used. MRI uses three main components to produce images from inside the body: a static magnetic field; a pulsed radio-frequency (RF) field and time-varying gradient electromagnetic field. The exact frequencies of these fields depend on the MRI system purchased; for example, a 0.5T scanner uses 21 MHz RF, a 1.5 T system uses 63 MHz and a 3 T system uses 127 MHz RF. There is also a wide range of options for gradient strengths and slew rates to be considered as well. The overall exposure for the foetus depends ultimately on the imaging sequence used and the area being scanned. This paper will discuss particular hazards that need to be addressed for pregnant women including biological effects of the static and time-varying magnetic fields, heating effects of the RF pulses and acoustic noise generated by the spatial encoding gradients. The circumstances for foetal In Austria the education for radiological technologists was first established by law in 1961. At this time the 'Matura' entrance-level standard for university education was implemented. In 1992 a special law was introduced. Professional academies were considered eligible for European programmes such as Erasmus-Socrates. This law still offers the basis for professional qualification. In 1994, Universities of Applied Sciences were founded in Austria in addition to universities. The students of both university types were equal and they received an academic degree. The structure was similar to our professional academies. As a consequence of this, the professional association (RTaustria) also joined in the task of converting academies into the university sector. The first bachelor study programme "Radiological Technology" started in 2005. The students finish with a BSc degree in health studies in Radiological Technology. The demands on teaching staff and students are already very large. A Bachelor' Degree is recognised as a university sector. Master's Degree programmes can be pursued immediately, e.g. MedTech (Master or Engineering for Functional Imaging, and Conventional Ion Radiotherapy), or Master of Science in Radiological Technology. This technology offers radiographers a starting point for medical computer science. Doctoral programmes for molecular and functional imaging, medical physics and computer science are well established and alumni are admitted. The strategic influence on the profession is a social benefit. If Radiological Technologists attain even more profound knowledge that more deeply penetrates their specialisation, the better their future patients will be treated. While resection and ablation are still the gold-standard for curative local treatment of the early stage HCC, several effective intra arterial procedures have been developed for advanced HCC stage. There is still no consensus on the best intra arterial local therapy; however, it offers great promise based upon the premise that HCC are fed mainly, if not exclusively, by arteries. Bland embolisation, chemoembolisation and radio-embolisation are some of the most common local treatments for patients with HCC and several embolic agents have been specifically developed for that purpose. Both TACE and TAE may shut-down the arterial blood flow to the tumour, leading for tumour ischaemia and, eventually, tumour cell death, if anoxia is induced. Association of local chemotherapy to the embolic effect represents the rationale for TACE. For this purpose, new embolic particles, which may precisely elute drugs, have been introduced (DEB-TACE= Drug Eluting Beads TACE). For Radioembolisation, micro-particles are injected into the feeding arteries as vehicles for delivering interstitial sources of radiation therapy. Small and precisely calibrated micro-particles have been introduced for a deeper a more effective TAE. Because hepatic tumours are supplied by several arterial feeders, complete tumour death may be obtained only if the entire vascular network supplying the tumour is treated. If even small feeding arteries are be missed, tumour mass will be not completely treated and it will relapse. For this reason, the knowledge of possible vascular abnormality is mandatory for a better outcome. Surgical resection remains one of the few treatments considered to be "curative" for malignancy contained within the liver (both primary and secondary). For resection to be effective all of the segments containing tumour must be removed. For a patient to survive such a procedure the remaining hepatic function must be sufficient to sustain life. In some circumstances so much of the liver is removed that the remaining segments are too small (or they were small in the first place). Pre-operative portal vein embolisation (POPE) is designed to stimulate enlargement in the segments that are intended to remain, so that when the cancer is removed, survival is improved. POPE is a technique where the portal venous supply to the diseased segments is blocked, so that growth factors are diverted to the healthy segments. Over a period of 4-6 weeks prior to surgery, these segments enlarge so that the diseased segments can be removed more safely. POPE is performed percutaneously, and a variety of embolic materials are used to occlude, usually the right portal vein. After POPE the liver is scanned to assess for hypertrophy of the healthy segments, and surgery scheduled to coincide with enlargement but before time has elapsed which would allow for tumour progression (local or distant). Surgery can usually be planned for 4-6 weeks after POPE (this time may vary depending upon factors such as the presence of cirrhosis and diabetes). Primitive malignant renal tumours comprise 6% of all childhood cancers. Wilm's tumour (WT) is the most frequent type accounting for more than 90%. Imaging alone cannot differentiate between these tumours with certainty but it plays an important role in screening, diagnostic workup, assessment of therapy response, preoperative evaluation and follow-up. The outcome of WT after therapy is excellent with an overall survival around 90%. This allows for a risk-based stratification maintaining excellent outcome in children with low-risk tumours while improving quality of life and decreasing toxicity and costs. The imaging issues for WT from the European perspective will be discussed as well as the characteristics of other paediatric malignant renal tumours. Primary adrenal malignant tumours can be categorised according to their origin. Adrenocortical neoplasms are rare in children. A size greater than 5-10 cm suggests malignancy as well as signs of local invasion or distant metastasis. The most frequent malignant medullary tumour is neuroblastoma. It accounts for 7-10% of all childhood cancers and has a survival rate between 5 and 80% depending on age at diagnosis, tumour spread, genetic markers, etc. It is related to borderline malignant ganglioneuroblastoma and benign ganglioneuroma. Imaging cannot distinguish between these tumours but it is essential in the diagnostic workup and during follow-up. The role of imaging: The main aims of imaging are confirmation of the hepatic origin of the tumour, diagnosis and staging. Differential diagnosis is facilitated by combining imaging findings with clinical parameters, such as serum alpha-fetoprotein (AFP). Children with malignant liver tumours often require three modalities (US, chest CT and MRI) for optimal imaging. Differential diagnosis: It is important to identify vascular tumours of infancy and mesenchymal hamartoma on imaging grounds if possible. These lesions rarely require biopsy. The major malignant primary tumours are hepatoblastoma, hepatocellular carcinoma, rhabdoid tumour, undifferentiated (embryonal) carcinoma and hepatocellular carcinoma variants (transitional liver cell tumour and fibrolamellar carcinoma). Malignant vascular tumours (epithelioid haemangioendothelioma and angiosarcoma) are rare. The distinction between multifocal primary liver tumours and metastases from an extrahepatic primary tumour (or multifocal infantile haemangioma) can almost always be made on a combination of imaging and clinical features. Staging: There are two major staging systems in current use. The Children's Oncology Group (COG) uses a surgical staging system, in which the main role of radiology is the detection of extrahepatic spread and preoperative surgical planning. The other major trials group, SIOPEL, uses the PRETEXT system, in which the role of imaging is much more important because surgery is delayed and chemotherapy is stratified according to clinical and radiological risk factors. Because COG now also collects data using the PRETEXT system, there is a global consensus that the ideal radiology report should include a description of each of its parameters. Learning Objectives:
1. To understand the role of US, CT and MRI. 2. To become familiar with the imaging findings and the main differential diagnoses. 3. To learn the imaging strategies for diagnosis and in staging.
C. Oncologic imaging in the paediatric brain G. Hahn; Dresden/DE (gabriele.hahn@uniklinikum-dresden.de) Brain tumours of children account for 15% to 20% of all primary brain tumours. Posterior fossa tumours and supratentorial tumours occur in nearly equal frequency. However, supratentorial tumours are more common in the first two to three years of life, whereas infratentorial tumours predominate from ages 4 to 10. The symptoms of children with brain tumours depend upon the age at the time of presentation and the location. MR is today the study of choice for diagnosis of intracranial neoplasms A-360 09:30 C. Exploring the benefits of European radiography networks: a personal and professional perspective of the Erasmus radiography group J. Portelli; Msida/MT (jonathan.portelli@um.edu.mt) Since its advent in 1987, ERASMUS has become the EU's leading education and training programme -enabling more than 2.2 million students to benefit from study and work experiences in different European countries. Furthermore, ERASMUS has also encouraged co-operation and staff mobility between higher education institutions across Europe and over the past 15 years about 250,000 higher education teachers and other staff have embarked on teaching exchanges and training opportunities abroad. The ERASMUS Radiography Group (ERG) was born from an initiative taken up by three European universities in 1990, who wanted to create a network that would facilitate the exchange of undergraduate radiography students. Ever since the success of the first student exchanges in 1994, the ERG has evolved considerably and today it is formed by 16 higher education institutions from 14 European countries. To date, more than 1,200 radiography students have successfully participated in ERASMUS exchange programmes organised by the different ERG institutions and more are expected to do so in the future. In this context, one may note just how such networks and groups can help bridge and strengthen the radiography profession across Europe. Apart from providing some historic details about the ERG and is underlying aims, this presentation will also seek to outline the benefits and opportunities that the ERASMUS programme may bring about on an individual and professional basis, as highlighted by the personal experience of a past ERASMUS student who today is one of the ERG coordinators. Learning Objectives:
1. To understand the history, structure, aims and objectives of the Erasmus radiography group. 2. To gain an insight into the Erasmus radiography group from a student-cumcoordinator's perspective. 3. To explore the potential for such groups or networks to strengthen the radiography professionals of the future. 4. To be aware of threats and opportunities in student and staff mobility in Europe. In the field of oncology, childhood cancer makes up only a minor amount of cases with less than 2% of all cases occurring in children. However, these 2% of cases do constitute, after trauma, the second most common cause of death in children.
In the past five decades the survival rate of children with cancer has increased significantly. In the beginning of the 1960s approximately 30% of children survived whereas this figure is approaching 80% nowadays. This will also have an impact on adult radiology as survivors of childhood cancer have shown to have an estimated 10% increased incidence of subsequent neoplasms. Paediatric radiology plays an important role in the diagnosis, staging, treatment (interventional radiology) and follow-up of childhood cancer. In order to fullfil its role the radiologist should have clinical knowledge about the epidemiology, presentation and treatment of childhood cancer. From a radiological point of view he/she should have knowledge of state-ofart imaging techniques and the implication of these techniques on cancer staging. The paediatric radiologist should play a pivotal role in imaging protocols, not only on a local level but also on an international level through, e.g. the International Society of Paediatric Oncology. With respect to reporting childhood cancer there is an ongoing debate on which measurements should be used. Historically paediactric cancer response assessments are based on 3-D measurements. However, with the introduction of new European guidelines it is expected that pharmaceutical companies will insist on the use of RECIST criteria in trials.
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Sunday structural changes in time and the multidisciplinary team management of cirrhotic nodules. Multislice CT evaluation of cirrhotic nodules involved a nonenhanced and enhanced multiphase CT during the arterial, portal and parenchymal phases to highlight early hypervascular lesions and for the analysis of time washout curve. MRI evaluation of the cirrhotic liver included conventional sequences without contrast (T1 and T2w, chemical shift artifact sequences, diffusion) in combination with multiphase dynamic 2D and 3D acquisition after iv. administration of liverspecific contrast agents including the hepatobiliary phase. From the total number of patients, 280 had "uncomplicated" liver cirrhosis, 578 patients had liver nodular regenerative cirrhosis and portal hypertension, 112 patients had regenerative and dysplastic nodules and 437 patients had hepatocellular carcinoma (multiple tumours in 278 cases and single tumour in 159 cases). Cross-sectional imaging with CT and MRI plays an important role in the evaluation and follow-up of patients with cirrhotic liver disease and its complications. Multidisciplinary dialogue between the clinician (gastroenterologist, surgeon, and oncologist), radiologist, medical laboratory scientist and anatomo-pathologist allows finding the optimal solutions concerning the monitoring and correct therapeutic approach of hepatic nodules developed in the cirrhotic liver. Learning Objectives:
1. To understand the particularities of CT and MR imaging techniques in liver cirrhosis. 2. To consolidate knowledge of CT and MR imaging appearance of regenerative nodules, dysplastic nodules, and hepatocarcinoma in cirrhosis. 3. To become familiar with the differential diagnosis in liver cirrhotic focal lesions considering the various enhancement patterns. 4. To discuss the importance of clinical, biochemical information and follow-up of small nodules by the same imaging modality that may be helpful in differential diagnosis of these lesions. This case-based lecture will present typical clinical cases of pelvic pain as well as some unusual but important causes. Cases of acute and of chronic pelvic pain and benign, as well as malignant disease will be included. The audience will have the opportunity to participate in case discussion by the use of interactive voting pads. The selection of imaging modality for each clinical presentation and the importance of knowing the clinical findings at the time of the radiological interpretation will be discussed. For each case, the key radiological features will be illustrated. The essential elements of the radiology report and the key information required by the clinician will be discussed. In each case, the differential diagnosis and the need for follow-up imaging will be considered. The key teaching points for each diagnosis will be reviewed. Learning Objectives:
1. To review typical cases illustrating the role of imaging modalities in the differential diagnosis of pelvic pain in female patients. 2. To get involved in the diagnostic process by the use of electronic voting pads. 3. To understand the conclusion that may be drawn on the basis of the discussed cases.
Interlude: Ten reasons to see Romania D. Negru; Iasi/RO
Imaging and guided biopsy in breast malignancies M. Lesaru; Bucharest/RO (mlesaru@hotmail.com)
The past decade in Romanian breast imaging was a time of progress. Training in breast imaging began to be organised systematically in 2001. Standards in quality assurance were set according to European standards in 2004 and 14 mammography centres were trained and equipped with materials in order to have consistent quality in mammography. For sustaining our perspective, we used the experience of two major centres in breast imaging, Cluj-Napoca and Bucharest, which initiated together the breast imaging "journey" in Romania. These centres at this moment account for over 13.000 breast examinations every year (mammography, ultrasound and MRI). The final result is a growing number of infraclinical breast carcinomas.
The main challenge we encountered along this period were the BIRADS 3 lesions and the number of these cases is analysed. In detected BIRADS 4 or BIRADS 5 lesions, the imaging methods were used for the inventory of the lesions and biopsy guidance. The biopsy performed by radiologists for these lesions is always done under imaging guidance obtaining histology specimens. The number of needle core biopsies done by radiologists was zero before 2001 and reached 757 in 2010. The clinical impact was high as we discovered cases other than breast carcinoma, i.e. haematological malignancies. In conclusion, we believe that Romania is able to prove at this moment its ability to have an appropriate radiological practice in breast imaging and breast biopsy, in line with the European standards. Pelvic pain is an important part of clinical practice for any clinician who provides health care for women. Pelvic pain may be acute, recurrent or chronic. Acute pelvic pain (APP) rarely lasts more than one month without crisis, resolution, or cure. Pain of more than 1 or 3 or 6 months of duration is considered as chronic pelvic pain (CPP) and in many settings may be considered and treated as an illness itself. Women who present with APP frequently exhibit nonspecific signs and symptoms. Diagnostic considerations encompass multiple organ systems, including obstetric, gynaecologic, urologic, gastrointestinal, and vascular aetiologies. As the first priority, urgent life-threatening conditions (e.g. ectopic pregnancy, appendicitis and ruptured ovarian cyst) and fertility-threatening conditions (e.g. pelvic inflammatory disease and ovarian torsion) must be considered. Adolescents and pregnant and postpartum women require unique considerations. CCP is a common and significant disorder of women, with a prevalence of 3.8-12%. Many disorders of the reproductive tract, gastrointestinal system, urological organs, musculoskeletal system, and psychoneurological system may be associated with CCP, the most common being endometriosis, adhesions, irritable bowel syndrome, and interstitial cystitis. Ultrasonography should be the initial imaging test because of its sensitivities across most aetiologies and its lack of radiation exposure. Computed tomography (CT) serves an important role in patients with nonlocalising symptoms, an indeterminate US evaluation, or in patients who require a wider search beyond the field of view available with US. Magnetic resonance imaging is an extremely useful second-line modality for problem solving after US or CT. Detection of intestinal inflammatory lesions is crucial for management of patients with Crohn's disease (CD). Awareness of the shortcomings of mere clinical evaluation for assessment of disease activity has grown. Ileocolonoscopy has been the gold standard for evaluation of lesions in the colon and terminal ileum. However, ileocolonoscopy cannot always be complete, there exist lacks in the evaluation of the complete small bowel and there are several drawbacks related to the invasiveness. Over the past few years, cross-sectional imaging techniques, including ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI) have been increasingly used for evaluation of patients with CD, allowing objective assessment of location, extension and severity of CD-related lesions. Imaging techniques are also the accepted reference for detection of complications including strictures and penetrating lesions such as fistulas and abscesses. Objective assessment of inflammatory lesions is required for guiding therapeutic interventions and for assessing the efficacy of these interventions. The choice between imaging techniques is often determined by local availability, expertise and technical details of these examinations which are also subject to considerable variation, which may affect accuracy. Chronic liver diseases represent a major public health problem. Their prognosis and management greatly depend on the amount and progression of liver fibrosis. Liver biopsy, traditionally considered as the reference examination, is currently less performed because of the development of non-invasive biological and imaging biomarkers. Serum biomarkers of liver fibrosis are neither very sensitive nor specific. Imaging biomarkers have the potential to more specifically quantify hepatic fibrosis. Anatomical imaging of liver fibrosis is hampered by its limited spatial resolution. Functional imaging is very promising because fibrosis is accompanied by changes of perfusion, diffusion, mechanical properties and metabolism that can be quantified with ultrasound or more comprehensively with MRI. Indeed, functional MRI can be used not only to stage liver fibrosis, but also to grade steatosis, necrosis and inflammation occurring in chronic liver diseases. Molecular imaging can be performed by targeting collagen or metalloproteases in fibrogenesis. However, molecular imaging remains currently an experimental tool. In conclusion, imaging biomarkers obtained at ultrasound and MRI are increasingly used to assess liver fibrosis. Functional MRI offers the opportunity of a global assessment of fibrosis, steatosis, necrosis and inflammation in chronic liver diseases. Atherosclerosis is a slow, progressive disease involving multiple if not all vascular territories and potentially results in disastrous events, such as myocardial infarction, stroke and renal failure, respectively. Frequently, more than one vascular territory is affected. Due to the demographic changes within developed countries and the dramatic increase in obesity and metabolic syndrome, atherosclerosis and its sequelae play an ever increasing socioeconomic role. Until recently imaging techniques have focused on depicting the degree of luminal stenosis. However, luminal stenosis occurs late in the atherosclerotic disease process, and several recent studies have shown that knowledge of luminal stenosis alone is insufficient to predict vulnerability of plaques. Thus, recent developments in imaging technology have focused to assess the vessel wall. With these techniques, qualitative and quantitative information of plaque composition and morphology can be obtained non invasively. Moreover, functional and molecular imaging techniques have emerged that allow assessing the biological activity of atherosclerotic plaques. While some of these methods are still in the realm of basic research, others are entering the field of clinical application. For radiologists and nuclear medicine specialists, as well as for imaging scientists knowledge and expertise in this important field has become mandatory. In the future, these techniques will most likely contribute to a personalised medicine by helping to tailor drug selection to an individual's atherosclerotic risk and to enable to assess an individual's response to therapy. Non invasive imaging of the vascular wall could transform clinical management for diagnosis, risk stratification, selection and efficacy assessment of anti-atherosclerotic therapeutics. If in the mid twentieth century. Much of our current understanding, and misunderstanding, of VTE, however, stems from the era before definitive imaging diagnosis was possible. The first definitive in vivo imaging diagnosis for VTE was lower extremity venography in the 1930s and pulmonary angiography in the 1960s. Since then, scintigraphy, CT, ultrasound and MRI have been developed. Each occupies a specific niche in the workup. CT currently has the largest role because of its sensitivity, specificity, availability and ability to provide alternative diagnosis. The first CT report of pulmonary infarction was in 1978 (Sinner) and the first use of helical scanning direct visualisation of central PE was in 1992 (Remy-Jardin 'Classical' imaging is used every day in clinics to guide diagnosis and therapy, but only gives access to anatomical and/or structural information. Molecular imaging will give access to the activity of a protein, the fate of a biomolecule and even to molecular pathways. The role of molecular imaging in the diagnosis of inflammation will be discussed in this lecture. Molecular imaging agents are classically designed as activatable or targeted agents. Activatable agents consist of chemically engineered substrates that undergo a physicochemical change after interaction with their intended target. This results in the activation of the contrast media. We will use the recruitment of monocytes/macrophages to the location of myocardial infarct to illustrate the concept of activatable probes. As monocytes/ macrophages are known to produce a substantial amount of myeloperoxidase, an activatable agent capable of imaging myeloperoxidase will be discussed. Imaging the recruitment of monocytes/macrophages with iron oxide-based MR contrast agent will also be illustrated in the case of inflammatory lesions. Lastly, MR-targeted probes consisting of nanoparticles with added targeting moieties will be discussed. Adding targeting moieties on the surface of probes allows to specifically target a receptor over-expressed by cells. To illustrate this specific targeting, neo-vessels over-expressing integrin and selectin will be used. Atherosclerosis is a chronic disease of the vessel wall which accounts for > 25% of ischaemic strokes and for the majority of myocardial infarctions and sudden cardiac deaths. Until recently little was known about the natural history of atherosclerosis due to difficulties to image the arterial wall. However, several new imaging methods, such as intravascular ultrasound (IVUS), high-resolution black blood magnetic resonance imaging (hr-bb-MRI) and Positron Emission Tomography / Computed Tomography (PET/CT) have emerged on the horizon. These techniques are able to depict the full vessel wall and are able to provide detailed information of plaque composition, morphology and activity non-invasively. In this lecture natural progression studies of atherosclerosis will be discussed and it will be demonstrated that certain plaque features, that develop over the years, such as thin-cap fibrous atheromas, intraplaque haemorrhage and inflammation, are associated with an increased risk of cerebro-or cardiovascular events. Data will be shown which demonstrate the effects of common atherosclerotic risk factors on the progression of the disease. In addition studies will be shown which suggest that certain plaque factors, such as intraplaque haemorrhage or plaque inflammation, accelerate the atherosclerotic disease process. Furthermore, the effects of anti-atherosclerotic drugs on plaque progression and plaque morphology will be discussed. Last but not least the pharmacological possibilities will be discussed to reverse or to decelerate the anti-atherosclerotic disease process. Lung cancer most often presents as a pulmonary nodule or mass, less commonly as a hilar mass or endobronchial lesion. Most small tumours radiologically present as pulmonary nodules. Chest radiography (CXR) is limited in demonstration of small nodules and even lesions as large as 3 cm may be missed. Digital tomosynthesis has been shown to markedly improve sensitivity for pulmonary nodules as compared with CXR but is not yet widely available. Magnetic resonance imaging (MRI) also is more sensitive for nodule detection than CXR, but is not routinely performed in most cases due to limitations in cost and availability. The gold standard for detection of pulmonary nodules as small as 1-2 mm is multidedector-computed tomography (MDCT). Technically, MDCT is able to demonstrate pulmonary nodules with sensitivity close to 100%; however, sensitivity of individual radiologists is much lower due to the task of analysing several hundred images, confusion between nodules and vessels imaged in cross section and other errors. Computer-assisted detection (CAD) software can improve sensitivity but may be limited by false-positive findings. Positron emission tomography (PET)-CT using 18 F Deoxyglucose (18 FDG) has a high sensitivity for lung cancer; however, false-negative findings may be due to well-differentiated adenocarcinoma, carcinoid and nodules < 10 mm, whereas false-positive findings may be due to inflammatory lesions. This interactive session will include examples of manifestations of lung cancer as well as data on the accuracy of different imaging methods for lung cancer detection. The majority of cardiovascular diseases arises from atherosclerosis resulting in coronary heart disease and its principal manifestations, angina pectoris, myocardial infarction, sudden cardiac death and heart failure or stroke. A hallmark of atherosclerosis is vascular inflammation initiated by an endotheial injury and driven by various noxes such as hypercholesteremia. The most dangerous lesions are unstable and prone to rupture. These plaques are often of lesser stenosis severity and thus would sometimes not impair blood flow at rest or during exercise. Unstable plaques are characterised by activated macrophages, mast cells and other cells being localised in the plaque shoulder which secrete a variety of matrix-degrading enzymes such as metalloproteinases (MMPs). Non-invasive molecular imaging of MMP activity in patients could help identify patients at high risk of major acute cardiovascular events. SPECT and PET provide the most sensitive and selective means for imaging molecular interactions non-invasively in the living body and could therefore prove a potent approach to the identification of the metabolically active plaque that is vulnerable to rupture. New radiopharmaceuticals addressing relevant targets in plaques, improvement in image acquisition and processing (e.g. motion correction), preclinical imaging in models and the first translational approaches will be discussed. Learning Objectives:
1. To learn about potential targets for molecular imaging of atherosclerosis. 2. To become acquainted with molecular imaging studies which have assessed atherosclerosis. 3. To understand the role of molecular imaging in current clinical practice and its potential role in the future.
Non-invasive imaging of the vulnerable atherosclerotic plaque J.H. Gillard; Cambridge/UK (jhg 21 @cam.ac.uk)
Until recently the risk of carotid disease in symptomatic patients was determined by simple luminal measurement-based conventional angiography, all on more subjective changes in Doppler ultrasound measurements which would establish whether a patient should undergo carotid endarterectomy or optimal medical therapy. Developments in MR and CT over the past decade have given us alternative tools to measure luminal stenosis which have reduced risks when compared with conventional X ray angiography. The current goal is to be better able to characterise plaque risk, whether it is in the carotid, coronary or peripheral vasculature. MR imaging of the carotid is feasible due to its size and superficial position, equivalent reproducible imaging of the coronary arteries being considerably more challenging.
Although the carotid artery may be viewed as a surrogate for disease in the coronaries, this is probably an oversimplification. Nevertheless it remains an important and practical target. Whilst the assessment of risk is aided by the quantification of individual morphological components in plaque viewed with MR, it has been difficult to image true plaque function. We can use MR to assess the contribution of local flow dynamics and the individual components of plaque to produce maps of stress in a specific patient. MR contrast media can contribute additional information with regard to plaque function. We are also able to image not only individual plaque structure, but also function allowing an improved understanding of why two patients with identical degrees of luminal stenosis may have completely different degrees of vulnerability. and midgut volvulus or with a water-soluble contrast enema in newborns with suspected meconium plug or microcolon. Ultrasound plays a complementary role in the neonates, but in all other ages US becomes the method of choice in the vomiting child because of its high sensitivity and specificity (e.g. in hypertrophic pyloric stenosis) and offers the advantage to avoiding exposure to the potential harmful radiation of x-rays. Computed tomography and/or magnetic resonance imaging in diagnosing the vomiting child will be reserved for less common causes such as intracerebral abnormalities or intracranial pressure. Learning Objectives:
1. To learn about useful and applicable imaging techniques, including their specific adaptations and the needs when applied in infants and children. 2. To become familiar with the respective imaging algorithms in applicable typical childhood scenarios. 3. To become familiar with the common imaging findings and their differential diagnoses and common pitfalls. The vomiting child can present the paediatrician and paediatric radiologist with a difficult diagnostic problem. As discussed in the previous lectures the differential diagnosis is based on age specific diseases. Both gastrointestinal as non-gastrointestinal diseases of childhood can cause vomiting in a child. During this interactive session a broad range of cases, the usual and unusual, will be presented. Each case will be introduced by a short summary of the clinical information. The question will not only be which diseases should be included in the differential diagnosis but also which imaging modality and protocol should be used in this case. The importance of choosing the correct imaging technique and protocol is of importance not only from a perspective of patient satisfaction but even more from an ALARA point of view. In paediatric radiology avoiding unnecessary (repeat) exams and choosing the right technique and protocol has the biggest impact on radiation reduction. Paraparesis is clinically defined as a weakness of the lower extremities. It is usually caused by an injury of the spinal cord in the thoracic, lumbar or sacral part of the spine or by toxic effects. An acute onset is a neurologic emergency, which requires an immediate clarification -mainly by an imaging study. There are several conditions in oncology that may cause an acute paraparesis. These include acute myelogenous leukaemia, metastasis, Hodkin´s disease, lymphomatosis meningitis, cervical or lumbar spinal cord tumour or metastasis, multiple myeloma, primary myelofibrosis/ myeloid metaplasia or metabolic and treatment-associated changes in the spinal cord. As the paraparesis starts in this context without a traumatic background, MR imaging is the method of choice in the workup of these patients. In this overview we will classify and describe the different oncologic conditions which may cause an acute paraparesis and highlight typical imaging findings in both intramedullary and extramedullary causes. Whilst the pathways for the detection and staging of lung cancer are well known, the use of imaging for the detection of disease relapse are less well established. Detection of relapse is now more complex, due to the use of multimodality therapy, including surgery, radiotherapy, chemotherapy and new ablative techniques. Each of these therapies may affect the site and appearance of recurrent disease. Additionally new techniques such as PET-CT and diffusion-weighted imaging have not yet been fully evaluated in this context. This presentation will focus on the timing of repeat imaging post treatment, the features of recurrent disease and suggest follow-up protocols, paying particular attention to imaging in the context of retreatment of disease relapse. Vomiting is a common symptom in childhood and can be caused by numerous conditions. In children we not only face difficulties in defining the conditions that may cause vomiting but also in differentiating true vomiting from physiological regurgitation, especially in younger children. In this session the clinico-radiological approach to a vomiting child will be discussed and illustrated by different clinical scenarios. This session will focus on the most common age-specific entities that may cause vomiting and how the approach to imaging in a vomiting child differs from in adults. It emphasises the importance of a good collaboration between the paediatrician and the radiologist to improve the diagnostic pathway. In detail, a multi-disciplinary approach is needed to avoid unnecessary diagnostic tests. The role of imaging not only in establishing the final diagnosis, but also in excluding underlying conditions that may need intervention will be discussed. This paper reviews the use of imaging techniques for the assessment of the vomiting infant and child. The differential diagnostic workup for the common clinical presentations of vomiting in the different age groups will be described and illustrated for age-specific diseases. Nausea and vomiting are common sequelae of multitude of disorders that can range from mild, self-limited illness to severe, life-threatening conditions. Bilious vomiting in the newborn is considered a radiological emergency and requires quick and exact diagnosis, whereas vomiting in children often is associated with gastroenteritis which not requires any imaging. In neonates with bilious vomiting, the first step in the imaging evaluation is the abdominal plain film which may reveal evidence of upper gastrointestinal tract obstruction. Direct imaging of the stomach and small bowel using the upper contrast gastrointestinal series or fluid-aided ultrasound answers the question if there is a mechanical obstruction. X-ray contrast enema may play a role in the evaluation of suspected malrotation The simple answer to this question is "yes" for most situations: and notably when surgery is not planned. The complicated answer is that US relying on morphologybased systems supplemented by Doppler evaluation cannot reliably provide the information used to direct women with a complex ovarian mass to appropriate surgery unless there are clear secondary signs of disseminated malignancy. The difficulty is in deciding when to use CT or MR imaging after US. That is the topic to be discussed in the context of new ESUR guidelines. Most women diagnosed with endometrial cancer are postmenopausal (~25% are premenopausal). Endovaginal ultrasound is widely used for initial assessment in menopausal patients, especially if bleeding is present. Measurement of the thickness of the endometrial stripe can help determine the need for D&C. After diagnosis, MRI is the best imaging modality for locoregional staging and detecting recurrence (preliminary data suggest FDG-PET may also be useful for follow-up). Surgery remains the primary treatment modality, but radiotherapy and chemotherapy are being used for advanced disease and patients at high risk of recurrence. In 2009, FIGO staging guidelines were revised. MRI is especially helpful for distinguishing stage IA from stage IB (≥ 50% myometrial invasion), which is associated with a 40% incidence of lymph node metastasis and a need for lymph node dissection and for identifying stage II (invasion of the cervical stroma, calling for radical hysterectomy). While pretreatment knowledge of these findings is helpful, cost-effectiveness studies are lacking, and routine use of MRI is controversial. MRI should include at least two T2-weighted sequences showing the short and long axes of the uterus (sagittal, axial oblique or coronal oblique). High-resolution imaging 2 min ± 30 s post-contrast injection is optimal for diagnosing myometrial invasion. If cervical invasion is suspected, an additional slice perpendicular to the endocervical channel is recommended. Pre-contrast sequences up the renal hilum enable retroperitoneal lymph node screening. Indications for MRI include high-grade, serous or clear-cell adenocarcinoma; suspicion of stage ≥IB; detection of enlarged nodes for sampling; inability to perform D&C or surgical staging. Posterior reversible encephalopathy syndrome (PRES) is characterised by headache, altered mental status, visual disturbances and seizures. Different conditions have been associated with PRES, such as for example eclampsia bone marrow transplantation and immunosuppressive treatment, hypertension, autoimmune disease and cancer chemotherapy. The pathophysiology of PRES is still unclear and different hypothesis will be discussed in this lecture. Typical radiological imaging findings which are best presented on MRI include signal abnormalities due to vasogenic oedema in predominately but not only white matter of the posterior regions of the cerebral hemispheres, like the occipital and posterior parietal lobes but also the frontal lobes often involved, whereas the cerebellum, the brainstem, the deep white matter and basal ganglia are more atypical locations. The incidence of radiation necrosis ranges from 5 to 24%. The delayed neurological symptoms include functional and cognitive impairments, including deficits in learning, working memory, executive function, vision, and motor function, and eventually dementia. A complete understanding of the pathophysiology of radiation therapy-induced changes to the central nervous system (CNS) is still lacking. MRI cannot reliably discriminate tumour recurrence/progression from the inflammatory or necrotic changes resulting from radiation, although the latter can be associated with more specific patterns of enhancement, such as "soap bubble"-or "Swiss cheese"-like with or without feathery margins and central necrosis. This lecture will focus on the pathophysiology, aetiology and imaging features, of PRES and radiation necrosis and their differential diagnosis in oncology patients. Percutaneous image-guided procedures can be applied for tumour management in cases of spinal tumours. They consist of the tumour ablation and/or vertebral consolidation. These procedures are usually not considered as acute emergency. However, in cases of impending vertebral collapse, spinal consolidation should be performed on an emergent basis. For the palliative treatment of painful spinal tumours (metastases), the therapeutic goal should not be the complete ablation of the tumour, but one or more of the following: tumour reduction, pain management, prevention of risk of pathological fractures and in some cases, decompression of spinal tumours extending towards the spinal canal. Precise clinical evaluation of the patient is mandatory: origin and location of pain, previous treatment, which anaesthesia the patient can tolerate, and life expectancy. For tumours causing any neurological signs, surgical decompression should be the first treatment option. Multidisciplinary decision is required to choose the more efficient and less disabling technique. To evaluate postsurgical patients it is important to know the primary clinical diagnosis, the surgical treatment, the interval since surgery and patients' current clinical symptoms. Radiography is the most common imaging modality to evaluate the postoperative ankle, particularly in traumatic cases; after reduction and fixation of a fracture or dislocation it is generally carried out as routine. Ultrasonography is highly sensitive and specific in postoperative tendon assessment, thanks to the superb resolution, and the opportunity for dynamic evaluation of tendon integrity. MRI has rapidly become important in post-operative assessment of the ankle, because it provides high soft-tissue contrast, multiplanar capability and osseous structures visualisation. It shows signal changes of ligaments and tendons, hypointense subchondral sclerosis, subchondral bone marrow oedema, joint effusion, capsular thickening, fibrosis and synovitis. MRI has also an important role in the evaluation of post-surgery ankle pain due to impingement syndrome and in the hindfoot chronic instability related to postoperated sinus tarsi syndrome; it demonstrates the anatomy of sinus tarsi, chronic synovitis and nonspecific inflammatory changes, synovial cysts, fibrosis and subtalar joint effusion. It is important also in the follow-up of tumours and tumour-like conditions of bone and soft tissues after surgery. Computed tomography is the most valuable method to define the osseous anatomy of the postoperative ankle, so it is important in the follow-up of the operated osteochondral lesions of the talus. CT allows the evaluation of irregularities or degenerative changes and progressive degenerative arthritis; however, CT usually fails to evaluate soft tissue ankle lesion. There are many surgical techniques to repair meniscal tear: focal cartilage defect, cruciate ligament tear, malalignment, fracture, osteoarthrosis,… Conventional radiography, CT-scan, CT-arthrography and MRI play an important role in evaluation of the knee after surgery or arthroscopy. Indications for postoperative imaging are infection, persistent pain and dysfunction. Every radiologist should be familiar with "normal" imaging findings after arthroscopy, osteosynthesis, ligament reconstruction, osteotomy, knee prosthesis and meniscal or (osteo)chondral repair, but also recognise the main complications after knee surgery or arthroscopy. Orthopaedic hardware is usually evaluated on plain radiography or CT, and only a relative contraindication for MRI. Microscopic metal artefacts and fibrotic scarring are frequently seen along the course of the instrumentation tract. After partial meniscectomy, an obtuse angle at the apex of the meniscus and increased signal intensity of the remnant part of the meniscus are normal findings, whereas fibrillation and recurrent tear may explain complaints of the patient. Various intra-and extra-articular reconstructive procedures exist for anterior and posterior cruciate ligament reconstruction. Besides the neoligament, an osseous tunnel, screws and metal artefacts are also visible. Postoperative findings of the extensor apparatus include a thickened patellartendon, focal myxoid degeneration, fibrosis and focal defects, e.g. after harvesting tendon tissue for ACL reconstruction or after release of the lateral patellar retinaculum for 'unstable' patella. Accelerated osteoarthritis may be a late postoperative finding. MRI very well depicts incorporation and alignment of osteochondral auto-or allografts, and the position, morphology and integrity of the meniscus after repair or transplantation. Even if the RECIST criteria based on anatomic size measurements of the tumours have been recently updated, difficulties are still often encountered in measuring lesions, both in primary tumours and metastases. The aim of this course is to analyse the major causes of these difficulties. Some are due to the tumours themselves, known to be a constant problem in assessing their response to treatment. Bone lesions for example are even considered non target lesion leading to a non-eligibility of the patients for a new therapy and the absence of correctly measurable disease in malignant pleural mesothelioma or ovarian cancer can affect patient management. Evolution under treatment of some tumours (even if they are easily evaluable before treatment) and /or the adjacent parenchyma may also be the cause of difficulties in assessing response. Modality used in the study, use of automated measurements, type of study and type of treatment (GISTS) may affect the measurements. The different criteria of response may also lead to discrepancies between observers. Panel reviews often offer the opportunity to realise how much intra-observer variability is important in these particular tumours. The second part of this course consists in searching either in our experience or in the literature, the new avenues to improve the quality of assessment of response in these kinds of tumours known as "difficult" and even to look forward to using new criteria, new techniques and also new endpoints The mainstay for image evaluation of response to oncological therapy is CT or MRI applying morphological criteria, generally RECIST, or in recent clinical trials RECIST 1.1. For some tumour types these criteria are not applicable and for others RECIST-like criteria have been developed depending on the specific characteristics of the tumour type. One example is the so-called Choi criteria for GIST that relies not only on changes in tumour size but also on alterations in lesion attenuation.
In functional imaging using FDG-PET the tumour uptake reflects the metabolic activity of the tumour and a decrease of FDG accumulation at follow-up during and/or after therapy as compared with the base-line examination indicates treatment response. For some tumours, such as lymphoma, a visual interpretation in this regard is often sufficient but for most cancers the FDG uptake in the tumours needs to be quantified in the PET images. Usually, the standardised uptake value (SUV) is used. SUV is a measure of the tumour radioactivity concentration that is normalised to the amount of FDG injected (the injected radioactivity, Bq) and A structured mammogram report avoids ambiguous communication of information and aids audit and data collection. It should contain the following information: an assessment of the breast density (fatty, mixed or dense or use the BIRADS system of 1 -glandular tissue < 25%, 2 -scattered fibroglandular tissue 25-50%, 3 -heterogeneously dense 51-75%, 4 -> 75% fibroglandular and fibrous tissue. A description of any mass seen in the breast with the size (maximum diameter together with perpendicular measurement), the shape (round, oval, irregular), margin (sharply or well defined, indistinct or illdefined, spiculated), density of the lesion, any associated features; presence of asymmetric density or architectural distortion. If calcifcation is present then the extent in two dimensions should be given, type of particles (amorphous or indistinct calcification with tea cupping) or fine linear, branching or casting. A description of the position of the abnormality naming the quadrant of the breast or the distance from the nipple. Coarse benign calification is not ususally reported. The presence or absence of any satellite lesions is important. Several different scoring systems are used. The UK uses a 5-point system -1 -normal, 2 -benign, 3 -indeterminate, 4 -suspicious, 5 -malignant. The American BIRADS system: 0 -more imaging required, 1 -normal, 2 -benign lesion, 3 -high probability of being benign, short interval follow-up recomended, 4 -suspicious, 5 -high probability of cancer and 6 -known malignancy. Breast ultrasound (US) examinations are sometimes reported separately from mammography and sometimes reported as part of a combined examination. In either case, the structure of the report should follow some general guidelines to make the report clear and concise. The report should start with a brief description of the technology used: transducer type and frequency, type of elastography when relevant. A brief description of clinical history will follow as long as the rationale for the use of US. Findings must be reported having in mind the diagnostic accuracy of US for different clinical and/or comparative studies (i.e., US of a cancer lesion will include findings relative to staging) and with comparison to prior studies when relevant. Reporting of findings must be followed by the assessment (BI-RADS categories should be preferred) with clear management recommendations. The report should be succinct, using terminology and descriptors internationally accepted. antiretroviral drugs has been established for many years. For both a successful long-term management as well as a potential eradication of HIV, future strategies have to be capable to battle the virus where it hides. The human immunodeficiency virus (HIV) enters the brain very early after exposure. The most probable way is through infected monocytes and lymphocytes that cross the blood-brain barrier (BBB). The introduction of antiretroviral therapies (cART) has resulted in significant declines in morbidity, mortality and prevalence of opportunistic infections. Despite treatment HIV-related CNS complications have remained common. HIV-associated dementia (HAD) is considered as the most severe form of HIV-related injury. Mild neurocognitive disorder (MND) represents a milder form of impairment. An asymptomatic neurocognitive impairment (ANI) was introduced to recognise individuals with impairment on neuropsychological testing who report functional limitations. Neuroimaging plays an important role in detection of HIVrelated changes in the brain. New imaging modalities (DTI, MRS) are increasingly used to measure injury within white matter tracts and regional changes in brain metabolites. This lecture will focus on the spectrum of "Neuro-AIDS disorders" and their imaging characteristics. Learning Objectives:
1. To review the most common CNS diseases in the HIV-positive population.
2. To learn how to use advanced imaging findings in distinguishing HIV-related brain disorders. 3. To consolidate knowledge on imaging-based therapy monitoring.
Understanding the role of immune activation and restoration in HIV infection A.G. Osborn; Salt Lake City, UT/US (anne.osborn@hsc.utah.edu) Immune reconstitution inflammatory syndrome (IRIS) occurs when restored immunity causes an abnormally robust response to infectious or noninfectious antigens. IRIS develops in 15-35% of AIDS patients beginning combination antiretroviral therapy (cART). IRIS develops in two distinct scenarios: "Unmasking" IRIS (u-IRIS) and "paradoxical" IRIS (p-IRIS). u-IRIS in AIDS occurs when beginning cART reveals a subclinical opportunistic infection against a living pathogen. p-IRIS occurs when a treated patient deteriorates after initiated of HAART. The recovering immune response targets persisting pathogen-derived antigens or self-antigens to cause tissue damage. This presentation discusses both types of IRIS and their imaging manifestations in the CNS. Neuro-IRIS affects only 1% of all IRIS cases but must be identified. u-IRIS should be distinguished from p-IRIS as disease management and prognosis differ significantly. PML-IRIS, TB-IRIS, fungal-and parasite-IRIS are illustrated. Non-HIV IRIS associated with natalizumab (Tysabri)-related PML will also be illustrated and insights into the roles of immune restoration in disease development in the setting of HIV-AIDS discussed. Since acquired immunodeficiency syndrome (AIDS) was first reported in the early 1980s, the lung was the organ most frequently affected by opportunistic lung infections (PCP) and noninfectious processes (Kaposi sarcoma and lymphoma). Since the beginning of the epidemic, more than 60 million people have contracted HIV and more than 25 million have died of AIDS-related causes. Actually, more than 33 million people live with HIV/AIDS and in 2009, 1.8 million people died from AIDS. The introduction of highly active antiretroviral therapy (HAART) has resulted in a considerable increase in the length of survival of HIV-infected patients. Although significant progress has been made in the past decade in the treatment Four randomised controlled trials (RCTs) comparing outcomes after carotid artery stenting (CAS) with carotid endarterectomy (CEA) have been published recently. Based on recent systematic reviews it has been recommended that CAS can no longer be justified for patients suitable for CEA. Indeed pooled data of the RCTs show higher peri-operative risk of performing CAS vs. CEA with comparable longterm efficacy in many centres. The inferiority of CAS to CEA as a method cannot be concluded from SPACE, EVA3S and ICSS because of limitations in study design and conduct. The goal of this presentation is not to discredit these trials but to develop a more differentiated and critical interpretation of the data and to create more discussion. I will discuss the necessity of randomised control trials (RCTs) for Interventional Neuroradiology in general and particular problems in study design (non-inferiority design and interpretation of results, clinical equipoise, study endpoints), practical study conduct difficulties (operator and centre experience, antiaggregation, timing of treatment) and the interpretation of the results (relation of internal and external validity, procedural complexity, the 68-years-surprise, longer-term outcome). A premature rejection of CAS based on the data from these studies could harm future patients who would have benefited from this procedure. The most common indication for PTA and stenting of extracranial arteries is atherosclerotic disease. Arterial dissection, spontaneous or traumatic, may also warrant such treatment. Other, less common, indications are Fibromuscular Dysplasia (FMD) and Takayasu´s arteritis (TA). Proximal stenosis of the internal carotid artery (ICA) is a common manifestation of atheroclerosis in the western world. It can be treated surgically with endarterectomy, but PTA/stenting has proven a good or better alternative for many patients as shown in recent multicentre randomised studies. It is today also possible in selected patients to reopen a completely occluded internal carotid artery. This may be a treatment option for patients with proven symptomatic hypoperfusion. Symptomatic atherosclerotic stenosis of the extracranial vertebral arteries (VA) is not as common, but PTA/stenting may be a treatment option also in this vascular territory. Arterial dissections usually heal but in a situation with haemodynamically significant impairment of flow, PTA/stenting may be needed in the acute situation. If the dissection causes a subarachnoidal haemorrhage, vessel sacrifice is often the safest treatment option. If that, however, for haemodynamic or other reasons is not possible, treatment with one or more stents may lead to a good outcome. When performing thrombectomy for acute stroke, a significant stenosis of the ipsilateral ICA or VA can be a complicating factor demanding procedural alterations including PTA/stenting. FMD may cause stenosis of the ICA, whereas TA predominately concerns the common carotid artery, both possible to treat with PTA/stenting if the patient suffers from thromboembolic or haemodynamic symptoms. Indications and contraindications of breast MRI will be discussed. Significant findings at breast MRI include foci (small contrastenhancing spot, NOS, < 5 mm), mass lesions, "non-mass-like enhancement" (no mass lesion, partly diffuse regional contrast enhancement of various size) and associated findings. As breast MRI is a functional study, different appearance patterns of kinetic contrast enhancement will be presented. The current status and the appropriate use of the BI-RADS MRI lexicon will be discussed. Any breast MRI report should not only follow these guidelines, but also express confidence, follow a red thread, be consistent and therefore be comprehensible for clinicians. The overall final BI-RADS assessment is based on the most worrisome finding, taking into account both breasts and all imaging methods (Mammography, Ultrasound, MRI) evaluated. Furthermore, adequate communication of the result as well as do's and don'ts of report wording will be discussed. MRI is more and more often used for PCa detection in patients with a negative series of biopsies and a persistent biological suspicion of PCa. Multiparametric MRI (mp-MRI) increases the accuracy of T2W-Imaging to localise PCa. The most widespread protocol includes dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI) which improve accuracy of T2W-MRI to detect P-Zone cancers with a tumour volume > 0.2-0.5 cc. DCE-MRI has a high sensitivity but a limited specificity related to a common enhancement of benign P-Zone sextants and of T-Zone BPH nodules. On DW-MRI, PCa shows a significantly lower Apparent Diffusion Coefficient (ADC) value than that of benign prostate tissue which increases the specificity of T2W-MRI. Chronic prostatitis and BPH stromal nodules can have a low ADC value close to that of PCa, but the use of ultra-high b values (b2000) allows for a better differentiation between the three conditions. Significant differences in tumour ADC values are observed between patients with low risk, and those with higher risk localised P-Zone PCa. Image fusion between MR and TRUS data set is a promising tool to increase the accuracy of targeted TRUS guided biopsies. There is more and evidence that a normal MRI is correlated with absence of cancer or presence of an insignificant tumour. PSA level and mp-MRI may thus select patients requiring immediate biopsy to detect significant tumours and those in whom biopsy could be deferred and indicated if follow-up shows signs of PSA level progression and/or occurrence of suspicious MRI findings. The role of imaging in patients with increased PSA level after radical prostatectomy or radiation therapy, which are the two main curative treatment options for prostate cancer, is to aid in differentiating locally recurrent disease which can be managed with local therapy from distant metastatic disease requiring systemic therapy. Although the majority of local recurrences in post-surgical patients can be detected by MRI in the perianastomotic region which can also be evaluated with TRUS and TRUS-guided biopsy, some recurrences can occur at pelvic sites that are beyond the range of TRUS; MRI has a role of labelling these sites for TRUS-guided biopsy. The combination of an external phased-array coil and endorectal coil is recommended for detecting local recurrent cancer. Current protocols involve T2-weighted MRI combined with functional techniques like dynamic contrast-enhanced MRI (DCE-MRI), magnetic resonance spectroscopy and diffusion-weighted MRI. In the post-prostatectomy bed, recurrences present as lobulated masses having low to intermediate signal intensity on T2-weighted images and showing early, nodular enhancement with early washout of gadolinium on DCE-MRI. After radiotherapy early contrast enhancement and early wash-out is seen recurrent tumors whereas the enhancement of post-radiation fibrosis is slow and low, offering a good contrast with the usually hypervascular recurrent cancer. The overall diagnostic efficacy of conventional MRI in combination with functional techniques is better than T2-weighted MRI alone which may play an important role in better assessment of locally recurrent disease. During the past decade we have observed a growing interest in in-vivo imaging techniques for small animals. On the shadow of the successful application of dualmodality imaging in the clinical environment, such as combined PET-Computed Tomography (CT) or SPETC/CT, the hybrid imaging modalities have been recently transferred to small animal scanners. Nowadays, the possibility of easy integration of nuclear imaging techniques with other modality such as PET/SPECT/CT, PET/ MR or PET/Optical has become a mandatory requirement in the design of small animal imaging systems. The combination of different imaging modalities in the same scanner offers the possibility to perform experiments more effectively than with a single modality only. This is especially true when two modality scans are performed sequentially or simultaneously and without moving the animal. Present technologies for the construction of hybrid systems for small animals will be presented together with examples of commercially available hybrid scanners. Looking at the next future, small animal PET/MR seems to be one of the most appealing perfect choice for hybrid imaging, combining the exquisite sensitivity of PET with the morphological/functional/spectroscopic high-resolution imaging of MR. The present major limitation on the development of simultaneous PET/MR scanners is the non-insensitivity of PMTs to magnetic fields. A reliable solution could be the development of magnetic field insensitive position-sensitive photodetectors. A brief overview of the present status of development of the detector technology for PET/ MRI will be also presented. The purpose of this lecture is to discuss the most common indications to perform sinonasal CT scans, to provide an understanding of the most common disease states and their impact on the choice of the respective imaging protocol and to provide a checklist of what the radiologist needs to report. First, the basic concepts of sinonasal CT and cone beam CT imaging including current protocols, radiation doses, 2D and 3D reconstructions and administration of contrast material will be discussed, followed by a brief discussion of the pertinent anatomy including the most relevant radiological anatomical landmarks. A systematic review will include key radiological features of acute and chronic sinusitis and related complications, polyposis, mycetomas, allergic fungal sinusitis and sinonasal tumours. Major emphasis will be put on the systematic approach, on the bottleneck areas where occlusion of normal passages may occur, on CT findings necessitating an additional MR and on how to report in a comprehensive way. CT examination of the temporal bone: Temporal bone CT scans should be performed in a reproducible way. Axial reconstructions are made in the plane of the lateral semicircular canal and coronal reconstructions exactly perpendicular to this plane. Thin sliced images are postprocessed using high-resolution algorithms. MR examination of the temporal bone: the challenge is to perform a complete examination. The standard version of such an exam starts with axial T2-weighted images of the minimal invasive focal therapy (laser, cryo, HiFu), and MR-guided radiotherapy will be discussed. Examples will be shown. In 1996, Hasegawa et al. presented a prototype SPECT/CT-design comprising a clinical SPECT-camera in tandem with a clinical single-slice CT. The combined device was used to perform a small number of clinical studies, such as for quantitative estimation of radiation-dosimetry in brain cancer, whereby the CT data were used also to generate the SPECT attenuation-correction-factors (ACF). Since then, SPECT/CT has benefited a great deal from the advances in CT-technology and several commercial system designs are available today. The clinical adoption of SPECT/CT has been rapid, particularly for oncology and cardiology. SPECT/ CT currently has a smaller installed base than PET/CT. The proposal to combine PET with CT was made in the early 1990s by Townsend, Nutt et al. In addition to intrinsic image alignment, the anticipated benefit of PET/CT was to use the CTimages to derive the PET-ACF's. The first prototype-PET/CT became operational in 1998 and was clinically evaluated at the University of Pittsburgh. Since then PET/ CT-technology has grown rapidly by incorporating new concepts of PET, available as PET/CT only, and multi-slice CT. Today's installed base is about 5,000 systems worldwide. The improvement in accuracy of PET/CT compared with PET or CT for re-/staging is statistically significant and averages 10-15% over all cancers. Image artefacts inherent to SPECT/CT and PET/CT can be detected, interpreted and corrected/avoided by well-trained users. By adopting more innovative acquisition schemes and data processing SPECT/CT and PET/CT will become faster and more dose-efficient imaging methods and manifest themselves as integral parts of state-of-the-art patient management. Radiographers have a key role to play in the provision of high-quality forensic imaging services and indeed forensic radiography should be viewed as an area of specialisation within the broader profession in keeping with other specialist areas.
The concept of optimisation has been established in clinical radiography practice across Europe and indeed in the forensic examination of living individuals, but remains an issue in many centres performing a variety of forensic examinations on the full or partial remains of deceased individuals along with specimen radiography. As with many other areas of service development barriers often exist to the development of forensic imaging services and there are key steps that must be considered in order to implement a high-quality service. Such barriers can include access to appropriate equipment and other resources, availability of education and training, a variety of medico-legal challenges and the voluntary nature of forensic work within a clinical setting. A guideline-driven, well-planned approach involving all key stakeholders is essential to the development and implementation of an excellent forensic imaging service. While many international centres of excellence in forensic imaging have now been established, and as more come on stream over the coming years, there remain many examples where the radiography-based forensic imaging service is not optimal and could be improved. A series of practical approaches and potential opportunities for radiographers to help develop their own local forensic imaging services will be explored. Radiography is now internationally regarded as a valuable tool in forensic and humanitarian investigations with continuous developments in this field. Despite this, there are currently no internationally recognised standards for education and training in forensic radiography. Training has traditionally been based on apprenticeship and as such, the use of clinical imaging techniques in the forensic context remains variable with little uniformity in service provision, training or education between countries. A 2006 study on international forensic radiography provision indicated a direct "correlation between the level of training given and its level of utilisation and perceived benefit to the investigation". Lack of standards in training and education can undermine good practice leading to poor record keeping, poor techniques in evidence provision and increased levels of anxiety in imaging personnel. This presentation further explores the role of international training and education in forensic radiography with recommendations for best practice including: 1. Development of international standards. 2. An increase in formally accredited, accessible education programmes. 3. Robust, sustainable national response teams of suitably trained personnel. 4. A database of trained personnel for international deployment. 5. The provision of practical simulation exercises (e.g. multidisciplinary mock disasters) encouraging experiential learning. 6. The provision of online professional development opportunities (e.g. modular e-learning programmes). There is nothing that can entirely prepare for the provision of forensic radiography services, particularly in response to a mass fatality incident. However, the international forensic radiography community has a responsibility to provide training opportunities for imaging practitioners to meet agreed international standards. Learning Objectives: 1. To understand the potential impact of a lack of standards in forensic imaging on forensic investigations. 2. To be informed about the role of education and training in key areas of forensic imaging practice. 3. To become familiar with examples of international best practice in forensic imaging education and training.
brain and posterior fossa, and axial T1-and heavily T2-weighted images covering the temporal bone. After intravenous injection of gadolinium axial and coronal T1weighted images are performed. Temporal bone report on CT and MRI: structured reporting is the result of structured viewing. In the evaluation of the middle ear on CT examinations it is best to follow the sound wave on the axial images (from tympanic membrane over malleus, incus and stapes to oval window), than inspect the fissula antefenestram region and the oval window. The inner ear is evaluated from cranially to caudally (semicircular canals, vestibule and vestibular aqueduct, cochlea and cochlear aqueduct). The facial canal, carotid canal and jugular fossa are finally evaluated. On the coronal images one should at least inspect the intactness of tegmen tympani and the bony cover of the lateral semicircular canal. On MRI, after viewing the brain images, one best first tries to detect abnormal enhancing inner ear structures on the gadolinium-enhanced T1-weighted images, and then explains them using the heavily T2-weighted images. The purpose of this lecture is to present how a good diagnostic head and neck scan can be achieved. Indications, technical problems and pitfalls, timing and contrast administration issues will be addressed. Tips and tricks regarding the best quality of the images will be discussed with appropriate examples. Afterwards, an example of a structured report will be presented, describing comprehensively all steps of radiological evaluation of the disease, followed by clear conclusion and staging. Emphasis will be placed on what the surgeon needs to know in order to plan the treatment. Next, attetion will be paid to lymph node evaluation: features of malignant infiltration will be discussed. As an endpoint, all steps including proper CT examination, radiological findings and structured reporting will be reviewed. Forensic imaging has grown slowly from its early radiographic applications starting before the year 1900, soon after the detection of x-rays, over the whole 20th century, before turning into an exponential growth after the introduction of multidetector CT and fast MRI techniques in the past ten years. Starting with a short summary of the history, this conference will address the advantages of virtual autopsy as compared with conventional autopsy: its non-destructive character, frozen objective documentation, ease of archiving and distribution, the lack of motion artefacts, and the lack of effects of ionising radiation. There are, however, significant disadvantages of postmortem imaging; these and approaches to overcome the limitations will be discussed as well as applications of forensic in vivo imaging. The differences between clinical and forensic imaging will finally be pointed out, and we will discuss some questions specific to forensic imaging, such as identification, detecting the cause and manner of death, vital reaction at imaging and reconstruction. Sunday markers of intraarticular disease, such as joint effusion, synovial hypertrophy and microvasculature and is able to detect subclinical synovitis. The ability of US to check multiple joints in one study and reveal subclinical synovitis seems to be particularly relevant in JIA, as definition of oligoarthritis and polyarthritis is based on the number of affected joints. US is an ideal means, therefore, for drawing a more correct patient classification, as well as for alerting the referring physicians towards the need of more aggressive treatment and closer monitoring. US is also an excellent means to distinguish between joint synovitis and tenosynovitis and to guide the aspiration and injection of steroids into joints that are difficult to reach blindly. Despite these advantages, US is inferior to MR imaging in depicting early inflammatory change and evaluating the articular cartilage. MR imaging is able to assess bone marrow abnormalities and can better evaluate the late manifestations of the disease, including erosions, joint space loss and ligamentous involvement. Both Doppler US and contrast-enhanced MR imaging are able to provide a measure of disease activity. The project aims at developing imaging tools able to improve the monitoring of cell therapy as well as the understanding of the fate of transplanted cells and the mechanism of action of cell-based therapies. Using the knowledge obtained, tools and treatment strategies can be further optimised to reap the full benefit of cell-based therapies. The project relies on the extensive collaboration of European experts for the development and implementation of novel imaging procedures with several working groups from different disciplines (physics, chemistry, biology, informatics, endocrinology, immunology, neurology, nuclear medicine, radiology). The work is organized in five subprojects:1) Novel Imaging Technologies (New imaging methods, post-processing techniques, visualisation tools); 2) Probe Chemistry (New MRI reporter probes that draw a more comprehensive picture of molecular imaging applications); 3) Novel Tools for Cell Imaging (Technologies enabling tracking of cell migration, proliferation, differentiation and death); 4)Pre-Clinical Validation (Tools to assess stem cell engraftment, cell differentiation in major disease areas); 5)Translation towards Clinical Applications (to increase the efficacy of cancer immunotherapy). Responding to an increasing level of interest and a great need for educational activities in the field of cellular and molecular imaging, ENCITE has elaborated a specific training initiative based on the creation of a Multi-Centre Cluster devoted to the implementation of a repository of training procedures and imaging reporters. The output of this activity is made available to all the project partners and, more in general, to all the Cellular Molecular Imaging community. Musculoskeletal sport injuries of children and adolescents differ from that of adults because they occur on a growing skeleton with particular characteristics. The bone and the cartilage are weaker than the muscles and the ligaments, resulting in specific lesions. The vulnerability of the growing bone and cartilage are increased during the rapid growth periods. There are two main types of sport injuries. The acute traumatic lesions are the most frequent and correspond to the specific fractures of the paediatric age. The chronic overused injuries are related to repetitive micro traumas and are more frequently encountered in young athletes. The specific acute sport injuries, more frequently seen in adolescents, are the metaphyso-epiphyseal fractures, the apophyseal avulsions and the patellar dislocation. Sport injuries in children can lead to specific complications when the physis is involved, resulting in growth disturbance, with the need of an extended follow-up. X-rays are the first imaging tool but indication should be selected according to the clinical findings. Ultrasonography is ideally suited for the evaluation of soft tissue, muscles, tendons and ligaments. CT is useful to better analyse complex articular fractures. MRI is the imaging of choice to look for ligaments, menisci, osteochondral injuries and overused injuries. MRI is also used to assess complications like epiphysiodesis or osteonecrosis. Developmental dysplasia of the hip (DDH) is the most common musculoskeletal disorder in childhood, with a reported prevalence of 1-4% according to method of ascertainment and definitions used. Ultrasound has enabled a detailed view of both neonatal hip stability (NHI) and morphology, and two different schools have developed: one arguing that NHI alone is the major pathology warranting splinting, the other including acetabular dysplasia as an important feature. Both static (Graf, Morin) and dynamic (Harcke) ultrasound techniques, as well as a combination of the two (modified Graf (Rosendahl)), have been described and are currently used. In Europe, Graf's ultrasound technique or a modification of this is commonly used within the German-speaking countries and areas, in parts of Scandinavia, the UK, Italy, France, Hungary and the Netherlands. Others use a modified Morin's method while Harcke's method is used only occasionally. Initially, universal ultrasound screening using Graf's method led to higher treatment and follow-up rates than that based on NHI alone, i.e. 3-5% versus 0.4-1.5% and 10-20% vs. 6-7%, respectively. However, improved examination techniques and a better understanding of the findings have led to a more tailored approach, and an extensive meta-analysis performed in 2000, including 534 papers, could not find any differences in treatment rates due to different ultrasound techniques. In this lecture I will present a crude status for US techniques used and also give recommendations for a worthwhile screening strategy based on present knowledge. Magnetic Resonance (MR) imaging has emerged as an important imaging modality for the assessment of cirrhosis and its complications, such as HCC. Concurrent technical improvement as well as implementation of advanced imaging sequence designs permits high-quality examination of the liver with high intrinsic soft-tissue contrast. The use of automated contrast detection methods combined with faster sequences allows to optimise the arterial phase, which is essential for the detection and characterisation of HCC. Finally, combining sequences, including T2 and T1 weighted images, diffusion-weighted sequences, dynamic gadolinium-enhanced 3D multiphasic imaging and liver-specific delayed phase sequences, allow a simultaneous evaluation of the background liver parenchyma and of the liver lesions. Morphologic features as well as the degree of fibrosis of the background liver parenchyma represent the most important criteria in the clinical setting for determining the presence of cirrhosis. Although liver biopsy is the standard of reference for diagnosis and staging of liver fibrosis, MR elastography, diffusionweighted imaging and MR perfusion imaging have been developed for non-invasive assessment of the liver fibrosis.
Learning Objectives: 1. To know how to perform a standardised study protocol that correctly assesses the cirrhotic changes in liver parenchyma. 2. To learn which contrast media are applicable for nodule characterisation and liver function evaluation. 3. To go through DW imaging, perfusion and MR elastography: help or hype?
A-438 16:30 B. Small bowel and colon: how to study a patient with suspected inflammatory bowel disease N. Papanikolaou; Iraklion/GR (nickolas.papanikolaou@gmail.com) MR Enteroclysis and MR Colonography examinations have been mainly utilised to study patients with Crohn's disease. Early, subtle lesions such as superficial ulceration, mucosal nodularity and thickening or distortion of the intestinal folds may be detected by MRE, although to a lesser extent comparing with conventional enteroclysis, due to its lower spatial resolution. To improve detection rate of these subtle lesions utilisation of dedicated ultrafast, high-resolution pulse sequences, coupled with strong gradients have to be considered. Fibrofatty proliferation can be easily depicted, while its composition regulates its signal intensity characteristics being of low signal intensity in case of fibrosis and of high signal intensity in case of excessive fat accumulation. Gadolinium uptake on FLASH images allows identification of hyperemic bowel segments due to inflammation and accompanying small inflammatory nodes. Certain MR imaging findings, including the presence of deep ulcers, wall thickness more than 7 mm and marked mesenteric lymph nodes gadolinium enhancement were well correlated with the clinical activity index (CDAI), and it is suggested that such findings may serve as indicators of Crohn's disease activity. Another important clinical challenge that MR Enteroclysis and MR Colonography must address is accurate disease subtype classification. By recruiting multiple contrast mechanisms like signal intensity on T2-weighted images, contrastenhancement patterns on T1-weighted post gadolinium gradient echo images and motility-related information as identified on cine-TrueFISP images the latter clinical questions can be answered in a certain extent. Learning Objectives:
1. To review the basic technical aspects of MR enteroclysis. 2. To become familiar with diagnostic imaging findings related to disease activity. 3. To understand the use of optional sequences depending on the clinical question in patients with Crohn's disease.
of implanted cells, and to optimize dendritic cell migration in vitro for clinical cancer vaccination studies. Finally, a multimodal imaging approach combining 19 F MRI with Bioluminescence Imaging (BLI) is presented which allows non-invasive imaging of the anatomical location (19 F MRI) and function (BLI) of neural stem cell grafts. In conclusion 19 F MRI will help to optimize pre-clinical protocols of cell therapy and possibly become clinically relevant with further advances in 19 F compound synthesis, development of MRI hardware, and MRI pulse sequence optimization.
Imaging of novel therapies in Gliblastomas using multiple biomarkers W. Reichardt; Freiburg/DE Glioblastomas still have an extremely poor prognosis up to now and new therapeutic concepts are urgently needed. One of them is Anti-angiogenesis, which has been approved recently and is now under clinical evaluation. Next to edema formation and tumor morphometry, MR imaging can provide a number of vascular characteristics for therapy assessment in the tumors, which could especially provide an early assessment of the therapy. Anti-angiogenic therapy in tumors can be monitored by dynamic contrast enhanced MRI (DCE-MRI) or dynamic susceptibility contrast imaging (DSC-MRI) and vessel size imaging (VSI) to detect changes in tumor microvasculature. DCE-MRI (yielding permeability) is based on the measurement of a concentration time curve after injection of a Gd-based contrast agent (CA), whereas vessel size imaging and DSC-MRI (yielding blood volume) is usually performed with iron-oxide particles. We could show that it is possible to acquire multiple biomarkers to assess therapy response simultaneously in vivo using a single shot gradient echo spin echo (GESE) EPI sequence with only one Gd-CA injection. To account for the typical tumor heterogeneity in gliomas, tumor segmentation was performed prior to the determination of the bloodflow characteristics. The determination of VSI and blood volume measurements using Gd-DTPA has already been demonstrated successfully.
Integrated image analysis of multi-modal pre-clinical imaging studies B. Lelieveldt; Leiden/NL
The rapid developments in in-vivo molecular imaging modalities such as fluorescence and bioluminescence imaging enables the live imaging of gene expression, cell fate and protein interactions. Combined with detailed structural imaging modalities such as magnetic resonance (MR), the biochemical onset of disease and therapy can be monitored in combination with structural and functional consequences over time. This presentation discusses a number of image analysis challenges emerging from longitudinal pre-clinical molecular imaging studies that have been addressed within the ENCITE project. Three steps towards a quantitative 3D analysis of follow-up small animal imaging are presented: whole-body registration, change visualization in follow-up data and fusion of optical and 3D structural imaging data. Several application examples are presented in the context of cell tracking and translational cancer research.
In vivo imaging of immune responses in cancer patients
We have been among the first to exploit dendritic cell (DC) therapy to treat cancer patients. Over the past years, immunological responses are increasingly reported and clinical responses have consistently been observed. Moreover, DC therapy often has much milder side effects than standard chemotherapy. A major hurdle in the development of the DC therapy is accurate delivery of the cells to lymph nodes (LNs), or their successful migration from the site of injection to LNs. In particular, tools for measuring cell migration in vivo are necessary. Ideally, we would be able to quantify the number of DCs at the relevant site, with high resolution anatomical context to allow differentiation of LNs and the possibility of longitudinal data acquisition. Furthermore, functional data on the ensuing immune response is also required. Towards these ends, we have been working on developing imaging techniques to study DCs in vivo, for example with scintigraphy on 111In-labeled DCs, and magnetic resonance imaging (MRI) on iron-labeled DCs. Scintigraphy is quantitative, but it is restricted to the relatively short half-life of the radioisotope and is unable to resolve individual LNs. MRI allows high resolution anatomic localisation, but the use of contrast agents such as iron oxide is not quantitative. Our recent work has focused on imaging the functionality of these DCs using positron emission tomography (PET) to study LN activation. Finally, we have also developed in vitro assays that closely mimic in vivo DC migration in 3D scaffolds imaged using quantitative 19F MRI, as a substitute for in vivo optimization. We plan on applying Patient transport into the radiology department, contact to other potentially infectious persons, and things like breath-holding are reasonable burdens and dangers to immunocompromised patients. When searching the focus of fever, imaging should help to identify an affected organ system in order to eventually guide invasive procedures to identify underlying micro-organism or non-infectious disease. Equally relevant is the exclusion of its involvement with a reasonable specificity. Depending on local epidemiology, organ system, and the clinical signs and symptoms, suspected differential diagnosis can be derived from image patterns. Some of these diagnoses might be exclusion diagnosis; others might require invasive procedures including time-consuming and costly analysis to be verified. Invasive procedures, however, require adequate heostasis, which is usually not available for a substantial duration due to pancytopenia in patients which underwent chemotherapy. If imaging fails to derive the underlying disease confidently and conclusively in a fast way, clinicians might need to treat on an empirical basis. Empirical treatment plays a major role in immunocompromised or severely ill patients at risk, since mortality rises within hours of untreated disease. On the other hand, empiric treatment causes relevant toxicity and substantial costs, while imaging might become cost effective. The appropriate investigational technique, frequently targeted differential diagnosis, and the special needs of immunocompromised patients need to be understood by the referring physician as well as by the radiologist. Thus, an intensive interdisciplinary co-operation on a patient basis, as well as on a department basis is essential. Learning Objectives: 1. To learn more about the clinical conditions that cause fever without an apparent origin. 2. To be informed about the clinician's way of thinking in the process of differential diagnosis. Diagnosis of fever of unknown origin (FUO) is a major challenge for internists. The spectrum of disease includes infections (28%), inflammatory diseases (21%), malignancies (17%), and "no diagnosis" (19%). Deep vein thrombosis (3%) and temporal arteritis in the elderly (16%-17%) are important considerations. Investigation of patients with FUO usually includes clinical and standard biological tests which give numerous clues. Imaging procedures depend on objectives and various imaging investigations should be organised in strategies. Early identification of the best tissue to be the site of biopsy is one of the most decisive procedures. First-line imaging studies usually include chest radiography, abdomen ultrasonography, and contrast-enhanced CT of the thorax abdomen pelvis in order to disclose common aetiologies such as infections ( Chronic pancreatitis (CP) is a continuing inflammatory process, characterised by irreversible morphological changes, pain and permanent loss of exocrine/endocrine function. Patients with suspected CP will undergo imaging and/or pancreatic function tests to rule out early CP. Standard MR protocols consist of: axial/coronal TSE T2-w, fat-suppression axial GRE T1-w and axial/coronal MRCP sequences. Early ductal changes, as ectatic secondary ducts (pathognomonic sign of early CP) may not be detected on standard MRCP. Secretin-enhanced MRCP (S-MRCP) studies, performed during exogenous secretin stimulation, increase the diagnostic accuracy in patients with suspicion of CP. The physiologic effect of secretin, i.e. transient increase of pancreatic exocrine juice in the ductal system, improves pancreatic duct visualisation on MRCP images. S-MRCP can quantitatively estimate pancreatic exocrine reserve. Significant differences were reported between normal and severe CP, while values obtained in mild CP were in the same range of normal. Normal pancreatic parenchyma appears bright on unenhanced fat-suppressed GRE T1-w images due to high protein content in the acini. On contrast-enhanced GRE T1-w studies normal pancreas enhances significantly on the arterial phase due to its rich arterial network. Parenchymal changes in CP patients are characterised by loss of normal functional parenchyma and vascularity, replaced by fibrosis. These changes induce the loss of pancreatic parenchyma hyper-intensity on unenhanced GRE T1-w images and delayed arterial enhancement on contrast-enhanced images. Recent published studies reported significant differences of apparent diffusion coefficients (ADCs) calculated at baseline and after secretin stimulation between normal and CP, with no differences among the different degree of severity. Sunday modalities of choice after renal transplantation. MSCT and MRI are valuable when these techniques are inconclusive or when post transplantation complications are suspicious. Renal transplant complications may result in impaired renal function or graft loss, most occurring at predictable times post transplantation. Post transplant complications can be divided into four major categories: parenchymal abnormalities, collecting system abnormalities, perinephric collections, and vascular abnormalities. Main vascular complications are renal artery stenosis/thrombosis, renal vein stenosis/thrombosis, arteriovenous fistula and pseudoaneurysm. Parenchymal complications are acute tubular necrosis, acute and chronic rejection, infarct, pyelonephritis, renal cell carcinoma and post transplant lymphoproliferative disorder. Perinephric collections are urinoma, haematoma, lymphocele and abscess. Main collecting system complications are hydronephrosis, calculi, urinary leak and obstruction. DUS is excellent tool not only to assess anatomic abnormalities and vascular status, but also to establish a baseline for future follow-up. Radionuclide scans help to assess renal perfusion and function. CT or MR urography allows imaging of the entire course of ureteral and periureteral abnormalities. US and CT are very useful for guidance of renal biopsy and drainage of large fluid collections. CT or MR angiography may be useful in doubtful cases to evaluate renal artery stenosis. Treatment of choice in diagnosis of the renal artery stenosis is percutaneous transluminal renal angioplasty (PTRA) or PTRA with stent. Learning Objectives:
1. To learn about the most common causes of transplanted kidney dysfunction. There are two types of AV shunts for haemodialysis: AV fistulas and AV by-pass. The former have the longest life duration and the latter are implanted when fistulas fail. No systematic follow-up of these shunts is required. But imaging plays a major role to define causes of dialysis dysfunction and to treat complications. Main dysfunctions are a decrease of pump flow, evocative for occlusive disease located up-stream the arterial needle, and an increased venous pressure, evocative for occlusive disease located down-stream the venous needle. Other problems are an increased dialysis time, difficulties in needle placement, clotting, etc. Clinical examination must focus on sites of anastomoses and along the shunt looking for decreased thrill, decrease of shunt tension, and abnormal thrill areas. Color Doppler must cover the entire shunt from the afferent artery to the subclavian vein, looking for stenoses, usually located on the venous side, aneurysms, mural thrombus, acute thrombosis, steel phenomenon or collections. When a stenosis is present, measurement of velocity ratio helps in quantifying its severity, being significant when > 3. The risk of thrombosis increases when flow decreases within the shunt. Angioplasty is required when stenosis is significant and/or when pressure increases or flow decreases on successive dialyses. Auto-expansive stents are implanted when stenoses recur. Acute thromboses are treated by mechanical thrombolysis using aspiration or mechanical devices. Contrast agents, both iodine-and gadolinium-based, are generally safe and are very widely utilised. However, there is concern about inducing contrast-induced nephropathy (CIN) associated with iodinated contrast media (ICM) and nephrogenic systemic fibrosis (NSF) following the administration of certain types of gadoliniumbased contrast media (Gd-CM) in patients with advanced renal impairment. CIN is associated with further deterioration in renal function, may speed the need for dialysis, and increase the morbidity and mortality of the affected patients. Unfortunately all ICM have the potential of inducing CIN in patients with reduced renal function. This complication can be minimised by volume expansion and reducing the dose of the contrast agent. Large volume of data indicates that NSF is caused by the release of free gadolinium from Gd-CM with low kinetic and thermodynamic stability such as the non-ionic linear agents. This complication can be avoided with the use of the lowest possible dose of the highly stable macrocyclic agents.
Learning Objectives: 1. To become familiar with the potential risks of using iodine or gadolinium-based contrast media in patients with renal impairment. 2. To know how to reduce the risk of these potential risks. The global prevalence of renal insufficiency (RI) in the population is increasing. RI may be classified depending on 1. the clinical type: acute, intermittent and chronic; 2 the stage: mild to severe; and 3. the cause: pre-renal, renal, post-renal or urinary obstruction. An appropriate management of RI may lead to decrease of morbidity and mortality, especially in intensive care units or among patients with end-stage renal disease (ESRD). The role of imaging is based on a multimodality approach, with ultrasound, CT, MRI and scintigraphy providing both morphological and functional information, including split renal function and glomerular filtration rate measurements. Several strategies in key clinical situations, including renal artery stenosis, parenchymal diseases, and obstructive uropathy will be discussed in the adult and paediatric population.In chronic ESRD, the development of acquired multicystic disease is frequently observed on native and transplanted kidneys. Primary malignancy of the kidney allograft is not an exceptional condition in the long term and may justify regular screening by imaging, with potential diagnostic difficulties in differentiating cysts and tumours. In case of a tumour, imaging is helpful in selecting the appropriate surgical or percutaneous procedure, with the objective of transplant nephron sparing. Total hip arthroplasty is one of the most effective orthopaedic reconstruction procedures and is predicted to have ever-increasing patient volumes. Outcome assessment has become a sub-specialty in its own right attracting a large volume of scientific research and coverage in leading orthopaedic journals. The goal is for the hip replacement to outlive its recipient! Imaging plays a pivotal role in the assessment of its clinical outcomes, detection of failure and assessment of complications. There are multiple risk factors including patient selection, patient age, gender, primary hip disease, body weight, geometry of prosthesis, surface texture, type of metal, durability of bearing surface, bearing surface coupling, type of prosthesis, etc., which the radiologist needs to be familiar with when called to interpret post-operative imaging. In addition, bone remodelling also takes place around the prosthetic components resulting in altered bone density and morphological changes, which although normal may also increase the risk of fracture. These risks do not only operate in isolation, but are also inter-related. Component position related to surgical technique and proficiency is crucial, as it has been consistently shown that proper positioning is critical for the stability of the implant, fixation durability and optimum bearing surfaces wear. The three speakers will address the role of imaging with a view of differentiating the expected "normal" changes and appearances from developing peri-prosthetic complications. Session Objectives:
1. To learn about the current state-of-the-art prostheses. 2. To be familiar with the common problems of hip replacement. 3. To define the strengths and weaknesses of the imaging modalities in assessing hip replacement.
Radiography and ultrasound: how far can you go? S. James; Birmingham/UK Hip arthroplasty is an extremely common orthopaedic procedure and there is a wide array of implants which are in current use. It is important to understand the normal radiographic appearances following hip joint replacement, so complications can be appreciated and accurately reported. Radiographs still provide the mainstay for the initial diagnosis of component failure, both in the immediate post-operative period and at long-term follow-up. The relative significance of different patterns of radiolucency, bone sclerosis and component position is discussed. The normal or pathological significance of these findings is correlated with design, surface and fixation of the prosthetic components. It is of vital importance that serial studies are compared to early post-operative x-rays during follow-up to report accurately any sign of prosthetic failure and trigger prompt specialist referral. Ultrasound is quick to perform, inexpensive, does not suffer from susceptibility artefacts and can be used to guide treatment. The common soft tissue complications following hip arthroplasty are reviewed and illustrated. Learning Objectives: 1. To become familiar with the radiographic appearances of different types of prosthesis design. 2. To recognise the normal radiographic appearances following hip joint replacement. 3 . To learn about the common abnormal radiographic findings following hip replacement. 4. To understand the role of ultrasound in assessing the post-operative hip. CT scan plays an increasing role in the evaluation of hip arthroplasty. This technique has wide indications, as it is more sensitive than radiographs in detecting osteolysis secondary to prosthesis loosening, it clearly shows the periprosthetic fluid collections suggesting infection, and is the best technique to guide their aspiration including the aorta and mesenteric, pelvic and cervical systems. In addition, in extremity trauma with suspicion for arterial injuries, CTA has proven accurate in helping diagnose significant vascular injuries. Yet digital subtraction angiography (DSA) is reserved for therapeutic intervention. This talk aims to discuss technical aspects as well as indications for CTA and DSA. In particular, the implementation of CTA in whole-body trauma CT is emphasised. In addition, typical imaging findings are reviewed. Learning Objectives:
1. To become familiar with the most important imaging features and their impact on patient management. 2. To understand technical details, potential difficulties and pitfalls of demonstrating these findings. 3. To be familiar with the pertinent findings to report. Thoraco-abdominal injuries are a significant cause of death in the polytraumatised patients. Early recognition and communication of life-threatening thoraco-abdominal injuries is the major task for the radiologists involved in the emergency room. Although, most of these patients reach the hospital prior to death, lethality continues to remain high. Heart, thoracic great vessels, trachea, bronchus, pleura, lung, diaphragm, abdominal/retroperitoneal vascular and solid organ injuries are potential causes of death. Any appropriate surgical/interventional management approach must be carried out "around the clock", before the thoraco-abdominal injuries reach the level of clinical evidence. On the other hand, non operative management has actually become the standard of care for the most serious thoraco-abdominal injuries. These goals become feasible if a correct contrast-enhanced MDCT diagnosis, in a dedicated facility in which the trauma team works effectively 24 hours a day, seven days a week, is performed. Thus, in this lecture, the most serious thoraco-abdominal injuries will be illustrated, with special emphasis on vascular injuries as well as the value of post-processing techniques, protocols, pitfalls, tips and tricks. Furthermore, the importance of a rational and integrated imaging approach will be pointed out and, finally, the role of the radiologist in emergency room will be emphasised. Learning Objectives:
1. To become familiar with the most important imaging features and their impact on patient management. 2. To understand technical details, potential difficulties and pitfalls of demonstrating these findings. 3. To be familiar with the pertinent findings to report.
A-453 17:00 C. Extremities U. Linsenmaier; Munich/DE (ulrich.linsenmaier@med.uni-muenchen.de) Extremity injuries in patients after polytrauma can be very complex and are often difficult to be diagnosed comprehensively when patients undergo only an initial whole body CT (WBCT). Extremity injuries comprise: fractures of (1) long bones, (2) articular joints, (3) complex fractures of hands and feet; (4) vascular, (5) soft tissue and (6) nerval and plexus injuries and (7) amputations. The use of MDCT could replace conventional radiographs (CR) in many indications. MDCT is indicated in all major and complex bony fractures and should be carried out early. Many institutions include those extremity scans in the WBCT workup using the arterial contrast phase as CT angiography (CTA) providing an excellent workup of the vascular system. CR remains an important diagnostic mean; however, these CRs should only be carried out after completion of the initial WBCT to avoid any dangerous delays in diagnosis of life-threatening injuries. CRs are especially suitable for follow-up and postoperative controls. Magnetic resonance imaging (MR) is an adjunct to MDCT and CR in the diagnosis of extremity injuries. It is indicated in complex injuries with unstable articular injuries, those involving tendons or major ligaments and nerval and plexus injuries. However, MR should only be carried out after complete stabilisation of the patient. Finally, it has to be mentioned that the operative treatment of extremity injuries must be priority oriented and carefully planned in the context of possible concomitant injuries and a possible risk of multi-organ failure (MOF). Sunday examples. Epidemiological, clinical and imaging findings of these diseases will be discussed, and differential diagnostic features will be proposed. It will also be discussed as to how these diseases insert into the broader general context of lung diseases, and how imaging can contribute to a better understanding of their respective clinical characteristics. Learning Objectives:
1. To learn about the imaging findings in pulmonary fibrosis. 2. To be able to recognise the imaging findings in pulmonary sarcoidosis. 3 . To learn about the imaging findings in pulmonary vasculitis. B S111 C D E F G H A European Congress of Radiology 2012 Sunday identified and treated in advance to avoid major coronary events. Cardiac CT allows for the exact assessment of coronary morphology including length, calcification and severity of lesions. Based on these morphological information, success of a percutaneous revascularisation procedure can be anticipated with high prognostic accuracy. Additionally, the possibility of identification of lesion at risk by means of coronary CT has been described recently. By this, dedicated treatment of only the relevant stenosis should become possible avoiding multiple, potentially unneeded, stents. Finally, even the assessment of myocardial viability by means of CT becomes possible. Using all the possibilities of cardiac CT optimised treatment plan can be established, and outcome can be estimated. This presentation should give an overview about potential applications of cardiac CT for optimised treatment decisions and planning. Potentially useful algorithms for improving the outcome of coronary interventions should be provided. Prediction of coronary revascularisation outcome represents a major clinical target since a large number of medical and surgical options have become available for chronic ischaemic cardiomyopathy with need to identify more rigorous criteria for patient selection. The combination in a single examination of function, stressperfusion and tissue characterisation with T2-weighted "oedema-sensitive" and late-gadolinium enhancement (LGE) techniques, supported the role of cardiac MR (CMR) as an important technique for the evaluation of patient candidates for revascularisation. Besides more "traditional" indicators like ejection fraction, enddiastolic wall thickness or end-systolic volumes, extent and distribution of myocardial scar depicted with LGE has been identified as one of the most important predictors of post-revascularisation outcome with direct influence on functional recovery and on major adverse cardiovascular events (MACE) due to the potential induction of arrhythmias from the scar. LGE technique has been shown to be superior to nuclear medicine for the assessment of myocardial viability due to the higher spatial resolution (up to 60-fold greater than SPECT) and an intrinsic high-contrast resolution. A further technique that could be adopted before revascularisation is stress imaging. Myocardial ischaemia detected by either CMR adenosine first-pass perfusion or dobutamine-induced wall motion abnormalities has been shown to predict subsequent cardiac death, whereas normal stress perfusion showed a high negative predictive value for MACE. In conclusions, although as a relatively new diagnostic modality prognostic evidence is predominantly derived from single-centre studies, CMR is increasingly becoming an important tool for risk stratification before revascularisation, offering indications about outcome and mortality. Cardiac valve diseases are an important public health problem, with an increasing incidence strongly linked to the increasing age of the Western population. The most frequent valve disease is aortic stenosis, for which percutaneous aortic valve replacement (PAVR) is currently evolving as an alternative therapy in high-risk patients. Nevertheless, careful evaluation of all aspects of this new approach is still required to avoid uncontrolled diffusion. Imaging plays a key role in selecting patients who may be eligible for PAVR, focusing on the evaluation of leaflet anatomy, severity of valve dysfunction, haemodynamic consequences and potential problems in the access route. While echocardiography is commonly used for both the anatomical and functional evaluation, multidetector CT (MDCT) has important intrinsic advantages providing state-of-the-art 3D imaging with a high spatial resolution over a large anatomic coverage. During this course, we will discuss the advantages and disadvantages of MDCT compared with other imaging modalities. The relevant anatomy of the aortic valve and annulus will be reviewed, with emphasis on correct alignment of the imaging planes, and its implications for Diffusion tensor imaging plays an important role in the diagnosis and management of paediatric brain disorders as it provides important physiological information. This physiological information comes from the fact that water molecules diffuse approximately 10 microns using typical diffusion parameters, allowing interrogation of tissue properties at a micron level with this information volume averaged over a 8 cm3 image voxel. In our clinical experience we have observed that decreased diffusivity occurs in situations where there is 1. Metabolic failure (ex. acute necrosis) or compromise (ex. Status epilepticus), 2. Myelin vacuolization (ex. Maple Syrup Urine Disease), 3. Increased cellular density (pus, medulloblastoma). However, in hypoxic, ischemic insults it is important to remember that normal diffusivity does exclude a significant brain injury in the first few days after an event. In this situation, delayed cell death mechanisms may be activated, resulting in delayed decreases in diffusivity (delayed necrosis) or just long term volume loss (delayed apoptosis).
Using the directional information, underlying tissue structure can be further interrogated. This is particularly helpful when assessing lesions of decreased diffusivity to determine if they are more likely to be metabolic compromise or myelin vacuolization as opposed to irreversible metabolic failure. In addition tractography can be used in preoperative planning, however, appreciation for limitations in regions of edema need to be understood to avoid underestimating the presence of axonal bundles.
Research approaches use diffusion tractography to better understand structural connectivity and changes that occur in networks with development and disease.
Learning Objectives: 1. To recognise the specificities of DTI in the paediatric brain. 2. To gain knowledge of the importance of DTI in the assessment of brain maturation and white matter diseases. 3. To learn the DTI value in the evaluation of brain destructive lesions. Advancements in CT and MRI technology have led to an increasing use of these modalities in the non-invasive assessment of coronary arteries, myocardial perfusion and cardiac function. While their role in detecting coronary artery disease and functional disorders has been widely accepted, it is still unclear whether they could be adopted in triaging patients for the best therapeutic approach. Large studies have already suggested that indication for surgery and percutaneous interventions cannot be solely based on the demonstration of morphological alterations and that such "cosmetic" interventions are not always leading to the expected outcomes. Therefore, non-invasive imaging techniques have to offer more than just the detection of grades of coronary artery stenosis, of areas of infarcted myocardium, or of valvular alterations. Adjustment of imaging protocols for additional evaluation of coronary flow reserve, of myocardial perfusion and contractility and of valve size, position and damage with subsequent quantification of degree of stenosis and/ or regurgitation are necessary in order to allow choosing the most appropriate therapeutic approach and thus become the "gold standard" for prognosis and pretherapeutic diagnosis of cardiac diseases.
A. Can CT predict the outcome of percutaneous intervention? C. Loewe; Vienna/AT (christian.loewe@meduniwien.ac.at)
The outcome of coronary revascularisation is not only defined by primary technical success, but also by improvement of symptoms and quality of life. Thus, despite the individual comorbidities, the outcome and thus the potential benefit of coronary revascularisation depends on many different factors, including morphology, distribution and severity of coronary lesions, myocardial viability, and ventricular function. Consequently, the detection of coronary stenosis is not sufficient for planning an optimised treatment, and more information is needed to personalise the treatment plan. It should be evaluated if the myocardial territory supported by the diseased artery is still vital and lesions at risk for plaque rupture (culprit lesions) should be the detector. These various approaches to obtaining energy-dependent measurements have different dose efficiency and different sensitivity to subject motion. The simplest method to process the multi-energy data reconstructs CT images from each spectrum and performs the multi-energy analysis on the reconstructed images. A somewhat preferred method performs energy-dependent processing on the raw projections prior to reconstruction. Hybrid methods are now available. Filtered backprojection (FBP) is currently the standard reconstruction algorithm in CT. FBP is fast and performs very well in many cases. FBP assigns the same certainty to all projections. In reality, the reliability of the projection data depends on the attenuation along the projection lines. As a result, FBP is suboptimal for noisy data. Performance in the presence of noise can be improved either by certainty based preprocessing of the projection data or by using iterative reconstruction algorithms based on a more realistic noise model. If the number of projection angles is limited, FBP suffers from artefacts. In contrast, iterative reconstruction is more robust against angular sampling issues. Nonetheless, if the number of projection angles is too low, there is not enough information for perfect reconstruction, and any algorithm will have problems finding the true solution. The problem can be alleviated by using prior knowledge about the object to be reconstructed. An example is the currently very popular total variation prior, which assumes that the reconstructed object should be piecewise uniform.
One can improve the performance of image reconstruction using more accurate models of the acquisition physics (e.g. finite resolution, energy spectrum: "MBIR"). However, this makes the reconstruction task also more complicated. Many methods have been proposed, which combine preprocessing based on physics with fast reconstruction using FBP. Even better reconstructions can be obtained by integrating the model into the iterative reconstruction algorithm, but at the cost of increased computation times. Major changes in the treatment of head and neck neoplasms encompass the advances of endoscopic-based surgical techniques, mainly for nasosinusal and laryngeal tumours, and the application of sophisticated radiation therapy techniques, combined with chemotherapy. As most tumours arise from the mucosa of the upper aero-digestive tract, clinical surveillance is necessary to detect superficial recurrences, while morphological and 'functional' imaging techniques are indispensable to detect subclinical extra-mucosal and nodal recurrences. How can imaging techniques discriminate recurrence, inflammation, necrosis or scar? Key points include the knowledge of the normal appearance of tissues (morphology & signals) on CT and MRI after surgery and chemo-radiotherapy. Specifically, when non-surgical treatment has been used, it means to become familiar with the expected changes both of tumour and adjacent tissues. Changes that can be asymmetric as extremely precise techniques of dose delivering are used, like tomotherapy. Morphologybased imaging techniques are often inadequate to discriminate small recurrences from vascularised scar tissue (enhancing). CT or MRI requires to be integrated by information provided by functional-based imaging techniques, FDG-PET-CT being the most established. Recently, a great interest among radiologists is focused on the application of DCE-CT or DCE-MRI and DWI-MRI in the follow-up of head and neck neoplasms. In fact, several studies have credited these techniques for providing functional information about tissues (perfusion, water exchange) that correct reporting of the necessary measurements targeted at the clinicians' need. Furthermore, MDCT scan protocol design will be reviewed, focusing not only on optimal implementation of common scan parameters but also on the need of ECG-triggering and its consequences. Finally, we will present the current status of evidence on using MDCT in PAVR procedures, and discuss future challenges and perspectives. CT remains a particular focus for efforts in radiological protection owing to its steadily increasing clinical application and the relatively high patient doses. Dosimetry is an essential element within good CT practice in order to allow the assessment of typical radiation risks in support of the justification of procedures and the routine monitoring and comparison of typical doses in pursuit of the optimisation of patient protection. The practical basis for dosimetry in contemporary CT remains the (updated) CT dose index (CTDI100), measured in either free air or the standard CT dosimetry phantoms. These latter measurements underpin the dose indicators commonly displayed by the CT scanner: volume-weighted CT dose index (CTDIvol) and dose-length product (DLP). Whereas these quantities are not patient doses, they nevertheless provide useful characterisation of each CT exposure in order to allow comparison of practice and facilitate improvements in patient protection. Typical levels of CTDIvol and DLP at each CT centre, periodically determined as the mean values observed for representative samples for each patient group and type of examination (and associated clinical indication), should be adopted as local diagnostic reference levels (DRLs). These should be subject to periodic review and compared with both corresponding national DRLs and also practice at other CT centres in pursuit of optimised patient protection. When required, estimates of typical organ and effective doses to reference patients from standard CT examinations can be made on the basis of appropriate dose coefficients normalised to the dose indicators (CTDIair, CTDIvol or DLP). Recent advances have generated renewed interest in dual energy CT. This presentation will discuss the basic principles, and the strengths and limitations of the techniques and implementations, of multi-energy methods for material characterisation. Conventional CT is at best an image of the linear attenuation coefficient at one energy and cannot uniquely identify tissue. Materials with different atomic numbers have different energy-dependent attenuation. Multi-energy imaging measures attenuation at different energies to more fully characterise materials. An important limitation is the fact that in the diagnostic energy range the attenuation of all materials is dominated by Compton scattering and photoelectric absorption, and unless there is a K-edge in the spectrum used, these phenomena have the same energy dependence for all materials. Thus, there are only two "basis functions" and spectral CT essentially measures effective atomic number and electron density. While there is residual ambiguity, it still provides important information. The main requirement for dual energy CT is to measure data using two spectra using multiple kVp and/ or filtration or methods in which the x-ray energy discrimination is performed at B S117 C D E F G H The spectrum of differential diagnoses is broad in the liver. Therefore, incidentally discovered liver lesions represent a challenging clinical situation. Fortunately, there are specific imaging features for the most common benign and malignant liver lesions (like, e.g. haemangioma, FNH, cysts, vascular pseudolesions, HCC, metastases) so that a minimal-invasive diagnosis with a biopsy in not needed in a lot of cases. In ultrasound the echogenity and recently also the contrast agent behaviour is used for liver lesion characterisation. In CT attenuation and also contrast agent behaviour are used for characterisation. MR imaging offers several options including T1-and T2 weighted images, use of chemical shift imaging, GRE sequences with long echoes and diffusion weighted images, so that tissue components like fat, water, glycogen, iron, etc. can be evaluated already in the pre-contrast examination. Beside the evaluation of dynamic signal characteristics in the early dynamic phase after contrast agent application, MR can utilise also tissue-specific contrast agents dedicated to the RES or to the haepatocytes. Other modalities like angiography, PET or other nuclear medicine methods usually only play a minor role nowadays in the evaluation of incidental liver lesions in non-oncological patients. In oncological patients the clinical consequences and also the range of diagnoses and pre-test probabilities are different from those of the non-oncological group; therefore, the demands to imaging are even higher. In case of atypical presentation of otherwise benign liver lesions like sclerosed haemangioma close follow-up or even biopsy can be necessary in such a setting. In recent years there has been a major change in the way patients presenting with haematuria are investigated, particularly in relation to imaging. This has been mainly influenced by the significant advances in CT and MR technology, and the introduction of CT Urography which is now considered one of the reliable imaging modalities. The role of IVU and ultrasound has been relegated to a complementary role, to some extent problem solving rather than their past essential role. The role of IVU in imaging patients with haematuria is therefore limited to very specific circumstances as follows: a) lack of access to CT or MR, b) stone localisation in some cases to demonstrate the calyceal anatomy and c) chronic pyelonephritis. Ultrasound imaging remains, however, the first line investigation for patients with microscopic haematuria and a useful adjunct to CT in the investigation of frank haematuria: a) characterisation of renal lesions, b) follow-up of known lesions and c) follow-up of renal trauma. diarrhoea, vomiting, constipation and abdominal pain. The differential diagnosis includes acute complications of treatment, complications or progression of the primary tumour, metastatic spread of tumour and problems related to co-morbid conditions. In this presentation I will illustrate these principles in two situations, one generic and acute and one more specific with subacute and chronic problems; the neutropaenic patient after chemotherapy or bone marrow transplantation; following radical chemoradiotherapy for cervical cancer.
Learning Objectives: 1. To be familiar with the range of complications that may arise following chemotherapy/radiotherapy. 2. To understand how these changes can simulate active disease. 3. To learn how to distinguish between these changes and active disease.
A-507 09:21 C. Complications of treatment in the CNS P. Demaerel; Leuven/BE (philippe.demaerel@uz.kuleuven.ac.be) Radiotherapy and chemotherapy are the common central nervous system cancer treatments. Other possible treatments, including recombinant humanised monoclonal antibodies and autologous dendritic cell-based immunotherapy have been used. Complications can occur soon after initiating a treatment, but delayed complications several months or years after treatment are increasingly being recognised. One of the difficulties concerns the differentiation between progressive disease/tumour recurrence and treatment-related changes, e.g. radionecrosis or inflammatory/immune-mediated changes. The term "pseudoprogression" is often used for the latter. The Response Assessment in Neuro-Oncology working group has recently updated the response criteria. Advanced MR imaging techniques are being used in an attempt to find imaging biomarkers and to gain more insight into this difficult differential diagnosis. Diffusion and perfusion imaging and MR spectroscopy have shown promising results. Elevation of choline and depression of N-acetyl aspartate is usually associated with the presence of tumour. Post-therapy necrosis is often characterised by the presence of lipid/lactate and the absence of other metabolites. Measurements of the apparent diffusion coefficient on diffusion imaging and cerebral blood volume on perfusion imaging have been investigated as potential biomarker too. Cancer treatments may also affect the central nervous system and cause neurotoxicity by direct injury to the glial cells, vascular injury or through immune-mediated mechanism. The imaging findings can include entities such as posterior reversible encephalopathy syndrome, progressive multifocal leukoencephalopathy and Wernicke encephalopathy. Learning Objectives:
1. To be aware of the complications of chemotherapy and radiotherapy on the CNS. 2. To be able to recognise the specific imaging features of these complications. 3. To understand the optimal use of imaging techniques for evaluating these complications. Patients being treated for malignancy are vulnerable to a unique set of complications that are often urgent and are first identified or clarified on radiologic imaging studies. As advances in cancer therapy have resulted in an improved prognosis for the patient, an awareness of the consequences of treatment becomes increasingly important. All cancer treatment modalities are associated with a spectrum of toxic effects that may involve all organ systems. This session will discuss and illustrate the complications of a range of cancer treatments and their effects on the lung, abdomen, pelvis and CNS. The panel will discuss common complications that develop after the completion of cancer treatment and emphasise the importance of radiologists' awareness of these complications. This will permit more effective patient surveillance, which may afford patients the opportunity for earlier intervention and an improved quality of life. In patients undergoing therapy for malignant disease with new pulmonary abnormalities, differentiation between pulmonary manifestations of malignancy, i.e. progressive disease and non-neoplastic changes associated with the therapy may be challenging. The consequences, however, are usually very important as therapy-associated abnormalities may be treated with discontinuation of the therapy whereas progressive malignancy often requires more intense therapy. The knowledge of typical imaging findings of therapy-induced changes as well as strategies to further clarify this dilemma are, therefore, of utmost importance. This presentation will demonstrate the chest radiographic and CT-findings of common therapy-associated pulmonary abnormalities including opportunistic infection with uncommon organisms due to immunosuppression, radiation pneumonitis, pulmonary toxicity due the chemotherapy, graft versus host disease in bone marrow transplant recipients, rejection in pulmonary transplantation and vascular disorders. Learning Objectives: 1. To be familiar with pulmonary complications that may arise in patients being treated for malignancy. 2. To be aware of the possible infectious aetiologies. 3. To learn the appearances of typical and atypical infections, including fungal infection.
B. Imaging the effects of cancer treatment in the abdomen and pelvis J.A. Spencer; Leeds/UK (johnaspencer 50 @hotmail.com)
The effects of cancer treatment can be desired, i.e. a treatment response or undesired, i.e. complications which may harm or even kill the patient. Complications are classified as acute, defined as those encountered up to 3 months; sub-acute, between 3 and 12 months; and delayed or late complications after 12 months. However, there is variation in susceptibility among individuals and there may be overlap of the acute, sub-acute and early delayed effects in the first few weeks to months following treatment. Radiological manifestations are often non-specific and thus the clinical context and temporal relationship to induction of therapy are important considerations. Good communication between the radiologist and the oncologist is vital for the early recognition and accurate diagnosis of complications related to treatment. The role of the radiologist is to help clinical colleagues to make a distinction between the effects of the cancer and the effects of its treatment so that ineffective or toxic therapies can be discontinued. Cancer patients with treatment complications usually present with common and non-specific symptoms such as Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions. Virus species in the Luteoviridae, including Cereal yellow dwarf virus (CYDV)-RPV, referred to herein as luteovirids, are each exclusively transmitted by one or a few species of aphids in a persistent, circulative, non-propagative manner. Aphids can transmit acquired virus for days or weeks and even after molting [1, 2, 3] . Virus is acquired when the virus moves from the aphid hindgut (HG) lumen to the hemolymph. Once in the hemolymph, the virus passes into the accessory salivary gland (ASG) and is released into the salivary duct. Aphids transmit the virus into phloem cells during salivation and feeding. Through specific interactions with putative cellular receptors [4, 5, 6] , luteovirids actively cross the membranes of the aphid HG and ASG. The HG and the ASG are the major barriers to the successful aphid acquisition and transmission of luteovirids. The movement of luteovirids across these barriers is under genetic control by an unlinked set of aphid genes [7] that are additive in effect [8] . These genes encode for proteins that are differentially expressed in aphids with varying virus transmission efficiency [9, 10] .
The virus encodes two structural proteins that make up the capsid. These proteins orchestrate systemic movement within host plants and transcytosis within aphids [11, 12, 13, 14] . The 22-24 kDa coat protein (CP) expressed from the viral open reading frame (ORF) 3 is the major capsid protein. Occasionally, readthrough translation of the CP stop codon results in the translational fusion of ORF3 with ORF5, resulting in the minor protein species component of the capsid called the readthrough protein (RTP). Virions can be assembled from the CP alone, but the RTP is required for luteovirid movement across the ASG as well as efficient systemic infection [13, 14, 15, 16, 17] . These activities in aphids and plants are regulated by virion topological features, specifically interactions between CP monomers and welldefined interfacial regions within the RTP [18] .
Although a number of aphid proteins that bind luteovirids have been identified, the molecular details of virus movement in aphids are not well understood. Due to the paucity of molecular tools for the study of aphids, most virus-binding proteins have been identified using proteomics approaches. In Sitobion avenae, four 2-D gel spots were differentially expressed between parthenogenetically-reproducing F1 clones that differed in their vectoring capacity for two isolates of the species Barley yellow dwarf virus (BYDV)-PAV [19] . Two proteins from head tissues of S. avenae were identified as potential receptors for BYDV-MAV based on virus overlay and 2-D immunoblot assays [5] . Other studies progressed beyond 2-D gel spot patterns to use mass spectrometry (MS) to investigate the identity of aphid proteins associated with virus transmission [9, 10, 20] in effort to link the protein function to vector biology. Membrane-associated actin, rack-1, and GAPDH-3 were identified to interact with a wild-type isolate of Beet western yellows virus (BWYV). Actin and GAPDH-3, but not rack-1, also interacted with two BWYV RTP mutants displaying a reduction in aphid transmission ability [20] indicating either (a) their assay for virus binding does not reflect the in vivo functions of these interactions in aphids, or (b), that these RTP mutants still retain the interaction interface for binding with these aphid proteins. Cyclophilin and a luciferase homologue were identified from the greenbug aphid Schizaphis graminum as binding to CYDV-RPV. These proteins were also differentially expressed among different S. graminum F2 genotypes with different CYDV-RPV vectoring capacity [9, 10] . Intriguingly, all studies investigating proteomic differences of aphid F1 [19] or F2 progeny genotypes [9, 10, 19] reported the differences between vectors and non-vectors were not related to gross changes in protein expression levels. Instead, vectoring capacity was associated with a small shift in the isoelectric point of only a small number of proteins, indicating that the control of luteovirid transmission in multiple aphid species was via the expression of allelic variants of proteins that either differentially bind to the virion or participate in the various steps of the virus transmission pathways.
In plants, luteovirids move from cell-to-cell and long distance as virions. However, in plants luteovirids must also replicate, assemble into virions, thwart the plant immune response and exhibit phloem-tissue tropism for host-to-host transmission by aphids, processes which likely involve the recruitment of a wide range of host factors to accomplish [21] . Interestingly, Bencharki and colleagues showed that the addition of soluble proteins to the aphid diet, including phloem-specific proteins that can interact with virions, can enhance transmission efficiency of the polerovirus, cucurbit aphid borne yellows virus [22] . Thus, we hypothesize that plant host proteins are also involved in aphid transmission. Serendipitously, we discovered that common additives to the plant tissue homogenization buffer during virus purification do not appear to alter the virus morphology or protein structure, but do render the virus non-transmissible by aphids. By comparing the host plant proteomes associated with transmissible and non-transmissible virions, proteins with potential involvement in transmission were identified. Moreover, our data indicate an increased accumulation of several virus-associated host proteins within aphids that have fed on infected plants, supporting a role for these host proteins in the circulative transmission process.
Sodium sulfite (Na 2 SO 3 ) and ethylenediaminetetraacetic acid (EDTA) are commonly added to infected plant homogenate during virus purification to minimize the effects of polyphenolic compounds and virion aggregation, respectively [23, 24] . When sodium sulfite and EDTA were added to the tissue homogenization buffer, and the subsequent purified CYDV-RPV virions were fed to aphids through ParafilmH membranes, the virus was not transmitted in two independent experiments (Table 1 ). In contrast, CYDV-RPV purified without these additives was highly transmissible by aphids (.95%) in six independent experiments (Table 1) . To determine whether treated virions were unstable and being degraded during membrane feeding by aphids, treated and non-treated virions were collected from the diet following the acquisition access period (AAP) and analyzed by transmission electron microscopy (TEM). Importantly, treated virions remained intact in the diet following the AAP (Fig. 1 ). Treated and untreated virions were morphologically indistinguishable at this point ( Fig. 1) . Broken capsids and capsid swelling were not observed, indicating virions were structurally stable for transmission (Fig. 1) .
To determine whether sodium sulfite or EDTA was responsible for the reduction in transmission efficiency, virions were purified with only EDTA or sodium sulfite as additives and transmissibility was determined. When only EDTA was added, the purified virus was transmissible at levels similar to virus prepared with neither of the additives, 92% compared to 95%, respectively (Table 1 ). In contrast, the sodium sulfite treatment reduced transmissibility to ,5%. The negative impact of sodium sulfite on virus transmission by aphids was independent of virus concentration since the results were consistent across four independent experiments where the 
Transmission of CYDV-RPV is dependent upon the virus circulating through the aphid. The interaction between the virions from each purification treatment and the aphid was evaluated, particularly to identify possible barriers to transmission. To determine if aphids ingested and acquired virus, aphids were collected following a 24-h AAP on membranes containing each type of purified virus: buffer only, EDTA-treated, or sodium-sulfite treated. Total RNA was extracted from half of the aphids from each treatment and the remaining aphids were transferred to healthy oat seedlings for a 3-day inoculation access period (IAP). Total RNA was also extracted from those aphids immediately following the 3-day IAP. Viral RNA (vRNA) was detected within aphids after a 24-h AAP for each virus treatment preparation (Fig. 2) using RT-PCR and CP-specific primers. After 24-h, vRNA can be detected in all three groups of aphids. At 24-h, virus could be in the gut lumen, and/or in the HG cells and possibly in the hemolymph. This indicates aphids were ingesting, and possibly acquiring, the virus under all conditions. For aphids collected after a 3-day IAP, vRNA was detected for the buffer only treatment (Fig. 2) indicating uptake of the virus into the hemolymph, which was supported by high level of transmission (92% transmission, Table 2 ). No vRNA was detected after 3 day IAP (Fig. 2) for EDTA-or sodium sulfite-treated virions in the aphids. EDTAtreated virions were still transmitted by aphids to oat seedlings (92% transmission efficiency, Table 2 ), suggesting there was virus uptake and persistence occurred within the aphid, albeit at levels not sensitive enough to detect using RT-PCR. However, the sodium sulfite treatment reduced the rate of virus transmission into oat seedlings (Tables 1 and 2) . Taken together with the RT-PCR results, these data suggest that sodium sulfite-treated virions may not be acquired. If the virus is not acquired, any virus remaining in the lumen after 24-h may flow out of the aphid in the honeydew [25] .
To determine if either additive affected the ability of virus to move into gut cells, we used TEM to visualize virion attachment and virus penetration across the gut membrane (Fig. 3) . Aphids were fed on sodium sulfite-or EDTA-treated as well as nontreated virions for a 48-hr AAP. Non-treated (Fig. 3A, B) and EDTA-treated virions (Fig. 3C , D) were observed to attach to apical plasmalemma lining the HG lumen, in coated pits invaginating and budding into the cytoplasm, as well as retained Figure 1 . Negative stained grids, coated with CYDV-RPV coat protein antibody, of purified virus from each virus preparation after purification and virus recovered from membranes fed on by R. padi for a 24 h AAP. Virion morphology was similar within each group and a representative picture for each is shown. Transmissible virions after purification (A) and after membrane feeding (B) look morphologically similar. Non-transmissible virions after purification (C) and after membrane feeding (D) look morphologically similar to each other and are indistinguishable from transmissible virions in shape and size. Scale bars = 100 nm. doi:10.1371/journal.pone.0048177.g001 in vesicles dispersed throughout the cytoplasm. In contrast, sodium sulfite-treated virions were rarely observed to interact with the apical plasmalemma nor were they observed within the cytoplasm of the HG. However, virions were observed in the HG lumen ( Fig. 3E , F). The patterns of virus transmission and virus distribution within the aphid for each purified virus (non-treated, EDTA-or sodium sulfite-treated) were consistent in five aphids fed on the different virus preparations ( Table 2) .
After virus egress from the HG, virions must remain stable in the hemolymph and pass through the ASG for transmission to occur. To determine whether the treatments had an effect on these latter steps in the circulative transmission pathway, treated and non-treated virions were microinjected into aphid hemolymph to bypass the HG barrier. Transmission efficiency was reduced when aphids were injected with sodium sulfite-treated or sodium sulfite plus EDTA-treated virions, 8% and 0%, respectively, relative to aphids microinjected with EDTA-treated or untreated virions, 78% and 91%, respectively (Table 3) . These data were consistent over multiple independent experiments, indicating the effect was reproducible and not dependent on the concentration of virus. Initial results from the membrane-feeding experiments indicated that the EDTA and sodium sulfite could be acting additively or synergistically to block CYDV-RPV transmission since no transmission was observed. An identical trend was observed in the microinjection experiments (Table 3) . We performed a loglinear analysis of a 3-way contingency table using sodium sulfite, EDTA, and transmissibility as the three variables. We were unable to conclusively show any interaction effects of EDTA and sodium sulfite on aphid transmission of CYDV-RPV with the number of replicates performed for either the feeding or microinjection assays.
To test the hypothesis that sodium sulfite treatment changes the host-virus interactome, LC-MS/MS was used to analyze tryptic digests of non-transmissible and transmissible virion preparations purified from infected oat plants. Although the genome of oat is not available, we were able to use predicted proteins from available genomes of related cereals for homology-based protein identification of the plant proteins found in the virus preparations. Twenty proteins were identified in the transmissible, but not the non-transmissible virus preparations (Table 4 ). Sixteen proteins ( Table 4 ) are candidate proteins that may interact with transmissible virus directly or in complex. These include three chloroplast proteins (326533372, 20302473, and 2565305), seven mitochondrial proteins (115472339, 115474041, 115471693, 326500100, 115477529, 115448577, and 357139868), two isoforms of remorin (357164942 and 115456099), one predicted cytoplasmic protein (115449199) and three proteins with predicted functions in the nucleus (357144283, 357121487, and 226531758). Six host proteins were found in both transmissible and non-transmissible virus preparations. These include a 33-kD secretory protein, thaumatin-like protein 5 (TLP-5), ATP synthase CF1 beta subunit, cellulose synthase, triosephosphate isomerase (TIM), and a cysteine-rich repeat secretory protein (Table 4 ). Two proteins, prophobilinogen synthase and adenosylhomocysteinase, were identified exclusively from preparations of sodium-sulfite treated, non-transmissible virions. Proteins homologous to most of these proteins are involved in plant defense and have been previously identified in plant phloem sap proteomes [26, 27, 28, 29] . Among the potential virus genome-derived protein products, peptides from only the two virus structural proteins (CP and RTP) were identified (not shown).
To rule out co-sedimentation of plant proteins with purified virus based on density alone, we used LC-MS/MS to thoroughly characterize sucrose gradient fractions of healthy plant homogenates (Table S1 ). Healthy plants were grown in the greenhouse alongside infected plants and the tissue was used in the virus purification protocol. Fractions from the gradient that would normally contain virus particles when infected plant tissue is used in the purification were subjected to trypsin digestion and LC-MS/MS to identify any plant proteins that could co-sediment with virus particles based on density and not a physical association. Four proteins ( Table 4 ) that are likely not associated with virions, Figure 2 . RT-PCR of total RNA extracted from R. padi aphids that fed on different virus preparations was performed to detect RPV using RPV coat protein primers, amplifying a 614 bp product. Aphids were collected after initially feeding for a 24 h AAP (A) and after 3 d IAP (B). Three independent replicates using RNA collected from small pools of aphids are shown for each treatment. doi:10.1371/journal.pone.0048177.g002 Table 2 . Summary of R. padi transmission and virion detection following membrane feeding of different cereal yellow dwarf virus-RPV preparations used for RT-PCR and TEM evaluations. including homologues of three ribosomal proteins and fructokinase, were also identified in the analysis of the gradient fractions from healthy plants. Other abundant proteins detected in the sucrose gradients from healthy tissue included Rubisco, pyrophosphate-dependent phosphofructo-1-kinase, 6-phosphofructokinase, ribosomal proteins, and histone proteins (Table S1 ). Although many of these proteins have been detected in phloem sap in other plant species [26] and in the gradients of purified virus, the presence of these proteins is probably unrelated to the transmissibility of virions since they were detected in the fractions from healthy tissue. Importantly, no peptides were identified as belonging to CYDV-RPV indicating that the plants used for the healthy controls were not infected. Analysis of purified sodium sulfite and EDTA-treated and nontreated CYDV-RPV using 1-D gel electrophoresis often showed distinct protein profiles (Fig. S1A ). These differences were initially thought to be due to degradation products of the RTP; since the C-terminal half of the RTP is truncated during purification [30, 31] . For both transmissible and non-transmissible virus, we analyzed gel bands corresponding to viral and host proteins. MS analysis of peptides recovered from an in-gel trypsin digestion of band containing the truncated RTP from purified virions produced nearly identical tryptic fragments representing both the CP and RTP from both treated (Fig. S1B ) and untreated virions (Fig. S1C ). These data indicate minimal or no differences in the virus protein composition of the virion particles. Consistent with previous reports of C-terminal truncation of the RTP during purification, no peptides were discovered in the C-terminal portion of the RTP from either type of purified virus. The highly abundant proteins (Table S1 ) were also found in analysis of the gel bands from Fig. S1 (data not shown) and enabled us to attribute some of the variability in the gel bands to differences in plant protein complexes that co-sediment based on their density alone.
The LC-MS/MS data suggest that treatment of plant tissue homogenate with sodium sulfite disrupts the host-virus interactome that is required for virus transmission by aphids. Here, the host-virus interactome is defined as the complement of host plant proteins binding directly to or in complex with the purified virus. The host proteins may play a direct role in virus uptake, maintaining virion stability, or help to provide enzymatic or cofactor activity during initial steps of entry into aphid cells. We hypothesized that if host plant proteins were involved in transmission, either via direct interactions with virions or by another indirect mechanism, we should be able to detect evidence of these proteins accumulating to higher levels in aphids that have fed on infected plants compared to aphids that have fed on healthy plants. A targeted proteomics approach called selective reaction monitoring (SRM) mass spectrometry [32, 33, 34, 35] was used to detect peptides derived from virus-interacting plant proteins in aphid protein homogenates. SRM detects and quantifies selected tryptic peptides within a total protein extract that are unique to the proteins of interest by monitoring specific intact tryptic peptides ions and their collisionally-induced dissociation (CID) fragments (ions derived from fragments of the tryptic peptides) based on in silico predictions of their mass:charge ratios. In contrast to the discovery approach that enabled us to ask ''What host proteins are in the different virus preparations?'' (Table 4) , SRM enables us to ask the hypothesis-driven question, ''Are virus-interacting plant proteins A, B, and C, present in our aphid protein sample?''.
The candidate host proteins considered for SRM studies included all of the proteins listed in Table 4 that were found to be exclusively associated with transmissible virus. Proteins found in both transmissible and non-transmissible virus were also considered since they may be found in different amounts in the aphid. All of these plant proteins were checked for similarity against all available aphid and aphid bacterial endosymbiont protein sequences. Peptides from proteins that were identical to aphid or endosymbiont proteins at the amino acid level were not considered for further analysis as only peptides unique to the plant proteins could be informative for the specific measurements of the plant proteins in aphids. Those that remained included cellulose synthase, thaumatin-like protein 5, 33-kD secretory protein, pyruvate dehydrogenase E1, pyruvate dehydrogenase E2, remorin, cysteine-rich repeat secretory protein 55-like, structural maintenance of chromosomes protein 3, and predicted ribosomal protein. We also included b-D-glucosidase which was abundant in healthy controls as well as the purified virus preparations. To perform relative quantification of these proteins, a method was created in Skyline [32] that identified peptides specific to each of the candidate virus-interacting host proteins and exported to a TSQ Vantage mass spectrometer operating in SRM mode. Total protein extracted from the efficient RPV-vector aphid species Rhopalosiphum padi (all developmental stages) fed on healthy or infected plants for 21 days was digested with trypsin and analyzed by SRM. Peptides from five of the plant proteins listed in Table 4 Figure 3. Effect of sodium sulfite and EDTA on CYDV-RPV virion (arrowhead) attachment to apical plasmalemma and endocytosis into the HG cells of R. padi following membrane acquisition. (A and B) no sulfite or EDTA (buffer only) virus is internalized into cells of R. padi HG and can be found in tubular vesicles; (C and D) EDTA only treatment shows no effect of EDTA on acquisition of virions into the aphid HG which is consistent with the transmission data presented in Tables 1 & 2 ; (E-F) sodium sulfite treatment prevents attachment and acquisition of virus into cells of the HG. APL, apical plasmalemma; HG, HG; L, lumen; TV, tubular vesicle; T, tubule; R, ribosome. doi:10.1371/journal.pone.0048177.g003 Table 3 . Effects of EDTA and sodium sulfite on R. padi transmission of purified cereal yellow dwarf virus-RPV using microinjection into the aphid hemolymph. [90] . Each unique peptide match is based on at least 2 distinct spectra, but in some cases, many more. In the case of cysteine-rich repeat secretory protein 55-like, more than 300 total spectra were matched, highlighting the limitations of a homology-based search strategy for protein identification [79] . doi:10.1371/journal.pone.0048177.t004
were detected in the R. padi homogenate (Table 5) . A new method was created containing only these peptides, and three biological replicate samples of digested homogenates from aphids reared on infected or healthy plants were analyzed. The chromatographic retention time for each peptide was highly reproducible among all six samples ( Table 5 ). The transitions are generally free from interference when they were detected (Fig. S2 , Table S2 ) in the digested homogenates that were derived from aphids fed on infected plant tissue. Only one peptide from thaumatin-like protein 5, 33-kD secretory protein and pyruvate dehydrogenase E1 and E2 was detected (Table 5) , whereas two peptides from cellulase were detected (Table 5 , Figure S2 ). Until the oat genome is sequenced, we do not know exactly what protein we are monitoring, i.e., a single plant protein isoform or a mixture of proteins sharing the same peptide. Total peak areas for the selected peptide ions and their CID fragments were used to calculate a fold-change, and the log [2] of the fold-change is reported so that a positive value indicates an enrichment of the peptide in the extract from aphids fed on CYDV-RPV infected plants, and a negative value indicates a lower level of the peptide in the extract from aphids fed on CYDV-RPV infected plants. Pyruvate dehydrogenase E1 and E2 were found to associate with only transmissible virions (Table 4 ). Peptides derived from both of these proteins were found to accumulate to higher levels in aphids fed on CYDV-RPV infected plant tissue (Table 5 ). In fact, the peptide from pyruvate dehydrogenase E2, GLGMIAEEVK, was only detected in aphids fed on infected plant tissue (Table 5) and not in aphids fed on healthy plants. Cellulose synthase, thaumatin-like protein, and the 33-kD secretory protein co-purified with both transmissible and non-transmissible virions (Table 4 ). Cellulose synthase was detected at similar levels in aphids reared on healthy and infected plants, with fold-changes consistent across two different peptides from this protein (20.3 and 0). In contrast, peptides specific to thaumatin-like protein 5 and 33-kD secretory protein were found to accumulate to higher levels in aphids that had fed on infected plants (Table 5 ). These data suggest that the amount of these proteins was increased in aphids fed on CYDV-RPV infected tissue, with fold-changes of 2.0 and 1.6, respectively. Peptides from remorin, cysteine-rich repeat secretory protein 55-like, structural maintenance of chromosomes protein 3, predicted ribosomal protein, and b-D-glucosidase were not detected. SRM is excellent at reproducibly monitoring peptide signals; however, upon finding interesting differences such as the ones described above, the next step is validation and absolute quantification via the use of stable isotope labeled peptides.
Successful virus purification depends on obtaining high titers of virus in host plants and having methods to efficiently extract biologically active virus from infected tissue. The latter is most critical since virus particles must retain their infectivity while facing the harsh oxidative environment during plant tissue homogenization. Ideally, purification methods should be optimized to obtain a high titer while maintaining biological activity for each virus species under investigation. Purified BYDV (later to be recognized as BYDV-MAV) from Coast black oats was first reported by Rochow and Brakke [36] , using 0.1 M pH 7 phosphate buffer with no additives included in the homogenization buffer. Pierpoint [37] identified a number of substances (ascorbate, ethyl xanthate diethyldithiocarbamate, cysteine, 2mercaptobenzothiazole) that prevented oxidation of polyphenolic compounds in homogenized, infected plant tissue (which can be observed as browning of the plant homogenate). As a result, reducing agents became commonplace additives to virus extraction buffers and, in many cases, with positive outcomes. In contrast to our current study, the addition of sodium sulfite and EDTA were necessary for successful purification of the potyvirus, peanut mottle virus [38] . When purifying BYDV-PAV, Hammond (1983) incorporated sodium sulfite in the extraction buffer to prevent browning, and its addition did not affect infectivity nor did reducing agents affect other luteovirids such as the potato leafroll virus (PLRV) [14] or soybean dwarf virus (B. Tian, personal communication). However these studies did not examine the virus transmissibility in the absence of reducing agents so it is unknown whether sodium sulfite imparts any negative (or positive) effects on the transmissibility of other purified luteovirids. Those reviewing plant virus purification protocols [24] cautiously warn against adoption of a one-size-fits-all protocol for purifying plant viruses. Together with the previous works, our data show such caution should be duly noted by those purifying luteovirids. The vectorvirus specificity that defines the luteovirids undoubtedly reflects the complex chemical nature of protein interactions mediated by the CP and RTP. Care should be taken in understanding how the chemistry of the purification protocol can impart biochemical changes in the virion that will alter virus-host and virus-vector protein interactions. The action of sulfite on disulfides (e.g. cystines) can be represented by the equilibrium equation: R.S.S.R + SO 3 22 « RS 2 + RS.SO 3 2 . Above pH 9, the equilibrium constant does not favor the total cleavage of the disulfide bond, and large excesses of sulfite are required to drive the reaction to completion. However, at lower pH values (e. g. pH 7 of the homogenization buffer used in these virus preparations) the thiol predominates over the thiolate ion resulting in more complex reaction kinetics and more favorable equilibriums [39] . In general, the equilibrium constant can be shifted to the right by any process that removes the thiol and in the presence of divalent metal ions, known to stabilize several icosahedral viruses [40] , and all thiol and disulfide groups can be readily converted to S-sulfonates by a process that reduces the metal ion [41] . The reaction of sulfite with protein disulfides is further complicated by an acute sensitivity to the ionic atmosphere in the neighborhood of the disulfide bond. For example, anionic disulfides have been shown to react much more slowly than neutral or cationic disulfides [42] . For cystines (disulfides) having two positively charged flanking amino acids, the rate constant for reaction of the negative disulfide is 132,000 s 21 M 21 as compared to one having two neutral neighbors, 367 s 21 M 21 , a 10 6 -fold range in rate constants [43] . Furthermore, the rate of reaction can be greatly affected by steric factors, and the disulfide bonds of many proteins show great variability in their susceptibility to cleavage by sulfite in the absence of denaturants such as guanidine hydrochloride or urea [44, 45, 46, 47] . Thus, it can be expected that in the absence of denaturants, the thiols and disulfides most susceptible to modification by sulfite would be those that are most exposed and that have positively charged amino acids as their nearest neighbors.
With these thoughts in mind, the three cysteines in the CP or RTP of CYDV-RPV may provide some clues (Fig. S2 ) as to why sodium sulfite treatment would affect the virus-plant interactome. Two of the cysteines are flanked by at least one basic amino acid. These are likely the most reactive cysteines in the CP or RTP with respect to the formation of S-sulfonates and would readily participate in thiol-disulfide exchange reactions initiated by the sulfolysis of other disulfide bonds. Blocking the critical thiol by converting it to a thiosulfonate or otherwise modifying critical interactions via the formation of a non-native disulfide could have negative consequences for the internalization of RPV into aphid cells, as is observed for a wide range of other viruses (enveloped and non-enveloped) that rely on disulfide bond formation for internalization into and transport through host cells [48, 49, 50] . In contrast to the sodium sulfite treatment, EDTA treatment did not prevent entry of virus into aphid cells or transmissibility, but it did appear to reduce the long term stability of the virus inside the aphid. Hence, in vivo interactions between divalent cations and the virus capsid may be required for long-term virion stability. Indeed, other icosahedral plant viruses such as rice yellow mottle virus, tomato bushy stunt virus, southern bean mosaic virus and cowpea chlorotic mottle virus are stabilized by divalent cations such as Ca 2+ and Mg 2+ , [40, 51, 52, 53, 54] .
These data support the hypothesis that transmission of CYDV-RPV requires the formation of a critical disulfide bond pairing either intramolecular, within the CP or RTP, or intermolecular with a specific host protein, and that treatment with sodium sulfite promotes a random process of thiol-disulfide exchange that creates structures that interfere with normal virus transmission. Sodium sulfite-treated virions did not enter into the HG or into the ASG and thus, a partial overlap in the biochemical mechanisms for virus entry may exist in these two aphid tissues. Previous work using infectious mutants of other luteovirids supports the role of cysteine residues within the capsid as contributing to virus-host specificity and even aphid transmission. PLRV mutants with cysteine deletions or modification of residues flanking cysteines residues show phenotypes in a host-dependent manner Two mutants D-P-K (which alters two cysteine-flanking residues) and H-C-K (which deletes the cysteine residue) showed defects in systemic infection in the host Physalis floridana, but not in Solanum tuberosom, Nicotiana benthamiana, or Nicotiana clevelandii [11] . Aphid transmission of the D-P-K mutant was severely impaired when acquired from or inoculated into P. floridana [11] .
LC-MS/MS enabled us to detect numerous host proteins copurifying with virion particles in the sucrose gradients. The SRM data indicate a complex picture on the roles of these plant proteins in luteovirid transmission by aphids. How might host proteins also participate in aphid transmission? As a result of natural selection, aphid acquisition of CYDV-RPV by aphids may have been favored from phloem cells with higher protein expression, a phenotype that could have evolved as a response from virus infection, the act of aphid feeding, or both in combination. Coingestion of soluble plant proteins that associate with virions may help stimulate endocytosis into the epithelial cells of the aphid HG in a host-dependent manner. These data are consistent with previous observations of host proteins as associated with insecttransmitted viruses [21, 22] and that addition of any soluble protein into the diet acquired together with virus can enhance transmission efficiency [22] . In tobacco BY-2 cells, sugar levels tightly regulate pyruvate dehydrogenase E1 and E2 promoters. Promoter activity is markedly increased by sugar depravation [55] . We detected pyruvate dehydrogenase E1 and E2 at increased levels in aphids fed on CYDV-RPV infected plants. In contrast, cellulose synthase was detected at similar levels in aphids fed on healthy or CYDV-RPV infected plants. This is consistent with a more general role for the latter protein in aphid-plant interactions that CYDV-RPV may exploit. In varieties of wheat that are susceptible to the phytotoxic Russian wheat aphid, Diuraphis noxia, the mRNA encoding for cellulose synthase is up-regulated 4 to7fold during aphid infestation [56] . Intriguingly though, evidence suggests that the Russian wheat aphid is a poor vector for yellow dwarf viruses [57, 58] . Cellulose synthase is a member of the glycosyltransferase family A protein family. These proteins synthesize glycoconjugates by transferring a sugar moiety to a donor molecule, such as a protein or lipid. Little is understood about the role of protein glycosylation in non-enveloped virusesspecifically plant viruses [59] and a role for glycosylation in luteovirid transmission is not well understood [59, 60] .
Independent of a direct role in aphid transmission, cellulose synthase and the other virus-associated host proteins may help to orchestrate virion functions in planta. Most plant viruses, including CYDV-RPV, move from cell-to-cell in host plants via plasmodesmata (PD, reviewed in [61] . Luteovirids are targeted to PD early during infection [62] . PD permeability is regulated by carbohydrate metabolism via callose deposition [63] . Callose turnover regulates PD size exclusion limit, as ectopic expression of m-type thioredoxin that is expressed in non-green plastids, which controls callose deposition, causes an increase in plasmodesmal permeability [63] . For cell-to-cell movement, plant viruses have evolved specialized mechanisms to tap into the plants endogenous system for controlling PD permeability for their own cell-to-cell transport. We propose cellulose synthase may be involved in modification of the cell-wall encasing the specialized PD to assist in cell-to-cell virion translocation. A number of other plant viruses have been known to directly interact with and use host enzymes involved in carbohydrate breakdown for cell-to-cell movement through plasmodesmata [21, 64, 65] . Susceptibility to virus infection is decreased in a class I beta-1,3-glucanase-deficient mutant of tobacco generated by stable transformation of tobacco with an antisense construct. The mutant exhibited delayed intercellular trafficking via PD of a tobamovirus (tobacco mosaic virus), of a potexvirus (potato virus X), and of the movement protein 3a of a cucumovirus (cucumber mosaic virus), as well displayed alterations in callose deposition. Through interactions with host beta-1,3 glucanase, the triple gene block protein of PVX, TGB 12 modulates plasmodesmal permeability, probably to mediate cellto-cell spread [66] . Viruses that move from cell-to-cell as ribonucleoproteins (RNPs) also recruit cell-wall modifying enzymes for cell-to-cell movement [64] , suggesting that enzymatic modulation of cell wall carbohydrates to alter PD permeability is a strategy widely used by plant viruses for intercellular trafficking.
Several of the proteins found associated with CYDV-RPV have well-described functions in nuclei, for example SMC1, SMC3 [67] , and NAP [68] . Luteovirids are positive-sense, single stranded RNA viruses. Although the coat protein and the RTP of the related luteovirid PLRV can localize to the nucleolar compartment of plant cell nuclei, this localization is lost in the presence of replicating vRNA [69] . Furthermore, in the early stages of infection of oats with BYDV, infected cell nuclei become morphologically distorted and filaments associated with cytoplasmic virions appear in the nucleoplasm and in nuclear pores [62] . Thus, a physical interaction between coat protein and these proteins, in vivo, is an intriguing possibility. However, the latter three proteins have been reported to have additional, non-nuclear functions and/or localization [70, 71, 72] . Furthermore, aphid SMC proteins are commonly found associating with purified PLRV in co-immunoprecipitation studies (Cilia, unpublished) . It is also possible that maturation of phloem sieve elements may release these proteins into the sap and provide a mechanism for functional protein interactions with luteovirids, or that these proteins are transported into the phloem sap to carry out functions yet to be defined. A detailed LC-MS/MS analysis of the pumpkin phloem sap proteome revealed numerous ribosomal proteins and homologues to NAP and many other proteins with nuclear functions [26] .
Excitingly, we found two remorin proteins to co-purify with transmissible virus (Table 3) . Consistent with remorin localization in plant cell membranes [73] , peptides derived from remorin were never detected from aphid homogenates (data not shown). Remorins are plant-specific proteins with unknown functions [74] but have been receiving wide attention because of their involvement in plant defense against viral, bacterial, and rhizobial infections [reviewed in [75] ]. In vivo, remorins cluster in the plasma membrane within PD and lipid rafts [73] . Remorin proteins accumulate in mature and aging tissues and in source tissue [76] where mature, branched PD are in the majority. Remorin can physically interact with the PVX movement protein TRIPLE GENE BLOCK PROTEIN 1. Remorin association with PVX is inversely proportional to the ability of PVX to move from cell-to-cell [73] . These data show that remorin proteins may function in vivo to retain virus within individual cells. Remorin association with luteovirids is particularly interesting because the luteovirid RTP retains virus in the phloem. Phloem-retention of luteovirids is critical for virus dispersal by aphids [13] . How this occurs is not known, but one possibility could be via protein-protein interactions between the C-terminal domain of the RTP and remorin. Luteovirid mutants that are no longer restricted to the phloem [13] will be particularly useful to probe direct interactions with remorin proteins.
There is a paucity of tools to study vector biology and vectorvirus-host interactions at the molecular level; however, mass spectrometry technologies are emerging as one of the most powerful tools to develop a comprehensive understanding of virusvector-host interactions [9, 18, 22, 77, 78, 79, 80, 81, 82, 83] . We used mass spectrometry to describe several host proteins that associate with virions and to show they may even be ingested by aphids during feeding. The mounting body of evidence is that luteovirids commandeer their hosts and vectors to ensure their own survival and transmission. For instance, luteovirids move from companion cells into sieve elements but natural selection has favored their retention in the phloem of host plants and hence, dispersal by aphids. Luteovirid infection may also exert changes in the phloem proteome, changes that may also facilitate virus dispersal by aphids. A critical next experiment would have to distinguish between the following two hypotheses a) that virion-host protein complexes are internalized into aphids or b) that the virus manipulates the phloem to have a higher concentration of these proteins during infection. Due to the dynamic nature of protein trafficking in plants [61, 63, 84, 85, 86, 87] and cell-type specific transcriptional regulation [88] , the latter experiment is not a trivial undertaking. The current work has laid the groundwork for these future experiments. The virus-host interactome we describe in this study provides critical insights into the biochemical mechanisms that luteovirids use for movement in plants and aphids. These plant proteins (and associated biochemical pathways) are novel targets for developing host-resistance to luteovirid infection in cereals and other crops. Furthermore, the exciting, serendipitous discovery that sodium sulfite reduces transmissibility of virions provides biochemical evidence that intra or intermolecular disulfide bonding may be required for luteovirid entry into aphid cells and may also be exploited as part of a strategy to disrupt aphid-virus interactions and ultimately to mitigate virus transmission.
CYDV-RPV was purified from oat plants (Avena byzantina K. Koch cv. Coast black) inoculated 7 to 8 weeks previously with viruliferous R. padi as described [10] . Infection was determined by yellowing and dwarfing symptoms. Tissue harvested was divided into 200-300 g batches, chopped into 2.5 cm pieces and frozen at 280uC until used. Virus was purified using a modified version of the protocol of Hammond et al. [89] . Tissue was homogenized using 0.1 M phosphate (K 2 HPO 4 ) buffer (pH adjusted to 7 using 0.1 M KH 2 PO 4 ) at 2.5 ml g 21 tissue. Tissue was homogenized with phosphate buffer containing 1% cellulase with and without 0.01 M EDTA and 0.5% sodium sulfite together or individually. Sucrose gradients were fractionated using a density-gradient fractionator (Teledyne-ISCO) at sensitivity 0.5 and chart speed set at 60 cm/h. Two milliliter fractions were collected along the entire gradient for each gradient. The virus fractions were collected as 4 ml gradient fractions and were concentrated by centrifugation for 4 h at 113,6136g in a type 70Ti rotor (Beckman Coulter). The remaining fractions were stored at 280uC until needed. Supernatant was discarded and the pellet was resuspended overnight in 0.01 M phosphate buffer, pH 7. Purified virus was equally divided among several tubes and stored at 280uC. As a control, 500 g of healthy tissue was homogenized in phosphate buffer containing 1% cellulase and purified following the above protocol.
Purified virus from each sample preparation after purification was evaluated by negative staining. In addition, virus was recovered from membranes after aphids fed for 24 h to assess the stability of the virus. 300 Mesh copper carbon-coated formvar grids were incubated for 30 min on a 10 ml drop of RPV coating antibody diluted 1:500 in phosphate buffered saline (PBS). Excess antibody was wicked off with a wedge of filter paper and grids were rinsed in 3 drops of PBS, wicking off excess with filter paper after each rinse. Antibody-coated grids were incubated for 1 h on a 10 ml drop of virus. Virus was wicked off using filter paper and grids were rinsed in 2 drops of PBS, followed by 3 drops of sterile distilled water, wicking excess after each rinse. Grids were stained by incubating for 3 min on a 20 ml drop of 2% aqueous uranyl acetate, excess stain was wicked off and grids were stored dry in a grid box. Grids were viewed on a Jeol 1200 TEM at the Electron Microscope Facility for The Huck Institute of the Life Sciences at The Pennsylvania State University.
For each purified virus preparation (buffer only; buffer including EDTA and sodium sulfite; buffer including EDTA only; buffer including sodium sulfite only), healthy R. padi aphids were allowed a 24-48 h AAP on stretched ParafilmH membranes made by standard protocol by stretching in two directions until very thin. Approximately 75 ml of the virus preparation, at 20-60 mg/ml concentration containing 15% sucrose was used in each membrane. After 24 h, aphids were allowed a 3-4 day IAP on 7 day old oat seedlings (Coast black), 5 aphids per plant, 12-16 plants per treatment. Plants were fumigated with Orthene to kill the aphids. The fumigated plants were placed in the greenhouse and observed for symptom expression 3-4 weeks later. Infected plants were evaluated by obvious yellowing and reddening of the leaves, and dwarfing of the plant. In addition, a randomly selected subset of the leaves (symptomatic and asymptomatic) was tested by double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) using anti-CYDV-RPV antibodies. Each type of virus preparation was tested 2-4 times.
In addition to membrane feeding, virus was directly injected into the hemocoel of the aphid to bypass the HG. For each purified virus preparation, 10 nl of a 20-60 mg/ml virus preparation was injected into an aphid using a Microinjector IM300 (Narishige). Three aphids were placed onto a 7 day old oat seedling (Coast black), 12-16 plants per virus treatment, for a 4 day IAP, after which plants were fumigated to terminate feeding. Plants were evaluated for infection 3-4 weeks later as described above. To test for synergistic effects of EDTA and sodium sulfite treatment on CYDV-RPV transmission by R. padi, we performed a log-linear analysis for a three-way contingency table on the transmission results following microinjection using +/2 EDTA, +/2 sodium sulfite and +/2 transmission as the three variables. Simulations and the experiment were run using the statistical software package JMP (SAS).
Healthy R. padi aphids were allowed a 24 h AAP on membranes, as described previously, which contained 40 mg/ml of purified virus in 15% sucrose buffered with 0.01 M phosphate buffer, pH 7. The following virus treatments used were preparations containing 1) phosphate buffer only, 2) phosphate buffer including EDTA only, and 3) phosphate buffer including Na 2 SO 3 only. To examine an aphid's ability to acquire virus, after 24 h, six aphids were collected, divided randomly into three groups of two for each treatment and stored at 280uC in 25 ml nuclease-free water in an RNase-free microcentrifuge tube. The remaining aphids were transferred to healthy oat seedlings (Coast black), placing five aphids per seedling. Aphids were allowed a 3 day IAP to test for transmissibility of the virus, and aphids were collected to examine the treatment effects on virus uptake into the hemocoel. For each virus treatment, six aphids were collected, divided randomly into three groups of two for each treatment and stored at 280uC in 25 ml nuclease-free water in an RNase-free microcentrifuge tube. Total RNA was isolated, five ml of RNA was used in an RT-PCR as described [14] , with the exception that primers specific for the coat protein of CYDV-RPV (RPV-CP For: 59-ATGAGTACGGTCGTCCTTAGATCC-39; RPV-CP Rev: 59-CTATTTTGGGTTTTGTAGCTGGAC-39) were used to amplify a 614 bp fragment.
TEM was also used to detect virus within the aphid. R. padi were allowed a 48 h AAP on membranes containing 40 mg/ml per virus preparation. For each virus preparation, ten aphids were collected after feeding, the head and cauda were removed using a razor blade, and heads and abdomens were fixed for TEM in 1% formaldehyde-2% gluteraldehyde in 0.02 M sodium cacodylate (pH 7.2) containing 10 mM calcium chloride and 0.05% sodium azide for 24 h. Aphids were subsequently prepared for TEM accordingly [13] . The HGs of five aphids were examined per virus treatment. Grids were viewed on a Jeol 1200 transmission electron microscope (TEM) at the Electron Microscope Facility for The Huck Institute of the Life Sciences at The Pennsylvania State University.
Electrophoresis 25 mg of virus preparations were separated on 10-20% Novex tricine gels (Invitrogen) according to the manufacturer's instructions, at 125 V for 2 hr at room temperature in the SureLock XCell minicell (Invitrogen). Gels were fixed for 30 min in 100 ml of a solution containing 50% methanol and 7% acetic acid. After 30 min, the fixing solution was replaced with fresh fixing solution. The gels were stained overnight with a 1:1 dilution of Sypro Ruby stain (Invitrogen) in nanopure water at room temperature in the dark. The gels were then washed for 30 min in a solution containing 10% methanol and 7% acetic acid. Gels were scanned on the Typhoon Variable Mode Imager (GE Healthcare) at 100 dpi and visualized with the 532 nm laser using the 610BP30 filter. The bands were excised from the gel using a bench top UV transilluminator at a wavelength of 302 nm.
Gel bands. Each gel band was subjected to an in gel tryptic digestion and extraction as described [77] . Dried samples were reconstituted with 12 mL of 3% acetonitrile (ACN) with 0.1% trifluoracetic acid. Nano-LC (nLC) separation of tryptic peptides was performed with a nanoACQUITY system (Waters), equipped with a Symmetry C 18 5 mm, 20 mm6180 mm trapping column and a UPLC BEH C 18 1.7 mm, 15 cm675 mm analytical column (Waters). The samples were transferred to the trapping column using a 5 mL partial loop injection with a 0.1% solution of formic acid (FA) in water at a flow rate of 7 mL/min for 3 min. Mobile phase A consisted of 0.1% FA in water and mobile phase B consisted of 0.1% FA in ACN. Following desalting and concentration, the trapping column was subjected to a reverse flush to the analytical column and separated with a gradient of 2-40% mobile phase B over 60 min at a flow rate of 300 nL/min, followed by a 5 min rinse with 95% of mobile phase B. The column was re-equilibrated at initial conditions for 20 min. Column temperature was maintained at 35uC. 100 fmol/mL [Glu 1 ]-fibrinopeptide B in 25% ACN with 0.1% FA was used as the lock mass compound and was delivered via the auxiliary pump of the LC system at a flow rate of 300 nL/min to the reference sprayer of the NanoLockSpray source of the mass spectrometer. The eluent from the analytical column was delivered to the analytical sprayer of the same source through a PicoTip emitter (New Objective, Woburn, MA) with 10 mm tip diameter.
Mass spectrometric analysis of tryptic peptides was performed using a Synapt HDMS mass spectrometer (Waters, Manchester, UK) or a 4700 Proteomics Analyzer (Applied Biosystems) as described in [78] and [77] , respectively.
In solution digests of purified virions. For analysis of purified CYDV-RPV virions in solution, disulfide bonds were reduced with 5 mM dithiothreitol (DTT) for 30 min, followed by alkylation with 10 mM iodoacetamide (IAA) for 30 min, in the dark. Reduction and alkylation were performed at 25uC. CYDV-RPV virions were then digested with trypsin (1:200 ratio, Promega) at 35uC for 16 h. The peptide mixture was desalted using a C18 sep-pak (Waters) and stored at 280uC for 1 week prior to MS analysis.
For nLC-MS/MS analysis on the LTQ-Orbitrap Velos (Thermo-Fisher Scientific, San Jose, CA), the tryptic digest was reconstituted in 10 mL of 2% ACN with 0.5% formic acid (FA). The mass spectrometer was equipped with a ''Plug and Play'' nano ion source device (CorSolutions LLC, Ithaca, NY). The nanoLC was performed using a Dionex UltiMate3000 MDLC system (Dionex, Sunnyvale, CA). The gel extracted peptides (5-10 mL) were injected using a ''User Defined Program'' onto a PepMap C18 trap column (5 mm, 300 mm65 mm, Dionex) at a 20 mL/min flow rate for on-line desalting and then separated on a PepMap C18 reverse phase (RP) nano column (3 mm, 75 mm615 cm, Dionex) which was installed in the ''Plug and Play'' device with a 10-mm spray emitter (NewObjective, Woburn, MA) mounted in front of the Orbitrap ion transfer tube. The peptides were then eluted in a 60 min gradient of 10% to 40% ACN in 0.1% FA at 300 nL/min. The Orbitrap Velos was operated as described previously [9] . For repeat injections of the same samples, an exclusion list containing m/z values identified in the previous DDA run were generated using Proteome Discovery 1.1 software and applied to prevent resampling of the same ions. All data were acquired using Xcalibur 2.1 software (Thermo-Fisher Scientific).
Tandem mass spectra from purified CYDV-RPV were converted mascot generic format (MGF) peak list files using Proteome Discovery 1.1. An in-house FASTA protein database was created from all NCBI entries, including all the translations of all the available cereal genomes, common contaminants, and viruses, was downloaded on January 31, 2012. This strategy was used as opposed to restricting the search to green plants to minimize false matches due to the presence of virus gene products or to the presence of other unanticipated sources of proteins (such as from plant-infecting bacteria of fungi) in the samples. All data were searched against this database using Mascot v2.3.02 (Matrix Science, Boston, MA) as follows. Fixed carbamidomethyl and variable methionine oxidation were used as modifications. Precursor ion tolerances were set to 30 parts per million (ppm), and fragment tolerance was 0.8 Dalton (Da). ESI-Trap was selected as the instrument type. The enzyme selected was trypsin with 1 missed tryptic cleavage permitted.
Mascot *.dat files were created in Mascot and loaded into Scaffold (version 3_00_05). Peptide and protein probabilities were calculated using PeptideProphet and ProteinProphet algorithms [90] . We reported protein accession numbers that could be identified on the basis of at least one peptide with a Mascot score exceeding the identity threshold and E-value ,0.05. The FDR was less than 1.0%. Spectral counts were normalized to the total and compared between treated and non-treated virions, as well as a healthy control. The healthy control consisted of the fraction in the sucrose gradient corresponding to the virus sedimentation position. Proteins were not reported if they were also detected in the healthy control (Table S1 ). To minimize redundancy due to effects of homology-based searching, only one protein per protein family were reported. Rubisco was abundant in all virus and healthy plant samples.
Parthenogenetically reproducing aphid colonies of R. padi were maintained on CYDV-RPV infected or healthy caged oats at 20uC with an 18-h photo-period for 21 days. Aphids were harvested from plants for protein extraction as described [77] . Proteins were extracted from aphids using the TCA-Acetone method as described [77] . The pellets were dried and stored at 280uC until used for mass spectrometry, approximately 2 weeks.
Protein pellets were prepared for mass spectrometry as previously described [80] . Pellets were solubilized by adding a volume of 50 mM ammonium bicarbonate (Sigma; St. Louis, MO)/0.1% RapiGest SF (Waters Corp.; Milford, MA) solution. Samples were left to stir overnight at 4uC and then centrifuged at 16,0006g for 5 min to pellet insoluble debris. Protein concentration of the supernatant was determined using a Quickstart Bradford assay (Biorad) and verified using 1-D SDS PAGE as described [77] . For each sample replicate, 100 mg of protein was diluted in 50 ml 50 mM ammonium bicarbonate/0.1% RapiGest SF (Waters Corp., Milford, MA) and used as the starting material for the digestion procedure. Samples were reduced with DTT at a 5 mM final concentration for 30 min at 50uC and then alkylated with IAA at a 15 mM final concentration for 30 min at room temperature, in the dark. For digestion, a 200 ng/ml trypsin (Promega; Madison, WI) solution was prepared using 0.01% acetic acid. Two mg of trypsin was added to each sample at a trypsin:protein ratio of 1:50 and incubated at 37uC for 3.5 h, with gentle vortexing every 15 min. To hydrolyze the RapiGest surfactant, samples were acidified with HCl to a pH #2, final HCl concentration of 200 mM, incubated at 37uC for 45 min, and centrifuged at 16,0006g for 10 min. The supernatant was transferred to new tubes and frozen at 280uC until mass spectrometry, approximately 48 h. Impurities were removed using mixed mode RP SCX SPE cation exchange cartridges (Waters Oasis 1cc MCX cartridge).
Nano-flow liquid chromatography was performed using an Eksigent 1D nanoLC system (Dublin, CA) with direct column injection. Tips were pulled from silica capillary (75 mm I.D. 6 360 mm O.D.) in-house using a commercial CO 2 laser puller (Sutter Instruments Co., Novato, CA), and then packed to a length of 15 cm with 4 mm C12 reverse phase particles (Phenomenex, Torrance, CA). Two mL of the 1 mg/mL digested aphid protein extracts were injected directly on the column and eluted with a flow-rate of 300 nL/mn. The gradient ramped from 2% B (80:20 ACN/H 2 0) to 37% B across 50 min, and then increased to 80% B and held constant for 5 min. Initial conditions were restored for the final 15 min of the run. Electrospray ionization (ESI) was initiated by applying 2.2 kV via a liquid junction distally from the ESI tip. The capillary voltage and temperature were 42 V and 275uC, respectively. MS analyses were performed using a TSQ Vantage (ThermoFisher, San Jose, CA) operating in SRM mode. For SRM-mass spectrometry, the doubly charged precursor ions were monitored in Q1 with a resolution of 0.7 full width at half-maximum (FWHM) and three to four singly charged y-ions for each peptide were monitored in Q3 at 0.7 FWHM. Each transition was monitored for 25 ms (dwell time) enabling a maximum duty cycle of 2.5 s.
Targeted protein sequences were imported into Skyline [32] and converted into trypsin fragments. Refinement was performed as described [91] . Briefly, to optimize collection of SRM data, we focused initial analysis on peptides from host proteins that could be detected in the matrix of total protein homogenates extracted from R. padi fed on CYDV-RPV infected tissues. From these samples, MS/MS data were collected for Skyline-predicted tryptic peptide ions from host proteins of interest. These data were imported back into Skyline for refinement of the SRM method. During refinement, we selected proteotypic peptides that ionized well (3-4 abundant y-ions for each peptide) and showed reproducible chromatographic retention properties and made a new SRM method. The new, data-driven, refined SRM method was exported to the mass spectrometer. Three biological replicates were analyzed using the refined SRM method, and a Student's Ttest was used to compare total peak areas. A normalization factor of 0.92 was calculated by monitoring for peptides derived from two different proteins that were not differentially expressed between treatments and applied to the peptides derived from healthy samples. Both raw and normalized peak areas for each transition are reported (Table S2 ). Figure S1 (A) 1-D PAGE of proteins from purified virus showing bands that were excised and digested with trypsin. Lane 1, Broad range molecular weight standards (Biorad) in kDa; Lane 2, transmissible virus; Lane 3; non-transmissible virus. The band containing the RTP is encircled in red. Other bands subjected to LC-MS/MS analysis are indicated with a red *. Multiple proteins were found in each lane. These proteins were abundant contaminants (reported in the text and Table S1) also found in sucrose gradients separating healthy tissue. Tryptic peptides matched to the full-length RTP in (B) non-transmissible virus purified with sodium sulfite and EDTA and (C) transmissible virus are highlighted in red. Virions were purified from the same infected source tissue (oats), the only difference was addition of sodium sulfite and EDTA in homogenization buffer. Peptides were identified from the same region of the protein in both treated and untreated virions indicating no cleavage of the RTP in the nontransmissible virion preparations. (TIF) Figure S2 Clustal W alignment showing CYDV-PRV cysteine residues in the RTP in the context of a multiple alignment of twelve luteovirid species. C136 and C373 are highly conserved among luteovirids whereas C112 is unique to CYDV-RPV. In CYDV-RPV, all three cysteine residues within the RTP are flanked by at least one basic amino acid, making them especially reactive and likely to be involved in disulfide bonding. (TIF) Figure S3 SRM transitions of the peptides from host plant proteins that were detected in tryptic digests of pooled, whole-body R. padi protein samples (from data shown in Table 5 ). One replicate per peptide is shown. Two peptides from cellulose synthase show no statistically significant fold-change in aphids reared on CYDV-RPV infected or healthy plants (A) SQTGDFDHNR detected aphids fed on CYDV-RPV infected plants or (B) healthy plants, (C) IPMFAYVSR detected in aphids fed on CYDV-RPV infected plants or (D) healthy plants. The peptide FGGDTYCCR from thaumatin-like protein 5 detected in aphids fed on CYDV-RPV infected plants in (E) or healthy plants in (F). The peptide VLYSSCYVR from 33-kD secretory protein detected in aphids fed on CYDV-RPV infected plants (G) or healthy plants (H). The peptide VLYSSCYVR was at the lower limit of detection in the samples derived from aphids reared on healthy plants. The peptide SDSIITAYR from pyruvate dehydrogenase E1 derived from samples of aphids collected from CYDV-RPV infected plants (I) or healthy plants (J). One peptide was detected from pyruvate dehydrogenase E2: GLGMIAEEVK was only detected in samples of aphids reared on CYDV-RPV infected tissues (K), and not in aphids reared on healthy tissue (L). The next step to confirm these differences in aphids fed on healthy or infected plants is validation and absolute quantification of these peptides via the use of stable isotope labeled peptides. (TIF)
Table S1 Plant proteins that were identified in the sucrose gradient fractions from healthy oat plants.
Table S2 Raw and normalized peak areas, T-test results, and retention time coefficient of variation for plant peptides detected using SRM in aphids fed on CRDV-RPV infected or healthy plants. (PDF)  Mortality, Severe Acute Respiratory Infection, and Influenza-Like Illness Associated with Influenza A(H1N1)pdm09 in Argentina, 2009 INTRODUCTION: While there is much information about the burden of influenza A(H1N1)pdm09 in North America, little data exist on its burden in South America. METHODS: During April to December 2009, we actively searched for persons with severe acute respiratory infection and influenza-like illness (ILI) in three sentinel cities. A proportion of case-patients provided swabs for influenza testing. We estimated the number of case-patients that would have tested positive for influenza by multiplying the number of untested case-patients by the proportion who tested positive. We estimated rates by dividing the estimated number of case-patients by the census population after adjusting for the proportion of case-patients with missing illness onset information and ILI case-patients who visited physicians multiple times for one illness event. RESULTS: We estimated that the influenza A(H1N1)pdm09 mortality rate per 100,000 person-years (py) ranged from 1.5 among persons aged 5–44 years to 5.6 among persons aged ≥65 years. A(H1N1)pdm09 hospitalization rates per 100,000 py ranged between 26.9 among children aged <5 years to 41.8 among persons aged ≥65 years. Influenza A(H1N1)pdm09 ILI rates per 100 py ranged between 1.6 among children aged <5 to 17.1 among persons aged 45–64 years. While 9 (53%) of 17 influenza A(H1N1)pdm09 decedents with available data had obesity and 7 (17%) of 40 had diabetes, less than 4% of surviving influenza A(H1N1)pdm09 case-patients had these pre-existing conditions (p≤0.001). CONCLUSION: Influenza A(H1N1)pdm09 caused a similar burden of disease in Argentina as in other countries. Such disease burden suggests the potential value of timely influenza vaccinations. Early during the 2009 pandemic, the number of deaths attributed to influenza A(H1N1)pdm09 in Argentina (population 40 million [1] ) was only surpassed by the number of deaths in United States (population 300 million [2] ). A case-series conducted during the first months of the pandemic suggested that influenza A(H1N1)pdm09 mortality rates were at least 0.5-1.1/100.000 and concentrated among middle-aged adults with comorbidities [3] . The case-fatality proportion among hospitalized case-patients was 18% during June, 2009 [3] . Such findings suggested that the burden of influenza A(H1N1)pdm09 in Argentina was greater than that elsewhere.
A subsequent review of surveillance data suggested that the apparent influenza-associated disease burden in Argentina was likely an artifact of the way that the initial severe case patients were preferentially sampled and reported to authorities. Indeed subsequent surveillance data from Argentina suggested that the proportion of persons infected with influenza A(H1N1)pdm09 who died as a result of their illness may have been similar to that elsewhere. In order to explore whether Argentina's influenza A(H1N1)pdm09 burden was higher or similar to the burden documented elsewhere, we use active facility-based influenza surveillance and health utilization surveys from three cities in Argentina to estimate rates of influenza A(H1N1)pdm09-associated mortality, hospitalization, and influenza-like illnesses.
This study was conducted in three cities in Argentina, Mar de Plata (central Argentina, population 701,096), Tucumán (the largest city in northern Argentina, population 1,493,488) , and Santa Fe (east-central Argentina, population 396,243) that comprised 6% of the total population of 40 million persons in Argentina. During April-December 2009 (epidemiological weeks , surveillance staff actively searched city and hospital ledgers for all decedents with a history, in the previous two-weeks, of influenza-like illness (ILI), defined as persons with sudden onset fever [$38uC], with cough or sore throat [4] . In addition, surveillance staff actively searched for all persons hospitalized with severe acute respiratory infection (SARI), defined as persons with sudden onset fever [$38uC] with cough or sore throat requiring hospitalization as a result of complications from ILI [4] . Last, because of limited personnel, surveillance staff actively searched for a convenience sample of ILI case-patients who sought care among all sentinel city providers. Clinicians in these sentinel cities were also mandated to report all ILI case-patients whom they tested for influenza to the national surveillance system.
For each SARI and ILI case patient, staff recorded the person's age; sex; date of illness onset; history of asthma or chronic obstructive pulmonary disease, diabetes, obesity, pregnancy; and survival status. As part of clinical care, a proportion of these casepatients provided both nasal and throat swabs for influenza testing during the course of their acute illness [4] . These were then tested by indirect immunofluorescence (sensitivity of ,95%) [5] and/or reverse transcription-polymerase chain reaction to identify influenza type A and B, universal swine (primers and probes designed to identify swine influenzas), and 2009 H1N1 at one of the National Influenza Centers in Argentina using methods previously described (sensitivity of ,98%) [4, 6, 7] .
We conducted door to door cross-sectional surveys in each of the sentinel city populations during May-November 2010, the seasonal influenza epidemic months in Argentina, to determine if household members had developed ILI, if they had sought care, and the proportion who visited a physician multiple times for a single illness event (and therefore generated multiple case-reports in the national surveillance system). We assumed that the proportion of case-patients visiting physicians multiple times were similar during 2009 and 2010 and used bootstrapping to determine the 95% confidence interval for this proportion. We also estimated the proportion of persons in the population who were pregnant, obese, diabetic, asthmatic, or had chronic obstructive pulmonary disease and developed ILI.
For each case-patient (decedent with a history of ILI, SARI, and ILI-case-patient) of a particular age group, we estimated the number of case-patients associated with influenza A(H1N1)pdm09 illness each week by adding case-patients which tested positive for influenza A(H1N1)pdm09 to the number of untested case-patients who may have tested positive for influenza A(H1N1)pdm09 if a respiratory sample had been obtained (Appendix S1). We obtained this number by multiplying the number of untested case-patients identified each week by the proportion of case-patients of the same age group which tested positive for influenza A(H1N1)pdm09 and this proportion's 95% confidence interval (Appendix S1). We calculated the number of ILI associated with influenza A(H1N1)pdm09 by adding the number ILI-case patients which tested positive for influenza A(H1N1)pdm09 to the number of untested ILI cases reported to the obligatory surveillance system multiplied by the proportion of ILI cases testing positive for influenza A(H1N1)pdm09 identified through active surveillance each week and this proportion's 95% confidence interval (Appendix S1).
We adjusted each numerator by the proportion of persons without information about their date of illness onset (i.e. epidemiologic week of illness) and, in the case of ILI case-patients, for the proportion of persons who sought care multiple times per illness event as estimated by the health utilization surveys (Appendix S1). Next, we estimated the rate of influenza A(H1N1)pdm09 -associated case-patients by dividing the sum of influenza-associated decedents, SARI-case patients, and ILI-casepatients by the age-specific census population in the sentinel sites catchment during 2009 (Appendix S1) [1] . Last, we compared the probability that decedents with a history of ILI, SARI, and ILI case-patients were more likely to be of a certain age or have a preexiting medical condition using rank-sum tests and Fisher's Exact tests.
The research protocol was reviewed and approved by the Argentina Ministry of Health and the Argentina office of the Pan American Health Organization. Health authorities strived to maintain case-patient confidentiality by protecting data elements that help third parties identify them. Health utilization survey participants provided verbal informed consent prior to participation.
Investigators identified 108 decedents with a history of ILI during the study period (Table 1 ) ( Figure 1 ). Sixty-five (61%) of 108 were males. The median age of the 74 decedents tested for influenza was 50 years compared to 61 years among the 34 untested decedents (p = 0.01). Laboratorians identified influenza A(H1N1)pdm09 among 49 (66%) of 74 decedents tested for influenza. Accounting for age and epidemiologic week, we estimated that 66 persons died with influenza A(H1N1)pdm09 illness within our study population. We divided this estimate by the age-appropriate census population, adjusted for the proportion of decedents without known symptom onset (4 [4%] of 108), and estimated that the influenza A(H1N1)pdm09-associated mortality rate per 100,000 ranged from 1.5 (95%CI 1.5-1.7) among persons aged 5-44 years to 5.6 (95%CI 5.6-5.6) among persons aged $65 years.
Investigators identified 1,622 SARI case-patients (Table 2 ) ( Figure 1 ). Eight-hundred and twenty-two (51%) were males. Among those with available age information, the median age of the 687 SARI case-patients tested for influenza A(H1N1)pdm09 was 27 years compared to 36 years among the 887 untested SARI case-patients (p,0.0001). Laboratorians identified influenza A(H1N1)pdm09 among 279 (45%) of 621 SARI case-patients with adequate laboratory samples. After accounting for age and epidemiologic week, we estimated that 600 persons of all ages developed SARI associated with influenza A(H1N1)pdm09. We divided these estimates by the age-appropriate census population and estimated that the influenza A(H1N1)pdm09-associated SARI rate per 100,000 ranged from 26. 9 
Clinicians at the three sentinel cities reported 101,179 ILI cases to the national obligatory surveillance system when physicians submitted samples for respiratory virus testing (Table 3) . Investigators at the sentinel cities also identified through active surveillance 22,474 ILI case-patients who sought medical care 
Staff interviewed 14,535 households with 22,066 household members (mean 1.5 persons per household). Seven hundred fiftytwo (3.4%) of the 22,066 household members reported a history of ILI during the month before the interview. The ILI case patients had a median age of 20 years (IQR 7-40 years) and 339 (45%) of 752 were male (Table 4 ). Of ILI case-patients with available data, 50 had asthma, 47 had chronic obstructive pulmonary disease, 22 had diabetes, and 13 had obesity. Nine of 164 women aged 15-50 
We estimated that the influenza A(H1N1)pdm09-associated ILI rates per 100 py were 1.6 (95% CI 3.8-0.32) among children aged ,5 years, 15.0 (95% CI 26.6-5.7) among persons aged 5-44 years, 17.1 (95% CI 42.8-1.7) among persons aged 45-64, and 7.1 (95% CI 9.5-5.2) among persons aged $65 years ( 
Our findings suggest that influenza A(H1N1)pdm09 caused a significant burden of disease in Argentina during 2009. If we assume that influenza activity was similar throughout country, we could multiply the age specific influenza-associated rates from Santa Fe, Tucumán, and Mar del Plata and their 95% confidence intervals by the census population of Argentina (3,240,001 persons aged ,5 years; 25,055,187 aged 5-44 years; 7,717,549 aged 45-64; and 4,121,684 aged $65 years) to estimate that approximately 1,300,000 (95% CI 10,000,000-1,800,000) persons visited clini-cians for ILI, 10,000 (95% CI 13,000-7,200) were hospitalized, and 990 died (95% CI 1,100-970) throughout the country as a result of the 2009 pandemic.
Our mortality pandemic rates were more conservative than those estimated using linear models of 2009 Argentina pneumonia and influenza mortality data (8.4/100,000py (95% CI 6.5-10.3/ 100,000py) a method that is not well suited to differentiate the impact of influenza from that or RSV [9] . Our estimates are similar, however, to age-adjusted influenza A(H1N1)pdm09associated respiratory and cardiovascular rates for the southern cone countries (2.1-4.0/100,000py) [8] . Indeed, our estimates were similar to those of diverse countries such as Bangladesh (4/ 100,000py) [9] and subpopulations within the United States (0.9-3.7/100,000py) [9, 10] .
Influenza A(H1N1)pdm09 mortality and hospitalization rates were also similar to those of seasonal influenza [11, 12, 13] . Influenza A(H1N1)pdm09 mortality was similar to Argentina's seasonal influenza mortality estimated using pneumonia and influenza diagnostic codes and Serfling models (2.3-10.6/100,000 person-years during 2002-2009) [9] and auto-regressive integrated moving averages models (0-4.6/100,000py during 1992-2002) [11, 12] . Our findings, therefore, suggest that early case-fatality proportions overestimated the actual burden of influenza A(H1N1)pdm09. Plausible explanations for this early overestimation include clinicians' preferentially identifying, sampling, and reporting severely ill case-patients. Nevertheless, it is important to note that while the overall influenza rates may be have been similar during 2009 and previous years, the years to life lost were likely greater during the pandemic because a greater proportion of ill persons were aged ,65 years than during a typical influenza season.
Influenza A(H1N1)pdm09-associated hospitalizations were similar to those reported in Australia during 2009 (23/100,000py) [14] but higher to those of low income countries such as Bangladesh (13/100,000py hospitalizations) [15] . The similarities between Argentina's medically attended ILI rates and those of Bangladesh (6.6/100py) [15] , however, suggests that differences in hospitalization rates may be a factor of health utilization. [16] . Such findings suggest that ministries of health may be justified in exploring the burden of seasonal influenza in these groups and whether measures used to control and prevent influenza during the pandemic [17] would be applicable to prevent and mitigate disease among subpopulations at high risk of complications from seasonal influenza illness [18] .
Our study found that the proportion of decedents with positive 2009 H1N1 samples (66%) was significantly higher than that of hospitalized SARI case-patients (42%) and ILI case-patients (25%). Such a finding suggests that while influenza was identified in a fraction of ILI case-patients [15] and community acquired pneumonias [19, 20] , influenza can be identified among a significant proportion (1/3-2/3) of severe hospitalized illness case-patients and decedents in during epidemic periods. If consistent among other surveillance platforms, such findings could have implications for countries exploring whether to empirically treat SARI case-patients with oseltamivir or other antivirals during influenza epidemic periods [21] .
This study had several important limitations. We assumed that after accounting for case-definition, age-group, and epidemiologic week, the proportion of tested and untested case-patients with influenza A(H1N1)pdm09 was likely similar. This may be incorrect if physicians were more likely to test severely ill younger case-patients without pre-existing medical conditions, if the laboratory used one assay preferentially to test severely ill casepatients, and if the probability of testing positive for influenza A(H1N1)pdm09 was greater among severely ill case-patients. Although improbable, it is mathematically feasible that all untested case-patients had influenza A(H1N1)pdm09 infection, (a theoretical scenario where our influenza A(H1N1)pdm09-associated ILI rates would have been 50/100py, the hospitalization rates 48/100,000py, and the mortality rates 3.3/100,000py). Conversely, all un-tested case-patients may have been infected with other pathogens and not influenza (a theoretical scenario where our influenza A(H1N1)pdm09-associated ILI rates would have been 1/100py, the hospitalization rates 11/100,000py, and the mortality rates 2.0/100,000py). Last, we assumed that proportion of persons seeking care multiple times for ILI was similar during the 2010 and 2009 epidemic periods.
Our study suggests that influenza A(H1N1)pdm09 burden in Argentina was similar to that elsewhere and caused a large number of deaths, hospitalizations, and cases of ILI. Indeed, the majority of decedents with a history of ILI identified during the pandemic tested positive for influenza A(H1N1)pdm09. Influenza-associated mortality and hospitalization rates were similar to those elsewhere and to those of Argentina during seasonal influenza epidemics. Such findings suggest that it may be prudent to examine interventions used during the pandemic to determine their potential value to prevent and mitigate Argentina's annual seasonal influenza burden.
Appendix S1 Equation for calculating the rates of influenza A(H1N1)pdm09-associated mortality among decedents with a history of influenza-like illness (ILI) at three sentinel cities (DOC) C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.
Spo0A is the key regulator for sporulation [1, 2] . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA [3] [4] [5] . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay [6] . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain (receiver domain), and the C-terminal DNA binding (effector) domain are separated by a hinge region that is relatively poorly conserved [7] . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization [8, 9] , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation [10] . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box [10, 11] . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence [12, 13] . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis [14] [15] [16] , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis [17] [18] [19] [20] [21] , and solvent production in Clostridium acetobutylicum [22, 23] .
C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection (CDI) (for recent reviews see [24, 25] ). Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed [26] . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 (BI/ NAP1) and 078 [27, 28] . Its main virulence factors are the major clostridial toxins A and B [29, 30] . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin [31, 32] . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow [24, 25] .
There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores [33] and the gene is required for persistence and transmission in mice [34] . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A [2] . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A [35] . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum [36] . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter [37] . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions.
Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes.
Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta(DE3) pLysS (Novagen). C. difficile strains were grown in a glucose-free trypton-yeast based medium (TTY; 3% w/v bactotrypton (BD), 2% yeast extract (Fluka), 0.1% w/v thioglycollate (Sigma) pH 7.4), supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates (Biomerieux).
All plasmids are listed in Table 1 . Primers (obtained from Sigma Aldrich) are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase (Fermentas) according to the instructions of the manufacturer.
Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO (Invitrogen), yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE (20 mM Tris Acetate, 0.5 mM EDTA) gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation (using a GeneJET Gel Extraction kit, Fermentas) and cloned into similarly digested pMF14 [10] that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 (see below).
Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a.
Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 (see below).
Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit (Macherey Nagel) according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry (Invitrogen) on an ABI3130 sequencer (Perkin Elmer), according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix (Invitrogen) in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid (Invitrogen) at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 (SciEd) and Geneious version 5.6.2 (Biomatters Ltd).
Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta(DE3) pLysS (Novagen). Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described [10] . In short, cells were disrupted in 4 mL lysis buffer (2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry (Clontech) in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column (BioRad) and washed with 2 mL wash buffer (20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column (identical to wash buffer but with 50, 100, 250 or 500 mM imidazole). The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT) using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa (Pierce).
Proteins were stored at 280uC in storage buffer (identical to dialysis buffer but containing 20% glycerol). Protein concentrations were determined using Bradford reagent (BioRad), according to the manufacturer's instructions.
DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase (Promega) and chromosomal DNA from B. subtilis JH642 (Bacillus Genetic Stock Center 1A96; http://www.bgsc.org), plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm [38] as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS) and a QIAExII kit (Qiagen), according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase (Invitrogen) according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter (Beckman).
EMSA conditions were based on previous studies [10] . In short, binding reactions were carried out in binding buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol) in the presence of 200 mg/mL bovine serum albumin (NEB) and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument (GE Healthcare).
The toxic effects of C. difficile culture supernatants on Vero cells (a kind gift of Eric Snijder [39] ) were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum), before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin (Techlab) was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB (Techlab) was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects (cell rounding) were observed.
Statistical significance was evaluated with an independent sample t-test.
Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute (Hinxton, UK). Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer (10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC (Roche)). After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer (0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB) was added, and samples were heated to 96uC for 5 mins. Total cell lysates (amounts corrected for OD 600 ) were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in PBST buffer (phosphate buffered saline with 0.1% v/v Tween-20) containing 5% membrane blocking reagent (Amersham Biosciences). To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection (Amersham Bioscience), or a goatanti-mouse-biotin-conjugated secondary antibody (Dako) followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody (Jackson). Detection was done using on a Typhoon instrument (GE Healthcare). Background corrected peak volumes were quantified using ImageQuant TL (Amersham Biosciences).
Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank (accession no JX050222). Consensus Spo0A boxes were identified using a Single string Search command in Genome2D [40] , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL (Amersham Biosciences), Adobe Photoshop CS3 (Adobe Systems Inc) and Corel Graphics Suite X5 (Corel Corporation).
In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain (DBD) were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli (Fig. 1A) and purified to near homogeneity using metal affinity chromatography ( Fig. 1A ; lanes P). Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays (see below).
We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium (TTY). We found a clear signal of the size expected for full length Spo0A (,31 kDa) as early as 3 hours post inoculation (exponential growth phase), through transition phase (8 h) as well as 24 and 48 hours post inoculation (stationary growth phase) ( Figure 1B; 630Derm) . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant (Fig. 1B , CT::spo0A). We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth (BHIS; data not shown).
To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation ( Figure 1C ).
Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase.
Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 (CD1214) and previous work demonstrated that a spo0A mutant (an insertional inactivation of cd1214) -as expected -no longer forms spores [41] . In silico analyses suggest a similar secondary structure for both proteins ( Fig. 2A) , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region [7, 12] .
We compared the sequence of CD1214 obtained from our lab strain 630Derm [38] to that of the published C. difficile 630 genome [42] . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 [33, 38] . The 630Derm spo0A sequence (Genbank accession no JX050222) was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab (data not shown). We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid (NVGNIE) duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation (hinge), flanking the highly conserved DNA binding domain ( Fig. 2A  and B) . We verified the absence of this duplication in strain 630 by PCR (Fig. 2C ) as well as sequencing from the chromosomal DNA of C. difficile 630 (data not shown), to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 (to which 630 and 630Derm belong) by PCR, but the duplication was found to be unique to 630Derm among the isolates tested (data not shown).
C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A (Spo0A-DBD) between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined [13] . We found that all these residues were conserved in the C. difficile protein sequence (Fig. 2B) , indicating that the protein likely recognizes a similar motif.
DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization [8, 9] . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein [10] . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene (PabrB) of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein [43, 44] . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB (Fig. 2D and E) . We performed electrophoretic mobility shift assays (EMSAs) using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control (a DNA fragment of B. subtilis citG [45] ) (Fig. 2E) , suggesting that binding was specific for the abrB promoter region.
B. subtilis Spo0A recognizes a distinct sequence (0A box), that is characterized by a 7 bp core motif (TGTCGAA) [10, 11] . Structural studies have revealed that the protein makes specific contacts with the G at position 2 (G2), and the C at position 4 (C4) and 5 (G5) of this motif [13] . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced (Fig. 2E) . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other (non-consensus) 0A boxes in the abrB promoter [44] .
Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD.
Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence (Fig. 2 ). Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A.
We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D [40] . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand (see Table S1 ). Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation [3, [46] [47] [48] . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH.
We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments (Fig. 3A) , but not of a spoVG fragment which did not contain a consensus 0A box (Fig. 3B) . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis [10] .
We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A (275) and sigH (278). We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA.
We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene (CD1654; box at 267) and the ssuA gene (CD1484; box at 282) (Fig. 3A) . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms.
Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile.
It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway [33] . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes (downstream of spo0A itself) as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A (such as spoIIAA and spoIIE) do not contain a 100% match to the core motif, but rather one or more near-consensus boxes [5, 49] . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above (Fig. 3B ). For spoIIAA (encoding an antianti sigma-factor) and spoIIE (encoding a serine phosphatase), we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA (encoding a sporulation specific protease) we observed the shifted species only at higher concentrations of protein (.200 nM). The negative control (spoVG) did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site ( Figure S1B-D) . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex.
Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions.
C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells [35] . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands.
We performed EMSAs using DNA upstream of tcdR (encoding a sigma factor responsible for the activation of toxin gene transcription), tcdB (encoding toxin B), tcdA (encoding toxin A). In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE (encoding a holin-like protein [50, 51] ) and tcdC (encoding a putative negative regulator of toxin production [52] [53] [54] ), even though this regulator does not have a significant effect on toxin levels under the conditions we used [55, 56] . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB ( Figure 4A ); the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site ( Figure S1E ). For tcdC, some smearing was observed at all concentrations of proteins tested ( Figure 4A ), and there did not seem to be a clear effect of the addition of unlabeled DNA fragments ( Figure S1F ). The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG.
We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type.
We grew three independent biological replicates of a wild type (630Derm) or Clostron-generated spo0A mutant (CT::spo0A -a kind gift of the Minton lab) in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase (approximately 7 hours post inoculation), the transition phase between exponential and stationary growth phase (approximately 9 hours post inoculation), as well as two time points in stationary phase (24 and 48 hours post inoculation) and determined the toxin endpoint titres (see Materials and Methods). In contrast to previous findings, we observed a small (#4-fold) increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant (p.0.05, independent sample t-test). In other medium (BHIS), we observed no differences at all (data not shown).
We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions.
The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box [5, 10, 11, 45] . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues [10, 11] . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures (e.g. AT content), modes of action (e.g. activation or repression) or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A [43, 57] .
For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved (Fig. 2B) , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A (Fig. 2D) , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA (Fig. 2E ) and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A (Fig. 3A) . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis [58] already suggests possible differences in the extended Spo0A motif (W.K. Smits, unpublished observations). These differences may relate to the much higher AT content of C. difficile compared to B. subtilis (71 vs. 56.5%, respectively), or phosphorylation dependent dimerization, for instance.
The initiation of sporulation in B. subtilis is subject to complex regulation (for review see ref [1, 59] ). The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues [60] and ensures a gradual increase in the level of phosphorylated Spo0A in the cell [57] . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter [46] , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter [48] .
In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay [2] and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases [35] . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H [37] , as it is in B. subtilis [61] . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile (Fig. 3A) , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis [48] . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase ( Figure 1C) .
Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA (Fig. 3B ). All these observations are consistent with direct regulation of these genes by Spo0A in other organisms [5, 45, 49, 62] , and the conservation of the sporulation pathway [2] .
Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology [10, [14] [15] [16] . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P [43] . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells.
Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation [22, 23] . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on ( Figure 1B) .
We observed direct binding of C. difficile Spo0A to the promoter region of sigH (Fig. 3A) . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well [37] . Moreover, we found significant levels of Spo0A from early stationary phase on ( Fig. 1B and unpublished observations) , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA (Fig. 3A) . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain (R20291), a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile.
It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments.
Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control [17] . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner [63] . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein [18, 21] . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A [64] and also the production of enterotoxin in this organism seems to be (indirectly) dependent on sporulation [65, 66] .
In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A (TcdA), both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B (TcdB) [35] . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC (Fig. 4A) . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant (a kind gift of the Minton lab; [33] ) did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type (#2fold in exponential phase cells up to 4-fold in late-stationary phase cells). The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 (a PCR ribotypes 027/BI/NAP1 epidemic strain) demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease [34] .
The differences between Underwood et al [35] on the one hand and our study as well as the study of Deakin and coworkers [34] on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously (data not shown). Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al [35] . In the absence of a complementation experiment and/or Southern blot data, this remains to be established.
In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays.
In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile (0A box, possible auto-regulation and binding to early sporulation promoters), whereas others are not (the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA). The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis [18, 21] . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism.
Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species (DNA:protein complexes). Titrations with PCR fragments of PabrB (containing a high affinity binding site) and PtcdA (lacking such a site) correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA). C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. (TIF) Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. (PDF) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001 Approximately 2 billion people have been infected with human hepatitis B virus (HBV) worldwide. Over 350 million people currently are chronically infected and are at high risk for progression to cirrhosis, liver failure, or cancer. More than 50% of liver cancers worldwide are attributed to HBV infection. HBV-related liver diseases remain a major public health problem, causing approximately 1 million deaths per year. Individuals coinfected with HBV and HDV are at greater risk for rapid progression and severe disease (Lavanchy, 2004; Hughes et al., 2011) . Despite its enormous medical and social relevance, progress in HBV research has been impeded by the lack of understanding of HBV entry by which the virus specifically infects human liver cells. HBV is an enveloped virus containing a small genome of 3.2 kb of partially double-stranded DNA encoding four overlapping reading frames. The HBV envelope consists of the small (S), middle (M), and large (L) envelope proteins, which are multiple transmembrane spanners sharing the same C-terminal domain corresponding to the S protein but differing at their N-terminal domains ( Figure 1A ) (Heermann et al., 1984; Seeger et al., 2007) . HDV is a small satellite RNA virus of HBV carrying all three HBV envelope proteins and can only propagate when coexisting with HBV. The mechanism of viral entry of HDV is believed to be similar to that of HBV, and HDV has been used as a surrogate for studying HBV infection at the entry level (Barrera et al., 2004; Sureau, 2006; Hughes et al., 2011) . The L protein and integrity of S protein are critical for HBV
Photoreactive ligand peptides for identification of interacting protein(s) of pre-S1 domain of L envelope protein To identify the pre-S1 interacting molecule(s), we employed a photo-cross-linking approach using a synthetic peptide derived from the native pre-S1 peptide with particular residues replaced by eLife digest Liver diseases related to the human hepatitis B virus (HBV) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. Some 15 million of these people are also infected with the hepatitis D virus (HDV), which is a satellite virus of HBV, and this places them at an even higher risk of liver diseases, including cancer. The viruses are known to enter liver cells by binding to receptors on their surface before being engulfed.
Both HBV and HDV have outer coats that consist of three kinds of envelope proteins, and a region called the pre-S1 domain in one of them is known to have a central role in the interaction between the viruses and the receptors and, therefore, in infecting the cells. However, the identity of the HBV receptor has remained a mystery. Now Yan et al. have identified this receptor to be sodium taurocholate cotransporting polypeptide. This protein, known as NTCP for short, is normally involved in the circulation of bile acids in the body.
In addition to humans, only two species are known to be susceptible to infection by human HBV and HDV-chimpanzees and a small mammal known as the treeshrew. Yan et al. started by isolating primary liver cells from treeshrews, and then used a combination of advanced purification and mass spectrometry analysis to show that the NTCP on the surface of the cells interacts with the pre-S1 domain in HBV.
The authors then performed a series of gene knockdown experiments on liver cells of both human and treeshrew origin: when the gene that codes for NTCP was silenced, HBV infection was greatly reduced. Moreover, they were able to transfect HepG2 cells-which are widely used in research into liver disease, but are not susceptible to HBV and HDV infection-with NTCP from humans and treeshrews to make them susceptible. Similarly, although monkeys are not susceptible to HBV, replacing just five amino acids in monkey NTCP with their human counterparts was enough to make the monkey NTCP a functional receptor for the viruses.
In the past, basic research into HBV and the development of antiviral therapeutics have both been hindered by the lack of suitable in vitro infection systems and animal models. Now, the work of Yan et al. means that it will be possible to use NTCP-complemented HepG2 cells for challenges as diverse as fundamental studies of basic viral entry/replication mechanisms and large-scale drug screening. It is also possible that HBV and HDV infection might interfere with some of the important physiological functions carried out by NTCP, so the latest work could also be of interest to medical scientists working on other diseases related to these infections. Research article Figure 1 . Developing photoreactive peptide ligands and an antibody for identifying pre-S1 binding partner(s) by zero distance cross-linking. (A) Schematic diagram of HBV envelope proteins and N-terminal peptides of pre-S1 domain. Pre-S1 (2-47): 2-47 th residues of the pre-S1 domain of the L Figure 1 . Continued on next page nonnatural amino acids (L-photo-leucine, L-2-amino-4,4-azi-pentanoic acid) ( Figure 1A) . L-photoleucine contains a photoactivatable diazirine ring. Irradiation of ultraviolet (UV) light at 365 nm induces a loss of nitrogen of the diazirine ring and yields a reactive carbene group with short half-life for covalent cross-linking at nearly zero distance (Suchanek et al., 2005) . Primary hepatocytes isolated from treeshrews (Tupaia belangeri), the only species susceptible to human HBV infection other than humans and chimpanzees (Su et al., 1987; Walter et al., 1996; Glebe et al., 2003) , were used as target cells.
To maximize the efficiency of photo-cross-linking, two residues (leu 11 and phe 14 ) in a region (aa 9-15) known to be critical for viral infection (Schulze et al., 2010) were chosen for substitution with L-photoleucine. Leu 11 is 100% conserved among HBV genotypes, and the 14th residue is a phenylalanine in most genotypes but a leucine in some HBV strains of genotypes F and G. Changing phe 14 to leucine (F14L) did not significantly affect the binding of HDV virion to primary Tupaia hepatocytes (PTHs) ( Figure 1B) . The activity of the synthesized peptide ligand Myr-47/WT b (or WT b hereafter) containing photo-leucines at positions 11 and 14 was also confirmed ( Figure 1C,D) . WT b inhibited HDV binding to PTHs with efficiency comparable to Myr-47/WT that is comprised of all natural amino acids ( Figure 1A,C) . A peptide Myr-47/N9K b (or N9K b hereafter) similar to WT b but with an additional mutation at the ninth residue (N9K) did not block HDV binding to PTHs ( Figure 1C ). WT b but not N9K b inhibited viral infection of HBV and HDV on PTHs ( Figure 1D ). Both WT b and N9K b peptides were myristoylated at the N-terminus and conjugated with a biotin tag on a C-terminal lysine residue ( Figure 1A ). N9K b differs from WT b by only one amino acid but completely lost these blocking activities. Thus, N9K b was used as a negative control for WT b . In addition, a monoclonal antibody (mAb) 2D3, which specifically recognizes an epitope adjacent to the critical receptor-binding region of the peptides and shared by both WT b and N9K b , was developed ( Figure 1E ).
Identification of NTCP as a specific binding protein of pre-S1
The WT b or control N9K b peptide at 200 nM was then applied to PTHs in culture and near zero distance cross-linking was induced by UV irradiation. The cross-linked peptide and associated partners were precipitated by streptavidin T1 beads and separated by SDS-PAGE. Western blotting using 2D3 as a probe revealed several bands including a major smeared band with apparent molecular weight of ∼65 kDa in the WT b but not N9K b cross-linked sample. The 65-kDa band shifted to ∼43 kDa upon treatment with the deglycosylation enzyme PNGase F (Figure 2A, left) , indicating that it is highly N-glycosylated. The WT b cross-linked protein apparently contained no intermolecular disulfide bonds as it migrated similarly under both nonreducing and reducing conditions (Figure 2A, right) . The non-photoreactive Myr-47/WT peptide but not its N9K mutant peptide effectively competed with WT b for cross-linking to the 65-kDa band ( Figure 2B) . The cross-linked protein from PTHs decreased in abundance rapidly over time during culture ( Figure 2C) . We also examined primary human hepatocytes (PHHs) in the crosslinking experiments. Bands with slightly smaller molecular weights than those seen in the PTH cells were also observed in PHHs ( Figure 2D) .
We then proceeded to identify the target protein(s) using affinity purification followed by mass spectrometry (MS) analysis. The purification procedure included three tandem steps after photo-cross-linking: protein of HBV (S472 strain, genotype C). Residue numbering is based on genotype D. Asterisk indicates highly conserved residues among genotypes. Epitope of mAb 2D3 was shaded in gray. (B) Effect of alterations of the critical N-terminal residues within pre-S1 region of L protein on HDV binding to PTHs. Both wild-type (WT) and mutant HDV virions carry HBV envelope proteins. Mutant HDV carries point mutation as indicated in the pre-S1 region of L protein. PTHs were incubated with HDV at 16°C for 4 hr and followed by extensive wash; bound virions were quantified by qRT-PCR for virus genome RNA copy, and the data are presented as percentage of virus binding, the binding of WT virus was set as 100%. (C) Myr-47/WT b bait peptide dosedependently inhibited HDV virion binding. The binding assay was performed similarly as panel B except that PTHs were pre-incubated with indicated peptides. (D) Inhibition of viral infection by the photoreactive peptides. Left: PTHs were pre-incubated with peptides at indicated concentrations at 37°C for 1 hr and then inoculated with HDV virus. Viral infection was examined by measuring viral RNA in infected cells with qRT-PCR 6 days post-infection (dpi). Data are presented as percentage HDV infection. Right: peptides at indicated concentrations were added to PTHs before HBV inoculation. The cell culture medium was replenished every 2 days. Secreted viral antigen HBeAg was measured by ELISA on 6 dpi, and the data are presented as percentage of that in the absence of peptides. (E) Antibody 2D3 recognizes residues 19-33 of pre-S1. Peptide NC36 (aa 4-36 of pre-S1, NLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNP) conjugated with keyhole limpet hemocyanin (KLH) was the immunogen peptide for generating mouse mAb 2D3. Binding activity of 2D3 with full-length pre-S1 protein was measured by ELISA in the presence of competition peptides at indicated concentrations. LD15 peptide compassing residues 19-33 of pre-S1 inhibited 2D3 binding in a dose-dependent manner, indicating that 2D3 recognizes an epitope within this region. HBV: hepatitis B virus; mAb: monoclonal antibody; HDV: hepatitis D virus; PTH: primary Tupaia hepatocytes; HBeAg: HBV e antigen. DOI: 10.7554/eLife.00049.003 ) or Myr-47/N9K b (N9K b ), followed by Streptavidin Dynal T1 beads precipitation and Western blot analysis using mAb 2D3. The protein cross-linked by WT b is sensitive to PNGase F treatment and shifted from ∼65 to ∼43 kDa. Right: WT b cross-linked samples were treated with 100 mM DTT and/or PNGase F as indicated and detected similarly as in the left panel. (B) Non-photoreactive Myr-47/WT peptide (WT) but not its N9K mutant competed with 200 nM of WT b peptide for cross-linking with PTHs in a dose-dependent manner. (C) The abundance of the target protein(s) in PTH cells decreased over time. PTHs on different days of in vitro culturing were photo-cross-linked with 200 nM WT b . The cross-linked samples were analyzed by Western blot. The two bands at ∼65 and ∼43 kDa were due to incomplete deglycosylation by PNGase F. (D) WT b cross-linking with primary human hepatocytes (PHH). Frozen PHH cells were thawed and plated 1 day before cross-linking. With same procedure as in panel A, 200 nM WT b but not N9K b cross-linked with a glycoprotein of molecular weight at ∼60 kDa, which shifted to ∼39 kDa upon PNGase F treatment. (E) Purification of target protein(s) for MS analysis. PTHs photo-cross-linked with 200 nM of WT b or N9K b peptide were lysed, then the peptides and their cross-linked proteins were purified in tandem with Streptavidin Dynal T1 beads, mAb 2D3 conjugated beads, and Streptavidin Dynal T1 beads in 1× RIPA buffer. Extensive wash was applied for each purification step. The samples were treated with or without PNGase F as indicated prior to the last step of Streptavidin beads precipitation. The final purified samples were subjected to SDS-PAGE followed by silver staining (left). Bracketed areas indicate the bands cut for MS analysis. Western blot analysis (right) of the same cross-linked samples were performed similarly as in panel A. The top 10 nonredundant proteins identified in the 3 samples by MS analysis are listed in Figure 2 -Source data 1. The common protein hit identified by MS analysis of the ∼65and ∼43-kDa bands cut from the WT b cross-linked sample was Tupaia NTCP (tsNTCP), and the representative MS/MS spectra and parameters of the peptide hits are shown in Figure 2 -figure supplement 5. The control band cut from N9K b cross-linked sample did not generate any hits on any of these peptides. (F) Predicted tsNTCP protein sequence. A 30-amino acid insertion unique to tsNTCP is underlined. Two peptides identified by LC-MS/MS were highlighted in green. All lysine and arginine are highlighted in red to Figure 2 . Continued on next page capturing all biotin-labeled proteins with streptavidin T1 beads, sorting out the target protein(s) with 2D3 antibody affinity beads, and then purifying with streptavidin T1 beads again to remove residual molecules that were not covalently cross-linked with the bait peptide. The purified samples were subsequently subjected to SDS-PAGE followed by silver staining. Similar to the Western blotting results with the 2D3 antibody, a ∼65-kDa protein band was visible by silver staining. The band was also shifted to ∼43 kDa upon PNGase F treatment ( Figure 2E ). Both the original 65-kDa and the shifted 43-kDa bands were subsequently excised from the gel and subjected to LTQ-Orbitrap Velos (Thermo Fisher Scientific, MA. USA) MS analysis after trypsin digestion. The tandem mass spectra were searched against a Tupaia hepatocyte protein database, which we had established by deep sequencing of the transcriptome (Figure 2-figure supplements 1-4) . Two different tryptic peptide fragments, which were identified from both the ∼65-kDa and ∼43-kDa bands ( Figure 2-figure supplement 5 ), matched to a protein homolog of human NTCP. Tupaia NTCP (tsNTCP) shares 83.9% protein sequence identity with its human counterpart and has an insertion of 30 aa near its C-terminus ( Figure 2F ). The peptide (TEETIPGTLGNSTH) containing 4 aa of this insertion (underlined) was one of the two peptides identified by the MS analysis at a high confidence level ( Figure 2-figure supplement 5) . These data suggest that NTCP is the protein specifically interacting with the WT b bait peptide.
Confirmation of NTCP as a specific binding protein of pre-S1
We next cloned human and Tupaia NTCPs and validated the binding of the exogenously expressed NTCPs with the WT b peptide and an N-terminal myristoylated pre-S1 peptide with native residues. Both human NTCP (hNTCP) and tsNTCP could be efficiently cross-linked by WT b but not N9K b when expressed in 293T cells as shown by Western blotting with the anti-WT b antibody 2D3 as well as an anti-C9 antibody recognizing the C-terminal C9 tag of the recombinant hNTCP and tsNTCP proteins ( Figure 3A ). WT b but not the control N9K b peptide bound to 293T cells expressing a green fluorescent protein (GFP)-tagged tsNTCP (tsNTCP-EGFP) and co-localized with tsNTCP-EGFP on the cell surface. This binding was readily competed off by the free Myr-47/WT peptide ( Figure 3B ). Moreover, a native pre-S1 peptide specifically recognized the human hepatocellular carcinoma Huh-7 cell line transfected with hNTCP ( Figure 3C ). Consistently, Huh-7 cells transfected with either tsNTCP or hNTCPs had markedly increased HDV binding to the cells. The Myr-47/WT peptide readily competed with binding of the wild-type HDV, whereas a noninfectious mutant HDV virus bearing a single N9K mutation in the pre-S1 domain of its L envelope protein failed to bind either hNTCP-or tsNTCPexpressing Huh-7 cells ( Figure 3D) . Collectively, these data demonstrated a specific interaction between NTCP and the pre-S1 domain of the L protein, which directly mediates the binding of HDV virions to target cells. 
To test the requirement of endogenous expression of NTCP for HBV and HDV infection, we first examined the effect of NTCP gene silencing on viral infection of PTHs. PTHs were transfected with tsNTCP-specific or a control small interfering RNA (siRNA) prior to viral inoculation. When tsNTCP Figure 3 . Binding of NTCP with N-terminal peptide of pre-S1 and HDV virions. (A) 293T cells transfected with an expression vector or plasmid containing cDNA of hNTCP or tsNTCP fused with a C9 tag at its C-terminus were cross-linked with 200 nM Myr-47/WT b or Myr-N9K b similarly as in Figure 2A at 24 hr post-transfection. Cross-linked protein samples were precipitated by Streptavidin Dynal beads followed by treatment with PNGase F as indicated, and then analyzed by Western blotting using mAb 2D3 or anti-C9 tag antibody. (B) 293T cells transfected with tsNTCP-EGFP or a control hSDC2-EGFP (encoding human heparan sulfate proteoglycan core protein fused with EGFP at C-terminus) expression plasmid were incubated with WT b or N9K b in the presence or absence of 200 nM non-photoreactive Myr-47/WT as indicated. Bound peptides were probed with PE-streptavidin and the colocalization of peptide and NTCP on cell surface was shown in the merged images. (C) FACS analysis of pre-S1 peptide binding with hNTCP transiently transfected Huh-7 cells. 24 hr post-transfection with hNTCP or a control plasmid, the cells were stained with 200 nM FITC-pre-S1 (FITC-labeled lipopeptide corresponding to the N-terminal 59-amino acid of pre-S1). The binding was analyzed by flow cytometry. (D) Huh-7 cells, after 24 hr of transfection of indicated plasmids, were incubated with wild-type HDV or HDV with a N9K mutation on its L protein. Bound virions were quantified by qRT-PCR. The result is presented as fold changes of binding over the background virus binding to pcDNA6-transfected cells. mAb: monoclonal antibody; tsNTCP: Tupaia NTCP; NTCP: sodium taurocholate cotransporting polypeptide. DOI: 10.7554/eLife.00049.011 mRNA level was reduced to ∼30% in tsNTCP siRNA-transfected cells ( Figure 4A , upper-left), total HDV RNA copies were markedly reduced in these cells comparing to those transfected with control siRNA. We further quantified the HDV genome and antigenome RNA copies using strand-specific reverse transcription followed by quantitative real-time polymerase chain reaction (qPCR). The HDV antigenome is a circular replication intermediate that is complementary to the genome. It is not present in the inoculum and only appears in infected cells (Chen et al., 1986) . As shown in Figure 4A (upper-middle panel), both HDV genomic and antigenomic RNA copies were greatly reduced in cells transfected with tsNTCP-specific siRNA but not the control siRNA, indicating that tsNTCP is required for de novo HDV infection. By contrast, lenti-VSV-G virus infection, for which viral entry is mediated by glycoprotein protein G of VSV, was not affected in the tsNTCP-and siRNA-transfected cells ( Figure 4A , upper-right). These data demonstrate that HDV viral entry requires NTCP. As HDV is enveloped by HBV envelope proteins and can only infect target cells in a single round in the absence of HBV, these data support that tsNTCP functions at entry level for viral infection mediated by HBV envelope proteins.
We then tested HBV infection on tsNTCP knockdown PTHs. Infection with HBV can be assessed by measuring secreted viral antigens HBV S antigen (HBsAg) and HBV e antigen (HBeAg). HBV inocula may contain residual HBsAg that can release and interfere with the detection of newly synthesized HBsAg during the first few days of infection. To differentiate de novo HBsAg synthesis from the contaminating inoculum, we assayed HBsAg secretion over time from days 6 to 12 after infection with the culture medium changed every 2-3 days. In addition, the kinetics of production of HBeAg with minimal or no residuals in the inoculum was also examined in the same time course experiment. As shown in Figure 4A (lower-left), both HBsAg and HBeAg levels were markedly reduced by transfection of tsNTCP-specific but not a control siRNA at all three time points tested, demonstrating that tsNTCP expression is required for bona fide HBV infection. To confirm that tsNTCP functions at the viral entry level for HBV as it does for HDV, we tested AAV8-HBV virus infection on tsNTCP knockdown PTHs. AAV8-HBV is a recombinant adenovirus-associated virus containing a 1.05× overlength HBV genome, for which viral entry is mediated by AAV8 capsid instead of HBV envelope proteins. AAV8-HBV infection of PTHs can nevertheless transduce the HBV genome into cells and lead to subsequent HBV viral antigen expression. NTCP knockdown did not affect AAV8-HBV infection in PTHs, as shown by the kinetics of HBeAg ( Figure 4A , lower-right). This result shows that NTCP has no effect on post-entry steps of HBV infection.
We next examined the effect of silencing human NTCP on HBV and HDV infections in human hepatocytes. Human hepatoma cell line HepaRG is the only cell line known to date to be susceptible to HBV and HDV infections upon differentiation into a mixture of hepatocyte-like and biliary-like cells (Gripon et al., 2002) . HepaRG differentiation requires a lengthy cell culture procedure, including maintaining undifferentiated cells for 2 weeks before induction, followed by induction with corticoids and DMSO for another 2-4 weeks (Gripon et al., 2002) . The NTCP mRNA level was low in HepaRG cells before induction when examined on days 5 and 10 after initial plating, but increased dramatically when the cells differentiated after induction (Figure 4B, . To examine if the acquired hNTCP expression on differentiated HepaRG cells is required for HDV and HBV infections, the cells were transfected with siRNAs targeting hNTCP. About 70% HDV infection was reduced by hNTCP knockdown as indicated by decreased levels of HDV viral RNAs ( Figure 4B , upper-right). Similarly, HBV infection was also inhibited as indicated by significantly reduced HBeAg at multiple time points ( Figure 4B , lower-left), as well as viral RNAs including the 3.5 kb RNA for HBV pre-C and pregenome RNA (pgRNA) and HBV total RNA ( Figure 4B , lower-right) quantified at the end of the experiment. We further validated the critical role of hNTCP on HBV infection in PHHs, the natural host of the virus. Consistently, knockdown of hNTCP significantly reduced HBV infection, which was correlated with the NTCP mRNA knockdown efficiency. Both viral antigens and viral RNAs were decreased in cells transfected with hNTCP-specific siRNAs but not with the control siRNA ( Figure 4C ). Taken together, these data demonstrate NTCP as a common key cellular receptor component necessary for HBV and HDV infections of hepatocytes.
We then investigated the ability of NTCP to render nonsusceptible cells susceptible to viral infection. NTCP mRNA expression is low in human hepatocarcinoma cell lines that are not susceptible to HBV or HDV infection. The levels of NTCP mRNA in Huh-7 and HepG2 cells were about 10,000 times lower than that in primary human and Tupaia hepatocytes ( Figure 5A ). We first examined if NTCP expression renders Huh-7 susceptible to HDV infection. Human NTCP-transfected Huh-7 cells supported HDV infection with an efficiency comparable to that of PTHs; nearly 10% of cells were infected as shown by staining of the HDV delta antigen that mainly locates in cell nuclei, whereas Huh-7 cells transfected with a vector plasmid allowed no HDV infection ( Figure 5B ). Moreover, the infection could be blocked by known HBV entry inhibitors, such as pre-S1 lipopeptide and hepatitis B immune globulin (HBIG), demonstrating a genuine infection of HDV mediated by HBV envelope proteins on these cells ( Figure 5C ). HDV RNAs, including antigenomic RNA that is only produced during HDV replication, rapidly increased over time in the infected cells ( Figure 5D ). Moreover, the infection efficiency correlated with both the inoculation dose of HDV ( Figure 5E ) and the expression level of hNTCP ( Figure 5F ). HDV also infected HepG2 cells transiently transfected with hNTCP ( Figure 5 -figure supplement 1) as well as a cell line established by G418 selection of HepG2 cells after hNTCP transfection, which expresses hNTCP stably and could be readily stained by the FITC-pre-S1 peptide ( Figure More efficient HBV infection was achieved on stable HepG2-hNTCP cells with about 5-10% of the cells being infected as revealed by intracellular staining of HBsAg, whereas there was no HBV infection in the parental HepG2 cells ( Figure 6A ). HBV infection in the HepG2-hNTCP stable cells was further evidenced by the continuously increased production of HBeAg during the testing period. HBV entry inhibitors, in particular the Myr-59 peptide and 17B9, efficiently inhibited the infection ( Figure 6B ). The infection efficiency as evidenced by HBV total and 3.5 kb RNA levels correlated with the inoculation dose. Moreover, the formation of HBV covalently closed circular DNA (cccDNA), which is a replicative intermediate and transcriptional template for production of viral RNAs, was confirmed by Southern blot analysis ( Figure 6D ). To further demonstrate that the replicative intermediates of HBV were synthesized de novo in HepG2-hNTCP cells after infection, we performed additional time course experiments. HBV viral replicative intermediates, including cccDNA, the 3.5 kb HBV RNA, as well as the total HBV RNA in the HBV-infected HepG2-hNTCP cells were quantified at different time points. The cccDNA became detectable at 24 hr post-infection. It markedly increased at day 3 post-infection and maintained a relatively stable level for the rest of the time points examined, whereas the formation of HBV cccDNA was completely abolished if entry inhibitor Myr-59 was included with the initial virus inoculation ( Figure 6E) . Consistently, HBV RNA levels of the 3.5 kb transcript and the total HBV viruses AAV8-HBV and Lenti-VSV-G. For HDV and HBV, PTHs were infected at 500 and 100 genome equivalent copies per cell, respectively. The level of HDV viral RNAs in infected cells was quantified by qRT-PCR on 6 dpi. Strand-specific primers were used to differentiate the HDV genomic and antigenomic RNAs (see 'Materials and methods'). For VSV-G control virus infection, recombinant lentivirus pseudotyped by VSV-G carrying a luciferase reporter was inoculated to PTHs 3 days after siRNA transfection. The luciferase activity was assessed on 6 dpi. For HBV infection, the kinetics of secreted viral antigens HBsAg and HBeAg were measured by ELISA. The medium was changed every 3 days. For AAV8-HBV infection, PTHs were infected with a recombinant AAV8 carrying 1.05× overlength HBV genome. Secreted HBeAg was assessed on indicated days post-infection. The effect of tsNTCP silencing in all viral infections was independently evaluated with a total of four siRNAs against tsNTCP (see 'Materials and methods'). The data shown are the result of a representative siRNA out of the four tested. (B) Differentiated HepaRG cells express high level of NTCP mRNA and knockdown NTCP in these cells inhibited HDV and HBV infections. HDV and HBV infection of siRNA-transfected HepaRG cells was conducted similarly as in panel A. HDV RNA levels in the infected cells were measured on 9 dpi. For HBV infection, secreted HBeAg was collected every 2 days as indicated and analyzed by ELISA. The copy numbers of HBV total RNA and 3.5 kb RNA in the infected cells were measured at the end of the experiment, 10 dpi. (C) Knockdown hNTCP in PHHs hampered HBV infection. Frozen PHHs were thawed and plated 1 day before transfecting with siRNAs against hNTCP or a control siRNA. Similar to panels A and B, 3 days after transfection, PHHs were inoculated with 100 genome equivalent copies of HBV per cell, and the levels of secreted HBeAg were determined at indicated dpi. HBV RNAs were quantified at the end of the experiment, 9 dpi. The knockdown efficiency of siRNA targeting tsNTCP or hNTCP shown in panels A-C was determined by real time RT-PCR on day 4 after transfection. NTCP: sodium taurocholate cotransporting polypeptide; HBV: hepatitis B virus; HDV: hepatitis D virus; PTH: primary Tupaia hepatocytes; tsNTCP: Tupaia NTCP; siRNA: small interfering RNA; dpi: days post-infection; hNTCP: human NTCP. DOI: 10.7554/eLife.00049.012 The Huh-7 was used to normalize the relative expression levels in other cells. (B) 1 × 10 5 Huh-7 cells were transfected with 100 ng hNTCP/pcDNA6 or a vector control in 24-well plate and maintained in PMM, 24 hr after transfection, transfected cells were infected with HDV at 500 genome equivalent copies per cell. On 8 dpi, HDV delta antigen, which typically locates in nuclei, was stained with 4G5 antibody in green, nuclei were stained with DAPI in blue. (C) Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B in the presence or absence of HBV entry inhibitors: HBIG (hepatitis B immune globulin), Myr-59, and anti-HBsAg mAb, 17B9. 4G5 was used as an antibody control. HDV RNA copies of infected cells were quantified by real-time RT-PCR on 6 dpi. (D) Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B. The HDV viral RNAs in infected cells at indicated time points were quantified by real-time RT-PCR. (E) HDV infection with increasing multiplicities of genome equivalents (mge). With 100 ng hNTCP/pcDNA6, 1 × 10 5 Figure 5 . Continued on next page transcripts in the infected HepG2-hNTCP cells gradually increased during first several days of infection and reached a steady level after day 5 ( Figure 6F ). Together these data show that NTCP contributes substantially to HBV infection.
We next compared the efficiency of HBV infection in HepG2-hNTCP cells with that in PHHs. As shown by intracellular staining of HBV core antigen (HBcAg) on day 8 post-infection, about 10% HepG2-NTCP cells were infected at multiplicities of genome equivalents (mge) of 100, which is comparable to the efficiency of HBV infection of PHHs ( Figure 6-figure supplement 2) . In contrast to PHHs, HepG2-NTCP cells propagate in cultures, thus the actual infection efficiency of HepG2-NTCP cells may be more likely than not underestimated by the observed end-point HBcAg staining. We also compared the levels of secreted HBeAg and intracellular viral RNAs in these two types of cells infected with three inoculation doses. The level of secreted HBeAg from HepG2-NTCP appeared to be higher than that in PHHs from two donors, whereas the levels of viral RNAs per nanogram of total cell RNA in both infected cell types are comparable ( Figure 6-figure supplement 3) . This may be partially explained by their different abilities in propagation and supporting viral replication and protein expression that would require more detailed studies.
Efficient HBV infection of PHHs or HepaRG cells in vitro normally requires high dose of virus inoculums, and only limited progeny viruses are produced after infection (Gripon et al., 1988; Gripon et al., 2002; Boehm et al., 2005) . To assess viral particles released from HBV-infected HepG2-NTCP cells, we first quantified viral DNA in the medium collected at different time points after the infection. As indicated by drastic decline of viral DNA level on day 4 post-infection, the majority of residual viruses from inocula were removed by changing the medium and washing during the first few days of infection. The levels of viral DNA in the media resulted from the ongoing infection during days 4-13 post-infection were low (equivalent to ∼1% of input viral DNA copies) despite significant amount of HBeAg secretion during this period. Similarly, only low levels of viral DNA were detected in the medium from HBV-infected PHHs ( Figure 6-figure supplement 4, right) . It is reasonable to speculate that some host factors that are lacking in cell cultures might be needed for efficient viral particles formation or releasing; or some cellular factors in cultures may hinder these processes during infection. The culture medium collected from infected HepG2-NTCP cells was subsequently tested for infection of PHHs. In line with the low HBV viral DNA level in the medium inoculum, very low number of intracellular HBV total RNA copies were detected in PHHs on day 13 post-infection ( Figure 6-figure  supplement 5) , indicating that only very limited HBV infection might have occurred, which may be attributed to the low multiplicity of infection.
Residues 157 to 165 of hNTCP are critical for pre-S1 binding and viral infections
We finally investigated the molecular determinants of NTCP for HBV and HDV infections. Crab-eating monkey (Macaca fascicularis) NTCP (mkNTCP) shares high protein sequence identity with hNTCP (96.3%) (Figure 7-figure supplement 1) . However, mkNTCP neither supports HDV infection nor pre-S1 peptide (Myr-59) binding ( Figure 7A) , consistent with the known narrow species specificity of the We then made a series of human NTCP variants to cover all the different amino acids between hNTCP and mkNTCP. In each variant, two or a few residues were mutated to their mkNTCP counterparts (Figure 7-figure supplement 1) . Whereas most mutations did not significantly interfere with Myr-59 binding or HDV infection, alteration of five residues of hNTCP between aa 157-165 and its monkey counterpart (from KGIVISLVL to GRIILSLVP, distinct residues are underlined) completely abolished Myr-59 binding and the ability to support HDV infection. Remarkably, replacing the motif of aa 167-156 in mkNTCP with the corresponding human residues converted mkNTCP to an efficient receptor for HDV infection ( Figure 7A) . All the NTCP variants tested were examined for NTCP expression, and comparable levels of cell surface expression were confirmed ( Figure 7B ). Similar to HDV, HBV infection was also abolished on HepG2 cells expressing hNTCP carrying monkey-like mutations, GRIILSLVP, while mkNTCP-bearing human residues KGIVISLVL within the motif of aa 157-165 largely restored HBV infection ( Figure 7C) . These data show that residues between 157 and 165 of NTCP are crucial for binding to the receptor-binding region of the pre-S1 domain of the L protein of HBV, and critically contribute to NTCP-mediated HBV and HDV infections.
In this study, by employing a unique approach of tandem affinity purification combined with MS analysis against a Tupaia hepatocyte proteome database established by deep sequencing, we revealed that the liver bile acid transporter, NTCP, specifically interacts with a key region in the pre-S1 domain of the HBV envelope L protein. By performing a series of virological analyses, we showed that silencing NTCP expression markedly inhibited viral infection of HBV and HDV in Tupaia as well as human hepatocytes. Exogenous expression of NTCP rendered nonsusceptible human hepatoma cells susceptible to the viral infections. The authentic viral infections in cells complemented with NTCP were shown by the kinetic analyses of several markers of viral infections, in particular the quantification of newly synthesized viral replicative intermediates. Moreover, the NTCP-rendered infections were blocked by known entry inhibitors. NTCP residues 157 to 165 were identified to be critical for pre-S1 binding and viral infections. These data clearly demonstrate that NTCP is a functional receptor for both HBV and HDV.
Identification of cellular receptor(s) of HBV and HDV has been challenging. In our study, we utilized a short peptide ligand, WT b , which was originated from the known receptor-binding domain of the L protein (Barrera et al., 2005; Glebe et al., 2005; Gripon et al., 2005; Engelke et al., 2006; Schulze et al., 2010) , but with specially designed properties suitable for photo-cross-linking and tandem purification. form) at the mutated positions of NTCP are shown for hNTCP, crab-eating monkey NTCP (mkNTCP), and tsNTCP. Huh-7 cells were transfected with plasmids encoding tsNTCP, hNTCP, mkNTCP, or NTCP mutants as indicated. The mutant NTCPs include hNTCP-bearing mutations of mkNTCP residues and mkNTCP-bearing mutations of human residues at indicated positions. The transfected cells were maintained in PMM for 24 hr and then either stained with 200 nM FITC-pre-S1 or infected with 500 mge HDV. HDV delta antigen in infected cells was detected with mAb 4G5 on 7 dpi. Replacing aa 157-165 of mkNTCP with human counterpart rendered mkNTCP an efficient receptor for pre-S1 binding and HDV infection. (B) All NTCP variants expressed comparable levels of NTCP. Huh-7 cells transfected as in panel A were biotinylated 24 hr after the transfection, then lysed and analyzed for cell surface NTCP expression (top), total NTCP expression (middle), and GAPDH (bottom), respectively. For cell surface expression, cell lysates were pulled down with streptavidin T1 Dynabeads and subsequently examined by western blot with mAb 1D4 recognizing a C9 tag at the C-terminus of each NTCP variant. For total NTCP expression, cell lysates were directly subjected to SDS-PAGE, followed by Western blot analysis with 1D4. (C) Effects of NTCP mutations on HBV infection. HepG2 cells were transfected with plasmids encoding hNTCP, mkNTCP, or hNTCP variants bearing the indicated monkey residues, or mkNTCP variants with the indicated human residues. Transfected cells were maintained in PMM for 24 hr, and subsequently infected with HBV at 100 mge. HBeAg and HBV 3.5 kb RNA were assayed on 6 dpi. Similar to panel B, comparable NTCP surface expression levels in the transfected HepG2 cells were confirmed for all the NTCP variants tested (Figure 7-figure supplement 2 Figure 7C were analyzed for total or cell surface NTCP expression at 24 hr post-transfection as described in Figure 7B . DOI: 10.7554/eLife.00049.025
Two photo-leucines were incorporated into the critical receptor-binding region of WT b without interfering with its receptor-binding activity, which allowed highly specific zero distance cross-linking of its direct binding partner(s) but not other neighboring molecules. A biotin moiety of WT b facilitated purification of the complex of WT b and its binding partner(s) by streptavidin beads. An mAb, 2D3, was developed to recognize WT b on an epitope outside the receptor-binding site, serving as a highly specific tool for detection as well as additional affinity purification of the complex. Thus, the binding partner(s) was first cross-linked by WT b , and then purified by using streptavidin and 2D3 beads in tandem. The covalent interaction between the WT b ligand and its partner(s) enabled a purification process under high-stringency conditions, and efficient isolation was achieved irrespective of the nature of the binding partner(s) even if it is a membrane protein(s) with multiple transmembrane domains, like NTCP identified here. NTCP (Slc10a1) is the founding member of the SLC10 family of solute carrier proteins. It is a hepatic Na + bile acid symporter and is responsible for cotransportation of sodium and bile acids across cellular membranes to maintain the enterohepatic circulation of bile acids Stieger, 2011) . NTCP is a multiple transmembrane glycoprotein presumed to span the cellular membrane up to 10 times with small extracellular loops (Mareninova et al., 2005; Hu et al., 2011) . It is mainly expressed in the liver (Stieger, 2011) , consistent with the liver tropism of HBV and HDV. NTCP localizes to the sinusoidal (basolateral) plasma membrane of hepatocytes (Stieger et al., 1994) , a location that fits well with its receptor role for blood-borne HBV and HDV. Whereas HBV first attaches to hepatocytes mainly through heparan sulfate (Schulze et al., 2007; Leistner et al., 2008) , our data demonstrate that the interaction between NTCP and L protein of HBV is highly specific, and NTCP is crucial for productive viral entry of hepatocytes. Consistent with previous reports on primary cultures of rat hepatocytes (Liang et al., 1993; Rippin et al., 2001) , NTCP expression rapidly decreased over time in cultured PTHs after isolation. This may at least partially explain the observations that primary hepatocytes typically remain susceptible to HBV infections in vitro for only a few days after isolation from liver tissues (Gripon et al., 1988; Seeger et al., 2007) .
NTCP is functionally conserved in mammalians, but protein sequences of NTCP vary among species, which is likely to contribute to the narrow species tropism of viral infection. Strikingly, despite the high level of protein sequence homology between human and monkey NTCP, the later did not support HBV and HDV infection. Replacing a small motif of aa 157-165 of mkNTCP with the corresponding hNTCP residues converted mkNTCP to a receptor for pre-S1 binding as well as HDV and HBV infection. Further studies are warranted to determine if and how NTCP contributes to the species specificity of HDV and HBV infection in other species. It also remains to be determined if other molecule(s) additional to NTCP contributes to the cellular entry of HBV and/or HDV as a coreceptor(s) or receptor component(s), and if other host factors such as the microenvironment or architecture of hepatocytes in liver, or soluble blood components like those that have been shown to involve in infections of other viruses (Shayakhmetov et al., 2005; Morizono et al., 2011) , contribute to HBV and/or HDV infection.
Expression and subcellular distribution of NTCP are precisely regulated under physiological conditions. NTCP accounts for most, if not all, hepatic Na + -dependent bile acid transport (Stieger, 2011) . NTCP expression is low and inversely correlated with the degree of dedifferentiation of cancer cells in human hepatocellular carcinoma (Kullak-Ublick et al., 1997; Zollner et al., 2005) and the severity of HBV-related liver cirrhosis (Lee and Kim, 2007) . The newly discovered role of NTCP as an entry receptor for HBV and HDV raises interesting questions regarding its involvement in viral pathogenesis. Identification of NTCP as a functional receptor for HBV and HDV advances our understanding of their entry into host cells and may lead to new prevention and treatment strategies against these viruses and related diseases.
Adult tree shrews (Tupaia belangeri chinensis) were housed in a Tupaia animal facility at the National Institute of Biological Science, Beijing. All studies were performed in accordance with institutionally approved protocols and adherent to guidelines of the National Institute of Biological Sciences Guide for the care and use of laboratory animals. PTH cells were obtained from anesthetized Tupaia (100-150 g) with a two-step perfusion method as previously described (Walter et al., 1996) . Cell suspensions after perfusion were filtered through a 70-μm cell strainer and centrifuged at 50 g for 3 min. The cell pellet containing PTHs was resuspended in plating medium of Williams E medium supplemented with 10% FBS, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were then plated on collagen-coated cell culture dishes or plates. 4 hr after plating, medium were changed to primary hepatocytes maintenance medium (PMM), that is, Williams E medium supplemented with 5 μg/ml transferrin, 10 ng/ml EGF, 3 μg/ml insulin, 2 mM L-glutamine, 18 μg/ml hydrocortisone, 40 ng/ml dexamethasone, 5 ng/ml sodium selenite, 2% DMSO, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in 5% CO 2 humidified incubator at 37°C with regular medium change every 2-3 days.
PHHs were purchased from Becton Dickinson (United States) or Shanghai RILD Inc. (Shanghai, China). The cells were cultured similarly as PTHs using the same plating medium and maintaining PMM medium as described above.
Human embryonic kidney cell lines 293 and 293T, human cervix carcinoma cell line Hela, and human hepatocellular carcinoma cell line HepG2 were from American Type Culture Collection (ATCC); human hepatocellular carcinoma cell lines Huh-7, SMMC-7721 (SMMC), and Bel-7404 (BEL) were from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences. The cells were cultured with Dulbecco's Modification of Eagle's Medium (DMEM; Invitrogen, United States) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO 2 humidified incubator except otherwise indicated. HepaRG cells were purchased from Biopredic International (Rennes, France) and were cultured following the product manual. Differentiated HepaRG cells were obtained following a two-step procedure as described by Gripon et al. (2002) .
A plasmid containing a head to tail trimer of 1.0× HDV cDNA of a genotype I virus (Genebank accession number: AF425644.1) under the control of a CMV promoter was constructed with de novo synthesized HDV cDNA for the production of HDV RNPs. A pUC18 plasmid containing nucleotide 2431-1990 of HBV (Genotype D, Genebank accession number: U95551.1), or the same plasmid bearing mutation generated by site-directed mutagenesis, was used for expressing HBV envelope proteins under the control of endogenous HBV promoter. HDV virions were produced by transfection of the plasmids in Huh-7 as previously described by Sureau et al. (1992) .
HBV HBV genotype B virus was obtained by ultracentrifugation of plasma from an HBV chronic carrier with written consent. HBV genotype D virus was produced by transfection of Huh-7 cells with a plasmid containing 1.05 copies of HBV genome under the control of a CMV promoter similarly as previously described by Blanchet and Sureau (2006) . The Genebank accession numbers for the viruses are JX978431 and U95501.1, respectively.
Recombinant adeno-associated virus 8 (AAV8) carrying 1.05 copies of HBV genome was produced similarly as previously described (Xiao et al., 1998) by cotransfection of 293 cells with plasmids for AAV8 packaging, 1.05× overlength HBV genome (genotype D) and adenovirus helper.
An HIV-1 genome-based lentivirus pseudotyped by glycoprotein of vesicular stomatitis virus and carrying a firefly luciferase reporter gene was produced by cotransfection of 293T cells with plasmids for VSV-G expression, HIV genome packaging, and luciferase reporter, respectively, as described (Sui et al., 2005) .
Virus-related experiments were conducted in a BSL-2 facility at the National Institute of Biological Sciences, Beijing.
Peptides with nonnatural amino acid L-2-amino-4,4-azi-pentanoic acid (L-photo-leucine) were synthesized by American Peptide Company Inc. (United States). Other peptides corresponding to the N-terminal of pre-S1 domain of HBV L protein (genotype C, strain S472, GeneBank EU554535.1) were synthesized by SunLight Peptides (Beijing, China). Mouse monoclonal antibodies (mAb) 2D3, 1C10, and 4G5 were generated in the laboratory; all are of IgG1 isotype. 2D3 specifically recognizes the 19-33 amino acids of the pre-S1 domain of HBV L protein; 1C10 recognizes HBcAg; 4G5 targets HDV delta antigen. 17B9, a mouse mAb-specific to HBV S protein, was provided by Dr. Lin Jiang, China National Biotec Group. Hepatitis B immune globulin (HBIG) was from the National Institutes for Food and Drug control, Beijing, China. 2D3 magnetic beads were prepared by covalently cross-linking 2D3 to Dynabeads M-270 Epoxy following manufacturer's instructions. Secondary antibodies for immunofluorescence staining and Western blot were purchased from Invitrogen or Sigma-Aldrich (United States).
ELISA kits for HBsAg and HBeAg measurement were purchased from Wantai Pharm Inc. (Beijing, China). SYBR Premix Ex Taq quantitative real-time PCR kit and Reverse Transcriptase (RT) kit were from Takara Inc. (Beijing, China). Streptavidin-coupled magnetic beads (Dynabeads MyOne Streptavidin T1) and magnetic beads coated in glycidyl ether (Epoxy) groups (Dynabeads M-270 Epoxy) were purchased from Invitrogen. Other reagents were purchased from New England Biolabs (United States), Life Technologies (United States), or Sigma-Aldrich.
HBV viral antigens HBsAg and HBeAg were examined using 50 μl supernatants with commercial ELISA Kits (Wantai Pharmacy, Beijing, China) following manufacturer's instructions. In most cases, HBsAg level was normalized with WHO HBsAg reference serum (kindly provided by Dr. Zhenglun Liang from the National Institutes for Food and Drug control, Beijing, China) and presented as international units per milliliter.
Quantification of HDV total RNA (genome equivalent) copies and HBV genome equivalent copies HDV Viral RNA was isolated with Trizol reagent following manufacturer's instructions. Total RNA was reverse transcribed into cDNA with random primers (PrimeScript RT kit; Takara) and 2 μl of the cDNA was used for real-time PCR assay. Primers for quantifying HDV total RNA or genome equivalent copies are complemented with the delta antigen coding region of HDV RNA genome: forward primer HDV-1184F, 5′-TCTTCCTCGGTCAACCTCTT-3′, and backward primer HDV-1307R, 5′-ACAAGGAGAGGCAGGATCAC-3′.
HBV Viral DNA was isolated by standard genomic DNA isolation method. The DNA was quantified using specific primers: 5′-GAGTGTGGATTCGCACTCC-3′ (forward) and 5′-GAGGCGAGGGAGTTCTTCT-3′ (backward) by real-time PCR. The viral genome equivalent copies were calculated based on a standard curve generated with known copy numbers. Real-time PCRs were performed using SYBR Premix Ex Taq kit on an ABI Fast 7500 real-time system instrument (Applied Biosystems, United States).
With 5 × 10 7 copies of genome equivalent HDV, 1 × 10 5 of target cells were incubated at 16°C for 4 hr in the presence of 4% PEG8000, followed by extensive wash with cold PBS for four times. The cells were then lysed directly with Trizol reagent and followed by reverse transcription. RNA copy numbers of viral genome and internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were determined by real-time PCR. For HDV binding inhibition assay, peptides were pre-incubated with target cells at 16°C for 1 hr before incubating with the virus; antibodies against viral envelope protein were pre-incubated with viruses before adding to target cells.
Viral infections of HDV and HBV were conducted in 48-well plates at multiplicities of genome equivalents of 500 and 100, respectively. Normally, 5 × 10 7 copies of genome equivalent HDV or 1 × 10 7 copies of genome equivalent HBV were inoculated in the presence or absence of entry inhibitors with 1 × 10 5 cells and incubated for 16 hr except otherwise indicated. Cells were then washed with medium for three times and maintained in PMM medium with medium change every 2-3 days. For HDV infection, 4% PEG 8000 was present during the 16 hours viral inoculation period similarly as described by Barrera et al. (2004) . Viral infection at different time points was analyzed by measuring viral DNA/ RNAs and viral antigen expression. Quantitative real time RT-PCR was used to quantify HDV total RNAs, strand-specific real time RT-PCR to determine copies of HDV genome and antigenome RNA (see below). For HBV viral infection on HepaRG cells and PHH, ∼ 4% PEG 8000 was present during the inoculation period as previously described by Gripon et al. (Gripon et al., 1993; Gripon et al., 2002; Schulze et al., 2007) . Viral infection of PTH was conducted in the absence of PEG8000. Culture medium was changed every 2-3 days. Secreted HBsAg and/or HBeAg were determined with commercial ELISA kits. Real-time PCR, with or without a prior reverse transcription step, was used for quantification of HBV-specific 3.5 kb pre-C and pregenomic RNA, total HBV sub-genomic RNA, and HBV cccDNA copies.
The strand-specific qRT-PCR was performed as previously described by Freitas et al. (2012) . Briefly, the genomic and antigenomic RNAs were reverse transcribed separately with strand-specific primers into cDNAs: primer HDV398R (5′-CGCTTCGGTCTCCTCTAACT-3′) for genomic RNA; primer HDV288F (5′-GCAGACAAATCACCTCCAGA-3′) for antigenomic RNA. The reverse transcribed cDNAs of genmomic or antigenmoic HDV RNAs were used as templates for real-time PCR using the HDV398R and HDV288F primer pair. TaqMan probe was 5′ FAM-AGAGCTCTGACGCGCGAGGAGTAAGC-TAMRA 3′. Real-time PCR assays were conducted with an ABI Fast 7500 real-time PCR instrument.
Total RNA from HBV-infected cells was isolated with Trizol reagent (Invitrogen). About 400 ng total RNA was reverse transcribed into cDNA with PrimeScript RT kit (Takara) in a 10 μl reaction. cDNA derived from 20 ng total RNA was used as template for real-time PCR amplification. In a separate realtime PCR reaction, 20 ng of total RNA was directly used as template to assess the possible HBV viral DNA contamination in the RNA preparation. Primers (HBV2270F: 5′-GAGTGTGGATTCGCACTCC-3′) and (HBV2392R: 5′-GAGGCGAGGGAGTTCTTCT-3′) were used for HBV 3.5 kb transcripts; (HBV1805F: 5′-TCACCAGCACCATGCAAC-3′) and (HBV1896R: 5′-AAGCCACCCAAGGCACAG-3′) were for total HBV-specific transcripts. Amplification of 123-bp fragment for 3.5 kb transcripts and 92-bp product for total HBV-specific transcripts were both conducted by denaturation at 95°C for 30 s, followed by 40 cycles of 95°C denaturation for 3 s, and 60°C annealing/elongation for 30 s. Real-time PCR was performed using SYBR Premix Ex Taq kit on an ABI Fast 7500 real-time system instrument. Real-time PCR using either set of the primers generated highly specific amplification product. HBV RNA copy numbers were deduced from a standard curve generated from known nucleic acid quantities. Then the HBV RNA copy number per nanogram RNA in the infected cell cultures was calculated by subtracting the background amplification noise derived from the viral DNA contamination in the RNA preparation from that cDNA amplification. The signal-to-noise ratio for HBV total transcripts is usually ≥50, and for 3.5 kb transcripts ≥20. The real-time PCR detection limits for total HBV-specific transcripts and 3.5 kb transcripts are ∼0.5 and ∼3.5 copies per nanogram cellular total RNA, respectively.
HBV cccDNA Southern blot was conducted following a similar procedure as described by Summers et al. (1990) with modifications. Briefly, to selectively extract HBV cccDNA, infected hepG2-NTCP cells in 6-cm dishes were lysed with 1 ml lysis buffer at 37°C for 60 min, followed by addition of 0.25 ml of 2.5 M KCl and incubation at 4°C overnight. The lysis buffer was not supplemented with proteinase K, containing 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1% SDS. The lysate was then clarified by centrifugation at 12,000 g for 30 min at 4°C and extracted with phenol and phenol:chloroform. DNA was precipitated with equal volume of isopropanol in the presence of 20 µg glycogen (Roche) and finally dissolved in TE buffer. The prepared DNA sample was then treated with plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease DNase (Epicentre Technologies) following manufacturer's instructions. For Southern blotting, the plasmid-safe DNase-treated DNA was separated on a 1.3% agarose gel and then transferred to a nylon membrane (Hybond-N + ; Amersham) using a standard neutral transfer procedure. A 3280-bp plasmid constructed by inserting a 588-bp HBV DNA fragment (from 1805 to 2392, genotype D, Southern blot probe) into a 2692 bp pMD18T vector (Takara) was also run on the same agarose gel to serve as the molecular marker for cccDNA in Southern blot analysis. The plasmid is of similar size of HBV genome and was mainly in supercoiled form; therefore it runs at similar size as HBV cccDNA in agarose gel. The nylon membrane was hybridized with a [α-32 P] dCTP-labeled HBV probe (genotype D HBV DNA fragment from 1805 to 2392) prepared by random primer DNA labeling kit (Ver.2.0; Takara). Hybridization was carried out in 7 ml of Perfect Hyb Plus Hybridization Buffer (Sigma) with 1 hr pre-hybridization, followed by overnight hybridization at 67°C. The membrane was then washed once with 2× SSC/0.1% SDS, 1× SSC/0.1% SDS, and 0.5× SSC/0.1% SDS at 67°C for 20 min, respectively. Finally, the membrane was subjected to autoradiographic exposure.
HBV cccDNA (double-stranded DNA without nick and gap) was quantified by real-time PCR using a protocol as previously described Werle-Lapostolle et al., 2004) with modifications. In particular, specific primers for cccDNA detection reported by Glebe et al. (2003) (ccc-1582F: 5′-TGCACTTCGCTTCACCT-3′; ccc-2316R: 5′-AGGGGCATTTGGTGGTC-3′) were validated and used for quantifying copy numbers of cccDNA using real-time PCR. Viral DNAs other than cccDNA, including single-stranded and relaxed circular DNAs, were degraded prior to amplification by treatment of the DNA templates with plasmid-safe adenosine triphosphate (ATP)dependent deoxyribonuclease DNase (Epicentre Technologies). In brief, HBV-infected cells were lysed for 4 hr at 65°C in lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 mM NaCl, 1% SDS) supplemented with proteinase K (200 μg/ml) and followed by phenol-chloroform extraction. A total of 250 ng of the extracted DNA was digested with 5-10 units plasmid-safe DNase in a 50 μl volume for 8 hr at 37°C followed by DNase inactivation at 70°C for 30 min. 2 μl of the 50 μl reaction was then added to 20 μl of a real-time PCR reaction. Amplification of 735 bp cccDNA product was conducted by denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s, 62°C annealing for 25 s, and 72°C elongation for 45 s. HBV cccDNA copy numbers were calculated with a standard curve from plasmid with known nucleic acid quantities. The detection limit for cccDNA is ∼10 copies cccDNA per reaction (equivalent to 10 ng of total cell lysate DNA). Realtime PCR was performed with SYBR Premix Ex Taq kit on an ABI Fast 7500 real-time system instrument.
PTH cDNA library construction, deep sequencing of Tupaia transcriptome, and bioinformatics analysis of Illumina deep sequencing-determined transcriptome Primary Tupaia hepatocytes were isolated as described above. PTH mRNA was purified from 10 μg of total RNA using Oligo-dT magnetic beads. The mRNA was fragmented into small pieces by incubation with divalent cations at 94°C for exactly 5 min. The first strand cDNA was synthesized using random primers and SuperScript II reverse transcriptase (Invitrogen) with fragmented mRNAs. RNA template was then removed by RNase H, and double-stranded cDNA was prepared with DNA polymerase I. cDNA with blunt ends was created by T4 DNA polymerase and Klenow DNA polymerase and an 'A' base was subsequently added to the 3′ end of the blunt phosphorylated DNA fragments by Klenow fragment (3′ to 5′ exo minus). The cDNA was then ligated with adapters and then ran on a 2% agarose gel. The fragments with a size range from 200 ± 25 bp were purified, followed by amplification using the manufacturer's primers. The PCR products were then purified using QIAquick PCR purification Kit (Qiagen), quantified and diluted for cluster generation and deep sequencing. The 72-cycle pair-end sequencing was performed with Sequencing Kits (Version 5) on an Illumina Genome Analyzer IIx (Illumina, San Diego, United States). Illumina CASAVA pipeline v1.8.1 was used for sequence extraction and filtering.
Bioinformatics analysis of Illumina deep sequencing-determined transcriptome of primary Tupaia hepatocytes De novo reconstruction of transcriptome from cDNA library deep sequencing data Total 253,919,616-pair 72 nt sequences with 36.6G base from the sequencing results of the hepatocyte cDNA library described above were fed to Trinity (Grabherr et al., 2011) r20110519 using pair end RNA-seq protocol, with which 209,063 transcripts of average length of 1421 nt (minimum 300 nt, maximum 21,043 nt, and scaffold N50 of 3674 nt) were generated.
Following assembly, GENSCAN (Burge and Karlin, 1997) was used with default parameters to identify coding sequences and the encoded protein sequences of these transcripts. A total of 91,479 protein sequences were identified. Each chosen protein sequence was first annotated with its corresponding blastp (Camacho et al., 2009 ) matches from NCBI human protein sequences. Those not annotated in the first step were then submitted for similar annotation process with UniprotKB human proteome and NCBI nonredundant protein sequence database. Protein sequences that were not annotated by previous steps were submitted for annotation with their corresponding transcripts. Protein sequences with their corresponding transcripts that can be annotated by the blastx (Camacho et al., 2009) hits of NCBI human protein sequences, UniProtKB human proteome or NCBI nonredundant protein sequence database were annotated with these hits from transcripts.
All identified protein sequences were included in the hepatocyte protein sequences database. The protein sequences were labeled with corresponding functional annotation results. Any identified protein sequences that were not successfully annotated are labeled with 'Uncharacterized protein'. Total 50,951 annotated and 40,528 uncharacterized protein sequences generated from the Tupaia hepatocytes transcriptome were combined into the database. A numeric ID (gi) was generated for each protein sequence in the database. The corresponding cDNA sequences were deposited to NCBI Transcriptome Shotgun Assembly (TSA) database of GenBank with accession number from JU120276 to JU170736 after removal of cDNAs shorter than 200 bp and a vector sequence.
PANTHER database (Mi et al., 2005) was used to determine the protein class distribution of annotated transcripts and protein sequences in primary Tupaia hepatocytes (PTHs) generated in this study and transcriptome from primary human hepatocytes (PHHs) reported by Hart et al. (2010). Photo-cross-linking with peptide ligand and tandem purification of the target molecule(s) L-photo-leucine-bearing wild-type bait peptide (WT b ) or control bait peptide (N9K b ) was dissolved in DMSO in dark. L-photo-leucine contains a photoactivatable diazirine ring, irradiation of UV light at 365 nm induces a loss of nitrogen of the diazirine ring, and yields a reactive carbene group with short half-life for covalent cross-linking at nearly zero distance. For tandem purification, WT b or N9K b at indicated concentrations was applied to ∼1 × 10 7 hepatocytes plated on collagen-coated dishes. Cells were cross-linked by UV irradiation and then washed to remove residual free peptides and subsequently lysed with 1 ml radioimmunoprecipitation assay (RIPA, pH 7.4) buffer containing 20 mM Tris, 150 mM NaCl, 0.1% SDS; 0.5% sodium deoxycholate, 1% NP40, and 1× protease inhibitor cocktail (Roche). The cell lysates were precipitated with 100 μl streptavidin T1 magnetic beads, eluted with 50 μl nonreducing SDS-PAGE loading buffer and then diluted with cold RIPA buffer to a final volume of 1 ml and was precipitated with 100 μl (1 × 10 8 ) 2D3-conjugated M-270 dynabeads, then eluted with 100 μl nonreducing loading buffer. The elute, with or without PNGase F treatment, was diluted to 1 ml with RIPA buffer and was precipitated again with 100 μl streptavidin T1 magnetic beads, followed by extensive washing, and finally eluted by boiling 5 min with 20 μl SDS-PAGE loading buffer. The samples were then analyzed with 12% SDS-PAGE and silver staining. For photocross-linking analysis of primary cells, cell lines, or NTCP-or control plasmid-transfected Huh-7 or 293T cells, WT b or N9K b bait peptide in the presence or absence of competing peptide was applied to 2 × 10 6 cells, photo-cross-linking was conducted similarly as described above. The cross-linked samples were precipitated with streptavidin T1 magnetic beads and separated by SDS-PAGE, and followed by Western blotting with mAb 2D3 (recognizing bait peptides) or mAb 1D4 against C-terminal tag C9.
Silver stained gel bands were cut, followed by in-gel reduction, alkylation, and trypsin digestion as previously described (Shevchenko et al., 2006) . Digested peptide mixtures containing 0.1% formic acid were loaded onto a 4 cm, 75-μm inner diameter fused silica capillary column packed with 10-μm YMC C18 material (YMC, Kyoto, Japan). After desalting, samples were separated with a Waters nano ACQUITY UltraPerformance LC (Waters, United States) and eluted to a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, United States). The UPLC separation gradient included a 30-min gradient from 0% to 30% acetonitrile, followed by a 10-min gradient to 80% acetonitrile, then 10 min of 80% acetonitrile and back to 0% acetonitrile within 5 min. The mass spectrometer was operated in the data-dependent mode. Survey MS scans were acquired in the orbitrap with the resolution set to a value of 60,000. Each survey scan (300-2000 m/z) was followed by four data-dependent CID tandem mass (MS/MS) scans at 35% normalized collision energy and four data-dependent HCD tandem mass (MS/MS) scans at 40% normalized collision energy with 15,000 resolution in orbitrap. AGC target values were 500,000 for the survey scan, 10,000 for the ion trap MS/MS scan, and 50,000 for the orbitrap MS/MS scan. Target ions already selected for MS/MS were dynamically excluded for 30 s. Tandem mass spectra were searched against the Illumina deep sequencing-determined Tupaia hepatocyte protein database that was concatenated with reversed sequences to estimate false positives and was supplemented with the sequence of bait peptide under the Linux operating system using the ProLuCID (Xu et al., 2006) protein database search algorithm with peptide mass tolerance of ±100 ppm, fragment ion mass tolerance of ±400 ppm, half tryptic specificity, and a static modification of 57.0215 on Cys due to carboxyamidomethylation. ProLuCID search results were then filtered with DTA Select 2.0 (Tabb et al., 2002) using a cutoff of 1% for peptide false identification rate (-fp 0.01). Peptides with DeltaMass > 10 ppm (-DM 10) were rejected; the minimum number of peptides to identify a protein was set to 1 (-p 1).
Immunofluorescence microscopy and FACS analysis of NTCP binding with pre-S1 peptides Plasmid encoding human, or Tupaia NTCP, with or without a tag, or a control plasmid was transfected into cells. The cells were washed and blocked with 3% BSA 24-36 hr after transfection and then stained with biotin-labeled peptides at 4°C about 1-2 hr, followed by fixation with 4% paraformaldehyde (PFA) for 10 min. Cells were then stained with PE-labeled Streptavidin (eBioscience). In some cases, Myr-59 peptide containing the first 59 residues of pre-S1 domain with an N-terminal myristoylation modification and labeled with FITC (FITC-pre-S1) was applied to cells directly. The cell images were captured with a Nikon Eclipse Ti Fluorescence Microscope or a Zeiss LSM 510 Meta Confocal Microscope. For FACS analysis, the FITC-pre-S1 stained cells without fixation were detached with 0.5 mM EDTA/PBS, washed and resuspended in PBS, and analyzed with a FACS LSRII instrument (BD).
Four siRNAs (tsNTCP-si1: 5′-CUAUGUAGGCAUUGUGAUAdTdT-3′, tsNTCP-si2: 5′-GUGUUAUCCU GGUGGUUAUdTdT-3′, tsNTCP-si3: 5′-GGACAUGAAUCUCAGCAUUdTdT-3′, tsNTCP-si4: 5′-GGGCAAGAGCAUCAUGUUUdTdT-3′) specifically targeting Tupaia slc10a1 were designed through siDESIGN Center (www.thermo.com/sidesign). The specificities of these siRNAs were examined by searching against NCBI cDNA databases and the in-house Tupaia hepatocyte transcriptome to ensure they are free of off-target. siRNA with sequence Ctrl-si: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ that is a scramble sequence with no known mammalian target sequence was used as a negative control. 20 nM siRNA were transfected into PTHs 24 h after initial seeding with lipofectamine 2000 (Invitrogen). tsNTCP mRNA level in the siRNA-transfected cells was quantified by real-time RT-PCR 3 days after siRNA transfection. Transfected PTH cells were infected on day 4 after siRNA transfection with HBV, HDV, AAV8-HBV (carrying 1.05 copies of HBV genome), or Lenti-VSV-G-Luc viruses. The secreted viral antigen HBsAg or HBeAg from HBV-and AAV8-HBV-infected cells were examined as indicated. For Lenti-VSV-G-Luc virus-infected cells, luciferase activity was determined on 6 days post-infection.
For gene knockdown experiment on HepaRG cells SLC10A1 from HepaRG cells was cloned, and the sequence was deposited to Genebank (accession number: JQ814895). Differentiated HepaRG cells in 48-well plate were transfected using RNAiMax (Invitrogen) with 20 nM of a siRNA pool (Qiagen) containing four specific siRNAs targeting different regions of human SLC10A1 (5′-GGAUCGUCCUCAAAUCCAAdTdT-3′, 5′-GGAGUCAGCCGGAGAACAAdTd T-3′, 5′-GGACAAGGUGCCCUAUAAAdTdT-3′, 5′-GGUGCUAUGAGAAAUUCAAdTdT-3′) or a negative control siRNA (Ctrl-si: 5′-UUCUCCGAACGUGUCACGUdTdT-3′) as indicated. Gene knockdown efficiency was examined 4 days after transfection. Cells were then inoculated for 16 hr with HBV in the presence of 3.6% PEG8000. The secreted HBeAg and the 3.5 kb HBV RNA were determined on indicated days after infection.
Frozen PHH cells were thawed and subsequently transfected as that of PTH with human SLC10A1specific siRNA 5′-CACAAGUGCUGUAGAAUUAdTdT-3′ (siR405) or a siRNA pool containing four SLC10A1 specific siRNAs from QIAGEN (5′-GGAUCGUCCUCAAAUCCAAdTdT-3′, 5′-GGAGUCAGCCGGAGAACAAdTdT-3′, 5′-GGACAAGGUGCCCUAUAAAdTdT-3′, 5′-GGUGCUA UGAGAAAUUCAAdTdT-3′). Control siRNA 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (Ctrl-si) has a scramble sequence with no known mammalian target sequence. Total 20 nM siRNA was transfected into ∼1.4 × 10 5 PHH cells per well in 48-well plate 24 h after initial cell seeding; the cells were then inoculated with HBV 72 hr after transfection. hNTCP mRNA level in the Research article siRNA-transfected cells was quantified by qRT-PCR 3 days after siRNA transfection. The secreted antigens HBsAg and HBeAg, and the intracellular HBV RNAs were determined on indicated days after infection.
HepG2 or Huh-7 cells were transfected with a plasmid expressing human, treeshrew, monkey NTCP, or an NTCP variant, or a vector control. Stable cell line expressing hNTCP was established by transfection of HepG2 cells with a plasmid encoding hNTCP (hNTCP/pcDNA3.1) followed by selection with 500 μg/ml G418 and maintained in DMEM supplemented with 10% FBS, 500 μg/ml G418, 100 U/ml penicillin, and 100 μg/ml streptomycin. The transfected cells, or HepG2-hNTCP stable cells, were cultured in PMM 24 hr before infection.
With 5 × 10 7 genome equivalent copies of HDV, 1 × 10 5 cells were incubated, or otherwise indicated, for 24 hr in the presence or absence of entry inhibitors. HDV inoculation was conducted in the presence of 4% PEG8000. PMM was replenished every 2 days. On indicated days postinfection, cells were treated with 100% methanol for 10 min, followed by incubating with 5 μg/ml FITC-labeled mAb 4G5 for 1 hr at RT to stain HDV delta antigen. The nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) before analyzing. HDV viral RNA copies in cell lysates were quantified by qPCR.
With ∼1 × 10 7 genome equivalent copies of HBV, 1 × 10 5 cells were inoculated, or indicated otherwise, in the presence of ∼4% PEG8000 as reported for primary human hepatocyte and HepaRG cell (Gripon et al., 1993; Gripon et al., 2002; Schulze et al., 2007) . The cells were maintained subsequently in PMM and the medium was changed every 2-3 days. For immunofluorescence microscopy analysis, HBV-infected cells, with or without replating on glass coverslips for imaging, were fixed with 4% Paraformaldehyde (PFA) and permeabilized with 0.5% TritionX-100, and then stained either with 10 μg/ml 17B9 against HBsAg followed by FITC-labeled goat anti-mouse IgG, or with 5 μg/ml 1C10 against HBcAg followed by Qdot 655 VIVID donkey anti-mouse IgG. 1 μg/ml of DAPI was added to stain the nucleus before analyzing. The cell images were captured with a Nikon Eclipse Ti Fluorescence Microscope or a Zeiss LSM 510 Meta Confocal Microscope. Secreted viral antigens and intracellular viral replication intermediates cccDNA and/or RNAs were examined on indicated days after infection.
For cell surface expression, the transfected cells expressing NTCPs or mutants were surfacebiotinylated with sulfo-NHS-LC-biotin (Pierce) following the manufacturer's instruction manual. The biotinylated cells were then lysed in 600 μl of 1× RIPA buffer supplemented with 1× protease inhibitor cocktail (Roche) and then treated with PNGaes F. Total cellular protein in the lysate was determined by using Bio-Rad DC Protein assay. About 300 μg Streptavidin T1 Dynabeads (Invitrogen) were then used to pull down surface-biotinylated proteins in the supernatants containing ∼160 μg of total cellular protein each sample. After extensive washing with 1× RIPA buffer, bound proteins were eluted, and separated by SDS-PAGE and subsequently examined with anti-C9 mAb 1D4 by Western blotting. For total NTCP expression, the same transfected cells expressing wild-type or mutant NTCPs were lysed with 1× RIPA buffer and treated with PNGase F. Each sample containing same amount of total cellular protein (∼8 μg) were loaded for SDS-PAGE followed by Western blotting analysis with mAb 1D4 that recognizes the C9 tag fused at the C-terminus of NTCPs.
All experiments were repeated 2-6 times with duplicate or triplicate samples for each condition unless indicated otherwise. A representative result of multiple independent experiments is present (n = 2-6) in each figure. Error bars shown in all figures represent standard deviation of the mean (n = 2-4). Dotted lines show detection limit except otherwise specified. Reporting standards: We followed the reporting standards for the Transcriptome Shotgun Assembly Sequence Database of NCBI, which are: Submitted sequences must be assembled from data experimentally determined by the submitter. Screened for vector contamination and any vector/linker sequence removed. This includes the removal of NextGen sequencing primers. Sequences cannot be less than 200 bp. Sequences should have no more than 10% n's or greater than 14 n's in a row. If the submission is a single-step, unannotated assembly and the output is a BAM file(s) these should be submitted as a TSA project to SRA. The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years. Influenza A viruses (IVs), which belong to the orthomyxoviridae family, consist of eight negative RNA strands. Hemagglutinin (HA) and neuraminidase (NA) are two glycoproteins that are encoded by the IV genome, expressed from segments 4 and 6, respectively. The selection due to various host immune systems and anti-flu drugs accelerate the mutation rates of viral proteins, especially for these two membrane proteins [1, 2] . There are 17 HA subtypes and 10 NA subtypes, designated H1-H17 and N1-N10, respectively. Over 118 combinations of IVs can be isolated from wild birds, which are also the natural reservoir of these viruses (except the H17N10 virus, which, until recently, was isolated only from bat) [3] [4] [5] . The species jumping ability of IVs can result in the infections of poultry and mammals, such as chicken, swine, equine or whale species, with different virulence levels [6] [7] [8] [9] . The H1N1, H2N2 and H3N2 viruses have been responsible for tens of millions deaths during the deadly history of human influenza epidemics. Furthermore, the H5N1, H7N7, H7N2, H7N3 and H9N2 viruses have been isolated from sporadic human infections and deaths [10] [11] [12] [13] . It is worth noting that the H5N1 virus is the most severe for human and avian species, with sudden onset and high mortality. The mortality rate in hundreds of patients who were hospitalized for H5N1 infections was roughly 59.05%, much higher than the mortality rates of the Spanish Flu or the 2009 influenza pandemic (H1N1) [11, 14, 15] .
As a requirement for infection, the homotrimeric HAs play a key role in binding to the host sialic acid (SA) receptors and membrane fusion. The nascent HA of all subtypes consists of conserved structures, including the signal peptide, the cytoplasm domain, the transmembrane domain and the extracellular domain [16] . The mature HA monomer can be cleaved by proteases into the global HA1 and stalk HA2 subunits [17] . When IVs are located in the host digestive tract or respiratory tract cell, the receptor binding domains (RBDs) at the tip of HA1 bind to the SAa2-3Gal or SAa2-6Gal receptors, which are essential for endocytosis [18, 19] . HA unfold and expose the interior HA2 subunits in the acid environment, then the fusion peptides in HA2 insert themselves into the host membrane (viral membrane fusion) [20, 21] .
Homotetrameric NA is a type II membrane protein, whereas HA is a type I membrane protein. The nascent NA consists of four parts: the cytoplasm tail (in amino-terminus), the transmembrane domain, the stalk domain and the global domain [22] . Different subtypes of NA are composed of 450,480 amino acids, displaying low sequence similarity. Although there is variable homology among the various NA subtype sequences, especially in the N1 and N2 subtypes with the deletion of 4,30 amino acids in the stalk domain [23] , NA subtypes display stable topologies: a six-bladed b-propeller fold makes an enzymatic activity domain that functions in the release of progeny virions [24, 25] .
HA and NA have a distribution ratio of 4:1 on the influenza viral envelop and maintain the basic functions of host recognition, infection and viral diffusion [26] . Various studies have reported that some of the factors that influence HA include the number of the basic residues in the HA0 cleavage site, the mutation of key residues in the RBD, the changing of antigenic sites or Nglycosylation sites (glycosites) and the variation of the topology of N-glycan structures [15, 27] . Meanwhile, the factors that influence NA include deletion of the stalk domain, the mutations drugresistance, as well as the changing of antigen sites or glycosites and the variation of the topology of N-glycan structures [28] . As a kind of glycan-binding protein (GBP), HA works with NA, which functions as an exoglycosidase, cooperatively. Only by achieving a dynamic equilibrium between attaching to the host and releasing progeny virions can the IVs gain a long-term mechanism for infection and diffusion.
The existence of N-glycosylation is necessary for viral membrane glycoproteins. The biosynthesis and modification of nascent secretory or membrane proteins occurs in the endoplasmic reticulum and Golgi, N-linked glycans encode crucial information for the folding, maturation, transport or degradation of proteins [29] . To escape both the host's humoral and cellular immune systems, the potential glycosites in viral envelope proteins can provide the identical glycans as those of the host's cells to mask the antigenic sites [28, 30] . Additionally, glycosylation also impacts the sensitivity of HA to temperature, the protection of cleavage sites and the stalk domain, and even the receptor-binding preferences [31] [32] [33] . As the ideal model for the influence of N-glycosylation in pathogen-host interaction, the present studies show that the envelope glycoproteins of IVs appear to only have N-glycosylation, with no O-glycosylation and GPI-anchors [34] ; hence, the glycosites discussed in this paper only pertain to N-glycosylation site. Figure 1 . The N-J trees of two glycoproteins in IVs with the corresponding distribution chart of glycosites. The phylogenetic trees of HAs and NAs were constructed using three to ten representative amino acid sequences in each subtype (File S1). The distribution charts of glycosites, colored according to the statistics of conservation in each HA or NA subtype (File S2), are shown in various strips. The red, green and blue color represent the levels of conservation of ''.95%'', ''5%,95%'' and '',5%'', respectively. The conserved cysteines are shown in yellow strips. (A) The N-J tree of HA subtypes with the corresponding distribution chart of glycosites. (B) The N-J tree of NA subtypes with the corresponding distribution chart of glycosites. doi:10.1371/journal.pone.0049224.g001
The Glycosylation Sites in Influenza Virus PLOS ONE | www.plosone.org Current studies have analyzed the evolutionary dynamics of Nglycosylation sites of select subtypes or HA/NA as a whole [35] [36] [37] . Although most influenza evolution can be accounted for by genetic drift, there is also evidence of adaptive evolution of mutations which are under positive selection [38, 39] . We have extended previous studies of basic similarity alignment scoring of similarity to analyze the position-specific glycosites that are under selective pressure in IVs [40] . Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, especially in the H5N1 virus.
Protein sequence data from all subtypes of HA and NA
The amino acid sequences of HA and NA were obtained from the NCBI (National Center for Biotechnology Information) Influenza Resource (http://www.ncbi.nlm.nih.gov/genomes/ FLU, accessed 15th March 2012) [5] . To fully understand the distributed regularity of the glycosites in each subtype, we downloaded the 29 sets of HA and NA with the customized definition ''.{serotype} {strain} {segname}'' using the following combinations: H1Nx,H17Nx and HyN1,HyN10 (where x and y represent ''any'' by default), in addition to the HA and NA from the H5N1 virus.
All the sequence alignments of the various HAs and NAs were performed using Clustal 2.0. Repetitive, incomplete and mixed sequences were removed. For the purpose of convenience, one representative sequence with the generally longest length from each subtype was chosen for further description (Table S1 and S2).
To assess the relationship between the distributed regularity of the glycosites in the envelope glycoproteins and the evolutionary position of various HAs and NAs, firstly, the preliminary trees for each HA and NA subtype were constructed using all available sequences; secondly ten representative sequences from discrepant clades, with consideration of host source and areas, were picked up (excluding H14, H17 and N10 due to limited records). Finally, the phylogenetic trees of HA and NA were constructed by these hundreds of sequences (File S1). The phylogenetic trees of the HA and NA were constructed by MEGA5.05 with N-J methods and a p-distance model with a bootstrap value of 1000 [41, 42] .
As is known, N-X-T/S (X cannot be a proline) is the glycosite motif. Although some researchers have concluded that not all the potential glycosites would be glycosylated in mature glycoproteins, Kelley WM et al. argued that the 14-sugar glycans would be transferred to all the glycosites in nascent proteins and be truncated and modified in the subsequent process [42] .
Due to the fixed pattern of glycosite, many programs and software provide the prediction of glycosites [43] , such as the ''NetNGlyc 1.0 Server'' (www.cbs.dtu.dk/services/NetNGlyc). By submitting the alignment files to a prediction server, we obtained a series scores for the potential glycosites. In consideration of experimental error, those occasional glycosites, which may result from the genomic sequencing or translated by different genetic codes, were excluded. For example, the 429NLS in the HA of A/ duck/Hunan/3315/2006(H5N1) only appears in H5 set once, and it is also embedded inside of HA trimer [44] . Statistics of the position, pattern and the levels of conservation in various subtypes were used for further discussion (Table S1 , File S2).
The available structures of HA and NA would help us to explore the distinctive function of various glycosites. Therefore, we collected the structures of various HA or NA subtypes from the PDB (Protein Data Bank, http://www.rcsb.org/pdb/home/ home.do). To obtain the whole multimer, these monomer structures were processed with a VMD 1.9 transformation matrix using a Tcl script [45] . The code for each representative structure of the various subtypes of HA and NA and the corresponding source of IVs are shown in Table S2 .
Most coordinate files of envelope glycoproteins are obtained from X-ray crystallography or NMR. Furthermore, complete larger glycans are too exible to yield sufficient electron density [46] . Isolation and purification of membrane glycoproteins by particular enzyme treatments lead to the lack of partial domains 
The H5N1 virus can be divided into 10 clades, according to the evolutionary position of the HAs defined by the WHO (World Health Organization) in 2008. These clades are numbered 0 to 9 [48, 49] . Clades 0,2 are responsible for all the human infections, resulting in 359 deaths since 2003 [11] . Many clades have not been reported for the last four years, while the dominant H5N1 viruses are concentrated in clade 0,2. Clade 2.1, clade 2.2 and clade 2.3 have further evolved into third-or fourth-order clades, and these newly formed viruses have become geographic strains.
For the purpose of investigating the co-evolution relationship between the glycosite patterns of HA and NA, we reconstructed a phylogeny tree of HAs and determined ten clades. When discrete monophyletic groups with a common node meet a bootstrap value of $60 at the clade-defining node and average percentage pairwise nucleotide distances between and within clades of .1.5% and ,1.5%, respectively, they could split into the second-, third-, or even fourth-order clades [50] . The statistics of the glycosites of each HA, as well as the corresponding NA, were recorded according to their clades (Files S3, S4 and Figure S1 ).
As shown in Figure 1A , the current 17 HA subtypes were concentrated in two evolutionary groups. One large group, represented by H1 and H5, contained H2, H6, H8, H9, H11, H12, H13 and H16; Another large group was represented by H3, and contained H4, H7, H10, H14 and H15. Although each subtype of HA had a lower similarity to each other and the distribution of the glycosites differed in sequential numbering, we found that the sequence alignment of all HA subtypes indicated that partial glycosites appeared in similar domains. Furthermore, the structure alignment of available HAs also showed that various HA subtypes had a highly conserved structure, and the distribution of glycosites was also regular ( Figure S2 ).
By analyzing the position of glycosites and their conservative rates, we conclude that two types of glycosites appear in HAs: one with a high level of conservation in all HA subtypes and another with various conservative rates in different HA subtypes. Two highly conserved glycosites are located near the HA0 cleavage site (e.g., the 27NNST in H1 or the 30NGT in H8) and the fusion peptide of the HA2 (e.g., the 498NGT in H1 or 500NGS in H5) respectively in all subtypes, and these two glycosites play necessary role in viral life cycle for protecting the HA0 cleavage sites and fusion peptide [34, 51] . In addition, another highly conserved glycosite appears at C-terminal part of the HA1 sequence, which is near the connection of the global and stalk domains, except in H7 and H15 sequences [52] . Three to ten characteristic glycosites were distinctive in each subtype. Their conservative rates were influenced by different internal evolution branches, ranked from 0.5% (e.g., 292NGS with 2.85% in H3) to 100% (e.g., 38NGT with 99.94% in H3), distributed mainly in the global domain. It is worth mentioning that some highly conserved glycosites were near to cysteines (Figure 1 ). Kozlov et al. hypothesized that ERp53, which is involved in the formation of disulfide bonds during the folding of nascent proteins, would form a complex with calreticulin/calnexin, which depends on precursor N-glycans [53] .
One most obvious characteristics of glycosites was that the increasing samples or cross-species reports would lead to more glycosites. There are many factors contributing to the existence of numerous glycosites in H1, H3 and H5 subtypes. Because the RNA virus is rapidly mutating, long-term cross-species infections have resulted in the accumulation of adaptive mutations and glycosites. Due to the selection pressure of different host immune systems, a disadvantageous mutation would be eliminated while a virus with the gains and losses of glycosites would be conserved.
For example, the HAs from avian H1N1 or H3N2 virus had less glycosites than those of the seasonal human H1N1 or H3N2 virus (e.g., 286NAS in A/Memphis/28/1983 (H1N1). Figure 2A and File 5S).
The H3 set had the most glycosites (16, as shown in File S6) among all HA subtypes, which were mainly contributed by H3N2 and H3N8 virus. The earliest H3 record had only one moderate conservative glycosite (79NCT, e.g. A/equine/Miami/1/1963 (H3N8)) except for six highly conserved glycosites, which is also conserved in most seasonal human H3N2 virus and mammalian H3N8 virus (e.g. A/Denmark/22/2011 (H3N2) or A/equine/ Yokohama/aq79/2011 (H3N8)), but not in subsequent avian H3N1, H3N9 virus (e.g. A/duck/Zhejiang/5/2011 (H3N3) or A/duck/Saitama/2/2009 (H3N8). File S5). Over the last four decades, seasonal human H3N2 viruses have gradually acquired additional glycosites within the globular HA1 [37, 40] . The occurrence of 294NSS has increased the glycosite numbers to 13 in parts of current HA of H3N2 virus from 2010 (A/Singapore/ GP5/2011(H3N2). File S6).
Not all the glycosites shared the same numbering in one subtype. For this reason, we chose a representative sequence for each subtype (Table S1 ). Most glycosites were influenced by the deletion or insertion of the mutants in the upstream sequence, which is also known as ''antigenic drift'' [54] . In the HAs of the H1N1 or H5N1 viruses, some nearby glycosites were determined by ''antigenic shift'' [55] . In the H1 subtypes, the 144NVT glycosite from the seasonal H1N1 flu, which were isolated from 1940s to 1980s (e.g., A/Memphis/1/1984 (H1N1)), was replaced by the 142NHT glycosite after the 1990s (e.g., A/California/04/ 2007 (H1N1)). Additionally, no such glycosylation appeared in those H1N1 viruses isolated from swine H1N1 flu (e.g., A/swine/ Guangdong/1604/2010 (H1N1)), Spanish flu (e.g., A/South Carolina/1/1918 (H1N1)) and the 2009 influenza pandemic (H1N1) (e.g., A/Mexico city/CIA1/2009 (H1N1)). It has been partially shown that vaccination of mice with the 1918 influenza strain protected against subsequent lethal infection by the 2009 virus; however, the 1918 strain did not protect against the seasonal H1N1 flu [56] . It is interesting to note that the migration of the 179NKS to 177NLS tracks with these effects. Almost all of the 179NKS glycosites identified were centralized in swine-origin influenza viruses (S-OIVs, e.g., A/Swine/Guandong/1604/2010 (H1N1)) and parts of the 2009 influeza pandemic H1N1 virus (e.g., A/Mexico City/014/2009 (H1N1)), while the 177NLS glycosite was mainly found in the swine or human H1N2 viruses (e.g., A/ New York/481/2003 (H1N2)) and most seasonal flu viruses (e.g., A/California/04/2007 (H1N1)). These date further confirm that the HA of the 2009 influenza pandemic (H1N1) originated from S-OIV, not previous seasonal virus [57] . Similar positional conversion of the glycosites in H5N1 caused by ''antigenic drift'' was the ''179NYT/181NNT''. The conserved 181NNT glycosites have been replaced by the 179NYT in parts of clade 7 viruses since 2005. It reminds us that although these glycosites were adjacent, their N-glycans would shield different antigenic sites, which would provide some suggestions for the development of influenza vaccine.
Moreover, in the smaller cluster of H7, H10 and H15, one highly conserved glycosite appeared in the long a-helix of HA2 (e.g., 431NWT in A/turkey/Chile/4418/02 (H7N3)). This uncommon glycosite requires further investigation (File S1).
It can be observed from Figure 1B that N10 is highly divergent from other subtypes [4] . The remainder of the current 9 NA subtypes are concentrated into two evolutionary groups: one group was represented by N2 and contains N3, N6, N7, and N9; and another group contains N1, N4, N5, and N8.
The glycosites of NA can be divided into two types according to the distributed region: two to four highly conserved glycosites are located in the stalk domain in each subtype; two conserved glycosites and most middle-low conserved glycosites are mainly located in the global domain, which are near the tip of NA, the connection of the global and stalk domains, or the antigenic sites. The glycosite of 146N is conservative in all NA subtypes (e.g., 146NDT in A/Boston/20/2008(H3N2), 144NGT in A/duck/ Taiwan/4201/99(H7N7) or 146NGT in /Viet Nam/1203/ 2004(H5N1)). It has been shown that the N-glycan at this glycosite affects NA enzymatic activity, causing a 20-fold decrease in activity [58] . Similar to the description of HAs, a large number of H1N1, H3N2, H5N1, H7N2 and H9N2 viruses have accumulated numerous glycosites in N1 and N2, especially in the global domain, mainly participating in immune evasion. Moreover, one conserved glycosite, 12NTT (Conservative rate: 93%, e.g., A/ turkey/Italy/3807/2004 (H7N3)), located in the transmembrane domain in N3 and N10, requires further investigation [59] .
The significance of the conserved glycosites in the stalk domain was providing the N-glycans to avoid the cleavage by host enzyme (e.g., Trypsin) [60, 61] . The variance of the glycosites was closely related to the deletion of the stalk domain. Although the threedimensional structure of stalk domain has not been determined yet, it is speculated that the presence of an a-helix motif in the uncrystallized structure has also been provided by cryoelectron microscopy [62] . Wagner et al. believed that a longer stem domain would enhance the replication capacity of the virus, while the deletion of the stem domain would decrease the enzymatic activity of NA [63] . Various subtypes had stalk domain deletions of 3 to 24 residues, except for N4, N8, N9 and N10. The numbers of deletions were also distinctive across different combinations of IVs and even within one subtype, such as the N2 subtype. Generally, no deletions were found in the NA of the H3N2 virus; deletion of 3 residues and one corresponding glycosite with the pattern ''E-R-61N-3-64T-V-H'' (meaning 3 residues missing between 61N and 64T, e.g., A/chicken/Zhejiang/611/ 2011 (H9N2)), appeared in most of the NA subtypes of the H9N2 virus. A similar deletion of 20 residues and two glycosites (I-E-60R-20-80N-I-I) appeared in most NA subtypes of the H6N2 virus (e.g., A/duck/Fujian/3193/2005 (H6N2)). The deletion of 16 residues and two glycosites as ''C-E-55P-16-72T-T-E'' were a distinctive part of the H7N2 virus (e.g., A/unknown/New York/19501-5/2006 (H7N2)). Parts of the H5N2 virus were characterized by the deletion of 20 residues and two glycosites (''R-N-62I-20-83G-Y-R'', e.g., A/chicken/Ibaraki/3/2005 (H5N2)). Moreover, in some avian H2N2 viruses, the deletion of 22 residues resulted in the loss of two glycosites; however, a new glycosite appeared in the newly created sequences (''P-A-47N-22-70N-T-V'', e.g., A/chicken/New York/Sg-00300/1997 (H2N2)). In all, the diversity of deletions in the stalk domain indicates that the glycosylation pattern of HA and NA has a complex relationship.
Globally, the researchers have been paying attention to the highly pathogenic avian influenza (HPAI) since the first human death caused directly by avian H5N1 virus in 1997. It is generally considered that the HPAI viruses were characterized by polybasic residues in the HA0 cleavage site in HA and the deletion of the stalk domain in NA [64] . However, these characteristics had been reported even before 1990s. The earliest H5N1 virus, ''A/ chicken/Scotland/1959 (H5N1)'', had four continuous basic residues in the HA0 cleavage site (compared to 5,6 continuous basic residues in most common H5N1 HAs); the stalked deletion of NA also existed in A/turkey/Ontario/84/1983 (H5N1). The records of H5N1 virus have increased rapidly since 2003. Since then, a number of new clades and subclades have emerged and resulted in various new glycosites ( Figure 3A and 3B) . The percentage of unconserved glycosites in HA also increased and diversified rapidly after 2003, meanwhile the percentage of unconserved glycosites in NA reduced gradually, regardless of those highly-conserved glycosites ( Figure 3C and 3D) .
Both twelve glycosites were found in the HA and NA of the H5N1 virus, shown in the Tables 1 and 2 and Files S3 and S4.
Most H5N1 viruses were grouped into clade 0 before it appeared in Hong Kong again in 2003. Various patterns of the glycosites in HA and NA had co-existed in these original viruses. These original NAs contained the known glycosites, including four highly conserved glycosites in the stalk domain and seven in the global domain (except the occasional 341NGT which only appeared in clade 1 and Thailand records during 2004,2010, e.g., A/chicken/Thailand/CU-354/2008 (H5N1)). In contrast, six highly conserved glycosites together with 170NST exist widely in clade 0. Since 2003, the WHO has recorded a three-wave epidemic of H5N1, which resulted in hundreds of deaths and huge economic losses [11] . Until recently, the glycosite patterns were highly conserved in all the avian clades except for clade 7 (e.g., clade 3, 4, 5, 6, 8 and 9), as shown in Figure 4 and Figure S2 .
Most currently recorded H5N1 viruses were concentrated in the fourth-order clades. During this decade, there have been increasing human infections and new glycosite patterns of HA and NA. It has been reported that the H5N1 virus of clade 2.2 was involved in the outbreak that occurred among the migratory bird population near Qinghai Lake in 2005. Since then clade 2.2 spread westward. This resulted in a number of deaths of wild birds in Europe [65] . In 2006, the H5N1 virus appeared in Africa for the first time, followed by hundreds of mortally infected humans in Egypt, Nigeria and Djibouti [66] . Figure 5D , File S3).
The 181NVT glycosite, which is located at the apical b-folding of HA, is conserved in all human IVs (e.g., A/Anhui/1/2007 (H5N1)). Previous statistics have indicated that this glycosite has a lower level of conservation in avian IVs (e.g., A/chicken/ Vietnam/NCVD-093/2008 (H5N1)). The results of the molecular dynamics simulation indicated that the a2-3-sialoglycans adopted a straight-like and outward topology structure while the a2-6sialoglycans were fishhook-like and inward; therefore, we inferred that the that the deficiency of the glycosite would benefit the binding of SAa2-3Gal sialoglycans [68] . Actually, all the viruses that had deficiencies of the 181N glycosite were isolated from the avian host, which were concentrated in clades 7.1, 7.2 and 2.2.1.1 ( Figure 6A, File S3) .
Interestingly, the deletion of the stalk domain in H5N1 NA is variable. As the first human death reported in 1997, most H5N1 viruses that belonged to clade 0 and isolated in Hong Kong were characterized by ''N-Q-S-I-54I-18-73N-F-Y'', which remained a glycosite: 50NQS. Although previous studies conjectured that the H6N1 virus (e.g., A/Teal/Hong Kong/W312/97 (H6N1)) was the donor of the NA gene in 1997 HPAI virus [69, 70] ; however, similar motif could be found even in 1983. Since 2000, the most common pattern of ''A-E-48P-20-69I-S-N'' with four glycosites missing has dominated in the H5N1 NAs ( Figure 6B, File S4 ).
There is one kind of glycoprotein that participates in the recognition and membrane fusion in most virus envelopes, such as the spike (S) protein in the SARS virus, the gp160 in HIV and HA in IVs [71, 72] . These glycoproteins can bind to one specific glycan structure which is known as the lectin or GBP. Other viral glycoproteins, such as HN in the Newcastle Disease Virus or NA in IVs, function as the exoglycosidase in the release of virus particles [73] .
The IVs have an innate capacity for high mutation rates because they are RNA viruses. Humans are under continuous attacks by newly emerging IVs which constantly undergo ''antigenic drift'' and ''antigenic shift''. Accumulation of substantial sequences and 3D coordinates of IV proteins have provided the ideal tools for the investigation of how mutations affect transfaunation, vaccine design and drug-resistance [74, 75] .
N-glycosylation not only influences the folding and secretion of glycoproteins such as HA and NA but also provides the same glycans (similar to the host's own glycans) to escape the host's immune system. As the key modification of biological significance in viral glycoproteins, we found the glycosites in 17 known HA subtypes and 10 known NA subtypes that have complex characteristics. In general, two highly conserved glycosites near the HA0 cleavage site and fusion peptide site may maintain the basic function of HA; these sites were seldom absent in the HA subtypes. More glycosites were identified in the global domain or in the connection of global and stalk domains, along with longterm and large-scale epidemics in several of the subtypes. The distributional regularity of the glycosites in the NA subtypes is also complex; two to four glycosites located in the stalk domain are highly conserved in various subtypes, and are affected by the deletion of the stalk domain. Another highly conserved glycosite was found at the tip of tetramer NA in all subtypes. Other glycosites were found to be mainly concentrated in the global domain, which surrounds the antigenic sites.
The HA or NA subtypes exhibited low similarities of amino acid sequences in all subtypes while maintaining identical structures, which revealed that the functions of various HA or NA subtypes remain conserved. Notably, some of the glycosites near the cysteines were found to also be conserved. The cysteines take the main role in stabilizing the tertiary structure; the conservation verifies that cysteines and N-glycans played an important role in the protein folding and quality control.
We have further investigated the H5N1 virus to elaborate the collaborative relationship of glycosites in HA and NA. Five highly conserved glycosites in HA had existed before the H5N1 virus first crossed species barriers and infected humans, as well as two additional glycosites, 181NVT and 170NST. Since 2003, the H5N1 virus has exhibited a rapid evolutionary dynamic. Under the selection pressure of different hosts and antigenic drift, the glycosite pattern of current H5N1 viruses in different geographical locations has been distinctive: the HA in clade 2.2.1.1, isolated from Egypt, lacked the 181N glycosite but had added the 88NVS glycosite. In addition, the HA in clade 2.3.2.1, isolated from China or Vietnam, lacked the 170N glycosite but had added the 152N glycosite. In contrast, as the glycosites in HA became more diverse in the H5N1 virus, the glycosylation in NA was impaired by a decreasing number of glycosites. The NAs of current H5N1 viruses lack a stalk domain and the four corresponding glycosites; except the four high conserved glycosites in the global domain, others glycosites have rarely been identified.
The envelope glycoproteins, which play a crucial role in virus recognition, invasion and spread. The analysis of the glycosites in HAs and NAs has provided basic information for vaccine design, host selection and changing virulence. However, infection is a complex process; the alternation of glycosites and glycan shapes may affect the functions of glycoproteins. In addition, there are other mutations also worthy of further consideration, such as the E627K in the PB2 protein that enhances the avian viral replication capacity in mammalian cells [76, 77] . Drug resistance is also related to viral proteins, such as the M2 protein [78] . Thus, to prevent the next influenza pandemics, more research needs to be done. Figure S1 The N-J trees of H5N1 HA. The phylogenetic tree was inferred from protein sequences by the Neighbor-Joining method and rooted using A/chicken/Scotland/1959. Estimates of the statistical significance of the phylogenies were calculated by performing 1,000 bootstrap replicates. The clades classified by the WHO are shown as colored bars. (TIF) Figure S2 The superposition of crystal structures from various HA and NA subtypes. The red, green, blue and yellow color represent the H1, H3, H5 and H7 in HAs or N1, N2, N8 and N9 in NA respectively. File S5 The analytic alignment file containing the 5973 protein sequences of H3 HA. As is shown in the output file, the red, green and blue color represent the levels of conservation of ''.95%'', ''5%,95%'' and '',5%'', respectively. (ZIP)
File S6 The N-J trees of H3 HA. The phylogenetic tree was inferred from H3 HA sequences by the N-J method and rooted using A/equine/Miami/1/1963(H3N8). Estimates of the statistical significance of the phylogenies were calculated by performing 1,000 bootstrap replicates. 633 representative H3 HA sequences were chosed in consideration of the age, hosts, areas, as well as the known HA sequences from H3N1 and H3N3,H3N7 viruses. Those unconserved glycosites are labeled (conservative rate ,95%).  Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals. 1 Supporting Text S1 infection (relative to control)  A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476–1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573–586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. dation by the proteasome due to the paucity of lysine residues in its structure. It then forms an allosterically activated complex by binding to an ADP ribosylation factor (ARF), and it ADP ribosylates the alpha subunit of the stimulatory G protein, leading to constitutive activation of adenylate cyclase. In the human intestinal cell line T84, an increased concentration of cyclic AMP elicits a Clsecretory response which can be measured electrophysiologically in real time as a change in short circuit current, I sc (11) .
A key step in the intoxication process is the transport of the toxin from the cell surface to the ER. How GM 1 confers the specificity for lipid trafficking has not yet been determined. Pentameric binding of GM 1 by the B subunit of CT may itself induce membrane curvature and induce invagination to begin the entry process, as is seen with simian virus 40 (SV40) (12) and Shiga toxin (13) (14) (15) . Interestingly, invasion by SV40 occurs only with GM 1 that has native long-chain acyl groups. Clustering of GM 1 may enable the toxin to associate with lipid rafts that serve as platforms for trafficking of CT through the retrograde pathway or parts of it. Lipid rafts are viewed as highly dynamic microdomains that may self-assemble in membranes from sphingomyelin, cholesterol, glycolipids, and proteins that favor a lipid-ordered microenvironment (reviewed in references 16 to 18). Some functions attributed to lipid rafts may require interactions with protein components or scaffolds to stabilize them, extend their lifetimes, or facilitate their coalescence into larger physiologically significant structures. By binding to and cross-linking five GM 1 molecules, CTB might serve as such a protein scaffold and promote the function of lipid rafts in toxin trafficking. Conversion of PM sphingomyelin to ceramide or acute depletion of membrane cholesterol both prevent endocytosis of CT (19, 20) , consistent with a role for lipid rafts in CT trafficking. A requirement for the lipid raftassociated proteins flotillin 1 and flotillin 2 in a zebra fish model of intoxication by cholera toxin (21) also supports the key role of lipid rafts in trafficking of CT.
Our previous study (22) showed that a mixture of CT holotoxins produced in vivo and having chimeric B pentamers with from zero to two wild-type (wt) GM 1 binding sites (BS) was still capable of intoxicating host cells. Because genetic methods, not chemical modifications, were used to prepare these toxin variants, the structures of their GM 1 BS were fully defined and were known to be either the same as wt sites or inactive. Thus, having at most 2 wt GM 1 BS was sufficient for CT to intoxicate host cells, albeit at reduced efficiency. Left unanswered, however, was the question of whether a holotoxin with only one binding site for the GM 1 receptor can function. A toxin molecule able to bind only a single GM 1 molecule would be completely unable to cluster GM 1 molecules or scaffold them into microdomains and would therefore be unable to induce membrane curvature (2, 23) . We designed the experiments reported here to produce defined holotoxin variants that have from zero to five native GM 1 BS in their B pentamers, and we compared their abilities to bind to GM 1 and intoxicate T84 cells. We found that holotoxins able to interact with only a single GM 1 molecule can nevertheless still complete the intoxication process, demonstrating directly that binding a single GM 1 molecule permits the toxin to enter the host cell, complete the trafficking process, and deliver the toxic CT-A1 fragment to the cytoplasm. However, we also found that eliminating even a single binding site from wt holotoxin produced a detectable attenuation in toxicity.
Production and purification of cholera toxin variants with 0, 1, 2, 3, 4, or 5 native GM 1 binding sites. To produce CT holotoxins with defined combinations of wt and mutant CTB subunits, we made three compatible plasmid constructs encoding either tagged wt CTB, CTB-G33D, or wt CTA and expressed them in the same Escherichia coli strain to produce a holotoxin pool with B pentamers consisting of tagged wt CTB and/or CTB-G33D, from which to purify all six possible holotoxin variants. The tag is a 34-aminoacid peptide (denoted GSH6) which is genetically appended after the Met103 codon of native ctxB and encodes glycosylation (bold) and sulfation sites (underlined) (24) , SSSGGGGSSH-PNNTSNNTSSAEDYEYPS, followed by six His residues. Each plasmid has a different replication origin, antibiotic selection, and combination of promoters ( Fig. 1A ; Table 1 ). The ctxA gene, encoding CTA, is expressed from dual pLac and pBAD promoters. Holotoxins and free B pentamers were purified from whole-cell lysates by metal-ion affinity chromatography on Talon resin, and free B pentamers were removed by passing the eluate pool over cation exchange resin, resulting in binding of the free CTB pentamers to the resin and recovery of holotoxin in the flowthrough (Fig. 1B) . Holotoxins with different numbers of BS were then separated by anion-exchange chromatography (Fig. 1C) . By varying the amount of each inducer, we sought to alter the ratio of wt and mutant B subunits in the holotoxin pool. However, for practical purposes, we found that a single condition of relatively low levels of arabinose (0.0005% to express GSH6-tagged wt-CTB) and high levels of isopropyl-␤-d-thiogalactopyranoside (IPTG) (400 M to express native-size CTB-G33D) gave acceptable yields of holotoxin with homopentameric and singly or doubly tagged heteropentameric B subunits (with zero, one, and two wt BS, respectively) and detectable but lower yields of the triple, quadruple, and homopentameric tagged B subunits as described in detail in the next paragraph. This production strain we designated AMBT (for A, mutant CTB, wt CTB-tagged). Simply by swapping the wt and G33D alleles in the respective CTB-encoding vectors and keeping the same inducer ratios, we were able to express the single or double tagged heteropentameric and homopentameric wt CTB subunits with three, four, and five wt BS, respectively, from the production strain designated ABMT (A, wt CTB, mutant CTBtagged). Using these strategies, from two production strains, we produced all six variant holotoxins, which had from zero to five wt BS and a maximum of two tagged subunits. A third production strain, designated ABBT (A, wt CTB, wt CTB-tagged), in which both the GSH6-tagged and untagged B polypeptides were wt CTB, was used under the same expression conditions to produce control holotoxins with five native BS and zero, one, or two tagged CTB subunits. Figure 1 shows the purification process for the AMBT strain (mutant CTB-G33D, wt CTB tagged). Since CTB pentamers naturally bind to metal affinity resins (25) , cholera toxin variants can be purified to near-homogeneity by a single passage over Talon resin. The imidazole eluate pool from the Talon resin contained a mixture of holotoxin and some free CTB pentamers. These free pentamers were removed by cation exchange chromatography, where at pH 8.0 in 50 mM Tris buffer, holotoxin passed through the column while free pentamers bound and could be eluted subsequently with a salt gradient (Fig. 1B) . The heterogenous mixture of holotoxins was further separated into its components by anion-exchange chromatography. The GSH6 tag not only changed the size of the B monomer but also changed its predicted pI from 8.24 for native CTB to 6.21 for CTB-GSH6. The respective predicted pIs for the CTB-G33D variant changed from 7.31 for the native length to 5.91 for the GSH6-tagged variant. All holotoxins bound to the anion exchange matrix, and at least five peaks could be discerned following elution with a salt gradient (Fig. 1C) . Samples from individual fractions were analyzed by SDS-PAGE either without (Fig. 1D , upper) or after (Fig. 1D , lower) boiling in sample loading buffer. The unboiled samples in lanes 4 to 13 showed a single band corresponding to assembled holotoxin (AB 5 ), with a small amount of contaminating protein of slower mobility in lanes 2 and 3. The major fractions from each peak are homogenous and are consistent with each peak containing a single species CTB-G33D-Linker-His 6 , pAra BAD , Cm r , Ori p15a This study of variant holotoxin, The assembled holotoxins in the unboiled samples bind less SDS than if they were fully denatured and therefore do not migrate in direct proportion to their molecular weights. The relative molecular weight (M r ) for each holotoxin variant is increased by the presence of one or more tagged CTB subunits. Each of these holotoxins completely disassembles into its component polypeptides after boiling (Fig. 1D , lower panel; CTA, 29 kDa; CTB-GSH6-tagged monomer, 14.4 kDa; and native CTB-G33D monomer, 11.5 kDa). The holotoxin in peak 0 ( Fig. 1C ) has homopentameric CTB-G33D subunits and no functional GM 1 BS, while holotoxins in peaks 1 and 2 have a mixture of wt CTB-GSH6 and CTB-G33D pentamers with 1 and 2 GM 1 BS, respectively. The fractions loaded in lanes 2 to 5 contain CTA plus 5 CTB-G33D (peak 0), and those in lanes 6 to 9 contain CTA plus 1 wt CTB-GSH6 and 4 CTB-G33D (peak 1), and those in lanes 10 to 13 contain CTA plus 2 wt CTB-GSH6 and 3 CTB-G33D (peak 2). Only these first three peaks were well enough separated to be purified efficiently from the extract of production strain AMBT. The fractions corresponding to each peak were pooled, dialyzed, and rerun over the same anion-exchange column to achieve a very high degree of purity. To make holotoxins with 3 and 4 native GM 1 BS, an expression and purification run was done using strain ABMT, which produced three peaks similar to those shown in Fig. 1C , containing holotoxins with zero, one, or two GSH6tagged CTB-G33D subunits and five, four, or three native GM 1 BS, respectively. Finally, as noted above, strain ABBT was used for expression and purification of wt control holotoxins, all with 5 GM 1 BS and zero, one, or two GSH6-tagged CTB subunits. Analysis of the eight final highly purified holotoxin preparations by SDS-PAGE and Coomassie blue staining is shown in Fig. 2 . The upper panel shows a gel run with unboiled samples, and the lower panel shows a gel run with samples boiled in sample buffer to dissociate the components. Without boiling, holotoxins with untagged or tagged wt CTB pentamers (Fig. 2 , upper panel, lanes 2 to 4) dissociated into free CTA and stable pentameric B subunits that ran near the 45-kDa marker (lane 1). Interestingly, the presence of one or more CTB-G33D polypeptides increased stability of the heterohexameric AB 5 holotoxins. Consequently, unboiled holotoxins with three or more CTB-G33D polypeptides (Fig. 2 , upper panel, lanes 5 to 7) remained fully associated and ran near the 66-kDa marker (lane 1), whereas unboiled holotoxins with one or two CTB-G33D subunits exhibited partial dissociation into B pentamer and free CTA (Fig. 2 , upper panel, lanes 8 and 9). The high stability of the heterohexameric AB 5 holotoxins containing five, four, or three CTB-G33D polypeptides is also evident in the upper panel of Fig. 1D . As expected, increasing the number of tagged CTB polypeptides resulted in moderate decreases in observed mobilities of the pentamer bands (Fig. 2 , upper panel, compare lanes 2, 3, and 4) and the holotoxin bands (Fig. 2 , upper panel, compare lanes 5, 6, and 7), and increasing the number of CTB-G33D polypeptides resulted in slight increases in mobilities of the holotoxin bands (Fig. 2 , upper panel, compare lanes 6 to 8 and lanes 7 to 9). Upon boiling, all holotoxins dissociated and resolved into their individual polypeptide components (Fig. 2, lower panel) . All holotoxins have one CTA subunit. Holotoxins in lanes 2 and 5 are predicted to have five native CTB monomers; holotoxins in lanes 3, 6, and 8 have are predicted to have one tagged CTB and 4 untagged CTB monomers; and holotoxins in lanes 4, 7, and 9 are predicted to have two tagged and three untagged CTB monomers.
Stoichiometry of the individual polypeptides in each purified holotoxin variant was confirmed experimentally by densitometric scanning of the gel in the lower panel of Fig. 2 . To adjust for differences in loading of the purified holotoxins (ranging from 0.6 g in lane 6 to 2.9 g in lane 2), the observed density for each band was expressed as a fraction of the total density for all bands in the same lane. The observed fractional densities were then compared with the expected values based on the predicted molecular mass of each polypeptide (CTA, 27.2 kDa; wt CTB, 11.6 kDa; and tagged CTB, 15.3 kDa) and the assumption that binding of Coomassie blue is proportional to the mass of each peptide. The results (see Table S1 in the supplemental material) were generally within 15% of expected values, but all observed values for tagged B subunits were higher than expected, suggesting that the tagged subunits bound proportionately more Coomassie blue stain than native CTA or CTB. Corrected for loading differences, the expected stoichiometric ratios for the tagged B subunit bands in lanes 3 versus 4, 6 versus 7, and 8 versus 9 were 1:2, and the corresponding observed ratios were 1:2.1, 1:1.7, and 1:1.9, respectively.
Ganglioside GM 1 -binding activities of cholera toxin variants. Relative binding activity of each holotoxin preparation with from zero to five binding sites (and with zero, one, or two tagged B subunits) to ganglioside GM 1 receptor was measured by ELISA with a fixed amount of toxin (5 ng, 60 fmol) added to individual wells coated previously with serial dilutions of GM 1 , and bound toxin was detected with polyclonal rabbit anti-CTB and horserad- ish peroxidase (HRP)-conjugated secondary antibody. Figure 3 shows that at high GM 1 density, toxins with more than one native GM 1 BS bound almost as well as native cholera toxin. The single-BS holotoxin showed a lower plateau signal at high GM 1 density than holotoxins with more than one BS, which we interpret to be due to a less favorable equilibrium between binding and release for the holotoxin with one BS for GM 1 . With more than one BS, a toxin molecule is expected to exhibit faster initial binding to immobilized GM 1 , multivalent binding as the density of immobilized GM 1 increases, and slower dissociation from GM 1 , resulting in increased avidity of binding. In wells coated with 75 nM GM 1 (50 l, 3.75 pmol), toxins with 2 or more native GM 1 BS bound at more than 90% of the wt level, toxin with a single BS bound significantly less at 60% of the wt level, and toxin with no BS gave a minimal signal (4% or less of the wt level). At lower GM 1 densities, there were significant differences between all variants, and in wells coated with 1.2 nM GM 1 (50 l, 60 fmol), toxins with four or three BS bound at 54 and 49% of wt levels, respectively, and toxins with 2, 1, or no BS bound at 25, 12, and 1% of wt levels, respectively. The amount of GM 1 required to coat each well and give 50% of the maximal signal was calculated to be 65, 100, 150, 240, and 850 fmol per well for wt holotoxin and for holotoxin variants with 4, 3, 2, or 1 wt BS, respectively.
Biological activities of the eight holotoxin preparations (the untagged and singly and doubly tagged native holotoxin controls and the variant holotoxins with zero to four wt BS and one or two tags) were initially tested in an overnight assay on mouse Y1 adrenal cell monolayers, on which cholera toxin causes an easily scorable morphological change (rounding of intoxicated cells). In this assay, approximately 2 ng of unnicked native cholera toxin caused rounding of 75 to 100% of the cells (48 U/100 ng). The G33D holotoxin showed some rounding at only the highest concentration tested (33 ng, extrapolating to less than 1 U/100 ng). All holotoxin variants with one or more BS showed rounding of Y1 cells at between 24 and 48 U/100 ng, showing that a single GM 1 binding site is sufficient to intoxicate mouse Y1 cells at near-wt levels. Failure to detect significantly less toxicity after overnight exposure of mouse Y1 adrenal cells to holotoxin variants with from 1 to 4 GM 1 binding sites suggests that any differences in delivery of the wt CT-A1 fragment from the cell surface to the cytosol by these holotoxin variants were not rate limiting for development of the morphological manifestations of intoxication.
To assess the biological consequences of decreased numbers of BS in a more quantitative manner, we determined the real-time electrophysiological effects of the holotoxin variants on polarized human intestinal cells (T84 cell line) by measuring the short circuit current required to eliminate the potential difference induced by the cAMP-dependent Clsecretory response resulting from CT-A1-mediated ADP ribosylation of Gs␣ and constitutive activation of adenylate cyclase. Figure 4A to 4F shows the results of these experiments. Panel A shows that loss of even a single GM 1 BS attenuated the activity of cholera toxin to some degree, an effect which increased as the number of native GM 1 BS was lost. The time of onset of intoxication was also delayed as the number of native GM 1 BS decreased. Nevertheless, toxin with a single native GM 1 BS had clearly detectable activity. As expected, the G33D holotoxin with no native GM 1 BS had almost no activity over the time frame of the experiment: 1.5% of wt toxin signal over baseline at 90 min and less than 9% of wt signal at 120 min. Since these variants have differing numbers of C-terminal GSH6 tags (zero, one, or two), we also examined the effect that the number of tags had on the activity of wt holotoxin with five BS (Fig. 4B) . The presence of the tags modestly affected both the time of onset of intoxication, a measure of toxin-GM 1 trafficking from the PM to the ER of host cells, and the rate of increase of I sc . The effect was greater for doubly tagged holotoxin, although it eventually showed a similar maximal I sc . To control for these effects of the B-subunit tags, toxins with different numbers of native GM 1 BS but the same number of tags were compared. For these analyses, data were normalized by setting the maximal signal for the five native GM 1 BS toxins in each comparison to 1.00 (Fig. 4C to F) . For the toxin variants with one tag on the B subunit, the holotoxin with four native GM 1 BS exhibited a slight (25%) attenuation of toxicity relative to holotoxin with five native GM 1 BS, seen as a decrease in maximal I sc but with no delay in onset of intoxication (Fig. 4C) . A similar result (30% decrease of maximal I sc ) was seen for comparisons of the holotoxin variants with three or five native GM 1 BS and two CTB-GSH6-tagged subunits (Fig. 4D) . When the number of native GM 1 BS was reduced to two or one in doubly or singly wt B-subunit-tagged holotoxin variants, the peak I sc decreased by more than 50% and also the onset of intoxication was delayed ( Fig. 4E and 4F) , suggesting defects in entry of CT into the cell or transport to the ER (or both).
As an initial step toward investigating whether CT variants with five and one native GM 1 BS trafficked from the cell surface to the ER by the same pathway, we examined the effects of brefeldin A (BFA) on their toxicity for T84 cell monolayers. We found that BFA completely inhibited the I sc (Fig. 5) induced by both single and five native GM 1 BS toxin variants. BFA-treated cells, however, still responded to addition of the cAMP agonist vasoactive intestinal polypeptide (VIP) at 90 min, showing that the toxin-treated cells retained viability and were competent for cAMP-dependent Clsecretion (I sc ). Thus, a toxin variant with a single native GM 1 BS, like native CT holotoxin, traffics through a BFA-sensitive pathway to exert its toxic effects.
Previous studies have shown that holotoxins with reduced numbers of GM 1 BS are still able to intoxicate host cells, albeit with attenuated activity. The initial studies (26, 27) used chemical modification and denaturation-renaturation to generate mixed populations of holotoxins predicted to have one or two (nonnative but active) GM 1 BS. In our previous study (22) , we used genetic methods to create populations of chimeric holotoxins with only one or two completely native GM 1 BS and showed that these chimeras were still capable of intoxicating host cells but with attenuated activity.
More recently, we studied how a membrane lipid might specify trafficking of toxin in the retrograde pathway by using fluorophore-labeled GM 1 and imaging toxin trafficking in live cells (28) . We found that a subset of GM 1 species, those with unsaturated ceramide domains, sorted efficiently from the PM to the trans-Golgi network and the ER. Cross-linking by toxin binding was dispensable for such GM 1 trafficking, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. Our results implicated an endogenous proteindependent mechanism of lipid sorting that is dependent on cer- Maximum signal for the tagged wt holotoxin in panels C through F was adjusted to 1.00, and all data points in the panel were normalized to that value. Data points show the mean response Ϯ SE; n ϭ 2 to 4 for A; n ϭ 3 to 4 for B; n ϭ 2 for C to F; each study was reproduced at least once. amide structure and could explain how the toxin gains access to the ER of host cells to induce disease (28) .
This work shows that multivalent binding to GM 1 is indeed dispensable for CT toxicity. To do this, we prepared and purified homogenous preparations of holotoxins with defined numbers of GM 1 BS. Monomeric binding of CT to single molecules of GM 1 permits the holotoxin to intoxicate the host cell. Our results confirm and expand our studies on trafficking of the single GM 1 lipids. Conversely, loss of even a single GM 1 BS resulted in a measurable diminishment of toxicity in the T84 line of polarized human intestinal cells. This result is also consistent with findings of our studies of trafficking of the non-cross-linked GM 1 molecules, where we found that GM 1 cross-linking by toxin binding enhanced entry of CT into the ER. Thus, although multivalent binding to GM 1 by CT is fundamentally dispensable, it does have a significant effect on retrograde trafficking from the cell surface to the ER and on toxicity.
One way that multivalent binding could affect CT function would be by enhancing the binding avidity for cell membranes containing GM 1 . In this and in our previous studies of the CT binding site mutants, we observe a loss of avidity for binding GM 1 when the GM 1 binding pockets are mutated. In the current studies, the loss of apparent avidity for the one-binding-site species is 10-fold, but the toxin still binds to GM 1 applied to the well at nanomolar concentrations, suggesting this may not fully explain the strong loss of toxin function.
It is possible that the attenuated toxicity seen with holotoxins with reduced numbers of native GM 1 BS could be enhanced by the presence of one or more CTB-G33D polypeptides that confer increased stability to the holotoxins (and thus might decrease the delivery of the CT-A1 subunit to the cytosol). We raise this idea because we observed that insertion of even a single CTB-G33D monomer into the pentameric B subunit rendered the holotoxin partially resistant to dissociation by SDS, and increasing the number of CTB-G33D monomers resulted in holotoxins that were completely resistant to dissociation by SDS. Conversely, there is no a priori reason to assume that the presence of one or more CTB-G33D polypeptides would affect chaperone-mediated release of CT-A1 from the nicked and reduced holotoxin in the ER, which appears to facilitate CT-A1 retrotranslocation from the ER to the cytosol (29) . Further experiments will be required to test these possibilities.
Another way that multivalent binding to GM 1 might affect CT function would be through reorganization of membrane structure and function induced by scaffolding GM 1 into ceramide-based nanodomains, as suggested by studies in vitro and in vivo (14, 15, (30) (31) (32) (33) . Significantly, studies of the closely related AB 5 subunit Shiga toxin show that multivalent binding to the glycosphingolipid GB3 spontaneously induces high-curvature membrane tubules by coupling the toxin-lipid complex to membrane shape (31) . Multivalent binding of CT to GM 1 in model membranes also induces spontaneous membrane curvature, implying a similar coupling of the toxin-glycosphingolipid complex formation to membrane shape (32, 34, 35) . This could allow partitioning of the CT-GM 1 complex into highly curved sorting tubules of the sorting endosome, which is required for retrograde transport, and explain why the toxins with more than 1 GM 1 binding site are more potent in intoxication. It is also possible that multivalent binding to GM 1 may be needed to activate intracellular signaling pathways that enhance uptake and trafficking, as for Shiga toxin (36) .
Effects either on binding avidity or on membrane structure and trafficking dynamics could underlie how the CTB subunit, and the other AB 5 toxins, evolved as pentameric structures. This and our other recent studies, however, show that the most fundamental function exploited by CT is to coopt an endogenous glycosphingolipid sorting pathway from the PM to the ER that is essential for toxin entry into the cytosol and the induction of disease.
Bacterial strains and construction of expression clones. All chimeric toxins in this study were produced by expression from recombinant plasmids in Escherichia coli BW27784 (araFGH pCP18-araE [37] ). This strain does not metabolize arabinose and constitutively expresses the arabinose transporter, and therefore a uniform degree of induction occurs in all cells of a culture at any arabinose concentration. To produce the expression strains, genes encoding native-length CTB or carboxy-terminally extended CTB tagged with a glycosylation-sulfation signal (24) followed by a hexahistidine peptide were cloned on compatible plasmids with different selectable markers and inducible promoters. These encoded resistance to ampicillin (Ap) with the IPTG-inducible lacUV5 promoter or resistance to chloramphenicol (Cm) and the arabinose-inducible araBAD promoter, along with the regulator araC. Variants of these CTB clones were also made with G33D substitutions that eliminate GM 1 binding. A separate plasmid (pSlacbadCTA) selectable with spectinomycin (Sp) and compatible (with a pSC101 origin) with both ctxB plasmids was used to independently express the ctxA gene under control of both the lac and araBAD promoters. Each expression strain thus contained three plasmids, encoding a native CTB subunit (wt or G33D variant, Ap, and lac promoter), a GSH6-tagged CTB subunit (G33D or wt, Cm, and ara promoter), and the wt CTA subunit (Sp and ara and lac promoters). Details of construction, including full DNA sequences, are available on request. Plasmids created or used in this study are listed in Table 1 . Plasmids pAlacCTB and pAlacCTB G33D encode Ap resistance and the wt and G33D native-length variants of CTB, respectively, under control of the lac promoter and have previously been published as pLMP1 and pLMP148, respectively (38) . Carboxy-terminally linker-hexahistidine-tagged variants of CTB and CTB-G33D were made in a compatible Cm r arabinoseinducible vector, pAR3 (39), using the hexa-His tag from pT7sh6 (40) and a glycine-rich repeat from the M13 phage PIII protein, encoding XX (EX) 4 XDPRVPSS (where X is GGGS) inserted between residues 102 and 103 of CTB. Initial experiments to make wt and CTB-G33D mixed pentamers were done using these plasmids, but we saw significant proteolysis of the polyglycine linker-tagged variants, and subsequent experiments were done with variants that had the linker replaced with the GSH6 tag-pCbadCTBGSH6 and pCbadCTB G33D GSH6.
Toxin expression and purification. A 400-ml LB culture at 30°C was inoculated with a 1/8 volume of an overnight culture with appropriate antibiotic selection (Sp and Ap at 100 g/ml and Cm at 25 g/ml) and grown to an A 600 of 1.2, when it was induced with 400 M IPTG and 0.0005% l-arabinose with incubation continued overnight. Cells were collected by centrifugation, resuspended in 20 ml phosphate-buffered saline (PBS), and lysed by mixing for 20 min at room temperature with 0.5 mg/ml lysozyme and Elugent detergent (EMD Biosciences, Inc., La Jolla, CA) to 2%. Viscosity was reduced by sonication four times (10 s each) on ice, and the lysate was cleared by centrifugation at 15,000 rpm for 20 min in an SS34 rotor. Toxin was purified from the supernatants by Talon chromatography as detailed by the manufacturer (Clontech Laboratories, Inc., Mountain View, CA). Imidazole eluates were dialyzed against 50 mM Tris-HCl (pH 8.0) (buffer A). All chromatographic separations were conducted on an Akta purifier (GE Healthcare Biosciences, Pittsburgh, PA) in 4.6-mm by 100-mm Poros perfusion chromatography columns packed with Poros HS 20 cation exchange medium or Poros HQ 20 anion exchange medium. For cation exchange, the column was equilibrated with 5 column volumes (CV) buffer A and washed with 5 CV buffer A after sample loading. Bound material was eluted with 10 CV of a linear gradient of 0 to 100% buffer B (50 mM Tris-HCl, pH 8.0, 1M NaCl). For anion exchange, the column was equilibrated with 5 CV buffer A, washed with 5 CV of buffer A after sample loading, and was eluted with a 40-CV (initial separation) or 10-CV (second purification) linear gradient of 0 to 100% buffer B. Anion exchange fractions were pooled and concentrated using Microcon Ultracel YM-10 filter devices (EMD Millipore, Billerica, MA).
Ganglioside GM 1 ELISA. Ninety-six-well microtiter plates were coated overnight with 50 l of 2-fold serial dilutions of ganglioside GM 1 (Supelco, Sigma-Aldrich, St. Louis, MO) in PBS, starting at 150 nM, and then blocked with 10% horse serum in PBS. All steps were performed at 37°C for 1 h, after which samples were aspirated off and wells were washed three times with PBS plus 0.05% Tween 20. Each holotoxin was assayed in triplicate by incubating 100 l of 50-ng/ml holotoxin per well (approximately 60 fmol), followed by 1/5,000 rabbit anti-cholera toxin B subunit (B10) and then 1/2,000 HRP-conjugated goat anti-rabbit serum (Thermo Fisher Scientific Inc., Rockford, IL), followed by o-phenylenediamine (OPD) substrate (Sigma-Aldrich), and the reaction was stopped with 3 M HCl. Absorbance was read at 450 nm. Y1 assay. Mouse Y1 adrenal cells (ATCC CCL-79) were grown at 37°C in a humidified 5% CO 2 atmosphere in RPMI medium with 5% fetal bovine serum with 1ϫ penicillin-streptomycin (Life Technologies), and 96-well plates were seeded with 10 4 cells per well. One-hundredmicroliter volumes of 2-fold serial dilutions of toxins were added to semiconfluent monolayers and incubated overnight, followed by scoring for toxin-induced rounding. One unit of toxin was defined as the amount contained in the last dilution giving 75 to 100% rounding of cells.
T84 cells and electrophysiology. Measurements of short-circuit current (I sc ) on monolayers (0.33-cm 2 inserts) of the polarized human intestinal cell line T84 were performed as previously described (41) . A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles BACKGROUND: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. METHODOLOGY/FINDINGS: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. CONCLUSIONS: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses. H5N1 influenza virus is highly pathogenic in poultry, certain bird populations, and has occasionally infected human causing severe clinical outcomes [1] [2] [3] . Since the first human outbreak in 1997, there have been more than 600 confirmed human cases of H5N1 infection with a mortality rate of approximately 60% [4] . To initiate an infection, like all other subtypes of influenza viruses, H5N1 virus first binds to cell surface glycan receptors via its surface glycoprotein hemagglutinin (HA) and is subsequently internalized via endocytic pathways [5] [6] [7] . HA is a homotrimeric type I transmembrane glycoprotein, which can be cleaved into HA1 and HA2 subunits [8] . A furin-dependent polybasic cleavage site has been shown to be characteristic of highly pathogenic avian influenza viruses [9, 10] , although not all H5-HAs contain the polybasic cleavage site. In cells infected by influenza virus, HA protein is first synthesized as a precursor (HA0), which is then oligomerized, glycosylated and ultimately transported to the plasma membrane where assembly and budding of progeny virions takes place [11] . Then, during the final stage of the virus life cycle, neuraminidase (NA), the second major envelope glycoprotein of influenza, cleaves the terminal sialic acids from the cell surface glycans to allow release of the virus from the host cell [12] .
Following the first report of H5N1 outbreak in 1996, the virus has evolved into different clades as shown by the phylogenetic analysis of H5-HA protein sequences [13] [14] [15] . Currently the spread of H5N1 virus in human population is limited. However, through mutation and reassortment, the virus may become more easily transmissible from bird to human or from human to human, posing a potential pandemic threat to public health worldwide [2, 3] . It is therefore important to fully understand the biology of H5N1 viruses and to develop sensitive and rapid diagnostic methods. However, an obstacle to the study of H5N1 viruses is the stringent safety requirement to work with them. Recently, we and other research groups have developed retroviral particles pseudotyped with H5-HA (H5pp) as an alternative strategy for large scale serological studies [16] [17] [18] [19] [20] [21] . Similar to the replication-competent virus, H5pp entry requires alpha-2,3 sialic acids, is pH-dependent, and can be neutralized by sera containing anti-H5N1 antibodies [18] , thus validating H5pp as very useful and safe tool for a wide range of applications, including entry mechanism studies, serodiagnosis and drug discovery [16, 18] .
In our previous work, we have produced H5pp using the H5-HA of A/Cambodia/40808/2005 (H5Cam), which was isolated from a patient with a lethal infection of H5N1 virus [18] . In the current study, we have analyzed the ability of H5-HAs from different clades of avian influenza virus to pseudotype lentiviral particles and have found that they do not give rise to the same level of efficient H5pp production when compared with H5Cam. In particular, we have carried out a detailed comparison of the expression and cleavage of two H5-HAs, i.e., H5-HA of A/Anhui/ 1/2005 (H5Anh) and H5Cam, and of their ability to pseudotype lentiviral vector in HEK293T cells. Through several independent lines of evidence we have identified the molecular determinants in H5-HA for efficient incorporation into H5pp envelope and have delineated the underlying mechanism. Our results are discussed in the context of the understanding of human host adaptation of avian influenza H5N1 viruses.
The ability of H5-HA to pseudotype lentiviral particles does not correlate with HA protein expression level in producer cells Similar to HA of other subtypes of influenza viruses, H5-HA is highly mutable as a result of antibody-selection pressure, leading to the rise of divergent H5N1 viruses that are categorized into various strains and clades [13, 14, 22] . To ascertain the flexibility and adaptability of H5pp production as an alternative approach for serological studies in the event of novel emerging H5N1 viruses, we sought to develop cladespecific H5pp and compared the ability of three other H5N1 viruses belonging to different clades to pseudotype lentiviral particles. H5-HA from clade 1 (H5Cam), clade 2.1 (H5Ind), clade 2.2 (H5Qin) or clade 2.3 (H5Anh) (see Table 1 ) was expressed in 293T cells together with lentiviral backbone plasmid to allow the production of H5-pseudotyped lentiviral particles (H5pp). Expression levels of H5-HAs in transfected 293T cells was monitored by Western blot using anti-FLAG antibody directed against the C-terminal tag (Fig. 1A , upper panel). Supernatants containing H5pp were harvested 48 hr post-transfection, and used to transduce MDCK cells for luciferase reporter activity assay (Fig. 1A , lower panel), as described in Materials and Methods. Unexpectedly, we observed significant differences in the transduction of MDCK cells by H5pp, depending on the clades of H5-HAs. In particular, H5Anh from A/Anhui/2005/01 resulted in very low luminescence levels after particle transduction in MDCK cells; whereas H5Cam from A/Cambodia/40808/2005 was the most efficient, inducing a consistent 3-4 log increase in luciferase activity compared with H5Anh ( Fig. 1A -B, lower panels). Analysis of cell lysates by Western blots, however, demonstrated that all H5-HAs tested were well expressed in the producer cells and, consequently, that luciferase reporter activity in MDCK target cells did not correlate with the level of HA protein expression in the cells (Fig. 1A) . Two main protein bands were detected, consistent with the expected electrophoretic mobility of the uncleaved protein (HA0) and the C-terminal portion of the cleaved form (HA2 subunit), whereas the N-terminal fragment (HA1 subunit) could not be recognized by the anti-FLAG antibody due to C-terminal tagging (Fig. 1A) . We next decided to compare in detail the behaviour of H5Cam and H5Anh. To determine whether the difference in luciferase reporter activity was due to the level of H5pp production, culture supernatants containing H5Cam-pp and H5Anh-pp were concentrated by ultracentrifugation, and the resulting H5pp pellets were analyzed by Western blotting. Our results showed that the number of particles produced in the culture supernatant was significantly less for H5Anh than for H5Cam in presence of soluble bacterial neuraminidase, as indicated by lower levels of p24 in concentrated supernatants to detect the lentiviral core and lower luciferase reporter activities in MDCK cells (Fig. 1B) . More importantly, incorporation of H5Anh into the pseudotyped lentiviral particles was not observed using anti-FLAG antibody (Fig. 1B, upper panel) . Altogether, these data suggest that H5Anh cannot be efficiently incorporated into pseudotyped particles and released into the cell culture supernatant.
Swapping of HA2 domain (including the polybasic cleavage site) does not increase production of H5Anh-pp Sequence analysis of the polybasic cleavage site reveals that H5Anh has a deletion of a lysine residue when compared to H5Cam and moreover, there is an additional amino acid difference in the HA2 region at position 533, which is located at the border between the ecto-domain and the transmembrane domain (TMD) ( Fig. 2A) . Thus, H5Cam has an isoleucine at position 533 (I533), while H5Anh has a threonine (T533). Cleavage of HA into HA1 and HA2 subunits by host protease is a critical step for influenza viruses to gain membrane fusion capability [23, 24] ; whereas the TMD of HA is important for its association with lipid rafts at the plasma membrane [25] . To test the potential influence of these differences in the cleavage site and at position 533, we generated several chimerical constructs in which either the entire HA2 region including the cleavage site was replaced with that of H5Cam (AnhCam1), or only the cleavage site (AnhCam2) or a single T533I amino acid change was introduced (AnhCam3). All constructs were FLAG-tagged at the C-terminal end of H5 sequences as described in the Materials and Methods section ( Fig. 2A) . When transfected into 293T cells, all three mutant H5Anh proteins were well expressed in the producer cell lysates (Fig. 2B) ; however, analysis of transduction levels of MDCK target cells by H5pp produced with these H5AnhCam chimerical proteins suggests that none of them was able to increase the production of pseudotyped particles (Fig. 2C) . These data indicate that differences in the HA2 domain cannot account for the reduced ability of H5Anh to form pseudotyped particles.
Two amino acid substitutions in the 130-loop of the receptor binding domain (RBD) are sufficient to confer H5Anh pseudotyping ability Sequence alignment of H5-HA proteins revealed a striking amino acid divergence at 9 positions over a short stretch of only 33-amino-acid-long region around the 130-loop of RBD (Table 1) , which accounted for over 30% of the amino acid differences found in the entire HA molecule (576 amino acids in length). Therefore this region was chosen for site-directed mutagenesis to generate a series of H5Anh mutants that were subsequently tested for their ability to pseudotype lentiviral vectors. The level of protein expression for all H5Anh mutants in producer cells was comparable, albeit slightly lower for AnhM1 and AnhM6 (Fig. 3A) . Interestingly, all H5Anh mutants that harbored residues alanine-valine at positions 133-134 (AnhM1-5, Table 1 ) displayed a largely restored ability of H5Anh to produce pseudotyped particles, despite other sequence differences at the 130-loop flanking region (Fig. 3B ). By contrast, AnhM6, which contains H5Cam-like 130-loop flanking sequences but serine-alanine at positions 133-134, did not generate efficiently H5Anh-pp in culture supernatant (Fig. 3B) . These data clearly demonstrate that amino acid residues at positions 133-134 are crucial for efficient H5pp production. More specifically, substitution of the two amino acids S133-A134 of H5Anh with A133-V134, which are unique to H5Cam sequence, confers H5Anh the ability to be incorporated into the lipid envelope of lentiviral particles and is essential for efficient production of transduction-competent H5Anh-pp.
A single A134V mutation in the 130-loop of the RBD of H5-HA is the critical determinant for H5pp production To further delineate the respective roles of A133 and V134 for efficient pseudotyping, two additional H5Anh mutants were generated with a single amino acid substitution either at position 133 (S133A, AnhM7) or 134 (A134V, AnhM8) ( Table 1) . These experiments revealed that the A134V mutation was sufficient to confer H5Anh the ability to be incorporated into transductioncompetent pseudo-particles; whereas the S133A mutation was not (Fig. 4A) . We also generated reciprocal mutants of H5Cam that contained either the two residues found at positions 133-134 of H5Anh (viz., S133-A134; CamM1), or only one single V134A change (CamM2), or A133S substitution (CamM3) ( Table 1) . Again, the presence of valine at position 134 was found to be crucial for efficient H5pp production, whereas the A133S substitution had only a marginal effect, consistent with the results obtained with H5Anh mutants (Fig. 4A ). To confirm that the effect of valine at position 134 was indeed on the production of H5pp, we analyzed by Western blot both cellular lysates and culture supernatants containing H5pp that were concentrated by ultracentrifugation (Fig. 4B ). This series of experiments showed that incorporation of the ''Anhui-like'' single mutant CamM2 into the pseudotyped lentiviral particles was below the antibody detection limit, as also seen for wild-type H5Anh; whereas the ''Cambodialike'' single mutant AnhM8 induced H5pp production with an efficiency similar to H5Cam ( Fig. 4A-B) . Altogether, these experiments demonstrate that valine at position 134 (V134) is a critical residue for efficient H5pp production.
The A134V mutation affects cell surface expression level of HA Because influenza virus, as well as particles pseudotyped with HA, buds from the plasma membrane [11, 18] , we reasoned that changes in surface expression of H5-HA could have an impact on the production of H5pp. Thus, we have compared by flow cytometry plasma membrane expression levels of HA protein in cells transfected with H5Anh and H5Cam. Cells were labelled with an anti-H5N1 antibody, fixed and then stained with a PEconjugated secondary antibody. As assessed by measuring mean fluorescence intensity (MFI), cell surface expression of H5Anh was significantly less compared to H5Cam ( Fig. 4C ; p,0.01). Interestingly, introduction of the A134V mutation into H5Anh (AnhM8) increased its cell surface expression ( Fig. 4C ; p,0.02), and conversely, a V134A mutation in H5Cam (CamM2) reduced transport to the plasma membrane to a level that was not significantly different from that measured with H5Anh (Fig. 4C ). These data demonstrate that the Ala to Val substitution at position 134 enhances surface expression of H5-HA.
As residue 134 is in the 130-loop of the receptor binding site, we next investigated the impact of A134V mutation on receptor binding properties. We employed a cell-based assay using soluble H5-HA proteins that were engineered by removing TMD and Ctail of HA (Fig. 5A ) as described in Material and Methods. Stable cell lines were generated to express sH5Anh, sH5Cam and their reciprocal single amino acid mutant forms (sH5AnhM8 with A134V and sH5CamM2 bearing V134A; see also Table 1) , and soluble HA proteins were affinity-purified as described under Material and Methods. When analyzed on native gels, purified soluble H5-HA proteins contained mostly the homotrimeric form ( Fig. 5A ) that can bind to the sialic acid-containing cellular receptors. We observed that sH5Anh bound strongly to MDCK cells, whereas the A134V mutation reduced the binding to a much lower level (Fig. 5B ). By contrast, sH5Cam bound weakly to MDCK cells and, as predicted, the single V134A change induced a major increase in the binding of sH5Cam to MDCK cells (Fig. 5B) .
The binding assay was also performed in MDCK-SIAT-1 cells which express two-fold higher amounts of alpha-2,6-link sialic acids than parental MDCK cells [26] . The results obtained were similar to that in parental MDCK cells ( Fig. 5C-D) . When cells were treated with bacterial neuraminidase NAvb before fixation with PFA, the binding of sHA proteins was diminished to background level in both MDCK and MDCK-SIAT-1 cells, indicating that the binding of sHA proteins is sialic acid dependent ( Fig. 5C-D) .
Because we had previously found that H5pp with HIVbackbone bud from the plasma membrane in 293T cells [18] , a reduced cell surface expression of viral envelope proteins (see Fig. 4C ) would be expected to influence the formation of pseudotyped particles and, hence, could account for the observed differences in pseudotyping. It has been reported that retroviruses including HIV and Murine Leukemia Virus (MLV) can also bud from intracellular compartments [27, 28] , depending on the cell type and Gag expression systems. Therefore, we also used MLVbased pseudotyping system to compare the efficiency of H5pp production between H5Anh and H5Cam. As demonstrated in Fig. 6A , the results obtained with the MLV-backbone were similar to those with HIV-backbone, thus, indicating that inefficiency of H5Anh-pp production is not a mere consequence of the lentiviral system used for pseudotyping. However, co-transfection of the viral NA from A/Cambodia/JP52a/2005, rescued the inefficiency of H5Anh-pp production (Fig. 6A ).
To further test whether reduced binding to sialic acid receptors, as a result of A134V mutation, is a major contributing factor for pseudotyping efficiency of H5-HA, we examined the production of H5Cam-pp and H5Anh-pp in Lec2 cells which are sialylationdeficient mutants of CHO cells [29] . As H5Cam binds weakly to sialic acid receptors (Fig. 5) , NAvb added exogenously post transfection was sufficient to release H5Cam-pp into culture supernatant in CHO cells; and the level of H5Cam-pp in CHO cells was not significantly different from that in Lec2 cells (Fig. 6B ). Similar to the results obtained in 293T cells, production of H5Anh-pp was lower than H5Cam-pp in CHO cells. By contrast, H5Anh-pp production in Lec2 cells was significantly increased in comparison to that in parental CHO cells and the level of H5Anhpp obtained in Lec2 cells was similar to that of H5Cam-pp, as indicated by the values of luciferase activity detected in MDCK cells 72 hr post H5pp transduction (Fig. 6B) . Together, these findings further suggest that binding of H5-HA to cellular sialic acid containing glycans is a major determinant of H5-HA incorporation into pseudo-particles.
H5N1 viruses carrying the A134V mutation exhibit reduced capability to agglutinate horse red blood cells
The reduced binding to sialic acid receptors as a result of the A134V mutation not only leads to changes in pseudotyping efficiency, but is also found to have an impact at the whole virus level. Reverse genetics generated RG-A/Cambodia/408008/2005 with the A134V mutation has been shown to agglutinate to the same degree both human red blood cells (RBCs), which express alpha-2,6-sialic acid, and guinea pig RBCs, which exhibit both alpha-2,3 and alpha2,6-linked sialic acid, but failed to agglutinate horse RBCs, which carry only alpha-2,3-sialic acid [30] . These observations provide an experimental evidence to support the notion that the A134V mutation leads to a reduced alpha-2,3sialic acid binding of the virus. To confirm the effect of A134V mutation on H5N1 viruses, we performed similar hemagglutination assays using another H5N1 virus strain A/Cambodia/ V0401301/2011, which also contains the same A134V mutation. 
In previous studies, we have reported the generation of H5pp and have characterized it as a safe alternative to the use of replicative H5N1 virus for sero-surveillance [16, 18] . Because H5pp mimics the entry mechanism of the avian virus while carrying only the H5-HA as envelope protein, it offers the advantage to be specifically neutralized only by anti-hemagglutinin antibodies, avoiding the confounding effect of antibodies directed against N1 neuraminidase due to infection of influenza virus subtypes other than H5N1. We report here that the efficiency to generate HA-only H5pp varies with HAs derived from different H5N1 virus clades, regardless of the lentiviral backbone used. Through serial mutagenesis of two H5-HAs, we have uncovered that differences in receptor binding ability, due to mutations in the receptor-binding domain of HA, may be the underlying mechanism.
It is widely believed that HA is targeted to lipid rafts at the plasma membrane and the transmembrane domain has been described to be important for lipid rafts association of HA [25] . Therefore, we first swapped the transmembrane regions between H5Anh and H5Cam. We also noticed that the cleavage of H5Cam appears to be more efficient (Fig. 1A, 2B) . Thus, mutants with or without sequence variations found at the poly-basic cleavage site (AnhCam1, AnhCam2 and AnhCam3) were generated and analysed. However, none of these H5Anh mutants showed appreciable improvement in their ability to generate H5pp, when compared with wild type H5Anh. In fact, the production of H1 and trypsin-dependent H5 pseudo-particles has been reported [31, 32] , hence indicating that HA cleavage is not a determining factor for pseudotyping efficiency. Then by multiple sequence alignment, we identified a small region around the 130-loop of the receptor binding site of HA which appeared to be a ''hot-spot'', harboring several sequence variations among different H5N1 clades. Through a series of mutagenesis studies, we have found that one single residue at position 134 is a critical switch to dictate the ability of H5 HA to pseudotype lentiviral vectors for the production of H5pp. Similar to influenza virus, H5pp generated with an HIVbackbone bud at the plasma membrane [18] ; therefore the simplest explanation is that the mutation at position 134 may result in a change in cell surface expression of HA. Indeed we have observed a small but consistent change in cell surface HA expression due to mutations at position 134 (Fig. 4C) . To exclude the possibility that this finding merely reflected a differential binding to the two HA of the rabbit anti-H5 polyclonal serum (described in Material and Methods), we used another polyclonal serum from a different source (a duck anti-H5 serum described in Ref. 18 ) and found that the results of cell surface HA staining were similar (data not shown). The fact that the A134V mutation increased cell surface expression of H5Anh, may partially explain the effect of this amino acid substitution on H5pp production. Considering that the variation between H5Cam-pp and H5Anhpp production resulted in a 3 to 4 log difference in luciferase activity, it is likely that A134V mutation may have an impact on other properties of H5-HA, including binding to sialic acid receptors, which contribute to the observed phenotype. It has been reported in the case of H3-HA pseudotyping that lentiviral particles which incorporate sialic acid binding-incompetent H3-HA (derived from A/Aichi/2/68) can be efficiently generated and released into culture supernatant in the absence of exogenous bacterial NA; whereas the wild-type Aichi-HA fails to do so [33] . Although the difference in pseudotyping observed with wild-type Aichi-HA and its receptor binding-incompetent mutant is diminished when bacterial NA is added, the study by Bosch et al. [33] implies that changes in receptor binding properties can affect pseudotyping efficiency of lentiviral vectors by influenza HA.
Regarding the potential influence of mutations at position 134 of H5-HA on receptor binding properties, there have been reports with contradictory results. First, Yamada et al. [34] found that A134T mutation did not change alpha-2,3 sialic acid binding preference of H5-HA. Then, Auewarakul et al. [35] reported that L129V/A134V allowed for dual binding to both alpha-2,3 and alpha-2,6-sialic acid receptors, although in their study, the effect of A134V mutation alone was not assessed. More recently, using virus elution assay, Imai and colleagues [36] found that H5N1 viruses containing alanine at position 134 (A134) show stronger binding than those harbouring threonine (T134) to both chicken erythrocytes (expressing both alpha-2,3 and alpha-2,6-sialic acid) and horse erythrocytes (expressing only alpha 2,3-sialic acid). Similar to the observation by Imai et al., we found in the current study that H5Anh which contains A134 displayed a strong binding to both MDCK and MDCK-SIAT-1 cells (expressing an increased level of alpha-2,6 and a decreased level of alpha-2,3-sialic acid than parental MDCK) [26] . As predicted by these observations, the A134V mutation reduced H5Anh binding to a dramatically lower level in both cell lines. It is likely that strong binding of H5Anh to cell surface sialic acid receptors makes it difficult to release H5pp from the producer cells even in the presence of exogenous bacterial NA; and the A134V mutation reduces binding, thus allowing for the release of H5pp. In keeping with this hypothesis, co-transfection with the viral NA gene from H5N1 led to the production of similar amounts of mixed HA-NA pseudoparticles for both H5Anh and H5Cam. Moreover, we did not observe an increase in binding to MDCK-SIAT-1 cells, which contain more alpha-2,6-sialic acids on the cell surface. In fact both sH5Anh and sH5CamM2 bind with slightly lower efficiency to MDCK-SIAT-1, compared with parental MDCK cells, suggesting that A134V mutation probably leads to a decreased binding of H5-HA to alpha-2,3-sialic acid rather than a switch to alpha-2,6sialic acid binding. Consistent with this notion, we observed an increased level of H5Anh-pp production in Lec2 sialylationdeficient cells, when compared with parental CHO cells (Fig. 6B) .
We have found that the A134V mutation not only exerts a critical influence in the determination of pseudotyping efficiency, but has also an impact on H5N1 viruses. Both A/Cambodia/ 408008/2005 and A/Cambodia/V0401301/2011, two different H5N1 isolates carrying the same A134V mutation could agglutinate human and guinea pig RBCs but failed to agglutinate horse RBCs [30] (also Figure 7 of this paper); whereas two other strains of H5N1 viruses without the A134V mutation could also agglutinate horse RBCs (Figure 7) . These observations indicate that A134V mutation in H5-HA reduces virus binding to alpha-2,3-sialic acid. Although co-transfection with viral NA enables efficient lentiviral pseudotyping by H5Anh (Fig. 6A) , the differential RBC binding properties observed at the whole virus level, when both HA and NA are present, support the idea that A134V mutation in H5-HA can be biologically relevant. Interestingly, alanine at position 134 (A134) is highly conserved in avian H5N1 viruses and so far A134V mutation has only been found in human isolates of H5N1 viruses, both clade 1 and clade 2 viruses isolated from 2004 to 2011. Almost all avian H5N1 isolates possess A134 in the HA. So far only one avian H5N1 virus in the NCBI database has serine instead at position 134 of the HA protein. Notably, more diversity is observed at this position for human isolates of H5N1 viruses: three H5N1 viruses isolated from human patients have a threonine and eleven a valine at position 134 [36] . At least in two cases (A/Cam/408008/2005 and A/ Thailand/676/2005), viruses found in the original patient specimens were mixtures of both wild type, containing A134 in the HA, and mutant virus, containing V134 [30, 35] . It is possible that other human isolates of H5N1 viruses may actually contain the A134V mutation but failed to be detected in the process of either virus isolation or traditional capillary sequencing of viral genomes. Thousands of H5-HA sequences are available in the NCBI Influenza Database (http://www.ncbi.nlm.nih.gov/ genomes/FLU/FLU.html) from non-human isolates of H5N1 viruses, none of which contains this particular mutation. Altogether, these observations suggest that a valine at residue 134 of the receptor-binding domain is unlikely to be a random sequence variation but may be selected as the avian H5N1 viruses adapt for replication in human hosts. We speculate in general terms that changes in cell surface receptor binding of H5-HA, as a result of A134V mutation, may lead to changes in virus entry and virus release and, therefore, be considered an important factor for determination of host range. It is not clear whether intracellular sialic acid content and distribution may also influence this feature. As our data focus on the pseudotyping system, further studies are required to understand more precisely the biological consequences of A134V mutation and its potential influence on the adaptation of H5N1 viruses in humans.
Our findings have also implications for the applicability of H5pp assay in serological surveys. H5pp has several advantages over the microneutralization method, which is the current gold standard serological assay for the detection of antibodies against avian influenza viruses [37, 38] . Pseudotyped particles are produced from synthetic genes without the need to have access to the virus and can be safely used in BSL-2 containment, making them ideal for widespread use, especially in areas where BSL-3 facilities are not available. Moreover, it has been reported that the H5pp assay is more sensitive than micro-neutralization [16, 39] . It appears, however, that its use in sero-epidemiological studies and to monitor the efficacy of candidate vaccines may be limited by the strain under investigation. Although we and others have found that production of mixed HA-NA pseudo-particles is consistently successful using N1 from either H1N1 or H5N1 [17, 20, 21] (also Fig. 6A of this paper) , the production of HA-only pseudotypes would be necessary to eliminate potential cross-reactivity that may be displayed by circulating anti-NA antibodies against N1 from avian H5N1 or seasonal influenza H1N1 [40] . We are cognizant that a single A134V mutation may result in a change of antigenicity but this limitation is not different from that of the microneutralization assay, which is the gold-standard to detect anti-H5 neutralization antibodies and utilizes an available H5N1 virus strain that may not be a perfect match of the viruses associated with the serum samples being tested. In fact, serologic surveys often use a collection of serum samples from human or animals without necessarily knowing the exact virus strain(s) involved. Moreover, if positive, samples shall contain polyclonal antibodies against multiple epitopes to an H5N1 virus, further minimizing the likelihood that a single amino acid substitution would compromise the usefulness of a pseudotype-based serological assay as a safer alternative to the microneutralization test. Careful assessment of the H5pp-based assay should obviously be performed when new strains emerge.
In conclusion, by comparing the ability of different H5-HA to produce pseudotyped particles, we have demonstrated that when a single A134V mutation is introduced in the receptor binding site, the ability of the usually inefficient H5Anh to generate H5pp is largely restored. It is likely that the A134V mutation leads to an increased level of cell surface HA expression and reduced binding to sialic acid receptors, both of which contribute to the production of H5pp. The A134V mutation has been reported as a naturally occurring mutation in human host; and importantly, this mutation is so far only found in human isolates of H5N1 viruses. Our data with hemagglutination assays further demonstrate that viral isolates from human cases with avian influenza carrying the A134V substitution exhibit a reduced binding to alpha-2,3 linked sialic acids. Therefore, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses. It is possible that other mutations leading to reduction in receptor binding may exist and cause a change in pseudotyping efficiency. Thus, H5pp production together with soluble HA protein cell binding analysis may serve as convenient functional assays to screen for mutations with potential consequences on receptor binding properties and host adaptations of H5N1 viruses. Although zoonotic transmission from poultry to humans remains inefficient for H5N1, it may be of importance to monitor closely mutations in regions of the receptor binding site of H5-HA.
293T, MDCK, CHO and Lec2 cell lines were obtained from ATCC (Manassas, VA, USA). MDCK-SIAT-1 cells were generated by stable transfection of human alpha-2,6-sialyltransferase in MDCK cells and was described elsewhere [26] . This cell line overexpresses alpha-2,6-linked sialic acid compared to parental MDCK [26] . 293T, MDCK and CHO cells were cultured at 37uC with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillinstreptomycin. MDCK-SIAT-1 cells were grown in DMEM containing 10% FBS and 1 mg/ml G418. Lec2 cells, which lack terminal sialic acid in their glycoproteins and gangliosides due to a defect in the CMP-sialic acid transporter [29] , were cultured in Minimum Essential Medium (MEM-alpha, Invitrogen) supplemented with 10% FBS and 40 ug/ml L-proline.
H5Cam (HQ664938, see also ref. 30 ), H5Anh (ABD28180), H5Ind (ABP51969), H5Qin (ABE68923)), mutants AnhM1-M6, CamM1-M3 (Table 1 ) and N1 gene (ABO10176) from A/ Cambodia/JP52a/2005 were synthesized as human codon optimized genes (GENEARTH, Regansburg, Germany) and subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). Mutants AnhM7 and AnhM8 were generated by sitedirected mutagenesis using QuikChange site-directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions. To generate soluble H5-HA constructs, the transmembrane domain (TMD) of the HA was removed, and the polybasic cleavage site was changed into a monobasic cleavage site RESR by site-directed mutagenesis to avoid the potential influence of H5-HA cleavage in cells on the purification of sHA proteins, which involve multiple steps. The truncated HAs were then subcloned into pcDNA3.1 (Invitrogen). All H5 plasmids were tagged with the FLAG-epitope at the C-terminal and sequenced to confirm that they contain only the expected mutations as indicated in Table 1 .
The production of lentiviral particles pseudotyped with H5 hemagglutinin was performed as previously described [18] . Briefly, HEK293T cells were co-transfected with a plasmid containing the coding sequence of the indicated H5-HA and a lentiviral backbone plasmid pNL-Luc E2 R2 which carries a modified proviral genome of HIV with env deleted and is engineered to express the firefly luciferase reporter. Alternatively, MLV-backbone plasmids (a kind gift from Dr. Michael Farzan, Harvard Medical School)described in [41] were used where indicated. To release particles into the culture medium, either soluble bacterial NA from Vibrio cholerae (NAvb; Roche, Mannheim, Germany) was added to the producer cells at a concentration of 6.25 mU/ml or co-transfection of N1 gene was used where indicated. Supernatants containing H5pp were harvested 48 hr post-transfection, filtered and used to transduce MDCK cells for luciferase reporter activity assay or concentrated by ultracentrifugation as indicated.
Luciferase reporter activity assay MDCK cells (4000 cells/well) were seeded in 96-well white assay plates one day before H5pp transduction. Luciferase reporter activity assay was performed 72 hr post transduction using Bright-Glow Luciferase substrate (Promega, Mandison, WI, USA) according to the manufacturer's instructions. Samples were measured using a Microbeta Luminometer (PerkinElmer, Waltham, MA, USA) and data were expressed as Relative Luminescence Units.
Equal amounts of protein from total cell lysates or equal volumes of H5pp concentrated by ultracentrifugation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto Hybond-P polyvinylidene difluoride (PVDF) membranes (Invitrogen) that were blocked with 5% milk for 30 min at room temperature. H5-HA was detected by incubation for 2 hr at room temperature with a mouse monoclonal anti-FLAG M2 antibody (Sigma, St.Louis, MO, USA; 1:1000 dilution) conjugated with horse radish peroxidase (HRP) (Sigma). The core protein in the pseudotyped particles was detected using an anti-p24 antibody (Abcam, Cambridge, UK) for 1 hr at room temperature at a 1:1000 dilution, followed by an additional 1 hr incubation with a goat-anti-mouse secondary antibody conjugated with HRP (ZymedH, Invitrogen) at a 1:5000 dilution. The levels of cyclophilin B (detected with a rabbit anti-cyclophilin B antibody from Abcam, 1:5000 dilution) or GAPDH (detected with a mouse anti-GAPDH antibody from Abcam, 1:10000 dilution) were measured on the same blots to verify that equal amount of samples had been transferred. Proteins were visualized by chemiluminescence using ECL Western blot detection reagents (Invitrogen). The relative electrophoretic mobility was estimated using NovexH Sharp Pre-stained Protein Standards (Invitrogen).
293T cells transfected with H5 HA were detached with and resuspended in PBS, blocked in 10% horse serum and then labelled with a polyclonal rabbit anti-H5N1 antibody (Sino Biologicals Inc., Beijing, China) at a 1:400 dilution for 1 hr at 4uC. Unbound antibodies were removed by washing three times with cold PBS, followed by staining with a phycoerythrin (PE)-conjugated, donkey-anti-goat secondary antibody (Jackson Immunoresearch Laboratories, Suffolk, UK) for 30 min at 4uC. Data were collected from at least 5000 cells on an LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and post-acquisition analyses of cell surface expression of H5-HA was performed using FlowJo software (TreeStar, Ashland, OR, USA).
Expression and purification of soluble H5-HA proteins HEK293T cells stably expressing soluble HA (sHA) proteins were generated by selection of transfected cells in culture medium containing 300 ug/ml hygromycin for at least 1 month. To purify sHA proteins, cells were grown in DMEM with 5% FBS until 90% confluence. Culture supernatant containing secreted sHA proteins was cleared by centrifugation at 4000 rpm for 15 minutes at 4uC, concentrated by Amicon Ultra-15 Centrifugal Filter Units with Ultracel-100 membrane (Millipore, Billerica, MA, USA; 100 kDa cut-off) and stored at 280uC until use. Soluble HA proteins were affinity purified from concentrated supernatant using anti-FLAG M2 affinity gel (Sigma). Because it has been shown that HA-bound sialic acid could interfere with the accessibility of the receptorbinding site to cellular receptors [42] , anti-FLAG M2 resins bound with sHA proteins were washed twice in cold PBS and subjected to treatment with NAvb (Roche, 62.5 mU/ml) at 37uC for 45 minutes to remove terminal sialic acid residues, followed by three washes in cold PBS. Bound sHA proteins were eluted with 150 ml FLAG peptide (International Laboratory, USA, 0.4 mg/ml in PBS) for four times. To remove the FLAG peptides, all eluates were pooled and concentrated using Amicon Ultra-0.5 mL with Ultracel-100 membrane (Millipore, 100 kDa cut-off). To examine the oligomeric state of sHAs, proteins were resolved on a discontinuous native PAGE (6% of acrylamide) followed by western blot detection using a HRP-conjugated anti-FLAG M2 antibody (Sigma).
Cell-based HA binding assay MDCK cells were grown in 96-well plate until complete confluence, then fixed with 4% paraformaldehyde (PFA, Sigma), washed three times in PBS and blocked for at least 2 hrs in 5% BSA. The indicated amount of purified soluble HA proteins, measured by the Bradford assay, were added to the wells in duplicates or triplicates and incubated overnight at 4uC. Cells were washed three times in PBS and then incubated with anti-FLAG antibody (Origene, Rockville, MD, USA; 1:1000 dilution for 2 hrs at room temperature) to detect sHA proteins bound to cell surface. After washing for three more times in PBS to remove unbound sHA proteins, cells were incubated with goat-anti-mouse secondary antibody conjugated with HRP (ZymedH, Invitrogen) at a 1:5000 dilution for 1 hr at room temperature. Unbound secondary antibody was removed by washing three times in PBS, and ABTS substrate (Invitrogen) was added to the plate according to the manufacturer's instructions. Forty minutes after the addition of substrate, absorbance at 415 nm (OD415) was measured using a Sunrise TM plate reader (Tecan, Mä nnedorf, Switzerland).
Cambodian H5N1 virus strains were isolated from human clinical specimens by inoculation in Madin-Darby canine kidney (MDCK) cells in the biosafety level 3 laboratory of the Institut Pasteur in Cambodia, according to conventional protocols [43] .
Hemagglutination titres were measured using 0.75% suspensions of human (type O), horse and guinea pig red blood cells, as previously described [30, 43] .
Results are presented as mean values 6 SD of the indicate number of observations. Statistical difference between groups was determined by the unpaired Students's t-test with a 0.05 significance level. Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation BACKGROUND: Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHODS: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca(2+) mobilization were also measured. RESULTS: PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca(2+)-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes. Ambient particulate matter (PM) poses a threat to national public health in urban environments and other polluted areas throughout the US and around the world. Epidemiological studies have shown associations of exposure to low levels of urban particulate matter with increased cardiopulmonary morbidity and mortality [1, 2] . The assessment of PM-induced health effects is challenging. Various mechanisms have been proposed to explain the cardiopulmonary health effects of PM including increased pulmonary and systemic oxidative stress and inflammation, enhanced coagulation, and altered cardiac autonomic function [3, 4] .
Airway epithelium represents a well-investigated target for environmental pollutants such as PM. Exposure of airway epithelium to airborne PM causes altered cytokine/chemokine gene expression and increased production of IL-1β, IL-6, IL-8 and TNF-α [5, 6] . Now, the lung endothelium is also gaining attention as a viable PM target tissue. Exposure of the endothelium to PM or its active components in the systemic circulation induces significant systemic endothelial inflammation and dysfunction, even at low levels of exposure [7, 8] . The water soluble fraction of PM (up to 35-50%) can easily diffuse through the epithelium/endothelial barrier to the systemic circulation. Bioavailable transition metals present in urban PM catalyze redox reactions in human lung endothelium, which cause oxidative stress, increase the production of inflammatory cytokines, and increase the activation of NF-κB signaling pathways, all of which trigger further endothelial damage [9] . Increased endothelial monolayer permeability is also observed in inflammatory pulmonary conditions such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis; devastating lung disorders with mortality exceeding 30%, as well as in more subacute and chronic inflammatory disorders such as asthma [10, 11] .
We recently described a murine asthma model with strong evidence for PM-mediated vascular barrier dysfunction with increased protein leakage into bronchoalveolar lavage (BAL), a marker of acute inflammatory lung damage [12] . By assessment of direct effects on endothelial barrier integrity in vitro, we further demonstrated that the vascular hyperpermeability mediated by intratracheal PM exposure is mainly dependent on acute endothelial barrier disruption by PM [13] . Exposure of human lung EC to PM resulted in significant ROS generation, which mediates p38beta MAPK activation, leading to the phosphorylation of HSP27 [13] . Phosphorylated HSP27 facilitates the synthesis of stress fibers and formation of paracellular gaps, which causes protein-containing fluid to leak from the microvessel lumen to the lung alveoli, leading to further pulmonary inflammation [13] . However, these previous findings did not explain the persistent character of PM-mediated endothelial barrier disruption.
To fully examine PM-mediated vascular hyperpermeability, we explored effector targets such as tight junction and adherens junction proteins, which are known to be critical in EC barrier maintenance. We demonstrated that PM exposure induced the relocation of tight junction protein Zona occludens-1 (ZO-1) from the cell periphery, which was accompanied by a significant reduction in the level of ZO-1 protein but not in the levels of adherens junction proteins (VE-cadherin and β-catenin). PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca 2+ -dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. These observations not only provide new information as to how PM disrupts endothelial tight junctions, but also represent the first evidence establishing the critical role of calpain signaling in modulating endothelial cell barrier function under oxidative stress. These results increase our understanding of PM-induced adverse cardiopulmonary outcomes. Moreover, as the newly characterized signaling cascade of ROS/TRPM2/Calpain/ ZO-1 likely has fundamental roles in regulating the cytoskeleton under oxidative stress, these novel observations may have broad applicability to vascular pathophysiology in a variety of cell types.
Molecular mass standards, polyacrylamide gels, and protein assay reagents were purchased from Bio-Rad (Hercules, CA 
Human lung microvascular ECs obtained from Lonza (Basel, Switzerland) were cultured as previously described [16] in EGMMV-2 complete medium (Lonza).
Endothelial cells were grown to confluence in polycarbonate plates containing evaporated gold microelectrodes, and TER measurements were continuously obtained using an electrical cell-substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY) as previously described in detail [17] .
Human microvascular lung ECs were transfected with siRNA using siPORT Amine (Ambion, Austin, TX) according to the manufacturer's protocol as we described previously [13] .
ECs plated on glass cover slips were loaded with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA) in 1 ml of basal medium as previously reported [18] . The cover slips with ECs were inserted diagonally into 1 cm acrylic cuvettes filled with 3 ml of basal medium at 37°C. Fura-2 fluorescence was measured with an Aminco-Bowman Series 2 luminescence spectrometer (SLM/Aminco, Urbana, IL) at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. The cover slips with ECs were then moved to 35 mm dishes, treated with PM suspension, and incubated for 15 minutes at 37°C in 95% O 2 and 5% CO 2 . After every 15-minute incubation, the coverslips were withdrawn from the dish and inserted back into the acrylic cuvettes for Fura-2 fluorescence measurement, then returned to the dish with the resuspended PM preparation for another incubation period (15-60 min).
Male A/J mice (10-12 weeks of age; Jackson Laboratories, Bar Harbor, ME) were housed in an environmentally controlled animal facility at the University of Illinois at Chicago (UIC) for the duration of the experiments. All animal procedures follow the guideline of the UIC Animal Care and Use Committee. PM (10 mg/kg, in 50 μl of saline) was delivered via intratracheal aspiration 1 hr after NAC or calpeptin treatment, as previously described [12, 15] . Animals were sacrificed 24 hr after PM treatment, and bronchoalveolar lavage (BAL) and lung tissue were collected [12, 19] . Total BAL cells were counted with a hemocytometer. The BAL fluid was used for protein and cytokine measurement (Bio-Rad, Hercules, CA) according to the user's manual.
Data are presented as group means ± SEM. We performed statistical comparisons among treatment groups by randomized-design two-way analysis of variance followed by the Newman-Keuls post hoc test for more than two groups, or by an unpaired Student's t-test for two groups. In all cases, we defined statistical significance as p < 0.05.
We assessed human lung microvascular EC barrier function as measured by transendothelial electrical resistance (TER), a highly sensitive measurement of permeability. PM challenge (10-100 μg/ml) induced doseand time-dependent reduction in TER (Additional file 1: Figure S1 ) or increases in FITC-dextran leakage through EC monolayer (Additional file 1: Figure S2 ) [20] , indicating a loss of EC barrier integrity. At the same time, PM induced a time-dependent (1-6 hr) reduction of the levels of tight junction proteins ZO-1 and ZO-2, but did not affect levels of adhesion junction proteins VE-cadherin or β-catenin ( Figure 1A ). No obvious cytotoxicity in the ECs was found after PM challenge (100 μg/ml, 0-16 hr) with MTT assay [13] or LDH release assay (Additional file 1: Figure S3 ).
PM (100 μg/ml) induced substantial time-dependent ROS production in microvascular ECs as measured by DCFDA oxidation, which peaked around 30-60 min (Additional file 1: Figure S4 ). EC pretreatment with N-acetyl-cysteine (NAC, 5 mM, 1 hr), an ROS scavenger, or PEG-catalase (PEG-CAT, 250 U/ml, 1 hr), which degrades H 2 O 2 , prevented PM-induced DCFDA oxidation by ROS (Additional file 1: Figure S4 ). PM produced a sustained timedependent decrease in TER, with a maximal effect observed at 100 μg/ml (80% decrease in TER), which is similar to its effects on other endothelial cell types, as we have reported previously [13] . NAC pretreatment (5 mM, 1 hr pretreatment) prevented PM-induced ZO-1 degradation ( Figure 1B ), while NAC (5 mM, 1-24 hr) does not change ZO-1 protein levels by itself (Additional file 1: Figure S5 ). After ECs were treated with PM (100 μg/ml, 6 hr), ZO-1 was relocated from the cell periphery and degraded, which was followed by gap formation between ECs. VE-cadherin, on the other hand, underwent no relocation or degradation ( Figure 1C ). NAC pretreatment (5 mM, 1 hr pretreatment) prevented PM-induced ZO-1 relocation and gap formation ( Figure 1C ). These data strongly indicate that PM causes EC barrier disruption selectively via oxidative tight junction protein degradation. NAC (5 mM) or PEG-CAT (250 U/ml) pretreatment significantly inhibited PM-induced EC barrier disruption as measured by TER ( Figure 1D ). We also examined the effects of another ROS scavenger EUK-134 on PMchallenged ECs. As NAC, EUK (5 μM, 1 hr pretreatment) attenuated PM-induced ZO-1 degradation and TER reduction (Additional file 1: Figure S6 ). These results further confirmed that PM induces ROS-dependent EC barrier disruption.
We previously demonstrated that high levels of ROS in endothelial cells activate calpain, a calcium-dependent protease [21] . We therefore investigated the role of calpain in PM-mediated ZO-1 degradation. PM (100 μM, 1 hr) induced a significant increase in calpain activity in ECs, which was inhibited by selective calpain inhibitors ALLN (30 μM, 1 hr pretreatment) and calpeptin (10 μM, 1 hr pretreatment) (Figure 2A ). PM-induced calpain activation was also inhibited by BAPTA-AM (50 μM, 1 hr pretreatment), a calcium chelator, and by NAC (5 mM, 1 hr pretreatment) (Figure 2A ). We next determined the role of calpain in PM-induced EC barrier disruption. PM-induced reduction in TER was partially inhibited by calpeptin or ALLN ( Figure 2B ). In parallel, ALLN and calpeptin also significantly prevented PM-induced ZO-1 degradation, as did chelation of intracellular calcium via BAPTA-AM ( Figure 2C ).
Addition of PM (100 μg/ml) to human lung microvascular ECs produced slow time-dependent Ca 2+ influx ( Figure 3A ; Ca 2+ influx is represented by an increase in the 340/380 ratio of Fura-2 AM.). Notably, this finding of increased intracellular Ca 2+ is in accordance with our previous finding of increased activated calpain. Under oxidative stress, a key member of the transient receptor potential (TRP) cation channel, member M2 (TRPM2), is activated and causes slow calcium influx [22] . We therefore examined the role of activated TRPM2 in PM-stimulated calcium influx. Anti-TRPM2 blocking antibody (5 μg/ml, 4 hr pretreatment) significantly prevented the PM-induced Ca 2+ transients compared to ECs treated with control IgG ( Figure 3B ). Reductions in TRPM2 protein expression (by siRNA) also inhibited the PM-induced Ca 2+ influx compared to ECs treated with control siRNA ( Figure 3C ).
We next investigated whether TRPM2 activation was ROS-dependent. Depletion of PM-induced ROS by NAC (5 mM, 1 hr pretreatment) significantly prevented PMmediated calcium influx ( Figure 3D ). Under oxidative stress, Poly ADP ribose polymerase (PARP) generates ADPribose [23, 24] , which activates TRPM2 by binding to its carboxyl terminus. The PARP inhibitor 3-aminobenzamide (3-AB, 1 mmol/L, 1 hr pretreatment)significantly reduced PM-induced Ca 2+ influx ( Figure 3E ), which further confirmed that TRPM2 is activated by PM via ROS and PARP.
We next investigated the role of TRPM2 activation in EC barrier function and ZO-1 degradation. TRPM2 neutralizing antibody (5 μg/ml, 4 hr pretreatment) significantly prevented ZO-1 degradation induced by PM ( Figure 4A ). TRPM2 siRNA (100 ng/ml), which downregulated TRPM2 protein level ( Figure 4B ), also inhibited PM-induced ZO-1 degradation ( Figure 4C ). In parallel, antagonizing TRPM2 by either TRPM2 antibody (5 μg/ml, 4 hr pretreatment) or TRPM2 siRNA (100 ng/ml) significantly prevented PM-induced EC barrier disruption ( Figure 4D-E) , as indicated by TER measurements.
A PM-mediated murine model of pulmonary inflammation has been well established [12] . We investigated the role of ROS and calpain in PM-induced pulmonary inflammation by examining protein leakage, white blood cell infiltration (inflammatory leukocytes), and the release of proinflammatory cytokines into BAL fluids ( Figure 5 ). Pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) 1 hr before PM challenge (10 mg/kg, 24 hr) significantly attenuated PM-induced protein leakage into BAL fluids (~50% reduction, Figure 5A ). PM challenge (10 mg/kg, 24 hr) resulted in an increase in inflammatory leukocytes, e.g. neutrophils and eosinophils [12, 15] , in BAL fluids ( Figure 5B ). Pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) attenuated PMinduced inflammatory leukocyte infiltration. Furthermore, NAC (150 mg/kg) or calpeptin (1 mg/kg) attenuated the release of PM-induced proinflammatory cytokines IL-6 and THF-α into BAL (~50% inhibition, Figure 5C-D) . These results suggest that ROS scavenging by NAC or Figure 2 Activated calpain is required for PM-mediated ZO-1 degradation and EC barrier disruption. (A) Human lung microvascular ECs grown in 60-mm dishes to approximately 95% confluence were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), NAC (5 mM), or BAPTA-AM (50 μM) for 1 hr, and then challenged with PM (100 μg/ml) for 1 hr. Cell lysates were subjected to calpain activity assay by using the calpain activity kit (Calbiochem). Calpain activity was normalized to protein concentration and expressed as fold changed compared to control. *p < 0.05 compared to control. **p < 0.05 compared to PM only group. (B) ECs grown on ECIS gold electrodes were treated with ALLN (30 μM), calpeptin (CALP, 10 μM) for 1 hr, and then challenged with PM (100 μg/ml). Changes in TER were measured with ECIS. *p < 0.05 compared to PM only group. (C) ECs grown on 6-well plates were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), or BAPTA-AM (50 μM) for 1 hr, and challenged with PM (100 μg/ml) for 1-6 hr. Cell lysates were analyzed by Western blotting with antibody to ZO-1. Changes in levels of ZO-1 are expressed as fold changes and normalized to β-actin. Shown are representative blots from three independent experiments. *p < 0.05 compared to PM-1 hr group. **p < 0.05 compared to PM-6 hr group. calpain activity inhibition by calpeptin leads to multiple protective effects including enhancement of lung endothelial barrier function, reduction of inflammatory cell infiltration, and prevention of proinflammatory cytokine release in the lungs of PM-challenged mice. We next examined tight junction ZO-1 levels in the PM-exposed lung. PM challenge (10 mg/kg, 24 hr) induced a significant reduction of ZO-1 protein levels in the murine lung, while pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) 1 hr before PM challenge attenuated PMinduced ZO-1 loss from lung tissues. Taken together, these data suggest a crucial role for the ROS-calpain-ZO-1 signaling pathway in the regulation of EC barrier disregulation in PM-mediated pulmonary inflammation.
To further confirm the critical role of ZO-1 degradation in EC barrier disruption in vitro and pulmonary inflammation in vivo, we next examined the beneficial effects of over-expressing ZO-1 protein in endothelial cells in vitro and in vivo. ZO-1 over-expression ( Figure 6A ) significantly (but not completely) attenuated PM-induced EC barrier disruption ( Figure 6B ). These facts demonstrate that endothelial ZO-1 loss contributes to PM-mediated EC barrier disruption. We next over-expressed ZO-1 in vivo by a liposome delivery system labeled with ACE antibody, which successfully over-expressed ZO-1 in Human lung microvascular ECs were plated on glass cover slips and loaded with 5 μM Fura-2 AM in 1 ml of basic medium. ECs were rinsed twice and treated with PM (100 μg/ml). Fura-2 fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm (0-60 min). Fura-2 has excitation wavelengths of 380 nm in its free form and 340 nm when it is bound to calcium; therefore, relative calcium concentrations are represented by the ratio of 340/ 380. (B) ECs plated on glass cover slips were treated with TRPM2 antibody or control IgG (5 μg/ml, 4 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). *p < 0.05 compared to control IgG group at the same time point. (C) ECs were transfected with TRPM2 siRNA or control siRNA for 48 hrs. ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). EC cell lysates were analyzed by Western blot with antibodies to TRPM2 and β-actin to confirm silencing. *p < 0.05 compared to control siRNA group at the same time point. (D-E) ECs plated on glass cover slips (95% confluent) were treated with NAC (5 mM, 1 hr pretreatment) or 3-AB (1 mM, 1 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0-60 min). *p < 0.05 compared to PM-only group at the same time point. murine lung tissues ( Figure 6C ). ZO-1 over-expression significantly attenuated BAL protein leakage ( Figure 6D) , BAL white blood cell infiltration ( Figure 6E) , and the release of proinflammatory cytokine IL-6 into BAL ( Figure 6F ), indicating the crucial role of ZO-1 loss in mediating PM-induced pulmonary inflammation and lung vascular hyperpermeability.
The most significant finding of the present study is the novel characterization of a ROS-dependent pathway that causes calpain-dependent endothelial ZO-1 degradation in response to PM. These data represent the first evidence that calpain signaling, via calcium leakage from activated TRPM2 by ROS, plays a critical role in modulating endothelial cell barrier function, resulting in tight junction protein ZO-1 degradation (Additional file 1 Figure S7 ). The consequence of ZO-1 degradation is sustained endothelial hyperpermeability and persistent lung inflammation, both of which contribute to variety of acute or chronic cardiovascular disorders [25, 26] . These effects were also observed with other types of PM samples (1648a from Human lung microvascular ECs grown in 6-well dishes to approximately 95% confluence were treated with TRPM2 antibody or control IgG (5 μg/ml) for 4 hr, and then challenged with PM (100 μg/ml) for 6 hr. Cell lysates were analyzed by Western blotting with ZO-1 antibody. Changes in levels of ZO-1 are expressed as fold changes and normalized to β-actin. Shown are representative blots from three independent experiments. *p < 0.05 compared to control. **p < 0.05 compared to PM challenge. (B) ECs grown in 6-well dishes to approximately 80% confluence were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and cell lysates were analyzed by Western blotting with ZO-1 antibody. (C) ECs grown in 6-well dishes to approximately 95% confluence were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and then challenged with PM (100 μg/ml) for 6 hr. Cell lysates were analyzed by Western blotting with ZO-1 antibody. Changes in levels of ZO-1 are expressed as fold changes and normalized to β-actin. Shown are representative blots from three independent experiments. *p < 0.05 compared to control. **p < 0.05 compared to PM challenge. (D) ECs grown on ECIS gold electrodes were treated with TRPM2 antibody or control IgG (5 μg/ml) for 4 hr, and then challenged with PM (100 μg/ml). Changes in TER were measured with ECIS. *p < 0.05 compared to PM-challenged group. (E) ECs grown on 100 mm dishes were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and then plated onto gold electrodes for ECIS measurement. 24 hours after replating, the ECs were challenged with PM (100 μg/ml) and changes in TER were measured with ECIS. *p < 0.05 compared to PM-challenged group.
National Institute of Standards and Technology, fine PM collected from New York city or Baltimore, data not shown), indicating a selective pathogenesis pathway by PM pollution.
Previous studies report that PM triggers the generation of reactive oxygen species or ROS mainly from dysfunctional mitochondria [27, 28] , and we also noticed the massive generation of ROS by this PM sample is also mainly from mitochondria (unpublished observation). The high iron level of this particular PM might also contribute to the ROS generated via Fenton reactions. ROS released endogenously, have been implicated in the pathophysiology of several lung diseases, including asthma and COPD, as the biochemical mechanisms underlying the urban PM-induced airway inflammation and toxicity [29] . ROS are highly reactive and cause deleterious gene, protein, and tissue effects. ROS are increased in BAL or exhaled breath condensate from patients with inflammatory lung injuries and from people with cardiopulmonary disease who have been exposed to PM [30, 31] . This response may reflect the high oxidative potential of fine and ultrafine particulates. Residual oil fly ash (ROFA) and PM1.7-3.5 cause pulmonary inflammation mediated by oxidative stress [32, 33] . In vivo, exposing rats to PM leads to the formation of free radicals in the lung [34] . Since cardiovascular disease is considered a risk factor of PMrelated mortality and morbidity, it is interesting to note that spontaneously hypertensive rats, when exposed to PM, were more susceptible to pulmonary (inflammatory injury) and cardiovascular complications (acute depression of ECG activity) in an oxidant-dependent manner [35] . Besides ROS, PM might trigger adverse outcomes via other potential mechanisms including nonselective phosphatase inhibition (by vanadium) or competitive ion channel inhibition (by nickel) due to the complex and variable chemical components.
In this study, we first define a novel pathway that mediates ROS-dependent tight junction disruption upon particulate matter challenge. Tight junctions, or zonula occludens, are the most apical component of the intercellular junctional complex, which also includes adherens junctions, desmosomes, and gap junctions [36] . ZO-1 was the first tight junction protein to be identified, and ZO-2 and ZO-3 were later isolated as proteins that coimmunoprecipitated with ZO-1 [37, 38] . ZO-1 is a peripheral membrane-associated component of the cytoplasmic plaque of tight junctions and is found ubiquitously within tight junctions of epithelial and endothelial cells [39] . ZO-1 interacts with many cellular proteins via its multiple protein-binding domains. ZO-1 has been reported to interact with other ZO family members or claudins via the PDZ domains [40, 41] . ZO-1 interacts with the C-terminus of occludin with its GuK domain and the acidic domain [42] . The proline-rich C-terminus of ZO-1 mediates its binding to F-actin in vitro, and thus links it to the cytoskeleton [43] . Clearly, ZO-1 interacts with a wide variety of cell skeleton components and plays a central role in orchestrating tight junction complexes. Any dysregulation of ZO-1 in endothelial cells by extracellular stimuli, such as virus shell proteins or alcohol, leads to persistent tight junction disruption and vascular hyperpermeability.
Calpain is a regulator of endothelial integrity which helps control fundamental cellular processes including cytoskeletal remodeling, membrane fusion, cell proliferation and differentiation, and activation of proteolytical cascades leading to apoptosis [44, 45] . Under oxidative stress, activated calpain cleaves eNOS and cytoskeletal proteins and induces apoptosis [21, [46] [47] [48] . Particulate matter induces endothelial cell intracellular oxidative stress, which leads to the activation of calpain, one of the major cytoskeletal regulators. Here we describe the cleavage of tight junction protein ZO-1 by activated calpain both in vitro and in vivo, indicating that calpain plays a central role in PM-induced endothelial barrier disruption and vascular hyperpermeability. In addition, as activated calpain cleaves other critical cytoskeletal proteins including ezrin and MARCKS protein, the contribution of the other cytoskeletal proteins to the EC hyperpermeability induced by PM needs to be further investigated.
Oxidative calcium influx is mediated by plasma membrane cation-permeable ion channels. The transient receptor potential protein (TRP) and its homologs are cation channels with a tetramer secondary structure which senses diverse stimuli from the extracellular and intracellular Figure 6 Over-expression of endothelial ZO-1 attenuates PM-induced EC barrier disruption in vitro and pulmonary inflammation in vivo. (A) Human lung microvascular EC were grown to 60% confluence and treated with ZO-1 expression plasmid with X-fect reagent for 48 hr, and over-expression of ZO-1 protein was confirmed by Western blot. (B) The ECs were then challenged with PM (100 μg/ml), and changes in TER after 6 hr were measured with ECIS. *p < 0.05 compared to PM-challenged group. AJ mice were treated with ZO-1 expression plasmid with an ACE antibody-conjugated liposome delivery system (5 mg/kg) for 3 days, then challenged with PM (10 mg/kg). After 24 hr of PM exposure, (C) lung ZO-1 levels were analyzed with Western blot. Shown is one of the three repeated blots. BAL was collected and (D) protein content, (E) total white blood cell, and (F) IL-6 levels were measured. N = 4. *p < 0.05 compared to PM-challenged group. **p < 0.05 compared to control. environments [49] . Mammalian TRPs comprise six major subfamilies. TRPM2, a member of the TRP channel M2 subtype, is a calcium-permeable channel activated by intracellular messengers such as ADP-ribose [50] . Massive ROS burden induced by PM contributes to DNA oxidation and damage, which activates poly-ADP ribose polymerase (PARP) to initiate DNA repair mechanisms. PARP binds to single-stranded and double-stranded DNA breaks and catalyses the breakdown of NAD into nicotinamide and ADPribose, the intracellular agonist of TRPM2 [22, 51, 52] . Oxidative stress-mediated activation of the PARP pathway serves as the major source of free ADP-ribose production in endothelial cells [53] . Intracellular ADP-ribose activates TRPM2, allowing calcium ions to enter the cell, which in turn trigger numerous physiological and pathological processes.
An important limitation of our study is the high dose of PM that we employed. With 10-30 μg/m 3 ambient PM level in the US or Europe, it is hardly to achieve a high level of acute PM exposure. While 100 μg/ml (in vitro) or 10 mg/kg (in vivo) are typical doses used in particulate matter toxicology studies [12, 13, 27, [54] [55] [56] . With an assumed ambient PM level of 20 μg/m 3 , one man with 70 kg body weight and 8 m 3 /minute respiration rate would receive a dose of 10 mg/kg corresponding to about 16 years of exposure with 50% deposition rate. As noted, a lot of cities in the developing countries still have high levels of ambient PM. A report by world bank [57] Extensive epidemiologic and experimental evidence has demonstrated that particulate air pollution directly causes cardiopulmonary damage. Our observations demonstrate a novel mechanism of PM-mediated disruption of endothelial barrier function which is attributable to ZO-1 degradation by calpain, which is activated by extracellular calcium leakage through oxidant-sensitive TRPM2 channels. Therefore, inhibition of ROS/TRPM2/calpain/ZO-1 degradation may provide useful therapeutic strategies for the treatment of endothelial barrier dysfunction and lung inflammation.
Additional file 1: Figure S1 . (A-B) PM induces dose-dependent reduction in transendothelial resistance (TER). (C) PM induces dosedependent (6 hr) reduction of ZO-1 protein levels. Figure S2 . PM induced FITC-dextran leakage across EC monolayer. Figure S3 . PM (100 μg/ml, 1-16 hr) does not induce LDH release from human ECs. Figure  S4 . NAC or PEG-CAT attenuates PM-induced ROS in ECs. Figure S5 . NAC (5 mM, 1-24 hr) does not change ZO-1 protein levels in human ECs. Figure S6 . EUK-134 (5 μM, 1 hr pre-treatment) attenuates PM (100 μg/ml, 6 hr)-induced ZO-1 degradation and TER reduction. Figure S7 . We hypothesize that PM induces EC barrier disruption in delayed phase (via ZO-1 degradation) and acute phase (via stress fiber formation). The expression of nicotinic receptor alpha7 during cochlear development Nicotinic acetylcholine receptor alpha7 expression was examined in the developing and adult auditory system using mice that were modified through homologous recombination to coexpress either GFP (alpha7GFP) or Cre (alpha7Cre), respectively. The expression of alpha7GFP is first detected at embryonic (E) day E13.5 in cells of the spiral prominence. By E14.5, sensory regions including the putative outer hair cells and Deiters' cells express alpha7GFP as do solitary efferent fibers. This pattern diminishes after E16.5 in a basal to apex progression, as Hensen's cells and cells of the spiral ligament acquire alpha7GFP expression. At birth and thereafter alpha7GFP also identifies a subset of spiral ganglion cells whose processes terminate on inner hair cells. Efferent fibers identified by peripherin or calcitonin gene-related protein do not coexpress alpha7GFP. In addition to cochlear structures, there is strong expression of alpha7GFP by cells of the central auditory pathways including the ventral posterior cochlear nucleus, lateral lemniscus, central inferior colliculus, and the medial geniculate nucleus. Our findings suggest that alpha7 expression by both neuronal and non-neuronal cells has the potential to impact multiple auditory functions through mechanisms that are not traditionally attributed to this receptor. Numerous neurotransmitter systems contribute to the normal development and function of the auditory sensory (cochlear) apparatus and the circuitry of the central nervous system. This includes members of the excitatory ligand-activated nicotinic acetylcholine receptor family (nAChR; Albuquerque et al. 2009 ). The nAChR subunit family consists of 16 distinct subunits that in various pentameric combinations form ligand-activated ion channels that each exhibit uniquely specialized pharmacological and functional properties (Albuquerque et al. 2009 ). One of these is the homomeric alpha7 nAChR (a7) whose functional uniqueness is in part due to its expression by both neuronal and non-neuronal cells in many tissues throughout the body and because it is responsive to multiple agonists (including acetylcholine and choline as well as nicotine). This results in its ability to modulate a diverse range of cellular functions including cell growth, cell survival, neurotransmission, and inflammation (Gahring and Rogers 2005; Levin et al. 2006; Albuquerque et al. 2009 ).
Members of the nAChR family contribute to essentially all aspects of the auditory sensory system function and development (Morley and Happe 2000; Morley 2005) . This includes widespread changes in expression during embryogenesis that optimizes their contribution to signal transduction, fine-tuning of sensory hair cells, and modulating central auditory circuit neurotransmission (Elgoyhen et al. 1994 (Elgoyhen et al. , 2001a Happe and Morley 1998; Vetter et al. 1999 Vetter et al. , 2007 Morley and Happe 2000; Katz et al. 2004; Morley 2005) . This functional diversity is in part accomplished through strict spatiotemporal control of different nAChR subunit expression, as has been extensively described for the nAChRs composed of either homomeric (a9) or heteromeric (a9 + a10) subunits (Elgoyhen et al. 1994; Vetter et al. 1999 Vetter et al. , 2007 Elgoyhen et al., 2001b; Murthy et al. 2009) . Less is known about the role of other nAChRs including a7, although this receptor is implicated in modifying longer lived stimulation by high-frequency sound and supporting survival of spiral ganglion cells during development (Morley and Happe 2000; Morley 2005) . Because the measurement of a7 expression and function can be compromised by low receptor expression levels or the absence of conditions that best reveal its modulatory role (Gahring and Rogers 2005; Albuquerque et al. 2009 ), the participation by this receptor as an important contributor to the development and normal auditory sensory function remains to be fully explored.
In this study, we examine a7 expression during development of the auditory sensory system. This was done using mice that were modified though methods of homologous recombination to introduce, at the a7 gene 3′ end, a hemagglutinin (HA) protein tag to the a7 receptor protein and a bicistronic IRES-driven tau + enhanced-GFP fusion protein reporter (a7 GFP ). An advantage of the tauGFP reporter construct is that the tau component directs GFP into the axon of cells expressing a7 GFP . Also generated was a mouse in which Cre-recombinase replaces the tauGFP. The expression of a7 GFP in these mice reveals extensive spatial and temporal remodeling of receptor expression during embryonic and postnatal development of the cochlear sensory structures. Furthermore, a7 GFP expression continues in both neuronal and non-neuronal cells of the adult cochlear structure and the central ascending auditory pathway. This suggests that a7 has the potential to impact functionally on auditory processes through multiple pathways and mechanisms that could impact upon the adult function in ways not traditionally attributed to this receptor.
All animals were used and housed in accordance with protocols approved in advance by the Institutional Animal Care and Use Committee at the University of Utah . This includes adherence to the Guide for the Care and use of Laboratory Animals of the National Institutes of Health.
Generation of alpha7-HA-IRES-tauGFP and alpha7-HA-IRES-Cre mice
The construction of the a7 protein and gene (Chrna7) reporter mouse lines; Chrna7-HA-IRES-tauGFP (a7 GFP ) and Chrna7-HA-IRES-Cre (a7 Cre ) have been described in detail . Briefly, as diagramed in Fig. 1A , the methods of homologous recombination were used to introduce an epitope hemagglutinin (HA) and stop codon extension to the a7 C-terminus and a bicistronic IRES-tauGFP reporter cassette ). This generated the a7 GFP mouse (Fig. 1A) , which expresses the tauGFP protein as a marker of Chrna7 transcription. The Speed Congenic Program of the Jackson Laboratory was used to achieve 98% C57BL/6 background congenicity . For conditional cell ablation of the cells expressing Cre as in the a7 Cre mouse, we crossed this mouse with the LoxP conditional diphtheria toxin (DTA) mouse lines as described previously .
Embryo (E) timing was based upon identification of coital plugs (equal to E0.5). Immunohistochemical methods were as described . Embryos were fixed in PBS/2% paraformaldehyde/5% sucrose, cryoprotected with sucrose in PBS to a final of 30%, embedded and sectioned using a Microm EM550 microtome. The 12-lm sections were mounted on glass slides, blocked, and permeabilized with 1% deoxycholate and 0.2% Triton X-100 in PBS, and then incubated overnight at 4°C with the appropriate primary antibodies. After washing, sections were incubated with secondary antibodies conjugated to fluorescent markers (Jackson Immuno-Research, West Grove, Pennsylvania) for 1 h at room temperature. The sections were again washed, and mounted in prolog gold antifade reagent (Invitrogen, Grand Island, New York; P36930) and cover-slipped before being photographed using fluorescence microscopy . Images were collected using a Microfire 24-bit CCD camera (Optronics, Goleta, California) and imported into Photoshop C2 for preparation of figures.
The antibodies used were commercially obtained. These were anti-calcitonin gene-related protein (CGRP; rabbit; 1:30; Chemicon/Millipore, Temecula, Californa AB5920), anti-GFP (chicken; 1:800, Aves Labs, Tigard, Oregon GFP-1020), anti-HA (rabbit; 1:200; HA.11 Covance, Princeton, New Jersey PRB-101P), anti-peripherin (rabbit; 1:100; Abcam, Cambridge, Massachusetts #1530), anti-S100beta (rabbit; 1:100; Abcam ab868), rabbit anti-beta-III tubulin (TUJ1; 1:3000; Covance MMS-435P). Detection of GFP offers superior sensitivity that is well over background fluorescence ( Fig. 1B and C) . For this study, some inconsistent signal detection or autoflourescence was occasionally observed and these sites identified in the individual figures. We find the expression of GFP and HA are similar, although anti-HA expression is detected predominantly on the surface of cells identified by anti-GFP expression (Fig. 1D ).
The expression of a7 exhibits distinct spatiotemporal patterning in developing cochlear structures. Previously, we demonstrated the earliest expression of a7 in the developing embryo to be in rhombomeres 3 and 5 of the E9.0 embryo ). Thus, we initiated studies of a7 GFP staining at this time. From E9.5 through approximately E12.5, the otic and cochlear structures did not express detectable a7 GFP ( Fig. 2A and not shown, see . The earliest detected expression of a7 GFP in the cochlear structures was at E13.5 in cells of the spiral prominence (SP; Fig. 2B ). The SP retains a7 GFP expression throughout embryonic and post-natal development (see below). By E14.5 ( Fig. 2C and D) , a7 GFP expression extends to cells in the sensory domain of the lesser epithelial ridge near the site of the presumptive outer hair cells (OHC) and Deiters' cells (Morsli et al. 1998; Lanford et al. 1999; Kiernan et al. 2005a,b) . Light staining of the greater epithelium ridge was also present from E14.5 and thereafter, although this staining is inconsistently observed (Fig. 1B and C and not shown).
Coincident with this expression was strong staining of pioneering efferents that become separated into individually distinguished processes as they progress through the spiral ganglion (SG) to reach the external face of this sensory domain ( Fig. 2C ; see below). The staining of the epithelial cells of the lesser epithelial ridge intensifies thereafter (e.g., E15.5 in Fig. 2E ). At this stage, expression of a7 GFP by cells of the SG was in general only weakly observed in scattered cells (Fig. 2E ). By E16.5, a7 GFP expression continues to increase in cells of the lesser epithelial ridge of the prosensory domain where OHC and Deiters' cells can now be distinguished (Fig 2F and G and insert) . Cells throughout the SG were also revealed by expression of a7 GFP by this developmental stage. Pillar cells do not express a7 GFP and there were no identifiable efferent processes labeled by the expression of this receptor at this stage or thereafter (see the following sections). the a7 gene (Chrna7) was modified using homologous recombination to add a C-terminus epitope tag (hemagglutinin [HA] ) and inserted into the 3′ terminus of Chrna7 a reporter bicistronic internal ribosome entry sequence (IRES)-tau fusion to enhanced green fluorescent protein (eGFP) fusion protein cassette (a7 GFP ; see Methods and ). This construct was subsequently altered by replacing the tau:GFP cassette with the Cre-recombinase gene (a7 Cre ). (B, C) The visualization of the Chrna7 transcription using immunological detection of GFP compared with background. Shown are sagittal sections of the cochlear sensory structures of an E16.5 a7 GFP embryo in (B) and at greater magnification in (C). The panels on the left are stained for GFP expression (see Methods), whereas the image on the right shows an adjacent serial section that received the same staining treatment, only primary antibody was omitted. Photographs were collected at the same gain and exposure. The asterisk identifies cochlear ducts and the arrow points to the spiral prominence and the arrow head points to cell giving rise to the outer hair cells and Deiters' cells. Abbreviations are SG, spiral ganglion; and tg, trigeminal ganglion. In (B), the bar = 100 lm and in (C), the bar = 400 lm. (D) Examples of colabeling for a7 GFP (green) and anti-HA (HA) in cells associated with the spiral ganglion at E16.5. Examples of double-labeled cells are identified by with arrows. Some processes are also colabeled (arrow head). Bar = 50 lm.
The pattern of a7 GFP expression in the E18.5 cochlear structure undergoes significant remodeling as both sensory hair cells and the associated supporting cells complete their differentiation ( Fig. 2H and I). This includes a decrease of a7 GFP expression by OHCs and underlying Deiters' cells that progresses away from the inner hair cells and proceeds in a basal-to-apical direction (next section). This is coincident with the appearance of signal in Hensen's cells that are most proximal to the outer line of OHCs (returned to below). Ganglionic afferent fibers ending at the base of the inner hair cells are also detected (see subsequent sections). In the postnatal mouse, as shown in the P6 cochlear sensory structure ( Fig. 2J and K), the expression of a7 GFP becomes limited to Hensen's cells immediately adjacent to the most distal OHC. Cells of the spiral ligament also acquire a7 GFP expression, while the spiral prominence remains unchanged. In the SG, the expression of a7 GFP is well established and the projections from these labeled cells can be followed to the vicinity of the inner hair cells (IHC) where their terminals appear to surround the base of the inner hair cell (IHC; Fig. 2J and K). A summary diagram illustrating the expression of the a7 GFP during these major developmental stages is shown in Fig. 2L . Remodeling of a7 GFP in the cochlear structure after E16.5 is in a basal-to-apical direction
The remodeling of the sensory cell region of the cochlear structure between E16.5 and E18.5 as suggested by the progression in changing a7 GFP expression was examined further. Through E16.5, all otic structures exhibit a similar a7 GFP expression pattern (Fig. 3A) . This was not the case in the E18.5 cochlear structure where the loss of a7 GFP expression by OHC and Deiters' cells and acquisition of staining by Hensen's cells was first observed in the most basal structures and it then appears in the more apical structures successive developmental stages ( Fig. 3B and C and not shown). This generates a striking contrast in a7 GFP expression between cochlear structures at the apex relative to the base with intermediary turns, exhibiting the progressive stages of this change in a7 GFP expression (Fig. 3B ). By P4, this gradient was not evident (not shown) and the mature a7 GFP expression pattern first observed in the E18.5 basal cochlear structures was present across the entire structure. In Fig. 3D , a diagram depicts the remodeling of a7 GFP expression seen in the E18.5 developing cochlear structure.
Nonsensory cells of the cochlear structure express a7 GFP As suggested by the preceding discussion, there was expression of a7 GFP by both neuronal and non-neuronal cells (Fig. 4) . This is particularly clear in the postnatal mouse (e.g., P6-P12), where the predominant expression of a7 GFP in neuronal cells was by cells of the SG (Fig. 4A) . The strongest labeling of cochlear structures was restricted to Hensen's cells and the spiral prominence ( Fig. 4A-E) . Evident at the P6 stage was a7 GFP signal in individual cells of the spiral ligament ( Fig. 4C and D) . Also evident were the extended branching that is characteristic of the morphology of type II fibrocytes located in this region (Fig. 4D ; Spicer and Schulte 1991; Sun et al. 2012 ). In the P12 cochlear structure, the branches were more abundant and form a 'feathered' structure that emanates from cell bodies defined by a7 GFP expression (Fig. 4E) . Cells of the stria vascularis or other members of the cell family composing the structures of the lateral wall and surrounding cochlear duct were not observed to express a7 GFP in these later stages of development (Fig. 4) .
The expression of a7 GFP during innervation of the developing cochlear structure
Innervation of cochlear sensory cells follows a series of distinct steps that were in part revealed by a7 GFP visualization (Fig. 5 ). As noted, the first detection of a7 expression was in the prominently labeled efferent processes that appear to form bundles upon entering the SG and then disperse into small solitary fibers (E14.5; Figs. 5A and 2C,D). These solitary processes exhibit a beaded structure as they proceed to the base of the developing sensory cells (Fig. 5B) . The origin of these efferent fibers was examined in serial sections of the E14.5 hind brain. These fibers appear to originate from a cell grouping in the basal brain stem caudal to trigeminal nucleus V that could be distinguished by their transient a7 GFP expression (Fig. 5C ). These cells occur in clusters (Fig. 5C insert) and their prominently labeled processes can be followed using serial section sets to the cochlear structure where they give rise to the fiber bundles and the point of dissemination associated with the SG (Fig. 5C and insert) . The anatomical location of these cells suggest that these cells are within the forming olive complex, which is consistent with the reports of pioneering fibers that originate from the developing olive complex and extend to the developing cochlea (Zuo et al. 1999 ). These fibers were not detected after E15.5. During the E15.5-16.5 period, there was essentially no labeling of neuronal processes by a7 GFP (Fig. 5D-F) .
However, ongoing innervation of cochlear sensory cells was identified using peripherin labeling ( Fig. 5E ; see Simmons et al. 1996; Hafidi 1998; Huang et al. 2007) or for olivocochlear efferents that were identified by labeling for calcitonin gene-related protein (CGRP; Fig. 5F , Fritzsch 2003). By E18.5, the SG a7 GFP signal was present in afferent processes that extend to the base or near vicinity of the IHCs (Fig. 5G) .
At birth and thereafter (P0-P12 analyzed), the expression of a7 GFP was strongly detected in SG afferent fibers where they terminate near or at the base of IHC sensory cells ( Fig. 5H and I) . This basic pattern of a7 GFP expression was reinforced during the remaining postnatal period as fibers continue to form a dense plexus that appears to surround the base of the IHCs. The other efferent fibers not detected by a7 GFP continue to be trimmed and also associate with their final targets (Merchan-Perez and Liberman 1996; Simmons et al. 1996; Hafidi 1998; Huang et al. 2007 ). The outcome of this remodeling was evident by P12 when the SG1 afferent terminals surrounding the IHC were distinguished by strong a7 GFP staining of the terminal clusters ( Fig. 5I and inset) . This was approximately the same time hearing onset occurs in mice (~P10; Kros et al. 1998 ). Processes originating from SG cells identified by peripherin expression that were not colabeled with a7 GFP form distinct efferent terminals on or very near OHCs cells and on the terminals that end on the IHC afferent terminals identified by a7 GFP labeling ( Fig. 5I ; Huang et al. 2007 ). While not entirely evident from the images shown, not all SG cells at P12 expressed a7 GFP , suggesting this could identify a functionally distinct subpopulation ( Fig. 5I ; Happe and Morley 1998) . Again, no a7 GFP labeling of olivocochlear efferents was detected. A diagram summarizing these findings is shown in Fig. 5J .
Ablation of the a7 Cre -expressing cell lineage confirms a7 GFP expression during cochlear development Although a7 GFP expression was not detected in the developing cochlear structures until E13.5 (Fig. 2B) , as reported previously the earliest a7 expression we have defined is at P9.0 in rhombomeres 3 and 5 . Because cochlear morphogenesis includes signaling from rhombomere 5 (Liang et al. 2010) , the possibility of a7 GFP contributing to the development of this structure was examined. This was done using embryos from a7 Cre mice crossed with mice harboring the conditional ROSA26-loxp (diphtheria-A toxin (DTA; termed a7 Cre: DTA ; . In these embryos, a7 Cre: DTA -expressing cells and their direct lineages were ablated, thus revealing expression that could have been be missed by a7 GFP measurements ). An example of the cochlear structure at E16.5 taken from a7 Cre:DTA crosses is shown in Fig. 6 . Because there is only occasional overlap with a7 GFP (see Fig. 5E ), we used peripherin expression to aid in examining the fate of non-a7-expressing cells (Fig. 6A and B) . The overall patterning of the cochlear structure and the formation of major boney structures of the cochlea inclusive of the otic capsule and modiolus were intact, albeit somewhat distorted. The cochlear ducts were collapsed (Fig. 6B) , probably due to the absence or severe thinning of the distal lateral wall. Also absent was the sensory cell domain Figure 5 . The a7 GFP expression during cochlear innervation. Innervation of the developing cochlear structure is revealed by a7 GFP labeling. (A) An E13.5 sagittal section shows a group of efferent processes (arrow) that distribute to solitary fibers that are strongly labeled for a7 GFP expression (arrow heads). Cells of the putative sensory region (sr) and the spiral prominence (SP) are identified. (B) At greater magnification, these fibers (arrow heads) have a beaded appearance and project towards the base of sr. (C) The possible origin of the pioneering efferent fibers is suggested by the intense expression of a7 GFP in the E13.5 cell groups (arrow) located caudal to the trigeminal sensory nucleus (V) consistent with the early olive in this horizontal section through the posterior brain stem. At increased magnification (Insert), the cell clustering (arrow) and their projections (arrowhead) are identified. Serial sections (not shown) reveal continuity between these cells and those entering the cochlear structures (arrow heads). (D) E15.5 a7 GFP expression and colabeling with other neuronal process markers (red). The processes that express peripherin (arrow) end mostly in the vicinity of the inner hair cells (IHC). Occasional solitary fibers (arrow) extend towards the base of the outer hair cells (OHC) at the dorsal boarder of the Deiters' cells (D). (E) The E16.5 cochlear innervation pattern looks much the same as E15.5, although the peripherinlabeled fibers (arrow) are more distinct. These processes lack detectable a7 GFP expression. (F) Olivocochlear efferents identified by calcitonin generelated proteins (CGRP; arrows). (G) The E18.5 embryo exhibits afferents detected by a7 GFP expression (arrows). These extend from SG cells that are not colabeled with peripherin (not shown). (H) At birth (P0), there are distinctly labeled a7 GFP afferents (arrowhead) and peripherin-labeled efferents that extend to the Deiters' cells (D) and then turn (arrows) to contact the base of the OHCs. Hensen's cells are noted (H). (I) The P12 innervation pattern is similar to the P0. In this merged image of a7 GFP expression (green) and peripherin (red), many spiral ganglion (SG) cells and processes are labeled, but the labels only rarely overlap in the same processes (see insert). The a7 GFP identify mostly processes reaching the IHCs (arrow). Peripherin-labeled processes mostly terminate at the base of the outer hair cells (OHC) or onto the a7 GFP -labeled afferent fiber near the base of the IHC. Hensen's cells expressing a7 GFP is identified (H). The inset shows the sensory cell region at increased magnification. The arrows identify the a7 GFP -expressing afferent ending at the base of nonlabeled IHC, whereas the double arrow heads point to the peripherin-labeled terminal. Other peripherin processes extend to the base of the OHCs (individual arrow heads). (J) Diagrams as in Fig. 2 depicting the basic innervation patterns observed in this study. Green is a7 GFP and red is peripherin. Afferent (af) and efferents (ef). Bars = 50 lm containing presumptive OHCs and Deiters' cells ( Fig. 6C and D), as expected from results of a7 GFP expression (Figs. 2, 5) .
The SG of a7 Cre:DTA embryos is reduced in size and the majority of cells remaining give rise to mostly peripherinlabeled efferents (see Fig. 5E ). These fibers also appear to be more densely aggregated relative to the a7 GFP control mouse ( Fig. 6A and B) . While peripherin-identified processes still project to the presumptive sensory cells (both IHC and OHC), they were less branched and those that did project to the former OHC target fields often turn and proceed backwards towards the vicinity of IHCs ( Fig. 6C and D) . These results are consistent with the earliest expression of a7 being after major cochlear structures are determined, and there was the expected selective ablation of OHCs and Deiters's cells. The necessity of the presence of the target sensory cell to coordinate the innervation process is also suggested by these findings.
The results of studies examining a7 expression using in situ hybridization and functional measurements using electrophysiology have shown that this receptor is an important contributor to various nuclei of the central auditory system (Happe and Morley 1998; Vetter et al. 1999 Vetter et al. , 2007 Morley and Happe 2000; Morley 2005 ). The a7 GFP mouse system offers an excellent opportunity to view these central systems and their connections as shown in Fig. 7 . The connections between the SG and the cochlear nuclei were strongly identified at E18.5, presumably due to the dense projections from SG cells expressing a7 GFP that extend processes both to the IHC (Fig. 2 ) and the developing cochlear nuclei of the brainstem (Fig. 7A) .
The expression of a7 GFP appears to intensify after P10, and by P12 signal is consolidated almost exclusively in the ventral-posterior cochlear nucleus (Fig. 7B ). This is in agreement with reports from in situ hybridization studies reporting the strong expression of a7 in this nucleus, whereas other major cochlear nuclear divisions exhibited only weak or sporadic labeling (Yao and Godfrey 1999; Morley and Happe 2000) . Also consistent with those studies was that the cells identified by a7 GFP expression resemble octopus cells (Fig. 7B, insert) . Essentially, no expression of a7 GFP was detected in the dorsal cochlear nucleus, although some dispersed and weakly stained cells were present in the granular aspect. Also evident was the strong staining of neuropil, presumably in part due to terminals of SG cells associated with the eighth cranial nerve (Fig. 7B, inset) . This strong labeling of the P12 SG and OHC afferents is consistent with other reports (Morley and Happe 2000) .
The expression of a7 GFP also persists into the adult animal. This is apparent in the ascending central auditory system nuclei and their fibers (Fig. 7C) . After the cochlear nucleus, a7 GFP is present in the ventral lateral lemniscus, on through the dorsal lateral lemniscus, and to the inferior colliculus where dense staining of a7 GFP is present ( Fig. 7C ; Morley and Happe 2000; Yao and Godfrey 1999) . The commissural fibers of the inferior colliculus are also identified by a7 GFP expression (Fig. 7D) . Thereafter, efferents follow the brachium of the inferior colliculus to the medial geniculate nucleus where scattered cells expressing a7 GFP were seen. Not shown is that the expression of a7 GFP in the adult auditory cortex appears restricted to cells of layer 1. Labeling of olivocochlear fibers was not detected.
This study extends the reports of spatiotemporal regulation of a7 expression during mouse embryonic development to include the cochlear sensory structure, as well as confirms the extensive expression of this nAChR in the ascending central auditory system. The novel finding that in addition to expression of a7 GFP in developing sensory cells of the cochlear structure and neuronal cells of the spiral ganglion, there is also considerable expression by nonsensory cells. Cells of the spiral prominence and ligament, Deiters' cells, and some Hensen's cells. Despite overall agreement between our studies and those using in situ hybridization (e.g., Happe and Morley 1998; Morley and Happe 2000) , these nonsensory cells were not reported previously to express a7. However, these comparisons are incomplete because the earlier studies did not necessarily show the comparable structures or the developmental stages at times where we observed peak a7 GFP expression. Also, our method of detecting GFP as a marker of a7 expression offers improved sensitivity and resolution that has previously not been available for this nAChR.
The nicotinic receptors a9 and a10 are particularly well characterized in the auditory system (Elgoyhen et al. 1994 (Elgoyhen et al. , 2001a Vetter et al. 1999 Vetter et al. , 2007 Katz et al. 2004; Morley 2005) . Comparing the expression of a7 GFP to the results from these studies of the sensory hair cells and the nonsensory cells of the cochlea indicate that there are significant spatiotemporal differences during development between the expression of a7 versus a9 and/or a10. The a9 KO mouse also exhibits auditory deficiencies that are not observed in the a7 KO mouse, which is largely devoid of a phenotype in this sensory system under normal physiological conditions (Liberman and Brown 1986; Simmons and Morley 1998; Morley 2005; Lustig 2006 ). The a7 GFP is not detected in IHCs, which is consistent with a9 nAChR being the principle target of alpha-bungarotoxin in this cell type (Uziel et al. 1981; Glowatzki and Fuchs 2000) . Collectively, this suggests that functional redundancy between these receptor subtypes is unlikely (see also . This is also supported by the extensive studies by the Morley group Morley 1998, 2004; Morley and Happe 2000; Simmons and Morley 2011) who showed that multiple receptor subtypes are expressed in the cochlear and central auditory systems, but each exhibits distinct spatiotemporal patterns that likely preclude substantial or sustained functional overlap.
Noteworthy is that the functional contribution of a7 towards modulating physiological systems may not be revealed unless the system is imbalanced as by genetic . Central auditory systems express a7 GFP . Central auditory nuclei identified by a7 GFP expression. (A) At E18.5 in this sagittal image of the entire otic complex and the adjacent basal brainstem is included. The cochlear nucleus (C) and the eighth cranial nerve (8n) are visible as is the fifth cranial nerve (5n), the trigeminal nucleus (TGN), and trigeminal ganglion (TGG). Also noted are cochlear ducts (asterisk) and a spiral ganglion (SG). (B) At P12, a7 GFP expression of cochlear complex reveals the strongly labeled cells of the ventralposterior cochlear nucleus (VCP). The dorsal cochlear nucleus (DC) and ventral-anterior cochlear nucleus (VCA) are identified and is the eighth nerve (8n) and a cochlear duct (asterisk). The inset shows the VCP at increased magnification. Cells clusters expressing a7 GFP (arrowhead) and individual cells that resemble the morphology of octopus cells described previously (Morley and Happe 2000; Morley 2005) to express a7 (arrow) are noted. (C) Another P12 sagittal section reveals a7 GFP expression in the ascending central auditory pathways. (D) The expression of a7 GFP in the inferior colliculus (CIC) of this horizontal section reveals staining of the commissural fibers (arrow). Structures identified are the brachium of the inferior colliculus (BIC); dentate gyrus (DG), inferior colliculus, central nucleus (CIC); lateral lemniscus, dorsal nucleus (DLL); lateral lemniscus, ventral nucleus (VLL); medial geniculate nucleus (MGN), and the substantia nigra (SN). Bars = 100 lm (A, B0; 20 lm (B-insert), and 1 mm (C, D). deficiencies, sustained exposure to pharmacological compounds, or other events such as inflammation (e.g., Faustman et al. 1992; Gahring and Rogers 2005; Venables et al. 2007; Albuquerque et al. 2009; Brown 2011; Severance et al. 2011) . For example, the dysfunction of a7 is implicated in several psychiatric syndromes associated with certain forms of autism and schizophrenia (particularly in patients who hallucinate) whose spectrum of disorders include abnormal sensitivity to sensory stimuli including an abnormal auditory gating phenotype (Khalfa et al. 2001; Veuillet et al. 2001; Araki et al. 2002; McEvoy and Allen 2002; Freedman et al. 2003; Lippiello 2006; Martin and Freedman 2007; Wallace and Porter 2011 and references therein) . Also, the association of certain auditory deficits and nicotine abuse, mostly associated with cigarette smoking, has further focused speculation on the role of a7 in these pathologies and the possible advantages of therapeutically targeting this receptor for symptomatic relief in these cases (Araki et al. 2002; McEvoy and Allen 2002; Simosky et al. 2002; Freedman et al. 2003; Levin et al. 2006; Lippiello 2006; Martin and Freedman 2007; Wallace and Porter 2011) . In this context, our results suggest additional lines of investigation. For example, in a 7Cre:DTA cell lineage ablation there are collapsed cochlear ducts and abnormal innervation indicating that the cells express a7 and the cells that do so contribute an obligatory role in the successful development and long-term function of these structures. The a7 receptor could also participate in auditory performance after birth, including functions related to the central auditory pathways. This study also adds the possibility of an effect by a7 on the performance of the spiral ligament. These cells exhibit a cholinergic response that is most often described in terms of muscarinic acetylcholine receptors (Khan et al. 2002; Maison et al. 2010) , and their dysfunction is related to several pathogenic auditory deficiencies (Spicer and Schulte 1991; Slepecky et al. 1995; Kikuchi et al. 2000; Sun et al. 2012) . The role of a7 has, to our knowledge, not been examined in these cells. Collectively, the potential for a7 functional pleiotropy in the auditory system is similar to other tissues we have recently examined . Thus, multiple defects that impact upon adult function could be expected depending upon the timing, duration, and nature of the receptor dysfunction. Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 μg/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1–200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH = 9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious swine diseases throughout the world [1] [2] [3] and is still having, more than two decades after its emergence, major impacts on pig health and welfare (reviewed by [4] ). The responsible agent is an enveloped, ca. 15 kb long positive-stranded RNA virus (PRRSV) that belongs to the Arteriviridae family [5] and that can cause late-term abortions in sows and respiratory symptoms and mortality in young or growing pigs. Once this virus has entered a herd it tends to remain present and active indefinitely causing severe economic losses and marketing problems due to high direct medication costs and considerable animal health costs needed to control secondary pathogens [6, 7] .
Pigs of all ages are susceptible to this highly infectious virus, which has been shown to be present in most pigs for the first 105 days post infection [8] . However clinical manifestations vary with physiological status and age [9] , as the virus uses several immune evasion ways to complicate the ability of the host to respond to the infection process [4, 10, 11] . Weaning piglets, in particular, are likely to be exposed to the infection. Although PRRSV viraemia is often asymptomatic in these piglets, their productive performance is significantly decreased. Indeed, despite being sero-negative, persistently infected piglets still harbor PRRSV and have been shown to be a source of virus for susceptible animals [12] .
SELDI-TOF MS analysis allows the comparison of protein profiles obtained from a large number of diverse biological samples by combining two principles, chromatography by retention on chip surface on the basis of defined properties (e.g. charge, surface hydrophobicity, or biospecific interaction with ligands) and mass spectrometry. It is thus distinct from common non-selective techniques, such as two-dimensional polyacrilamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionisation (MALDI) MS. SELDI-TOF MS has been widely used for diagnostic biomarker discovery and validation across studies in blood serum/plasma, particularly in cancer research (reviewed by [13] ), but also to characterize and identify biomarkers associated with viral and other infectious diseases [14] [15] [16] [17] [18] [19] . The protein signatures identified by SELDI-TOF MS analysis have thus many potential applications in animal health, including early diagnosis of diseases, prediction of disease states, as well as monitoring of disease progression, recovery, and response to vaccination. Few reports have been published for livestock applications [19] [20] [21] [22] .
Current needs in veterinary medicine and animal husbandry include the identification of tools that allow the early warning of diseases, especially during the incubation periods and before the onset of clinical signs. Therefore, the objective of this study was to identify by SELDI-TOF MS a proteomic profile able to differentiate PPRSV-positive from -negative weaning piglets raised in commercial farms and without clinical symptoms of the disease. We optimized the experimental conditions previously described [20] and validated 47 statistically significant discriminatory biomarkers. Among these, a combination of 14 biomarkers identified in F1 on CM10 at low focus mass permitted to correctly assign the piglets to the PPRSV-positive or PRRSV-negative groups with sensitivity and specificity of 77% and 73%, respectively.
To enable identification of medium-low abundant proteins, only samples with a total content of hemoglobin lower than 4.52 μg/mL were included in the study. Total hemoglobin absorbance and the resulting hemoglobin content were calculated for all the piglet sera in both discovery (n = 50) and validation (n = 70) phases of the study [Additional file 1: Table S1 and Additional file 2:  Table S2 , respectively].
Fractioning of the sera resulted in six different pH fractions; F1 = pH9, F2 = pH7, F3 = pH5, F4 = pH4, F5 = pH3, and F6 = organic solvent. The fractions F1, F4, and F6 were analyzed on the three surfaces CM10, IMAC30, and H50 at both low and high focus masses. Fractions F2 and F3 were excluded from further analyses because preliminary data with 3 serum samples showed that they still contained elevated quantities of abundant proteins (such as albumin), as well as the quality of the spectra and the number of signals detected were very low. Fraction F5 was excluded because no signals were detected.
The fractions F1, F4, and F6 on the surfaces CM10, IMAC30, and H50 showed generally good signal intensities and low coefficient of variation (CV) values (< 30%) in both the discovery and validation phases. Exceptions were fraction F1 on IMAC30 (analyzed at high focus mass) and H50 (both low and high focus masses), as well as fraction F4 on H50 (low focus mass), which were therefore excluded from further analyses.
A total of 50 pig sera, 25 from PRRSV-positive and 25 from PRRSV-negative piglets were analyzed during the discovery phase of the study [Additional file 1: Table S1 ].
We found a total of 785 protein peaks in the sera of all samples (Table 1 ). The most represented pH fraction was F6 (n = 381), followed by F4 (n = 223), and F1 (n = 181). On surface CM10 we identified 317 peaks, on IMAC30 302 peaks, and on H50 166 peaks. Furthermore, a much higher number of peaks (n = 512) was found on low mass range (1-20 kDa) compared to the high (n = 273; 20-200 kDa).
Of the total 785 peaks, 200 were statistically significant (p < 0.05) and permitted to discriminate between PRRSV-positive and PRRSV-negative piglets. Discriminatory peaks were found in F1 (n = 80), F4 (n = 49), and F6 (n = 71) on the surfaces CM10 (n = 107), IMAC50 (n = 58), and H50 (n = 35), as well with low (n = 110) and high (n = 90) focus masses ( Table 1) .
The highest sensitivity (80%) and specificity (76%) were obtained with the 22 discriminatory peaks of F1 on CM10 at low focus mass. Higher sensitivities were found with the 18 peaks of F4 on CM10 at low focus mass (87%), the 7 peaks of F6 on CM10 at low focus mass (85%), and the 12 peaks of F6 on CM10 at high focus mass (87%), however the specificities of these peaks were lower (64%, 66%, and 66%, respectively).
The validation phase was performed on 35 new PRRSVpositive and 35 new PRRSV-negative piglets using the same experimental conditions applied in the discovery phase [Additional file 2: Table S2 ]. Of the total 200 peaks that were significant in the discovery phase, 47 were confirmed in the validation phase (Table 2 ).
In particular, 28 peaks were confirmed on CM10, 19 on IMAC30, whereas none of the peaks could be validated on the surface H50. In the 3 fractions with different pH tested, F1 contained 28 peaks, F4 3 peaks, and F6 16 peaks. A higher number of peaks (n = 36) corresponded to small peptides (acquired at low focus mass 1-20 kDa), compared to big peptides (n = 11) that were acquired at high focus mass (Table 2 ). In line with the results of the discovery phase, the combination of peaks with the highest sensitivities (77% and 64.5%) and specificities (73% and 69.7%) were found on CM10 at low focus mass with the 14 discriminatory peaks of F1 and the 6 discriminatory peaks of F6, respectively ( Table 2 ). The correctly and incorrectly assigned piglets using these peaks are graphically illustrated in the heat map of Figure 1 ; part 1A shows the 14 peaks of F1 and part 1B the 6 peaks identified in F6.
Principal component analysis (PCA) was performed on the profiles of the 47 discriminatory peaks identified during the discovery and confirmed during the validation phase to identify and quantify independent sources of variation observed in the data. PCA analysis showed that 58.2% (PCA1), 17.9% (PCA2), and 12.9% (PCA3) of the total variability within the data was accounted for the X, Y, and Z axes, respectively. These axes were used to plot the data ( Figure 2 ) and they provide an overview of the variation between the individual samples and show how samples grouped. Figure 2A showed three-dimensionally that the PCA peak profiles of piglets positive to PRRSV differed from piglets negative to PRRSV and revealed a good separation among the profiles of the two different groups, especially considering the high heterogeneity of the samples included in the study, as reported in the MM section and in [Additional file 1: Table S1 and Additional file 2: Table S2 ]. Furthermore, with the exception of few The 785 total number of peaks detected and the 200 statistically significant (p < 0.05) discriminatory peaks associated with PRRS infection that were identified by the Ciphergen Express software are reported with the fraction, the array surface, and the acquisition focus mass (low: 1-20 kDa; high: 20-200 kDa). outliers, PCA1 combined with PCA2 also separated well the two piglet populations ( Figure 2B ).
To provide an overview of the current literature and to try to correlate the discriminatory peaks identified in this study with relevant proteins, we summarized in Table 3 the molecular weights of several peaks that have been shown to be related to PRRSV infection. First of all, we summarized the available information on the PRRS viral proteins. The PRRSV genome is ca. 15 kb in size and consists of the 5' untranslated region (UTR), at least nine open reading frames (ORFs), and Table 3 , along with the MW of the closest discriminatory peak identified in the current study. Interestingly, the MW of the viral proteins ORF2b, ORF4, and ORF7 were very similar (difference of MW ≤0.3 kDa) to up-regulated discriminatory peaks identified here ( Table 3) .
As next, we compared proteins related to PRRSV infection that were identified in additional studies (Table 3) ; interestingly, all the 9 peaks found by [28] , and in particular the only up-regulated in PRRSV infected (corresponding to the Alpha 1 S (a1S)-subunit of porcine Haptoglobin), showed minimal MW differences (≤0.3 kDa) with up-regulated peaks identified in this study (Table 3) .
Additional discriminatory peaks found in the current study were very similar (MW differences ≤0.3 kDa) to those identified in other PRRS-related proteomic studies (Table 3) . They corresponded to the following proteins: Glyceraldehyde-3-phosphate dehydrogenase, Proteasome activator hPA28 subunit beta, S100 calcium binding protein A10, Galectin 1, and Gastric-associated differentially expressed protein YA61P [26] ; Heat shock 27 kDa protein 1, Superoxide dismutase 2, Myoglobin, and Vacuolar protein sorting 29 [29] ; Heat shock protein 27 kDa and Nucleoside diphosphate kinase A [30] ; Heat shock 27 kDa protein 1, Galectin 1, and Ubiquitin [31] .
In the present work, we show that proteomic fingerprint profiling is useful in researches on PRRS immuno-pathogenesis and might also be a robust, large scale diagnostic tool for the assessment of the proportion of PRRSV-positive weaning piglets without clinical symptoms in a herd. Indeed, we confirmed that the high-throughput capacity of the SELDI-TOF MS technology allows the screening for disease biomarkers of hundred of samples in a relative short-time period and with minimal sample preparation (as previously also reported by [32] ).
Our results indicate that from the 200 significant peaks found in the discovery phase, a total of 47 could be confirmed in the validation phase. These values are comparable with another study where similar experimental conditions were applied to ovine sera [19] .
Our findings also show that the combination of 14 discriminatory peaks in F1 on CM10 at low focus mass provided the highest sensitivity of 77% and specificity of 73% to correctly assign the piglets to the PPRSVpositive or PRRSV-negative groups. These percentages are in line with recent studies in humans using the [33, 34] . Also the PCA results showed a good separation of the piglets in the two groups under examination. This was reached even though the tested piglets had large variability and heterogeneity, as they were collected from several farms located in different regions, and underwent high environmental pressures, typical of the field conditions. This is mainly due to the careful choice of the serum samples, where we tried to minimize the environmental differences by using same experimental parameters (e.g. sample collection procedures, storage, handling) and by including a similar number of pigs from the same breed (Large White) and with very similar sex ratios and ages (at weaning).
In a preliminary work [20] we had successfully transferred the experimental conditions used in profiling experiments of human sera to pig sera. However, in that work, none of the potential biomarkers identified in the discovery phase could be validated in the subsequent validation phase, because of high samples heterogeneity and high content of serum (e.g. albumin) and contaminant proteins (e.g. hemoglobin), having a negative effects on the detection of significant biomarkers, particularly those corresponding to the medium-low abundant proteins. It has been reported that low abundant proteins constitute about 1% of the entire human serum proteome, with the remaining 99% being comprised of only 22 proteins [35] .
As it was therefore necessary to reduce the level of abundant proteins, in this follow up study, particular relevance was given to the content of the contaminant protein hemoglobin. Only non-hemolytic samples with similar, low contents of hemoglobin were included in the study. Additionally, to further increase the likelihood to identify statistically significant discriminatory biomarkers, we introduced a fractioning step based on anion-exchange chromatography. In a similar study performed with MALDI-TOF [28] , where serum samples were analyzed in the first weeks (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) Table 3 ). Furthermore, two peaks identified in this study (23. 162 and 14.843 kDa) were similar to peaks identified elsewhere (corresponding to Heat shock 27 kDa protein 1 [29] [30] [31] and Galectin 1 [26, 31] , respectively). In accordance with [31] , the identified peak corresponding to Heat shock 27 kDa protein 1 was upregulated, while the peak corresponding to Galectin 1 was down-regulated. Thus, these proteins seem to be very interesting and suitable candidates for future investigations. The preponderance of the significant biomarkers had a molecular mass lower than 20 kDa, confirming that small peptides are a rich source of relevant biomarkers in SELDI-TOF MS analyses as previously reported in human [36] and ovine [19] sera. This may also partly be caused by the fact that the low molecular weight region (LMW) of the serum proteome, called peptidome, is an assortment of small intact proteins and proteolytic fragments of larger proteins, including several classes of physiologically important proteins like peptide hormones and components of both the innate and adaptive immune systems (i.e. cytokines and chemokines) [35, 37] . This is particularly interesting as the patho-physiological state of the body's tissue is predominantly reflected in the LMW and low abundance region of the serum proteome, and specific protein fragments of the serum peptidome have been shown to contain a rich source of disease-specific diagnostic information and they have been correlated with disease stages in several studies (reviewed by [37] ).
In agreement with other studies [29, 31] , we found that the majority of the discriminatory biomarkers were up-regulated in PRRSV-positive piglets. This seems to suggest that the corresponding proteins might be of viral origin or related to the innate or adaptive immune responses (e.g. cytokines, chemokines, acute phase proteins, toll like receptors). In fact, several peaks showed high similarities (MW differences ≤0.3 kDa) with previous works, in particular regarding viral proteins ( Table 3 ). The assignment of the discriminatory peak to a specific protein will require additional work, because the SELDI-TOF technology can only detect masses/peaks of proteins that are differentially expressed between samples but can not directly identify the proteins. This represents one of the major drawbacks of this technology compared to other methods. However, an advantage of the SELDI-TOF MS in this regard is that the results of this technique might lead to the identification of new proteins that were previously not correlated to the disease, and this might hopefully lead to the identification of new biomarkers representing the field situation. The interpretation of these results and the continuation of this project will benefit from the very imminent termination and publication of the sequence of the swine genome [38] , which will definitely contribute to a more precise annotation and a better identification of genes and proteins and thus will greatly facilitate genome wide mapping association studies.
Although a combination of peaks identified with different experimental conditions (e.g. using different fractions and different surfaces) might have provided higher discriminatory power, here we developed a PRRSV diagnostic test based on peaks identified with the same experimental conditions (e.g. fraction, surface, and focus mass), which can be reproduced at high-throughput at reasonable costs. These results provide a set of proteomic biomarkers and related, optimized experimental conditions for high-throughput profiling of pig populations by SELDI-TOF MS for whole genome association studies, where identification of proteins underlying the phenotype can be made a posteriori. SELDI-TOF MS might therefore represent a complementary test or a possible alternative to classical (PCR) and more recent diagnostic methods (e.g. antibody detection in saliva) for profiling large flocks of pigs at reasonable costs, using blood samples that are routinely collected for general veterinary inspections. As well, these SELDI-TOF MS based tests could complement and provide a broader reference for emerging diagnostic methods and have potential applications for the detection of relevant proteins having highly heritable traits (e.g. acute phase proteins).
A total of 120 serum samples of Large White piglets were selected from a well defined and characterized repository database, presently containing more than 20,000 swine samples from 18 different farms of the Lombardy region, Italy. Selection of the piglets aimed to minimize environmental factors and experimental conditions that might influence the results [39] . Hence, all piglets were from the same breed (Large White), had similar ages (weaning: 45-50 days), and their sera showed a low and comparable amount of hemoglobin (calculated as shown below).
In the discovery phase of the study, a total of 50 pig sera, 25 from PRRSV-positive and 25 from PRRSVnegative piglets, as determined by PCR (see below), were analyzed [Additional file 1: Table S1 ]. The validation phase was performed with the same experimental conditions as the discovery phase. A total of 35 new PRRSVpositive and 35 new PRRSV-negative piglets were examined [Additional file 2: Table S2 ]. The actual duration of infection for each individual PRRSV-positive piglet was unknown, as sera were collected and analyzed once for each piglet (at weaning: 45-50 days of age). None of the piglets was treated, as they did not show any symptom of the disease.
To ensure large variability and heterogeneity of the samples and minimize environmental differences, we included in the PRRSV-positive and -negative groups similar numbers of piglets with the same sex that originated from several farms located in different regions. In fact, PRRSV-positive piglets originated from 6 farms of the Lodi region (n = 8) and 7 farms of the Mantua region (n = 52), while PRRSV-negative piglets were collected in 5 farms around Lodi (n = 19) and 9 farms around Mantua (n = 41). Sex ratios males/females (44/76) were very similar in PRRSV-positive (21 vs. 39) and -negative (23 vs. 37) piglets, respectively.
Veterinary inspections of the overall clinical status of the piglets at the day of serum collection did not evidence any clinical symptoms of PRRS, including respiratory distress or sneezing.
All the serum samples were collected, stored, and handled in the same way. They were obtained for each piglet by storing two mL of whole blood without anticoagulants at room temperature (RT) for 4 h followed by centrifugation at 3,500 rpm for 4 min. As suggested in a previous work [20] , an abundant quantity of hemoglobin in the serum can hide early diagnostic biomarkers of PRRSV by competing with the other serum components for the binding site of the chromatographic surfaces. To avoid the consequent signal suppression of the medium-low abundant proteins, only non-hemolytic samples were included in the present study.
A total of 200 clear, transparent sera without red pigmentation (low hemoglobin content) were first selected by visual screening from the total sera available in the database. Hemoglobin content of each serum sample was then determined according to [40] with minor modifications. A calibration curve was generated using five standard solutions (concentrations: 1.8, 3.6, 5.4, 7.2, and 9 μg/ml) of porcine hemoglobin diluted in 400 μL commercially available porcine serum (Sigma Aldrich, St Louis, MO, USA). Triplicate samples were incubated for 5Ámin at RT, then absorbance (E) was measured at 380, 415, and 440 nm. Absorbance at 380 and 440 nm was used to discern background absorbance flanking the absorbance peak (415Ánm) of oxygenated hemoglobin. Absorbance due to hemoglobin was calculated as: E415-[(E380 + E440)/2]. Hemoglobin absorbance values of the samples were converted to μg/mL of hemoglobin by means of the calibration curve. Of the 200 initial samples, a total of 120 samples having an absorbance ≤ 0.085 (corresponding to a hemoglobin content below 4.52 μg/mL) were included in the study; 50 in the discovery and 70 in the validation phases, respectively. Viral RNA extraction from the sera was performed following standard Roche procedures (High Pure Viral RNA Kit, Roche Diagnostics GmbH, Germany). Presence or absence of PRRSV was determined by multiplex PCR of conserved regions of viral ORF7 using primers and conditions previously described [41, 42] . The test also enabled to discriminate European and American genotypes and could detect all the different viral strains present in the Lombardy region at the time of sample collection.
All the detailed steps of the SELDI-TOF MS process performed here are schematically represented [see Additional file 3: Figure S1 ]. The protocol follows the manufacturer's instructions with minor modifications (Bio-Rad Laboratories, ProteinChip W Serum Fractionation Kit manual).
Briefly, serum samples were pre-fractionated with U9 buffer (9 M urea, 2% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM Tris-HCl, pH = 9) to favor dissociation of protein complexes [Additional file 3: Figure S1A ].
Sera were fractionated using a ProteinChip Q strong anion-exchange resin filtration plate (Bio-Rad Laboratories, Hercules, CA). The filtration plate was re-hydrated and equilibrated with rehydration buffer (50 mM Tris-HCl, pH = 9) and the resin washed with rehydration buffer and U1 solution (1 M urea, 0.2% CHAPS, 50 mM Tris-HCl, pH = 9) [Additional file 3: Figure S1B ]. Serum samples were then mixed with U1 solution and added to the equilibrated filtration plate. Successive elutions with different buffers with decreasing pH and a final organic solvent (= different fractions) were collected by centrifugation. The buffers used included pH = 9 (50 mM Tris-HCl, 0.1% n-octyl β-D-glucopyranoside (OGP)), pH = 7 (50 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 0.1% OGP), pH = 5 (100 mM Na acetate, 0.1% OGP), pH = 4 (100 mM Na acetate, 0.1% OGP), pH = 3 (100 mM Na citrate, 0.1% OGP), and organic solvent (33.3% isopropanol, 16.7% acetonitrile, 0.1% trifluoroacetic acid) [Additional file 3: Figure S1C ].
The six pH fractions obtained (F1 = pH9, F2 = pH7, F3 = pH5, F4 = pH4, F5 = pH3, and F6 = organic solvent) were profiled on weak cation-exchange (CM10), immobilized metal affinity capture-copper (IMAC30-CU), and reverse-phase (H50) ProteinChip W arrays. The arrays were initially placed in a Bioprocessor (C50-30011, Bio-Rad Laboratories) and then treated according to their surface [Additional file 3: Figure S1D ]. Each sample fraction was then bound/spotted randomly to the different Pro-teinChip W arrays using array-specific binding buffers [Additional file 3: Figure S1E ]. A 50% saturated sinapinic acid (SPA) matrix solution was finally added to each spot on the ProteinChip array prior to the final analysis [Additional file 3: Figure S1F ].
ProteinChip arrays were read using a Ciphergen Protein-Chip Reader PCS4000 model and data were analyzed with Ciphergen Express Software (Ciphergen Biosystems).
Profiles were collected in the range 1-200 kDa at the two different ion focus mass 10 kDa ("low focus mass") and 50 kDa ("high focus mass"). The instrument was calibrated for dataset collection using all-in-one peptide standard (Bio-Rad Laboratories) in the 1-20 kDa range for 10 kDa low ion focus mass and all-in-one protein standard in the 20-200 kDa range for 50 kDa high ion focus mass [Additional file 3: Figure S1G ].
Spectra were normalized by total ion current, starting and ending at the M/Z of the collection ranges (1-20 or 20-200 kDa) after baseline subtraction and noise calculation. Outlier spectra were removed. The spectra were aligned to a reference spectrum with the normalization factor nearest 1.0. The spectra were aligned only if the percentage coefficient of variation was reduced after the alignment. Peaks from the different spectra were aligned using the cluster wizard function of the Ciphergen Express 3.0.6 software. The peak detection was automated within the M/Z range of analysis. Peaks were detected on the first pass when the signal-to-noise (S/N) ratio was 7 and the peak was 5 times the valley depth. Peaks below threshold were deleted and all first-pass peaks were preserved. Clusters were created within 0.15% of M/Z for each peak detected in the first pass. The clusters were completed by adding peaks with S/N ratio of 2 and two times the valley depth. P-values and ROC/AUC (Receiver Operating Characteristic/ Area Under Curve) values were calculated by using the P-value wizard.
A 2-tailed t-test was used for statistical analysis of differences in peak intensity between groups. P-values below 0.05 were considered statistically significant. Principal component analysis (PCA) and agglomerative hierarchical clustering algorithm were applied to investigate the pattern among the different statistically significant peaks.
PCA is a multivariate data analysis that transforms without a loss of essential information a number of correlated variables into a smaller number of uncorrelated variables called principal components (PCs), which can explain sufficiently the data structure. PCA transformation allows studying many variables simultaneously, showing how similar samples are correlated and grouped together. The data structure is visualized directly in a graphical way by projection of objects onto the space defined by the selected PCAs (for details see [43] ).
Finally, to evaluate the influence of external variables (e.g. sample processing and acquisition) on the system under study and to calculate the dispersion of the acquired data, the coefficient of variation (CV), which is the normalized measure of dispersion of a probability distribution and shows the% dispersion of the data in rapport to the media (intensity variation), was also calculated. Six serum samples commercially available were prepared and analyzed in parallel with the pig samples of both, discovery and validation phases. The CV was calculated for all fractions and surfaces by choosing 6 peaks evenly distributed along the entire range.
Additional file 1: Table S1 . Pigs tested with SELDI-TOF MS during the discovery phase of the study. List of the 25 positive and 25 negative pigs to PRRS (PCR-tested) analyzed with SELDI-TOF MS during the discovery phase of the study. The pig ID is reported with the total absorbance and the total amount of hemoglobin present in the sample, the status regarding the PRRS virus, as well as the sex and the number and location of the farm (MA = Mantua region, LO = Lodi region).
Additional file 2: Table S2 . Pigs tested with SELDI-TOF MS during the validation phase of the study. List of the 35 positive and 35 negative pigs to PRRS (PCR-tested) analyzed with SELDI-TOF MS during the validation phase of the study. The pig ID is reported with the total absorbance and the total amount of hemoglobin present in the sample, the status regarding the PRRS virus, as well as the sex and the number and location of the farm (MA = Mantua region, LO = Lodi region). Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region. The non-covalently associated surface (gp120) and transmembrane (gp41) subunits of the envelope glycoprotein are decorated on the surface of Human Immunodeficiency Virus Type-1 (HIV-1) as a trimeric spike [1] , and serve as a target for broadly neutralizing monoclonal antibodies (bNAbs) [2] [3] [4] . Because of its involvement in the primary steps of receptor [5] and co-receptor binding [6] , the envelope gp120 has been identified as a major target for HIV-1 NAbs [2, [7] [8] [9] [10] . However, the antigenic variability of exposed regions and low immunogenicity of conserved domains on gp120 impose great challenges to identify the vulnerable targets on HIV-1 [2, 4, 11] . Nevertheless, the conserved epitopes on gp120 have been identified using antibodies from neutralizing sera [12] [13] [14] and bNAbs [9, 10, [15] [16] [17] , which include the antibodies directed to the CD4 receptor binding site (CD4bs) and co-receptor binding site mainly the third variable region (V3) [10, [18] [19] [20] [21] [22] .
The crystal structure of V3 resolved recently shows that V3 protrudes~30 Å from the CD4-bound gp120 core, and this extended structure can be divided into three regions: the base (residues 1-8 and [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] , the stem (9-14 and 18-24) and the crown (residues 15-17) (residue numbering w.r.t. V3) [23, 24] . The V3 loop of gp120 is highly antigenic in humans [25] [26] [27] [28] , and was previously recognized as the principal neutralizing domain of HIV-1 [29] . However its role was shown to be restricted to type specific viruses [30, 31] and such an observation was supported by the extensive sequence variation in V3 from different viral isolates [32, 33] . Given the critical interaction with the co-receptors (CXCR4 or CCR5) on host cells, V3 conventionally must retain structurally conserved elements required for binding [34, 35] . More recently, studies have revealed that the V3 domain possesses conserved structural motifs despite the sequence variation, and is often accessible on the virus surface as a target for bNAbs [21, [36] [37] [38] .
Although, the V3 loop displays high structural conservation, yet the degree of cross reactive anti-V3 antibody response in individuals infected with diverse HIV-1 subtypes varies substantially [39] . This difference in antibody response to V3 loop, has been shown to be primarily determined by the four amino residues in the V3 crown (GPGQ or GPGR), which mostly form a type II β-turn [19, 40] . Interestingly, the anti-V3 monoclonal antibodies (mAbs) isolated from non-clade B infected individuals, bearing GPGQ at the tip of V3 display better neutralization capacity than subtype-B (having GPGR) derived anti-V3 mAbs [19] . Such an observation was substantiated by a study showing a high neutralization potential of the anti-V3 mAbs derived from Cameroon subjects infected with viruses harboring GPGQ (subtype-AG) at the V3 crown [21] . Further, in an immunization study carried out in rabbits with a gp120 DNA prime followed by a boost with various cholera toxin B (CTB) fusion proteins containing the V3 sequences from different HIV-1 subtypes, the CTB fusion protein containing a consensus-C (con-C) V3 sequence (V3C-CTB) (having a GPGQ motif at V3 crown), elicited a highly potent HIV-1 neutralizing response compared to the other V3-CTB constructs [41] .
A limited number of mAbs have been generated so far against the HIV-1 subtype-C viruses including mAbs against the V3 loop despite the fact that subtype-C accounts for more than 50% of the global HIV-1 infections [42] . The only known human anti-V3 bNAb is from a clade B infected patient [15] . Keeping in view the above facts, we generated here three anti-V3 human mAbs from HIV-1 infected Indian patients, using the EBV immortalization method of human hybridoma technology. The functional analysis of the HIV-1 antibodies generated in this study revealed cross reactive binding and neutralization of the viruses tested.
A total of 3321 culture wells (96 well plate) of PBMCs derived from 33 HIV-1 infected patients were established. After 2-3 weeks, the culture supernatants of approximately 6% transformed wells tested positive for reactivity with V3C-CTB (Table 1) . Three heterohybridomas producing anti-V3 mAbs were generated from different individuals (IDs; 277, 903 and 904). The mAbs belong to IgG1 subclass with one (904) using a lambda and the other two (277 and 903) with kappa light chain genes. Interestingly, the kappa light chain Abs displayed the same immunoglobulin heavy chain variable (IGHV) gene usage (3-30*03) while the other Ab (904) showed 1-8*01 gene usage ( Table 2 ). The complementarity determining region 3 for the heavy chain (CDRH3) were different for each mAb indicating their uniqueness ( Table 2) .
Cross reactive binding and epitope mapping of anti-V3 mAbs by ELISA All the plasma samples from patients recruited for this study, were previously screened for their relative binding to V3C and V3B peptides (Andrabi et al., submitted) and the data is here shown for only three samples from which anti-V3 mAbs were isolated ( Figure 1C ). The peptide binding assays revealed the cross reactive binding potential of the anti-V3 Abs in the plasma sample 277 and 904 while 903 showed subtype-C V3 specific binding.
In order to determine the specificity of the anti-V3 mAbs generated from these three patients, we performed the ELISA binding titration of the anti-V3 mAbs with recombinant envelope gp120 proteins and HIV-1 derived peptides (Table 3 ). In addition to three Indian anti-V3 mAbs, which were generated in this study, we also tested the binding of an anti-V3 bNAb (447-52D), isolated from a subtype-B HIV-1 infected individual living in USA. The binding curves of mAbs to con-C and B V3 peptides and subtype-C (Du156.12) and B (JRFL) gp120 proteins are depicted in the representative Figure 1 . The mAbs were also tested with a series of peptides and proteins for quality control purpose and the 50% maximal binding (Max50) binding titers are summarized (Table 3) . Overall, the mAbs 277 and 903 showed binding to subtype-A or C V3 while the antibody 904 also reacted with subtype-B V3 region ( Figure 1A -B, Table 3 ). We further tested the mAbs by ELISA binding assays using Three anti-V3 monoclonal Abs listed were derived from HIV-1 infected Indian donors. 2 Immunoglobulin gene usage for heavy (IGHV) and amino acid sequences of 3 CDRH3 domains was determined using IMGT system; an asterisk indicates allele. Figure 1 Relative binding affinity of anti-V3 antibodies to HIV-1 derived peptides and proteins. The binding pattern of anti-V3 mAbs derived from Indian donors (red) and 447-52D (an anti-V3 Ab isolated from HIV-1 subtype-B infected American individual) (green) to consensus-C and B V3 peptides (1A), and subtype-C (Du156.12) and subtype-B (JRFL) derived envelope gp120 proteins (1B). The binding of anti-V3 mAbs was tested by ELISA using mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). Human anti-parvovirus B19 mAb 1418 was used as negative control. Relative reactivity of anti-V3 plasma antibodies to consensus-B and C V3 peptides is shown in terms of 50% ELISA binding titers (Max50) for three patients from whom antibodies were generated (1C). Two plasma samples 277 and 904 showed cross clade reactive binding while 903 displayed subtype-C specific binding.
linear overlapping peptides encompassing mainly V3 region flanked with second constant (C2) and third constant (C3) region of gp120, to identify the core epitope recognized and found that all these anti-V3 mAbs, including mAb 447-52D, bind to the crown of V3 loop ( Table 4) .
The binding of Abs to HIV-1 derived peptides or proteins does not necessarily mean that these mAbs will be able to bind intact viruses, which essentially present a more native conformation. In order to address this possibility, we tested the anti-V3 mAbs for binding with Du156.12 (subtype-C) and JRFL (subtype-B) viruses in an intact virion binding assay. The two viruses (Du156.12 and JRFL) were chosen for this experiment on the basis of their V3 sequence similarity with the corresponding con-C and B V3 sequence ( Figure 2C ). Consistent with the binding of anti-V3 mAbs to Du156.12 and JRFL derived envelope gp120 proteins, the three mAbs showed differential binding affinity to intact virions, 904 displaying high affinity as compared with 277 and 903. In addition, the mAb 904 retained the crossreactive binding potential to the intact viruses, evident from the binding pattern ( Figure 2A ). The experiment was validated by testing the intact virion binding of anti-V3 mAb 447-52D to SF162 virus, known to have a well exposed V3 region and allows accessibility to Ab without any interference [43] [44] [45] . The binding of 447-52D to SF162 intact virus revealed a very high binding affinity (more than five folds) of mAb 447-52D as compared to binding of three anti-V3 mAbs to Du156.12 and JRFL at equivalent viral concentration (i,e 25 ng/ml) (Figure 2A -B). This differential binding of mAbs to intact virions could also be attributed to the number of potential N- Table 3 Binding of mAbs to subtype-A, B and C derived HIV-1 proteins and peptides 2 anti-V3 mAbs 1 Protein/peptide Subtype I 277 I 903 I 904 A 447-52D 1418
List of recombinant proteins and peptides derived from subtype-A, B and C HIV-1 viruses, NA: Not applicable. 2 Four anti-V3 antibodies, three from Indian I (277, 903 and 904) and one from American A (447-52D) HIV-1 infected donor were tested for their relative binding. The binding activity of anti-V3 mAbs against proteins and peptides was tested by ELISA using mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). The 50% binding titers (Max50, conc. μg/ml) of each antibody against the corresponding protein or peptide is depicted as numerical values in Bold (high affinity), Italic (low affinity) and >10, where Max50 value was not reached. Human anti-parvovirus B19 mAb 1418 was used as negative control. Each experiment was performed at least two independent times. Amino acid sequences of linear overlapping peptides encompassing the third variable region (V3: Underlined (middle)), flanked by second (C2: (left)) and third (C3: (right)) constant regions and are aligned with the corresponding consensus-C gp120 sequence. 2 Four anti-V3 antibodies, three from Indian I (277, 903 and 904) and one from American A (447-52D) HIV-1 infected donor were tested for their binding to overlapping peptides by ELISA using mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). The 50% binding titers (Max50, conc. μg/ml) of each antibody against the corresponding peptides is depicted as numerical values in Bold (high affinity), Italic (low affinity) and >10, where Max50 value was not reached. Human anti-parvovirus B19 mAb 1418 was used as negative control. Each experiment was performed at least two independent times.
glycosylation sites (PNGS) within the V1/V2 region (3 for SF162, while 6 and 7 PNGS for Du156.12 and JRFL respectively) of these viruses [46] . Overall the results suggest that the V3 epitopes recognized by three anti-V3 mAbs were exposed on both Du156.12 and JRFL, however mAbs 277 and 903 were not able to bind JRFL due to their subtype-C specific binding activity.
The anti-V3 mAbs were tested to assess their capacity to neutralize a panel of eleven tier 1 and 2 viruses from different HIV-1 subtypes using the standard TZM-bl cell assay. The anti-V3 mAbs showed effective neutralization against two, a subtype-A (DJ263) and a subtype-C (MW965), out of five tier 1 viruses while only one virus (a subtype-C, HIV-001428) out of six tier 2 viruses was neutralized by two of the mAbs (903 and 904) with relatively low efficiency ( Table 5) . None of the Indian anti-V3 mAbs were able to neutralize any of the subtype-B viruses, despite antibody 904 showing cross reactive binding, nevertheless, the number of viruses tested here was limited. In contrast, the mAb 447-52D reached IC50 neutralization titers with 5/11 viruses tested, consistent with the previous studies [15, 19] . 
Human monoclonal antibodies against the HIV-1 envelope glycoproteins are useful tools in the structural and functional analysis of the viral envelope and have crucial roles in guiding the design of prophylactic anti-HIV vaccines. Despite a huge expansion of HIV-1 subtype-C worldwide, the clade-C viruses remain to be one of the least studied subtypes especially in terms of HIV-1 neutralizing antibodies. Using the rationale from previous studies showing that viruses with GPGQ residues at the tip of the V3 crown of the HIV-1 envelope induce potent and cross reactive NAbs as compared to viruses with GPGR motif, we generated here three anti-V3 mAbs from Indian donors presumably infected with subtype-C viruses bearing GPGQ at the V3 crown [42] . The functional analysis of the Abs generated reveals a potent neutralization potential with tier 1 viruses while such activity was limited with the tier 2 viruses tested. The anti-V3 Abs were selected from EBV-transformed B-cell cultures of 33 HIV-1 infected antiretroviral drug naïve patients using V3C-CTB fusion protein [47] . The advantage of using a conformationally constrained instead of a linear V3 peptide for selection of mAbs from cultures or as animal immunogens has been previously demonstrated [36, 41, [48] [49] [50] . We found 1-25% (mean = 6%) of the transformed wells positive for binding with V3C-CTB in the first screening. The characteristic nature of the B-cells from HIV-1 infected subjects and the conditions used to immortalize them apparently affects the number and type of Ab-producing cell lines that grow out [51] . The overall positive percentage of Ab secreting culture wells was relatively good and could be attributed to the high titers of anti-V3 Ab reactivity of the corresponding plasma [28] . In contrast to a high percentage of positive secretors in the initial screening, we were able to stabilize only three (277, 903 and 904) anti-V3 Ab producing B-cell clones. This loss could be in part due to the outgrowth of the non-secretor B cells over the secretor B cells in subsequent steps of secondary screening, cell fusion and dilution cloning process. Moreover, the B cells from HIV-1 infected patients are mostly dysfunctional and polyclonally activated [52] , and such properties have been associated with a low persistence of EBV infectivity [51, 53, 54] .
The amino acid sequence variation of V3 across the various HIV-1 subtypes is often related to a differential immune response focused to V3 which is expected to originate due to the subtype specific conformational differences in the V3 region [55, 56] . For instance, the HIV-1 V3 crown residues GPGQ in non-clade B and GPGR in clade B viruses respectively are the major determinants of Ab binding and neutralization [19, 40] . Epitope mapping of the anti-V3 Abs with overlapping V3 peptides reveal that their core epitopes lie in the crown region only. Indeed, the recent immunological and structural studies of anti-V3 mAbs have observed similar pattern of binding, wherein essentially all the anti-V3 mAbs bind to~14 residues in the V3 crown [57] [58] [59] . Two of our anti-V3 mAbs (277, 903) showed binding to subtype-A or C but not to subtype-B derived proteins and peptides while mAb 904 displayed cross reactive binding with subtype-B as well. The binding pattern (in context of clade specific or cross reactive V3 antibodies) of the two anti-V3 mAbs 903 and 904 was similar to binding of polyclonal anti-V3 plasma antibodies from the respective patients, however it was different for mAb 277 in the context of the plasma Abs of this patient ( Figure 1A-C) . The finding highlights the importance of pre-screening of plasma for binding to peptides from different viral subtypes prior to isolation of mAbs. One plausible reason for the non-binding of mAb 277 to the V3B peptide in contrast to its corresponding polyclonal plasma may be attributed to the higher number of subtype-C specific B-cell clones in the B cell repertoire of this patient, as indicated by its very high binding to V3C ( Figure 1C ). It may also partly be ascribed to biased selection with a CTB construct containing only V3C sequence, which might allow it to preferentially pick up the B cell clone with clade-C V3 specificity. Interestingly, the anti-V3 mAbs 277 and 903, which show clade-A or C (both having a common GPGQ crown motif ) specific binding use the same variable heavy chain gene (VH3-30) whereas the cross reactive mAb 904 uses a different VH gene ( Table 1) . The finding suggests a possible association of antibody gene usage with epitope specificity, however the number of the mAbs generated in this study is too small for comparison. Remarkably, a recent analysis of anti-HIV Abs has pointed out a preferential usage of VH5-51 gene usage of anti-V3 Abs [60] , and such a preference was later shown to define a conserved antigenic structure within the V3 [61] .
The Ab accessibility on the HIV-1 native virus is often challenged by the glycosylation pattern and epitope masking [39, 62, 63] . This effect has been particularly recognized for the V3 region wherein the neighboring regions including V1/V2 shield the epitopes recognized by anti-V3 Abs [46] . Although studies have suggested that the HIV-1 V3 loop remains accessible on most of the viruses [36] , however this information is limited to subtype-B viruses and remains to be explored for other subtypes. Consistent with the binding to gp120 proteins (Du156.12 and JRFL), the three anti-V3 mAbs were able to bind intact native virions with a similar binding pattern (Figure 2A) . The results suggest that V3 epitopes are well exposed over the intact trimeric viruses (Du156.12 and JRFL), and these findings are highly supported by previous work in the literature [36, 43] . The rationale of using same proteins (gp120) and its corresponding viruses (intact virion) for the binding assays was to minimize the effect of both, the V3 loop sequence and the neighboring regions, on the local and global orientation of V3 and on the subsequent binding of Abs.
The anti-V3 mAbs showed potent neutralizing activity against subtype-A and C tier 1 viruses, however this activity was restricted for tier 2, especially the subtype-B viruses. The finding was intriguing given the ability of the anti-V3 mAbs to bind to two representative intact virions of subtype-B (JRFL) and subtype-C (Du156. 12) , and yet failing to reach IC50 neutralization titers. However, it should be noted that mAbs 903 and 904 which display a better affinity than 277, showed neutralization of up to 19% at 30 μg/ml, though not reaching IC50 neutralization titers, with these viruses (data not shown). Overall, the data suggest that higher concentrations of these mAbs may be effective in neutralization, however that needs to be confirmed in detail. Together, these results suggest that effective concentrations for binding and neutralization may vary substantially, and the high affinity binding by mAb might be critical for neutralization. The data are highly supported by various studies conducted previously [15, 36] .
We isolated here three anti-V3 mAbs from HIV-1 infected donors from India which can effectively neutralize tier 1 viruses but are less effective with tier 2 viruses, however this needs to be confirmed by testing them with a broader panel of viruses from different HIV-1 subtypes. This study demonstrates the importance of pre-screening of plasma Abs for cross reactive binding, for production of mAbs and the idea can likewise be employed for other antigenic regions. Also the study highlights the significance of the antibody affinity, which may probably be equally important as its epitope accessibility, for effective viral neutralization. Furthermore, the analysis of the mAbs generated in this study will allow us to identify epitopes that are unique to clade C viruses and also those that are shared with other subtypes in the context of V3 loop, and the data may provide useful information for HIV-1 vaccine design.
The study was approved by the ethics committee of All India Institute of Medical Sciences (AIIMS) New Delhi, and the written informed consent for research and publication of the data was obtained from all the participants.
Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20-57 years (median = 33 years) were recruited in this study from the Regional STD Teaching Training & Research centre, Safdarjang Hospital, New Delhi, India during the period 2008-2011. The patients had a median CD4 count of 449 (range = 203-966) cells/cubic millimeter (Additional file 1: Table S1 ). The whole blood samples of HIV-1 positive donors were collected in EDTA vacutainers, plasma was separated by centrifugation at 300 g and stored in aliquots at -80°C until use. The plasma samples were heat inactivated at 56°C for 1 h before using in the assays. The patients have been previously shown to have high titers of anti-V3 Abs in their plasma and a good proportion of these V3 directed Abs displayed cross reactivity [28] , (Andrabi et al., submitted) . Presumably, the patients were infected with subtype-C viruses, which is a major subtype in India [64, 65] . Indeed, the envelope sequences (partial C2-C5 of envelope gp120) of a few patients revealed that majority of the patients were infected with subtype-C viruses (Andrabi et al., submitted) .
The mAbs were generated using cellular techniques as previously described [66] . Briefly, peripheral blood mononuclear cells (PBMCs) were EBV transformed in 96-well plates and cultured with a polyclonal B cell activator, CpG [67] , which enhanced EBV infection and B cell transformation. The wells containing Ab-producing cells were identified by testing the culture supernatants for binding activity to V3C-CTB [47] . Cells from wells that test positive were expanded and fused with the heteromyeloma cell line SHM-D33 (ATCC; catalog no. 1668). The fused cells that continued to make functional Abs were repeatedly cloned until monoclonality was achieved. The Abs were purified from culture supernatants using Protein G affinity columns (GE Healthcare) and concentration of the mAbs was determined by noncommercial ELISA. The anti-V3 mAb 447-52D generated previously from a clade B infected individual and a mAb 1418, specific to parvovirus B19 [68] , were used as control Abs in this study.
Nucleotide sequence of Ig variable genes of human anti-V3 mAbs was determined as previously described [60, 61] . The messenger RNA was extracted from hybridoma cell lines producing anti-V3 mAbs and reverse transcribed into cDNA using oligo dT primer. Amplification of the variable fragment was performed by PCR using different gene family-specific sets of primers and cDNA as template. were located at 5 0 end of V genes. Reverse primer [5 0 -CTTGGTGGARGCTGARGAGACGGTGACC-3 0 ] was located at the 3 0 end of JH segment and up to 12 nucleotides at the 5 0 of the constant region of IgG [69] . PCR amplification was performed using cycling program of 2 min at 94°C, 35 cycles of 60s at 94°C, 60s at 56°C, and 90s at 72°C, followed 8 min at 72°C. Ethidium bromidestained 0.8% agarose gels were used to visualize the PCR products. The bands of the appropriate size were excised and cleaned with GeneElute Minus EtBr Spin Column (Sigma, USA). PCR products were sequenced (Macrogen, South Korea) in both directions using the primers applied for amplification. The sequence data were analyzed using the International ImMunoGeneTics (IMGT) information system (http://imgt.cines.fr).
Five recombinant gp120s representing sequences of primary HIV-1 isolates from clade A, B and C (produced in 293 cells) and a p24 protein were purchased from Immune Technology Corp. (New York, NY). A set of 12 linear overlapping peptides (each 15mer with an 11 amino acid overlap or a 4 amino acid walk) corresponding to the sequence of consensus subtype-C V3 gp120 and CEF Control Peptide Pool (PP) (Cat. No. 9808) were obtained from the NIH AIDS Research and Reference Reagent Program (NIH, ARRRP). Two full length (35mer) peptides corresponding to the consensus-B (CTRPNNNTRKSIHIGPGRAFYTTGEIIG DIRQAHC) (V3B) and con-C (CTRPNNNTRKSIRI GPGQTFYATGDIIGDIRQAHC) (V3C) of V3 gp120, a 24mer con-C MPER (DLLALDSWKNLWNWFDITNW LWYIK) and a 19mer con-C IDR (LGIWGCSGKLICT TAVPWN) peptides of gp41 were selected from Los Alamos HIV-1 sequence database (http://hiv.lanl.gov), and were synthesized from Sigma Genosys, USA. The peptides were HPLC purified to >95% purity (based on information provided by company). The V3-cholera toxin B (CTB) fusion protein (V3C-CTB) and wild type CTB (WT-CTB) used for screening of antibody cultures were kindly provided by Prof. Susan Zolla Pazner from New York University School of Medicine.
The binding activity of anti-V3 mAbs against gp120 proteins and peptides (including the V3 overlapping peptides) were tested by enzyme-linked immunosorbent assay (ELISA) as described [18] . Briefly, ELISA plates were coated overnight with protein or peptide at 1.0 μg/ml, blocked with 2% bovine serum albumin (BSA) in PBS, and then incubated with mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). The bound mAbs were detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG (γ specific) (SouthernBiotech, Birmingham, AL) followed by adding substrate to develop color and the plates were read at 405 nm. The relative affinities of mAbs were determined by measuring the concentration of mAbs required for 50% maximal binding (Max50), defined when the binding curve reached the saturation level as described [70] .
The binding of mAbs to intact virions was determined with a capture assay as previously described [71] . Briefly, a 96-well plate was coated overnight at 4°C with goat anti-human immunoglobulin G (IgG) Fc Abs (Sigma) at 4.0 μg/ml, and then anti-V3 human mAbs at a saturating level of 10 μg/ml were added for 1.5 h incubation at 37°C. The plate was blocked with 0.5% BSA in phosphatebuffered saline (PBS) containing 10% goat serum and 10 μg of human IgG/ml. The culture supernatant, containing pseudotyped viruses (Du156.12 and JRFL) at a p24 concentration of 25 ng/ml, and the two fold diluted primary isolate (SF162) with a starting p24/ml concentration of 50 ng were added. The plate was incubated overnight at room temperature. Viruses captured by immobilized mAbs were lysed with 1% Triton-X in PBS. Between each step of the assay, the plate was washed with PBS containing 0.05% Tween 20, pH 7.4. The p24 in the virus lysate was quantified by using a commercial ELISA. The anti-V3 Ab 447-52D and SF162, a subtype-B virus known to be sensitive to most of anti-V3 Abs were used as assay control while the human mAb, 1418, was used as a negative control.
Eleven HIV-1 subtype A, B and C viruses including five primary isolates (MW965, DJ263, SF162, JR-CSF and 92RW009) and six pseudotyped viruses (HIV-001428, ZM109F.PB4, ZM233M.PB6, Du156.12, JRFL and RHPA4259.7) were used for this study. All the primary viruses and envelope clones were obtained from the NIH, ARRRP. The HIV-1 isolates were expanded by only one cycle of growth on phytohemagglutinin (PHA) and interleukin-2 (IL-2)-stimulated PBMCs, as described previously [45] to avoid alterations in env sequences due to multiple rounds of expansion. Pseudotyped viruses were produced by co-transfection of the rev/env expression plasmid and an env-deficient HIV-1 backbone vector (pSG3ΔEnv) into exponentially dividing 293 T cells (ATCC; catalog no. 11268), in 6-well tissue culture plates (Corning Inc) using calcium phosphate (Promega Inc) method. Pseudovirus-containing culture supernatants were harvested 48 hours post transfection filtered (0.45 μm pore size) and stored at −80°C in 1 ml aliquots. The 50% tissue culture infectious dose (TCID50) was determined in TZM-bl cells.
Neutralization of viruses by anti-V3 mAbs was measured as a reduction in luciferase gene expression after a single round of infection of JC53bl-13 cells, also known as TZM-bl cells (NIH, ARRRP; catalog no. 8129), with viruses [72, 73] . Briefly, 200 TCID50 of pseudovirus was pre-incubated for 1 hr at 37°C, 5% CO 2 in 96-well flatbottom culture plates, with serial dilutions of mAbs, starting from 30 μg/ml. Freshly trypsinized TZM-bl cells (10,000 cells in 100 μl of growth medium containing DEAE Dextran and protease inhibitor indianavir (in case of primary isolates only), were added to each well of the mAb/virus mixtures in duplicates. One set of control wells received cells plus pseudovirus (virus control) and another set received cells only (background control). After 48 hours of incubation at 37°C, 5% CO 2 , luciferase activity was measured by using the Bright-Glo Luciferase Assay System (Promega Inc.). The 50% inhibitory concentration of mAb (IC50) was determined at which relative luminescence units (RLU) were reduced 50% compared to virus control wells. Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study. The development of a safe and effective prophylactic vaccine against human immunodeficiency virus type I (HIV-1) is a global health priority. During the past two decades, significant efforts have been made to develop such a vaccine. Among the few candidate HIV-1 vaccines tested in large Phase IIb or III clinical trials, only the RV144 trial, evaluating a recombinant canarypox vector prime and HIV-1 gp120 protein boost, showed a modest efficacy (31 %) against HIV-1 acquisition (reviewed by McElrath and Haynes 2010; McMichael et al. 2010) . Although ultimately, a preventive vaccine against HIV-1 inducing sterile immunity would be optimal, vaccines that would reduce viral load and disease progression by induction of strong and polyfunctional T cell responses should also prove beneficial (McMichael et al. 2010) .
Live attenuated measles virus (MV) vaccine strains, such as the widely used and strongly immunogenic Schwarz strain (Griffin 2007) , are replicating RNA viruses (Paramyxoviridae family), capable of inducing long-lived antibody and memory T cell responses (Ovsyannikova et al. 2003; Vandermeulen et al. 2007 ). Besides being highly efficacious, these vaccines are also recognized as safe (WHO 2009) , as MV replicates in the cytoplasm and does not integrate into the host cell genome. Moreover, reversion of the vaccine genome into a pathogenic form has never been observed. The experience accumulated with these vaccines in the past 50 years, and their capacity to induce both CD4 + and CD8 + T cells, render recombinant MV vectors an attractive platform for vaccines aimed to induce T cell responses specific for the HIV-1 transgene. For several of these vaccines expressing HIV-1 antigens, the immunogenicity has been preclinically demonstrated (Combredet et al. 2003; Guerbois et al. 2009; Liniger et al. 2009; Lorin et al. 2004) .
We constructed the HIV-1 vaccine candidate MV1-F4, using an in vivo replication-competent MV vector (Combredet et al. 2003 ) derived from the Schwarz vaccine strain, to generate recombinant MV expressing the F4 antigen. F4 is a fusion protein comprising the clade B viral antigens p17, p24, reverse transcriptase and the regulatory protein Nef. Combined with AS01 (a liposome-based Adjuvant System containing 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and QS21; Garçon et al. 2007 ), this F4 antigen was shown to induce potent polyfunctional CD4 + T cell responses in HIVseronegative volunteers (Van Braeckel et al. 2011 ).
We conducted a study of the biodistribution, shedding and single-and repeated-dose toxicity of one or three intramuscular (IM) immunizations with MV1-F4 in cynomolgus macaques. The potential intrinsic toxicity of MV1-F4 was studied, as well as the potential immune-mediated toxicity resulting from the host response to the vaccine. The resulting toxicity and biodistribution profiles, including any target organs identified, could be used to guide clinical safety monitoring, while the shedding profile is crucial for determining the potential of infectious viral dissemination by future vaccine recipients, and thus of person-to-person transmission of the virus. The biodistribution, shedding and toxicity profiles of the MV1-F4 vaccine were compared to those of either the reference vaccine (the live attenuated monovalent Schwarz MV vaccine Rouvax), or saline.
Humans are the natural hosts of MV. Measles pathogenesis has traditionally been studied in non-human primates as no suitable alternative models exist. Live attenuated MV vaccines are generally non-infectious in rodents, except for cotton rats (in which MV replication is restricted to the lungs) and transgenic mice (which reproduce only limited aspects of MV pathogenesis) (de Swart 2008) . In the current study, we used (MV-seronegative) cynomolgus monkeys. This species is highly sensitive to MV infection and able to develop pathologic lesions and clinical symptoms comparable to those in human MV infections (Kobune et al. 1996) . Moreover, their size permits the administration of a full human vaccine dose.
As none of the known Schwarz strain virus traits, including viral envelope proteins, were altered for the construction of the MV1-F4 vector, we hypothesized that the toxicological profile, tropism and shedding capacity of MV1-F4 is similar to those of the parental strain (as represented by the reference vaccine). In addition, we hypothesized that both vaccine viruses have the potential to spread into a large variety of organs, as attenuated MV strains can use the CD46 receptor (which is ubiquitously expressed in both humans and cynomolgus macaques; Sakurai et al. 2008) , in addition to the receptors used by wild-type MV (Dorig et al. 1993) . Wildtype MV strains use primarily the signaling lymphocyte activation molecule (SLAM/CD150) expressed on certain immune cells, and likely other low-affinity receptors to enter CD150-negative epithelial cells (Tatsuo et al. 2000b; Watanabe et al. 2010) . As a result, these strains replicate predominantly in lymphoid organs and epithelial tissues (Griffin 2007; Takeda 2008) . Consequently, MV1-F4 and Rouvax were expected to spread not only to the latter organs and tissues, but also to other parts of the body.
The MV vector pTM-MV-Schw that was used to construct the MV1-F4 recombinant virus has been described previously (Combredet et al. 2003) . This vector contains an infectious MV cDNA corresponding to the anti-genome of the Schwarz MV vaccine strain. An additional transcription unit (ATU) was inserted into pTM-MV-Schw in order to sub-clone the F4 fusion protein sequence. F4 has been described previously (Van Braeckel et al. 2011 ) and comprises HIV-1 subtype B antigens p24 (BH10), RT (HXB2), Nef (Bru-Lai) and p17 (BH10). The ATU contains a cloning-site cassette inserted into a copy of the original N-P intergenic region of the MV genome. This region contains the cis-acting sequences required for transcription of the viral P gene, enabling the recombinant MV to express the F4 protein like a measles gene. The ATU was introduced into the plasmid backbone by site-directed mutagenesis between the MV P and M genes, resulting in the plasmid pTM-MVSchw-ATU2_F4co_mut. MV1-F4 virus was rescued from the pTM-MVSchw-ATU2_F4co_mut plasmid using a helper cell-based system developed at the Institut Pasteur. Briefly, helper HEK293 cells expressing both the T7-RNA polymerase and the Schwarz MV N and P proteins were co-transfected with the pTM-MVSchw-ATU2_F4co_mut cDNA and a plasmid expressing the Schwarz MV polymerase L. Subsequently, transfected HEK293-T7-MV helper cells were gently harvested and co-cultured with MRC-5 cells for the amplification of the MV1-F4 virus. Virus titers were determined by endpoint titration on Vero cells and were expressed as 50 % cell culture infectious dose (CCID 50 )/ml.
The selected route of administration (IM) and dose were identical to those intended to be used in the first MV1-F4 clinical trial, with the dose-level representing the highest intended human dose. One-dose and three-dose schedules were used, with the three-dose schedule representing the number of doses to be used in the clinical study, plus one. One milliliter of MV1-F4 vaccine (viral titer 1.6 × 10 4 CCID 50 /ml) was injected intramuscularly. The reference vaccine, the commercial (Schwarz) MV vaccine Rouvax (Sanofi-Aventis, Paris, France) was used according to the manufacturer's procedure for IM administration, with each vaccine dose containing 3.9×10 3 CCID 50 of the Schwarz measles vaccine. This virus titer was determined with the same assay as was used for the MV1-F4 vaccine (endpoint titration on Vero cells). All injections were administered in the thigh muscle.
The study included purpose-bred cynomolgus monkeys (Macaca fascicularis), aged either 2-3 years (males and females) or 7-12 years (sexually mature males), obtained from Noveprim Ltd (Port Louis, Mauritius) that were seronegative for anti-MV antibodies. During the full experimentation period, the animals were housed in a dedicated primate unit under controlled environmental conditions. Due to the biosafety level of the MV1-F4 candidate vaccine (class 2, group II), they were housed individually in stainless steel cages (level A2, L2 confinement) and appropriate precautions were established. The study was conducted in compliance with the European regulations regarding the protection of animals used for experimental and other scientific purposes, and an ethical committee reviewed the study plan before the initiation of the study.
The study design followed the guidelines published by the European Medicines Agency (EMA) as well as other relevant guidelines (EMEA 1995 (EMEA , 2000 WHO 2005) . All experiments were performed under good laboratory practices (GLP) conditions. Animals were inoculated and monitored at the laboratories of the Centre International de Toxicologie (CIT; Evreux, France). Of the 42 monkeys included in the study, 18 young males and 18 young females were allocated to treatment groups 1-6 (Table 1) . Animals were allocated to groups (by sex) using a computerized randomization procedure (CITOX software, developed in-house at CIT, Evreux, France). In addition, six sexually mature males (three per group) were allocated to treatment groups 7 and 8 using the same randomization procedure, and were only used for collection of semen and peripheral blood mononuclear cells (PBMC), as well as for selected clinical observations (body weight, body temperature and food consumption). Treatments included 3.9 × 10 3 CCID 50 of Rouvax, 1.6 × 10 4 CCID 50 of MV1-F4 vaccine, or saline, and were administered according to either a one-dose schedule (injection at day 1) or a three-dose schedule (injections at days 1, 29 and 57). Monkeys were monitored clinically until sacrifice (groups 1-6) or until return to laboratory stock at day 85 (groups 7 and 8). Upon completion of the observation period, animals of groups 1-6 were sedated with ketamine hydrochloride (Imalgène, Mérial, Lyon, France), then anesthetized with thiopental and sacrificed by exsanguination. Necropsy was performed at two time-points, either within the expected peak of viremia (10 days after the first vaccine dose (day 11) for groups 1-3) or when clearance of infectious MV was expected to have occurred (represented by 28 days after the third dose [day 85] for groups 4-6) (Auwaerter et al. 1999; Pan et al. 2005; Permar et al. 2003) . Single-and repeated-dose toxicity was assessed at various time points (Table 2 ). Shedding and post-mortem analyses were conducted at Texcell (Evry, France). All other procedures were conducted at CIT.
Animals were monitored at least twice daily for mortality, morbidity and clinical signs of toxicity. Toxicological parameters included dermal reactions at the injection sites, body weight, food consumption, rectal body temperature, electrocardiography and ophthalmology. Complete clinical examinations were performed pre-treatment and thereafter once weekly. Sedation, used for electrocardiography, ophthalmology and occasionally for weighing, was done by IM administered ketamine hydrochloride (Imalgène).
At 3 and 24 h after each immunization, dermal reactions, including edema and erythema formation, were evaluated using the Draize scale, and any other lesions were noted. Reactions persisting for 48 h after immunization were evaluated daily until disappearance.
Electrocardiographic examinations were performed using a Cardioline Delta 3 Plus and Cardiovit AT-6 (Schiller AG, Baar, Switzerland) with standard leads I-III, starting with determining the heart rate, PQ and QT intervals and the QRS-complex duration on lead II.
Ophthalmology included assessment of pupillary light reflexes using tropicamide (Mydriaticum, Théa, Clermont-Ferrand, France), examination of appendages, optic media and fundus by indirect ophthalmoscopy (Oméga 180, Heine, Germany) and of anterior segments and lenses (portable slitlamp biomicroscope, model SL-15; Kowa, Japan).
Peripheral blood samples were collected without sedation at different time points after the first and the third dose (Table 2), in tubes containing EDTA, sodium citrate or lithium heparin (for haematology, coagulation parameters or blood biochemistry, respectively).
Haematology (i.e., erythrocyte count, haemoglobin (HB), mean and packed cell volumes, mean cell HB concentration, mean cell HB, thrombocytes, leucocytes (differential) and reticulocytes) was determined by ADVIA 120 haematology analyser (Siemens, Saint-Denis, France). Leukocyte differential analysis (with cell morphology) was assessed in blood smears stained with May-Grünwald-Giemsa. Coagulation parameters (i.e., prothrombin time, activated partial thromboplastin time and fibrinogen) were measured with an ACL300 coagulation analyzer (Beckman Coulter, Instrumentation Laboratory, France). Complete blood biochemistry was assessed by the ADVIA 1650 Chemistry System (Siemens) using whole blood.
Urinalysis (including volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood (HB) and urobilinogen) was done using a Clinitrek 500 urine chemistry analyzer (Siemens).
Blood samples for assessment of humoral responses were taken at prevaccination and days 11, 29, 56, 67 and 85. Anti-MV humoral responses in sera were measured using an anti-MV enzyme-linked immunosorbent assay (ELISA) adapted from the commercial immunoassay Enzygnost Anti-Measles Virus/Ig (Dade-Behring, Illinois, USA). A peroxidase-labelled anti-monkey immunoglobulin (Ig) secondary antibody (Rockland, Pennsylvania, USA) was used in the assay. Animals were considered to be responders if they were seropositive for anti-MV antibodies, i.e., if the absorbance (measured at 490/620 nm) exceeded the cut-off value, for a serum dilution of 1/500. The cut-off values (percentile 100) were determined per gender for sera collected prior to vaccination (serum dilution 1/500), and were found to be equal to 1.74 and 1.22 for sera from female and male monkeys, respectively.
At necropsy, organs were weighed and subjected to macroscopic and microscopic examination. The selection of organs to be examined for gross pathology and histopathology analyses followed the applicable European and international guidelines (EMEA 1995 (EMEA , 2000 WHO 2005) . Of this selection, the following organs were weighed: adrenals, brain, epididymides, heart, kidneys, cervical and iliac lymph nodes, liver, lungs with bronchi, ovaries, pituitary gland, prostate, spleen, testes, thymus, thyroid with parathyroid and uterus. Ratios of organ weight to body weight (at necropsy) were calculated. For gross pathology, the external surface of the body, orifices, the neck with organs and tissues, the cranial cavity with surfaces of the brain and spinal cord and the thoracic, abdominal and pelvic cavities with their contents were examined. For histopathological examination, 4-μm-thick sections of tissues were stained with haematoxylin and eosin. Sample processing for biodistribution and shedding analyses
The selection of organs to be examined for biodistribution analyses followed the applicable European and international guidelines (EMEA 1995 (EMEA , 2000 WHO 2005) . For biodistribution analyses, tissue samples (3×3×3 mm 3 ) were dissected out, rinsed with cold phosphate-buffered saline (PBS) and snap-frozen in cryotubes in liquid nitrogen. For shedding analyses, selected excretions and biological fluids (i.e., semen [only for groups 7 and 8], serum, urine, throat and nasal swabs, saliva, vaginal secretion and faeces) and PBMC were collected. Throat swabs, saliva, nasal secretions and vaginal secretions were conserved in M4RT tubes (Remel, Oxoid, Cedex, France) at −70°C. Semen and faeces were directly frozen in cryotubes and conserved at −70°C. Immediately after collection, urine was centrifuged (800×g, 10 min, 4°C) and the pellet was resuspended in PBS and stored at −70°C. PBMC were isolated from EDTA blood samples by gradient centrifugation and frozen in PBS (−70°C). To obtain serum, blood was collected in plain tubes, incubated (20 min, room temperature) and centrifuged (3,000×g, 10 min, 4°C).
Samples of organs, tissues, excretions and biological fluids were used for RNA extraction and MV N-specific, quantitative reverse transcriptase (RT) PCR analysis (RT-qPCR). Briefly, cDNA was synthesized from the RNA templates, and then amplified by qPCR specific for the MV N gene. This gene is identical in both vaccine viruses used in this study.
Frozen organ samples were homogenized using a Tissue Lyser (30 Hz for ≥2 min; Qiagen, Valencia, CA) and kept on ice. Samples were then lysed using 1 ml of Qiazol lysis reagent (Qiagen). Frozen PBMC, excretions and biological fluids were directly (for urine, serum, throat swabs, nasal swabs and vaginal secretion), or with a 1/45 dilution factor (for semen) submitted to RNA extraction. Faeces was resuspended into PBS to 1/10 (v/v), centrifuged, and the supernatant was filtered (0.22 μm). 
a Body weights: recorded at pre-treatment, days 1 and 4 and then once weekly until study end. Food consumption: estimated daily from 5 days before start throughout the study b Dermal observations at the injection sites were recorded at 3 and 24 h after each vaccination c Performed for groups 4-6 only d On days 1 and 57, tests were done just before and 1 h post immunization. A test at day 85 was done before sacrifice for only one animal e Blood sampling for clinical pathology performed for groups 1-6 only f At day 55 or day 56, blood samples for haematology, coagulation parameters and blood biochemistry were taken before immunisation. On day 85, samples were taken before necropsy g Day 11, day 85 0 time points of termination of the animals of groups 1-3, groups 4-6 respectively Total RNA was extracted from the samples using an RNeasy 96 kit with the Biorobot 3000 (both Qiagen) and reagents and protocols recommended by the manufacturer. Briefly, the lysed samples in 0.2 ml of chloroform were homogenized (15 s), left at room temperature for 2-3 min and centrifuged (12,000×g, 15 min, 5°C). The aqueous phase was transferred to 96-well plates and placed on the Biorobot for automatic extraction (by vacuum or centrifugation). Ethanol (600 μl, 70 % (v/v)) was added to each well. After mixing, samples were placed in the column for RNA adsorption on the membranes. After three wash steps (once with 800 μl RW1 buffer and twice with 800 μl RPE buffer), columns were centrifuged (5,600×g, 10 min). RNA was then eluted with 60 μl elution buffer (RNase free water) and stored at temperatures below −70°C.
RT and qPCR were performed using the AgPath-ID Onestep RT-PCR kit (Applied Biosystems, Inc., Foster City, CA) and the LightCycler 480 and software version 1.5.0.39 (Roche Applied Sciences). The forward primer was MeN_TMF(5′-GCGAGAGCTGCCCATCTTC), the reverse primer was MeN_TMR (5′-ACTCCGTTGCAGTGTCAATGTC) and the forward MeN_TMP probe was (6-FAM)~AACCGGCA CACCC~(NFQ), with the fluorogenic probe located in the region bracketed by forward and reverse primers. Cycling conditions were 45°C for 10 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 45 s, followed by 40°C for 2 min. The standard curve and RT-qPCR parameter determination was performed using an in vitro transcript RNA template with the sequence of the N gene of the measles vector. MV1-F4 suspension (GSK; EF4MA001A, viral titer 4.2 log CCID 50 /ml, 7.2 log genome equivalent (geq)/ml, diluted to 10 4 copies geq), was used as positive extraction control. Negative (RNase/DNase free H 2 O) and positive extraction controls were run in quadruplicate and duplicate reactions, respectively. MV1-F4 RNA recovery was assessed by N-specific RT-qPCR after spiking each type of tissue with MV1-F4 at various concentrations, and ranged from 90 % to 100 %. The presence of MV1-F4 RNA was considered positive if the measurements exceeded the limit of detection (LOD) of 100 geq/reaction, and if the acceptance criteria (error <0.2, efficiency>90 % and crossing point <45) were fulfilled. The LOD was defined as the lowest amount of copies that can be detected (but not necessarily quantified as an exact value) with 95 % probability. All samples were tested once.
In vitro infectivity and immunofluorescence assays All samples in which a RT-qPCR signal was detected (either confirmed [above LOD], or at a very low level [below LOD]) were submitted to an in vitro infectivity assay. The presence of infectious viral particles for all fluids except semen was considered positive for values exceeding the LOD of 10 CCID 50 /well (with 95 % probability). Nevertheless, the assay was designed to detect one infected cell that propagates the virus to its adjacent cells, resulting in a plaque forming unit (as aided by the presence of agarose in the culture medium). Therefore, it was assumed that the system was able to detect 1 CCID 50 /well (but with a probability of detection of less than 95 %). However, for this to occur at least 10 CCID 50 should be present in the inoculum, in order to allow the adsorbtion onto the cells. For semen, validation of the infectivity assay could not be conducted due to high bacterial contamination.
Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-5 % fetal calf serum (FCS)-4 mM glutamine. Before seeding the cells, a sterilized (ethanol, UV) coverslip (18×18 mm 2 ) was placed in each well. One day before inoculation, 6-well plates were seeded with 4×10 5 Vero cells/well. Biological fluid samples were diluted (1: 15.625 for serum, 1:10 for all other fluids) in dilution medium (DMEM-1 % antibiotics) and 700 μl of each fluid sample was added to Vero cells for 2 h of inoculation (37°C, 5 % CO 2 ). Inoculum was then removed and 2 ml of survival medium (DMEM-2 % FCS-4 mM of glutamine-1 % antibiotics) supplemented with 1 % (v/v) low-melting agarose was added. The plates were incubated (37°C, 5 % CO 2 ) for 6 days. As a positive control, Vero cells were inoculated with100 CCID 50 of MV1-F4 viral suspension (as described above) and diluted in 700 μl dilution medium. As a negative control, Vero cells were inoculated with 700 μl dilution medium.
The presence of infectious viral particles was revealed by immunofluorescence assay (IFA), using an antibody directed against the measles N protein. The acceptance criteria of the assay were the absence or presence of fluorescence, respectively, in the negative or the positive controls.
After 6 days of incubation, the culture medium was removed and 2 ml of 4 % paraformaldehyde was added (15 min, 5°C). The slides were washed with PBS and incubated overnight with 2 % goat serum at 5°C. Next, 100 μl of the primary antibody (mouse anti-MV-N monoclonal antibody [# MAB8906; Chemicon, Temecula, CA, USA]), diluted 1:200 in PBS-2 % goat serum-0.1 % saponin, was added to each well (60 min, room temperature). After washing twice with PBS, 100 μl of the secondary antibody (goat anti-mouse F(ab′) 2 -Cy3 [# LU1513615; Jackson ImmunoResearch, West Grove, PA, USA]) diluted to 1:500 in PBS-2 % goat serum-0.1 % saponin, was added to each well. After incubation (45 min, room temperature), coverslips were washed twice with PBS, rinsed with water, mounted on a slide using Vectashield DAPI mounting medium (Vector Laboratories, Peterborough, UK) and inspected under fluorescence microscope. Positivity or negativity was checked by the presence or absence of fluorescence, respectively.
The following sequence was used for statistical analyses of body weights, body temperatures and electrocardiography, haematology, blood biochemistry and urinalysis data. A test of normality using the Kolmogorov-Lilliefors test was performed, followed by log-transformation of the data if normality assumptions were not satisfied. Assessment of homogeneity of variance was performed using the Bartlett test. If the homogeneity of variance was not rejected, the treatment groups were compared to the control group using the Dunnett test, in other cases the Dunn test was applied. The same procedure, but without logtransformation of the data, was performed for the statistical analysis of organ weights using PathData software (version 6.2b5). Statistical significance was expressed as p<0.05 or p<0.01.
No mortality or morbidity was observed in any of the groups (Table 3 ). The only clinical effect recorded for a vaccine group was marked dehydration (without diarrhoea) in a Rouvax-treated male on days 56-62, which resolved within the following 2 days. Scab formation was noted in one Rouvax-treated male each of the one-dose and three-dose groups. These effects were incidental, self-limiting and of a mild-to-moderate nature, and therefore considered toxicologically irrelevant. In the controls of group 4, incidental vomiting (day 1; one male) and very slight erythema (days 1-3, one female) were noted.
For both sexes, mean body weights of vaccine and control groups were not significantly different and remained virtually Organ weights -Elevated absolute and relative spleen weights on days 11 (in males) and day 85 (in both genders, statistically significant in Rouvax-treated females).
No changes -Elevated mean absolute and relative weights of cervical and iliac lymph nodes, mainly in treated males (both vaccines; days 11 and 85).
-Lowered absolute and relative thymus weights in treated males (both vaccines; days 11 and 85).
Histopathology -Increased size of white pulp for both sexes on day 11 (MV1-F4) and day 85 (both treatments, corresponding with higher spleen weights).
Increased size of white pulp in females (day 11).
-Increased sizes/numbers of germinal centers in mandibular lymph nodes in MV1-F4 treated males (day 11).
Increased sizes/numbers of germinal centers in mandibular lymph nodes in females (day 85).
M/F male/female, IS Injection site, aPTT activated partial thromboplastin time, CK creatine kinase constant, with minor increases between days 0 and 85. Apart from scheduled fasting days prior to procedures, the individual food consumption was generally 75-100 % of the daily ration across groups. On days when animals were sedated, the consumption was typically 25-50 % of the daily ration, and occasionally 0 % (on days 1 and 57 for nearly all females of groups 4-6, and on day 84 for two Rouvax-treated males). These fasting periods had no impact on body weights and were similar across control and vaccine groups, and thus considered to be unrelated to immunization. Mean rectal temperatures of groups of vaccinated males were not significantly different from those of the control groups at all time points. For females, the sporadically noted statistical differences between groups remained within the ranges commonly recorded in animals of this age housed in similar experimental conditions. Consequently, no treatment-related effects on body temperature were observed in the study.
There were no treatment-related abnormalities in the qualitative and quantitative electrocardiography parameters. Although low-amplitude R and P waves and high-or lowamplitude, biphasic and negative T waves were noted, these findings were isolated, observed both prior to and post treatment, and occurred in both controls and immunized groups. They were also commonly observed in non-treated cynomolgus monkeys kept under similar laboratory conditions. No statistical differences in quantitative parameters (PQ, QRS and QT intervals and heart rates) were recorded between any of the groups. Sinusal arrhythmia was observed prior to treatment in one female in the control group.
No relevant ophthalmological findings were observed in any treatment group. In two control females, a hypopigmented fundus was noted at all three observation time points.
No treatment-related effects were noted for haematological, blood clinical chemistry and urinalysis parameters in any of the vaccine groups. At all time points, values for control and immunized animals of both genders were similar. On day 56, the mean activated partial thromboplastin time (aPTT) for MV1-F4treated males was slightly (15 %) shorter than for control males (i.e., 17.4 vs. 20.4 s; p<0.01), which was not observed for females of this group or for other treatment groups. Prothrombin times (PT) were unaffected by immunization. For blood biochemistry parameters, the only relevant finding was a higher mean creatine kinase (CK) activity in Rouvax-treated males compared to control males (i.e., 3,760 vs. 1,010 IU/l; p<0.05) on day 85, due to elevated values recorded for two animals.
To evaluate the humoral responses induced by both vaccines, anti-MV (Ig) antibody responses were measured in sera. Ten days after a single dose (day 11), responses were undetectable in the majority (87 %) of immunized animals (Fig. 1) . However, a 100 % seroconversion rate was observed in the animals of the three-dose Rouvax or MV1-F4 groups (groups 5-8) from 28 days after the first dose (day 29) onwards. This suggests that the anti-MV humoral responses may not have been fully developed at day 11, as confirmed by the kinetics of these responses in the vaccinated animals.
To assess the potential release of infectious MV1-F4 viral particles in PBMC, excretions and biological fluids (including serum, urine, throat-and nasal swabs, saliva, vaginal secretions, semen and faeces), samples were first tested for the presence of MV viral sequences by N-specific RT-qPCR assay. All samples in which viral RNA was detected, even if below the defined LOD, were then tested further using an in vitro infectivity assay.
Of the eight time points at which shedding analysis was performed, MV viral RNA was only detected at day 11 in some of the vaccine groups. At this time point, all signals in animals of all vaccine groups were below the LOD (Table 4) , with the exception of one positive result in the three-dose Rouvax group. In the MV1-F4 groups, viral RNA was detected (below the LOD) in faeces from two males of the one-dose group and in urine and vaginal secretions from one female of the three-dose group. In the Rouvax groups, viral RNA was detected in throat secretions (above the LOD, three-dose group), in faeces, throat swabs, vaginal secretion and serum (below the LOD, three-dose group), in nasal secretions (below LOD, both groups) and in PBMC (below the LOD, three-dose group and group of mature males). No positive results were detected in any of the samples of the control groups or in saliva samples of any treatment group.
In the in vitro infectivity assay and at the LOD of 10 CCID 50 /well, no infectious viral particles were recovered from any sample in which viral RNA had been detected, with acceptance criteria (i.e., negative and positive controls showing respectively negative and positive results) being fulfilled.
The potential presence of viral RNA in organs and tissues from vaccinated monkeys was detected by RT-qPCR specific for the N gene. This gene is identical in both vaccine viruses used in this study.
At day 11, MV viral RNA was detected in a large number of animals and tissues from both vaccine groups (Table 5) , confirming the expected time point of the peak of viral replication. In animals of both one-dose vaccine groups, viral RNA was mainly detected in secondary lymphoid organs (lymph nodes, spleen, Peyer's patches) and in the non-lymphoid tissues of the intestine (with the majority of signals detected above the LOD) and to a lesser extent in the liver, trachea, larynx and urinary bladder. Viral RNA was also detected in few animals in lungs, eyes, adrenals (for animals in both vaccine groups), in ureters and salivary glands (for the MV1-F4 group) and in several other organs for the Rouvax group. Importantly, no viral RNA was detected in the brain, cerebellum, cerebrum, spinal cord respectively. a Young animals (N06/group) were immunized at day 1 and sacrificed at day 11 (groups 1, 2 and 3). Blood samples were taken at days 0 and 11. b Young animals (N06/group) were immunized at days 1, 29 and 57, and sacrificed at day 85 (groups 4, 5 and 6). Blood samples were taken at days 0, 11, 29, 56, 67 and 85. c Sexually mature animals (N03/group) were immunized at days 1, 29 and 57 (groups 7 and 8). Blood samples were taken at days 0, 11, 29, 56, 67 and 85 regions, thalamus and hypothalamus of any animal. No gender-determined patterns were detected. At day 85 (28 days after the third dose), viral RNA was still detectable in the spleen and lymph nodes of some animals (both vaccine groups), and also in Peyer's patches and the intestine (Rouvax group). Low signals were also detected in the larynx and the trachea (Rouvax group).
No viral RNA was detected in the controls at both time points of necropsy.
Gross pathology, organ weights and histopathology Necropsy after either one or three injections revealed no macroscopic findings attributable to either treatment. Recorded findings were sporadic or common for cynomolgus monkeys and therefore considered toxicologically irrelevant.
At day 11, the mean absolute and relative spleen weights of males of both vaccine groups tended to be higher than for control males (without reaching statistical significance), 
Pia maters (cervical/lumbar/thoracic) -1/6<LOD --Spleen 4/6+; 1/6<LOD 5/6+ 1/6+; 2/6<LOD 3/6+; 1/6<LOD Sternum with bone marrow -2/6<LOD --
Urinary bladder 1/6+; 2/6<LOD 1/6<LOD --
No detections were made in the remaining organs (aorta, brain, cerebellum, cerebrum, epididymides, femoral bone with articulation, heart, hypothalamus, injection sites, knee joint, optic nerve, oesophagus, ovaries, oviducts, pituitary gland, prostate, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord regions, stomach (fundus), testes, thalamus, thyroids and parathyroids) for these groups and time points N no. of animals with positive signal in tissue sample/no. of tested animals in that group; +/−: + 0 positive signal (signal > defined limit of detection [LOD] of the RT-qPCR assay [with 95 % probability]). -: no RNA signal detected; <LOD: RNA signal detected but the number of copies was less than the defined LOD of 100 geq/reaction (with 95 % chance to detect a positive signal) which was only observed to a minor extent for the females of these groups (Table 6 ). Similar effects were observed for the cervical lymph nodes. For the iliac lymph nodes, which were the draining lymph nodes for the injection sites (the thighs), there was a slight tendency for higher weights in all Rouvax-treated animals. Mean thymus weights tended to be lower in both treated male groups than in control groups, possibly due to high absolute and relative weights of one control male (129 % and 118 % of group mean values, respectively). No clear effects of immunization were observed on the thymus weights of females of any group. At day 85, mean absolute and relative spleen weights of both vaccine groups were higher than those of controls for both sexes, which reached statistical significance (p<0.05) in the Rouvax-treated females. Mean absolute and relative weights of cervical and iliac lymph nodes tended to be higher in both groups of vaccinated males as compared to the control groups, while a high variability in values were noted for the female groups. Mean thymus weights tended to be lower in both groups of vaccinated males and in Rouvax-treated females, as compared to control groups.
None of these differences between groups or sexes, observed on either day of necropsy, were supported by macroscopic observations.
For the majority of immunized animals that were sacrificed at days 11 or 85, the increase in the absolute and/or relative spleen weights could not be directly correlated to the detection of viral RNA in these spleens in the biodistribution analyses. An increased spleen weight was noted in some animals for which no viral RNA was detected in the spleen, and conversely, no increased spleen weight was observed in some animals for which viral RNA was detected in the spleen.
Microscopically, enlargements of the white pulp in the spleen were occasionally noted in the animals sacrificed at day 11, in one of the three males and one of the three females treated with MV1-F4, and in one of the three control females. This enlargement was primarily due to generation of germinal centers, and was also noted in the animals sacrificed on day 85 (in one of the three MV1-F4treated males and two of the three Rouvax-treated males). At both time points, the increased sizes of the white pulp and/or germinal centers were not necessarily linked to the detection of viral RNA in the spleens. In addition, no enlargement was observed in some animals for which viral RNA was detected in the spleen. Furthermore, minimally increased germinal center sizes and/or numbers were occasionally noted in mandibular lymph nodes in two of the three MV1-F4treated males sacrificed on day 11. Although this was also seen in one of three control females sacrificed on day 85, a relationship to treatment could not be excluded. This effect was rarely seen in iliac, cervical or popliteal lymph nodes. The higher absolute and relative weights of cervical and iliac lymph nodes recorded in males of both vaccine groups on day 85 did not correlate with any consistent microscopic changes.
No relevant microscopic changes were observed at the injection sites on both days of necropsy. All other microscopic findings occurred at equal frequency across groups and/or were within the normal laboratory ranges for this species, and were therefore considered as toxicologically irrelevant. Moreover, no effects suggestive of MV infection (follicular necrosis within the hair follicles, proliferative and necrotizing bronchointerstitial pneumonia, thymus atrophy or Warthin-Finkeldey cells within the lymph nodes or spleen) were found. In addition, there was no evidence of subacute sclerosing panencephalitis (a rare syndrome in humans occurring after infection with wild-type MV stains, characterized by progressive gliosis, demyelination and neuronal loss).
In order to support the clinical development of the MV1-F4 vaccine, we have compared the toxicology, biodistribution and shedding profiles of the HIV-1 candidate vaccine MV1-F4 to those of the parental Schwarz vaccine strain, using a cynomolgus macaque model. In addition, the results may serve to increase our knowledge of the toxicity or in vivo tropism of MV vectors or vaccine strains, which has been the focus of few preclinical studies to date (e.g., de Vries et al. 2010; Lemon et al. 2011; Myers et al. 2007; Peng et al. 2003) .
Our data show that no shedding of infectious virus was observed for either of the two vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines. One or three IM injections of a full human dose of MV1-F4 or of the reference vaccine were well tolerated and induced no clinical symptoms of local or systemic reactogenicity. For both vaccines, virus replication was predominantly observed in secondary lymphoid organs (including spleens, Peyer's patches and all major lymph nodes) and to a lesser extent in epithelium-rich tissues (including intestine, larynx, trachea and urinary bladder) and liver, which were thus designated as potential target organs for intrinsic toxicity of the vaccines. However, no gross or histopathological effects indicative of toxicity were observed in these target organs, or in any other organs. Mean spleen weights were increased after three doses of either vaccine, which corresponded in some animals with enlargements of the white pulp, due to generation or growth of germinal centers. This effect likely resulted from immune activation of the secondary lymphoid organs by the vaccines, which has also been observed in other preclinical studies (Sheets et al. 2006; Speijers et al. 1988) . It is less likely that local virus replication in these organs has caused the increased spleen weights, as no clear relationship between increased spleen weights, enlargements of the white pulp and the presence of viral RNA in the spleens could be established. All other observations were considered to be unrelated to treatment, as they occurred incidentally, without correlation between genders, or at similar frequencies in control and immunized animals.
The occasional reductions in food consumption had no impact on body weights and were not considered to be indicative of systemic toxicity. For DNA plasmid vaccines, it has been observed that food consumption data and body weights did not correlate with toxicity or other findings (Sheets et al. 2006) . A similar conclusion could potentially be drawn for recombinant live-attenuated vaccines such as MV1-F4.
The only clinical pathological finding for MV1-F4 treatment groups was a slight but statistically significant decrease of the aPTT (but not of the PT) for males on day 56. However, shortening of the aPTT has generally little clinical relevance and may be associated with suboptimal sample collection or processing (Adcock et al. 1998; Awad et al. 2006) . Therefore this is not considered indicative of coagulation abnormalities of toxicological relevance. We also observed that on day 85, the mean CK activity for Rouvax-treated males was significantly higher than for control males. Elevated CK values are used as a marker of injury, heart attack, severe muscle breakdown, muscular dystrophy or acute renal failure (Lott and Abbott 1986) . None of these conditions were observed for the two males involved, and urinalysis showed no treatment-related effect for either vaccine. In addition, no elevated CK activity was found in these animals on previous time points. Possibly these elevations reflect the effects of IM-delivered sedation for ophthalmology on day 84. This effect is commonly seen in preclinical studies (Stewart et al. 2008 ) and is therefore not considered to be an adverse reaction to treatment.
Cell tropism for vaccine and wild-type MV strains is largely determined by virus entry (Tatsuo et al. 2000a ). Wild-type MV, which use predominantly SLAM/CD150 expressed on activated immune cells (Tatsuo et al. 2000b) , is known to mainly replicate in lymphoid organs and epithelial tissues such as the skin, respiratory tract, intestine and urinary bladder (Griffin 2007; Takeda 2008) . Recent studies suggest that after aerosol infection, wild-type MV initially targets macrophages and dendritic cells in alveolar tissues, which is followed by replication in regional lymph nodes and systemic spreading to lymphoid organs (Lemon et al. 2011) . How MV infects CD150-negative epithelial cells remains largely unclear, although putative epithelial receptors have recently been described (Watanabe et al. 2010) . As MV vaccine strains use the ubiquitous CD46 receptor in addition to the receptors used by wild-type MV (Dorig et al. 1993) , the vaccine strains were expected to exhibit a wider tropism than that documented previously for wild-type MV (Griffin 2007; Lemon et al. 2011; Takeda 2008) . However, in macaques infected intratracheally or by aerosol with wild-type or attenuated (Edmonston tag) recombinant MV, only the pathogenic MV caused significant viremia and widespread distribution. The attenuated MV had a restricted systemic spread and was rarely detected in lymphoid tissues (de Vries et al. 2010) , in contrast to observations in the current study. Our data seem to be more aligned with studies in mice immunized intraperitoneally or intravenously with an attenuated oncolytic (Edmonston) recombinant strain, in which infected cells were mainly detected in secondary lymphoid organs, liver and lungs (Myers et al. 2007; Peng et al. 2003) . These disparities between studies may be explained by differences in administration routes, MV strains or methods of MV detection.
The RT-qPCR results for tissues and biological fluids confirm a peak of replication around 10 days after the first dose, supporting other studies in monkeys with wild-type or attenuated MV strains (Auwaerter et al. 1999; Pan et al. 2005; Permar et al. 2003) . This resembles the clinical situation, as vaccine-induced peaks are known to occur a few days before those induced by natural infection taking place between 11 and 14 days after exposure (Strebel et al. 2004) . We observed that viral clearance for both vaccines was not fully completed at 28 days after the third dose, since viral RNA was still detectable in lymphoid and epithelium-rich tissues. This is consistent with an earlier report showing that MV RNA remained detectable in monkeys for 4-5 months (in this case, in PBMCs), even though clearance of viremia occurred within 14 or 29 days (Pan et al. 2005 ).
An additional study objective was to assess the potential of virus shedding of the MV1-F4 vaccine (in parallel to that of the reference vaccine). In natural infection, MV can be isolated from urine up to 10 days after rash onset (Gresser and Katz 1960) , and viral RNA can be detected in PBMC and nasopharingeal specimens up to 100 days after rash onset (Riddell et al. 2007 ). The few known studies of shedding by attenuated MV vectors report detection of (Edmonston) viral RNA in buccal swabs from monkeys (after intravenous administration; Myers et al. 2007 ), but no detection in human urine or saliva (after intraperitoneal administration; Galanis et al. 2010) . Consistent with reports of detection of viral RNA from attenuated MV vaccine strains in human urine up to 14 days after vaccination (Rota et al. 1995) , we detected MV1-F4-derived RNA in urine at day 11. Infectious viral particles from MV vaccines have rarely been found in human throat or nasopharyngeal secretions, and there is only one reported case of isolation of (Schwarz) MV vaccine virus from throat swabs (Morfin et al. 2002) . While we detected viral RNA from both vaccines at the peak of viremia in a few samples such as throat and nasopharingal swabs, none of these contained infectious virus at a LOD of 10 CCID 50 /well (with 95 % probability). Therefore, transmission of MV1-F4 virus between persons is considered unlikely to occur, and has also never been documented previously for any of the current measles vaccines (WHO 2009) .
The immunogenicity of the MV1-F4 candidate vaccine was not investigated in detail in the current study, as our study was primarely designed to assess the toxicology, biodistribution and shedding profiles of the candidate vaccine. However, immune responses directed against the F4 transgene were characterized in a separate study using the same animal model (MV-seronegative cynomolgus macaques). In the latter study, the MV1-F4 candidate vaccine was shown to induce F4specific CD4 + and CD8 + T cell responses, as well as antibody responses to F4 (manuscript in preparation).
In conclusion, while viral dissemination was observed in various organs, no shedding of infectious viral particles was noted, and no toxic effect in relation to the MV vaccination was found following one or three IM injections with a clinically relevant dose of MV1-F4, with the same outcomes for the (Schwarz) measles comparator vaccine. Moreover, either vaccine virus replicated predominantly in secondary lymphoid organs and, to a lesser extent, in epithelium-rich tissues. Thus, as expected, introduction of the F4 transgene did not change the toxicological profile, shedding capacity or tropism of the parental strain. Forty-Five Years of Marburg Virus Research In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology. Marburg virus (MARV) first appeared in August 1967, when laboratory workers in Marburg and Frankfurt, Germany and Belgrade, Yugoslavia (now Serbia) were infected with a previously unknown infectious agent. The 31 patients (25 primary, six secondary infections) developed severe disease that progressed to a fatal outcome in seven of the cases. An additional case showing symptoms of disease was diagnosed retrospectively (reviewed in [1] ). The source of infection was traced back to African green monkeys (Chlorocebus aethiops) that had been imported from Uganda and were shipped to all three locations. The primary infections ironically occurred when the monkeys were necropsied for the purpose of obtaining kidney cells to culture poliomyelitis vaccine strains. In the remarkable period of less than three months the etiologic agent was isolated, characterized, and identified by the joint effort OPEN ACCESS of scientists in Marburg and Hamburg [2] and was later confirmed by Kunz and colleagues [3] and Kissling and colleagues [4] . The pathogen was named Marburg virus after the city with the most cases and represented the first isolation of a filovirus. Erroneously, a study published in 'The Lancet' claiming that the mysterious disease was caused by rickettsia or chlamydia has frequently been cited as the first report on the causative agent of Marburg virus disease (MVD) [5] .
It was not until 1976 that the now better-known member of the family, Ebola virus (EBOV), first emerged in Africa [6, 7] . Shortly thereafter marburgviruses and ebolaviruses were classified together in a newly established family termed Filoviridae, so-named after their distinctive thread-like structure (filum being Latin for thread).
MARV had not been heard of for eight years, when a young Australian who had traveled throughout Zimbabwe was admitted to a hospital in Johannesburg, South Africa with symptoms reminiscent of those observed during the 1967 outbreak in Europe [8] . When he died and the infection spread to his travel companion and later also to a nurse, Lassa fever was initially suspected resulting in strict barrier nursing techniques and isolation of the patients and their primary contacts. This lead to a quick containment of the outbreak, and while the secondary cases recovered, MARV was identified as the causative agent of the disease. In the following years from 1975 through 1985, only sporadic outbreaks that affected small numbers of individuals were caused by MARV on the African continent (Table 1, Figure 1a ). As the case fatality rates associated with MVD were also lower than those seen in the devastating outbreaks associated with EBOV disease that reached up to 90%, MARV was long thought to be less threatening (Table 1) . However, this view had to be revised as MARV reemerged in two large outbreaks occurring in the Democratic Republic of the Congo (DRC) in 1998-2000 [9] and then, for the first time also in Western Africa, in Angola, in 2004-2005 [10] . The total number of 406 cases and the high fatality rates (83% in DRC and 90% in Angola) revealed that MARV was as big of a threat for public health as EBOV [1, 11] . The variation observed in disease severity and case fatality rates between these outbreaks versus the initial one in 1967 may depend on many complicating/mitigating factors. These include quality and availability of medical care, infectious dose and route of infection, differences in host population susceptibility (depending on immune and nutritional status) and genetics, inherent differences in viral variant virulence, and the prevalence of co-infections (particularly malaria and AIDS in patients from sub-Saharan Africa) [9] . The assumption that MARV Angola might be inherently more virulent than other MARV variants has been proposed mainly based on infection studies with nonhuman primates (NHP) [12] [13] [14] but is a matter of debate [15] . The genomes of the Angolan isolates differ about 7% at nucleotide level from the majority of the East African MARV isolates, including the ones from 1967 [10] . There is no evidence so far that the observed genetic differences result in higher virulence in humans.
The DRC outbreak was unique, as there were at least nine different virus variants circulating in the tested patients indicative of several different spillover events from the natural reservoir to the human population [9] . In contrast, sequence data from the Angolan outbreak suggested a single introduction of MARV to an unidentified index patient and subsequent spread via person-to-person contact. The viral genomes showed a remarkably high genetic stability within this outbreak. Identical MARV genomes were isolated from patients even after two to three human to human transmissions [10] . 
MVD is considered a zoonotic disease that is thought to persist in a healthy reservoir host in the endemic areas in Africa. Humans and NHPs are spillover hosts and show a high rate of fatal disease outcomes. Several large-scale attempts to identify the natural host of filovirus infection throughout sub-Saharan Africa had been undertaken in the years since filoviruses first emerged with frustratingly little success [28] [29] [30] [31] . Consistent with ecologic niche modeling of outbreaks and epidemiological patterns, isolated cases have suggested that EBOV is endemic in the rain forests of central and western Africa while MARV is more prevalent in open, dry areas of eastern, south-central Africa [32, 33] . Almost all of the primary infections of natural MVD outbreaks so far have been linked to human entry into caves inhabited by bats (e.g., cave visitors, mine workers) (Table 1) . Thus, bats have long been suspected to play an important role in the transmission cycle of the disease [31, 32, 34] . In 2007, evidence was detected for MARV infection of the common Egyptian fruit bat (Rousettus aegyptiacus) [35, 36] (Figure 2 ), and MARV was isolated from healthy infected R. aegyptiacus bats caught in the same year in Uganda [22] . The bats were collected in Kitaka cave around the same time as human infections occurred that had been linked to the cave (Table 1 and see above, 1. Epidemiology). Genomic analysis of the few isolates of MARV acquired from bats showed that the sequences matched closely to the MARV genomes isolated from patient samples (Figure 1b) . This was also the case for partial MARV sequences isolated from bats inhabiting the Goroumbwa mine in the DRC that was suspected to be the major location for several independent spillover events to gold miners between 1998 and 2000. The bat MARV sequences were closely related to the distinct isolates that had been reported during these outbreaks in humans [36] .
A study analyzing MARV prevalence in bat populations in Gabon found MARV-specific nucleic acids in R. aegyptiacus bats in several local caves [37] . Together with previous data showing a high prevalence of MARV-specific antibodies in Gabonese bat populations [38] and with an observed relation of the isolated sequences with previously reported Gabonese bat isolates [35] this study Viruses 2012, 4 1886 suggests that MARV is enzootic in Gabon and raises the concern of further spread of MARV into other countries. Therefore, close ecological as well as serological surveillance of the bat populations in sub-Saharan Africa could help to predict and prevent further MVD outbreaks especially in areas where bats are still used as a food source.
It is not currently clear whether R. aegyptiacus bats are the exclusive reservoir for MARV or if other bat species reported to be positive for viral antibodies and RNA are also natural reservoirs or merely intermediate hosts [36] .
The genus Marburgvirus includes a single species, Marburg marburgvirus (formerly referred to as Lake Victoria marburgvirus) [39, 40] . Phylogenetic analysis based on genomic sequence data suggests that the known members of this species can be assigned to at least five different lineages of which four are very closely related (nucleotide sequences differ up to 7%) while the fifth is divergent (a nucleotide difference of 21%) ( Figure 1b) [10, 22, 39] . As the genomic divergence between all isolates is below 30%-the cutoff for the classification of the five different ebolaviruses into five different species-the five marburgvirus lineages were recently reclassified as two viruses. Ravn virus (RAVV) is represented by the Ravn isolates from 1987, one isolate from the DRC outbreak in 1998-2000, and one human and several bat isolates from infections that took place in Uganda in 2007. Marburg virus (MARV) is represented by all other sequenced isolates ( Figure 1b , Table 1 ) [39] . For the sake of simplicity, in this review the abbreviation "MARV" is used for all marburgviruses and the abbreviation "EBOV" for all ebolaviruses.
Initial MVD patients are believed to contract the virus via exposure to an infected animal: either a reservoir host (several bat species) or a spill-over host such as NHPs as described in the first MVD outbreak (see above, 1. Epidemiology) [23, 41] . Following transmission to humans, spread of the virus between individuals is the result of direct contact with blood or other body fluids (saliva, sweat, stool, urine, tears, and breast milk) from infected patients. Typical risks of exposure include administration of medical care to infected individuals as well as handling of corpses without use of proper protection [34] . Of particular note, virus has been found in tears, semen, and in a liver biopsy weeks to months following the onset of symptoms highlighting the importance of monitoring convalescent patients [20, [42] [43] [44] .
Much of what we know about typical MVD symptoms comes primarily from clinical data obtained during the three largest recorded MVD outbreaks: the 1967 outbreak in Germany and Yugoslavia, the 1998-2000 outbreak in the DRC, and the 2004-2005 outbreak in Angola. Although the case fatality rates were significantly higher in the latter outbreaks, most of the clinical symptoms observed were similar.
Based on the most reliable documented cases of exposure and subsequent illness, MVD has an incubation period ranging from 3 to 21 days (typically 5 to 10 days), which is likely modulated by factors such as infectious dose and possibly by route of infection. The course of MVD has conventionally been broken down into three phases [45] : an initial generalization phase, an early organ phase, and either a late organ phase or convalescence phase depending upon disease outcome [45] . A summary of MVD symptoms is reviewed below [16, [46] [47] [48] [49] .
The onset of illness begins with generic flu-like symptoms; a characteristic high fever (typically 39-40 o C), severe headache, chills, myalgia, prostration, and malaise. For many patients (50-75%) this is followed by rapid debilitation characterized by gastrointestinal symptoms including anorexia, abdominal pain, severe nausea, vomiting, and watery diarrhea. Starting on day four to five patients commonly develop enanthem, dysphasia, and pharyngitis. Additionally, a characteristic maculopapular rash is typically the first distinctive feature indicating a filovirus infection versus influenza or malaria. Other common symptoms include lymphadenopathy, leukopenia, and thrombocytopenia.
Many of the initial symptoms may persist in the early organ phase, and patients may sustain a high fever. They may additionally display neurological symptoms including encephalitis, confusion, delirium, irritability, and aggression. Patients can also develop dyspnea and abnormal vascular permeability, particularly conjunctival injection and edema. During the latter part of this phase more than 75% of patients present with some form of clear hemorrhagic manifestation such as petechiae, mucosal bleeding, melena, bloody diarrhea, hematemesis, and ecchymoses. Due to the unusualness of hemorrhagic symptoms, diseases caused by filoviruses have sometimes been referred to as hemorrhagic fevers (Marburg Hemorrhagic Fever (MHF) and Ebola Hemorrhagic Fever (EHF)), although these terms are currently disfavored since not all patients display hemorrhagic symptoms. At this stage, multiple organs are affected including the pancreas, kidney, and liver. Elevated serum activity of a number of liver enzymes including SGOT and SGPT have been observed in most patients sampled.
The late stages of MVD result in one of two potential outcomes: patients either succumb to the disease or enter a prolonged phase of recuperation. Typical preagonal symptoms include restlessness, obtundation, confusion, dementia, convulsions, reduced circulation due to severe dehydration, metabolic disturbances, severe diffuse coagulopathy, multiorgan failure, shock, and coma. Fatalities typically occur 8-16 days following the onset of symptoms, with death usually the resulting of shock and multiorgan failure. Non-fatal cases are typified by an extensive convalescent period during which myalgia, exhaustion, sweating, peeling of the skin at the sites of rash, partial amnesia, and secondary infections are all common. 
Prevention of newly emerging MARV infections and effective containment during ongoing outbreaks is both essential and challenging, as there is currently no licensed vaccine or treatment available for general use.
Following the 1967 outbreak of MVD in Europe and cases of infection with ebolavirus Reston in imported crab-eating macaques (Macaca fascicularis) in the USA in 1989/1990 and 1996 as well as 1992 in Italy (reviewed in [50] ), strict quarantine procedures have been put in place that have so far prevented infections acquired by imported NHPs into non-endemic countries [51, 52] . To avoid the spread of filoviruses by tourists, Python cave was closed to the public following the diagnosis of the Dutch patient in 2008.
The prevention and control of outbreaks and infections in endemic countries is much more challenging. In the past, joint efforts of teams from the WHO, Doctors Without Borders, the Red Cross, the CDC and others in collaboration with the local ministries of health have been undertaken to cease the spread of MVD. The main focus of outbreak control is the prevention of secondary transmission and further primary infections.
The first measures in response to a MVD outbreak include setting up isolation wards in hospitals to assure rapid isolation of MARV-infected patients and prevent person-to-person transmission (Figure 3b ). Proper and fast laboratory diagnosis of suspected cases is key to eliminate further spread. Nosocomial infections were commonly seen in earlier outbreaks [8, 9, 23] . However, reinforcement of barrier nursing techniques and education of health care workers have limited these infections in recent outbreaks ( Figure 3a ). Epidemiological surveillance has been crucial in the identification of index cases as well as the predominant modes of transmission. In endemic areas, secondary infections mainly occurred while taking care of ill patients and family members or during traditional burial practices involving close contact to corpses [53] . Therefore, the execution of safe burial and disinfection techniques and information campaigns to educate the local population are essential in order to contain the spread of infections in endemic areas ( Figure 3a ) [27, 53, 54] .
Biosafety and epidemiological efforts alone were not sufficient for efficient outbreak control during large outbreaks, emphasizing the need for additional psychosocial support of the affected communities [53] . The fast progression and high lethality rates associated with MVD even-and especially-after hospital admission resulted in a high level of fear and suspicion by the resident population. The fact that health care workers wearing recommended personal protective equipment (PPE) were fully masked and not identifiable further increased anxiety (Figure 3b ). This resulted in the hiding of infected family members and verbal and in some cases physical aggression towards members of aid organizations [53] . Communicating necessary protective measures while respectfully considering the affected families' and communities' traditions and culture during ongoing outbreaks is therefore essential for successful outbreak management.
The recent identification of bats as the potential reservoirs for MARV as well as EBOV [22, 35, 36, 55, 56] will help to increase not only the public awareness, but also the effectiveness of the preventive measures taken in endemic areas to minimize contact with infected animals (i.e. closing of bat inhabited caves for the public, serosurveillance of bat populations) [18, 19] . This is a challenging task, emphasized by the fact that during the last cluster of MARV infections linked to a gold mine in Uganda, the miner hired to enforce the restricted access to the mine got infected. The mine had been closed in response to the ongoing outbreak and even though he was aware of the risk, he had entered the mine without the suggested PPE [19] .
Later, the bat population of this mine was eliminated by the owner by means of fumigation [19] . As bats of most species are endangered, this does not seem a viable option and educational campaigns aimed at villagers living close to bat-inhabited caves as well as tourist groups and tour operators might prove more sustainable in the future. 
In 1967, during the first reported filovirus disease outbreak in Europe, the identification of the previously unknown causative agent of the deadly disease was performed by electron microscopy (EM) (Figure 4 ). The unusual filamentous structure of the particles led to some confusion and it was even suggested that the causative agent of the disease might be related to the spiral-shaped Leptospira, a genus of the spirochaetes bacteria [58] . Others concluded that the observed particles were viruses morphologically related to rhabdoviruses and named the newly discovered pathogen Marburg virus [23, 59] . Marburg virions are pleomorphic particles, which appear as rod-or ring-like, crook-or sixshaped, or branched structures. Cryo-EM analysis of purified virions showed that about 30% of viral particles released from infected Vero cells were filamentous, 37% were six-shaped, and 33% were round [60] . The same study revealed a mean particle length of 892 nm and a mean diameter of 91 nm. Previous conventional EM studies showed that the MARV particles were uniformly 80 nm in diameter, whereas the length varied widely with virions measuring up to 14,000 nm. The average particle length was 790 nm [61] [62] [63] . The reported differences in particle size might be due to experimental differences between cryo-EM and conventional EM [60] . Notably, MARV particles are considerably shorter than EBOV virions, although MARV genomes are slightly longer than EBOV genomes [62, 63] . MARV particles are surrounded by a host-derived membrane that is coated with spikes of 5-10 nm in length, which are formed by trimers of the viral glycoprotein (GP) ( Figure 5 ) [60] [61] [62] [63] [64] [65] . The central core of the viral particles is the ribonucleoprotein complex (nucleocapsid) formed by the viral RNA genome and tightly associated nucleocapsid proteins ( Figure 5 ). The nucleocapsids are highly organized tubular structures with an outer diameter of 45-50 nm and an electron-dense central axis of 19-25 nm. The central axis is surrounded by a helical capsid with cross-striations at a 5 nm interval [61] [62] [63] . A recently published detailed cryo-electron tomography analysis of MARV virions has shed some light on the structural organization of the nucleocapsids. Reconstructions of virion-associated nucleocapsids using subtomogram-averaging analysis revealed that the MARV nucleocapsids form a left-handed helix with a pitch of 7.5 nm and a flexible average symmetry of 14.96 protrusions per turn with two inner lobes of density per protrusion. The inner lobes represent the nucleoprotein (NP), suggesting that the MARV nucleocapsid contains an average number of 29.92 NP molecules per turn with each NP molecule packaging six RNA bases [60] . MARV nucleocapsids show directionality, having a pointed and a barbed tip [60] .
The nonsegmented negative-sense (NNS) RNA genomes of the various MARV isolates range in size from 19,111 to 19,114 nts and contain seven monocistronic genes in a linear order ( Figure 5 ) [66, 67] . Each gene is composed of a highly conserved transcription start and stop signal, an unusually long 3' and 5' untranslated region, and the open reading frame (ORF). The genes are either separated by short intergenic regions that range from 4 to 97 nts, or the transcription stop signal of the upstream gene and the transcription start signal of the downstream gene overlap, sharing five highly conserved nts ( Figure 5 ). The structure of this gene overlap is found among all filoviruses and is unique among members of the order Mononegavirales (for review see [68] ). The 3' and 5' genome ends are extracistronic regulatory regions that contain cis-acting signals essential for transcription and replication, including transcription and replication promoters.
There are generally two types of genomic replication promoters for NNS RNA viruses: a bipartite promoter found in members of the paramyxovirus subfamily Paramyxovirinae and one continuous more compact replication promoter for rhabdo-and pneumoviruses [69] . The bipartite promoter structure of the Paramyxovirinae subfamily is associated with the "rule of six", i.e., the total genome length must be a multiple of six [70] . Given that filoviruses do not obey the rule of six, it was surprising that mapping of the MARV genomic replication promoter revealed a bipartite structure. The 3' genome end, the leader, comprises 48 nts and contains the first promoter element of the bipartite genomic replication promoter. The second promoter element consists of a (UNNNNN) 3 motif with three conserved uridine residues separated from each other by five non-conserved nucleotides. The UN (5) hexamers are located within the 3' untranslated region of the first MARV gene, the NP gene, and are separated from the first promoter region by the 12 nts long transcription start signal. Substitutions in the NP transcription start signal do not affect replication activity but do interfere with transcription initiation [71] . The 5' extracistronic region, the trailer, spans the last 75 nts of the MARV genome and contains the complement of the antigenomic replication promoter (see below, 8.2. Transcription and Replication). The structure of the MARV antigenomic promoter has not yet been determined. However, due to the presence of UN (5) hexamers it is likely that it is of bipartite nature, similar to the genomic promoter. A common feature of the leader and trailer regions of all NNS RNA viruses is a high degree of complementarity of the 10-15 most terminal 3' and 5' nucleotides [72] . Although filoviruses share this feature, both the leader and the trailer also have the capability to form an internal secondary structure, which is not the case for the leaders and trailers of other NNS RNA viruses [71, [73] [74] [75] .
The MARV genome encodes seven structural proteins listed in Table 2 . MARV has a single surface protein, GP, which is encoded by the fourth gene and mediates attachment to target cells and virus entry [76] . GP is a Type I transmembrane protein which is inserted into the viral envelope in the form of homotrimeric spikes [65] . In contrast to ebolaviruses, which use transcriptional editing to express the membrane-bound GP and at least two nonstructural glycoproteins [77] [78] [79] [80] , the MARV GP gene contains a single open reading frame (ORF) encoding the full-length GP. During its transport from the endoplasmic reticulum (ER) to the plasma membrane via the secretory pathway, the precursor GP is the target of various posttranslational modifications including glycosylation [65, 81] , acylation [82] , and phosphorylation [83] . GP is heavily glycosylated by complex and high mannose-type N-linked glycans as well as by mucin-type O-linked glycans, with the carbohydrates contributing about 50% of the apparent molecular weight of the protein [65, 84, 85] . Similar to EBOV GP, the O-linked glycans and many of the N-linked oligosaccharides are clustered in a mucin-like domain [76] . After synthesis in the ER, the precursor GP is cleaved at amino acid 435 by furin or a furin-like protease in the trans Golgi network, resulting in two disulfide-linked subunits, GP 1 (160 kD) and GP 2 (38 kD) [86] . While the ectodomain, which is mainly formed by GP 1 , mediates binding to entry factors and receptors [87] [88] [89] [90] [91] [92] [93] [94] [95] , the transmembrane subunit GP 2 contains the fusion peptide and is presumed to mediate fusion of the viral and the cellular membrane based on similarity to EBOV GP 2 both at the amino acid and structural level [96, 97] . The 30 amino acid long transmembrane domain of GP 2 is required for the incorporation of GP into virions [98] . In addition, the cytoplasmic tail of GP 2 is involved in enhancing the efficiency of viral entry by maintaining the structure of the ectodomain [99] . The receptor binding domain of MARV GP was mapped to the aminoterminal region of GP 1 spanning amino acids 38 to 188 [100] , whereas the highly glycosylated mucin-like domain is not essential for virus entry [101] . An important step in MARV entry is the proteolytic activation of GP 1 by endosomal proteases, facilitating binding of the receptor binding region to the endosomal entry factor Niemann-Pick C1 protein (see below, 8.1. Entry) [94, 95] .
Besides its function in entry and budding, GP may also play a role in immune evasion. The IFN-inducible antiviral protein tetherin was shown to block the release of VP40-induced virus-like MARV and EBOV particles, suggesting that tetherin might act as a restriction factor for filovirus release [102, 103] . However, co-expression of GP was sufficient to counteract the antiviral activity of tetherin by a yet unknown mechanism [104, 105] . It is possible that GP not only subverts innate immune responses but also suppresses the adaptive immune response. Filoviral GP 2 subunits, including MARV GP 2 , contain a domain resembling an immunosuppressive motif found in retroviral envelope proteins [106] . A 17-mer peptide corresponding to the putative immunosuppressive domain of MARV GP was shown to induce lymphocyte death and suppression of cytokine responses [107] . It is not yet known if this motif plays a role in the induction of lymphocyte apoptosis observed in MARV infection. Finally, it has been suggested that shedding of the ectodomain of membrane-bound EBOV GP by tumor necrosis factor α-converting enzyme (TACE) may play a role in blocking the activity of neutralizing antibodies during infection [108] . It has been reported for MARV that considerable amounts of GP shed from infected cells, although it is not clear if MARV GP is a target for TACE cleavage [108, 109] .
The matrix protein VP40 is encoded by the third MARV gene and is the counterpart of the M proteins of other NNS RNA viruses. VP40 plays a major role in the formation of virions by redistributing nucleocapsids from the perinuclear region to the plasma membrane, recruiting GP to the sites of budding, and mediating particle release [110] [111] [112] . Overexpression of VP40 led to reduced reporter gene expression of MARV minigenomes, suggesting a regulatory role of VP40 in transcription and/or replication [113] .
As a peripheral membrane protein, VP40 coats the inner side of the virion's membrane ( Figure 5 ) [114] . Cryo-EM tomography studies suggest that VP40 associates with the nucleocapsid through flexible interactions [60] . It can be easily removed from the nucleocapsid by salt dissociation, indicating that it is only loosely connected to the nucleocapsid [115] . After synthesis in the cytoplasm of the infected cell, VP40 associates rapidly with cellular membranes and accumulates in membranous structures of the late endosomal compartment, the multivesicular bodies. A minor portion of VP40 is also found in association with viral nucleocapsids and in inclusions. Additionally, VP40 appears in patches beneath the plasma membrane where it is transported via the retrograde late endosomal pathway [62, 114, 116] . Similar to EBOV VP40, MARV VP40 is the major factor in particle formation and budding. Expression of VP40 in the absence of other viral proteins leads to the formation and release of filamentous virus-like particles (VLPs) resembling authentic virions. This process is enhanced in the presence of GP [113, [117] [118] [119] . The role of VP40 during budding is described in more detail below (see 8.3. Budding). Compared to EBOV VP40, little is known about the structure of MARV VP40. The N-terminal domain of MARV VP40 folds into ring-like structures, which have the tendency to polymerize into rod-like structures. While EBOV VP40 has been shown to form hexamers and octamers, the stoichiometry of MARV VP40 oligomers is not known [120] . MARV VP40 is phosphorylated at several tyrosine residues located in the N-terminal region of the protein. A non-phosphorylatable mutant of VP40 is impaired in its ability to recruit nucleocapsids to the sites of budding, but is still able to efficiently induce particle release [112] . VP40 also possesses a PPPY late domain motif in its amino terminus which is important for its interaction with components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in order to mediate budding, including Tumor susceptibility gene 101 (Tsg101) and the membrane-bound E3 ubiquitin ligase Nedd4.1 [119, 121, 122] . Besides the PPPY motif, other motifs and single amino acids have been found to be important for particle release [123, 124] .
Besides its role as a classical matrix protein, MARV VP40 also acts as a virulence factor by counteracting the innate immune response and determining the host tropism for MARV [125, 126] . MARV VP40 blocks the phosphorylation of Janus kinases, which play an important role in multiple signaling pathways by phosphorylating and activating STAT proteins ( Figure 6 ).
When MARV-infected cells were treated with various stimuli, including IFNα, IFNγ, and IL6, it was shown that the STAT proteins were neither phosphorylated nor translocated into the nucleus [126, 127] . It was then shown that in MARV-infected cells treated with exogenous stimuli, Janus kinases were also not phosphorylated and VP40 was identified as the viral protein inhibiting IFN signaling. It is believed that Jak1 is the target for VP40, however, the mechanism of VP40-induced inhibition is not completely understood [126] . Intriguingly, EBOV is also able to block IFN signaling by employing a completely different mechanism. EBOV VP24 blocks the nuclear translocation of phosphorylated STAT proteins by binding to STAT1 and importins involved in the nuclear transport of specific STAT proteins ( Figure 6 ) ( [128] , reviewed in [129] ).
When MARV was adapted to non-or less permissive animals, such as mouse and guinea pig, the adapted viruses showed mutations in VP40. Two of the amino acid changes in the mouse-adapted MARV VP40 have been shown to be essential for the inhibition of IFN signaling in mouse cells, underlining the importance of IFN suppression for the virulence and host specificity of MARV [125, 130, 131] . The protein product of the sixth gene, VP24, is unique to the filovirus family. VP24 is generally addressed as a second, minor matrix protein. However, cryo-electron tomography analysis of viral particles showed that VP24 is located in close proximity to the nucleocapsid proteins, suggesting that it might be part of the nucleocapsid complex [60] . VP24 can easily be released from virion-associated nucleocapsids by treatment with increasing salt concentrations, indicating that it is only loosely connected to the nucleocapsid [115] . Intracellular localization studies of VP24 showed that a minor part of the protein (approx. 10%) is weakly bound to cellular membranes, including filopodia enriched with VP40. VP24 is also distributed diffusely in the cytoplasm, relocalizes to nucleocapsid-containing inclusions, and is found in association with free nucleocapsids. Co-expression of NP and VP24 is sufficient to direct VP24 to NP inclusions in the cytoplasm [132] . Functional studies on MARV VP24 suggest that the protein is important for the release of viral particles in the context of infection, although it influences neither the morphology of VP40-derived VLPs nor the efficiency of VLP release. In addition, RNAi-mediated knockdown of VP24 in MARV-infected cells had no impact on viral genome replication, indicating that VP24 is involved in a step after replication and before budding [132] . According to the model that has been proposed based on these data, VP24 is involved in the maturation of transport-competent nucleocapsids and/or mediates the interaction between nucleocapsids and budding sites at the plasma membrane [132] . There is also evidence that MARV VP24 affects transcription and replication in a transcription and replication competent VLP system [113] .
Structural information about MARV VP24 is very limited. It has been shown that it forms oligomers, preferentially tetramers [132] . Structure prediction studies have proposed an ancestral link between VP24 and the Armadillo repeat family [133] .
The MARV nucleocapsid complex consists of the genomic RNA and four tightly associated proteins, NP, VP35, VP30, and L ( Figure 5 ). Encapsidation of the viral RNA by the nucleocapsid proteins protects it from both RNase degradation and detection by cellular pattern recognition receptors. Similar to the genomic RNA, the antigenomic RNA, a replicative intermediate, is also encapsidated by the nucleocapsid proteins (see below, 8.2. Transcription and Replication). In contrast, the viral mRNAs are not encapsidated [134] . The nucleocapsid rather than naked RNA serves as the template for viral transcription and replication. In a MARV minigenome system, NP, VP35, and L are essential for transcription and replication [113, 134] . The role of VP30 in MARV transcription and replication is not well understood and the steps in genome amplification that require, or do not require, VP30 are not yet defined.
The nucleoprotein NP enwraps the genomic and antigenomic RNAs. Replication and transcription activity in a MARV minigenome system depends on the presence of NP [134] . When NP is expressed in the absence of other nucleocapsid proteins, it self-assembles into highly organized helical tubular structures that resemble the nucleocapsids in infected cells, indicating that it is the driving force for nucleocapsid formation [60, 135, 136] . Recently, it has been shown that the conserved 390 N-terminal residues of MARV NP are sufficient to form the helical structure of the nucleocapsid core [60] . Indeed, NP serves as a viral hub protein. It forms interactions with most of the other viral proteins, leading to the subcellular redistribution of these proteins. The strong binding of NP to the nucleocapsid proteins VP35 and VP30 redirects both proteins into NP-derived inclusions [115] . A bipartite coiled-coil motif in the central part of NP has been shown to play an important role for self-assembly and NP-VP35 interaction [137] . As mentioned above, there is also a weak interaction between NP and VP24, leading to the partial relocalization of VP24 into NP-containing inclusions [60, 132] . In addition, NP interacts with VP40, which is important for the transport of newly synthesized nucleocapsids to the plasma membrane [110, 111, 138, 139] . Interestingly, NP contains a C-terminal late domain motif, PSAP, which has been shown to be required for budding. NP recruits Tsg101, a component of the ESCRT I complex, through its late domain motif, leading to enhanced VP40-induced budding [111] .
NP is heavily phosphorylated at serine and threonine residues clustered in seven regions in the C-terminal part of the protein. Only the phosphorylated form of NP is incorporated into virions [140, 141] . Recent studies suggest that the phosphorylation level in Region II modulates transcription and/or replication activity [142] .
VP35 is a polymerase cofactor and essential for transcription and replication. Together with the catalytic subunit L, VP35 forms the RNA-dependent RNA polymerase complex [134, 143] . VP35 is tightly associated with NP and serves as a bridging protein between the nucleocapsid complex and L. Without VP35, L is not associated with the nucleocapsids which serve as the templates for viral transcription and replication [115, 134] . VP35 forms homo-oligomers mediated by a coiled-coil motif located in the N-terminal part of the protein. Homo-oligomerization of VP35 is essential for its interaction with L but not needed for redistribution of VP35 into NP-derived inclusions [144] . VP35 shares many features with the phospho (P) proteins of other NNS RNA viruses, including its position as the second gene in the viral genome and its role in transcription and replication. However, in contrast to the P proteins, VP35 is either not or only very weakly phosphorylated [145] .
Besides its function in transcription and replication, MARV VP35 acts as an IFN antagonist. While the impact of EBOV VP35 on the host's antiviral response has been intensively investigated (reviewed in [129] ), much less information is available about similar functions of MARV VP35. When we tested MARV VP35 for its ability to block IFN induction in a reporter gene assay, it blocked reporter gene expression as efficiently as EBOV VP35 (unpublished data). In addition, Bosio and colleagues [146] reported that expression of MARV VP35 in the absence of other viral proteins was sufficient to completely block the induction of IFNα in stimulated human dendritic cells. Besides its ability to inhibit the induction of Type I IFN, EBOV VP35 has been shown to block the activation of the antiviral protein PKR and to interfere with RNA silencing pathways. Importantly, EBOV VP35 is a dsRNA binding protein. The C-terminus of EBOV VP35 contains a domain with patches of basic amino acids which is important for dsRNA binding and the protein's inhibitory functions (for review see [129] ). This C-terminal region, the so-called IFN inhibitory domain, is conserved in MARV VP35 [147] , suggesting that MARV VP35 possesses similar inhibitory functions.
MARV and EBOV VP30 proteins show many structural similarities. Both MARV and EBOV VP30 proteins are tightly associated with the nucleocapsid via their binding to NP ( Figure 5 ) [115, 148] . Both are highly phosphorylated at N-terminally located serine and threonine residues, and phosphorylation is crucial for their interaction with NP [148, 149] . Both contain an unusual C3H1 Zn binding domain, which is essential for the function of EBOV VP30 as transcription initiation factor, but whose functional relevance for MARV VP30 is not known [150] . It has also been shown that EBOV VP30 forms hexamers [151, 152] , binds single-stranded RNA [153] , and interacts with L [154] . However, to date, similar data for MARV VP30 are not available.
The role of MARV VP30 in viral transcription and replication is not well understood. In contrast to EBOV VP30, which plays an important role in regulating transcription initiation [68, 143, [155] [156] [157] , MARV VP30 is not essential for transcription or replication activity in a MARV minigenome system [113, 134] . Nevertheless, it seems to play an important role in viral amplification, since rescue of a full-length MARV clone is only successful in the presence of VP30 [158] . In addition, down-regulation of VP30 by RNA interference in MARV-infected cells led to the reduction of both viral protein synthesis and virion production [159] . Among the NNS RNA viruses, only the members of the subfamily Pneumovirinae possess a protein similar to VP30, M2-1, which functions as a transcription processivity factor [160] .
The major component of the MARV polymerase complex, L, has an estimated molecular weight of 267 kD [67] . It is essential for transcription and replication and together with VP35 forms the RNA-dependent RNA polymerase complex (see above, 7.3. Viral Proteins, VP35). L contains the enzymatic functions of the polymerase. The binding site for VP35 has been mapped to the N-terminal 530 amino acid residues of L [115, 134] . The L proteins of the NNS RNA viruses are highly conserved multifunctional proteins, which are organized in functional domains [161] . Based on this conservation with other NNS RNA polymerases, MARV L is believed to carry out RNA synthesis, capping, and polyadenylation of viral mRNAs although these functions have not been shown experimentally.
To date most of the studies characterizing the MARV replication cycle have utilized recombinant systems, allowing for these experiments to be performed in a biosafety level 2 (BSL-2) context, unfettered by the restrictions of a BSL-4 setting. Surrogate systems mimicking specific steps in the MARV replication cycle include MARV GP-pseudotyped retroviruses or recombinant vesiculoviruses expressing GP to study entry, VLPs to study budding, and minigenome systems to study replication and transcription. While such experiments allow for the more facile examination of the MARV replication cycle, the findings must be recapitulated with infectious MARV since all surrogate systems lack elements of the infectious virus such as the distinct morphological features and virion protein composition of MARV.
Marburg virus entry consists of three distinct phases: cellular attachment, endocytosis, and fusion ( Figure 7) . Based on the sequence similarity between EBOV and MARV GPs many investigators have presumed identical functions and characteristics between the filovirus glycoproteins. This is presumptuous given the differences in glycosylation and sialic acid linkages [85] and dependence upon endosomal proteases (see below, 8.1.2. Endocytosis). Despite the existence of a number of detailed studies and structural analyses of EBOV GP [162] [163] [164] [165] , relatively few mechanistic studies of MARV GP have been performed [101] , although one recent post-fusion structure of MARV GP 2 has been reported [97] . The structure of MARV GP 2 is nearly identical to that of EBOV GP 2 , indicating that the mechanisms of fusion between the two viruses is likely conserved [97] . (2), GP 1 is cleaved by endosomal proteases (3) facilitating binding to NPC1, the entry receptor (4). Fusion is mediated in a pH-dependent manner by GP 2 . Following release of viral nucleocapsid into the cytosol (5), transcription of the viral genome takes place (6) . mRNA is subsequently translated by the host cell machinery (7) . Synthesis of GP takes place at the ER and undergoes multiple posttranslational modifications on its way through the classical secretory pathway (8) . Positive sense antigenomes are synthesized from the incoming viral genomes (9) . These intermediate products then serve as templates to replicate new negative sense genomes (10) . After cleavage in the Golgi, GP is transported to multivesicular bodies (MVB) and to the cell membrane where budding takes place (11) . Nucleocapsids and VP24 are also recruited to sites of viral budding (12) , which is driven by VP40 (13).
MARV GP mediates both cell attachment and fusion of the virus. There is convincing evidence that initial virus attachment at the cell surface can occur via the binding of GP carbohydrates to various cellular C-type lectins, including the hepatocyte-specific ASGP-R [87] , DC-SIGN and DC-SIGNR (also known as L-SIGN) [89, 92, 166] , hMGL [91, 92] , and LSECtin [166, 167] . Other cell surface proteins have also been implicated in facilitating MARV entry including the TAM receptor protein kinases Ax1, Dtk, and Mer [90] , and TIM-1 [93] . However, although these proteins may play a role in attachment or entry of certain cell types, the ability of MARV to infect cells lacking these receptors [92, 93, 168] indicates that there might be redundancy in cellular molecules required for MARV attachment to cells.
A number of key residues of EBOV GP that are involved in virion incorporation and GP-mediated entry have been identified [163] and found to play a similar role in MARV GP [101] , indicating that the viruses might utilize similar mechanisms to enter the cell.
Following attachment, Marburg virions undergo endocytosis mediated through a mechanism that currently remains undetermined. (Figure 7 ) [16, 62] Initial studies investigating caveolin-mediated endocytosis showed that depletion of host cell cholesterol reduced viral infectivity but presented no direct evidence of caveolae involvement [169] . In addition, studies examining the role of caveolae in EBOV endocytosis are conflicting [169, 170] . A major role for clathrin in MARV entry has also been proposed based upon the ability of chlorpromazine (an inhibitor of both clathrin-mediated endocytosis and macropinocytosis) as well as RNAi-knockdown of clathrin heavy chain to inhibit MARV GP-pseudotyped HIV-1 entry [171] . A caveat to these analyses of MARV endocytosis is that they were performed only in the context of MARV GP-pseudotyped retroviruses which lack the characteristic filamentous morphology and size of Marburg virions.
While other reports have verified that cholesterol is important for live MARV particle uptake [172] , canonical caveolae-and clathrin-mediated mechanisms are unlikely to be the primary mechanism of MARV entry due to steric issues. The typical MARV particle size (average 790 nm) is much larger than canonical caveolae (50-100 nm) or clathrin-coated pits (up to 200 nm) whereas pseudotyped murine leukemia virus (MLV) (100 × 100 nm) and vesicular stomatitis virus (VSV) (70 × 180 nm) are not [173] . These findings indicate that involvement of the caveolae-and clathrin-mediated endocytic pathways for virus entry may therefore be the result of the artificial nature of the pseudotyped virions and highlights the need to confirm such experiments with live MARV.
Macropinocytosis has been identified as a major entry pathway of EBOV by research using the morphologically more relevant VLPs and live EBOV [174] [175] [176] [177] . Although none of these analyses examined the role of this pathway during MARV entry, it remains an intriguing possibility given the cholesterol-dependence and large size of macropinocytotic vesicles (up to 3-5 µm) [178] .
Another important process in MARV entry is believed to occur while virions are being trafficked within endocytic vesicles; the proteolytic cleavage of GP 1 . Endosomal cleavage of GP has been shown to be critical for the efficient entry of MARV [179, 180] . The current model for MARV entry involves the cleavage of GP 1 by host endosomal cysteine proteases. This removal of a large portion of GP 1 (including the mucin-like domain) is believed to expose the putative receptor-binding domain based on studies conducted with EBOV GP [181, 182] .
Studies examining the roles of endosomal proteases on the entry of MARV and EBOV have produced mixed results. Experiments analyzing recombinant VSV expressing EBOV GP indicate a primary role for Cathepsin B (CatB) and minor role for Cathepsin L (CatL) [181] . Entry of recombinant VSV particles containing MARV GP was inhibited when cells were treated with an inhibitor of both CatB and CatL [92] . These reports are confounded by a report conducted with infectious Marburg and Ebola viruses in which CatB and CatL inhibitors greatly reduced EBOV infection but showed mixed results with MARV [172] . Yet two other, more recent analyses determined that CatB was not required for MARV entry (although over-expression did enhance infectivity) and that CatL was required for entry into mouse embryonic fibroblasts but not Vero cells, 293T cells, or human macrophages [179, 180] . These data as well as the ability of other proteases to greatly diminish MARV infectivity [179, 180] , indicate that although CatB and CatL likely play a role in cleavage and activation of GP 1 in certain cell types, other endosomal proteases may also be able to facilitate GP 1 activation via cleavage.
Recently, two independent studies elegantly showed the requirement of the endosomal cholesterol transporter Niemann-Pick C1 (NPC1) for the entry of both MARV GP-pseudotyped viruses (VSV and MLV) as well as infectious MARV [94, 95] . It was also shown that NPC1 catalytic activity is not required for EBOV infection indicating that specific binding to NPC1 rather than its role in cholesterol transport is required, although this was not tested for MARV [95] . In one of the studies identifying NPC1 as the MARV entry receptor, it was also determined that members of the homotypic fusion and vacuole protein-sorting (HOPS) complex were important for EBOV entry, although they appeared to play a less important role in MARV entry [94] .
The current model of EBOV and MARV fusion is that GP 1 cleavage by endosomal proteases removes heavily glycosylated domains, exposing the receptor binding domain on GP 1 and enabling binding to NPC1 [95] . The membrane-bound fusogenic GP 2 undergoes a low pH-dependent rearrangement to an extended conformation resulting in the fusion of virion and endo-lysosomal membranes [96] . In support of the pH-dependence of GP-mediated fusion, pre-treatment of cells with ammonium chloride prevented entry of a MARV GP-pseudotyped virus [183] . A recent report with live MARV showed that ammonium chloride inhibited entry and replication, but that Bafilomycin A 1 , which specifically inhibits vacuolar-type H(+) ATPase and prevents re-acidification of vesicles of the central vacuolar system, surprisingly had no effect [172] . Following viral fusion with the endosomal membrane, the nucleocapsid is released into the cytoplasm (Figure 7 ).
After the nucleocapsid is released into the cytoplasm of the infected cell, transcription and replication of the viral RNA genome takes place (Figure 7 ). The first morphological sign of viral replication observed by EM analysis is the appearance of granular material containing RNA and viral proteins in the cytoplasm of the infected cells at 12 h post infection. Later on, tubular structures can be detected in the granular material representing the newly synthesized nucleocapsids embedded in the viral inclusions [63] . While experimental data on the sites of MARV replication and transcription are not available, recent studies on EBOV have shown that viral replication takes place in the inclusions, while transcription was observed prior to inclusion formation [184] .
The encapsidated negative-sense RNA genome is transcribed resulting in seven monocistronic mRNAs by the viral polymerase. They are co-transcriptionally capped and polyadenylated and subsequently translated by the cellular machinery (Figure 7) . The genomic RNA also serves as the template for the production of positive-sense antigenomes, which are complementary copies of the genomes. The antigenomes are encapsidated by the nucleocapsid proteins and are in turn used as templates for genome synthesis (Figure 7 ) (for review see [68] ). As mentioned above, NP, VP35, L, and probably VP30 are needed for viral transcription and replication. Analogous to EBOV, it is conceivable that VP40 and VP24 inhibit transcription and replication [113, 185, 186] . It is hypothesized that negative regulators of replication convert the active polymerase complex into an inactive state, resulting in mature and transport-competent nucleocapsids.
Following assembly, newly synthesized nucleocapsids are recruited to the sites of virus budding (Figure 7) . Release of viral particles is mainly mediated by VP40 via recruitment of nucleocapsids from the inclusions to the plasma membrane, recruiting GP to the sites of budding, and inducing the formation and release of filamentous VLPs. VP40-induced budding is enhanced by NP, GP, and VP24 [98, 111, 132] . As is the case with many other viruses, MARV exploits the vesicular transport machinery of the infected cell for viral egress, including the COPII vesicular transport system and the ESCRT machinery. The COPII vesicular transport system is used by VP40 for its intracellular trafficking to the multivesicular bodies, where MARV budding takes place [118, 187] . Cellular proteins that promote particle release and are linked to the ESCRT machinery include Tsg101, Vps4A/B, and Nedd4.1 [111, 119, 121, 122] . MARV budding not only takes place at internal membranes but also at the plasma membrane [63, 118, 188] . In cell culture, MARV particles are preferentially released at filopodia, filamentous cellular protrusions [60, 188, 189] . Filopodia are used by cells to explore the extracellular environment, which includes neighboring cells, and it is believed that viral particles can bud directly into adjacent cells via filopodia-mediated cell-to-cell contact [188, 190] . Budding at filopodia depends on actin and is not sensitive to the depolymerization of microtubules [188] . MARV budding was observed at the basolateral membrane of polarized epithelial cells and hepatocytes [109, 191] , whereas viral particles were predominantly released from the apical membrane of infected endothelial cells [64] , suggesting that cell-type specific components determine the sites of virus release.
Electron tomography studies of MARV-infected cells led to the following model for MARV particle release: The budding process is initiated when intracellular nucleocapsids associate laterally with the plasma membrane. Starting from one end, the nucleocapsids are then subsequently wrapped by the plasma membrane until the viral particles protrude vertically from the cell surface. The release of infectious filamentous MARV from cultured cells peaked at 1-2 days post infection, when the cells were still intact. At 4 d post infection, when most of the cells were vesiculated, the released virions were round or bent and infectivity was decreased [189] . Determination of the nucleocapsid orientation at the sites of budding by 3-D reconstructions revealed that the pointed tip of the budding nucleocapsids is oriented towards the membrane, indicating that MARV budding is directional [60] .
MARV infections usually occur by direct contact with infected body fluids or direct personal contact with infected animals or humans. The viruses enter the body through small skin lesions or mucosal membranes (reviewed in [47] ). Cells of the mononuclear phagocyte system, including monocytes, macrophages and dendritic cells, are early target cells of MARV, as shown in different experimental animal models [14, [192] [193] [194] [195] [196] [197] . MARV replication was observed as early as 24 hours post infection in macrophages of infected guinea pigs [63] , and infected monocytes have been found in cynomolgus macaques at 2 days post infection [193] . Monocytes and macrophages were also identified as early target cells in human patients [197] . This has been confirmed by cell culture experiments showing that primary human monocytes and macrophages are highly susceptible to MARV infection and produce infectious virus [198] [199] [200] . In addition, primary human monocyte-derived dendritic cells (mDCs) and endothelial cells support MARV replication [64, 146, 201] .
Early sites of virus replication are the lymph nodes, liver, and spleen where the most severe necrotic lesions are observed [8, 13, 14, 41, 130, 194, 202] .
These organs contain high numbers of monocytes and macrophages. Migration of infected monocytes and macrophages into surrounding tissues or transport of free virus via the lymph-or bloodstream is believed to facilitate the dissemination to multiple organs, resulting in a systemic infection [203, 204] . Cell-free virus has been observed in the tissue and organs of infected animals, and high levels of virus have been detected in the blood [12] [13] [14] 130, 194, 195, 205, 206] . Besides monocytes, macrophages, and dendritic cells, a wide range of cell types including hepatocytes, adrenal cortical and medullary cells and fibroblasts are permissive to MARV infection [12, 14, [192] [193] [194] [195] 197, 201, 207] . Endothelial cells are late target cells during MARV infection in multiple tissues. Whether or not replication of MARV in endothelial cells is associated with the observed vascular impairment during MVD is a matter of debate [64, 194] . Only low numbers of infected endothelial cells are observed in NHP infection and therefore changes in the endothelium are likely caused by paracrine effects of cytokines [14] .
In late stages of infection MARV particles can be isolated from nearly every organ [12, 14, 130, 194, 208] . Despite high viral load and necrotic lesions, only minor inflammation is observed in infected tissues and organs, indicating a dysregulated immune response [8, 14, 41, 194] . Strong liver pathology is observed, including increased serum activity of liver enzymes. This might influence synthesis of clotting factors and contribute to the observed coagulation defects in MVD [12, 41, 130, 194] . These factors together with systemic virus replication and associated pathology probably trigger the multiorgan failure associated with fatal cases.
Although lymphocytes are not susceptible to MARV infection [14, 193, 194, 201] , massive bystander lymphocyte apoptosis is a hallmark of MVD [8, 14, 130, 194, 195, 201] . However, the molecular mechanisms for lymphocyte depletion and the role it may play in the pathogenesis of MVD are far from being understood.
Cytokine secretion may play a role in the induction of lymphocyte apoptosis, since MARV-infected cells secrete cytokines known to induce apoptosis, including TNFα [194, 198, 200, 209] . Increased levels of TNFα have been observed in infected rhesus macaques [210] and mice [130] , although no increase was observed in infected cynomolgus macaques [14] . Elevated TNFα levels may also play a role in the formation of endothelial gaps in the context of MARV infection [198, 199] . In addition, increased survival of MARV-infected guinea pigs treated with anti-TNFα antibodies suggests that TNFα indeed plays an important role in MVD pathogenesis [209] .
Increased serum levels of additional proinflammatory cytokines and chemokines have been observed in infected NHPs and in mice, but the reported data are not completely consistent [12, 14, 130, 193, 210] . Cytokine and chemokine secretion has also been observed in infected primary human monocytes and macrophages [200, 211] . However, data about the cytokine levels in the serum of MARV-infected patients are not available, but high levels of cytokines have been observed in EBOV-infected patients [212] [213] [214] [215] .
Upregulation of the proinflammatory cytokines IL6 (mediator of fever and acute inflammatory response) and IL8 (chemoattraction of neutrophils and macrophages) is consistently found in infected NHPs, with macrophages and plasmacytoid dendritic cells (pDCs) serving as the main sources of IL6 secretion in the spleen [12, 14, 193] . Primary human monocytes and macrophages produce both IL6 and IL8 after infection [200] . Elevated levels of IL6 have also been detected in MARV-infected mice [130] . Increased levels of IL1β mRNA and secreted protein were detected in primary human cells [200, 211] , but contradictory data have been reported for the NHP model. One study reported elevated IL1β levels in final disease stages [12] , whereas no change was observed in another study [14] .
IFNα levels were elevated in infected NHPs and mice [130, 193, 210] . However, no change in IFNα levels was detected in another study of infected NHPs [14] . It is unclear whether or not the observed differences are due to different MARV variants being used for the studies.
Serum levels of several chemokines were also found to be elevated during MARV infection of NHPs and mice, including macrophage inflammatory proteins (MIP) and monocyte chemotactic protein 1 (MCP-1) [12, 14, 130] .
The involvement of multiple cell types along with the possible role of non-infected cells in the secretion of cytokines further complicates the analysis of existing data. Primary human monocytes and macrophages are activated by MARV infection inducing the secretion of cytokines. Induction of cytokines has also been described using UV-inactivated MARV, suggesting that viral replication might not be needed for the observed cytokine increase [200] . In contrast, MARV-infected mDCs show no upregulation of activation markers, do not secrete cytokines, and fail to stimulate T cells [146] . mDCs treated with VLPs containing MARV VP40 and GP show functional mDC responses including cytokine secretion indicating that MARV replication is required to inhibit mDC activation [216] . However, infection of mDCs with MARV did not prevent LPS-induced TNFα production whereas dsRNA-dependent IFNα secretion was inhibited [146] , suggesting differential regulation of cytokines by MARV. Interestingly, pDCs in the spleen were identified as the major source of secreted IFNα in MARV infected NHPs, but secretion most likely occurs from non-infected cells [193] . It has been shown for EBOV that pDCs are not productively infected due to impairment of viral entry [217] .
These results suggest that secretion of cytokines by non-infected bystander cells might play an important role during MARV pathogenesis. Taken together, MARV infection induces both an increase in the production of proinflammatory cytokines and high levels of chemokines, but the molecular mechanisms causing these changes are not well understood.
To date four different animal models have been established for MARV infection: NHPs, mice, guinea pigs, and hamsters. The NHP model best reflects the symptoms and pathology observed in human cases (described in 5. Clinical Manifestations and reviewed in [46, 48] ) with uniform lethality in cynomolgus and rhesus macaques as well as African green monkeys [12, 14, 26, 193, 194, 206, [218] [219] [220] . The disease symptoms are generally the same for all types of NHPs. The animals develop febrile illness with high fever, anorexia, weight loss and unresponsiveness. Death is observed after 6-13 days and thrombocytopenia, lymphopenia, blood coagulation abnormalities and hemorrhages are observed. Squirrel monkeys have also been successfully infected with MARV, showing typical disease symptoms [206] . Recently, a small NHP model using marmosets has been developed recapitulating the features of human infections except for the typical maculopapular rash development that is observed in other NHPs and humans infected with MARV [221] . Rodents with an intact immune system do not develop disease after infection with MARV. MARV variants Musoke and Ci67 and RAVV variant Ravn were adapted to severe-combined immunodeficiency (scid) mice by serial passaging, reducing the time to death from 50-70 days to 7-10 days for all tested virus variants [195] . Further passaging of the scid mouse-adapted marburgviruses in immunocompetent mice was used to establish mouse models for RAVV Ravn and MARV Ci67 [130, 131] . Successful adaptation by serial passaging was also used to generate lethal infection models for both guinea pig [218, 222] and hamster [206, 208] . Coagulation abnormalities, typical rash development, and hemorrhagic manifestations (especially in mice) are not as pronounced as in the NHP model [130] ; (reviewed in [223] ). Neuropathogenicity, recapitulating the CNS involvement described during the first human MVD outbreak in Germany [41, 224, 225] , has only been observed in the hamster model [208] . It is not clear if CNS pathology is developed in other animal models as no brain pathology has been observed in mice [195] and cynomolgus macaques [14] . Nevertheless, virus has been isolated from brain from MARV-infected marmosets, showing micro-hemorrhages [221] .
Sequence comparison of the rodent-adapted viruses to the human MARV isolates revealed several mutations. Sixty-one nucleotide changes in the mouse-adapted RAVV Ravn variant were detected in the ORFs of NP, VP35, VP40, and VP30 (14 amino acid changes in total) or untranslated regions (VP35, VP40, GP, VP30) [130] . In a second study analyzing genome changes during mouse adaptation, 75 nucleotide changes during adaptation of RAVV Ravn and 33 changes for MARV Ci67 were described, with most amino acid changes occurring in VP40 [131] . During guinea pig adaptation, only 11 nucleotide changes were observed, resulting in four amino acid changes. One of these changes was located in VP40 and the other mutations were detected in the viral polymerase L [222] . The only amino acid exchange detected in both the mouse-and the guinea pig-adapted MARV is amino acid 184 in VP40 (Asp to Asn). This was also the first mutation detected during mouse adaptation for both RAVV Ravn and MARV Ci67, as analyzed by sequencing of serial passages [131] . This is of particular interest because VP40 has been shown to function as an inhibitor of IFN signaling (see above, 7.3. Viral Proteins, VP40) [125, 126] . Both IFN receptor-and STAT1-deficient mice develop disease using non-adapted MARV, highlighting the importance of the IFN pathway for the control of MVD [226] [227] [228] .
Virological, serological, and molecular diagnostic methods for the detection for MARV are available, including virus isolation, ELISA, RT-PCR, EM, and immunohistochemistry (summarized in [16, 46] ). During outbreak settings, mobile laboratories commonly use PCR and/or ELISA analysis for rapid screening. Sensitive ELISA assays have been developed for detection of viral antigen or virus-specific antibodies using overexpressed MARV NP or GP [229] [230] [231] [232] . Detection of filoviruses by PCR is the only assay currently available to distinguish between different virus variants for a variety of tissue and fluid specimens. The use of a combination of virus-specific primer sets for conventional RT-PCR makes the detection of all know filoviruses in a single assay possible [233] . For more sensitive and quantitative detection, real-time RT-PCR-based assays have been developed for the detection of MARV [10, [234] [235] [236] [237] [238] . Feasibility of real-time RT-PCR in the field was successfully proven during the MARV outbreak in Uíge, Angola using an improved field laboratory-adapted RNA isolation protocol [239] . A network of European BSL-4 facilities in collaboration with a company (QIAGEN) developed the first commercial prototype of a real-time-RT-PCR assay for the detection of filoviruses [240] . A recently developed assay, RT loop-mediated isothermal amplification (LAMP), has the potential to significantly improve field diagnosis of MARV infections, by eliminating the need of PCR machines [241] .
Initial approaches using inactivated virus to develop a vaccine against MARV were unsuccessful or had contradictory results [16] . In addition, successful protection of rodents did not always translate into protection of NHPs. For example, inactivated MARV protects guinea pigs from lethal MARV challenge but only 50% of challenged NHPs survived [210, [242] [243] [244] . A panel of different approaches has been used in order to develop successful vaccines for MARV (reviewed in [245, 246] ).
Recombinant GP expressed from insect cells or a DNA vaccine based on GP only partially protected guinea pigs, but use of a combination of both vaccines resulted in 100% survival of guinea pigs [243] . In another study, complete protection of guinea pigs using a different GP DNA vaccine was reported, but only four of six vaccinated NHPs survived the challenge with MARV, showing incomplete protection [247] . Increased doses of a codon-optimized DNA vaccine resulted in 100% survival of NHPs, although some animals developed symptoms before recovering. In comparison to other vaccine candidates, a poor induction of virus-specific antibodies was observed using a DNA vaccine [248] . A codon-optimized DNA vaccine elicited a strong antibody response and resulted in complete protection of mice with no clinical symptoms observed [249] . Vaccine candidates based on the Venezuelan equine encephalitis virus (VEEV) replicon system expressing either MARV GP along with NP or GP alone completely protected guinea pigs and NHPs [250] .
A vaccine based on VLPs represents an additional candidate for protection against MARV [251] . Complete protection of guinea pigs has been demonstrated with a VLP-based vaccine containing MARV GP, with induction of virus-specific antibodies. Protection with this vaccine relied on a functional CD4+ T cell response, whereas depletion of CD8+ T cells did not ablate the protective response [244] . VLPs containing MARV Musoke GP provided cross-protection in animals challenged with MARV Ci67 or RAVV Ravn in both guinea pigs and NHPs [252, 253] .
Another approach to MARV vaccines is the use of viral vectors expressing MARV GP. To date, two different systems have been established based on replication-defective adenoviral vectors or recombinant VSV expressing MARV GP. The adenovirus-based vaccine successfully protects guinea pigs and NHPs, and provides cross-protection. High levels of cross-reactive MARV-specific IgG and T cell responses are induced, indicating an induction of an immune response [248, 254] . Preexisting immunity against the adenovirus strain Ad5 might pose a problem for its successful use in humans (reviewed in [245] ).
The VSV-based vaccine completely protects NHPs and additionally has proven successful in post-exposure treatment (reviewed in [255] ). A single immunization with recombinant VSV expressing MARV Musoke GP resulted in 100% protection of cynomolgus macaques challenged by intramuscular injection or aerosol exposure and protected against RAVV Ravn and MARV Angola [13, 256, 257] . Although MARV-specific IgG were produced, only low levels of neutralizing antibodies were detected [13, 257] . Surprisingly, T cell-mediated responses were not observed in NHPs vaccinated with recombinant VSV expressing MARV GP [13, 256] . Safety is a concern for this vaccine, especially for immunocompromised individuals, as it is a replication-competent VSV vector. However, in all VSV-based filovirus vaccine studies VSV viremia was observed only shortly after immunization. Additionally, the VSV-based filovirus GP vaccine was well tolerated and protective in immunocompromised mice and NHPs and lacked neurovirulence in NHPs [258] [259] [260] (reviewed in [255] ).
Cross-protection has not been observed in animals vaccinated with MARV-based vaccines and subsequently challenged with EBOV, while combined MARV and EBOV vaccines have been successful in protection against both viruses [252, 261, 262] .
To date no approved treatment is available for MARV infection. Supportive care (fluids, anti-microbials, blood transfusions) has been the primary treatment of patients during MVD outbreaks. In the guinea pig model various treatments had some success as reflected by prolonged survival or increased survival rates. Applied treatments included cytokine inhibition, IFN treatment, or antibody transfer. The tested treatments were unsuccessful in the NHP model (reviewed in [16, 47] ).
A third of EBOV-infected NHPs survived, however, following treatment with recombinant nematode coagulant protein 2, while only one of six MARV-infected animals survived [12, 263] . Treatment using antisense technology to block viral protein expression using phosphorodiamidate morpholino oligomers (PMO) beginning 30 to 60 minutes after MARV infection completely protected NHPs [264] . Additionally, a small molecule inhibitor showed complete protection of MARV-infected mice when administered 24h after infection but has not been tested in NHPs [265] .
The VSV-based vaccine expressing MARV GP has also been demonstrated to be effective as a post-exposure treatment. A hundred percent survival of NHPs was observed when the vaccine was administered 20 to 30 minutes after MARV infection [266] . Delaying the time before treatment results in incomplete protection, although five of six animals or two of six animals still survived when given the treatment 1 or 2 days after MARV exposure, respectively [205] . These post-exposure treatments may be useful to prevent disease after known exposure to MARV, such as a laboratory accident, but the effective time frame during an outbreak might be too short and alternatives are needed. The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system. Most replicative DNA polymerases include a catalytic subunit, responsible for DNA polymerization, and a processivity factor that holds the catalytic subunit on DNA to allow continuous DNA synthesis. One of the best-studied processivity factors is proliferating cell nuclear antigen (PCNA) of eukaryotic DNA polymerases d and e [1] . PCNA, which belongs to the family of so-called ''sliding clamps'', has no inherent DNA-binding capacity, but with the aid of clamp loader proteins is assembled onto DNA as a toroidal homotrimer [2] . In addition to DNA replication, PCNA has been implicated in DNA recombination and repair, as well as in DNA methylation, chromatin remodeling, and cell cycle regulation [1] . Consistent with its pleiotropic functions, it interacts with a plethora of proteins [3] and undergoes a number of posttranslational modifications, including phosphorylation, acetyla-tion, ubiquitination and sumoylation, which are believed to regulate its subcellular localization, stability and protein binding specificity [4, 5, 6] .
The human cytomegalovirus (HCMV) DNA polymerase includes a catalytic subunit, UL54 (the UL54 gene product), and an accessory, homodimeric subunit, UL44 (the UL44 gene product), that binds DNA without the aid of clamp loaders [7] yet wraps around DNA akin to PCNA [8] . While UL44 shows no apparent sequence homology with PCNA, there is striking structural similarity between UL44 and PCNA monomers [2, 9] . Similarly to PCNA, UL44 is a phosphoprotein [10] . Intriguingly, the phosphorylation state of UL44 has been shown to regulate its nuclear import rate by controlling its interaction with host cell factors [11, 12, 13] . The best-characterized function of UL44 during HCMV infection is that of binding to UL54 through a region named connector loop [14, 15, 16] , stimulating its activity and conferring processivity to the holoenzyme [17, 18] . However, UL44 continues to accumulate to strikingly high levels at late times after infection, when DNA replication is accomplished [19, 20] . Its early-late kinetics of transcription and the high level of expression suggest that UL44 might play additional roles during the viral life cycle.
To investigate this possibility, we conducted yeast two-hybrid (Y2H) screenings to search for cellular partners of UL44. To our surprise, Ubc9, an enzyme involved in the sumoylation process, was identified as a UL44 protein interaction partner. Sumoylation is a post-translational protein modification analogous to ubiquitination. It consists of reversible and covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to a protein target [21, 22] . In the sumoylation cascade, the C-terminus of SUMO is activated by an activating enzyme (E1), transferred to a conjugating enzyme (E2, that is Ubc9), and linked to a lysine residue of the substrate protein with the aid of a ligase (E3). Mainly, three SUMO paralogs (SUMO-1, -2, -3) have been identified so far [23, 24] . SUMO-2 and SUMO-3 are highly homologous to one another (95% identity) while they differ from SUMO-1 by 50%. Conjugation of SUMO-1 has been shown to play a functional role in a number of biological processes, ranging from nucleocytoplasmic transport to transcription, the maintenance of genome stability, nucleic acid DNA metabolism, cell signaling, and many others [21] , whereas the role of SUMO-2/23 modification is less clear.
Here we report that the association of Ubc9 and UL44 leads to conjugation of SUMO molecules on multiple lysine residues. Both SUMO-1 and SUMO-2/3 were found to be conjugated to UL44. Sumoylation of UL44 was detected not only in vitro and in transiently transfected cells but, more importantly, also in HCMVinfected human cells during virus replication. Interestingly, we observed that binding of UL44 to DNA greatly stimulates SUMO conjugation to the protein both in vitro and in cells. In addition, we show that overexpression of SUMO-1 alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both viral DNA replication and virus production in an Ubc9-dependent manner. These data represent the first report of sumoylation of a viral processivity factor and show that there is a complex interplay between the HCMV UL44 protein and the cellular sumoylation system.
The Y2H plasmids expressing LexA-UL44 and LexA-Ubc9 were generated by cloning the UL44 and Ubc9 coding sequences from pRSET44 (a gift of P. F. Ertl, GlaxoSmithKline, UK) and pACT2-Ubc9 (from G. Gao, Chinese Academy of Sciences, Beijing, China) respectively, in pBTMK, derived from pBTM116 [25] . The pACT-UL44 and pACT2-Ubc9 plasmids, encoding GAD-UL44 and GAD-Ubc9 fusions, respectively, have been described in [26, 27] . The plasmid expressing GAD-UL54 was created by cloning the UL54 coding sequence from pRSET-Pol (a gift of P. F. Ertl) in pACT2 (Clontech). Plasmid pRSET44 was used to express 6His-UL44 in Escherichia coli. Plasmid pRSET-Ubc9 was constructed by cloning the Ubc9 coding sequence from pACT2-Ubc9 in pRSET (Invitrogen). Plasmid pCDNA3-PB1, used for in vitro transcription of the PB1 subunit of influenza A virus RNA polymerase, was described previously [28] . Plasmids pD15-GST and pD15-UL44, which express GST and GST-UL44, respectively, have been described in [29] . Plasmid pTE1E2S1 [30] was provided by H. Saitoh (Kusamoto University, Japan). Plasmids GFP-UL44, pDESTnV5-UL44, and pDESTnV5-UL44DNLS have been described previously [11, 31] . Plasmids pDsRed2-Ubc9 [27] and pDsRed2-UL53 [32] were kindly provided by G. Gao (Chinese Academy of Sciences, Beijing, China) and D. Camozzi (University of Bologna, Italy), respectively. Plasmid pCDNA3.1-UL44-FLAG (from M. Marschall, Universitat Erlangen-Numberg, Germany) was used to express C-terminally FLAG-tagged UL44 [33] , while plasmid pDEST-nFLAG [34] was used to express N-terminally FLAG-tagged UL44 [35] , UL44Dloop [36] and UL44L86A/L87A in mammalian cells. Plasmids pCDNA3-Ubc9, pCDNA3-Ubc9C93S, pCDNA3-HA-SUMO-1, pCDNA3-HA-SUMO-2, and pCDNA3-HA-SUMO-3 used in overexpression experiments in mammalian cells were a gift of R. T. Hay (University of Dundee, UK). The deletion mutants LexA-UL44 1-100 , LexA-UL44 1-200 , LexA-UL44 1-300 , LexA-UL44 1-350 , LexA-UL44 1-390 , and LexA-UL44 1-420 were generated by PCR amplification of plasmid pBTMK-UL44 with appropriate primers (Table S1 ). The LexA-UL44 114-433 , LexA-UL44 201-433 , and LexA-UL44 313-433 constructs were generated by deleting part of the UL44 coding sequence from pBTMK-UL44 with restriction enzymes. Plasmids pDESTnFLAG-UL44(1-300) and pDESTnFLAG-UL44(313-433) were generated using the Gateway Technology (see Supplementary Material and Methods in Text S1). All other UL44 mutants and the Ubc9C93S mutant were obtained by using the QuikChange mutagenesis kit (Stratagene) with primers containing appropriate nucleotide change(s). More details on plasmid construction and mutagenesis are given in Supplementary Material and Methods in Text S1. The sequences of all primers used in this work are reported in Table S1 . All DNA sequences were confirmed by sequencing.
Y2H screenings and interaction assays. Growth media and standard methods for manipulating yeast cells were as described [37] . Saccharomyces cerevisiae strain L40 was transformed [38] with the bait plasmid pBTMK-UL44 and subsequently with either of two cDNA libraries fused to GAD (see Supplementary Material and Methods in Text S1). Primary transformants were selected for growth on -His-Leu-Trp dropout plates. His + colonies were thereafter analyzed for b-galactosidase activity by filter lift experiments [39] . Double positive clones were subjected to another cycle of screening (for further details see Supplementary Material and Methods in Text S1). cDNA inserts of interactor plasmids were sequenced and analyzed with BLAST (www.ncbi/ blastn). To quantify b-gal expression, the method of Breeden and Nasmyth [40] was used.
Proteins. E. coli-expressed, purified GST and GST-or 6Histagged UL44 proteins were obtained as previously described [29] , with modifications (Supplementary Material and Methods in Text S1). In some preparations, samples were treated with polymin P as described [41] to eliminate residual bacterial nucleic acids. Preparation of UL44 SUMO-modified in E. coli was accomplished as described in Supplementary Material and Methods in Text S1.
GST-pulldown assays. Assays were performed using GST and GST-UL44 and in vitro-translated UL54, PB1, or Ubc9 as previously described [29] , with modifications (Supplementary Material and Methods in Text S1). In vitro transcription-translation of proteins was performed from the appropriate plasmid by using the TNT T7 coupled transcription-translation system (Promega). The translation products were labeled with [ 35 S]methionine (Amersham Pharmacia Biotech).
In vitro sumoylation assays. The assays to test in vitro sumoylation of UL44 by SUMO-1, -2, and -3 were performed using purified wild-type and mutant 6His-UL44 or GST-UL44 fusion proteins and the SUMOlink SUMO-1 kit from Active Motif or the SUMOylation Kit from Enzo Life Science according to the manufacturer's suggestions. In some experiments, double stranded (ds) DNA (e.g., activated calf thymus DNA from Amersham Pharmacia Biotec or salmon testes DNA from Sigma) or singlestranded (ss) DNA (e.g., single-stranded calf lung DNA, from Crinos, Como, Italy) was added to the reaction mixture at a final concentration of 500 nM.
Human foreskin fibroblasts (HFF; from the American Type Culture Collection [ATCC]), HeLa (from ATCC), eco Phoenix (a generous gift from G. P. Nolan, Stanford, USA; [42] ), and Human Embryonic Kidney 293T (HEK 293T; from ATCC) cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Biotechnologies) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin (P/S). COS-1 cells (from ATCC) were maintained in DMEM with 5% FCS and P/S. The U373-SUMO-1 cell line, which constitutively expresses FLAG-tagged SUMO-1 [43] , and the control U373-Neo cell line, stably transfected with an empty vector carrying a Neomycin resistance marker [44] , were kindly provided by G. S. Hayward (Johns Hopkins University School of Medicine, Baltimore, USA) and were maintained in medium containing 0.5 mg/ml Neomycin (G418, Gibco-BRL). HCMV strain AD169 was purchased from ATCC.
To analyze UL44 sumoylation in transient expression assays, HeLa or Phoenix cells were transfected for 48 h with appropriate plasmids using the calcium phosphate precipitation method. For analysis of UL44 sumoylation during HCMV replication, HFF, U373-Neo, and U373-SUMO-1 cells were mock-infected or infected with HCMV at a multiplicity of infection (MOI) of 5 PFU/cell. Cells were harvested at different time points after infection and analyzed by western blotting as described below.
Transfected or infected cells were lysed in an appropriate volume of buffer I (5% SDS, 0.15 M Tris-HCl pH 6.8, 30% glycerol) diluted 1:3 in buffer II (25 mM Tris-HCl pH 8, 50 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete protease inhibitors (Roche Molecular Biochemicals) and 5 mM N-ethylmaleimide (NEM). Lysates were then incubated on ice for 20 minutes and boiled at 95uC for 10 min. Proteins were separated by SDS-PAGE, electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad), and analyzed by western blotting with indicated antibodies (details are given in Supplementary Material and Methods in Text S1).
For immunoprecipitation analysis, lysates were diluted 1:5 in E1A buffer (50 mM Hepes pH 7.5, 250 mM NaCl, 0.1% NP-40) supplemented with Complete protease inhibitors and 5 mM NEM. Immunoprecipitation was performed with 2-3 mg of total lysate using a ratio of 3-7 mg antibody/mg of total proteins (see Supplementary Material and Methods in Text S1 for details on antibodies) and protein A-Sepharose beads. For co-immunoprecipitation analysis, cells were lysed in E1A buffer supplemented with Complete protease inhibitors and 5 mM NEM, and successively co-immunoprecipitations were performed with 1.5-5 mg of total lysate and 50 ml of 50% slurry of anti-FLAG-M2-Agarose beads (Sigma).
Mass spectrometric identification of sumoylated lysine residues within UL44 was performed after in-gel-digestion of E. coliexpressed and SUMO-modified UL44 with endoproteinase Trypsin. Extracted peptides were analyzed by LC-MSMS on an Orbitrap Velos (ThermoFisherScientific) exactly under the conditions described in Hsiao et al. [45] . Data analysis was performed by the use of software ''ChopNSpice'' [45] in combination with MASCOT as search engine. See Supplementary Material and Methods in Text S1 for details.
For confocal laser-scanning microscopy (CLSM) analysis, COS-1 were transfected using the Arrest-IN TM (Biosystems) reagent, according to the manufacturer's recommendations. At 24 h posttransfection, cells were fixed with 4% paraformaldehyde. Cells were imaged using a Leica TCS-SP2 confocal microscope equipped with a 636 oil immersion objective.
For analysis of UL44 intranuclear localization in HCMVinfected U373-SUMO-1 and U373-Neo cell lines, cells were seeded at 2.5 6 10 5 /well on glass coverslips in 6-well plates and allowed to attach. The next day, cells were infected with HCMV AD169 at an MOI of 1 or of 5 PFU/cell. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, and then permeabilized with acetone for 2 min at -20uC. After washing extensively with PBS, cells were incubated first with 4% FBS in PBS for 1 h at room temperature and then with a primary mouse monoclonal antibody against UL44 (10-C50, Fitzgerald Industries International) at a dilution of 1:100 in FBS 4% in PBS for 1 h at 37uC. Cells were then washed extensively with 4% FBS in PBS and incubated with a secondary goat anti-mouse fluorescein-conjugated antibody (Ig-FITC, Chemicon International) at a dilution of 1:1000 for 1 h at 37uC. Cells were successively washed with PBS and mounted in 70% glycerol in PBS. For better visualization, cells were counterstained with Evans Blue and analyzed also for red fluorescence. CLMS analysis was then performed as described above.
To analyze the effects of SUMO-1 overexpression on viral DNA synthesis, U373-Neo and U373-SUMO-1 cells transduced with either shUbc9 or non-silencing lentiviral particles (see below) or non-transduced, were seeded at a density of 5610 4 per well in 24well plates. The next day, cells were infected with HCMV AD169 at an MOI of 1 PFU/cell. At 72 h post-infection (p.i.), cells were collected and total DNA was extracted using the QiAmp DNA Extraction Kit (Qiagen). The levels of viral DNA were then determined by qPCR and normalized to the cellular b-globin gene copies as described [46] .
To analyze the effects of SUMO-1 overexpression on virus production, virus yield assays were performed as described previously [47] , with some modifications. Briefly, U373-Neo and U373-SUMO-1 cells transduced with either shUbc9 or nonsilencing lentiviral particles (see below) or non-transduced, 5 610 4 cells per well were seeded in 24-well plates, incubated overnight, and infected with HCMV AD169 at an MOI of 1. At 120 h p.i., cells were subjected to one cycle of freezing and thawing, and titers were determined by transferring 100-ml aliquots from each of the wells to a fresh 96-well monolayer culture of HFF cells followed by 1:5 serial dilution across the plate. After incubation at 37uC for 7 days, cell monolayers were stained with crystal violet and plaques were counted.
Lentiviral particles were produced by transient transfection of HEK 293T cells with packaging plasmids helper D8.9 (Addgene) and helper Ampho (kindly provided by the Tissue Culture Facility at the IEO, Milan, Italy) and either pTRIPZ-shUBC9 vector (Open Biosystems) or non-silencing pTRIPZ control vector (Open Biosystems) by the calcium phosphate precipitation method. Supernatants were collected at 48 h post-transfection and used for the transduction of U373-SUMO-1 and U373-Neo target cells in the presence of polybrene (8 mg/ml, Sigma). Selection was done in puromycin (0.5 mg/ml, Invitrogen) for two weeks prior to 4-day doxycycline (1 mg/ml, Sigma) induction to obtain Ubc9 silencing. Ubc9-knocked-down cells were screened for Red Fluorescent Protein expression and used for further experiments.
To identify cellular proteins that interact with UL44, Y2H screens were carried out with a bait consisting of full-length UL44 protein (amino acids 1-433) fused to the E. coli LexA protein.
Control experiments demonstrated that the LexA-UL44 protein did not activate expression of either HIS3 or lacZ reporter gene by itself and, as expected [9, 14] , could both interact with UL54 and dimerize (Table 1 and data not shown). Thus, this bait was used to screen two different cellular cDNA libraries fused to S. cerevisiae GAL4 activation domain (GAD), one derived from human B lymphocytes [48] and the other from promyelocytic HL-60 cells [49] . A total of 167 or 85 colonies, respectively, were positive for both HIS3 and lacZ reporter genes. Plasmids encoding putative interactors of UL44 were isolated from double-positive clones and retransformed into yeasts expressing LexA-UL44 in order to confirm the interaction. The 28 and 13 positive clones after this retransformation, respectively, were sequenced.
In total, from the two screenings we identified 7 cellular UL44binding proteins; here we report the identification of human Ubc9 as a specific interaction partner of UL44. In particular, 15 out of 28 clones of B lymphocytes library and 6 out of 13 clones of the HL-60 cells library contained the whole Ubc9 coding sequence, plus 59 and 39 untranslated regions which varied in length in different clones. By co-transformation experiments with each individual interactor clone expressing Ubc9 and the LexA vector, it was excluded that Ubc9 could activate the reporter genes in the absence of the bait protein (Table 1) . Ubc9 specifically interacted with UL44 in Y2H assays also in the reverse combination, i.e., with Ubc9 fused to LexA and UL44 fused to GAD (Table 1 ). In addition, in quantitative assays the b-galactosidase activity of yeasts expressing both UL44 and Ubc9 turned out to be comparable to that of yeasts expressing UL44 and UL54 (Table 1) , which served as a positive control.
Having identified Ubc9 as a potential interaction partner of UL44 in Y2H screens, we wished to confirm their physical interaction by an independent experimental approach. For this purpose, pulldown assays with a purified GST-UL44 protein and in vitro-translated, 35 S-labeled Ubc9 were performed. As positive and negative controls, we also assayed the interaction between UL44 and in vitro-translated UL54 or the PB1 subunit of influenza A virus RNA polymerase. As expected, we could detect the interaction of UL54 [14, 29] , but not of PB1, with GST-UL44 (Fig. 1A) . Consistent with the Y2H results, Ubc9 specifically associated with GST-UL44, while no interaction with GST was observed (Fig. 1A) .
Since our data suggested that UL44 can interact with Ubc9 both in vitro and in yeast cells, we sought to examine whether UL44 and Ubc9 could also co-localize in mammalian cells. To this end, aggregates distributed on a diffuse background fluorescence throughout the nucleus (Fig. 1B) . As previously reported [50] , when expressed alone Ubc9 exhibited a nuclear punctate pattern (Fig. 1B) . Upon co-expression of GFP-UL44 and DsRed2-Ubc9, co-localization of UL44 we analyzed the subcellular localization of GFP-UL44 when transiently expressed either alone or in the presence of DsRed2-Ubc9. As a negative control, we also expressed the DsRed2-UL53 fusion protein, which localizes to the cell nucleus but does not interact with UL44 [32] . In GFP-UL44-transfected cells, UL44 localized in a large number of discrete nuclear with Ubc9 was observed (Fig. 1B) . UL44 also colocalized with a catalytically impaired Ubc9 mutant (Ubc9C93S) [51] , but not with UL53 [32] . In addition, co-immunoprecipitation experiments confirmed that UL44 could physically interact with endogenous Ubc9 in mammalian cells. The specificity of the interaction was confirmed by the inability of UL44 to coimmunoprecipitate with Cyclin D1 (CycD1; Fig. 1C ).
To further explore the interaction between UL44 and Ubc9, we sought to map the domain of UL44 that interacts with Ubc9. To this aim, several N-and C-terminal deletion mutants of UL44 fused to LexA were generated and tested for the ability to interact with GAD-Ubc9 by Y2H assays. Control western blot experiments with an anti-LexA antibody evidenced protein bands of the expected molecular mass for all mutants (data not shown). As shown in Fig. 1D , the truncated protein UL44 1-300 , lacking most of the C-terminal disordered region of UL44, exhibited interaction with Ubc9. Further C-terminal truncation of UL44 revealed that a protein fragment corresponding to the first 200 amino acids of UL44 (UL44 ) was still capable of interacting with Ubc9 ( Fig. 1D ). In contrast, the N-terminal 100 residues of UL44 (UL44 1-100 ) exhibited no interaction with Ubc9 (Fig. 1D ). Although unfolding of the UL44 1-100 mutant protein cannot be excluded, these results suggested that this region of UL44 may not contain sequences important and/or sufficient for Ubc9 binding. Therefore, we analyzed the effects of N-terminal truncations. Deletion of the N-terminal 113 residues of UL44 (UL44 114-433 ) did not impair the ability of UL44 to bind Ubc9. Similarly, the UL44 201-433 mutant, lacking the N-terminal 200 amino acids, interacted with Ubc9. Interestingly, a mutant that only expresses the C-terminal 121 residues of UL44 (UL44 313-433 ) still retained the ability to bind Ubc9 (Fig. 1D) . Control Y2H experiments showed that none of the truncated UL44 proteins was able to activate transcription by itself (data not shown). Mapping studies in mammalian cells expressing UL44 deletion mutants showed that both the UL44 1-300 and UL44 313-433 mutants could immunoprecipitate endogenous Ubc9, similarly to wild-type UL44 (Fig. S1 ). Thus, our results suggest that UL44 contains two domains capable of independently binding to Ubc9, located at the N-and Cterminus of the protein (likely within residues 1-200 and 313-433, respectively).
The observation that UL44 interacts with the SUMO-conjugating enzyme Ubc9 prompted us to investigate the possibility that UL44 may be sumoylated. This hypothesis was first tested in a cellfree system by incubating a purified 6His-UL44 fusion protein with purified Aos1/Uba2 (E1), Ubc9 (E2), and SUMO-1. As a control, sumoylation of human p53 was also examined by the same assay. As expected [52, 53] , p53 was readily modified to give mainly a single mono-sumoylated product that reacted with both anti-p53 ( Fig. 2A , left bottom panel) and anti-SUMO-1 ( Fig. 2A , right bottom panel) antibodies (the bands appearing at high molecular weight, which are particularly visible in the anti-SUMO-1 panel, correspond to SUMO-E1 and -E2 enzymes conjugates). In a western blot analysis with an anti-UL44 antibody ( Fig. 2A , left top panel), three main slower migrating forms of UL44 were observed (lane 3). The appearance of the ,65, 80, and 95-kDa forms of UL44 was strictly dependent on the presence of SUMO-1, as the substitution of wild-type SUMO-1 with a mutant form which bears the Gly97-to-Ala change (SUMO-1 mut) and hence cannot be attached to target proteins, eliminated formation of these products ( Fig. 2A, left top panel, lane 4 ). In addition, SUMO-1 modification was abolished if either SUMO-1, Aos1/ Uba2, or Ubc9 was omitted from the reaction (not shown). A western blot analysis with an anti-SUMO-1 antibody confirmed that the slower migrating UL44 bands contained SUMO-1 ( Fig. 2A, right top panel) . Taken together, the above results established that UL44 is a substrate for in vitro SUMO-1 conjugation.
Having shown that UL44 can be modified by SUMO-1, we wondered whether it could be conjugated also to SUMO-2 and SUMO-3. Thus, the in vitro sumoylation system was applied to purified 6His-UL44 in the presence of sumoylation enzyme components and activated forms of SUMO-2 or SUMO-3. In these assays, the RanGTPase-activating protein RanGAP1 [54] was used as a positive control and as previously observed [55] , was modified in the presence of SUMO-2 and SUMO-3 (data not shown). As shown in Fig. 2B , UL44 was also modified by either of the two SUMO peptides.
To verify whether UL44 could also be sumoylated in mammalian cells, we expressed UL44-FLAG and HA-SUMO-1 fusion proteins, in the presence of either wild-type Ubc9 or the catalytically impaired mutant Ubc9C93S in Phoenix cells and analyzed cell lysates by western blotting with anti-FLAG, anti-HA, and anti-Ubc9 antibodies. As expected, several bands corresponding to SUMO-1-conjugated proteins that reacted with an anti-HA antibody were detected upon co-expression of wild-type Ubc9, while less SUMO-1 conjugation was observed in the presence of the Ubc9C93S mutant (Fig. 3A) . This was not due to differences in expression of the two Ubc9 variants, as evidenced by western blot analysis with the anti-Ubc9 antibody (Fig. 3A) . When UL44-FLAG was expressed in the absence of HA-SUMO-1 and Ubc9, a single band with an apparent molecular mass of ,50 kDa was detected using the anti-FLAG antibody. In contrast, slower migrating bands similar to those observed in in vitro sumoylated products ( Fig. 2A) were observed upon co-expression of UL44-FLAG with HA-SUMO-1 and wild-type Ubc9 (Fig. 3A) . These bands were significantly reduced in the presence of the Ubc9C93S mutant (Fig. 3A) , demonstrating that they were dependent on the catalytic activity of Ubc9, with the residual slower migrating bands due to the activity of wild-type endogenous Ubc9.
To confirm that the observed products were indeed sumoylated forms of UL44, we expressed UL44-FLAG either in the absence or in the presence of HA-SUMO-1 and Ubc9, immunoprecipitated UL44-FLAG using an anti-FLAG antibody and analyzed the immunoprecipitated proteins by western blot. As expected, three slower migrating bands were detected by the anti-FLAG antibody from lysates of cells expressing UL44-FLAG in the presence of both HA-SUMO-1 and Ubc9 (Fig. 3B, left panel) . The ,50-kDa non-sumoylated form of UL44 could be immunoprecipitated both from cells expressing UL44-FLAG alone and from cells expressing UL44-FLAG together with HA-SUMO-1 and Ubc9, but not from Table 1 . UL44 interacts with human Ubc9 in yeast two-hybrid assays. LexA-UL44 / 2(,1)
UL44, UL54, and Ubc9 proteins were fused to the C-terminus of LexA protein and/or of GAL4 activation domain (GAD). Fusion proteins were then assayed for interaction by qualitative b-galactosidase (b-gal) filter assays and by quantitative b-gal liquid assays. Ubc9, but not in immunoprecipitates obtained from cells expressing only UL44-FLAG (Fig. 3B, right panel) . Similar results were obtained in HeLa cells (Fig. S2) . Altogether, these results demonstrate that Ubc9 can mediate the conjugation of SUMO-1 to UL44 in mammalian cells. Moreover, sumoylation of UL44 in mammalian cells by both SUMO-2 and SUMO-3 could also be detected (Fig. S3) .
Since sumoylation mainly occurs in the nucleus of mammalian cells [56] and UL44 is translocated to the host cell nucleus during HCMV infection [11] , we decided to investigate whether nuclear localization might be a prerequisite for conjugation of SUMO-1 to UL44. We therefore analyzed the ability of UL44bDNLS, a derivative of UL44 bearing point mutations within the nuclear localization signal (NLS, 425 PNTKKQK 431 ) which prevent UL44 nuclear accumulation [11] , to be modified by SUMO-1. Interestingly, mutations of UL44 NLS impaired the sumoylation of a V5-UL44 fusion protein (V5-UL44DNLS, Fig. 3C ), suggesting that SUMO-1 modification of UL44 most likely occurs into the nucleus or during nuclear import.
We next sought to identify the SUMO-1 acceptor sites of UL44. A prediction analysis with the SUMOplot program (Abgent) identified seven residues in UL44 with a certain probability to be sumoylated: K73, K172, K224, K339, K371, K410, and K431. To test whether one or more of these lysines could be a SUMO-1 acceptor site, each of the candidate residues was conservatively mutated to arginine, both individually and in combination, and the mutant proteins were tested for in vitro sumoylation. None of the single point mutants exhibited a consistently altered SUMO-1 modification pattern as compared to wild-type UL44 (Fig. S4A ). Mutants carrying two or three K/R substitutions also showed a SUMO-1 modification pattern identical to that of wild-type UL44 ( Fig. S4A and data not shown) . Similar results were obtained when in vitro sumoylation reactions with SUMO-2/23 were performed (data not shown). We decided to also test the ability of these K/R mutants to be modified by SUMO-1 in mammalian cells. Consistent with the in vitro data, none of the tested mutations significantly affected the ability of UL44 to undergo SUMO-1 conjugation ( Fig. S4B and data not shown) .
These results suggested that UL44 might possess multiple lysine residues that could alternatively serve as SUMO-1 acceptors, as reported for other proteins [57, 58] , and/or that UL44 might be sumoylated on lysine residues other than those predicted by SUMOplot. UL44 contains 31 lysines, most of which are solventexposed in the crystal structure [9] and therefore potentially accessible to SUMO molecules, which makes it difficult to identify the target lysines by mutational approaches. Therefore, we attempted to map the sites of UL44 where SUMO-1 is conjugated by mass spectrometry analysis. To this end, we expressed UL44 in an E. coli expression/modification system that produces SUMOconjugated proteins [30] . The 6His-tagged UL44 construct was co-expressed in E. coli with the pTE1E2S1 plasmid, which contains a linear fusion of genes for E1 and E2 enzymes and SUMO-1 under the control of an IPTG-inducible promoter [30] . To confirm sumoylation of UL44 with this system, UL44 was purified from the bacterial cultures expressing UL44 alone or in combination with the SUMO conjugation system and analyzed by western blotting with both the anti-UL44 (Fig. S5, left panel) and the anti-SUMO-1 (Fig. S5, right panel) antibody. Shifted bands with an apparent molecular mass of ,65, 80, and 95 kDa, similar to those detected in in vitro reactions ( Fig. 2A) and in cells (Fig. 3) , were observed in UL44 purified from bacteria cotransformed with pTE1E2S1, but not in UL44 purified from bacteria expressing only UL44 (Fig. S5) . Then, to identify lysine acceptor sites by mass spectrometry, sumoylated UL44 was separated by SDS-PAGE, in-gel-digested with trypsin and peptides were analyzed by LC-MSMS. Upon application of the software ''ChopNSpice'' for database search, we identified 16 sumoylation sites in UL44, including the predicted sites K172, K339, K371, K410, and K431. Table 2 summarizes the UL44 peptides that have been found to be sumoylated. The corresponding MS spectra are shown in Fig. S6 .
These results indicated that UL44 possesses multiple SUMO target lysines that are located throughout the protein, in accordance with the observation that Ubc9 could bind to both N-and C-terminal portions of UL44 ( Fig. 1D and Fig. S1 ). Mutagenesis of several of these lysines in combination caused a strong decrease of UL44 expression (data not shown), likely due to protein misfolding and/or instability, making impossible to analyze the sumoylation state of the mutated protein and to compare it to that of wild-type UL44.
UL44 sumoylation is stimulated by binding to DNA. As mentioned above, UL44 possesses a structural fold similar to that of the eukaryotic processivity factor PCNA [9] . In addition, like UL44, PCNA is SUMO-conjugated and its sumoylation involves both a consensus and a non-consensus site [4] . Since it has been shown that PCNA needs DNA to be sumoylated efficiently [59] , we wished to investigate whether UL44 might behave similarly and its sumoylation could be stimulated by the presence of DNA. Thus, we performed in vitro sumoylation experiments in the absence and in the presence of DNA using as a substrate a purified 6His-UL44 fusion protein treated with polymin P to eliminate residual bacterial nucleic acids. In a western blot analysis with an anti-UL44 antibody (Fig. 4A, left panel) , only a faint band corresponding to mono-sumoylated UL44 was observed in the absence of DNA (lane 2). Upon addition of dsDNA (e.g., activated calf thymus DNA) to the reaction mixture, a marked increase of the mono-sumoylated product and the appearance of bi-and trisumoylated forms of UL44 were observed (Fig. 4A , lane 4 of left panel). A western blot analysis with an anti-SUMO-1 antibody confirmed that these bands indeed contained SUMO-1 (data not shown). Similar results were obtained when GST-UL44 was used as a substrate (Fig. S7) . Thus, like PCNA sumoylation, UL44 sumoylation is strongly stimulated by the presence of DNA.
To investigate whether the nature of DNA could influence the stimulation of UL44 sumoylation, we also performed in vitro sumoylation reactions in the presence of different DNA substrates. Similar stimulation levels were obtained when different dsDNAs were added, regardless of their sequence (data not shown), in keeping with previous observations that UL44 binds DNA in a sequence-independent manner [7] . In contrast, consistently less stimulation of UL44 sumoylation was observed in the presence of ssDNA (Fig. 4A, right panel) , for which UL44 has been shown to possess an apparent affinity lower than for dsDNA [7] . This suggested that UL44 sumoylation could depend on binding to To analyze UL44 sumoylation in vitro, purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either wild-type SUMO-1 (SUMO-1 wt) or a mutant form of SUMO-1 (SUMO-1 mut) which cannot be covalently linked to substrates. The reaction products were analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. As a positive control, in vitro sumoylation of p53 was also analyzed. (B) Purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either SUMO-2 or SUMO-3 and analyzed by western blotting with anti-UL44 and anti-SUMO-2/23 antibodies. For all panels, the arrowhead indicates the unmodified form of UL44 or p53 and the asterisks indicate the respective sumoylated forms. doi:10.1371/journal.pone.0049630.g002 DNA. To test this hypothesis, we analyzed the ability of two UL44 mutants, FLAG-UL44Dloop and FLAG-UL44L86A/L87A, which are defective for DNA binding [36] , to be modified by SUMO-1 in mammalian cells. FLAG-UL44Dloop contains three point mutations within a UL44 flexible loop ( 163 HTRVKRNVKKAP 174 ) involved in UL44-DNA interaction [8, 9] , and is therefore impaired in DNA binding [36] . FLAG-UL44L86A/L87A carries two point mutations preventing dimerization of UL44 and strongly impairing the UL44-DNA interaction both in vitro and in vivo [9, 36, 60] . Both FLAG-UL44Dloop (Fig. 4B) and FLAG-UL44L86A/L87A (Fig. 4C) , when coexpressed with HA-SUMO-1 and Ubc9, exhibited strongly reduced sumoylation levels when compared to FLAG-UL44. Importantly, co-immunoprecipitation experiments demonstrated that the reduced sumoylation of FLAG-UL44Dloop and FLAG-UL44L86A/L87A was not due to an impairment of binding of the mutant proteins to Ubc9, since the two mutants precipitated with endogenous Ubc9 at levels comparable to those of the wild-type protein (Fig. 4D ). In addition, the presence of DNA did not stimulate the sumoylation of the UL44Dloop or UL44L86A/L87A mutants in in vitro reactions (Fig. 4E) . Finally, since the UL44Dloop mutant contains a substitution (K167N) involving a potential SUMO target lysine, we wished to exclude the possibility that the reduced sumoylation levels of FLAG-UL44Dloop might be due to alteration of a putative SUMO acceptor site rather than an impairment of DNA binding. To this end, the K167 residue was conservatively mutated to arginine and the mutant protein was tested for in vitro sumoylation in the presence of DNA. The K167R mutant showed a SUMO-1 modification pattern identical to that of wild-type UL44 (Fig. 4F) .
Altogether, these results suggest that UL44 is preferentially modified by SUMO-1 when it is bound to DNA as a dimer.
Having demonstrated that UL44 is sumoylated by Ubc9 in vitro and in transfected cells, we sought to investigate whether a similar modification also occurs naturally in HCMV-infected cells. Protein lysates of HFFs infected with HCMV and collected at different times post-infection (p.i.) were analyzed by western blotting with an anti-UL44 antibody. Two main bands of 65 and 80 kDa were observed above the primary UL44 band of 50 kDa during the whole time course of lytic infection, being detectable already at 24 h p.i. (Fig. 5A) . A third band of ,95 kDa was also visible from 48 h p.i. These subforms were similar in electrophoretic mobility to the UL44 bands covalently modified by SUMO-1 in transfected cells (Fig. 3A) . A densitometric analysis of the protein bands (Fig. 5B ) revealed that the relative amount of the sumoylated forms increased during the course of HCMV infection, becoming ,50% of total UL44 protein at 120 h p.i.
To confirm that the slower migrating forms indeed represent UL44 molecules conjugated to SUMO-1, lysates from HCMVinfected cells at 120 h p.i. or from mock-infected cells were immunoprecipitated with an antibody against UL44. Subsequent- ly, the immunocomplexes were analyzed by western blotting with an anti-SUMO-1 antibody. Three main bands of the expected molecular mass (,65, 80, and 95 kDa) were recognized in the anti-UL44 immunoprecipitate (Fig. 5C, left panel) . Finally, the same immunocomplexes were analyzed by western blotting with the anti-UL44 antibody to demonstrate that the SUMO-1 crossreactive proteins were indeed modified UL44 forms (Fig. 5C, right  panel) . Thus, these results clearly indicate that UL44 is covalently modified by SUMO-1 in HCMV-infected cells.
Considering the difficulties in expressing a UL44 mutant completely impaired in sumoylation, whose activities could be compared to that of wild-type UL44, to gain some insight on the functional role of UL44 sumoylation in the context of HCMV replication we sought to undertake a different approach. We overexpressed SUMO-1 in virus-infected cells and analyzed the effects on the intracellular distribution of UL44, as the targeting to specific subcellular domains is one of most common biological effect exerted by the conjugation of SUMO to a substrate protein.
It has been previously shown that during HCMV replication UL44 localizes to large globular intranuclear structures that correspond to viral DNA replication compartments [61, 62, 63] . A U373-MG cell line that constitutively overexpresses FLAG-SUMO-1 and control U373-Neo cells were mock-infected or infected with HCMV at an MOI of 1 or of 5 PFU/cell and the intracellular localization of UL44 was successively analyzed by indirect immunofluorescence with an anti-UL44 antibody. Control western blotting experiments (Fig. S8A ) confirmed that UL44 is sumoylated in the HCMV-infected U373 cells. In fact, slower migrating bands of the expected molecular mass and similar to the UL44 sumoylated forms observed in infected HFFs (Fig. 5A) were detected. Furthermore, as expected, they increased upon SUMO-1 overexpression. In immunofluorescence assays, the nuclei of mock-infected cells were oval-shaped with no anti-UL44 staining (Fig. 6A, upper panels) . Control HCMV-infected U373-Neo cells showed deformed nuclei, many of which exhibited a kidney shape (Fig. 6A, upper panels) . Indeed, it has been observed that infection by HCMV causes this kind of distortions in nuclear shape [64, 65] . Moreover, UL44 showed a globular fluorescent pattern consistent with previously described viral replication compartments in HCMV-infected cells [61, 66] . In contrast, in HCMV-infected U373-SUMO-1 cells UL44 staining was more distributed throughout the nucleus and, especially at the lower MOI (MOI 1), also failed to coalesce into any recognizable globular structures (Fig. 6A, upper panels) . Therefore, overexpression of SUMO-1 during HCMV replication appears to alter the intranuclear distribution of UL44, likely leading to significantly decreased localization of UL44 in viral DNA replication compartments. Importantly, the intranuclear distribution of another HCMV protein localizing to the replication compartments, i.e. UL57, the single-stranded DNA (ssDNA)-binding protein [61, 67] , appeared not to change upon SUMO-1 overexpression (Fig. 6A, lower  panels) .
To investigate whether sumoylation is indeed involved in the altered intranuclear distribution of UL44, an RNAi approach was employed to suppress the sumoylation system of the cells by silencing Ubc9 since Ubc9 is the only unique and essential enzyme in the SUMO-conjugating pathway [21] . U373-Neo and U373-SUMO-1 cells were transduced with a lentivirus expressing shUbc9, followed by selection with puromycin and induction with doxycyline to establish Ubc9-knocked-down cell lines (U373-Neo shUbc9 and U373-SUMO-1 shUbc9, respectively). As a control, U373-SUMO-1 cells were also transduced with a lentivirus expressing a non-silencing shRNA sequence (U373-SUMO-1 NS). As shown in Figure 6B , Ubc9 was almost completely silenced in cells infected with the shUbc9 lentivirus (U373-SUMO-1 shUbc9) as compared to the cells transduced with the control lentivirus (U373-SUMO-1 NS) and to nontransduced cells (U373-Neo and U373-SUMO-1). To examine the effect of Ubc9 knock-down on intranuclear distribution of UL44, these cell lines were infected with HCMV. As shown in Fig. 6A (upper panels) , upon doxycycline induction, the nuclear UL44 staining in Ubc9-knocked-down U373-SUMO-1 cells was similar to that of U373 cells not overexpressing SUMO-1 (U373-Neo and U373-Neo shUbc9; Fig. 6A , upper panels, and Fig.  S8B ). In contrast, the U373-SUMO-1 cells transduced with the non-silencing lentivirus (U373-SUMO-1 NS, Fig. 6A , upper panels) retained an altered intranuclear distribution of UL44 similar to that observed in the non-transduced cells (U373-SUMO-1) or in the shUbc9-transduced U373-SUMO-1 cells with no doxycycline induction (data not shown). Altogether, these results established that the altered intranuclear distribution of UL44 in HCMV-infected cells upon SUMO-1 overexpression depends on Ubc9-mediated sumoylation.
These observations raised the question whether the altered intranuclear distribution of UL44 observed upon SUMO-1 overexpression affected the viral replication efficiency. To examine the effects of SUMO-1 overexpression on viral DNA replication, U373-Neo and U373-SUMO-1 cells were infected with HCMV at an MOI of 1 and viral DNA levels were measured by quantitative real-time PCR. Viral DNA production from U373-SUMO-1 cells was ,two-fold higher than that from the control cells (U373-Neo and U373-Neo shUbc9; Fig. 6C ). This increase was not observed in Ubc9-knocked down cells (U373-SUMO-1 shUbc9), while the U373-SUMO-1 cells transduced with the non-silencing lentivirus (U373-SUMO-1 NS) exhibited augmented viral DNA levels like the non-transduced U373-SUMO-1 cells (Fig. 6C) .
We also examined the effects of SUMO-1 overexpression on virus yield. The titers of viral particles produced from nontransduced U373-SUMO-1 and from transduced U373-SUMO-1 shUbc9 and U373-SUMO-1 NS cells after infection with HCMV at an MOI of 1 were determined and compared to those produced from infected U373-Neo and U373-Neo shUbc9 control cells. A 2-3-fold increase in viral progeny titers was observed in U373-SUMO-1 and U373-SUMO-1 NS with respect to U373-Neo, while the U373-SUMO-1 shUbc9 cells exhibited yields of Ubc9, and anti-vinculin antibodies (left panel). Cell lysates were incubated with anti-FLAG-M2-Agarose beads and the immunoprecipitated samples were analyzed by western blotting with anti-UL44 and anti-Ubc9 antibodies (right panel). (E) The sumoylation in vitro of wild-type 6His-UL44 and mutant 6His-UL44Dloop and 6His-UL44L86A/L87A proteins was carried out as in (A) and analyzed by western blotting with an anti-UL44 antibody. (F) The sumoylation in vitro of a UL44 mutant bearing the K167R substitution in the flexible loop of UL44 involved in DNA binding was carried out in the presence of DNA and compared to that of wild-type UL44. For all panels, the arrowhead indicates the unmodified form of UL44 or free SUMO-1 and the asterisks indicate the sumoylated forms. doi:10.1371/journal.pone.0049630.g004 infectious virus similar to those of the U373-Neo and U373-Neo shUbc9 cells (Fig. 6D) .
Thus, the altered intranuclear distribution of UL44 upon SUMO-1 overexpression appears not to compromise HCMV replication, but conversely, SUMO-1 overexpression causes a positive effect on virus production.
In this study we report that UL44, a viral ortholog of PCNA, is sumoylated on multiple lysines by the cellular factor Ubc9. Importantly, a consistent portion of UL44 is SUMO-modified in HCMV-infected human cells, resulting in ,50% of the protein being modified at late times during virus replication. From a structural point and functional of view, UL44 and PCNA share blotting with anti-SUMO-1 (left panel) and anti-UL44 (right panel) antibodies. For all panels, the arrowhead indicates the unmodified form of UL44, the arrow indicates the immunoglobulin G heavy chain (IgG hc) and the asterisks indicate the sumoylated UL44 forms. doi:10.1371/journal.pone.0049630.g005 some remarkable similarities and some differences. Monomers of UL44 and PCNA are structurally very similar, despite having extremely different primary sequences [2, 9] . However, while PCNA forms toroidal-homotrimers, UL44 binds to dsDNA as a head-to-head homodimer [7, 9] . In addition, PCNA must be loaded onto DNA in an ATP-dependent process by so-called clamp loaders [68] ; in contrast, UL44 directly binds DNA without the need for ATP hydrolysis or accessory proteins [7, 14, 18] .
Similarities and differences also emerge from the comparison of the sumoylation processes of UL44 and PCNA. The most striking similarity is the DNA-dependence of such post-translational modification: in the case of PCNA, clamp loading rather than the mere presence of DNA was shown to be important for stimulation, implying a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated [59] . This could also be the case for UL44: in fact, point mutations preventing DNA binding [8, 9, 36, 60] strongly impaired UL44 sumoylation in cells (Fig. 4C) and abolished the ability of dsDNA to stimulate UL44 sumoylation in vitro (Fig. 4E) . Furthermore, the ability of DNA to stimulate UL44 sumoylation appears to correlate with the efficiency of the UL44-DNA interaction. In fact, addition of dsDNA -for which UL44 has a ,3-to 8-fold higher affinity than for ssDNA [7] -caused a much stronger increase of UL44 sumoylation as compared to ssDNA (Fig. 4A) . In this context, it is important to note that point mutations impairing DNA binding and sumoylation in cells did not compromise the UL44-Ubc9 interaction (Fig. 4D) , suggesting that binding to DNA does not promote UL44 sumoylation by facilitating its binding to Ubc9.
Another similarity between UL44 and PCNA sumoylation is that both proteins are modified on canonical (K127 for PCNA, and K410 for UL44) and non-canonical residues (K164 for PCNA, and all other sumoylation sites for UL44, see Table 2 ). However, while PCNA is exclusively sumoylated at the Nterminus, both N-and C-terminal residues of UL44 can be modified. In addition, according to our MS-analysis UL44 can be alternatively sumoylated at 16 different sites, while K127 and K164 appear to be the only target sites in PCNA. This flexibility of UL44 in terms of sumoylation target sites, which is reminiscent of the ones described for Daxx and the small hepatitis delta antigen [57, 58] , is arguably the main difference from PCNA. This makes it extremely difficult to study the physiological importance of UL44 sumoylation, also because mutation of several of these lysines caused protein instability. Currently it is therefore impossible to test if, like in the case of budding yeast PCNA, SUMOmodification of UL44 also plays a role in DNA repair/ recombination [69] .
In terms of functional effects, sumoylation is known to regulate protein activity and/or intracellular location [21, 22] . As for the latter, the targeting to specific subcellular domains is one of the best-characterized biological effects exerted by the conjugation of SUMO to a substrate protein. This effect is exemplified by the targeting of cellular protein RanGAP1 to the cytosolic side of the nuclear pore complex upon sumoylation [54, 70] . As a first, preliminary attempt to characterize the role of UL44's sumoylation, here we show that overexpression of SUMO-1 in the context of HCMV replication alters the intranuclear distribution of UL44 as it appears to result in a more diffuse pattern and in decreased localization of UL44 in viral DNA replication compartments (Fig. 6A) , suggesting that sumoylation of UL44 may retarget the protein to other nuclear site(s). Importantly, Ubc9 knock-down studies confirmed that sumoylation is responsible for such altered intranuclear pattern of UL44. The observation that SUMO-1 overexpression causes a positive effect on HCMV replication suggests that sumoylation of UL44 could be important for its function(s) in the context of the virus life cycle, although effects on HCMV replication mediated by sumoylation of other viral or cellular proteins cannot be excluded. However, at the moment understanding the molecular details of how SUMO alters the intranuclear distribution of UL44 is rather difficult. Most likely, sumoylation does not affect the functions of UL44 as a DNA polymerase processivity factor -the only role currently well established for UL44. In fact, several reports have shown that the UL44 protein expressed in E. coli, which is non-sumoylated, is capable of performing all known biochemical activities related to this role (e.g., [7, 14] ). In addition, our observation that UL44 sumoylation peaks at late times during virus replication, once the viral DNA replication has been accomplished (Fig. 5) , suggests that SUMO-conjugation might enable UL44 to fulfill some role(s) in HCMV replication other than that of conferring processivity to the viral DNA polymerase. A role of UL44 in late gene expression has been previously suggested [71, 72] ; however, no conclusive demonstration has been provided yet.
Intriguingly, higher molecular mass forms of the Epstein-Barr virus DNA polymerase processivity factor BMRF1 compatible with sumoylated products have been observed [73] , suggesting that such post-translational modification could be a general feature of the DNA polymerase accessory subunit in herpesviruses. In addition, sumoylation of Vaccinia Virus G8R protein has been recently predicted on the basis of structural similarities to PCNA [74] , but not yet experimentally demonstrated. Thus, our findings could stimulate further studies on sumoylation of DNA polymerase subunits in other herpesviruses or, more in general, in other viral systems. Figure S5 Sumoylation of UL44 in E. coli. The pRSET44 plasmid, encoding 6His-tagged UL44, was introduced into E. coli together with the pTE1E2S1 plasmid, which expresses E1 and E2 sumoylation enzymes and SUMO-1. As a control, bacteria were also transformed only with pRSET44. The 6His-tagged UL44 was purified from bacterial cultures expressing UL44 alone or in combination with the SUMO conjugation system and analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. (TIF) Figure S6 Mass spectrometry analysis of sumoylation sites of UL44. MSMS analysis of tryptic peptides conjugated to a tryptic peptide of SUMO-1 (ELGMEEEDVIEVYQEQTGG or IADNHTPKELGMEEEDVIEVYQEQTGG, 1 tryptic miscleavage) derived from E. coli-expressed, sumoylated UL44. Sequence of the conjugate is listed in each table above the spectra. Y-type and b-type fragment ions are assigned in the spectra. The table below each spectrum summarizes the database search. Highlighted are the m/z values that match the fragment ions obtained from in silico fragmentation of the conjugate. Conjugated lysine residues to SUMO-1 are highlighted in red and the position in UL44 is listed. Modifications, measured (observed) m/z values, the actual mass (in Da), the charge state, and the mass error (ppm, parts per million) are listed as well in the table above each spectrum. The different colors represent the various measured and calculated fragment ions of each conjugate in the spectrum and table underneath, respectively. The question marks in some of the spectra indicate fragment ions that do not match to any calculated y-and b-type ions of the conjugate. (PDF) Figure S7 Sumoylation in vitro of a GST-UL44 fusion is stimulated by DNA. E. coli-expressed, purified GST-tagged UL44 was incubated with purified sumoylation enzymes in the absence or presence of SUMO-1 and/or DNA. The samples were analyzed by western blotting with an anti-UL44 antibody. (TIF) Figure S8 UL44 is sumoylated in HCMV-infected U373 cells. (A) U373-Neo and U373-SUMO-1 cells were either mockinfected or infected with HCMV at MOI of 5 PFU/cell for 72 h. Cell lysates were then analyzed by western blotting with an anti-UL44 antibody. The arrowhead indicates the unmodified form of UL44 and the asterisks indicate the sumoylated UL44 forms. (B) Control U373-Neo cells, and U373-Neo cells transduced with lentiviral particles expressing either a Ubc9-silencing shRNA (U373-Neo shUbc9) or a non-silencing shRNA sequence (U373-Neo NS) were mock-infected or infected with HCMV at an MOI of 5 or 1 PFU/cell. At 72 h p.i., cells were fixed and stained with a primary antibody against UL44 and successively with a secondary fluorescein-conjugated antibody (green) which contained Evans Blue to counterstain cells (red). Cell samples were then analyzed by CLSM.
(TIF)  Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice. Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS) remain important clinical problems in the United States, with an incidence rate of 38.3 cases per 100,000 person-years and a mortality rate of 45% [1] . ALI/ARDS is characterized clinically by sudden respiratory failure with impaired oxygenation and non-cardiogenic pulmonary edema [2] . Pathologically ALI/ARDS is associated with an early inflammatory phase with neutrophilic alveolitis and destruction of the alveolar/capillary permeability barrier, followed by a late fibroproliferative phase with abnormal repair and collagen deposition. There are no specific treatments for ARDS and the main supportive treatment, mechanical ventilation, can be harmful to the lungs when delivered at high tidal volumes [3] .
A growing body of experimental evidence suggests that in addition to the injury caused by high tidal volumes, even moderate or low tidal volumes markedly enhance injury when the lungs are exposed to pathogens or their products [4] [5] [6] . For example, mechanical ventilation synergistically enhances lung injury in response to low doses of bacterial lipopolysaccharide (LPS) and this is associated with expression of specific sets of genes that aren't expressed with LPS or ventilation alone [4, 5, 7] . A computational analysis has mapped the biological processes that are activated by the combination of mechanical ventilation and LPS [8] . One of these biological processes is apoptosis, and a known mediator of apoptosis in injured lungs is the Fas/FasL system.
The Fas/FasL system is composed of the membrane surface receptor Fas and its cognate ligand, FasL. FasL exists in a membrane-bound form, and also in a soluble form that is present in the lungs during acute lung injury [9] . Binding of Fas to sFasL in alveolar epithelial cells independently activates apoptotic and inflammatory pathways that result in death of the cells but also in release of pro-inflammatory cytokines [10] . Although the Fas/ FasL system is better known for its pro-apoptotic function, the pro-inflammatory function is also important in the development of lung injury, and mice deficient in Fas (lpr) have an impaired neutrophil recruitment in response to LPS installation and bacterial infections [11] .
Several lines of evidence suggest that the Fas/FasL system plays a role in ALI/ARDS. First, bioactive sFasL is present in the lungs of patients with ARDS, and this is associated with increased mortality [9, 12] . Second, genetic variations in the Fas gene are associated with increased risk for ALI/ARDS in humans [13] . Third, activation of the Fas/FasL system in mammals leads to acute inflammatory injury followed by fibrosis [14] [15] [16] [17] . And finally, mice lacking Fas are protected in a number of models of lung injury [11, [18] [19] [20] . Thus, the apoptotic and inflammatory responses induced by Fas activation in the lungs are one important factor in the development of ALI/ARDS.
One of the most important neutrophil chemoattractants in mice is the chemokine KC (CXCL1), which is the murine functional homologue of the human chemokine IL8 (CXCL8). Activation of Fas in alveolar epithelial cells is followed by a marked increase in KC expression [10] . Interestingly, recent studies suggest that in injured lungs, IL8 in humans and KC in mice form immune complexes with autoantibodies, and these immune complexes can in turn bind Fc receptors such as FcγRIIa and FcγRIII that amplify the inflammatory response [21, 22] . These studies suggest that, in addition to the absolute levels of chemokines, the formation of autoimmune complexes and their association with Fc receptors is important for the development of inflammatory responses in the lungs.
In this study, we ask whether the Fas/FasL system plays a role in the amplification of inflammatory responses that occur early in the course of mechanical ventilation. The specific hypothesis to be tested is that the Fas/FasL system is required for the development of lung injury in mechanically ventilated mice exposed to LPS. To test this hypothesis, we investigate whether the combination of a non-injurious mechanical ventilation strategy with a minimal dose of intratracheal LPS results in an acute lung injury, and whether this injury is attenuated in Fas-deficient lpr mice. We further investigate whether the development of injury is associated with formation and deposition of anti-KC:KC immune complexes.
All of the animal experiments were approved by the institutional animal research committee of the University of Washington. Mice were housed in a pathogen-free environment according to University of Washington animal use guidelines. Male C57BL/6 mice ("B6") and mice carrying a spontaneous mutation in the Fas gene that impairs Fas signaling (B6. MRL-Fas lpr /J, "lpr") were obtained from the Jackson Laboratories and studied at 7-13 weeks of age. Briefly, the mice were anesthetized with 5% inhaled isoflurane and then intubated endotracheally with a 20-gauge angiocatheter. Placement of the catheter in the trachea was verified by visualizing the movement of a 100 μl bubble of water in response to respiratory efforts. After confirming intubation, the trachea was instilled with either E. coli LPS, 15 ng/kg or PBS. The instillate was suspended in 2.5% colloidal carbon to allow later confirmation of the extent and distribution of the instillation macro and microscopically. After the installations some of the mice were extubated, returned to their cages, and allowed free access to food and water; other mice where kept intubated and subjected to mechanical ventilation with the following settings: tidal volume (Tv) 10 ml/kg; respiratory rate (RR) 150 breaths/minute; fraction of inspired oxygen (FiO 2 ) 0.21; and positive end-expiratory pressure (PEEP) of 3 cm H 2 O. The heart rate, airway pressures, rectal temperatures and EtCO 2 were monitored continuously using a computerized monitoring system (Chart 4, AD Instruments, Colorado Springs, CO). The RR was adjusted to maintain the EtCO 2 between 30 -40 mmHg. The body temperature was maintained between 37 and 38°C with external heating. The mice were hydrated with a continuous intraperitoneal infusion of lactated ringer solution at 500 μl/hour. Muscle relaxation was attained with pancuronium bromide, 1 μg/g i.p followed by 0.5 μg/g i.p. every hour. After four hours of mechanical ventilation the mice were euthanized with 0.30 ml/kg i.p. of Beuthanasia-D (Schering-Plough Animal Health, Union, NJ). The thorax was rapidly opened and the mouse was exsanguinated by direct cardiac puncture. The left lung was removed and flash-frozen in liquid nitrogen. The right lung was lavaged with 0.6 mM EDTA in PBS; an aliquot of the bronchoalveolar lavage fluid (BALF) was removed for cell counts and differentials, the remaining fluid was spun at 1200 x g, and the supernatants stored at -80°C in individual aliquots. Following the BAL, the right lung was fixed in 4% buffered paraformaldehyde at an inflation pressure of 15 cm H 2 O for histological evaluation.
We studied four groups of mice. Two groups consisted of B6 and lpr mice instilled with PBS and allowed to breathe spontaneously ("SB") (n = 7 for B6 mice, 4 for lpr). The other two groups consisted of B6 and lpr mice instilled with LPS and exposed mechanical ventilation (MV + LPS) (n = 10 for B6 mice, 6 for lpr). The main experimental comparison was between the B6 and lpr mice instilled with LPS and exposed to ventilation.
Total cell counts in the BALF were performed with a hemacytometer. Differential counts were performed on cytospin preparations using the Diff-quick method (Fisher Scientific Company L.L.C., Kalamazoo, MI). BALF total protein was measured with the bicinchoninic acid method (BCA assay, Pierce, Rockford, IL). BALF IgM (Bethyl Laboratories, Montgomery, TX) and α-macroglobulin (Life Diagnostics, West Chester, PA) were measured with immunoassays. Lung homogenate TNF-α, KC, IL1β, and IL6 were measured using a multiplex fluorescent bead assay (R&D Systems, Minneapolis, MN).
As a measurement of the total content of PMN in the lungs we measured myeloperoxidase (MPO) activity in lung homogenates prepared in 50 mM K 2 HPO 4 , pH 6.0 with 5% CH 3 (CH 2 ) 15 N(Br)(CH 3 ) 3 , 5 mM EDTA. Active caspase-3 and Poly ADP ribose polymerase (PARP) activity were measured in lung homogenates prepared on a 1:20 ratio of a lysis buffer (0.5% Triton-X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl, and 1 mM MgCl, pH7.4). The lung homogenate was spun at 10,000 x g for 20 min at 4°C and the supernatant used for measurements of active caspase-3 and PARP using the CPP32/ Caspase-3-Fluorometric Protease Assay Kit (BioVision, Mountain View, CA) and a PARP activity kit (Cell Signaling, Boston MA). Serum Creatinine, ALT and bilirubin were measured at a commercial laboratory. Anti-KC autoantibody:KC immune complexes were measured in BAL fluids using an ELISA assay according to a previously described protocol [21] . Briefly, 96-well microtiter plates were coated with antibody against KC (Peprotech). After blocking, the plates were incubated with BAL fluid samples obtained from mice. Then, the plates were washed and incubated with biotinylated horse antibody against mouse immunoglobulins (Vector Laboratories) followed by HRP-conjugated streptavidin and the substrate tetramethyl benzidine (Sigma).
C57BL/6 and lpr mice were euthanized by exposure to CO 2 followed by cervical dislocation. The femur and tibia of both hind legs were isolated and freed of all soft tissue, and then the ends of both bones were removed. The femur and tibia were placed proximal end down in a 0.6 mL Eppendorf tube, which had been punctured at its lower tip with an 18-gauge needle and placed inside a 1.5 mL Eppendorf tube. The tubes were spun at 2000 X g for 30 seconds and neutrophils were isolated as previously described [23] . After isolation, neutrophils were labeled with calcein-AM (5 μg/ml; Molecular Probes, Eugene, OR) for 30 minutes at 37°C, washed two times in PBS and resuspended at a concentration of 1 x 10 6 /mL.
Neutrophil chemotaxis was assessed using the Neuro Probe ChemoTx W Disposable Chemotaxis system (Neuro Probe Inc. Gaithersburg, MD). The wells of the 96-well plate were filled with various concentrations of KC. A polycarbonate filter (8 μm pores) with a hydrophobic ring around the area over each well was placed on the 96-well plate and calcein-labelled neutrophils were added to each ring. The chemotaxis chamber, consisting of the polycarbonate filter and 96-well plate, was incubated for 30 min at 37°C in 5% CO 2 and then non-migrating neutrophils were removed from the upper side of the filter. The chemotaxis chamber was placed in a multi-well fluorescent plate reader (Synergy 4, BioTek, Winooski, VT) and the migrated cells were measured using the calcein fluorescence signal (excitation -485 nm, emission -530 nm). Neutrophil migration was expressed as a percent of the total number of neutrophils that were placed on the topside of the filter (% Total).
Lung sections were embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin.
Lung tissue sections were deparaffinized, washed in xylene, rehydrated, permeabilized with proteinase K, and incubated with the TUNEL reaction mixture according to manufacturer instructions (In Situ Cell Death Detection kit, AP, Roche Applied Science, Indianapolis, IN). Negative controls were treated with labeling solution without terminal transferase. Immediately after TUNEL labeling the slides were washed three times in PBS, blocked with Protein Block (Dako, Carpinteria CA) and incubated for 2 hr in the dark at 37°C with the mouse monoclonal pan-cytokeratin antibody C11 (Abcam, Cambridge, UK) previously labeled with Alexa Fluor 555 (Invitrogen, Eugene, OR). Negative control slides were incubated with an isotype control mouse IgG1k labeled with Alexa Fluor 555 (BD Pharmingen, San Diego, CA). The slides were washed and immediately visualized with a fluorescent microscope.
Lung tissue sections from B6 and lpr mice exposed to MV + LPS were processed as previously described [21] . The sections were incubated with anti-KC antibody (Peprotech, Rocky Hill, NJ) followed by chicken antirabbit secondary antibody (Alexa 647, pseudocolor red) (Invitrogen), and then with anti-FcγRIII antibody (R&D Systems) followed by chicken anti-rat secondary antibody (Alexa 488, green), and finally with biotynylated anti-Ly-6 G antibody (eBioscience, San Diego, CA), used as a neutrophil marker, followed by streptavidin (Alexa 568, pseudocolor magenta). Lung tissues were counterstained with Hoechst 33342 (Calbiochem, Gibbstown, NJ). The slides were evaluated using a PerkinElmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope using PlanApox20 objective and PlanApox60 or x100 immersion oil objective (numerical aperture [NA] 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.
Statistical analysis was performed using two-factor ANOVA followed by the Bonferroni post-hoc analysis. One factor was "treatment" (SB or MV + LPS) and the other factor was "strain" (B6 or lpr). The analysis was designed to determine the overall effect of each of the factors, the presence of an interaction effect, and the comparison of "strain" for each level of "treatment". Data was generated using GraphPad Prism. A p value of less than 0.05 was considered significant.
The neutrophil response to MV + LPS was attenuated in the lpr mice There were significantly less PMN in the BAL of lpr mice exposed to LPS + MV than in the B6 mice (2.2 x 0.5 x 10 3 vs 1.3 ± 0.2 x 10 4 cells, p < 0.05) (Figure 1-A) . A similar pattern was seen for lung MPO activity (Figure 1-B) . However, the lung concentrations of the PMN chemoattractants KC and MIP2 (CXCL2), although increased in response to MV + LPS, were similar in the lpr and B6 mice; this was also true for the pro-inflammatory cytokine TNFα (Figure 1 , C-F). Interleukin 6, GM-CSF, and VEGF were not affected by the administration of MV + LPS and were similar in the B6 and lpr mice (data not shown). These data suggest that both lung PMN recruitment and airspace PMN migration were impaired in the absence of functional Fas, but the difference in neutrophil recruitment seen in this model cannot be explained by differences in KC or MIP-2 release.
Other parameters of lung injury were similar in B6 and lpr mice Permeability response
We and others have postulated that the Fas/FasL system leads to lung injury by inducing apoptosis of pneumocytes, resulting in disruption of the alveolar-epithelial barrier and non-cardiogenic pulmonary edema. However, in the present study the BAL concentration of total protein and of the serum protein α-macroglobulin were similar in the lpr and B6 mice, despite the difference in PMN numbers ( Figure 2 ). The lack of difference may be due to a low level of injury, as supported by the lack of overall change in the MV + LPS group as compared with the SB group; indeed the model was designed so that the ventilatory pattern and the LPS dose would cause minimal or no injury by themselves, so as to determine the very first components of the injury response. The data suggest that neutrophil changes precede permeability changes in ventilated mice exposed to LPS, and that the role of the Fas/FasL system on neutrophil recruitment precedes its disruptive effect on the alveolar-capillary barrier.
Interestingly, the activity of caspase-3, often used as a surrogate for apoptosis, was increased in the spontaneously breathing lpr mice, and this increase reached significance in the MV + LPS group (185 ± 24 vs 480 ± 137, p < 0.05) (Figure 3-A) . PARP, a downstream target of active caspase-3, showed similar activity in the lpr and B6 mice (Figure 3-B) . Double labeling of tissue sections for TUNEL and cytokeratin revealed that in both the lpr and the B6 mice, the apoptotic cells were located primarily in the alveolar walls, but were cytokeratin-negative (Figure 3-C) . Thus, contrary to our expectations, there was increased caspase-3 activity in the lpr mice exposed to MV + LPS, and the apoptotic cells were mostly cytokeratin-negative cells localized to the alveolar walls.
The lungs of spontaneously breathing B6 and lpr mice showed normal architecture (Figure 4 ). The lungs of B6 mice exposed to MV + LPS showed thickening of the alveolar walls, intra-alveolar neutrophilic infiltrates, and intra-alveolar fibrin strand deposition. In contrast, the lungs of lpr mice exposed to MV + LPS retained their normal architecture. The LPS was administered with inert colloidal carbon, which on the tissue sections was taken up by macrophages and appears as black granulate material in the cytoplasm. This indicates that the Figure 1 Inflammatory response. C57BL/6 (B6) and Fas-deficient lpr mice received intratracheal installations of either PBS followed by four hours of spontaneous breathing (SB), or E. coli LPS, 15 ng/kg, followed by 4 hrs of mechanical ventilation (MV) with tidal volumes of 10 mL per kilogram, FiO 2 of 0.21, PEEP = 3 and respiratory rate = 150 breaths per minute. In response to the combination of MV and LPS, the B6 mice showed significantly more total neutrophils (PMN) in the BAL fluid than the lpr mice (A). A similar pattern was seen for the lung MPO activity, which is a measure of the total neutrophil content in the lung (B). MV + LPS was associated with increases in the lung homogenate concentrations of the cytokinesTNF-α, MIP-2, and KC, and these similar in the B6 and the lpr mice; MV + LPS had no effect on IL-1β (C-F). n = at least 6/group. sections depicted were all exposed to instillate -an important point as intratracheal instillations are patchy and normal tissue can simply reflect a non-instilled area.
We have previously reported that the combination of MV + LPS for 6 hours (2 hr longer than the present study) is associated with distal organ injury, in particular biochemical and histologic evidence of kidney and liver damage [24] . In this study, we also noticed an increase in serum creatinine in the mice exposed to MV + LPS, but this increase was strain-independent ( Figure 5-A) . Interestingly, the lpr mice had increased serum AST concentrations at baseline, and this was not further increased by the addition of MV + LPS (Figure 5-B) . Total bilirubin was similar in lpr and B6 mice, and was not affected by MV + LPS (Figure 5-C) . Thus, at this early time the evidence of distal organ injury was limited to increased creatinine, and this increase was not dependent on the Fas/FasL system.
The differences in BAL neutrophils are not explained by differences in B6 and lpr neutrophil chemotaxis An impairment in lpr neutrophil chemotaxis towards KC could explain the attenuation of BAL neutrophils seen in the lpr mice exposed to MV + LPS. Therefore, we compared the chemotactic ability of neutrophils isolated from B6 and lpr mice to KC in vitro. However, we noticed no impairment of chemotaxis in the lpr neutrophils; if anything, chemotaxis was slightly increased in the lpr neutrophils as compared with the B6 neutrophils ( Figure 6 ).
A new line of research suggests that mammals form autoantibodies against chemokines such as IL8 in humans and KC in mice, and immune complexes derived from these autoantibodies can induce inflammation by binding to Fcγ receptors on leukocytes, endothelial cells and other cells. Measurement of anti-KC:KC complexes in the BALF revealed an increase in B6 mice treated with MV + LPS, which was not seen in the lpr mice (Figure 7) We also used immunofluorescence staining and confocal microscopy to investigate if there were differences in the deposition of anti-KC:KC complexes in ventilated B6 and lpr mice. The alveolar walls of the ventilated B6 mice contained abundant leukocytes, identified based on Gr-1 positivity ( Figure 8 ). The leukocytes expressed the receptor FcγRIII on their membrane surface. KC co-localized with the FcγRIII receptors. In contrast, there were, in general, Caspase-3 activity was significantly higher in the lpr mice exposed to MV + LPS. n = at least 6/group. Double-labeling for TUNEL (green) and cytokeratin (red) reveals that the TUNEL positive cells are located in the alveolar wall, but most of them are cytokeratin negative.
less Gr-1 positive cells in the lungs of the lpr mice, and there was also decreased deposition of KC in the tissue. Thus, the formation of anti-KC:KC IC was attenuated in lpr mice, and this was associated with decreased KC deposition in the tissues.
To confirm the specificity of the anti-KC:KC labeling, mouse PMN were purified and incubated with either anti-KC-KC immune complexes (Figure 9 , first two columns) or with KC alone (Figure 9 , third column). Then, the cells were labelled with either the Peprotech antibody, which detected the immune complexes, or an R&D antibody, which detected only KC.
Finally, we tested expression of FcγRIII in WT and lpr mice treated with MV + LPS, and found it to be lower in the lpr mice ( Figure 10 ).
The goal of this study was to determine whether Fas deficiency is protective in mechanically ventilated mice exposed to intratracheal LPS. The main finding was that Fas-deficient lpr mice showed decreased neutrophil recruitment in response to combined mechanical ventilation and LPS, even though the cytokine, permeability, and apoptotic responses were similar to those of B6 mice. Interestingly, despite the presence of similar concentrations of KC in the BAL of lpr and B6 mice, only the B6 mice showed extensive KC deposition in the lungs, and this was associated with the presence of anti-KC:KC complexes in the BALF and lung tissue.
In this study, we focused on the initial events of the injury response by studying mice four hours after the initiation of injury, which was induced by combining a very low dose of endotoxin with mechanical ventilation. The main finding was that a functional Fas/FasL system was required for neutrophil migration into the airspaces of the lungs of mechanically ventilated mice exposed to LPS. The changes in BAL neutrophils seen in this study were associated with changes in lung myeloperoxidase (MPO) activity, and because lung MPO activity is a measurement of the total content of neutrophils in the lungs, the data suggests that the Fas/FasL system is required for both neutrophil migration into the airspaces and for neutrophil recruitment into the lungs. This observation confirms other studies that suggest a key role for the Fas/FasL system in neutrophil migration into the airspaces; for example, activation of Fas in the lungs of mice and rabbits results in a neutrophilic alveolitis (1, 2) , whereas pharmacologic blockade of the Fas/FasL system attenuates the neutrophilic response to bacteria and bacterial products (3) (4) (5) (6) . Tissue respons. C57BL/6 or Fas-deficient lpr mice were exposed to either intratracheal installations of PBS followed by spontaneous breathing or to intratracheal installation of LPS, 15 ng/kg, followed by four hours of mechanical ventilation (MV) with tidal volumes of 10 mL per kilogram. Lung tissue sections were stained with hematoxylin and eosin to demonstrate the pattern of injury. The instillate contained 2.5% colloidal carbon to identify the instilled areas. Spontaneously breathing mice instilled with PBS showed normal lung architecture, regardless of strain (A, C). The B6 mice exposed to MV and LPS showed intra-alveolar and interstitial neutrophilic infiltrates, as well as thickening of the alveolar walls and occasional fibrin deposition (B).
One potential explanation for the decreased migration of neutrophils into the airspaces of the Fas-deficient mice is that Fas ligation induces release of neutrophilic cytokines such as KC in macrophages and in lung epithelial cells in vitro (7-9). Thus, we had expected to see lower concentrations of KC in the mechanically ventilated lpr mice exposed to LPS, as compared with the B6 animals. However, this was not the case, and the difference in neutrophil migration was not due to differences in soluble KC concentrations.
Another potential mechanism that would explain differences in neutrophil migration with similar concentrations of soluble KC is a difference in neutrophil chemotaxis of lpr and B6 neutrophils. However, our chemotaxis studied showed that, if anything, neutrophils from lpr mice have slightly increased chemotaxis to KC, strongly suggesting that differences in chemotaxis are not the reason for the attenuation in the neutrophilic response seen in the lpr mice. Neutrophil recruitment and migration appears to be partly dependent on the formation of anti-KC:KC immune complexes, which can bind Fcγ receptors in local leukocytes, enhancing the inflammatory response [22] . For example, the generation of anti-KC:KC immune complexes in the lungs of mice is followed by acute inflammatory lung injury, and this injury requires the presence of Fcγ receptors [21] . Furthermore, mice lacking γ chains show attenuated injury in response to LPS, suggesting that this process is relevant for inflammation secondary to bacterial products [21] . The human equivalent of anti-KC:KC complexes are anti-IL8:IL8 complexes, and these are present in the lungs of patients with ARDS [25, 26] . In our study, we found that there are less anti-KC:KC complexes in the lungs of the lpr mice, even though the concentrations of KC were similar to those in the B6 mice. Thus, it is possible that the lower numbers of neutrophils seen in the lpr mice were due to decreased formation and deposition of anti-KC:KC complexes in the lungs. Additional studies are necessary to confirm this hypothesis, and it remains possible that differences in other unmeasured chemotactic agents account for the differences in neutrophil recruitment. The mechanism linking the Fas/FasL system with impaired formation and deposition of immune complexes remains unclear. To date, lpr mice, particularly the MRL/ lpr strain are known to generate autoantibodies that can result in a lupus-like syndrome [27] .
A surprising finding in the present study was that there was no increase in the markers of permeability or apoptosis in the B6 mice exposed to mechanical ventilation and LPS, as compared to the lpr mice; instead, the injury response was limited to neutrophilic inflammation and cytokine release. One explanation is that neutrophil recruitment precedes the development of tissue injury in our model, in which the mice were studied relatively early, four hours after the onset of ventilation. Less clear is the finding that the lpr mice actually had increased caspase-3 activity and TUNEL (See figure on previous page.) Figure 8 Anti-KC autoantibody:KC complex deposition in the lungs. FcγRIII (green) and KC (red) in tissue sections from B6 (A) and lpr (B) mice treated with intratracheal E. coli LPS, 15 ng/kg, and exposed to 4 hours of mechanical ventilation (MV) at Tv = 10 cc/kg, FiO2 = 0.21, PEEP = 3. FcγRIII is shown in green, KC is shown in red, and Gr1 (staining leukocytes) is shown in pink. Nuclei are shown in blue. There is an increase signal for both KC and FcγRIII in the B6 mice as compared with the lpr mice. Note the colocalization of KC and FcγRIII. Figure 9 The detection of anti-KC:KC immune complex is specific. Mouse PMN were purified and incubated with either anti-KC-KC immune complexes (first two columns) or with KC alone (third column). Note that the Peprotech antibody, detected only anti-KC-KC immune complexes, whereas the R&D antibody detected only KC. The FcγRIII was labeled green and KC was labeled red.
positive cells compared with the B6 mice. We do not have a clear explanation for this finding, but they seem to be specific to the LPS + MV model, because in another model using mechanical ventilation in which WT and lpr mice were exposed to pneumonia virus of mice (PVM) prior to four hours of MV, we found a decrease in caspase-3 activity in the lpr mice [28] .
It is important to emphasize that the decrease in neutrophils seen in the lpr mice does not imply "protection". It is unclear whether the lung neutrophilic response is directly associated with negative outcomes in ALI/ARDS or not. Most studies of lung injury presume that neutrophilia or increased concentrations of cytokine are deleterious, but all of these studies are limited in that they do not effectively reproduce the series of events seen in the clinical setting, where patients with multiple comorbidities are intubated for prolonged periods of time. Our study should be interpreted as knowledge on the mechanisms of that initiate lung injury, but not extrapolated to the ultimate results of that injury.
In summary, we report that in B6 mice, the combination of mechanical ventilation and LPS is associated with recruitment of neutrophils to the lungs and with deposition of anti-KC:KC immune complexes. In comparison, mice deficient in Fas recruited lower numbers of neutrophils, and this was associated with less deposition of immune complexes in the lungs. We conclude that a functioning Fas/FasL system is required for a full neutrophilic response to LPS in mechanically ventilated mice. Evolutionary Dynamics of the Interferon-Induced Transmembrane Gene Family in Vertebrates Vertebrate interferon-induced transmembrane (IFITM) genes have been demonstrated to have extensive and diverse functions, playing important roles in the evolution of vertebrates. Despite observance of their functionality, the evolutionary dynamics of this gene family are complex and currently unknown. Here, we performed detailed evolutionary analyses to unravel the evolutionary history of the vertebrate IFITM family. A total of 174 IFITM orthologous genes and 112 pseudogenes were identified from 27 vertebrate genome sequences. The vertebrate IFITM family can be divided into immunity-related IFITM (IR-IFITM), IFITM5 and IFITM10 sub-families in phylogeny, implying origins from three different progenitors. In general, vertebrate IFITM genes are located in two loci, one containing the IFITM10 gene, and the other locus containing IFITM5 and various numbers of IR-IFITM genes. Conservation of evolutionary synteny was observed in these IFITM genes. Significant functional divergence was detected among the three IFITM sub-families. No gene duplication or positive selection was found in IFITM5 sub-family, implying the functional conservation of IFITM5 in vertebrate evolution, which is involved in bone formation. No IFITM5 locus was identified in the marmoset genome, suggesting a potential association with the tiny size of this monkey. The IFITM10 sub-family was divided into two groups: aquatic and terrestrial types. Functional divergence was detected between the two groups, and five IFITM10-like genes from frog were dispersed into the two groups. Both gene duplication and positive selection were observed in aquatic vertebrate IFITM10-like genes, indicating that IFITM10 might be associated with the adaptation to aquatic environments. A large number of lineage- and species-specific gene duplications were observed in IR-IFITM sub-family and positive selection was detected in IR-IFITM of primates and rodents. Because primates have experienced a long history of viral infection, such rapid expansion and positive selection suggests that the evolution of primate IR-IFITM genes is associated with broad-spectrum antiviral activity. First discovered by cDNA library screening in 1984 [1] , the interferon-induced transmembrane (IFITM) gene family plays critical roles in a variety of cellular processes and contains IFITM1, IFITM2, IFITM3, IFITM5, IFITM6, IFITM7, IFITM10 and some IFITM-like genes [2] . Except for IFITM5 that is specifically expressed in bone cells in an interferon (IFN)independent way [3, 4] , all IFITM genes can be stimulated by IFN [5, 6] , and are widely expressed in tissues and organs [2] .
IFITM family members contain a conservative CD225 domain and two terminal hypervariable regions [2] . The CD225 domain accounts for more than half of the protein in length, containing one intact transmembrane domain (TMD), two S-palmitoylation sites regions and partial TMD in the C-terminus of the protein. The S-palmitoylation sites have been demonstrated to play important roles in post-translational processing and stability of IFITM proteins [7] . The N-terminal hypervariable region generally contains 21 amino acid residues and the C-terminal one includes a TMD (Fig. 1) [8] .
In different vertebrates, the functions of different IFITM members diverge. IFITM1, IFITM2 and IFITM3 are involved in cell adhesion [9] , antiproliferation [9] , tumor suppression [10, 11] , and germ cell and embryonic development [12] . More recently, these genes were identified as novel types of antiviral restriction factors with a wide spectrum of antiviral activity against influenza A viruses (e.g. H1N1 viruses), West Nile virus, dengue virus, filoviruses, HIV-1, HCV, venezuelan equine encephalitis virus (VEEV), chikungunya virus (CHIKV), vesicular stomatitis virus (VSV) and even SARS-coronavirus [8, [13] [14] [15] [16] [17] [18] . The main function of IFITM5 is associated with bone development in vertebrates [4, 19, 20] . IFITM6 seems to be involved in macrophage functions in tumor suppression [20] . To date, however, there is no information about the functions of IFITM7 and IFITM10.
Several antiviral restriction factors (e.g. APOBEC3G, Tetherin, and SAMHD1) have been demonstrated to evolve under positive selective pressure from viruses [21] [22] [23] [24] [25] [26] [27] [28] [29] . As important virus inhibitors, IFITM1, IFITM2 and IFITM3 may have also undergone a similar co-evolutionary process, such as other antiviral restriction factors do. Despite this connection, relationships between antiviral functions and adaptive evolution in IFITM family have seldom been reported and although previous reports had illustrated the phylogenetic history of IFITM family in some eukaryotic species [2, 30, 31] , there has been no detailed information about IFITM genes in vertebrates. In this study, we performed detailed evolutionary analyses not only to test whether the primate IFITM genes evolved under positive selection throughout primate evolution, but also to unravel the evolutionary history of vertebrate IFITM family.
To characterize the IFITM gene repertoires in vertebrates, we searched 27 vertebrate genome sequences with high genome coverage ($66) or representing the major evolutionary lineages in vertebrate phylogeny (such as opossum, lizard, platypus, etc.), using previously described IFITM sequences as queries. The taxa included ten non-mammalian vertebrates (five fishes: stickleback, tetraodon, medaka, fugu, zebrafish; one amphibian: frog; one nonavian reptile: lizard; three birds: chicken, turkey, zebra finch) and 17 mammals covering six primates (human, chimpanzee, gorilla, orangutan, macaque, marmoset), four glires (mouse, guinea pig, rat, and rabbit), five other mammals (tree shrew, cow, horse, dog and elephant), 1 metatherian (opossum) and 1 prototherian (platypus). We divided the newly identified genes into three types based on the following criteria: (i) functional genes, which contain full-length ORFs with intact CD225 domain and C-terminal TMDs; (ii) putative functional genes, which have CD225 domains but contain incomplete ORFs; and (iii) pseudogenes, which are sequences with pre-mature stop codons.
We identified a total of 286 IFITM-related sequences ( Table 1 ). The number of IFITM genes varies considerably between mammals and non-mammalian vertebrates. A total of 27 functional genes were identified from 10 non-mammalian species, ranging from one gene in stickleback or tetraodon to six genes in frog. By contrast, 134 functional genes were identified from 17 mammalian species, ranging from one in platypus or rabbit to 26 in marmoset. Additionally, 14 putative functional genes including 13 in mammals and one in non-mammalian vertebrates were identified. Among the 10 non-mammalian and 17 mammalian species, 10 and 102 pseudogenes were identified, respectively. Interestingly, among 10 non-mammalian vertebrates, frog has the highest number (six functional genes and eight pseudogenes) of IFITM-related sequences. Among the 17 mammals, marmoset has the highest number (40) of IFITM-related sequences, of which 26 are functional genes. Human has the second highest number (29) of IFITM-related sequences, of which 18 are pseudogenes, the maximum in vertebrates.
To understand the phylogenetic relationship of IFITM gene family, 160 functional IFITM genes identified from 27 vertebrate species (Table S1 ) were subjected to phylogenetic analyses using Bayesian inference, maximum likelihood (ML) and maximum parsimony (MP) methods. The Bayesian, ML and MP trees show consistent topological structures (Figs. 2A and S1). All functional IFITM genes were well divided into three clades I, II and III with Bayesian posterior probabilities of $97. These analyses indicate that the IFITM family most likely originated from three progenitors.
The clades I, II and III contain 114, 26, and 20 IFITM genes, respectively. All IFITM10 and IFITM5 genes are clustered in clades II and III, respectively. All other functional IFITM genes, including IFITM1, IFITM2, IFITM3, IFITM6 and IFITM7, are grouped in clade I, forming the biggest sub-family in the IFITM family. Because the expression of the IFITM genes in clade I can be induced by IFN and their functions are associated with immunity [8, 13, 15] , they are defined as immunity-related IFITM (IR-IFITM) sub-family.
In each clade, eutherian IFITM genes form a separate group from opossum and bird genes, consistent with the species phylogeny. Besides IFITM5 and IFITM10, IR-IFITM genes also have orthologs in both marsupials and eutherians, arguing against previous observation that only IFITM5 and IFITM10 orthologs could be identified in marsupials and eutherians [30] . In addition, clade II and III contain homologous IFITM sequences from teleosts and amphibians, but clade I does not ( Fig. 2A) , indicating that IR-IFITM originated later than IFITM5 and IFITM10.
Phylogenetic Analyses of the Vertebrate Immunityrelated IFITM Sub-family IR-IFITM genes can be divided into two groups: one consisting of eutherian homologs and the other including homologs from metatheria and bird. We further constructed a sub-tree to show the phylogenetic relationship of 109 IR-IFITM genes from eutheria (Fig. 3) . The sequences from elephant are located on the basal position. All IR-IFITM genes from the primate lineages form a sub-clade, and those from rodents form another sub-clade. Four genes from tree shrew form a species-specific cluster located between the sub-clades of the primates and the rodents. Three mammal species, dog, horse and cow, form several species-specific IR-IFITM gene clusters, which further compose a sub-clade in accordance with the phylogeny of these three species. These suggest that IR-IFITM genes evolved via gene duplication after species separation.
Interestingly, the majority of IR-IFITM genes from rodents do not form species-specific clusters as each of IFITM1, IFITM2 and IFITM3 clusters together in a lineage-specific manner. This clustering indicates that IR-IFITM genes have diverged into different IFITM isoforms prior to the split of rodents from other mammals. Additionally, in the lineage-specific clusters, more than one IR-IFITM gene was observed from certain species, indicating that gene duplication of IR-IFITM genes continued until after species separation of the rodents. Furthermore, rat IFITM7 clusters closely with rat IFITM3, indicating that they are a pair of duplicated genes. Similarly, mouseIFITM7, mouseIFITM1 and mouseIFITM-like3 (IFITMac) are another group of duplicated genes, suggesting that IFITM7 might have similar biological function to IFITM3 or IFITM1.
Within the primate sub-clade, three separate clusters of IFITM1, IFITM2, and IFITM3 were observed. The IFITM1 cluster contains the sequences from all analyzed primates, excluding marmoset and orangutan, and is located at the basal position of the primate sub-clade, indicating that IFITM1 separated earlier than other IR-IFITM genes, including IFITM2 and IFITM3, during the primate evolution. The IFITM2 and IFITM3 clusters only contain sequences from three hominids (human, chimpanzee and gorilla), indicating that IFITM2 and IFITM3 originated prior to the separation of these three hominids.
Likewise, similar to the rodent sub-clade, primate IR-IFITM genes also form different clusters, which contain orthologous IFITM genes from different species, indicating that most IR-IFITM members diverged prior to species separation. Furthermore, some IR-IFITM genes from same species cluster together and form species-specific sub-clusters, indicating that the IR-IFITM subfamily experienced a rapid expansion through gene duplications after the divergence of primates. Interestingly, a species-specific cluster was formed by the 25 IR-IFITM genes from marmoset, suggesting a rapid expansion of IR-IFITM genes by gene duplication. This speculation is supported by the four pairs of marmoset IFITM genes (IFITM-like8 and IFITM-like24, IFITM-like10 and IFITM-like29, IFITM-like14 and IFITM-like26, and IFITM-like11 and IFITM-like27) that exhibit very close genetic relationships, possibly indicating relatively recent gene duplication events. Additionally, the two genes in each pair are located in different chromosomes (Fig. 4B ), indicating their origination by segmental duplication rather than tandem duplication.
In total, 20 IFITM5 genes were identified in 20 vertebrates, covering species from teleosts to eutherians (Fig. 2B ). No IFITM5 gene was identified in gorilla, marmoset, tree shrew, rabbit, dog, fugu, or medaka. Due to low sequencing coverage, we cannot rule out the possibility that the lack of IFITM5 in these genomes should be ascribed to relatively low quality of the genome sequences. The phylogenetic relationships of the available IFITM5 genes are consistent with the known species phylogeny. Only one IFITM5 gene was identified in each species, indicating that no gene duplication occurred in IFITM5 gene during the evolution of vertebrates.
In 19 vertebrates, 26 IFITM10 genes were identified, covering species from teleosts to eutherians (Fig. 2C ). Each terrestrial vertebrate we surveyed possesses one IFITM10 gene, implying that no gene duplication occurred during the evolution of terrestrial vertebrates. In semi-aquatic frog, one IFITM10 and four IFITM10-like genes were identified. In aquatic vertebrates, three and two IFITM10 or IFITM10-like genes were identified in fugu and medaka, respectively. These indicate that species-specific gene duplications occurred in lower vertebrates. Within the IFITM10 clade, two groups were observed. One includes four IFITM10-like genes from frog and various numbers of IFITM10like genes from teleosts. The other one comprises the frog IFITM10 gene and all IFITM10 genes from terrestrial vertebrates. Such division might distinguish aquatic-and terrestrial-type of IFITM10 and accordingly the amphibian frog possesses both types. These findings suggest that functional divergence of IFITM10 in the evolution of vertebrates may have occurred during the transition from an aquatic to a terrestrial environment. 
In order to further understand the evolutionary scenario of IFITM family, we investigated the chromosomal distribution of well-defined IFITM genes including IFITM1, IFITM2, IFITM3, IFITM5, IFITM6, and IFITM10. Because IFITM7 has chromosome location independent from any other IFITM genes, and only mouse and rat have IFITM7 gene, we did not take it into account in this analysis. Orthologous relationships between IFITM family members were well confirmed with conserved syntenies (Fig. 4A ). All these genes are located in one chromosome and form two loci in terrestrial vertebrates, except in cow that has two loci in two different chromosomes. With the exception of IFITM10, that is located at one locus, all other IFITM genes gather together and form a gene-cluster in the other locus. In teleosts (e.g. zebrafish), the two loci are dispersed in two different chromosomes. Although we identified two loci in two scaffolds of frog, whether they are dispersed in two different chromosomes is still unknown. We investigated genes flanking both sides of the two IFITM loci, and found that the two loci have almost completely same flanking genes from lower (e.g. zebrafish) to higher (e.g. human) vertebrate species. These findings suggest that chromosomal fusion might have occurred during the vertebrate evolution from aquatic to terrestrial species. Furthermore, all these IFITM genes have two exons, and the IFITM gene-clusters exhibit consistent gene order in four hominids, two rodents and three birds, supporting the syntenic relationship of IFITM genes.
The IFITM gene-clusters of different vertebrate species contain a variety of IFITM gene numbers (Fig. 4B) . Due to incomplete genome information, the gene-cluster of some species including tree shrew, platypus and some teleosts are not determined. In zebrafish, the orthologous IFITM gene-cluster only contains IFITM5 in chromosome 25. In bird and lizard, two IFITM genes IFITM3 and IFITM5 are included in the gene-cluster. The hominid IFITM gene-clusters contain 3-4 IFITM genes, and the two rodent (mouse and rat) clusters contain five IFITM genes. In other mammals (cow, dog, horse, and elephant), gene-clusters have 4-6 IFITM or IFITM-like genes, including IFITM5 and various number of IFITM-like genes.
The IFITM gene family was divided into three clades. Clade I was further divided into several sub-clades. To test whether there is functional divergence between different clades or between different sub-clades, we estimated type I divergence using DI-VERGE v2.0 [32] and detected significant functional divergence between IR-IFITM and IFITM5, and between IFITM10 and IFITM5 (P,0.01). However, functional divergence signal was not detected between the IR-IFITM genes and IFITM10 (P = 0.0655) ( Fig. 5 and Table 2 ). IFITM10 genes are divided into two subgroups, terrestrial-type and aquatic-type and significant functional divergence was also detected between the two sub-groups (Fig. 5D ). Among IR-IFITM genes, although IFITM2 and IFITM3 might originate from IFITM1 via gene duplication, there is no functional divergence observed between IFITM1 and IFITM2&3 genes (data not shown).
Conversely, crucial amino acid residues responsible for the functional divergence of IFITM genes among the three clades were predicted using a posterior-based site-specific profile (Fig. 5) . Surprisingly, almost all sites of IFITM CD225 domains are crucial for the functional divergence between IR-IFITM and IFITM5. Some residues located in the CD225 domain and the C-terminal 
To investigate whether positive selection drove the evolution of the vertebrate IFITM gene family, we calculated the nonsynonymous substitution (dN) and synonymous substitution (dS) distances [33] between each pair of the sequences from the three clades. To exclude false signals caused by recombination, we first evaluated the effect of gene conversion using GENECONV [34] . Gene conversions were found in some species which are under species-specific duplication including dog, cow, horse, etc. (Table  S2 ). Those sequences were removed from our datasets for subsequent analyses.
There is no significantly higher dN than dS in the pairwise comparisons of the sequences from clade III, suggesting that no positive selection acted on IFITM5 (Fig. 6A) . Further site-specific analyses using PAML (Table 3 ) and HyPhy (data not shown) confirmed no positive selection acting on IFITM5 genes. There are three pairs of IFITM10 genes (in fugu: IFITM10-like3 vs. IFITM10-like2, and IFITM10-like1 vs. IFITM10-like2; in medaka: IFITM10-like1 vs. IFITM10-like2) with dN/dS.1 (Fig. 6A) , indicating positive selection acting on aquatic IFITM10 gene. Because gene expansion of IFITM10 was observed in frog (Fig. 2C) , we analyzed the five IFITM10 related genes (including one IFITM10 and four IFITM10-like genes) using the site-specific model. A strong signal of positive selection was detected. Eight sites were identified as under positive selection at the level of posterior probability.0.8 (Table 3) . We further constructed the phylogeny of frog IFITM10-related genes and counted the numbers of non-synonymous (n) and synonymous (s) substitutions on each branch (Fig. S6) . The gn/gs ratio (1.75) is significantly smaller than the N/S ratio (2.96) (P = 0.0112, chi-square test), showing no positive selection. However, a very strong signal of positive selection (v = 999, P = 0.0077, chi-square test) was Within clade I, 12 of 5996 pairwise comparisons exhibit significantly higher dN than dS (Fig. 6B and Table S3 ), indicating the presence of positive selection. Among 12 pairwise comparisons with significantly higher dN than dS, eight occur between the primate sequences (Fig. 5C) , three between the rodent sequences (Fig. 5D) , and one between cow sequences. Further site-specific analysis detected two positively selected sites (PSS) in the primate IR-IFITM genes and one in the rodent IR-IFITM genes ( Table 3) . No PSS was detected in the primate IFITM1 and IFITM2&3 subgroups. Because positive selection is generally associated with the occurrence of gene duplication [35] , and a large number of gene duplications were observed in the marmoset IR-IFITM genes, we also performed site-specific analysis on marmoset IR-IFITM genes. As expected, one significant PSS was detected in marmoset IR-IFITM genes.
To confirm the results from PAML, similar positive selection analyses were performed using MEME on the DATAMONKEY server. The results reveal that there are five, four, seven and one sites underwent significant positive selection in primate, rodent, marmoset IR-IFITM groups and frog IFITM10 group, respectively (Table S4) . PSSs identified by MEME method are consistent with those identified by PAML. Furthermore, positive selection can be also confirmed by branch-site REL method in both IR-IFITM and IFITM10 clades (Fig. S7) . The major lineages that have undergone positive selection in the IR-IFITM clade are primate and rodent (Fig. S7A) . Additionally, IFITM10 in lower vertebrate lineages also experienced strong selective pressure. Positively selected branches were detected in both teleost and frog lineages (Fig. S7B ).
IFITM family contains seven members (IFITM1, IFITM2, IFITM3, IFITM5, IFITM6, IFITM7 and IFITM10), as well as some IFITM-like genes. All vertebrate IFITM genes are divided into three clades ( Fig. 2A) , implying origins from three progenitors. Clades I, II and III contain IR-IFITM, IFITM5 and IFITM10 genes, respectively. Substantial functional divergences occurred between IR-IFITM, IFITM5 and IFITM10 genes, indicating that IR-IFITM, IFITM5 and IFITM10 experienced individual evolutions. IR-IFITM, IFITM5 and IFITM10 genes are usually located in two loci (Fig. 4) . One locus contains only the IFITM10 gene, and the other locus contains various numbers of IR-IFITM genes with a syntenic location with IFITM5, forming an IFITM genecluster. The two loci can be used as good markers to trace the evolutionary history of IFITM family. They are located in two different chromosomes in lower aquatic vertebrates, and evolved to lie in one chromosome by chromosomal fusion in higher mammals (Fig. 4A) . The syntenic relationship of IR-IFITM genes and the presence of more IR-IFITM genes in mammals than other vertebrates suggest that IR-IFITM gene sub-family experienced a rapid expansion via tandem duplication during evolution from lower vertebrates to mammals.
Different IFITM members exhibit various functions. IFITM5 is specifically expressed in bone cells, but could not be induced by IFN stimulation [3, 19] . IFITM5 is involved in bone formation and considered as a bone-specific modulator of mineralization. The vertebrate IFITM5 genes form an independent clade in IFITM family ( Fig. 2A) . Neither gene duplication nor positive selection was identified in IFITM5 sub-family ( Fig. 6B and Table 3 ), implying the functional conservation of IFITM5 in vertebrate evolution. Interestingly, no IFITM5 gene was found in the genomic data of two primate species, gorilla and marmoset. Gorilla has a close phylogenetic relationship with human and chimpanzee. In human and chimpanzee, IFITM5 is located upstream of the IFITM1-IFITM2-IFITM3 gene-cluster in chromosome 11. IFITM1, IFITM2 and IFITM3 in gorilla are also located in chromosome 11, and form a similar gene-cluster to those of human and chimpanzee (Fig. 4) . Therefore, gorilla is also presumed to have an IFITM5 gene upstream of the IFITM1-IFITM2-IFITM3 gene-cluster. In fact, we found a region with incomplete sequencing at the corresponding locus of human or chimpanzee IFITM5 genes in chromosome 11 of gorilla, explaining why we could not identify IFITM5 gene locus in current gorilla genome dataset. In contrast, marmoset has special IFITM gene organization and experienced rapid gene expansion, thus forming unique species-specific gene cluster (Fig. 3) . Considering that marmoset is one of the smallest monkeys in the world, as well as that IFITM5 plays a crucial role in bone formation [19, 36] , we inferred that marmoset most likely lost its IFITM5 during the long period of evolution. Nevertheless, whether the loss of IFITM5 gene contributes to the tiny size of the marmoset needs to be determined by future studies.
So far, there is no study reporting the function of IFITM10. All various vertebrate IFITM10 genes can be divided into two subgroups, one of which contains IFITM10 from terrestrial vertebrates with frog IFITM10 at the basal position, suggesting that terrestrial vertebrates and frog share a common IFITM10 ancestor. The other one contains IFITM10 from aquatic vertebrates, as well as four frog IFITM10-like genes. Significant signal of functional divergence was observed between the two subgroups (Table 2) , possibly suggesting an association with terrestrial and aquatic environments. In particular, both gene duplication and positive selection can be detected in IFITM10 or IFITM10like genes from the aquatic vertebrates ( Fig. 6D and Table 3 ), indicating that IFITM10 is associated with the adaptation to aquatic environments. There are one IFITM10 gene and four IFITM10-like genes in frog (Fig. 2C ). An episodic adaptive evolution was found on the branch leading to three frog IFITM10like genes (Fig. S6) , supporting the association of IFITM10 with terrestrial and/or aquatic environments. However, what the function of IFITM10 is and how it helps the aquatic vertebrates to adapt to aquatic environments still need to be determined. Distinct from IFITM5 and IFITM10, the IR-IFITM sub-family contains IFITM1, IFITM2, IFITM3, IFITM6, IFITM7, as well as a large number of IFITM-like genes [37, 38] . This sub-family forms a large clade in the phylogenetic tree (Fig. 3) . The IR-IFITM genes from same mammalian species, such as dog, horse, cow, elephant, guinea pig and tree shrew, cluster together and form species-specific IR-IFITM gene sub-clusters (Fig. 3) , in-dicating that gene duplication occurred after the separation of these mammalian species in a species-specific pattern. The same IFITM member from mouse and rat cluster together, indicating that the gene duplication there occurred prior to the separation of the two species in a lineage-specific pattern. The rodent IFITM1, IFITM2 and IFITM3 sub-groups cluster together (Fig. 3) , suggesting a close phylogenetic relationship and similar functions. IFITM6 and IFITM7 are specific for the rodents. The rat and mouse IFITM6 genes cluster together and further group with the guinea pig IR-IFITM gene sub-cluster. Mouse IFITM7 clusters closely with mouse IFITM1 gene and rat IFITM7 clusters closely Evolutionary Dynamics of IFITM Gene Family with rat IFITM3 gene (Fig. 3) , which might indicate IFITM7 has similar functions to IFITM1 or IFITM3.
Lineage-specific gene duplications were also observed in the primate IR-IFITM genes. The IFITM1 genes from some primate species form an individual sub-group located at the basal position of the primate IR-IFITM sub-clade (Fig. 3) . IFITM1, IFITM2 and IFITM3 have miscellaneous functions including cell adhesion, antiproliferation, tumor suppression and embryonic development. Apart from these biological functions, human IFITM1, IFITM2 and IFITM3 have a broad-spectrum of antiviral activity, possibly brought on by inhibiting the viral entry processes [14, 18, 39] . In particular, human IFITM3 and IFITM2 appear to have higher antiviral activity than IFITM1 [14] and IFITM3 has been reported to inhibit virus replication in other mammals [40] . These findings suggest that after IFITM1 occurrence, the generation of IFITM2 and IFITM3 might be associated with host defense against various virus infections.
Primate and rodent IFITM1, IFITM2 and IFITM3 have similar functions [2] , but do not form a monophyletic cluster (Fig. 3) , indicating that they do not share the most recent common ancestor (MRCA) and moreover suggesting convergent evolution of IFITM1, IFITM2 and IFITM3 in primates and rodents. Convergently evolved amino acids between primates and rodents were found in the C-terminus of IFITM2 and IFITM3 (data not shown), a crucial region for antiviral activity, supporting the association between viral infections and the evolution of IR-IFITM genes.
The largest scale of gene expansion of IR-IFITM genes was observed among the primates in a complex pattern that includes both lineage-and species-specific gene duplication events. The species-specific pattern mainly seems to have occurred in macaque and marmoset, giving rise to seven IFITM2&3-like genes in macaque and 29 IR-IFITM genes in marmoset (Fig. 3) . Large scale of duplication and pseudogenization events suggest that the IR-IFITM clade evolved under birth-and-death model [41, 42] . Interestingly, however, marmoset does not possess any one of IFITM1, IFITM2 or IFITM3, and macaque possesses IFITM1 but not IFITM2 and IFITM3. Why marmoset and macaque evolved so many IR-IFITM genes remains unclear and should be explored in more detail in future studies.
The Red Queen hypothesis presumes that the antagonistic coevolutionary dynamics of host-virus systems can generate selection Table 3 . Site-specific tests for positive selection on different IFITM sub-families. for accelerated evolution of host antiviral restriction factors, just like the observations on the primate antiviral restriction factors APOBEC3G, Tetherin, and SAMHD1 [21] [22] [23] [24] [25] [26] [27] [28] [29] . We detected positive selection acting on marmoset IR-IFITM genes (Table 3) . Macaque and marmoset are susceptible to infection by many contemporary viruses and are often used as suitable non-human primate models for viral infectious disease studies [9, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] . Cell lines (e.g. kidney cells) from marmoset can be infected by most primate viruses, such as flaviviridae family of viruses (GBV-B), lassa virus, peste des petitis ruminants (PPR) virus and so on [59, 60, 61] . These imply that marmoset and macaque might be able to be infected by the ancestors of contemporary primate viruses and/or some unknown viruses during early evolution of primates. Additionally, relative to pig IFITM3 that has been demonstrated to have an antiviral activity [40] , IFITM1, IFITM2 and IFITM3 genes from macaque and marmoset have higher similarity to human IFITM1, IFITM2 and IFITM3 in sequence and domain organization. These imply that although lacking experimental support, the IR-IFITM genes from macaque and marmoset might have similar antiviral activity to human IFITM1, IFITM2 and IFITM3. Therefore, the rapid expansion of IR-IFITM genes might be ascribed to the infection of marmosets by viruses.
In this study, we demonstrated the evolutionary dynamics of IFITM genes that diversifies in different sub-clades, probably in accordance with their distinct functions. Future studies on immunology, developmental biology and comparative biology to determine IFITM functions would better clarify the relationship between divergence and functions and likewise extend our knowledge on IFITM function and evolutionary mechanisms.
Functional IFITM gene sequences were gained based on orthologous and paralogous relationships by querying the Ensembl genome assemblies (http://www.ensembl.org/index.html) using known authentic IFITM genes [17] . Collected IFITM genes were used as queries to search against known IFITM gene datasets using tBLASTn or BLASTn searches to make sure the best hit is an functional IFITM genes with E value,10 210 . The presence of CD225 domain in each obtained IFITM protein sequence was confirmed using P-fam database (http://pfam.sanger.ac.uk).
Annotated IFITM genes were retrieved from Ensembl databases (http://www.ensembl.org/). To identify additional putative IFITM functional genes and pseudogenes, tBLASTn searches were performed in Ensembl using human IFITM1, IFITM2, IFITM3 and IFITM5 as queries against the available genome sequences of species listed in Table 1 with E value,10 25 [62] . After deleting the redundancies and merging overlapping sequences, the remained sequences with.150 nt were analyzed by GENSCAN to identify the putative coding sequence. Each candidate IFITM gene was used as a query to BLAST against GenBank non-redundant protein database to make sure the best hit is an IFITM gene. The exons and intron of the remained sequences were detected with GeneWise. The presences of CD225 domain and C-terminal TMD in candidate sequences were identified using the online tools SMART (http://smart.emblheidelberg.de) and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/ ), respectively. If the sequence did not have complete CD225 domain and the C-terminal TMD, or its open reading frame (ORF) was disrupted, this sequence was regarded as a pseudogene. To avoid the possible error in pseudogene identification, the PSEDOPIPE approach (http://pseudofam.pseudogene.org/ pages/main/about.jsf) [63] was further used to confirm identified IFITM pseudogenes. On the other hand, because authentic IFITM proteins are in a range of 102 to 157 amino acids [2] , the candidates that are outside above range and have complete open reading frame, complete CD225 domain and the C-terminal TMD were identified as functional genes and referred to as IFITM-like genes.
Conserved motifs in the complete amino acid sequences of the mammalian IFITM proteins were analyzed by MEME/MAST software (http://meme.sdsc.edu/meme/website/intro.html). The secondary structure of IFITM proteins was predicted using SMART (http://smart.embl-heidelberg.de/smart). The logo pictures were generated by Weblogo (http://weblogo.berkeley.edu).
Multiple sequence alignments were performed using Muscle in MEGA 5.0 [64] and were refined manually in Bioedit (http:// www.mbio.ncsu.edu/BioEdit/BioEdit.html).
Unambiguously aligned positions (Figs. S2, S3 and S4) were used for subsequent phylogenetic analyses. Maximum likelihood (ML) tree of IFITM gene family was reconstructed by PHYML2.4 implemented in Jmodeltest 0.1 package with the best-fitting model of F81+I+G that is selected using JmodelTest in the same package [65, 66] . The bootstrap analysis was performed with 1000 replications. Bayesian inference (Bayes) tree was reconstructed using MrBayes v3.1.2 [67] . Four independent Markov Chain Monte Carlo (MCMC) chains were used with the default temperature of 0.01. Four repetitions were run for 8,000,000 generations with tree and parameter sampling occurring every 1,000 generations. The first 25% of trees were discarded as burn-in, leaving 750 trees per run. Posterior probabilities for internal node were calculated from the posterior density of trees. Maximum parsimony (MP) tree was reconstructed by PAUP 4.0 with a bootstrap value of 1,000 repetitions [68] . To investigate whether gene conversion occurred in mammalian IR-IFITM genes, an analysis was performed using the GENECONV program [34] .
To detect whether positive selection acted on IFITM family, the CODEML program implemented in PAML 4.2 package was used. The site-specific model was performed by comparing the models M2a (positive selection) and M8 (beta & v) vs. the null models M1a (nearly neutral) and M7 (beta), respectively. Likelihood ratio tests (LRT) of different models were used to find the best fit model for the data [65, 66, 69] . We also used the MEME method and the branch-site REL model implemented in DATAMONKEY (http://www.datamonkey.org/) to confirm the results by PAML analyses [70, 71] . MEME is the latest method to identify PSS and can find signatures of episodic selection even when the majority of lineages are subject to purifying selection.
In the phylogenetic tree, IR-IFITM, IFITM5 and IFITM10 genes from different species form three independent clusters. We investigated type I functional divergence among IR-IFITM, IFITM5 and IFITM10 using Diverge v2.0 with the Maximum-Likelihood Estimation (MLE) and Model-Free Method (MFE) [32] . Type I sites represent amino acids conservation in one cluster, but high variability in another, suggesting that these residues have been subjected to different functional constraints. The coefficient of functional divergence, h, ranging from 0 to 1, was used to test the statistical significance of functional divergence that has occurred between different clusters. A null hypothesis of h = 0 indicates that the evolutionary rate is virtually the same between two clusters at each site. When h.0.5, the null hypothesis is considered to be significantly rejected. The important amino acid residues most likely to be responsible for functional divergence were then predicted by calculating the site-specific profile based on posterior analysis for all pairs of clusters with functional divergence. Figure S2 Sequence alignment of the vertebrate IFITM5 genes. Alignment was used to reconstruct the Bayesian tree in Figure 2B . (PDF) Figure S3 Sequence alignment of the vertebrate IFITM10 genes. Alignment was used to reconstruct the Bayesian tree in Figure 2C . (PDF) Figure S4 Sequence alignment of the mammalian IR-IFITM genes. Alignment was used to reconstruct the phylogenetic trees shown in Figure 3 . The abbreviations were adopted from the species names in Table 1  Filovirus Research in Gabon and Equatorial Africa: The Experience of a Research Center in the Heart of Africa Health research programs targeting the population of Gabon and Equatorial Africa at the International Center for Medical Research in Franceville (CIRMF), Gabon, have evolved during the years since its inception in 1979 in accordance with emerging diseases. Since the reemergence of Ebola virus in Central Africa, the CIRMF “Emerging Viral Disease Unit” developed diagnostic tools and epidemiologic strategies and transfers of such technology to support the response of the National Public Health System and the World Health Organization to epidemics of Ebola virus disease. The Unit carries out a unique investigation program on the natural history of the filoviruses, emergence of epidemics, and Ebola virus pathogenesis. In addition, academic training is provided at all levels to regional and international students covering emerging conditions (host factors, molecular biology, genetics) that favor the spread of viral diseases. The International Centre for Medical Researches of Franceville (CIRMF) was founded in 1974 by His Excellency El Hadj Omar Bongo Ondimba, President of the Gabonese Republic, and Mr. Pierre Guillaumat, the chairman of the petroleum company, Total Gabon. The Centre was inaugurated on December 5th, 1979 with the participation of numerous internationally eminent scientists (Figure 1) . In the 1990s, viral hemorrhagic fevers became a focus of attention in Equatorial. The decision to develop a high security laboratory for the study of Ebola virus disease came after a 1996 Ebola virus disease outbreak in Mayibout area, Gabon. The main objective was to develop the potential for rapid and specific diagnosis on viral hemorrhagic fevers and to have a backup for field investigation of severe viral hemorrhagic fever epidemics. Because the unique expertise and interaction of the CIRMF team along with the international World Health Organization (WHO) teams for Ebola virus disease, the Gabonese government agreed to such a project. The first BSL3+ (including negative pressure and glove box) laboratory was built in 1997, mostly financed by the Foreign Ministry of France ( Figure 2 ). This laboratory was built in a period of quasi "emergence" of successive Ebola virus disease outbreaks in Gabon, and the plans to upgrade the BSL3+ laboratory were not efficient. A specific research unit was founded in 1998 to study emerging infectious diseases: The Emerging Viral Disease Unit (UMVE). Thanks to work done between 1996 and 1998, both in the field and in research, CIRMF became a National Reference Laboratory and a WHO Collaborating Center in the Equatorial African region. A second high security laboratory for Risk Group 3/4 Agents, mostly funded by the TOTAL Gabon oil company and the Gabonese Government, was built between 2003 and 2008 on CIRMF campus ( Figure 3 ). This BSL-4 laboratory was commissioned by a combined team of experts from the Pasteur Institute of Paris, National Institute of Health and Medical Research, France, and Jean Mérieux P4 laboratory of Lyon. 
On 45 hectares, the CIRMF campus has a working space of 2,500 square meters composed of a main building, laboratories, service buildings, and living accommodations (Figure 1 ). The present high containment and high security laboratory, operated by the Emerging Viral Diseases Unit (UMVE), is one of 2 laboratories in Africa that can manipulate Risk Group 3/4 Agents (i.e., Ebola, Marburg, and Crimean-Congo hemorrhagic fever viruses). Research, including isolation and characterization of these highly pathogenic viruses is performed in accordance with international rules defined by WHO on the handling Risk Group 3/4 Agents Updated equipment includes a double door autoclave, thermo regulated cabinet, a high security centrifuge system, a conventional photonic microscope, a virus isolation unit, and two independent rooms the can be shut down alternatively after decontamination when necessary. An uninterruptable controlled electrical supply for refrigeration, computer systems, and other systems is ensured by two back-up power plants. The telecommunication network consists of mobile phones and the Internet through a dedicated satellite antenna. Other service units consist of a Primate Center, the Gorilla and Chimpanzee Study Station in Lopé National Park, and the Dienga Health Observatory. Investigators from this observatory conduct field studies on the prevalence of viral and parasitic diseases and their implications for public health [1] Dedicated to medical research, the Primate Center houses more than 450 primates belonging to ten different African species (e.g., chimpanzees (Pan troglodytes), gorillas (Gorilla sp), mandrills (Mandrillus sphinx), guenons (Cercopithecus sp.), collared mangabeys (Cercocebus torquatus), greater spot-nosed monkeys (Cercopithecus nictitans), vervet monkeys (Chlorocebus pygerythrus) and an Asian macaque (Macaca sp.) colony. One of the largest primate centers in Africa, the Primate Center is equipped with level A2 and A3 animal facilities for scientific research protocols. The Great Apes are housed in large open-air facilities. Semi-free living colony of twelve forested hectares harbor about half of the primates at the Primate center. At the Gorillas and Chimpanzee Study Station, researchers study ecological approaches to the emergence of zoonotic diseases, inter-species transmission of pathogens, and disease outbreaks in humans and wild animals [2] . 
Running costs are funded by the Ministry of Economy, Gabon, the national petroleum company of Total-Gabon, and the Ministry of Foreign and European Affairs, France. Several international agencies participate in a variety of financial supports including scientists' salaries, equipment, research projects, and academic grants (e.g., IRD, WHO, United States Agency for International Development, National Center for Scientific Research, France).
Technical laboratory training support of Gabonese teams and other African countries has been one of the major aims of CIRMF. The UMVE actively participates in the academic training at the Regional Graduate School and the different State universities of Equatorial Africa. A special relationship with the "Health Sciences University" of Libreville and the "Sciences and Technology University" of Masuku in Franceville encourages collaborative research projects with teachers and supports students of the Faculties of Medicine and Sciences in the preparation of doctoral theses. CIRMF receives doctoral and post-doctoral scientists from other universities of developed countries (e.g., Bonn, Marseille, Montpellier, and Tübingen Universities). Continuing medical education in the form of post-doctoral workshops are held at the CIRMF for discussion and demonstration of modern techniques.
As a National reference laboratory, CIRMF has the following roles: diagnosis of suspected cases during outbreaks of viral hemorrhagic fevers or severe clinical infectious syndromes; development of new methods for diagnosing such infections; surveillance of animal fatalities in reservoir or intermediate hosts; and intervention during outbreaks of unknown etiology. CIRMF diagnosed infections of more than 70 pathogens that could not be identified in other biology laboratories throughout the country. CIRMF maintains close ties to several components of the National Healthcare system, such as Amissa Bongo Regional Hospital in Franceville or the Sino-Gabonese Friendship Hospital.
In order to facilitate national and international scientific exchanges including scientists, equipment, biological specimens, the capital of Gabon, Libreville, is part of CIRMF operational system. Hosted by the University of Health Sciences, Libreville, one laboratory is now operational. Tight connections with other scientific teams in Libreville are under development (i.e.,: Military Hospital, Libreville; General Hospital, Libreville; A. Schweitzer Lambarene Foundation).
The Emerging Viral Diseases Unit, CIRMF, proposes forming a research partnership to study infectious diseases transmitted by animals of the tropical rain forests regions of Equatorial Africa. The proposed partnership builds on such existing collaborations of several years between the major research centers of 2 other French speaking Equatorial African countries, namely the National Public Health Laboratory in Brazzaville, Republic of the Congo, and the Institute for Biomedical Research in Kinshasa, Democratic Republic of Congo. An international partnership with the IRD, Marseille, France, and the Institute of Virology, Bonn, Germany, will assist in the development of this regional partnership.
With the WHO Regional Office, Brazzaville, Republic of the Congo, the UMVE-CIRMF field team participates along with other international partners (e.g., Centers for Disease Control and Prevention, USA; Laboratory Centre for Disease Control and National Microbiology Laboratory, Winnipeg, Canada; P4 Laboratory of the National Institute for Communicable Diseases, South Africa) to respond to all Ebola virus disease epidemics in Africa. CIRMF aims to use laboratory and field expertise be a regional focal resource in conjunction with local health authorities to organize epidemic responses. CIRMF expertise is also offered from by entering into laboratory-to-laboratory agreements. Also, an informal international laboratory network for the diagnosis and surveillance of severe infectious clinical syndromes includes: the Institut National de Recherche Biomédicale, Democratic Republic of the Congo; Laboratoire National de Santé Publique de Brazzaville, Republic of the Congo; Metabiota/Laboratoire des Maladies Emergentes, Yaoundé, Cameroon; Pasteur Institute, Bangui, Central African Republic; Institute of Virology, Bonn University, Germany; and P4 Jean Mérieux Lyon, France. Exchange of materials, equipment, and personnel is facilitated through memorandums of understanding.
CIRMF holds more than 250,000 samples of various origins in a biological repository, which is accessible to the International scientific community. The UVME assists the National Public Health System in consolidating and formalizing microbiological monitoring of the Equatorial African sub-region. Ultimately, CIRMF will be positioned as a Center of Excellence for microbiological surveillance and research in a global network.
Developing diagnostic tools and strategies is the main driver to improve surveillance and research of emerging viral diseases. A strategic choice was made to link syndromes to an etiological agent, including hemorrhagic syndromes. To isolate and diagnose highly pathogenic viruses, a progressive and diversified methodology was applied. The first approach used real-time virus-specific PCR (qRT-PCR). If the first approach was not successful, conventional RT-PCR was implemented using degenerate consensus primers targeting conserved regions of the genome. Ultimately, random amplification of nucleotide sequences was directly applied to the original biological material (DNA chip re-sequencing, meta-genomic pyrosequencing (454 Life Sciences, Branford, CT)).
UMVE studied the factors implicated in the three steps that led to Ebola virus and Marburg virus diseases emergence in humans. These steps include: the identification of reservoir species, the circulation within the natural host, the crossing to intermediary animal species, and finally the direct transmission to humans from great apes and fruit bats. Antibodies and nucleotide sequences specific for Ebola virus were detected in the liver and spleen of fruit bat belonging to three species (Hypsignathus monstrosus, Epomops franqueti, Myonycteris torquata) in Gabon and Republic of the Congo (Figure 4 ). Antibodies and nucleotide sequences specific for Marburg virus were found in the Egyptian fruit bat (Rousettus aegyptiacus) in Gabon, suggesting that bats might be reservoirs for filoviruses [3] [4] [5] . We showed that Ebola virus caused extensive epizootics among gorillas and chimpanzees, killing thousands of animals during the last decade in parts of Gabon and Republic of the Congo [4] . We characterized the viral variants associated with all Ebola virus disease outbreaks that occurred between 1996 and 2008 and developed new epidemiological models of Ebola virus disease epidemics, based on the identification of several independent epidemic chains. The identification of multiple variants during the 2001 Gabon/Republic of Congo outbreak and two phylogenetically divergent lineages suggest independent introductions into great ape and human populations following multiple viral spillovers from a reservoir host [6] [7] . In this "multi-emergence" hypothesis, Ebola virus disease outbreaks would occur episodically during certain ecological conditions caused by habitat disturbances or climatic phenomena. This hypothesis also implicitly assumes that Ebola virus was present in Equatorial Africa long before the first documented disease outbreak in 1976, as supported by various serological surveys. Furthermore, we recently showed that the 2007 Luebo outbreak in the Democratic Republic of the Congo was linked to massive fruit bat migration, strongly suggesting that humans could be infected directly by bats or by consumption of bats [8] .
In the study of immunological mechanisms of Ebola virus disease humans, we showed that fatal infection is associated with aberrant innate immunity and global suppression of adaptive immunity [, [9] [10] [11] [12] . The innate immune reaction is characterized by a 'cytokine storm', with a hyper secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, and by the noteworthy absence of antiviral interferon (IFN)-α [9, 13, 14] . Immunosuppression of adaptive immunity is characterized by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of peripheral CD4 and CD8 lymphocytes, probably through Fas/FasL-mediated apoptosis. Finally, we hypothesized that a viral protein with super-antigen activity might be involved in the massive T cell apoptosis [15] . In striking contrast with fatal outcome, effective control of Ebola virus infection is associated with balanced immune responses in survivors. Asymptomatic Ebola virus infection was demonstrated in humans during the 1996-97 disease outbreak in Gabon [16] . Asymptomatic infection was associated with an early strong inflammatory response that may be involved in the early inhibition of viral replication [16, 17] . Consistent with this discovery, we showed a decade later that a large fraction of the human population living in forested areas of Gabon has both humoral and cellular immunity to Ebola virus [18] . In the absence of identified risk factors, the high prevalence of 'immune' individuals suggests a common source of human exposure such as fruits contaminated by bat saliva.
Initially focused on Ebola virus disease, UMVE science policy was redirected since 2007 by expanding the main research themes to other emerging viral diseases that could threaten public health in the Congo basin (Table 1) [19] [20] [21] [22] .
CIRMF is geographically isolated from the capital of Gabon, Libreville. The Libreville office is essential to the Franceville headquarters as it coordinates visits from staff on field missions, and forwards imported equipment to headquarters. The capital is accessible by a 12-hour ride in a four-wheel drive vehicle or in a train (three times/week schedule) covering 641 km. Due to tropical weather, four weekly domestic plane rotations often fly on an inconsistent schedule. Ultimately CIRMF needs to be largely autonomous in term of electrical power (i.e.,: unexpected fuel supply disruption), cold chain with the necessity to maintain in situ a unit of liquid nitrogen production (repository), and purified water supply. 
CIRMF is uniquely suited to study infectious diseases of the Congolese tropical rain forest, the second world's largest rain forest. As a central point of a North-South transect of the rain forest, the Center is able to study the biodiversity of Africa including animal species, microbes, and parasites.
CIRMF is dedicated to conduct medical research of the highest standard, and is the only facility of its type in Equatorial Africa. With unrivalled infrastructure, multiple sites, and multidisciplinary teams, the Center promotes a modern healthcare system in Gabon. CIRMF teams are engaged in trans-disciplinary projects bringing together specialists from the health sciences, biological sciences, veterinary medicine, conservation, the humanities, and environmental sciences. The Center welcomes partnerships from around the world to work on global human health issues. Reproductive Number and Serial Interval of the First Wave of Influenza A(H1N1)pdm09 Virus in South Africa BACKGROUND/OBJECTIVE: Describing transmissibility parameters of past pandemics from diverse geographic sites remains critical to planning responses to future outbreaks. We characterize the transmissibility of influenza A(H1N1)pdm09 (hereafter pH1N1) in South Africa during 2009 by estimating the serial interval (SI), the initial effective reproductive number (initial R(t)) and the temporal variation of R(t). METHODS: We make use of data from a central registry of all pH1N1 laboratory-confirmed cases detected throughout South Africa. Whenever date of symptom onset is missing, we estimate it from the date of specimen collection using a multiple imputation approach repeated 100 times for each missing value. We apply a likelihood-based method (method 1) for simultaneous estimation of initial R(t) and the SI; estimate initial R(t) from SI distributions established from prior field studies (method 2); and the Wallinga and Teunis method (method 3) to model the temporal variation of R(t). RESULTS: 12,360 confirmed pH1N1 cases were reported in the central registry. During the period of exponential growth of the epidemic (June 21 to August 3, 2009), we simultaneously estimate a mean R(t) of 1.47 (95% CI: 1.30–1.72) and mean SI of 2.78 days (95% CI: 1.80–3.75) (method 1). Field studies found a mean SI of 2.3 days between primary cases and laboratory-confirmed secondary cases, and 2.7 days when considering both suspected and confirmed secondary cases. Incorporating the SI estimate from field studies using laboratory-confirmed cases, we found an initial R(t) of 1.43 (95% CI: 1.38–1.49) (method 2). The mean R(t) peaked at 2.91 (95% CI: 0.85–2.91) on June 21, as the epidemic commenced, and R(t)>1 was sustained until August 22 (method 3). CONCLUSIONS: Transmissibility characteristics of pH1N1 in South Africa are similar to estimates reported by countries outside of Africa. Estimations using the likelihood-based method are in agreement with field findings. During 2009, the emergence and worldwide spread of influenza A(H1N1)pdm09 (pH1N1) was observed [1] . While a rapid and timely estimation of the transmission parameters of this novel virus played an important role in informing transmission potential and mitigation interventions during the 2009 pandemic period, the post-pandemic documentation of these parameters is equally important as many previous estimates were established from analyses conducted during the early stages of epidemics and often from preliminary data [2, 3] . Additionally enhancing our knowledge of past pandemics assists in providing greater insight to prepare and respond in future outbreaks.
Four key measures are typically used to describe the transmissibility of an infectious disease. First, the serial interval (SI) describes the mean time between illness onset of two successive cases in the chain of transmission. Second, the secondary attack rate (SAR) describes the proportion of susceptible contacts that acquire infection from an infectious person. Third, the basic reproductive number (R 0 ) is defined as the average number of secondary cases per primary case in an idealised entirely susceptible population in the absence of control measures. Finally, the effective reproductive number (R t ) at any given time point represents the actual average number of secondary cases per primary case observed in a population. R t reflects the impact of control measures and the depletion of susceptible persons over time. The initial R t may approximate R 0 in pandemic situations. [2] [3] [4] [5] .
Previously published estimates of pH1N1 transmission parameters vary by study setting and methods employed. The majority of studies found the mean SI of pH1N1 to range from 2.5-3.3 days [2, [6] [7] [8] [9] [10] [11] ; however, Canada and Texas reported a longer SI of 4-5 days, respectively [12, 13] . Estimates of the R 0 of pandemic influenza from the USA range from 1.3-2.3 [2, 9, 11] . Estimates from Mexico range from 1.4-2.9 [2, 14, 15] . Outside of North America, R 0 estimates include: Australia (mean 2.4) [16] , Canada (mean 2.62) [12] , Thailand (mean 2.07) [17] , Peru (range 1.2-1.7) [18] and New Zealand (mean 1.96) [19] . Finally, Japan revised their mean R 0 estimates from 2.3 to 1.28 after repeating analyses later in the pandemic [20] ; thus demonstrating a need to revisit revised and more complete datasets. A variation in R t with progression of the pandemic was observed in Mexico, averaging at 1.47 (based on a negative binomial model) [14] , but peaking between 2.1-4.0 depending on the generation interval chosen [21] .
In a previous work, we estimated the SAR and SI of pH1N1 among the first 100 cases detected in South Africa by prospectively examining virus transmission between household contacts [22] . We found a SAR of 10% and a mean SI of 2.3 days (SD 61.3, range 1-5) between successive laboratory-confirmed cases in the transmission chain. When additionally including suspected secondary cases into the analysis, the SAR increased to 17% and the SI to 2.7 days (SD 61.5, range 1-6). In this work we incorporate data collected on all laboratory-confirmed cases detected during the 2009 pH1N1 epidemic in South Africa with the aim of describing the transmissibility characteristics (initial R t and temporal variation of R t ) of the epidemic in the country and compare its dynamics with those observed in other countries in the same year.
During 2009, the National Institute for Communicable Diseases (NICD), of the National Health Laboratory Service (NHLS), South Africa, maintained a central registry of all pH1N1 laboratory-confirmed cases detected throughout the country. The methodology of collating this data has previously been described in detail [23] . Briefly, we collated individual case-based data from all laboratories offering pH1N1 testing throughout South Africa, which included patient age, sex, dates of illness onset and specimen collection, and the administrative location (province) of the healthcare facility where the patient presented. Testing was performed by accredited laboratories, including: the National Influenza Centre (NICD-NHLS), NHLS public-sector laboratories or private-sector laboratories. All testing laboratories performed detection and characterisation of pH1N1 virus by real-time PCR by either the protocol developed by the WHO Collaborating Centre for Influenza, U.S. Centers for Disease Control and Prevention [24] , or using commercially available kits.
Wherever the date of symptom onset was missing, we estimated it from the date of specimen collection using a multiple imputation approach. Firstly, we modelled the lag time from date of symptoms onset to date of specimens collection from cases with complete data via a Poisson regression model using predictors significant at p,0.05. The covariates assessed in the model were patient age, gender, province, date of specimen collection, and collection of a specimen on a weekend day (i.e. Saturday or Sunday). Secondly we obtained an estimated lag-time for each observation with missing date of symptoms onset using a random sampling process from a Poisson distribution centred on the predicted value from the Poisson regression model. A Poisson distribution was selected to model count data. Thirdly we imputed missing dates of symptoms onset by subtracting the estimated lag-time from the date of specimen collection. The imputation process was repeated 100 times for each missing value, creating 100 datasets with information on the onset date (imputed or observed) for 12,630 laboratory-confirmed cases.
We based the estimation of initial R t and temporal variation of R t on date of symptoms onset (observed and imputed). In all analyses we modelled the SI via a multinomial distribution. When estimating initial R t , we focus our analysis on the exponential growth phase of the epidemic in South Africa (i.e. the period from the first occurrence of five consecutive days with confirmed cases reported to the epidemic peak). The parameters were estimated using three methods:
Method 1. We make use of the likelihood-based method for the simultaneous estimation of initial R t and the SI described by [25] . This method is well suited for estimation of initial R t and SI in real-time with observed aggregated daily counts of new cases, denoted by N = (N 0 , N 1 …,N T ) where T is the last day of observation and N 0 are the initial number of seed cases that begin the outbreak. The N i are assumed to be composed of a mixture of cases that were generated by the previous k days, where k is the maximal value of the serial interval. We denote these as X j , the number of cases that appear on day i that were infected by individuals with onset of symptoms on day j. We assume that the number of infectees generated by infectors with symptoms on day j follows a Poisson distribution with parameter R t N j . Additionally, X j = (X j,j+1 , X j,j+2 …,X j,j+k+1 ), the vector of cases infected by the N j individuals, follows a multinomial distribution with parameters p, k and X j . Here p is a vector of probabilities that denotes the serial interval distribution. Using these assumptions, the following likelihood is obtained:
where m i~Rt ( P k j~1 p j N i{j ). Parameter estimates are obtained using maximum likelihood methods. For this method we used 6 days as the maximal value of the SI (k), which is consistent with the length of the SI observed in field investigations in South Africa [22] . In addition we implemented a sensitivity analysis to assess the variation of the initial R t estimates vis-à-vis k values of 4 days and 8 days, respectively.
Method 2. We assume a known distribution of the SI in South Africa and we estimate the initial R t using the maximum likelihood estimator for known SI described by [9, 25] . The estimator of initial R t in this case is a modification of Method 1 and is given by:
For this analysis we use the two SI distributions observed from investigations of the first 100 pH1N1 cases in South Africa [22] : (1) the SI distribution between primary cases and laboratoryconfirmed secondary cases only (39%, 24%, 14%, 17%, 3% and 3% for day 1 to 6 respectively), and (2) the SI distribution between primary cases and suspected plus laboratory-confirmed secondary cases (30%, 17%, 20%, 23%, 7% and 3% for day 1 to 6 respectively). We consider suspected secondary cases, individuals that developed ILI symptoms within 14 days from the symptom onset of a confirmed index case within the same household.
Method 3. We make use of the Wallinga and Teunis' method for estimation of R t from the imputed data [26] . This method uses the daily case counts of cases and assumes the serial interval is known. We make the same assumptions for the serial interval as in method 2. The method calculates the relative probability a case on day i infects a case on day j as: Table 1 . Observed lag-time between date of symptom onset and date of specimen collection, incidence rate ratio (IRR) and significance value of the covariates significant in the Poisson regression model. where p k is the probability of a serial interval of length k. Then the estimate for the reproductive number for case i, is:
This method requires that we make use of the entire epidemic curve. We calculate R t as the average of the R i when i is in the epidemic period, as previously defined.
Estimates are reported as the means across the 100 imputations. For all estimates, we calculate bootstrap confidence intervals as has been described previously [9, 26] . We combine the results from all 100 imputations to obtain a confidence interval that incorporates both imputation error, as well as random error [27] .
All analyses were performed using R version 2.14.
12,630 laboratory-confirmed pH1N1 cases were captured by the South African central registry during 2009. The overall demographic, spatial and temporal distribution of these cases has been previously described [23] . Data on date of symptom onset was available for 758 (6%) cases and date of specimen collection for 12,500 (99%) cases. The first case reported illness onset of June 12, 2009 The lag-time between symptom onset and specimen collection was significantly associated with the provincial location of specimen collection, as well as the collection of a specimen on a weekend day (Table 1) . We used these two covariates in the multiple-imputation to predict the date of symptom onset where missing for all cases (Figure 1 ). Other available variables, including date of specimen collection (period during the epidemic), patient age and sex were not significantly associated with the lag-time between symptom onset and specimen collection and, therefore, not included in the final model. Analyses to simultaneously estimate initial R t and serial interval, and estimate initial R t given a known serial interval, were performed over the exponential growth phase of the epidemic from June 21 to August 3, 2009.
Using the likelihood-based method to simultaneously estimate initial R t and the SI across 100 imputations of the dataset (Method 1), we estimated aR R t of 1.47 (95% CI: 1.30-1.72) and a mean SI of 2.78 days (95% CI: 1.80-3.75) (Figure 2 ).R R t estimates ranged from 1.31 (95% CI: 1.21-1.48) to 1.54 (95% CI: 1.37-2.03) when the maximal value of the SI ranged from 4 to 8 days.
We first utilised the SI established from the aforementioned field investigations of the initial 100 cases in estimating R t , as described in method 2. When performing the analysis using the SI distribution observed for laboratory-confirmed pH1N1 secondary cases only (mean 2.3 days, SD 61.3, range 1-5) [22] , we found an initialR R t of 1.43 (95% CI: 1.38-1.49) ( Figure 3A) . When performing the analysis using the SI distribution observed for both confirmed and suspected secondary cases (mean 2.7 days, SD 61.5, range 1-6) [22] , we found an initialR R t of 1.49 (95% CI: 1.44-1.55) ( Figure 3B ). Figure 4 shows the variation inR R t with the progression of the outbreak over time. We observed relatively highR R t values following the introduction of pH1N1 virus into South Africa, corresponding to high rates of transmission and exponential growth of the local epidemic during this period.R R t peaked on the first day of the epidemic growth period (June 21) at 2.91 (95% CI: 0.85-3.99).R R t began to drop from July 27 onward and remained consistently below one after August 22. This corresponds with the decline in the daily incidence of new cases detected. Averaging the R t values obtained during the epidemic growth period (June 21 to August 3, 2009), we estimate initial R t to be 1.42 (95% CI: 1.20-1.71). 
Utilising temporal data on illness onset and specimen collection, and the epidemic curve derived from these data, we provide estimates of the transmissibility parameters of pH1N1 during the first wave experienced in South Africa. Our results focus primarily on the use of analytical techniques to estimate initial R t and SI without incorporating contact tracing or household transmission studies. However, when parameters from field studies are available, we show that these can be incorporated to provide robust estimates of transmission parameters. We found that initial R t estimates established using the likelihood-based method for the simultaneous estimation of R t and SI (method 1: initialR R t : 1.47, SI: 2.78 days) are in agreement with those obtained using SI observed in field investigations [22] (method 2: initialR R t : 1.43 and 1.49 using observed SI for laboratory confirmed or laboratory confirmed and suspected cases respectively). In addition, the mean SI estimate obtained with method 1 (2.78 days) is in agreement with field findings (SI: 2.3-2.7 days using observed SI for laboratory confirmed or laboratory confirmed and suspected cases respectively). Previous estimates of initial R t and the mean SI for pH1N1 have ranged between 1.3-2.9 and 2.5-3.3 days, respectively [2, [6] [7] [8] [9] [10] [11] [14] [15] [16] [17] [18] [19] . Our estimates are consistent with these findings, regardless of the method used for the analysis and despite difference in climate, demography and health systems across these countries. It appears that once established, the transmission characteristics of pH1N1 are very consistent. Differences in transmission rates may occur within smaller subgroups of the overall population; however, this has not been well-studied.
Previous estimates of the epidemiological parameters of seasonal influenza epidemics found a SI = 2-4 days [28] [29] [30] , and a R t a little over 1 with slight variation between climates; R t = 1.03 in Brazil [31] versus R t = 1.1-1.3 in more temperate climates [32] . A number of studies have retrospectively estimated the transmissibility of influenza pandemics. During the 1918 Spanish influenza A(H1N1) pandemic, when assuming a SI = 4 days, R 0 estimates range from 2.0-4.3 in community settings [33, 34] , and even higher values (R 0 = 2.6-10.6) in confined settings such as ships and prisons [34] . A separate analysis predicted a slightly lower SI of 3.3 in community settings and a SI of 3.81 in confined settings during the 1918 pandemic, and subsequently estimated R 0 values of 1.34-3.21 and 4.97 in these respective settings [35] . R 0 estimates from the 1957 Asian influenza A(H2N2) pandemic range from 1.65-1.68 [36, 37] . During the first wave of the 1968-1969 Hong Kong influenza A(H3N2) pandemic, estimates of R 0 range from 1.06-2.06 and increased to 1.21-3.58 during the second wave [38] .
Given our findings, the overall transmissibility of pH1N1 in South African during 2009 was more similar to that of seasonal influenza strains than the 1918 pandemic, and comparable to lower end estimates of the latter pandemics. However, by showing variation in transmissibility with time, we demonstrate that shortly after introduction of pH1N1 into the country, transmission of the virus reached anR R t of 2.9, resulting in exponential growth of the local epidemic and widespread illness. Nonetheless, we show that after a period of less than 2 months of heightened transmission,R R t dropped below 1, corresponding to a decline in the incidence of new cases; likely a result of a combination of herd immunity, There are several limitations in this analysis which merit discussion. First, we assume that all cases are known and reported. It has been shown previously that, if cases are not reported, this may bias estimates generated using this method [39] . If the proportion of cases reported remains consistent over the study, then the estimates of transmissibility will not be biased; however, if the reporting fraction varies through time, then biased estimates of the reproductive number and serial interval may result. Likewise, variation in case ascertainment with time may bias our estimates of the temporal variation of R t . Generally higher reporting rates may be anticipated in the early phase, with reporting fatigue later becoming a factor. Secondly, data for this study are derived from laboratory-based surveillance data from several regions across South Africa; a large and diverse country. Our findings do not incorporate heterogeneities (such as spatial and demographic differences) that likely exist in transmission patterns, or assess the degree to which these impact aggregate measures of initial R t . Methodologies that incorporate heterogeneities inherent in public health data warrant further study.
Despite these limitations, the post-pandemic estimates presented here add to the body of knowledge of pH1N1 transmissibility parameters, which were previously dominated by estimates from developed nations and often based on preliminary data. It remains important that revised parameters, from complete datasets and diverse geographies, are incorporated into planning mitigation strategies for future pandemics. Nonetheless, the methods used in this study would be adaptable to generating real-time estimates during future epidemics. As we continue to build epidemiological capacity in developing nations, including South Africa, we must keep in mind the need for rapid assessments of transmissibility of novel pathogens, in addition to disease severity, to better inform public health interventions. Measuring healthcare preparedness: an all-hazards approach In a paper appearing in this issue, Adini, et al. describe a struggle familiar to many emergency planners—the challenge of planning for all scenarios. The authors contend that all-hazards, or capabilities-based planning, in which a set of core capabilities applicable to numerous types of events is developed, is a more efficient way to achieve general health care system emergency preparedness than scenario-based planning. Essentially, the core of what is necessary to plan for and respond to one kind of disaster (e.g. a biologic event) is also necessary for planning and responding to other types of disasters, allowing for improvements in planning and maximizing efficiencies. While Adini, et al. have advanced the science of health care emergency preparedness through their consideration of 490 measures to assess preparedness, a shorter set of validated preparedness measures would support the dual goals of accountability and improved outcomes and could provide the basis for determining which actions in the name of preparedness really matter. Despite years of planning and billions of dollars spent on disaster preparedness and response activities worldwide, the science of preparedness is in its infancy. The empirical evidence for much of health emergency preparedness is scant. As a result, it is challenging to define what it means to be "fully prepared." Collectively, the world has dealt with numerous disasters, but only a handful of nations have confronted many. This has made it challenging to convincingly link the structures and processes for health preparedness to outcomes, particularly mitigation of morbidity and mortality.
Israel, in part because it has confronted numerous mass casualty events, has been at the forefront of medical preparedness planning, and has developed sophisticated structures and processes for dealing with such medical emergencies. As described by Adini, et al. [1] , Sarpy, et al. [2] , and Einav, et al. [3] , elements of this system include standard operating procedures, drills and exercises for most conceivable events, and measures to inform continuous improvement following each drill, exercise, and actual event. Given the state of the evidence, these authors have relied on a rigorous use of expert opinion to develop their measures; many of the experts involved have had frequent and direct response experience.
In a paper appearing in this issue, Adini et al. [4] describe a struggle familiar to many planners-the challenge of planning for all scenarios. They contend that allhazards, or capabilities-based planning is a more efficient way to achieve general health care system preparedness than scenario-based planning. The authors describe a systematic investigation of the components of their preparedness system that impact hospital preparedness. The paper represents an important advance on several fronts. First, it describes a system of measurement which is consistently applied to assess and improve the preparedness of hospitals. Second, it uses thoughtful analytic methods to answer the question of whether an all-hazards approach is an appropriate methodology for preparedness. Supporting this, the authors found moderate to strong correlations between preparedness measures for various kinds of disasters-including mass casualty, toxicologic, and biological events. In other words, the core of what is necessary to plan for and respond to one kind of disaster (e.g. biologic event) assists with for planning and responding to other types of disasters. This finding allows for improvements and greater efficiencies in planning and time-consuming and expensive drills and exercises; an important consideration given the current fiscal climate in countries worldwide. Third, the authors found that SOPs, training and drills made more of a contribution to overall preparedness than equipment and preparedness knowledge of personnel-an important observation that facilitates effective resource allocation.
The authors' findings are consistent with recent releases of a US Presidential Policy Directive [5] , and related guidance from the US Department of Health and Human Services [6, 7] , which shift the focus of preparedness planning in the US toward an all-hazards, capabilities-based approach.
One challenge faced by most countries, including Israel and the US, is finding a parsimonious set of measures to assess and improve preparedness. While Adini, et al., initially considered 490 measures to assess preparedness, we recognize that countries and systems may not be capable of consistently deploying and analyzing such a large volume of measures. Interestingly, the findings by Adini, et al., support a shift to an all-hazards approach which, enables a reduction in the number of measures. Future work should examine the correlations between measures, and could make use of techniques such as factor analysis to identify a short set of measures, or even scales, that could serve as proxys for a longer, more complex measurement set.
This paper does not address another critical issuemeasuring the preparedness of the public health system. As public health and medical care are so interdependent, we hope that similarly rigorous work regarding the public health system is ongoing.
Adini, et al., have advanced the measurement of health care emergency preparedness. With limited resources, the necessity to find commonalities of approach and efficiency in all we do, including measurement, is critical. In the end, a set of validated preparedness measures would support the dual goals of accountability and improved outcomes and could provide the basis for determining which actions in the name of preparedness really make a difference. Presentation of hemophagocytic lymphohistiocytosis due to a novel MUNC 13–4 mutation masked by partial therapeutic immunosuppression Hemophagocytic lymphohistiocytosis is a potentially fatal disease characterized by excessive macrophage and lymphocyte activity. Patients can be affected following immune activation after an oncologic, autoimmune or infectious trigger. An associated gene mutation may be found which impairs cytolytic lymphocyte function. We describe a pediatric case of hemophagocytic lymphohistiocytosis with a novel mutation of MUNC 13–4 whose diagnosis was confounded by concurrent immunosuppression. Clinical reassessment for hemophagocytic lymphohistiocytosis is necessary in persistently febrile patients with laboratory derangements in the setting of immunosuppressive agent exposure. Hemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome of abnormal immune activation causing excess inflammation. There is increased ectopic migration and proliferation of T cells, tissue infiltration by activated macrophages (histiocytes), hyper-activation of lymphocytes, and prolonged release of pro-inflammatory cytokines [1] . This leads to uncontrolled inflammation manifesting as fever, cytopenias and organ dysfunction. The syndrome is fatal without prompt symptom recognition and treatment.
Primary HLH, also known as familial HLH, is often considered a pediatric disease. Affected individuals have mutations in genes leading to impaired lytic activity of lymphocytes, including NK cells and cytotoxic T lymphocytes [2] . These individuals are at risk of developing HLH. Mutations are inherited in an autosomal recessive pattern, but can also result from de novo mutations, making family history less reliable for diagnosis.
Acquired HLH, also known as secondary HLH, occurs more commonly in adults. Nonetheless, it is also described in children. There are presently no identifiable genetic mutations linked to this phenotype. Currently, secondary HLH is attributed to an abnormal immune response triggered by an infectious, oncologic or autoimmune etiology. As such, other names for secondary HLH include Virus-Associated Hemophagocytic Syndrome and Malignancy-Associated Hemophagocytic Syndrome [3] .
The diagnosis of HLH is based on clinical criteria. The most recent diagnostic guidelines were revised in 2004 [4] . HLH is diagnosed if either a molecular diagnosis consistent with HLH is made, or five of the eight following diagnostic criteria are met: 1.) fever; 2.) splenomegaly; 3.) cytopenias affecting at least two of three lineages in the peripheral blood (haemoglobin <90 g/L, platelets <100 × 10 9 /L, or neutrophils <1 × 10 9 /L); 4.) hypertriglyceridemia (fasting triglycerides ≥ 3 mmol/L (≥ 265 mg/ dL)) and/or hypofibrinogenemia (fibrinogen ≤ 1.5 mg/dL); 5.) hemophagocytosis in bone marrow, spleen, or lymph nodes (excluding signs of malignancy); 6.) low or absent NK-cell activity; 7.) hyperferritinemia (ferritin >500 μg/L); and 8.) high levels of sIL-2R (sIL-2R ≥ 2400 U/ml).
Here we present a pediatric case of HLH with a novel mutation in MUNC 13-4 whose diagnosis of HLH was confounded by low dose treatment with immunosuppressive agents thereby complicating her clinical picture.
Our patient is a 3 year-old female who was previously healthy. Four months prior to hospitalization she began experiencing fatigue, recurrent fevers, progressive muscle weakness, and behavioral changes. The week prior to admission, she had decreased urine output, increased abdominal girth and respiratory distress. She was admitted to a community hospital for possible pneumonia. After 3 days, she was transferred to a tertiary center for oncologic assessment. There, her physical examination was concerning for a tender right axillary lymph node and hepatosplenomegaly. A chest radiograph demonstrated diffuse airspace opacities. PCR studies identified both rhinovirus and mycoplasma in nasal pharyngeal swabs. Twenty-four hours after arrival she developed hypoxemic respiratory failure requiring tracheal intubation and mechanical ventilation. This was complicated by an aspiration event and subsequent cardiopulmonary arrest, requiring resuscitation. Multiple subspecialties convened to facilitate an underlying diagnosis. HLH was considered given her hepatosplenomegaly, cytopenias, elevated CRP and normal ESR in the context of documented infection. Table 1 presents key HLH laboratory markers ordered at onset and their results. MAS, possibly associated with sJIA, was also considered despite the absence of arthritis, because of an intermittent, pink, net-like rash in the preceding weeks. Bone marrow evaluation was performed soon after her admission and demonstrated only rare hemophagocytes, similar to those seen in children with an underlying infection [5] .
Over the next month our patient continued to be dependent upon mechanical ventilation and developed acute respiratory distress syndrome (ARDS). Other complications included respiratory syncitial virus (RSV) infection and central line-related deep vein thrombosis requiring a six-week course of heparin sulfate. A brain MRI showed diffuse patchy white matter lesions most prominent in the right frontal lobe, suggestive of ischemia. Concern arose for adrenal insufficiency and she received several courses of stress dose corticosteroids with intermittent tapers. Intermittent episodes of fever and worsening cytopenias continued over the next month and HLH, possibly MAS, remained a consideration. For this reason an 18-day trial of anakinra was undertaken. However, fevers continued and abnormalities in liver enzymes along with hematologic abnormalities continued.
At this point, genetic testing results became available and identified two mutations in MUNC13-4: a known pathogenic mutation 1389(+1) G > A, a splice donor site of intron 15, and a second previously unidentified mutation, 1847 A > G. This second mutation was located in the splice donor site of exon 20, and the A > G change at this position would likely cause splicing error. It was unclear if this represented a compound heterozygous mutation or if the two variants were on a single chromosome. Parental testing was initiated. Given the genetic results and her persistent symptoms, the laboratory and bone marrow evaluations for HLH were reconsidered. Investigations relevant to HLH before, during and after anakinra use were evaluated (Tables 1 and 2) . When compared to the initial assessment, there was subsequent marked reduction in natural killer (NK) cell function, CD107a upregulation and an elevation in soluble IL-2 (soluble CD25) receptor. Importantly, a drop in ANC ( Figure 2a ) and platelet count (Figure 2b ) was identified during the end of the anakinra trial. Children with active HLH have been noted to have elevated white cell and platelet counts initially that decrease over time [6] , as illustrated in our case. As a confounding feature, however, cytopenia is an uncommon but known side effect of anakinra. It is thus possible that addition of anakinra in our patient, with underlying MUNC 13-4 mutations, magnified the drop in platelet count and ANC that would have been seen with HLH alone. In addition, when ferritin was measured on the 15 th day of anakinra therapy, the level was 629 ng/mL (above the aforementioned acceptable limit of 500 ng/mL), whereas one day after anakinra treatment was terminated, the ferritin level increased to 7129 ng/mL ( Figure 2c ). IL-1 is known to increase the synthesis of ferritin subunits in vitro [7] . We hypothesize, therefore, that anakinra administration inhibited the surge of ferritin that would have otherwise been identified in association with clinical progression of HLH.
A repeat bone marrow biopsy and the first and second bone biopsies were compared ( Figure 2 and Figure 3 ). The first and second bone marrow biopsies were also critically compared (Figure 3) . The latter showing a substantive increase in hematophagocytes compared to the former. In addition, a lumbar puncture showed an elevated protein level (109 mg/dL) with a lymphocytic pleocytosis (12 WBC with 100% lymphocytes). These findings suggested central nervous system HLH involvement. Table 1: Initial laboratory values  Table 2 : Evolution of laboratory values Figure 1 : Illustration of key laboratory value evolution Figure 2 : Bone marrow aspirate; Wright stain postanakinra Figure 3 : CD 163 immunohistochemical stain of bone marrow biopsy
With this collective evidence, the HLH-2004 clinical protocol was initiated [4] . Our patient was treated with dexamethasone and etoposide. Cyclosporine was not initially utilized because of concern of abnormal kidney and liver function, but was added later as she improved.
Parental genetic testing was performed in order to determine if her case was likely primary HLH with familial genetic etiology. Testing confirmed that each parent carried one of the MUNC 13-4 mutations found in the patient. One parent possessed the novel variant identified while the other carried the known splice site mutation thus defining the patient as a compound UNC13D heterozygote consistent with FHL3.
Our patient was hospitalized for almost 4 months prior to transfer to a rehabilitation institution. She received a tracheostomy tube prior to discharge because of her inability to be weaned from respiratory support. She developed mild hypertension which was attributed to cyclosporine. Her liver enzymes continue to decrease although she has a persistently enlarged liver and spleen. She has not had any recent fevers and has an age-appropriate neurologic exam. However, cognitive limitations cannot be predicted. An unrelated HLA-matched donor has been identified and she will be proceeding to bone marrow transplant.
Hemophagocytic lymphohistiocytosis is a potentially fatal disease characterized by excessive macrophage and lymphocyte activity. The onset of HLH in a susceptible individual typically follows either a documented or presumed viral infection. Presently, HLH is often classified into primary (familial) and secondary (acquired) HLH.
The incidence of primary HLH is approximately 1:50,000 live born children [8] . However, HLH incidence varies in clinical studies, most likely due to a difference in prevalence across ethnic groups and/or emerging awareness [9] . Although HLH often presents between birth and 18 months of age, onset in older age groups is possible and has been shown to be a feature of particular gene mutations [10] . Familial HLH has a median survival of less than 2 months after diagnosis if it remains untreated [9] . It is likely that many cases are misdiagnosed as severe fatal infection.
In both primary and secondary HLH, Epstein Barr virus is the most commonly identified inciting infection, although cases associated with cytomegalovirus, human herpes virus 8, influenza, parvovirus, enterovirus and human immunodeficiency virus have also been described [11] . In secondary HLH, associated malignancies include neuroblastoma, Non-Hodgkin's lymphoma and Langerhans' cell histiocytosis. Macrophage activation syndrome (MAS) can be considered a form of secondary HLH syndrome associated with autoimmune disease. MAS complicates an estimated 10% of Systemic Juvenile Idiopathic Arthritis (sJIA) cases [12] , and approximately 1-5% of Systemic Lupus Erythematosus (SLE) cases [13] . Clinical features of MAS similar to primary HLH include high unremitting fever, hepatosplenomegaly, hepatic dysfunction, lymphadenopathy, encephalopathy, cytopenia, elevated ferritin and coagulopathy [12, 14] . Laboratory similarities include depressed natural killer (NK) cell cytotoxic function, elevated soluble IL-2 receptor levels and soluble CD163. In its early stages, MAS can be a diagnostic challenge due to the overlap of symptoms with the underlying autoimmune disease. Factors potentially differentiating HLH from MAS include: 1.) change from quotidian to persistent fever pattern; 2.) sudden change from cytosis to cytopenia; 3.) coagulopathy; and 4.) decreasing ESR. Preliminary diagnostic criteria have been examined for MAS in sJIA, which may help to improve diagnosis of this condition in these patients [13, 15] .
As mentioned before, HLH is diagnosed through clear clinical criteria. Despite awareness of the diagnostic criteria, HLH diagnosis can be challenging because of the variability in presentation. Symptoms, especially if infection associated, may spontaneously remit [3] . Individual diagnostic criteria can be observed at distinct points in the disease course and can also remit and recur. Moreover, many of the criteria are non-specific for HLH, making it a diagnosis of exclusion in many cases. Finally, criteria such as NK cell function and genetic testing can take weeks to be finalized. Given all these challenges, it is important to consider HLH as a diagnosis both at onset and in the early stages of the disease process. In a review of familial and acquired HLH, key laboratory findings in establishing a diagnosis were identified as negative or decreased NK function as well as elevated soluble CD25 levels in all patients [16] , suggesting these studies must be evaluated early on in the clinical course. In some centers, CD107a upregulation, a marker of NK cell degranulation is used as a surrogate for NK cell cytotoxicity [17] . However, as depressed NK cell cytotoxicity is seen in many conditions secondary to inflammation or immunosuppressive factors, other symptoms must be considered. Fever and splenomegaly occur in approximately 70% of HLH patients [16] . Fevers may be protracted, variable and may even resolve spontaneously. About 50% of patients initially present with elevated triglycerides, high ferritin, high LDH and/or a combination of anemia, neutropenia and thrombocytopenia [8, 16] . Of note, ferritin is rarely > 200 μg/L in pediatric patients with infections outside of the context of HLH [16] and levels higher than this should raise suspicion for HLH. Lastly, hemophagocytosis is seen in less than 40% of patients at onset but is present in >80% of patients at time of diagnosis [16] .
There are presently five types of familial HLH (FHL) [9] , all of which impair the lytic activity of cytotoxic lymphocytes. Specifically, cytotoxic lymphocytes mediate contact-dependent elimination of cells perceived as dangerous by secreting preformed destructive molecules contained within specialized organelles termed lytic granules [2] . In order to mediate cytotoxicity, lytic granules must contain appropriate effector molecules and be localized to the contact site with the cell targeted for destruction. Once localized to that intercellular interface, the lytic granules dock at the cell membrane. The membrane of the granule and the cytotoxic lymphocyte are fused, allowing the release of the lytic effector molecules onto the targeted cell.
Familial HLH type 1 (FHL1) is linked to chromosome 9q21, but the exact gene remains unknown [9] . Perforin (PRF-1) was the first identified FHL gene (located on chromosome 10q21-22), and is responsible for familial HLH type 2 (FHL2).
Perforin protein is an effector molecule found in lytic granules. Once secreted, perforin inserts into the membrane of the target cell and facilitates the uptake of granzyme B and other cytolytic molecules contained in the lytic granules, into the target cell to induce cell death [1, 9] .
In perforin-deficient mice infected with high doses of lymphocytic choriomeningitis virus (LMCV), haemophagocytic lymphohistiocytosis could be induced, similar to human FHL [18] . This suggests that failure to clear virus will lead to persistence of viral antigens and prolonged CD8 T cell activation and cytokine production. Other animal model studies demonstrate perforin's role in regulating lymphocyte number in autoimmunity [19] , after microbial infection [20] and when other cell-death pathways are impaired [21] . The extent that these mechanisms are involved in the control of immune responses, however, is still speculative.
PRF-1 mutations account for approximately 20-40% of FHL cases. It is important to note however that CD107a up-regulation is usually normal in FHL2 as CD107a represents a measure of degranulation [22] .
FHL3 is caused by mutations in the UNC13D gene located on chromosome 17q25. UNC13D encodes the protein MUNC 13-4. Found on the surface of lytic granules, MUNC 13-4 is required for priming the lytic granules for docking at the cytotoxic cell membrane [9, 12] . FHL 4 is caused by mutations in STX11 on chromosome 6q24 that encodes the syntaxin 11 protein. Syntaxin-11 is a member of the SNARE protein family and facilitates the fusion of the lytic granule membrane with that of the cytotoxic lymphocyte [1, 23] . FHL5 is caused by mutations in MUNC 18-2 located on chromosome 19p13. MUNC 18-2 encodes the syntaxin binding protein 2. It is a partner of syntaxin 11 and is required for SNARE complex-mediated fusion of the lytic granule with the cytolytic cell membrane [9, 22] .
In addition to genetic defects associated with FHL, there are also immunodeficiency syndromes associated with HLH that impair the secretion of lytic granule contents. These include Griscelli syndrome type 2, Hermansky Pudlak type II, Chediak Higashi and X-linked lymphoproliferative disease Type 1. Griscelli syndrome type 2 and The orange triangles indicate the first and last day that daily anakinra was given. In Figure 2a and 2b the red line denotes the threshold below which diagnostic criteria is met for HLH while in Figure 2c this line denotes the threshold above which HLH criteria is met.
Chediak Higashi syndrome are typically associated with albinism due to an effect upon melanocyte pigment secretion. Griscelli syndrome type 2 is identified by mutations in RAB27A while Chediak Higashi syndrome is associated with LYST mutations. Mutations of both these gene impair proteins important for formation and/or trafficking of secretory lysosomes [24] . In these syndromes, a gene defect interferes with lytic granules reaching the cytotoxic lymphocyte membrane, thus leading to impaired NK cell cytotoxicity. Patients with X-linked lymphoproliferative disease have difficulty clearing Epstein-Barr virus (EBV) infected B-cells, with subsequent extensive lymphocytic expansion into multiple organs. The SH2D1A mutation, which encodes for the signaling lymphocyte activation molecule (SLAM)-associated protein SAP, is identified in XLP1. Impaired cytolytic function in XLP1 is thought to cause accumulation EBV-infected B cells and persistence of reactive inflammatory cells, which combine to produce an exaggerated immune response [25] . Treatment for HLH is described in the HLH 2004 revised guidelines [14] and is divided into acute and long-term management. Initial treatment with an immunomodulatory regimen is recommended. Patients with primary HLH who fail to reach disease resolution within 8 weeks of treatment should continue on this regimen for an additional treatment course. Hematopoetic stem cell transplantation should be pursued as soon as a suitable donor is available for all patients with primary HLH, relapsed HLH, or those failing to progress on therapy [4] . There is no standard for the treatment of secondary HLH or MAS in the context of rheumatologic disease. High dose corticosteroids, biologic agents, cyclosporine and high dose intravenous immunoglobulin have been used with varying success. Anakinra, a recombinant IL-1 receptor antagonist, has been increasingly used for sJIA patients with MAS [24] . If initial treatment fails, HLH salvage therapy may be pursued according to the HLH 2004 chemotherapeutic regimens [14, 12] . Confirmation of a genetic mutation is not needed for immediate management but is important in differentiating familial HLH and secondary HLH for long-term patient management and family genetic counseling.
While anakinra has been successfully utilized in MAS, it has not been formally studied in HLH associated with bonafide mutations. In our patient with two identified UNC13D mutations, anakinra was utilized as an immunomodulator while the diagnosis was evolving. Although the progression of our patient's disease was not florid while receiving anakinra, there were decreased platelet and neutrophil counts during therapy. However, when anakinra was stopped, there was a clear surge in ferritin levels. It is unclear from our experience if anakinra magnifies certain abnormalities found in primary HLH while moderately quelling others. A more likely explanation is that the anakinra functioned in partially blocking the HLH-associated inflammation in our patient thus allowing for the masking of certain phenotypic associations of HLH such as ferritin, but not others (i.e., platelet counts). While we were not situated to immunologically prove the partial blockade of inflammation in our patient while receiving anakinra, our experience suggests that its use be reserved for the more mild secondary forms of Figure 3 Comparison of CD163 immunohistochemical stain of bone marrow one month before trial of anakinra trial (3a), and the day after discontinuation (3b). Shown at 40x magnification. CD163 is an immunohistochemical stain that is specific for monocytes and macrophages and the positive cells show brown surface staining. Prior to anakinra, there are slit-like macrophages and some with a few ingested cells (blue arrow). On repeat biopsy and stain done after anakinra, there is an increase in the number of macrophages and the CD163 stain demonstrates numerous hemophagocytic macrophages containing multiple ingested cells (blue arrows).
HLH and that therapy of primary HLH be limited to more wide-ranging immunosuppression such as that provided through the HLH-2004 protocol [4, 14] .
HLH is a clinical syndrome that remains difficult to diagnose. Our patient's case demonstrates that use of immunosuppressive agents can cloud diagnosis. As clinicians, it is important to be aware of this in order to avoid delay in diagnosis and life-saving therapy.
"Written informed consent was obtained from the parent of the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal."  Relative cost and outcomes in the intensive care unit of acute lung injury (ALI) due to pandemic influenza compared with other etiologies: a single-center study BACKGROUND: Critical illness due to 2009 H1N1 influenza has been characterized by respiratory complications, including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), and associated with high mortality. We studied the severity, outcomes, and hospital charges of patients with ALI/ARDS secondary to pandemic influenza A infection compared with ALI and ARDS from other etiologies. METHODS: A retrospective review was conducted that included patients admitted to the Cleveland Clinic MICU with ALI/ARDS and confirmed influenza A infection, and all patients admitted with ALI/ARDS from any other etiology from September 2009 to March 2010. An itemized list of individual hospital charges was obtained for each patient from the hospital billing office and organized by billing code into a database. Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student’s t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient subgroups. Data that were not normally distributed were compared with the Wilcoxon rank-sum test. RESULTS: Forty-five patients were studied: 23 in the H1N1 group and 22 in the noninfluenza group. Mean ± SD age was similar (44 ± 13 and 51 ± 17 years, respectively, p = 0.15). H1N1 patients had lower APACHE III scores (66 ± 20 vs. 89 ± 32, p = 0.015) and had higher Pplat and PEEP on days 1, 3, and 14. Hospital and ICU length of stay and duration of mechanical ventilation were comparable. SOFA scores over the first 2 weeks in the ICU indicate more severe organ failure in the noninfluenza group (p = 0.017). Hospital mortality was significantly higher in the noninfluenza group (77 vs. 39%, p = 0.016). The noninfluenza group tended to have higher overall charges, including significantly higher cost of blood products in the ICU. CONCLUSIONS: ALI/ARDS secondary to pandemic influenza infection is associated with more severe respiratory compromise but has lower overall acuity and better survival rates than ALI/ARDS due to other causes. Higher absolute charges in the noninfluenza group are likely due to underlying comorbid medical conditions. The spread of a novel H1N1 strain of the Influenza A virus represents the first pandemic of the 21 st century and the first influenza pandemic since 1968 [1] . Compared with seasonal influenza, this strain was more prevalent in younger-aged individuals, obese patients, and pregnant women [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . Severe cases of pandemic H1N1 resulted in respiratory failure thought to be secondary to direct cell damage and systemic cytokine release resulting in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) requiring prolonged ventilatory assistance and the frequent use of rescue therapies [4, 5, 8, [13] [14] [15] [16] [17] .
Limited data exist that compare the clinical differences between ALI in H1N1 patients and ALI arising from other etiologies. Furthermore, whereas a number of studies have assessed different aspects of the economic impact of the recent pandemic [18] [19] [20] [21] , few have focused on the health care cost of the pandemic, particularly the utilization of limited ICU resources.
We report the severity, clinical outcomes, and hospital charges of ALI/ARDS secondary to pandemic influenza A infection compared with ALI/ARDS from other etiologies during a similar period of time. Based on clinical bedside observations and published reports [4, 5, 8] , we hypothesize that ALI/ARDS secondary to pandemic influenza is associated with similar ICU outcomes but increased resource utilization and higher hospital charges due to the frequent need for rescue interventions and prolonged ventilatory assistance.
The study was approved by the Human Investigation Committee of the Cleveland Clinic Foundation (CCF) (Institutional Review Board approval # 10-229) as a retrospective, single-center study at the CCF Medical ICU.
Patients were identified from a unit-based acute lung injury screening database (Cleveland Clinic is one of the centers participating in the ARDSnetwork) and the H1N1 patient log maintained during the fall-winter season of 2009-2010. Patients were included if they met criteria for ALI (PaO 2 /FiO 2 ≤300; acute bilateral infiltrates; positive pressure ventilation via endotracheal tube; and no clinical evidence of left atrial hypertension or congestive heart failure) between the months of September 2009 to March 2010-the time that influenza infection was most prevalent. Diagnostic methods for influenza A virus detection consisted of rapid antigen testing, polymerase chain reaction (rtPCR), and viral culture from nasopharyngeal swabs, tracheal aspirates, and bronchioalveolar lavage specimens. The patients were grouped into two categories: those with laboratoryproven H1N1 infection; and those in whom H1N1was not clinically suspected. Only patients with confirmed infection were included in the influenza group to ensure that the clinical course of the disease was accurately captured. Patients were excluded from the study if they did not meet the above criteria for ARDS, or if clinical suspicion pointed to a likely pandemic viral infection with negative diagnostics.
A Research Electronic Data Capture (REDCap) database was constructed with a complete listing of the patient's demographic and clinical information, including age, gender, height, weight, body mass index (BMI), presenting symptoms, past medical history, primary reason for admission to the ICU, vital signs, presence of vasopressors, laboratory values, ventilator settings and respiratory parameters, Acute Physiology and Chronic Health Evaluation (APACHE) III and Sequential Organ Failure Assessment (SOFA) scores on admission to the MICU, number of intubated days, duration of ICU and hospital stay, mortality, and rescue therapies (namely inhaled nitric oxide, proning, high-frequency oscillatory ventilation, and extracorporeal membrane oxygenation [ECMO]) [22] . The data collection was de-identified and collected in accordance with HIPAA guidelines.
As part of the routine MICU respiratory therapy protocol, mechanical ventilation parameters are recorded every 4 hours. All patients are managed according to a mechanical ventilation protocol that incorporates the use of nonconventional modes when a lung protective strategy on conventional modes failed to provide adequate oxygenation. The following criteria were used to define the analyzed parameters: 1) mode of ventilation: the mode of ventilation that was used for the longest time for a given day; 2) PaO 2 /FiO 2 : worst daily ratios were recorded; 3) plateau pressure (Pplat): for patients on volume control ventilation the airway pressure was measured after a 5-second inspiratory hold without concomitant active inspiratory efforts, and for patients on pressure control ventilation (PCV) the highest total system pressure (PEEP + inspiratory pressure) was recorded; 4) positive end expiratory pressure (PEEP): the value corresponding to the highest PEEP for the day was recorded; 5) tidal volume (Vt): the largest daily volume was recorded. Respiratory data were captured on the first day of intubation (day 1) and then on subsequent days 3, 7, and 14 of mechanical ventilation. There were no differences in ventilator protocols or management between the two groups.
An itemized bill of individual charges for each patient was obtained from the hospital billing office and was organized by billing code into the following categories: room/board, pharmacy, supplies, laboratory, radiology, surgical (including procedures performed under general anesthesia), blood products, respiratory services, dialysis, and miscellaneous (which included some professional fees, nonsurgical procedures and phlebotomy, and diagnostics not included in the other categories, such as electroencephalograms, electrocardiograms, echocardiograms, cardiac catheterizations, and vascular studies). The values represent the hospital charges for the aforementioned services rather than the actual reimbursement, which may be subject to more variability. The single-center nature of the study removes interfacility differences in clinical and billing practices.
Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student's t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient groups. In instances where the data were not normally distributed, the groups were compared with the Wilcoxon rank-sum test. Differences were considered statistically significant if the p value was <0.05.
Fifty-one patients were identified in the acute lung injury screening database between September 2009 and March 2010. Twenty-two met criteria for ALI and did not have confirmed or suspected H1N1 infection and were thus included in the noninfluenza group (ALI/ARDS secondary to noninfluenza etiologies). Thirty-six patients in the H1N1 patient log had confirmed influenza A testing. Of those, 23 had ALI requiring mechanical ventilation (MV) during their MICU stay and were included in our analysis.
Demographics, presenting symptoms, past medical history, and acuity on admission are shown in Table 1 . Patients in the influenza group tended to be younger with a higher BMI. Patients in the influenza group presented more often with lower respiratory infection (100 vs. 73%, p = 0.135) and had increased requirement for mechanical ventilation on admission to the ICU (96 vs. 68%, p = 0.022). On the other hand, the noninfluenza group had a higher propensity to present with shock requiring vasopressors (45 vs. 22%, respectively, p = 0.07). The primary cause of ALI in the H1N1 group was pneumonia (n = 23), whereas in the noninfluenza group the etiologies were more varied, including pneumonia (n = 9), sepsis (n = 5), aspiration of gastric contents (n = 2), transfusion reaction (n = 1), and other (n = 5). Whereas seven patients (30%) in the H1N1 group were considered healthy, only one patient (5%) in the noninfluenza group had no comorbid medical conditions on admission to the ICU (Table 1) . This difference is reflected in the lower mean APACHE III score on admission to the ICU in the H1N1 group (66 ± 20 vs. 89 ± 32, p = 0.015), despite similar SOFA scores (8.3 ± 3.4 and 9.2 ± 4.1, p = 0.44).
There were no statistically significant differences between the two groups for initial laboratory data, including white blood cell count, platelets, serum creatinine, bilirubin, and creatinine kinase. The number of patients who developed acute renal failure that required dialysis throughout their ICU stay was the same (n = 8) in both groups. SOFA scores on days 1, 3, 7, and 14 of mechanical ventilation indicate that patients in the noninfluenza group had more severe organ failure during their ICU stay (p = 0.017; Table 2 ). Table 3 shows oxygenation index and mechanical ventilation related parameters on days 1, 3, 7, and 14. There was a nonsignificant trend toward worsening hypoxia in the H1N1 group, despite significantly higher PEEP and Pplat on days 1, 3, and 14. Tidal volumes were comparable throughout. Plateau pressures in the H1N1 group were high due to the relative decrease in pulmonary compliance in H1N1-related lung injury. Four patients in both groups were ventilated with airway pressure release ventilation (APRV). More patients in the influenza group required rescue therapies on day 1 of mechanical ventilation (4 vs. 0, respectively, p = 0.108); however, similar numbers of patients in both groups required rescue therapies over the duration of MV (7 and 5 patients, respectively). Rescue therapies in the H1N1 group included inhaled NO (n = 4), ECMO (n = 2), prone ventilation (n = 3), and high-frequency ventilation (n = 1), and in the noninfluenza group only inhaled NO (n = 3) and prone ventilation (n = 2).
Mechanical ventilation days were comparable between groups (22 ± 17 vs. 19 ± 15 days for groups I and II, respectively, p = 0.53) as were 28-day ventilator-free days (5 ± 7.6 and 4.6 ± 9, p = 0.88). Four patients in the H1N1 group and seven in the noninfluenza group underwent a tracheostomy procedure. Hospital and ICU LOS were comparable (median ± IQR: 16 ± 22 vs. 24.5 ± 26.5 and 12 ± 15 vs. 17 ± 25.5 days for the influenza group and II, respectively, Wilcoxon p = 0.17 and 0.45). Mortality was significantly higher for patients in the noninfluenza group (77 vs. 39%, p = 0.016). Interestingly, a Kaplan-Meier curve of ICU mortality (Figure 1) indicates that patients in the H1N1 group were more likely to be discharged alive from the ICU when the length of stay was greater than 25 days, despite a trend toward higher mortality within the first 2 weeks.
Even though all charges were higher in the noninfluenza group, only the difference in blood products utilized in the ICU was significant (4 ± 6 vs. 21 ± 25 thousands of U.S. dollars, Wilcoxon p < 0.001; Table 4 ). Differences in ICU charges in pharmacy (p = 0.23), supplies (p = 0.09), radiology (p = 0.08), and miscellaneous (p = 0.09) were large but not significant due to considerable variation. The proportion of charges in each of the major categories was similar between the groups (Figure 2 ). The average total ICU cost per patient (253 ± 193 vs. 350 ± 270 thousands of U.S. dollars, Wilcoxon p = 0.19) and the average ICU cost per patient per day (13 ± 4 vs. 15 ± 6 thousands of U.S. dollars, Wilcoxon p = 0.06) tended to be higher in the noninfluenza group.
The fall of 2009 heralded the influx of patients suffering from severe hypoxic respiratory complications secondary to the pandemic H1N1 influenza to ICUs across the country. Due to the severity of pulmonary disease that many of these patients experienced, perception among treating clinicians was that these patients would have 
a All values expressed as mean ± SD. Using mixed models, the overall p value comparing the influenza and noninfluenza groups is 0.017. The trend over time was not significant (p = 0.1). worse outcomes and consume more resources, as measured by hospital charges, than patients who developed ALI from other etiologies. We demonstrated that, contrary to what was perceived, pandemic influenza A ALI/ ARDS was associated with a lower acuity and, consequently, lower hospital mortality that ALI/ARDS from other etiologies, and had a similar ICU and hospital LOS. ICU and total hospital charges reflected a trend toward higher overall charges for room and board, blood products, pharmacy, and overall charge per patient in the noninfluenza group.
In accordance with other descriptive reports of pandemic influenza [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] , patients who tested positive for H1N1 infection, tended to be young (no patients >64 years old), obese (15 had BMI >30 kg/m 2 ), and in relatively good health (30% with no comorbid medical conditions). There were no pregnant patients in either group. Compared with other studies of pandemic influenza patients who required mechanical ventilation, SOFA scores (mean 8.3) were similar, although APACHE II (25 ± 9) scores were higher [5] [6] [7] [8] 14, 16, 17, 23] . The degree of respiratory compromise in our patients was more severe than other reports judging by the higher PEEP requirements and longer duration of mechanical ventilation, which was roughly double that reported in other studies [4] [5] [6] 8, 11, 13, 14, 16, 23] . Plateau pressures in these studies were not consistently reported. However, despite significantly longer ventilation duration and prolonged ICU and hospital stays, the mortality in our cohort was not higher than that seen in other studies, which ranged from 22-41% in patients who required mechanical ventilation [4] [5] [6] 8, 11, 13, 14, 16, 23] .
Looking at the different patient characteristics between groups, it may be tempting to postulate that the higher rate of patients with pulmonary ARDS in the H1N1 group, in contrast to prevalent nonpulmonary ARDS in the noninfluenza group, would correlate with a higher PEEP response among the latter [24] . Our findings suggest the contrary. Patients in the H1N1 group had higher mean plateau pressure, likely indicative of lower compliance. The similarity of PaO 2 /FiO 2 ratios in the two groups may be a reflection of higher PEEP values used in the H1N1 group for lung recruitment, rather than being indicative of comparable degrees of lung injury. Although assessing recruitability from this retrospective analysis is difficult and may be inaccurate, the higher PEEP used and the implication of lower compliance observed are predictors of potentially recruitable lung [24] . These observations support the recent call for a reevaluation of the ALI and ARDS criteria to account for this heterogeneity in the patient population [25] .
A number of important differences between the two cohorts emerged as well. As expected, the noninfluenza group was older, had more comorbid medical conditions, and less often presented to the ICU with respiratory failure. The degree of ventilator support was significantly higher in the H1N1 group on days 1, 3, and 14, and there was a trend to more severe hypoxemia during that time as well. Nevertheless, the use of use of APRV and rescue therapies was comparable in both groups. Despite more severe respiratory compromise, H1N1 patients did not have longer time on the ventilator, longer ICU or hospital stays, or higher mortality. Although SOFA scores were similar, the noninfluenza group had significantly higher APACHE III scores, likely secondary to points assigned to comorbid medical conditions. The high acuity of illness, as well as the presence of severe comorbidities, such as solid and hematologic oncologic conditions (7 patients), chronic renal insufficiency (6 patients), and cirrhosis of the liver (4 patients), likely contributed to the poor outcomes in the noninfluenza group. Conversely, despite more severe respiratory compromise, patients in the H1N1 group were more likely to recover due to their younger age and better overall health histories.
The 77% mortality in the noninfluenza group was much higher than typically reported in clinical trials, with one notable exception [26] . However, reports from tertiary care centers involving patient cohorts with similar underlying comorbid conditions have reported equally high mortality rates [27] . Our observation brings up an interesting point, namely the difference between the reported mortality in clinical trials and the observed mortality in a similar clinical condition affecting patients that would have been excluded from such trials due to coexisting comorbidities. A Kaplan-Meier plot of ICU mortality (Figure 1) indicates that although patients in the H1N1 group were less likely to survive the first 14 days of ICU care, those that did survive past day 25 were more likely to be discharged alive from the hospital. Patients in the noninfluenza group were unlikely to survive if their ICU length of stay exceeded 3 weeks. ARDS is among the most expensive conditions encountered in the ICU [28] . In 1984, Bellamy and Oye described the charges of patients with ARDS, with the most expensive being room and board (30%), clinical laboratory (24%), pharmacy (14%), and inhalation therapy and ventilation (8%) [27] . Twenty-five years later, our study indicates that the aforementioned categories continue to represent the most expensive charges incurred by ARDS patients in the ICU.
The overall similarity of charges in room and board and respiratory therapy between the two groups is likely indicative of the comparative durations of hospitalization and mechanical ventilation. Interestingly, despite higher ventilatory requirements and more severe hypoxemia in the H1N1 group, respiratory charges were similar between the two groups, suggesting that the high cost of maintaining a patient on mechanical ventilation is independent of the degree of ventilator support necessary. Thus, respiratory charges are more likely a reflection of duration of mechanical ventilation rather than the degree of ventilator support necessary. Absolute ICU charges for room and board, blood products, pharmacy, radiology, average daily charge, and overall charge per patient were larger in the noninfluenza group. ICU charges for blood products in the noninfluenza group were greater by a factor of four, and pharmacy charges double that of the H1N1 group. This finding is likely a reflection of the higher prevalence of underlying comorbid medical conditions in the noninfluenza group, such as malignancy and cirrhosis, which require expensive medications and predispose to anemia. Moreover, the high mortality in this cohort likely precluded even higher hospital charges. Nevertheless, the H1N1 cohort amassed charges of similar magnitude to the most ill and expensive patients in the ICU, indicating the abundant health care resources consumed by severe pandemic influenza infection.
There are a number of limitations to our study. As a retrospective chart review rather than a prospective investigation, the information was culled from sources that were at times incomplete. Second, the study contained a relatively small number of patients, and measures taken to ensure internal validity of each group, such as limiting the influenza group to confirmed H1N1 infection and the noninfluenza group to the duration of the influenza season, further limited its size. Additionally, whereas our study provides descriptive information relevant to the patient population of our institution and tertiary referral centers with similar acuity, other ICUs may be exposed to a different cohort of patients. On the other hand, as a single-center study, potential differences in clinical and billing practices could be minimized. Although a comprehensive charge profile of each patient was generated, trends in the timing of charges could not be obtained. Finally, the hospital charge data were mined from an extensive database divided by charge coding, and therefore, some charges may have been mislabeled or inappropriately categorized.
Our study provides interesting observations about the clinical course, outcomes, and cost of the H1N1 influenza pandemic. Although patients with severe pulmonary complications of pandemic influenza infection have poor oxygenation and require significant ventilatory support and rescue therapies, their younger age and tendency to have fewer comorbid medical conditions contribute to their improved prognosis compared with patients with ALI from other causes. Both groups of patients consume enormous amounts of hospital resources, and physicians and policy makers must be aware of this when future pandemics arise. Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. Reticuloendotheliosis viruses (REVs) are a group of viruses in the family Retroviridae, specifically gammaretroviruses in the same genus as mammalian C-type retroviruses [1] . The REV group includes defective REV-T [2, 3] , non-defective REV-A [4, 5] , chick syncytial virus (CSV) [6] , duck infectious anemia virus [7] , and spleen necrosis virus (SNV) [8] . Except for the defective REV-T, all isolated REV strains belong to a single serotype [5] and their genetic sequences show little variation [9] .
REV genome consists of three structural genes (gag, pol and env) flanked by long-terminal repeats (LTRs) [10] . The major mature env gene products of REVs are the surface glycoprotein (gp90) and the transmembrane protein (gp20) [11, 12] . The gp90 protein containing both continuous and discontinuous epitopes functions as the immunodominant protein [13] and is responsible for eliciting REV antibodies. Previous studies indicated that the Cterminal epitope of gp90 was exposed on the outer surface of the REV-A-infected cell [12] . However, the epitope identified in REV gp90 protein has not been finely mapped, and the core sequence of the epitope needs to be determined.
Detailed analysis of epitopes is important for the understanding of immunological events, and the development of epitopebased marker vaccines and diagnostic tools for various diseases [14, 15] . In this study, we prepared a neutralizing monoclonal antibody (mAb) against gp90 protein from the REV-A strain HLJ07I, and used it to screen a phage-displayed random 12mer peptide library for the linear B-cell epitope. This study describes the first identification of the precise location of the epitope on gp90 protein. The information provided in this study will facilitate the development of specific serological diagnosis of REV infection, and will contribute to the rational design of vaccines by further understanding of the antigenic structure of gp90.
Care of laboratory animals and animal experimentation were performed in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032).
REV-A Strain HLJ07I (GenBank accession No. GQ375848) was isolated from Heilongjiang Province in China in 2007. Chicken embryo fibroblasts (CEFs) were prepared as primary cultures from 10-day-old chicken embryos as previously described [16] and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum plus antibiotics. Viruses were grown in CEFs and incubated at 37uC with 5% CO 2 for 5 days. The suspension was frozen and thawed three times to disrupt cells and release virus, and then clarified by two centrifugation steps (2000 g for 15 min, and 10,000 g for 60 min). Virus present in the upper phase was precipitated with 10% (w/v) polyethylene glycol 6000 (PEG 6000) for 4 hours at 4uC. Precipitates were collected by centrifugation at 9,000 g for 30 minutes and resuspended in TNE buffer (50 mM tris-HC1, pH 7.5; 0.1 M NaC1, 10 mM EDTA). Finally, they were centrifuged through a 30% (w/v) sucrose cushion for 90 minutes at 200,000 g and resuspended in TNE buffer. The purified virus was analyzed in SDS-PAGE.
Six-week-old female BALB/c mice were subcutaneously immunized with 100 mg of the purified recombinant gp90 protein emulsified with an equal volume of Freund's complete adjuvant (Sigma, St. Louis, MO, USA). Two boosters of the Freund's incomplete adjuvant (Sigma, St. Louis, MO, USA) emulsified antigen were given at two week interval. Two weeks after the third immunization, the mice were intraperitoneally boosted with 100 mg antigen alone. Three days later, the spleen cells from immunized mice were fused with myeloma cells SP2/0 (SP2/0-Agl4; ATCC CRL 1581) [17] , using 50% (wt/vol) polyethylene glycol and 10% dimethyl sulfoxide (DMSO) (vol/vol) (Sigma, St Louis, MO, USA). Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA). The hybridomas producing mAbs were cloned three times by limiting dilution of the cells. Antibody subtype identification was performed using SBA Clonotyping TM System/HRP Kit (Southern Biotech, Birmingham, AL, USA).
Plates were coated with 100 mL/well of purified REV gp90 antigen diluted in carbonate-bicarbonate buffer (pH 9.6) for incubation overnight at 4uC. Following 4 washes with 200 mL/ well of PBS/0.05% Tween-20, the plates were blocked with 200 mL/well of blocking buffer (PBS containing 5% skim milk) for 1 h at 37uC. The supernatant of hybridoma culture (100 mL/well) was added in duplicate and the plates were incubated for 1 h at 37uC. After washing three times with PBS, 100 mL of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG, 1:5,000 dilution,Sigma, St Louis, MO, USA) was added to each well and incubated for 1 h at 37uC. Plates were washed three times with PBS and incubated with 100 mL/well of o-phenylenediamine dihydrochloride (OPD, Sigma, St Louis, MO, USA) containing 0.3% H 2 O 2 for 5 minutes at room temperature in the dark. The reaction was stopped with 50 mL/well of 2 M H 2 SO 4 and the absorbance measured at 492 nm.
About 70-80% confluent CEF cells in 96-well plates were infected with REV-A HLJ07I at a MOI of 0.2. At 5 days postinfection, the infected cells were fixed with icy cold ethanol absolute for 15 min at 4uC, and air dried. The fixed cells were incubated with mAb A9E8, REV-A-positive chicken serum, antiporcine IFN-c mAb (Sigma, St Louis, MO, USA), or REV-Anegative chicken serum for 1 h at 37uC. After washing three times with PBS, 50 mL/well of FITC-conjugated goat anti-mouse IgG or FITC-conjugated rabbit anti-chicken IgG (Sigma, St Louis, MO, USA) at 1:100 dilutions were added and incubated for 1 h at 37uC. The cells were rinsed three times with PBS and once with deionized water, and mounted in 50 mL of 90% glycerol in PBS, and then observed under the Nikon Eclipse Ti-E microscope equipped with NIS-Elements AR software.
The micro-neutralization assay was modified from a previously described procedure [18] . The ascitic fluid was heat inactivated for 30 min at 56uC, and two fold serial dilutions were incubated with 2610 3 tissue culture infective doses 50% (TCID 50 /mL ) of REV-A in a 96-well micro-plate. Four uninfected control wells were included on each plate as control wells. After 2 h incubation at 4uC, 100 mL of CEF cells at 1.5610 5 cells/mL was added to each well. The plates were incubated for 5 days at 37uC and 5% CO 2 . The monolayers were washed with PBS and fixed in icy cold ethanol for 15 minutes. The presence of viral gp90 protein was detected by ELISA with the mAb A9E8. The absorbance was measured at 492 nm with an ELISA microplate reader. The average A492 was determined for quadruplicate wells of virusinfected and uninfected control wells, and a neutralizing endpoint was determined by using a 50% specific signal calculation. The endpoint titer was expressed as the reciprocal of the highest dilution of ascitic fluid with A492 value less than X, where 6= [(average A492 of infected wells) 2 (average A492 of control wells)]/2+ (average A492 of control wells).
The Ph.D.-12 TM Phage Display Peptide Library Kit was purchased from New England BioLabs Inc. The dodecapeptide library consisted of 2.7610 9 electroporated sequences (1.5610 13 pfu/mL). The mAb was purified from the ascites uid of mice inoculated with the hybridma cells secreting A9E8 by affinity chromatography using rProtein G Agorose (Invitrogen, Carlsbad, CA,USA) according to the manufacturer's instructions. The concentration of the purified protein was determined using the Bradford Protein Assay Kit (Beyotime, Shanghai, China). Three successive rounds of biopanning were carried out according to the manufacturer's instruction manual. Briey, one well of a 96well microtiter plate was coated with 10 mg/mL of mAb A9E8 in coating buffer (0.1 M NaHCO 3 , pH 8.6) overnight at 4uC, followed by blocking with blocking buffer (0.1 M NaHCO 3 , pH 8.6, 0.02% NaN3, and 5 mg/ml BSA) for 2 h at 4uC. The phage library (1.5610 11 phages/100 mL) was added to the blocked wells and the plate incubated for 1 h at room temperature. The unbound phages were removed by successive washings with TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing gradually increased concentrations (0.1%, 0.3%, and 0.5%) of Tween-20, and the bound phages were eluted by 0.2 M glycine-HCl containing 1 mg/mL BSA (pH 2.2) and immediately neutralized with 1 M Tris-HCl (pH 9.1). The eluted phages were amplified by infecting E. coli (ER2738), and were titered on LB/IPTG/Xgal plates for the subsequent rounds of selection. The output to input ratio was calculated as follows: (titer of the amplified eluent phages/titer of the input phages (1.5610 11 ))6100%.
After three rounds of biopanning, eight individual phage clones were selected for target binding in ELISA as described in the manufacturer's instructions. Briey, 96-well plates were coated with 100 ng of purified mAb A9E8, or anti-porcine IFN-c mAb (Sigma, St Louis, MO, USA) as negative controls overnight at 4uC. The coated wells were blocked for 2 h at room temperature and then the phages (10 10 pfu/100 mL/well) diluted in blocking solution were added. The plates were incubated for 1 h at room temperature followed by washing ten times with TBST. Bound phages were subjected to reaction with horseradish peroxidase (HRP)-conjugated sheep anti-M13 antibody (Pharmacia, Piscataway, NY, USA), followed by color development with substrate solution containing o-phenylenediamine (OPD).
The positive phage clones identified by phage ELISA were sequenced with the 296 gIII sequencing primer 59-TGA GCG GAT AAC AAT TTC AC-39 as described in the manufacturer's instructions.
A series of complementary oligonucleotides (Table 1) coding for wild-type and truncated motif SVQYHPL were synthesized, annealed, and cloned into the BamHI/XhoI sites of the prokaryotic expression vector pGEX-6p-1 (Pharmacia, Piscataway, NY, USA), producing a group of recombinant plasmids. All the resulting recombinant plasmids were validated by restriction analysis and DNA sequencing. Expression plasmids were transformed into BL21 (DE3) competent cells, followed by the addition of 1 mM isopropyl-D-thioga-lactopyranoside (IPTG; GE Healthcare, USA) for induction.
Approximately equivalent amount of each GST fusion protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE). The gel was either stained with commassie blue staining solution or electrophoretically transferred to nitrocellulose membrane. After being blocked with 5% skim milk in PBS overnight at 4uC, the membrane was incubated with mAb A9E8 (diluted 1:2,000 in PBS) or REV-A-positive chicken serum (diluted 1:100 in PBS) at 37uC for 1 h. After being washed three times with PBST, the membrane was probed with a 1:5,000 dilution of HRP-conjugated goat anti-mouse IgG 
To investigate the conservation of the epitope among REV viruses, sequence alignment of the epitope and the corresponding regions on gp90 proteins of 32 REV-A strains, one REV-T strain, four SNV strains and one CSV strain was performed using the DNASTAR Lasergene program (Windows version; DNASTAR Inc., Madison, WI, USA).
Purified gp90 protein was used to immunize BALB/c mice. After cell fusion and screening, several hybridoma cell lines were generated, which produced gp90-reactive mAbs. One monoclonal antibody produced by the line designated as A9E8 was selected for strong reactivity with recombinant gp90 protein in Western blot ( Figure 1A ) and in an indirect ELISA (data not shown). It also showed strong reactivity with purified whole virus in Western blot ( Figure 1B) and could be used to detect REV-A antigen by an indirect immunofluorescence assay (IFA; Figure 1C ). The mAb A9E8 was compose of an IgG2b heavy chain paired with a k-type light chain, as determined using the SBA Clonotyping TM System/ HRP Kit. The titers of antibody in hybridoma cell culture supernatants and in ascites were measured by indirect ELISA and determined to be 1:3,200 and 1:128,000, respectively.
The neutralizing activities of the mAb A9E8 were then determined by a micro-neutralization assay on CEF cells using REV-A HLJ07I. The mAb A9E8 neutralized the virus with a neutralization titer (NT 50 ) of 100.
To determine the epitope recognized by mAb A9E8, biopanning of a phage displayed 12-mer random peptide library was performed using the affinity purified mAb A9E8. After three rounds of biopanning, an enrichment of phages bound to the mAb A9E8 was obtained. The output to input ratios of the three rounds of biopanning were 0.00008%,0.038% and 0.79%.
Eight phage clones were selected for reactivity with the mAb A9E8 after three rounds of biopanning and enrichment of the phages binding to the mAb A9E8. These selected clones were further evaluated by Phage ELISA for reactivity with the mAb A9E8 and a negative control mAb (anti-porcine IFN-c). As shown in Figure 2 , all the selected eight phage clones (A1-A8) showed specific reactivity with A9E8 (OD492 nm .1.10), but not with anti-porcine IFN-c mAb (OD492 nm ,0.15). The eight phage clones were sequenced, and were shown to display a consensus sequence SVQYHPL, which was identical to the motif 213 SVQYHPL 219 at the C-terminus of the gp90 protein of REV-A strain HLJ07I (Table 2) .
To verify whether the identified motif represented an epitope recognized by the mAb A9E8, a DNA fragment coding for the motif SVQYHPL was expressed as a GST fusion protein (GST-H7wt) in E. coli. Western blot analysis showed that the fusion protein was recognized by the mAb A9E8 ( Figure 3A ) and REV-A infected chicken antiserum ( Figure 3B ), indicating that the motif represented a linear B-cell epitope.
To define the epitope precisely, four mutants with deletions at C-and N-termini of the motif SVQYHPL (Table 1) were constructed to express the GST fusions GST-H7DS, GST-H7DL, GST-H7DSV, and GST-H7DPL representing -VQYHPL, SVQYHP-, -QYHPL and SVQYH-(deletions were shown as dashes) in E. coli, respectively. We found that only the full-length SVQYHPL polypeptide (GST-H7wt) was recognized by the mAb A9E8 ( Figure 3A ). Removal of one or more amino acids at either the amino or carboxyl terminus of the peptide abolished antibody binding, indicating that the peptide SVQYHPL represented the minimal requirement for the reactivity of the epitope with A9E8. Minimal Unit of the Epitope with the Maximal Binding Activity to mAb A9E8
To investigate minimal unit of the epitope with the maximal binding activity to mAb A9E8, a series of GST-fusion proteins were expressed with extended amino acid residues at both N and C termini of the motif SVQYHPL (Table 1) . These GST-fusion proteins were subjected to SDS-PAGE and testing for reactivity with mAb A9E8 in Western blot. Fusion proteins GST-R1 (SVQYHPLA), GST-R2 (SVQYHPLAL) and GST-R3 (SVQYH-PLALP) reacted strongly with mAb A9E8 in Western blot ( Figure 4) . The GST-R2 and GST-R3 showed similar binding activity to the GST-R1, indicating that alanine alone significantly increased binding activity of the core epitope to mAb A9E8. In contrast, GST-fusion proteins with extended amino acid residues at the N terminus of the motif SVQYHPL showed no increased binding activity compared with GST-H7wt in Western blot (data not shown). Taken together, these results showed that SVQYH-PLA was the minimal unit of the epitope with the maximal binding activity to mAb A9E8.
To investigate the conservation of the SVQYHPL epitope, we aligned the epitope identified in this study with REVs gp90 coding regions available in GenBank. The alignment results showed that all amino acids in the motif were identical among all REV strains ( Figure 5 ), indicating that the motif represented a conserved epitope on the gp90 protein of REVs. Figure 2 . Detection of the selected phages for antibody binding by Phage ELISA. Eight phage clones selected after three rounds of biopanning were added to the microplate wells (10 10 pfu/100 mL/well) coated with the mAb A9E8 or anti-porcine IFN-c mAb (negative control) (100 ng/well ), and incubated for 1 h at room temperature. Bound phages were subjected to reaction with horseradish peroxidase (HRP)-conjugated anti-M13 antibody, followed by color development with substrate solution containing o-phenylenediamine (OPD). Three independent assays were performed for each selected phage. doi:10.1371/journal.pone.0049842.g002 Table 2 . Sequence comparison of random peptide inserts displayed on the positive phages.
Amino acid sequence of the insert a
Conservative amino acid motifs are bold and underlined. doi:10.1371/journal.pone.0049842.t002
The gp90 protein of REV is an important antigenic protein and is associated with virus neutralization, which is the major candidate antigen for vaccine development and disease serological diagnosis [12, 13] . Studies showed that recombinant gp90 protein expressed in Pichia pastoris induced a protective immune response against REV in chickens [19] . Precise mapping of epitopes in gp90 is important for understanding antibody-mediated protection and developing epitope-based marker vaccines and diagnostic tools. Cui et al. [20] reported the generation and partial characterization of a panel of 11 mAbs against the nondefective REV Strain T, and showed that the epitope was on the viral envelope glycoprotein. However, they only identified the relative regions in REV envelope glycoprotein recognized by the mAbs, and did not map the fine locations of the epitopes. To our knowledge, there has been no report on linear epitope mapping of the gp90 of REV.
Mapping epitopes using monoclonal antibodies has become a powerful tool to study protein structure and has been used to diagnose diseases and design marker vaccines [21, 22, 23] . In this study, we described the generation and epitope mapping of a gp90 protein specific mAb, and demonstrated that the epitope was conserved among the REV group. Precise analysis of REV-A gp90 protein epitope will provide the fundamental information for development of epitope-based vaccines and diagnostic tools for REV-A and/or other REV group infection.
Phage display is an in vitro selection technique in which a peptide or protein is genetically fused to a coat protein of bacteriophage and the fused peptide or protein is displayed on the exterior surface of the phage virion. The phage displayed random peptide library is a powerful and high throughput tool for rapid mapping of epitopes [24] .
In this study, we generated a gp90-specific mAb A9E8 using recombinant gp90 protein expressed in E. coli. The mAb A9E8 showed strong reactivity against purified whole virus in Western blot and could be used to detect REV-A antigen by an indirect immunofluorescence assay. The linear epitope recognized by the mAb A9E8 was defined as SVQYHPL by screening a random phage display peptide library. This peptide sequence was identical to 213 SVQYHPL 219 of the gp90 protein of REV-A. N-or Cterminal deletions of amino acids of this epitope demonstrated that 213 SVQYHPL 219 is the minimal requirement for recognition by A9E8. Fusion proteins GST-R1 with extended amino acid residues at the C terminus of the motif SVQYHPL showed increased binding activity compared with that of GST-H7wt in Western blot, indicating that alanine alone significantly increased binding activity of the core epitope to mAb A9E8. Thus, the peptide SVQYHPLA was determined to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8.
The peptide was also recognized by REV-A-positive chicken serum, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding reactivity. Sequence alignments of REV-A strains, REV-T strain and five other REV strains demonstrated that the motif was highly conserved among REV viruses, indicating that it is a broad group-specific epitope. Since A9E8 was identified as a neutralizing mAb, the epitope identified with A9E8 in this study was a neutralizing epitope.
Many neutralizing epitopes have been mapped in the variable regions of the proteins of viruses, including infectious bursal disease virus [25] , infectious bronchitis virus [26] , hepatitis C virus [27] , and HIV [28] . Some neutralizing epitopes, however, are highly conserved across most of the viruses in the same group [29, 30] . A novel epitope was mapped within the highly conserved flavivirus fusion loop peptide 98 DRXW 101 by phage-display biopanning and structure modeling using mAb 2A10G6 that had broad cross-reactivity with dengue virus (DENV) 1-4, yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) viruses. This mAb potently neutralized DENV 1-4, YFV, and WNV and conferred protection against lethal challenge with DENV 1-4 and WNV in murine model. Further functional studies revealed that 2A10G6 blocked infection at a step after viral attachment. These results show that the broad cross-reactivity epitope recognized by neutralizing mAb 2A10G6 is highly conserved among DENV 1-4, YFV and WNV [29] . An epitope recognized by mAb 51 belonging to isotype IgM was mapped to 215 KQEKD 219 of the VP1 capsid protein of Enterovirus 71 (EV71), which possessed neutralizing activity in vitro and provided 100% in vivo passive protection against lethal challenge with EV71 strain HFM 41. BLAST analyses of the neutralizing epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 [30] . In this study, the epitope recognized by neutralizing mAb A9E8 was mapped to a highly conserved region of the gp90 protein among REVs, which would be useful for development of REV marker vaccines and diagnostic techniques.
In summary, a highly conserved neutralizing linear B-cell epitope on the gp90 protein of REV-A was identified in this study. The identified conserved epitope may have potential for development of REV specific diagnostic assays and epitope-based marker vaccines. Identification of an Epitope on REV gp90 Protein PLOS ONE | www.plosone.org Lower Respiratory Tract Infection in a Renal Transplant Recipient: Do not Forget Metapneumovirus Human metapneumovirus (hMPV) is emerging as a cause of a severe respiratory tract infection in immunocompromised patients. hMPV pneumonia has only been seldom reported in nonpulmonary solid organ transplanted patients, such as renal transplant recipients. We report here a case of a 39-year-old patient presenting with fever, cough, and interstitial opacities on CT scan diagnosed as a nonsevere hMPV pneumonia 11 years after a renal transplantation. Infection resolved spontaneously. Differential diagnosis with Pneumocystis pneumonia was discussed. We review the medical literature and discuss clinical presentation and detection methods that can be proposed in solid organ transplant recipients. Respiratory viruses such as respiratory syncytial virus (RSV), influenza, parainfluenza viruses (PIV), and adenovirus are commonly associated with mild to severe symptoms, depending on the immune status. Human Metapneumovirus (hMPV) was the sixth most frequent viral infection in patients hospitalized for respiratory illness [1] . hMPV is a nonsegmented, enveloped, negative single-stranded RNA virus [2] responsible for lower respiratory tract infections (LRI), especially in extreme ages [3, 4] . It has a seasonal distribution and occurs mainly in winter and spring, with an incubation period usually between 4 and 6 days [5] . hMPV is now widely recognized as an opportunistic infection in immunocompromised hosts such as hematopoietic stem cell transplant (HSCT) and pulmonary transplant recipients, leading to a significant respiratory morbidity [6] . Although its detection is not yet routinely performed, hMPV appears to account for 9% of acute pneumonia in patients with haematologic malignancies (including HSCT), in a similar proportion to RSV [7] . This rate is close to that reported in lung transplant recipients, ranging from 6% to 12% of LRI [6, 8] .
In contrast, it has been seldom reported in other SOT settings such as renal transplantation [9] .
A 39-year-old patient with an 11-year history of kidney transplantation for severe amyloidosis was referred to the Centre d'Infectiologie Necker Pasteur for acute fever for 2 days. After 8 years of transplantation, he was treated for graft rejection by corticosteroids. Clinical course was uneventful, except for recurrent prostatitis. His current immunosuppressive regimen consisted of mycophenolate mofetil except for sore throat and rhinorrhea. Biological analyses showed an elevated C-reactive protein blood level (157 mg/L, normal <5 mg/L) but normal blood leukocytes and neutrophil counts. Blood lymphocyte count was low (0.94 G/L), with CD4+ T cells accounting for 39.5% of total lymphocytes (0.372 G/L). HIV serology was negative. Blood and urine cultures were sterile and initial chest radiograph was normal. Nonproductive cough without dyspnea or chest pain appeared on day 3 of hospitalization. Oxygen saturation in ambient air was 92%. Chest auscultation was normal. As the cough increased, a thoracic computed tomography (CT) scan was performed on day 6 and revealed bilateral ground glass infiltrates mainly located in subpleural and peripheral areas, associated with bilateral pleural effusion ( Figure 1 ). No mediastinal adenopathy was seen. Because of clinical and radiological presentation suggesting Pneumocystis jirovecii pneumonia, trimethoprim-sulfamethoxazole was initiated the same day. Nasopharyngeal aspirates were screened by direct immunofluorescence with specific monoclonal antibodies to RSV, influenza virus A and B, PIV, adenovirus, and hMPV (Argène, Verniolle, France) on day 7. Immunofluorescence was strongly positive for hMPV and negative for other viruses. Blood cultures and S. pneumoniae and L. pneumophila urinary antigen detections were negative. As Pneumocystis pneumonia was initially suspected, bronchoalveolar lavage (BAL) fluid analysis performed on day 11 demonstrated 450.10 3 cells/mL (macrophages 72%, neutrophils 17%, and lymphocytes 11%). Microbiological studies did not reveal any bacterial or fungal microorganisms. Gomori-Grocott staining for Pneumocystis jiroveci detection, indirect immunofluorescence, and polymerase chain reaction (PCR) for P. jiroveci were negative. hMPV was also detected in BAL fluid by direct immunofluorescence. As all microbiological investigations were negative except for hMPV, antibiotics were discontinued; respiratory symptoms spontaneously improved within 6 days. Thus, decreased immunosuppression or other medications such as ribavirin or intravenous immunoglobulin were not considered. The patient was discharged on day 14.
This is the second hMPV pneumonia in a kidneytransplanted recipient described in the literature. The first reported case was a severe LRI requiring transient intensive care unit stay [9] . It occurred three years after kidney transplantation, while receiving immunosuppressive regimen consisting of ciclosporine (125 mg b.i.d), azathioprine (75 mg/d), and prednisone (10 mg/d). Compared to this case, our patient had mild symptoms, mainly cough and upper respiratory symptoms. He was also less immunosuppressed without corticosteroids regimen.
In solid organ transplanted patients, hMPV is responsible for LRI and may lead to hospitalization and significant respiratory illness in up to 63% of cases [6, 8] . As initial clinical symptoms are nonspecific, thoracic CT scan can be more helpful than chest X-ray, which is less sensitive. Consolidation, nodular infiltrates, and pleural effusions may be seen. Subpleural and basal areas are usually observed, and bilateral locations are seen in 50% of cases, as in our case [10] . Whereas crazy paving, network of a smooth linear pattern superimposed on an area of ground-glass opacity, is unusual, bronchiectasis is common, up to 68% in the series by Wong et al. [10] .
Of note, lymphopenia, as noticed in our patient, is the most common feature reported in HSCT patients with hMPV, accounting for 73% of patients in one series [7] . This illustrates that although innate immune responses are stimulated upon hMPV exposure, adaptive immunity also appears important to control hMPV. As for other paramyxoviruses, the matrix proteins are involved in the induction of proinflammatory and Th1 responses by dendritic cells and macrophages (i.e., production of interleukin-2 and interferon-γ) [11] . Inflammation may cause diffuse alveolar damage and hyaline membrane formation as shown by histopathology investigations [12] .
Apart from other respiratory viral infections occurring in SOT recipients, differential diagnoses of hMPV-associated LRI include severe bacterial and fungal pneumonitis, particularly Pneumocystis pneumonia. Ribavirin, previously shown active in a mouse model of infection [13] , has been suggested as a potential antiviral therapy in HSCT and lung transplant recipients with hMPV-associated LRI [14, 15] . In our case and in the other case of the literature [9] , ribavirin was not used because the diagnosis was made retrospectively after the patient's spontaneous clinical improvement.
In conclusion, hMPV has to be considered as a potential cause of LRI in kidney transplant recipients and may mimic Pneumocystis pneumonia. A prompt recognition would have avoided antibiotic use and further diagnostic studies such as bronchoscopy. Its early detection using immunofluorescence and/or RT-PCR must be proposed routinely in transplantation settings. In addition, early recognition could improve the implementation of appropriate infection control practices to prevent viral spread of this potential lifethreatening infection in immunocompromised patients. What was the primary mode of smallpox transmission? Implications for biodefense The mode of infection transmission has profound implications for effective containment by public health interventions. The mode of smallpox transmission was never conclusively established. Although, “respiratory droplet” transmission was generally regarded as the primary mode of transmission, the relative importance of large ballistic droplets and fine particle aerosols that remain suspended in air for more than a few seconds was never resolved. This review examines evidence from the history of variolation, data on mucosal infection collected in the last decades of smallpox transmission, aerosol measurements, animal models, reports of smallpox lung among healthcare workers, and the epidemiology of smallpox regarding the potential importance of fine particle aerosol mediated transmission. I introduce briefly the term anisotropic infection to describe the behavior of Variola major in which route of infection appears to have altered the severity of disease. Controversy exists regarding the best method of protecting the public against the potential release of smallpox as a biological weapon (Bicknell, 2002; Fauci, 2002; Halloran et al., 2002; Kaplan et al., 2002; Mack, 2003) . Infectious disease modeling plays an important role in this dialog, and the biology of the transmission pathway, the focus of this review, is critical to producing appropriate predictive models and understanding which controls will work best under varying conditions (Ferguson et al., 2003) .
The rapidity with which smallpox would spread in a developed nation is not known and is a major source of uncertainty in models used for public health planning (Ferguson et al., 2003) . The basic reproductive number (R 0 ), which describes the tendency of a disease to spread, has been estimated for smallpox from historical data and outbreaks in developing countries (Gani and Leach, 2001; Eichner and Dietz, 2003) . Because R 0 is a function of the contact rate between individuals, it can be affected by changes in the environment (Anderson and May, 1991) . A potentially important difference between contemporary environments and those used to estimate R 0 is that today many buildings, including hospitals, mechanically recirculate air. If smallpox was almost entirely transmitted by mucosal contact with large droplets (aerodynamic diameters >10 µm), which can only occur following "face-toface" exposure over distances of a few feet, then change in the built environment would not change the contact rate between individuals. If, however, smallpox was frequently transmitted from person-to-person by airborne droplet nuclei [fine particles with aerodynamic diameters of ≤2.5 µm capable of remaining suspended in air for hours and of depositing in the lower lung (Hinds, 1999) ] then mechanically recirculated air systems would increase the contact rate, R 0 , the risk of epidemic spread, and the difficulty of hospital infection control. Unfortunately, leading authorities disagree regarding the relative importance of fine and large particle routes of transmission; some state that smallpox was transmitted primarily via airborne droplet nuclei, (Henderson et al., 1999) while others emphasize "face-to-face" contact and state that, airborne transmission was rare (Centers for Disease Control, 2002; Mack, 2003) . This paper reviews the evidence for each of these modes of transmission.
Prior to Jenner, variolation, (Fenner et al., 1988) inoculation of variola into the skin or nasal mucosa, was used to reduce the risk of smallpox. Jenner himself was variolated as a child. Skin inoculation with a small amount of fresh pustule fluid, likely to have contained large numbers of infectious virions, produced a local lesion with satellite pustules, but generalized rash was reported to be less severe and mortality rates were usually 10-fold lower than with naturally acquired disease (Fenner et al., 1988) . In China, variolation was frequently performed by inoculation of the nasal mucosa. Some accounts describe blowing carefully aged scabs compounded with plant material into the nose (MacGowan, 1884). Other reports suggests that nasal insufflation was considered relatively ineffective and that nasal insertion of cotton pledgets impregnated with powdered scabs or smeared with vesicle contents was preferred (Wong and WU, 1936; Miller, 1957) . Descriptions of the latter method do not include ageing infectious material before use.
Because natural infection was thought to occur via large droplets deposited on the upper respiratory mucosal, the success of nasal inoculation in producing low mortality rates has been hard to understand. A theory suggested by Henderson to the author of a smallpox history, (Hopkins, 1983, p. 114 ) "is that virus inhaled naturally was in sufficiently small particles to be deposited deep within the lung, whereas particles inoculated by nasal insufflation may have been much larger and were likely to implant in the nose or throat where [only] a local lesion might be produced." The relative importance of age and health of inoculated subjects, infectious dose, and route of exposure are not known.
However, it appears that inoculation via the skin or nasal mucosa tended to produce modified disease. If true, this would indicate that natural transmission did not occur via direct skin or mucosal contact. Figure 1 shows graphically a how these different routes of exposure may have produced altered patterns of viral replication within the host and resulted in different risks of extensive viremia and severe disease.
If natural smallpox was initiated through the upper respiratory mucosa, then an early asymptomatic mucosal infection would be expected. To investigate this, Sarkar and colleagues performed pharyngeal swab surveys of household contacts (Sarkar et al., 1973a 4-8 days following onset of rash in the index cases. They found that contacts with positive throat cultures often did not develop smallpox. In one survey, (Sarkar et al., 1973a) 10% (Westwood et al., 1966) of 328 contacts had positive swabs, but only 12% (Kaplan et al., 2002) of those with positive swabs developed smallpox. Among 59 unvaccinated contacts 27% (Miller, 1957) were culture positive, but only one developed smallpox. All subjects were vaccinated at the time of examination. However, vaccination four or more days after exposure is usually considered to be too late to prevent disease. The observation that disease did not develop in 94% of persons with mucosal infection suggests that, even in unvaccinated contacts, mucosal infection may not have been sufficient to initiate disease.
Sarkar and colleagues also showed that the oropharyngeal excretion of virus was greatest during the first days after the rash erupted and generally resolved at most 2 weeks following onset of rash (Sarkar et al., 1973b) . Rao et al. found that oropharyngeal excretion was greatest in the most severe, hemorrhagic cases and corresponded with the period of infectiousness (Rao et al., 1968) . In contrast to oropharyngeal excretion, scabs contained large quantities of virus regardless of disease severity and were shed for another week or more after throat cultures were negative. Scabs alone, however, were not associated with further cases (Rao et al., 1968; Mitra et al., 1974) .
The apparent lack of infectiousness of scab associated virus has been attributed to encapsulation with inspissated pus (Fenner et al., 1988 ). Henderson's theory about the importance of small particles may provide a straightforward mechanism for why encapsulated virus, simply by entrapment in large particles, had low infectious potential. Sarkar et al. (1973a) were concerned that asymptomatic contacts could have been infectious because their throat swab viral titers were similar to those of milder smallpox cases. A paradox arose from these data because there was never evidence of infection arising from asymptomatic household contacts. Yet, oropharyngeal secretions were thought to be the primary source of infectious virus particles. An explanation may be that oropharyngeal excretion of virus was merely temporally correlated with excretion of virus from elsewhere in the respiratory tract and not the actual source of fine particles virus aerosols.
The large spray of particles from sneezing visualized by high speed photography consists of particles down to about 10 µm in diameter (Papineni and Rosenthal, 1997) . Smaller particles may also be dislodged from the upper airways by the turbulence of sneezing, coughing, and talking, but will mostly be larger than 2.5 µm in diameter. Recent studies, however, show that the healthy lung generates abundant fine particles (100-1000/l with size <0.3 µm diameter) during normal breathing (Fairchild and Stampfer, 1987 ) that do not arise from the oropharynx; condensates of these particles are the subject of recent reviews (Mutlu et al., 2001; Hunt, 2002) . Such particles could carry variola virus (0.2-0.3 µm diameter), would remain airborne in indoor air for many hours, and would be deposited primarily in the lower airways after inhalation.
There is some evidence that variola was present in the lung and potentially available for aerosolization. Animals infected by inhalation produced high concentrations of variola in the lung (Hahon and Wilson, 1960) . Fenner et al. (1988) regarded bronchitis and pneumonitis as a part of the normal smallpox syndrome, especially in the more severe cases which were also the most infectious, (Rao et al., 1968) although specific lesions were less frequent in the lower trachea and bronchi. Systematic evaluations of viral excretion in the lower respiratory tract of non-fatal cases were not reported. Thus, if some degree of pneumonitis with pulmonary excretion of virus and exhalation of fine particle variola aerosols was a feature of clinical smallpox but was not a feature asymptomatic household contact with positive throat cultures, then the paradox would be resolved.
Air sampling for viruses is a difficult undertaking and the literature on the subject remains sparse in comparison with that for bacteria and fungi (Sattar and Ijaz, 2002) . Only three attempts to detect airborne variola were published. The earliest attempt used highly inefficient methods and was negative (Meiklejohn et al., 1961) . In a subsequent study, Downie and colleagues used short duration, low volume air sampling with liquid impingers and obtained 5 positive samples out of 47 attempts to sample exhaled breath of patients (Downie et al., 1965) . Assuming that each positive sample represented a single infectious particle, the concentration of airborne infectious particles was 0.85/m 3 ; higher concentrations were observed close to shaken bed sheets. Concentrations were likely to have been underestimated because of several frequently encountered problems with air sampling for viruses including failure of impingers to retain particles less than 1 µm in diameter that represent the majority of particles in exhaled breath, culture of only a portion of the impinger fluid, uncertain suitability of sampling fluid for virus survival, and loss of infectivity due to sampling trauma (Spendlove and Fannin, 1982) .
In the 1970s, Thomas Figure 3 .1] appears to have frequently been less extensive after dermal inoculation and nasal insufflation compared with naturally acquired infection. This may have been due to less extensive lymphatic replication of virus and limited viremia by dermal and nasal routes as compared with infection via lower respiratory tract deposition. The size of the arrows represents the historically reported proportions of cases following each pathway. The size of the X on each image represents the reported mortality rate from each pathway. For natural infection, the ordinary-type rash and flat and hemorrhagic rashes are shown.
efficiency for submicrometer particles) for long duration large air volume viral sampling (Thomas, 1970a) . He showed that 23% of naturally airborne rabbit pox particles were ≤2.5 µm and 71% were between 2.5 and 10 µm (Thomas, 1970b) . Both Thomas and Westwood et al. (1966) in a room supplied with 10 ACH containing 7-9 infected rabbits. Westwood et al. probably obtained higher concentrations because they used an electrostatic precipitator allowing higher efficiency collection of submicrometer particles compared with Thomas's slit sampler. Thomas also studied convalescent cases of variola minor (Thomas, 1974) . One patient with relatively active lesions produced an average concentration of approximately 1 PFU/m 3 . Unfortunately the samples were collected late in the disease when the patient was probably minimally infectious, based on comparison with epidemiological data (Rao et al., 1968; Eichner and Dietz, 2003) . The airborne virus observed appears to have been due to resuspension and is unlikely to be representative of the airborne concentration of respirable variola earlier in the course of the infection. The method used would also not have been able to collect submicrometer viral aerosol particles.
Overall, the air sampling studies suggest that animals and people infected with poxviruses generated respirable aerosols, but that air concentrations may have been low, or airborne virus was present in submicrometer particles that could not be collected the instruments available. Because detection of virus aerosols is subject to potentially large losses in sampling equipment, especially when sampling dilute natural aerosols over extended periods, and because plaque assays may not accurately represent the infectivity of virus deposited in human airways at 100% relative humidity, (Spendlove and Fannin, 1982; Ijaz, 1987, 2002) the available data can be considered a lower limit on concentration of infectious natural poxvirus aerosols.
Experimental aerosol data suggested that poxvirus, which survived the trauma of artificial aerosolization, remained infectious for significant periods of time. Aerosols of vaccinia demonstrated a half-life of about 6 h at 22 • C and relative humidity ≤50% with reduced stability at higher relative humidity and temperature (Harper, 1961) . Variola appeared to have a similar half-life and not to be affected by relative humidity at 26.67 • C (Mayhew and Hahon, 1970) . Other experiments demonstrated that airborne vaccinia is highly sensitive to inactivation by germicidal ultraviolet light (Edward et al., 1943; Jensen, 1964) . Westwood et al. (1966) demonstrated that inhalation of a single PFU of a submicrometer vaccinia aerosol was sufficient to infect rabbits. Airborne rabbit pox was similarly infectious. They demonstrated rabbit-to-rabbit airborne transmission of rabbit pox in each of seven trials by placing uninfected rabbits in separate cages in the same room with infected animals. They also infected rhesus monkeys using submicrometer aerosols of variola.
In one of the earliest extensive animal models of smallpox, Brinckerhoff and Tyzzer (1906) reported the effect of inoculating cynomologus monkeys with variola at different sites. Inoculation of mucus membranes of the lip, palate, and nose produced local lesions, but generalized rash occurred in only 10% of animals. Inoculation through the skin produced a local lesion and a generalized eruption in 70-80% of animals. Animals inoculated by scratching the tracheal mucosa through a rigid bronchoscope all developed a generalized rash, and one developed a variolous bronchitis and pneumonia. Laryngeal instillation of dry pustule contents produced infections while instillation of powdered crusts did not. Inhalation exposures to an atomizer spray of vesicle contents infected only one of five monkeys; however, the particle size distribution and type of atomizer were not reported.
Hahon and Wilson demonstrated that infection of Macaca irus with high dose [5 × 10 5 PFU] fine particle (<5 µm) variola aerosols produced a disease that simulated human smallpox (Hahon and Wilson, 1960; Hahon, 1961) . The initial site of virus replication was the lung, with subsequent appearance of virus in the nasopharynx and nares. Peak concentrations of virus per gram of tissue were higher in the lung than in the upper respiratory tract; the peak in lung tissue occurred during the incubation period and lung levels declined during the secondary viremia and exanthem. Whether the time course and viral concentrations in lung in this animal model produced by inhalation of high dose aerosols mimicked that in humans with natural infection is doubtful. However, it may be relevant to the first generation of cases exposed to concentrated aerosols in a biological attack. In a relatively recent experiment, (Kalter et al., 1979) a female chimpanzee became infected with variola while housed in the same room, but without direct contact, with two infected chimpanzees. She developed a generalized rash and was reported to have had more severe constitutional symptoms than the other chimpanzees infected by dermal inoculation or direct contact. The authors concluded that she was infected via aerosol.
The animal data show that artificial respirable aerosols were effective means of producing poxvirus infections, that the infectious dose by the airborne route could be very low, and that animal-to-animal airborne transmission of rabbitpox and variola was observed. They also suggest that inoculation of mucus membranes was less effective at producing a generalized rash than was exposure of the lower respiratory tract.
Two reports, one from the 1940s and one from the 1960s showed that, during epidemics, staff in smallpox hospitals who had been repeatedly vaccinated sometimes developed malaise, fever, and pneumonitis without evidence of infection with smallpox or other viruses, and without evidence of allergic reaction to other agents (Howat and Arnott, 1944; Morris Evans and Foreman, 1963) . In one outbreak, after investigation of other possible causes, the authors attributed the phenomena to an allergic reaction to inhaled variola. The pulmonary focus of the reaction suggests that there were significant concentrations of respirable variola in the vicinity of smallpox patients. Concentrations of respirable variola high enough to elicit allergic reactions, if true, raise a significant concern for the likelihood of airborne transmission.
Fomites, particularly exposure of laundry workers to contaminated bedding, were implicated in a few reported outbreaks (Cramb, 1951) . However, during the eradication campaign careful epidemiologic investigation rarely implicated fomites as a source of infection (Fenner et al., 1988) . Laundry was contaminated by scabs containing large amounts of virus, and with respiratory secretions containing virus in smaller particles (Downie et al., 1965) . Very large particles with diameters greater than 50-100 µm are easily reaerosolized. Thus, the rarity of clear evidence of transmission due to fomites would be surprising, if exposure of upper respiratory mucosa to virus in large particles were an efficient means of initiating infection. However, the probability of reaerosolizing particles ≤10 µm from surfaces is extremely low because surface forces tend to bind particles more avidly the smaller the particle (Hinds, 1999) . Thus, the rarity of smallpox transmission via fomites suggests that mucosal exposure was not the primary means of transmission and is consistent with a preference for infection via the lower respiratory tract. The rarity of transmission on crowded buses and trains could be evidence that airborne transmission was not important. However, Fenner et al. (1988) state that transmission on public transport was rare because patients seldom traveled after becoming ill. They showed that transmission did occur on public transport by reporting a case of confluent smallpox who traveled early in her illness and infected five persons on a bus. If most patients who traveled were convalescent so that they no longer had virus in respiratory secretions and only shed virus in large particles from scabs, which were rarely associated with transmission of infection, (Rao et al., 1968 ) then lack of transmission on buses and trains was consistent with a preference for airborne transmission. Mack (1972) emphasized that 85% of cases had clear-cut exposures to known cases. However, the remaining 15% had no obvious exposure suggesting that a small number of more distant or casual contacts transmitted infection as would be expected if smallpox were transmitted by dilute virus aerosols. For example, in the 1947 New York outbreak one secondary case was seven floors away in the hospital (Weinstein, 1947) . Dispersal of smallpox downwind of hospitals was the only obvious explanation for a small number of cases in a British outbreak (Bradley, 1963; Westwood, 1963) . Unexplained introductions of smallpox into Pakistani towns was greatest in towns with facilities for treatment of smallpox, (Thomas et al., 1972) which may suggest that relatively casual contact, or down wind dispersal were capable of occasionally spreading infection.
Some well-known hospital-associated outbreaks make it clear that airborne transmission at a distance of more than a few feet did occur occasionally (Wehrle et al., 1970) . But, these examples were rare. However, because highly infectious disseminators are rare in other airborne infectious diseases, (Riley, 1980; Olsen et al., 2003) the rarity of superspreaders in smallpox is not an indication that transmission by less infectious cases was necessarily by a different route.
To examine whether the available data on variola aerosols is consistent with Mack's observation regarding known contacts, we can apply a standard Poisson probability model of airborne infection to estimate how long a susceptible person would need to be in a patient's room to have a reasonably high probability of contracting disease (Riley et al., 1978; Rudnick and Milton, 2003) . If, we assume that inhalation and lower respiratory deposition of one PFU of variola was sufficient to cause infection, as for rabbits exposed to vaccinia and rabbit pox, (Westwood et al., 1966) and if a patient's room contained between 0.5 and 5 PFU/m 3 in particles with a 25% lower respiratory deposition fraction (consistent with the literature discussed above), a susceptible individual breathing at 8 l/min would have needed to spend between 1.7 and 16.7 h in the patient's room to have a 63% probability of becoming infected. Outside of the patient's room, aerosol concentrations would have been much lower. If most patients stayed at home in small buildings or in hospitals without mechanically recirculated air, the risk of infection would have been significantly lower outside of patients' rooms, consistent Mack's (1972) observation that 85% of cases arose from identifiable contacts. Thus, a predominance of identifiable face-to-face contacts among cases is not strong evidence against transmission by fine particle aerosols.
The weight of evidence suggests that fine particle aerosols were the most frequent and effective mode of smallpox transmission because this would explain the relatively low mortality after variolation, the rarity of transmission by fomites, resolve the paradox of mucosal infection, and be consistent with "smallpox handler's lung" and with animal and virus aerosol experimental data. Certainly other modes of transmission occurred; fullblown disease could result from inoculation through the skin, the nasal mucosa, or the conjunctiva. Thus, smallpox cannot be classified as an "obligate" airborne infectious disease, such as tuberculosis (Riley et al., 1995) (sometimes referred to as a "true" airborne infection), because it was capable of initiating disease via infection of tissues outside of the lower respiratory tract. However, smallpox also cannot be classified as an isotropic infection (formerly termed "opportunistically" airborne infectious disease) because it appeared not to have been transmitted with equal effectiveness and virulence by all routes, whether aerosol, large droplet, or direct contact and skin inoculation. Smallpox appears to have been most effectively and virulently transmitted by fine particle aerosols and therefore should be classified as an anisotropic infection; an infection where route of transmission influences either virulence and or probability of infection, formerly called a "preferentially" airborne infectious disease.
Current recommendations for control of secondary smallpox infections emphasize transmission "by expelled droplets to close contacts (those within 6-7 feet)" (Centers for Disease Control, 2002 Control, , 2003 . Recommendations include vigilant maintenance of standard, droplet, and airborne precautions. However, emphasis on spread via large droplets may reduce the vigilance with which more difficult airborne precautions are maintained. High concentrations of variola in the lung during the incubation and prodromal periods in monkeys after simulated use of variola as a bioweapon (Hahon, 1961 ) may indicate that first generation cases after an attack with a concentrated aerosol may be more infectious than expected based on historical data. Moreover, because airborne precautions are not routine for all hospitalized patients, and because first generation cases will probably not be initially suspected to have smallpox, it is likely that they will not be placed on airborne precautions until well into their infectious period. Therefore, the extent of transmission to a second generation in the contemporary hospital environment may be greater than expected based on historical estimates.
These considerations suggest that models of a potential smallpox attack should incorporate an aerobiological perspective to predict how the infection might propagate in the modern environment. It is particularly important to examine smallpox transmission in hospitals because hospitals have previously been identified as the major site of transmission in developed countries and ill patients will inevitably gravitate to hospitals, at least early in the outbreak before alternatives exist (Mack, 1972 (Mack, , 2003 . Additional attention to prevention of airborne transmission in hospitals from unrecognized cases may not only be an important aspect of public health preparedness for smallpox, but may also benefit society by reduced morbidity and disruption from SARS and other emergent airborne infections. Chitinase Dependent Control of Protozoan Cyst Burden in the Brain Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack. In the case of Toxoplasma gondii, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Currently, little is known about the nature of the immune response stimulated by the presence of these cysts or how they are able to propagate. Here we establish a novel chitinase-dependent mechanism of cyst control in the infected brain. Despite a dominant Th1 immune response during Toxoplasma infection there exists a population of alternatively activated macrophages (AAMØ) in the infected CNS. These cells are capable of cyst lysis via the production of AMCase as revealed by live imaging, and this chitinase is necessary for protective immunity within the CNS. These data demonstrate chitinase activity in the brain in response to a protozoan pathogen and provide a novel mechanism to facilitate cyst clearance during chronic infections. The brain has unique structures in place to limit access of immune cells and molecules. Although this can provide protection against an overambitious inflammatory response it may also lead to the high prevalence of latent and chronic infections that can persist at this site. Removal of such pathogens has its own particular problems in an organ dense with sensitive neurons and stringent gateways for immune cell infiltration. Toxoplasma gondii is a common intracellular protozoan parasite that forms a chronic infection in the brain for the lifetime of the host. The infection is controlled, in part, through the effector mechanisms of macrophages that result in the conversion of fast replicating tachyzoites to the slow replicating, cyst forming bradyzoites [1] [2] [3] . Cysts can form in all tissues but exist predominantly in the brain for the lifetime of the host requiring a continuous immune response to prevent cyst reactivation and Toxoplasmic encephalitis, a common cause of AIDS related fatalities [4, 5] . The infection-induced immune response in the brain consists of activated CNS resident cells including astrocytes and microglia, infiltrating CD4+ and CD8+ T cells, peripheral macrophages and substantial tissue remodeling [6] [7] [8] . Such immune activity in the brain is often associated with a pathological outcome yet despite the high prevalence of infection Toxoplasma is seemingly controlled without adverse neurological damage. The mechanisms that are involved in the trafficking and control of such a potentially pathological immune response within the CNS are only beginning to be understood [6, [8] [9] [10] [11] . The cyst and cyst-forming bradyzoites are poorly immunogenic [12, 13] and although we have known for some time that T cells are required to prevent cyst reactivation [4, 5, 14] , very little is understood about the biology of this structure in the brain. Although anti-Toxoplasma drugs are available that efficiently control the tachyzoite, there are as yet no therapies available that can effectively remove the cyst form of the parasite. Thus, the continuous presence of Toxoplasma cysts in the brain presents a critical and constant danger for the immune compromised patient.
It is widely believed that cysts remain intracellular within neurons possibly minimizing their contact with host defense systems [15] . However it has been known for some time that cyst burden reaches a peak, declines and becomes stable over time pointing to some form of effector mechanism that can target this stage of the parasite [16] . Studies have implicated CD8+ T cell production of perforin in cyst clearance with perforin deficient mice exhibiting higher cyst burden and susceptibility at the chronic stage of infection [17, 18] . Nevertheless, histological analysis from these studies as well as recent live imaging of cell interactions in the CNS [19] demonstrates monocyte accumulation and contact with cysts.
In recent years, our understanding of macrophages has expanded and we now appreciate these cells' remarkable plasticity. Thus, although whole populations of macrophages can become polarized to classical or alternative phenotypes associated with protection against protozoan and helminth pathogens respectively, the ability to respond and adapt to local stimuli in the environment is paramount [20] [21] [22] [23] [24] . The role of classically activated macrophages in the control of T. gondii infection is well documented. These cells are a source of IL-12, reactive oxygen and nitrogen species, and GTPases that enable the direct killing of the parasite [6, [25] [26] [27] [28] [29] [30] . However, here we describe a population of CXCR3+ macrophages in the brain following T. gondii infection. These cells express the scavenger receptors MMR and stabilin-1 and produce arginase in response to the presence of Toxoplasma cysts. In addition to these traditional signs of alternative activation, these studies demonstrate that macrophages respond to chitin present in the cyst wall and produce the true mammalian chitinase, AMCase. Finally we show that this chitinase activity destroys cysts and is essential for the control of cyst burden within the chronically infected brain.
Recent studies have identified a substantial increase in tissue remodeling in the brain during chronic T. gondii infection [8] . Additionally, there is a continuous need for the clearance of debris from ruptured cysts and dead cells in the brain [31] . To investigate if AAMØ, known for their role in tissue remodeling and homeostatic clearance, are present during such an event in the CNS, macrophage populations from the infected brain were phenotypically analyzed for the expression of known markers of alternative activation. One of the key molecules that has been associated with a tissue remodeling macrophage phenotype in the CNS is the expression of CXCR3 on microglia [32, 33] . CXCR3 is required for protective immune responses to T. gondii primarily due to its role in Th1 cell recruitment and most recently for T cell search strategies in the brain [34] [35] [36] [37] . Indeed CXCR3 and its ligands are significantly upregulated in the brain at a timepoint associated with significant T cell influx into the CNS following infection ( Figure S1A , B) with ,35% of T cells expressing CXCR3 ( Figure 1A ). However, in addition to this well characterized role on T cells, there is a small but distinct population of macrophages that express high levels of CXCR3 (,10% of total macrophages)( Figure 1B ). There is also constitutive, although lower, expression of CXCR3 by CNS resident microglia, which remains unchanged following infection ( Figure S1C ).
To confirm that expression of CXCR3 is associated with alternative activation of macrophages the expression of the scavenger receptor 'macrophage mannose receptor' (MMR; also known as Mrc1 and CD206), a key indicator of the AAMØ phenotype [38] was analyzed. Here we show that MMR expression is limited to macrophages and microglia that also express CXCR3 (Figures 1C and S1D ). In contrast these cells did not express IL-10 ruling out an anti-inflammatory phenotype ( Figure S1E ) [39] . Depletion using blocking antibodies to CXCR3 or its ligand, CXCL10 led to a significant decrease in T cell recruitment and a reciprocal increase in parasite burden ( Figure  S2 ). However, in addition, the proportion of macrophages in the brain was significantly reduced ( Figure 1D ) despite no defect in macrophage-attracting chemokines ( Figures 1E, F) , confirming a role for CXCR3 in the maintenance of this cell population.
To quantify MMR expression by macrophages and microglia in the infected brain, qRT-PCR was performed on magnetically isolated CD11b+ cells from the brains of naive and infected animals. Our results show an approximate 3-fold increase in MMR expression in macrophage populations from infected mice over naïve ( Figure 1G ). Confirmation of this population in the brain was revealed by immunohistochemical analysis. MMR+ macrophages were observed as small and discrete populations of IBA-1+ or tomato lectin+ cells confirming the source of MMR on macrophages or microglia ( Figures 1H, I) . A further functional marker of alternative activation is the scavenger receptor stabilin-1 [40] . Stabilin-1 is involved in the clearance of cell corpses as well as the uptake of extracellular matrix components [41, 42] . Expression of MMR co-localized with stabilin-1 and microglia/ macrophage markers, confirming that these cells display an alternatively activated phenotype ( Figures 1H, I) . These cell populations were frequently found in close proximity with intact and degrading T. gondii cysts in the CNS ( Figure 1J and S3 ).
An important feature of AAMØ is the cell's ability to produce arginase-1, which acts on its substrate, L-arginine to produce Lornithine, a precursor to collagen [43] . L-arginine is also the substrate for NO synthase and the two enzymes compete for substrate availability and are regulated by Th1 and Th2 type cytokines [44, 45] . Previous studies have demonstrated that direct infection of macrophages by T. gondii tachyzoites can induce arginase expression via STAT-6 dependent and independent pathways [46] [47] [48] . Furthermore these studies imply that such an induction is a survival strategy enlisted by the parasite to inhibit killing via NO. To assess whether or not macrophages and microglia in the infected brain produce arginase, CD11b+ BMNCs from infected mice were isolated and analyzed for arginase-1 expression by qRT-PCR. Our results show almost a 2fold increase in arginase-1 expression in cells from infected brains over naïve ( Figure 1K ). Thus, during chronic T. gondii infection there is a population of AAMØ in the CNS characterized by expression of CXCR3, MMR, stabilin-1 and the production of arginase-1.
Alternatively activated macrophages secrete an active chitinase in the CNS in response to chitin in the cyst wall During chronic infection there are several forms of the parasite that could be the source of the infection-associated stimulus for
Described here is a novel mechanism of protozoan cyst clearance in the CNS during chronic infection. These data show the presence of a population of alternatively activated macrophages in the brain that secrete the active chitinase, AMCase, in response to chitin in the cyst wall. Using both chemical and genetic inhibition in vitro, it is revealed that this enzyme is required for efficient degradation and destruction of the cyst. The necessity for AMCase is demonstrated in vivo, as the absence of the enzyme resulted in a significant increase in cyst burden and decrease in survival during chronic infection. Together, these data identify an important mechanism of parasite control and cyst clearance in the CNS. Currently, no therapies exist that lead to the total clearance of this parasite from the brain. Therefore, developing an understanding of the natural mechanisms of cyst clearance has the potential to lead to new and effective therapies for this and other chronic infections. alternative activation of macrophages in the CNS. Since latent cysts are the most prevalent form of infection in the brain, an attractive candidate for the source of this stimulus is the presence of chitin in the cyst wall [49, 50] as it has been shown that the presence of chitin induces the recruitment of macrophages that have an alternatively activated phenotype [51, 52] . To determine if sources of T. gondii can induce alternative activation, tachyzoites, bradyzoites, and cysts were added to bone marrow derived macrophage (BMDM) cultures and the production of urea, a downstream product of arginase activity, was measured [53] . In addition, soluble antigen derived from freeze-thawed tachyzoites (sTAg) and whole cysts (cystAg) was assessed for their ability to induce urea (Figure 2A) . Our results show a baseline production of urea in unstimulated (media) macrophage cultures, possibly due to the presence of M-CSF [54] . This significantly increased (p,0.001) during AAMØ polarization with IL-4. Despite the known ability of tachyzoites to induce arginase production [46] , tachyzoite infection of macrophages did not lead to significant production of urea ( Figure 2A ). This can be attributed to the use of a type II strain which is a weak inducer of arginase-1 [47, 48] . Importantly, addition of cysts or cyst antigen but not ''naked'' bradyzoites, did lead to a significant increase in urea production although not as great as induction of alternative activation by IL-4 [38, 55] . This points to components of the cyst wall as the stimulus for AAMØ. Taken together, these data demonstrate that macrophages can be alternatively activated by the presence of T. gondii cysts, but not free replicating parasites.
Chitinase activity has been demonstrated in certain populations of AAMØ in both mice and humans [52, 56, 57] . Chitinolytic activity by macrophages has also been implicated in host defense against chitin-containing fungal pathogens [56, 58] . To test whether or not chitinase activity is induced by Toxoplasma infection, a chitinase assay was performed on whole brain lysates from naïve and infected animals. Three chitin substrates labeled with 4-methylumbelliferone (4MU) were used to assess the type of chitinase activity present. Upon hydrolysis, 4MU is released and can be measured fluorometrically to determine chitolytic activity. Our data reveal that chitinase activity is significantly increased in the brains of infected mice as compared to the naïve group in only one of the three substrates ( Figure 2B ). This substrate, 4MU Nacetyl-b-D-glucosaminide, is suitable for detection of exochitinase activity where the enzyme degrades the non-reducing end of the chitin [59] . Several studies have linked chitinases and chitinase-like proteins to inflammation [58] [59] [60] . This family of 18 glycosyl hydrolases is typically induced during Th2 type immune responses and plays a role in tissue remodeling, fibrosis, and the modulation of both the innate and adaptive immune response [60] . Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) are unique members of this family in that they possess an enzymatically active domain that hydrolyzes the b 1-4 linkages that exist in chitin [56, 58] . Analysis using qRT-PCR demonstrated a significant upregulation of AMCase but not CHIT1. In addition, the chitinase-like protein, Ym-1 (Chi313) was also upregulated following infection ( Figure S4 ). This molecule is known to inhibit IL-12 production and induce alternative activation in macrophages [25, 61, 62] . In contrast to Ym-1, that has been associated with AMCase production by macrophages in the lung and airway [63] , Ym-2 and BRP-39 were not upregulated in infected brains ( Figure 2C , S4). Since chitin has been shown to activate and recruit AAMØ, it is possible that the cyst wall may serve as the stimulus for chitinase activity in this population of cells. To test this further, BMDM were co-cultured with different forms of the parasite. The addition of tachyzoites, bradyzoites or sTAg did not lead to chitinase production ( Figure 2D ). In contrast, live cysts and cyst antigen led to a significant increase in chitinase activity that was abolished following chitinase treatment of cysts ( Figure 2D , S4). Furthermore, treatment with IL-4 to induce alternative activation in macrophages did not lead to increased chitinase activity. Indeed measurement of IL-4, IL-4Ra and the IL-4dependent RELM-a [64] [65] [66] in the brains of chronically infected mice showed no significant increase over naïve mice ( Figure S4 ). These data suggest that the presence of chitin in the cyst wall induces a phenotype of macrophage characterized by the production of the enzymatically active chitinase, AMCase and is distinct from IL-4 induced activation.
Previous work has shown macrophages in close association with rupturing cysts [17, 31] and the presence of an active chitinase could point to a role for these cells in the breakdown of cysts within the brain. Recognition of chitin by macrophages is size dependent and likely contact dependent [49, 58, 67] . To test this, we cocultured cysts separated from macrophages using 5 mm transwell inserts and assayed for urea and chitinase activity as previously described ( Fig. 2A, 2D ). Our results show no increase in either urea production or chitinase activity from macrophages that have been separated from cysts, confirming that the observed alternative activation is dependent on contact with cysts or cyst antigen.
Immunohistochemical analysis of the location of AMCase secreting macrophages in the infected brain shows them in close proximity with tissues cysts (Figures 3A-C) . As a proportion of macrophages and microglia in the brain, alternatively activated cells are in the minority and it was not possible to find such cells in the naïve brain. However, cysts are easily identifiable with a highly spherical distinct morphology, can stain non-specifically and specifically with antibodies and individual bradyzoites within the cyst are visible by DAPI staining. Examination of chitinase localization in the infected brain revealed distinct cytoplasmic staining of several cells, nearly all of which were within 75 mm of a cyst (Fig. 3A) . Although there were cells that were AMCase positive yet did not stain positively for macrophage/microglial markers, there were clearly several macrophages in close association with cysts that displayed chitinase activity polarized to the cyst wall ( Figure S5 , arrows). AmCase activity was also observed in macrophages surrounding cysts that seemed to be in the process of lysing or cysts that had been lysed (Figures 3B,C and Video S1). The examples provided show the destruction of the spherical cyst (Video S1) and escaping parasites visualized using anti-Toxoplasma antibodies. Directly at the point of rupture there are AMCase expressing macrophages (Figures 3B). Taken together, these data suggest that the induction of chitinase activity in macrophages occurs in close proximity with the cyst wall and that this distinct population of macrophages is responsible for attacking the long-term chronic cyst stage of Toxoplasma via chitinase activity.
The prevailing view is that cysts in the brain remain intracellular within neurons and that CD8+ T cell production of perforin is responsible for cyst clearance in the brain although the exact mechanism of cyst destruction has yet to be described [17, 18] . In order to determine whether or not macrophage chitinase activity could be responsible for the direct lysis of cysts, BMDM were co-cultured with cysts; with and without the chitinase inhibitor allosamidin. Cultures were observed for 14 hours capturing images every 10 minutes. Cysts observed in the absence of macrophages remained intact for the entire time course suggesting no parasite intrinsic mechanism of cyst destruction ( Figure 4A ; Video S2). In contrast, the addition of 20 mg/ml trichoderma chitinase to cyst cultures led to rapid rupture of the cysts within an average of 4 hrs, releasing bradyzoites into the media ( Figure 4B ; Video S3). Strikingly, cysts that were cultured with macrophages came under vigorous attack. This involved efficient and rapid migration of macrophages toward the cyst creating clusters of macrophages that could be seen pulling at the cyst wall ( Figure 4C ; Video S4). In these cultures most of the cysts were destroyed during the observation period with the average survival time of 9.5 hours ( Figure 4E ). In contrast cysts cultured in the presence of macrophages and the chitinase inhibitor allosamidin survived significantly longer than in untreated cultures. Although there appeared to be no defect in the recruitment and activity of macrophages to cysts with similar clustering and 'pulling' of the cyst wall, the majority of cysts survived the entire 14 hour period when treated with either 100 mM or 10 mM allosamidin ( Figure 4D ; Video S5). Decreasing concentrations of allosamidin led to a dose dependent decrease in cyst survival time ( Figure 4E ). These results demonstrate that macrophages can induce cyst lysis in a chitinase dependent manner.
Although both AMCase and CHIT1 are upregulated in certain bacterial and nematode infections [68, 69] only AMCase was significantly increased in the brain following Toxoplasma infection ( Figure 2C ). To confirm that AMCase is responsible for the observed chitinase activity, we performed a chitinase assay similar to that in Figure 1D using BMDM from WT and AMCase null mice ( Figure 5A ). Our results reveal a severe defect in chitinase production by AMCase null macrophages. Indeed, these cells showed a significantly lower baseline level of chitinase and were unresponsive to the addition of cysts. To test whether the ability to destroy cysts is dependent on this enzyme, BMDM from WT and AMCase2/2 mice were fluorescently labeled and cultured with Me49-RFP expressing cysts and cyst lysis time imaged as before. Using fluorescently labeled parasites enhanced the ability to see escaping parasites from lysing cysts. Results, as before, demonstrated that WT non-polarized macrophages were able to lyse cysts in ,10 hours ( Figures 5B and 5D ; Videos S6 and S10). In contrast, cysts cultured with AMCase2/2 macrophages had a significantly increased survival time over WT macrophages consistent with AMCase being the source of chitinase activity required to lyse cysts ( Figures 5C and 5D ; Videos S7 and S11). To determine the requirement for macrophage polarization in their ability to lyse cysts, macrophages were treated with cytokines to polarize them to classical or alternative phenotypes prior to cyst addition. In line with the lack of chitinase induction, IL-4 priming had no effect on cyst survival time, suggesting that cytokineinduced alternative activation does not enhance the ability to destroy cysts ( Figures 5D; Video S9) . In contrast, macrophages that were classically polarized with LPS and IFN-c showed a defect in chitinase activity and cyst destruction (Figures 5D and  5E ; Video S9). Suggesting that polarization of macrophages is required but that chitin is the most likely source of alternative activation and not IL-4. Taken together, these data demonstrate that macrophages lyse cysts in an AMCase-dependent manner in vitro.
The consequences of chitinase dependent cyst lysis in the CNS could potentially benefit either the host or the parasite. If the escaping bradyzoites were quickly killed by macrophages or associated immune cells, we would expect this mechanism to benefit the host and result in a lower parasite burden. Conversely, if bradyzoites are able to propagate and infect new cells, this could be a mechanism that promotes the persistence of the parasite in the brain. To investigate the role of AMCase in the brain in vivo, we infected WT and AMCase deficient mice and analyzed the immune response and parasite burden in the absence of chitinase activity. To determine if AMCase is required during the acute stage of infection, tissue samples from lungs were taken at day 7 and analyzed for parasite burden by qPCR. No significant differences in lung parasite load were found and serum cytokine concentrations were equivalent throughout acute infection ( Figure 6A-F) . Thus a lack of AMCase does not lead to deficient immune responses early on during infection in the periphery. At 5 weeks post infection, when systemic inflammation has been controlled and parasites are located solely in the brain predominantly as cysts containing bradyzoites [67, 70] parasite burden was evaluated. In the absence of AMCase, there was a significant increase (p = 0.0014) of approximately 2-fold in the total number but not the size of cysts in the brain (Figures 6G and 6H) . Differences in cyst burden were not observed at 3 weeks post infection ( Figure S6 ), a period representing the transition between acute and chronic infection, further suggesting that the increase in cyst burden is occurring due to events within the CNS during chronic infection. In addition, total parasite burden in the CNS as measured by qPCR was significantly greater by more than 2-fold (p = 0.0055) ( Figure 6I ) correlating with the appearance of more cysts histologically ( Figure 6J ). In addition, parasite burden was evaluated using RT-qPCR with stage-specific primers identifying tachyzoite (SAG1), bradyzoite (SAG4), and cyst (MAG1) specific transcripts [71] (Figure S6 ). Our results show similar increases in parasite burden for all three transcripts, suggesting that cyst lysis is also an important mechanism to control the cell invasive forms of the parasite. Flow cytometric analysis revealed no differences in infiltrating CD4+, CD8+ T cells, or macrophage populations ( Figure 6K) . Therefore, the increase in parasite burden is not due to a defect in infiltrating effector immune cells. Furthermore, AMCase2/2 mice failed to survive and succumbed to infection beginning at six weeks (p = 0.0177) ( Figure 6L ). Although some acute mortality was noted over several experiments significance was only achieved when chronic mortality was included. These results demonstrate that AMCase activity is required for the protective immune response to T. gondii during chronic infection in the brain and that AMCase mediated cyst lysis in the CNS is a beneficial mechanism for the host to control parasite burden at non-lethal levels.
Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites and fungal pathogens have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack [72] [73] [74] . In the case of Toxoplasma, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Here we describe three novel findings 1) despite a dominant Th1 immune response during Toxoplasma infection there exists a population of macrophages in the infected brain which display a distinct alternatively activated phenotype; 2) these cells are responsible for chitinase dependent lysis of Toxoplasma cysts and 3) this chitinase activity is through the production of AMCase which is required for protective immune responses.
Multiple studies have demonstrated the role of CXCR3 and its ligands in the migration of activated T cells during Th1 immune responses including to sites of infection [75, 76] . It is also known that the chemokines CXCL9 and CXCL10 are induced by the presence of the proinflammatory cytokine, IFN-c [34, 36, 37, 77] . More recently, however, the function of this family of chemokines has expanded to include neural-glial signaling following brain lesion where injured neurons upregulate CXCL10 and recruit CXCR3 expressing microglia to phagocytose denervated dendrites [33] . Consistent with this, another recent study has implicated CXCR3 in the function of perivascular macrophages and their ability to remodel the vasculature during stress [78] . We noted upregulation of CXCR3, CXCL9 and CXCL10 in the brain during chronic Toxoplasma infection. Furthermore, CXCR3 was preferentially expressed on macrophages expressing the scavenger receptors MMR and stabilin-1, suggesting an alternatively activated phenotype for these cells. Previous studies have established important functions of AAMØ in the context of helminth infection and wound healing [43, 79, 80] however not during an infection that generates such a polarized Th1 immune response such as Toxoplasma. T. gondii is known to exploit the arginine metabolic pathway and induce arginase-1 expression in macrophages thereby suppressing nitric oxide production but this does not lead to the alternative activation of these cells [46] [47] [48] . Instead our data point to a role for the cyst being the source of alternative activation of macrophages and the subsequent ability of these cells to lyse cysts via destruction of the chitin in the cyst wall. Thus, we observed a contact dependent significant increase in arginase activity following treatment with cysts and cystAg suggesting that this induction is not a result of infection by the replicating parasite, but rather by the presence of chitin in the cyst wall. The weak induction of arginase activity observed also points to a limited role for arginase-1 in the chitin-induced phenotype.
Chitin is found in the exoskeletons of insects, fungal cell walls, sheaths of parasitic nematodes, and is a component of the T. gondii cyst wall [49, 50] . The presence of this exogenous molecule can induce the recruitment of AAMØ, basophils, neutrophils, and eosinophils [52, [81] [82] [83] [84] . Active chitinases such as AMCase and chitotriosidase are secreted by macrophages in response to chitincontaining pathogens and has been shown to inhibit hyphal growth of chitinous fungi such as Candida and Aspergillus [56, 58] . Despite the link between chitin and the recruitment of type 2 inflammation in the lung [52] , a recent study has demonstrated no role for AMCase in the generation of allergic airway pathology [85] . In this study we demonstrate for the first time, macrophage chitinase activity in response to a protozoan pathogen. Chitin recognition is thought to be a size dependent process and involve a combination of TLR2 and scavenger receptors such as MMR and dectin-1 [49, 58] . Here we have demonstrated the presence of such scavenger receptors in association with cysts and it will be of interest in future studies to determine the role of these molecules in cyst containment during Toxoplasma infection. Independent of the receptors involved it is likely that this is a contact-dependent process and indeed, cysts were unable to induce urea production or chitinase activity in macrophages when separated by transwell membranes. Furthermore, analysis of the location of AMCase producing cells in the brain finds them reliably close to cysts and often in direct contact with reactivating or rupturing cysts. These images show that despite the presence of many DAPI positive cells surrounding the cyst structure, the escape of parasites through the cyst wall occurs juxtaposed to the macrophage or AMCase activity. Our data suggest that the presence of cyst antigens induces alternative activation of macrophages and that these antigens are required for macrophages to produce chitinase even in the presence of IL-4. Thus alternative activation of macrophages is not sufficient for AMCase production and chitinase activity. The significant increase of the non-enzymatically active chitinase-like molecule YM-1 in infected brains is consistent with previous reports of AMCase and YM-1 being co-expressed specifically in macrophages and not epithelial cells [63] .
Live imaging in vitro demonstrated AMCase dependent degradation of cysts as shown using both a chitinase inhibitor and AMCase2/2 macrophages. Although there is no evidence of an active chitinase produced by T. gondii (ToxoDB), the similar cyst survival times observed for AMCase-null macrophages and chitinase-inhibited macrophages exclude the possibility that bradyzoites are the source of enzymatic activity and are breaking out of the cyst. The chitin dependent induction of chitinase activity implies that macrophages have access to the chitin in the cyst wall Figure 6 . AMCase2/2 mice have a higher parasite burden in the brain and succumb to infection during the chronic stage. (A-F) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii. Serum was isolated from whole blood samples at days 3, 7 and 14 post infection and analyzed for (A) IFN-c, (B) IL-6, (C) MCP-1, (D) TNF-a, (E) IL-12p70 (F) At day 7 DNA was isolated from the lungs and analyzed for parasite burden using qPCR. Results are displayed as parasites per mg tissue. (G-K) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii and sacrificed at 5 weeks following infection. Brains were harvested and analyzed for cyst burden, cellular composition and histology. (G) Cyst counts were obtained from homogenized whole brain samples. (H) Cyst area, 20 cysts from each mouse were photographed microscopically and cyst area was determined using ImageJ software. (I) DNA from brains of WT and AMCase2/2 was isolated and analyzed for parasite burden using qPCR. (J) Whole brains were fixed, frozen and stained for H&E to examine cyst burden and pathology. (K) BMNCs were isolated and analyzed for expression of CD4+ T cells, CD8+ T cells, macrophages (CD45 hi /CD11b+) and microglia (CD45 hi /CD11b+) by flow cytometry. Significance was determined using log rank test with p = 0.0177. Data are representative of at least 2 individual experiments with a minimum of n = 4 and are represented as mean 6 SEM, ns = not significant, * p,0.05, ** p,0.01. (L) Survival data from C57Bl/6 (squares, n = 7) and AMCase2/2 (triangles, n = 7). Data are representative of 4 individual experiments with C57Bl/6 (n.40) and AMCase2/2 (n = 40) and significance tested using Logrank (Mantel-Cox) and Gehan-Breslow Wilcoxan test * p,0.05. doi:10.1371/journal.ppat.1002990.g006 prior to chitinase-mediated cyst destruction. The prevailing view from ultrastructure studies is that cysts remain intracellular within neurons [15, 86, 87] (A. Koshy and J. Boothroyd personal communication) yet analysis of cyst burden over time shows a reduction in cyst numbers implying some form of effector mechanism in place [16] . Several studies have demonstrated perforin dependent control of cyst burden during chronic infection [17, 18] . We suggest that instead of a direct effect of perforin on cysts, it is most likely that perforin production by CD8+ T cells may initiate this process by lysing the cyst infected cell, thus exposing the cyst wall to chitinase activity from macrophages. This model would explain the many observations of macrophages in close association with rupturing cysts [8, 17, 19] . Of note, we found that BALB/c macrophages that are more easily alternatively activated had enhanced cyst lysis activity when compared to C57Bl/6 ( Figure S7 ). This may be one explanation for the increased resistance to toxoplasmic encephalitis in BALB/c mice [30, 88] .
Although AMCase activity is not required for protective immunity during acute infection it is required for protection during the chronic stage of infection. Our observation of a higher cyst count and parasite burden as well as decreased survival in AMCase-null mice points to a specific and important role for chitinase mediated cyst lysis in the brain. Thus, within the brain, cyst containment seems as important as the killing of free parasites in the control of pathology. In addition, continuous chitinmediated attack by macrophages and the release of parasites from latent cysts will provide a constant source of antigenic stimulation for the immune response. This latter discovery may provide an explanation for the continuous recruitment of T cells into the brain.
It has been apparent for some time that cyst numbers in the brain can be controlled endogenously yet identification of the exact effector mechanisms has not been so apparent. In these studies we demonstrate the presence of a distinct population of macrophages in the brain during chronic Toxoplasma infection, which express CXCR3, MMR, stabilin-1 and arginase-1. Furthermore these macrophages have chitinase activity, are localized to cysts and are observed in association with cyst degradation. The mechanism of cyst lysis is dependent on AMCase and this enzyme is required for survival during chronic infection to reduce parasite burden. The continuous presence of Toxoplasmic cysts in the brain and other tissues presents a constant threat of reactivation to the immune compromised patient. Mechanisms that enhance cyst removal or prevent their reactivation during Toxoplasma or other protozoan infections would provide a novel line of anti-parasitic therapies.
The experiments in this study were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at University of California, Riverside. All efforts were made to minimize animal suffering during the course of these studies.
T. gondii Pruigniund and RH strains were maintained in vitro as previously described [89] . Soluble toxoplasma antigen (sTAg) was prepared from RH strain tachyzoites as previously described [90] . The Pruigniud strain was used for in vitro tachyzoite infections at a ratio of 3:1. The Me49 strain of T. gondii was maintained in infected Swiss Webster and CBA/CaJ mice. For infection, brains from infected mice were removed placed in 3 ml sterile 16PBS and passed 3-5 times through an 18.5 gauge followed by 20.5 and 22.5 gauge needle. The number of cysts in a 30 ml aliquot was determined microscopically. Brain suspensions were adjusted to 100 cysts/ml and mice were infected each with 20 cysts intraperitoneally. Infection studies of C57Bl/6 and AMCase null mice were conducted at least four times with a minimum of 7 biological controls. C57Bl/6, CBA/CaJ (Jackson, Bar Harbor, ME) and Swiss Webster mice (Charles River, Wilmington, MA) were maintained in a pathogen free environment under IACUC established protocols at the University of California Riverside. AMCase-null mice were generated by targeting exon 5 using loxP/CRE recombination as previously described [85] . The AMCase gene deleted mice were of a mixed background, C57BL/6NTac:129SvEvBrd, and were backcrossed to C57/BL6 for at least 10 generations. These mice were generated and maintained under IACUC protocols established by Pfizer.
A single cell suspension from spleens was prepared by passing through a nylon 40 mm cell strainer (BD, San Jose, CA). Suspensions were washed with RPMI complete (10% FCS, 1%Pennicilin/Streptomycin, 1% Glutamine, 1% Sodium Pyruvate, 1% nonessential amino acids, 0.1% B-mercaptoethanol) (Life Technologies, Grand Island, NY) and centrifuged for 5 minutes at 1200 RPM at 4uC. Red Blood Cells were lysed using 0.86% ammonium chloride solution, centrifuged and resuspended in RPMI complete. BMNCs were prepared as previously described [89] .
BMNCs or splenic cells were incubated with various conjugated antibodies against CXCR3, CD3, CD4, CD8, CD11b, IL-10, and CD45, (eBioscience, San Diego, CA) and MMR (Biolegend, San Diego, CA). Cells were analyzed using the BD FACSCanto II flowcytometer (BD Biosciences, San Jose, CA) and FlowJo analysis software v.8.7.3 (Treestar Software, Ashland, OR). Cell populations were determined by gating on CD4+, CD8+, CD45 hi / CD11b+ (macrophages) and CD45 int /CD11b+ (microglia) from live cell gate.
Total RNA from brain tissue samples was extracted with TRIzol reagent (Life Technologies, Grand Island, NY) according to manufacturer's instructions. DNase1 treatment and first strand cDNA synthesis was performed using cDNA synthesis kit (BioRad, Hercules, CA) according to the manufacturer's instructions. CXCR3, CXCL9, CXCL10, CCL2, CCL5, AMCase, Arg1, and Chit1 specific primers for Real Time PCR were purchased from IDT's primer Quest (http://www.idtdna.com/Scitools/ Applications/Primerquest/). Primer sequences were as follows: 
Immediately following excision, brains were bisected sagitally and flash-frozen in cold isopentane. Frozen brains were then put into standard Tissue-Tek cryomold and filled with Optimal Cutting Temperature (OCT) solution (Tissue-Tek, Torrance, CA) and put on dry ice and subsequently stored at 280uC. Serial sections of 10-20 mm were prepared on a standard Cryostat machine (LEICA/CM1850, Simi Valley, CA). Frozen tissue sections were fixed 75% acetone/25% ethanol then blocked in 10% donkey serum prior to incubation with purified antibodies. Purified primary antibodies for Iba-1 (Wako, Richmond, VA), CXCR3 (Life Technologies, Grand Island, NY), MMR (AbD Serotec, Raleigh, NC) arginase-1, AMCase and stabilin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) as well as biotinylated tomato lectin (Sigma-Aldrich, St. Louis, MO) were incubated with tissue samples for 2 h at RT or overnight at 4uC, and followed with appropriate secondary antibodies conjugated to Alexa 488, Alexa 568, or Alexa 647 at 2 mg/mL (Life Technologies, Grand Island, NY). Samples were mounted in Prolong Gold with DAPI (Life Technologies, Grand Island, NY) for nuclear counterstaining. Images were collected on a Leica SP2 scanning confocol microscope (Leica Optics, Germany), and analyzed using Improvision Volocity 5.0 (Perkin-Elmer, Waltham, MA). Distance of cells from cysts were calculated from confocal images of at least 12 cysts and at least 6 AMCase expressing cells per cyst.
Parasite burden was measured by amplifying the T. gondii genes B1, SAG1, SAG4, or MAG1 by real-time PCR as previously described [71, 89] .
In vivo peptide blocking C57BL/6 mice were infected i.p. with 10 4 Pruigniund tachyzoites. At day 21, 23, 25, and 27 post infection the animals were injected i.p. with either 0.5 ml a-CXCL10 (0.5 mg/mL), 0.5 mL a-CXCR3 (polyclonal), or 0.5 ml PBS as previously published [76, 91] . The mice were sacrificed on day 28 p.i. and brains were excised for flow cytometric analysis, and parasite burden as described above. Blocking studies were conducted twice with at least 5 biological replicates.
Supernatants from infected macrophage cultures were added to a 96 well UV plate at 50 ml per sample in triplicates. Urea reagents A and B were mixed from quantichrom urea assay kit (Bioassay systems, Hayward, CA) and 200 ml of mixture added to each well. Included standard was used starting at 50 mg/ml and diluted 2 fold. Samples were incubated for 30 min at room temperature and plates were read at 520 nm to determine urea concentration. 
Femurs and tibias were obtained from 6-12 week old C57BL/6 mice. After euthanasia, the mice were sprayed with 70% ethanol and the femurs and tibias were dissected using scissors. Muscles connected to the bone were removed using scissors, and the femurs were placed into a 50 mL tube containing sterile DMEM on ice. In a tissue culture hood, the bones were washed in sterile DMEM and then both epiphyses were removed using sterile scissors and forceps. The bone marrow was flushed out with a 10 ml syringe filled with BM20 differentiation media (DMEM supplemented with 10% fetal bovine serum, 20% L929 supernatant, 5% horse serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine) (Life Technologies, Grand Island, NY) into a 50 mL sterile tube. The tube was vortexed gently and topped off to 50 mL with fresh BM20. 10 mL of cell suspension was plated out on 100 cm untreated dishes and incubated for 7 days at 37uC, 5% CO 2 with fresh media added at day 4. Cells were then washed, counted and plated at 10 6 cells/mL in BM10 media (DMEM supplemented with 10% fetal bovine serum 10% L929 supernatant, 5% horse serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine) (Life Technologies, Grand Island, NY) into a 50 mL sterile tube and allowed to rest for 3 days. Macrophages were stimulated overnight with either recombinant IL-4 (10 ng/ml), LPS (50 ng/ml) or IFN-c (100 U/ml) (all from R&D Systems, Minneapolis, MN), stAg (100 mg/ml) or cystAg (100 mg/ml) in complete DMEM.
To observe the interaction of macrophages and cysts in vitro, cysts were isolated from the brains of chronically infected mice as described above. 50 cysts were added per well to 96 well plates containing 2610 5 bone marrow derived macrophages. Cysts and cells were viewed using a BD HT Pathway 855 microscope (BD Biosciences, San Jose, CA) in a climate-controlled chamber (37uC, 5% CO 2 ). Nine cysts were identified per condition and photographed every 10 minutes for 14 or 16 hours. Movies were compiled using ImageJ software (NIH, Bethesda, MD) and cyst survival time was determined.
For statistical analysis of survival data, the log-rank and Gehan-Breslow Wilcoxon test was used and involved over 40 C57Bl/6 and 40 AMCase2/2 mice. Acute (0-14 days) and chronic deaths (.14days) were analyzed individually. For all other data, an unpaired, two-tailed Student's t test, or ANOVA test with a 95% confidence interval was used (Prism; GraphPad Software, Inc., La Jolla, CA). All data are represented as means 6 SEM. Figure S3 AAMØ associated with cyst lysis. Confocal fluorescence microscopy of 20 mm brain slices taken from mice at 4 weeks post infection. Imunohistochemical analysis of alternatively activated macrophage (Iba-1, red) as judged by its expression of stabilin-1 (green), adhering closely to a large round cyst. Polarized, to the site of macrophage 'attachment', bradyzoites are seen escaping in an organized fashion towards or into the AAMØ (arrows). (TIF) Figure S4 Chitinase activity is dependent on the presence of chitin and is independent of IL-4 activation. A) Bone marrow derived macrophages were analyzed for chitinase activity. Macrophages were cultured with whole cysts, cysts treated with trichoderma chitinase or media alone. Data are representative of at least 2 individual experiments with a minimum of n = 3 and are represented as mean 6 SEM. B) qRT-PCR was conducted on BMNC to measure YM-1, YM-2, RELM-a, BRP39, IL-4 and IL4Ra. Data are presented as fold increase over naïve. (TIF) Figure S5 AMCase activity associated with cyst. Confocal fluorescence microscopy of 20 mm brain slices taken from mice at 4 weeks post infection. Immunohistochemical analysis of macrophage (Iba-1, green) and AMCase (red), arrows point to AMCase polarized to the cyst wall. (TIF) Figure S6 AMCase2/2 polarization and infection studies. BMDM from WT and AMCase2/2 mice were polarized to A) M2 and B) M1 phenotype as measured by Urea and Greiss assays respectively. C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii and sacrificed at C) 3 weeks for cyst counts and D) 5 weeks following infection. RNA was isolated from infected brains, reverse transcribed, and the resulting cDNA was analyzed for SAG1, SAG4, and MAG1 transcript levels using qRT-PCR to measure gene expression from tachyzoites, bradyzoites and cysts, respectively. Results are shown as absolute quantitation of copy number using standard curve. Data are representative of at least 3 individual experiments with a minimum of n = 3 and are represented as mean 6 SEM, ** p,0.01, *** p,0.001. (TIF) Figure S7 BALB/c macrophages lyse cysts more quickly than C57Bl/6 macrophages. BMDM from BALB/c and C57Bl/6 mice were cultured with cysts and imaged using an HT pathway microscope for 16 hours. Images were collected every 10 minutes and cyst survival time was calculated. (TIF) Video S1 3D colocalization of AMCase-secreting macrophages and parasite cysts. Three dimensional z-plane progression of 20 mm confocal image taken from mice at 4 weeks post infection. Green: tomato lectin labeling macrophages; red: AmCase; blue: DAPI labeling nuclei; white: anti-Toxoplasma. (MOV) Video S2 Cysts do not lyse in the absence of macrophages. 14 hour time-lapse movie of cysts cultured in the absence of BMDM. Images were collected every 10 minutes and movies were compiled using ImageJ. (MOV) Video S3 Rupture of T. gondii cysts in the presence of chitinase. 14 hour time-lapse movie of cysts co-cultured with WT BMDM and pretreated with 10 mg/ml trichoderma chitinase. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S4 Untreated Cysts cultured with bone marrowderived macrophages. 14 hour time-lapse movie of cysts co-cultured with untreated BMDM. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S5 Cysts cultured with BMDM and treated with allosamidin. 14 hour time-lapse movie of cysts co-cultured with WT BMDM and treated with 100 mM allosamidin. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S6 Cysts cultured with untreated WT BMDM. 16 hour time-lapse movie of cysts co-cultured with BMDM from WT mice. Images were collected every 10 minutes and movies were compiled using ImageJ. (MOV)
Video S7 Cysts cultured with untreated AMCase-null BMDM. 16 hour time-lapse movie of cysts co-cultured with BMDM from AMCase-null mice. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S8 Cysts cultured with WT BMDM pretreated with LPS/IFN-c. 16 hour time-lapse movie of cysts co-cultured with BMDM from WT mice, pre-treated with LPS/IFN-c. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S9 Cysts cultured with WT BMDM pretreated with IL-4. 16 hour time-lapse movie of cysts co-cultured with BMDM from WT mice, pre-treated with IL-4. Images were collected every 10 minutes and movies were compiled using ImageJ.
Video S10 Me49-RFP cysts cultured with WT BMDM labeled with CellTracker green. 16 hour time-lapse movie of Me49-RFP cysts co-cultured with CellTracker green labeled BMDM from WT mice. Images were collected every 10 minutes and movies were compiled using ImageJ. (MOV)
Video S11 Me49-RFP cysts cultured with AMCase-null BMDM labeled with CellTracker green. 16 hour time-lapse movie of Me49-RFP cysts co-cultured with CellTracker green labeled BMDM from AMCase-null mice. Images were collected every 10 minutes and movies were compiled using ImageJ. (MOV) Automatic Detection and Quantification of Tree-in-Bud (TIB) Opacities from CT Scans This study presents a novel computer-assisted detection (CAD) system for automatically detecting and precisely quantifying abnormal nodular branching opacities in chest computed tomography (CT), termed tree-in-bud (TIB) opacities by radiology literature. The developed CAD system in this study is based on 1) fast localization of candidate imaging patterns using local scale information of the images, and 2) Möbius invariant feature extraction method based on learned local shape and texture properties of TIB patterns. For fast localization of candidate imaging patterns, we use ball-scale filtering and, based on the observation of the pattern of interest, a suitable scale selection is used to retain only small size patterns. Once candidate abnormality patterns are identified, we extract proposed shape features from regions where at least one candidate pattern occupies. The comparative evaluation of the proposed method with commonly used CAD methods is presented with a dataset of 60 chest CTs (laboratory confirmed 39 viral bronchiolitis human parainfluenza CTs and 21 normal chest CTs). The quantitative results are presented as the area under the receiver operator characteristics curves and a computer score (volume affected by TIB) provided as an output of the CAD system. In addition, a visual grading scheme is applied to the patient data by three well-trained radiologists. Interobserver and observer–computer agreements are obtained by the relevant statistical methods over different lung zones. Experimental results demonstrate that the proposed CAD system can achieve high detection rates with an overall accuracy of 90.96%. Moreover, correlations of observer–observer [Formula: see text] , [Formula: see text] and observer–CAD agreements [Formula: see text] , [Formula: see text] validate the feasibility of the use of the proposed CAD system in detecting and quantifying TIB patterns. I NFECTIOUS lung diseases, such as novel swine-origin H1N1 influenza, tuberculosis (TB), etc., are among the leading causes of disability and death all over the world [1] - [3] , [5] . Computed tomography (CT) examination of the lungs during acute respiratory tract infections has become an important part of patient care, both at diagnosis and monitoring progression or response to therapy. Although CT examination serves as a primary (imaging) diagnostic tool for assessing lung infections, visual analysis of CT images is restricted by low specificity for causal infectious organisms and a limited capacity to assess severity and predict patient outcomes [2] .
Common CT findings associated with respiratory tract infections include tree-in-bud (TIB) nodularity, ground-glass opacities (GGO), random distribution of pulmonary nodules, linear interstitial/bronchovascular thickening, and consolidations [6] . Although none of these visual patterns are specific for one pathogen, the amount of lung volume exhibiting these features could provide insights into the extent or severity of infection. Among these patterns, TIB opacities, represented by thickened bronchial structures surrounded locally by clusters of 2-3 mm micronodules, are associated with inflammation of the small airways (bronchioles), such as in viral or bacterial bronchiolitis, and the increasing sizes of abnormal regions on CT can suggest the progression of disease [6] . Often considered to have a limited differential diagnosis-M TB infection, infection with nontuberculous mycobacteria, viral infection, cystic fibrosis, this pattern is recognized as a CT appearance of many different entities. Unlike the other imaging patterns such as GGO and consolidations, it is an extremely challenging task to detect and quantify the regions with TIB opacities due to interobserver variations and inconsistent visual scoring methods [2] . Therefore, an accurate method for detecting TIB is a critical in computer-assisted detection (CAD) schemes from chest CT. Although the correct diagnosis for TIB pattern is very important, it is also one of the most difficult tasks for radiologists because the contrast of lesions is often low and the disease patterns are very complex. All these limitations suggest that CAD could make a valuable contribution to the management of respiratory tract infections by assisting in the early recognition of pulmonary parenchymal lesions and providing quantitative measures of disease severity. 
Respiratory tract infections, caused by viruses, bacteria, fungi, and parasites, are a major component of global infectious disease mortality. TIB patterns, in particular, usually represent the disease of the small airways such as infectious-inflammatory bronchiolitis as well as bronchiolar luminal impaction with mucus, pus, cells, or fluid causing normally invisible peripheral airways to become visible on CT [7] . Fig. 1 shows typical TIB patterns in a chest CT ( Fig. 1 shows labeled TIB patterns with blue). As its name implies, this pattern resembles a budding tree in CT due to the branching opacities with adjacent centrilobular nodularity [2] . It is not specific for a single disease entity, but suggests pathology in the peripheral airways, which can be associated with air trapping or subsegmental consolidation in the surrounding alveolar airspaces. Because any organism that infects the small airways can cause a TIB pattern, pulmonary infections are its most common cause.
TIB is difficult to be detected with conventional CAD systems due to high complexity of their irregular shapes, as well as strong textural similarity of micronodules and thickened airways to other normal and abnormal lung structures. Currently, no reported CAD system is capable of automatically detecting a TIB pattern, therefore, which warrants a need for the development of such a system to improve the diagnostic decision process and quantitative measurement of respiratory tract infections. In this paper, we develop a new CAD system to evaluate respiratory tract infections by automatically detecting and quantifying TIB patterns on CT images.
The main contributions of this study are twofold. 1) A candidate selection method that locates possible abnormal patterns in the images. This process comes from a learning perspective such that the size, shape, and textural characteristics of TIB patterns are learned a priori. The candidate selection process removes large homogeneous regions from consideration which results in a rapid localization of candidate TIB patterns. The local regions enclosing candidate TIB patterns are then used to extract shape and texture features for automatic detection; 2) another novel aspect in this study is to extract Möbius invariant local shape features (i.e., Willmore energy-based features). Extracted local shape features are combined with statistical texture features to classify lung tissues. In addition, we also investigate the extraction and use of different local shape features as compared to the proposed shape features to facilitate local structure analysis. To the best of our knowledge, this is the first study that uses automatic detection of TIB patterns for a CAD system in infectious lung diseases. Since there is no published work on automatic detection of TIB patterns in the literature, we compare our proposed CAD system on the basis of different feature sets previously shown to be successful in detecting lung diseases in general. Early version of this study appeared in [3] , and can be accessed in [8] .
This paper is organized as follows. Section II explains the methods of the proposed CAD system. We discuss our proposed and conventional feature extraction methods in Section III. Next, we present the feasibility of the proposed CAD system by evaluating the detection and quantification performances in Section IV followed by a discussion and conclusion in Section V and Section VI, respectively.
The proposed CAD methodology is illustrated in Fig. 2 . First, lungs are segmented from chest CTs. Second, we use locally adaptive scale-based filtering method to detect candidate TIB patterns. Third, segmented lung is divided into local patches in which we extract Möbius invariant shape features and statistical texture features followed by support vector machine (SVM) classification. We extract features from local patches of the segmented lung only if there are candidate TIB patterns in the patches. The details of the proposed methods are described in the following. 
Prior to detection, segmentation is often the first step in CAD systems. In this study, fuzzy connectedness (FC) image segmentation algorithm is used to achieve successful delineations [9] . In FC framework, as illustrated in Fig. 3 , left and right lungs are "recognized" by user-defined or automatically assigned seeds, which initiate FC segmentation. In this study, one seed per lung volume (i.e., left or right) is automatically set by only considering the locations of small intensity valued voxels inside the body region (see [9] for a detailed description of the use of FC in anatomy segmentation). Fig. 3 (middle and right) shows the resulting segmentation of the chest CT given on the left. Although we use FC algorithm to segment lung regions, there are many well-established lung segmentation methods in the literature [9] - [11] , [29] , and [41] such that they could possibly be used as well to accomplish the delineation step. In that sense, we do not have any strict restriction on the choice of segmentation algorithm prior to detection system as long as it successfully segments the lung regions.
The size/volume of a region occupied by a typical TIB pattern does not usually exceed a few mm 2 /mm 3 . Together with the fact that TIB pattern has a complex shape with varying intensities over discontinues branches (i.e., buds), TIB patterns have intensity characteristics with high variation toward nearby voxels (see Fig. 1 ). In other words, TIB patterns do not constitute sufficiently large homogeneous regions. Thus, TIB patterns are localized only in the vicinity of small homogeneous regions, and their boundaries have high curvatures due to the nature of its complex shape. In the next section, we use these two observations to extract novel characteristic features to detect TIB patterns.
Our candidate selection method comes from a learning perspective such that we assign every internal voxel of the lung a membership value reflecting the size (i.e., scale) of the homogeneous region that the voxel belongs to. To do this, we use a locally adaptive scale-based filtering method called ball-scale (or b-scale for short) [9] , [16] , [17] . The b-scale is the simplest form of a locally adaptive scale where the scene is partitioned into several scale levels. Every voxel in each scale is assigned the size of the local structure it belongs. For instance, voxels within the large homogeneous objects have highest scale values, and the voxels nearby the boundary of objects have small-scale values. Voxels on the boundary of objects have smallest scale values. Because of these observations, we conclude that TIB Fig. 4(c) . We describe the computation of b-scale patterns and the details of the candidate selection process in the next section.
There are several advantages to the local scale-based approach. For instance, boundary-and region-based representations of objects are explicitly contained in the scale-based methods. Based on continuity of homogeneous regions, geometric properties of objects (i.e., size information) can be identified, and this new representation is called scale images, i.e., b-scale, tensor-scale (t-scale), generalized-scale (g-scale) images [16] , [18] , [19] . The b-scale model has been shown to be extremely useful in object recognition [17] , image segmentation [9] , [41] , filtering [16] , inhomogeneity correction [20] , and image registration [20] , [21] . In this study, on the other hand, we show how to use b-scale encoding together with a proper scale selection method for detecting candidate abnormality patterns. The main idea in b-scale encoding is to determine the size of local structures at every voxel as the radius of the largest ball centered at the voxel within which intensities are homogeneous under a prespecified region-homogeneity criterion.
Although the conventional b-scale encoding method is well established for nD images (n ≥ 2), we use 2-D b-scale encoding method in this study because low-resolution CT data do not allow continuous analysis of TIB patterns through lowresolution imaging direction. In the 2-D digital space (Z 2 , ν), a scene C = (C, f ) is represented by a pair where C is a rectangular array of voxels, ν = (ν 1 , ν 2 ) indicates the size of the voxels, and f is a function that assigns to every voxel an image intensity value. A ball B k (c) of radius k ≥ 0 and with center at
The fraction of object is denoted by F O k (c) and indicates the fraction of the ball boundary occupied by a region which is sufficiently homogeneous with c.
and W ψ is a homogeneity function [9] . In all experiments, we use a zero-mean unnormalized Gaussian function for W ψ . The size of the local structure is estimated using appearance information of the gray-level images, i.e., region-homogeneity criterion; b-scale scenes contain rough geometric information.
A detailed description of W ψ and F O k,ν is presented in [9] . The b-scale algorithm works as follows: the ball radius k is iteratively increased starting from one, and the algorithm checks for F O k,ν (c), the fraction of the object containing c that is contained in the ball. When this fraction falls below a predefined threshold, it is considered that the ball contains an object region different from that to which c belongs [16] . This process is repeated for every voxel within the scene. Voxels are assigned their b-scale values discreetly from 1 to r max . 1 In principle, b-scale partitions the scene into several levels based on the size of local structures from 1 to r max . Computing b-scale values for every voxel leads b-scale scenes as shown in Fig. 4 (a). Note also that locally adaptive scale in regions with fine details or in the vicinity of boundaries is small, while it is large in the interior of large homogeneous objects.
Developing a successful CAD system for infectious lung diseases requires acquisition of representative features characterizing shape and texture of TIB patterns efficiently. Since TIB is a complex shape pattern consisting of curvilinear structures with nodular structures nearby, we propose to use local shape features (derived from geometry of the local structures) combined with gray-level statistics derived from a given local patch (i.e., local window with a predefined size).
The shape operator is the second-order invariant (or curvature) which determines the original surface. Since it is usually more convenient to work with scalar quantities rather than vectorial shape quantities, symmetric functions of local Hessian matrices are usually used to extract geometric meaning of the surface/shape of interest. Therefore, curvatures play an important role in the representation and recognition of intrinsic shapes. However, similarity of curvature values may not necessarily be equivalent to intrinsic shape similarities, which causes a degradation in recognition and matching performance.
To overcome this difficulty, we propose to use Willmore energy functional [22] and several different affine invariant shape features parametrically related to the Willmore energy functional. While local shape features characterize the curvilinear and small nodular structures (via Willmore energy), gray-level features characterize background and foreground intensity variation with objects' pose and size for a given local window. Moreover, for comparison purpose, we use different feature sets previously shown to be successful in detecting lung diseases in general. Fig. 5 enlists all the features that we extracted for the proposed CAD system and for the experimental comparison. Details of extracted features are defined in the following.
The Willmore energy of surfaces plays an important role in digital geometry, elastic membranes, and image processing [23] . It is closely related to Canham-Helfrich model [24] , where a surface energy is defined as
where α, β, and γ are some constants, H is the mean curvature vector on Σ (area space), K is the Gaussian curvature on ∂Σ (boundary space), and dA is the induced area metrics on Σ. This model is curvature driven, invariant under the group of Möbius transformations (in particular, under rigid motions and scaling of the surface) and shown to be very useful in energy minimization problems [25] . Invariance of the energy under rigid motions leads to conservation of linear and angular momenta, and invariance under scaling plays a role in setting the size of complex parts of the intrinsic shapes (i.e., corners, wrinkles, folds, etc.). In other words, the position, gray-level characteristics, size, and orientation of the pattern of interest have minimal effect on the extracted features as long as the suitable patch is reserved for the analysis. In order to have simpler and more intuitive representation of the given model, we simply set α = 0 and β = γ = 1, and the equation turns into Willmore energy functional
where ds is the length metric on ∂Σ. The resultant energy of a surface can be regarded as a function H and K, and captures the deviation of a surface from local sphericity [22] such that a sphere has zero Willmore energy. Note also that the Willmore energy is always nonnegative. Since a homogeneity region that a typical TIB pattern appears is small in size, total curvature (or energy) of that region is high and can be used as a discriminative feature.
The main motivation in describing intrinsic shapes by Willmore energy is due to its ability to encode surface (i.e., image area in 2-D) with Möbius invariant features (translation, contrast, rotation, and inversion invariant). In addition to Willmore energy features that we adapt from Canham-Helfrich surface model, we have included seven different local shape features, which are parametrically related to Willmore energy formulation, into the proposed CAD system due to their some invariant properties and discriminative powers. Assume κ 1 and κ 2 indicate eigenvalues of the local Hessian matrix H e for any given local patch L , the following shape features are extracted: 1) shape index (SI), 2) Gaussian curvature, 3) mean curvature, 4) elongation, 5) distortion, 6) shear, 7) compactness.
The SI is a statistical measurement and used to define intrinsic shape of the localized structure within the image [26] , [27] . SI values are encoded as a continuous range of values between −1 and 1, with zero SI indicates saddle-like local structures, +1 and −1 SI values indicate umbilical minima and maxima (i.e., cap and cup, respectively), and midpoints of the two half-intervals (+0.5 and −0.5) indicate concave and convex parabolic or line-like structures (i.e., rut and ridge, respectively). SI can simply be computed through principal curvatures (κ 1 , κ 2 ) as follows:
where κ 1 ≥ κ 2 . As suggested in [26] , we obtain principal curvatures from the eigenvalues of the local Hessian matrix (H e ) as
where L xx , L xy = L y x , and L y y are second-order derivatives of local image patch L , and eig() denotes eigenvalue decomposition. We choose to use SI because of its invariance property with respect to rotation, absolute gray value, and translation.
2) Gaussian Curvature: Gaussian curvature (K) is an intrinsic measure and simply the product of the principal curvatures as K = κ 1 κ 2 for a given point on a surface, equivalent to the determinant of local Hessian matrix H e . Note that K is unchanged even by bending the surface without stretching it, meaning that the Gaussian curvature is independent of the choice of unit normal and it gives three types of classified local shapes: elliptic shape (K > 0), hyperbolic shape (K < 0), parabolic shape (K = 0) with one of the κ is equal to zero, planar shape (K = 0) with both κ are equal to zero. Gaussian curvature is translation and rotation invariant, but not scale invariant.
3) Mean Curvature: Mean curvature (H) is an extrinsic measure and it describes the curvature as H = (κ 1 + κ 2 )/2. Unlike K, H is defined in the distributional sense. Note that mean curvature measure is the trace of local Hessian matrix H e . Mean curvature can be thought as a negative gradient (as a Laplacian) of the area functional due to its nice variational interpretation over the surface. This does not only give insights into the size of the local shape but also into the total symmetrical deviation from the sphere. Mean curvature is translation and rotation invariant, but not scale invariant. 4) Elongation: Shape elongation is one of the basic shape descriptors and it indicates flatness measure of the local shape [28] . In this paper, we used the ratio of principal curvatures to measure elongation as κ 2 /κ 1 with κ 2 ≤ κ 1 . Elongation measure is invariant with respect to a similarity transformation, and therefore, it is a robust feature that helps to identify curvilinear shapes. Elongation varies from −1 to +1, from hyperbolic to elliptic points.
Distortion is an algebraic quantity defined as the difference of eigenvalues (i.e., |κ 1 − κ 2 |) of the local Hessian matrix H e . Distortion is a valuable image analysis property revealed by magnitude difference of principal curvatures. Distortion measure captures the deviation of principal curvatures, thus nonplanarity of a region. Together with Gaussian or mean curvature, distortion measure brings further information into encoding of local shape. Distortion measure is translation and rotation invariant, but not scale invariant. 6) Shear: The shear is another algebraic distortion quantity defined as proportional to the normalized distortion: (κ 1 − κ 2 ) 2 /4. The physical information contained in the shear is basically the same as that of the distortion; it is related to distortion with powers of the difference of principal curvatures. Different than distortion, shear descriptor captures higher degree of nonplanarity of a region due to having more robustness against noise. 7) Compactness: Compactness feature measures the similarity between shape of interest and a perfect ellipse, and is defined as 1/(4π √ κ 1 κ 2 ). Note that this ratio is a dimensionless ratio between the area of the shape (1 for a normalized shape) and the area of the best ellipse fitting the shape. Note also that the compactness measure is invariant to affine transformations and parametrically related to Gaussian curvature. Given a single-axial CT slice of left lung, Fig. 6 (b) indicates a thresholded (i.e., selected candidate patterns) b-scale scene encoded from the corresponding gray-level CT slice shown in Fig. 6(a) . Furthermore, Fig. 6 (c) and (d) shows mean and Gaussian curvature maps from which all the other local shape features are extracted, respectively. In addition, Fig. 6 (e) and (f) shows Willmore energy maps using both mean and Gaussian curvature maps as formulated in (4) and shown in Fig. 6(c) and (d) .
Based on the observation in training step where we analyzed the appearance and shape of TIB patterns, TIB patterns most likely occur in the regions inside the lung with high variability of intensity values over a small number of voxels and with certain size (i.e., a few millimeter in length). These observations (size and high intensity variation) facilitate one practically useful fact in the algorithm that, in the feature extraction process, we only extract features if and only if at least "one" small b-scale pattern exists in the local regions (i.e., blue local regions in Fig. 4) .
We also explore the use of alternative local shape features as a comparison to Willmore energy-based features. Based on the observations of spatial properties of the selected candidate patterns, it becomes apparent that instead of using conventional high-dimensional feature extraction methods such as Gabor wavelets, steerable wavelets, etc., one may extract much fewer and more reliable statistical features to discriminate the pattern of interest. Motivated from the fact that TIB patterns consist of numerous small (or micro-) nodules nearby the main curvilinear structure and those small structures have varying opacities, the location and distribution of those small structures can be obtained by simple thresholding method which has been popular in estimation for more than two decades [30] . However, since the opacities are varying through different nodular structures, it is challenging to find an optimum threshold value. Therefore, instead of using one single threshold level, we empirically choose n = 10 different threshold levels (λ j ) to obtain local statistics of those structures in a hierarchical way, where λ j = 10j, 1 ≤ j ≤ 10 [31] . This process is named LGS because we extract different statistical measurements in gradient of the images. Note also that we confine ourselves into the local patches where at least one b-scale pattern occupies.
To obtain shape statistics over local patches, we use gradient fields because boundary information can be used much more effectively in that sense. Fig. 7 shows an example thresholding process over a candidate TIB pattern centered at c (only for four levels are shown for demonstration purpose). After different threshold levels are applied over the local regions of b-scale images, resultant thresholded local patches are used to extract the following features: mean SI values of the local patch for each thresholding level (one feature), and the number of bscale patterns left after thresholding process (one feature). Since we use ten different thresholding levels, we extract 20 features totally. Moreover, for a local region centered at a voxel c of a candidate TIB pattern, we extract one global feature as an SI value of the voxel c, three features as the maximum, minimum, and mean SI values over the local region prior to thresholding. Therefore, a total of 24 features (LGS+SI) are extracted from a typical local patch to be used in CAD system [4] . Although n and λ j are chosen empirically based on the observations of shape and textural characteristics of normal and TIB patterns during the training step, one may propose to use cross validation, control of the global and local false discovery rate, and uncertainty principles to decide those parameters near-optimally [30] .
Steerable Features: It has been well documented in the literature that decomposition of images by using basis functions localized in spatial position, orientation, and scale (e.g., wavelets) are extremely useful in object recognition and detection [32] , [33] . Since steerable filters are rotation and translation invariant, they accurately represent the underlying image structure [34] . In this study, we use steerable derivative of Gaussian filters to decompose local regions around each candidate pattern into several oriented basis. These basis are used as features in voxelwise classification for TIB identification. We extract steerable features (i.e., directional derivatives) from one scale and six different orientations.
Gray-Level Co-Occurrence Matrix (GLCM) Features: Spatial statistics based on GLCM [35] are shown to be very useful in discriminating and quantifying patterns pertaining to lung diseases. As texture can give a lot of insights into the classification and characterization problem of poorly defined lesions, regions, and objects, we combine our proposed shape-based invariants with Haralick's popular GLCM-based features [35] . We extract 18 features from each local patch including autocorrelation, contrast, entropy, variance, dissimilarity, homogeneity, cluster shade, energy, maximum probability, sum of squares of variance, sum of averages, sum of variance, sum of entropy, difference of entropy, difference of variance, normalized inverse difference moment, cluster prominence, and mutual information. Readers are encouraged to refer to [35] for further details on these well-established features in machine learning, and [12] - [15] for particular CAD systems in identification of lung abnormalities from CT scans in general.
Laboratory confirmed (with pathology identification tests) 39 CTs of human parainfluenza (HPIV) infection and 21 normal lung CTs were collected for the experiments. All patients were imaged at our institution using a 64-detector row Philips Brilliance or a 320-detector row Toshiba Aquilion CT scanner. The noncontrasted chest CT studies were performed at end inspiration with 1.0 or 2.0 collimation obtained at 10-or 20-mm intervals from the base of the neck to upper abdomen with a tube voltage of 120 kV and a current of 200-320 mA depending on the subject's weight. Imaging data were constructed to 512 × 512 matrices with slice thickness of 5 mm. The in-plane resolution was affected by patients' size and varied from 0.62 to 0.82 mm. All 60 CT scans (both HPIV and normal) were collected from different subjects (no multiple scans from subjects).
A well-trained radiologist [with more than nine years experience (DMJ)] carefully examined the complete scan (i.e., 60 CTs) and labeled the lung regions as normal and abnormal (with TIB patterns) (see Fig. 1 ). As many regions as possible showing abnormal lung tissues from 39 HPIV patients were labeled (see Table I for details of the number of regions used in the experiments). Those 39 patients do not include only TIB opacities, but also GGO, nodules, consolidations, and linear thickening such that only TIB regions are labeled in training step. Note also that the control group consisting of 21 subjects with no observed lung abnormalities was constructed and lung tissues pertaining to this group were labeled carefully.
In the training step, we also explored how the number of b-scale patterns change for normal and diseased subjects. Our observations from detail analysis in candidate selection part showed that only 21-40% of the segmented lung volumes were chosen as candidate TIB patterns. This interval was subject to change based on the severity of the diseases. For patients without having infections (i.e., control group), for instance, the percentage of the candidate regions was smaller than the patients with infections; therefore, an increase in the amount of small-sized b-scale patterns is observed. In any case, local scale could be used as a quantitative measure validating the sensitivity and specificity of the classification rates as we describe it in Section IV-E.
Occurrences of TIB abnormality and normality of subjects were noted for each CT scan. To analyze existence and severity of abnormality as well as normality of subjects, a visual grading system was adapted from studies examining CT findings in other infections [36] - [38] . Each lung was divided into three zones (for a bilateral total of six) as shown in Fig. 8 . Zone 1 included the apex to the carina. Zone 2 extended from the tracheal carina to the left atrium's junction with inferior pulmonary veins. Zone 3 included the remainder of the lungs below the level of the inferior pulmonary veins atrial junction. A severity score (0 to 5 such that 0 indicates no abnormality) was assigned to each zone based on the percentage of the zone occupied as listed in Fig. 8 (second row). A total score was also extracted by considering all zones during visual grading. Consensus visual scores 2 from participating radiologists [one with more than nine years of experience (DMJ) and one with more than one year of experience (AW)] on a scale of 0-5 over lungs were recorded and compared with computer scores (of the proposed CAD system). Following the same visual scoring scheme, another participating radiologist [with more than seven years of experience (OA)], who was blinded to the consensus visual scores previously obtained, was involved in the visual grading process to provide information on interobserver variability.
To measure and evaluate the detection capabilities of a CAD system quantitatively, the area under the receiver operator characteristic (ROC) curves is often used [39] . After the proposed CAD system was tested via twofold cross validations with labeled dataset, we presented ROC curves of the system performances. Table I summarizes the performance of the proposed CAD system as compared to other feature sets. The performances are reported as the areas under the ROC curves (A z ). Note that proposed shape features (i.e., Willmore energy and parametrically related local shape features) alone are superior to other methods even though the dimension of the proposed shape feature is only 8. The best performance is obtained when we combine the proposed shape and GLCM features. This is to be expected because spatial statistics are incorporated into the shape features such that texture and shape features are often complementary to each other. On the other hand, compared to the proposed shape features, the LGS and SI features have lower detection rates because they are not affine (and Möbius) invariant and eventually having difficulty in appreciating the large amount of details of TIB patterns. Another reason is that there is no optimal choice of thresholding process and this may yield less remarkable statistical measurements over local patches. However, the LGS and SI features alone perform better than the high-dimensional conventional features similar to the proposed shape features. This result itself suggests the use of local shape features and their adapted extensions in detection of TIB patterns.
In what follows, we selected the best window size for each feature set and plotted their ROC curves all in Fig. 9 . Superiority of the proposed shape features is clear in all cases. To have a valid comparison, we repeated candidate selection step for all the methods because we observed that the CAD performances of compared conventional feature sets had much lower accuracies if the candidate selection part was not applied (i.e., proposed method's accuracy was decreased to A z = 0.6803, while the best result of all compared methods were decreased to A z < 0.5281). To show whether the proposed method was significantly different than the other methods, we compared the performances through paired t-tests. p-values of the tests indicate that none of the feature set are significantly correlated with the proposed CAD features such that highest and smallest pvalues are reported as 0.0195 (p < 0.05) and 0.0053 (p < 0.01), respectively.
Visual scoring by radiologists still lies at the heart of diagnostic decisions, and often used as a validation tool. In this section, we explore the correlation between computer score (i.e., CAD score) and visual scores by participating radiologists. Furthermore, we investigate the effectiveness of the proposed method's ability to roughly discriminate normal and diseased patients by only considering the size of the structures pertaining to lung anatomy.
Based on the visual grading scheme explained in Section IV-C, we compared the consensus reading of two expert observers (AW and DJM) to another expert observer (OA), who was blinded to the consensus scores. We used Pearson product-moment correlation coefficients to determine interobserver agreement over each zone, left, right, and all lung volumes. The reported correlation ratios are shown in Fig. 10(a) . Note that interobserver agreement correlation values for all TIB measurements were high for all zones and the lung. The lowest agreement seen on the zone 1 may be because subtle abnormalities in this zone may have been given greater visual assessment variance among the observers. Nevertheless, an overall correlation coefficient of R 2 = 0.8848 (p < 0.01) indicates an excellent agreement on the existence of TIB patterns.
We further analyzed the variability of change of scores of expert radiologists for each subject. For this, we constructed Bland-Altman plot [40] where the limits of observer agreements were indicated by bias ± 1.96 std (bias: average difference, std: standard deviation). In Bland-Altman plot, the difference of the performances was plotted against the average of the performances as shown in Fig. 10(b) . It was noted from this figure that the largest disagreement of the scoring between observers To obtain an overall computer score from the proposed CAD system, on the other hand, TIB regions detected by the CAD system were first labeled automatically during the detection process. Then, a computer score was calculated by averaging the volume occupied by the labeled TIB regions over the whole lung volume. Calculated computer score was then normalized to fit the visual grading scheme explained in Section IV-C. Linear regression model was fitted to all subjects' scores both from computer and the consensus scores of the participating radiologists (DMJ and AW) and Pearson product-moment correlation coefficient was computed for this model. A scatter plot of the linear regression model and the computer-observer agreement correlation is shown in Fig. 11 . It is clear from this plot that visual and quantitative assessments correlate well as indicated by the Pearson product-moment correlation of R 2 = 0.824 (p < 0.01). Finally, we illustrate an example of TIB and non-TIB region classification by expert annotation and computer quantification by our proposed method in Fig. 12(a) and (b) , respectively.
Scale-based analysis: In addition to visual scoring scheme, we also show the effectiveness of the proposed scale-based method on quantification of the disease extent and identification. Scale-based analysis of the regions occupied by TIB patterns is illustrated in Fig. 13 . A CT slice of a patient with HPIV shows fewer large homogeneous regions (green) with respect to a normal control. It also shows a greater number of small homogeneous regions (yellow and red). (from 1 to 10), we recorded the average number of b-scale patterns. As readily seen from both curves, the existence of TIB patterns was indicated through the small number of highly homogeneous regions (i.e., small number of large b-scale patterns) and large number of less homogeneous regions (i.e., large number of small b-scale patterns). This figure validated the qualitative Fig. 13 . The difference between two curves was at statistically significant level (p < 0.01).
All programs used in this study were developed using gcc 4.5 (Copyrigth (C) 2010 Free Software Foundation) on a Linux platform (Pardus), and all statistical computations were processed in R (Version 2.12.2) and MATLAB (Copyright (C) 2010 Mathworks). All the programs were executed on an Intel (R) Core(TM) i7 CPU 930 at 2.80 GHz with 12 GB RAM workstation. While segmentation of lung regions from CT scans takes only about 10 s, the b-scale encoding algorithm takes a couple of minutes (average 2 min, at most 5 min). The time required to compute b-scale scenes changes from patient to patient due to different number of slices in CT scans. Details of the computational cost analysis for segmentation of lungs, and feature extractions for particular algorithms are enlisted in Table II. A further feature selection method such as a principal component analysis might be used to reduce the dimension of steerable features that we used only for comparison purposes. Note that the proposed features are having a small number of dimension per local patch; there is not necessarily an additional feature selection method needed; hence, it is outside the scope of this paper.
Briefly, the whole dataset was randomly divided into training and test sets of 30 CT scans (20 HPIV-10 Normal versus 19 HPIV-11 Normal). Parameters of the SVM classifier were learned based on the CT scans pertaining to training set. SVM regression was based on pixel-wise classification [42] . Followed by feature extraction step, the trained SVM classifier was applied to the test set. Note also that we have used twofold cross-validation technique for training and testing; therefore, we changed the role of training and testing dataset in the second fold. We also noticed that there was no significant changes in training and test performances of SVM classifications if twofold cross validation was changed into n-fold cross-validation system with n > 2. In addition, we have used Efron's bootstrap [43] method (i.e., repeating the experiments 100 times based on the actual data) to assess the variability of the estimated classifications derived from SVM regressions, and provide confidence intervals for ROC curves.
We used radial basis functions as kernel of SVM, and set to epsilon parameter of SVM as 0.1 [42] . Resulting SVM values of pixels are ranging from 0 to 1. This value indicates the likelihood of a local patch belonging to a certain class (TIB or non-TIB); low ratings indicate a non-TIB region, and high ratings indicate a TIB region. Soon after the SVM values were computed for the entire lung, we changed the cutoff values of SVM (0.5 as default) several times to obtain ROC curves.
In this paper, we studied a very particular, yet important, pattern of lung abnormality observed in chest CTs. Our proposed detection system is tuned to detect TIB regions from non-TIB regions; therefore, a multiclass classifier (with specifically tuned detection filters for each abnormality class) might be needed as an extension of this study to detect as much abnormality as possible in a whole system. Although such a system will bring its unique challenges into the CAD platform, it would be a valuable second opinion tool for radiologists. As a further step, we are currently investigating combining different imaging patterns pertaining to lung abnormalities as well as clinical laboratory information into our CAD system.
One question arises as to the use of high-resolution CT (HRCT) scans instead of conventional CT scans in detecting TIB patterns, as well as the effect of using HRCT scans in this process. Although HRCT scans appreciate detection of small nodular patterns, they have more noise and lungs might not be fully covered due to large gaps between slices (i.e., 10-30 mm). Furthermore, at our institution and in many other institutions, the protocol for acute pulmonary infection is 5 mm contiguous slice images of the chest without IV contrast, for which we adapted our CAD method. Nevertheless, the method we present is not data dependent and can be used for HRCT scans as well.
Considering 2-D computation of b-scale scenes, one may doubt if the algorithm can be extended into 3-D. Based on our observations on appearance and location of TIB patterns over the lung regions and experiences on feature extraction in 3-D, as we stated previously, TIB patterns rarely extend in depth direction for more than a few slices due to constraints of lowresolution imaging direction. Therefore, there is no significant classification rate changes in 3-D; however, there is an increase in computational cost. Nevertheless, 3-D b-scale encoding and feature extraction for a similar pattern detection problem or the same problem with high-resolution images (with thinner slice thickness compared to low-resolution CT images) can readily be combined and used with similar accuracies reported in this study.
Number of large and small b-scale patterns might perhaps be used to identify other type of abnormality patterns such as GGO and consolidations where we expect to have more large b-scale patterns than small b-scale patterns. Therefore, as an extension of this study, we will tune our proposed methodology with different types of abnormalities to generalize the CAD systems for infectious lung diseases in general.
Our proposed method is capable of detecting and quantifying TIB patterns very accurately as validated by the statistical tests compared to the expert annotations (i.e., ground truth). Therefore, both in detection and quantification steps, the proposed CAD system will highly possibly be helpful for clinicians as a second opinion tool in routine clinical examinations.
In this study, we have proposed b-scale-based binary classification approach for automatic TIB pattern detection and quantification from chest CTs. The proposed system integrates 1) fast localization of candidate TIB patterns through b-scale filtering and scale selection, and 2) combined shape and textural features to identify TIB patterns. Note that texture-based recognition methods offer a complementary view to shape-based methods; therefore, the integration of spatial information and the proposed shape features achieves high detection rates. Moreover, our proposed local shape features illustrate the usefulness of the invariant properties, Willmore energy in particular, to analyze TIB patterns in chest CT. We have also compared computer scoring of the proposed CAD system with subjective visual grading. A high correlation between objective (CAD) and subjective (visual grading) scores is obtained, which implies highly satisfactory accuracy of the proposed CAD system. Mannose-binding lectin deficiency and acute exacerbations of chronic obstructive pulmonary disease BACKGROUND: Mannose-binding lectin is a collectin involved in host defense against infection. Whether mannose-binding lectin deficiency is associated with acute exacerbations of chronic obstructive pulmonary disease is debated. METHODS: Participants in a study designed to determine if azithromycin taken daily for one year decreased acute exacerbations had serum mannose-binding lectin concentrations measured at the time of enrollment. RESULTS: Samples were obtained from 1037 subjects (91%) in the trial. The prevalence of mannose-binding lectin deficiency ranged from 0.5% to 52.2%, depending on how deficiency was defined. No differences in the prevalence of deficiency were observed with respect to any demographic variable assessed, and no differences were observed in time to first exacerbation, rate of exacerbations, or percentage of subjects requiring hospitalization for exacerbations in those with deficiency versus those without, regardless of how deficiency was defined. CONCLUSION: In a large sample of subjects with chronic obstructive pulmonary disease selected for having an increased risk of experiencing an acute exacerbation of chronic obstructive pulmonary disease, only 1.9% had mannose-binding lectin concentrations below the normal range and we found no association between mannose-binding lectin concentrations and time to first acute exacerbation or frequency of acute exacerbations during one year of prospective follow-up. Mannose-binding lectin (MBL) is a pattern-recognition collectin that is related to surfactant proteins A and D and has two roles in host defense. MBL activates complement via serine proteases, particularly MBL-associated serine protease-2, with which it circulates. 1,2 MBL is also involved in opsonophagocytosis, binding several types of pathogens to phagocytes via its carbohydrate recognition domain, triggering release of a number of proinflammatory cytokines [3] [4] [5] and facilitating clearance of apoptotic cells. 6 MBL is secreted primarily by the liver and circulates in the serum. Low MBL concentrations occur as a result of one of three single nucleotide polymorphisms on exon 1, but the most common cause of deficiency in Caucasians is the LXP haplotype resulting from polymorphisms of the promoter region of the gene, presenting either as a homozygous mutation or in combination with other haplotypes. [7] [8] [9] Several observations suggest that MBL deficiency may compromise host defense in the lungs. MBL binds to carbohydrates on the surface of a number of respiratory pathogens that are associated with acute exacerbations of chronic obstructive pulmonary disease (COPD), including Haemophilus influenzae, 10, 11 Mycoplasma pneumoniae, 12 and influenza. 13 Patients with cystic fibrosis and low MBL concentrations have decreased lung function, an increased prevalence of Burkholderia cepacia infection, and reduced survival relative to patients with cystic fibrosis and normal MBL concentrations. The difference in survival is particularly notable in those with chronic Pseudomonas aeruginosa infection and those with homozygous mutations in the MBL-2 gene. [14] [15] [16] [17] Low MBL concentrations have been associated with an increased risk of respiratory infections in immunocompetent subjects, [18] [19] [20] [21] with an increased frequency of respiratory syncytial virus infections, 22 and with worse outcomes in patients with community-acquired pneumonia and Streptococcus pneumoniae infections. [23] [24] [25] [26] MBL concentrations in bronchoalveolar lavage fluid obtained from a small number of current and former smokers with COPD are lower than those in fluid from healthy controls and tend to be higher in former smokers than in current smokers, 27, 28 but no association has been found between genotypes producing low MBL concentrations and the prevalence of COPD. 29 However, studies assessing the association of low MBL concentrations with acute exacerbations of COPD (AECOPDs) report conflicting results. [30] [31] [32] Accordingly, we prospectively measured MBL concentrations in a large cohort of subjects with COPD who were at increased risk of experiencing acute exacerbations and followed them for one year while tracking the number of acute exacerbations that occurred. Our hypothesis was that subjects with COPD who had an increased risk of experiencing an AECOPD would have more frequent acute exacerbations during one year of follow-up if they were deficient in MBL than if they were not.
Subjects were men and women enrolled in a multicenter randomized trial designed to determine if azithromycin, taken daily for one year, decreased the frequency of AECOPDs. 33 Eligibility criteria included age $ 40 years, a clinical diagnosis of COPD, and an increased risk of experiencing an AECOPD based on criteria defined by Niewoehner et al. 34 Patients had to be free of AECOPDs for a minimum of 4 weeks prior to enrollment. Exclusion criteria included a diagnosis of asthma or bronchiectasis, among others. 33 Acute exacerbations were defined as "a complex of respiratory symptoms (increased or new onset) of more than one of the following: cough, sputum, wheezing, dyspnea, or chest tightness with a duration of at least three days requiring treatment with antibiotics or systemic steroids". 34 
Serum was collected at the time subjects were enrolled in the study when they had not experienced an AECOPD for a minimum of 4 weeks and was stored at −80°C until MBL concentrations were assayed by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, MN). Samples were diluted 1/500 for this assay and assayed in duplicate wells. At this dilution, the range of the standard curve corresponds to concentrations ranging from 78 ng/mL to 5000 ng/mL. When concentrations were extrapolated above 5450 (n = 7) or below 60 (n = 6), the samples were reassayed at either a 1/2500 or a 1/20 dilution and these new values used as MBL serum concentrations. In one case, a sample was still less than the detection limit at 1/20 dilution and this sample was assigned the concentration of less than 3 pg/mL.
The MBL concentration that def ines MBL def iciency is debated. Some define deficiency as a serum concentration , 500 ng/mL. 24, 31, 35, 36 Others def ine it as #100 ng/mL, and still others define severe deficiency as #50 ng/mL and partial deficiency as .50 ng/mL but ,1000 ng/mL. 9, 10, 21, 25, 31, [37] [38] [39] [40] The normal value reported by the manufacturer of the assay is 1135 ng/mL with a range of 103-3308 ng/mL (R&D Systems). Because of these uncertainties, we defined MBL deficiency in four ways, ie, #50 ng/mL, #100 ng/mL, #500 ng/mL, and .50 but #1000 ng/mL.
Azithromycin increases expression of the mannose receptor, and uptake of apoptotic cells by human alveolar macrophages, and decreases recovery of apoptotic bronchial epithelial cells. 27 Accordingly, MBL concentrations from patients randomized to receive azithromycin or placebo were analyzed both separately and together.
A Cox proportional-hazards model analysis was used with time-to-first-exacerbation as the outcome variable and MBL group (ie, below versus above specified limits) as the primary variable of interest. Bootstrap methods were used to compute confidence intervals for median survival times. Rates of AECOPDs were determined by dividing the number of AECOPDs by person-years of follow-up and were analyzed as a function of MBL concentration using a negative binomial model. MBL concentrations are presented as medians and interquartile ranges. P , 0.05 was considered to be statistically significant. The study (ClinicalTrials.gov number NCT00325897) was approved by the institutional review boards at all participating centers. submit your manuscript | www.dovepress.com
MBL assays were performed at baseline in 1037 (91%) of the 1142 subjects enrolled in the azithromycin trial. Of these, 909 had experienced one or more AECOPDs in the year prior to enrollment. Duplicate measurements of MBL had coefficients of variations ,15% in all cases and ,5% in most.
The median MBL concentration for all patients was 918 ng/mL (interquartile range 508-1683 ng/mL, inclusive range 0-8194 ng/mL). The median MBL concentration in subjects randomized to receive azithromycin or placebo was 1008 ng/mL (95% confidence interval [CI] 909-1082) and 850 ng/mL (95% CI 776-929), respectively (P = 0.017).
Patient demographics and clinical characteristics, stratified by MBL concentration, are summarized in Table 1 . The prevalence of MBL deficiency was 0.5%, 1.9%, 24.2%, or 52.3%, when deficiency was defined as #50, #100, #500, or .50 and #1000 ng/mL, respectively (Table 1) . Regardless of the concentration of MBL used to define MBL deficiency, no difference was observed with respect to the prevalence of MBL deficiency by gender, race, or age (with the exception that a greater fraction of women had MBL deficiency defined as .50 ng/mL and #1000 ng/mL than was seen with the other definitions), smoking status, chronic bronchitis, or steroid use, and there was no suggestion that airflow limitation was worse or that GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage was higher in subjects with MBL deficiency ( Table 1 ). The same findings were also observed for the subgroups of subjects receiving azithromycin or placebo (data not shown).
When analyzing time-to-first AECOPD using a model that included treatment group (ie, azithromycin versus placebo) and log-transformed MBL concentration stratified by clinic, treatment group was significant (P , 0.0001) and log-transformed MBL concentration was not (P = 0.629). The hazard ratio for a one-unit increase in log-transformed MBL concentration was 1.02 (95% CI 0.94-1.12). For the rate per person-year of AECOPDs, a negative-binomial analysis of a model that included treatment group and log-transformed MBL concentration found that treatment group was significant (P = 0.010) but log-transformed MBL concentration was not (P = 0.470). The coefficient for log-transformed MBL concentration in this model was 0.031 (95% CI, −0.053 to +0.115).
The median time to first AECOPD and the rate of AECOPD per patient-year are shown in Figures 1-3 Table 2 relative to the various definitions of MBL deficiency (only five patients had MBL concentrations # 50 ng/mL, precluding life table analyses for patients in this subgroup). Regardless of the MBL concentration used to define deficiency, no association between the time to first AECOPD, or the rate of AECOPDs and MBL concentration was observed in the population as a whole, or in either treatment subgroup. Nonsignificant trends favoring a longer time to first AECOPD were seen in the subgroup of subjects with MBL concentrations , 100 ng/ mL compared with those with concentrations $ 100 ng/mL ( Figure 1A and C). No difference was observed in MBL concentrations in subjects experiencing no, one, two, or at least three AECOPDs during the course of the study in the population as a whole, or in either treatment group (Table 3) .
Of the 1037 subjects in the study, 220 (21%) required hospitalization for AECOPDs. The median MBL concentration in these 220 subjects was 1055 (95% CI 861-1213) ng/mL [1091 (958-1450) for those on azithromycin and 891 (816-1276) for those on placebo]. The median MBL concentration in the 817 subjects who were not hospitalized was 904 (833-980, P = 0.17, Table 4 ).
The important findings of this study are that, in this large sample of subjects with COPD selected for having an increased risk of experiencing an AECOPD within one year, only 1.9% had MBL concentrations below the normal range reported by the manufacturer of the assay, and regardless of the MBL concentration used to define MBL deficiency, we found no association between deficiency and time to first AECOPD, rate of AECOPDs, or need for subjects to be hospitalized for AECOPDs.
Two studies concluded that MBL deficiency was associated with an increased incidence of AECOPDs and one concluded that it was not. The age, spirometry, and smoking histories of the subjects in these three studies were similar to those we evaluated, with the exception that none of the three studies selected patients who were at increased risk of experiencing acute exacerbations as we did. Yang et al 30 found that 24 of 82 (29%) subjects with COPD had MBL-deficient genotypes. These subjects had lower MBL concentrations than those with the wild-type genotype (107 ng/mL, IQR 30-246, range 21-7675 versus 920 ng/mL, IQR 398-1355, range 21-2256, P , 0.001). MBL concentrations were not presented in a fashion that allowed determination of the prevalence of MBL deficiency based on the definitions used in the literature. Forty of the 82 patients (49%) had one or more admissions for AECOPDs during a two-year follow-up period and 18 of these (45%) had MBL-deficient genotypes. Forty-two patients had no AECOPDs and only six (14%) of these had MBL-deficient genotypes (odds ratio 4.9 95% CI 1.7-14.4, P = 0.0037). We did not determine MBL genotypes but genotypes do not COPD (P = 0.23 and P = 0.10, respectively). No association between MBL concentration and a history of AECOPDs was observed, but no information was provided with respect to how AECOPDs were defined. Eagan et al 31 also noted that subjects with GOLD stage 3 disease had a higher prevalence of MBL deficiency (defined as #100 ng/mL). We found no demographic or COPD severity indicators that were more or less common in subjects with MBL deficiency, regardless of how deficiency was defined ( Table 1) . The strengths of our study include the large sample size, the multicenter design, and the prospective ascertainment of AECOPDs using an event-based (ie, health care utilization) definition. Our study population was enriched by enrolling subjects whom we anticipated would be at increased submit your manuscript | www.dovepress.com risk of experiencing an AECOPD within the one-year follow-up period, based on previous predictors identified by Niewoehner et al. 34 This should have increased the likelihood of finding a higher prevalence of MBL deficiency, regardless of how deficiency was defined, compared with the prevalence in healthy controls. Our study has a number of limitations. First, we only measured MBL concentrations on one occasion. Several groups have demonstrated that MBL is an acute phase reactant. [41] [42] [43] However, in clinically stable patients, MBL concentrations are constant over time, 42, 44 and our patients had to be free of AECOPDs for at least 4 weeks before meeting inclusion criteria. In addition, even during acute phase responses, MBL concentrations in patients with low concentrations only increase by 1.5-4.3-fold and do not reach normal values. 45, 46 Accordingly, we suggest that there is a low likelihood of our data being confounded by spuriously elevated concentrations of MBL resulting in an underestimate of the prevalence of MBL deficiency.
We did not confirm by genotyping that low MBL concentrations were the result of variant alleles. Approximately 30% and 4%-8% of the normal population have heterozygous or homozygous genetic mutations, respectively, associated with low MBL concentrations. 35 Although MBL concentrations are well correlated with genotypes, (eg, MBL concentrations , 50 ng/mL are 100% sensitive and 83% specific for variant exon-1 polymorphisms 43 ) subjects with wild-type MBL genes may still have low concentrations of MBL, 35 and MBL concentrations may vary as much as 10-fold in patients with identical genotypes for all of the MBL variants described to date. 38 Accordingly, assessing relationships between MBL concentrations and AECOPDs is likely to be a more sensitive approach than genotyping. 21 Similarly, we did not assess MBL function using a complement deposition assay. Bouwman et al 47 and Eisen 48 suggested that while MBL function was a better way of defining MBL deficiency than determining MBL concentrations, assessing MBL concentrations was "most appropriate" for defining MBL deficiency in studies seeking associations with infections. Differences between MBL binding and complement activation may vary depending on the method of assessment, 49 and MBL concentrations from 500 ng/mL to 1000 ng/mL are associated with decreases in function by up to 90%. 9 While we saw no association between MBL deficiency and AECOPDs, MBL deficiency could still be associated with AECOPDs that result from infection with one or more specific pathogens if the frequency with which these specific pathogens affected our subjects was too low to discern the association. However, we think this possibility is unlikely, because the two studies documenting the association between MBL deficiency and AECOPDs found an association with AECOPDs that were not otherwise defined by cause or potential infecting organism. 30, 32 In addition, Lin et al 32 found no difference in the distribution of pathogens in those subjects with AECOPD and without MBL-deficient genotypes.
Because we only found 20 subjects (1.9%) with MBL concentrations below the lower range of normal reported by the manufacturer of the assay, we cannot exclude the possibility that very low concentrations of MBL have an association with AECOPDs. However, the infrequency of this finding implies that even if this association were found, it would pertain only to a small minority of patients suffering AECOPDs.
In conclusion, we found a very low prevalence of MBL deficiency in subjects who were at increased risk for experiencing AECOPDs and no association between MBL deficiency and time to first acute exacerbation, frequency of acute exacerbations, or percent of subjects requiring hospitalization for acute exacerbations in subjects with MBL deficiency regardless of how deficiency was defined. Accordingly, our data do not support the idea that MBL might be a therapeutic target to reduce the incidence of AECOPDs. Rather, they imply that, while COPD is an inflammatory disorder with systemic manifestations, the fundamental pathophysiology of COPD differs from conditions in which MBL deficiency seems to be a clear risk factor (ie, childhood pneumonia, rheumatoid arthritis, systemic lupus). The changing phenotype of microglia from homeostasis to disease It has been nearly a century since the early description of microglia by Rio-Hortega; since then many more biological and pathological features of microglia have been recognized. Today, microglia are generally considered to be beneficial to homeostasis at the resting state through their abilities to survey the environment and phagocytose debris. However, when activated microglia assume diverse phenotypes ranging from fully inflamed, which involves the release of many pro-inflammatory cytokines, to alternatively activated, releasing anti-inflammatory cytokines or neurotrophins, the consequences to neurons can range from detrimental to supportive. Due to the different experimental sets and conditions, contradictory results have been obtained regarding the controversial question of whether microglia are “good” or “bad.” While it is well understood that the dual roles of activated microglia depend on specific situations, the underlying mechanisms have remained largely unclear, and the interpretation of certain findings related to diverse microglial phenotypes continues to be problematic. In this review we discuss the functions of microglia in neuronal survival and neurogenesis, the crosstalk between microglia and surrounding cells, and the potential factors that could influence the eventual manifestation of microglia. I. Introduction II. The origin of microglia III.Microlgia the dual natures of neurotoxicity and neuroprotection IV.Crosstalk between microglia and other brain cells 1. Cross talk between microglia and neurons: neurons as regulators of microglial activation 2. Cross talk between astrocytes and microglia: reciprocal influences 3. Microglia-T cell crosstalk:key determinants for the Introduction Microglia are generally considered the immune cells of the central nervous system (CNS) and account for 10% of the total glial cell population in the brain. In a normal physiological environment, they work as sentinel cells by continually screening the brain tissue; they actively participate in pathological processes by changing morphology, expressing various antigens and becoming phagocytic. During the past 20 years, thousands of papers have been published describing both the detrimental and beneficial roles of microglia in various brain disorders, from acute infection or stroke to the long and chronic process of neurodegeneration. Microglia have been firmly established as a key cellular component involved in the eventual outcome of inflammation and eventually contribute to the chronic neurodegeneration; The physiology and signaling of microglia have been comprehensively reviewed by Kettenmann's series papers [1] [2] [3] [4] [5] [6] , however, the regulation of microglial activity is a highly complex system, and the responses of microglia are tailored in a multi-factor dependent manner, and which are the focus we try to review in this paper.
The precise origin and cell lineage of microglia has been a long time debate. So far two most important hypotheses for microglial origin have been held: "neuroectodermal" or "myeloid-monocytic". Even though the latter has been more widely accepted now, the neuroectodermal hypothesis remains interesting. Skoff [7] detected "multipotential glia cell" with a rat model of optic nerve degeneration and optic nerve development, these cells were demonstrated to originate from neuroectodermal matrix cells, and Kitamura later confirmed this result by describing a continuous morphological transition between glioblasts and ramified microglia in the developing gray matter of hippocampus [8] . The hematopoietic origin of microglia also received a lot attention, the presence of bone marrow Mac-1 positive cells were demonstrated in the brain of embryonic and adult mice, and these cells were proved to be the progenitors for microglial cells [9] , also transplantation of GFP + mice bone marrow cells in GFP-host mice revealed the presence of many GFP + microglia throughout developing and/or inflamed CNS [10, 11] , which strongly suggest the hematopoietic stem cells as one of the origins for replenishment of microglia in the neuropathology. Additionally due to the high similarity in marker expression and phagocytosis behavior between circulating monocytes and microglia, people speculate the monocytic origin of microglia, and a couple of experiments have been performed to show the appearance of labeled monocytes in the developing [12] or inflamed brain [13] . In many cases, the peripheral macrophages are considered to be the orthologue [14, 15] or backup of microglia and infiltrate the brain to supplement microglia, thus to some extent peripheral macrophages mirror the behavior of microglia in the brain and Monocytederived Macrophages (MDMs) from patients have been used as a substitute of microglia in many studies [16] [17] [18] .
Neuroinflammation has long been considered a mediator of secondary damage following a small injury to the CNS. As the primary immune cells in the brain, microglia are expected to take active roles in the damage process. The presence of activated microglia within injured brain regions and in post-mortem tissue from patients having various neurodegenerative disorders has led to the assumption that all reactive microglia contribute to an adverse and degenerative process. Further studies describe destructive roles for microglia by demonstrating the release of a range of neurotoxins from microglia that includes pro-inflammatory cytokines [19] [20] [21] , nitric oxide [22, 23] and reactive oxygen species [24, 25] ; the inhibition of microglial activation in various experiments results in the attenuation of neurotoxic events and improves neuronal survival. In various neurodegenerative disorders, the over-activation of microglia is considered to be a key causative factor in the process or, at a minimum, to promote the neuropathology. For example, in Alzheimer's disease, microglia activated by amyloid-β(Aβ) protein, the hallmark of the disease, release neurotoxins and potentiate neuronal damage, and this microglial over-activation is an early event that precedes neuropil destruction [26] . The activated microglia cluster around or penetrate the neuritic plaques [27] , supporting a critical role of microglial activation in the pathogenesis and progression of the disease. In Parkinson's disease (PD), an increased number of activated microglia are present in the vicinity of degenerating neurons [28] in the substantia nigra [29] , which is particularly deleterious to dopaminergic neurons due to their glutathione deficiency [30] . A single injection of lipopolysaccharide (LPS) to activate microglia in the substantia nigra region led to a progressive, preferential and irreversible loss of dopaminergic neurons [31] [32] [33] , even though LPS itself did no direct harm to the neurons, indicating that the over-activation of microglia is capable of inducing neuronal death in the absence of other pathological stimulation. All of the evidence described above supports the hypotheses of the neurotoxic features of microglia.
However, as the sentinel and essential cells of the CNS, it is unlikely that microglia would function to damage neurons in all scenarios. Once stimulated the microglia migrate rapidly to the injury site along the chemokine gradients in vitro [34] and also in response to chemoattractants including ATP and NO released directly or indirectly by the injury [35] to exert effect on the survival of neurons. In fact, some specifically designed experiments have begun to uncover the neuroprotective roles of microglia, and more studies are emerging to show beneficial functions of microglia. Firstly, studies have demonstrated instructive roles for microglia in the developing brain for neuronal differentiation [36, 37] and in the regulation of neuronal apoptosis [38] through the production of neurotrophins [39] . Secondly, in the adult brain, resting microglia, which are characterized by many fine perpendicular processes extending from a few long prolongations, have been regarded as sensor cells for the detection of abnormalities or changes in the brain [40] and help to maintain environmental homeostasis. Lastly but most importantly, activated microglia have also been shown to perform neurotrophic functions following neuronal injury. One compelling study supporting this finding involves the axotomy of peripheral nerves (facial or optic), where a rapid microglial response is exhibited with the efficient clearance of myelin debris that contained inhibitory molecules of axon growth, finally leading to successful axonal regeneration [41] ; the inhibition of this microglial response to facial nerve axotomy impairs neuronal survival [42] . In addition, in neonatal mice administered MPTP, highly activated microglia show neurotrophic potential towards dopamine neurons [43] and after traumatic injury, clear glutamate without evoking inflammatory mediators [44] . The benefits of microglial activation are further demonstrated by the exacerbation of neuropathology in inducible mouse models that are deficient in microglia [45, 46] , the finding of protective microglia in cases of cerebral ischemia [47] and multiple sclerosis [48] and the fact that transplantation of microglia can help to enhance neurite growth and functional recovery after CNS injury [49, 50] . The bunch of factors that can activate microglia and the differential behavior of microglia in various conditions have been listed in Table 1 & 2. The above studies clearly demonstrate that microglia can be neurotrophic in the proper situations; there might be a third possibility that microglia are activated by simply reacting to pathogenic stimulation and takes very limited roles in the neurological disorders, in such case the activation of microglia is solely a result of pathogenic stimulation and work as a bystander that either involved passively during the whole process or even go to apoptosis by some other signals. Thus These activated microglia might have different phenotypes. However, the details of what conditions induce microglia to take beneficial phenotypes remain unknown. Many factors are likely involved in determining the eventual outcome of the manifestation of microglia, including their interaction with neurons or astrocytes in the same environment, age-related dysfunction of microglia, activation timing, and the activation state of the microglia, which we will be discussing below.
Microglia have been considered to be the first line of defense in the CNS [91] , a hypothesis that has been supported by the finding that microglia actively screen their microenvironment with highly motile processes; thus, the brain is under continual surveillance by microglia. To do this with high efficiency, microglia must be variable, adaptive to their environment and capable of integrating various inputs and responding appropriately [92, 93] . All of these processes require significant interactions with other components within the same environment, including neurons and astrocytes.
When we talk about whether microglia are neuroprotective or neurotoxic, we only refer to the influence of microglia on neurons. However, many studies indicate that neurons are not merely passive targets of microglia but rather exert control over microglial activities [94] . There are considerable interactions between neurons and microglia. For example, Polazzi hypothesized that activation of microglia as a consequence of neuronal injury is primarily aimed at neuroprotection, with the loss of specific communications between neurons and microglia leading to the neurotoxic behavior of microglia [95] . Accumulating evidence demonstrates that there is significant information exchange between neurons and microglia. Depending on whether they are healthy or injured, neurons send "on" or "off" signals to influence microglial activation. On one hand, the activation of [81, 82] microglia by neuronal injury or degeneration has been widely reported [91, 96] . On the other hand, in the healthy brain, microglial activation is tightly restricted by signaling from neurons. CD200-CD200R has been identified as one of the critical pathways in attenuating microglial activation. CD200 is a member of the immunoglobulin superfamily and is expressed on the neuronal membrane surface, while the CD200 receptor (CD200R) is primarily present in the macrophage lineage, which includes microglia [97] . The disruption of CD200-CD200R interactions results in an accelerated microglial response, whereas intensified CD200-CD200R interactions contribute to an attenuation in neurodegeneration [98] . In mice that have had CD200 selectively removed from neurons, microglia exhibited an activated phenotype and were numerous upon facial nerve transaction; damaged CD200-deficient neurons elicited an accelerated microglial response, which demonstrated a loss of the neuronal inhibitory signal for microglial response [97] . Apart from direct interactions through receptor-ligand combinations, electrical activity and soluble factors released from intact neurons also maintain microglial quiescence. In a neuron-glia co-culture, the blockade of neuronal electrical activity by tetrodotoxin or a glutamate receptor antagonist facilitated microglial activation induced by IFN-γ [99] . Soluble molecules from neurons such as neurotrophins and anti-inflammatory agents downregulate antigen expression on cultured rat microglia [99, 100] . Additionally, released factors from neurons can also influence the survival of microglia. Fukui et al. demonstrated that treatment with conditioned media from mature neurons significantly induced the death of microglial cells independent of LPS, while heated neuron-conditioned media or low-calcium-ion media prevented the death of microglia [101] , indicating that specific factors released from neurons exert detrimental effects on microglia. It has been demonstrated that microglial cells undergo apoptosis following peripheral nerve injury [102] [103] [104] or in cases of experimental autoimmune encephalomyelitis(EAE) [105] Injured neurons induced either neuroprotective or neurotoxic behaviors in microglia depending on the manner of injury [91, [106] [107] [108] [109] , providing strong evidence to support the hypothesis of crosstalk between neurons and microglia. Thus, microglia are not merely surveyors of brain tissue but also receive and actively respond to signals from neurons.
Although less obvious than the crosstalk with neurons, the interactions between microglia and astrocytes are far from simple and are also crucial for our understanding of how microglia respond to their environment and exert influence on neuronal degeneration or regeneration. Several studies have demonstrated the substantial influence of astrocytes on microglial activation [110] . The induction of microglia by Trimethyltin or Borna disease virus-infected neurons is dependent on the presence of astrocytes [111, 112] . Astrocytes play neuroprotective roles by modulating microglial cell activity and decreasing their cytotoxicity [113, 114] . The expression of IL-12 and the production of inducible nitric oxide synthase (iNOS) in activated microglia have been reported to be suppressed by astrocytes or conditioned media from astrocytes [82, 111, [115] [116] [117] , delineating the signals from astrocytes that affect the activities of microglia. Furthermore, the communication between these two types of cells is two-way; microglia both receive and give signals, as proinflammatory cytokines released from microglia inhibit gap junctions and down-regulate connexin 43 expression in astrocytes [118] [119] [120] , which enhances astrocyte survival. In another study, comparative proteome analysis was performed on astrocytes that were treated with conditioned media from quiescent or activated microglia. Following culture in activated-microglial media, the anti-oxidative enzymes expressed in astrocytes were up-regulated, and these astrocytes were protected against oxidative stress. This result gave insight into the complex intercellular events that take place during neurological disorders [121] . Alzheimer's Disease Internalize and degrade amyloid beta [87] Multiple sclerosis Secrete soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration [48] As in many pathological conditions in the central nervous system such as in neurodegeneration [122] , microglia, activated earlier than astrocytes, promote astrocytic activation through IL-1which is mostly from microglia [123] . On the other hand, activated astrocytes not only facilitate activation of distant microglia via calcium wave [124, 125] , but also inhibit microglial activities [126] . Additionally, it was observed that activated-microglial-conditioned media increased astroglial proliferation [127] , down-regulated the astroglial metabotropic glutamate receptor [128] and induced astroglial brain-derived neurotrophic factor (BDNF) and IL-6 gene expression [129] . Taken together, the importance of microglial activities lies in that they not only exert direct effects on neuronal survival, but they also affect the responses of other supporting cells in the same environment.
Microglia-T cell crosstalk: key determinants for the trend of immune response
The entire immune response consists of the cooperation of the innate and adaptive immune systems. In the brain, it has been postulated that the beneficial or destructive outcome of the local microglial (innate) response is determined by a well-controlled dialogue between the innate and the adaptive immune players, which are, in most cases, the microglia and T cells. Activated T cells can cross the bloodbrain barrier and interact with resident microglia in the parenchyma [130] ; these microglia have been characterized as myeloid progenitor cells that can differentiate into macrophage-like or dendritic-like cells [131] and thus work crucially as the principal APCs [85] in the CNS. Monsonego et al. demonstrated that IFN-γ-treated microglia serve as efficient Aβ antigen-presenting cells (APCs) of both Aβ1-40 and Aβ1-42, mediating CD86-dependent proliferation of Aβ-reactive T cells [132] . The activated T cells then exert effects in the injured neural tissues by altering the reactive microglial phenotypes and inducing the astrocytic expression of growth factors or modulating microglia to act as glutamate scavengers [44] to improve neuronal survival [133, 134] . In a model for optic nerve injury, the passive transfer of regulatory CD4 + CD25+ T cells was either destructive or beneficial depending on the genetic background of the mice tested, which determines the differential interaction of T cells with microglia and thus the different T cellmediated microglial phenotypes [133] . Kipnis even observed that both the suppressor and the effector activities of T cells could be mediated through dialogue with microglia in the condition of neurodegneration [135] , The entire scenario of crosstalk between T cells and microglia could be described as the following: microglia are initially activated by pathological stimuli during acute or chronic injury to the brain; if the activation occurs with the proper timing and mode and is well-controlled, the activated microglia will work as APCs [133] to stimulate Treg cells that eventually modulate the microglial activation directly or indirectly and affect the milieu balance between neurotrophism and cytotoxicity [44, 136, 137] .
Whether microglial activation is neurotrophic or neurotoxic is context-dependent
After considerable time and research, we have recognized the "double-edged sword" nature of microglial cells. On one hand, significant evidence from in vitro and in vivo studies has associated neuronal injury with microglial activation [138] [139] [140] [141] . This evidence results from an inflammatory phenotype of microglia releasing neurotoxic factors, mediators and reactive oxygen species [138] [139] [140] [141] . On the other hand, several other studies have highlighted the beneficial and important roles of microglia in neuronal regeneration, repair and neurogenesis [142] [143] [144] [145] [146] . These seemingly paradoxical results cannot be directly compared, because they come from different experimental sets that vary in terms of the stimulus, timing of microglial activation and age of animals. Thus, whether microglia have positive or negative effects on neuronal survival is contextdependent.
There are studies suggesting that senescence in microglia causes them to function abnormally and that the destructive roles of activated microglia in the aged neurodegenerative brain may result from age-associated microglia senescence, causing a failure of the aged microglia to respond correctly to stimuli [147, 148] and eventually promoting neurodegeneration [149] (Figure 1 ). The most prominent and also the initially identified feature of microglial senescence is the morphological alteration described as "dystrophy" [150] . Characteristics of "dystrophic" microglia observed in the aged brain include de-ramification (the loss of finely branched cytoplasmic processes), cytoplasmic beading/ spheroid formation, shortened and twisted cytoplasmic processes, and instances of partial or complete cytoplasmic fragmentation [150] . Such dystrophic microglia were prevalent and extensively distributed in the brain of older human subjects [150, 151] , whereas normally ramified microglial morphology with only rare instances of dystrophic microglia is observed in the young brain [148] . These observations provide initial evidence for the age-associated changes in microglia in the healthy elderly brain. Telomere shortening, a marker of aging, has also been demonstrated in microglia in the aged brain in Flanary's study, who reported that microglial cells in rats exhibit significant telomere shortening and a reduction in telomerase activity during normal aging [152] . More importantly, microglial senescence is also manifested by functional alterations, such as an altered inflammatory profile, increased immunophenotypic expression, and the switch from neuroprotective Luo and Chen Translational Neurodegeneration 2012, 1:9 Page 5 of 13 http://www.translationalneurodegeneration.com/content/1/1/9 in the young brain to neurotoxic in the aged brain upon activation [147] . Also, the timing of microglial proliferation and presentation in the injured aged brain is distinct from that in the young brain. For example, Conde et al. reported that microglial proliferation rates in the aged rat brain were significantly higher than in the young rat brain four days after axotomy of the facial nerve [148] . The distinct pattern of the microglial response to injury in the aged brain has also been recorded in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model of neurotoxicity [153] , the model of controlled cortical impact (CCI) [154] , cortical stab injury [155] and transient retinal ischemia [156] . Although more attention has been paid to the dysfunction of aged microglia, many critical questions remain unanswered. Some of these questions are: whether the activated state of microglia in the aging brain is concurrent with or secondary to microglial dystrophy; which specific function of microglia is primarily affected by microglial dystrophy, how it is affected and what is the direct consequence of the affected function; and whether the deterioration of a specific microglial function is more related to neurodegeneration than others. Clearly, more research is needed to answer these questions.
Another important element that critically determines the destructive or neuroprotective role of microglia is the timing of their activation. Because large and very complicated communications pathways exist between immunocompetent cells and cytokines in the CNS, the timing of microglial activation leads to diverse trends and outcomes related to the entire inflammation event. In a model of optic nerve crush injury, Shaked et al. found that an earlier onset of phagocytic activity and antigen presentation by microglia results in a resistance to injury and neurons survived [133] ; the early, moderate, transient and well-controlled activation of local microglia caused them to function as APCs, leading to the communication with Treg cells that subsequently proves to be neuroprotective through the modulation of microglial activation states [133] . In a multiple sclerosis (MS) Luo and Chen Translational Neurodegeneration 2012, 1:9
Page 6 of 13 http://www.translationalneurodegeneration.com/content/1/1/9 model of experimental allergic encephalomyelitis (EAE) [157] , the inhibition of microglial activation through tPA knockout (tissue plasminogen activator, an essential element for microglia activation) leads to a delayed onset of the disease but increased severity and delayed recovery from the neurological dysfunction, which suggests that microglial activation is harmful during the onset of the disease but beneficial in the recovery phase [157] . Furthermore, when microglial activation was either stimulated or inhibited at different stages, the disease progression was attenuated or exacerbated accordingly [158] . For example, the inhibition of microglial activation at EAE onset, rather than prior to EAE induction, markedly decreased EAE progression, while the stimulation of microglial activation prior to the onset of EAE promotes lower-level EAE and an earlier recovery from symptoms. Together, these findings suggest different roles for microglial activation during various phases of the disease and that different timing of microglial activation dramatically affects whether microglia will be neuroprotective or deleterious [158] . Similarly, in an oxygen-glucose deprivation model, the time window of microglial neuroprotection has been estimated to up to 48 hour after injury, while the pre-stimulation of microglia with LPS before the injury fails to induce microglial-mediated neuroprotection [86] . It has been proposed that the effects of the early activation of microglia on disease progression could be beneficial through phagocytic activity and antigen presentation, recruitment and interactions with the adaptive immune response and the induction of protective autoimmunity [133] . Furthermore, the balance between protective autoimmunity and autoimmune disease may be determined by the timing and intensity of microglial activation [133] . As the immuno-competent cells in the CNS, microglia are critical determinants of the outcome of injury, and the timing of microglial activation appears to be crucial to the outcome of the injury. Thus, any interference with microglial activation in an attempt to affect the disease course clearly must be temporally-restricted.
Two distinct phenotypes of macrophages have long been known to play different roles in the inflammatory context. Classically-activated macrophages, characterized by the involvement of T Helper type 1 (Th-1) cytokines such as interferon-γ, promote the release of various proinflammatory cytokines and thus exacerbate the inflammation. Alternatively, activated macrophages predominate in the T Helper type 2 (Th-2) microenvironment and tend to soothe the inflammation. Thus, the behavior of macrophages is dictated by their phenotype, which may eventually affect the beneficial or detrimental roles of macrophages during inflammation. Similarly, research over the past few years has established that microglia do not constitute a single, uniform cell population, but rather comprise a family of cells with diverse phenotypes; some are neuroprotective while others are destructive [92] . So far, three distinct functions have been proposed for microglia. The first is the classical activation state of microglia, which, accompanied by the induction of receptors that participate in the innate immune response [159] , is responsible for the pro-inflammatory milieu, and has been linked to neurotoxic effects in the brain. The second is alternatively activated microglia, which are associated with the production of anti-inflammatory cytokines in the resolution phase of the inflammatory response. Recently, the third activation state of microglia has been identified: it overlaps with and is complementary to the alternative activation and is called acquired deactivation [160, 161] . This is another activation state that promotes immunosuppression and is associated with the anti-inflammatory and functional repair phenotype .Both alternative activation and acquired deactivation down-regulate innate immune responses and have similar gene profiles; the most prominent difference is that acquired deactivation is induced by the exposure of microglia to apoptotic cells or to TGF-β or IL-10, while IL-4 and IL-13 induce alternative activation [160, 161] . It has been observed that multiple activation states of microglia coexist in certain chronic inflammations due to parasitic disease [162] , in which the balance between classical activation and alternative activation/acquired deactivation states is of "benefit" to both host and parasite: the host benefits from reduced self-damage, and the parasite eventually survives within the host. Neurodegenerative disorders are also associated with chronic inflammation and the coexistence of various activation states. For example, in AD, some levels of classical activation may be required to limit the brain levels of Aβ despite the risk of self-damage [163] , while alternative activation of microglia in AD may foster the protection of the surrounding tissue from immune damage even though it may facilitate Aβ deposits. Similar studies [164] [165] [166] have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical activation in AD, while Colton et al. found increased mRNA expression of alternative activation-associated gene profiles in microglia in both the AD brain and an AD mouse model [167] , suggesting the presence of multiple activation states of microglia during neurodegeneration. However, the recognition of heterogeneous phenotypes of microglia only raises more questions: what instructs microglia to acquire a particular phenotype; can any conversion occur between these phenotypes; and is it possible to avoid or at least change the commitment to a destructive phenotype? All of these questions are difficult to answer with our current knowledge of microglia; more extensive work is warranted before we can reach a conclusion.
As an active sensor in the brain, microglia respond to even minor stimuli; however, different types of stimulation may also lead to different actions of microglia and thus be either harmful or beneficial to neuronal survival. In a neonatal mouse MPTP-induced brain injury model, microglia activated by systemic administration of LPS were shown to be neuroprotective. In contrast to the MPTP model, LPS-activated microglia in neonatal mice receiving a stereotaxic injection of ethanol into the striatum were shown to be neurotoxic, and systemic LPS administration in the ethanol-injury model caused a marked increase both in the volume of necrotic lesions and in the number of degenerating neurons in the striatum [168] . Even with the same stimuli, the degree can also determine microglial release of toxic versus protective effectors [169] ; neurotoxic cytokines and ROS were released from microglia only in response to mild neuronal injuries, while trophic microglial effectors such as BDNF and GDNF were up-regulated in response to all degrees of neuronal injury [169] . Additionally, different types of pain resulted in differing activations of microglia [170] .
So far, what we know is that not all microglia respond in the same way, even to the same stimulus, and microglial function is tailored in a context-specific manner [171] . Numerous elements are involved in this context; most likely there are many more beyond what we have discussed here. Identifying these elements and clarifying their interactions or crosstalk with microglia is essential before we are able to design a strategy to control inflammation through the manipulation of microglia. The simple therapy of inhibiting all microglia without differentiating their function in a context-dependent manner surely should be abandoned.
It has been long recognized that the birth of new neurons within the postnatal brain continues throughout life and remains as a potential source of replacement cells in the CNS for the treatment of disease. The microenvironment or the niche in which neural progenitor cells live critically influences the process of neurogenesis, which spans several steps including the proliferation of stem or progenitor cells; the survival of immature or mature neurons; the migration of new neuroblasts to their appropriate locations; and the differentiation of neuroblasts to a neuronal phenotype and the construction of synaptic connectivity [172] . As an important component of the brain microenvironment and due to their invariant participation in most pathological processes in the CNS, microglia are increasingly implicated as a potential non-neural regulator of neurogenesis, as demonstrated by circumstantial evidence [144, 172] . However, just as in the debate over the neuroprotective or neurotoxic nature of microglial activation, whether microglia support or damage the survival and development of neural progenitor cells also remains controversial. On one hand, microglia were shown to play instructive roles during postnatal neurogenesis in the neurogenic niche either by influencing the differentiation of stem cells toward a neuronal phenotype or by directing their migration [144, [173] [174] [175] . On the other hand, multiple studies have demonstrated the deleterious effect of microglial activation on neurogenesis [176, 177] and the effective restoration of neurogenesis though the blockade of microglial activation.
In the two situations of neurogenesis and neuronal survival, similar factors are shared, leading microglia to take supportive or detrimental roles. Among these factors, the most prominent is the microglial activation phenotype that is associated with different cytokine profiles. When acutely activated by either LPS or injury, microglia that release the pro-inflammatory cytokines IL-6, TNF-α or IL-1β usually down-regulate the differentiation or proliferation of neural stem cells or induce the aberrant migration of newborn neurons [178] . This group of inflammatory cytokines has been proven to inhibit neurogenesis [176, 177, 179] ; conversely, blocking antibodies to these pro-inflammatory cytokines (such as IL-6 [177] ) or the use of monocycline to mitigate the microglial activation simply restores neurogenesis [176] . In contrast, microglia that are activated by anti-inflammatory cytokines such as IL-4 or TGF-β increase neurogenesis in vitro or the differentiation of neural stem cells (NSCs) in vivo [180, 181] . Neurotrophins, such as IGF-1, were identified [181] in anti-inflammatory cytokineactivated microglia and were proposed to be one of the mechanisms underlying this pro-neurogenic activity of microglia [182, 183] . However, just like the dual roles in neuroprotection, whether a specific cytokine-activated microglial cell will take a pro-or anti-neurogenic role is also context-dependent. For example, microglial cells activated by IFN-γ, a pro-inflammatory cytokine can be neurotoxic or supportive of neurogenesis, depending on the concentration of IFN-γ [184] . TGF-β, which is considered to be beneficial to neurogenesis, can actually exert a negative influence on neurogenesis when it is chronically produced in the aged brain [185] . Additionally, if other cytokines exist in the same niche simultaneously, the outcome will be determined by the balance among the various cytokines; some authors have concluded that activated microglia are not pro-or anti-neurogenic per se, but the balance between pro-and anti-inflammatory secreted molecules influences the final effect of microglial activation [172, 180] . However, in which situations the microglia will release pro-or anti-inflammatory cytokines is complicated and is affected by multiple factors such as the injury type, the phase of disease or inflammation, and crosstalk with other regulating components, including neural precursors; this is similar to the question of whether microglia will be neuroprotective or neurotoxic. Most likely the same inflammatory scenario that induces neurodegeneration would also inhibit neurogenesis, while a situation that favors neuronal survival would also support neurogenesis. Interestingly, even in a high-inflammation environment, such as two days after a Trimethyltin-induced acute injury in the hippocampus, significant neurogenesis can be detected [186, 187] , suggesting a complicated system of neurogenesis regulation beyond the inflammation scenario.
Cumulative studies have found an age-related decline in neurogenesis, both in the aged adult and in the diseased brain. Because aging may contribute to microglial dysfunction and neurotoxicity, as we discussed previously in this review, one could assume that microglial dysfunction may also be involved in the downregulation of neurogenesis in the aged or diseased brain [188, 189] . Even though very few studies have focused on the effect of microglial dysfunction on neurogenesis, we can still find a clue from Zhu's study that the difference in microglia function patterns between the immature and juvenile brain might be related to a decrease in neurogenesis in the juvenile brain [190] ; however, stronger evidence from the direct comparison of microglia-associated neurogenesis between aged and young brains is needed to support this view.
Another important element regulating the activities of microglia is the T cell, which comes from the peripheral adaptive immune system and enters the CNS by extravasating across the endothelium of the choroid plexus into the cerebrospinal fluid [191] . The interaction of T cells with microglia in the injured spinal cord correlates with enhanced neuronal survival [184] , and rapidly recruited T cells in the middle cerebral artery obstruction (MCAO) model increased hippocampal and cortical neurogenesis by modulating the microglial response and through the production of IGF in the sub-acute phase [192] . Hippocampal neurogenesis was associated with the recruitment of T cells and microglial activation. Immune-deficient mice show impaired neurogenesis in the hippocampus, but this deficiency was attenuated and neurogenesis boosted by T cells recognizing a specific CNS antigen [193] . The cellular source of IFN-γ and IL-4 in vivo is likely to be T cells, therefore it is reasonable to assume that the T cell-mediated immune response is an integral part of the regulation of microglial phenotype or function, and thus can influence neuronal survival or neurogenesis directly or indirectly.
From an increasing number of studies of diverse microglial activity in different experimental sets, we are beginning to appreciate the heterogeneity of microglial functions that have either beneficial or detrimental roles in specific physiological or pathological environments. Whether microglia are committed to one function from the very beginning or if there is any conversion between different phenotypes remains elusive and the factors that initiate this commitment or promote its conversion are far from being clarified. Due to the invariant critical participation of microglia in most diseases, ongoing research to uncover these questions is warranted; before we are sure about the answer, any potential strategies targeting microglia to manipulate inflammation and modify a disease course are unrealistic. Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis BACKGROUND: Salmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed. RESULTS: The microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria. CONCLUSIONS: These results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes. Salmonella spp. found in water buffalo (Bubalus bubalis) herds are a matter of concern since they are responsible for serious economic losses in livestock and are a zoonotic agent responsible for foodborne illness [1] . As for bovine calves, Salmonella-induced diseases in water buffalo calves are characterized by severe gastrointestinal lesions, profuse diarrhea, and severe dehydration [1] . Acute salmonellosis generally induces diarrhea, mucous at first, later becoming bloody and fibrinous, often containing epithelial casts. Ingestion is the main route of infection, although it can also occur through the mucosa of the upper respiratory tract and conjunctiva. The major source of infection in the herd is represented by asymptomatic older animals shedding heavy loads of bacteria through feces. Other sources of infection are contaminated forages and water, as well as rodents, wild winged animals, insects and man [1, 2] . The disease can also cause sudden death without symptoms. Occasionally, the infection is systemic, affecting joints, lungs and/or the central nervous system (CNS) [1] . Moreover, several Salmonella serovars seem to be able to infect water buffalo, mainly affecting 1-12 week old calves, even though reports on salmonellosis in B. bubalis are scarce [1, 3] .
Water buffalo calves are more frequently affected by gastroenteritis than bovine calves, with mortality rates as high as 70% in water buffalo species vs. 50% in bovine [1, 4] . This difference might be due to a greater susceptibility of water buffalo to gastroenteric pathogens, although it also may reflect the lack of appropriate management practices for this animal species. Therefore, water buffalo represents a suitable model to study causative agents of gastroenteritis. In water buffalo, S. enterica serovar Typhimurium can induce a variety of clinical syndromes with different anatomopathological lesions [1, 3] . The severity of the disease can depend on several factors, including host-pathogen interactions, which is highly influenced by the route of infection, the infectious dose, natural or acquired host resistance factors, and the possible presence of other pathogens. Moreover, specific Salmonella virulence factors, frequently located on Salmonella pathogenicity islands (SPIs), prophage regions or virulence plasmids, play a key role in the pathogenesis of the gastroenteritis [5] .
The current study investigated the intestinal contents collected from 248 water buffalo calves affected by gastroenteritis with lethal outcome to: (i) evaluate the prevalence of Salmonella spp., and (ii) perform a polyphasic characterization of the collected isolates of S. Typhimurium.
Salmonella spp. were isolated from 25% of the intestinal contents collected from 248 water buffalo calves affected by gastroenteritis with lethal outcome. Positive samples were detected in subjects bred in 37 of 58 farms (interherd prevalence, 64%). The S. enterica serovars most frequently isolated were Typhimurium (n=13), Muenster (n=7) and Give (n=7). Other recovered serovars were: Derby (n=5), 4 Bovismorbificans (n=4), Newport (n=4), monophasic S. Typhimurium (B:4,12:i:-; n=3), Blockley (n=2), Meleagridis (n=2), Umbilo (n=2), Altona (n=1), Anatum (n=1), Bredeney (n=1), Enterica (−;i;1,2; n=1), Gaminara (n=1), Haardt (n=1), Hadar (n=1), Infantis (n=1), Isangi (n=1), Kottbus (n=1), London (n=1), Muenchen (n=1), and S.II:41;z;1,5 (n=1). Phage-typing of the S. Typhimurium and monophasic Typhimurium strains (Table 1 ) indicated a variable distribution of phage types among strains with nine different phage types of 13 Typhimurium strains, and three different phage types out of three monophasic Typhimurium strains.
This study reports a significant prevalence of Salmonella spp. (25%) in diarrheic water buffalo calves, that are more relevant than those reported in previous studies (11 and 0.8%) [3, 6] . Moreover, in contrast with bovine species where salmonellosis results primarily associated with serovars Dublin and Typhimurium [5] , the extremely variable distribution of the observed serovars confirms the absence of a serovar specifically adapted to water buffalo, as previously suggested [1] . These data provide therefore evidence that Salmonella, particularly S. Typhimurium, can be potentially considered an important pathogen for this animal species. The definitive phage type 104 (DT104), which has often been associated with multiple-antibiotic-resistant strains with ascertained zoonotic potential and, in many countries, has increased over the past two decades [5] , does not seem to be widely spread in water buffalo. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found that are S. Typhimurium lacking phase two flagellar antigens that have a rapid emergence and dissemination in food animals, companion animals, and humans. More significantly, the public health risk posed by these emerging monophasic S. Typhimurium strains is considered comparable to that of other epidemic S. Typhimurium [7] .
The diagnostic investigation indicated that non-Typhimurium Salmonella isolates were detected with at least another potential pathogen in 78% of cases ( Figure 1A ). In 35% of cases Salmonella was linked with pathogenic Escherichia coli that were characterized for the presence of virulence factors. Other frequent associations were found with Cryptosporidium spp. (20%) and Rotavirus (10%) ( Figure 1A) . Remarkably, S. Typhimurium was never associated with pathogenic E. coli, while it was isolated sporadically with Clostridium perfringens (strain #82280), Rotavirus (strain #107025), and Cryptosporidium spp. (strain #112) ( Figure 1B) . The presence of more pathogens in the same subject might suggest that, as for other animal species [5] , diarrhea in water buffalo calves can be characterized by a multifactorial etiology. Data from necroscopic examinations of tissues indicated that the lesions caused by S. Typhimurium were characterized by severe damage of the intestine, ranging from congestive to necrotic-ulcerative enterocolitis. In particular, the strains isolated from animals exhibiting the most severe lesions were #16, #92, #233, and #83528. Among these strains, the two DT104 strains were also found, thus supporting the pathogenic role of this phage type. The other Salmonella serovars were instead isolated from subjects exhibiting a variety of different lesions, mostly minor lesions confined to the jejunum, and often (78% of cases) associated with other pathogens. Similarly, the monophasic S. Typhimurium strains were detected either with Rotavirus (strain #154) or st-positive E. coli (strains #175 and #188). These data confirm the pathogenic potential of the serovar Typhimurium for water buffalo calves. On the other hand, the scarcity of observed lesions and the frequent presence of more than one microorganism in the same subject hamper a clear understanding of the potential pathogenic role of the non-Typhimurium Salmonella serovars included in this study.
S. Typhimurium and monophasic S. Typhimurium strains were further characterized by the molecular detection of 24 genes coding for virulence factors. The genetic characterization (Table 2) included five loci (avrA, ssaQ, mgtC, siiD, and sopB) located on SPI 1-5, respectively [8] , eight loci (gipA, gtgB, sopE, sodC1, gtgE, gogB, sspH1, and sspH2) of prophage origin [9] [10] [11] [12] [13] , the gene spvC, located on a virulence plasmid [12] , and nine genes (stfE, safC, csgA, ipfD, bcfC, stbD, pefA, fimA, and agfA) coding for bacterial fimbriae, involved in surface adhesion and gut colonization [5] . As a positive control for the PCR assay, amplification of the chromosomal gene invA was carried out for each strain. All the S. Typhimurium and monophasic Typhimurium isolates displayed the presence of avrA, ssaQ, mgtC, siiD, sopB, sspH2, stfE, ipfD, bcfC, stbD, and fimA genes, and the absence of the sopE gene. Other loci were variably distributed among the strains, with frequency values ranging from 38-92% (Table 1) . On the basis of the presence or absence of the 24 loci included in the study, the 13 strains of S. Typhimurium were subdivided into 10 different genotypes (Table 1) ; however, the isolates with identical genotype displayed different phage types suggesting the presence of 13 different strains. Interestingly, the three monophasic S. Typhimurium strains exhibited three different genotypes (Table 1) . 
The following loci: invA, sspH2, stfE, ipfD, bcfC, stbD, fimA, avrA, ssaQ, mgtC, siiD, sopB were present in all the strains; the sopE gene was not found in any of these strains. b NT = not typeable.
The 24 loci-genetic characterization was also extended to the S. Muenster and S. Give isolates to investigate their pathogenic potential because of their large presence in water buffalo calves. In addition they have already been reported to cause saepticemic salmonellosis in cattle and calves [14, 15] . The molecular results (Table 3) indicated that the loci invA, safC, bcfC, fimA and ssaQ were present in all the strains, the genes gipA, gogB, sspH2, sodC1, gtgE, spvC, stfE, ipfD and pefA were not found in any of these isolates, while the remaining loci were variably distributed, with frequency values ranging from 14-86%. In particular, the prophage genes were scarcely present (2 loci in the Muenster serovar, 1 locus in the Give serovar), the plasmidic spvC locus was absent in all the analyzed isolates, while the fimbrial genes and the SPI 1-5 genetic markers were discretely represented (6 loci for the former genes in both serovars, 5 and 4 loci for the latter genes in the serovar Muenster and Give, respectively). Moreover, the molecular profiles allowed to identify 6 different genotypes out of the 7 S. Muenster isolates, and 5 different genotypes out of the 7 S. Give isolates (Table 3) .
Our data confirm the high variability of the Typhimurium serovar [9, 10] , mostly related to virulence factors, and highlight the high discriminating potential of the genotyping technique performed. Our data also suggest that monophasic Typhimurium strains are likely to possess a similarly high degree of genetic variability, particularly linked to virulence markers. Moreover, the presence of virulence markers in the isolated strains of monophasic S. Typhimurium, S. Muenster and S. Give could further support their pathogenic potential. The products of the genes included in the virulotyping assay performed here are known to be important during different stages of infection (Table 2) . However, the distribution of these factors among the tested strains highlights the complexity and the variety of potential mechanisms used by Salmonella to induce disease in the host.
The avrA, ssaQ, mgtC, siiD, and sopB genes are genetic markers for the presence of the SPI 1-5 in all S. Typhimurium strains tested, although their presence does not necessarily implicate the presence of the entire SPI. SPIs are clusters of genes on the chromosome, likely to be horizontally acquired, and variably associated with enhanced invasion and intracellular survival within both phagocytic and non-phagocytic cells. In particular, SPI-5 has been largely associated with the ability to produce enteritis [5] . The S. Typhimurium strains included in this study all displayed the presence of the investigated SPI markers. Interestingly, these loci appeared widely distributed also among the serovars Muenster and Give. The sopE gene is known to favor the entry of Salmonella into host cells and its presence has been correlated with disease in humans [16] and with the epidemic potential of S. Typhimurium strains in cattle [17] . This gene was absent in all the S. Typhimurium strains included in the present study, while was present in all the S. Muenster strains analyzed.
The pefA (plasmid encoded fimbria), agfA (aggregative fimbria A) and spvC (Salmonella plasmid of virulence gene C) genes are all located on plasmids [18] . Five S. Typhimurium isolates tested in the current study possessed both pefA and spvC, two isolates were positive for only spvC, and three isolates were positive for only agfA (Table 1) . These results confirm the presence of more than one virulence plasmid among S. Typhimurium strains isolated from diarrheic water buffalo calves, and suggest horizontal exchange of virulence factors. However, the loci pefA and spvC were absent in all the monophasic S. Typhimurium, S. Muenster and S. Give strains tested. Prophage genes are known to account for most of the variability of closely-related S. Typhimurium strains. Moreover, lysogenic bacteriophages promote changes in the composition of genomic DNA often altering the phenotype of the host [9, 10] . The prophage virulence genes included in this study exhibited a variable distribution among the isolates tested, thus suggesting synergistic and/or redundant effects of these loci on the pathogenicity of Salmonella, likely contributing to the ACTGCGAAAGATGCCACAGA phenotypic variability of this pathogen. These loci were mostly present in S. Typhimurium and monophasic S. Typhimurium rather than in S. Muenster and S. Give isolates. Fimbrial genes appeared widely distributed among all the serovars tested, particularly in S. Typhimurium strains, with frequency values ≥92%, except for the plasmid-borne pefA and agfA genes (with frequency values of 38% and 54%, respectively). These data are consistent with the essential functions of adhesion factors for the attachment and internalization processes that occur during pathogenesis.
To better characterize in vivo virulence, three strains representative of all S. Typhimurium isolates were chosen to perform mixed infections in mice. Animal experiments included the two strains exhibiting the highest and the lowest number of virulence factors (strains #92 and #112, respectively), and strain #16, carrying the same virulotype as strain #92, but that does not harbor the agfA locus (Table 1 ). In the competition assay, strain #92 outcompeted both strains #112 and #16 (CI 0.004; P<0.001, and CI 0.031; P<0.001, respectively). These results were confirmed in a gastrointestinal mouse model of infection, which better resembles the clinical form of salmonellosis in livestock. Using oral inoculation, in the competition assay, again strain #92 outcompeted both strains #112 and #16 (CI 0.009; P<0.001, and CI 0.186; P<0.01, respectively). Our data indicate that among those strains included in the experiment, strain #92 was the most virulent in mice. These competition assays in mice suggest a key role of the agfA gene coding for a thin aggregative fimbria involved in the colonization of host intestinal epithelial cells by attachment to glycoprotein or glycolipid receptors on epithelial cell surfaces. Indeed, the strain which was more virulent in in vivo experiments was characterized by a high number of virulence factors and by the presence of the agfA locus. Moreover, it was isolated from one of the subjects with necrotic-ulcerative enterocolitis.
The presence of this type of fimbria has been reported in clinical human and animal isolates of Salmonella +  +  ----+  ---2   15228  -+  --------3   66761  -+  --------3 72827 -
Freq. a The following loci: invA, safC, bcfC, fimA and ssaQ were present in all the strains; the genes gipA, gogB, sspH2, sodC1, gtgE, spvC, stfE, ipfD and pefA were not found in any of these strains. [19, 20] . The data presented here suggest that agfA might increase bacterial pathogenicity. Nevertheless, we cannot reject the hypothesis that the mouse model chosen for in vivo experiments could have influenced the virulence phenotype of the tested strains originally isolated from water buffalo calves. Therefore, future studies will be necessary to exclude the possibility that the phenotypic differences observed among the tested Salmonellae are dependent on the animal model or on other virulence factors not included in this study. However, in vivo experiments carried out in mouse models represent a good preliminary source of information on the expression of traits associated with pathogenicity of Salmonella in mammalian species.
This study showed a significant (25%) prevalence of Salmonella spp. in water buffalo calves affected by gastroenteritis with lethal outcome. However, our results did not indicate the existence of a Salmonella serovar specifically adapted to water buffalo and highlighted that S. Typhimurium is the most frequently found serovar. The molecular and phenotypic characterization of the S. Typhimurium isolates provided evidence that within this serovar there are different pathotypes potentially responsible for different clinical syndromes, therefore requiring prophylaxis protocols including the use of specific vaccines for the effective control of salmonellosis in water buffalo calves and possible contamination of the food chain.
This study was carried out in the Campania region, Southern Italy, during 2008-2009, using samples taken from 248 water buffalo calves bred in 58 different farms. The animals were aged between 1-12 weeks old and were all affected by gastroenteritis with lethal outcome. During necropsy, the intestinal lesions were evaluated and the intestinal content of the involved sections was collected and tested for the presence of Salmonella spp. In addition, the presence of E. coli, Eimeria spp., Cryptosporidium spp., Giardia spp., Coronavirus, Rotavirus, and C. perfringens were also determined to investigate their association with Salmonella spp. The isolation of Salmonella spp. was performed according to ISO 6579:2002 [21] . The isolated Salmonella spp. were serotyped according to the Kaufmann-White scheme [22] . Phage-typing of the isolated S. Typhimurium strains was performed by the Italian National Reference Centre for Salmonellosis (Istituto Zooprofilattico Sperimentale delle Venezie).
The presence of Rotavirus and Coronavirus was detected by polymerase chain reaction (PCR) amplification [23, 24] .
Cryptosporidium spp. and Giardia spp. antigens were detected by chromatographic immunoassay (Oxoid, Basingstoke, UK). The presence of Eimeria spp. was examined by flotation technique using saturated saline [25] . E. coli and C. perfringens were isolated according to the protocol reported by Quinn et al. [2] . E. coli hemolytic activity was evaluated by growing colonies on blood agar base, while virulence factors (lt-heat-labile toxin, st-heatstable toxin, stx1-Shiga toxin 1, stx2-Shiga-toxin 2, eaeintimin, cnf-cytotoxic necrotizing factor, and cdt-cytolethal distending toxin) were detected by molecular assays, as previously reported [26] [27] [28] .
Bacterial DNA was extracted from 1 mL of overnight cultures using Chelex 100 Resin (BioRad, Hercules, CA) and used as the template for the PCR detection of genes listed in Table 2 , as described previously [8] [9] [10] [11] [12] [13] 18] . The primers used to amplify the genes sspH1, sspH2, ssaQ, sopB, siiD, stfE, safC, csgA, ipfD, bcfC, stbD, and fimA were designed using the Primer3 software (version 0.4.0; http:// frodo.wi.mit.edu/), and PCR was performed in a final volume of 25 μL containing HotStar Taq Master Mix (Qiagen, Valencia, CA) 1×, 0.4 μM each primer and 1 μL of extracted DNA. The thermal profile included an initial denaturation step at 95°C for 15 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, and a final extension step at 72°C for 5 min. Amplification products were visualized under ultraviolet (UV) light after electrophoresis on 3% agarose gels and staining with SYBRsafe (Invitrogen, Carlsbad, CA).
Groups of five age matched (8-10 weeks old) female BALB/c mice used in this study were purchased from Charles River (Calco, Italy). Three strains (S. Typhimurium #16, S. Typhimurium #92, S. Typhimurium #12), representative of the 13 genotypically characterized S. Typhimurium isolates, were selected for an in vivo analysis of virulence by using the Competitive Index (CI) resulting from mixed infections [29] . In particular, two strains were selected that exhibited the highest and lowest number of virulence factors (strains #92 and #112, respectively), and strain #16, carrying the same virulotype as strain #92, but without the locus agfA (Table 1) .
Bacteria were grown overnight at 37°C in Brain Heart Infusion medium (Oxoid, Basingstoke, UK), washed, and diluted in sterile saline. Cultures were alternatively combined in a mixture of equivalent numbers (1:1 ratio) of two of the three selected strains (input). Mice were inoculated intraperitoneally (IP) with a dose of 2×10 4 bacteria or received 20 mg of streptomycin orally (200 μL of sterile solution or sterile saline) 24 h prior of being intragastrically administered with 2×10 7 bacteria. The number of colony-forming units (CFU) contained in the inocula were confirmed by plating serial dilutions and counting colony growth. At 4 (IP) or 7 (os) days after infection, mice were sacrificed, spleens were aseptically removed, and bacteria were counted by plating serial dilutions (output). The ratio of two strains in the input and in the output was evaluated by picking and transferring 200 colonies on selective plates. Antibiotics used were streptomycin and sulfonamide, for which strain 92 and strains 16 or 112 were naturally resistant. The CI was calculated using the formula: CI = output (strain A/strain B)/inoculum (strain A/strain B). Statistical differences between outputs and inputs were determined by Student's t test. All animal handling and sampling procedures were performed under the conditions of the local ethics committee meeting the requirements of Italian legislation. Severe Childhood Malaria Syndromes Defined by Plasma Proteome Profiles BACKGROUND: Cerebral malaria (CM) and severe malarial anemia (SMA) are the most serious life-threatening clinical syndromes of Plasmodium falciparum infection in childhood. Therefore it is important to understand the pathology underlying the development of CM and SMA, as opposed to uncomplicated malaria (UM). Different host responses to infection are likely to be reflected in plasma proteome-patterns that associate with clinical status and therefore provide indicators of the pathogenesis of these syndromes. METHODS AND FINDINGS: Plasma and comprehensive clinical data for discovery and validation cohorts were obtained as part of a prospective case-control study of severe childhood malaria at the main tertiary hospital of the city of Ibadan, an urban and densely populated holoendemic malaria area in Nigeria. A total of 946 children participated in this study. Plasma was subjected to high-throughput proteomic profiling. Statistical pattern-recognition methods were used to find proteome-patterns that defined disease groups. Plasma proteome-patterns accurately distinguished children with CM and with SMA from those with UM, and from healthy or severely ill malaria-negative children. CONCLUSIONS: We report that an accurate definition of the major childhood malaria syndromes can be achieved using plasma proteome-patterns. Our proteomic data can be exploited to understand the pathogenesis of the different childhood severe malaria syndromes. Human malaria caused by Plasmodium falciparum has an estimated annual global disease burden of 300 million clinical episodes, leading to one million deaths [1] [2] [3] [4] . Eighty-five per cent of the cases and 90% of the mortality occurs in sub-Saharan Africa, mostly amongst children [5, 6] . Recent reports point to a reduction of malaria cases in parts of Africa [7] . However, Nigeria, the most populous country of Africa, accounts for a quarter of the global cases and a third of the malaria-attributable childhood deaths [2, 8, 9] .
Cerebral malaria (CM) and severe malarial anemia (SMA) are the major severe disease syndromes in African children with a high level of mortality in the under-five age group. The current WHO case definitions for severe malaria combine P. falciparum blood stage parasitemia with coma, severe anemia or respiratory distress [10] , and it is well documented that there is significant overlap across these syndromes [11] . Despite the fact that these WHO case definitions are sensitive and useful for clinical diagnosis, the pathogenesis of severe disease is not well understood. One disadvantage of the WHO clinical definitions is that they lack the specificity required to carry out studies aimed at understanding the pathogenesis of clinically different forms of childhood malaria.
Previous studies have attempted to define malaria syndromes by studying plasma correlates of severity using reductionist approaches with variable success [12] [13] [14] . Small sample sizes, a lack of validation cohorts and a focus on a small selection of host plasma proteins have limited these studies. To overcome such limitations we use a systems approach to define the plasma proteome profile during malaria infection and identify distinctive patterns that are characteristic of different disease states. Contrary to other proteomic approaches, high-throughput plasma proteome profiling enables simultaneous analysis of a large number of samples. Therefore plasma proteome profiling allows the use of statistical pattern-recognition methods to discover and validate proteome-patterns that discriminate disease states.
We hypothesized that the plasma proteome during malaria infection reflects the molecules that are modulated as the severe status is established. In the present study we show that distinctive plasma proteome-patterns distinguish the different severe presentations of P. falciparum childhood malaria from the uncomplicated cases and also from well or unwell children without malaria.
Parents or guardians of study participants gave informed written consent. This research was approved by the joint ethics committee of the College of Medicine of the University of Ibadan and the University College Hospital Ibadan.
All study participants were recruited under the auspices of the Childhood Malaria Research Group (CMRG) at the 600-bed tertiary hospital University College Hospital (UCH) in the city of Ibadan, Nigeria in west sub-Saharan Africa. Ibadan is a densely populated urban setting with a population of 2.5 million inhabitants. Ibadan has a lengthy 8 months rainy season from March to October with malaria transmission and severe disease present all year round (holoendemic).
The study site is located in the UCH Ibadan Department of Paediatrics. We screen about 12,000 children attending the hospital (ill and well) for malaria parasites per year. Our studies report 11.3% SMA and 19.7% CM admissions in the parasitized children under five years of age [9] .
The participants in this study were recruited during 2006 to 2009 as part of a larger prospective case-control study of childhood severe malaria currently ongoing under the auspices of the CMRG. This case-control study was divided into a Discovery Cohort consisting of those patients recruited during 2006 to 2008 and a Validation Cohort made up of those recruited in the 2008 to 2009 period.
Malaria parasites were detected and counted by microscopy following Giemsa staining of thick and thin blood films [15] . Children with severe malaria were recruited on admission from the Otunba Tunwase Children's Emergency Ward (OTCHEW). Children with uncomplicated malaria were recruited as part of a daily routine malaria parasite screening at the Children's Outpatient Clinics (CHOP). Malaria-negative ill children were recruited either at admission from OTCHEW or from the Department of Paediatrics In-patient wards. Malaria-negative healthy community control children were recruited from local vaccination clinics as well as during school visits across several Ibadan districts.
We recruited children aged from 6 months to 13 years using five participant definitions. The malaria-positive children, the cases, are Uncomplicated Malaria (UM), Severe Malarial Anemia (SMA) and Cerebral Malaria (CM). The malaria-negative children, the controls, are Disease Control (DC) and Community Controls (CC). We followed the WHO criteria for severe P. falciparum malaria [10] . Cerebral malaria cases were defined as children in unrousable coma for at least one hour in the presence of asexual P. falciparum parasitemia with normal cerebrospinal fluid. A Blantyre coma score less than 2 was used to assess coma status. Children with hypoglycemia were excluded from the study. Added to the Table 1 . Characteristics of discovery and validation study groups. strict clinical and laboratory definitions of CM, our study patients recover consciousness after effective antimalarial therapy. We excluded from this study those CM patients who died. Our overall mortality rate for CM is of the order of 10%. Severe malarial anemia cases were defined as conscious children with Packed Cell Volume (PCV) less than 16% in the presence of P. falciparum parasitemia. We excluded from this study those SMA patients who died. Our overall mortality rate for SMA is less than 1%. Uncomplicated malaria cases were defined as febrile children with P. falciparum parasitemia who did not require hospital admission. Our study was designed to discover and validate plasma proteome changes in dichotomous cases for which we only included those children with CM and UM with PCV greater than 20% (Table 1) . We excluded from the study blood culture positive cases. Although we did not carry out blood cultures in all severe malaria patients, the cases recruited into this study are those in whom septicemia was not suspected and who were successfully treated with antimalarial alone. The DC group consists of malaria-negative children with infectious diseases such as meningitis, otitis media, diarrhea and upper respiratory tract infections. It also includes mild to moderately anemic children and children admitted for surgery.
Participants's clinical data were collected using a malariatailored questionnaire designed by the CMRG. A 2.5 ml blood sample was obtained from each participant in an EDTA blood collection tube for subsequent plasma separation. Blood samples were kept on ice and transferred to the central malaria laboratory. Plasma for this study was harvested by centrifugation (1000 g, 10 
Packed cell volume (PCV) was measured using the microhaematocrit method [15] . Briefly, Blood was obtained in capillary tubes. Tubes were centrifuged at 12,000 g for 5 minutes. The percentage cell volume compared to the whole tube volume was calculated (i.e. PCV). Mean (6 standard deviation, sd), minimum and maximum PCV for each clinical group are tabulated in Table 1 . For discovery and validation cohort, these data were compared using a one-way multiple ANOVA test (p,0.05).
Malaria parasites were detected and counted by microscopy following Giemsa staining of thick and thin blood films [15] . Malaria Parasite (MP) densities were calculated as follows MP/ Table 1) . The microscopic criterion for declaring a participant to be free of malaria was the absence of parasites in 100 high-power (1000X) fields. One in 10 thick blood films were randomly selected and independently reviewed by local experienced microscopists not part of the research team.
Crude plasma was profiled using Surface Enhanced Laser Desorption/Ionization-Time Of Flight (SELDI-TOF) mass spectrometry. All plasma samples underwent two freeze-thaw cycles prior to analysis. Plasma samples were coded, blinded and randomized before application onto the following solid-phase fractionation surfaces (ProteinChipH arrays Bio-Rad): weak-cation exchange (CM10), strong-anion exchange (Q10) and reverse phase (H50) as previously described [16] . Liquid handling steps were automated using a Biomek 3000 Laboratory Automation Workstation (Beckman Coulter) and a 96 well BioprocessorH (Bio-Rad). Each ProteinChipH 96 well BioprocessorH included 1 quality control plasma standard derived from a single healthy individual, placed at random. Mass spectra were generated on a System 4000 Bio-Rad ProteinChipH mass spectrometer. Spectral peaks corresponding to mass/charge (m/z) clusters were detected and clustered using ProteinChipH Datamanager Client 4.1 software (BioRad). Mass spectrometer calibration was performed using Allin-1 Peptide and Protein calibrants (Bio-Rad). Reproducibility was determined by measuring the inter-ProteinChipH coefficient of variation (CV) for the quality control spectra, based on all peaks in the spectrum with intensity .1 mA. Overall interchip CV for the quality control sample was 20%, consistent with similar studies.
Liquid-phase anion-exchange fractionation of plasma samples was carried out using the ProteinChipH Fractionation Kit (Bio-Rad) according to the manufacturer's instructions with a Biomek 3000 Laboratory Automation Workstation. Six fractions were obtained from each sample eluting at pH 9.0 (f1), pH 7.0 (f2), pH 5.0 (f3), pH 4.0 (f4), pH 3.0 (f5) and organic phase (f6).
We selected subsets of the most relevant mass clusters in the discovery cohort groups using the weighted Kernel-based Iterative Estimation of Relevance Algorithm [17] (wKIERA) that combines a stochastic-search estimation of distribution algorithm with a kernel pattern-recognition method. We then used discovered relevant subsets of mass clusters to build discriminatory predictive models. We adopted a supervised learning approach to derive a classification rule using the Support Vector Machine (SVM) method [18] . Briefly, we used 10-fold cross validation to select parameters for the SVM. For the final model parameters, we selected those that gave the overall highest accuracy across the whole 10-fold cross validation. To obtain robust accuracy estimates for the classifier on the discovery data, we took 100 random re-samplings of the data, using 80% for training and 20% for testing. We selected as a final classifier the one that produced the highest accuracy and was then tested on the validation cohort data. Results were expressed as sensitivity, specificity and accuracy (proportion of correct classifications) and plotted on Receiver Operator Characteristic (ROC) space plots.
Our multivariate statistical tests included testing against age or sex to ascertain that significant pattern changes in the proteome were not dependent on those variables in the population studied.
To visualize the covariance within the mass spectral profiles we used Principal Component Analysis (PCA). PCA encapsulates the covariance within a set of variables by extracting a ranked set of independent factors or principal components. The first 3 components encompass a high proportion (,95%) of the informational content of a multivariate dataset. We plotted each patient with respect to the first 3 components, in 3-dimensional space, color-coding according to patient group.
A total of 946 children participated in this study as part of the discovery and validation case-controlled cohorts. The discovery cohort comprised of 367 malaria-positive children with either Cerebral Malaria (CM), Severe Malarial Anemia (SMA) or Uncomplicated Malaria (UM), and 289 malaria-negative children who were either Disease Controls (DC) or Community Controls (CC) ( Table 1 ). The validation cohort was prospectively recruited after the discovery cohort and comprised 160 malaria-positive children with either CM, SMA or UM, and 130 malaria-negative DC or CC children (Table 1) . PCV and malaria parasite (MP) densities are presented in Table 1 . Consistent with the recruitment criteria, both discovery and validation SMA groups had PCVs below 16% (Table 1 ). There was mild anemia across CM, UM and DC groups in both cohorts, whereas CC had normal mean hematocrit (Table 1) . Parasite densities across all the infected groups were similar (Table 1) .
To compare the proteome-patterns of the study groups, we fractionated plasma samples by three different chromatography procedures on solid-phase surfaces (weak-cationic and stronganionic ion-exchange, and reverse-phase) followed by Time-Of-Flight mass spectrometry. The resulting mass spectra from each of the surfaces contained a series of mass/charge ratio (m/z) peak clusters, each representing a protein of a particular mass. A set of proteins that are present, absent or at a different level in the samples defines a proteome-pattern that may discriminate between two or more of the study groups. To discover such patterns we applied statistical pattern recognition algorithms to the profiles and the selected number of discriminating proteins for each of the pairwise group comparisons is shown in Figure 1 , as the numbers in parentheses (Data S1). We built predictive models with the selected proteome-pattern for each study group comparison using a non-parametric supervised learning statistical framework. The discriminatory accuracy of these predictive models in the discovery cohort groups is shown in Figure 1a . To determine differences for malaria-positive children from healthy malaria-negative children we compared individually the plasma proteome of CM, SMA and UM groups with that of the CC group.
Overall, 22 to 33 proteins composed the discriminatory patterns with accuracies above 90% across the three comparisons (Figure 1a, blue bars) . Twenty-six proteins discriminated healthy from ill (hospital admitted) malaria-negative children (CC vs. DC) with similar accuracy (Figure 1a, green bar) . To examine proteins that are specific to malaria infection we compared each of the malaria-positive groups (CM, SMA, UM) to the DC group, obtaining discrimination accuracies above 80% (Figure 1a . orange bars). Finally, to assess differences between defined malaria syndromes we compared the malaria-positive groups (Figure 1a . yellow bars). In the comparison between CM and SMA, the two major severe syndromes, the accuracy was 70% (24 proteins). Higher accuracies between 70 to 80% were observed when samples from either CM or SMA groups were compared to UM children, using 36 and 54 proteins, respectively.
To validate the accuracy of the discrimination for the discovered plasma proteome-patterns, we tested the predictive models on the validation cohort groups (Figure 1b) . The best predictive model for each group comparison in the discovery cohort was asked to predict the group class in the validation cohort. We found that the predictive models obtained using the discovery cohort had similar accuracy for discrimination in the different group comparisons for the validation cohort (Figure 1b) . We compared the sensitivity and specificity of the predictive models for both discovery and validation cohort groups in ROC space and found them to be similar ( Figure 2) .
We then used Principal Component Analysis (PCA) on the selected proteins to visualize the separation of patient groups. The CC group clustered tightly together (Figure 3 , green spheres). Individual malaria-positive groups showed good separation from the malaria-negative CC group (Figure 3a-c) indicating that regardless of disease severity there are significant differences in the proteomes of the groups. The heterogeneous DC group had a more dispersed cluster pattern with little overlap with the CC group (Figure 3d ). The DC group, despite being distinct, showed different degrees of overlap with the malaria-positive groups (Figure 4a-c) . Of these comparisons, the CM vs. DC patient groups showed the greatest level of cluster dispersion (Figure 4a ) indicating greater covariance in the proteins that define these groups. We then compared the malaria-positive patient groups among themselves (Figure 4d-f ). CM and SMA groups showed overlap at the cluster interface and clearer segregation at the periphery; in the comparison of both severe forms (CM and SMA) with UM we observed that the severe patient groups had compact center clusters surrounded by a more disperse cluster of the UM patient group.
We simplified further the complexity of the plasma proteome by high-throughput liquid-phase anion-exchange fractionation followed by solid-phase weak-cation exchange fractionation prior to protein mass determination in the spectrometer on a subset of the samples. We assessed the discriminatory accuracy of relevant proteins obtained from each of the six anion-exchange fractions ( Figure 5 , f1 to f6) (Data S2). The reduction in the complexity of each fraction of the plasma samples resulted in a larger subset of proteins that improved discrimination between the malaria syndromes. Sets of proteins that distinguish between SMA and CM groups (Figure 5a , f1 to f6 in brackets) slightly outperformed the proteome-pattern from non-fractionated plasma. Sets of proteins differentiated the CM and UM groups with accuracies ranging from 70 to 80% (Figure 5b , f1 to f6 in brackets) and distinguished between SMA and UM with comparable accuracy (Figure 5c, f1 to f6 in brackets) .
We carried out an overall analysis of plasma proteome pattern overlap by comparing the discovered sets of proteins that discriminate UM, CM, SMA (malaria-positive) and DC (malaria-negative) ill children from the malaria-negative well children CC (Figure 6 , f1 to f6). We show that each plasma fraction (f1 to f6) contains a set of proteins that clearly define both the malariapositive and malaria-negative ill children to those malaria-negative well children in the community. Furthermore, we also show that the set of proteins that discriminate SMA and CM from UM have very little overlap across the six plasma fractions (Figure 6 , f1 to f6).
In the present study we carried out a large case-control study of severe childhood malaria, using a discovery cohort to define discriminatory plasma proteome-patterns and a second cohort to validate our findings, at the main tertiary hospital of the city of Ibadan, Nigeria.
We show that proteome-patterns from both crude and prefractionated plasma samples accurately define childhood malaria syndromes in the discovery cohort. We confirmed these findings using a prospectively collected validation cohort. Malaria infection introduces distinguishable changes in the plasma proteome of children as seen by the striking differences between the malarianegative CC and the malaria-positive children groups. The plasma proteome differences are specific for the malaria disease process and not surrogate markers of acute illness, as we are able to accurately distinguish between malaria-negative ill children and malaria-positive groups independently of their disease severity. We have also discovered plasma proteome differences that are specific to each of the childhood malaria syndromes assessed in the present study. Our findings provide a starting point to refine the current WHO definitions of these syndromes, which lack the necessary specificity to further study severe malaria pathogenesis.
We show that assessing the plasma proteome of the major malaria syndromes provides an unbiased discovery of combination of proteins that could be used to deepen our understanding of the pathogenesis of childhood malaria. This is supported by the finding that we can discriminate children with uncomplicated malaria from those with severe malarial anemia or cerebral malaria in both discovery and validation cohorts. These proteomepatterns encapsulate what changes differentiate uncomplicated malaria from the severe cases.
Overall, accuracy of discrimination between the CM and SMA was lower than that in the comparison of each of these syndromes with the UM group. The degree of overlap between CM and SMA goes beyond that expected from strict application of the WHO case definitions used in this study. Nevertheless, the plasma proteome-pattern discriminated with over 70% accuracy between the severe groups. This suggests that beyond common underlying mechanisms, such as acute inflammation, there are significant differences in the pathogenesis of the severe syndromes studied.
Our large cohorts allowed us to statistically validate the patternbased proteome definitions of the major childhood malaria syndromes. Although the mass spectrometry platform used in our study does not provide direct molecular identification, the chromatographic chemistry used and the mass-to-charge (m/z) ratio can be exploited to guide the identification of the set of proteins relevant for discrimination between syndromes. Plasma proteome profiling has been used to define a variety of disease states [16, [19] [20] [21] [22] [23] [24] as there is growing recognition of the advantages of using 'omics'-based methods to achieve sufficient levels of accuracy [24] . Our study showed that complex plasma protein patterns were necessary to discriminate between the different malaria syndromes. This further underlines the advantage of using unbiased high-throughput pattern recognition based methods.
In many infectious diseases, there are clinically important distinctions to be made between different manifestations associated with the same underlying pathogen and malaria clinical syndromes are a clear case in point. The pathogenesis of malaria due to its erythrocytic cycle occurs in the cardiovascular system and it is plausible that proteome changes in organs such as brain, spleen, kidney and bone marrow can be reflected in the plasma proteome. Our study confirms that there are proteome changes characteristic of the clinical malarial syndromes with different level of accuracy. Furthermore, host modulation by the pathogen is likely to generate changing patterns of protein expression associated with the progression of severe malaria syndromes and our current studies are designed to address such changes.
The lack of specific childhood malaria definitions has limited the progress on understanding the pathology of the major severe syndromes. To the best of our knowledge this study is the first to show that a panel of proteins, defined as a proteome-pattern, dissects clinical malaria syndromes. Further identification of the proteins that comprise the proteome-patterns will provide hints to the underlying pathogenesis on each of the syndromes. Furthermore, these proteome-patterns provide a reference point to facilitate the identification of other complex and overlapping severe childhood malaria syndromes.
Data S1 Solid-phase fractionation data. (XLS) Data S2 Liquid-phase fractionation data. (XLS) Predicting pseudoknotted structures across two RNA sequences Motivation: Laboratory RNA structure determination is demanding and costly and thus, computational structure prediction is an important task. Single sequence methods for RNA secondary structure prediction are limited by the accuracy of the underlying folding model, if a structure is supported by a family of evolutionarily related sequences, one can be more confident that the prediction is accurate. RNA pseudoknots are functional elements, which have highly conserved structures. However, few comparative structure prediction methods can handle pseudoknots due to the computational complexity. Results: A comparative pseudoknot prediction method called DotKnot-PW is introduced based on structural comparison of secondary structure elements and H-type pseudoknot candidates. DotKnot-PW outperforms other methods from the literature on a hand-curated test set of RNA structures with experimental support. Availability: DotKnot-PW and the RNA structure test set are available at the web site http://dotknot.csse.uwa.edu.au/pw. Contact: janaspe@csse.uwa.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. Macromolecules such as DNA, RNA and proteins have the ability to form diverse tertiary structures, which enable functionality and thus, life. For many decades, proteins were deemed the global players in the cell until RNA entered the spotlight. For example, RNA structures have been found to be catalytically active, which was assumed to be the privilege of proteins. Furthermore, small RNAs are known to regulate gene expression and RNA viruses employ a plethora of structure elements to invade the host cell.
To gain insight into macromolecule function, one must investigate the structure. The first step in RNA folding is stable base pairing that leads to a secondary structure. As RNA structure formation is of hierarchical nature, secondary structure is the basis for the tertiary fold that produces the functional structure. Especially for RNAs, structure determination by experimental means is an intricate and expensive task. Computational RNA structure prediction is therefore an invaluable tool for biologists. Comparative structure prediction is considered the most reliable approach for computational RNA structure prediction. Single sequence structure prediction is always limited by the accuracy of the underlying folding model. Three main streams have been identified for comparative RNA secondary structure prediction: (i) predict a structure from a pre-computed sequence alignment; (ii) simultaneously compute an alignment and a structure and (iii) alignment-free methods (Gardner and Giegerich, 2004) .
Tools for multiple sequence alignments such as ClustalW (Thompson et al., 1994) are readily available and thus, structure prediction from an alignment is a tempting approach [e.g. RNAalifold (Hofacker et al., 2002) ]. Such methods heavily depend on the sequence conservation and quality of the underlying alignment. However, ncRNAs are conserved rather on the structure level than on the sequence level. The gold standard of RNA comparative structure prediction is the Sankoff approach as it does not rely on a high-quality sequence alignment and captures the structural conservation of ncRNAs. Sankoff (1985) introduced a theoretical dynamic programming algorithm for simultaneous folding and aligning for a set of N sequences that takes O(n 3N ) time and O(n 2N ) space. Practical variants have been derived which more or less retain the Sankoff principle by sacrificing optimality. Alignment-free methods aim to avoid the pragmatic restrictions made in a practical Sankoff approach as well as the reliance on a high-quality alignment [e.g. CARNAC (Perriquet et al., 2003) ]. Note that all of these comparative structure prediction methods exclude the prediction of RNA pseudoknots.
RNA pseudoknots are crossing structure elements with diverse functions. The principle of pseudoknot formation is that bases within a loop region pair with complementary unpaired bases outside the loop. From an algorithmic point of view, even the simplest type of pseudoknot adds considerable computational demands due to crossing base pairs. In fact, the majority of comparative RNA structure prediction methods exclude pseudoknots. Biologists have delivered a wealth of studies, which show that pseudoknots have an astonishing number of diverse functions and occur in most classes of RNA (Staple and Butcher, 2005) . RNA viruses use pseudoknots for hijacking the replication apparatus of the host (Brierley et al., 2007) .
A limited number of RNA comparative structure prediction methods can handle pseudoknots due to the computational complexity. Several of these methods take a sequence alignment as an input. ILM is an algorithm that takes as an input either individual sequences or a sequence alignment (Ruan et al., 2004) . A base pair score matrix is prepared initially and helices are added to the structure in an iterative fashion. In the approach hxmatch, a maximum weighted matching algorithm with combined thermodynamic and covariance scores is used (Witwer et al., 2004) . This program gives the option to be combined with RNAalifold.
KNetFold is a machine learning method, which takes a sequence alignment as an input and outputs a consensus structure allowing pseudoknots (Bindewald and Shapiro, 2006) . Simulfold takes an alignment as an input and simultaneously calculates a structure including pseudoknots, a multiple-sequence alignment and an evolutionary tree by sampling from the joint posterior distributions (Meyer and Miklos, 2007) . Tfold combines stem stability, covariation and conservation to search for compatible stems and subsequently for pseudoknots for a set of aligned homologous sequences (Engelen and Tahi, 2010) . Several comparative structure prediction methods including pseudoknots do not rely on an initial sequence alignment. The graph-theoretical approach comRNA computes stem similarity scores and uses a maximum clique finding algorithm to find pseudoknotted structures (Ji et al., 2004) . SCARNA performs pairwise structural alignment of stem fragments with fixed lengths derived from the probability dot plot (Tabei et al., 2008) .
In the following, a novel comparative approach for predicting structures including H-type pseudoknots called DotKnot-PW will be introduced. The input consists of two unaligned, evolutionarily related RNA sequences. Similarity scores between structure elements will be calculated. Statistically significant pairs will be used to find the set of conserved structure elements common to two sequences, which maximize a combined thermodynamic and similarity score. Using a hand-curated test set of pseudoknotted structures with experimental support, the prediction accuracy of DotKnot-PW will be compared with methods from the literature.
Pseudoknots are functional elements in RNA structures and therefore, the most promising approach for comparative prediction is a structure comparison with less focus on exact sequence matching. In fact, perfect conservation on the sequence level can be more of a curse than a blessing. Especially ncRNAs are known to evolve quickly and so-called consistent and compensatory base pairs in both sequences will give much more confidence for structure conservation than a sequence alignment. One strong point of the DotKnot method for single sequence pseudoknot prediction (Sperschneider and Datta, 2010; Sperschneider et al., 2011) is that the set of possible H-type pseudoknot candidates (and secondary structure elements) is explicitly computed and thus readily available for further investigation. The main steps in the pairwise pseudoknot prediction approach DotKnot-PW are as follows ( Fig. 1) :
(1) Run DotKnot for two unaligned sequences Seq x and Seq y . This returns secondary structure element and Htype pseudoknot candidate dictionaries.
(2) Calculate pairwise base pair similarity scores for secondary structure elements and H-type pseudoknot candidates. Keep significant pairs that have a low estimated P-value.
(3) Use significant pairs to calculate the set of conserved structure elements and pseudoknots for the two sequences that maximizes a combined free energy and similarity score.
The key point of the DotKnot-PW approach is how to score the similarity of stems, secondary structure elements and H-type pseudoknot candidates derived from sequences Seq x and Seq y . Related work has been done for stem finding in unaligned sequences, where stem candidates are assigned a matching score across unaligned sequences, e.g. in SCARNA. Another point is how to assess the significance of a similarity score using Pvalues. These points will be explained in detail in the following section.
For two unaligned RNA sequences Seq x and Seq y , the single sequence prediction method DotKnot (Sperschneider and Datta, 2010; Sperschneider et al., 2011) returns two stem dictionaries D s (x) and D s (y) derived from the probability dot plot. It also returns secondary structure element dictionaries D L s ðxÞ, D L s ðyÞ and D M s ðxÞ, D M s ðyÞ and H-type pseudoknot candidate dictionaries D p (x) and D p (y) (Fig. 1) . To detect conserved structure elements for the two sequences, a pairwise structural comparison is performed. Instead of a full structure-tostructure alignment, which takes O(n 4 ) time and O(n 3 ) space, pairwise base pair similarity scores are calculated using the RIBOSUM85-60 matrix for base pair substitutions (Klein and Eddy, 2003) .
For two given stems s i (x) and s j (y) with fixed lengths in sequences Seq x and Seq y , respectively, the base pair similarity score sim[s i (x), s j (y)] is calculated using an ungapped local structure alignment of the base pairs with the RIBOSUM85-60 matrix. As an example, consider the following optimal ungapped local structure alignment of the two stems with base pair similarity score of sim[s 1 (x), s 2 (y)] ¼ 22.04 using the RIBOSUM85-60 matrix.
)))))--To evaluate the significance of base pair similarity scores instead of the raw score, one has to find out what the underlying probability distribution is. Similar to the case of ungapped local sequence alignments (Karlin and Altschul, 1990) , it is assumed here that the base pair similarity scores follow an extreme value distribution. However, the main difference is that a comparison between fixed-length stem fragments is made. It is important to remember that parameters and K describe the extreme value distribution of optimal local alignment scores in the asymptotic limit of long sequences (Altschul et al., 2001) . Here, the parameters for the generalized extreme value distribution are pre-calculated using maximum Fig. 1 . For two unaligned RNA sequences Seq x and Seq y , DotKnot-PW produces structure element dictionaries derived from the probability dot plot. Similarity scores and P-values are computed to detect conserved elements likelihood fitting of a distribution to the histogram of a large sample of random base pair similarity scores. The maximum likelihood fitting was performed using the ismev package of the R statistical language for a range of stem lengths (see Supplementary Material). The P-value is defined as the probability to obtain a score greater than or equal to the observed score strictly by chance. A stem s i (x) in sequence Seq x and a stem s j (x) in sequence Seq y are a significant pair if the score sim[s i (x), s j (y)] has an estimated P-value less than . Stem pairs with a P-value larger than are not considered in the following.
For two interrupted stems, the base pair similarity score is calculated by deleting bulges and internal loops and scoring stems as consecutive base pairs. Base pair similarity scores for regular and interrupted stems are also calculated if the difference in number of base pairs is less than 5. For example, a stem with one bulge might be a conserved match with a regular stem. A stem s i (x) in sequence Seq x and a stem s j (y) in sequence Seq y are a significant pair if the score sim[s i (x), s j (y)] has an estimated P-value less than .
Calculating the base pair similarity score for two multiloop structures is complex due to the variety of inner loop elements, which may be regular or interrupted stems. A multiloop s M i ðxÞ can be decomposed into an outer stem s o i ðxÞ and a set of inner structure elements
The base pair similarity score sim½s o i ðxÞ, s o j ðyÞ for the outer stems of two multi-loops can be easily obtained from the previously calculated base pair similarity scores. If the outer stem is a conserved match, a local alignment on the set of inner structure elements is used to find the base pair similarity score. Here, gaps are allowed in the local alignment of inner structure elements; however, no gap penalty is used. Let two sets of inner structure elements S i (x) ¼ [s 1 (x),. . ., s n (x)] and S j (y) ¼ [s 1 (y),. . ., s m (y)] be given. Let H(i, j) be the maximum similarity score between a suffix of S i (x) and a suffix of S j (y). The optimal local alignment is calculated as follows:
Hði, 0Þ ¼ 0, 0 i n Hð0, jÞ ¼ 0, 0 j m Hði, jÞ ¼ max 0 Hði À 1, jÞ Hði À 1, j À 1Þ þ sim½s i ðxÞ, s j ðyÞ Hði, j À 1Þ
A multiloop s M i ðxÞ in sequence Seq x and a multiloop s M j ðyÞ in sequence Seq y are a significant pair if the similarity score sim½s M i ðxÞ, s M j ðyÞ has an estimated P-value less than .
A H-type pseudoknot has two pseudoknot stems S 1 and S 2 . The prerequisite for a conserved pseudoknot pair is that both core H-type pseudoknot stem pairs [S 1 (x), S 1 (y)] and [S 2 (x), S 2 (y)] are significant. The base pair similarity score for two H-type pseudoknots p i (x) and p j (y) in sequences Seq x and Seq y , respectively, is the sum of base pair similarity scores for the core pseudoknot stems as well as the base pair similarity score from a gapped local alignment of the recursive secondary structure elements in the loops (as described for multiloops). A pseudoknot p i (x) in sequence Seq x and a pseudoknot p j (y) in sequence Seq y are a significant pseudoknot pair if the similarity score sim[p i (x), p j (y)] has an estimated P-value less than .
The base pair similarity score calculated in the previous sections might not be powerful enough to distinguish true positive conserved structure element pairs from false-positive structure element pairs due to the finite lengths of stems and exclusion of loop sequences in the alignment. Therefore, a dissimilarity score is also used to confirm whether a pair is significant. The dissimilarity for two given structure elements s i (x) and s j (y) in sequences Seq x and Seq y is defined as:
where dissim 1 is the difference in the stem lengths and dissim 2 is the difference in the number of loop lengths. As an example, consider the pseudoknot pair p 1 (x) and p 1 (y) in sequences Seq x and Seq y , respectively, with stems S 1 , S 2 and loops L 1 , L 2 , L3. The pseudoknot pair has dissimilarity of 6. [[[[[[.) )))))) [.[[[[[[[.) ))))). .
A weight is assigned to a significant pair, which is a combination of the free energy, covariation and dissimilarity. The overall weight s of a significant structure element pair [s i (x), s j (y)] in sequences Seq x and Seq y is a combination of the free energy weights w[s i (x)] and w[s j (y)], base pair similarity score sim[s i (x), s j (y)] and dissimilarity dissim[s i (x), s j (y)]: s½s i ðxÞ, s j ðyÞ ¼ Â sim½s i ðxÞ, s j ðyÞ À Â fw½s i ðxÞ þ w½s j ðyÞg À Â dissim½s i ðxÞ, s j ðyÞ Only structure element pairs with positive score s are allowed in the following dynamic programming algorithm. Here, and are set to 0.5 and is set to 1.
Let p x 1 , . . . , p x n be the number of structure elements in the first sequence Seq x and p y 1 , . . . , p y m be the number of structure elements in the second sequence Seq y . Each structure element has a left and right endpoint in the sequence and is a stem, interrupted stem, multiloop or H-type pseudoknot. Structure elements can also be represented as nodes in a graph. In each sequence, the structure elements are ordered by their right endpoints. An edge is drawn between two structure elements in the first and the second sequence if their base pair similarity score has a P-value less than . Given the set of edges between nodes p x 1 , . . . , p x n and p y 1 , . . . , p y m , the goal is to find the set of edges with maximum weight that are non-crossing. This relates to finding the set of non-overlapping structure elements in the two sequences that maximize the score under the requirement that the interval ordering is preserved. A set of structure elements in the first and second sequence, which preserves the interval ordering is called a feasible structure element alignment and must satisfy the following two requirements. Each structure element can be aligned with at most one other structure element in the other sequence. The order of structure elements must be preserved with respect to the alignment. That is, if structure elements p x i and p x j in the first sequence are aligned with p y a and p y b in the second sequence, respectively, the pairs may never overlap: p x i 5p x j^p x a 5p x b (Fig. 2) . Given nodes p x 1 . . . , p x n in the first sequence Seq x and p y 1 , . . . , p y m in the second sequence Seq y , let f(i, a) be the maximum sum of edge weights for nodes between 1 and i in the first sequence and 1 and a in the second sequence such that the edges are non-crossing (i n and a m). The nodes that maximize the sum of edge weights are called an optimal structure element alignment for the two sequences. The optimal structure element alignment is calculated using dynamic programming. For a given structure element p i with start point a i and end point b i , let pre(i) be the non-overlapping predecessor. For each structure element, its predecessor is pre-computed using the sorted list of structure elements. The recursion for calculating the optimal structure element alignment is as follows:
Furthermore, nested structures are taken into account for significant outer stem pairs, which have estimated P-value less than . For each significant outer hairpin loop pair, the optimal structure element alignment of inner elements is computed.
For two unaligned RNA sequences Seq x and Seq y , the single sequence prediction method DotKnot returns structure element dictionaries derived from the probability dot plot. Let n and m be the number of structure elements in sequences Seq x and Seq y , respectively. Calculating the similarity scores and the optimal structure element alignment takes O(nm) time. Furthermore, nested structures are taken into account for significant outer stem pairs, which have estimated P-value less than . Let a be the number of significant stem pairs, where both stems are hairpin loops. For each significant outer hairpin loop pair, the optimal structure alignment of inner elements is computed. In the worst case, this increases time requirements to O(a  nm). The number of structure elements depends on the base composition of the sequence. Empirically, n and m can be observed to grow linearly with the length of the sequence for uniform base distribution (see Supplementary Material). In practice, DotKnot-PW can be expected to run in the order of minutes for sequences shorter than 500 nt.
Many pseudoknot prediction programs have been evaluated using all the entries in the PseudoBase database (van Batenburg et al., 2000) . There are several caveats in this approach. First, the sequences given in PseudoBase are those which exactly harbor the pseudoknot. However, in practice structure prediction algorithms will be applied to longer sequences without prior knowledge of the pseudoknot location. Second, long-range pseudoknot entries appear in a truncated version in the database. Third, some classes of pseudoknots have a large number of entries (such as short H-type pseudoknots in the 3 0 -untranslated regions of plant viruses), whereas more complex types of pseudoknots only have one representative (such as long-range rRNA pseudoknots). Therefore, a hand-curated dataset of pseudoknot structures will be used here.
When it comes to pseudoknots, many structures have been published based on a secondary structure predicted by free energy minimization. These predicted secondary structures are used as a working model and refined using experimental techniques such as chemical and enzymatic probing. However, the native structure remains unsolved unless tertiary structure determination methods such as X-ray crystallography are used. Testing structures that are based on computer predictions with no experimental support creates a bias in the benchmark and will be avoided in this evaluation.
A total of 16 pseudoknotted reference structures from different RNA types were collected, which have strong experimental support. For each reference structure, a supporting set of 10 evolutionarily related sequences was obtained from the RFAM database (Gardner et al., 2010) . Note that for the vast majority of supporting sequences, no experimentally determined structures are available. The average pairwise sequence identities vary from 55% to 99%. Given a reference structure, the performance of prediction algorithms is evaluated in terms of sensitivity (S), i.e. the percentage of base pairs in the reference structure, which are predicted correctly, as well as positive predictive value (PPV), i.e. the percentage of predicted pairs, which are in the reference structure. The Matthews correlation coefficient (MCC) is also reported and is in the range from À1 to 1, where 1 corresponds to a perfect prediction and À1 to a prediction that is in total disagreement with the reference structure. The performance of each method for predicting the reference structure was evaluated as described in Gardner and Giegerich (2004) .
DotKnot-PW was compared with methods that are freely available and use standard input and output formats. The comparative methods are CARNAC, Tfold and hxmatch (with the -A option using RNAalifold). All of these methods return structure predictions for only the reference structure with regards to the support set of evolutionarily related sequences. Tfold and hxmatch take a sequence alignment as the input. ClustalW with the default parameters was used to produce the initial sequence alignment. DotKnot-PW and CARNAC take a set of unaligned sequences as the input. Furthermore, prediction results for the reference sequence (not the supporting sequences) were obtained from the single sequence methods DotKnot (Sperschneider and Datta, 2010; Sperschneider et al., 2011), ProbKnot (Bellaousov and Mathews, 2010) , IPknot (Sato et al., 2011) and RNAfold (Hofacker et al., 1994) . Note that all methods except CARNAC and RNAfold allow pseudoknot prediction.
The results are shown in Table 1 . DotKnot-PW has the highest average MCC of 0.75 for the test sequences. For each reference structure with the 10 support sequences from the corresponding RFAM family, 10 predictions are returned ordered by the combined free energy and similarity score. If only the Fig. 2 . A set of edges with positive scores is given between nodes p 1 ,. . .,p 7 in the first sequence and p 1 ,. . .,p 6 in the second sequence. The goal is to find the best set of non-overlapping structure elements in the two sequences such that the interval ordering is preserved. The optimal structure element alignment, which preserves the interval ordering includes structure elements p 1 , p 4 , p 7 in the first sequence and p 1 , p 4 , p 6 in the second sequence pairwise prediction with highest combined free energy and similarity score is taken, DotKnot-PW has an improved average MCC of 0.81. Tfold and hxmatch have average MCC of 0.6 and 0.59, respectively. CARNAC has average MCC of 0.45 with much higher average specificity than sensitivity. The prediction results for single sequence structure prediction for each of the reference sequences with experimentally determined structures are also shown in Table 1 . Note that this does not include the prediction for the support sequences from RFAM, as no experimentally determined structures are available. All single sequence pseudoknot prediction methods show improved results over using RNAfold. DotKnot has the highest average MCC of 0.76, followed by IPKnot and ProbKnot.
As an example, consider the S15 mRNA pseudoknot that binds to specific proteins in the autoregulation mechanism of ribosomal protein S15 synthesis (Philippe et al., 1995) . For the reference sequence S15 and 10 support sequences from the corresponding RFAM family, DotKnot-PW returns pairwise predictions ordered by the combined free energy and similarity score. The top two pairwise predictions with the highest scores are shown in Figure 3 .
We presented DotKnot-PW for prediction of structures common to two RNA sequences, including H-type pseudoknots. Both DotKnot and DotKnot-PW have been designed as dedicated pseudoknot prediction tools. In the following, important aspects of pseudoknot prediction will be discussed.
Single sequence prediction methods are always limited by the underlying RNA folding model. This may be the set of free energy parameters used by free energy minimization methods or the underlying methodological framework such as maximum expected accuracy methods. DotKnot-PW shows excellent results on H-type pseudoknots with short interhelix loops. For this type of pseudoknots, DotKnot-PW uses free energy pseudoknot parameters by Chen (2006, 2009 ) based on polymer statistical mechanics. Improvements of the accuracy of free energy parameters, both for secondary structures and pseudoknots, will lead to more accurate prediction methods. However, one has to keep in mind that the algorithms themselves must be designed in such a fashion that novel parameters can be efficiently incorporated. The heuristic framework of DotKnot-PW has been designed such that it can incorporate sophisticated free energy parameters for pseudoknots, secondary structures and coaxial stacking. In the future, DotKnot-PW could also use contributions from basic tertiary structure elements such as base triples around the pseudoknot junction or stem-loop interactions.
Pseudoknot prediction algorithms come in two flavors: either they can predict a certain, restricted class of pseudoknots or they do not have a restriction on the type of pseudoknot that can be predicted. For methods using free energy parameters, the inclusion of general types of pseudoknots might be more of a 
Each reference structure is given by its ID (see Supplementary Material for dot-bracket notation). The following column gives the method of experimental support (NMR, NMR spectroscopy; X-ray, X-ray crystallography; SC, sequence comparison; MG, mutagenesis; SP, structure probing), length of the sequence and number of pseudoknots. For each reference structure, the corresponding RFAM family ID, average sequence length and average pairwise sequence identity is shown. The * symbol means that the method failed to run. The 'first' prediction for DotKnot-PW is the pairwise prediction with highest combined free energy and similarity score.
curse than a blessing, as no reliable free energy parameters for complex pseudoknots are available. DotKnot-PW has restrictions on the type of pseudoknot that can be predicted. However, this does not always lead to poor prediction results in practice. For example, DotKnot-PW shows the best result for the HDV ribozyme, which is a complex double nested pseudoknot.
The results from the benchmark for structure prediction in Table 1 must be interpreted with care. First, the tested methods can be run with different parameters, possibly producing better results. However, as a typical user has no prior knowledge about the structure, the default parameters for each method are used. Of course, a comprehensive benchmark should include a larger number of structures to obtain a more reliable evaluation. However, in this study, the focus has been on a test set where the structures are supported experimentally. Many structures have been published, which were determined using computational tools and this will inevitably create a bias in a benchmark, and thus they were excluded here.
Here, an extension of the single sequence prediction method DotKnot was presented based on the pairwise comparison of structure elements. This approach called DotKnot-PW is designed as an algorithm for finding the structure including H-type pseudoknots common to two sequences. As shown in Table 1 , DotKnot-PW can greatly improve structure predictions for RNA families when compared with the single sequence prediction using DotKnot. In some cases, a comparative approach might have lower sensitivity than a single sequence prediction; however, this should not generally be judged as 'inferior'. For example, ncRNAs might preserve some integral base pairs throughout evolution and only these will be detected by a comparative approach, which returns the set of base pairs common to a set of evolutionarily related sequences. DotKnot-PW uses a set of unaligned sequences as the input; therefore, no expert user intervention is required. In the future, DotKnot-PW will be extended to include intramolecular kissing hairpins. Furthermore, constrained folding will be implemented to predict a structure subject to constraints, e.g. enforce certain base pairs or regions, which must remain unpaired.
DotKnot-PW has been designed as a dedicated pseudoknot prediction tool and should be applied to RNA sequences where pseudoknotted interactions are suspected in the structures. Prediction accuracy will inevitably decrease for sequences, which are longer than say 400 nt for any single sequence structure prediction method (Reeder et al., 2006) . To achieve reliable results, short sequences should be folded using DotKnot and predictions should be compared with results from other methods from the literature. To gain confidence in predictions, subsequent comparative prediction using DotKnot-PW and other comparative methods is highly recommended. Ideally, experimental verification of computationally predicted pseudoknots should be sought. Fig. 3 . Pairwise prediction results for the S15 mRNA pseudoknot (RFAM family RF00114) with the top two combined free energy and similarity scores. The reference structure is shown at the top and folds into two conformations in dynamic equilibrium: a H-type pseudoknot or a series of hairpins.
For the pairwise prediction with highest score, the pseudoknot structure is returned. For the second-best pairwise prediction, the alternative hairpin loop structure is returned